Combinatorial bioactive botanicals re-sensitize tamoxifen treatment in ER-negative breast cancer via epigenetic reactivation of ERα expression.

Conventional cancer prevention has primarily focused on single chemopreventive compounds that may not be sufficiently efficacious. We sought to investigate potential combinatorial effects of epigenetic bioactive botanicals including epigallocatechin-3-gallate (EGCG) in green tea polyphenols (GTPs) and sulforaphane (SFN) in broccoli sprouts (BSp) on neutralizing epigenetic aberrations in estrogen receptor-α (ERα) leading to enhanced anti-hormone therapeutic efficacy in ERα-negative breast cancer. Our results showed that this combinatorial treatment re-sensitized ERα-dependent cellular inhibitory responses to an estrogen antagonist, tamoxifen (TAM), via at least in part, epigenetic reactivation of ERα expression in ERα-negative breast cancer cells. Further in vivo studies revealed the combinatorial diets of GTPs and BSp significantly inhibited breast tumor growth in ERα-negative mouse xenografts, especially when combined with TAM treatment. This novel treatment regimen can lead to remodeling of the chromatin structure by histone modifications and recruitment changes of transcriptional factor complex in the ERα promoter thereby contributing to ERα reactivation and re-sensitized chemotherapeutic efficacy of anti-hormone therapy. Our studies indicate that combinatorial bioactive botanicals from GTPs and BSp are highly effective in inhibiting ERα-negative breast cancer due at least in part to epigenetic reactivation of ERα, which in turn increases TAM-dependent anti-estrogen chemosensitivity in vitro and in vivo.

Synthesis of ß-Carboline-Based N-Heterocyclic Carbenes and Their Antiproliferative and Antimetastatic Activities against Human Breast Cancer Cells.

A series of novel ß-carboline-based N-heterocyclic carbenes was prepared via Mannich reaction between methyl 1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylate, formaldehyde, and primary amines. All compounds were evaluated for their antiproliferative activity using human breast cancer and lung cancer cell lines. Three compounds, 3c, 3j, and 3h, were discovered to display IC50 less than 10 µM against human breast cancer MDA-MB-231 cells at 24 h of treatment. Pharmacologically these compounds lead to G2/M phase cell cycle arrest and induction of cellular apoptosis by triggering intrinsic apoptotic pathway through depolarization of mitochondrial membrane potential and activation of caspases. At lower concentrations, these compounds also showed antimigratory and antiinvasive effects against highly metastatic human breast cancer MDA-MB-231 cells via aberration of MAP-kinase signaling and by the inhibition of matrix metalloproteinases. However, these analogues lack in vivo effect in mouse model which may be attributed to their strong affinity to HSA that was investigated spectroscopically with compound 3h.

Epigenetic reactivation of p21CIP1/WAF1 and KLOTHO by a combination of bioactive dietary supplements is partially ERα-dependent in ERα-negative human breast cancer cells.

Available treatment strategies against estrogen receptor (ER)-negative breast cancer patients are limited due to their poor response to hormonal therapy. We have shown previously that the combinations of green tea polyphenols (GTPs), a dietary DNA methyltransferase inhibitor, and sulforaphane (SFN), a dietary histone deacetylase inhibitor, reactivate ERα expression in ERα-negative MDA-MB-231 cells. Here, we investigated the functional significance of ERα reactivation in the reactivation of silenced tumor suppressor genes (TSGs) in ERα-negative human breast cancer cells. We found that the treatment of MDA-MB-231 cells with the combinations of GTPs and SFN leads to the reactivation of silenced TSGs such as p21(CIP1/WAF1) and KLOTHO through active chromatin modifications. Further, GTPs- and SFN-mediated reactivation of TSGs was, at least in part, dependent on ERα reactivation in ERα-negative MDA-MB-231 cells. Collectively, our findings suggest that a novel combination of bioactive dietary supplements could further be explored as an effective therapeutic option against hormonal refractory breast cancer.

Epigenetic regulation by selected dietary phytochemicals in cancer chemoprevention.

The growing interest in cancer epigenetics is largely due to the reversible nature of epigenetic changes which tend to alter during the course of carcinogenesis. Major epigenetic changes including DNA methylation, chromatin modifications and miRNA regulation play important roles in tumorigenic process. There are several epigenetically active synthetic molecules such as DNA methyltransferase (DNMTs) and histone deacetylases (HDACs) inhibitors, which are either approved or, are under clinical trials for the treatment of various cancers. However, most of the synthetic inhibitors have shown adverse side effects, narrow in their specificity and also expensive. Hence, bioactive phytochemicals, which are widely available with lesser toxic effects, have been tested for their role in epigenetic modulatory activities in gene regulation for cancer prevention and therapy. Encouragingly, many bioactive phytochemicals potentially altered the expression of key tumor suppressor genes, tumor promoter genes and oncogenes through modulation of DNA methylation and chromatin modification in cancer. These bioactive phytochemicals either alone or in combination with other phytochemicals showed promising results against various cancers. Here, we summarize and discuss the role of some commonly investigated phytochemicals and their epigenetic targets that are of particular interest in cancer prevention and cancer therapy.

Epigenetic reactivation of estrogen receptor-α (ERα) by genistein enhances hormonal therapy sensitivity in ERα-negative breast cancer.

BACKGROUND: Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and normally does not respond to conventional estrogen target-directed therapies. The soybean isoflavone, genistein (GE), has been shown to prevent and inhibit breast cancer and recent studies have suggested that GE can enhance the anticancer capacity of an estrogen antagonist, tamoxifen (TAM), especially in ERα-positive breast cancer cells. However, the role of GE in ERα-negative breast cancer remains unknown. METHODS: We have evaluated the in vitro and in vivo epigenetic effects of GE on ERα reactivation by using MTT assay, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, western-blot assay, immunoprecipitation (ChIP) assay, immunohistochemistry and epigenetic enzymatic activity analysis. Preclinical mouse models including xenograft and spontaneous breast cancer mouse models were used to test the efficacy of GE in vivo. RESULTS: We found that GE can reactivate ERα expression and this effect was synergistically enhanced when combined with a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), in ERα-negative MDA-MB-231 breast cancer cells. GE treatment also re-sensitized ERα-dependent cellular responses to activator 17ß-estradiol (E2) and antagonist TAM. Further studies revealed that GE can lead to remodeling of the chromatin structure in the ERα promoter thereby contributing to ERα reactivation. Consistently, dietary GE significantly prevented cancer development and reduced the growth of ERα-negative mouse breast tumors. Dietary GE further enhanced TAM-induced anti-cancer efficacy due at least in part to epigenetic ERα reactivation. CONCLUSIONS: Our studies suggest that soybean genistein can epigenetically restore ERα expression, which in turn increases TAM-dependent anti-estrogen therapeutic sensitivity in vitro and in vivo. The results from our studies reveal a novel therapeutic combination approach using bioactive soybean product and anti-hormone therapy in refractory ERα-negative breast cancer which will provide more effective options in breast cancer therapy.

Bioactive dietary supplements reactivate ER expression in ER-negative breast cancer cells by active chromatin modifications.

Breast cancer is the most common cancer and the leading cause of cancer death in women. Although tamoxifen therapy is successful for some patients, it does not provide adequate benefit for those who have estrogen receptor (ER)-negative cancers. Therefore, we approached novel treatment strategies by combining two potential bioactive dietary supplements for the reactivation of ERα expression for effective treatment of ERα-negative breast cancer with tamoxifen. Bioactive dietary supplements such as green tea polyphenols (GTPs) and sulforaphane (SFN) inhibit DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), respectively, which are of central importance to cancer prevention. In the present study, we have observed that treatment of ERα-negative breast cancer cells with GTPs and SFN alone or in combination leads to the reactivation of ERα expression. The combination of 20 µg/mL GTPs and 5 µM SFN was found to be the optimal dose of ERα-reactivation at 3 days in MDA-MB-231 cells. The reactivation of ERα expression was consistently correlated with ERα promoter hypomethylation and hyperacetylation. Chromatin immunoprecipitation (ChIP) analysis of the ERα promoter revealed that GTPs and SFN altered the binding of ERα-transcriptional co-repressor complex thereby contributing to ERα-reactivation. In addition, treatment with tamoxifen in combination with GTPs and SFN significantly increased both cell death and inhibition of cellular proliferation in MDA-MB-231 cells in comparison to treatment with tamoxifen alone. Collectively, our findings suggest that a novel combination of bioactive-HDAC inhibitors with bioactive-demethylating agents is a promising strategy for the effective treatment of hormonal refractory breast cancer with available anti-estrogens.

Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism.

Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells.

A novel prodrug of epigallocatechin-3-gallate: differential epigenetic hTERT repression in human breast cancer cells.

Epigallocatechin-3-gallate (EGCG), a major component of green tea polyphenols (GTP), has been reported to downregulate telomerase activity in breast cancer cells thereby increasing cellular apoptosis and inhibiting cellular proliferation. However, the major concerns with GTPs are their bioavailability and stability under physiologic conditions. In the present study, we show that treatments with EGCG and a novel prodrug of EGCG (pro-EGCG or pEGCG) dose- and time-dependently inhibited the proliferation of human breast cancer MCF-7 and MDA-MB-231 cells but not normal control MCF10A cells. Furthermore, both EGCG and pro-EGCG inhibited the transcription of hTERT (human telomerase reverse transcriptase), the catalytic subunit of telomerase, through epigenetic mechanisms in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 cells. The downregulation of hTERT expression was found to be because of hTERT promoter hypomethylation and histone deacetylations, mediated at least partially through inhibition of DNA methyltransferase and histone acetyltransferase activities, respectively. In addition, we also observed that EGCG and pEGCG can remodel chromatin structures of the hTERT promoter by decreasing the level of acetyl-H3, acetyl-H3K9, and acetyl-H4 to the hTERT promoter. EGCG and pEGCG induced chromatin alterations that facilitated the binding of many hTERT repressors such as MAD1 and E2F-1 to the hTERT regulatory region. Depletion of E2F-1 and MAD1 by using siRNA reversed the pEGCG downregulated hTERT expression and associated cellular apoptosis differently in ER-positive and ER-negative breast cancer cells. Collectively, our data provide new insights into breast cancer prevention through epigenetic modulation of telomerase by using pro-EGCG, a more stable form of EGCG, as a novel chemopreventive compound.

Synergistic epigenetic reactivation of estrogen receptor-α (ERα) by combined green tea polyphenol and histone deacetylase inhibitor in ERα-negative breast cancer cells.

BACKGROUND: The status of estrogen receptor-α (ERα) is critical to the clinical prognosis and therapeutic approach in breast cancer. ERα-negative breast cancer is clinically aggressive and has a poor prognosis because of the lack of hormone target-directed therapies. Previous studies have shown that epigenetic regulation plays a major role in ERα silencing in human breast cancer cells. Dietary green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), is believed to be an anticancer agent in part through its regulation of epigenetic processes. RESULTS: In our current studies, we found that EGCG can reactivate ERα expression in ERα-negative MDA-MB-231 breast cancer cells. Combination studies using EGCG with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), revealed a synergistic effect of reactivation of ERα expression in ERα-negative breast cancer cells. Reactivation of ERα expression by EGCG and TSA treatment was found to sensitize ERα-dependent cellular responses to activator 17ß-estradiol (E2) and antagonist tamoxifen in ERα-negative breast cancer cells. We also found that EGCG can lead to remodeling of the chromatin structure of the ERα promoter by altering histone acetylation and methylation status thereby resulting in ERα reactivation. A decreased binding of the transcription repressor complex, Rb/p130-E2F4/5-HDAC1-SUV39H1-DNMT1, in the regulatory region of the ERα promoter also contributes to ERα transcriptional activation through treatment with EGCG and/or TSA. CONCLUSIONS: Collectively, these studies show that green tea EGCG can restore ERα expression by regulating epigenetic mechanisms, and this effect is enhanced when combined with an HDAC inhibitor. This study will facilitate more effective uses of combination approaches in breast cancer therapy and will help to explore more effective chemotherapeutic strategies toward hormone-resistant breast cancer.

Sulforaphane causes epigenetic repression of hTERT expression in human breast cancer cell lines.

BACKGROUND: Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a common dietary component that has histone deacetylase inhibition activity and exciting potential in cancer prevention. The mechanisms by which SFN imparts its chemopreventive properties are of considerable interest and little is known of its preventive potential for breast cancer. PRINCIPAL FINDINGS: We found that SFN significantly inhibits the viability and proliferation of breast cancer cells in vitro while it has negligible effects on normal breast cells. Inhibition of telomerase has received considerable attention because of its high expression in cancer cells and extremely low level of expression in normal cells. SFN treatment dose- and time-dependently inhibited human telomerase reverse transcriptase (hTERT), the catalytic regulatory subunit of telomerase, in both MCF-7 and MDA-MB-231 human breast cancer cells. DNA methyltransferases (DNMTs), especially DNMT1 and DNMT3a, were also decreased in SFN-treated breast cancer cells suggesting that SFN may repress hTERT by impacting epigenetic pathways. Down-regulation of DNMTs in response to SFN induced site-specific CpG demethylation occurring primarily in the first exon of the hTERT gene thereby facilitating CTCF binding associated with hTERT repression. Chromatin immunoprecipitation (ChIP) analysis of the hTERT promoter revealed that SFN increased the level of active chromatin markers acetyl-H3, acetyl-H3K9 and acetyl-H4, whereas the trimethyl-H3K9 and trimethyl-H3K27 inactive chromatin markers were decreased in a dose-dependent manner. SFN-induced hyperacetylation facilitated the binding of many hTERT repressor proteins such as MAD1 and CTCF to the hTERT regulatory region. Depletion of CTCF using siRNA reduced the SFN-induced down-regulation of hTERT mRNA transcription in these breast cancer cells. In addition, down-regulation of hTERT expression facilitated the induction of cellular apoptosis in human breast cancer cells. SIGNIFICANCE: Collectively, our results provide novel insights into SFN-mediated epigenetic down-regulation of telomerase in breast cancer prevention and may open new avenues for approaches to SFN-mediated cancer prevention.

Proanthocyanidins inhibit in vitro and in vivo growth of human non-small cell lung cancer cells by inhibiting the prostaglandin E(2) and prostaglandin E(2) receptors.

Overexpression of cyclooxygenase-2 (COX-2) and prostaglandins (PG) is linked to a wide variety of human cancers. Here, we assessed whether the chemotherapeutic effect of grape seed proanthocyanidins (GSP) on non-small cell lung cancer (NSCLC) cells is mediated through the inhibition of COX-2 and PGE(2)/PGE(2) receptor expression. The effects of GSPs on human NSCLC cell lines in terms of proliferation, apoptosis, and expression of COX-2, PGE(2), and PGE(2) receptors were determined using Western blotting, fluorescence-activated cell sorting analysis, and reverse transcription-PCR. In vitro treatment of NSCLC cells (A549, H1299, H460, H226, and H157) with GSPs resulted in significant growth inhibition and induction of apoptosis, which were associated with the inhibitory effects of GSPs on the overexpression of COX-2, PGE(2), and PGE(2) receptors (EP1 and EP4) in these cells. Treatment of cells with indomethacin, a pan-COX inhibitor, or transient transfection of cells with COX-2 small interfering RNA, also inhibited cell growth and induced cell death. The effects of a GSP-supplemented AIN76A control diet fed to nude mice bearing tumor xenografts on the expression of COX-2, PGE(2), and PGE(2) receptors in the xenografts were also evaluated. The growth-inhibitory effect of dietary GSPs (0.5%, w/w) on the NSCLC xenograft tumors was associated with the inhibition of COX-2, PGE(2), and PGE(2) receptors (EP1, EP3, and EP4) in tumors. This preclinical study provides evidence that the chemotherapeutic effect of GSPs on lung cancer cells in vitro and in vivo is mediated, at least in part, through the inhibition of COX-2 expression and subsequently the inhibition of PGE(2) and PGE(2) receptors.

Green tea polyphenols prevent UV-induced immunosuppression by rapid repair of DNA damage and enhancement of nucleotide excision repair genes.

UV radiation-induced immunosuppression has been implicated in the development of skin cancers. Green tea polyphenols (GTP) in drinking water prevent photocarcinogenesis in the skin of mice. We studied whether GTPs in drinking water (0.1-0.5%, w/v) prevent UV-induced immunosuppression and (if so) potential mechanisms of this effect in mice. GTPs (0.2% and 0.5%, w/v) reduced UV-induced suppression of contact hypersensitivity (CHS) in response to a contact sensitizer in local (58-62% reductions; P < 0.001) and systemic (51-55% reductions; P < 0.005) models of CHS. Compared with untreated mice, GTP-treated mice (0.2%, w/v) had a reduced number of cyclobutane pyrimidine dimer-positive (CPD(+)) cells (59%; P < 0.001) in the skin, showing faster repair of UV-induced DNA damage, and had a reduced (2-fold) migration of CPD(+) cells from the skin to draining lymph nodes, which was associated with elevated levels of nucleotide excision repair (NER) genes. GTPs did not prevent UV-induced immunosuppression in NER-deficient mice but significantly prevented it in NER-proficient mice (P < 0.001); immunohistochemical analysis of CPD(+) cells indicated that GTPs reduced the numbers of UV-induced CPD(+) cells in NER-proficient mice (P < 0.001) but not in NER-deficient mice. Southwestern dot-blot analysis revealed that GTPs repaired UV-induced CPDs in xeroderma pigmentosum complementation group A (XPA)-proficient cells of a healthy person but did not in XPA-deficient cells obtained from XPA patients, indicating that a NER mechanism is involved in DNA repair. This study is the first to show a novel NER mechanism by which drinking GTPs prevents UV-induced immunosuppression and that inhibiting UV-induced immunosuppression may underlie the chemopreventive activity of GTPs against photocarcinogenesis.

Epigenetic targets of bioactive dietary components for cancer prevention and therapy.

The emergent interest in cancer epigenetics stems from the fact that epigenetic modifications are implicated in virtually every step of tumorigenesis. More interestingly, epigenetic changes are reversible heritable changes that are not due to the alteration in DNA sequence but have potential to alter gene expression. Dietary agents consist of many bioactive ingredients which actively regulate various molecular targets involved in tumorigenesis. We present evidence that numerous bioactive dietary components can interfere with various epigenetic targets in cancer prevention and therapy. These agents include curcumin (turmeric), genistein (soybean), tea polyphenols (green tea), resveratrol (grapes), and sulforaphane (cruciferous vegetables). These bioactive components alter the DNA methylation and histone modifications required for gene activation or silencing in cancer prevention and therapy. Bioactive components mediate epigenetic modifications associated with the induction of tumor suppressor genes such as p21(WAF1/CIP1) and inhibition of tumor promoting genes such as the human telomerase reverse transcriptase during tumorigenesis processes. Here, we present considerable evidence that bioactive components and their epigenetic targets are associated with cancer prevention and therapy which should facilitate novel drug discovery and development. In addition, remarkable advances in our understanding of basic epigenetic mechanisms as well as the rapid progress that is being made in developing powerful new technologies, such as those for sensitive and quantitative detection of epigenetic and epigenomic changes in cancer biology, hold great promise for novel epigenetic approaches to cancer prevention and therapy.

IL-12 deficiency suppresses 12-O-tetradecanoylphorbol-13-acetate-induced skin tumor development in 7,12-dimethylbenz(a)anthracene-initiated mouse skin through inhibition of inflammation.

Interleukin (IL)-12 deficiency exacerbates tumorigenesis in ultraviolet (UV) radiation-induced skin. Here, we assessed the effects of IL-12 deficiency on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated mouse skin. Using this two-stage chemical carcinogenesis protocol, we found that the development of DMBA/TPA-induced skin tumors was diminished in IL-12p40-knockout mice than in their wild-type counterparts. At the termination of the experiment (at 24 weeks), the skin tumor incidence and tumor multiplicity were significantly lower (P < 0.005) in interleukin-12-knockout (IL-12 KO) mice than in their wild-type counterparts, as was the malignant transformation of DMBA/TPA-induced papillomas to carcinomas (P < 0.01). Analysis of samples collected at the termination of the experiments for biomarkers of inflammation by immunohistochemical analysis, western blotting, enzyme-linked immunosorbent assay and real-time polymerase chain reaction revealed significantly lower levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E(2), proliferating cell nuclear antigen, cyclin D1 and the proinflammatory cytokines (tumor necrosis factor-alpha, IL-1beta and IL-6) in the DMBA/TPA-treated tumors and tumor-uninvolved skin of IL-12 KO mice than the skin and tumors of DMBA/TPA-treated wild-type mice. Analysis of the skin 6 h after TPA treatment showed that the TPA-induced promotion of skin edema, inflammatory leukocyte infiltration, COX-2 expression and PGE(2) production was significantly lower in the skin of the IL-12-KO mice than their wild-type counterparts. These results indicate that DMBA/TPA-induced skin tumor development differs from UVB-induced skin tumor development in that endogenous IL-12 acts to inhibit UVB-induced skin tumor development and malignant progression of the skin tumors to carcinoma. In the case of DMBA/TPA-induced skin tumor development, the endogenous IL-12 modulates the tumor promoter stimulation of inflammatory responses.

Loss of endogenous interleukin-12 activates survival signals in ultraviolet-exposed mouse skin and skin tumors.

Interleukin-12 (IL-12)-deficiency promotes photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated. Here, we report that long-term exposure to ultraviolet (UV) radiation resulted in enhancement of the levels of cell survival kinases, such as phosphatidylinositol 3-kinase (PI3K), Akt (Ser(473)), p-ERK1/2, and p-p38 in the skin of IL-12p40 knockout (IL-12 KO) mice compared with the skin of wild-type mice. UV-induced activation of nuclear factor-kappaB (NF-kappaB)/p65 in the skin of IL-12 KO mice was also more prominent. The levels of NF-kappaB-targeted proteins, such as proliferating cell nuclear antigen (PCNA), cyclooxygenase-2, cyclin D1, and inducible nitric oxide synthase, were higher in the UV-exposed skin of IL-12 KO mice than the UV-exposed skin of wild types. In short-term UV irradiation experiments, subcutaneous treatment of IL-12 KO mice with recombinant IL-12 (rIL-12) or topical treatment with oridonin, an inhibitor of NF-kappaB, resulted in the inhibition of UV-induced increases in the levels of PCNA, cyclin D1, and NF-kappaB compared with non-rIL-12- or non-oridonin-treated IL-12 KO mice. UV-induced skin tumors of IL-12 KO mice had higher levels of PI3K, p-Akt (Ser(473)), p-ERK1/2, p-p38, NF-kappaB, and PCNA and fewer apoptotic cells than skin tumors of wild types. Together, these data suggest that the loss of endogenous IL-12 activates survival signals in UV-exposed skin and that may lead to the enhanced photocarcinogenesis in mice.