Synergistic Anticancer Activity And Mechanism Of Action Between Alkannin And ABT-263

According to recent decades’research,cancer cells produce many obvious metabolic pathways and malignant transformation of life process,such as glycolysis activation,Warburg effect,etc.,which make cancer cells survive or proliferate rapidly under adverse conditions.At the same time,the fragile point of cancer cells is exposed:because of the rapid proliferation of cancer cells,the cells themselves need to cope with high pressure metabolic rate,which makes cancer cells highly dependent on certain biochemical pathways,while normal cells are independent or less dependent.A large number of studies have found that reactive oxygen species（ROS）,as a byproduct of normal metabolism,produces much higher rates and accumulates in cancer cells than in normal cells.ROS can have serious side effects on intracellular biomolecules,such as G:A mismatch caused by the insertion of oxidized guanine triphosphate nucleotides（8-oxo-d GTP）into the DNA chain.A large number of such mutations can cause biochemical and metabolic changes.Severe cytotoxicity can kill cells.Since cancer cells need to cope with such a high level of ROS pressure,the tolerance to ROS is lower than that of normal cells.Therefore,additional treatment to increase the ROS level in cancer cells or weakening the antioxidant ability of cancer cells can be used as a research direction to develop broad-spectrum anticancer drugs.Purple grass is a traditional Chinese medicine with a long history of use in China,which has a good therapeutic effect on various burns,herpes and so on.One of the main extracts of shikonin has been found to inhibit thioredoxin reductase,antibacterial,immunomodulation and other functions.However,theβ,β-dimethyl acryloylalkanin（hereinafter referred to as alkanin or DAL）is rarely studied for one of the other main extracts.At present,most of the chemotherapy methods for tumor cells are to inhibit cell DNA replication,target inhibition of key regulatory factors as an effective treatment,with low toxicity,high efficiency,strong specificity and other characteristics.However,with the increase of clinical application,cancer cell mutation and other reasons,the probability of drug resistance is increasing,and the increase of drug dose will bring serious side effects.In order to overcome this phenomenon,modern medical research takes advantage of the characteristics of cancer cells with more mutation,rapid proliferation and high metabolism compared with normal cells,and focuses on the combination of drugs and blocking the effects of several pathways.This method has achieved good results at the cell level and in clinical trials,so it has become one of the hot research directions.This study mainly takes colon cancer cell SW480 as the experimental object to explore the effect and mechanism of DAL on cancer cells,and then selects non-lethal concentration combined with targeted inhibitor ABT-263（hereinafter referred to as ABT）to achieve the effect of synergistic lethal cancer cells.Verify its combined action and explore its mechanism on a variety of cancer cells.The SW480 was first treated by DAL through MTT experiment.The cytotoxicity of the 48 h-treatment was measured(IC₅₀=3.9μM),and the non-lethal concentration of2μM was selected as the study concentration.It was found that DAL could cause the SW480 cells to accumulate ROS to a higher level in just 3 h and increase with time.The accumulation of ROS in SW480 cells also caused a significant increase in 8-oxo G insertion in the DNA chain,DNA double-strand breaks（DSBs）are further increased（γH2A.X accumulation is evident）,DNA Damage Response Pathway（DDR）activation（increased CHK1 phosphorylation）,with oxidation inhibitor N-acetylcysteine（NAC）this phenomenon can be blocked.Treatment of SW480 cells with a nonlethal concentration of 3μM,the colony forming experiment showed that the cell proliferation was inhibited with the time of treatment.Throughβ-galactosidase（β-gal）staining,with the increase of DAL processing time,β-gal positive cells can be increased,therefore,DAL can cause SW480 cell senescence.Secondly,we chose to use senescent cell scavenger ABT-263 combined with DAL to act on SW480 cells,and found that they had a good synergistic lethal effect,and were verified by colony formation experiments.The combination index（CI）of the two combinations can be calculated to find that the CI values of the multiple combinations are lower than 1,which proves that the two drugs have a good combined lethal effect.Then,to explore whether the effect has a broad spectrum of combined lethal effect,the DAL combined with ABT was used on the other seven cancer cells and normal cells,and the results were found to be divided into sensitive and insensitive groups.The detection of MCL1,BCL-2 and BCL-XL of anti-apoptotic proteins of two groups of cells found out that the expression of these proteins in the cells of the sensitive group was very high.The expression levels of these proteins in the insensitive group were low.Therefore,the combined effect is better for cancer cells dependent on anti-apoptotic proteins.Then we found that the combination of drugs caused more than 53 BP1 andγH2A.X aggregation,and a longer tail of comets,indicating a higher proportion DSBs.The proportion of cleaved caspase3 positive cells triggered by DSBs increased,green fluorescence of JC-1 staining also increased,and cleaved caspase3 protein upregulation,whic prove that DAL combined with ABT can induce apoptosis.This is the mechanism of lethality of cancer cells.The results of the above studies showed that the accumulation of ROS in cancer cells caused by low dose DAL is the basis of its selective toxic effect on cancer cells.The DSBs is produced but not fatal because of the activation of cell senescence.When combined with senescent scavenger ABT,the synergistic effect can significantly increase the intracellular DSBs and activate the endogenous apoptosis pathway to kill cancer cells,and the combined effect has a better effect on the cells with high expression of anti-apoptotic proteins.These findings can provide a reference for the further development of DAL medicinal value and the anticancer effect of combined use.