All-trans Retinoic Acid And Fucoidan On The Tumor Chemopreventive Mechanism

Objective: Cervical cancer is one of the common malignant tumors, which is seriously treathening women health. The study is to explore the relationship between TopoIIαand chemotherapy of cervical carcinoma; to understand the mechanism of ATRA-trigged regulation of TopoIIαin human cervical carcinoma HeLa cells and to learn whether the alternation of TopoIIαhas the influence on cell differentiation and drug resistance in HeLa cells.Method: Immunohistochemistry was used to detect the level of TopoIIαprotein in cervical carcinoma tissure from patients before and after chemotherapy. The alternation of endogenous and exogenous TopoIIαprotein caused by various dosages of ATRA was detected by western blot. TopoIIαmRNA level was detected by revers transcription-PCR. The protein synthesis inhibitor cycloheximide (CHX) was used to confirm the change of protein turnover rate. The alternation of TopoIIαwas examined after treated with MG-132 and leupeptin. Immunoprecipitation and western blot were used to detect whether ubiquitination or ISG15ylation were involved in modification of TopoIIα. The nitroblue tetrazolium analysis was carried out to detect differentiation of HeLa cell after the cells were transiently transfected with TopoII? plasimid or siRNA targeting TopoII?. Meanwhile, cyclin D1 expression was also examined by western blot. The alteration of TopoIIαlocalization was detected by immunofluorescence. The expression of P-gp level in the cells treated with ATRA or etoposide was detected by western blot.Results:1. TopoIIαis a chemotherapeutic target for cervical carcinomaImmunohistochemistry assay showed that the immunostaining intensity of TopoIIαwas much higher in cervical carcinoma tissue than in normal cervix tissue. TopoIIαlevel was remarkably decreased after chemotherapeutic treatment.2. In human cervical carcinoma HeLa cells, ATRA promoted degradation of TopoIIαATRA decreased endogenous and exogenous TopoIIαprotein level in a dose-dependent way.3. The declines of TopoIIαoccured in a translation or post-translation levelATRA did not change the mRNA level of TopoIIα. The rapid degradation of ATRA-induced TopoIIαdecline was occurred after CHX treatment. This suggests that declines of TopoIIαoccured in a translation or post-translation level.4. ATRA triggered degradation of TopoIIαvia proteasomal pathwayWhen cells were treated with ATRA in the presence of leupeptin, a lysosome inhibitor, TopoIIαlevel still went down. However, MG-132, a proteasome inhibitor, was able to reverse the decreased TopoIIα, compared with only ATRA treatment. Polyubiqutination and ISG15 conjugation were observed when the cells were co-transfected with TopoIIαand Ub or ISG15.5. TopoIIαwas involved in HeLa cell differentiationIt suggested that ATRA increased cell differentiation by more than 20%. Overexpression of TopoIIαresulted in cell de-differentiation. The de-differentiation was recovered in the presence of ATRA. However, knockdown of TopoIIαcaused more than 20% of cells differentiation. Overexpression of TopoIIαincreased the expression of cyclinD1, while knockdown of TopoIIαreduced the level of cyclinD1.6. ATRA had an effect on location of TopoIIαand drug resistanceImmunocytochemistry analysis was performed followed by ATRA treatment. The results revealed that only nuclear fluorescence faded with the treatment of ATRA, while the fluorescence intensity within the cytoplasm did not change.Western blot showed that ATRA treatment could reduce P-gp level, while the TopoIIa inhibitor epotoside treatment did not.Conclusion: TopoIIαcould be a specific target for diagnosis and therapy of cervical cancer. ATRA-induced TopoIIαdecline is occurred in the level of post-translation, which ubiquitination could be involved. ISG15ylation may have an effect on the expression of TopoIIα. It is a novel standpoint that TopoIIαis related with tumor cell defferientiation. ATRA-induced TopoIIαdegradation was occured in cell nucleus but not cytoplasm. ATRA could induce the decline of P-gp. Taken together, these data suggest ATRA may be a good candidate for treatment of cervical cancer. Objective: To explore the effect of fucoidan on hepatoma cell proliferation and the related mechanisms.Method: Proliferation inhibition rate of hepatoma HepG2 cells was measured by MTT. HepG2 cells were treated with a variety of dosage of fucoidan (0μg/ml,10μg/ml,100μg/ml and 500μg/ml). The morphology change of the cells was observed under microscopy. Apoptosis was detected by Hoechst 33258 staining and DNA Ladder analysis. CyclinD1 and TopoIIαwere used as the proliferation biomarker, and their protein levels were examined by western blot.Result: Fucoidan inhibited HepG2 proliferation in a dose-dependent manner. There was a remarkable morphological change when cells were treated of 500μg/ml of fucoidan. Apoptosis was occurred when the cells were incubated with 100μg/ml and 500μg/ml of fucoidan. In addition, fucoidan also suppressed the expression of cyclinD1 and TopoIIαin HepG2 cells.Conclusion: Fucoidan can inhibit the proliferation of HepG2 cells and induce apoptosis. Meanwhile, fucoidan also has the ability to suppress the protein expression of cyclin D1 and topoIIα, which may be one of the mechanisms of inhibition of hepatoma cells proliferation.