Pharmacological Double Inhibition of Glycolysis in glioblastoma Multiforme Cells Maximizes Cancer Cell Killing: A Synergism between Citrate and 3-bromopyruvate

Faten M. Omran¹, Hussam Baghdadi², Tamer M. Soliman³, Osama Alhadramy⁴, Momen El-shazley^{5, 6}, Samer A. El-Sawy⁷, Hytham Mahmoud Abdel-Latif¹, Wafaa A. Abdellah¹, Azza Mahmoud Ahmed Abouelella¹, Amr El-Dardear⁸, Mohamed Abdel-haleem⁹, Elhussainy MA Elhussainy¹⁰, Hassan El-Alaf¹¹, Nassar Ayoub¹², Sayed Mostafa El Sayed^{13, 14}, Hesham I. Abdallah^{13, 14}, Salah Mohamed El Sayed^{2, 7,} , Mongi Ayat¹⁵ and Ahmed Mohamed Shehata^{16, 17}

¹Department of Medical Pharmacology, Sohag Faculty of Medicine, Sohag University, Egypt

²Department of Clinical Biochemistry and Molecular Medicine, Taibah Faculty of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia

³Department of Clinical Pathology, Sohag Faculty of Medicine, Sohag University, Egypt

⁴Division of Cardiology, Department of Medicine, Taibah College of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia

⁵Department of Medicine, Taibah College of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia

⁶Department of Occupational Diseases and Toxigenomics, Sohag Faculty of Medicine, Sohag University, Egypt

⁷Department of Medical Biochemistry, Sohag Faculty of Medicine, Sohag University, Egypt

⁸Department of Pediatrics, Taibah Faculty of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia

⁹Department of Ear, Nose and Throat, Taibah Faculty of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia

¹⁰Department of Medical Physiology, Kafr Elshekh Faculty of Medicine, Kafr Elsheikh University, Egypt

¹¹Department of Medical Physiology, Sohag Faculty of Medicine, Sohag University, Egypt

¹²Anatomy and Embryology Department, Faculty of Medicine, Sohag University, Egypt

¹³Anatomy and Embryology Department, Faculty of Medicine, Ain Shams University, Egypt

¹⁴4Anatomy and Embryology Department, Faculty of Medicine, Taibah University, Saudi Arabia

¹⁵Division of Hematology, Taibah College of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia

¹⁶Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia

¹⁷7Department of Pharmacology and toxicology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt

Cite this paper:
Faten M. Omran, Hussam Baghdadi, Tamer M. Soliman, Osama Alhadramy, Momen El-shazley, Samer A. El-Sawy, Hytham Mahmoud Abdel-Latif, Wafaa A. Abdellah, Azza Mahmoud Ahmed Abouelella, Amr El-Dardear, Mohamed Abdel-haleem, Elhussainy MA Elhussainy, Hassan El-Alaf, Nassar Ayoub, Sayed Mostafa El Sayed, Hesham I. Abdallah, Salah Mohamed El Sayed, Mongi Ayat and Ahmed Mohamed Shehata. Pharmacological Double Inhibition of Glycolysis in glioblastoma Multiforme Cells Maximizes Cancer Cell Killing: A Synergism between Citrate and 3-bromopyruvate. American Journal of Pharmacological Sciences. 2020; 8(1):1-6. doi: 10.12691/ajps-8-1-1

Abstract

Glioblastoma multiform (GBM) tumors are the most aggressive and diffusely infiltrating brain tumors in human patients. Despite surgery, modern chemotherapy and anti-angiogenesis treatments, GBM tumors regrow rapidly and end patients’ lives in a relatively short time. GBM tumors aggressively invade and infiltrate adjacent normal brain tissues. GBM cells are driven by glycolysis (glucose oxidation to produce ATP and lactate in cancer cells). In this study, we cultured rat C6 GBM cells and human GBM cells (U373MG) to be treated using glycolysis inhibitors. Citrate is a natural product enormously available in citrus fruits and has many pharmacological uses e.g. to treat urate renal stones. Citric acid was recently reported to exert cardioprotective effects on myocardial ischemia/reperfusion injury. Serial doses of citrate (glycolytic inhibitor of phosphofructokinase) were lethal to human GBM cell lines at relatively high doses (in millimolar range). Using the combination drug index, low effective doses of citrate (1, 3 and 5 mM) exerted a synergistic anticancer effect with low effective doses of 3-bromopyruvate (15 and 25 µM). Combination index was < 1 and denoted strong synergism between citrate and 3-bromopyruvate. As 3-bromopyruvate is another glycolysis inhibitor (hexokinase II inhibitor) in addition to citrate itself, double inhibition of glycolysis was evident when combining both citrate and 3-bromopyruvate. Both drugs benefited from tumor biology as citrate is acidic in solution and does better in acidic tumor microenvironment and same thing applies to 3-bromopyruvate that is also acidic in solution. In addition, 3-bromopyruvate is a structural analog and competitive antagonist of lactate (The Warburg effect) to deprive cancer cells of vital benefits of lactate. Lactate-based benefits to cancer cells include enhancing angiogenesis, metastasis, invasion, proliferation, migration, chemoresistance, radioresistance and acidic tumor microenvironment. Our data confirmed that pharmacological glycolysis double inhibition significantly, maximally and synergistically distorted GBM cells morphology and reduced cellular viability. This may carry a lot of hope for treating the dismal outcome in those patients.

Keywords:
Glycolysis double inhibition hexokinase pharmacological antagonist phosphofructokinase glioblastoma viability

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/

References:

[1]	Piao Y, Lu L, de Groot J. AMPA receptors promote perivascular glioma invasion via beta1 integrin-dependent adhesion to the extracellular matrix. Neuro Oncol. 2009 Jun; 11(3): 260-73.
[2]	Kurhanewicz J, Dahiya R, Macdonald JM, Chang LH, James TL, Narayan P. Citrate alterations in primary and metastatic human prostatic adenocarcinomas: 1H magnetic resonance spectroscopy and biochemical study. Magn Reson Med. 1993 Feb; 29(2): 149-57.
[3]	Costello LC, Franklin RB, Narayan P. Citrate in the diagnosis of prostate cancer. Prostate. 1999 Feb 15; 38(3): 237-45.
[4]	Kline EE, Treat EG, Averna TA, Davis MS, Smith AY, Sillerud LO. Citrate concentrations in human seminal fluid and expressed prostatic fluid determined via 1H nuclear magnetic resonance spectroscopy outperform prostate specific antigen in prostate cancer detection. J Urol. 2006 Nov; 176(5): 2274-9.
[5]	Seymour ZA, Panigrahy A, Finlay JL, Nelson MD Jr, Blüml S. Citrate in pediatric CNS tumors? AJNR Am J Neuroradiol. 2008 May; 29(5): 1006-11.
[6]	Roelcke U, Leenders KL, von Ammon K, Radü EW, Vontobel P, Günther I, Psylla M. Brain tumor iron uptake measured with positron emission tomography and 52Fe-citrate. J Neurooncol. 1996 Aug; 29(2): 157-65.
[7]	Yousefi S, Owens JW, Cesario TC. Citrate shows specific, dose-dependent lympholytic activity in neoplastic cell lines. Leuk Lymphoma. 2004 Aug; 45(8): 1657-65.
[8]	Islam MR, Samadi AR, Ahmed SM, Bardhan PK, Ali A.Oral rehydration therapy: efficacy of sodium citrate equals to sodium bicarbonate for correction of acidosis in diarrhoea. Gut. 1984 Aug; 25(8): 900-4.
[9]	Byer K, Khan SR. Citrate provides protection against oxalate and calcium oxalate crystal induced oxidative damage to renal epithelium. J Urol. 2005 Feb; 173(2): 640-6.
[10]	Seltzer MA, Low RK, McDonald M, Shami GS, Stoller ML. Dietary manipulation with lemonade to treat hypocitraturic calcium nephrolithiasis. J Urol. 1996 Sep; 156(3): 907-9.
[11]	Pak CY, Peterson R. Successful treatment of hyperuricosuric calcium oxalate nephrolithiasis with potassium citrate. Arch Intern Med. 1986 May; 146(5): 863-7.
[12]	El Sayed SM, Abou El-Magd RM, Shishido Y, Chung SP, Sakai T, Watanabe H, Kagami S, Fukui K. D-amino acid oxidase gene therapy sensitizes glioma cells to the antiglycolytic effect of 3-bromopyruvate. Cancer Gene Ther. 2012 Jan; 19(1): 1-18.
[13]	El Sayed SM, El-Magd RM, Shishido Y, Chung SP, Diem TH, Sakai T, Watanabe H, Kagami S, Fukui K. 3-Bromopyruvate antagonizes effects of lactate and pyruvate, synergizes with citrate and exerts novel anti-glioma effects. J Bioenerg Biomembr. 2012 Feb; 44(1): 61-79.
[14]	El Sayed SM. Enhancing anticancer effects, decreasing risks and solving practical problems facing 3-bromopyruvate in clinical oncology: 10 years of research experience. Int J Nanomedicine. 2018 Aug 15; 13: 4699-4709.
[15]	El Sayed SM, El-Magd RM, Shishido Y, Yorita K, Chung SP, Tran DH, Sakai T, Watanabe H, Kagami S, Fukui K. D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects. J Bioenerg Biomembr. 2012 Oct; 44(5): 513-23.
[16]	El Sayed SM, Mahmoud AA, El Sawy SA, Abdelaal EA, Fouad AM, Yousif RS, Hashim MS, Hemdan SB, Kadry ZM, Abdelmoaty MA, Gabr AG, Omran FM, Nabo MM, Ahmed NS. Warburg effect increases steady-state ROS condition in cancer cells through decreasing their antioxidant capacities (anticancer effects of 3-bromopyruvate through antagonizing Warburg effect). Med Hypotheses. 2013 Nov; 81(5): 866-70.
[17]	LOEWE S. The problem of synergism and antagonism of combined drugs. Arzneimittelforschung. 1953 Jun; 3(6): 285-90.
[18]	Roell KR, Reif DM, Motsinger-Reif AA. An Introduction to Terminology and Methodology of Chemical Synergy-Perspectives from Across Disciplines. Front Pharmacol. 2017 Apr 20; 8: 158.
[19]	El Sayed SM, Baghdadi HH, Abdellatif NA, Ahmad AN, Hablas NM, Fakhreldin AR, Mariah RA, El-Shazley M, Ayat M, Mostafa S, Abu-el Naga M, Abdel-Halim M. The Antioxidant Glycolysis Inhibitor (Citric Acid) Induces a Dose-dependent Caspase-mediated Apoptosis and Necrosis in Glioma Cells. Journal of Cancer Research and Treatment, 2018, 6(1), 18-24.
[20]	Cardone RA, Casavola V, Reshkin SJ. The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer. 2005 Oct; 5(10): 786-95.
[21]	Mahoney BP, Raghunand N, Baggett B, Gillies RJ. Tumor acidity, ion trapping and chemotherapeutics. I. Acid pH affects the distribution of chemotherapeutic agents in vitro. Biochem Pharmacol. 2003 Oct 1; 66(7): 1207-18.
[22]	Chan CP, Jeng JH, Hsieh CC, Lin CL, Lei D, Chang MC. Morphological alterations associated with the cytotoxic and cytostatic effects of citric acid on cultured human dental pulp cells. J Endod. 1999 May; 25(5): 354-8.
[23]	Nagoba BS, Punpale AS, Ayachit R, Gandhi RC, Wadher BJ. Citric acid treatment of postoperative wound in an operated case of synovial sarcoma of the knee. Int Wound J. 2011 Aug; 8(4): 425-7.
[24]	Salama N, Kishimoto T, Kanayama HO, Kagawa S. The mobile phone decreases fructose but not citrate in rabbit semen: a longitudinal study. Syst Biol Reprod Med. 2009 Dec; 55(5-6): 181-7.
[25]	Marín-Hernández A, Rodríguez-Enríquez S, Vital-González PA, Flores-Rodríguez FL, Macías-Silva M, Sosa-Garrocho M, Moreno-Sánchez R. Determining and understanding the control of glycolysis in fast-growth tumor cells. Flux control by an over-expressed but strongly product-inhibited hexokinase. FEBS J. 2006 May; 273(9): 1975-88.
[26]	Warburg O. 1956 On the origin of cancer cells. Science 123, 309-314.