Regulation Of Monocarboxylate Transporter 1 (MCT1) By Protein Kinase A (PKA), The Canonical Wnt/Beta-Catenin, Notch Signaling Pathways, And By A MCT Metabolic Blocker

Abstract

Monocarboxylate transporters (MCTs) are responsible for the bi-directional diffusion of monocarboxylic acids, such as lactate, pyruvate, & beta-hydroxybutyrate and acetate, across cellular membrane. As a result, they play essential roles in cellular metabolism and various biological processes. In this thesis, we first reported an update on the new developments and advances of MCTs families in the past decade, with an emphasis on their functional roles and regulation mechanisms. Then, we studied MCT1 and its regulation by protein kinase A (PKA), the canonical Wnt/beta-catenin and Notch signaling pathways in an immortalized rat brain endothelial (RBE4) cell line. Our results showed that cAMP-dependent PKA activation caused dephosphorylation and subsequent internalization of MCT1 from plasma membrane into early endosomes. Wnt/beta-catenin pathway failed to affect Mct1 mRNA level, but increased its protein's expression on cellular surface. MCT1 in RBE4 cells is regulated by endosomal-lysosomal, but not proteasomal degradation. Inhibition of deubiquitinases (DUBs) caused an upregulation of MCT1, implying a possible involvement of ubiquitination in MCT1's regulation by Wnt pathway. Notch inhibition by a gamma-secretase inhibitor (DAPT) also elevated MCT1 protein level, and an intact Notch activity was required for the upregulation of MCT1 by Wnt/beta-catenin signaling, indicating a crosstalk between the two pathways. Furthermore, we tested a novel MCT inhibitor (MD1), a substantially more potent analog derived from alpha-cyano-4-hydroxycinnamic acid (alpha-CHC), in suppressing human peripheral blood mononuclear cells (hPBMCs) and selected tumor cells proliferation. Our findings showed that MD1 inhibited hPBMCs proliferation at concentrations no less than 50 micromolar. Under hypoxic culturing condition, it suppressed SW480 colorectal cancer cells and H4IIE hepatoma cells proliferation starting at concentrations of 25 micromolar and 5 micromolar, respectively. The same effects were not observed under normoxia. Besides, hypoxic culturing may sensitize tumor cells to metabolic inhibition of MCT1. A potential combinatorial usage of MD1 with other chemotherapeutic drugs is implied to achieve improved clinical outcomes. In summary, our results showed, for the first time, the regulation of brain endothelial MCT1 by PKA, Wnt/beta-catenin and Notch signaling pathways; we also evaluated the inhibitory role of MD1 in suppressing hPBMCs and tumor cells by blocking lactate transport.