DNA fragmentation and caspase-independent programmed cell death by modulated electrohyperthermia.

BACKGROUND AND PURPOSE: The electric field and the concomitant heat (electrohyperthermia) can synergistically induce cell death in tumor tissue, due to elevated glycolysis, ion concentration, and permittivity in malignant compared with nonmalignant tissues. Here we studied the mechanism and time course of tumor destruction caused by electrohyperthermia. MATERIAL AND METHODS: Bilateral implants of HT29 colorectal cancer in the femoral regions of Balb/c (nu/nu) mice were treated with a single 30-min shot of modulated, 13.56-MHz, radiofrequency-generated electrohyperthermia (mEHT). Tumors at 0, 1, 4, 8, 14, 24, 48, and 72 h posttreatment were studied for morphology, DNA fragmentation, and cell death response-related protein expression using tissue microarrays, immunohistochemistry, Western immunoblots, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. RESULTS: Modulated EHT treatment induced significant tumor destruction in HT29 xenografts with a peak of a sevenfold increase compared with the untreated controls. The significant treatment-related elevation of DNA fragmentation--detected with TUNEL assay--and apoptotic bodies between 24 and 72 h posttreatment was proof of a programmed cell death response. This was associated with significant mitochondrial accumulation of bax and mitochondrial-to-cytoplasmic release of cytochrome c proteins between 8 and 14 h. Cleaved caspase-3 levels were low and mainly localized to inflammatory cells. The substantial cytoplasmic-to-nuclear translocation of apoptosis-inducing factor (AIF) and its 57-kDa activated fragment detected between 14 and 24 h after treatment indicated AIF as an effector for DNA fragmentation. CONCLUSION: Modulated EHT treatment can induce programmed cell death-related tumor destruction in HT29 colorectal adenocarcinoma xenografts, which dominantly follows a caspase-independent subroutine.

Selective in situ protein expression profiles correlate with distinct phenotypes of basal cell carcinoma and squamous cell carcinoma of the skin.

Non-melanoma skin cancer is the most common malignancy that shows increasing incidence due to our cumulative exposure to ultraviolet irradiation. Its major subtypes, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) differ in pathobiology, phenotype and clinical behavior, which must be reflected at the molecular level. In this study, protein expression profiles of BCC and SCC were tested in tissue microarrays and correlated with that of actinic keratosis, Bowen's disease, seborrheic keratosis and normal epidermis by detecting 22 proteins involved in cell interactions, growth, cell cycle regulation or apoptosis. The significantly more reduced collagen XVII, CD44v6, pan-Desmoglein levels and more evident E-Cadherin delocalization in BCC compared to SCC correlated with the de novo dermal invasion of BCC against the progressive invasion from in situ lesions in SCC development. EGFR was also expressed at a significantly higher level in SCC than in BCC. The upregulated cell communication protein connexin43 in BCC could contribute to the protection of BCC from metastatic invasion. Elevated cell replication in BCC was underlined by the increased topoisomerase IIα and reduced p21(waf1) and p27(kip1) positive cells fractions compared to SCC. Compared to differentiated keratinocytes, caspase-8 and -9 were equally upregulated in skin carcinoma subtypes for either mediating apoptosis induction or immune escape of tumor cells. Hierarchical cluster analysis grouped SCC and actinic keratosis cases exclusively together in support of their common origin and malignant phenotype. BCC cases were also clustered fully together. Differentially expressed proteins reflect the distinct pathobiology of skin carcinoma subtypes and can serve as surrogate markers in doubtful cases.

Collagen XVII is expressed in malignant but not in benign melanocytic tumors and it can mediate antibody induced melanoma apoptosis.

The 180 kDa transmembrane collagen XVII is known to anchor undifferentiated keratinocytes to the basement membrane in hemidesmosomes while constitutively shedding a 120 kDa ectodomain. Inherited mutations or auto-antibodies targeting collagen XVII cause blistering skin disease. Collagen XVII is down-regulated in mature keratinocytes but re-expressed in skin cancer. By recently detecting collagen XVII in melanocyte hyperplasia, here we tested its expression in benign and malignant melanocytic tumors using endodomain and ectodomain selective antibodies. We found the full-length collagen XVII protein in proliferating tissue melanocytes, basal keratinocytes and squamous cell carcinoma whereas resting melanocytes were negative. Furthermore, the cell-residual 60 kDa endodomain was exclusively detected in 62/79 primary and 15/18 metastatic melanomas, 8/9 melanoma cell lines, HT199 metastatic melanoma xenografts and atypical nests in 8/63 dysplastic nevi. The rest of 19 nevi including common, blue and Spitz subtypes were also negative. In line with the defective ectodomain, sequencing of COL17A1 gene revealed aberrations in the ectodomain coding region including point mutations. Collagen XVII immunoreaction-stained spindle cell melanomas, showed partly overlapping profiles with those of S100B, Melan A and HMB45. It was concentrated at vertical melanoma fronts and statistically associated with invasive phenotype. Antibody targeting the extracellular aa507-529 terminus of collagen XVII endodomain promoted apoptosis and cell adhesion, while inhibiting proliferation in HT199 cells. These results suggest that the accumulation of collagen XVII endodomain in melanocytic tumors is associated with malignant transformation to be a potential marker of malignancy and a target for antibody-induced melanoma apoptosis.