Effects Of Arsenic Trioxide On Malignant Tumors And The Molecular Mechanisms Involved

1Arsenic trioxide inhibiting catalase gene expression to promote the apoptosis ofhuman osteosarcoma MG63cells and the mechanisms involvedOsteosarcoma is the most common malignant bone tumor in children and adolescents.Many patients are not cured by the current osteosarcoma therapy consisting of combinationchemotherapy along with surgery and thus new treatments are urgently needed. Consideringthat the majority of high-grade osteosarcoma patients are children and adolescents who havea long life expectancy, the long-term effects of chemotherapy must be taken into account.Therefore, new regents that have no or less accumulation toxicity for the body are criticalrequired for the effective chemotherapy.Arsenic trioxide has been successfully used to treat leukemia and solid malignanttumors. Little is known on the therapeutic effects of arsenic trioxide on p53mutantosteosarcomas. To explore the possibility for arsenic trioxide to be developed as atherapeutic reagent and to clarify its molecular mechanisms, the studies were carried out byanalyzing the effects of arsenic on cell apoptosis, intracellular reactive oxygen species(ROS), and activity and the mRNA expression level of catalase in human osteosarcomaMG63cells.Objective: To explore the possibility for arsenic trioxide to be developed as atherapeutic reagent and to clarify its molecular mechanisms. Method: We uesd AnnexinV-FITC/PI to detect the apoptotic cells, ROS detection reagent kit for intracellular reactiveoxygen species level, catalase activity kit for the activity of intracellular hydrogen peroxideenzyme and real-time PCR assay for intracellular catalase mRNA level. Results: Exposure ofMG63cells to arsenic resulted in significant increases of apoptotic cells in a dose-dependentmanner compared with that of non-treated cells. The lowest concentration for arsenic toinduce significant cell apoptosis is10μM, suggesting the possibility for it to be developed asa therapeutic reagent in treating p53mutant osteosarcomas. The cell apoptosis wascompletely prevented by the presence of a general anti-oxidative regent, NAC (10mM) in theculture. Interestingly, catalase (10mM) has the same protective role as NAC did; Followingexposure to the arsenic, intracellular ROS levels were found to be significantly increased in adose-dependent manner. The concentration of arsenic causing significant increase of intracellular ROS level is5μM, which is lower than that resulting cell apoptosis (10μM).Importantly, arsenic-induced intracellular ROS increase correlated well with it-mediated cellapoptosis. Meanwhile, the presence of NAC (10mM) or catalase (10mM) effectivelyprevented the ROS accumulation as well as the protective role on cell apoptosis, describedabove; arsenic in both lower (5μM) and higher (20μM) concentration significantly decreasesenzyme activities and the transcript of catalase. Conclusion: The suppression on catalase isthe major mechanism for intracellular ROS accumulation and resulted apoptosis resultedfrom arsenic exposure.2Arsenic trioxide inhibiting melanoma B16cell proliferation and promotingapoptosisDue to the aggravation of environmental pollution, ozone layer are destroyed, humanexposure to ultraviolet rate rises ceaselessly, the risk of melanoma increased significantly,thus annual incidence rates of melanoma are rising rapidly, especially prevalent in youngadults in more outdoor activities. Once the progression advanced, melanoma is very easy tobe invasion and metastasis, but at present there is no effective solutions and the prognosis ispoor. It also requires long-term effective chemotherapy with little toxic side effects. Thisstudy showed that arsenic trioxide in low concentration can cause malignant MG63cellssignificantly apoptosis. Therefore, arsenic trioxide has the potential to be a long-termchemotherapy regimen.Objective: We examined the effect of arsenic trioxide on melanoma B16cells and themolecular mechanisms involved. Methods: We use the CCK-8kit for cell proliferation, cellcycle detection kit for the detection of the cell cycle, Annexin V-FITC/PI for detection ofapoptosis. Results: With the5μM concentration of arsenic trioxide, compared with thecontrol group, cell proliferation rates began to decrease (P<0.05), with a dose-effect relationand the IC50value was65.3±6.5μM; With the5μM concentration of arsenic trioxide in24h,compared with0h group, cell proliferation rate was significantly reduced (P<0.05), and theresults are consistent. In48h, the cell proliferation inhibition effect was more significantly(P<0.01), and similar with the results followed; Compared with untreated groups arsenicdecreased the number of cells in S phase, increased G0/G1phase cells, and the values ofSPF, PI were significantly lower than the control group(P<0.05); With the5μMconcentration of arsenic trioxide, the apoptosis rate increased significantly(P<0.05), with adose-effect manner, and the IC50value was70.4±3.6μM. Conclusion: Arsenic Trioxide cansignificantly inhibit the proliferation of B16cells by blocking the G1→S process; Another important mechanism is significantly promoting cell apoptosis.3Effects of arsenic trioxide on overexpression of Oct4, Sox2, Klf4and c-Myc B16cells by inhibiting proliferationIn2006, according to Takahashi and yamanaka reported with retroviral mediated fourtranscription factor Oct4, Sox2, Klf4and c-Myc(OSKM) transducting to the differentiationand maturation of mouse fibroblast cells, made the differentiation cells reprogramming toembryonic stem-like pluripotent stem cells. More recently it has also been used tumor celllines as a receptor, take exogenous transcription factor Oct4into isolated tumor cells toinduce tumor stem cells. Tumor stem cells are tumor initiating cells, determining the tumoroccurrence, development, metastasis and prognosis of recurrence. We will transfect the fourfactors into B16cells to detect the relationship between the transfected cells and cancer stemcells. While making these factors into tumor cells, requires a suitable carrier.3.1The adjustable carrier mediating four factors expression in B16cellsObjective: To detect what effect of the four factors system transfected in tumor cells canbe induced. Methods: We use the plasmid transfection techniques, TetO-FUW-OSKM andFUW-M2rtTA two plasmid was transfected into B16cells together, by real-time PCR,Western blot, immunochemical method for identification. Results: Compared with parentcells, the Oct4, Sox2, Klf4and c-Myc expression were significantly higher in the clones withthe DOX induced, while the gene expression did not change significantly in the parent cellswith DOX; of intracellular Oct4and c-Myc protein was expressed in the DOX inducedgroup, compared with parent cells; Compared with the control group, Oct4and c-Mycprotein expressed in cytoplasmic and nuclear expression were significantly enhanced inDOX group. Conclusion: The four factors plasmid system transfected in B16cells weresuccessful. It is successful that we establish a DOX regulated and mutual control cell model.3.2Four factors promoting the cell proliferation of B16cells and inhibiting theapoptosisObjective: To find whether there are some differences between the cells with overexpression of four factors has and its parent cells. Methods: We use the CCK-8assay for cellproliferation, cell cycle detection kit for the detection of the cell cycle, Annexin V-FITC/PIfor detection of apoptosis, real-time PCR assay for the detection of mRNA gene expressionlevel. Results: The cell proliferation rate of+DOX group and the parent group wassignificantly increased since24h (P<0.05); In+DOX group, cells in S phase increased, G0/G1phase decreased and the values of SPF, PI was significantly greater than that of the control group (P<0.05); The apoptosis rate of+DOX cells was significantly lower(P<0.05);The mRNA level of three proliferation related gene were significantly higher in+DOX cells(P<0.05), respectively, they were cyclinB1, JAK1and Stat3. The apoptosis inhibition relatedgene Bcl2was significantly higher (P<0.01) and apoptosis promotion related gene Baxreduced significantly; under normal culture conditions, the cell morphology of two groupswere no differences. By adding cell factors, cell spheres were significantly increased in+DOX cells(P<0.05). Conclusion: the four factors:1can promote cell proliferation byincreasing the number of cells in S phase cells.2can inhibit cell apoptosis.3can upregulatethe mRNA level of cyclinB1, JAK1, Stat3and Bcl2and reduced Bax.4can increase thecancer stem-like cell spheres.3.3Over expression of four factors in B16cells increasing the tumor formation rate invivoObjective: In order to test the situation of over expression of four factors in cells in vivo.Method: We use two groups of DOX induced and non induced cells in C57mice as testmodels in vivo. We use HE staining, real-time PCR method for the detection of two groupsof cells in vivo proliferation. Results:7d after inoculation, subcutaneous tumor can betouched in-DOX cells in group, and in+DOX group at5days the tumor formation can beobserved, and the tumor volume and tumor weight has significant difference (P<0.01);Compared with the control group, tumor tissues over expression of four factors under themicroscope shows: more visible karyokinesis can be found(P <0.05); The mRNA levels ofthe four factors were significantly up-regulated in+DOX cells; The mRNA levels of cellproliferation related genes cyclinB1and JAK1rised significantly. Conclusion: The overexpression of four factors cells formed the transplanted tumor rate significantly increased inmice, and four factors enhanced cell proliferation ability is consistent in vitro experiment;Over expression of four factors in cells formed more karyokinesis increased significantly;The mRNA levels of cell proliferation related genes cyclinB1and JAK1rised significantly,supported that four factor enhanced cell proliferation ability in vitro and the tumor formingspeed in vivo.3.4Arsenic trioxide inhibiting cell proliferation and promoting apoptosis on overexpression of four factors in B16cellsObjective: We examined the effect of arsenic trioxide on over expression of four factorsin B16cells and the molecular mechanisms involved. Methods: We use the CCK-8kit forcell proliferation, cell cycle detection kit for the detection of the cell cycle, Annexin V-FITC/PI for detection of apoptosis. Results: With the5μM concentration of arsenictrioxide, compared with the control group, cell proliferation rates began to decrease (P<0.05), with a dose-effect relation and the IC50value was23.1±6.3μM; With the5μMconcentration of arsenic trioxide in24h, compared with0h group, cell proliferation rate wassignificantly reduced (P <0.05), and the results are consistent. In48h, the cell proliferationinhibition effect was more significantly (P<0.01), and similar with the results followed;Compared with untreated groups arsenic decreased the number of cells in S phase, increasedG0/G1phase cells, and the values of SPF, PI were significantly lower than the control group(P<0.05); With the5μM concentration of arsenic trioxide, the apoptosis rate increasedsignificantly (P<0.05), with a dose-effect manner, and the IC50value was69.2±3.9μM.Conclusion: Arsenic Trioxide can significantly inhibit the proliferation of over expression offour factors in B16cells by blocking the G1→S process; Another important mechanism issignificantly promoting cell apoptosis.4Four factors promotes arsenic trioxide on proliferation inhibitionObjective: To study the effect of four factors on cell cytotoxicity of arsenic trioxide andits molecular mechanism involved. Methods: We used CCK-8for cell proliferation, cellcycle detection kit for the detection of the cell cycle, Annexin V-FITC/PI for detection ofapoptosis. Results: With different concentrations of arsenic trioxide, the+DOX group ofcells in the IC50was23.1±6.3μM, significantly lower than the IC5065.3±6.5μM of controlgroup (P <0.01); With the5μM concentration of arsenic trioxide in24h, compared with-DOX group, cell proliferation rate was significantly reduced (P <0.01), and similar with theresults followed.; Compared with-DOX group, arsenic decreased the number of cells in Sphase, increased G0/G1phase cells, and the values of SPF, PI were significantly lower inthe+DOX group (P <0.05); With different concentrations of arsenic trioxide, the apoptosisrates of the two groups had no significant difference, the same with the IC50values, and theapoptosis rate has no significant changed. Conclusion: Four factors significantly enhancedthe arsenic trioxide induced cells arrest in G1→S phase, to enhance the sensitivity of cells toarsenic trioxide, and the four factors promoting the sensitivity of cells to arsenic trioxide wasnot through the promotion of arsenic trioxide inducing apoptotic.