Effects And Molecular Mechanisms Of The Transcription Factor CRBE In Taxol-induced Anti-tumor Activity

Background Recent reports showed CREB was involved in tumorigenesis. Evidences showed thattranscriptional factor CREB was essential for cell proliferation and apoptosis through regulating a series ofenzyme-linked reaction activated by external stimulis. Further studies showed that cell cycle andcytoskeleton were marked changes in the process of CREB-regulating tumorigenesis, however, themechanisms of CREB in regulting processes needed further exploration.In the present study, we were aimed to explore the cell cycles and cytoskeleton changes in the processof cell proliferation and apoptosis after taxol treatment. Firstly we studied the roles of CREB in regulationof cyclins and CDKs, especially in the processes of taxol-induced cell apoptosis. Secondly, we used theCaSR agonist(Gdcl3)/antagonist(NPS2390) to explore the effects of CaSR on regulation of intracellularcalcium concentration([Ca2+]i) and CREB expression. Thirdly, we further explored the effect of CaSR inregulation of cytoskeleton arrangements and subtype gene expression. Finally, we investigated whetherCREB was involved in mediation the CaSR-regulated cytoskeleton.Methods（1）MTT method was applied to assay the effective treatment concentration and time；（2）With PCR method to construct the recombinant plasmid pCI neo/CREB（PN）and its mutation ofS133A pCI neo/CREB-M（PM）. After transfection into cells, we detected the changes of cyclins,CDKs and cytoskeleton subtype gene expressions；（3）Flow cytometry was applied to detect the cell cycle；（4）Western blot method to detect the expressions of pCREB、CREB、cyclins and CDKs；（5）Immunofluorescence was applied to detect the changes of [Ca2+]i and cytoskeleton arrangement；（6）Use real-time PCR method to detect the cytoskeleton subtype gene expression；（7）Use immunohistochemistry to assay the expression of pCREB.Results（1）Taxol induced cylin A decrease, cyclin B1, D1increase and G2/M phase arrest, accompanied with asignificant decrease of G0/G1and S phase, with a manner of time and concentration-dependent.However, Combined treatment with CREB dominant negative plasmid and PM reversed the cyclin Adecrease, cylcin B1and D1increase, and ultimately dimished the G2/M arrest.（2）Taxol induced a significant increase of [Ca2+]i, which was accompanied with a rigid and bundle ofMTs around the nucleus, and a marked assemble of MFs in the inner membrane. Further studyrevealed that taxol induce a significant decrease of β-actin, γ-actin, α-tubulin and β-tubulin subtypegenes expression, however, these phenomena were reversed/enhanced by addition of NPS2390/Gdcl3.In addition, PM reversed the effect of Gdcl3-inhibited γ-actin, α-tubulin and β-tubulin expression.（3）Taxol induced a significant increase of pCREB in HeLa cells, with a CaSR activity-dependent manner. In addition, pCREB has a higher expression in tumor tissues/cells than normal.Conclusion（1）Taxol-decreased cyclin A, and increased cyclin B1and cyclin D1expression were mediated byphosphorylation of CREB, finally induced HeLa cells G2/M arrest, accompanied with a significantdecrease of G0/G1and S phase.（2）CaSR was involved in regulation of taxol-induced cytoskeleton arrangement and subtype genechanges, however, this process was mediated by transcription factor CREB.SignificanceThis study illustrated the mechanisms of CREB in regulation of cancer cells proliferation andapoptosis, according to results of CREB-regulated cell cycle, cytoskeleton arrangement and its relatedprotein in apoptotic cells. These findings will provide important evidences on CREB-mediated targettherapy of cancer.