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Morin, a flavonoid from moraceae, induces apoptosis by induction of BAD protein in human leukemic cells.

Park C, Lee WS, Go SI, Nagappan A, Han MH, Hong SH, Kim GS, Kim GY, Kwon TK, Ryu CH, Shin SC, Choi YH - Int J Mol Sci (2014)

Bottom Line: An anti-cancer effect of morin was screened with several human leukemic cell lines.In conclusion, morin induced caspase-dependent apoptosis through an intrinsic pathway by upregulating BAD proteins.This study provides evidence that morin might have anticancer properties in human leukemic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, College of Natural Sciences, Dongeui University, Busan 614-714, Korea. parkch@deu.ac.kr.

ABSTRACT
Evidence suggests that phytochemicals can safely modulate cancer cell biology and induce apoptosis. Here, we investigated the anti-cancer activity of morin, a flavone originally isolated from members of the Moraceae family in human leukemic cells, focusing on apoptosis. An anti-cancer effect of morin was screened with several human leukemic cell lines. U937 cells were most sensitive to morin, where it induced caspase-dependent apoptosis in a dose-dependent manner. It also induced loss of MMP (ΔΨm) along with cytochrome c release, down-regulated Bcl-2 protein, and up-regulated BAX proteins. The apoptotic activity of morin was significantly attenuated by Bcl-2 augmentation. In conclusion, morin induced caspase-dependent apoptosis through an intrinsic pathway by upregulating BAD proteins. In addition, Bcl-2 protein expression is also important in morin-induced apoptosis of U937 cells. This study provides evidence that morin might have anticancer properties in human leukemic cells.

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Effects of Bcl-2 overexpression on morin-induced apoptosis. U937/vector or U937/Bcl-2 cells were treated with morin for 48 h, and effects of Bcl-2 overexpression on morin-induced apoptosis. (A) MTT assay. The data are shown as means ± SD of three independent experiments. *p < 0.05 vs. control; (B) DNA fragmentation test; (C) Cell cycle analysis; The cells harboring sub-G1 DNA content represents the fractions undergoing apoptotic DNA degradation by morin treatment; (D) Flow cytometry for the dual staining of Annexin V and PI. The proportion was expressed by percentage and (E) The effects of Bcl-2 overexpression on the expression of Bcl-2 family members and caspases in the cells treated with morin. The expression of BAD protein was not induced in U937/Bcl-2 cells suggesting that the up-regulation of BAD expression by morin may be associated with inhibition of BAD protein degradation. The results are from one representative of two independent experiments that showed similar patterns. The expression of the indicated proteins were measured by densitometry and expressed as average relative ratio compared to actin, from two or three different experiments.
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ijms-16-00645-f005: Effects of Bcl-2 overexpression on morin-induced apoptosis. U937/vector or U937/Bcl-2 cells were treated with morin for 48 h, and effects of Bcl-2 overexpression on morin-induced apoptosis. (A) MTT assay. The data are shown as means ± SD of three independent experiments. *p < 0.05 vs. control; (B) DNA fragmentation test; (C) Cell cycle analysis; The cells harboring sub-G1 DNA content represents the fractions undergoing apoptotic DNA degradation by morin treatment; (D) Flow cytometry for the dual staining of Annexin V and PI. The proportion was expressed by percentage and (E) The effects of Bcl-2 overexpression on the expression of Bcl-2 family members and caspases in the cells treated with morin. The expression of BAD protein was not induced in U937/Bcl-2 cells suggesting that the up-regulation of BAD expression by morin may be associated with inhibition of BAD protein degradation. The results are from one representative of two independent experiments that showed similar patterns. The expression of the indicated proteins were measured by densitometry and expressed as average relative ratio compared to actin, from two or three different experiments.

Mentions: BAD forms a heterodimer with Bcl-2 and Bcl-xL, inactivating them and thus inducing apoptosis [10]. Free forms of Bcl-2 inhibits BAD-triggered apoptosis [4]. In addition, Bcl-2 is the founding member of family proteins that regulate apoptosis. Previous data suggested that BAD protein can induce apoptosis in the cells with Bcl-2 overexpression without loss of MMP (ΔΨm) [11]. To answer the question whether morin-induced BAD can induce apoptosis without triggering the loss of MMP (ΔΨm), we evaluated the effects of high level of Bcl-2 on morin-induced apoptosis, by comparing U937/vector with U937/Bcl-2 cells that constitutively express high levels of Bcl-2. Unexpectedly, overexpressed Bcl-2 significantly inhibited morin-induced cell death (Figure 5A), DNA fragmentation (Figure 5B), and apoptotic cell death (Figure 5C). In addition, Bcl-2 overexpression reduced morin-induced loss of MMP (ΔΨm) (Figure 5D). Western blot analysis revealed that Bcl-2 expression level in U937/Bcl-2 cells was significantly higher (five-fold) than that in U937/vector cells, and that BAD expression was slightly lower than that in U937/vector cells (Figure 5E). The overexpressed Bcl-2 suppressed the activation of caspase 3 and subsequent PARP cleavages (Figure 5E). Interestingly, morin did not induce upregulation of BAD protein in U937/Bcl-2 cells. These finding suggests that Bcl-2 overexpression may suppress morin-induced apoptosis through inhibition of loss of MMP (ΔΨm) and/or suppression of BAD protein expression.


Morin, a flavonoid from moraceae, induces apoptosis by induction of BAD protein in human leukemic cells.

Park C, Lee WS, Go SI, Nagappan A, Han MH, Hong SH, Kim GS, Kim GY, Kwon TK, Ryu CH, Shin SC, Choi YH - Int J Mol Sci (2014)

Effects of Bcl-2 overexpression on morin-induced apoptosis. U937/vector or U937/Bcl-2 cells were treated with morin for 48 h, and effects of Bcl-2 overexpression on morin-induced apoptosis. (A) MTT assay. The data are shown as means ± SD of three independent experiments. *p < 0.05 vs. control; (B) DNA fragmentation test; (C) Cell cycle analysis; The cells harboring sub-G1 DNA content represents the fractions undergoing apoptotic DNA degradation by morin treatment; (D) Flow cytometry for the dual staining of Annexin V and PI. The proportion was expressed by percentage and (E) The effects of Bcl-2 overexpression on the expression of Bcl-2 family members and caspases in the cells treated with morin. The expression of BAD protein was not induced in U937/Bcl-2 cells suggesting that the up-regulation of BAD expression by morin may be associated with inhibition of BAD protein degradation. The results are from one representative of two independent experiments that showed similar patterns. The expression of the indicated proteins were measured by densitometry and expressed as average relative ratio compared to actin, from two or three different experiments.
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Related In: Results  -  Collection

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ijms-16-00645-f005: Effects of Bcl-2 overexpression on morin-induced apoptosis. U937/vector or U937/Bcl-2 cells were treated with morin for 48 h, and effects of Bcl-2 overexpression on morin-induced apoptosis. (A) MTT assay. The data are shown as means ± SD of three independent experiments. *p < 0.05 vs. control; (B) DNA fragmentation test; (C) Cell cycle analysis; The cells harboring sub-G1 DNA content represents the fractions undergoing apoptotic DNA degradation by morin treatment; (D) Flow cytometry for the dual staining of Annexin V and PI. The proportion was expressed by percentage and (E) The effects of Bcl-2 overexpression on the expression of Bcl-2 family members and caspases in the cells treated with morin. The expression of BAD protein was not induced in U937/Bcl-2 cells suggesting that the up-regulation of BAD expression by morin may be associated with inhibition of BAD protein degradation. The results are from one representative of two independent experiments that showed similar patterns. The expression of the indicated proteins were measured by densitometry and expressed as average relative ratio compared to actin, from two or three different experiments.
Mentions: BAD forms a heterodimer with Bcl-2 and Bcl-xL, inactivating them and thus inducing apoptosis [10]. Free forms of Bcl-2 inhibits BAD-triggered apoptosis [4]. In addition, Bcl-2 is the founding member of family proteins that regulate apoptosis. Previous data suggested that BAD protein can induce apoptosis in the cells with Bcl-2 overexpression without loss of MMP (ΔΨm) [11]. To answer the question whether morin-induced BAD can induce apoptosis without triggering the loss of MMP (ΔΨm), we evaluated the effects of high level of Bcl-2 on morin-induced apoptosis, by comparing U937/vector with U937/Bcl-2 cells that constitutively express high levels of Bcl-2. Unexpectedly, overexpressed Bcl-2 significantly inhibited morin-induced cell death (Figure 5A), DNA fragmentation (Figure 5B), and apoptotic cell death (Figure 5C). In addition, Bcl-2 overexpression reduced morin-induced loss of MMP (ΔΨm) (Figure 5D). Western blot analysis revealed that Bcl-2 expression level in U937/Bcl-2 cells was significantly higher (five-fold) than that in U937/vector cells, and that BAD expression was slightly lower than that in U937/vector cells (Figure 5E). The overexpressed Bcl-2 suppressed the activation of caspase 3 and subsequent PARP cleavages (Figure 5E). Interestingly, morin did not induce upregulation of BAD protein in U937/Bcl-2 cells. These finding suggests that Bcl-2 overexpression may suppress morin-induced apoptosis through inhibition of loss of MMP (ΔΨm) and/or suppression of BAD protein expression.

Bottom Line: An anti-cancer effect of morin was screened with several human leukemic cell lines.In conclusion, morin induced caspase-dependent apoptosis through an intrinsic pathway by upregulating BAD proteins.This study provides evidence that morin might have anticancer properties in human leukemic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, College of Natural Sciences, Dongeui University, Busan 614-714, Korea. parkch@deu.ac.kr.

ABSTRACT
Evidence suggests that phytochemicals can safely modulate cancer cell biology and induce apoptosis. Here, we investigated the anti-cancer activity of morin, a flavone originally isolated from members of the Moraceae family in human leukemic cells, focusing on apoptosis. An anti-cancer effect of morin was screened with several human leukemic cell lines. U937 cells were most sensitive to morin, where it induced caspase-dependent apoptosis in a dose-dependent manner. It also induced loss of MMP (ΔΨm) along with cytochrome c release, down-regulated Bcl-2 protein, and up-regulated BAX proteins. The apoptotic activity of morin was significantly attenuated by Bcl-2 augmentation. In conclusion, morin induced caspase-dependent apoptosis through an intrinsic pathway by upregulating BAD proteins. In addition, Bcl-2 protein expression is also important in morin-induced apoptosis of U937 cells. This study provides evidence that morin might have anticancer properties in human leukemic cells.

Show MeSH
Related in: MedlinePlus