Monia Lenzi

Natural isothiocyanates: genotoxic potential versus chemoprevention.

Isothiocyanates, occurring in many dietary cruciferous vegetables, show interesting chemopreventive activities against several chronic-degenerative diseases, including cancer, cardiovascular diseases, neurodegeneration, diabetes. The electrophilic carbon residue in the isothiocyanate moiety reacts with biological nucleophiles and modification of proteins is recognized as a key mechanism underlying the biological activity of isothiocyanates. The nuclear factor-erythroid-2-related factor 2 system, which orchestrates the expression of a wide array of antioxidant genes, plays a role in the protective effect of isothiocyanates against almost all the pathological conditions reported above. Recent emerging findings suggest a further common mechanism. Chronic inflammation plays a central role in many human diseases and isothiocyanates inhibit the activity of many inflammation components, suppress cyclooxygenase 2, and irreversibly inactivate the macrophage migration inhibitory factor. Due to their electrophilic reactivity, some isothiocyanates are able to form adducts with DNA and induce gene mutations and chromosomal aberrations. DNA damage has been demonstrated to be involved in the pathogenesis of various chronic-degenerative diseases of epidemiological relevance. Thus, the genotoxicity of the isothiocyanates should be carefully considered. In addition, the dose-response relationship for genotoxic compounds does not suggest evidence of a threshold. Thus, chemicals that are genotoxic pose a greater potential risk to humans than non-genotoxic compounds. Dietary consumption levels of isothiocyanates appear to be several orders of magnitude lower than the doses used in the genotoxicity studies and thus it is highly unlikely that such toxicities would occur in humans. However, the beneficial properties of isothiocyanates stimulated an increase of dietary supplements and functional foods with highly enriched isothiocyanate concentrations on the market. Whether such concentrations may exert a potential health risk cannot be excluded with certainty and an accurate evaluation of the toxicological profile of isothiocyanates should be prompted before any major increase in their consumption be recommended or their clinical use suggested.

Sulforaphane as a promising molecule for fighting cancer.

Cancer is a complex disease characterized by multiple genetic and molecular alterations involving transformation, deregulation of apoptosis, proliferation, invasion, angiogenesis, and metastasis. To grow, invade, and metastasize, tumors need host components and primary dysfunction in the tumor microenvironment, in addition to cell dysfunction, can be crucial for carcinogenesis. A great variety of phytochemicals have been shown to be potentially capable of inhibiting and modulating several relevant targets simultaneously and is therefore non-specific. Because of the enormous biological diversity of cancer, this pleiotropism might constitute an advantage. Phytochemicals, in particular diet-derived compounds, have therefore been proposed and applied in clinical trials as cancer chemopreventive/chemotherapeutic agents. Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables. SFN has proved to be an effective chemoprotective agent in cell culture, in carcinogen-induced and genetic animal cancer models, as well as in xenograft models of cancer. It promoted potent cytostatic and cytotoxic effects orchestrated by the modulation of different molecular targets. Cell vulnerability to SFN-mediated apoptosis was subject to regulation by cell-cycle-dependent mechanisms but was independent of a mutated p53 status. Moreover, combination of SFN with cytotoxic therapy potentiated the cytotoxic effect mediated by chemotherapy in vitro, thus suggesting its potential therapeutic benefit in clinical settings. Overall, SFN appears to be an effective and safe chemopreventive molecule and a promising tool to fight cancer.

Chemoprevention of Cancer by Isothiocyanates and Anthocyanins: Mechanisms of Action and Structure-Activity Relationship

Carcinogenesis is a multi-step, multi-path and multi-focal process, which involves a series of epigenetic and genetic alterations that begin with genomic instability and end with the development of cancer. This long and complex process presents opportunities for the development of interventions both in preventing cancer initiation and in treating the neoplasm during its premalignant stages. Failure and high systemic toxicity of conventional cancer therapies have accelerated the search for newer agents, which could prevent and/or slow-down cancer growth and have more human acceptability by being less or non-toxic. Now, it is recognized that diets rich in fruits and vegetables are associated with lower risk of cancer. Taking cue from these observations, there is a strong interest in isolating and characterizing the nutritive and non-nutritive components of fruits and vegetables as potential chemopreventive agents. Isothiocyanates and anthocyanins, present in widely consumed fruits and vegetables, are two such agents. In recent years, increasing body of evidence has underscored the cancer preventive efficacy of isothiocyanates and anthocyanins in both in vitro and in vivo animal models. In this review article, we will provide detailed insight into the chemopreventive efficacy of isothiocyanates and anthocyanins based on the evidence generated from various studies performed using cell culture or animal models of epithelial cancers. Moreover, we will discuss the potential clinical relevance of the observed chemopreventive effects of these agents.

Sulforaphane induces DNA single strand breaks in cultured human cells.

Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 microM SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value of SFR in anticancer drug protocols.

Interaction of the isothiocyanate sulforaphane with drug disposition and metabolism: pharmacological and toxicological implications.

Isothiocyanates (ITCs) are sulfur-containing compounds that are broadly distributed among cruciferous vegetables such as cabbages and broccoli. The consumption of ITCs is expected to rise due to the use of dietary supplements and public health initiatives promoting the consumption of more fruits and vegetables. Sulforaphane (SFN) is by far the most widely studied and characterized ITC. SFN is extensively metabolized and can therefore compete with other substrates of Phases I, II, III enzymes and transporters. In addition, it has an unusually high potency as an inducer of phase II enzymes and regulates the expression and function of different cytochrome P-450 genes. Such effects can be beneficial and may indicate a mechanism for the preventive role that SFN is believed to play against the degenerative events of aging and chronic diseases. Furthermore, these gene induction effects and the interaction with detoxification responses can modify bioavailability and in vivo bioactivity of drugs. This review will discuss 1) the metabolism of ITCs using SFN as an example, 2) inhibition of drug metabolism by SFN, and 3) induction of drug metabolizing enzymes by SFN. The potential pharmacological and toxicological implications of these effects on drug metabolism will also be discussed.

Protective effect of creatine against RNA damage

It is well documented that damage to DNA could be very harmful for all cells and is the source of several consequences such as cancer development, apoptosis or genetic diseases. In contrast, RNA damage is a poorly examined field in biomedical research, despite its potential to affect cell physiology. For example, a significant loss of RNA integrity has been demonstrated in advanced human atherosclerotic plaques as compared with non-atherosclerotic mammary arteries, and oxidative RNA damage has been described in several neurodegenerative diseases including Alzheimer disease. In the present study, we investigated whether RNA damage could be related to the exposure of particular xenobiotics and then we studied the potential protective activity of creatine against RNA-damaging activity of a series of chemicals with different mechanisms of action [ethyl methanesulfonate (EMS), H(2)O(2), doxorubicin, spermine NONOate, S-nitroso-N-acetylpenicillamine (SNAP)]. Since the protective effect against RNA damage can be mediated by different mechanisms, such as alterations of the rates of toxic agent absorption and uptake, trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA, we used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of creatine. We demonstrated that total RNA is susceptible to chemical attack by doxorubicin, H(2)O(2), spermine and SNAP. Creatine significantly reduced the RNA-damaging activity of only two of the toxic tested agents (H(2)O(2) and doxorubicin), while it lacked activity in counterstaining the RNA damage induced by the NO donors spermine and SNAP. Its inhibitory activity could be at least partially dependent on its capacity to directly scavenge free radicals and/or to maintain phosphocreatine store and ATP regeneration.

Combination of doxorubicin and sulforaphane for reversing doxorubicin-resistant phenotype in mouse fibroblasts with p53Ser220 mutation.

Abstract:  Chemoresistance in cancer therapy is a multifactorial process, which includes alterations in drug accumulation, increased activity of gluthatione S‐transferases, loss of function, and mutations of p53, etc. One strategy for reversing chemoresistance is the use of chemopreventive agents alongside standard chemotherapeutic protocols. Sulforaphane is one of the most promising chemopreventive agents. Sulforaphane inhibits cell proliferation and induces apoptosis in different tumor cell lines. Its proapoptotic potential could make it effective either alone or in combination with other therapeutic strategies in reversing chemoresistance. We investigated the effects of sulforaphane on mouse fibroblasts bearing a different p53 status (wild‐type, knockout, mutated) for understanding whether its activity is prevented by a mutated p53 status. p53‐knockout fibroblasts from newborn mice transfected with the p53Ser220 mutation, observed in different human cancers, were used as a model of mutated p53 status. Moreover, since p53Ser220 mutation fibroblasts showed a doxorubicin‐resistant phenotype, we treated the cells with a combination of doxorubicin plus sulforaphane. Taken together, our results suggest that a mutated p53 status did not prevent the induction of apoptosis by sulforaphane and that sulforaphane was able to reverse the resistance to doxorubicin. The association of sulforaphane–doxorubicin may therefore allow doxorubicin to be administered at lower doses, thereby reducing its potential toxicity.

Mitochondrial Pathway Mediates the Antileukemic Effects of Hemidesmus Indicus, a Promising Botanical Drug

Background Although cancers are characterized by the deregulation of multiple signalling pathways, most current anticancer therapies involve the modulation of a single target. Because of the enormous biological diversity of cancer, strategic combination of agents targeted against the most critical of those alterations is needed. Due to their complex nature, plant products interact with numerous targets and influence several biochemical and molecular cascades. The interest in further development of botanical drugs has been increasing steadily and the FDA recently approved the first new botanical prescription drug. The present study is designed to explore the potential antileukemic properties of Hemidesmus indicus with a view to contributing to further development of botanical drugs. Hemidesmus was submitted to an extensive in vitro preclinical evaluation. Methodology/Principal Findings A variety of cellular assays and flow cytometry, as well as a phytochemical screening, were performed on different leukemic cell lines. We have demonstrated that Hemidesmus modulated many components of intracellular signaling pathways involved in cell viability and proliferation and altered the protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential and increased Bax/Bcl-2 ratio. ADP, adenine nucleotide translocator and mitochondrial permeability transition pore inhibitors did not reverse Hemidesmus-induced mitochondrial depolarization. Hemidesmus induced a significant [Ca2+]i raise through the mobilization of intracellular Ca2+ stores. Moreover, Hemidesmus significantly enhanced the antitumor activity of three commonly used chemotherapeutic drugs (methotrexate, 6-thioguanine, cytarabine). A clinically relevant observation is that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemic patients. Conclusions/Significance These results indicate the molecular basis of the antileukemic effects of Hemidesmus and identify the mitochondrial pathways and [Ca2+]i as crucial actors in its anticancer activity. On these bases, we conclude that Hemidesmus can represent a valuable tool in the anticancer pharmacology, and should be considered for further investigations.

Sweet Chestnut (Castanea sativa Mill.) Bark Extract: Cardiovascular Activity and Myocyte Protection against Oxidative Damage

This work was aimed at evaluating the cardioprotective effects of Castanea sativa Mill. (CSM) bark extract characterized in its phenolic composition by HPLC-DAD-MS analysis. The study was performed using primary cultures of neonatal rat cardiomyocytes to investigate the antioxidant and cytoprotective effects of CSM bark extract and isolated guinea pig left and right atria, left papillary muscle, and aorta to evaluate its direct effect on cholinergic and adrenergic response. In cultured cardiomyocytes the CSM bark extract reduced intracellular reactive oxygen species formation and improved cell viability following oxidative stress in dose-dependent manner. Moreover, the extract decreased the contraction induced by noradrenaline (1 μM) in guinea pig aortic strips and induced transient negative chronotropic and positive inotropic effects without involvement of cholinergic or adrenergic receptors in the guinea pig atria. Our results indicate that CSM bark extract exhibits antioxidant activity and might induce cardioprotective effect.

Apoptosis and Modulation of Cell Cycle Control by Bile Acids in Human Leukemia T Cells

Depending on the nature of chemical structures, different bile acids exhibit distinct biological effects. Their therapeutic efficacy has been widely demonstrated in various liver diseases, suggesting that they might protect hepatocytes against common mechanisms of liver damage. Although it has been shown to prevent apoptotic cell death in certain cell lines, bile acids significantly inhibited cell growth and induced apoptosis in cancer cells. To better understand the pharmacological potential of bile acids in cancer cells, we investigated and compared the effects of deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and their taurine‐derivatives [taurodeoxycholic acid (TDCA) and tauroursodeoxycholic acid (TUDCA), respectively] on the induction of apoptosis and inhibition of cell proliferation of a human T leukemia cell line (Jurkat cells). All the bile acids tested induced a delay in cell cycle progression. Moreover, DCA markedly increased the fraction of apoptotic cells. The effects of TDCA, UDCA, and TUDCA were different from those observed for DCA. Their primary effect was the induction of necrosis. These distinctive features suggest that the hydrophobic properties of DCA play a role in its cytotoxic potential and indicate that it is possible to create new drugs useful for cancer therapy from bile acid derivatives as lead compounds.