Cep70 drives taxol drug resistance through regulating the acetylation α-tubulin to reduce microtubule stability

Abstract

Objective To explore the Cep70 by adjusting the stability of acetylated alpha tubulin, participate in breast cancer drug resistance mechanisms. Methods ⑴ In order to induce taxol drug resistance cell line Michigan cancer foundation-7 (MCF-7)/pac, high-dose shock treatments taxol MCF-7 was used for 6 months, until the cells can grow in 3.5 μmol/L of paclitaxel. ⑵ The 3-(4, 5-dimenthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method was used to detect inhibition rate by taxol to MCF-7 and MCF-7/pac cell. ⑶ Immunofluorescence and Western blot were used to test acetylated alpha-tubulin and Cep70 expression levels in MCF-7 and MCF-7/pac cells. ⑷ Chemical intervention was used to acetylate apha-tubulin expression, Western blot and polymerase chain reaction (PCR) were used to detect the change of acetylated alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac groups. Flow cytometry and Western blot were used to detect the change of cell cycle. Results ⑴ IC50 of MCF-7 and MCF-7/pac was 22.47 μmol/L and 31.38 μmol/L, respectively. ⑵ Immunofluorescence and Western blot results showed that the expression of acetylation of alpha-tubulin in resistant MCF-7 cell/pac was obviously decreased. ⑶ Real time polymerase chain reaction (RT-PCR) and Western blot showed Cep70 expression was consistent of acetylation of alpha-tubulin. ⑷ After incubation with paclitaxel for 24 hours, the expressions of acetylation of alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac were increased, but the extent of MCF-7 cell was much higher. Instead, incubation with nocodazole after 24 hours, the acetylation of alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac cells were obviously lowered. ⑸ After paclitaxel intervention, compared to the same group MCF-7 cells, the G2 phase ratio in MCF-7/pac cells was lower. In addition, given nocodazole after the intervention, compared to the same group MCF-7 cells, the ratio of G2 phase in MCF-7 cell/pac was significantly decreased. Conclusions Cep70 decreased the expression of the acetylated alpha-tubulin, reduced the stability of microtubules, which could be an important mechanism of taxol drug resistance. Key words: Centrosome; Tubulin; Breast neoplasms/DT/ME; Taxoids; Drug resistance, neoplasm