Study On The Synergistic Killing Effect Of Photodynamic Therapy Combined With Pirarubicin On Bladder Cancer Cells And Their Invasive Abilit

Objective:The aim of this study is to investigate the cytotoxic effect and inhibition of invasion and migration of bladder cancer cell lines in vitro through the combination of Hexylaminolaevulinate(HAL)-mediated photodynamic therapy and Pirarubicin.Furthermore,the underlying mechanisms will be elucidated.Method:The bladder cancer cell viability was assessed by CCK-8 assay,Hochest33342/PI double staining,and the Bcl-2、Bax gene expression was assessed by RT-q PCR.A co-culture model of normal urothelial cells SV-HUC-1 and bladder cancer T24 and RT4 cells was constructe,and the wound healing and transwell assays were used to detect the migration and invasion abilities of the RT4 and T24 cells.The expression of EMT markers and integrinα3β1,the levels of MMP-2,MMP9 and Smad2/3 in T24 cells and RT4 cells were measured by Western blot assay.The study will also use combined therapy in bladder cancer organoids to observe the growth of organoids,changes in morphological structure,HE staining,and the expression of integrin integrin α3β1 and N-cadherin.Results:1.When the cells were treated with 12.500ug/ml,10 min of HAL-PDT in combination with 0.500μg/ml or 1.250μg /ml of pirarubicin for 24 h,the cell viability of T24 cells treated with photodynamic therapy,pirarubicin,and combination therapy was 65.79 ± 6.33%,64.42 ± 8.72%,and 27.52% ± 6.46%,respectively.The cell viability of RT4 cells in the corresponding groups was 52.38 ± 2.39%,60.09 ± 3.55%,and 17.72 ± 4.23%,respectively.Further,the down-regulation of P-gp and Bcl-2 gene expression,up-regulation of Bax gene expression were observed.2.In the cell migration and Matrigel invasion assay,both HAL-PDT(12.500μg/ml,5 min)and pirarubicin(0.125μg/ml)suppressed the invasion and migration of RT4 and T24 cells significantly.The OD value of RT4 and T24 cells migrating through the pored membrane of the Transwell cell culture chamber was significantly reduced compared to the control cells both in HAL-PDT-treated cells and in pirarubicin treated cells.In addition,the combined effect of HAL-PDT and pirarubicin was much stronger than the effect of single treatment.Similar results were observed in the Matrigel invasion assay.Moreover,the migrating distance of the cells in each group showed that both HAL-PDT or pirarubicin alone inhibited the wound gap closure.The effect of the combination group was still the most obvious.To further verify the mechanism,Western blot assay was used to determine the key mediators in the migration and invasion process.The results showed a revealed decreasing expression of phospho-Smad2/3,integrin α3β1,N-cadherin,vinment,MMP-2,MMP-9,in PDT or pirarubicin alone and combination groups,and increasing expression of E-cadherin in these groups.There were weaker expressions of phospho-smad2/3,integrin α3β1,N-cadherin,vinment,MMP-2,MMP-9 in the combination group than in the PDT or pirarubicin alone groups,but stronger expressions of E-cadherin.3.The combined treatment was able to suppress the growth of organoids,reduce the maximum size and surface area of the organoids,and make the edges of the organoids rough and defective.The expression of integrin α3β1 and mesenchymal marker N-cadherin was also reduced.Conclusion:Taken together,our findings demonstrated that HAL-PDT combined with pirarubicin could significantly kill bladder cancer cells by inhibiting P-gp protein and Bcl-2 gene,and effectively suppress invasion and migration of bladder cancer cells,which might be ascribed to the reversal of EMT progress as well as the lower expression of MMP-2 and MMP-9 by inhibiting the Smad2/3 pathway.These results suggested that the combination of HAL-PDT and pirarubicin may provide a new and promising therapeutic strategy for bladder cancer recurence.