Downregulation of miR-221/222 sensitizes glioma cells to temozolomide by regulating apoptosis independently of p53 status

Abstract

Abstract.A previous study showed that miR-221/222 can regulate cell apoptosis. p53 is a well known tumor suppressor which can influence the chemosensitivity of glioma cells. However, the effect of miR-221/222 in gliomas with different p53 status is unknown. Here, we demostrate that knockdown of miR-221/222 increases apoptosis in human gliomas of different p53 types (U251 cells, p53 mutant-type; LN308 cells, p53 null-type; and U87 cells, p53 wild-type). Furthermore, the effect of miR-221/22 caused no change of p53 expression in the glioma cells studied. In addition, when a specific siRNA against p53 was employed in U87 cells, no attenuation of apoptosis was found after knockdown of miR-221/222. Importantly, we found that As-miR-221/222-treated cells increased expression of Bax, cytochrome c, Apaf-1 and cleaved-caspase-3. Our results showed that low expression of miR-221/222 sensitized glioma cells to temozolomide (TMZ); in addition, ectopic expression of PUMA by pcDNA-PUMA had a similar effect. Taken together, our study indicates that downregulated miR-221/222 can sensitize glioma cells to TMZ by regulating apoptosis independently of p53 status. IntroductionGlioblastoma, the most aggressive type of tumor arising in the central nervous system (CNS), is considered to be one of the deadliest of human cancers. Despite the advances in surgery, radiation therapy, and chemotherapy, the 1-year overall survival rate is less than 30%, and the median survival time of patients with high-grade glioma is only approximately 15 months (1,2). To develop more optimized and effective treatment strategies for glioblastomas, it is critical to gain deeper understanding of the molecular mechanisms underlying gliomagenesis and to identify targets for therapeutic intervention (3,4).MicroRNAs are small regulatory RNA molecules that in recent years have been identified in the progression of various cancers and proposed as novel targets for anticancer therapies (5,6). By negatively regulating their mRNA targets to either degradation or translational repression, they can act as both tumor suppressors and oncogenes (7). Recent studies showed frequent deregulation of miR-221/222 in glioblastoma and astrocytoma cell lines (8). Overexpression of miR-221/222 increased glioma cell proliferation and invasion in vitro and induced glioma growth in a subcutaneous mouse model (9). In addition, blockade of miR-221/222 cluster suppressed human glioma cell growth, suggesting the cosuppression of miR-221/222 cluster might be a potential therapeutic strategy in glioma (10). Our previous study demostrated that miR-221/222 can directly target PUMA inducing apoptosis (11). p53, an upstream gene of PUMA, is a well known tumor-suppressor. However, the role of miR-221/222 in glioma of different p53 status is not well elucidated.In this study, we identified the involvement of miR-221/-222 in mediating apoptosis in glioma regardless of p53 status. Furthermore, we confirmed that low expression of miR-221/222 sensitizes glioma cells to the alkylating agent TMZ partly...

A 13 mer LNA-i-miR-221 Inhibitor Restores Drug Sensitivity in Melphalan-Refractory Multiple Myeloma Cells

“…More importantly, this anti-tumor activity has been also shown in vivo following systemic treatment with LNA-i-miR-221 plus melphalan in SCID/NOD mice bearing melphalan-resistant MM xenografts without any evidence of toxicity or side effects in treated mice. Our results further support the role of miR-221/222 as crucial mediator of tumor cell resistance to alkylating agents such as cisplatin and temozolamide (28, 31), and we here provide proof-of principle that LNA-i-miR-221 is a potent sensitizing-agent in melphalan-refractory MM.…”

“…In particular, miRNAs have been shown to regulate drug efflux transporters, induction of apoptosis, cell cycle progression, DNA repair mechanisms, and other alterations of drug targets (27). Among miRNAs involved in development of drug-resistance, miR-221/222 plays a key role: inhibition of miR-221/222 has been reported to overcome resistance to cisplatin (28), tamoxifene (29), fulvestrant (30), temozolamide (31), tirosin Kinase Inhibitors (32), and TRAIL (33) in a variety of cancers. We recently provided the first evidence that silencing of miR-221/222 by specific inhibitors exerts anti-tumor activity in MM cells bearing t(4;14) translocations in vitro and in vivo (34), and that naked LNA-inhibitors of miR-221 (LNA-i-miR-221) are suitable for systemic delivery in animals (35).…”

Is There a Best Radiosensitizing Agent in the Treatment of Locally Advanced Rectal Cancer?

“…Overall, cetuximab did not improve the primary outcome (ypCR), thus it was not felt to have contributed significantly to increased radiation-induced cytotoxicity. Given the role of TP53 wild-type status and a functional p53 tumor suppressor gene in radiosensitization [65,66], a retrospective analysis in EXPERT-C noted that TP53 wild-type status was a predictive biomarker in favor of cetuximabbased therapy, albeit not for ypCR, with 5-year DFS and OS of 93 vs. 89 and 68 vs. 65 %, respectively (p = 0.02 each), in favor of the cetuximab arm [67]. Another phase II study tested RT with capecitabine (500 mg/m 2 twice daily), irinotecan (40 mg/m 2 weekly) and cetuximab (400 mg/m 2 on day 1 followed by 250 mg/m 2 weekly) [26].…”

MicroRNAs in glioblastoma multiforme pathogenesis and therapeutics

“…Thus, forced expression of miR‐221/222 and the subsequent downregulation of PUMA, promotes cell survival. Therefore, the knockdown of miR‐221/222 is able to induce cell death and decrease tumor growth 48, 49. miR‐221/222 can also target the cell growth‐suppressive CDK inhibitors P27 and P57 50 and thus are tightly linked to cell cycle checkpoints for initiation of S phase.…”

“…When human glioblastoma U251 cells are treated with antisense miR‐221/222, the cell cycle is arrested in G0 or G1 phase 51. Moreover, treatment with antisense oligonucleotides for miR‐221/222 enhances the effects of both temozolomide (TMZ) and radiation 49, 52.…”