Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.     

Spy1 induces de-ubiquitinating of RIP1 arrest and confers glioblastoma's resistance to tumor necrosis factor (TNF-α)-induced apoptosis through suppressing the association of CLIPR-59 and CYLD

Glioblastoma multiforme (GBM), a grade-IV glioma, is resistant to TNF-α induced apoptosis. CLIPR-59 modulates ubiquitination of RIP1, thus promoting Caspase-8 activation to induce apoptosis by TNF-α. Here we reported that CLIPR-59 was down-regulated in GBM cells and high-grade glioma tumor samples, which was associated with decreased cancer-free survival. In GBM cells, CLIPR-59 interacts with Spy1, resulting in its decreased association with CYLD, a de-ubiquitinating enzyme. Moreover, experimental reduction of Spy1 levels decreased GBM cells viability, while increased the lysine-63-dependent de-ubiquitinating activity of RIP1 via enhancing the binding ability of CLIPR-59 and CYLD in GBM, thus promoting Caspase-8 and Caspase-3 activation to induce apoptosis by TNF-α. These findings have identified a novel Spy1-CLIPR-59 interplay in GBM cell''s resistance to TNF-α-induced apoptosis revealing a potential target in the intervention of malignant brain tumors.     

TRIM32 protein sensitizes cells to tumor necrosis factor (TNFα)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP)

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.     

APRIL depletion induces cell cycle arrest and apoptosis through blocking TGF-β1/ERK signaling pathway in human colorectal cancer cells

It is well documented that a proliferation-inducing ligand (APRIL), a newly found member of tumor necrosis factor superfamily, overexpressed in the majority of malignancies, plays a potential role in the occurrence and development of these tumors. Herein, we demonstrated that APRIL depletion by using RNA interference in human colorectal cancer (CRC) COLO 205 and SW480 cells resulted in cell proliferation inhibition and evoked cell cycle arrest in G0/G1 phase and apoptosis, coupled with decrease in CDK2, Cyclin D1, Bcl-2 expression and an increase of p21 and Bax expression. In addition, the decreased expression of transforming growth factor-β1 (TGF-β1) and p-ERK was also showed in siRNA-APRIL transfected COLO 205 and SW480 cells, whereas the protein expression levels of Smad2/3, p-Smad2/3, and ERK were not significantly changed. Taken together, our results indicate that APRIL depletion induces cell cycle arrest and apoptosis partly through blocking noncanonical TGF-β1/ERK, rather than canonical TGF-β1/Smad2/3, signaling pathway in CRC cells. Moreover, our study highlights APRIL as a potential molecular target for the therapy of CRC.     

Involvement of both tumor necrosis factor-α-induced necrosis and p53-mediated caspase-dependent apoptosis in nephrotoxicity of cisplatin

We previously reported that necrosis occurs predominantly in porcine renal tubular LLC-PK₁ cells, when the cells were exposed transiently to a high concentration of cisplatin. Moreover, we demonstrated that generation of reactive oxygen species and subsequent production of tumor necrosis factor-α (TNF-α) through phosphorylation of p38 MAPK are implicated in the pathogenesis of cisplatin-induced renal cell injury. However, some TUNEL-positive cells appeared in renal proximal tubules of rats after systemic injection of cisplatin, suggesting an involvement of apoptosis. In the present study, we found in LLC-PK₁ cells that both apoptosis and necrosis were elicited when the cells were exposed to 200 μM cisplatin for 1 h followed by incubation for 24 h in the presence of 20 μM cisplatin. The cisplatin-induced necrosis was largely attenuated by the antioxidant N-acetylcysteine, while apoptosis was prevented by the specific inhibitors for caspases-2, -8, and -3 and a p53 inhibitor pifithrin-α but not by the p38 MAPK inhibitor SB203580. On the other hand, SB203580 attenuated the cisplatin-induced increase in TNF-α production. These findings suggest that p53-mediated activations of caspases-2, -8 and -3 play a key role in cisplatin-induced renal cell apoptosis, while oxidative stress-induced TNF-α synthesis via p38 MAPK phosphorylation contributed to the necrosis.     

Green tea constituent (−)-Epigallocatechin-3-gallate inhibits hep G2 cell proliferation and induces apoptosis through p53-dependent and fas-mediated pathways

(-)-Epigallocatechin-3-gallate (EGCG) is a polyphenolic compound found in green tea. It has been reported to possess a wide range of pharmacological properties, and is one of the most promising chemopreventive agents for cancer. To provide a better understanding of the preventive effect of EGCG on liver cancer, we examined EGCG for its effect on proliferation and cell cycle progression in a human liver cancer cell line, Hep G2. The results showed that EGCG inhibited the proliferation of Hep G2 by inducing apoptosis and blocking cell cycle progression in the G1 phase. ELISA showed that EGCG significantly increased the expression of p53 and p21/WAF1 protein, and this contributed to cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), as well as Bax protein, was responsible for the apoptotic effect induced by EGCG. Taken together, our study suggests that the induction of p53 and the activity of the Fas/FasL apoptotic system play major roles in the antiproliferative activity of EGCG in Hep G2 cells.     

Phytoglycoprotein (38 kDa) induces cell cycle (G₀/G₁) arrest and apoptosis in HepG2 cells

Styrax japonica Siebold et al Zuccarini (SJSZ) has been used to heal inflammation and bronchitis as folk medicine in Korea. Firstly, glycoprotein isolated from SJSZ (SJSZ glycoprotein) has a molecular weight with 38 kDa and consists of carbohydrate (57.64%) and protein (42.35%). In the composition of SJSZ glycoprotein, carbohydrate mostly consists of glucose (28.17%), galactose (21.85%), and mannose (2.62%) out of 52.64%, respectively. The protein consists of Trp (W, 7.01%), Pro (P, 6.72%), and Ile (I, 5.42%) out of 42.35% as three major amino acids, while total amount of other amino acids is 23.20%. The purpose of this study is to know whether the SJSZ glycoprotein (38 kDa) induces the cell cycle arrest and apoptosis in HepG2 cells. Cytotoxicity was evaluated using MTT and lactate dehydrogenase assay and amount of intracellular reactive oxygen species (iROS) and nitric oxide (NO) was measured using fluorescence microplate reader. Activities of cell cycle-related proteins [p53, p21, p27, Cyclin D1, and cyclin-dependent kinase (CDK)4] and apoptosis-related factors [iNOS, Bid, Bcl-2/bax, cytochrome c, caspase-9, caspase-3, and poly-(ADP-ribose) polymerase (PARP)] were assessed by Western blot and fluorescence-activated cell sorter (FACS) analysis. In the cell cycle-related proteins, SJSZ glycoprotein (50 μg/ml) significantly enhances the expression of p53, p21, and p27, whereas it suppressed the activity of cyclin D1/CDK4. In the apoptosis-related factors, SJSZ glycoprotein (50 μg/ml) stimulates to increase iROS, and NO, to activate iNOS, Bid, Bcl-2/bax, cytochrome c, caspase-9, caspase-3, and PARP. SJSZ glycoprotein (50 μg/ml) has potent effect to arrest cell cycle from G(0) /G(1) to S and to induce apoptosis in HepG2 cells.     

Isoflavone genistein protects human vascular endothelial cells against tumor necrosis factor-α-induced apoptosis through the p38β mitogen-activated protein kinase

Isoflavone genistein may have beneficial effects on vascular function, but the mechanism is unclear. Here, we investigated whether genistein protects vascular endothelial cells against apoptosis induced by tumor necrosis factor-α. We show that genistein significantly inhibited TNF-α-induced apoptosis in human aortic endothelial cells as determined by caspase-3 activation, 7-amino actinomycin D staining, in situ apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of Bcl-2 protein and its promoter activity. Inhibition of extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptors had no effect on the cytoprotective effect of genistein. However, inhibition of p38 mitogen-activated protein kinase (p38) completely abolished this genistein effect. Accordingly, stimulation of HAECs with genistein resulted in rapid activation of p38β, but not p38α. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Preservation of the functional integrity of the endothelial monolayer may represent an important mechanism by which genistein exerts its vasculoprotective effect.     

Calpeptin suppresses tumor necrosis factor-α-induced death and accumulation of p53 in L929 mouse sarcoma cells

The cytokine tumor necrosis factor (TNF) induces caspase-dependent cell death in a subset of tumor cells. In this report, we show a novel suppressive effect of calpeptin, a calpain inhibitor, on TNF-induced cell death and accumulation of p53 in L929 mouse fibrosarcoma. Exposure to 10 ng/ml TNF induced cell death in >50% of L929 cells within 12 h and stimulated accumulation of p53 (8-fold). Preincubation of cells with calpeptin blocked both TNF-induced cell death and accumulation of p53 as examined with Western blot. TNF-induced accumulation of p53 was in part contributed by increase of p53 mRNA level (2.2-fold) in a calpeptin-insensitive manner. Interestingly, other calpain inhibitors tested did not show these effects like calpeptin and TNF treatment did not increase apparent calpain activity in L929 cells, suggesting that calpeptin may have another function besides targeting calpain. Expression of dominant negative mutant p53Val¹³⁵ reduced the incidence of TNF-mediated cell death. Taken together, our findings suggest that TNF induces calpeptin-dependent, but calpain-independent accumulation of p53 protein as a necessary step leading to death in L929 cells.     

The ruthenium(II)–arene compound RAPTA-C induces apoptosis in EAC cells through mitochondrial and p53–JNK pathways

An investigation of the molecular mechanism of the anticancer activity demonstrated by the ruthenium(II)–arene compound [Ru(η⁶-p-cymene)Cl₂(pta)] (pta is 1,3,5-triaza-7-phosphaadamantane), termed “RAPTA-C”, in Ehrlich ascites carcinoma (EAC) bearing mice is described. RAPTA-C exhibits effective cell growth inhibition by triggering G₂/M phase arrest and apoptosis in cancer cells. Cell cycle arrest is associated with increased levels of p21 and reduced amounts of cyclin E. RAPTA-C treatment also enhances the levels of p53, and its treatment triggers the mitochondrial apoptotic pathway, as shown by the change in Bax to Bcl-2 ratios, resulting in cytochrome c release and caspase-9 activation. c-Jun NH₂-terminal kinase (JNK) is a critical mediator in RAPTA-C-induced cell growth inhibition. Activation of JNK by RAPTA-C increases significantly during apoptosis. Overall, these results suggest a critical role for JNK and p53 in RAPTA-C-induced G₂/M arrest and apoptosis of EAC-bearing mice. Consequently, RAPTA-C treatment results in a significant inhibition in the progression of cancer in an animal model, which emulates the human disease, and does so with remarkably low general toxicity; hence, RAPTA-C has potential for clinical application.     

Docosahexaenoic acid-induced unfolded protein response, cell cycle arrest, and apoptosis in vascular smooth muscle cells are triggered by Ca²⁺-dependent induction of oxidative stress

Proliferation of vascular smooth muscle cells is a characteristic of pathological vascular remodeling and represents a significant therapeutic challenge in several cardiovascular diseases. Docosahexaenoic acid (DHA), a member of the n-3 polyunsaturated fatty acids, was shown to inhibit proliferation of numerous cell types, implicating several different mechanisms. In this study we examined the molecular events underlying the inhibitory effects of DHA on proliferation of primary human smooth muscle cells isolated from small pulmonary artery (hPASMCs). DHA concentration-dependently inhibited hPASMC proliferation, induced G1 cell cycle arrest, and decreased cyclin D1 protein expression. DHA activated the unfolded protein response (UPR), evidenced by increased mRNA expression of HSPA5, increased phosphorylation of eukaryotic initiation factor 2α, and splicing of X-box binding protein 1. DHA altered cellular lipid composition and led to increased reactive oxygen species (ROS) production. DHA-induced ROS were dependent on both intracellular Ca(2+) release and entry of extracellular Ca(2+). Overall cellular ROS and mitochondrial ROS were decreased by RU360, a specific inhibitor of mitochondrial Ca(2+) uptake. DHA-induced mitochondrial dysfunction was evidenced by decreased mitochondrial membrane potential and decreased cellular ATP content. DHA triggered apoptosis as found by increased numbers of cleaved caspase-3- and TUNEL-positive cells. The free radical scavenger Tempol counteracted DHA-induced ROS, cell cycle arrest, induction of UPR, and apoptosis. We conclude that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of UPR, cell cycle arrest, and apoptosis in DHA-treated hPASMCs.     

2′-epi-2′-O-Acetylthevetin B extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

2′-epi-2′-O-Acetylthevetin B (GHSC-74) is a cardiac glycoside isolated from the seeds of Cerbera manghas L. We have demonstrated that GHSC-74 reduced the viability of HepG2 cells in a time- and dose-dependent manner. The present study was designed to explore cellular mechanisms whereby GHSC-74 led to cell cycle arrest and apoptosis in HepG2 cells. Cell cycle flow cytometry demonstrated that HepG2 cells treated with GHSC-74 (4 μM) resulted in S and G2 phase arrest in a time-dependent manner, as confirmed by mitotic index analysis. G2 phase arrest was accompanied with down-regulation of CDC2 and Cyclin B1 protein. Furthermore, GHSC-74-induced apoptotic killing, as demonstrated by DNA fragmentation, DAPI staining, and flow cytometric detection of sub-G1 DNA content in HepG2 cells. GHSC-74 treatment resulted in a significant increase in reactive oxygen species, activation of caspase-9, dissipation of mitochondrial membrane potential, and translocation of apoptosis-inducing factor (AIF) from the mitochondrion to the nucleus in HepG2 cells. Nevertheless, after GHSC-74 exposure, no significant Fas and FasL up-regulation was observed in HepG2 cells by flow cytometry. In addition, treatment with antioxidant N-acetyl-l-cysteine (NAC) and broad-spectrum caspase inhibitor z-VAD-fmk partially prevented apoptosis but did not abrogate GHSC-74-induced nuclear translocation of AIF. In conclusion, we have demonstrated that GHSC-74 inhibited growth of HepG2 cells by inducing S and G2 phase arrest of the cell cycle and by triggering apoptosis via mitochondrial disruption including both caspase-dependent and -independent pathways, and ROS generation.     

The dietary phytochemical 3,3'-diindolylmethane induces G2/M arrest and apoptosis in oral squamous cell carcinoma by modulating Akt-NF-κB, MAPK, and p53 signaling

In light of the growing incidence of oral cancer in Taiwan, this study is aimed at investigating the antitumor activity of 3,3'-diindolylmethane (DIM), an active metabolite of the phytochemical indole-3-carbinol (I3C), in oral squamous cell carcinoma (OSCC). DIM exhibited substantially higher antiproliferative potency than I3C in three OSCC cell lines with IC(50) values in SCC2095, SCC9, and SCC15 cells, respectively, of 22 versus 168μM, 25 versus 176μM, and 29versus 300μM. Flow cytometric analysis and Comet assay indicated that DIM suppressed the viability of SCC2095 cells by inducing apoptosis and G2/M arrest. Western blot analysis of various signaling markers revealed the ability of DIM to target pathways mediated by Akt, mitogen-activated protein (MAP) kinases, nuclear factor (NF)-κB, and p53, of which the concerted action underlined its antitumor efficacy. The concomitant inactivation of Akt and MAP kinases in response to DIM facilitated the dephosphorylation of the proapoptotic protein Bad at Ser-136 and Ser-112, respectively. Through endoplasmic reticulum (ER) stress, DIM stimulated the activation of p53 via Ser-15 phosphorylation, leading to increased expression of the BH3-only proapoptotic Bcl-2 members Puma and Noxa. Together, these changes decreased the mitochondrial threshold for apoptosis. G2/M arrest might be attributable to the suppressive effect of DIM on the expression of cyclin B1 and cdc25c. As many downstream effectors of the Akt-NF-κB pathway, including glycogen synthase kinase 3β, IκB kinase α, and cyclooxygenase-2, have been shown to promote oral tumorigenesis, the ability of DIM to inhibit this signaling axis underscores its chemopreventive potential in oral cancer.