Mechanism Of NF-κB/HIF-1α Pathway Induced Chemotherapy Resistant And EGCG Enhanced Chemotherapy Sensitivity To Cisplatin In Human Lung Adenocarcinoma A549Cells Under Hypoxia

Background:As a pathological type of non-small cell lung cancer, the incidence rate of Lung adenocarcinoma showed a significant upward trend in many countries and regions in the world. It has a characteristic of early distant metastasis. Therefore, platinum-based combination chemical is one of its main methods of treatment. However, due to to drug resistance or toxicity, it often leads to chemotherapy failure. The mechanisms involved in drug-resistant are multi-factorial and complex. Recently, growing studies reported that tumor hypoxia micro-environment is the initiating factor of drug resistance.Tumor hypoxia is one of the characteristics of the solid tumor and unregulated HIF-1α expression. HIF-1α is the most critical transcription factor that transcriptional activates hypoxia response gene. It is not only related to keep cells survival, but also induced tumor cell resistance to radio and chemo-therapy resistant through up-regulation expression of multi-drug resistance protein and anti-apoptosis protein. Hence, it becomes target to reverse hypoxic induced radio and chemo-therapy resistant. However, with the advanced research of tumor hypoxia and HIF-1α, it is found that NF-κB was activated in hypoxia; in addition, it regulates the expression of HIF-la both at transcription and translation levels. Therefore, inhibition activation of NF-κB is expected to down-regulate expression of HIF-la and its pathway and becomes a new target for reversal of hypoxic induced chemo-resistance. At the same time, a natural and safe substance is urgent to be found out to reverse hypoxic induced chemo-resistance.Object:To investigate the effect of expression of NF-κB, HIF-1α on chemosensitivity and whether EGCG can enhance the chemosensitivity to cisplatin in human lung adenocarcinoma A549cells under hypoxia conditions.Method:CoCl2was used to establish chemical hypoxia model of human lung adenocarcinoma A549cells. Part I:A549cells is divided into normoxia control group (Control group), CoCl2(100,200uM) experimental group. The expression of HIF-la was examined by Immune-chemistry and Western blot. Then, the morphology and structure of A549cells was observed through light and electron microscope. Proliferation and cell cycle were measured by MTT and flow cytometry; IC50of cisplatin and apoptosis rate of A549cells in the absence or presence of cisplatin were detected by MTT and Annexin V-FITC/PI, respectively. Part Ⅱ:A549cells were divided into normoxic control group (control group), and200uM CoCl2hypoxia group (CoCl2group). First of all, the expression of NF-κBp65and HIF-1α were measured by Immuno fluorescence cytochemistry, Real-time PCR,and Western-blot; Then,A549cells was transiently transfected p65-siRNA、FITC-conjugated control-siRNA by LipofectamineTM2000, or added with HIF-1-specific inhibitor YC-1in normoxia conditions; Nextly, they were coultured in200μM CoCl2hypoxia conditions, and divided into normoxic control group (control group), the200uM CoCl2hypoxia group (CoCl2groups), p65-siRNA+200uM CoCl2hypoxia group (P65-siRNA group), the Control-siRNA+200uM CoCl2hypoxia group (Control-siRNA group), the Lip+200uM CoCl2hypoxia group (Lip group) and YC-1+200uM CoCl2hypoxia group (YC-1group). Finally, the expression of NF-KBp65and HIF-la were measured by Real-timePCR and Western-blot. Part Ⅲ: A549cells were divided into normoxic control group (control group), the200uM CoCl2hypoxia group (CoCl2groups), p65-siRNA+200uM CoCl2hypoxia group (P65-siRNA group), the Control-siRNA+200uM CoCl2hypoxia group (Control-siRNA group), the Lip+200uM CoCl2hypoxia group (Lip group) and YC-1+200uM CoCl2hypoxia group (YC-1group).Firstly, IC50of cisplatin and apoptosis rate of A549cells in the absence or presence of cisplatin were detected by MTT and Annexin V-FITC/PI, respectively; Then, the expression of Bcl-2、p27were detected by Western-blot, Subsequently, A549cell growth curve、cell cycle were measured by MTT and flow cytometry. Finally, the relation between cell cycle and cisplatin induced apoptosis rate was measured by regression. Part IV:A549cells were divided into normoxic control group (control group),200uM CoCl2hypoxia group (CoCl2groups), after cultured for24h, EGCG was added. Then, the growth and apoptosis of A549cells were measured by MTT assay、Hoechst staining and Annexin V-FITC/PI under EGCG alone or in combination with cisplatin, Bcl-2expression was examined by Western-blot analysis.Results:Part I:HIF-1α was over-expressed in A549nucleus of CoCl2group, compared with control group. There were fewer cells and mitochondrial swelling, endoplasmic reticulum expansion and autophagic vacuoles in A549cells of CoCl2group through light and electron microscope, compared with control group. Growth curve of A549cell in control group was S-shaped, while, in CoCl2group, growth curve is low and flat. There were more G1phase cells and less S phase cells in CoCl2group, compared with control group. CoCl2alone didn’t induce more apoptosis rate, however, when treated with cisplatin, the survival rate of the A549cells in the same concentration of cisplatin and IC50of cisplatin were higher, while cisplatin-induced apoptosis rate was lower in CoCl2group, compared with control group. Part Ⅱ:NF-KBp65mRNA and total protein expression of NF-KBp65in CoCl2group were nearly as same as control group. However, NF-κBp65and HIF-1α protein expressed in nucleus, together with HIF-1αmRNA were increased in CoCl2group, compared with control group; p65-siRNA could successfully inhibit CoCl2induced activation of NF-κB, and down-regulate HIF-1α both at mRNA and protein levels. While, YC-1could only decrease HIF-1α protein, and had no effect on NF-κBp65. Part Ⅲ:survival rate of A549cells in the same concentration of cisplatin and IC50of cisplatin were lower in p65-siRNA group or YC-1group, compared with CoCl2group; p65-siRNA or YC-1alone could induced more cells apoptosis,when combined them with cisplatin, apoptosis rate was significantly increased, meanwhile, Bcl-2, p27expression were both down-regulated, and a short period of accelerated proliferation, more S-phase cells were found in p65-siRNA group or YC-1group, compared with CoCl2group; Furthermore, S-phase was positively regression with cisplatin-induced apoptosis. Part Ⅳ:the growth of A549cells was inhibited in an EGCG dose dependent manner, when combined EGCG with cisplatin, a synergistic effect on inhibition the growth of A549cells was produced under200uM CoCl2chemical hypoxia. More apoptosis cells could be observed in EGCG group or treated with cisplatin, Bcl-2over-expressed in CoCl2chemical hypoxia was down-regulated in EGCG group, when combined with cisplatin, Bcl-2was significantly down-regulated in EGCG group, compared with CoCl2group.Conclusion:NF-κB/HIF-1α pathway activated resulted in chemo-resistance and EGCG enhanced chemosensitivity to cisplatin in human lung adenocarcinoma A549cells under CoCl2induced chemical hypoxia conditions.