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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

Combined cytotoxic effects of plumbagin (PL) and zoledronic acid (ZA) on MDA-MB-231SArfp breast cancer cells. (A) Combined cytotoxicity after treatment with various ZA concentrations plus 0, 2.5, 5, and 10 μmol/L PL for up to 96 h. (B) Combined cytotoxicity after treatment with various PL concentrations plus 0, 12.5, 25, 50, and 100 μmol/L ZA for up to 96 h. (C) Synergistic analysis is presented with Fa-CI plot (a), Fa-log (CI) (b), Fa-DRI (c), and Fa-log (DRI) plot (d) generated using Compusyn software, with the method based on the median effect principle of Chou and Talalay. All data represent the means±SD of at least three independent experiments. bP<0.05, cP<0.01.
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fig2: Combined cytotoxic effects of plumbagin (PL) and zoledronic acid (ZA) on MDA-MB-231SArfp breast cancer cells. (A) Combined cytotoxicity after treatment with various ZA concentrations plus 0, 2.5, 5, and 10 μmol/L PL for up to 96 h. (B) Combined cytotoxicity after treatment with various PL concentrations plus 0, 12.5, 25, 50, and 100 μmol/L ZA for up to 96 h. (C) Synergistic analysis is presented with Fa-CI plot (a), Fa-log (CI) (b), Fa-DRI (c), and Fa-log (DRI) plot (d) generated using Compusyn software, with the method based on the median effect principle of Chou and Talalay. All data represent the means±SD of at least three independent experiments. bP<0.05, cP<0.01.

Mentions: To explore the possibility of synergistic cytotoxicity, MDA-MB-231SArfp cells were treated with various concentrations of PL and ZA, either alone or in combination. As shown in Figure 1, PL and ZA at up to 20 and 100 μmol/L, respectively, each exhibited dose-dependent cytotoxicity in MDA-MB-231SArfp cells from 24 to 96 h. In the PL-treatment group, the 24-h IC50 was 12.18 μmol/L, whereas in the ZA-treatment group, the 24-h IC50 was exceeded 100 μmol/L, and the 48-h IC50 was 67.36 μmol/L. Only high concentrations (10 and 5 μmol/L) inhibited cell viability in the PL group, which was similar to that observed in the ZA group, in which cytotoxicity was demonstrated only for the 100-μmol/L treatment. Following consecutive drug interventions, the cytotoxic effect increased as the effective administration dose dropped to the minimum value of 0.625 μmol/L in the PL group and 3.125 μmol/L in the ZA group at 96 h. When administered simultaneously, the treatment efficacy increased relative to either reagent alone. As shown in Figures 2A and 2B, during the first 24-h treatment, only 10 μmol/L PL exhibited a synergistic effect to every ZA concentration, which was similar to that of 100 μmol/L ZA in each PL group. Furthermore, a synergistic effect could be observed in the 5-μmol/L PL group with all concentrations of ZA. No synergism was evident in the 10-μmol/L PL group, irrespective of the actual concentration of ZA. From 48 to 96 h, both 5 and 10 μmol/L PL were capable of significantly reinforcing the cytotoxicity of ZA, although 2.5 μmol/L PL only showed an effect after 96 h of treatment. Furthermore, none of the PL concentrations were shown to be instrumental in enhancing the cytotoxicity of 50 and 100 μmol/LZA treatments at 96 h. For the PL treatment group combined with various concentrations of ZA, synergistic efficacy was observed for almost all doses of ZA, despite the relative absence of synergism of 10 μmol/L PL with ZA.


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)

Combined cytotoxic effects of plumbagin (PL) and zoledronic acid (ZA) on MDA-MB-231SArfp breast cancer cells. (A) Combined cytotoxicity after treatment with various ZA concentrations plus 0, 2.5, 5, and 10 μmol/L PL for up to 96 h. (B) Combined cytotoxicity after treatment with various PL concentrations plus 0, 12.5, 25, 50, and 100 μmol/L ZA for up to 96 h. (C) Synergistic analysis is presented with Fa-CI plot (a), Fa-log (CI) (b), Fa-DRI (c), and Fa-log (DRI) plot (d) generated using Compusyn software, with the method based on the median effect principle of Chou and Talalay. All data represent the means±SD of at least three independent experiments. bP<0.05, cP<0.01.
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Related In: Results  -  Collection

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fig2: Combined cytotoxic effects of plumbagin (PL) and zoledronic acid (ZA) on MDA-MB-231SArfp breast cancer cells. (A) Combined cytotoxicity after treatment with various ZA concentrations plus 0, 2.5, 5, and 10 μmol/L PL for up to 96 h. (B) Combined cytotoxicity after treatment with various PL concentrations plus 0, 12.5, 25, 50, and 100 μmol/L ZA for up to 96 h. (C) Synergistic analysis is presented with Fa-CI plot (a), Fa-log (CI) (b), Fa-DRI (c), and Fa-log (DRI) plot (d) generated using Compusyn software, with the method based on the median effect principle of Chou and Talalay. All data represent the means±SD of at least three independent experiments. bP<0.05, cP<0.01.
Mentions: To explore the possibility of synergistic cytotoxicity, MDA-MB-231SArfp cells were treated with various concentrations of PL and ZA, either alone or in combination. As shown in Figure 1, PL and ZA at up to 20 and 100 μmol/L, respectively, each exhibited dose-dependent cytotoxicity in MDA-MB-231SArfp cells from 24 to 96 h. In the PL-treatment group, the 24-h IC50 was 12.18 μmol/L, whereas in the ZA-treatment group, the 24-h IC50 was exceeded 100 μmol/L, and the 48-h IC50 was 67.36 μmol/L. Only high concentrations (10 and 5 μmol/L) inhibited cell viability in the PL group, which was similar to that observed in the ZA group, in which cytotoxicity was demonstrated only for the 100-μmol/L treatment. Following consecutive drug interventions, the cytotoxic effect increased as the effective administration dose dropped to the minimum value of 0.625 μmol/L in the PL group and 3.125 μmol/L in the ZA group at 96 h. When administered simultaneously, the treatment efficacy increased relative to either reagent alone. As shown in Figures 2A and 2B, during the first 24-h treatment, only 10 μmol/L PL exhibited a synergistic effect to every ZA concentration, which was similar to that of 100 μmol/L ZA in each PL group. Furthermore, a synergistic effect could be observed in the 5-μmol/L PL group with all concentrations of ZA. No synergism was evident in the 10-μmol/L PL group, irrespective of the actual concentration of ZA. From 48 to 96 h, both 5 and 10 μmol/L PL were capable of significantly reinforcing the cytotoxicity of ZA, although 2.5 μmol/L PL only showed an effect after 96 h of treatment. Furthermore, none of the PL concentrations were shown to be instrumental in enhancing the cytotoxicity of 50 and 100 μmol/LZA treatments at 96 h. For the PL treatment group combined with various concentrations of ZA, synergistic efficacy was observed for almost all doses of ZA, despite the relative absence of synergism of 10 μmol/L PL with ZA.

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