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Inhibition of HIF-1α by PX-478 enhances the anti-tumor effect of gemcitabine by inducing immunogenic cell death in pancreatic ductal adenocarcinoma.

Zhao T, Ren H, Jia L, Chen J, Xin W, Yan F, Li J, Wang X, Gao S, Qian D, Huang C, Hao J - Oncotarget (2015)

Bottom Line: We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice.Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects.Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Department of Pancreatic Cancer, Tianjin, China.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is the worst prognoses among all the malignancies. Now, gemcitabine (Gem) is the first line chemotherapeutic drug for advanced pancreatic cancer. However, Gem is usually ineffective to the PDAC because of high degree of drug resistance. Hypoxia and immune suppressive milieu are the best-described hallmarks of PDAC; therefore, we investigated the impact of hypoxia inducible factor-1 (HIF-1) inhibitor, PX-478, in combination with Gem on the induction of immunogenic cell death (ICD). We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice. Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects. Pancreatic cell lines treated with Gem and PX-478 can induce an increase in eIF2α phosphorylation was correlated with down-regulation of HIF-1α and elicited exposure of CRT and release of HMGB1 and ATP. Only co-treated cells induced DC maturation/phagocytosis and IFN-γ secretion by cytotoxic T lymphocytes. Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization. We propose that inhibition HIF-1α elicits Gem-induced immune response and eliminates PDAC cells by inducing ICD.

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Negative correlation between HIF-1α expression and eIF2α phosphorylationPDAC cell lines were incubated with saline, Gem (1.0 μM), PX-478 (25 μM), Gem/PX-478, OXP (oxaliplatin) (300μM) or OXP/PX-478 for 24 hours. (A) CRT surface exposure on CFPAC-1, BxPC-3 and Panc02 cell lines. Non-permeabilized cells were co-stained with anti-CRT antibody and Hoechst 33342 and determined by confocal microscopy (magnification, 600×). Green colour indicates ecto-CRT. (B) Five PDAC cell lines were treated with GEM, PX-478, OXP (300μM), Gem/PX-478, or Gem/OXP for 24 hours. Expression of HIF-1α, CRT, P-eIF2α, and eIF2α was determined by Western blotting. β-tubulin was used as a loading control and OXP was served as a positive control. (C) Negative correlation between levels of HIF-1α and P-eIF2α. Expression levels of both HIF-1α and P-eIF2α were analysed by densitometry and represented as ratios of HIF-1α/β-tubulin and P-eIF2α/β-tubulin. Data were collected from 5 cell lines and expressed as mean ± SD. Correlation between HIF-1α and P-eIF2α was analysed by Pearson's correlation method. **P<0.01, γ=-0.943. (D) Pancreatic cancer cell lines (CFPAC-1 and BxPC-3) were treated with siHIF-1 and 2-ME and then evaluate the expression HIF-1α, CRT and P-eIF2α by Western blotting experiment.
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Figure 4: Negative correlation between HIF-1α expression and eIF2α phosphorylationPDAC cell lines were incubated with saline, Gem (1.0 μM), PX-478 (25 μM), Gem/PX-478, OXP (oxaliplatin) (300μM) or OXP/PX-478 for 24 hours. (A) CRT surface exposure on CFPAC-1, BxPC-3 and Panc02 cell lines. Non-permeabilized cells were co-stained with anti-CRT antibody and Hoechst 33342 and determined by confocal microscopy (magnification, 600×). Green colour indicates ecto-CRT. (B) Five PDAC cell lines were treated with GEM, PX-478, OXP (300μM), Gem/PX-478, or Gem/OXP for 24 hours. Expression of HIF-1α, CRT, P-eIF2α, and eIF2α was determined by Western blotting. β-tubulin was used as a loading control and OXP was served as a positive control. (C) Negative correlation between levels of HIF-1α and P-eIF2α. Expression levels of both HIF-1α and P-eIF2α were analysed by densitometry and represented as ratios of HIF-1α/β-tubulin and P-eIF2α/β-tubulin. Data were collected from 5 cell lines and expressed as mean ± SD. Correlation between HIF-1α and P-eIF2α was analysed by Pearson's correlation method. **P<0.01, γ=-0.943. (D) Pancreatic cancer cell lines (CFPAC-1 and BxPC-3) were treated with siHIF-1 and 2-ME and then evaluate the expression HIF-1α, CRT and P-eIF2α by Western blotting experiment.

Mentions: We questioned whether the sensitization effect of PX-478 on ICD inducers is due to inhibition of HIF-1α. Induction of surface exposure of CRT (ecto-CRT) is one of the hallmarkers of ICD [28-30] and it occurs as a consequence of ER stress-induced phosphorylation of eIF2α [18, 19, 31]. Ecto-CRT was stained on non-permeabilized cells which had been treated individually as indicated. Gem or PX-478 alone had weaker effect on triggering CRT exposure but treatment with Gem/PX-478 showed strong induction of CRT surface exposure (Figure 4A). To determine the association between HIF-1α inhibition and eIF2α phosphorylation, treatment-induced alterations of HIF-1α, CRT, P-eIF2α and eIF2α protein expression were monitored in 5 PDAC cell lines. A typical ICD inducer, OXP (oxaliplatin) [18, 19] was used as a positive control. While none of these reagents induced changes in CRT and eIF2α expression, all of them mediated down-regulation of HIF-1α and up-regulation of P-eIF2α. Combination of PX-478 with either Gem or OXP led to maximum inhibition of HIF-1α and up-regulation of P-eIF2 α (Figure 4B). Treatment-induced changes in HIF-1α and P-eIF2α showed strong negative correlation, P<0.01, γ=-0.943 (Figure 4C). To further evaluate the effect of HIF-1 inhibition on ICD, PDAC cells were either transfected with HIF-1α-siRNA or treated with another HIF-1α inhibitor 2-Methoxyestradiol (2-ME) (Selleck. cn). As expected, both HIF-1α-siRNA and 2-ME induced eIF2α phosphorylation in PDAC cells (Figure 4D). These results demonstrate that inhibition of HIF-1α expression could trigger ICD by up-regulation P-eIF2α.


Inhibition of HIF-1α by PX-478 enhances the anti-tumor effect of gemcitabine by inducing immunogenic cell death in pancreatic ductal adenocarcinoma.

Zhao T, Ren H, Jia L, Chen J, Xin W, Yan F, Li J, Wang X, Gao S, Qian D, Huang C, Hao J - Oncotarget (2015)

Negative correlation between HIF-1α expression and eIF2α phosphorylationPDAC cell lines were incubated with saline, Gem (1.0 μM), PX-478 (25 μM), Gem/PX-478, OXP (oxaliplatin) (300μM) or OXP/PX-478 for 24 hours. (A) CRT surface exposure on CFPAC-1, BxPC-3 and Panc02 cell lines. Non-permeabilized cells were co-stained with anti-CRT antibody and Hoechst 33342 and determined by confocal microscopy (magnification, 600×). Green colour indicates ecto-CRT. (B) Five PDAC cell lines were treated with GEM, PX-478, OXP (300μM), Gem/PX-478, or Gem/OXP for 24 hours. Expression of HIF-1α, CRT, P-eIF2α, and eIF2α was determined by Western blotting. β-tubulin was used as a loading control and OXP was served as a positive control. (C) Negative correlation between levels of HIF-1α and P-eIF2α. Expression levels of both HIF-1α and P-eIF2α were analysed by densitometry and represented as ratios of HIF-1α/β-tubulin and P-eIF2α/β-tubulin. Data were collected from 5 cell lines and expressed as mean ± SD. Correlation between HIF-1α and P-eIF2α was analysed by Pearson's correlation method. **P<0.01, γ=-0.943. (D) Pancreatic cancer cell lines (CFPAC-1 and BxPC-3) were treated with siHIF-1 and 2-ME and then evaluate the expression HIF-1α, CRT and P-eIF2α by Western blotting experiment.
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Related In: Results  -  Collection

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Figure 4: Negative correlation between HIF-1α expression and eIF2α phosphorylationPDAC cell lines were incubated with saline, Gem (1.0 μM), PX-478 (25 μM), Gem/PX-478, OXP (oxaliplatin) (300μM) or OXP/PX-478 for 24 hours. (A) CRT surface exposure on CFPAC-1, BxPC-3 and Panc02 cell lines. Non-permeabilized cells were co-stained with anti-CRT antibody and Hoechst 33342 and determined by confocal microscopy (magnification, 600×). Green colour indicates ecto-CRT. (B) Five PDAC cell lines were treated with GEM, PX-478, OXP (300μM), Gem/PX-478, or Gem/OXP for 24 hours. Expression of HIF-1α, CRT, P-eIF2α, and eIF2α was determined by Western blotting. β-tubulin was used as a loading control and OXP was served as a positive control. (C) Negative correlation between levels of HIF-1α and P-eIF2α. Expression levels of both HIF-1α and P-eIF2α were analysed by densitometry and represented as ratios of HIF-1α/β-tubulin and P-eIF2α/β-tubulin. Data were collected from 5 cell lines and expressed as mean ± SD. Correlation between HIF-1α and P-eIF2α was analysed by Pearson's correlation method. **P<0.01, γ=-0.943. (D) Pancreatic cancer cell lines (CFPAC-1 and BxPC-3) were treated with siHIF-1 and 2-ME and then evaluate the expression HIF-1α, CRT and P-eIF2α by Western blotting experiment.
Mentions: We questioned whether the sensitization effect of PX-478 on ICD inducers is due to inhibition of HIF-1α. Induction of surface exposure of CRT (ecto-CRT) is one of the hallmarkers of ICD [28-30] and it occurs as a consequence of ER stress-induced phosphorylation of eIF2α [18, 19, 31]. Ecto-CRT was stained on non-permeabilized cells which had been treated individually as indicated. Gem or PX-478 alone had weaker effect on triggering CRT exposure but treatment with Gem/PX-478 showed strong induction of CRT surface exposure (Figure 4A). To determine the association between HIF-1α inhibition and eIF2α phosphorylation, treatment-induced alterations of HIF-1α, CRT, P-eIF2α and eIF2α protein expression were monitored in 5 PDAC cell lines. A typical ICD inducer, OXP (oxaliplatin) [18, 19] was used as a positive control. While none of these reagents induced changes in CRT and eIF2α expression, all of them mediated down-regulation of HIF-1α and up-regulation of P-eIF2α. Combination of PX-478 with either Gem or OXP led to maximum inhibition of HIF-1α and up-regulation of P-eIF2 α (Figure 4B). Treatment-induced changes in HIF-1α and P-eIF2α showed strong negative correlation, P<0.01, γ=-0.943 (Figure 4C). To further evaluate the effect of HIF-1 inhibition on ICD, PDAC cells were either transfected with HIF-1α-siRNA or treated with another HIF-1α inhibitor 2-Methoxyestradiol (2-ME) (Selleck. cn). As expected, both HIF-1α-siRNA and 2-ME induced eIF2α phosphorylation in PDAC cells (Figure 4D). These results demonstrate that inhibition of HIF-1α expression could trigger ICD by up-regulation P-eIF2α.

Bottom Line: We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice.Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects.Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Department of Pancreatic Cancer, Tianjin, China.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is the worst prognoses among all the malignancies. Now, gemcitabine (Gem) is the first line chemotherapeutic drug for advanced pancreatic cancer. However, Gem is usually ineffective to the PDAC because of high degree of drug resistance. Hypoxia and immune suppressive milieu are the best-described hallmarks of PDAC; therefore, we investigated the impact of hypoxia inducible factor-1 (HIF-1) inhibitor, PX-478, in combination with Gem on the induction of immunogenic cell death (ICD). We verified that combined treatment with Gem/PX-478 significantly enhanced the anti-tumor effect and increased proportion of tumor infiltrating T-lymphocytes in Panc02-bearing immune-competent but not in immune-deficient mice. Vaccination using Panc02 cell line treated with single agent or in combination showed significant anti-tumor effects. Pancreatic cell lines treated with Gem and PX-478 can induce an increase in eIF2α phosphorylation was correlated with down-regulation of HIF-1α and elicited exposure of CRT and release of HMGB1 and ATP. Only co-treated cells induced DC maturation/phagocytosis and IFN-γ secretion by cytotoxic T lymphocytes. Altogether, combined treatment with Gem/PX-478 showed significantly inhibition on tumor growth and anti-tumor immunization. We propose that inhibition HIF-1α elicits Gem-induced immune response and eliminates PDAC cells by inducing ICD.

Show MeSH
Related in: MedlinePlus