Effects of Lactate Transport Inhibition by AZD3965 in Muscle-Invasive Urothelial Bladder Cancer

Abstract

The Warburg Effect is characterized by high rates of glucose uptake and lactate production. Monocarboxylate transporters (MCTs) are crucial to avoid cellular acidosis by internal lactate accumulation, being largely overexpressed by cancer cells and associated with cancer aggressiveness. The MCT1-specific inhibitor AZD3965 has shown encouraging results in different cancer models. However, it has not been tested in urothelial bladder cancer (UBC), a neoplasm where rates of recurrence, progression and platinum-based resistance are generally elevated. We used two muscle-invasive UBC cell lines to study AZD3965 activity regarding lactate production, UBC cells' viability and proliferation, cell cycle profile, and migration and invasion properties. An "in vivo" assay with the chick chorioallantoic membrane model was additionally performed, as well as the combination of the compound with cisplatin. AZD3965 demonstrated anticancer activity upon low levels of MCT4, while a general lack of sensitivity was observed under MCT4 high expression. Cell viability, proliferation and migration were reduced, cell cycle was arrested, and tumor growth "in vivo" was inhibited. The compound sensitized these MCT4-low-expressing cells to cisplatin. Thus, AZD3965 seems to display anticancer properties in UBC under a low MCT4-expression setting, but additional studies are necessary to confirm AZD3965 activity in this cancer model.

Keywords: AZD3965; Warburg effect; lactate; monocarboxylate transporters; urothelial bladder cancer.

Figure 1

Western blot (A) and immunofluorescence (B) results showing expression of glycometabolism-related biomarkers in muscle-invasive urothelial bladder carcinoma cell lines (HT1376R, 253J, BFTC-905 and TCCSUP). Western blot (A) represents similar blots from three independent cell lysates (shown in Figure S1; quantification of the results shown in Figure S2); β-Actin, β-Tubulin and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) were used as loading controls. Immunofluorescence images (B) show co-localization of selected biomarkers; pictures were captured at 200× amplification.

Figure 2

Effect of AZD3965 on metabolic functions of HT1376R and 253J urothelial bladder carcinoma cell lines, detected by quantification of lactate (A,B) and glucose (C) levels (colorimetric kit (A) and high-performance liquid chromatography (B,C)), mitochondrial activity (D; flow cytometry of mitotrackers red vs. green) and ATP production (E, bioluminescence kit) at 24 h and 48 h post-treatment. * p < 0.05, *** p < 0.005 and **** p < 0.001 for AZD3965 treatment versus control condition.

Figure 3

Effect of AZD3965 on the viability of HT1376R and 253J urothelial bladder carcinoma cell lines, detected by the Sulforhodamine B assay at 24, 48 and 72 h post single-dose treatment (A) and at 24 h post triple-dose treatment (given at 24 h, 48 h and 72 h post cells’ incubation, (C)), and by Western blot analysis of proteins associated with cell death pathways at 24 h post-treatment (B). Western blot (B) is representative of similar blots from three independent cell lysates (shown in Figure S4; quantification of the results shown in Figure S5); β-Actin and β-Tubulin were used as loading controls. * p < 0.05, ** p < 0.01 *** p < 0.005 and **** p < 0.001 for AZD3965 treatment versus control condition.

Figure 4

Effect of AZD3965 on the proliferation of HT1376R and 253J urothelial bladder carcinoma cell lines, detected by the BrdU incorporation assay (A) and by quantification of the cells in distinct phases of the cell cycle by flow cytometry analysis of propidium iodide (PI) (B) at 24, 48 and 72 h post-treatment. In (B), a representative cell cycle profile of each condition is shown. * p < 0.05, ** p < 0.01 ***, p < 0.005 and **** p < 0.001 for AZD3965 treatment versus control condition.

Figure 4

Effect of AZD3965 on the proliferation of HT1376R and 253J urothelial bladder carcinoma cell lines, detected by the BrdU incorporation assay (A) and by quantification of the cells in distinct phases of the cell cycle by flow cytometry analysis of propidium iodide (PI) (B) at 24, 48 and 72 h post-treatment. In (B), a representative cell cycle profile of each condition is shown. * p < 0.05, ** p < 0.01 ***, p < 0.005 and **** p < 0.001 for AZD3965 treatment versus control condition.

Figure 5

Effect of AZD3965 on the migration (A) and invasion (B) abilities of HT1376R and 253J urothelial bladder carcinoma cell lines, detected by the wound-healing assay at 24 and 48 h post-treatment (A), and by matrigel invasion chambers at 72 h post-treatment (B). Representative pictures of the assays for each condition are shown. * p < 0.05, ** p < 0.01 and *** p < 0.005 for AZD3965 treatment versus control condition.

Figure 5

Effect of AZD3965 on the migration (A) and invasion (B) abilities of HT1376R and 253J urothelial bladder carcinoma cell lines, detected by the wound-healing assay at 24 and 48 h post-treatment (A), and by matrigel invasion chambers at 72 h post-treatment (B). Representative pictures of the assays for each condition are shown. * p < 0.05, ** p < 0.01 and *** p < 0.005 for AZD3965 treatment versus control condition.

Figure 6

Effect of AZD3965 on tumor growth and angiogenesis of HT1376R urothelial bladder carcinoma cell lines “in vivo”, detected by the chicken chorioallantoic membrane (CAM) assay at 96 h post-treatment. In (A), quantification of tumor growth and blood vessels formation is shown, followed by representative images at 13th (“in ovo”—start of treatment) and 17th (“in ovo” and “ex ovo”—end of treatment) days post egg incubation (4th and 8th days post cells’ injection, respectively) of the control (0.0 nM AZD3965, 0.01% DMSO) and treated (100.0 and 1000.0 nM AZD3965) conditions. In (B), representative images of the excised CAM tissue sections of each condition, immunostained for lectin and Ki67, are shown. Positive controls in insets (CAM section with known positivity for lectin; lymphoma section with known positivity for Ki67). Original magnifications of 100 or 200×. ** p < 0.01 and *** p < 0.0005 for AZD3965 treatment versus control condition.

Figure 7

Effect of cisplatin (C), administered alone and in combination (at indicated dosages) with AZD3965 (A), on the viability (A) and proliferation (B) of HT1376R and 253J urothelial bladder carcinoma cell lines, detected by the Sulforhodamine B assay (A) and by the BrdU incorporation assay (B) at 72 h post-treatment. **** p < 0.0001 for cisplatin treatment (alone or combined) versus control condition. ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.005 and ⁺⁺⁺⁺ p > 0.001 for combined treatment (at indicated dosages) versus cisplatin treatment.