Carmela Fimognari

Broad targeting of resistance to apoptosis in cancer

Apoptosis or programmed cell death is natural way of removing aged cells from the body. Most of the anti-cancer therapies trigger apoptosis induction and related cell death networks to eliminate malignant cells. However, in cancer, de-regulated apoptotic signaling, particularly the activation of an anti-apoptotic systems, allows cancer cells to escape this program leading to uncontrolled proliferation resulting in tumor survival, therapeutic resistance and recurrence of cancer. This resistance is a complicated phenomenon that emanates from the interactions of various molecules and signaling pathways. In this comprehensive review we discuss the various factors contributing to apoptosis resistance in cancers. The key resistance targets that are discussed include (1) Bcl-2 and Mcl-1 proteins; (2) autophagy processes; (3) necrosis and necroptosis; (4) heat shock protein signaling; (5) the proteasome pathway; (6) epigenetic mechanisms; and (7) aberrant nuclear export signaling. The shortcomings of current therapeutic modalities are highlighted and a broad spectrum strategy using approaches including (a) gossypol; (b) epigallocatechin-3-gallate; (c) UMI-77 (d) triptolide and (e) selinexor that can be used to overcome cell death resistance is presented. This review provides a roadmap for the design of successful anti-cancer strategies that overcome resistance to apoptosis for better therapeutic outcome in patients with cancer.

Creatine as an antioxidant

Creatine monohydrate (Cr), the most diffuse supplement in the sports industry, is receiving greater attention because of its beneficial effects in a wide number of human degenerative diseases and conditions. These effects can be barely explained on the basis of the sole ergogenic role of the Cr/CrP system. Indeed, a wide number of research articles indicate that Cr is capable of exerting multiple, non-energy related, effects on diverse and relevant cellular targets. Among these effects, the antioxidant activity of Cr emerges as an additional mechanism which is likely to play a supportive role in the Cr-cytoprotection paradigm.

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.

Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in non-transformed human T lymphocytes.

Abstract. Despite experimental evidence that sulforaphane can exert chemopreventive effects, whether these effects are specific for neoplastic cells is not known. Following our previous demonstration that sulforaphane induces cell cycle arrest and apoptosis in human T lymphoblastoid Jurkat leukemia cells and increases p53 and bax protein expression, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human lymphocytes. Here, we demonstrate that sulforaphane arrested cell cycle progression in G1 phase, through a decrease in the protein expression of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53. These findings suggest that sulforaphane is a growth modulator for T cells. Our in vitro evidence that sulforaphane is active and even cytotoxic in normal as well as transformed lymphocytes raises important questions regarding its suitability for cancer chemoprevention.

Potential Effects of Pomegranate Polyphenols in Cancer Prevention and Therapy

Cancer is the second leading cause of death and is becoming the leading one in old age. Vegetable and fruit consumption is inversely associated with cancer incidence and mortality. Currently, interest in a number of fruits high in polyphenols has been raised due to their reported chemopreventive and/or chemotherapeutic potential. Pomegranate has been shown to exert anticancer activity, which is generally attributed to its high content of polyphenols. This review provides a comprehensive analysis of known targets and mechanisms along with a critical evaluation of pomegranate polyphenols as future anticancer agents. Pomegranate evokes antiproliferative, anti-invasive, and antimetastatic effects, induces apoptosis through the modulation of Bcl-2 proteins, upregulates p21 and p27, and downregulates cyclin-cdk network. Furthermore, pomegranate blocks the activation of inflammatory pathways including, but not limited to, the NF-κB pathway. The strongest evidence for its anticancer activity comes from studies on prostate cancer. Accordingly, some exploratory clinical studies investigating pomegranate found a trend of efficacy in increasing prostate-specific antigen doubling time in patients with prostate cancer. However, the genotoxicity reported for pomegranate raised certain concerns over its safety and an accurate assessment of the risk/benefit should be performed before suggesting the use of pomegranate or its polyphenols for cancer-related therapeutic purposes.

The New Isothiocyanate 4-(Methylthio)Butylisothiocyanate Selectively Affects Cell-Cycle Progression and Apoptosis Induction of Human Leukemia Cells

We investigated proliferation and apoptosis induction in Jurkat T-leukemia cells by the new isothiocyanate 4-(methylthio)butylisothiocyanate (MTBITC). To help elucidate whether the effects of MTBITC are specific for cancer cells, we tested MTBITC on freshly isolated, non-transformed human peripheral T lymphocytes. The effects of MTBITC are leukemic-cell-specific and consist of derangements in a critical point of cell-cycle control (G2/M transition). In fact, an increase in the proportion of G2 cells (from about 18% to 50%) was apparent following 24h of treatment, associated with a decrease in the protein expression of cyclin B1. The expression of cyclin-dependent kinase (CDK) 1 was more mildly attenuated by MTBITC. Our results demonstrated that high concentrations of MTBITC can also induce apoptosis, through an increase of p53 and bax, but not bcl-2, protein expression. No effects of MTBITC were demonstrated on non-transformed T lymphocytes. Taking into account its in vitro antineoplastic activity and selectivity toward leukemia cells, MTBITC can be viewed as a conceptually promising agent in cancer therapy.

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.

Cytogenetic effects of Metalaxyl on human and animal chromosomes

The purpose of this study was to assess the cytogenetic effects in vitro and in vivo of a commonly used fungicide, Metalaxyl. Chromosome damage in vitro, quantified by cultured human peripheral blood lymphocytes, demonstrated dose-related effects not associated with mitotic inhibition or cell death. Significant induction of chromosomal aberrations was observed with between 300 and 1000 micrograms/ml Metalaxyl in the absence of microsomal activation. Incubation in the presence of S9 mix produced less cytogenetic damage. Single i.p. injections of 75-300 mg/kg Metalaxyl had no effect on the frequency of micronuclei, detected in murine polychromatic erythrocytes. Micronuclei results were not compromised by direct evidence of cytotoxicity in the bone marrow of treated animals. The results in the present study indicated that genotoxicity of Metalaxyl was detected only in vitro and not in vivo. Available data reported that Metalaxyl was non-carcinogenic and gave negative results in a battery of genotoxicity tests. So, clastogenicity of Metalaxyl may not be evidence for DNA reactivity, but it may indicate alterations in cell homeostasis which are well implicated in the process of carcinogenesis.