Apoptosis In Saos-2 Cells Research Articles

Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis

Doxorubicin (DOX) is a common anticancer drug. The mechanisms of DOX induced apoptosis and the involvement of reactive oxygen species (ROS) in apoptotic signaling were investigated in p53-null human osteosarcoma Saos-2 cells. Accumulation of pre-G1 phase cells and induction of DNA laddering, which are the hallmarks of apoptosis, were detected in cells at 48 h upon DOX treatment. Furthermore, DOX increased the intracellular hydrogen peroxide and superoxide levels, followed by mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, prior to DNA laddering in Saos-2 cells. In addition, DOX treatment also upregulated Bax and downregulated Bcl-2 levels in the cells. The role of ROS in DOX induced cell death was confirmed by the suppression effect of catalase on DOX induced ROS formation, mitochondrial cytochrome c release, procaspase-3 cleavage, and apoptosis in Saos-2 cells. The catalase treatment however only suppressed DOX induced Bax upregulation but had no effect on Bcl-2 downregulation. Results from the present study suggested that ROS might act as the signal molecules for DOX induced cell death and the process is still functional even in the absence of p53.

Oncogenes Induce and Activate Endogenous p73 Protein

The identification of upstream pathways that signal to TP73 is crucial for understanding the biological role of this gene. Since some evidence suggests that TP73 might play a role in tumorigenesis, we asked whether oncogenes can induce and activate endogenous TP73. Here, we show that endogenous p73 alpha and beta proteins are up-regulated in p53-deficient tumor cells in response to overexpressed E2F1, c-Myc, and E1A. E2F1, c-Myc, and E1A-mediated p73 up-regulation leads to activation of the p73 transcription function, as shown by p73-responsive reporter activity and by induction of known endogenous p73 target gene products such as p21 and HDM2. Importantly, E2F1-, c-Myc-, and E1A-mediated activation of endogenous p73 induces apoptosis in SaOs-2 cells. Conversely, inactivation of p73 by a dominant negative p73 inhibitor (p73DD), but not by a mutant p73DD, inhibits oncogene-induced apoptosis. These data show that oncogenes can signal to TP73 in vivo. Moreover, in the absence of p53, oncogenes may enlist p73 to induce apoptosis in tumor cells.

Inhibitors of protein synthesis and RNA synthesis protect against okadaic acid-induced apoptosis in human osteosarcoma cell line MG63 cells but not in Saos-2 cells.

In a previous study, we demonstrated that the protein phosphatase inhibitors, okadaic acid and calyculin A, induced apoptosis in human osteosarcoma cell lines, Saos-2 and MG63 cells. In the present study, to determine if new gene transcription and protein synthesis are required for okadaic acid-induced apoptosis in Saos-2 and MG63 cells, the cells were treated for 48h with varying concentrations of the inhibitors of protein or RNA synthesis, i.e., cycloheximide, actinomycin D, and puromycin, in the presence of a fixed dose of okadaic acid. All these reagents in different concentrations prevented the okadaic acid-induced apoptosis in MG63 cells in a dose-dependent fashion. The same concentrations of cycloheximide, actinomycin D, or puromycin alone did not induce any apoptotic features in MG63 cells. However, not all the aforementioned reagents affected okadaic acid-induced apoptosis in Saos-2 cells. Okadaic acid-induced and cycloheximide-prevented apoptosis was shown by phase-contrast microscopy, WST-1 assay, direct visualization of nuclear condensation and fragmentation of chromatin, and the characteristic DNA ladder formation on agarose gel electrophoresis. The present results indicate that the induction of new cell death genes and ongoing protein synthesis may have a role in okadaic acid-induced apoptosis in MG63 cells and that such proteins are not required in Saos-2 cells.

Paclitaxel Induces Apoptosis in Saos-2 Cells with CD95L Upregulation and Bcl-2 Phosphorylation

We examined the effect of paclitaxel on human osteoblastic cells Saos-2 to determine if paclitaxel can affect proliferation and apoptosis. We used a p53-negative cell line in order to mimic the loss of function frequently observed at the clinical level. Paclitaxel induced cell death in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were observed by Hoechst 33258 stain, DNA ladder formation, electron microscopy, and flow cytometry at concentrations as low as 100 nM, a concentration which can be achieved by infusion in human plasma. At 100 nM, paclitaxel induced a G2 arrest at 8 h of treatment. The cells then continued to accumulate in G2 until 72 h when the percentage of apoptotic events reached 54%. At the molecular level, Bcl-2 protein was phosphorylated at 16 h and PARP protein was cleaved, indicating the activation of caspase-3-like proteases. Caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK rescued Saos-2 cells from paclitaxel-induced apoptosis. CD95 expression was constantly high, while CD95L showed a threefold increase in expression. This suggests that, following the G2 arrest, apoptosis is induced through the CD95/CD95L system.

Prilocaine induces apoptosis in osteoblastic cells.

To determine whether prilocaine, a local anesthetic, induces apoptosis in osteoblastic cells. After reaching subconfluence, human osteoblastic Saos-2 and MG63 cells and mouse osteoblastic MC3T3-E1 cells were exposed for 48 hr to varying concentrations of prilocaine up to 10 mM and the cytotoxicity of the cells was analyzed by phase-contrast microscopy and WST-1 assay. Saos-2 cells treated for 48 hr with 5 mM prilocaine were stained with Hoechst 33342 and nuclear fragmentation was examined under a fluorescence microscope. DNA was extracted from the cells treated with 5 mM prilocaine and DNA ladder formation (a hallmark of apoptosis) was analyzed by agarose gel electrophoresis. Prilocaine induced cell death in Saos-2 cells in a dose- and time-dependent manner up to the concentration of 10 mM. Marked nuclear condensation and fragmentation of chromatin were observed in the prilocaine-treated cells. DNA ladder formation also was induced by prilocaine treatment. Prilocaine-induced DNA ladder formation was dose-dependent with maximal effect at a concentration of 5 mM and was time-dependent from 12 to 48 hr. DNA ladder formation was also induced by prilocaine treatment in human osteoblastic MG63 cells and mouse osteoblastic MC3T3-E1 cells. Cycloheximide prevented prilocaine-induced apoptosis in Saos-2 cells in a dose-dependent fashion up to 20 microM as determined by WST-1 assay and DNA ladder formation in agarose gel electrophoresis. Osteoblastic cells treated with prilocaine exhibit both morphological and biochemical features indicative of apoptosis. The apoptotic mechanisms involve transcriptional regulation of specific proteins or protein synthesis.

プリロカインによる培養骨肉腫細胞のアポトーシス誘導

Local anesthetics cause cytotoxicity to various types of mammalian cells. To determine whether local anesthetics can induce apoptosis in osteoblastic cells, the effects of prilocaine were examined on cultured human osteoblastic cell lines, Saos-2 and MG63 cells, and mouse MC3T3-E1 cells. The cells in confluence were exposed to varying concentrations of prilocaine. In Saos-2 cells, prilocaine induced cell damage in a dose- and time-dependent manners up to the concentration of 10mM as determined by phase-contrast microscopy and WST-1 assay. Marked nuclear condensation and fragmentation of chromatin were observed in the prilocaine-treated cells by using Hoechst 33342 staining. Prilocaine-induced DNA ladder formation in agarose gel electrophoresis was dose-dependent with maximal effect at a concentration of 5mM and was time-dependent from 12 h to 48 h. Prilocaine induced also DNA ladder formation in both human osteoblastic MG63 cells and mouse osteoblastic MC3T3-E1 cells. Other local anesthetics, including dibcaine, tetracaine, lidocaine, and procaine, also induced DNA ladder formation in Saos-2 cells. Cycloheximide and Actinomycin D prevented prilocaine-induced apoptosis in Saos-2 cells in a dose-dependent fashion up to 10μM and 40nM, respectively, as determined by WST-1 assay and DNA ladder formation. The present results showed that prilocaine and other local anesthetics caused both morphological and/or biochemical features indicative of apoptosis in osteoblastic cells, and that the synthesis of specific proteins would be involved in prilocaine-induced apoptosis.

Anticancer Quinones Induce pRb-Preventable G 2/M Cell Cycle Arrest and Apoptosis

Reactive oxygen species generated during the metabolism of the antitumor quinone 3,6-diaziridinyl-1,4-benzoquinone (DZQ) in human colonic carcinoma HCT116 cells lead to the induction of p21 (WAF1, Cip1, or sdi1), an upstream regulator of the retinoblastoma gene product pRb involved G 1 cell cycle control. We here demonstrate that the cell cycle was arrested in G 2/M phase following supplementation with DZQ of human osteosarcoma Saos-2 cells (lacking both p53 and pRb) and HCT116 cells. DZQ also induced p21 and apoptosis in Saos-2 cells. The transfection of the Rb gene into Saos-2 cells did not alter the level of p21 induction, but changed cell cycle arrest into G 1 phase and prevented apoptosis. These findings suggest that quinones may lead to a p53-independent and pRb-preventable G 2/M arrest and apoptosis, which correlate with p21 induction.