Analysis Of The Efficacy Of Different Targeted Microtubule Chemotherapeutics Mediated By Op18/stathmin In NSCLC Cells

Objective:Taxol(paclitaxel)and vinblastine(VBL)are both efficacious chemotherapeutic agents that target the microtubules of tumor cells,but each functions in a mutual antagonistic manner.Op18/stathmin is a small molecular phosphoprotein which promotes depolymerization of microtubules,has a close relationship with the tumor malignant phenotypes maintaining and the sensitivity of chemotherapeutics.Non-small cell lung cancer(NSCLC)NCI-H1299 cells were employed to compare the curative effects of VBL and Taxol and explore the correlation between drug sensitivity and Op 18/stathmin signaling.Methods:The NSCLC NCI-H1299 cell lines were chosen to analyze the differences of sensitivity and regulation of Op 18/stathmin signaling for induction of Taxol or VBL.NCI-H1299 cells were treated with Taxol and VBL at the same concentration,and DMSO was used as control.Flow cytometry was used to detect the apoptotic ratios of the treated NCI-H1299 cells and the expressions of caspase 3,8,9 were detected by westernblot.MTT assays and colony formation assays were detected to analysis the cell proliferation viability.The cell migration in a 2-dimensional plane and 3-dimensional space was detected through wound healing assays and transwell assays.To explore the difference of molecular mechanisms on Op 18/stathmin signaling mediated by Taxol or VBL,the expression of Op 18/stathmin and the total phosphorylation of Op18/stathmin as well as the phosphorylation of four serine sites were examined.Meanwhile,the cell proliferation was tested after silencing the expression of Op18/stathmin by target siRNA.The expression of PP2A(Protein phosphatase 2A),NF-?B(Nuclear Factor ?B),Bcl-2(B-cell lymphoma-2)and IL-10(interleukin-10)in the tumor cells and the autocrine of IL-10 were also detected.The association between the expression of Op18/stathmin and drug sensitivity was compared between the two different non-small cell lung cancer cells.Results:The cells were treated with Taxol or VBL for 24 h,and the solvent DMSO was used as control.Cell apoptotic ratios were 8.65,14.95 and 22.66%following treatment with the solvent(DMSO),Taxol and VBL,respectively,in the NCI-H1299 cells as detected by FCM.Westernblot analysis showed that both Taxol and VBL increased the expression levels of caspase 3 and 9,but to a greater extent following VBL treatment,while no obvious changes in caspase 8 were observed in the three treatment groups.By contrast with DMSO,the relative cell proliferation percentages were 84.24,55.11 and 32.00%,respectively,for VBL treatment at time points of 24,48 and 72 h,while these percentages were 95.86,91.47 and 86.46%,respectively,following Taxol treatment.Statistical differences existed in the proliferation percentages of each group(P<0.01).The mean colony formation efficiency was 11.60,9.47 and 1.07%,respectively,in the DMSO,Taxol and VBL treatment group(P<0.05).The scratch-wound trails were completely recovered at time point of 36 h in the DMSO treatment group,while the scratched areas nearly healed for Taxol induction.However,a large area of blank region still remained in the cells treated with VBL.Transwell assay analysis revealed that the mean number of invading cells was 27,16 and 8,respectively,in the DMSO,Taxol and VBL treatment group(P<0.01).Exploring the correlation between drug sensitivity and Op 18/stathmin signaling,we found that the expression of Op 18/stathmin was decreased by Taxol,but was not affected by VBL.IP-westernblot showed that Taxol decreased the phosphorylation of Op 18/stathmin,however,VBL upregulated the total level of phosphorylated Op 18/stathmin.Meanwhile,VBL universally increased the level of phosphorylated Op18/stathmin at all 4 serine sites including Ser63,Ser38,Ser25 and Ser16,while Taxol mainly reduced phosphorylated Op 18/stathmin at Ser25 and Ser63 sites.Op 18/stathmin RNAi reduces the difference in cell proliferation inhibition between VBL and Taxol.The regulation mechanism of Taxol and VBL on Op 18/stathmin signal was further studied.PP2A is an important serine/threonine phosphatase acting to dephosphorylate Op 18/stathmin.The present study showed that the expression of PP2A was increased by Taxol,but decreased by VBL,while both Taxol and VBL inhibited the expression levels of NF-?B,Bcl-2 and IL-10 as well as autocrine IL-10 in NCI-H1299 cells,but the inhibitory effects were more obvious for VBL than for Taxol.The correlation between the expression of Op18/stathmin and the drug sensitivity in different non small cell lung cancer cell lines was analyzed.Western blot analysis demonstrated that NCI-H1299 cells highly expressed Op 18/stathmin compared with the A549 cells.FACS showed that cellular apoptotic percentages were 6.07,13.47 and 21.81%,respectively,in the NCI-H1299 cells in the DMSO,Taxol and VBL treatment groups,while they were 6.95,21.59 and 19.21%respectively in A549 cells in the corresponding groups.NCI-H 1299 cells presented notable resistance to Taxol when compared with VBL,but the sensitivity of Taxol was similar to VBL in the A549 cells.Conclusions:NCI-H 1299 cells with high expression of Op 18/stathmin are more sensitive to VBL than to Taxol.High expression of Op 18/stathmin was found to be negatively correlated with the sensitivity of Taxol in the NSCLC cells,but had a minor impact on VBL cytotoxicity.These findings revealed that both VBL and Taxol induce cell apoptosis through Op 18/stathmin,but the mechanisms are completely different.VBL is an attractive alternative to the treatment of Taxol-resistant tumors with high expression of Op 18/stathmin.