Thyroid hormone (T3), like many other ligands of the steroid/thyroid hormone nuclear receptor superfamily is a strong inducer of liver cell proliferation in rats and mice. However, the molecular basis of its mitogenic activity, which is currently unknown, must be elucidated if its use in hepatic regenerative medicine is to be considered. F-344 rats or C57BL/6 mice were fed a diet containing T3 for 2-7 days. In rats, administration of T3 led to an increased cytoplasmic stabilization and nuclear translocation of β-catenin in pericentral hepatocytes with concomitant increase in Cyclin-D1 expression. T3 administration to wild-type (WT) mice resulted in increased hepatocyte proliferation, however no mitogenic response in hepatocytes to T3 was evident in the hepatocyte-specific β-catenin knockout mice (KO). In fact, T3 induced β-catenin-TCF4 reporter activity both in vitro and in vivo. Livers from T3-treated mice demonstrated no changes in Ctnnb1 expression, activity of Glycogen synthase kinase-3β known to phosphorylate and eventually promote β-catenin degradation, or E-cadherin-β-catenin association. However, T3 treatment increased β-catenin phosphorylation at Ser675, an event downstream of protein kinase A (PKA). Administration of PKA inhibitor during T3 treatment of mice and rats as well as in cell culture abrogated Ser675-β-catenin and simultaneously decreased Cyclin-D1 expression to block hepatocyte proliferation. Conclusion We have identified T3-mediated hepatocyte mitogenic response to be mediated by PKA-dependent β-catenin activation. Thus, T3 may be of therapeutic relevance to stimulate β-catenin signaling to in turn induce regeneration in selected cases of hepatic insufficiency.

Fasting-mimicking diets delay tumor progression and sensitize a wide range of tumors to chemotherapy, but their therapeutic potential in combination with non-cytotoxic compounds is poorly understood. Here we show that vitamin C anticancer activity is limited by the upregulation of the stress-inducible protein heme-oxygenase-1. The fasting-mimicking diet selectivity reverses vitamin C-induced up-regulation of heme-oxygenase-1 and ferritin in KRAS-mutant cancer cells, consequently increasing reactive iron, oxygen species, and cell death; an effect further potentiated by chemotherapy. In support of a potential role of ferritin in colorectal cancer progression, an analysis of The Cancer Genome Atlas Database indicates that KRAS mutated colorectal cancer patients with low intratumor ferritin mRNA levels display longer 3-and 5-year overall survival. Collectively, our data indicate that the combination of a fasting-mimicking diet and vitamin C represents a promising low toxicity intervention to be tested in randomized clinical trials against colorectal cancer and possibly other KRAS mutated tumors.

Mesoporous silica nanoparticles (MSNs), based on the MCM-41 matrix, were functionalized with amino groups, and then with hyaluronic acid (HA) or chitosan (CHIT) to fabricate bioactive conjugates. The role of the functional groups toward cytotoxicity and cellular uptake was investigated using 3T3 mouse fibroblast cells. A very high biocompatibility of MSN-NH2, MSN-HA and MSN-CHIT matrices was assessed through the MTS biological assay and Coulter counter evaluation. No significant differences in cytotoxicity data arise from the presence of different functional groups in the investigated MSNs. Fluorescence microscopy experiments performed using fluorescein isothiocyanate-conjugated MSN-NH2, MSN-HA, and MSN-CHIT, and transmission electron microscopy experiments performed on slices of the investigated systems embedded in epoxy resins give evidence of significant differences due to type of functionalization in terms of cellular uptake and stability of the particles in the biological medium. MSN-NH2 and MSN-HA conjugates are easily internalized, the uptake of the HA-functionalized MSNs being much higher than that of the -NH2-functionalized MSNs. Differently, MSN-CHIT conjugates tend to give large aggregates dispersed in the medium or localized at the external surface of the cell membranes. Both fluorescence microscopy and TEM images show that the MSNs are distributed in the cytoplasm of the cells in the case of MSN-NH2 and MSN-HA, whereas only a few particles are internalized in the case of MSN-CHIT. Flow cytometry experiments confirmed quantitatively the selectively high cellular uptake of MSN-HA particles.

The fruit fly Drosophila melanogaster (Dm) mutant for PTEN-induced putative kinase 1 (PINK1B9) gene is a powerful tool to investigate physiopathology of Parkinson's disease (PD). Using PINK1B9 mutant Dm we sought to explore the effects of Mucuna pruriens methanolic extract (Mpe), a L-Dopa-containing herbal remedy of PD. The effects of Mpe on PINK1B9 mutants, supplied with standard diet to larvae and adults, were assayed on 3–6 (I), 10–15 (II) and 20–25 (III) days old flies. Mpe 0.1% significantly extended lifespan of PINK1B9 and fully rescued olfactory response to 1-hexanol and improved climbing behavior of PINK1B9 of all ages; in contrast, L-Dopa (0.01%, percentage at which it is present in Mpe 0.1%) ameliorated climbing of only PINK1B9 flies of age step II. Transmission electron microscopy analysis of antennal lobes and thoracic ganglia of PINK1B9 revealed that Mpe restored to wild type (WT) levels both T-bars and damaged mitochondria. Western blot analysis of whole brain showed that Mpe, but not L-Dopa on its own, restored bruchpilot (BRP) and tyrosine hydroxylase (TH) expression to age-matched WT control levels. These results highlight multiple sites of action of Mpe, suggesting that its effects cannot only depend upon its L-Dopa content and support the clinical observation of Mpe as an effective medication with intrinsic ability of delaying the onset of chronic L-Dopa-induced long-term motor complications. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the properties of Mucuna pruriens for PD treatment.

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A better understanding of the complex relationship between aging and cancer will provide important tools for the prevention and treatment of neoplasia. In these studies, the hypothesis was tested that aging may fuel carcinogenesis via alterations imposed in the tissue microenvironment. Preneoplastic hepatocytes isolated from liver nodules were orthotopically injected into either young or old syngeneic rats and their fate was followed over time using the dipeptidyl-peptidase type IV (DPPIV) system to track donor-derived-cells. At 3 months post-Tx, the mean size of donor-derived clusters was 11±3 cells in young vs. 42±8 in old recipients. At 8 months post-Tx, no visible lesion were detected in any of 21 young recipients, while 17/18 animals transplanted at old age displayed hepatic nodules, including 7 large tumors. All tumors expressed the DPPIV marker enzyme, indicating that they originated from transplanted cells. Expression of senescence-associated β-galactosidase was common in liver of 18-month old animals, while it was a rare finding in young controls. Finally, both mRNA and IL6 protein were found to be increased in the liver of aged rats compared to young controls. These results are interpreted to indicate that the microenvironment of the aged liver promotes the growth of pre-neoplastic hepatocytes.

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Hepatocellular carcinoma (HCC), the third most common cause of cancer-related deaths worldwide, lacks effective medical therapy. Large subsets of HCC demonstrate Wnt/β-catenin activation, making this an attractive therapeutic target. We report strategy and characterization of a novel small-molecule inhibitor, ICG-001, known to affect Wnt signaling by disrupting β-catenin-CREB binding protein interactions. We queried the ZINC online database for structural similarity to ICG-001 and identified PMED-1 as the lead compound, with ≥70% similarity to ICG-001. PMED-1 significantly reduced β-catenin activity in hepatoblastoma and several HCC cells, as determined by TOPflash reporter assay, with an IC50 ranging from 4.87 to 32 μmol/L. Although no toxicity was observed in primary human hepatocytes, PMED-1 inhibited Wnt target expression in HCC cells, including those with CTNNB1 mutations, and impaired cell proliferation and viability. PMED-1 treatment decreased β-catenin-CREB binding protein interactions without affecting total β-catenin levels or activity of other common kinases. PMED-1 treatment of Tg(OTM:d2EGFP) zebrafish expressing GFP under the β-catenin/Tcf reporter led to a notable decrease in β-catenin activity. The PMED effect on β-catenin signaling lasted from 12 to 24 hours in vitro and 6 to 15 hours in vivo. Thus, using a rapid and cost-effective computational methodology, we have identified a novel and specific small-molecule inhibitor of Wnt signaling that may have implications for HCC treatment.