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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 1. Q6 has potent antitumor efficacy and triggers caspase-dependent apoptosis in 2 HCC cell lines. (A and B) Two HCC cell lines, HepG2 (left) and Bel-7402 (right), were treated with Q6 or TPZ (0 to 12.5 μM) for 72 h under normoxia or hypoxia. Cell viability was determined by the MTT assay. Data are representative of 3 independent experiments and are expressed as the means ± SD (C and D) HepG2 and Bel-7402 cells were treated with Q6 or TPZ (0, 10 μM) or Q6 (0 to 10 μM) +Z-VAD-FMK (40 μM) for 24 h under normoxia or hypoxia. (C) Percentage of apoptosis was measured by flow cytometry using the ANXA5/PI apoptosis detection kit. Three independent experiments were performed and the values were expressed as the mean ± SD (D) Protein levels of PARP1 were detected by western blot analysis. ACTB was measured as the loading control. Data are representative of 3 independent experiments.
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Figure 1: Figure 1. Q6 has potent antitumor efficacy and triggers caspase-dependent apoptosis in 2 HCC cell lines. (A and B) Two HCC cell lines, HepG2 (left) and Bel-7402 (right), were treated with Q6 or TPZ (0 to 12.5 μM) for 72 h under normoxia or hypoxia. Cell viability was determined by the MTT assay. Data are representative of 3 independent experiments and are expressed as the means ± SD (C and D) HepG2 and Bel-7402 cells were treated with Q6 or TPZ (0, 10 μM) or Q6 (0 to 10 μM) +Z-VAD-FMK (40 μM) for 24 h under normoxia or hypoxia. (C) Percentage of apoptosis was measured by flow cytometry using the ANXA5/PI apoptosis detection kit. Three independent experiments were performed and the values were expressed as the mean ± SD (D) Protein levels of PARP1 were detected by western blot analysis. ACTB was measured as the loading control. Data are representative of 3 independent experiments.

Mentions: As a promising HAP, Q6 (Fig. S1) has shown broad antitumor activity and hypoxia-selectivity in a variety of cancer cell lines.16 Thus, in the current study, we speculated that Q6 may inhibit HCC cell survival, especially under hypoxic conditions. To address this question, HepG2 and Bel-7402 cells were exposed to Q6/TPZ for 72 h under normoxia and hypoxia conditions, and cell viability was determined using the MTT assay. As expected, the survival rate of Q6-exposed cells under hypoxia was much lower than that of the cells under normoxia, with the average IC50 values of 1.99 ± 0.25 μM in hypoxia and 13.90 ± 5.15 μM in normoxia, a 7-fold selectivity (Fig. 1A). In addition, we compared the antiproliferative effects of Q6 and TPZ, in HepG2 and Bel-7402 cells under hypoxic conditions. The results showed that Q6 appeared to be more potent than TPZ with the IC50 values of 2.24 ± 0.18 and 1.74 ± 0.32 μM, respectively, compared with corresponding values of 24.92 ± 6.79 and 6.55 ± 1.78 μM for TPZ (Fig. 1B). In comparison to malignant cells, 2 normal human liver cell lines, HL-7702 and Chang liver cells, were resistant to both compounds (Fig. S2).


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 1. Q6 has potent antitumor efficacy and triggers caspase-dependent apoptosis in 2 HCC cell lines. (A and B) Two HCC cell lines, HepG2 (left) and Bel-7402 (right), were treated with Q6 or TPZ (0 to 12.5 μM) for 72 h under normoxia or hypoxia. Cell viability was determined by the MTT assay. Data are representative of 3 independent experiments and are expressed as the means ± SD (C and D) HepG2 and Bel-7402 cells were treated with Q6 or TPZ (0, 10 μM) or Q6 (0 to 10 μM) +Z-VAD-FMK (40 μM) for 24 h under normoxia or hypoxia. (C) Percentage of apoptosis was measured by flow cytometry using the ANXA5/PI apoptosis detection kit. Three independent experiments were performed and the values were expressed as the mean ± SD (D) Protein levels of PARP1 were detected by western blot analysis. ACTB was measured as the loading control. Data are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389865&req=5

Figure 1: Figure 1. Q6 has potent antitumor efficacy and triggers caspase-dependent apoptosis in 2 HCC cell lines. (A and B) Two HCC cell lines, HepG2 (left) and Bel-7402 (right), were treated with Q6 or TPZ (0 to 12.5 μM) for 72 h under normoxia or hypoxia. Cell viability was determined by the MTT assay. Data are representative of 3 independent experiments and are expressed as the means ± SD (C and D) HepG2 and Bel-7402 cells were treated with Q6 or TPZ (0, 10 μM) or Q6 (0 to 10 μM) +Z-VAD-FMK (40 μM) for 24 h under normoxia or hypoxia. (C) Percentage of apoptosis was measured by flow cytometry using the ANXA5/PI apoptosis detection kit. Three independent experiments were performed and the values were expressed as the mean ± SD (D) Protein levels of PARP1 were detected by western blot analysis. ACTB was measured as the loading control. Data are representative of 3 independent experiments.
Mentions: As a promising HAP, Q6 (Fig. S1) has shown broad antitumor activity and hypoxia-selectivity in a variety of cancer cell lines.16 Thus, in the current study, we speculated that Q6 may inhibit HCC cell survival, especially under hypoxic conditions. To address this question, HepG2 and Bel-7402 cells were exposed to Q6/TPZ for 72 h under normoxia and hypoxia conditions, and cell viability was determined using the MTT assay. As expected, the survival rate of Q6-exposed cells under hypoxia was much lower than that of the cells under normoxia, with the average IC50 values of 1.99 ± 0.25 μM in hypoxia and 13.90 ± 5.15 μM in normoxia, a 7-fold selectivity (Fig. 1A). In addition, we compared the antiproliferative effects of Q6 and TPZ, in HepG2 and Bel-7402 cells under hypoxic conditions. The results showed that Q6 appeared to be more potent than TPZ with the IC50 values of 2.24 ± 0.18 and 1.74 ± 0.32 μM, respectively, compared with corresponding values of 24.92 ± 6.79 and 6.55 ± 1.78 μM for TPZ (Fig. 1B). In comparison to malignant cells, 2 normal human liver cell lines, HL-7702 and Chang liver cells, were resistant to both compounds (Fig. S2).

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