Jacob Junco

Effects of combined phytochemicals on skin tumorigenesis in SENCAR mice.

The purpose of our study was to determine the effect of the combined action of phytochemicals on the early stages of skin tumorigenesis, i.e. initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (UA) were applied topically. The 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted multistage skin carcinogenesis model in SENCAR mice was used. Mice received one topical dose of DMBA, then after one month, two weekly doses of TPA for 14 weeks until sacrifice. RES or UA were applied 20 min prior to DMBA or TPA treatment and 2% dietary CG was given from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. UA applied alone and in combination with CG during the promotion stage was the only inhibitor of tumor multiplicity and tumor incidence. A number of combinations reduced epidermal proliferation, but only UA and the combination UA+CG applied during promotion significantly reduced epidermal hyperplasia. DMBA/TPA application resulted in significant increases in c-jun and p50, which were reversed by a number of different treatments. DMBA/TPA treatment also strongly increased mRNA levels of inflammation markers COX-2 and IL-6. All anti-promotion treatments caused a marked decrease in COX-2 and IL-6 expression compared to the DMBA/TPA control. These results show that UA is a potent inhibitor of skin tumor promotion and inflammatory signaling and it may be useful in the prevention of skin cancer and other epithelial cancers in humans.

Resveratrol and P-glycoprotein Inhibitors Enhance the Anti-Skin Cancer Effects of Ursolic Acid

Ursolic acid, present in apples, rosemary, and other sources, is known to inhibit tumor formation and tumor cell viability in multiple systems, including skin. However, various cancers are resistant to ursolic acid treatment. Herein, skin carcinoma cells (Ca3/7) as compared with skin papilloma cells (MT1/2) displayed more resistance to ursolic acid-induced cytotoxicity. Interestingly, Ca3/7 cells had elevated levels of P-glycoprotein (P-gp), an ATP-dependent efflux pump that mediates resistance to chemotherapy in preclinical and clinical settings, and not only accumulated less but also more rapidly expelled the P-gp substrate rhodamine 123 (Rh123) indicating ursolic acid is transported by P-gp. To determine whether P-gp inhibition can enhance ursolic acid-mediated cytotoxicity, cells were challenged with P-gp inhibitors verapamil or cyclosporin A. Alternatively, cells were pretreated with the natural compound resveratrol, a known chemotherapy sensitizer. Verapamil and resveratrol enhanced the effects of ursolic acid in both cell lines, whereas cyclosporin A only did so in Ca3/7 cells. Similarly, verapamil inhibited Rh123 efflux in both lines, whereas cyclosporin A only inhibited Rh123 efflux in Ca3/7 cells. Resveratrol did not inhibit Rh123 efflux in either line, indicating the synergistic effects of resveratrol and ursolic acid are not manifest by inhibition of P-gp–mediated efflux of ursolic acid. These results indicate that the anti-skin cancer effects of ursolic acid are enhanced with P-gp inhibitors. In addition, resveratrol and ursolic acid interact synergistically, but not through inhibition of P-gp. Implications: Resveratrol and/or p-glycoprotein inhibitors in combination with ursolic acid are an effective anti-skin cancer regimen. Mol Cancer Res; 11(12); 1521–9. ©2013 AACR.

Ursolic acid and resveratrol synergize with chloroquine to reduce melanoma cell viability

Malignant melanoma is associated with a 5-year survival rate of less than 20% once metastasized. Malignant melanoma cells exhibit increased levels of autophagy, a process of intracellular digestion that allows cells to survive various stresses including chemotherapies, resulting in reduced patient survival. Autophagy can be inhibited by chemicals like chloroquine (CQ), which prevents fusion of autophagosomes to lysosomes, resulting in autophagosome accumulation in most systems. Here, we describe how tested CQ to see whether it could sensitize B16F10 metastatic mouse melanoma cells to the anticancer activities of the natural compounds ursolic acid (UA) and resveratrol (RES). CQ with UA or RES strongly and synergistically reduced the viability of B16F10 mouse melanoma and A375 human melanoma cells. Surprisingly, flow cytometry of acridine orange-stained cells showed that UA or RES in combination with CQ significantly reduced autophagosome levels. Western blotting analysis revealed that CQ plus UA or RES paradoxically increased LC3II, indicative of autophagosome accumulation. In addition, CQ plus RES synergistically decreased the levels of both autophagy initiator beclin-1 and autophagy supporter p62. These results indicate that CQ with UA or RES strongly and synergistically reduces the viability of B16F10 and A375 melanoma cells. However, studies on B16F10 cells have shown that the synergistic effect was not mediated by inhibition of autophagy induced by UA or RES. These compounds are well-tolerated in humans, and CQ has shown promise as an adjuvant therapy. These combinations may be valuable treatment strategies for melanoma.

Differential effects on lung cancer cell proliferation by agonists of glucocorticoid and PPARα receptors

Glucocorticoids (GCs) are well‐known anti‐inflammatory compounds, but they also inhibit cell proliferation depending on cell type. Similarly, peroxisome proliferator‐activated receptors (PPARα, PPARδ, and PPARγ) also possess anti‐proliferation properties beyond their canonical roles as metabolic mediators. In the present study, we investigated the potential additive or synergistic inhibitory effects on cancer cell proliferation by simultaneous application of fenofibrate and budesonide, agonists for PPARα and glucocorticoid receptor, respectively. We observed differential effects on cell proliferation in A549 and SK‐MES‐1 lung cancer cells by budesonide and fenofibrate. Fenofibrate inhibited cell proliferation in both TP53 wild type and deficient lung cancer cells. The anti‐proliferation effect of budesonide in TP53 wild type A549 cells was abolished in SK‐MES‐1 cells that do not have wild type TP53 protein. An additive effect against cell proliferation by budesonide and fenofibrate combination was observed only in TP53 wild type A549 cancer cells. Analysis of cell cycle distribution and cyclin profile indicated that the inhibition of cell proliferation was associated with G1 cell cycle arrest. The suppression of NF‐κB activity and ERK signaling may contribute to the inhibition of cell proliferation by budesonide and or fenofibrate. The additive inhibitory effect on cell proliferation by budesonide and fenofibrate combination suggests that the same or greater therapeutic effect could be achieved with reduced dosage and side effects when the two compounds are applied simultaneously.

DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells

Timely removal of oxidatively damaged proteins is critical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modification that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 (deleted in split hand/split foot 1), an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSS1-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin-proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as a modifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.

The effects of dissociated glucocorticoids RU24858 and RU24782 on TPA‐induced skin tumor promotion biomarkers in SENCAR mice

Glucocorticoids (GCs) are very effective at preventing carcinogen‐ and tumor promoter‐induced skin inflammation, hyperplasia, and mouse skin tumor formation. The effects of GCs are mediated by a well‐known transcription factor, the glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA‐binding (transactivation) and DNA binding‐independent protein–protein interactions between GR and other transcription factors, such as nuclear factor kappa B (NF‐κB) or activator protein 1 (AP‐1; transrepression). We hypothesize that the transrepression activities of the GR are sufficient to suppress skin tumor promotion. We obtained two GCs (RU24858 and RU24782) that have dissociated downstream effects and induce only transrepression activities of the GR in a number of systems. These compounds bind the GR with high affinity and repress AP‐1 and NF‐κB activities while showing a lack of GR transactivation. RU24858, RU24782, or control full GCs desoximetasone (DES) and fluocinolone acetonide (FA) were applied to the dorsal skin of SENCAR mice prior to application of the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), two times per week for 2 weeks. DES, FA and RU24858 reversed TPA‐induced epidermal hyperplasia and proliferation, while RU24782 treatment had no effect on these markers of skin tumor promotion. All tested compounds decreased TPA‐induced c‐jun mRNA levels in skin. DES, FA, and RU24858, but not RU24782, were also able to reverse TPA‐induced increases in the mRNA levels of COX‐2 and iNOS. These findings show that RU24858 but not RU24782 reduced TPA‐induced epidermal hyperplasia, proliferation, and inflammation, while both compounds reversed c‐jun mRNA increases in the skin.

Role of AMPK and PPARα in the anti-skin cancer effects of ursolic acid

The phytonutrient ursolic acid (UA), present in apples, rosemary, and other plant sources, has anti‐cancer properties in a number of systems, including skin cancers. However, few reports have examined upstream mechanisms by which UA may prevent or treat cancer. Recent reports have indicated UA induces death of cancer cell lines via AMP‐activated protein kinase (AMPK), an energy‐sensing kinase which possesses both pro‐metabolic and anti‐cancer effects. Other studies have shown UA activates peroxisome proliferator activated receptor α (PPARα) and the glucocorticoid receptor (GR). Here, we found the cytotoxic effect of UA in skin carcinoma cells required AMPK activation. In addition, two inhibitors of PPARα partially reversed the cytotoxic effects of UA, suggesting its effects are at least partially mediated through this receptor. Finally, inhibition of the GR did not reverse the effects of UA nor did this compound bind the GR under the conditions of experiments performed. Overall, studies elucidating the anti‐cancer effects of UA may allow for the development of more potent analogues utilizing similar mechanisms. These studies may also reveal the mediators of any possible side effects or resistance mechanisms to UA therapy.

Harnessing the gatekeepers of glucocorticoids for chemoprevention of non-melanoma skin cancer

Despite effective surgical methods for non‐melanoma skin cancer (NMSC), patients suffer from tissue damage, scarring, or even disfigurement; thus, there is a need for chemopreventive approaches. Because of the complex interplay between glucocorticoids (GCs), inflammation, and cancer, we sought to determine the role of 11β‐hydroxysteroid dehydrogenase 1 and 2 (11βHSD1 and 2) in regulating GCs during skin cancer development and progression. 11βHSDs modulate the activation of GCs in a tissue‐specific manner and have been reported to play a role in development and progression of other types of cancer, but their role has not yet been reported in NMSC. Here, we found a significant upregulation of 11βHSD2 protein in skin cancer cells when compared to normal skin cells, suggesting a role for this enzyme in the multifactorial process of skin cancer development. In addition, inhibition of 11βHSD2 with siRNA resulted in significant reduction in colony formation in vitro. Finally, our in vivo study elucidated that inhibition of 11βHSD2 with pharmacological inhibitor, Glycyrrhetinic acid (GA) could significantly diminish tumorigenesis in a well‐studied in vivo mouse model of NMSC. Overall, these studies highlight for the first time a potential novel role for 11βHSD2 in NMSC development and may allow for new GC treatment approaches capable of avoiding deactivation by the enzyme. If 11βHSD2 can be inhibited as we have done here, or circumvented using modified GCs, this may lead to more efficacious outcomes for NMSC patients by preventing deactivation of the GC and minimizing resistance.

Abstract 1899: Anti-skin tumor promoting effects of pentacyclic triterpenes found in Perilla frutescens

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA In this study, we conducted a comprehensive examination of a series of pentacyclic tritperpenes found in P. frutescens, including ursolic acid (UA) oleanolic acid (OA), augustic acid (AA), corosolic acid (CA), 3-epi-corosolic acid (3-epiCA), maslinic acid (MA), and 3-epi-maslinic acid (3-epiMA) for their effects on epidermal cell signaling, proliferation, and skin inflammation in relation to their ability to inhibit skin tumor promotion by TPA. For these experiments, female ICR mice (7-9 weeks of age) were treated with either acetone or 2 μmol of UA, OA, AA, CA, 3-epiCA, MA, or 3-epiMA 30 min prior to TPA (6.8 nmol) treatment The treatment protocol involved twice weekly treatments over a two week period. Mouse epidermis (6 hr time point) and whole skin (48 hr time point) were collected for Western blot analysis and histological analysis, respectively. Pretreatment with these compounds inhibited the activation of JNK1/2, Src, and Stat3 as well as the induction of Cox-2 protein. Pretreatment with the compounds also reversed the effect of TPA on PDCD4 and p27, and increased the activation of AMPK and LKB1. We also examined the effect of the compounds on TPA-induced epidermal hyperproliferation as assessed by epidermal thickness and epidermal labeling index (LI). All of the compounds examined significantly inhibited TPA-induced epidermal hyperproliferation (significant reductions in both epidermal thickness and LI). In addition, the effect of the triterpenes on TPA-induced infiltration of mast cells in dermis was evaluated. UA significantly inhibited the infiltration of dermal mast cells induced by TPA. Notably, OA, AA, CA, 3-epiCA, MA, and 3-epiMA produced a greater inhibitory effect than that seen with UA on the number of infiltrated mast cells. Thus, UA and a series of related triterpenes differentially inhibited multiple signaling pathways, epidermal hyperproliferation, and a marker of skin inflammation (infiltrated mast cells) induced by TPA. Several of the compounds including CA, 3-epiCA, MA, and 3-epiMA were particularly effective in these experiments. We are currently testing the ability of this group of compounds to inhibit skin tumor promotion by TPA in a two-stage skin carcinogenesis experiment. Collectively, the current data suggest that several pentacyclic triterpenes, in addition to UA, may have potent chemopreventive activity. Research supported by CA164159. Citation Format: Jiyoon Cho, Okkyung Rho, Jacob Junco, Thomas J. Slaga, Andrew M. Camelio, Dionicio Siegel, John DiGiovanni. Anti-skin tumor promoting effects of pentacyclic triterpenes found in Perilla frutescens. [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 1899. doi:10.1158/1538-7445.AM2015-1899

Abstract 1596: Effect of combined treatment with ursolic acid (UA) and resveratrol (Res) on skin tumor promotion by TPA

In recent years, emerging evidence suggests that cancer chemopreventive agents when combined might be more effective than when given alone. Both UA and Res have been shown to reduce inflammation and tumor development in various tissues. In mouse skin, both compounds have been shown to block skin tumor promotion by TPA and reduce TPA-induced inflammation. In this study, we have further evaluated the effect of UA and Res on skin tumor promotion with emphasis on examining combinations of these agents. For these experiments, UA, Res and combinations of UA + Res were applied topically (various doses) prior to TPA treatment on mouse skin. The combination of UA + Res showed additive and possibly synergistic inhibition of TPA-induced epidermal hyperproliferation as assessed by both epidermal hyperplasia and BrdU labeling index. The effect of UA, Res and the combination of UA + Res on epidermal signaling pathways was also evaluated. The combination of UA + Res significantly inhibited activation of epidermal Akt, NF-κB, p38 MAPK, and JNK in response to TPA treatment compared to the treatment with the individual compounds. The combination of UA + Res also dramatically reduced the induction of epidermal Cox-2 by TPA. Notably, combined treatment with UA and Res induced a dramatic increased phosphorylation of AMPK-α suggesting that AMPK activation may also play a critical role in the ability of the combination to alter skin tumor promotion. Combination treatment during skin tumor promotion showed greater inhibition of tumor multiplicity than with either agent alone. In further experiments, the effect of UA, Res and UA + Res on proliferation of bulge-region keratinocyte stem cells (KSCs) was evaluated. Both UA and Res treatment inhibited TPA-induced proliferation and migration of bulge-region KSCs. Furthermore, the combination of UA + Res appeared to be more effective at blocking proliferation of these cells from the hair follicle when compared with the single compound-treated groups. Taken together, these data suggest that the combination of UA + Res has either additive or synergistic inhibitory effects on keratinocyte proliferation (including KSCs) and various signaling pathways. These effects may explain the greater inhibition of skin tumor promotion by the combination of UA and Res. 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 1596. doi:1538-7445.AM2012-1596