Normal Epidermal Cells Research Articles

Qa-1b functions as an oncogenic factor in mouse melanoma cells

BackgroundMalignant melanoma is one kind of rare cancer in human and animals, which occurs not only in skin but also in the mucous membranes of the nose, mouth, anus, digestive tract, and in the uvea (choroid) of the eye. In order to develop therapies, a better understanding of the genetic landscape and signal pathways are needed. Numerous studies highlighted the importance of NKG2A-HLA-E in tumor immunotherapy, but the function and mechanism of HLA-E in tumor cells have seldom been studied alone. The statistical analyses from publicly available database Oncomine and The Cancer Genome Atlas (TCGA) showed that HLA-E is highly expressed in melanoma compared to non-transformed counterparts. In addition, melanoma patients with HLA-E high expression had a worse OS rate than the patients with low expression. These data indicate the importance of HLA-E in human melanoma. Qa-1b is the homolog of HLA-E in mouse. ObjectiveTo investigate the function and mechanism of Qa-1b in mouse melanoma. MethodsMouse melanoma cell line B16-F10 and allogenic melanoma model were used to investigate the function and mechanism of Qa-1b in melanoma. ResultsQa-1b was highly expressed in B16-F10 compared with normal mouse epidermal cells. Qa-1b knockdown inhibited B16-F10 cell proliferation and migration in vitro and allogenic melanoma process in vivo. Furthermore, Qa-1b knockdown promoted cell cycle arrest at G0/G1 phase and cell apoptosis through Ras-Raf-MAPK signal pathway. ConclusionQa-1b functions as an oncogenic factor and it can be used as a new therapeutic target in melanoma.

Combinational Anti-tumor Effects of Chemicals from Paeonia lutea Leaf Extract in Oral Squamous Cell Carcinoma Cells.

We identified chemical components that exhibited antitumor activity against oral squamous cell carcinoma (OSCC) cells and examined their effective concentrations and additive and/or synergistic effects in combinational usage on the proliferation, apoptosis and cell cycle of OSCC cells. Using high-performance liquid chromatography, nuclear magnetic resonance spectroscopy and electrospray ionization-mass spectrometry, we identified the main chemical components of the methanol extracts from Paeonia lutea. We investigated the pharmaceutical effects of those components on the proliferation, apoptosis, and cell cycle of an OSCC cell line, SAS, using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and caspase assays, as well as flow cytometry cell cycle analysis. We also examined the effects of those components on the mitogen-activated protein kinase signal transduction pathway by western blotting. Finally, the effects on normal human epidermal keratinocyte cells were also examined in similar experiments. Three chemicals have been identified in P. lutea leaves using high performance liquid chromatography: gallic acid methyl ester (GAME), pentagalloyl glucose (PGG) and paeoniflorin (PF). Both GAME and PGG significantly suppressed cell proliferation, and their combined effects were synergistic, while the effect of PF was minimal. However, those chemicals did not induce apoptosis. Cell cycle and western blotting analysis showed that the suppressive effects on cell proliferation resulted from G2 arrest and the suppression of phosphorylation of Akt/PKB. No effect was identified on normal human epidermal keratinocyte cells. These results indicate that GAME and PGG are the main chemical components of P. lutea leaves that have potential anti-cancer therapeutic effects.

TCF21 regulates miR-10a-5p/LIN28B signaling to block the proliferation and invasion of melanoma cells.

Background and aimSome research has suggested that miRNA-10a (miR-10a-5p) had an inhibitory function in proliferation and invasion of cancers. Whereas the role of miR-10a-5p in melanoma has not been fully explored. This study aims to confirm LIN28B as the targeted gene of miR-10a-5p which was explored in melanoma cells. In addition, upstream regulatory molecule of miR-10a-5p was also investigated in melanoma cells.MethodsReal-time Quantitative polymerase chain reaction (RT-qPCR) was adopted to analyze miR-10a-5p expression level in melanoma and the normal human epidermal melanocyte cells. Several biological assays were performed to evaluate miR-10a-5p influences on cell proliferation, migration and invasion ability in A375 and B16-F10 cells. Gene prediction of miRNA targeting and a dual luciferase assay were applied to assess miR-10a-5p-targeted LIN28B. Western blot assessed the impacts of miR-10a-5p on the protein expression of LIN28B. Western blot analyzed the TCF21 effects on the expression of LIN28B and RT-qPCR assessed the influence of TCF21 on the expression level of miRNA-10a. In addition, Chromatin Immunoprecipitation (ChIP) Assay and JASPAR databases were employed to explore the regulatory relationship between TCF21 and miR-10a-5p.ResultsWe discovered that miR-10a-5p expression was lower in melanoma cells and high expression of miR-10a-5p suppressed the proliferation, migration and invasion abilities of melanoma cells. We also discovered that miR-10a-5p targeted the LIN28B mRNA 3′UTR area and diminished LIN28B protein expression. We found that LIN28B expression was strongly decreased by TCF21 upregulation in the two melanoma cells. The qRT-PCR assay showed that miR-10a-5p expression level was obviously boosted by increased TCF21 expression. The results also demonstrated that TCF21 directly regulated miR-10a-5p at transcript levels.ConclusionTCF21 induced miRNA-10a targeting LIN28B could affect the progression and growth of melanoma.

Epitope Mapping of an Antihuman EGFR Monoclonal Antibody (EMab-134) Using the REMAP Method.

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that plays an important role in normal epidermal cell physiology. EGFR is overexpressed in cancer cells and has a number of mutations that implicate tumor malignancy, development, and poor patient prognosis; thus, EGFR is an attractive target for cancer therapy. At present, anti-EGFR monoclonal antibodies (mAbs) have been approved and are used for treating patients with a variety of EGFR-expressing cancers. Epitope mapping is important in identifying the therapeutic mechanism of anti-EGFR mAbs; however, the development of epitope mapping techniques lags behind the development of antimolecular target mAbs, including anti-EGFR mAbs. Hence, in this study, a novel epitope mapping method, RIEDL insertion for epitope mapping (REMAP) method, was developed. The results of this study demonstrated that the critical epitope of anti-EGFR mAb EMab-134 is Gly378, Asp379, Ser380, Phe381, Thr382, His383, Thr384, Pro385, and Pro386 of EGFR. The REMAP method could be useful for determining the critical epitope of functional mAbs against many target molecules.

Differential expression of phosphorylated MEK and ERK correlates with aggressive BCC subtypes.

Basal cell carcinoma (BCC) is associated with aberrant Hedgehog (HH) signalling through mutational inactivation of PTCH1; however, there is conflicting data regarding MEK/ERK signalling in BCC and the signalling pathway interactions in these carcinomas. To address this, expression of active phospho (p) MEK and ERK was examined in a panel of 15 non-aggressive and 14 aggressive BCCs. Although not uniformly expressed, both phospho-proteins were detected in the nuclei and/or cytoplasm of normal and tumour-associated epidermal cells however, whereas phospho-MEK (pMEK) was present in all non-aggressive BCCs (14/14), phospho-ERK (pERK) was rarely expressed (2/14). In contrast pERK expression was more prevalent in aggressive tumours (11/14). Interestingly, pMEK was only localized to the tumour mass whereas pERK was expressed in tumours and stroma of aggressive BCCs. Similarly, pERK (but not pMEK) was absent in mouse BCC-like tumours derived from X-ray irradiated Ptch1+/- mice with stromal pERK observed in myofibroblasts of the aggressive variant as well as in the tumour mass. RNA sequencing analysis of tumour epithelium and stroma of aggressive and non-aggressive BCC revealed the upregulation of epidermal growth factor receptor- and ERK-related pathways. Angiogenesis and immune response pathways were also upregulated in the stroma compared with the tumour. PTCH1 suppressed NEB1 immortalized keratinocytes (shPTCH1) display upregulated pERK that can be independent of MEK expression. Furthermore, epidermal growth factor pathway inhibitors affect the HH pathway by suppressing GLI1. These studies reveal differential expression of pERK between human BCC subtypes that maybe active by a pathway independent of MEK.

Senescent cell removal via JAG1-NOTCH1 signalling in the epidermis.

Emerging evidence has pointed to the noxious effects of senescent cells in various tissues, and senescent cells in the epidermis are known to accumulate with age. We hypothesized that there is a mechanism by which senescent cells in the epidermis are preferentially removed and that the function of such removal mechanism declines as age increases. In this study, we investigated whether Notch signalling is involved in such senescent cell removal. We found that Notch1 receptor was expressed more highly in p16INK4a-positive senescent cells than in surrounding cells in human epidermis both in young and old subjects. On the other hand, the expression of its ligand JAG1 was decreased in the epidermis of aged subjects. When normal epidermal cells and UVB-irradiated senescent cells were mixed and three-dimensional reconstructed epidermis was developed in vitro, the senescent cells were preferentially removed from the basal layer and located in the upper layer. We also found that the depletion of senescent cells from the basal layer was suppressed by JAG1 knockdown in normal cells or using a Notch signalling inhibitor. From these results, Notch signalling may be involved in senescent cell removal in the epidermis and the age-related decrease of JAG1 expression in the basal layer may lead to accumulation of senescent cells owing to reduced activation of Notch signalling.

Sunitinib decreases the expression of KRT6A and SERPINB1 in 3D human epidermal models.

Hand-foot skin reaction (HFSR) is a common side effect caused by several tyrosine kinase inhibitors, including sunitinib. However, the nature of the cornifying factors related to the molecular biological mechanisms underlying HFSR remains poorly understood. We used human keratinocyte models to investigate the key cornifying factors for dermatological and biological abnormalities induced by sunitinib. On the basis of the results of microarray analysis using the three-dimensional (3D) human epidermal model, keratin (KRT)6A, serine protease inhibitor (SERPIN)B1, KRT5, and SERPIN Kazal-type 6 were selected as candidate genes related to HFSR. Sunitinib treatment significantly decreased the expression of SERPINB1 and KRT6A in the immunohistochemical staining of the 3D epidermal model. In PSVK1 cells, but not in normal human epidermal keratinocyte cells, both of which are human normal keratinocyte cell lines, sunitinib decreased the expression of KRT6A with a concomitant decrease in levels of phosphorylated extracellular signal-regulated kinases (ERK)1/2 and phosphorylated p38 mitogen-activated protein kinase (MAPK). Inhibitors of the ERK and p38 MAPK signal pathways also significantly decreased KRT6A expression. Sunitinib-induced decrease in KRT6A expression was suppressed by the inhibition of glycogen synthase kinase-3β by enhancing ERK1/2 and p38 MAPK phosphorylation. Thus, sunitinib reduces the expression of KRT6A and SERPINB1 by inhibiting the ERK1/2 and p38 MAPK signalling pathways in the skin model. These changes in expression contribute to the pathology of HFSR.

MiR-143 acts as an inhibitor of migration and proliferation as well as an inducer of apoptosis in melanoma cancer cells in vitro.

Melanoma is a serious form of skin cancers begins in the melanocyte. Micro-RNAs are small noncoding RNA with 19 to 25 nucleotides in length involves in the regulation of a wide range of biological processes. MicroRNAs are affected by an aberrant epigenetic alteration in the tumors that may lead to their dysregulation and formation of cancer. Recently, dysregulation of numerous microRNAs has been reported in different types of cancer. The present study focused on the role of miR-143 in carcinogenesis of melanoma cancer. Here, we evaluated the expression level of miR-143 in three melanoma cell lines in comparison with the normal human epidermal melanocyte cell line. Then, miR-143 gene plasmid transfected into the WM115 cell line, for having the lowest expression of miR-143. In addition, the effect of miR-143 transfection on mRNA and protein levels of metastasis-related genes was performed along with MTT assay, wound healing assay, and flow cytometry. The results showed that mRNA and protein expression levels of metastasis-related genes including MMP-9, E-cadherin, Vimentin, and CXCR4 have been reduced following transfection of miR-143. Moreover, the results of the scratch test showed that miR-143 re-expression inhibited cell migration. Also, the role of miR-143 in the induction of apoptosis and inhibition of proliferation by flow cytometry and MTT was confirmed. As a result, the present study showed that miR-143 was involved in metastatic and apoptotic pathways, suggesting that miR-143 acts as a tumor-suppressor microRNA in melanoma cancer.

146 Global phosphoproteomic analysis of protein kinase C delta activation reveals signaling pathways involved in triggering keratinocyte growth arrest

Activation of multiple protein kinase C (PKC) isoforms have been associated with keratinocyte (KC) cell cycle withdrawal during differentiation. To study how PKC activation may induce KC growth arrest, we evaluated the effects of the PKC agonist TPA on normal human epidermal KCs (NHEKs) and HaCaT cells, an immortalized human KC cell line. Treating NHEKs with 10 nM TPA for as little as 15 minutes induced complete growth arrest, while untreated KCs underwent a 93-fold increase in cell number over 7 days (p<0.005). HaCaT cells were completely resistant to TPA-induced growth arrest, indicating that immortalized KCs have defective PKC-dependent growth arrest signaling. To test if PKCδ activation was able to enforce growth arrest in HaCaT cells, we transduced HaCaT cells with retroviruses encoding either EGFP or a PKCδ-EGFP fusion and sorted EGFP positive cells. HaCaT-EGFP cells behaved similar to HaCaTs and were not growth arrested by TPA treatment, while HaCaT-PKCδ-EGFP cells were 80% growth inhibited by TPA over a 4 day period. TPA-induced growth inhibition in HaCaT-PKCδ-EGFP cells was due to arrest in G1 and G2/M, not apoptosis. Global phosphoproteomics analysis of HaCaT-EGFP and HaCaT-PKCδ-EGFP cells exposed to TPA for 30 minutes identified 667 proteins whose phosphorylation was significantly changed >2-fold, with 605 proteins having increased phosphorylation in TPA-treated PKCδ-EGFP cells. Surprisingly, only 54 of these proteins were phosphorylated at the PKCδ consensus motif, with phosphorylated motifs for the kinases MAPKAP2, AKT1 and RSK2 most abundant. Gene set enrichment analysis revealed increased phosphorylation of proteins involved in translation (eIF4, mTOR, eIF2 signaling), Rho, tight junctions and ERK/MAPK signaling. Thus, global phosphoproteomics identified PKCδ signaling pathways that can overcome the defective growth arrest in immortalized KCs and may have therapeutic benefit for the treatment of actinic keratosis and squamous cell carcinomas.

Role of SPRED1 in keratinocyte proliferation in psoriasis.

Psoriasis is a recurrent inflammatory skin disease, affecting approximately 2% of the population. Previous studies have demonstrated that psoriatic dermal mesenchymal stem cells (DMSC) stimulated keratinocyte (KC) proliferation and that psoriasis exhibited missense SPRED1 mutations. To further investigate the molecular mechanism by which psoriatic DMSC stimulate KC proliferation, and the role of missense SPRED1 mutations in psoriasis, we assessed expression levels of miRNA, and both mRNA and protein of SPRED1 in normal human epidermal keratinocyte cells (NHEK) cocultured with either psoriatic or control DMSC. Expression levels of miRNA and mRNA were determined by RNA sequencing. Expression levels of spred1 protein were assessed using western blot analysis. Moreover, the variation in SPRED1 was also examined by whole-genome sequencing in 665 psoriatic patients, and verified by Sanger sequencing. Our results showed that coculture of NHEK with psoriatic DMSC induced 32 differentially expressed miRNA, in which expression levels of miR-1 increased approximately 16-fold over control DMSC-treated NHEK (P<0.05). Likewise, expression levels of miR-21-3p increased over twofold (P<0.05). Moreover, coculture of NHEK with psoriatic DMSC induced marked increase in expression levels of mRNA for MAPK3, CDC25B and CDC25C, while decreasing expression levels of SPRED1 mRNA and protein in comparison with control DMSC treatment (P<0.05 for all between cocultured with control and psoriatic DMSC). Furthermore, psoriasis displayed non-synonymous mutation of SPRED1 enriched in exon 7: c.A881T:p.Y294F (chr15:38351210). These results suggest that dysregulation and mutations of SPRED1 may participate in the pathogenesis of psoriasis, including epidermal hyperproliferation.

Grape skin extract prevents UV irradiation induced DNA damage of normal human epidermal keratinocytes cells

BACKGROUND: There is concern that DNA damage may occur in skin cells due to UV irradiation. In this study, we investigated whether extracts from grape skin, which should be discarded, can suppress DNA damage caused by UV radiation. OBJECTIVE: This study aims to investigate the effectivity of GSE in diminishing UV-induced cytotoxicity in normal human epidermal keratinocytes (NHEK) cells. METHODS: The polyphenol content in GSE was carried out using the HPLC instrument. UV dose was selected by measurement of cell viability, and ELISA results of DNA photoproduct. Optimum extracts condition of grape skin selected by DNA photoproduct content as well as at this condition; Bax/Bcl-2 ratio and cytochrome c gene expressions were evaluated by Western blotting. RESULTS: In this study, we confirmed that GSE protect against DNA damage-induced cell death. Condition for 80% EtOH for 24 h at 60 °C was suitable to extract for grape skin. Red grape skin (Zweigelt) was more effective than white grape skin (Niagara) in preventing (before irradiation) and repairing (after irradiation). UV-induced upregulation of Bax/Bcl-2 ratio and cytochrome c expression were reduced by GSE treatment. CONCLUSIONS: The study demonstrated a promising potential of GSEs in skin therapeutics application.

Synthesis and in vitro cytotoxicity evaluation of star-shaped polymethacrylic conjugates with methotrexate or acitretin as potential antipsoriatic prodrugs

Water-soluble polymer-drug conjugates were obtained and analyzed towards their potential use as prodrugs for two hydrophobic antipsoriatic agents, including methotrexate (MTX) and acitretin (AC). The conjugation efficacy of MTX decreased with a decreasing molar ratio of N,N-dimethylaminoethyl methacrylate (DMAEMA) repeating units in the polymethacrylic chains. Cytotoxicity of positively charged (from +5 to +10 mV) nano- and microparticles (3–1500 nm in DMEM at 37 °C) were estimated by in vitro MTT and Annexin-V apoptosis assays on Me45, NHDF, HaCaT and BEAS-2B cell lines. Further, cell cycle analysis revealed arrest in G0/G1 phase in melanoma cells, while neither apoptosis induction nor cell cycle arrest occurred in normal epidermal and epithelial cells. Tested conjugates displayed a novel cytostatic effect in Me45 cells and a pro-apoptotic effect in HaCaT cells. Epithelial BEAS-2B cells were the most sensitive to the tested conjugates and responded via induction of necrosis. Cell line models allowed for characterization of the biologically relevant potential action of pro-drugs. Additionally, a skin in vitro evaluation assay provided the first known evidence of side-effect reduction with pro-drug use. Histological examinations confirmed the lack of negative effects of conjugates on the skin and showed no irritating properties.

Jasmonic Acid Induces Endoplasmic Reticulum Stress with Different Outcome in Cultured Normal and Tumor Epidermal Cells.

Plant hormones produce cytotoxic effect on human cells and can trigger the processes unrelated to cell death, e.g., biosynthetic system stress. The goal of this study was to investigate activation of the endoplasmic reticulum (ER) stress by jasmonic acid (JA) and to distinguish between the responses of cultured immortalized non-tumorigenic HaCaT cells and epidermoid carcinoma A431 cells to this plant hormone. JA was used in the concentration of 2 mM, as it suppressed cell proliferation in both cell lines. We analyzed expression of genes associated with the activation of ER stress (GRP78, ATF4, CHOP), the structure of the ER and Golgi complex, and synthetic processes in the HaCaT and A431 cell lines. JA induced expression of genes responsible for the activation of ER stress and caused hypertrophic changes in the Golgi complex in both cell lines. However, the patterns of gene expression in the HaCaT and A431 cells were different, and higher levels of involucrin synthesis were observed in A431 but not in HaCaT cells, suggesting that JA activated differentiation of the tumor A431 cells only. Therefore, JA induced ER stress in both cell lines, but the consequences of ER stress were different for the epidermal immortalized non-tumorigenic and tumor cells.

Surface modification of oriented polysaccharide scaffolds using biotic nanofibers for epidermal cell culture

In this study, a novel scaffold was fabricated from nematically ordered polysaccharide molecules such as cellulose, chitin and cellulose-chitin blend (50–50%), termed as NOC, NOCh, NOC-Ch, by deposition of biotic nanofibers such as bacterial cellulose (BC) and collagen, which were prepared by aqueous counter collision method to allow the fiber width to reduce into nano-scales by high pressure impinging of aqueous suspension jets. The resultant aqueous dispersion of the nanofibers was spread over the templates using the spin coating technique and its surface morphology, roughness, strength, and hydrophobicity were characterized by atomic force microscopy followed by the tensile strength and contact angle measurements. The results indicated that, after initial deposition of BC nanofibers, the surface wettability of the scaffolds switched from hydrophobic to hydrophilic, whereas for collagen nanofibers, the surface hydrophobicity was increased. Repeated coating also improved the strength of the scaffold. Cell culture studies were carried out to explore the potentials of the modified scaffolds using the normal human epidermal cells. The results showed that the number of nanofiber coating cycles and surface morphology on the scaffolds played a role in cell elongation and its morphology. Particularly in this study, we have focussed on the changes in the morphogenesis such as cell adhesion and cell size grown on 1, 3 and 5 times coated scaffolds. The result indicated that the morphological changes depending on the coating nanofibers in the surface of scaffolds were related to increase in the epidermal cell size and altering cell adhesion.

Water-soluble variant of human Lynx1 induces cell cycle arrest and apoptosis in lung cancer cells via modulation of α7 nicotinic acetylcholine receptors

Lynx1 is the first three-finger prototoxin found in the mammalian central nervous system. It is a GPI-anchored protein modulating nicotinic acetylcholine receptors (nAChRs) in the brain. Besides the brain, the Lynx1 protein was found in the lung and kidney. Endogenous Lynx1 controls the nicotine-induced up-regulation of the expression of α7 type nAChRs in lung adenocarcinoma A549 cells as well as the cell growth. Here, we analyzed the Lynx1 expression in the set of human epithelial cells. The Lynx1 expression both at the mRNA and protein level was detected in normal oral keratinocytes, and lung, colon, epidermal, and breast cancer cells, but not in embryonic kidney cells. Co-localization of Lynx1 with α7-nAChRs was revealed in a cell membrane for lung adenocarcinoma A549 and colon carcinoma HT-29 cells, but not for breast adenocarcinoma MCF-7 and epidermoid carcinoma A431 cells. The recombinant water-soluble variant of Lynx1 without a GPI-anchor (ws-Lynx1) inhibited the growth of A549 cells causing cell cycle arrest via modulation of α7-nAChRs and activation of different intracellular signaling cascades, including PKC/IP3, MAP/ERK, p38, and JNK pathways. A549 cells treatment with ws-Lynx1 resulted in phosphorylation of the proapoptotic tumor suppressor protein p53 and different kinases participated in the regulation of gene transcription, cell growth, adhesion, and differentiation. Externalization of phosphatidylserine, an early apoptosis marker, observed by flow cytometry, confirmed the induction of apoptosis in A549 cells upon the ws-Lynx1 treatment. Our data revealed the ability of ws-Lynx1 to regulate homeostasis of epithelial cancer cells.

Propofol suppresses proliferation, invasion, and migration of human melanoma cells via regulating microRNA-137 and fibroblast growth factor 9.

Propofol is an intravenous anesthetic widely used in clinical surgeries, such as tumor resection. Propofol affects the growth of many cancers, though its effect on melanoma is unknown. Our study aimed to explore how propofol affects melanoma cells. Melanoma cells A2058 and WM793B were cultured with propofol for 24 hr. Propofol significantly suppressed proliferation, migration, and invasion of A2058 and WM793B cells. Lower miR-137 level was observed in A2058 and WM793B cells, compared with normal human epidermal melanocyte HEMa-LP cells. Propofol-induced miR-137 upregulation and decreased proliferation, invasive ability, and migrated ability of A2058 and WM793B cells. Transfection with the miR-137 inhibitor reversed these effects. Additionally, miR-137 was verified to target and negatively regulate fibroblast growth factor 9 (FGF9) expression. Propofol efficiently downregulated FGF9 protein expression by upregulating miR-137. Furthermore, FGF9 overexpression abrogated propofol's repressive effects on the malignant potential of A2058 and WM793B cells. These findings indicate that propofol suppressed melanoma cell proliferation, invasion, and migration by regulating miR-137 and FGF9.

Proliferation of MicroRNA-365 and E74-like Factor 4 in Cervical Cancer Cells and Its Clinical Significance

Objective To investigate the expressions,roles,and clinical significance of microRNA-365(miR-365)and E74-like factor 4(ELF4)in cervical cancer. Methods The expressions of miR-365 in normal cervical tissues(n=34),cervical intraepithelial neoplasia 1(CIN 1)(n=31),cervical intraepithelial neoplasia2-3(CIN 2-3)(n=37),squamous cell carcinoma of the cervix(SCC)(n=33),and three cervical cancer cell lines(C33A cells,Hela cells,and SiHa cells)were detected by real-time quantitative polymerase chain reaction(qPCR).Bioinformatic prediction and luciferase reporter gene assay were performed to verify whether ELF4 was a direct target of miR-365.Western blot and immunohistochemistry were used to detect ELF4 expression in cervical cancer cells and in different pathological cervix tissues.CCK8 assay was used to detect the effect of overexpression or inhibition of miR-365 on the proliferation of cervical cancer cells at different time points.The relationships among the miR-365 expression,ELF4 expression,and clinicopathological parameters of cervical cancer were analyzed by correlation analysis. Results qPCR results showed that compared with the normal cervical cell HcerEpic,the expressions of miR-365 in CIN1,CIN2-3,and cervical cancer tissues gradually decreased with the increased pathologic grade,and its expressions also decreased in different cervical cancer cell lines.The luciferase reporter gene assay confirmed that ELF4 was the direct target of miR-365.Western blot showed that the expression of ELF4 increased in all three cervical cancer cell lines compared with normal cervical epidermal cell(P=0.013,P=0.002,P=0.004).Immunohistochemistry showed that ELF4 expression was up-regulated in CIN and cervical cancer tissues.CCK8 assay showed that overexpression of miR-365 inhibited cell proliferation,while inhibition of miR-365 promoted the proliferation of three cervical cancer cells(P<0.05).Further analysis confirmed that there was a negative correlation between the expression levels of miR-365 and ELF4 in CIN2-3 and SCC(r=-0.351,P=0.045;r=-0.349,P=0.035).Clinical analysis showed that the expressions of both miR-365 and ELF4 were correlated with tumor size,pathological grade,and clinical stage in SCC(all P < 0.05).<b>Conclusion</b> The decreased expression of miR-365 in human cervical cancer cells relieves its inhibitory effect on ELF4,which promotes the proliferation of cervical cancer cells and the formation of tumor.

Synthesis and Biological Evaluation of 4β-N-Acetylamino Substituted Podophyllotoxin Derivatives as Novel Anticancer Agents.

A series of novel podophyllotoxin derivatives obtained by 4β-N-acetylamino substitution at C-4 position was designed, synthesized, and evaluated for in vitro cytotoxicity against four human cancer cell lines (EC-9706, HeLA, T-24 and H460) and a normal human epidermal cell line (HaCaT). The cytotoxicity test indicated that most of the derivatives displayed potent anticancer activities. In particular, compound 12h showed high activity with IC50 values ranging from 1.2 to 22.8 μM, with much better cytotoxic activity than the control drug etoposide (IC50: 8.4 to 78.2 μM). Compound 12j exhibited a promising cytotoxicity and selectivity profile against T24 and HaCaT cell lines with IC50 values of 2.7 and 49.1 μM, respectively. Compound 12g displayed potent cytotoxicity against HeLA and T24 cells with low activity against HaCaT cells. According to the results of fluorescence-activated cell sorting (FACS) analysis, 12g induced cell cycle arrest in the G2/M phase accompanied by apoptosis in T24 and HeLA cells. Furthermore, the docking studies showed possible interactions between human DNA topoisomerase IIα and 12g. These results suggest that 12g merits further optimization and development as a new podophyllotoxin-derived lead compound.

158 Triggering immortalized keratinocyte growth arrest by protein kinase C delta activation

Normal epidermal keratinocyte (KC) differentiation is marked by irreversible cell cycle exit and a step-wise transition through the suprabasal layers of the epidermis. In contrast, KCs in both actinic keratosis and squamous cell carcinomas (SCCs) continue to proliferate in the suprabasal compartment, indicating a defect in their growth arrest signaling. Activation of multiple protein kinase C (PKC) isoforms have been associated with KC cell cycle withdrawal. To study the involvement of PKC in KC growth arrest, we evaluated the effects of the PKC agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on normal human epidermal keratinocytes (NHEKs) and HaCaT cells, an immortalized, non-tumorigenic human keratinocyte cell line. Treating NHEKs with 10 nM TPA for as little as 15 minutes induced complete growth arrest over a 7 day period while untreated KCs underwent a 93-fold increase in cell number (p<0.005). 10 nM TPA treatment of HaCaT cells did not induce any growth arrest, indicating that immortalized KCs have defective PKC-dependent growth arrest signaling. To test if PKCδ activation was able to enforce growth arrest in HaCaT cells, we transduced HaCaT cells with retroviruses encoding either EGFP or a PKCδ-EGFP fusion and sorted EGFP positive cells by flow cytometry. HaCaT-EGFP cells behaved similar to HaCaTs and were not growth arrested by TPA treatment. In contrast, HaCaT-PKCδ-EGFP treated with TPA increased in cell number only 3-fold compared to a 15-fold increase in untreated HaCaT-PKCδ-EGFP over a 4 day growth assay. Activation of PKCδ in HaCaT-PKCδ-EGFP cells was confirmed by observing TPA-induced translocation of PKCδ-EGFP to the plasma membrane and increased S299 autophosphorylation. TPA-induced growth inhibition in HaCaT-PKCδ-EGFP cells was due to arrest in G1 and G2/M, not apoptosis. Global phosphoproteomics are being employed to identify PKCδ signaling pathways that can overcome the defective growth arrest in immortalized keratinocytes and may have therapeutic benefit for the treatment of actinic keratosis and SCCs.

UVB-induced inactivation of manganese-containing superoxide dismutase promotes mitophagy via ROS-mediated mTORC2 pathway activation

Mitochondria are major sites of energy metabolism that influence numerous cellular events, including immunity and cancer development. Previously, we reported that the mitochondrion-specific antioxidant enzyme, manganese-containing superoxide dismutase (MnSOD), has dual roles in early- and late-carcinogenesis stages. However, how defective MnSOD impacts the chain of events that lead to cell transformation in pathologically normal epidermal cells that have been exposed to carcinogens is unknown. Here, we show that UVB radiation causes nitration and inactivation of MnSOD leading to mitochondrial injury and mitophagy. In keratinocytes, exposure to UVB radiation decreased mitochondrial oxidative phosphorylation, increased glycolysis and the expression of autophagy-related genes, and enhanced AKT Ser/Thr kinase (AKT) phosphorylation and cell growth. Interestingly, UVB initiated a prosurvival mitophagy response by mitochondria-mediated reactive oxygen species (ROS) signaling via the mammalian target of the mTOR complex 2 (mTORC2) pathway. Knockdown of rictor but not raptor abrogated UVB-induced mitophagy responses. Furthermore, fractionation and proximity-ligation assays reveal that ROS-mediated mTOC2 activation in mitochondria is necessary for UVB-induced mitophagy. Importantly, pretreatment with the MnSOD mimic MnTnBuOE-2-PyP5+ (MnP) attenuates mTORC2 activation and suppresses UVB-induced mitophagy. UVB radiation exposure also increased cell growth as assessed by soft-agar colony survival and cell growth assays, and pretreatment with MnP or the known autophagy inhibitor 3-methyladenine abrogated UVB-induced cell growth. These results indicate that MnSOD is a major redox regulator that maintains mitochondrial health and show that UVB-mediated MnSOD inactivation promotes mitophagy and thereby prevents accumulation of damaged mitochondria.