sj-docx-1-ict-10.1177_15347354231198090 – Supplemental material for Comparison of Plasma Metabolites From Patients With Non-Small Cell Lung Cancer by Erlotinib Treatment and Skin Rash

Supplemental material, sj-docx-1-ict-10.1177_15347354231198090 for Comparison of Plasma Metabolites From Patients With Non-Small Cell Lung Cancer by Erlotinib Treatment and Skin Rash by Won Kil Lee, Jisoo Myong, Eunbin Kwag, Younmin Shin, Ji Woong Son, Byong Chul Yoo, Byoung-Soo Kim, Hwa-Seung Yoo and Jeong June Choi in Integrative Cancer Therapies</p

Effect of cordycepin on the expression of MKK7, c-FLIP_L, and p-JNK in renal tumor tissues.

<p>(A) Cordycepin induces tumor growth suppression. Nude mice were injected with TK-10 cells and then treated with either PBS or 20 mg/kg cordycepin once daily for 20 days. Representative dissected tumors are shown on the right. (B) Immunohistochemical analysis of tumor tissues. Data are the mean ± SD of three independent experiments performed in triplicate. *<i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Cordycepin activates the NF-κB pathway in TNF-α-stimulated TK-10 cells.

<p>(A) Inhibition of TNF-α-induced NO production by cordycepin. (B) Effect of cordycepin on IκB-α (S32) phosphorylation in TNF-α-treated TK-10 cells. The data represent the mean ± SE from three independent experiments. **<i>p</i> < 0.01 and *** <i>p</i> < 0.001 (C) Immunofluorescence micrographs of NF-κB (p65) translocation in TK-10 cells. White arrows indicate p65 retained in the cytoplasm. The data represent the mean ± SE from three independent experiments. **<i>p</i> < 0.01 and *** <i>p</i> < 0.001.</p

Cordycepin inhibits the IκB/NF-κB signaling pathway.

<p>(A) Effect of NF-κB on GADD45B and MKK7 expression in cordycepin-treated TK-10 cells. (B) Effect of cordycepin on NO production in TNF-α-stimulated TK-10 cells. Each column represents the mean ± SD of three independent experiments. (C) Estimation of NF-κB promoter activity in TK-10 cells by luciferase assay. Luciferase activity was determined by measuring the luminescence levels of both firefly and Renilla luciferase with a luminometer. **<i>p</i> < 0.01 and *** <i>p</i> < 0.001.</p

Cordycepin promotes apoptosis in renal carcinoma cells by activating the MKK7-JNK signaling pathway through inhibition of c-FLIP_L expression

<div><p>Cellular FLICE inhibitory protein (c-FLIP) is a key anti-apoptotic regulator that associates with the signaling complex downstream of NF-κB, negatively interfering with apoptotic signaling. The role of c-FLIP downregulation by negative regulation of NF-κB signaling during apoptosis is poorly understood. Here, we demonstrate that NF-κB-mediated c-FLIP_L negatively regulates the JNK signaling pathway, and that cordycepin treatment of human renal cancer cells leads to apoptosis induction through c-FLIP_L inhibition. TNF-α-induced inflammatory microenvironments stimulated NF-κB signaling and the c-FLIP long form (c-FLIP_L) in TK-10 cells. Specifically, cordycepin inhibited TNF-α-mediated NF-κB activation, which induced renal cancer cell apoptosis. Cordycepin downregulated GADD45B and c-FLIP_L, but upregulated MKK7 and phospho-JNK, by preventing nuclear mobilization of NF-κB. Furthermore, siRNA-mediated knockdown of GADD45B in cordycepin-treated TK-10 cells considerably increased MKK7 compared to cordycepin alone. siRNA-mediated knockdown of c-FLIP_L prevented TNF-α-induced JNK inactivation, whereas c-FLIP_L overexpression inhibited cordycepin-mediated JNK activation. The JNK inhibitor SP600125 strongly inhibited Bax expression. In nude mice, cordycepin significantly decreased tumor volume. Taken together, the results indicate that cordycepin inhibits TNF-α-mediated NF-κB/GADD45B signaling, which activates the MKK7-JNK signaling pathway through inhibition of c-FLIP_L expression, thus inducing TK-10 cell apoptosis.</p></div

Cordycepin induces apoptosis in renal cancer cells.

<p>(A) Inhibition of renal cancer cell growth by cordycepin. TK-10 and UO-31 cancer cells were treated with the indicated concentrations of cordycepin for 24, 48 and 72 h. HEK293, Human embryonic kidney cell line was used as normal cells. Data are presented as the mean ± standard deviation (SD) from triplicate experiments. *<i>p</i> < 0.05 and ** <i>p</i> < 0.01 vs. untreated control (B) Flow-cytometric analysis of TK-10, UO-31 and HEK293 cells post cordycepin treatment stained with PI and Annexin-V. The data are representative of three independent experiments.</p

Cordycepin enhances JNK-mediated Bax activation and reduces tumor cell migration.

<p>(A) Effect of p-JNK on Bax expression in cordycepin-treated TK-10 cells. (B) Effects of treatment with cordycepin or a combination of cordycepin and MKK7 siRNA on TK-10 cell migration. Migrating cells were photographed under an inverted microscope (magnification 100×). The migration data are presented as the mean ± SD of three independent experiments performed in triplicate. *<i>p</i> < 0.05 and ***<i>p</i> < 0.001, vs. the control (0 h).</p

Cordycepin induces genes involved in inflammation and apoptosis.

<p>(A) Gene ontology analysis of genes differentially expressed in cordycepin-treated (60 μg/ml) TK-10 cells versus untreated cells. (B) Gene lists (>2-fold, <2-fold, and apoptosis-related genes) and Venn diagrams of the numbers of genes up- and downregulated in each experimental group. (C) Hierarchical clustering of apoptotic genes deregulated in response to cordycepin. (D) Signaling network of the apoptotic genes in response to cordycepin. Nodes colored using IPA are the genes in the apoptotic regulatory network in cordycepin-treated TK-10 cells (red: upregulated genes, green: downregulated genes).</p

Cordycepin mediates the regulation of MKK7 by GADD45B via NF-κB suppression.

<p>(A) Effect of GADD45B on MKK7 expression in cordycepin-treated TK-10 cells. (B) Effect of MKK7 on JNK expression in cordycepin-treated TK-10 cells. (C) Effect of TNF-α-induced NF-κB on c-FLIP_L expression. (D) Effect of c-FLIP_L on JNK expression in cordycepin-treated TK-10 cells. The data represent the mean ± SE from three independent experiments. **<i>p</i> < 0.01 and *** <i>p</i> < 0.001.</p