Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+ content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients' samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA-Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver. Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.

Department of Experimental Biology Faculty of Science Masaryk University 625 00 Brno Czech Republic

Department of Functional and Evolutionary Ecology Archaea Physiology and Biotechnology Group Universität Wien 1030 Wien Austria

Department of Human and Animal Physiology Faculty of Biology Ivan Franko National University of Lviv 79005 Lviv Ukraine

Department of Plant Origin Food Sciences Faculty of Veterinary Hygiene and Ecology University of Veterinary Sciences Brno 612 42 Brno Czech Republic

Department of Therapy No 1 Medical Diagnostic and Hematology and Transfusiology of Faculty of Postgraduate Education Danylo Halytsky Lviv National Medical University 79010 Lviv Ukraine

Zobrazit více v PubMed

Dröse S., Altendorf K. Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases. J. Exp. Biol. 1997;200:1–8. doi: 10.1242/jeb.200.1.1. PubMed DOI

Sun J., Li Y., Yang X., Dong W., Yang J., Hu Q., Zhang C., Fang H., Liu A. Growth differentiation factor 11 accelerates liver senescence through the inhibition of autophagy. Aging Cell. 2022;21:e13532. doi: 10.1111/acel.13532. PubMed DOI PMC

Park H.-S., Song J.-W., Park J.-H., Lim B.-K., Moon O.-S., Son H.-Y., Lee J.-H., Gao B., Won Y.-S., Kwon H.-J. TXNIP/VDUP1 attenuates steatohepatitis via autophagy and fatty acid oxidation. Autophagy. 2021;17:2549–2564. doi: 10.1080/15548627.2020.1834711. PubMed DOI PMC

Lee D.H., Park J.S., Lee Y.S., Han J., Lee D.-K., Kwon S.W., Han D.H., Lee Y.-H., Bae S.H. SQSTM1/p62 activates NFE2L2/NRF2 via ULK1-mediated autophagic KEAP1 degradation and protects mouse liver from lipotoxicity. Autophagy. 2020;16:1949–1973. doi: 10.1080/15548627.2020.1712108. PubMed DOI PMC

Lu X., Chen L., Chen Y., Shao Q., Qin W. Bafilomycin A1 inhibits the growth and metastatic potential of the BEL-7402 liver cancer and HO-8910 ovarian cancer cell lines and induces alterations in their microRNA expression. Exp. Ther. Med. 2015;10:1829–1834. doi: 10.3892/etm.2015.2758. PubMed DOI PMC

Wang R., Wang J., Hassan A., Lee C.-H., Xie X.-S., Li X. Molecular basis of V-ATPase inhibition by bafilomycin A1. Nat. Commun. 2021;12:1782. doi: 10.1038/s41467-021-22111-5. PubMed DOI PMC

Futai M., Sun-Wada G.-H., Wada Y., Matsumoto N., Nakanishi-Matsui M. Vacuolar-type ATPase: A proton pump to lysosomal trafficking. Proc. Jpn. Acad. Ser. B. 2019;95:261–277. doi: 10.2183/pjab.95.018. PubMed DOI PMC

Jeger J.L. Endosomes, lysosomes, and the role of endosomal and lysosomal biogenesis in cancer development. Mol. Biol. Rep. 2020;47:9801–9810. doi: 10.1007/s11033-020-05993-4. PubMed DOI

Mauvezin C., Neufeld T.P. Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion. Autophagy. 2015;11:1437–1438. doi: 10.1080/15548627.2015.1066957. PubMed DOI PMC

Wu Y.C., Wu W.K.K., Li Y., Yu L., Li Z.J., Wong C.C.M., Li H.T., Sung J.J.Y., Cho C.H. Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells. Biochem. Biophys. Res. Commun. 2009;382:451–456. doi: 10.1016/j.bbrc.2009.03.051. PubMed DOI

Yan Y., Jiang K., Liu P., Zhang X., Dong X., Gao J., Liu Q., Barr M.P., Zhang Q., Hou X., et al. Bafilomycin A1 induces caspase-independent cell death in hepatocellular carcinoma cells via targeting of autophagy and MAPK pathways. Sci. Rep. 2016;6:37052. doi: 10.1038/srep37052. PubMed DOI PMC

Li F., Hu Y., Hu Y., Zhou R., Mao Z. Bafilomycin A1 Induces Caspase-Dependent Apoptosis and Inhibits Autophagy Flux in Diffuse Large B Cell Lymphoma. Med. Pharmacol. 2021:2021070520. doi: 10.20944/preprints202107.0520.v1. DOI

Hreniukh V., Bychkova S., Kulachkovsky O., Babsky A. Effect of bafilomycin and NAADP on membrane-associated ATPases and respiration of isolated mitochondria of the murine Nemeth-Kellner lymphoma: Mitochondria and ATPase activities in lymphoma. Cell Biochem. Funct. 2016;34:579–587. doi: 10.1002/cbf.3231. PubMed DOI

Bychkova S.V., Stasyshyn A.R., Bychkov M.A. The role of bafilomycin as a therapeutic agent in the modulation of endo-lysosomal store of rat hepatocytes. Med. Perspekt. 2022;27:22–26. doi: 10.26641/2307-0404.2022.3.265768. DOI

Morgan A.J. Ca2+ dialogue between acidic vesicles and ER. Biochem. Soc. Trans. 2016;44:546–553. doi: 10.1042/BST20150290. PubMed DOI

Faris P., Pellavio G., Ferulli F., Di Nezza F., Shekha M., Lim D., Maestri M., Guerra G., Ambrosone L., Pedrazzoli P., et al. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Induces Intracellular Ca2+ Release through the Two-Pore Channel TPC1 in Metastatic Colorectal Cancer Cells. Cancers. 2019;11:542. doi: 10.3390/cancers11040542. PubMed DOI PMC

Kushkevych I., Bychkov M., Bychkova S., Gajdács M., Merza R., Vítězová M. ATPase Activity of the Subcellular Fractions of Colorectal Cancer Samples under the Action of Nicotinic Acid Adenine Dinucleotide Phosphate. Biomedicines. 2021;9:1805. doi: 10.3390/biomedicines9121805. PubMed DOI PMC

Bychkova S.V., Chorna T.I. NAADP-sensitive Ca2+ stores in permeabilized rat hepatocytes. Ukr. Biochem. J. 2014;86:65–73. doi: 10.15407/ubj86.05.065. PubMed DOI

Chen J., Sitsel A., Benoy V., Sepúlveda M.R., Vangheluwe P. Primary Active Ca2+ Transport Systems in Health and Disease. Cold Spring Harb. Perspect. Biol. 2020;12:a035113. doi: 10.1101/cshperspect.a035113. PubMed DOI PMC

Peters A.A., Milevskiy M.J.G., Lee W.C., Curry M.C., Smart C.E., Saunus J.M., Reid L., da Silva L., Marcial D.L., Dray E., et al. The calcium pump plasma membrane Ca2+-ATPase 2 (PMCA2) regulates breast cancer cell proliferation and sensitivity to doxorubicin. Sci. Rep. 2016;6:25505. doi: 10.1038/srep25505. PubMed DOI PMC

Aung C.S., Ye W., Plowman G., Peters A.A., Monteith G.R., Roberts-Thomson S.J. Plasma membrane calcium ATPase 4 and the remodeling of calcium homeostasis in human colon cancer cells. Carcinogenesis. 2009;30:1962–1969. doi: 10.1093/carcin/bgp223. PubMed DOI

Aung C.S., Kruger W.A., Poronnik P., Roberts-Thomson S.J., Monteith G.R. Plasma membrane Ca2+-ATPase expression during colon cancer cell line differentiation. Biochem. Biophys. Res. Commun. 2007;355:932–936. doi: 10.1016/j.bbrc.2007.02.050. PubMed DOI

Bychkova S., Hreniuh V. Activity of ATPases in postmitichondtial fraction of lymphoma NK/Ly cells under bafilomicine and NAADP presence. Biol. Stud. 2015;9:31–38. doi: 10.30970/sbi.0902.413. DOI

Papp B., Launay S., Gélébart P., Arbabian A., Enyedi A., Brouland J.-P., Carosella E.D., Adle-Biassette H. Endoplasmic Reticulum Calcium Pumps and Tumor Cell Differentiation. Int. J. Mol. Sci. 2020;21:3351. doi: 10.3390/ijms21093351. PubMed DOI PMC

Chung F.-Y., Lin S.-R., Lu C.-Y., Yeh C.-S., Chen F.-M., Hsieh J.-S., Huang T.-J., Wang J.-Y. Sarco/Endoplasmic Reticulum Calcium-ATPase 2 Expression as a Tumor Marker in Colorectal Cancer. Am. J. Surg. Pathol. 2006;30:969–974. doi: 10.1097/00000478-200608000-00006. PubMed DOI

Da Silva C.I., Gonçalves-de-Albuquerque C.F., de Moraes B.P.T., Garcia D.G., Burth P. Na/K-ATPase: Their role in cell adhesion and migration in cancer. Biochimie. 2021;185:1–8. doi: 10.1016/j.biochi.2021.03.002. PubMed DOI

Song Y., Lee S.-Y., Kim S., Choi I., Kim S.-H., Shum D., Heo J., Kim A.-R., Kim K.M., Seo H.R. Inhibitors of Na+/K+ ATPase exhibit antitumor effects on multicellular tumor spheroids of hepatocellular carcinoma. Sci. Rep. 2020;10:5318. doi: 10.1038/s41598-020-62134-4. PubMed DOI PMC

Zhang F., Xia M., Li P.-L. Lysosome-dependent Ca2+ release response to Fas activation in coronary arterial myocytes through NAADP: Evidence from CD38 gene knockouts. Am. J. Physiol. Cell Physiol. 2010;298:C1209–C1216. doi: 10.1152/ajpcell.00533.2009. PubMed DOI PMC

Chemaly E.R., Troncone L., Lebeche D. SERCA control of cell death and survival. Cell Calcium. 2018;69:46–61. doi: 10.1016/j.ceca.2017.07.001. PubMed DOI PMC

Rüschoff J.H., Brandenburger T., Strehler E.E., Filoteo A.G., Heinmöller E., Aumüller G., Wilhelm B. Plasma Membrane Calcium ATPase Expression in Human Colon Multistep Carcinogenesis. Cancer Investig. 2012;30:251–257. doi: 10.3109/07357907.2012.657817. PubMed DOI

Rüschoff J., Hanna W., Bilous M., Hofmann M., Osamura R.Y., Penault-Llorca F., van de Vijver M., Viale G. HER2 testing in gastric cancer: A practical approach. Mod. Pathol. 2012;25:637–650. doi: 10.1038/modpathol.2011.198. PubMed DOI

Brouland J.-P., Gélébart P., Kovàcs T., Enouf J., Grossmann J., Papp B. The Loss of Sarco/Endoplasmic Reticulum Calcium Transport ATPase 3 Expression Is an Early Event during the Multistep Process of Colon Carcinogenesis. Am. J. Pathol. 2005;167:233–242. doi: 10.1016/S0002-9440(10)62968-9. PubMed DOI PMC

Ribiczey P., Tordai A., Andrikovics H., Filoteo A.G., Penniston J.T., Enouf J., Enyedi Á., Papp B., Kovács T. Isoform-specific up-regulation of plasma membrane Ca2+ ATPase expression during colon and gastric cancer cell differentiation. Cell Calcium. 2007;42:590–605. doi: 10.1016/j.ceca.2007.02.003. PubMed DOI PMC

Varga K., Hollósi A., Pászty K., Hegedűs L., Szakács G., Tímár J., Papp B., Enyedi Á., Padányi R. Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells. BMC Cancer. 2018;18:1029. doi: 10.1186/s12885-018-4945-x. PubMed DOI PMC

Faris P., Casali C., Negri S., Iengo L., Biggiogera M., Maione A.S., Moccia F. Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells. Front. Cell Dev. Biol. 2022;10:874043. doi: 10.3389/fcell.2022.874043. PubMed DOI PMC

Fameli N., Ogunbayo O.A., van Breemen C., Evans A.M. Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling. F1000Research. 2014;3:93. doi: 10.12688/f1000research.3720.1. PubMed DOI PMC

Ronco V., Potenza D.M., Denti F., Vullo S., Gagliano G., Tognolina M., Guerra G., Pinton P., Genazzani A.A., Mapelli L., et al. A novel Ca2+-mediated cross-talk between endoplasmic reticulum and acidic organelles: Implications for NAADP-dependent Ca2+ signalling. Cell Calcium. 2015;57:89–100. doi: 10.1016/j.ceca.2015.01.001. PubMed DOI

Yoshida H., Shimamoto C., Ito S., Daikoku E., Nakahari T. HCO3−-dependent transient acidification induced by ionomycin in rat submandibular acinar cells. J. Physiol. Sci. 2010;60:273–282. doi: 10.1007/s12576-010-0095-x. PubMed DOI PMC

Hwang S.-M., Lee J.Y., Park C.-K., Kim Y.H. The Role of TRP Channels and PMCA in Brain Disorders: Intracellular Calcium and pH Homeostasis. Front. Cell Dev. Biol. 2021;9:584388. doi: 10.3389/fcell.2021.584388. PubMed DOI PMC

Kushkevych I., Fafula R., Parák T., Bartoš M. Activity of Na+/K+-activated Mg2+-dependent ATP-hydrolase in the cell-free extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9. Acta Vet. Brno. 2015;84:3–12. doi: 10.2754/avb201585010003. DOI

Kosterin S.O., Veklich T.O., Pryluts’kyĭ I.I., Borysko P.O. Kinetic interpretation of the original pH-dependence of enzymatic activity of “basal” Mg(2+)-ATPase of the smooth muscle sarcolemma. Ukr. Biokhim. Zh. (1999) 2005;77:37–45. PubMed

Sakai H., Suzuki T., Maeda M., Takahashi Y., Horikawa N., Minamimura T., Tsukada K., Takeguchi N. Up-regulation of Na+, K+-ATPase α3-isoform and down-regulation of the α1-isoform in human colorectal cancer. FEBS Lett. 2004;563:151–154. doi: 10.1016/S0014-5793(04)00292-3. PubMed DOI

Khajah M.A., Mathew P.M., Luqmani Y.A. Na+/K+ ATPase activity promotes invasion of endocrine resistant breast cancer cells. PLoS ONE. 2018;13:e0193779. doi: 10.1371/journal.pone.0193779. PubMed DOI PMC

Nakamura K., Shiozaki A., Kosuga T., Shimizu H., Kudou M., Ohashi T., Arita T., Konishi H., Komatsu S., Kubota T., et al. The expression of the alpha1 subunit of Na+/K+-ATPase is related to tumor development and clinical outcomes in gastric cancer. Gastric Cancer. 2021;24:1278–1292. doi: 10.1007/s10120-021-01212-6. PubMed DOI

Wu D., Yu H.-Q., Xiong H.-J., Zhang Y.-J., Lin X.-T., Zhang J., Wu W., Wang T., Liu X.-Y., Xie C.-M. Elevated Sodium Pump α3 Subunit Expression Promotes Colorectal Liver Metastasis via the p53-PTEN/IGFBP3-AKT-mTOR Axis. Front. Oncol. 2021;11:743824. doi: 10.3389/fonc.2021.743824. PubMed DOI PMC

Brown A.M., Lew V.L. The effect of intracellular calcium on the sodium pump of human red cells. J. Physiol. 1983;343:455–493. doi: 10.1113/jphysiol.1983.sp014904. PubMed DOI PMC

Arai K., Shimaya A., Hiratani N., Ohkuma S. Purification and characterization of lysosomal H(+)-ATPase. An anion-sensitive v-type H(+)-ATPase from rat liver lysosomes. J. Biol. Chem. 1993;268:5649–5660. doi: 10.1016/S0021-9258(18)53369-X. PubMed DOI

Bychkova S. Influence of NAADP and bafilomycine A1 on activity of ATPase in liver postmitochondrial fraction. Biol. Stud. 2015;9:31–40. doi: 10.30970/sbi.0903.445. DOI

Hohmann J., Kowalewski H., Vogel M., Zimmerman H. Isolation of a Ca2+ or Mg2+-activated ATPase (ecto-ATPase) from bovine brain synaptic membranes. Biochim. Biophys. Acta BBA Biomembr. 1993;1152:146–154. doi: 10.1016/0005-2736(93)90241-Q. PubMed DOI

Konno Y., Kato K., Dairaku N., Koike T., Iijima K., Imatani A., Sekine H., Ohara S., Shimosegawa T. Expression of Mg2+-dependent, HCO3−-stimulated adenine triphosphatase in the human duodenum. J. Gastroenterol. Hepatol. 2004;19:18–23. doi: 10.1111/j.1440-1746.2004.03175.x. PubMed DOI

Wang H., Gilles-Baillien M. Ca2+-ATPase and Mg2+-ATPase activities distinct from alkaline phosphatase in rat jejunal brush-border membranes. Arch. Int. Physiol. Biochim. Biophys. 1993;101:387–393. doi: 10.3109/13813459309046997. PubMed DOI

Jacob J., Chandran D., Sasidharan R., Kuruvila L., Madhusudan U.K., Rao N.L., Banerjee D. Chlortetracycline, a fluorescent probe for pH of calcium stores in cells. Curr. Sci. 2003;2003:671–674.

Colacurcio D.J., Nixon R.A. Disorders of lysosomal acidification—The emerging role of v-ATPase in aging and neurodegenerative disease. Ageing Res. Rev. 2016;32:75–88. doi: 10.1016/j.arr.2016.05.004. PubMed DOI PMC

Kim H.-K., Lee G.-H., Bhattarai K.R., Lee M.-S., Back S.H., Kim H.-R., Chae H.-J. TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium. Autophagy. 2021;17:761–778. doi: 10.1080/15548627.2020.1732161. PubMed DOI PMC

Fitzpatrick J.S., Hagenston A.M., Hertle D.N., Gipson K.E., Bertetto-D’Angelo L., Yeckel M.F. Inositol-1,4,5-trisphosphate receptor-mediated Ca2+ waves in pyramidal neuron dendrites propagate through hot spots and cold spots: Ca2+ waves propagate through hot and cold spots. J. Physiol. 2009;587:1439–1459. doi: 10.1113/jphysiol.2009.168930. PubMed DOI PMC

Giacomello M., Drago I., Bortolozzi M., Scorzeto M., Gianelle A., Pizzo P., Pozzan T. Ca2+ Hot Spots on the Mitochondrial Surface Are Generated by Ca2+ Mobilization from Stores, but Not by Activation of Store-Operated Ca2+ Channels. Mol. Cell. 2010;38:280–290. doi: 10.1016/j.molcel.2010.04.003. PubMed DOI

Tepikin A.V. Mitochondrial junctions with cellular organelles: Ca2+ signalling perspective. Pflügers Arch. Eur. J. Physiol. 2018;470:1181–1192. doi: 10.1007/s00424-018-2179-z. PubMed DOI PMC

Hulsurkar M.M., Lahiri S.K., Karch J., Wang M.C., Wehrens X.H.T. Targeting calcium-mediated inter-organellar crosstalk in cardiac diseases. Expert Opin. Ther. Targets. 2022;26:303–317. doi: 10.1080/14728222.2022.2067479. PubMed DOI PMC

Calcraft P.J., Ruas M., Pan Z., Cheng X., Arredouani A., Hao X., Tang J., Rietdorf K., Teboul L., Chuang K.-T., et al. NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature. 2009;459:596–600. doi: 10.1038/nature08030. PubMed DOI PMC

Mándi M., Tóth B., Timár G., Bak J. Ca2+ release triggered by NAADP in hepatocyte microsomes. Biochem. J. 2006;395:233–238. doi: 10.1042/BJ20051002. PubMed DOI PMC

Blaauboer B.J., Boobis A.R., Castell J.V., Coecke S., Groothuis G.M.M., Guillouzo A., Hall T.J., Hawksworth G.M., Lorenzon G., Miltenburger H.G., et al. The Practical Applicability of Hepatocyte Cultures in Routine Testing: The Report and Recommendations of ECVAM Workshop 1. Altern. Lab. Anim. 1994;22:231–241. doi: 10.1177/026119299402200404. DOI

Ferents I.M., Bychkova S.V., Bychkov M.A. Peculiarities of the effects of bile acids on atpase activity of the colon mucosa in patients with overweight and irritable bowel syndrome. Wiad. Lek. 2020;73:574–577. doi: 10.36740/WLek202003133. PubMed DOI

Bailey N.T.J. Statistical Methods in Biology. 3rd ed. Volume 75. Cambridge University Press; Cambridge, UK: 1995. p. 515.255p

The impact of 3-sulfo-taurolithocholic acid on ATPase activity in patients' colorectal cancer and normal colon tissues, and its hepatic effects in rodents