| [AIM] This investigation aims at approaching the existence of micro-environmental hypoxia in human pancreatic carcinoma tissues, and the correlations and molecular mechanisms with the drug resistance to Gemcitabine during chemo-therapy, proving if targeting hypoxia and HIF-1a could reverse the resistance, and finally apply a new theoretical way for the iateria of human pancreatic carcinoma.[ METHODS ] 34 tumor samples from patients diagnosed with pancreatic adenocarcinoma were stained with immunohistochemical staining to observe the expression of HIF-1a, P-Gp, Bcl-Xl and Bax protein, and then the correlation between HIF-1a and P-Gp, Bcl-Xl and Bax was analyzed. The expression of HIF-la in tumor tissues was compared with that in normal pancreatic tissues as well. Resistance to Gemcitabine was measured by IC50 with MTT assay in human pancreatic carcinoma cell lines SW1990 and Panc-1, and the cell apoptosis and cell cycle analyses were measured by flow cytometry by PI and PI combined Annexin-V detections and the expression of HIF-1a, mdr1/P-Gp, Bcl-Xl, and Bax were detected by RT-PCR and western-bolt assays under normoxia, hypoxia and YC-1-added circumstances in vitro.[RESULTS] The positive rate of HIF-la in tumor tissues was 76.4 %( 26/34), comparing a totally negative expression in normal pancreatic tissues. There were significant positive correlations between the expression of HIF-1a and P-Gp, Bcl-Xl, Bax (P=0.005, P=0.009, P=0.002). Under hypoxia atmosphere, there were certain micro-structural changes occurred which might have certain relations with the molecular changes under hypoxia circumstance. The IC50 of SW1990 and Panc-1 under hypoxia atmosphere were 85.050±3.407μg/ml and 208.805±15.882μg/ml respectively, compared with 4.896±0.051μg/ml and 51.727±3.152μg/ml under normoxia atmosphere, with 17-times-fold and 4-times-fold increasing. Flow cytometry indicates apoptosis activation in SW1990 and inhibition in Panc-1, and G2/M accumulation in SW1990 and G1/S arrest in Panc-1 in cell cycle analyses.When YC-1 was added into each group with a final concentration of 20μmol/L, both cell lines showed a partially decrease of IC50 to Gemcitabine, resulting in 37.218±3.690μg/ml in SW1990, and 164.002±11.057μg/ml in Panc-1. With thehypoxia time extensions, both cell lines show time-dependent up-regulation of mdr1/P-Gp, Bcl-XL, and Bax in both mRNA and protein levels, and up-regulation of HIF-la in protein levels, but in SW1990 the transcription and translation levels of Bcl-Xl remain the same as normoxia. And the mdrl, Bcl-Xl, and Bax mRNAs and proteins were down-regulated significantly when YC-1 was added. [CONCLUSIONS] There is a definitely significant over-expression of HIF-la, which means there is micro-environmental hypoxia in human pancreatic adenocarcinoma tissues.The micro-environmental hypoxia may leads to the resistance to Gemcitabine in human pancreatic carcinoma cell lines, and the mechanisms may include hypoxia-mediated apoptosis inhibition and cell cycle arrest which may cause Gemcitabine to lose the affecting targets and result in resistance. There may be such HIF-1a-mediated pathways which activate the expression of mdrl/P-Gp, Bcl-Xl, and Bax, leading to multidrug resistance and inhibition of apoptosis that result in resistance to chemotherapy, especially Gemcitabine. A Gemcitabine and YC-1 combined therapy may improve the effective rate of chemotherapy in human pancreatic carcinoma, and targeting HIF-la would possibly become a brand new way to reverse the chemo-resistance to Gemcitabine in human pancreatic carcinoma. |
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