Cytokeratin, filaggrin, and p63 expression in reepithelialization during human cutaneous wound healing.

Cytokeratin (CK), filaggrin (filament aggregating protein), and p63 expression and cellular distribution during reepithelialization has not been systemically studied in the healing stage of human cutaneous wounds. We examined these proteins by immunohistochemical methods in 12 cases of skin ulcer, using seven anti-keratin antibodies, anti-filaggrin, and anti-p63 antibody. At the edge of the wound in skin ulcers, CK1 and 10 expression was reduced, while CK14, 16, and 17 expression was raised. Beneath the wound bed, all layers of the epidermal tongue, deriving from sweat ducts, were positive for CK14 and 17. Both cytokeratins were also found in basal and luminal cells of the dermal duct. CK expression by epithelia continuous with hair follicles showed that, CK14, 16, and 17 were present, and CK1 and 10 were absent. Filaggrin expression was elevated in reepithelialized epithelium. Expression of p63 expression was verified in the suprabasal layer in reepithelialized epithelia. CK, filaggrin, and p63 expression in the reepithelialization stage at the wound edge and at epidermal appendages remaining in the wound bed is undifferentiated and hyperproliferative. The presence of CK14 and 17 in the remaining epidermal appendages in the pathological wound may be important in epidermal replacement.

Comparison of transforming growth factor-beta/Smad signaling between normal dermal fibroblasts and fibroblasts derived from central and peripheral areas of keloid lesions.

BACKGROUND: Keloids are benign proliferations of fibroblasts, but their exact etiology and molecular pathogenesis are unknown. MATERIALS AND METHODS: Fibroblasts were isolated from the central and peripheral regions of keloids, and the growth behavior and molecular characteristics of the keloid fibroblasts were compared with those of age-adjusted normal dermal fibroblasts. RESULTS: Central (but not peripheral) keloid fibroblasts exhibited significantly increased growth and high levels of expression of transforming growth factor-beta (TGF-beta) receptor 1 and Smad 2/3. CONCLUSION: Proliferation of central keloid fibroblasts, which results in keloid formation, appears to mainly involve the TGF-beta/Smad pathway.

Enterolactone induces apoptosis and inhibits growth of Colo 201 human colon cancer cells both in vitro and in vivo.

BACKGROUND: The mammalian lignan enterolactone (ENL) is produced from plant lignans which are present in large amounts in flaxseed (linseed). The effect of ENL on colon cancer cell growth in vitro and in vivo, and its mechanisms of action, have not been studied in detail. MATERIALS AND METHODS: The growth of the colo 201 human colon cancer cell line was examined by colorimetric 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2- (4-sulphophenyl)-2H-tetrazolium (MTS) assay, while the expression of apoptosis- and proliferation-related proteins (p53, Bax, Bcl-xL and S, Bcl-2, Caspase-8, Caspase-3 and proliferating cell nuclear antigen (PCNA)) were examined by Western blotting. In vivo tumor growth was examined by transplanting colo 201 cells into ENL-treated and placebo-treated athymic mice. RESULTS: The MTS assay showed that ENL suppressed colo 201 cell growth (IC50 for 72 h: 118.4 microM) in vitro. On flow cytometry, induction of apoptosis was confirmed by the appearance of subG1 populations, while cell cycle progression was not affected. The expression of an apoptosis-suppressing protein (Bcl-2) was down-regulated, an apoptosis-enhancing protein (cleaved form of Caspase-3) was up-regulated, proliferation-related PCNA protein was down-regulated and p53, Bax, Bcl-xL and S and Caspase-8 levels were unchanged. ENL, at a dose of 10 mg/kg given 3 times per week by subcutaneous injection, significantly inhibited the growth of colo 201 cells transplanted into athymic mice without any adverse effects. CONCLUSION: ENL suppressed colo 201 human colon cancer cell growth both in vitro and in vivo. The tumor-suppressing mechanisms included apoptosis and decreased cell proliferation.

Basal cell carcinoma arising from a keratinous cyst of the skin: a case report and review of the literature.

Although keratinous cysts of the skin are frequently seen, malignant transformation is a rare event. Here we report a case of basal cell carcinoma arising in the wall of the keratinous cyst, and we review 12 other such Japanese cases.

Effects of prepubertal exposure to xenoestrogen on development of estrogen target organs in female CD-1 mice.

BACKGROUND: There have been no previous reports comparing the effects of prepubertal xenoestrogen exposure on development of the reproductive tract and mammary glands in female mice. The effects of genistein (GEN), resveratrol (RES), zearalenone (ZEA), zeranol (ZER), bisphenol A (BPA) and diethylstilbestrol (DES) were examined. MATERIALS AND METHODS: Beginning at 15 days of age, female CD-1 mice were administered 4 daily subcutaneous injections of 10 mg/kg/day of GEN, RES, ZEA, ZER or BPA, or 10 microg/kg/day of DES dissolved in dimethylsulfoxide (DMSO), or DMSO vehicle. Vaginal opening was checked; estrous cyclicity was monitored from 5, 9 or 21 weeks of age for 21 consecutive days; 6 animals per group were autopsied at 4, 8 and 24 weeks of age. RESULTS: Prepubertal exposure to GEN, ZEA, ZER and DES (but not RES or BPA) accelerated puberty onset (vaginal opening). Vaginal smears indicated that all xenoestrogen-treated mice were cycling, but ZEA-, ZER- and DES-treated mice spent more time in estrus. At 4 weeks of age, absence of corpora lutea (anovulatory ovary) was observed in the untreated controls (33%, 2/6) and the GEN (50%, 3/6), RES (50%, 3/6), ZEA (100%, 6/6), ZER (100%, 6/6), BPA (83%, 5/6) and DES groups (100%, 6/6). At 8 weeks of age, absence of corpora lutea was observed in the ZEA (33%, 2/6) group. Corpora lutea were present in all mice sacrificed at 24 weeks of age. Groups that received prepubertal xenoestrogen injections exhibited no morphological abnormalities of the uterus and vagina, and exhibited mammary gland growth similar to that of the untreated controls at all time-points. CONCLUSION: GEN, ZEA, ZER and DES (but not RES or BPA) caused early vaginal opening; mice exposed to ZEA, ZER or DES spent more time in the estrus phase; ZEA-treated mice had a longer period of anovulatory ovary than other xenoestrogen-treated mice; however, none of the xenoestrogens tested altered the uterine or vaginal morphology or mammary gland growth.

Conjugated docosahexaenoic acid is a potent inducer of cell cycle arrest and apoptosis and inhibits growth of colo 201 human colon cancer cells.

The effect of conjugated docosahexaenoic acid (CDHA) on the inhibition of colon cancer cell growth was examined in the colo 201 human colon cancer cell line, and the effect was compared with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). CDHA was a more potent tumor cell growth inhibitor than DHA and EPA by colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (IC50 for 72 h: 31.6 microM, 46.8 microM, and 56.6 microM, respectively). CDHA inhibited cell cycle progression, due to accumulation of cells in G1 phase, which involved increased p21Cip1/Waf1 and decreased cyclin D1, cyclin E, and proliferating cell nuclear antigen expression; the p53 and cyclin A levels were unchanged. Induction of apoptosis was confirmed by the appearance of sub-G1 populations, and apoptosis cascade involved upregulation of the apoptosis-enhancing proteins (Bak and Bcl-xS) and downregulation of the apoptosis-suppressing proteins (Bcl-xL and Bcl-2). CDHA modulated cell cycle regulatory proteins and apoptosis-related proteins, similar to the effects of DHA. CDHA at a dietary dose of 1.0% significantly inhibited growth of colo 201 cells transplanted in nude mice.

Effects of maternal xenoestrogen exposure on development of the reproductive tract and mammary gland in female CD-1 mouse offspring.

The objective of this study was to examine the effects of maternal exposure to xenoestrogen, at levels comparable to or greater than human exposure, on development of the reproductive tract and mammary glands in female CD-1 mouse offspring. Effects of genistein (GEN), resveratrol (RES), zearalenone (ZEA), bisphenol A (BPA) and diethylstilbestrol (DES) were examined. Beginning on gestational day 15, pregnant CD-1 mice were administered four daily subcutaneous injections with 0.5 or 10 mg/kg/day of GEN, RES, ZEA or BPA, 0.5 or 10 microg/kg/day of DES dissolved in dimethylsulfoxide (DMSO), or DMSO vehicle (n = 6). Vaginal opening was monitored, 6 animals per group were autopsied at 4, 8, 12 and 16 weeks of age and estrous cyclicity was monitored from 9 to 11 weeks of age. Maternal exposure to xenoestrogen accelerated puberty onset (vaginal opening) and increased the length of the estrous cycle; mice treated with GEN, RES, BPA or DES spent more time in diestrus, and ZEA-treated mice spent more time in estrus. Lack of corpora lutea and vaginal cornification were observed at 4 weeks of age in the high-dose GEN (33%) and RES (17%) groups, and in the high- and low-dose BPA groups (33 and 50%, respectively) and DES groups (83 and 100%, respectively). Lack of corpora lutea and vaginal cornification was observed in the high-dose ZEA group at 4, 8, 12 and 16 weeks of age (83, 100, 83 and 33%, respectively). Mammary gland differentiation was accelerated in ZEA- and BPA-treated mice with corpora lutea at 4 weeks of age. ZEA-treated mice without corpora lutea showed mammary growth arrest at 8, 12 and 16 weeks of age; their mammary glands consisted only of a dilatated duct filled with secreted fluid. Mammary gland growth was similar with xenoestrogens other than ZEA or BPA to that of the controls at all time points. High-dose GEN and RES and high- and low-dose BPA and DES exerted transient effects on the reproductive tract and mammary glands, whereas ZEA exerted prolonged effects.

Conjugated docosahexaenoic acid suppresses KPL-1 human breast cancer cell growth in vitro and in vivo: potential mechanisms of action.

INTRODUCTION: The present study was conducted to examine the effect of conjugated docosahexaenoic acid (CDHA) on cell growth, cell cycle progression, mode of cell death, and expression of cell cycle regulatory and/or apoptosis-related proteins in KPL-1 human breast cancer cell line. This effect of CDHA was compared with that of docosahexaenoic acid (DHA). METHODS: KPL-1 cell growth was assessed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; cell cycle progression and mode of cell death were examined by flow cytometry; and levels of expression of p53, p21Cip1/Waf1, cyclin D1, Bax, and Bcl-2 proteins were examined by Western blotting analysis. In vivo tumor growth was examined by injecting KPL-1 cells subcutaneously into the area of the right thoracic mammary fat pad of female athymic mice fed a CDHA diet. RESULTS: CDHA inhibited KPL-1 cells more effectively than did DHA (50% inhibitory concentration for 72 hours: 97 micromol/l and 270 micromol/l, respectively). With both CDHA and DHA growth inhibition was due to apoptosis, as indicated by the appearance of a sub-G1 fraction. The apoptosis cascade involved downregulation of Bcl-2 protein; Bax expression was unchanged. Cell cycle progression was due to G0/G1 arrest, which involved increased expression of p53 and p21Cip1/Waf1, and decreased expression of cyclin D1. CDHA modulated cell cycle regulatory proteins and apoptosis-related proteins in a manner similar to that of parent DHA. In the athymic mouse system 1.0% dietary CDHA, but not 0.2%, significantly suppressed growth of KPL-1 tumor cells; CDHA tended to decrease regional lymph node metastasis in a dose dependent manner. CONCLUSION: CDHA inhibited growth of KPL-1 human breast cancer cells in vitro more effectively than did DHA. The mechanisms of action involved modulation of apoptosis cascade and cell cycle progression. Dietary CDHA at 1.0% suppressed KPL-1 cell growth in the athymic mouse system.

Immunohistochemical profiles of Mallory body by a panel of anti-cytokeratin antibodies.

Mallory bodies (MBs) are hyaline inclusions found in a variety of liver diseases. Because the major components of MBs are cytokeratins (CKs), CK profiles of MBs were examined immunohistochemically in 37 autopsied liver specimens (including a variety of nontumor pathological livers and hepatocellular carcinomas), using 13 antibodies against specific CKs: 34betaB4 (CK 1), OV-TL12/30 (CK 7), 34betaH11 (CK 8), LHP1 (CK 10), KS-1A3 (CK 13), LL002 (CK 14), LHK15 (CK 15), LL025 (CK 16), E3 (CK 17), DC10 (CK 18), b170 (CK 19), Ks20.8 (CK 20), and 34betaE12 (CKs 1, 5, 10, and 11). Positive staining rates of MBs in 37 cases were as follows: CK 8, 100% (37/37); CK 18, 100% (37/37); CK 19, 57% (21/37). CK 7, 49% (18/37); CK 20, 35% (13/37); CK 1, CK 5, CK 10, CK 11, CK 13, CK 14, CK 15, CK 16, and CK 17 were all negative in MBs. CK expression patterns in MBs found in tumor or non-tumor hepatocytes was basically similar. Thus, all present MBs were composed of similar material with common antigenic determinants, regardless of underlying disease; in particular, they consistently contained CK 8 and CK 18, and frequently contained CK 7, CK 19, and CK 20.

Perillyl alcohol inhibits human breast cancer cell growth in vitro and in vivo.

The effect of monoterpene perillyl alcohol (POH) on cell growth, cell cycle progression, and expression of cell cycle-regulatory proteins in estrogen receptor (ER)-positive (KPL-1 and MCF-7) and ER-negative (MKL-F and MDA-MB-231) human breast cancer cell lines was examined. POH inhibited cell proliferation in a dose-dependent manner in all cell lines tested. POH at a dose of 500 micro M had a cytostatic effect, in which growth inhibition was due to accumulation of cells in G1-phase. Cell cycle progression was preceded by a decrease in G1 cyclins (cyclin D1 and E), followed by an increase in p21(Cip1/Waf1) and a decrease in proliferating cell nuclear antigen level. Levels of p53 and cyclin A were unchanged. POH at a dose of 75 mg/kg administered intraperitoneally three times a week throughout the entire 6-week experimental period suppressed orthotopically transplanted KPL-1 tumor cell growth and regional lymph node metastasis in a nude mouse system. POH inhibited both ER-positive and -negative human breast cancer cell growth in vitro, and suppressed growth and metastasis in vivo.

Dietary docosahexaenoic acid suppresses N-methyl-N-nitrosourea-induced mammary carcinogenesis in rats more effectively than eicosapentaenoic acid.

We compared the effects of identical amounts but different proportions of dietary n-3 polyunsaturated fatty acids (PUFAs) on N-methyl-N-nitrosourea (MNU)-induced mammary cancer in a rat model. The ability of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to suppress mammary cancer was evaluated. Female Sprague-Dawley rats were randomly assigned to three groups and maintained on diets containing 10% fatty acid consisting of EPA, a 1:1 mixture of EPA-plus-DHA, or DHA. The experimental diet was started after administration of MNU at 49 days of age, and the rats were maintained on the respective diets until the largest mammary tumor reached >1 cm in diameter or until the end of the study period (20 wk after MNU). All histologically detected mammary carcinomas were evaluated, irrespective of size. The DHA diet was associated with significant suppression of the carcinogenic effect of MNU compared with the EPA and EPA-plus-DHA diets: tumor incidence decreased to 23% (3/13) compared with 73% (11/15) and 65% (12/17) (P < 0.01 and P < 0.05, respectively); tumor multiplicity decreased to 0.23 compared with 1.67 and 1.59 (P < 0.01 and P < 0.05, respectively). There was no significant difference in tumor latency among the DHA, EPA, and EPA-plus-DHA groups (119, 105, and 117 days, respectively). Over 20 wk, the fatty acid composition of serum and mammary fat tissue reflected differences in the dietary n-3 PUFAs. Although DHA suppressed MNU-induced mammary carcinogenesis more effectively than EPA, generalized steatosis including mammary fat tissue appeared in all three groups.

p63 expression in normal human epidermis and epidermal appendages and their tumors.

BACKGROUND: p63, a member of the p53 gene family, is expressed in basal cells of several different organs. METHODS: The immunoreactivity of p63 was examined in normal human epidermis and epidermal appendages and their tumors, and compared with proliferative activity as evaluated by Ki-67. RESULTS: In normal skin, p63 expression was seen in basal/suprabasal cells of the epidermis, outer root sheath and hair matrix cells of the hair follicle, seboblast situated in the outermost layer of sebaceous glands, and outer layer cells of the ductal portion and myoepithelial cells of the secretory portion of the sweat glands. p63 expression was confined to the cells forming a continuous basal rim along the normal epithelial structure. In tumors, p63 expression resembled that in normal tissue in that tumor components originating from p63-positive cells were constantly positive for p63. In normal and tumor tissues, not all p63-positive cells were positive for Ki-67. CONCLUSIONS: p63 expression may be a marker of basal/progenitor cells in tumors of epidermis and epidermal appendages, and may be a diagnostic marker of these tumors.