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The effects of sulforaphane on canine osteosarcoma proliferation and invasion
Abstract
Recent evidence in in vitro and in vivo models suggests that sulforaphane (SFN), found in raw cruciferous vegetables, may have utility in chemoprevention, as an antineoplastic agent and as a free radical scavenger. The effects of SFN alone or with doxorubicin on cell viability were examined, as well as cell cycle kinetics, invasion capabilities and apoptosis in three canine osteosarcoma cell line (D17, OS 2.4 and HMPOS). Results showed that SFN could not induce cell death at potentially physiological concentrations (<50 μM), but significantly diminished cell invasion and downregulation of focal adhesion kinase (FAK) signaling. Modest cell cycle changes were observed in each cell line. When doxorubicin was used in conjunction with SFN, there was a protective effect to doxorubicin-induced cytotoxicity in D17 and OS 2.4 cells. Further studies examining SFN as a supplement are warranted, particularly in light of pro-proliferative and cytoprotective properties in canine osteosarcoma.
... Sulforaphane (SFN) is another natural chemical that has been studied for its potential use in the treatment of OS (osteosarcoma) [97]. This isothiocyanate chemical is derived from vegetables such as broccoli, Brussels sprouts, and cabbage. ...
MAPK (mitogen-activated protein kinase) or ERK (extracellular-signal-regulated kinase) pathway is an important link in the transition from extracellular signals to intracellular responses. Because of genetic and epigenetic changes, signal-ing cascades are altered in a variety of diseases, including cancer. Extant studies on the homeostatic and pathologic behavior of MAPK signaling have been conducted; however, much remains to be explored in preclinical and clinical research in terms of regulation and action models. MAPK has implications for cancer therapy response, more specifically in response to experimental MAPK suppression, compensatory mechanisms are activated. The current study investigates MAPK as a very complex cell signaling pathway that plays roles in cancer treatment response, cellular normal conduit maintenance, and compensatory pathway activation. Most MAPK inhibitors, unfortunately, cause resistance by activating compensatory feedback loops in tumor cells and tumor microenvironment components. As a result, innovative combinatorial treatments for cancer management must be applied to limit the likelihood of alternate pathway initiation as a possibility for generating novel therapeutics based on incorporation in translational research. We summarize current knowledge about the implications of ERK (MAPK) in cancer, as well as bioactive products from plants, microbial organisms or marine organisms, as well as the correlation with their chemical structures, which modulate this pathway for the treatment of different types of cancer.
... As far as FAK protein in humans is concerned, it is well-established that FAK serves as a scaffold for multiple protein signaling complexes, and its scaffolding function is very important for tumor progression (131). In canine oncology, interesting results were shown by Rizzo et al., demonstrating that the treatment of highly invasive D17 cells and other two OSA cell lines with Sulforaphane significantly decreased the phosphorylated state of FAK, also diminishing the invasion ability of cells cultured on Matrigel (132). These findings indirectly suggest a correlation between FAK activity and VM, considering that the inhibition of D17 OSA cell invasiveness corresponds to a decrease of VM features in vitro (14). ...
Canine tumors are valuable comparative models for human counterparts, especially to explore novel biomarkers and to understand pathways and processes involved in metastasis. Vasculogenic mimicry (VM) is a unique property of malignant cancer cells which promote metastasis. Thus, it represents an opportunity to investigate both the molecular mechanisms and the therapeutic targets of a crucial phenotypic malignant switch. Although this biological process has been largely investigated in different human cancer types, including osteosarcoma, it is still largely unknown in veterinary pathology, where it has been mainly explored in canine mammary tumors. The presence of VM in human osteosarcoma is associated with poor clinical outcome, reduced patient survival, and increased risk of metastasis and it shares the main pathways involved in other type of human tumors. This review illustrates the main findings concerning the VM process in human osteosarcoma, search for the related current knowledge in canine pathology and oncology, and potential involvement of multiple pathways in VM formation, in order to provide a basis for future investigations on VM in canine tumors.
... The mortality rate is also linked to late diagnosis resulting in a 5-year survival rate with ongoing chemotherapy and surgical treatments (Osborne & Khanna, 2012). The impact of SFN was recorded for pro-proliferation and cryoprotective characteristics were observed for canine osteosarcoma cell line in D17, OS 2.4, and HMPOS (Rizzo et al., 2017). The combination of SFN with radiation treatment was reportedly researched in LM8 murine osteosarcoma cells (Sawai et al., 2013). ...
Sulforaphane belongs to the active class of isothiocyanates capable of delivering various biological benefits for health promotion and disease prevention. This compound is considered vital to curtail numerous metabolic disorders. Various studies have proven its beneficial effects against cancer prevention and its possible utilization as a therapeutic agent in cancer treatment. Understanding the mechanistic pathways and possible interactions at cellular and subcellular levels is key to design and develop cancer therapeutics for humans. In this respect, a number of mechanisms such as modulation of carcinogen metabolism & phase II enzymatic activities, cell cycle arrest, activation of Nrf2, cytotoxic, proapoptotic and apoptotic pathways have been reported to be involved in cancer prevention. This article provides sufficient information by critical analysis to understand the mechanisms involved in cancer prevention attributed to sulforaphane. Furthermore, various clinical studies have also been included for design and development of novel therapies for cancer prevention and cure.
Practical applications
Diet and dietary components are potential tools to address various lifestyle-related disorders. Due to plenty of environmental and cellular toxicants, the chances of cancer prevalence are quite large which are worsen by adopting unhealthy lifestyles. Cancer can be treated with various therapies but those are acquiring side effects causing the patients to suffer the treatment regime. Nutraceuticals and functional foods provide safer options to prevent or delay the onset of cancer. In this regard, sulforaphane is a pivotal compound to be targeted as a potential agent for cancer treatment both in preventive and therapeutic regimes. This article provides sufficient evidence via discussing the underlying mechanisms of positive effects of sulforaphane to further the research for developing anticancer drugs that will help assuage this lethal morbidity.
... Both studies showed that Vorinostat was able to induce histone H3 acetylation in these canine cancer cells. The effects of another HDACi, sulforaphane, has been shown on canine osteosarcoma cells, significantly decreasing cell invasion and downregulating focal adhesion kinase (FAK) signaling (203). ...
A plethora of data has highlighted the role of epigenetics in the development of cancer. Initiation and progression of different cancer types are associated with a variety of changes of epigenetic mechanisms, including aberrant DNA methylation, histone modifications, and miRNA expression. At the same time, advances in the available epigenetic tools allow to investigate and reverse these epigenetic changes and form the basis for the development of anticancer drugs in human oncology. Although human and canine cancer shares several common features, only recently that studies emerged investigating the epigenetic landscape in canine cancer and applying epigenetic modulators to canine cancer. This review focuses on the existing studies involving epigenetic changes in different types of canine cancer and the use of small-molecule inhibitors in canine cancer cells.
... 34 Sulforaphane also reduces cell invasion and decreases focal adhesion kinase phosphorylation in canine osteosarcoma cell lines. 35 However, to our knowledge, the impact of SFN in cancer-bearing dogs has not been explored. ...
Background:
Lymphoma (LSA) is a common malignancy in dogs. Epigenetic changes are linked to LSA pathogenesis and poor prognosis in humans, and LSA pathogenesis in dogs. Sulforaphane (SFN), an epigenetic-targeting compound, has recently gained interest in relation to cancer prevention and therapy.
Objective:
Examine the impact of oral supplementation with SFN on the lymph node proteome of dogs with multicentric LSA.
Animals:
Seven client-owned dogs with multicentric LSA.
Methods:
Prospective, nonrandomized, noncontrolled study in treatment-naïve dogs with intermediate or large cell multicentric LSA. Lymph node cell aspirates were obtained before and after 7 days of oral supplementation with SFN, and analyzed via label-free mass spectrometry, immunoblots, and Gene Set Enrichment Analysis.
Results:
There was no clinical response and no adverse events attributed to SFN. For individual dogs, the expression of up to 650 proteins changed by at least 2-fold (range, 2-100) after supplementation with SFN. When all dogs where analyzed together, 14 proteins were significantly downregulated, and 10 proteins were significantly upregulated after supplementation with SFN (P < .05). Proteins and gene sets impacted by SFN were commonly involved in immunity, response to oxidative stress, gene transcription, apoptosis, protein transport, maturation and ubiquitination.
Conclusions and clinical importance:
Sulforaphane is associated with major changes in the proteome of neoplastic lymphocytes in dogs.
... However, conflicting data on the effects of SFN when used in association with Doxo impose caution. Rizzo and coworkers showed that SFN can decrease Doxo's antitumor potential depending on the specific redox status of the cell line (Rizzo et al., 2017). SFN sensitized cells characterized by high basal Nrf2 expression to Doxo, whereas it reduced Doxo's anticancer effects in cells with very low Nrf2 basal levels (Hu et al., 2010) ( Figure 1). ...
The success of cancer therapy is often compromised by the narrow therapeutic index of many anticancer drugs and the occurrence of drug resistance. The association of anticancer therapies with natural compounds is an emerging strategy to improve the pharmaco-toxicological profile of cancer chemotherapy. Sulforaphane, a phytochemical found in cruciferous vegetables, targets multiple pathways involved in cancer development, as recorded in different cancers such as breast, brain, blood, colon, lung, prostate, and so forth. As examples to make the potentialities of the association chemotherapy raise, here we highlight and critically analyze the information available for two associations, each composed by a paradigmatic anticancer drug (cisplatin or doxorubicin) and sulforaphane.
... In veterinary medicine, SFN has been investigated, in vitro, alone and with doxorubicin against osteosarcoma cell lines (Rizzo et al. 2017). The results indicate that SFN significantly decreased cell invasion and downregulation of focal adhesion kinase signalling. ...
The role of epigenetic alterations during cancer has gained increasing attention and has resulted in a paradigm shift in our understanding of mechanisms leading to cancer susceptibility. Sulforaphane (SFN) is a naturally occurring isothiocyanate derived from the precursor glucosinolate, glucoraphanin (GFN), which is found in cruciferous vegetables such as broccoli. Sulforaphane has been shown to suppress tumour growth by several mechanisms including inhibiting histone deacetylases. The objective of this study was to provide a detailed analysis of sulforaphane absorption following a single oral dose of a broccoli sprout supplement in normal dogs. A single dose of broccoli sprout supplement (with active myrosinase) was orally administered to 10 healthy adult dogs. Blood and urine samples were collected prior to dosing, and at various time points post‐dosing. Plasma total SFN metabolite levels peaked at 4 h post‐consumption and were cleared by 24 h post‐consumption. Urinary SFN metabolites peaked at 4 h post‐consumption, and remained detectable at 24 and 48 h post‐supplement consumption. A trend for decrease in histone deacetylase activity was observed at 1 h post‐consumption and a significant decrease was observed at 24 h post‐consumption. The data presented herein indicate that oral SFN is absorbed in dogs, SFN metabolites are detectable in plasma and urine post‐dosing, and SFN and its metabolites have some effect on histone deacetylase activity following a single dose.
... Using bladder cancer cell lines, Abbaoui, et al. [68] demonstrated that downregulation of survivin, epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (HER2/neu) induced G2/M cell cycle accumulation and apoptosis [68]. In osteosarcoma cell lines, SFN decreased cell invasion and also focal adhesion kinase (FAK), both of which are important in cancer progression [114]. SFN has been shown to inhibit inflammatory responses, including downregulation of cyclooxygenase-2 (Cox-2) and reduction of prostaglandin E2 levels [84,85]. ...
The clinical course for both early and late stage Bladder Cancer (BC) continues to be characterized by significant patient burden due to numerous occurrences and recurrences requiring frequent surveillance strategies, intravesical drug therapies, and even more aggressive treatments in patients with locally advanced or metastatic disease. For these reasons, BC is also the most expensive cancer to treat. Fortunately, BC offers an excellent platform for chemoprevention interventions with potential to optimize the systemic and local exposure of promising agents to the bladder mucosa. However, other than smoking cessation, there is a paucity of research that systematically examines agents for chemoprevention of bladder cancers. Adopting a systematic, molecular-mechanism based approach, the goal of this review is to summarize epidemiological, in vitro, and preclinical studies, including data regarding the safety, bioavailability, and efficacy of agents evaluated for bladder cancer chemoprevention. Based on the available studies, phytochemicals, specifically isothiocyanates such as sulforaphane, present in Brassicaceae or "cruciferous" vegetables in the precursor form of glucoraphanin are: (a) available in standardized formulations; (b) bioavailable- both systemically and in the bladder; (c) observed to be potent inhibitors of BC carcinogenesis through multiple mechanisms; and (d) without toxicities at these doses. Based on available evidence from epidemiological, in vitro, preclinical, and early phase trials, phytochemicals, specifically isothiocyanates (ITCs) such as sulforaphane (SFN) represent a promising potential chemopreventitive agent in bladder cancer.
In numerous experimental models, sulforaphane (SFN) is shown herein to induce hormetic dose responses that are not only common but display endpoints of biomedical and clinical relevance. These hormetic responses are mediated via the activation of nuclear factor erythroid- derived 2 (Nrf2) antioxidant response elements (AREs) and, as such, are characteristically biphasic, well integrated, concentration/dose dependent, and specific with regard to the targeted cell type and the temporal profile of response. In experimental disease models, the SFN-induced hormetic activation of Nrf2 was shown to effectively reduce the occurrence and severity of a wide range of human-related pathologies, including Parkinson’s disease, Alzheimer’s disease, stroke, age-related ocular damage, chemically induced brain damage, and renal nephropathy, amongst others, while also enhancing stem cell proliferation. Although SFN was broadly chemoprotective within an hormetic dose-response context, it also enhanced cell proliferation/cell viability at low concentrations in multiple tumor cell lines. Although the implications of the findings in tumor cells are largely uncertain at this time and warrant further consideration, the potential utility of SFN in cancer treatment has not been precluded. This assessment of SFN complements recent reports of similar hormesis-based chemoprotections by other widely used dietary supplements, such as curcumin, ginkgo biloba, ginseng, green tea, and resveratrol. Interestingly, the mechanistic profile of SFN is similar to that of numerous other hormetic agents, indicating that activation of the Nrf2/ARE pathway is probably a central, integrative, and underlying mechanism of hormesis itself. The Nrf2/ARE pathway provides an explanation for how large numbers of agents that both display hormetic dose responses and activate Nrf2 can function to limit age-related damage, the progression of numerous disease processes, and chemical- and radiation- induced toxicities. These findings extend the generality of the hormetic dose response to include SFN and many other chemical activators of Nrf2 that are cited in the biomedical literature and therefore have potentially important public health and clinical implications.
Drugs used in clinical oncology have narrow therapeutic indices with adverse toxicity often involving oxidative damage. Chemoresistance to these conventional antineoplastics is usually mediated by oxidative stress-upregulated pathways such as those of nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor-1 alpha (HIF-1α). Accordingly, the use of antioxidants in combinational approaches has begun to be considered for fighting cancer because of both the protective role against adverse effects and the ability to sensitize chemoresistant cancer cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been identified as a mediator of the cytoprotection but it is not regularly associated with tumor chemosensitization. However, some Nrf2 inducers could be exerting cytoprotective and chemosensitizing roles through a simple integrated mechanism in which the cellular level of reactive oxygen species is controlled, thus inhibiting the oxidative damage in non-target tissues and the tumor chemoresistance mediated by NF-κB or HIF-1α. As examples to show the general idea of this antioxidant combination chemotherapy, this review explores the preclinical information available for four combinations, each composed by a paradigmatic oncological drug (cisplatin or doxorubicin) and a recognized antioxidant (sulforaphane or curcumin). The issues for translating these outcomes to clinical trials are briefly discussed.
The answer to chemoprevention has perhaps been available to the general public since the dawn of time. The epigenetic diet is of extreme interest, for research suggests that cruciferous vegetables are not only an important source of nutrients, but perhaps a key to eliminating cancer as a life-threatening disease. Cruciferous vegetables such as kale, cabbage, Brussels sprouts, and broccoli sprouts contain chemical components, such as sulforaphane (SFN) and indole-3-carbinol (I3C), which have been revealed to be regulators of microRNAs (miRNAs) and inhibitors of histone deacetylases (HDACs) and DNA methyltransferases (DNMTs). The mis-regulation and overexpression of these genes are responsible for the uncontrolled cellular proliferation and viability of various types of cancer cells. The field of epigenetics and its incorporation into modern medicinal investigation is an exponentially growing field of interest and it is becoming increasingly apparent that the incorporation of an epigenetic diet may in fact be the key to chemoprevention.
Sulforaphane is a dietary isothiocyanate found in cruciferous vegetables showing antileukemic activity. With the purpose of extending the potential clinical impact of sulforaphane in the oncological field, we investigated the antileukemic effect of sulforaphane on blasts from patients affected by different types of leukemia and, taking into account the intrinsically hypoxic nature of bone marrow, on a leukemia cell line (REH) maintained in hypoxic conditions. In particular, we tested sulforaphane on patients with chronic lymphocytic leukemia, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, and blastic NK cell leukemia. Sulforaphane caused a dose-dependent induction of apoptosis in blasts from patients diagnosed with acute lymphoblastic or myeloid leukemia. Moreover, it was able to cause apoptosis and to inhibit proliferation in hypoxic conditions on REH cells. As to its cytotoxic mechanism, we found that sulforaphane creates an oxidative cellular environment that induces DNA damage and Bax and p53 gene activation, which in turn helps trigger apoptosis. On the whole, our results raise hopes that sulforaphane might set the stage for a novel therapeutic principle complementing our growing armature against malignancies and advocate the exploration of sulforaphane in a broader population of leukemic patients.
Rhadbomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and is subdivided in the embryonal (ERMS) and alveolar (ARMS) subtypes, the latter being associated with the worst prognosis. We report that sulforaphane (SFN), a broccoli-derived anticancer isothiocyanate, causes dose- and time-dependent growth inhibition and apoptosis in both ERMS and ARMS cells. In ARMS, SFN induced the modulation of expression of crucial genes and proteins: mRNA and protein levels of PAX3-FKHR, MYCN, and MET decreased, while those of p21 and TRAIL-receptor DR5 (but not DR4) increased. Since DR5 expression increased specifically in ARMS, we treated ARMS cells with TRAIL, SFN, or their combination. While ARMS cells (RH30 and RH4) proved to be TRAIL-resistant, SFN restored their sensitivity to TRAIL-induced cell-growth inhibition, leading to a stronger effect in combination with TRAIL. ARMS cells transfected with siDR5 showed that SFN-induced DR5 acts as a key regulator, being directly related to the TRAIL-induced cell-growth inhibition. The in vivo anti-tumor activity of SFN and TRAIL was evaluated in a xenograft murine model of ARMS through microPET. The results showed that the systemic treatment (3 wk) of mice with SFN or TRAIL as single agents only delayed tumor evolution, while the combined treatment of SFN and TRAIL led to tumor elimination. These findings indicate that SFN triggers the apoptotic pathway in both alveolar and embryonal rhabdomyosarcomas and that combined treatment with SFN and TRAIL might be a promising therapy for the aggressive alveolar subtype.
Sulforaphane (SFN) is a naturally-occurring isothiocyanate best known for its role as an indirect antioxidant. Notwithstanding, in different cancer cell lines, SFN may promote the accumulation of reactive oxygen species (ROS) and cause cell death e.g. by apoptosis. Osteosarcoma often becomes chemoresistant, and new molecular targets to prevent drug resistance are needed. Here, we aimed to determine the effect of SFN on ROS levels and to identify key biomarkers leading to ROS unbalance and apoptosis in the p53-null MG-63 osteosarcoma cell line. MG-63 cells were exposed to SFN for up to 48 h. At 10 μM concentration or higher, SFN decreased cell viability, increased the%early apoptotic cells and increased caspase 3 activity. At these higher doses, SFN increased ROS levels, which correlated with apoptotic endpoints and cell viability decline. In exposed cells, gene expression analysis revealed only partial induction of phase-2 detoxification genes. More importantly, SFN inhibited ROS-scavenging enzymes and impaired glutathione recycling, as evidenced by inhibition of glutathione reductase (GR) activity and combined inhibition of glutathione peroxidase (GPx) gene expression and enzyme activity. In conclusion, SFN induced oxidative stress and apoptosis via a p53-independent mechanism. GPx expression and activity were found associated with ROS accumulation in MG-63 cells and are potential biomarkers for the efficacy of ROS-inducing agents e.g. as co-adjuvant drugs in osteosarcoma.
Glioblastoma has highly invasive potential, which might result in poor prognosis and therapeutic failure. Hence, the key we study is to find effective therapies to repress migration and invasion. Sulforaphane (SFN) was demonstrated to inhibit cell growth in a variety of tumors. Here, we will further investigate whether SFN inhibits migration and invasion and find the possible mechanisms in human glioblastoma U87MG and U373MG cells.
First, the optimal time and dose of SFN for migration and invasion study were determined via cell viability and cell morphological assay. Further, scratch assay and transwell invasion assay were employed to investigate the effect of SFN on migration and invasion. Meanwhile, Western blots were used to detect the molecular linkage among invasion related proteins phosphorylated ERK1/2, matrix metalloproteinase-2 (MMP-2) and CD44v6. Furthermore, Gelatin zymography was performed to detect the inhibition of MMP-2 activation. In addition, ERK1/2 blocker PD98059 (25 µM) was integrated to find the link between activated ERK1/2 and invasion, MMP-2 and CD44v6.
The results showed that SFN (20 µM) remarkably reduced the formation of cell pseudopodia, indicating that SFN might inhibit cell motility. As expected, scratch assay and transwell invasion assay showed that SFN inhibited glioblastoma cell migration and invasion. Western blot and Gelatin zymography showed that SFN phosphorylated ERK1/2 in a sustained way, which contributed to the downregulated MMP-2 expression and activity, and the upregulated CD44v6 expression. These molecular interactions resulted in the inhibition of cell invasion.
SFN inhibited migration and invasion processes. Furthermore, SFN inhibited invasion via activating ERK1/2 in a sustained way. The accumulated ERK1/2 activation downregulated MMP-2 expression and decreased its activity and upregulated CD44v6. SFN might be a potential therapeutic agent by activating ERK1/2 signaling against human glioblastoma.
Cruciferous vegetables are widely acknowledged to provide chemopreventive benefits in humans, but they are not generally consumed at levels that effect significant change in biomarkers of health. Because consumers have embraced the notion that dietary supplements may prevent disease, this review considers whether an appropriately validated sulforaphane-yielding broccoli sprout supplement may deliver clinical benefit. The crucifer-derived bioactive phytochemical sulforaphane is a significant inducer of nuclear factor erythroid 2-related factor 2 (Nrf2), the transcription factor that activates the cell's endogenous defenses via a battery of cytoprotective genes. For a broccoli sprout supplement to demonstrate bioactivity in vivo, it must retain both the sulforaphane-yielding precursor compound, glucoraphanin, and the activity of glucoraphanin's intrinsic myrosinase enzyme. Many broccoli sprout supplements are myrosinase inactive, but current labeling does not reflect this. For the benefit of clinicians and consumers, this review summarizes the findings of in vitro studies and clinical trials, interpreting them in the context of clinical relevance. Standardization of sulforaphane nomenclature and assay protocols will be necessary to remove inconsistency and ambiguity in the labeling of currently available broccoli sprout products.
Despite numerous published studies describing adjuvant chemotherapy for canine appendicular osteosarcoma, there is no consensus as to the optimal chemotherapy protocol. The purpose of this study was to determine whether either of two protocols would be associated with longer disease-free interval (DFI) in dogs with appendicular osteosarcoma following amputation. Dogs with histologically confirmed appendicular osteosarcoma that were free of gross metastases and underwent amputation were eligible for enrollment. Dogs were randomized to receive either six doses of carboplatin or three doses each of carboplatin and doxorubicin on an alternating schedule. Fifty dogs were included. Dogs receiving carboplatin alone had a significantly longer DFI (425 versus 135 days) than dogs receiving alternating carboplatin and doxorubicin (P = 0.04). Toxicity was similar between groups. These results suggest that six doses of carboplatin may be associated superior DFI when compared to six total doses of carboplatin and doxorubicin.
Malignant tumors are the single most common cause of death and the mortality rate of ovarian cancer is the highest among gynecological disorders. The excision of benign tumors is generally followed by complete recovery; however, the activity of cancer cells often results in rapid proliferation even after the tumor has been excised completely. Thus, clinical treatment must be supplemented by auxiliary chemotherapy or radiotherapy. Sulforaphane (SFN) is an extract from the mustard family recognized for its anti-oxidation abilities, phase 2 enzyme induction, and anti-tumor activity.
This study investigated the cell cycle arrest in G2/M by SFN and the expression of cyclin B1, Cdc2, and the cyclin B1/CDC2 complex in PA-1 cells using western blotting and co-IP western blotting.
This study investigated the anticancer effects of dietary isothiocyanate SFN on ovarian cancer, using cancer cells line PA-1. SFN-treated cells accumulated in metaphase by CDC2 down-regulation and dissociation of the cyclin B1/CDC2 complex.
Our findings suggest that, in addition to the known effects on cancer prevention, SFN may also provide antitumor activity in established ovarian cancer.
Acute lymphoblastic leukemia (ALL) is the most common hematological cancer in children. Although risk-adaptive therapy, CNS-directed chemotherapy, and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs are needed as frontline treatments in high-risk disease and as salvage agents in relapsed ALL. In this study, we report that purified sulforaphane, a natural isothiocyanate found in cruciferous vegetables, has anti-leukemic properties in a broad range of ALL cell lines and primary lymphoblasts from pediatric T-ALL and pre-B ALL patients. The treatment of ALL leukemic cells with sulforaphane resulted in dose-dependent apoptosis and G2/M cell cycle arrest, which was associated with the activation of caspases (3, 8, and 9), inactivation of PARP, p53-independent upregulation of p21(CIP1/WAF1), and inhibition of the Cdc2/Cyclin B1 complex. Interestingly, sulforaphane also inhibited the AKT and mTOR survival pathways in most of the tested cell lines by lowering the levels of both total and phosphorylated proteins. Finally, the administration of sulforaphane to the ALL xenograft models resulted in a reduction of tumor burden, particularly following oral administration, suggesting a potential role as an adjunctive agent to improve the therapeutic response in high-risk ALL patients with activated AKT signaling.
Sulforaphane (SUL), an isothiocyanate found in broccoli and other cruciferous vegetables, has been shown to induce phase II detoxification enzymes, inhibit chemically induced mammary tumors in rats, and more recently to induce cell cycle arrest and apoptosis in cancer cells of the colon. Here, we provide evidence that SUL also acts as a breast cancer anti-proliferative agent. The BALB/c mouse mammary carcinoma cell line F3II was treated with SUL at concentrations up to 15 microM and examined for markers of cell cycle arrest and apoptosis. Treatment of asynchronous F3II cells with 15 microM SUL resulted in G2/M cell cycle arrest, elevated p34cdc2 (cdc2) kinase activity, Bcl-2 down-regulation, evidence of caspase activation, and aggregation of condensed nuclear chromatin. Subsequent exposure of synchronized cells to 15 microM SUL resulted in elevated numbers of prophase/prometaphase mitotic figures, indicating cell cycle progression beyond G2 and arrest early within mitosis. Moreover, cells treated with 15 microM SUL displayed aberrant mitotic spindles, and higher doses of SUL inhibited tubulin polymerization in vitro. In addition, BALB/c mice injected s.c. with F3II cells and subsequently injected daily i.v. with SUL (15 nmol/day for 13 days) developed significantly smaller tumors (approximately 60% less in mass) than vehicle-treated controls. Western blot analysis of tumor proteins demonstrated significantly (P<0.05) reduced PCNA and elevated PARP fragmentation in samples from animals dosed with SUL. Taken together, these results indicate that SUL has mammary cancer suppressive actions both in cell culture and in the whole animal. Inhibition of mammary carcinogenesis appears in part to involve perturbation of mitotic microtubules and early M-phase block associated with cdc2 kinase activation, indicating that cells arrest prior to metaphase exit.
Consumption of fruits and vegetables is recognized as an important part of a healthy diet. Increased consumption of cruciferous vegetables in particular has been associated with a decreased risk of several degenerative and chronic diseases, including cardiovascular disease and certain cancers. Members of the cruciferous vegetable family, which includes broccoli, Brussels sprouts, cauliflower, and cabbage, accumulate significant concentrations of glucosinolates, which are metabolized in vivo to biologically active isothiocyanates (ITCs). The ITC sulforaphane, which is derived from glucoraphanin, has garnered particular interest as an indirect antioxidant due to its extraordinary ability to induce expression of several enzymes via the KEAP1/Nrf2/ARE pathway. Nrf2/ARE gene products are typically characterized as Phase II detoxification enzymes and/or antioxidant (AO) enzymes. Over the last decade, human clinical studies have begun to provide in vivo evidence of both Phase II and AO enzyme induction by SF. Many AO enzymes are redox cycling enzymes that maintain redox homeostasis and activity of free radical scavengers such as vitamins A, C, and E. In this review, we present the existing evidence for induction of PII and AO enzymes by SF, the interactions of SF-induced AO enzymes and proposed maintenance of the essential vitamins A, C, and E, and, finally, the current view of genotypic effects on ITC metabolism and AO enzyme induction and function.
Cisplatin (cis-diamminedichloroplatinum II, CDDP) is a chemotherapeutic agent that induces nephrotoxicity associated with oxidative/nitrosative stress. Sulforaphane (SFN) is an isothiocyanate produced by the enzymatic action of myrosinase on glucorophanin, a glucosinolate contained in cruciferous vegetables. SFN is able to induce cytoprotective enzymes through the transcription factor Nrf2. The purpose of this study was to evaluate whether SFN induces a cytoprotective effect on the CDDP-induced nephrotoxicity. Preincubation of LLC-PK1 cells with 0.5-5 microM SFN by 24 h was able to prevent, in a concentration-dependent way, CDDP-induced cell death. Immunofluorescent staining confirmed the nuclear translocation of Nrf2 after treatment with SFN. In the in vivo studies, CDDP was given to Wistar rats as a sole i.p. injection at a dose of 7.5 mg/kg. SFN (500 microg/kg i.v.) was given two times (24 h before and 24 after CDDP-injection). Animals were killed three days after CDDP-injection. SFN attenuated CDDP-induced renal dysfunction, structural damage, oxidative/nitrosative stress, glutathione depletion, enhanced urinary hydrogen peroxide excretion and the decrease in antioxidant enzymes (catalase, glutathione peroxidase and glutathione-S-transferase). The renoprotective effect of SFN on CDDP-induced nephrotoxicity was associated with the attenuation in oxidative/nitrosative stress and the preservation of antioxidant enzymes.
Approximately 200 studies that examined the relationship between fruit and vegetable intake and cancers of the lung, colon, breast, cervix, esophagus, oral cavity, stomach, bladder, pancreas, and ovary are reviewed. A statistically significant protective effect of fruit and vegetable consumption was found in 128 of 156 dietary studies in which results were expressed in terms of relative risk. For most cancer sites, persons with low fruit and vegetable intake (at least the lower one-fourth of the population) experience about twice the risk of cancer compared with those with high intake, even after control for potentially confounding factors. For lung cancer, significant protection was found in 24 of 25 studies after control for smoking in most instances. Fruits, in particular, were significantly protective in cancers of the esophagus, oral cavity, and larynx, for which 28 of 29 studies were significant. Strong evidence of a protective effect of fruit and vegetable consumption was seen in cancers of the pancreas and stomach (26 of 30 studies), as well as in colorectal and bladder cancers (23 of 38 studies). For cancers of the cervix, ovary, and endometrium, a significant protective effect was shown in 11 of 13 studies, and for breast cancer a protective effect was found to be strong and consistent in a meta analysis. It would appear that major public health benefits could be achieved by substantially increasing consumption of these foods.
This paper gives an overview of the epidemiological data concerning the cancer-preventive effect of brassica vegetables, including cabbage, kale, broccoli, Brussels sprouts, and cauliflower. The protective effect of brassicas against cancer may be due to their relatively high content of glucosinolates. Certain hydrolysis products of glucosinolates have shown anticarcinogenic properties. The results of 7 cohort studies and 87 case-control studies on the association between brassica consumption and cancer risk are summarized. The cohort studies showed inverse associations between the consumption of cabbage, cauliflower, and broccoli and risk of lung cancer; between the consumption of brassicas and risk of stomach cancer; between broccoli consumption and risk of all cancers taken together; and between brassica consumption and the occurrence of second primary cancers. Of the case-control studies, 67% showed an inverse association between consumption of total brassica vegetables and risk of cancer at various sites. For cabbage, broccoli, cauliflower, and Brussels sprouts, these percentages were 70, 56, 67, and 29%, respectively. Although the measured effects might have been distorted by various types of bias, it is concluded that a high consumption of brassica vegetables is associated with a decreased risk of cancer. This association appears to be most consistent for lung, stomach, colon, and rectal cancer and least consistent for prostatic, endometrial, and ovarian cancer. It is not yet possible to resolve whether associations are to be attributed to brassica vegetables per se or to vegetables in general. Further epidemiological research should separate the anticarcinogenic effect of brassica vegetables from the effect of vegetables in general.
Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.
Glucosinolates (GL) can inhibit, retard or reverse experimental multistage carcinogenesis. When brassica plant tissue is broken, GLs are hydrolyzed by the endogenous enzyme myrosinase (Myr), releasing many products including isothiocyanates (ITC). Synthetic ITCs like sulforaphane exert chemopreventive effects against chemically induced tumors in animals, modulating enzymes required for carcinogens' activation/detoxification and/or the induction of cell-cycle arrest and apoptosis in tumor cell lines. To investigate the chemopreventive potential of ITCs while reproducing the circumstances of dietary contact with sulforaphane, we studied proliferation, apoptosis induction and p53, bcl-2 and bax protein expression in Jurkat T-leukemia cells by sulforaphane, the ITC generated in situ in a quantitative manner by Myr starting from glucoraphanin (GRA). Jurkat cells were treated with different doses of GRA-Myr mixture. Effects on cell growth or survival were evaluated by counting trypan blue-excluding cells. Cell-cycle progression, apoptosis and expression of p53, bax and bcl-2 proteins were analyzed by flow cytometry. Results were analyzed by two-sided Fisher's exact test. Sulforaphane, but not GRA, caused G(2)/M-phase arrest (P = 0.028) and increase of apoptotic cell fraction (P < 0.0001) in a time- and dose-dependent manner. Necrosis was observed after prolonged exposure to elevated sulforaphane doses. Moreover, it markedly increased p53 and bax protein expression, and slightly affected bcl-2 expression. These findings indicate that sulforaphane but not the native GL GRA can exert both protective and toxic effects inhibiting leukemic cell growth. Sulforaphane therefore deserves study as a potential chemopreventive/chemotherapeutic antileukemic agent.
Cytochrome p450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the p450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome p450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome p450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome p450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to p450 research. While human cytochrome p450 databases have swelled with a flood of new human sequence variants, however, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome p450 structure/function. This review examines the latest advances in our understanding of human cytochrome p450s.
We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.
Focal-adhesion kinase (FAK) is an important mediator of growth-factor signalling, cell proliferation, cell survival and cell migration. Given that the development of malignancy is often associated with perturbations in these processes, it is not surprising that FAK activity is altered in cancer cells. Mouse models have shown that FAK is involved in tumour formation and progression, and other studies showing that FAK expression is increased in human tumours make FAK a potentially important new therapeutic target.
A series of sulforaphane analogues were synthesized with various amines by treatment of carbon disulfide followed by Boc2O and DMAP. These synthesized sulforaphane analogues were tested on cisplatin treated cultured LLC-PK1 kidney cell line. Among these analogues, several compounds including SF5 show a potent effect on kidney cell protection assay at the concentration of 2.5 μM. Further studies with compound SF5 revealed that the kidney cell protection effect was related by inhibiting the apoptosis pathway through JNK-p53-caspase apoptotic cascade. Compound SF5 may be considered as a promising candidate for the development of new kidney protection agent against drug induced acute kidney disease.
In malignant melanoma complex reprogramming of cell death and survival pathways leads to increased chemoresistance and poor longer-term survival. Sulforaphane (SF) is a promising isothiocyanate compound occurring in cruciferous plants with reported antiproliferative and proapoptotic activity in several tumor cell lines including melanoma. In this work we investigated the effects of SF in several melanoma cell lines and fresh melanoma cultivates. We found that SF is cytotoxic and induces mitochondrial, caspase-dependent apoptosis in our study model, however with lower efficiency in fresh melanoma cultivates. Moreover, our results indicate that in melanoma cell lines and fresh melanoma cultivates SF induces multiple signaling including oxidative stress-mediated activation of DNA-damage response pathway, changes in p38 kinase activity and enhanced expression of Bax and Puma proapoptotic proteins. In addition, in SF-exposed p53-mutant melanoma cells Puma expression seem to be under p38 control and acts as a compensatory proapoptotic mechanism. Conversely, decreased apoptosis in SF-exposed melanoma cultivates might be attributed to Akt-mediated suppression of p38 as well as p53 activity. Together, our results suggest that SF inhibits growth and proliferation and induces mitochondrial apoptosis both in melanoma cell lines as well as in fresh melanoma cultivates. This proapoptotic effect might be enhanced in combination with Akt inhibitors, in particular in melanoma samples. SF is thus commendable for further preclinical testing, both as a single agent as well as in combination regimens.
Sulforaphane (a potent anticarcinogenic isothiocyanate derived from glucoraphanin) is widely considered responsible for the protective effects of broccoli consumption. Broccoli is typically purchased fresh or frozen and cooked before consumption. We compared the bioavailability and metabolism of sulforaphane from portions of lightly cooked fresh or frozen broccoli, and investigated the bioconversion of sulforaphane to erucin.
Eighteen healthy volunteers consumed broccoli soups produced from fresh or frozen broccoli florets that had been lightly cooked and sulforaphane thio-conjugates quantified in plasma and urine. Sulforaphane bioavailability was about tenfold higher for the soups made from fresh compared to frozen broccoli, and the reduction was shown to be due to destruction of myrosinase activity by the commercial blanching-freezing process. Sulforaphane appeared in plasma and urine in its free form and as several thio-conjugates forms. Erucin N-acetyl-cysteine conjugate was a significant urinary metabolite, and it was shown that human gut microflora can produce sulforaphane, erucin, and their nitriles from glucoraphanin.
The short period of blanching used to produce commercial frozen broccoli destroys myrosinase and substantially reduces sulforaphane bioavailability. Sulforaphane was converted to erucin and excreted in urine, and it was shown that human colonic flora were capable of this conversion.
Forty-eight dogs with histologically confirmed appendicular osteosarcoma (OSA) entered a prospective clinical trial evaluating treatment with amputation and up to 4 doses of carboplatin given every 21 days. The median disease-free interval (DFI) was 257 days, with 31.2% of the dogs disease-free at 1 year. The median survival time was 321 days, with 35.4% of the dogs alive at 1 year. Dogs with proximal humeral OSA had shorter DFI (P = .016) and survival (P = .037) times than dogs with OSA at other locations. Dogs with lower body weights (40 kg) had longer DFI (P = .0056) and survival (P = .007) times than larger dogs. Survival times for dogs that received carboplatin were statistically longer than those previously reported for amputation alone (P < .001). DFI and survival times are similar to those previously reported for 2 to 4 doses of cisplatin. Carboplatin appears to be a well-tolerated chemotherapeutic drug that can be given safely every 21 days at a dose of 300 mg/m2. Neutropenia was the dose-limiting toxicity in this study. J Vet Intern Med 1996;10:76–81. Copyright © 1996 by the American College of Veterinary Internal Medicine.
The consumption of cruciferous vegetables has long been associated with a reduced risk in the occurrence of cancer at various sites, including the prostate, lung, breast and colon. This protective effect is attributed to isothiocyanates present in these vegetables, and sulforaphane (SF), present in broccoli, is by far the most extensively studied to uncover the mechanisms behind this chemoprotection. The major mechanism by which SF protects cells was traditionally thought to be through Nrf2-mediated induction of phase 2 detoxification enzymes that elevate cell defense against oxidative damage and promote the removal of carcinogens. However, it is becoming clear that there are multiple mechanisms activated in response to SF, including suppression of cytochrome P450 enzymes, induction of apoptotic pathways, suppression of cell cycle progression, inhibition of angiogenesis and anti-inflammatory activity. Moreover, these mechanisms seem to have some degree of interaction to synergistically afford chemoprevention.
In this study, the homozygous tubby (tub/tub) mutant mouse, with an early progressive hearing loss and photoreceptor degeneration, was used as a model system to examine the effects of systemic administration of a naturally occurring isothiocyanate, sulforaphane (SF), on photoreceptor degeneration. Several novel observations have been made: (i) the mRNA and protein expression of thioredoxin (Trx), thioredoxin reductase (TrxR) and NF-E2-related factor-2 (Nrf2) were significantly reduced even prior to photoreceptor cell degeneration in the retinas of tub/tub mice, suggesting that retinal expression of the Trx system is impaired and that Trx regulation is involved in the pathogenesis of retinal degeneration in this model, (ii) intraperitoneal injection with SF significantly up-regulated retinal levels of Trx, TrxR, and Nrf2, and effectively protected photoreceptor cells in tub/tub mice as evaluated functionally by electroretinography and morphologically by quantitative histology, and (iii) treatment with PD98059, an inhibitor of extracellular signal-regulated kinases (ERKs), blocked SF-mediated ERKs activation and up-regulation of Trx/TrxR/Nrf2 in the retinas of tub/tub mice. This suggests that ERKs and Nrf2 are involved in the mechanism of SF-mediated up-regulation of the Trx system to protect photoreceptor cells in this model. These novel findings are significant and could provide important information for the development of a unique strategy to prevent sensorineural deafness/retinal dystrophic syndromes and also other forms of inherited neurological disorders.
Sulforaphane is a predominant isothiocyanate in Brassica oleracea, a family of cruciferous vegetables, and is known to be inversely related to the risk of various types of human carcinomas. Studies using oral carcinoma cell lines are scarce, however, and the role of sulforaphane on oral carcinoma cell metastasis is yet to be determined. In this study, the growth inhibition of oral carcinoma cell lines by sulforaphane was determined using aqueous soluble tetrazolium salts, and the growth of various oral cancer cell lines was attenuated. The migration and invasion activities of the cells also decreased, as observed in monolayer scratch assays and transwell invasion experiments. The molecular change behind the impairment of the migration and invasion was investigated via secreted metalloprotease level detection using Multiplex protein analysis kits. At the molecular level, the secreted forms of MMP-1 and MMP-2 were down-regulated. The expressions of MMP-1 and MMP-2 did not change when a conventional tumoricidal agent paclitaxel was used. These findings indicate that sulforaphane may have therapeutic potential as an inhibitor of metastasis in oral carcinoma patients.
To determine outcomes and prognostic factors for those outcomes in dogs with appendicular osteosarcoma treated with curative-intent surgery and adjuvant carboplatin.
Retrospective case series.
65 client-owned dogs with appendicular osteosarcoma and no evidence of gross metastatic disease at the time of diagnosis.
Medical records of dogs that underwent limb amputation or distal ulnectomy and adjuvant carboplatin treatment for appendicular osteosarcoma were reviewed. Adverse effects of chemotherapy and findings regarding preoperative biopsy specimens and postoperative diagnostic imaging were recorded. Signalment, clinical history, and chemotherapy variables were evaluated for associations with outcome. Histologic grade and other variables were evaluated for association with outcome for 38 tumors that were retrospectively graded.
The median disease-free interval was 137 days (95% confidence interval [CI], 112 to 177 days). Median survival time was 277 days (95% CI, 203 to 355 days). The 1-, 2-, and 3-year survival rates were 36%, 22%, and 19%, respectively. None of the chemotherapy variables were associated with outcome. Preoperative proteinuria was the only clinical variable associated with poor outcome. Histologic features of tumors associated with a poor outcome were intravascular invasion, mitotic index > 5 in 3 microscopic hpfs, and grade III classification.
Carboplatin administration was well tolerated and resulted in a disease-free interval and median survival time similar to those of other published protocols.
Sulforaphane [1-isothiocyanate-(4R)-(methylsulfinyl)butane] is a natural dietary isothiocyanate produced by the enzymatic action of the myrosinase on glucopharanin, a 4-methylsulfinylbutyl glucosinolate contained in cruciferous vegetables of the genus Brassica such as broccoli, brussel sprouts, and cabbage. Studies on this compound is increasing because its anticarcinogenic and cytoprotective properties in several in vivo experimental paradigms associated with oxidative stress such as focal cerebral ischemia, brain inflammation, intracerebral hemorrhage, ischemia and reperfusion induced acute renal failure, cisplatin induced-nephrotoxicity, streptozotocin-induced diabetes, carbon tetrachloride-induced hepatotoxicity and cardiac ischemia and reperfusion. This protective effect also has been observed in in vitro studies in different cell lines such as human neuroblastoma SH-SY5Y, renal epithelial proximal tubule LLC-PK1 cells and aortic smooth muscle A10 cells. Sulforaphane is considered an indirect antioxidant; this compound is able to induce many cytoprotective proteins, including antioxidant enzymes, through the Nrf2-antioxidant response element pathway. Heme oxygenase-1, NAD(P)H: quinone oxidoreductase, glutathione-S-transferase, gamma-glutamyl cysteine ligase, and glutathione reductase are among the cytoprotective proteins induced by sulforaphane. In conclusion, sulforaphane is a promising antioxidant agent that is effective to attenuate oxidative stress and tissue/cell damage in different in vivo and in vitro experimental paradigms.
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays critical roles in integrin-mediated signal transductions and also participates in signaling by other cell surface receptors. In integrin-mediated cell adhesion, FAK is activated via disruption of an auto-inhibitory intra-molecular interaction between its amino terminal FERM domain and the central kinase domain. The activated FAK forms a complex with Src family kinases, which initiates multiple downstream signaling pathways through phosphorylation of other proteins to regulate different cellular functions. Multiple downstream signaling pathways are identified to mediate FAK regulation of migration of various normal and cancer cells. Extensive studies in cultured cells as well as conditional FAK knockout mouse models indicated a critical role of FAK in angiogenesis during embryonic development and cancer progression. More recent studies also revealed kinase-independent functions for FAK in endothelial cells and fibroblasts. Consistent with its roles in cell migration and angiogenesis, increased expression and/or activation of FAK are found in a variety of human cancers. Therefore, small molecular inhibitors for FAK kinase activity as well as future development of novel therapies targeting the potentially kinase-independent functions of FAK are promising treatments for metastatic cancer as well as other diseases.
The anticancer activity of sulforaphane is known to be mediated at least partly by apoptosis induction and associated with the presence of the -N=C=S moiety. The present study explored how oxidation of sulphur in the side chain of sulforaphane affected apoptosis induction to provide the chemical basis of sulforaphane effects. Sulforaphane analogues containing oxidised sulphur (alyssin, sulforaphane, erysolin and alyssin sulfone) exerted a superior growth inhibitory effect compared with sulforaphane analogues with nonoxidised sulphur (erucin and berteroin) in human colon cancer cell lines. Furthermore, erysolin was a more potent inducer of reactive oxygen species (ROS) and apoptosis compared with erucin. Erysolin-induced ROS generation and subsequent apoptosis were inhibited by pretreatment with the antioxidant N-acetyl-cysteine. Erysolin induced caspase-8 activation, while blockade of caspsase 8 activation inhibited apoptosis induced by erysolin. Taken together, sulforaphane analogues with oxidised sulphur were the most efficient apoptosis inducers, likely due to high-level ROS induction.
This work was designed to further study the mechanism by which sulforaphane (SFN) exerts a renoprotective effect against cisplatin (CIS)-induced damage. It was evaluated whether SFN attenuates the CIS-induced mitochondrial alterations and the impairment in the activity of the cytoprotective enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1) and γ glutamyl cysteine ligase (γGCL). Studies were performed in renal epithelial LLC-PK1 cells and in isolated renal mitochondria from CIS, SFN or CIS+SFN treated rats. SFN effectively prevented the CIS-induced increase in reactive oxygen species (ROS) production and the decrease in NQO1 and γGCL activities and in glutathione (GSH) content. The protective effect of SFN on ROS production and cell viability was prevented by buthionine sulfoximine (BSO), an inhibitor of γGCL, and by dicoumarol, an inhibitor of NQO1. SFN was also able to prevent the CIS-induced mitochondrial alterations both in LLC-PK1 cells (loss of membrane potential) and in isolated mitochondria (inhibition of mitochondrial calcium uptake, release of cytochrome c, and decrease in GSH content, aconitase activity, adenosine triphosphate (ATP) content and oxygen consumption). It is concluded that the protection exerted by SFN on mitochondrial alterations and NQO1 and γGCL enzymes may be involved in the renoprotection of SFN against CIS.
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.
Development of cancer is a long-term and multistep process which comprises initiation, progression, and promotion stages of carcinogenesis. Conceivably, it can be targeted and interrupted along these different stages. In this context, many naturally occurring dietary compounds from our daily consumption of fruits and vegetables have been shown to possess cancer preventive effects. Phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) are two of the most widely investigated isothiocyanates from the crucifers. Both have been found to be very potent chemopreventive agents in numerous animal carcinogenesis models as well as cell culture models. They exert their chemopreventive effects through regulation of diverse molecular mechanisms. In this review, we will discuss the molecular targets of PEITC and SFN potentially involved in cancer chemoprevention. These include the regulation of drug-metabolizing enzymes phase I cytochrome P450s and phase II metabolizing enzymes. In addition, the signaling pathways including Nrf2-Keap 1, anti-inflammatory NFkappaB, apoptosis, and cell cycle arrest as well as some receptors will also be discussed. Furthermore, we will also discuss the similarities and their potential differences in the regulation of these molecular targets by PEITC and SFN.
Despite the reported cytotoxicity and apoptosis-inducing properties of sulforaphane (SF) in colon cancer cells, the details concerning individual mechanisms and signaling cascades underlying SF-mediated apoptosis remain unclear. To understand different aspects of SF-induced proapoptic signaling in advanced colon carcinoma, we investigated its mechanisms in metastatic SW620 cell line. Our results indicate that in SW620 cells SF acts to induce multivariate cascades including DNA-damage response pathway whose proapoptotic signaling is nevertheless reduced owing to the mutant status of p53 and caspase-2-JNK pathway which seems to complement and enhance p53-dependent signaling, however only in wild-type p53. Furthermore, both pathways require the active role of mitochondria and do not depend on generation of ROS, making SF an attractive chemopreventive agent whose antitumor properties should be further investigated in colon cancer.
The epidemiologic literature on the relationship between vegetable and fruit consumption and human cancer at a variety of sites was reviewed systematically in Part I. It was concluded that consumption of higher levels of vegetables and fruit is associated consistently, although not universally, with a reduced risk of cancer at most sites, and particularly with epithelial cancers of the alimentary and respiratory tracts. Possible mechanisms by which vegetable and fruit intake might alter risk of cancer are addressed here. A large number of potentially anticarcinogenic agents are found in these food sources, including carotenoids, vitamins C and E, selenium, dietary fiber, dithiolthiones, glucosinolates and indoles, isothiocyanates, flavonoids, phenols, protease inhibitors, plant sterols, allium compounds, and limonene. These agents have both complementary and overlapping mechanisms of action, including the induction of detoxification enzymes, inhibition of nitrosamine formation, provision of substrate for formation of antineoplastic agents, dilution and binding of carcinogens in the digestive tract, alteration of hormone metabolism, antioxidant effects, and others. It appears extremely unlikely that any one substance is responsible for all the associations seen. Possible adverse effects of vegetable and fruit consumption are also examined. One way to consider the relationships reviewed here is to hypothesize that humans are adapted to a high intake of plant foods that supply substances crucial to the maintenance of the organism, but only some of which are currently called 'essential nutrients.' Cancer may be the result of reducing the level of intake of foods that are metabolically necessary--it may be a disease of maladaptation.
The epidemiologic literature on the relationship between vegetable and fruit consumption and human cancer at a variety of sites is reviewed systematically. A total of 13 ecologic studies, nine cohort studies, and 115 case-control studies are included. Cancer of all sites, cancers of lung, breast, colon, rectum, esophagus, larynx, oral cavity and pharynx, stomach, pancreas, prostate, bladder, ovary, endometrium, cervix, and thyroid, as well as mesothelioma and gestational trophoblastic disease, are considered. Relevant data from clinical trials, animal, and in vitro studies are included. It is concluded that consumption of higher levels of vegetables and fruit is associated consistently, although not universally, with a reduced risk of cancer at most sites. The association is most marked for epithelial cancers--particularly those of the alimentary and respiratory tracts--and, currently, is weak to nonexistent for hormone-related cancers. The association exists for a wide variety of vegetables and fruit with some suggestion that raw forms are associated most consistently with lower risk. Possible mechanisms by which vegetable and fruit intake might alter risk of cancer and possible adverse effects of vegetable and fruit consumption will be considered in Part II of this review.
Organic isothiocyanates block the production of tumors induced in rodents by diverse carcinogens (polycyclic aromatic hydrocarbons, azo dyes, ethionine, N-2-fluorenylacetamide, and nitrosamines). Protection is afforded by alpha-naphthyl-, beta-naphthyl-, phenyl-, benzyl-, phenethyl-, and other arylalkyl isothiocyanates against tumor development in liver, lung, mammary gland, forestomach, and esophagus. Many isothiocyanates and their glucosinolate precursors (beta-thioglucoside, N-hydroxysulfate) occur naturally and sometimes abundantly in plants consumed by humans, e.g., cruciferous vegetables. Nevertheless, the possible contributions of isothiocyanates and glucosinolates to the well recognized protective effects against cancer of high consumptions of vegetables are unclear. The anticarcinogenic effects of isothiocyanates appear to be mediated by tandem and cooperating mechanisms: (a) suppression of carcinogen activation by cytochromes P-450, probably by a combination of down-regulation of enzyme levels and direct inhibition of their catalytic activities, which thereby lower the levels of ultimate carcinogens formed; and (b) induction of Phase 2 enzymes such as glutathione transferases and NAD(P)H: quinone reductase, which detoxify any residual electrophilic metabolites generated by Phase 1 enzymes and thereby destroy their ability to damage DNA. Since isothiocyanates block carcinogenesis by dual mechanisms and are already present in substantial quantities in human diets, these agents are ideal candidates for the development of effective chemoprotection of humans against cancer.
Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl)butane] was recently isolated from one variety of broccoli as the major and very potent inducer of phase 2 detoxication enzymes in murine hepatoma cells in culture. Since phase 2 enzyme induction is often associated with reduced susceptibility of animals and their cells to the toxic and neoplastic effects of carcinogens and other electrophiles, it was important to establish whether sulforaphane could block chemical carcinogenesis. In this paper we report that sulforaphane and three synthetic analogues, designed as potent phase 2 enzyme inducers, block the formation of mammary tumors in Sprague-Dawley rats treated with single doses of 9,10-dimethyl-1,2-benzanthracene. The analogues are exo-2-acetyl-exo-6-isothiocyanatonorbornane, endo-2-acetyl-exo-6-isothiocyanatonorbornane, and exo-2-acetyl-exo-5-isothiocyanatonorbornane. When sulforaphane and exo-2-acetyl-exo-6-isothiocyanatonorbornane were administered by gavage (75 or 150 mumol per day for 5 days) around the time of exposure to the carcinogen, the incidence, multiplicity, and weight of mammary tumors were significantly reduced, and their development was delayed. The analogues endo-2-acetyl-exo-6-isothiocyanatonorbornane and exo-2-acetyl-exo-5-isothiocyanatonorbornane were less potent protectors. Thus, a class of functionalized isothiocyanates with anticarcinogenic properties has been identified. These results validate the thesis that inducers of phase 2 enzymes in cultured cells are likely to protect against carcinogenesis.
Forty-eight dogs with histologically confirmed appendicular osteosarcoma (OSA) entered a prospective clinical trial evaluating treatment with amputation and up to 4 doses of carboplatin given every 21 days. The median disease-free interval (DFI) was 257 days, with 31.2% of the dogs disease-free at 1 year. The median survival time was 321 days, with 35.4% of the dogs alive at 1 year. Dogs with proximal humeral OSA had shorter DFI (P = .016) and survival (P = .037) times than dogs with OSA at other locations. Dogs with lower body weights ( < 40 kg) had longer DFI (P = .0056) and survival (P = .007) times than larger dogs. Survival times for dogs that received carboplatin were statistically longer than those previously reported for amputation alone (P < .001). DFI and survival times are similar to those previously reported for 2 to 4 doses of cisplatin. Carboplatin appears to be a well-tolerated chemotherapeutic drug that can be given safely every 21 days at a dose of 300 mg/m2. Neutropenia was the dose-limiting toxicity in this study.
The relation between the consumption of cruciferous vegetables and reduced prostate cancer occurrence has been documented, although the responsible phytochemicals are unknown. The effects of sulforaphane (SFN) which occurs as the precursor glucosinolate in broccoli and other cruciferous vegetables, and its metabolite N-acetylcysteine conjugate (SFN-NAC) on prostate cancer cells were investigated. SFN and SFN-NAC were analyzed with the androgen-dependent human prostate cancer LNCaP cell line model. Cell growth and apoptosis were determined with the expression of androgen receptor and prostate specific antigen, DNA synthesis, cell cycle progression, DNA strand breaks and caspase activation to ascertain the effects and mechanism. SFN and SFN-NAC were demonstrated for the first time to mediate a dose-dependent apoptosis and growth arrest in the prostate cancer cells. Caspases were activated and DNA strand breaks were detected in apoptotic cells. The expression of phosphorylated and dephosphorylated androgen receptors, and the production of prostate specific antigen were attenuated. The expression of cyclin D1 and DNA synthesis were inhibited along with G1 cell cycle block, causing decreased cell density and growth. SFN and its metabolite SFN-NAC have similar activities to induce growth arrest and apoptosis, indicating that the effects of SFN are maintained through the metabolic processes. SFN as a dietary component of cruciferous vegetables active in the prevention of prostate cancer is discussed.
Death receptor- or mitochondrion-dependent apoptosis is initiated by the recruitment and activation of apical caspases in the apoptosis signaling pathways. In death receptor-mediated apoptosis, engagement of death receptors leads to the formation of the death-inducing signaling complex (DISC) containing the death receptors, adaptor proteins, caspase-8 and caspase-10. In mitochondrion-dependent apoptosis, release of cytochrome C into the cytosol results in the formation of apoptosome containing cytochrome C, Apaf-1 and caspase-9. Caspase-8, caspase-10 and caspase-9 are believed to be the initiator caspases at the top of the caspase signaling cascade. Recruitment of caspases to DISC and apoptosome leads to their activation by dimer formation. Recent biochemical and structural analyses of components in the DISC and apoptosome shed new lights on their roles in inducing the onset of apoptosis signaling.
Diets rich in fruit and vegetables have been recommended for preventing cancer. The evidence supporting this recommendation is based on observational studies, although results of several prospective studies have cast some doubts on whether fruit and vegetables are associated with cancer risk reduction.
We sought to summarize evidence from case-control and prospective studies on fruit and vegetable intake and cancer risk with a meta-analytic approach.
Published case-control and cohort studies that reported on total vegetable and fruit intake and risk of cancer of several sites were included. Relative risks were estimated by using linear logistic regression models.
Case-control studies overall support a significant reduction in the risks of cancers of the esophagus, lung, stomach, and colorectum associated with both fruit and vegetables; breast cancer is associated with vegetables but not with fruit; and bladder cancer is associated with fruit but not with vegetables. The overall relative risk estimates from cohort studies suggest a protective effect of both fruit and vegetables for most cancer sites considered, but the risk reduction is significant only for cancers of the lung and bladder and only for fruit.
Prospective studies provide weaker evidence than do case-control studies of the association of fruit and vegetable consumption with reduced cancer risk. The discrepancies may be related to recall and selection biases in case-control studies. In contrast, the association may have been underestimated in prospective studies because of the combined effects of imprecise dietary measurements and limited variability of dietary intakes within each cohort.
There is increasing evidence that a variety of natural substances derived from the diet may act as potent chemopreventive agents. In this work, we show that DAOY cells, a widely used model of metastatic medulloblastoma (MBL), are highly sensitive to sulforaphane, a naturally occurring isothiocyanate from Brassica vegetables. Sulforaphane induced DAOY cell death by apoptosis, as determined by DNA fragmentation and chromatin condensation. DAOY apoptosis correlates with the induction of caspase-3 and -9 activities, resulting in the cleavage of PARP and vimentin. Both the cytotoxic effect and apoptotic characteristics induced by sulforaphane were reversed by zVAD-fmk, a broad spectrum caspase inhibitor, demonstrating the important role of caspases in its cytotoxic effect. These results identify sulforaphane as a novel inducer of MBL cell apoptosis, supporting the potential clinical usefulness of diet-derived substances as chemopreventive agents.
Cancer chemoprevention is a new approach in the management of cancer. Traditional cytotoxic chemotherapeutic approaches cannot cure most advanced solid malignancies. Chemoprevention can be defined as the use of non-cytotoxic drugs and natural agents to block the progression to invasive cancer. Recently, isothiocyanates, natural products found in the diet of humans, has been shown to function as cancer chemopreventive agents. They are strong inhibitors of phase I enzymes and inducers of phase II enzymes. They can also induce apoptosis and modulate cell-cycle progression of highly proliferating cancer cells. This commentary will review the mechanism of apoptosis and growth inhibition mediated by different isothiocyanates. Particular attention will be given to the effects of the new isothiocyanate 4-(methylthio)butylisothiocyanate (MTBITC). Since selective targeting and low toxicity for normal host tissues are fundamental requisites for proposed chemopreventive agents, we will also review the effects of different isothiocyanates on non-transformed human cells.
Anticancer effects of the dietary isothiocyanate sulforaphane were investigated in the human pancreatic cancer cell lines MIA PaCa-2 and PANC-1. Sulforaphane-treated cells accumulated in metaphase as determined by flow cytometry [4C DNA content, cyclin A(-), cyclin B1(+), and phospho-histone H3 (Ser(10))(+)]. In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-8, loss of mitochondrial membrane potential, and loss of plasma membrane integrity. The initial detection of caspase-3 cleavage occurring in G(2)-M arrest was independent of a change in phospho-cdc2 (Tyr(15)) protein; consequently, sulforaphane treatment combined with UCN-01 had no significant impact on cellular toxicity. Incubations at higher sulforaphane doses (>10 micromol/L) resulted in cleavage of caspase-3 in the G(1) subpopulation, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated. Cellular toxicity in MIA PaCa-2, and to a greater extent in PANC-1, was positively correlated with a decrease in cellular glutathione levels, whereas sustained increases in glutathione observed in MIA PaCa-2 cells or the simultaneous incubation with N-acetyl-L-cysteine in PANC-1 cells were associated with resistance to sulforaphane-induced apoptosis. Daily sulforaphane i.p. injections (375 micromol/kg/d for 3 weeks) in severe combined immunodeficient mice with PANC-1 s.c. tumors resulted in a decrease of mean tumor volume by 40% compared with vehicle-treated controls. Our findings suggest that, in addition to the known effects on cancer prevention, sulforaphane may have activity in established pancreatic cancer.
A central question in cell biology is how membrane-spanning receptors transmit extracellular signals inside cells to modulate cell adhesion and motility. Focal adhesion kinase (FAK) is a crucial signalling component that is activated by numerous stimuli and functions as a biosensor or integrator to control cell motility. Through multifaceted and diverse molecular connections, FAK can influence the cytoskeleton, structures of cell adhesion sites and membrane protrusions to regulate cell movement.
Isothiocyanate sulforaphane (SFN) is a potent cancer chemopreventive agent. We investigated the mechanisms underlying the anti-proliferative effects of SFN in the human colon carcinoma cell line, HT-29. We demonstrate that SFN inhibits the growth of HT-29 cells in a dose- and time-dependent manner. Treatment of serum-stimulated HT-29 cells with SFN suppressed the re-initiation of cell cycle by inducing a G(1) phase cell cycle arrest. At high doses (>25 microM), SFN dramatically induces the expression of p21(CIP1) while significantly inhibits the expression of the G(1) phase cell cycle regulatory genes such as cyclin D1, cyclin A, and c-myc. This regulation can be detected at both the mRNA and protein levels as early as 4 h post-treatment of SFN at 50 microM. Additionally, SFN activates MAPKs pathways, including ERK, JNK and p38. Exposure of HT-29 cells with both SFN and an antioxidant, either NAC or GSH, completely blocked the SFN-mediated activation of these MAPK signaling cascades, regulation of cyclin D1and p21(CIP1) gene expression, and G(1)phase cell cycle arrest. This finding suggests that SFN-induced oxidative stress plays a role in these observed effects. Furthermore, the activation of the ERK and p38 pathways by SFN is involved in the upregulation of p21(CIP1) and cyclin D1, whereas the activation of the JNK pathway plays a contradictory role and may be partially involved in the downregulation of cyclin D1. Because cyclin D1 and p21(CIP1) play opposing roles in G(1) phase cell cycle progression regulation, blocking the activation of each MAPK pathway with specific MAPK inhibitors, is unable to rescue the SFN-induced G(1) phase cell cycle arrest in HT-29 cells.