1α,25-dihydroxyvitamin D3 inhibits C4-2 prostate cancer cell growth via a retinoblastoma protein (Rb)-independent G1 arrest.

BACKGROUND: The active metabolite of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25D) reduces the growth of several prostate cancer cell lines, most commonly by inducing a cell-cycle arrest in G(1). This is mediated, in part, through down-regulation of c-Myc, a positive regulator of the transcription factor, E2F. There is evidence that prostate cancer cells lacking functional retinoblastoma protein (Rb), a negative regulator of E2F activity, are poorly responsive to 1,25D treatment. Since up to 60% of prostate cancers demonstrate a loss of heterozygosity for Rb, we sought to determine whether Rb is required for the growth inhibitory effects of 1,25D. METHODS: Using siRNA, Rb was reduced in C4-2 prostate cancer cells, and the response of cells to 1,25D treatment or depletion of c-myc measured by [(3)H]-thymidine incorporation and flow cytometry. The effects of 1,25D treatment on E2F levels and activity, and E2F target gene expression were also measured. RESULTS: 1,25D treatment and c-Myc depletion both cause a G(1) arrest inhibiting C4-2 cell proliferation independently of Rb. 1,25D reduces c-Myc expression and causes a decrease in E2F and E2F target genes. Bcl-2, an E2F target and positive regulator of C4-2 cell growth, also is down-regulated by 1,25D independently of Rb. CONCLUSIONS: Redundant growth inhibitory pathways compensate for the loss of Rb, and tumors lacking functional Rb may be responsive to 1,25D.

Decitabine and suberoylanilide hydroxamic acid (SAHA) inhibit growth of ovarian cancer cell lines and xenografts while inducing expression of imprinted tumor suppressor genes, apoptosis, G2/M arrest, and autophagy.

BACKGROUND: Epigenetic therapy has had a significant impact on the management of hematologic malignancies, but its role in the treatment of ovarian cancer remains to be defined. The authors previously demonstrated that treatment of ovarian and breast cancer cells with DNA methyltransferase and histone deacetylase (HDAC) inhibitors can up-regulate the expression of imprinted tumor suppressors. In this study, demethylating agents and HDAC inhibitors were tested for their ability to induce re-expression of tumor suppressor genes, inhibiting growth of ovarian cancer cells in culture and in xenografts. METHODS: Ovarian cancer cells (Hey and SKOv3) were treated with demethylating agents (5-aza-20-deoxycytidine [DAC] or 5-azacitidine [AZA]) or with HDAC inhibitors (suberoylanilide hydroxamicacid [SAHA] or trichostatin A [TSA]) to determine their impact on cellular proliferation, cell cycle regulation, apoptosis, autophagy, and re-expression of 2 growth inhibitory imprinted tumor suppressor genes: guanosine triphosphate-binding Di-RAS-like 3 (ARHI) and paternally expressed 3 (PEG3). The in vivo activities of DAC and SAHA were assessed in a Hey xenograft model. RESULTS: The combination of DAC and SAHA produced synergistic inhibition of Hey and SKOv3 cell growth by apoptosis and cell cycle arrest. DAC induced autophagy in Hey cells that was enhanced by SAHA. Treatment with both agents induced re-expression of ARHI and PEG3 in cultured cells and in xenografts, correlating with growth inhibition. Knockdown of ARHI decreased DAC-induced autophagy. DAC and SAHA inhibited the growth of Hey xenografts and induced autophagy in vivo. CONCLUSIONS: A combination of DAC and SAHA inhibited ovarian cancer growth while inducing apoptosis, G2/M arrest, autophagy, and re-expression of imprinted tumor suppressor genes.

Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor.

The constitutive androstane receptor (CAR, NR1I3) is a central regulator of xenobiotic metabolism. CAR activation induces hepatic expression of detoxification enzymes and transporters and increases liver size. Here we show that CAR-mediated hepatomegaly is a transient, adaptive response to acute xenobiotic stress. In contrast, chronic CAR activation results in hepatocarcinogenesis. In both acute and chronic xenobiotic responses, hepatocyte DNA replication is increased and apoptosis is decreased. These effects are absent in CAR null mice, which are completely resistant to tumorigenic effects of chronic xenobiotic stress. In the acute response, direct up-regulation of Mdm2 expression by CAR contributes to both increased DNA replication and inhibition of p53-mediated apoptosis. These results demonstrate an essential role for CAR in regulating both liver homeostasis and tumorigenesis in response to xenobiotic stresses, and they also identify a specific molecular mechanism linking chronic environmental stress and tumor formation.

Sulindac sulfide inhibits epidermal growth factor-induced phosphorylation of extracellular-regulated kinase 1/2 and Bad in human colon cancer cells.

Colorectal cancer is the second leading cause of cancer death in the United States. Nonsteroidal anti-inflammatory drugs including sulindac are promising chemopreventive agents for colorectal cancer. Sulindac and selective cyclooxygenase (COX)-2 inhibitors cause regression of colonic polyps in familial polyposis patients. Sulindac induces apoptotic cell death in cancer cells in vitro and in vivo. In tumor cells, activation of extracellular-regulated kinase (ERK) 1/2 results in phosphorylation of several ERK1/2 effectors, including the proapoptotic protein Bad. Phosphorylation of Ser112 by ERK1/2 inactivates Bad and protects the tumor cell from apoptosis. Sulindac metabolites and other nonsteroidal anti-inflammatory drugs selectively inhibit ERK1/2 phosphorylation in human colon cancer cells. In this study we show that epidermal growth factor (EGF) strongly induces phosphorylation of ERK1/2 and Bad in HT29 colon cancer cells. EGF-stimulated phosphorylation of ERK and Bad is blocked by pretreatment with U0126, a selective MAP kinase kinase (MKK)1/2 inhibitor. Similarly, pretreatment with sulindac sulfide blocks the ability of EGF to induce ERK1/2 and Bad phosphorylation, but also down-regulates total Bad but not ERK1/2 protein levels. The ability of sulindac to block ERK1/2 signaling by the EGF receptor may account for at least part of its potent growth-inhibitory effects against cancer cells.

1{alpha},25-Dihydroxyvitamin D3 inhibits growth of VCaP prostate cancer cells despite inducing the growth-promoting TMPRSS2:ERG gene fusion.

Vitamin D receptor (VDR) agonists have been shown to reduce the growth of several prostate cancer cell lines. However, the effects of VDR activation have not been examined in the presence of the recently identified androgen-regulated TMPRSS2:ERG gene fusions, which occur in a high percentage of prostate cancers and play a role in growth and invasiveness. In a previous microarray study, we found that VDR activation induces TMPRSS2 expression in LNCaP prostate cancer cells. Here we show that the natural VDR agonist 1alpha,25-dihydroxyvitamin D(3) and its synthetic analog EB1089 increase expression of TMPRSS2:ERG mRNA in VCaP prostate cancer cells; this results in increased ETS-related gene (ERG) protein expression and ERG activity as demonstrated by an increase in the ERG target gene CACNA1D. In VCaP cells, we were not able to prevent EB1089-mediated TMPRSS2:ERG induction with an androgen receptor antagonist, Casodex, although in LNCaP cells, as reported for some other common androgen receptor and VDR target genes, Casodex reduces EB1089-mediated induction of TMPRSS2. However, despite inducing the fusion gene, VDR agonists reduce VCaP cell growth and expression of the ERG target gene c-Myc, a critical factor in VDR-mediated growth inhibition. Thus, the beneficial effects of VDR agonist treatment override some of the negative effects of ERG induction, although others remain to be tested.