Project description:INTRODUCTION: Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines. METHODS: Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response. RESULTS: Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade. CONCLUSIONS: These studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991
Project description:S phase kinase-associated protein 2 (Skp2) has been shown to be required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb) deficient mice. Here we have demonstrated that flavokawain A (FKA), a novel chalcone from the kava plant, selectively inhibited the growth of pRb deficient cell lines and resulted in a proteasome-dependent and ubiquitination-mediated Skp2 degradation. Degradation of Skp2 by FKA was found to be involved in a functional Cullin1, but independent of Cdh1 expression. Further studies have demonstrated that FKA docked into the ATP binding pocket of the precursor cell-expressed developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) complex, inhibited NEDD8 conjugations to both Cullin1 and Ubc12 in PC3 cells and Ubc12 NEDDylation in an in vitro assay. Finally, dietary feeding of the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with FKA inhibited the formation of high-grade prostatic intra-epithelial neoplasia lesions (HG-PIN) and prostate adenocarcinomas, reduced the tumor burden and completely abolished distant organ metastasis. Immunohistochemistry studies revealed that dietary FKA feeding resulted in marked anti-proliferative and apoptotic effects via down-regulation of Skp2 and NEDD8 and up-regulation of p27/Kip1 in the prostate of TRAMP mice. Our findings therefore provide evidence that FKA is a promising NEDDylation inhibitor for targeting Skp2 degradation in prostate cancer prevention and treatment.
Project description:Aberrant activation of Cyclin D-Cdk4/6 signaling pathway is commonly found in pancreatic ductal adenocarcinoma (PDAC). Here, we show that PD-0332991, a highly specific inhibitor for Cdk4 and Cdk6, exerted growth inhibitory effects on three human PDAC cell lines. Microarray analysis revealed that PD-0332991 downregulated cell-cycle-related genes, but upregulated genes implicated in extracellular matrix (ECM) remodeling and pancreatic cancer cell invasion and metastasis. Moreover, PD-0332991 enhanced invasion in TGF-?-responsive PDAC cell lines that harbor a wild-type SMAD4 gene (COLO-357, PANC-1), but not in TGF-?-resistant AsPC-1 cells that harbor a mutated SMAD4. PD-0332991 also induced epithelial-mesenchymal transition (EMT) in COLO-357 and PANC-1, but not in AsPC-1 cells. Inhibition of CDK4/6 using shRNA mimicked the effects of PD-0332991 on EMT induction. Furthermore, PD-0332991 increased Smad transcriptional activity in luciferase readout assays and activated TGF-? signaling. SB-505124, an inhibitor of the type-I TGF-? receptor (T?RI) kinase, completely blocked EMT induction by PD-0332991. When combined with PD-0332991, SB-505124 inhibited the growth of COLO-357 and PANC-1 cells. Taken together, these data suggest that anti-Cdk4/6 therapy could induce EMT and enhance pancreatic cancer cell invasion by activating Smad-dependent TGF-? signaling, and that combining PD-0332991 and SB-505124 may represent a novel therapeutic strategy in PDAC.
Project description:This study was undertaken to investigate the role of mouse double minute 2 (MDM2) oncogene in prostate cancer growth and the potential of MDM2 as a target for prostate cancer therapy. An antisense anti-human-MDM2 mixed-backbone oligonucleotide was tested in human prostate cancer models with various p53 statuses, LNCaP (p53wt/wt), DU145 (p53mt/mt), and PC3 (p53null). In a dose- and time-dependent manner, it specifically inhibited MDM2 expression and modified expression of several genes, at both mRNA and protein levels. In LNCaP cells, p53, p21, Bax, and hypophosphorylated retinoblastoma tumor suppressor protein (pRb) levels increased, whereas Bcl2, pRb protein, and E2F transcription factor 1 (E2F1) levels decreased. In DU145 cells, p21 levels were elevated and E2F1 levels decreased, although mutant p53, Rb, and Bax levels remained unchanged. In PC3 cells, MDM2 inhibition resulted in elevated p21, Bax, and pRb levels and decreased ppRb and E2F1 levels. In all three cell lines, MDM2 inhibition reduced cell proliferation, induced apoptosis, and potentiated the effects of the chemotherapeutic agents 10-hydroxycamptothecin and paclitaxel. The anti-MDM2 oligonucleotide showed antitumor activity and increased therapeutic effectiveness of paclitaxel in both LNCaP and PC3 xenografts, causing changes in gene expression similar to those seen in vitro. In summary, this study demonstrates that MDM2 has a role in prostate cancer growth via p53-dependent and p53-independent mechanisms and that multiple genes are involved in the process. MDM2 inhibitors such as second-generation antisense oligonucleotides have a broad spectrum of antitumor activities in human cancers regardless of p53 status, providing novel approaches to therapy of human prostate cancer.
Project description:Aberrant activation of the cyclin D1-cyclin-dependent kinase 4/6 (CDK4/6)-Rb signalling pathway is common in oesophageal squamous cell carcinoma (ESCC). PD-0332991, a highly specific inhibitor of CDK4/6, has potent antitumour activity against many types of cancer. The purpose of this study was to examine the in vitro and in vivo antineoplastic effect of PD-0332991 against the growth and metastasis of ESCC cells.Cell viability and any synergy between PD-0332991 and 5-fluorouracil or cisplatin were measured by MTS assay and CalcuSyn software respectively. Cell migration and invasion were detected by wound healing and transwell assays. Apoptosis was evaluated by flow cytometry after staining annexin V-FITC/PI. Cellular senescence was assessed by measuring SA-?-gal activity. Nude mouse xenograft models of ESCC were employed to determine the in vivo activity of PD-0332991 against tumour growth and lung metastasis.PD-0332991 inhibited cellular growth and induced mitochondrial-dependent apoptosis in ESCC cells. PD-0332991 also suppressed migration, invasion and the expression of MMP-2 in ESCC cells. Furthermore, PD-0332991 treatment caused cell senescence in a FOXM1-dependent manner. In addition, there was synergy between PD-0332991 and cisplatin or 5-fluorouracil. Importantly, the xenografted tumour experiments demonstrated that PD-0332991 potently inhibits ESCC cell growth and lung metastasis.PD-0332991 can elicit a strong antitumour activity against ESCC growth and metastasis and may be a promising candidate drug for the treatment of patients with ESCC. Our results warrant a clinical trial to further evaluate the efficacy of PD-0332991 in ESCC patients, even those with metastasis.
Project description:Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a major challenge to targeted therapy for non-small cell lung cancer (NSCLC). We investigated whether a cyclin D kinase 4/6 (CDK4/6) inhibitor, PD 0332991, could reverse EGFR-TKI resistance in human lung cancer cells and explored the underlying mechanisms. We found that PD 0332991 potentiated gefitinib-induced growth inhibition in both EGFR-TKI-sensitive (PC-9) and EGFR-TKI-resistant (PC-9/AB2) cells by down-regulating proliferation and inducing apoptosis and G0/G1 cell cycle arrest. Tumor xenografts were then used to verify the effects of PD 0332991 in vivo. Mice treated with a combination of PD 0332991 and gefitinib had the fastest tumor regression and delayed relapse. Tumors from mice receiving the combination treatment exhibited down-regulated proliferation, up-regulated apoptosis, and less angiogenesis. Finally, lung adenocarcinoma patients with acquired resistance to EGFR-TKIs were given an exploratory treatment of PD 0332991. One patient with gefitinib resistance exhibited clinical remission after treatment with PD 0332991. These findings suggest PD 0332991 reverses acquired EGFR-TKI-resistance in NSCLC cells, and may provide a novel treatment strategy for NSLSC patients with EGFR-TKI resistance.
Project description:BACKGROUND:PD-0332991, the selective cyclin-dependent kinase 4/6 inhibitor palbociclib, causes cell cycle arrest by inhibiting phosphorylation of retinoblastoma (Rb) protein. The aim of this study was to evaluate the therapeutic potential of PD-0332991 in endometrial cancer. METHODS AND FINDINGS:Four human endometrial cancer cell lines, ECC, HEC1A, HEC108 and TEN, were treated with PD-0332991 and their function was evaluated. In vivo, the therapeutic efficacy was evaluated in a model of subcutaneous endometrial cancer. An immunohistochemical analysis was performed in 337 endometrial cancer specimens. A proliferation assay revealed that 2 of the 4 cell lines that expressed Rb were sensitive to PD-0332991 with an IC50 of 0.65 ?M (HEC1A) and 0.58 ?M (HEC108), respectively. Both cell lines had G0/G1 cell cycle arrest after treatment with PD-0332991 according to flow cytometry. In vivo, PD-0332991 had antitumoral efficacy with a reduction in the activity of Ki67 and phosphorylation of Rb. Immunohistochemical analyses revealed that the positive rate of Rb was 67.7%, however, there was no significant relationship between the expression levels of Rb and the tumor grade. CONCLUSIONS:PD-0332991 had therapeutic potential against endometrial cancer cell lines expressing Rb protein. Our immunohistochemical analysis revealed that approximately 70% of patients with endometrial cancer might have therapeutic indications for PD-0332991. Of note, the tumor grade had no impact on the indications for treatment.
Project description:The n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), found in fish oil, exert a number of beneficial effects, and they are used in the treatment of hyperlipidemia. In recent years, EPA and DHA have been found to affect cancer cell proliferation. In the present study, PC3 cells, which are androgen-independent prostate cancer cells that resemble castration-resistant prostate cancer cells, were used to investigate a possible novel treatment for castration-resistant prostate cancer. The PC3 cells were cultured and incubated with various concentrations of EPA or DHA. Cancer proliferation was confirmed by trypan blue microscopy. Invasion and migration assays were used in the upper chamber in PC3 cells, and serum-free medium and various concentrations of EPA or DHA were placed in the lower chamber in serum-containing medium. EPA and DHA decreased PC3 cell proliferation, invasion and migration. The effect of EPA on PC3 cells was dose-dependent and significant differences were observed at concentrations of 100 and 200 µg/ml. The effect of DHA on PC3 cells was similar to that of EPA. In the migration assay, EPA exerted almost no effects at 25 µg/ml, but migration was reduced at 50 µg/ml. Similar to EPA, DHA exerted almost no effects at 25 µg/ml, but further reduction was observed at the 50 µg/ml concentration. In the invasion assay, EPA at 25 µg/ml was not significantly different from the control, but suppressed invasion at 50 µg/ml. DHA decreased invasion compared with the control at 25 µg/ml, whereas invasion was significantly reduced at a DHA concentration of 50 µg/ml. In conclusion, it was demonstrated that EPA and DHA were effective in decreasing the proliferation, invasion and migration of prostate PC3 cancer cells. However, the detailed underlying mechanisms have not yet been fully elucidated.