Song et al. Cell Death and Disease (2018) 9:202 DOI 10.1038/s41419-017-0193-2 Cell Death & Disease ARTICLE Open Access Temozolomide–perillyl alcohol conjugate downregulates O6- methylguanin DNA methltransferase via inducing ubiquitination-dependent proteolysis in non-small cell lung cancer Xingguo Song1,LiXie1,2,MinghuiChang2,3,XinranGeng4,XingwuWang1,ThomasC.Chen5 and Xianrang Song 1,2 Abstract The DNA repair enzyme O6-methylguanin-DNA-methltransferase (MGMT) is able to remove products of alkylating agent such as O6-meG and emerges as a central determinant of cancer resistance to temozolomide (TMZ). Temozolomide–perillyl alcohol conjugate (TMZ–POH), a novel TMZ analog developed based on the conjugation of TMZ and POH, displayed strong anticancer potency in multiple cancer types, but seemed not to experience the chemoresistance even in cells with high MGMT expression unlike TMZ and other alkylating agents. In this study, we demonstrated TMZ–POH inhibited MGMT dependent on proteasomal pathway and this inhibition is a significant factor in its toxic effect in the non-small cell lung cancer (NSCLC) cells. 1234567890():,; 1234567890():,; Introduction such as O6-methyl-guanine (O6-meG), which trigger cell Nowadays, chemotherapy has been traditionally con- cycle-dependent DNA damage and cell death4. Although sidered as one of the standard treatment options for TMZ chemotherapy may enhance survival of cancer cancer patients, but the chemoresistance dramatically patients, intrinsic or acquired resistance to TMZ is also hinders its clinical application, especially for alkylating common and accounts for many treatment failures5, agents like temozolomide (TMZ)1. TMZ, an imidazote- because TMZ-induced DNA alkylation damage can be trazine derivative of the alkylating agent dacarbazine, was repaired by O6-methylguanin-DNA-methltransferase approved by the US FDA (Food and Drug Administration) (MGMT)6. and European Union2. Its cytotoxicity is mainly due to its MGMT, an evolutionary conserved enzyme whose high capability to react with DNA to form methyl adducts3, expression indicates poor prognosis in multiple cancers7, plays a crucial role in the defense against alkylating agents8. MGMT is able to remove O6-meG which cova- 9 Correspondence: Xianrang Song ([email protected]) lently is attached to the protein and inactivates it , and 1Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer emerges as a central determinant of tumor resistance to fi Hospital Af liated to Shandong University, Shandong Academy of Medical alkylating agents. Ongoing studies have intended to Sciences, Jinan, Shandong, China 2Department of Clinical Laboratory, Shandong Cancer Hospital Affiliated to inhibit MGMT activity to enhance the therapeutic effect Shandong University, Shandong Academy of Medical Sciences, Jinan, of TMZ10. Despite the development and application Shandong, China of many MGMT inhibitors including O6-benzylguanine Full list of author information is available at the end of the article Xingguo Song and Li Xie contributed equally to this work Edited by A. Stephanou © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Song et al. Cell Death and Disease (2018) 9:202 Page 2 of 10 (O6-BG)11, lomeguatrib12, and their corresponding deri- in A549, SPC-A1, and H460 cells. TMZ–POH treatment vatives, the efficacy of MGMT inhibitors combined with resulted in endogenous MGMT protein downregulation alkylating agents remains controversial due to their compared to its individual constituents and their combi- – hematologic toxicity and inefficiency13 15. nation (Fig. 1b). Moreover, H520 was employed and TMZ–perillyl alcohol conjugate (TMZ–POH), a novel subject to infection of recombinant adenoviral vector TMZ analog16, is developed based on the conjugation of carrying the human MGMT gene (Ad-MGMT). Although TMZ and POH, the latter is a naturally occurring adenoviral infection led to sustained MGMT expression, monoterpene which has the amazing capability to TMZ–POH downregulated the exogenous MGMT pro- enhance the cytotoxicity of TMZ in several tumors17. tein after 48 and 72 h of treatment significantly (Fig. 1c). Previous studies had revealed that TMZ–POH displayed In addition, the phenomenon TMZ–POH-inhibited stronger anticancer potency than its individual con- MGMT protein expression was also observed in other stituents to several types of malignancy such as triple- cancer-derived cells, including ovarian carcinoma-derived negative breast cancer (TNBC)16, non-small cell lung cell A2780, human NPC-derived cell lines CNE2, and cancer (NSCLC)18, human nasopharyngeal carcinoma glioma-derived cell line T98G as shown in Figure S1. (NPC)19, and even TMZ-resistant gliomas20. Unlike TMZ These results suggest TMZ–POH inhibited both endo- and other alkylating agents, TMZ–POH seems not to genous and exogenous MGMT protein, independent of experience the chemoresistance even in cells with high cell type. MGMT expression. To illuminate this issue, in this study Besides, A549 and SPC-A1 cells were exposed to we aimed to explore the mechanisms of TMZ–POH- TMZ–POH with different concentrations, and MGMT regulating MGMT. protein level was detected. As shown in Fig. 1d, MGMT Accumulating evidences prove that MGMT is regulated protein was not affected by low concentration (12.5 and by multiple mechanisms. MGMT is epigenetically 25 μM) of TMZ–POH, but suppressed significantly by silenced by its promoter methylation in many cancer high concentration (50 and 100 μM) TMZ–POH, indi- types21,22, and its expression is regulated by Wnt/β-cate- cating TMZ–POH-downregulated MGMT dependent on nin signaling as the direct transcriptional target of β- concentration. In addition, we delineated the appropriate catenin7. Besides, MGMT protein can be modified by time for TMZ–POH treatments following MGMT multiple pathways. In mammals, it is a short-lived protein, downregulation. The presence of 100 µM TMZ–POH degraded via ubiquitination-dependent proteolysis23, and caused a significant decrease of MGMT protein from 12 h acts as a proteolytic target for the E6 human papilloma until 24 h in A549 and SPC-A1 cells (Fig. 1e). It has been virus oncoprotein24. In yeast, MGMT is a physiological reported that MGMT performs a stoichiometric reaction substrate of both the Ubr1-dependent N-end rule path- to accomplish the DNA repair and is inactivated and way and the Ufd4-dependent Ub fusion degradation degraded, it is not recycled to its active form but restored (UFD) pathway25. due to fresh translation in cells26,27. Therefore, Given the importance and complicated regulation of TMZ–POH was withdrawn to allow MGMT regeneration MGMT, in this paper we focused on the role of TMZ–POH for another 24, 48, and 72 h after incubation for 24 h, and in MGMT regulation, and that of MGMT in TMZ–POH’s then MGMT protein levels were analyzed by western blot. cytotoxicity in multiple cancer cells. We show that As expected, MGMT protein level was restored when TMZ–POH inhibits MGMT dependent on proteasomal TMZ–POH was evacuated (Fig. 1e), suggesting pathway and this inhibition is a significant factor in its toxic TMZ–POH functioned as a transient inhibitor of MGMT. effect in NSCLC cells, thus proposing TMZ–POH as a Taken together, our data support TMZ–POH down- potential therapeutic candidate for NSCLC. regulates MGMT in a concentration and time-dependent manner. Results TMZ–POH downregulates MGMT protein in a MGMT downregulation is required for TMZ–POH’s concentration and time-dependent manner cytotoxicity In order to illuminate the effect of TMZ–POH on Our previous study has revealed that TMZ–POH MGMT protein, we detected MGMT protein expression exhibited its cytotoxic effect on NSCLC18. To explore the in four different NSCLC cell, including A549, SPC-A1, influence of MGMT on TMZ–POH’s cytotoxicity, O6- NCI-H460, and NCI-H520. As shown Fig. 1a, A549, SPC- benzylguanine (O6-BG), an established inhibitor of A1, and NCI-H460 expressed high level of MGMT pro- MGMT and currently undergoing clinical trials15, was tein whereas H520 expressed low. Next, endogenous administrated and combined with TMZ–POH. Cell via- MGMT protein level followed by 100 μM TMZ, POH, bility was assayed by the MT assay. As shown in Fig. 2a, TMZ + POH (100 μM TMZ plus 100 μM POH), combination of TMZ–POH (50 and 100 μM) and O6-BG TMZ–POH or DMSO for 48 h, respectively, was detected (25 μM) suppressed cell proliferation in A549 and SPC-A1 Official journal of the Cell Death Differentiation Association Song et al. Cell Death and Disease (2018) 9:202 Page 3 of 10 Fig. 1 TMZ–POH downregulates MGMT protein in a concentration-dependent and time-dependent manner. a MGMT protein level was evaluated by western blots in A549, SPC-A1, H460, and H520 cells. b MGMT protein level was detected in A549, SPC-A1, and H460 cells following treatment with 100 μM TMZ, POH, TMZ + POH (100 μM TMZ + 100 μM POH), TMZ–POH, or DMSO for 48 h, respectively. c H520 cells were infected by Ad-MGMT or control for 24 h followed by 100 μM TMZ–POH treatment for indicated time. MGMT protein level was detected by western blots.