Heme oxygenase 1 facilitates cell proliferation via the B-Raf-ERK signaling pathway in melanoma.

BACKGROUND: Despite therapeutic advancements (e.g. B-RAF inhibitors) targeting cutaneous melanoma, many cellular processes, including inducible heme oxygenase 1 (HO-1), counteract treatments for malignancies. So there is an urgent need to find biological treatment targets, develop new therapeutic approaches and achieve longer responses. This study aimed to explore the relationship of HO-1 and B-Raf via mediating ERK1/2 signaling on cell cycle in melanoma. METHODS: Immunohistochemistry was applied to evaluate the levels of HO-1 and B-Raf expression in melanoma tissues and adjacent healthy tissues. Co-immunoprecipitation (Co-IP) assessed the interaction of HO-1 with B-Raf. Further study overexpression and knock-down of HO-1 in A375 cell lines, especially knockout HO-1 using CRISPR-Cas9, verified HO-1 regulate cell proliferation in vivo and in vitro. Finally, Western blot analysis and qRT-PCR were performed to investigate the mechanisms by which HO-1 mediates cell cycle by B-RAF-ERK1/2 signaling. RESULTS: First, histology and Co-IP show that HO-1 interacts with B-Raf directly in melanoma tissue. Further study illustrated that HO-1 overexpression promotes melanoma cell proliferation while HO-1 reduction represses melanoma cell proliferation because of HO-1 affects cell cycle. Mechanistic studies revealed that HO-1 was associated with a marked activation of B-RAF-ERK1/2 signaling and led to CDK2/cyclin E activation, thereby promoting melanoma proliferation. CONCLUSIONS: Our result reveals a previously unknown mechanism that the HO-1-B-RAF-ERK axis plays an important role in melanoma cell proliferation. Therapeutic target on HO-1 could be a novel method for treating melanoma.

A novel heme oxygenase-1 splice variant, 14kDa HO-1, promotes cell proliferation and increases relative telomere length.

Alternative splicing is a routine phenomenon which greatly increases the diversity of proteins in eukaryotic cells. In humans, most multi-exonic genes are alternatively spliced and their splice variants confer distinct functions. Heme oxygenase-1 (HO-1, 32 kDa) is an inducible stress responsive protein, which possesses multiple functions in many cellular processes. In the current study, we identified a novel alternative splice isoform of 14 kDa HO-1 generated through exclusion of exon 3, and it is highly expressed in immortalized cells. In contrast to nuclear accumulation of the full-length 32 kDa HO-1, the novel 14 kDa HO-1 isoform is retained in the cytoplasm under ultraviolet (UV) irradiation. Interestingly, the 14 kDa HO-1 is shown to promote cell proliferation and an increase in relative telomere lengths in vivo and in vitro. Thus, we are pioneer to report and confirm the presence of a novel splice form of HO-1 and its distinct role in modulating telomere length and tumor growth.

MicroRNA let-7b inhibits keratinocyte differentiation by targeting IL-6 mediated ERK signaling in psoriasis.

BACKGROUND: The extensive involvement of microRNA (miRNA) in the pathophysiology of psoriasis is well documented. However, in order for this information to be useful in therapeutic manipulation of miRNA levels, it is essential that detailed functional mechanisms are elucidated. This study aimed to explore the effects of IL-6 targeting by let-7b and ERK1/2 mediated signaling on keratinocyte differentiation in psoriasis. METHODS: Following imiquimod cream (IMQ) application to let-7bTG (keratinocyte-specific let-7b overexpression mouse) and control mice for 7 days, we analyzed erythema, scaling and thickening of skin. A dual luciferase reporter assay and bioinformatics was carried out to detect target gene of let-7b. Additionally, the differentiation markers were measured. Immunohistochemistry analyses demonstrate a relationship of let-7b with IL-6 and ERK signaling. RESULTS: we found let-7bTG inhibits acanthosis and reduces the disease severity by treatment with IMQ compared to wild-type mice. Further study illustrated that let-7b promotes differentiation of keratinocytes in vivo and in vitro. Using bioinformatics and reporter gene assays, we found that IL-6 is a target gene of let-7b. In psoriasis, high expression levels of IL-6 lead to increased acivation of p-ERK1/2. High levels of let-7bTG transgene expression suppresses IL-6 expression and leads to increased keratinocyte differentiation. Moreover, let-7b acts as an upstream negative regulator of the ERK signaling pathway in keratinocytes of psoriasis. CONCLUSIONS: Our result reveals a previously unknown mechanism for regulation of IL-6 levels during psoriasis by let-7b and highlights a critical role for the ERK1/2 signaling pathway in epidermal differentiation during psoriasis. TRIAL REGISTRATION: The ethical approval for this study was from the Affiliated Hospital of Medical University of Anhui _ Fast_ PJ2017-11-14.

Nrf2- and Bach1 May Play a Role in the Modulation of Ultraviolet A-Induced Oxidative Stress by Acetyl-11-Keto-ß-Boswellic Acid in Skin Keratinocytes.

BACKGROUND: Exposure of human skin to solar ultraviolet A (UVA) irradiation causes severe oxidative stress with damage to various cellular components and concomitant inflammation and carcinogenesis. OBJECTIVE: The aim of this study is to investigate the protective effect of acetyl-11-keto-ß-boswellic acid (AKBA) against UVA radiation on human skin keratinocytes. METHODS: HaCaT cells were pretreated with AKBA followed by UVA irradiation. Radiation effects on cell morphology, cell viability, intracellular reactive oxygen species (ROS) levels, and antioxidant enzymes were examined. RESULTS: AKBA reduces UVA irradiation-induced cell viability loss, accompanied by a decreased production of UVA-induced ROS, decreased malondialdehyde, and increased superoxide dismutase expression. In addition, AKBA increased basal and UVA-induced levels of Nrf2 (NF-E2-related factor 2), the redox-sensitive factor, and its target genes NQO1 and heme oxygenase-1 (HO-1), whereas expression of the transcriptional repressor Bach1 (BTB and CNC homology 1) was reduced. Furthermore, the cytoprotective effects of AKBA against UVA-derived oxidative damage were accompanied by modulating expression of inflammatory mediators (i.e., cyclooxygenase-2 and nuclear factor-κB) and NOX1. CONCLUSIONS: AKBA protects skin cells from UVA-induced damage by modulating inflammatory mediators and/or ROS production. Therefore, AKBA has potential in the development of skin care products.

CCT6A promotes cell proliferation in colon cancer by targeting BIRC5 associated with p53 status.

Chaperonin-containing TCP1 (CCT) is a multi-subunit complex, known to participate the correct folding of many proteins. Currently, the mechanism underlying CCT subunits in cancer progression is incompletely understood. Based on data analysis, the expression of CCT subunit 6 A (CCT6A) is found higher than the other subunits of CCT and correlated with an unfavorable prognosis in colon cancer. Here, we find CCT6A silencing suppresses colon cancer proliferation and survival phenotype in vitro and in vivo. CCT6A plays a role in cellular process, including the cell cycle, p53, and apoptosis signaling pathways. Further investigations have shown direct binding between CCT6A and both Wtp53 and Mutp53, and BIRC5 is found to act downstream of CCT6A. The highlight is that CCT6A inhibition significantly reduces BIRC5 expression independent of Wtp53 levels in Wtp53 cells. Conversely, in Mutp53 cells, downregulation of BIRC5 by CCT6A inhibition mainly depends on Mutp53 levels. Additionally, combined CCT6A inhibition and Wtp53 overexpression in Mutp53 cell lines effectively suppresses cell proliferation. It is concluded CCT6A is a potential oncogene that influences BIRC5 through distinct pathways in Wtp53 and Mutp53 cells.

Cloning, expression and characterization of the putative nuclear transport factor 2 (NTF2) gene from moss Conocephalum conicum(L.) Dum.

Biomacromolecules import into the nucleus is a complex progress which requires the participation of several cytosolic factors, and nuclear transport factor 2 (NTF2) is one of essential components in nuclear trafficking. Its main role is to transport RanGDP from cytoplasm to nucleus by interacting with FxFG nucleoporin repeats. In the study a putative new gene, designated as CcNTF2, was obtained from the moss (Conocephalum conicum) cDNA library we have constructed. The full-length cDNA sequence is 913 bp in size contains a 372 bp open reading frame (ORF) flanked by a 195 bp 5'-untranslated sequence and a long 346 bp 3'-non-coding region, encoding 123 amino acids of 13,575.3 Da. Part of the genomic sequence was also cloned and sequenced, which is 1,602 bp long and possesses two exons and one intron. Alignment analysis showed that the CcNTF2 protein is high conserved among plant NTF2 and shares 81% similarity with the ones from Arabidopsis thaliana and Brassica rapa. The expression of wild-type CcNTF2 was detected by immunoblotting of extraction of C. conicum and it indicated the putative protein is integral. Through functional expression of CcNTF2-green fluorescent protein (GFP) in tobacco, it was demonstrated that CcNTF2 can accumulate at the nuclear rim. Site-directed mutagenesis analysis confirmed CcNTF2 P71K has influence on the protein import into nucleus. In addition, overexpression of CcNTF2 P71K was observed to be deleterious for the plant cell. It is the first illumination of NTF2 in moss, and our study established the primary foundation for further research on moss NTF2.

The p75NTR and its carboxyl-terminal fragment exert opposing effects on melanoma cell proliferation and apoptosis via modulation of the NF-κB pathway.

The p75 neurotrophin receptor (p75NTR ), a member of the tumor necrosis factor superfamily of receptors, is sensitive to proteolysis and has been observed to be expressed in various cancers. However, the roles of p75NTR and its proteolytic fragments in tumorigenesis remain incompletely understood. Here, we report that the proportion of the p75NTR carboxyl-terminal fragment (p75NTR -CTF) is much higher than that of the full-length p75NTR (p75NTR -FL) in melanoma cells. Whereas p75NTR -FL positively regulates apoptosis, p75NTR -CTF promotes cell proliferation and survival, as well as increasing sorafenib resistance in vivo and in vitro. Moreover, p75NTR -CTF activates the nuclear factor kappa B pathway and enhances the mRNA and protein levels of its downstream genes c-IAP1/2, FLIP, bFGF, IL8 and VEGF. On the contrary, p75NTR -FL inhibits these processes. Taken together, these findings demonstrate that p75NTR -CTF and p75NTR -FL have opposing functions in melanoma cells, suggesting that the ratio of the two proteins affects the balance between cell death and survival. The presence of distinct p75NTR proteolytic fragments may affect biological outcomes in tumor cells.

UVA Irradiation Enhances Brusatol-Mediated Inhibition of Melanoma Growth by Downregulation of the Nrf2-Mediated Antioxidant Response.

Brusatol (BR) is a potent inhibitor of Nrf2, a transcription factor that is highly expressed in cancer tissues and confers chemoresistance. UVA-generated reactive oxygen species (ROS) can damage both normal and cancer cells and may be of potential use in phototherapy. In order to provide an alternative method to treat the aggressive melanoma, we sought to investigate whether low-dose UVA with BR is more effective in eliminating melanoma cells than the respective single treatments. We found that BR combined with UVA led to inhibition of A375 melanoma cell proliferation by cell cycle arrest in the G1 phase and triggers cell apoptosis. Furthermore, inhibition of Nrf2 expression attenuated colony formation and tumor development from A375 cells in heterotopic mouse models. In addition, cotreatment of UVA and BR partially suppressed Nrf2 and its downstream target genes such as HO-1 along with the PI3K/AKT pathway. We propose that cotreatment increased ROS-induced cell cycle arrest and cellular apoptosis and inhibits melanoma growth by regulating the AKT-Nrf2 pathway in A375 cells which offers a possible therapeutic intervention strategy for the treatment of human melanoma.

Label-free electrochemical sensor to investigate the effect of tocopherol on generation of superoxide ions following UV irradiation.

BACKGROUND: Generation of reactive oxygen species (ROS), triggered by ultraviolet radiation (UVR), is associated with carcinogenesis of the skin. UV irradiation induced superoxide anion (O2•-) is the key ROS involved in the cellular damage. The cytoprotective efficacy of an unknown anti-oxidant compound can be evaluated by analyzing the production of O2•- from treated cells. METHODS: In this study, a glass carbon electrode functionalized with nanotube@DNA-Mn3(PO4)2 composite was applied to quantitative determination of generation of highly unstable O2•- from the melanoma A375 cell line following UVR(UV, UVA and UVB). In addition, the cytoprotective efficacy of anti-oxidant α-tocopherol was evaluated by quantifying the production of O2•-. RESULTS: The results showed that, UVR triggers generation of O2•- in melanoma A375 cells, and α-tocopherol is effective in diminishing the production of O2•- following UV irradiation. By comparing the conventional cell-survival assays results, we found that our simple and quick electrochemical sensing method can quantify O2•- generation through the biological activity of an anti-oxidant compound (α-tocopherol). CONCLUSION: Our label-free electrochemical quantification method for ROS (O2•- major) in cells facing UVR stress demonstrates its potential application for high-throughput screening of anti-oxidation compounds.

Telomerase reverse transcriptase mediates EMT through NF-κB signaling in tongue squamous cell carcinoma.

Locoregional lymph nodes metastasis in oral tongue squamous cell carcinoma represents one of important and common prognostic factors for poor clinical outcome. The human Telomerase Reverse Transcriptase (hTERT) is one of key players in cancer metastasis and stemness, but its exact function in tongue squamous cell carcinoma remains unknown. Here, we aim to understand the role of hTERT by utilizing the CRISPR/Cas9 gene editing system to deplete hTERT in the SCC-15 cell line. Functional comparison of SCC-15 control and knockout cells (hTERT-/-) showed that loss of hTERT suppressed cell proliferation and migration/invasion. Furthermore, hTERT depletion significantly decreased tumorigenesis, including alterations in cellular morphology that areindicative for epithelial-mesenchymal transition (EMT). Mechanistically we demonstrated that the hTERT knockout attenuates NF-κB signaling via a negative feedback regulation in tumorprogression. From these results we propose a novel molecular mechanism of hTERT to promote SCC-15 invasion and metastasis via NF-κB activation. We conclude that targeting hTERT may represent a new therapeutic strategy to improve therapy and survival of tongue squamous cell carcinoma patients.

Development of refractoriness of HO-1 induction to a second treatment with UVA radiation and the involvement of Nrf2 in human skin fibroblasts.

UVA treatment of cultured human skin fibroblasts (FEK4) has been shown previously to reduce transcriptional activation of heme oxygenase 1 (HO-1) following a second dose of UVA radiation, a phenomenon known as refractoriness. This study demonstrates that the levels of HO-1 protein are also reduced after a second dose of UVA radiation as are Nrf2 levels, and there is less accumulation of Nrf2 in the nucleus where as Bach1 does accumulate in the nucleus. Cell viability is further reduced and cell membrane damage increased as compared with a single UVA treatment when an initial UVA treatment was followed by a second dose. Knockdown of Nrf2 by siRNA (siNrf2) targeting caused additional refractoriness of HO-1 protein induction to a second UVA or heme treatment and this treatment also further enhanced cell damage by a second dose of UVA radiation. However, transfection with Nrf2 caused less refractoriness of HO-1 to a second dose of UVA and reduced cell damage by a second dose of UVA radiation. These findings are consistent with the proposal that Nrf2 is involved in HO-1 refractoriness and could serve as a cytoprotective factor against cell damage caused by repeated exposure to moderate doses of UVA radiation. We propose that protection by the Nrf2-HO-1 pathway protection may have clinical relevance since human skin is exposed repeatedly to UVA radiation.