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Effects of SFN on the levels of the proteins involved in the regulation of G2/M transition. (A) immunoblotting for p21, cyclin B and cyclin A using lysates from control and SFN-treated KB cells. (B) The data points in the graph are the mean ± SD of three independent experiments. *p<0.05 compared to the control group. (C) Transactivation activity of pWWP (p21 promoter construct) induced by SFN in KB cells. (D) immunobloting for sp1 protein using lysates from control and SFN-treated KB cells.
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Previously, our group reported that sulforaphane (SFN), a naturally occurring chemopreventive agent from cruciferous vegetables, effectively inhibits the proliferation of KB and YD-10B human oral squamous carcinoma cells by causing apoptosis. In this study, treatment of 20 and 40 microM of SFN for 12 h caused a cell cycle arrest in the G(2)/M phase...
Contexts in source publication
Context 1
... of the cells were arrested at the G2/M phase, experiments were carried out to determine whether the SFN treatment also affects the expression level of G2/M phase cell cycle regulatory proteins. KB cells were treated with 20 μM of SFN for 12 h and the expression of these proteins (p21, cyclin A and cyclin B) were examined by western blot analysis. Fig. 3A and B showed a significant increase in the level of p21 protein expression and the level of cyclin B expression was significantly reduced by SFN whereas cyclin A was not changed. Furthermore, SFN induced the transactivation in KB cells transfected with pWWP (Fig. 3C). To find whether sp1 protein regulates p21 protein expression, we ...Context 2
... proteins (p21, cyclin A and cyclin B) were examined by western blot analysis. Fig. 3A and B showed a significant increase in the level of p21 protein expression and the level of cyclin B expression was significantly reduced by SFN whereas cyclin A was not changed. Furthermore, SFN induced the transactivation in KB cells transfected with pWWP (Fig. 3C). To find whether sp1 protein regulates p21 protein expression, we evaluated the level of sp1 protein expression. Unfortunately, sp1 protein was not changed by SFN compared to control (Fig. 3D). These results suggest that SFN modulates the expression level of the G2/M phase cell cycle regulatory proteins and its transactivation in KB ...Context 3
... was significantly reduced by SFN whereas cyclin A was not changed. Furthermore, SFN induced the transactivation in KB cells transfected with pWWP (Fig. 3C). To find whether sp1 protein regulates p21 protein expression, we evaluated the level of sp1 protein expression. Unfortunately, sp1 protein was not changed by SFN compared to control (Fig. 3D). These results suggest that SFN modulates the expression level of the G2/M phase cell cycle regulatory proteins and its transactivation in KB oral carcinoma ...Context 4
... molecule causes a number of different cell lines to arrest in both G1 and G2 checkpoint. SFN was reported to induce G2/M arrest in a variety of cancer cell lines and p21 expression is associated with G2/M phase cell cycle arrest [31,35,36]. Our study showed that SFN induces the p21 expression and its trans- activation to induce G2/M phase arrest (Fig. 3). There results are fully consistent with other studies. We also previously reported that SFN decreased tumor growth in athymic nude mice bearing KB cells as xenograft. Fig. 4 shows that p21 was also induced in the tumor sections from the same animals demonstrating comparable induction of this pro- apoptotic protein in both in vivo and ...Context 5
... GC-rich or GT-rich DNA and regulates the promoters of several genes [37,38]. The human p21 gene contains six Sp1 binding sites [39]. Due to close relationship between Sp1 and p21, we evaluated Sp1 protein expression level in this system. Unfortunately, Sp1 protein was not affected by SFN indi- cating that Sp1 is not an upstream protein of p21 (Fig. 3D). Of several CDK inhibitors, p21 is an important mediator of cell cycle arrest regulated by tumor suppressor p53 in response to DNA damage [40]. In addition to being induced by p53, p21 is also induced by other factors in a p53- independent pathway [41,42]. Recently, Traka et al. (2009) reported that the induction of p21 protein by SFN ...Citations
... This implied differential action by SFN: low concentrations causing cell cycle arrest at G2/M and high concentrations hindering cell division itself and presenting with increased sub G1 phase cells. Similarly, G2/M phase arrest has been observed in other oral cancer cell lines also (KB and YD-10 B). 11 This pattern was cited in bladder cancer cell lines too: 15 mmol/L causing G2/M arrest and 30 mmol/L disrupting cell division altogether. 20 Prostate cancer cell lines (PC-3) also demonstrated this trend: G2/M arrest (25%e38%) at 20 mM SFN and increase of sub G1 cell population (4%e24%) at 40 mM. ...
Background: Biologic compounds have recently generated interest in cancer chemoprevention.
Sulforaphane (SFN), an isothiocyanate from cruciferous vegetables, has profound
epigenetic actions. Since epigenetic aetiology is crucial for oral cancer, this study evaluated
the role of SFN in oral cancer prevention.
Methods: Oral squamous cell carcinoma cells (UPCI-SCC-172) were treated with SFN in three
concentrations: 10 mM, 20 mM and 30 mM for two time periods: 24 h and 48 h. MTT assay
assessed cell proliferation. Histone deacetylase (HDAC) enzyme activity was colorimetrically
estimated in the nuclear extracts. Flow cytometry determined cell cycle stages, reactive
oxygen species (ROS) generation and mitochondrial membrane potential (MMP) changes.
Extrinsic and intrinsic apoptotic pathways were evaluated from caspase enzyme assays.
Results: Cell proliferation and HDAC activity (44% in 24 h and 40% in 48 h) were significantly
inhibited (p < 0.01). For 10 mM concentration, G2/M cell cycle arrest was found with a
reduction in G1 phase cell population at 24 h and 48 h. Concentrations of 20 mMand 30 mMSFN
presented cells in apoptosis marked by increased sub G1 cells at 48 h. Concentrations of
10 mMand 20 mMSFN showed a 1.3 to 2.8-fold increase in ROS generation at 24 h and 48 h. The
concentration of 30 mM SFN showed a drop in ROS production, denoting cells already in
apoptosis. Fall in MMP was also dose- and time-dependent. Caspase enzyme assays
(p < 0.001) demonstrated activation of both extrinsic and intrinsic apoptotic pathways.
Conclusion: Inhibitory action of SFN on oral cancer cell proliferation and HDAC activity
led to cell cycle arrest and apoptosis. These effects marked by increase in ROS, a decrease in MMP and activation of apoptotic pathways offer exciting therapeutic
options.
... To exemplify, more than 50 CTs have investigated the beneficial properties of broccoliderived molecules (mainly sulforaphane and glucoraphanin) in cancer disease remission [102,103]. Nevertheless, only a few of them have brought into focus the molecular mechanisms involving the downregulation of cell cycle-related proteins such as cyclins and CDKs [104], or the induction of CDKIs and correlated pathways involving signaling cascades such as the mammalian target of rapamycin (mTOR) [105] or STAT 3 ( Table 2, [106]). Indole-3-carbinol (I3C) represents an additional natural anticancer agent belonging to the same broccoli vegetable family (Brassicaceae). ...
Simple Summary
This review aims to provide an outline of the potential use of plant-based foods, nutraceuticals, and derived micronutrients—particularly those typically found in the Mediterranean diet—as anticancer agents, with a focus on their mechanism of action as cyclin-dependent kinase inhibitors (CDKIs) by inactivating the CDK 4/6 pathway and the regulation of the cell-cycle cascade. We discuss the preclinical and pharmacological significance of some already approved CDK blockers as a promising therapeutic approach against breast and ovarian cancers.
Abstract
Overweight and obesity constitute the most impactful lifestyle-dependent risk factors for cancer and have been tightly linked to a higher number of tumor-related deaths nowadays. The excessive accumulation of energy can lead to an imbalance in the level of essential cellular biomolecules that may result in inflammation and cell-cycle dysregulation. Nutritional strategies and phytochemicals are gaining interest in the management of obesity-related cancers, with several ongoing and completed clinical studies that support their effectiveness. At the same time, cyclin-dependent kinases (CDKs) are becoming an important target in breast and ovarian cancer treatment, with various FDA-approved CDK4/6 inhibitors that have recently received more attention for their potential role in diet-induced obesity (DIO). Here we provide an overview of the most recent studies involving nutraceuticals and other dietary strategies affecting cell-cycle pathways, which might impact the management of breast and ovarian cancers, as well as the repurposing of already commercialized chemotherapeutic options to treat DIO.
... Several research groups have reported that SF can arrest cancer cells at G1 phase [83,84] , S phase [85] , or G2/M phase [86][87][88] in different cell lines. The main cell cycle phase when SF interfere is G2/M as this cell cycle arrest has been reported in PC-3 and DU-145 human prostate cancer cells [89,90] , HCT-116, HT29, and Caco-2 human colon cancer cells [89,91,92] , MCF-7 human breast cancer cells [86] , U2-OS human osteosarcoma cells [93] , KB and YD-10B human OSCC [94] , even in non-solid tumors as acute lymphoblastic leukemia (ALL) [95] and in primary myeloma tumor cells [96] . ...
... Another suggested mechanism is inhibition of tubulin polymerization, resulting in mitotic arrest, as was reported after SF treatment results in lack of equatorial metaphase alignment of condensed chromosomes in MCF-7 cells [86] , and aberrant and absent mitotic microtubules in F311 [97] and MCF-7 cells [86] . In KB and YD-10B human OSCC cells, the reported mechanism was the induction of p21, a potent cyclin-dependent kinase inhibitor, which led to a decrease in the cyclin B levels [94] . In all, similar mechanism was reported with upregulation of p21 and inhibition of the Cdc2/ Cyclin B1 complex [95] . ...
Tumorigenicity-inhibiting compounds have been identified in our daily diet. For example, isothiocyanates (ITCs) found in cruciferous vegetables were reported to have potent cancer-prevention activities. The best characterized ITC is sulforaphane (SF). SF can simultaneously modulate multiple cellular targets involved in carcinogenesis, including (1) modulating carcinogen-metabolizing enzymes and blocking the action of mutagens; (2) inhibition of cell proliferation and induction of apoptosis; and (3) inhibition of neo-angiogenesis and metastasis. SF targets cancer stem cells through modulation of nuclear factor kappa B (NF-κB), Sonic hedgehog (SHH), epithelial-mesenchymal transition, and Wnt/β-catenin pathways. Conventional chemotherapy/SF combination was tested in several studies and resulted in favorable outcomes. With its favorable toxicological profile, SF is a promising agent in cancer prevention and/or therapy. In this article, we discuss the human metabolism of SF and its effects on cancer prevention, treatment, and targeting cancer stem cells, as well as providing a brief review of recent human clinical trials on SF.
... The redox stress-sensitive, Nrf2-antioxidant response element (ARE)-mediated antioxidant pathway is the major cellular defense against several internal and external environmental stresses. Oxidative stress and many compounds with electrophilic properties, such as heavy metals, tBOOH and naturally occurring phytochemicals such as SFN, have been reported to yield therapeutic benefits by modulating Nrf2-ARE pathway [1,27,67,[94][95][96][97][98]. At physiological conditions, Nrf2 is controlled via Keap1, an adopter protein for CULLI3-based ubiquitin E3 ligase that ubiquitinates Nrf2 for proteasomal degradation. ...
Sulforaphane (SFN), an activator of transcription factor Nrf2 (NFE2-related factor), modulates antioxidant defense by Nrf2-mediated regulation of antioxidant genes like Peroxiredoxin 6 (Prdx6) and affects cellular homeostasis. We previously observed that dose levels of SFN are crucial in determining life or death of lens epithelial cells (LECs). Herein, we demonstrated that higher doses of SFN (>6 μM) activated death signaling by overstimulation of Nrf2/ARE (antioxidant response element)-mediated Kruppel-like factor (Klf9) repression of Prdx6 expression, which increased reactive oxygen species (ROS) load and cell death. Mechanistically, Klf9 bound to its repressive Klf9 binding elements (RKBE; 5-CA/GCCC-3) in the Prdx6 promoter, and repressed Prdx6 transcription. Under the condition of higher dose of SFN, excessive Nrf2 abundance caused death signaling by enforcing Klf9 activation through ARE (5-RTGAYnnnGC-3) in Klf9 promoter that suppress antioxidant genes such as Prdx6 via a Klf9-dependent fashion. Klf9-depletion showed that Klf9 independently caused ROS reduction and subsequent cell survival, demonstrating that Klf9 upregulation caused cell death. Our work revealed the molecular mechanism of dose-dependent altered activity of SFN in LECs, and demonstrated that SFN activity was linked to levels of Nrf2/Klf9/Prdx6 axis. We proposed that in the development of therapeutic interventions for aging/oxidative disorders, combinations of Klf9-ShRNA and Nrf2 inducers may prove to be a promising strategy.
... In this work, treatment of HT29 colon cancer cells with an inhibitor specific for p38 activity attenuated the PEITC-induced (25 μM) G1 arrest, while MAPK activation induced the activity of various other cyclins and, consequently, blocked growth progression [100]. Other studies have also shown that ITCs induced cell cycle arrest in various cancer cell lines, an effect associated mainly with the regulation of the expression levels of p21, GADD45 (growth arrest and DNA damage) and various other cyclins [101][102][103][104]. Finally, only recently, it was proposed that the deregulation of the cell cycle may gradually lead to apoptotic induction [93]. ...
Many studies have shown evidence in support of the beneficial effects of phytochemicals in preventing chronic diseases, including cancer. Among such phytochemicals, sulphur-containing compounds (e.g., isothiocyanates (ITCs)) have raised scientific interest by exerting unique chemo-preventive properties against cancer pathogenesis. ITCs are the major biologically active compounds capable of mediating the anticancer effect of cruciferous vegetables. Recently, many studies have shown that a higher intake of cruciferous vegetables is associated with reduced risk of developing various forms of cancers primarily due to a plurality of effects, including (i) metabolic activation and detoxification, (ii) inflammation, (iii) angiogenesis, (iv) metastasis and (v) regulation of the epigenetic machinery. In the context of human malignant melanoma, a number of studies suggest that ITCs can cause cell cycle growth arrest and also induce apoptosis in human malignant melanoma cells. On such basis, ITCs could serve as promising chemo-therapeutic agents that could be used in the clinical setting to potentiate the efficacy of existing therapies.
... Numerous studies reported the anti-neoplastic effect of SF against solid tumors such as breast tumors, hepatic tumors, brain tumors, pancreatic tumors, prostate tumors, and skin tumors [13]. Recently, it was shown that SF has comparable cytotoxic effects on the squamous cell carcinoma of the head and neck [33][34][35]. Our results showed that SF decreased the SCCHN cell lines viability through increasing treatment dosage and duration. ...
The efficacy of cisplatin (CIS) and 5-fluorouracil (5-FU) against squamous cell carcinomas of the head and neck (SCCHN) remains restricted due to their severe toxic side effects on non-cancer (normal) tissues. Recently, the broccoli extract sulforaphane (SF) was successfully tested as a combination therapy to target cancer cells. However, the effect of lower doses of CIS or 5-FU combined with SF on SCCHN remained unknown. This study tested the chemotherapeutic efficacies of SF combined with much lower doses of CIS or 5-FU against SCCHN cells aiming to reduce cytotoxicity to normal cells. Titrations of SF standalone or in combination with CIS and 5-FU were tested on SCCHN human cell lines (SCC12 and SCC38) and non-cancerous human cells (fibroblasts, gingival, and salivary cells). Concentrations of SF tested were comparable to those found in the plasma following ingestion of fresh broccoli sprouts. The treatment effects on cell viability, proliferation, DNA damage, apoptosis, and gene expression were measured. SF reduced SCCHN cell viability in a time- and dose-dependent manner. SF-combined treatment increased the cytotoxic activity of CIS by twofolds and of 5-FU by tenfolds against SCCHN, with no effect on non-cancerous cells. SF-combined treatment inhibited SCCHN cell clonogenicity and post-treatment DNA repair. SF increased SCCHN apoptosis and this mechanism was due to a down-regulation of BCL2 and up-regulation of BAX, leading to an up-regulation of Caspase3. In conclusion, combining SF with low doses of CIS or 5-FU increased cytotoxicity against SCCHN cells, while having minimal effects on normal cells.
... It has also been reported to be down-regulated in osteosarcoma [26] and colon carcinoma cells [27]. However, down-regulation has not been reported in oral carcinoma cells [28]. The cell cycle regulator, p19, was up-regulated in both Caki-1 par and Caki-1 res by SFN. ...
Although the mechanistic target of rapamycin (mTOR) inhibitor, everolimus, has improved the outcome of patients with renal cell carcinoma (RCC), improvement is temporary due to the development of drug resistance. Since many patients encountering resistance turn to alternative/complementary treatment options, an investigation was initiated to evaluate whether the natural compound, sulforaphane (SFN), influences growth and invasive activity of everolimus-resistant (RCCres) compared to everolimus-sensitive (RCCpar) RCC cell lines in vitro. RCC cells were exposed to different concentrations of SFN and cell growth, cell proliferation, apoptosis, cell cycle, cell cycle regulating proteins, the mTOR-akt signaling axis, adhesion to human vascular endothelium and immobilized collagen, chemotactic activity, and influence on surface integrin receptor expression were investigated. SFN caused a significant reduction in both RCCres and RCCpar cell growth and proliferation, which correlated with an elevation in G2/M- and S-phase cells. SFN induced a marked decrease in the cell cycle activating proteins cdk1 and cyclin B and siRNA knock-down of cdk1 and cyclin B resulted in significantly diminished RCC cell growth. SFN also modulated adhesion and chemotaxis, which was associated with reduced expression of the integrin subtypes α5, α6, and β4. Distinct differences were seen in RCCres adhesion and chemotaxis (diminished by SFN) and RCCpar adhesion (enhanced by SFN) and chemotaxis (not influenced by SFN). Functional blocking of integrin subtypes demonstrated divergent action on RCC binding and invasion, depending on RCC cell sensitivity to everolimus. Therefore, SFN administration could hold potential for treating RCC patients with established resistance towards everolimus.
... Resveratrol and sulforaphane are two examples of natural compounds that have attracted attention for their selective toxicity to cancer cells and for their ability to sensitize cancer cells to other therapies (7,8). Both Resveratrol and sulforaphane have been shown to inhibit growth and induce apoptosis in tumor cells as well as in tumor animal models (9)(10)(11)(12). Sulforaphane is in clinical trial phase II treating patients with recurrent prostate cancer (13). ...
... The latter types of cancer cells cannot survive, when there is not enough extra glutamine and are therefore said to be "addicted" to glutamine (17). Both resveratrol and sulphoraphane have been shown to act by means of the PI3K signaling pathway (12,13,18). PI3K occupies a central role in several cellular processes critical for cancer progression, including metabolism, growth, survival, and motility. ...
Glutamine metabolism is, with its many links to oncogene expression, considered a crucial step in cancer metabolism and it is thereby a key target for alteration in cancer development. In particular, strong correlations have been reported between oncogene expression and expression and activity of the enzyme glutaminase. This mitochondrial enzyme, which is responsible for the deamidation of glutamine to form glutamate, is overexpressed in many tumour tissues. In animal models, glutaminase expression is correlated with tumour growth rate and it is readily possible to limit tumour growth by suppression of glutaminase activity. In principle, hyperpolarized (13)C MR spectroscopy can provide insight to glutamine metabolism and should hence be a valuable tool to study changes in glutaminase activity as tumours progress. However, no such successful in vivo studies have been reported, even though several good biological models have been tested. This may, at least partly, be due to problems in preparing glutamine for hyperpolarization. This paper reports a new and improved preparation of hyperpolarized [5-(13)C]glutamine, which provides a highly sensitive (13)C MR marker. With this preparation of hyperpolarized [5-(13)C]glutamine, glutaminase activity in vivo in a rat liver tumour was investigated. Moreover, this marker was also used to measure response to drug treatment in vitro in cancer cells. These examples of [5-(13)C]glutamine used in tumour models warrant the new preparation to allow metabolic studies with this conditionally essential amino acid.
... In the present study, SFN inhibited the growth of articular chondrocytes, as determined by trypan blue exclusion assay (Fig. 2B). Various checkpoint mechanisms function to regulate the cell cycle, ensuring appropriate cellular response to external stresses, including abnormal mitogenic signaling (14). ...
Sulforaphane (SFN), a natural compound extracted from cruciferous vegetables, exhibits potent anticancer activity in various types of tumor cells. However, the effect of SFN on the proliferation of chondrocytes is not well understood. In the present study, we addressed the mechanism of action by which SFN suppresses proliferation. We demonstrate that SFN causes an irreversible arrest in cell proliferation, as determined by trypan blue dye exclusion assay and flow cytometric analysis. SFN induced cell cycle arrest at the G2/M phase by downregulation of cyclin B, cdc2 and cdc25c and upregulation of p21WAF1/CIP1 and p53 in a dose- and time-dependent manner, as determined by western blot analysis. Our data suggest that SFN regulates cell cycle arrest at the G2/M phase in rabbit articular chondrocytes.
... Elucidation of the mechanism underlying cancer chemopreventive response to SFN has been the topic of intense research over the past decade. Seminal contributions from colleagues around the world concerning mechanisms of cancer chemoprevention by SFN include: inhibition of CYP2E1 [Barcelo et al., 1996], cell cycle arrest [Gamet-Payrastre et al., 2000; Singh et al., 2004a; Kim et al., 2010], apoptosis induction [Gamet-Payrastre et al., 2000; Singh et al., 2004b; Singh et al., 2005], inhibition of angiogenesis [Bertl et al., 2006] induction of autophagy as a protective mechanism against apoptotic cell death [HermanAntosiewicz et al., 2006], inhibition of histone deacetylase [Myzak et al., 2004], protein binding [Mi et al., 2007], induction of phase 2 enzymes [Li et al., 2006; Kensler and Wakabayashi, 2010], epigenetic repression of hTERT [Meeran et al., 2010], and inhibition of breast cancer stem cells [Li et al., 2010]. Mechanism by which SFN causes cell cycle arrest and apoptotic cell death continues to expand. ...
... For example, SFN-induced G2/M phase cell cycle arrest in human prostate cancer cells was mediated by checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C [Singh et al., 2004a]. In KB and YD-10B human oral squamous carcinoma cells, G2/M phase cell cycle arrest resulting from SFN exposure was associated with a significant increase in the p21 protein level [Kim et al., 2010]. Furthermore, SFN treatment increased the p21 promoter activity and resulted in induction of p21 expression in tumor xenograft in vivo [Kim et al., 2010]. ...
... In KB and YD-10B human oral squamous carcinoma cells, G2/M phase cell cycle arrest resulting from SFN exposure was associated with a significant increase in the p21 protein level [Kim et al., 2010]. Furthermore, SFN treatment increased the p21 promoter activity and resulted in induction of p21 expression in tumor xenograft in vivo [Kim et al., 2010]. SFN-mediated suppression of many cellular pathways implicated in apoptosis control have also been described, including nuclear factor-κB, Akt, anti-apoptotic proteins (Bcl-2, Bcl-xL), and signal transducer and activator of transcription 3 [Xu et al., 2005; Singh et al., 2005; Hahm and Singh, 2010]. ...
Cancer chemopreventive response to D,L-sulforaphane (SFN), a synthetic racemic analogue of broccoli constituent L-sulforaphane, is partly attributable to apoptosis induction, but the mechanism of cell death is not fully understood. The present study demonstrates a critical role for adapter protein p66(Shc) in SFN-induced apoptosis. Immortalized mouse embryonic fibroblasts (MEF) derived from p66(shc) knockout mice were significantly more resistant to SFN-induced apoptosis, collapse of mitochondrial membrane potential, and reactive oxygen species (ROS) production compared with MEF obtained from the wild-type mice. Notably, a spontaneously immortalized and non-tumorigenic human mammary epithelial cell line (MCF-10A) was resistant to SFN-induced ROS production and apoptosis. Stable overexpression of manganese superoxide dismutase in MCF-7 and MDA-MB-231 human breast cancer cells conferred near complete protection against SFN-induced apoptosis and mitochondrial membrane potential collapse. SFN treatment resulted in increased S36 phosphorylation and mitochondrial translocation of p66(shc) in MDA-MB-231 and MCF-7 cells, and SFN-induced apoptosis was significantly attenuated by RNA interference of p66(shc) in both cells. SFN-treated MDA-MB-231 and MCF-7 cells also exhibited a marked decrease in protein level of peptidyl prolyl isomerase (Pin1), which is implicated in mitochondrial translocation of p66(shc) . However, stable overexpression of Pin1 failed to alter proapoptotic response to SFN at least in MCF-7 cells. Finally, SFN-induced S36 phosphorylation of p66(Shc) was mediated by protein kinase Cβ (PKCβ), and pharmacological inhibition of PKCβ significantly inhibited apoptotic cell death resulting from SFN exposure. In conclusion, the present study provides new insight into the mechanism of SFN-induced apoptosis involving PKCβ -mediated S36 phosphorylation of p66(shc).
