| Background:With lung cancer mortality rates increasing,the lung cancer population is expected to reach 1million in 2025 and our country,china,is expected to be the first in the world.It is urgent that lung cancer require prevention and treatment.There are many ways to treat lung cancer,most patients still receiving conventional treatment: chemotherapy.With the increase of drugs in drug resistance,clinical chemotherapy effect is increasingly unsatisfactory.Therefore,it is important that finding efficient,low toxicity of drugs to enhance lung cancer chemotherapy.Okadaic acid is a kind of cell permeable polyether complex whith is protein phosphatase PP1,PP2 A inhibitor,low concentration can induce normal cell cancer,very low concentration can be used as a drug for cancer treatment,and in the treatment of lung cancer rarely reported.Curcumin is considered to be the third generation of anti-tumor drugs.It was approved by the FDA in 1996 for the treatment of lung cancer,colon cancer and other treatment,and the study in lung cancer of curcumin combination with okadaic acid has not been reported.In this paper,we selected two drugs,marine algal toxins okadaic acid which is polyether and curcumin which is a traditional china drug,to investigate their’s effects on the proliferation,apoptosis mechanism of human lung adenocarinoma cell line A549.Looking for clinical chemotherapy agents to provide a new theoretical basis is our parpose.Methods:1、The inhibitory rate of cell proliferation by MTT assay,to analyze the effect of okadaic acid,curcumin and combination on A549 cells.The relationship of tow drugs and the combination of them in the inhibitory rate of A549 through jin zheng jun formula analysis.Trypan blue dye exclusion test for the viable cell number.2、The morphological changes of A549 cells were observed by inverted phase contrast microscope,Giemsa staining,Hoecjst33258 staining,transmission electron microscopy and scanning electron microscopy.3、Fluorescence spectrophotometry was used to determine ROS content and mitochondrial depolarization.Fluorescence inverted microscope was used to observe the change of mitochondrial membrane potential of Rhodamine123.Western blot and agarose gel electrophoresis were used to detect the expression of Bax,Bcl-2 and cytochrome C on A549 cells in mitochondrial pathway,and A549 cells genetic material DNA.Results:1、The result of MTT method and the trypan blue dye exclusion testshow that okadaic acid and curcumin both inhibited the proliferation of A549,and the inhibition of drugs was enhanced with the prolongation of time and concentration,within a certain concentration range.The effect of combination was significantly better than that of OA or CUR.The results of the combination of okadaic acid and curcumin were the same as above,but it was more significant.The calculation results of jin formula showed the relationship of okadaic acid and curcumin was additive effect or synergistic effect.2、The result of morphological observation showed that cell morphology became round,cell volume reduction,intercelluar contact disappeared and chromatin condensation,serious vacuolation,apoptotic bodies appear on the periphery of the membrane,part of A549 were broken,when the cells were treated with 20 ng/m L okadaic acid,20 μg/m L curcumin and 20ng/m L okadaic acid + 20 μg/m L curcumin for 48 h.The morphological observation of two drugs was more serious than that of single drug.3、After the cells were treated with 20 ng/m L okadaic acid,20 μg/m L curcumin and 20 ng/m L okadaic acid + 20 μg/m L curcumin for 48 h,drug-induced apoptosis of A549 cells may be through ROS,intracellular signaling pathways involved in the mitochondria.The experimental results showed that Drugs increased intracellular ROS content,Rhodamine 123 detection of mitochondrial membrane potential also founded A549 cell endometrial potential declined.Western Blot showed that,cytochrome C,Bax increased significantly and Bcl-2 was significantly reduced in Mitochondrial apoptotic pathway,explaining okadaic acid and curcumin can induce the apoptosis of A549 cells through mitochondrial apoptosis pathway.The degree of apoptosis okadaic acid combined curcumin is stronger than that of single drug.Agarose gel electrophoresis showed the DNA was damaged in A549 cells after drugs trentment,and ladder-like bright bands appeared.it indicated that the role of drugs in A549 cells apoptosis.The DNA bands of the combined treatment group were the longest and brightest.The extent of apoptosis under the combined effects of drugs is stronger than single-agent role.Conclusion:These results suggest that okadaic acid and curcumin can inhibit the proliferation of human lung adenocarcinoma A549 cells and induce apoptosis by mitochondrial apoptosis. |
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