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Cistanche tubulosa induces reactive oxygen species-mediated apoptosis of primary and metastatic human colon cancer cells

Cistanche tubulosa induces reactive oxygen species-mediated apoptosis of primary and metastatic... Journal of Applied Pharmaceutical Science Vol. 7 (05), pp. 039-045, May, 2017 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2017.70507 ISSN 2231-3354 Cistanche tubulosa induces reactive oxygen species-mediated apoptosis of primary and metastatic human colon cancer cells 1 1 1 2 1 Afnan Saleh Al-Menhali , Safya Ali Jameela , Aishah A. Latiff , Mohamed A. Elrayess , Mohammed Alsayrafi , Morana Jaganjac * Anti Doping Lab Qatar, Toxicology and Multipurpose, Doha, Qatar. Anti Doping Lab Qatar, Life Science Research, Doha, Qatar. ABSTRACT ARTICLE INFO Article history: Colon cancer is the third most common cancer worldwide. Conventional therapies have shown moderate Received on: 01/11/2016 efficacy with severe adverse effects, therefore there is an urgent need for safer alternatives. In this study, Accepted on: 07/01/2017 Cistanche tubulosa, local name Thanoon, was considered as a potential phytotherapeutic strategy because of its Available online: 30/05/2017 known high therapeutic potential in traditional medicine and wide abundance in the Middle East region. Bioactive compounds were extracted from powdered Cistanche tubulosa and tested for their anticancer Key words: properties against four colon cancer cell lines including two derived from primary tumor (CaCo2 and HCT116) Cistanche tubulosa, colon and two derived from metastatic site (LoVo and SW620). Effect of Cistanche tubulosa on induction of apoptosis cancer, redox homeostasis, and cellular redox homeostasis were also investigated. Cistanche tubulosa exhibited a concentration and time- anticancer bioactivity. dependent inhibition of proliferation of all tested cancer cell lines by more than 60% upon 72 hours treatment with 1 mg/mL of crude extract. Inhibition of proliferation was marked by induction of apoptosis, intracellular reactive oxygen species production and mitochondrial superoxides. This data suggest that Cistanche tubulosa is a promising candidate for additive anti-colon cancer therapy. This is the first study showing anticancer bioactivity of Cistanche tubulosa against colon cancer cells. Abbreviations: 7-AAD, 7-Amino-Actinomycin D; CTE, Cistanche tubulosa extract; EMEM, Eagle's Minimum Essential Medium; FBS, fetal bovine serum; DCF, 2,7-dichlorofluorescein; DCFH-DA, 2,7- dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethylsulfoxide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium; PE, Phycoerythrin; RFU, relative fluorescence units; ROS, reactive oxygen species Phytotherapeutics (Weidner et al., 2015). Phytotherapy, the use of INTRODUCTION medicinal plants to treat diseases, has been an inevitable part of Colorectal cancer is one of the most common cancers ancient human history. Medicinal plants have long been utilized as worldwide (Brenner et al., 2014), and its incidence is constantly alternative treatment sources for cancers, representing more than increasing with an estimated 2.4 million cases in 2035, due to sixty percent of anticancer agents used in conventional medicine modern diet and lifestyle, along with reduced physical activity. (Balunas and Kinghorn, 2005; Saibu et al., 2015).Some of the best Current efforts are not sufficient to combat the present epidemic known examples include extracts from Catharanthus roseus G. of colorectal cancer and therefore novel approaches are needed Don. (Apocynaceae), Taxus baccata L. (Taxaceae) and for effective prevention and treatment including changes in life Camptotheca acuminate Decne (Nyssaceae) (Cragg and Newman, style in combination with safer alternative interventions such as 2005; da Rocha et al., 2001). Several herbal extracts and phytochemicals were shown to exert antitumor effects in colorectal * Corresponding Author cancer attributed to the induction of reactive oxygen species (ROS) Morana Jaganjac, PhD, Senior Scientist, Toxicology and Multipurpose production and associated apoptosis of cancer cells as the case of Labs, Anti Doping Lab Qatar, Sport City Road, Doha, Qatar. Phone: +97444132846; Fax: +97444132997 extracts from Melissa officinalis (Weidner et al., 2015). © 2016 Pachacama Al-Menhali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). 040 Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 Among phytotherapeutic candidates, Cistanche tubulosa, at 37°C in respective medium supplemented with 10% fetal bovine an Orobanchaceae parasitic desert plant (Jiang et al., 2009) that is serum (FBS, SIGMA, Germany) and 1% Penicillin/Streptomycin widely distributed in arid and semi-arid regions of Africa, Asia (SIGMA, Germany) in a humidified atmosphere containing 5% and the Mediterranean region, has been shown to have valuable carbon dioxide. medicinal properties. Cistanche tubulosa has been extensively used in traditional medicine, and suggested to have curative effects Cell viability assay in kidney deficiency, morbid leucorrhea, metrorrhagia, female The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium infertility, and senile constipation (Jiang et al., 2009).In addition to (MTT) assay was used to evaluate the cytotoxic activity of CTE its traditional medicinal uses, important medicinal properties of similarly as described earlier (Jaganjac et al., 2010). The seeding Cistanche Tubulosa have been intensively studied during the last density of CaCo2, HCT116, SW620 and LoVo cells cultured in decade including vasorelaxant (Yoshikawa et al., 2006), 96-well plates was 10 cells per well. Cells were plated in hepatoprotective (Morikawa et al, 2010), anti-hyperglycemic and respective medium supplemented with 10% FBS 24 hours prior to hypolipidemic effects (Xiong et al., 2013). Cistanche Tubulosa treatment. After 24 hours, the medium was removed and cells were was also suggested as a potent enhancer of the immune system, treated with 0, 0.25, 0.5, 1 and 2 mg/mL of CTE for 24, 48 and 72 promoter of bone formation, and an anti-aging and anti-fatigue hours at 37°C in a humidified atmosphere containing 5% CO . agent (Xu et al., 2014). Furthermore, the Cistanche tubulosa Upon CTE treatment, the medium was removed and 40 µL of extract has been shown to block amyloid deposition in MTT solution (0.5 mg/mL) added to each well. Alzheimer’s disease model (Wu et al., 2015). Despite its various After 3 h of incubation, MTT solution was removed, the therapeutic uses, the effect of Cistanche Tubulosa as a potential formazan product dissolved in dimethylsulfoxide (DMSO, anticancer agent has not been studied yet. SIGMA, Germany), and the absorbance measured at 590 nm with In the present work we have investigated the microplate reader (Infinite 200 PRO NanoQuant, Tecan Trading anticancer effect of Cistanche Tubulosa on two primary and two AG, Switzerland). metastatic colon cancer cell lines and the potential mechanisms Apoptosis assay underlying this effect. Apoptosis in CaCo2, HCT116, SW620 and LoVo cells was detected using PE Annexin V Apoptosis Detection Kit I with MATERIALS AND METHODS 7-Amino-Actinomycin D (7-AAD) as a vital dye (Becton Collection and preparation of plant extract Dickinson International, Belgium) according to the manufacturer's The samples of Cistanche tubulosa were collected from instruction. Briefly, cells were seeded in 24-well plates at a density desert area in Qatar during 2014 and the authenticity of the plant of 5×10 cells/well in a respective media supplemented with 10% was confirmed by herbatologist. Voucher samples are archived in FBS for 24 h prior to the addition of CTE (0, 0.5 or 1 mg/mL). the Toxicology and Multipurpose Department at ADLQ. The sun- Following 24 CTE incubation, cells were harvested, washed twice dried plant samples were grinded with Retsch Knife Mill with cold phosphate-buffered saline and stained with Grindomix GM300 into fine powder. Twenty grams of powder Phycoerythrin (PE) Annexin V and 7-AAD for 15 min at room were extracted with 200 mL ultrapure water over night at 37 °C on temperature in the dark. Stained cells were analyzed within 1 hour a rotary shaker at 200 rpm. The crude Cistanche tubulosa extracts by flow cytometry using FACS. Aria III flow cytometer and (CTE) were centrifuged for 30 min at 8000 rpm to pellet non- FACSDiva software (Becton Dickinson) at a low flow rate with a soluble compounds, supernatant collected and freeze dried using minimum of 10 cells. Treatment of cells for 4 hours with 6 µM Labconco Freezone 6 plus Freeze dryer. The dried extract was camptothecin (SIGMA) was used as a positive control for the reconstituted in Dulbecco’s Modified Eagle’s Medium (DMEM, assay. SIGMA, Germany) to a concentration of 20 mg/mL and sterile filtered through 0.2 micron membrane filter. Intracellular ROS production Intracellular ROS production was examined using 2,7- Cell lines and cell maintenance dichlorodihydrofluorescein diacetate (DCFH-DA, SIGMA, Human colon carcinoma cell lines CaCo2, SW620 and Germany). DCFH-DA is a nonfluorescent probe, which is oxidized LoVo were obtained from Cell Lines Service (CLS, Eppelheim, with intracellular ROS to the fluorescent compound 2,7- Germany) while HCT 116 cell line was a kind gift from the dichlorofluorescein (DCF) (Poljak-Blazi et al., 2011). The DCFH- Biological and Environmental Sciences, Department at Qatar DA assay was performed similarly as we have described before University. CaCo2 and HCT11 were derived from the primary site (Cindric et al, 2013; Poljak-Blazi et al., 2011). Briefly, the seeding of colon carcinoma while SW620 and LoVo derived from density of CaCo2, HCT116, SW620 and LoVo cells cultured in metastatic site. SW620, HCT116 and LoVo cells were cultured 96-well black plates was 10 cells per well. Cells were plated in and maintained in DME Medium while CaCo2 cells were respective medium supplemented with 10% FBS for 24 hours. maintained in Eagle's Minimum Essential Medium (EMEM, Prior to treatment cells were incubated with 10 µM DCFH-DA at SIGMA, Germany). The cells were grown as monolayer cultured 37°C for 30 min in 5% CO / 95% air. Cells were then washed and 2 Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 041 treated with 0, 0.5 and 1 mg/mL of CTE in the medium w/o phenol tested showed a strong inhibitory effect on CaCo2 cell line in a red. The intracellular ROS formation was monitored continuously concentration and time dependent manner (Figure 1A). Seventy throughout 25 hours at 37°C and 5% CO using microplate reader two hours of CTE treatment inhibited the growth of CaCo2 cells with top fluorescence and gas control module (Infinite 200 PRO, by more than 60% compared to control (p < 0.05 for all Tecan Trading AG, Switzerland). The fluorescence intensity was concentrations). Significant impact of CTE treatment on HCT116 measured with an excitation wavelength of 500 nm and emission cell growth was also detected at all-time points at the two highest detection at 529 nm. The arbitrary units, relative fluorescence units concentrations (1 mg/mL and 2 mg/mL), reaching greater than (RFU), were based directly on fluorescence intensity. 70% reduction at latter concentration (Figure 1B, p < 0.05). Although the two lower CTE concentrations (0.25 and 0.5 mg/mL) Mitochondrial superoxide generation significantly reduced HCT116 growth of cells after 24 hours The ability of CTE to induce superoxide generation by (p<0.05), they had no significant effect following 72 hours mitochondria was estimated using cell-permeable, mitochondria treatment compared to control (p>0.05). Time and concentration targeted MitoSOX Red probe (Life Technologies) and Hoechst dependent inhibition of proliferation with CTE was further 33342 for nuclear staining (Life Technologies). CaCo2, HCT116, confirmed in LoVo cells by more than 60% at highest SW620 and LoVo cells were seeded in 96-well plates at a density concentration (p < 0.05) (Figure 1C), and all four CTE 10 cells per well in a respective medium supplemented with 10% concentrations tested reduced the growth of SW620 cells after 48 FBS for 24 hours. Cells were then loaded with 4 µM of MitoSOX hours (Figure 1D, p < 0.05 for all). After 72 hours of treatment the and 2 µM of Hoechst 33342 for 20 min, excess dye washed and same effect was observed only for the two highest concentrations wells treated with 0, 0.5 and 1 mg/mL of CTE for 24 hours at 37°C (p < 0.05) while 0.25 and 0.5 mg/mL concentrations showed no and 5% CO . The fluorescence intensity was measured with an significant effect compared to control (p > 0.05). The impact of 0.5 excitation wavelength of 510 nm and emission detection at 580 nm and 1 mg/mL CTE treatment on induction of apoptosis was further for MitoSOX and an excitation wavelength of 350 nm and tested in all four cell lines (Figure 2). Increased number of cells in emission detection at 461 nm for Hoechst 33342 using microplate early apoptosis was detected in HCT116 and LoVo following 24 reader with top fluorescence (Infinite 200 PRO, Tecan Trading hours treatment with 0.5 mg/mL (p<0.05, Figure 2B and 2C) and AG, Switzerland) in all cell lines at 1 mg/mL (p<0.05). Significant increase in necrotic or late apoptotic cell number was further observed in Statistical analysis CaCo2 and SW620 cell lines (p<0.05, Figure 2A and 2D). Ability Descriptive statistics were shown as the mean +/− SD. of CTE to induce intracellular ROS production is demonstrated in The significance of differences between groups was assessed using Figure 3. Three hours following CTE treatment there was a strong the Student t-test and Chi-square test. When more than two groups increase in intracellular ROS production in all cell lines (p<0.05). were compared, we used one sided ANOVA with appropriate post The intracellular ROS production increased progressively hoc testing. The SPSS 11.01 for Mircosoft Windows were used. throughout the 25 hours treatment in a time and concentration Differences with P less than 0.05 were considered statistically dependent manner. Furthermore, staining of cells with significant. mitochondria targeted probe revealed a strong impact of CTE on mitochondrial superoxide production in a concentration dependent RESULTS manner (Figure 4). Highest increase in superoxide production by mitochondria was observed in HCT116 (69%, Figure 4B) and The effect of CTE on proliferation of human colon LoVo cells (82%, Figure 4C) following 24 hours treatment with 1 cancer cell lines is shown in Figure 1. All CTE concentrations mg/mL of CTE. 042 Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 Fig. 1: Effect of Cistanche tubulosa on the viability of colon cancer cell lines. Cell viability measured by MTT assay of (A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells are presented as percentage of control untreated colon cancer cell line. Mean values (±SD) for 5-replicates of representative experiment is given: (*) significance p<0.05 in comparison to control untreated respective cells. Fig. 2: Cistanche tubulosa water extract induces apoptosis in human colon cancer cells. Annexin-V-FITC flow cytometry analyses of (A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells are presented as percentage of control untreated c olon cancer cell line. Mean values (±SD) for 3 -replicates of representative experiment is given: (*) significance p<0.05 in comparison to control untreated respective cells. Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 043 Fig. 3: Cistanche tubulosa water extract induces intracellular ROS production in a time- and dose-dependent. ROS production measured by DCFH-DA assay in (A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells is presented as mean RFU values (±SD) for the respective 5 -replicates of representative experiment. (*) Significance p<0.05 in comparison to control untreated colon cancer cells. Fig. 4: Cistanche tubulosa water extract induces mitochondrial superoxide production in human colon cancer cells. Fluorescence intensity of mitochondria targeted MitoSOX Red probe in A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells are presented as percentage of control untreated colon cancer cell line. Mean values (±SD) for 5-replicates of representative experiment is given: (*) significance p<0.05 in comparison to control untreated colon cancer cells. 044 Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 DISCUSSION is at least in part mediated through mitochondria induced ROS mechanism. Although earlier studies have reported numerous medicinal properties of Cistanche tubulosa, this is the first report CONCLUSIONS of its anti proliferative effect in malignant cells. Cistanche In conclusion, our data suggests that the water extract of tubulosa bioactive compounds extracted with water, a highly polar desert plant Cistanche tubulosa may represent a promising solvent, showed strong anticancer bioactivity. We have previously candidate for anticancer approach in combination with other compared the efficiency of Cistanche tubulosa solubilized in water conventional therapies for prevention and treatment of colon against other solvents such as methanol and ethyl acetate, but cancer. We also demonstrate that the toxicity of the plant extract water extracts exhibited the most promising anticancer activities against cancer cells is mediated by increased intracellular ROS (data not shown). production and, at least in part, by mitochondrial-dependent We have demonstrated the ability of CTE at 1 mg/mL apoptosis. Further studies are ongoing to isolate and characterize and 2 mg/mL to inhibit 60% of the growth of both primary and the individual biologically active constituents responsible for metastatic colon cancer cell lines, revealing a potential important anticancer activity. role of Cistanche tubulosa as a colon cancer treatment. Compared More research is needed to evaluate the potential use of to normal cells, cancer cells are generally characterized by a this extract as an effective chemopreventive agent and to disturbance in redox homeostasis and a common strategy of understand the mechanisms of action on colon cancer cells at the current anticancer therapies is to increase cellular oxidative stress molecular level. Further preclinical and clinical studies are also (Yang et al, 2013). Although physiologically low levels of ROS needed to confirm the observed beneficial health effects of have important role as signaling molecules, the excessive ROS Cistanche tubulosa for cancer prevention. production can contribute to cancer instability and malignancy (Liou and Storz, 2010). Paradoxically, this imbalance in cellular ACKNOWLEDGEMENTS redox homeostasis renders cancer cells more vulnerable to ROS- induced cell death (Jaganjac et al., 2008; Nogueira and Hay, Financial support and sponsorship: This study was supported by 2013). The anti-proliferative effect of CTE reported in this study the Anti Doping Lab Qatar. can be mediated by various extra- and intracellular mechanisms of Conflict of Interests: The authors’ declare no conflict of interest. known and unknown compounds within the extract, targeting multiple pathways that play essential roles in apoptosis. In order to REFERENCES differentiate between different modes of cell death, we investigated the potential mechanism responsible for the observed Balunas MJ, Kinghorn, AD. Drug discovery from medicinal CTE-induced cytotoxicity. plants. Life Sci. 2005;78:431-41. Brenner H, Kloor M, Pox CP. Colorectal cancer. 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Indeed, our data also indicate that CTE induced Jaganjac M, Matijevic T, Cindric M, Cipak A, Mrakovcic L, phosphatidylserine externalization, a common effect in apoptosis, Gubisch W, Zarkovic N. Induction of CMV-1 promoter by 4-hydroxy-2- in both primary and metastatic cancer cell lines, suggesting that nonenal in human embryonic kidney cells. Acta Biochim Pol. 2010;57:179-83. the mechanism of CTE induced death is mediated by apoptosis Jaganjac M, Poljak-Blazi M, Zarkovic K, Schaur RJ, Zarkovic rather than necrosis. The activation of apoptosis in cancer cells is a N. The involvement of granulocytes in spontaneous regression of Walker corrective strategy and many anticancer drugs may exert apoptotic 256 carcinoma. Cancer Lett. 2008;260:180-6. effects in cancer cells. JiangY, Tu PF. Analysis of chemical constituents in Cistanche species. J Chromatogr A. 2009;1216:1970-9. Compounds or extracts with pro-apoptotic activities in Liou GY, Storz P. Reactive oxygen species in cancer. Free cancer cells are therefore potentially useful in anticancer drug Radic Res. 2010;44:479-96. research (Wong, 2011). In order to determine whether CTE Morikawa T, Pan Y, Ninomiya K, Imura K, Matsuda H, Yoshikawa M, Yuan D, Muraoka O. Acylated phenylethanoid induced pro-apoptotic effect is mediated by mitochondria-induced oligoglycosides with hepatoprotective activity from the desert plant ROS mechanism, we measured superoxide production using Cistanche tubulosa. Bioorg Med Chem. 2010;18:1882-90. mitochondria targeted fluorescent probe in CTE treated cells.Our Nogueira V, Hay N. Molecular pathways: reactive oxygen data clearly shows that CTE stimulates mitochondrial superoxide species homeostasis in cancer cells and implications for cancer therapy. Clin Cancer Res. 2013;19:4309-14. production suggesting that Cistanche tubulosa anticancer activity Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 045 Poljak-Blazi M, Jaganjac M, Sabol I, Mihaljevic B, Matovina Xu R, Sun S, Zhu W, Xu C, Liu Y, Shen L, Shi Y, Chen J. M, Grce M. Effect of ferric ions on reactive oxygen species formation, Multi-step infrared macro-fingerprint features of ethanol extracts from cervical cancer cell lines growth and E6/E7 oncogene expression. different Cistanche species in China combined with HPLC fingerprint. J ToxicolIn Vitro. 2011;25:160-6. Mol Struct. 2014;1069:236-244. Saibu GM, Katerere DR, Rees DJG, Meyer M. In vitro Yang Y, Karakhanova S, Werner J, Bazhin AV. Reactive cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea oxygen species in cancer biology and anticancer therapy. Curr Med Chem. leaves and bulbs. J Ethnopharmacol. 2015;164:203-9. 2013;20:3677-92. Sinha K, Das J, Pal PB, Sil PC. Oxidative stress: the Yoshikawa M, Matsuda H, Morikawa T, Xie H, Nakamura S, mitochondria-dependent and mitochondria-independent pathways of Muraoka O. Phenylethanoid oligoglycosides and acylated oligosugars with apoptosis. Arch Toxicol.2013; 87:1157-80. vasorelaxant activity from Cistanche tubulosa. Bioorg Med Chem. Trachootham D, Lu W, Ogasawara MA, Nilsa RD, Huang P. 2006;14:7468-75. Redox regulation of cell survival. Antioxid Redox Signal. 2008;10:1343- Weidner C, Rousseau M, Plauth A, Wowro SJ, Fischer C, Abdel-Aziz H, Sauer S. Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species. Phytomedicine. 2015;22:262-70. Wong RS. Apoptosis in cancer: from pathogenesis to treatment. J ExpClin Cancer Res. 2011;30:87. How to cite this article: Wu S-H, Chou F-P, Chyau C-C, Chen J-H, Tu S-F, Lin H-H. Anti-cancerous effects of Wasabia japonica extract in Hep3B liver cancer Al-Menhali AS, Jameela SA, Latiff AA, Elrayess MA, Alsayrafi cells via ROS accumulation, DNA damage and p73-mediated apoptosis. J M, Jaganjac M. Cistanche tubulosa induces reactive oxygen Funct Foods. 2015;14:445-55. species-mediated apoptosis of primary and metastatic human colon Xiong W-T, Gu L, Wang C, Sun H-X, Liu X. Anti- cancer cells. J App Pharm Sci, 2017; 7 (05): 039-045. hyperglycemic and hypolipidemic effects of Cistanche tubulosa in type 2 diabetic db/db mice. J Ethnopharmacol. 2013;150:935-45. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Pharmaceutical Science Unpaywall

Cistanche tubulosa induces reactive oxygen species-mediated apoptosis of primary and metastatic human colon cancer cells

Journal of Applied Pharmaceutical ScienceJan 1, 2017

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Journal of Applied Pharmaceutical Science Vol. 7 (05), pp. 039-045, May, 2017 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2017.70507 ISSN 2231-3354 Cistanche tubulosa induces reactive oxygen species-mediated apoptosis of primary and metastatic human colon cancer cells 1 1 1 2 1 Afnan Saleh Al-Menhali , Safya Ali Jameela , Aishah A. Latiff , Mohamed A. Elrayess , Mohammed Alsayrafi , Morana Jaganjac * Anti Doping Lab Qatar, Toxicology and Multipurpose, Doha, Qatar. Anti Doping Lab Qatar, Life Science Research, Doha, Qatar. ABSTRACT ARTICLE INFO Article history: Colon cancer is the third most common cancer worldwide. Conventional therapies have shown moderate Received on: 01/11/2016 efficacy with severe adverse effects, therefore there is an urgent need for safer alternatives. In this study, Accepted on: 07/01/2017 Cistanche tubulosa, local name Thanoon, was considered as a potential phytotherapeutic strategy because of its Available online: 30/05/2017 known high therapeutic potential in traditional medicine and wide abundance in the Middle East region. Bioactive compounds were extracted from powdered Cistanche tubulosa and tested for their anticancer Key words: properties against four colon cancer cell lines including two derived from primary tumor (CaCo2 and HCT116) Cistanche tubulosa, colon and two derived from metastatic site (LoVo and SW620). Effect of Cistanche tubulosa on induction of apoptosis cancer, redox homeostasis, and cellular redox homeostasis were also investigated. Cistanche tubulosa exhibited a concentration and time- anticancer bioactivity. dependent inhibition of proliferation of all tested cancer cell lines by more than 60% upon 72 hours treatment with 1 mg/mL of crude extract. Inhibition of proliferation was marked by induction of apoptosis, intracellular reactive oxygen species production and mitochondrial superoxides. This data suggest that Cistanche tubulosa is a promising candidate for additive anti-colon cancer therapy. This is the first study showing anticancer bioactivity of Cistanche tubulosa against colon cancer cells. Abbreviations: 7-AAD, 7-Amino-Actinomycin D; CTE, Cistanche tubulosa extract; EMEM, Eagle's Minimum Essential Medium; FBS, fetal bovine serum; DCF, 2,7-dichlorofluorescein; DCFH-DA, 2,7- dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethylsulfoxide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium; PE, Phycoerythrin; RFU, relative fluorescence units; ROS, reactive oxygen species Phytotherapeutics (Weidner et al., 2015). Phytotherapy, the use of INTRODUCTION medicinal plants to treat diseases, has been an inevitable part of Colorectal cancer is one of the most common cancers ancient human history. Medicinal plants have long been utilized as worldwide (Brenner et al., 2014), and its incidence is constantly alternative treatment sources for cancers, representing more than increasing with an estimated 2.4 million cases in 2035, due to sixty percent of anticancer agents used in conventional medicine modern diet and lifestyle, along with reduced physical activity. (Balunas and Kinghorn, 2005; Saibu et al., 2015).Some of the best Current efforts are not sufficient to combat the present epidemic known examples include extracts from Catharanthus roseus G. of colorectal cancer and therefore novel approaches are needed Don. (Apocynaceae), Taxus baccata L. (Taxaceae) and for effective prevention and treatment including changes in life Camptotheca acuminate Decne (Nyssaceae) (Cragg and Newman, style in combination with safer alternative interventions such as 2005; da Rocha et al., 2001). Several herbal extracts and phytochemicals were shown to exert antitumor effects in colorectal * Corresponding Author cancer attributed to the induction of reactive oxygen species (ROS) Morana Jaganjac, PhD, Senior Scientist, Toxicology and Multipurpose production and associated apoptosis of cancer cells as the case of Labs, Anti Doping Lab Qatar, Sport City Road, Doha, Qatar. Phone: +97444132846; Fax: +97444132997 extracts from Melissa officinalis (Weidner et al., 2015). © 2016 Pachacama Al-Menhali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). 040 Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 Among phytotherapeutic candidates, Cistanche tubulosa, at 37°C in respective medium supplemented with 10% fetal bovine an Orobanchaceae parasitic desert plant (Jiang et al., 2009) that is serum (FBS, SIGMA, Germany) and 1% Penicillin/Streptomycin widely distributed in arid and semi-arid regions of Africa, Asia (SIGMA, Germany) in a humidified atmosphere containing 5% and the Mediterranean region, has been shown to have valuable carbon dioxide. medicinal properties. Cistanche tubulosa has been extensively used in traditional medicine, and suggested to have curative effects Cell viability assay in kidney deficiency, morbid leucorrhea, metrorrhagia, female The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium infertility, and senile constipation (Jiang et al., 2009).In addition to (MTT) assay was used to evaluate the cytotoxic activity of CTE its traditional medicinal uses, important medicinal properties of similarly as described earlier (Jaganjac et al., 2010). The seeding Cistanche Tubulosa have been intensively studied during the last density of CaCo2, HCT116, SW620 and LoVo cells cultured in decade including vasorelaxant (Yoshikawa et al., 2006), 96-well plates was 10 cells per well. Cells were plated in hepatoprotective (Morikawa et al, 2010), anti-hyperglycemic and respective medium supplemented with 10% FBS 24 hours prior to hypolipidemic effects (Xiong et al., 2013). Cistanche Tubulosa treatment. After 24 hours, the medium was removed and cells were was also suggested as a potent enhancer of the immune system, treated with 0, 0.25, 0.5, 1 and 2 mg/mL of CTE for 24, 48 and 72 promoter of bone formation, and an anti-aging and anti-fatigue hours at 37°C in a humidified atmosphere containing 5% CO . agent (Xu et al., 2014). Furthermore, the Cistanche tubulosa Upon CTE treatment, the medium was removed and 40 µL of extract has been shown to block amyloid deposition in MTT solution (0.5 mg/mL) added to each well. Alzheimer’s disease model (Wu et al., 2015). Despite its various After 3 h of incubation, MTT solution was removed, the therapeutic uses, the effect of Cistanche Tubulosa as a potential formazan product dissolved in dimethylsulfoxide (DMSO, anticancer agent has not been studied yet. SIGMA, Germany), and the absorbance measured at 590 nm with In the present work we have investigated the microplate reader (Infinite 200 PRO NanoQuant, Tecan Trading anticancer effect of Cistanche Tubulosa on two primary and two AG, Switzerland). metastatic colon cancer cell lines and the potential mechanisms Apoptosis assay underlying this effect. Apoptosis in CaCo2, HCT116, SW620 and LoVo cells was detected using PE Annexin V Apoptosis Detection Kit I with MATERIALS AND METHODS 7-Amino-Actinomycin D (7-AAD) as a vital dye (Becton Collection and preparation of plant extract Dickinson International, Belgium) according to the manufacturer's The samples of Cistanche tubulosa were collected from instruction. Briefly, cells were seeded in 24-well plates at a density desert area in Qatar during 2014 and the authenticity of the plant of 5×10 cells/well in a respective media supplemented with 10% was confirmed by herbatologist. Voucher samples are archived in FBS for 24 h prior to the addition of CTE (0, 0.5 or 1 mg/mL). the Toxicology and Multipurpose Department at ADLQ. The sun- Following 24 CTE incubation, cells were harvested, washed twice dried plant samples were grinded with Retsch Knife Mill with cold phosphate-buffered saline and stained with Grindomix GM300 into fine powder. Twenty grams of powder Phycoerythrin (PE) Annexin V and 7-AAD for 15 min at room were extracted with 200 mL ultrapure water over night at 37 °C on temperature in the dark. Stained cells were analyzed within 1 hour a rotary shaker at 200 rpm. The crude Cistanche tubulosa extracts by flow cytometry using FACS. Aria III flow cytometer and (CTE) were centrifuged for 30 min at 8000 rpm to pellet non- FACSDiva software (Becton Dickinson) at a low flow rate with a soluble compounds, supernatant collected and freeze dried using minimum of 10 cells. Treatment of cells for 4 hours with 6 µM Labconco Freezone 6 plus Freeze dryer. The dried extract was camptothecin (SIGMA) was used as a positive control for the reconstituted in Dulbecco’s Modified Eagle’s Medium (DMEM, assay. SIGMA, Germany) to a concentration of 20 mg/mL and sterile filtered through 0.2 micron membrane filter. Intracellular ROS production Intracellular ROS production was examined using 2,7- Cell lines and cell maintenance dichlorodihydrofluorescein diacetate (DCFH-DA, SIGMA, Human colon carcinoma cell lines CaCo2, SW620 and Germany). DCFH-DA is a nonfluorescent probe, which is oxidized LoVo were obtained from Cell Lines Service (CLS, Eppelheim, with intracellular ROS to the fluorescent compound 2,7- Germany) while HCT 116 cell line was a kind gift from the dichlorofluorescein (DCF) (Poljak-Blazi et al., 2011). The DCFH- Biological and Environmental Sciences, Department at Qatar DA assay was performed similarly as we have described before University. CaCo2 and HCT11 were derived from the primary site (Cindric et al, 2013; Poljak-Blazi et al., 2011). Briefly, the seeding of colon carcinoma while SW620 and LoVo derived from density of CaCo2, HCT116, SW620 and LoVo cells cultured in metastatic site. SW620, HCT116 and LoVo cells were cultured 96-well black plates was 10 cells per well. Cells were plated in and maintained in DME Medium while CaCo2 cells were respective medium supplemented with 10% FBS for 24 hours. maintained in Eagle's Minimum Essential Medium (EMEM, Prior to treatment cells were incubated with 10 µM DCFH-DA at SIGMA, Germany). The cells were grown as monolayer cultured 37°C for 30 min in 5% CO / 95% air. Cells were then washed and 2 Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 041 treated with 0, 0.5 and 1 mg/mL of CTE in the medium w/o phenol tested showed a strong inhibitory effect on CaCo2 cell line in a red. The intracellular ROS formation was monitored continuously concentration and time dependent manner (Figure 1A). Seventy throughout 25 hours at 37°C and 5% CO using microplate reader two hours of CTE treatment inhibited the growth of CaCo2 cells with top fluorescence and gas control module (Infinite 200 PRO, by more than 60% compared to control (p < 0.05 for all Tecan Trading AG, Switzerland). The fluorescence intensity was concentrations). Significant impact of CTE treatment on HCT116 measured with an excitation wavelength of 500 nm and emission cell growth was also detected at all-time points at the two highest detection at 529 nm. The arbitrary units, relative fluorescence units concentrations (1 mg/mL and 2 mg/mL), reaching greater than (RFU), were based directly on fluorescence intensity. 70% reduction at latter concentration (Figure 1B, p < 0.05). Although the two lower CTE concentrations (0.25 and 0.5 mg/mL) Mitochondrial superoxide generation significantly reduced HCT116 growth of cells after 24 hours The ability of CTE to induce superoxide generation by (p<0.05), they had no significant effect following 72 hours mitochondria was estimated using cell-permeable, mitochondria treatment compared to control (p>0.05). Time and concentration targeted MitoSOX Red probe (Life Technologies) and Hoechst dependent inhibition of proliferation with CTE was further 33342 for nuclear staining (Life Technologies). CaCo2, HCT116, confirmed in LoVo cells by more than 60% at highest SW620 and LoVo cells were seeded in 96-well plates at a density concentration (p < 0.05) (Figure 1C), and all four CTE 10 cells per well in a respective medium supplemented with 10% concentrations tested reduced the growth of SW620 cells after 48 FBS for 24 hours. Cells were then loaded with 4 µM of MitoSOX hours (Figure 1D, p < 0.05 for all). After 72 hours of treatment the and 2 µM of Hoechst 33342 for 20 min, excess dye washed and same effect was observed only for the two highest concentrations wells treated with 0, 0.5 and 1 mg/mL of CTE for 24 hours at 37°C (p < 0.05) while 0.25 and 0.5 mg/mL concentrations showed no and 5% CO . The fluorescence intensity was measured with an significant effect compared to control (p > 0.05). The impact of 0.5 excitation wavelength of 510 nm and emission detection at 580 nm and 1 mg/mL CTE treatment on induction of apoptosis was further for MitoSOX and an excitation wavelength of 350 nm and tested in all four cell lines (Figure 2). Increased number of cells in emission detection at 461 nm for Hoechst 33342 using microplate early apoptosis was detected in HCT116 and LoVo following 24 reader with top fluorescence (Infinite 200 PRO, Tecan Trading hours treatment with 0.5 mg/mL (p<0.05, Figure 2B and 2C) and AG, Switzerland) in all cell lines at 1 mg/mL (p<0.05). Significant increase in necrotic or late apoptotic cell number was further observed in Statistical analysis CaCo2 and SW620 cell lines (p<0.05, Figure 2A and 2D). Ability Descriptive statistics were shown as the mean +/− SD. of CTE to induce intracellular ROS production is demonstrated in The significance of differences between groups was assessed using Figure 3. Three hours following CTE treatment there was a strong the Student t-test and Chi-square test. When more than two groups increase in intracellular ROS production in all cell lines (p<0.05). were compared, we used one sided ANOVA with appropriate post The intracellular ROS production increased progressively hoc testing. The SPSS 11.01 for Mircosoft Windows were used. throughout the 25 hours treatment in a time and concentration Differences with P less than 0.05 were considered statistically dependent manner. Furthermore, staining of cells with significant. mitochondria targeted probe revealed a strong impact of CTE on mitochondrial superoxide production in a concentration dependent RESULTS manner (Figure 4). Highest increase in superoxide production by mitochondria was observed in HCT116 (69%, Figure 4B) and The effect of CTE on proliferation of human colon LoVo cells (82%, Figure 4C) following 24 hours treatment with 1 cancer cell lines is shown in Figure 1. All CTE concentrations mg/mL of CTE. 042 Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 Fig. 1: Effect of Cistanche tubulosa on the viability of colon cancer cell lines. Cell viability measured by MTT assay of (A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells are presented as percentage of control untreated colon cancer cell line. Mean values (±SD) for 5-replicates of representative experiment is given: (*) significance p<0.05 in comparison to control untreated respective cells. Fig. 2: Cistanche tubulosa water extract induces apoptosis in human colon cancer cells. Annexin-V-FITC flow cytometry analyses of (A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells are presented as percentage of control untreated c olon cancer cell line. Mean values (±SD) for 3 -replicates of representative experiment is given: (*) significance p<0.05 in comparison to control untreated respective cells. Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 043 Fig. 3: Cistanche tubulosa water extract induces intracellular ROS production in a time- and dose-dependent. ROS production measured by DCFH-DA assay in (A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells is presented as mean RFU values (±SD) for the respective 5 -replicates of representative experiment. (*) Significance p<0.05 in comparison to control untreated colon cancer cells. Fig. 4: Cistanche tubulosa water extract induces mitochondrial superoxide production in human colon cancer cells. Fluorescence intensity of mitochondria targeted MitoSOX Red probe in A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells are presented as percentage of control untreated colon cancer cell line. Mean values (±SD) for 5-replicates of representative experiment is given: (*) significance p<0.05 in comparison to control untreated colon cancer cells. 044 Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 DISCUSSION is at least in part mediated through mitochondria induced ROS mechanism. Although earlier studies have reported numerous medicinal properties of Cistanche tubulosa, this is the first report CONCLUSIONS of its anti proliferative effect in malignant cells. Cistanche In conclusion, our data suggests that the water extract of tubulosa bioactive compounds extracted with water, a highly polar desert plant Cistanche tubulosa may represent a promising solvent, showed strong anticancer bioactivity. We have previously candidate for anticancer approach in combination with other compared the efficiency of Cistanche tubulosa solubilized in water conventional therapies for prevention and treatment of colon against other solvents such as methanol and ethyl acetate, but cancer. We also demonstrate that the toxicity of the plant extract water extracts exhibited the most promising anticancer activities against cancer cells is mediated by increased intracellular ROS (data not shown). production and, at least in part, by mitochondrial-dependent We have demonstrated the ability of CTE at 1 mg/mL apoptosis. Further studies are ongoing to isolate and characterize and 2 mg/mL to inhibit 60% of the growth of both primary and the individual biologically active constituents responsible for metastatic colon cancer cell lines, revealing a potential important anticancer activity. role of Cistanche tubulosa as a colon cancer treatment. Compared More research is needed to evaluate the potential use of to normal cells, cancer cells are generally characterized by a this extract as an effective chemopreventive agent and to disturbance in redox homeostasis and a common strategy of understand the mechanisms of action on colon cancer cells at the current anticancer therapies is to increase cellular oxidative stress molecular level. Further preclinical and clinical studies are also (Yang et al, 2013). Although physiologically low levels of ROS needed to confirm the observed beneficial health effects of have important role as signaling molecules, the excessive ROS Cistanche tubulosa for cancer prevention. production can contribute to cancer instability and malignancy (Liou and Storz, 2010). Paradoxically, this imbalance in cellular ACKNOWLEDGEMENTS redox homeostasis renders cancer cells more vulnerable to ROS- induced cell death (Jaganjac et al., 2008; Nogueira and Hay, Financial support and sponsorship: This study was supported by 2013). The anti-proliferative effect of CTE reported in this study the Anti Doping Lab Qatar. can be mediated by various extra- and intracellular mechanisms of Conflict of Interests: The authors’ declare no conflict of interest. known and unknown compounds within the extract, targeting multiple pathways that play essential roles in apoptosis. In order to REFERENCES differentiate between different modes of cell death, we investigated the potential mechanism responsible for the observed Balunas MJ, Kinghorn, AD. Drug discovery from medicinal CTE-induced cytotoxicity. plants. Life Sci. 2005;78:431-41. Brenner H, Kloor M, Pox CP. Colorectal cancer. 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Published: Jan 1, 2017

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