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Alkalization of cellular pH leads to cancer cell death by disrupting autophagy and mitochondrial function

Abstract

We previously found that lactic acidosis in the tumor environment was permissive to cancer cell surviving under glucose deprivation and demonstrated that neutralizing lactic acidosis restored cancer cell susceptibility to glucose deprivation. We then reported that alternate infusion of bicarbonate and anticancer agent into tumors via tumor feeding artery markedly enhanced the efficacy of transarterial chemoembolization (TACE) in the local control of hepatocellular carcinoma (HCC). Here we sought to further investigate the mechanism by which bicarbonate enhances the anticancer activity of TACE. We propose that interfering cellular pH by bicarbonate could induce a cascade of molecular events leading to cancer cell death. Alkalizing cellular pH by bicarbonate decreased pH gradient (ΔpH), membrane potential (ΔΨm), and proton motive force (Δp) across the inner membrane of mitochondria; disruption of oxidative phosphorylation (OXPHOS) due to collapsed Δp led to a significant increase in adenosine monophosphate (AMP), which activated the classical AMPK-mediated autophagy. Meanwhile, the autophagic flux was ultimately blocked by increased cellular pH, reduced OXPHOS, and inhibition of lysosomal proton pump in alkalized lysosome. Bicarbonate also induced persistent mitochondrial permeability (MPT) and damaged mitochondria. Collectively, this study reveals that interfering cellular pH may provide a valuable approach to treat cancer.

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Fig. 1: Bicarbonate decreases the ΔpH across the inner membrane of mitochondria, depolarizes ΔΨm, reduces Δp, and disrupts OXPHOS in SK-HEP-1 cells.
Fig. 2: Bicarbonate increases cellular AMP concentration and activates AMPK-mediated autophagy.
Fig. 3: Bicarbonate concentration-dependent effect on lysosome alkalization and autophagic flux blockage.
Fig. 4: Bicarbonate alkalizes lysosome and autolysosome and blocks autophagic flux induced by bicarbonate.
Fig. 5: The effect of Cyclophilin D knockdown on the ΔΨm, MPT, mitochondrial respiration, and autophagy of SK-HEP-1 cells treated with bicarbonate.
Fig. 6: The contribution of bicarbonate-induced autophagy and MPT to cell death and combined effect of bicarbonate with pirarubicin on cell cytotoxicity.
Fig. 7: Contrast-enhanced MRI scans of patients with hepatocellular carcinoma before and after treatment with TILA-TACE or cTACE.
Fig. 8: Bicarbonate induces a cascade of molecular events that lead to cancer cell death.

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References

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71:209–49.

    Article  PubMed  Google Scholar 

  2. Bruix J, Llovet JM. Prognostic prediction and treatment strategy in hepatocellular carcinoma. Hepatology. 2002;35:519–24.

    Article  PubMed  Google Scholar 

  3. Galle PR, Forner A, Llovet JM, Mazzaferro V, Piscaglia F, Raoul JL, et al. EASL Clinical Practice Guidelines: Management of hepatocellular carcinoma. J Hepatol. 2018;69:182–236.

    Article  Google Scholar 

  4. Piscaglia F, Bolondi L. The intermediate hepatocellular carcinoma stage: Should treatment be expanded? Dig Liver Dis. 2010;42:S258–263.

    Article  PubMed  Google Scholar 

  5. Raoul JL, Sangro B, Forner A, Mazzaferro V, Piscaglia F, Bolondi L, et al. Evolving strategies for the management of intermediate-stage hepatocellular carcinoma: available evidence and expert opinion on the use of transarterial chemoembolization. Cancer Treat Rev. 2011;37:212–20.

    Article  PubMed  Google Scholar 

  6. Piscaglia F, Ogasawara S. Patient Selection for Transarterial Chemoembolization in Hepatocellular Carcinoma: Importance of Benefit/Risk Assessment. Liver Cancer. 2018;7:104–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lencioni R, de Baere T, Soulen MC, Rilling WS, Geschwind JF. Lipiodol transarterial chemoembolization for hepatocellular carcinoma: A systematic review of efficacy and safety data. Hepatology. 2016;64:106–16.

    Article  CAS  PubMed  Google Scholar 

  8. Jansen MC, van Hillegersberg R, Chamuleau RAFM, van Delden OM, Gouma DJ, van Gulik TM. Outcome of regional and local ablative therapies for hepatocellular carcinoma: a collective review. Eur J Surg Oncol. 2005;31:331–47.

    Article  CAS  PubMed  Google Scholar 

  9. Llovet JM, Real MI, Montana X, Planas R, Coll S, Aponte J, et al. Arterial embolisation or chemoembolisation versus symptomatic treatment in patients with unresectable hepatocellular carcinoma: a randomised controlled trial. Lancet. 2002;359:1734–9.

    Article  PubMed  Google Scholar 

  10. Xie J, Wu H, Dai C, Pan Q, Ding Z, Hu D, et al. Beyond Warburg effect−dual metabolic nature of cancer cells. Sci Rep. 2014;4:4927.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Hu X, Chao M, Wu H. Central role of lactate and proton in cancer cell resistance to glucose deprivation and its clinical translation. Signal Transduct Target Ther. 2017;2:16047.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Wu H, Ding Z, Hu D, Sun F, Dai C, Xie J, et al. Central role of lactic acidosis in cancer cell resistance to glucose deprivation-induced cell death. J Pathol. 2012;227:189–99.

    Article  CAS  PubMed  Google Scholar 

  13. Chao M, Wu H, Jin K, Li B, Wu J, Zhang G, et al. A nonrandomized cohort and a randomized study of local control of large hepatocarcinoma by targeting intratumoral lactic acidosis. Elife. 2016;5:e15691.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Poburko D, Santo-Domingo J, Demaurex N. Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations. J Biol Chem. 2011;286:11672–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Oakhill JS, Steel R, Chen ZP, Scott JW, Ling N, Tam S, et al. AMPK is a direct adenylate charge-regulated protein kinase. Science. 2011;332:1433–5.

    Article  CAS  PubMed  Google Scholar 

  16. Hawley SA, Ford RJ, Smith BK, Gowans GJ, Mancini SJ, Pitt RD, et al. The Na+/Glucose Cotransporter Inhibitor Canagliflozin Activates AMPK by Inhibiting Mitochondrial Function and Increasing Cellular AMP Levels. Diabetes. 2016;65:2784–94.

    Article  CAS  PubMed  Google Scholar 

  17. Xiao B, Sanders MJ, Underwood E, Heath R, Mayer FV, Carmena D, et al. Structure of mammalian AMPK and its regulation by ADP. Nature. 2011;472:230–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Liu Y, Shi Y. Mitochondria as a target in cancer treatment. MedComm (2020). 2020;1:129–39.

    Google Scholar 

  19. Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science. 2011;331:456–61.

    Article  CAS  PubMed  Google Scholar 

  20. Zois CE, Koukourakis MI. Radiation-induced autophagy in normal and cancer cells: towards novel cytoprotection and radio-sensitization policies? Autophagy. 2009;5:442–50.

    Article  CAS  PubMed  Google Scholar 

  21. Ju JS, Varadhachary AS, Miller SE, Weihl CC. Quantitation of “autophagic flux” in mature skeletal muscle. Autophagy. 2010;6:929–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu B, Fang M, Hu Y, Huang B, Li N, Chang C, et al. Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation. Autophagy. 2014;10:416–30.

    Article  CAS  PubMed  Google Scholar 

  23. Yue W, Hamaï A, Tonelli G, Bauvy C, Nicolas V, Tharinger H, et al. Inhibition of the autophagic flux by salinomycin in breast cancer stem-like/progenitor cells interferes with their maintenance. Autophagy. 2014;9:714–29.

    Article  CAS  Google Scholar 

  24. Gowans GJ, Hawley SA, Ross FA, Hardie DG. AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net phosphorylation. Cell Metab. 2013;18:556–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ahmad F, Leake DS. Lysosomal oxidation of LDL alters lysosomal pH, induces senescence, and increases secretion of pro-inflammatory cytokines in human macrophages. J Lipid Res. 2019;60:98–110.

    Article  CAS  PubMed  Google Scholar 

  26. Caricato R, Giordano ME, Schettino T, Lionetto MG. Functional Involvement of Carbonic Anhydrase in the Lysosomal Response to Cadmium Exposure in Mytilus galloprovincialis Digestive Gland. Front Physiol. 2018;9:319.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Pasquier A, Vivot K, Erbs E, Spiegelhalter C, Zhang Z, Aubert V, et al. Lysosomal degradation of newly formed insulin granules contributes to β cell failure in diabetes. Nat Commun. 2019;10:3312.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Yoshii SR, Mizushima N. Monitoring and Measuring Autophagy. Int J Mol Sci. 2017;18:1865.

    Article  PubMed Central  CAS  Google Scholar 

  29. Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 2007;3:452–60.

    Article  CAS  PubMed  Google Scholar 

  30. Beyenbach KW, Wieczorek H. The V-type H+ ATPase: molecular structure and function, physiological roles and regulation. J Exp Biol. 2006;209:577–89.

    Article  CAS  PubMed  Google Scholar 

  31. Bartel K, Müller R, von Schwarzenberg K. Differential regulation of AMP-activated protein kinase in healthy and cancer cells explains why V-ATPase inhibition selectively kills cancer cells. J Biol Chem. 2019;294:17239–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bernardi P, Vassanelli S, Veronese P, Colonna R, Szabó I, Zoratti M. Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations. J Biol Chem. 1992;267:2934–9.

    Article  CAS  PubMed  Google Scholar 

  33. Malhi H, Gores GJ, Lemasters JJ. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology. 2006;43:S31–44.

    Article  CAS  PubMed  Google Scholar 

  34. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J. 1999;341:233–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Tsujimoto Y, Shimizu S. Role of the mitochondrial membrane permeability transition in cell death. Apoptosis. 2007;12:835–40.

    Article  CAS  PubMed  Google Scholar 

  36. Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H, et al. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature. 2005;434:652–8.

    Article  CAS  PubMed  Google Scholar 

  37. Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, et al. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature. 2005;434:658–62.

    Article  CAS  PubMed  Google Scholar 

  38. Rasola A, Sciacovelli M, Chiara F, Pantic B, Brusilow WS, Bernardi P. Activation of mitochondrial ERK protects cancer cells from death through inhibition of the permeability transition. Proc Natl Acad Sci. 2010;107:726–31.

    Article  CAS  PubMed  Google Scholar 

  39. Vaseva AV, Marchenko ND, Ji K, Tsirka SE, Holzmann S, Moll UM. p53 opens the mitochondrial permeability transition pore to trigger necrosis. Cell. 2012;149:1536–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wu YT, Tan HL, Shui G, Bauvy C, Huang Q, Wenk MR, et al. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem. 2010;285:10850–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 2012;8:445–544.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Perera RM, Stoykova S, Nicolay BN, Ross KN, Fitamant J, Boukhali M, et al. Transcriptional control of autophagy-lysosome function drives pancreatic cancer metabolism. Nature. 2015;524:361–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Russo M, Russo GL. Autophagy inducers in cancer. Biochem Pharm. 2018;153:51–61.

    Article  CAS  PubMed  Google Scholar 

  44. Goodall ML, Fitzwalter BE, Zahedi S, Wu M, Rodriguez D, Mulcahy-Levy JM, et al. The Autophagy Machinery Controls Cell Death Switching between Apoptosis and Necroptosis. Dev Cell. 2016;37:337–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Liu J, Kuang F, Kroemer G, Klionsky DJ, Kang R, Tang D. Autophagy-Dependent Ferroptosis: Machinery and Regulation. Cell Chem Biol. 2020;27:420–35.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Shlomovitz I, Speir M, Gerlic M. Flipping the dogma - phosphatidylserine in non-apoptotic cell death. Cell Commun Signal. 2019;17:139.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Ibrahim-Hashim A, Cornnell HH, Abrahams D, Lloyd M, Bui M, Gillies RJ, et al. Systemic buffers inhibit carcinogenesis in TRAMP mice. J Urol. 2012;188:624–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Martin NK, Robey IF, Gaffney EA, Gillies RJ, Gatenby RA, Maini PK. Predicting the safety and efficacy of buffer therapy to raise tumour pHe: an integrative modelling study. Br J Cancer. 2012;106:1280–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Martin NK, Gaffney EA, Gatenby RA, Gillies RJ, Robey IF, Maini PK. A mathematical model of tumour and blood pHe regulation: The HCO3-/CO2 buffering system. Math Biosci. 2011;230:1–11.

    Article  CAS  PubMed  Google Scholar 

  50. Gong YN, Guy C, Olauson H, Becker JU, Yang M, Fitzgerald P, et al. ESCRT-III Acts Downstream of MLKL to Regulate Necroptotic Cell Death and Its Consequences. Cell. 2017;169:286–300.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Zargarian S, Shlomovitz I, Erlich Z, Hourizadeh A, Ofir-Birin Y, Croker BA, et al. Phosphatidylserine externalization, “necroptotic bodies” release, and phagocytosis during necroptosis. PLoS Biol. 2017;15:e2002711.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. de Vasconcelos NM, Van Opdenbosch N, Van Gorp H, Parthoens E, Lamkanfi M. Single-cell analysis of pyroptosis dynamics reveals conserved GSDMD-mediated subcellular events that precede plasma membrane rupture. Cell Death Differ. 2019;26:146–61.

    Article  PubMed  CAS  Google Scholar 

  53. Klöditz K, Fadeel B. Three cell deaths and a funeral: macrophage clearance of cells undergoing distinct modes of cell death. Cell Death Disco. 2019;5:65.

    Article  Google Scholar 

  54. Chen J, Kuroki S, Someda M, Yonehara S. Interferon-γ induces the cell surface exposure of phosphatidylserine by activating the protein MLKL in the absence of caspase-8 activity. J Biol Chem. 2019;294:11994–2006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Jin C, Zhu X, Wu H, Wang Y, Hu X. Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells. J Biol Chem. 2020;295:6425–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Mauvezin C, Nagy P, Juhasz G, Neufeld TP. Autophagosome-lysosome fusion is independent of V-ATPase-mediated acidification. Nat Commun. 2015;6:7007.

    Article  PubMed  Google Scholar 

  57. Yang K, Lu Y, Xie F, Zou H, Fan X, Li B, et al. Cationic liposomes induce cell necrosis through lysosomal dysfunction and late-stage autophagic flux inhibition. Nanomed . 2016;11:3117–37.

    Article  CAS  Google Scholar 

  58. Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques. 2011;50:98–115.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work has been supported in part by China Natural Sciences Foundation projects (82073038, 81772947 to XH), a key project (2018C03009) funded by Zhejiang Provincial Department of Sciences and Technologies (to XH & MC), and the Fundamental Research Funds for the Central Universities (2017XZZX001-01, 2019FZJD009, to XH), National Ministry of Education, China. We thank Dr. Guo-Hua Fong (University of Connecticut School of Medicine, USA) for critical readings of our manuscript and constructive comments, Dr. Wei Liu (Zhejiang University) for the kind gift of plasmids GFP-LC3, Cherry-GFP-LC3, and Dr. Shanrong Cai for the statistical assistance (Zhejiang University).

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XH conceived the project, designed the study, analyzed the data, wrote the manuscript. CY & CJ. performed the experiments, CY, CJ, SZ. analyzed the data, MC, analysis of MRI images.

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Correspondence to Ming Chao or Xun Hu.

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Ying, C., Jin, C., Zeng, S. et al. Alkalization of cellular pH leads to cancer cell death by disrupting autophagy and mitochondrial function. Oncogene 41, 3886–3897 (2022). https://doi.org/10.1038/s41388-022-02396-6

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