Melanoma is the most dangerous form of skin cancer, with poor prognosis in advanced stages. Vitamin D, also produced by ultraviolet radiation, is known for its anti-proliferative properties in some cancers including mel-anoma. While vitamin D deficiency has been associated with advanced melanoma stage and higher levels of vitamin D have been associated with better outcomes, the role for vitamin D in melanoma remains unclear. Vitamin D synthesis is initiated upon UVB exposure of skin cells and results in formation of the active metabolite 1,25-dihydroxyvitamin D3 (1,25D). We have previously demonstrated that 1,25D plays a role in protection against ultraviolet radiation-induced DNA damage, immune suppression, and skin carcinogenesis. In this study 1,25D significantly reduced cell viability and increased caspase levels in human melanoma cell lines. This effect was not present in cells that lacked both phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a well-known tumour suppressor, and the vitamin D receptor (VDR). PTEN is frequently lost or mutated in mel-anoma. Incubation of selected melanoma cell lines with 1,25D resulted in significant increases in PTEN levels and downregulation of the AKT pathway and its downstream effectors. This suggests that 1,25D may act to reduce melanoma cell viability by targeting PTEN.

Susceptibility to photoimmune suppression and photocarcinogenesis is greater in male than in female humans and mice and is exacerbated in female estrogen receptor-beta knockout (ER-β−/−) mice. We previously reported that the active vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), applied topically protects against the ultraviolet radiation (UV) induction of cutaneous cyclobutane pyrimidine dimers (CPDs) and the suppression of contact hypersensitivity (CHS) in female mice. Here, we compare these responses in female versus male Skh:hr1 mice, in ER-β−/−/−− versus wild-type C57BL/6 mice, and in female ER-blockaded Skh:hr1 mice. The induction of CPDs was significantly greater in male than female Skh:hr1 mice and was more effectively reduced by 1,25(OH)2D in female Skh:hr1 and C57BL/6 mice than in male Skh:hr1 or ER-β−/− mice, respectively. This correlated with the reduced sunburn inflammation due to 1,25(OH)2D in female but not male Skh:hr1 mice. Furthermore, although 1,25(OH)2D alone dose-dependently suppressed basal CHS responses in male Skh:hr1 and ER-β−/− mice, UV-induced immunosuppression was universally observed. In female Skh:hr1 and C57BL/6 mice, the immunosuppression was decreased by 1,25(OH)2D dose-dependently, but not in male Skh:hr1, ER-β−/−, or ER-blockaded mice. These results reveal a sex bias in genetic, inflammatory, and immune photoprotection by 1,25(OH)2D favoring female mice that is dependent on the presence of ER-β.

Sunlight has the potential to induce DNA damage in the skin. If these photolesions are left unrepaired, they can greatly increase the risk of skin cancer. This process is facilitated by another UV-induced insult, photoimmune suppression. Vitamin D metabolites have demonstrated a marked ability to mitigate these negative effects. The photoprotective pathway still remains elusive. Recent research, however, points to the important role of the vitamin D receptor and the endoplasmic reticulum protein ERp57 in mediating the actions of vitamin D compounds. Determining the functional relationship between vitamin D metabolites, the VDR/ERp57 complex, and downstream DNA repair mechanisms will contribute to the greater understanding of this photoprotective system. Furthermore, in light of evidence suggesting that CYP27B1 is not necessary for photoprotection, strong emphasis must now be placed on examining other vitamin D metabolic pathways and the actions of new metabolites in photoprotection. Recent literature points to 20(OH)D3 as a possible alternative to 1,25(OH)2D3. However, further work must be conducted to determine the photoprotective pathway of this vitamin D derivative. Keywords: 1, 25-dihydroxyvitaminD3, 20-hydroxyvitamin D3, photoprotection, DNA damage, photoimmune suppression, VDR, ERp57.

Topical 1,25-dihydroxyvitamin D (1,25D) and other vitamin D compounds have been shown to protect skin from damage by ultraviolet radiation (UVR) in a process that requires the vitamin D receptor. Yet, while mice which do not express the vitamin D receptor are more susceptible to photocarcinogenesis, mice unable to 1α-hydroxylate 25-hydroxyvitamin D to form 1,25D do not show increased susceptibility to UVR-induced skin tumors. A possible explanation is that an alternative pathway, which does not involve 1α-hydroxylation, may produce photoprotective compounds from vitamin D. The cholesterol side chain cleavage enzyme CYP11A1 is expressed in skin and produces 20-hydroxyvitamin D3 (20OHD) as a major product of vitamin D3. We examined whether topical 20OHD would affect UVR-induced DNA damage, inflammatory edema or immune suppression produced in Skh:hr1 mice. Photoprotection by 20OHD at 23 or 46 pmol/cm2 against cyclobutane pyrimidine dimers (DNA lesions) after UVR in mice was highly effective, up to 98 ± 0.8%, (p < 0.001) and comparable to that of 1,25D. Sunburn edema measured as skinfold thickness 24 h after UVR was also significantly reduced by 20OHD (p < 0.001). In studies of contact hypersensitivity (CHS), which is suppressed by UVR, topical application of 20OHD to mice protected against UVR-induced immunosuppression (p < 0.05), similar to the effect of 1,25D at similar doses (46 ± 0.6% protection with 20OHD, 44 ± 0.5% with 1,25D). Both UVR-induced DNA damage and immunosuppression contribute to increased susceptibility to UVR-induced skin tumors. This study indicates a potentially anti-photocarcinogenic role of the naturally occurring vitamin D metabolite, 20OHD, which does not depend on 1α-hydroxylation for generation. This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’.

AbstractThe actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers. Cancer Res; 73(16); 5169–82. ©2013 AACR.

Vitamin D production requires UVB. In turn, we have shown that vitamin D compounds reduce UV-induced damage, including inflammation, sunburn, thymine dimers, the most frequent type of cyclobutane pyrimidine dimer, immunosuppression, and photocarcinogenesis. Our previous studies have shown most of the photoprotective effects by 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) occurred through the nongenomic pathway because similar protection was seen with an analog, 1 alpha,25-dihydroxylumistrol(3) (JN), which has little ability to alter gene expression and also because a nongenomic antagonist of 1,25(OH)(2)D-3 abolished protection. In the current study, we tested whether this photoprotective effect would extend to other types of DNA damage, and whether this could be demonstrated in human ex vivo skin, as this model would be suited to pre-clinical testing of topical formulations for photoprotection. In particular, using skin explants, we examined a time course for thymine dimers (TDs), the most abundant DNA photolesion, as well as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is a mutagenic DNA base lesion arising from UV-induced oxidative stress, and 8-nitroguanosine (8-NG). Nitric oxide products, known markers for chronic inflammation and carcinogenesis, are also induced by UV. This study showed that 1,25(OH)(2)D-3 significantly reduced TD and 8-NG as early as 30 min post UV, and 8-oxodG at 3 h post UV, confirming the photoprotective effect of 1,25(OH)(2)D-3 against DNA photoproducts in human skin explants. At least in part, the mechanism of photoprotection by 1,25(OH)(2)D-3 is likely to be through the reduction of reactive nitrogen species and the subsequent reduction in oxidative and nitrosative damage.This article is part of a Special Issue entitled 'Vitamin D Workshop'. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

As skin cancer is one of the most costly health issues in many countries, particularly in Australia, the possibility that vitamin D compounds might contribute to prevention of this disease is becoming increasingly more attractive to researchers and health communities. In this article, important epidemiologic, mechanistic and experimental data supporting the chemopreventive potential of several vitamin D-related compounds are explored. Evidence of photoprotection by the active hormone, 1α,25dihydroxyvitamin D3, as well as a derivative of an over-irradiation product, lumisterol, a fluorinated analog and bufalin, a potential vitamin D-like compound, are provided. The aim of this article is to understand how vitamin D compounds contribute to UV adaptation and potentially, skin cancer prevention.

Hypoxia inducible factor-1α (HIF-1α), a ubiquitous inducible oxygen-sensing transcription factor, promotes cell survival under hypoxic conditions, including the early pre-angiogenic period of tumorigenesis, and is known to contribute to many malignancies. However HIF-1α can also be activated by inflammatory mediators, and can activate inflammation-modulating proteins itself, including heme oxygenase-1 (HO-1) and the cytokine IL-6. Recently HIF-1α was reported to be induced by UVB (290–320 nm) radiation in cultured human keratinocytes, acting as a stress protein associated with the release of reactive oxygen species. In this in vivo murine study we demonstrate that HIF-1α protein is an early responder to UV radiation in the skin, and its activation can be attenuated by treating mice with its post-translational inhibitor, YC-1. Treatment with YC-1 following UV-irradiation of mice has revealed the involvement of HIF-1α in UV-induced inflammation, IL-6 production, and epidermal hyperplasia. In addition, upregulated cutaneous HIF-1α was found to be an important factor in the UV-suppression of T cell-mediated immunity, measured by contact hypersensitivity (CHS). The mechanism remains unclear, however it did not appear to involve the immunosuppressive cutaneous photoproduct cis -urocanic acid, but HIF-1α induction was inhibited by irradiation with photoimmune protective UVA (320–400 nm), implicating a negative correlation between the two stress proteins, HIF-1α and the photoimmune protective UVA responder HO-1.

Epidermal squamous cell carcinoma (SCC) is the most aggressive non-melanoma skin cancer and is dramatically increased in patients undergoing immunosuppression following solid organ transplantation, contributing substantially to morbidity and mortality. Recent clinical studies show that use of the mammalian target of rapamycin (mTOR) inhibitor rapamycin as a post-transplantation immunosuppressive significantly reduces SCC occurrence compared with other immunosuppressives, though the mechanism is not fully understood. We show that rapamycin selectively upregulates epidermal Akt1, while failing to upregulate epidermal Akt2. Rapamycin increases epidermal Akt1 phosphorylation via inhibition of the mTOR complex 1-dependent regulation of insulin receptor substrate-1. Epidermal Akt1 is commonly downregulated in SCC while Akt2 is upregulated. We now demonstrate similar Akt1 downregulation and Akt2 upregulation by ultraviolet (UV) radiation, the most important skin carcinogen. Hence, rapamycin's upregulation of Akt1 signaling could potentially oppose the effects of UV radiation and/or tumor-associated changes on Akt1 signaling. We show in skin culture that rapamycin does enhance restoration of Akt1 phosphorylation in skin recovering from UV radiation, suggesting a mechanism for rapamycin's antitumor activity in epidermis in spite of its efficient immunosuppressive properties.

Topical application of lotions containing the phytoestrogenic isoflavonoid equol have been reported to protect mice against UV radiation-induced inflammation, immune suppression and photocarcinogenesis. The photoimmune protective property was shown to depend on equol’s activation of oestrogen receptor signalling in the skin. However, isoflavones are also recognised for their antioxidant properties in biological systems. As endogenous cutaneous antioxidant enzymes including the inducible stress protein haem oxygenase (HO)-1, have photoprotective efficacy, this study in the Skh:hr-1 hairless mouse seeks evidence for an antioxidant role for equol in contributing to its photoimmune protection. Oxidative stress has been measured as UVA-induced lipid peroxidation in the mouse skin, and was dose-dependently inhibited by topical equol. Inhibition of the UVA (320-400 nm)-inducible HO activity significantly reduced the level of equol protection against lipid peroxidation, thereby attributing a component of equol’s lipid protection capacity to this stress enzyme. It was consistent that topical equol enhanced the level of HO induction by UVA irradiation in both skin and liver. Subsequently, the dose-dependent protection by topical equol lotions against solar simulated UV radiation induced immunosuppression, measured by the contact hypersensitivity reaction, was found also to be partially reduced by the inhibition of HO activity. Therefore, in addition to the activation by equol of oestrogenic signalling pathways for photoprotection, this isoflavonoid also provides UV-protective antioxidant effects that depend partially on HO-1 induction.

Abstract The actin cytoskeleton is fundamental in the regulation of cellular processes involved in tumourigenesis and is therefore a highly desirable chemotherapeutic target. However, the non-specific nature of actin-targeting drugs makes them ineffective due to their toxic action on the heart. We have designed a class of drugs that target the second core component of actin filaments, tropomyosin. Tm5NM1, a cytoskeletal tropomyosin isoform, is upregulated in a variety of tumour cells and patient samples, and is known to promote tumour cell growth. Our lead drug, TR100, targets Tm5NM1 and potentially other isoforms, reducing cell proliferation in vitro in a panel of melanoma and neuroblastoma cell lines and decreases the rate of tumour growth in vivo in melanoma and neuroblastoma mouse models without affecting heart integrity. The difficulty in improving the specificity of these anti-tropomyosin compounds for Tm5NM1 was the lack of a suitable cell-based system to test the drugs. NIH3T3 stable cells overexpressing fluorophore-tagged Tm5NM1 or the muscle-specific isoform αfastTm now allows us to determine the impact of our drugs on functionally distinct actin populations. Since tropomyosin is a structural protein with no known measurable catalytic activity it has previously been challenging to design a high throughput assay to quantitate specific changes in actin filament architecture. High content imaging provides a system to image actin filament integrity which can be quantified using an algorithm designed in collaboration with the CSIRO. Using this algorithm we can calculate the impact of the anti-tropomyosin compounds on actin filaments in co-cultured stable cells. Development of these assays will contribute to the design of more specific drugs which target defined tropomyosin containing actin populations highly expressed in cancer without showing toxicity in muscle, thereby improving the therapeutic index of anti-tropomyosin drugs for the clinical treatment of a wide array of cancers. 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 5230. doi:1538-7445.AM2012-5230

UVR produces vitamin D in skin, which is hydroxylated locally to 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). 1,25(OH)(2)D(3) protects skin cells against UVR-induced DNA damage, including thymine dimers, but the mechanism is unknown. As DNA repair is inhibited by nitric oxide (NO) products but facilitated by p53, we examined whether 1,25(OH)(2)D(3) altered the expression of nitrotyrosine, a product of NO, or p53 after UVR in human keratinocytes. 1,25(OH)(2)D(3) and the nongenomic agonist 1α,25-dihydroxylumisterol(3) reduced nitrotyrosine 16 hours after UVR, detected by a sensitive whole-cell ELISA. p53 was enhanced after UVR, and this was further augmented in the presence of 1,25(OH)(2)D(3). DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a chloride channel blocker previously shown to prevent 1,25(OH)(2)D(3)-induced chloride currents in osteoblasts, had no effect on thymine dimers on its own but prevented the 1,25(OH)(2)D(3)-induced protection against thymine dimers. Independent treatment with DIDS, at concentrations that had no effect on thymine dimers, blocked UVR-induced upregulation of p53. In contrast, reduction of nitrotyrosine remained in keratinocytes treated with 1,25(OH)(2)D(3) and DIDS at concentrations shown to block decreases in post-UVR thymine dimers. These results suggest that 1,25(OH)(2)D(3)-induced chloride currents help protect from UVR-induced thymine dimers, but further increases in p53 or reductions of nitrotyrosine by 1,25(OH)(2)D(3) are unlikely to contribute substantially to this protection.

UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.

Table 1. 4 minutes of frequency 40/116 at (200microamps) immediately after painting of AA gives a 70% reduction in ear swelling. 4 minutes of frequency 40/355 at (200microamps) immediately after painting AA gives a 70% reduction in ear swelling Test hypothesis using an ear inflammation model – Arachadonic acid induced swelling. Male SKh-1 albino hairless mice aged 6 weeks. Ears were measured with a micrometer, 5l of 25mg/ml of Arachadonic acid in ethanol, was painted on both sides of the ear and both ears were treated. FSM was applied immediately after it had dried and measurements were taken at 1 hour post application. The mean ear swelling for the change in thickness was determined and presented in a table.

Abstract Background: The actin cytoskeleton is an important regulator of a variety of cellular functions including cell motility, adhesion, and proliferation, making it an ideal chemotherapeutic target. Despite this promise there are still no anti-actin compounds used in current chemotherapy, primarily due to the inability of existing anti-actin agents to discriminate between the actin cytoskeleton of tumor cells and the actin filaments of the muscle sarcomere. We have previously shown that tropomyosin, an integral component of the actin cytoskeleton, defines functionally distinct populations of actin filaments. We have identified a specific tropomyosin isoform common to all tumor cells tested to date which regulates cell proliferation and have designed a new class of compounds to target this filament population. Summary of Results: Anti-tropomyosin (Tm) compounds were selected based their ability to target the actin cytoskeleton and their efficacy against a panel of neuroblastoma and melanoma cell lines. The lead compound, TR100, was shown to be effective against a panel of tumor cell lines with an average EC50 of 3μM. When tested in 3D melanoma spheroid models, which more accurately mimic the tumor microenvironment, TR100 inhibited melanoma cell growth and motility. This effect translated to a reduction in tumor cell growth in vivo in both neuroblastoma and melanoma xenograft models. In vitro data using isolated rat cardiomyocytes demonstrated that TR100 had minimal impact on contractile function. In vivo data from the drug treated animals also showed no evidence of cardiac damage as measured by blood Troponin I levels and no changes in the intraventricular septum thickness of isolated hearts. These results demonstrate that it is possible to target distinct actin filament populations based on the tropomyosin composition. Next generation anti-Tm compounds with improved efficacy and specificity have now been developed. Preliminary data demonstrate that these compounds exhibit increased selectivity for transformed cells. Conclusions: Taken together, our findings suggest that the anti-Tm compounds show a significant improvement in the therapeutic window compared to existing anti-actin agents. This novel approach and the development of the new class of anti-Tm compounds may be the key for disabling a long sought after target, the actin cytoskeleton, and may lead to a new class of chemotherapeutics active against a broad range of cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A178.

Immunosuppression attributed mainly to the UVB (290–320 nm) waveband is a prerequisite for skin cancer development in mice and humans. The contribution of UVA (320–400 nm) is controversial, but in mice UVA irradiation has been found to antagonise immunosuppression by UVB. In other studies of photoimmune regulation, protection mediated via oestrogen receptor-β signalling was identified as a normal endogenous defence in mice, and was shown to depend on UVA irradiation. A gender bias in photoimmune responsiveness was thus suggested, and is tested in this study by comparing the UV-induced inflammatory and immune responses in male and female hairless mice. We report that male mice, which show greater skin thickness than females, developed a less intense but slower resolving sunburn inflammatory oedema, correlated with reduced epidermal expression of pro-inflammatory IL-6 than females following solar simulated UV (SSUV, 290–400 nm) exposure. On the other hand, the contact hypersensitivity reaction (CHS) was more severely suppressed by SSUV in males, correlated with increased epidermal expression of immunosuppressive IL-10. Exposure to the UVB waveband alone, or to cis -urocanic acid, suppressed CHS equally in males and females. However, whereas UVA irradiation induced immunoprotection against either UVB or cis -urocanic acid in females, this protection was significantly reduced or abrogated in males. The results indicate that males are compromised by a relative unresponsiveness to the photoimmune protective effects of UVA, alone or as a component of SSUV. This could explain the known gender bias in skin cancer development in both mice and humans.

Exposure to ultraviolet radiation (UVR) can lead to a range of deleterious responses in the skin. An important form of damage is the DNA photolesion cyclobutane pyrimidine dimer (CPD). CPDs can be highly mutagenic if not repaired prior to cell division and can lead to UV-induced immunosuppression, making them potentially carcinogenic. UVR exposure also produces vitamin D, a prehormone. Different shapes of the steroid hormone 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] can produce biological responses through binding either to its cognate nuclear receptor (VDR) to regulate gene transcription or to the VDR associated with plasma membrane caveolae to produce, via signal transduction, nongenomic physiologic responses. Here, we show that both 1,25(OH)₂D₃ and 1α,25(OH)₂-lumisterol (JN), a conformationally restricted analogue that can generate only nongenomic responses, are effective inhibitors of UV damage in an immunocompetent mouse (Skh:hr1) model susceptible to UV-induced tumors. Both 1,25(OH)₂D₃ and JN significantly reduced UVR-induced CPD, apoptotic sunburn cells, and immunosuppression. Furthermore, these compounds inhibited skin tumor development, both papillomas and squamous cell carcinomas, in these mice. The observed reduction of these UV-induced effects by 1,25(OH)₂D₃ and JN suggests a role for these compounds in prevention against skin carcinogenesis. To the best of our knowledge, this is the first comprehensive report of an in vivo long-term biological response generated by chronic dosing with a nongenomic-selective vitamin D steroid.