The Combined Effects Of Okadaic Acid And Hematein On Proliferation And Apoptosis Of Human Lung Adenocarcinoma A549 Cells

Background:The toll death from lung cancer ranks in human-cancer-related diseases have the first status and that is the leading cause of cancer-related deaths in humans,with more than 1 million deaths each year.Of the cancer cases diagnosed each year,the number of new lung cancer cases is as high as more than 1.6 million.Most patients with lung cancer do not like the traditional chemotherapy or radiotherapy treatment program,that advanced patients with lung cancer worse prognosis.The care of current standard is to extend patient life indefinitely while preserving the original quality of life and reducing unwanted side effects.In this thesis,the hematein which is traditional Chinese medicine and Okadaic acid which is seaweed toxins combined action to explore the two drugs and their combined effects on human lung adenocarcinoma A549 cell proliferation and apoptosis mechanism,with our country's medical and scientific research contribute to a power.Methods:1.MTT experimental study of Okadaic acid,hematein and the combined effect of the two on A549 cell inhibition;LDH leak test to determine the size of the cytotoxic effect of the drug.2.Trypan blue staining experiments to determine the number of living cells;Inverted phase contrast microscope and scanning electron microscope to observe changes in cell surface morphology after dosing.3.Acridine orange staining and agarose gel electrophoresis were used to observe the morphological changes of A549 cells and DNA content of genetic material.4.Coomassie brilliant blue G250 and Western Blot detection of drug treatment after the changes in total cellular protein content.5.The changes of mitochondrial membrane potential were observed by Rhodamine123staining;mitochondrial depolarization was observed by JC-1 probe;the changes of intracellular ROS level and MDA content were detected by fluorescence spectrophotometer.Results:1.MTT experiments showed that: the solvent content of 0.2% had no significant effect on cell viability;combination of two drugs was significantly better than single drug,compared with the control group,the two drugs both have additive effect or synergistic effect.LDH leak experiments show that: the combined effect of the best cytotoxicity,the best inhibitory effect.2.Trypan blue staining: The number of cells was significantly decreased in a time-dependent manner.Inverted phase contrast microscopy and scanning electron microscopy: 48 h after drug treatment,the control group of cells were spindle-shaped,arranged in close,drug-treated cells round the surface,cell contact disappeared,in part have apoptotic bodies.3.Acridine orange staining and agarose gel electrophoresis showed that DNA staining was found in A549 cells treated with drug for 48 h,Showing ladder-like bands,and the combined treatment of DNA decreased,the obvious trailing band,the band brightness weakened.4.Coomassie brilliant blue G250 staining and Western Blot results showed that: the drug-treated A549 cells for 48 h,the chief protein decreased,the two drugs combined treatment compared with the control group were significantly different.Cytc and Bax in mitochondria pathway increased and Bax decreased.CHOP,Calpain2,JNK1 and IRE1 which are endoplasmic reticulum pathway-related have increased.Both Okadaic acid and hematein can induce apoptosis,that it is through mitochondrial pathway and endoplasmic reticulum pathway.5.Rhodamine123 staining,JC-1 probe,fluorescence spectrophotometer detection of intracellular ROS levels and MDA content changes showed that both Okadaic acid and hematein mitochondrial membrane potential changes and the degree of lipid peroxidation,and the combined effect of the two drugs changes in cell membrane potential more obvious,a higher degree of LPO of cells.Conclusion:Okadaic acid and hematein can cause apoptosis,and in a time which are dose dependent.Both monotherapy and its combination can induce apoptosis through extrinsic apoptosis of mitochondrial pathway and endoplasmic reticulum pathway.The combination effect of two drugs is obviously better than the other single drug,and there is obvious difference.