Pharmacophore-Based Virtual Screening for Identification of Negative Modulators of GLI1 as Potential Anticancer Agents.

Starting from known GLI1 inhibitors, a pharmacophore-based virtual screening approach was applied to databases of commercially available compounds with the aim of identifying new GLI1 modulators. As a result, three different chemical scaffolds emerged that were characterized by a significant ability to reduce the transcriptional activity of the endogenous Hedgehog-GLI pathway and GLI1 protein level in murine NIH3T3 cells. They also showed a micromolar antiproliferative activity in human melanoma (A375) and medulloblastoma (DAOY) cell lines, without cytotoxicity in non-neoplastic mammary epithelial cells.

Mesenteric lymph nodes as alternative site for pancreatic islet transplantation in a diabetic rat model.

BACKGROUND: Islet transplantation has progressively become a safe alternative to pancreas transplantation for the treatment of type 1 diabetes. However, the long-term results of islet transplantation could be significantly increased by improving the quality of the islet isolation technique even exploring alternative islet transplantation sites to reduce the number of islets required to mitigate hyperglycemia. The goal of the study was to test the lymph node as a suitable anatomical location for islet engraftment in a rodent model. METHODS: Forty Lewis rats, 6-8 weeks old, body weight 250-300 g, have been used as islet donors and recipients in syngeneic islet transplantation experiments. Ten rats were rendered diabetic by one injection of 65 mg/Kg of streptozotocin. After pancreas retrieval from non diabetic donors, islet were isolated and transplanted in the mesenteric lymph nodes of 7 diabetic rats. Rats were followed for 30 days after islet transplantation. RESULTS: A total of 7 islet transplantations in mesenteric lymph nodes have been performed. Two rats died 24 and 36 h after transplantation due to complications. No transplanted rat acquired normal glucose blood levels and insulin independence after the transplantation. However, the mean blood levels of glycemia were significantly lower in transplanted rats compared with diabetic rats (470.4 mg/dl vs 605 mg/dl, p 0.04). Interestingly, transplanted rats have a significant weight increase after transplantation compared to diabetic rats (mean value 295 g in transplanted rats vs 245 g in diabetic rats, p < 0.05), with an overall improvement of social activities and health. Immunohistochemical analysis of the 5 mesenteric lymph nodes of transplanted rats demonstrated the presence of living islets in one lymph node. CONCLUSIONS: Although islet engraftment in lymph nodes is possible, islet transplantation in lymph nodes in rats resulted in few improvements of glucose parameters.

Targeted inhibition of Hedgehog-GLI signaling by novel acylguanidine derivatives inhibits melanoma cell growth by inducing replication stress and mitotic catastrophe.

Aberrant activation of the Hedgehog (HH) signaling is a critical driver in tumorigenesis. The Smoothened (SMO) receptor is one of the major upstream transducers of the HH pathway and a target for the development of anticancer agents. The SMO inhibitor Vismodegib (GDC-0449/Erivedge) has been approved for treatment of basal cell carcinoma. However, the emergence of resistance during Vismodegib treatment and the occurrence of numerous side effects limit its use. Our group has recently discovered and developed novel and potent SMO inhibitors based on acylguanidine or acylthiourea scaffolds. Here, we show that the two acylguanidine analogs, compound (1) and its novel fluoride derivative (2), strongly reduce growth and self-renewal of melanoma cells, inhibiting the level of the HH signaling target GLI1 in a dose-dependent manner. Both compounds induce apoptosis and DNA damage through the ATR/CHK1 axis. Mechanistically, they prevent G2 to M cell cycle transition, and induce signs of mitotic aberrations ultimately leading to mitotic catastrophe. In a melanoma xenograft mouse model, systemic treatment with 1 produced a remarkable inhibition of tumor growth without body weight loss in mice. Our data highlight a novel route for cell death induction by SMO inhibitors and support their use in therapeutic approaches for melanoma and, possibly, other types of cancer with active HH signaling.

Abdominal CT Scan Findings of Decompression Sickness: A Case Report.

This case report depicts the radiologic findings of a 51-year-old male presenting with Decompression Sickness. Decompression Sickness is diagnosed clinically, therefore radiologic imaging of this disease entity is limited. Our patient's history includes a scuba dive to depth of 110 feet with a descending time of 24 minutes and an ascending time of 8 minutes. The patient subsequently presented to the Emergency Room with symptoms of shortness of breath and abdominal pain. The abdominal pain prompted physicians to explore further, and hence computed tomography (CT) imaging of the abdomen and pelvis was performed. This case report demonstrates a striking and unique gas pattern in both the systemic and portal venous system of our patient and provides an excellent example of the imaging findings of Decompression Sickness in the literature.

Psychological perspective of medication adherence in transplantation.

AIM: To identify the risk factors and the post-transplant psychological symptoms that affect adherence to therapy in a population of kidney transplant recipients. METHODS: The study examined the psychological variables likely responsible for the non-adherent behavior using a psychological-psychiatric assessment, evaluation of the perception of patients' health status, and an interview regarding the anti-rejection drug therapy assumption. The study included 74 kidney transplant recipients. RESULTS: Individuals with a higher level of education and more years since transplantation showed better mental balance. Regarding gender, women appeared to be less adherent to therapy. Further, the years since transplantation adversely affected the proper pharmacological assumption. Adherence to therapy did not significantly change with the mental health index. CONCLUSION: The biopsychosocial illness model provides a conceptual frame of reference in which biological, psychological, and social aspects take on the same importance in the adherence to treatment protocols. For effective management, it is necessary to understand the patients' personal experiences, their assumptions about the disease, health status perception, and mood, and to identify any "barriers" that could cause them to become noncompliant.

Hypoxia-resistant profile implies vulnerability of cancer stem cells to physiological agents, which suggests new therapeutic targets.

We have previously shown that peculiar metabolic features of cell adaptation and survival in hypoxia imply growth restriction points that are typical of embryonic stem cells and disappear with differentiation. Here we provide evidence that such restrictions can be exploited as specific antiblastic targets by physiological factors such as pyruvate, tetrahydrofolate, and glutamine. These metabolites act as powerful cytotoxic agents on cancer stem cells (CSCs) when supplied at doses that perturb the biochemical network, sustaining the resumption of aerobic growth after the hypoxic dormant state. Experiments were performed in vivo and in vitro using CSCs obtained from various anaplastic tumors: human melanoma, leukemia, and rat hepatoma cells. Pretreatment of melanoma CSCs with pyruvate significantly reduces their self-renewal in vitro and tumorigenicity in vivo. The metabolic network underlying the cytotoxic effect of the physiological factors was thoroughly defined, principally using AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem stage. This network, based on a tight integration of aerobic glycolysis, cellular redox state, and folate metabolism, is centered on the cellular NADP/NADPH ratio that controls the redox pathway of folate utilization in purine synthesis. On the whole, this study indicates that pyruvate, FH 4, and glutamine display anticancer activity, because CSCs are committed to survive and maintain their stemness in hypoxia. When CSC need to differentiate and proliferate, they shift from anaerobic to aerobic status, and the few mitochondria available makes them susceptible to the injury of the above physiological factors. This vulnerability might be exploited for novel therapeutic treatments.

Culture and isolation of melanoma-initiating cells.

Melanoma is the most aggressive skin cancer. This unit illustrates protocols for culture and isolation of human melanoma cancer stem cells/tumor-initiating cells (CSC/TIC). We describe two complementary methods to enrich for melanoma CSC/TIC. The first approach exploits the ability of CSC/TIC to grow as tumor spheres in low-adherent culture conditions, as previously shown for neural stem cells and human embryonic stem cells. As a second approach, melanoma CSC/TIC are enriched by fluorescence-activated cell sorting for the aldehyde dehydrogenase (ALDH) enzyme activity. We previously showed that melanoma cells with high ALDH activity (ALDH(high)) are endowed with higher self-renewal and tumorigenic abilities than the population with low activity (ALDH(low)), suggesting that ALDH might be a good marker to select for melanoma CSC/TIC. This unit will also describe how to functionally test melanoma CSC/TIC by determining self-renewal in vitro and tumor-forming abilities in vivo using orthotopic xenograft assay.

Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.

The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDH(high)), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH(low) population. A good correlation between the number of ALDH(high) cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDH(high) melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH(high) melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment.