The Combination Of ADR And NaBu Inhibits Telomerase Activity In Hela Cells

IntrouductionTelomerase is the ribonucleoprotein responsible for de novo synthesis and maintenance of telomeres. Using RNA as template, the repeat sequence of TTAGGG was synthesized and then linked to the end of chromosome. Telomerase is composed of the constructive RNA, TEP1 and hTERT, which functions as reverse transcription. The activity of telomerase is related to hTERT mRNA level. Telomerase is active in most cancer cells and immortalized cell . The inhibiting of telomerase now becomes a new strategy in cancer therapy.Telomere can form Guanine - quadruplex by its guanine - rich single strings at the 3' end and then inhibits the polymeration of telomere. It was reported that anthraquinone derivates like Adriamycin ( ADR) could induce the formation of guanine — quadruplex and inhibit the activity of telomerase. Histone deacetylase inhibitors, HDIs, is a kind of anti - tumour drug. HDIs induce apoptosis in cancer cell by changing the structure of chromosome and gene transcription through regulating its histone acetylation.Cervical cancer is one of the main cancers in woman. ADR and other drugs are used on chemotherapy at late stage of this cancer. More and more works are focused on how to improve those drugs' activities at low concentration and reduce their side effects. This work studies the combining inhibition of ADR and sodium butyrate ( NaBu) on the cell growth of Hela cell, telomerase activities and hTERT mRNA expression. The association is observed between telomerase activity and sensitivity of cancer cell to chemotherapeatic agents.Materials and MethodsIn our study ,HeLa cells were cultured in 5 ml of RPMI1640 medium supplemented with 10% fetal bovine serum in a 5% CO2 incubator with 100% humidity at 37X1. MTT assay was used to study the inhibition of Hela cell growth treated by ADR and combination of NaBu and ADR. Apoptosis was observed by flow cytometry with double staining of PI and Annexin V - FITC. Telomerase activities were evaluated with PAGE and silver staining after the TRAP (telomeric repeat amplification protocol) assay. RT - PCR were used to assess the telomerase activities and hTERT mRNA expression in Hela cells after the agents treatment. The significance of difference was analyzed by statistics software, SPSS 11.5. The comparisons of multiple samples were determined by Kruskal - Wallis H Test ( Equal Variances Not Assumed) and the comparison of two samples was determined by Wilcoxon Test (Equal Variances Not Assumed).ResultsAfter 48hr culture, Hela cells growth were inhibited at different levels when applied combining lmmol/L NaBu with ADR and applied ADR alone at different concentration of ADR. This inhibition is dose - dependant as the differences of treatment at variable concentration are significant (P <0. 05). IC50 was reduced from 6000nmol/L with ADR alone to 320nmol/L when combined with NaBu. This showed ADR and Nabu coordinately inhibited telomerase activity.After 48hr treatment with lOOnmol/L ADR, most of Hela cells were dying or at late apoptosis based on flow cytometry with double staining of PI and Annexin V FITC. The early apoptosis was induced when combining treatment with lmmol/L NaBu, which is time - and dose - dependant. DNA ladder was also observed after 48hr treatment of combining lmmol/L NaBu and lnmol/L , lOnmol/L ,100nmol/L ADR.The telomerase activities of Hela cells were reduced after treated 48 hr with ADR alone or with NaBu. The difference of both treatments with ADR at theconcentration of lOOnmol/L is significant ( p <0. 05). hTERT mRNA of HeLa cells was reduced in RT - PCR when treated with ADR with/without NaBu. The difference of hTERT mRNA expression levels between ADR treatment with/without NaBu is significant ( P <0.05) , which is consistent with the temomerase activity.DiscussionTelomerase is a DNA polymerase using RNA as template. Its RNA component acts as template for elongating and maintaining telomeric DNA through the addition of TTAGGG repeats to the end of chromosome and maintains the length of telomere. Unlimited proliferation in cancer cell requires this enzyme to maintain chromosomal stability. Telomerase inhibitor was used to study the relation between telomerase acitivity and its induced apoptosis. HDIs, like NaBu, and ADR are involved the regulation the telomerase activity. However, it remains unclear that the combining effect of NaBu and ADR on temomerase activity and induced apoptosis in cancer cells.MTT assay in this study showed that the combining treatment of lmmol/L NaBu and lnmol/L ADR could significantly inhibit Hela cell growth after 48hr, which is much higher than ADR treatment alone. ADR treatment with/without NaBu is dose - dependant. The IC50 of Hela cells is decreased from from 6000nmol/L with ADR alone to 320nmol/L when combined with NaBu. This suggested that combination of those two drugs could inhibit Hela cell growth. NaBu might enhance the sensitivity to ADR in Hela cell, which provide a potential application in anticancer therapy.The induced apoptosis was proved by electrophoresis and double staining. Based on double staining of PI and Annexin V - FITC, the percentage of late apoptosis is 23. 56% in Hela cells after 48hr treatment with lOOnmol/L ADR a-lone, and early apopstosis is just about 12.93%. The early apoptosis in HeLa cell treated with lmmol/L NaBu is about 6.68% and increased damatically to 22. 01% after combining treatment of the two drugs. The combining effect is dependant on the dose of ADR. The induced apoptosis in HeLa cells by lmmol/LNaBu and 100 nmol/L ADR is also time - dependant. Agarose gel result also showed the induced apoptosis in Hela cell after 48hr treatment with lmmol/L NaBu and 1 nmol/L ,10nmol/L ,100nmol/L ADR.Previous reports showed that both of ADR and NaBu could inhibit telomerase activities in various cancer cells, but no reports on their combining effect. This study showed the combining inhibition of NaBu and ADR on telomerase activity during the process of induced apoptosis. The inhibition of lmmol/L NaBu and lOOnmol/L ADR on telomerase activity in Hela cell is much higher than those drugs used alone. RT - PCR also showed the reduced expression hTERT mRNA in both drugs treatment, which is consistent with the telomerase activities. It is concluded that the combining of NaBu and ADR treatment might reduce hTERT mRNA expression and inhibit the telomerase activity.Conclusions1. From 1 nmol/L to 30000nmol/L ADR treatment with lmmol/L NaBu inhibit Hela cell growth and is dose - dependand.2. The treatment of NaBu and ADR reduced the telomerase activityies, which is dose — dependant. The down - regulation of hTERT mRNA by NaBu and ADR is time - and dose - dependant.3. The combining effects of NaBu and ADR induced apoptosis are related to inhibition of telomerase activities and down — regulation hTERT mRNA.