DISCUSSION: The findings of present studies of reveal that Carissa carandas plant extract suppress DMBA/TPA induced two stage skincarcinogenesis due to its anticarcinogenic effect. Application of 7,12 dimethylbenz(a) anthracene (DMBA) and 12- O-tetra decanoylphorbol- 13-acetate (TPA) constitute of croton oil induced two stage skincarcinogenesis through three sequential steps of tumor initiation, promotion, and progression, also helpful for study of genetic and biochemical alterations caused by them. In the present investigation DMBA + TPA (Group III) used as topical application because it was reported that fastest way of absorption of DMBA carcinogen was found in skin tissue. In present experiment carcinogen treated animals exhibited 100% tumor incidence and highest cumulative number of tumors. However, treatment with CCE (Group IV and V) showed regressed of tumor incidence and cumulative number of tumor.
Aim & Method: The current study was designed to elucidate the protective effect of Syzygium cumini seed extract (SCE) on skincarcinogenesis induced by a single topical application of 7,12-dimethylbenz(a)anthracene (100 μg/100 μl of acetone) and 2 weeks later promoted by repeated application of croton oil (1% in acetone/three times a week) till the end of the experiment (16 weeks). Result: Oral administration of SCE at a dose of 125 mg/kg b.wt./day for 15 days at the peri-initiational stage (i.e., 7 days before & 7 days after DMBA application) and for 14 weeks at the promotional stage (i.e., from the time of croton oil application), revealed a significant reduction in lipid peroxidation (p<0.05-0.001) along with an elevation in the activities of enzymatic antioxidants (superoxide dismutase, p<0.05-0.001 & catalase, p<0.05- 0.001), non-enzymatic antioxidant (reduced glutathione, p<0.05-0.01 & vitamin-C, p<0.01- 0.001) and total proteins levels (p<0.01-0.001) when compared to the carcinogen treated control animals. Histopathological study revealed that dyskeratosis of the epidermis, deposition of keratinous pearl and epidermal hyperplasia in skin tumors of DMBA treated control and the same were found to be of lesser degree in both the SCE treated experimental animals. Conclusions: These results demonstrate that SCE ameliorate the DMBA/croton oil induced adverse biochemical and histopathological alterations during skincarcinogenesis in mice.
Long-term exposure to arsenic is associated with cancers of lung, urinary bladder, kidney, liver and skin. Arsenic car- cinogenesis might result from oxidative stress, altered growth factors, chromosomal abnormality, immune dysregula- tion, and aberrant epigenetic regulations. Bowen’s disease (As-BD) is the most common form of arsenic-induces skin cancers and is characterized by chronicity, multiplicity, and predisposition in sun-spare skin. However, only about 1% of the population exposed to arsenic developped skin cancers, indicating the host immune response plays an important modulatory role in skincarcinogenesis. In this review, we review the pathomechanisms of arsenic skincarcinogenesis and the immune interactions. Arsenic affects innate and adaptive immune responses through CD4+ T cells, monocytes, macrophages, and Langerhans cells. In skin of As-BD, CD4+ T cells undergo selective and differential apoptosis via Fas-FasL interaction. Numbers and dendrites of Langerhans cells are reduced in As-BD lesions. There is a defective homeostasis and aberrant trafficking of Langerhans cells. Such information is essential to understand the molecular mechanism for arsenic carcinogenesis in both skin and in internal organs.
The principal aim of the present work was to evaluate the chemopreventive effect of hydro-alcoholic extract of the whole plant of Aegle marmelos, in 6-7 weeks old male Swiss albino mice, on two stage process of skincarcinogenesis induced by a single topical application of 7, 12-dimethylbenz (a) anthracene (DMBA) and two weeks later promoted by repeated application of croton oil till the end of experiment (i.e. 16 weeks).The oral administration of A. marmelos at 50 mg/kg/b.wt./day during peri- initiational and post-initiational phases of papillomagenesis significantly inhibited the decrease in reduced glutathione (p ≤ 0.001), catalase (p ≤ 0.001) and proteins (p ≤ 0.001) as well as the increase in lipid peroxidation (p ≤ 0.001) levels in skin and liver of experimental mice while compared to carcinogen treated control. The results from the present study suggest the chemopreventive effect of Aegle marmelos fruit extract in DMBA induced skin papillomagnesis.
These ﬁndings and the fact that the mutant: normal Ha-ras gene ratio is increased during pro- gression from the benign (papilloma) to the malig- nant (squamous–spindle) stage of the mouse skin model prompted us to examine the activation sta- tus of its downstream effector ERK1/2. Phosphory- lated ERK1/2 showed a progressive increase from the papilloma to the squamous and spindle cell lines implying that ERK1/2 signaling is involved in malignant progression of mouse skin tumors. In ac- cordance are the results of Sivaraman et al. (37), who demonstrated that primary human breast carcino- mas exhibited a 5- to 10-fold increase ERK1/2 activ- ity over benign conditions, such as ﬁbroadenomas and ﬁbrocystic disease. Interestingly, this gradual el- evation of ERK1/2 activation was also observed within the squamous PDV:PDVC57 cell pair. The squamous PDV and PDVC57 cell lines are derived from tumors originated from the same progenitor cell but differ, as we mentioned, in the normal:mu- tated Ha-ras ratio (5). Given that PDVC57 has more malignant characteristics than PDV, our ﬁnding im- plies that ERK1/2 signaling may play a role in the degree of aggressiveness during the squamous stage of mouse skincarcinogenesis. To prove that this in- crease in ERK1/2 signaling was related to the qual- itative changes of the endogenous Ha-ras, we at- tempted to reproduce the putative effects of ras by introducing in the PDV cells an activated Ha-ras al- lele (16). Indeed, the phosphorylation pattern of ERK1/2 in the PDVras clones was comparable to that of PDVC57, conﬁrming our initial hypothesis. Although several groups have shown that ras is re- quired for the activation of the MAP kinase pathway in several cell types (38–40), this is the ﬁrst study to demonstrate a gradual increase of the ras/MEK/ERK pathway during the various stages of mouse skincarcinogenesis.
Both Rac1 and HPV of the Beta Genus, such as HPV-8, have been implicated in skincarcinogenesis. While HPV-8 was found to be involved in the development of SCCs in sun exposed skin of patients with epidermodysplasia verruciformis, over-expression and activation of Rac1 have been reported in SCCs of mucosa and cornified skin [6, 12, 13]. In our study we identified, in addition, a role of Rac1 in the development of HPV-8 related skin papillomas in mice: inhibition of Rac1 activity or epidermis specific deletion of Rac1 led to a strong decrease in both spontaneous and UV-light induced skin papilloma formation. These results are in line with those of Wang et al. 2010, who found that deletion of Rac1 in the epidermis inhibits chemically induced formation of skin papillomas which are thought to be precursors of SCCs in mice [9, 15]. On the other hand, constitutive activation of Rac1, in addition to expression of HPV-8, led to the development of papillomas, but not SCCs, in 100% of the double transgenic K14 HPV-8/K14 L61Rac1 mice, whereas K14 L61Rac1 mice without HPV- 8 did not develop skin papillomas. This shows that Rac1 activity essentially supports HPV-8 induced papilloma
Mutations in the tumor suppressor p53 are detectable in over 50% of all human malignancies. Mutant p53 protein is incapable of transactivating its downstream target genes that are required for DNA repair and apoptosis. Chronic exposure to UVB induces p53 mutations and is carcinogenic in both murine and human skin. CP-31398, a styrylquinazoline compound, restores the tumor suppressor functions of mutant forms of p53 in tumor cells. However, its effectiveness in vivo remains unclear. Here, we demonstrate that CP-31398 blocked UVB-induced skincarcinogenesis and was associated with increases in p53, p21, and BclXs. CP-31398 downregulated Bcl2, proliferating nuclear cell antigen, and cyclin D1. Activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase also occurred in both tumor and perilesional skin following treatment. CP-31398 induced the expression of p53-dependent target proteins, and this was followed by apoptosis in UVB-irradiated wild-type mice but not in their p53-deficient littermates. Similar effects were observed in human skin carcinoma A431 cells expressing mutant p53. In addition, CP-31398 induced mitochondrial translocation of p53, leading to changes in mitochondrial membrane permeability pore transition (MPT) and consequent cytochrome c release in these cells. Blocking MPT diminished p53 translocation and apoptosis. These studies indicate that reconstituting p53 tumor suppressor functions in vivo by small molecular weight compounds may block the pathogenesis and progression of skin cancer.
Actively proliferating Lgr5+ skin stem cells are found deep in the hair follicle (HF). These cells renew the HF and drive its expansion in anagen phase. Their long residence and continuous mitotic activity make them prime candidates to transform into skin tumor-initiating cells. This was investigated by subjecting Lgr5-EGFP-Ires- CreERT2/R26R-LacZ mice (haired and hairless) to chemical and UV carcinogenic regimens. In the course of these regimens Lgr5+ cells (EGFP+) remained exclusively located in HFs, and in deep-seated cysts of hairless skin. In haired mice, progeny of Lgr5+ stem cells (LacZ+ after a pulse of tamoxifen) appeared in the interfollicular epidermis upon UV-induced sunburn and in TPA-induced hyperplasia. In hairless mice the progeny remained located in deep-seated cysts and in HF remnants. Progeny in hairless skin was only detected interfollicularly at a late stage, in between outgrowing tumors. Lgr5+ stem cells were absent in the ultimate tumor masses, and no tumor appeared to be a (clonal) expansion of Lgr5+ cells (52 tumors with tamoxifen at the start of carcinogenesis, 42 tumors with tamoxifen late during tumor outgrowth). In contrast to CD34/K15+ quiescent bulge stem cells, actively proliferating Lgr5+ stem cells do therefore not appear to be tumor drivers in experimental skincarcinogenesis.
inflammatory process of a number of diseases such as cancer, atherosclerosis, or rheumatoid arthritis [80– 82]. These facts correlated with our results revealing that IL-32γ suppressed cancer stemness through the inhibition of ITGAV and TIMP-1 in A431 and SK-Mel-28 skin cancer cells. Additionally, knockdown of ITGAV suppressed cancer stemness with decreased expression of cancer stem cell markers. Furthermore, expression of ITGAV was downregulated in DMBA/ TPA-induced skin tissues of IL-32γ mice. Tissue microarray data showed that ITGAV expression was elevated in skin cancer tissues from patients. These data indicated that the downregulation of the anti-stemness and anti-inflammatory effects of ITGAV could be associated with the reducing effect of IL-32γ on skincarcinogenesis. TIMP-1 is a glycoprotein and plays a role in extracellular matrix composition, wound healing, and pregnancy by regulating matrix metalloproteinases. Previous studies revealed that TIMP-1 inhibited a disintegrin and metalloprotease (ADAM)-10 activity, which regulates cancer stem-like cells and tumor growth through activation of Notch signaling in colon cancer [83, 84]. However, TIMP-1 was involved in the development of various cancer, such as melanoma, colon, and acute myeloid leukemia, through exerting an inflammatory network in the tumor microenvironment [39, 40, 85]. Melan- oma cells overexpressing TIMP-1 had increased anchorage-independent growth and in vivo cancer progression [40, 86]. Increased serum level of TIMP-1 was correlated with an unfavorable prognosis in pa- tient with advanced stage melanoma . In this study, we found that IL-32 γ inhibited cancer sphere formation and its effect was associated with the downregulation of TIMP-1 expression in skin cancer cells. We also found that inhibition of TIMP-1 showed reduced cancer sphere formation and expres- sion of CSC markers. The expression of TIMP-1 was also reduced in DMBA/TPA-induced skin tissues of IL-32γ mice. These data indicated that anti-inflammatory and anti-stemness effects by down-regulation of TIMP-1 could contribute to the inhibitory effect of IL-32 γ .
Lgr6+ cells have been identified as a novel class of proliferating (Ki67+) stem cells in mouse epidermis. We investigated their response to UV exposure in Lgr6- EGFP-Ires-CreERT2/R26R-LacZ haired and hairless mice and whether they become initiating cells of UV- or chemically induced skin tumors. UV overexposure erased Lgr6+ cells (EGFP+) from the interfollicular epidermis (IFE), but - as after wounding - they apparently repopulated the IFE from the hair follicles. Under sub-sunburn chronic UV exposure, Lgr6+ cells and their progeny (LacZ+ after pulse of tamoxifen) diminished strongly in the IFE. Although the inter-tumoral IFE clearly showed Lgr6 progeny, none of the UV- or chemically induced tumors (n = 22 and 41, respectively) appeared to be clonal expansions of Lgr6+ stem cells; i.e. no Lgr6+ cells or progeny in the proliferating tumor bulk. In checking for promoter methylation we found it to occur stochastically for the EGFP-Cre cassette. Lgr6 mRNA measured by qPCR was found to be diminished in skin tumors (also in UV tumors from wt type mice). The ratio of Lgr6/Ki67 was significantly reduced, pointing at a loss of Lgr6+ cells from the proliferative pool. Our data show that Lgr6+ cells are not major tumor-initiating cells in skincarcinogenesis.
Flaxseed is an economically important oilseed crop grown around the world for its oil and fibers. The physiological benefits of flax oil are attributed primarily to the high α-linoleic acid content, chlorophyll pigments, tocopherol, plastochromanol-8, phenolic acids and flavanoids which may play significant and/or synergistic role in the pharmacological quality of the oil. 12,13 Therefore, the present study has been undertaken to evaluate the anticancer activity of Flaxseed oil on DMBA and croton oil induced skincarcinogenesis in Swiss albino mice.
TREX2 is a 3′-DNA exonuclease specifically expressed in keratinocytes. Here, we investigated the relevance and mechanisms of TREX2 in ultraviolet (UV)-induced skincarcinogenesis. TREX2 expression was up-regulated by chronic UV exposure whereas it was de-regulated or lost in human squamous cell carcinomas (SCCs). Moreover, we identified SNPs in the TREX2 gene that were more frequent in patients with head and neck SCCs than in healthy individuals. In mice, TREX2 deficiency led to enhanced susceptibility to UVB-induced skincarcinogenesis which was preceded by aberrant DNA damage removal and degradation as well as reduced inflammation. Specifically, TREX2 loss diminished the up-regulation of IL12 and IFNγ, key cytokines related to DNA repair and antitumor immunity. In UV-treated keratinocytes, TREX2 promoted DNA repair and passage to late apoptotic stages. Notably, TREX2 was recruited to low- density nuclear chromatin and micronuclei, where it interacted with phosphorylated H2AX histone, which is a critical player in both DNA repair and cell death. Altogether, our data provide new insights in the molecular mechanisms of TREX2 activity and establish cell autonomous and non-cell autonomous functions of TREX2 in the UVB-induced skin response.
membrane-associated E-cadherin/catenin. These results suggest that even though loss of membrane-associated E-cadherin is required for EMT, this event is dispensable for tumor metastasis. In addition to being involved in EMT, the Rho/Rac pathway is documented as a metastasis-associated pathway (39), and elevated levels of Erk and JNK have been observed in invasive skin SCCs (40, 41). Specifically, both the RhoA/Rac and the MAPK pathways have been documented to significantly affect cell motility (42, 43). Once cancer cells have Figure 7
Notch1 signaling is essential for proper skin differentiation through induction of p21 (WAF1/Cip1) [37,40]. We speculated that Notch2 signaling might not be required for this process since it is expressed mainly by differentiated keratinocytes. p21 is a cyclin-dependent kinase inhibitor that induces cell cycle arrest , predictably its loss is commonly associated with skin malignancies, particularly in an active Ras context . We found that p21 expression was highly reduced in Notch1 ablated cells whereas no significant differences were noted in Notch2 deficient keratinocytes both on mRNA and protein level (Fig. 6A, Figure 2. Pdx1 is physiologically expressed in the adult mouse epidermis. A: Immunohistochemical PDX1 staining of normal wildtype epidermis (i, ii) reveals that PDX1 is expressed in suprabasal keratinocytes (black arrowheads) and only rarely in basal cells (black arrows). Pdx1- Cre;Kras;N1ko papilloma (iii) is strongly positive for PDX1. Inclusion (iii) shows positive staining of pancreatic islet cells. Nuclei were contrastained with methyl green (i, ii) or hematoxilin (iii). B: Immunofluorescent PDX1 staining (i) indicates positive keratinocytes in the suprabasal (white arrowheads) and the basal (arrow) layer of the skin. Signal strength is comparable to that in duodenum cells (ii, arrowheads) and weaker than in pancreatic islet cells (ii, inclusion). Double immunofluorescence (iii) demonstrates that the majority of PDX1 + cells co-localize with a suprabasal marker Keratin10 (arrowheads) however, a small subset of PDX1 + cells can be found in the basal layer of the epidermis (arrow). Asterisks indicate unspecific staining of stratum corneum. C: Pdx1 expression in cultured keratinocytes is increased during Ca ++ -induced differentiation. Quantitative RT-PCR of Pdx1, Keratin10, Loricrin and p63 transcripts in induced primary keratinocytes in vitro. D: Schematic representation of PDX1 expression in the epidermal layers: (SC) Stratum Corneum, (GL) Granular Layer, (SL) Spinous Layer, (BL) Basal Layer, (BM) Basement Membrane, (D) Dermis and their markers: Loricrin, K1/10, K5/14. The scale bars represent 50 mm.
Vitamin D (VD) is a secosteroid hormone that is mainly synthesized in the skin upon exposure to UVB radiation. VD is widely known for its role in calcium metabolism; however, multiple endocrine, paracrine and autocrine functions of VD have been described, including a prominent role on carcinogenesis. In recent years, multiple associations between VD deficiency and different types of cancer have been described, supported by evidence of anti-proliferative, anti-angiogenic, pro-apoptotic, cell-differentiating and anti-invasive effects of this hormone. An immunomodulatory role of VD associated to cancer microenvironment has also been suggested. Regarding skin cancer, it has been shown that VD inhibits tumor development in basal cell carcinoma, squamous cell carcinoma, and melanoma in vitro. Some studies have suggested that lower VD levels may be a risk factor for skin cancer, while others have shown the opposite; there is also preliminary evidence on the role of VD supplementation for the prevention of melanoma in vivo. In this review, we explore the mechanisms of VD effects on carcinogenesis and the available scientific evidence of the interplay between VD and the genesis of both non-melanoma and melanoma skin cancer.
Skin bioassay protocol: The animals were randomly divided in to 8 groups. Each group comprises of 6 animals. Mice were shaved in 2cm 2 area with the help of hair removing cream in interscapular region initially and after every 2 weeks hair were removed with the help of scissors. The treatment was provided topically on shaved area using the following protocol.
Figure 29. CDK6 distribution in mouse keratinocytes. CDK6 expression was detected by immunofluorescence analysis in paraffin sections of mouse papillomas at 20 weeks of promotion (A) and untreated mouse skin (B). Nuclear localization of CDK6 was observed in mouse inter follicular epidermis (B, I), whereas cytoplasmic distribution of CDK6 was observed in papillomas (A) and hyperproliferative areas of the hair follicle (B, II).
Objective: This study was designed to estimate the percentage of non‑malignant skin tumours (papillomas) pro‑ gressing to malignant squamous cell carcinomas (SCCs) in a carcinogenesis study using established transgenic mouse models. In our skin cancer model, we conditionally induced oncogenic point mutant alleles of p53 and k‑ras in undif‑ ferentiated, basal cells of the epidermis.
p53 is a protein that causes cell cycle arrest, apoptosis or senescence, being crucial in the process of tumor suppression in several cell types. Different in vitro and animal models have been designed for the study of p53 role in skin cancer. These models have revealed opposing results, as in some experimental settings it appears that p53 protects against skin cancer, but in others, the opposite conclusion emerges. We have generated cohorts of mice with efficient p53 deletion restricted to stratified epithelia and control littermates expressing wild type p53 and studied their sensitivity to both chemically-induced and spontaneous tumoral transformation, as well as the tumor types originated in each experimental group. Our results indicate that the absence of p53 in stratified epithelia leads to the appearance, in two-stage skincarcinogenesis experiments, of a higher number of tumors that grow faster and become malignant more frequently than tumors arisen in mice with wild type p53 genotype. In addition, the histological diversity of the tumor type is greater in mice with epidermal p53 loss, indicating the tumor suppressive role of p53 in different epidermal cell types. Aging mice with p53 inactivation in stratified epithelia developed spontaneous carcinomas in skin and other epithelia. Overall, these results highlight the truly protective nature of p53 functions in the development of cancer in skin and in other stratified epithelia.
For skincarcinogenesis experiments, the backs of 6-8 week-old female mice of the indicated genotypes were shaved. Two days later, the two-step DMBA/TPA carcinogenesis protocol was initiated using a single topical application of DMBA (100 µg in 200 µl acetone; Sigma). The promotion phase consisted of biweekly TPA applications (10 µg in 200 µl acetone; Sigma) for 19 weeks. Mice were examined regularly for tumour appearance and from week 15 tumour growth was measured with a calliper. Mice were sacrificed at week 29 and skin samples were processed for further analysis. Control mice were treated with acetone alone. This treatment was performed twice, with similar results. For short-term in vivo studies of epidermal apoptosis and DNA repair, dorsal skin of 6-8 week old female mouse was treated with a single DMBA application (100 µg in 200 µl acetone) or acetone (200 µl) 2 days after shaving and analysed 24 h later. For inflammation and proliferation assays, mouse dorsal skin was treated with a single topical TPA application (10 µg in 200 µl acetone) or acetone 2 days after shaving and analysed at different times post-challenge. For proliferation assays, mice received intraperitoneal injections of 100 mg BrdU/kg body weight in sterile PBS 2 h before sacrifice.