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The novel histone deacetylase inhibitor, N-hydroxy-7-(2-naphthylthio) hepatonomide, exhibits potent antitumor activity due to cytochrome-c-release-mediated apoptosis in renal cell carcinoma cells.

Park KC, Heo JH, Jeon JY, Choi HJ, Jo AR, Kim SW, Kwon HJ, Hong SJ, Han KS - BMC Cancer (2015)

Bottom Line: Renal tumors have been shown to have a higher global methylation percentage and reduced histone acetylation.An in vivo study showed that HNHA had greater anti-tumor and pro-apoptotic effects on RCC xenografts than the established HDAC inhibitors.These results suggest that HNHA may offer a new therapeutic approach to RCC.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epigenetic modifications play a critical role in the regulation of all DNA-based processes, such as transcription, repair, and replication. Inappropriate histone modifications can result in dysregulation of cell growth, leading to neoplastic transformation and cell death. Renal tumors have been shown to have a higher global methylation percentage and reduced histone acetylation. Preclinical models have revealed that histone gene modifiers and epigenetic alterations play important roles in renal cell carcinoma (RCC) tumorigenesis. Recently, a novel HDAC inhibitor, N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), has been introduced as an example of a new class of anti-cancer agents. The anti-cancer activity of HNHA and the underlying mechanisms of action remain to be clarified.

Methods: The MTS assay using a panel of RCC cells was used to evaluate the anti-proliferative effects of HNHA. The established HDAC inhibitors, SAHA and TSA, were used for comparison. Western blotting analysis was performed to investigate the acetylation of histone H3 and the expression of apoptotic markers in vitro and in vivo. Subcellular fractionation was performed to evaluate expression of Bax and cytochrome c in the cytosol and mitochondria, and also translocation of cytochrome c from the cytoplasm to the nucleus. A confocal microscopic evaluation was performed to confirm inhibition of cell proliferation, induction of apoptosis, and the nuclear translocation of cytochrome c in RCC cells.

Results: In this study, we investigated the apoptosis-inducing activity of HNHA in cultured kidney cancer cells. Apoptosis in the HNHA-treated group was induced significantly, with marked caspase activation and Bcl-2 suppression in RCC cells in vitro and in vivo. HNHA treatment caused cytochrome c release from mitochondria, which was mediated by increased Bax expression and caspase activation. HNHA also induced nuclear translocation of cytochrome c, suggesting that HNHA can induce caspase-independent nuclear apoptosis in RCC cells. An in vivo study showed that HNHA had greater anti-tumor and pro-apoptotic effects on RCC xenografts than the established HDAC inhibitors.

Conclusions: HNHA has more potent anti-tumor activity than established HDAC inhibitors. Its activities are mediated by caspase-dependent and cytochrome-c-mediated apoptosis in RCC cells. These results suggest that HNHA may offer a new therapeutic approach to RCC.

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HNHA reduced tumor burden and induced caspase-dependent apoptotic cell death in anin vivomodel. Immunoblot analysis for Bcl-2, phospho-IκB, and caspases-3 and -7 in Caki-1 and A-498 cells (A, B). Cytochrome c protein levels were determined by immunohistochemistry to decide when to sacrifice the animal (C, D). Negative control slides (IgG control) were treated with isotype-matched IgG. Error bars show SEs (n = 7). *P < 0.05, vs. control, **P < 0.01, vs. control, ***P < 0.005, vs. control. The MetaMorph 4.6 image-analysis software was used to quantify the immunostained target protein.
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Fig7: HNHA reduced tumor burden and induced caspase-dependent apoptotic cell death in anin vivomodel. Immunoblot analysis for Bcl-2, phospho-IκB, and caspases-3 and -7 in Caki-1 and A-498 cells (A, B). Cytochrome c protein levels were determined by immunohistochemistry to decide when to sacrifice the animal (C, D). Negative control slides (IgG control) were treated with isotype-matched IgG. Error bars show SEs (n = 7). *P < 0.05, vs. control, **P < 0.01, vs. control, ***P < 0.005, vs. control. The MetaMorph 4.6 image-analysis software was used to quantify the immunostained target protein.

Mentions: To determine whether the potent antitumor effect of HNHA in the in vivo animal model was related to the induction of apoptosis, we examined the expression of apoptosis-related proteins in xenograft tissues harvested after treatment. All HDAC inhibitors downregulated Bcl-2 expression and activation of IκB, induced the ‘pro’ forms of caspases-3 and -7, and activated caspases-3 and -7 by cleavage in Caki-1 and A-498 xenografts (Figure 7A, B). HNHA showed the most potent inhibition of Bcl-2 and p-IκB and activation of caspase in RCC cells. To examine whether the induction of apoptosis was related to cytochrome c release, we performed immunohistochemical staining for cytochrome c in the harvested xenografts (Figure 7C, D). Cytochrome c was stained focally in the cytoplasm in the control group but diffusely throughout the cytoplasm in the groups treated with HDAC inhibitors. The staining was most intense in the HNHA-treated xenografts. In summary, these data indicate that HNHA induced more potent RCC tumor suppression in an animal model by activation of caspase-dependent apoptotic signals and cytochrome c release from mitochondria.Figure 7


The novel histone deacetylase inhibitor, N-hydroxy-7-(2-naphthylthio) hepatonomide, exhibits potent antitumor activity due to cytochrome-c-release-mediated apoptosis in renal cell carcinoma cells.

Park KC, Heo JH, Jeon JY, Choi HJ, Jo AR, Kim SW, Kwon HJ, Hong SJ, Han KS - BMC Cancer (2015)

HNHA reduced tumor burden and induced caspase-dependent apoptotic cell death in anin vivomodel. Immunoblot analysis for Bcl-2, phospho-IκB, and caspases-3 and -7 in Caki-1 and A-498 cells (A, B). Cytochrome c protein levels were determined by immunohistochemistry to decide when to sacrifice the animal (C, D). Negative control slides (IgG control) were treated with isotype-matched IgG. Error bars show SEs (n = 7). *P < 0.05, vs. control, **P < 0.01, vs. control, ***P < 0.005, vs. control. The MetaMorph 4.6 image-analysis software was used to quantify the immunostained target protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4318161&req=5

Fig7: HNHA reduced tumor burden and induced caspase-dependent apoptotic cell death in anin vivomodel. Immunoblot analysis for Bcl-2, phospho-IκB, and caspases-3 and -7 in Caki-1 and A-498 cells (A, B). Cytochrome c protein levels were determined by immunohistochemistry to decide when to sacrifice the animal (C, D). Negative control slides (IgG control) were treated with isotype-matched IgG. Error bars show SEs (n = 7). *P < 0.05, vs. control, **P < 0.01, vs. control, ***P < 0.005, vs. control. The MetaMorph 4.6 image-analysis software was used to quantify the immunostained target protein.
Mentions: To determine whether the potent antitumor effect of HNHA in the in vivo animal model was related to the induction of apoptosis, we examined the expression of apoptosis-related proteins in xenograft tissues harvested after treatment. All HDAC inhibitors downregulated Bcl-2 expression and activation of IκB, induced the ‘pro’ forms of caspases-3 and -7, and activated caspases-3 and -7 by cleavage in Caki-1 and A-498 xenografts (Figure 7A, B). HNHA showed the most potent inhibition of Bcl-2 and p-IκB and activation of caspase in RCC cells. To examine whether the induction of apoptosis was related to cytochrome c release, we performed immunohistochemical staining for cytochrome c in the harvested xenografts (Figure 7C, D). Cytochrome c was stained focally in the cytoplasm in the control group but diffusely throughout the cytoplasm in the groups treated with HDAC inhibitors. The staining was most intense in the HNHA-treated xenografts. In summary, these data indicate that HNHA induced more potent RCC tumor suppression in an animal model by activation of caspase-dependent apoptotic signals and cytochrome c release from mitochondria.Figure 7

Bottom Line: Renal tumors have been shown to have a higher global methylation percentage and reduced histone acetylation.An in vivo study showed that HNHA had greater anti-tumor and pro-apoptotic effects on RCC xenografts than the established HDAC inhibitors.These results suggest that HNHA may offer a new therapeutic approach to RCC.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epigenetic modifications play a critical role in the regulation of all DNA-based processes, such as transcription, repair, and replication. Inappropriate histone modifications can result in dysregulation of cell growth, leading to neoplastic transformation and cell death. Renal tumors have been shown to have a higher global methylation percentage and reduced histone acetylation. Preclinical models have revealed that histone gene modifiers and epigenetic alterations play important roles in renal cell carcinoma (RCC) tumorigenesis. Recently, a novel HDAC inhibitor, N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), has been introduced as an example of a new class of anti-cancer agents. The anti-cancer activity of HNHA and the underlying mechanisms of action remain to be clarified.

Methods: The MTS assay using a panel of RCC cells was used to evaluate the anti-proliferative effects of HNHA. The established HDAC inhibitors, SAHA and TSA, were used for comparison. Western blotting analysis was performed to investigate the acetylation of histone H3 and the expression of apoptotic markers in vitro and in vivo. Subcellular fractionation was performed to evaluate expression of Bax and cytochrome c in the cytosol and mitochondria, and also translocation of cytochrome c from the cytoplasm to the nucleus. A confocal microscopic evaluation was performed to confirm inhibition of cell proliferation, induction of apoptosis, and the nuclear translocation of cytochrome c in RCC cells.

Results: In this study, we investigated the apoptosis-inducing activity of HNHA in cultured kidney cancer cells. Apoptosis in the HNHA-treated group was induced significantly, with marked caspase activation and Bcl-2 suppression in RCC cells in vitro and in vivo. HNHA treatment caused cytochrome c release from mitochondria, which was mediated by increased Bax expression and caspase activation. HNHA also induced nuclear translocation of cytochrome c, suggesting that HNHA can induce caspase-independent nuclear apoptosis in RCC cells. An in vivo study showed that HNHA had greater anti-tumor and pro-apoptotic effects on RCC xenografts than the established HDAC inhibitors.

Conclusions: HNHA has more potent anti-tumor activity than established HDAC inhibitors. Its activities are mediated by caspase-dependent and cytochrome-c-mediated apoptosis in RCC cells. These results suggest that HNHA may offer a new therapeutic approach to RCC.

Show MeSH
Related in: MedlinePlus