Overexpression Of Osteopontin Promotes Resistance To Cisplatin Treatment In HCC

Background and Aim:Hepatocellular carcinoma (HCC) constitute a frequent types of cancer in China. HCC is highly malignant, most of the patients present with invasion and metstasis at diagnosis, which leads to the lost of the opportunity for surgical resection, and the recurrent rate is relatively high (60-70%). Transarterial chemoembolization (TACE) is considered as the most commonly used initial treatment for unresectable HCC. One important limitation in the clinical battle against these tumours is their marked refractivity to the available chemotherapy and the development of resistance to drugs during treatment.The resistance of HCC is a complex process involving multiple genes, processes, and stages, as well as great individual difference. It is thus important to find out an effective target to predict and interfere the chemoresistance. OPN is a multifunctional cytokine involved in survival, cell migration and adhesion. Increasing evidence have clarified its role in tumor development, progression and metastasis. OPN gene localized on human chromosome 4, ql3. The final synthetic protein of OPN after gene transcription and translation contains 314 amino acids. OPN participate in many physiological and pathological processes. These processes include bone remodeling, angiogenesis, tissue repair, atherosclerosis. Previous studies have found that OPN was closely related to the occurrence and development of tumor. It is highly expressed in prostate cancer, breast cancer, liver cancer. OPN binds to its receptor CD44 or integrins, transduces signal in tumor cells, promotes cell chemotaxis and migration, and mediates cancer cell invasion and metastasis. Therefore, evidence elucidates OPN’s role in recurrence and metastasis. However, the role of OPN in chemoresistance of HCC has not yet been investigated.Materials and Methods:Patients diagnosed with HCC were enrolled from Tongji Hospital, Huazhong University of Science and Technology, were treated with TACE. All the patients did not receive any previous treatment. The clinical data including patient name, sex, age, status of hepatitis virus infection, clinical staging (TNM staging) (in accordance with the sixth revision of the TNM staging from International Union against cancer 2002 (UICC)), pathological grading of hepatocellular carcinoma (according to the Edmondson standard) were collected. In this study, pathological specimens before and after TACE were collected from a total of 8 patients. The expression of OPN was determined using immunohistochemical SP method and comapred.Human hepatocellular carcinoma cell lines HepG2, Huh-7 and PLC/PRF/5 in medium containing 10%fetal bovine serum,100 U/ml penicillin and streptomycin 100mg/mL DMEM medium cultured box air cells are placed in 37 or 5% C, and 95% of the carbon dioxide. The sensitivity of hepatocellular carcinoma cells to cisplatin were analyzed using cytotoxicity assay and flow cytometry. OPN mRNA expression before and after treatment with cisplatin in three hepatocellular carcinoma cell line were studied using real-time quantitative PCR. The expression of OPN protein before and after treatment with cisplatin in three hepatocellular carcinoma cell line were studied using western blot assay. The phosphorylated and total PI3K, total AKT, Caspase-3 protein expression were detected by western blot. OPN is added in the medium, the cisplatin sensitivity in different group were detect,cisplatin toxicity curve were depicted. The cell apoptosis were measured by flow cytometry. OPN pathway was blocked by CD44 antibody and alpha v beta 3 antibodies and PI3K/AKT pathway inhibitor LY294002, the apoptosis ratio and reaction of cisplatin resistance were observed. Each experiment was performed independently at least three times. Values were expressed as mean ± SD. A two-tailed Student’s t-test was used to estimate intergroup differences if not otherwise stated. P<0.05 was considered to be statistically significant.Results:1. In the present study, we examined the expression of OPN in human HCC samples before and after cisplatintreatment, the results showed that OPN was significantly increased in cisplatin-resistant specimens.2. We then studied the effect of cisplatin on OPN expression in HCC cells. The expression of OPN in HCC cells was elevated compared to control cells after exposure to cisplatin. We also found that PI3K/AKT signaling pathway was also activated by cisplatin and this effect was induced by OPN pathway. To study the effect of OPN on chemoresistance, HCC cells were treated with cisplatin along with OPN. Incubation with OPN enchanced the chemoresistance of HCC cells to cisplatin.3. In contrast, blockage of OPN pathway promoted the chemosensitivity of HCC cells to cisplatin.Conclusion:Our results suggest that OPN enhance chemoresistance of cisplatin in HCC cells by activating PI3K/AKT signaling pathway, blocking OPN pathway might be a novel way to overcome the disease.