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NPC-26 kills human colorectal cancer cells via activating AMPK signaling

View Article: PubMed Central - PubMed

ABSTRACT

NPC-26 is novel mitochondrion-interfering compound. The current study tested its potential effect against colorectal cancer (CRC) cells. We demonstrated that NPC-26 induced potent anti-proliferative and cytotoxic activities against CRC cell lines (HCT-116, DLD-1 and HT-29). Activation of AMP-activated protein kinase (AMPK) signaling mediated NPC-26-induced CRC cell death. AMPKα1 shRNA knockdown or dominant negative mutation abolished NPC-26-induced AMPK activation and subsequent CRC cell death. NPC-26 disrupted mitochondrial function, causing mitochondrial permeability transition pore (mPTP) opening and reactive oxygen species (ROS) production. ROS scavengers (NAC or MnTBAP) and mPTP blockers (cyclosporin A or sanglifehrin A) blocked NPC-26-induced AMPK activation and attenuated CRC cell death. Significantly, intraperitoneal injection of NPC-26 potently inhibited HCT-116 tumor growth in severe combined immuno-deficient (SCID) mice. Yet, its anti-tumor activity was significantly weakened against AMPKα1-silenced HCT-116 tumors. Together, we conclude that NPC-26 kills CRC cells possibly via activating AMPK signaling.

No MeSH data available.


AMPKα1 mutation inhibits NPC-26-induced killing of HCT-116 cellsPuromycin-selected HCT-116 cells, expressing dominant negative AMPKα1 (“dn-AMPKα1”, T172D) or empty vector, were treated with/out NPC-26 (10 μM) for applied time, listed proteins were shown A.; Cell death B. were tested. HT-29 cells or FHC colon epithelial cells were treated with designated concentration of NPC-26 for 4 hours, expression of listed proteins was shown C. and D. For each assay, n=5. Experiments in this figure were repeated three times, and similar results were obtained each time. AMPKα1/ACC phosphorylation (vs. total protein, or vs. tubulin when mentioned) was quantified (A). * p <0.05 vs. “C”. #p <0.05 vs. “Vector”.
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Figure 3: AMPKα1 mutation inhibits NPC-26-induced killing of HCT-116 cellsPuromycin-selected HCT-116 cells, expressing dominant negative AMPKα1 (“dn-AMPKα1”, T172D) or empty vector, were treated with/out NPC-26 (10 μM) for applied time, listed proteins were shown A.; Cell death B. were tested. HT-29 cells or FHC colon epithelial cells were treated with designated concentration of NPC-26 for 4 hours, expression of listed proteins was shown C. and D. For each assay, n=5. Experiments in this figure were repeated three times, and similar results were obtained each time. AMPKα1/ACC phosphorylation (vs. total protein, or vs. tubulin when mentioned) was quantified (A). * p <0.05 vs. “C”. #p <0.05 vs. “Vector”.

Mentions: The above shRNA results imply that AMPK activation mediates NPC-26-induced cytotoxicity against HCT-116 cells. To further support this hypothesis, a dominant negative AMPKα1 (“dn-AMPKα1”, T172D) construct [27, 31, 32] was introduced to the HCT-116 cells. Via puromycin selection, two stable HCT-116 cell lines with this construct were established [“dn-AMPKα1 (L1/2)”]. Western blot assay results in Figure 3A confirmed expression of dn-AMPKα1 (Flag-tagged) in the stable cells. Significantly, dn-AMPKα1 expression almost completely blocked NPC-26-induced AMPK activation (Figure 3A). As a result, NPC-26-induced HCT-116 cell death was also attenuated (Figure 3B). Interestingly, treatment with NPC-26 (10 μM, 4h) induced significant AMPK activation in HT-29 cells (Figure 3C), but not in the FHC colon epithelial cells (Figure 3D). As a matter of fact, expression of total AMPKα1 and ACC was also extremely low in the epithelial cells (Figure 3C and 3D). These results could at least in part explain why these epithelial cells were not killed by NPC-26 (Figure 1F). The above AMPKα1 shRNA and mutation experiments were also repeated in HT-29 cells, and similar results were obtained (Data not shown). Together, these results suggest that NPC-26-induced killing of CRC cells requires AMPK activation.


NPC-26 kills human colorectal cancer cells via activating AMPK signaling
AMPKα1 mutation inhibits NPC-26-induced killing of HCT-116 cellsPuromycin-selected HCT-116 cells, expressing dominant negative AMPKα1 (“dn-AMPKα1”, T172D) or empty vector, were treated with/out NPC-26 (10 μM) for applied time, listed proteins were shown A.; Cell death B. were tested. HT-29 cells or FHC colon epithelial cells were treated with designated concentration of NPC-26 for 4 hours, expression of listed proteins was shown C. and D. For each assay, n=5. Experiments in this figure were repeated three times, and similar results were obtained each time. AMPKα1/ACC phosphorylation (vs. total protein, or vs. tubulin when mentioned) was quantified (A). * p <0.05 vs. “C”. #p <0.05 vs. “Vector”.
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Related In: Results  -  Collection

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Figure 3: AMPKα1 mutation inhibits NPC-26-induced killing of HCT-116 cellsPuromycin-selected HCT-116 cells, expressing dominant negative AMPKα1 (“dn-AMPKα1”, T172D) or empty vector, were treated with/out NPC-26 (10 μM) for applied time, listed proteins were shown A.; Cell death B. were tested. HT-29 cells or FHC colon epithelial cells were treated with designated concentration of NPC-26 for 4 hours, expression of listed proteins was shown C. and D. For each assay, n=5. Experiments in this figure were repeated three times, and similar results were obtained each time. AMPKα1/ACC phosphorylation (vs. total protein, or vs. tubulin when mentioned) was quantified (A). * p <0.05 vs. “C”. #p <0.05 vs. “Vector”.
Mentions: The above shRNA results imply that AMPK activation mediates NPC-26-induced cytotoxicity against HCT-116 cells. To further support this hypothesis, a dominant negative AMPKα1 (“dn-AMPKα1”, T172D) construct [27, 31, 32] was introduced to the HCT-116 cells. Via puromycin selection, two stable HCT-116 cell lines with this construct were established [“dn-AMPKα1 (L1/2)”]. Western blot assay results in Figure 3A confirmed expression of dn-AMPKα1 (Flag-tagged) in the stable cells. Significantly, dn-AMPKα1 expression almost completely blocked NPC-26-induced AMPK activation (Figure 3A). As a result, NPC-26-induced HCT-116 cell death was also attenuated (Figure 3B). Interestingly, treatment with NPC-26 (10 μM, 4h) induced significant AMPK activation in HT-29 cells (Figure 3C), but not in the FHC colon epithelial cells (Figure 3D). As a matter of fact, expression of total AMPKα1 and ACC was also extremely low in the epithelial cells (Figure 3C and 3D). These results could at least in part explain why these epithelial cells were not killed by NPC-26 (Figure 1F). The above AMPKα1 shRNA and mutation experiments were also repeated in HT-29 cells, and similar results were obtained (Data not shown). Together, these results suggest that NPC-26-induced killing of CRC cells requires AMPK activation.

View Article: PubMed Central - PubMed

ABSTRACT

NPC-26 is novel mitochondrion-interfering compound. The current study tested its potential effect against colorectal cancer (CRC) cells. We demonstrated that NPC-26 induced potent anti-proliferative and cytotoxic activities against CRC cell lines (HCT-116, DLD-1 and HT-29). Activation of AMP-activated protein kinase (AMPK) signaling mediated NPC-26-induced CRC cell death. AMPK&alpha;1 shRNA knockdown or dominant negative mutation abolished NPC-26-induced AMPK activation and subsequent CRC cell death. NPC-26 disrupted mitochondrial function, causing mitochondrial permeability transition pore (mPTP) opening and reactive oxygen species (ROS) production. ROS scavengers (NAC or MnTBAP) and mPTP blockers (cyclosporin A or sanglifehrin A) blocked NPC-26-induced AMPK activation and attenuated CRC cell death. Significantly, intraperitoneal injection of NPC-26 potently inhibited HCT-116 tumor growth in severe combined immuno-deficient (SCID) mice. Yet, its anti-tumor activity was significantly weakened against AMPK&alpha;1-silenced HCT-116 tumors. Together, we conclude that NPC-26 kills CRC cells possibly via activating AMPK signaling.

No MeSH data available.