13-Methyltetradecanoic Acid Exhibits Anti-Tumor Activity on T-Cell Lymphomas <i>In Vitro</i> and <i>In Vivo</i> by Down-Regulating p-AKT and Activating Caspase-3

<div><p>13-Methyltetradecanoic acid (13-MTD), a saturated branched-chain fatty acid purified from soy fermentation products, induces apoptosis in human cancer cells. We investigated the inhibitory effects and mechanism of action of 13-MTD on T-cell non-Hodgkin’s lymphoma (T-NHL) cell lines both <i>in vitro</i> and <i>in vivo</i>. Growth inhibition in response to 13-MTD was evaluated by the cell counting kit-8 (CCK-8) assay in three T-NHL cell lines (Jurkat, Hut78, EL4 cells). Flow cytometry analyses were used to monitor the cell cycle and apoptosis. Proteins involved in 13-MTD-induced apoptosis were examined in Jurkat cells by western blotting. We found that 13-MTD inhibited proliferation and induced the apoptosis of T-NHL cell lines. 13-MTD treatment also induced a concentration-dependent arrest of Jurkat cells in the G₁-phase. During 13-MTD-induced apoptosis in Jurkat cells, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP, a caspase enzymolysis product) were detected after incubation for 2 h, and increased after extending the incubation time. However, there was no change in the expression of Bcl-2 or c-myc proteins. The appearance of apoptotic Jurkat cells was accompanied by the inhibition of AKT and nuclear factor-kappa B (NF-κB) phosphorylation. In addition, 13-MTD could also effectively inhibit the growth of T-NHL tumors <i>in vivo</i> in a xenograft model. The tumor inhibition rate in the experimental group was 40%. These data indicate that 13-MTD inhibits proliferation and induces apoptosis through the down-regulation of AKT phosphorylation followed by caspase activation, which may provide a new approach for treating T-cell lymphomas.</p></div

Figure 5

<div><p>The therapeutic effect of 13-MTD on Jurkat cell xenografts.</p> <p>The tumor volumes of xenografts were measured with calipers every 3 days for a total of 30 days after the start of treatment. After 30 days of treatment, mice were sacrificed and the tumors removed and photographed. (<b>A</b>) Tumor growth was significantly suppressed with 13-MTD treatment. The respective tumor volumes from the solvent control and 13-MTD treatment groups were 2325.43±318.32 mm³ and 1000.54±156.78 mm³ (n = 5, <i>P</i><0.01, Student’s <i>t</i>-test) (<b>B</b>) Tumors were then fixed and stained with H&E to examine tumor cell morphology. IHC showed decreased phosphorylation of AKT and NF-κB in the tumor tissue after treatment with 13-MTD. (Original magnification×100) (<b>C</b>) 13-MTD enhanced the activation of caspase-3 and PARP proteins in tumor xenografts compared with controls. Tumor lysates were subjected to the analysis of protein levels using western blot analysis. GAPDH was used as a loading control. Representative blots are shown from independent experiments with six different tumors in each treatment group.</p></div

The effects of 13-MTD in the G₁ phase arrest in T-NHL cells.

<p>Cells at a density of 2 × 10⁶ cells per well were treated with 13-MTD at different concentrations (0, 20, 40, 60, 80 µg/ml) for 48 h and harvested to evaluate the cell cycle distribution. The percentage of cells in G₁, S, and G₂ phases in Jurkat (<b>A</b>), EL4 (<b>B</b>) and Hut78 (<b>C</b>) cells are shown. *<i>P</i>>0.05, ** <i>P</i><0.05 compared with the solvent group. All data are derived from three individual experiments with triplicate wells.</p

Western blot analysis of the expression of Bcl-2, c-myc, AKT, p-Akt, p-NF-κB in Jurkat EL4 and Hut78 cells treated with 60 µg/ml solvent control or 13-MTD for 2, 6, 12, 24 h.

<p><b>GAPDH was used as a loading control.</b> There was no change in the expression of Bcl-2, c-myc or AKT proteins. The expression of phosphorylated AKT and NF-κB were decreased in a time-dependent manner with 13-MTD treatment. All data are derived from three individual experiments with triplicate wells. (B) These three cell lines were treated with solvent or 13-MTD (60 mg/ml) for 24 hours, followed by Akt inhibitor V (40 uM) exposure for 30 min. The phosphorylation of AKT was significantly inhibited by Akt inhibitor V. The anti-growth (C) and apoptosis-promoting (D) effects of 13-MTD on T-NHL cells almost disappeared once the phosphorylation of AKT was inhibited. Result are representative of there independent experiments. All the values represent means ±standard deviation (S.D.).</p

13-MTD induces apoptosis, pro-caspase-3 and PARP cleavage in T-NHL cells.

<p>Apoptotic cells were detected by flow cytometry after Annexin V/PI staining, and caspase-3 and PARP were detected by Western blot analysis. (NC, negative control) (<b>A</b>) 13-MTD induced apoptosis in Jurkat, Hut78, and EL4 cells after 48 h of 13-MTD treatment at different concentrations (0, 20, 40, 60, 80 µg/ml). The number of apoptotic cells increased in a dose-dependent manner. Values represent the means ± standard deviation (SD) of three independent experiments. (<b>B</b>) 13-MTD induced apoptosis in Jurkat cells at different incubation time points (24, 48, 72 h). Values represent the means ± SD of three independent experiments. (<b>C</b>) Jurkat, EL4 and Hut78 cells were incubated with 13-MTD or solvent control at 60 µg/ml for the indicated time periods. Each sample was stained with Annexin V/PI and analyzed by flow cytometry. The ratio of cells is shown in each quadrant. The percentage written behind the braces refers to the ratio of apoptosis cells in each sample. Data shown are representative of three independent experiments. (<b>D</b>) Jurkat,EL4 and Hut78 cells were treated with 60 µg/ml 13-MTD or solvent control for 2, 6, 12 and 24 h. Cells were then collected, lysed and subjected to western blot analysis with PARP and caspase-3 antibodies that can detect cleaved PARP and cleaved caspase-3. GAPDH was used as a loading control. All data are derived from three individual experiments with triplicate wells.</p

Inhibition of proliferation of Jurkat, Hut78 and EL4 cells by 13-MTD treatment.

<p>(<b>A</b>) Cultivation with 13-MTD for 48 hours at different concentrations (10, 20, 40, 60, 80 µg/ml) inhibited the proliferation of Jurkat, Hut78 and EL4 cells in a dose-dependent manner. (<b>B</b>) Cell viability of 13-MTD-treated Jurkat cells decreased in a time-dependent manner at different incubation time points (24, 48, 72 h).</p

13-MTD induced apoptosis in Jurkat, Hut78 and EL4 cells.

<p>13-MTD: 13-Methyltetradecanoic acid. Each value represents the mean ± SD of three independent experiments.</p>*<p><i>P</i>>0.05,</p>**<p><i>P</i><0.01,</p>***<p><i>P</i>>0.01 compared with the solvent control group.</p

DataSheet_1_m6A methylation regulators as predictors for treatment of advanced urothelial carcinoma with anti-PDL1 agent.doc

PurposeImmune checkpoint blockade agents were shown to provide a survival advantage in urothelial carcinoma, while some patients got minimal benefit or side effects. Therefore, we aimed to investigate the prognostic value of m6A methylation regulators, and developed a nomogram for predicting the response to atezolizumab in urothelial carcinoma patients.MethodsA total of 298 advanced urothelial carcinoma patients with response data in the IMvigor210 cohort were included. Differential expressions of 23 m6A methylation regulators in different treatment outcomes were conducted. Subsequently, a gene signature was developed in the training set using the least absolute shrinkage and selection operator (LASSO) regression. Based on the multivariable logistic regression, a nomogram was constructed by incorporating the gene signature and independent clinicopathological predictors. The performance of the nomogram was assessed by its discrimination, calibration, and clinical utility with internal validation.ResultsSix m6A methylation regulators, including IGF2BP1, IGF2BP3, YTHDF2, HNRNPA2B1, FMR1, and FTO, were significantly differentially expressed between the responders and non-responders. These six regulators were also significantly correlated with the treatment outcomes. Based on the LASSO regression analysis, the gene signature consisting of two selected m6A methylation regulators (FMR1 and HNRNPA2B1) was constructed and showed favorable discrimination. The nomogram integrating the gene signature, TMB, and PD-L1 expression on immune cells, showed favorable calibration and discrimination in the training set (AUC 0.768), which was confirmed in the validation set (AUC 0.755). Decision curve analysis confirmed the potential clinical usefulness of the nomogram.ConclusionsThis study confirmed the prognostic value of FMR1 and HNRNPA2B1, and constructed a nomogram for individualized prediction of the response to atezolizumab in patients with urothelial carcinoma, which may aid in making treatment strategies.</p

DataSheet_2_m6A methylation regulators as predictors for treatment of advanced urothelial carcinoma with anti-PDL1 agent.docx

PurposeImmune checkpoint blockade agents were shown to provide a survival advantage in urothelial carcinoma, while some patients got minimal benefit or side effects. Therefore, we aimed to investigate the prognostic value of m6A methylation regulators, and developed a nomogram for predicting the response to atezolizumab in urothelial carcinoma patients.MethodsA total of 298 advanced urothelial carcinoma patients with response data in the IMvigor210 cohort were included. Differential expressions of 23 m6A methylation regulators in different treatment outcomes were conducted. Subsequently, a gene signature was developed in the training set using the least absolute shrinkage and selection operator (LASSO) regression. Based on the multivariable logistic regression, a nomogram was constructed by incorporating the gene signature and independent clinicopathological predictors. The performance of the nomogram was assessed by its discrimination, calibration, and clinical utility with internal validation.ResultsSix m6A methylation regulators, including IGF2BP1, IGF2BP3, YTHDF2, HNRNPA2B1, FMR1, and FTO, were significantly differentially expressed between the responders and non-responders. These six regulators were also significantly correlated with the treatment outcomes. Based on the LASSO regression analysis, the gene signature consisting of two selected m6A methylation regulators (FMR1 and HNRNPA2B1) was constructed and showed favorable discrimination. The nomogram integrating the gene signature, TMB, and PD-L1 expression on immune cells, showed favorable calibration and discrimination in the training set (AUC 0.768), which was confirmed in the validation set (AUC 0.755). Decision curve analysis confirmed the potential clinical usefulness of the nomogram.ConclusionsThis study confirmed the prognostic value of FMR1 and HNRNPA2B1, and constructed a nomogram for individualized prediction of the response to atezolizumab in patients with urothelial carcinoma, which may aid in making treatment strategies.</p

Table_1_m6A methylation regulators as predictors for treatment of advanced urothelial carcinoma with anti-PDL1 agent.xlsx

PurposeImmune checkpoint blockade agents were shown to provide a survival advantage in urothelial carcinoma, while some patients got minimal benefit or side effects. Therefore, we aimed to investigate the prognostic value of m6A methylation regulators, and developed a nomogram for predicting the response to atezolizumab in urothelial carcinoma patients.MethodsA total of 298 advanced urothelial carcinoma patients with response data in the IMvigor210 cohort were included. Differential expressions of 23 m6A methylation regulators in different treatment outcomes were conducted. Subsequently, a gene signature was developed in the training set using the least absolute shrinkage and selection operator (LASSO) regression. Based on the multivariable logistic regression, a nomogram was constructed by incorporating the gene signature and independent clinicopathological predictors. The performance of the nomogram was assessed by its discrimination, calibration, and clinical utility with internal validation.ResultsSix m6A methylation regulators, including IGF2BP1, IGF2BP3, YTHDF2, HNRNPA2B1, FMR1, and FTO, were significantly differentially expressed between the responders and non-responders. These six regulators were also significantly correlated with the treatment outcomes. Based on the LASSO regression analysis, the gene signature consisting of two selected m6A methylation regulators (FMR1 and HNRNPA2B1) was constructed and showed favorable discrimination. The nomogram integrating the gene signature, TMB, and PD-L1 expression on immune cells, showed favorable calibration and discrimination in the training set (AUC 0.768), which was confirmed in the validation set (AUC 0.755). Decision curve analysis confirmed the potential clinical usefulness of the nomogram.ConclusionsThis study confirmed the prognostic value of FMR1 and HNRNPA2B1, and constructed a nomogram for individualized prediction of the response to atezolizumab in patients with urothelial carcinoma, which may aid in making treatment strategies.</p