| Micro-enviromental hypoxia is a characteristic feature of most solid tumors and has been associated with poor treatment response. The microenvironment of rapidly growing tumors is associated with increased energy demand and diminished vascular supply, resulting center areas of prominent hypoxia. Hypoxia inducible factor-1 (HIF-1) is the major transcription factor and key regulator of adoptive responses to hypoxia. During hypoxia cells undergo a variety of adoptive responses including activation of signaling pathways, which promote cell survival, especially in the sensitivity for chemotherapy. It was reported that hypoxia decreased the efficacy of chemotherapeutic drugs in solid tumors. In our study, we show that CoCl2 mediated mimic hypoxia induce some multidrug resistant genes and some antiapopotic factors change a lot,compared with the normoxic cells. So, further study to illuminate the exact mechanism of HIF-1 in hypoxia-induced drug resisitence and anti-apoptosis will provide us some new target moleculars for target treatment in human colon cancer treatment.Objective: The purposes of our research have three aspects. The first is to study the proliferation and the chemotherapeutic drug sensitivity under micro-enviromental hypoxia. The second is to exploit the mechanism of hypoxia-induced drug resistance and anti-apoptosis. The third is to discuss the realationship between hypoxia and the human colon cancer therapy.Methods:1 The hypoxia model was set up by the usage of cobalt chloride(CoCl2) and MTT assay was used to observe the hypoxia of Lovo cell growth curve;2 Human colon cancer cell exposed to hypoxia, MTT assay was used to observe the sensitivity of treated cells toward chemotherapeutic drug;3 RT-PCR was applied to examine the expression of HIF-1α, Mdr1, MRP, BCRP and LRP mRNA in hypoxia;4 Human colon cancer cell exposed to hypoxia, flow cytometry (FCM) Adriamycin accumulation and retention were performed to analyze intracellular drugs transport;5 Under different hypoxia condition, HIF-1αand The pro- and antiapopotic members of the Bcl-2 protein family: Bcl-2, Bax and Bad were investigate by western-blot.Results:1 MTT assay was found that Lovo cell proliferared at a slow rate as the concentration of CoCl2 increasing;2 Under hypoxia,at the concentration of 100μmol/L,150μmol/L and 200μmol/L CoCl2 human colon cancer Lovo cell had a strong resistance to FU with increase of CoCl2 compare with normoxia group(p<0.05), but 250μmol/L made Lovo cell more sensitive to FU, compared with normoxia group(p<0.05);3 Semiquantitative RT-PCR show that the up-regulated expression of mdr1, MRP and BCRP mRNA was consistent with dose-effect curve with the same time(p<0.05),but the mRNA expression of HIF-1a and LRP remained at the same level;4 Lovo cells represent a decrease retention of ADR under hypoxic, but the accumulation have no different between hypoxia and normoxia;5 Western-blot results reveal that hypoxia increased HIF-1a protein expression, however decreased Bax and Bad protein expression, compare with normoxia group(p<0.05).Conclusion:1 The hypoxia induced by CoCl2 can inhibit the proliferation of human colon cancer Lovo cells;2 The appropriate hypoxia can decrease the sensitivity of the cell to FU;3 Under the hypoxia condition there are two aspects to decrease the sentivity of chemotherapy;3.1 HIF-1a play a important role in drug resistance by up-regulating mdr1, BCRP and MRP, by which decreases the retention of chemotherapeutic drug;3.2 Under hypoxia HIF-1a mediate the resistance of apoptosis by decreasing Bax and Bad expression, which is a possible mechanism of escaping form apoptosis mediate by chemotherapeutic drug. |
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