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Interferonα enhances etoposide-induced apoptosis in human osteosarcoma U2OS cells by a p53-dependent pathway
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Life Sciences 82 (2008) 393 – 401 www.elsevier.com/locate/lifescie
Interferonα enhances etoposide-induced apoptosis in human osteosarcoma U2OS cells by a p53-dependent pathway Xiang-Wei Yuan a,b , Xiao-Feng Zhu c,⁎, Sheng-Gen Liang b , Quan Fan b , Zhong-Xian Chen b , Xiu-Fang Huang d , Pu-Yi Sheng a , Ai-Shan He a , Zi-Bo Yang a , Rong Deng c , Gong-Kan Feng c , Wei-Ming Liao a,⁎ a
Department of Orthopedic Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China Department of Orthopedic Surgery, The Affiliated Jiangmen Hospital, Sun Yat-sen University, Jiangmen, 529030, China c State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou, 510060, China d Department of Pathology, The Affiliated Jiangmen Hospital, Sun Yat-sen University, Jiangmen, 529030, China
b
Received 2 March 2007; accepted 24 November 2007
Abstract Interferonα (IFNα) induces cell cycle arrest and triggers apoptosis and chemosensitivity. But the mechanism of IFNα in regulating chemosensitivity has not been fully understood. To study whether IFNα affected chemosensitivity of osteosarcoma cells, we treated p53-wild U2OS cells and p53-mutant MG63 cells with IFNα and etoposide, alone or in combination, and then examined growth inhibition, cell cycle arrest and apoptosis. IFNα enhanced etoposide-induced growth inhibition and apoptosis in p53-wild U2OS cells but not p53-mutant MG63 cells in a dose- and time-dependent manner. Etoposide-induced G2/M phase arrest was also enhanced by IFNα. The enhanced apoptosis was associated with the accumulation of transcriptionally active p53 accompanied with increased Bax and Mdm2, as well as decreased Bcl-2. IFNα also activated caspases-3, -8 and -9 protein kinases and PARP cleavage in response to etoposide in U2OS cells. Moreover, the combination-induced cytotoxicity and PARP cleavage were significantly reduced by caspase pan inhibitor and p53 siRNA. Thus we conclude that IFNα enhances etoposide-induced apoptosis in human osteosarcoma U2OS cells by a p53-dependent and caspase-activation pathway. The proper combination of IFNα and conventional chemotherapeutic agents may be a rational strategy for the treatment of human osteosarcoma with functional p53. © 2007 Elsevier Inc. All rights reserved. Keywords: Interferonα; Etoposide; Apoptosis; p53; Osteosarcoma
Introduction Osteosarcoma is the most common primary malignant tumor of the skeletal system, and occurs most frequently in children and adolescents (Young and Miller, 1975). The therapeutic modalities are combined treatments with surgery and neoadjuvant chemotherapy (Bruland and Pihl, 1997). The chemother⁎ Corresponding authors. W.M. Liao is to be contacted at Department of Orthopedic Surgery, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan Road 2, Guangzhou 510080, China. Tel.: +86 20 87755766 8898; fax: +86 20 87750632. X.F. Zhu, State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, 651 Dongfeng Road East, Guangzhou 510060, China. Tel.: +86 20 87343149; fax: +86 20 87343392. E-mail addresses: [email protected] (W.-M. Liao), [email protected] (X.-F. Zhu). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.11.025
apy results in significant improvement in patient survival. However, the frequent acquisition of drug-resistant phenotypes and unwanted side effects are often associated with chemotherapy and remain as serious problems (de Saint Aubain Somerhausen and Fletcher, 1999). Therefore, new therapeutic strategies which can improve the effect of current chemotherapy need to be developed. Interferons (IFNs), grouped into two families: type I and type II, are multifunctional proteins that share immunomodulatory, antiviral and antitumor activities (Mogensen et al., 1999; Prejean and Colamonici, 2000; Stark et al., 1998). IFNα, which belongs to the type I IFNs, interacts with IFN-α/β receptor on the cell surface to induce activation of JAK1 and TYK2, leading to phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1). The activated STAT1
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forms homodimers or heterodimers which translocate into nucleus and subsequently bind specific promoter regions (interferon-stimulated response elements (ISRE) or gammaactivating sequence (GAS)) to regulate transcription of the genes responsible for antiproliferative effects (Stark et al., 1998). The tumor suppressor p53, which is often disrupted in human malignancies including osteosarcoma (Chandar et al., 1992), induces cell cycle arrest and apoptosis in response to DNA-damaging agents including etoposide (Hande, 1998; Levine et al., 1991), therefore playing a key role in cancer control. Previous reports have shown that p53 induction contributes to type I IFN-induced apoptosis in some cancer cells (Porta et al., 2005; Takaoka et al., 2003). Such observations indicate that IFNα-treated cells may be more sensitive to p53-dependent apoptosis in response to DNAdamaging chemotherapeutic agents. Thus we hypothesize that IFNα may increase etoposide sensitivity in human osteosarcoma cells. To test this hypothesis, we utilized the p53-wild and p53-mutant osteosarcoma cells to examine whether IFNα increases etoposide sensitivity by modulating p53-mediated apoptosis. We show here that IFNα enhances etoposide-induced apoptosis in osteosarcoma p53-wild U2OS but not p53-mutant MG63 cells and define a p53-dependent pathway as the molecular mechanism. This work also supports the rational clinical utility of IFNα combination therapy for treatment of osteosarcoma with functional p53. Materials and methods Cell culture and reagents The human osteosarcoma U2OS cells (Ponten and Saksela, 1967) containing wild type p53 and MG63 cells (Heremans et al., 1978) containing mutant p53 were maintained in DMEM (Life Technologies, Inc.) and supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a 5% CO2 humidified atmosphere. MG63 cells had a rearrangement mutation of the p53 gene, which results in disruption of p53 protein function (Chandar et al., 1992). Etoposide, Hoechst 33258 and MTT were obtained from Sigma. Human recombinant IFNα2a was purchased from Peprotech. ZVAD-FMK (pan caspase inhibitor) was purchased from Calbiochem Co. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The logarithmically growing U2OS and MG63 cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After an overnight growth, cells were treated with IFNα and etoposide, alone or in combination. After indicated times, 10 μl of 5 mg/ml MTT was added into each well followed by incubation for an additional 4 h. Supernatants were removed and 200 μl of DMSO was added. After crystals were dissolved, absorbance at 450 nm was measured in the microplate reader.
Flow cytometric analysis Cells were collected, washed twice with ice-cold PBS, resuspended in cold PBS, and fixed with 70% ethanol. After incubation overnight and subsequent rehydration in PBS for 30 min at 4 °C, the samples were stained for 30 min in the dark with 50 μg/ml propidium iodide (Sigma) containing 125 U/ml protease-free RNase, both diluted in PBS and then were analyzed in a FACS flow cytometer. Morphological analysis of apoptosis Cells were collected, washed twice with PBS and stained with 10 μg/ml of Hoechst 33258 for 15 min, followed by examination on an Olympus fluorescence microscope. DNA fragmentation assay Cells (3 × 106) were collected and washed once with PBS. DNA was extracted using DNAZol reagent (Invitrogen) according to the manufacturer's instructions and electrophoresed on 2% agarose gel containing 0.5 μg/ml ethidium bromide. The gel was photographed with UV illumination. Western blot analysis Cells were collected and lysed with the lysis buffer (10 mM Tris–HCl pH 7.4, 5 mM MgCl2, 1 mM EDTA, 25 mM NaF, 100 μM Na3VO4 and 1 mM dithiothreitol). An equal amount of protein determined by Bradford assay was resolved by SDS–PAGE and transferred onto PVDF membranes (Roche). Blots were incubated with primary antibody overnight at 4 °C, followed by 3 washes in TBST for 5 min, and then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Signals were detected using ECL and exposed to X-ray film (Kodak). Anti-caspase-8 antibody was purchased from Cell Signaling Technologies. The other antibodies were obtained from Santa Cruz. RT-PCR Total RNA was extracted with TriZol reagent (Invitrogen) according to the manufacturer's recommendations. For RT reaction, 2 µg RNA was combined with 0.5 µg Oligo(dT)15 with 15 µl total volume, and the mixture was incubated at 70 °C for 5 min and placed on ice. Then 5 µl 5× M-MLV reaction buffer, 1.25 µl 4× dNTP (10 mM), 1 µl M-MLV (Promega, 200 U/µl), and 0.625 µl RNaseOUTTM (40 U/µl) were added to 25 µl total volume, and the tube was incubated at 42 °C for 60 min and then at 75 °C for 10 min for termination. PCR reaction was performed in the presence of Taq DNA polymerase, dNTP mix, PCR buffer (all from Invitrogen), and primers. After denaturation at 94 °C for 2 min, the samples underwent 30 cycles of amplification (1 min at 94 °C, 1 min at 58 °C for p53 or 55 °C for β-actin, 1 min at 72 °C) with a 10 min extension at 72 °C following
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the last cycle. Sense and antisense primers were: 5′-CAG CCA AGT CTG TGA CTT GCA CGT AC-3′ and 5′-CTA TGT CGA AAA GTG TTT CTG TCA TC-3′ for p53 and 5′ACT ACC TCA TGA AGA TCC TC-3′ and 5′-CTA AAG ATT GCG TGG CGA GG-3′ for β-actin. Products were electrophoresed on 1.5% agarose gels containing 0.5 μg/ml ethidium bromide.
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Statistical analysis SPSS 10.0 software (SPSS Inc, IL, US) was used for statistical analysis. The unpaired t test was used to determine statistically significant differences between treatment effects. Significance was determined as P b 0.05. Results
Small interfering RNA transfection U2OS cells were plated onto six-well plates at a density of 3 × 105 cells/well with growth medium without antibiotics. After an overnight incubation, transfection was performed at a confluency of 50% by using Opti-MEM media, lipofectamine 2000, and specific or nonspecific siRNA for p53 according to manufacturer's recommendations. Six hours later, the medium was replaced with growth medium without antibiotics. After transfection for 20 h, cells were trypsinized and sub-seeded onto 96-well plates. After incubation for another 4 h, cells were treated as described for MTT assay. The p53-siRNA duplexes were synthesized by Genepharma Co., Ltd. (Shanghai, China). The mRNA sequence selected to be targeted by p53-siRNA is 5′-CTA CTT CCT GAA AAC AAC G-3′.
IFNα enhances etoposide-induced growth inhibition in human osteosarcoma p53-wild U2OS but not p53-mutant MG63 cells To study whether IFNα could enhance etoposide-induced growth inhibition, cell viability was examined using MTT assay. Fig. 1A and B shows that in p53-wild U2OS cells, IFNα alone did not induce growth inhibition at concentrations of 50 U/ml, 500 U/ml and 5000 U/ml, but significantly enhanced etoposideinduced cytotoxicity in a dose- and time-dependent manner. Similar results were induced by treatment with IFNα combined with cisplatin or doxorubicin, both DNA-damaging agents (data not shown). In contrast, such effects were not observed in p53mutant MG63 cells (Fig. 1C and D). These results show that IFNα enhances etoposide-induced growth inhibition in p53-
Fig. 1. Effect of IFNα on etoposide-induced growth inhibition in U2OS and MG63 cells. U2OS cells (A) and MG63 cells (C) were treated with a range of doses of IFNα (50 to 5000 U/ml) and etoposide (2.5 μg/ml for U2OS and 0.625 μg/ml for MG63), alone or in combination. U2OS cells (B) and MG63 cells (D) were treated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml for U2OS and 0.625 μg/ml for MG63) alone or in combination for different times. Cell viability was examined after the indicated treatment using MTT assay and shown as means ± SD. The experiment was repeated three times with similar results.
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wild U2OS cells but not p53-mutant MG63 cells and imply that p53 may be involved in this effect. IFNα enhances etoposide-induced apoptosis in p53-wild U2OS but not p53-mutant MG63 cells To test whether IFNα enhanced etoposide-induced apoptosis, flow cytometry analysis was applied. The fraction of subG1 cells represents cells undergoing DNA fragmentation, a reliable manifestation of apoptosis (Ormerod, 1998). IFNα did not induce obvious apoptosis, but markedly increased etoposide-induced apoptosis in p53-wild U2OS cells (Fig. 2A) but not p53-mutant MG63 cells (Fig. 2B). A higher level of apoptotic morphological changes, such as nuclear condensation and fragmentation, and internucleosomal DNA fragmentation – hallmarks of apoptosis – were observed in U2OS cells treated for 72 h with IFNα/etoposide combination than in those treated
with either alone (Fig. 3A and B). Similar results were observed after treatment with IFNα combined with cisplatin or doxorubicin (data not shown). In contrast, such results were not obtained in p53-mutant MG63 cells (data not shown). These results show that IFNα enhances etoposide-induced apoptosis in p53-wild U2OS cells but not p53-mutant MG63 cells. IFNα enhances etoposide-induced cell cycle arrest in p53-wild U2OS cells Etoposide-induced growth inhibition is caused by the induction of both apoptosis and cell cycle arrest (Hande, 1998). Next we used FACS analysis to determine the effect of IFNα on etoposide-induced cell cycle arrest in U2OS cells. After 72 h of etoposide treatment, there was an obvious accumulation of cells in G2/M phase and a decline of cells in S phase, compared with control cells. IFNα further increased cells
Fig. 2. Effect of IFNα on etoposide-induced apoptosis in U2OS and MG63 cells. U2OS cells (A) and MG63 cells (B) were treated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml for U2OS and 0.625 μg/ml for MG63) alone or in combination for 72 h. Apoptotic cells were examined by flow cytometry. The experiment was repeated three times with similar results.
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Fig. 3. IFNα enhances etoposide-induced apoptosis in U2OS cells. U2OS cells were treated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml) alone or in combination for 72 h. (A) Apoptotic cells were determined by Hoechst 33258 staining of nuclei as a result of chromatin condensation and nuclear fragmentation. (B) DNA fragmentation assay was examined to confirm the ladder change representing apoptosis. Lane M: DNA molecular marker; Lane 1: control cells; Lane 2: IFNα-treated cells; Lane 3: etoposide-treated cells; Lane 4: IFNα/etoposide combination-treated cells.
in G2/M phase and decreased cells in S phase in response to etoposide (P b 0.05, Fig. 4). These results show that IFNα also enhances etoposide-induced cell cycle arrest in U2OS cells. IFNα activates caspases-3, -8, -9 and PARP cleavage in response to etoposide in U2OS cells Caspases are key executors of apoptotic cell death signals by cascade activation and selectively cleaving the substrates such as poly(ADP-ribose) polymerase (PARP). To further elucidate the signaling pathway underlying the enhanced apoptosis, we detected activation of caspases-3, -8 and -9 and PARP using Western blot. As shown in Fig. 5, caspases-3, -8 and -9 were cleaved to yield 17 kD, 43/41 kD and 18 kD, as well as 37 kD fragments, respectively in response to IFNα/etoposide combination in U2OS cells. Moreover, the combination promoted the 85 kD active fragment of PARP, compared with either alone.
Fig. 4. Effect of IFNα on etoposide-induced cell cycle arrest in U2OS cells. U2OS cells were treated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml) alone or in combination for 72 h. Cell cycle distribution was examined by flow cytometry analysis. The experiment was repeated three times with similar results. An unpaired t test was used to statistically compare etoposide-induced cell cycle arrest in the presence and absence of IFNα. ⁎, P b 0.05.
These results further prove that IFNα enhances etoposideinduced apoptosis. Caspase inhibitor prevents the enhanced growth inhibition and apoptosis induced by IFNα/etoposide combination in U2OS cells The results shown above indicated that caspase activation may be involved in the enhanced apoptosis. To determine whether caspase activation is required for the facilitated apoptosis, U2OS cells was pretreated with pan caspase inhibitor
Fig. 5. IFNα activates caspases-3, -8 and -9 and PARP cleavage in response to etoposide after treatments for 72 h in U2OS cells by Western blot. Proteins were resolved by SDS–PAGE and blotted with antibodies for caspases-3,-8,-9 and PARP. GAPDH was used as loading control.
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Z-VAD-FMK for 2 h. We determined whether Z-VAD-FMK inhibited the sensitization of growth inhibition and PARP cleavage induced by IFNα/etoposide combination. As shown in Fig. 6A and B, growth inhibition and PARP cleavage were greatly inhibited by Z-VAD-FMK. This suggests that the enhanced apoptosis induced by IFNα/etoposide combination is caspase-dependent. IFNα regulates p53 pathway in response to etoposide in U2OS cells Next we asked whether IFNα could affect etoposide-induced activation of p53 pathway. Fig. 7A shows that p53 protein level was enhanced by etoposide alone and further augmented by IFNα/etoposide combination, but unaltered by IFNα alone. The expression of Bax, Bcl-2 and Mdm2, well-known downstream genes of p53, was examined to determine the transcriptional activity of the elevated p53. The pro-apoptotic Bax expression was further increased by IFNα/etoposide combination than either alone. Mdm2 expression was also increased. Conversely, the anti-apoptotic Bcl-2 expression was further decreased by IFNα/etoposide combination compared to either alone. Such effects were not observed in MG63 cells (Fig. 7A). To further explore the mechanisms leading to the p53 protein upregulation, RT-PCR was applied to examine mRNA level of p53. Fig. 7B shows that p53 mRNA level was increased greatly by etoposide but not further augmented by IFNα/etoposide combination in
Fig. 7. Effect of IFNα on expression of p53, Bax, Mdm2 and Bcl-2 in response to etoposide in U2OS and MG63 cells. (A) Cells were treated with IFNα and etoposide as indicated for 48 h. Expression of the indicated proteins was detected in U2OS and MG63 cells by Western blot. (B) mRNA level of p53 was examined at 48 h post-treatment in U2OS cells using RT-PCR.
U2OS cells, suggesting that the p53 protein boost is mediated in a post-transcriptional manner. siRNA-mediated p53 knockdown in U2OS cells results in decrease of growth inhibition and apoptosis induced by IFNα/etoposide combination To examine whether the enhanced apoptosis was p53dependent, p53-specific siRNA was transfected into U2OS cells; this significantly suppressed p53 expression after treatment with the IFNα/etoposide combination (Fig. 8A). The p53 knockdown resulted in a substantial reduction in the combination-induced cell death (Fig. 8B). This finding was supported by the loss of cleaved PARP in U2OS cells where p53 expression had been suppressed, compared with nonspecific control (Fig. 8C). These results show that p53 function is required for the enhanced apoptosis induced by IFNα/etoposide combination. Discussion
Fig. 6. Caspase pan inhibitor Z-VAD-FMK prevents the enhanced growth inhibition and apoptosis induced by IFNα/etoposide combination in U2OS cells. U2OS cells were pretreated with caspase pan inhibitor Z-VAD-FMK (80 μM) for 2 h, then incubated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml) alone or in combination for 72 h. Cell viability (A) and PARP cleavage (B) were examined using MTT assay and Western blot, respectively. The experiment was repeated three times with similar results.
IFNα is a multifunctional cytokine with antitumor effects including suppressing cell growth, inducing apoptosis and increasing chemosensitivity (Kondo et al., 2005; RodriguezVillanueva and McDonnell, 1995; Roos et al., 1984). However, the molecular basis remains unclear. Here we found that IFNα alone did not alter cell growth but promoted etoposide-induced cytotoxicity pronouncedly in osteosarcoma U2OS cells. Further examinations revealed a significantly enhanced apoptosis in response to IFNα/etoposide combination, compared with etoposide
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Fig. 8. siRNA-mediated p53 silencing in U2OS cells results in a decrease of growth inhibition and apoptosis induced by IFNα/etoposide combination. (A) U2OS cells were transfected with p53 specific or nonspecific siRNA duplexes and treated with IFNα/etoposide combination as described in Materials and methods. p53 protein level was examined by Western blot. (B) U2OS cells were treated as described. Cell viability was determined by MTT assay at 72 h post-treatment. The data are shown as means ± SD that is representative of three separate experiments. (C) U2OS cells were transfected with siRNA duplexes and treated as described for 72 h. Cleavage of PARP was detected by Western blot.
alone. However, the cell growth inhibition was higher than the apoptotic rate in U2OS cells treated with IFNα/etoposide combination, which indicates that, besides the apoptosis, cell cycle arrest is possibly involved in the IFNα/etoposide combination-induced cytotoxicity. This was consistent with our result that IFNα enhances etoposide-induced G2/M phase arrest in U2OS cells, albeit weakly. Therefore, the enhancing effect of the combination is based on the induction of both apoptosis and cell cycle arrest. However, together with the result shown in Fig. 2A, it is suggested that IFNα enhances etoposide-induced growth inhibition mainly through its enhancing effect on apoptosis rather than cell cycle arrest. Interestingly, similar effects were induced by IFNα combined with cisplatin or doxorubicin, implying that it may be common for IFNα to enhance the apoptosis induced by DNAdamaging agents in appropriate cell types. In contrast, such an enhanced cytotoxicity and apoptosis were not seen in p53mutant MG63 cells. Although IFNα can induce apoptosis in a large group of tumor cells, such an effect was not observed in U2OS and MG63 cells. This is not surprising because IFNαinduced apoptosis requires an ARF pathway in some cell types (Sandoval et al., 2004) and U2OS and MG63 cells both lack ARF expression (Park et al., 2002). Etoposide is one of the most widely used agents in treating osteosarcoma (Hande, 1998). It induces DNA damage and leads to cell cycle arrest or apoptosis by activating p53 (Hande, 1998). As a tumor suppressor gene which often mutates in human malignancies including osteosarcoma (Chandar et al., 1992), p53 gene product also plays a role in type I IFNs-mediated apoptotic pathway (Porta et al., 2005; Takaoka et al., 2003). These findings prompted us to explore the role of p53 in IFNα/etoposide combination-induced apoptosis. Our results showed that the enhanced apoptosis induced by combination in U2OS cells was associated with
an accumulation of p53 protein. Bax and Bcl-2, members of Bcl-2 family, exert pro-apoptotic or anti-apoptotic functions respectively to regulate p53-dependent apoptosis (Hockenbery et al., 1993; Thornborrow et al., 2002). In this study, the combination-induced p53 upregulation was accompanied with increasing Bax and decreasing Bcl-2 expression, which may induce the mitochondrial permeability transition, an event that releases cytochrome c and culminates in the apoptosis of the cells (Cory and Adams, 2002). p53 is implicated in the induction of both intrinsic and extrinsic apoptotic signaling pathways that lead to the activation of the caspases that mediate apoptosis (Haupt et al., 2003). Our results of activation of caspase-8 and caspase-9, special markers of intrinsic mitochondrial pathway and extrinsic death receptor pathway, respectively (Riedl and Shi, 2004), by IFNα/etoposide combination also support this concept. Caspase-3 is a key executor of the apoptotic machinery. The cleaved caspases-8 and -9 sequentially induce caspase-3 activation which can activate its substrates such as PARP, resulting in activation of DNA fragmentation of apoptosis (Shi, 2002). Furthermore, we demonstrated the requirement for caspase activation in the sensitized cell death, because pretreatment with the caspase pan inhibitor Z-VAD-FMK could largely inhibit growth inhibition and PARP cleavage induced by IFNα/etoposide combination in U2OS cells. Taken together, these results provide strong evidence that this enhanced apoptosis is mediated by caspase activation. Mdm2 regulates p53 level through a negative feedback loop (Moll and Petrenko, 2003). When nuclear p53 level is elevated, it activates Mdm2 transcription. Mdm2 binds to p53 and then induces p53 degradation. Our result that combination-mediated p53 upregulation was accompanied by an increase in Mdm2 expression was possibly due to this negative feedback loop in which p53 upregulation induced Mdm2 expression. p53 mRNA level did not further increase
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following IFNα/etoposide combination, implying that p53 upregulation is probably mediated by one of the IFNα/ etoposide combination-induced products in a post-transcriptional manner. STAT1, a key mediator of the IFNα-induced pathway, is such a candidate. It can promote p53 protein level through downregulating Mdm2 and increase apoptosis in response to DNA damage (McDermott et al., 2005; Thomas et al., 2004; Townsend et al., 2004). Also some other products of IFN-stimulated genes, such as promyelocytic leukemia gene (PML) (Gottifredi and Prives, 2001), dsRNAdependent protein kinase (PKR) (Cuddihy et al., 1999) and IFN-regulatory factor 1 (IRF1) (Tanaka et al., 1996), have been reported to participate in modulating p53 activity in response to DNA damage. It is possible that such candidates may contribute to the effects we observed. Although p53 is involved in type I IFNs-mediated apoptosis (Porta et al., 2005; Takaoka et al., 2003), its precise role in apoptosis induced by combination of IFNs and chemotherapeutic drugs remains unclear. We show here that knockdown of p53 expression markedly decreased the apoptotic response to IFNα/etoposide combination, suggesting that p53 function is required in this process. At present, the major problem in osteosarcoma treatment is the resistance against chemotherapy and drug-induced side effects (de Saint Aubain Somerhausen and Fletcher, 1999). Our results suggest that combination of IFNα and standard chemotherapeutic agents may reduce the therapeutic doses of chemotherapy to limit the side effects and achieve an enhanced chemosensitivity as well as a greater tumor remission. In conclusion, we demonstrated that IFNα enhanced etoposide-induced growth inhibition, cell cycle arrest and apoptosis in human osteosarcoma U2OS cells, and defined a p53-dependent and caspase-activation pathway as the underlying mechanism. The ability of IFNα to sensitize osteosarcoma U2OS cells to chemotherapy may also be exploited to improve treatment of osteosarcoma with functional p53. Acknowledgement This work was supported by awards from the National Natural Science Foundation of China (NO. 30070766) to Wei-Ming Liao. References Bruland, O.S., Pihl, A., 1997. On the current management of osteosarcoma. A critical evaluation and a proposal for a modified treatment strategy. European Journal of Cancer 33 (11), 1725–1731. Chandar, N., Billig, B., McMaster, J., Novak, J., 1992. Inactivation of p53 gene in human and murine osteosarcoma cells. British Journal of Cancer 65 (2), 208–214. Cory, S., Adams, J.M., 2002. The Bcl2 family: regulators of the cellular life-ordeath switch. Nature Reviews Cancer 2 (9), 647–656. Cuddihy, A.R., Li, S., Tam, N.W., Wong, A.H., Taya, Y., Abraham, N., Bell, J.C., Koromilas, A.E., 1999. Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. Molecular and Cellular Biology 19 (4), 2475–2484. de Saint Aubain Somerhausen, N., Fletcher, C.D., 1999. Soft-tissue sarcomas: an update. European Journal of Surgical Oncology 25 (2), 215–220.
Gottifredi, V., Prives, C., 2001. P53 and PML: new partners in tumor suppression. Trends in Cell Biology 11 (5), 184–187. Hande, K.R., 1998. Etoposide: four decades of development of a topoisomerase II inhibitor. European Journal of Cancer 34 (10), 1514–1521. Haupt, S., Berger, M., Goldberg, Z., Haupt, Y., 2003. Apoptosis — the p53 network. Journal of Cell Science 116 (Pt 20), 4077–4085. Heremans, H., Billiau, A., Cassiman, J.J., Mulier, J.C., de Somer, P., 1978. In vitro cultivation of human tumor tissues. II. Morphological and virological characterization of three cell lines. Oncology 35 (6), 246–252. Hockenbery, D.M., Oltvai, Z.N., Yin, X.M., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75 (2), 241–251. Kondo, M., Nagano, H., Wada, H., Damdinsuren, B., Yamamoto, H., Hiraoka, N., Eguchi, H., Miyamoto, A., Yamamoto, T., Ota, H., Nakamura, M., Marubashi, S., Dono, K., Umeshita, K., Nakamori, S., Sakon, M., Monden, M., 2005. Combination of IFN-alpha and 5-fluorouracil induces apoptosis through IFN-alpha/beta receptor in human hepatocellular carcinoma cells. Clinical Cancer Research 11 (3), 1277–1286. Levine, A.J., Momand, J., Finlay, C.A., 1991. The p53 tumour suppressor gene. Nature 351 (6326), 453–456. McDermott, U., Longley, D.B., Galligan, L., Allen, W., Wilson, T., Johnston, P.G., 2005. Effect of p53 status and STAT1 on chemotherapy-induced, Fas-mediated apoptosis in colorectal cancer. Cancer Research 65 (19), 8951–8960. Mogensen, K.E., Lewerenz, M., Reboul, J., Lutfalla, G., Uze, G., 1999. The type I interferon receptor: structure, function, and evolution of a family business. J Interferon Cytokine Research 19 (10), 1069–1098. Moll, U.M., Petrenko, O., 2003. The MDM2–p53 interaction. Molecular Cancer Research 1 (14), 1001–1008. Ormerod, M.G., 1998. The study of apoptotic cells by flow cytometry. Leukemia 12 (7), 1013–1025. Park, Y.B., Park, M.J., Kimura, K., Shimizu, K., Lee, S.H., Yokota, J., 2002. Alterations in the INK4a/ARF locus and their effects on the growth of human osteosarcoma cell lines. Cancer Genetics and Cytogenetics 133 (2), 105–111. Ponten, J., Saksela, E., 1967. Two established in vitro cell lines from human mesenchymal tumours. International Journal of Cancer 2 (5), 434–447. Porta, C., Hadj-Slimane, R., Nejmeddine, M., Pampin, M., Tovey, M.G., Espert, L., Alvarez, S., Chelbi-Alix, M.K., 2005. Interferons alpha and gamma induce p53-dependent and p53-independent apoptosis, respectively. Oncogene 24 (4), 605–615. Prejean, C., Colamonici, O.R., 2000. Role of the cytoplasmic domains of the type I interferon receptor subunits in signaling. Seminars in Cancer Biology 10 (2), 83–92. Riedl, S.J., Shi, Y., 2004. Molecular mechanisms of caspase regulation during apoptosis. Nature Reviews Molecular Cell Biology 5 (11), 897–907. Rodriguez-Villanueva, J., McDonnell, T.J., 1995. Induction of apoptotic cell death in non-melanoma skin cancer by interferon-alpha. International Journal of Cancer 61 (1), 110–114. Roos, G., Leanderson, T., Lundgren, E., 1984. Interferon-induced cell cycle changes in human hematopoietic cell lines and fresh leukemic cells. Cancer Research 44 (6), 2358–2362. Sandoval, R., Xue, J., Pilkinton, M., Salvi, D., Kiyokawa, H., Colamonici, O.R., 2004. Different requirements for the cytostatic and apoptotic effects of type I interferons. Induction of apoptosis requires ARF but not p53 in osteosarcoma cell lines. Journal of Biological Chemistry 279 (31), 32275–32280. Shi, Y., 2002. Mechanisms of caspase activation and inhibition during apoptosis. Molecular Cell 9 (3), 459–470. Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H., Schreiber, R.D., 1998. How cells respond to interferons. Annual Review of Biochemistry 67, 227–264. Takaoka, A., Hayakawa, S., Yanai, H., Stoiber, D., Negishi, H., Kikuchi, H., Sasaki, S., Imai, K., Shibue, T., Honda, K., Taniguchi, T., 2003. Integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence. Nature 424 (6948), 516–523. Tanaka, N., Ishihara, M., Lamphier, M.S., Nozawa, H., Matsuyama, T., Mak, T.W., Aizawa, S., Tokino, T., Oren, M., Taniguchi, T., 1996. Cooperation
X.-W. Yuan et al. / Life Sciences 82 (2008) 393–401 of the tumour suppressors IRF-1 and p53 in response to DNA damage. Nature 382 (6594), 816–818. Thomas, M., Finnegan, C.E., Rogers, K.M., Purcell, J.W., Trimble, A., Johnston, P.G., Boland, M.P., 2004. STAT1: a modulator of chemotherapy-induced apoptosis. Cancer Research 64 (22), 8357–8364. Thornborrow, E.C., Patel, S., Mastropietro, A.E., Schwartzfarb, E.M., Manfredi, J.J., 2002. A conserved intronic response element mediates direct p53-
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Interferonα enhances etoposide-induced apoptosis in human osteosarcoma U2OS cells by a p53-dependent pathway Xiang-Wei Yuan a,b , Xiao-Feng Zhu c,⁎, Sheng-Gen Liang b , Quan Fan b , Zhong-Xian Chen b , Xiu-Fang Huang d , Pu-Yi Sheng a , Ai-Shan He a , Zi-Bo Yang a , Rong Deng c , Gong-Kan Feng c , Wei-Ming Liao a,⁎ a
Department of Orthopedic Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China Department of Orthopedic Surgery, The Affiliated Jiangmen Hospital, Sun Yat-sen University, Jiangmen, 529030, China c State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou, 510060, China d Department of Pathology, The Affiliated Jiangmen Hospital, Sun Yat-sen University, Jiangmen, 529030, China
b
Received 2 March 2007; accepted 24 November 2007
Abstract Interferonα (IFNα) induces cell cycle arrest and triggers apoptosis and chemosensitivity. But the mechanism of IFNα in regulating chemosensitivity has not been fully understood. To study whether IFNα affected chemosensitivity of osteosarcoma cells, we treated p53-wild U2OS cells and p53-mutant MG63 cells with IFNα and etoposide, alone or in combination, and then examined growth inhibition, cell cycle arrest and apoptosis. IFNα enhanced etoposide-induced growth inhibition and apoptosis in p53-wild U2OS cells but not p53-mutant MG63 cells in a dose- and time-dependent manner. Etoposide-induced G2/M phase arrest was also enhanced by IFNα. The enhanced apoptosis was associated with the accumulation of transcriptionally active p53 accompanied with increased Bax and Mdm2, as well as decreased Bcl-2. IFNα also activated caspases-3, -8 and -9 protein kinases and PARP cleavage in response to etoposide in U2OS cells. Moreover, the combination-induced cytotoxicity and PARP cleavage were significantly reduced by caspase pan inhibitor and p53 siRNA. Thus we conclude that IFNα enhances etoposide-induced apoptosis in human osteosarcoma U2OS cells by a p53-dependent and caspase-activation pathway. The proper combination of IFNα and conventional chemotherapeutic agents may be a rational strategy for the treatment of human osteosarcoma with functional p53. © 2007 Elsevier Inc. All rights reserved. Keywords: Interferonα; Etoposide; Apoptosis; p53; Osteosarcoma
Introduction Osteosarcoma is the most common primary malignant tumor of the skeletal system, and occurs most frequently in children and adolescents (Young and Miller, 1975). The therapeutic modalities are combined treatments with surgery and neoadjuvant chemotherapy (Bruland and Pihl, 1997). The chemother⁎ Corresponding authors. W.M. Liao is to be contacted at Department of Orthopedic Surgery, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan Road 2, Guangzhou 510080, China. Tel.: +86 20 87755766 8898; fax: +86 20 87750632. X.F. Zhu, State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, 651 Dongfeng Road East, Guangzhou 510060, China. Tel.: +86 20 87343149; fax: +86 20 87343392. E-mail addresses: [email protected] (W.-M. Liao), [email protected] (X.-F. Zhu). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.11.025
apy results in significant improvement in patient survival. However, the frequent acquisition of drug-resistant phenotypes and unwanted side effects are often associated with chemotherapy and remain as serious problems (de Saint Aubain Somerhausen and Fletcher, 1999). Therefore, new therapeutic strategies which can improve the effect of current chemotherapy need to be developed. Interferons (IFNs), grouped into two families: type I and type II, are multifunctional proteins that share immunomodulatory, antiviral and antitumor activities (Mogensen et al., 1999; Prejean and Colamonici, 2000; Stark et al., 1998). IFNα, which belongs to the type I IFNs, interacts with IFN-α/β receptor on the cell surface to induce activation of JAK1 and TYK2, leading to phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1). The activated STAT1
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forms homodimers or heterodimers which translocate into nucleus and subsequently bind specific promoter regions (interferon-stimulated response elements (ISRE) or gammaactivating sequence (GAS)) to regulate transcription of the genes responsible for antiproliferative effects (Stark et al., 1998). The tumor suppressor p53, which is often disrupted in human malignancies including osteosarcoma (Chandar et al., 1992), induces cell cycle arrest and apoptosis in response to DNA-damaging agents including etoposide (Hande, 1998; Levine et al., 1991), therefore playing a key role in cancer control. Previous reports have shown that p53 induction contributes to type I IFN-induced apoptosis in some cancer cells (Porta et al., 2005; Takaoka et al., 2003). Such observations indicate that IFNα-treated cells may be more sensitive to p53-dependent apoptosis in response to DNAdamaging chemotherapeutic agents. Thus we hypothesize that IFNα may increase etoposide sensitivity in human osteosarcoma cells. To test this hypothesis, we utilized the p53-wild and p53-mutant osteosarcoma cells to examine whether IFNα increases etoposide sensitivity by modulating p53-mediated apoptosis. We show here that IFNα enhances etoposide-induced apoptosis in osteosarcoma p53-wild U2OS but not p53-mutant MG63 cells and define a p53-dependent pathway as the molecular mechanism. This work also supports the rational clinical utility of IFNα combination therapy for treatment of osteosarcoma with functional p53. Materials and methods Cell culture and reagents The human osteosarcoma U2OS cells (Ponten and Saksela, 1967) containing wild type p53 and MG63 cells (Heremans et al., 1978) containing mutant p53 were maintained in DMEM (Life Technologies, Inc.) and supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a 5% CO2 humidified atmosphere. MG63 cells had a rearrangement mutation of the p53 gene, which results in disruption of p53 protein function (Chandar et al., 1992). Etoposide, Hoechst 33258 and MTT were obtained from Sigma. Human recombinant IFNα2a was purchased from Peprotech. ZVAD-FMK (pan caspase inhibitor) was purchased from Calbiochem Co. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The logarithmically growing U2OS and MG63 cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After an overnight growth, cells were treated with IFNα and etoposide, alone or in combination. After indicated times, 10 μl of 5 mg/ml MTT was added into each well followed by incubation for an additional 4 h. Supernatants were removed and 200 μl of DMSO was added. After crystals were dissolved, absorbance at 450 nm was measured in the microplate reader.
Flow cytometric analysis Cells were collected, washed twice with ice-cold PBS, resuspended in cold PBS, and fixed with 70% ethanol. After incubation overnight and subsequent rehydration in PBS for 30 min at 4 °C, the samples were stained for 30 min in the dark with 50 μg/ml propidium iodide (Sigma) containing 125 U/ml protease-free RNase, both diluted in PBS and then were analyzed in a FACS flow cytometer. Morphological analysis of apoptosis Cells were collected, washed twice with PBS and stained with 10 μg/ml of Hoechst 33258 for 15 min, followed by examination on an Olympus fluorescence microscope. DNA fragmentation assay Cells (3 × 106) were collected and washed once with PBS. DNA was extracted using DNAZol reagent (Invitrogen) according to the manufacturer's instructions and electrophoresed on 2% agarose gel containing 0.5 μg/ml ethidium bromide. The gel was photographed with UV illumination. Western blot analysis Cells were collected and lysed with the lysis buffer (10 mM Tris–HCl pH 7.4, 5 mM MgCl2, 1 mM EDTA, 25 mM NaF, 100 μM Na3VO4 and 1 mM dithiothreitol). An equal amount of protein determined by Bradford assay was resolved by SDS–PAGE and transferred onto PVDF membranes (Roche). Blots were incubated with primary antibody overnight at 4 °C, followed by 3 washes in TBST for 5 min, and then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Signals were detected using ECL and exposed to X-ray film (Kodak). Anti-caspase-8 antibody was purchased from Cell Signaling Technologies. The other antibodies were obtained from Santa Cruz. RT-PCR Total RNA was extracted with TriZol reagent (Invitrogen) according to the manufacturer's recommendations. For RT reaction, 2 µg RNA was combined with 0.5 µg Oligo(dT)15 with 15 µl total volume, and the mixture was incubated at 70 °C for 5 min and placed on ice. Then 5 µl 5× M-MLV reaction buffer, 1.25 µl 4× dNTP (10 mM), 1 µl M-MLV (Promega, 200 U/µl), and 0.625 µl RNaseOUTTM (40 U/µl) were added to 25 µl total volume, and the tube was incubated at 42 °C for 60 min and then at 75 °C for 10 min for termination. PCR reaction was performed in the presence of Taq DNA polymerase, dNTP mix, PCR buffer (all from Invitrogen), and primers. After denaturation at 94 °C for 2 min, the samples underwent 30 cycles of amplification (1 min at 94 °C, 1 min at 58 °C for p53 or 55 °C for β-actin, 1 min at 72 °C) with a 10 min extension at 72 °C following
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the last cycle. Sense and antisense primers were: 5′-CAG CCA AGT CTG TGA CTT GCA CGT AC-3′ and 5′-CTA TGT CGA AAA GTG TTT CTG TCA TC-3′ for p53 and 5′ACT ACC TCA TGA AGA TCC TC-3′ and 5′-CTA AAG ATT GCG TGG CGA GG-3′ for β-actin. Products were electrophoresed on 1.5% agarose gels containing 0.5 μg/ml ethidium bromide.
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Statistical analysis SPSS 10.0 software (SPSS Inc, IL, US) was used for statistical analysis. The unpaired t test was used to determine statistically significant differences between treatment effects. Significance was determined as P b 0.05. Results
Small interfering RNA transfection U2OS cells were plated onto six-well plates at a density of 3 × 105 cells/well with growth medium without antibiotics. After an overnight incubation, transfection was performed at a confluency of 50% by using Opti-MEM media, lipofectamine 2000, and specific or nonspecific siRNA for p53 according to manufacturer's recommendations. Six hours later, the medium was replaced with growth medium without antibiotics. After transfection for 20 h, cells were trypsinized and sub-seeded onto 96-well plates. After incubation for another 4 h, cells were treated as described for MTT assay. The p53-siRNA duplexes were synthesized by Genepharma Co., Ltd. (Shanghai, China). The mRNA sequence selected to be targeted by p53-siRNA is 5′-CTA CTT CCT GAA AAC AAC G-3′.
IFNα enhances etoposide-induced growth inhibition in human osteosarcoma p53-wild U2OS but not p53-mutant MG63 cells To study whether IFNα could enhance etoposide-induced growth inhibition, cell viability was examined using MTT assay. Fig. 1A and B shows that in p53-wild U2OS cells, IFNα alone did not induce growth inhibition at concentrations of 50 U/ml, 500 U/ml and 5000 U/ml, but significantly enhanced etoposideinduced cytotoxicity in a dose- and time-dependent manner. Similar results were induced by treatment with IFNα combined with cisplatin or doxorubicin, both DNA-damaging agents (data not shown). In contrast, such effects were not observed in p53mutant MG63 cells (Fig. 1C and D). These results show that IFNα enhances etoposide-induced growth inhibition in p53-
Fig. 1. Effect of IFNα on etoposide-induced growth inhibition in U2OS and MG63 cells. U2OS cells (A) and MG63 cells (C) were treated with a range of doses of IFNα (50 to 5000 U/ml) and etoposide (2.5 μg/ml for U2OS and 0.625 μg/ml for MG63), alone or in combination. U2OS cells (B) and MG63 cells (D) were treated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml for U2OS and 0.625 μg/ml for MG63) alone or in combination for different times. Cell viability was examined after the indicated treatment using MTT assay and shown as means ± SD. The experiment was repeated three times with similar results.
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wild U2OS cells but not p53-mutant MG63 cells and imply that p53 may be involved in this effect. IFNα enhances etoposide-induced apoptosis in p53-wild U2OS but not p53-mutant MG63 cells To test whether IFNα enhanced etoposide-induced apoptosis, flow cytometry analysis was applied. The fraction of subG1 cells represents cells undergoing DNA fragmentation, a reliable manifestation of apoptosis (Ormerod, 1998). IFNα did not induce obvious apoptosis, but markedly increased etoposide-induced apoptosis in p53-wild U2OS cells (Fig. 2A) but not p53-mutant MG63 cells (Fig. 2B). A higher level of apoptotic morphological changes, such as nuclear condensation and fragmentation, and internucleosomal DNA fragmentation – hallmarks of apoptosis – were observed in U2OS cells treated for 72 h with IFNα/etoposide combination than in those treated
with either alone (Fig. 3A and B). Similar results were observed after treatment with IFNα combined with cisplatin or doxorubicin (data not shown). In contrast, such results were not obtained in p53-mutant MG63 cells (data not shown). These results show that IFNα enhances etoposide-induced apoptosis in p53-wild U2OS cells but not p53-mutant MG63 cells. IFNα enhances etoposide-induced cell cycle arrest in p53-wild U2OS cells Etoposide-induced growth inhibition is caused by the induction of both apoptosis and cell cycle arrest (Hande, 1998). Next we used FACS analysis to determine the effect of IFNα on etoposide-induced cell cycle arrest in U2OS cells. After 72 h of etoposide treatment, there was an obvious accumulation of cells in G2/M phase and a decline of cells in S phase, compared with control cells. IFNα further increased cells
Fig. 2. Effect of IFNα on etoposide-induced apoptosis in U2OS and MG63 cells. U2OS cells (A) and MG63 cells (B) were treated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml for U2OS and 0.625 μg/ml for MG63) alone or in combination for 72 h. Apoptotic cells were examined by flow cytometry. The experiment was repeated three times with similar results.
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Fig. 3. IFNα enhances etoposide-induced apoptosis in U2OS cells. U2OS cells were treated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml) alone or in combination for 72 h. (A) Apoptotic cells were determined by Hoechst 33258 staining of nuclei as a result of chromatin condensation and nuclear fragmentation. (B) DNA fragmentation assay was examined to confirm the ladder change representing apoptosis. Lane M: DNA molecular marker; Lane 1: control cells; Lane 2: IFNα-treated cells; Lane 3: etoposide-treated cells; Lane 4: IFNα/etoposide combination-treated cells.
in G2/M phase and decreased cells in S phase in response to etoposide (P b 0.05, Fig. 4). These results show that IFNα also enhances etoposide-induced cell cycle arrest in U2OS cells. IFNα activates caspases-3, -8, -9 and PARP cleavage in response to etoposide in U2OS cells Caspases are key executors of apoptotic cell death signals by cascade activation and selectively cleaving the substrates such as poly(ADP-ribose) polymerase (PARP). To further elucidate the signaling pathway underlying the enhanced apoptosis, we detected activation of caspases-3, -8 and -9 and PARP using Western blot. As shown in Fig. 5, caspases-3, -8 and -9 were cleaved to yield 17 kD, 43/41 kD and 18 kD, as well as 37 kD fragments, respectively in response to IFNα/etoposide combination in U2OS cells. Moreover, the combination promoted the 85 kD active fragment of PARP, compared with either alone.
Fig. 4. Effect of IFNα on etoposide-induced cell cycle arrest in U2OS cells. U2OS cells were treated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml) alone or in combination for 72 h. Cell cycle distribution was examined by flow cytometry analysis. The experiment was repeated three times with similar results. An unpaired t test was used to statistically compare etoposide-induced cell cycle arrest in the presence and absence of IFNα. ⁎, P b 0.05.
These results further prove that IFNα enhances etoposideinduced apoptosis. Caspase inhibitor prevents the enhanced growth inhibition and apoptosis induced by IFNα/etoposide combination in U2OS cells The results shown above indicated that caspase activation may be involved in the enhanced apoptosis. To determine whether caspase activation is required for the facilitated apoptosis, U2OS cells was pretreated with pan caspase inhibitor
Fig. 5. IFNα activates caspases-3, -8 and -9 and PARP cleavage in response to etoposide after treatments for 72 h in U2OS cells by Western blot. Proteins were resolved by SDS–PAGE and blotted with antibodies for caspases-3,-8,-9 and PARP. GAPDH was used as loading control.
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Z-VAD-FMK for 2 h. We determined whether Z-VAD-FMK inhibited the sensitization of growth inhibition and PARP cleavage induced by IFNα/etoposide combination. As shown in Fig. 6A and B, growth inhibition and PARP cleavage were greatly inhibited by Z-VAD-FMK. This suggests that the enhanced apoptosis induced by IFNα/etoposide combination is caspase-dependent. IFNα regulates p53 pathway in response to etoposide in U2OS cells Next we asked whether IFNα could affect etoposide-induced activation of p53 pathway. Fig. 7A shows that p53 protein level was enhanced by etoposide alone and further augmented by IFNα/etoposide combination, but unaltered by IFNα alone. The expression of Bax, Bcl-2 and Mdm2, well-known downstream genes of p53, was examined to determine the transcriptional activity of the elevated p53. The pro-apoptotic Bax expression was further increased by IFNα/etoposide combination than either alone. Mdm2 expression was also increased. Conversely, the anti-apoptotic Bcl-2 expression was further decreased by IFNα/etoposide combination compared to either alone. Such effects were not observed in MG63 cells (Fig. 7A). To further explore the mechanisms leading to the p53 protein upregulation, RT-PCR was applied to examine mRNA level of p53. Fig. 7B shows that p53 mRNA level was increased greatly by etoposide but not further augmented by IFNα/etoposide combination in
Fig. 7. Effect of IFNα on expression of p53, Bax, Mdm2 and Bcl-2 in response to etoposide in U2OS and MG63 cells. (A) Cells were treated with IFNα and etoposide as indicated for 48 h. Expression of the indicated proteins was detected in U2OS and MG63 cells by Western blot. (B) mRNA level of p53 was examined at 48 h post-treatment in U2OS cells using RT-PCR.
U2OS cells, suggesting that the p53 protein boost is mediated in a post-transcriptional manner. siRNA-mediated p53 knockdown in U2OS cells results in decrease of growth inhibition and apoptosis induced by IFNα/etoposide combination To examine whether the enhanced apoptosis was p53dependent, p53-specific siRNA was transfected into U2OS cells; this significantly suppressed p53 expression after treatment with the IFNα/etoposide combination (Fig. 8A). The p53 knockdown resulted in a substantial reduction in the combination-induced cell death (Fig. 8B). This finding was supported by the loss of cleaved PARP in U2OS cells where p53 expression had been suppressed, compared with nonspecific control (Fig. 8C). These results show that p53 function is required for the enhanced apoptosis induced by IFNα/etoposide combination. Discussion
Fig. 6. Caspase pan inhibitor Z-VAD-FMK prevents the enhanced growth inhibition and apoptosis induced by IFNα/etoposide combination in U2OS cells. U2OS cells were pretreated with caspase pan inhibitor Z-VAD-FMK (80 μM) for 2 h, then incubated with IFNα (5000 U/ml) and etoposide (2.5 μg/ml) alone or in combination for 72 h. Cell viability (A) and PARP cleavage (B) were examined using MTT assay and Western blot, respectively. The experiment was repeated three times with similar results.
IFNα is a multifunctional cytokine with antitumor effects including suppressing cell growth, inducing apoptosis and increasing chemosensitivity (Kondo et al., 2005; RodriguezVillanueva and McDonnell, 1995; Roos et al., 1984). However, the molecular basis remains unclear. Here we found that IFNα alone did not alter cell growth but promoted etoposide-induced cytotoxicity pronouncedly in osteosarcoma U2OS cells. Further examinations revealed a significantly enhanced apoptosis in response to IFNα/etoposide combination, compared with etoposide
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Fig. 8. siRNA-mediated p53 silencing in U2OS cells results in a decrease of growth inhibition and apoptosis induced by IFNα/etoposide combination. (A) U2OS cells were transfected with p53 specific or nonspecific siRNA duplexes and treated with IFNα/etoposide combination as described in Materials and methods. p53 protein level was examined by Western blot. (B) U2OS cells were treated as described. Cell viability was determined by MTT assay at 72 h post-treatment. The data are shown as means ± SD that is representative of three separate experiments. (C) U2OS cells were transfected with siRNA duplexes and treated as described for 72 h. Cleavage of PARP was detected by Western blot.
alone. However, the cell growth inhibition was higher than the apoptotic rate in U2OS cells treated with IFNα/etoposide combination, which indicates that, besides the apoptosis, cell cycle arrest is possibly involved in the IFNα/etoposide combination-induced cytotoxicity. This was consistent with our result that IFNα enhances etoposide-induced G2/M phase arrest in U2OS cells, albeit weakly. Therefore, the enhancing effect of the combination is based on the induction of both apoptosis and cell cycle arrest. However, together with the result shown in Fig. 2A, it is suggested that IFNα enhances etoposide-induced growth inhibition mainly through its enhancing effect on apoptosis rather than cell cycle arrest. Interestingly, similar effects were induced by IFNα combined with cisplatin or doxorubicin, implying that it may be common for IFNα to enhance the apoptosis induced by DNAdamaging agents in appropriate cell types. In contrast, such an enhanced cytotoxicity and apoptosis were not seen in p53mutant MG63 cells. Although IFNα can induce apoptosis in a large group of tumor cells, such an effect was not observed in U2OS and MG63 cells. This is not surprising because IFNαinduced apoptosis requires an ARF pathway in some cell types (Sandoval et al., 2004) and U2OS and MG63 cells both lack ARF expression (Park et al., 2002). Etoposide is one of the most widely used agents in treating osteosarcoma (Hande, 1998). It induces DNA damage and leads to cell cycle arrest or apoptosis by activating p53 (Hande, 1998). As a tumor suppressor gene which often mutates in human malignancies including osteosarcoma (Chandar et al., 1992), p53 gene product also plays a role in type I IFNs-mediated apoptotic pathway (Porta et al., 2005; Takaoka et al., 2003). These findings prompted us to explore the role of p53 in IFNα/etoposide combination-induced apoptosis. Our results showed that the enhanced apoptosis induced by combination in U2OS cells was associated with
an accumulation of p53 protein. Bax and Bcl-2, members of Bcl-2 family, exert pro-apoptotic or anti-apoptotic functions respectively to regulate p53-dependent apoptosis (Hockenbery et al., 1993; Thornborrow et al., 2002). In this study, the combination-induced p53 upregulation was accompanied with increasing Bax and decreasing Bcl-2 expression, which may induce the mitochondrial permeability transition, an event that releases cytochrome c and culminates in the apoptosis of the cells (Cory and Adams, 2002). p53 is implicated in the induction of both intrinsic and extrinsic apoptotic signaling pathways that lead to the activation of the caspases that mediate apoptosis (Haupt et al., 2003). Our results of activation of caspase-8 and caspase-9, special markers of intrinsic mitochondrial pathway and extrinsic death receptor pathway, respectively (Riedl and Shi, 2004), by IFNα/etoposide combination also support this concept. Caspase-3 is a key executor of the apoptotic machinery. The cleaved caspases-8 and -9 sequentially induce caspase-3 activation which can activate its substrates such as PARP, resulting in activation of DNA fragmentation of apoptosis (Shi, 2002). Furthermore, we demonstrated the requirement for caspase activation in the sensitized cell death, because pretreatment with the caspase pan inhibitor Z-VAD-FMK could largely inhibit growth inhibition and PARP cleavage induced by IFNα/etoposide combination in U2OS cells. Taken together, these results provide strong evidence that this enhanced apoptosis is mediated by caspase activation. Mdm2 regulates p53 level through a negative feedback loop (Moll and Petrenko, 2003). When nuclear p53 level is elevated, it activates Mdm2 transcription. Mdm2 binds to p53 and then induces p53 degradation. Our result that combination-mediated p53 upregulation was accompanied by an increase in Mdm2 expression was possibly due to this negative feedback loop in which p53 upregulation induced Mdm2 expression. p53 mRNA level did not further increase
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following IFNα/etoposide combination, implying that p53 upregulation is probably mediated by one of the IFNα/ etoposide combination-induced products in a post-transcriptional manner. STAT1, a key mediator of the IFNα-induced pathway, is such a candidate. It can promote p53 protein level through downregulating Mdm2 and increase apoptosis in response to DNA damage (McDermott et al., 2005; Thomas et al., 2004; Townsend et al., 2004). Also some other products of IFN-stimulated genes, such as promyelocytic leukemia gene (PML) (Gottifredi and Prives, 2001), dsRNAdependent protein kinase (PKR) (Cuddihy et al., 1999) and IFN-regulatory factor 1 (IRF1) (Tanaka et al., 1996), have been reported to participate in modulating p53 activity in response to DNA damage. It is possible that such candidates may contribute to the effects we observed. Although p53 is involved in type I IFNs-mediated apoptosis (Porta et al., 2005; Takaoka et al., 2003), its precise role in apoptosis induced by combination of IFNs and chemotherapeutic drugs remains unclear. We show here that knockdown of p53 expression markedly decreased the apoptotic response to IFNα/etoposide combination, suggesting that p53 function is required in this process. At present, the major problem in osteosarcoma treatment is the resistance against chemotherapy and drug-induced side effects (de Saint Aubain Somerhausen and Fletcher, 1999). Our results suggest that combination of IFNα and standard chemotherapeutic agents may reduce the therapeutic doses of chemotherapy to limit the side effects and achieve an enhanced chemosensitivity as well as a greater tumor remission. In conclusion, we demonstrated that IFNα enhanced etoposide-induced growth inhibition, cell cycle arrest and apoptosis in human osteosarcoma U2OS cells, and defined a p53-dependent and caspase-activation pathway as the underlying mechanism. The ability of IFNα to sensitize osteosarcoma U2OS cells to chemotherapy may also be exploited to improve treatment of osteosarcoma with functional p53. Acknowledgement This work was supported by awards from the National Natural Science Foundation of China (NO. 30070766) to Wei-Ming Liao. References Bruland, O.S., Pihl, A., 1997. On the current management of osteosarcoma. A critical evaluation and a proposal for a modified treatment strategy. European Journal of Cancer 33 (11), 1725–1731. Chandar, N., Billig, B., McMaster, J., Novak, J., 1992. Inactivation of p53 gene in human and murine osteosarcoma cells. British Journal of Cancer 65 (2), 208–214. Cory, S., Adams, J.M., 2002. The Bcl2 family: regulators of the cellular life-ordeath switch. Nature Reviews Cancer 2 (9), 647–656. Cuddihy, A.R., Li, S., Tam, N.W., Wong, A.H., Taya, Y., Abraham, N., Bell, J.C., Koromilas, A.E., 1999. Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. Molecular and Cellular Biology 19 (4), 2475–2484. de Saint Aubain Somerhausen, N., Fletcher, C.D., 1999. Soft-tissue sarcomas: an update. European Journal of Surgical Oncology 25 (2), 215–220.
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