N6-isopentenyladenosine a new potential anti-angiogenic compound that targets human microvascular endothelial cells in vitro.

N6-isopentenyladenosine is an anti-proliferative and pro-apoptotic atypical nucleoside for normal and tumor cells. Considering the role of angiogenesis in various diseases, we investigated the cytotoxic effect of N6-isopentenyladenosine on human microvascular endothelial cells, protagonists in angiogenesis. Our results show that N6-isopentenyladenosine induced a significant reduction of cell viability, upregulated p21 and promoted caspase-3 cleavage in a dose dependent manner leading to apoptotic cell death as detected by FACS analysis. To understand structure-function relationship of N6-isopentenyladenosine, we investigated the effect of some N6-isopentenyladenosine analogs. Our results suggest that N6-isopentenyladenosine and some of its derivatives are potentially novel angiostatic agents and might be associated with other anti-angiogenic compounds for a better outcome.

N6-isopentenyladenosine affects cytotoxic activity and cytokines production by IL-2 activated NK cells and exerts topical anti-inflammatory activity in mice.

N6-isopentenyladenosine (iPA) is a modified adenosine with an isopentenyl moiety derived from the mevalonate pathway which displays pleiotropic biological effects, including anti-tumor and anti-angiogenic activity. Previous evidence revealed a biphasic effect of iPA on phytohemagglutinin-stimulated lymphocytes, being pro-proliferative at low doses and anti-proliferative at high doses. Analogously, we have recently shown that low iPA concentrations (<1µM) increased the immune response of natural killer (NK) cells against cancer targets. In the present study, we evaluated the effect of iPA at high concentration (10µM) on IL-2-activated NK cells. iPA, inhibited NK cell proliferation and cytotoxicity against their conventional tumor target, human K562 cells. This inhibition was associated with decreased expression and functionality of NK cell activating receptors NKp44 and NKG2D as well as impaired cyto/chemokines secretion (RANTES, MIP-1α, TNF-α and IFN-γ). ERK/MAPK and STAT5 activation in IL-2-activated NK cells were inhibited by iPA. The results obtained in vitro were validated in vivo in the inflammatory murine model of croton oil-induced ear dermatitis. The topical application of iPA significantly reduced mouse ear oedema, thus suggesting anti-inflammatory properties of this molecule. These results show the ability of iPA to exert anti-inflammatory effects both in vitro and in vivo directly targeting NK cells, providing a novel pharmacological tool in those diseases characterized by a deregulated immune-response, such as cancer or inflammatory conditions.

N(6)-isopentenyladenosine and analogs activate the NRF2-mediated antioxidant response.

N(6)-isopentenyladenosine (i(6)A), a naturally occurring modified nucleoside, inhibits the proliferation of human tumor cell lines in vitro, but its mechanism of action remains unclear. Treatment of MCF7 human breast adenocarcinoma cells with i(6)A or with three synthetic analogs (allyl(6)A, benzyl(6)A, and butyl(6)A) inhibited growth and altered gene expression. About 60% of the genes that were differentially expressed in response to i(6)A treatment were also modulated by the analogs, and pathway enrichment analysis identified the NRF2-mediated oxidative stress response as being significantly modulated by all four compounds. Luciferase reporter gene assays in transfected MCF7 cells confirmed that i(6)A activates the transcription factor NRF2. Assays for cellular production of reactive oxygen species indicated that i(6)A and analogs had antioxidant effects, reducing basal levels and inhibiting the H2O2- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced production in MCF7 or dHL-60 (HL-60 cells induced to differentiate along the neutrophilic lineage) cell lines, respectively. In vivo, topical application of i(6)A or benzyl(6)A to mouse ears prior to TPA stimulation lessened the inflammatory response and significantly reduced the number of infiltrating neutrophils. These results suggest that i(6)A and analogs trigger a cellular response against oxidative stress and open the possibility of i(6)A and benzyl(6)A being used as topical anti-inflammatory drugs.

Enhancement of the antitumor activity of N6-(delta 2-isopentenyl)adenosine against cultured L-1210 leukemia cells by pentostatin using a polymeric delivery system.

The adenosine deaminase inhibitor pentostatin (I), recently shown to be effective in the treatment of several types of acute and chronic human leukemias, was impregnated in a silicone polymer monolithic disk device for release in vitro in the presence of the antitumor nucleoside N6-(delta 2-isopentenyl)adenosine (II) against mouse L-1210 lymphocytic leukemia cells. Although I is ineffective alone against L-1210 cells, controlled release from the polymeric delivery matrix potentiates the antiproliferative effects of II during the midlog phase of growth (48 hr). Cytotoxicity is prolonged, leading to total cell death during the stationary phase of growth (96 hr). The present study suggests that polymeric delivery systems be used for controlled release of oncologic agents, alone or in combination with inhibitors, especially where liability is a concern.

Antineoplastic effects of N6-(delta 2-isopentenyl)adenosine against L-1210 mouse lymphocytic leukemic cells using a polymeric delivery system.

N6-(delta 2-Isopentenyl)adenosine (I), a nucleoside previously shown to be cytotoxic against several types of tumor cells, was impregnated in silicone polymer monolithic disc devices for release in vitro against lymphocytic mouse leukemia cells. Plotting the cumulative amount of N6-(delta 2-isopentenyl)adenosine released per unit area of the device versus the square root of time revealed a linear relationship. However, the higher loading dose tended to rapidly release any drug deposited on the polymer surface. The optimum loading dose of the device for the most effective antileukemic activity in 24 hr was calculated based on a plot of the release rate versus the square root of an initial loading dose. The silicone polymer--I delivery system enabled a sustained and controllable release of additional agent. It was thus possible to achieve virtually total inhibition of leukemia cell replication using the polymeric delivery system. Increased concentrations of I, without the use of the polymeric system, resulted in maximum 24 hr inhibition of only approximately 81%, followed by a decline in overall antileukemic activity. It is possible to achieve a more predictable release rate of N6-(delta 2-isopentenyl)adenosine and corresponding antileukemic activity using a polymeric delivery system against L-1210 mouse leukemic cells in vitro. The relative data indicate the ED50 concentrations to be considerably less using the polymeric delivery system.