Perspective on this Article from Green Tea Polyphenols Prevent UV-Induced Immunosuppression by Rapid Repair of DNA Damage and Enhancement of Nucleotide Excision Repair Genes

Macrophages are important in mounting an innate immune response to injury as well as in repair of injury. Gene expression of Rho proteins is known to be increased in fibrotic models; however, the role of these proteins in idiopathic pulmonary fibrosis (IPF) is not known. Here, we show that BAL cells from patients with IPF have a profibrotic phenotype secondary to increased activation of the small GTPase Rac1. Rac1 activation requires a posttranslational modification, geranylgeranylation, of the C-terminal cysteine residue. We found that by supplying more substrate for geranylgeranylation, Rac1 activation was substantially increased, resulting in profibrotic polarization by increasing flux through the mevalonate pathway. The increased flux was secondary to greater levels of acetyl-CoA from metabolic reprogramming to β oxidation. The polarization mediated fibrotic repair in the absence of injury by enhancing macrophage/fibroblast signaling. These observations suggest that targeting the mevalonate pathway may abrogate the role of macrophages in dysregulated fibrotic repair.

Ultraviolet B (UVB) radiation induces regulatory T cells (Treg cells) and depletion of these Treg cells alleviates immunosuppression and inhibitsphotocarcinogenesis in mice.Here, we determined the effects of dietary grape seed proanthocyanidins (GSPs) on the development and activity of UVB-induced Treg cells.C3H/HeN mice fed a GSPs (0.5%, w/w)-supplemented or control diet were exposed to UVB (150 mJ/cm 2 ) radiation, sensitized to 2,4-dinitrofluorobenzene (DNFB) and sacrificed 5 days later.FACS analysis indicated that dietary GSPs decrease the numbers of UVB-induced Treg cells.ELISA analysis of cultured sorted Treg cells indicated that secretion of immunosuppressive cytokines (interleukin-10, TGF-β) was significantly lower in Treg cells from GSPs-fed mice.Dietary GSPs also enhanced the ability of Treg cells from wild-type mice to stimulate production of IFNγ by T cells.These effects of dietary GSPs on Treg cell function were not found in XPA-deficient mice, which are incapable of repairing UVB-induced DNA damage.Adoptive transfer experiments revealed that naïve recipients that received Treg cells from GSPs-fed UVB-irradiated wild-type donors that had been sensitized to DNFB exhibited a significantly higher contact hypersensitivity (CHS) response to DNFB than mice that received Treg cells from UVB-exposed mice fed the control diet.There was no significant difference in the CHS response between mice that received Treg cells from UVB-irradiated XPAdeficient donors fed GSPs or the control diet.Furthermore, dietary GSPs significantly inhibited UVB-induced skin tumor development in wild-type mice but not in XPAdeficient mice.These results suggest that GSPs inactivate Treg cells by promoting DNA repair in dendritic cells in UVB-exposed skin.

Melanoma is a highly aggressive form of skin cancer with poor survival rate. Aberrant activation of Wnt/β-catenin has been observed in nearly one-third of human melanoma cases thereby indicating that targeting Wnt/β-catenin signaling could be a promising strategy against melanoma development. In the present study, we determined chemotherapeutic effect of grape seed proanthocyanidins (GSPs) on the growth of melanoma cells and validated their protective effects in vivo using a xenograft mouse model, and assessed if β-catenin is the target of GSP chemotherapeutic effect. Our in vitro data show that treatment of A375 and Hs294t human melanoma cells with GSPs inhibit the growth of melanoma cells, which was associated with the reduction in the levels of β-catenin. Administration of dietary GSPs (0.2 and 0.5%, w/w) in supplementation with AIN76A control diet significantly inhibited the growth of melanoma tumor xenografts in nude mice. Furthermore, dietary GSPs inhibited the xenograft growth of Mel928 (β-catenin-activated), while did not inhibit the xenograft growth of Mel1011 (β-catenin-inactivated) cells. These observations were further verified by siRNA knockdown of β-catenin and forced overexpression of β-catenin in melanoma cells using a cell culture model.

Cutaneous malignant melanoma is the leading cause of death from skin diseases and is often associated with activating mutations of the proto‐oncogene BRAF. To develop more effective strategies for the prevention or treatment of melanoma, we have examined the inhibitory effects of silymarin, a flavanoid from Silybum marianum, on melanoma cells. Using A375 (BRAF‐mutated) and Hs294t (non BRAF‐mutated but highly metastatic) human melanoma cell lines, we found that in vitro treatment with silymarin resulted in a dose‐dependent: (i) reduction in cell viability; (ii) enhancement of either Go/G1 (A375) or G2‐M (Hs294t) phase cell cycle arrest with corresponding alterations in cyclins and cyclin‐dependent kinases; and (iii) induction of apoptosis. The silymarin‐induced apoptosis of human melanoma cells was associated with a reduction in the levels of anti‐apoptotic proteins (Bcl‐2 and Bcl‐xl), an increase in the levels of pro‐apoptotic protein (Bax), and activation of caspases. Further, oral administration of silymarin (500 mg/kg body weight/2× a week) significantly inhibited (60%, P < 0.01) the growth of BRAF‐mutated A375 melanoma tumor xenografts, and this was associated with: (i) inhibition of cell proliferation; (ii) induction of apoptosis of tumor cells; (iii) alterations in cell cycle regulatory proteins; and (iv) reduced expression of tumor angiogenic biomarkers in tumor xenograft tissues. These results indicate that silymarin may have a chemotherapeutic effect on human melanoma cell growth and warrant its further evaluation. © 2014 Wiley Periodicals, Inc.

Cigarette smoking is the major cause of lung cancer. It is therefore important to develop effective strategies that target molecular abnormalities induced by cigarette smoke condensate (CSC). Cigarette smoking increases oxidative stress particularly via activation of NADPH oxidase (NOX), a key source of superoxide anion production. Here, we report that grape seed proanthocyanidins (GSPs) exert an inhibitory effect on the CSC‐induced migration of non‐small cell lung cancer (NSCLC) cells (A549, H460, and H1299). Using an in vitro invasion assay, we found that treatment of NSCLC cells with CSC increased NSCLC cell migration by enhancing NOX mediated‐oxidative stress. Treatment of NSCLC cells with GSPs inhibited the CSC‐induced cell migration through reduction in oxidative stress levels and a reduction in the epithelial‐to‐mesenchymal transition. To identify the molecular targets of GSPs, we examined the effects of GSPs on CSC‐induced alterations in the levels of key NOX components, namely p22 phox and p47 phox proteins, using A549 cells. We also determined the effect of GSPs on CSC‐induced interaction/binding between these proteins, which is a key event in NOX activation. We found that treatment of A549 cells with GSPs not only inhibited the CSC‐induced increase in the expression levels of p22 phox and p47 phox , but also reduced the binding of p22 phox to p47 phox proteins. This new insight into the anti‐lung cancer cell migration activity of GSPs could serve as a basis for development of improved chemopreventive or therapeutic strategies for lung cancer. © 2014 Wiley Periodicals, Inc.

Melanoma is a highly aggressive form of skin cancer and a leading cause of death from skin diseases mainly due to its propensity to metastasis. Due to metastatic tendency, melanoma is often associated with activation of Wnt/β-catenin signaling mechanism. Blocking β-catenin activation may be a good strategy to block melanoma-associated mortality. We have shown earlier that grape seed proanthocyanidins (GSPs) inhibit melanoma cell migration via targeting cyclooxygenase-2 (COX-2) overexpression. Here we explored further whether inhibition of inflammatory mediators-mediated activation of β-catenin by GSPs is associated with the inhibition of melanoma cell migration. Our study revealed that PGE2 receptors (EP2 and EP4) agonists promote melanoma cell migration while PGE2 receptor antagonist suppressed the migration capacity of melanoma cells. GSPs treatment inhibit butaprost (EP2 agonist) or Cay10580 (EP4 agonist) induced migration of melanoma cells. Western blot analysis revealed that GSPs reduced cellular accumulation of β-catenin, and decreased the expressions of matrix metalloproteinase (MMP)-2, MMP-9 and MITF, downstream targets of β-catenin in melanoma cells. GSPs also reduced the protein expressions of PI3K and p-Akt in the same set of experiment. To verify that β-catenin is a specific molecular target of GSPs, we compared the effect of GSPs on cell migration of β-catenin-activated (Mel1241) and β-catenin-inactivated (Mel1011) melanoma cells. GSPs inhibit cell migration of Mel1241 cells but not of Mel1011 cells. Additionally, in vivo bioluminescence imaging data indicate that dietary administration of GSPs (0.5%, w/w) in supplementation with AIN76A control diet inhibited the migration/extravasation of intravenously injected melanoma cells in lungs of immune-compromised nude mice, and that this effect of GSPs was associated with an inhibitory effect on the activation of β-catenin and its downstream targets, such as MMPs, in lungs as a target organ.

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5'-untranslated regions (5'-UTR) due to differential splicing. The 5'-UTR variant ACD' is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication.

Colon cancer development and malignant progression are driven by genetic and epigenetic alterations in tumor cells and by factors from the tumor microenvironment. Cancer cells become reliant on the activity of specific oncogenes and on prosurvival and proliferative signals they receive from the abnormal environment they create and reside in. Accordingly, the response to anticancer therapy is determined by genetic and epigenetic changes that are intrinsic to tumor cells and by the factors present in the tumor microenvironment. Recent advances in the understanding of the involvement of the tumor microenvironment in tumor progression and therapeutic response are optimizing the application of prognostic and predictive factors in colon cancer. Moreover, new targets in the tumor microenvironment that are amenable to therapeutic intervention have been identified. Because stromal cells are with rare exceptions genetically stable, the tumor microenvironment has emerged as a preferred target for therapeutic drugs. In this review, we discuss the role of stromal fibroblasts and macrophages in colon cancer progression and in the response of colon cancer patients to therapy.

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

Previously, we showed that administration of a high-fat diet (HF-diet) to C57BL/6 mice exacerbates their response to short-term UVB radiation-induced inflammation in the skin. To explore the effects of an HF-diet on UVB-induced tumorigenesis, we have used the SKH-1 hairless mouse model in which the mice are exposed to UVB radiation (180 mJ/cm2) three times a week for 24 weeks. The development of UVB-induced skin tumors was rapid and the tumor multiplicity and tumor size were significantly higher (P < 0.01–0.005) in the mice fed an HF-diet than the mice fed a control-diet (C-diet). Moreover, the malignant progression of UVB-induced papillomas to carcinomas was higher in HF-diet-fed mice. On analysis of tumors and tumor-uninvolved skin samples from the tumor-bearing mice, we found that administration of an HF-diet significantly enhanced the levels of UVB-induced expression of cyclooxygenase-2 (COX-2), prostaglandin E2 (P < 0.01), and PGE2 receptors, and activation of NF-κB in the UVB-exposed skin as well as in tumors. In addition the HF-diet enhanced the expression of proinflammatory cytokines, including tumor necrosis factor-α (P < 0.01), interleukin (IL)-1β (P < 0.01) and IL-6 (P < 0.05) in the UVB-exposed skin as well as in tumors. Western blot analysis revealed that HF-diet enhanced the levels of epidermal cell proliferation, phosphatidylinositol 3-kinase and phosphorylation of Akt at Ser473 in UVB-exposed skin and skin tumors. Collectively, these data demonstrate that the regular consumption of an HF-diet increases the risk of photocarcinogenesis in mice and that this is associated with enhanced expression of inflammatory mediators in the UVB-exposed skin and tumors.

Abstract Immunosuppressive effects of solar ultraviolet (UV) radiation have been implicated in the risk of non-melanoma and melanoma skin cancers in humans. Previously, we have shown that dietary grape seed proanthocyanidins (GSPs) inhibit photocarcinogenesis in mice and inhibit UV-induced suppression of allergic contact hypersensitivity (CHS) response, a prototypic T-cell-mediated immune response. Studies have shown that UV-induced T regulatory (Treg) cells or suppressor T cells have a role in immune suppression and UV-induced skin carcinogenesis is indicated by the demonstration that depletion of UV-induced T-suppressor cells can inhibit UV-induced carcinogenesis. In our continued efforts to investigate the molecular targets responsible for the prevention of immunosuppression in UV exposed mice by GSPs, we have investigated whether GSPs inhibit UV-induced immunosuppression by inhibiting the development or activity of UV-induced Treg cells in mice. To understand the mechanism, C3H/HeN mice were exposed to UV (150 mJ/cm2) radiation on 4 consecutive days with and without treatment of mice with dietary GSPs, mice were sensitized with 2,4-dinitrofluorobenzene (DNFB) 48 h after the last UV exposure, and then sacrificed 5 days after DNFB sensitization. The flow cytometry analysis of lymph node cells indicated that the numbers of Treg cells were reduced in the mice which were given GSPs in diet compared to non-GSPs-treated UV-exposed mice. We then analyzed effects of dietary GSPs (0.5%, w/w) on secretion of cytokines by Treg cells that were isolated from lymph nodes and spleens of mice that were exposed to UVB (150 mJ/cm2). Our ELISA data showed that in comparison to Treg cells isolated from control mice (GSPs-untreated), Treg cells isolated from mice that received GSPs in diet secreted a significantly higher level of IFNγ, while significantly lower levels of interleukin (IL)-10 and TGF-β, which are immunosuppressive in nature. Finally, we used an adoptive transfer approach, wherein draining lymph nodes were harvested from mice that were exposed to UVB on 4 consecutive days with and without GSPs supplementation in diet and sensitized by DNFB onto the UVB-exposed skin. Treg cells were positively selected using magnetic beads and MACS system and transferred into naïve mice that were subsequently challenged by application of DNFB on the ear skin. Naïve recipients that received Treg cells from GSPs-treated, UVB-irradiated donors exhibited CHS response, whereas no significant CHS was observed in mice which were injected Treg cells from UVB alone-exposed mice compared to control group. These data suggest that dietary GSPs inhibit UVB-induced immunosuppression by suppressing the development as well as the functional activation of Treg cells in UV-exposed mice. Citation Format: Santosh K. Katiyar, Tripti Singh, Mudit Vaid. Dietary grape seed proanthocyanidins inhibit UV-induced immune suppression by targeting the development of regulatory T cells in mice. [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 1239. doi:10.1158/1538-7445.AM2014-1239

Ultraviolet (UV) radiation-induced immunosuppression has been implicated in skin carcinogenesis. Grape seed proanthocyanidins (GSPs) have anti-skin carcinogenic effects in mice and GSPs-fed mice exhibit a reduction in UV-induced suppression of allergic contact hypersensitivity (CHS), a prototypic T-cell–mediated response. Here, we report that dietary GSPs did not inhibit UVB-induced suppression of CHS in xeroderma pigmentosum complementation group A (XPA)-deficient mice, which lack nucleotide excision repair mechanisms. GSPs enhanced repair of UVB-induced DNA damage (cyclobutane pyrimidine dimers) in wild-type, but not XPA-deficient, dendritic cells (DC). Co-culture of CD4+ T cells with DCs from UVB-irradiated wild-type mice resulted in suppression of T-cell proliferation and secretion of T-helper (TH) 1-type cytokines that was ameliorated when the DCs were obtained from GSP-fed mice, whereas DCs obtained from GSP-fed XPA-KO mice failed to restore T-cell proliferation. In adoptive transfer experiments, donor DCs were positively selected from the draining lymph nodes of UVB-exposed donor mice that were sensitized to 2,4,-dinitrofluorobenzene were transferred into naïve recipient mice and the CHS response assessed. Naïve recipients that received DCs from UVB-exposed wild-type donors that had been fed GSPs exhibited a full CHS response, whereas no significant CHS was observed in mice that received DCs from XPA-KO mice fed GSPs. These results suggest that GSPs prevent UVB-induced immunosuppression through DNA repair–dependent functional activation of dendritic cells in mice. Cancer Prev Res; 6(3); 242–52. ©2013 AACR.

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

Silymarin inhibits UVB-induced immunosuppression in mouse skin. To identify the molecular mechanisms underlying this effect, we used an adoptive transfer approach in which dendritic cells (DCs) from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) were transferred into naïve recipient mice. The contact hypersensitivity (CHS) response of the recipient mice to DNFB was then measured. When DCs were obtained from UVB-exposed donor mice that were not treated with silymarin, the CHS response was suppressed confirming the role of DCs in the UVB-induced immunosuppression. Silymarin treatment of UVB-exposed donor mice relieved this suppression of the CHS response in the recipients. Silymarin treatment was associated with rapid repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in DCs and silymarin treatment did not prevent UV-induced immunosuppression in XPA-deficient mice which are unable to repair UV-induced DNA damage. The CHS response in mice receiving DCs from silymarin-treated UV-exposed donor mice also was associated with enhanced secretion of Th1-type cytokines and stimulation of T cells. Adoptive transfer of T cells revealed that transfer of either CD8(+) or CD4(+) cells from silymarin-treated, UVB-exposed donors resulted in enhancement of the CHS response. Cell culture study showed enhanced secretion of IL-2 and IFNγ by CD8(+) T cells, and reduced secretion of Th2 cytokines by CD4(+) T cells, obtained from silymarin-treated UVB-exposed mice. These data suggest that DNA repair-dependent functional activation of DCs, a reduction in CD4(+) regulatory T-cell activity, and stimulation of CD8(+) effector T cells contribute to silymarin-mediated inhibition of UVB-induced immunosuppression.

Abstract Ultraviolet (UV) radiation-induced immunosuppression has been implicated in the risk of skin cancers, including melanoma and non-melanoma. Topical treatment of mouse skin with silymarin, a bioactive phytochemical from milk thistle (Silybum marianum L. Gaertn.), inhibits UVB radiation-induced skin tumor development and immunosuppression in mice. To identify the molecular mechanisms underlying its effect on immune system, we used an adoptive transfer approach in which dendritic cells (DCs) from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) were transferred into naïve recipient mice. The contact hypersensitivity (CHS) response of the recipient mice to DNFB was then measured. When DCs were obtained from UVB-exposed donor mice that were not treated with silymarin, the CHS response was suppressed confirming the role of DCs in the UVB-induced immunosuppression. CHS response is considered as a prototypic T-cell mediated immune response. Silymarin treatment of UVB-exposed donor mice relieved this suppression of the CHS response in the recipients. Silymarin treatment was associated with rapid repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in DCs and silymarin treatment did not prevent UV-induced immunosuppression in xeroderma pigmentosum complementation Group A (XPA)-deficient mice which are unable to repair UV-induced DNA damage. The CHS response in mice receiving DCs from silymarin-treated UV-exposed donor mice also was associated with enhanced secretion of Th1-type cytokines and stimulation of T cells. Adoptive transfer of T cells revealed that transfer of either CD8+ or CD4+ cells from silymarin-treated, UVB-exposed donors resulted in enhancement of the CHS response. Cell culture study showed enhanced secretion of IL-2 and IFNγ by CD8+ T cells, and reduced secretion of Th2 cytokines by CD4+ cells, obtained from silymarin-treated UVB-exposed mice. These data suggest that DNA repair-dependent functional activation of DCs, a reduction in CD4+ regulatory T-cell activity, and stimulation of CD8+ effector T cells contribute to silymarin-mediated inhibition of UVB-induced immunosuppression in mice. This property of silymarin can be used as an alternative strategy to augment immune system and that may help to prevent or delay the risk of skin cancers in high-risk human population. Citation Format: Mudit Vaid, Ram Prasad, Tripti Singh, Craig A. Elmets, Hui Xu, Santosh K. Katiyar. Silymarin, a phytochemical from milk thistle, inhibits UVB-induced immune suppression through DNA repair-dependent activation of dendritic cells and stimulation of effector T 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 1082. doi:10.1158/1538-7445.AM2013-1082

The importance of epigenetic alterations in the development of various diseases including the cancers has been realized. As epigenetic changes are reversible heritable changes, these can be utilized as an effective strategy for the prevention of cancers. DNA methylation is the most characterized epigenetic mechanism that can be inherited without changing the DNA sequence. Although limited available data suggest that silencing of tumor suppressor genes in ultraviolet (UV) radiation‐exposed epidermis leads to photocarcinogenesis and is associated with a network of epigenetic modifications including alterations in DNA methylation, DNA methyltransferases and histone acetylations. Various bioactive dietary components have been shown to protect skin from UV radiation‐induced skin tumors in animal models. The role of bioactive dietary components, such as, (−)‐epicatechins from green tea and proanthocyanidins from grape seeds has been assessed in chemoprevention of UV‐induced skin carcinogenesis and underlying epigenetic mechanism in vitro and in vivo animal models. These bioactive components have the ability to block UV‐induced DNA hypermethylation and histone modifications in the skin required for the silencing of tumor suppressor genes ( e.g. Cip1/p21 , p16 INK4a ). This information is of importance for understanding the role of epigenetic modulation in UV‐induced skin tumor and the chemopreventive mechanism of bioactive dietary components.

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, p16(INK4a) 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 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<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