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Q6, a novel hypoxia-targeted drug, regulates hypoxia-inducible factor signaling via an autophagy-dependent mechanism in hepatocellular carcinoma.

Liu XW, Cai TY, Zhu H, Cao J, Su Y, Hu YZ, He QJ, Yang B - Autophagy (2013)

Bottom Line: Autophagic degradation of HIF1A was further confirmed by the observation that HIF1A coimmunoprecipitated with the ubiquitin-binding adaptor protein, SQSTM1, which is degraded through autophagy.These findings suggest that the novel hypoxia-targeted agent, Q6, has potential clinical value in the therapy of HCC.Furthermore, the identification of autophagy as a crucial regulator of HIF1A provides new insights into hypoxia-related treatments.

View Article: PubMed Central - PubMed

Affiliation: Zhejiang Province Key Laboratory of Anti-Cancer Drug Research; Institute of Pharmacology and Toxicology; College of Pharmaceutical Sciences; Zhejiang University; Hangzhou, China.

ABSTRACT
Tumor hypoxia underlies treatment failure and yields more aggressive and metastatic cancer phenotypes. Although therapeutically targeting these hypoxic environments has been proposed for many years, to date no approaches have shown the therapeutic value to gain regulatory approval. Here, we demonstrated that a novel hypoxia-activated prodrug, Q6, exhibits potent antiproliferative efficacy under hypoxic conditions and induces caspase-dependent apoptosis in 2 hepatocellular carcinoma (HCC) cell lines, with no obvious toxicity being detected in 2 normal liver cell lines. Treatment with Q6 markedly downregulated HIF1A [hypoxia inducible factor 1, α subunit (basic helix-loop-helix transcription factor)] expression and transcription of the downstream target gene, VEGFA (vascular endothelial growth factor A). This dual hypoxia-targeted modulation mechanism leads to high potency in suppressing tumor growth and vascularization in 2 in vivo models. Intriguingly, it is the autophagy-dependent degradation pathway that plays a crucial role in Q6-induced attenuation of HIF1A expression, rather than the proteasome-dependent pathway, which is normally regarded as the predominant mechanism underlying posttranslational regulation of HIF1A. Inhibition of autophagy, either by short interfering RNA (siRNA) or by chemical inhibitors, blocked Q6-induced HIF1A degradation. Autophagic degradation of HIF1A was further confirmed by the observation that HIF1A coimmunoprecipitated with the ubiquitin-binding adaptor protein, SQSTM1, which is degraded through autophagy. Additionally, silencing of SQSTM1 inhibited Q6-induced HIF1A degradation. These findings suggest that the novel hypoxia-targeted agent, Q6, has potential clinical value in the therapy of HCC. Furthermore, the identification of autophagy as a crucial regulator of HIF1A provides new insights into hypoxia-related treatments.

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Figure 4. ATG5 and LC3B are required for Q6-induced autophagy and HIF1A degradation. (A and B) After transfection with specifically targeted siRNA (ATG5 or LC3B) for 36 h, HepG2 and Bel-7402 cells were treated with Q6 (0, 2.5, 5 μM) for 6 h, after which HIF1A, ATG5, and LC3B protein levels were measured by western blot analysis. ACTB was measured as the loading control. (C and D) After transfection with specifically targeted siRNA (ATG5 or LC3B) for 36 h, HepG2 and Bel-7402 cells were treated with Q6 (5 μM) for different times, after which HIF1A protein levels were measured by western blot analysis. ACTB was measured as the loading control. (E) HepG2 cells were treated with Q6 (0, 5 μM) for 6 h under hypoxic conditions, after which HIF1A protein was detected by immunoelectron microscopy analysis. Arrows indicate HIF1A. In the lower panel, the number of immuno-gold labeled HIF1A is presented for HepG2 cells. Twenty cross sections were counted in each experiment and a total of 40 autophagosomes were detected. Data shown are means ± SD of 3 independent experiments. **P < 0.01 compared with the control group.
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Figure 4: Figure 4. ATG5 and LC3B are required for Q6-induced autophagy and HIF1A degradation. (A and B) After transfection with specifically targeted siRNA (ATG5 or LC3B) for 36 h, HepG2 and Bel-7402 cells were treated with Q6 (0, 2.5, 5 μM) for 6 h, after which HIF1A, ATG5, and LC3B protein levels were measured by western blot analysis. ACTB was measured as the loading control. (C and D) After transfection with specifically targeted siRNA (ATG5 or LC3B) for 36 h, HepG2 and Bel-7402 cells were treated with Q6 (5 μM) for different times, after which HIF1A protein levels were measured by western blot analysis. ACTB was measured as the loading control. (E) HepG2 cells were treated with Q6 (0, 5 μM) for 6 h under hypoxic conditions, after which HIF1A protein was detected by immunoelectron microscopy analysis. Arrows indicate HIF1A. In the lower panel, the number of immuno-gold labeled HIF1A is presented for HepG2 cells. Twenty cross sections were counted in each experiment and a total of 40 autophagosomes were detected. Data shown are means ± SD of 3 independent experiments. **P < 0.01 compared with the control group.

Mentions: In addition, we characterized whether autophagy-related genes are involved in Q6-induced HIF1A reductions and RNA interference approaches were employed. Autophagosome formation requires 2 ubiquitin-like conjugation systems, the ATG12–ATG5 conjugate and the MAP1LC3 (LC3) systems, which are tightly associated with the expansion of autophagosomal membranes. Depletion of ATG5 and LC3B expression in these cells inhibited Q6-induced autophagy, and prolonged the half-life of HIF1A protein in a concentration and time-dependent manner (Fig. 4A–D; Fig. S6C). These data further suggest that autophagy regulates Q6-induced HIF1A turnover in HCC cells.


Q6, a novel hypoxia-targeted drug, regulates hypoxia-inducible factor signaling via an autophagy-dependent mechanism in hepatocellular carcinoma.

Liu XW, Cai TY, Zhu H, Cao J, Su Y, Hu YZ, He QJ, Yang B - Autophagy (2013)

Figure 4. ATG5 and LC3B are required for Q6-induced autophagy and HIF1A degradation. (A and B) After transfection with specifically targeted siRNA (ATG5 or LC3B) for 36 h, HepG2 and Bel-7402 cells were treated with Q6 (0, 2.5, 5 μM) for 6 h, after which HIF1A, ATG5, and LC3B protein levels were measured by western blot analysis. ACTB was measured as the loading control. (C and D) After transfection with specifically targeted siRNA (ATG5 or LC3B) for 36 h, HepG2 and Bel-7402 cells were treated with Q6 (5 μM) for different times, after which HIF1A protein levels were measured by western blot analysis. ACTB was measured as the loading control. (E) HepG2 cells were treated with Q6 (0, 5 μM) for 6 h under hypoxic conditions, after which HIF1A protein was detected by immunoelectron microscopy analysis. Arrows indicate HIF1A. In the lower panel, the number of immuno-gold labeled HIF1A is presented for HepG2 cells. Twenty cross sections were counted in each experiment and a total of 40 autophagosomes were detected. Data shown are means ± SD of 3 independent experiments. **P < 0.01 compared with the control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Figure 4. ATG5 and LC3B are required for Q6-induced autophagy and HIF1A degradation. (A and B) After transfection with specifically targeted siRNA (ATG5 or LC3B) for 36 h, HepG2 and Bel-7402 cells were treated with Q6 (0, 2.5, 5 μM) for 6 h, after which HIF1A, ATG5, and LC3B protein levels were measured by western blot analysis. ACTB was measured as the loading control. (C and D) After transfection with specifically targeted siRNA (ATG5 or LC3B) for 36 h, HepG2 and Bel-7402 cells were treated with Q6 (5 μM) for different times, after which HIF1A protein levels were measured by western blot analysis. ACTB was measured as the loading control. (E) HepG2 cells were treated with Q6 (0, 5 μM) for 6 h under hypoxic conditions, after which HIF1A protein was detected by immunoelectron microscopy analysis. Arrows indicate HIF1A. In the lower panel, the number of immuno-gold labeled HIF1A is presented for HepG2 cells. Twenty cross sections were counted in each experiment and a total of 40 autophagosomes were detected. Data shown are means ± SD of 3 independent experiments. **P < 0.01 compared with the control group.
Mentions: In addition, we characterized whether autophagy-related genes are involved in Q6-induced HIF1A reductions and RNA interference approaches were employed. Autophagosome formation requires 2 ubiquitin-like conjugation systems, the ATG12–ATG5 conjugate and the MAP1LC3 (LC3) systems, which are tightly associated with the expansion of autophagosomal membranes. Depletion of ATG5 and LC3B expression in these cells inhibited Q6-induced autophagy, and prolonged the half-life of HIF1A protein in a concentration and time-dependent manner (Fig. 4A–D; Fig. S6C). These data further suggest that autophagy regulates Q6-induced HIF1A turnover in HCC cells.

Bottom Line: Autophagic degradation of HIF1A was further confirmed by the observation that HIF1A coimmunoprecipitated with the ubiquitin-binding adaptor protein, SQSTM1, which is degraded through autophagy.These findings suggest that the novel hypoxia-targeted agent, Q6, has potential clinical value in the therapy of HCC.Furthermore, the identification of autophagy as a crucial regulator of HIF1A provides new insights into hypoxia-related treatments.

View Article: PubMed Central - PubMed

Affiliation: Zhejiang Province Key Laboratory of Anti-Cancer Drug Research; Institute of Pharmacology and Toxicology; College of Pharmaceutical Sciences; Zhejiang University; Hangzhou, China.

ABSTRACT
Tumor hypoxia underlies treatment failure and yields more aggressive and metastatic cancer phenotypes. Although therapeutically targeting these hypoxic environments has been proposed for many years, to date no approaches have shown the therapeutic value to gain regulatory approval. Here, we demonstrated that a novel hypoxia-activated prodrug, Q6, exhibits potent antiproliferative efficacy under hypoxic conditions and induces caspase-dependent apoptosis in 2 hepatocellular carcinoma (HCC) cell lines, with no obvious toxicity being detected in 2 normal liver cell lines. Treatment with Q6 markedly downregulated HIF1A [hypoxia inducible factor 1, α subunit (basic helix-loop-helix transcription factor)] expression and transcription of the downstream target gene, VEGFA (vascular endothelial growth factor A). This dual hypoxia-targeted modulation mechanism leads to high potency in suppressing tumor growth and vascularization in 2 in vivo models. Intriguingly, it is the autophagy-dependent degradation pathway that plays a crucial role in Q6-induced attenuation of HIF1A expression, rather than the proteasome-dependent pathway, which is normally regarded as the predominant mechanism underlying posttranslational regulation of HIF1A. Inhibition of autophagy, either by short interfering RNA (siRNA) or by chemical inhibitors, blocked Q6-induced HIF1A degradation. Autophagic degradation of HIF1A was further confirmed by the observation that HIF1A coimmunoprecipitated with the ubiquitin-binding adaptor protein, SQSTM1, which is degraded through autophagy. Additionally, silencing of SQSTM1 inhibited Q6-induced HIF1A degradation. These findings suggest that the novel hypoxia-targeted agent, Q6, has potential clinical value in the therapy of HCC. Furthermore, the identification of autophagy as a crucial regulator of HIF1A provides new insights into hypoxia-related treatments.

Show MeSH
Related in: MedlinePlus