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BACKGROUND/AIMS: Low 25-hydroxyvitamin D serum levels have been associated with the severity of liver fibrosis in genotype 1 chronic hepatitis C patients (G1CHC), and experimental evidence suggested a hepatoprotective role of vitamin D via interaction with hepatic vitamin D receptor (VDR). We assessed the hepatic expression of VDR protein and its association with liver disease severity. METHODS: Ninety-one consecutive patients with biopsy-proven G1CHC and available frozen liver tissue were evaluated. Ten subjects without chronic liver diseases and nine patients with autoimmune hepatitis served as controls. The hepatic expression of VDR protein was assessed by Western blot for quantification and by immunohistochemistry for morphological distribution. RESULTS: Liver VDR protein was mainly localized in hepatocytes and cholangiocytes, and its expression by a Western blot was similar in chronic hepatitis C (CHC) and controls (1.83 +/- 0.97 vs 2.18 +/- 0.62, P = .14) but was lower in autoimmune hepatitis (0.84 +/- 0.14, P < .001). The expression was lower in CHC with severe necroinflammatory activity (1.44 +/- 0.87) vs both controls and CHC with grade 1-2 inflammation (1.94 +/- 0.97, P = .01 and P = .03, respectively) but higher compared with autoimmune hepatitis (P = .007). A similar difference was observed in CHC patients with F3-F4 fibrosis whose VDR expression (1.51 +/- 1.07) was also lower compared with controls and CHC with F0-F2 fibrosis (1.98 +/- 0.89, P = .02 and P = .04, respectively) but higher vs autoimmune hepatitis (P = .003). At multivariate logistic regression analysis, low VDR protein expression remained associated with severe necroinflammatory activity and severe fibrosis (odds ratio 0.543,95% confidence interval 0.288-0.989, P = .04; and odds ratio 0.484,95% confidence interval 0.268-0.877, P = .01, respectively) in CHC after correction for clinical, biochemical, and histological features. CONCLUSION: In a cohort of G1CHC patients, the hepatic expression of VDR protein is associated with the severity of both liver fibrosis and inflammation.

There is convincing epidemiological and clinical evidence that, independent of aging, lifestyle and, notably, nutrition are associated with development or progression of major human cancers, including breast, prostate, colorectal tumors, and an increasingly large collection of diet-related cancers. Mechanisms underlying this association are mostly related to the distinct epigenetic effects of different dietary patterns. In this context, Mediterranean diet has been reported to significantly reduce mortality rates for various chronic illnesses, including cardiovascular diseases, neurodegenerative diseases and cancer. Although many observational studies have supported this evidence, dietary intervention studies using a Mediterranean dietary pattern or its selected food components are still limited and affected by a rather large variability in characteristics of study subjects, type and length of intervention, selected end-points and statistical analysis. Here we review data of two of our intervention studies, the MeDiet study and the DiMeSa project, aimed at assessing the effects of traditional Mediterranean diet and/or its component(s) on a large panel of both plasma and urine biomarkers. Both published and unpublished results are presented and discussed.

With the aim of enhancing the structural complexity and diversity of an existing collection of bi- and terphenyl compounds, we synthesized hybrid molecules comprising of spirocyclic ketones (a complexity-bearing core) and bi/terphenyls (privileged fragments). Compounds 1, 3, 4, and 6 showed well-defined activity on apoptosis and differentiation, making them potential leads for development as new anticancer agents and chemical probes to study signaling networks in neoplastic cells.

Resistance to imatinib mesylate is an emergent problem in the treatment of Bcr-Abl expressing myelogenous leukemias and additional therapeutic strategies are required. We observed that galangin, a non-toxic, naturally occurring flavonoid was effective as anti-proliferative, and apoptotic agent in Bcr-Abl expressing K562 and KCL22 cells and in imatinib mesylate resistant K562-R and KCL22-R cells. Galangin induced an arrest of cells in G0–G1phase of cell cycle and a decrease in pRb, cdk4, cdk1, cycline B levels; moreover, it was able to induce a monocytic differentiation of leukemic Bcr-Abl+ cells. Of note, galangin caused a decrease in Bcl-2 levels and markedly increased the apoptotic activity of imatinib both in sensitive or imatinib-resistant Bcr-Abl+ cell lines. In contrast, flavonoids unable to modify the Bcl-2 intracellular levels, such as fisetin and chrysin, did not increase the apoptotic effect of imatinib. These data suggest that galangin is an interesting candidate for a combination therapy in the treatment of imatinib-resistant leukemias.

To further explore the SAR of resveratrol-related trans-stilbene derivatives, here we describe the synthesis of (a) a series of 3,5-dimethoxy analogues in which a variety of substituents were introduced at positions 2', 3', 4', and 5' of the stilbene scaffold and (b) a second group of derivatives (2-phenylnaphthalenes and terphenyls) that incorporate a phenyl ring as a bioisosteric replacement of the stilbene alkenyl bridge. We thoroughly characterized all of the new compounds with respect to their apoptosis-inducing activity and their effects on the cell cycle. One of the new derivatives, 13g, behaved differently from the others, as it was able to block the cell cycle in the G(0)-G(1) phase and also to induce differentiation in acute myelogenous leukemia HL60 cells. Compared to resveratrol, the synthetic terphenyl 13g showed a more potent apoptotic and differentiating activity. Moreover, it was active on both multidrug resistance and Bcr-Abl-expressing cells that were resistant to resveratrol.

Pterostilbene and 3,5-hydroxypterostilbene are the natural 3,5-dimethoxy analogs of trans-resveratrol and piceatannol, two compounds which can induce apoptosis in tumor cells. In previous studies we demonstrated the importance of a 3,5-dimethoxy motif in conferring pro-apoptotic activity to stilbene based compounds so we now wanted to evaluate the ability of pterostilbene and 3,5-hydroxypterostilbene in inducing apoptosis in sensitive and resistant leukemia cells. When tested in sensitive cell lines, HL60 and HUT78, 3'-hydroxypterostilbene was 50-97 times more potent than trans-resveratrol in inducing apoptosis, while pterostilbene appeared barely active. However, both compounds, but not trans-resveratrol and piceatannol, were able to induce apoptosis in the two Fas-ligand resistant lymphoma cell lines, HUT78B1 and HUT78B3, and the multi drug-resistant leukemia cell lines HL60-R and K562-ADR (a Bcr-Abl-expressing cell line resistant to imatinib mesylate). Of note, pterostilbene-induced apoptosis was not inhibited by the pancaspase-inhibitor Z-VAD-fmk, suggesting that this compound acts through a caspase-independent pathway. On the contrary, 3'-hydroxypterostilbene seemed to trigger apoptosis through the intrinsic apoptotic pathway: indeed, it caused a marked disruption of the mitochondrial membrane potential delta psi and its apoptotic effects were inhibited by Z-VAD-fmk and the caspase-9-inhibitor Z-LEHD-fmk. Moreover, pterostilbene and 3'-hydroxypterostilbene, when used at concentrations that elicit significant apoptotic effects in tumor cell lines, did not show any cytotoxicity in normal hemopoietic stem cells. In conclusion, our data show that pterostilbene and particularly 3'-hydroxypterostilbene are interesting antitumor natural compounds that may be useful in the treatment of resistant hematological malignancies, including imatinib, non-responsive neoplasms.

Given our interest in finding potential antitumor agents and in view of the multifactorial mechanistic nature of cancer, in the present work, taking advantage of the multifunctional ligands approach, new chimeric molecules were designed and synthesized by combining in single chemical entities structural features of SAHA, targeting histone deacetylases (HDACs), with substituted stilbene or terphenyl derivatives previously obtained by us and endowed with antiproliferative and pro-apoptotic activity. The new chimeric derivatives were characterized with respect to their cytotoxic activity and their effects on cell cycle progression on different tumor cell lines, as well as their HDACs inhibition. Among the other, trans -6 showed the most interesting biological profile, as it exhibited a strong pro-apoptotic activity in tumor cell lines in comparison with both of its parent compounds and a marked HDAC inhibition.

Over the past years, we synthesized a series of new molecules that are hybrids of spirocyclic ketones as complexity-bearing cores with bi- and ter-phenyls as privileged fragments. Some of these newly-shaped small molecules showed antiproliferative, pro-apoptotic and differentiating activity in leukemia cell lines. In the present study, to investigate more in depth the mechanisms of action of these molecules, the protein expression profiles of K562 cells treated with or without the compounds IND_S1, MEL_T1, IND_S7 and MEL_S3 were analyzed using two-dimensional gel electrophoresis coupled with mass spectrometry. Proteome comparisons revealed several differentially expressed proteins, mainly related to cellular metabolism, chaperone activity, cytoskeletal organization and RNA biogenesis. The major results were validated by Western blot and qPCR. To attempt integrating findings into a cellular signaling context, proteomic data were explored using MetaCore. Network analysis highlighted relevant relationships between the identified proteins and additional potential effectors. Notably, qPCR validation of central hubs showed that the compound MEL_S3 induced high mRNA levels of the transcriptional factors EGR1 and HNF4-alpha; the latter to our knowledge is reported here for the first time to be present in K562 cells. Consistently with the known EGR1 involvement in the regulation of differentiation along megakaryocyte lineage, MEL_S3-treated leukemia cells showed a marked expression of glycoprotein IIb/IIIa (CD41) and glycoprotein Ib (CD42), two important cell markers in megakaryocytic differentiation, together with morphological aspects of megakaryoblasts and megakaryocytes.

The identification of novel compounds modulating the expression/activity of molecular targets downstream to BCR-ABL could be a new approach in the treatment of chronic myeloid leukemias (CMLs) resistant to imatinib or other BCR-ABL-targeted molecules. Recently, we synthesized a new class of substituted 2-(3,4,5-trimethoxybenzoyl)-2-N,N-dimethylamino-benzo[b]furans, and among these 3-iodoacetylamino-6-methoxybenzofuran-2-yl(3,5-trimethoxyphenyl)methanone (TR120) showed marked cytotoxic activity in BCR-ABL-expressing cells. Interestingly, TR120 was more potent than imatinib in cell growth inhibition and apoptosis induction in both BCR-ABL-expressing K562 and KCL22 cells. Moreover, it showed antitumor activity in imatinib-resistant K562-R and KCL22-R cells at concentrations similar to those active in the respective sensitive cells. Further, TR120 induced a marked decrease in signal transducer and activator of transcription 5 (STAT5) expression in K562 cells. Consistent with this effect, it determined a block of cells in the G0-G1 phase of the cell cycle, a decrease in the level of cyclin D1, and a reduction in Bcl-xL expression; however, it did not cause modifications in the Bcl-2 level. Of interest, TR120 had synergistic effects when used in combination with imatinib in both sensitive and resistant cells. Considering that STAT5 is a BCR-ABL molecular target that plays a key role in the pathogenesis of CML as well as in BCR-ABL-mediated resistance to apoptosis, TR120 could potentially be a useful novel agent in the treatment of imatinib-resistant CML.

Background. Polymorphisms in the interleukin-28B are important determinants in the spontaneous and drug-induced control of hepatitis C virus infection. Design and Methods. We assessed the association of rs8099917 and rs12979860 polymorphisms with spontaneous viral clearance, severity of liver fibrosis, and response to interferon-monotherapy in 245 Thalassemia Major patients with Hepatitis C Virus infection. Results. Ninety-eight patients (40%) had a spontaneous viral clearance, while 147 patients (60%) developed a chronic infection. Spontaneous viral clearance was more frequent among patients with the T/T genotype of rs8099917 polymorphism (OR 2.130; p=0.008) or C/C genotype of rs12979860 polymorphism (OR 2.425; p=0.001). During observation, 131 patients with chronic infection underwent a liver biopsy, age (OR 1.058; p=0.01) G/T or G/G genotypes of rs8099917 polymorphism (OR 3.962; p=0.001) and C/T or T/T genotypes of rs12979860 polymorphism (OR 3.494; p=0.005) were associated with severe liver fibrosis, independent of liver iron concentration. Finally, T/T genotype of rs8099917 polymorphism (OR 3.014; p=0.03) or C/C genotype of rs12979860 polymorphism (OR 3.285; p=0.01), age (OR 0.902; p=0.001), female gender (OR 3.418; p=0.01) and 2 or 3 virus C genotypes (OR 4.700; p=0.007 ) were independently associated with sustained virological response in 114 patients treated with alpha- interferon . Conclusions. Polymorphisms in the interleukin-28B are associated with the control of hepatitis C virus infection in Thalassemia Major patients, and the knowledge of allelic patterns has a relevant role in determining the prognosis and therapeutic management.

Background Genotype 1 (G1) chronic hepatitis C (CHC) patients achieving a rapid virological response (RVR) on pegylated interferon (PEG-IFN) plus ribavirin have a high chance of sustained virological response (SVR), influenced by IL28B status, viral load, fibrosis and insulin resistance. We assessed whether 25-hydroxyvitamin D (25[OH]D) serum levels are linked to RVR and can be used together with IL28B to construct a pretreatment model to predict RVR. Methods A total of 117 consecutive patients with G1 CHC were evaluated by biopsy and anthropometric and metabolic measurements. 25(OH)D serum levels were measured by HPLC. IL28B rs12979860 and rs8099917 polymorphisms were also evaluated. All patients underwent antiviral therapy with PEG-IFN-α2a plus ribavirin. HCV RNA was assessed at baseline, week 4, week 12, at the end of therapy and after 6 months of follow-up. Results Mean ±sd 25(OH)D serum levels were 26.3 ±10.6 μg/l (range 8.0–58.0) and 31 (26.5%) patients had the rs12979860 CC polymorphism. RVR was achieved in 35 (29.9%) patients, and 32 (91.4%) of them had an SVR, compared to 26 of 82 (31.7%) without RVR. The rs12979860 CC polymorphism (OR 4.575, 95% CI 1.761, 11.889; P=0.002) and higher 25(OH)D levels (OR 1.055, 95% CI 1.010, 1.101; P=0.01) were independently associated with the achievement of RVR by multivariate analysis. The likelihood of RVR progressively increased from patients in the worst class (vitamin DConclusions In patients with G1 CHC, 25(OH)D serum levels and IL28B status are independently associated with the likelihood to achieve RVR and SVR. When incorporated into a pretreatment predictive model they can assist in further discriminating patients with a high likelihood of achieving RVR and SVR.

During a screening for compounds that could act against Mycobacterium tuberculosis, a series of new cellular antiproliferative agents was identified. The most cytotoxic molecules were evaluated against a panel of human cell lines derived from hematological and solid human tumors. In particular, (E)-2-(1H-benzo[d] [1,2,3]triazol-1-yl)-3-(4-methoxyphenyl)acrylonitrile (1) was found to be of a potency comparable to etoposide and greater than 6-mercaptopurine in all cell lines tested. Accordingly, a synthesis of a new series of (E)-2-(5,6-dichloro-1H-benzo[d] [1,2,3]triazol-1-yl)-3-(4-R-phenyl)acrylonitriles was conducted in order to extend the studies of structure-activity relationship (SAR) for this class of molecules. With the aim to evaluate if 3-aryl-2-[1H-benzotriazol-1-yl]acrylonitriles were able to act like tubulin binding agents, the effects on cell cycle distribution of the most active compounds (1, 2a, 3 and 4) were analyzed in K562 cells. A detailed molecular modeling study of the putative binding mode of this series of compounds on tubulin is also reported.

Microtubules are dynamic structures that play a crucial role in cellular division and are recognized as an important target for cancer therapy. In search of new compounds with strong antiproliferative activity and simple molecular structure, a new series of 2-amino-3-(3',4',5'-trimethoxybenzoyl)-5-(hetero)aryl ethynyl thiophene derivatives was prepared by the Sonogashira coupling reaction of the corresponding 5-bromothiophenes with several (hetero)aryl acetylenes. When these compounds were analyzed in vitro for their inhibition of cell proliferation, the 2- and 3-thiophenyl acetylene derivatives were the most powerful compounds, both of which exerted cytostatic effects at submicromolar concentrations. In contrast, the presence of a more flexible ethyl chain between the (hetero)aryl and the 5-position of the thiophene ring resulted in significant reduction in activity relative to the 5-(hetero)aryl acetylene substituted derivatives. The effects of a selected series of compounds on cell cycle progression correlated well with their strong antiproliferative activity and inhibition of tubulin polymerization. We found that the antiproliferative effects of the most active compounds were associated with increase of the proportion of cells in the G(2)/M and sub-G(1) phases of the cell cycle. ispartof: Bioorganic & Medicinal Chemistry Letters vol:21 issue:9 pages:2746-51 ispartof: location:England status: published

Several new 2-{[(2E)-3-phenylprop-2-enoyl]amino}benzamides 12a–s and 17t–v were synthesized by stirring in pyridine the (E)-3-(2-R1-3-R2-4-R3-phenyl)acrylic acid chlorides 11c–k and 11t–v with the appropriate anthranilamide derivatives 10a–c or the 5-iodoanthranilic acid 13. Some of the synthesized compounds were evaluated for their in vitro antiproliferative activity against the full NCI tumor cell line panel derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast). COMPARE analysis, effects on tubulin polymerization in cells and with purified tubulin, and effects on cell cycle distribution for 17t, the most active of the series, indicate that these new antiproliferative compounds act as antitubulin agents.

Background and aim To investigate the hepatic expression of retinol-binding protein-4 (RBP4) in chronic hepatitis C (CHC) and nonalcoholic steatohepatitis (NASH) patients, and its association with biochemical and histological patterns of liver damage. Materials and methods Sixty-six genotype 1 CHC and 32 NASH patients were tested for hepatic RBP4 expression. Liver expression at immunostaining was scored as 0 (slight), 1 (mild), 2 (moderate), and 3 (intense). In addition, the mRNA and the quantitative protein expressions of RBP4 were tested by PCR and by western blot, respectively, in 12 NASH and 28 CHC patients. Twelve subjects undergoing elective cholecystectomy served as controls. Results Ten (31%), 16 (50%) and 6 (19%) NASH patients, and 21 (32%), 31 (47%) and 14 (21%) CHC patients had scores of 1, 2 and 3, respectively. All control subjects scored 0. In both CHC and NASH liver RBP4 scores were directly related to western blot ( p = 0.001 and p = 0.03), not to mRNA expression ( p = 0.77 and p = 0.40). Older age (OR, 1.07; 95%CI, 1.01–1.13), RBP4 score (4.26; 1.27–14.21) and HOMA (2.26; 1.15–4.42) were independently associated with steatosis ≥10% in CHC patients. In NASH lobular inflammation (OR, 3.77; 95%CI, 1.01–24.22) and RBP4 score (4.87; 1.003–23.65) were the only risk factors for fibrosis ≥2 at logistic regression analysis. Conclusion Hepatic storage of RBP4, unrelated to its expression, could cause liver damage both in NASH and CHC.

The central role of microtubules in cell division and mitosis makes them a particularly important target for anticancer agents. On our early publication, we found that a series of 2-(3',4',5'-trimethoxybenzoyl)-3-aminobenzo[b]thiophenes exhibited strong antiproliferative activity in the submicromolar range and significantly arrested cells in the G2-M phase of the cell cycle and induced apoptosis. In order to investigate the importance of the amino group at the 3-position of the benzo[b]thiophene skeleton, the corresponding 3-unsubstituted and methyl derivatives were prepared. A novel series of inhibitors of tubulin polymerization, based on the 2-(3,4,5-trimethoxybenzoyl)-benzo[b]thiophene molecular skeleton with a methoxy substituent at the C-4, C-5, C-6 or C-7 position on the benzene ring, was evaluated for antiproliferative activity against a panel of five cancer cell lines, for inhibition of tubulin polymerization and for cell cycle effects. Replacing the methyl group at the C-3 position resulted in increased activity compared with the corresponding 3-unsubstituted counterpart. The structure-activity relationship established that the best activities were obtained with the methoxy group placed at the C-4, C-6 or C-7 position. Most of these compounds exhibited good growth inhibition activity and arrest K562 cells in the G2-M phase via microtubule depolymerization. ispartof: Bioorganic & Medicinal Chemistry vol:18 issue:14 pages:5114-5122 ispartof: location:England status: published

A series of new 3-amino-N-phenyl-1H-indazole-1-carboxamides 10 have been prepared from commercially available phenyl isocyanate precursors 8 and 3-aminoindazole 9. Some of the synthesized compounds were evaluated for their in vitro antineoplastic activity against 60 human cell lines derived from seven clinically isolated cancer types (lung, colon, melanoma, renal, ovarian, brain, and leukemia) according to the NCI standard protocol. The test results indicated that 3-amino-1H-indazole-1-carboxamides 10 were endowed with an interesting antiproliferative activity. The most active compounds of this series, 10d,e, were able to inhibit cell growth of many neoplastic cell lines at concentrations lower than 1 μM (0.0153 μM in SR leukemia) causing a block in G0–G1 phase of cell cycle. Analysis of pRb expression showed that these two compounds increased the ratio between underphosphorylated pRb and total pRb. The X-ray structure of 10w, confirmed the 3-amino-N-phenyl-1H-indazole-1-carboxamide structure of compounds 10.

The biological importance of microtubules in mitosis and cell division makes them an interesting target for the development of anticancer agents. Small molecules such as benzo[b]furans are attractive as inhibitors of tubulin polymerization. Thus, a new class of inhibitors of tubulin polymerization based on the 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[b]furan molecular skeleton, with electron-donating (Me, OMe or OH) or electron-withdrawing (F, Cl and Br) substituents on the benzene ring, was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell cycle effects. Adding a methyl group at the C-3 position resulted in increased activity. The most promising compound in this series was 2-(3′,4′,5′-trimethoxybenzoyl)-3-methyl-6-ethoxy-benzo[b]furan, which inhibits cancer cell growth at nanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.

In this continuation of our research on derivatives containing the stilbene privileged structure or that are derived from it, we report the results of further studies carried out on the previously initiated collection of compounds. We used a parallel synthetic approach to rapidly obtain small sets of compounds and started the annotation of the library in progress by calculating some physicochemical properties to be eventually correlated with biological activities. A pharmacophore for the antiproliferative activity was also built to summarize the features of the library. We evaluated the antiproliferative and pro-apoptotic activities of all compounds as well as the cell-cycle effects of some representative compounds. After in-depth investigations, 3'-phenyl- [1, 1';4', 1"]terphenyl-4,3",5"-triol showed the most interesting biological profile, as it interferes with cell-cycle progression at the G(1) -> S transition, acting on retinoblastoma phosphorylation and inducing cell differentiation.

A new study on terphenyl and diaryl-isoxazole and -isoxazoline derivatives, maintaining a common 3-adamantyl-4-hydroxyphenyl moiety, has been conducted to find compounds with growth supporting and antiapoptotic properties. Unexpectedly, diphenyisoxazole derivatives bearing a nitro group replacing the carboxylic function have been found with the highest cell protective activity within the series, in complete and in serum-free conditions. Inhibition of apoptosis induced by daunorubicin has also been observed for the most active compound.

Microtubules are among the most successful targets of compounds potentially useful for cancer therapy. A new series of inhibitors of tubulin polymerization based on the 2-amino-3-(3,4,5-trimethoxybenzoyl)-4,5,6,7-tetrahydrothieno[b]pyridine molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. The most promising compound in this series was 2-amino-3-(3,4,5-trimethoxybenzoyl)-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[b]pyridine, which inhibits cancer cell growth with IC(50)-values ranging from 25 to 90 nM against a panel of four cancer cell lines, and interacts strongly with tubulin by binding to the colchicine site. In this series of N(6)-carbamate derivatives, any further increase in the length and in the size of the alkyl chain resulted in reduced activity.

Several stilbenes, related to known resveratrol, have been synthesized and tested for their anticancer effect on HL60 leukemia cell line, taking particular care of the cell cycle analysis. The most potent compound was the known (Z)-3,4',5-trimethoxystilbene (6b) which was active as apoptotic agent at 0.24 microM. Differently from other stilbenes (including resveratrol) that induced a prevalent recruitment of cells in S phase of cell cycle, we found a peculiar behavior of 6b that caused a decrease of cells in all phases of cell cycle (G0-G1, S, and G2-M) and a proportional increase of apoptotic cells. The potent pro-apoptotic activity shown by compound 6b and its effects on cell cycle make this compound of great interest for further investigations.

New terphenyl derivatives have been synthesized and tested for their effect on cell survival in serum-free cultures. These compounds protected HL60 cells from death and supported their growth with an activity higher than that of the natural 14-hydroxy-retro-retinol. Terphenyls 26 and 28 also possess antiapoptotic activity on neuronal cells, proving them as possible candidates for the treatment of neurodegenerative and ischemic diseases.

Two new series of combretastatin (CA-4) analogues have been prepared. The alkenyl motif of CA-4 was replaced either by a five-membered heterocyclic (isoxazoline or isoxazole) or by a six-membered ring (pyridine or benzene). The new compounds have been evaluated for their effects on tubulin assembly and for cytotoxic and apoptotic activities. Five compounds (18b, 20a, 21a, 34b, and 35b) demonstrated an attractive profile of cytotoxicity (IC501 microM) and apoptosis-inducing activity but poor antitubulin activity. The isoxazoline derivatives 18b, 20a, and 21a, demonstrated potent apoptotic activity different from that of natural CA-4. Their ability to block most cells in the G2 phase suggests that these compounds could act on targets different from the mitotic spindle. This would indicate activation of both the intrinsic and the extrinsic apoptotic pathways. The data suggest unambiguously that structural alteration of the stilbene motif of CA-4 can be extremely effective in producing potent apoptosis-inducing agents.

Gap-junction-mediated intercellular communication (GJIC) is required for completion of embryonic development, tissue homeostasis, and regulation of cell proliferation and death. Although, as emphasized in several reports, defects or disruption of GJIC may be important in carcinogenesis, the potential role of GJIC in the onset and progression of human prostate cancer remains ill-defined. The gap junction channel-forming connexins (Cx) comprise a multigene family of highly conserved proteins that are differentially expressed in a tissue- and development-specific manner; changes in connexin expression are also commonly seen during cellular differentiation. However, when multiple connexins are concurrently expressed, gap junction channels may consist of more than one connexin species. This is important, because only certain pairings give rise to functional channels. In our studies, we investigated GJIC in a panel of both nontumorigenic (RWPE-1) and malignant (RWPE-2, LNCaP, DU-145) human prostate epithelial cells, compared to a normal rat liver epithelial F344 (WB-1) cell line, as it was found to be junctionally proficient. In addition, expression and regulation of Cx43 and Cx32 were also inspected using western blot analysis. The ability of hormones, antihormones, and the antihypertensive drug forskolin to restore GJIC in nontumorigenic and malignant human prostate epithelial cells was examined by the scrape-loading/dye transfer (SL/DT) or fluorescence recovery after photobleaching (FRAP) methods using an Ultima laser cytometer. Results from both assays showed that neither nontumorigenic nor malignant prostate cells have functional GJIC. However, both estrone (E1) and forskolin (FK) induced a significant increase (4.4- and 2.8-fold, respectively) in cell-cell communication only in the RWPE-1 cells. Interestingly, the use of Matrigel, a solubilized basement membrane, as substrate for cell attachment and growth resulted in the rescue of GJIC activity in RWPE-1 cells, as revealed by the SL/DT method. Furthermore, E1 induced a twofold increase in connexin 43 (Cx43), whereas forskolin caused a 50% reduction in Cx32 expression in RWPE-1 cells. These data suggest that agents that increase Cx43:Cx32 ratio may restore GJIC in junctionally deficient cells, providing a basis for the development of new strategies for the prevention and treatment of human prostate cancer.

Expression of gap-junction proteins connexins (Cx), specifically Cx43, Cx32, and Cx26, in both nontumorigenic (RWPE-1) and tumorigenic (RWPE-2) human prostate epithelial cells as well as in two cell clones (WPEI-7 and WPEI-10) originating from the RWPE-1 cell line was investigated. The aim was to determine whether individual connexins are differentially expressed in cultured cells. Western blot analysis revealed striking differences in the expression of individual connexins in the cell lines studied. In particular, Cx43 is largely expressed in RWPE-1 and WPEI-10 cells, whereas Cx32 is expressed predominantly in RWPE-2 and WPEI-7 cells. In addition, both forskolin and estrone increase Cx43 expression levels in WPEI-10 cells, with no apparent effect on WPEI-7 cells. Conversely, forskolin and especially estrone induce a marked increase of Cx32 in WPEI-7 cells, whereas Cx32 expression is limitedly affected by both agents in WPEI-10 cells. Overall, expression levels of Cx43 and Cx32 appear to be inversely related, with RWPE-1 and WPEI-10 cells having a significantly higher Cx43 to Cx32 ratio than that observed in RWPE-2 and WPEI-7 cells. We recently reported that junctional communication could be rescued in RWPE-1 cells by either forskolin or estrone and that restoration of GJIC is associated with an increase of Cx43 or a decrease of Cx32, or both, eventually leading to a marked rise of the Cx43 to Cx32 ratio. Studies are currently ongoing in our laboratories to assess the potential effect of agents increasing the Cx43 to Cx32 ratio on GJIC activity in these systems.