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Antiproliferative effects of cucurbitacin B in breast cancer cells: down-regulation of the c-Myc/hTERT/telomerase pathway and obstruction of the cell cycle.

Duangmano S, Dakeng S, Jiratchariyakul W, Suksamrarn A, Smith DR, Patmasiriwat P - Int J Mol Sci (2010)

Bottom Line: Our results show that the cucurbitacin B inhibits growth and telomerase activity in the three breast cancer cell lines and exerts an obvious inhibitory effect in the estrogen receptor (ER)-negative breast cancer SKBR-3 cells.The expression of hTERT and c-Myc were also inhibited by cucurbitacin B, In addition, a clear reduction of c-Myc protein was observed after treatment in SKBR-3 cells even with a concentration of cucurbitacin B that was ten-times lower compared to the concentration used for HBL-100.Our findings imply that cucurbitacin B exerts an anticancer effect by inhibiting telomerase via down regulating both the hTERT and c-Myc expression in breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medical Technology, Mahidol University Main Campus, 999 Puttamonthon 4 street, Salaya, Nakornpathom 73170, Thailand; E-Mails: sduangmano@hotmail.com (S.Du.); sdakeng@yahoo.com (S.Da.).

ABSTRACT
Naturally occurring cucurbitacins have been shown to have anticancer, antimicrobial and anti-inflammatory activities. In this study, we determined the effects of cucurbitacin B extracted from the Thai herb Trichosanthes cucumerina L. on telomerase regulation in three human breast cancer cell lines (T47D, SKBR-3, and MCF-7) and a mammary epithelium cell line (HBL-100). Cell viability after treatment with cucurbitacin B, which is an active ingredient of this herb, was assessed. Telomeric Repeat Amplification Protocol (TRAP) assays and RT-PCR (qualitative and realtime) were performed to investigate activity of telomerase as well as expression of human telomerase reverse transcriptase (hTERT) and c-Myc. The c-Myc protein level was also determined in SKBR-3 and HBL-100 cells. Our results show that the cucurbitacin B inhibits growth and telomerase activity in the three breast cancer cell lines and exerts an obvious inhibitory effect in the estrogen receptor (ER)-negative breast cancer SKBR-3 cells. The expression of hTERT and c-Myc were also inhibited by cucurbitacin B, In addition, a clear reduction of c-Myc protein was observed after treatment in SKBR-3 cells even with a concentration of cucurbitacin B that was ten-times lower compared to the concentration used for HBL-100. Our findings imply that cucurbitacin B exerts an anticancer effect by inhibiting telomerase via down regulating both the hTERT and c-Myc expression in breast cancer cells.

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Effect of cucurbitacin B on telomerase activity in breast cancer cells. (A) SKBR-3 cells were treated with cucurbitacin B at final concentrations of 0, 2, 4, 6, 8 and 10 μg/mL while T47D, MCF-7 and HBL-100 cells were treated with cucurbitacin B at final concentrations of 0, 20, 40, 60, 80 and 100 μg/mL. Telomerase activity was examined by the TRAP assay and the decrease in telomerase activity was interpreted by means of reduced intensities of bands (stepwise 6 bp (TTAGGG) repetitive ladder bands). (B) Relative telomerase activities of the cells after cucurbitacin B treatment were determined by the TRAPEZE-ELISA assay, and the relative telomerase levels are expressed as % relative activity. * P < 0.05 vs. control (untreated) group.
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f3-ijms-11-05323: Effect of cucurbitacin B on telomerase activity in breast cancer cells. (A) SKBR-3 cells were treated with cucurbitacin B at final concentrations of 0, 2, 4, 6, 8 and 10 μg/mL while T47D, MCF-7 and HBL-100 cells were treated with cucurbitacin B at final concentrations of 0, 20, 40, 60, 80 and 100 μg/mL. Telomerase activity was examined by the TRAP assay and the decrease in telomerase activity was interpreted by means of reduced intensities of bands (stepwise 6 bp (TTAGGG) repetitive ladder bands). (B) Relative telomerase activities of the cells after cucurbitacin B treatment were determined by the TRAPEZE-ELISA assay, and the relative telomerase levels are expressed as % relative activity. * P < 0.05 vs. control (untreated) group.

Mentions: The effect of cucurbitacin B on telomerase activity was investigated. The human breast cancer cell lines were treated with cucurbitacin B at varying concentrations for 48 h. According to the markedly greater sensitivity of SKBR-3 cells to cucurbitacin B compound, the SKBR-3 cells were, therefore, incubated at 10-fold lower concentrations than the other three cell lines. After incubation, telomerase activity was determined by the TRAP assay. As shown in Figure 3A, the band intensities of the 6 bp (TTAGGG) repeat length were reduced after incubation with cucurbitacin B in all cell lines tested, and the results are consistent with a dose dependent reduction of telomerase activity in the four cell lines. To confirm this, relative telomerase activity levels were determined using the TRAPEZE-ELISA detection assay system, and a dose dependent reduction in telomerase levels was observed in all four cell lines (Figure 3B). Both the assays for telomerase activity showed a greater effect of the cucurbitacin B in the estrogen receptor negative cell lines (SKBR-3 and HBL-100) as compared to the estrogen receptor positive cell lines (MCF-7 and T47D), consistent with the results from the cell viability assays. Moreover, telomerase activity was obviously reduced in ER negative breast cancer SKBR-3 cells, even though concentrations of cucurbitacin B used for this cell type were 10-times less than the non-malignant mammary epithelium HBL-100.


Antiproliferative effects of cucurbitacin B in breast cancer cells: down-regulation of the c-Myc/hTERT/telomerase pathway and obstruction of the cell cycle.

Duangmano S, Dakeng S, Jiratchariyakul W, Suksamrarn A, Smith DR, Patmasiriwat P - Int J Mol Sci (2010)

Effect of cucurbitacin B on telomerase activity in breast cancer cells. (A) SKBR-3 cells were treated with cucurbitacin B at final concentrations of 0, 2, 4, 6, 8 and 10 μg/mL while T47D, MCF-7 and HBL-100 cells were treated with cucurbitacin B at final concentrations of 0, 20, 40, 60, 80 and 100 μg/mL. Telomerase activity was examined by the TRAP assay and the decrease in telomerase activity was interpreted by means of reduced intensities of bands (stepwise 6 bp (TTAGGG) repetitive ladder bands). (B) Relative telomerase activities of the cells after cucurbitacin B treatment were determined by the TRAPEZE-ELISA assay, and the relative telomerase levels are expressed as % relative activity. * P < 0.05 vs. control (untreated) group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100835&req=5

f3-ijms-11-05323: Effect of cucurbitacin B on telomerase activity in breast cancer cells. (A) SKBR-3 cells were treated with cucurbitacin B at final concentrations of 0, 2, 4, 6, 8 and 10 μg/mL while T47D, MCF-7 and HBL-100 cells were treated with cucurbitacin B at final concentrations of 0, 20, 40, 60, 80 and 100 μg/mL. Telomerase activity was examined by the TRAP assay and the decrease in telomerase activity was interpreted by means of reduced intensities of bands (stepwise 6 bp (TTAGGG) repetitive ladder bands). (B) Relative telomerase activities of the cells after cucurbitacin B treatment were determined by the TRAPEZE-ELISA assay, and the relative telomerase levels are expressed as % relative activity. * P < 0.05 vs. control (untreated) group.
Mentions: The effect of cucurbitacin B on telomerase activity was investigated. The human breast cancer cell lines were treated with cucurbitacin B at varying concentrations for 48 h. According to the markedly greater sensitivity of SKBR-3 cells to cucurbitacin B compound, the SKBR-3 cells were, therefore, incubated at 10-fold lower concentrations than the other three cell lines. After incubation, telomerase activity was determined by the TRAP assay. As shown in Figure 3A, the band intensities of the 6 bp (TTAGGG) repeat length were reduced after incubation with cucurbitacin B in all cell lines tested, and the results are consistent with a dose dependent reduction of telomerase activity in the four cell lines. To confirm this, relative telomerase activity levels were determined using the TRAPEZE-ELISA detection assay system, and a dose dependent reduction in telomerase levels was observed in all four cell lines (Figure 3B). Both the assays for telomerase activity showed a greater effect of the cucurbitacin B in the estrogen receptor negative cell lines (SKBR-3 and HBL-100) as compared to the estrogen receptor positive cell lines (MCF-7 and T47D), consistent with the results from the cell viability assays. Moreover, telomerase activity was obviously reduced in ER negative breast cancer SKBR-3 cells, even though concentrations of cucurbitacin B used for this cell type were 10-times less than the non-malignant mammary epithelium HBL-100.

Bottom Line: Our results show that the cucurbitacin B inhibits growth and telomerase activity in the three breast cancer cell lines and exerts an obvious inhibitory effect in the estrogen receptor (ER)-negative breast cancer SKBR-3 cells.The expression of hTERT and c-Myc were also inhibited by cucurbitacin B, In addition, a clear reduction of c-Myc protein was observed after treatment in SKBR-3 cells even with a concentration of cucurbitacin B that was ten-times lower compared to the concentration used for HBL-100.Our findings imply that cucurbitacin B exerts an anticancer effect by inhibiting telomerase via down regulating both the hTERT and c-Myc expression in breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medical Technology, Mahidol University Main Campus, 999 Puttamonthon 4 street, Salaya, Nakornpathom 73170, Thailand; E-Mails: sduangmano@hotmail.com (S.Du.); sdakeng@yahoo.com (S.Da.).

ABSTRACT
Naturally occurring cucurbitacins have been shown to have anticancer, antimicrobial and anti-inflammatory activities. In this study, we determined the effects of cucurbitacin B extracted from the Thai herb Trichosanthes cucumerina L. on telomerase regulation in three human breast cancer cell lines (T47D, SKBR-3, and MCF-7) and a mammary epithelium cell line (HBL-100). Cell viability after treatment with cucurbitacin B, which is an active ingredient of this herb, was assessed. Telomeric Repeat Amplification Protocol (TRAP) assays and RT-PCR (qualitative and realtime) were performed to investigate activity of telomerase as well as expression of human telomerase reverse transcriptase (hTERT) and c-Myc. The c-Myc protein level was also determined in SKBR-3 and HBL-100 cells. Our results show that the cucurbitacin B inhibits growth and telomerase activity in the three breast cancer cell lines and exerts an obvious inhibitory effect in the estrogen receptor (ER)-negative breast cancer SKBR-3 cells. The expression of hTERT and c-Myc were also inhibited by cucurbitacin B, In addition, a clear reduction of c-Myc protein was observed after treatment in SKBR-3 cells even with a concentration of cucurbitacin B that was ten-times lower compared to the concentration used for HBL-100. Our findings imply that cucurbitacin B exerts an anticancer effect by inhibiting telomerase via down regulating both the hTERT and c-Myc expression in breast cancer cells.

Show MeSH
Related in: MedlinePlus