Study On The Biological Mechanism Of The Alcohol Extracts Of Two Meconopsis Plants In Inhibiting The Proliferation Of Leukemia Cells

Malignant tumors(cancers)have become frequently-occurring and common diseases that seriously threaten human health.According to statistics of the WHO,there are more than 12 million people diagnosed with malignant tumors each year in the world,and the global cancer incidence rate will increase by 50%in 2020.At present,the main therapy methods for malignant tumors still are resection surgery,radiotherapy and chemotherapy.Traditional chemotherapy drugs have poor selectivity for cells in treating tumors.When killing tumor cells,they also have a toxic effect on normal cells,especially for the cells in the hematopoietic tissues and immune system.They can cause myelosuppression and immunocompromise.At the same time,the occurrence of drug resistance for tumor cells is still a very pivotal issue in the treatment of tumors.Therefore,searching for therapeutic drugs with good curative effects and low toxic and side effects has become an urgent need for clinical treatment of cancer.Chinese herbal medicine has the advantages of wide range of sources,low prices,long history of application,low adverse reactions,and low genotoxicity.It can fundamentally regulate and restore the function of the body when treating cancer,enhance the immunity of patients,and prolong survival period.It has become a hot spot in the current researches for developing new drugs for tumor treatment.In this study,we used the Tibetan medicines(Meconopsis integrifolia(Maxim.)Franch.;Meconopsis.horridula Hook.f.et Thoms)collected from the Qinghai-Tibet Plateau as the research materials.The L1210 and K562 cells as in vitro cell models,and the S180 tumor-bearing mice as the in vivo models.We systematically study the anti-tumors effects of ethanol extracts of Meconopsis integrifolia(Maxim.)Franch.and Meconopsis.horridula Hook.f.et Thoms,as well as potential underlying molecular mechanisms.The main results are as follows:1.GC-MS was used to analyze the chemical constituents of the ethanol extracts of Meconopsis integrifolia(Maxim.)Franch.and Meconopsis horridula Hook.f.et Thoms.16 and 11 different chemical components were obtained,respectively.Furthermore,the cytotoxic effects of the ethanol extracts of Meconopsis integrifolia(Maxim.)Franch and Meconopsis horridula Hook.f.et Thoms on K562 and L1210 cell viability were measured by MTT method.The corresponding IC50 values were obtained.2.The proliferation inhibitory effects of ethanol extracts of Meconopsis integrifolia(Maxim.)Franch.and Meconopsis horridula Hook,f.et Thoms,on K562 cells,L1210 cells,peripheral blood PBMCs and human umbilical vein endothelial cells(HUVEC)were detected using MTT assay.The results showed that the ethanol extract of Meconopsis integrifolia(Maxim.)Franch.inhibited the proliferation of K562 cells in time-and concentration-dependent manners,but almost no effect on PBMCs.The ethanol extract of Meconopsis horridula Hook.f.et Thoms,suppressed the proliferation of L1210 in time-and concentration-dependent manners,but had no effects on HUVEC.3.Scanning electron microscope technology was performed to detect the surface membrane structure of K562 cells after ethanol extract of Meconopsis integrifolia(Maxim.)Franch.treatment,as well as the surface membrane structure of L1210 cells after ethanol extract of Meconopsis horridula Hook.f.et Thoms.treatment.4.The damage of DNA in K562 cells after ethanol extract of Meconopsis integrifolia(Maxim.)Franch.treatment was determined using DNA gel electrophoresis assay and HO staining.The results showed that the ethanol extract of Meconopsis integrifolia(Maxim.)Franch damage the DNA of K562 cells in time-and concentration-dependent manner.The result of AnnexinV-FIT/PI double staining displayed that ethanol extract of Meconopsis integrifolia(Maxim.)Franch.induced K562 cell apoptosis.The results of cell cycle analysis presented that the ethanol extract of Meconopsis integrifolia(Maxim.)Franch.arrested the cell cycle of K562 cells in G2/M phase in a concentration-dependent manner.5.The ROS production,change of mitochondrial membrane potential,and location of cytochrome C of K562 cells after ethanol extract of Meconopsis integrifolia(Maxim.)Franch.treatment were used to analyze the potential molecular mechanism of anti-tumor effects of ethanol extract of Meconopsis integrifolia(Maxim.)Franch.6.Western blotting was conducted to measure the expression levels of p53,Caspase-3,cleaved Caspase-9,and PARP cleavage fragments in K562 cells after ethanol extract of Meconopsis integrifolia(Maxim.)Franch.treatment.The results showed that the ethanol extract of Meconopsis integrifolia(Maxim.)Franch.could induce p53-dependent mitochondria-dependent cell apoptosis.7.The DNA damage of L1210 cells after ethanol extract of Meconopsis horridula Hook.f.et Thoms.treatment was also detected in this research.The results displayed that the ethanol extract of Meconopsis horridula Hook.f.et Thoms.induced L1210 cell DNA fragmentation.Flow cytometer analysis showed that the ethanol extract of Meconopsis horridula Hook.f.et Thoms.arrested the cell cycle of L1210 cells in the G2/M phase.8.The effects of the ethanol extract of Meconopsis horridula Hook.f.et Thoms.on L1210 cell ROS production,change of mitochondrial membrane potential were also analyzed.Results pointed out that the ethanol extract of Meconopsis horridula Hook.f.et Thoms.enhanced the ROS production and reduced the mitochondrial membrane potential in L1210 cells.9.Taking S180 tumor-bearing mice as an in vivo research model,the anti-tumor mechanism of the ethanol extract of Meconopsis horridula Hook.f.et Thoms was studied.The results are as follows:The ethanol extract of Meconopsis horridula Hook.f.et Thoms had no significant effects on S180 tumor-bearing mice weight,living condition and organ index.Moreover,the ethanol extract of Meconopsis horridula Hook f.et Thoms had significant tumor-killing and inhibitory effects on the S180 tumor-bearing mice.Its tumor inhibitory growth may be achieved through activation of pro-apoptotic protein Bax,tumor suppressor gene p53 and Caspase family proteins in tumor cells.Overall,the ethanol extract of Meconopsis.horridula Hook.f.et Thoms had low-toxic to the immune system of S180 tumor-bearing mice.Taken together,these above results provide a new theoretical basis for the reasonable development and use of traditional Tibetan medicine.