| Hepatocellular carcinoma(HCC) is one of the most common and aggressive malignancy worldwide. It has been currently ranked the fifth common solid carcinoma and the third leading cause of cancer-related death worldwide, as well as the second leading cause of death due to cancer. Despite tremendous achievements being made basically and clinically especially in the surgical resection during past decades, the prognosis of HCC remains dismal. Surgery is currently the only curative treatment but it is only feasible in a minority of patients. Even after curative resection, the 5-year recurrence rate was more than 60 percent.the high recurrence rate becomes the major obstacle of improving prognosis. For those patients who have advanced stage disease, poor liver function, or recurrent tumors after local treatments, systemic pharmacologic treatment is the final and main therapy. Unfortunately, the response rate to traditional chemotherapy for HCC patients is quite low and the outcome is also poor. Traditional drugs,such as 5-FU,cisplatin,ADM, show low curative effect.Hence, new drugs with a higher effect and hypotoxic or chemotherapy method which combined traditional and new drugs are urgently needed key.Nuclear factor kappa B is widely distributed in eukaryotic cells. In recent years,NFκB has become hotspot of studying. It has been reported that NFκB plays a key role in cell proliferation, apoptosis, invasiveness, metastasis, tumorigenesis, and angiogenesis. extracellular matrix plays an important role in tumor metastasis.MMPs can be upregulated expression through NFκB pathway. Recently, it has been reported that the resistance of tumor chemotherapy is related with NFKB.It is activated by diverse stimuli, which include pro-inflammatory cytokines, cellular stress as well as growth factors; and also known to be activated by chemotherapeutic drugs, including daunorubicin, adriamycin, as well as cisplatin.NF-κB is also a key regulator of apoptosis, where it suppresses apoptotic cell death by inducing the expression of several anti-apoptotic factors. Its activation is tightly regulated by Inhibitor of kappa B (IκB).Hence, optimal inhibitor of NFκB combined with traditional drugs,which is blocking NFκB pathway and suppressing expression of related-proteins may decrease local recurrence and improve patient survival. Pyrrolidine dithiocarbamate (PDTC) is a low molecular-weight thiol compound with a variety ofbiochemical activities such as redox state alternation, heavy metal chelation,and enzyme inhibition. PDTC was initially regarded as a an antioxidant compound both to counteractthe toxic effects of free radicals and to interferewith the generation of pro-inflammatory cytokines,and it has beenused as potent inhibitor of nuclear factor-KB (NF-KB).It has been reported that the protective effects of PDTC has the potential to increase the expressions of genes for endogenous antioxidants, such as superoxide dismutase, it also has attributed to its ability to inhibit NF-κB.We design vitro experiment to study th impact of pyrrolidine dithiocarbamate on the proliferation of hepatocellular carcinoma cell HepG2 and on adhering and invasive abilities which are closely relative to metastasis and correlating mechanism. Meantime, we study the synergistic effect of pyrrolidine dithiocarbamate combined with adriamycin in resisting tumor proliferation, apoptosis and metastasis, and correlating mechanism of congenerous effect.PARTâ… The vitro experiment of pyrrolidine dithiocarbamate on the proliferative,apoptosis and invasive ability of HepG2 cellsObjective:To approach the impact of pyrrolidine dithiocarbamate (PDTC) on the proliferative, and apoptosis of HepG2 cells, and on adhering,invasive abilities of HepG-2 cells which are closely related to tumor metastasis and mechanism. Methods:Take human hepatocellular carcinoma cell HepG2 as the research object, detect the impact of PDTC of different concentration on the growth of HepG2 cells using MTT. Detect cell cycle and cell apoptosis variation after treated with PDTC with flow cytometry. Observe the variation of cell adhering, invasion experiment and detect the influence of PDTC on the expression of tumor metastasis relative factor MMP-2,TIMP-2 and NFκBp65 with immunocytochemical methods. Results:(1)In PDTC-treated group, cell survival rate decreased compared to control group, snd this effect became more significant with the increase of drug concentration. (2) In PDTC 50μmol-L-1-group, apoptosis rate was 16.1% and cells were blocked G0/G1 stage, the differences between control and PDTC 50μmol-L"1-treated group were statistical significant. (3)After treated PDTC,NFκBp65 and MMP-2 score were(5.80±0.80) and (2.75+0.74) respectively,while score of the control group is (5.25+0.72).The differences were statistical significant. (4)Cells in PDTC-treated group show adhere inhibition ratio to Matrigel after 60,90 min as (68.02±12.14)% and (58.23+10.42)% respectively. In Transwell trial, the invasive cell number of PDTC-treated group were (67.31±9.53),The differences compared with control group were statistical significant. Conclusion:PDTC could inhibit the growth of HepG-2 cell,and the inhibiting effect is time-concentration dependent. PDTC could detain HepG-2cell at G0/G1 stage and increase the apoptosis rate. PDTC could also inhibit the adhering,invasive abilities which are relative to tumor metastasis,this is because PDTC could decrease the expression of NFκBp65 and MMP-2 through inhibiting the activation of NFκB.PART II Effect and mechanism of nuclear factor-κB inhibitor PDTC combined with adriamycin on the hepatocellular carcinoma cell HepG 2Objective To study the effect and possible synergistic mechanism of pyrrolidine dithiocarbamate (PDTC),a potent inhibitor of nuclear factor kappa B (NFκB),combined with adriamycin (ADM)on human hepatocellular carcinoma cell HepG2. Methods HepG2 cells were incubated and divided into control group, ADM group,PDTC group, and PDTC+ADM group. The cells growth activity was measured by growth curve and the proliferation and cell apoptosis rates were evaluated by MTT reduction assay and the median-effect principle was also used. Use flow cytometry to detect cell cycle and cell apoptosis variation after treated with PDTC combined with Adriamycin. Detect the influence of different group on the expression of NFκBP65 of HepG2 cells with immunocytochemical methods.Results:(1)In PDTC combining with Adriamycin group, cell inhibiting rate increased more than signal-drug group,and it presented dose dependent. When they were combined,the median-effect dose of Adriamycin and PDTC were 58.31ng·ml-1 and 29.11μmol·L"1 respectively. While Adriamycin and PDTC were used, the dose were 109.52ng·ml-1 and 87.42μmol·L-1 respectively. The median-effect dose of combined group was less than signal-drug group.From the dose-effect curve,,when the effect was less than 0.82, they performed synergism when they used together. (2)In signal Adriamycin group,cell apoptosis rate was (85.00+5.00)%,cells were blocked at G2/M stage,whose cell occupied (36.67+6.65)%. In signal PDTC group,cell apoptosis rate was(11.00±2.65)%,the ratio of G0/G1 stage cells was (78.33+9.07)%. While in the combination group, apoptosis rate increased to (31.33±8.62)%, G0/G1 stage cells occupied (85.00±5.00)%.The differences of G0/G1 stage cells and cell apoptosis between them are statistical significant.(3)The score of NFκB P65 expression rate after treated with PDTC and Adriamycin combined was (2.35+0.54),while the score after treated with PDTC and Adriamycin respectively were (5.45+0.78), (3.05±0.62).The difference between combined group and control group whose score was (5.35±0.83)are statistical significant.Conclusions Both PDTC and Adriamycin can cause human hepatocellular carcinoma cell HepG2 growth inhibition. The effect of inducing cell growth was more significant.and they could produce the synergism effect in vitro experiment. Combining PDTC and Adriamycin could block cell at G0/G1 stage and they could promote the increase of apoptosis rate. NFκB P65 expression was inhibited after treated PDTC and Adriamycin. The mechanism of repression may be that PDTC could inhibit the activition of NFκB which was brought by Adriamycin,... |
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