Studio dell'attività antiproliferativa e dello stato di stress ossidativo nelle cellule tumorali di prostata (PC3) dopo trattamento con alfa-tocoferolo e naringenina

Flavonoids are a group of polyphenolic compounds occurring in plants, where they play a role in many physiological process. They possess a remarkable spectrum of biological activities, including antiallergic, antiinflammatory, antioxidant, antimutagenic and anticarcinogenic properties. Compelling data from in vivo and in vitro studies, epidemiological investigations and human clinical trials have evidenced the important role of flavonoids in cancer chemoprevention and therapy. The chemopreventive properties of flavonoids are generally believed to reflect their ability to scavenge endogenous ROS. However their antioxidant action may be an mechanism dealing with the anticancer and apoptosis-inducing properties, as ROS can mediate apoptotic DNA fragmentation. Naringenin is a flavonoid that is considered to have a bioactive effect on human health as antioxidant, free radical scavenger, anti-inflammatory, carbohydrate metabolism promoter, and immune system modulator. It is the predominant flavanone in grapefruit. Unfortunately, this bioflavonoid is difficult to absorb on oral ingestion. In the best case scenario, only 15% of ingested naringenin will get absorbed in the human gastrointestinal tract. Tocopherols are a class of chemical compounds of which many have vitamin E activity. It is a series of organic compounds consisting of various methylated phenols. Because the vitamin activity was first identified in 1936 from a dietary fertility factor in rats, it was given the name "tocopherol" from the Greek words “τόκος” [birth], and “φέρειν”, [to bear or carry] meaning in sum "to carry a pregnancy," with the ending "-ol" signifying its status as a chemical alcohol. Tocotrienols, which are related compounds, may also have vitamin E activity. All of these various derivatives with vitamin activity may correctly be referred to as "vitamin E." Tocopherols and tocotrienols are fat-soluble antioxidants but also seem to have many other functions in the body. The aim of this study was to highlight the role of naringenina and α-tocopherol as potential chemopreventive and chemotherapic molecules. Moreover, the possible induction of tumor cell differentiation and antimetastaticity have been investigated. The effects of naringen (10μM) and tocopherol (100μM) on proliferation rate of PC-3, human prostate cancer cells, was investigated. Treatments with the two molecules were carried out for 24, 48 and 72 hours and cell growth decreased after treatment, with respect to the control. Established the anti-proliferative role of naringenina and tocopherol, the possible effect of these molecules on another characteristic process of cancer cells, the metastatic process, was studied. First of all, the action on cell migration was evaluated. The migration ability of treated cells remarkably decreased. The in vitro invasion assay, using Boyden Chamber and Matrigel, was performed to highlight the modulation of antimetastatic properties of cancerous cells by naringenina and vitamin E. Porous filters employed in this experiment were analysed by a computer-assisted image analysis method: the invasive power of treated cells was reduced, with respect to the control (in particular after combined treatment). For the quantification of secretory metalloproteinase (MMPs) levels, we performed a zymographic technique. Densitometric analysis of gel spots showed, with respect to the control, a decrease of MMP-9 activity. On the contrary, MMP-2 activity was not influenced by the treatment with the same molecules. The next step was to verify if the anti-proliferative capacity of these molecules could be associated to the induction of cell differentiation; we have analysed the in vivo activity of transglutaminase (TGase, E.C. 2.3.2.13), marker of terminal differentiation. TGase increased its activity, with respect to the control. Additionally to elucidate the mode of inhibition of PC-3 cell growth by naringenina and α-tocopherol, we measured DNA content using flow cytometry after propidium iodide staining of nuclei. The proportion of subG0/G1 cell population was change after drugs exposure (in particular after combined treatments). To further confirm this result, we analyzed apoptotic cell death using APOAC apoptosis detection kit. To elucidate an effect of these molecules on p16 expression and its relation to p21, western blot analyses was performed on PC-3 cells. Increases in the levels of p16 protein in PC-3 after combined treatment occurred. The reduced expression of p21 was also observed. These results indicate that together with p21, p16 protein may play an important role for apoptosis response to naringenin and tocopherol combined treatment. Furthermore, it was investigated the effects of alpha-tocopherol and naringenin on antioxidant enzymes of PC3 cell line: catalase, total SOD, GPx, GR, Glyoxalase I and II, GST as well as on the concentration of total glutathione and lipid peroxide (T-BARS). T-BARS, GST, Glyoxalase I, GR, SOD are biomarkers of oxidative stress for naringenin. GPx is a biomarker of oxidative stress for tocopherol.