Giuseppe Portella

Antitumor Activity of Plant Cannabinoids with Emphasis on the Effect of Cannabidiol on Human Breast Carcinoma

Δ9-Tetrahydrocannabinol (THC) exhibits antitumor effects on various cancer cell types, but its use in chemotherapy is limited by its psychotropic activity. We investigated the antitumor activities of other plant cannabinoids, i.e., cannabidiol, cannabigerol, cannabichromene, cannabidiol acid and THC acid, and assessed whether there is any advantage in using Cannabis extracts (enriched in either cannabidiol or THC) over pure cannabinoids. Results obtained in a panel of tumor cell lines clearly indicate that, of the five natural compounds tested, cannabidiol is the most potent inhibitor of cancer cell growth (IC50 between 6.0 and 10.6 μM), with significantly lower potency in noncancer cells. The cannabidiol-rich extract was equipotent to cannabidiol, whereas cannabigerol and cannabichromene followed in the rank of potency. Both cannabidiol and the cannabidiol-rich extract inhibited the growth of xenograft tumors obtained by s.c. injection into athymic mice of human MDA-MB-231 breast carcinoma or rat v-K-ras-transformed thyroid epithelial cells and reduced lung metastases deriving from intrapaw injection of MDA-MB-231 cells. Judging from several experiments on its possible cellular and molecular mechanisms of action, we propose that cannabidiol lacks a unique mode of action in the cell lines investigated. At least for MDA-MB-231 cells, however, our experiments indicate that cannabidiol effect is due to its capability of inducing apoptosis via: direct or indirect activation of cannabinoid CB2 and vanilloid transient receptor potential vanilloid type-1 receptors and cannabinoid/vanilloid receptor-independent elevation of intracellular Ca2+ and reactive oxygen species. Our data support the further testing of cannabidiol and cannabidiol-rich extracts for the potential treatment of cancer.

One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes.

A system of epithelial cells is described in which it is possible to study the number and the nature of genes capable of conferring the malignant phenotype. Two fully differentiated, hormone-responsive cell lines from rat thyroid glands are presented which are susceptible to one-step or two-step transformation upon infection with several murine acute retroviruses. After infection, both cell lines became independent from their thyrotropic hormone requirement for growth. However, complete transformation was achieved with one of the cell lines (FRTL-5 Cl 2), whereas the other cell line (PC Cl 3) failed to grow in agar and to give rise to tumors in vivo. The latter cell line was susceptible to complete transformation upon cooperation of the v-ras-Ha and the human c-myc oncogenes.

Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis

Stimulation of cannabinoid CB1 receptors by 2‐methyl‐arachidonyl‐2′‐fluoro‐ethylamide (Met‐F‐AEA) inhibits the growth of a rat thyroid cancer cell‐derived tumor in athymic mice by inhibiting the activity of the oncogene product p21ras. Here we report that Met‐F‐AEA also blocks the growth of tumors previously induced in nude mice by the s.c. injection of the same rat thyroid carcinoma cells. Met‐F‐AEA significantly inhibited, in tumors as well as transformed cells, the expression of the vascular endothelial growth factor, an angiogenetic factor known to be up‐regulated by p21ras, as well as of one of its receptors, flt‐1/VEGFR‐1. The levels of the cyclin‐dependent kinase inhibitor p27(kip1), which is down‐regulated by p21ras, were instead increased by Met‐F‐AEA. All these effects were antagonized by the selective CB1 receptor antagonist SR141716A. Met‐F‐AEA inhibited in vitro the growth of a metastasis‐derived thyroid cancer cell line more potently than a primary cancer cell line. Therefore, the hypothesis that CB1 receptor stimulation interferes not only with angiogenesis but also with metastatic processes was tested in a widely used model of metastatic infiltration in vivo, the Lewis lung carcinoma (3LL) in C57Bl/6 mice. Three weeks from the paw injection of 3LL cells, Met‐F‐AEA reduced significantly the number of metastatic nodes, in a way antagonized by SR141716A. Our findings indicate that CB1 receptor agonists might be used therapeutically to retard tumor growth in vivo by inhibiting at once tumor growth, angiogenesis, and metastasis.

A new strategy to block tumor growth by inhibiting endocannabinoid inactivation

Endocannabinoid signaling has been shown to be enhanced in several cancer tissues and malignant cells, and studies in cell lines have shown that this up‐regulation might serve the purpose of providing transformed cells with a further means to inhibit their proliferation. Here we investigated the effect of inhibitors of endocannabinoid degradation on the growth of rat thyroid tumor xenografts induced in athymic mice. VDM‐11, a selective inhibitor of endocannabinoid cellular re‐uptake, and arachidonoyl‐serotonin (AA‐5‐HT), a selective blocker of endocannabinoid enzymatic hydrolysis, both inhibited the growth in vivo of tumor xenografts induced by the subcutaneous injection of rat thyroid transformed (KiMol) cells. This effect was accompanied by significantly enhanced endocannabinoid concentrations in the tumors excised at the end of the in vivo experiments. Endocannabinoids, as well as VDM‐11 and AA‐5‐HT, inhibited the growth in vitro of the transformed rat thyroid cells used to induce the tumors in vivo, and their effect was reversed at least in part by the cannabinoid CB1 receptor antagonist SR141716A. This compound, however, when administered alone, did not enhance, but instead slightly inhibited, the growth of rat thyroid transformed cells both in vitro and in tumor xenografts induced in vivo. These findings indicate that endocannabinoids tonically control tumor growth in vivo by both CB1‐mediated and non‐CB1‐mediated mechanisms and that, irrespective of the molecular mechanism of their anti‐proliferative action, inhibitors of their inactivation might be used for the development of novel anti‐cancer drugs.

Control by the endogenous cannabinoid system of ras oncogene-dependent tumor growth

We investigated the effect of 2‐methyl‐arachidonyl‐2′‐fluoro‐ethylamide (Met‐F‐AEA), a stable analog of the endocannabinoid anandamide, on a rat thyroid epithelial cell line (FRTL‐5) transformed by the K‐ras oncogene, and on epithelial tumors derived from these cells. Met‐F‐AEA effect in vivo was evaluated in a nude mouse xenograft model, where K‐ras‐transformed (KiMol) cells were implanted subcutaneously. Met‐F‐AEA (0.5 mg/kg/dose) induced a drastic reduction in tumor volume. This effect was inhibited by the CBi receptor antagonist SR141716A (0.7 mg/kg/dose) and was accompanied by a strong reduction of K‐ras activity. Accordingly, KiMol cells and tumors express CB1 receptors. Met‐F‐AEA inhibited (IC50 ~5 μM) the proliferation in vitro and the transition to the S phase of KiMol cells and it reduced K‐ras activity; these effects were antagonized by SR141716A. Met‐F‐AEA cytostatic action was significantly smaller in nontransformed FRTL‐5 cells than in KiMol cells. Met‐F‐AEA treatment exerted opposite effects on the expression of CB1 receptors in KiMol and FRTL‐5 cells, with a strong up‐regulation in the former case and a suppression in nontransformed cells. The data suggest that: 1) Met‐F‐AEA inhibits ras oncogene‐dependent tumor growth in vivo through CB1 cannabinoid receptors; and 2) responsiveness of FRTL‐5 cells to endocannabinoids depends on whether or not they are transformed by K‐ras.

Analysis of the HMGI nuclear proteins in mouse neoplastic cells induced by different procedures

Four malignant tumors induced in mouse by different experimental procedures were compared as regards their high-mobility-group (HMG) proteins. All tumors showed the complete set of three HMG proteins which we call HMGI-C, I-D, and I-E. The presence of the three HMGI proteins is a characteristic of the transformed phenotype regardless of whether the tumor was chemically, virally, or spontaneously derived. However, the level of expression of the HMGI proteins is not constant in the four tumors. Using reverse-phase HPLC, the individual HMGI proteins were isolated from the spontaneously derived tumor (Lewis lung carcinoma) and shown by amino acid analysis to be similar to those previously obtained from a tumor grown in nude mice by inoculation of in vitro-transformed cells.

The TRK-T1 fusion protein induces neoplastic transformation of thyroid epithelium.

Genetic analysis of human papillary thyroid carcinomas (PTC) has revealed unique chromosomal translocations that form oncogenic fusion proteins and promote thyroid tumorigenesis in up to 60% of tumors examined. Although, the majority of thyroid specific translocations involve the growth factor receptor c-RET, variant rearrangements of the receptor for nerve growth factor, NTRK1 have also been described. One such translocation, TRK-T1, forms a fusion protein composed of the carboxyl terminal tyrosine kinase domain of NTRK1 and the amino terminal portion of TPR (Translocated Promoter Region). To determine if TRK-T1 expression can cause thyroid cancer in vivo, we developed transgenic mice that express the human TRK-T1 fusion protein in the thyroid. Immunohistochemical analysis of TRK-T1 transgenic mouse thyroids revealed TRK-T1 staining within the thyroid follicular epithelium. In contrast to nontransgenic littermates, 54% of transgenic mice developed thyroid abnormalities that included follicular hyperplasia and papillary carcinoma. Furthermore, all transgenic mice examined greater than 7 months of age developed thyroid hyperplasia and/or carcinoma. These data support the conclusion that TRK-T1 is oncogenic in vivo and contributes to the neoplastic transformation of the thyroid.

Overexpression of the ped/pea-15 Gene Causes Diabetes by Impairing Glucose-Stimulated Insulin Secretion in Addition to Insulin Action

ABSTRACT Overexpression of the ped/pea-15 gene is a common feature of type 2 diabetes. In the present work, we show that transgenic mice ubiquitously overexpressing ped/pea-15 exhibited mildly elevated random-fed blood glucose levels and decreased glucose tolerance. Treatment with a 60% fat diet led ped/pea-15 transgenic mice to develop diabetes. Consistent with insulin resistance in these mice, insulin administration reduced glucose levels by only 35% after 45 min, compared to 70% in control mice. In vivo, insulin-stimulated glucose uptake was decreased by almost 50% in fat and muscle tissues of the ped/pea-15 transgenic mice, accompanied by protein kinase Cα activation and block of insulin induction of protein kinase Cζ. These changes persisted in isolated adipocytes from the transgenic mice and were rescued by the protein kinase C inhibitor bisindolylmaleimide. In addition to insulin resistance, ped/pea-15 transgenic mice showed a 70% reduction in insulin response to glucose loading. Stable overexpression of ped/pea-15 in the glucose-responsive MIN6 beta-cell line also caused protein kinase Cα activation and a marked decline in glucose-stimulated insulin secretion. Antisense block of endogenous ped/pea-15 increased glucose sensitivity by 2.5-fold in these cells. Thus, in vivo, overexpression of ped/pea-15 may lead to diabetes by impairing insulin secretion in addition to insulin action.

Thyroid targeting of the N-ras(Gln61Lys) oncogene in transgenic mice results in follicular tumors that progress to poorly differentiated carcinomas

Ras oncogenes are frequently mutated in thyroid carcinomas. To verify the role played by N-ras in thyroid carcinogenesis, we generated transgenic mice in which a human N-ras(Gln61Lys) oncogene (Tg-N-ras) was expressed in the thyroid follicular cells. Tg-N-ras mice developed thyroid follicular neoplasms; 11% developed follicular adenomas and ∼40% developed invasive follicular carcinomas, in some cases with a mixed papillary/follicular morphology. About 25% of the Tg-N-ras carcinomas displayed large, poorly differentiated areas, featuring vascular invasion and forming lung, bone or liver distant metastases. N-ras(Gln61Lys) expression in cultured PC Cl 3 thyrocytes induced thyroid-stimulating hormone-independent proliferation and genomic instability with micronuclei formation and centrosome amplification. These findings support the notion that mutated ras oncogenes could be able to drive the formation of thyroid tumors that can progress to poorly differentiated, metastatic carcinomas.

Bevacizumab Increases Viral Distribution in Human Anaplastic Thyroid Carcinoma Xenografts and Enhances the Effects of E1A-Defective Adenovirus dl922-947

Purpose: Anaplastic thyroid carcinoma is a prime target for innovative therapy because it represents one of the most lethal human neoplasms and is refractory to conventional treatments such as chemotherapy and radiotherapy. We have evaluated a novel therapeutic approach based on the oncolytic replication-selective adenovirus dl922-947. Experimental Design: The antitumor efficacies of the E1AΔCR2 (dl922-947) and ΔE1B55K (dl1520) mutants were compared in human thyroid anaplastic carcinoma cells in culture and in xenografts in vivo. To enhance the effects of dl922-947, anaplastic thyroid carcinoma tumor xenografts were treated with dl922-947 in combination with bevacizumab. Results: We showed that the efficacy of dl922-947 exceeded that of dl1520 in all tested anaplastic thyroid carcinoma cells in vitro and in vivo. Furthermore, bevacizumab in combination with dl922-947 significantly reduced tumor growth compared with single treatments alone. Bevacizumab treatment significantly improved viral distribution in neoplastic tissues. Conclusions: Our data showed that dl922-947 had a higher oncolytic activity compared with dl1520 in anaplastic thyroid carcinoma cell lines and might represent a better option for virotherapy of anaplastic thyroid carcinoma. Moreover, bevacizumab increased the oncolytic effects of dl922-947 by enhancing viral distribution in tumors. The results described herein encourage the use of the dl922-947 virus in combination with bevacizumab.