Ambivalent Effects of Tumor Necrosis Factor Alpha on Apoptosis of Malignant and Normal Human Keratinocytes

Introduction: Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that may paradoxically induce either apoptosis or cell survival. It mediates its activity through binding of TNF-receptor (TNFR) 1 or 2. TNFR1 is mainly responsible for transmitting apoptotic signals. The activation of apoptotic mechanisms can either be intrinsic (mitochondrial) or extrinsic (death receptors). Death ligands such as TNF-related apoptosis-inducing ligand (TRAIL) specifically induce extrinsic apoptosis, while cytostatic drugs such as 5-fluorouracil (5FU) induce intrinsic apoptosis. Objectives: To investigate the effects of TNFα on apoptosis in malignant and normal human keratinocytes. Methods: Human cutaneous squamous cell carcinoma (SCC) cell line SCC-13 and immortalized human keratinocytes HaCaT as well as primary normal human keratinocytes (PNHK) were stimulated with TNFα and then treated either with TRAIL or 5FU. Cell viability and cell proliferation, DNA fragmentation, apoptosis, and cytotoxicity were determined by WST-1 proliferation assay, ELISA, flow cytometry, and colorimetric analysis of lactate dehydrogenase, respectively. In addition, Western blotting was performed for analysis of caspase-3. Results: TNFα affected viability of SCC-13 and HaCaT cells in combination with 5FU or TRAIL. In contrast, TNFα did not influence cell viability of PNHK. It enhanced the apoptotic effects of both extrinsic and intrinsic stimuli in SCC-13 and HaCaT. In clear contrast, TNFα protected PNHK against TRAIL- and 5FU-induced apoptosis. The effects were dose-dependent and TNFα-specific; furthermore, the apoptosis pathway was caspase-dependent. Conclusions: In summary, opposing effects of TNFα in malignant versus normal human keratinocytes were observed with possibly relevant clinical implications, when patients are treated with TNFα inhibitors.

Trichohyalin-like 1 protein plays a crucial role in proliferation and anti-apoptosis of normal human keratinocytes and squamous cell carcinoma cells

Epidermal differentiation is a complex process that requires the regulated and sequential expression of various genes. Most fused-type S100 proteins are expressed in the granular layer and it is hypothesized that these proteins may be associated with cornification and barrier formation. We previously identified a member of the fused-type S100 proteins, Trichohyalin-like 1 (TCHHL1) protein. TCHHL1 is distributed in the basal layer of the normal epidermis. Furthermore, the expression is markedly increased in cancerous/non-cancerous skin samples with the hyperproliferation of keratinocytes. We herein examined the role of TCHHL1 in normal human keratinocytes (NHKs) and squamous cell carcinoma (SCC). The knockdown of TCHHL1 by transfection with TCHHL1 siRNA significantly inhibited proliferation and induced the early apoptosis of NHKs. In TCHHL1-knockdown NHKs, the level of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was markedly decreased. In addition, the slight inhibition of v-akt murine thymoma viral oncogene homolog (AKT) phosphorylation and upregulation of forkhead box-containing protein O1(FOXO1), B-cell lymphoma2 (BCL2) and Bcl2-like protein 11 (BCL2L11) was observed. Skin-equivalent models built by TCHHL1-knockdown NHKs showed a markedly hypoplastic epidermis. These findings highlight that TCHHL1 plays an important role in homeostasis of the normal epidermis. TCHHL1 was expressed in the growing cells of cutaneous SCC; therefore, we next examined an association with the cell growth in HSC-1 cells (a human SCC line). In HSC-1 cells, the knockdown of TCHHL1 also suppressed cell proliferation and induced apoptosis. These cells showed an inhibition of phosphorylation of ERK1/2, AKT and signal transducers and activator of transcription 3, and the significant upregulation of FOXO1, BCL2, and BCL2L11. Accordingly, TCHHL1 is associated with survival of cutaneous SCC. In addition, we hypothesize that TCHHL1 may be a novel therapeutic target in cutaneous SCC.

Resveratrol enhances apoptosis induced by the heterocyclic aromatic amines in p53-wt LoVo cells, but not in p53-deficient HaCaT cells

In the present study, we investigated the influence of resveratrol on PhIP treated human colon cancer cells and compared the effect to HaCaT cells considered as normal, human keratinocytes. Our results show that resveratrol decreases DNA damage in both cell types, it increases the sensitivity of LoVo cells to apoptosis and has no effect on PhIP-treated HaCaT cells. We confirm that PhIP-induced apoptosis is p53 and caspase 3/7 dependent. Interestingly, normal cells such as HaCaT, which lack functional p53 are more resistant to PhIP treatment.

Molecular Mapping of Hydrogen Sulfide Targets in Normal Human Keratinocytes

Although sulfur-rich thermal waters have ancestrally been used in the context of dermatological conditions, a global mapping of the molecular effects exerted by H2S on human keratinocytes is still lacking. To fill this knowledge gap, we subjected cultured human keratinocytes to distinct amounts of the non-gaseous hydrogen sulfur donor NaHS. We first checked that H2S accumulated in the cytoplasm of keratinocytes under our experimental conditions andused a combination of proteomics, genomics and biochemical approaches to unravel functionally relevant H2S targets in human keratinocytes. We found that the identified targets fall into two main categories: (i) the oxidative stress response molecules superoxide dismutase 2 (SOD2), NAD(P)H quinone dehydrogenase 1 (NQO1) and culin 3 (CUL3) and (ii) the chemokines interleukin-8 (IL-8) and CXCL2. Interestingly, NaHS also stimulated the caspase-1 inflammasome pathway, leading to increased secretion of the pro-inflammatory molecule interleukin-18 (IL-18). Interestingly, the secretion of interleukin-1 beta (IL-1β) was only modestly impacted by NaHS exposure despite a significant accumulation of IL-1β pro-form. Finally, we observed that NaHS significantly hampered the growth of human keratinocyte progenitors and stem cells cultured under clonogenic conditions or as epidermal cell sheets. We conclude that H2S exerts specific molecular effects on normal human keratinocytes.

Stem Cell Properties of Normal Human Keratinocytes Determine Transformation Responses to Human Papillomavirus 16 DNA

ABSTRACT Human papillomavirus (HPV) infection of the genital tract is common; however, only about 10 to 15% of infections persist, and approximately 10 to 15% of these persistent infections result in cancer. Basal epidermal stem cells are the presumed target cells for HPV infection, providing a reservoir of latently infected cells that persist over time and initiate lesions. However, it is not known whether stem cell density has any influence on transformation of human keratinocytes by HPV. We explored the relationship between stem cell properties of normal human keratinocytes and their susceptibility to transformation by HPV16 DNA. Normal human keratinocyte isolates (NHKc) derived from different donors were cultured in three-dimensional anchorage-free suspension to assess their spheroid-forming ability. NHKc spheroids were then plated back into plastic monolayer culture and transfected with full-length HPV16 DNA, which we have previously shown to integrate into the host cell genome upon transfection. Spheroid-derived NHKc (SD-NHKc) and fluorescence-activated cell sorting-purified populations of basal stem-like keratinocytes, expressing low levels of epidermal growth factor receptor and high levels of integrin alpha 6 (EGFR lo /ITGα6 hi ), responded to transfection with HPV16 DNA with more vigorous proliferation, greater immortalization efficiency, and faster progression to differentiation resistance than autologous mass-cultured cells. Conversely, cells committed to terminal differentiation (EGFR hi /ITGα6 lo ) grew slowly after transfection with HPV16 and failed to generate immortalized or DR clones. HPV16 DNA induced stem cell properties in mass-cultured NHKc. We conclude that HPV16 preferentially immortalizes basal keratinocytes with stem cell properties and that these cells readily achieve a differentiation-resistant phenotype upon immortalization by HPV16. IMPORTANCE This paper explores the relationship between the stem cell properties of normal human epidermal cells in culture and these cells' susceptibility to transformation by HPV16 DNA, the HPV type present in about 50% of cervical cancers. We report variable susceptibilities to HPV16-mediated transformation among different keratinocyte isolates derived from neonatal foreskin. Our findings provide strong experimental evidence that HPV16 preferentially transforms basal keratinocytes with stem cell properties. Insights gained from these studies increase our understanding of the host cell-specific factors influencing individual susceptibility to HPV-driven transformation and the contributing factors leading to preneoplastic and neoplastic progression of HPV-positive lesions.