Modulation of cell-cycle regulatory signaling network by 2-methoxyestradiol in prostate cancer cells is mediated through multiple signal transduction pathways

2-Methoxyestradiol (2-ME(2)), a promising anticancer drug, induces growth arrest and apoptosis in various androgen-dependent (LNCaP) and -independent (DU145 and PC-3) prostate cancer cell lines. Moreover, flow cytometric analysis indicated a novel dual impact of 2-ME(2) on the cell division cycle of prostate cancer cells. Chronic exposure of high doses of 2-ME(2) enhance the accumulation of cells in S and G2/M phases, while cell numbers in the G1 phase were reduced significantly by this treatment. Because cyclin B1 overexpression, induction of cdc2 phosphorylation, and its regulatory proteins wee1 and phospho-cdc25C (interphase and mitotic forms) by 2-ME(2) treatment correlated with the induction of apoptosis, growth arrest at the G2/M phase, and accumulation of the S phase, we reasoned that cyclin B1 and cdc2 phosphorylation and its upstream regulatory molecular networks may be associated with the ultimate impacts of 2-ME(2). Because phosphorylation of cdc2 and upregulation of wee1 by 2-ME(2) can be abolished by both extracellular receptor kinase (ERK) inhibitor (U0126) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), our studies indicate that the 2-ME(2)-induced upregulation of wee1 and subsequent cdc2 phosphorylation are mediated through mitogen-activated protein kinase (MAPK)-ERK-JNK signaling pathways.     

Mitotic catastrophe and apoptosis induced by docetaxel in hormone-refractory prostate cancer cells

Studies performed in different experimental and clinical settings have shown that Docetaxel (Doc) is effective in a wide range of tumors and that it exerts its activity through multiple mechanisms of action. However, the sequence of events induced by Doc which leads to cell death is still not fully understood. Moreover, it is not completely clear how Doc induces mitotic catastrophe and whether this process is an end event or followed by apoptosis or necrosis. We investigated the mechanisms by which Doc triggers cell death in hormone-refractory prostate cancer cells by analyzing cell cycle perturbations, apoptosis-related marker expression, and morphologic cell alterations. Doc induced a transient increase in G2/M phase followed by the appearance of G0/1 hypo- and hyperdiploid cells and increased p21 expression. Time- and concentration-dependent apoptosis was induced in up to 70% of cells, in concomitance with Bcl-2 phosphorylation, which was followed by caspase-2 and -3 activation. In conclusion, Doc would seem to trigger apoptosis in hormone-refractory prostate cancer cells via mitotic catastrophe through two forms of mitotic exit, in concomitance with increased p21 expression and caspase-2 activation.     

Caffeine induces G2/M arrest and apoptosis via a novel p53-dependent pathway in NB4 promyelocytic leukemia cells

Methylxantine derivative, caffeine, is known to prevent the p53-dependent apoptosis pathway via inhibition of ATM (ataxia telangiectasia mutated) kinase, which activates p53 by phosphorylation of the Ser-15 residue. In contrast, it has been reported that caffeine induces p53-mediated apoptosis through Bax protein in non-small-cell lung cancer cells. Therefore, the effects of caffeine on cellular growth in malignant cells are controversial. We investigated the effects of caffeine on cell proliferation, cell cycle progression, and induction of apoptosis in NB4 promyelocytic leukemia cells containing wild-type p53. Caffeine suppressed the cellular growth of NB4 cells in a dose- and time-dependent manner. Caffeine induced G(2)/M phase cell cycle arrest in NB4 cells in association with the induction of phosphorylation at the Ser-15 residue of p53 and induction of tyrosine phosphorylation of cdc2. Expression of Bax protein was increased in NB4 cells after treatment with caffeine. Interestingly, the antisense oligonucleotides for p53 significantly reduced p53 expression and caffeine-induced G(2)/M phase cell cycle arrest in NB4 cells. These results suggest that caffeine induces cell cycle arrest and apoptosis in association with activation of p53 by a novel pathway to phosphorylate the Ser-15 residue and induction of phosphorylation of cdc 2 in leukemic cells with normal p53.     

Sustained activation of p34(cdc2) is required for noscapine-induced apoptosis.

Mitotic arrest and subsequent apoptosis has been observed in many types of cells treated with anti-microtubule agents. However, the molecular mechanisms underlying the two events as well as their relationship are not well understood; on the contrary, there has been increasing evidence indicating that anti-microtubule agents might induce apoptosis via signaling pathways independent of mitosis. In this study, we found that apoptosis induced by noscapine, an anti-microtubule drug previously shown to cause both mitotic arrest and apoptotic cell death, was blocked by inhibiting p34(cdc2) activity with olomoucine in FM3A murine mammary carcinoma cells or by reducing the level and activity of p34(cdc2) in a mutant cell line FT210 derived from FM3A. Furthermore, transfection of the mutant FT210 cells with wild-type p34(cdc2) restored their ability to undergo mitotic arrest and then apoptosis in response to noscapine. Thus, we conclude that sustained activation of the p34(cdc2) kinase during mitotic arrest is required for subsequent apoptosis induced by noscapine, establishing a link between the two events.     

Evaluation of 4-phenylamino-substituted naphthalene-1,2-diones as tubulin polymerization inhibitors

A series of 4-phenylamino-substituted naphthalene-1,2-dione derivatives were prepared and evaluated as effective antiproliferative agents. MTT assays showed that the compounds with a methyl group on the nitrogen linker exhibited potent antiproliferative activities against human cancer cells. The mechanistic study revealed that these compounds could induce mitochondrial depolarization, which resulted in intracellular ROS production, and they also acted as tubulin polymerization inhibitors. Moreover, the typical compound could arrest A549 cells in the G2/M phase, resulting in cellular apoptosis and induced mitotic arrest in A549 cells through disrupting microtubule dynamics.     

KUD773, a phenylthiazole derivative,displays anticancer activity in human hormone-refractory prostate cancers through inhibition of tubulin polymerization and anti-Aurora A activity

Background

Hormone-refractory prostate cancer (HRPC), which is resistant to hormone therapy, is a major obstacle in clinical treatment. An approach to inhibit HRPC growth and ultimately to kill cancers is highly demanded.

Results

KUD773 induced the anti-proliferative effect and subsequent apoptosis in PC-3 and DU-145 (two HRPC cell lines); whereas, it showed less active in normal prostate cells. Further examination showed that KUD773 inhibited tubulin polymerization and induced an increase of mitotic phosphoproteins and polo-like kinase 1 (PLK1) phosphorylation, indicating a mitotic arrest of the cell cycle through an anti-tubulin action. The kinase assay demonstrated that KUD773 inhibited Aurora A activity. KUD773 induced an increase of Cdk1 phosphorylation at Thr¹⁶¹ (a stimulatory phosphorylation site) and a decrease of phosphorylation at Tyr¹⁵ (an inhibitory phosphorylation site), suggesting the activation of Cdk1. The data were substantiated by an up-regulation of cyclin B1 (a Cdk1 partner). Furthermore, KUD773 induced the phosphorylation and subsequent down-regulation of Bcl-2 and activation of caspase cascades.

Conclusions

The data suggest that KUD773 induces apoptotic signaling in a sequential manner. It inhibits tubulin polymerization associated with an anti-Aurora A activity, leading to Cdk1 activation and mitotic arrest of the cell cycle that in turn induces Bcl-2 degradation and a subsequent caspase activation in HRPCs.     

Human Parvovirus B19 Induces Cell Cycle Arrest at G2 Phase with Accumulation of Mitotic Cyclins

Human parvovirus B19 infects specifically erythroid progenitor cells, which causes transient aplastic crises and hemolytic anemias. Here, we demonstrate that erythroblastoid UT7/Epo cells infected with B19 virus fall into growth arrest with 4N DNA, indicating G(2)/M arrest. These B19 virus-infected cells displayed accumulation of cyclin A, cyclin B1, and phosphorylated cdc2 and were accompanied by an up-regulation in the kinase activity of the cdc2-cyclin B1 complex, similar to that in cells treated with the mitotic inhibitor. However, degradation of nuclear lamina and phosphorylation of histone H3 and H1 were not seen in B19 virus-infected cells, indicating that the infected cells do not enter the M phase. Accumulation of cyclin B1 was persistently localized in the cytoplasm, but not in the nucleus, suggesting that B19 virus infection of erythroid cells raises suppression of nuclear import of cyclin B1, resulting in cell cycle arrest at the G(2) phase. The B19 virus-induced G(2)/M arrest may be the critical event in the damage of erythroid progenitor cells seen in patients with B19 virus infection.     

Effectiveness of the Histone Deacetylase Inhibitor (S)-2 against LNCaP and PC3 Human Prostate Cancer Cells

Histone deacetylase inhibitors (HDACi) represent a promising class of epigenetic agents with anticancer properties. Here, we report that (S)-2, a novel hydroxamate-based HDACi, shown previously to be effective against acute myeloid leukemia cells, was also a potent inducer of apoptosis/differentiation in human prostate LNCaP and PC3 cancer cells. In LNCaP cells (S)-2 was capable of triggering H3/H4 histone acetylation, H2AX phosphorylation as a marker of DNA damage and producing G₀/G₁ cell cycle arrest. Consistently, (S)-2 led to enhanced expression of both the protein and mRNA p21 levels in LNCaP cells but, contrary to SAHA, not in normal non-tumorigenic prostate PNT1A cells. Mechanistic studies demonstrated that (S)-2-induced apoptosis in LNCaP cells developed through the cleavage of pro-caspase 9 and 3 and of poly(ADP-ribose)-polymerase accompanied by the dose-dependent loss of mitochondrial membrane potential. Indeed, the addition of the pan-caspase inhibitor Z-VAD-fmk greatly reduced drug-mediated apoptosis while the antioxidant N-acetyl-cysteine was virtually ineffective. Importantly, preliminary data with nude mice xenografted with LNCaP cells showed that (S)-2 prompted a decrease in the tumor volume and an increase in H2AX phosphorylation within the cancer cells. Moreover, the highly metastatic prostate cancer PC3 cells were also sensitive to (S)-2 that: i) induced growth arrest and moderate apoptosis; ii) steered cells towards differentiation and neutral lipid accumulation; iii) reduced cell invasiveness potential by decreasing the amount of MMP-9 activity and up-regulating TIMP-1 expression; and iv) inhibited cell motility and migration through the Matrigel. Overall, (S)-2 has proven to be a powerful HDACi capable of inducing growth arrest, cell death and/or differentiation of LNCaP and PC3 prostate cancer cells and, due to its low toxicity and efficacy in vivo, might also be of clinical interest to support conventional prostate cancer therapy.     

The cell cycle control gene cdc2+ of fission yeast encodes a protein kinase potentially regulated by phosphorylation

The cdc2+ gene function has an important role in controlling the commitment of the fission yeast cell to the mitotic cycle and the timing of mitosis. We have raised antibodies against the cdc2+ protein using synthetic peptides and have demonstrated that it is a 34 kd phosphoprotein with protein kinase activity. The protein level and phosphorylation state remain unchanged during the mitotic cycle of rapidly growing cells. When cells cease to proliferate and arrest in G1 the protein becomes dephosphorylated and loses protein kinase activity. Exit from the mitotic cycle and entry into stationary phase may be controlled in part by modulation of the cdc2 protein kinase activity by changes in its phosphorylation state.     

Survivin phosphorylation and M-phase promoting factor in oral carcinogenesis

Survivin is a recently described inhibitor of apoptosis and mitotic regulator which is selectively over-expressed in human tumors. Its expression rate is predictive of disease progression, early recurrences and resistance to therapy. Up-regulation of survivin in oral pre-malignant lesions (OPL) and in oral squamous cell carcinoma (OSCC) has already been demonstrated in previous studies. A critical step for activation of survivin has been identified in the phosphorylation on Thr34 by the main mitotic kinase p34cdc2-cyclin B1. The aim of this work was to investigate the relationship between survivin, its phosphorylated active form (p-survivin) and M-phase promoting factor (MPF), p34cdc2-cyclin B1 in oral carcinogenesis. 32 OSCCs and 17 OPLs from surgical specimens were studied for cyclin B1, p-survivin, survivin, and p34cdc2 expression by immunohistochemistry. All cases of OSCC expressed survivin and its expression rate was correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 80% of cases, while p-34cdc2 was over-expressed in all OSCCs. All OPLs associated with OSCC expressed survivin and its levels were correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 70% of cases, while p-34cdc2 was positive in all OPLs. In conclusion, this study demonstrated that MPF, survivin and p-survivin are expressed during early and late phase of oral carcinogenesis. MPF proteins, which are co-expressed on mitotic apparatus, could represent a potential target for therapies based on manipulation of survivin phosphorylation, which would induce apoptosis in cancer cells.     

Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae.

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.     

Checkpoint kinase 1 regulates diallyl trisulfide-induced mitotic arrest in human prostate cancer cells

We have shown previously that diallyl trisulfide (DATS), a constituent of processed garlic, inhibits proliferation of PC-3 and DU145 human prostate cancer cells by causing G(2)-M phase cell cycle arrest in association with inhibition of cyclin-dependent kinase 1 activity and hyperphosphorylation of Cdc25C at Ser(216). Here, we report that DATS-treated PC-3 and DU145 cells are also arrested in mitosis as judged by microscopy following staining with anti-alpha-tubulin antibody and 4',6-diamidino-2-phenylindole and flow cytometric analysis of Ser(10) phosphorylation of histone H3. The DATS treatment caused activation of checkpoint kinase 1 and checkpoint kinase 2, which are intermediaries of DNA damage checkpoints and implicated in Ser(216) phosphorylation of Cdc25C. The diallyl trisulfide-induced Ser(216) phosphorylation of Cdc25C as well as mitotic arrest were significantly attenuated by knockdown of check-point kinase 1 protein in both PC-3 and DU145 cells. On the other hand, depletion of checkpoint kinase 2 protein did not have any appreciable effect on G(2) or M phase arrest or Cdc25C phosphorylation caused by diallyl trisulfide. The lack of a role of checkpoint kinase 2 in diallyl trisulfide-induced phosphorylation of Cdc25C or G(2)-M phase cell cycle arrest was confirmed using HCT-15 cells stably transfected with phosphorylation-deficient mutant (T68A mutant) of checkpoint kinase 2. In conclusion, the results of the present study suggest existence of a checkpoint kinase 1-dependent mechanism for diallyl trisulfide-induced mitotic arrest in human prostate cancer cells.     

Two S-phase checkpoint systems, one involving the function of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit NIMA and prevent premature mitosis.

We demonstrate that there are at least two S-phase checkpoint mechanisms controlling mitosis in Aspergillus. The first responds to the rate of DNA replication and inhibits mitosis via tyrosine phosphorylation of p34cdc2. Cells unable to tyrosine phosphorylate p34cdc2 are therefore viable but are unable to tolerate low levels of hydroxyurea and prematurely enter lethal mitosis when S-phase is slowed. However, if the NIMA mitosis-promoting kinase is inactivated then non-tyrosine-phosphorylated p34cdc2 cannot promote cells prematurely into mitosis. Lack of tyrosine-phosphorylated p34cdc2 also cannot promote mitosis, or lethality, if DNA replication is arrested, demonstrating the presence of a second S-phase checkpoint mechanism over mitotic initiation which we show involves the function of BIME. In order to overcome the S-phase arrest checkpoint over mitosis it is necessary both to prevent tyrosine phosphorylation of p34cdc2 and also to inactivate BIME. Lack of tyrosine phosphorylation of p34cdc2 allows precocious expression of NIMA during S-phase arrest, and lack of BIME then allows activation of this prematurely expressed NIMA by phosphorylation. The mitosis-promoting NIMA kinase is thus a target for S-phase checkpoint controls.     

Critical role of cyclin B1/Cdc2 up-regulation in the induction of mitotic prometaphase arrest in human breast cancer cells treated with 2-methoxyestradiol

Earlier studies showed that 2-methoxyestradiol (2ME(2)), an endogenous nonpolar metabolite of estradiol-17β, is a strong inducer of G(2)/M cell cycle arrest (based on analysis of cellular DNA content) in human cancer cell lines. The present study sought to investigate the molecular mechanism underlying 2ME(2)-induced cell cycle arrest. We found that 2ME(2) can selectively induce mitotic prometaphase arrest, but not G(2) phase arrest, in cultured MDA-MB-435s and MCF-7 human breast cancer cells. During the induction of prometaphase arrest, there is a time-dependent initial up-regulation of cyclin B1 and Cdc2 proteins, occurring around 12-24h. The strong initial up-regulation of cyclin B1 and Cdc2 matches in timing the 2ME(2)-induced prometaphase arrest. The 2ME(2)-induced prometaphase arrest is abrogated by selective knockdown of cyclin B1 and Cdc2, or by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or by co-treatment of cells with cycloheximide, a protein synthesis inhibitor that was found to suppress the early up-regulation of cyclin B1 and Cdc2. In addition, we provided evidence showing that MAD2 and JNK1 are important upstream mediators of 2ME(2)-induced up-regulation of cyclin B1 and Cdc2 as well as the subsequent induction of mitotic prometaphase arrest. In conclusion, treatment of human cancer cells with 2ME(2) causes up-regulation of cyclin B1 and Cdc2, which then mediate the induction of mitotic prometaphase arrest.     

p56(chk1) protein kinase is required for the DNA replication checkpoint at 37 degrees C in fission yeast.

Fission yeast p56(chk1) kinase is known to be involved in the DNA damage checkpoint but not to be required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU). For this reason, p56(chk1) is considered not to be necessary for the DNA replication checkpoint which acts through the inhibitory phosphorylation of p34(cdc2) kinase activity. In a search for Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive DNA polymerase delta strain at 37 degrees C, we isolated two chk1 alleles. These alleles are proficient for the DNA damage checkpoint, but induce mitotic catastrophe in several S phase thermosensitive mutants. We show that the mitotic catastrophe correlates with a decreased level of tyrosine phosphorylation of p34(cdc2). In addition, we found that the deletion of chk1 and the chk1 alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells are grown at 37 degrees C. These findings suggest that chk1 is important for the maintenance of the DNA replication checkpoint in S phase thermosensitive mutants and that the p56(chk1) kinase must possess a novel function that prevents premature activation of p34(cdc2) kinase under conditions of impaired DNA replication at 37 degrees C.     

Pretreatment of docetaxel enhances TRAIL-mediated apoptosis in prostate cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether treatment of docetaxel (DTX) can enhance apoptotic cell death by TRAIL against androgen-independent prostate cancer (AIPC). The cell death effect of combinations of TRAIL and docetaxel on prostate cancer cell lines (androgen-dependent LNCaP and its derived androgen-independent, metastatic C4-2B) was evaluated by synergisms of apoptosis. Western blot assay and DNA fragmentation assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL induced apoptosis in the presence of docetaxel. In addition, we investigated the in vitro anti-tumor effects of combined docetaxel and TRAIL using MAP kinase inhibitors. Docetaxel itself could not induce apoptotic cell death in 24 h even in high concentration. Apoptotic cell death, however, was drastically enhanced by pretreatment of docetaxel 20 h before TRAIL treatment. Docetaxel enhanced the PARP-1 cleavage and caspases activation by TRAIL especially in androgen-independent, metastatic C4-2B cell line, mainly by phosphorylation of Bcl-2 by JNK activation. It appears that apoptotic cell death was protected by the JNK inhibitor SP600125. The results of our study show that pretreatment of docetaxel is able to enhance the apoptosis produced by TRAIL in prostate cancer cells, especially in hormone-refractory prostate cancer (HRPC).