食管癌Antitumor activity of Lobaplatin against esophageal squamous cell carcinoma through caspase-dependent apoptosis and increasing the Bax-Bcl2 ratio

Contents lists available at ScienceDirect Biomedicine Received in revised form 26 August 2017; Accepted 28 August 2017 Corresponding author at: Department of Oncology, Zhejiang Hospital, No.12 Lingyin Road, Hangzhou, Zhejiang 310013, PR China. 1Contributed equally to this work. E-mail address: zjhown (N. Wei). Biomedicine & Pharmacotherapy 95 (2017) 447452 0753-3322/ 2017 Elsevier Masson SAS. All rights reserved. MARK cisplatin/5-fl uorouracil-based concurrent chemoradiotherapy of ad- vanced inoperable oesophageal cancer 16. However, its role and molecular mechanism in ESCC has seldom been studied and not clearly understood. Therefore, the aim of present study was to investigate the anti- tumor activity of Lobaplatin against ESCC in vitro and in vivo, and clarify the underlying molecular mechanism, which could provide evidence for clinical application. 2. Materials and methods 2.1. Compounds and reagents Lobaplatin was obtained from Hainan Changan International Pharmaceutical Co., Ltd. (Hainan, China). RPMI 1640 and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). MTT was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies against cleaved caspase-3, cleaved caspase-8, cleaved cas- pase-9, Bax, Bcl-2 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondly antibodies were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). Annexin V-FITC/ PI apoptosis detection kits was purchased from Becton Dickinson &Co. (Franklin Lakes, NJ, USA). 2.2. Cell lines and culture Human ESCC cell lines KYSE-410 and EC-109 were obtained from Cellcook Cell Biotechnology, Ltd. (Guangzhou, China) and Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 g/ml streptomycin and 100IU/ml penicillin in a humidifi ed incubator con- taining 5% CO2at 37 C. 2.3. Cell viability assay MTT assay was performed to assess the anti-tumor activity of Lobaplatin in esophageal squamous cancer cells. KYSE-410 and EC-109 cells were seeded in 96-well plates and incubated overnight, then treated with diff erent concentrations of Lobaplatin (0.25, 0.5, 1, 2, 4, 8, 16 and 32 g/ml) for 24 h, 48 h and 72 h. Solution absorbance (wa- velength of 570 nm) was detected by Microplate reader (Thermo Fisher Scientifi c Inc., Waltham, MA, USA). Then the cell viability curves were generated. 2.4. Clonogenic assay KYSE-410 and EC-109 cells were seeded in 6-well plates and in- cubated overnight. The cells were exposed to Lobaplatin (0.25, 1, 4 and 16 g/ml) for 48 h. Then the Lobaplatin medium were replaced by fresh RPMI 1640 medium and the cells were cultured for 2 weeks. After being fi xed and stained, colonies (more than 50 cells) were scored by optical microscope (Olympus Co., Tokyo, Japan). 2.5. Annexin V-FITC/PI apoptosis assay KYSE-410 and EC-109 cells were seeded in 6-well plates and in- cubated overnight, then exposed to Lobaplatin (0.25, 1, 4 and 16 g/ ml) for 48 h. Cells were harvested, washed with PBS, and resuspended in binding buff er, then stained with 5 l Annexin V-FITC and PI in the dark for 15 min. FACSCalibur fl ow cytometer was used to detect apoptosis and the data was analyzed by CellQuest software (Becton- Dickinson, San Jose, CA, USA). 2.6. Western blot analysis Cells were exposed to diff erent concentration of Lobaplatin for 48 h. Then the cells were harvested, washed with PBS and lysed in lysis buff er. Total protein was extracted and loaded onto 10% sodium do- decyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were subsequently electrotransferred to polyvinylidene di- fl uoride (PVDF) membranes and blocked with 5% nonfat milk in TBST. After blocking, the membranes were incubated with primary antibodies (cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, Bcl-2) (1:1000) at 4 C overnight, then incubated with HRP-conjugated sec- ondary antibodies (1:10000) at 37 C for 1 h. Reactive bands were identifi ed using an enhanced chemiluminescence reaction system (Amersham, Arlington Heights, IL, USA). 2.7. Animal study Male BALB/c nude mice aged 46 weeks were obtained from Zhejiang Academy of Medical Sciences (Hangzhou, China). EC-109 cells (1 106) were suspended in PBS (0.2 ml) and injected subcutaneously into the fl ank of each nude mice. When the average tumor volume reached 200 mm3, the mice were randomly assigned to control and treatment groups. Mice were intraperitoneally injected with 0.9% saline in control group (d1, d8, d15, n = 5) and Lobaplatin (d1, d8, d15, 5 mg/kg and d1, d8, d15, 10 mg/kg, n = 10) in treatment group. Tumor volume (mm3) and mice body weight were routinely measured every 5 days and calculated by the formula (width)2 length/2. Mice were sacrifi ced after 21 days, and the tumors were excised and stored at 80 C, or embedded in paraffi n for further use. The present experi- ment was performed according to the NIH guidelines and the protocol was approved by the Animal Ethics Committee of Zhejiang Hospital. 2.8. Immunohistochemistry Tumor tissue sections were deparaffi nized using diff erent con- centration of graded ethanol series and xylene series. Then epitope retrieval was performed by boiling at 100 C in citrate buff er. The sections were incubated with 3% H2O2to reduce the endogenous per- oxidase activity, then blocked with 5% skim milk for 1 h and incubated with primary antibodies (Bax, Bcl-2) at 4 C overnight. Horseradish peroxidase secondary antibody was added and incubated for 1 h, then incubated with the DAB substrate for 10 min. Images were captured under a light microscopy (Olympus Co., Tokyo, Japan) and the average density of each image was analyzed. 2.9. Statistical analysis Data were obtained from triplicate independent experiments and expressed as mean standard deviation. Students t-test or one way analysis of variance (ANOVA) and SNK-q test were performed by using SPSS 17.0 (SPSS Inc., Chicago,IL, USA) and p 0.05 was considered statistically signifi cant. 3. Results 3.1. Lobaplatin inhibits the proliferation of ESCC cell lines The anti-proliferative eff ect of Lobaplatin in the ESCC cell lines KYSE-410 and EC-109 was evaluated by MTT assay and clonogenic assay. To explore the pharmacological potential of Lobaplatin in the ESCC cell viability, the cells were fi rst exposed to diff erent concentra- tions of Lobaplatin (0.25, 0.5, 1, 2, 4, 8, 16 and 32 g/ml) for 24 h, 48 h and 72 h, and then assessed using MTT assay. The results showed that Lobaplatin inhibited the growth of KYSE-410 and EC-109 cells in a dose- and time-dependent manner (Fig. 1A). Clonogenic assay was performed to further determine whether Lobaplatin could inhibit the proliferation of ESCC cell lines. It was found that Lobaplatin inhibited the clone formation activity of KYSE-410 and EC-109 cells in a dose- dependent manner (Fig. 1B). These results indicated that Lobaplatin L. Du et al.Biomedicine & Pharmacotherapy 95 (2017) 447452 448 showed promising anti-proliferative activity in the ESCC cell lines. 3.2. Lobaplatin induces ESCC cell apoptosis Annexin V-FITC/PI apoptosis assay was performed to assess the apoptosis induced by Lobaplatin in KYSE-410 and EC-109 cells. The cells were exposed to Lobaplatin (0.25, 1, 4 and 16 g/ml) for 48 h, and the percentage of cell apoptosis was evaluated by fl ow cytometry. The results showed that the percentage of apoptotic cells signifi cantly in- creased after treatment of Lobaplatin in a dose-dependent manner (p 0.01) (Fig. 2). Compared to the control group, the Lobaplatin treatment showed signifi cant apoptosis and these results indicated that Lobaplatin could eff ectively induce apoptosis in the ESCC cell lines. 3.3. Lobaplatin modulates expression of key apoptosis-related proteins Key proteins of apoptosis including caspase-3, caspase-8, caspase-9, Bax and Bcl-2 were further investigated in KYSE-410 and EC-109 cells by Western blot analysis. The results showed that increasing expres- sions of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax, while decreasing expression of Bcl-2 were found in the Lobaplatin group compared with the control group (Fig. 3A). The ratio of Bax to Bcl-2 was signifi cantly enhanced (p 0.01) (Fig. 3B). 3.4. Lobaplatin suppresses tumor growth of ESCC xenograft In vivo study was further performed to evaluate the anti-tumor ef- fects of Lobaplatin in ESCC xenograft. EC-109 cells were used to es- tablish xenograft model. The results showed that tumor volumes were suppressed signifi cantly in the Lobaplatin group compared with control group (p 0.05). However, mice treated by high dose Lobaplatin showed a signifi cant body weight loss compared with the mice in control group (p 0.05) (Fig. 4). Tumor samples were analyzed to verify the expression of Bax and Bcl-2 by immunohistochemical analysis. The expression of Bax Fig. 1. Lobaplatin inhibits the proliferation of ESCC cell lines. (A) Diff erent concentration of Lobaplatin inhibits the cell viability of KYSE-410 and EC-109 cells at 24 h, 48 h and 72 h by MTT assay. (B) Diff erent concentration of Lobaplatin inhibits the clonogenic ability of KYSE-410 and EC-109 cells (*p 0.01 versus control group). Fig. 2. Lobaplatin induces ESCC cell apoptosis. Diff erent concentration of Lobaplatin increases the apoptotic rates of KYSE-410 and EC-109 cells by AnnexinV-FITC/PIapoptosisassay(*p 0.01 versus control group). L. Du et al.Biomedicine & Pharmacotherapy 95 (2017) 447452 449 increased and Bcl-2 expression decreased in the Lobaplatin group (5 mg/kg) compared with the control group (Fig. 5). 4. Discussion Platinum drugs are widely used to treat numerous malignancies, however, their clinical application is limited by severe toxicity and intrinsic or acquired resistance 17. Cisplatin is the fi rst-generation platinum drug against a wide spectrum of solid tumors. However, cis- platin medication is accompanied by dose-limiting nephrotoxicity, gastrointestinal toxicity, ototoxicity and neurological toxicity. Re- sistance to cisplatin also represents a major problem for clinical use. Carboplatin is the second-generation platinum compound with reduced neurotoxicity and ototoxicity, but it has limited effi cacy due to severe myelosuppression, especially thrombocytopenia. The third-generation drug, Lobaplatin, just received only regional approval 18, however, it has shown great anti-tumor activity for diff erent malignancies with its less side eff ects and incomplete cross-resistance to cisplatin and car- boplatin. Several preclinical studies have reported the anti-tumor activity of Lobaplatin and investigated the underlying mechanism in most of ma- lignancies. Lobaplatin might arrest cell cycle progression, induce apoptosis, alter the proteome, impair migration and invasion, reduce Cyclin D1, CDK4, CDK6, MMP-2, MMP-9 and Bcl-2 expression, upre- gulate the expressions of p53, Bax, PARP, caspase-3, -8, and -9 710. Clinical Studies on Lobaplatin based regimen in treating esophageal cancer patients have showed a signifi cant anti-tumor eff ect to ESCC with manageable toxicity 1416. But there is no preclinical study available focus on the molecular mechanism of Lobaplatin in ESCC. In the present study, it has been found that Lobaplatin is a potent anti-tumor agent in ESCC cell lines. Cell viability and clonogenic assays were performed to confi rm the cytotoxic activity of Lobaplatin against ESCC cells. The results demonstrated that Lobaplatin inhibited the growth of KYSE-410 and EC-109 cells in a dose- and time-dependent manner, and the clone formation activity of KYSE-410 and EC-109 cells was inhibited signifi cantly. Previous studies have shown that tumor- igenesis is associated with malfunction of cell apoptosis 1921. However, apoptosis is one of the critical mechanisms of the anti-tumor agents. The activity of inducing apoptosis and the degree of apoptosis is evaluated for anti-tumor drugs. Lobaplatin has been reported to induce Fig. 3. Lobaplatinmodulates expression ofkey apoptosis-related proteins. (A) Western blot analysis shows upregulation of cleaved-caspase-3, cleaved- caspase-8,cleaved-caspase-9,Baxanddown- regulation of Bcl-2 by treatment of diff erent con- centration of Lobaplatin in KYSE-410 and EC-109 cells. (B) The ratio of Bax to Bcl-2 (*p 0.01 versus control group). Fig. 4. Lobaplatin suppresses tumor growth of EC- 109 xenograft. (A) Tumor volumes of EC-109 xeno- graft treated by diff erent concentration of Lobaplatin (*p 0.05 be- tween Lobaplatin groups). (B) Body weight of EC- 109 xenograft treated by diff erent concentration of Lobaplatin(*p 0.05 versus control group). L. Du et al.Biomedicine & Pharmacotherapy 95 (2017) 447452 450 signifi cant apoptosis in several cancer cell lines with diff erent con- centrations 712. In the present study, it has been also observed that the apoptosis rate signifi cantly increased after Lobaplatin treatment in a dose-dependent manner in the ESCC cell lines. Tumor cell apoptosis has been regulated by a series of apoptosis- related proteins. Among them, the most important one is caspase fa- mily. Intracellular cysteine protease caspases are activated at diff erent stages and pathways to induce apoptosis 22. In a typical apoptosis process, caspase-3, 8, 9 are crucial mediators in apoptosis. The initiator caspases, such as caspase-8 or 9, are in charge of activating the eff ector caspase, caspase-3 2325. Caspase-8 is activated through self-cleavage to activate its downstream eff ector caspase-3, while cas- pase-9 subsequently triggers apoptosis by caspase-3 activation 26,27. The caspase cascade can be regulated by either pro-apoptotic factors or anti-apoptotic factors 28. Bcl-2 family proteins play an important role in the regulation of the mitochondria-mediated pathway of apoptosis. Bax and Bcl-2 are the major factors that control the apoptosis 29,30. Bax can activate or inhibit Bcl-xl and Bad, while Bcl-22 can inhibit Bax. It is believed that the ratio of Bax/Bcl-2 activity level is a critical de- terminant of susceptibility to apoptosis, rather than the levels of in- dividual proteins 31,32. The present study found that the expressions of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax increased, while the expression of Bcl-2 decreased by the Lobaplatin treatment, and the ratio of Bax to Bcl-2 was signifi cantly enhanced in vitro an in vivo. The results indicated that Lobaplatin-induced cell death is under control of caspase-dependent apoptosis and Bax/Bcl-2. 5. Conclusions In summary, Lobaplatin signifi cantly inhibited the growth of ESCC cells and xenograft in a dose- and time-dependent manner by inducing apoptosis through the caspase-dependent pathway and modulating Bax/Bcl-2 ratio. Thus, Lobaplatin might be an eff ective drug against ESCC, and further investigation and clinical evaluation need to be carried out. Confl ict of interest The Authors have no confl icts of interest to declare. Acknowledgements This study was supported by a grant from the Natural Science Foundation of Zhejiang (grant no. LY15H280013) and Wenzhou Science and Technology Bureau Foundation (Grant No. Y20160121). References 1 R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA. Cancer J. Clin. 67 (1) (2017) 730.5. 2 M.J. Domper Arnal, . Ferrndez Arenas, . Lanas Arbeloa, Esophageal cancer: risk factors, screening and endoscopic treatment in Western and Eastern countries, World J. Gastroenterol. 21 (26) (2015) 79337943. 3 H. Kuwano, Y. Nishimura, T. Oyama, H. 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