Detection of Pancreatic Cancer-Induced Cachexia Using a Fluorescent Myoblast Reporter System and Analysis of Metabolite Abundance.

The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body "holistic" approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice-bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real-time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight losses of only 1.2% to 2.7% and tumor burdens of only approximately 79 to 170 mm(3). Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma into a reporter-carrying mouse may be able to provide a risk assessment of cachexia, with possible implications for therapeutic development.

Potent genistein derivatives as inhibitors of estrogen receptor alpha-positive breast cancer.

The estrogen receptor (ER) is a major target for the treatment of breast cancer cells. Genistein, a soy isoflavone, possesses a structure similar to estrogen and can both mimic and antagonize estrogen effects although at high concentrations it inhibits breast cancer cell proliferation. Hence, to enhance the anti-cancer activity of Genistein at lower concentrations, we have synthesized seven structurally modified derivatives of Genistein (MA-6, MA-8, MA-11, MA-19, MA-20, MA-21 and MA-22) based on the structural requirements for an optimal anti-cancer effect. Among those seven, three derivatives (MA-6, MA-8 and MA-19) showed high antiproliferative activity with IC(50) levels in the range of 1-2.5 µM, i.e., at much lower concentrations range than Genistein itself, in three ER-positive breast cancer cell lines (MCF-7, 21PT and T47D) studied. In our analysis, we noticed that at IC(50) concentrations, the MA-6, MA-8 and MA-19 Genistein derivatives induced apoptosis, inhibited ER-α messenger RNA expression and increased the ratio of ER-ß to ER-α levels in a manner comparable to the parent compound Genistein. Of note, these three modified Genistein derivatives exerted their effects at concentrations 10-15 times lower than the parent compound, decreasing the likelihood of significant ER- α pathway activation, which has been a concern for Genistein. Hence these compounds might play a useful role in breast cancer chemoprevention.

DNA methylation-related vitamin D receptor insensitivity in breast cancer.

Calcitriol (1α, 25(OH)(2)-Vitamin D3) binds to the vitamin D receptor (VDR) and regulates differentiation of the normal mammary gland, and may therefore be useful in breast cancer treatment or prevention. Many breast cancer cells are, however, resistant to Calcitriol. In this study, we investigated the resistance mechanism and the role of epigenetic silencing of VDR by promoter hypermethylation. Bisulfite sequencing of the VDR promoter region revealed methylated CpG islands at -700 base pairs (bp) upstream and near the transcription start site. VDR CpG islands were demethylated by 5'deoxy-azacytidine treatment, and this was accompanied by a parallel increase in VDR mRNA levels in breast cancer cell lines. Quantitative methylation-specific PCR analyses confirmed hypermethylation of these CpG islands in primary tumors, and its absence in normal breast tissue. VDR transcripts detected in breast cancers were predominantly 5'-truncated, while normal breast tissue expressed full-length transcripts. Consistent with this observation, genes containing the VDR-responsive element (VDRE), such as cytochrome p450 hydroxylases, p21 or C/EBP were underexpressed in breast cancers compared to normal breast samples. Expression of the active longer transcripts of VDR was restored with 5'deoxy-Azacytidine (AZA) treatment, with a concurrent increase in expression of VDRE-containing genes. Thus, promoter methylation-mediated silencing of expression of the functional variants of VDR may contribute to reduced expression of downstream effectors of the VDR pathway and subsequent Calcitriol insensitivity in breast cancer. These data suggest that pharmacological reversal of VDR methylation may re-establish breast cancer cell susceptibility to differentiation therapy using Calcitriol.

Differentiating alternative splice variant patterns of human telomerase reverse transcriptase in thyroid neoplasms.

BACKGROUND: Although fine-needle aspiration (FNA) biopsy of thyroid nodules is very sensitive in detecting thyroid malignancy, it remains ambiguous in 20-30% of cases. Current biomarkers for thyroid cancer lack either the sensitivity or specificity to substantially address this clinical problem. The aim of this study was to investigate the gene expression patterns of human telomerase reverse transcriptase (hTERT) alternative splice variants in benign and malignant thyroid tumors in an attempt to find a more reliable biomarker in the differential diagnosis of thyroid nodules. METHODS: One hundred and thirty-three thyroid tumors from eight histopathological tumor types were collected from patients undergoing thyroid surgery at Johns Hopkins Hospital. Gene expression patterns of hTERT alternative splice variants were investigated in the tumors by nested reverse transcriptase-PCR. Telomerase enzyme activity was evaluated in a subset of 16 samples associated with the different hTERT patterns. Association of c-myc expression and hTERT patterns was also examined. RESULTS: Malignant thyroid tumors exhibited a greater proportion of the active full-length hTERT transcript (0.57 +/- 0.15) than inactive splice variants, alpha(-) (0.13 +/- 0.02), or beta(-)/alpha(-)beta(-) deletion transcripts (0.30 +/- 0.11; p < 0.001). The opposite was observed in benign tumors, which exhibited greater proportions of beta(-)/alpha(-)beta(-) deletion transcripts (0.64 +/- 0.08) than either the full-length (0.19 +/- 0.06) or alpha(-) deletion transcripts (0.17 +/- 0.02; p < 0.001). Similar results were observed among a diagnostically challenging subset of 50 thyroid tumors that were suspicious for malignancy on FNA. Further, increased telomerase enzymatic activity was only associated with expression of the full-length hTERT isoform. In contrast, c-myc expression, which has been implicated in hTERT regulation, correlated with overall hTERT transcription without specificity for expression of the full-length isoform. CONCLUSIONS: These differences in gene expression patterns of hTERT alternative splice variants may provide a useful adjunct to FNA diagnosis of suspicious thyroid tumors.

Mechanism of impairment of cytochrome P450-dependent metabolism in hamster liver during leishmaniasis.

The mechanism of impairment of cytochrome P450 (P450)-dependent metabolism in hamster liver during leishmaniasis is reported. A significant decrease in the level of P450 was observed on the 20th day of infection when the parasite load in the liver was maximum. The decrease in P450 level was accompanied by a significant increase in the level of marker enzymes of liver and degeneration of liver tissue. The impairment was isozyme-specific and concomitant with the induction of nitric oxide synthase. The results of in vitro experiments with generated nitric oxide and with scavengers demonstrated that the impairment is mediated by NO. Treatment of the infected animals with a combination therapy showed reduction in parasite load, reversal of P450 impairment, and recovery of liver enzymes and tissue close to normal.