MRI to assess chemoprevention in transgenic adenocarcinoma of mouse prostate (TRAMP)

<p>Abstract</p> <p>Background</p> <p>The current method to determine the efficacy of chemoprevention in TRAMP mouse model of carcinoma of prostate (CaP) is by extracting and weighing the prostate at different time points or by immunohistochemistry analysis. Non-invasive determination of volumes of prostate glands and seminal vesicles before, during and after treatment would be valuable in investigating the efficacy of newer chemopreventive agents in CaP. The purpose of this study was to determine whether <it>in vivo </it>magnetic resonance imaging (MRI) using a 3 tesla clinical MRI system can be used to follow the effect of chemoprevention in TRAMP model of mouse CaP.</p> <p>Methods</p> <p>Mice were randomized into control and treated groups. The animals in treated group received 10 µmol/kg of CDDO, 5 days a week for 20 weeks. Animals underwent <it>in vivo </it>MRI of prostate gland and seminal vesicles by a clinical 3 Tesla MRI system just before (at 5 weeks), during and at the end of treatment, at 25 weeks. T1-weighted and fat saturation (FATSAT) multiecho fast spin echo T2- weighted images (T2WI) were acquired. Volume of the prostate glands and seminal vesicles was determined from MR images. T2 signal intensity changes in the seminal vesicles were determined by subtracting higher echo time (TE) from lower TE T2WI. Following treatments all animals were sacrificed, prostate and seminal vesicles collected, and the tissues prepared for histological staining. All data were expressed as mean ± 1 standard deviation. Two-way or multivariate analysis of variance followed by post-hoc test was applied to determine the significant differences. A p-value of <0.05 was considered significant.</p> <p>Results</p> <p>Histological analysis indicated tumor in 100% of control mice, whereas 10% of the treated mice showed tumor in prostate gland. Both MRI and measured prostate weights showed higher volume/weight in control mouse group. MRI showed significantly higher volume of seminal vesicles in control animals and T2 signal intensity changes in seminal vesicles of control mice indicating higher number of tumor foci, which was also proven by histology.</p> <p>Conclusions</p> <p><it>In vivo </it>MRI is helpful in determining the efficacy of chemoprevention of prostate cancer in TRAMP mice.</p

Inhibition of Telomerase Activity by Oleanane Triterpenoid CDDO-Me in Pancreatic Cancer Cells is ROS-Dependent

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a synthetic derivative of oleanolic acid, a triterpene, with apoptosis-inducing activity in a wide range of cancer cells. Induction of apoptosis by CDDO-Me is associated with the generation of reactive oxygen species (ROS) and inhibition of telomerase activity. In the present study, we investigated the role of ROS in inhibition of telomerase by CDDO-me. Treatment of MiaPaCa-2 and Panc-1 pancreatic cancer cell lines with CDDO-Me induced the production of hydrogen peroxide and superoxide anions and inhibited the telomerase activity. Pretreatment of cells with N-acetylcycsteine, a general purpose antioxidant or overexpression of glutathione peroxidase (GPx) or superoxide dismutase-1 (SOD-1) blocked the telomerase inhibitory activity of CDDO-Me. Furthermore, blocking ROS generation also prevented the inhibition of hTERT gene expression, hTERT protein production and expression of a number of hTERT–regulatory proteins by CDDO-Me (e.g., c-Myc, Sp1, NF-κB and p-Akt). Data also showed that Akt plays an important role in the activation of telomerase activity. Together, these data suggest that inhibition of telomerase activity by CDDO-Me is mediated through a ROS-dependent mechanism; however, more work is needed to fully understand the role of ROS in down-regulation of hTERT gene and hTERT-regulatory proteins by CDDO-Me

CDDO-Me: A Novel Synthetic Triterpenoid for the Treatment of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancy with dismal prognosis and few effective therapeutic options. Novel agents that are safe and effective are urgently needed. Oleanolic acid-derived synthetic triterpenoids are potent antitumorigenic agents, but their efficacy or the mechanism of action for pancreatic cancer has not been adequately investigated. In this study, we evaluated the antitumor activity and the mechanism of action of methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a oleanane-derived synthetic triterpenoid for human pancreatic cancer cell lines. CDDO-Me inhibited the growth of both K-ras mutated (MiaPaca2, Panc1 and Capan2) and wild-type K-ras (BxPC3) pancreatic cancer cells at very low concentrations. The growth inhibitory activity of CDDO-Me was attributed to the induction of apoptosis characterized by increased annexin-V-FITC binding and cleavage of PARP-1 and procaspases-3, -8 and-9. In addition, CDDO-Me induced the loss of mitochondrial membrane potential and release of cytochrome C. The antitumor activity of CDDO-Me was associated with the inhibition of prosurvival p-Akt, NF-κB and mammalian target of rapamycin (mTOR) signaling proteins and the downstream targets of Akt and mTOR, such as p-Foxo3a (Akt) and p-S6K1, p-eIF-4E and p-4E-BP1 (mTOR). Silencing of Akt or mTOR with gene specific-siRNA sensitized the pancreatic cancer cells to CDDO-Me, demonstrating Akt and mTOR as molecular targets of CDDO-Me for its growth inhibitory and apoptosis-inducing activity

Role of telomerase in anticancer activity of pristimerin in prostate cancer cells

Pristimerin (PM) is a quinonemethide triterpenoid present in various plant species with strong antiprolifertive and proapoptotic activities in cancer cells. The effect of PM on telomerase which is reactivated in most cancers including carcinoma of the prostate (CaP) has not been studied. We investigated the effect of PM on the expression of human telomerase reverse transcriptase (hTERT) gene that codes for the catalytic subunit of the telomerase holoenzyme complex in prostate cancer cell lines LNCaP and PC-3 cells. The inhibition of cell proliferation and induction of apoptosis by PM in both cell lines was associated with the inhibition of hTERT mRNA expression, suppression of native and phosphorylated hTERT protein and hTERT telomerase activity. The ablation of hTERT expression increased the sensitivity of cancer cells to PM. In addition, results also revealed that the inhibition of hTERT expression is attributed to the inhibition of transcription factors SP1, c-Myc and STAT3 and protein kinase B/Akt which regulate hTERT transcriptionally and post-translationally, respectively. These data provide evidence that telomerase is a potential target of PM in prostate cancer

Article Telomerase Reverse Transcriptase (TERT) is a Therapeutic Target of Oleanane Triterpenoid CDDO-Me in Prostate Cancer

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CDDO-Me inhibits tumor growth and prevents recurrence of pancreatic ductal adenocarcinoma

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) has shown potent antitumorigenic activity against a wide range of cancer cell lines in vitro and inhibited the growth of liver, lung and prostate cancer in vivo. In the present study, we examined the antitumor activity of CDDO-Me for pancreatic ductal adenocarcinoma (PDAC) cells with and without activating K-ras mutations. Treatment of K-ras mutant MiaPaCa-2 and K-ras normal BxPC-3 cells with CDDO-Me elicited strong antiproliferative and proapoptopic responses in both cell lines in culture. The inhibition of cell proliferation and induction of apoptosis was accompanied by the inhibition of antiapoptotic/prosurvival p-Akt, NF-кB and p-mTOR signaling proteins. For testing efficacy of CDDO-Me in vivo heterotopic and orthotopic xenografts were generated by implanting BxPC-3 and MiaPaCa-2 cells subcutaneously and in the pancreatic tail, respectively. Treatment with CDDO-Me significantly inhibited the growth of BxPC-3 xenografts and reduced the levels of p-Akt and p-mTOR in tumor tissue. In mice with orthotopic MiaPaCa-2 xenografts, treatment with CDDO-Me prolonged the survival of mice when administered following the surgical resection of tumors. The latter was attributed to the eradication of residual PDAC remaining after resection of tumors. These preclinical data demonstrate the potential of CDDO-Me for treating primary PDAC tumors and for preventing relapse/recurrence through the destruction of residual disease

The Functional Role of Atypical Chemokine Receptor 1 in Immune Cell Regulation of Breast Cancer

Breast cancer (BC) is a heterogeneous disease that leads to varied molecular subtypes and distinct clinical outcomes. Tumor heterogeneity, which is in part influenced by genetic ancestry, contributes to racial disparities in BC. This disparity has persisted for over 30 years, despite improvements in detection, screening, and treatment methods. When investigating race-specific differences in the breast tumor microenvironment (TME), we believe that chemokine receptors, such as atypical chemokine receptor 1 (ACKR1/DARC), play an integral role in regulating chemokine and immune cell migration in circulation and the TME, ultimately influencing tumor progression. ACKR1/DARC specifically plays a role in ancestry-related differences in BC due to an African-specific ancestral allele (“Duffy-null” mutation) that is fixed in Sub-Saharan African populations, and present in 60-70% of African-Americans. Those that harbor the mutation do not express ACKR1/DARC on erythrocytes, ultimately affecting immune cell migration in these populations only. To better understand the effects of this variant in circulation and the TME, we performed immunohistochemistry (IHC) on breast tumors from a cohort of patients with matched blood samples. From our IHC analysis, we quantified levels of ACKR1/DARC, in addition to levels of target pro-inflammatory chemokines, and distinct immune populations of T-cells and macrophages. In addition, individuals were genotyped to determine Duffy-null status, and a correlative Luminex multiplex assay was performed on patient plasma to determine concentrations of pro-inflammatory chemokines in circulation. We determined that those with low expression of ACKR1/DARC in tumors exhibited a unique signature of immune cell infiltrates that would encourage a more aggressive TME. In addition, we observed variable levels of circulating chemokines, where those with the Duffy-null mutation exhibited significantly decreased levels of CCL2. Overall, our analyses support the hypothesis that tumor-specific DARC/ACKR1 plays a vital role in immune cell regulation in women with BC

The inhibition of cell proliferation and induction of apoptosis in pancreatic ductal adenocarcinoma cells by verrucarin A, a macrocyclic trichothecene, is associated with the inhibition of Akt/NF-кB/mTOR prosurvival signaling

Pancreatic ductal adenocarcinoma (PDA) remains one of the most difficult to treat of all malignancies. Multimodality regimens provide only short-term symptomatic improvement with minor impact on survival, underscoring the urgent need for novel therapeutics and treatment strategies for PDA. Trichothecenes are powerful mycotoxins that inhibit protein synthesis and induce ribotoxic stress response in mammalian cells. Verrucarin A (VC-A) is a Type D macrocyclic mycotoxin which inhibited cell proliferation and induced apoptosis in breast cancer cells. However, the antitumor activity of VC-A for PDA cells has not been investigated. Here we show potent antitumor activity and the mechanism of action of VC-A in PDA cell lines. VC-A strongly inhibited the proliferation and arrested cells in the S phase of the cell cycle. The blocking of cell cycle progression by VC-A was associated with the inhibition of cell cycle regulatory proteins cyclin D1, cyclin E, cyclin-dependent kinases (cdks) cdk2, cdk4 and cdk inhibitor WAF1/21. VC-A induced apoptosis in PDA cells as indicated by the increased Annexin V FITC-binding, cleavage of poly(ADP-ribose) polymerase‑1 (PARP-1) and procaspases-3, -8 and -9. VC-A also induced mitochondrial depolarization and release of cytochrome c and it inhibited Bcl-2 family proteins that regulate apoptosis (Bcl-2, Bcl-xL, Bax and Bad). In addition, VC-A reduced the levels of inhibitors of apoptosis survivin and c-IAP-2. Finally, VC-A downregulated the expression of prosurvival phospho-Akt (p-Akt), nuclear factor κB (NF-κB) (p65) and mammalian target of rapamycin (p-mTOR) signaling proteins and their downstream mediators. Together, these results demonstrated strong antiproliferative and apoptosis-inducing activity of verrucarin A for PDA cells through cell cycle arrest and inhibition of the prosurvival (antiapoptotic) AKT/NF-κB/mTOR signaling

Inhibition of Telomerase Activity by Oleanane Triterpenoid CDDO-Me in Pancreatic Cancer Cells is ROS-Dependent

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Inhibition of hTERT in pancreatic cancer cells by pristimerin involves suppression of epigenetic regulators of gene transcription

Previously we have shown that the inhibition of proliferation and induction of apoptosis in pancreatic ductal adenocarcinoma (PDA) cells by pristimerin (PM), a quinonemethide triterpenoid, was associated with the inhibition of human telomerase reverse transcriptase (hTERT) mRNA and hTERT protein. Herein we show that PM inhibits transcription factors and epigenetic processes that regulate hTERT expression. Treatment with PM inhibited transcription factors c-Myc, Sp1, NF-κB and kinases p-Akt and p-mTOR that regulate hTERT post-translationally. PM also downregulated DNA methyl transferases DNMT1 and DNMT3a and transcriptionally active chromatin markers, such as acetylated histone H3 (Lys9), acetylated histone H4, di-methyl H3 (Lys4) and tri-methyl H3 (Lys9). In addition, chromatin immunoprecipitation (ChIP) analysis showed decrease in c-Myc and Sp1 transcription factors, but not repressive factors CTCF, E2F or Mad1 in the regulatory region of the hTERT promoter after treatment with PM. PM also reduced acetylated histone 3 and 4 and methylated H3 at hTERT promoter. Collectively, these results indicated that PM downregulates hTERT/telomerase through the inhibition of the genetic and epigenetic regulators of hTERT gene expression