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Quercetin, the major constituent of flavonoid and widely present in fruits and vegetables, is an attractive compound for cancer prevention due to its beneficial anti proliferative effects, showing a crucial role in the regulation of apoptosis and cell cycle signaling. In vitro studies have demonstrated that quercetin specifically influences colon cancer cell proliferation. Our experiments, using human colon adenocarcinoma cells, confirmed the anti proliferative effect of quercetin and gave intriguing new insight in to the knowledge of the mechanisms involved. We observed a significant increase in the expression of the endocannabinoids receptor (CB1-R) after quercetin treatment. CB1-R can be considered an estrogen responsive receptor and quercetin, having a structure similar to that of the estrogens, can interact with CB1-R leading to the regulation of cell growth. In order to clarify the contribution of the CB1-R to the quercetin action, we investigated some of the principal molecular pathways that are inhibited or activated by this natural compound. In particular we detected the inhibition of the major survival signals like the PI3K/Akt/mTOR and an induction of the pro apoptotic JNK/JUN pathways. Interestingly, the metabolism of β-catenin was modified by flavonoid both directly and through activated CB1-R. In all the experiments done, the quercetin action has proven to be reinforced by anandamide (Met-F-AEA), a CB1-R agonist, and partially counteracted by SR141716, a CB1-R antagonist. These findings open new perspectives for anticancer therapeutic strategies.

The endocannabinoid system, through cannabinoid receptor signaling by endocannabinoids, is involved in a wide range of functions and physiopathological conditions. To date, very little is known concerning the role of the endocannabinoids in the control and regulation of cell proliferation. An anti-proliferative action of CB1 signaling blockade in neurogenesis and angiogenesis argues in favor of proliferation-promoting functions of endocannabinoids through CB1 receptors when pro-growth signals are present. Furthermore, liver regeneration, a useful in vivo model of synchronized cell proliferation, is characterized by a peak of anandamide that elicits through CB1 receptor, the expression of critical mitosis genes. The aim of this study was to focus on the timing of endocannabinoid signaling changes during the different phases of the cell cycle, exploiting the rat liver regeneration model following partial hepatectomy, the most useful to study synchronized cell cycle in vivo. Hepatic regeneration led to increased levels of anandamide and endocannabinoid-like molecules oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in the G1 phase of the cell cycle, with a concomitant increase in CB1 mRNA levels, whose protein expression peaked later during the S phase. Blocking of CB1 receptor with a low dose of the selective antagonist/inverse agonist SR141716 (0.7 mg/kg/dose) affected cell cycle progression reducing the expression of PCNA, and through the inhibition of pERK and pSTAT3 pathways. These results support the notion that the signaling mediated by anandamide through CB1 receptor may be important for the entry and progression of cells into the cell cycle and hence for their proliferation under mitogenic signals.

Increasing evidence suggest the role of the cannabinoid receptors (CBs) in the control of cell survival or death and signaling pathways involved in tumor progression. Cancer cell lines are characterized by a subtle modulation of CB levels which produces a modified responsiveness to specific ligands, but the molecular mechanisms underlying these events are poorly and partially understood. We previously provided evidence that the endocannabinoid (EC) anandamide (AEA) exerts anti-proliferative effect likely by modulation of the expression of genes involved in the cellular fate. In this study we focused on the role of the CB1 receptor, ECs, and steroids in the mechanisms involved in colorectal cancer (CRC) cell growth inhibition in vitro. We demonstrated that, in DLD1 and SW620 cells, 17β-estradiol induced a specific and strong up-regulation of the CB1 receptor by triggering activation of the CB1 promoting region, localized at the exon 1 of the CNR1 gene. Moreover, treatment of DLD1 and SW620 cells with Met-F-AEA, a stable AEA-analogous, or URB597, a selective inhibitor of FAAH, induced up-regulation of CB1 expression by co-localization of PPARγ and RXRα at the promoting region. Finally, increased availability of AEA, of both exogenous and endogenous sources, induced the expression of estrogen receptor-beta in both cell lines. Our results partially elucidated the role of EC system in the molecular mechanisms enrolled by steroids in the inhibition of colon cancer cell growth and strongly suggested that targeting the EC system could represent a promising tool to improve the efficacy of CRC treatments.

Activating mutations in RAS genes and p21 Ras overactivation are common occurrences in a variety of human tumors. p21 Ras oncoproteins deregulate a number of signaling pathways, dedifferentiating the thyroid cell, and negatively regulating the expression of thyroid specific genes. In rat thyroid cells, Ras oncoproteins inhibit the TSH pathway by reducing PKA activity and thus the expression of thyroid specific genes, while in mouse melanocytes, Ras oncoproteins reduce the αMSH-stimulated cAMP signaling by increasing the expression of the phosphodiesterase-4B. Given these cell-dependent differences, we investigated if and how the TSH/CREB pathway is modulated by Ras oncoprotein in a human thyroid cell line. CREB phosphorylation was stimulated by TSH and forskolin in TAD-2 cells. Ras(V12) expression negatively regulated the TSH-stimulated CREB phosphorylation but was ineffective on forskolin-stimulated CREB phosphorylation. Phosphodiesterase inhibition by IBMX enhanced TSH-stimulated CREB phosphorylation, but did not restore TSH-stimulated CREB phosphorylation inhibited by Ras oncoprotein. These data indicate that Ras oncoprotein disrupts the TSH/CREB pathway, upstream adenylyl cyclase, and highlight the existence of mechanisms of interaction between Ras and the cAMP pathway different in human and in rat thyroid cells.

We examined the synthesis of extracellular matrix macromolecules by the differentiated rat thyroid epithelial cell line FRTL-5. As shown by electron microscopy, the extracellular material produced by these cells is deposited at the basolateral surface and focally organized in the form of a basement membrane. Biochemical and biosynthetic studies demonstrated that laminin, type IV collagen, and fibronectin are synthesized and deposited in the culture monolayer. Secretion of fibronectin into the culture medium also occurred. By immunofluorescence we observed some peculiarities in the distribution patterns of the basement membrane glycoproteins; while fibronectin and laminin had an almost superimposable distribution, type IV collagen displayed a rather different pattern. Type IV collagen and laminin localization at sites where extracellular material was detected was confirmed by immuno electronmicroscopy using the protein A-colloidal gold technique. The results indicate that under appropriate culture conditions the differentiated thyroid epithelial cell line FRTL-5 synthesizes, secretes and organizes an extracellular matrix where some basement membrane glycoproteins are present.