Apoptosis In Saos-2 Cells Research Articles

Knockdown of TRAF4 expression suppresses osteosarcoma cell growth in vitro and in vivo.

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is an adapter molecule that is overexpressed in certain cancers. TRAF4 is overexpressed in osteosarcoma tissues and osteosarcoma cells. Using the technique of RNA interference, the expression of TRAF4 in the human osteosarcoma Saos-2 cell line was shown to be downregulated. The proliferation, cell cycle arrest and apoptosis ability of Saos‑2 cells were examined, as was tumor development in a xenograft mouse model. The results showed that the TRAF4 knockdown exerts inhibitory effects on the proliferation ability of Saos-2 cells and tumor development in a xenograft mouse model. Simultaneously, it was found that TRAF4 knockdown led to cell cycle arrest in the G1 phase and promoted Saos-2 cell apoptosis. Following TNF-α treatment, the expression of nuclear factor κB was significantly reduced in the TRAF4‑small interfering RNA group. These results indicate that TRAF4 regulated osteosarcoma cell growth in vitro and in vivo, and offers a candidate molecular target for osteosarcoma prevention and therapy.

Epigenetic silencing of methyl-CpG-binding protein 2 gene affects proliferation, invasion, migration, and apoptosis of human osteosarcoma cells.

Methyl-CpG-binding protein 2 (MeCP2) is a DNA methylation-related gene of the methyl-CpG-binding protein family. Here, we investigated the epigenetic function of the MeCP2 in SaOS2 and U2OS cell lines, and explored the antitumor effects of the gene silencing for osteosarcoma. In this study, chromatin immunoprecipitation assay was used to detect MeCP2 binding activity with TSSC3 gene. RT-PCR and western blot assay were used to analyze the MeCP2 expression in osteosarcoma cell lines after transfection with LV-MECP2-RNAi. Transwell invasion and migration assays were used to detect the cell invasion and migration. The cell apoptosis was examined by using the flow cytometry assay. The tumor size was also assessed to determine the therapeutic effects of gene silencing. The results indicated that MeCP2 indicated the highest combining power with TSSC3 gene. LV-MECP2-RNAi could decrease MeCP2 level in tumor cells compared with the untreated cells (P < 0.05). LV-MECP2-RNAi inhibited the U2OS and SaOS2 cells invasion and migration compared with the control cells (P < 0.05). LV-MECP2-RNAi triggered the U2OS and SaOS2 cell apoptosis, and inhibited the cell proliferation significantly compared with the control cells (P < 0.05). The gene silencing of RNAi could also decreased the tumor size significantly compared with untreated cells (P < 0.05). In conclusion, silencing the MeCP2 gene could block the MeCP2 expression and inhibit the tumor cell migration, invasion, and proliferation, and decreases the tumor size by inducing the apoptosis of the tumor cells.

Icaritin suppresses the proliferation of human osteosarcoma cells in vitro by increasing apoptosis and decreasing MMP expression

To explore whether icaritin, a prenylflavonoid derivative of the Chinese tonic herb Epimedium, could suppress the proliferation of human osteosarcoma cells in vitro, and to elucidate the mechanisms of the action. Human osteosarcoma SaOS2 cell line was used in the present study. The proliferation of the cells was examined using MTT assay and immunofluorescence DAPI staining. Cell motility was studied with the scratch assay. Cell apoptosis was determined by Annexin V-FITC and PI double staining using flow cytometry. Western blotting and RT-PCR were used to measure the expression of mRNAs and proteins in the cells. Icaritin (5-15 μmol/L) suppressed the proliferation of SaOS2 cells in vitro in a dose-dependent manner. Furthermore, the cell motility was significantly decreased after exposure to icaritin. Moreover, icaritin (5 μmol/L) time-dependently induced the apoptosis of SaOS2 cells, markedly suppressed MMP-2 and MMP-9 expression, upregulated caspase-3 and caspase-9 expression, and increased the level of cleaved caspase-3 in the cells. Co-exposure to the caspase-3 inhibitor zVAD-fmk (10 μmol/L) compromised the icaritin-induced caspase-3 expression and apoptosis in SaOS2 cells. Icaritin suppresses the proliferation of SaOS2 human osteosarcoma cells by increasing apoptosis and downregulating MMP expression.

Mediator of RNA polymerase II transcription subunit 19 promotes osteosarcoma growth and metastasis and associates with prognosis

Osteosarcoma (OS) is the most common primary malignant tumour of bone. Nearly 30–40% of OS patients have a poor prognosis despite multimodal treatments. Because the carcinogenesis of OS remains unclear, the identification of new oncogenes that control the tumourigenesis and progression of OS is crucial for developing new therapies. Here, we found that the expression of Mediator of RNA polymerase II transcription subunit 19 (Med19) was increased in OS samples from patients compared to normal bone tissues. Cyclin D1 and cyclin B1 are upregulated in Med19 positive OS tissues. Importantly, among 97 OS patients of Enneking stage IIB or IIIB, Med19 expression was correlated with metastasis (P<0.05) and poor prognosis (P<0.01). Med19 knockdown significantly induced growth inhibition, reduced colony-forming ability and suppressed migration in the OS cell lines Saos-2 and U2OS, along with the downregulated expression of cyclin D1 and cyclin B1. Med19 knockdown also induced apoptosis in Saos-2 cells via induction of caspase-3 and poly ADP-ribose polymerase (PARP). In addition, Med19 knockdown significantly suppressed tumour growth in an OS xenograft nude mouse model via suppression of cyclin D1 and cyclin B1. Simultaneously, Med19 downregulation decreased the expression of Ki67 and proliferating cell nuclear antigen (PCNA) in tumour samples from OS xenograft nude mice. Med19 depletion remarkably reduced tumour metastasis in a model of OS metastatic spreading. Taken together, our data suggest that Med19 acts as an oncogene in OS via a possible cyclin D1/cyclin B1 modulation pathway.

Zinc finger X-chromosomal protein promotes growth and tumorigenesis in human osteosarcoma cells

To investigate the role of Zinc finger X-chromosomal protein (ZFX) in oncogenesis of Osteosarcoma tumor. Here, we first conducted an expression analysis of ZFX in Osteosarcoma cell lines. Then, we constructed ZFX-specific small interfering RNA (siRNA)-lentiviral vector that is capable of effectively inhibiting the expression of ZFX gene in human Osteosarcoma Saos-2 cells, and investigated systemically the impacts of ZFX silence on the growth and invasive ability of the cancer cells in vitro. Furthermore, we determined the effects of ZFX knockdown on the cell cycle distribution and apoptosis of Saos-2 cells. We found that ZFX inhibition resulted in significantly impaired proliferation and colony formation as well as mitigated invasiveness of Saos-2 cells. Importantly, si-ZFX infected cells exhibited a greater portion of cells at G1 phase, but a minor portion of S and G2/M phase cells. Moreover, a greater portion of sub-G1 apoptotic cells was observed in si-ZFX infected cells. These results strongly suggest that ZFX is a novel proliferation regulator that promotes growth of Osteosarcoma cells, and downregulation of ZFX expression induces growth suppression of Saos-2 cells via arrested G0/G1 phase cell cycle and apoptosis pathways, thereby indicating that ZFX may serve as a new molecular target for Osteosarcoma tumor therapy.

Roles and mechanisms of E2F-1 and PAC1 in signaling oxidative stress-induced apoptosis of Saos-2 cells

To explore the roles and mechanisms of E2F-1 and PAC1 in signaling apoptosis of Saos-2 cells under oxidative stress. siRNAs were used to construct the cell clones of expressing siE2F-1 and siPAC1. E2F-1/PAC1-mediated induction of apoptotic cell death in response to H2O2 were examined by trypan blue exclusion and the expression levels of related target factors detected by Western blot. The cell viabilities of Saos-2/siE2F-1 and Saos-2/siPAC1 increased markedly comparing with the control cells after the treatment of H2O2. And the expression level of p-ERK1/2 was higher than that of the control cells. The pathway of E2F-1/PAC1/MAPKase is a specific cascade for apoptotic signaling.

Effect of C23 down-regulation on proliferation inhibition and apoptosis induced by cisplatin in human osteosarcoma SaOS-2 cells

To investigate the effect of C23 down-regulation on proliferation inhibition and apoptosis induced by cisplatin in human osteosarcoma SaOS-2 cells. SaOS-2 cells were randomly divided in to 5 groups: a control group, a DDP group, an S+DDP group, an AS+DDP group, and an R+DDP group. Cell growth suppression of each group was analyzed by MTT assay. RT-PCR and Western blot were used to detect the expression of C23, Bcl-2 and Bax mRNA and protein in each group. Morphologic character of apoptosis was evaluated by DAPI staining. The percentage of apoptotic cells was analyzed by flowcytometer assay. C23 antisense oligonucleotides remarkably inhibited the expression of C23 mRNA and protein in SaOS-2 cells (P<0.01). The proliferation of SaOS-2 cells was remarkably inhibited in the AS+DDP group compared with the DDP group (P<0.01). The expression of C23, Bcl-2 mRNA and protein was weaker and that of Bax mRNA and protein was stronger in the AS+DDP group compared with the DDP group (P<0.01). Cisplatin-mediated apoptosis neclei in SaOS-2 cells and the rate of apoptotic cells were higher in the AS+DDP group than those of the DDP group (P<0.01). Down-regulation of C23 can contribute to cisplatin-mediated proliferation inhibition and apoptosis in SaOS-2 cells.

Inhibition of the STAT3 signaling pathway is involved in the antitumor activity of cepharanthine in SaOS2 cells

To investigate the molecular mechanisms underlying the antitumor activity of cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata. Human osteosarcoma cell line SaOS2 was used. MTT assay, Hoechst 33342 nuclear staining, flow cytometry, Western blotting and nude mouse xenografts of SaOS2 cells were applied to examine the antitumor activity of CEP in vitro and in vivo. The expression levels of STAT3 and its downstream signaling molecules were measured with Western blotting and immunochemistry analysis. The activity of STAT3 was detected based on the phosphorylation level of STAT3, luciferase gene reporter assay and translocation of STAT3 to the nucleus. Treatment of SaOS2 cells with CEP (2.5-20 μmol/L) inhibited the cell growth in a concentration- and time-dependent manner. CEP (10 μmol/L) caused cell cycle arrest at G(1) phase and induced apoptosis of SaOS2 cells. CEP (10 and 15 μmol/L) significantly decreased the expression of STAT3 in SaOS2 cells. Furthermore, CEP (5 and 10 μmol/L) significantly inhibited the expression of target genes of STAT3, including the anti-apoptotic gene Bcl-xL and the cell cycle regulators c-Myc and cyclin D1. In nude mouse xenografts of SaOS2 cells, CEP (20 mg·kg(-1)·d(-1), ip for 19 d) significantly reduced the volume and weight of the tumor. Our findings suggest that inhibition of STAT3 signaling pathway is involved in the anti-tumor activity of CEP.

Perturbation of 14q32 miRNAs-cMYC gene network in osteosarcoma

Osteosarcoma (OS) is the common histological form of primary bone cancer and one of the leading aggressive cancers in children under age fifteen. Although several genetic predisposing conditions have been associated with OS the understanding of its molecular etiology is limited. Here, we show that microRNAs (miRNAs) at the chr.14q32 locus are significantly downregulated in osteosarcoma compared to normal bone tissues. Bioinformatic predictions identified that a subset of 14q32 miRNAs (miR-382, miR-369-3p, miR-544 and miR-134) could potentially target cMYC transcript. The physical interaction between these 14q32 miRNAs and cMYC was validated using reporter assays. Further, restoring expression of these four 14q32 miRNAs decreased cMYC levels and induced apoptosis in Saos2 cells. We also show that exogenous expression of 14q32 miRNAs in Saos2 cells significantly downregulated miR-17-92, a transcriptional target of cMYC. The pro-apoptotic effect of 14q32 miRNAs in Saos2 cells was rescued either by overexpression of cMYC cDNA without the 3'UTR or with miR-17-92 cluster. Further, array comparative genomic hybridization studies showed no DNA copy number changes at 14q32 locus in OS patient samples suggesting that downregulation of 14q32 miRNAs are not due to deletion at this locus. Together, our data support a model where the deregulation of a network involving 14q32 miRNAs, cMYC and miR-17-92 miRNAs could contribute to osteosarcoma pathogenesis.

Effects of thymoquinone on growth and apoptosis of osteosarcoma SaOS-2 cell line in vitro

Objective To investigate the inhibitory effect of thymoquinone on the growth of human osteosarcoma cell line SaOS-2 in vitro.Methods After human osteosarcoma SaOS-2 cells were treated with different concentrations of thymoquinone, the proliferation was measured by Cell Counting Kit-8 (CCK-8) assay. The flowcytometry (FCM) was used to determine apoptosis of SaOS-2 cells. The morphological changes of SaOS-2 cells were observed under the fluorescence microscopy after DAPI staining. Western blotting was used to detect the protein expression of Caspase-3, X-linked inhibitor of apoptosis protein (XIAP) and Smac.Results Thymoquinone obviously suppressed proliferation of SaOS-2 cells in a dose-dependent manner, and the apoptosis rate was (8.1±0.7)%, (13.2±1.1)% and (20.2±1.7)% respectively when the concentrations of thymoquinone exposed were 20, 40 and 80 μmol/L. After treatment with thymoquinone, the expression of Caspase-3 and Smac was up-regulated in SaOS-2 cells, and thymoquinone treatment significantly decreased the XIAP levels in SaOS-2 cells.Conclusion Thymoquinone has the anti-tumor effect on the human osteosarcoma SaOS-2 cells in vitroprobably by up-regulating the expression of Caspase-3 and Smac and down-regulating the expression of XIAP. Key words: Thymoquinone; Osteosarcoma; Apoptosis

Abstract 4836: RB/E2F1 in the crossroad of autophagy and apoptosis

Abstract Autophagy is a protective mechanism that renders cells viable in stressful conditions. Mounting evidence suggests that this cellular process is also a tumor suppressor pathway. We hypothesized that retinoblastoma protein (RB), a key tumor suppressor, induces autophagy. To test this hypothesis, we first transduced RB into RB-defective human cancer cells: sarcoma osteogenic (Saos-2) cells, hepatoma (Hep3B) cells, and brain tumor stem cells (MDNSC23). The ectopic RB induced autophagy as demonstrated by significantly increased percentage of cells with acidic vesicular organelles (from less than 10% to more than 30%, P &amp;lt; 0.05), and the lipidation of LC3-I leading to the formation of autophagosomes/autolysosomes that were visualized by EGFP-LC3 punctation (from less than 20% to more than 80%, P = 0.003). These double-membraned vesicles were clearly shown by ultrastructural study via transmission electron microscopy. However, when Beclin 1 was silenced with siRNA in Saos-2 cells, the RB-mediated autophagy was blocked. In addition, study of autophagy flux with Saos-2 cells expressing double-tagged mRFP-EGFP-LC3 fusion protein revealed that RB stimulated the formation and maturation of autophagosomes. Consistently, RB activators p16INK4a and p27/kip1 caused autophagy in an RB-dependent manner. Importantly, we found RB mutants Delta-22 and R661W deficient for binding E2F failed to induce autophagy. Moreover, E2F1 overexpression antagonized RB-mediated autophagy, leading to apoptosis in Saos-2 cells. In agreement with the above observations, silencing E2F1 with siRNA resulted in autophagy in U-87 MG cells; and autophagy levels increased from 4% in wild-type MEFs to 40% in E2F1 knockout MEFs (P = 0.002). Collectively, our data reveal that RB/E2F1 plays a key role in the decision of a cell to undergo autophagy or apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4836.

RUNX2 regulates the effects of TNFα on proliferation and apoptosis in SaOs-2 cells

The runt-related transcriptional factor RUNX2 is an essential mediator of the osteoblast phenotype and plays a pivotal role in the process of osteoblast differentiation. The involvement of RUNX2 includes the regulation of genes that are important in committing cells to the osteoblast lineage. Increasing evidences are consistent with a requirement of RUNX2 for stringent control of osteoblast proliferation and recent data even suggested that RUNX2 might act as a proapoptotic factor. Among the cytokines described as modulators of osteoblast functions, TNFα affects both apoptosis and the differentiation rate from mesenchymal precursor cells of osteoblast. Thus we evaluated on the human osteosarcoma cell line SaOs-2 stably transfected with a RUNX2 dominant negative construct (ΔRUNX2) the effects of serum and TNFα on proliferation and apoptosis. In this study we showed that SaOs-2 clones expressing high levels of ΔRUNX2 presented a higher proliferation rate than clones transfected with an empty vector. This increase in cell growth was accompanied by a rise in cyclins A1, B1 and E1 expression and a decrease in the cyclin inhibitor p21. Moreover we observed that the expression of the RUNX2 transgene protected the SaOs-2 cells from the antiproliferative and the apoptotic effects induced by TNFα. This was accompanied by the inhibition of Bax and activation of Bcl2 expression. Experiments done on SaOs-2 cells transiently transfected with siRNA confirmed that RUNX2 represents a critical link between cell fate, proliferation and growth control. This study also suggested that RUNX2 might control osteoblastic growth depending on the differentiation stage of the cells by regulating expression of elements involved in hormones and cytokines sensitivity.

Effect of human granulocyte macrophage-colony stimulating factor on differentiation and apoptosis of the human osteosarcoma cell line SaOS-2

We investigated the effects of human granulocyte macrophage-colony stimulating factor (GM-CSF) on the relation between differentiation and apoptosis in SaOS-2 cells, an osteoblast-like cell line. To determine the relationship between these cellular processes, SaOS-2 cells were treated in vitro for 1, 7 and 14 days with 200 ng/mL GM-CSF and compared with untreated cells. Five nM insulin-like growth factor (IGF-I) and 30 nM okadaic acid were used as negative and positive controls of apoptosis, respectively. Effects on cell differentiation were determined by ECM (extracellular matrix) mineralization, morphology of some typical mature osteoblast differentiation markers, such as osteopontin and sialoprotein II (BSP-II), and production of bone ECM components such as collagen I. The results showed that treatment with GM-CSF caused cell differentiation accompanied by increased production of osteopontin and BSP-II, together with increased ECM deposition and mineralization. Flow cytometric analysis of annexin V and propidium iodide incorporation showed that GM-CSF up-regulated apoptotic cell death of SaOS-2 cells after 14 days of culture in contrast to okadaic acid, which stimulated SaOS-2 apoptosis only during the early period of culture. Endonucleolytic cleavage of genomic DNA, detected by "Aúladdering analysis"Aù, confirmed these data. The results suggest that GM-CSF induces osteoblastic differentiation and long-term apoptotic cell death of the SaOS-2 human osteosarcoma cell line, which in turn suggests a possible in vivo physiological role for GM-CSF on human osteoblast cells.

Antiproliferative and proapoptotic activities of new pyrazolo[3,4‐d]pyrimidine derivative Src kinase inhibitors in human osteosarcoma cells

Osteosarcoma is the most frequent primitive malignant tumor of the skeletal system, characterized by an extremely aggressive clinical course that still lacks an effective treatment. Src kinase seems to be involved in the osteosarcoma malignant phenotype. We show that the treatment of human osteosarcoma cell lines with a new pyrazolo[3,4-d]pyrimidine derivative Src inhibitor, namely SI-83, impaired cell viability, with a half-maximal inhibitory concentration of 12 microM in nonstarved cells and a kinetic different from that known for the Src inhibitor PP2. Analysis by terminal deoxynucleotidyl transferase-mediated nick end labeling, Hoechst, and flow cytometric assay showed that SI-83 induced apoptosis in SaOS-2 cells. Moreover, SI-83, by inhibiting Src phosphorylation, decreased in vivo osteosarcoma tumor mass in a mouse model. Finally, SI-83 showed selectivity for osteosarcoma, since it had a far lower effect in primary human osteoblasts. These results show that human osteosarcoma had Src-dependent proliferation and that modulation of Src activity may be a therapeutic target of this new compound with low toxicity for nonneoplastic cells.

Enhanced Deacetylation of p53 by the Anti-apoptotic Protein HSCO in Association with Histone Deacetylase 1

HSCO (hepatoma subtracted-cDNA library clone one, also called ETHE1) was originally identified by its frequent overexpression in hepatocellular carcinomas. HSCO inhibits function of NF-kappaB by binding to RelA and accelerating its export from the nucleus. We show here that HSCO exhibits anti-apoptotic activity in cells exposed to DNA-damaging agents by suppressing transcriptional activity of p53. Induction of pro-apoptotic genes, Noxa, Perp, PIG3, and Bax were suppressed in cells over-expressing HSCO. By increasing ubiquitylation and degradation of p53, HSCO reduces p53 protein levels. HSCO specifically associates with histone deacetylase 1 (HDAC1) independently of Mdm2 and facilitates deacetylation of p53 at Lys-373/382 by HDAC1. The metallo-beta-lactamase family consensus sequence in HSCO is important for its effect on p53 deacetylation. Co-immunoprecipitation and immunofluorescence studies suggested that HSCO, HDAC1, and p53 form a complex in the nucleus. Thus, HSCO is a cofactor that increases the deacetylase activity of HDAC1 toward p53, leading to suppression of apoptosis. Treatment of hepatocellular carcinomas that retain wild-type p53 and overexpress HSCO with anti-HSCO agents might re-establish the p53 response and revert chemoresistance.

Cadmium Induces Apoptosis in the Human Osteoblast-like Cell Line Saos-2

Human exposure to the heavy metal cadmium has been associated with the development of bone diseases, including osteoporosis and osteomalacia. The mechanisms by which cadmium exerts a direct effect on bone remain unclear. Bone cells go through apoptosis for proper bone remodeling; therefore, it was hypothesized that cadmium disrupts this normal balance by inducing apoptosis. Human osteoblast-like cells (Saos-2) were treated with 10–200 μM cadmium chloride (CdCl2) and evaluated by trypan blue staining and phase-contrast microscopy. Exposure to CdCl2 resulted in decreased cell viability and changes in cell morphology characteristic of apoptosis. The role of apoptosis in cadmium-induced toxicity was further evaluated using the fluorescent marker annexin V, which detects externalization of cell membrane phosphatidylserine. Nuclear changes associated with apoptosis were assessed by Hoechst staining and a DNA fragmentation assay. A significant increase in annexin V-positive cells was observed following CdCl2 treatment. Nuclear changes associated with apoptosis, including marginalization and condensing of chromatin and DNA fragmentation, were also observed following CdCl2 treatment. Cadmium-induced apoptosis in Saos-2 cells was also accompanied by an increase in caspase-3 activity. The addition of the caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) or the known cadmium chelating agent potassium bis(2-hydroxyethy)dithiocarbamate, (K[bhedtc]), blocked caspase-3 activation induced by cadmium. Collectively, this study has identified a role for apoptosis in cadmium-induced toxicity in bone cells, and provides insight for future studies on mechanisms underlying the disruption of apoptotic signaling cascades in bone and the relationship to bone disease.

Green tea polyphenols-induced apoptosis in human osteosarcoma SAOS-2 cells involves a caspase-dependent mechanism with downregulation of nuclear factor-κB

Development of chemotherapy resistance and evasion from apoptosis in osteosarcoma, a primary malignant bone tumor, is often correlated with constitutive nuclear factor-kappaB (NF-kappaB) activation. Here, we investigated the ability of a polyphenolic fraction of green tea (GTP) that has been shown to have antitumor effects on various malignant cell lines to inhibit growth and induce apoptosis in human osteosarcoma SAOS-2 cells. Treatment of SAOS-2 cells with GTP (20-60 microg/ml) resulted in reduced cell proliferation and induction of apoptosis, which correlated with decreased nuclear DNA binding of NF-kappaB/p65 and lowering of NF-kappaB/p65 and p50 levels in the cytoplasm and nucleus. GTP treatment of cells reduced IkappaB-alpha phosphorylation but had no effect on its protein expression. Furthermore, GTP treatment resulted in the inhibition of IKK-alpha and IKK-beta, the upstream kinases that phosphorylate IkappaB-alpha. The increase in apoptosis in SAOS-2 cells was accompanied with decrease in the protein expression of Bcl-2 and concomitant increase in the levels of Bax. GTP treatment of SAOS-2 cells also resulted in significant activation of caspases as was evident by increased levels of cleaved caspase-3 and caspase-8 in these cells. Treatment of SAOS-2 cells with a specific caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) and general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) rescued SAOS-2 cells from GTP-induced apoptosis. Taken together, these results indicate that GTP is a candidate therapeutic for osteosarcoma that mediates its antiproliferative and apoptotic effects via activation of caspases and inhibition of NF-kappaB.

Increased radiation‐induced apoptosis of Saos2 cells via inhibition of NFκB: A role for c‐Jun N‐terminal kinase

To elucidate the possible effect of NFkappaB on radioresistance, we used the osteosarcoma cell line Saos2, stably expressing the NFkappaB constitutive inhibitor, mIkappaB (Saos2-mIkappaB) or stably transfected with the empty vector (Saos2-EV). Ionizing radiation induced "intrinsic" apoptosis in Saos2-mIkappaB cells but not in Saos2-EV control cells, with intact NFkappaB activity. We find as expected, that this NFkappaB activity was enhanced following irradiation in the Saos2-EV control cells. On the other hand, inhibition of NFkappaB signaling in Saos2-mIkappaB cells led to the upregulation of the pro-apoptotic systems, such as Bax protein and c-Jun N-terminal Kinase (JNK)/c-Jun/AP1 signaling. Inhibition of NFkappaB resulted in decreased expression of the DNA damage protein GADD45beta, a known inhibitor of JNK. Subsequently, JNK activation of c-Jun/AP-1 proteins increased radiation-induced apoptosis in these mutants. Radiation-induced apoptosis in Saos2-mIkappaB cells was inhibited by the JNK specific inhibitor SP600125 as well as by Bcl-2 over-expression. Furthermore, release of cytochrome-c from mitochondria was increased and caspase-9 and -3 were activated following irradiation in Saos2-mIkappaB cells. Antisense inhibition of GADD45beta in Saos2-EV cells significantly enhanced apoptosis following irradiation. Our results demonstrate that radioresistance of Saos2 osteosarcoma cells is due to NFkappaB-mediated inhibition of JNK. Our study brings new insight into the mechanisms underlying radiation-induced apoptosis of osteosarcoma, and may lead to development of new therapeutic strategies against osteosarcoma.

Lack of Correlation between p53-Dependent Transcriptional Activity and the Ability to Induce Apoptosis among 179 Mutant p53s

Tumor suppressor p53-dependent apoptosis is thought to be one of the most important tumor-suppressive functions in human tumorigenesis. However, whether the major mechanism underlying the p53-dependent apoptosis is transactivation dependent or independent remains unclear. Using 179 mutant p53s with diverse transcriptional activities for distinct p53-binding sequences in yeast, we evaluated both their sequence-specific transcriptional activities on six p53 target genes and their ability to induce apoptosis in Saos-2 cells. These mutant p53s also represented diversity in their ability to both transactivate target genes and induce apoptosis. We identified 17 mutant p53s with superior ability to induce apoptosis than wild-type p53 that tend to cluster at residues 121 or 290 to 292. There was no significant correlation between the two functional properties on any single target gene examined. Furthermore, the 17 mutant p53s were not classified in a specific cluster by hierarchical cluster analysis on their diverse transcriptional activities, indicating that these mutant p53s were not similar in the transcriptional activity of downstream genes. These results suggested that transactivation-dependent apoptosis does not always play a major role in p53-dependent apoptosis, indirectly supporting the importance role of the transactivation-independent mechanism.

Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins.

The adenovirus E4orf6 is a viral oncoprotein known to cooperate with the E1A gene product in transforming primary murine cells. It has been shown to inhibit the apoptotic activities of p53 and p73 through direct binding to these proteins. Here, we demonstrate that the adenovirus E4orf6 protein inhibits apoptosis mediated by BNIP3 and Bik, which are BH3-only proteins of the Bcl-2 family. This activity was not mediated by p53 and p73 because E4orf6 had the same effect on the apoptosis in Saos-2 cells that do not express p53-related genes. It was also ascertained that E4orf6 could change the mitochondrial localization of BNIP3 and Bik. A mutant lacking the nuclear export signal of E4orf6 failed to inhibit apoptosis and to translocate BNIP3 protein from the mitochondria. Moreover, it was also established that E4orf6 was able to interact with BNIP3 and Bik. In BNIP3 protein, the region required for the interaction included the transmembrane domain, which is required for the localization of BNIP3 to the mitochondria. These results suggest that E4orf6 is exported from the nucleus to the cytoplasm, enabling it to interact with BH3-only proteins, eventually leading to the inhibition of apoptotic activity.