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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

Cell apoptosis induced in MDA-MB-231SArfp cells by plumbagin and zoledronic acid alone or in combination. (A, B) MDA-MB-231SArfp cells were incubated with the indicated concentrations of PL and ZA alone for 24 h, followed by annexin V-FITC/PI double staining to evaluate apoptotic cell death. (C, D) Similar procedures were performed in MDA-MB-231SArfp cells after combined treatment with PL and ZA for 24 h. All data represent the means±SD of at least three independent experiments. cP<0.01 vs vehicle.
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fig3: Cell apoptosis induced in MDA-MB-231SArfp cells by plumbagin and zoledronic acid alone or in combination. (A, B) MDA-MB-231SArfp cells were incubated with the indicated concentrations of PL and ZA alone for 24 h, followed by annexin V-FITC/PI double staining to evaluate apoptotic cell death. (C, D) Similar procedures were performed in MDA-MB-231SArfp cells after combined treatment with PL and ZA for 24 h. All data represent the means±SD of at least three independent experiments. cP<0.01 vs vehicle.

Mentions: The translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane during the early stages of apoptosis can be assessed using the phospholipid-binding protein annexin V. Flow cytometry revealed that PL and ZA, either individually or in combination, were able to generate a dose-dependent increase in the apoptotic and post-apoptotic populations of MDA-MB-231SArfp breast cancer cells (Figure 3). In the PL-treatment group, independent treatments at 2.5, 5.0, and 7.5 μmol/L resulted in total apoptotic rates of 7.5%±0.8%, 29.7%±1.2%, and 35.9%±0.8%, respectively. Similarly, the dose-dependent effect observed in the ZA-treatment group demonstrated apoptosis rates of 26.9%±0.2%, 33.9%±1.3%, and 37.2%±1.2% in the 12.5, 25, and 50 μmol/L groups, respectively. In combination, 5 and 7.5 μmol/L PL significantly augmented the apoptotic efficacy of 12.5, 25, and 50 μmol/L ZA from 51.3%±2.3% to 78.1%±0.8%, 59.3%±1.3% to 80.9%±2.0% and 67.5%±0.15% to 84.5%±0.4%, respectively. Furthermore, ZA treatment increased the apoptotic potential of PL as well. The rates of apoptosis achieved by 5 and 7.5 μmol/L PL were increased to 51.3%±2.3%, 59.3%±1.2%, and 59.3%±1.3% as well as to 78.1%±0.5%, 80.9%±1.5%, and 84.5%±0.4% with a ZA treatment range of 12.5 to 50 μmol/L. The induction of apoptosis was dose dependent, as demonstrated by the increase in the number of MDA-MB-231SArfp cells in the late stages of apoptosis, indicating that synergism between PL and ZA not only promoted apoptotic cell death but also enhanced the rate of apoptosis.


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)

Cell apoptosis induced in MDA-MB-231SArfp cells by plumbagin and zoledronic acid alone or in combination. (A, B) MDA-MB-231SArfp cells were incubated with the indicated concentrations of PL and ZA alone for 24 h, followed by annexin V-FITC/PI double staining to evaluate apoptotic cell death. (C, D) Similar procedures were performed in MDA-MB-231SArfp cells after combined treatment with PL and ZA for 24 h. All data represent the means±SD of at least three independent experiments. cP<0.01 vs vehicle.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4561967&req=5

fig3: Cell apoptosis induced in MDA-MB-231SArfp cells by plumbagin and zoledronic acid alone or in combination. (A, B) MDA-MB-231SArfp cells were incubated with the indicated concentrations of PL and ZA alone for 24 h, followed by annexin V-FITC/PI double staining to evaluate apoptotic cell death. (C, D) Similar procedures were performed in MDA-MB-231SArfp cells after combined treatment with PL and ZA for 24 h. All data represent the means±SD of at least three independent experiments. cP<0.01 vs vehicle.
Mentions: The translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane during the early stages of apoptosis can be assessed using the phospholipid-binding protein annexin V. Flow cytometry revealed that PL and ZA, either individually or in combination, were able to generate a dose-dependent increase in the apoptotic and post-apoptotic populations of MDA-MB-231SArfp breast cancer cells (Figure 3). In the PL-treatment group, independent treatments at 2.5, 5.0, and 7.5 μmol/L resulted in total apoptotic rates of 7.5%±0.8%, 29.7%±1.2%, and 35.9%±0.8%, respectively. Similarly, the dose-dependent effect observed in the ZA-treatment group demonstrated apoptosis rates of 26.9%±0.2%, 33.9%±1.3%, and 37.2%±1.2% in the 12.5, 25, and 50 μmol/L groups, respectively. In combination, 5 and 7.5 μmol/L PL significantly augmented the apoptotic efficacy of 12.5, 25, and 50 μmol/L ZA from 51.3%±2.3% to 78.1%±0.8%, 59.3%±1.3% to 80.9%±2.0% and 67.5%±0.15% to 84.5%±0.4%, respectively. Furthermore, ZA treatment increased the apoptotic potential of PL as well. The rates of apoptosis achieved by 5 and 7.5 μmol/L PL were increased to 51.3%±2.3%, 59.3%±1.2%, and 59.3%±1.3% as well as to 78.1%±0.5%, 80.9%±1.5%, and 84.5%±0.4% with a ZA treatment range of 12.5 to 50 μmol/L. The induction of apoptosis was dose dependent, as demonstrated by the increase in the number of MDA-MB-231SArfp cells in the late stages of apoptosis, indicating that synergism between PL and ZA not only promoted apoptotic cell death but also enhanced the rate of apoptosis.

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