JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice

Xinxin Song, Shan Zhu, Yangchun Xie, Jiao Liu, Lingyi Sun, Dexing Zeng, Pengcheng Wang, Xiaochao Ma, Guido Kroemer, David L. Bartlett, Timothy R. Billiar, Michael T. Lotze, Herbert J. Zeh, Rui Kang, Daolin Tang

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Background & Aims: Maintenance of acid–base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. Methods: We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRasG12D/+;Tp53R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;KrasG12D/+ mice crossed with Hmgb1flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. Results: Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax-/-/Bak-/- cells), necroptosis (Ripk1-/-, Ripk3-/-, or Mlkl-/- cells), ferroptosis (Gpx4-/- cells), or autophagy (Atg3-/-, Atg5-/-, Atg7-/-, or Sqstm1-/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34+ progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. Conclusions: In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer.

Original languageEnglish (US)
Pages (from-to)1480-1493
Number of pages14
JournalGastroenterology
Volume154
Issue number5
DOIs
StatePublished - Apr 1 2018
Externally publishedYes

Fingerprint

Growth
Neoplasms
Cell Death
Cell Line
Adenocarcinoma
Pancreatic Neoplasms
G-Protein-Coupled Receptors
N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide
Genes
Autophagy
phospholipid-hydroperoxide glutathione peroxidase
Apoptosis
Inbred C57BL Mouse
Heterografts
Fibroblasts
Immunohistochemistry
B-Cell Activating Factor
Neoplasm Metastasis
Hydroxychloroquine
Tissue Survival

Keywords

  • Drug Development
  • Pancreas
  • Targeted Therapy
  • Tumor Microenvironment

ASJC Scopus subject areas

  • Hepatology
  • Gastroenterology

Cite this

JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice. / Song, Xinxin; Zhu, Shan; Xie, Yangchun; Liu, Jiao; Sun, Lingyi; Zeng, Dexing; Wang, Pengcheng; Ma, Xiaochao; Kroemer, Guido; Bartlett, David L.; Billiar, Timothy R.; Lotze, Michael T.; Zeh, Herbert J.; Kang, Rui; Tang, Daolin.

In: Gastroenterology, Vol. 154, No. 5, 01.04.2018, p. 1480-1493.

Research output: Contribution to journalArticle

Song, X, Zhu, S, Xie, Y, Liu, J, Sun, L, Zeng, D, Wang, P, Ma, X, Kroemer, G, Bartlett, DL, Billiar, TR, Lotze, MT, Zeh, HJ, Kang, R & Tang, D 2018, 'JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice', Gastroenterology, vol. 154, no. 5, pp. 1480-1493. https://doi.org/10.1053/j.gastro.2017.12.004
Song, Xinxin ; Zhu, Shan ; Xie, Yangchun ; Liu, Jiao ; Sun, Lingyi ; Zeng, Dexing ; Wang, Pengcheng ; Ma, Xiaochao ; Kroemer, Guido ; Bartlett, David L. ; Billiar, Timothy R. ; Lotze, Michael T. ; Zeh, Herbert J. ; Kang, Rui ; Tang, Daolin. / JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice. In: Gastroenterology. 2018 ; Vol. 154, No. 5. pp. 1480-1493.
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abstract = "Background & Aims: Maintenance of acid–base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. Methods: We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRasG12D/+;Tp53R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;KrasG12D/+ mice crossed with Hmgb1flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. Results: Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax-/-/Bak-/- cells), necroptosis (Ripk1-/-, Ripk3-/-, or Mlkl-/- cells), ferroptosis (Gpx4-/- cells), or autophagy (Atg3-/-, Atg5-/-, Atg7-/-, or Sqstm1-/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34+ progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. Conclusions: In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer.",
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author = "Xinxin Song and Shan Zhu and Yangchun Xie and Jiao Liu and Lingyi Sun and Dexing Zeng and Pengcheng Wang and Xiaochao Ma and Guido Kroemer and Bartlett, {David L.} and Billiar, {Timothy R.} and Lotze, {Michael T.} and Zeh, {Herbert J.} and Rui Kang and Daolin Tang",
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TY - JOUR

T1 - JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice

AU - Song, Xinxin

AU - Zhu, Shan

AU - Xie, Yangchun

AU - Liu, Jiao

AU - Sun, Lingyi

AU - Zeng, Dexing

AU - Wang, Pengcheng

AU - Ma, Xiaochao

AU - Kroemer, Guido

AU - Bartlett, David L.

AU - Billiar, Timothy R.

AU - Lotze, Michael T.

AU - Zeh, Herbert J.

AU - Kang, Rui

AU - Tang, Daolin

PY - 2018/4/1

Y1 - 2018/4/1

N2 - Background & Aims: Maintenance of acid–base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. Methods: We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRasG12D/+;Tp53R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;KrasG12D/+ mice crossed with Hmgb1flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. Results: Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax-/-/Bak-/- cells), necroptosis (Ripk1-/-, Ripk3-/-, or Mlkl-/- cells), ferroptosis (Gpx4-/- cells), or autophagy (Atg3-/-, Atg5-/-, Atg7-/-, or Sqstm1-/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34+ progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. Conclusions: In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer.

AB - Background & Aims: Maintenance of acid–base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. Methods: We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRasG12D/+;Tp53R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;KrasG12D/+ mice crossed with Hmgb1flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. Results: Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax-/-/Bak-/- cells), necroptosis (Ripk1-/-, Ripk3-/-, or Mlkl-/- cells), ferroptosis (Gpx4-/- cells), or autophagy (Atg3-/-, Atg5-/-, Atg7-/-, or Sqstm1-/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34+ progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. Conclusions: In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer.

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