Anti-tumor Activity And Mechanism Of Gemcitabine Derivative SZY-200 On Human Bladder Cancer Cells

Gemcitabine is a hydrophilic pyrimidine nucleoside anticancer drug,which plays an anti-tumor role by interfering with DNA synthesis,and it is a first-line drug for the treatment of non-small cell lung cancer,pancreatic cancer and bladder cancer etc.However,its instability,drug resistance and toxicity severely limit its utilization in clinics.Therefore,it is urgent for researchers to further modify and design novel gemcitabine derivatives.In order to facilitate gemcitabine uptake and prolong retention in cells,various derivatives containing fatty acid side chains have been developed,such as CP-4126,which has completed phase II clinical trials.In this paper,we studied the anti-tumor effect and mechanism of a new derivative of gemcitabine named SZY-200 on bladder cancer cells,SZY-200 was synthesized by introducing the moiety of lauric acid(a saturated medium-chain fatty acid)at 5’ position on the sugar moiety of gemcitabine.Firstly,we used gemcitabine and CP-4126 as controls to evaluate the effect of SZY-200 on the proliferation of various cells in vitro using CCK-8 assay.Our data showed that SZY-200 could significantly inhibit the proliferation of a variety of cancer cells in a dose-dependent manner and has a certain selectivity to normal cells,the three drugs showed similar cytotoxic effects at the same dose and the human bladder cancer cells were more sensitive to these three drugs;Moreover,we found that elaidic acid,one of the hydrolyzed products of CP-4126,and lauric acid produced by hydrolysis of SZY-200 have no significant difference on normal cells,but clonogenic survival assay showed that lauric acid could significantly inhibit the proliferation of UM-UC-3 cells in a dose-dependent manner,but elaidic acid has no inhibitory effect;Subsequently,qPCR analyses showed the differential expressions of hENT1 mRNA in different cells,and the expression levels were basically consistent with the sensitivities of the cells to these three drugs.After adding nucleoside transporter inhibitors(NBMPR),we found that SZY-200 and CP-4126 were independent of the membrane transport system,that is,they did not rely on hENT1 to help them enter bladder cancer cells.Next,we explored the anti-tumor mechanism of SZY-200 in the bladder cancer cells.PI staining showed that SZY-200,gemcitabine and CP-4126 could induce cycle arrest of bladder cancer cells;By Hoechst 33258 staining,it showed that the DNA of bladder cancer cells was concentrated and densely stained after treatment with the three drugs,and the nuclei were deformed,concentrated,fragmented to form apoptotic bodies,and the numbers of apoptotic bodies were increased with the doses of the three drugs;Annexin V-FITC/PI staining showed that the three drugs induced the comparable apoptosis effects;Western Blotting confirmed that three drugs affected the expressions of cell cycle and apoptosis-related proteins.These data suggest that the three drugs could inhibit the proliferation of bladder cancer cells by inducing cell cycle arrest and apoptosis.In addition,the results showed that three drugs could significantly reduce the migration abilities of bladder cancer cells.Finally,we used the BATMAN-TCM database to predict the target genes of gemcitabine and lauric acid,we found that the target genes of lauric acid,PPARG and PTGS2,were related to the occurrence and development of bladder cancer;The GEPIA database was used to explore the biological effects of the two genes in bladder cancer.Real time PCR analyses showed that the three drugs could downregulate the expressions of the two genes in bladder cancer cells.The reason may be that these genes are located downstream of the target genes of gemcitabine.In summary,these results indicated that the inhibitory effects of SZY-200 on the proliferation of bladder cancer cells are comparable to gemcitabine and CP-4126,additionally,similar to CP-4126,SZY-200 is expected to overcome gemcitabine resistance induced by low hENT1 expression.We reported for the first time that SZY-200 could inhibit the proliferation of bladder cancer cells by inducing cell cycle arrest and apoptosis.Overall,SZY-200 has the same or more advantages as CP-4126 and will be an ideal preclinical drug for further in vivo investigation.