The Inhibitory Effects And Mechanisms Of ART On Chinese Colorectal Cancer Cell Line

Artemisinin is a sesquiterpene isolated from Artemisia annua L.,which is used in traditional Chinese medicine for the treatment of fever and chills.Artemisinin reveals profound activity against Plasmodium falciparum and Plasmodium vivax.In addition to their antimalarial activity,artemisinin and its derivatives are also active against cancer cells. And leukemia and colorectal cancer cell lines are most sensitive to their cytotoxic effects. Too many researches have focused on the anticancer activity of artemisinins against leukemia.However,there were few investigations about the anticancer effects on colorectal cancer.Colorectal cancer(CRC)with liver metastasis is a fatal disease with rapid progression and poor patient outcome.However,the molecular mechanisms involved in liver metastasis of CRC remains essentially unknown,largely because of the presence of few available CRC cell lines with liver metastasis origin and spontaneous metastatic potentials in nude mice.In this study,we established a novel metastatic CRC cell line,CLY,derived from liver metastasis of a 64-year-old Chinese CRC patient.The cell line was characterized by morphology,growth kinetics,tumorigenicity,spontaneous liver metastatic potential,and cytogenetics.In addition,immunofluorescence analysis ofβ-catenin and E-cadherin and methylation-specific PCR(MSP)of E-cadherin gene(CDH1)promoter were also used to compare CLY with conventional CRC cell lines(HT-29 and Lovo).The findings are as follows:CLY exhibited compact and polygonal-shaped epithelial cells in vitro and its population doubling time was 29.5 hours;subcutaneous transplantation of CLY into nude mice resulted in subcutaneous tumors formation and spontaneous liver metastasis in all of 10 nude mice;Cytogenetic analysis identified aneuploidy karyotypes with a modal chromosome number of 60;and in immunofluorescence analysis,CLY exhibited a low expression but high restricted nuclear localization ofβ-catenin and a silenced expression of E-cadherin,which were hallmarks of hyperactive Wnt/β-catenin signaling pathway and the epithelial to mesenchymal transition(EMT),respectively.Therefore,CLY cell line was poorly-differentiated on molecular levels.However,β-catenin and E-cadherin had cytoplasmic expressions in Lovo cells and membranous distributions in HT-29 cells,which implied the two cell lines were moderately- and well-differentiated,respectively.Therefore, CLY is an ideal cell model for further exploring the metastatic mechanisms of CRC and testing new therapeutic reagents for CRC with liver metastasis.Artesunate(ART),a semi-synthetic derivative of artemisinin with improved pharmacological features,also reveals profound cytotoxic activity.In the present investigation,we compared the anticancer effects of ART on three colorectal cancer(CRC) cell lines and analyzed the relationship between drug sensitivity and malignant phenotype of tumor cells.Poorly-differentiated was CRC cell line CLY showing nuclearβ-catenin accumulation and loss of E-cadherin;moderately-differentiated were Lovo cells with cytoplasmic distribution of the two proteins;while well-differentiated were HT-29 cells with membranous localization of them.As determined by MTT assay,flow cytometry analysis,proliferation-associated marker Ki67 analysis and the growth inhibition analysis of xenograft tumors,poorly- or moderately- differentiated CLY and Lovo were more susceptible to the anticancer effects of ART than well-differentiated HT-29.Furthermore,as determined by immunofluorescence and western blot analysis onβ-catenin and E-cadherin and RT-PCR analysis on Wnt/β-catenin target genes,the sensitive response of CLY and Lovo to ART was associated with membranous translocation ofβ-catenin and increased expression of E-cadherin,which indicated the inhibition of hyperactive Wnt/β-catenin signaling pathway and the reversion of the epithelial to mesenchymal transition(EMT), respectively.Due to vital roles of Wnt/β-catenin pathway and EMT in tumor differentiation, we thought ART might promote the re-differentiation and apoptosis of CRC cells by inhibition of hyperactive Wnt/β-catenin pathway and reversion of EMT.In addition, bioluminescent imaging also revealed ART decreased the physiological activity of CLY tumor xenografts and delayed spontaneous liver metastasis.These results and the known low toxicity are clues that ART might be a promising candidate drug for the treatment of colorectal carcinoma.We hope these results and further extensive studies will benefit individualized CRC treatment in clinical research.In summary,we established and characterized a novel metastatic CRC cell line derived from liver metastasis of a Chinese CRC patient.Furthermore,the cell line was used to elucidate the anticancer effects and mechanisms of artesunate.And we found the Wnt/β-catenin pathway and the EMT status of CRC cells were closely associated with the chemosensitivity to artesunate,which had never been reported.