Tumor suppressor genes are often silenced in human cancer; this can occur by transcriptional repression by deacetylation in the promoter regions, mediated by histone deacetylase (HDAC). HDAC inhibitors can block cancer cell growth by restoring expression of tumor suppressor genes. In this study, we investigated the effects of a HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) on pancreatic cancer cells. SAHA inhibited the growth of 6 pancreatic cancer cell lines in a dose-dependent manner as measured by MTT and clonogenic assays (ED 50 10 26 M) associated with induction of apoptosis, G2 cell cycle arrest and also induced differentiation as indicated by morphology and increased expression of cytokeratin 7. It increased expression of p21 WAF1 (independent of the mutational status of p53), C/EBPa, RARa and E-cadherin; these genes have been associated with decreased proliferation in other cancers. SAHA decreased cyclin B1 expression; this cyclin normally promotes progression through G2 of the cell cycle. SAHA mediated acetylation of histone H3 globally, as well as, associated with the p21 WAF1 promoter, as measured by chromatin immunoprecipitation. SAHA also decreased levels of c-myc and cyclin D1, independent of an active b-catenin pathway. In further studies, the combination of SAHA and an inhibitor of DNA methylation, 5-Aza-2 0 -deoxycytidine, had an enhanced antiproliferative effect on pancreatic cancer cells. In summary, SAHA inhibited the growth of human pancreatic cancer cells by inducing apoptosis, differentiation and cell cycle arrest, as well as increase in the expression of several tumor suppressor genes. SAHA is a novel, promising therapeutic agent for human pancreatic cancers. ' 2007 Wiley-Liss, Inc.

Cucurbitacins are a group of diverse triterpenoid substances isolated from plants with medicinal properties. One particularly potent family member is cucurbitacin B (CuB). The antiproliferative effects of CuB against human breast cancer cells were tested. Six human breast cancer cell lines were examined because they represent a diverse mix of breast cancer subtypes varying in expression of estrogen receptor (ER), Her2/neu, and p53 mutation. The antiproliferative effect of CuB were also studied in vivo. The effective dose inhibiting 50% growth (ED 50 ) was between 10 -8 M and 10 -7 M for this collection of breast cancer cell lines. These cells underwent rapid morphologic changes after 15-20 min exposure to CuB (5´10 -7 M), which was associated with disruption of the microtubules and F-actin, as observed by confocal microscopy. C ucurbitacins were originally isolated from Cucurbitaceae plants. They are a group of diverse triterpenoid molecules containing a cucurbitane skeleton characterized by a 19-(10→9β)-abeo-10α-lanost-5-ene (Fig. 1).(1) They have been reported to possess a broad range of biological effects such as chemoprevention and hepatoprotection, as well as anti-inflammatory, antimicrobial and antitumor activities.(2-10) Cucurbitacins have been reported to inhibit several types of cancers including those originating in the prostate, (9) lung and breast (11) as well as choriocarcinoma.(12) Among the various cucurbitacins, the most abundant is cucurbitacin B (CuB) and its analog dihydrocucurbitacin B.(13) Our preliminary data suggest that CuB is one of the most potent compounds in this family.Breast cancer afflicts about one in nine women, with approximately 43 000 deaths per year in the USA from this disease. Although great progress has been made in both controlling the disease and preventing recurrence, additional novel therapies are needed leading us to focus on investigating the effects of CuB on breast cancer in vitro and in vivo. Materials and Methods Cells and compounds. MCF-7, MDA-MB-231, MDA-MB-453, T47D, BT474, SK-BR-3 and ZR-75-1 cells (American TypeCulture Collection, Manassas, VA, USA) were maintained at 37°C in 5% CO 2 in Roswell Park Memorial Institute media (RPMI)-1640 (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Invitrogen), 10 U/mL penicillin and 10 mg/ mL streptomycin (Invitrogen). CuB was kindly provided by CK Life Sciences International Inc., Hong Kong.Cytoskeletal staining for b-tubulin and F-actin. After incubation with CuB, culture media were removed and cells were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS, and incubated with fluorescein isothyanate (FITC)-conjugated antiβ-tubulin antibody (Sigma Chemical, St. Louis, MO, USA) and/or rhodaminephalloidin (Invitrogen) to detect filamentous (F)-actin. Confocal images were collected on a Leica microscope (Leica Microsystems, Wetzlar, Germany).Animal experiments. MDA-MB-231 (1 × 10 6 ) breast cancer cells were resuspended in 100-μL matrigel and were inocul...

Glioblastoma Multiforme (GBM) is almost inevitably a fatal tumor of the brain with most individuals dying within 1 year of diagnosis. It is the most frequent brain tumor in adults. Dose-response studies showed that Cucurbitacin B inhibited 50% growth (ED 50 ) of 5 human GBM cell lines in liquid culture at 10 27 M. Soft-gel assays demonstrated that nearly all of the GBM clonogenic cells were inhibited at 10 28 M of Cucurbitacin B. FACS analysis found that the compound (10 27 M, 24 hr) caused G2/M arrest. The GBM cells underwent profound morphologic changes within 15-30 min after exposure to Cucurbitacin B (10 27 M), rounding up and losing their pseudopodia associated with disruption of actin and microtubules, as observed by immunoflourescence. Cucurbitacin B (10 27 M) caused prominent multinucleation of the cells after they were pulse-exposed (48 hr) to the drug, washed and cultured in normal medium for an additional 2 days. The drug (10 27 M, 3-24 hr) increased levels of p-p38, p-JNK and p-JUN in U87 and T98G GBM cell lines as seen by Western blot. Interestingly, alterations in cell morphology caused by Cucurbitacin B (10 27 M) were blocked by the JNK inhibitor SP600125. In summary, Cucurbitacin B has a prominent anti-proliferative activity on GBM cells; and at least in part, the mode of action is by affecting the cytoskeleton, as well as, the JNK pathway. Clinical trails of this drug should be pursued in GBM.

Deficiency of the cholesteryl ester transfer protein (CETP) in humans is characterized by markedly elevated plasma concentrations of HDL cholesterol and apoA-I. To assess the metabolism of HDL apolipoproteins in CETP deficiency, in vivo apolipoprotein kinetic studies were performed using endogenous and exogenous labeling techniques in two unrelated homozygotes with CETP deficiency, one heterozygote, and four control subjects. All study subjects were administered '3C6-labeled phenylalanine by primed constant infusion for up to 16 h. The fractional synthetic rates (FSRs) of apoA-I in two homozygotes with CETP deficiency (0.135, 0.134 /d) were found to be significantly lower than those in controls (0.196±0.041 /d, P < 0.01).Delayed apoA-I catabolism was confirmed by an exogenous radiotracer study in one CETP-deficient homozygote, in whom the fractional catabolic rate of 125I-apoA-I was 0.139/d (normal 0.216±0.018/d). The FSRs of apoA-II were also significantly lower in the homozygous CETP-deficient subjects (0.104, 0.112/d) than in the controls (0.170±0.023/d, P < 0.01). The production rates of apoA-I and apoA-II were normal in both homozygous CETP-deficient subjects. The turnover of apoA-I and apoA-II was substantially slower in both HDL2 and HDL3 in the CETP-deficient homozygotes than in controls. The kinetics of apoA-I and apoA-II in the CETP-deficient heterozygote were not different from those in controls.These data establish that homozygous CETP deficiency causes markedly delayed catabolism of apoA-I and apoA-II without affecting the production rates of these apolipoproteins. (J.