Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression

Abstract

As the result of genetic alterations and tumor hypoxia, many cancer cells avidly take up glucose and generate lactate through lactate dehydrogenase A (LDHA), which is encoded by a target gene of c-Myc and hypoxia-inducible factor (HIF-1). Previous studies with reduction of LDHA expression indicate that LDHA is involved in tumor initiation, but its role in tumor maintenance and progression has not been established. Furthermore, how reduction of LDHA expression by interference or antisense RNA inhibits tumorigenesis is not well understood. Here, we report that reduction of LDHA by siRNA or its inhibition by a small-molecule inhibitor (FX11 [3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid]) reduced ATP levels and induced significant oxidative stress and cell death that could be partially reversed by the antioxidant N-acetylcysteine. Furthermore, we document that FX11 inhibited the progression of sizable human lymphoma and pancreatic cancer xenografts. When used in combination with the NAD(+) synthesis inhibitor FK866, FX11 induced lymphoma regression. Hence, inhibition of LDHA with FX11 is an achievable and tolerable treatment for LDHA-dependent tumors. Our studies document a therapeutical approach to the Warburg effect and demonstrate that oxidative stress and metabolic phenotyping of cancers are critical aspects of cancer biology to consider for the therapeutical targeting of cancer energy metabolism.

Fig. 1.

Reduction of LDHA expression by siRNA leads to increased oxygen consumption and oxidative stress-induced cell death of P493 human lymphoma B cells. siRNAs targeting human LDHA (SMARTpool) were transfected via electroporation to knock down the LDHA expression transiently. (A) Immunoblotting was performed on whole-cell lysates, probed with rabbit monoclonal anti-LDHA, and reprobed with anti-α-tubulin as a loading control. (B) Oxygen consumption of P493 cells was determined by the use of a Clark-type oxygen electrode at 72 h posttransfection with siLDHA (slope = −1.7) or siControl (slope = −0.7). (C) Intracellular ROS production was detected with DCFDA fluorescence and monitored by flow cytometry at 72 h posttransfection with siLDHA or siControl in the presence or absence of NAC. (D) Cell death was determined by flow cytometry of annexin V- and 7-AAD-stained cells at 96 h posttransfection with siLDHA or siControl in the presence or absence of NAC (20 mM added 24 h after transfection). The number in each figure represents the average percentage (±SEM) of dead cells. The number of dead cells treated with siLDHA compared with the control group has a P value of 0.0002 using the Student’s t test; those treated with siLDHA and NAC compared with the siLDHA group have a P value of 0.001. (E) Cell population growth of siControl cells compared with cells treated with siLDHA grown in the presence or absence 5 mM NAC added daily starting 24 h after transfection. Relative cell numbers at day 4 for siControl vs. siLDHA and siLDHA vs. siLDHA + NAC have a P value of 0.008 and 0.004, respectively. For siControl vs. siControl + NAC, the P value is 0.63.

Fig. 2.

Inhibition of LDHA by FX11 resulted in increased oxygen consumption, ROS production, and cell death. (A) Oxygen consumption of P493 cells was determined by a Clark-type oxygen electrode in the presence (slope = −2.4) and absence (slope = −1.7) of FX11. Data are representative of duplicate experiments. (B) ROS levels were determined by DCFDA fluorescence in P493 cells treated with FX11 or FK866. Data are representative of triplicate samples of two separate experiments. (C) Cell death was determined by flow cytometry of Annexin V- and 7-AAD-stained cells after 24 h of FX11 treatment as compared with control. The number in each figure represents the average percentage of dead cells. The FX11-treated cells compared with the control group have a P value of 8.92e-06. (D and E) Cell population growth of control cells compared with cells treated with FX11 or FK866 in the presence or absence of 20 mM NAC. All cells were grown at 1 × 10⁵ cells/mL. Cell counts were performed in triplicate and shown as mean ± SD, and the entire experiment was replicated, with similar results.

Fig. 3.

(A) FK866 enhances FX11-induced loss of mitochondrial membrane potential. P493 cells treated with control vehicle, FK866, FX11, or both inhibitors were stained with JC-1 and subjected to flow cytometric analysis, with FL2 representing red fluorescence intensity and FL1 representing green fluorescence intensity, which is reflective of cells with decreased mitochondrial membrane. The average percentage (±SEM) of cells with decreased membrane potential is indicated in each panel. The P values were 0.03, 0.002, and 0.0008 when comparing FX11-treated cells, FK866-treated cells, or cells treated with both, respectively, with the control group. (B) Effect of FK866 and FX11 on P493-6 cell proliferation. Live cells were counted using trypan blue dye exclusion. Data are shown as the mean ± SD of triplicate samples. (C) Effect of FX11 or FK866 on ATP levels. P493 cells were treated with 9 μM FX11 or 0.5 nM FK866 for 20 h and counted. ATP levels (mean ± SEM, n = 5 experiments) were determined by luciferin–luciferase-based assay on aliquots containing an equal number of live cells. *P = 0.008; **P = 0.003. (D) Immunoblot of phosphor-AMP kinase (PAMPK) in lysates of cell treated with FX11 or FK866. Tubulin serves as a loading control. AICAR, an AMP analogue that activates AMPK, was used to treat the cells as a positive control. (E) FX11 increases the NADH/NAD⁺ ratio. NADH/NAD⁺ ratio in P493 cells treated with 9 μM FX11 for 24 h as compared with vehicle control. *P = 0.028. (F) FX11 inhibits lactate production. Lactate levels in the media of P493 human B cells treated with 9 μM FX11 or 0.5 nM FK866 for 24 h as compared with control. Control RPMI contained 10.7 mmol/L glucose and no detectable lactate. *P = 6.9E-06.

Fig. 4.

In vivo efficacy of FX11 as an antitumor agent. (A) Immunohistochemical staining to detect hypoxic regions (dark brown) of spleen, liver, and P493 lymphoma by pimonidazole labeling. (B) Effect of FX11 on growth of palpable human P493 B-cell xenografts. Control animals were treated with daily i.p. injection of vehicle (2% (vol/vol) DMSO), and doxycycline (0.8 mg/day) was used as a positive control because it inhibits Myc expression and tumorigenesis in P493 cells. (C) Effect of FX11 and/or FK866 daily treatment as compared with control or compound E (a weak LDHA inhibitor) on established human lymphoma xenografts. (Inset) Photographs of representative animals treated with control vehicle or FX11. (D) FX11 inhibited P198 human pancreatic cancer xenografts as compared with compound E. For experiments in all panels, 2.0 × 10⁷ P493 cells or 5 × 10⁶ P198 cells were injected s.c. into SCID mice or athymic nude mice, respectively. When the tumor volume reached 200 mm³, 42 μg of FX11 and/or 100 μg of FK866 was injected i.p. daily and observed for 10–14 days. The tumor volumes were measured using digital calipers every 4 days and calculated using the following formula: [length (mm) × width (mm) × width (mm) × 0.52]. The results represent the average ± SEM.