Monocarboxylate Transporter Inhibitor Research Articles

Abstract 2330: MCT inhibition as a therapeutic strategy to target Enzalutamide-resistant prostate cancer

Abstract Sayani Bhattacharjee, Jonathan Doan, Rebecca Wynn, Nagalakshmi Nadiminty Enzalutamide is a second-generation anti-androgen used for the treatment of prostate cancer. However, almost all patients develop resistance to this drug, thereby creating an urgent need of identifying new approaches that can overcome said resistance. Using parental and Enzalutamide-resistant C4-2B and 22RV1 cells, we targeted cellular metabolism to beat therapeutic resistance. Prostate cancers are more heterogeneous in their use of energy sources compared with other solid tumors and the most common metabolic end product is lactate, which is toxic to cancer cells upon accumulation. Hence, cancer cells upregulate the expression of monocarboxylate transporters (MCTs) that play a pivotal role in lactate efflux. Different MCT isoforms are differentially expressed across prostate cancer progression and presumably according to cellular demands at different stages. Gradual progression from benign prostate, to prostatic intraepithelial neoplasia, to in-situ carcinoma showed an increase in the expression of MCT2 and MCT4, with MCT4 being expressed in invasive prostate cancers. Based on our preliminary data that the expression of MCTs is higher in prostate cancer cells resistant to enzalutamide, we hypothesized that MCT inhibition may be an attractive therapeutic strategy against enzalutamide-resistant prostate cancer cells. Using cell survival, cell proliferation and clonogenic-ability assays, we found that the suppression of MCT activity using the MCT1/2 antagonists AR-C155858 or AZD3965 and the MCT 4 antagonist Syrosingopine not only diminishes the proliferation and survival of prostate cancer cells, but also re-sensitizes resistant cells to treatment with enzalutamide. We also found that treatment with AR-C155858 or AZD3965 either singly, or in combination with Enzalutamide suppressed prostate cancer cell xenograft growth in SCID mice bearing subcutaneously injected parental or Enzalutamide-resistant C4-2B cells. These findings imply that the MCTs have an intrinsic role in the acquisition of resistance to therapy. Citation Format: MCT inhibition as a therapeutic strategy to target Enzalutamide-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2330.

Multifunctional Spiky Topological Nanocapsules for the Discrimination and Differential Inhibition of Inflammation and Cancer.

Accurate discrimination of inflammations and cancers as well as differential inhibition of cancers are significant for early diagnoses and timely treatments. Nanoparticles have become new modalities for diagnosis and therapy. However, they are still challenged by the efficient delivery of multiple reagents into living cells, discriminating multisignals without any interference, and differential treatments of different diseases. Here, multifunctional spiky topological nanocapsules (STNs) are prepared for the discrimination and differential inhibition of inflammation and cancer. With unique spiky hollow architectures, STNs' advantages including excellent loading capacity, enhanced cellular uptake, DNAs' protection against degradation, target-controlled drug release, and efficient endo-/lysosome escape are demonstrated. Therefore, sequential detection of inflammation-related miR-155 (by external modified hairpin DNAs) and the cancer target of monocarboxylate transporter 1 (MCT1) (by internal loaded pH-sensitive carbon dots and MCT1 inhibitor-AZD3965) are achieved. Furthermore, the release of AZD3965 from the cavities of STNs is controlled by the miR-155 amount (first target). Therefore, the released drug of AZD3965 realizes the stage-dependent differential treatment of diseases via cellular acidosis induced by MCT1 inhibition. Via in vivo evaluations of normal, inflammatory, and liver cancer cells/mice, as well as the efficient inhibition of tumor growth, the possibility of STN-based discrimination and differential treatment is confirmed. This would encourage new strategies for multidiagnosis and differential treatment of early-stage cancer.

Toxicokinetic/Toxicodynamic Interaction Studies in Rats between the Drugs of Abuse γ-Hydroxybutyric Acid and Ketamine and Treatment Strategies for Overdose.

γ-hydroxybutyric acid (GHB) is widely abused alone and in combination with other club drugs such as ketamine. GHB exhibits nonlinear toxicokinetics, characterized by saturable metabolism, saturable absorption and saturable renal reabsorption mediated by monocarboxylate transporters (MCTs). In this research, we characterized the effects of ketamine on GHB toxicokinetics/toxicodynamics (TK/TD) and evaluated the use of MCT inhibition and specific receptor antagonism as potential treatment strategies for GHB overdose in the presence of ketamine. Adult male Sprague-Dawley rats were administered GHB 600 mg/kg i.v. alone or with ketamine (6 mg/kg i.v. bolus plus 1 mg/kg/min i.v. infusion). Plasma and urine samples were collected and respiratory parameters (breathing frequency, tidal and minute volume) continuously monitored using whole-body plethysmography. Ketamine co-administration resulted in a significant decrease in GHB total and metabolic clearance, with renal clearance remaining unchanged. Ketamine prevented the compensatory increase in tidal volume produced by GHB, and this resulted in a significant decline in minute volume when compared to GHB alone. Sleep time and lethality were also increased after ketamine co-administration when compared to GHB. L-lactate and AR-C155858 (potent MCT inhibitor) treatment resulted in an increase in GHB renal and total clearance and improvement in respiratory depression. AR-C155858 administration also resulted in a significant decrease in GHB brain/plasma ratio. SCH50911 (GABAB receptor antagonist), but not naloxone, improved GHB-induced respiratory depression in the presence of ketamine. In conclusion, ketamine ingestion with GHB can result in significant TK/TD interactions. MCT inhibition and GABAB receptor antagonism can serve as potential treatment strategies for GHB overdose when it is co-ingested with ketamine.

γ-Hydroxybutyric Acid-Ethanol Drug-Drug Interaction: Reversal of Toxicity with Monocarboxylate Transporter 1 Inhibitors.

The drug of abuse, γ-hydroxybutyric acid (GHB), is commonly co-ingested with ethanol, resulting in a high incidence of toxicity and death. Our laboratory has previously reported that GHB is a substrate for the monocarboxylate transporters (MCTs), necessary for its absorption, renal clearance, and tissue distribution, including across the blood-brain barrier. Our goal was to investigate the drug-drug interaction (DDI) between GHB and ethanol and to evaluate MCT1 inhibition as a strategy to reverse toxicity. The toxicokinetics of this DDI were investigated, including brain-to-plasma concentration ratios, in the presence and absence of ethanol. The toxicodynamic parameters examined were respiratory depression (breathing frequency, tidal volume) and sedation (time of return-of-righting reflex). Ethanol was administered (2 g/kg i.v.) 5 minutes before the intravenous or oral administration of GHB, and MCT1 inhibitors AZD-3965 and AR-C155858 (5 mg/kg i.v.) were administered 60 minutes after GHB administration. Ethanol administration did not alter the toxicokinetics or respiratory depression caused by GHB after intravenous or oral administration; however, it significantly increased the sedation effect, measured by return-to-righting time. AZD-3965 or AR-C155858 significantly decreased the effects of the co-administration of GHB and ethanol on respiratory depression and sedation of this DDI and decreased brain concentrations and the brain-to-plasma concentration ratio of GHB. The results indicate that ethanol co-administered with GHB increases toxicity and that MCT1 inhibition is effective in reversing toxicity by inhibiting GHB brain uptake when given after GHB-ethanol administration. SIGNIFICANCE STATEMENT: These studies investigated the enhanced toxicity observed clinically when γ-hydroxybutyric acid (GHB) is co-ingested with alcohol and evaluated strategies to reverse this toxicity. The effects of the novel monocarboxylate transporter 1 (MCT1) inhibitors AR-C155858 and AZD-3965 on this drug-drug interaction have not been studied before, and these preclinical studies indicate that MCT1 inhibitors can decrease brain concentrations of GHB by inhibiting brain uptake, even when administered at times after GHB-ethanol. AZD-3965 represents a potential treatment strategy for GHB-ethanol overdoses.

Monocarboxylate transporter 1 (MCT1) function modulates adipocyte cell cycle and insulin sensitivity

Enlarged, hypertrophic, insulin resistant adipocytes are a hallmark feature of obesity and contribute to many of the negative metabolic consequences of excess adipose tissue. Although the precise mechanisms remain unclear, adipocyte size is directly correlated with insulin sensitivity wherein smaller adipocytes retain insulin sensitivity, and larger adipocytes often develop insulin resistance, a phenomenon referred to as hypertrophy. It is well established that preadipocytes become growth arrested prior to differentiation and the accumulation of triglyceride lipid droplets that can expand overtime leading to hypertrophic expansion. Therefore, pharmacological strategies aimed at limiting adipocyte hypertrophy and prolonged lipid accumulation may improve insulin sensitivity and have therapeutic benefits in the context of metabolic disorders characterized by insulin resistance. Here, using RNA sequencing and in vitro characterization, we explore the functional consequences of inhibiting the Monocarboxylate Transporter 1 (MCT1), a key regulator of cellular metabolism and proliferation, in adipocytes. We have identified a mechanism through which MCT1 inhibition improves insulin sensitivity in differentiated adipocytes by inducing the re-entry of these cells into the cell cycle. Finally, we provide compelling evidence that warrants the need for the further investigation of the role MCT1 plays in adipocyte metabolism and its potential as a therapeutic drug target for insulin resistance in obesity and other metabolic disorders.

HCAR1/MCT1 Regulates Tumor Ferroptosis through the Lactate-Mediated AMPK-SCD1 Activity and Its Therapeutic Implications

Ferroptosis is a recently discovered form of programed cell death caused by the metabolically regulated lipid peroxidation and holds promise for cancer treatment, but its regulatory mechanisms remain elusive. In this study, we observe that lactate-rich liver cancer cells exhibit enhanced resistance to the ferroptotic damage induced by common ferroptosis inducers such as Ras-selective lethalsmall molecule3 (RSL3) and Erastin and that the monocarboxylate transporter 1 (MCT1)-mediated lactate uptake could promote ATP production in hepatocellular carcinoma (HCC) cells and deactivate the energy sensor AMP-activated protein kinase (AMPK), leading to the upregulation of sterol regulatory element-binding protein 1 (SREBP1) and the downstream stearoyl-coenzyme A (CoA) desaturase-1 (SCD1) to enhance the production of anti-ferroptosis monounsaturated fatty acids. Additionally, blocking the lactate uptake via hydroxycarboxylic acid receptor 1 (HCAR1)/MCT1 inhibition promotes ferroptosis by activating the AMPK to downregulate SCD1, which may synergize with its acyl-coenzyme A synthetase 4 (ACSL4)-promoting effect to amplify the ferroptotic susceptibility. Invitro and invivo evidence confirms that lactate regulates the ferroptosis of HCC cells and highlights its translational potential as a therapeutic target for ferroptosis-based tumor treatment.

Butyrate Permeation across the Isolated Ovine Reticulum Epithelium

Simple SummaryShort-chain fatty acids are the main source of energy for ruminants. The effective uptake of these substrates from the forestomach is a prerequisite for the health and performance of these animals. Thus far, the mechanisms of uptake have been investigated almost exclusively in the epithelium of the largest forestomach section, the rumen. Previous research suggests that the reticulum is also involved in the uptake of short-chain fatty acids, but the mechanisms involved have not been studied and may differ from those known from the rumen epithelium due to the different milieu in this compartment. To investigate this, ovine reticulum epithelium was mounted in Ussing chambers, and the transport of radiolabeled butyrate (as a representative of short-chain fatty acids) across the tissue was measured with and without the addition of inhibitors of particular transport proteins. Our results show that butyrate can be taken up effectively across the reticulum epithelium via pathways that are energized by the Na+/K+-ATPase and may involve monocarboxylate transporters, sodium-proton exchangers, and anion channels. However, our results are not completely congruent to those obtained in the rumen epithelium. These modifications could assure the effective uptake of short-chain fatty acids from the reticulum lumen under the particular conditions (p. e. high pH) of this forestomach compartment.We hypothesized that, due to the high pH of this compartment, the reticulum epithelium displays particular features in the transport of short-chain fatty acids (SCFA). Ovine reticulum epithelium was incubated in Ussing chambers using a bicarbonate-free buffer solution containing butyrate (20 mmol L−1). p-hydroxymercuribenzoic acid (pHMB), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), or ouabain were added to the buffer solution as inhibitors of monocarboxylate transporters, sodium-proton-exchangers, or the Na+/K+-ATPase, respectively. The short-circuit current (Isc) and transepithelial conductance (Gt) were monitored continuously while the flux rates of 14C-labelled butyrate were measured in the mucosal-to-serosal (Jmsbut) or serosal-to-mucosal direction (Jsmbut). Under control conditions, the mean values of Isc and Gt amounted to 2.54 ± 0.46 µEq cm−2 h−1 and 6.02 ± 3.3 mS cm−2, respectively. Jmsbut was 2.1 ± 1.01 µmol cm−2 h−1 on average and about twice as high as Jsmbut. Incubation with ouabain reduced Jmsbut, while Jsmbut was not affected. The serosal addition of EIPA did not affect Jmsbut but reduced Jsmbut by about 10%. The addition of pHMB to the mucosal or serosal solution reduced Jmsbut but had no effect on Jsmbut. Mucosally applied pHMB provoked a transient increase in the Isc. The serosal pHMB sharply reduced Isc. Our results demonstrate that butyrate can be effectively transported across the reticulum epithelium. The mechanisms involved in this absorption differ from those known from the rumen epithelium.

Exogenous pyruvate alleviates UV-induced hyperpigmentation via restraining dendrite outgrowth and Rac1 GTPase activity

BackgroundMelanin is synthesized in melanocytes and transferred to keratinocytes through dendrites. Endogenous pyruvate is a key metabolite for ATP production in glycolysis, and the tricarboxylic acid (TCA) cycle and exogenous pyruvate provide protection against oxidative stress and acidosis in the intercellular space. The function of pyruvate in the regulation of dendrite outgrowth remains to be elucidated. ObjectiveWe examined the effect of pyruvate on dendritic elongation and skin pigmentation MethodsMurine B16F10 melanoma cells and human primary melanocytes were used for in vitro analysis. Melanin quantitation and histochemical staining were performed in a 3D pigmented human skin model. ResultsWe demonstrated the participation of monocarboxylate transporters (MCTs) responsible for the membrane transport of pyruvate in B16F10 melanoma cells. The accumulation of pyruvate occurred in a pH-dependent manner, which was highly sensitive to a specific MCT inhibitor (α-cyano-4-hydroxycinnamic acid). α-MSH-induced morphological changes, including dendrite elongation and growth-cone-like structure, were diminished in B16F10 cells upon treatment with pyruvate. In addition, the number of dendrite branches was reduced in normal human epidermal melanocytes. As the Rho-subfamily of monomeric GTP-binding proteins modulates dendrite formation, we subsequently examined the suppression of Rac1 activation by pyruvate, but not RhoA and Cdc42. Furthermore, pyruvate showed anti-melanogenic effects against UV-induced pigmentation in reconstructed pigmented epidermis, established by co-seeding autologous melanocytes and keratinocytes, which act similar to in vivo skin tissue. ConclusionThese results suggest that pyruvate treatment may be an alternative or additive therapeutic strategy to prevent hyperpigmentation.

The Potential of Lonidamine in Combination with Chemotherapy and Physical Therapy in Cancer Treatment.

Simple SummaryThe unique characteristics of tumor energy metabolism (highly dependent on aerobic glycolysis, namely, the Warburg effect) make it an interesting and attractive target for drug discovery. Radio- and chemoresistance are closely associated with the Warburg effect. Lonidamine (LND), as a glycolytic inhibitor, although having low anticancer activity when used alone, exhibits selectivity to various tumors, and its adverse effects do not overlap when combined with other chemotherapeutic drugs. Therefore, LND may be very promising as a sensitizer of tumors to chemotherapeutic agents and physical therapies. This review summarizes the advance of LND in combination with chemotherapy and physical therapy over the past several decades, as well as the promising LND derivative adjudin (ADD). The underlying sensitizing mechanisms were also analyzed and discussed, which may contribute to an improved therapeutic effect in future clinical cancer treatment.Lonidamine (LND) has the ability to resist spermatogenesis and was first used as an anti-spermatogenic agent. Later, it was found that LND has a degree of anticancer activity. Currently, LND is known to target energy metabolism, mainly involving the inhibition of monocarboxylate transporter (MCT), mitochondrial pyruvate carrier (MPC), respiratory chain complex I/II, mitochondrial permeability transition (PT) pore, and hexokinase II (HK-II). However, phase II clinical studies showed that LND alone had a weak therapeutic effect, and the effect was short and reversible. Interestingly, LND does not have the common side effects of traditional chemotherapeutic drugs, such as alopecia and myelosuppression. In addition, LND has selective activity toward various tumors, and its toxic and side effects do not overlap when combined with other chemotherapeutic drugs. Therefore, LND is commonly used as a chemosensitizer to enhance the antitumor effects of chemotherapeutic drugs based on its disruption of energy metabolism relating to chemo- or radioresistance. In this review, we summarized the combination treatments of LND with several typical chemotherapeutic drugs and several common physical therapies, such as radiotherapy (RT), hyperthermia (HT), and photodynamic therapy (PDT), and discussed the underlying mechanisms of action. Meanwhile, the development of novel formulations of LND in recent years and the research progress of LND derivative adjudin (ADD) as an anticancer drug were also discussed.

Cyclosporine protects from intestinal epithelial injury by modulating butyrate uptake via upregulation of membrane monocarboxylate transporter 1 levels

Background and aimsA relationship between treatment outcomes and intestinal microbiota in patients with inflammatory bowel diseases has been demonstrated. Cyclosporine treatment leads to rapid improvement in severe ulcerative colitis. We hypothesized that the potent effects of cyclosporine would be exerted through relationships between intestinal epithelial cells (IECs) and the host microbiota. The present study was designed to elucidate the effects of cyclosporine on monocarboxylate transporter 1 (MCT1) regulation and butyrate uptake by IECs. MethodsColitis was induced in C57BL6 mice via the administration of 4% dextran sulfate sodium in drinking water, following which body weights, colon lengths, and histological scores were evaluated. To examine the role of butyrate in the protective effects of cyclosporine, MCT1 inhibitor and an antibiotic cocktail was administered and tributyrin (TB; a prodrug of butyrate) was supplemented; MCT1 protein expression and acetylated histone 3 (AcH3) signals in IECs, as well as the MCT1-membrane fraction of Caco-2 cells, were evaluated. To explore butyrate uptake, as s butyrate derivatives, 3-bromopyruvic acid (3-BrPA) and 1-pyrenebutyric acid were used. ResultsTreatment with cyclosporine inhibited body weight loss and colon length shortening. However, treatment with MCT1 inhibitor and the antibiotic cocktail negated the efficacy of cyclosporine, whereas TB supplementation restored its protective effect. Furthermore, cyclosporine upregulated MCT1 expression in the membrane and the AcH3 signal in IECs, while also inducing higher anti-inflammatory cytokine production compared to that in the vehicle-treated mice. The transcription level of MCT1 mRNA in IECs and Caco-2 cells did not increase with cyclosporine treatment; however, cyclosporine treatment increased membrane MCT1 expression in these cells and uptake of butyrate derivative. ConclusionCyclosporine treatment modulates butyrate uptake via the post-transcriptional upregulation of membrane MCT1 levels in IECs.

Lactate Exposure Promotes Immunosuppressive Phenotypes in Innate Immune Cells.

Lactate secreted by tumors is not just a byproduct, but rather an active modulator of immune cells. There are few studies aimed at investigating the true effect of lactate, which is normally confounded by pH. Such a knowledge gap needs to be addressed. Herein, we studied the immunomodulatory effects of lactate on dendritic cells (DCs) and macrophages (MΦs). Bone marrow-derived innate immune cells were treated with 50mM sodium lactate (sLA) and incubated for 2days or 5days at 37°C. Controls included media, lipopolysaccharide (LPS), MCT inhibitors (α-cyano-4-hydroxycinnamic acid and AR-C15585). Flow cytometric analysis of immune phenotypes were performed by incubating cells with specific marker antibodies and viability dye. Differential expression analyses were conducted on R using limma-voom and adjusted p-values were generated using the Bejamini-Hochberg Procedure. Lactate exposure attenuated DC maturation through the downregulation of CD80 and MHCII expression under LPS stimulation. For MΦs, lactate exposure resulted in M2 polarization as evidenced by the reduction of M1 markers (CD38 and iNOS), and the increase in expression of CD163 and Arg1. We also revealed the role of monocarboxylate transporters (MCTs) in mediating lactate effect in MΦs. MCT4 inhibition significantly boosted lactate M2 polarization, while blocking of MCT1/2 failed to reverse the immunosuppressive effect of lactate, correlating with the result of gene expression that lactate increased MCT4 expression, but downregulated the expression of MCT1/2. This research provides valuable insight on the influence of metabolic products on tumor immunity and will help to identify novel metabolic targets for augmenting cancer immunotherapies.

From Warburg effect to Reverse Warburg effect; the new horizons of anti-cancer therapy

An old ideology of killing the cancer cells by starving them is the underlying concept of the Warburg effect. It is the process of aerobic glycolysis exhibited by the cancer cells irrespective of anaerobic glycolysis or mitochondrial oxidative phosphorylation following by their healthy counterparts. Dr Otto Heinrich Warburg proposed this abnormal metabolic behaviour of tumour cells in 1920. This phenomenon illustrates the metabolic switching in tumour cells from oxidative phosphorylation to aerobic glycolysis triggered by an injury to the mitochondrial respiration. A modernised perspective of the Warburg hypothesis termed the Reverse Warburg effect introduced in 2009, with a two-compartment model describing the metabolic symbiosis between cancer cells and its neighbouring stromal cells or cancer-associated fibroblasts. This theory is elucidating the aerobic glycolysis occurring in cancer-associated fibroblasts which leads to the generation and deposition of the lactate in tumour microenvironment along with its significance. The transportation of lactate to and from the cancer cell and extracellular space is facilitated by the lactate transporters called monocarboxylate transporters. This lactate generated irrespective of the hypoxic or aerobic conditions acts as a primary metabolic fuel for the cancer cells. Besides, it will create a tumour microenvironment that is favouring the progression and metastasis of malignancy through several means. Overall, the lactate produced through this metabolic reprogramming is supporting and worsening the conditions of cancer. The concept of the Reverse Warburg effect proposes a new anti-cancer treatment modality by preventing the generation and transport of lactate through the inhibition of monocarboxylate transporters and in turn, defeating the cancer disease by arresting the cancer cells along with silencing tumour microenvironment.

T-cells produce acidic niches in lymph nodes to suppress their own effector functions

The acidic pH of tumors profoundly inhibits effector functions of activated CD8 + T-cells. We hypothesize that this is a physiological process in immune regulation, and that it occurs within lymph nodes (LNs), which are likely acidic because of low convective flow and high glucose metabolism. Here we show by in vivo fluorescence and MR imaging, that LN paracortical zones are profoundly acidic. These acidic niches are absent in athymic Nu/Nu and lymphodepleted mice, implicating T-cells in the acidifying process. T-cell glycolysis is inhibited at the low pH observed in LNs. We show that this is due to acid inhibition of monocarboxylate transporters (MCTs), resulting in a negative feedback on glycolytic rate. Importantly, we demonstrate that this acid pH does not hinder initial activation of naïve T-cells by dendritic cells. Thus, we describe an acidic niche within the immune system, and demonstrate its physiological role in regulating T-cell activation.

Reducing monocarboxylate transporter MCT1 worsens experimental diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most common complications in diabetic patients. Though the exact mechanism for DPN is unknown, it clearly involves metabolic dysfunction and energy failure in multiple cells within the peripheral nervous system. Lactate is an alternate source of metabolic energy that is increasingly recognized for its role in supporting neurons. The primary transporter for lactate in the nervous system, monocarboxylate transporter-1 (MCT1), has been shown to be critical for peripheral nerve regeneration and metabolic support to neurons/axons. In this study, MCT1 was reduced in both sciatic nerve and dorsal root ganglia in wild-type mice treated with streptozotocin (STZ), a common model of type-1 diabetes. Heterozygous MCT1 null mice that developed hyperglycemia following STZ treatment developed a more severe DPN compared to wild-type mice, as measured by greater axonal demyelination, decreased peripheral nerve function, and increased numbness to innocuous low-threshold mechanical stimulation. Given that MCT1 inhibitors are being developed as both immunosuppressive and chemotherapeutic medications, our results suggest that clinical development in patients with diabetes should proceed with caution. Collectively, our findings uncover an important role for MCT1 in DPN and provide a potential lead toward developing novel treatments for this currently untreatable disease.

Tracing Nutrient Flux Following Monocarboxylate Transporter-1 Inhibition with AZD3965.

The monocarboxylate transporter 1 (MCT1) is a key element in tumor cell metabolism and inhibition of MCT1 with AZD3965 is undergoing clinical trials. We aimed to investigate nutrient fluxes associated with MCT1 inhibition by AZD3965 to identify possible biomarkers of drug action. We synthesized an 18F-labeled lactate analogue, [18F]-S-fluorolactate ([18F]-S-FL), that was used alongside [18F]fluorodeoxyglucose ([18F]FDG), and 13C-labeled glucose and lactate, to investigate the modulation of metabolism with AZD3965 in diffuse large B-cell lymphoma models in NOD/SCID mice. Comparative analysis of glucose and lactate-based probes showed a preference for glycolytic metabolism in vitro, whereas in vivo, both glucose and lactate were used as metabolic fuel. While intratumoral L-[1-13C]lactate and [18F]-S-FL were unchanged or lower at early (5 or 30 min) timepoints, these variables were higher compared to vehicle controls at 4 h following treatment with AZD3965, which indicates that inhibition of MCT1-mediated lactate import is reversed over time. Nonetheless, AZD3965 treatment impaired DLBCL tumor growth in mice. This was hypothesized to be a consequence of metabolic strain, as AZD3965 treatment showed a reduction in glycolytic intermediates and inhibition of the TCA cycle likely due to downregulated PDH activity. Glucose ([18F]FDG and D-[13C6]glucose) and lactate-based probes ([18F]-S-FL and L-[1-13C]lactate) can be successfully used as biomarkers for AZD3965 treatment.

In Vitro and In Vivo Efficacy of AZD3965 and Alpha-Cyano-4-Hydroxycinnamic Acid in the Murine 4T1 Breast Tumor Model

Monocarboxylate transporter 1 (MCT1) represents a potential therapeutic target in cancer. The objective of this study was to determine the efficacy of AZD3965 (a specific inhibitor of MCT1) and α-cyano-4-hydroxycinnamic acid (CHC, a nonspecific inhibitor of MCTs) in the murine 4T1 tumor model of triple-negative breast cancer (TNBC). Expression of MCT1 and MCT4 in 4T1 and mouse mammary epithelial cells were determined by Western blot. Inhibition of MCT1-mediated L-lactate uptake and cellular proliferation by AZD3965 and CHC was determined. Mice bearing 4T1 breast tumors were treated with AZD3965 100mg/kg i.p. twice-daily or CHC 200mg/kg i.p. once-daily. Tumor growth, metastasis, intra-tumor lactate concentration, immune function, tumor MCT expression, and concentration-effect relationships were determined. AZD3965 and CHC inhibited cell growth and L-lactate uptake in 4T1 cells. AZD3965 treatment resulted in trough plasma and tumor concentrations of 29.1 ± 13.9 and 1670 ± 946nM, respectively. AZD3965 decreased the tumor proliferation biomarker Ki67 expression, increased intra-tumor lactate concentration, and decreased tumor volume, although tumor weight was not different from untreated controls. CHC had no effect on tumor volume and weight, or intra-tumor lactate concentration. AZD3965 treatment reduced the blood leukocyte count and spleen weight and increased lung metastasis, while CHC did not. These findings indicate AZD3965 is a potent MCT1 inhibitor that accumulates to high concentrations in 4T1 xenograft tumors, where it increases tumor lactate concentrations and produces beneficial effects on markers of TNBC; however, overall effects on tumor growth were minimal and lung metastases increased.

Targeted sensitization of tumor cells for radiation through monocarboxylate transporters 1 and 4 inhibition in vitro.

Monocarboxylate transporters (MCT) 1, 2 and 4 play an important role in tumor metabolism. The amount of lactate transported by MCT's highly correlates with overall survival. Furthermore, glycolysis and hypoxia are possible causes for radiation resistance. An oral squamous cell carcinoma cell line (CAL27, ATCC) was analyzed in an in vitro cell assay. After incubation with two different inhibitors for MCT1 (AR-C122982/SR-13800 and AR-C155858/SR-13801, Tocris) or for MCT4 (simvastatin, Sigma-Aldrich and 2-cyano-3-(4-hydroxyphenyl)-2-propenoic acid (CHC), Tocris), cells were irradiated with six gray with a Gammacell 2000 (Nuklear Data). For analysis, cell counting assay, wound healing assay, MTT assay and clonogenic assay were applied. Cell counting assay showed significant lower results for simvastatin, CHC and for the highest concentrations of AR-C122982 and AR-C155858 (p < 0.03). Additionally, cell counts decreased significantly with irradiation after 72 hours (p < 0.05) only for AR-C122982, CHC and simvastatin. The clonogenic assay confirmed these results with substantially reduced growth when incubated with CHC, simvastatin and AR-C155858 (p < 0.002). Furthermore, MCT1 and 4 inhibition led to highly reduced migration (p < 0.05). There again, comparing the wound healing assay of irradiated to non-irradiated tests showed contrary results (controls: p < 0.001; AR-C155858: p > 0.05; AR-C122982: p > 0.32; CHC: p > 0.1; simvastatin p > 0.1). The MTT assay presented significant effects with MCT1 and 4 inhibition (simvastatin/AR-C122982/CHC: p < 0.007). Irradiated cells showed significantly lower expression after only 48 h compared to non-irradiated cells (simvastatin/AR-C122982/CHC: p < 0.02). Inhibition of MCT, especially MCT4 may represent a possible tool to overcome radiation resistance in tumor cell lines. MCT Inhibitors may be used as a possible therapeutic approach to sensitize OSCC to radiation.

Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in triple-receptor negative breast cancer

BackgroundGlutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients.MethodsA panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity.ResultsThe anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness.ConclusionThis study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.

Tumor-Targeted Inhibition of Monocarboxylate Transporter 1 Improves T-Cell Immunotherapy of Solid Tumors.

Export of lactic acid from glycolytic cancer cells to the extracellular tumor milieu has been reported to enhance tumor growth and suppress antitumor immunity. In this study, a pH-activatable nanodrug is reported for tumor-targeted inhibition of monocarboxylate transporter-1 (MCT1) that reverses lactic acid-induced tumor immunosuppression. The nanodrug is composed of an MCT1 inhibitor (AZD3965) loaded inside the ultra-pH-sensitive nanoparticles (AZD-UPS NPs). AZD-UPS NP is produced by a microfluidics method with improved drug loading efficiency and optimal nanoparticle size over sonication methods. The nanodrug remains as intact micelles at pH 7.4 but rapidly disassembles and releases payload upon exposure to acidic pH. When combined with anti-PD-1 therapy, AZD-UPS NP leads to potent tumor growth inhibition and increases survival in two tumor models over oral administration of AZD3965 at dramatically reduced dose (>200-fold). Safety evaluations demonstrate reduced drug distribution in heart and liver tissues with decrease in toxic biomarkers such as cardiac troponin by the nanodrug. Increased T-cell infiltration and reduced exhaustive PD1+ Tim3+ T cells are found in tumors. These data illustrate that tumor-targeted inhibition of MCT1 can reverse the immune suppressive microenvironment of solid tumors for increased safety and antitumor efficacy of cancer immunotherapy.

Stiripentol alleviates neuropathic pain in L5 spinal nerve-transected mice.

Antiepileptic drugs are used not only for the treatment of epilepsy but also for that of neuropathic pain. However, their action mechanisms have not always been well explained. Stiripentol, an effective antiepileptic drug indicated as a therapeutic for Dravet syndrome, was recently shown to act as an inhibitor of lactate dehydrogenase in astrocytes. In this present study, we examined the effect of stiripentol on neuropathic pain in L5 spinal nerve-transected mice. We carried out behavioral tests using calibrated von Frey filaments and the immunohistochemistry of glial fibrillary acidic protein, an astrocyte marker, in L5 spinal nerve-transected mice after intrathecal administration of drugs. Like other anticonvulsants such as gabapentin and carbamazepine, stiripentol alleviated mechanical hyperalgesia induced by L5 spinal nerve transection in a dose-dependent manner, when intrathecally administered to mice 7, 14, and 28days after L5 spinal nerve transection. Likewise, α-cyano-4-hydroxycinnamic acid, a broad inhibitor of monocarboxylate transporters, diminished mechanical hyperalgesia induced by L5 spinal nerve transection. Simultaneous administration of L-lactate negated the analgesic effect elicited by stiripentol, carbamazepine or α-cyano-4-hydroxycinnamic acid, but not that by gabapentin. None of the anticonvulsants affected the immunoreactivity of glial fibrillary acidic protein. This present study demonstrated that stiripentol was effective against neuropathic pain and suggested that the astrocyte-neuron lactate shuttle was involved in such pain.