ANIMAL CELLS AND SYSTEMS 2019, VOL. 23, NO. 3, 176–183 https://doi.org/10.1080/19768354.2019.1595139 a b Dan Li and Yong Xu a b Department of Geriatrics, The Second Xiangya Hospital of Central South University, Changsha, People’s Republic of China; Blood Puriﬁcation Center, The Third Xiangya Hospital of Central South University, Changsha, People’s Republic of China ABSTRACT ARTICLE HISTORY Received 29 September 2018 The inhibitory eﬀect of buforin IIb on diﬀerent types of cancer, although not liver cancer, has been Revised 23 January 2019 demonstrated previously. The aim of the present study was to investigate the eﬀects of buforin IIb Accepted 29 January 2019 on the progression of liver cancer. The human liver cancer cell line HepG2 was treated with puriﬁed buforin IIb and the cell activity was determined by MTT, colony formation and transwell assays. The KEYWORDS protein expression levels of cyclin-dependent kinases (CDKs) and cyclins were analyzed by western Buforin IIb; liver cancer; blotting and immunoﬂuorescent cell staining. A tumor growth model was constructed using nude apoptosis; cell cycle mice, and buforin IIb treatment was administered. The levels of CDK2 and cyclin A in the tumor tissues were detected by western blotting. Buforin IIb treatment depressed cell viability and colony formation and induced apoptosis signiﬁcantly, and 1.0 µM concentration of buforin IIb was found to be the optimal dosage. The cell cycle was arrested at the G2/M phase following buforin IIb treatment. CDK2 and cyclin A were downregulated by treatment of the cells with 1.0 µM buforin IIb for 24 h. Treatment with buforin IIb also inhibited the migration of liver cancer cells in vitro. Furthermore, 50 nmol buforin IIb injection suppressed HepG2 cell subcutaneous tumor growth in the nude mouse model. Similar to the in vitro results, buforin IIb injection reduced the expression of CDK2 and cyclin A in the tumor tissue. these results demonstrate that buforin IIb inhibited liver cancer cell growth via the regulation of CDK2 and cyclin A expression. Introduction antimicrobial activity against a variety of microorgan- Metastatic liver tumors represent the most prevalent isms, including bacteria and fungi without hemolytic cancers in adults (Bosch et al. 2004). As a leading cause activity (Park et al. 1998). Due to its unique structure, of morbidity and mortality worldwide, liver cancer buforin II is able to rapidly cross the membranes of bac- aﬀects survival and the quality of life (Bosch et al. teria without lysing cells and kills the bacteria by the 2004). Currently, resection surgery, radiation therapy destruction of intracellular macromolecules (Park et al. and chemotherapy are the standard treatments used 2000). An analog of buforin II, known as buforin IIb has for patients with liver cancer (Baskar et al. 2012). been developed, which contains an α-helical sequence However, severe side eﬀects arise following radiation at the end of the C-terminus and has a stronger cytolytic therapy and chemotherapy (Lawrence et al. 1995; Wulf activity than buforin II in microorganisms (Jang et al. et al. 2006). Furthermore, these treatments have shown 2011; Jang et al. 2012). Buforin IIb exhibits antitumor limited curative eﬀects and poor prognosis in clinical activities by speciﬁcally targeting cancer cells via inter- practice (Bruix and Sherman 2011). The development action with cell surface gangliosides (Lee et al. 2008). In of novel agents that speciﬁcally target liver cancer is, a study conducted by Lee et al., the eﬀects of buforin therefore, urgently required to improve the treatment IIb on cancer cell lines, including leukemia, central of liver cancer. nervous system tumor, non-small cell lung cancer, mela- Several cationic antimicrobial peptides have been noma and renal cancer cell lines were demonstrated (12). reported to display anticancer activity (Hancock 2001). The authors also used a NCI-H460 lung cancer cell line Buforin II is an antimicrobial peptide derived from transplant to form tumor xenografts and demonstrated histone H2A with a helix-hinge-helix structure, which that buforin IIb treatment was able to suppress cancer was ﬁrst isolated from the stomach tissue of the Asian development in vivo (Lee et al. 2008). These ﬁndings indi- toad (Bufo bufo gargarizans) (Park et al. 1996). The pep- cate that buforin IIb is a potential novel therapeutic tide contains 21 amino acids and exhibits a strong agent for the treatment of cancers. CONTACT Yong Xu firstname.lastname@example.org Blood Puriﬁcation Center, The Third Xiangya Hospital of Central South University, Tongzipo Road No. 138, Changsha, Hunan 410013, People’s Republic of China © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MOLECULAR & CELLULAR BIOLOGY ANIMAL CELLS AND SYSTEMS 177 Although antitumor eﬀects have been illustrated in (DMSO) were used as control. After treatment, the cells leukemia, central nervous system tumors, non-small were stained using 0.5 mg/ml MTT solution (Sigma- cell lung cancer, melanoma and renal cancer, the poten- Aldrich, St. Louis, MO, USA) for 4 h. The medium was tial curative eﬀect of buforin IIb on liver cancer has not then replaced with 500 ml DMSO and the optical yet been unveiled. Therefore, the present study investi- density (OD) was measured with a microplate reader gated the anticancer activity of buforin IIb in liver (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The cell cancer and analyzed the mechanism of cancer cell- viability was calculated using the formula: % viability = killing in vitro and in vivo. The purpose of this study (OD treated sample/OD untreated sample) × 100 using was to explore the curative value of buforin IIb in the the OD570 value. treatment of liver cancer. Colony formation assay Materials and methods For each well, 2 × 10 cells/well were seeded into 6-well Peptides plates and incubated for 7 days (n = 3 for each group). After washing three times using phosphate-buﬀered Buforin IIb [amino acid sequence, RAGLQFPVG(RLLR)3] saline (PBS), the cells were ﬁxed in methanol and was supplied in crude form by GL Biochem Ltd. (Shang- stained with crystal violet for 10 min. The colonies hai, China). The crude peptide was then puriﬁed to near were detected using an IX71 inverted microscope homogeneity by reversed phase high-performance (Olympus Corporation, Tokyo, Japan). liquid chromatography using a 0.46 × 25-cm Vydac 214TP54 C4 column equilibrated with acetonitrile, water and triﬂuoroacetic acid (35.0:64.9:0.1, v/v/v) at a Cell cycle analysis ﬂow rate of 6 ml/min. The acetonitrile concentration Cells were treated with buforin IIb for diﬀerent times or was increased to 65% (v/v) after 60 min using a linear diﬀerent dosages (n = 3 for each group) as described in gradient. The purity of the peptides was N98%, as deter- the MTT assay. DMSO was used as the control. The mined by electrospray mass spectrometry. The peptide cells were then stained using 0.05 mg/ml Propidium was highly soluble in physiological buﬀers. Iodide (PI) (Jiamay Biotech, Beijing, China). Brieﬂy, the cells were ﬁrst washed with 10 mM PBS and centrifuged Cell lines and animal models at room temperature for 5 min. The sediment was resus- pended in binding buﬀer and then incubated with PI for The human liver cancer cell line HepG2 was purchased 30 min. Finally, the cells were analyzed using a Cytomics from the Shanghai Cell Bank at the Chinese Academy FC 500 MPL cytometer (Beckman Coulter, Inc., Miami, FL, of Sciences (Shanghai, China). The culture conditions USA). for the cells were a temperature of 37°C in a 5% CO2:95% air-humidiﬁed atmosphere in Dulbecco’s modiﬁed Eagle’s medium supplemented with 10% fetal Western blotting bovine serum and 2 mM L-glutamine. The cell passage The protein expression of cyclin-dependent kinases was performed using 0.5% trypsin-ethylenediamine (CDKs) and cyclins was analyzed using western blot tetraacetic acid. Five-week-old female BALB/c nude assays. Primary rabbit polyclonal antibodies against mice were purchased from the Chinese Academy of CDKs and cyclins were purchased from Sigma-Aldrich. Medical Sciences. Experiments were approved by the The secondary antibody was horseradish peroxidase ethics committee of the Second Xiangya Hospital of (HRP)-conjugated anti-rabbit IgG (Sigma-Aldrich). After Central South University (Changsha, China). homogenization using radioimmunoprecipitation assay buﬀer (Beyotime Institute of Biotechnology, Wuhan, 3-(4,5-Dimethylthiazol-2-yl)-2,5- China), the proteins from tissues or cells were extracted diphenyltetrazolium bromide (MTT) assay and quantiﬁed using a BCA protein assay kit (Beyotime The cells were ﬁrst seeded into 96-well plates at a density Institute of Biotechnology). For each sample, 10 µg of 1 × 103 cells/well. After 24 h culture, the cells were total proteins were electrophoresed on 15% sodium treated using buforin IIb for diﬀerent times (0, 4, 8, 12, dodecyl sulfate-polyacrylamide gel and transferred to a 24 and 48 h treatment at 1.0 µM concentration; n =3 poly-vinylidene diﬂuoride membrane (EMD Millipore, for each group) or diﬀerent dosages (24 h treatment at Billerica, MA, USA). The membrane was then blocked 0.1, 0.5, 1.0, 5.0 and 10.0 µM concentration; n = 3 for using 4% skimmed milk for 1 h at room temperature each group). Cells incubated with dimethylsulfoxide and incubated with primary antibodies against CDK1 178 D. LI AND Y. XU (1:1000), CDK2 (1:1000), CDK4 (1:800), cyclin (1:500), Terminal deoxynucleotidyl-transferase-mediated cyclin B (1:1000) and glyceraldehyde 3-phosphate dehy- dUTP nick end labeling (TUNEL) staining assay drogenase (GAPDH; 1:2000) at 4°C overnight. After To detect cell apoptosis in the tumor tissues, tissue sec- washing with Tris-buﬀered saline and 84 Tween 20 tions were prepared as described above. TUNEL staining (TBST) buﬀer (pH 7.6, 20 mM Tris-HCl, 137 mM NaCl, was performed according to the manufacturer’s protocol 0.01% Tween-20), the membranes were incubated with (Roche Diagnostics, Indianapolis, IN, USA). The nuclei secondary antibody and visualized using enhanced che- were stained using DAPI. Signals of TUNEL-stained cells miluminescence reagents (ECL; EMD Millipore). were detected using a laser scanning confocal micro- scope (Olympus Fluoview FV1000). Immunoﬂuorescent cell staining HepG2 cells were seeded onto coverslips coated with Statistical analysis poly-L-lysine at a density of 5 × 10 per coverslip. After 24 h culture, the cells were washed with PBS and All experiments were repeated three times. Data are treated with 1.0 µM buforin IIb for 24 h at 37°C. The expressed as mean ± standard error of the mean (SEM). cells were then washed with PBS to clear the buforin Diﬀerences among groups were analyzed by one-way IIb, and ﬁxed with 4% paraformaldehyde. After incu- analysis of variance conducted using the SPSS version bation with primary antibodies against CDK2 (1:200) 17 software package (SPSS, Inc., Chicago, IL, USA). P < and cyclin A (1:100) at room temperature for 2 h, the 0.05 was considered to indicate a statistically signiﬁcant cells were co-stained using 4’,6-diamidino-2-phenylin- diﬀerence. dole (DAPI) to stain the cell nuclei. The signals were observed under a laser scanning confocal microscope (Olympus Fluoview FV1000; Olympus Corporation). Results Buforin IIb depresses the tumorigenicity of liver In vitro migration assay cancer cells Cell migration was detected using Transwell analysis (24- The peptide buforin IIb has been demonstrated to have well insert; pore size, 8 mm; BD Biosciences, Franklin antitumor eﬀects on several types of cancer, although Lakes, NJ, USA; n = 3 for each group). The cells were not on liver cancer. In order to investigate the potential ﬁrst treated with 1.0 µM buforin IIb for 24 h and then of buforin IIb as a drug for liver cancer, buforin IIb was plated (5 × 10 ) into the upper chambers in serum-free obtained and puriﬁed. A structural representation of medium. The lower chambers were ﬁlled with serum- this peptide is shown in Figure 1(A). The anticancer containing medium. After 24 h culture, the cells were activity of buforin IIb was evaluated in HepG2 liver stained using crystal violet and observed using an IX71 cancer cells. Following 4, 8, 12, 24 and 48 h treatment inverted microscope. with 1.0 µM buforin IIb, cell viability was decreased sig- niﬁcantly and in a time-dependent manner (P < 0.05; Figure 1(B)). The 24-h treatment with buforin IIb at 0.1, Tumor growth in a nude mouse model 0.5, 1.0, 5.0 and 10.0 µM concentrations also signiﬁcantly A HepG2 cell subcutaneous tumor model was estab- decreased the cell viability (Figure 1(C)). However, no lished in mice as described below and used to investi- dosage-dependent eﬀect was observed. The 1.0 µM con- gate the eﬀect of buforin IIb on tumor growth (n =3 centration of buforin IIb exhibited the optimal depressive for each group). The tumor formation model was gener- eﬀects on cell viability. Similarly, treatment with buforin ated by the injection of HepG2 cells (2 × 10 cells in IIb after 4, 8, 12, 24 and 48 h suppressed colony for- 0.1 ml PBS) subcutaneously into the right shoulder of mation in liver cancer cells and 24 h of treatment with the mouse. After the tumors reached 50 mm in buforin IIb exhibited the greatest inhibitory eﬀect on volume, buforin IIb (50 nmol in 100 ml PBS) or injection colony formation (Figure 1(D)). Buforin IIb at 0.1, 0.5, buﬀer (DMSO in 100 ml PBS) was administered by injec- 1.0, 5.0 and 10.0 µM also inhibited colony formation. tion into the tail vein every 2 days. Tumor volumes were Therefore, 1.0 µM concentration of buforin IIb had the calculated every 3 days using the following formula: greatest inhibitory eﬀect on colony formation (Figure 1 Volume = length × width2 × 0.52. After weeks, mice (E)). Next, Flow cytometry was used to detect the apop- were sacriﬁced and tumor tissues were assayed by tosis and cell cycle distribution following buforin IIb western blot analysis and immunoﬂuorescent cell treatment. Considering 1.0 µM was the optimal dosage staining. of buforin IIb, the cells were assayed after treatment ANIMAL CELLS AND SYSTEMS 179 Figure 1. In vitro antitumor eﬀect of buforin IIb on liver cancer cells. (A) Structural model of buforin IIb predicted and generated by the I-TASSER server. Eﬀect of buforin IIb on cell viability at various (B) times and (C) concentrations (n =3). *P < 0.05 vs. the control. Eﬀect of buforin IIb on colony formation at varying (D) times and (E) concentrations. (F) Phases of the cell cycle following treatment with buforin IIb (n =3). *P < 0.05 vs. the control. with 1.0 µM buforin IIb for 24 h. Cell cycle analysis indi- (Figure 2(B)). Therefore, buforin IIb suppress cell cycle cated that the cells were signiﬁcantly arrested at the by regulation CDK2 and cyclin A expression in the liver G2/M phase following buforin IIb treatment compared cancer. with the control (Figure 1(F)). These results suggested that buforin IIb inhibits cell proliferation and promote Buforin IIb inhibits liver tumor cell migration in apoptosis in liver cancer. vitro The generation of metastatic tumors is dependent upon Buforin IIb regulates CDK2 and cyclin A the migration of tumor cells. The eﬀect of buforin IIb on expression. cell migration was thus investigated. HepG2 cells were treated with 1.0 µM buforin IIb for 24 h and then the Previous study showed Buforin IIb has an inhibitory ability of the cells to migrate was detected using a Trans- eﬀect on cell cycle. The cell cycle is controlled by CDKs well assay system. The results indicated that at a concen- and cyclins, including CDK1, CDK2, cyclin A, cyclin B tration of 1.0 µM, buforin IIb inhibited cell migration and cyclin D. Thus, to understand the molecular mechan- signiﬁcantly compared with that of the control group ism by which buforin IIb arrests cells at the G2/M phase, (Figure 3(A)). Cell migration was decreased by 47.62% the expression levels of these proteins were analyzed fol- in the buforin IIb group compared with that in the lowing buforin IIb treatment. A 1.0 µM concentration of control group (Figure 3(B)). Our results suggest an inhibi- buforin IIb was used to treat the HepG2 cells for 24 h. tory role of buforin IIb in cell migration. The results of western blot analysis showed that, with the exception of CDK2 and cyclin A, these proteins underwent no signiﬁcant changes after buforin IIb treat- Buforin IIb inhibits tumor growth in vivo ment. However, CDK2 and cyclin A levels decreased notably following treatment with 1.0 µM buforin IIb for Following determination of the eﬀect of Buforin IIb on 24 h (Figure 2(A)). Similarly, the immunoﬂuorescent cell liver tumor cells in vitro, whether this peptide could staining showed that after treatment, the protein levels inhibit tumor growth in vivo was next determined. In of CDK2 and cyclin A were suppressed by buforin IIb order to investigate the eﬀect of Buforin IIb in tumor 180 D. LI AND Y. XU Figure 2. Buforin IIb reduced the expression of CDK2 and cyclin A in liver cancer cells. (A) Western blot analysis showing the expression of CDK1, CDK2, CDK4, cyclin A and cyclin B after buforin IIb treatment. (B) Immunoﬂuorescent cell staining conﬁrmed the inhibitory eﬀect of buforin IIb on CDK2 and cyclin A. Figure 3 Buforin IIb inhibited the migration of liver cancer cells in vitro. HepG2 cells were treated with 1.0 μM buforin IIb for 24 h. (A) Representative images of migrated liver cancer cells. Scale bar = 50 μM. (B) Quantiﬁcation of the percentage of the migrated cells. n = 3. *P < 0.05 vs. the control. growth of HepG2 xenografts, 6-week-old male mice were in 100 ml PBS) as control were injected by tail vein injec- inoculated with HepG2. After the tumor volume reached tion every 2 days. The tumor volume and tumor weight by 50 mm , 50 nmol buforin IIb or injection buﬀer (DMSO were signiﬁcantly inhibited in the buforin IIb peptide- ANIMAL CELLS AND SYSTEMS 181 Figure 4. Tumor formation and TUNEL assay of the tumor tissues. Changes in (A) tumor volume and (B) tumor weight after treatment with 50 nmol buforin IIb, (n = 3). *P < 0.05 vs. the control. (C) TUNEL assay results showed that 50 nmol buforin IIb promoted apoptosis in tumor tissues. TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling. expression of CDK2 and cyclin A in xenograft tumor tissues (Figure 5). Discussion The present study ﬁrst determined the primary ﬁnding that buforin IIb suppressed the progression of liver cancer. Treatment with buforin IIb inhibited the expression of CDK2 and cyclin A in HepG2 cells, which contributed to disruption of the cell cycle and arrested Figure 5. Buforin IIb repressed the expression of CDK2 and the cell cycle at the G2/M phase. Furthermore, buforin cyclin A in tumor tissues in vivo. IIb also suppressed cell migration in vitro. The results of the in vivo experiments showed that buforin IIb inhibited tumor growth, and similar to the in vitro ﬁndings, the treated mice compared with control from days 6 to 21 of expression levels of CDK2 and cyclin A in the tumor treatment (P < 0.05; Figure 4(A,B)), which is consistent tissue were inhibited by buforin IIb treatment. with the in vitro ﬁndings. Furthermore, the TUNEL Previous studies have identiﬁed several peptides that assay results showed that buforin IIb treatment are able to signiﬁcantly depress tumor progression and induced cell apoptosis in the tumor tissue (Figure 4(C)). could 17 be potential antitumor therapies(Cao and Lin 2006; Hoskin and Ramamoorthy 2008). However, until now, all the investigated peptides have shown either Evaluation of CDK2 and cyclin A expression limited eﬀects on tumors or non-speciﬁcity for tumor following buforin IIb treatment in vivo cells, which leads to injury of normal cells. The emer- The eﬀects of buforin IIb on CDK2 and cyclin A in xeno- gence of buforin II, however, provides a promising thera- graft tumor tissues were analyzed by western blotting peutic strategy. In addition to exhibiting strong and immunohistochemistry. The western blotting antitumor eﬀects, buforin II has also been revealed to results were consistent with the results of the in vitro be highly speciﬁc against tumor cells in comparison experiments; buforin IIb treatment repressed the with normal cells (Park et al. 1998; Park et al. 2000). On 182 D. LI AND Y. XU the basis of buforin II, Lee et al. developed a new buforin non-small cell lung cancer, melanoma and renal cancer II analog, buforin IIb, which had a stronger cytolytic (Lee et al. 2008), the inhibition of liver cancer by activity against cancer cells than does buforin II (Lee buforin IIb may have been expected. However, the et al. 2008). The authors demonstrated cytolytic activity present study is the ﬁrst to demonstrate that buforin for buforin IIb against several types of tumor cells but IIb inhibits cell proliferation by regulating the cell cycle not liver cancer. In the present study, it was demon- via mediating the expression of CDK2 and cyclin A. This strated that buforin IIb is able to depress tumor prolifer- information extends the scope of the understanding of ation and cell migration. buforin IIb’seﬀects on cancer progression. According to previous studies, buforin IIb is able to In conclusion, the present study demonstrates that exert eﬀects on several types of cancer cells, with remark- buforin IIb suppresses the progression of liver cancer able selectivity for cancer cells, and it has been indicated by inducing cell apoptosis, and inhibiting cell viability, that the anticancer action of buforin IIb involves the colony formation and cell migration. Furthermore, it induction of cancer cell apoptosis (Jang et al. 2011; found that buforin IIb treatment depresses CDK2 and Jang et al. 2012; Lee et al. 2008; Wang et al. 2011). cyclin A expression. In vivo experiments using a nude However, the mechanism of the apoptosis induced by mouse model suggested that buforin IIb inhibits tumor buforin IIb remains unclear. The present study demon- formation. These results indicate that buforin IIb could strated that buforin IIb is a potential anti-cancer drug be a potent therapeutic drug for liver cancer. that can accelerate the apoptosis of liver cancer cells as a result of arresting the cell cycle at the G2/M phase. Disclosure statement The molecular mechanism of aberrant cell cycles may be associated with the abnormal expression of cell- No potential conﬂict of interest was reported by the authors. cycle genes, such as CDKs and cyclins. The ﬁndings of the current study are consistent with References this; the expression levels of CDK2 and cyclin A were sig- niﬁcantly decreased following buforin IIb treatment, Baskar R, Lee KA, Yeo R, Yeoh KW. 2012. Cancer and radiation therapy: current advances and future directions. Int J Med which may aﬀect the cell cycle and arrest the cell cycle Sci. 9:193–199. at the G2/M phase. CDK2 and cyclin A are key factors Bosch FX, Ribes J, Diaz M, Cleries R. 2004. 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Animal Cells and Systems
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Published: May 4, 2019
Keywords: Buforin IIb; liver cancer; apoptosis; cell cycle