Bicarbonate and dichloroacetate: evaluating pH altering therapies in a mouse model for metastatic breast cancer

Abstract

Background: The glycolytic nature of malignant tumors contributes to high levels of extracellular acidity in the tumor microenvironment. Tumor acidity is a driving force in invasion and metastases. Recently, it has been shown that buffering of extracellular acidity through systemic administration of oral bicarbonate can inhibit the spread of metastases in a mouse model for metastatic breast cancer. While these findings are compelling, recent assessments into the use of oral bicarbonate as a cancer intervention reveal limitations.

Methods: We posited that safety and efficacy of bicarbonate could be enhanced by dichloroacetate (DCA), a drug that selectively targets tumor cells and reduces extracellular acidity through inhibition of glycolysis. Using our mouse model for metastatic breast cancer (MDA-MB-231), we designed an interventional survival study where tumor bearing mice received bicarbonate, DCA, or DCA-bicarbonate (DB) therapies chronically.

Results: Dichloroacetate alone or in combination with bicarbonate did not increase systemic alkalosis in mice. Survival was longest in mice administered bicarbonate-based therapies. Primary tumor re-occurrence after surgeries is associated with survival rates. Although DB therapy did not significantly enhance oral bicarbonate, we did observe reduced pulmonary lesion diameters in this cohort. The DCA monotherapy was not effective in reducing tumor size or metastases or improving survival time. We provide in vitro evidence to suggest this outcome may be a function of hypoxia in the tumor microenvironment.

Conclusions: DB combination therapy did not appear to enhance the effect of chronic oral bicarbonate. The anti-tumor effect of DCA may be dependent on the cancer model. Our studies suggest DCA efficacy is unpredictable as a cancer therapy and further studies are necessary to determine the role of this agent in the tumor microenvironment.

Figure 1

Comparison of urine and serum pH in treatment groups. A) Urine samples were collected from tumor bearing mice at eight different time points over a 3 week period from 3-10 mice in each treatment group. At time zero before starting treatment, urine pH in all groups averaged 5.59 ± 0.2 with no statistical difference between groups. The first collection time after starting treatment was at 24 hours. Urine pH measurements from all collection times were averaged. Asterisks (_*) designate average urine pH values that were statistically significant from untreated mouse group by student's two-tailed t-test (p < 0.001). The cross (†) designates average urine pH values between the DCA and DB treated groups were statistically significant (p < 0.03). B) Serum pH was measured in euthanized mice. Differences in serum pH between groups were not statistically significant. Error bars are expressed as SEM.

Figure 2

Effect of bicarbonate, DCA and DB treatments on primary tumor growth. Growth rates of primary tumors are expressed in mm³/time (days). Plots indicate none of the treatments exhibited a measurable effect on primary tumor growth at the beginning of the study (A) or from the time of the tumor resections to study termination (B). Error bars are expressed as SEM. Two-tailed, unpaired t-test, between these groups yielded a p > 0.9.

Figure 3

Primary tumor re-occurrence after survival surgeries. Survival surgeries occurred between day 28 and 50. Average resection day occurred on day 37 ± 8. Primary tumor sizes in all treatment groups averaged 490 ± 12 mm³. Primary tumors started to re-grow around day 50. Primary tumor re-occurrence is plotted over the survival time course as Kaplan-Meier curve. A plot point represents the day when a primary tumor was first measured after surgery. In mice where no primary tumor was observed in post-study necropsy experiments, a value of zero was recorded for day 120. A) Plots for all experimental cohorts (p = 0.29). B) Curves comparing groups that were not administered bicarbonate (untreated and DCA) and groups treated with bicarbonate (bicarbonate and DB) (p = 0.046).

Figure 4

Effect of bicarbonate, DCA, and DB on and survival. MDA-MB-231 cells were stably transfected to express neomycin-resistant pcDNA3/EGFP (16). MDA-MB-231/eGFP cells (5 × 10⁶) were injected into inguinal mammary fat pads of animals that were randomized into bicarbonate, DCA, DB, and control groups (n = 16 per group) 6 days post inoculation. Tumors were allowed to grow for 5 to 6 wk (to a volume of approximately 500 mm³), at which time they were surgically removed. After survival surgeries, the disease model was allowed to progress until tumor burdens or morbidity criteria warranted the mice to be euthanized. The survival experiment proceeded to day 120. At time of sacrifice, mice were necropsied by examination with a fluorescence dissecting scope. Data from this experiment are plotted as Kaplan-Meier survival curves (A) bicarbonate (p = 0.03), (B) DCA (p = 0.2), and (C) DB (p = 0.01). Treated mice are represented as solid lines compared to the untreated group represented as a dashed line. The difference in the survival curve for the treated versus untreated animals was evaluated by log-rank test.

Figure 5

Representative pulmonary lesions from the four study cohorts. Green fluorescent lung tumor metastases from necropsies were detected by the Illumatool Bright Light System (LT-9500) using a 470 nm/40 nm excitation filter (Lightools Research). Whole lung images were captured in the frame of view at the same focal plane in the presence of 480-nm excitation and > 490-nm filtered emission.

Figure 6

MDA-MB-231 breast tumor cells were cultured for 24 hours (n ≥ 3) with titrating doses of DCA starting from 80 mM and diluted 2-fold to the lowest concentration of 2.5 mM. Percent viability for all groups of replicates was normalized by dividing absorbance (590 nm) values by the absorbance values obtained from sham treated (growth media) control cells. Cytotoxicity by DCA in a crystal violet assay was carried out under A) normoxia (O₂ = 20%) and B) hypoxia (O₂ = 1%). Asterisks (_*) designate viable DCA treated cells percentage that were significantly different from 100% viable sham treated cells. Error bars are expressed as SEM.

Figure 7

MDA-MB-231 breast tumor cells were cultured for 24 hours with titrating doses of DCA starting from 80 mM and diluted 2-fold to the lowest concentration of 2.5 mM (n ≥ 3). Supernatant lactate was measured as a ratio of the total cellular protein. Lactate production was measured under A) normoxia (O₂ = 20%) and B) hypoxia (O₂ = 1%). Asterisks (_*) designate lactate production in DCA treated cells percentage that were significantly different from untreated control cells. Error bars are expressed as SEM.