Polypodium leucotomos extract decreases UV-induced Cox-2 expression and inflammation, enhances DNA repair, and decreases mutagenesis in hairless mice.

UV-irradiated skin and UV-induced tumors overexpress the inducible isoform of cyclooxygenase-2 (Cox-2), and Cox-2 inhibition reduces photocarcinogenesis. To evaluate photoprotective effects of Polypodium leucotomos extract (PL), hairless Xpc(+/-) mice were fed for 10 days with PL (300 mg/kg) or vehicle then UV-irradiated, once. By 24 hours, UV-induced Cox-2 levels were increased in vehicle-fed and PL-fed mice, whereas by 48 and 72 hours, Cox-2 levels were four- to fivefold lower in PL-fed mice (P < 0.05). p53 expression/activity was increased in PL-fed versus vehicle-fed then UV-irradiated mice. UV-induced inflammation was decreased in PL-fed mice, as shown by approximately 60% decrease (P < 0.001) in neutrophil infiltration at 24 hours, and macrophages by approximately 50% (<0.02) at 24 and 48 hours. By 72 hours, 54 +/- 5% cyclobutane pyrimidine dimers remained in vehicle-fed versus 31 +/- 5% in PL-fed skin (P < 0.003). The number of 8-hydroxy-2'-deoxyguanosine-positive cells were decreased before UV irradiation by approximately 36% (P < 0.01), suggesting that PL reduces constitutive oxidative DNA damage. By 6 and 24 hours, the number of 8-hydroxy-2'-deoxyguanosine-positive cells were approximately 59% (P < 0.01) and approximately 79% (P < 0.03) lower in PL-fed versus vehicle-fed mice. Finally, UV-induced mutations in PL-fed-mice were decreased by approximately 25% when assessed 2 weeks after the single UV exposure. These data demonstrate that PL extract supplementation affords the following photoprotective effects: p53 activation and reduction of acute inflammation via Cox-2 enzyme inhibition, increased cyclobutane pyrimidine dimer removal, and reduction of oxidative DNA damage.

Topical thymidine dinucleotide treatment reduces development of ultraviolet-induced basal cell carcinoma in Ptch-1+/- mice.

Treatment with thymidine dinucleotide (pTT) has well documented DNA-protective effects and reduces development of squamous cell carcinoma in UV-irradiated mice. The preventive effect of pTT on basal cell carcinoma (BCC) was evaluated in UV-irradiated Ptch-1(+/-) mice, a model of the human disease Gorlin syndrome. Topical pTT treatment significantly reduced the number and size (P < 0.001) of BCCs in murine skin after 7 months of chronic irradiation. Skin biopsies collected 24 hours after the final UV exposure showed that pTT reduced the number of nuclei positive for cyclobutane pyrimidine dimers by 40% (P < 0.0002) and for 8-hydroxy-2'-deoxyguanosine by 61% (P < 0.01 compared with vehicle control). Immunostaining with an antibody specific for mutated p53 revealed 63% fewer positive patches in BCCs of pTT-treated mice compared with controls (P < 0.01), and the number of Ki-67-positive cells was decreased by 56% (P < 0.01) in pTT-treated tumor-free epidermis and by 76% (P < 0.001) in BCC tumor nests (P < 0.001). Terminal dUTP nick-end labeling staining revealed a 213% increase (P < 0.04) in the number of apoptotic cells in BCCs of pTT-treated mice. Cox-2 immunostaining was decreased by 80% in tumor-free epidermis of pTT-treated mice compared with controls (P < 0.01). We conclude that topical pTT treatment during a prolonged period of intermittent UV exposure decreases the number and size of UV-induced BCCs through several anti-cancer mechanisms.

T-oligos augment UV-induced protective responses in human skin.

We have shown that DNA oligonucleotides substantially homologous to the telomere 3-prime overhang sequence (T-oligos) increase DNA repair capacity (DRC) in cultured human cells and decrease UV-induced mutation rate and photocarcinogenesis in mouse skin. To investigate the protective effects of T-oligos in intact human skin, paired skin explants obtained from adult donors were treated with T-oligos or diluent alone for 24 h, then UVB- or sham-irradiated, and processed after 6, 24, 48, 72, and 96 h for histological analysis. After UV irradiation apoptotic epidermal cells were comparable in diluent- and T-oligo-treated skin. Proliferating (Ki67+) cells were sparse in sham-irradiated skin and for 24 h after UV in both diluent- and T-oligo-treated specimens. However, compared to diluent controls, at 48 and 72 h T-oligos significantly inhibited UV-induced rebound hyperproliferation. Maximum and comparable cyclobutane pyrimidine dimers (CPDs) were detected immediately after UV irradiation in diluent- and T-oligo-treated skin, but CPDs were strikingly reduced in T-oligo- vs. diluent-treated skin at 24, 48, and 72 h. Total and activated p53 protein was increased in T-oligo- vs. diluent-pretreated skin at the time of irradiation, and up to 3-fold increases persisted for 24 h post-UV. Over 5 days, UV irradiation and T-oligo comparably increased expression of melanogenic proteins and each increased epidermal melanin content 3- to 5-fold, with distinct nuclear capping in many keratinocytes. In combination, these findings predict that T-oligo treatment will increase melanogenesis, prolong epidermal arrest, and increase DNA repair rate after UV irradiation, thus decreasing the severity of acute and chronic photodamage in human skin. Moreover, the data document an inducible SOS-like response consisting of increased melanogenesis and increased DNA repair capacity in human skin following UV-induced damage that is also produced by T-oligos in the absence of initial damage.

Clostridium botulinum type A toxin for the treatment of upper face animation lines: an Iranian experience.

The purpose of this study was to evaluate the treatment of vertical glabellar frown lines, frontal lines and crow's feet by means of direct injection of Dysport (Clostridium botulinum type A toxin - hemagglutinin complex) in Iranian patients. Dysport is one of the commercially available type A subtypes (Ipsen Ltd, Maidenhead Berkshire, UK). It is the one used most often in Iran. Botox, the other form of commercially available botulinum type A toxin, is used most often in the United States and Canada. Dysport is two to four times less effective, in similar unit doses, than Botox, which is why it is manufactured in larger vials (500-U vials for Dysport vs. 100-U vials for Botox). The authors have been using botulinum toxin type A in their practice for cosmetic purposes since 1995. An experience of over 1295 injections to the upper face animation (dynamic) lines in 108 patients is presented. This study included 108 patients and 208 injection sessions. We used Dysport containing 500 U toxin. Toxin was diluted with 2.5 mL of sterile normal saline and yielded 20 U for each 0.1 mL. A dose of 76 to 90 U was injected into each muscle. There were 95 women and 13 men in this study, ranging in age from 20 to 79 years. Doses ranged from 10 to 20 mL per injection, varying according to the severity of wrinkles, intensity of muscle contraction, and the mass of the muscle. The more bulky or greater intensity of muscle contraction, the higher the dose required to obtain good results. Therefore, the more bulky corrugator or frontalis muscle requires more than the thinner orbicularis. Dysport seemed to be a safe and effective alternative to Botox, giving good to excellent cosmetic results lasting at least 4 months in the majority of the patients.

Quantification of inducible SOS-like photoprotective responses in human skin.

To document and quantify inducible photoprotective effects in human skin, explant cultures were treated once with thymidine dinucleotide (pTT) or diluent alone or UV-irradiated. Both pTT and UV increased the melanogenic protein levels on days 1-5 and comparably increased melanocyte dendricity and epidermal melanin content. Explants treated with pTT or UV but not with diluent alone showed initial inhibition of epidermal proliferation followed by mild reactive hyperplasia; melanocyte proliferation was minimal. To determine whether pTT and UV provide comparable protection against subsequent UV-induced DNA damage, explants were pTT- or diluent-treated or UV-irradiated. All explants were then irradiated with the same UV dose 72 hours later. Compared to diluent alone, pTT or UV pretreatment decreased the number of epidermal cells positive for cyclobutane pyrimidine dimers (CPDs) 50% immediately post-irradiation. In pTT- and UV- versus diluent-pretreated explants, the rate of CPD removal was also more rapid, approximately 80 vs 45% of the initial burden within 72 hours. These data confirm and quantify comparable SOS-like responses in human skin after pTT or UV irradiation, attributable to both increased epidermal melanin and increased DNA repair rate, in the case of pTT in the absence of initial damage.

T-oligo treatment decreases constitutive and UVB-induced COX-2 levels through p53- and NFkappaB-dependent repression of the COX-2 promoter.

Chronically irradiated murine skin and UV light-induced squamous cell carcinomas overexpress the inducible isoform of cyclooxygenase (COX-2), and COX-2 inhibition reduces photocarcinogenesis in mice. We have reported previously that DNA oligonucleotides substantially homologous to the telomere 3'-overhang (T-oligos) induce DNA repair capacity and multiple other cancer prevention responses, in part through up-regulation and activation of p53. To determine whether T-oligos affect COX-2 expression, human newborn keratinocytes and fibroblasts were pretreated with T-oligos or diluent alone for 24 h, UV-irradiated, and processed for Western blotting. In both cell types, T-oligos transcriptionally down-regulated base-line and UV light-induced COX-2 expression, coincident with p53 activation. In fibroblasts with wild type versus dominant negative p53 (p53(WT) versus p53(DN)), T-oligos decreased constitutive expression of a COX-2 reporter plasmid by >50%. We then examined NFkappaB, a known positive regulator of COX-2 transcription. In p53(WT) but not in p53(DN) fibroblasts and in human keratinocytes, T-oligos decreased readout of an NFkappaB promoter-driven reporter plasmid and decreased NFkappaB binding to DNA. After T-oligo treatment and subsequent UV irradiation, binding of the transcriptional co-activator protein p300 to NFkappaB was decreased, whereas binding of p300 to p53 was increased. Human skin explants provided with T-oligos had markedly decreased COX-2 immunostaining both at base-line and post-UV light, coincident with increased p53 immunostaining. We conclude that T-oligos transcriptionally down-regulate COX-2 expression in human skin via activation and up-regulation of p53, at least in part by inhibiting NFkappaB transcriptional activation. Decreased COX-2 expression may contribute to the observed ability of T-oligos to reduce photocarcinogenesis.