Study Of The Antitumor Activity And Mechanism Of Bisimidazolium Salt Glycosyltransferase Inhibitors C20 And C22

Cancer exhibits extremely high morbidity and mortality worldwide.The incidence of cancer was increasing and the estimated numbers of new cancer cases in China was 4.57million annually.The number of cancer-caused death is as high as 3 million per year.However,little is known about the molecular mechanism of the development and progression of cancer.Studies have shown that the cancer progression is accompanied with the enhancement of the proliferation and metastasis of cancer cells.Therefore,re-establishing the apoptosis program,preventing the abnormal proliferation,and inhibiting the migration and invasion of cancer cells are promising strategies to eliminate cancer.The current clinical treatment methods for cancer are mainly surgery and chemotherapy,among which chemotherapy tolerance and toxic side effects are important factors that lead to the low quality of life and poor survival rate of cancer patients.Therefore,establishing a method to selectively inhibit cancer cell proliferation and induce apoptosis has become an important research direction in the field of cancer research.Recent research indicated that glycosylation plays a key regulatory role in physiological and pathological processes.The glyco-phenotype of cancer cells as well as the gene expression and activity of related biosynthetic enzymes,glycosyltransferases,are often abnormal.Changes in glycosylation lead to increased affinity of cancer cells to endothelium and promote cancer cell metastasis.For example,sialylated Lewis x(SLe~x)has been found overexpressed on the surface of cancer cells,is an important biomarker of cancer metastasis and poor prognosis among cancer patients.Interations between SLe~x and E/P-selectin on the surface of endothelial cells is an important step to promote cancer cells to enter the blood and lymphatic circulatory system,which followed by the transfer to the new metastatic sites.Therefore,inhibiting the abnormal expression of SLe~x on the surface of cancer cells is believed to be a new therapeutic strategy for treating cancer.Aberrant glycosylation also significantly affects cell surface receptor expression,trafficking,and susceptibility to the signaling-inducing factors.Altering glycan structure shows influence in the inherent biological activities of these cell surface receptors.In addition,abnormal glycosylation has been shown to closely relate to the drug resistance of cancer cells.These studies revealed the effects of glycosylation on cell signaling,which play important regulatory roles in cancer cell proliferation,invasion,extracellular interactions,angiogenesis,immune regulation,metastasis formation,and the resulting drug-resistant phenotype.Meanwhile,abnormal glycosylation can be used as the"biomarker of cancer",and glycosyltransferases involved in the glycosylation process can also be used as important biomarkers and provide a series of specific targets for the development of cancer therapy.Glycosyltransferase inhibitors are expected to interfere with the pathological processes caused by the abnormal expressions of glycosyltransferases,which have attracted great interest and wide recognition in the field of drug development,but their specific mechanisms of action remain to be elucidated.Previous studies have shown that bisimidazolium salts C20 and C22 could serve as glycosyltransferase inhibitors.The in vitro cell homogenate experiments demonstrated that C20/C22 could effectively inhibit the activity of glycosyltransferases that involved in the synthesis of cancer antigen SLe~x.Therefore,this study further explored the tumor suppressor effects and the underlying mechanism of C20 and C22.Studies indicated that(1)C20 and C22could significantly reduce the viability of cancer cells.While,compared with cancer cells,C20/C22 showed lower toxicity against normal cells such as the immortalized human umbilical vein endothelial cells(HUVEC),human embryonic kidney cells(HEK293A)and immortalized mouse hippocampal cells(HT22).(2)The cytotoxicity assays showed that the estrogen-dependent and independent breast cancer cell lines MCF-7 and MDA-MB-231 as well as liver cancer cell line Hep G2 were more sensitive to the C20 and C22 treatment.Therefore,the anticancer effects of C20 and C22 in inhibiting cancer cell proliferation,metastasis,angiogenesis and inducing apoptosis were further investigated with the above three cancer cell lines and xenograft mouse models.(3)The results of cell adhesion assays and transwell assays showed that incubation of cancer cell lines(MCF-7,MDA-MB-231 and Hep G2)with C20/C22 could significantly reduce the adhesion between cancer cells and HUVEC and weaken the migration and invasion capabilities of cancer cells.(4)Treatment of C20/C22 led to changes in the glycosylation of cancer cells,which significantly down-regulated the expression of SLe~x on the surface of cancer cells,thereby inhibiting selectin-mediated cancer cell metastasis.(5)C20/C22 up-regulated the expression and cell surface distribution of cancer cell apoptosis receptors DR4 and DR5,enhanced the susceptibility of cancer cells to the apoptosis inducing factor TRAIL,and promoted cancer cells to enter the extrinsic apoptosis program.(6)C20/C22-treatment promoted cancer cell cycle arrest,induced endoplasmic reticulum stress,upregulated ROS levels in cancer cells,and induced cancer cell apoptosis through the mitochondrial pathway.(7)The results of in vivo experiments showed that C20 and C22 could effectively inhibit tumor growth and angiogenesis in xenograft mice without causing the weight loss and pathological changes in major organs.(8)Based on the western blot and immunohistochemical analysis of tumor tissues harvested from the xenograft mice,the effects and the underlying mechanism of C20 and C22 in inhibiting tumor in vivo were consistent with the observations in the in vitro cell experiments.In conclusion,C20 and C22 glycosyltransferase inhibitors have shown influences on the glycosylation process in cancer cells,inhibited the metastasis of cancer cells,and regulated the proliferation and apoptosis network of cancer cells.C20 and C22 are worthy of further development as a new generation of anticancer drugs.