In Vitro Antitumor Effects of Bapst, a Repositioned Regimen for the Metabolic Therapy of Cancer

In Vitro Antitumor Effects of Bapst, a Repositioned Regimen for the Metabolic Therapy of Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In Vitro Antitumor Effects of Bapst, a Repositioned Regimen for the Metabolic Therapy of Cancer Guadalupe Dominguez-Gomez, Alma D. Chavez-Blanco, Romo-Perez Adriana, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7768473/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose . Key metabolism pathways altered in cancer are glycolysis, glutaminolysis, fatty acid synthesis, cholesterol synthesis, and beta-oxidation. Cancer cells reprogram their metabolism to resist cancer treatment. The BAPST regimen (Benserazide, Apomorphine, Pantoprazole, Simvastatin, Trimetazidine) is a repurposed drug combination that inhibits the key enzymes involved in these five pathways (HK2, GLS1, FASN, HMGCR, and 3-KAT). This study evaluates its vitro antitumor effects. Methods: We evaluated 20 human and three murine/rat cancer cell lines. The cells were treated with BAPST (Benserazide: 65 μM, Apomorphine: 8.49 μM, Pantoprazole: 84.3 μM, Simvastatin: 0.162 μM, Trimetazidine: 2.7 μM) or individual drugs for 24–120 hours. Cell viability and clonogenicity were assessed with crystal violet staining. Results: BAPST reduced viability across all cell lines. Major effects were observed in gastric (100%), pancreatic (>90%), and breast (65–90%) cells. Clonogenicity was entirely inhibited in five of six tested cell lines. Individually, benserazide and apomorphine, showed the highest effect, while pantoprazole a minor one. Simvastatin and trimetazidine had minimal/no inhibition or even increased viability and clonogenicity. Conclusion: BAPST demonstrates potent in vitro antitumor effects across diverse cancer cell lines, warranting further studies to confirm that its effect arises from metabolism enzyme inhibition and impeding metabolic reprogramming, as well as in vivo antitumor efficacy. BAPST Benserazide Apomorphine Pantoprazole Simvastatin Trimetazidine Cancer metabolism polypharmacotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Precision medicine in oncology with targeted pharmacology has advanced. However, the bar for drug approval has been lowered [1]. Many approved oncological therapies provide only marginal benefits and do not significantly extend overall survival (OS) or increase the chance of cure. Thus, the average increase in survival of the 124 drugs approved by the FDA from 2003 to 2021 is a mere 2.8 months [2]. While we must continue deciphering the molecular basis of cancer and developing targeted drugs, it is crucial to acknowledge the limitations of targeted therapy, particularly those arising from the paradoxes found in the somatic mutation theory [3] and search for novel schedules of multitargeted polypharmacotherapy to attack cancer as a robust system [4]. We recently published the theoretical principles of the BAPST (Benserazide, Apomorphine, Pantoprazole, Simvastatin, and Trimetazidine) regimen [5]. The regimen BAPST is based on fourth grounds: i) cancer can be seen as a robust system, and in experimental models, multiple hits are more effective than a single one in attacking a robust system [6]; ii) polypharmacotherapy implies the use of at least five drugs administered concomitantly mainly for complex diseases including neurological [7] and cancer [8] combination therapy has allowed for the cure of malignant diseases [9]; iii) altered cancer metabolism is a cancer hallmark [10] and its targeting is an active cancer therapy research area [11], and iv) the knowledge gained on the molecular basis of cancer has uncovered that many non-cancer drugs hit cancer targets, which has led to the study of cancer drug repurposing [12]. The malignant metabolic phenotype includes high rates of glycolysis [13], [14], glutaminolysis[15], increased synthesis of fatty acids [16], [17] and cholesterol[18], [19], as well as increased beta-oxidation of fatty acids (FAO)[20], [21]. The hyperactivity of these five metabolic pathways is not exclusive to malignant cells [22], [23]; however, the malignant cell preference to overuse these five metabolic pathways over normal cells hypothetically would provide a certain level of "specificity" for the metabolic treatment of cancer. Moreover, malignant tumors show metabolic heterogeneity and metabolic reprogramming. Tumors exhibit metabolic flexibility in response to the availability of nutrients, oxygen concentration, and damaging external factors [24]. Therefore, it is necessary to carry out a multiple metabolic blockade, explicitly blocking the primary five metabolic pathways that metabolize glucose, glutamine, fatty acid synthesis, cholesterol synthesis, and catabolism of fatty acids (FAO). Building on these principles, we proposed that the BAPST regimen could exert antitumor effects by targeting critical metabolic processes [5]. Benserazide and apomorphine, used for Parkinson's disease, inhibit Hexokinase-2 (HK2) and Glutaminase-1 (GLS1) respectively, which are crucial enzymes in glycolysis and glutaminolysis. Pantoprazole and simvastatin, used for peptic ulcer disease and hypercholesterolemia, respectively, inhibit fatty acid synthase (FASN) and 3-hydroxy-3-methyl-glutaryl coenzyme-A reductase (HMGCR), enzymes that catalyze fatty acids and cholesterol synthesis respectively. Trimetazidine, used for ischemic heart disease, inhibits 3-ketoacyl-CoA thiolase (3-KAT), an enzyme of the mitochondrial trifunctional multiprotein complex HADHA that catalyzes the last three reactions of the mitochondrial beta-oxidation pathway [5]. Here we report the preliminary results on the in vitro antitumor effects of the BAPST regimen in several cancer cell lines. MATERIALS AND METHODS Cell lines All the cell lines were obtained from the American Type Culture Collection (ATCC). Cells were plated in DMEM/F12 or RPMI-1640 (both from Gibco), supplemented with 10% fetal bovine serum (Corning) and 1% streptomycin/amphotericin (Gibco) (complete medium), at 37 ºC in a humidified 5% CO 2 atmosphere. Drugs Benserazide, apomorphine, simvastatin and trimetazidine were obtained from (Sigma-Aldrich Ò ), and Pantoprazole from (Takeda Ò ). Apomorphine and simvastatin were dissolved in DMSO (Sigma-Aldrich Ò ), Benserazide and Trimetazidine in sterile water for injection (PiSA Ò ) and Pantoprazole in saline solution 0.9% (PiSA Ò ). The compounds were administered individually or the five combined (BAPST). Doses The concentrations used in this work [5] were calculated according to the daily dose used in pharmacological therapy in humans. The concentrations used for viability and clonogenicity assays were the following: Benserazide, 65 mM; Apomorphine, 8.49 mM; Pantoprazole, 84.3mM; Trimetazidine, 2.7 mM; Simvastatin, 0.162 mM. Viability assays Each cell line was seeded with 0.5 mL of its respective medium in 24 cell plates, during a pre-incubation period of 24 h. Then, cells were treated during 24, 48, 72, 96 and 120 h with the BAPST scheme or its vehicle (DMSO, sterile water for injection or saline solution 0.9%). Fresh complete medium containing drugs/vehicle was changed every 24 h. After that, the medium with treatment or vehicle was removed and the wells were stained with a crystal violet (Sigma-Aldrich®) solution (EtOH (50%), formaldehyde (1.75%), crystal violet (0.75%), NaCl (0.25%), for 30 min. After this time, the crystal violet solution was retired, and the plates were washed with tap water and the plates were dried upside down for 24 hours. The cytotoxic effect was expressed as the percentage of cell viability relative to control cells. Clonogenicity assays of BAPST‑treated cells Cells treated for 120 hours with the BAPST scheme were used for this experiment. Therefore, 1000 cells/condition were recovered and plated in new 6-well plates with 2 mL of drug-free complete medium for 14 days. Subsequently, colonies were fixed stained with crystal violet solution. Colonies on culture dish were counted using the ImageJ software (4.0 version). Statistical analysis Unless otherwise specified, all experiments were independently performed in triplicate, with three internal replicates. Statistical analyses were calculated using one-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test for viability over the 21 cell lines and for clonogenicity assay. The results were analyzed with GraphPad Prism V6 (GraphPad, San Diego, CA, USA). Data were expressed as means ± SD. p < 0.05 was considered statistically significant. RESULTS Colon The human colon cancer cell lines SW480 and Caco-2 showed high inhibition with the BAPST treatment. In SW480 cells, the inhibition was significantly observed at 24 hours, and at 120h, the viability was around 5%. CACO-2 cells were also significantly inhibited for 24 hours, reaching 78% inhibition at 120 hours ( Figure 1 A, B ). Breast. Three human breast cancer cell lines were evaluated. The triple-negative MDA-MB-231 cells were around 90% inhibited at 120 hours of treatment. Major inhibition of estrogen receptor-positive MCF-7 cells occurred at 72 hours, reaching 80%, which was further decreased to 85% at 120 hours. The Her2-positive SKBR-3 cell line also showed inhibition, which was significant at 48 hours, reaching a maximum inhibition of 65% at 120 hours ( Figure 1 C, D, E). Cervix. BAPST was also evaluated in HeLa, SiHa, Ca sKi, and C33-A cervical cancer cell lines, which are HPV-18, HP-16, HP-16-positive, and HPV-negative respectively. All these cell lines showed statistically significant inhibition at 24 hours. The maximum inhibitions at 120 hours were 120 hours of 75%, 80%, 68% and 60%, respectively. ( Figure 1 F,G,H,I ). Gastric. Three human gastric cancer cell lines were evaluated: AGS NCI-CN87 and SNU-1. All three cell lines exhibited remarkable inhibition, which reached around 100% at 120 hours. These cell lines only differed in the speed of inhibition ( Figure 1 J, K, L ). Prostate. The human androgen receptor-dependent LnCaP and androgen receptor-independent DU-145 and PC3 cell lines were evaluated after 120 hours of treatment. The results show LnCaP reached 67% inhibition, the inhibition of DU-145 was 80%, while the PC3 cells had almost 90% of inhibition ( Figure 1 M, N, O ). Pancreas. PANc-1, BxPc-3, and AsPC-1 human pancreatic cancer cell lines were evaluated. All three cell lines were highly sensitive to the BAPST treatment, which reached more than 90% inhibition at 120 hours. Of note, a major effect was seen as soon as 24 hours of treatment, which was almost 80% in AsPc-1 cells ( Figure 1 P, Q, R ). Glioma and melanoma. Human glioma D54MG and human melanoma A375 cells were also evaluated. Both cell lines showed a similar inhibition pattern, achieving around 80% at 120 hours. ( Figure 1 S, T ). Murine and rat cancer cell lines. The mouse lines CT26.WT (colon carcinoma), LL/2-LLC1 (lung carcinoma) and the Walker 256 rat breast carcinosarcoma cell line were evaluated. All three cell lines showed high sensitivity to the regimen, with an almost total inhibition in LL/2(LCC1) to 85% inhibition for Walker cells at 120 hours. ( Figure 2 A, B,C ). Individual effects of the regimen BAPST in human cancer cells. These five drugs were individually evaluated in the gastric cancer cell lines AGS andNCI-N87, and the breast cancer cells MDA-MB-231. As shown in Figure 3 , in AGS cells apomorphine and benserazide were the most active drugs. At 120 hours, apomorphine showed around 95% inhibition, benserazide inhibited almost 90% but no viability was observed with BAPST. In NCI-N87, the effects of benserazide are very similar to those in AGS; however, apomorphine, despite showing inhibition in the first 72 hours, causes cells to regrow to almost the untreated control, nevertheless almost complete inhibition was observed with BAPST. In the breast cancer cells, the effect of benserazide and apomorphine were somehow like that observed in AGS but observing around 90% inhibition with benserazide alone and BAPST as well. Of note, pantoprazole, simvastatin, and trimetazidine have no inhibitory effects; indeed, the cell viability slightly increased over the control at different times which was more pronounced in AGS cells. However, in both cell lines, the complete regimen showed complete inhibition ( Figure 3 A, B, C ). Individual effects of the regimen BAPST in murine and rat cancer cells. The effect of BAPST and each individual drug somehow were similar to the human cancer cells with some small differences. In CT26-WT benserazide and apomorphine were the most effective drugs, indeed at 120 hours their individual effect was higher that the complete BAPST, the full regimen keeps more than 80% inhibition. Pantoprazole in these cells almost reached 50% inhibition at 96 hours whereas almost none effect was observed for trimetazidine and simvastatin. Similar effects occurred in LL/2(LLC1) but apomorphine increase the viability at 96 and 120 hours after being diminished at 72 hours. At 120 hours there no differences between benserazide alone and BAPST. In rat cells, both benserazide and apomorphine showed the higher inhibition and at 120 hours comparable effects of benserazide alone and BAPST were observed ( Figure 4 A, B, C ) Clonogenicity of BAPST To further delve into the effect of this regimen, clonogenicity assays were performed in both human and murine cancer cell lines. Remarkable, as observed in Figure 5 A, B, C, D, E, F ) a total inhibition was observed in five out of the six cell lines in comparison to control and vehicle treated cells. In less sensitive NCI-N87 the inhibition reached almost around 80%. Clonogenicity of individual drugs The analysis of the effect of each drug of the regimen BAPST confirms that benserazide in six out the seven human cancer cell lines tested completely inhibits clonogenicity at 120 as did the BAPST and this inhibition while major (around 80%) was not complete in NCI-N87. Likewise, apomorphine the second mas active after benserazide totally inhibited clonogenicity in four out the seven cell lines, in two exhibited between 30% and 60% decrease, but actually increased clonogenicity in the murine cancer cells. Pantoprazole on the other hand showed major inhibition in two cell lines, small effect in one, no effect in two but an increase in two of them. Trimetazidine lead to increased clonogenicity in four cell lines, and no effect in three. Simvastatin on the other hand increased the clonogenicity in all seven cell lines which was around four-fold higher in Ca Ski cells ( Figure 6 ). DISCUSSION The results of this preliminary report on the inhibitory effects of the regimen BAPST upon 20 human, two mice and one rat cancer cell line, including colon, breast, gastric, pancreas, cervix, prostate, melanoma, and glioma, show that this 5-drug regimen induces potent inhibition of viability as well as a clonogenicity. This effect is time-dependent, starting as early as 24 hours and reaching a maximum at 120 hours. The time-dependent effect is significant as it provides insights into the optimal treatment duration for maximum efficacy. Remarkably, the individual assessment of each of these five drugs shows that the two more active are benserazide and apomorphine, with the effect of pantoprazole somewhat intermediate. Interestingly, no inhibitory cellular effects of simvastatin and trimetazidine are observed. In 1890, by studying the Nitrogen content in the urine of cancer patients, simple starvation, and other wasting diseases, Muller reported that in cancer patients, the excreted amount of Nitrogen in the urine far exceeded the amount ingested [25]. In the early 1900s, Warburg's work sparked the field of cancer metabolism [26], which led Hanahan to propose it as an emerging hallmark of cancer in 2011 [10]. While most agree on the therapeutic implications of exploiting the knowledge on tumor metabolism, so far, no anticancer drug that directly targets cancer metabolism has been approved, yet several candidate drugs are in preclinical and clinical stages of development. The significance of our findings lies in two aspects. First, the proposal, as presented in our previous work [4], [5], is based on polypharmacotherapy, which historically provides better results than single-agent therapy [27], [28]. Second, the proposal to use repositioned drugs whose clinical safety is widely documented, providing a reassuring foundation for our approach [29]. In particular, polypharmacotherapy is of special interest when targeting tumor metabolism, given the basal metabolic heterogeneity of tumors and the widely documented metabolic reprogramming, which suggests the importance of simultaneously blocking as many metabolic pathways as possible [4], [5], [30], [31]. There is an extensive list of novel or repurposed drugs that inhibit enzymes of glycolysis, glutaminolysis, fatty acid synthesis, cholesterol synthesis, and beta-oxidation, some of which are being clinically evaluated. Among these are 2-deoxy-D-glucose[32], telaglenastat[33], and denifanstat [34], which are inhibitors of glycolysis, glutaminolysis, and fatty acid synthesis, respectively. Though no novel inhibitors of cholesterol synthesis are being clinically developed in cancer, simvastatin and other statins are being widely tested with variable results, mostly in combination with other cytotoxics [35]. Regarding inhibitors of beta-oxidation, the only drug clinically approved is trimetazidine for ischemic heart disease [36]; however, its testing as cancer therapy is yet to be done. A single study proved to be well tolerated and effective for protecting breast cancer patients from anthracycline-induced cardiotoxicity [37]. Despite the clinical availability of these inhibitors, no preclinical studies have used them in combination. Although the inhibition exerted by each of the drugs in the BAPST regimen on the corresponding enzymes has been consistently demonstrated: benserazide - HK2 [38], apomorphine - GLS1 [39], pantoprazole-FASN [40], simvastatin – HMGCR [41], and trimetazidine - (3-KAT) [42] - the antitumor effects observed in this large number of malignant cell lines cannot be explicitly attributed to the inhibition of each of these enzymes since this would require the evaluation of the enzymatic activity of these targets in our model. However, it is notable that the combination of the five drugs is very effective in all cell lines and supports the experimental data of 12 cell lines including breast, ovary, cervix, prostate, osteosarcoma, melanoma, bladder, colon and stomach where the expression of HK2, GLS1 and FASN were found hyperexpressed at the mRNA and protein [43]. Similarly, several tumors exhibit either deficient feedback control of HMGCR or increased HMGCR expression and activity [44]–[46] as well as abnormal expression of 3-KAT [45,46]. On the other hand, when analyzing the inhibition behavior with each of the drugs, it is pretty evident that of the five drugs, the two with the most significant cellular inhibitory activity are the glucolytic benserazide and the glutaminolytic apomorphine, which supports the evidence that glucose and glutamine are the two main anaplerotic molecules that provide precursors and energy to malignant cells [47,49]. The cellular inhibition by pantoprazole is somewhat intermediate, which could reflect tumor-specific sensitivity to FASN inhibitors [52] and that tissue culture cells can utilize serum triglycerides by their intake with no prior hydrolysis [53], while the small or not effect of simvastatin could be because, under basal conditions and optimal in vitro growth conditions, existing intracellular cholesterol levels could suffice. The lack of cell effects of trimetazidine may stem because the competition between glycolysis and FAO is conserved in some cancers and that may explain why trimetazidine does not reduce cell viability as most tumors are glycolytic [54]. To all possible explanations, it must be added that the doses here used are those that are clinically relevant, which differ from most in vitro studies, where inhibitory concentrations in culture are higher and determined without caring on their clinical relevance; and that these drugs are not target-selective and therefore exhibit many off-target effects. Analyzed simplistically, it could be assumed that in this scheme, the use benserazide alone in most cases, or even apomorphine in some, provides the same cell inhibition, which is correct. However, the rationale for the use of the five drugs lies in several aspects: i. The significant basal heterogeneity in the preferential use of each of these five pathways by different types of neoplasia and even within the same neoplasia, ii) the highly documented metabolic reprogramming, a process where cancer cells alter their metabolism to support rapid growth and proliferation. In colon cancer blocking glycolysis leads to compensatory glutaminolysis [55]. Not only that occurs with glucose and glutamine, but also in lipid metabolism. Impeding lipid oxidation with the FAO inhibitor etomoxir increases glycolysis in mouse xenografts [56] and blocking glutaminolysis increases glycolysis and lipid synthesis [57]. Likewise compensatory glutaminolysis and ketone metabolism are induced when lung cancer cells are treated with the FASN inhibitors [58]. The most compelling in vivo evidence comes from a study in which deletion of GLS1 is compensated by glycolysis requiring co-inhibition of both pathways to significantly affect the Krebs cycle activity and tumor formation [59]. Thus, the rationale behind using the BAPST regimen is that it will theoretically prevent or impede in some degree, the metabolic reprogramming, a highly documented resistance mechanism in cancer treatment [60]. It must be noted that polypharmacy (five drugs) has a negative connotation as they usually occur in multimorbid older, and frail patients, indeed, the prevalence of polypharmacy can be as high as 89% [61], [62]. However, polypharmacy is used to treat more than one condition whereas polypharmacotherapy has here described all drugs are intended as cancer treatment and on a strict sense, this is not polypharmacy. Moreover, in the context of multifactorial diseases such as cancer, polypharmacotherapy is an approach using multiple drugs against multiple cancer targets. In the case of the BAPST regimen, the rational is backed on the need to target these main five metabolic pathways, which is crucial in the context of the metabolic reprogramming that often occurs in cancer cells. There are no epidemiological or clinical studies describing patients with any condition who concomitantly receive the five drugs of the BAPST regimen; however, it is intuitive that this could occur in the clinical setting. Considering patients with Parkinson's disease as an example, 1) the combination of oral dopamine agonists plus benserazide (because benserazide alone is not clinically used) with apomorphine is clinically indicated for a subset of Parkinson´s disease patients [63]. 2) Gastrointestinal disorders such as gastroesophageal reflux and gastritis are common comorbidities in patients with Parkinson's disease [64], [65]. 3) Hypercholesterolemia and statin use are prevalent in Parkinson's disease [66], [67]; 4) Coronary artery disease is prevalent in Parkinson's disease, and the use of trimetazidine in these patients has been reported [68], [69]; and, 5) 40% of patients with Parkinson's disease use polypharmacy [70]. 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Heal Aging Clin Care Elder, 5:1–8. https://doi.org/10.4137/HACCE.S11173 Magne FO, Drapier S, Marques AR, et al (2025) A delphi consensus statement about French practical management of continuous apomorphine infusion in patients with Parkinson’s disease and motor fluctuations. Parkinsonism Relat Disord, p. 107866. https://doi.org/10.1016/j.parkreldis.2025.107866 Schrag A, Bohlken J, Dammertz L, et al (2023) Widening the Spectrum of Risk Factors, Comorbidities, and Prodromal Features of Parkinson Disease. JAMA Neurol, 80(2): 161-171. https://doi.org/ 10.1001/jamaneurol.2022.3902 Tan AH, Chuah KH, Beh YY, Schee JP, Mahadeva S, and Lim SY, (2023) Gastrointestinal Dysfunction in Parkinson’s Disease: Neuro-Gastroenterology Perspectives on a Multifaceted Problem. J Mov Disord, 16(2):138–151. https://doi.org/ 10.14802/jmd.22220 Gao X, Simon KC, Schwarzschild MA, et al (2012) Prospective Study of Statin Use and Risk of Parkinson Disease. Arch Neurol,69(3):380-384. https://doi.org/10.1001/archneurol.2011.1060 G. Hu (2010) Total Cholesterol and the Risk of Parkinson’s Disease: A Review for Some New Findings. Parkinsons Dis, 2010:836962. https://doi.org/10.4061/2010/836962 Chua SKK, Saffari SE, Lee SJY, and Tan EK (2022) Association Between Parkinson’s Disease and Coronary Artery Disease: A Systematic Review and Meta-Analysis. J Parkinsons Dis 12(6):1737–1748. https://doi.org/10.3233/JPD-223291 Pintér D, Bereczki D, Ajtay A, Oberfrank F, Janszky J, and Kovács N (2021) Trimetazidine Use in Parkinson’s Disease: Is It a Resolved Problem? eNeuro, vol. 8(3): ENEURO.0452-20.2021. https://doi.org/10.1523/ENEURO.0452-20.2021 Bhagavathula SA, Tesfaye W, Vidyasagar K, and Fialova D (2022) Polypharmacy and Hyperpolypharmacy in Older Individuals with Parkinson’s Disease: A Systematic Review and Meta-Analysis. Gerontology 68(10):1081–1090. https://doi.org/ 10.1159/000521214 Additional Declarations No competing interests reported. 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0.0001, *** \u003cem\u003ep\u003c/em\u003e \u003cem\u003e\u0026lt;\u003c/em\u003e 0.001, and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs \u003c/em\u003econtrol.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7768473/v1/e54efc1e0df565b353998f37.png"},{"id":95179058,"identity":"e517464e-0cdd-47b6-b505-5a980967db6f","added_by":"auto","created_at":"2025-11-05 08:06:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112300,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of BAPST in cell viability of two murine and one rata cancer cell lines. **** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001, *** \u003cem\u003ep\u003c/em\u003e \u003cem\u003e\u0026lt;\u003c/em\u003e 0.001, and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs \u003c/em\u003econtrol.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7768473/v1/c4e700fc7e9b2425c61974b2.png"},{"id":95179062,"identity":"ac04fbbd-e929-4432-8c5c-ff3744b54500","added_by":"auto","created_at":"2025-11-05 08:06:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":168726,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of cell viability by BAPST and each individual drug treatment in three human cancer cell lines. **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, *** \u003cem\u003ep\u003c/em\u003e \u003cem\u003e\u0026lt;\u003c/em\u003e 0.001, and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs \u003c/em\u003econtrol.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7768473/v1/bca07ab4e73af08690000ae5.png"},{"id":95227084,"identity":"4f8319f1-a5d3-4402-9a22-67bf4f0ad69a","added_by":"auto","created_at":"2025-11-05 16:32:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":446515,"visible":true,"origin":"","legend":"\u003cp\u003eThe individual effect on viability of each drug and the BAPST regimen\u003cstrong\u003e \u003c/strong\u003ein two murine and one rat cancer cell lines. **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, *** \u003cem\u003ep\u003c/em\u003e \u003cem\u003e\u0026lt;\u003c/em\u003e 0.001, and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs \u003c/em\u003econtrol.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7768473/v1/55c1779bdd592b416894f1ee.png"},{"id":95179060,"identity":"f6f7c6f7-a687-4035-8ae7-51a20e2e3a14","added_by":"auto","created_at":"2025-11-05 08:06:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":284202,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of clonogenicity of the effects of BAPST treatment in seven human and two murine cancer cell lines. **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 \u003cem\u003evs \u003c/em\u003econtrol.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7768473/v1/3c9978da99e607fea134f19f.png"},{"id":95179059,"identity":"3bd9746b-20f4-4658-9258-8b0726c4b585","added_by":"auto","created_at":"2025-11-05 08:06:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":489779,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of individual drugs and BAPST upon clonogenicity in six human cell lines. **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 and *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs \u003c/em\u003econtrol.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7768473/v1/163af8f5b52a5f339428e068.png"},{"id":95230536,"identity":"b6a81761-7a4c-43ad-acd5-f0d967baf2e6","added_by":"auto","created_at":"2025-11-05 16:37:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2736776,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7768473/v1/2fece111-f790-4599-a028-de6ccc1f5c9e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eIn Vitro Antitumor Effects of Bapst, a Repositioned Regimen for the Metabolic Therapy of Cancer\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePrecision medicine in oncology with targeted pharmacology has advanced. However, the bar for drug approval has been lowered [1]. Many approved oncological therapies provide only marginal benefits and do not significantly extend overall survival (OS) or increase the chance of cure. Thus, the average increase in survival of the 124 drugs approved by the FDA from 2003 to 2021 is a mere 2.8 months [2]. While we must continue deciphering the molecular basis of cancer and developing targeted drugs, it is crucial to acknowledge the limitations of targeted therapy, particularly those arising from the paradoxes found in the somatic mutation theory [3] and search for novel schedules of multitargeted polypharmacotherapy to attack cancer as a robust system [4].\u003c/p\u003e\n\u003cp\u003eWe recently published the theoretical principles of the BAPST (Benserazide, Apomorphine, Pantoprazole, Simvastatin, and Trimetazidine) regimen [5]. The regimen BAPST is based on fourth grounds: i) cancer can be seen as a robust system, and in experimental models, multiple hits are more effective than a single one in attacking a robust system [6]; ii) polypharmacotherapy implies the use of at least five drugs administered concomitantly mainly for complex diseases including neurological [7] and cancer [8] combination therapy has allowed for the cure of malignant diseases [9]; iii) altered cancer metabolism is a cancer hallmark [10] \u0026nbsp;and its targeting is an active cancer therapy research area [11], and iv) the knowledge gained on the molecular basis of cancer has uncovered that many non-cancer drugs hit cancer targets, which has led to the study of cancer drug repurposing [12].\u003c/p\u003e\n\u003cp\u003eThe malignant metabolic phenotype includes high rates of glycolysis [13], [14], glutaminolysis[15], increased synthesis of fatty acids [16], [17] and cholesterol[18], [19], as well as increased beta-oxidation of fatty acids (FAO)[20], [21]. The hyperactivity of these five metabolic pathways is not exclusive to malignant cells \u0026nbsp;[22], [23]; however, the malignant cell preference to overuse these five metabolic pathways over normal cells hypothetically would provide a certain level of \u0026quot;specificity\u0026quot; for the metabolic treatment of cancer. Moreover, malignant tumors show metabolic heterogeneity and metabolic reprogramming. Tumors exhibit metabolic flexibility in response to the availability of nutrients, oxygen concentration, and damaging external factors [24]. Therefore, it is necessary to carry out a multiple metabolic blockade, explicitly blocking the primary five metabolic pathways that metabolize glucose, glutamine, fatty acid synthesis, cholesterol synthesis, and catabolism of fatty acids (FAO).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBuilding on these principles, we proposed that the BAPST regimen could exert antitumor effects by targeting critical metabolic processes [5]. Benserazide and apomorphine, used for Parkinson\u0026apos;s disease, inhibit Hexokinase-2 (HK2) and Glutaminase-1 (GLS1) respectively, which are crucial enzymes in glycolysis and glutaminolysis. Pantoprazole and simvastatin, used for peptic ulcer disease and hypercholesterolemia, respectively, inhibit fatty acid synthase (FASN) and 3-hydroxy-3-methyl-glutaryl coenzyme-A reductase (HMGCR), enzymes that catalyze fatty acids and cholesterol synthesis respectively. Trimetazidine, used for ischemic heart disease, inhibits 3-ketoacyl-CoA thiolase (3-KAT), an enzyme of the mitochondrial trifunctional multiprotein complex HADHA that catalyzes the last three reactions of the mitochondrial beta-oxidation pathway [5]. Here we report the preliminary results on the in vitro antitumor effects of the BAPST regimen in several cancer cell lines.\u0026nbsp;\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell lines\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the cell lines were obtained from the American Type Culture Collection (ATCC). Cells were plated in DMEM/F12 or RPMI-1640 (both from Gibco), supplemented with 10% fetal bovine serum (Corning) and 1% streptomycin/amphotericin (Gibco) (complete medium), at 37 \u0026ordm;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDrugs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBenserazide, apomorphine, simvastatin and trimetazidine were obtained from (Sigma-Aldrich\u003csup\u003e\u0026Ograve;\u003c/sup\u003e), and Pantoprazole from (Takeda\u003csup\u003e\u0026Ograve;\u003c/sup\u003e). Apomorphine and simvastatin were dissolved in DMSO (Sigma-Aldrich\u003csup\u003e\u0026Ograve;\u003c/sup\u003e), Benserazide and Trimetazidine in sterile water for injection (PiSA\u003csup\u003e\u0026Ograve;\u003c/sup\u003e) and Pantoprazole in saline solution 0.9% (PiSA\u003csup\u003e\u0026Ograve;\u003c/sup\u003e). The compounds were administered individually or the five combined (BAPST).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDoses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentrations used in this work [5] were calculated according to the daily dose used in pharmacological therapy in humans. The concentrations used for viability and clonogenicity assays were the following: Benserazide, 65 mM; Apomorphine, 8.49 \u0026nbsp; mM; Pantoprazole, 84.3mM; Trimetazidine, 2.7 \u0026nbsp;mM; Simvastatin, 0.162 \u0026nbsp; mM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eViability assays\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach cell line was seeded with 0.5 mL of its respective medium in 24 cell plates, during a pre-incubation period of 24 h. Then, cells were treated during 24, 48, 72, 96 and 120 h with the BAPST scheme or its vehicle (DMSO, sterile water for injection or saline solution 0.9%). Fresh complete medium containing drugs/vehicle was changed every 24 h. After that, the medium with treatment or vehicle was removed and the wells were stained with a crystal violet (Sigma-Aldrich\u0026reg;) solution (EtOH (50%), formaldehyde (1.75%), crystal violet (0.75%), NaCl (0.25%), for 30 min. After this time, the crystal violet solution was retired, and the plates were washed with tap water and the plates were dried upside down for 24 hours. The cytotoxic effect was expressed as the percentage of cell viability relative to control cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eClonogenicity assays of\u0026nbsp;BAPST‑treated cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells treated for 120 hours with the BAPST scheme were used for this experiment. Therefore, 1000 cells/condition were recovered and plated in new 6-well plates\u0026nbsp;with 2\u0026nbsp;mL of drug-free complete medium for 14\u0026nbsp;days. Subsequently, colonies were fixed stained with crystal violet solution. Colonies on culture dish were counted using the ImageJ software (4.0 version).\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eUnless otherwise specified, all experiments were independently performed in triplicate, with three internal replicates. Statistical analyses were calculated using one-way analysis of variance (ANOVA) followed by the Dunnett\u0026rsquo;s multiple comparison test for viability over the 21 cell lines and for clonogenicity assay. The results were analyzed with GraphPad Prism V6 (GraphPad, San Diego, CA, USA). Data were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eColon\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human colon cancer cell lines SW480 and Caco-2 showed high inhibition with the BAPST treatment. In SW480 cells, the inhibition was significantly observed at 24 hours, and at 120h, the viability was around 5%. CACO-2 cells were also significantly inhibited for 24 hours, reaching 78% inhibition at 120 hours (\u003cstrong\u003eFigure 1 A, B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBreast.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree human breast cancer cell lines were evaluated. The triple-negative MDA-MB-231 cells were around 90% inhibited at 120 hours of treatment. Major inhibition of estrogen receptor-positive MCF-7 cells occurred at 72 hours, reaching 80%, which was further decreased to 85% at 120 hours. The Her2-positive SKBR-3 cell line also showed inhibition, which was significant at 48 hours, reaching a maximum inhibition of 65% at 120 hours (\u003cstrong\u003eFigure 1 C, D, E).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCervix.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBAPST was also evaluated in HeLa, SiHa, Ca sKi, and C33-A cervical cancer cell lines, which are HPV-18, HP-16, HP-16-positive, and HPV-negative respectively. All these cell lines showed statistically significant inhibition at 24 hours. The maximum inhibitions at 120 hours were 120 hours of 75%, 80%, 68% and 60%, respectively. (\u003cstrong\u003eFigure 1 F,G,H,I\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGastric.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree human gastric cancer cell lines were evaluated: AGS NCI-CN87 and SNU-1. All three cell lines exhibited remarkable inhibition, which reached around 100% at 120 hours. These cell lines only differed in the speed of inhibition (\u003cstrong\u003eFigure 1 J, K, L\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProstate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human androgen receptor-dependent LnCaP and androgen receptor-independent DU-145 and PC3 cell lines were evaluated after 120 hours of treatment. The results show LnCaP reached 67% inhibition, the inhibition of DU-145 was 80%, while the PC3 cells had almost 90% of inhibition (\u003cstrong\u003eFigure 1 M, N, O\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePancreas.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePANc-1, BxPc-3, and AsPC-1 human pancreatic cancer cell lines were evaluated. All three cell lines were highly sensitive to the BAPST treatment, which reached more than 90% inhibition at 120 hours. Of note, a major effect was seen as soon as 24 hours of treatment, which was almost 80% in AsPc-1 cells (\u003cstrong\u003eFigure 1 P, Q, R\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlioma and melanoma.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman glioma D54MG and human melanoma A375 cells were also evaluated. Both cell lines showed a similar inhibition pattern, achieving around 80% at 120 hours. (\u003cstrong\u003eFigure 1 S, T\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMurine and rat cancer cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse lines CT26.WT (colon carcinoma), LL/2-LLC1 (lung carcinoma) and the Walker 256 rat breast carcinosarcoma cell line were evaluated. All three cell lines showed high sensitivity to the regimen, with an almost total inhibition in LL/2(LCC1) to 85% inhibition for Walker cells at 120 hours. (\u003cstrong\u003eFigure 2 A, B,C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIndividual effects of the regimen BAPST in human cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese five drugs were individually evaluated in the gastric cancer cell lines AGS andNCI-N87, and the breast cancer cells MDA-MB-231. \u0026nbsp;As shown in \u003cstrong\u003eFigure 3\u003c/strong\u003e, in AGS cells apomorphine and benserazide were the most active drugs. At 120 hours, apomorphine showed around 95% inhibition, benserazide inhibited almost 90% but no viability was observed with BAPST. \u0026nbsp;In NCI-N87, the effects of benserazide are very similar to those in AGS; however, apomorphine, despite showing inhibition in the first 72 hours, causes cells to regrow to almost the untreated control, nevertheless almost complete inhibition was observed with BAPST. In the breast cancer cells, the effect of benserazide and apomorphine were somehow like that observed in AGS but observing around 90% inhibition with benserazide alone and BAPST as well. \u0026nbsp;Of note, pantoprazole, simvastatin, and trimetazidine have no inhibitory effects; indeed, the cell viability slightly increased over the control at different times which was more pronounced in AGS cells. However, in both cell lines, the complete regimen showed complete inhibition (\u003cstrong\u003eFigure 3 A, B, C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIndividual effects of the regimen BAPST in murine and rat cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of BAPST and each individual drug somehow were similar to the human cancer cells with some small differences. In CT26-WT benserazide and apomorphine were the most effective drugs, indeed at 120 hours their individual effect was higher that the complete BAPST, the full regimen keeps more than 80% inhibition. Pantoprazole in these cells almost reached 50% inhibition at 96 hours whereas almost none effect was observed for trimetazidine and simvastatin. Similar effects occurred in LL/2(LLC1) but apomorphine increase the viability at 96 and 120 hours after being diminished at 72 hours. At 120 hours there no differences between benserazide alone and BAPST. In rat cells, both benserazide and apomorphine showed the higher inhibition and at 120 hours comparable effects of benserazide alone and BAPST were observed (\u003cstrong\u003eFigure 4 A, B, C\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClonogenicity of BAPST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further delve into the effect of this regimen, clonogenicity assays were performed in both human and murine cancer cell lines. Remarkable, as observed in \u003cstrong\u003eFigure 5 A, B, C, D, E, F\u003c/strong\u003e) a total inhibition was observed in five out of the six cell lines in comparison to control and vehicle treated cells. In less sensitive NCI-N87 the inhibition reached almost around 80%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClonogenicity of individual drugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of the effect of each drug of the regimen BAPST confirms that benserazide in six out the seven human cancer cell lines tested completely inhibits clonogenicity at 120 as did the BAPST and this inhibition while major (around 80%) was not complete in NCI-N87. Likewise, apomorphine the second mas active after benserazide totally inhibited clonogenicity in four out the seven cell lines, in two exhibited between 30% and 60% decrease, but actually increased clonogenicity in the murine cancer cells. Pantoprazole on the other hand showed major inhibition in two cell lines, small effect in one, no effect in two but an increase in two of them. Trimetazidine lead to increased clonogenicity in four cell lines, and no effect in three. Simvastatin on the other hand increased the clonogenicity in all seven cell lines which was around four-fold higher in Ca Ski cells (\u003cstrong\u003eFigure 6\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe results of this preliminary report on the inhibitory effects of the regimen BAPST upon 20 human, two mice and one rat cancer cell line, including colon, breast, gastric, pancreas, cervix, prostate, melanoma, and glioma, show that this 5-drug regimen induces potent inhibition of viability as well as a clonogenicity. This effect is time-dependent, starting as early as 24 hours and reaching a maximum at 120 hours. The time-dependent effect is significant as it provides insights into the optimal treatment duration for maximum efficacy. Remarkably, the individual assessment of each of these five drugs shows that the two more active are benserazide and apomorphine, with the effect of pantoprazole somewhat intermediate. Interestingly, no inhibitory cellular effects of simvastatin and trimetazidine are observed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn 1890, by studying the Nitrogen content in the urine of cancer patients, simple starvation, and other wasting diseases, Muller reported that in cancer patients, the excreted amount of Nitrogen in the urine far exceeded the amount ingested [25]. In the early 1900s, Warburg\u0026apos;s work sparked the field of cancer metabolism [26], which led Hanahan to propose it as an emerging hallmark of cancer in 2011 [10]. While most agree on the therapeutic implications of exploiting the knowledge on tumor metabolism, so far, no anticancer drug that directly targets cancer metabolism has been approved, yet several candidate drugs are in preclinical and clinical stages of development.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe significance of our findings lies in two aspects. First, the proposal, as presented in our previous work [4], [5], is based on polypharmacotherapy, which historically provides better results than single-agent therapy [27], [28]. Second, the proposal to use repositioned drugs whose clinical safety is widely documented, providing a reassuring foundation for our approach [29]. In particular, polypharmacotherapy is of special interest when targeting tumor metabolism, given the basal metabolic heterogeneity of tumors and the widely documented metabolic reprogramming, which suggests the importance of simultaneously blocking as many metabolic pathways as possible [4], [5], [30], [31].\u003c/p\u003e\n\u003cp\u003eThere is an extensive list of novel or repurposed drugs that inhibit enzymes of glycolysis, glutaminolysis, fatty acid synthesis, cholesterol synthesis, and beta-oxidation, some of which are being clinically evaluated. Among these are 2-deoxy-D-glucose[32], telaglenastat[33], and denifanstat [34], which are inhibitors of glycolysis, glutaminolysis, and fatty acid synthesis, respectively. Though no novel inhibitors of cholesterol synthesis are being clinically developed in cancer, simvastatin and other statins are being widely tested with variable results, mostly in combination with other cytotoxics [35]. Regarding inhibitors of beta-oxidation, the only drug clinically approved is trimetazidine for ischemic heart disease [36]; however, its testing as cancer therapy is yet to be done. A single study proved to be well tolerated and effective for protecting breast cancer patients from anthracycline-induced cardiotoxicity [37]. Despite the clinical availability of these inhibitors, no preclinical studies have used them in combination.\u003c/p\u003e\n\u003cp\u003eAlthough the inhibition exerted by each of the drugs in the BAPST regimen on the corresponding enzymes has been consistently demonstrated: benserazide - HK2 [38], apomorphine - GLS1 [39], pantoprazole-FASN [40], simvastatin \u0026ndash; HMGCR [41], and trimetazidine - (3-KAT) [42] - the antitumor effects observed in this large number of malignant cell lines cannot be explicitly attributed to the inhibition of each of these enzymes since this would require the evaluation of the enzymatic activity of these targets in our model. However, it is notable that the combination of the five drugs is very effective in all cell lines and supports the experimental data of 12 cell lines including breast, ovary, cervix, prostate, osteosarcoma, melanoma, bladder, colon and stomach where the expression of HK2, GLS1 and FASN were found hyperexpressed at the mRNA and protein [43]. Similarly, several tumors exhibit either deficient feedback control of HMGCR or increased HMGCR expression and activity [44]\u0026ndash;[46] as well as abnormal expression of 3-KAT [45,46].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, when analyzing the inhibition behavior with each of the drugs, it is pretty evident that of the five drugs, the two with the most significant cellular inhibitory activity are the glucolytic benserazide and the glutaminolytic apomorphine, which supports the evidence that glucose and glutamine are the two main anaplerotic molecules that provide precursors and energy to malignant cells [47,49].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cellular inhibition by pantoprazole is somewhat intermediate, which could reflect tumor-specific sensitivity to FASN inhibitors [52] and that tissue culture cells can utilize serum triglycerides by their intake with no prior hydrolysis [53], while the small or not effect of simvastatin could be because, under basal conditions and optimal in vitro growth conditions, existing intracellular cholesterol levels could suffice. The lack of cell effects of trimetazidine may stem because the competition between glycolysis and FAO is conserved in some cancers and that may explain why trimetazidine does not reduce cell viability as most tumors are glycolytic [54]. To all possible explanations, it must be added that the doses here used are those that are clinically relevant, which differ from most in vitro studies, where inhibitory concentrations in culture are higher and determined without caring on their clinical relevance; and that these drugs are not target-selective and therefore exhibit many off-target effects.\u003c/p\u003e\n\u003cp\u003eAnalyzed simplistically, it could be assumed that in this scheme, the use benserazide alone in most cases, or even apomorphine in some, provides the same cell inhibition, which is correct. However, the rationale for the use of the five drugs lies in several aspects: i. The significant basal heterogeneity in the preferential use of each of these five pathways by different types of neoplasia and even within the same neoplasia, ii) the highly documented metabolic reprogramming, a process where cancer cells alter their metabolism to support rapid growth and proliferation. In colon cancer blocking glycolysis leads to compensatory glutaminolysis [55]. Not only that occurs with glucose and glutamine, but also in lipid metabolism. Impeding lipid oxidation with the FAO inhibitor etomoxir increases glycolysis in mouse xenografts [56] and blocking glutaminolysis increases glycolysis and lipid synthesis [57]. Likewise compensatory glutaminolysis and ketone metabolism are induced when lung cancer cells are treated with the FASN inhibitors [58]. The most compelling in vivo evidence comes from a study in which deletion of GLS1 is compensated by glycolysis requiring co-inhibition of both pathways to significantly affect the Krebs cycle activity and tumor formation [59]. \u0026nbsp;Thus, the rationale behind using the BAPST regimen is that it will theoretically prevent or impede in some degree, the metabolic reprogramming, a highly documented resistance mechanism in cancer treatment [60].\u003c/p\u003e\n\u003cp\u003eIt must be noted that polypharmacy (five drugs) has a negative connotation as they usually occur in multimorbid older, and frail patients, indeed, the prevalence of polypharmacy can be as high as 89% [61], [62]. However, polypharmacy is used to treat more than one condition whereas polypharmacotherapy has here described all drugs are intended as cancer treatment and on a strict sense, this is not polypharmacy. Moreover, in the context of multifactorial diseases such as cancer, polypharmacotherapy is an approach using multiple drugs against multiple cancer targets. In the case of the BAPST regimen, the rational is backed on the need to target these main five metabolic pathways, which is crucial in the context of the metabolic reprogramming that often occurs in cancer cells.\u003c/p\u003e\n\u003cp\u003eThere are no epidemiological or clinical studies describing patients with any condition who concomitantly receive the five drugs of the BAPST regimen; however, it is intuitive that this could occur in the clinical setting. Considering patients with Parkinson\u0026apos;s disease as an example, \u0026nbsp;1) the combination of oral dopamine agonists plus benserazide (because benserazide alone is not clinically used) with apomorphine is clinically indicated for a subset of Parkinson\u0026acute;s disease patients [63]. 2) Gastrointestinal disorders such as gastroesophageal reflux and gastritis are common comorbidities in patients with Parkinson\u0026apos;s disease [64], [65]. 3) Hypercholesterolemia and statin use are prevalent in Parkinson\u0026apos;s disease [66], [67]; 4) Coronary artery disease is prevalent in Parkinson\u0026apos;s disease, and the use of trimetazidine in these patients has been reported [68], [69]; and, 5) 40% of patients with Parkinson\u0026apos;s disease use polypharmacy [70]. This indirectly suggests that this regimen is not only feasible but has likely been used in a clinical setting, suggesting that it is well tolerated.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the results of this study demonstrate that the BAPST regimen is effective in a large number of cell lines in vitro using clinically relevant doses. Furthermore, information in the literature suggests that drugs in this regimen may have already been administered and tolerated in patients with Parkinson\u0026apos;s disease. However, the results can be considered as the initial step on the investigation of this regimen. Its \u003cem\u003ein vivo\u003c/em\u003e activity needs to be investigated as well as whether its antitumor effects are due to metabolic enzymes inhibition and whether or not indeed impede the reprogramming of metabolism. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSchnog JJB, Samson MJ, Gans ROB, and Duits AJ (2021) An urgent call to raise the bar in oncology. Br J Cancer125(11):1477\u0026ndash;1485. https://doi.org/10.1038/s41416-021-01495-7\u003c/li\u003e\n \u003cli\u003eMichaeli DT and Michaeli T (2022) Overall survival, progression-free survival, and tumor response benefit supporting initial US Food and Drug Administration approval and indication extension of new cancer drugs, 2003-2021. J Clin Oncol 40 (35): 4095\u0026ndash;4106. https://doi.org/10.1200/JCO.22.00535\u003c/li\u003e\n \u003cli\u003eBr\u0026uuml;cher BLDM and Jamall IS (2016) Somatic Mutation Theory - Why it\u0026rsquo;s Wrong for Most Cancers. 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Gerontology 68(10):1081\u0026ndash;1090. https://doi.org/ 10.1159/000521214\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"BAPST, Benserazide, Apomorphine, Pantoprazole, Simvastatin, Trimetazidine, Cancer metabolism, polypharmacotherapy","lastPublishedDoi":"10.21203/rs.3.rs-7768473/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7768473/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e. Key metabolism pathways altered in cancer are glycolysis, glutaminolysis, fatty acid synthesis, cholesterol synthesis, and beta-oxidation. Cancer cells reprogram their metabolism to resist cancer treatment. The BAPST regimen (Benserazide, Apomorphine, Pantoprazole, Simvastatin, Trimetazidine) is a repurposed drug combination that inhibits the key enzymes involved in these five pathways (HK2, GLS1, FASN, HMGCR, and 3-KAT). This study evaluates its vitro antitumor effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e We evaluated 20 human and three murine/rat cancer cell lines. The cells were treated with BAPST (Benserazide: 65 μM, Apomorphine: 8.49 μM, Pantoprazole: 84.3 μM, Simvastatin: 0.162 μM, Trimetazidine: 2.7 μM) or individual drugs for 24–120 hours. Cell viability and clonogenicity were assessed with crystal violet staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e BAPST reduced viability across all cell lines. Major effects were observed in gastric (100%), pancreatic (\u0026gt;90%), and breast (65–90%) cells. Clonogenicity was entirely inhibited in five of six tested cell lines. Individually, benserazide and apomorphine, showed the highest effect, while pantoprazole a minor one. Simvastatin and trimetazidine had minimal/no inhibition or even increased viability and clonogenicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e BAPST demonstrates potent \u003cem\u003ein vitro\u003c/em\u003e antitumor effects across diverse cancer cell lines, warranting further studies to confirm that its effect arises from metabolism enzyme inhibition and impeding metabolic reprogramming, as well as \u003cem\u003ein vivo \u003c/em\u003eantitumor efficacy.\u003c/p\u003e","manuscriptTitle":"In Vitro Antitumor Effects of Bapst, a Repositioned Regimen for the Metabolic Therapy of Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 08:06:52","doi":"10.21203/rs.3.rs-7768473/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4c9a2405-c699-4038-b93d-c706ce449682","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-05T08:06:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 08:06:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7768473","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7768473","identity":"rs-7768473","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}