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siRNA inhibition of telomerase enhances the anti-cancer effect of doxorubicin in breast cancer cells.

Dong X, Liu A, Zer C, Feng J, Zhen Z, Yang M, Zhong L - BMC Cancer (2009)

Bottom Line: The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control.The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 microM doxorubicin treatment had a comparable effect but with a slower kinetics.When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Molecular Medicine Center of Shaoxing People's Hospital, The First Affiliate Hospital of Shaoxing University, Shaoxing, PR China. dxj9666@163.com

ABSTRACT

Background: Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant administration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, we examined the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase on breast cancer cells. We found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, we observed an enhanced and sustained apoptosis induction in breast cancer cells.

Methods: siRNA targeting the mRNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT mRNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay. The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied in vivo by injection of the siRNA-transfected breast cancer cells into nude mice. The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examined in vitro by MTT assay, FACS and SA-beta-galactosidase staining.

Results: The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control. In vivo, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells. The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 microM doxorubicin treatment had a comparable effect but with a slower kinetics. The combination of hTERT siRNA and 0.5 microM doxorubicin killed twice as many cancer cells, showing a cumulative effect of the two treatments.

Conclusion: The study demonstrated the potential of telomerase inhibition as an effective treatment for breast cancer. When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.

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Effects of hTERT-siRNA knock-down in breast cancer cells. (A and B) hTERT mRNA expression levels quantified by RT-PCR in MCF-7 (A) and MDA-MB-453 (B) cells at 48 h after a 6 h exposure in hTERT siRNA, control siRNA, or untransfected, respectively. Relative quantification of hTERT mRNA expression levels was accomplished by the Pfaffl method of  Data are shown as mean ± SD (error bar) of 3 experiments; *, p = 0.001, two-tailed student's t-test. (C and D) representative western blots showing the expression of hTERT protein expression levels at 48 h after a 6 h exposure in hTERT siRNA (right lane; hTERT), control siRNA (middle lane; control), or untransfected (left lane; blank), respectively, in MCF-7 (C) and MDA-MB-453 (D) cells. β-actin was the internal loading control. (E and F) Telomerase activity levels quantified by TRAP assay in MCF-7 (E) and MDA-MB-453 (F) cells at 48 h after a 6 h exposure in hTERT siRNA, control siRNA, or untransfected, respectively. Data are shown as mean ± SD (error bar) of 3 experiments; *, p = 0.001, two-tailed student's t-test.
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Figure 1: Effects of hTERT-siRNA knock-down in breast cancer cells. (A and B) hTERT mRNA expression levels quantified by RT-PCR in MCF-7 (A) and MDA-MB-453 (B) cells at 48 h after a 6 h exposure in hTERT siRNA, control siRNA, or untransfected, respectively. Relative quantification of hTERT mRNA expression levels was accomplished by the Pfaffl method of Data are shown as mean ± SD (error bar) of 3 experiments; *, p = 0.001, two-tailed student's t-test. (C and D) representative western blots showing the expression of hTERT protein expression levels at 48 h after a 6 h exposure in hTERT siRNA (right lane; hTERT), control siRNA (middle lane; control), or untransfected (left lane; blank), respectively, in MCF-7 (C) and MDA-MB-453 (D) cells. β-actin was the internal loading control. (E and F) Telomerase activity levels quantified by TRAP assay in MCF-7 (E) and MDA-MB-453 (F) cells at 48 h after a 6 h exposure in hTERT siRNA, control siRNA, or untransfected, respectively. Data are shown as mean ± SD (error bar) of 3 experiments; *, p = 0.001, two-tailed student's t-test.

Mentions: The hTERT siRNA was transiently transfected into the breast cancer cell lines MCF-7 and MDA-MB-453. After 48 hours, the hTERT mRNA and protein levels were quantified by real time RT-PCR and western blots, respectively. As shown in Fig. 1A and 1B, hTERT siRNA transfection significantly reduced the amount of hTERT mRNA. The level of hTERT mRNA in the hTERT siRNA-treated group was about 40% of the blank group in MCF -7 cells and about 38% in MDA-MB-453 cells. The control siRNA had no effect on the hTERT mRNA level in either cell line. The results between MCF-7 and MDA-MB-453 cells were not significantly different, indicating that the effect of the hTERT siRNA was specific. The hTERT siRNA was also successful in knocking down hTERT protein expression. As shown in Fig. 1C and 1D, while the β-actin internal control showed equal loading among the three groups, the level of hTERT protein was noticeably lower in the hTERT siRNA-treated group compared to both the blank and the negative siRNA-treated groups, suggesting that the hTERT siRNA treatment could effectively reduce the hTERT protein level.


siRNA inhibition of telomerase enhances the anti-cancer effect of doxorubicin in breast cancer cells.

Dong X, Liu A, Zer C, Feng J, Zhen Z, Yang M, Zhong L - BMC Cancer (2009)

Effects of hTERT-siRNA knock-down in breast cancer cells. (A and B) hTERT mRNA expression levels quantified by RT-PCR in MCF-7 (A) and MDA-MB-453 (B) cells at 48 h after a 6 h exposure in hTERT siRNA, control siRNA, or untransfected, respectively. Relative quantification of hTERT mRNA expression levels was accomplished by the Pfaffl method of  Data are shown as mean ± SD (error bar) of 3 experiments; *, p = 0.001, two-tailed student's t-test. (C and D) representative western blots showing the expression of hTERT protein expression levels at 48 h after a 6 h exposure in hTERT siRNA (right lane; hTERT), control siRNA (middle lane; control), or untransfected (left lane; blank), respectively, in MCF-7 (C) and MDA-MB-453 (D) cells. β-actin was the internal loading control. (E and F) Telomerase activity levels quantified by TRAP assay in MCF-7 (E) and MDA-MB-453 (F) cells at 48 h after a 6 h exposure in hTERT siRNA, control siRNA, or untransfected, respectively. Data are shown as mean ± SD (error bar) of 3 experiments; *, p = 0.001, two-tailed student's t-test.
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Figure 1: Effects of hTERT-siRNA knock-down in breast cancer cells. (A and B) hTERT mRNA expression levels quantified by RT-PCR in MCF-7 (A) and MDA-MB-453 (B) cells at 48 h after a 6 h exposure in hTERT siRNA, control siRNA, or untransfected, respectively. Relative quantification of hTERT mRNA expression levels was accomplished by the Pfaffl method of Data are shown as mean ± SD (error bar) of 3 experiments; *, p = 0.001, two-tailed student's t-test. (C and D) representative western blots showing the expression of hTERT protein expression levels at 48 h after a 6 h exposure in hTERT siRNA (right lane; hTERT), control siRNA (middle lane; control), or untransfected (left lane; blank), respectively, in MCF-7 (C) and MDA-MB-453 (D) cells. β-actin was the internal loading control. (E and F) Telomerase activity levels quantified by TRAP assay in MCF-7 (E) and MDA-MB-453 (F) cells at 48 h after a 6 h exposure in hTERT siRNA, control siRNA, or untransfected, respectively. Data are shown as mean ± SD (error bar) of 3 experiments; *, p = 0.001, two-tailed student's t-test.
Mentions: The hTERT siRNA was transiently transfected into the breast cancer cell lines MCF-7 and MDA-MB-453. After 48 hours, the hTERT mRNA and protein levels were quantified by real time RT-PCR and western blots, respectively. As shown in Fig. 1A and 1B, hTERT siRNA transfection significantly reduced the amount of hTERT mRNA. The level of hTERT mRNA in the hTERT siRNA-treated group was about 40% of the blank group in MCF -7 cells and about 38% in MDA-MB-453 cells. The control siRNA had no effect on the hTERT mRNA level in either cell line. The results between MCF-7 and MDA-MB-453 cells were not significantly different, indicating that the effect of the hTERT siRNA was specific. The hTERT siRNA was also successful in knocking down hTERT protein expression. As shown in Fig. 1C and 1D, while the β-actin internal control showed equal loading among the three groups, the level of hTERT protein was noticeably lower in the hTERT siRNA-treated group compared to both the blank and the negative siRNA-treated groups, suggesting that the hTERT siRNA treatment could effectively reduce the hTERT protein level.

Bottom Line: The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control.The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 microM doxorubicin treatment had a comparable effect but with a slower kinetics.When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Molecular Medicine Center of Shaoxing People's Hospital, The First Affiliate Hospital of Shaoxing University, Shaoxing, PR China. dxj9666@163.com

ABSTRACT

Background: Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant administration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, we examined the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase on breast cancer cells. We found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, we observed an enhanced and sustained apoptosis induction in breast cancer cells.

Methods: siRNA targeting the mRNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT mRNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay. The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied in vivo by injection of the siRNA-transfected breast cancer cells into nude mice. The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examined in vitro by MTT assay, FACS and SA-beta-galactosidase staining.

Results: The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control. In vivo, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells. The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 microM doxorubicin treatment had a comparable effect but with a slower kinetics. The combination of hTERT siRNA and 0.5 microM doxorubicin killed twice as many cancer cells, showing a cumulative effect of the two treatments.

Conclusion: The study demonstrated the potential of telomerase inhibition as an effective treatment for breast cancer. When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.

Show MeSH
Related in: MedlinePlus