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Methyl gallate isolated from Spondias pinnata exhibits anticancer activity against human glioblastoma by induction of apoptosis and sustained extracellular signal-regulated kinase 1/2 activation.

Chaudhuri D, Ghate NB, Singh SS, Mandal N - Pharmacogn Mag (2015 Apr-Jun)

Bottom Line: Spondias pinnata has been reported for its efficient anticancer effects, but the studies were mostly focused on its extract.MG treatment also induced the expression of p53 and B-cell lymphoma-2-associated X and cleavage of BH3 interacting-domain with a concomitant decrease in B-cell lymphoma-2 expression.Moreover, MG-induced sustained phosphorylation of extracellular signal-regulated kinase (ERK1/2) in U87 cells with no change in the phosphorylation of other mitogen-activated protein kinases (c-Jun N-terminal of stress-activated protein kinases, p38).

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, West Bengal, India.

ABSTRACT

Background: Spondias pinnata has been reported for its efficient anticancer effects, but the studies were mostly focused on its extract.

Objective: Since its bioactive compounds are largely unknown, this study was designed to characterize the lead components present in it and their anticancer activity against human glioblastoma cell line (U87).

Materials and methods: Major compounds from the ethyl acetate fraction were isolated by column chromatography and their anticancer potentials against U87 cells were evaluated. Furthermore, flow cytometric and immunoblotting analyses were performed to demonstrate the mechanism of apoptosis inducing activity of methyl gallate (MG) against U87 cell line.

Results: Four major compounds were isolated from the ethyl acetate fraction. Amongst these, two compounds showed promising activities and with the help of different spectroscopic methods they were identified as gallic acid and MG. Flow cytometric studies revealed that MG-induced apoptosis in U87 cells dose-dependently; the same was confirmed by activation of caspases through cleavage of endogenous substrate poly (adenosine diphosphate-ribose) polymerase. MG treatment also induced the expression of p53 and B-cell lymphoma-2-associated X and cleavage of BH3 interacting-domain with a concomitant decrease in B-cell lymphoma-2 expression. Moreover, MG-induced sustained phosphorylation of extracellular signal-regulated kinase (ERK1/2) in U87 cells with no change in the phosphorylation of other mitogen-activated protein kinases (c-Jun N-terminal of stress-activated protein kinases, p38).

Conclusion: MG is a potent antioxidant and it induces sustained ERK1/2 activation and apoptosis in human glioblastoma U87, and provide a rationale for evaluation of MG for other brain carcinoma cell lines for the advancement of glioblastoma therapy.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of caspases and poly(adenosine diphosphate-ribose) polymerase (PARP) of U87 cells treated with 50 μM methyl gallate. Graphs adjoining the blots represent the expression levels of corresponding proteins for indicated time intervals: (a) Pro and cleaved caspase-9, (b) Pro and cleaved caspase-3, (c) Pro and cleaved caspase-8, (d) Native and cleaved PARP
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Figure 5: Western blot analysis of caspases and poly(adenosine diphosphate-ribose) polymerase (PARP) of U87 cells treated with 50 μM methyl gallate. Graphs adjoining the blots represent the expression levels of corresponding proteins for indicated time intervals: (a) Pro and cleaved caspase-9, (b) Pro and cleaved caspase-3, (c) Pro and cleaved caspase-8, (d) Native and cleaved PARP

Mentions: Mitochondria play a pivotal role in the activation of caspase cascade and thereby signal transduction of apoptosis.[19] Effects of MG on the proteolytic activation of caspase-9 and caspase-3 were then examined. As shown in Figure 5a and b, MG treatment resulted in a significant increase in the active form of caspase-9 and caspase-3 in U87 cells. Furthermore, procaspse-8 level decreased, caspase-8 level elevated [Figure 5c]. The activation of caspases in MG treated U87 cells was further confirmed by detecting the cleavage of PARP, an endogenous substrate of activated caspase-3 and a hallmark of apoptosis. As shown in Figure 5d, treatment of U87 cells with MG resulted in the cleavage of PARP to a 25 kDa fragment. The observation of MG-mediated activation of caspase-9, caspase-3, subsequent cleavage of PARP in U87 cells, suggesting that mitochondrial-mediated caspase cascade pathway plays a very important role in MG-induced apoptosis in U87 cells.[20] Bcl-2 family proteins, including Bcl-2 and Bcl-2-related family members such as Bax, Bid, Bcl-extra-large and Bcl-2-associated death promoter, play an important role in the regulation of apoptosis. The balance between the expression levels of Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), is critical for cell survival and death as the increase in Bax/Bcl-2 ratio contributes to the release of cytochrome C from mitochondria and decides the susceptibility of cells to undergo apoptosis.[21] As shown in Figure 6a, MG treatment increased the level of Bax expression while reduced Bcl-2 expression in a time-dependent manner. A densitometric analysis of the bands revealed that MG increased the Bax/Bcl-2 ratio, which is responsible for MG-induced apoptosis in U87 cells. As cleaved caspase-8 level was elevated there was an appearance of the t-Bid [Figure 6b] after the treatment with MG. Previously it is reported that, caspase-8 links intrinsic pathway with extrinsic pathway by cleaving Bid into truncated-Bid and plays an important role in activation of both these pathways.[22] It is possible that p53 molecule plays a role in the alteration of the expression of Bax and Bcl-2. Our results showed that MG treatment induces the expression of p53 in U87 cells [Figure 7a]. This suggests that p53 is responsible for the upregulation of Bax and down regulation of Bcl-2 in MG treated U87 cells. The ability of wild type p53 to upregulate Bax and downregulate Bcl-2 and proceed to the apoptosis is demonstrated previously.[23]


Methyl gallate isolated from Spondias pinnata exhibits anticancer activity against human glioblastoma by induction of apoptosis and sustained extracellular signal-regulated kinase 1/2 activation.

Chaudhuri D, Ghate NB, Singh SS, Mandal N - Pharmacogn Mag (2015 Apr-Jun)

Western blot analysis of caspases and poly(adenosine diphosphate-ribose) polymerase (PARP) of U87 cells treated with 50 μM methyl gallate. Graphs adjoining the blots represent the expression levels of corresponding proteins for indicated time intervals: (a) Pro and cleaved caspase-9, (b) Pro and cleaved caspase-3, (c) Pro and cleaved caspase-8, (d) Native and cleaved PARP
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4378123&req=5

Figure 5: Western blot analysis of caspases and poly(adenosine diphosphate-ribose) polymerase (PARP) of U87 cells treated with 50 μM methyl gallate. Graphs adjoining the blots represent the expression levels of corresponding proteins for indicated time intervals: (a) Pro and cleaved caspase-9, (b) Pro and cleaved caspase-3, (c) Pro and cleaved caspase-8, (d) Native and cleaved PARP
Mentions: Mitochondria play a pivotal role in the activation of caspase cascade and thereby signal transduction of apoptosis.[19] Effects of MG on the proteolytic activation of caspase-9 and caspase-3 were then examined. As shown in Figure 5a and b, MG treatment resulted in a significant increase in the active form of caspase-9 and caspase-3 in U87 cells. Furthermore, procaspse-8 level decreased, caspase-8 level elevated [Figure 5c]. The activation of caspases in MG treated U87 cells was further confirmed by detecting the cleavage of PARP, an endogenous substrate of activated caspase-3 and a hallmark of apoptosis. As shown in Figure 5d, treatment of U87 cells with MG resulted in the cleavage of PARP to a 25 kDa fragment. The observation of MG-mediated activation of caspase-9, caspase-3, subsequent cleavage of PARP in U87 cells, suggesting that mitochondrial-mediated caspase cascade pathway plays a very important role in MG-induced apoptosis in U87 cells.[20] Bcl-2 family proteins, including Bcl-2 and Bcl-2-related family members such as Bax, Bid, Bcl-extra-large and Bcl-2-associated death promoter, play an important role in the regulation of apoptosis. The balance between the expression levels of Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), is critical for cell survival and death as the increase in Bax/Bcl-2 ratio contributes to the release of cytochrome C from mitochondria and decides the susceptibility of cells to undergo apoptosis.[21] As shown in Figure 6a, MG treatment increased the level of Bax expression while reduced Bcl-2 expression in a time-dependent manner. A densitometric analysis of the bands revealed that MG increased the Bax/Bcl-2 ratio, which is responsible for MG-induced apoptosis in U87 cells. As cleaved caspase-8 level was elevated there was an appearance of the t-Bid [Figure 6b] after the treatment with MG. Previously it is reported that, caspase-8 links intrinsic pathway with extrinsic pathway by cleaving Bid into truncated-Bid and plays an important role in activation of both these pathways.[22] It is possible that p53 molecule plays a role in the alteration of the expression of Bax and Bcl-2. Our results showed that MG treatment induces the expression of p53 in U87 cells [Figure 7a]. This suggests that p53 is responsible for the upregulation of Bax and down regulation of Bcl-2 in MG treated U87 cells. The ability of wild type p53 to upregulate Bax and downregulate Bcl-2 and proceed to the apoptosis is demonstrated previously.[23]

Bottom Line: Spondias pinnata has been reported for its efficient anticancer effects, but the studies were mostly focused on its extract.MG treatment also induced the expression of p53 and B-cell lymphoma-2-associated X and cleavage of BH3 interacting-domain with a concomitant decrease in B-cell lymphoma-2 expression.Moreover, MG-induced sustained phosphorylation of extracellular signal-regulated kinase (ERK1/2) in U87 cells with no change in the phosphorylation of other mitogen-activated protein kinases (c-Jun N-terminal of stress-activated protein kinases, p38).

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, West Bengal, India.

ABSTRACT

Background: Spondias pinnata has been reported for its efficient anticancer effects, but the studies were mostly focused on its extract.

Objective: Since its bioactive compounds are largely unknown, this study was designed to characterize the lead components present in it and their anticancer activity against human glioblastoma cell line (U87).

Materials and methods: Major compounds from the ethyl acetate fraction were isolated by column chromatography and their anticancer potentials against U87 cells were evaluated. Furthermore, flow cytometric and immunoblotting analyses were performed to demonstrate the mechanism of apoptosis inducing activity of methyl gallate (MG) against U87 cell line.

Results: Four major compounds were isolated from the ethyl acetate fraction. Amongst these, two compounds showed promising activities and with the help of different spectroscopic methods they were identified as gallic acid and MG. Flow cytometric studies revealed that MG-induced apoptosis in U87 cells dose-dependently; the same was confirmed by activation of caspases through cleavage of endogenous substrate poly (adenosine diphosphate-ribose) polymerase. MG treatment also induced the expression of p53 and B-cell lymphoma-2-associated X and cleavage of BH3 interacting-domain with a concomitant decrease in B-cell lymphoma-2 expression. Moreover, MG-induced sustained phosphorylation of extracellular signal-regulated kinase (ERK1/2) in U87 cells with no change in the phosphorylation of other mitogen-activated protein kinases (c-Jun N-terminal of stress-activated protein kinases, p38).

Conclusion: MG is a potent antioxidant and it induces sustained ERK1/2 activation and apoptosis in human glioblastoma U87, and provide a rationale for evaluation of MG for other brain carcinoma cell lines for the advancement of glioblastoma therapy.

No MeSH data available.


Related in: MedlinePlus