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. 2013:2013:461487.
doi: 10.1155/2013/461487. Epub 2013 Jul 7.

The Synthetic Compound Norcantharidin Induced Apoptosis in Mantle Cell Lymphoma In Vivo and In Vitro through the PI3K-Akt-NF- κ B Signaling Pathway

Hongyan Lv  1 , Yan Li, Hengfei Du, Jie Fang, Xiaoning Song, Jinqiao Zhang
Affiliations

The Synthetic Compound Norcantharidin Induced Apoptosis in Mantle Cell Lymphoma In Vivo and In Vitro through the PI3K-Akt-NF- κ B Signaling Pathway

Hongyan Lv et al. Evid Based Complement Alternat Med. 2013.

Abstract

This study aimed to elucidate the antitumor activity of norcantharidin (NCTD) against human mantle cell lymphoma (MCL). Cell proliferation and apoptosis were examined by MTS and flow cytometry. Caspase-3, -8, and -9 activities were detected with a colorimetric caspase protease assay. Apoptotic proteins-including PARP, cyclin D1, Bcl-2 family proteins, XIAP, and cIAP I-were studied by western blot. The phosphoinositide 3 kinase (PI3K) inhibitor LY294002 was used to investigate the involvement of the PI3K/Akt signaling pathway. In vivo studies were performed using Z138 cell xenografts in nude mice. NCTD inhibited proliferation and induced apoptosis of Z138 and Mino cells, both in vitro and in vivo. PI3Kp110 α and p-Akt expressions were downregulated by NCTD treatment. NCTD downregulated NF- κ B activity by preventing NF- κ B phosphorylation and nuclear translocation. This effect was correlated with the suppression of NF- κ B-regulated gene products, such as cyclin D1, BAX, survivin, Bcl-2, XIAP, and cIAP. This phenomenon was blocked by the PI3K inhibitor LY294002. Our results demonstrated that NCTD can induce growth arrest and apoptosis in MCL cells and that the mechanism may involve the PI3K/Akt/NF- κ B signaling pathway. NCTD may have therapeutic and/or adjuvant therapeutic applications in the treatment of MCL.

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Figures

Figure 1
Figure 1
NCTD inhibited proliferation and induced apoptosis in MCL cells. (a) Z138 and Mino cells were treated with various concentrations of NCTD for 24, 48, and 72 h, and then cell viability was measured by MTS assay. (b) Primary MCL cells from six patients were treated with NCTD for 48 h and then by MTS assay. (c) Z138 and Mino cells were treated with different concentration of NCTD (0–20 μM) for 24 h, and then apoptosis was detected using an annexin V/PI assay followed by flow cytometry. (d) PARP and cleaved PARP were determined by western blot. Experiments were performed in triplicate. Data are representative of three independent experiments. *P < 0.05 versus control.
Figure 2
Figure 2
NCTD-induced caspase activities and G2/M arrest in MCL cells. (a) Z138 and Mino cells were treated with different concentrations of NCTD (0–40 μM) for 24 h or with 5 μM NCTD for 0, 12, 24, 36, and 48 h. Spectrophotometry was used to determine the activities of caspase-3, -8, and -9. (b) Z138 and Mino cells were preincubated with the pancaspase inhibitor Z-VAD-FMK (50 μM) for 1 h before treatment with 5 μM NCTD for 24 h, followed by the annexin V-PI assay. (c) Z138 cells were treated with different concentrations of NCTD (0, 5, 10, and 20 μM) for 24 h, and then cell cycle analysis was performed using flow cytometry. Experiments were performed in triplicate. Data are represented as the mean ± SD (n = 3). *P < 0.05 versus control group.
Figure 3
Figure 3
NCTD downregulated NF-κB by preventing NF-κB phosphorylation and nuclear translocation. Z138 and Mino cells were treated with 5 μM NCTD for 24 h. (a) The protein expressions of NF-κB p65, p-P65, IκBα, and p-IκBα were detected by western blot analysis. (b) Quantitative data of (a). Statistical analysis was carried out using the ANOVA and Bonferroni tests. *P < 0.05 versus control.
Figure 4
Figure 4
NF-κB-regulated gene products were downregulated by NCTD. Z138 and Mino cells were treated with 0, 5, and 10 μM NCTD for 24 h. (a) Western blot was used to analyze the expressions of Bcl-2, BAX, cyclin-D1, survivin, XIAP, and cIAP1. (b) Quantitative data of (a). (c) Z138 cells were treated with different concentrations of NCTD (0, 5, and 10 μM) for 24 h, and then RT-qPCR was used to determine the mRNA expressions of Bcl-2, BAX, cyclin-D1, survivin, XIAP, and cIAP1. Statistical analysis was carried out using the ANOVA and Bonferroni tests. *P < 0.05 versus control.
Figure 5
Figure 5
The PI3K/Akt survival pathway was associated with NCTD-mediated apoptosis. Z138 and Mino cells were treated with 5 μM NCTD for 24 h. (a) Left, western blot was used to analyze the expressions of PI3Kp110α, p-Akt, and Akt. Right, quantitative data from these western blots for three independent experiments. (b) Inhibition of PI3Kp110α expression was confirmed at the mRNA level. (c) Z138 and Mino cells were preincubated with 50 μmol/L LY294002 for 1 h before treatment of 5 μM NCTD for 24 h. NCTD combined with LY294002 led to significant inhibition of the expression of PI3Kp110α, p-Akt, NF-κB p-P65, and cyclin D1 proteins. (d) Quantitative data of (c). *P < 0.05 versus control.
Figure 6
Figure 6
NCTD significantly inhibited Z138 nude mice xenografts. (a) Median tumor volumes of xenografts in mice receiving vehicle control or 20 mg/kg NCTD. Data represent mean ± SD of eight nude mice in each group. Statistical analysis was carried out performed using the ANOVA and Bonferroni tests. *P < 0.05 versus control. (b) Cell apoptosis determined by TUNEL method in MCL tumor samples. (c) Immunohistochemical analysis of NF-κB expression in MCL mouse xenografts (400x). (d) Quantitative data of (b). (e) Quantitative data of (c). *P < 0.05 versus control.
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