Antiproliferative activity of rosamultic acid is associated with induction of apoptosis,cell cycle arrest,inhibition of cell migration and caspase activation in human gastric cancer (SGC-7901) cells

BackgroundGastric cancer is the second leading cause of cancer related deaths after lung cancer globally. Among natural products, natural triterpenes represent a structurally diverse group of organic compounds with potent antitumor activity.PurposeThe objective of the present research work demonstrated the antiproliferative and apoptotic activity of rosamultic acid, a natural triterpenoid, in human gastric cancer (SGC-7901) cells. Its effect on cellular morphology, cell cycle arrest, DNA fragmentation and expression levels of caspase-3, caspase-8 and caspase-9 were also determined.MethodsAntiproliferative activity of rosamultic acid was evaluated by MTT assay. Phase contrast, fluorescence microscopy as well as flow cytometry using Hoechst 33342, acridine orange/ethidium bromide and Annexin V-FITC as cellular probes were used to evaluate the induction of apoptosis by rosamultic acid. Protein level expressions were analyzed by western blot analysis.ResultsThe results revealed that rosamultic acid induced dose-dependent as well as time dependent cytotoxic effects in SGC-7901 gastric cancer cells. It also led to a reduction in clonogenic activity along with inhibiting the cell migration. Characteristic features of apoptosis induced by rosamultic acid were observed and quantified. Cell cycle arrest at sub-G1 phase was induced by rosamultic acid along with downregulation of expression levels of CDK4, CDK6 and cyclin D1. Rosamultic acid also significantly led to the activation of caspase-3, -8 and -9 during the 48 h treatment along with cleaving PARP in a dose-dependent manner. DNA fragmentation following rosamultic acid treatment was also observed in these cells.ConclusionThe current study strongly reveals that rosamultic acid inhibits gastric cancer proliferation by inducing apoptosis mediated through cell cycle arrest, downregulation of cell cycle related protein expressions, inhibition of cell migration, DNA damage, and activation of caspases.     

Geraniol inhibits prostate cancer growth by targeting cell cycle and apoptosis pathways

The progression of prostate cancer is associated with escape from cell cycle arrest and apoptosis under androgen-depleted conditions. Here, we found that geraniol, a naturally occurring monoterpene, induces cell cycle arrest and apoptosis in cultured cells and tumor grafted mice using PC-3 prostate cancer cells. Geraniol modulated the expression of various cell cycle regulators and Bcl-2 family proteins in PC-3 cells in vitro and in vivo. Furthermore, we showed that the combination of sub-optimal doses of geraniol and docetaxel noticeably suppresses prostate cancer growth in cultured cells and tumor xenograft mice. Therefore, our findings provide insight into unraveling the mechanisms underlying escape from cell cycle arrest and apoptosis and developing therapeutic strategies against prostate cancer.     

Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells

BackgroundPrevious studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown.MethodsWe generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics.ResultsPC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP.ConclusionsSuppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells.General significanceOur results highlight the possibility of the use of PC as an anti-cancer drug target.     

Geranium thunbergii extract-induced G1 phase cell cycle arrest and apoptosis in gastric cancer cells

ABSTRACT

Geranium thunbergii is a traditional East Asian medicine for stomach diseases including dysentery and stomach ulcers in East Asia and has been reported to possess biological activity. The benefits of G. thunbergii in gastric cancer are unknown. In this study, we demonstrate that G. thunbergii extract suppresses proliferation and induces death and G1/S cell cycle arrest of gastric cancer cells. Proliferation was significantly inhibited in a time- and dose-dependent manner. Cell cycle arrest was associated with significant decreases in CDK4/cyclinD1 complex and CDK2/cyclinE complex genes expression. In addition, the protein expression of caspase-3 was decreased and that of activated poly (ADP-ribose) polymerase (PARP) was increased, which indicated apoptosis. The expressions of the Bax and Bcl-2, which are apoptosis related proteins, were upregulated and down-regulated, respectively. The results indicate that G. thunbergii extract can inhibit proliferation and induce both G/S cell cycle arrest and apoptosis of gastric cancer cells. Also, the induction of apoptosis involved the intrinsic pathways of the cells. Take the results, we suggest that G. thunbergii extract has anti-gastric cancer activity and may be a potential therapeutic candidate for gastric cancer.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

A novel indoloquinoline derivative, IQDMA, induces S-phase arrest and apoptosis in promyelocytic leukemia HL-60 cells

N′-(11H-indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine (IQDMA), an indoloquinoline compound, was identified in our laboratory as a novel antineoplastic agent with a broad spectrum of antitumor activity against many human cancer cells. Cell cycle analysis showed S-phase arrest and induction of apoptosis in HL-60 cells following 24 h exposure to IQDMA. Analysis of the cell cycle regulatory proteins demonstrated that IQDMA did not change the steady-state levels of cyclin B1, cyclin D3, and p21, but decreased the protein levels of Cdk1, Cdk2, and cyclin A. IQDMA also caused a marked increase in apoptosis, which was accompanied by increased levels of Bax, activated caspase-3, -8, and -9, and cleaved PARP. These molecular alterations provide an insight into IQDMA-caused growth inhibition, S-phase arrest, and apoptotic death of HL-60 cells.     

The p75(NTR) tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

The p75 neurotrophin receptor (p75(NTR)) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75(NTR) retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (DeltaDD) dominant-negative antagonist of p75(NTR) showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75(NTR)-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75(NTR) expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75(NTR) rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75(NTR) was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75(NTR)-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75(NTR) expressing prostate cancer cells.     

A methylene chloride fraction of Saururus chinensis induces apoptosis through the activation of caspase-3 in prostate and breast cancer cells

The aerial parts of Saururus chinensis (SC) have been used for the treatment of edema, fever, jaundice, and inflammatory diseases in Korean folk medicine for centuries. However, the mechanism by which SC exerts these anti-tumorigenic activities in human prostate and breast cancer cells has not yet been fully understood. In this study, we report on the methylene chloride fraction from SC exerting cytotoxicity against prostate and breast cancer cells in a dose-dependent manner. Specifically, SC exerted the most potent cytotoxicity in LNCaP and MCF-7 cells. SC was shown to down-regulate various angiogenetic (VEGF), proliferative (Cyclin D₁), anti-apoptotic (Bcl-2) gene products in these cells. SC also increased the number of annexin V-positive apoptotic bodies and the sub-G1 DNA contents of the cell cycle undergoing apoptosis through caspase-3 activation in both LNCaP and MCF-7 cells. We further confirmed that caspase-3 plays an important role in SC-induced apoptosis in LNCaP and MCF-7 cells through the use of the caspase-3 inhibitor. Moreover, we observed that SC potentiated paclitaxel-induced apoptosis in MCF-7 cells and sauchinone is a major active constituent of SC, which could induce apoptosis in the cells. Taken together, our data provide the evidence that SC induces apoptosis depending on caspase-3 activation and overcomes the natural biological resistance to chemotherapy found in human prostate and breast cancer cells.     

Zebrafish embryo proteins induce apoptosis in human colon cancer cells (Caco2)

Previous studies have shown that proteins extracted from Zebrafish embryo share some cytostatic characteristics in cancer cells. Our study was conducted to ascertain the biological properties of this protein network. Cancer cell growth and apoptosis were studied in Caco2 cells treated with embryonic extracts. Cell proliferation was significantly inhibited in a dose-dependent manner. Cell-cycle analysis in treated cells revealed a marked accumulation in the G₂/M phase preceding induction of apoptosis. Embryo proteins induced a significant reduction in FLIP levels, and increased caspase-3 and caspase-8 activity as well as the apoptotic rate. Increased phosphorylated pRb values were obtained in treated Caco2 cells: the modified balance in pRb phosphorylation was associated with an increase in E2F1 values and c-Myc over-expression. Our data support previous reports of an apoptotic enhancing effect displayed by embryo extracts, mainly through the pRb/E2F1 apoptotic pathway, which thus suggests that Zebrafish embryo proteins have complex anti-cancer properties.     

Human immunodeficiency virus type 1 Vpr induces cell cycle arrest at the G(1) phase and apoptosis via disruption of mitochondrial function in rodent cells

Vpr of human immunodeficiency virus type 1 causes cell cycle arrest at the G(2)/M phase and induces apoptosis after G(2)/M arrest in primate cells. We have reported previously that Vpr also induces apoptosis independently of G(2)/M arrest in human HeLa cells. By contrast, Vpr does not induce G(2)/M arrest in rodent cells, but it retards cell growth. To clarify the relationship between cell cycle arrest and apoptosis, we expressed Vpr endogenously in rodent cells and investigated cell cycle profiles and apoptosis. We show here that Vpr induces cell cycle arrest at the G(1) phase and apoptosis in rodent cells. Vpr increased the activity of caspase-3 and caspase-9, but not of caspase-8. Moreover, Vpr-induced apoptosis could be inhibited by inhibitors of caspase-3 and caspase-9, but not by inhibitor of caspase-8. We also showed that Vpr induces the release of cytochrome c from mitochondria into the cytosol and disrupts the mitochondrial transmembrane potential. Finally, we showed that apoptosis occurred in HeLa cells through an identical pathway. These results suggest that disruption of mitochondrial functions by Vpr induces apoptosis via cell cycle arrest at G(1), but that apoptosis is independent of G(2)/M arrest. Furthermore, it appears that Vpr acts species-specifically with respect to induction of cell cycle arrest but not of apoptosis.     

Ganoderic acid Mf and S induce mitochondria mediated apoptosis in human cervical carcinoma HeLa cells

In this work, the effects of a pair of positional isomer of ganoderic acids (GAs), namely ganoderic acid Mf (GA-Mf) and ganoderic acid S (GA-S) purified from the fermented mycelia of Ganoderma lucidum, on induction of cell apoptosis and the apoptotic pathway in HeLa cells were investigated. The results demonstrate that both isomers decreased cell population growth on various human carcinoma cell lines by MTT assay, while GA-Mf had better selectivity between normal and cancer cells. The flow cytometry analysis indicated that treatment of HeLa cells with GA-S caused cell cycle arrest in the S phase, while GA-Mf caused cell cycle arrest in the G1 phase. Compared with GA-S, GA-Mf had more potent increase in the number of early and late apoptotic cells. Treatment of HeLa cells with each isomer decreased the mitochondria membrane potential and caused the release of cytochrome c from mitochondria into the cytosol. In addition, stimulation of caspase-3 and caspase-9 activity was observed. The Bax/Bcl-2 ratio was also increased in GA-treated HeLa cells. The results demonstrated that both isomers GA-Mf and GA-S induced apoptosis of human HeLa cells through a mitochondria mediated pathway, but they had the different cell cycle arrest specificity. The findings will be helpful to the development of useful cancer chemopreventive compounds from G. lucidum.     

The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells

Abstract

Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-κB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs.     

Involvement of caspase-3 in apoptosis induced by Viscum album var. coloratum agglutinin in HL-60 cells

A cytotoxic lectin (Viscum album L. coloratum agglutinin, VCA) from Korean mistletoe was isolated by affinity chromatography on Sepharose 4B immobilized with asialofetuin. In HL-60 cells, addition of VCA resulted in a dose- and time-dependent growth suppression, morphological changes of apoptotic nuclei, and DNA fragmentation characteristics of apoptosis. To investigate how caspase-3 activation during VCA-induced apoptosis induces cleavages of PARP, the expression of PARP and the pattern of caspase-3 activation in HL-60 cells were investigated. The native and processed PARP forms typically seen in apoptotic cells were observed, and a decrease in expression of the 32-kDa form of caspase-3 in a dose-dependent manner was observed. The VCA-induced apoptosis was significantly inhibited by a caspase-3 specific inhibitor, z-DEVD-FMK, and the PARP processing and caspase-3 activation were also inhibited by the inhibitor. A possible involvement of cell cycle arrest in VCA-induced apoptosis was investigated by flow cytometry and the results suggested that the apoptotic effect of VCA is not involved in the induction of cell cycle arrest.     

Enhanced G2/M Arrest,Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells

Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133^+high/CD44^+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC₅₀ dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in prostate CSCs; and may be a potential effective therapeutic agent against prostate cancer.     

Endogenous HIV-1 Vpr-mediated apoptosis and proteome alteration of human T-cell leukemia virus-1 transformed C8166 cells

HIV-1 viral protein R (Vpr) can induce cell cycle arrest and cell death, and may be beneficial in cancer therapy to suppress malignantly proliferative cell types, such as adult T-cell leukemia (ATL) cells. In this study, we examined the feasibility of employing the HIV-vpr gene, via targeted gene transfer, as a potential new therapy to kill ATL cells. We infected C8166 cells with a recombinant adenovirus carrying both vpr and GFP genes (rAd-vpr), as well as the vector control virus (rAd-vector). G₂/M phase cell cycle arrest was observed in the rAd-vpr infected cells. Typical characteristics of apoptosis were detected in rAd-vpr infected cells, including sub-diploid peak exhibition in DNA content assay, the Hoechst 33342 accumulation, apoptotic body formation, mitochondrial membrane potential and plasma membrane integrity loss. The proteomic assay revealed apoptosis related protein changes, exhibiting the regulation of caspase-3 activity indicator proteins (vimentin and Rho GDP-dissociation inhibitor 2), mitochondrial protein (prohibitin) and other regulatory proteins. In addition, the up-regulation of anti-inflammatory redox protein, thioredoxin, was identified in the rAd-vpr infected group. Further supporting these findings, the increase of caspase 3&7 activity in the rAd-vpr infected group was observed. In conclusion, endogenous Vpr is able to kill HTLV-1 transformed C8166 cells, and may avoid the risks of inducing severe inflammatory responses through apoptosis-inducing and anti-inflammatory activities.     

Effects of diallyl disulfide (DADS) on expression of apoptosis associated proteins in androgen independent human prostate cancer cells (PC-3)

Prostate cancer is a leading cause of death among the aging men. Surgical or radiotherapy is effective when the cancer is confined to the prostate gland but once the cancer spreads beyond the pelvis even chemotherapy and hormonal ablation therapy fails in curing this disease. Our previous studies have shown that diallyl disulfide (DADS) induces cell cycle arrest and also induces apoptosis in PC-3 cells. And now the present study is focused to see whether there is an activation of caspase cascade pathway. Hence, in the present study the apoptotic effect of DADS is studied by Western blot analysis of caspase-3, -9, -10 and Bcl-2, Bad, and Bax protein. The Apoptotic cells were assessed by Hoechst 33342 staining with 25 and 40 microM concentrations of DADS for 24 h. The results have shown that DADS at 25 and 40 microM concentrations has induced the activation of caspases. There is a significant increase in the expression of caspases (3, 9, and 10). The proapoptotic protein Bax has significantly increased at 40 microM of DADS treatment and there is significant increase of Bad protein at both the concentration. Bcl-2 protein has significantly decreased in DADS treated cells. Therefore, the present investigation serves as evidence that DADS may be a therapeutic drug in the treatment of prostate cancer.     

Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70–Bax interaction in prostate cancer

Dysfunction of the apoptotic pathway in prostate cancer cells confers apoptosis resistance towards various therapies. A novel strategy to overcome resistance is to directly target the apoptotic pathway in cancer cells. Apigenin, an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms which are not fully explored. In the present study we provide novel insight into the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment resulted in significant decrease in cell viability and apoptosis induction with the increase of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. These results suggest that apigenin targets inhibitor of apoptosis proteins and Ku70–Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy.     

Apoptosis and cell recovery in response to oxidative stress in p53-deficient prostate carcinoma cells

We have studied the effects of different concentrations of H(2)O(2) on the proliferation of PC-3 prostate carcinoma cells. Since this cell line lacks functional p53, we sought to characterize whether apoptotic response to the oxidative insult was altered such that, unlike in cells containing functional p53 apoptosis may be reduced and replaced by other mechanisms of cellular arrest and death. We did not observe necrosis in PC-3 cells treated with H(2)O(2) concentrations of up to 500 microM. In the presence of 50 microM H(2)O(2), arrest was observed in the G2-phase of the cell cycle, along with p53-independent apoptosis. In the presence of 500 microM H(2)O(2), addition of l-buthionine sulfoximine increased the percentage of apoptotic cell death. Senescence-associated cell arrest was never observed. Moreover, some of the treated cells seemed to be resistant to oxidative damage. These cells re-entered the cell cycle and proliferated normally. Analysis of the expression of p21(waf1) and of p21 protein levels, as well as the activity of caspase-3 and caspase-8, allowed us to characterize some aspects of the arrest of PC-3 cells in G2 and the apoptotic response to oxidative stress in the absence of functional p53.