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Human intervention trials have suggested that supplemental beta-carotene resulted in more cancer in smokers, whereas it was protective in non-smokers. However, the mechanisms underlying these effects are still unknown. The aim of this study was to evaluate the effects of an association of cigarette smoke condensate (tar) and beta-carotene on DNA oxidative damage and molecular pathways involved in cell cycle progression and apoptosis in cultured cells. In RAT-1 fibroblasts, tar caused increased levels of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and this effect was enhanced by the concomitant presence of beta-carotene (0.5-4.0 microM) in a dose- and time-dependent manner. In contrast, beta-carotene alone did not significantly modify it. Fibroblasts treated with tar alone decreased their cell growth with respect to control cells through an arrest of cell cycle progression in the G0/G1 phase and an induction of apoptosis. These effects were accompanied by an increased expression of p53, p21 and Bax and by a decreased expression of cyclin D1. In contrast, fibroblasts treated with tar and beta-carotene, after an initial arrest of cell growth at 12 h, re-entered in cell cycle and were unable to undergo apoptosis at 36 h. Concomitantly, their p53 expression, after an increase at 12 h, progressively returned at basal levels at 36 h by a mechanism independent of Mdm2. Such a decrease was followed by a decrease in p21 and Bax expression and by an increase in cyclin D1 expression. Moreover, the presence of the carotenoid remarkably enhanced cyclooxygenase-2 expression induced by tar. During tar treatment, a depletion of beta-carotene was observed in fibroblasts. The effects of tar and beta-carotene on 8-OHdG levels, cell growth and apoptosis were also observed in Mv1Lu lung, MCF-7 mammary, Hep-2 larynx and LS-174 colon cancer cells. This study supports the evidence for potential detrimental effects of an association between beta-carotene and cigarette smoke condensate.

Although an antioxidant mechanism has been involved in the beneficial effects of ferulic acid in human diseases, there are few reports on the antioxidant properties of this compound in isolated membranes and intact cells. Here, we evaluated the ability of ferulic acid in inhibiting lipid peroxidation in rat liver microsomal membranes and reactive oxygen species production in NIH-3T3 fibroblasts, induced by both tert-BOOH and AAPH. We also compared its antioxidant efficiency with that of other antioxidants, such as alpha-tocopherol, beta-carotene, and ascorbic acid, added alone or in combination. Ferulic acid acted as a potent antioxidant in our models, being more effective in protecting from tert-BOOH than from AAPH. Moreover, the compound was the most effective among the antioxidants tested. Synergistic interactions were observed when the compound was used in combination with the other antioxidants, suggesting that they can cooperate in preserving physiological integrity of cells exposed to free radicals.

There is a growing body of interest on the role of beta-carotene and other carotenoids in human chronic diseases, including cancer. While epidemiological evidence shows that people who ingest more dietary carotenoids exhibit a reduced risk for cancer, results from intervention trials indicate that supplemental beta-carotene enhances lung cancer incidence and mortality among smokers. A possible mechanism which can explain the dual role of beta-carotene as both a beneficial and a harmful agent in cancer as well as in other chronic diseases is its ability in modulating intracellular redox status. beta-Carotene may serve as an antioxidant or as a prooxidant, depending on its intrinsic properties as well as on the redox potential of the biological environment in which it acts. This review summarizes the available evidence for a prooxidant activity of beta-carotene in cultured cells, focusing on biochemical and molecular markers of oxidative stress, which have been reported to be enhanced by the carotenoid.

We demonstrated previously that beta-carotene may affect cell growth by a redox mechanism. The purpose of this study was to determine whether the redox-sensitive transcription factor nuclear factor (NF)-kappaB may be involved in the growth-inhibitory and proapoptotic effects of the carotenoid. To test this hypothesis, human leukemic cells (HL-60) and colon adenocarcinoma cells (LS-174 and WiDr) were treated with beta-carotene, alone or in combination with alpha-tocopherol or N-acetylcysteine, and changes in 1) cell oxidative status, 2) cell growth and apoptosis, 3) DNA-binding activity of NF-kappaB and 4) expression of c-myc, a NF-kappaB target gene involved in apoptosis, were evaluated. In HL-60 cells, beta-carotene induced a significant increase in reactive oxygen species (ROS) production (P < 0.001) and in oxidized glutathione (GSSG) content (P < 0.005) at concentrations >/=10 micro mol/L. These effects were always accompanied by a sustained elevation of NF-kappaB and by a significant inhibition (P < 0.002) of cell growth. NF-kappaB DNA-binding activity increased at 3 h and persisted for at least 48 h. Colon adenocarcinoma cells displayed substantial differences in their sensitivity to beta-carotene, exhibiting increased ROS levels and activation of NF-kappaB at concentrations much lower in LS-174 cells (2.5-5.0 micro mol/L) than in WiDr cells (50-100 micro mol/L). In all cell lines studied, alpha-tocopherol and N-acetylcysteine inhibited the effects of beta-carotene on NF-kappaB, cell growth and apoptosis, and normalized the increased expression of c-myc induced by the carotenoid. These data suggest that the redox regulation of NF-kappaB induced by beta-carotene is involved in the growth-inhibitory and proapoptotic effects of the carotenoid in tumor cells.

In this study, we examined possible mechanisms of caspase activation during carotenoid-induced apoptosis in tumor cells. We found that beta-Carotene induces apoptosis by the activation of caspase-3 in human leukemia (HL-60), colon adenocarcinoma (HT-29) as well as melanoma (SK-MEL-2) cell lines. This activation is dose dependent and follows that of caspase-8 and caspase-9. Although caspase-8 cleavage is an early event, reaching its maximum activation at 3 h, caspase-9 reaches its maximum activation only at 6 h. The addition of IETD-CHO, a caspase-8-specific inhibitor, completely prevents beta-Carotene-induced apoptosis, whereas only a partial prevention was observed in the presence of LEHD-CHO, a caspase-9-specific inhibitor. beta-Carotene activates caspase-9 via cytochrome c release from mitochondria and loss of mitochondrial membrane potential (Dym). Concomitantly, a dose-dependent decrease in the antiapoptotic protein Bcl-2 and a dose-dependent increase in the cleaved form of BID (t-BID) are observed. Moreover, NF-kB activation is involved in beta-Carotene-induced caspase cascade. These results support a pharmacological role for beta-Carotene as a candidate antitumor agent and show a possible sequence of molecular events by which this molecule may induce apoptosis in tumor cells.

Although the pharmacological role of beta-carotene in the prevention and treatment of colon cancer has received increasing attention, little is known about the molecular mechanisms of action of this carotenoid. The present study demonstrates that beta-carotene, a natural pigment widely present in fruit and vegetables, inhibits the growth of several human colon adenocarcinoma cell lines (COLO 320 HSR, LS-174, HT-29 and WiDr) by inducing cell cycle arrest in G(2)/M phase and apoptosis. These effects were dose and time dependent and strictly related to cell ability to accumulate the carotenoid. COLO 320 HSR cells incorporated beta-carotene to a greater extent than LS-174, HT-29 and WiDr cells and, concomitantly, they exhibited a higher sensitivity to the growth inhibitory effects of the carotenoid. At inhibitory concentrations beta-carotene reduced the expression of cyclin A, a key regulator of G(2)/M progression. Neither p21 nor p27, two cyclin kinase inhibitors, were significantly modified by carotenoid treatment. With respect to apoptosis induction, decreased levels of the apoptosis blocking proteins Bcl-2 and Bcl-xL were also observed. On the other hand, no changes in expression of the apoptosis promoter protein Bax were detected. This study represents a novel aspect of the biological profile of beta-carotene and a new step in elucidating the underlying molecular mechanisms of its antitumor action. In addition, since cell growth inhibitory effects were reached at beta-carotene concentrations achievable in vivo following its supplementation, this study provides a rational approach for the use of beta-carotene in colon cancer.