The premise for these experiments was based on mathematical models that suggest the buffering capacity of bicarbonate against tumor acidity could be safely enhanced by addition of DCA (Martin N, Robey I, Gaffney E, Gillies R, Gatenby R, Maini P: Predicting the Safety and Efficacy of Buffer Therapy to Raise Tumor pHe: An Integrative Modeling Study, submitted). The model indicated that inhibition of tumor proton production by a relatively small amount could cause substantial reductions in tumor acidity, without any adverse effects on blood pH. A reagent like DCA fits this criterion because it can reduce extracellular tumor acidity by inhibiting lactic acid production. Moreover, DCA has been shown to selectively target tumor cells [17, 18]. The design of this study was to treat our mouse model for breast cancer as similar to a patient study as possible by carrying out a survival experiment with surgical tumor resections.
We confirmed from both urine and serum pH measurements that addition of chronic DCA at the doses administered did not cause significant changes in either urine or serum pH. The urine and serum pH results from DB treated mice were statistically comparable to the measurements in the bicarbonate treated group. We conclude from these findings that DCA does not induce any systemic perturbations that could lead to systemic alkalosis.
Our studies show that none of the treatments reduced primary tumor growth. Our bicarbonate treatment group results were consistent with earlier studies . Other studies have shown that DCA significantly inhibits tumor growth rates in vivo . We expected to see this result as well, but found that DCA and DB treated mice had similar primary tumor growth rates to untreated and bicarbonate treated mice. This difference may be explained by the variable effect DCA can have in different tumor cell lines [20, 21]. With respect to the metastases results, our findings were similar to a study that administered 5 mM DCA orally to rats injected with a metastatic breast tumor line through the tail vein. There was no measurable effect of the 5 mM dose .
It is unknown why DCA treatment did not provide therapeutic benefit against tumors in mice, but the in vitro studies may help to explain our findings. The viability studies under normoxic conditions demonstrate that tumor cells have a biphasic response to titrating DCA concentrations. This occurrence is known as hormesis, where low doses can be agonistic and higher doses are toxic to cancer cells . The normoxic in vitro viability results suggest DCA concentrations at 20 mM or lower could have a stimulatory effect in vivo, though this was not measured. Only at concentrations exceeding 20 mM did DCA reduce viability under normoxia. Under hypoxia, DCA concentrations at 20 mM or lower had no effect on viability. There was a marginal, but significant reduction in viability at DCA concentrations greater than 20 mM under hypoxia. An 80 mM concentration was required to reduce viability 50%.
In the lactate production studies, DCA doses at 20 mM or lower significantly reduced MDA-MB-231 cell glycolytic metabolism under normoxia by approximately 19%. Doses higher than 20 mM inhibited lactate production by 29% under normoxia. No concentration of DCA tested reduced cellular lactate production under hypoxia. The in vitro studies suggest; 1) that DCA concentrations greater than 20 mM may have a non-specific effect on MDA-MB231 cells and 2) hypoxia may be a factor contributing to the in vivo findings.
MDA-MB-231 mammary xenografts are known to develop regions of hypoxia due to the development of necrotic lesions. Also, unpublished experiments (Robert Gillies laboratory at the University of Arizona) imaging MDA-MB-231 tumors in mice with bioluminescent reporters linked to promoter regions for hypoxia response elements such as VEGF or CAIX demonstrate that tumors develop regions of hypoxia (exhibit bioluminescence) as soon as tumors become palpable (between 100-200 mm3). Other studies have shown the link between DCA function and hypoxia. DCA inhibits pyruvate dehydrogenase kinase-1 (PDK1) which inhibits cellular metabolism and causes oxygen consumption resulting in increased hypoxia [23, 24].
Although the metastatic load observed in bicarbonate treated mice appeared markedly lower than reported in DB treated mice (especially mesentery and intestinal), the data is insufficient to conclude that bicarbonate monotherapy was more effective at reducing the spread of metastases. The major impact on survival in our cancer model is lung metastases. As seen in the untreated group, most (79%) of the animals showed evidence of metastatic lung lesions. Only one of the bicarbonate treated animals in the analysis was found to have lung metastases, but there were two (of the four) spontaneous deaths that occurred in this group and one in the untreated group where we were unable to analyze the lungs. Nonetheless, a two-tailed Fischer's exact analysis shows that including the bicarbonate treated group spontaneous deaths as lung metastases positive (23%) and the untreated as negative for lung metastases, frequency of lung metastases would still be significantly lower in the bicarbonate treated mouse group (p = 0.02). It was surprising that the percentage of DB treated mice with lung metastases was 60%, and not significantly different from the occurrence of lung metastases in the untreated mouse group, indicating the bicarbonate monotherapy as a more favorable treatment modality at least in this tumor model. The findings suggest a potential risk in the DB treatment that would need to be investigated further in other models for cancer.
Analysis of the pulmonary lesions revealed that DB treatment resulted in significantly smaller mean tumor metastases compared to all other groups. The mean lesion diameter in DB treated mouse lungs was no larger than 30% of the lesion diameters in the other treatment groups. In a previous study it was reported that smaller tumor metastases in lungs was correlated significantly with bicarbonate treatment alone, but this was a 30 day experiment using β-galactosidase expressing MDA-MB-231 cells and the mean tumor diameters were about 87% of the lesion diameters measured in the untreated group . The comparison of tumor lesion sizes between the metastasized lungs in all the study groups appears to suggest that DB treatment inhibits the growth of these metastases (Table 3). However, given the prevalence of non-metastatic lungs in the bicarbonate treated group, it could be argued that DCA has more of a stimulatory effect, especially if O2 concentrations are more normoxic, and may even compete with bicarbonate therapy. This conclusion is supported by the in vitro findings which show that lower concentrations of DCA increase cell viability under normoxia.
Improved survival was a significant outcome in all groups treated with bicarbonate. There seemed to be little measurable therapeutic effect in DCA treated mice, suggesting bicarbonate was a driving component in the survival study. The results are consistent with earlier findings , but the mechanisms are not well understood. More than half the primary tumors in bicarbonate and DB treated mice did not return after surgical resections. We attribute the lower frequency of primary tumor re-occurrence to the improved survival rates in these groups. The difference between bicarbonate treated groups (bicarbonate and DB) and non-bicarbonate treated groups (untreated and DCA) in tumor re-occurrence after surgeries is statistically significant (p < 0.05). It may be possible that the effect of systemic bicarbonate could potentiate the wound healing process after surgical disruption of the tumor region. The role of bicarbonate ions (HCO3
-) in wound repair was first reported in a study investigating gastric mucosal repair in cats systemically administered sodium bicarbonate. It was concluded that systemic excess of HCO3
- facilitated superficial mucosal repair . The molecular mechanisms driving this effect are unknown, but other reports have shown that blocking the Na+-HCO3
- co-transport (NBC) with isothiocyanate (ITC) inhibits epithelial restitution, the process of epithelial migration involved in wound healing. Additionally, cell migration during restitution is dependent on glycolysis for energy . This is notable because NBC functionally cooperates with monocarboxylate lactate transporter (MCT-1), a glycolysis-associated enzyme. Extracellular HCO3
- increases NBC activity, which in turn enhances MCT activity leading to an increase in intracellular pH , a physiological condition conducive to glucose metabolism . It is thought that the upregulated glycolytic activity of tumor cells offers a competitive advantage over surrounding host cells . The epithelial wound healing processes after tumor resection in conjunction with an increase in extracellular bicarbonate may improve the competitive state of some host cells in the tumor microenvironment, and result in prevention of primary tumor re-growth. Although further evidence for these processes is required, it suggests that an optimal time to administer systemic bicarbonate would be after a tumor surgery.