Knockdown of Mad2 induces osteosarcoma cell apoptosis-involved Rad21 cleavage

Knockdown of Mad2 induces osteosarcoma cell apoptosis-involved Rad21 cleavage

J Orthop Sci (2011) 16:814–820 DOI 10.1007/s00776-011-0156-x ORIGINAL ARTICLE Knockdown of Mad2 induces osteosarcoma cell apoptosis-involved Rad21 c...

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J Orthop Sci (2011) 16:814–820 DOI 10.1007/s00776-011-0156-x

ORIGINAL ARTICLE

Knockdown of Mad2 induces osteosarcoma cell apoptosis-involved Rad21 cleavage Ling Yu • Weichun Guo • Shenghao Zhao Jin Tang • Jianhua Liu



Received: 11 December 2010 / Accepted: 16 August 2011 / Published online: 7 September 2011 Ó The Japanese Orthopaedic Association 2011

Abstract Background Besides Mad2’s role in carcinogenesis, recent study has shown that it is essential in cell survival. Here we found that knockdown of Mad2 causes osteosarcoma cell death through apoptosis, with the apoptotic signal resulting from Rad21 cleavage. Methods U2OS and MG63 cells were divided into three groups: the Mad2 siRNA group, mock group and normal control group; the Mad2 siRNA group and mock group are transfected with Mad2 shRNA plasmid and mock plasmid, respectively. G418 was used to increase the transfection efficacy, which was evaluated by GFP fluorescence. Quantitative PCR and Western blotting analyses were used to detect the transcription and expression of Mad2, Rad21 and caspase-3, respectively. Flow cytometry assay using PE-labeled Annexin-V and PI, TUNEL assay and Hoechst 33258 staining were used to evaluate cell apoptosis. Results We successfully achieved knockdown of Mad2 expression in cancer cells using RNA interference. We observed obvious apoptosis in the Mad2 siRNA group compared with the Mock and control group. We found that the apoptosis induced by Mad2 knockdown correlated with Rad21 cleavage. Conclusion These results confirmed that knockdown of Mad2 causes osteosarcoma cell death through apoptosis and provides evidence that the apoptotic signal resulted L. Yu  W. Guo (&)  S. Zhao  J. Tang Department of Orthopedics, Renmin Hospital, Wuhan University, Wuhan, Hubei, China e-mail: [email protected] J. Liu Department of Orthopedics, Renmin Hospital of Dongxihu District, Wuhan, Hubei, China

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from Rad21 cleavage. This study suggested that Mad2 has potential to be a novel target for cancer therapy.

Introduction Osteosarcoma is the most prevalent malignancy in bone and principally affects adolescents [1]. Chromosomal instability is commonly seen in patients with osteosarcoma, as well as in those with various other malignancies [2–5]. Mitotic arrest defective protein 2 (Mad2) is a key component in the spindle checkpoint in which malfunction leads to chromosomal instability, and its important role in tumorigenesis has been well established [6–8]. We previously reported that Mad2 was upregulated in osteosarcoma compared with normal bone tissue and that its expression level correlated with patients’ prognosis [9]. Recently it has been demonstrated that Mad2 plays an important role in cell survival [10, 11]; however, the mechanism of action is still unclear. Mad2 potentially inhibits the activation of anaphase-promoting complex/ cyclosome (APC/C) by Cdc20, while securing is potentially polyubiquitinated by activated APC/C, thus inhibiting the cleavage of Rad21 [12]. Rad21 is highly similar to the gene product of Schizosaccharomyces pombe rad21, which is involved in the repair of DNA double-strand breaks and in chromatid cohesion during mitosis. Hence, Mad2 may promote Rad21 accumulation, whose cleavage product has been reported to be an intrinsic apoptotic component. We aimed to elucidate Mad2’s role in cell survival and Rad21 cleavage. In this study, we found that Mad2 expression correlates with Rad21 in tumor tissues and that the knockdown of Mad2 causes increased apoptosis of osteosarcoma cells accompanied by Rad21 cleavage.

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was added the following day after transfection. All the cells were harvested on the 3rd day after transfection.

Materials and methods Cell culture

RNA extraction and quantitative PCR The human osteosarcoma cell lines U2OS and MG63 were purchased from Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. They were cultured in DMEM (high glucose) supplemented with 10% fetal bovine serum. The cells were cultured in a 37°C humidified incubator with a mixture of 95% air and 5% CO2. siRNA constructs and transfection The siRNA sequences were reported previously by Shi et al. [13], and the siRNA sequences were inserted into pGCsilencerTM plasmid, whose detailed information of the plasmid is shown in Fig. 1. The recombined clone was made by Shanghai GeneChem Co., Ltd. The recombinant plasmid was confirmed by sequencing. According to the manufacturer’s instruction, the siRNA plasmid and mock plasmid were transfected into U2OS and MG63 using TM Lipofectamine 2000 reagent (Invitrogen, USA), The human osteosarcoma cells transfected with Mad2 siRNA plasmid and mock plasmid were named Mad2-siRNA and mock, respectively. Selective medium containing G418

Fig. 1 Characteristics of the pGCsilencer. Vector map showing the shRNA cassette expression is driven by a H1 promoter system, and a transcript of GFP is driven by another CMV promoter. Detailed information is provided

Total RNA was isolated from the cultured cells by TRIzol reagent according to the manufacturer’s protocol (Invitrogen). The quality and quantity of the RNA prepared from each sample were determined by UV absorbance spectroscopy. cDNA was made by reverse transcription using RevertAidTM First Strand cDNA Synthesis Kit (Fermentas). The PCR reactions were performed using a Real-time PCR Detection System (ABI 7900). The primers for Mad2 were 50 -GGTGCAGAAATACGGACTCACCTT-30 and 30 -TTCCAGGACCTCACCACTTTCA-30 . The primers for Rad21 were 50 -TGGGTTGTGTTTGTGTTCTG-30 and 50 -TCAAGAGGGTGACCATTGTT-30 . Melting curve analysis was used to ensure the purity of the amplified PCR product. The relative mRNA level was calculated using the 2-DDCt method. Western blotting analysis Cells were washed with ice-cold PBS after being trypsinized and centrifuged at 1,000 rpm for 10 min at room

Vector description shRNA: 9 - 55 CMV promoter: 105 - 704 GFP: 709 - 1428 SV40 promoter: 2098 2475 Neomycin: 2503 - 3297 pUC ori: 3534 - 4702 Ampicillin: 4805 - 5659 H1 promoter: 6077- 6169

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temperature. The pellet containing *106 was lysed in 100 ll of RIPA cell lysis buffer containing protease inhibitors and quantified by BCA method. One hundred micrograms of protein was separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking by 5% skim milk, the membranes were incubated with the following primary antibodies: polyclonal rabbit anti-Mad2 (1:1,000; Abcam), polyclonal rabbit anti-caspase-3 (1:500; Abcam), monoclonal mouse anti-Rad21 (H-12) (1:500, Santa Cruz) and monoclonal mouse anti-b-actin (1:2,500; Sigma). The blots were then incubated with horseradish peroxidaseconjugated secondary antibody. Enhanced chemiluminescence was used for detection, and they were developed by X-ray film. Flow cytometry assay The siRNA-transfected cells, mock cells, and normal U2OS or MG63 cells were seeded in 6-well plates. The cells were harvested using 0.25% trypsin without EDTA. The cells were then stained with PE-labeled Annexin-V and PI. PBS was added as negative control, and compensation was done by PE-labeled Annexin-V and PI stain, respectively. The transfection efficiency was calculated by detecting GFP expression compared with untreated cells. Apoptotic cells were measured by a FACScalibur, and data analysis was performed with the standard cell Quest software. All the experiments were repeated three times. TUNEL assay TUNEL assay was performed by using the ‘‘In Situ Cell Death Detection Kit, POD’’ (Roche). In brief, cells cultured in 6-well plate were fixed for 1 h in freshly prepared 4% paraformaldehyde and then were incubated with blocking solution for 10 min at 20°C. After two washes in PBS, slides were incubated in permeabilization solution for 2 min on ice. Slides were rinsed twice with PBS, 50 ll TUNEL reaction mixture was added to the sample, and they were incubated for 60 min at 37°C in a humidified atmosphere. Then 50 ll Converter-POD was added to the samples, which were incubated for 30 min at 37°C in a humidified chamber. They were rinsed with PBS, and then the DAB substrate was added for 10 min incubation at 20°C. They were rinsed with PBS, and the results were analyzed under an inverted light microscope. Hoechst 33258 staining of nuclear chromatin Cells mounted on coverslips were fixed with 4% formaldehyde in PBS at 37°C for 15 min, permeabilized with ethanol/acetic acid at 20°C for 15 min and washed with PBS. Cells were then exposed to 1 lg/ml Hoechst 33258

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(Sigma) in PBS at room temperature for 15 min. Cultures were washed three times in PBS, and ProLong Anti-Fade reagent (Molecular Probes, Leiden, The Netherlands) was added to the cells according to the manufacturer’s instructions. Cells were visualized with an Axiovert 135 fluorescence microscope (Zeiss, Oberkochen, Germany) with UV illumination using a 409 fluorescence objective. Nuclei of control cells appeared round to oval, with blue fluorescence; apoptotic nuclei were characterized by increasing brightness, decreased in size and fragmented into apoptotic bodies. Statistical analysis Statistical analyses were carried out by one-way ANOVA test; P values \0.05 were considered significant.

Results Knockdown of Mad2 expression in cancer cells by siRNA We established a U2OS cell line with Mad2 depletion using a siRNA plasmid. The vector was cloned by inserting siRNA sequences into pGCsilencer plasmid. The pGCsilencer plasmid has one GFP and one Neomycin open reading frame; Neomycin resistance was used to improve transfection efficiency, while GFP fluorescence was used to evaluate the transfection efficiency (Fig. 1). The transfection efficacy was evaluated both by fluorescence microscopy and flow cytometry. The transfection efficiency after plating in selective medium for 2 days was around 55–65%. We evaluated Mad2 expression by quantitative PCR (Fig. 2a, b) and Western blot (Fig. 2c), and both methods showed that siRNA reduced the level of endogenous Mad2 mRNA and protein. Knockdown of Mad2 expression elevated cell apoptosis To verify the effect of depletion of Mad2 on apoptosis, we used flow cytometry. GFP fluorescence divided the mock or siRNA group into transfected and untransfected groups. The apoptosis rate regarding the Mad2-RNAi group was determined from the GFP-positive group, which shows G418 resistance. In this case, G418 won’t influence the apoptosis rate. The apoptotic rate in different cells is shown in Table 1. The apoptotic rate of U2OS cells transfected with Mad2 siRNA reached 3.9% compared with 0.4% in U2OS nontransfected cells and 0.3% in mock-transfected cells (Fig 3a); the data for MG63 are provided in Table 1. TUNEL assay and Hoechst 33258 staining showed the

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Fig. 2 Depletion of Mad2 by RNA interference. a Melting curve and b relative mRNA levels of Mad2. c Expression of Mad2 by immunoblot analysis. d Sample of fluorescence image of U2OS cells transfected with Mad2-siRNA

Table 1 The apoptosis rate detected by FCM from different osteosarcoma cells Negative control group

Mock control group

Mad2-siRNA group

U2OS (n = 3)

0.26 ± 0.15

0.50 ± 0.20

3.23 ± 0.70**

MG63 (n = 3)

0.33 ± 0.15

0.40 ± 0.20

3.77 ± 0.90**

The apoptosis rate is summarize in the table above; the data are shown as mean ± SD **P \ 0.01

same tendency. We also found that the Western blot assay resulted in decreased Mad2 and Rad21 accompanied by elevated caspase-3 (Fig 4c). The apoptosis induced by Mad2 knockdown correlated with Rad21 cleavage As is evident in Fig. 4, the Rad21 protein level was decreased in Mad2-siRNA-transfected U2OS cells, whereas no difference was found in the normal or mock-

transfected group. Quantitative PCR showed that the mRNA level of Rad21 was no different regardless of the decrease in Mad2. The disalignment of mRNA and protein level indicates that the regulatory mechanism during protein degradation is affected.

Discussion As Rad21 is located downstream of Mad2, Mad2 is able to inhibit Rad21 cleavage and arrest the cell cycle when chromosome mis-segregation occurs, and thus there is sufficient time to repair the mis-segregation of sister chromatids. According to Nasmyth [12], it can be inferred that upregulation of Mad2 expression causes decreased Rad21 cleavage; in other words, the more Mad2 accumulated, the more intact Rad21 exists. The efficiency of the RNAi was confirmed using both quantitative PCR and Western blot. We observed significant apoptosis compared with the normal and mocktransfected group using both Annexin-V-PE/PI stain, TUNEL assay, and Hoechst 33258 stain. The elevated expression of caspase-3 also exhibited the activation of

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Fig. 3 Apoptosis induced by Mad2 depletion. a Flow cytometric analysis of Annexin V-PE/PI double-stained cells (mock and siRNA groups were analyzed from GFP-positive cells). b TUNEL assay. c Hoechst 33258 stain

programmed death. Similar results were obtained in the MG63 osteosarcoma cell line. Taken together, the data indicated that Mad2 depletion induced apoptosis. This was in concordance with reports by two other research groups [10, 11]. We found that Rad21 protein was downregulated when Mad2 was decreased. Rad21 protein expression was detected using Western blot with an antibody against an internal region of Rad21 of human origin. As it has been reported [14] that Asp279 is the key cleavage site of Rad21 during apoptosis, the antibody raised against amino acids 143–352 would be an ideal indicator for detection of Rad21 degradation during apoptosis. However, we found no difference between the different groups of Rad21 mRNA using quantitative PCR. Importantly, the difference between the level of mRNA and protein suggested that

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downregulation of Rad21 was a result of protein cleavage, but not transcriptional regulation. Rad21 has been reported to be an intrinsic apoptotic factor [15, 16], which is involved downstream of the Mad2 pathway. Yang et al. [14] reported cleavage of Mcd1 by protease Esp1 in budding yeast. This indicates there is the possibility of Rad21’s cleavage by separase in humans. In the present study, our data indicated that Mad2 depletion induced apoptosis is tightly related to the Mad2 pathway. However, downregulation of Mad2 was thought to play an important role in tumorigenesis. Many human cancers has been reported to have low levels of MAD2, such as nasopharyngeal carcinoma, ovarian cancer and hepatocellular carcinoma [17–19]. Michel et al. [20] also reported that Mad?/- mice had a high tendency to form lung tumors

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Fig. 4 Depletion of Mad2 accompanied by Rad21 cleavage. a Melting curve and b relative mRNA levels of Rad21. c Expression of Mad2, caspase-3 and Rad21 by Western blot analysis

after long latencies. Wang et al. [21] reported that the depression of MAD2 inhibits apoptosis and increases proliferation and multi-drug resistance in gastric cancer cells. In fact, exquisite expression of Mad2 is essential for normal cell maintenance. Either up- or downregulation would play an essential part in carcinogenesis. We have provided evidence demonstrating that Mad2 can be used as a target in tumor therapy. In this scenario, initial high activity of Mad2 and subsequently low activity of Mad2 was the key point in the strategy we proposed. Our results indicated that the accumulation of Mad2 caused an increase in Rad21. Once Mad2 was depleted, Rad21 suffered cleavage, and the degradation product was transferred to the mitochondria as an apoptotic signal. The difference between the apoptosis rate of controls and that of Mad2siRNA in our study is relatively low. The main reason for the low difference may be because the depletion of Mad2 function was not complete. The RNA interference only inhibits the synthesis of Mad2, while it cannot destruct the existing Mad2. Remaining Mad2 decreased the cleaving rate of Rad21. This cause of the effect of Rad21’s apoptotic fragments was neutralized by the anti-apoptotic component inside cell. Finding a synthesized or biomaterial substance

that can effectively inhibit the Mad2’s function is a potential way to solve this problem. The accumulation of Mad2 depends on two main factors: the cell division rate and the incidence of chromosome mis-segregation. Normal cells with an average proliferative rate and little chromosome mis-segregation accumulate little Mad2 compared with many cancer cell types. Concerning the clinical potential, our strategy selectively targets dividing cells so as to protect non-dividing cells from damage, such as neurons, heart and muscles. Also, this method attacks cells with many chromosome mis-segregations, which distinguishes cancer cells from normal somatic cells. Many researchers have shown that different cancer types present different abnormal expression patterns. In osteosarcoma, gastric, colon and hepatocellular carcinomas, high levels of Mad2 are commonly seen [9, 22–24], while downregulation of Mad2 expression has also been reported in many other human cancers, such as nasopharyngeal carcinoma, ovarian cancer and hepatocellular carcinoma [17–19]. Therefore, we presume that Mad2 could be a perspective target for cancers exhibiting excessively high Mad2 expression levels.

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In conclusion, the results of this study provide evidence that Mad2 depletion causes programmed cell death and that this apoptosis is related to Rad21. We have suggested one possible strategy for combating cancer in which Mad2’s expression level is the critical element. To verify this strategy, further in vivo study is needed. Acknowledgments We thank Dr. Zan Tong (Wuhan University, Wuhan, China) for her critical reading of this manuscript. This work was supported by a grant from the Natural Science Foundation of Hubei Province, China (no. 302-131702). Conflict of interest None of the authors of this manuscript have received any type of support, benefits or funding from any commercial party related directly or indirectly to the subject of this article.

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