G. Timothy Bowden

Chemoprevention of human skin cancer.

The incidence of skin cancer has been rising in recent years with significant effects on public health. Primary prevention has proven inadequate in impacting the incidence of skin cancer, thus stimulating the development of chemopreventive strategies. The majority of skin cancer chemoprevention studies focus on occurrence of new nonmelanoma skin cancers (NMSC) in individuals with a previous NMSC, or on reduction in the number of premalignant skin lesions such as actinic keratoses (AK). Dysplastic nevi, a likely precursor of melanoma, are also potential targets for chemoprevention strategies. Premalignant lesions are especially attractive as endpoints since they are more common than frank cancer, resulting in reduced sample size, length, and cost of clinical trials. Development of new agents that affect the pathogenesis of skin cancer will be discussed, from elucidation of molecular targets to implementation of trials designed to determine the effects of chemopreventive interventions on human skin cancer.

The Role of JNK and p38 MAPK Activities in UVA-Induced Signaling Pathways Leading to AP-1 Activation and c-Fos Expression

To further delineate ultraviolet A (UVA) signaling pathways in the human keratinocyte cell line HaCaT, we examined the potential role of mitogen-activated protein kinases (MAPKs) in UVA-induced activator protein-1 (AP-1) transactivation and c-Fos expression. UVA-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK) proteins was detected immediately after irradiation and disappeared after approximately 2 hours. Conversely, phosphorylation of extracellular signal-regulated kinase was significantly inhibited for up to 1 hour post-UVA irradiation. To examine the role of p38 and JNK MAPKs in UVA-induced AP-1 and c-fos transactivations, the selective pharmacologic MAPK inhibitors, SB202190 (p38 inhibitor) and SP600125 (JNK inhibitor), were used to independently treat stably transfected HaCaT cells in luciferase reporter assays. Both SB202190 and SP600125 dose-dependently inhibited UVA-induced AP-1 and c-fos transactivations. SB202190 (0.25-0.5 microM) and SP600125 (62-125 nM) treatments also primarily inhibited UVA-induced c-Fos expression. These results demonstrated that activation of both JNK and p38 play critical role in UVA-mediated AP-1 transactivation and c-Fos expression in these human keratinocyte cells. Targeted inhibition of these MAPKs with their selective pharmacologic inhibitors may be effective chemopreventive strategies for UVA-induced nonmelanoma skin cancer.

Pharmacology of mitoxantrone: mode of action and pharmacokinetics

SummaryAlthough a number of investigators have established that mitoxantrone (Novantrone®; dihydroxyanthracenedione) inhibits RNA and DNA synthesis and intercalates with DNA in vitro, its exact mechanism of action is unclear. Mitoxantrone is structurally related to a series of substituted anthraquinones and has features known to be essential for DNA intercalation; however, we have determined recently that mitoxantrone binds DNA in intact L1210 leukemia cells by a non-intercalative, electrostatic interaction and induces both protein associated and non-protein associated DNA strand scissions. The difference between mitoxantrone and doxorubicin with respect to their interactions with DNA could account for their relative lack of cross-resistance in the treatment of lymphoma and acute leukemia.Distribution and half-life data provide a pharmacological rationale for the use of mitoxantrone on an intermittent dosing schedule. Considerable evidence exists to suggest that mitoxantrone undergoes extensive metabolism, probably in the liver. Preliminary data show that abnormal liver function leads to decreased rates of total body mitoxantrone clearance, suggesting a possible need for dose reduction in patients with severe liver dysfunction. The most important route of mitoxantrone elimination appears to be fecal. Because of the relatively low urinary excretion it is unlikely that the standard drug dose must be reduced in the presence of compromised renal function.

Skin Cancer Chemoprevention: Strategies to Save Our Skin

There are over 1 million cases of skin cancer diagnosed yearly in the United States. The majority of these are nonmelanoma (NMSCs) and are associated with chronic exposure to ultraviolet light (UV). Actinic keratosis (AK) has been identified as a precursor for SCC, but not for BCC. AKs are far more common than SCC, making them excellent targets for chemoprevention. Cancer chemoprevention can prevent or delay the occurrence of cancer in high-risk populations using dietary or chemical interventions. We have developed strategies that have rational mechanisms of action and demonstrate activity in preclinical models of skin cancer. Promising agents proceed to phase I-III trials in subjects at high risk of skin cancer. UV light induces molecular signaling pathways and results in specific genetic alterations (i.e., mutation of p53) that are likely critical to skin cancer development. UVB-induced changes serve as a basis for the development of novel agents. Targets include inhibition of polyamine or prostaglandin synthesis, specific retinoid receptors, and components of the Ras and MAP kinase signaling pathways. Agents under study include: epigallocatechin gallate (EGCG), a green tea catechin with antioxidant and sunscreen activity, as well as UVB signal transduction blocking activity; perillyl alcohol, a monoterpene derived from citrus peel that inhibits Ras farnesylation; difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase and polyamines; retinoids that target retinoid X receptors and AP-1 activity; and nonsteroidal anti-inflammatory agents that inhibit cylooxygenase and prostaglandin synthesis. We performed a series of Phase I-II trials in subjects with multiple AK. For example, a phase II randomized trial of topical DFMO reduced AK number, suppressed polyamines, and reduced p53 protein. Our goal is to develop agents for use in combination and/or incorporation into sunscreens to improve chemoprevention efficacy and reduce skin cancer incidence.

Functional protein pathway activation mapping of the progression of normal skin to squamous cell carcinoma.

Reverse phase protein microarray analysis was used to identify cell signaling derangements in squamous cell carcinoma (SCC) compared with actinic keratosis (AK) and upper inner arm (UIA). We analyzed two independent tissue sets with isolation and enrichment of epithelial cells by laser capture microdissection. Set 1 served as a pilot and a means to identify protein pathway activation alterations that could be further validated in a second independent set. Set 1 was comprised of 4 AK, 13 SCC, and 20 UIA. Set 2 included 15 AK, 9 SCCs, and 20 UIAs. Activation of 51 signaling proteins, known to be involved in tumorigenesis, were assessed for set 1 and showed that the MEK–ERK [mitogen-activated protein (MAP)/extracellular signal-regulated (ERK; MEK)] pathway was activated in SCC compared with AK and UIA, and that epidermal growth factor receptor (EGFR) and mTOR pathways were aberrantly activated in SCC. Unsupervised two-way hierarchical clustering revealed that AK and UIA shared a common signaling network activation architecture while SCC was dramatically different. Statistical analysis found that prosurvival signaling through phosphorylation of ASK and 4EBP1 as well as increased Bax and Bak expression was higher in AK compared with UIA. We expanded pathway network activation mapping in set 2 to 101 key signaling proteins, which corroborated activation of MEK–ERK, EGFR, and mTOR pathways through discovery of a number of upstream and downstream signaling molecules within these pathways to conclude that SCC is indeed a pathway activation–driven disease. Pathway activation mapping of SCC compared with AK revealed several interconnected networks that could be targeted with drug therapy for potential chemoprevention and therapeutic applications. Cancer Prev Res; 5(3); 403–13. ©2012 AACR.

Ultraviolet A-induced Modulation of Bcl-XL by p38 MAPK in Human Keratinocytes POST-TRANSCRIPTIONAL REGULATION THROUGH THE 3′-UNTRANSLATED REGION

We examined the effect of inhibiting p38 MAPK on UVA-irradiated HaCaT cells, a spontaneously immortalized human keratinocyte cell line. Recent work from our laboratory has shown that UVA (250 kJ/m2) induces a rapid phosphorylation of p38 MAPK in the HaCaT cell line. Inhibition of p38 MAPK activity through the use of a specific inhibitor, SB202190, in combination with UVA treatment induced a rapid cleavage of caspase-9, caspase-8, and caspase-3, whereas UVA irradiation alone had no effect. Similarly, cleavage of the caspase substrate poly(ADP-ribose) polymerase was observed in UVA-irradiated HaCaT cells treated with SB202190 or in cells expressing a dominant-negative p38 MAPK. No effect of p38 MAPK inhibition upon caspase cleavage was observed in mock-irradiated HaCaT cells. In addition, increases in apoptosis were observed in UVA-irradiated cells treated with SB202190 by morphological analysis with no significant apoptosis occurring from UVA irradiation alone. Similar results were obtained by using normal human epidermal keratinocytes. UVA induced expression of the anti-apoptotic Bcl-2 family member, Bcl-XL, with abrogation of expression by using the p38 MAPK inhibitor SB202190. Overexpression of Bcl-XL prevented poly(ADP-ribose) polymerase cleavage induced by the combination of UVA and p38 MAPK inhibition. UVA enhanced the stability of Bcl-XL mRNA through increases in p38 MAPK activity. We determined that increases in UVA-induced expression of Bcl-XL occur through a posttranscriptional mechanism mediated by the 3′-untranslated region (UTR). We used Bcl-XL 3′-UTR luciferase constructs to determine the mechanism by which UVA increased Bcl-XL mRNA stability. Additionally, RNA binding studies indicate that UVA increases the binding of RNA-binding proteins to Bcl-XL 3′-UTR mRNA, which can be decreased by using SB202190. In conclusion, p38 MAPK and Bcl-XL expression play critical roles in the survival of UVA-irradiated HaCaT cells.

Inhibition of Activator Protein-1 by Sulforaphane Involves Interaction with Cysteine in the cFos DNA-Binding Domain: Implications for Chemoprevention of UVB-Induced Skin Cancer

Sulforaphane is an isothiocyanate derived from cruciferous vegetables that has been linked to decreased risk of certain cancers. Although the role of sulforaphane in the induction of the transcription factor Nrf2 has been studied extensively, there is also evidence that inhibition of the transcription factor activator protein-1 (AP-1) may contribute to the chemopreventive properties of this compound. In this study, we show for the first time that sulforaphane is effective at reducing the multiplicity and tumor burden of UVB-induced squamous cell carcinoma in a mouse model using cotreatment with the compound and the carcinogen. We also show that sulforaphane pretreatment is able to reduce the activity of AP-1 luciferase in the skin of transgenic mice after UVB. Chromatin immunoprecipitation analysis verified that a main constituent of the AP-1 dimer, cFos, is inhibited from binding to the AP-1 DNA binding site by sulforaphane. Electrophoretic mobility shift assay analysis of nuclear proteins also shows that sulforaphane and diamide, both known to react with cysteine amino acids, are effective at inhibiting AP-1 from binding to its response element. Using truncated recombinant cFos and cJun, we show that mutation of critical cysteines in the DNA-binding domain of these proteins (Cys(154) in cFos and Cys(272) in cJun) results in loss of sensitivity to both sulforaphane and diamide in electrophoretic mobility shift assay analysis. Together, these data indicate that inhibition of AP-1 activity may be an important molecular mechanism in chemoprevention of squamous cell carcinoma by sulforaphane.

Phase II enzyme inducer, sulforaphane, inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism.

Ultraviolet (UV) light‐induced activation of activator protein‐1 (AP‐1), resulting at least in part from oxidative stress, promotes skin carcinogenesis. It has not yet been determined whether elevating cellular phase II enzymes and glutathione (GSH) levels inhibits the AP‐1 activation. We have, therefore, examined the effects of two well‐known inducers of phase II enzymes, sulforaphane (SF) and tert‐butylhydroquinone (tBHQ), on UVB‐induced AP‐1 activation, with an AP‐1‐luciferase reporter plasmid that was stably transfected into human HaCaT keratinocytes (HCL14 cells). Exposure of HCL14 cells to SF or tBHQ led to the induction of quinone reductase‐1 (QR‐1), a marker of global cellular phase II enzymes, as well as elevation of cellular GSH levels. Incubation of the cells with 1–10 μM SF or 11–45 μM tBHQ for 24 h resulted in up to 1.4‐fold and 1.7‐fold increase of QR‐1 activity, respectively, and up to 1.5‐fold and 1.6‐fold increases in cellular GSH levels, respectively. AP‐1 activation was dramatically enhanced by irradiating HCL14 cells with 250 J/m2 of UVB. While the above SF treatment dose‐dependently reduced the UVB‐induced AP‐1 activation in HCL14 cells, the tBHQ treatment did not, suggesting that elevating cellular phase II enzymes and GSH levels may not lead to inhibition of UVB‐induced AP‐1 activation. Indeed, depleting cellular GSH by 80% did not affect UVB‐induced AP‐1 activation either. Subsequent electrophoretic mobility shift assays (EMSA) showed that SF added directly to the EMSAs inhibited AP‐1 DNA binding activity, whereas tBHQ was ineffective. Taken together, our results indicated that elevating phase II enzymes and GSH levels in human keratinocytes does not lead to significant inhibition of UVB‐induced AP‐1 activation. The inhibitory effect of SF on UVB‐induced AP‐1 activation appears to be at least partly due to the direct inhibition of AP‐1 DNA binding activity. This direct effect of SF on AP‐1 DNA binding is a novel mechanism for the action of a drug inhibitor of AP‐1 activation.

A Phase 2a Study of Topical Perillyl Alcohol Cream for Chemoprevention of Skin Cancer

The chemopreventive and antitumor properties of perillyl alcohol (POH) that were studied preclinically indicate that topical POH inhibits both UVB-induced murine skin carcinogenesis (squamous cell tumor models) and 7,12-dimethylbenz(a)anthracene–induced murine melanoma (transgenic models involving tyrosinase-driven Ras). A previous phase 1 clinical trial in participants with normal-appearing skin showed that topical POH cream was well tolerated at a dose of 0.76% (w/w). Here, we performed a 3-month, double-blind, randomized, placebo-controlled phase 2a trial of two different doses of topical POH in individuals with sun-damaged skin. Participants applied POH cream twice daily to each dorsal forearm. Baseline and end-of-study biopsies were taken from each participant to evaluate whether the topical application of POH was effective in reversing actinic damage as evidenced by normalization of quantitative skin histopathologic scores and change in nuclear chromatin pattern as measured by karyometric analysis. There was a borderline reduction in the histopathologic score of the lower-dose POH group compared with the placebo (P = 0.1), but this was not observed in the high-dose group. However, in the high-dose group, a statistically significant reduction in the proportion of nuclei deviating from normal was observed by the use of karyometric analysis (P < 0.01). There was no statistical significance shown in the lower-dose group. No changes were observed in p53 expression, cellular proliferation (by proliferating cell nuclear antigen expression), or apoptosis in either treatment group compared with the placebo group. These results suggest that whereas our karyometric analyses can detect a modest effect of POH in sun-damaged skin, improved delivery into the epidermis may be necessary. Cancer Prev Res; 3(2); 160–9

Cross-validation of Murine UV Signal Transduction Pathways in Human Skin

Acute UVB irradiation of mouse skin results in activation of phospatidyinositol‐3 (PI‐3) kinase and mitogen‐activated protein kinase (MAPK) pathways leading to altered protein phosphorylation and downstream transcription of genes. We determined whether activation of these pathways also occurs in human skin exposed to 4× minimal erythemic dose of UVB in 23 volunteers. Biopsies were taken prior to, at 30 min, 1 and 24 h post‐UVB. In agreement with mouse studies, the earliest UV‐induced changes in epidermis were seen in phospho‐CREB (two‐ and five‐fold at 30 min and 1 h) and in phospho‐MAPKAPK‐2 (three‐fold at both 30 min and 1 h). At 1 h, phospho‐c‐JUN and phospho‐p38 were increased five‐ and two‐fold, respectively. Moreover, phospho‐c‐JUN and phospho‐p38 were further increased at 24 h (12‐ and six‐fold, respectively). Phospho‐GSK‐3β was similarly increased at all time points. Increases in phospho‐p53 (12‐fold), COX‐2 (four‐fold), c‐FOS (14‐fold) and apoptosis were not seen until 24 h. Our data suggest that UVB acts through MAPK p38 and PI‐3 kinase with phosphorylation of MAPKAPK‐2, CREB, c‐JUN, p38, GSK‐3β and p53 leading to marked increases in c‐FOS, COX‐2 and apoptosis. Validation of murine models in human skin will aid in development of effective skin cancer chemoprevention and prevention strategies.