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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 6. Q6 arrests tumor growth in vivo. (A–E) Nude mice bearing established Bel-7402 tumors were treated with TPZ or Q6 at 25 or 12.5 mg/kg by daily i.p. injection for 27 d. (A) Tumor volumes are expressed as the mean ± SEM ***P < 0.001 vs. vehicle-treated controls (n = 5 to 8 per group). (B) Relative body weights are expressed as the mean ± SEM **P < 0.01 vs. vehicle-treated controls (n = 5 to 8 per group). (C) Western blot analysis of Bel-7402 cell-derived tumors treated with Q6 (12.5 mg/kg) or vehicle for expression of PARP1, CASP3, cleaved-CASP3, HIF1A, and VEGFA. ACTB was measured as the loading control. (D and E) Effects of Q6 on expression levels of HIF1A and VEGFA in Bel-7402 cell-derived tumors were determined by hematoxylin and eosin staining and immunohistochemical and immunofluorescence analysis. (F) Schematic diagrams depicting the mechanism by which Q6 exerts its anticancer effects. On the one hand, Q6 triggers the caspase-dependent apoptosis pathway and induces HCC cell death both in vitro and in vivo. On the other hand, Q6 activates the ATG5 and LC3B-dependent autophagy pathway, which plays an essential role in HIF1A degradation and the anti-angiogenesis and anti-metastatic activities of Q6. Furthermore, interaction with SQSTM1 regulates the degradation of HIF1A. Thus, these data strongly support the conclusion that Q6 is a novel hypoxia-targeted drug for HCC therapy, and we propose that autophagy acts as a tumor suppressor by accelerating HIF1A degradation and arresting angiogenesis and metastatic processes.
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Figure 6: Figure 6. Q6 arrests tumor growth in vivo. (A–E) Nude mice bearing established Bel-7402 tumors were treated with TPZ or Q6 at 25 or 12.5 mg/kg by daily i.p. injection for 27 d. (A) Tumor volumes are expressed as the mean ± SEM ***P < 0.001 vs. vehicle-treated controls (n = 5 to 8 per group). (B) Relative body weights are expressed as the mean ± SEM **P < 0.01 vs. vehicle-treated controls (n = 5 to 8 per group). (C) Western blot analysis of Bel-7402 cell-derived tumors treated with Q6 (12.5 mg/kg) or vehicle for expression of PARP1, CASP3, cleaved-CASP3, HIF1A, and VEGFA. ACTB was measured as the loading control. (D and E) Effects of Q6 on expression levels of HIF1A and VEGFA in Bel-7402 cell-derived tumors were determined by hematoxylin and eosin staining and immunohistochemical and immunofluorescence analysis. (F) Schematic diagrams depicting the mechanism by which Q6 exerts its anticancer effects. On the one hand, Q6 triggers the caspase-dependent apoptosis pathway and induces HCC cell death both in vitro and in vivo. On the other hand, Q6 activates the ATG5 and LC3B-dependent autophagy pathway, which plays an essential role in HIF1A degradation and the anti-angiogenesis and anti-metastatic activities of Q6. Furthermore, interaction with SQSTM1 regulates the degradation of HIF1A. Thus, these data strongly support the conclusion that Q6 is a novel hypoxia-targeted drug for HCC therapy, and we propose that autophagy acts as a tumor suppressor by accelerating HIF1A degradation and arresting angiogenesis and metastatic processes.

Mentions: Given the superior antitumor activity of Q6 in vitro, we hypothesized that it should be capable of slowing tumor growth in vivo. As depicted in Figure 6A and Table S2, Q6 administration significantly inhibited growth of Bel-7402 xenograft tumors and H22 hepatoma. Furthermore, it is worth noting that Q6 treatment did not cause obvious weight loss in either of the 2 in vivo models (Fig. 6B; Table S2). Next, we performed western blot analysis of tumor xenografts and investigated that Q6 administration remarkably activated the caspase-dependent apoptosis pathway in Bel-7402 cell-derived tumors. Finally, western blot and immunohistochemical and immunofluoresence analyses verified that Q6 treatment inhibited HIF1A, VEGFA and CD31 expression in tumors from Q6-treated mice, indicating that Q6 suppresses HIF1A activity and related signaling mechanisms in vivo (Fig. 6D and E;Fig. S7).


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 6. Q6 arrests tumor growth in vivo. (A–E) Nude mice bearing established Bel-7402 tumors were treated with TPZ or Q6 at 25 or 12.5 mg/kg by daily i.p. injection for 27 d. (A) Tumor volumes are expressed as the mean ± SEM ***P < 0.001 vs. vehicle-treated controls (n = 5 to 8 per group). (B) Relative body weights are expressed as the mean ± SEM **P < 0.01 vs. vehicle-treated controls (n = 5 to 8 per group). (C) Western blot analysis of Bel-7402 cell-derived tumors treated with Q6 (12.5 mg/kg) or vehicle for expression of PARP1, CASP3, cleaved-CASP3, HIF1A, and VEGFA. ACTB was measured as the loading control. (D and E) Effects of Q6 on expression levels of HIF1A and VEGFA in Bel-7402 cell-derived tumors were determined by hematoxylin and eosin staining and immunohistochemical and immunofluorescence analysis. (F) Schematic diagrams depicting the mechanism by which Q6 exerts its anticancer effects. On the one hand, Q6 triggers the caspase-dependent apoptosis pathway and induces HCC cell death both in vitro and in vivo. On the other hand, Q6 activates the ATG5 and LC3B-dependent autophagy pathway, which plays an essential role in HIF1A degradation and the anti-angiogenesis and anti-metastatic activities of Q6. Furthermore, interaction with SQSTM1 regulates the degradation of HIF1A. Thus, these data strongly support the conclusion that Q6 is a novel hypoxia-targeted drug for HCC therapy, and we propose that autophagy acts as a tumor suppressor by accelerating HIF1A degradation and arresting angiogenesis and metastatic processes.
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Related In: Results  -  Collection

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Figure 6: Figure 6. Q6 arrests tumor growth in vivo. (A–E) Nude mice bearing established Bel-7402 tumors were treated with TPZ or Q6 at 25 or 12.5 mg/kg by daily i.p. injection for 27 d. (A) Tumor volumes are expressed as the mean ± SEM ***P < 0.001 vs. vehicle-treated controls (n = 5 to 8 per group). (B) Relative body weights are expressed as the mean ± SEM **P < 0.01 vs. vehicle-treated controls (n = 5 to 8 per group). (C) Western blot analysis of Bel-7402 cell-derived tumors treated with Q6 (12.5 mg/kg) or vehicle for expression of PARP1, CASP3, cleaved-CASP3, HIF1A, and VEGFA. ACTB was measured as the loading control. (D and E) Effects of Q6 on expression levels of HIF1A and VEGFA in Bel-7402 cell-derived tumors were determined by hematoxylin and eosin staining and immunohistochemical and immunofluorescence analysis. (F) Schematic diagrams depicting the mechanism by which Q6 exerts its anticancer effects. On the one hand, Q6 triggers the caspase-dependent apoptosis pathway and induces HCC cell death both in vitro and in vivo. On the other hand, Q6 activates the ATG5 and LC3B-dependent autophagy pathway, which plays an essential role in HIF1A degradation and the anti-angiogenesis and anti-metastatic activities of Q6. Furthermore, interaction with SQSTM1 regulates the degradation of HIF1A. Thus, these data strongly support the conclusion that Q6 is a novel hypoxia-targeted drug for HCC therapy, and we propose that autophagy acts as a tumor suppressor by accelerating HIF1A degradation and arresting angiogenesis and metastatic processes.
Mentions: Given the superior antitumor activity of Q6 in vitro, we hypothesized that it should be capable of slowing tumor growth in vivo. As depicted in Figure 6A and Table S2, Q6 administration significantly inhibited growth of Bel-7402 xenograft tumors and H22 hepatoma. Furthermore, it is worth noting that Q6 treatment did not cause obvious weight loss in either of the 2 in vivo models (Fig. 6B; Table S2). Next, we performed western blot analysis of tumor xenografts and investigated that Q6 administration remarkably activated the caspase-dependent apoptosis pathway in Bel-7402 cell-derived tumors. Finally, western blot and immunohistochemical and immunofluoresence analyses verified that Q6 treatment inhibited HIF1A, VEGFA and CD31 expression in tumors from Q6-treated mice, indicating that Q6 suppresses HIF1A activity and related signaling mechanisms in vivo (Fig. 6D and E;Fig. S7).

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