M-phase Cell Cycle Arrest Research Articles

Evodiamine inhibits ESCC by inducing M-phase cell-cycle arrest via CUL4A/p53/p21 axis and activating noxa-dependent intrinsic and DR4-dependent extrinsic apoptosis

BackgroundEsophageal squamous cell carcinoma (ESCC) is a malignancy with high incidence in several regions of China, and the prognosis of patients with ESCC is unfavorable. Evodiamine (Evo), a small molecule derived from the traditional Chinese herb Evodia rutaecarpa, has shown anti-cancer efficacy in numerous human malignancies but not in ESCC. PurposeTo determine whether Evo induces cell-cycle arrest and apoptosis in ESCC in vitro and in vivo and elucidate the underlying mechanisms. MethodsATPlite and colony formation assay were used to validate the inhibiting effect of Evo on three ESCC cells in vitro; Two subcutaneous tumor models of ESCC cells were used to evaluate the anti-ESCC effect of Evo and assess the biosafety of Evo in vivo; RNAseq and Database of KEGG pathway analysis provided a direction for the mechanistic study of Evo; FACS was used to detect Evo-induced cell cycle arrest and cell apoptosis in ESCC cells; Western blot and QPCR were respectively used to detect the level of related genes and proteins in Evo-treated ESCC cells; SiRNA and other experimental techniques were used to identify the molecular mechanism of Evo-induced ESCC cell cycle arrest and cell apoptosis. ResultsEvo significantly suppressed the growth of ESCC both in vitro and in vivo. Mechanistically, Evo induced M-phase cell-cycle arrest by inactivation of CUL4A E3 ligase, which mediates degradation blockage of p53 and transcriptional activation of p21. With the prolonged treatment time, Evo triggered both Noxa-dependent intrinsic and DR4-dependent extrinsic cell apoptosis in two ESCC cell lines. ConclusionOur findings revealed the anti-tumor efficacy and mechanisms of Evo, providing a solid scientific basis for Evo as an attractive choice for ESCC treatment.

Minnelide synergizes with conventional chemotherapy by targeting both cancer and associated stroma components in pancreatic cancer

Addition of nab-paclitaxel to gemcitabine offers a survival benefit of only 6 weeks over gemcitabine alone at a cost of increased toxicity in PDAC. The goal of the present study is to evaluate the efficacy of Minnelide, a water-soluble prodrug of triptolide, in combination with the standard of care regimen for chemotherapy with the added advantage of reducing the doses of these drugs to minimize toxicity. Pancreatic cancer cell lines were implanted subcutaneously or orthotopically in athymic nude or C57BL/6J mice. Subsequently, animals were randomized and received saline or minnelide or full dose chemotherapy or low dose chemotherapy or minnelide in combination with low dose chemotherapy. Our results show that a combination of low doses of Minnelide with Gemcitabine + nab-paclitaxel significantly inhibited tumor progression and increased the survival of tumor-bearing mice in comparison with conventional chemotherapy alone. Moreover, combination therapy significantly reduced cancer-related morbidity by decreasing ascites and metastasis and effectively targeted both cancer and the associated stroma. In vitro studies with a combination of low doses of triptolide and paclitaxel significantly decreased the cell viability, increased apoptosis and led to significantly increased M-phase cell cycle arrest in various pancreatic cancer cell lines as compared to either drug alone. Our results show that Minnelide synergizes with conventional chemotherapy leading to a significant reduction in the doses of these toxic drugs, all the while achieving better efficacy in the treatment of PDAC. This combination effectively targeted both the cancer and the associated stromal components of pancreatic cancer.

Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells

Scoulerine is an isoquinoline alkaloid, which indicated promising suppression of cancer cells growth. However, the mode of action (MOA) remained unclear. Cytotoxic and antiproliferative properties were determined in this study. Scoulerine reduces the mitochondrial dehydrogenases activity of the evaluated leukemic cells with IC50 values ranging from 2.7 to 6.5 µM. The xCELLigence system revealed that scoulerine exerted potent antiproliferative activity in lung, ovarian and breast carcinoma cell lines. Jurkat and MOLT-4 leukemic cells treated with scoulerine were decreased in proliferation and viability. Scoulerine acted to inhibit proliferation through inducing G2 or M-phase cell cycle arrest, which correlates well with the observed breakdown of the microtubule network, increased Chk1 Ser345, Chk2 Thr68 and mitotic H3 Ser10 phosphorylation. Scoulerine was able to activate apoptosis, as determined by p53 upregulation, increase caspase activity, Annexin V and TUNEL labeling. Results highlight the potent antiproliferative and proapoptotic function of scoulerine in cancer cells caused by its ability to interfere with the microtubule elements of the cytoskeleton, checkpoint kinase signaling and p53 proteins. This is the first study of the mechanism of scoulerine at cellular and molecular level. Scoulerine is a potent antimitotic compound and that it merits further investigation as an anticancer drug.

A Flavone Constituent from Myoporum bontioides Induces M-Phase Cell Cycle Arrest of MCF-7 Breast Cancer Cells.

Myoporum bontioides is a traditional medicinal plant in Asia with various biological activities, including anti-inflammatory and anti-bacterial characteristics. To identify the bioactive constituents from M. bontioides, a newly-identified flavone, 3,4′-dimethoxy-3′,5,7-trihydroxyflavone (compound 1), along with eight known compounds, were investigated in human MCF-7 breast cancer, SCC4 oral cancer, and THP-1 monocytic leukemia cells. Among these compounds, compound 1 exhibited the strongest antiproliferative activity with half-maximal inhibitory concentration (IC50) values ranging from 3.3 μM (MCF-7) to 8.6 μM (SCC4). Flow cytometric analysis indicated that compound 1 induced G2/M cell cycle arrest in MCF-7 cells. Mechanistic evidence suggests that the G2/M arrest could be attributable to compound 1’s modulatory effects on the phosphorylation and expression of numerous key signaling effectors, including cell division cycle 2 (CDC2), CDC25C, and p53. Notably, compound 1 downregulated the expression of histone deacetylase 2 (HDAC2) and HDAC4, leading to increased histone H3 acetylation and p21 upregulation. Together, these findings suggest the translational potential of compound 1 as a breast cancer treatment.

Abstract B23: Preclinical evaluation of a PARP inhibitor in mice representing genetically different subtypes of pancreatic cancers

Abstract The incredibly short survival of patients diagnosed with pancreatic cancer underscores one of the greatest failures of the medical community: our inability to identify effective treatments for pancreatic ductal adenocarcinoma. Most new compounds in clinical trials fail because of highly toxic side effects in normal tissues and limited efficacy in tumor response. An ideal drug would kill only tumor cells and spare the normal cells. In this regard, poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated a promising therapeutic potential, particularly in patients with BRCA-deficient tumors. BRCA-deficient cells have an impaired homology-directed repair (HDR) pathway, a mechanism critical for resolving DNA double strand breaks (DSBs). When PARP1 is inhibited, DNA single strand breaks are not repaired and they mature to double strand breaks, which are very toxic for the cell. Hence, inhibition of PARP1 is selectively toxic to cells with an impaired HDR pathway, such as BRCA-deficient tumor cells, but not to normal cells. Because up to 10% of pancreatic cancer patients carry mutations in either BRCA2 or other proteins involved in the HDR pathway, PARP inhibitors may also prove effective in this setting. However, a number of subtleties remain that call this conclusion into question, including poor drug delivery, loss of heterozygosity, and BRCA-independent alteration of the HDR pathway. We designed a preclinical study to evaluate the efficacy of niraparib, a potent PARP inhibitor currently in clinical trials, in three different genetically engineered mouse models representing different pancreatic cancer patient populations. The KPC mice carry mutations in genes that are frequently mutated in pancreatic cancer patients and develop tumors that fully recapitulate the biology of human pancreatic ductal adenocarcinoma. These mice will represent the broad population of patients with spontaneous pancreatic tumors. A second model with heterozygous mutations in BRCA2 (KPCB2+/-) will represent patients with hereditary BRCA2 mutations. A third model with homozygous conditional deletion of BRCA2 (KPCB2f/f) will represent spontaneous tumors that have completely lost HDR functions. We show that BRCA2-deficient KPC tumor cells strongly respond to treatment with niraparib in vitro and in vivo. Niraparib efficiently inhibits PARP activity, increases double strand break formation and induces M-phase cell cycle arrest in tumors of KPCB2f/f mice. In the future, we will use pre- and post-treatment tumor biopsies to determine the molecular mechanisms of response to PARP inhibition. The ultimate goal of this study is to identify genetic determinants of PARP sensitivity and validate our results in a clinical setting. Identification of a biomarker would be an extremely valuable clinical tool to classify pancreatic cancer patients that could respond to the treatment with PARP inhibitors, a tool that is currently not available. Citation Format: Barbara Orelli, Stephen A. Sastra, Carmine F. Palermo, Thomas Ludwig, Kenneth P. Olive. Preclinical evaluation of a PARP inhibitor in mice representing genetically different subtypes of pancreatic cancers. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B23.

Abstract 2195: Preclinical evaluation of a PARP inhibitor in mice representing genetically different subtypes of pancreatic cancers.

Abstract The incredibly short survival of patients diagnosed with pancreatic cancer underscores one of the greatest failures of the medical community: our inability to identify effective treatments for pancreatic ductal adenocarcinoma. Most new compounds in clinical trials fail because of highly toxic side effects in normal tissues and limited efficacy in tumor response. An ideal drug would kill only tumor cells and spare the normal cells. In this regard, poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated a promising therapeutic potential, particularly in patients with BRCA-deficient tumors. BRCA-deficient cells have an impaired homology-directed repair (HDR) pathway, a mechanism critical for resolving DNA double strand breaks (DSBs). When PARP1 is inhibited, DNA single strand breaks are not repaired and they mature to double strand breaks, which are very toxic for the cell. Hence, inhibition of PARP1 is selectively toxic to cells with an impaired HDR pathway, such as BRCA-deficient tumor cells, but not to normal cells. Because up to 10% of pancreatic cancer patients carry mutations in either BRCA2 or other proteins involved in the HDR pathway, PARP inhibitors may also prove effective in this setting. However, a number of subtleties remain that call this conclusion into question, including poor drug delivery, loss of heterozygosity, and BRCA-independent alteration of the HDR pathway. We designed a preclinical study to evaluate the efficacy of MK-4827, a potent PARP inhibitor, in three different genetically engineered mouse models representing different pancreatic cancer patient populations. The KPC mice carry mutations in genes that are frequently mutated in pancreatic cancer patients and develop tumors that fully recapitulate the biology of human pancreatic ductal adenocarcinoma. These mice will represent the broad population of patients with spontaneous pancreatic tumors. A second model with heterozygous mutations in BRCA2 (KPCB2+/-) will represent patients with hereditary BRCA2 mutations. A third model with homozygous conditional deletion of BRCA2 (KPCB2f/f) will represent spontaneous tumors that have completely lost HDR functions. We show that BRCA2-deficient KPC tumor cells strongly respond to treatment with MK-4827 in vitro and in vivo. MK-4827 efficiently inhibited PARP activity, increased double strand break formation and induced M-phase cell cycle arrest in tumors of KPCB2f/f mice. In the future, we will use pre- and post-treatment tumor biopsies to determine the molecular mechanisms of response to PARP inhibition. The ultimate goal of this study is to identify genetic determinants of PARP sensitivity and validate our results in a clinical setting. Identification of a biomarker would be an extremely valuable clinical tool to classify pancreatic cancer patients that could respond to the treatment with PARP inhibitors, a tool that is currently not available. Citation Format: Barbara Orelli, Stephen A. Sastra, Carmine F. Palermo, Thomas Ludwig, Kenneth P. Olive. Preclinical evaluation of a PARP inhibitor in mice representing genetically different subtypes of pancreatic cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2195. doi:10.1158/1538-7445.AM2013-2195

Cullin-4A·DNA Damage-binding Protein 1 E3 Ligase Complex Targets Tumor Suppressor RASSF1A for Degradation during Mitosis

Tumor suppressor RASSF1A (RAS association domain family 1, isoform A) is known to play an important role in regulation of mitosis; however, little is known about how RASSF1A is regulated during the mitotic phase of the cell cycle. In the present study, we have identified Cullin-4A (CUL4A) as a novel E3 ligase for RASSF1A. Our results demonstrate that DNA damage-binding protein 1 (DDB1) functions as a substrate adaptor that directly interacts with RASSF1A and bridges RASSF1A to the CUL4A E3 ligase complex. Depletion of DDB1 also diminishes intracellular interactions between RASSF1A and CUL4A. Our results also show that RASSF1A interacts with DDB1 via a region containing amino acids 165-200, and deletion of this region abolishes RASSF1A and DDB1 interactions. We have found that CUL4A depletion results in increased levels of RASSF1A protein due to increased half-life; whereas overexpression of CUL4A and DDB1 markedly enhances RASSF1A protein ubiquitination resulting in reduced RASSF1A levels. We further show that CUL4A-mediated RASSF1A degradation occurs during mitosis, and depletion of CUL4A markedly reverses mitotic-phase-stimulated RASSF1A degradation. We also note that overexpression of CUL4A antagonizes the ability of RASSF1A to induce M-phase cell cycle arrest. Thus, our present study demonstrates that the CUL4A·DDB1 E3 complex is important for regulation of RASSF1A during mitosis, and it may contribute to inactivation of RASSF1A and promoting cell cycle progression.

Mechanisms for the activity of heterocyclic cyclohexanone curcumin derivatives in estrogen receptor negative human breast cancer cell lines

Estrogen receptor (ER)-negative breast cancer is an aggressive form that currently requires more drug treatment options. Thus, we have further modified cyclohexanone derivatives of curcumin and examined them for cytotoxicity towards ER-negative human breast cancer cells. Two of the analogs screened elicited increased cytotoxic potency compared to curcumin and other previously studied derivatives. Specifically, 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91) elicited EC(50) values of 1.54 and 1.10 µM, respectively, in MDA-MB-231 cells and EC(50) values of 0.51 and 0.23 in SKBr3 cells. All other new compounds examined were less potent than curcumin, which elicited EC(50) values of 7.6 and 2.4 µM in MDA-MB-231 and SKBr3 cells, respectively. Mechanistic analyses demonstrated that RL90 and RL91 significantly induced G(2)/M-phase cell cycle arrest and apoptosis. RL90 and RL91 also modulated the expression of key cell signaling proteins, specifically, in SKBr3 cells, protein levels of Her-2, Akt, and NFκB were decreased in a time-dependent manner, while activity of stress kinases JNK1/2 and P38 MAPK were increased. Signaling events in MDA-MB-231 cells were differently implicated, as EGFR protein levels were decreased and activity of GSK-3β transiently decreased, while β-catenin protein level and activity of P38 MAPK, Akt, and JNK1/2 were transiently increased. In conclusion replacement of the phenyl group of cyclohexanone derived curcumin derivatives with heterocyclic rings forms a class of second-generation analogs that are more potent than both curcumin and other derivatives. These new derivatives provide a platform for the further development of drugs for the treatment of ER-negative breast cancer.

A G-quadruplex Stabilizer Induces M-phase Cell Cycle Arrest

G-quadruplex stabilizers such as telomestatin and HXDV bind with exquisite specificity to G-quadruplexes, but not to triplex, duplex, or single-stranded DNAs. Studies have suggested that the antiproliferative and possibly anti-tumor activities of these compounds are linked to their inhibitory effect on telomerase and/or telomere function. In the current studies, we show that HXDV, a synthetic analog of telomestatin, exhibits antiproliferative activity against both telomerase-positive and -negative cells and induces robust apoptosis within 16 h of treatment, suggesting a mode of action independent of telomerase. HXDV was also shown to inhibit cell cycle progression causing M-phase cell cycle arrest, as evidenced by accumulation of cells with 4 n DNA content, increased mitotic index, separated centrosomes, elevated histone H3 phosphorylation at Ser-10 (an M-phase marker), and defective chromosome alignment and spindle fiber assembly (revealed by time-lapse microscopy). The M-phase arrest caused by HXDV paralleled with reduction in the expression level of the major M-phase checkpoint regulator Aurora A. All these cellular effects appear to depend on the G-quadruplex binding activity of HXDV as its non-G-quadruplex binding analog, TXTLeu, is completely devoid of all these effects. In the aggregate, our results suggest that HXDV, which exhibits anti-proliferative and apoptotic activities, is also a novel M-phase blocker, with a mode of action dependent on its G-quadruplex binding activity.

PI3K/Akt is involved in bufalin-induced apoptosis in gastric cancer cells

Bufalin is the active ingredient of the Chinese medicine Chan Su, and it has been reported that bufalin induces apoptosis in some human leukemia and solid cancer cell lines. The exact mechanism of bufalin-induced apoptosis is, however, still not clear. In this study, we demonstrated that bufalin inhibited the proliferation of gastric cancer MGC803 cells in a dose-dependent and time-dependent manner. At a low concentration (20 nmol/l), bufalin induced M-phase cell cycle arrest, whereas at a high concentration (80 nmol/l) it induced apoptosis in MGC803 cells. Bufalin increased the Bax/Bcl-2 ratio and activated caspase-3 during the apoptotic process of MGC803 cells. It should be noted that bufalin transiently activated the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and then inhibited it completely, and upregulated the Casitas B-lineage lymphoma (Cbl) family of ubiquitin ligases, upstream modulators of PI3K. A combination of bufalin and LY294002, a PI3K-specific inhibitor, enhanced apoptosis, but PD98059, an extracellular-regulated protein kinase-specific inhibitor, had no significant effect on bufalin-induced apoptosis. These results suggested that the PI3K/Akt pathway might play a key role in bufalin-induced apoptosis in gastric cancer MGC803 cells.

The combination of epigallocatechin gallate and curcumin suppresses ERα‐breast cancer cell growth in vitro and in vivo

Both epigallocatechin gallate (EGCG) and curcumin have shown efficacy in various in vivo and in vitro models of cancer. This study was designed to determine the efficacy of these naturally derived polyphenolic compounds in vitro and in vivo, when given in combination. Studies in MDA-MB-231 cells demonstrated that EGCG + curcumin was synergistically cytotoxic and that this correlated with G(2)/M-phase cell cycle arrest. After 12 hr, EGCG (25 microM) + curcumin (3 microM) increased the proportion of cells in G(2)/M-phase to 263 +/- 16% of control and this correlated with a 50 +/- 4% decrease in cell number compared to control. To determine if this in vitro result would translate in vivo, athymic nude female mice were implanted with MDA-MB-231 cells and treated with curcumin (200 mg/kg/day, po), EGCG (25 mg/kg/day, ip), EGCG + curcumin, or vehicle control (5 ml/kg/day, po) for 10 weeks. Tumor volume in the EGCG + curcumin treated mice decreased 49% compared to vehicle control mice (p < 0.05), which correlated with a 78 +/- 6% decrease in levels of VEGFR-1 protein expression in the tumors. Curcumin treatment significantly decreased tumor protein levels of EGFR and Akt, however the expression of these proteins was not further decreased following combination treatment. Therefore, these results demonstrate that the combination of EGCG and curcumin is efficacious in both in vitro and in vivo models of ER alpha-breast cancer and that regulation of VEGFR-1 may play a key role in this effect.

Mitotic kinase Aurora-A phosphorylates RASSF1A and modulates RASSF1A-mediated microtubule interaction and M-phase cell cycle regulation

RASSF1A (RAS-association domain family 1, isoform A) is a newer tumor suppressor that binds to and stabilizes microtubules as well as induces M-phase cell cycle arrest. Several other proteins that interact with and stabilize microtubules also undergo mitotic phase phosphorylation to regulate microtubule dynamics and M-phase cell cycle progression. Currently, however, there is a paucity of information regarding the phosphorylation status of RASSF1A and its regulation during mitosis. In this study, for the first time, we demonstrate that Aurora-A is a RASSF1A kinase and, to the best of our knowledge, this is also the first study reporting the identification of a kinase for RASSF1A. We show that the mitotic kinase Aurora-A directly interacts with and phosphorylates RASSF1 and that RASSF1A is phosphorylated by Aurora-A during mitosis. These findings therefore link an important oncogenic mitotic kinase to regulate RASSF1A tumor suppressor. Aurora-A appears to phosphorylate RASSF1A at Threonine202 and/or Serine203 that reside within the known microtubule-binding domain of RASSF1A. Substitutions of these residues with glutamic acid at both positions, mimicking constitutive phosphorylation of RASSF1A, disrupt RASSF1A interactions with microtubules and abolish its ability to induce M-phase cell cycle arrest. Our results further demonstrate that Aurora-A overexpression also interferes with RASSF1A-mediated growth suppression. In view of our results, we propose that Aurora-A-mediated phosphorylation of RASSF1A is a novel mechanism that regulates the ability of this tumor suppressor to interact with microtubules and modulate M-phase cell cycle progression.

WP760, a melanoma selective drug

Our goal was to perform studies on the specificity and antimelanoma mechanism of a novel bis-anthracycline, WP760. WP760 initially identified in the NCI 160 screen as anti-melanoma. The methyl thiazolyl tetrazolium reduction (MTT) assay was used to test tumor cell growth inhibition; confocal microscopy to view WP760 intracellular distribution; flow cytometry for cell-cycle arrest and apoptosis; and Western blotting was employed to identify and compare quantities and kinetics of cell growth related molecule levels. WP760 induced G(2)/M-phase cell-cycle arrest and apoptosis in melanoma cell lines and short-term melanoma explants established from clinical specimens in a time and concentration dependent manner at nM concentrations. In contrast, effects on fibroblasts and A549 lung cancer cells required higher concentrations, suggesting that WP760 possesses selectivity for melanoma. Molecular studies indicated that WP760 induced p53 stabilization, checkpoint kinase 2 and p27(Kip1) protein upregulation, and activation of caspase-3. Endogenous nitric oxide (NO) production has been implicated in the chemoresistance of melanoma; WP760 caused inhibition of the inducible nitric oxide synthase (iNOS) protein as well as inhibition of phosphorylation of ERK, known to drive the iNOS pathway. Based on WP760 localization into mitochondria, and caspase-3 inhibitor block the killing of WP760, the intrinsic pathway of apoptosis appears to have been activated. Our results indicate that WP760 affects a critical and unique set of growth regulatory effects in melanoma, and is a promising candidate for further preclinical studies.

Quercetin Inhibits eNOS, Microtubule Polymerization, and Mitotic Progression in Bovine Aortic Endothelial Cells

Quercetin (QRN), one of the most abundant flavonoids in the human diet, is a known antioxidant and inhibitor of cancer cell cycle progression. Here, we provide the first evidence that QRN inhibits angiogenesis via a mechanism involving both suppression of endothelial nitric oxide synthase (eNOS) and early M-phase cell cycle arrest. Bovine aortic endothelial (BAE) cells were exposed to doses of up to 100 micromol/L QRN and assayed for eNOS activity and phosphorylation status. Phosphorylation of eNOS at Ser 617 (bovine sequence) is thought to occur in response to Akt stimulation and to be required for eNOS activity. Together with basal eNOS activity, eNOS phosphorylation at Ser 617 and Akt Ser 473 phosphorylation were dose dependently and concomitantly suppressed by QRN within 30 min. Furthermore, although the significant (P < 0.05) inhibitory effect of a single 100 micromol/L QRN dose on eNOS activity was overcome within approximately 24 h, chronic QRN exposures (24-48 h) led to early M-phase arrest and disruption of mitotic microtubule polymerization. In vivo, QRN administered i.p. to female Balb/C mice bearing both syngeneic mammary tumors and Matrigel implants suppressed angiogenesis as measured by endothelial cell immunohistochemistry and hemoglobin concentration. Taken together, these findings suggest a dual mechanism by which QRN suppresses endothelial cell proliferation, both acutely via inhibition of eNOS Ser 617 phosphorylation, and chronically via perturbation of mitotic microtubule polymerization. This novel mechanism of QRN in endothelial cells may in part explain its inhibitory action on angiogenesis and further discern a potential role of QRN as a chemopreventive agent.

The sulindac derivatives OSI-461, OSIP486823, and OSIP487703 arrest colon cancer cells in mitosis by causing microtubule depolymerization

Exisulind (sulindac sulfone) and three highly potent derivatives, OSI-461 (CP461), OSIP486823 (CP248), and OSIP487703, inhibit growth and induce apoptosis in SW480 human colon cancer cells, with IC(50)s of 200, 2, 0.1, and 0.003 micromol/L, respectively. The latter three compounds, but not exisulind, induce marked M-phase cell cycle arrest in these cells. This effect seems to be independent of the known ability of these compounds to cause activation of protein kinase G. When tested at twice their IC(50) concentration for growth inhibition, OSI-461, OSIP486823, and OSIP487703 cause depolymerization of microtubules in interphase cells, inhibit spindle formation in mitotic cells, and induce multinucleated cells. In vitro tubulin polymerization assays indicate that all three compounds interact with tubulin directly to cause microtubule depolymerization and/or inhibit de novo tubulin polymerization. These results suggest that the dual effects of OSI-461, OSIP486823, and OSIP487703 on impairment of microtubule functions and protein kinase G activation may explain the potent antiproliferative and apoptotic effects of these compounds in cancer cells.

Effector mechanisms of norcantharidin-induced mitotic arrest and apoptosis in human hepatoma cells.

NCTD is a demethylated form of cantharidin with antitumor properties, which is now in use as a routine anticancer drug against hepatoma. However, there is limited information on the effect of NCTD on human cancer cells. In the present study, NCTD inhibited proliferation, caused mitotic arrest, then progressed to apoptosis within 96 hr in 3 human hepatoma cell lines: HepG2, Hep3B and Huh-7. NCTD treatment (5 microg/ml) enhanced the expression of Cdc25C and p21(Cip1/Waf1), increasing the phosphorylation of these 2 proteins. In addition, NCTD treatment induced an earlier increase in cyclin B1-associated histone H1 kinase activity within 48 hr, but an approximately 70% reduction of both protein level and kinase activity of cyclin B1 was observed at 72 hr. Treatment with NCTD significantly decreased the expression of p53 protein but did not affect the expression of Cdk1 and p27(Kip1). Moreover, NCTD treatment also increased the phosphorylation of Bcl-2 and Bcl-X(L) but did not affect the expression of Bax or Bad. Bcl-2 phosphorylation appears to inhibit its binding to Bax since less Bax was detected in immunocomplex with Bcl-2 in NCTD-treated HepG2 cells. In addition, NCTD treatment caused activation of caspase-9 and caspase-3, preceding DNA fragmentation and morphologic features of apoptosis. Pretreatment with the broad-spectrum caspase inhibitor z-VAD-fmk markedly inhibited NCTD-induced caspase-3 activity and cell death. These results suggest that phosphorylation of p21(Cip1/Waf1) and Cdc25C and biphasic regulation of cyclin B1-associated kinase activity may contribute to NCTD-induced M-phase cell-cycle arrest. Furthermore, the increase of p21(Cip1/Waf1), phosphorylation of Bcl-2 and Bcl-X(L), activation of caspase-9 and caspase-3 may be the molecular mechanism through which NCTD induces apoptosis.

Decreased pulmonary radiation resistance of manganese superoxide dismutase (MnSOD)-deficient mice is corrected by human manganese superoxide dismutase-Plasmid/Liposome (SOD2-PL) intratracheal gene therapy.

The pulmonary ionizing radiation sensitivity of C57BL/6 Sod2(+/-) mice heterozygous for MnSOD deficiency was compared to that Sod2(+/+) control littermates. Embryo fibroblast cell lines from Sod2(-/-) (neonatal lethal) or Sod2(+/-) mice produced less biochemically active MnSOD and demonstrated a significantly greater in vitro radiosensitivity. No G(2)/M-phase cell cycle arrest after 5 Gy was observed in Sod2(-/-) cells compared to the Sod2(+/-) or Sod2(+/+) lines. Subclonal Sod2(-/-) or Sod2(+/-) embryo fibroblast lines expressing the human SOD2 transgene showed increased biochemical activity of MnSOD and radioresistance. Sod2(+/-) mice receiving 18 Gy whole-lung irradiation died sooner and had an increased percentage of lung with organizing alveolitis between 100 and 160 days compared to Sod2(+/+) wild-type littermates. Both Sod2(+/-) and Sod2(+/+) littermates injected intratracheally with human manganese superoxide dismutase-plasmid/liposome (SOD2-PL) complex 24 h prior to whole-lung irradiation showed decreased DNA strand breaks and improved survival with decreased organizing alveolitis. Thus underexpression of MnSOD in the lungs of heterozygous Sod2(+/-) knockout mice is associated with increased pulmonary radiation sensitivity and parallels increased radiation sensitivity of embryo fibroblast cell lines in vitro. The restoration of cellular radioresistance in vitro and in lungs in vivo by SOD2-PL transgene expression supports a potential role for SOD2-PL gene therapy in organ-specific radioprotection.

Mos‐induced p42 mitogen‐activated protein kinase activation stabilizes M‐phase in Xenopus egg extracts after cyclin destruction

Previously, we have shown that the addition of a constitutively-active mitogen-activated protein kinase kinase protein (MAPKK = MEK) to cycling Xenopus egg extracts activates the p42MAPK pathway, leading to a G2 or M-phase cell cycle arrest. The stage of the arrest depends on the timing of p42MAPK activation. If p42MAPK is activated prior to M-phase, or after exit from M-phase, the extract is arrested in G2. If p42MAPK is activated during entry into M-phase, the extract is arrested in M-phase. In this study, we show that the addition of recombinant Mos protein (which directly phosphorylates and activates MEK) to cycling egg extracts has the same effect as those described for MEK. The addition of Mos to the extract at the start of incubation leads to a G2 arrest with large interphase nuclei with intact nuclear envelopes. If Mos is added at later times, however, the activation of p42MAPK leads to an M-phase arrest with condensed chromosomes and mitotic arrays of microtubules. Moreover, the extent of M-phase specific phosphorylations is shown by the sustained presence of phosphoproteins that are detected by the monoclonal antibody MPM-2. Unexpectedly, in certain M-phase arrested extracts, histone H1 kinase activity levels reach a peak on entry into M-phase but then fall abruptly to interphase levels. When these extracts are analyzed by immunoblotting, Cyclin B2 is destroyed in those samples containing low maturation promoting factor activity (MPF, cyclin B/Cdc2), yet chromosomes remain condensed with associated mitotic arrays of microtubules and M-phase-specific phosphorylations are sustained. These results suggest that although MPF is required for entry into M-phase, once established, M-phase can be maintained by the p42MAPK pathway after the proteolysis of mitotic cyclins.

Mos-induced p42 mitogen-activated protein kinase activation stabilizes M-phase in Xenopus egg extracts after cyclin destruction

Previously, we have shown that the addition of a constitutively-active mitogen-activated protein kinase kinase protein (MAPKK = MEK) to cycling Xenopus egg extracts activates the p42MAPK pathway, leading to a G2 or M-phase cell cycle arrest. The stage of the arrest depends on the timing of p42MAPK activation. If p42MAPK is activated prior to M-phase, or after exit from M-phase, the extract is arrested in G2. If p42MAPK is activated during entry into M-phase, the extract is arrested in M-phase. In this study, we show that the addition of recombinant Mos protein (which directly phosphorylates and activates MEK) to cycling egg extracts has the same effect as those described for MEK. The addition of Mos to the extract at the start of incubation leads to a G2 arrest with large interphase nuclei with intact nuclear envelopes. If Mos is added at later times, however, the activation of p42MAPK leads to an M-phase arrest with condensed chromosomes and mitotic arrays of microtubules. Moreover, the extent of M-phase specific phosphorylations is shown by the sustained presence of phosphoproteins that are detected by the monoclonal antibody MPM-2. Unexpectedly, in certain M-phase arrested extracts, histone H1 kinase activity levels reach a peak on entry into M-phase but then fall abruptly to interphase levels. When these extracts are analyzed by immunoblotting, Cyclin B2 is destroyed in those samples containing low maturation promoting factor activity (MPF, cyclin B Cdc2 ), yet chromosomes remain condensed with associated mitotic arrays of microtubules and M-phase-specific phosphorylations are sustained. These results suggest that although MPF is required for entry into M-phase, once established, M-phase can be maintained by the p42MAPK pathway after the proteolysis of mitotic cyclins.