Abstract
Chemoresistance to cisplatin is a principal cause of treatment failure and mortality of advanced bladder cancer (BC). The underlying mechanisms remain unclear, which hinders the development of preventive strategies. Recent data indicate that pyruvate kinase M2 (PKM2), a glycolytic enzyme for Warburg effect, is strongly upregulated in BC. This study explores the role of PKM2 in chemoresistance and whether inhibiting PKM2 augments the chemosensitivity to cisplatin and reduces BC growth and progression. We found that Shikonin binds PKM2 and inhibits BC cell survival in a dose-dependent but pyruvate kinase activity-independent manner. Down-regulation of PKM2 by shRNA blunts cellular responses to shikonin but enhances the responses to cisplatin. Shikonin and cisplatin together exhibit significantly greater inhibition of proliferation and apoptosis than when used alone. Induced cisplatin-resistance is strongly associated with PKM2 overexpression, and cisplatin-resistant cells respond sensitively to shikonin. In syngeneic mice, shikonin and cisplatin together, but not as single-agents, markedly reduces BC growth and metastasis. Based on these data, we conclude that PKM2 overexpression is a key mechanism of chemoresistance of advanced BC to cisplatin. Inhibition of PKM2 via RNAi or chemical inhibitors may be a highly effective approach to overcome chemoresistance and improve the outcome of advanced BC.
Highlights
Bladder cancer (BC) or urothelial carcinoma of the bladder is the fourth most prevalent cancer in men and the costliest cancer to manage[1,2]
With the equal amounts of total protein input as illustrated by Western blotting of GAPDH (Fig. 1A, upper panel), shikonin pulled down, upon SDS-PAGE and silver-nitrate staining, a 55-kDa protein species from T24 cells stably expressing a non-specific, control shRNA, but not from T24 cells stably expressing an shRNA of pyruvate kinase M2 (PKM2) (Fig. 1A, middle panel)
An independent pull-down experiment reproduced the results (Fig. 1B, left panel), and further showed that shikonin did not pull down PKM1, MAPK or AKT, even though these proteins were present in the protein input (Fig. 1B, right panel)
Summary
Bladder cancer (BC) or urothelial carcinoma of the bladder is the fourth most prevalent cancer in men and the costliest cancer to manage[1,2]. At the concentration that markedly inhibited cell proliferation (0.5 μM; panel C), shikonin did not significantly affect the pyruvate kinase activity (E). Using an unbiased proteomic profiling we found PKM2 to be a principally upregulated protein during urothelial tumor formation in low-grade non-invasive pathway of BC in transgenic mice expressing an activated HRAS19. We subsequently extended this finding into human BC cell lines and tumors, establishing PKM2 overexpression in both low-grade non-invasive and high-grade invasive human BC19. The present study was designed to test this idea by inhibiting PKM2 alone or in combination with conventional chemotherapeutic approach using in vitro and in vivo models
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