Regulation Mechanism Of Telomerase In Joint Action Of Selenium And Cadmium On Carcinogenesis

Cadmium(Cd) is an important environmental pollutant and is widely dispersed in the work place and living environments. Acute and chronic exposure to cadmium can induce functional disturbance of liver, kidney, prostate and lung etc. Because of its characteristics as a lung carcinogen, cadmium has been classified as a category 1 carcinogen (human carcinogen) by IARC in 1993. Mechanisms of cadmium in carcinogenesis remain unclear though much study has been investigated on its toxicological effects.Telomerase is a specialized RNA template-containing reverse transcriptase that allows the replication of the telomeric repeats present at the end of all eukaryotic chromosomes. The human telomerase complex comprises several components, human telomerase RNA component (hTR), which is used as a template in DNA replication; human telomerase reverse transcriptase (hTERT), a human telomerase catalytic subunit; and human telomerase-associated protein 1 (hTEP1). Being involved in cellular immortality, the telomerase activity is present in most types of human tumors and absent in noncancerous cells. Concomitant up-regulation or down-regulation of the hTERT mRNA expression and telomerase activity during cell immortalization or differentiation is observed, suggesting that control of the hTERT expression at the mRNA level mainly contributes to the regulation of telomerase enzymatic activity. Thus hTERT is the key component for the control of telomerase activity. Telomerase is a molecule marker of malignant cell and it becomes a new target of anticarcinogenic agent. We assume that cadmium through activate telomerase activity to induce tumorigenesis because many factors that can regulate hTERT also can be influenced by cadmium.Selenium(Se) is an essential trace element with several important biological functions and has been received considerable attention for its possible role as an effective, naturally occurring, anticarcinogenic agent. Therefore it's important to understand character of selenium and its mechanism on carcinoma prevention and anticancer. The tumor preventive effect of selenium compounds has also been shown in human cancers especially in decreasing the morbidity of cancer in gastrointestinal tract, liver and respiratory system. It has two purposes why we chose selenium to study: the first one is that selenium is an anticarcinogenic agent, and the second, selenium has antagonistic effect on cadmium.In this study, we investigated the inhibitory effect of sodium selenite on the cadmium-transformed 16HBE cells, effect of cadmium on telomerase and antagonistic effect of selenium on the changes induced by cadmium using TRAP-PCR, RT-PCR and immunohistochemistry methods. Moreover, this research would shed light on carcinogenesis mechanism of cadmium, and these data provided a basis for further comprehending the anticarcinogenic effects of selenium, and antagonistic effects of selenium on cadmium in particular.Part 1 Study on telomerase activity and regulatory factors in cadmium-transformed 16HBE cells on exposure to seleniumSection 1Telomerase activity and apoptosis in cadmium-transformed 16HBE cells onexposure to seleniumObjective To investigate the effects of sodium selenite on telomerase activity andapoptosis in cadmium-transformed 16HBE cells.Methods Telomerase activity and apoptosis were measured after culturedcadmium-transformed 16HBE cells were exposed to sodium selenite at different doses (0.625, 1.25, 2.50, 5.00μM) for 24 hours. We use MTT to detect cell viability, TRAP-fluorescence to study telomerase activity and flow cytometry (FCM) to detect apoptosis. Results Cell viability was decreased and there existed significant difference at 1.25-5.0μmol/L selenium group compared to control group. There existed an obvious dose-effect relationship between the selenium concentration and these changes. Selenium caused decrease in telomerase activity in cadmium-transformed 16HBE cells. Apoptosis induced by selenium was increased on cadmium-transformed 16HBE cell and the rates of apoptosis in selenium groups at the doses of 1.25-5.0μmol/L were higher than that of control group.Conclusion It was suggested that cell viability, telomerase activity of cadmium-transformed 16HBE cell was decreased and apoptosis was increased after incubated with sodium selenite for 24 hours. And there exists a significant correlation between selenium concentration and these changes.Section 2Expression of hTERT and regulatory factors in Cadmium-transformed 16HBECells on Exposure to SeleniumObjective To investigate the effects of sodium selenite on expression of hTERT mRNAand regulatory factors in cadmium-transformed 16HBE cells.Methods Expression of hTERT and regulatory factors were measured by RT-PCR aftercultured cadmium-transformed 16HBE cells were exposed to sodium selenite at differentdoses (0.625, 1.25, 2.50, 5.00μM) for 24 hours.Results Selenium caused decrease in hTERT expression in cadmium-transformed 16HBE cells. There existed an obvious dose-effect relationship between the selenium concentration and these changes. And the expression of c-myc, p53, bcl-2 mRNA also decreased but the expression of mad1 mRNA was increased after exposure to selenium for 24 hours. No difference was found on expression of bax mRNA after incubated with sodium selenite for 24 hours compared with control group. Conclusion It was suggested that selenium can decrease hTERT, c-myc, p53, bcl-2 mRNA and increase mad1 mRNA in cadmium-transformed 16HBE cells and there exists a significant correlation between selenium concentration and these changes.Section 3Expression of hTRF1 and hTRF2 in cadmium-transformed 16HBE cells onexposure to seleniumObjective To investigate the effects of sodium selenite on expression of hTRF1 andhTRF2 mRNA in cadmium-transformed 16HBE cells.Methods Expression of hTRF1 and hTRF2 mRNA were measured by RT-PCR aftercultured cadmium-transformed 16HBE cells were exposed to sodium selenite at differentdoses (0.625, 1.25, 2.50, 5.00μM) for 24 hours.Results No difference was found on expression of hTRF1 and hTRF2 mRNA after incubated with sodium selenite for 24 hours compared with control group. Conclusion It was suggested that selenium has no effect on hTRF1 and hTRF2 mRNA after incubated with sodium selenite for 24 hours.Part Two Effects of selenium and cadmium and their joint action on telomerase activity and regulatory factors in rat liverSection 1Effects of selenium and cadmium and their joint action on telomerase activity andapoptosis in rat liverObjective To study the effects of sodium selenite, cadmium chloride and their jointaction on telomerase activity and apoptosis in rat liver.Methods Male SD rats were divided randomly to 16 groups, each group had 5 animals.The groups include: control group, Se groups (Na₂SeO₃ at the doses of 2.5, 5 and10μmol/kg), Cd groups (CdCl₂ at the doses of 5, 10 and 20μmol/kg), Se(Na₂SeO₃ at thedoses of 2.5, 5 and 10μmol/kg) + Cd(CdCl₂ at the doses of 5, 10 and 20μmol/kg) groups.After 48h of the first injection, the telomerase was measured with TRAP- fluorescenceand apoptosis was detected by flow cytometry (FCM). Results No difference was found on telomerase activity after Se intragastric administration at the doses of 2.5, 5 and 10μmol/kg for 48h. Apoptosis was increased at the doses of 5, 10μmol/kg selenium compared to control group. Cd at the doses of 5, 10 and 20μmol/kg could increase the telomerase activity and apoptosis in rat liver, compared with control group, statistical analysis yielded P<0.01. There existed an obvious dose-effect relationship between the Cd concentration and these changes. In the joint action groups of selenium and cadmium, selenium at the doses of 2.5, 5 and 10μmol/kg could decrease telomerase activity and apoptosis over expressions induced by cadmium at the doses of 5, 10 and 20μmol/kg respectively. But statistical analysis showed that telomerase activity expressions in 2.5μmol/kg Na₂SeO₃+ 5μmol/kg CdCl₂ group, 2.5μmol/kg Na₂SeO₃+ 20μmol/kg CdCl₂ group and μmol/kg Na₂SeO₃+ 5μmol/kg CdCl₂ group were lower than those of correspondent Cd group respectively and significantly. And apoptosis rate in 2.5μmol/kg Na₂SeO₃+ 5umol/kg CdCl₂ group, 2.5μmol/kg Na₂SeO₃+ 10μmol/kg CdCl₂ group, 2.5μmol/kg Na₂SeO₃+ 20μmol/kg CdCl₂ group, 5μmol/kg Na₂SeO₃+ 10μmol/kg CdCl₂ and 5μmol/kg Na₂SeO₃+ 20μmol/kg CdCl₂ group were lower than those of correspondent Cd group respectively and significantly. The telomerase activity and apoptosis in 10μmol/kg Na₂SeO₃ + 5, 10 and 20μmol/kg CdCl₂ groups were no significant different from correspondent Cd groups, but higher than that of control significantly.Conclusion It was suggested that selenium at the dose of 10μmol/kg can enhance apoptosis, and cadmium at the dose of 5～20μmol/kg can increase telomerase activity and apoptosis. Selenium at the doses of 2.5～5μmol/kg have antagonistic effect on over-expression of telomerase activity and apoptosis induced by cadmium in rat liver.Section 2Effects of selenium and cadmium and their joint action on expression of TERT andc-myc, p53 in rat liverObjective To study the effects of sodium selenite, cadmium chloride and their jointaction on expression of TERT and c-myc, p53 in rat liver. Methods Male SD rats were divided randomly to 16 groups, each group had 5 animals. The groups include: control group, Se groups (Na₂SeO₃ at the doses of 2.5, 5 and 10μmol/kg), Cd groups (CdCl₂ at the doses of 5, 10 and 20μmol/kg), Se(Na₂SeO₃ at the doses of 2.5, 5 and 10μmol/kg) + Cd(CdCl₂ at the doses of 5,10 and 20μmol/kg) groups. After 48h of the first injection, the expression of TERT, c-myc, p53 mRNA was measured with RT-PCR, c-Myc and P53 proteins were measured by immunohistochemistry method.Results No difference was found on expression of TERT mRNA induced by sodium selenite at the doses of 2.5, 5 and 10μmol/kg compared with control group. Se at the dose of 10μmol/kg could increase TERT mRNA, c-myc mRNA, c-Myc protein and p53 mRNA expression in rat liver, compared with control group, statistical analysis yielded P<0.05. Se at the doses of 5, 10μmol/kg could induce P53 protein over-expression compared with control group. Cd at the doses of 5, 10 and 20μmol/kg could induce TERT mRNA, c-myc mRNA, p53 mRNA and P53 protein over-expression in rat liver compared with control group statistical analysis yielded P<0.05 or P<0.01. Cd at the doses of 10, 20μmol/kg could induce c-Myc protein over-expression compared with control group. In the joint action groups of selenium and cadmium, selenium at the doses of 2.5, 5 and 10μmol/kg could decrease TERT mRNA, c-myc mRNA and protein, p53 mRNA and protein over expressions induced by cadmium at the doses of 5, 10 and 20μmol/kg respectively. But statistical analysis showed that TERT mRNA expressions in 2.5μmol/kg Na₂SeO₃+ 5μmol/kg CdCl₂group, 2.5μmol/kg Na₂SeO₃+ 10μmol/kg CdCl₂ group and 5μmol/kg Na₂SeO₃+ 10μmol/kg CdCl₂ group were lower than those of correspondent Cd group respectively and significantly, c-myc mRNA in 2.5μmol/kg Na₂SeO₃+ 5μmol/kg CdCl₂ group, 2.5μmol/kg Na₂SeO₃+ 10μmol/kg CdCl₂ group and 5μmol/kg Na₂SeO₃+ 5μmol/kg CdCl₂ group were lower than those of correspondent Cd group respectively and significantly. p53 mRNA in 2.5μmol/kg Na₂SeO₃+ 5μmol/kg CdCl₂ group, 2.5μmol/kg Na₂SeO₃+ 10μmol/kg CdCl₂ group, 5μmol/kg Na₂SeO₃+ 5μmol/kg CdCl₂ group and 5μmol/kg Na₂SeO₃+ 10μmol/kg CdCl₂ group were lower than those of correspondent Cd group respectively and significantly. c-Myc protein in 2.5μmol/kg Na₂SeO₃+10μmol/kg CdCl₂ group, 2.5μmol/kg Na₂SeO₃+ 20μmol/kg CdCl₂ group, and 5μmol/kg Na₂SeO₃+10μmol/kg CdCl₂ group were lower than those of correspondent Cd group respectively and significantly. P53 protein in 2.5μmol/kg Na₂SeO₃+ 5μmol/kg CdCl₂ group, 2.5μmol/kg Na₂SeO₃+10μmol/kg CdCl₂ group, 2.5μmol/kg Na₂SeO₃+ 20μmol/kg CdCl₂ group, 5umol/kg Na₂SeO₃+ 5μmol/kg CdCl₂ group and10μmol/kg Na₂SeO₃+ 5μmol/kg CdCl₂ group were lower than those of correspondent Cd group respectively and significantly. The expression of TERT RNA in10μmol/kg Na₂SeO₃ + 5, 10 and 20μmol/kg CdCl₂ groups were no significant different from correspondent Cd groups, but higher than that of control significantly.Conclusion It was suggested that selenium at the dose of10μmol/kg can induce c-myc mRNA, p53 mRNA and P53 protein over expression, and cadmium at the dose of 5～ 20μmol/kg can increase TERT mRNA, c-myc mRNA and protein, p53 mRNA and protein over expressions. Selenium at the doses of 2.5～5p.mol/kg have antagonistic effect on over-expression of TERT mRNA, c-myc mRNA and protein, p53 mRNA and protein induced by cadmium in rat liver.