Archive of Medical Research

Pretreatment of docetaxel enhances TRAIL-mediated apoptosis in prostate cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether treatment of docetaxel (DTX) can enhance apoptotic cell death by TRAIL against androgen-independent prostate cancer (AIPC). The cell death effect of combinations of TRAIL and docetaxel on prostate cancer cell lines (androgen-dependent LNCaP and its derived androgen-independent, metastatic C4-2B) was evaluated by synergisms of apoptosis. Western blot assay and DNA fragmentation assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL induced apoptosis in the presence of docetaxel. In addition, we investigated the in vitro anti-tumor effects of combined docetaxel and TRAIL using MAP kinase inhibitors. Docetaxel itself could not induce apoptotic cell death in 24 h even in high concentration. Apoptotic cell death, however, was drastically enhanced by pretreatment of docetaxel 20 h before TRAIL treatment. Docetaxel enhanced the PARP-1 cleavage and caspases activation by TRAIL especially in androgen-independent, metastatic C4-2B cell line, mainly by phosphorylation of Bcl-2 by JNK activation. It appears that apoptotic cell death was protected by the JNK inhibitor SP600125. The results of our study show that pretreatment of docetaxel is able to enhance the apoptosis produced by TRAIL in prostate cancer cells, especially in hormone-refractory prostate cancer (HRPC). J. Cell. Biochem. (c) 2008 Wiley-Liss, Inc.

Yoo J, Park SS, Lee YJ.

Department of Surgery and Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.

Hypermethylation of RAS effector related genes and DNA methyltransferase 1 expression in endometrial carcinogenesis.

Epigenetic aberration is known to be important in human carcinogenesis. Promoter methylation status of RAS effector related genes, RASSF1A, RASSF2A, hDAB2IP (m2a and m2b regions) and BLU, was evaluated in 76 endometrial carcinomas and their non-neoplastic endometrial tissue by methylation specific PCR. Hypermethylation of at least one of the 5 genes was detected in 73.7% of carcinomas. There were significant correlations between methylation of hDAB2IP and RASSF1A, RASSF2A (p = 0.042, p = 0.012, respectively). Significantly, more frequent RASSF1A hypermethylation was found in Type I endometrioid carcinomas than Type II carcinomas (p = 0.049). Among endometrioid cancers, significant association between RASSF1A hypermethylation and advanced stage, as well as between methylation of hDAB2IP at m2a region with deep myometrial invasion (p < 0.05) was observed. mRNA expression of RASSF1A, RASSF2A and BLU in endometrial cancer cell lines significantly increased after treatment with the demethylating agent 5-Aza-2\’-deoxycytidine supporting the repressive effect of hypermethylation on their transcription. Immunohistochemical study of DNMT1 on eight normal endometrium, 16 hyperplastic endometrium without atypia, 40 atypical complex hyperplasia and 79 endometrial carcinomas showed progressive increase in DNMT1 immunoreactivity from normal endometrium to endometrial hyperplasia and endometrioid carcinomas (p = 0.001). Among carcinomas, distinctly higher DNMT1 expression was observed in Type I endometrioid carcinomas (p < 0.001). DNMT1 immunoreactivity correlated with RASSF1A and RASSF2A methylation (p < 0.05). The data suggested that hypermethylation of RAS related genes, particularly RASSF1A, was involved in endometrial carcinogenesis with possible divergent patterns in different histological types. DNMT1 protein overexpression might contribute to such aberrant DNA hypermethylation of specific tumor suppressor genes in endometrial cancers. (c) 2008 Wiley-Liss, Inc.

Liao X, Siu MK, Chan KY, Wong ES, Ngan HY, Chan QK, Li AS, Khoo US, Cheung AN.

Department of Pathology, the University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong.

A Comparison of the Aldosterone-blocking Agents Eplerenone and Spironolactone.

Improved understanding of the adverse pharmacological properties of aldosterone has prompted investigation of the clinical benefits of blocking aldosterone at the receptor level. This article reviews the pharmacology, clinical efficacy, and tolerability of the two available blocking agents, spironolactone and eplerenone. A Medline search identified clinical studies assessing spironolactone and eplerenone. Priority was given to large, well-controlled, clinical trials and comparative studies. Pharmacological differences between spironolactone and eplerenone include lower affinity of eplerenone for progesterone, androgen, and glucocorticoid receptors; more consistently demonstrated nongenomic properties for eplerenone; and the presence of long-acting metabolites for spironolactone. Both agents effectively treat hypertension and heart failure but comparisons are complicated by the deficiency of head-to-head trials and differences between patient populations. There are differences in the tolerability profiles; spironolactone is associated with dose-dependent sexual side effects. Both agents produce dose-dependent increases in potassium concentrations, although the effect with spironolactone appears to be greater when both agents are administered at recommended doses. Choice of a specific agent should be based on individual patient issues, such as the nature of heart failure and patient concerns about adverse events. Copyright (c) 2008 Wiley Periodicals, Inc.

Struthers A, Krum H, Williams GH.

Division of Medicine and Therapeutics, Ninewells Hospital and Medical School, Dundee, UK;

Silencing of hSlo potassium channels in human osteosarcoma cells promotes tumorigenesis.

Potassium channels, the most diverse superfamily of ion channels, have recently emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large conductance Ca(2+)-activated K(+) channels, often referred to as BK channels, are at the crossroads of several tumor-associated processes such as cell proliferation, survival, secretion and migration. Despite the high BK channel expression in osteosarcoma (OS), their function has not yet been investigated in this malignant bone pathology. Here, using stable RNA interference to reduce the expression of hSlo, the human pore-forming alpha-subunit of the BK channel, in human Cal72 OS cells, we show that BK channels play a functional role in carcinogenesis. Our results reveal for the first time that BK channels exhibit antitumoral properties in OS in vivo and affect the tumor microenvironment through the modulation of both chemokine expression and leukocyte infiltration. (c) 2008 Wiley-Liss, Inc.

Cambien B, Rezzonico R, Vitale S, Rouzaire-Dubois B, Dubois JM, Barthel R, Soilihi BK, Mograbi B, Schmid-Alliana A, Schmid-Antomarchi H.

Université de Nice Sophia Antipolis, UFR Sciences, Nice F‐06002, France.

Pooled analysis of the accuracy of five cervical cancer screening tests assessed in eleven studies in Africa and India.

Cervical cancer is the main cancer among women in sub-Saharan Africa, India and other parts of the developing world. Evaluation of screening performance of effective, feasible and affordable early detection and management methods is a public health priority. Five screening methods, naked eye visual inspection of the cervix uteri after application of diluted acetic acid (VIA), or Lugol\’s iodine (VILI) or with a magnifying device (VIAM), the Pap smear and human papillomavirus testing with the high-risk probe of the Hybrid Capture-2 assay (HC2), were evaluated in 11 studies in India and Africa. More than 58,000 women, aged 25-64 years, were tested with 2-5 screening tests and outcome verification was done on all women independent of the screen test results. The outcome was presence or absence of cervical intraepithelial neoplasia (CIN) of different degrees or invasive cervical cancer. Verification was based on colposcopy and histological interpretation of colposcopy-directed biopsies. Negative colposcopy was accepted as a truly negative outcome. VIA showed a sensitivity of 79% (95% CI 73-85%) and 83% (95% CI 77-89%), and a specificity of 85% (95% CI 81-89%) and 84% (95% CI 80-88%) for the outcomes CIN2+ or CIN3+, respectively. VILI was on average 10% more sensitive and equally specific. VIAM showed similar results as VIA. The Pap smear showed lowest sensitivity, even at the lowest cutoff of atypical squamous cells of undetermined significance (57%; 95% CI 38-76%) for CIN2+ but the specificity was rather high (93%; 95% CI 89-97%). The HC2-assay showed a sensitivity for CIN2+ of 62% (95% CI 56-68%) and a specificity of 94% (95% CI 92-95%). Substantial interstudy variation was observed in the accuracy of the visual screening methods. Accuracy of visual methods and cytology increased over time, whereas performance of HC2 was constant. Results of visual tests and colposcopy were highly correlated. This study was the largest ever done that evaluates the cross-sectional accuracy of screening tests for cervical cancer precursors in developing countries. The merit of the study was that all screened subjects were submitted to confirmatory investigations avoiding to verification bias. A major finding was the consistently higher sensitivity but equal specificity of VILI compared with VIA. Nevertheless, some caution is warranted in the interpretation of observed accuracy measures, since a certain degree of gold standard misclassification cannot be excluded. Because of the correlation between visual screening tests and colposcopy and a certain degree of over-diagnosis of apparent CIN2+ by study pathologists, it is possible that both sensitivity and specificity of VIA and VILI were overestimated. Gold standard verification error could also explain the surprisingly low sensitivity of HC2, which contrasts with findings from other studies. (c) 2008 Wiley-Liss, Inc.

Arbyn M, Sankaranarayanan R, Muwonge R, Keita N, Dolo A, Mbalawa CG, Nouhou H, Sakande B, Wesley R, Somanathan T, Sharma A, Shastri S, Basu P.

Scientific Institute of Public Health, Unit of Cancer Epidemiology, Brussels, Belgium.

Comparative analysis of immunohistochemical markers with invasiveness and histologic differentiation in squamous cell carcinoma and basal cell carcinoma of the skin.

BACKGROUND: This study evaluates several tumor-related markers to examine the expression pattern of markers according to the invasiveness and histopathologic differentiation of squamous cell carcinoma and basal cell carcinoma. METHODS: Ninety-four cases of squamous cell carcinoma and 108 cases of basal cell carcinoma using tissue array in order to determine correlations between the expression of Ki-67, p53, EGFR, CD44v6, MMP-1 and MMP-3, invasiveness and histologic differentiation. In order to determine invasiveness, we measured the depth of invasion in resected tissues. RESULTS: The depth of invasion showed a correlation with CD44v6 expression of tumor cell in both squamous cell carcinoma and basal cell carcinoma (P = 0.009, P = 0.036, respectively) and with the MMP-1 expression of stromal cell in squamous cell carcinoma (P = 0.010). The differentiation of squamous cell carcinoma was correlated with Ki-67 index. The loss of palisading arrangement in basal cell carcinoma was correlated with the MMP-1 expression of stromal cells (P = 0.045). CONCLUSIONS: CD44v6 and MMP-1, expressed in tumor cells and stromal cells respectively, are significant markers associated with the invasiveness of tumors in squamous cell carcinoma and basal cell carcinoma of the skin and that it will be helpful to evaluate the invasiveness by measuring the expression of these markers. J. Surg. Oncol. (c) 2008 Wiley-Liss, Inc.

Son KD, Kim TJ, Lee YS, Park GS, Han KT, Lim JS, Kang CS.

Department of Plastic and Reconstructive Surgery, Kangnam St. Mary\’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.

Regulation of signaling pathways involved in lupeol induced inhibition of proliferation and induction of apoptosis in human prostate cancer cells.

Prostate cancer (PCa) is the most frequently diagnosed noncutaneous cancer and the leading cause of cancer related deaths in men in the United States and many other Asian countries. Dietary factors are considered as a strategic agent to control the risk of PCa. Lupeol, a triterpene, present in fruits and medicinal plants, has been shown to possess many pharmacological properties including anticancer effects. Here, effect of lupeol on cell proliferation and cell death was evaluated using human PCa cells, PC-3. In MTT assay, lupeol inhibited the cell proliferation (12-71%) in dose (50-800 microM) and time dependent manner. Flow-cytometric analysis of cell-cycle revealed that an antiproliferative effect of lupeol (400-600 microM) is associated with an increase in G(2)/M-phase arrest (34-58%). RT-PCR analysis showed that lupeol-induced G2/M-phase arrest was mediated through the inhibition of cyclin regulated signaling pathway. Lupeol inhibited the expression of cyclin B, cdc25C, and plk1 but induced the expression of 14-3-3sigma genes. However no changes were observed in the expression of gadd45, p21(waf1/cip1) and cdc2 genes. Results of western blot showed that lupeol regulates the phosphorylation of cdc2 (Tyr15) and cdc25C (Ser198). Further, on increase of lupeol exposure to PC-3 cells an induction of apoptosis was recorded, which was associated with upregulation of bax, caspase-3, -9, and apaf1 genes and down regulation of antiapoptotic bcl-2 gene. The role of caspase-induced apoptosis was confirmed by increase in reactive oxygen species, loss of mitochondrial membrane potential followed by DNA fragmentation. Thus, our study suggests that lupeol possess novel antiproliferative and apoptotic potential against PCa. (c) 2008 Wiley-Liss, Inc.

Prasad S, Nigam N, Kalra N, Shukla Y.

Proteomics Laboratory, Indian Institute of Toxicology Research, Lucknow, India.

Rhesus glycoprotein and urea transporter genes are expressed in early stages of development of rainbow trout (Oncorhynchus mykiss).

The objective of this study was to determine if the genes for the putative ammonia transporters, Rhesus glycoproteins (Rh) and the facilitated urea transporter (UT) were expressed during early development of rainbow trout, Oncorhynchus mykiss Walbaum. We predicted that the Rh isoforms Rhbg, Rhcg1 and Rhcg2 would be expressed shortly after fertilization but UT expression would be delayed based on the ontogenic pattern of nitrogen excretion. Embryos were collected 3, 14 and 21 days postfertilization (dpf), whereas yolk sac larvae were sampled at 31 dpf and juveniles at 60 dpf (complete yolk absorption). mRNA levels were quantified using quantitative polymerase chain reaction and expressed relative to the control gene, elongation factor 1alpha. All four genes (Rhbg, Rhcg1, Rhcg2, UT) were detected before hatching (25-30 dpf). As predicted, the mRNA levels of the Rh genes, especially Rhcg2, were relatively high early in embryonic development (14 and 21 dpf), but UT mRNA levels remained low until after hatching (31 and 60 dpf). These findings are consistent with the pattern of nitrogen excretion in early stages of trout development. We propose that early expression of Rh genes is critical for the elimination of potentially toxic ammonia from the encapsulated embryo, whereas retention of the comparatively benign urea molecule until after hatch is less problematic for developing tissues and organ systems. J. Exp. Zool. 309A, 2008. (c) 2008 Wiley-Liss, Inc.

Hung CC, Nawata CM, Wood CM, Wright PA.

Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada.

Cytology and receptor architecture of human anterior cingulate cortex.

Anterior cingulate cortex (ACC) is involved in emotion, mood, and autonomic regulation. Although a subgenual part of ACC (sACC) may be vulnerable in depression and area 25 is cytologically unique, there are no assessments that contrast this region with pregenual ACC (pACC). Thus, we undertook independent multimodal verifications of architectural differences among subregions and areas. Areas 24a and 24b have pregenual and subgenual components. The latter have a thin layer III. Area 24c has dorsal (pd24c) and ventral (pv24c) parts. Area pd24c has larger neurofilament-expressing neurons in layer Va, and neurons in Vb form aggregates in area pv24c. Area pd24c occupies both banks of the cingulate sulcus, with pv24c on the ventral bank. Layer III of pd24cd has many larger neurofilament-expressing neurons and a richer dendritic plexus. Area 32 has pregenual (p32) and subgenual (s32) components. Layer II in s32 is of particular note because it has a neuron-dense IIa and sparse IIb. Area 25 has anterior (25a) and posterior (25p) parts; 25p has the thinnest layer III in the cingulate gyrus. Area 25a contains significantly higher AMPA, kainate, NMDA, GABA(A), GABA(B), and alpha(1) densities than 25p. Area 33 continues around the genu and ventrally to encompass the full caudal extent of area 25. Subgenual ACC has significantly higher GABA(A), GABA(B), benzodiazepine (BZ), alpha(1), and 5-HT(1A) densities than pACC. GABA(B), BZ, and alpha(1) binding confirms the subdivision of area pd24c. In conclusion, ACC comprises two parts that are unique in terms of their cytoarchitecture and neurotransmitter receptor organization. J. Comp. Neurol. 508:906-926, 2008. (c) 2008 Wiley-Liss, Inc.

Palomero-Gallagher N, Mohlberg H, Zilles K, Vogt B.

Institute of Neurosciences and Biophysics—Medicine, Research Centre Jülich, 52425 Jülich, Germany.

TGF-beta1 and WISP-1/CCN-4 can regulate each other\’s activity to cooperatively control osteoblast function.

Wnt-induced secreted protein-1 (WISP-1), like other members of the CCN family, is expressed in skeletal tissues. Its mechanism of action remains unknown. Expression of WISP-1 was analyzed in human bone marrow stroma cells (hBMSC) by RT-PCR. We identified two major transcripts corresponding to those of full-length WISP-1, and of the splice variant WISP-1va which lacks a putative BMP/TGF-beta binding site. To investigate the function of WISP-1 in bone, hBMSC cultures were treated with recombinant human (rh)WISP-1 and analyzed for proliferation and osteogenic differentiation. WISP-1 treatment increased both BrdU incorporation and alkaline phosphatase (AP) activity. Considering the known functional synergy found between the TGF-beta super-family and members of the CCN family, we next tested the effect of WISP-1 on TGF-beta1 activity. We found that rhWISP-1 could reduce rhTGF-beta1 induced BrdU incorporation. Similarly, rhTGF-beta1 inhibited rhWISP-1 induction of AP activity. To explore functional differences between the WISP-1 variants, WISP-1 or WISP-1va were transfected into hBMSC. Both variants could strongly induce BrdU incorporation. However, there were no effects of either variant on AP activity without an additional osteogenic stimulus such as TGF-beta1. Taken together our results suggest a functional relationship between WISP-1 and TGF-beta1. To further define this relationship we analyzed the effect of WISP-1 on TGF-beta signaling. rhWISP-1 significantly reduced TGF-beta1 induced phosphorylation of Smad-2. Our data indicates that full-length WISP-1 and its variant WISP-1va are modulators of proliferation and osteogenic differentiation, and may be novel regulators of TGF-beta1 signaling in osteoblast-like cells. J. Cell. Biochem. (c) 2008 Wiley-Liss, Inc.

Inkson CA, Ono M, Kuznetsov SA, Fisher LW, Robey PG, Young MF.

Craniofacial and Skeletal Diseases Branch, National Institutes of Craniofacial and Dental Research, National Institutes of Heath, DHHS, Bethesda, Maryland 20892.