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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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HMGB1 and ATP release in response to treatmentPDAC cell lines were incubated with saline, Gem (1.0 μM), PX-478 (25 μM), or Gem/PX-478, OXP (oxaliplatin) (300μM) or OXP/PX-478 for 24 hours. (A) Determination of treatment-induced HMGB1 re-localization and release in three PDAC cell lines by fluorescent microscopy (magnification, 1000×). After treatment, cells on slides were fixed/permeablized and stained with both mouse anti-HMGB1 antibody/Alexa Fluor 568-conjugated secondary anti-mouse antibody (showing red) and DAPI (showing blue). (B) Determination of treatment-induced HMGB1 release in five PDAC cell lines by Western blotting. After treatment, 50 μl of conditioned medium was taken for detecting HMGB1 in the supernatants. (C) Chemiluminescence detection of treatment-induced ATP release in five PDAC cell lines. After treatment, 10 μl of conditioned medium was taken for ATP assay using chemiluminescence ELISA kit. Significantly increased ATP release by Gem/PX-478 (***P<0.001) was compared with those treated with single agent.
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Figure 5: HMGB1 and ATP release in response to treatmentPDAC cell lines were incubated with saline, Gem (1.0 μM), PX-478 (25 μM), or Gem/PX-478, OXP (oxaliplatin) (300μM) or OXP/PX-478 for 24 hours. (A) Determination of treatment-induced HMGB1 re-localization and release in three PDAC cell lines by fluorescent microscopy (magnification, 1000×). After treatment, cells on slides were fixed/permeablized and stained with both mouse anti-HMGB1 antibody/Alexa Fluor 568-conjugated secondary anti-mouse antibody (showing red) and DAPI (showing blue). (B) Determination of treatment-induced HMGB1 release in five PDAC cell lines by Western blotting. After treatment, 50 μl of conditioned medium was taken for detecting HMGB1 in the supernatants. (C) Chemiluminescence detection of treatment-induced ATP release in five PDAC cell lines. After treatment, 10 μl of conditioned medium was taken for ATP assay using chemiluminescence ELISA kit. Significantly increased ATP release by Gem/PX-478 (***P<0.001) was compared with those treated with single agent.

Mentions: Release of HMGB1 and ATP from dying or dead cells are also crucial hallmarkers of ICD [18-20]. HMGB1 release from nucleus was determined by both immuno-fluorescent microscopy (Figure 5A) and Western blotting (Figure 5B). In control cells, HMGB1 is mainly expressed in the nucleus. Treatment with either Gem or PX-478 alone induced a translocation of HMGB1 from the nucleus to the cytoplasm. Co-treatment with Gem/PX-478 caused a loss of HMGB1 in the nucleus (Figure 5A). Western blotting confirmed that treatment with Gem/PX-478 induced maximum HMGB1 release compared with other treatment (Figure 5B). Similarly to HMGB1 release, Gem/PX-478 induced a significantly increased ATP release (P<0.001) in all 5 PDAC cell lines compared with other groups (Figure 5C). In summary, these data demonstrate that in the presence of HIF-1α inhibitor PX-478, GEM gains its ICD-inducing potential on triggering the exposure and release of ICD markers in pancreatic cancer cells.


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)

HMGB1 and ATP release in response to treatmentPDAC cell lines were incubated with saline, Gem (1.0 μM), PX-478 (25 μM), or Gem/PX-478, OXP (oxaliplatin) (300μM) or OXP/PX-478 for 24 hours. (A) Determination of treatment-induced HMGB1 re-localization and release in three PDAC cell lines by fluorescent microscopy (magnification, 1000×). After treatment, cells on slides were fixed/permeablized and stained with both mouse anti-HMGB1 antibody/Alexa Fluor 568-conjugated secondary anti-mouse antibody (showing red) and DAPI (showing blue). (B) Determination of treatment-induced HMGB1 release in five PDAC cell lines by Western blotting. After treatment, 50 μl of conditioned medium was taken for detecting HMGB1 in the supernatants. (C) Chemiluminescence detection of treatment-induced ATP release in five PDAC cell lines. After treatment, 10 μl of conditioned medium was taken for ATP assay using chemiluminescence ELISA kit. Significantly increased ATP release by Gem/PX-478 (***P<0.001) was compared with those treated with single agent.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385849&req=5

Figure 5: HMGB1 and ATP release in response to treatmentPDAC cell lines were incubated with saline, Gem (1.0 μM), PX-478 (25 μM), or Gem/PX-478, OXP (oxaliplatin) (300μM) or OXP/PX-478 for 24 hours. (A) Determination of treatment-induced HMGB1 re-localization and release in three PDAC cell lines by fluorescent microscopy (magnification, 1000×). After treatment, cells on slides were fixed/permeablized and stained with both mouse anti-HMGB1 antibody/Alexa Fluor 568-conjugated secondary anti-mouse antibody (showing red) and DAPI (showing blue). (B) Determination of treatment-induced HMGB1 release in five PDAC cell lines by Western blotting. After treatment, 50 μl of conditioned medium was taken for detecting HMGB1 in the supernatants. (C) Chemiluminescence detection of treatment-induced ATP release in five PDAC cell lines. After treatment, 10 μl of conditioned medium was taken for ATP assay using chemiluminescence ELISA kit. Significantly increased ATP release by Gem/PX-478 (***P<0.001) was compared with those treated with single agent.
Mentions: Release of HMGB1 and ATP from dying or dead cells are also crucial hallmarkers of ICD [18-20]. HMGB1 release from nucleus was determined by both immuno-fluorescent microscopy (Figure 5A) and Western blotting (Figure 5B). In control cells, HMGB1 is mainly expressed in the nucleus. Treatment with either Gem or PX-478 alone induced a translocation of HMGB1 from the nucleus to the cytoplasm. Co-treatment with Gem/PX-478 caused a loss of HMGB1 in the nucleus (Figure 5A). Western blotting confirmed that treatment with Gem/PX-478 induced maximum HMGB1 release compared with other treatment (Figure 5B). Similarly to HMGB1 release, Gem/PX-478 induced a significantly increased ATP release (P<0.001) in all 5 PDAC cell lines compared with other groups (Figure 5C). In summary, these data demonstrate that in the presence of HIF-1α inhibitor PX-478, GEM gains its ICD-inducing potential on triggering the exposure and release of ICD markers in pancreatic cancer cells.

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