Human Cell DOI 10.1007/s13577-017-0167-9 RESEARCH ARTICLE Enhanced Stim1 expression is associated with acquired chemo- resistance of cisplatin in osteosarcoma cells 1 2 3 2 2 Xilong Sun • Qiang Wei • Jie Cheng • Yanzhu Bian • Congna Tian • 2 4 Yujing Hu • Huijie Li Received: 18 January 2017 / Accepted: 1 March 2017 Ó The Author(s) 2017. This article is an open access publication Abstract Osteosarcoma is the most common primary resistant variants, which was evidenced by the activation of malignant bone tumor. Although cisplatin is the primary molecular markers of ER stress, GRP78, CHOP and ATF4. chemotherapy used in osteosarcoma treatment, the cisplatin Knockdown of Stim1 using siRNA remarkably enhanced resistance remains a big challenge for improving overall cisplatin-induced apoptosis and ER stress in MG63/CDDP survival. The store-operated calcium (Ca2?) entry (SOCE) cells, thereby sensitizing cancer cells to cisplatin. On the and its major mediator Stim1 have been shown to be other hand, overexpression of Stim1 markedly reversed implicated in a number of pathological processes typical apoptosis and ER stress following cisplatin treatment. for cancer. In this study, we showed that Stim1 expression Taken together, these results demonstrate that Stim1 as was significantly increased in chemo-resistant osteosar- well as Ca2? entry contributes cisplatin resistance via coma tissues compared with chemo-sensitivity tissues. inhibition of ER stress-mediated apoptosis, and provide Patients with Sitm1 expression exhibited poorer overall important clues to the mechanisms involved in cisplatin survival than Stim1-negative patients. Moreover, un-regu- resistance for osteosarcoma treatment. Stim1 represents as lation of Stim1 expression and SOCE were also observed a target of cisplatin and blockade of Stim1-mediated Ca2? in cisplatin-resistant MG63/CDDP cells compared with entry may be a useful strategy to improve the efficacy of their parental cells. Cisplatin treatment obviously reduced cisplatin to treat osteosarcoma. Stim1 expression and SOCE in cisplatin-sensitivity MG63 cells, but had no effects on MG63/CDDP cells. In addition, Keywords Cisplatin resistance Á Osteosarcoma Á cisplatin resulted in a more pronounced increase of endo- Endoplasmic reticulum Á Apoptosis Á Stim1 plasmic reticulum (ER) stress in MG63 cells than in their Introduction Electronic supplementary material The online version of this article (doi:10.1007/s13577-017-0167-9) contains supplementary material, which is available to authorized users. Osteosarcoma is the most common primary malignant bone tumor in children and adolescents [1]. It accounts for about & Huijie Li 2.4% of all malignancies in pediatric patients and 20% of [email protected] primary bone cancer [2]. Patients with osteosarcoma will receive a three-drug chemotherapy regimen consisting of 1 Department of Orthopaedics, Hebei General Hospital, Shijiazhuang, People’s Republic of China cisplatin, doxorubicin and methotrexate, followed by sur- gical resection of the primary tumor in which higher sur- 2 Department of Nuclear Medicine, Hebei General Hospital, Shijiazhuang, People’s Republic of China vival rates have been achieved [3, 4]. However, the development of chemo-resistance is a serious problem that 3 Department of Stomatology, Hebei General Hospital, Shijiazhuang, People’s Republic of China largely limits the efficacy of chemotherapy. While many efforts have been made in the past [5, 6], little is known 4 Department of Orthopedics, The Third Hospital of Hebei Medical University, No. 139, Ziqiang Road, Qiaoxi District, about the molecular mechanisms underlying osteosarcoma Shijiazhuang 050051, Hebei, People’s Republic of China chemo-resistance for clinical therapy. 123 X. Sun et al. Multiple studies have emphasized the critical role of died within 3 years because of requiring chemo-resistance calcium (Ca2?) and Ca2? ion channel in the regulation of (n = 32). The chemo-resistance was associated with less cancer cell death and apoptosis [7–10]. Store-operated than 25% of tumor shrinkage and 10% of tumor cell Ca2? entry (SOCE) has been confirmed as the major Ca2? necrotic rate. All tissue specimens were paraffin-embedded influx pathway in non-excitable cells [11, 12]. Stim1, for immunohistochemistry staining, or stored in liquid located in the membrane of endoplasmic reticulum (ER), is nitrogen for western blotting and real-time PCR analysis. major component of store-operated Ca2? channels and servers as a sensor of ER Ca2? level [11, 13]. To note, Cell culture previous studies have suggested a critical role of Stim1 in cancerogenesis. Knockdown of Stim1 or pharmacological Human osteosarcoma cell line MG63 was obtained from inhibition of SOCE suppressed tumor metastasis in animal American Type Culture Collection (ATCC, Rockville, models of breast cancer [14]. Moreover, blockade of Stim1 MD, USA) and cultured in RPMI1640 containing 10% inhibited hepatocarcinoma cell migration and sensitized FBS, 100 U/ml penicillin and 100 lg/ml streptomycin (all prostate cancer cells to therapy [15]. However, the possible from Invitrogen, Grand Island, NY, USA) in a humidified role of Stim1 in osteosarcoma is still inconclusive. atmosphere of 5% CO2 at 37 °C. The cisplatin-resistant The ER is an essential organelle that is critical for the MG63 (MG63/CDDP) cells were developed from the par- biological processes required for cell survival and death ental MG63 cells using an intermittent stepwise selection [16]. Upon multiple stimuli, unfolded and incompletely protocol as previously described [22, 23]. folded proteins are accumulated in ER, leading to unfolded protein response and ER stress [16, 17]. Importantly, ER Immunohistochemistry plays a crucial role in Ca2? homeostasis and Ca2? sig- naling [18]. Moreover, previous studies have reported that Immunohistochemistry was used to detect the expression of ER stress can be induced by cisplatin and evidenced by the Stim1 in all tissue specimens. The paraffin-embedded activation of molecular markers of ER stress, such as specimens were cut into 8-lm-thick sections, and GRP78, CHOP and ATF4 [19, 20]. Considering the deparaffinized and rehydrated in a graded series of alco- important role of Stim1 and SOCE in ER stress and hols. After antigen retrieval, the sections were incubated apoptosis [18, 21], we proposed that Stim1 and SOCE with 3% hydrogen peroxide for 10 min to block endoge- might be involved in cisplatin resistance in cancer cells. nous peroxidase activity, and then were immunostained Therefore, in the present study, we explored the corre- with Stim1 antibody (1:50) (Santa Cruz Biotechnology, lation between Stim1 and ER stress under cisplatin treat- Inc., Paso Robles, CA, USA) at 4 °C overnight. After ment in osteosarcoma cells, demonstrating that increased rinsing with PBS, the sections were incubated with horse- Stim1 as well as Ca2? entry contributes to cisplatin resis- radish peroxidase-conjugated secondary antibody (Zhong- tance by inhibition of ER stress-mediated apoptosis. Our shan Jinqiao Co., Beijing, China) for 1 h. 3,3- findings suggest that targeting Stim1 may be a potential diaminobenzidine tetrahydrochloride (DAB, Zhongshan strategy to improve the efficacy of chemotherapy for Jinqiao Co.) was used as the substrate for the final visu- osteosarcoma. alization. Counterstaining was carried out with hema- toxylin. The negative controls were processed in a same protocol with PBS instead of primary antibody. The sec- Methods and materials tions were observed under a light microscope (Olympus, Tokyo, Japan). Patients and specimen preparation RNA extraction and real-time PCR The specimens of tumor tissues were collected from patients with osteosarcoma who underwent surgical To quantify mRNA expression of Stim1 in tissue speci- resection between 2010 and 2013 in the Department of mens, total RNA was prepared using Trizol reagent (In- Orthopedics of The Third Hospital of Hebei Medical vitrogen) according to the manufacturer’s instruction. The University. These patients were received standardized isolated RNA was reverse transcribed using SuperScript II protocol consisting of neoadjuvant chemotherapy followed RT kit (Invitrogen). Stim1 mRNA expression was deter- by appropriate surgical management. The specimens from mined by real-time PCR on an ABI PrismÒ 7300 (Applied 60 cases of osteosarcoma were divided into the following Biosystems, Foster City, CA, USA) with SYBR Green Real groups based on the follow-up visits from 2011: (1) sub- Time PCR kit (Invitrogen). b-Actin was used as an internal jects with a good response and a survival of more than control. The following primers were used: Stim1 sense 50- 3 years (n = 28); (2) subjects with a poor response, who GAATTGACAAGCCCCTGTGT-30 and antisense 50- 123 Enhanced Stim1 expression is associated with acquired chemo-resistance of cisplatin in… ATGACTTCCATGCCTTCCAC-30; b-actin sense 50- Transfection GGCGGCACCACCATGTACCCT-30 and antisense 50- AGGGGCCGGACTCGTCATACT-30. Full-length cDNAs for human STIM1 (NM_003156) were purchased from OpenBioSystems (Huntsville, AL, USA), and then were subcloned into the expression vector Western blotting analysis pSMCV (OpenBioSystems). The plasmid was confirmed by sequencing. To overexpress Stim1 in osteosarcoma, Tissue specimens or cells were lysed using Western & IP cells were transfected transiently with Stim1 plasmid for Cell lysis Kit (Beyotime, Jiangsu, China) supplemented 24 h using the Lipofectamine Plus reagent (Invitrogen) in with protease inhibitor cocktail (Merck, CA, USA). The