PDF – DCA Inhibits Metastic Breast Cancer Australian National University Medical School

DCA Prevents Metastic Breast Cancer, PDF Medical School at Australian National University

BRIEF REPORT Dichloroacetate suppresses the development of metastatic breast cancer cells both in vitro and in vivo by reversing the glycolytic phenotype. Jane E. Dahlstrom, Christopher R. Parish, Philip G. Board, Anneke C. Blackburn, Ramon C. Sun, Mitali Fadia 2009 Springer Science+Business Media, LLC. received it on April 17 and accepted it on June 2. Abstract Several solid cancers, including breast cancer, exhibit the glycolytic phenotype. Recently, it has been shown that the novel anti-cancer compound dichloroacetate (DCA) can reverse the glycolytic phenotype in cancer cells by inhibiting pyruvate dehydrogenase kinase. We’ve looked at how DCA affects breast cancer cells, including in an in vivo model with a high level of metastatic spread.

In vitro testing revealed that DCA suppressed the growth of a number of breast cancer cell lines. When the glycolytic phenotype was reversed by DCA in 13762 MAT rat mammary cancer cells, there was an inhibition of proliferation without an increase in cell death, according to further investigation. Despite a little but considerable increase in caspase 3/7 activity, which may make cancer cells more susceptible to other apoptotic stimuli, this was the case. After injecting 13762 MAT cells into the tail vein of rats, DCA resulted in a 58% decrease in the number of lung metastases visible macroscopically in vivo (P = 0.0001, n C 9 per group). These findings underscore DCA’s potential for clinical application by demonstrating that it possesses anti-proliferative as well as pro-apoptotic effects and can be beneficial in vivo against highly metastatic illness. 

Keywords Dichloroacetate Mammary cancer Metastasis of glycolysis in an animal model Introduction High rates of glucose uptake and glycolysis take place in the majority of cancer forms, known as the glycolytic phenotype, whereas mitochondrial respiration is suppressed despite the presence of oxygen. This phenomenon is frequently referred to as the Warburg effect. During the evolution of anaerobic tumors, it is thought that this metabolic trait was acquired for the production of ATP. However, mounting data suggests that the glycolytic phenotype is also accompanied by gene expression changes that are directly related to tumorigenic processes, such as increased metastatic potential and resistance to apoptosis [1, 2]. 

The glycolytic phenotype in breast cancer has been extensively discussed. In comparison to paired normal breast tissue samples, breast tumors have been shown to have an altered bioenergetic cellular index (BEC) and a significant shift toward an improved glycolytic phenotype, which has been linked to the patients’ overall and disease-free survival [3, 4]. Numerous breast cancer cell lines’ invasiveness has been linked to increased lactate generation, decreased activation of the transcription factor HIF-1a in hypoxia, and greater constitutive levels of HIF-1a in normoxic circumstances [5]. In microinvasive foci of ductal carcinoma in situ (DCIS), immunohistochemical analysis revealed overexpression of two markers of the glycolytic phenotype (glucose transporter GLUT1 and Na/H exchanger NHE-1), suggesting that adaption Email: R. C. Sun P. G. Board & Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra 2601, Australia Fadia M Canberra Hospital and Australian National University Medical School, Woden, ACT 2606, Australia, J. E. Dahlstrom Department of Anatomical Pathology Australian National University’s John Curtin School of Medical Research, C. R. Parish Cancer and Vascular Biology Group, Canberra, ACT 0200, Australia 123 DOI: 10.1007/s10549-009-0435-9 for Breast Cancer Res Treat The progression from in situ to invasive breast cancer may be aided by hypoxia and acidosis [6]. 

Therefore, reversing the glycolytic phenotype to prevent breast cancer spread and recurrence is an appropriate therapy plan. Since pyruvate dehydrogenase (PDH) controls the process of turning pyruvate into acetyl Co-A, it has the power to regulate the flow of metabolites from glycolysis to the citric acid cycle and, in turn, the mitochondria’s ability to produce ATP. Pyruvate dehydrogenase kinase (PDK), which phosphorylates and inactivates PDH, controls PDH [7]. A novel and generally non-toxic anti-cancer drug, dichloroacetate (DCA), has recently been proposed as it inhibits PDK and inhibits PDK activity [8]. In several cancer cell lines, DCA has been demonstrated to restore the glycolytic phenotype by depolarizing the hyperpolarized inner mitochondrial membrane potential to normal values and boosting mitochondrial metabolism [8, 9]. 

DCA can be effective against cancer cells without having an adverse effect on healthy cells because it targets a change that occurs during carcinogenesis. DCA has the potential to enter the clinic swiftly for various uses as it has cleared phase I/II toxicity testing in humans [12] and is currently in phase III clinical studies for the treatment of chronic lactic acidosis in congenital mitochondrial diseases [10, 11]. Clinical trials assessing its toxicity in cancer patients are currently underway (; nevertheless, to identify which tumors and which people are most suitable for DCA treatment, controlled tests to comprehend the anti-cancer effects of DCA are required. In this work, the impact of DCA was investigated both in vitro and in vivo using a rat mammary cancer cell line. 

The findings show that DCA can be successful in vivo in reducing the proliferation of metastatic cancer cells, increasing its relevance to the treatment of breast cancer. They also point to a mechanism of action for DCA in these cells as an anti-proliferative agent rather than an apoptosis-inducing agent. Resources and techniques culture of cells As previously mentioned [13], 13762 MAT rat mammary adenocarcinoma cells (MAT cells) were cultured in vitro. A spontaneous mammary cancer that developed in a BALB/c-Trp53?/- animal provided the source of the V14 cells [14]. cell expansion Cell viability was determined by neutral red absorption after cells were treated to 1–5 mM DCA (Sigma Chemical Co., St. Louis, MO) for 1-4 days in 96-well plates with daily medium and DCA replacements [15]. 

Apoptosis According to the manufacturer’s instructions, the Caspase-Glo 3/7 assay (Promega Corp., Madison, WI) was used to measure the activity of caspases 3 and 7 in MAT cells. After labeling cells with FITC-labeled Annexin-V (BD Pharmingen, NJ) and propidium iodide (PI), the amount of apoptosis was measured by flow cytometry (Sigma Chemical Co St. Louis, MO). Proliferation Fluorescence-activated cell-sorting analysis was used to assess the stained cells after applying 5 lM carboxyfluorescein succinimidyl ester (CFSE) [16]. cellular metabolism According to the manufacturer’s instructions, the CellTiter-Glo assay (Promega Corp., Madison, WI) was used to measure the internal ATP levels in MAT cells. By detecting the conversion of NAD to NADH at 340 nm by lactate dehydrogenase in neutralized perchloric acid extracts of medium, extracellular lactate levels were quantified spectrophotometrically [17].

 Metastasis of MAT cells in vivo at 13762 Under the rules established by the Australian National Health and Medical Research Committee, animal experiments were carried out with the agreement of the Australian National University Animal Ethics Experimentation Committee. The lateral tail veins of three groups of female Fischer 344 rats (10–13 weeks old) were injected with 2 9 105 13762 MAT cells [13]. Rats in group 1 (control) received no treatment. To reduce GSTZ1 activity and increase DCA bioavailability prior to the injection, groups 2 (low dose) and 3 (high dosage) received DCA given orally in drinking water at 0.2 g/l (23 mg/kg) for 7 days [18]. 

To achieve a daily dosage of 86 mg/kg without significantly changing water consumption (control: 120 ml/kg/day), the oral dose was increased to 0.75 g/l on the day of cell injection (average water consumption: 115 ml/kg/day). A second round of DCA therapy (high dose) was administered to the rats in group 3; they received 200 mg/kg/day of the drug intraperitoneally (i.p.) in phosphate buffered saline (neutralized and filter sterilized), with the first injection given about two hours before cell injection. Using extrapolation from data that has already been published [18, 19], it is predicted that oral dosing will produce plasma concentrations of DCA that are in the range of 0.5–1 mM, whereas Breast Cancer Res Treat 123 additional 200 mg/kg i.p. will increase this concentration by about three times, to 1.5–3.0 mM. 

The lungs of rats were fixed in Bouins solution after they were killed 14 days after receiving tumor cell injection. A dissecting microscope was used to count the number of lung metastases. Next, each lung’s two largest lobes were paraffin-embedded, dyed, and used for microscopic analysis. It was determined how many microscopic lesions, how big they were, and how many mitoses there were in each high power field (hpf). The degree of tumor-associated lymphocyte density (occasional/mild/moderate/severe) and the presence or absence of tumoral necrosis were both observed. 

The glutathione transferase GSTZ1-1 in the liver converts DCA to glyoxylate, but DCA can impede this process by combining with GSTZ1 to produce an inactive enzyme-substrate complex [20]. In order to make sure that DCA was delivered effectively, GSTZ1 activity in rat liver was assessed using the previously mentioned technique [21]. Statistic evaluation The Cell Quest software suite (BD Bioscience, Rockville, MD) was used to collect the FACS data, and FlowJo was used to analyze it (Tree Star Inc, OR). The GraphPad Prism software program was used to do the calculations, and the Student’s t-test was used to evaluate differences between the DCA-treated and control groups. Statistical significance was defined as a P value less than 0.05. The data is shown as mean standard deviation. 

Results DCA slows the proliferation of breast cancer cells. We subjected a variety of breast cancer cell lines to 5 mM DCA treatment to examine the susceptibility of breast cancer cells to the drug (Fig. 1a). On day 4 of treatment, MCF-7, T-47D, 13762 MAT, and V14 cells all displayed a 60–80% reduction in cell number, however 4T1 cells were insensitive. On the other hand, MCF-10A, a non-cancerous control cell line, grew unaffected by DCA. Both in vitro and in vivo studies were conducted to better evaluate DCA’s impact on MAT cells. Time and dose both affected how MAT cells responded to DCA (Fig. 1b, c). On day 4, when MAT cells treated with 5 mM DCA had 68 5% fewer cells than control cultures (n = 3, P0.0001), the effect was highest. Cell proliferation and apoptosis were assessed to ascertain the cause of the decline in cell numbers. MAT cells treated with 5 mM DCA displayed considerably increased fluorescence compared to the untreated cells after 3 days (P = 0.0009; n = 3), indicating inhibited cell division, according to the CFSE cell proliferation experiment (Fig. 2a). This was clear from Fig. 1. DCA’s impact on cell proliferation a panel of breast cancer cell lines with varying sensitivity to 5 mM DCA treatment over the course of 4 days. A non-cancerous control is MCF-10A. b The manner in which DCA inhibited the development of MAT cells. c Breast Cancer Res Treat 123 even at 1 mM DCA, dose response of MAT cells toward DCA on day 4 of DCA treatment. In contrast, DCA had no effect on the rate of apoptosis (Fig. 2b–d). When compared to the staurosporine positive control, treatment with 5 mM DCA resulted in a modest (15%) but statistically significant increase in caspase 3/7 activation after 3 hours (Fig. 2b) (2.2-fold). Additionally, 5 mM DCA treatment failed to cause apoptosis in MAT cells even after 24 hours of incubation, according to annexin V and PI staining (Fig. 2c, d). 

The glycolytic phenotype is reversed by DCA Total ATP levels increased by 18 3% after MAT cells were treated with 5 mM DCA after 30 min (n = 3, P = 0.009), and this effect sustained for three hours. Extracellular lactate levels dropped by 16.3 5.3% (n = 4, P = 0.01) after 12 hours of DCA administration. These results attest to DCA’s ability to reverse the glycolytic phenotype of MAT cells. In vivo, DCA slowed tumor development. The number of metastases in the rats in the low dose DCA group (getting *86 mg/kg DCA by the oral route alone) did not alter when MAT cells were injected intravenously. However, rats in the high dosage DCA group (getting daily i.p. injections of 200 mg/kg/day DCA in addition to the oral DCA) significantly reduced the number of lung tumors visible macroscopically by 58 17% (P = 0.0001, n C 9 per group) (Fig. 3).

 Microscopically, however, there were the same amounts of lesions in each of the three groups (control, low, and high dose, respectively: 6.4 2.8, 7.1 3.4, and 6.2 3.2 per 5 high power fields). Lesions underwent reduced tumoral necrosis and had a higher mitotic count in high dosage DCA-treated rats (9.4 7.0 vs 20.2 9.2 per 5 hpf in control versus high dose DCA, respectively, P = 0.03). (Fig. 4). None of the groups contained any apoptotic bodies. A considerable lymphocytic infiltration, particularly around the borders of the tumors, was also induced by DCA therapy, but in the control group, tumor-associated lymphocytes were only occasionally detected (Fig. 4). To validate that the rats had actually been exposed to DCA, GSTZ1-1 activity was examined in the liver of tumor-bearing, DCA-treated rats. Following DCA therapy, the liver GSTZ1-1 activity in the low and high dose treatment groups decreased by 93% and 95%, respectively, demonstrating nearly full elimination of GSTZ1-1 activity at both doses.

 Discussion Breast carcinomas with the glycolytic character nearly always have microinvasive foci and have worse survival rates [2–5]. As a PDK inhibitor, DCA can stop the glycolytic process. Fig. 2 Effect of DCA on MAT cell markers for cell proliferation A and apoptosis B-D. Following three days of DCA therapy, the average CFSE cell fluorescence. activity of caspase 3/7 three hours after treatment. percent of cells that have early- and late-stage apoptosis. Breast Cancer Res Treat 123: Staurosporine is a positive control for apoptotic induction. Fig. 3 vivo research. Rat lungs demonstrating metastases developed after tail vein injection of MAT cells in control (a) and high dosage (b) Fischer rats. c Rats in the control, low, and high dose DCA treatment groups each had one lung metastasis. d The same rats’ liver GSTZ1 activity Fig. 4 Photomicrographs of a control rat demonstrating a central necrosis and the absence of a lymphocytic infiltration. 

Tumors b and d in high dose DCA-treated rats showed reduced necrosis development and a moderate lymphocytic infiltration, respectively. Breast Cancer Res Treat 123 phenotype (H and E stained, original magnification (a, b) 9100, (c, d) 9400). We find that a number of breast cancer cell lines are susceptible to DCA in our investigation, with growth suppression seen after several days of treatment (Fig. 1). With no evidence of apoptosis or cell death, in vitro tests on MAT cells clearly showed that this was caused by suppression of growth (Fig. 2). In investigations on DCA treatment of endometrial, prostate, and lung cancer cells that have been published thus far, enhanced apoptosis with no effect on cell cycle distribution [9, 22] or increased apoptosis along with reduced proliferation [8] were found in the majority of cases. Out of the six cell lines that were studied, only two (one with G0/G1 arrest and the other with some S and some G2/M arrest) showed any change in cell cycle distribution after DCA treatment [9, 22]. 

While DCA slows down cell development in a variety of cancer cells, the exact mechanism seems to vary on the cell line. This response is not only shown in MAT cells, but may also be a trait of breast cancer cells, as it was also seen that the human breast cancer cell line T-47D did not undergo apoptosis after DCA treatment (data not shown). We will look at this option more because it’s intriguing. Alternately, the presence of apoptosis may be prevented by the high concentrations of cell survival proteins found in the studied cell lines, including Bcl-2, survivin, and PUMA. In prostate, lung, and endometrial cancer cells, DCA treatment decreased the expression of these survival factors [8, 22], which may have influenced the apoptotic response seen. 

With 4T1 cells being the least sensitive, the sensitivity of breast cancer cell lines to DCA ranged from 20 to 80% suppression of cell growth after 4 days of 5 mM treatment (Fig. 1). Multiple variables, such as the capacity to metabolize DCA via GSTZ1 or the overexpression of certain PDK isoforms, may influence sensitivity to DCA. There are four different isoforms of PDK, and their corresponding Kis for DCA are 1, 0, 8, and 0.5 mM [23]. The doses of DCA utilized in this investigation would inhibit PDK1, 2, and 4, but not PDK3. Although PDK3 expression and stimulation by hypoxia have been reported for various cancer cell lines [24], PDK3 is typically only expressed in the testes [23]. Studies are being conducted to identify the tumor types that DCA targets most successfully and to connect PDK expression with DCA sensitivity. Additionally, there was a slight rise in caspase 3/7 activity. 

This may be caused by the electron transport chain’s reactivation by DCA and an increase in the production of reactive oxygen and nitrogen species in the mitochondria [25]. This rise in baseline caspase activity might not be enough to cause apoptosis, but it might suggest that DCA could be employed to make cancer cells more susceptible to other apoptotic triggers like hypoxia, radiation, or other chemotherapeutic drugs. More research has to be done on these potential synergies. Targeting the pyruvate to acetyl CoA linkage of glycolysis and mitochondrial respiration has previously been shown to be effective against primary tumor growth in two models when the glycolytic phenotype is reversed in vivo [1, 8]. In the MAT cell model, we have shown that DCA has the potential to be effective against metastatic illness in vivo. 

High dosage DCA reduced the number of macroscopic lung lesions but did not affect the number of microscopic lesions, indicating that the main effect of DCA was on the size of the tumors rather than a decrease in the number of cells that may become lung tumors. A growth suppression mechanism as was seen in vitro is also compatible with the observation of a decreased incidence of necrosis in tumors treated with DCA. The greater number of mitoses initially seems to be at odds with this, indicating higher rates of proliferation. However, we propose that this might be because DCA arrests the cell cycle prior to anaphase, causing an accumulation of cells to be seen as mitotic figures. Intriguingly, the high dose DCA-treated rats had a rise in tumor-associated lymphocytes. A decrease in tumor lactate levels brought about by DCA treatment may encourage a better immunological response against the tumors because high lactic acid concentrations have been proven to impair T cell activity [26]. Experiments with V14 cells in vivo indicate that these in vivo effects are not specific to MAT cells and that V14 cells are responsive to DCA in vivo in a manner similar to the MAT cells, as evidenced by reduced primary tumor growth and increased lymphocyte presence (data not shown). 

Agents at millimolar concentrations are frequently thought to be unsuitable for use. However, by giving patients oral DCA at a dose of 25 mg/kg/day, millimolar blood levels of DCA (0.3–1 mM) have been sustained over time [27]. As much as 80 mg/kg intravenously has been tolerated in acute patient therapy during liver transplantation [28]. Despite the high effective DCA dose in vivo in this rat model, the estimated plasma concentration obtained in vivo (1.5–3 mM; see Methods) is comparable to the human dose of 25 mg/kg/day and is therefore applicable to the clinical situation. This plasma concentration range also aligns with the Ki for PDK inhibition by DCA [23] and the suppression of proliferation in vitro (as low as 1 mM, Fig. 2a), supporting the idea that PDK is the target responsible for the anti-cancer actions of DCA. DCA is a suitable candidate agent for combination therapy because, despite the fact that longterm DCA treatment in MELAS patients caused some reversible peripheral neurotoxicity [10], DCA’s toxicity to tissues susceptible to conventional cytotoxic agents is modest [8, 29]. For instance, despite the fact that DCA irreversibly inhibits GSTZ1 (Fig. 2d), DCA-treated animals did not exhibit the lymphocyte depletion seen in mice with GSTZ genetic defects [21, 30]. 

Breast Cancer Treatment Research 123 Therefore, inhibiting PDK with DCA is a viable anticancer method for reversing the glycolytic phenotype in breast cancer and also suggests a possible utility for different PDK inhibitors now under development as drugs [31]. To effectively target cancer cell metabolism for therapeutic purposes, additional mechanistic investigations to comprehend the causal link between the glycolytic phenotype and tumor features are necessary. Acknowledgments The National Breast Cancer Foundation of Australia and the NHMRC 366787 R.D. Wright Career Development Award both provided funding for this study. References 1. Attenuation of LDH-a expression reveals a connection between glycolysis, mitochondrial physiology, and tumor maintenance in Fantin VR, St-Pierre J, and Leder (2006). 

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