Two dichloric compounds inhibit in vivo U87 xenograft tumor growth

ABSTRACT

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK) that has been shown to reverse the Warburg effect and cause tumor cell death. Clinical research into the anti-cancer activity of DCA revealed high dosage requirements and reports of toxicity. While there have been subsequent mechanistic investigations, a search for DCA alternatives could result in a safer and more effective anticancer therapy. This study evaluates eight small compounds with a conserved dichloric terminal and their in vitro and in vivo potential for anticancer activity. Initial viability screening across six cancer cell lines reveals even at 10 mg/mL, compound treatments do not result in complete cell death which suggests minimal compound cytotoxicity. Furthermore, in vivo data demonstrates that cationic dichloric compounds DCAH and DCMAH, which were selected for further testing based on highest in vitro viability impact, inhibit tumor growth in the U87 model of glioblastoma, suggesting their clinical potential as accessible anti-cancer drugs. Immunoblotting signaling data from tumor lysates demonstrates that the mechanism of actions of cationic DCAH and DCMAH are unlikely to be consistent with that of the terminally carboxylic DCA and warrants further independent investigation.

Introduction

Based on 2016 data, cancer is the second leading cause of death in the United States, resulting in nearly 9 million deaths a year worldwide.¹ Substantial obstacles in the development of safe anti-cancer therapeutics include the remarkable adaptability of cancer cells in a stressed environment, which includes evasion of apoptosis and alteration of major signaling pathways, along with the relative inability of conventional chemotherapies to target only malignant cells while leaving normal healthy cells unharmed. Dichloroacetic acid, the salt form of dichloroacetate (DCA), is a widely-accessible and orally-administered drug originally used to treat lactic acidosis.^2,3 Intravenous DCA treatment of lactic acidosis in humans has been administered at a C_max exceeding 100 µg/ml, demonstrating high tolerability.⁴

In recent years, DCA (sodium salt) has been proposed for clinical trials as an anti-cancer treatment.^5,6 A mixture of in vitro and case studies has linked the compound to the inhibition of breast cancer,⁷ colon cancer,⁸ prostate cancer,⁹ glioblastoma,¹⁰ and lung cancer,¹¹ as well as other forms of cancer.^12–17 The mechanism of action of DCA is suggested to be metabolic modulation that, for tumor cells, targets the Warburg effect via pyruvate dehydrogenase kinase (PDK) inhibition.^5,18

The Warburg effect, where cancer cells exhibit a metabolic preference towards anaerobic glycolysis for energy production,¹⁹ is accompanied by cancer-promoting phenotypes including ample anabolic pathway support, extracellular acidosis facilitating metastatic potential, and mitochondrial damage that inhibits both oxidative phosphorylation and pro-apoptotic pathways.^20–22 These phenotypes can be traced through the dual fate of pyruvate, where its cytosolic fermentation into lactate promotes cancer-supporting phenotypes and where its mitochondrial conversion into acetyl-CoA promotes a healthy, normal cell function.^23–25 Pyruvate metabolism is ultimately controlled by the gatekeeping mitochondrial protein pyruvate dehydrogenase (PDH), which catalyzes the rate-limiting decarboxylation of pyruvate into acetyl-CoA. PDH is inhibited through phosphorylation by PDK, and this inhibition can be reversed via dephosphorylation by pyruvate dehydrogenase phosphatase (PDP).²⁶ PDK has been shown to be upregulated in several cancer types,^27,28 and while it has gained traction as an anti-cancer target the only known inhibitors of PDK are pyruvate and DCA.^29–32 Due to structural similarities, DCA is hypothesized to bind and inhibit PDK via the same mechanism as pyruvate³³ without fueling anaerobic glycolysis and subsequent tumor growth. Unfortunately, reports of cytotoxicity and high dosage requirements prevent DCA from being developed into an anti-cancer drug.^13,34,35 Additionally, DCA studies have mainly focused on finding mechanistic pathways, with a few targeted efforts to modify its chemical structure.

In this study, we tested eight DCA-like compounds, dichloroacetate structurally modified analogs (DCASMAs), for potential anti-tumor effects (Figure 1). Of note is the identification of two promising DCASMAs, DCAH and DCMAH, that would benefit from further validation. We demonstrated the minimal apparent cytotoxicity of these compounds as evidenced by in vitro viability assays, where complete cell death was not achieved even at concentrations as high as 10 mg/mL. While we selected the DCASMAs for their conserved dichloric terminal and resulting similarity to DCA, we acknowledged the potential for a different mechanism due to the varying properties of the DCASMAs’ side groups. A preliminary probing into DCA-related protein levels including PDH, PDK and PDP did implicate a DCASMA mechanism distinct from DCA, prompting the need for further studies. Finally, we demonstrated the anti-tumor effect of DCAH and DCMAH on U87 xenograft growth inhibition that is comparable in effect and toxicity to both DCA and the well-known chemotherapy drug bevacizumab.

Figure 1.

Compound structures. (a) Pyruvate and DCA, the only known PDK inhibitors. (b) The salt form of the eight selected DCASMAs tested in this study with conserved dichloric terminals. The first two amines, DCAH and DCMAH, were designed as cations to investigate the effects switching polarity from that of DCA. All remaining compounds preserved the anionic nature of DCA but incorporated other changes. DCTP and DCPA have opposite effects on electron distribution from DCA. The CH₃ of DCPA is an electron donor group, and the CF₃ of DCTP is an electron acceptor group. The remaining compounds have similar functional groups but differ by replacing the hydrogen of DCA, which varies the spatial arrangement of the overall molecule.

Materials and methods

Compounds

2,2-dichloroethan-1-amine hydrochloride (DCAH), (2,2-dichloroethyl)(methyl)amine hydrochloride (DCMAH), 2-(2,2-dichlorocyclopropyl)acetic acid (DCCP), and 2,2-dichlorocyclopropane-1-carboxylic acid (DCPC) were purchased from Enamine (Monmouth Jct., NJ); 2,2-Dichloropropionic acid (DCPA), 2,2-Dichloro-3,3,3-trifluoropropionic acid (DCTP), and 2,2-Dichloro-1-methyl-cyclopropanecarboxylic acid (DCMC) were purchased from Sigma Aldrich (St. Louis, MO); 10,10-dichlorotricyclo[7.1.0.0 ~ 4,6~]decane-5-carboxylic acid (DCTDC) was purchased from Chem Bridge (San Diego, CA).

Cell culture

Human glioblastoma cells (U87), human hepatocellular carcinoma cells (HUH7), and human pancreatic cancer cell lines (PANC-1 and Mia-PaCa2) were cultivated in Dulbecco Modified Eagle Medium‎ (DMEM) containing 10% fetal bovine serum (FBS). Human gastric cancer cells (KATO-III) were cultivated in Iscove’s Modified Dulbecco’s Medium containing 20% FBS. Human colon cancer cells (HCT116) were cultivated in McCoy’s 5a medium containing 10% FBS. Cell lines were maintained under a humidified atmosphere of 5% CO₂ at 37°C and split 1:4 at 70–90% confluence. All cell lines, media, and FBS were obtained from the American Type Culture Collection (ATCC; Manassas, VA). 0.05% Trypsin-EDTA solution was purchased from Thermo Fisher Scientific; Waltham, MA).

Cytotoxicity assays

Each compound was tested in each of the cultured cell lines. Ten concentrations of each compound were tested: 10 mg/ml, 2 mg/ml, 0.4 mg/ml, 0.08 mg/ml, 0.016 mg/ml, 0.0032 mg/ml, 0.00064 mg/ml, 0.000128 mg/ml, 0.0000256 mg/ml, and 0.00000512 mg/ml. For reference, molecular weights of compounds are included in supplementary materials (Table S1). 5–10 × 10³ cells were cultured and seeded with 100 µL into 96-well plates 24 h prior to treatment. After compound addition the cells were incubated for 72 hours. The cellular toxicity was quantified with alamarBlue cell viability indicator or Trypan blue indicator (both were purchased from Thermo Fisher Scientific). For trypan blue assay, cells were trypsinized for 2–3 min in a humidified 37°C incubator and suspended in complete growth medium with trypan blue stock solution (50:50); cells were counted immediately using a hemacytometer. IC-50 values were calculated by performing non-linear regression in GraphPad Prism 8.0. Replicates were evaluated using the Student’s t-test (p < .05).

Animal studies

Altogen Labs received Institutional Animal Care and Use Committee (IACUC) approval for the LC03341 study protocol on October 2, 2017 (Altogen Labs IACUC protocol number 4–05719). Female Mus Musculus NU(NCr)-Foxn1nu nude mice (9–10 weeks old) were purchased from the Harlan Laboratories (Indianapolis, IN). Mice underwent a mandatory 1-week acclimatization period. The animals were housed in individual ventilated cages (up to 5 mice per cage) under the following conditions: temperature, 22–25°C; humidity, 40–60%; light cycle, 12 hours of light and 12 hours of darkness; irradiated sterile individually ventilated cages; irradiated corncob bedding; immunocompromised mouse diet (i.e., irradiation sterilized dry granule food); and sterile autoclaved water. All animal procedures and maintenance were conducted in accordance with the institutional guidelines. Mice were housed at the animal facility at Altogen Labs. Phosphate buffered saline (PBS) was purchased from BioRad (Hercules, CA), Matrigel was purchased from Corning Life Sciences (Tewksbury, MA), and additional reagents were purchased from Thermo Fisher Scientific unless otherwise specified.

Cell culture

Tumor cells were maintained in vitro as a monolayer culture in DMEM medium (ATCC) supplemented with 10% FBS at 37ºC in an atmosphere of 5% CO₂ in air. Tumor cells were sub-cultured twice a week using trypsin-EDTA treatment (0.25% Trypsin-EDTA). Cells in exponential growth phase were harvested and counted for tumor inoculation.

Tumor inoculation

Prior to xenotransplantation, tumor cells were analyzed for cell count and cell viability (99%) using GUAVA PCA flow cytometry (Sigma Aldrich, St. Louis, MO). Each mouse was inoculated subcutaneously at the flank with either U87, HUH7, or HCT116 tumor cells. The injection consisted of 1.0 × 10⁶ cells suspended in 0.1 ml of 1 × PBS mixed with Matrigel (1:1) according to the 50% Matrigel protocol (Altogen Labs SOP 6.012). Measurable tumors (50–100 mm³) developed 96 hours post-xenotransplantation, and 25 animals with approximately 50–100 mm³ tumors were selected for follow-up experiments.

Group assignment

The animals were randomized into five groups as described below. The date of tumor cell inoculation was denoted as Day 0. Compound administration started on Day 5. Before grouping and treatment, all animals were weighed, and the tumor volumes were confirmed (approx. 50–100 mm³) using an electronic caliper. Because the tumor volume can affect the effectiveness of any given treatment, mice were assigned into groups using a randomized block design: 1) the experimental animals were divided into homogeneous blocks based on their tumor volume and 2) within each block, experimental animals were randomization to different treatment groups. This randomization method ensured that each animal had the same probability of being assigned to any given treatment group, minimizing systematic error.

Compound administration

Intravenous (IV) administration of compounds was performed using a Genie Touch Syringe Pump (Kent Scientific; Torrington, CT), metered to deliver tail vein IV injections. Terumo Surshield Safety Winged Infusion Sets (S25BLS, 25G × 3/4ʺ, Terumo Medical Products; Somerset, NJ) were used for tail vein administration. Subjects were treated with DCASMA (25 mg/kg), bevacizumab (10 mg/kg), or PBS (control) every three days, receiving a total of four doses of their respective treatment. Compounds were prepared, and an injection volume 20% larger than the delivery volume was loaded into the syringes (Kent Scientific), which ensured the injection volume was sufficient once the tubing was filled. All work was performed in a biological safety cabinet (Kent Scientific), including compound preparation and syringe loading. A new syringe was used for each individual compound and control group samples.

Observation and data collection

After tumor cell inoculation, animals were checked daily for morbidity and mortality. At the time of routine monitoring, animals were visually checked for any adverse effects of treatment on tumor growth, normal behavior (mobility, posture, etc.), food and water consumption, body weight gain/loss, and eye/hair matting, as well as any other abnormal effects. Death and observed clinical signs were recorded in accordance with Altogen Labs IACUC protocols.

Tumor volumes were measured three times a week in two dimensions using an electronic caliper. The volumes were expressed in mm³ using the formula V = 0.5 a × b², where a and b are the long and short diameters of the tumor, respectively. Dosing and tumor volume measurement were conducted in a laminar flow cabinet. Statistical analysis of the difference in tumor volumes among the experimental groups and the analysis of compound activity were conducted from the data obtained after the final treatment.

Immunoblotting

Mouse xenografts of the HUH7, U87, and HCT116 cell lines treated with DCAH and DCMAH were quantitatively analyzed for the presence of PDH, PDK2, PDP1, p53, phospho-PDH, caspase-3 and HIF-1α using the Wes instrument (Protein Simple; San Jose, CA) according to the manufacturer’s protocol (n = 2). Images were acquired with the contrast set to white −100 and black 4000 for standardization. Mice treated with PBS served as controls. Thirty days post xenotransplantation tumor extracts were retrieved surgically and immediately lysed in RIPA buffer supplemented with protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO). Biological replicates were obtained for each sample and control.

A BCA assay (BioRad) was used to normalize the protein concentration from each sample obtained from the xenografts. Each sample was diluted to a final protein concentration of 0.2 mg/ml for PDH, PDP1, and p53 detection and 2 mg/ml for HIF-1α, PDK2, phospho-PDH and caspase-3 detection. The dilution buffer was 91% PBS, 1% phosphatase inhibitors (Phosphatase Inhibitor Cocktail B; Santa Cruz Biotechnology; Dallas, TX), and 8% Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Six primary antibodies were diluted in the Wes Antibody Diluent II solution (Protein Simple): 1:20 anti-rabbit PDH (Cell Signaling Technology [CST]; Danvers, MA; Cat# 2784S, RRID:AB_2162928), 1:20 anti-rabbit PDK2 (Proteintech Group; Rosemont, IL; Cat# 15647–1-AP, RRID:AB_2268006), 1:20 anti-rabbit PDP1 (CST; Cat# 65575), 1:20 anti-mouse p53 (Santa Cruz Biotechnology; Cat# sc-126, RRID:AB_628082), 1:20 anti-mouse total and phospho-PDH E1 subunit (Abcam; Cambridge, United Kingdom; Cat# 92696 & 110334), 1:20 anti-rabbit caspase-3 (Neomarkers; Freemont, CA; Cat# 1197) and 1:20 anti-rabbit HIF-1α (Millipore; Burlington, MA; Cat# 07–1585, RRID:AB_10807017). Target proteins were detected using a horseradish peroxidase (HRP)-conjugated secondary antibody (Protein Simple). Statistical analysis was conducted using Compass software (Protein Simple) and Microsoft Excel.

Statistical analysis

Data points were obtained in a minimum of duplicates and were presented as mean ± SEM. Statistical differences between and among the experimental groups were determined using a two-tailed t-test. A p-value less than 0.05 was considered statistically significant.

Results

DCASMAs were cytotoxic to in vitro tumor cells at high concentrations

DCA is a small dicarbon molecule with one dichloric and one carboxylic terminal, each being a distinct functional group. We incorporated the fundamental characteristics of DCA, including structure, charge, spatial parameters, negatively charged polar configuration, the C-Cl group with high reaction potential, and C-H groups into our design of eight compounds, referred to as DCASMAs (Figure 1). For our initial screen we studied the effects of the DCASMAs on cell viability across six human cancer cell lines: Kato-3, PANC1, U87, HUH7, Mia-PaCa2, and HCT116.

A viability dose-response curve was performed to assess compound potency using two methods of metabolically active cells (alamarBlue) and a cell death assay (trypan blue exclusion). However, even at the maximum doses (up to 70 mM), most treatments did not substantially impact viability (Table 1). Because DCAH and DCMAH had the highest impact on viability consistent across both methods, we selected these compounds for further examination.

Table 1.

Lowest observed percent viability.

	DCCP	DCPC	DCAH	DCMAH	DCPA	DCTDC	DCTP	DCMC
A
PANC1	69.0	73.9	50.7	57.7	76.9	81.1	66.2	76.4
Kato-3	78.8	75.1	60.7	56.0	67.6	90.1	70.1	77.6
U87	62.7	61.1	35.8	37.2	46.9	67.2	56.0	50.2
HUH7	58.7	63.3	53.2	52.6	62.5	65.5	70.1	62.3
MiaPaCa-2	56.7	55.7	55.6	57.1	57.4	63.1	65.9	60.4
HCT-116	48.2	70.9	44.9	40.0	60.2	77.3	61.2	45.3
B
PANC1	74.0	81.3			81.7	86.3	75.7	84.0
Kato-3	69.7	75.3			75.0	89.7	75.0	80.3
U87	72.0	70.0			58.7	71.0	73.0	65.7
HUH7	64.7	75.3			75.3	71.7	79.7	72.7
MiaPaCa-2	73.0	78.0			75.3	78.0	77.0	75.0
HCT-116	65.0	77.3			70.7	82.0	72.3	68.7

DCASMA dose-response curves were performed on six cell lines (n = 3). Concentrations ranged from 10 mg/ml to 0.00000512 mg/ml. A) A viability indicator measuring metabolically active cells was used; the values reported are the lowest viability achieved with corresponding treatment normalized to the highest observed viability. B) A trypan blue exclusion assay was performed; the values reported reflect highest cell death observed reported as percent of alive cells.

DCA and the DCASMAs were submitted to the NCI-60 human tumor lines screen which was performed by the NIH National Cancer Institute’s Developmental Therapeutics Program. This standard protocol screen involves applying compounds at 0.01 mM concentrations to more than 60 human tumor cell lines and monitoring for growth inhibition or death.¹⁵ Because our compounds just started to show effects on viability in the 10 mM range, the screen did not provide conclusive results (Fig. S1).

DCAH and DCMAH had varying effects on protein expression in xenografted tumors

To assess whether DCMAH and DCAH affect cell function in a manner similar to DCA, we analyzed mouse xenografts for changes in seven key protein levels: PDH, phospho-PDH, PDP1, PDK2, p53, caspase 3, and HIF-1α.

We used the most sensitive cell lines, HUH7, U87 and HCT116, for our xenograft models (Figure 2). DCAH and DCAH had varying effects across cell lines, and each treatment was interpreted individually. Phospho-PDH levels were considered to be negative indicators of oxidative phosphorylation flux (with the exception of DCMAH treatments which were inconclusive regarding phospho-PDH levels, not shown). While other related protein levels (total PDH, PDK2, and PDP1) were often conflicting, the ratio of inactive PDH was considered the closest reflection of acetyl-CoA production and subsequent ETC activity. Caspase-3 was a measure of apoptosis; HIF-1α was a measure of both positive tumor health and an upstream activator of PDK as PDK expression is reportedly increased via trans-activation by hypoxia inducible factor 1 (HIF-1).^26,36

Figure 2.

Changes of relative protein levels in tumors following DCASMA compound treatment. (A-C) Mouse xenograft models of HUH7, U87, and HCT116 cell lines were treated with 25 mg/kg of compound as previously described (n = 2). (a) Normalized tumor lysate protein levels were analyzed by Western immunoblotting. PDH levels included both the α1 and α2 subunit levels. (b) Quantified results normalized to the respective control are reported in table form ± SEM. PDH inactivation was assessed using a phospho-specific antibody and comparing those levels with total PDH levels. Phospho-PDH levels for DCMAH treatment was inconclusive (not shown). (c) A diagram of the observed results of DCASMA treatment. Dotted lines indicate the protein expression levels that were altered by DCASMAs DCAH and DCMAH treatment (in select cell lines). Molecules are shown as plain text and processes are represented by bubbles.

In the HCT116 colorectal cancer cell line, DCAH treatment yielded an overall suggested decrease in ETC flux, caspase-3 levels were unaffected and both HIF-1α and p53 levels increased. This suggests an active Warburg effect but potentially increased tumor suppressor activity. DCMAH treatment in the same cell line also demonstrated unchanged apoptosis and an increase in p53, yet HIF-1α levels decreased, which suggests an impact on tumors via an apoptosis-independent pathway.

In HUH7 hepatoma cells DCAH treatment resulted in a decrease in ETC flux and HIF-1α, a slight increase in p53 and a nearly three-fold increase in caspase-3, which suggests a largely apoptosis-mediated effect on tumor cells with a potential impact on the cell cycle and growth support. DCMAH treatment in HUH7 cells caused comparable changes in HIF-1α and p53 levels, yet caspase-3 levels decreased, suggesting an apoptosis-independent effect on tumors.

Lastly, DCAH treatment in the U87 glioblastoma cell line resulted in decreased HIF-1α and caspase-3 with increased p53 and ETC flux, as evidenced by the two-fold higher ratio of inactive/phosphorylated to total PDH in the control. This demonstrates a potential anti-tumor mechanism of DCAH mediated through metabolism, HIF-1α or p53, but independent of apoptosis. Similar expression levels were observed with DCMAH treatment in U87 cells; HIF-1α and caspase-3 decreases were accompanied by unchanged p53 levels. This indicates DCMAH activity may be mediated through HIF-1α but is unlikely to affect p53 or caspase-3 pathways.

Overall, depending on the cell line, DCASMAs DCAH and DCMAH have affected PDH and associated enzymes PDK and PDP, p53, caspase-3, and HIF-1α; this means the two compounds have the potential to alter cellular pathways including oxidative phosphorylation, cell cycle arrest, tumor growth support and apoptosis (Figure 2(c)). The data do not validate any mechanism of action for either compound, and primarily serve to demonstrate a cellular effect distinct from that of DCA.

DCAH and DCMAH decreased tumor growth in a xenograft mouse model

To more thoroughly analyze the effects of our compounds in vivo, we compared DCAH and DCMAH to an established chemotherapy drug, bevacizumab. We used the U87 xenograft mouse model to study the effects of DCAH, DCMAH, DCA, and bevacizumab on xenograft tumor size and determine if our two most potent DCA analogs elicited a substantial phenotype in vivo. The U87 xenograft model was chosen because its protein levels were the most consistently affected following treatment. We used bevacizumab because it is a well-established chemotherapy drug that is FDA-approved for clinical treatment of multiple cancer types.

The tumor volumes of all compound-treated mice decreased compared with controls (Figure 3). The tumors of mice treated with DCAH and DCA showed similar volume reductions of 50% and 54%, respectively, compared with those of untreated control mice. DCMAH and bevacizumab treatment elicited similar tumor volume reductions of 38% and 32%, respectively, compared with untreated controls. These data suggest that DCMAH has the higher efficacy of the two tested DCASMAs. Overall, our DCASMAs were as effective as DCA and bevacizumab in reducing tumor growth, supporting the hypothesis that that DCAH and DCMAH may affect tumors comparably to DCA while having potential for minimized cytotoxicity; however, further study is needed.

Figure 3.

DCAH and DCMAH decreased tumor volumes in the U87 xenograft mouse model. U87 xenograft mouse models were dosed with DCAH, DCMAH, DCA (25 mg/kg), bevacizumab (10 mg/kg), or PBS (control) (n = 5). A) DCMAH (grey) decreased tumor volume to a similar degree as the chemotherapy drug bevacizumab (green). DCA (red) and DCAH (blue) decreased tumor volumes to a similar degree. B) All compounds decreased tumor sizes relative to the control.

Additionally, we performed an LD-50 study of all eight DCASMAs in nude mice, in which cumulative doses were administered over 10 days (50 mg/kg/day, total dose 500 mg/kg). There were no deaths or observable phenotypes following treatment except for of DCPA, a known herbicide. DCPA caused moderate body weight loss in mice (data not shown).

Discussion

Cationic analogs have the largest effect on in vitro tumor viability

Identifying and characterizing novel anti-cancer agents with limited cytotoxicity is a cornerstone of cancer research. Studies have shown that inhibition of overexpressed PDK can restore the metabolic profile of tumor cells and subsequently induce apoptosis.^6,14,37 This led us to a pilot study of investigating the effects of eight structural analogs, derived from the known PDK inhibitor DCA, on various tumor cell lines. Conventional chemotherapies exhibit substantial cellular toxicity in the sub-millimolar range,³⁸ so comparatively speaking, our compounds were extremely well tolerated with minimal cytotoxicity. This indicates that our compounds may be administered safely at prolonged high doses, which is therapeutically advantageous if they demonstrate anti-tumor effects.

Of the three primary characteristics we varied in our DCASMAs (charge/polarity, electron distribution, and spatial arrangement), reversing the charge compared with DCA produced the largest effect on tumor viability. The remaining six compounds apart from DCAH and DCMAH had significantly larger side groups. Crystal structures of PDK have shown that pyruvate and DCA both bind an N-terminal regulatory (R) domain of the kinase which is compact and not easily accessible (Figure 4).^30,39 This could explain the low affinity of DCASMAs in general and the attenuated potency of the larger compounds. Another property to consider is the dichloric terminal of DCAH and DCMAH, which is not as restricted by steric strain compared with the other analogs. The cationic amine group, small size, and dichloric terminal sterics all could potentially contribute to the binding interactions of DCAH and DCMAH with PDK, which could affect their ability to modulate downstream pathways.

Figure 4.

DCA-bound PDK1. The N-terminal domain of PDK contains the lipoyl-binding pocket, which is essential for PDH binding. The C-terminus contains the conserved kinase domain. The N-terminus comprises eight α-helices that bundle to form a four-helix core (left). DCA (magenta, red arrow) binds inside this helix bundle. Skeletal structures of amino acids within 4 Å of DCA are shown. It has been hypothesized that the conformational changes required to accommodate DCA interfere with ligand binding and kinase activity. PDK1 is the only isoform to have a crystal structure with DCA bound; however, studies suggest this mechanism is similar among isoforms.^24,30,33 (Image rendered with Open-Source PyMOL.).

DCASMAs affected metabolic protein expression in tumor cells

We saw minimal impact from in vitro cytotoxicity assays, while in vivo data demonstrated the significant ability of DCAH and DCMAH treatment to inhibit U87 xenograft tumor growth. To analyze potential mechanisms we treated three xenograft models with DCAH and DCMAH and surveyed expression levels of several key proteins and pathways: 1) Oxidative phosphorylation flux as initiated by production of acetyl-CoA by active PDH, 2) p53-mediated tumor suppressing processes such as apoptosis and cell cycle arrest, 3) HIF-1α mediated tumor promoting processes such as glycolysis and growth support, and 4) pro-apoptotic caspase-3 levels. These protein levels were selected due to their relation to DCA and potential for gauging metabolic remodeling. It is also worth noting the mutation status of individual cell lines that may contribute to differences in cellular pathways and protein levels. HCT116 cells possess a mutated KRAS gene, encoding a protein that contributes to the Warburg effect via upregulation of glucose transport, though it is not fully understood how this affects overall glucose metabolism.^40,41 Higher intracellular ROS levels from aerobic glycolysis could also contribute to reduced growth; U87 lack PTEN function (often implicated in tumor formation) which has led to cellular senescence in the presence of ROS.⁴² HUH7 cells are known to have a mutation in the NRAS oncogene, empirically correlated with tumor formation.⁴³

As binding interactions are largely polarity and charge based, we do not expect the cationic amine groups of DCAH and DCMAH to have a comparable molecular mechanism to the anionic carboxyl group of DCA. This notion is supported by our data; we were able to confirm that both DCAH and DCMAH treatments result in altered protein levels, but in varying capacities across cell lines. For this reason, results were reported individually based on treatment and cancer type; a more unifying mechanism of action would likely be better elucidated in pathways outside of the ones we examined here. Taken together, this study suggests that a more thorough investigation of DCAH and DCMAH effects on tumor growth would be beneficial.

Clinical relevance

In recent years, DCA has grown in popularity as a potential anti-cancer agent. While DCA has been shown to selectively kill tumor cells in vitro and slow tumor growth in patients in several case studies, side effects (e.g., reversible peripheral neuropathy, tremors, hallucinations, depression, memory loss, nausea, fatigue, confusion) combined with large-dose requirements (10–25 mg/kg/day) are substantial obstacles in its path to clinical trials.^{8,13,16,17,34,35,44–47} Here, we present two alternative compounds, DCMAH and DCAH, which demonstrate minimal cell toxicity in vitro, impact cellular pathways in tumor samples and effectively slow tumor growth in a xenograft model to a level comparable to the known chemotherapy bevacizumab. While DCMAH and DCAH do not appear to be identical to DCA in cellular effect, these results present a case for future investigations into the efficacies and mechanisms of the two novel dichloric compounds as potential anticancer drugs.

Funding Statement

The study was sponsored by Altogen Labs.

Acknowledgments

The authors wish to thank TuAnh Dang (Protein Simple) for consultation on data analysis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.