Barbara Bucci

3,5,3′-triiodothyronine (T3) is a survival factor for pancreatic β-cells undergoing apoptosis

3,5,3′‐triiodothyronine (T3) is essential for the growth and the regulation of metabolic functions, moreover, the growth‐stimulatory effect of T3 has largely been demonstrated and the pathways via which T3 promotes cell growth have been recently investigated. Type 1 diabetes (T1D) is due to the destruction of β‐cells, which occurs even through apoptosis. Aim of our study was to analyze whether T3 could have an antiapoptotic effect on cultured β‐cells undergoing apoptosis. We have demonstrated that T3 promotes cell proliferation in islet β‐cell lines (rRINm5F and hCM) provoking an increment in cell number (up to 55%: rRINm5F and 45%: hCM), cell viability, and BrdU incorporation, and regulating the cell cycle‐related molecules (cyc A, D1, E, and p27kip1). T3 inhibited the apoptotic process induced by streptozocin, S‐Nitroso‐N‐Acetylpenicylamine (SNAP), and H2O2 via regulation of the pro‐ and anti‐apoptotic factors Bcl‐2, Bcl‐XL, Bad, Bax, and Caspase 3. The T3 protective effect was PI‐3 K‐, but not MAPK‐ or PKA‐mediated, involving pAktThr308. Thus, T3 could be considered a survival factor protecting islet β‐cells from apoptosis. J. Cell. Physiol. 206: 309–321, 2006.

Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27 kip1 levels

In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the melanoma pathogenesis. We have previously demonstrated that treatment of melanoma cells with c-myc antisense oligodeoxynucleotides can inhibit cell proliferation and activate apoptosis. To gain insight into the mechanisms activated by Myc down-regulation, we have now developed an experimental model that allows modulating Myc protein expression in melanoma cells. This was achieved by originating stable melanoma cell clones expressing ecdysone-inducible c-myc antisense RNA. We show that the induction of c-myc antisense RNA in M14 melanoma cells leads to an inhibition of cell proliferation characterized by accumulation of cells in the G1 phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27kip1 levels and a significant reduction of the cdk2-associated kinase activity. In addition, we show that an ectopic overexpression of p27kip1 in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G1 arrest and activates apoptosis by increasing p27kip1 content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G1 checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells.

Rosiglitazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells

Thiazolidinediones, specific peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-γ-inhibitor, showed that rosiglitazone acts through both PPAR-γ-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-γ. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPKα and beclin-1. The autophagy seems to be independent of PPAR-γ activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.

Low correspondence between K-ras mutations in pancreatic cancer tissue and detection of K-ras mutations in circulating DNA.

Objective: K-ras is the most frequently mutated gene in pancreatic cancer; reported rates range from 70% to 90%. The aim of this study was to evaluate the correspondence between K-ras mutations in pancreatic cancer tissue and in circulating DNA and the value of K-ras mutations as serological marker. Methods: The research was conducted in 30 patients with pancreatic cancer in whom both plasma and neoplastic tissues were available. Such research was extended to circulating DNA isolated from 40 patients with chronic pancreatitis. Mutations in codon 12 were examined by mutant allele-specific amplification method and by direct sequencing. Serum values of routinely used tumor markers such as carbohydrate antigen (Ca) 19.9, carcinoembryonic antigen, Ca 50, and Ca 242 have been tested in all the patients enrolled in this study. Results: K-ras mutations were detected in 70% of neoplastic tissue samples, but no mutated DNA resulted in circulating DNA samples. The 60% of patients with tissue K-ras mutation showed elevation of some tumor markers among Ca 19.9, carcinoembryonic antigen, Ca 50, and Ca 242. As a whole, these last showed low sensitivity (20%-56.67%) and specificity (56.67%-77.5%) when compared with chronic pancreatitis. Conclusion: Over the years, there has been no change in the direction of an earlier diagnosis by serological markers, and also, these data indicate that K-ras mutation in serum is an unsatisfactory method for the detection in patients with pancreatic cancer as well as in patients with high risk of progression toward neoplastic pancreatic disease.

Myc Down-Regulation Sensitizes Melanoma Cells to Radiotherapy by Inhibiting MLH1 and MSH2 Mismatch Repair Proteins

Purpose: Melanoma patients have a very poor prognosis with a response rate of <1% due to advanced diagnosis. This type of tumor is particularly resistant to conventional chemotherapy and radiotherapy, and the surgery remains the principal treatment for patients with localized melanoma. For this reason, there is particular interest in the melanoma biological therapy. Experimental Design: Using two p53 mutant melanoma models stably expressing an inducible c-myc antisense RNA, we have investigated whether Myc protein down-regulation could render melanoma cells more susceptible to radiotherapy, reestablishing apoptotic p53-independent pathway. In addition to address the role of p53 in the activation of apoptosis, we studied the effect of Myc down-regulation on radiotherapy sensitivity also in a p53 wild-type melanoma cell line. Results: Myc down-regulation is able per se to induce apoptosis in a fraction of the cell population (∼40% at 72 hours) and in combination with γ radiation efficiently enhances the death process. In fact, ∼80% of apoptotic cells are evident in Myc down-regulated cells exposed to γ radiation for 72 hours compared with ∼13% observed after only γ radiation treatment. Consistent with the enhanced apoptosis is the inhibition of the MLH1 and MSH2 mismatch repair proteins, which, preventing the correction of ionizing radiation mismatches occurring during DNA replication, renders the cells more prone to radiation-induced apoptosis. Conclusions: Data herein reported show that Myc down-regulation lowers the apoptotic threshold in melanoma cells by inhibiting MLH1 and MSH2 proteins, thus increasing cell sensitivity to γ radiation in a p53-independent fashion. Our results indicate the basis for developing new antitumoral therapeutic strategy, improving the management of melanoma patients.

3,5,3′‐Triiodo‐L‐thyronine enhances the differentiation of a human pancreatic duct cell line (hPANC‐1) towards a β‐cell‐Like phenotype

The thyroid hormone, 3,5,3′‐Triiodo‐L‐thyronine (T3), is essential for growth, differentiation, and regulation of metabolic functions in multicellular organisms, although the specific mechanisms of this control are still unknown. In this study, treatment of a human pancreatic duct cell line (hPANC‐1) with T3 blocks cell growth by an increase of cells in G0/G1 cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic β‐cell differentiation revealed a time‐dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a β‐cell‐like phenotype, we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion, our results suggest, for the first time, that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC‐1) into pancreatic‐β‐cell‐like cells. These findings have important implications in cell‐therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes.

The TRβ1 Is Essential in Mediating T3 Action on Akt Pathway in Human Pancreatic Insulinoma Cells

Thyroid hormone action, widely recognized on cell proliferation and metabolism, has recently been related to the phosphoinositide 3 kinase (PI3K), an upstream regulator of the Akt kinase and the involvement of the thyroid hormone receptor β1 has been hypothesized. The serine‐threonine kinase Akt can regulate various substrates that drive cell mass proliferation and survival. Its action has also been characterized in pancreatic β‐cells. We previously demonstrated that Akt activity and its activation in the insulinoma cell line hCM could be considered a specific target of the non‐genomic action of T3. In this study we analyzed the molecular pathways involved in the regulation of cell proliferation, survival, size, and protein synthesis by T3 in a stable TRβ1 interfered insulinoma cell line, derived from the hCM, and evidenced a strong regulation of both physiological and molecular events by T3 mediated by the thyroid hormone receptor β1. We showed that the thyroid receptor β1 mediates the T3 regulation of the cdk4·cyc D1·p21CIP1·p27KIP1 complex formation and activity. In addition TRβ1 is essential for the T3 upregulation of the Akt targets β‐catenin, p70S6K, and for the phosphorylation of Bad and mTOR. We demonstrated that the β1 receptor mediates the T3 upregulation of protein synthesis and cell size, together with the cell proliferation and survival, playing a crucial role in the T3 regulation of the PI3K/Akt pathway. J. Cell. Biochem. 106: 835–848, 2009.

Temozolomide induced c-Myc-mediated apoptosis via Akt signalling in MGMT expressing glioblastoma cells.

Purpose: We investigated the molecular mechanisms underlying the cytotoxic effect of Temozolomide (TMZ) in both O6-methylguanine-DNA methyl transferase (MGMT) depleted as well as undepleted glioblastoma cell lines. Since TMZ is used in clinics in combination with radiotherapy, we also studied the effects of TMZ in combination with ionising radiation (IR). Methods: Cell colony-forming ability was measured using a clonogenic assay. Cell cycle analysis and apoptosis were evaluated by Flow Cytometry (FCM). Proteins involved in cell cycle control were detected by Western blot and co-immunoprecipitation assays. Results: Our data showed that TMZ, independent of MGMT expression, inhibited glioblastoma cell growth via an irreversible G2 block in MGMT depleted cells or the induction of apoptosis in MGMT normal expressing cells. When TMZ was administered in combination with IR, apoptosis was greater than observed with either agent separately. This TMZ-induced apoptosis in the MGMT expressing cells occurred through Akt/Glycogen-Synthase-Kinase-3ß (GSK3ß) signalling and was mediated by Myelocytomatosis (c-Myc) oncoprotein. Indeed, TMZ phosphorylated/activated Akt led to phosphorylation/inactivation of GSK3ß which resulted in the stabilisation of c-Myc protein and subsequent modulation of the c-Myc target genes involved in the apoptotic processes. Conclusion: c-Myc expression could be considered a good indicator of TMZ effectiveness.

Myc down‐regulation affects cyclin D1/cdk4 activity and induces apoptosis via Smac/Diablo pathway in an astrocytoma cell line

Objectives:  We investigated the antiproliferative effect of Myc down‐regulation via cell proliferation inhibition, cell cycle perturbation and apoptosis in two human astrocytoma models (T98G and ADF) steadily expressing an inducible c‐myc Anti‐sense RNA.

Thyroid hormones induce cell proliferation and survival in ovarian granulosa cells COV434

Numerous evidences indicate that thyroid hormones exert an important role in the regulation of the reproductive system in the adult female. Although a clear demonstration of the thyroid–ovarian interaction is still lacking, it is conceivable that thyroid hormones might have a direct role in ovarian physiology via receptors in granulosa cells. In this study we analyzed if thyroid hormone treatment could affect cell proliferation and survival of COV434 cells. To this aim cell growth experiments and cell cycle analyses by flow cytometry were performed. Secondly the T3 survival action was tested by TUNEL assay and MD30 cleavage analysis. We showed that T3, and not T4, can protect ovarian granulosa cells COV434 from apoptosis, regulating cell cycle and growth in the same cells. The increase in cell growth resulted in an augmented percentage of the cells in the S phase and, in a reduction of the doubling time (18%). Subsequently apoptotic pathway induced by serum deprivation has been evaluated in the cells exposed or not to thyroid hormone treatment. The T3 treatment was able to remarkably counteract the apoptotic process. Even at the ultrastructural level there was an evident protective effect of T3 in the cells that, besides the maintenance of the original morphology and, the absence of basophilic cytoplasm, conserved normal junctional areas. Furthermore, the protective T3 effect evaluated by FACS analysis in the presence of a PI3K inhibitor revealed, as also confirmed by Western Blot on pAkt, that the PI3K pathway is crucial in T3 survival action. J. Cell. Physiol. 221: 242–253, 2009.