Lactate Dehydrogenase B Is Associated with the Response to Neoadjuvant Chemotherapy in Oral Squamous Cell Carcinoma

<div><p>Oral squamous cell carcinoma (OSCC) comprises a subset of head and neck squamous cell carcinoma (HNSCC) with poor therapeutic outcomes and high glycolytic dependency. Neoadjuvant chemotherapy regimens of docetaxel, cisplatin and 5-fluorouracil (TPF) are currently accepted as standard regimens for HNSCC patients with a high risk of distant metastatic spread. However, the antitumor outcomes of TPF neoadjuvant chemotherapy in HNSCC remain controversial. This study investigated the role of lactate dehydrogenase B (LDHB), a key glycolytic enzyme catalyzing the inter-conversion between pyruvate and lactate, in determining chemotherapy response and prognosis in OSCC patients. We discovered that a high protein level of LDHB in OSCC patients was associated with a poor response to TPF regimen chemotherapy as well as poor overall survival and disease-free survival. Our in-depth study revealed that high LDHB expression conferred resistance to taxol but not 5-fluorouracil or cisplatin. LDHB deletion sensitized OSCC cell lines to taxol, whereas the introduction of LDHB decreased sensitivity to taxol treatment. Taxol induced a pronounced impact on LDHB-down-regulated OSCC cells in terms of apoptosis, G2/M phase cell cycle arrest and energy metabolism. In conclusion, our study highlighted the critical role of LDHB in OSCC and proposed that LDHB could be used as a biomarker for the stratification of patients for TPF neoadjuvant chemotherapy and the determination of prognosis in OSCC patients.</p></div

Concentration-response relationship for proton with or without the pre-application of WIN55,212-2.

<p>A. Sequential currents evoked by different pH in the absence of WIN55,212-2 (WIN) and presence of WIN. B. The concentration-response curves for proton with or without WIN55,212-2 (10⁻⁷ M) pre-application. The concentration-response curve for proton with WIN55,212-2 pretreatment shifted downwards. Each point represents the mean ± SEM of 7–11 neurons. All current values were normalized to the current response induced by pH 4.5 applied alone (marked with asterisk).</p

LDHB impacts the efficacy of taxol.

<p>(A, B) KB and HN12 cells were transfected with scrambled or LDHB siRNA. (A) After 72 h, LDHB expression was examined, and LDH activity was measured. (B) After 48 h, cells were washed with PBS twice followed by the addition of fresh serum-free media and extracellular lactate amount was measured at 12 h post-incubation. (C, D) KB and HN12 cells were transfected with scrambled or LDHB siRNA and then treated with DMSO or taxol for 72 h at the indicated concentrations, and cell viability was measured. (E) The LDHB stable-interference strains of KB cells were established and verified by western blotting; the clonogenic assay was conducted in shCON and shLDHB KB cells exposed to 0.5 nM taxol. (F) HN30 cells were transfected with empty vector or LDHB and treated with DMSO or taxol for 72 h at the indicated concentrations. *<i>P</i> < 0.05, compared to the control group. Mean ± SE (n = 3).</p

Effect of WIN55,212-2 on proton-evoked action potentials of rat DRG neurons.

<p>A. Original current and action potentials recordings from the same DRG neuron. Left panel, pH 5.5 induced an inward current with voltage-clamp recording. Right panel, pH 5.5 produced action potentials with current-clamp recording in the same neuron. The pretreatment of WIN55,212-2 (WIN, 10⁻⁷ M) decreased the acid-induced the number of action potentials. B. Bar graph shows the effect of WIN55,212-2 on the number of action potentials produced by pH 5.5 acid perfusions. After 30 min washout of WIN55,212-2, acid-evoked action potentials recovered to control condition. *P<0.05, paired t-test, compared with control, n = 8/each column.</p

Inhibition of the three types of proton-gated currents by WIN55,212-2.

<p>Pre-application of WIN55,212-2 (WIN, 10⁻⁷ M) for 60-s decreased the peak phases of all three types of proton-induced currents (Original current traces in left). Bar graphs in right panel show currents normalized to control (100%, white column). Data in all bar graphs are shown relative to control. Error bars show ± SEM. Statistical tests were performed on raw data using pairing <i>t</i>-test, and significance is shown as follows: *P<0.05, **P<0.01. n = 6/each column.</p

Combination of taxol and LDHB down-regulation exhibits a synergistic effect on cell metabolism.

<p>KB and HN12 cells were transfected with scramble or LDHB siRNA and then treated with DMSO or taxol of 10 nM for 24 h. (<b>A, B</b>) ECAR was measured by an XF96 analyzer. (<b>C, D</b>) OCR was measured by an XF96 analyzer. (<b>E, F</b>) Cell lysates were prepared, and the intracellular ATP level was determined. *<i>P</i> < 0.05, compared to the control group; ^#<i>P</i> < 0.05, compared to the taxol group. Mean ± SE (n = 3).</p

Knockdown of LDHB enhances taxol-induced apoptosis.

<p>(<b>A, B</b>) KB cells and HN12 cells were transfected with scrambled or LDHB siRNA and treated with DMSO or 10 nM taxol for 48 h. <b>(C)</b> HN30 cells were transfected with empty vector or LDHB and treated with DMSO or taxol for 48 h at the indicated concentrations. Quantification results of three independent experiments are shown on the right side.</p

Effect of WIN55,212-2 on nociceptive responses to injection of acetic acid in rats.

<p>Intraplantar injection acetic acid (0.6%, 20 µl) evoked a flinch/shaking response. The bar graph in (A) shows acid-evoked pain was blocked by pretreatment of 200 µM amiloride, and partly blocked by pretreatment of 30 nM PcTx1. In contrast, the acid-evoked pain did not obviously change with pretreatment of 10 µM AMG 9810, *P<0.05, **P<0.01, unpaired t-test, compared with control. n = 8/each column. The bar graph in (B) shows the pretreatment of WIN55,212-2 (WIN) decreased flinching behavior induced by acetic acid in a dose-dependent manner. The effect of WIN55,212-2 was blocked by AM281 (10⁻⁶ M), a selective CB1 receptor antagonist. *P<0.05, **P<0.01, one way analysis of variance followed by <i>post hoc</i> Bonferroni’s test, compared with control; & P<0.01, <i>post hoc</i> Bonferroni’s test, compared with WIN (10⁻⁷ M) column. n = 8/each column. Flinching shaking of paw was recorded as the number of flinches per observation period (5 min).</p

Knockdown of LDHB induces the mitochondrion-dependent apoptosis pathway.

<p>KB cells were transfected with scrambled or LDHB siRNA and then treated with DMSO or 10 nM taxol for 24 h. (<b>A</b>) The mitochondria and the cytoplasm were separated and measured for cytochrome C by western blotting. COX IV was used as a marker for mitochondria. (<b>B</b>) Mitochondrial cytochrome C was measured by FACS analysis. (<b>C</b>) Cell lysates were prepared for western blotting. GAPDH was used as a loading control.</p

Correlations between LDHB and pathological parameters.

<p>*Statistically significant difference.</p><p>Correlations between LDHB and pathological parameters.</p