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Isoliensinine induces apoptosis in triple-negative human breast cancer cells through ROS generation and p38 MAPK/JNK activation.

Zhang X, Wang X, Wu T, Li B, Liu T, Wang R, Liu Q, Liu Z, Gong Y, Shao C - Sci Rep (2015)

Bottom Line: We found that both p38 MAPK and JNK signaling pathways were activated by isoliensinine treatment and contributed to the induction of apoptosis.Furthermore, inhibitors or specific siRNAs of p38 MAPK and JNK could attenuate apoptosis induced by isoliensinine.However, only the p38 inhibitor or p38-specific siRNA blocked the elevation of ROS in isoliensinine-treated cells.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Experimental Teratology, Ministry of Education and Department of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012, China.

ABSTRACT
Isoliensinine, liensinine and neferine are major bisbenzylisoquinoline alkaloids in the seed embryo of lotus (Nelumbo nucifera), and exhibit potential anti-cancer activity. Here, we explored the effects of these alkaloids on triple-negative breast cancer cells and found that among the three alkaloids isoliensinine possesses the most potent cytotoxic effect, primarily by inducing apoptosis. Interestingly, isoliensinine showed a much lower cytotoxicity against MCF-10A, a normal human breast epithelial cell line. Further studies showed that isoliensinine could significantly increase the production of reactive oxygen species (ROS) in triple-negative breast cancer cells, but not in MCF-10A cells. The isoliensinine-induced apoptosis could be attenuated by radical oxygen scavenger N-acetyl cysteine, suggesting that the cytotoxic effect of isoliensinine on cancer cells is at least partially achieved by inducing oxidative stress. We found that both p38 MAPK and JNK signaling pathways were activated by isoliensinine treatment and contributed to the induction of apoptosis. Furthermore, inhibitors or specific siRNAs of p38 MAPK and JNK could attenuate apoptosis induced by isoliensinine. However, only the p38 inhibitor or p38-specific siRNA blocked the elevation of ROS in isoliensinine-treated cells. Our findings thus revealed a novel antitumor effect of isoliensinine on breast cancer cells and may have therapeutic implications.

No MeSH data available.


Related in: MedlinePlus

p38 MAPK and JNK pathways mediate isoliensinine-induced apoptosis in triple-negative breast cancer cells.A, time course of p38 MAPK and JNK actvation. MDA-MB-231 cells were treated with 20 μM isoliensinine for various lengths of time. B, MDA-MB-436 and MDA-MB-468 cells were incubated with 20 μM isoliensinine for 24 h. C,D, MDA-MB-231 cells were treated with 20 μM isoliensinine alone or in combination with 10 μM SB203580 (C) or 10 μM SP600125 (D) for 24 h. Cell lysates were prepared and analyzed by western blotting for cleaved PARP-1. β-actin was used as a loading control. E, p38 siRNA rescued isoliensinine-induced apoptosis. MDA-MB-231 cells were transfected with mock siRNA or p38 siRNA, and incubated with 20 μM isoliensinine for 24 h. F, JNK siRNA blocked isoliensinine-induced apoptosis. MDA-MB-231 cells were transfected with mock siRNA or JNK siRNA, and treated with 20 μM isoliensinine for 24 h. Western blot analysis was performed using total cell lysates to examine the phosphorylation levels of p38 and JNK and cleaved caspase-3 and PARP-1 protein levels. β-actin was used as a loading control. For cleaved caspase-3 and cleaved PARP-1, band intensities were quantified by ImageJ and normalized to β-actin. For p-JNK1 and p-p38, band intensities were normalized to JNK1 and p38, respectively. Data are expressed as a fold change relative to the control. Data shown are representative of three independent experiments. The full-length blots are included in the supplementary information (Figure S7, S8 and S9).
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f5: p38 MAPK and JNK pathways mediate isoliensinine-induced apoptosis in triple-negative breast cancer cells.A, time course of p38 MAPK and JNK actvation. MDA-MB-231 cells were treated with 20 μM isoliensinine for various lengths of time. B, MDA-MB-436 and MDA-MB-468 cells were incubated with 20 μM isoliensinine for 24 h. C,D, MDA-MB-231 cells were treated with 20 μM isoliensinine alone or in combination with 10 μM SB203580 (C) or 10 μM SP600125 (D) for 24 h. Cell lysates were prepared and analyzed by western blotting for cleaved PARP-1. β-actin was used as a loading control. E, p38 siRNA rescued isoliensinine-induced apoptosis. MDA-MB-231 cells were transfected with mock siRNA or p38 siRNA, and incubated with 20 μM isoliensinine for 24 h. F, JNK siRNA blocked isoliensinine-induced apoptosis. MDA-MB-231 cells were transfected with mock siRNA or JNK siRNA, and treated with 20 μM isoliensinine for 24 h. Western blot analysis was performed using total cell lysates to examine the phosphorylation levels of p38 and JNK and cleaved caspase-3 and PARP-1 protein levels. β-actin was used as a loading control. For cleaved caspase-3 and cleaved PARP-1, band intensities were quantified by ImageJ and normalized to β-actin. For p-JNK1 and p-p38, band intensities were normalized to JNK1 and p38, respectively. Data are expressed as a fold change relative to the control. Data shown are representative of three independent experiments. The full-length blots are included in the supplementary information (Figure S7, S8 and S9).

Mentions: Many anticancer compounds induce ROS formation and activate MAPK signaling, and ultimately cause apoptosis in cancer cells1920. In our previous study, we have shown that neferine could inhibit proliferation of human osteosarcoma cells and activate p38 MAPK and JNK pathways5. We next examined the phosphorylation (activation) status of the p38 MAPK and JNK proteins. MDA-MB-231cells were exposed to 20 μM isoliensinine for various lengths of time (3 to 24 h) and the activations of the p38 MAPK and JNK pathways were evaluated by immunoblotting. The levels of phosphorylated p38 MAPK and JNK were gradually increased after the isoliensinie treatment (Fig. 5A). Meanwhile, the levels of cleaved caspase-3 and PARP-1 were elevated in a time-dependent manner in MDA-MB-231 cells (Fig. 5A). In addition, MDA-MB-436 cells and MDA-MB-468 cells also showed activation of p38 MAPK and JNK in response to 20 μM isoliensinine treatment for 24 h (Fig. 5B). These results suggested that p38 MAPK and JNK pathways might mediate isoliensinine-induced apoptosis.


Isoliensinine induces apoptosis in triple-negative human breast cancer cells through ROS generation and p38 MAPK/JNK activation.

Zhang X, Wang X, Wu T, Li B, Liu T, Wang R, Liu Q, Liu Z, Gong Y, Shao C - Sci Rep (2015)

p38 MAPK and JNK pathways mediate isoliensinine-induced apoptosis in triple-negative breast cancer cells.A, time course of p38 MAPK and JNK actvation. MDA-MB-231 cells were treated with 20 μM isoliensinine for various lengths of time. B, MDA-MB-436 and MDA-MB-468 cells were incubated with 20 μM isoliensinine for 24 h. C,D, MDA-MB-231 cells were treated with 20 μM isoliensinine alone or in combination with 10 μM SB203580 (C) or 10 μM SP600125 (D) for 24 h. Cell lysates were prepared and analyzed by western blotting for cleaved PARP-1. β-actin was used as a loading control. E, p38 siRNA rescued isoliensinine-induced apoptosis. MDA-MB-231 cells were transfected with mock siRNA or p38 siRNA, and incubated with 20 μM isoliensinine for 24 h. F, JNK siRNA blocked isoliensinine-induced apoptosis. MDA-MB-231 cells were transfected with mock siRNA or JNK siRNA, and treated with 20 μM isoliensinine for 24 h. Western blot analysis was performed using total cell lysates to examine the phosphorylation levels of p38 and JNK and cleaved caspase-3 and PARP-1 protein levels. β-actin was used as a loading control. For cleaved caspase-3 and cleaved PARP-1, band intensities were quantified by ImageJ and normalized to β-actin. For p-JNK1 and p-p38, band intensities were normalized to JNK1 and p38, respectively. Data are expressed as a fold change relative to the control. Data shown are representative of three independent experiments. The full-length blots are included in the supplementary information (Figure S7, S8 and S9).
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Related In: Results  -  Collection

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f5: p38 MAPK and JNK pathways mediate isoliensinine-induced apoptosis in triple-negative breast cancer cells.A, time course of p38 MAPK and JNK actvation. MDA-MB-231 cells were treated with 20 μM isoliensinine for various lengths of time. B, MDA-MB-436 and MDA-MB-468 cells were incubated with 20 μM isoliensinine for 24 h. C,D, MDA-MB-231 cells were treated with 20 μM isoliensinine alone or in combination with 10 μM SB203580 (C) or 10 μM SP600125 (D) for 24 h. Cell lysates were prepared and analyzed by western blotting for cleaved PARP-1. β-actin was used as a loading control. E, p38 siRNA rescued isoliensinine-induced apoptosis. MDA-MB-231 cells were transfected with mock siRNA or p38 siRNA, and incubated with 20 μM isoliensinine for 24 h. F, JNK siRNA blocked isoliensinine-induced apoptosis. MDA-MB-231 cells were transfected with mock siRNA or JNK siRNA, and treated with 20 μM isoliensinine for 24 h. Western blot analysis was performed using total cell lysates to examine the phosphorylation levels of p38 and JNK and cleaved caspase-3 and PARP-1 protein levels. β-actin was used as a loading control. For cleaved caspase-3 and cleaved PARP-1, band intensities were quantified by ImageJ and normalized to β-actin. For p-JNK1 and p-p38, band intensities were normalized to JNK1 and p38, respectively. Data are expressed as a fold change relative to the control. Data shown are representative of three independent experiments. The full-length blots are included in the supplementary information (Figure S7, S8 and S9).
Mentions: Many anticancer compounds induce ROS formation and activate MAPK signaling, and ultimately cause apoptosis in cancer cells1920. In our previous study, we have shown that neferine could inhibit proliferation of human osteosarcoma cells and activate p38 MAPK and JNK pathways5. We next examined the phosphorylation (activation) status of the p38 MAPK and JNK proteins. MDA-MB-231cells were exposed to 20 μM isoliensinine for various lengths of time (3 to 24 h) and the activations of the p38 MAPK and JNK pathways were evaluated by immunoblotting. The levels of phosphorylated p38 MAPK and JNK were gradually increased after the isoliensinie treatment (Fig. 5A). Meanwhile, the levels of cleaved caspase-3 and PARP-1 were elevated in a time-dependent manner in MDA-MB-231 cells (Fig. 5A). In addition, MDA-MB-436 cells and MDA-MB-468 cells also showed activation of p38 MAPK and JNK in response to 20 μM isoliensinine treatment for 24 h (Fig. 5B). These results suggested that p38 MAPK and JNK pathways might mediate isoliensinine-induced apoptosis.

Bottom Line: We found that both p38 MAPK and JNK signaling pathways were activated by isoliensinine treatment and contributed to the induction of apoptosis.Furthermore, inhibitors or specific siRNAs of p38 MAPK and JNK could attenuate apoptosis induced by isoliensinine.However, only the p38 inhibitor or p38-specific siRNA blocked the elevation of ROS in isoliensinine-treated cells.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Experimental Teratology, Ministry of Education and Department of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012, China.

ABSTRACT
Isoliensinine, liensinine and neferine are major bisbenzylisoquinoline alkaloids in the seed embryo of lotus (Nelumbo nucifera), and exhibit potential anti-cancer activity. Here, we explored the effects of these alkaloids on triple-negative breast cancer cells and found that among the three alkaloids isoliensinine possesses the most potent cytotoxic effect, primarily by inducing apoptosis. Interestingly, isoliensinine showed a much lower cytotoxicity against MCF-10A, a normal human breast epithelial cell line. Further studies showed that isoliensinine could significantly increase the production of reactive oxygen species (ROS) in triple-negative breast cancer cells, but not in MCF-10A cells. The isoliensinine-induced apoptosis could be attenuated by radical oxygen scavenger N-acetyl cysteine, suggesting that the cytotoxic effect of isoliensinine on cancer cells is at least partially achieved by inducing oxidative stress. We found that both p38 MAPK and JNK signaling pathways were activated by isoliensinine treatment and contributed to the induction of apoptosis. Furthermore, inhibitors or specific siRNAs of p38 MAPK and JNK could attenuate apoptosis induced by isoliensinine. However, only the p38 inhibitor or p38-specific siRNA blocked the elevation of ROS in isoliensinine-treated cells. Our findings thus revealed a novel antitumor effect of isoliensinine on breast cancer cells and may have therapeutic implications.

No MeSH data available.


Related in: MedlinePlus