Supplemental Figure 4 from Inhibition of the EGFR/STAT3/CEBPD Axis Reverses Cisplatin Cross-resistance with Paclitaxel in the Urothelial Carcinoma of the Urinary Bladder
Gefitinib and S3I-201 attenuate ABCB1 transcripts of and enhance CDDP sensitivity of J82 cells. A, cells were treated with CDDP alone or in combination with gefitinib or S3I-201 for 24 h. A qPCR assay was conducted with specific primers for ABCB1 gene. ***, significant difference (P < 0.001). B, J82 cells were then transfected with ABCB1 reporter (-21~+917) (AB-I, as shown in Fig. 3D) and treated with CDDP alone or in combination with gefitinib or S3I-201. After 24 h. A Luciferase assay was conducted using lysates of transfectants. ***, significant difference (P < 0.001). C, gefitinib and S3I-201 attenuate ABCB1 pump activity and enhance CDDP sensitivity of J82 cells. J82 cells were treated with CDDP alone or CDDP combining gefitinib or S3I-201 for 24 h, MDR dye-loading solution was added to each well for 4 h, and fluorescence intensities were detected by ELISA reader. ***, significant difference (P < 0.001). D, J82 cells were treated with CDDP alone or CDDP combining gefitinib or S3I-201 for 24 h. Death of experimental cells was examined by PI staining. ***, significant difference (P < 0.001). E, CEBPD contributes to drug resistance of cisplatin (CDDP) in nasopharyngeal carcinoma HONE1/R (CDDP-resistant HONE) cells. HONE1/R cells were resistant to CDDP treatment. HONE1 and HONE1/R cells were treated with CDDP in dose-dependent manner. Cell viability was examined by CCK8 assay. *, significant difference (P < 0.05). **, significant difference (P < 0.01). ***, significant difference (P < 0.001). F, CDDP induces expression of CEBPD and ABCB1. HONE1/R cells were treated with CDDP for 24 h and then lysates of experimental cells were analyzed by Western blot. G, HONE1/R cells were treated with gefitinib, S3I-201 or CDDP or in combination as indicated for 24 h, MDR dye-loading solution was added to each well for 4 h and fluorescence intensities were detected by ELISA reader. ***, significant difference (P < 0.001). H, S3I-201 enhances sensitivity of CDDP in HONE1/R cells. HONE1/R cells were treated with CDDP (20 μM) alone or in combination with S3I-201 (100 μM) for 24 h and death of experimental cells was examined by PI staining. ***, significant difference (P < 0.001).
Funding
Ministry of Science and Technology
Ministry of Health and Welfare
History
ARTICLE ABSTRACT
Purpose: Cisplatin (CDDP) is frequently used in combination chemotherapy with paclitaxel for treating urothelial carcinoma of the urinary bladder (UCUB). CDDP cross-resistance has been suggested to develop with paclitaxel, thus hindering successful UCUB treatment. Therefore, elucidating the mechanisms underlying CDDP-induced anticancer drug resistance is imperative and may provide an insight in developing novel therapeutic strategy.Experimental Design: Loss-of-function assays were performed to elucidate the role of the EGFR and STAT3 in CDDP-induced CCAAT/enhancer-binding protein delta (CEBPD) expression in UCUB cells. Reporter and in vivo DNA-binding assays were employed to determine whether CEBPD directly regulates ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily C member 2 (ABCC2) activation. Finally, a xenograft animal assay was used to examine the abilities of gefitinib and S3I-201 (a STAT3 inhibitor) to reverse CDDP and paclitaxel sensitivity.Results: CEBPD expression was maintained in postoperative chemotherapy patients, and this expression was induced by CDDP even in CDDP-resistant UCUB cells. Upon CDDP treatment, CEBPD activated ABCB1 and ABCC2. Furthermore, the EGFR/STAT3 pathway contributed to CDDP-induced CEBPD expression in UCUB cells. Gefitinib and S3I-201 treatment significantly reduced the expression of CEBPD and enhanced the sensitivity of CDDP-resistant UCUB cells to CDDP and paclitaxel.Conclusions: Our results revealed the risk of CEBPD activation in CDDP-resistant UCUB cells and suggested a therapeutic strategy for patients with UCUB or UCUB resisted to CDDP and paclitaxel by combination with either gefitinib or S3I-201. Clin Cancer Res; 23(2); 503–13. ©2016 AACR.Usage metrics
