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Synergistic suppression of human breast cancer cells by combination of plumbagin and zoledronic acid In vitro.

Qiao H, Wang TY, Yan W, Qin A, Fan QM, Han XG, Wang YG, Tang TT - Acta Pharmacol. Sin. (2015)

Bottom Line: Synergism was evaluated using Compusyn software, and the combination index (CI) and drug reduction index (DRI) values were determined.The DRI values also showed a synergistic effect between PL and ZA, with actual values of 5.52 and 3.59, respectively.Moreover, the combination of ZA and PL decreased the expression of Notch-1, cleaved PARP, Bcl-2 and Bcl-xl, and increased the expression of cleaved caspase-3, CDKN1A and ID1.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

ABSTRACT

Aim: Zoledronic acid (ZA), a bisphosphonate, is currently used in combination with chemotherapeutic agents to suppress breast cancer cell proliferation or breast cancer-induced osteolysis. The aim of this study was to investigate the effects of ZA combined with a natural anticancer compound plumbagin (PL) against human breast cancer cells in vitro.

Methods: Human breast cancer MDA-MB-231SArfp cells were treated with ZA, PL or a combination of ZA and PL. The cell growth, apoptosis and migration were evaluated using CCK-8 assay, flow cytometry and transwell assay, respectively. The expression of apoptosis-related proteins was measured using real-time PCR and Western blotting. Synergism was evaluated using Compusyn software, and the combination index (CI) and drug reduction index (DRI) values were determined.

Results: PL or ZA alone caused mild cytotoxicity (the IC50 value at 24 h was 12.18 and above 100 μmol/L, respectively). However, the combination of ZA and PL caused a synergistic cytotoxicity (CI=0.26). The DRI values also showed a synergistic effect between PL and ZA, with actual values of 5.52 and 3.59, respectively. Furthermore, PL and ZA synergistically induced apoptosis and inhibited migration of the breast cancer cells. Moreover, the combination of ZA and PL decreased the expression of Notch-1, cleaved PARP, Bcl-2 and Bcl-xl, and increased the expression of cleaved caspase-3, CDKN1A and ID1. When the breast cancer cells were transfected with specific siRNA against Notch-1, the combination of ZA and PL markedly increased the expression of Bcl-2.

Conclusion: Combination of ZA and PL synergistically suppresses human breast cancer MDA-MB-231SArfp cells in vitro. PL can inhibit ZA-induced activation of the Notch-1 signaling pathway and subsequently reduce the expression of Bcl-2, thus potentiating cancer cell apoptosis.

No MeSH data available.


Related in: MedlinePlus

siRNA against Notch-1 and subsequent Bcl-2 expression alteration after combined reagent treatment. (A, B, and C) Specific siRNA silencing of Notch-1. RT-PCR and Western blotting were used to evaluate the efficacy of this approach. (D, E) MDA-MB-231SArfp cells were treated with the indicated agents after specific siRNA-3916 silencing against Notch-1. Both the combination and individual PL treatments resulted in increased Bcl-2 expression compared with the non-siRNA group, suggesting that the absence of upstream Notch-1 inhibition may lead to increased Bcl-2 production after the combination treatment. Protein expression levels (relative to GAPDH) were calculated. All data represent the means±SD of at least three separate experiments.
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fig7: siRNA against Notch-1 and subsequent Bcl-2 expression alteration after combined reagent treatment. (A, B, and C) Specific siRNA silencing of Notch-1. RT-PCR and Western blotting were used to evaluate the efficacy of this approach. (D, E) MDA-MB-231SArfp cells were treated with the indicated agents after specific siRNA-3916 silencing against Notch-1. Both the combination and individual PL treatments resulted in increased Bcl-2 expression compared with the non-siRNA group, suggesting that the absence of upstream Notch-1 inhibition may lead to increased Bcl-2 production after the combination treatment. Protein expression levels (relative to GAPDH) were calculated. All data represent the means±SD of at least three separate experiments.

Mentions: To further examine the mechanisms by which changes in Notch-1 and Bcl-2 expression alter apoptosis upon synergistic treatment of PL and ZA, three individual Notch-1 siRNA duplexes were synthesized. To determine the effectiveness of siRNA silencing, RT-PCR and Western blot analyses were performed at 24 and 48 h, respectively, after transfection of Notch-1 siRNA into MDA-MB-231SArfp cells (Figure 7A and 7B). Notch-1 expression was downregulated by siRNA-3916 within 24 h, and all three siRNAs were effective in reducing Notch-1 expression after 48 h of knockdown, indicating the efficacy of this approach. Because siRNA-3916 exhibited the most efficient mRNA knockdown within 24 h, as well as favorable efficiency at the protein level, this siRNA was applied in further experiments.


Synergistic suppression of human breast cancer cells by combination of plumbagin and zoledronic acid In vitro.

Qiao H, Wang TY, Yan W, Qin A, Fan QM, Han XG, Wang YG, Tang TT - Acta Pharmacol. Sin. (2015)

siRNA against Notch-1 and subsequent Bcl-2 expression alteration after combined reagent treatment. (A, B, and C) Specific siRNA silencing of Notch-1. RT-PCR and Western blotting were used to evaluate the efficacy of this approach. (D, E) MDA-MB-231SArfp cells were treated with the indicated agents after specific siRNA-3916 silencing against Notch-1. Both the combination and individual PL treatments resulted in increased Bcl-2 expression compared with the non-siRNA group, suggesting that the absence of upstream Notch-1 inhibition may lead to increased Bcl-2 production after the combination treatment. Protein expression levels (relative to GAPDH) were calculated. All data represent the means±SD of at least three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4561967&req=5

fig7: siRNA against Notch-1 and subsequent Bcl-2 expression alteration after combined reagent treatment. (A, B, and C) Specific siRNA silencing of Notch-1. RT-PCR and Western blotting were used to evaluate the efficacy of this approach. (D, E) MDA-MB-231SArfp cells were treated with the indicated agents after specific siRNA-3916 silencing against Notch-1. Both the combination and individual PL treatments resulted in increased Bcl-2 expression compared with the non-siRNA group, suggesting that the absence of upstream Notch-1 inhibition may lead to increased Bcl-2 production after the combination treatment. Protein expression levels (relative to GAPDH) were calculated. All data represent the means±SD of at least three separate experiments.
Mentions: To further examine the mechanisms by which changes in Notch-1 and Bcl-2 expression alter apoptosis upon synergistic treatment of PL and ZA, three individual Notch-1 siRNA duplexes were synthesized. To determine the effectiveness of siRNA silencing, RT-PCR and Western blot analyses were performed at 24 and 48 h, respectively, after transfection of Notch-1 siRNA into MDA-MB-231SArfp cells (Figure 7A and 7B). Notch-1 expression was downregulated by siRNA-3916 within 24 h, and all three siRNAs were effective in reducing Notch-1 expression after 48 h of knockdown, indicating the efficacy of this approach. Because siRNA-3916 exhibited the most efficient mRNA knockdown within 24 h, as well as favorable efficiency at the protein level, this siRNA was applied in further experiments.

Bottom Line: Synergism was evaluated using Compusyn software, and the combination index (CI) and drug reduction index (DRI) values were determined.The DRI values also showed a synergistic effect between PL and ZA, with actual values of 5.52 and 3.59, respectively.Moreover, the combination of ZA and PL decreased the expression of Notch-1, cleaved PARP, Bcl-2 and Bcl-xl, and increased the expression of cleaved caspase-3, CDKN1A and ID1.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

ABSTRACT

Aim: Zoledronic acid (ZA), a bisphosphonate, is currently used in combination with chemotherapeutic agents to suppress breast cancer cell proliferation or breast cancer-induced osteolysis. The aim of this study was to investigate the effects of ZA combined with a natural anticancer compound plumbagin (PL) against human breast cancer cells in vitro.

Methods: Human breast cancer MDA-MB-231SArfp cells were treated with ZA, PL or a combination of ZA and PL. The cell growth, apoptosis and migration were evaluated using CCK-8 assay, flow cytometry and transwell assay, respectively. The expression of apoptosis-related proteins was measured using real-time PCR and Western blotting. Synergism was evaluated using Compusyn software, and the combination index (CI) and drug reduction index (DRI) values were determined.

Results: PL or ZA alone caused mild cytotoxicity (the IC50 value at 24 h was 12.18 and above 100 μmol/L, respectively). However, the combination of ZA and PL caused a synergistic cytotoxicity (CI=0.26). The DRI values also showed a synergistic effect between PL and ZA, with actual values of 5.52 and 3.59, respectively. Furthermore, PL and ZA synergistically induced apoptosis and inhibited migration of the breast cancer cells. Moreover, the combination of ZA and PL decreased the expression of Notch-1, cleaved PARP, Bcl-2 and Bcl-xl, and increased the expression of cleaved caspase-3, CDKN1A and ID1. When the breast cancer cells were transfected with specific siRNA against Notch-1, the combination of ZA and PL markedly increased the expression of Bcl-2.

Conclusion: Combination of ZA and PL synergistically suppresses human breast cancer MDA-MB-231SArfp cells in vitro. PL can inhibit ZA-induced activation of the Notch-1 signaling pathway and subsequently reduce the expression of Bcl-2, thus potentiating cancer cell apoptosis.

No MeSH data available.


Related in: MedlinePlus