A Study Of The Apoptosis Effects And Mechanisms Induced By Cinobufagin And Alantolactone On Human Colorectal Cancer Cells

Colorectal cancer is a kind of common malignant tumor that seriously threatens human health.In USA,the morbidity and mortality of colorectal cancer are 4th and 2nd among all cancers respectively.The morbidity and mortality of colorectal cancer in western countries are reducing while it is increasing and tending to be younger among Chinese people.Therefore,developing anti-cancer drugs with more efficiency and lower toxicity is the key point of reducing the mortality of colorectal cancer.In recent years,molecular targeted treatment become an efficient therapy and has more advantages than traditional chemotherapy and radiotherapy.Molecular targeted drugs specially target cancer cells and have less effect on normal tissues.Although the researching of molecular targeted drugs has achieved some success,problems appear at the same time.Cell signaling transduction is multi-target and multi-processing while single-target drugs may cause drug tolerance and even cancer relapse or metastasis.Therefore,discovering efficient tumor specific targets and developing multi-targeted anti-cancer drugs may be an efficient method to the treatment of cancers.Cancer cells have a higher metabolic rate than normal cells and afford more oxidative stress.With the development of cancer,the reactive oxygen species(ROS)level increased significantly.More than a hundred kinds of DNA oxidative damage can be induced by ROS.8-oxo G and 2-OH-d ATP are the most common oxidative DNA modifications of d GTP and d ATP in nucleotide pool or DNA single chain.Base excision repair and mismatch repair caused by inserting 8-oxo G and 2-OH-d ATP into DNAs can cause gaps and fractures on its single-chain,which cause fracture and damage on DNA double-chain and lead to cells apoptosis.Previous studies find that MTH1,a homologous enzyme of Mut T,which locates in mitochondria and nuclei and contributes to DNA repair,is highly expressed in cancer cells with a positive correlation between its expression and cancer malignancy.MTH1 in cancer cells acts as an “eradicator” to hydrolyze oxidative nucleotide.The MTH1 protein sanitizes oxidized d NTP pool,converting 8-oxo G and 2-OH-d ATP into 8-oxod GMP and 2-OH-d AMP,avoiding incorporating of these oxidative nucleotides into DNA.Researches showed that depleted using si RNA on MTH1 protein in several cancer cell lines resulting in oxidative DNA and therefore cancer cells apoptosis.Notably,the survival of human normal cells is independent to MTH1 and no effects is observed with MTH1 knocked out in normal cells.In conclusion,increasing the ROS level or specifically inhibiting the activity of MTH1 could increase the oxidative stress in cancer cells,followed by increment of oxidative nucleotide inserted into DNA and cancer cell apoptosis.It is a long history of anti-cancer treatment with Chinese traditional medicine.A Chinese herb is complicated and contains many inefficient even toxic constituents.Therefore,it is of great importance to develop a method of extraction,separation and purification of efficient monomer of Chinese herb and sieving an anti-cancer chemical compound from the purified monomer.Thus,we organized our technical route: First,we use high-flux enzymatic screening assay to screen monomers that could inhibit MTH1 activity from herb monomer bank.Second,we use human colorectal cancer cell to verify that the monomer can cause the accumulation of 8-oxo G,oxidative damage of DNA and apoptosis of colorectal cancer cell via inhibiting MTH1 in vitro.Finally,we use computer simulation technology to confirm the combination of selected herb monomer and MTH1.In our study,cinobufagin was selected as a MTH1 inhibitor from 175 herb monomers with its IC50 of 11.51μm by enzymatic screening experiment.In our experiment,we find cinobufagin could inhibit the proliferation and induce the apoptosis of human colorectal SW480 cells in vitro.Under the same dosage,cinobufagin significantly inhibits SW480 cell proliferation while has no effects on human normal cells.Cinobufagin could induce the cell cycle arrest on G1/G0 and the expression of p21Cip/WAF1 is up-regulated.Results of immunofluorescence and single cell gel electrophoresis show that 8-oxo G and 53BP-1,a DNA damage marker,increase after treated with cinobufagin.Cinobufagin could also decrease the mitochondrial membrane potential and increase ROS level.We get the conclusion that cinobufagin could induce the apoptosis of colorectal cancer cell via both specific inhibiting the activity of MTH1 and increasing cellular ROS level.To confirm the combination of cinobufagin and MTH1,we use computer technology to simulate the molecular docking process.Molecular docking results show that cinobufagin bound in the catalytic site of MTH1 and cinobufagin-MTH1 formed a stable complex via hydrogen bond and pi-pi interaction.Additionally,we predict and analysis the pharmacokinetics,pharmacodynamics and toxicity of cinobufagin and results show that cinobufagin has a good druggability.Next,we find alantolactone could induce the accumulation of 8-oxo G and DNA damage although without MTH1 inhibition effect.We find alantolactone could induce the apoptosis of colorectal cell,arrestment of cell cycle on G1/G0 and up-regulation of p21Cip/WAF1.Alantolactone could decrease the mitochondrial membrane potential and increase ROS level.We also detect mitochondrial signaling changes characterized by decreased Bcl-2 while increasing in Bax and activated caspase-3.We find that alantolactone could increase the level of ROS and activate the mitochondrial apoptosis signaling via endogenous pathways to induce the apoptosis of SW480 cells.Our study demonstrates the molecular mechanisms of apoptosis on human colorectal cell induced by cinobufagin and alantolactone,especially cinobufagin,with the effects of both specific inhibiting of MTH1 and increasing cellular ROS.Compared to alantolactone with IC50 of 21.63μM,IC50 of cinobufagin is as low as 110.3n M.Computer simulation demonstrates that cinobufagin docks on the catalysis site of MTH1 and predicts a good druggability.In conclusion,our study demonstrates that cinobufagin and alantolactone could inhibit the proliferation and induce the apoptosis of human colorectal cancer cells with potential of further development.Our study provides techniques and evidences for further researches in formulated pesticide and clinical usage of cinobufagin and alantolactone.