The Study Of Mechanisms Of Apoptosis Induced By Actinomycin V On Human Colorectal Cancer Cells

Ten billion people died of cancer each year approximately,which make cancer a huge problem that threaten people’s health worldwide.Due to the increasing usage of alcohol and high fat low fiber diet habit,the rate of getting colon cancer is increasing yearly,making it a top three high incidence cancer.Cancer can be treated in different therapies depending on its type and severity,and most people chose a combination therapy which will bring toxic and side effect to organs,and drug resistance is also a main factor that fails chemotherapy.Therefore,searching for new medicine with high efficacy and low toxicity remains highlight in pharmacy field.Traditional cancer-preventing compounds are always from natural origin and most of them are originated from terrestrial ecosystems.Marine ecosystem species account for more than 80%of the world’s bio-diversity,making it a fertile field for potential cancer preventing compounds.Actinomycin D was approved by FDA to treat Wilms tumor and other solid tumors in 1960s.However,due to its hepatotoxicity and side effects,its clinical use is limited.While actinomycin V,a natural compounds derived from marine actinomycetes,has a similar chemical structure with actinomycin D.It has a higher efficacy in anti-cancer bioactivity and a lower toxicity to liver and kidney,which was proved by our group previously.In this paper,we dig into its mechanism against colorectal cancer to provide more pre-clinical data considering its high efficacy.Actinomycin V has quite remarkable anti-cancer activity,however its function and mechanism to colon cancer remains unreported.We compared actinomycin V and actinomycin D on their function to colon cancer cells and revealed that actinomycin V is more effective than actinomycin D.Therefore,we undergo research on its mechanism on treating colon cancer cells.Our results are:1.Actinomycin V inhibits the proliferation of colorectal cancer cellUsing MTT assay,we tested the viability of colorectal cancer cell lines treated with actinomycin V and actinomycin D at 24 h,48 h and 72 h.With the increase of concentration and dosing,the cell viability decreased and it was time-dependent and dose-dependent.The results show that the IC50 on HCT-116 is 2.85±0.10 nmol/L and 6.43±0.16 nmol/L for SW620.Furthermore,the toxicity on human normal lung and kidney cells are lower than actinomycin D,which suggested actinomycin’s potential to replace actinomycin D.2.Actinomycin V induces the apoptosis of colorectal cancer cellsActinomycin V’s inhibitory effect on cells is reflected in the induction of apoptosis.Flow cytometry detected and analyzed the apoptotic cell proption in the treatment group,and it could be concluded that the apoptotic effect is dose-dependent.DAPI staining,fluorescence microscope was used,and it was found that the nucleus of the treated cells was shrivelled and apoptotic bodies appeared.Further,Western blot confirming the expression trends of protein Bax and Bcl-2 in the treated cells,proved that apoptosis occurred at the microscopic protein level.3.Actinomycin V induces apoptosis on colon cancer cells associated with mitochondrial pathway and PI3K/Akt pathwayActinomycin V decreased mitochondrial membrane potential in HCT-116 cells,which was confirmed by JC-1 staining and flow cytometry and it suggested a mitochondrial damage.The result showed Cytochrome C are abundant in cytoplasm along with an increased cleaved caspase-9,resulting in increased apoptosis effector protein cleaved caspase-3.Immunofluorescence staining also confirmed the location and abundance of P-Akt in nucleus.Western blot further proved that the expression of PI3K and P-Akt/Akt protein decreased.Further,molecular docking and HTRF confirmed actinomycin V’s affinity to PI3K protein.Thus,we can conclude that the apoptosis effect that actinomycin V induced on colorectal cancer cell was associated with mitochondrial pathway and PI3K/AKT pathway.