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Salternamide A Suppresses Hypoxia-Induced Accumulation of HIF-1α and Induces Apoptosis in Human Colorectal Cancer Cells.

Bach DH, Kim SH, Hong JY, Park HJ, Oh DC, Lee SK - Mar Drugs (2015)

Bottom Line: In addition, SA suppresses the upstream signaling of HIF-1α, such as PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3 signaling under hypoxic conditions.Furthermore, we found that SA induces cell death by stimulating G2/M cell cycle arrest and apoptosis in human colorectal cancer cells.Taken together, SA was identified as a novel small molecule HIF-1α inhibitor from marine natural products and is potentially a leading candidate in the development of anticancer agents.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Seoul National University, Seoul 151-742, Korea. bdhiep90@snu.ac.kr.

ABSTRACT
Hypoxia inducible factor-1α (HIF-1α) is an essential regulator of the cellular response to low oxygen concentrations, activating a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1α is overexpressed in various cancers and therefore represents a considerable chemotherapeutic target. Salternamide A (SA), a novel small molecule that is isolated from a halophilic Streptomyces sp., is a potent cytotoxic agent against a variety of human cancer cell lines. However, the mechanisms by which SA inhibits tumor growth remain to be elucidated. In the present study, we demonstrate that SA efficiently inhibits the hypoxia-induced accumulation of HIF-1α in a time- and concentration-dependent manner in various human cancer cells. In addition, SA suppresses the upstream signaling of HIF-1α, such as PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3 signaling under hypoxic conditions. Furthermore, we found that SA induces cell death by stimulating G2/M cell cycle arrest and apoptosis in human colorectal cancer cells. Taken together, SA was identified as a novel small molecule HIF-1α inhibitor from marine natural products and is potentially a leading candidate in the development of anticancer agents.

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Effects of SA on apoptosis and autophagy in HCT116 human colorectal cancer cells. (A) HCT116 cells (1 × 105 cells/mL) were treated with SA for 48 h. Following treatment, HCT116 cells were harvested and stained with Annexin V-FITC and PI and analyzed using flow cytometry as described in the experimental section. (B) HCT116 cells were treated at the indicated time points in the presence or absence of SA (10 μM) prior to Western blotting. (C) Effects of SA on the expression of apoptosis- and autophagy-related proteins in HCT116 colorectal cancer cells. HCT116 cells (2 × 105 cells/mL) were treated with SA for 48 h; subsequently, the protein expression levels of apoptosis- and autophagy-related proteins were analyzed by Western blotting.
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marinedrugs-13-06962-f005: Effects of SA on apoptosis and autophagy in HCT116 human colorectal cancer cells. (A) HCT116 cells (1 × 105 cells/mL) were treated with SA for 48 h. Following treatment, HCT116 cells were harvested and stained with Annexin V-FITC and PI and analyzed using flow cytometry as described in the experimental section. (B) HCT116 cells were treated at the indicated time points in the presence or absence of SA (10 μM) prior to Western blotting. (C) Effects of SA on the expression of apoptosis- and autophagy-related proteins in HCT116 colorectal cancer cells. HCT116 cells (2 × 105 cells/mL) were treated with SA for 48 h; subsequently, the protein expression levels of apoptosis- and autophagy-related proteins were analyzed by Western blotting.

Mentions: To further confirm whether the induction of the sub-G1 peak by SA (10 μM) at 48 h is related to apoptotic cell death, the cells were treated with SA (10 μM) for 48 h, and the quantification of Annexin-V/PI staining, a marker for apoptosis, was determined by flow cytometry. The number of cells that were positive for the double staining of Annexin-V/PI was significantly increased by SA treatment, suggesting that SA is able to induce apoptotic cell death in cancer cells (Figure 5A). To further unveil the mechanism of SA-induced apoptosis in HCT116 cells, the expression of apoptosis-associated proteins was analyzed by Western blotting. As shown in Figure 5B,C, the expression of cleaved caspase-8, cleaved caspase-3, and cleaved PARP was upregulated, but the expression of pro-caspase-8, caspase-3, pro-PARP, Bcl-2 and Bcl-xL was downregulated in a time- and concentration-dependent manner. In addition, LC3-II is an autophagy-specific marker, and LC3-II formation (LC3 lipidation) is also a key step in autophagy-associated cell death [23]. To evaluate whether the cytotoxic activity of SA is related, in part, to the induction of autophagy, the expression level of LC3-II was determined by 48 h of SA (10 μM) treatment. SA also significantly induced the expression of LC3-II in a time- and concentration-dependent manner. Taken together, these results suggest that the anti-proliferative activity of SA might be due, in part, to the G2/M phase cell cycle arrest and the induction of apoptosis and autophagic cell death in HCT116 cells.


Salternamide A Suppresses Hypoxia-Induced Accumulation of HIF-1α and Induces Apoptosis in Human Colorectal Cancer Cells.

Bach DH, Kim SH, Hong JY, Park HJ, Oh DC, Lee SK - Mar Drugs (2015)

Effects of SA on apoptosis and autophagy in HCT116 human colorectal cancer cells. (A) HCT116 cells (1 × 105 cells/mL) were treated with SA for 48 h. Following treatment, HCT116 cells were harvested and stained with Annexin V-FITC and PI and analyzed using flow cytometry as described in the experimental section. (B) HCT116 cells were treated at the indicated time points in the presence or absence of SA (10 μM) prior to Western blotting. (C) Effects of SA on the expression of apoptosis- and autophagy-related proteins in HCT116 colorectal cancer cells. HCT116 cells (2 × 105 cells/mL) were treated with SA for 48 h; subsequently, the protein expression levels of apoptosis- and autophagy-related proteins were analyzed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663561&req=5

marinedrugs-13-06962-f005: Effects of SA on apoptosis and autophagy in HCT116 human colorectal cancer cells. (A) HCT116 cells (1 × 105 cells/mL) were treated with SA for 48 h. Following treatment, HCT116 cells were harvested and stained with Annexin V-FITC and PI and analyzed using flow cytometry as described in the experimental section. (B) HCT116 cells were treated at the indicated time points in the presence or absence of SA (10 μM) prior to Western blotting. (C) Effects of SA on the expression of apoptosis- and autophagy-related proteins in HCT116 colorectal cancer cells. HCT116 cells (2 × 105 cells/mL) were treated with SA for 48 h; subsequently, the protein expression levels of apoptosis- and autophagy-related proteins were analyzed by Western blotting.
Mentions: To further confirm whether the induction of the sub-G1 peak by SA (10 μM) at 48 h is related to apoptotic cell death, the cells were treated with SA (10 μM) for 48 h, and the quantification of Annexin-V/PI staining, a marker for apoptosis, was determined by flow cytometry. The number of cells that were positive for the double staining of Annexin-V/PI was significantly increased by SA treatment, suggesting that SA is able to induce apoptotic cell death in cancer cells (Figure 5A). To further unveil the mechanism of SA-induced apoptosis in HCT116 cells, the expression of apoptosis-associated proteins was analyzed by Western blotting. As shown in Figure 5B,C, the expression of cleaved caspase-8, cleaved caspase-3, and cleaved PARP was upregulated, but the expression of pro-caspase-8, caspase-3, pro-PARP, Bcl-2 and Bcl-xL was downregulated in a time- and concentration-dependent manner. In addition, LC3-II is an autophagy-specific marker, and LC3-II formation (LC3 lipidation) is also a key step in autophagy-associated cell death [23]. To evaluate whether the cytotoxic activity of SA is related, in part, to the induction of autophagy, the expression level of LC3-II was determined by 48 h of SA (10 μM) treatment. SA also significantly induced the expression of LC3-II in a time- and concentration-dependent manner. Taken together, these results suggest that the anti-proliferative activity of SA might be due, in part, to the G2/M phase cell cycle arrest and the induction of apoptosis and autophagic cell death in HCT116 cells.

Bottom Line: In addition, SA suppresses the upstream signaling of HIF-1α, such as PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3 signaling under hypoxic conditions.Furthermore, we found that SA induces cell death by stimulating G2/M cell cycle arrest and apoptosis in human colorectal cancer cells.Taken together, SA was identified as a novel small molecule HIF-1α inhibitor from marine natural products and is potentially a leading candidate in the development of anticancer agents.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Seoul National University, Seoul 151-742, Korea. bdhiep90@snu.ac.kr.

ABSTRACT
Hypoxia inducible factor-1α (HIF-1α) is an essential regulator of the cellular response to low oxygen concentrations, activating a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1α is overexpressed in various cancers and therefore represents a considerable chemotherapeutic target. Salternamide A (SA), a novel small molecule that is isolated from a halophilic Streptomyces sp., is a potent cytotoxic agent against a variety of human cancer cell lines. However, the mechanisms by which SA inhibits tumor growth remain to be elucidated. In the present study, we demonstrate that SA efficiently inhibits the hypoxia-induced accumulation of HIF-1α in a time- and concentration-dependent manner in various human cancer cells. In addition, SA suppresses the upstream signaling of HIF-1α, such as PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3 signaling under hypoxic conditions. Furthermore, we found that SA induces cell death by stimulating G2/M cell cycle arrest and apoptosis in human colorectal cancer cells. Taken together, SA was identified as a novel small molecule HIF-1α inhibitor from marine natural products and is potentially a leading candidate in the development of anticancer agents.

Show MeSH
Related in: MedlinePlus