Lobaplatin induces apoptosis in T24 and 5637 bladder cancer cells by regulating Bcl-2 and Bax expression and inhibiting the PI3K/Akt signaling pathway

Abstract

Background: Lobaplatin (LBP) is a third-generation platinum-based drug that has been approved only in China for the treatment of several cancer types. Nonetheless, its efficacy in treating bladder cancer (BC) is unclear thus far. Through in vitro and in vivo experiments, this study aimed to explore whether LBP has an antitumor effect on T24 and 5637 BC cells and whether the effect is related to B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and regulation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway.

Methods: For in vitro experiments, the cell counting kit-8 (CCK-8) method was used to determine how different concentrations of LBP affect the viability of two types of BC cells. A wound healing assay was used to test the inhibitory effect of LBP on the migration of the two cell lines. Annexin V-fluorescein isothiocyanate isomer I (V-FITC)/propidium iodide (PI) staining was used to detect changes in cell apoptosis before and after LBP treatment, and Western blotting was used to detect the expression of apoptosis-related proteins and PI3K/Akt pathway proteins. For in vivo experiments, a cell-derived xenograft (CDX) model was employed, and the weight of nude mice and the tumor size were measured. Immunohistochemistry was used to detect the effect of LBP on the expression of apoptosis-related proteins in tumor xenografts.

Results: In vitro, LBP reduced proliferation (P<0.05), inhibited migration (P<0.05), and induced apoptosis in T24 (31.25%±1.20%, P<0.01) and 5637 (14.3%±2.24%, P<0.05) BC cells, in a dose-dependent manner (P<0.05); increased the expression of proapoptotic proteins, including Bax, caspase-3 and cleaved caspase-3 (P<0.05); and suppressed the expression of antiapoptotic proteins, including Bcl-2, PI3K, Akt and phosphorylated Akt (p-Akt). The in vivo experiment confirmed that LBP can reduce the size of subcutaneous tumors in nude mice (P<0.05), increase the expression levels of Bax and cleaved caspase-3 and lower the expression of Bcl-2 (P<0.05) in bladder tumor tissue.

Conclusions: The results obtained from both experiments suggest that LBP can inhibit the proliferation of T24 and 5637 BC cells, which might be credited to its effects in regulating Bcl-2 and Bax expression and inhibiting the PI3K/Akt pathway.

Keywords: Lobaplatin (LBP); apoptosis; bladder cancer (BC); phosphoinositide 3-kinase/protein kinase B pathway (PI3K/Akt pathway).

Figure 1

Effects of different concentrations of LBP on T24 and 5637 cell viability after 24, 48 and 72 hours. Compared with the control group, *, P<0.05, **, P<0.01. LBP, lobaplatin.

Figure 2

Effect of LBP on cell (T24 and 5637) migration ability. Compared with the control group, *, P<0.05, **, P<0.01. LBP, lobaplatin. 40×, microscopic observation.

Figure 3

Flow cytometry was used to detect the effect of LBP on the proportion of apoptotic cells. *, P<0.05; **, P<0.01, compared with the control group (the Y-axis is PI and the X-axis is annexin-V FITC). LBP, lobaplatin; PI, propidium iodide; FITC, fluorescein isothiocyanate isomer I.

Figure 4

LBP regulates bladder cancer cell growth through the PI3K/AKT signalling pathway. (A-C) The effect of LBP on the expression of apoptosis-related proteins and PI3K/Akt pathway proteins in T24 cells. (D-F) The effect of LBP on apoptosis-related proteins and PI3K/Akt pathway expression in 5637 cells. *, P<0.05; **, P<0.01, compared with the control group. LBP, lobaplatin; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

In vivo efficacy of LBP treatment in nude mouse models of T24 and 5637 tumour xenografts. (A,B) Photo of the tumors removed from mice in the two experimental groups after three weeks. (C) Body weight curves of T24 tumor xenografts in the nude mice in the two groups. (D) Body weight of 5637 tumor xenografts in the nude mice in the two groups. (E) Comparison of the tumor volume of T24 tumor xenografts in the two groups of nude mice. (F) Comparison of the tumor volume of 5637 tumor xenografts in the two groups of nude mice. Scale bars, 10 mm. *, P<0.05; **, P<0.01. LBP, lobaplatin.

Figure 6

IHC staining images of the bladder tumor tissue in mice and analyses. Effect of LBP on the expression of Bax, Bcl-2 and cleaved caspase-3 in tumour sections using immunohistochemical staining of tissue wax blocks (×200). (A,B) The effect of LBP on the expression of apoptosis-related proteins in T24 metastatic tumors; (C,D) the effect of LBP on the expression of apoptosis-related proteins in 5637 metastatic tumors. *, P<0.05; **, P<0.01. LBP, lobaplatin; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2.