| To increase the therapeutic efficacy for tumors so as to achieve thorough therapy finally is one of essential targets of current biology and medical science.As one of the main approaches to tumor treatment,chemotherapy itself had some defaults which are determined by the mechanisms of chemotherapeutic agents themselvees, such as the deficiency of selectivity and general deleterious side effect for patients. Thus,the therapeutic efficacies are limited.The improvement of therapeutic methods and the exploration of new chemotherapeutic agents little side effect have been an essential task for researchers.The hot topics about current chemotherapy include following:one,to elevate the specificity and chemotherapeutic efficacy through switching on or off the activity of cell signaling pathway related to proliferation, apoptosis and metabolism by investigating and developing specific small compounds or molecules with biological activities,which is the most promising and potential approaches;two,to sensitize the tumor cells to chemicals(mainly through overcoming the drug-resistance of cells) by means of combination chemotherapy of traditional chemotherapeutic agents and new drugs(including newly-developed chemicals and natural drugs that had inhibitive effect of tumors),which is an important approach for better chemotherapeutic efficacy;three,to amend the medication ways by various means of targeting topical solid tumors directly and releasing slowly in order to inhibit tumors longer and kill the tumor cells.It is shown that some of signaling pathway of animal embryonic development correlated closely with tumorigenesis and tumor development,such as Wnt,Hh, Notch,BMP signaling pathways,etc.The relationship between Hh signaling pathway and tumors is one of hot spots in research in recent years.Hh signaling pathway is not only extremely important for the patterning and cell fate decision,but also crucial in tumorigenesis and tumor development.Recently,the role and significance of Hh signaling in human hepatocellular cancer(HCC) is highlighted by and by.It is demonstrated that SHh(Sonic Hedgehog) signaling pathway is frequently activated in HCC and participate in carcinogenesis and development of HCC.Thus,the correlated research on Hh signaling pathway has essential values in specific treatment strategy targeted by cell signaling pathway.By far,the research on HCC seldom involved the alteration of SHh signaling pathway with chemotherapeutic treatment.The research materials in present dissertation is based on Hep3B cells which has activated SHh signaling pathway,and what we cared is whether the alterations of Hh signaling activity has relationship with the growth and migration ability of HCC cells.The present study confirmed proper work concentration by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay,which is 50% inhibition concentration for Hep3B cells treated for 48 h.When treatment elapsed by 6 h,12 h,24 h and 48 h,the results from semi-quantitative RT-PCR found the down regulation of target genes PTC1(Patched 1) and Gli1(glioma-associated oncogene homolog 1),ligand gene SHH(Sonic Hedgehog) and receptor gene SMO (Smoothened),indicated the influence of 5-FU on main components in SHh signaling pathway.While in the case of HepG2 cells,there is no expression of PTC1 and Gli1,and the expression of SHH ligand was not affected obviously.To confirm whether the cell growth arrest and apoptosis induced by 5-FU involves the activity of SHh signaling pathway,the present study made Gli1 overexpressed in Hep3B cells and when exposed to the same concentration of 5-FU, the transfection groups had higher survival curve than non-transfection groups in MTT assay.The results in MTT assay was also demonstrated directly by Trypan Blue assay;it is observed under fluorescent microscope in acridine dyeing assay that the nuclei in part of Hep3B cells happened to be of gragmentation,marginalization and chromatic agglutination with apoptotic body scattered around,and besides these, it was found that 5-FU treated transfection groups at 48 h had significant decrease comparing with non-transfection groups.As for Hochest33258 fluorescent assay, except for obvious apoptotic characteristics of nuclei,we found the average fluorescent intensity in 5-FU treated groups was much stronger than control groups (~*,p＜0.05),while the average fluorescent intensity in transfection groups is also stronger than that in control groups,but with no significant difference.TUNEL assay and corresponding fluorescent intensity assay with statistical analysis showed remarkable decrease trend in transfection groups with 5-FU treated,comparing with non-transfection groups with 5-FU treated.So,in present study the overexpression of Gli1 in trasfection group was found to antagonize the cell growth arrest and apoptosis induced by 5-FU.The overexpression of Gli1 in Hep3B cells can also elevate the activity of SHh signaling pathway,thus it decreased the sensitivity of Hep3B cells with 5-FU treatment and increased antagonistic effect in Hep3B cells.Our study found through scratch wound closure assay showed that the overexpression of Gli1 can rescue the decrease in motility of Hep3B cells induced by 5-FU,and this indicated that 5-FU may have influenced motility of SHh-activated tumor cells,which is involved inhibition of the activity of pathway.Besides,the overexpression of Gli1 in Hep3B cells may antagonize the change of subcellular location of PTC1.In this study,we detected the subcellular location of PTC1 in Hep3B cells through immunocytochemistry(ICC) assay,and found most of the PTC1 lost their location on membrane and distributed dispersedly with 5-FU treated for certain period.With time passed by and apoptosis progression,the signal from PTC1 became weaker and distribution became more scattered.After the treatment for 48 h,the expression of PTC1 was almost disappeared,this is consistent with the results in Western Blot.What's more,the overexpression of Gli1 may antagonize the alteration of subcellular location for PTC1 in Hep3B cells treated with 5-FU.Unexpectedly,the unique intracellular distribution of PTC1 in telophase of mitosis was found in our study,which possibly meant the isolation of PTC1 from their location near nuclear envelope and the recruitment of a major of PTC1 on cell membrane.Especially,near the equatorial plate region where the membrane was going to invaginate to form daughter cells,PTC1 was expressed in large amount,and this implies that PTC1 may involve the regulation of mitosis.Furthermore,the study detected the expression of cyclin E,the cyclin that can function in late G1 phase,in ICC assay.We found significant down regulation of cyclin E induced by 5-FU in Hep3B cells.While in transfection groups,the overexpression of Gli1 in Hep3B cells can inhibit the down regulation of cyclin E. Thus,the overexpression of Gli1 may partially inhibit the cell growth arrest of Hep3B.In conclusion,our study for the first time reported that down-regulation of SHh signaling pathway activity is involved in 5-fluorouracil-induced apoptosis and motility inhibition in Hep3B cells.The overexpression of Gli1 can antagonize the effect of the cell growth arrest and apoptosis,and can also rescue motility inhibition of Hep3B cells induced by 5-FU,down regulation of receptor PTC1 with distribution change and cell cycle related protein,cyclin E.Besides,we found a unique distribution character of PTC1 at telophase in mitosis unexpectedly,which implies its involvement in regulation of mitosis.The study provides a new therapeutic strategy for 5-FU based therapy.As for HCC patients with SHh signaling pathway activated,the combination of inhibitor of SHh signaling pathway and other chemotherapeutic agents may represent a more promising therapy for HCC patients.In addition,the study made the research on combination chemotherapy of a new chemical,TV(tetrazolium violet) and traditional nitrosourea chemotherapeutic agents,ACNU(nimustine hydrochloride),which can cause synergistic effect in combination chemotherapy.One of important ways to promote synergy in chemotherapy is to increase the sensitivity of therapeutic agents,especially for traditional clinical drugs,such as ACNU.And this have become to important topics for researchers.Targeting to different mechanisms of chemotherapeutic agents,researchers have increased the sensitivity of different therapeutic agents with different ways,but all the destinations are to increase the chemotherapeutic effect of drugs or decrease their deleterious effect to normal cells.For the sake of limited research depth and selective range,in present,the common way to reverse various possible drug-resistance mechanisms is combination chemotherapy.TV is a tetrazolium salts that can elicit cell growth arrest at G1 phase and apoptosis for tumor cells.Recent research indicated that TV can cooperate with BCNU(carmustine) to elevate the sensitivity of tumor ceils to BCNU. Therefore,TV is a promising chemotherapeutic agent.But whether the combination chemotherapy of ACNU and TV can cause synergy in treatment for gliomas,and whether this combination chemotherapeutic approaches for gliomas will be improved is unknown.Thus,we perform the tests on C6 rat glioma cells which is an ideal model in vitro for gliomas in mammals and we investigate the synergistic effect of this combination chemotherapy and possible synergistic mechanism,which may have essential significancy in therapy in gliomas.The present study indicated that the combination chemotherapy of ACNU and TV can promote proliferative inhibition of in C6 cells and promote apoptosis, comparing with treatment in single drug;at the same time,this combination chemotherapy will cause up regulation in wild type p53 protein,cell cycle related protein p21 and p27;furthermore,we measured the activity of drug-resistant protein, only to find little relevance.Thus,the synergistic mechanism of this combination needs further tests and other analysis in future.Since the synergistic effect of combination chemotherapy for gliomas is a complicated process with multiple factors,it is hard to elucidate the practical problems with the function of only one gene or one protein.Thus,the research on gliomas must seek the drug-resistant mechanisms from multiple genes and approaches in order to reverse the drug-resistance and improve the chemotherapeutic efficacy.On the whole,the two parts of this study improved basic research on chemotherapeutic efficacy and expand the investigation field.Although the specific mechanisms is yet required to further investigation,these data provide the helpful clues to further investigation on chemotherapeutic mechanism of action,and to enrich the chemotherapeutic approaches.Innovations:To report for the first time that down-regulation of SHh signaling pathway activity is involved in 5-fluorouracil-induced apoptosis and motility inhibition in Hep3B cells,which provides new clues to chemotherapeutic synergy in future, combined with alterations of cell signaling pathways.To report for the first time that combination chemotherapeutic of TV and ACNU will bring about synergistic effect on C6 cells,which will provide effective scheme to combination chemotherapy clinically. |
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