Enhancement Of Telomerase And NF-Kappa B Activation In Vincristine-resistant Human Gastric Cancer Cells

Background and aims: Gastric carcinoma, one of the most common tumors in China, shows little or no response to the chemotherapy. One important reason is the emergence of drug resistance, which usually presents as multiple drug resistance (MDR). The occurrence of MDR greatly reduces the effect of antineoplastic agents and often leads to the failure of chemotherapy. It has been known the mechanism of MDR involves the upregulation of some MDR genes and alteration of enzyme targeted by antineoplastic agents. But it is still difficult to explain the complexity of MDR clearly. Resent studies showed telomerase and NF-kappa B (NF-kB) might play important roles in acquirement of the drug resistance. Telomerase is a ribonucleoprotein complex that adds telomere repeats at the end of chromosomes, which participates in cell proliferation and immortalization through stabilization of chromosomal structure. Human telomerase reverse transcriptase (hTERT) has been generally accepted as the major determinant of human telomerase activity. Telomerase is highly expressed in the majority of human tumors, but low and difficult to detect in somatic cells. People had found the levels of telomerase activity might correlate with the invasive capability of carcinomas. Moreover cancer tissues and cell lines with high level of telomerase activity showed little response to antineoplastic agents, which strongly suggested there was possible relationship lying between telomerase activity and chemosensitivity. NF-kB is an important family of dimeric transcription factors with p65/p50 as the most common member. In resting cells, NF-kB is localized in the cytoplasm and associated with the inhibitory protein IkB. Upon stimulation, IkB degrades and NF-kB migrates into the nucleus. Then NF-kB binds with the target genes and regulates their functions. Recent studies have shown the role of NF-kB gene products on cell transformation and tumor development and NF-kB has been found constitutively activated in several tumors. People also found NF-kB activation could inhibited the apoptosis induced by chemotherapy and radiotherapy through upregulating the anti-apoptotic gene at transcription level. Sequence analysis showed the promoter of hTERT contained several binding sites for transcription factors, such as Sp1 AP-2. So it is interesting to known whether NF-kB involves in the regulation of hTERTand telomerase activity. Up to now, however, there is little information about the roles of telomerase and NF-kB in gastric cancer resistant to vincristine (VCR). So we undertook the present study to investigate whether telomerase and NF-kB activation involved in the development of VCR-resistance in human gastric cancer cells and whether telomerase activation was mediated by NF-KB-dependent way. In the field of MDR reversal, investigations on NF-kB inhibitors are few and there is lack of study on telomerase inhibitors. So we also investigated the effect of MG-132 (an inhibitor for NF-kB) and AZT (an inhibitor for telomerase) on the sensitivity of vincristine-resistant human gastric cancer cells and the parent sensitive clone.Methods: (1) Vincristine-resistant human gastric cancer SGC7901 cells (SGC7901/VCR) and the parent sensitive clone (SGC7901) were grown with routine cell cultivation and grown for 36-40h before treatments. Both cells were treated with VCR, MG-132 (pretreating for 30 min before adding VCR) or AZT (pretreating for 90 min before adding VCR), respectively. (2) The sensitivity of both cells to VCR was determined by the MTT assay. Cell survival and resistance index (RI) were determined by the following formula respectively: Cell survival (%) = (OD value of sample) / (OD value of control) x100% and RI = IC50 (SGC79Ol/VCR)/IC50(SGC7901). (3) Telomerase activity was measured using the telomeric repeat amplification protocol ELIS A kit and the activity was expressed as an OD value. (4) NF-kB-DNA binding activity was determined by electrophoretic mobility shift assay (EMSA). Intensities of bands were quantified using phosphorimager...