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Melittin restores PTEN expression by down-regulating HDAC2 in human hepatocelluar carcinoma HepG2 cells.

Zhang H, Zhao B, Huang C, Meng XM, Bian EB, Li J - PLoS ONE (2014)

Bottom Line: Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4.Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression.These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: School of pharmacy, Anhui key laboratory of bioactivity of natural products, Anhui Medical University, Hefei, Anhui Province, China; Institute for Liver Diseases of Anhui Medical University (AMU), Hefei, Anhui Province, China.

ABSTRACT
Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2) expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

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Effects of PTEN on Akt activation in HepG2 cells.HepG2 cells were treated with 1, 4, 8 µg/ml of melittin and 0.5 µmol/ml TSA for 24 h. The p-Akt and Akt proteins expression were analyzed by Western blot (a). *P<0.05, **P<0.01 compared to cells without melittin treatment. Silencing of HDAC2 genes in HepG2 cells was performed and phosphorylation of Akt was checked by Western blot (b) as described in the Materials and methods section. Representative images of at least three independent experiments are shown and results of densitometric analyses (mean±SE) are shown. **P<0.01 vs controls and scrambled siRNA. HepG2 cells were treated with pEGFP-C2, pEGFP-C2-HDAC2 plasmid, pEGFP-C2-HDAC2 plasmid and melittin, pEGFP-C2-HDAC2 plasmid and TSA for 24 h. Phosphorylation of Akt was checked by Western blot (c). **P<0.01 vs control and vector group. #P<0.05 vs pEGFP-C2-HDAC-2 plasmid group.
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pone-0095520-g008: Effects of PTEN on Akt activation in HepG2 cells.HepG2 cells were treated with 1, 4, 8 µg/ml of melittin and 0.5 µmol/ml TSA for 24 h. The p-Akt and Akt proteins expression were analyzed by Western blot (a). *P<0.05, **P<0.01 compared to cells without melittin treatment. Silencing of HDAC2 genes in HepG2 cells was performed and phosphorylation of Akt was checked by Western blot (b) as described in the Materials and methods section. Representative images of at least three independent experiments are shown and results of densitometric analyses (mean±SE) are shown. **P<0.01 vs controls and scrambled siRNA. HepG2 cells were treated with pEGFP-C2, pEGFP-C2-HDAC2 plasmid, pEGFP-C2-HDAC2 plasmid and melittin, pEGFP-C2-HDAC2 plasmid and TSA for 24 h. Phosphorylation of Akt was checked by Western blot (c). **P<0.01 vs control and vector group. #P<0.05 vs pEGFP-C2-HDAC-2 plasmid group.

Mentions: Akt is a downstream receptor of PI3K that is known to mediate survival signaling. To determine whether melittin-inhibited cell proliferation is closely related to an Akt signal, we examined the phosphorylation levels of Akt in cells following melittin treatment for 24 h. It was demonstrated that (Fig 8 a), melittin did not affect total Akt protein levels; however, Akt phosphorylation decreased in comparison to control after treatment of melittin for 24 h. Knockdown of HDAC2 significantly prevented the activation of Akt comparied to control or scrambled siRNA (Fig 8 b). To further investigate the interaction between HDAC2 and melittin in the regulation of activation of PI3K/Akt signaling, we overexpressed HDAC2 and treated with melittin or TSA in HepG2 cells. The study revealed that the phosphorylation level of Akt was obviously elevated, while the total protein expression level of Akt did not changed. However, the phosphorylation level of Akt was reduced under melittin or TSA in comparison to pEGFP-C2-HDAC2 plasmid, while it was still higher than the control and vector groups(Fig 8 c). These results suggest that there might be a deacetylation occurred in the PTEN gene which contributes to the increase in PI3K/Akt activation and melittin downregulated the expression of phosphorylation Akt, suppressed the activation of Akt.


Melittin restores PTEN expression by down-regulating HDAC2 in human hepatocelluar carcinoma HepG2 cells.

Zhang H, Zhao B, Huang C, Meng XM, Bian EB, Li J - PLoS ONE (2014)

Effects of PTEN on Akt activation in HepG2 cells.HepG2 cells were treated with 1, 4, 8 µg/ml of melittin and 0.5 µmol/ml TSA for 24 h. The p-Akt and Akt proteins expression were analyzed by Western blot (a). *P<0.05, **P<0.01 compared to cells without melittin treatment. Silencing of HDAC2 genes in HepG2 cells was performed and phosphorylation of Akt was checked by Western blot (b) as described in the Materials and methods section. Representative images of at least three independent experiments are shown and results of densitometric analyses (mean±SE) are shown. **P<0.01 vs controls and scrambled siRNA. HepG2 cells were treated with pEGFP-C2, pEGFP-C2-HDAC2 plasmid, pEGFP-C2-HDAC2 plasmid and melittin, pEGFP-C2-HDAC2 plasmid and TSA for 24 h. Phosphorylation of Akt was checked by Western blot (c). **P<0.01 vs control and vector group. #P<0.05 vs pEGFP-C2-HDAC-2 plasmid group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4008415&req=5

pone-0095520-g008: Effects of PTEN on Akt activation in HepG2 cells.HepG2 cells were treated with 1, 4, 8 µg/ml of melittin and 0.5 µmol/ml TSA for 24 h. The p-Akt and Akt proteins expression were analyzed by Western blot (a). *P<0.05, **P<0.01 compared to cells without melittin treatment. Silencing of HDAC2 genes in HepG2 cells was performed and phosphorylation of Akt was checked by Western blot (b) as described in the Materials and methods section. Representative images of at least three independent experiments are shown and results of densitometric analyses (mean±SE) are shown. **P<0.01 vs controls and scrambled siRNA. HepG2 cells were treated with pEGFP-C2, pEGFP-C2-HDAC2 plasmid, pEGFP-C2-HDAC2 plasmid and melittin, pEGFP-C2-HDAC2 plasmid and TSA for 24 h. Phosphorylation of Akt was checked by Western blot (c). **P<0.01 vs control and vector group. #P<0.05 vs pEGFP-C2-HDAC-2 plasmid group.
Mentions: Akt is a downstream receptor of PI3K that is known to mediate survival signaling. To determine whether melittin-inhibited cell proliferation is closely related to an Akt signal, we examined the phosphorylation levels of Akt in cells following melittin treatment for 24 h. It was demonstrated that (Fig 8 a), melittin did not affect total Akt protein levels; however, Akt phosphorylation decreased in comparison to control after treatment of melittin for 24 h. Knockdown of HDAC2 significantly prevented the activation of Akt comparied to control or scrambled siRNA (Fig 8 b). To further investigate the interaction between HDAC2 and melittin in the regulation of activation of PI3K/Akt signaling, we overexpressed HDAC2 and treated with melittin or TSA in HepG2 cells. The study revealed that the phosphorylation level of Akt was obviously elevated, while the total protein expression level of Akt did not changed. However, the phosphorylation level of Akt was reduced under melittin or TSA in comparison to pEGFP-C2-HDAC2 plasmid, while it was still higher than the control and vector groups(Fig 8 c). These results suggest that there might be a deacetylation occurred in the PTEN gene which contributes to the increase in PI3K/Akt activation and melittin downregulated the expression of phosphorylation Akt, suppressed the activation of Akt.

Bottom Line: Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4.Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression.These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: School of pharmacy, Anhui key laboratory of bioactivity of natural products, Anhui Medical University, Hefei, Anhui Province, China; Institute for Liver Diseases of Anhui Medical University (AMU), Hefei, Anhui Province, China.

ABSTRACT
Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2) expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

Show MeSH
Related in: MedlinePlus