ABSTRACT
Objective@#To investigate the effect of ubiquitous mitochondrial creatine kinase 1(CKMT1) on the sensitivity of human nasopharyngeal carcinoma cell line CNE-1 to DDP. @*Methods@#CNE-1 cells were transiently transfected with CKMT1 overexpression (CKMT1) or empty vector (EV). The growth curve and DDP IC50 were developed by MTT assay, plate clone formation assay was performed by gradient concentration of DDP treatment, cell cycle and apoptosis were detected by flow cytometry, levels of apoptosis related protein Bax/Bcl-2/C-PARP and the transcription factor p-STAT3-Tyr705 were detected by Western Blot. @*Results@#The transfection efficiencies of CKMT1 and EV were more than 90% with a higher proliferation rate in the CKMT1-transfected cells. However, the CKMT1-transfected cells had a DDP IC50 of 2.76 μmol/L, which was significantly lower than that of 4.60 μmol/L in the EV-transfected cells (P<0.01). With the treatment of certain concentration of DDP, the CKMT1-transfected cells had a lower clone formation rate, the cell cycle arrested more obviously in G2/M phase, and the apoptosis rate was higher (P<0.01), with higher levels of Bax/C-PARP (P<0.05 or P<0.01), but lower levels of Bcl-2 (P<0.01) and p-STAT3-Tyr705 (P<0.01), compare with the EV-transfected cells. @*Conclusions@#CKMT1 may inhibit the activation of STAT3, increasing the sensitivity of CNE-1 to chemotherapeutic drug DDP.
ABSTRACT
Objective:To clone the full-length cDNA of human ubiquitious mitochondrial creatine kinase(uMtCK) and express its protien in E. coli. The protein was used to immunize rabbit to prepare anti-uMTCR polyclonal antibody which was then used to detect the expression of uMtck in gastrointestinal carcinoma. Methods: A 1 062 bp segement was amplified from human HaLa cell by RT-PCR,which was then inserted into pMD18-T plasmid and proved to be the coding sequence of uMtCK by endonuclease digestion and sequencing. The coding sequence of uMtCK was inserted into pQE30 plasmid and recombinant plasmid pQE30-uMtCK was transformed into E. coli. The expressed protein was purified with a His. Bind column and was analyzed with SDS-PAGE and Westen blot and the activity of uMtCK was tested. An antiserum against uMtCK was perpetrated by immunizing rabbit with purified uMtCK. The expression of uMtCK in 59 patients with gastric and colonic cancer was detected by immunohistochemistry. Results:The coding sequence of uMtCK was successfully obtained by RT-PCR and the product was successfully inserted into pQE30 to construct a recombinant vector pQE30-uMtCK. The expression of uMtCK was effective and soluble, A good specific antiserum against uMtCK was proved by Western blot. The expression of uMtCK of gastric and colonic cancer was 76. 5%. Conclusion:The coding sequence of uMtCK is coined and expressed in E.coli. uMtCK is a prospective marker for the diagnosis of gastric and colonic cancer.