Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0753 Small Molecule Therapeutics Molecular Cancer Therapeutics Triptolide Induces Cell Killing in Multidrug- Resistant Tumor Cells via CDK7/RPB1 Rather than XPB or p44 Jun-Mei Yi1, Xia-Juan Huan1, Shan-Shan Song1, Hu Zhou2, Ying-Qing Wang1, and Ze-Hong Miao1 Abstract Multidrug resistance (MDR) is a major cause of tumor treat- induced by 72-hour treatment of triptolide, which may be due ment failure; therefore, drugs that can avoid this outcome are to partial rescue of RPB1 degradation. We suggest that a precise urgently needed. We studied triptolide, which directly kills MDR phosphorylation site on RPB1 (Ser1878) was phosphorylated by tumor cells with a high potency and a broad spectrum of cell CDK7 in response to triptolide. In addition, XPB and p44, two death. Triptolide did not inhibit P-glycoprotein (P-gp) drug efflux transcription factor TFIIH subunits, did not contribute to tripto- and reduced P-gp and MDR1 mRNA resulting from transcription lide-driven RPB1 degradation and cell killing, although XPB was inhibition. Transcription factors including c-MYC, SOX-2, OCT- reported to covalently bind to triptolide. Several clinical trials are 4, and NANOG were not correlated with triptolide-induced cell underway to test triptolide and its analogues for treating cancer killing, but RPB1, the largest subunit of RNA polymerase II, was and other diseases, so our data may help expand potential clinical critical in mediating triptolide's inhibition of MDR cells. Tripto- uses of triptolide, as well as offer a compound that overcomes lide elicited antitumor and anti-MDR activity through a universal tumor MDR. Future investigations into the primary molecular mechanism: by activating CDK7 by phosphorylating Thr170 in target(s) of triptolide responsible for RPB1 degradation may both parental and MDR cell lines and in SK-OV-3 cells. The suggest novel anti-MDR target(s) for therapeutic development. CDK7-selective inhibitor BS-181 partially rescued cell killing Mol Cancer Ther; 15(7); 1495–503. Ó2016 AACR. Introduction overcoming activities of natural products were associated with the regulation of certain transcription factors. However, these Drug resistance, especially multidrug resistance (MDR), is a compounds do not hold promise for chemotherapeutic use, as major cause of tumor treatment failure (1). Clinically, few drugs they have relatively poor anticancer activity either in vitro are available that do not produce resistance, and resistance- (methyl spongoate and tanshinone I), in vivo (pseudolaric acid reversal agents are not available because most tested compounds B, YCH337, and MT series), or in clinical trials (salvicine). reverse MDR by inhibiting drug transporter function, such as Triptolide (Fig. 1A), a principle ingredient of Tripterygium P-glycoprotein (P-gp), which mediates tumor MDR. However, wilfordii Hook F, is a unique transcription inhibitor (13) with drug transporters are critical for physiologic processes at the potent anticancer activity. Its analogue minnelide is in clinical blood brain barrier, intestine, kidney, and liver (2). trials for cancer therapy, and several others are also in We reported that specific compounds can directly kill MDR clinical development, such as LLDT8 for rheumatoid arthritis and tumor cells without affecting the function of P-gp, such as the PG490-88 and WilGraf for graft rejection after organ transplan- natural products salvicine (3, 4), pseudolaric acid B (5, 6), methyl tation (14). Triptolide has been reported to covalently bind to the spongoate (7), tanshinone I (2, 8), and synthetic small molecules Cys342 residue of XPB, one subunit of TFIIH, a general transcrip- YCH337 (9) and MT series (10–12). Among them, the MDR- tion factor that regulates RNA polymerase I and II (RNAPII; refs. 15–17). Our previous work indicates that CDK7 and p44, two subunits of TFIIH, may contribute to the degradation of 1Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of RPB1 (the largest subunit of RNAPII) and therefore to tumor Sciences, Shanghai, P.R. China. 2CAS Key Laboratory of Receptor cell killing (13), but these relationships require clarification. Research, Shanghai Institute of Materia Medica, Chinese Academy of Here, we report that triptolide directly kills various tumor MDR Sciences, Shanghai, P.R. China. cells without inhibiting P-gp drug-efflux function. Reduced P-gp Note: Supplementary data for this article are available at Molecular Cancer by triptolide was due to transcription inhibition. Transcription Therapeutics Online (http://mct.aacrjournals.org/). factors including c-MYC, SOX-2, OCT-4, and NANOG do not Corresponding Authors: Ze-Hong Miao, Shanghai Institute of Materia Medica, contribute to proliferative inhibition of triptolide, but RPB1 Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, does facilitate this in MDR sublines and in parental tumor Shanghai 201203, China. Phone: 8621-5080-6820; Fax: 8621-5080-6820; E-mail: cell lines. We report that triptolide leads to the phosphorylation [email protected]; and Ying-Qing Wang, [email protected] of CDK7 at its Thr170 and RPB1 at its Ser1878. XPB and p44 doi: 10.1158/1535-7163.MCT-15-0753 appear not to be correlated with triptolide-driven RPB1 degrada- Ó2016 American Association for Cancer Research. tion and cell killing. www.aacrjournals.org 1495 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0753 Yi et al. Materials and Methods pended in PBS for 90 minutes and assessed with flow cytometry with a FACSCalibur cytometer (BD Biosciences; ref. 2). Drugs, chemicals, and reagents Triptolide was purchased from Sigma-Aldrich, and BS-181 was from Selleck. Verapamil, doxorubicin (DX), and vincristine (VCR) Plasmid transfection were obtained from Melonepharma. RIPA lysis buffer, Protein Plasmid-expressing pMSCV-c-MYC (#18775) was obtained AþG beads, and Rhodamine 123 (Rh123) were from the Beyo- from Addgene. Transfection was conducted as reported previously time Institute of Biotechnology (Haimen, China). All antibodies in the published literature (18). were commercially available: RPB1 [carboxy-terminal domain (CTD) repeat], p-S5-RPB1, XPB, OCT-4, SOX-2, NANOG, ubiqui- RNA interference tin, and p-Thr170-CDK7 were from Abcam, GAPDH and IgG were XPB gene expression was reduced with specific siRNA duplexes from Beyotime Institute of Biotechnology (Haimen, China), from Santa Cruz Biotechnology. siRNA transfection was per- CDK7 and p44 were from Santa Cruz Biotechnology, and c-MYC formed with RNAiMAX Transfection Reagent (Invitrogen) accord- was from BD Biosciences. ing to the manufacturer's instructions. Cell culture qRT-PCR Human cancer KB, IM-9, and MES/SA cell lines and doxoru- Total RNA was prepared with the TRIzol reagent (Invitrogen) bicin-selected resistant MES-SA/DX5 cell line were purchased and reverse transcribed into cDNA with a PrimeScript RT Reagent from the ATCC. Human cancer K562 cells and adriamycin-select- Kit (TaKaRa). cDNA was amplified with the SYBR Premix EX TaqII ed resistant K562/A02 cells were purchased from the Institute of Kit (TaKaRa) in a 7500 Fast Real-Time PCR System (Applied Hematology, Chinese Academy of Medical Sciences (Tianjin, Biosystems). The PCR program was as follows: 95 C, 30 seconds; China). Human cancer SK-OV-3 cells were obtained from the 40 cycles (for each cycle 95 C, 5 seconds; 64 C, 20 seconds; 72 C, Japanese Foundation of Cancer Research (Tokyo, Japan). 15 seconds); 72 C, 10 minutes. All primers were synthesized by GM21071 and GM02252 cells were purchased from the Coriell 0 0 Sangon as follows: 5 -GTATTCAACTATCCCACCC-3 (forward) Institute (Camden, NJ). A vincristine-selected resistant KB/VCR 0 0 0 and 5 - GCTTTATTTCTTTGCCATC-3 (reverse) for MDR1;5- subline was from the Sun Yat-Sen University of Medical Sciences 0 0 TCTACAATGAGCT GCGTGTG-3 (forward) and 5 -GGTGAG- (Guangzhou, China). During this study, all cell lines were authen- 0 0 GATCTTCAT-GAGGT-3 (reverse) for b-actin;5-CGTCTCCACA- ticated using the short tandem repeat (STR) profiling at Shanghai 0 0 CATCAGCACAA-3 (forward) and 5 -TGTTGGCAGC AGGA- Genesky Bio-Tech CO., LTD (KB and KB/VCR, May 2013; MES/SA 0 TAGTCCTT-3 (reverse) for c-MYC. and MES-SA/DX5, June 2013; SK-OV-3, August 2013; K562, March 2013; K562/A02 and IM-9, February 2014). Cells were – also periodically authenticated with morphologic inspection and Eliminating p44 expression with transcription activator like tested for mycoplasma contamination. Cell lines were cultured effector nuclease technique – according to the manufacturer's instructions. The transcription activator like effector nuclease technique (TALEN) technique is a new method for genome editing and fi Proliferative inhibition assays genetic modi cations by inducing DNA double-strand breaks IC values of different agents in adherent and suspended that stimulate error-prone nonhomologous end joining or 50 fi cells were measured using a sulforhodamine B (SRB; Sigma) homology-directed repair at speci c genomic locations (19). assay and the Cell Counting Kit-8 (Dojindo Laboratories) To eliminate p44 expression, TALEN was performed with a fi assay, respectively. Cells were seeded into 96-well plates, cul- FASTALE TALEN Kit (SiDanSai Biotechnology) speci cally tar- tured overnight, and treated with gradient concentrations of the geting p44. Transfected positive TALEN plasmids into SK-OV-3 m tested agents for 72 hours. Optical density for both assays was cells and screened with 1.5 g/mL puromycin. After puromycin read with a SpectraMax 190 (Molecular Devices). Averaged IC screening, surviving cells were cultured and selected for p44- 50 fi values were calculated using logit method from three indepen- de cient monoclonal cells. dent experiments (9). Immunoprecipitation Colony formation assays SK-OV-3 cells were treated with 1 mmol/L triptolide for the indi- KB and KB/VCR cells were plated into 6-well plates (200 cells/ cated times, and cells were treated as described previously (10).