Rotenone-induced G2/M cell cycle arrest and apoptosis in a human B lymphoma cell line PW.

Concentrations of rotenone (ROT) that block electron flow through mitochondrial complex I (100 nM) did not significantly alter either cell viability or the growth of PW cells. However, 10- to 50-fold higher concentrations (1-5 microM) were found to induce a dose-dependent cell cycle arrest predominantly at the G2/M stage of the cycle and apoptosis. Apoptosis was dependent on the cell cycle arrest, since apoptosis but not the G2/M arrest was prevented with the broad spectrum caspase inhibitor zVADfmk. Biochemical features of apoptosis included mitochondrial cytochrome c release, reactive oxygen species generation, and the activation of procaspase 3. Thus, ROT inhibition of mitochondrial electron transport may be insufficient to induce apoptosis in PW cells. Instead, apoptosis in these cells occurs as a consequence of disruption of the cell cycle and is only indirectly dependent upon mitochondrial electron transport.     

Anthraquinone derivatives induce G2/M cell cycle arrest and apoptosis in NTUB1 cells

Thirteen anthraquinone derivatives 5-17 including two 3-(3-alkylaminopropoxy)-9,10-anthraquinone (NHA) derivatives 5 and 6, and 11 1-hydroxy-3-(3-alkylaminopropoxy)-9,10-anthraquinone (MHA) derivatives 7-17 were synthesized, evaluated for cytotoxicities against two cancer cell lines, and assayed the generation of reactive oxygen species (ROS) in NTUB1 cells (a human bladder carcinoma cell line). Compound 9 bearing a pyrrolidinyl group induced the stronger cytotoxic effect than those of other synthesized NHA and MHA derivatives. Exposure of NTUB1 cells to 9, 13, and 17 for 24h significantly increased the production of ROS, respectively. Flow cytometric analysis exhibited that the exposure of NTUB1 cells to the selective 9 led to the G2/M phase arrest accompanied by an increase of apoptotic cell death after the incubation for 24h. Compound 9 induced up-regulation of cyclinB1 and p21 expressions. Biological results suggested that the induction of G2/M arrest, apoptosis, and cell death by 9 may associate with increased expression of p21 and cyclin B1, elevation of Bax and p53 levels, and generation of ROS in the cell. In conclusion, these series of compounds may be used as anticancer agents.     

Estrogen and progesterone lower cyclin B1 AND D1 expression, block cell cycle in G2/M, and trigger apoptosis in human adrenal carcinoma cell cultures.

The effects of 17 beta-estradiol and progesterone were evaluated separately and in combination, on the growth, survival, and cell cycle dynamics of SW-13 human adrenal carcinoma cells in culture. Both hormones significantly decreased cell survival, with dose response curves at four days demonstrating EC (50)s estimated at 1.2 x 10 (-5) M for 17 beta-estradiol and 4.8 x 10 (-6) M for progesterone. Flow cytometry studies of these cultures indicated a strong G2/M blocking effect of both steroids, either individually or in combination; the effects of progesterone and of both agents together were substantially greater than the effect with 17 beta-estradiol alone. The sub-G1 region of the flow cytometry profile was significantly enhanced by exposure to 17 beta-estradiol and even more by progesterone. Sub-G1 "apoptosis" was confirmed by fragmented and condensed nuclear chromatin staining using a standard DAPI fluorescence assay. The expression of the critical cell cycle regulatory proteins cyclin B1 and D1 were significantly decreased by each hormone, with the influence of progesterone again predominating. These data demonstrate that high doses of 17 beta-estradiol and progesterone have inhibitory and apoptotic effects on SW-13 human adrenal carcinoma cells IN VITRO. The observed effects are associated with declines in cyclin B1 and D1 expression as well as a block in G2/M.     

A resveratrol analog, phoyunbene B, induces G2/M cell cycle arrest and apoptosis in HepG2 liver cancer cells

Among the seven natural resveratrol analogs separated and identified from Pholidota yunnanensis R(OLFE), we found phoyunbene B (PYB, trans-3,4'-dihydroxy-2',3',5-trimethoxystilbene) was more effective in inhibiting the growth of HepG2 hepatocellular carcinoma cells than resveratrol. The inhibitory effect of PYB in HepG2 cells was due to its induction of G2/M cell cycle arrest and apoptosis. Induction of G2/M phase cell cycle arrest by PYB was associated with its up-regulation of Cyclin B1, while its induction of apoptosis was accompanied with its down-regulation of Bcl-2 and up-regulation of Bax. Our in vitro invasion/migration assays also showed that PYB could inhibit the invasion of hepatocellular carcinoma cells.     

Flavonoids from tartary buckwheat induce G2/M cell cycle arrest and apoptosis in human hepatoma HepG2 cells

The cytotoxicity and antioxidant activity on human hepa toma cell line HepG2 of three flavonoids homogenous com pounds from tartary buckwheat seeds and bran, namely quercetin, isoquercetin, and rutin, were investigated. The total antioxidant competency detection results indicated that the antioxidant capacity of quercetin was the strongest in a biological response system. A [3-（4,5-dimethylthiazol-2-yl）-2,5diphenyltetrazolium bromide] assay showed that quercetin exhibited the strongest cytotoxic effects against the HepG2 cell line. Flow cytometric analysis indicated that quercetin significantly increased the production of reactive oxygen species, and led to the G2/M phase arrest accom panied by an increase of apoptotic cell death after 48 h of incubation. Quercetininduced cell apoptosis was shown to involve p53 and p21 upregulation, Cyclin D1, Cdk2, and Cdk7 downregulation. These results suggested that the in duction of G2/M arrest, apoptosis, and cell death by quer cetin may associate with increased expression of p53 and p21, decrease of Cyclin D1, Cdk2, and Cdk7 levels, and generation of reactive oxygen species in cells. This study will help to better understand and fully utilize medicinal resources of plant flavonoids.     

Involvement of the role of Chk1 in lithium-induced G2/M phase cell cycle arrest in hepatocellular carcinoma cells

Lithium, a therapeutic agent for bipolar disorder, can induce G2/M arrest in various cells, but the mechanism is unclear. In this article, we demonstrated that lithium arrested hepatocellular carcinoma cell SMMC-7721 at G2/M checkpoint by inducing the phosphorylation of cdc2 (Tyr-15). This effect was p53 independent and not concerned with the inhibition of glycogen synthase kinase-3 and inositol monophosphatase, two well-documented targets of lithium. Checkpoint kinase 1 (Chk1), a critical enzyme in DNA damage-induced G2/M arrest, was at least partially responsible for the lithium action. The lithium-induced phosphorylation of cdc2 and G2/M arrest was abrogated largely by SB218078, a potent Chk1 inhibitor, as well as by Chk1 siRNA or the over-expression of kinase dead Chk1. Furthermore, lithium-induced cdc25C phosphorylation in 7721 cells and in vitro kinase assay showed that the activity of Chk1 was enhanced after lithium treatment. Interestingly, the increase of Chk1 activity by lithium may be independent of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) kinase. This is because no elevated phosphorylation on Chk1 (Ser-317 and Ser-345) was observed after lithium treatment. Moreover, caffeine, a known ATM/ATR kinase inhibitor, relieved the phosphorylation of cdc2 (Tyr-15) by hydroxyurea, but not that by lithium. Our study's results revealed the role of Chk1 in lithium-induced G2/M arrest. Given that Chk1 has been proposed to be a novel tumor suppressor, we suggest that the effect of lithium on Chk1 and cell cycle is useful in tumor prevention and therapy.     

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.     

G2/M cell cycle arrest and induction of apoptosis by a stilbenoid, 3,4,5-trimethoxy-4'-bromo-cis-stilbene, in human lung cancer cells

Stilbenoids, including resveratrol (3,5,4'-trihydroxy-trans-stilbene) which is a naturally occurring phytoalexin abundant in grapes and several plants, have been shown to be active in inhibiting proliferation and inducing apoptosis in human cancer cell lines. Using resveratrol as the prototype, we have synthesized various analogs and evaluated their growth inhibitory effects in cultured human cancer cells. In the present study, we show that one of the stilbenoids, 3,4,5-trimethoxy-4'-bromo-cis-stilbene (BCS), was more effective than its corresponding trans-isomer and resveratrol on the inhibition of cancer cell growth. Prompted by the strong growth inhibitory activity of BCS (IC50; 0.03 microM) compared to its trans-isomer (IC50; 6.36 microM) and resveratrol (IC50; 33.0 microM) in cultured human lung cancer cells (A549), we investigated its mechanism of action. BCS induced arrest at the G2/M phase cell cycle in the early time and subsequently increased in the sub-G1 phase DNA contents in a time-dependent manner, indicating induction of apoptosis. Morphological observation with round-up shape and DNA fragmentation was also revealed the apoptotic phenomena. BCS treatment elevated the expression levels of the pro-apoptotic protein p53, the cyclin-dependent kinase inhibitor p21, and the release of cytochrome c in the cytosol. The down-regulation of checkpoint protein cyclin B1 by BCS was well correlated with the cell cycle arrest at G2/M. These data suggest the potential of BCS to serve as a cancer chemotherapeutic or chemopreventive agent by virtue of arresting the cell cycle and induction of apoptosis of human lung cancer cells.     

Tetrandrine-induced cell cycle arrest and apoptosis in Hep G2 cells

The effects of tetrandrine in the human hepatoblastoma G2 (Hep G2) cell line were investigated in this study. The results showed that tetrandrine not only inhibited Hep G2 growth but also induced apoptosis and blocked cell cycle progression in the G1 phase. ELISA assay demonstrated that tetrandrine significantly increased the expression of p53 and p21/WAF1 protein, which caused cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by tetrandrine. Taken together, p53 and Fas/FasL apoptotic system possibly participated in the antiproliferative activity of tetrandrine in Hep G2 cells.     

Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c(551) from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c(551), the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G(1) to S phase, as well as at the G(2)/M phase at higher concentrations. Unlike P. aeruginosa cytochrome c(551) which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G(2)/M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 degrees C, suggesting energy requirement in the entry process.     

Genistein-induced G2/M cell cycle arrest of human intestinal colon cancer Caco-2 cells is associated with Cyclin B1 and Chk2 down-regulation

Genistein is an isoflavonic phyto-oestrogen contained in soya beans. It is thought to display anti-cancer effects. This study was designed to investigate its effect on human intestinal colon cancer Caco-2 cells. MTT assay, flow cytometric analysis and western blotting were used to investigate the effect of genistein on cell proliferation, cell cycle progression and protein alterations of selected cell cycle-related proteins in Caco-2 cells. Our results showed that genistein and daidzein significantly suppressed cell proliferation. Genistein treatment was demonstrated to modulate cell cycle distribution through accumulation of cells at G2/M phase, with a significant decreasing effect of Cyclin B1 and Serine/threonine-protein kinase 2 (Chk2) proteins expression. However, daidzein did not alter the cell cycle progression in Caco-2 cells. All these observation strongly indicate that genistein has anti-proliferative effect in human intestinal colon cancer Caco-2 cells through the down-regulation of cell cycle check point proteins, Cyclin B1 and Chk2.     

Polycystin-1 induced apoptosis and cell cycle arrest in G0/G1 phase in cancer cells

Studies have shown that polycystin-1, encoded by PKD1, the major ADPKD, may have a central role in regulating both apoptosis and proliferation, which could prevent the malignant transformation of affected cells. However, as a putative tumor suppressor, direct studies on the possibility that polycystin-1 may play a role in cancer cells' biological properties have not yet been reported. We have demonstrated that the apoptosis of cancer cells was induced by overexpression of polycystin-1. After transfection with polycystin-1, three cancer cell lines, HepG2, A549, and SW480, showed significantly increased apoptosis compared with the respective control groups. This was accompanied by cell cycle arrest at G(0)/G(1) phase, whereas cell proliferation was not significantly affected. Overexpression of polycystin-1 induces apoptosis in cancer cells, at least partially, through Wnt and a caspase-dependent pathway.     

Mycoepoxydiene, a fungal polyketide, induces cell cycle arrest at the G2/M phase and apoptosis in HeLa cells

Mycoepoxydiene (MED) is a polyketide isolated from a marine fungus associated with mangrove forests. It contains an oxygen-bridged cyclooctadiene core and an α,β-unsaturated δ-lactone moiety. MED induced the reorganization of cytoskeleton in actively growing HeLa cells by promoting formation of actin stress fiber and inhibiting polymerization of tubulin. MED could induce cell cycle arrest at G2/M in HeLa cells. MED-associated apoptosis was characterized by the formation of fragmented nuclei, PARP cleavage, cytochrome c release, activation of caspase-3, and an increased proportion of sub-G1 cells. Additionally, MED activated MAPK pathways. Interestingly, the time of JNK, p38, and Bcl-2 activation did not correlate with the release of cytochrome c. This study is the first report demonstrating the action mechanism of MED against tumor cell growth. These results provide the potential of MED as a novel low toxic antitumor agent.     

Molecular mechanisms of luteolin-7-O-glucoside-induced growth inhibition on human liver cancer cells: G2/M cell cycle arrest and caspase-independent apoptotic signaling pathways

Luteolin-7-O-glucoside (LUT7G), a flavone subclass of flavonoids, has been found to increase anti-oxidant and anti-inflammatory activity, as well as cytotoxic effects. However, the mechanism of how LUT7G induces apoptosis and regulates cell cycles remains poorly understood. In this study, we examined the effects of LUT7G on the growth inhibition of tumors, cell cycle arrest, induction of ROS generation, and the involved signaling pathway in human hepatocarcinoma HepG2 cells. The proliferation of HepG2 cells was decreased by LUT7G in a dose-dependent manner. The growth inhibition was due primarily to the G2/M phase arrest and ROS generation. Moreover, the phosphorylation of JNK was increased by LUT7G. These results suggest that the anti-proliferative effect of LUT7G on HepG2 is associated with G2/M phase cell cycle arrest by JNK activation. [BMB Reports 2013; 46(12): 611-616]     

2ME and 2OHE2 exhibit growth inhibitory effects and cell cycle arrest at G2/M in RL95-2 human endometrial cancer cells through activation of p53 and Chk1

Evidence is accumulating that estradiol (E2) may play a dual role in carcinogenic and anticarcinogenic effects by different metabolic pathways. It has been shown that some metabolites of E2 exert proliferative and others anti-proliferative properties on human cancer cells. In the present study, the effects of E2 and its four primary metabolites including 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME), and 4-methoxyestradiol (4ME) on proliferation and cell cycle in RL95-2 human endometrial cells were investigated. Our results indicate that 2ME and 2OHE2, but not E2, 4ME, and 4OHE2, exhibit the inhibitory effect through cell cycle arrest at G2/M. 2ME and 2OHE2-induced G2/M cell cycle arrest associated with activation of p53 (Ser15), upregulation of p21(WAF1/Cip1) (p21) and GADD45, inactivation of Cdc2 (Tyr15), as well as downregulation of Cyclin B1. 2ME and 2OHE2-mediated cell cycle arrest at G2/M was also related to activation of protein kinase Chk1 which is associated with p53 (Ser20) activation and downstream responses.     

Fluoroquinolone-mediated inhibition of cell growth, s-g(2)/m cell cycle arrest, and apoptosis in canine osteosarcoma cell lines

Despite significant advancements in osteosarcoma research, the overall survival of canine and human osteosarcoma patients has remained essentially static over the past 2 decades. Post-operative limb-spare infection has been associated with improved survival in both species, yet a mechanism for improved survival has not been clearly established. Given that the majority of canine osteosarcoma patients experiencing post-operative infections were treated with fluoroquinolone antibiotics, we hypothesized that fluoroquinolone antibiotics might directly inhibit the survival and proliferation of canine osteosarcoma cells. Ciprofloxacin or enrofloxacin were found to inhibit p21(WAF1) expression resulting in decreased proliferation and increased S-G(2)/M accumulation. Furthermore, fluoroquinolone exposure induced apoptosis of canine osteosarcoma cells as demonstrated by cleavage of caspase-3 and PARP, and activation of caspase-3/7. These results support further studies examining the potential impact of quinolones on survival and proliferation of osteosarcoma.