Sequence-Dependent Synergistic Inhibition of Human Glioma Cell

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Sequence-Dependent Synergistic Inhibition of Human Glioma Cell Lines by Combined Temozolomide and miR-21 Inhibitor Gene Therapy Xiaomin Qian,†,⊥ Yu Ren,*,‡,⊥ Zhendong Shi,§,∥,⊥ Lixia Long,† Peiyu Pu,§,∥ Jing Sheng,† Xubo Yuan,*,† and Chunsheng Kang*,§,∥ †

School of Materials Science & Engineering, Tianjin University, Tianjin 300072, China Tianjin Research Center of Basic Medical Science, Tianjin Medical University, Tianjin 300070, China § Department of Neurosurgery, Tianjin Medical University General Hospital, Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government, Tianjin 300052, China ∥ Chinese Glioma Cooperative Group ‡

ABSTRACT: Down-regulation of microRNA-21 (miR-21) can induce cell apoptosis and reverse drug resistance in cancer treatments. In this study, we explored the most effective schedule of the miR-21 inhibitor (miR-21i) and Temozolomide (TMZ) combined treatment in human glioma cells. Three tumor cell lines, U251 phosphatase and tensin homologue (PTEN) mutant, LN229 (PTEN wild-type), and U87 (PTEN loss of function), were subjected to evaluate the antitumor effects of deigned treatments (a predose of miR-21i for 4/8 h and then a subsequent TMZ treatment, a predose of TMZ for 4/8 h and then a subsequent miR-21i treatment, or a concomitant treatment) in vitro. A synergistic antiproliferative and proapoptotic activity was only obtained in U251 and U87 cells when a predose was administered for 4 h before the treatment of the other therapeutic agent, while the best antitumor effect in LN229 cells was achieved by using the concomitant treatment. Our data indicate that the effect of sequence and timing of administration is dependent on the PTEN status of cell lines. The best suppression effect was achieved by a maximal inhibition of STAT3 and phosphorylated STAT3, in PTEN loss of function cells. Our results reveal that both the sequence and the timing of administration are crucial in glioma combination therapy. KEYWORDS: glioma, Temozolomide, microRNA-21 inhibitor, sequence, STAT

1. INTRODUCTION Glioblastoma multiforme (GBM) is the most common and deadly primary tumor of the central nervous system. Despite numerous therapeutic advances, the prognosis for patients afflicted with this disease has improved little in recent decades, and the median survival remains less than 15 months.1 The current standard therapy for GBM includes surgery with maximum feasible resection, radiotherapy, and treatment with the monofunctional alkylating agent, Temozolomide (TMZ), also referred to as Temodar.2 Although the use of TMZ has improved overall and progression-free survival, 70% of patients still experience disease progression within 1 year.3 The response to TMZ is heterogeneous, which induces wide debates on the efficacy of TMZ. Drug insensitivity or resistance © 2012 American Chemical Society

also develops in many cases. Therefore, approaches designed to optimize TMZ efficacy may present an opportunity to extend the survival of patients. The formation of TMZ resistance is commonly considered to be mediated by the DNA repair enzyme, O-6-methylguanine methyltransferase (MGMT), which can reverse the methylation damage induced by alkylating agents.4 Although several studies have shown that a deficiency of MGMT can increase the sensitivity of high-grade glioma to alkylating agents, such as TMZ, many tumors with Received: Revised: Accepted: Published: 2636

April 12, 2012 June 20, 2012 August 1, 2012 August 1, 2012 dx.doi.org/10.1021/mp3002039 | Mol. Pharmaceutics 2012, 9, 2636−2645

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2. MATERIALS AND METHODS Human glioma cell lines LN229, U251, and U87 were obtained from the China Academia Sinica cell repository (Shanghai, China). The methanol solution of poly(amido amine) (PAMAM) dendrimer (generation five) containing 128 surface amino groups (G5-PAMAM, G5D) was purchased from SigmaAldrich (St. Louis, MO). TMZ dissolved in dimethyl sulfoxide (DMSO) was also purchased from Sigma-Aldrich. The 2′-Omethyl (2′-O-Me) miR-21i and the FITC-labeled miR-21i were chemically synthesized by Shanghai GenePharma (Shanghai, China). 2′-O-Me oligos were composed entirely of 2′-O-methyl bases and had the following sequences: miR-21i, 5′-GTC CAC TCT TGT CCT CAA TG-3′; scrambled sequences, 5′-AAG GCA AGC UGA CCC UGA AGU-3′. 2′-O-Me oligos were purified using a high-pressure liquid chromatography system. They were then dissolved in diethylpyrocarbonate (DEPC) water and frozen at −20 °C. 2.1. Preparation and Characterization of the miR-21i/ PAMAM Polyplex. The G5 PAMAM dendrimers were first dialyzed against PBS (MW cutoff 7000) for 1 day and then against deionized water for 2 days to remove methanol. The miR-21i solution (20 μmol/L) was incubated with the PAMAM solution at different N/P ratios for 30 min. The ability of the miR-21i to bind to PAMAM was examined by gel electrophoresis. Briefly, agarose gels (4%, w/v) containing 0.5 μg/mL ethidium bromide (EB) were prepared in TAE (tris-acetateEDTA) buffer. Each sample (10 μL) was mixed with 2 μL of 6 × loading dye, and the mixture was loaded onto an agarose gel. Gels were run at 80 V for 15 min. The locations of RNA bands were visualized using a UV Illuminator. The morphologies of miR21i/PAMAM complexes were observed by scanning electron microscopy (SEM, model S-2250n, Hitachi, Japan). 2.2. Cell Culture. Cells were maintained in DMEM supplemented with 10% FBS (Invitrogen, United States), 50 U/mL penicillin G, and 250 μg/mL streptomycin in an atmosphere containing 5% CO2 at 37 °C. 2.3. MiR-21i Uptake by Tumor Cells. The ability of PAMAM to transfer miR-21i into glioma cells was detected by flow cytometry and confocal fluorescence microscopy. U87 cells were treated with PAMAM/FITC-labeled miR-21i alone or combined with TMZ for 4 h. Cells were trypsinised and washed three times with PBS. Then, the FITC fluorescence was detected by flow cytometry. Confocal fluorescence microscopy was used to assess the intracellular trafficking of the miR-21i. U87 cells grown on glass coverslips in a six-well plate were incubated with the PAMAM/FITC-miR-21i alone or combined with TMZ for 4 h. At the end of the incubation period, the cells were washed three times with PBS and then fixed in paraformaldehyde in PBS for 10 min. For nucleus labeling, fixed cells were washed with PBS and then incubated with DAPI (Molecular Probes, Invitrogen, OR) for 10 min and examined by confocal microscopy. 2.4. MiR-21 Detection by in Situ Hybridization. miR-21 expression was detected by in situ hybridization using antisense locked nucleic acid (LNA)-modified oligonucleotide probes. LNA/DNA oligos contained locked nucleic acids at eight consecutive centrally located bases (indicated by underlined sequence) and had the following sequences: LNA-miR-21 5′TCAACATCAGTCTGATAAGCTA-3′. Cells were fixed with freshly prepared 4% paraformaldehyde (containing 0.1% DEPC) after drug treatment according to the manufacturer's protocol (Boster, Wuhan, China). MiR-21 was labeled by Cy3-

low levels of MGMT are nevertheless chemoresistant. This suggests that other mechanisms are also involved in the formation of resistance to chemotherapy. For example, mutations in p53 or overexpression of the antiapoptotic protein BCL-2 or Bcl-XL can both cause resistance to proapoptotic signals. Currently, microRNA (miRNA)-targeted therapy is a promising alternative for malignant tumors. miRNAs are small, noncoding RNAs that repress gene expression by interacting with the 3′ untranslated regions (3′ UTRs) of mRNAs. miRNAs are predicted to target over 50% of human protein-coding genes, enabling them to play numerous regulatory roles in physiological and developmental processes. Global dysregulation of miRNA expression is an emerging feature in cancer, and the specific deregulation of certain miRNAs is seen in specific tumor types.5 The evasion of apoptosis, a characteristic of many cancers,6 underlies both tumorigenesis and drug resistance.7 Large amounts of evidence point to miRNAs regulating malignancy and apoptosis and further conclude feasible mechanisms for cancer development and resistance to cancer therapy. Furthermore, recent studies support the role of miRNAs in modulating sensitivity to anticancer therapy.8,9 Inhibition of miR-21 and miR-200b sensitized cholangiocytes to gemcitabine;10 repression of miR451 leads to increased metabolism of DOX;11 likewise, downregulation of miR-328 resulted in enhanced mitoxantrone sensitivity.12 Recent reports suggest that overexpressed miR-21 functions as an oncogene in the pathogenesis of glioblastoma.13 miR-21 is considered to activate PI3K/Akt through reducing phosphatase and tensin homologue (PTEN) protein levels, resulting in the resistance of glioblastoma cells to apoptosis and chemotherapy treatment. Conversely, reduced miR-21 expression leads to the inhibition of proliferation and to apoptosis in multiple glioblastoma cell lines.14,15 Furthermore, miR-21 inhibition was demonstrated to sensitize gliomas to cytotoxic therapy in vivo. Suppression of miR-21 by specific antisense oligonucleotides caused enhanced cytotoxicity of VM-26,16 taxol,17 and 5Fu18 against U373, U251, and LN229 glioblastoma cells, respectively. The significance of miRNAs has attracted wide attention from the biomedical research community. Several preclinical and clinical trials have been initiated for miRNA-based therapeutics.19 An extensive number of investigations focus on miRNA regulation of tumor cell chemosensitivity; however, little is known about the sequence-dependent antitumoral effects of combining miRNA-targeted therapies and chemotherapy. Therefore, determination of the optimum inhibitory effect for tumor therapy is urgent and necessary. In the present study, we speculated that administration sequence-dependent antitumor effects may exist when combining an miR-21 inhibitor (miR-21i) with TMZ and that these effects may be, in part, based on modulation of PTEN and its downstream effector pathways. Because different phenotypic contexts might lead to inconsistent results, we selected three cell lines, LN229 (PTEN-wild), U87 (PTEN-lost), and U251 (PTEN mutant).20 We show that miR-21 affects the potency of TMZ, revealing a pervasive role in drug response. A greater understanding of the combinatorial effects of chemotherapy and agents targeting specific signaling pathways in cancer may lead to strategies of chemopotentiation that are distinct from those currently being tested in the clinic. 2637

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Figure 1. (A) Agarose gel electrophoresis of miR-21i/PAMAM complexes with different N/P ratios. (B) SEM images of polyplexes at an N/P of 16:1. (C) Cell uptake detected by confocal microscopy and flow cytometry of U87 cells after treatment with miR-21i, PAMAM/miR-21i, or different treatment combinations.

as the internal control. Data are presented as fold changes (2−ΔΔCt) and were analyzed using Opticon Monitor Analysis Software V2.02 (MJ Research, United States). 2.6. Evaluation of Cytotoxicity and Combination Effects. Cell proliferation was assessed by the MTT assay. Briefly, 4000 cells/well were seeded in 96-well plates and allowed to attach overnight. Cancer cells were exposed to different concentrations of TMZ (1.875, 7.5, 15, and 30 μM/L) for 72 h. In the combination treatment experiments, cancer cells were exposed to drugs as described in section 2.5. At the end of the treatment period, 20 μL of MTT (5 mg/mL) was added to each well, and the cells were incubated at 37 °C for 4 h. The reaction was then stopped by dissolving the cells in 200 μL of DMSO for 15 min. Quantification measurements (optical density) were obtained at a wavelength of 570 nm using spectrophotometric analysis. The results of the combined treatment were analyzed according to the Zheng-Jun Jin method. Briefly, this method supplied a “Q” value, according to which the combination effect between two drugs can be classified as a synergistic effect (Q > 1.15), an additive effect (0.85 < Q < 1.15), or an antagonistic effect (Q < 0.85). The formula is Q = Ea+b/(Ea + Eb − Ea × Eb), where Ea+b is the average effect of the combination treatment, Ea is the effect of the miR-21i only, and Eb is the effect of TMZ only. 2.7. Protein Extraction and Western Blotting. Glioma cells were treated with each drug or drug combination sequence as described above. Then, each group of cells was washed with prechilled phosphate-buffered saline (PBS) three times and then solubilized in 1% Nonidet P-40 lysis buffer. Homogenates were clarified by centrifugation at 20000g for 15 min at 4 °C, and protein concentrations were determined with a Bicinchoninic Acid Protein Assay Kit (Pierce Biotechnology). Total protein lysates were separated by SDS-PAGE on 8% SDS-

avidin at a concentration of 0.5 mg/mL, and nuclei were dyed with DAPI (Genmed, Boston, MA). Then, the fluorescence was visualized using a FluoView confocal laser scanning microscope-FV1000 (Olympus, Tokyo, Japan) and analyzed by IPP5.1 software (Olympus). 2.5. RNA Extraction and Real-Time PCR. The three glioma cell lines were treated with PAMAM/miR-21i alone or concurrently with TMZ. In the combination treatment experiments, cancer cells were treated according to each of the following sequences: (a) concurrent treatment: cells were exposed to both miR-21i (0.05 μM) and TMZ (7.5, 15, and 22.5 μmol/L) for the first 3 days. (b) miR-21i (0.05 μmol/L) followed by TMZ: cells were exposed to miR-21i for 4 or 8 h, then the medium was removed, and cells were treated with TMZ for an additional 2 days. (c) TMZ followed by miR-21i (0.05 μmol/L): cells were exposed to TMZ (7.5, 15, and 22.5 μmol/L) for 4 or 8 h, then the medium was removed, and cells were treated with miR-21i for an additional 2 days. The RNA of different groups was extracted using Trizol reagent (Invitrogen) according to the standard protocol. To detect the concentration of total mRNA, a nanodrop spectrophotometer (Gene, United States) was used. Reverse transcription (RT) was conducted with the mir-Vana qRT-PCR miRNA detection kit (Ambion, United States) in a 10 μL reaction, comprising 2 μL of mirVana 5 × RT buffer, 1 μL of mirVana 1 × RT primer, 25 ng of total miRNA, 0.4 μL of ArrayScript enzyme mix, and DDW (deuterium depleted water) up to 10 μL. MJ-real-time PCR (BioRad, United States) was used to perform the amplification reaction, and the protocol was carried out for 40 cycles, comprising 95 °C for 3 min, 95 °C for 15 s, and 60 °C for 30 s. Both RT and PCR primers were purchased from Ambion. Relative quantification was conducted using amplification efficiencies derived from cDNA standard curves. U6 was used 2638

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Figure 2. (A) LN229, U251, and U87 cells were treated with various concentrations of TMZ, and after 72 h of incubation, an MTT assay was performed. IC50 values were assessed by MTT assays. Each value represents the mean ± SD from triplicate determinations. (B) LN229, U251, and U87 cell lines were treated with various concentrations of TMZ, miR-21i, and combinations of the two drugs as described in the Materials and Methods. MiR-21i after TMZ 8h or reverse sequence vs miR-21i after TMZ 4h (*p < 0.05 for all cell lines). MiR-21i after TMZ 4h or reverse sequence vs simultaneously treat (*p < 0.05 for all cell lines). (C) Q values were calculated according to the Zheng-Jun Jin method for drug interactions using the formula in which Q is a quantitative measure of the degree of interaction between different drugs. Q = Ea+b/(Ea + Eb − Ea × Eb), where Ea+b is the average effect of the combination treatment, Ea is the effect of the miR-21i only, and Eb is the effect of TMZ only. Each value represents the mean ± SD from triplicate determinations.

were designated as apoptotic, and Annexin V+ and PI+ cells were necrotic. The experiments were performed in triplicate. 2.9. Statistical Analysis. Results were analyzed by analysis of variance and the w2 test using SPSS software 11.0. Data are presented as the mean ± standard deviation (SD) of separate experiments (n ≥ 3), and a P value of 0.05 was considered significant.

acrylamide gels, transferred to PVDF membranes (Millipore), and then incubated with primary antibodies detecting STAT3, PSTAT3, or BCL2 (Zhongshan Bio Corp., Beijing, China) followed by incubation with an HRP-conjugated secondary antibody (Zhongshan Bio Corp.). Specific protein was detected using a SuperSignal protein detection kit (Pierce, Rockford, IL). The membrane was stripped and reprobed with a primary antibody against GAPDH (Santa Cruz; 1:1000 dilution). 2.8. Flow Cytometric Analysis of Cell Apoptosis Distribution. Cells were cultured in six-well plates, and after overnight incubation, cells were exposed to different drug administration combinations, as described in section 2.5. Flow cytometry (Becton Dickinson, United States) was then performed. For apoptosis assays, cells were washed with cold PBS and resuspended in binding buffer at a concentration of 1 × 105 cells/100 μL suspension. Cells were transferred to a Falcon tube, and 5 μL of annexin V-FITC and 5 μL of propidium iodide (PI) were added into each tube, which was then incubated at room temperature for 15 min. Annexin V− and PI− cells were used as controls. Annexin V+ and PI− cells

3. RESULTS 3.1. Characterization of miR-21i/PAMAM Nanoparticles and MiR-21i Uptake in Tumor Cells. To improve the transfection of miR-21i, PAMAM was chosen as the vector due to its ability to confer high efficiency of gene transfer.21 The formation of miR-21i/PAMAM polyplexes was controlled with different N/P ratios using gel retardation assays, and the complete retardation of miR-21i by PAMAM was achieved at an N/P ratio of 16:1 (Figure 1A). SEM images demonstrated that miR-21i/PAMAM polyplexes had a well-defined spherical shape with a homogeneous diameter of ∼50 nm (Figure 1B). 2639

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Figure 3. In situ hybridization (A) and real-time PCR analysis (B) detected relative miR-21 expression levels following different drug regimens. Each experiment was done in duplicate. MiR-21i after TMZ 8h or reverse sequence vs miR-21i after TMZ 4h (*p < 0.05 for all cell lines). MiR-21i after TMZ 4h or reverse sequence vs simultaneously treat (*p < 0.05 for all cell lines).

Confocal microscopy and flow cytometry were performed to test the uptake efficiencies of miR-21i/PAMAM. As shown in Figure 1C, clear miR-21i fluorescence was observed in cytoplasm when miR-21i was complexed with PAMAM, whereas miR-21i alone gave almost no detectable fluorescence in the cells. These results were further confirmed by flow cytometry. The cell uptake of miR-21i dramatically increased from 0.5 to 56.1% by binding to PAMAM. Interestingly, simultaneously using TMZ had no significant effect on the uptake of miR-21i (58.2%). In addition, the endocytosis of miR-21i was not affected by TMZ administration and uptake efficiency maintained above 50% with either predose or postdose of TMZ. 3.2. Sequence Dependence of miR-21i and TMZ on Proliferation of Glioma Cells. MTT assays were performed to evaluate the antiproliferative effects of TMZ and miR-21i separately and in combination with different treatment sequences. Treatment with TMZ alone showed a dosedependent inhibition with IC50 values of 27.5 μmol/L for LN229 cells, 30 μmol/L for U251 cells, and 25 μmol/L for U87 cells (Figure 2A). We then assessed the antiproliferation effect of TMZ and miR-21i in combination in the three different exposure sequences of administration. MTT assays were used to compare the proliferation of LN229, U251, and U87 cells (Figure 2B). The sensitivity to TMZ was increased by the specific inhibition of miR-21, of which the maximal inhibition differed for the three glioma cell lines. For LN229, the maximal inhibition occurred with concurrent miR-21i and TMZ treatment, while for U251 and U87 cells, the best inhibition obtained when miR-21i was administered 4 h before TMZ. However, for all cell lines, increasing the interval time between the two therapeutic agents up to 8 h did not yield an improved suppression on tumor cell growth. Our results indicated that

the sequence and timing of administration lead to various tumor suppression effects depending on the cell line, which correlated to PTEN status. To further understand of the effect of the miR-21i on sensitizing LN229, U251, and U87 cells to TMZ, we used the Zheng-Jun Jin22 method to evaluate the effect of different administration regimens and to analyze the cytotoxicity data for antagonism, additivity, or synergy. As shown in Figure 2C, for LN229 cells, concomitant exposure of the two agents resulted in clear synergy with a Q value between 1.15 and 1.46 for the three different concentrations of TMZ. As compared with the concomitant treatment, Q values of both 4 h TMZ predose or postdose systems ranged from 0.90 to 1.10, indicating a clear additive inhibitory effect on cell growth. In U251 and U87 cells, the synergy effect appeared when administration of the miR-21i was followed 4 h later by TMZ or vice versa with Q values between 1.18 and 1.71 (P < 0.01). For concomitant treatments, the Q value ranged from 0.90 to 1.13, showing apparently additive effects. However, for all cell lines, when the dosing interval reached 8 h, a slightly antagonistic effect occurred with Q values between 0.71 and 0.84. 3.3. Drug Regimen Dependence of miR-21 Expression in Glioma Cell Lines. In situ hybridization and PCR were employed to test the expression level of miR-21, with the red fluorescence of Cy-3 indicating the miR-21 expression level. As shown in Figure 3B, different sequences of drug treatment resulted in different miR-21 expression levels. In LN229 cells, the lowest level of miR-21 expression observed by RT-PCR was achieved by concomitant treatment of miR-21i and TMZ. On the other hand, in U251 and U87 cells, miR-21i followed by TMZ 4 h later, produced the lowest level of miR-21 expression. TMZ alone also down-regulated miR-21 expression in the three cell lines. When the interval time between two agents reached 8 h or more, no obvious improvement in down-regulation of 2640

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Figure 4. STAT3 expression level analyzed by Western blot at 4 and 8 h of treatment by TMZ or miR-21i alone. The analysis was done in triplicate. GAPDH protein was used as a protein loading control. In the column chart, 1.0 represented the ratio of STAT3 expression and GAPDH expression in three glioma cell lines with nothing added and was used as a control, respectively. Other values were ratios of STAT3 expression and GAPDH expression in different groups as compared with control. Control vs TMZ or miR-21 treatment at 4 h (*P < 0.05). TMZ or miR-21 treatment at 4 h vs TMZ or miR-21 treatment at 8 h (*P < 0.05).

Figure 5. Evaluation of STAT3 and p-STAT3 levels in LN229, U251, and U87 cell lines. Western blot of protein extracts from cells treated with the miR-21i or TMZ, alone or in different combinations. The expression of GAPDH was examined to ensure uniform protein loading in all lanes. MiR21i after TMZ 8h or reverse sequence vs miR-21i after TMZ 4h (*p < 0.05 for all cell lines). MiR-21i after TMZ 4h or reverse sequence vs simultaneously treat (*p < 0.05 for all cell lines).

either miR-21i or TMZ for 4 h, STAT3 expression was significantly reduced as compared with that of the control and the cells with the other time interval, indicating a clear synergistic effect of this combination. These results clearly demonstrated that STAT3 plays a crucial role in proliferation and apoptosis under combined miR-21i and TMZ treatment, especially in glioma cell lines with either mutant or loss of function PTEN. That is to say, when STAT3 levels were downregulated to a minimal level by a drug, then adding another drug will lead to further suppression. To further verify this conclusion, the cells were treated with TMZ and miR-21i using different sequences and timing of administration and then harvested 72 h later. Western blot analysis was used to detect STAT3 and P-STAT3 levels. As shown in Figure 5, STAT3 and p-STAT3 expression were significantly down-regulated. This clearly indicated that the treatment schedule indeed affected the status of STAT3, which caused a distinct increase in cell proliferation when miR-21i was combined with TMZ. In other words, for all three glioma cell lines, the best antiproliferative

miR-21 expression was seen. These results were corroborated with in situ hybridization experiments (Figure 3A), in which significantly different expression was seen in the distinctive groups. In summary, these data suggested that miR-21 expression was affected by the drug regimen, indicating potential mechanisms for the observed antiproliferative effects on different cell lines. 3.4. TMZ and miR-21i Interaction Associated with PTEN and STAT3 Expression. STAT3 is a convergent point of many signaling pathways and plays a leading role in tumorigenesis and chemoresistance.23 We further investigated the involvement of STAT3 between the interaction of TMZ and miR-21i. As depicted in Figure 4, STAT3 protein showed different levels of reduction with the treatment of TMZ (22.5 μM) or miR-21i. There was no significant difference of STAT3 levels in LN229 cells as compared with the control, which indicated that STAT3 expression did not play a leading role in the cells treated with different combinations of miR-21i and TMZ. However, when U251 and U87 cells were treated with 2641

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Figure 6. LN229 U251 and U87 cells were treated with TMZ (22.5 μmol/L) or miR-21i alone or with the two treatment schedules. Both adherent and detached cells were harvested. Then, flow cytometric analysis of cell death (left) and Western blot analysis of BCL-2 protein (right) were performed as described in the Materials and Methods. In each panel, the number represents the percent of apoptotic cells. Each experiment was done in duplicate. MiR-21i after TMZ 8h or reverse sequence vs miR-21i after TMZ 4h (*p < 0.05 for all cell lines). MiR-21i after TMZ 4h or reverse sequence vs simultaneously treat (*p < 0.05 for all cell lines).

effect occurred when the expression level of STAT3 and PSTAT3 expression level was lowest. 3.5. TMZ-Induced Apoptosis of Glioma Cells Is Potentiated by miR-21i. Next, we assessed the effect of administration sequence on apoptosis and apoptotic regulatory proteins. As expected, treatment with TMZ or miR-21i alone for 72 h resulted in an increase in apoptosis as compared with the control (Figure 6A). In the combined treatment, a significant induction of apoptosis was observed with greater level than that of TMZ or miR-21i alone treatment. However, there were notable differences among the different regimens in three different cell lines. For LN229 cells, the best induction of apoptosis was observed following simultaneous treatment. For U251 and U87 cells, the highest rates of apoptosis appeared when miR-21i was followed 4 h later by TMZ or vice versa, indicating sequence-dependent effects on cell apoptosis by the combined treatment of TMZ and miR-21i. To further elucidate the mechanism of these proapoptotic effects by the synergistic treatment, we evaluated the expression of BCL-2 in the three cell lines. We hypothesized that TMZ would induce cell death through DNA damage by increasing

the expression of antiapoptotic proteins. Furthermore, we hypothesized that miR-21i would modulate the TMZ-induced changes in expression of apoptotic proteins in a sequencedependent manner. Consistent with our hypothesis, the antiapoptotic protein, BCL-2, was induced by TMZ or miR21i alone, whereas the induction of the combined treatment was markedly different in different cell lines (Figure 6B). In LN229 cells, the reduction in BCL-2 levels was most pronounced by the concomitant treatment, while in U251 and U87 cells, the greatest reduction appeared when treatment with miR-21i was followed 4 h later by TMZ or vice versa. These results suggested that the effects of combined TMZ and miR-21i treatment on apoptosis can be partially explained by the different expression of apoptosis proteins in the three glioma cell lines.

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DISCUSSION The combination of conventional cytotoxic drugs with novel agents, which specifically interfere the genes controlling cancer cell survival, proliferation, invasion, and/or metastasis, has generated widespread interest. This has become a promising 2642

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Figure 7. Mechanism of combination therapy for miR-21i and TMZ. MiR-21i was delivered by PAMAM into the cells and coordinated with miR-21, leading to inactivation of miR-21. For LN229 cells (PTEN-wild), the inactivation of miR-21, which is inhibited by the miR-21i and TMZ simultaneous treatment, targets and reduces the expression of PTEN, blocks the PI3K-pAKT pathway, induces an increase of apoptosis, and leads to synergistic antiproliferative activity. While for U251 (PTEN mutant) and U87 (PTEN-lost) cell lines with high STAT3 expression, the synergistic effect was achieved by sequence treatment of the two drugs. When miR-21i or TMZ was added into the cells, STAT3 expression was greatly reduced 4 h later and then combined with another drug, reaching the best antitumor effect.

possible administration sequence-dependent interaction between molecularly targeted and cytotoxic agents. For example, Di Gennaro et al. demonstrated a synergistic effect with 5-FU and either simultaneous or 24 h pretreatment with vorinostat.29 Ma et al. found that simultaneous treatment with Ad-delE1B55 and 5-FU achieved better cytotoxicity than sequential treatments in esophageal carcinoma.30 Morelli et al. found that the combination of a cytotoxic drug with an EGFR inhibitor caused different antiproliferative effects on KYSE30 cancer cells depending on the treatment schedule. Morgillo et al.24 clarified that a synergistic antiproliferative and proapoptotic activity was only obtained when chemotherapy was followed by enzastaurin treatment 48 h later in nonsmall cell lung cancer. In our previous study, we used antisense-miR-21 to inhibit the growth of LN229 and U251 cells, and we found that the growthinhibitory effect of decreased miR-21 levels reached a maximum 3 days after transfection.20 Here, we set the time between TMZ and miR-21i treatment from 0 to 24 h, and the proliferative effect was detected 3 days after the second agent was added. It was found that synergy, addition, and antagonism could be seen depending on administration schedule and cell lines. In LN229 cells (PTEN wild-type), concomitant treatment by miR-21i and TMZ gave a synergetic effect, while an additive effect was only seen in PTEN mutant and loss of function cell lines (U251 and U87), and the synergy appeared with the 4 h interval between miR-21i and TMZ treatments. In addition, an antagonism effect was found when the time between TMZ and miR-21i treatments extended to 8 h or more. It is well-known that PTEN is a direct target of miR-21.31 Thus, it seems reasonable for the LN229 cell lines to acquire a maximum inhibition when miR-21i and TMZ were used

therapeutic approach because the activity of genes regulating mitogenic signals can not only directly cause perturbation of cell growth but also affect the sensitivity of cancer cells to conventional chemotherapy and radiotherapy.24 TMZ is considered to be the most effective drug in the treatment of glioma and the standard chemotherapeutic drug in combination with surgery and radiotherapy.25 However, its efficacy is often limited by tumor recurrence due to the development of drug resistance.26 Recently, it was reported that miR-21 protected human glioblastoma U87MG cells from TMZ-induced apoptosis. Here, we assessed the chemosensitivity of glioma cells LN229, U251, and U87 to TMZ by down-regulating miR21 using an inhibitor. Three glioma cell lines with different PTEN backgrounds were exposed to a gradually increasing TMZ concentration in combination with miR-21i. PAMAM dendrimers were chosen as the vector of miR-21i because of their advantages in gene delivery, such as high degree of molecular uniformity, narrow molecular weight distribution, and specific size and shape characteristics.27 Especially, the cationic surface charge of the PAMAM dendrimer provides a suitable surface to bind anionic miR-21i and can feasibly form a complex through their charge-based interactions, which is effectively transferred into a variety of cultured mammalian cells.28 MTT assays indicated that miR-21i significantly decreased the IC50 of glioma cells to TMZ (Figure 2A). These observations are in agreement with our previous results that miR-21i enhanced glioma cell sensitivity to Taxol and 5-Fu, demonstrating the powerful ability of miR-21 to promote cell apoptosis.17,18 We then investigated the cytotoxic effects of sequence-dependent administration of miR-21i and TMZ combination. There is growing experimental evidence of a 2643

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concurrently. However, for U251 and U87 cell lines, in which PTEN function is lost, only when TMZ or miR-21i was regulated to an optimum state by certain cell signaling pathways, it is possible to yield synergistic effects. Several studies have shown that STAT3 is associated with drug resistance as well as tumor cell proliferation and apoptosis. Our previous study showed that the expression of STAT3 and p-STAT3 decreased to relatively low levels after miR-21i treatment.17 Moreover, STAT3 can contribute to TMZ resistance in gliomas and is a potential target for the reversal of TMZ resistance in patients with a recurrent glioma.26 Our present study demonstrated that STAT3 expression did not change significantly in LN229 cells with treatment of miR-21i followed 4 h later by TMZ, while in U251 and U87 cells, STAT3 expression was reduced to various levels following different administration sequences (Figures 4 and 5). We deduced that when the function of PTEN was lost, STAT3 became the key protein to determine the effect of miR-21i and TMZ combination therapy. STAT3 is not the direct target of miR-21; therefore, it took a longer time to achieve the synergistic combination effect in U251 and U87 cells than that in LN229, and this can explain the mechanism for the effects of administration sequence and timing (Figure 7). STAT3-dependent overexpression of BCL-2 confers a survival advantage to breast cancer cells and contributes to their chemoresistance.32 Therefore, we further investigated the effects of miR-21i and TMZ with different exposure sequences on miR-21 expression and cell apoptosis related signaling. The result of in situ hybridization and PCR corresponded well with that of cell proliferation. Interestingly, TMZ alone also downregulated miR-21 expression in the three cell lines, while the molecular mechanism was unidentified at present. Recently, studies have demonstrated that miR-21 could regulate many signaling pathways, including the EGFR/AKT, wnt/β-catenin signaling pathways. BCL-2, a downstream protein of the two signaling pathways, can be regulated by miR-21 indirectly.33 Further more, it was reported that miR-21 could modulate BCL-2 expression by directly targeting 3′ UTR of BCL-2 mRNA.34 The expression level of the antiapoptotic protein, BCL-2, with treatment of both miR-21i and TMZ was proportional to that of miR-21, strongly indicating that miR21 has a robust role in glioma cell resistance to chemotherapy.

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oncogenic pathway signatures to guide the use of targeted therapeutics.

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AUTHOR INFORMATION

Corresponding Author

*Y.R.: Tianjin Research Center of Basic Medical Science, Tianjin Medical University, Tianjin 300070, China. E-mail: [email protected]. Tel: +86-22-83336866. Fax: +8622-83336866. X.Y.: School of Materials Science & Engineering, Tianjin University, Tianjin 300072, China. E-mail: xbyuan@tju. edu.cn. Tel: +86-22-87401832. Fax: +86-22-27404704. C.K.: Department of Neurosurgery, Tianjin Medical University General Hospital, Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government, Tianjin 300052, China. E-mail: [email protected]. Tel: +86-22-60817499. Fax: +86-2227813550. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was financially supported by the National Nature Science Foundation of China (Grant Nos. 51073118, 51103107, and 81172406); the Program for New Century Excellent Talents in Universities (Grant No. NCET-08-0393); the National High Technology Research and Development Program 863 (2012AA02A508), and Specialized Research Fund for the Doctoral Program of Higher Education (20111202110004).

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REFERENCES

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CONCLUSIONS

In conclusion, our study shows that the combination of miR21i and TMZ acts in a sequence- and time-dependent manner of administration in suppressing human glioma cell activity in vitro. For U251 (PTEN mutant) and U87 (PTEN lost) cell lines, a synergistic antiproliferative and proapoptotic activity was only obtained when miR-21i was given after the 4 h of TMZ treatment or reverse, while in LN229 cells, the best antitumor effect was achieved by using concomitant treatment. Our data indicate that the effect of administration sequence and timing depend on the PTEN status of cell lines. The best suppression effect was achieved by a maximum inhibition of STAT3 and phosphorylated STAT3 levels, when PTEN function was lost. Furthermore, at the lowest level of STAT3 expression, codelivery of TMZ and miR-21i had the best suppression effect on proliferation and apoptosis. Our discovery provides a novel, mechanism-based, therapeutic strategy to explore new approaches to the codelivery of drug and gene therapies and provides an opportunity to make use of 2644

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