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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 3. Q6 accelerates HIF1A protein degradation via the autophagy-lysosome pathway. (A) HepG2 (left) and Bel-7402 (right) cells were exposed to Q6 (0 to 5 μM) for 6 h in hypoxia. Total RNA was extracted and HIF1A 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 (B) HepG2 and Bel-7402 cells exposed to hypoxia were treated with CHX in the presence or absence of Q6 (5 μM) for different times, and HIF1A protein levels were then measured by western blot analysis. ACTB was measured as the loading control. (C) HepG2 and Bel-7402 cells were pretreated with MG132 (a proteasome inhibitor) or 3-MA (an autophagy-lysosome inhibitor) for 30 min to allow functional inhibition of proteasomes and lysosomes. Cells were then exposed to hypoxia in the presence or absence of Q6 (5 μM) for 6 h, after which HIF1A protein levels were determined by western blot analysis. ACTB was measured as the loading control. (D) Ultrastructural features of HepG2 and Bel-7402 cells with or without Q6 treatment (5 μM) for 6 h were analyzed by electron microscopy. The typical images of autophagosomes (arrows) and autolysosomes (arrowheads) were shown at higher magnification. In the lower panel, the number of autophagosomes (AP) and autolysosomes (AL) were presented for HepG2 and Bel-7402 cells. Twenty cross sections were counted in each experiment. Data shown are means ± SD of 3 independent experiments. *P < 0.05 compared with HepG2 control group. **P < 0.01 compared with HepG2 control group. ##P < 0.01 compared with Bel-7402 control group. (E) HepG2 and Bel-7402 cells were treated with Q6 (0 to 5 μM) for 6 h and LC3B-I and LC3B-II protein levels were measured by western blot analysis. ACTB was measured as the loading control. Data are representative of 2 independent experiments.
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Figure 3: Figure 3. Q6 accelerates HIF1A protein degradation via the autophagy-lysosome pathway. (A) HepG2 (left) and Bel-7402 (right) cells were exposed to Q6 (0 to 5 μM) for 6 h in hypoxia. Total RNA was extracted and HIF1A 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 (B) HepG2 and Bel-7402 cells exposed to hypoxia were treated with CHX in the presence or absence of Q6 (5 μM) for different times, and HIF1A protein levels were then measured by western blot analysis. ACTB was measured as the loading control. (C) HepG2 and Bel-7402 cells were pretreated with MG132 (a proteasome inhibitor) or 3-MA (an autophagy-lysosome inhibitor) for 30 min to allow functional inhibition of proteasomes and lysosomes. Cells were then exposed to hypoxia in the presence or absence of Q6 (5 μM) for 6 h, after which HIF1A protein levels were determined by western blot analysis. ACTB was measured as the loading control. (D) Ultrastructural features of HepG2 and Bel-7402 cells with or without Q6 treatment (5 μM) for 6 h were analyzed by electron microscopy. The typical images of autophagosomes (arrows) and autolysosomes (arrowheads) were shown at higher magnification. In the lower panel, the number of autophagosomes (AP) and autolysosomes (AL) were presented for HepG2 and Bel-7402 cells. Twenty cross sections were counted in each experiment. Data shown are means ± SD of 3 independent experiments. *P < 0.05 compared with HepG2 control group. **P < 0.01 compared with HepG2 control group. ##P < 0.01 compared with Bel-7402 control group. (E) HepG2 and Bel-7402 cells were treated with Q6 (0 to 5 μM) for 6 h and LC3B-I and LC3B-II protein levels were measured by western blot analysis. ACTB was measured as the loading control. Data are representative of 2 independent experiments.

Mentions: In order to explore the mechanisms underlying Q6-induced HIF1A suppression, we first examined whether reduction of HIF1A by Q6 occurs at the transcriptional level. Real-time PCR analysis showed that HIF1A mRNA levels were not significantly altered after Q6 treatment in Bel-7402 and HepG2 cells (Fig. 3A). Furthermore, we found that Q6 had no effect on EGFR, PIK3CA-AKT1, or MAPK signaling pathways, which have been recently shown to control the protein synthesis of HIF1A (Fig. S4; Table S1). On the basis of these findings, we hypothesized that a degradative mechanism may be involved in Q6-induced reductions in HIF1A. To examine this possibility, cycloheximide (CHX, an inhibitor of protein synthesis) was used to prevent de novo protein synthesis; thus, changes in HIF1A levels would primarily reflect protein degradation. We exposed HepG2 and Bel-7402 cells to CHX under hypoxic conditions in the presence or absence of Q6 at different time points and measured expression of HIF1A. As shown in Figure 3B, although the intensity of the HIF1A signal was not obviously changed in Q6 untreated cells, the reduction of HIF1A protein levels were observed in Q6-treated cells in a time-dependent manner. Together, these results indicate that Q6 downregulates HIF1A protein expression through accelerating its degradation.


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 3. Q6 accelerates HIF1A protein degradation via the autophagy-lysosome pathway. (A) HepG2 (left) and Bel-7402 (right) cells were exposed to Q6 (0 to 5 μM) for 6 h in hypoxia. Total RNA was extracted and HIF1A 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 (B) HepG2 and Bel-7402 cells exposed to hypoxia were treated with CHX in the presence or absence of Q6 (5 μM) for different times, and HIF1A protein levels were then measured by western blot analysis. ACTB was measured as the loading control. (C) HepG2 and Bel-7402 cells were pretreated with MG132 (a proteasome inhibitor) or 3-MA (an autophagy-lysosome inhibitor) for 30 min to allow functional inhibition of proteasomes and lysosomes. Cells were then exposed to hypoxia in the presence or absence of Q6 (5 μM) for 6 h, after which HIF1A protein levels were determined by western blot analysis. ACTB was measured as the loading control. (D) Ultrastructural features of HepG2 and Bel-7402 cells with or without Q6 treatment (5 μM) for 6 h were analyzed by electron microscopy. The typical images of autophagosomes (arrows) and autolysosomes (arrowheads) were shown at higher magnification. In the lower panel, the number of autophagosomes (AP) and autolysosomes (AL) were presented for HepG2 and Bel-7402 cells. Twenty cross sections were counted in each experiment. Data shown are means ± SD of 3 independent experiments. *P < 0.05 compared with HepG2 control group. **P < 0.01 compared with HepG2 control group. ##P < 0.01 compared with Bel-7402 control group. (E) HepG2 and Bel-7402 cells were treated with Q6 (0 to 5 μM) for 6 h and LC3B-I and LC3B-II protein levels were measured by western blot analysis. ACTB was measured as the loading control. Data are representative of 2 independent experiments.
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Figure 3: Figure 3. Q6 accelerates HIF1A protein degradation via the autophagy-lysosome pathway. (A) HepG2 (left) and Bel-7402 (right) cells were exposed to Q6 (0 to 5 μM) for 6 h in hypoxia. Total RNA was extracted and HIF1A 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 (B) HepG2 and Bel-7402 cells exposed to hypoxia were treated with CHX in the presence or absence of Q6 (5 μM) for different times, and HIF1A protein levels were then measured by western blot analysis. ACTB was measured as the loading control. (C) HepG2 and Bel-7402 cells were pretreated with MG132 (a proteasome inhibitor) or 3-MA (an autophagy-lysosome inhibitor) for 30 min to allow functional inhibition of proteasomes and lysosomes. Cells were then exposed to hypoxia in the presence or absence of Q6 (5 μM) for 6 h, after which HIF1A protein levels were determined by western blot analysis. ACTB was measured as the loading control. (D) Ultrastructural features of HepG2 and Bel-7402 cells with or without Q6 treatment (5 μM) for 6 h were analyzed by electron microscopy. The typical images of autophagosomes (arrows) and autolysosomes (arrowheads) were shown at higher magnification. In the lower panel, the number of autophagosomes (AP) and autolysosomes (AL) were presented for HepG2 and Bel-7402 cells. Twenty cross sections were counted in each experiment. Data shown are means ± SD of 3 independent experiments. *P < 0.05 compared with HepG2 control group. **P < 0.01 compared with HepG2 control group. ##P < 0.01 compared with Bel-7402 control group. (E) HepG2 and Bel-7402 cells were treated with Q6 (0 to 5 μM) for 6 h and LC3B-I and LC3B-II protein levels were measured by western blot analysis. ACTB was measured as the loading control. Data are representative of 2 independent experiments.
Mentions: In order to explore the mechanisms underlying Q6-induced HIF1A suppression, we first examined whether reduction of HIF1A by Q6 occurs at the transcriptional level. Real-time PCR analysis showed that HIF1A mRNA levels were not significantly altered after Q6 treatment in Bel-7402 and HepG2 cells (Fig. 3A). Furthermore, we found that Q6 had no effect on EGFR, PIK3CA-AKT1, or MAPK signaling pathways, which have been recently shown to control the protein synthesis of HIF1A (Fig. S4; Table S1). On the basis of these findings, we hypothesized that a degradative mechanism may be involved in Q6-induced reductions in HIF1A. To examine this possibility, cycloheximide (CHX, an inhibitor of protein synthesis) was used to prevent de novo protein synthesis; thus, changes in HIF1A levels would primarily reflect protein degradation. We exposed HepG2 and Bel-7402 cells to CHX under hypoxic conditions in the presence or absence of Q6 at different time points and measured expression of HIF1A. As shown in Figure 3B, although the intensity of the HIF1A signal was not obviously changed in Q6 untreated cells, the reduction of HIF1A protein levels were observed in Q6-treated cells in a time-dependent manner. Together, these results indicate that Q6 downregulates HIF1A protein expression through accelerating its degradation.

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