Failure of Elevating Calcium Induces Oxidative Stress Tolerance and Imparts Cisplatin Resistance in Ovarian Cancer Cells
Ma Liwei1, Wang Hongjun1,2, Wang Chunyan1, Su Jing3, Xie Qi3, Xu Lu1, Yu Yang1, Liu Shibing1, Li Songyan1, Xu Ye1,*, Li Zhixin2,*
1Medical Research Laboratory, Jilin Medical University, Jilin 132013, China 2Department of Histology and Embryology, Jilin Medical University, Jilin 132013, China 3Department of Pathophysiology, Basic College of Medicine, Jilin University, Changchun, 130021, China
Cisplatin is a commonly used chemotherapeutic drug, used for the treatment of malignant ovarian cancer, but acquired resistance limits its application. There is therefore an overwhelming need to understand the mechanism of cisplatin resistance in ovarian cancer, that is, ovarian cancer cells are insensitive to cisplatin treatment. Here, we show that failure of elevating calcium and oxidative stress tolerance play key roles in cisplatin resistance in ovarian cancer cell lines. Cisplatin induces an increase in oxidative stress and alters intracellular Ca2+ concentration, including cytosolic and mitochondrial Ca2+ in cisplatin-sensitive SKOV3 cells, but not in cisplatin-resistant SKOV3/DDP cells. Cisplatin induces mitochondrial damage and triggers the mitochondrial apoptotic pathway in cisplatin-sensitive SKOV3 cells, but rarely in cisplatin-resistant SKOV3/DDP cells. Inhibition of calcium signaling attenuates cisplatin-induced oxidative stress and intracellular Ca2+ overload in cisplatin-sensitive SKOV3 cells. Moreover, in vivo xenograft models of nude mouse, cisplatin significantly reduced the growth rates of tumors originating from SKOV3 cells, but not that of SKOV3/DDP cells. Collectively, our data indicate that failure of calcium up-regulation mediates cisplatin resistance by alleviating oxidative stress in ovarian cancer cells. Our results highlight potential therapeutic strategies to improve cisplatin resistance.
Ma Liwei,Wang Hongjun,Wang Chunyan, et al. Failure of Elevating Calcium Induces Oxidative Stress Tolerance and Imparts Cisplatin Resistance in Ovarian Cancer Cells[J]. Aging and disease,
2016, 7(3): 254-266.
Figure 1. Cisplatin inhibits proliferation and induces cell death of ovarian cancer cells
(A) SKOV3 and SKOV3/DDP cells were treated with varying doses of cisplatin for 24 or 48 h. Cell viability was determined by the MTT assay. Data are presented as means ± SD, n=3. (B) Cells were treated with 6 μg/ml cisplatin for 0 h and 16 h, and then stained with Hoechst 33342 and PI. Then, cells were observed by confocal microscopy (bar, 20 μm). (C) Quantitation of cell death ratio. Data are presented as means ± SD, n=3, **P<0.01 vs. control.
Figure 2. Alteration of cytosolic Ca2+ is significantly induced in SKOV3 cells by cisplatin or ATP, but not in SKOV3/DDP cells
(A) Cells were treated with ATP and time-lapse imaging was used to detect changes in cytosolic Ca2+ levels. Data were obtained by confocal laser microscopy. (B) After cells treated with 6 μg/mL cisplatin for 0 h, 8 h and 16 h, cytosolic Ca2+ levels was detected by Fluo-4/AM (bar, 20 μm).
Figure 3. Cisplatin or ATP treatment induces mitochondrial Ca2+ influx in SKOV3, but not SKOV3/DDP cells
(A) Both cell lines were treated with ATP and time-lapse scanning was used to detect the changes of mitochondrial Ca2+. Data were obtained by confocal laser microscopy. (B) After cells treated with 6 μg/ml cisplatin for 0 h, 8 h and 16 h, mitochondrial Ca2+ was detected by confocal microscopy (bar, 20 μm).
Figure 4. Mitochondrial Ca2+ overload induces cell apoptosis through mitochondrial-dependent pathway in SKOV3 cells
(A) Representative transmission electron microscopy photomicrographs of both cell lines treated with 6μg/ml cisplatin for 8 h. Mitochondrial morphologies are normal in control cells (1,500x). Exposure to 6μg/ml cisplatin for 8 h resulted in mitochondrial damage (1,500x; arrows indicate mitochondrial damage). (B) Western blot analysis of cytosolic cytochrome c, caspase-3, and cleaved caspase-3 expression in cells treated with cisplatin for 0 h, 8 h, and 16 h. (C) Quantification of cytosolic cytochrome c and cleaved caspase-3 protein. Data are presented as means ± SD, n=3. **P<0.01 vs. control.
Figure 5. Inhibition of cisplatin-induced cytosolic Ca2+ influx and mitochondrial Ca2+ overload reduces intracellular ROS production in SKOV3 cells
(A) Cells were treated with 6 μg/ml cisplatin for 0 h, 8 h and 16 h, and stained with DCFH-DA (bar, 20 μm). (B) After cells treated with 6 μg/ml cisplatin with or without 2-APB (50 μM) and BAPTA/AM (30 μM) for 16 h, cells were stained with DCFH-DA (bar, 20 μm). (C) After the same treatment with (A), cytosolic Ca2+ levels were detected by Fluo-4/AM and mitochondrial Ca2+ was detected by Rhod-2/AM (bar, 20 μm).
Figure 6. Inhibition of cisplatin-induced cytosolic Ca2+ influx and mitochondrial Ca2+ overload protect SKOV3 cells from cisplatin-induced apoptosis
(A) After cells treated with 6 μg/ml cisplatin with or without 2-APB (50 μM) and BAPTA/AM (30μM) for 16 h, cells were then stained with Annexin-V. Data are presented as the mean ± SD, n = 3. (B) After the same treatment with (A), the expression of caspase-3, cleaved caspase-3, and cytosolic cytochrome c in both cell lines is detected by western blotting. (C) Quantitation of cleaved caspase-3, and cytosolic cytochrome c protein levels. Data are presented as the mean ± SD, n = 3. **P < 0.01 vs. control, #P < 0.05 vs. cisplatin.
Figure 7. Cisplatin displays anti-tumor activity in xenograft mouse models bearing tumors originating from SKOV3 cells, but not SKOV3/DDP cells
(A) The average volume of tumor originating from SKOV3 cells were obviously diminished by cisplatin, but not in SKOV3/DDP cells. (B) BALB/c femal nude mice subcutaneous transplant tumor model was established using SKOV3 and SKOV3/DDP cells. After treatment with cisplatin, tumors in mice were excised and photographed for each group. Data are presented as the mean ± SD, n = 3. **P < 0.01 vs. control.
Mei L, Chen H, Wei DM, Fang F, Liu GJ, Xie HY, Wang X, Zou J, Han X, Feng D (2013). Maintenance chemotherapy for ovarian cancer. Cochrane Database Syst Rev, 6:CD007414.
Pils D, Bachmayr-Heyda A, Auer K, Svoboda M, Auner V, Hager G, Obermayr E, Reiner A, Reinthaller A, Speiser Pet al (2014). Cyclin E1 (CCNE1) as independent positive prognostic factor in advanced stage serous ovarian cancer patients - a study of the OVCAD consortium. Eur J Cancer, 50(1):99-110.
Shank JJ, Yang K, Ghannam J, Cabrera L, Johnston CJ, Reynolds RK, Buckanovich RJ (2012). Metformin targets ovarian cancer stem cells in vitro and in vivo. Gynecol Oncol, 127(2):390-397.
Agarwal R, Kaye SB (2003). Ovarian cancer: strategies for overcoming resistance to chemotherapy. Nat Rev Cancer, 3(7):502-516.
Samuel P, Pink RC, Caley DP, Currie JM, Brooks SA, Carter DR (2015). Over-expression of miR-31 or loss of KCNMA1 leads to increased cisplatin resistance in ovarian cancer cells. Tumour Biol.
Gomez-Roman N, McGregor F, Wheate NJ, Plumb JA (2015). Cucurbit uril encapsulated cisplatin overcomes resistance to cisplatin induced by Rab25 overexpression in an intraperitoneal ovarian cancer model. J Ovarian Res, 8(1):62.
Liu M, Qi Z, Liu B, Ren Y, Li H, Yang G, Zhang Q (2015). RY-2f, an isoflavone analog, overcomes cisplatin resistance to inhibit ovarian tumorigenesis via targeting the PI3K/AKT/mTOR signaling pathway. Oncotarget, 6:25281-94.
Chtourou Y, Aouey B, Kebieche M, Fetoui H (2015). Protective role of naringin against cisplatin induced oxidative stress, inflammatory response and apoptosis in rat striatum via suppressing ROS-mediated NF-kappaB and P53 signaling pathways. Chem Biol Interact, 239:76-86.
Gunes DA, Florea AM, Splettstoesser F, Busselberg D (2009). Co-application of arsenic trioxide (As2O3) and cisplatin (CDDP) on human SY-5Y neuroblastoma cells has differential effects on the intracellular calcium concentration ([Ca2+]i) and cytotoxicity. Neurotoxicology, 30(2):194-202.
Chandra S (2010). Quantitative imaging of chemical composition in single cells by secondary ion mass spectrometry: cisplatin affects calcium stores in renal epithelial cells. Methods Mol Biol, 656:113-130.
Krebs J, Agellon LB, Michalak M (2015). Ca(2+) homeostasis and endoplasmic reticulum (ER) stress: An integrated view of calcium signaling. Biochem Biophys Res Commun, 460(1):114-121.
Berridge MJ, Bootman MD, Lipp P (1998). Calcium--a life and death signal. Nature, 395(6703):645-648.
Marin J, Encabo A, Briones A, Garcia-Cohen EC, Alonso MJ (1999). Mechanisms involved in the cellular calcium homeostasis in vascular smooth muscle: calcium pumps. Life Sci, 64(5):279-303.
Ermak G, Davies KJ (2002). Calcium and oxidative stress: from cell signaling to cell death. Mol Immunol, 38(10):713-721.
Xu D, Su C, Song X, Shi Q, Fu J, Hu L, Xia X, Song E, Song Y (2015). Polychlorinated biphenyl quinone induces endoplasmic reticulum stress, unfolded protein response, and calcium release. Chem Res Toxicol, 28(6):1326-1337.
Splettstoesser F, Florea AM, Busselberg D (2007) IP(3) receptor antagonist, 2-APB, attenuates cisplatin induced Ca2+-influx in HeLa-S3 cells and prevents activation of calpain and induction of apoptosis. Br J Pharmacol, 151(8):1176-1186.
Ding X, Yu Q, Zhang B, Xu N, Jia C, Dong Y, Chen Y, Xing L, Li M (2014). The type II Ca2+/calmodulin-dependent protein kinases are involved in the regulation of cell wall integrity and oxidative stress response in Candida albicans. Biochem Biophys Res Commun, 446(4):1073-1078.
Barzilai A, Rotman G, Shiloh Y (2002). ATM deficiency and oxidative stress: a new dimension of defective response to DNA damage. DNA Repair (Amst), 1(1):3-25.
Ikner A, Shiozaki K (2005). Yeast signaling pathways in the oxidative stress response. Mutat Res, 569(1-2):13-27.
Lin L, Zheng J, Zhu W, Jia N (2015). Nephroprotective effect of gelsemine against cisplatin-induced toxicity is mediated via attenuation of oxidative stress. Cell Biochem Biophys, 71(2):535-541.
Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, Castedo M, KroemerG (2012). Molecular mechanisms of cisplatin resistance. Oncogene, 31(15):1869-1883.
Yang Y, Parsons KK, Chi L, Malakauskas SM, Le TH (2009). Glutathione S-transferase-micro1 regulates vascular smooth muscle cell proliferation, migration, and oxidative stress. Hypertension, 54(6):1360-1368.
Xia M, Yu H, Gu S, Xu Y, Su J, Li H, Kang J, Cui M (2014). p62/SQSTM1 is involved in cisplatin resistance in human ovarian cancer cells via the Keap1-Nrf2-ARE system. Int J Oncol, 45(6):2341-2348.
Parihar P, Solanki I, Mansuri ML, Parihar MS (2015). Mitochondrial sirtuins: emerging roles in metabolic regulations, energy homeostasis and diseases. Exp Gerontol, 61:130-141.
Pinton P, Giorgi C, Siviero R, Zecchini E, Rizzuto R (2008). Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis. Oncogene, 27(50):6407-6418.
Lakhani SA, Masud A, Kuida K, Porter GAJr., Booth CJ, Mehal WZ, Inayat I, Flavell RA (2006). Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science, 311(5762):847-851.
Kuribayashi K, Mayes PA, El-Deiry WS (2006). What are caspases 3 and 7 doing upstream of the mitochondria? Cancer Biol Ther, 5(7):763-765.
Fan Z, Yu H, Cui N, Kong X, Liu X, Chang Y, Wu Y, Sun L, Wang G (2015). ABT737 enhances cholangiocarcinoma sensitivity to cisplatin through regulation of mitochondrial dynamics. Exp Cell Res, 335(1):68-81.
Xu Y, Yu H, Qin H, Kang J, Yu C, Zhong J, Su J, Li H, Sun L (2012). Inhibition of autophagy enhances cisplatin cytotoxicity through endoplasmic reticulum stress in human cervical cancer cells. Cancer Lett, 314(2):232-243.
Basu A, Krishnamurthy S (2010). Cellular responses to Cisplatin-induced DNA damage. J Nucleic Acids.
Ramachandran S, Quist AP, Kumar S, Lal R (2006). Cisplatin nanoliposomes for cancer therapy: AFM and fluorescence imaging of cisplatin encapsulation, stability, cellular uptake, and toxicity. Langmuir, 22(19):8156-8162.
Seo YH, Jo YN, Oh YJ, Park S (2015). Nano-mechanical reinforcement in drug-resistant ovarian cancer cells. Biol Pharm Bull, 38(3):389-395.
Marullo R, Werner E, Degtyareva N, Moore B, Altavilla G, Ramalingam SS, Doetsch PW (2013). Cisplatin induces a mitochondrial-ROS response that contributes to cytotoxicity depending on mitochondrial redox status and bioenergetic functions. PLoS One, 8(11):e81162.
Mandic A, Hansson J, Linder S, Shoshan MC (2003). Cisplatin induces endoplasmic reticulum stress and nucleus-independent apoptotic signaling. J Biol Chem, 278(11):9100-9106.
Gonzalez VM, Fuertes MA, Alonso C, Perez JM (2001). Is cisplatin-induced cell death always produced by apoptosis? Mol Pharmacol, 59(4):657-663.
Du A, Huang S, Zhao X, Zhang Y, Zhu L, Ding J, Xu C (2016). Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells. J Neuroimmunol, 290:109-114.
Ozcan L, Tabas I (2012). Role of endoplasmic reticulum stress in metabolic disease and other disorders. Annu Rev Med, 63:317-328.
Ron D, Walter P (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol, 8(7):519-529.
Tian F, Schrodl K, Kiefl R, Huber RM, Bergner A (2012). The hedgehog pathway inhibitor GDC-0449 alters intracellular Ca2+ homeostasis and inhibits cell growth in cisplatin-resistant lung cancer cells. Anticancer Res, 32(1):89-94.
Liang X, Huang Y (2000). Intracellular free calcium concentration and cisplatin resistance in human lung adenocarcinoma A549 cells. Biosci Rep, 20(3):129-138.
Moreau B, Nelson C, Parekh AB (2006). Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration. Curr Biol, 16(16):1672-1677.
Kruidering M, Van de Water B, de Heer E, Mulder GJ, Nagelkerke JF (1997).Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain. J Pharmacol Exp Ther, 280(2):638-649.
Pourahmad J, Hosseini MJ, Eskandari MR, Shekarabi SM, Daraei B (2010). Mitochondrial/lysosomal toxic cross-talk plays a key role in cisplatin nephrotoxicity. Xenobiotica, 40(11):763-771.
Ozaki T, Ishiguro S, Itoh H, Furuhama K, Nakazawa M, Yamashita T (2013). Cisplatin binding and inactivation of mitochondrial glutamate oxaloacetate transaminase in cisplatin-induced rat nephrotoxicity. Biosci Biotechnol Biochem, 77(8):1645-1649.
Zsengeller ZK, Ellezian L, Brown D, Horvath B, Mukhopadhyay P, Kalyanaraman B, Parikh SM, Karumanchi SA, Stillman IE, Pacher P (2012). Cisplatin nephrotoxicity involves mitochondrial injury with impaired tubular mitochondrial enzyme activity. J Histochem Cytochem, 60(7):521-529.
Gaona-Gaona L, Molina-Jijon E, Tapia E, Zazueta C, Hernandez-Pando R, Calderon-Oliver M, Zarco-Marquez G, Pinzon E, Pedraza-Chaverri J (2011). Protective effect of sulforaphane pretreatment against cisplatin-induced liver and mitochondrial oxidant damage in rats. Toxicology, 286(1-3):20-27.
Santos NA, Catao CS, Martins NM, Curti C, Bianchi ML, Santos AC (2007). Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Arch Toxicol, 81(7):495-504.
Motiani RK, Stolwijk JA, Newton RL, Zhang X, Trebak M (2013). Emerging roles of Orai3 in pathophysiology. Channels (Austin), 7(5):392-401.
Fan W, Chang J, Fu P (2015). Endocrine therapy resistance in breast cancer: current status, possible mechanisms and overcoming strategies. Future Med Chem, 7(12):1511-1519.
Lipskaia L, Lompre AM (2004). Alteration in temporal kinetics of Ca2+ signaling and control of growth and proliferation. Biol Cell, 96(1):55-68.
Wang C, Li T, Tang S, Zhao D, Zhang C, Zhang S, Deng S, Zhou Y, Xiao X (2015). Thapsigargin induces apoptosis when autophagy is inhibited in HepG2 cells and both processes are regulated by ROS-dependent pathway. Environ Toxicol Pharmacol, 41:167-179.