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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 combined with doxorubicin on the proliferation of breast cancer cells. The MCF-7 (A) and MDA-MB-453 (B) breast cancer cells were either untreated or treated with 0.5 μM doxorubicin 12 h after the siRNA transfection. Cell viability was measured by the MTT assay every day for 4 days. Data are shown as mean ± SD from three independent experiments. Panel C and D show the inhibition effect of hTERT siRNA on the tumorigenic potential of human breast cancer cells. 2 × 106 MCF-7 cells (C) or MDA-MB-453 cells (D) that were untreated or transfected with control siRNA or hTERT siRNA were injected subcutaneously into each flank of athymic nude mice. The tumor dimensions were measured every 3 days. The mean tumor volume (mm3) was calculated according to the formula: (d2 × D)/2, where d and D are the shortest and longest diameters of the tumor, respectively. All measurements were performed in a coded, blinded fashion. Data are shown as mean ± SD with 5 mice per treatment group.
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Figure 2: Effects of hTERT-siRNA combined with doxorubicin on the proliferation of breast cancer cells. The MCF-7 (A) and MDA-MB-453 (B) breast cancer cells were either untreated or treated with 0.5 μM doxorubicin 12 h after the siRNA transfection. Cell viability was measured by the MTT assay every day for 4 days. Data are shown as mean ± SD from three independent experiments. Panel C and D show the inhibition effect of hTERT siRNA on the tumorigenic potential of human breast cancer cells. 2 × 106 MCF-7 cells (C) or MDA-MB-453 cells (D) that were untreated or transfected with control siRNA or hTERT siRNA were injected subcutaneously into each flank of athymic nude mice. The tumor dimensions were measured every 3 days. The mean tumor volume (mm3) was calculated according to the formula: (d2 × D)/2, where d and D are the shortest and longest diameters of the tumor, respectively. All measurements were performed in a coded, blinded fashion. Data are shown as mean ± SD with 5 mice per treatment group.

Mentions: Decreased telomerase activity is associated with arrested cell growth; we thus sought to determine whether or not the hTERT siRNA-induced reduction in telomerase activity would affect cell viability in the two breast cancer cell lines. The cells were transfected with the hTERT siRNA as previously, and the amount of viable cells was determined by the MTT assay every 24 hours for four days. As shown in Fig. 2A and 2B, the hTERT siRNA significantly decreased the percentage of viable cells in both cell lines. The effect of the siRNA was similar in both cell lines. The decrease was rapid: only ~63% of cells were alive after 24 hours, and only 50% of cells survived after 48 hours. The number of surviving cells remained at about 50% from there on and seemed to be heading into a recovery trend, consistent with the transient nature of the siRNA transfection.


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 combined with doxorubicin on the proliferation of breast cancer cells. The MCF-7 (A) and MDA-MB-453 (B) breast cancer cells were either untreated or treated with 0.5 μM doxorubicin 12 h after the siRNA transfection. Cell viability was measured by the MTT assay every day for 4 days. Data are shown as mean ± SD from three independent experiments. Panel C and D show the inhibition effect of hTERT siRNA on the tumorigenic potential of human breast cancer cells. 2 × 106 MCF-7 cells (C) or MDA-MB-453 cells (D) that were untreated or transfected with control siRNA or hTERT siRNA were injected subcutaneously into each flank of athymic nude mice. The tumor dimensions were measured every 3 days. The mean tumor volume (mm3) was calculated according to the formula: (d2 × D)/2, where d and D are the shortest and longest diameters of the tumor, respectively. All measurements were performed in a coded, blinded fashion. Data are shown as mean ± SD with 5 mice per treatment group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691745&req=5

Figure 2: Effects of hTERT-siRNA combined with doxorubicin on the proliferation of breast cancer cells. The MCF-7 (A) and MDA-MB-453 (B) breast cancer cells were either untreated or treated with 0.5 μM doxorubicin 12 h after the siRNA transfection. Cell viability was measured by the MTT assay every day for 4 days. Data are shown as mean ± SD from three independent experiments. Panel C and D show the inhibition effect of hTERT siRNA on the tumorigenic potential of human breast cancer cells. 2 × 106 MCF-7 cells (C) or MDA-MB-453 cells (D) that were untreated or transfected with control siRNA or hTERT siRNA were injected subcutaneously into each flank of athymic nude mice. The tumor dimensions were measured every 3 days. The mean tumor volume (mm3) was calculated according to the formula: (d2 × D)/2, where d and D are the shortest and longest diameters of the tumor, respectively. All measurements were performed in a coded, blinded fashion. Data are shown as mean ± SD with 5 mice per treatment group.
Mentions: Decreased telomerase activity is associated with arrested cell growth; we thus sought to determine whether or not the hTERT siRNA-induced reduction in telomerase activity would affect cell viability in the two breast cancer cell lines. The cells were transfected with the hTERT siRNA as previously, and the amount of viable cells was determined by the MTT assay every 24 hours for four days. As shown in Fig. 2A and 2B, the hTERT siRNA significantly decreased the percentage of viable cells in both cell lines. The effect of the siRNA was similar in both cell lines. The decrease was rapid: only ~63% of cells were alive after 24 hours, and only 50% of cells survived after 48 hours. The number of surviving cells remained at about 50% from there on and seemed to be heading into a recovery trend, consistent with the transient nature of the siRNA transfection.

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