Studies Of The Effects And The Related Mechanisms Of Hypoxia On The Culturing And The Biological Characteristics Of Mammospheres

PartⅠ The effects of hypoxia on the culturing ofmammospheresObjective: To study the effects of hypoxia on the speed and efficacy ofmammospheres’(MSs) culturing.Methods: The human breast cancer tissues cells were cultured in thenormal oxygen and hypoxia serum-free media supplemented with growthfactors. The culture times and sizes of the MSs were recorded. The ratio ofCD44＋CD24－/Lowcells of each group MSs and MDA-MB-231cells wereevaluated by the flow cytometry. And the expressions of CD44,CD24andALDH1in each group MSs and MDA-MB-231cells were detected byWestern blotting assay.Results: The hypoxia group MSs had more bigger size and shorterculture time than the normal oxygen MSs, P＜0.05. The flow cytometryresult showed the ratio of CD44＋CD24－/Lowcells in the hypoxia MSs was 82.35±6.28%,and the ratio of which in normal oxygen MSs was1.17±0.05%,P＜0.05. The Western blotting assays revealed that the expressions of CD44and ALDH1in the hypoxia MSs were higher than the expressions of them innormal oxygen MSs and MDA-MB-231cells, P＜0.05. There was nostatistic difference of the expressions of CD24between hypoxia and normaloxygen MSs, P＞0.05.Conclusion: Hypoxia could promote the speed of the MSs culturing,and raise the ratio of CD44＋CD24－/Lowcells in MSs. The combination ofhypoxia and MSs cultured in serum-free media supplemented with growthfactors could be useful for classification of breast cancer stem cells.Part ⅡThe effects of hypoxia on MSs’ migration, invasiveand animal tumor formation characteristics, and preliminarydiscussion of the related mechanismObjective: To study the effects of hypoxia on the migration, invasiveand animal tumor formation characteristics. And discuss the relatedmechanism of the effects preliminarily.Methods: The MSs’ abilities about migration and invasion were testedby Wound healing assay, Transwell invasive assay. The animal tumorformation characteristics of the MSs were evaluated by the nude mice tumorformation assay. The transplantation tumors’ sizes and growth times were recorded. The expressions of HIF-2α and ABCG2were tested by Immuno-histochemistry assay. The expressions of HIF-2α, ABCG2, EMT(epithelial-mesenchymal transition) marker gene including E-Cadherin, Twist,Vimentin, N-Cadherin in hypoxia and normal oxygen MSs were evaluatedby Western blotting and Real time RT-PCR. The expressions of VEGF andEGF in the hypoxia and normal oxygen nude transplantation tumors wereevaluated by Western blotting and Real time RT-PCR assay.Results: The wound healing index of hypoxia MSDCs(92.52±9.36%)was higher than which of normal MSDCs(31.75±6.21%), P＜0.05.In theTranswell assay the number of the cells going through the membrane inhypoxia MSDCs was76.24±10.35, and the number of which in normalMSDCs was27.38±8.18, P＜0.01. The hypoxa MSDCs transplantationtumors had a much shorter growth time and bigger size than what of normalMSDCs transplantation tumor, P＜0.05. The Immuno-histochemistry assayshowed that a higher expressions of HIF-2α and ABCG2in hypoxia MSDCsthan normal oxygen MSDCs. Western blotting and Real time RT-PCRshowed that the expressions of HIF-2α, ABCG2, Twist, Vimentin, andN-Cadherin in hypoxia MSDCs were all higher than what in normal oxygenMSDCs and MDA-MB-231cells, P＜0.05. The expressions of E-Cadherinin both hypoxia MSDCs and normal MSDCs were low, and there was nostatistic difference between the2groups, P＞0.05. In addition, theexpressions of VEGF and EGF in hypoxia MSDCs nude mice transplantation tumors were higher than what in normal oxygen MSDCstransplantation tumors, P＜0.05.Conclusion: Hypoxia could promote the abilities of MSs’ migration,invasive and animal tumor formation abilities. And the mechanism of theeffects might be that hypoxia activated HIF-2α, and then HIF-2α inducedEMT occurring and raise the expressions of VEGF, EGF in MSDCs, finallyenhanced some MSs’ characteristics like cancer stem cells.Part ⅢEstablishment of recombinant lentiviral overexpression vector carrying ABCG2and its transfected onhuman breast cancer MDA-MB-231cellsObjective: To develop a lentiviral over expression vector carryingABCG2and establish a stable expression model of human breast cancerMDA-MB-231cells.Methods: The lentiviral over expression vector carrying ABCG2wastransduced into MDA-MB-231cells, and the transfection efficiency wascalculated. The expression of ABCG2in the MDA-MB-231cells transfectedafter subculturing was detected by Western blotting assay.Results: The lentiviral over expression vector carrying ABCG2wasconstructed successfully, and the transfection efficiency was95.6±3.5%.The expression of ABCG2in the MDA-MB-231cells transfected after sub-culturing was higher than which of the control group, P＜0.05.Conclusion: The lentiviral over expression vector carrying ABCG2can be expressed stably in MDA-MB-231cells.Part Ⅳ Studies of the influence and the relatedmechanisms of hypoxia on mammospheres’ resistance tochemotherapyObjective: To study the effect of hypoxia on human breast cancer cellmicrospheres’ resistance to chemotherapy and discuss the relatedmechanisms, to provide a experimental basis for the breast cancer stem cells’chemotherapy resistance.Methods: MDA-MB-231cells were cultured in the normal oxygen andhypoxia serum-free media supplemented with growth factors. The culturetimes and sizes of the MSs were observed. The survival rates of each groupof MSs and each group of MDA-MB-231cells under various chemotherapydrugs with various concentrations were detected by MTT colorimetricmethod.The expressions of HIF-2α and ABCG2in each group MSs wereobserved by Immunofluorescence technique. The expressions of ABCG2ofeach group were detected by Western blotting. The expressions of HIF-2α,ABCG2, P-gP and MDR1in each group were detected by Western blottingand Real-time RT-PCR. Results: The hypoxia group MSs had much bigger size and shorterculture time than the normal oxygen MSs. The survival rates of the hypoxiagroup MSs under various chemotherapy drugs with various concentrationswere higher than both the normal oxygen group MSs and MDA-MB-231cells (P＜0.05). However, the survival of the hypoxia group of MSs showeda similar trend with the MDA-MB-231cells transfected.Immunofluorescence showed that the fluorescence of HIF-2α and ABCG2inthe hypoxia group MSDCs was stronger than which in normal group MSs.The proteins’ expressions of HIF-2α,ABCG2and P-gP in hypoxia groupwere higher than what in the normal oxygen group and MDA-MB-231cells，P＜0.05. There was no statistic difference between the expressions ofABCG2in the hypoxia group MSDCs and what in the MDA-MB-231cellstransfected，P＞0.05. The mRNA’ expressions of HIF-2α, ABCG2andMDR1in the hypoxia group were all higher than which in other groups，P＜0.05..Conclusion: The pathway containing HIF-2α as the promoter andABCG2, MDR1as the effecters may be the mechanism of hypoxiaenhancing the chemotherapy resistance of MSs.