Effect Of Grape Seed Proanthocyanidins Research Articles

Grape Seed Proanthocyanidins Protect N2a Cells against Ischemic Injury via Endoplasmic Reticulum Stress and Mitochondrial-associated Pathways.

Grape seed proanthocyanidins (GSPs) are a group of polyphenolic bioflavonoids, which possess a variety of biological functions and pharmacological properties. We studied the neuroprotective effects of GSP against oxygen-glucose deprivation/reoxygenation (OGD/R) injury and the potential mechanisms in mouse neuroblastoma N2a cells. OGD/R was conducted in N2a cells. Cell viability was evaluated by CCK-8 and LDH release assay. Apoptosis was assessed by TUNEL staining and flow cytometry. Protein levels of cleaved caspase-3, Bax and Bcl-2 were detected by Western blotting. CHOP, GRP78 and caspase-12 mRNA levels were assessed by real-time PCR. JC-1 dying was used to detect mitochondrial membrane potential. ROS levels, activities of endogenous antioxidant enzymes and ATP production were examined to evaluate mitochondrial function. GSP increased cell viability after OGD/R injury in a dose-dependent manner. Furthermore, GSP inhibited cell apoptosis, reduced the mRNA levels of CHOP, GRP78 and caspase-12 (ER stressassociated genes), restored mitochondrial membrane potential and ATP generation, improved activities of endogenous anti-oxidant ability (T-AOC, GXH-Px, and SOD), and decreased ROS level. Our findings suggest that GSP can protect N2a cells from OGD/R insult. The mechanism of anti-apoptotic effects of GSP may involve attenuating ER stress and mitochondrial dysfunction.

Grape seed proanthocyanidin inhibits monocrotaline-induced pulmonary arterial hypertension via attenuating inflammation: in vivo and in vitro studies

Inflammation in pulmonary arterioles initiates and maintains pathological processes in pulmonary arterial hypertension (PAH), and inhibition of it attenuates PAH development. Grape seed proanthocyanidin (GSP) is believed to be effective in protecting vascular system via inhibiting inflammation, while its effect on pulmonary circulation remains inconclusive. In this study, we made observations in monocrotaline (MCT)-induced PAH rats and found decreases in mean pulmonary arterial pressure, pulmonary vessel resistance, right ventricular hypertrophy index, percentage of medial wall thickness, percentage of medial wall area, and lung weight of wet and dry tissue ratio after GSP administration in vivo. At the cellular and molecular levels, we also found several effects of GSP on MCT-induced PAH: (a) endothelial nitric oxide synthase expression in lung tissue and plasma NO level were increased; (b) Ca2+ level in pulmonary arterial smooth muscle cell (PASMC) was decreased; (c) transcription of inflammatory factors such as myeloperoxidase, interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α) was down-regulated in lung tissue; (d) nuclear factor-κB pathway was inhibited as IκBα was less phosphorylated; (e) TNFα-induced PASMC overproliferation could be inhibited. These results indicated a possible mechanism of GSP reversing pulmonary vascular remodeling and vascular contraction by inhibiting inflammation, and it may be useful for preventing PAH development.

Abstract 5442: Down-regulation of miRNA-214 expression by bioactive molecules mitigates invasive potential of melanoma cells through ALCAM/TFAP2 signaling

Abstract Melanoma is responsible for 80% of skin cancer related deaths in the United States and represents fifth most common neoplasia in humans. Over the past decades, the incidence of melanoma has increased by 3% to 8% per year in western countries. Therefore, it is essential to understand the molecular regulatory pathways that contribute to melanoma aggressiveness and metastatic dissemination. MicroRNAs (miRNAs) are small noncoding regulatory RNAs that contribute to tumor formation and progression, for their ability to regulate gene expression post-transcriptionally by either inhibiting protein translation or degrading target mRNAs. miR-214 is overexpressed in various types of cancers and is associated with the regulation of numerous carcinogenic processes including cell differentiation, senescence, angiogenesis and cell migration/invasion, etc. In the present study, we demonstrated that miR-214 shows distinct over expression in melanoma cell lines (A375, Hs294t, SK-Mel119, and SK-Mel28) as compared to its expression in normal human epidermal melanocytes (NHEM), and this overexpression of miR-214 in melanoma cells was associated with the invasive potential than NHEM. Treatment of melanoma cells with an inhibitor of miR-214 (50 nM) resulted in inhibition of cell invasion ability. We also determined the effects of bioactive phytochemical grape seed proanthocyanidins (GSPs) on the expression levels of miRNA-214 in melanoma cell lines (A375 and Hs294t) and its underlying molecular targets. Treatment of A375 and Hs294t cells with GSPs resulted in suppression of the levels of miRNA-214, which leads to decrease in migration potential of melanoma cell lines. By using 3D melanoma progression model, we also verified this suppressive effect of GPSs on melanoma invasion. The transition from the non-invasive to the invasive and metastatic stage is accompanied by the loss of AP-2 transcription factors (TFAP-2) and expression of activated leukocyte cell adhesion molecule (ALCAM) and those are associated with miRNA-214 overexpression. Here, we observed that the treatment of GSPs decreases ALCAM expression and restore TFAP-2 levels in melanoma cell lines. Further, dietary administration of GSPs significantly reduce the metastasis potential of A375 cells in different vital organs such as lung, liver, kidney, and spleen, and this suppressive effect of GSPs on invasive potential of A375 cells was associated with the reduction in the levels of miRNA-214 and ALCAM. Our results indicate that modulation of miR-214 influences in vitro cell movement and extravasation from blood vessels in vivo. Together, these studies suggest that an identification of metastasis related miRNAs may provide potential diagnostic biomarkers and better understanding of the complex functions of miRNAs, as well as therapeutic targets for clinical application. Note: This abstract was not presented at the meeting. Citation Format: Ram Prasad, Santosh K. Katiyar. Down-regulation of miRNA-214 expression by bioactive molecules mitigates invasive potential of melanoma cells through ALCAM/TFAP2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5442. doi:10.1158/1538-7445.AM2017-5442

Protective effect of Grape seed proanthocyanidin on learning disabilities in a rat model with radiation brain injury

Objective To observe the protective effect of grape seed proanthocyanidin extract (GSPE) on learning disabilities after radiation injury brain in rats. Methods 120 male Wistar rats were divided into control group, model group, high and low dose GSPE groups. The radiation injury brain models were established using a method of linear accelerator irradiation in 22 Gy. The morphological changes of neurons in hippocampus were observed with HE staining; the expression of phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) and growth associated protein-43(GAP-43) were detected by immunohistochemistry; the contents of malonalde hyde(MDA) and superoxide dismutase(SOD) were measured respectively by thiobarbituric acid(TBA) and xanthine oxidation(XTO) methods; the learning ability was assessed with shuttle box. Results Compared with the model group, the GSPE groups showed a decreased degree of nerve cell morphological injury; and increased ERK1/2 activities((13.20±1.45)/view, (27.40±2.52)/view, (19.80±1.30)/view), GAP-43 expression level((52.93±2.07)/view, (66.50±0.77)/view, (73.05±2.40)/view) and SOD activities((79.82±5.26) U/mg, (76.20±6.86) U/mg, (80.12±5.76) U/mg)(P<0.05); and decreased the contents of MDA((71.62±1.88) μmol/g, (76.41±1.94) μmol/g, (72.32±1.98) μmol/g)(P<0.05). Shuttle box testing showed that the active avoidance reaction rate was increased(7 d: (56.23±2.56)%; 14 d: (44.66±2.79)%; 28 d: (50.40±2.16)%)(P<0.05)and passive avoidance latency was shorted(7 d: (34.11±1.57)s; 14 d: (45.52±1.88)s; 28 d: ( 39.52±1.79)s)(P<0.05) in GSPE groups.The above mentioned changes were more significant in high dose of GSPE(P<0.05). Conclusion GSPE has protective effect on learning disabilities of radiation injuries brain, which is related to enhance ERK1/2 activity and the expression of GAP-43. Key words: Radiation injuries brain; Learning; Extracellular signal regulated kinase; Growth associated protein-43

Abstract 1916: Proanthocyanidins from grape seeds inhibit growth of head and neck squamous cell carcinoma by decreasing dna methylation and reactivation of silenced tumor suppressors

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in diagnostic and therapeutic methods, survival of HNSCC patients remains unchanged over the last 30 years. Epigenetic alterations are commonly associated with several types of cancers including HNSCC. Studies have shown that DNA hypermethylation plays important roles in the risk of cancers by inactivating the tumor suppressor genes/proteins. Thus, epigenetic changes are considered as promising therapeutic targets for cancer treatment. Dietary phytochemicals offer promising options for the development of effective strategies for the prevention of cancer, and thus can be utilized as complementary and alternative medicine. In the present study, we investigated the effect of grape seed proanthocyanidins (GSPs) on DNA methylation and growth of HNSCC cells in vitro and in vivo models. Our analytical data revealed that the levels of global DNA methylation and DNA methyltransferase (DNMT) activity were higher in HNSCC cells compared with normal human bronchial epithelial cells (NHBEC). Treatment of HNSCC cells (FaDu and SCC-1) with GSPs for 6 days results in significant decrease in global DNA methylation (70-80%, P&amp;lt;0.001) level, DNMT activity (60-80%) as well as in levels of DNMT proteins (DNMT 3A and DNMT 3B) compared to non-GSPs-treated control cells. The observation on DNA methylation levels was verified by analyzing the levels of 5-methylcytosine (5-mC) using dot-blot analysis. Additionally, GSPs treatment also reactivated tumor suppressor proteins, such as Cip1/WAF1/p21 and p27 in HNSCC cells. Treatment of HNSCC cells with 5-Aza-dc, an inhibitor of DNA methylation, also resulted in suppression of DNA methylation as well as DNMT protein expression and DNMT activity in these cells. Tumor xenograft studies were performed to further determine the effect of GSPs on the growth of HNSCC cells using athymic nude mouse model. Our in vivo study demonstrated that administration of GSPs (0.5%, w/w) in supplementation with AIN76A control diet resulted in inhibition of xenograft growth of FaDu cells in nude mice (80%; P&amp;lt;0.01) compared to the tumor xenograft growth in mice fed with non-GSPs-supplemented control diet. The growth inhibitory effect of dietary GSPs on the HNSCC tumor xenografts was associated with the inhibition of DNA methylation, DNMT activity and reactivation of silenced tumor suppressor proteins in tumor tissues. Together, this preclinical study provides evidence that GSPs act as a DNA demethylating agent and able to reactivate an epigenetically silenced tumor suppressors to inhibit growth of HNSCC cells. Citation Format: Santosh K. Katiyar, Ram Prasad. Proanthocyanidins from grape seeds inhibit growth of head and neck squamous cell carcinoma by decreasing dna methylation and reactivation of silenced tumor suppressors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1916. doi:10.1158/1538-7445.AM2015-1916

Abstract 1240: Bioactive proanthocyanidins from grape seeds inhibit cigarette smoke condensate-enhanced invasion of human non-small cell lung cancer cells by targeting NADPH oxidase and epithelial-mesenchymal transition

Abstract Lung cancer is the leading cause of cancer-related deaths in the United States, and the dismal 5-year survival rate of approximately 14% has shown no improvement over the past three decades. Cigarette smoke (CS) is the cause of ∼90% of lung cancer cases, and promotes drug resistance in lung cancer patients and is even known to limit effectiveness of treatment of cancers of other organs. It is therefore important to develop more effective strategies targeting CS-induced molecular abnormalities. Cigarette smoking increases oxidative stress particularly via activation of NADPH oxidase (NOX), a key source of superoxide anion production. Here we report the inhibitory effects of bioactive phytochemicals, grape seed proanthocyanidins (GSPs), on the migration potential of human non-small cell lung cancer (NSCLC) cells (A549, H460 and H1299) and assessed whether CS-induced NOX activation is their target. Using an in vitro invasion assay, we found that treatment of NSCLC cells with CS condensate (CSC) increased the migration of NSCLC cells in a dose-dependent manner, via enhancing NOX mediated-reactive oxygen species (ROS) accumulation. Treatment of NSCLC cells with GSPs inhibited the cell migration via reduction in ROS accumulation or oxidative stress and subsequent reduction of epithelial-to-mesenchymal transition. To examine the molecular targets of GSPs, we studied the effect of GSPs on CSC-induced alterations in the levels of key NOX components, namely, p22phox and p47phox, using A549 cells. We also determined the effect of GSPs on CSC-induced interaction between p22phox and p47phox proteins, a key event in NOX activation. Our results indicate that treatment of A549 cells with GSPs not only reverse CSC-induced expression levels of p22phox and p47phox, but also inhibit CSC-induced NOX activation by decreasing the binding of p22phox and p47phox proteins. This new insight into the anti-lung cancer cell migration activity of GSPs could serve as a basis for chemoprevention or therapy of advanced stage of lung cancer in human patients. Citation Format: Mudit Vaid, Santosh K. Katiyar. Bioactive proanthocyanidins from grape seeds inhibit cigarette smoke condensate-enhanced invasion of human non-small cell lung cancer cells by targeting NADPH oxidase and epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1240. doi:10.1158/1538-7445.AM2014-1240

Effect of grape seed proanthocyanidin on mitochondrial injury in hippocampus and cognition impairment in a rat model of obstructive sleep apnea hypoxia

Objective To investigate the effects of grape seed proanthocyanidin(GSPE) on mitochondrial injury in hippocampus and learning-memory impairment after obstructive sleep apnea hypoxia in rats. Methods Male SD rats(n=80) were randomly divided into control group, model group, low dose of GSPE treatment group and high dose of GSPE treatment group.Rats in control group were exposed in air, the model group were suffered from intermittent hypoxia conditions(50 ml/L, 8-hour-intermittent hypoxia everyday, and the duration of experiment 2 and 6 weeks, respectively). Mitochondrion pathology in hippocampal region was observed using electron microscope; malondialdehyde (MDA) contents and superoxide dismutase activity were detected by colorimetry and apoptotic cells was measured by TUNEL method. The cognitive function of rats in each group was assessed with the Morris water maze (MWM). Results After hypoxia, mitochondrion was significantly injured.The MDA contents were increased(79.86±2.52, 88.26±2.86) and SOD level decreased(70.67±6.70, 64.26±7.86). The number of neural apoptotic cells was significantly enhanced (9.68±0.79, 15.9±2.92). MWM test showed that the escaping latency was prolonged and the frequency of crossing the platform was decreased(P<0.05). Compared with that in the model group, low dose of GSPE decreased MDA contents (76.38±1.96, 82.16±2.02), increased SOD level(76.20±6.86, 70.58±6.86), and decreased apoptotic cells(6.60±0.69, 9.54±1.36). MWM test showed that the escaping latency was shortened and the frequency of crossing the platform was increased in GSPE treatment groups(P<0.05). Compared with low dose of GSPE, high dose of GSPE decreased MDA contents increased SOD level and decreased apoptotic cells.MWM test showed that the escaping latency was shortened and the frequency of crossing the platform was increased (P<0.05). Conclusion GSPE can attenuate mitochondrial injury and improve learning-memory function after obstructive sleep apnea hypoxia. Key words: Sleep apnea; Learning memory; Mitochondrion; Grape seed proanthocyanidin

Abstract 3683: Bioactive phytochemical proanthocyanidins target β-catenin signaling in preventing invasive potential of human melanoma cells.

Abstract Melanoma is the leading cause of death from skin diseases due to its propensity to metastasize. The overall incidence of melanoma is increasing in US, and is increasing rapidly in children. Since, melanoma is a highly malignant cancer with a potent capacity to metastasize distantly, an approach that decreases its metastatic potential may facilitate the development of an effective strategy for its prevention or treatment. Metastatic melanoma is often associated with activation of Wnt/β-catenin signaling pathway. To develop newer and more effective strategies for the prevention of melanoma metastasis, we have examined the inhibitory effect of proanthocyanidins, bioactive components of grape seeds, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t) and assessed whether Wnt/β-catenin signaling is the target of grape seed proanthocyanidins (GSPs). Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with GSPs resulted in a dose-dependent inhibition of cell migration, which was associated with the degradation of cytosolic β-catenin, and subsequently reducing the nuclear accumulation of β-catenin (i.e., inactivation of β-catenin) and reducing the levels of matrix metalloproteinase (MMP) -2 and MMP-9 which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. GSPs increased the: (i) levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical serine residues (Ser45, Ser33/37 and Thr41), and (ii) binding of β-transducin repeat-containing proteins (β-TrCP) with phospho forms of β-catenin in melanoma cells. These molecular events lead to degradation and/or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of GSPs, the effect of GSPs was determined on β-catenin-activated (Mel1241) and β-catenin-inactivated (Mel1011) melanoma cells. Treatment of Mel1241 cells with GSPs or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of GSPs and FH535 was not found in Mel1011 melanoma cells. These results indicate for the first time that grape seed proanthocyanidins inhibit the invasive potential of melanoma cells by targeting β-catenin signaling. This new insight into the anti-melanoma cell migration activity of GSPs could serve as the basis for chemoprevention or therapy of malignant melanoma in high risk human population. Citation Format: Santosh K. Katiyar, Mudit Vaid. Bioactive phytochemical proanthocyanidins target β-catenin signaling in preventing invasive potential of human melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3683. doi:10.1158/1538-7445.AM2013-3683

Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16INK4a and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans.

Abstract 1614: Grape seed proanthocyanidins inhibit pancreatic cancer cell growth in vitro and in vivo through induction of apoptosis and inhibition of PI3K/Akt pathway

Abstract The American Cancer Society reported that 35,420 Americans died in 2009 because of pancreatic cancer. It is an aggressive malignancy that is frequently diagnosed at an advanced stage with poor prognosis. Phosphoinositide 3-kinase (PI3K)/Akt is a potent survival pathway that may mediate resistance to chemotherapeutic drugs and conventional therapies in pancreatic cancer patients. Thus, PI3K/Akt is considered as a promising therapeutic target for this cancer. Dietary phytochemicals offer promising options for the development of effective strategies for the prevention of cancer, and thus can be utilized as complementary and alternative medicine. Therefore, to develop newer and more effective chemotherapeutic agent for pancreatic cancer, we have examined the effect of grape seed proanthocyanidins (GSPs) on the growth of pancreatic cancer cell lines and the molecular mechanisms underlying this effect using PANC-1, AsPC-1 and Miapaca-2 cell lines as a model. The effect of GSPs on pancreatic cancer cell lines was studied on cell proliferation, cell cycle regulation, apoptosis and PI3K/Akt pathway using MTT assay, western blotting and FACS analysis. In vitro treatment of pancreatic cancer cells (Miapaca-2, PANC-1, and AsPC-1) with GSPs resulted in: (i) growth inhibition, which was associated with G2/M-phase arrest, and includes the inhibition of the expressions of cyclin B1, Cdc25B and Cdc25c proteins. (ii) Induction of apoptosis of pancreatic cancer cells by GSPs was associated with the enhanced expression of Bax, and caspase-3 activation while reduced levels of anti-apoptotic proteins (Bcl2, Bcl-xl). (iii) Blocking of PI3K/Akt pathway. Exposure of Miapaca-2 and PANC-1 cancer cells to PI3K inhibitor (wortmannin) also resulted in a dose-dependent induction of apoptosis in these cells. The in vitro data were further verified using in vivo tumor xenograft model. Administration of GSPs (0.2% and 0.5%, w/w) as a supplement of an AIN76A control diet resulted in a dose-dependent inhibition of the growth of Miapaca-2 tumor xenografts in athymic nude mice (35-65%; P&amp;lt;0.05-0.01) compared to non-GSPs-treated controls. The growth inhibitory effect of dietary GSPs on the pancreatic xenograft tumors was associated with the induction of apoptotic cell death of tumor cells, and induction of Bax expression and activation of caspase-3 while decreased the expression of anti-apoptotic proteins. Further, the levels of phospho-Akt and the proteins of PI3K family (PI3K 110 and PI3K 85) were markedly decreased in the xenograft tumors treated with GSPs compared with controls. Together, this preclinical study provides evidence that the chemotherapeutic effect of GSPs on the growth of pancreatic cancer cells in vitro and in vivo is mediated, at least in part, through the induction of apoptosis, G2/M phase arrest and blocking of PI3K/Akt cell survival pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1614. doi:1538-7445.AM2012-1614

Abstract 1599: Grape seed proanthocyanidins inhibit UV-induced tolerogenic T-cell response through DNA repair-dependent functional activation of Langerhans cells

Abstract It is well documented that ultraviolet (UV) radiation-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) diminishes the antigen presenting capacity of epidermal Langerhans cells (LCs) and this leads to the suppression of immune system. Previously, we have shown that dietary grape seed proanthocyanidins (GSPs) inhibit UVB-induced immunosuppression in mice via activation of CD8+ T cells. However, it is not understood whether (1) GSPs target LCs while exerting its immunomodulatory effects on T cells in response to the application of cutaneous contact sensitizer, 2,4-dinitrofluorobenzene (DNFB) onto the UV-exposed skin, and whether (2) the ability of GSPs to activate dendritic cells/LCs correlates with their DNA repair properties. To understand this mechanism, we first determined DNA repair ability of GSPs in UV-exposed dendritic cells for which CD11c+ cells were prepared from bone marrow of xeroderma pigmentosum complementation group A-knockout (XPA-KO) mice which are deficient in DNA repair, and their wild-type (WT) counterparts. CD11c+ cells were exposed to UVB radiation with or without pretreatment with GSPs. Treatment of GSPs resulted in significant reduction in the number of CPD-positive cells in CD11c+ cells from WT mice, but this DNA repair effect of GSPs was not seen in the CD11c+ cells from XPA-KO mice. We then analyzed effects of dietary GSPs (0.5%, w/w) on secretion of cytokines by CD11c+ cells that were isolated from lymph nodes and spleens of mice (WT and XPA-KO) that were exposed to UVB [150 mJ/cm2 (WT), 20 mJ/cm2 (XPA-KO)]. Our results showed that in comparison to CD11c+ cells isolated from WT control mice (GSP-untreated), CD11c+ cells isolated from mice that received GSPs in diet secreted a significantly higher level of Th1 cytokines (IL-12, IFNα), while the levels of Th2 cytokines (IL-4, IL-10) were significantly lower, but this effect of GSPs was not observed in CD11c+ cells isolated from XPA-KO mice. Finally, we used an adoptive transfer approach, wherein draining lymph nodes were harvested from mice that were exposed to UVB with and without GSPs supplementation in diet and sensitized by DNFB onto the UVB-exposed skin. CD11c+ cells were positively selected and transferred into naïve mice that were subsequently challenged by application of DNFB on the ear skin. Naïve recipients that received CD11c+ cells from WT, GSPs-treated, UVB-irradiated donors exhibited full contact hypersensitivity (CHS) response, whereas no significant CHS was observed in XPA-KO mice. These data suggest that dietary GSPs inhibit UVB-induced immunosuppression in mice by improving the ability of LCs to mount an immune response via activation of T cells in a manner which correlates with its capacity to enhance repair of UVB-induced DNA damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1599. doi:1538-7445.AM2012-1599

Abstract 1597: Grape seed proanthocyanidins inhibit the invasive potential of head and neck squamous cell carcinoma cells by targeting EGFR expression and epithelial-to-mesenchymal transition

Abstract Head and neck squamous cell carcinoma (HNSCC) is responsible for over 20,000 deaths every year in the United States, and often generates from critical organs sites including the oral cavity, larynx, pharynx, and tongue. Most of the deaths are due, in large part, to its propensity to metastasize. Therefore, an approach that decreases the metastatic ability of HNSCC cells may facilitate the development of an effective strategy for its treatment and/or prevention. Naturally occurring bioactive phytochemicals may serve as appropriate candidates for the prevention of HNSCC metastasis. We have examined the effect of bioactive component grape seed proanthocyanidins (GSPs) on the invasive potential of human HNSCC cells and determined the molecular mechanisms underlying these effects using various cell lines from multiple sub-sites, such as SCC1 (oral cavity), SCC5 (larynx), FaDu (pharynx) and OSC19 (tongue). Using in vitro cell invasion assays, we observed that OSC19 cells, which was metastasis-specific, have significantly higher (P&amp;lt;0.001) invasive potential than SCC1, SCC5, and FaDu cell lines. Further studies were extended with OSC19 cell line to determine the effect of GSPs on cell migration. Treatment of OSC19 cells with GSPs (10, 20 and 40 μg/ml) for 48 h resulted in a concentration-dependent inhibition of cell migration of these cells. As EGFR is overexpressed in approximately 90% of HNSCC tumors, the EGFR is considered as a promising target of HNSCC therapy. It was found that the inhibitory effect of GSPs on cell migration was associated with a reduction in the levels of EGFR. Treatment of cells with gefitinib and erlotinib, small molecule inhibitors of EGFR, also inhibited migration of these cells. EGFR pathway has been involved in epithelial-mesenchymal transition (EMT), an early event that occurs during metastasis of cancers of an epithelial origin. Therefore, we determined the effect of GSPs on EMT, and was found that the inhibition of HNSCC cell migration by GSPs was associated with reversal of EMT, which resulted in an increase in the levels of epithelial biomarker (E-cadherin) while loss of mesenchymal biomarkers (vimentin, fibronectin and N-cadherin) in cells. Similar effect on EMT biomarkers was also observed when OSC19 cells were treated with erlotinib. As activation of nuclear factor-βB (NF-κB) has been implicated in stimulation of EMT, we further checked the effect of GSPs on NF-κB activation. GSPs inhibited the activation of NF-κB/p65 in OSC19 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited cell migration. Together these results indicate that GSPs have the ability to inhibit the invasive potential of HNSCC cells by targeting EGFR expression and reversing epithelial-to-mesenchymal transition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1597. doi:1538-7445.AM2012-1597

Abstract 810: Dietary grape seed proanthocyanidins inhibit UV-induced immunosuppression through stimulation of CD8+ effector T cells and inactivation of regulatory CD4+ T cells

Abstract The immunosuppressive effects of solar ultraviolet (UV) radiation, particularly UVB (290-320 nm), are well recognized. Experimental evidence indicates that UV-induced immunosuppression is a risk factor for skin cancer development, including melanoma and non-melanoma, in mice and clinical evidence suggests that it is also a risk factor in humans. Conversely, animals with an enhanced immune response exhibit a lower risk of skin tumor development on chronic UV exposure. Dietary grape seed proanthocyanidins (GSPs) have been shown to inhibit UVB-induced immunosuppression in mice; however, the cellular and molecular mechanisms underlying this inhibitory effect of GSPs are not yet clearly understood. To characterize the cell population responsible for the GSPs-mediated inhibition of UVB-induced immunosuppression, we used an adoptive transfer approach. Splenocytes and draining lymph nodes were harvested from mice that had been administered dietary GSPs, exposed to UVB, and sensitized by topical application of 2,4-dinitorfluorobenzene (DNFB) onto the UVB-exposed skin. CD8+ and CD4+ T cells were positively selected and transferred into naïve mice that were subsequently challenged by application of DNFB on the ear skin. Naïve recipients that received CD8+ T cells from GSPs-treated, UVB-irradiated donors exhibited full contact hypersensitivity (CHS) response. CHS is considered to be a prototypic T-cell mediated immune response. Naïve mice that received CD4+ regulatory T cells from GSPs-treated, UVB-exposed mice were able to mount a CHS response after sensitization and subsequent challenge with DNFB. On culture, the CD8+ T cells from GSPs-treated, UVB-exposed mice secreted higher levels of Th1 cytokines (IL-2, IFNγ) than CD8+ T cells from UVB-irradiated mice not treated with GSPs. CD4+ T cells from GSPs-treated, UVB-exposed mice secreted significantly lower levels of Th2 cytokines (IL-4 and IL-10) than CD4+ T cells from UVB-exposed mice not treated with GSPs. These data suggest that dietary GSPs inhibit UVB-induced immunosuppression by stimulating CD8+ effector T cells and diminishing regulatory CD4+ T cells in mice. This property of GSPs can be used as an alternative strategy to augment the induction of CD8+ effector T cells and to diminish the development of CD4+ regulatory T cells and that may lead to the prevention of skin cancer risk in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 810. doi:10.1158/1538-7445.AM2011-810

Proanthocyanidins Inhibit Photocarcinogenesis through Enhancement of DNA Repair and Xeroderma Pigmentosum Group A–Dependent Mechanism

Dietary grape seed proanthocyanidins (GSP) inhibit photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated. As ultraviolet B (UVB)-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) has been implicated in skin cancer risk, we studied whether dietary GSPs enhance repair of UVB-induced DNA damage and, if so, what is the potential mechanism? Supplementation of GSPs (0.5%, w/w) with AIN76A control diet significantly reduced the levels of CPD(+) cells in UVB-exposed mouse skin; however, GSPs did not significantly reduce UVB-induced CPD(+) cells in the skin of interleukin-12p40 (IL-12) knockout (KO) mice, suggesting that IL-12 is required for the repair of CPDs by GSPs. Using IL-12 KO mice and their wild-type counterparts and standard photocarcinogenesis protocol, we found that supplementation of control diet with GSPs (0.5%, w/w) significantly reduced UVB-induced skin tumor development in wild-type mice, which was associated with the elevated mRNA levels of nucleotide excision repair genes, such as XPA, XPC, DDB2, and RPA1; however, this effect of GSPs was less pronounced in IL-12 KO mice. Cytostaining analysis revealed that GSPs repaired UV-induced CPD(+) cells in xeroderma pigmentosum complementation group A (XPA)-proficient fibroblasts from a healthy individual but did not repair in XPA-deficient fibroblasts from XPA patients. Furthermore, GSPs enhance nuclear translocation of XPA and enhanced its interactions with other DNA repair protein ERCC1. Together, our findings reveal that prevention of photocarcinogenesis by GSPs is mediated through enhanced DNA repair in epidermal cells by IL-12- and XPA-dependent mechanisms.