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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 2. Q6 downregulates hypoxia-induced HIF1A protein expression and HIF1A-mediated signal transduction. (A and C) HepG2 (left) and Bel-7402 (right) cells were exposed to hypoxia or normoxia and treated with Q6 (0 to 5 μM) for 6 h. (A) Protein levels of HIF1A, EPAS1, and VEGFA were detected by western blot analysis. ACTB was analyzed as the loading control. Data are representative of 3 independent experiments. (B) An HRE-dependent reporter assay was used to determine the effect of Q6 on HIF1A transcriptional activity. Five independent experiments were performed and the values were expressed as the mean ± SD **P < 0.01 and ***P < 0.001, compared with untreated controls in hypoxia. (C) Total RNA was extracted and VEGFA mRNA expression was analyzed by RT-PCR, using GAPDH as a control gene. Five independent experiments were performed and the values were expressed as the mean ± SD **P < 0.01, compared with untreated controls in hypoxia.
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Figure 2: Figure 2. Q6 downregulates hypoxia-induced HIF1A protein expression and HIF1A-mediated signal transduction. (A and C) HepG2 (left) and Bel-7402 (right) cells were exposed to hypoxia or normoxia and treated with Q6 (0 to 5 μM) for 6 h. (A) Protein levels of HIF1A, EPAS1, and VEGFA were detected by western blot analysis. ACTB was analyzed as the loading control. Data are representative of 3 independent experiments. (B) An HRE-dependent reporter assay was used to determine the effect of Q6 on HIF1A transcriptional activity. Five independent experiments were performed and the values were expressed as the mean ± SD **P < 0.01 and ***P < 0.001, compared with untreated controls in hypoxia. (C) Total RNA was extracted and VEGFA mRNA expression was analyzed by RT-PCR, using GAPDH as a control gene. Five independent experiments were performed and the values were expressed as the mean ± SD **P < 0.01, compared with untreated controls in hypoxia.

Mentions: Previous studies have demonstrated the relationship between drug resistance and tumor progression with HIF1A overexpression; we observed that Q6 could significantly inhibit HIF1A and related signaling transduction. First, we investigated that treatment with Q6 (0 to 5 μM) for 6 h dramatically decreased hypoxia-induced HIF1A protein expression in HepG2 and Bel-7402 cells in a concentration-dependent manner. (Fig. 2A; Fig. S5A). Given that the inhibition of HIF1A accumulation in hypoxic cells could be correlated with Q6-induced cytotoxicity, parallel studies of cell viability were performed. The results showed that there was no significant alteration in cell viability after Q6 treatment (Fig. S4A), suggesting that Q6-induced reductions in HIF1A levels are not a result of its cytotoxic actions. Following this, we evaluated that Q6 could inhibit HIF1A-mediated transcriptional activity in a concentration-dependent manner in HepG2 and Bel-7402 cell lines, as determined using a hypoxia-responsive reporter construct, containing a luciferase gene under the control of HREs (Fig. 2B).


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 2. Q6 downregulates hypoxia-induced HIF1A protein expression and HIF1A-mediated signal transduction. (A and C) HepG2 (left) and Bel-7402 (right) cells were exposed to hypoxia or normoxia and treated with Q6 (0 to 5 μM) for 6 h. (A) Protein levels of HIF1A, EPAS1, and VEGFA were detected by western blot analysis. ACTB was analyzed as the loading control. Data are representative of 3 independent experiments. (B) An HRE-dependent reporter assay was used to determine the effect of Q6 on HIF1A transcriptional activity. Five independent experiments were performed and the values were expressed as the mean ± SD **P < 0.01 and ***P < 0.001, compared with untreated controls in hypoxia. (C) Total RNA was extracted and VEGFA mRNA expression was analyzed by RT-PCR, using GAPDH as a control gene. Five independent experiments were performed and the values were expressed as the mean ± SD **P < 0.01, compared with untreated controls in hypoxia.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Figure 2. Q6 downregulates hypoxia-induced HIF1A protein expression and HIF1A-mediated signal transduction. (A and C) HepG2 (left) and Bel-7402 (right) cells were exposed to hypoxia or normoxia and treated with Q6 (0 to 5 μM) for 6 h. (A) Protein levels of HIF1A, EPAS1, and VEGFA were detected by western blot analysis. ACTB was analyzed as the loading control. Data are representative of 3 independent experiments. (B) An HRE-dependent reporter assay was used to determine the effect of Q6 on HIF1A transcriptional activity. Five independent experiments were performed and the values were expressed as the mean ± SD **P < 0.01 and ***P < 0.001, compared with untreated controls in hypoxia. (C) Total RNA was extracted and VEGFA mRNA expression was analyzed by RT-PCR, using GAPDH as a control gene. Five independent experiments were performed and the values were expressed as the mean ± SD **P < 0.01, compared with untreated controls in hypoxia.
Mentions: Previous studies have demonstrated the relationship between drug resistance and tumor progression with HIF1A overexpression; we observed that Q6 could significantly inhibit HIF1A and related signaling transduction. First, we investigated that treatment with Q6 (0 to 5 μM) for 6 h dramatically decreased hypoxia-induced HIF1A protein expression in HepG2 and Bel-7402 cells in a concentration-dependent manner. (Fig. 2A; Fig. S5A). Given that the inhibition of HIF1A accumulation in hypoxic cells could be correlated with Q6-induced cytotoxicity, parallel studies of cell viability were performed. The results showed that there was no significant alteration in cell viability after Q6 treatment (Fig. S4A), suggesting that Q6-induced reductions in HIF1A levels are not a result of its cytotoxic actions. Following this, we evaluated that Q6 could inhibit HIF1A-mediated transcriptional activity in a concentration-dependent manner in HepG2 and Bel-7402 cell lines, as determined using a hypoxia-responsive reporter construct, containing a luciferase gene under the control of HREs (Fig. 2B).

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