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The antitumor effect of lobaplatin against Ishikawa endometrial cancer cells in vitro and in vivo
Biomedicine & Pharmacotherapy 114 (2019) 108762
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
The antitumor effect of lobaplatin against Ishikawa endometrial cancer cells in vitro and in vivo Jingchao He, Haijun Zhang
T
⁎
Department of Oncology, Zhongda Hospital, Medical School, Southeast University, 87 Dingjiaqiao, Nanjing 210009, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lobaplatin Endometrial carcinoma Apoptosis Cell cycle arrest Platinum compounds
The new effective chemotherapeutic drugs are required urgently for advanced and recurrent endometrial carcinoma (EC), which is one of the most common gynaecological tumors among women worldwide. Preclinical studies have shown that lobaplatin, one of the third-generation platinum compounds, has possessed powerful anti-cancer efficacy on a series of tumors. The purpose of this study is to investigate its effect and molecular mechanism on the growth of endometrial cancer cell line Ishikawa in vitro and in vivo. The results of cell counting kit-8 (CCK-8) shown that lobaplatin concentration-dependent inhibited cell proliferations in human endometrial carcinoma ishikawa cells. Flow cytometry (FCM) assay demonstrated that lobaplatin affected the survival of endometrial carcinoma cell by arresting cell cycle at S phase and G2/M phase and inducing apoptosis in dose-dependent manner. Moreover, Western blot analysis also showed that the apoptosis-inducing effects of lobaplatin was associated with the reduction of Bcl-2 expression while upregulation of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax. Meanwhile, lobaplatin significantly suppressed tumor growth of human Ishikawa xenograft models and terminal deoxynucleotidyl trans-ferase dUTP nick end labeling confirmed the significant occurrence of lobaplatin-treated tumor tissues of apoptosis. Therefore, lobaplatin could be an effective chemotherapeutic agent for human endometrial carcinoma and warrants further clinical investigation.
1. Introduction Endometrial carcinoma(EC)is the most common gynaecological tumor among women worldwide, with increasing incidence and mortality rates [1]. Based on the global cancer statistics, about 320,000 patients of EC were newly clinically diagnosed and approximately 70,000 deaths result from EC in 2012 worldwide [2]. Although most women diagnosed with EC are early stage disease, which is usually cured with surgery, others with advanced or recurrent disease have poor prognosis and lack successful treatment options [3]. According to the National Comprehensive Cancer Network (NCCN) guidelines, platinum-based chemotherapy is regard as first-line therapy and ocuppies an important place in the comprehensive treatment of EC. However, advanced EC patients have frequently suffered from severe side effects and acquired resistance induced by cisplatin and carboplatin-based treatment [4]. Moreover, antiangiogenic agents such as bevacizumab and molecularly targeted therapies including mTOR inhibitors have been studied in clinical trial, but these are not approved by FDA for the treatment of EC so far [5]. Therefore, finding novel and effective platinum drugs become more and more important, which could improve the overall survival of EC patients.
⁎
As the third-generation platinum compound, lobaplatin was approved in the People’s Republic of China for the treatment of metastatic breast cancer, chronic myelogenous leukemia and small cell lung cancer. Lots of researches and clinical studies have shown that lobaplatin possessed minor side effects and no cross-resistance with cisplatin and carboplatin [6–8]. Meanwhile, it has demonstrated that lobaplation had good antitumor activities in various cancers including human hepatocellular carcinoma, nasopharyngeal carcinoma, esophageal cancer, non-small cell lung cancer, colorectal carcinoma, melanoma, prostate cancer, cervical cancer and ovarian cancer, etc [9–15]. However, there is limited study evaluating the therapeutic effects of lobaplatin on EC and its underlying molecular mechanisms remains not understood. Therefore, the purpose of this study was to investigate the antitumor efficacy of lobaplatin on human endometrial carcinoma cells in vitro and in vivo, and explored the underlying mechanisms of action to offer evidence for its further clinical treatment of patients with EC.
Corresponding author. E-mail address: [email protected] (H. Zhang).
https://doi.org/10.1016/j.biopha.2019.108762 Received 9 January 2019; Received in revised form 6 March 2019; Accepted 6 March 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 114 (2019) 108762
J. He and H. Zhang
2. Materials and methods
Pharmacia Biotech, Amersham, UK).
2.1. Compounds and reagents
2.7. Analysis of the xenograft
Lobaplatin was obtained from Hainan Chang’an International Pharmaceutical Co., Ltd. (Hainan, China). Cell Counting Kit-8 (CCK-8), Annexin V-FITC Apoptosis Detection Kit, propidium iodide (PI) was purchased from Nanjing KeyGen Biotech Co. (Nanjing, China). 0.25% trypsin-EDTA and Fetal Bovine Serum(FBS) was purchased from Gibco Chemical Co. (Carlsbad, CA, USA). Phosphate-buffered saline(PBS) was purchased from HyClone (Healthcare Life Sciences, USA). Antibodies against Bcl-2, Bax, caspase-3, caspase-8, caspase-9 was from Abcam, Inc. (USA).
Female BALB/c nude mice aged 4–5 weeks were obtained from Shanghai slike experimental animal co (Shanghai, People’s Republic of China). The present animal experiments were conducted according to the protocols approved by the animal ethics committee of the Medical School of Southeast University. Ishikawa cells (1 × 106/100 μL) were subcutaneously injected onto the right flanks of each mouse. After tumors reaching a volume of 80–100mm3, mice were randomly assigned into the control group (n = 6) and treatment group (n = 6) randomly. The mice in treatment group were injected with lobaplatin at 12 mg/kg dosage and in control group were treated with only saline via tail vein at the first and eighth days, respectively. The tumor diameter and the mice body weights were measured every other day for a total of 21 days. Tumor volumes was calculated as the formula:[1/2×(width) 2×length]. After mice being sacrificed by inhalation anesthesia, the tumors were removed and stored at −80 °C for further analysis. The relative tumor volume (RTV) = [Vn the volume on each measurement /Vi the volume on initial treatment].T/C (%) calculated as [RTV the average treated group/ RTV the average control group] ×100%, which was used to assess anti-tumor efficacy of a given compound.
2.2. Cell culture The human endometrial carcinoma cell line Ishikawa was purchased from Nanjing Key Gen Biotech Co. (Nanjing, China). Cells were propagated in RPMI 1640 containing 10% heat-inactivated FBS and 1% antibiotics (penicillin and streptomycin) in a humidified atmosphere with 5% CO2 at 37 °C. 2.3. Cell viability assay CCK-8 assay was carried out to measure the cytotoxicity of lobaplatin to Ishikawa. Cells were plated onto a 96-well culture plate and incubated 37 °C for overnight. After that, the cells were treated with various concentrations of lobaplatin (0, 0.125, 0.25, 0.5, 1, 2, 4, 8 and 16 μg /mL) for 48 h and followed by incubation with CCK-8 solution (10 μL) for additional 4 h. Absorbance at 450 nm was detected by scanning with ELX 800 Microplate Reader (BioTek Instruments, Inc, Winooski, VT, USA).
2.8. TUNEL analysis The tumors were excised, immobilized with Formalin and then embedded in paraffin. After Sections being cut, according to the manufacturer’s protocol, apoptosis of tumor sections was assessed by Transferase dUTP nick end labeling (TUNEL) system (Nanjing KeyGen Biotech Co. Nanjing, China). The images were gained by an Olympus IX73 optical microscope (400×).
2.4. Apoptosis analysis
2.9. Statistical analysis
Ishikawa cells were treated with different concentrations of lobaplatin (0, 1.5, 3 and 6 μg/mL) for 48 h. Then, cells were trypsinized with EDTA-free trypsinogen, washed with PBS, centrifuged and resuspended in 500μLbinding buffer. After staining with annexin V-FITC (5 μL) and PI (5 μL) solution for 15 min in the dark at room temperature, cells were analyzed by a FACSCalibur™ flow cytometer.
Statistical calculations were performed using GraphPad Prism 5.0 (GraphPad software; San Diego, CA, USA). The results were represented as mean ± standard deviation (SD). Statistical significance was determined using a Student’s t-test, and P-values < 0.05 were considered statistically significant. 3. Results
2.5. Cell cycle distribution analysis
3.1. Lobaplatin inhibited proliferation of Ishikawa cells
Following the treatment with different lobaplatin concentrations (0, 1.5, 3 and 6 μg /mL) for 48 h, cells were harvested and fixed with icecolded 70% ethanol at 4 °C overnight. After being washed by cold PBS, cells was stained in PI/RNase staining solution for 30 min and then analyzed by a flow cytometry.
The CCK-8 assay was used to evaluate anti-proliferative effect of lobaplatin on Ishikawa cells. After the treatment of lobaplatin for 48 h, the proliferation of Ishikawa cells was markedly attenuated and represented a dose-dependent manner (Fig. 1A). the inhibition rates were 34.73 ± 0.79% and 56.3 ± 0.63% respectively at 2ug and 4ug/mL dosage, which led to a significant inhibition of proliferation. Thus, working concentration (1.5, 3 and 6 μg/ mL) was safely chosen for subsequent experiments.
2.6. Western blot analysis Ishikawa cells were exposed with designed concentration of lobaplatin (0, 1.5, 3 and 6 μg/mL) for 48 h. After being harvested and washed, the cells were lysed in radioimmuno precipitation assay buffer and cells protein was extracted. The protein concentrations were tested using the bicinchoninic acid method, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% milk, the membranes were probed with the following primary antibody: caspase-3, caspase-8, and caspase-9,Bax, Bcl-2 and GAPDH overnight at 4 °C. After incubation with the corresponding secondary antibodies, the reactive bands were measured by an enhanced electrochemiluminescence detection system (Amersham
3.2. Lobaplatin induced S and G2/M phase arrest The effect of lobaplatin on Ishikawa cell cycle distribution was assessed using flow cytometry analysis. After treatment with lobaplatin for 48 h, Ishikawa cell cycle progression was disrupted (Fig. 1B). The treatment of lobaplatin notably increased the population of cells in S phase in a dose-dependent manner and in G2/M phase at low concentration. Meanwhile, the percentage of cells in G1 phase was decreased in a dose-dependent manner. The results suggested that lobaplatin could induce S and G2/M phase arrest in Ishikawa cells. 2
Biomedicine & Pharmacotherapy 114 (2019) 108762
J. He and H. Zhang
Fig. 1. The lobaplatin inhibited proliferation and arrested cell cycle progression in human endometrial carcinoma cell line. Ishikawa cells were exposed with various concentrations (0.125–16 μg /mL) of lobaplatin for 48 h and then analyzed by CCK assay(A). Ishikawa cells were exposed to different concentrations of lobaplatin (0, 1.5, 3, 6 μg /mL) for 48 h and then analyzed cell cycle by flow cytometry (B). The cell cycle distribution was treated statistically (* P < 0.05, *** P < 0.001). Data expressed are mean ± SD.
exposed with 6 μg/ml (38.94 ± 2.17%) was higher than that of exposed with 1.5 μg/ml lobaplatin (19.17 ± 1.85%), which indicated that lobaplatin could lead to the occurrence of apoptosis in a concentration-dependent manner.
3.3. Lobaplatin induced cell apoptosis After treated with different concentration of lobaplatin (0, 1.5, 3 and 6 μg/mL) for 48 h, the Ishikawa cells were stained with Annexin VFITC/PI solutions and then tested using flow cytometry. As shown in Fig. 2, the apoptotic rates of lobaplatin-treated cells were significant higher than the control group. The apoptotic rates of Ishikawa cells
Fig. 2. The effect of lobaplatin on cell apoptosis in Ishikawa cells. (A): Ishikawa cells were treated with different concentrations of lobaplatin (0, 1.5, 3, 6 μg /ml) for 48 h followed by Annexin V-FITC/PI apoptosis assay. (B): The statistical images of apoptosis rate was presented (*** P < 0.001). Data expressed are mean ± SD. 3
Biomedicine & Pharmacotherapy 114 (2019) 108762
J. He and H. Zhang
Fig. 3. The effects of lobaplatin on apoptosis-related proteins in Ishikawa cells. (A) Lobaplatin upregulated Bax and cleaved - caspase-3, -8 and -9 expression, and reduced Bcl-2 expression. GADPH was employed as a control. (B–C) The relative intensity of apoptosis-related proteins was treated statistically (**P < 0.01, *** P < 0.001). Data are expressed as mean ± SD.
and play an vital important role in the treatment of EC. However, because of their serious side effects, intrinsic or acquired resistance, cisplatin and carboplatin frequently affected patients' quality of life and resulted in treatment failure [16]. Meanwhile, there was no standard the second-line chemotherapy regimens of platinum-resistant patients with EC [4]. As one of the third-generation platinum antineoplastic drug, lobaplatin was developed by ASTA (Germany) and has been approved in clinical application of several cancers by china [17]. Many studies have proved that lobaplatin induced apoptosis, blocked cell cycle progression, and inhibited migration and invasion, of which the antitumor activity was involved in inducing the formation of DNA-drug adducts, the down-regulation expression of PI3K, phosphorylation-AKT, cyclin B, CDK1, CDK6, Bcl-2, MMP-2, MMP-9 and upregulating p53, p21, and p27,caspase-3, -8, and -9, Bax and PARP expression [15,18–21]. Moreover, because of unique advantages including incomplete cross-resistance to cisplatin and carboplatin, fewer side effects, promising radiosensitizer [14,22,23], lobaplatin has attracted more and more attention. So, the aim of this study is firstly to explore the anti-tumor efficacy of lobaplatin on human endometrial cancer cell line Ishikawa and the potential mechanisms, and to further provide evidence for the lobaplatin’s clinical application in EC therapy. To assess the cytotoxic effects of lobaplatin in Ishikawa cells, the inhibition rate of lobaplatin on human endometrial cancer cell was firstly analyzed. The results showed that lobaplatin exerted powerful anti-proliferative efficacy on Ishikawa cells, which provided powerful evidence that lobaplatin could be a new anti-endometrial carcinoma platinum antineoplastic agent. Cell cycle arrest is vital for cancer cell growth suppression, which is one of main anti-cancer mechanisms of antineoplastic drugs including platinum compounds cisplatin, carboplatin and oxaliplatin [24]. In the present study, we found the population of cells in S phase significantly increased in a concentration-dependent behavior and in G2/M phase obviously increased at low concentration, which indicated that cytotoxicity of lobaplatin on Ishikawa cells might partly be caused by
3.4. Lobaplatin regulated expression of main apoptosis-related proteins As apoptosis is served as the critical anti-tumor mechanism, western blot analysis was used to evaluate key proteins of apoptosis expression. After lobaplatin treatment, compared with the control group, western blotting results showed that the protein expression levels of cleavedcaspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax were markedly up-regulated, while the protein expression levels of Bcl2 was significantly down-regulated. Meanwhile, lobaplatin regulated the protein expression levels of the above proteins in a concentration-dependent behavior (Fig. 3). 3.5. Lobaplatin inhibited tumor growth in vivo As shown in Fig. 4A, groups injected with lobaplatin represented a significant body weight loss compared with the control group. However, the body weight of the treatment group and the control group showed an increasing trend. The growth of Ishikawa tumors were obviously suppressed by lobaplatin (Fig. 4B–E).The average tumor volume of treatment group (0.706 ± 0.071cm3) was significantly lower than that of the control group (1.625 ± 0.283cm3). Meanwhile, compared with the control group, the average tumor weight of the lobaplatintreated group was significantly lighter. When finishing the experiment, the T/C (%) value was 44.75%. TUNEL analysis was analyzed using the percentage of apoptotic cells compared to the mean number of cells per field. The results showed that, in the treatment group, the apoptotic rate was increased significantly when compared with the apoptotic rate of control group (Fig. 4F–G). 4. Discussion Up to date, advanced or recurrent EC lacks effective treatment options and has poor prognosis [5]. According to NCCN guidelines, platinum-based chemotherapy was considered as the first-line treatment 4
Biomedicine & Pharmacotherapy 114 (2019) 108762
J. He and H. Zhang
Fig. 4. Lobaplatin markedly inhibited tumor growth of Ishikawa xenograft. (A): Body weight of Ishikawa xenograft with treatment of Lobaplatin. (B): Tumor volume of Ishikawa xenograft treated with Lobaplatin. (C): Tumor weight of Ishikawa xenograft treated with Lobaplatin. (D-E): Photos of Ishikawa xenograft tumors. (F-G): representative photographs of tumor tissues stained with TUNEL. Magnification, ×400. The apoptosis rate of each tumor stained with TUNEL was treated statistically (*P < 0.05, **P < 0.01, *** P < 0.001). Data are expressed as mean ± SD.
activity and low side effects for the treatment of the recurrent and refractory ovarian carcinoma patients who have received cisplatin- or carboplatin- based chemotherapy [6]. Therefore, lobaplatin could be a potentially effective anti-cancer drug fo r endometrial cancer, warrant urgently further clinical investigation, and have a special significance in the treatment of patients developed secondary resistance to cisplatin or carboplatin.
inducing the arrest of S and G2/M cell cycle. As a programmed cell death, apoptosis is regarded as the critical anti-tumor mechanism and used to evaluate the performance of anticancer drugs [25–27]. Lobaplatin has been confirmed to induce obviously apoptosis by regulating the expression of many apoptosis-related proteins in a series of tumors [19,20,28]. In the current study, the results showed that the apoptotic rates of lobaplatin-treated cells were significantly increased in a dose-dependent manner by flow cytometry. Meanwhile, our study showed that tumor tissues of xenografts treated with lobaplatin possess significant cell apoptosis analyzed by TUNEL assay compared to the control group. Apoptosis pathways mainly includes the mitochondrial-mediated intrinsic pathway and the cell death receptor-mediated extrinsic pathway [29]. The proapoptotic protein Bax and anti-apoptotic Bcl-2 are the vital factors of controlling the apoptosis [30], which have been proved to participate in the regulation of intrinsic apoptosis [29]. The caspase family is a pivotal central link and executor during the apoptosis process. As an essential apoptotic executive caspase, caspase-3 is activated through caspase-8 and -9, which are in charge of the external and internal apoptotic pathways, respectively [31]. The present study found that after treatment with lobaplatin, the expressions level of cleaved-caspase-3, cleaved-caspase8, cleaved-caspase-9 and Bax were elevated, while the expression level of Bcl-2 was reduced. Our results indicated that lobaplatin induced apoptosis of human endometrial cancer cell line Ishikawa though involving in intrinsic and extrinsic apoptotic pathways. In our study, lobaplatin could significantly inhibit the tumor growth in the Ishikawa xenograft model. Clinical trials have proved that lobaplatin plus other chemotherapy drugs has encouraging antitumor
5. Conclusion Lobaplatin possessed a high inhibitory activity against Ishikawa cells in vitro and in vivo. Lobaplatin blocked the cell cycle in S and G2/ M phase and induced apoptosis, of which the possible mechanisms could be upregulating the expression level of Bax,cleaved-caspase-3,8,and-9 and downregulating the expression level of Bcl-2.Therefore, lobaplatin could be a potentially effective antineoplastic drug for the treatment of EC. Disclosure The authors declare no conflicts of interest in the article. Conflict of interest We guarantee that all authors do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. 5
Biomedicine & Pharmacotherapy 114 (2019) 108762
J. He and H. Zhang
Acknowledgements
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The work was financially supported by the National Nature Science Foundation of People's Republic of China (81371678) and Hainan Changan International Pharmaceutical Co., Ltd (China). References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2017, CA Cancer J. Clin. 67 (1) (2017) 7–30. [2] P. Morice, A. Leary, C. Creutzberg, N. Abu-Rustum, E. Darai, Endometrial cancer, Lancet 387 (10023) (2016) 1094–1108. [3] C.M. Bestvina, G.F. Fleming, Chemotherapy for endometrial cancer in adjuvant and advanced disease settings, Oncologist 21 (10) (2016) 1250–1259. [4] G.F. Fleming, Second-line therapy for endometrial cancer: the need for better options, J. Clin. Oncol. 33 (31) (2015) 3535–3540. [5] T. Van Nyen, C.P. Moiola, E. Colas, D. Annibali, F. Amant, Modeling endometrial cancer: past, present, and future, Int. J. Mol. Sci. 19 (8) (2018). [6] X. Li, Y. Li, Clinical study on chemotherapy of lobaplatin combined with docetaxel in patients with relapsed ovarian cancer, Zhong Nan Da Xue Xue Bao Yi Xue Ban 39 (11) (2014) 1131–1136. [7] J.J. Kavanagh, C.L. Edwards, R.S. Freedman, M.B. Finnegan, O. Balat, D. Tresukosol, K. Burk, S. Loechner, M. Hord, J.L. Franklin, et al., A trial of lobaplatin (D-19466) in platinum-resistant ovarian cancer, Gynecol. Oncol. 58 (1) (1995) 106–109. [8] A. Harstrick, C. Bokemeyer, M. Scharnofkse, G. Hapke, D. Reile, H.J. Schmoll, Preclinical activity of a new platinum analogue, lobaplatin, in cisplatin-sensitive and -resistant human testicular, ovarian, and gastric carcinoma cell lines, Cancer Chemother. Pharmacol. 33 (1) (1993) 43–47. [9] L. Du, Z. Fei, S. Song, N. Wei, Antitumor activity of Lobaplatin against esophageal squamous cell carcinoma through caspase-dependent apoptosis and increasing the Bax/Bcl-2 ratio, Biomed. Pharmacother. 95 (2017) 447–452. [10] Y. Li, B. Liu, F. Yang, Y. Yu, A. Zeng, T. Ye, W. Yin, Y. Xie, Z. Fu, C. Zhao, Lobaplatin induces BGC-823 human gastric carcinoma cell apoptosis via ROS- mitochondrial apoptotic pathway and impairs cell migration and invasion, Biomed. Pharmacother. 83 (2016) 1239–1246. [11] X. Wu, P. Tang, S. Li, S. Wang, Y. Liang, L. Zhong, L. Ren, T. Zhang, Y. Zhang, A randomized and open-label phase II trial reports the efficacy of neoadjuvant lobaplatin in breast cancer, Nat. Commun. 9 (1) (2018) 832. [12] H. Zhang, R. Chen, S. Yang, W. Liu, K. Li, H. Zhang, X. Zhu, B. Chen, Lobaplatin for the treatment of SK-MES-1 lung squamous cell line in vitro and in vivo, Oncol. Ther. 9 (2016) 4215–4224. [13] L.R. Ke, W.X. Xia, W.Z. Qiu, X.J. Huang, J. Yang, Y.H. Yu, H. Liang, G.Y. Liu, Y.F. Ye, Y.Q. Xiang, X. Guo, X. Lv, Safety and efficacy of lobaplatin combined with 5-fluorouracil as first-line induction chemotherapy followed by lobaplatin-radiotherapy in locally advanced nasopharyngeal carcinoma: preliminary results of a prospective phase II trial, BMC Cancer 17 (1) (2017) 134. [14] W.P. Li, H. Liu, L. Chen, Y.Q. Yao, E.F. Zhao, A clinical comparison of lobaplatin or cisplatin with mitomycine and vincristine in treating patients with cervical squamous carcinoma, Asian Pac. J. Cancer Prev. 16 (11) (2015) 4629–4631.
6
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
The antitumor effect of lobaplatin against Ishikawa endometrial cancer cells in vitro and in vivo Jingchao He, Haijun Zhang
T
⁎
Department of Oncology, Zhongda Hospital, Medical School, Southeast University, 87 Dingjiaqiao, Nanjing 210009, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lobaplatin Endometrial carcinoma Apoptosis Cell cycle arrest Platinum compounds
The new effective chemotherapeutic drugs are required urgently for advanced and recurrent endometrial carcinoma (EC), which is one of the most common gynaecological tumors among women worldwide. Preclinical studies have shown that lobaplatin, one of the third-generation platinum compounds, has possessed powerful anti-cancer efficacy on a series of tumors. The purpose of this study is to investigate its effect and molecular mechanism on the growth of endometrial cancer cell line Ishikawa in vitro and in vivo. The results of cell counting kit-8 (CCK-8) shown that lobaplatin concentration-dependent inhibited cell proliferations in human endometrial carcinoma ishikawa cells. Flow cytometry (FCM) assay demonstrated that lobaplatin affected the survival of endometrial carcinoma cell by arresting cell cycle at S phase and G2/M phase and inducing apoptosis in dose-dependent manner. Moreover, Western blot analysis also showed that the apoptosis-inducing effects of lobaplatin was associated with the reduction of Bcl-2 expression while upregulation of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax. Meanwhile, lobaplatin significantly suppressed tumor growth of human Ishikawa xenograft models and terminal deoxynucleotidyl trans-ferase dUTP nick end labeling confirmed the significant occurrence of lobaplatin-treated tumor tissues of apoptosis. Therefore, lobaplatin could be an effective chemotherapeutic agent for human endometrial carcinoma and warrants further clinical investigation.
1. Introduction Endometrial carcinoma(EC)is the most common gynaecological tumor among women worldwide, with increasing incidence and mortality rates [1]. Based on the global cancer statistics, about 320,000 patients of EC were newly clinically diagnosed and approximately 70,000 deaths result from EC in 2012 worldwide [2]. Although most women diagnosed with EC are early stage disease, which is usually cured with surgery, others with advanced or recurrent disease have poor prognosis and lack successful treatment options [3]. According to the National Comprehensive Cancer Network (NCCN) guidelines, platinum-based chemotherapy is regard as first-line therapy and ocuppies an important place in the comprehensive treatment of EC. However, advanced EC patients have frequently suffered from severe side effects and acquired resistance induced by cisplatin and carboplatin-based treatment [4]. Moreover, antiangiogenic agents such as bevacizumab and molecularly targeted therapies including mTOR inhibitors have been studied in clinical trial, but these are not approved by FDA for the treatment of EC so far [5]. Therefore, finding novel and effective platinum drugs become more and more important, which could improve the overall survival of EC patients.
⁎
As the third-generation platinum compound, lobaplatin was approved in the People’s Republic of China for the treatment of metastatic breast cancer, chronic myelogenous leukemia and small cell lung cancer. Lots of researches and clinical studies have shown that lobaplatin possessed minor side effects and no cross-resistance with cisplatin and carboplatin [6–8]. Meanwhile, it has demonstrated that lobaplation had good antitumor activities in various cancers including human hepatocellular carcinoma, nasopharyngeal carcinoma, esophageal cancer, non-small cell lung cancer, colorectal carcinoma, melanoma, prostate cancer, cervical cancer and ovarian cancer, etc [9–15]. However, there is limited study evaluating the therapeutic effects of lobaplatin on EC and its underlying molecular mechanisms remains not understood. Therefore, the purpose of this study was to investigate the antitumor efficacy of lobaplatin on human endometrial carcinoma cells in vitro and in vivo, and explored the underlying mechanisms of action to offer evidence for its further clinical treatment of patients with EC.
Corresponding author. E-mail address: [email protected] (H. Zhang).
https://doi.org/10.1016/j.biopha.2019.108762 Received 9 January 2019; Received in revised form 6 March 2019; Accepted 6 March 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 114 (2019) 108762
J. He and H. Zhang
2. Materials and methods
Pharmacia Biotech, Amersham, UK).
2.1. Compounds and reagents
2.7. Analysis of the xenograft
Lobaplatin was obtained from Hainan Chang’an International Pharmaceutical Co., Ltd. (Hainan, China). Cell Counting Kit-8 (CCK-8), Annexin V-FITC Apoptosis Detection Kit, propidium iodide (PI) was purchased from Nanjing KeyGen Biotech Co. (Nanjing, China). 0.25% trypsin-EDTA and Fetal Bovine Serum(FBS) was purchased from Gibco Chemical Co. (Carlsbad, CA, USA). Phosphate-buffered saline(PBS) was purchased from HyClone (Healthcare Life Sciences, USA). Antibodies against Bcl-2, Bax, caspase-3, caspase-8, caspase-9 was from Abcam, Inc. (USA).
Female BALB/c nude mice aged 4–5 weeks were obtained from Shanghai slike experimental animal co (Shanghai, People’s Republic of China). The present animal experiments were conducted according to the protocols approved by the animal ethics committee of the Medical School of Southeast University. Ishikawa cells (1 × 106/100 μL) were subcutaneously injected onto the right flanks of each mouse. After tumors reaching a volume of 80–100mm3, mice were randomly assigned into the control group (n = 6) and treatment group (n = 6) randomly. The mice in treatment group were injected with lobaplatin at 12 mg/kg dosage and in control group were treated with only saline via tail vein at the first and eighth days, respectively. The tumor diameter and the mice body weights were measured every other day for a total of 21 days. Tumor volumes was calculated as the formula:[1/2×(width) 2×length]. After mice being sacrificed by inhalation anesthesia, the tumors were removed and stored at −80 °C for further analysis. The relative tumor volume (RTV) = [Vn the volume on each measurement /Vi the volume on initial treatment].T/C (%) calculated as [RTV the average treated group/ RTV the average control group] ×100%, which was used to assess anti-tumor efficacy of a given compound.
2.2. Cell culture The human endometrial carcinoma cell line Ishikawa was purchased from Nanjing Key Gen Biotech Co. (Nanjing, China). Cells were propagated in RPMI 1640 containing 10% heat-inactivated FBS and 1% antibiotics (penicillin and streptomycin) in a humidified atmosphere with 5% CO2 at 37 °C. 2.3. Cell viability assay CCK-8 assay was carried out to measure the cytotoxicity of lobaplatin to Ishikawa. Cells were plated onto a 96-well culture plate and incubated 37 °C for overnight. After that, the cells were treated with various concentrations of lobaplatin (0, 0.125, 0.25, 0.5, 1, 2, 4, 8 and 16 μg /mL) for 48 h and followed by incubation with CCK-8 solution (10 μL) for additional 4 h. Absorbance at 450 nm was detected by scanning with ELX 800 Microplate Reader (BioTek Instruments, Inc, Winooski, VT, USA).
2.8. TUNEL analysis The tumors were excised, immobilized with Formalin and then embedded in paraffin. After Sections being cut, according to the manufacturer’s protocol, apoptosis of tumor sections was assessed by Transferase dUTP nick end labeling (TUNEL) system (Nanjing KeyGen Biotech Co. Nanjing, China). The images were gained by an Olympus IX73 optical microscope (400×).
2.4. Apoptosis analysis
2.9. Statistical analysis
Ishikawa cells were treated with different concentrations of lobaplatin (0, 1.5, 3 and 6 μg/mL) for 48 h. Then, cells were trypsinized with EDTA-free trypsinogen, washed with PBS, centrifuged and resuspended in 500μLbinding buffer. After staining with annexin V-FITC (5 μL) and PI (5 μL) solution for 15 min in the dark at room temperature, cells were analyzed by a FACSCalibur™ flow cytometer.
Statistical calculations were performed using GraphPad Prism 5.0 (GraphPad software; San Diego, CA, USA). The results were represented as mean ± standard deviation (SD). Statistical significance was determined using a Student’s t-test, and P-values < 0.05 were considered statistically significant. 3. Results
2.5. Cell cycle distribution analysis
3.1. Lobaplatin inhibited proliferation of Ishikawa cells
Following the treatment with different lobaplatin concentrations (0, 1.5, 3 and 6 μg /mL) for 48 h, cells were harvested and fixed with icecolded 70% ethanol at 4 °C overnight. After being washed by cold PBS, cells was stained in PI/RNase staining solution for 30 min and then analyzed by a flow cytometry.
The CCK-8 assay was used to evaluate anti-proliferative effect of lobaplatin on Ishikawa cells. After the treatment of lobaplatin for 48 h, the proliferation of Ishikawa cells was markedly attenuated and represented a dose-dependent manner (Fig. 1A). the inhibition rates were 34.73 ± 0.79% and 56.3 ± 0.63% respectively at 2ug and 4ug/mL dosage, which led to a significant inhibition of proliferation. Thus, working concentration (1.5, 3 and 6 μg/ mL) was safely chosen for subsequent experiments.
2.6. Western blot analysis Ishikawa cells were exposed with designed concentration of lobaplatin (0, 1.5, 3 and 6 μg/mL) for 48 h. After being harvested and washed, the cells were lysed in radioimmuno precipitation assay buffer and cells protein was extracted. The protein concentrations were tested using the bicinchoninic acid method, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% milk, the membranes were probed with the following primary antibody: caspase-3, caspase-8, and caspase-9,Bax, Bcl-2 and GAPDH overnight at 4 °C. After incubation with the corresponding secondary antibodies, the reactive bands were measured by an enhanced electrochemiluminescence detection system (Amersham
3.2. Lobaplatin induced S and G2/M phase arrest The effect of lobaplatin on Ishikawa cell cycle distribution was assessed using flow cytometry analysis. After treatment with lobaplatin for 48 h, Ishikawa cell cycle progression was disrupted (Fig. 1B). The treatment of lobaplatin notably increased the population of cells in S phase in a dose-dependent manner and in G2/M phase at low concentration. Meanwhile, the percentage of cells in G1 phase was decreased in a dose-dependent manner. The results suggested that lobaplatin could induce S and G2/M phase arrest in Ishikawa cells. 2
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Fig. 1. The lobaplatin inhibited proliferation and arrested cell cycle progression in human endometrial carcinoma cell line. Ishikawa cells were exposed with various concentrations (0.125–16 μg /mL) of lobaplatin for 48 h and then analyzed by CCK assay(A). Ishikawa cells were exposed to different concentrations of lobaplatin (0, 1.5, 3, 6 μg /mL) for 48 h and then analyzed cell cycle by flow cytometry (B). The cell cycle distribution was treated statistically (* P < 0.05, *** P < 0.001). Data expressed are mean ± SD.
exposed with 6 μg/ml (38.94 ± 2.17%) was higher than that of exposed with 1.5 μg/ml lobaplatin (19.17 ± 1.85%), which indicated that lobaplatin could lead to the occurrence of apoptosis in a concentration-dependent manner.
3.3. Lobaplatin induced cell apoptosis After treated with different concentration of lobaplatin (0, 1.5, 3 and 6 μg/mL) for 48 h, the Ishikawa cells were stained with Annexin VFITC/PI solutions and then tested using flow cytometry. As shown in Fig. 2, the apoptotic rates of lobaplatin-treated cells were significant higher than the control group. The apoptotic rates of Ishikawa cells
Fig. 2. The effect of lobaplatin on cell apoptosis in Ishikawa cells. (A): Ishikawa cells were treated with different concentrations of lobaplatin (0, 1.5, 3, 6 μg /ml) for 48 h followed by Annexin V-FITC/PI apoptosis assay. (B): The statistical images of apoptosis rate was presented (*** P < 0.001). Data expressed are mean ± SD. 3
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Fig. 3. The effects of lobaplatin on apoptosis-related proteins in Ishikawa cells. (A) Lobaplatin upregulated Bax and cleaved - caspase-3, -8 and -9 expression, and reduced Bcl-2 expression. GADPH was employed as a control. (B–C) The relative intensity of apoptosis-related proteins was treated statistically (**P < 0.01, *** P < 0.001). Data are expressed as mean ± SD.
and play an vital important role in the treatment of EC. However, because of their serious side effects, intrinsic or acquired resistance, cisplatin and carboplatin frequently affected patients' quality of life and resulted in treatment failure [16]. Meanwhile, there was no standard the second-line chemotherapy regimens of platinum-resistant patients with EC [4]. As one of the third-generation platinum antineoplastic drug, lobaplatin was developed by ASTA (Germany) and has been approved in clinical application of several cancers by china [17]. Many studies have proved that lobaplatin induced apoptosis, blocked cell cycle progression, and inhibited migration and invasion, of which the antitumor activity was involved in inducing the formation of DNA-drug adducts, the down-regulation expression of PI3K, phosphorylation-AKT, cyclin B, CDK1, CDK6, Bcl-2, MMP-2, MMP-9 and upregulating p53, p21, and p27,caspase-3, -8, and -9, Bax and PARP expression [15,18–21]. Moreover, because of unique advantages including incomplete cross-resistance to cisplatin and carboplatin, fewer side effects, promising radiosensitizer [14,22,23], lobaplatin has attracted more and more attention. So, the aim of this study is firstly to explore the anti-tumor efficacy of lobaplatin on human endometrial cancer cell line Ishikawa and the potential mechanisms, and to further provide evidence for the lobaplatin’s clinical application in EC therapy. To assess the cytotoxic effects of lobaplatin in Ishikawa cells, the inhibition rate of lobaplatin on human endometrial cancer cell was firstly analyzed. The results showed that lobaplatin exerted powerful anti-proliferative efficacy on Ishikawa cells, which provided powerful evidence that lobaplatin could be a new anti-endometrial carcinoma platinum antineoplastic agent. Cell cycle arrest is vital for cancer cell growth suppression, which is one of main anti-cancer mechanisms of antineoplastic drugs including platinum compounds cisplatin, carboplatin and oxaliplatin [24]. In the present study, we found the population of cells in S phase significantly increased in a concentration-dependent behavior and in G2/M phase obviously increased at low concentration, which indicated that cytotoxicity of lobaplatin on Ishikawa cells might partly be caused by
3.4. Lobaplatin regulated expression of main apoptosis-related proteins As apoptosis is served as the critical anti-tumor mechanism, western blot analysis was used to evaluate key proteins of apoptosis expression. After lobaplatin treatment, compared with the control group, western blotting results showed that the protein expression levels of cleavedcaspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax were markedly up-regulated, while the protein expression levels of Bcl2 was significantly down-regulated. Meanwhile, lobaplatin regulated the protein expression levels of the above proteins in a concentration-dependent behavior (Fig. 3). 3.5. Lobaplatin inhibited tumor growth in vivo As shown in Fig. 4A, groups injected with lobaplatin represented a significant body weight loss compared with the control group. However, the body weight of the treatment group and the control group showed an increasing trend. The growth of Ishikawa tumors were obviously suppressed by lobaplatin (Fig. 4B–E).The average tumor volume of treatment group (0.706 ± 0.071cm3) was significantly lower than that of the control group (1.625 ± 0.283cm3). Meanwhile, compared with the control group, the average tumor weight of the lobaplatintreated group was significantly lighter. When finishing the experiment, the T/C (%) value was 44.75%. TUNEL analysis was analyzed using the percentage of apoptotic cells compared to the mean number of cells per field. The results showed that, in the treatment group, the apoptotic rate was increased significantly when compared with the apoptotic rate of control group (Fig. 4F–G). 4. Discussion Up to date, advanced or recurrent EC lacks effective treatment options and has poor prognosis [5]. According to NCCN guidelines, platinum-based chemotherapy was considered as the first-line treatment 4
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Fig. 4. Lobaplatin markedly inhibited tumor growth of Ishikawa xenograft. (A): Body weight of Ishikawa xenograft with treatment of Lobaplatin. (B): Tumor volume of Ishikawa xenograft treated with Lobaplatin. (C): Tumor weight of Ishikawa xenograft treated with Lobaplatin. (D-E): Photos of Ishikawa xenograft tumors. (F-G): representative photographs of tumor tissues stained with TUNEL. Magnification, ×400. The apoptosis rate of each tumor stained with TUNEL was treated statistically (*P < 0.05, **P < 0.01, *** P < 0.001). Data are expressed as mean ± SD.
activity and low side effects for the treatment of the recurrent and refractory ovarian carcinoma patients who have received cisplatin- or carboplatin- based chemotherapy [6]. Therefore, lobaplatin could be a potentially effective anti-cancer drug fo r endometrial cancer, warrant urgently further clinical investigation, and have a special significance in the treatment of patients developed secondary resistance to cisplatin or carboplatin.
inducing the arrest of S and G2/M cell cycle. As a programmed cell death, apoptosis is regarded as the critical anti-tumor mechanism and used to evaluate the performance of anticancer drugs [25–27]. Lobaplatin has been confirmed to induce obviously apoptosis by regulating the expression of many apoptosis-related proteins in a series of tumors [19,20,28]. In the current study, the results showed that the apoptotic rates of lobaplatin-treated cells were significantly increased in a dose-dependent manner by flow cytometry. Meanwhile, our study showed that tumor tissues of xenografts treated with lobaplatin possess significant cell apoptosis analyzed by TUNEL assay compared to the control group. Apoptosis pathways mainly includes the mitochondrial-mediated intrinsic pathway and the cell death receptor-mediated extrinsic pathway [29]. The proapoptotic protein Bax and anti-apoptotic Bcl-2 are the vital factors of controlling the apoptosis [30], which have been proved to participate in the regulation of intrinsic apoptosis [29]. The caspase family is a pivotal central link and executor during the apoptosis process. As an essential apoptotic executive caspase, caspase-3 is activated through caspase-8 and -9, which are in charge of the external and internal apoptotic pathways, respectively [31]. The present study found that after treatment with lobaplatin, the expressions level of cleaved-caspase-3, cleaved-caspase8, cleaved-caspase-9 and Bax were elevated, while the expression level of Bcl-2 was reduced. Our results indicated that lobaplatin induced apoptosis of human endometrial cancer cell line Ishikawa though involving in intrinsic and extrinsic apoptotic pathways. In our study, lobaplatin could significantly inhibit the tumor growth in the Ishikawa xenograft model. Clinical trials have proved that lobaplatin plus other chemotherapy drugs has encouraging antitumor
5. Conclusion Lobaplatin possessed a high inhibitory activity against Ishikawa cells in vitro and in vivo. Lobaplatin blocked the cell cycle in S and G2/ M phase and induced apoptosis, of which the possible mechanisms could be upregulating the expression level of Bax,cleaved-caspase-3,8,and-9 and downregulating the expression level of Bcl-2.Therefore, lobaplatin could be a potentially effective antineoplastic drug for the treatment of EC. Disclosure The authors declare no conflicts of interest in the article. Conflict of interest We guarantee that all authors do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. 5
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Acknowledgements
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