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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

Notch receptor and Bcl-2 expression after drug and DAPT administration. (A) Real-time PCR demonstrated that Notch-1 expression decreased in the PL and combination group but increased in the ZA group, irrespective of the insignificant fluctuation of Notch-2/3 expression. (B, C) Western blotting revealed similar alterations in Notch receptor expression with the different treatments. (D, E) Compared with ZA treatment alone, Bcl-2 expression decreased upon treatment with ZA plus PL and DAPT, likely indicating a similar mechanism of action between PL and DAPT combined with ZA. e Indicates at least a two-fold mRNA change relative to the control group. n=3. Protein expression levels (relative to GAPDH) were calculated. All data represent the means±SD of at least three separate experiments.
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fig6: Notch receptor and Bcl-2 expression after drug and DAPT administration. (A) Real-time PCR demonstrated that Notch-1 expression decreased in the PL and combination group but increased in the ZA group, irrespective of the insignificant fluctuation of Notch-2/3 expression. (B, C) Western blotting revealed similar alterations in Notch receptor expression with the different treatments. (D, E) Compared with ZA treatment alone, Bcl-2 expression decreased upon treatment with ZA plus PL and DAPT, likely indicating a similar mechanism of action between PL and DAPT combined with ZA. e Indicates at least a two-fold mRNA change relative to the control group. n=3. Protein expression levels (relative to GAPDH) were calculated. All data represent the means±SD of at least three separate experiments.

Mentions: Based on the above results, we intended to further unravel the underlying mechanisms of the synergistic antitumor effect of PL and ZA. Because synergistic apoptosis regulation was more evident than growth inhibition and because an apoptosis rate of approximately 80% was achieved after 24 h with lower drug concentrations, we focused on the mechanisms of apoptosis. Western blot analysis revealed that the expression of t-caspase-3, clv caspase-3, CDKN1A, and ID1 increased in the combination group, whereas the expression of clv PARP, Bcl-2, and Bcl-xL decreased (Figure 5A), indicating the induction of apoptosis through the combination treatment. Because Notch downregulation is often associated with the inhibition of cell viability and the induction of cell apoptosis, real-time PCR was used to evaluate the effects of individual or combined PL and ZA treatment on the Notch signaling pathway in MDA-MB-231SArfp cells. As shown in Figure 6A, both PL and synergistic treatment downregulated Notch-1 expression, whereas ZA upregulated Notch-1 mRNA expression compared with the vehicle treatment group. However, the three isoforms of Notch-2/3 failed to exhibit any disparity with respect to the control, especially in the synergism group. Therefore, Western blotting was performed to examine Notch receptor family production at the protein level. Figure 6B shows that Notch-1 protein expression undergoes a similar decrease in the PL- and combination-treatment groups and an increase in the ZA-treatment group. Additionally, three bands corresponding to Notch-2 and Notch-3 exhibited only insignificant fluctuations upon Western blot analysis, consistent with reports that Notch-1 downregulation is frequently related to apoptosis induction36.


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)

Notch receptor and Bcl-2 expression after drug and DAPT administration. (A) Real-time PCR demonstrated that Notch-1 expression decreased in the PL and combination group but increased in the ZA group, irrespective of the insignificant fluctuation of Notch-2/3 expression. (B, C) Western blotting revealed similar alterations in Notch receptor expression with the different treatments. (D, E) Compared with ZA treatment alone, Bcl-2 expression decreased upon treatment with ZA plus PL and DAPT, likely indicating a similar mechanism of action between PL and DAPT combined with ZA. e Indicates at least a two-fold mRNA change relative to the control group. n=3. Protein expression levels (relative to GAPDH) were calculated. All data represent the means±SD of at least three separate experiments.
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Related In: Results  -  Collection

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fig6: Notch receptor and Bcl-2 expression after drug and DAPT administration. (A) Real-time PCR demonstrated that Notch-1 expression decreased in the PL and combination group but increased in the ZA group, irrespective of the insignificant fluctuation of Notch-2/3 expression. (B, C) Western blotting revealed similar alterations in Notch receptor expression with the different treatments. (D, E) Compared with ZA treatment alone, Bcl-2 expression decreased upon treatment with ZA plus PL and DAPT, likely indicating a similar mechanism of action between PL and DAPT combined with ZA. e Indicates at least a two-fold mRNA change relative to the control group. n=3. Protein expression levels (relative to GAPDH) were calculated. All data represent the means±SD of at least three separate experiments.
Mentions: Based on the above results, we intended to further unravel the underlying mechanisms of the synergistic antitumor effect of PL and ZA. Because synergistic apoptosis regulation was more evident than growth inhibition and because an apoptosis rate of approximately 80% was achieved after 24 h with lower drug concentrations, we focused on the mechanisms of apoptosis. Western blot analysis revealed that the expression of t-caspase-3, clv caspase-3, CDKN1A, and ID1 increased in the combination group, whereas the expression of clv PARP, Bcl-2, and Bcl-xL decreased (Figure 5A), indicating the induction of apoptosis through the combination treatment. Because Notch downregulation is often associated with the inhibition of cell viability and the induction of cell apoptosis, real-time PCR was used to evaluate the effects of individual or combined PL and ZA treatment on the Notch signaling pathway in MDA-MB-231SArfp cells. As shown in Figure 6A, both PL and synergistic treatment downregulated Notch-1 expression, whereas ZA upregulated Notch-1 mRNA expression compared with the vehicle treatment group. However, the three isoforms of Notch-2/3 failed to exhibit any disparity with respect to the control, especially in the synergism group. Therefore, Western blotting was performed to examine Notch receptor family production at the protein level. Figure 6B shows that Notch-1 protein expression undergoes a similar decrease in the PL- and combination-treatment groups and an increase in the ZA-treatment group. Additionally, three bands corresponding to Notch-2 and Notch-3 exhibited only insignificant fluctuations upon Western blot analysis, consistent with reports that Notch-1 downregulation is frequently related to apoptosis induction36.

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