| BackgroundBladder urothelial carcinoma (UC) is the most common malignancy in urogenital system, which comprises non-muscle invasive bladder cancer and muscle invasive bladder cancer. Non-muscle invasive bladder cancer present as 75%, which mainly confined to the mucosa or subepithelial layer. Although there arestandard treatment methods including transurethral bladder tumor resectionand installing intravesical chemotherapeutic drugs, about two-thirds of patients will have recurrence after surgery.Further, some patients will progress to a higher grade. Muscleinvasive bladder cancer refers to the bladder cancer in clinical stage of T2-T4. Muscleinvasive bladder cancer accounts for 25% of all bladder cancers, and has high degree of malignancy, fast progression and poor prognosis. Radical cystectomy is currently the standard treatment for muscle invasive bladder cancer, and cisplatin (CDDP)-based chemotherapy has been adopted for advanced urothelial carcinoma to improve the prognosis, combined with radical cystectomy. It is an effective method for enhancing advanced stage bladder cancer patients’ survival rate, decreasing tumor recurrence and metastasis. Nowadays, the traditional combination chemotherapies for muscle invasive bladder cancer are MVAC (methotrexate, vinblastine, doxorubicin and cisplatin)and GC (gemcitabine, cisplatin). Cisplatin, as a representative of platinum anticancer drugs, is the main drug in chemotherapy of bladder cancer.Although these combination therapies, there is significant toxicity and long-term survival in only 15-20% patients due to resistance to chemotherapy. So, to overcome chemoresistance and improve chemotherapeutic efficacy, novel molecular marker for chemosensitivity is highly desirable.Akt, another name is Kinase B, is one of the major downstream targets in the PI3K/Akt signaling pathway, and comprises three isoforms:Aktl, Akt2, Akt3.Akt plays a key role in the regulation of the diverse biological effects, including cell growth, proliferation, survival and metabolism.Dysregulation of Akt is related with several human diseases, including cancer, diabetes, cardiovascular and neurological disease. A lot of studies have demonstrated that Akt activation or overexpression is closely related with many tumors growh, progression and metastasis, such as lung cancer, ovarian cancer, breast cancer, prostate cancerand hematological disease. So Akt is the crucial target in PI3K/Akt signaling pathway. People could regulate Akt expression to control the balance between cell proliferation and apoptosis of tumor so as to inhibit tumor growth and proliferation, induce apoptosis of tumor. Activation of PI3K could stimulate Akt, and Akt phosphorylation will occur.Two of the Akt phosphorylation sites contain Thr308 and Ser473. Activation of Akt will induce cell proliferation and inhibit apoptosis of cancer cells, which play a role in promoting the progress of cancer. According to this mechanism, Akt inhibitors have been applied to suppress tumor growth and proliferation by inhibiting Akt and its downstream targets. Moreover, Akt activation is often associated with resistance to chemotherapy or radiotherapy. In urogenital tumors, some studies using immunohistochemistry have demonstrated that there was Akt pathway overexpression in bladder cancer tissues. Meanwhile, activation of Akt pathway in bladder cancer has the correlation with tumor progression, and patients with phosphorylatedAkt has reduced survival with a higher risk of mortality. Researchers Kreisberg and Shimizu studied the role of Akt in prostate cancer prognosis. The results showed that Akt and phosphorylated Akt expression were associated with malignant biological features of prostatecance and was an excellent predictor of poor clinical outcome in prostate cancer.As the important role of Akt pathway in tumor, Akt pathway becomes the focus of cancer targeted therapy, and Akt inhibitors are produced with the development of research work. Through blocking the expression of phosphorylated Akt, Akt inhibitors could inhibit the activity of many downstream effectors in Akt pathway, then suppress tumor growth and progression.In this study, we choose MK2206, an effective allosteric Akt inhibitor and it has been in I clinical phase.Akt pathway has been an appealing target for cancer therapy with Akt inhibitors, combined with chemotherapeutic agents. Akt inhibitors combined with chemotherapeutic agents could reach synergistic effect of killing tumor cells, and antoganize chemoresistance and enhance chemosensitivity by inhibiting Akt signaling pathway. However, to our knowledge, there was no data on Akt inhibitor MK2206 combined with cisplatin for the treatment of bladder cancer, and no studies on improving cisplation sensitivity and overcoming cisplatin resistance by inhibiting Akt pathway. In the present research, the researcher got the support from the overseas project of Shongdong University, and examined the important role of Akt pathway in bladder cancer progression, invasion and chemoresistance. Furthermore, we explored the molecular mechanisms in enhancing chemosensitivity. We hope that would provide a novel way for improving the efficiency of combined chemotherapy of urothelial cancers.ObjectivesTo improve conventional chemotherapeutic efficacy, it is important to detect new molecular markers for chemosensitivityand possible accelerating cell-killing mechanisms. In this study, we explored the relationship between the expression of Akt and its downstream targets and bladder cancer progression. Meanwhile, we investigated how MK2206, an allosteric Akt inhibitor, enhances thecisplatin (CDDP)-induced apoptosis and cytotoxicity in urothelial cancer cells.Materials and methods(1) We examined bladder cancer cell lines (including RT112, RT4,253J, J82, T24, UMUC3) for the expression of phospho-Akt (p-Akt) and its downstream targets by Western blot.(2) The potential antitumor effects were analyzed by MTT assay in vitro, and we used MTT to check the inhibition of cell growth of bladder cancder cell lines when they were exposed to MK2206 and CDDP, respectively. Then the IC50 of CDDP was calculated.The expression of Akt pathway targets and apoptosis related proteins were examined by Western blot, when exposed to MK2206 or CDDP. In combination therapy (MK2206+CDDP), the activities of the Akt signaling pathway and expression of apoptosis-relatedproteins were measured by Western blot, and we explored the molecular mechanism about how MK2206 enhanced CDDP-induced bladder cancer cytotoxicity and apoptosis.(3) The cellinvasion of MK2206 was examined by transwell invasion assay, and the activities of invasion related proteins, such as snail, slug and ZEB1, were measured by Western blot. Through this study, we examined the mechanism of MK2206 inhibiting cell invasion.(4) Establishing the xenograft of mice bearing RT112,we injected control (DMSO), CDDP, MK2206 and MK2206+CDDP subcutaneously to check the antitumor effects. Furthermore, Ki67 staining was examined to check the status of cell proliferation.ResultsThe expression of p-Akt and its downstream targets was increased in invasive bladder cancer cell lines vs. in noninvasivebladder cancer cell lines. MK2206 (500 nM) inhibited cell invasion and the expression invasion related proteins in UMUC3 cell line and significantly increased the susceptibility ofbladder cancer cell lines to CDDP. When used in combination with CDDP, MK2206 (500 nM) enhanced CDDP-induced cytotoxicity andapoptosis, with suppressed expression of p-Akt and its downstream targets. In vivo MK2206 combined with CDDP effectively suppressedtumor growth in subcutaneous xenograft models, and the expression of Ki-67 staining was inhibited in combination therapy group.ConclusionsAkt and its downstream targets overexpression correlate with progression of bladder cancer. The concomitant use of MK2206 could promote the CDDP-induced cytotoxicity and apoptosis inurothelial cancer cell lines through the inhibited expression of the Akt pathway. This combined treatment may provide a new therapeuticoption to overcome chemoresistance and enhance chemosensitivity in bladder cancer. |
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