Maspin enhances cisplatin chemosensitivity in bladder cancer T24 and 5637 cells and correlates with prognosis of muscle-invasive bladder cancer patients receiving cisplatin based neoadjuvant chemotherapy

Abstract

Background: Maspin, a non-inhibitory member of the serine protease inhibitor superfamily, has been characterized as a tumor suppressor gene in multiple cancer types. Chemotherapeutic insensitivity is one of major obstacles to effectively treating muscle invasive bladder cancer (MIBC). This study was conducted to investigate the role and probable mechanism of Maspin enhancing cisplatin chemosensitivity of bladder cancer in vitro and MIBC patients.

Methods: Maspin expression was quantified by qRT-PCR in two MIBC cell lines (T24 and 5637). After successful established Maspin overexpression model by lipidosome transfection, MTT and cell apoptosis assay were used to assess the MIBC's cisplatin sensitivity. Western blot method was used to test PI3K/ AKT/mTOR signal passway and apoptosis related molecules Caspase3 and Bcl-2. Additionally, we evaluated Maspin expression and prognosis in 62 MIBC cases who underwent cisplatin based neoadjuvant chemotherapy (NACT) using immunohistochemistry.

Result: Upregulate Maspin expression could enhance the chemosensitivity induced by cisplatin in T24 and 5637 cell lines. The cell viability, cloning ability and IC50 were reduced while apoptosis rate was upregulated when cells were transfected Maspin. Phospho(p)-AKT, PI3K, mTOR, and Bcl-2 expression were significantly decreased, whereas Caspase3 was greatly increased in the Maspin group. In the clinic study, there was significant correlation between Maspin expression and overall survival (OS) and progression-free survival (PFS) rate in MIBC patients who received cisplatin based NACT.

Conclusion: Maspin could enhance cisplatin chemosensitivity in T24 and 5637 cell lines. Its expression correlated with prognosis of MIBC patients who received cisplatin based neoadjuvant chemotherapy.

Fig. 1

The protein level of Maspin in bladder cancer T24, 5637 and normal bladder epithelial SV-HUC-1 cell lines. Data were shown in the grayscale (a) and bar chart (b), respectively. Maspin expression was significantly decreased in bladder cancer T24 and 5637 cell lines as compared the control group SV-HUC-1. *P < 0.05. Three independent experiments were performed (n = 3)

Fig. 2

Expression levels of Maspin post-transfection in bladder cancer T24 and 5637 cells. The mRNA and protein levels of Maspin in bladder cancer T24 and 5637 cells transfected with pcDNA3.1-Maspin or negative control (pcDNA3.1-con) were detected by RT-PCR and western blot. a and b were showed mRNA level of T24 and 5637 cells, respectively. And c and d were showed protein expression level. *P < 0.05. Three independent experiments were performed (n = 3)

Fig. 3

Effects of Maspin on proliferation and sensitivity to cisplatin of T24 and 5637 cells. a was showed the cell viability with different cisplatin measured by MTT assay. b was showed the IC50 value in each group. Cloning efficiency was calculated after cultured for 2 weeks. c and d showed the representative results of colony formation assay. Overexpression of Maspin dramatically decreased the cell viability, IC50 value and clone-formation, respectively. *P < 0.05. Three independent experiments were performed (n = 3)

Fig. 4

Effects of Maspin on cisplatin-induced cell apoptosis in T24 and 5637 cells. Apoptotic cells were detected by Annexin V-FITC and PI double staining and analyzed by flow cytometry. a and b were showed maspin overexpression enhanced cell apoptosis induced by cisplatin in T24 and 5637 cells, respectively. *P < 0.05. Three independent experiments were performed (n = 3)

Fig. 5

Western blotting analysis of proteins related to apoptosis in T24 and 5637 cells. a was showed AKT, p-AKT, PI3K, mTOR, Caspase3 and Bcl-2 expression in T24 and 5637 cells measured by immunoblotting. And β-actin was used as an internal control. b and c were showed the relative protein levels normalized to β-actin in T24 and 5637 cells, including Con, NC and Maspin group, respectively. AKT expression did’t have significant changes between each three group (P > 0.05). p-AKT, PI3K, mTOR, and Bcl-2 expression were significantly decreased, whereas Caspase3 was greatly increased in the Maspin group. *P < 0.05. Three independent experiments were performed (n = 3)

Fig. 6

Immunohistochemistry of bladder cancer tissue with Maspin antibody. a showed negative(−) stain of Maspin in MIBC sample. b showed positive(+) stain of Maspin in MIBC sample. c showed bladder cancer cells invaded into micro-vessels. d showed the lymph node metastasis of bladder cancer cells. (a, b, c and d, magnification, ×400)

Fig. 7

The relationship between Maspin staining and survival rate in MIBC patients receiving NACT plus RC-PLND. a and b showed Kaplan-Meier survival curves of the OS and PFS in the 62 patients, respectively. The results indicated Maspin expression was related to OS and PFS (P = 0.0036 and 0.0005, respectively)