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Cellular Levels of Oxidative Stress Affect the Response of Cervical Cancer Cells to Chemotherapeutic Agents

Cellular Levels of Oxidative Stress Affect the Response of Cervical Cancer Cells to... Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 574659, 14 pages http://dx.doi.org/10.1155/2014/574659 Research Article Cellular Levels of Oxidative Stress Affect the Response of Cervical Cancer Cells to Chemotherapeutic Agents 1 1 1 2 Maria Filippova, Valery Filippov, Vonetta M. Williams, Kangling Zhang, 1 1 1 Anatolii Kokoza, Svetlana Bashkirova, and Penelope Duerksen-Hughes Department of Basic Sciences, Loma Linda University School of Medicine, 11021 Campus Street, 101Alumni Hall,LomaLinda,CA92354,USA Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA Correspondence should be addressed to Penelope Duerksen-Hughes; pdhughes@llu.edu Received 12 June 2014; Revised 13 August 2014; Accepted 21 August 2014; Published 16 November 2014 Academic Editor: Wan-Liang Lu Copyright © 2014 Maria Filippova et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Treatment of advanced and relapsed cervical cancer is frequently ineffective, due in large part to chemoresistance. To examine the pathways responsible, we employed the cervical carcinoma-derived SiHa and CaSki cells as cellular models of resistance and sensitivity, respectively, to treatment with chemotherapeutic agents, doxorubicin, and cisplatin. We compared the proteomic profiles of SiHa and CaSki cells and identified pathways with the potential to contribute to the differential response. We then extended these findings by comparing the expression level of genes involved in reactive oxygen species (ROS) metabolism through the use of a RT- PCR array. eTh analyses demonstrated that the resistant SiHa cells expressed higher levels of antioxidant enzymes. Decreasing or increasing oxidative stress led to protection or sensitization, respectively, in both cell lines, supporting the idea that cellular levels of oxidative stress aeff ct responsiveness to treatment. Interestingly, doxorubicin and cisplatin induced different profiles of ROS, and these differences appear to contribute to the sensitivity to treatment displayed by cervical cancer cells. Overall, our findings demonstrate that cervical cancer cells display variable profiles with respect to their redox-generating and -adaptive systems, and that these different profiles have the potential to contribute to their responses to treatments with chemotherapy. 1. Introduction that they produce response rates of 15%–20%. Recent and ongoing trials are also likely to identify additional active Worldwide, cervical cancer is the second most common drugs [3]. eTh lowresponseratetothese agents canbe cancer in women; approximately 400 000 new cases of this attributed to the fact that invasive cervical cancer appears to disease are diagnosed each year, of which approximately half be relatively chemoresistant, as compared to other gyneco- will lead to death. The causative agents of most cases of logic tumors such as those of the breast or ovaries [3]. Studies cervical carcinomas are the high-risk types of human papil- in breast, prostate, and colorectal cancers have shown that lomaviruses (HPV). When cervical carcinomas are detected many factors can contribute to chemoresistance, including an at earlyorpreinvasive stages,theyare oeft n curablewithlocal individual’s geneticbackgroundaswellasepigeneticfactors treatments, most of which are based on ablative approaches. [4]. However, such studies have not yet analyzed the causes Unfortunately, a significant proportion of patients diagnosed of chemoresistance in cervical cancer. An understanding of with invasive cervical cancer sueff r relapses following initial the molecular events that lead to chemoresistance in the cells treatment. For this reason, the development of novel and comprising cervical carcinomas may lead to the discovery of effective therapeutic interventions, such as those based on new targets for chemical intervention. molecular techniques, remains an important priority [1, 2]. CaSki and SiHa cells represent useful cellular models More than 20 different chemotherapeutic agents are now for cervical carcinoma, as both lines contain an integrated considered active in the treatment of cervical carcinomas, in form of HPV16. Interestingly, however, they respond quite 2 BioMed Research International dieff rently to treatment with agents that induce cell death recommended by the TMT Mass Tagging Kit (Thermo Fisher through both intrinsic [5, 6] and extrinsic [7]apoptotic Scientific) protocol. Trypsin was added at a protein/enzyme pathways. In spite of the significant differences in the molec- ratio of 30 : 1 by mass and the digestion was performed at ular pathways involved (e.g., DNA-damaging agents versus 37 C overnight. Peptides were labeled with TMT (tandem- ligands that induce ligand-mediated apoptosis), one common mass-tagging)reagentsaccordingtothe manufacturer’s pro- observation stands out: CaSki cells are more sensitive to each tocol in duplicate and equal amounts of labeled peptides of these treatments than are SiHa cells. eTh reason(s) for these were combined to obtain one sample, which was separated dramatically different responses have not yet been identified. into 9 fractions by strong cation exchange chromatography It has been suggested that dieff rences in the levels of p53 [ 8, 9] using TopTip columns (PolyLC). Elution was performed and/or procaspase 8 [7, 10]could contribute. with increasing concentrations of KCl (from 0 to 0.5 M). In the current study, we compared the proteomic profiles Eluates were dried using a SpeedVac and then desalted of SiHa and CaSki cells and identified pathways with the using C18/hypercarb TopTip columns (PolyLC). Samples potential to contribute to the dieff rential response to chemo- from each fraction were runintriplicateonanOrbitrapPro therapeutic agents. We then extended these findings by ana- mass spectrometer that was coupled to a nanoLC (Thermo lyzing and comparing the expression level of genes involved Fisher Scientific), and the spectra obtained were analyzed in reactive oxygen species (ROS) metabolism through the use with Proteome Discoverer 1.3 software against the Human of an RT-PCR array. Both sets of analyses demonstrated that International Protein Index (IPI) database. Peptides were the resistant SiHa cells expressed higher levels of antioxidant identiefi dwithaFDR(falsediscovery rate)oflessthan1%. enzymes. Decreasing or increasing oxidative stress using Proteins were considered differentially expressed if the fold pharmacological agents led to protection or sensitization, ratio was more than 1.5. Protein data were further analyzed respectively, in both cell lines, supporting the idea that using Ingenuity Pathway Analysis (IPA) software to identify cellular levels of oxidative stress affect responsiveness to treat- differences in pathways and networks between cell lines. ment. Interestingly, the two agents tested, doxorubicin (DOX) and cisplatin, induced different profiles of ROS, and these 2.5. Measurement of ROS in Cells by Flow Cytometry. Intra- differences appear to contribute to the differential sensitivity cellular generation of hydrogen peroxide (H O ), hydroxyl 2 2 observed. and peroxyl radicals (DCFDA), and superoxide (O )(DHE) 󸀠 󸀠 was estimated using either the 5-(and-6)-carboxy-2 ,7 - dichlorodihydrofluorescein diacetate (DCFDA) or dihy- 2. Materials and Methods droethidium (DHE) membrane permeable probes (Life Bio- 2.1. Reagents. Monoclonal𝛼 -NQO1,𝛼 -OXR1, and𝛼 -𝛽 -actin sciences). Reagents were diluted into culture media and then were obtained from Sigma-Aldrich, monoclonal 𝛼 -SOD1, added to cells to a n fi al concentration of 10 𝜇 M. After treat- and𝛼 -GPX 1/2 from Santa Cruz Biotechnology, monoclonal ment, the cells were collected in 1x PBS and analyzed using 𝛼 -SOD2 from BD Biosciences, and monoclonal 𝛼 -PARP1 the Becton-Dickinson FACSCalibur flow cytometer (Becton- (Ab-2) from Millipore Corporation (Calbiochem). tert-Butyl Dickinson, San Francisco, CA). DCFDA was detected in the hydroperoxide solution (tBHP), N-Acetyl-L-cysteine (NAC), FL-1 channel, while DHE was detected in the FL-2 channel. cis-diammineplatinum(II) dichloride (cisplatin), doxorubi- Data was collected in log scale and analyzed using Flow-Jo cin hydrochloride (DOX), and DL-buthionine-(S-R)-sulfox- software. imine (BSO) were purchased from Sigma-Aldrich. 2.6. Microscopy. CaSki and SiHa cells were seeded onto Cul- 2.2. Cell Culture. CaSki, SiHa, HeLa, and C33A cells (derived tureSlides (Falcon) one day prior to treatment with DOX or from human cervical carcinomas) were obtained from the cisplatin. The following day, cells were stained with DCFDA ATCC (Manassas VA). All cells were cultured in modied fi and DHE as described above (measurement of ROS in cells Eagle medium (MEM) (CellGro). The medium was supple- by flow cytometry). Fluorescent images were recorded using mented with 10% fetal bovine serum (Life Biosciences) and a Biorevo microscope (Keyence) at the same magnifications with penicillin (100𝜇 g/mL) andstreptomycin(100𝜇 g/mL) and the same settings. (Sigma-Aldrich). 2.7. Immunoblot Assays. For immunoblot analysis, 10 cells 2.3. Cell Treatments and Cell Viability Assay. Cells (1 or 1.5× were lysedin100𝜇 L of Laemmli lysis buffer and lysates 10 cells per well) were seeded onto a 96-well plate and were sonicated and separated by SDS-PAGE. Aeft r transfer allowed to incubate for 24 h, aer ft which they were treated of protein onto Immobilon P membranes (Millipore Corpo- with the indicated concentrations of drugs or tBHP. NAC ration) and blocking of the membrane with 1% bovine serum or BSO was also added where indicated. Cell viability was albumin dissolved in TBST, primary antibodies were applied monitored using crystal violet staining. The absorbance of overnight. Secondary ImmunoPure antibody (𝛼 -mouse or each well was determined at 590 nm using a plate reader. 𝛼 -rabbit), conjugated with horseradish peroxidase (Thermo Fisher Scientific), was applied onto the membrane for 1 h and 2.4. Proteomic Analysis. SiHa and CaSki cells (10 )were the detection of signal was performed using the chemilumi- lysed in RIPA lysis bueff r (Sigma-Aldrich) and sonicated. nescent SuperSignal West Dura or Pico maximum-sensitivity Cleared lysates were denatured, reduced, and alkylated as substrate (Thermo Fisher Scientific). BioMed Research International 3 2.8. RNA Isolation, RT-PCR, and qRT-PCR. Cells were plated an LTQ-Orbitrap mass spectrometer. eTh total number of onto a10cmtissueculture plateand allowedtogrowtosemi- proteins in which the level of expression between SiHa confluency. RNA was isolated using Tri Reagent according and CaSki cells dieff red by more than 1.5-fold was 430 to the manufacturer’s protocol (Sigma-Aldrich). cDNA was (Table 1; see Supplementary Material available online at synthesized using ImPromII reverse transcriptase (Promega) http://dx.doi.org/10.1155/2014/574659) and the detected range 󸀠 󸀠 and an oligo(dT) primer. Primers for the 5 and 3 ends of the of differences in protein levels between these cells ranged indicated genes were used to amplify PCR products. from−6.0 to 6.9 fold. Seventy-six of these proteins were found to be upregulated, while the remainder was downregulated in Quantitative qRT-PCR was conducted using the Abso- SiHa cells as compared to CaSki cells. lute QPCR Sybr green kit according to the manufacturer’s To gain insight into the functions of these differentially protocol (ABgene). eTh observed gene concentrations were expressed proteins, we employed the online IPA analysis normalized using PGK1 expression levels. (Ingenuity Systems) tool to group them into functionally related networks and pathways. Figure 2(a) summarizes 2.9. Oxidative Stress and Antioxidant Defense PCR Array. the 9 functions for which protein expression differs most The PCR Microarray was performed according to the manu- between these two lines. o Th ugh HPV16 E6 accelerates the facturer’s instructions (SABiosciences, a QIAGEN company, degradation of p53 [12], thereby significantly lowering its Valencia CA). Gene expression was compared according to cellular level, our mass spectroscopy-based method was able the𝐶 value. Normalization was performed for each cDNA to detect and quantify p53 in both SiHa and CaSki cells, sample using the average of ve fi housekeeping genes provided demonstrating a 2.5-fold higher level of p53 in CaSki than by manufacture. in SiHa cells (Table 1, Supplementary Material). IPA analysis revealed that additional proteins within the p53 signaling 2.10. Statistics. All assays were repeated at least three times pathways were downregulated in SiHa cells as compared to andthe resultsreportedasmean ± standard deviation. CaSki (Figure 2(a)). One downstream consequence of these Differences were analyzed by the Student’s 𝑡 -test.𝑃 ≤ 0.05 differences in p53-linked pathways is the difference in the was regarded as significant. expression of proteins involved in G2/M DNA damage check- point regulation (Figure 2(a) and Table 1, Supplementary 3. Results Material). Some of the more remarkable differences in protein levels 3.1. SiHa Cells Are More Resistant an Th CaSki Cells to Dox- between SiHa and CaSki cells were detected in proteins orubicin- and Cisplatin-Induced Cell Death. Doxorubicin involved in mitochondrial functions such as mitochondrial (DOX) and cisplatin are chemotherapeutic agents used to depolarization, swelling of mitochondria, and the biogenesis treat solid tumors, including cervical carcinomas [3]. To of mitochondria (Figure 2(a)). One important outcome of evaluate and compare the sensitivity of CaSki and SiHa cells proper mitochondrial functioning involves the production to these chemotherapeutic drugs, cells were treated with and safe transport of radicals, as well as the maintenance increasing concentrations of DOX and cisplatin. For the of free radical homeostasis in the cell. Another group of initial set of experiments, relatively high concentrations were pathways dieff rentially activated between these cell lines is applied (10–40𝜇 Mfor DOXand 16–128𝜇 Mfor cisplatin) connected to DNA repair and the DNA damage response and crystal violet staining was used to monitor cell viability (Figure 2(a) and Table 1, Supplementary Material). Differ- following treatment for 20 h (Figures 1(a) and 1(b)). With encesinthe expressionofproteinsinvolvedinmitochondrial both treatments, we found that SiHa cells were more resistant status and DNA repair were accompanied by changes in to treatment than were the CaSki cells. For example, cisplatin thelevelsofproteinsinvolvedinthe regulation of ROS at a concentration of∼30𝜇 M killed 50% of CaSki cells, while levels (Figure 2(a)). For example, NAD(P)H dehydrogenase, alossof50% viabilityfor SiHa cellswas observed at 128𝜇 M quinine 1 (NQO1), peroxiredoxin 2 (PRDX2), and superoxide cisplatin (Figure 1(b)). Similar results were observed for DOX dismutase 1 (SOD1), which are responsible for inactivation of (Figure 1(a)). These experiments were then repeated using superoxide radicals, were found in higher levels in SiHa than lower, more physiologically relevant concentrations (0.05𝜇 M in CaSki cells (Table 1, Supplementary Material). to 2𝜇 Mfor DOXand 0.2𝜇 Mto5𝜇 Mfor cisplatin) [11], for a To verify this proteomicdataaswellasthe dieff rences longer period of time (72 h) (Figures 1(c) and 1(d)). Again, we between these two cell lines with regards to expression of found that SiHa cells were more resistant to treatment than proteins involved in ROS metabolism, we evaluated the were CaSki cells. expression levels of a subset of the proteins involved in antiox- idant defense by immunoblot (Figure 2(b)). Consistent with 3.2. Proteomic Analysis Identified Differences in Pathways our proteomic data, the immunoblot analysis confirmed Connected to p53 Activation, Mitochondrial Function, and higher levels of NQO1 and SOD1 in SiHa cells and added Oxidative Stress. To identify differences in the pathways glutathione peroxidase 1/2 (Gpx1/2) and SOD2 to the list of differentially-expressed genes ( Figure 2(b)). A marker of through which sensitive CaSki cells and the more resistant SiHa cells responded to drug treatment, we performed a DNAdamagecausedbyoxidative stress,PARP1 wasalso comparative proteomic analysis. Identicfi ation and quanti-fi detected at higher level in CaSki than in SiHa cells as assessed cation of proteins were done by simultaneously running both by proteomic (Table 1, Supplementary Material) and TMT-labeled trypsinized CaSki and SiHa lysates through immunoblot analyses (Figure 2(b)). 4 BioMed Research International 016 32 64 128 0 102040 Cisplatin (𝜇 M) DOX (𝜇 M) CaSki CaSki SiHa SiHa (a) (b) 0 0.2 0.5 2 5 0 0.05 0.2 0.5 2 Cisplatin (𝜇 M) DOX (𝜇 M) CaSki CaSki SiHa SiHa (c) (d) Figure 1: SiHa cells are more resistant than CaSki cells to treatment with the chemotherapeutic drugs DOX (a and c) and cisplatin (b and d). (a and b) SiHa and CaSki cells (1.5× 10 cells per well) were seeded into a 96-well plate, allowed to incubate overnight, and then treated with the indicated concentrations of drugs for 20 h. (c and d) SiHa and CaSki cells (0.5× 10 cells per well) were seeded into a 96-well plate, allowed to incubate overnight, and then treated with the indicated concentrations of drugs for 72 h. Viability was measured by crystal violet and the viability of untreated cells was set at 100%. Each measurement was done in triplicate and error bars indicate the standard deviations of the means. To further identify ROS-related genes with differential alpha polypeptide (CYBA)isacomponentofmitochondrial expression between SiHa and CaSki cells, we employed the Complex III, which is involved in the transfer of electrons Oxidative Stress and Antioxidant Defense PCR Array (SA to Complex IV so that water can be formed (Figure 2(c) and Table 1). Other genes belong to various antioxidant systems. Biosciences), which profiles the expression of 84 genes related For example, SODs, GPXs, and PRDXs catalyze reactions to oxidative stress. We found that several of these genes were that inactivate superoxide radicals (SODs) or H O (GPXs upregulated in SiHa (as compared to CaSki) cells and that a 2 2 and PRDXs). Overall, our immunoblot (Figure 2(b)), semi- few were downregulated (Table 1). Genes whose expression quantitative RT-PCR (Figure 2(c)), and quantitative qRT- was consistently downregulated in SiHa (relative to CaSki) PCR (Figure 2(d)) confirmed our initial results from the PCR cells included aldehyde oxidase 1 (AOX1)(Figure 2(c)), array profiles. In summary, our data demonstrated significant NADPH oxidase complex (NCF2)(Figure 2(d)), and oxi- differences between SiHa and CaSki cells with regards to dation resistance protein (OXR1)(Figure 2(d)); products of the expression levels of genes and proteins involved in ROS these genes are responsible for the production of reactive metabolism and homeostasis. oxygen radicals. A reduced level of OXR1 expressioninSiHa cells as compared to CaSki cells was also confirmed by immunoblot (Figure 2(b)). Several genes that were upreg- 3.3. Levels of ROS and Oxidative Stress-Induced Cell Death ulated in SiHa (Table 1) participate in scavenging radicals AreHigherinCaSki aTh ninSiHaCells. The differential in one way or another. For example, cytochrome b-245, expression of pro- and antioxidant enzymes in CaSki and % of control (viable cells) % of control (viable cells) % of control (viable cells) % of control (viable cells) BioMed Research International 5 CaSki SiHa NQO1 SOD2 SOD1 GPx 1/2 PARP1 𝛽 -Actin OXR1 40 kDa isoform No overlap with dataset Upregulated 𝛽 -Actin Downregulated (a) (b) CaSki SiHa AOX1 CYBA PGK1 −5 (c) (d) Figure 2: SiHa and CaSki cells differ in expression levels of proteins involved in the regulation of ROS. (a) Differential regulation of nine pathways in SiHa and CaSki cells. The percentage of genes downregulated in SiHa as compared to CaSki cells is shown in black, upregulated in grey. (b) Immunoblot analysis confirms differential expression. Lysates prepared from 10 CaSki and SiHa cells using Laemmli lysis bueff r were subjected to SDS-PAGE. Immunoblots were performed using antibodies directed against the indicated proteins. Loading was normalized by blotting for𝛽 -actin. (c and d) Transcription levels of genes related to oxidative stress as determined by semiquantitative RT-PCR (c) and by quantitative qRT-PCR (d) differ between SiHa and CaSki cells. Total RNA was isolated from 10 cells of each cell line using Trizol Reagent (Sigma Aldrich) and cDNA was synthetized using oligo-dT. PCR and qPCR were performed using specific primers for the genes of interest. The PCR and qPCR products obtained using primers for the PGK1 transcript were used to normalize to cDNA input. (d) Differences in gene expression are presented as fold changes (SiHa versus CaSki). Each measurement was done in triplicate and error bars indicate the standard deviations of the means. SiHa cells is expected to inu fl ence the baseline levels of ROS emission spectra of 495 nm and 529 nm, respectively [13]. in these cells. To test this idea, SiHa and CaSki cells were In the case of DHE, its oxidation by superoxide results in stained with the DCFDA and DHE uo fl rescent dyes and hydroxylation at the 2-position. 2-hydroxyethidium exhibits the intensity of staining monitored by flow cytometry. Once a uo fl rescence excitation peak at ∼400 nm [14]. Data pre- DCFDA enters a cell, it is deacetylated by cellular esterases sented in Figure 3(a) clearly demonstrates that SiHa cells to form a nonfluorescent compound. ROS radicals such as display lower levels of the reactive oxygen species detected H O , hydroxyl, and peroxyl radicals then oxidize this non- by both DCFDA and DHE than do CaSki cells. 2 2 󸀠 󸀠 u fl orescent substrate into 2 ,7 -dichlorofluorescein, which is a These data suggested that cells with an elevated level highly u fl orescent compound with maximum excitation and of oxidative stress should be more susceptible to treatment (%) Hypoxia-inducible factor signaling Increases depolarization of mitochondria Oxidative stress Swelling of mitochondria NRF2-mediated oxidative stress response p53 signaling Fold changes SiHa/CaSki G2/M DNA damage checkpoint regulation Negative acute phase response proteins PGK1 Biogenesis of mitochondria OXR1 NCF2 PRDX1 GPX1 SOD1 SOD2 6 BioMed Research International Table 1: Relative expression (SiHa versus CaSki) of genes involved to DOX and to cisplatin (Figures 4(d) and 4(e)), with a greater in oxidative stress and antioxidant defense. eect ff seen with DOX than with cisplatin, and on CaSki cells with theirhigherbasal levelofROS than on SiHa cells. Genes Fold St. dev. Together, these experiments demonstrate that manipulating AOX1 −3.91951 2.099233 thelevel of cellular oxidativestresscan modify thecellular Downregulated genes NCF2 −73.405 14.30477 response to chemotherapy agents, such that higher levels of OXR1 −3.49989 0.529737 ROS level sensitize cells to chemotherapy-induced cell death. CYBA 4.275 2.269813 DUSP1 6.58 2.870854 3.5. C33A and HeLa Cells Display Dier ff ential Sensitivities to GPX1 1.6 0.381838 DOX and Cisplatin. The results described above demonstrate GPX5 2.085 1.421285 that higher intracellular levels of ROS increase sensitivity to GPX6 2.625 2.043539 DOX and cisplatin in the CaSki and SiHa cervical carcinoma cell lines. To further explore the connection between ROS GSS 2.085 1.421285 Upregulated genes levels and the cellular response to such agents, two additional PRDX1 4.315 0.982878 cervical cancer cell lines, HeLa and C33A, were analyzed PRDX2 13.195 11.10865 for their response to DOX and cisplatin as well as for their SELS 5.635 1.916259 intercellular levels of ROS. Cell viability following treatment SOD1 7.745 4.249712 with these agents is presented in Figures 5(a) and 5(b). SOD2 3.84 0.127279 Interestingly, of these four tested cell lines, CaSki cells remain SOD3 3.055 2.38295 the most sensitive and SiHa the most resistant to both agents. C33A and HeLa cells displayed an intermediate sensitivity, with C33A cells more sensitive than the HeLa cells to both with agents that further increase oxidative stress than cells agents. Interestingly, the resistance displayed by HeLa cells with intrinsically lower levels of oxidative stress. To test this to cisplatin treatment was very similar to that seen in SiHa idea, we compared the cellular response to additional external cells (Figure 5(b)), although HeLa cells were more sensitive oxidative stress by treating cells with tBHP for 20 h and then to DOXthanwereSiHacells (Figure 5(a)). estimating the level of cell death. We found that CaSki cells, Differences in the level of ROS as measured by DCFDA with their higher baseline levels of oxidative stress, were more andDHE betweenthese four cell lineswerealsonoted susceptible to cell death caused by additional oxidative stress (Figures 5(c) and 5(d)). In particular, while C33A cells than were SiHa cells (Figure 3(b)). displayed intermediate levels of ROS as assessed by both We then exposed CaSki and SiHa cells to an antioxidant, DCFDA and DHE, HeLa cells displayed the lowest levels of NAC, prior to treatment with t-BHP. Cellular levels of ROS ROS as detected by DCFDA and the highest levels of ROS as before and aeft r pretreatment with NAC, as estimated by detected by DHE. flow cytometry, are shown in Figure 3(c) and demonstrate that exposure to NAC decreased ROS levels as detected by 3.6. DOX and Cisplatin Treatments Induce Different ROS DCFDA and DHE in both cell lines. Importantly, pretreat- Proles. fi One question raised by the previous experiments ment with NAC increased the viability of the tBHP-sensitive concerned the differential response of HeLa cells to treat- CaSki cells. Pretreatment of SiHa cells with the same NAC ment with DOX (intermediate sensitivity, similar to that concentration (140𝜇 M) did not aeff ct their viability, presum- seen with the C33A cells) versus treatment with cisplatin ably becausetheywerealready tBHP-resistant (Figure 3(d)). (quite resistant, similar to that seen with SiHa cells). The Together, the data presented above suggest that internal mechanisms by which these two agents induce cytotoxicity levels of oxidative stress may affect cellular sensitivity to cytotoxic agents. differ significantly; cisplatin cross-links the DNA, while DOX intercalates into the DNA and produces DNA lesions. This dieff renceinmechanism suggestedthatthese twoagents 3.4. Changes in Cellular Oxidative Stress Aeff ct the Response of likely triggered different cell death pathways, with different Cells to Chemotherapeutic Agents. We next asked whether a effects on oxidative stress. To ask if this were indeed the case, change in ROS levels would also affect the cellular response CaSki and SiHa cells were treated with DOX or cisplatin and to actual chemotherapeutic drugs. Cells were pretreated with changes in ROS levels were assessed by flow cytometry. eTh NAC and then exposed to DOX (Figure 4(a))ortocisplatin results (Figure 6) showed, somewhat unexpectedly, that DOX (Figure 4(b)). Pretreatment with NAC for 4 h protected both CaSki and SiHa cells from cell death induced by DOX and and cisplatin induced different profiles of ROS. Cisplatin cisplatin. treatment, in both cell lines, primarily increased the levels of We also asked whether increasing ROS would aeff ct agents detected by DCFDA (H O , hydroxyl and peroxyl rad- 2 2 the cellular response to chemotherapy agents by pretreating icals (Figure 6(b)), while DOX treatment increased primarily CaSki and SiHa cells with BSO, an inhibitor of glutathione the levels of superoxides as detected by DHE (Figure 6(a)). A [15]. Flow cytometry (Figure 4(c)) demonstrated that the similar eeff ct, showing DOX-induced increases in superoxide ability of BSO to reduce glutathione levels did indeed lead to (DHE) and cisplatin-induced increase in hydrogen peroxide, an increase in cellular ROS as detected by DCFDA and DHE. hydroxyl radicals, and peroxyl radicals (DCFDA), was noted Pretreatment with BSO sensitized both CaSki and SiHa cells in HeLa and C33A cells (data not shown). BioMed Research International 7 100 100 80 80 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 DHE DCFDA CaSki CaSki SiHa SiHa (a) 0 100 140 160 200 tBHP (𝜇 M) CaSki SiHa (b) ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ CaSki SiHa NAC (mM) tBHP DHE DCFDA DHE DCFDA No NAC CaSki 2 mM NAC SiHa (c) (d) Figure 3: CaSki cells display higher levels of ROS than do SiHa cells (a), the cell viability of CaSki cells decreases more than in SiHa cells aer ft treatment with tBHP (b). Pretreatment with NAC decreases ROS in CaSki and SiHa cells (c) and pretreatment with NAC protects cells from death induced by treatment with tBHP (d). (a) 10 cells of each line were treated with 10𝜇 MDCFDA or 10𝜇 M DHE in media and then incubated in the dark at 37 C for 30 minutes. eTh cells were washed and, aer ft trypsinization, were resuspended in 1x PBS and analysed by flow cytometry. A total of 10 000 events were measured per sample. (b) SiHa and CaSki cells (1.5 × 10 cells per well) were seeded into a 96-well plate, allowed to incubate overnight, and then treated with the indicated concentrations of tBHP for 20 h. Viability was measured by crystal violet. The viability of untreated cells was set at 100%. Each me asurement was done in triplicate and error bars indicate the standard deviations of the means. (c and d) Pretreatments with NAC were begun 24 h prior to treatment with tBHP (d). (c) eTh measurement of ROS levels was performed as described in (a) and (d) the measurements of cell viability were performed as described in (b). Mean fluorescence intensity Cell number % of control (viable cells) Cell number % of control (viable cells) 8 BioMed Research International ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ 160 ∗∗ ∗∗ ∗ ∗∗ ∗∗ ∗∗ 140 250 20 20 0 0 0 10 20 0 10 20 0.2 𝜇 M DOX 0.5 𝜇 M DOX 2𝜇 M 5𝜇 M CaSki SiHa cisplatin cisplatin BSO (𝜇 M) SiHa CaSki SiHa CaSki 0 mM NAC DCFDA 0.5 mM NAC 0 mM NAC DHE 1 mM NAC 0.5 mM NAC 1 mM NAC (c) (a) (b) ∗∗ ∗∗ ∗∗ ∗∗ ∗ 120 120 100 100 80 80 20 20 2𝜇 M cisplatin 5𝜇 M cisplatin 0.2 𝜇 M DOX 0.5 𝜇 M DOX CaSki SiHa CaSki SiHa 0𝜇 m BSO 0𝜇 M BSO 10 𝜇 M BSO 10 𝜇 M BSO 20 𝜇 M BSO 20 𝜇 M BSO (d) (e) Figure 4: Pretreatment of CaSki and SiHa cells with NAC decreases oxidative stress and protects them from death induced by DOX (a) and cisplatin (b), while pretreatment with BSO increases ROS (c) and sensitizes cells to cell death induced by DOX (d) and cisplatin (e). (a and b) SiHa and CaSki cells (1× 10 cells per well) were seeded into 96-well plate and then pretreated with the indicated concentrations of NAC for 4 h followed by treatment with DOX for 48 h (a) or cisplatin for 48 h (b). The viability of cells untreated with drugs in the presence or absenceofNAC cellswas setat100%. (c)10 CaSkiorSiHacells were treatedwith10or20𝜇 M BSO for 24 h and ROS measurements were performed as described in Figure 3(a).(dand e) Cells(1× 10 cells per well) were seeded onto a 96-well plate and then pretreated with indicated concentrations of BSO for 24 h followed by treatment with DOX (d) or cisplatin (e) for 48 h. Viability was assessed by crystal violet staining. The viability of cells untreated with drugs in the presence or absence of BSO was set at 100%. To visualizethe dieff renceinthe levels of ROSradicals oxygen species as well as the mechanism through which these detected by DCFDA and DHE staining after DOX or cisplatin agents exert cytotoxicity. treatment of CaSki and SiHa cells, microscopic analysis was performed. Fluorescent images on microphotographs 4. Discussion (Figure 7) confirmed the differences previously detected by flow cytometry, showing that (1) overall levels of ROS are Resistance to anticancer agents is a major concern in the higher in CaSkithaninSiHacells;(2) treatmentwith treatment of cervical and other cancers. Frequently, this DOX preferentially increases radicals detected by DHE; and resistance appears to be due to alterations in the activation of (3) treatment with cisplatin preferentially increases radicals survival pathways that enable escape from treatment-induced detected by DCFDA. Together, these results demonstrate that the cellular cell death. Identification of these events has the potential to identify new therapeutic targets and sets of biomarkers that response to chemotherapeutic agents depends not only on the overall or total levels of ROS, but also on levels of specicfi could guide clinicians in their selection of treatment options. % of control (viable cells) % of control (viable cells) % of control (viable cells) % of control (viable cells) Mean fluorescence intensity (%) BioMed Research International 9 100 100 60 60 40 40 20 20 0 0 010 20 40 0 5 25 100 DOX (𝜇 M) Cisplatin (𝜇 M) CaSki CaSki HeLa HeLa SiHa C33A SiHa C33A (a) (b) 100 100 60 60 40 40 20 20 0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 DHE DCFDA CaSki HeLa CaSki HeLa SiHa SiHa C33A C33A (c) CaSki SiHa HeLa C33A DCFDA DHE (d) Figure 5: CaSki, SiHa, HeLa, and C33A cervical cancer cells display differential responses to treatment with DOX (a) and cisplatin (b), as well as different baseline levels and distributions of ROS (c and d). (a and b) SiHa, CaSki, HeLa, and C33A cells (1 × 10 cells per well) were seeded into a 96-well plate, allowed to incubate overnight, and then treated with the indicated concentrations of DOX (a) or cisplatin (b) for 48 h. Viability was measured by crystal violet staining and the viability of untreated cells was set at 100%. Each measurement was done in triplicate and error bars indicate the standard deviations of the means. (c and d) 10 cells of each line was treated with 10𝜇 MDCFDA or 10𝜇 M DHE in media and then incubated in the dark at 37 C for 30 minutes. eTh cells were then washed, and following trypsinization were resuspended in 1x PBS and were analysed by flow cytometry. A total of 10 000 events were measured per sample. (c) DCFDA was detected in the FL-1 channel, while DHE was detected in the FL-2 channel. (d) Bar graphs show triplicate measurements of mean uo fl rescence intensity of DCFDA or DHE in SiHa, CaSki, HeLa, and C33A cells. Error bars represent the standard deviation. % of control (viable cells) Cell number Mean fluorescence intensity % of control (viable cells) Cell number 10 BioMed Research International 1400 350 1200 300 800 200 0 0.5 1 0 0.5 1 0 60 100 0 60 100 DOX (𝜇 M) Cisplatin (𝜇 M) CaSki SiHa SiHa CaSki DCFDA DHE DCFDA DHE (a) (b) Figure 6: Treatment of CaSki and SiHa cells with DOX increases the level of ROS detected by DHE (a), while treatment with cisplatin increases the level of ROS detected by DCFDA (b). 10 CaSki or SiHa cells were treated with 0.5 or 1𝜇 MDOX for2h(a) or with 60 or 100𝜇 M cisplatin for 4 h (b). DCFDA or DHE was added to the media to a final concentration of 10 𝜇 M and cells were incubated in the dark at 37 Cfor 30 minutes. Cells were then washed, resuspended in 1x PBS, and analysed using flow cytometry. A total of 10 000 events were measured per sample. DCFDA was detected in the FL-1 channel, while DHE was detected in the FL-2 channel. Bar graphs show triplicate measurements of the mean uo fl rescence intensity of DCFDA or DHE in SiHa and CaSki cells, expressed as 100% of the value observed in untreated cells. In this report, we asked which pathways had the potential other hand, the expression of proteins with antioxidant func- to contribute to the drug resistance observed for some tions was higher in SiHa than in CaSki cells. Examples of such cervical cancers. As our initial model, we selected two cell antioxidant enzymes include SOD1, SOD2, NQO1, PRDX, lines, CaSki and SiHa, as representatives of invasive cervi- and GPX (Figure 2(b), Table 1 and Table 1, Supplementary calcarcinoma.Althoughbothcelllines result from HPV- Material). These differences have downstream consequences, mediated transformation, the two lines respond quite differ- as we observed that the differences between SiHa and CaSki ently to treatments with chemotherapeutic drugs (Figure 1). cellsinthe expressionlevelsofproteinsinvolvedinROS Proteomicanalysisallowed us to identify severalpathways metabolism were reflected in the cellular levels of ROS with the potential to impact these differential responses as measured by flow cytometry ( Figure 3(a)). Furthermore, (Figure 2(a)). First, we were able to detect a 2.5-fold difference these differences were also reflected in the difference in levels in the level of p53 between SiHa and CaSki cells. However, of expression of proteins involved in DNA damage recogni- since the absolute levels of p53 are low in both cell lines due tion and response (Table 1, Supplementary Material), since to accelerated degradation of p53 by HPV16 E6 [12], this these processes are activated during oxidative stress [19, 20]. cannot be the only contributor to the observed differential The connection between chemoresistance and a high level drugresistance.Likewise,thepresenceofmutantp53inC33A of antioxidant defense has been shown previously for other cells [16] means that the p53 response is unlikely to contribute types of cancer, especially for those in advanced stages. An significantly to the C33A response to treatment. Other dif- upregulated antioxidant capacity not only allows cells to ferences identified by our proteomic analysis pointed toward survive under conditions of oxidative stress, but also provides proteins involved in mitochondrial functioning, as these have a mechanism for adapting to exogenous stresses such as thepotential to inufl ence theproductionoffreeradicals treatment with anticancer agents. For example, resistance to and ROS homeostasis. PCR analysis of an array of genes arsenic trioxide by bladder urothelial carcinoma cell lines involved in ROS regulation confirmed differences in the [21] and myeloma cells [22]was foundtobeassociated expression levels of several of these genes and also identified with an upregulation of heme oxygenase (decycling)1, SOD1, differences in the expression of additional genes involved in and glutathione reductase. Also, resistance to agents that the regulation of ROS (Table 1). These findings are consistent induce intracellular ROS production, such as paclitaxel, with the previous observation that, in the absence of p53, ROS DOX, or platinum-based drugs, is correlated with increased itself may act to signal DOX-induced cell death [17]. antioxidant capacity in hepatoma cells [23, 24]. One of the eTh majorsourceofROS production in cellsisthe mito- most important antioxidant enzymes is SOD2, also known chondria, where enzymes involved in the electron transport as MnSOD, which catalyzes the conversion of superoxide chainand theproductionofsuperoxideare located[18]. radicals to H O . SOD2 is also considered to function as a 2 2 ROS-producing enzymes identified in the present study were negative modulator of cellular apoptosis and as a prosurvival expressed at higher levels in CaSki cells (Table 1). On the factor for cancer cells [25]. Mean fluorescence intensity (%) Mean fluorescence intensity (%) BioMed Research International 11 CaSki SiHa DCFDA DHE DCFDA DHE DOX DOX Cisplatin Cisplatin (a) (b) Figure 7: Microphotograph of CaSki (left two panels) and SiHa (right two panels) cells treated with DOX and cisplatin. The majority of anticancer drugs in clinical use are been known to involve oxidative events as well. In fact, thought to act primarily by way of DNA damage or micro- oxidative damage is now considered an important factor tubule disruption. For example, cisplatin and mitomycin in the antitumor activity of DOX [30]. In fact, strategies C are DNA-damaging agents that form bifunctional DNA designed to manipulate levels of ROS are now considered a adducts, leading to activation of the cellular response to major focus of cancer chemotherapy [31, 32]. DNA-damage or to DNA damage-induced apoptosis. How- We found that treatment with both DOX and cisplatin led ever,cisplatin mayalsobeabletoinduceapoptosis in the to increased levels of ROS (Figures 6 and 7). Unexpectedly, absence of nuclear DNA [26], and cisplatin-induced cell however, we found that the two agents induced quite dieff rent deathinrenalcorticalcellswasshowntoinvolveperoxidation profiles of these reactive oxygen species (Figures 6 and andthe releaseofcalcium from intracellularstores[27]. 7). DOX increased the DHE signal (superoxides), but did Basedonthese andother studies, theantitumor eeff ctof not change the levels of those species detected by DCFDA cisplatin is now considered to be due to a combination of (H O , hydroxyl, and peroxyl radicals) (Figures 6(a) and 2 2 nuclear and nonnuclear effects including ROS induction, 7). In contrast, cisplatin considerably increased the DCFDA peroxidation, and lethal cell injury [28, 29]. Anthracyclines, signal, but did not significantly increase the DHE signal such as DOX, are classiefi d as inhibitors of topoisomerase- (Figures 6(b) and 7). Consistent with these ndin fi gs, previous II; however, the toxic side effects of doxorubicin have long work had found that treatment with DOX increased the level 1.0 M 0.5 M No treatment 100 M 60 M 12 BioMed Research International of superoxides, but not of H O , hydroxyl, or peroxyl rad- similartothatofHeLacells,for example,might favorDOX 2 2 icals in HaCaT keratinocytes [33, 34]. In contrast, cisplatin over cisplatin. Our experimental results demonstrated that primarily increased the level of hydroxyl radicals but not of different cervical cancer cell lines differ not only in their total superoxides in human hair follicle dermal papilla cells and in level of ROS, but also in the levels of specific free radicals. Ide- HaCaT keratinocytes [35]. ally, clinical selection of chemotherapeutic treatments should The mechanism of ROS generation induced by DOX consider the types of ROS induced, which will be related to is controversial and not yet fully understood [36, 37]. It the mechanism of drug action at the molecular level. is known that in the presence of molecular oxygen, the The data in this report provides evidence that differences derivatives from “redox cycle” are acted upon by a number in the sensitivity of cervical cancers to chemotherapeutic of NAD(P)H-oxidoreductases cytochrome P450 or cyto- treatments is likely due, at least in part, to differences in chrome-b5 reductases, mitochondrial NADH dehydroge- the relative levels of pro- and antioxidant enzymatic activity nase, xanthine dehydrogenase, endothelial nitric oxide syn- that lead to different baseline levels of oxidative stress. thase (reductase domain) to generate superoxides [38–40]. Chemoresistance is undoubtedly a multifactoral process and One-electron “redox cycling” of DOX also produces super- factors in addition to high levels of antioxidant defense are oxide; this process is accompanied by the release of iron likely to contribute. Such factors may include differences in from intracellular stores and results in formation of drug-iron angiogenesis, which could aeff ct the penetration of agents complexes that release superoxides and hydrogen peroxides into tumor tissue, increased drug eu ffl x from the cancer [41], which can then be decomposed by antioxidant systems. cells, reduced uptake of drugs, interactions of cancer cells The higher levels of superoxide observed during DOX treat- with their surrounding microenvironment, and other factors ment arebelievedtobedue to adecreaseinthe activity [43–45]. Such factors are also likely to contribute to the of superoxide-decomposing enzymes such as MnSOD and chemoresistance of cervical carcinomas. A better under- catalase [34]. standing of how and when these various factors, including ROS generation by platinum-based drugs is also not fully the baseline levels of oxidative stress, affect how a particular understood. eTh predominant formation of hydroxyl radicals tumor will respond to a specific treatment has the potential is believed to be the result of peroxynitrite decomposition in to improve our treatment of patients suffering from cervical cisplatin-treated cells [35]. After penetration into the cells, malignancies. alkylating agents such as cisplatin bind to glutathione, and this interaction leads to removal of this complex from cells 5. Conclusions through an ATP-binding cassette. Depletion of glutathione then results in increased H O and hydroxyl radicals [35, 42]. 2 2 We utilized a proteomic approach to identify the path- The difference in the mechanism of action between DOX ways involved in resistance to the chemotherapeutic agents and cisplatin is supported by experiments in which cells cisplatin and doxorubicin and then extended these find- were sensitized by BSO pretreatment prior to exposure to the ings by analyzing and comparing the expression level of drugs (Figures 4(d) and 4(e)). The lower level of sensitization genes involved in reactive oxygen species (ROS) metabolism to cisplatin can be explained on the basis that BSO and through the use of an qRT-PCR array. These data enabled cisplatin act on the same substrate, glutathione. eTh refore, us to demonstrate that pathways involved in oxidative stress we speculatethatglutathione depletion as aresultofBSO and antioxidant defense contribute to drug resistance. In treatment does not change cell viability dramatically aeft r particular, the sensitive CaSki cells expressed lower levels of administration of cisplatin, because the substrate for cisplatin antioxidant enzymes, resulting in higher levels of ROS, than toxicity is already depleted. did the resistant SiHa cells. Decreasing or increasing oxidative To examine the dependence of the cellular response to stress using pharmacological agents led to protection or sen- therapeutic agents on oxidative stress, we manipulated ROS sitization, respectively, in both cell lines, supporting the idea levels by either depleting ROS through pretreatment with that cellular levels of oxidative stress affect responsiveness to NAC or increasing ROS through pretreatment with BSO. treatment. Interestingly, the two agents tested, doxorubicin Pretreatment with NAC or BSO, reducing or increasing, (DOX) and cisplatin, induced different profiles of ROS, and respectively, the levels of ROS (Figures 3(c) and 4(c))were these differences appear to contribute to the differential able to protect or to sensitize cells to both agents, respectively sensitivity displayed by cervical cancer cells to treatment. (Figures 4(a), 4(b), 4(d),and 4(e)). We also demonstrated that cells with higher baseline levels of ROS experienced a more rapid loss of viability following drug treatment than Conflict of Interests did cells with lower baseline levels of ROS. In particular, eTh authors declare that there is no conflict of interests CaSki and C33A cells, which display higher levels of ROS, regarding the publication of this paper. died faster than did SiHa cells following treatment with either DOX or with cisplatin (Figures 1 and 5). 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Cellular Levels of Oxidative Stress Affect the Response of Cervical Cancer Cells to Chemotherapeutic Agents

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Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 574659, 14 pages http://dx.doi.org/10.1155/2014/574659 Research Article Cellular Levels of Oxidative Stress Affect the Response of Cervical Cancer Cells to Chemotherapeutic Agents 1 1 1 2 Maria Filippova, Valery Filippov, Vonetta M. Williams, Kangling Zhang, 1 1 1 Anatolii Kokoza, Svetlana Bashkirova, and Penelope Duerksen-Hughes Department of Basic Sciences, Loma Linda University School of Medicine, 11021 Campus Street, 101Alumni Hall,LomaLinda,CA92354,USA Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA Correspondence should be addressed to Penelope Duerksen-Hughes; pdhughes@llu.edu Received 12 June 2014; Revised 13 August 2014; Accepted 21 August 2014; Published 16 November 2014 Academic Editor: Wan-Liang Lu Copyright © 2014 Maria Filippova et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Treatment of advanced and relapsed cervical cancer is frequently ineffective, due in large part to chemoresistance. To examine the pathways responsible, we employed the cervical carcinoma-derived SiHa and CaSki cells as cellular models of resistance and sensitivity, respectively, to treatment with chemotherapeutic agents, doxorubicin, and cisplatin. We compared the proteomic profiles of SiHa and CaSki cells and identified pathways with the potential to contribute to the differential response. We then extended these findings by comparing the expression level of genes involved in reactive oxygen species (ROS) metabolism through the use of a RT- PCR array. eTh analyses demonstrated that the resistant SiHa cells expressed higher levels of antioxidant enzymes. Decreasing or increasing oxidative stress led to protection or sensitization, respectively, in both cell lines, supporting the idea that cellular levels of oxidative stress aeff ct responsiveness to treatment. Interestingly, doxorubicin and cisplatin induced different profiles of ROS, and these differences appear to contribute to the sensitivity to treatment displayed by cervical cancer cells. Overall, our findings demonstrate that cervical cancer cells display variable profiles with respect to their redox-generating and -adaptive systems, and that these different profiles have the potential to contribute to their responses to treatments with chemotherapy. 1. Introduction that they produce response rates of 15%–20%. Recent and ongoing trials are also likely to identify additional active Worldwide, cervical cancer is the second most common drugs [3]. eTh lowresponseratetothese agents canbe cancer in women; approximately 400 000 new cases of this attributed to the fact that invasive cervical cancer appears to disease are diagnosed each year, of which approximately half be relatively chemoresistant, as compared to other gyneco- will lead to death. The causative agents of most cases of logic tumors such as those of the breast or ovaries [3]. Studies cervical carcinomas are the high-risk types of human papil- in breast, prostate, and colorectal cancers have shown that lomaviruses (HPV). When cervical carcinomas are detected many factors can contribute to chemoresistance, including an at earlyorpreinvasive stages,theyare oeft n curablewithlocal individual’s geneticbackgroundaswellasepigeneticfactors treatments, most of which are based on ablative approaches. [4]. However, such studies have not yet analyzed the causes Unfortunately, a significant proportion of patients diagnosed of chemoresistance in cervical cancer. An understanding of with invasive cervical cancer sueff r relapses following initial the molecular events that lead to chemoresistance in the cells treatment. For this reason, the development of novel and comprising cervical carcinomas may lead to the discovery of effective therapeutic interventions, such as those based on new targets for chemical intervention. molecular techniques, remains an important priority [1, 2]. CaSki and SiHa cells represent useful cellular models More than 20 different chemotherapeutic agents are now for cervical carcinoma, as both lines contain an integrated considered active in the treatment of cervical carcinomas, in form of HPV16. Interestingly, however, they respond quite 2 BioMed Research International dieff rently to treatment with agents that induce cell death recommended by the TMT Mass Tagging Kit (Thermo Fisher through both intrinsic [5, 6] and extrinsic [7]apoptotic Scientific) protocol. Trypsin was added at a protein/enzyme pathways. In spite of the significant differences in the molec- ratio of 30 : 1 by mass and the digestion was performed at ular pathways involved (e.g., DNA-damaging agents versus 37 C overnight. Peptides were labeled with TMT (tandem- ligands that induce ligand-mediated apoptosis), one common mass-tagging)reagentsaccordingtothe manufacturer’s pro- observation stands out: CaSki cells are more sensitive to each tocol in duplicate and equal amounts of labeled peptides of these treatments than are SiHa cells. eTh reason(s) for these were combined to obtain one sample, which was separated dramatically different responses have not yet been identified. into 9 fractions by strong cation exchange chromatography It has been suggested that dieff rences in the levels of p53 [ 8, 9] using TopTip columns (PolyLC). Elution was performed and/or procaspase 8 [7, 10]could contribute. with increasing concentrations of KCl (from 0 to 0.5 M). In the current study, we compared the proteomic profiles Eluates were dried using a SpeedVac and then desalted of SiHa and CaSki cells and identified pathways with the using C18/hypercarb TopTip columns (PolyLC). Samples potential to contribute to the dieff rential response to chemo- from each fraction were runintriplicateonanOrbitrapPro therapeutic agents. We then extended these findings by ana- mass spectrometer that was coupled to a nanoLC (Thermo lyzing and comparing the expression level of genes involved Fisher Scientific), and the spectra obtained were analyzed in reactive oxygen species (ROS) metabolism through the use with Proteome Discoverer 1.3 software against the Human of an RT-PCR array. Both sets of analyses demonstrated that International Protein Index (IPI) database. Peptides were the resistant SiHa cells expressed higher levels of antioxidant identiefi dwithaFDR(falsediscovery rate)oflessthan1%. enzymes. Decreasing or increasing oxidative stress using Proteins were considered differentially expressed if the fold pharmacological agents led to protection or sensitization, ratio was more than 1.5. Protein data were further analyzed respectively, in both cell lines, supporting the idea that using Ingenuity Pathway Analysis (IPA) software to identify cellular levels of oxidative stress affect responsiveness to treat- differences in pathways and networks between cell lines. ment. Interestingly, the two agents tested, doxorubicin (DOX) and cisplatin, induced different profiles of ROS, and these 2.5. Measurement of ROS in Cells by Flow Cytometry. Intra- differences appear to contribute to the differential sensitivity cellular generation of hydrogen peroxide (H O ), hydroxyl 2 2 observed. and peroxyl radicals (DCFDA), and superoxide (O )(DHE) 󸀠 󸀠 was estimated using either the 5-(and-6)-carboxy-2 ,7 - dichlorodihydrofluorescein diacetate (DCFDA) or dihy- 2. Materials and Methods droethidium (DHE) membrane permeable probes (Life Bio- 2.1. Reagents. Monoclonal𝛼 -NQO1,𝛼 -OXR1, and𝛼 -𝛽 -actin sciences). Reagents were diluted into culture media and then were obtained from Sigma-Aldrich, monoclonal 𝛼 -SOD1, added to cells to a n fi al concentration of 10 𝜇 M. After treat- and𝛼 -GPX 1/2 from Santa Cruz Biotechnology, monoclonal ment, the cells were collected in 1x PBS and analyzed using 𝛼 -SOD2 from BD Biosciences, and monoclonal 𝛼 -PARP1 the Becton-Dickinson FACSCalibur flow cytometer (Becton- (Ab-2) from Millipore Corporation (Calbiochem). tert-Butyl Dickinson, San Francisco, CA). DCFDA was detected in the hydroperoxide solution (tBHP), N-Acetyl-L-cysteine (NAC), FL-1 channel, while DHE was detected in the FL-2 channel. cis-diammineplatinum(II) dichloride (cisplatin), doxorubi- Data was collected in log scale and analyzed using Flow-Jo cin hydrochloride (DOX), and DL-buthionine-(S-R)-sulfox- software. imine (BSO) were purchased from Sigma-Aldrich. 2.6. Microscopy. CaSki and SiHa cells were seeded onto Cul- 2.2. Cell Culture. CaSki, SiHa, HeLa, and C33A cells (derived tureSlides (Falcon) one day prior to treatment with DOX or from human cervical carcinomas) were obtained from the cisplatin. The following day, cells were stained with DCFDA ATCC (Manassas VA). All cells were cultured in modied fi and DHE as described above (measurement of ROS in cells Eagle medium (MEM) (CellGro). The medium was supple- by flow cytometry). Fluorescent images were recorded using mented with 10% fetal bovine serum (Life Biosciences) and a Biorevo microscope (Keyence) at the same magnifications with penicillin (100𝜇 g/mL) andstreptomycin(100𝜇 g/mL) and the same settings. (Sigma-Aldrich). 2.7. Immunoblot Assays. For immunoblot analysis, 10 cells 2.3. Cell Treatments and Cell Viability Assay. Cells (1 or 1.5× were lysedin100𝜇 L of Laemmli lysis buffer and lysates 10 cells per well) were seeded onto a 96-well plate and were sonicated and separated by SDS-PAGE. Aeft r transfer allowed to incubate for 24 h, aer ft which they were treated of protein onto Immobilon P membranes (Millipore Corpo- with the indicated concentrations of drugs or tBHP. NAC ration) and blocking of the membrane with 1% bovine serum or BSO was also added where indicated. Cell viability was albumin dissolved in TBST, primary antibodies were applied monitored using crystal violet staining. The absorbance of overnight. Secondary ImmunoPure antibody (𝛼 -mouse or each well was determined at 590 nm using a plate reader. 𝛼 -rabbit), conjugated with horseradish peroxidase (Thermo Fisher Scientific), was applied onto the membrane for 1 h and 2.4. Proteomic Analysis. SiHa and CaSki cells (10 )were the detection of signal was performed using the chemilumi- lysed in RIPA lysis bueff r (Sigma-Aldrich) and sonicated. nescent SuperSignal West Dura or Pico maximum-sensitivity Cleared lysates were denatured, reduced, and alkylated as substrate (Thermo Fisher Scientific). BioMed Research International 3 2.8. RNA Isolation, RT-PCR, and qRT-PCR. Cells were plated an LTQ-Orbitrap mass spectrometer. eTh total number of onto a10cmtissueculture plateand allowedtogrowtosemi- proteins in which the level of expression between SiHa confluency. RNA was isolated using Tri Reagent according and CaSki cells dieff red by more than 1.5-fold was 430 to the manufacturer’s protocol (Sigma-Aldrich). cDNA was (Table 1; see Supplementary Material available online at synthesized using ImPromII reverse transcriptase (Promega) http://dx.doi.org/10.1155/2014/574659) and the detected range 󸀠 󸀠 and an oligo(dT) primer. Primers for the 5 and 3 ends of the of differences in protein levels between these cells ranged indicated genes were used to amplify PCR products. from−6.0 to 6.9 fold. Seventy-six of these proteins were found to be upregulated, while the remainder was downregulated in Quantitative qRT-PCR was conducted using the Abso- SiHa cells as compared to CaSki cells. lute QPCR Sybr green kit according to the manufacturer’s To gain insight into the functions of these differentially protocol (ABgene). eTh observed gene concentrations were expressed proteins, we employed the online IPA analysis normalized using PGK1 expression levels. (Ingenuity Systems) tool to group them into functionally related networks and pathways. Figure 2(a) summarizes 2.9. Oxidative Stress and Antioxidant Defense PCR Array. the 9 functions for which protein expression differs most The PCR Microarray was performed according to the manu- between these two lines. o Th ugh HPV16 E6 accelerates the facturer’s instructions (SABiosciences, a QIAGEN company, degradation of p53 [12], thereby significantly lowering its Valencia CA). Gene expression was compared according to cellular level, our mass spectroscopy-based method was able the𝐶 value. Normalization was performed for each cDNA to detect and quantify p53 in both SiHa and CaSki cells, sample using the average of ve fi housekeeping genes provided demonstrating a 2.5-fold higher level of p53 in CaSki than by manufacture. in SiHa cells (Table 1, Supplementary Material). IPA analysis revealed that additional proteins within the p53 signaling 2.10. Statistics. All assays were repeated at least three times pathways were downregulated in SiHa cells as compared to andthe resultsreportedasmean ± standard deviation. CaSki (Figure 2(a)). One downstream consequence of these Differences were analyzed by the Student’s 𝑡 -test.𝑃 ≤ 0.05 differences in p53-linked pathways is the difference in the was regarded as significant. expression of proteins involved in G2/M DNA damage check- point regulation (Figure 2(a) and Table 1, Supplementary 3. Results Material). Some of the more remarkable differences in protein levels 3.1. SiHa Cells Are More Resistant an Th CaSki Cells to Dox- between SiHa and CaSki cells were detected in proteins orubicin- and Cisplatin-Induced Cell Death. Doxorubicin involved in mitochondrial functions such as mitochondrial (DOX) and cisplatin are chemotherapeutic agents used to depolarization, swelling of mitochondria, and the biogenesis treat solid tumors, including cervical carcinomas [3]. To of mitochondria (Figure 2(a)). One important outcome of evaluate and compare the sensitivity of CaSki and SiHa cells proper mitochondrial functioning involves the production to these chemotherapeutic drugs, cells were treated with and safe transport of radicals, as well as the maintenance increasing concentrations of DOX and cisplatin. For the of free radical homeostasis in the cell. Another group of initial set of experiments, relatively high concentrations were pathways dieff rentially activated between these cell lines is applied (10–40𝜇 Mfor DOXand 16–128𝜇 Mfor cisplatin) connected to DNA repair and the DNA damage response and crystal violet staining was used to monitor cell viability (Figure 2(a) and Table 1, Supplementary Material). Differ- following treatment for 20 h (Figures 1(a) and 1(b)). With encesinthe expressionofproteinsinvolvedinmitochondrial both treatments, we found that SiHa cells were more resistant status and DNA repair were accompanied by changes in to treatment than were the CaSki cells. For example, cisplatin thelevelsofproteinsinvolvedinthe regulation of ROS at a concentration of∼30𝜇 M killed 50% of CaSki cells, while levels (Figure 2(a)). For example, NAD(P)H dehydrogenase, alossof50% viabilityfor SiHa cellswas observed at 128𝜇 M quinine 1 (NQO1), peroxiredoxin 2 (PRDX2), and superoxide cisplatin (Figure 1(b)). Similar results were observed for DOX dismutase 1 (SOD1), which are responsible for inactivation of (Figure 1(a)). These experiments were then repeated using superoxide radicals, were found in higher levels in SiHa than lower, more physiologically relevant concentrations (0.05𝜇 M in CaSki cells (Table 1, Supplementary Material). to 2𝜇 Mfor DOXand 0.2𝜇 Mto5𝜇 Mfor cisplatin) [11], for a To verify this proteomicdataaswellasthe dieff rences longer period of time (72 h) (Figures 1(c) and 1(d)). Again, we between these two cell lines with regards to expression of found that SiHa cells were more resistant to treatment than proteins involved in ROS metabolism, we evaluated the were CaSki cells. expression levels of a subset of the proteins involved in antiox- idant defense by immunoblot (Figure 2(b)). Consistent with 3.2. Proteomic Analysis Identified Differences in Pathways our proteomic data, the immunoblot analysis confirmed Connected to p53 Activation, Mitochondrial Function, and higher levels of NQO1 and SOD1 in SiHa cells and added Oxidative Stress. To identify differences in the pathways glutathione peroxidase 1/2 (Gpx1/2) and SOD2 to the list of differentially-expressed genes ( Figure 2(b)). A marker of through which sensitive CaSki cells and the more resistant SiHa cells responded to drug treatment, we performed a DNAdamagecausedbyoxidative stress,PARP1 wasalso comparative proteomic analysis. Identicfi ation and quanti-fi detected at higher level in CaSki than in SiHa cells as assessed cation of proteins were done by simultaneously running both by proteomic (Table 1, Supplementary Material) and TMT-labeled trypsinized CaSki and SiHa lysates through immunoblot analyses (Figure 2(b)). 4 BioMed Research International 016 32 64 128 0 102040 Cisplatin (𝜇 M) DOX (𝜇 M) CaSki CaSki SiHa SiHa (a) (b) 0 0.2 0.5 2 5 0 0.05 0.2 0.5 2 Cisplatin (𝜇 M) DOX (𝜇 M) CaSki CaSki SiHa SiHa (c) (d) Figure 1: SiHa cells are more resistant than CaSki cells to treatment with the chemotherapeutic drugs DOX (a and c) and cisplatin (b and d). (a and b) SiHa and CaSki cells (1.5× 10 cells per well) were seeded into a 96-well plate, allowed to incubate overnight, and then treated with the indicated concentrations of drugs for 20 h. (c and d) SiHa and CaSki cells (0.5× 10 cells per well) were seeded into a 96-well plate, allowed to incubate overnight, and then treated with the indicated concentrations of drugs for 72 h. Viability was measured by crystal violet and the viability of untreated cells was set at 100%. Each measurement was done in triplicate and error bars indicate the standard deviations of the means. To further identify ROS-related genes with differential alpha polypeptide (CYBA)isacomponentofmitochondrial expression between SiHa and CaSki cells, we employed the Complex III, which is involved in the transfer of electrons Oxidative Stress and Antioxidant Defense PCR Array (SA to Complex IV so that water can be formed (Figure 2(c) and Table 1). Other genes belong to various antioxidant systems. Biosciences), which profiles the expression of 84 genes related For example, SODs, GPXs, and PRDXs catalyze reactions to oxidative stress. We found that several of these genes were that inactivate superoxide radicals (SODs) or H O (GPXs upregulated in SiHa (as compared to CaSki) cells and that a 2 2 and PRDXs). Overall, our immunoblot (Figure 2(b)), semi- few were downregulated (Table 1). Genes whose expression quantitative RT-PCR (Figure 2(c)), and quantitative qRT- was consistently downregulated in SiHa (relative to CaSki) PCR (Figure 2(d)) confirmed our initial results from the PCR cells included aldehyde oxidase 1 (AOX1)(Figure 2(c)), array profiles. In summary, our data demonstrated significant NADPH oxidase complex (NCF2)(Figure 2(d)), and oxi- differences between SiHa and CaSki cells with regards to dation resistance protein (OXR1)(Figure 2(d)); products of the expression levels of genes and proteins involved in ROS these genes are responsible for the production of reactive metabolism and homeostasis. oxygen radicals. A reduced level of OXR1 expressioninSiHa cells as compared to CaSki cells was also confirmed by immunoblot (Figure 2(b)). Several genes that were upreg- 3.3. Levels of ROS and Oxidative Stress-Induced Cell Death ulated in SiHa (Table 1) participate in scavenging radicals AreHigherinCaSki aTh ninSiHaCells. The differential in one way or another. For example, cytochrome b-245, expression of pro- and antioxidant enzymes in CaSki and % of control (viable cells) % of control (viable cells) % of control (viable cells) % of control (viable cells) BioMed Research International 5 CaSki SiHa NQO1 SOD2 SOD1 GPx 1/2 PARP1 𝛽 -Actin OXR1 40 kDa isoform No overlap with dataset Upregulated 𝛽 -Actin Downregulated (a) (b) CaSki SiHa AOX1 CYBA PGK1 −5 (c) (d) Figure 2: SiHa and CaSki cells differ in expression levels of proteins involved in the regulation of ROS. (a) Differential regulation of nine pathways in SiHa and CaSki cells. The percentage of genes downregulated in SiHa as compared to CaSki cells is shown in black, upregulated in grey. (b) Immunoblot analysis confirms differential expression. Lysates prepared from 10 CaSki and SiHa cells using Laemmli lysis bueff r were subjected to SDS-PAGE. Immunoblots were performed using antibodies directed against the indicated proteins. Loading was normalized by blotting for𝛽 -actin. (c and d) Transcription levels of genes related to oxidative stress as determined by semiquantitative RT-PCR (c) and by quantitative qRT-PCR (d) differ between SiHa and CaSki cells. Total RNA was isolated from 10 cells of each cell line using Trizol Reagent (Sigma Aldrich) and cDNA was synthetized using oligo-dT. PCR and qPCR were performed using specific primers for the genes of interest. The PCR and qPCR products obtained using primers for the PGK1 transcript were used to normalize to cDNA input. (d) Differences in gene expression are presented as fold changes (SiHa versus CaSki). Each measurement was done in triplicate and error bars indicate the standard deviations of the means. SiHa cells is expected to inu fl ence the baseline levels of ROS emission spectra of 495 nm and 529 nm, respectively [13]. in these cells. To test this idea, SiHa and CaSki cells were In the case of DHE, its oxidation by superoxide results in stained with the DCFDA and DHE uo fl rescent dyes and hydroxylation at the 2-position. 2-hydroxyethidium exhibits the intensity of staining monitored by flow cytometry. Once a uo fl rescence excitation peak at ∼400 nm [14]. Data pre- DCFDA enters a cell, it is deacetylated by cellular esterases sented in Figure 3(a) clearly demonstrates that SiHa cells to form a nonfluorescent compound. ROS radicals such as display lower levels of the reactive oxygen species detected H O , hydroxyl, and peroxyl radicals then oxidize this non- by both DCFDA and DHE than do CaSki cells. 2 2 󸀠 󸀠 u fl orescent substrate into 2 ,7 -dichlorofluorescein, which is a These data suggested that cells with an elevated level highly u fl orescent compound with maximum excitation and of oxidative stress should be more susceptible to treatment (%) Hypoxia-inducible factor signaling Increases depolarization of mitochondria Oxidative stress Swelling of mitochondria NRF2-mediated oxidative stress response p53 signaling Fold changes SiHa/CaSki G2/M DNA damage checkpoint regulation Negative acute phase response proteins PGK1 Biogenesis of mitochondria OXR1 NCF2 PRDX1 GPX1 SOD1 SOD2 6 BioMed Research International Table 1: Relative expression (SiHa versus CaSki) of genes involved to DOX and to cisplatin (Figures 4(d) and 4(e)), with a greater in oxidative stress and antioxidant defense. eect ff seen with DOX than with cisplatin, and on CaSki cells with theirhigherbasal levelofROS than on SiHa cells. Genes Fold St. dev. Together, these experiments demonstrate that manipulating AOX1 −3.91951 2.099233 thelevel of cellular oxidativestresscan modify thecellular Downregulated genes NCF2 −73.405 14.30477 response to chemotherapy agents, such that higher levels of OXR1 −3.49989 0.529737 ROS level sensitize cells to chemotherapy-induced cell death. CYBA 4.275 2.269813 DUSP1 6.58 2.870854 3.5. C33A and HeLa Cells Display Dier ff ential Sensitivities to GPX1 1.6 0.381838 DOX and Cisplatin. The results described above demonstrate GPX5 2.085 1.421285 that higher intracellular levels of ROS increase sensitivity to GPX6 2.625 2.043539 DOX and cisplatin in the CaSki and SiHa cervical carcinoma cell lines. To further explore the connection between ROS GSS 2.085 1.421285 Upregulated genes levels and the cellular response to such agents, two additional PRDX1 4.315 0.982878 cervical cancer cell lines, HeLa and C33A, were analyzed PRDX2 13.195 11.10865 for their response to DOX and cisplatin as well as for their SELS 5.635 1.916259 intercellular levels of ROS. Cell viability following treatment SOD1 7.745 4.249712 with these agents is presented in Figures 5(a) and 5(b). SOD2 3.84 0.127279 Interestingly, of these four tested cell lines, CaSki cells remain SOD3 3.055 2.38295 the most sensitive and SiHa the most resistant to both agents. C33A and HeLa cells displayed an intermediate sensitivity, with C33A cells more sensitive than the HeLa cells to both with agents that further increase oxidative stress than cells agents. Interestingly, the resistance displayed by HeLa cells with intrinsically lower levels of oxidative stress. To test this to cisplatin treatment was very similar to that seen in SiHa idea, we compared the cellular response to additional external cells (Figure 5(b)), although HeLa cells were more sensitive oxidative stress by treating cells with tBHP for 20 h and then to DOXthanwereSiHacells (Figure 5(a)). estimating the level of cell death. We found that CaSki cells, Differences in the level of ROS as measured by DCFDA with their higher baseline levels of oxidative stress, were more andDHE betweenthese four cell lineswerealsonoted susceptible to cell death caused by additional oxidative stress (Figures 5(c) and 5(d)). In particular, while C33A cells than were SiHa cells (Figure 3(b)). displayed intermediate levels of ROS as assessed by both We then exposed CaSki and SiHa cells to an antioxidant, DCFDA and DHE, HeLa cells displayed the lowest levels of NAC, prior to treatment with t-BHP. Cellular levels of ROS ROS as detected by DCFDA and the highest levels of ROS as before and aeft r pretreatment with NAC, as estimated by detected by DHE. flow cytometry, are shown in Figure 3(c) and demonstrate that exposure to NAC decreased ROS levels as detected by 3.6. DOX and Cisplatin Treatments Induce Different ROS DCFDA and DHE in both cell lines. Importantly, pretreat- Proles. fi One question raised by the previous experiments ment with NAC increased the viability of the tBHP-sensitive concerned the differential response of HeLa cells to treat- CaSki cells. Pretreatment of SiHa cells with the same NAC ment with DOX (intermediate sensitivity, similar to that concentration (140𝜇 M) did not aeff ct their viability, presum- seen with the C33A cells) versus treatment with cisplatin ably becausetheywerealready tBHP-resistant (Figure 3(d)). (quite resistant, similar to that seen with SiHa cells). The Together, the data presented above suggest that internal mechanisms by which these two agents induce cytotoxicity levels of oxidative stress may affect cellular sensitivity to cytotoxic agents. differ significantly; cisplatin cross-links the DNA, while DOX intercalates into the DNA and produces DNA lesions. This dieff renceinmechanism suggestedthatthese twoagents 3.4. Changes in Cellular Oxidative Stress Aeff ct the Response of likely triggered different cell death pathways, with different Cells to Chemotherapeutic Agents. We next asked whether a effects on oxidative stress. To ask if this were indeed the case, change in ROS levels would also affect the cellular response CaSki and SiHa cells were treated with DOX or cisplatin and to actual chemotherapeutic drugs. Cells were pretreated with changes in ROS levels were assessed by flow cytometry. eTh NAC and then exposed to DOX (Figure 4(a))ortocisplatin results (Figure 6) showed, somewhat unexpectedly, that DOX (Figure 4(b)). Pretreatment with NAC for 4 h protected both CaSki and SiHa cells from cell death induced by DOX and and cisplatin induced different profiles of ROS. Cisplatin cisplatin. treatment, in both cell lines, primarily increased the levels of We also asked whether increasing ROS would aeff ct agents detected by DCFDA (H O , hydroxyl and peroxyl rad- 2 2 the cellular response to chemotherapy agents by pretreating icals (Figure 6(b)), while DOX treatment increased primarily CaSki and SiHa cells with BSO, an inhibitor of glutathione the levels of superoxides as detected by DHE (Figure 6(a)). A [15]. Flow cytometry (Figure 4(c)) demonstrated that the similar eeff ct, showing DOX-induced increases in superoxide ability of BSO to reduce glutathione levels did indeed lead to (DHE) and cisplatin-induced increase in hydrogen peroxide, an increase in cellular ROS as detected by DCFDA and DHE. hydroxyl radicals, and peroxyl radicals (DCFDA), was noted Pretreatment with BSO sensitized both CaSki and SiHa cells in HeLa and C33A cells (data not shown). BioMed Research International 7 100 100 80 80 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 DHE DCFDA CaSki CaSki SiHa SiHa (a) 0 100 140 160 200 tBHP (𝜇 M) CaSki SiHa (b) ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ CaSki SiHa NAC (mM) tBHP DHE DCFDA DHE DCFDA No NAC CaSki 2 mM NAC SiHa (c) (d) Figure 3: CaSki cells display higher levels of ROS than do SiHa cells (a), the cell viability of CaSki cells decreases more than in SiHa cells aer ft treatment with tBHP (b). Pretreatment with NAC decreases ROS in CaSki and SiHa cells (c) and pretreatment with NAC protects cells from death induced by treatment with tBHP (d). (a) 10 cells of each line were treated with 10𝜇 MDCFDA or 10𝜇 M DHE in media and then incubated in the dark at 37 C for 30 minutes. eTh cells were washed and, aer ft trypsinization, were resuspended in 1x PBS and analysed by flow cytometry. A total of 10 000 events were measured per sample. (b) SiHa and CaSki cells (1.5 × 10 cells per well) were seeded into a 96-well plate, allowed to incubate overnight, and then treated with the indicated concentrations of tBHP for 20 h. Viability was measured by crystal violet. The viability of untreated cells was set at 100%. Each me asurement was done in triplicate and error bars indicate the standard deviations of the means. (c and d) Pretreatments with NAC were begun 24 h prior to treatment with tBHP (d). (c) eTh measurement of ROS levels was performed as described in (a) and (d) the measurements of cell viability were performed as described in (b). Mean fluorescence intensity Cell number % of control (viable cells) Cell number % of control (viable cells) 8 BioMed Research International ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ 160 ∗∗ ∗∗ ∗ ∗∗ ∗∗ ∗∗ 140 250 20 20 0 0 0 10 20 0 10 20 0.2 𝜇 M DOX 0.5 𝜇 M DOX 2𝜇 M 5𝜇 M CaSki SiHa cisplatin cisplatin BSO (𝜇 M) SiHa CaSki SiHa CaSki 0 mM NAC DCFDA 0.5 mM NAC 0 mM NAC DHE 1 mM NAC 0.5 mM NAC 1 mM NAC (c) (a) (b) ∗∗ ∗∗ ∗∗ ∗∗ ∗ 120 120 100 100 80 80 20 20 2𝜇 M cisplatin 5𝜇 M cisplatin 0.2 𝜇 M DOX 0.5 𝜇 M DOX CaSki SiHa CaSki SiHa 0𝜇 m BSO 0𝜇 M BSO 10 𝜇 M BSO 10 𝜇 M BSO 20 𝜇 M BSO 20 𝜇 M BSO (d) (e) Figure 4: Pretreatment of CaSki and SiHa cells with NAC decreases oxidative stress and protects them from death induced by DOX (a) and cisplatin (b), while pretreatment with BSO increases ROS (c) and sensitizes cells to cell death induced by DOX (d) and cisplatin (e). (a and b) SiHa and CaSki cells (1× 10 cells per well) were seeded into 96-well plate and then pretreated with the indicated concentrations of NAC for 4 h followed by treatment with DOX for 48 h (a) or cisplatin for 48 h (b). The viability of cells untreated with drugs in the presence or absenceofNAC cellswas setat100%. (c)10 CaSkiorSiHacells were treatedwith10or20𝜇 M BSO for 24 h and ROS measurements were performed as described in Figure 3(a).(dand e) Cells(1× 10 cells per well) were seeded onto a 96-well plate and then pretreated with indicated concentrations of BSO for 24 h followed by treatment with DOX (d) or cisplatin (e) for 48 h. Viability was assessed by crystal violet staining. The viability of cells untreated with drugs in the presence or absence of BSO was set at 100%. To visualizethe dieff renceinthe levels of ROSradicals oxygen species as well as the mechanism through which these detected by DCFDA and DHE staining after DOX or cisplatin agents exert cytotoxicity. treatment of CaSki and SiHa cells, microscopic analysis was performed. Fluorescent images on microphotographs 4. Discussion (Figure 7) confirmed the differences previously detected by flow cytometry, showing that (1) overall levels of ROS are Resistance to anticancer agents is a major concern in the higher in CaSkithaninSiHacells;(2) treatmentwith treatment of cervical and other cancers. Frequently, this DOX preferentially increases radicals detected by DHE; and resistance appears to be due to alterations in the activation of (3) treatment with cisplatin preferentially increases radicals survival pathways that enable escape from treatment-induced detected by DCFDA. Together, these results demonstrate that the cellular cell death. Identification of these events has the potential to identify new therapeutic targets and sets of biomarkers that response to chemotherapeutic agents depends not only on the overall or total levels of ROS, but also on levels of specicfi could guide clinicians in their selection of treatment options. % of control (viable cells) % of control (viable cells) % of control (viable cells) % of control (viable cells) Mean fluorescence intensity (%) BioMed Research International 9 100 100 60 60 40 40 20 20 0 0 010 20 40 0 5 25 100 DOX (𝜇 M) Cisplatin (𝜇 M) CaSki CaSki HeLa HeLa SiHa C33A SiHa C33A (a) (b) 100 100 60 60 40 40 20 20 0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 DHE DCFDA CaSki HeLa CaSki HeLa SiHa SiHa C33A C33A (c) CaSki SiHa HeLa C33A DCFDA DHE (d) Figure 5: CaSki, SiHa, HeLa, and C33A cervical cancer cells display differential responses to treatment with DOX (a) and cisplatin (b), as well as different baseline levels and distributions of ROS (c and d). (a and b) SiHa, CaSki, HeLa, and C33A cells (1 × 10 cells per well) were seeded into a 96-well plate, allowed to incubate overnight, and then treated with the indicated concentrations of DOX (a) or cisplatin (b) for 48 h. Viability was measured by crystal violet staining and the viability of untreated cells was set at 100%. Each measurement was done in triplicate and error bars indicate the standard deviations of the means. (c and d) 10 cells of each line was treated with 10𝜇 MDCFDA or 10𝜇 M DHE in media and then incubated in the dark at 37 C for 30 minutes. eTh cells were then washed, and following trypsinization were resuspended in 1x PBS and were analysed by flow cytometry. A total of 10 000 events were measured per sample. (c) DCFDA was detected in the FL-1 channel, while DHE was detected in the FL-2 channel. (d) Bar graphs show triplicate measurements of mean uo fl rescence intensity of DCFDA or DHE in SiHa, CaSki, HeLa, and C33A cells. Error bars represent the standard deviation. % of control (viable cells) Cell number Mean fluorescence intensity % of control (viable cells) Cell number 10 BioMed Research International 1400 350 1200 300 800 200 0 0.5 1 0 0.5 1 0 60 100 0 60 100 DOX (𝜇 M) Cisplatin (𝜇 M) CaSki SiHa SiHa CaSki DCFDA DHE DCFDA DHE (a) (b) Figure 6: Treatment of CaSki and SiHa cells with DOX increases the level of ROS detected by DHE (a), while treatment with cisplatin increases the level of ROS detected by DCFDA (b). 10 CaSki or SiHa cells were treated with 0.5 or 1𝜇 MDOX for2h(a) or with 60 or 100𝜇 M cisplatin for 4 h (b). DCFDA or DHE was added to the media to a final concentration of 10 𝜇 M and cells were incubated in the dark at 37 Cfor 30 minutes. Cells were then washed, resuspended in 1x PBS, and analysed using flow cytometry. A total of 10 000 events were measured per sample. DCFDA was detected in the FL-1 channel, while DHE was detected in the FL-2 channel. Bar graphs show triplicate measurements of the mean uo fl rescence intensity of DCFDA or DHE in SiHa and CaSki cells, expressed as 100% of the value observed in untreated cells. In this report, we asked which pathways had the potential other hand, the expression of proteins with antioxidant func- to contribute to the drug resistance observed for some tions was higher in SiHa than in CaSki cells. Examples of such cervical cancers. As our initial model, we selected two cell antioxidant enzymes include SOD1, SOD2, NQO1, PRDX, lines, CaSki and SiHa, as representatives of invasive cervi- and GPX (Figure 2(b), Table 1 and Table 1, Supplementary calcarcinoma.Althoughbothcelllines result from HPV- Material). These differences have downstream consequences, mediated transformation, the two lines respond quite differ- as we observed that the differences between SiHa and CaSki ently to treatments with chemotherapeutic drugs (Figure 1). cellsinthe expressionlevelsofproteinsinvolvedinROS Proteomicanalysisallowed us to identify severalpathways metabolism were reflected in the cellular levels of ROS with the potential to impact these differential responses as measured by flow cytometry ( Figure 3(a)). Furthermore, (Figure 2(a)). First, we were able to detect a 2.5-fold difference these differences were also reflected in the difference in levels in the level of p53 between SiHa and CaSki cells. However, of expression of proteins involved in DNA damage recogni- since the absolute levels of p53 are low in both cell lines due tion and response (Table 1, Supplementary Material), since to accelerated degradation of p53 by HPV16 E6 [12], this these processes are activated during oxidative stress [19, 20]. cannot be the only contributor to the observed differential The connection between chemoresistance and a high level drugresistance.Likewise,thepresenceofmutantp53inC33A of antioxidant defense has been shown previously for other cells [16] means that the p53 response is unlikely to contribute types of cancer, especially for those in advanced stages. An significantly to the C33A response to treatment. Other dif- upregulated antioxidant capacity not only allows cells to ferences identified by our proteomic analysis pointed toward survive under conditions of oxidative stress, but also provides proteins involved in mitochondrial functioning, as these have a mechanism for adapting to exogenous stresses such as thepotential to inufl ence theproductionoffreeradicals treatment with anticancer agents. For example, resistance to and ROS homeostasis. PCR analysis of an array of genes arsenic trioxide by bladder urothelial carcinoma cell lines involved in ROS regulation confirmed differences in the [21] and myeloma cells [22]was foundtobeassociated expression levels of several of these genes and also identified with an upregulation of heme oxygenase (decycling)1, SOD1, differences in the expression of additional genes involved in and glutathione reductase. Also, resistance to agents that the regulation of ROS (Table 1). These findings are consistent induce intracellular ROS production, such as paclitaxel, with the previous observation that, in the absence of p53, ROS DOX, or platinum-based drugs, is correlated with increased itself may act to signal DOX-induced cell death [17]. antioxidant capacity in hepatoma cells [23, 24]. One of the eTh majorsourceofROS production in cellsisthe mito- most important antioxidant enzymes is SOD2, also known chondria, where enzymes involved in the electron transport as MnSOD, which catalyzes the conversion of superoxide chainand theproductionofsuperoxideare located[18]. radicals to H O . SOD2 is also considered to function as a 2 2 ROS-producing enzymes identified in the present study were negative modulator of cellular apoptosis and as a prosurvival expressed at higher levels in CaSki cells (Table 1). On the factor for cancer cells [25]. Mean fluorescence intensity (%) Mean fluorescence intensity (%) BioMed Research International 11 CaSki SiHa DCFDA DHE DCFDA DHE DOX DOX Cisplatin Cisplatin (a) (b) Figure 7: Microphotograph of CaSki (left two panels) and SiHa (right two panels) cells treated with DOX and cisplatin. The majority of anticancer drugs in clinical use are been known to involve oxidative events as well. In fact, thought to act primarily by way of DNA damage or micro- oxidative damage is now considered an important factor tubule disruption. For example, cisplatin and mitomycin in the antitumor activity of DOX [30]. In fact, strategies C are DNA-damaging agents that form bifunctional DNA designed to manipulate levels of ROS are now considered a adducts, leading to activation of the cellular response to major focus of cancer chemotherapy [31, 32]. DNA-damage or to DNA damage-induced apoptosis. How- We found that treatment with both DOX and cisplatin led ever,cisplatin mayalsobeabletoinduceapoptosis in the to increased levels of ROS (Figures 6 and 7). Unexpectedly, absence of nuclear DNA [26], and cisplatin-induced cell however, we found that the two agents induced quite dieff rent deathinrenalcorticalcellswasshowntoinvolveperoxidation profiles of these reactive oxygen species (Figures 6 and andthe releaseofcalcium from intracellularstores[27]. 7). DOX increased the DHE signal (superoxides), but did Basedonthese andother studies, theantitumor eeff ctof not change the levels of those species detected by DCFDA cisplatin is now considered to be due to a combination of (H O , hydroxyl, and peroxyl radicals) (Figures 6(a) and 2 2 nuclear and nonnuclear effects including ROS induction, 7). In contrast, cisplatin considerably increased the DCFDA peroxidation, and lethal cell injury [28, 29]. Anthracyclines, signal, but did not significantly increase the DHE signal such as DOX, are classiefi d as inhibitors of topoisomerase- (Figures 6(b) and 7). Consistent with these ndin fi gs, previous II; however, the toxic side effects of doxorubicin have long work had found that treatment with DOX increased the level 1.0 M 0.5 M No treatment 100 M 60 M 12 BioMed Research International of superoxides, but not of H O , hydroxyl, or peroxyl rad- similartothatofHeLacells,for example,might favorDOX 2 2 icals in HaCaT keratinocytes [33, 34]. In contrast, cisplatin over cisplatin. Our experimental results demonstrated that primarily increased the level of hydroxyl radicals but not of different cervical cancer cell lines differ not only in their total superoxides in human hair follicle dermal papilla cells and in level of ROS, but also in the levels of specific free radicals. Ide- HaCaT keratinocytes [35]. ally, clinical selection of chemotherapeutic treatments should The mechanism of ROS generation induced by DOX consider the types of ROS induced, which will be related to is controversial and not yet fully understood [36, 37]. It the mechanism of drug action at the molecular level. is known that in the presence of molecular oxygen, the The data in this report provides evidence that differences derivatives from “redox cycle” are acted upon by a number in the sensitivity of cervical cancers to chemotherapeutic of NAD(P)H-oxidoreductases cytochrome P450 or cyto- treatments is likely due, at least in part, to differences in chrome-b5 reductases, mitochondrial NADH dehydroge- the relative levels of pro- and antioxidant enzymatic activity nase, xanthine dehydrogenase, endothelial nitric oxide syn- that lead to different baseline levels of oxidative stress. thase (reductase domain) to generate superoxides [38–40]. Chemoresistance is undoubtedly a multifactoral process and One-electron “redox cycling” of DOX also produces super- factors in addition to high levels of antioxidant defense are oxide; this process is accompanied by the release of iron likely to contribute. Such factors may include differences in from intracellular stores and results in formation of drug-iron angiogenesis, which could aeff ct the penetration of agents complexes that release superoxides and hydrogen peroxides into tumor tissue, increased drug eu ffl x from the cancer [41], which can then be decomposed by antioxidant systems. cells, reduced uptake of drugs, interactions of cancer cells The higher levels of superoxide observed during DOX treat- with their surrounding microenvironment, and other factors ment arebelievedtobedue to adecreaseinthe activity [43–45]. Such factors are also likely to contribute to the of superoxide-decomposing enzymes such as MnSOD and chemoresistance of cervical carcinomas. A better under- catalase [34]. standing of how and when these various factors, including ROS generation by platinum-based drugs is also not fully the baseline levels of oxidative stress, affect how a particular understood. eTh predominant formation of hydroxyl radicals tumor will respond to a specific treatment has the potential is believed to be the result of peroxynitrite decomposition in to improve our treatment of patients suffering from cervical cisplatin-treated cells [35]. After penetration into the cells, malignancies. alkylating agents such as cisplatin bind to glutathione, and this interaction leads to removal of this complex from cells 5. Conclusions through an ATP-binding cassette. Depletion of glutathione then results in increased H O and hydroxyl radicals [35, 42]. 2 2 We utilized a proteomic approach to identify the path- The difference in the mechanism of action between DOX ways involved in resistance to the chemotherapeutic agents and cisplatin is supported by experiments in which cells cisplatin and doxorubicin and then extended these find- were sensitized by BSO pretreatment prior to exposure to the ings by analyzing and comparing the expression level of drugs (Figures 4(d) and 4(e)). The lower level of sensitization genes involved in reactive oxygen species (ROS) metabolism to cisplatin can be explained on the basis that BSO and through the use of an qRT-PCR array. These data enabled cisplatin act on the same substrate, glutathione. eTh refore, us to demonstrate that pathways involved in oxidative stress we speculatethatglutathione depletion as aresultofBSO and antioxidant defense contribute to drug resistance. In treatment does not change cell viability dramatically aeft r particular, the sensitive CaSki cells expressed lower levels of administration of cisplatin, because the substrate for cisplatin antioxidant enzymes, resulting in higher levels of ROS, than toxicity is already depleted. did the resistant SiHa cells. Decreasing or increasing oxidative To examine the dependence of the cellular response to stress using pharmacological agents led to protection or sen- therapeutic agents on oxidative stress, we manipulated ROS sitization, respectively, in both cell lines, supporting the idea levels by either depleting ROS through pretreatment with that cellular levels of oxidative stress affect responsiveness to NAC or increasing ROS through pretreatment with BSO. treatment. Interestingly, the two agents tested, doxorubicin Pretreatment with NAC or BSO, reducing or increasing, (DOX) and cisplatin, induced different profiles of ROS, and respectively, the levels of ROS (Figures 3(c) and 4(c))were these differences appear to contribute to the differential able to protect or to sensitize cells to both agents, respectively sensitivity displayed by cervical cancer cells to treatment. (Figures 4(a), 4(b), 4(d),and 4(e)). We also demonstrated that cells with higher baseline levels of ROS experienced a more rapid loss of viability following drug treatment than Conflict of Interests did cells with lower baseline levels of ROS. In particular, eTh authors declare that there is no conflict of interests CaSki and C33A cells, which display higher levels of ROS, regarding the publication of this paper. died faster than did SiHa cells following treatment with either DOX or with cisplatin (Figures 1 and 5). 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