Despite its lower efficiency in ATP production in comparison to oxidative phosphorylation, glycolysis is a quick pathway to provide energy and metabolic precursors for fast proliferating cells. Tumor cells rely on high glucose consumption and also on the ability to continuously pump out the large amounts of lactate and H + generated by their metabolism to maintain the glycolytic process and to avoid intracellular acidification and metabolic inhibition caused by lactate accumulation. In this study, we attempted to force intracellular acidosis of esophageal tumor cells and, as a consequence, cell death, by increasing glucose concentration in culture medium and simultaneously inhibiting the removal of H + and lactate produced by using the selective MCT1 inhibitor AZD3965. Although treatment of cells with AZD3965 induced a significant decrease of pHi and cell proliferation and an increase of cell apoptosis in one cell line, higher glucose concentration in culture medium did not have any effect on the cytotoxicity of the MCT1 inhibitor in any of the cell lines used. It is possible that our cell lines exhibit a phenotype that makes them insensitive to glucose overload as it has been previously described that multidrug-resistant cancer cells were unaffected by high glucose levels while drug-sensitive cells exhibited increased proliferation rates in response to glucose as a consequence of low PKC-βII expression29.
Monocarboxylate influx and efflux is an active process carried out by MCTs, of which MCT1 and MCT4 have shown to play a key role in maintaining an alkaline pHi in tumors11. Lactate is the main substrate for MCTs, and in vitro and in vivo studies have demonstrated that a blockade of lactate export and the subsequent pHi disruption is an effective strategy to target highly glycolytic tumor cells15,23,24,28,30. In this context, the pharmacological MCT1 inhibitor AZD3965 is currently undergoing a phase I study in patients with advanced cancer (NCT01791595).
Our data demonstrate that sensitivity to the cytotoxic effects of MCT1 inhibition varies among cell lines and also depends on oxygen levels. In normoxia, metastatic EAC cells were affected by MCT1 inhibition whereas the non-metastatic cell line remained unaffected, and both cell lines were insensitive to AZD3965 under hypoxia. Previous works showed that co-expression of both MCT1 and MCT4 transporters was related to resistance to MCT1 inhibition, which is consistent with functional redundancy of MCTs23,30,31. Moreover, MCT4 knockdown or silencing in different highly aggressive cancer models in vivo and in vitro has been reported to make them sensitive to MCT1 inhibition22,31. In our study, immunocytochemistry showed that metastatic OACM5.1C cells in normoxia expressed high levels of MCT1, with only a small proportion of the cells expressing MCT4, while OE33 cells expressed both MCT1 and MCT4 and remained unaffected by AZD3965 treatment. According to these results, we next sought to evaluate whether incubation under hypoxic conditions was able to increase MCT4 expression in our cell lines, as previous studies demonstrated that hypoxia induces MCT4 expression through HIF1-α17,32. In line with these studies, both OE33 and OACM5.1C cells showed an increase in MCT4 expression after 48 hours growing in hypoxia, and thus AZD3965 effects on the metastatic cell line were completely abolished, suggesting that complete inhibition of MCTs is required to exert antineoplastic effects. Thus, empirical results reported in the present study should be considered in the light of the limitation of the lack of simultaneous pharmacological inhibition of both MCTs, since to date there are no commercially available MCT4 selective inhibitors.
In this work, we decided to evaluate apoptosis and proliferation after 48 h of treatment based on previous studies with our cell model in which we observed only a slight effect in apoptosis when apoptotic inducers were used for less than 48 hours.
Our results showed that MCT1 inhibition in normoxia induced growth arrest and had little effect on apoptosis on AZD3965 sensitive cells, effects which could be induced at least partially by the intracellular acidification observed in these cells. To further elucidate the cellular mechanisms involved in the observed cytotoxic effects, in the present work we also evaluated pHi and lactate after AZD3965 addition. Under normoxic conditions MCT1 inhibition decreased pHi of OACM5.1C cells but not OE33 cells and raised intracellular lactate levels in both EAC cell lines. However, this increase in lactate was remarkably higher in the metastatic cell line, which together with the effects on pHi seemed to be a consequence of the different levels of expression of MCTs between the cell lines. It should be noted that intracellular acidification in OACM5.1C cells may have been softened by the increase in MCT4 and NHE-1 expression, a proton pump implicated in pHi regulation in several tumors33–36, observed after AZD3965 addition. Previous studies showed that acidification triggers NHE-1 expression and this response allow cells to fight the lethal cell acidosis37. Since OE33 cells expressed both MCT1 and MCT4 transporters, there was no variation on pH levels and thus expression of NHE-1 remained unaffected after AZD3965 addition.
As previously seen, intracellular acidification affects a wide range of cellular processes like cell growth, through a blockade in G2/M entry and completion of S phase due to mTORC1 inhibition in response to intracellular acidic stress 5,30,38−43. Although the magnitude of internal pH changes observed in the metastatic cells after treatment with AZD3965 was small, previous reports observed on melanoma cells showed that even a slight variation on intracellular pH seems to be able to induce cell growth arrest44.
Intracellular acidification has also been linked to increased apoptosis in cancer cells, creating the optimal conditions for the activation of caspases and different apoptotic pathways45,46. In this study MCT1 inhibition increased apoptosis in OACM5.1C cells, but these effects may have been lowered as a consequence of the small drop observed in intracellular pH.
In line with these results, it is feasible that we did not observe effects on intracellular pH, apoptosis nor cell proliferation upon MCT1 blockade in both cell lines growing under hypoxia as a consequence of MCT4 increased expression. Results also showed that in these conditions, lactate increased to a lesser extent than in normoxia.
Finally, we evaluated the effects of MCT1 inhibition on intracellular ROS levels. High intracellular lactate levels have been previously linked with cytotoxic effects as a consequence of an increase in oxidative stress. It has been proposed that lactate accumulation might disrupt glycolytic flux and, thus, increase ROS production through enhanced mitochondrial respiration to maintain ATP homeostasis 22,33,47. This was not the case for OE33 and OACM5.1C cells, in which treatment with MCT1 inhibitor at different times did not affect ROS levels, a result consistent with the antioxidant character of lactate48, suggesting that in our model AZD3965-induced cytotoxic effects might be independent of ROS generation.
The present work has shown that targeting MCTs is an effective strategy to inhibit tumor growth of EAC cells by inducing an increase in intracellular lactate levels and a decrease in intracellular pH when effective inhibition of lactate and protons efflux is achieved. In this sense, and considering the fact that within a tumor there is a continuous shuttling of lactate among highly glycolytic hypoxic tumor cells, pumping out lactate through MCT4, and oxidative tumor cells which can import and use the monocarboxylate as a source of energy, in addition to the cytotoxic effects that intracellular acidification exerts on tumor cells, the need for developing selective and potent proton and lactate transporters inhibitors arises as a valuable anticancer therapy.