Cell Cycle Regulatory Proteins Cyclin Research Articles

Expression of cell-cycle regulatory proteins BUBR1, MAD2, Aurora A, cyclin A and cyclin E in invasive ductal breast carcinomas.

Cyclin A, cyclin E, BUBR1, MAD2 and Aurora A are all cell-cycle regulatory proteins and have been proven to play crucial roles in carcinogenesis. However, their expression patterns in invasive ductal breast carcinoma (IDBC) are controversial and unclear. In this study, we examined the expression status of these candidate proteins in a set of 117 invasive ductal carcinomas, and evaluated their associations with known clinicopathological parameters and the expressions of estrogen receptor, progesterone receptor, Ki-67 and Her-2. Univariate and multivariate data analyses both displayed that positive BUBR1 expression was associated with a high Ki-67 labeling index, and negative MAD2 expression was associated with Her-2 overexpression. Positive BUBR1 expression was also associated with a high histological tumor grade in univariate analysis, but not in multivariate analysis. In addition, high Aurora A expression was weakly associated with lymph node metastasis, and cyclin A was strongly associated with the expression of cyclin E in both univariate and multivariate models. In conclusion, this study suggests that evaluation of BUBR1, MAD2 and Aurora A expression levels is likely to improve accuracy of prognostic predictions in IDBC.

Abstract 2379: Cyclin D3 is important for ErbB2-induced mammary cancer initiation and progression in the absence of Cyclin D1

Abstract The cell cycle regulatory protein Cyclin D1 has been reported to be essential for both normal mammary gland development during pregnancy and the malignant transformation of the mammary epithelium in response to receptor tyrosine kinase (RTK) signaling. We have recently reported that the prolactin receptor-associated kinase Jak2 controls the transcriptional activation and, more importantly, the nuclear accumulation of Cyclin D1 in normal mammary epithelial cells via expression and activation of Akt1. We also demonstrated that the functional ablation of Jak2 and Stat5 in ErbB2-expressing mammary epithelial cells protects against the onset of mammary tumorigenesis. Jak2, however, is not required for the survival and proliferation of neoplastic cells. The constitutive activation of ErbB2 signaling, which is an initial event in the formation of mammary cancer in this model, was able to override the functional role of Jak2 in regulating the expression of Akt1 and Cyclin D1. To further define the role of the downstream effector Cyclin D1 during cancer initiation and progression in response to Jak2 and RTK signaling, we generated an ErbB2-associated mammary cancer model that allows a functional ablation of Cyclin D1 either prior to or following neoplastic transformation. Surprisingly, and in sharp contrast to previous reports, nearly all Cyclin D1-deficient females expressing wildtype ErbB2 developed mammary tumors. While Cyclin D1 deficiency extended the tumor-free survival, this particular cell cycle regulatory protein was clearly not required for tumor maintenance and progression. Growth and proliferation of the vast majority of Cyclin D1-deficient primary mammary cancers was accompanied by an upregulation of Cyclin D3. Since Cyclin D2 expression remained very low, knocking down the last remaining D-type cyclin (i.e. Cyclin D3) in tumor-derived primary cancer cells using three different shRNA constructs was sufficient to attenuate the proliferation rates. Interestingly, the ablation of the pool of all three D-type cyclins did not result in an upregulation of Cyclin E. Collectively, the results of this study show that Cyclin D1 is dispensable for neoplastic transformation of mammary epithelial cells and it might therefore not serve as a sole therapeutic target to treat ErbB2-associated breast cancers as previously suggested. In addition, these observations indicate that Cyclin D1 is most likely not the only downstream effector of Jak2 signaling that mediates the protective effects of a functional ablation of Jak2 in ErbB2-induced mammary carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2379. doi:10.1158/1538-7445.AM2011-2379

Abstract 4546: Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

Abstract The synthetic steroid hormone mifepristone (MF) was originally developed as a glucocorticoid receptor (GR) antagonist, but the capacity of this compound to modulate the activity of the progesterone receptor (PR) led to an intensive study of its potential applications as an antiprogestin. The emphasis on the interaction between MF and PR has been translated to its application as a potential therapeutic agent in the field of oncology; however, it remains unclear whether the expression of PR is required for MF to act as anti-cancer agent. Our laboratory has previously shown that MF is a potent inhibitor of ovarian cancer (OvCa) cell growth, and that the cytostatic action of MF occurs through an arrest in the G1 phase of the cell cycle characterized by a decrease in cyclin dependent kinase (Cdk) 2 activity. In this study, we questioned whether the growth inhibitory properties of MF observed in OvCa cells would be effectively translated to other cancers of reproductive and non-reproductive origin, and, importantly, whether the efficacy of MF as an antiproliferative agent was dependent upon PR expression. Dose-response experiments were conducted with a panel of cancer cell lines of the brain (meningioma IOMM-Lee and glioblastoma U87MG cells), breast (estradiol-responsive MCF-7 and estradiol-unresponsive MDA-MB-231 cells), prostate (androgen-responsive LNCaP and androgen-unresponsive PC-3 cells), ovary (OVCAR-3 and SK-OV-3 cells), and bone (osteosarcoma U-2OS and SAOS-2 cells). Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and then analyzed for cell number and cell cycle traverse. For all cell lines, expression of steroid hormone receptors and cell cycle regulatory proteins controlling the G1-S phase transition in cells treated with vehicle or MF for 24 h was studied by Western blot analysis. The activity of Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate its substrate histone H1. MF growth inhibited all cancer cell lines included in this study, regardless of tissue of origin and hormone responsiveness. Changes in the abundance of the cell cycle regulatory proteins p21cip1, p27kip1, cyclin E, and Cdk2 along with changes in Cdk2 activity following 24 h exposure to MF suggested that MF induces a G1 phase arrest in the majority of these cell lines, consistent with our previous results in OvCa cells. Importantly, studies of the basal expression of steroid hormone receptors in this panel of cell lines showed that while all cell lines were growth inhibited by MF, only MCF-7 cells expressed PR. This study supported our previous results with MF-treated OvCa cells suggesting a mechanism of action involving G1 arrest and a corresponding decrease in Cdk2 activity. Contrary to common opinion, growth inhibition of this panel of cancer cell lines by MF was not dependent upon PR expression. NCI K22CA121991. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4546. doi:10.1158/1538-7445.AM2011-4546

Change of MAX Interactor 1 Expression in an Anti-Thy1 Nephritis Model and its Effect on Mesangial Cell Proliferation

Background/Aims: During the disease process of mesangial proliferative glomerulonephritis, the expression of various factors that influence mesangial proliferation is altered. MAX interactor 1 (Mxi1) antagonizes the transcription factor Myc and is believed to be a tumor suppressor. However, no studies have investigated its effect on mesangial cell proliferation. Methods: To investigate the effect of Mxi1 on renal mesangial cell proliferation, we established a classic rat anti-Thy1 mesangial proliferative glomerulonephritis model. Mesangial proliferation was estimated by immunohistochemical analysis of Ki67. Mxi1 expression at each time point was assessed by real-time RT-PCR and Western blot analyses. Furthermore, we altered the expression level of Mxi1 by a plasmid and siRNA to detect its effect on rat mesangial cell proliferation in vitro. Results: Mxi1 expression decreased significantly during the proliferative period of anti-Thy1 nephritis model and then gradually increased as proliferation declined, indicating that Mxi1 may be linked to mesangial cell proliferation. Upregulation of Mxi1 expression via plasmid transfection in vitro reduced the expression of the positive-acting cell cycle regulatory proteins cyclin B1, cyclin D1, cyclin E, CDC2 and CDK2; significantly reduced mesangial cell proliferation; reduced the percentage of S phase cells; and increased the percentage of G2/M phase cells. Inhibition of Mxi1 expression by siRNA in vitro produced the opposite effects: increased expression of cyclin B1, cyclin D1, cyclin E, CDC2 and CDK2; markedly increased cell proliferation; higher percentage of S phase cells; and dramatically lower percentage of G2/M phase cells. Transcription factor c-myc protein expression showed no obvious difference after Mxi1 plasmid and siRNA transfection. The expressions of cell cycle regulatory proteins mentioned above were negative correlated with Mxi1 expression in anti-Thy1 nephritis model. Conclusion: These results suggest that Mxi1 expression levels were inversely correlated with proliferation in anti-Thy1 nephritis rats and it may influence cell cycle progression and thus the rate of mesangial cell proliferation by regulating the expression of c-myc target cell cycle regulatory proteins.

In CD28-costimulated human naïve CD4+ T cells, I-κB kinase controls the expression of cell cycle regulatory proteins via interleukin-2-independent mechanisms.

Stimulation of naïve CD4(+) T cells through engagement of the T-cell receptor (TCR) and the CD28 co-receptor initiates cell proliferation which critically depends on interleukin (IL)-2 secretion and subsequent autocrine signalling via the IL-2 receptor. However, several studies indicate that in CD28-costimulated T cells additional IL-2-independent signals are also required for cell proliferation. In this study, using a neutralizing anti-human IL-2 antibody and two selective, structurally unrelated, cell-permeable I-κB kinase (IKK) inhibitors, BMS-345541 and PS-1145, we show that in human naïve CD4(+) T cells stimulated through a short engagement of the TCR and the CD28 co-receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin-dependent kinase 2 (CDK2) and the stability of the F-box protein S-phase kinase-associated protein 2 (SKP2) and its co-factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL-2-independent mechanisms.

Mixed Tocotrienols Inhibit Prostate Carcinogenesis in TRAMP Mice

The biological activities of tocotrienols are receiving increasing attention. Herein, we report the efficacy of a mixed-tocotrienol diet against prostate tumorigenesis in the transgenic adenocarcinoma mouse prostate (TRAMP) mouse model. Male TRAMP mice, 8 wk old, were fed 0.1%, 0.3%, or 1% mixed tocotrienols in AIN-76A diet up to 24 wk old. Likewise, a positive control group consisting of male TRAMP mice and a negative control group consisting of wild-type nontransgenic mice were fed regular AIN-76A diet up to 24 wk old. Our results show that mixed-tocotrienol-fed groups had a lower incidence of tumor formation along with a significant reduction in the average wet weight of genitourinary apparatus. Furthermore, mixed tocotrienols significantly reduced the levels of high-grade neoplastic lesions as compared to the positive controls. This decrease in levels of high-grade neoplastic lesions was found to be associated with increased expression of proapoptotic proteins BAD (Bcl2 antagonist of cell death) and cleaved caspase-3 and cell cycle regulatory proteins cyclin dependent kinase inhibitors p21 and p27. In contrast, the expression of cyclins A and E were found to be decreased in mixed-tocotrienol groups. Taken together, our results show that by modulating cell cycle regulatory proteins and increasing expression of proapoptotic proteins, mixed tocotrienols suppress prostate tumorigenesis in the TRAMP mice.

Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

Artemisinin is a plant-derived anti-malarial drug that has relatively low toxicity in humans and is activated by heme and/or intracellular iron leading to intracellular free radical formation. Interestingly, artemisinin has displayed anti-cancer activity, with artemisinin dimers being more potent than monomeric artemisinin. Intracellular iron uptake is regulated by the transferrin receptor (TfR), and the activity of artemisinin depends on the availability of iron. We examined the level of TfR in prostate cancer (PCa) tumor cells, synthesized two new artemisinin dimers, and evaluated the effect of dihydroartemisinin and artemisinin dimers, ON-2Py and 2Py, on proliferation and apoptosis in PCa cells. TfR was expressed in the majority of PCa bone and soft tissue metastases, all 24 LuCaP PCa xenografts, and PCa cell lines. After treatment with dihydroartemisinin, ON-2Py, or 2Py all PCa cell lines displayed dose-dependent decrease in cell number. 2Py was most effective in decreasing cell number. An increase in apoptotic events and growth arrest was observed in the C4-2 and LNCaP cell lines. Growth arrest was observed in PC-3 cells, but no significant change was observed in DU 145 cells. Treatment with 2Py resulted in a loss of the anti-apoptotic protein survivin in all four cell lines. 2Py treatment also decreased androgen receptor and prostate-specific antigen expression in C4-2 and LNCaP cells, with a concomitant loss of cell cycle regulatory proteins cyclin D1 and c-Myc. This study shows the potential use of artemisinin derivatives as therapeutic candidates for PCa and warrants the initiation of preclinical studies.

Synergistic activity of letrozole and sorafenib on breast cancer cells

Estrogens induce breast tumor cell proliferation by directly regulating gene expression via the estrogen receptor (ER) transcriptional activity and by affecting growth factor signaling pathways such as mitogen-activated protein kinase (MAPK) and AKT/mammalian target of rapamycin Complex1 (mTORC1) cascades. In this study we demonstrated the preclinical therapeutic efficacy of combining the aromatase inhibitor letrozole with the multi-kinase inhibitor sorafenib in aromatase-expressing breast cancer cell lines. Treatment with letrozole reduced testosterone-driven cell proliferation, by inhibiting the synthesis of estrogens. Sorafenib inhibited cell proliferation in a concentration-dependent manner; this effect was not dependent on sorafenib-mediated inhibition of Raf1, but involved the down-regulation of mTORC1 and its targets p70S6K and 4E-binding protein 1 (4E-BP1). At concentrations of 5-10 μM the growth-inhibitory effect of sorafenib was associated with the induction of apoptosis, as indicated by release of cytochrome c and Apoptosis-Inducing Factor into the cytosol, activation of caspase-9 and caspase-7, and PARP-1 cleavage. Combination of letrozole and sorafenib produced a synergistic inhibition of cell proliferation associated with an enhanced accumulation of cells in the G(0)/G(1) phase of the cell cycle and with a down-regulation of the cell cycle regulatory proteins c-myc, cyclin D1, and phospho-Rb. In addition, longer experiments (12 weeks) demonstrated that sorafenib may be effective in preventing the acquisition of resistance towards letrozole. Together, these results indicate that combination of letrozole and sorafenib might constitute a promising approach to the treatment of hormone-dependent breast cancer.

Combination of ginsenoside Rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of NF-κB

Ginsenoside Rg3 has been a subject of interest for use as a cancer preventive or therapeutic agent. Nuclear factor-kappa (NF-kappaB) is constitutively activated in prostate cancer, and gives cancer cells resistance to chemotherapeutic agents. To investigate whether Rg3 can suppress the activation of NF-kappaB, and thus increase susceptibility of prostate (LNCaP and PC-3, DU145) cells against chemotherapeutics, prostate cancer cell growth as well as activation of NF-kappaB was examined. We found that a combination treatment of Rg3 (50 microM) with a conventional agent docetaxel (5 nM) was more effective in the inhibition of prostate cancer cell growth and induction of apoptosis as well as G(0)/G(1) arrest accompanied with the significant inhibition of NF-kappaB activity than those by treatment of Rg3 or docetaxel alone. It was also found that NF-kappaB target gene expression of Bax, caspase-3, and caspase-9 was much more significantly enhanced, but the expression of Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP), and the expression of cell cycle regulatory proteins cyclin B, D1 and E, and cyclin dependent kinases 2 and 4 was also much more significantly inhibited by the combination treatment. The combination of Rg3 (50 microM) with cisplatin (10 microM) and doxorubicin (2 microM) was also more effective in the inhibition of prostate cancer cell growth and NF-kappaB activity than those by the treatment of Rg3 or chemotherapeutics alone. These results indicate that ginsenoside Rg3 inhibits NF-kappaB, and enhances the susceptibility of prostate cancer cells to docetaxel and other chemotherapeutics. Thus, ginsenoside Rg3 could be useful as an anti-cancer agent.

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

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

Anti-CD20 Antibody GA101 in Combination with Chemotherapy or Flavopiridol in Mantle Cell Lymphoma.

Abstract 4781 BackgroundMantle cell lymphoma (MCL) responds only transiently to conventional chemotherapy resulting in a dismal long-term prognosis. At a molecular level it is characterised by the chromosomal translocation t(11;14)(q13;q32), which leads to constitutive over-expression of the cell cycle regulatory protein cyclin D1. GA101 is a third generation, glycoengineered type II IgG1 anti-CD20 monoclonal antibody with superior direct cell death induction by targeting a type II epitope and enhanced antibody dependent cellular cytotoxicity (ADCC). High efficacy in lymphoma cell lines has led to combination experiments with various chemotherapeutic compounds or the CDK-inhibitor Flavopiridol. MethodsUsing a MCL cell line panel (Granta-519, HBL-2, Jeko-1, Rec-1 and Z-138) and a Diffuse Large B-Cell Lymphoma cell line (Karpas-422) we determined the effect of GA101 (1 μg/ml) monotherapy as well as in combination with Fludarabine (0,25 μg/ml), Bendamustine (5 μg/ml), Mitoxantrone (0,25 and 0,5 μg/ml) and Flavopiridol (100nM) on cell proliferation and viability. Trypan-blue exclusion tests were used to analyze cell viability at 0h, 24h, 48h and 72h. The panel of MCL cell lines was treated to determine potential synergism of agent combinations. Accordingly, fractional product was calculated: synergism > 0,1; additive effect -0,1<x<0,1; antagonism < -0,1. ResultsAfter mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (Granta: 65-75% cell reduction, Rec-1: 30-45%). Intermediate results were achieved for HBL-2 (20-30%), Z-138 and Karpas-422 (10-15%), Jeko-1 (5%). Fludarabine alone resulted in a 20-40% cell reduction. Bendamustine showed a higher efficacy in Jeko-1, Rec-1 and Z-138 (40-90%) than in Granta-519, Karpas-422 and HBL-2 (10%). Mitoxantrone treatment demonstrated a high impact on all cell lines (80-95% cell reduction). Flavopiridol induced a 65-85% cell reduction in Jeko-1, Rec-1 and Karpas-422in comparison to 30-45% in Granta-519, HBL-2 and Z-138. Additional experiments showed additive effects of all GA101 combinations resulting in 40-80% cell reduction (Fludarabine), 30-90% (Bendamustine), 85-95% (Mitoxantrone) and 60-80% (Flavopiridol). ConclusionsThese in vitro results demonstrate that the anti-CD20 monoclonal antibody GA101 alone or in combination with various chemotherapeutical compounds or the CDK-inhibitor (Flavopiridol) show a promising efficacy in MCL cell lines (additive in combination), supporting the clinical evaluation of such an innovative immuno-chemotherapy in mantle cell lymphoma. Disclosures:Klein:Roche (Glycart): Employment, Equity Ownership, Patents & Royalties. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.

Suppression of the inflammatory response by triterpenes isolated from the mushroom Ganoderma lucidum

Ganoderma lucidum is a popular medicinal mushroom, which has been used in the Traditional Chinese medicine for the prevention or treatment of a variety of diseases. In the present study we evaluated the anti-inflammatory effects of the triterpene extract from G. lucidum (GLT) in LPS-stimulated macrophages. Here we show that GLT markedly suppressed the secretion of inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and inflammatory mediator nitric oxide (NO) and prostaglandin E 2 (PGE 2) from lipopolysaccharide (LPS)-stimulated murine RAW264.7 cells. GLT also down-regulated LPS-dependent expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW264.7 cells. The anti-inflammatory effects of GLT were mediated by the inhibition of transcription factor NF-κB as demonstrated by decreased NF-κB-DNA binding activity, and the suppression of p65 phosphorylation in LPS-stimulated macrophages treated with GLT. Moreover, GLT inhibited LPS-dependent AP-1-DNA binding activity and down-regulated expression of AP-1 subunit c-Jun. In addition, GLT suppressed the activity of MAP kinases as observed by the down-regulation of LPS-induced phosphorylation of ERK1/2 and JNK but not p38. In vivo experiments clearly demonstrated that GLT also inhibited the production of TNF-α and IL-6 in LPS-induced endotoxemic mice. Apart from its anti-inflammatory activity, GLT suppressed cell proliferation of RAW264.7 cells through cell cycle arrest at G0/G1–G2M, which was mediated by the down-regulation of expression of cell cycle regulatory proteins cyclin D1, CDK4 and cyclin B1, respectively. In conclusion, the anti-inflammatory and anti-proliferative effects of GLT on macrophages are mediated through the inhibition of NF-κB and AP-1 signaling pathways.

ZD6474 in combination with paclitaxel and UVB irradiation enhances the antiproliferative effect and apoptosis on breast carcinoma

Breast cancer is the leader of cancer deaths among women. Despite developments in surgery, chemotherapy and radiotherapy, the long-term survival of patients with metastatic and higher staging breast cancer has remained very low. Breast cancers and their aggressive nature are characterized by overexpression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR). There is a common downstream signaling molecule involved in both EGFR and VEGFR mediated pathways. There are reports that cancer cells acquired resistance to anti-EGF/EGFR therapy by increased tumor-induced angiogenesis due to the constitutive overexpression of VEGF. Hence, the inhibition of both EGFR and VEGFR signaling pathways simultaneously may provide greater benefit than blockade of either pathway alone. There are also reports that breast cancer acquired resistance to conventional chemotherapy and radiotherapy by increased phosphorylation of EGFR and VEGFR. Moreover, patients suffering from breast cancer have a poor prognosis because of a lack of effective treatment strategies. We hypothesized that inhibiting the phosphorylation of the EGFR and VEGFR by ZD6474, a dual tyrosine kinase inhibitor of EGFR and VEGFR, in combination with paclitaxel or radiation would inhibit breast cancer cell growth. We tested this hypothesis using the human breast cancer cell lines MCF-7 and MDA-MB-231. In culture, ZD6474 in combination with paclitaxel or UVB had a synergistic effect in inhibition of cell proliferation, and lowered the IC50 by 10-fold and fivefold, respectively, compared with paclitaxel/UVB alone. Flow cytometry data suggest that combination therapy of ZD6474 with paclitaxel increases apoptosis from 20% to 45% in MDA-MB-231 and from 16% to 38% in MCF-7 compared with paclitaxel alone. This is further supported by a decrease in cell cycle regulatory protein cyclin D1 and an increase in apoptosis marker poly(ADP-ribose) polymerase in combination therapy compared with paclitaxel and UVB irradiation alone. Moreover, combination therapy sensitizes the paclitaxel resistance MCF-7/PTX and MDA-MB-231/PTX. ZD6474 along with paclitaxel or radiation can therefore be used for treatment in drug-insensitive advanced breast cancers.

Expression of cell cycle regulatory proteins in endometrial adenocarcinoma: variations in conventional tumor areas and in microcystic, elongated and fragmented glands

Endometrial adenocarcinomas may show a distinctive pattern of invasion characterized by the presence of microcystic, elongated and fragmented glands, often most evident along the advancing tumor margin. Earlier, we have shown that these changes appear restricted to low-grade endometrioid carcinomas, many of which show focal mucinous differentiation and lymphovascular space invasion. However, the molecular alterations associated with this morphological alteration are not known. In this study, we have examined immunoreactivity for the cell cycle regulatory proteins cyclin D1, p16 and β-catenin in 22 endometrial carcinomas, specifically comparing the results in conventional tumor areas and in foci in which the glands exhibited microcystic, elongated and fragmented appearances. The conventional neoplastic glands exhibited cyclin D1 and p16 expression in most cases, with >50% tumor cells positive in 8 cases and 11 tumors, respectively. Membranous expression of β-catenin was usually preserved, with variable cytoplasmic and nuclear staining. Cyclin D1 and β-catenin predominantly stained cells at the peripheral or basal aspect of the conventional glands, whereas p16 was more uniformly expressed centrally. Tumor foci composed of microcystic, fragmented and elongated glands showed strong expression of cyclin D1 and p16, sometimes in contrast to unstained contiguous or adjacent conventional neoplastic elements, and there was also loss or fragmentation of membranous β-catenin staining. Intravascular tumor cells also expressed cyclin D1 and p16 and therefore the immunostains often highlighted subtle foci of lymphovascular invasion. The heterogenous expression of cell cycle regulatory proteins within endometrial adenocarcinoma illustrates the importance of assessing microanatomical variations in immunoreactivity, particularly at the advancing margin of tumors. The upregulation of cyclin D1 and p16, together with loss of membranous β-catenin expression in microcystic, fragmented and elongated glands, is similar to epithelial–mesenchymal transitions observed in other malignancies and suggests that this pattern of invasion represents an active rather than a degenerative cellular process.

Anesthetic pentobarbital inhibits proliferation and migration of malignant glioma cells

Malignant gliomas are common and aggressive brain tumors in adults. The rapid proliferation and diffuse brain migration are main obstacles to successful treatment. Here we show that pentobarbital, a central depressant introduced clinically a century ago, is capable of suppressing proliferation and migration of C6 malignant glioma cells in a concentration-dependent manner. Pentobarbital also leads to a G1 phase cell cycle arrest accompanied by suppressed G1 cell cycle regulatory proteins Cyclin D1, Cyclin D3, CDK2 and phosphorylated Rb. In addition, noticeable morphological changes and interrupted α-tubulin microtubule assembly are induced by pentobarbital exposure. Intracellular signal pathways involved in the effect of pentobarbital is concerned with inactivation of ERK, c-Jun and Akt. Together, these findings suggest anti-proliferation and anti-migration effects of pentobarbital on malignant gliomas, most likely by arresting cell cycle and interfering microtubule. ERK, c-Jun MAPK and PI3K/Akt are possible signaling pathways involved.

Interactions between PTEN and the c-Met pathway in glioblastoma and implications for therapy

The tyrosine kinase receptor c-Met and its ligand hepatocyte growth factor (HGF) are frequently overexpressed and the tumor suppressor PTEN is often mutated in glioblastoma. Because PTEN can interact with c-Met-dependent signaling, we studied the effects of PTEN on c-Met-induced malignancy and associated molecular events and assessed the potential therapeutic value of combining PTEN restoration approaches with HGF/c-Met inhibition. We studied the effects of c-Met activation on cell proliferation, cell cycle progression, cell migration, cell invasion, and associated molecular events in the settings of restored or inhibited PTEN expression in glioblastoma cells. We also assessed the experimental therapeutic effects of combining anti-HGF/c-Met approaches with PTEN restoration or mTOR inhibition. PTEN significantly inhibited HGF-induced proliferation, cell cycle progression, migration, and invasion of glioblastoma cells. PTEN attenuated HGF-induced changes of signal transduction proteins Akt, GSK-3, JNK, and mTOR as well as cell cycle regulatory proteins p27, cyclin E, and E2F-1. Combining PTEN restoration to PTEN-null glioblastoma cells with c-Met and HGF inhibition additively inhibited tumor cell proliferation and cell cycle progression. Similarly, combining a monoclonal anti-HGF antibody (L2G7) with the mTOR inhibitor rapamycin had additive inhibitory effects on glioblastoma cell proliferation. Systemic in vivo delivery of L2G7 and PTEN restoration as well as systemic in vivo deliveries of L2G7 and rapamycin additively inhibited intracranial glioma xenograft growth. These preclinical studies show for the first time that PTEN loss amplifies c-Met-induced glioblastoma malignancy and suggest that combining anti-HGF/c-Met approaches with PTEN restoration or mTOR inhibition is worth testing in a clinical setting.

Cyclin overexpression inhibits Smad 3 tumor suppression in breast cancer cells.

Abstract Abstract #5033 Introduction: The transcription factor Smad 3, a member of the TGFβ signaling cascade, contributes to G1 cell cycle arrest in breast cancer cell lines. Overexpression of the cell cycle regulatory protein cyclin E, and its low molecular weight (LMW) counterparts, has been associated with poor prognosis in breast cancer. Cyclin E has been shown to mediate phosphorylation within or near the linker region of Smad 3 by cyclin dependent kinase 2 (CDK 2), and to inhibit Smad 3 activity in normal mouse fibroblasts and epithelial cells. We hypothesize that overexpression of cyclin E may exert tumorigenic effects in breast cancer cell lines through the functional inhibition of Smad 3 mediated by CDK 2 phosphorylation.&amp;#x2028; Methods: Endogenous levels of Smad 3 and phosphorylated Smad 3 in parental, vector only control, full length (FL), and two LMW forms of cyclin E overexpressing MCF-7 breast cancer cell lines were determined by western blotting. To elucidate the impact of cyclin E overexpression on Smad 3 function, constructs containing an empty vector, WT Smad 3, Smad 3 with a CDK phosphorylation site mutation within the linker region (Thr178), and outside the linker region (Thr8) were co-transfected with a Smad 3 reporter into the panel of MCF-7 cells. CDK2 or scrambled control siRNA was also transfected into the study panel of cells. Smad 3 function was then evaluated by luciferase reporter assays.&amp;#x2028; Results: MCF-7 cells overexpressing FL or LMW cyclin E had a higher level of P- SMAD 3 as compared to the parental and vector only control cells. Total SMAD 3 levels were similar in all cells examined. Transfection of WT Smad 3 elicited an increase in reporter activity in parental and vector control cells, while both FL and LMW forms of cyclin E overexpressing cells were resistant to WT Smad 3 reporter induction. In parental and vector control MCF-7 cells, expression of the Smad 3 Thr178 mutation resulted in a three-fold induction in Smad 3 reporter activity as compared to the WT Smad 3 response. In FL and LMW cyclin E overexpressing cells, induction by the Thr178 mutation was more pronounced, as seen by a 5-fold and 8-fold induction of reporter activity in FL and LMW cyclin E cells, respectively. Transient transfection of the Thr8 mutation failed to evoke a reporter response in any of the panel of MCF-7 cells. MCF-7, FL, and LMW cyclin E cells demonstrated the greatest fold induction of Smad 3 reporter activity when transfected with CDK 2 siRNA.&amp;#x2028; Conclusions: Overexpression of cyclin E may circumvent the growth inhibitory effects of Smad 3 signal transduction through the modulation of Smad 3 function. Specifically, this work indicates that cyclin E mediates Smad 3 inhibition in a CDK 2 dependent fashion in breast cancer cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5033.

Notch-1 regulates Akt signaling pathway and the expression of cell cycle regulatory proteins cyclin D1, CDK2 and p21 in T-ALL cell lines

Gain-of-function mutations in Notch-1 are common in T-cell lymphoblastic leukemia (T-ALL), making this receptor a promising target for drugs such as gamma-secretase inhibitors (GSIs). However, GSIs seem to be active in only a small fraction of T-ALL cell lines with constitutive Notch-1 activity and the downstream response of Notch signaling is only partially understood. To further investigate the molecular mechanisms underlying proliferation suppression and apoptosis and explore effective downstream target genes, we used RNA interference (RNAi) technology to down-regulate the expression of Notch-1 in GSIs-resistant T-ALL cell lines. Results showed that down-regulation of Notch-1 by transfection of a small interfering RNA (siRNA) could cause SupT1 cells proliferation inhibition by inducing G 0/G 1 cell cycle arrest and apoptosis. The proliferation inhibitory and apoptotic effects resulting from down-regulation of Notch-1 may be mediated through regulating the expression of cell cycle regulatory proteins cyclin D1, CDK2 and p21 and the activity of Akt signaling. In addition, our results demonstrated that down-regulation of Notch-1 signaling could sensitize SupT1 cells to adriamycin. Taken together, cell cycle regulatory proteins and Akt signaling may be attractive targets in T-ALL.

Follicle-Stimulating Hormone Inhibits Adenosine 5′-Monophosphate-Activated Protein Kinase Activation and Promotes Cell Proliferation of Primary Granulosa Cells in Culture through an Akt-Dependent Pathway

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 microm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-beta-D-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway.

Regulation of cell proliferation by intermediate-conductance Ca2+-activated potassium and volume-sensitive chloride channels in mouse mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine; however, their cellular physiology is not fully understood. The present study aimed at exploring the potential roles of the two dominant functional ion channels, intermediate-conductance Ca(2+)-activated potassium (IK(Ca)) and volume-sensitive chloride (I(Cl.vol)) channels, in regulating proliferation of mouse MSCs. We found that inhibition of IK(Ca) with clotrimazole and I(Cl.vol) with 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) reduced cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific short interference (si)RNAs significantly reduced IK(Ca) or I(Cl.vol) density and channel protein and produced a remarkable suppression of cell proliferation (by 24.4 +/- 9.6% and 29.5 +/- 7.2%, respectively, P < 0.05 vs. controls). Flow cytometry analysis showed that mouse MSCs retained at G(0)/G(1) phase (control: 51.65 +/- 3.43%) by inhibiting IK(Ca) or I(Cl.vol) using clotrimazole (2 microM: 64.45 +/- 2.20%, P < 0.05) or NPPB (200 microM: 82.89 +/- 2.49%, P < 0.05) or the specific siRNAs, meanwhile distribution of cells in S phase was decreased. Western blot analysis revealed a reduced expression of the cell cycle regulatory proteins cyclin D1 and cyclin E. Collectively, our results have demonstrated that IK(Ca) and I(Cl.vol) channels regulate cell cycle progression and proliferation of mouse MSCs by modulating cyclin D1 and cyclin E expression.