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Annually, 48,000 people die from pancreatic ductal adenocarcinoma (PDAC), ranking it the fourth among cancer-related deaths in the United States. Currently, anti-cancer drugs are not effective against PDAC, and only extends survival by 3 months. Aberrant DNA methylation has been shown to play an important role during carcinogenesis in PDAC, with approximately 80% of tumor overexpressing the DNA methyltransferase 1 (DNMT1) protein. In the present study, we used DNMTs as a screening platform to find a new DNMT inhibitor, n-butylidenephthalide (n-BP), which is identified from a Chinese herbal drug. n-BP could inhibit DNMT1 expression in both dose-dependent and time-dependent manner. It also displays an effect in suppressing growth of PDAC cells and inducing cell cycle arrest at G0/G1 phase leading apoptosis. Growth suppression can be restored by the overexpression of DNMT1 in PDAC cells. Furthermore, we found n-BP-mediated DNMT1 suppression influenced the protein stability rather than changing the RNA expression. Through microarray studies, we found that the patched domain contained 4 (PTCHD4) is the potential downstream gene of DNMT1. Following silencing of PTCHD4 expression by siRNA, n-BP decreased tumor growth inhibition. Finally, in vivo, two animal models were used to evaluate the efficacy and survival after n-BP treatment by interstitial control release polymer delivery. The results show that n-BP could effectively inhibit PDAC tumor volume growth and extend animal survival. In summary, n-BP may inhibit the growth of human PDAC cells though reducing DNMT1 and increasing the expression of PTCHD4 both in vitro and in vivo.

Introduction and aim. Developing reliable biomarkers for hepatocellular carcinoma (HCC) patients who are at a high risk of recurrence after curative hepatic resection is very important for determining subsequent therapeutic strategies. We investigated the role of the cell cycle factor NIMA-related kinase 2 (NEK2) in HCC progression in hepatoma cells and post-surgery patients. Material and methods. The effects of NEK2 on proliferation, invasion and migration of hepatoma HuH7 and SK-Hep1 cells were evaluated. In a post-surgery HCC cohort (N = 97), the Nek2 induction levels in the tumors were examined with real-time RT-PCR analysis, and the results were analyzed for their correlations with recurrence. Results. NEK2 promoted G1 to S phase cell cycle progression by causing increases in cyclin D1 and AKT phosphorylation and decreases in the cyclin-dependent kinase inhibitor p27, indicating that NEK2 plays an important role during interphase in addition to its previously identified role in M phase. NEK2 also enhanced the proliferation, migration and invasion of hepatoma cells and regulated the expression of E-cadherin and MMP9. The Nek2 mRNA levels in the tumors were highly correlated with recurrence rates in the post-surgery HCC patients. Combined evaluation of the tumor AJCC stage and the Nek2level can serve as a reliable method for predicting the relative risk of HCC recurrence in these patients. Conclusions. NEK2 plays a significant role in cell cycle progression in the inter- and M-phases. NEK2 enhances HCC metastasis and is correlated with recurrence and thus can potentially serve a promising high-risk biomarker for HCC.

BACKGROUND The androgen receptor (AR) is a main therapeutic target for treatment of prostate cancer (PCa). The natural compound isochaihulactone (K8), which has a chiral center ring and two racemic forms (E-K8 and Z-K8), has anti-tumor effects on multiple cancer types both in vitro and in vivo. Here, we determined which form of K8 contains significant tumor cytotoxicity and examined how this form regulates AR expression in PCa cells and xenografts. METHODS We chose the androgen-dependent human PCa cell line LNCaP and the androgen-independent cell lines DU145 and PC-3 to study the anti-tumor potency and AR regulation mediated by Z-K8. We measured cell viability and used flow cytometry, RT-PCR, and Western blotting. Growth inhibition in vivo was evaluated with an LNCaP xenograft animal model. RESULTS In LNCaP cells, Z-K8 significantly repressed cell proliferation, induced apoptosis, repressed AR mRNA and protein expression in a time-dependent manner, and induced JNK phosphorylation. Furthermore, treatment with a JNK inhibitor significantly abolished Z-K8-induced AR downregulation. Z-K8 did not significantly inhibit reporter gene expression of constructs containing the AR promoter when it contained a mutated Sp1 binding site. Z-K8 also showed anti-tumor effects in the xenograft animal model. CONCLUSION Z-K8 not only induced LNCaP apoptosis but also reduced AR expression. These results indicate that Z-K8 may be a potential anti-tumor drug for PCa therapy. Prostate 73: 531–541, 2013. © 2012 Wiley Periodicals, Inc.

Following a stroke, the administration of stem cells that have been treated with granulocyte colony-stimulating factor (GCSF) can ameliorate functional deficits in both rats and humans. It is not known, however, whether the application of GCSF-mobilized peripheral blood stem cells (PBSCs) to human skin can function as an antiaging treatment. We used a Lanyu pig ( Sus scrofa) model, since compared with rodents, the structure of a pig's skin is very similar to human skin, to provide preliminary data on whether these cells can exert antiaging effects over a short time frame. GCSF-mobilized PBSCs from a young male Lanyu pig (5 months) were injected intradermally into the cheek skin of aged female Lanyu pigs, and tissues before and after the cell injections were compared to determine whether this treatment caused skin rejuvenation. Increased levels of collagen, elastin, hyaluronic acid, and the hyaluronic acid receptor CD44 were observed in both dermal and subcutaneous layers following the injection of PBSCs. In addition, the treated skin tissue was tighter and more elastic than adjacent control regions of aged skin tissue. In the epidermal layer, PBSC injection altered the levels of both involucrin and integrin, indicating an increased rate of epidermal cell renewal as evidenced by reductions in both cornified cells and cells of the spinous layers and increases in the number of dividing cells within the basal layer. We found that the exogenous PBSCs, visualized using fluorescence in situ hybridization, were located primarily in hair follicles and adjacent tissues. In summary, PBSC injection restored young skin properties in the skin of aged (90 months) pigs. On the basis of our preliminary data, we conclude that intra dermal injection of GCSF-mobilized PBSCs from a young pig can rejuvenate the skin in aged pigs.

Cellular oncogenes have been shown to play crucial roles in the cell death process induced by cytotoxic agents. In this study, we have demonstrated that v-H-ras transformed NIH 3T3 cells but not other transformants (v-raf, v-src, v-erbB-2, v-fes and v-mos) exhibited a survival advantage to treatment by a DNA-damaging agent, methylmethanesulfonate (MMS). Subsequently, the biochemical and morphologic criteria of MMS-treated cells were examined. It was found that MMS induced v-H-ras transformants to go through necrosis, but it induced other transformed cells to undergo apoptosis. The levels of glutathione (GSH) within each transformant as well as in NIH 3T3 cells, were determined. The results showed that GSH levels within ras transformants were 2- to 7-fold higher than the levels in other transformants and normal NIH 3T3 cells. By using the GSH synthesis inhibitor buthionine sulfoximine, GSH levels were artificially reduced. This depletion, however, made ras transformed cells more sensitive to MMS killing, but the mode of cell death was still necrosis. Western blot analysis demonstrated that the anti-apoptotic protein Bcl-2 was constitutively expressed in ras transformed cells but not in NIH 3T3 or other transformed cells. The level of Bcl-2 was correlated with the resistant phenotype of ras transformants during MMS treatment. These observations suggest that GSH and Bcl-2 levels may cooperatively confer the resistant phenotype of ras transformants in response to MMS. In addition, the mode of cell death may possibly be determined at least in part by Bcl-2 protein.

Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan. A rapid and efficient detection method is required to identify this foodborne pathogen. A 0.76-Kb HindIII DNA fragment was cloned from the chromosomal DNA of V. parahaemolyticus strain no. 93, designated as pR72H fragment, was used as a polynucleotide probe. It was labeled with digoxigenin-11-dUTP (DIG) by the random primer-labeling method. The sensitivity and specificity of the digoxigenin-labeled 0.76-Kb DNA probe was determined by colony hybridization assay. Under stringent hybridization conditions, 122 of 124 isolates of V. parahaemolyticus showed positive hybridization reaction with DIG-0.76-Kb DNA probe; the negative strains were attributed to slow growth. The DIG-0.76-Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms. The sensitivity and specificity of this DIG probe are 98% and 100%, respectively. This nonisotopic colony hybridization assay can be very useful for routine monitoring of V. parahaemolyticus in the food industry, environmental analysis and clinical laboratories.