| ObjectiveAccording to International Agency for Research on Cancer(IARC),clorectal cancer is the third common cancer worldwide while it is the fourth common reason for cancer-related death.China has the highest clorectal cancer population and clorectal cancer-related deaths among world.By the Chinese National Cancer Center,the increasing incidence and mortality of clorectal cancer in China make the country face a remarkable challenge in the prevention and treatment of clorectal cancer.The main strategies for clorectal cancer treatment are surgery,radiotherapy as well as chemotherapy.Clinical researches have reported that 80%of clorectal cancer are looking preventable which can be achieved via several approaches such as clorectaloscopy,clorectal polypectomy and drug intervention.Common chemicals that used for cancer therapy are not the good drugs for prevention of clorectal cancer during its early stagy due to the serious side effects off them.Seeking safe and effective drugs for clorectal cancer prevention is of great importance in reducing the incidence of clorectal cancer and in slowing down the development of clorectal cancer.Artemisinin and the derivatives are the most common anti-malarial drugs worldwide with high and fast efficacy as well as low toxicity.More and more reports have indicated that artemisinin and its derivatives exhibit good anti-tumor activity.Some of them have been in clinical trials and have got considerable results.A clinical study reported in 2015revealed that artesunate can significantly reduce the risk of clorectal cancer recurrence.This result suggests that artemisinin and its derivatives may be good drugs for clorectal cancer prevention and treatment,but the molecular mechenisms by which artemisinin and its derivatives inhibit clorectal cancer are not clear enough yet.Epigenetic theory can explain the p Henomena that cannot be done by genetic theory during the development of human diseases.Abnormal epigenetic alterations occur during the full course of clorectal cancer.Epigenetic modifications of some genes have become markers for the diagnosis,treatment,and prediction of clorectal cancer.Numerous studies show that natural active products have good regulatory effects on tumor epigenetics and are an important source for the development of epigenetic drugs.Previous results observed by other colleagues in our team revealed that dihydroartemisinin and artesunate may regulate the epigenomes in lung cancer and inhibit the development and growth of lung caner.In this study,we evaluate the effects of artemisinin,dihydroartemisinin,artesunate on clorectal cancer.Furthermore,we investigate the molecular mechanisms of the best compound.Also,we investigate the regulatory effect on abnormal epigenetic alterations in clorectal cancer.Method1.Effects of artemisinin and its derivatives on cell viabilities.Clorectal cancer cell lines SW480,HCT116 and HT29 were treated with different concentrations of artemisinin(0-100μM),dihydroartemisinin(0-64μM)and artesunate(0-64μM)for 72 h.Cell viabilities was detected by MTT assay.Cell viability rate and IC50were calculated.Clorectalformation assay was performed to observe the effects of artemisinin and its derivatives on cell viabilities of SW480 and HCT116 during a long-term administration.2.Effects of artemisinin and its derivatives on cell division and DNA synthesis.SW480 and HCT116 were stained with 5μM CFSE for 3-6 h to detect the cell division after SW480 and HCT116 being treated with artemisinin(12.5,25,50μM),dihydroartemisinin(1,2,4μM),artesunate(1,2,4μM)for 72 h.Ed U assay was employed to observe the DNA synthesis in SW480 and HCT116 after they being treated with dihydroartemisinin(1,2,4μM),artesunate(1,2,4μM)for 72 h.3.Effects of artesunate on cell cycle in SW480 and HCT116PI staining was applied to analyze the cell cycle of SW480 and HCT116 cells after they being treated with artesunate(1,2,4μM)for 72 h.Western blot was employed to investigate the protein levels of cell cycle-related proteins in SW480 and HCT116 which were treated with artesunate(1,2,4μM)for 72 h.4.Effects of artesunate on apoptosis in SW480 and HCT116The apoptosis of SW480 and HCT116 cells was detected by Anexin V-FITC/PI staining and flow cytometry.SW480 and HCT116 cells were treated with artesunate(1,2,4μM)for24 h,48 h,72 h,respectively.The protein levels of apoptosis-related proteins are detected by western blot.5.Effects of artesunate on PI3K-AKT sinaling,p53 signaling,MAPK signaling and endoplasmic reticulumSW480 cells were treated with artesunate(2μM)for 72 h.Total RNA was extracted for RNA-seq to screen differentially expressed genes in SW480.Pathway Enrichment Analysis was performed to predict the potential signaling pathway affected by artesunate.The prediction result was verified by q PCR.SW480 and HCT116 cells were treated with artesunate(1,2,4μM)for 72 h to extract total protein.The levels of PI3K-AKT signaling pathway and p53 signaling pathway-related proteins in SW480 and HCT116 cells were detected by western blot.According to the reported studies,ER stress and MAPK signaling are the reported upstream of PI3K-AKT signaling and p53 signaling.Western blot was used to detect the levels of key proteins of MAPK signaling and of ER stress in SW480 and HCT116 cells.6.Effect of KDM4B on cell cycle and cell proliferation in SW480 and the effect of ARTS on KDM4BAccording to The Cancer Genome Atlas(TCGA),KDM4B is associated with the survival of colorectal cancer patients.SW480 cells were treated with artesunate(1,2,4μM)for 72 h.The m RNA and protein level of KDM4B was detected by q PCR and western blot respectively.The levels of catalytic substrates H3K9me1,H3K9me2,and H3K9me3 were also detected by western blot.Endogenous KDM4B in SW480 was silenced by liposome-mediated transfection of small RNA interference.Cell cycle was detected by PI staining.Cell proliferation was detected by CFSE staining.The protein levels of proteins in PI3K-AKT signaling、p53signaling、MAPK signaling and ER stress signaling were detected by western blot to search for potentially KDM4B-targeting genes.7.The effect of artesunate on growth of clorectal cancer in vivo.SW480-derived xenograft was established via subcutaneously injecting SW480 cell suspension into the right armpits of nude mice.Artesunate administration was started when the tumor volume was 50 to 100 mm~3.Artesunate was administered 5 times per week for 4weeks at a concentration of 60 mg/kg.During the administration,body weight was measured every 3 days and meanwhile the tumor volume was measured.When the administration finished,tumor tissue,heart,liver,spleen,lung,and kidney were detached and weighed.The organ index was calculated.Total protein was extracted and the protein levels of proteins in PI3K-AKT signaling,p53 signaling,MAPK signaling,endoplasmic reticulum signaling,as well as KDM4B and H3K9me1,H3K9me2,H3K9me3 were detected by western blot.Result1.Artemisinins inhibited cell viabilities in SW480,HCT116 and HT29.The 72 h-IC50 values of artemisinin,dihydroartemisinin,and artesunate in SW480cells were 31.59,2.637,and 3.159μM,respectively,while in HCT116 were 31.69,4.628,and 2.736μM,respectively.The IC50 values in HT29 were>100,11.54,and 9.543μM,respectively.The relative clorectaly formation rates of SW480 and HCT116 were significantly reduced by the 14 day-treatment of artemisinin,dihydroartemisinin and artesunate(p<0.001).2.Aretiminins inhibited cell division and DNA synthesis in SW480 and HCT116.The fluorescence intensity of CFSE in untreated SW480 was 1014±38.The fluorescence intensity of CFSE in artemisinin(12.5,25,50μM)-treated SW480 were1007±47(p>0.05),1067±38(p>0.05),1041±38(p>0.05).The fluorescence intensity of CFSE in dihydroartemisinin(1,2,4μM)-treated SW480 were 1492±35(p<0.001),1725±51(p<0.001),and 1803±52(p<0.001).The fluorescence intensity of CFSE in artesunate(1,2,4μM)-treated SW480 were 1353±44(p<0.001),1590±36(p<0.001),and1694±53(p<0.001).The fluorescence intensity of CFSE in untreated-HCT116 was 838±36.The fluorescence intensity of CFSE in artemisinin(12.5,25,50μM)-treated HCT116 were963±31(p<0.001),976±35(p<0.001),and 1116±46(p<0.001).The fluorescence intensity of CFSE in dihydroartemisinin(1,2,4μM)-treated HCT116 were 1689±122(p<0.001),2025±197(p<0.001),and 1901±130(p<0.001),respectively.The fluorescence intensity of CFSE in artesunate(1,2,4μM)-treated HCT116 treated were 1393±77(p<0.001),1998±177(p<0.001),and 1929±162(p<0.001).The incorporation rate of Ed U in untreated-SW480 cells was 36.57%±6.85%,and the incorporation rate of Ed U in dihydroartemisinin-treated SW480(1,2,4μM)was 14.43%±2.54%(p<0.05),9.79%±2.29%(p<0.01),7.18%±4.82%(p<0.01),respectively.The incorporation rate of Ed U in artesunate(1,2,4μM)-treated SW480 was 10.45%±3.63%(p<0.05),4.62%±3.44%(p<0.05),4.06%±2.47%(p<0.01).The incorporation rate of Ed U in untreated-HCT116 was 64.23%±9.21%,and the incorporation rate of Ed U in dyhroartemisnin(1,2,4μM)-treated HCT116 was 22.63%±3.37%(p<0.01),17.85%±4.01%(p<0.01),11.51%±2.63%(p<0.01).The incorporation rate of Ed U in ARTS-treated HCT116(1,2,4μM)HCT116 was 10.51%±4.17%(p<0.001),9.97%±4.97%(p<0.01),2.92%±1.67%(p<0.001).3.Artesunate caused cell cycle arrest at G0/G1 p Hase in SW480 and HCT116.In artesunate-treated SW480,the percentage of G1 p Hase cells increased from57.38%±3.98%to 73.21%±1.86%(2μM,p<0.01),and 70.19%±4.57%(4μM,p<0.05).1μM ARTS showed no significant effect on the percentage of G1 cells in SW480 cells(56.15%±2.98%,p>005).After artesunate(1,2,4μM)treatment,the percentage of G1 p Hase cells increased from 47.02%±31.44%to 59.90%±0.76%(p<0.001),72.85%±3.89%(p<0.001),68.16%±2.22%(p<0.001).Protein levels of CDKs(CDK2,CDK4,CDK6),Cyclins(Cyclin D1,Cyclin E1)and CDKIs(p16,p21)in SW480 and HCT116 were downregulated(p<0.01)while CDKIs(p16,p21)in SW480 and HCT116 were upregulated(p<0.001).4.Artesunate induced apoptosis in SW480 and HCT116 slowly and moderately.In 24h-and 48h-treated SW480,the ratio of Anexin V-FITC+/PI-cells,Anexin V-FITC+/PI+cells,and Anexin V-FITC+/PI+cells were all lower than 1%.After treated with artesunate(1,2,4μM)for 72 h,the ratio of Anexin V-FITC+/PI-cells in untreated-and treated-SW480 was 1.89%±0.06%(p<0.001),2.48%±0.04%(p<0.001),2.15%±0.06%(p<0.001),respectively,while that in untreated-SW480 was 0.21%±0.07%.The percentages of Anexin V-FITC+/PI+cells in the untreated-and treated-SW480 were0.33%±0.08%,3.53%±0.06%(p<0.001),4.84%±0.16%(p<0.001)and 6.28%±0.10%(p<0.001).The Anexin V-FITC-/PI+cells in the treated-group increased while the concentrations increased,which were 4.60%±0.05%,4.94%±0.03%(p<0.05),and5.83%±0.01%(p<0.001),respectively.,higher than the control group(4.67%±0.26%).In 24h-and 48h-treated SW480,the ratio of Anexin V-FITC+/PI-cells,Anexin V-FITC+/PI+cells,and Anexin V-FITC+/PI+cells were all lower than 1%.After treatment with artesunate(1,2,4μM)for 72 h,the ratio of Anexin V-FITC+/PI-cells in untreated-and treated-HCT116 was 1.41%±0.22%,1.04%±0.07%,2.28%±0.18%(p<0.01),and3.39%±0.21%(p<0.001),respectively.The percentages of Anexin V-FITC+/PI+cells in untreated-and treated-HCT116 were 1.68%±0.11%,0.77%±0.09%,2.13%±0.03%(p<0.01),3.10%±0.04%(p<0.001),respectively.The proportions of the Anexin V-FITC-/PI+cells in untreated-and treated-HCT116 were 5.58%±0.20%,2.85%±0.14%,4.86%±0.36%,and 5.61%±0.23%,respectively.5.Artesunate regulates endoplasmic reticulum stress,MAPK signaling and PI3K-AKT-p53 signalingThe Pathway Enrichment Analysis results showed that artesunate(2μM)can regulate PI3K-AKT signal and p53 signal in SW480 cells.The results of q PCR showed that the relative level of AKT m RNA in treated-SW480 cells was decreased to 0.36±0.01 times of that in untreated-SW480(p<0.001).The PTEN and TP53 m RNA levels were increased to1.22±0.04(p<0.001),2.13±0.26(p<0.001),respectively.Western blot results showed that the protein levels of AKT and PI3K in artesunate-treated SW480,HCT116,and HT29 cells decreased(p<0.001)while the levels of PTEN and p53 protein were increased(p<0.001).Western blot results show that ERK1/2 in SW480,HCT116,and HT29 cells were decreased.The protein levels of endoplasmic reticulum stress-related protein such as BIP,IRE1α,PDI,and CHOP were increased(p<0.001).Tauroursodeoxycholic acid(TUDCA)is an inhibitor of endoplasmic reticulum stress.IRE1αprotein level was downregulated(p<0.05)in TUDCA treated-SW480 while PI3K protein level was upregulated(p<0.001).In artesunat-treated HCT116,TUDCA attenuated the regulation of artesunate on the expression of IRE1α,PI3K(p<0.001).TUDCA impaired the antiproliferation effect of artesunate(p<0.05).6.Effect of KDM4B on cell cycle and cell proliferation in SW480 and the regulatory effect of artesunate on KDM4B.After treated with artesunate(1,2,4μM)for 72 h,the m RNA and protein levels of KDM4B in the cells were decreased(p<0.001),and the level of H3K9me2,which is a substrate of KDM4B,was increased(p<0.001).After KDM4B being silenced by si RNA,KDM4B m RNA level in SW480 was reduced to 0.26±0.03 times of that in negative control-transfected SW480(p<0.001),and the protein level was reduced to 21.01%±1.59%(p<0.001),and H3K9me1,H3K9me2levels were increased(p<0.001).Compared with negative control-transfected SW480,the fluorescence intensity of CFSE in KDM4B-silenced cells increased from 996±23 to1507±33(p<0.01),and the proportion of G0/G1 p Hase cells increased from 58.93%±1.92to 70.29.%±3.43%(p<0.01),and p16,p21 protein levels were increased(p<0.001),similar to the effect of artesnate on cell proliferation and cell cycle in SW480.The protein level of PI3K in KDM4B-silenced SW480 was downregulated(p<0.001).Inhibition ER stress by TUDCA in HCT116 upregulated KDM4B protein levels(p<0.001).Artesunate inhibited the expression of KDM4B induced by tauroursodeoxycholic acid(p<0.001).7.Artesunate inhibits clorectal cancer proliferation in vivo.Artesunate significantly inhibited the growth of SW480-derived xenografts in nude mice with no significant effect on body weight and organ index(p<0.05).The tumor weights in the control group and artesunate group were 1.13g±0.34 g and 0.49g±0.05g(p<0.05),respectively.The tumor inhibition rate was 56.61%±4.74%.Compared with the control group,the protein levels of PI3K,AKT,and ERK1/2 in tumor tissues from artesunate-treated mice were decreased(p<0.001)and the protein levels of p53,PTEN,BIP,IRE1α,and CHOP were increased(p<0.001).Compared with the control group,the KDM4B protein level in in tumor tissues from artesunate-treated mice was decreased(p<0.001),and the levels of its substrates,namely,H3K9me1 and H3K9me2,were increased(p<0.001).ConclusionsArtemisinin,dihydroartemisinin and artesunate can significantly inhibit the proliferation of clorectal cancer cells,and artesunate has the best inhibitory effect.Artesunate inhibits the proliferation of clorectal cancer cells by significantly inducing endoplasmic reticulum stress,thereby down-regulating PI3K and AKT protein levels,and up-regulating PTEN and p53 protein levels.KDM4B is a potential downstream target for endoplasmic reticulum stress,and PI3K is a potential downstream target of KDM4B.Endoplasmic reticulum stress-KDM4B-PI3K signaling axis may be involved in artesunate-caused anti-proliferation against clorectal cancer.The mechanisms for endoplasmic reticulum stress regulating KDM4B and KDM4B regulating PI3K need to be investigated. |
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