An acidic environment and hypoxia within the tumour are hallmarks of cancer that contribute to cell resistance to therapy. Deregulation of the PI3K/Akt pathway is common in colon cancer. Numerous Akt-targeted therapies are being developed, the activity of Akt-inhibitors is, however, strongly pH-dependent. Combination therapy thus represents an opportunity to increase their efficacy. In this study, the cytotoxicity of the Akt inhibitor perifosine and the Bcl-2/Bcl-xL inhibitor ABT-737 was tested in colon cancer HT-29 and HCT-116 cells cultured in monolayer or in the form of spheroids. The efficacy of single drugs and their combination was analysed in different tumour-specific environments including acidosis and hypoxia using a series of viability assays. Changes in protein content and distribution were determined by immunoblotting and a "peeling analysis" of immunohistochemical signals. While the cytotoxicity of single agents was influenced by the tumour-specific microenvironment, perifosine and ABT-737 in combination synergistically induced apoptosis in cells cultured in both 2D and 3D independently on pH and oxygen level. Thus, the combined therapy of perifosine and ABT-737 could be considered as a potential treatment strategy for colon cancer.

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

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

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Sung H, Ferlay J, Siegel RL, 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(3):209‐249. doi:10.3322/caac.21660 PubMed DOI

Jensen KH, Izarzugaza JMG, Juncker AS, et al. Analysis of a gene panel for targeted sequencing of colorectal cancer samples. Oncotarget. 2018;9(10):9043‐9060. doi:10.18632/oncotarget.24138 PubMed DOI PMC

Molinari C, Marisi G, Passardi A, Matteucci L, De Maio G, Ulivi P. Heterogeneity in colorectal cancer: a challenge for personalized medicine? Int J Mol Sci. 2018;19(12):3733. doi:10.3390/ijms19123733 PubMed DOI PMC

Danielsen SA, Eide PW, Nesbakken A, Guren T, Leithe E, Lothe RA. Portrait of the PI3K/AKT pathway in colorectal cancer. Biochim Biophys Acta. 2015;1855(1):104‐121. doi:10.1016/j.bbcan.2014.09.008 PubMed DOI

Malinowsky K, Nitsche U, Janssen KP, et al. Activation of the PI3K/AKT pathway correlates with prognosis in stage II colon cancer. Br J Cancer. 2014;110(8):2081‐2089. doi:10.1038/bjc.2014.100 PubMed DOI PMC

Ebrahimi S, Hosseini M, Shahidsales S, et al. Targeting the Akt/PI3K signaling pathway as a potential therapeutic strategy for the treatment of pancreatic cancer. Curr Med Chem. 2017;24(13):1321‐1331. doi:10.2174/0929867324666170206142658 PubMed DOI

Richardson PG, Eng C, Kolesar J, Hideshima T, Anderson KC. Perifosin, an oral, anti‐cancer agent and inhibitor of the Akt pathway: mechanistic actions, pharmacodynamics, pharmacokinetics, and clinical activity. Expert Opin Drug Metab Toxicol. 2012;8(5):623‐633. doi:10.1517/17425255.2012.681376 PubMed DOI PMC

Kondapaka SB, Singh SS, Dasmahapatra GP, Sausville EA, Roy KK. Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation. Mol Cancer Ther. 2003;2(11):1093‐1103. PubMed

Barnes EME, Xu Y, Benito A, et al. Lactic acidosis induces resistance to the pan‐Akt inhibitor uprosertib in colon cancer cells. Br J Cancer. 2020;122(9):1298‐1308. doi:10.1038/s41416-020-0777-y PubMed DOI PMC

Pavlatovská B, Machálková M, Brisudová P, et al. Lactic acidosis interferes with toxicity of Perifosine to colorectal cancer spheroids: multimodal imaging analysis. Front Oncol. 2020;10:1‐18. doi:10.3389/fonc.2020.581365 PubMed DOI PMC

Wojtkowiak JW, Verduzco D, Schramm KJ, Gillies RJ. Drug resistance and cellular adaptation to tumor acidic pH microenvironment. Mol Pharm. 2011;8(6):2032‐2038. doi:10.1021/mp200292c PubMed DOI PMC

Warren CFA, Wong‐Brown MW, Bowden NA. BCL‐2 family isoforms in apoptosis and cancer. Cell Death Dis. 2019;10(3):177. doi:10.1038/s41419-019-1407-6 PubMed DOI PMC

Hikita H, Takehara T, Shimizu S, et al. The Bcl‐xL inhibitor, ABT‐737, efficiently induces apoptosis and suppresses growth of hepatoma cells in combination with sorafenib. Hepatology. 2010;52(4):1310‐1321. doi:10.1002/hep.23836 PubMed DOI

Ryder C, McColls K, Zhongs F, Distelhorst CW. Acidosis promotes Bcl‐2 family‐mediated evasion of apoptosis: involvement of acid‐sensing G protein‐coupled receptor GPR65 signaling to MEK/ERK. J Biol Chem. 2012;287(33):27863‐27875. doi:10.1074/jbc.M112.384685 PubMed DOI PMC

Shamas‐Din A, Brahmbhatt H, Leber B, Andrews DW. BH3‐only proteins: orchestrators of apoptosis. Biochim Biophys Acta Mol Cell Res. 2011;1813(4):508‐520. doi:10.1016/j.bbamcr.2010.11.024 PubMed DOI

Flacke JP, Kumar S, Kostin S, Reusch HP, Ladilov Y. Acidic preconditioning protects endothelial cells against apoptosis through p38‐ and Akt‐dependent Bcl‐xL overexpression. Apoptosis. 2009;14(1):90‐96. doi:10.1007/s10495-008-0287-5 PubMed DOI PMC

Lopez JS, Banerji U. Combine and conquer: challenges for targeted therapy combinations in early phase trials. Nat Rev Clin Oncol. 2017;14(1):57‐66. doi:10.1038/nrclinonc.2016.96 PubMed DOI PMC

Avan A, Narayan R, Giovannetti E, Peters GJ. Role of Akt signaling in resistance to DNA‐targeted therapy. World J Clin Oncol. 2016;7(5):352‐369. doi:10.5306/wjco.v7.i5.352 PubMed DOI PMC

Konopleva M, Contractor R, Tsao T, et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT‐737 in acute myeloid leukemia. Cancer Cell. 2006;10(5):375‐388. doi:10.1016/j.ccr.2006.10.006 PubMed DOI

Tahir SK, Yang X, Anderson MG, et al. Influence of Bcl‐2 family members on the cellular response of small‐cell lung cancer cell lines to ABT‐737. Cancer Res. 2007;3:1176‐1184. doi:10.1158/0008-5472.CAN-06-2203 PubMed DOI

Tagscherer KE, Fassl A, Campos B, et al. Apoptosis‐based treatment of glioblastomas with ABT‐737, a novel small molecule inhibitor of Bcl‐2 family proteins. Oncogene. 2008;27(52):6646‐6656. doi:10.1038/onc.2008.259 PubMed DOI

Harrison LRE, Micha D, Brandenburg M, et al. Hypoxic human cancer cells are sensitized to BH‐3 mimetic‐induced apoptosis via downregulation of the Bcl‐2 protein Mcl‐1. J Clin Invest. 2011;121(3):1075‐1087. doi:10.1172/JCI43505 PubMed DOI PMC

Klymenko T, Brandenburg M, Morrow C, Dive C, Makin G. The novel Bcl‐2 inhibitor ABT‐737 is more effective in hypoxia and is able to reverse hypoxia‐induced drug resistance in neuroblastoma cells. Mol Cancer Ther. 2011;10(12):2373‐2383. doi:10.1158/1535-7163.MCT-11-0326 PubMed DOI PMC

Rosko AE, McColl KS, Zhong F, et al. Acidosis sensing receptor GPR65 correlates with anti‐apoptotic Bcl‐2 family member expression in CLL cells: potential implications for the CLL microenvironment. J Leuk (Los Angel). 2014;2(5):160. doi:10.4172/2329-6917.1000160 PubMed DOI PMC

Zhang F, Yu X, Liu X, et al. ABT‐737 potentiates cisplatin‐induced apoptosis in human osteosarcoma cells via the mitochondrial apoptotic pathway. Oncol Rep. 2017;38(4):2301‐2308. doi:10.3892/or.2017.5909 PubMed DOI

Mason KD, Khaw SL, Rayeroux KC, et al. The BH3 mimetic compound, ABT‐737, synergizes with a range of cytotoxic chemotherapy agents in chronic lymphocytic leukemia. Leukemia. 2009;23(11):2034‐2041. doi:10.1038/leu.2009.151 PubMed DOI

Gills JJ, Dennis PA. Perifosine: update on a novel Akt inhibitor. Curr Oncol Rep. 2009;11(2):102‐110. doi:10.1007/s11912-009-0016-4 PubMed DOI PMC

Zhang J, Hong Y, Shen J. Combination treatment with perifosine and MEK‐162 demonstrates synergism against lung cancer cells in vitro and in vivo. Tumour Biol. 2015;36(7):5699‐5706. doi:10.1007/s13277-015-3244-2 PubMed DOI

Locatelli SL, Giacomini A, Guidetti A, et al. Perifosine and sorafenib combination induces mitochondrial cell death and antitumor effects in NOD / SCID mice with Hodgkin lymphoma cell line xenografts. Leukemia. 2013;27(8):1677‐1687. doi:10.1038/leu.2013.28 PubMed DOI

Souza AG, Silva IBB, Campos‐Fernandez E, et al. Comparative assay of 2D and 3D cell culture models: proliferation, gene expression and anticancer drug response. Curr Pharm Des. 2018;24(15):1689‐1694. doi:10.2174/1381612824666180404152304 PubMed DOI

Kapałczyńska M, Kolenda T, Przybyła W, et al. 2D and 3D cell cultures – a comparison of different types of cancer cell cultures. Arch Med Sci. 2018;14(4):910‐919. doi:10.5114/aoms.2016.63743 PubMed DOI PMC

Machálková M, Pavlatovská B, Michálek J, et al. Drug penetration analysis in 3D cell cultures using fiducial‐based semiautomatic Coregistration of MALDI MSI and immunofluorescence images. Anal Chem. 2019;91(21):13475‐13484. doi:10.1021/acs.analchem.9b02462 PubMed DOI

Nunes AS, Barros AS, Costa EC, Moreira AF, Correia IJ. 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs. Biotechnol Bioeng. 2019;116(1):206‐226. doi:10.1002/bit.26845 PubMed DOI

Navrátilová J, Karasová M, Kohutková Lánová M, et al. Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate. J Cell Mol Med. 2017;21(9):1859‐1869. doi:10.1111/jcmm.13106 PubMed DOI PMC

Navrátilová J, Hankeová T, Beneš P, Šmarda J. Acidic pH of tumor microenvironment enhances cytotoxicity of the disulfiram/Cu2+ complex to breast and colon cancer cells. Chemotherapy. 2013;59(2):112‐120. doi:10.1159/000353915 PubMed DOI

Michálek J, Štěpka K, Kozubek M, et al. Quantitative assessment of anti‐cancer drug efficacy from Coregistered mass spectrometry and fluorescence microscopy images of multicellular tumor spheroids. Microsc Microanal. 2019;25(6):1311‐1322. doi:10.1017/S1431927619014983 PubMed DOI

Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006;58(3):621‐681. doi:10.1124/pr.58.3.10 PubMed DOI

Feng LX, Li M, Liu YJ, Yang SM, Zhang N. Synergistic enhancement of cancer therapy using a combination of ceramide and docetaxel. Int J Mol Sci. 2014;15(3):4201‐4220. doi:10.3390/ijms15034201 PubMed DOI PMC

Rogakou EP, Nieves‐Neira W, Boon C, Pommier Y, Bonner WM. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J Biol Chem. 2000;275(13):9390‐9395. doi:10.1074/jbc.275.13.9390 PubMed DOI

Vaupel P, Thews O, Hoeckel M. Treatment resistance of solid tumors: role of hypoxia and anemia. Med Oncol. 2001;18(4):243‐259. doi:10.1385/MO:18:4:243 PubMed DOI

Wang G, Wang JJ, Yin PH, et al. New strategies for targeting glucose metabolism–mediated acidosis for colorectal cancer therapy. J Cell Physiol. 2018;234(1):348‐368. doi:10.1002/jcp.26917 PubMed DOI

Shen J, Xu L, Zhao Q. Perifosine and ABT‐737 synergistically inhibit lung cancer cells in vitro and in vivo. Biochem Biophys Res Commun. 2016;473(4):1170‐1176. doi:10.1016/j.bbrc.2016.04.035 PubMed DOI

Chen JLY, Lucas JE, Schroeder T, et al. The genomic analysis of lactic acidosis and acidosis response in human cancers. PLoS Genet. 2008;4(12):e1000293. doi:10.1371/journal.pgen.1000293 PubMed DOI PMC

Opydo‐Chanek M, Rak A, Cierniak A, Mazur L. Combination of ABT‐737 and resveratrol enhances DNA damage and apoptosis in human T‐cell acute lymphoblastic leukemia MOLT‐4 cells. Toxicol In Vitro. 2017;42:38‐46. doi:10.1016/j.tiv.2017.03.013 PubMed DOI

Howard AN, Bridges KA, Meyn RE, Chandra J. ABT‐737, a BH3 mimetic, induces glutathione depletion and oxidative stress. Cancer Chemother Pharmacol. 2009;65(1):41‐54. doi:10.1007/s00280-009-1001-1 PubMed DOI

Song JH, Kandasamy K, Zemskova M, Lin YW, Kraft AS. The BH3 mimetic ABT‐737 induces cancer cell senescence. Cancer Res. 2011;71(2):506‐515. doi:10.1158/0008-5472.CAN-10-1977 PubMed DOI PMC

Ichim G, Lopez J, Murphy DJ, et al. Limited mitochondrial Permeabilization causes DNA damage and genomic instability in the absence of cell death. Mol Cell. 2015;57(5):860‐872. doi:10.1016/j.molcel.2015.01.018 PubMed DOI PMC

Liu Q, Turner KM, Yung WKA, Chen K, Zhang W. Role of AKT signaling in DNA repair and clinical response to cancer therapy. Neuro Oncol. 2014;16(10):1313‐1323. doi:10.1093/neuonc/nou058 PubMed DOI PMC

Song Z, Tu X, Zhou Q, et al. A novel UCHL3 inhibitor, perifosine, enhances PARP inhibitor cytotoxicity through inhibition of homologous recombination‐mediated DNA double strand break repair. Cell Death Dis. 2019;10(6):398. doi:10.1038/s41419-019-1628-8 PubMed DOI PMC

Bindra RS, Schaffer PJ, Meng A, et al. Down‐regulation of Rad51 and decreased homologous recombination in hypoxic cancer cells. Mol Cell Biol. 2004;24(19):8504‐8518. doi:10.1128/mcb.24.19.8504-8518.2004 PubMed DOI PMC

Edmondson R, Broglie JJ, Adcock AF, Yang L. Three‐dimensional cell culture systems and their applications in drug discovery and cell‐based biosensors. Assay Drug Dev Technol. 2014;12(4):207‐218. doi:10.1089/adt.2014.573 PubMed DOI PMC

Fu F, Nowak MA, Bonhoeffer S. Spatial heterogeneity in drug concentrations can facilitate the emergence of resistance to cancer therapy. PLoS Comput Biol. 2015;11(3):e1004142. doi:10.1371/journal.pcbi.1004142 PubMed DOI PMC

Moreno‐Gamez S, Hill AL, Rosenbloom DI, Petrov DA, Nowak MA, Pennings PS. Imperfect drug penetration leads to spatial monotherapy and rapid evolution of multidrug resistance. Proc Natl Acad Sci U S A. 2015;112(22):E2874‐E2883. doi:10.1073/pnas.1424184112 PubMed DOI PMC

Dheda K, Lenders L, Magombedze G, et al. Drug‐penetration gradients associated with acquired drug resistance in patients with tuberculosis. Am J Respir Crit Care Med. 2018;198(9):1208‐1219. doi:10.1164/rccm.201711-2333OC PubMed DOI PMC