Mechanism Of Dihydroartemisinin On The Enhancement Of Therapeutic Effect Of Cisplatin And On The Regulation Of HIF-1α Mediated Vascularize In Adenocarcinoma Of Lung

Hypoxia is usual in tumor,while hypoxia-inducible factor-lalpha(HIF-1α) is frequently overexpressed in tumors and mediates the adaptive response to numerous hypoxia-inducible genes.Several approaches have proved that vascular endothelial growth factor(VEGF) can be induced by HIF-1α.VEGF is the potent endothelial cell mitogen which is very important in tumor angiogenesis.Besides series of hypoxia-adaptive response in tumor cells,the macrophage in hypoxia area of tumor is also significant.Cycloxygenase-2(COX-2) as an inflammatory factor is usually produced by macrophage and monocyte when hypoxia or injured.The metabolic product of COX-2,PGE2 and PGI2 can active VEGF which can direct stimulus tumor angiogenesis.Angiogenesis is an important process in tumor.Cancer cells in solid tumors require access to blood vessels for growth and metastasis.Tumor vessels are structurally and functionally abnormal,with defective endothelium,basement membrane,and pericyte coverage.Generally considered that antiangiogenic drugs can breakdown tumor vessel net and accompany with hypoxia and malnutrition.Jain first proposed that anti-angiogenic therapy could normalize tumor vasculature before its destruction.The transient normalization of tumor vessels produces a temporary increase in oxygen and nutrient delivery to the cancer cells that surround these "normalized" vessels.So when antiangiogenic drugs are given in combination with chemotherapy will produce an unprecedented increase in therapy efficacy.Artemisinin is a sesquiterpene lactone endoperoxide found in the traditional chinese medicinal plant Artemisia annua.Dihydroartemisinin(DHA) is the main derivatives of artemisinin.There is growing evidence that DHA has some impact against tumors.In our previous study,we have found dihydroartemisinin inhibits the VEGF expression in LLC tumor xenograft,RPMI8226 cells,K562 cells,HL60 cells and exhibits the potent antiangiogenesis effect.However,to our knowledge,whether dihydroartemisinin can inhibit VEGF through HIF-1αhas not been reported yet and the enhancement of dihydroartemisinin to cyclophosphamide drugs has been demonstrated on Lewis lung carcinoma in vivo.In present study,we observed the therapeutic effect of dihydroartemisinin combined with cisplatin in the A549 and A549/DDP lung carcinoma in vivo and also explored the effective of HIF-1αmediated vascularize in adenocarcinoma of lung.Besides,we assessed the expression of HIF-1αand VEGF in A549 and A549/DDP ceils in vitro to seek the mechanism of dihydroartemisinin combined with cisplatin.This date would provide a clear rationale for investigation in future clinical trials.Results:1.Dihydroartemisinin enhances the therapeutic effect of cisplatin in vivo1.1 Antitumor effect of dihydroartemisinin in combination with cisplatinBoth A549 and A549/DDP carcinoma xenograft study showed dihydroartemisinin have the ability to enhance the chemotherapeutic effect of cisplatin.Dihydroartemisinin combined with cisplatin resulted in the significant regression of A549 and A549/DDP tumor compared with either therapy alone. However,the therapeutic efficacy in A549/DDP tumor model was better than A549 tumor model.A549/DDP tumors of drug combine used groups were lighter than single dihydroartemisinin and cisplatin administration groups(p＜0.05).1.2 Reduction of toxic reactionRegarding toxic reaction,all tumor-bearing mice behaved well and drug-induced body weight loss was not significantly different in any of the therapeutic groups.So the drug schedule we used was appropriate. 1.3 The content of cisplatin in A549/DDP tumor and blood plasmICP-MS analysis was performed to determine the content of cisplatin in A549/DDP tumor and blood plasm.The content of cisplatin progressively increased in three different dose of dihydroartemisinin combined with cisplatin.When the dose of dihydroartemisinin was between 0～100mg/kg,the content of cisplatin in tumor and the dose of dihydroartemisinin were in linear relationship(r=0.9993).The content of cisplatin in tumor xenograft was higher,while the weight of tumor was lighter.2.Dihydroartemisinin enhances the growth inhibition effect of cisplatin on A549 and A549/DDP cells under hypoxia condition2.1 The anti-proliferation effect of dihydroartemisinin combined with cisplatin in A549 and A549/DDP cellsTreated with dihydroartemisinin and cisplatin at different concentrations for 48h inhibited the growth of A549 and A549/DDP cells in a dose dependent manner as shown in MTT assay.Subsequently,CI values were calculated using Calcusyn at the fixed-ratio concentrations of dihydroartemisinin and cisplatin.It had demonstrated that cisplatin plus dihydroartemisinin showed distinct synergy in the two tested cancer cell lines,with the average CI value in A549 cell line was 0.6706 and in A549/DDP cell line was 0.5674.These results determined the synergistic effects of dihydroartemisinin and cisplatin on the proliferation of A549 and A549/DDP cell lines.2.2 Percentage of A549 and A549/DDP cells undergoing apoptosisFlow cytometry analysis after PI staining was used to determine the effects of dihydroartemisinin and cisplatin combination on the apoptosis-inducing abilities on A549 cells and A549/DDP cells.PI staining for Sub-G1 content analysis is used to characterize the apoptosis process in A549 cells treated with 3.75mg/L dihydroartemisinin,3.75mg/L cisplatin or the combination for 48h.About 65.70%of A549 cells were detected to be apoptotic(sub-G1) following the 48h treatment of dihydroartemisinin in combination with cisplatin;while the mono-treatment groups drove little cell to experience apoptosis.A549/DDP cells were treated with 11.36mg/L dihydroartemisinin,7.5mg/L cisplatin or the combination for 48h.About 60.00%of A549/DDP cells were under apoptosis.3.The regulation of dihydroartemisinin on HIF-1αmediated vascularize in lung cancer3.1 Down regulation of expression of HIF-1αand VEGF in A549 and A549/DDP cells under hypoxia conditionWestern blot was exploited to detect the expression of HIF-1αand VEGF in A549 and A549/DDP cells affected by dihydroartemisinin combined with cisplatin. The result showed that dihydroartemisinin plus cisplatin could lead to a stepwise reduction in HIF-1αand VEGF expression in a time dependent manner.Results presented that in A549 cells the expression of HIF-1αand VEGF had no difference with control group when drugs only administrated 12h,while after drugs used 24h the levels of HIF-1αand VEGF were decreased to 42.7%,and 44.3%(p＜0.001, p＜0.01).After treated with dihydroartemisinin and cisplatin for 48h,the levels of HIF-1αand VEGF were decreased to 61.6%,and 78.0%compared with vehicle control(p＜0.001,p＜0.01).However,in A549/DDP cells the expression of HIF-1αand VEGF had no difference with vehicle control group when drugs only administrated 12h.After administration of drugs 24h,the levels of HIF-1αand VEGF were decreased to 37.2%, and 29.6%(p＜0.05,p＜0.05),while after administration 48h the levels of HIF-1αand VEGF were decreased to 54.1%,and 41.9%compared with vehicle control(p＜0.01, p＜0.05).3.2 Down regulation of expression of HIF-1αand VEGF in A549 and A549/DDP xenograft tumor model The result of immunohistochemistry was that the expression of HIF-1αand VEGF in control group of A549 tumor model was the strongest.The expression of these two proteins in cisplatin single used group were weaker than in the control group,while in dihydroartemisinin single used groups and drug combine used groups the expression of HIF-1αand VEGF was the weakest.The expression of HIF-1αand VEGF in control group of A549/DDP tumor model was also the strongest,while in cisplatin single used group the expression was as strong as in the control group.But the expression of HIF-1αand VEGF in dihydroartemisinin single used groups and drug combine used groups was very weak.3.3 Down regulation of expression of COX-2 in A549 and A549/DDP xenograft tumor modelThe expression of COX-2 in control group of A549 tumor model was moderate. This protein in cisplatin single used group was a little less than in control group,while in dihydroartemisinin single used groups and drug combine used groups COX-2 was very low expression.The expression of COX-2 in control group of A549/DDP tumor model was strong positive.In cisplatin single used group and dihydroartemisinin single used groups the expression of COX-2 was moderate positive.,while the expression in drug combine used groups was very weak.3.4 Inhibition of tumor microvessel densitySpecific marker of endothelial cell CD31 was used as the blood capillary quantitate index.Compared with control group there was significant decrease of microvessel density in dihydroartemisinin single used groups in both A549 and A549/DDP tumor model,P＜0.05 or P＜0.01.At the same time the microvessel density in drug combine used groups decreased significantly compared with drug single used groups(P＜0.05 or P＜0.01).The proteoglycan NG2 in tumor model was used as mature blood vessels quantitate index.The result was that the mature blood vessels density in both A549 and A549/DDP tumor model decreased significantly compared with control group(P＜0.05 or P＜0.01),while in drug combine used groups the decrease was also significant compared with control group and cisplatin single used group(P＜0.05 or P＜0.01).Conclusion:Overall,the results of the current study demonstrate that dihydroartemisinin can enhance the therapeutic effects of cisplatin by active suppressing the A549 and A549/DDP carcinoma xenografts;and also can inhibit tumor vascularization by decreasing the expression of HIF-1αand VEGF protein.The combination of dihydroartemisinin and cisplatin exerted strong synergistic anti-proliferative effect against human cancer cells including A549 and A549/DDP cells and causation of tumor cells'apoptosis.These studies indicate that dihydroartemisinin and cisplatin have synergistic effect through tumor angiogenesis, providing a clear rationale for investigation in future clinical trials.