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Berberine in Combination with Cisplatin Induces Necroptosis and Apoptosis in Ovarian Cancer Cells

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Berberine in Combination with Cisplatin Induces Necroptosis and Apoptosis in Ovarian Cancer Cells Liu et al. Biol Res (2019) 52:37 https://doi.org/10.1186/s40659-019-0243-6 Biological Research RESEARCH ARTICLE Open Access Berberine in combination with cisplatin induces necroptosis and apoptosis in ovarian cancer cells Li Liu1†, Jingyan Fan2†, Guihai Ai1, Jie Liu1, Ning Luo1, Caixia Li1 and Zhongping Cheng1* Abstract Background: Berberine (BBR), a compound extracted from a variety of medicinal herbs, has been shown multiple pharmacological efects against cancer cells of diferent origins. Cisplatin (DDP) is known as an efective chemothera- peutic agent against cancer by inducing DNA damage and cell apoptosis. However, the efect of the combined used of BBR and DDP on cell necroptosis in ovarian cancer has not been reported. Methods: OVCAR3 and three patient-derived primary ovarian cancer cell lines (POCCLs) were chosen as the experi- mental objects. To determine the potential anti-cancer activity of BBR and DDP in combination, we frstly treated OVCAR3 and POCCLs cells with BBR and/or DDP. The cell viability of OVCAR3 and POCCLs with treatment of BBR or DDP for diferent hours was measured by CCK-8 assay. Flow cytometry was used to analyze cell cycle distribution and changes in apoptotic cells after treatment with BBR and/or DDP. The morphological changes of OVCAR3 cells were observed by using Transmission electron microscopy (TEM) analysis. Proliferation, apoptosis and necroptosis related markers of OVCAR3 and POCCLs with treatment of BBR or DDP were measured by RT-qPCR, western blotting and immunofuorescence assay. Results: Our results demonstrated that BBR signifcantly inhibited the proliferation of OVCAR3 and primary ovarian cancer cells in a dose- and time-dependent manner. The combination treatment of BBR and DDP had a prominent inhibitory efect on cancer cell growth and induced G0/G1 cell cycle arrest. TEM revealed that the majority of cells after BBR or DDP treatment had an increasing tendency of typical apoptotic and necrotic cell death morphology. Besides, BBR and DDP inhibited the expression of PCNA and Ki67 and enhanced the expression and activation of Caspase-3, Caspase-8, RIPK3 and MLKL. Conclusion: This study proposed that the combination therapy of BBR and DDP markedly enhanced more ovarian cancer cell death by inducing apoptosis and necroptosis, which may improve the anticancer efect of chemotherapy drugs. The apoptosis involved the caspase-dependent pathway, while the necroptosis involved the activation of the RIPK3–MLKL pathway. We hope our fndings might provide a new insight for the potential of BBR as a therapeutic agent in the treatment of ovarian cancer. Keywords: Ovarian cancer, Berberine, Cisplatin, Apoptosis, Necroptosis *Correspondence: [email protected] †Li Liu and Jingyan Fan are co-frst authors 1 Department of Obstetrics and Gynecology, Shanghai Tenth People’s Hospital, Tongji University, 301 Yanchang Road, Shanghai 200072, China Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Biol Res (2019) 52:37 Page 2 of 14 Background Materials and methods Ovarian cancer (OC) remains the most lethal form of all Reagents gynecologic malignancies worldwide. More than two- Reagents used in this study were BBR and DDP (Sigma, thirds of patients are diagnosed at an advanced stage St. Louis, MO, USA); antibodies for Ki67, PCNA, Cas- due to insidious onset, leading to poor prognosis [1, 2]. pase3, Clv C3, Caspase8, Clv C8, RIPK3, p-RIPK3 and Cytoreductive surgery followed by chemotherapy is the p-MLKL (Abcam, Cambridge, MA, USA); antibodies for current standard of care for patients. Cyclin D1 and glyceraldehyde-3-phosphate dehydroge- Cisplatin (DDP) and its derivatives are commonly used nase (GAPDH) (Cell Signaling Technologies, Danvers, agents for OC chemotherapy. Te therapeutic mecha- MA, USA); antibody for MLKL (Novus Biologicals, Lit- nism of these DNA-damaging agents, particularly DDP, tleton, CO, USA); and secondary antibodies of donkey seems to be associated with their efcacy in inhibit- anti-goat HRP, goat anti-rabbit HRP and goat anti-mouse ing the proliferation and apoptosis of cancer cells [3–5]. HRP (Beyotime Institute of Technology, China). However, serious side efects after administration of DDP was found in patients, including bone marrow suppres- Ethics statement sion, nephrotoxicity, neurotoxicity and gastrointestinal All three patients enrolled in this study underwent sur- reactions. To tackle this problem, researchers pinned gery of primary OC at Yangpu Hospital afliated to their hope on combination therapy. Several researches Tongji University School of Medicine (Shanghai, China). have been done in striving to fnd drugs fnd drugs that Te study was approved by the Research Ethics Commit- enhance the anticancer efect of DDP without increasing tee of Tongji University, and in accordance with the ten- side efects, but failed. ets and guidelines of the Declaration of Helsinki. Written Berberine (BBR), an isoquinoline derivative alkaloid informed consent was obtained from all patients. isolated from Huang Lian and other Chinese medici- nal herbs, has been extensively used for the treatment Cell collection and culture of various clinical diseases such as diabetes, metabolic Human OC OVCAR3 cells were obtained from the Chi- syndrome and diarrhea [6–8]. Recent evidence suggests nese Type Culture Collection of the Chinese Academy that BBR possesses anticancer activities involving mul- of Sciences (Shanghai, China). All three patient-derived tiple biological mechanisms for a wide variety of can- POCCLs were established after sterile processing of the cers, including liver cancer, glioblastoma, breast cancer samples from surgical biopsies obtained at diagnostic and OC [9–12]. BBR exhibits an inhibitory efect on cell radical surgeries of OC patients who were diagnosed as proliferation and induces apoptosis in OC cells. In addi- malignant Fesddration Internationale des Gynaecolo- tion, BBR has been demonstrated to induce cell apopto- gistes et Obstetristes (FIGO) stage III serous adenocarci- sis through directly binding with DNA and interfering nomas. Te detailed procedures for the isolation of these with DNA replication as DNA topoisomerase I inhibitor. cell lines were as previously described [19–21]. OVCAR3 Another study showed that BBR sensitized OC cells to cells and POCCLs were cultured in DMEM (hyclone) DDP through miR-93/PTEN/Akt signaling pathway, and and D/F12 (hyclone) respectively, supplemented with BBR enhanced DDP induced apoptosis [12–14]. 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicil- Programmed cell death, such as apoptosis, necroptosis lin sodium, and 100 mg/mL streptomycin sulfate (Solar- and autophagy, plays crucial roles in the physiopatho- bio) at 37 °C in a humidifed air atmosphere containing logical processes of the organism, as well as in the patho- 5% CO2. Cells were used when they were in the logarith- genesis and progress of cancer [15, 16]. Previous studies mic growth phase. were mainly focused on the mechanism of apoptosis in cancer, and little attention has been paid to the roles of Cell proliferation assay 3 necroptosis and autophagy in the pathogenesis and pro- Cells (5.0 × 10 /well) were plated, treated in 96-well gress of cancer [17, 18]. However, whether BBR exerts plates, and stimulated with diferent concentrations of the anticancer efect by inducing tumor cell necroptosis BBR (50, 100, 200, 500 μM) or/and DDP (5 mg/L) for remains unanswered. Te aim of the present study was to 24, 48 and 72 h [22]. Cell proliferation was detected by investigate the efect of BBR and DDP on human ovar- using the Cell Count Kit-8 (CCK-8, Signalway Antibody) ian cell line OVCAR3 and three patient-derived primary according to the manufacturer’s instructions. Absorb- OC cell lines (POCCLs) by simultaneous observation of ance was detected at a wavelength of 450 nm with a the ability of either BBR or DDP alone or both in induc- microplate reader (Bio-Rad). Half-maximal inhibitory ing apoptosis and necroptosis in these cells, hoping that concentration (IC50) of OVCAR3 cells after BBR treat- our study could provide new insights into the anticancer ment 24 h was calculated from an experimentally derived mechanism of BBR and DDP. dose–response curve for each concentration by using Liu et al. Biol Res (2019) 52:37 Page 3 of 14 GraphPad Prism 6.0 software for Windows (GraphPad Table 1 List of designed primers for real-time polymerase Software, La Jolla, CA, USA) [23]. Each assay was carried chain reaction out in triplicate. Primer name Primer sequence Cell cycle analysis Caspase3 (NM_004346.3) Forward: 5′ GTT TGA GCC TGA GCA GAG AC 3′ Reverse: 5′ TGG CAG CAT CAT CCA CAC 3′ Te cell cycle was evaluated by fow cytometry using Caspase-8 Forward: 5′ TGT GCC CAA ATC AAC AAG AG 3′ PI (7 Seabiotech, Shanghai, China) staining on a fow Reverse: 5′ TTC AAA GGT CGT GGT CAA AG 3′ cytometer (BD Biosciences). Cells were plated in 6-well Ki67 Forward: 5′ GTG CGA AGG TTC TCA TGC 3′ plates, treated with BBR (100 μM) or/and DDP (5 mg/L) Reverse: 5′ CTT GAC ACT CCG CGT TAC 3′ for 24 h, washed in PBS, and re-suspended in stain- PCNA Forward: 5′ GCT CTT CCC TTA CGC AAG TC 3′ Reverse: 5′ AGT GCC TCC AAC ACC TTC 3′ ing solution containing 20 μg/mL PI and 100 μg/mL RIPK3 Forward: 5 CGC CTG CTG AAA GAA GTG 3 RNase A.
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