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Inhibition of STAT3 and ErbB2 Suppresses Tumor Growth, Enhances Radiosensitivity, and Induces Mitochondria-Dependent Apoptosis in Glioma Cells
Int. J. Radiation Oncology Biol. Phys., Vol. 77, No. 4, pp. 1223–1231, 2010 Copyright Ó 2010 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/$–see front matter
doi:10.1016/j.ijrobp.2009.12.036
BIOLOGY CONTRIBUTION
INHIBITION OF STAT3 AND ERBB2 SUPPRESSES TUMOR GROWTH, ENHANCES RADIOSENSITIVITY, AND INDUCES MITOCHONDRIA-DEPENDENT APOPTOSIS IN GLIOMA CELLS LING GAO, PH.D., FENGSHENG LI, PH.D., BO DONG, B.S., JUNQUAN ZHANG, PH.D., YALAN RAO, PH.D., YUE CONG, PH.D., BINGZHI MAO, PH.D., AND XIAOHUA CHEN, PH.D. Department of Experimental Therapy of ARS, Beijing Institute of Radiation Medicine, Beijing, China Purpose: Constitutively activated signal transducer and activator of transcription 3 (STAT3) and ErbB2 are involved in the pathogenesis of many tumors, including astrocytoma. Inactivation of these molecules is reported to result in radiosensitization. The purpose of this study was to investigate whether inhibition of STAT3, ErbB2, or both could enhance radiotherapy in the human glioma model (U251 and U87 cell lines). Methods and Materials: The RNAi plasmids targeting STAT3 or ErbB2 were constructed, and their downregulatory effects on target proteins were examined by immunoblotting. After combination treatment of RNAi with or without irradiation, the cell viability was determined using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and clonogenic assays. The in vivo effect of combined treatment was determined using the U251 xenograft model. The apoptosis caused by the inhibition of STAT3 and ErbB2 was detected, and the mechanism involved in the apoptosis was investigated, including increases in caspase proteins, mitochondrial damage, and the expression of key modulating protein of different apoptosis pathways. Results: Transfection of U251 cells with STAT3 or ErbB2 siRNA plasmids specifically reduced their target gene expressions. Inhibition of STAT3 or ErbB2 greatly decreased glioma cell survival after 2, 4, or 6 Gy irradiation. Inhibition of STAT3 and ErbB2 also enhanced radiation-induced tumor growth inhibition in the U251 xenograft model. Furthermore, the suppression of either STAT3 or ErbB2 could induce U251 cell apoptosis, which was related primarily to the mitochondrial apoptotic pathway. Conclusions: These results indicated that simultaneous inhibition of STAT3 and ErbB2 expression can promote potent antitumor activity and radiosensitizing activity in human glioma. Ó 2010 Elsevier Inc. Signal transducers and activators of transcription, ErbB2, siRNA, Radiation, Apoptosis.
INTRODUCTION Malignant glioma is a highly lethal tumor characterized by aggressive proliferation and expansion into surrounding brain tissue (1, 2). Currently, the major approaches to treating the disease are surgical resection, radiotherapy, and adjuvant chemotherapy (3). Inasmuch as a single method usually cannot obtain an ideal therapeutic effect, combinations of surgery, radiotherapy, and chemotherapy are routinely used. The effect of radiation therapy and chemotherapy will greatly influence the prognosis of the patient. Therefore, increasing cell sensitivity to radiation and chemotherapy is an important research focus. Inasmuch as normal tissue surrounding the tumor will be damaged unavoidably during the course of routine treatment, an ideal approach to treating cerebral tumor would selectively kill tumor cells with little influence on normal tissue. Signal transducers and activators of transcription (STATs) are a family of transcription factors that directly transmit
signals from cell surface receptors to the nucleus (4). They play a critical role in mediating the signals of cytokines and growth factors involved in cell growth, differentiation, and survival (5). Whereas normal STAT activation is highly regulated and transient, one member of the STAT family, STAT3, is constitutively activated in diverse human tumors largely because of hyperactive tyrosine kinases (6, 7). It has been reported that inhibition of STAT3 expression could suppress cell growth and induce cell apoptosis in many kinds of cancer cells (8–10). Furthermore, STAT3 is also involved in the resistance to ionizing radiation in many tumor cells. U87 glioblastoma multiforme cells can be sensitized to the cytotoxic effects of ionizing radiation by the expression of dominant-negative STAT3 (11). It has also been found that STAT3 is correlated with the radiosensitization of anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody in head and neck cancers (12).
Reprint requests to: Xiaohua Chen, Ph.D., Department of Experimental Therapy of ARS, Beijing Institute of Radiation Medicine, Beijing 100850, China. Tel: (+86) 10-66931232 Fax: (+86) 1088219030; E-mail: [email protected]
Ling Gao, Fengsheng Li contributed equally to this article. Conflict of interest: none. Received Jan 2, 2009, and in revised form Dec 5, 2009. Accepted for publication Dec 9, 2009. 1223
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Overexpression of ErbB2 has also been reported in human glioma biopsy specimens (13). In fact, the earliest indication that this receptor might be involved in gliomagenesis came from a rat model of primary brain tumor and was later found to be overexpressed on the surface of 20–25% of breast cancer cells (14). The prognosis of cancer patients with overexpressed ErbB2 is poor. In the 1980s, trastuzumab, a monoclonal antibody against ErbB2, was developed, and in 1998, it was approved for the treatment of metastatic breast cancer (15). Furthermore, the monoclonal antibody to the ErbB2 receptor can also enhance the radiosensitivity of human breast cancer cells overexpressing ErbB2 (16). ErbB2 protein is being explored further as a potential target for novel therapeutics. Given the relationships between STAT3, ErbB2, and cancer cell proliferation and radiosensitivity, we presume that simultaneous inhibition of the two proteins may be an effective approach in addition to radiation therapy for glioma. In the present study, we examined the effects of radiosensitization in a glioma model via inhibiting STAT3 and ErbB2 using RNA interference plasmids. We found that suppression of STAT3 and/or ErbB2 reduced survival of glioma cells after 2, 4, or 6 Gy irradiation in culture and enhanced radiationinduced growth inhibition of tumor in a U251 xenograft model when combined with radiation. Furthermore, we found that the inhibition of STAT3 or ErbB2 could induce the decrease of Bcl-2 expression and mitochondria damage, indicating the occurrence of mitochondria-dependent apoptosis. MATERIALS AND METHODS Cell lines The U251 and U87 cell lines were obtained from the Beijing Xiehe Cell Culture Center and maintained in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum.
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TBST (Tris-Buffered Saline Tween-20) buffer (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.05% Tween 20) containing 5% skimmed milk, incubated with monoclonal antibodies to STAT3 (Cell Signaling, Beverly, MA), ErbB2 (Santa Cruz, CA), bcl-2 (Cell Signaling), c-FLIP (Santa Cruz), and actin (Santa Cruz), overnight, and followed by the addition of horseradish peroxidase–linked antimouse IgG (Zhongshan Goldenbridge Biotechnology Co., Beijing, China) and ECL (Enhanced Chemiluminescence) visualization of the bands.
Clonogenic assay Irradiated and/or siRNA-transfected cells were inoculated into 60-mm cell culture dishes (300 cells/dish) and incubated at 37 C in a humidified atmosphere containing 5% CO2 for 14 days. After incubation, the cells were fixed in methanol for 15 minutes and then stained with Giemsa dye for 10 minutes and washed with water, after which visible colonies were counted.
Tumor growth in nude mice Male athymic nu/nu mice weighing 16–18 g were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. U251 cells (1 107) in phosphate-buffered saline (PBS) were subcutaneously inoculated into the armpits of the mice. Tumor diameters were measured every 2 days, and each tumor volume in cubic millimeters was calculated by the following formula: V = 0.5 D d2 (V, volume; D, longitudinal diameter; d, latitudinal diameter). After 7 days, when the average tumor volume reached 0.2 cm3, the mice were stratified into groups (4 mice per group), so that the mean tumor volume in each group was comparable. Treatment groups were divided as follows: pSilence2.1GFP; pSilence2.1-STAT3; pSilence2.1-ErbB2; pSilence2.1-STAT3 + pSilence2.1-ErbB2; radiation + pSilence2.1-GFP; radiation + pSilence2.1-STAT3; radiation + pSilence2.1-ErbB2; and radiation + pSilence2.1-STAT3 + pSilence2.1-ErbB2. The radiation groups received 10 Gy of radiation fractionated over 5 consecutive days (from day 1 to day 5) using a cobalt irradiator to the tumor with the remainder of the body shielded with lead. The plasmid groups received daily intratumorally injections (from day 1 to day 5). After 2 weeks of treatment, the mice were killed, necropsy was performed, and the tumors were weighed.
Construction and transfection of siRNA plasmids The pSilencer2.1-U6 hygro plasmid was a gift from Dr. Yuwen Cong (Beijing Institute of Radiation Medicine). Appropriate sequences from the STAT3 (Medline: 213662) and ErbB2 (Medline:1003817) coding sequences were selected to design the gene-specific RNAi. The targeting sequence of STAT3 RNAi was as follows: 50 -ATCATCATGGGCTATAAGA-30 . The targeting sequence of ErbB2 RNAi was as follows: 50 -GAGATCACAG GTTACCTAT-30 The negative control was the green fluorescent protein (GFP) sequence 50 -GGTTATGTACAGGAACGCA-30 The double-stranded siRNA oligonucleotides with these sequences were cloned into pSilencer2.1-U6 hygro plasmids to generate pSilence2.1-STAT3, pSilence2.1-ErbB2, or pSilence2.1-GFP. The plasmids were transfected into cells using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Immunoblotting The U251 cells were removed 48 hours after transfection, and protein concentrations in total cell lysates were measured by the Bradford method. Aliquots of cell lysates containing 50 mg of proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose filters. The filters were blocked with
Immunohistochemistry Formalin-fixed, paraffin-embedded tissue sections were treated with anti-STAT3 or anti-ErbB2 antibody at a dilution of 1:100 for 1 hour and stained using the LSAB + System-HRP kit (DakoCytomation) according to the manufacturer’s instructions.
Isolation of primary astrocytes The cerebral cortices from 1-day old Wistar rats were removed using sterile techniques and cut into small pieces with scissors, to which 5 mL of warmed (37 C) trypsin solution (2.5mg/mL) in DHanks’ solution was added for the digestion. After incubation in 37 C for 10 minutes, the tissue was transferred into a sterile conical centrifuge tube and centrifuged at 1000 rcf for 5 minutes. The precipitation was resuspended in 2 mL of fresh DMEM-F12. The cell suspension was left for 1 to 2 minutes, and then the supernatant was transferred into another sterile tube. Two milliliters of fresh DMEM-F12 were added into the conical centrifuge tube again, and this process was repeated seven times. The supernatants were pooled and passed through a 21-gauge needle to make a single cell suspension. The cells were counted and inoculated at 1 104 cells/cm2 in plastic tissue culture dishes.
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Determining purity of primary astrocytes by indirect immunofluorescence assay To assess the purity of the primary astrocytes we isolated, an indirect immunofluorescence assay detecting Glial fibrillary acidic protein (GFAP), a specific marker of astrocytes, was performed. Briefly, the primary astrocytes were placed on a slide and incubated with anti-GFAP monoclonal antibody (Santa Cruz), followed by fluorescein-conjugated mouse-specific goat secondary antibody (Zhongshan Goldenbridge Biotechnology Co., Beijing, China). The labeled cells were then observed by fluorescence microscopy.
Nuclear damage detected by Hoechst 33258 staining Apoptotic nuclear morphology was assessed using Hoechst 33258 (Sigma, USA). Briefly, at the indicated time points after transfection, Hoechst 33258 (100 mM) was added to the cells in 96-well plates. After incubation at 37 C for 30 minutes, the cells were washed with PBS and observed by epifluorescence microscopy.
Cell apoptosis assay Apoptosis was quantified using the Annexin V-FITC (Fluorescein Isothiocyanate) Apoptosis Detection Kit (Bosai, China) according to the manufacturer’s instructions. Briefly, the treated cells were trypsinized, pelleted by centrifugation, and resuspended in Annexin V binding buffer. FITC-conjugated Annexin V (1 mg/mL) and propidium iodide (PI, 50 mg/mL) were added to the cells and incubated for 30 minutes at room temperature in the dark. Analyses were performed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA).
Caspase-3/7 assay A total of 5 103 cells in 100 mL of medium were plated in 96well plates and transfected with the siRNA plasmids. The caspase-3/ 7 levels were detected using Caspase-Glo 3/7 Reagent (Promega). The assay was performed according to the manufacturer’s instructions, and the luminescence was measured by a luminometer (Thermo Electron, Finland). The caspase-8 and caspase-9 assays were performed similarly to this assay.
Measurement of mitochondrial membrane potential U251 cells were plated in 12-well plates at 5 104 cells per well and treated without or with different siRNA plasmids. At the indicated time points after transfection, the transfected cells were removed. The cell suspensions were incubated with JC-1 dye (5 mg/mL) for 15 minutes at 37 C, and green (FL-1) and red (FL-2) fluorescence was measured by flow cytometry after washing the cells with PBS twice.
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Statistical analysis All experiments were done in triplicate or quadruplicate. Triplicate measurements were done per data point in each experiment. A two-tailed Student’s t test was used for statistical analysis of comparative data using SPASS software. Values of p < 0.05 were considered to be significant.
RESULTS STAT3 and ErbB2, constitutively expressed in the U251 human glioma cell line, could be specifically downregulated by RNAi U251 cells were transfected with pSilence2.1-STAT3, pSilence2.1-ErbB2, or the control pSilence2.1-GFP to determine their ability to silence STAT3 or ErbB2. Western blot analysis was performed 48 hours after transfection, and the results revealed that STAT3 (Fig. 1A) and ErbB2 (Fig. 1B) expressions were specifically suppressed up to 90% at this time point in cells transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2, respectively, but not with pSilence2.1-GFP. Knockdown of STAT3 or ErbB2 sensitized glioma cells to radiation by decreasing cell viability To determine whether inhibition of STAT3 or ErbB2 could sensitize U251 cells to radiotherapy, the U251 cells were transfected with the pSilence2.1-STAT3, pSilence2.1ErbB2, or both, with or without subsequent immediate radiation treatment of 2 Gy, which is the single radiation dose used in conventional fractionated radiotherapy. To determine cell viability after the various treatments described, the clonogenic assay was performed after treatment. As shown in Fig. 2A, radiation alone did not affect the proliferative activity/viability, whereas pSilence2.1-STAT3 and pSilence2.1-ErbB2 decreased viability significantly, compared with untreated or pSilence2.1-GFP, and the decrease in viability was more marked after STAT3/ErbB2 RNAi. However, the irradiation of cells transfected with either pSilence2.1-STAT3 or pSilence2.1-ErbB2 decreased cell viability more significantly than STAT3 or ErbB2 RNAi alone. Interestingly, the maximal reduction in activity was achieved in cells after simultaneous inhibition of STAT3 and ErbB2 and irradiation in comparison with those with STAT3/ErbB2 RNAi but without irradiation. Similar results were obtained after 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays (Fig. E1).
Fig. 1. siRNA targeting signal transducer and activator of transcription 3 (STAT3) or ErbB2 specifically inhibits their expression. (A) STAT3 expression 48 hours after transfection with siRNA targeting STAT3 in U251 cells, detected by immunoblotting. (B) ErbB2 expression 48 hours after transfection with siRNA targeting ErbB2 in U251 cells, detected by immunoblotting. b-actin expression was monitored as the normalizing control. The ratios of STAT3/b-actin and ErbB2/b-actin were calculated using densitometry.
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Fig. 2. Knockdown of signal transducer and activator of transcription 3 (STAT3) or ErbB2 sensitizes U251 cells to radiation by decreasing cell viability. (A–C) Colony formation assay of U251 cells after doses of 2 (A), 4 (B), and 6 Gy (C) irradiation with or without transfection. The surviving fraction (%) = (CFE of active treatment)/(CFE of control) 100%, CFE (colony form efficiency) (%) = (number of colonies)/(number of inoculation cells) 100%. In A–C, *p < 0.05 (compared with pSilence2.1-GFP transfection alone); #p < 0.05 (compared with irradiation plus pSilence2.1-GFP transfection); &p < 0.05 (compared with the same siRNA transfection without irradiation).
We further investigated whether the radiosensitizing effect would occur at higher irradiation doses of 4 Gy and 6 Gy. The clonogenic assays showed that the inhibition of STAT3, ErbB2, or both also sensitized U251 cells to 4 or 6 Gy radiation, which was similar to the results obtained with 2 Gy (Fig. 2C and D). Remarkably, the radiosensitizing effects of STAT3 and/or ErbB2 RNAi were weakest after 6 Gy was used as the radiation dose. This result might be due to the fact that the effect of RNAi was masked by the strong effect of the 6-Gy dose on cell viability. To investigate whether the radiosensitizing effect of STAT3 and/or ErbB2 RNAi could also occur in another glioma cell line, we repeated the clonogenic assay using 2 Gy as the radiation dose in the U87 cell line. The radiosensitizing effects was also observed in the U87 cell line and were very similar to results obtained with the U251 cell line (Fig. E2).
when both genes were downregulated. STAT3 RNAi plus 2 Gy radiotherapy or ErbB2 RNAi plus 2 Gy radiotherapy could further inhibit the tumor growth compared with STAT3 RNAi or ErbB2 RNAi, respectively. Among the different treatments, combining STAT3 and ErbB2 RNAi with 2 Gy radiotherapy led to the most significant inhibition of tumor growth. To determine whether the intratumoral injection of pSilence2.1-STAT3 or pSilence2.1-ErbB2 effectively inhibited the expression of their targeted proteins in vivo, the tumor tissues were removed, fixed, and prepared for immunohistochemical analysis as described in Materials and Methods. As represented in Fig. 3C, the expression of STAT3 in the tumor was markedly suppressed after pSilence2.1-STAT3 treatment in comparison with pSilence2.1-GFP treatment. A similar inhibitory effect on ErbB2 expression was observed after pSilence2.1-ErbB2 treatment.
Inhibition of STAT3 and ErbB2 enhanced radiationinduced inhibition of tumor growth in vivo To determine whether the knockdown of STAT3 or ErbB2 enhances radiation-induced growth inhibition of tumors, the U251 xenograft model was established by inoculating mice with U251 cells, and the tumor volumes were measured by calipers. The tumor-bearing mice were treated with 2 Gy daily for 5 days at the same time for radiotherapy, and the interference plasmids were given daily for 5 days at a dose of 5 mg/kg by intratumoral injections. As shown in Fig. 3A and B, both STAT3 RNAi and ErbB2 RNAi could inhibit the tumor growth. The effect was more pronounced
Downregulation of STAT3 or ErbB2 induced U251 cell apoptosis The change in tumor size is the result of a dynamic balance between cellular proliferation and apoptosis. Becuase downregulation of STAT3 or ErbB2 could decrease the proliferative abilities of U251 and U87 cells, we asked whether they could affect apoptosis in U251 cells. To clarify this, the apoptosis of U251 cells was determined by various assays after the different treatments. As shown in Fig. 4A, the cells treated with pSilence2.1-STAT3 or pSilence2.1-ErbB2 showed significant nuclear condensation at different time points, compared with the pSilence2.1-GFP control.
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Fig. 3. Inhibition of signal transducer and activator of transcription 3 (STAT3) and ErbB2 enhanced radiation-induced inhibition of tumor growth in vivo. Mice bearing U251 tumors with an average volume of 0.2 cm3 were stratified into groups of four and treated with siRNA plasmid via intratumoral injection daily for 5 days, with or without 2 Gy daily. (A) Tumor volumes every 4 days after the first administration. (B) Weight of tumors resected from nude mice at day 14 after treatment. (A and B) *p < 0.05 (compared with GFP RNAi (negative control)); #p < 0.05 (compared with GFP RNAi + 2Gy); &p < 0.05 (compared with the same siRNA transfection without irradiation). (C) Immunohistochemistry of tumor tissue for STAT3 and ErbB2 in specific siRNA-treated groups with or without radiation. The monoclonal mouse anti-STAT3 or -ErbB2 antibodies were used to stain paraffin sections.
Additionally, quantitative detection of cellular apoptosis was performed using the Annexin-V kit and flow cytometry. The results in Figure 4B show that pSilence2.1-GFP did not lead to significant apoptosis of U251 cell at any time point, compared with untreated cells. However, pSilence2.1STAT3 led to significant apoptosis of U251 cells in a timedependent manner. pSilence2.1-ErbB2 could also induce
U251 cell apoptosis significantly at 24 hours and to a greater degree at 48 hours after transfection. STAT3 and ErbB2 RNAi do not affect normal astrocytes An ideal approach to tumor therapy should have no toxic effects on normal cells while efficiently killing the tumor cells. To detect the effect of RNAi on normal astrocytes,
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Fig. 4. Down-regulation of signal transducer and activator of transcription 3 (STAT3) or ErbB2 induced U251 cell apoptosis. At indicated time points after transfection, the apoptosis of transfected U251 cells was analyzed by fluorescence microscopy after Hoechst 33258 staining (A) or analyzed by flow cytometry after Annexin V/PI double staining (B). *p < 0.05 (compared with GFP [negative control]).
the primary astrocytes from the brain of 1-day-old Wistar rats were cultured. As shown in Fig. 5A, B, and C, the purity of astrocytes was assessed by indirect immunofluorescence using GFAP antibody. Unlike U251 cells, Hoechst 33258 staining (Fig. 5D) and Annexin V/PI double staining–based flow cytometry analysis (Fig. 5E) showed no apoptosis in both pSilence2.1-STAT3 and pSilence2.1-ErbB2 groups at any time point. Apoptosis induced by inhibition of STAT3 and ErbB2 is primarily mitochondria-dependent pSilence2.1-STAT3or pSilence2.1-ErbB2-treated U251 cells were cotreated with the pan-caspase inhibitor Z-VAD-FMK to investigate whether their induction of apoptosis was dependent on caspase activation. Interestingly, the pSilence2.1-STAT3- and pSilence2.1-ErbB2-induced apoptosis was completely abrogated by the addition of Z-VAD-FMK (Fig. 6A). Furthermore, caspase-3 and caspase-7 are two effector caspases that play a key role in
the execution of apoptosis, and pSilence2.1-STAT3 or pSilence2.1-ErbB2 significantly activated caspase-3 and caspase-7 levels after treatment of U251 cells (Fig. 6B) but not of primary astrocytes (Fig. 6C). This result was consistent with the observations above that STAT3 and ErbB2 RNAi showed no notable apoptotic effects on normal astrocytes. Although the activation of caspase-3 and caspase-7 was shown to be involved in the apoptosis induced by STAT3 RNAi and ErbB2 RNAi, the exact molecular mechanisms were still not clear. To investigate it, we tested the activity of caspase-8 and caspase-9, which represent the death receptor pathway and the mitochondrial pathways, respectively, after the inhibition of STAT3 and ErbB2. As shown in Fig. 6D and E, the activities of caspase-8 and caspase-9 both increased. However, the apoptosis was almost completely inhibited when the specific inhibitor of caspase-9, Z-LEHD-FMK, was added simultaneously (Fig. 6F), indicating that caspase-9 plays a more important role in the apoptotic process.
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Fig. 5. Signal transducer and activator of transcription 3 (STAT3) and ErbB2 RNAi showed no notable effects on normal astrocytes. (A–C) Assessment of purity of primary astrocyte by indirect immunofluorescence assay using a monoclonal antibody against GFAP, the astrocyte-specific marker. (A) Primary astrocytes in a bright field were recorded, and (B) the green fluorescence of the same field was observed. (C) Finally, the fluorescence images and bright images were merged. At indicated time points after transfection, the apoptosis of transfected primary astrocytes was analyzed (D) by fluorescence microscopy after Hoechst 33258 staining or (E) by flow cytometry after annexin V/PI double staining.
Activation of caspase-9 is the result of cytochrome c release, which is caused by the mitochondrial transmembrane potential (DJm) reduction. Therefore, we subsequently examined the mitochondrial transmembrane potential using the cationic lipophilic dye JC-1. After the treated U251 cells were stained with JC-1, qualitative assessment using fluorescence microscopy and quantitative detection by flow cytometric analysis were performed. Both assays detected a time-dependent dissipation of DJm in pSilence2.1STAT3- or pSilence2.1-ErbB2-treated U251 cells (Fig. 6G and E3). Furthermore, the expressions of c-FLIP and Bcl-2, which serve as critical regulators of the death receptor pathway and the mitochondrial pathway, respectively, were examined by Western blotting. As shown in Fig. 6H, the expression of Bcl-2 decreased significantly 24 hours after STAT3 RNAi or ErbB2 RNAi treatment, compared with GFP RNAi treatment or untreated control, which was consist with the previous finding that STAT3 and ErbB2 could upregulate the expression of Bcl-2. However, the expression of c-FLIP, unlike Bcl-2, was inhibited only slightly. Inasmuch as Bcl-2 plays a critical role in maintaining DJm, the result indicated that the reduction of DJm, which caused the increased activity of caspase-9, might be the result of Bcl-2 suppression.
DISCUSSION STAT3 and ErbB2 are upregulated and overexpressed in various types of cancers. Previous studies suggest that they are both related to the oncogenesis and radiation resistance of cancer cells. In this study, we demonstrated the enhanced effects of radiosensitization in a glioma model by RNAimediated inhibition of STA T3 and ErbB2, suggesting that synchronous inhibition of STAT3 and ErbB2 could be an effective addition to radiation therapy of glioma. Specifically, we demonstrated that the inhibition of STAT3 or ErbB2 could decrease the survival of U251 and U87 cells in culture, and the radiation-induced growth inhibition of tumors in the U251 xenografts model was enhanced when combined with radiation. Currently, the main approaches to glioma treatment are surgery, radiotherapy, and chemotherapy. However, the prognosis of glioma is poor because of the existence of the blood–brain barrier and radiation resistance. Therefore, increasing the sensitivity of tumors to radiotherapy and chemotherapy would provide a significant advance in effective treatment for glioma patients. Some genes contributing to radiation resistance are known. In some previous studies, radiotherapy combined
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Fig. 6. The apoptosis induced by inhibition of signal transducer and activator of transcription 3 (STAT3) and ErbB2 is primarily mitochondrial-dependent. (A) U251 cells were treated with or without Z-VAD-FMK after transfection, and the apoptosis was determined by annexin V/PI double staining. The Annexin V-positive cells were counted. (B) The enzyme activities of caspases-3/7 in the transfected U251 cells or (C) treated primary astrocytes were determined. (D and E) U251 cells were treated with or without Z-VAD-FMK after transfection, and the enzyme activities of caspase-8 (D) and caspase-9 (E) were determined. A–E, *p < 0.05 (compared with GFP). (F) U251 cells were treated with or without Z-LEHD-FMK after transfection, and the apoptosis was determined by annexin V/PI double staining. The Annexin V-positive cells was counted. *p < 0.05 (compared with GFP of the same time). #p < 0.05 (compared with the same transfection without Z-LEHD-FMK). (G) The transfected U251 cells were removed from 12-well plates. DJm was detected by flow cytometry using JC-1, and the percentage of cells with a low DJm is indicated. *p < 0.05 (compared with GFP). (H) The expression of Bcl-2 and c-FLIP 48 hours after transfection with STAT3 or ErbB2 siRNA in U251 cells, detected by immunoblotting. b-actin expression was monitored as the control. The ratios of Bcl-2/b-actin and c-FLIP/b-actin were calculated using densitometry.
with the knockdown of a single gene contributing to radiation resistance showed promising results (17–19). However, as we know, significant crosstalk exists among different signaling pathways (20), and when one cell proliferation pathway is blocked, another might come into
play to maintain tumor cell viability. The hope is that when two signaling molecules involved in cell proliferation are knocked out simultaneously, the crosstalk may be blocked, and then the cells would be more sensitized to treatments.
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Both STAT3 and ErbB2 play an important role in cell proliferation. Their levels are upregulated in malignancies, and this contributes to the persistent proliferation of cancer cells. Furthermore, the upregulated STAT3 and ErbB2 can help cancer cells resist the cytotoxic effects of chemotherapy or radiotherapy (11, 16). Thus, a logical treatment approach is to target these promoters of cell proliferation and radiation resistance to sensitize cancer cells to cytotoxic therapies. Our data demonstrated that the combination of RNA interference with irradiation resulted in significantly increased cytotoxicity in glioma cells. We showed that downregulation of STAT3 and ErbB2 could induce apoptosis in U251 cells. Nuclear condensation, a feature typical of apoptotic cells, was observed after the downregulation of STAT3 or ErbB2. We further demonstrated that initiator caspase-8 and caspase-9 and effector caspase-3 and caspase-7 are concomitantly acti-
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vated after the downregulation of the STAT3 or ErbB2 target proteins, and the inhibition of caspase activation by Z-VADFMK could block the induced apoptosis. Although caspase-8 and caspase-9 were both activated after STAT3 or ErbB2 RNAi, we further showed that the activation of caspase-9 played the more significant role during the apoptosis induced by STAT3 or ErbB2 RNAi. Finally, we found that the expression of Bcl-2, which regulates the mitochondrial outer membrane permeabilization, decreased significantly. Together, these results suggest that downregulation of STAT3 or ErbB2 can decrease the viability of glioma cells through the induction of apoptosis via the mitochondria pathway, but with little toxicity to normal astrocytes. Such a strategy may be particularly beneficial to glioma therapy. However, further clinical studies are needed to validate the two proteins as targets for improving radiotherapy in glioma.
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12. Bonner JA, Raisch KP, Trummell HQ, et al. Enhanced apoptosis with combination C225/radiation treatment serves as the impetus for clinical investigation in head and neck cancers. J Clin Oncol 2000;18:47s–53s. 13. Kristt DA, Reedy E, Yarder Y. Receptor tyrosine kinase expression in astrocytic lesions: Similar features in gliosis and glioma. Neurosurgery 1993;33:106–115. 14. Drebin JA, Link VC, Stern DF, et al. Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies. Cell 1985; 41:697–706. 15. Chang JC. HER2 inhibition: From discovery to clinical practice. Clin Cancer Res 2007;13:1–3. 16. Pietras RJ, Poen JC, Gallardo D, et al. Monoclonal antibody to HER-2/neu receptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cells overexpressing this oncogene. Cancer Res 1999; 59:1347–1355. 17. Collis SJ, Swartz MJ, Nelson WG, et al. Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors. Cancer Res 2003;63:1550–1554. 18. Cordes N. Overexpression of hyperactive integrin-linked kinase leads to increased cellular radiosensitivity. Cancer Res 2004;64: 5683–5692. 19. Kim I-A, Bas S-S, Fernandes A, et al. Selective inhibition of Ras, phosphoinositide 3 kinase, and Akt isoforms increases the radiosensitivity of human carcinoma cell lines. Cancer Res 2005;65:7902–7910. 20. Bhuiyan MMR, Li Y, Banerjee S, et al. Down-regulation of androgen receptor by 3,3’-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in both hormone-sensitive LNCaP and insensitive C4-2B prostate cancer cells. Cancer Res 2006;66:10064–10072.
doi:10.1016/j.ijrobp.2009.12.036
BIOLOGY CONTRIBUTION
INHIBITION OF STAT3 AND ERBB2 SUPPRESSES TUMOR GROWTH, ENHANCES RADIOSENSITIVITY, AND INDUCES MITOCHONDRIA-DEPENDENT APOPTOSIS IN GLIOMA CELLS LING GAO, PH.D., FENGSHENG LI, PH.D., BO DONG, B.S., JUNQUAN ZHANG, PH.D., YALAN RAO, PH.D., YUE CONG, PH.D., BINGZHI MAO, PH.D., AND XIAOHUA CHEN, PH.D. Department of Experimental Therapy of ARS, Beijing Institute of Radiation Medicine, Beijing, China Purpose: Constitutively activated signal transducer and activator of transcription 3 (STAT3) and ErbB2 are involved in the pathogenesis of many tumors, including astrocytoma. Inactivation of these molecules is reported to result in radiosensitization. The purpose of this study was to investigate whether inhibition of STAT3, ErbB2, or both could enhance radiotherapy in the human glioma model (U251 and U87 cell lines). Methods and Materials: The RNAi plasmids targeting STAT3 or ErbB2 were constructed, and their downregulatory effects on target proteins were examined by immunoblotting. After combination treatment of RNAi with or without irradiation, the cell viability was determined using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and clonogenic assays. The in vivo effect of combined treatment was determined using the U251 xenograft model. The apoptosis caused by the inhibition of STAT3 and ErbB2 was detected, and the mechanism involved in the apoptosis was investigated, including increases in caspase proteins, mitochondrial damage, and the expression of key modulating protein of different apoptosis pathways. Results: Transfection of U251 cells with STAT3 or ErbB2 siRNA plasmids specifically reduced their target gene expressions. Inhibition of STAT3 or ErbB2 greatly decreased glioma cell survival after 2, 4, or 6 Gy irradiation. Inhibition of STAT3 and ErbB2 also enhanced radiation-induced tumor growth inhibition in the U251 xenograft model. Furthermore, the suppression of either STAT3 or ErbB2 could induce U251 cell apoptosis, which was related primarily to the mitochondrial apoptotic pathway. Conclusions: These results indicated that simultaneous inhibition of STAT3 and ErbB2 expression can promote potent antitumor activity and radiosensitizing activity in human glioma. Ó 2010 Elsevier Inc. Signal transducers and activators of transcription, ErbB2, siRNA, Radiation, Apoptosis.
INTRODUCTION Malignant glioma is a highly lethal tumor characterized by aggressive proliferation and expansion into surrounding brain tissue (1, 2). Currently, the major approaches to treating the disease are surgical resection, radiotherapy, and adjuvant chemotherapy (3). Inasmuch as a single method usually cannot obtain an ideal therapeutic effect, combinations of surgery, radiotherapy, and chemotherapy are routinely used. The effect of radiation therapy and chemotherapy will greatly influence the prognosis of the patient. Therefore, increasing cell sensitivity to radiation and chemotherapy is an important research focus. Inasmuch as normal tissue surrounding the tumor will be damaged unavoidably during the course of routine treatment, an ideal approach to treating cerebral tumor would selectively kill tumor cells with little influence on normal tissue. Signal transducers and activators of transcription (STATs) are a family of transcription factors that directly transmit
signals from cell surface receptors to the nucleus (4). They play a critical role in mediating the signals of cytokines and growth factors involved in cell growth, differentiation, and survival (5). Whereas normal STAT activation is highly regulated and transient, one member of the STAT family, STAT3, is constitutively activated in diverse human tumors largely because of hyperactive tyrosine kinases (6, 7). It has been reported that inhibition of STAT3 expression could suppress cell growth and induce cell apoptosis in many kinds of cancer cells (8–10). Furthermore, STAT3 is also involved in the resistance to ionizing radiation in many tumor cells. U87 glioblastoma multiforme cells can be sensitized to the cytotoxic effects of ionizing radiation by the expression of dominant-negative STAT3 (11). It has also been found that STAT3 is correlated with the radiosensitization of anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody in head and neck cancers (12).
Reprint requests to: Xiaohua Chen, Ph.D., Department of Experimental Therapy of ARS, Beijing Institute of Radiation Medicine, Beijing 100850, China. Tel: (+86) 10-66931232 Fax: (+86) 1088219030; E-mail: [email protected]
Ling Gao, Fengsheng Li contributed equally to this article. Conflict of interest: none. Received Jan 2, 2009, and in revised form Dec 5, 2009. Accepted for publication Dec 9, 2009. 1223
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Overexpression of ErbB2 has also been reported in human glioma biopsy specimens (13). In fact, the earliest indication that this receptor might be involved in gliomagenesis came from a rat model of primary brain tumor and was later found to be overexpressed on the surface of 20–25% of breast cancer cells (14). The prognosis of cancer patients with overexpressed ErbB2 is poor. In the 1980s, trastuzumab, a monoclonal antibody against ErbB2, was developed, and in 1998, it was approved for the treatment of metastatic breast cancer (15). Furthermore, the monoclonal antibody to the ErbB2 receptor can also enhance the radiosensitivity of human breast cancer cells overexpressing ErbB2 (16). ErbB2 protein is being explored further as a potential target for novel therapeutics. Given the relationships between STAT3, ErbB2, and cancer cell proliferation and radiosensitivity, we presume that simultaneous inhibition of the two proteins may be an effective approach in addition to radiation therapy for glioma. In the present study, we examined the effects of radiosensitization in a glioma model via inhibiting STAT3 and ErbB2 using RNA interference plasmids. We found that suppression of STAT3 and/or ErbB2 reduced survival of glioma cells after 2, 4, or 6 Gy irradiation in culture and enhanced radiationinduced growth inhibition of tumor in a U251 xenograft model when combined with radiation. Furthermore, we found that the inhibition of STAT3 or ErbB2 could induce the decrease of Bcl-2 expression and mitochondria damage, indicating the occurrence of mitochondria-dependent apoptosis. MATERIALS AND METHODS Cell lines The U251 and U87 cell lines were obtained from the Beijing Xiehe Cell Culture Center and maintained in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum.
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TBST (Tris-Buffered Saline Tween-20) buffer (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.05% Tween 20) containing 5% skimmed milk, incubated with monoclonal antibodies to STAT3 (Cell Signaling, Beverly, MA), ErbB2 (Santa Cruz, CA), bcl-2 (Cell Signaling), c-FLIP (Santa Cruz), and actin (Santa Cruz), overnight, and followed by the addition of horseradish peroxidase–linked antimouse IgG (Zhongshan Goldenbridge Biotechnology Co., Beijing, China) and ECL (Enhanced Chemiluminescence) visualization of the bands.
Clonogenic assay Irradiated and/or siRNA-transfected cells were inoculated into 60-mm cell culture dishes (300 cells/dish) and incubated at 37 C in a humidified atmosphere containing 5% CO2 for 14 days. After incubation, the cells were fixed in methanol for 15 minutes and then stained with Giemsa dye for 10 minutes and washed with water, after which visible colonies were counted.
Tumor growth in nude mice Male athymic nu/nu mice weighing 16–18 g were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. U251 cells (1 107) in phosphate-buffered saline (PBS) were subcutaneously inoculated into the armpits of the mice. Tumor diameters were measured every 2 days, and each tumor volume in cubic millimeters was calculated by the following formula: V = 0.5 D d2 (V, volume; D, longitudinal diameter; d, latitudinal diameter). After 7 days, when the average tumor volume reached 0.2 cm3, the mice were stratified into groups (4 mice per group), so that the mean tumor volume in each group was comparable. Treatment groups were divided as follows: pSilence2.1GFP; pSilence2.1-STAT3; pSilence2.1-ErbB2; pSilence2.1-STAT3 + pSilence2.1-ErbB2; radiation + pSilence2.1-GFP; radiation + pSilence2.1-STAT3; radiation + pSilence2.1-ErbB2; and radiation + pSilence2.1-STAT3 + pSilence2.1-ErbB2. The radiation groups received 10 Gy of radiation fractionated over 5 consecutive days (from day 1 to day 5) using a cobalt irradiator to the tumor with the remainder of the body shielded with lead. The plasmid groups received daily intratumorally injections (from day 1 to day 5). After 2 weeks of treatment, the mice were killed, necropsy was performed, and the tumors were weighed.
Construction and transfection of siRNA plasmids The pSilencer2.1-U6 hygro plasmid was a gift from Dr. Yuwen Cong (Beijing Institute of Radiation Medicine). Appropriate sequences from the STAT3 (Medline: 213662) and ErbB2 (Medline:1003817) coding sequences were selected to design the gene-specific RNAi. The targeting sequence of STAT3 RNAi was as follows: 50 -ATCATCATGGGCTATAAGA-30 . The targeting sequence of ErbB2 RNAi was as follows: 50 -GAGATCACAG GTTACCTAT-30 The negative control was the green fluorescent protein (GFP) sequence 50 -GGTTATGTACAGGAACGCA-30 The double-stranded siRNA oligonucleotides with these sequences were cloned into pSilencer2.1-U6 hygro plasmids to generate pSilence2.1-STAT3, pSilence2.1-ErbB2, or pSilence2.1-GFP. The plasmids were transfected into cells using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Immunoblotting The U251 cells were removed 48 hours after transfection, and protein concentrations in total cell lysates were measured by the Bradford method. Aliquots of cell lysates containing 50 mg of proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose filters. The filters were blocked with
Immunohistochemistry Formalin-fixed, paraffin-embedded tissue sections were treated with anti-STAT3 or anti-ErbB2 antibody at a dilution of 1:100 for 1 hour and stained using the LSAB + System-HRP kit (DakoCytomation) according to the manufacturer’s instructions.
Isolation of primary astrocytes The cerebral cortices from 1-day old Wistar rats were removed using sterile techniques and cut into small pieces with scissors, to which 5 mL of warmed (37 C) trypsin solution (2.5mg/mL) in DHanks’ solution was added for the digestion. After incubation in 37 C for 10 minutes, the tissue was transferred into a sterile conical centrifuge tube and centrifuged at 1000 rcf for 5 minutes. The precipitation was resuspended in 2 mL of fresh DMEM-F12. The cell suspension was left for 1 to 2 minutes, and then the supernatant was transferred into another sterile tube. Two milliliters of fresh DMEM-F12 were added into the conical centrifuge tube again, and this process was repeated seven times. The supernatants were pooled and passed through a 21-gauge needle to make a single cell suspension. The cells were counted and inoculated at 1 104 cells/cm2 in plastic tissue culture dishes.
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Determining purity of primary astrocytes by indirect immunofluorescence assay To assess the purity of the primary astrocytes we isolated, an indirect immunofluorescence assay detecting Glial fibrillary acidic protein (GFAP), a specific marker of astrocytes, was performed. Briefly, the primary astrocytes were placed on a slide and incubated with anti-GFAP monoclonal antibody (Santa Cruz), followed by fluorescein-conjugated mouse-specific goat secondary antibody (Zhongshan Goldenbridge Biotechnology Co., Beijing, China). The labeled cells were then observed by fluorescence microscopy.
Nuclear damage detected by Hoechst 33258 staining Apoptotic nuclear morphology was assessed using Hoechst 33258 (Sigma, USA). Briefly, at the indicated time points after transfection, Hoechst 33258 (100 mM) was added to the cells in 96-well plates. After incubation at 37 C for 30 minutes, the cells were washed with PBS and observed by epifluorescence microscopy.
Cell apoptosis assay Apoptosis was quantified using the Annexin V-FITC (Fluorescein Isothiocyanate) Apoptosis Detection Kit (Bosai, China) according to the manufacturer’s instructions. Briefly, the treated cells were trypsinized, pelleted by centrifugation, and resuspended in Annexin V binding buffer. FITC-conjugated Annexin V (1 mg/mL) and propidium iodide (PI, 50 mg/mL) were added to the cells and incubated for 30 minutes at room temperature in the dark. Analyses were performed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA).
Caspase-3/7 assay A total of 5 103 cells in 100 mL of medium were plated in 96well plates and transfected with the siRNA plasmids. The caspase-3/ 7 levels were detected using Caspase-Glo 3/7 Reagent (Promega). The assay was performed according to the manufacturer’s instructions, and the luminescence was measured by a luminometer (Thermo Electron, Finland). The caspase-8 and caspase-9 assays were performed similarly to this assay.
Measurement of mitochondrial membrane potential U251 cells were plated in 12-well plates at 5 104 cells per well and treated without or with different siRNA plasmids. At the indicated time points after transfection, the transfected cells were removed. The cell suspensions were incubated with JC-1 dye (5 mg/mL) for 15 minutes at 37 C, and green (FL-1) and red (FL-2) fluorescence was measured by flow cytometry after washing the cells with PBS twice.
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Statistical analysis All experiments were done in triplicate or quadruplicate. Triplicate measurements were done per data point in each experiment. A two-tailed Student’s t test was used for statistical analysis of comparative data using SPASS software. Values of p < 0.05 were considered to be significant.
RESULTS STAT3 and ErbB2, constitutively expressed in the U251 human glioma cell line, could be specifically downregulated by RNAi U251 cells were transfected with pSilence2.1-STAT3, pSilence2.1-ErbB2, or the control pSilence2.1-GFP to determine their ability to silence STAT3 or ErbB2. Western blot analysis was performed 48 hours after transfection, and the results revealed that STAT3 (Fig. 1A) and ErbB2 (Fig. 1B) expressions were specifically suppressed up to 90% at this time point in cells transfected with pSilence2.1-STAT3 or pSilence2.1-ErbB2, respectively, but not with pSilence2.1-GFP. Knockdown of STAT3 or ErbB2 sensitized glioma cells to radiation by decreasing cell viability To determine whether inhibition of STAT3 or ErbB2 could sensitize U251 cells to radiotherapy, the U251 cells were transfected with the pSilence2.1-STAT3, pSilence2.1ErbB2, or both, with or without subsequent immediate radiation treatment of 2 Gy, which is the single radiation dose used in conventional fractionated radiotherapy. To determine cell viability after the various treatments described, the clonogenic assay was performed after treatment. As shown in Fig. 2A, radiation alone did not affect the proliferative activity/viability, whereas pSilence2.1-STAT3 and pSilence2.1-ErbB2 decreased viability significantly, compared with untreated or pSilence2.1-GFP, and the decrease in viability was more marked after STAT3/ErbB2 RNAi. However, the irradiation of cells transfected with either pSilence2.1-STAT3 or pSilence2.1-ErbB2 decreased cell viability more significantly than STAT3 or ErbB2 RNAi alone. Interestingly, the maximal reduction in activity was achieved in cells after simultaneous inhibition of STAT3 and ErbB2 and irradiation in comparison with those with STAT3/ErbB2 RNAi but without irradiation. Similar results were obtained after 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays (Fig. E1).
Fig. 1. siRNA targeting signal transducer and activator of transcription 3 (STAT3) or ErbB2 specifically inhibits their expression. (A) STAT3 expression 48 hours after transfection with siRNA targeting STAT3 in U251 cells, detected by immunoblotting. (B) ErbB2 expression 48 hours after transfection with siRNA targeting ErbB2 in U251 cells, detected by immunoblotting. b-actin expression was monitored as the normalizing control. The ratios of STAT3/b-actin and ErbB2/b-actin were calculated using densitometry.
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Fig. 2. Knockdown of signal transducer and activator of transcription 3 (STAT3) or ErbB2 sensitizes U251 cells to radiation by decreasing cell viability. (A–C) Colony formation assay of U251 cells after doses of 2 (A), 4 (B), and 6 Gy (C) irradiation with or without transfection. The surviving fraction (%) = (CFE of active treatment)/(CFE of control) 100%, CFE (colony form efficiency) (%) = (number of colonies)/(number of inoculation cells) 100%. In A–C, *p < 0.05 (compared with pSilence2.1-GFP transfection alone); #p < 0.05 (compared with irradiation plus pSilence2.1-GFP transfection); &p < 0.05 (compared with the same siRNA transfection without irradiation).
We further investigated whether the radiosensitizing effect would occur at higher irradiation doses of 4 Gy and 6 Gy. The clonogenic assays showed that the inhibition of STAT3, ErbB2, or both also sensitized U251 cells to 4 or 6 Gy radiation, which was similar to the results obtained with 2 Gy (Fig. 2C and D). Remarkably, the radiosensitizing effects of STAT3 and/or ErbB2 RNAi were weakest after 6 Gy was used as the radiation dose. This result might be due to the fact that the effect of RNAi was masked by the strong effect of the 6-Gy dose on cell viability. To investigate whether the radiosensitizing effect of STAT3 and/or ErbB2 RNAi could also occur in another glioma cell line, we repeated the clonogenic assay using 2 Gy as the radiation dose in the U87 cell line. The radiosensitizing effects was also observed in the U87 cell line and were very similar to results obtained with the U251 cell line (Fig. E2).
when both genes were downregulated. STAT3 RNAi plus 2 Gy radiotherapy or ErbB2 RNAi plus 2 Gy radiotherapy could further inhibit the tumor growth compared with STAT3 RNAi or ErbB2 RNAi, respectively. Among the different treatments, combining STAT3 and ErbB2 RNAi with 2 Gy radiotherapy led to the most significant inhibition of tumor growth. To determine whether the intratumoral injection of pSilence2.1-STAT3 or pSilence2.1-ErbB2 effectively inhibited the expression of their targeted proteins in vivo, the tumor tissues were removed, fixed, and prepared for immunohistochemical analysis as described in Materials and Methods. As represented in Fig. 3C, the expression of STAT3 in the tumor was markedly suppressed after pSilence2.1-STAT3 treatment in comparison with pSilence2.1-GFP treatment. A similar inhibitory effect on ErbB2 expression was observed after pSilence2.1-ErbB2 treatment.
Inhibition of STAT3 and ErbB2 enhanced radiationinduced inhibition of tumor growth in vivo To determine whether the knockdown of STAT3 or ErbB2 enhances radiation-induced growth inhibition of tumors, the U251 xenograft model was established by inoculating mice with U251 cells, and the tumor volumes were measured by calipers. The tumor-bearing mice were treated with 2 Gy daily for 5 days at the same time for radiotherapy, and the interference plasmids were given daily for 5 days at a dose of 5 mg/kg by intratumoral injections. As shown in Fig. 3A and B, both STAT3 RNAi and ErbB2 RNAi could inhibit the tumor growth. The effect was more pronounced
Downregulation of STAT3 or ErbB2 induced U251 cell apoptosis The change in tumor size is the result of a dynamic balance between cellular proliferation and apoptosis. Becuase downregulation of STAT3 or ErbB2 could decrease the proliferative abilities of U251 and U87 cells, we asked whether they could affect apoptosis in U251 cells. To clarify this, the apoptosis of U251 cells was determined by various assays after the different treatments. As shown in Fig. 4A, the cells treated with pSilence2.1-STAT3 or pSilence2.1-ErbB2 showed significant nuclear condensation at different time points, compared with the pSilence2.1-GFP control.
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Fig. 3. Inhibition of signal transducer and activator of transcription 3 (STAT3) and ErbB2 enhanced radiation-induced inhibition of tumor growth in vivo. Mice bearing U251 tumors with an average volume of 0.2 cm3 were stratified into groups of four and treated with siRNA plasmid via intratumoral injection daily for 5 days, with or without 2 Gy daily. (A) Tumor volumes every 4 days after the first administration. (B) Weight of tumors resected from nude mice at day 14 after treatment. (A and B) *p < 0.05 (compared with GFP RNAi (negative control)); #p < 0.05 (compared with GFP RNAi + 2Gy); &p < 0.05 (compared with the same siRNA transfection without irradiation). (C) Immunohistochemistry of tumor tissue for STAT3 and ErbB2 in specific siRNA-treated groups with or without radiation. The monoclonal mouse anti-STAT3 or -ErbB2 antibodies were used to stain paraffin sections.
Additionally, quantitative detection of cellular apoptosis was performed using the Annexin-V kit and flow cytometry. The results in Figure 4B show that pSilence2.1-GFP did not lead to significant apoptosis of U251 cell at any time point, compared with untreated cells. However, pSilence2.1STAT3 led to significant apoptosis of U251 cells in a timedependent manner. pSilence2.1-ErbB2 could also induce
U251 cell apoptosis significantly at 24 hours and to a greater degree at 48 hours after transfection. STAT3 and ErbB2 RNAi do not affect normal astrocytes An ideal approach to tumor therapy should have no toxic effects on normal cells while efficiently killing the tumor cells. To detect the effect of RNAi on normal astrocytes,
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Fig. 4. Down-regulation of signal transducer and activator of transcription 3 (STAT3) or ErbB2 induced U251 cell apoptosis. At indicated time points after transfection, the apoptosis of transfected U251 cells was analyzed by fluorescence microscopy after Hoechst 33258 staining (A) or analyzed by flow cytometry after Annexin V/PI double staining (B). *p < 0.05 (compared with GFP [negative control]).
the primary astrocytes from the brain of 1-day-old Wistar rats were cultured. As shown in Fig. 5A, B, and C, the purity of astrocytes was assessed by indirect immunofluorescence using GFAP antibody. Unlike U251 cells, Hoechst 33258 staining (Fig. 5D) and Annexin V/PI double staining–based flow cytometry analysis (Fig. 5E) showed no apoptosis in both pSilence2.1-STAT3 and pSilence2.1-ErbB2 groups at any time point. Apoptosis induced by inhibition of STAT3 and ErbB2 is primarily mitochondria-dependent pSilence2.1-STAT3or pSilence2.1-ErbB2-treated U251 cells were cotreated with the pan-caspase inhibitor Z-VAD-FMK to investigate whether their induction of apoptosis was dependent on caspase activation. Interestingly, the pSilence2.1-STAT3- and pSilence2.1-ErbB2-induced apoptosis was completely abrogated by the addition of Z-VAD-FMK (Fig. 6A). Furthermore, caspase-3 and caspase-7 are two effector caspases that play a key role in
the execution of apoptosis, and pSilence2.1-STAT3 or pSilence2.1-ErbB2 significantly activated caspase-3 and caspase-7 levels after treatment of U251 cells (Fig. 6B) but not of primary astrocytes (Fig. 6C). This result was consistent with the observations above that STAT3 and ErbB2 RNAi showed no notable apoptotic effects on normal astrocytes. Although the activation of caspase-3 and caspase-7 was shown to be involved in the apoptosis induced by STAT3 RNAi and ErbB2 RNAi, the exact molecular mechanisms were still not clear. To investigate it, we tested the activity of caspase-8 and caspase-9, which represent the death receptor pathway and the mitochondrial pathways, respectively, after the inhibition of STAT3 and ErbB2. As shown in Fig. 6D and E, the activities of caspase-8 and caspase-9 both increased. However, the apoptosis was almost completely inhibited when the specific inhibitor of caspase-9, Z-LEHD-FMK, was added simultaneously (Fig. 6F), indicating that caspase-9 plays a more important role in the apoptotic process.
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Fig. 5. Signal transducer and activator of transcription 3 (STAT3) and ErbB2 RNAi showed no notable effects on normal astrocytes. (A–C) Assessment of purity of primary astrocyte by indirect immunofluorescence assay using a monoclonal antibody against GFAP, the astrocyte-specific marker. (A) Primary astrocytes in a bright field were recorded, and (B) the green fluorescence of the same field was observed. (C) Finally, the fluorescence images and bright images were merged. At indicated time points after transfection, the apoptosis of transfected primary astrocytes was analyzed (D) by fluorescence microscopy after Hoechst 33258 staining or (E) by flow cytometry after annexin V/PI double staining.
Activation of caspase-9 is the result of cytochrome c release, which is caused by the mitochondrial transmembrane potential (DJm) reduction. Therefore, we subsequently examined the mitochondrial transmembrane potential using the cationic lipophilic dye JC-1. After the treated U251 cells were stained with JC-1, qualitative assessment using fluorescence microscopy and quantitative detection by flow cytometric analysis were performed. Both assays detected a time-dependent dissipation of DJm in pSilence2.1STAT3- or pSilence2.1-ErbB2-treated U251 cells (Fig. 6G and E3). Furthermore, the expressions of c-FLIP and Bcl-2, which serve as critical regulators of the death receptor pathway and the mitochondrial pathway, respectively, were examined by Western blotting. As shown in Fig. 6H, the expression of Bcl-2 decreased significantly 24 hours after STAT3 RNAi or ErbB2 RNAi treatment, compared with GFP RNAi treatment or untreated control, which was consist with the previous finding that STAT3 and ErbB2 could upregulate the expression of Bcl-2. However, the expression of c-FLIP, unlike Bcl-2, was inhibited only slightly. Inasmuch as Bcl-2 plays a critical role in maintaining DJm, the result indicated that the reduction of DJm, which caused the increased activity of caspase-9, might be the result of Bcl-2 suppression.
DISCUSSION STAT3 and ErbB2 are upregulated and overexpressed in various types of cancers. Previous studies suggest that they are both related to the oncogenesis and radiation resistance of cancer cells. In this study, we demonstrated the enhanced effects of radiosensitization in a glioma model by RNAimediated inhibition of STA T3 and ErbB2, suggesting that synchronous inhibition of STAT3 and ErbB2 could be an effective addition to radiation therapy of glioma. Specifically, we demonstrated that the inhibition of STAT3 or ErbB2 could decrease the survival of U251 and U87 cells in culture, and the radiation-induced growth inhibition of tumors in the U251 xenografts model was enhanced when combined with radiation. Currently, the main approaches to glioma treatment are surgery, radiotherapy, and chemotherapy. However, the prognosis of glioma is poor because of the existence of the blood–brain barrier and radiation resistance. Therefore, increasing the sensitivity of tumors to radiotherapy and chemotherapy would provide a significant advance in effective treatment for glioma patients. Some genes contributing to radiation resistance are known. In some previous studies, radiotherapy combined
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Fig. 6. The apoptosis induced by inhibition of signal transducer and activator of transcription 3 (STAT3) and ErbB2 is primarily mitochondrial-dependent. (A) U251 cells were treated with or without Z-VAD-FMK after transfection, and the apoptosis was determined by annexin V/PI double staining. The Annexin V-positive cells were counted. (B) The enzyme activities of caspases-3/7 in the transfected U251 cells or (C) treated primary astrocytes were determined. (D and E) U251 cells were treated with or without Z-VAD-FMK after transfection, and the enzyme activities of caspase-8 (D) and caspase-9 (E) were determined. A–E, *p < 0.05 (compared with GFP). (F) U251 cells were treated with or without Z-LEHD-FMK after transfection, and the apoptosis was determined by annexin V/PI double staining. The Annexin V-positive cells was counted. *p < 0.05 (compared with GFP of the same time). #p < 0.05 (compared with the same transfection without Z-LEHD-FMK). (G) The transfected U251 cells were removed from 12-well plates. DJm was detected by flow cytometry using JC-1, and the percentage of cells with a low DJm is indicated. *p < 0.05 (compared with GFP). (H) The expression of Bcl-2 and c-FLIP 48 hours after transfection with STAT3 or ErbB2 siRNA in U251 cells, detected by immunoblotting. b-actin expression was monitored as the control. The ratios of Bcl-2/b-actin and c-FLIP/b-actin were calculated using densitometry.
with the knockdown of a single gene contributing to radiation resistance showed promising results (17–19). However, as we know, significant crosstalk exists among different signaling pathways (20), and when one cell proliferation pathway is blocked, another might come into
play to maintain tumor cell viability. The hope is that when two signaling molecules involved in cell proliferation are knocked out simultaneously, the crosstalk may be blocked, and then the cells would be more sensitized to treatments.
Inhibition of STAT3 and ErbB2 in glioma cells d L. GAO et al.
Both STAT3 and ErbB2 play an important role in cell proliferation. Their levels are upregulated in malignancies, and this contributes to the persistent proliferation of cancer cells. Furthermore, the upregulated STAT3 and ErbB2 can help cancer cells resist the cytotoxic effects of chemotherapy or radiotherapy (11, 16). Thus, a logical treatment approach is to target these promoters of cell proliferation and radiation resistance to sensitize cancer cells to cytotoxic therapies. Our data demonstrated that the combination of RNA interference with irradiation resulted in significantly increased cytotoxicity in glioma cells. We showed that downregulation of STAT3 and ErbB2 could induce apoptosis in U251 cells. Nuclear condensation, a feature typical of apoptotic cells, was observed after the downregulation of STAT3 or ErbB2. We further demonstrated that initiator caspase-8 and caspase-9 and effector caspase-3 and caspase-7 are concomitantly acti-
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vated after the downregulation of the STAT3 or ErbB2 target proteins, and the inhibition of caspase activation by Z-VADFMK could block the induced apoptosis. Although caspase-8 and caspase-9 were both activated after STAT3 or ErbB2 RNAi, we further showed that the activation of caspase-9 played the more significant role during the apoptosis induced by STAT3 or ErbB2 RNAi. Finally, we found that the expression of Bcl-2, which regulates the mitochondrial outer membrane permeabilization, decreased significantly. Together, these results suggest that downregulation of STAT3 or ErbB2 can decrease the viability of glioma cells through the induction of apoptosis via the mitochondria pathway, but with little toxicity to normal astrocytes. Such a strategy may be particularly beneficial to glioma therapy. However, further clinical studies are needed to validate the two proteins as targets for improving radiotherapy in glioma.
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