Human Keratinocyte Cell Line HaCaT Research Articles

1162 BMAL1 and CLOCK proteins in regulating the sensitivity of human keratinocytes to UVB-induced apoptosis

A diverse array of biological processes is under circadian controls. It has been reported that UV-induced apoptosis and DNA damage responses in mouse skin are time-of-day dependent and regulated by core clock proteins, including BMAL1, CLOCK, PERs, and CRYs. The aim of this study is to investigate the circadian controls of UV responses in human keratinocytes, using the siRNA-mediated loss-of-function approach. We found that low-dose (5 mJ/cm2) of UVB triggered an oscillation of BMAL1 and CLOCK mRNA expression in the immortal HaCat human keratinocyte cell line, as well as primary human keratinocytes. In HaCat cells, depletion of BMAL1 or CLOCK reduced the percentage of UVB-induced apoptotic cells from 43% (in controls) to 29% (p < 0.001) and 31% (P < 0.05), respectively. Moreover, knockdown of either clock gene strongly blocked UVB-induced expression of tumor necrosis factor α (TNFα) and phosphorylation of NF-κB. These results suggest that BMAL1-CLOCK proteins may promote cell death by regulating the TNFα-NF-κB mediated extrinsic apoptotic pathway in UVB-irradiated HaCat keratinocytes. Given the critical impact of UVB on photo-aging and photo-carcinogenesis, mechanistic elucidation and understanding of circadian controls on UVB effects in human skin will be beneficial for prevention and treatment of skin-related diseases including cancers.

Effects of 7-MEGA\u2122 500 on Oxidative Stress, Inflammation, and Skin Regeneration in H2O2-Treated Skin Cells

Environmental stimuli can lead to the excessive accumulation of reactive oxygen species (ROS), which is one of the risk factors for premature skin aging. Here, we investigated the protective effects of 7-MEGA™ 500 (50% palmitoleic acid, 7-MEGA) against oxidative stress-induced cellular damage and its underlying therapeutic mechanisms in the HaCaT human skin keratinocyte cell line (HaCaT cells). Our results showed that treatment with 7-MEGA prior to hydrogen peroxide (H2O2)-induced damage significantly increased the viability of HaCaT cells. 7-MEGA effectively attenuated generation of H2O2-induced reactive oxygen species (ROS), and inhibited H2O2-induced inflammatory factors, such as prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). In addition, cells treated with 7-MEGA exhibited significantly decreased expression of matrix metalloproteinase-1 (MMP-1) and increased expression of procollagen type 1 (PCOL1) and Elastin against oxidative stress by H2O2. Interestingly, these protective activities of 7-MEGA were similar in scope and of a higher magnitude than those seen with 98.5% palmitoleic acid (PA) obtained from Sigma when given at the same concentration (100 nL/mL). According to our data, 7-MEGA is able to protect HaCaT cells from H2O2-induced damage through inhibiting cellular oxidative stress and inflammation. Moreover, 7-MEGA may affect skin elasticity maintenance and improve skin wrinkles.These findings indicate that 7-MEGA may be useful as a food supplement for skin health.

3-Acetyl-8-methoxy-2[H]-chromen-2-one derived Schiff bases as potent antiproliferative agents: Insight into the influence of 4(N)-substituents on the in vitro biological activity

A series of 3-acetyl-8-methoxycoumarin appended thiosemicarbazones (1–4) was prepared from the reaction of 3-acetyl-8-methoxycoumarin with 4(N)-substituted thiosemicarbazides in a view of ascertaining their biological properties with the change of N-terminal substitution in the thiosemicarbazide moiety. Comprehensive characterization was brought about by various spectral and analytical methods. The molecular structures of all the compounds were determined by single crystal X-ray diffraction analysis. Binding studies with Calf thymus DNA (CT-DNA) and proteins such as Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) indicated an intercalative mode of binding with DNA and static quenching mechanism with proteins. The compounds cleaved plasmid DNA (pBR322) and acted well as free radical scavengers. A good spectrum of antimicrobial activity was observed against four bacterial and five fungal pathogens. The compounds exhibited profound antiproliferative activity on MCF-7 (human breast cancer) and A549 (human lung carcinoma) cell lines. Assay on human normal keratinocyte cell line HaCaT showed that the compounds were non-toxic to normal cells.

Ashwagandha root extract exerts anti‑inflammatory effects in HaCaT cells by inhibiting the MAPK/NF‑κB pathways and by regulating cytokines.

A paste composed of the boiled leaves and roots of the Ashwagandha plant is used to cure ulcer and swelling in Ayurvedic medicine. However, the effects of the hot water extract of Ashwagandha roots (ASH‑WEX), which is also used in Ayurveda, on skin have not been fully elucidated. Therefore, the present study investigated the anti‑inflammatory activity of ASH‑WEX on skin, by using the human keratinocyte cell line HaCaT. The results indicated that ASH‑WEX significantly inhibited mRNA expression of inflammatory cytokines, including interleukin (IL)‑8, IL‑6, tumor necrosis factor (TNF‑α), IL‑1β and IL‑12, and promoted the mRNA expression of the anti‑inflammatory cytokine transforming growth factor (TGF)‑β1 in HaCaT cells. In addition, ASH‑WEX inhibited the lipopolysaccharide‑induced phosphorylation of p38 and c‑Jun N‑terminal kinase, as well as the nuclear translocation of nuclear factor (NF)‑κB p65. Downregulation of TNF‑α mRNA and upregulation of TGF‑β1 mRNA were also observed invivo following ASH‑WEX treatment of mouse skin. In conclusion, the present study demonstrated that the anti‑inflammatory effect of ASH‑WEX may be due to its ability to suppress the NF‑κB and mitogen‑activated protein kinase pathways, and to modulate cytokine expression. These results suggest that ASH‑WEX can potentially protect against skin inflammation.

Cytostatic resistance profile of the sulfur mustard resistant keratinocyte cell line HaCaT/SM

BackgroundThe cell line HaCaT/SM was developed as a sulfur mustard (SM) resistant cell line from the human keratinocyte cell line HaCaT. This cell line was established to learn more about the effect of SM and possible therapeutic approaches to counteract the cytotoxic effects of SM. The aim of this study was to clarify whether the SM-resistant cell line HaCaT/SM exhibit also resistance to other alkylating agents or cytotoxic drugs with different mechanism of action. Material and methodThe chemosensitivity of SM-resistant human keratinocyte cell line HaCaT/SM and the original cell line HaCaT were tested using the XTT assay. Nine cytotoxic drugs from five different substance groups were investigated. ResultsHaCaT/SM showed a significant increase in resistance against all tested drugs. From the substance class of the alkylating agents, HaCaT/SM showed the strongest resistance increase against chlorambucil (1.7 fold increase). Whereas over all substances strongest increase was observed against cisplatin (5.1 fold increase). DiscussionThe highest resistance was observed for cisplatin. The SM resistant cells revealed changes in the miRNA profile as described before. The resistance to cisplatin is also connected to a specific miRNA profile. Interestingly, changes of miRNA-203 and miRNA-21 levels were found in HaCaT/SM as well as in cisplatin resistant cells. It is therefore conceivable that the same resistance pathways are involved for both substances.

空气细颗粒物PM2.5对HaCaT细胞增殖、细胞周期及凋亡的影响

Objective To evaluate effects of fine particulate matter PM2.5 in ambient air on the proliferation, cell cycle and apoptosis of a human keratinocyte cell line HaCaT. Methods PM2.5 in haze-fog episodes during the heating season was collected in Beijing from 2015 to 2016, and processed into PM2.5 suspensions. HaCaT cells were divided into several groups to be treated with culture medium alone (control group) , PM2.5 suspensions at different concentrations of 100-400 mg/L (experiment groups, 50-800 mg/L for observation of cellular morphology and analysis of cell proliferation) for 24 hours, or cell culture medium without cells or PM2.5 suspensions (blank group) . Cellular morphological changes were observed under an inverted microscope. Cell counting kit-8 (CCK-8) assay was performed to determine cell survival rate, flow cytometry to determine the cell cycle distribution and detect cell apoptosis, and Western blot analysis to determine the protein expression of cyclin A2 and cyclin-dependent kinase1 (CDK1) . Results Along with the increase of PM2.5 concentration, HaCaT cells lost their normal shape gradually, and the number of viable cells gradually decreased. Compared with the control group (100% ± 4.95%) , the 50-mg/L PM2.5 group showed no changes in cell survival rates (P > 0.05) , while the 100-, 200-, 400- and 800-mg/L PM2.5 group showed significantly lower survival rates (91.77% ± 2.04%, 80.01% ± 1.57%, 57.80% ± 1.56%, 21.98% ± 0.86%, respectively, all P < 0.05) . Flow cytometry revealed that the 100-, 200- and 400-mg/L PM2.5 groups showed gradually increased proportion of cells at S phase, but gradually decreased proportion of cells at G2/M phase compared with the control group (all P < 0.05) . As Western blot analysis showed, the protein expression of cyclin A2 and CDK1 significantly decreased in the 100-, 200- and 400-mg/L PM2.5 groups compared with the control group, which was lowest in the 200-mg/L PM2.5 group (all P < 0.05) . In addition, the 100-, 200- and 400-mg/L PM2.5 groups showed significantly higher total apoptosis rates (9.98% ± 0.21%, 12.56% ± 0.74%, 16.74% ± 1.48%, respectively) compared with the control group (6.24% ± 0.17%, all P < 0.05) . Conclusion PM2.5 can inhibit cell proliferation and promote apoptosis of HaCaT cells, likely by downregulating the expression of cyclin A2 and CDK1 and arresting HaCaT cells at S phase. Key words: Cell proliferation; Apoptosis; Particulate matter; Air; HaCaT cells; PM2.5

Downregulation of estrogen-related receptor alpha inhibits human cutaneous squamous cell carcinoma cell proliferation and migration by regulating EMT via fibronectin and STAT3 signaling pathways

Estrogen-related receptor alpha (ERRα), one of orphan nuclear receptors, has been recently revealed as an oncogenic regulator in a variety of cancers. However, the linking gain of ERRα expression and cancer progression in cutaneous squamous cell carcinoma (cSCC) is largely unknown. Here, we showed that the mRNA and protein expression levels of ERRα were markedly higher in A431 cells compared with human keratinocyte cell line HaCaT, and targeted inhibition of ERRα by shRNA or its inverse agonist XCT790 significantly suppressed A431 cells proliferation and migration, while overexpression of ERRα promoted cell proliferation and migration. In addition, the data revealed that ERRα downregulation markedly inhibited the epithelial mesenchymal transition (EMT) of A431 cells with increasing the expression of E-cadherin and decreasing fibronectin (FN) and vimentin. Results from further experiments using Western blot suggested that ERRα suppression inhibited signal transducer and activator of transcription (STAT3) protein expression. In contrast, overexpression of ERRα promoted EMT and the activation of STAT3 pathway. Moreover, treatment with specific STAT3 inhibitor reversed EMT markers in ERRα-overexpressing A431 cells. In tumor xenografts of A431 cells, we further showed that ERRα depletion inhibited cSCC tumor growth in vivo. Taken together, these results demonstrate, for the first time, that ERRα may function as an oncoprotein in cSCC to accelerate tumor aggressiveness by promoting EMT via FN and STAT3 pathway, and it could be a novel target for cSCC therapy.

Anti-Inflammatory Effect of Melittin on Porphyromonas Gingivalis LPS-Stimulated Human Keratinocytes.

Periodontitis is a chronic inflammatory disease that contributes to the destruction of the gingiva. Porphyromonas gingivalis (P. gingivalis) can cause periodontitis via its pathogenic lipopolysaccharides (LPS). Melittin, a major component of bee venom, is known to have anti-inflammatory and antibacterial effects. However, the role of melittin in the inflammatory response has not been elucidated in periodontitis-like human keratinocytes. Therefore, we investigated the anti-inflammatory effects of melittin on a P. gingivalis LPS (PgLPS)-treated HaCaT human keratinocyte cell line. The cytotoxicity of melittin was measured using a human keratinocyte cell line, HaCaT, and a Cell Counting Kit-8. The effect of melittin on PgLPS-induced inflammation was determined with Western blot, real-time quantitative PCT, and immunofluorescence. PgLPS increased the expression of toll-like receptor (TLR) 4 and proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and interferon-γ (IFN-γ). Moreover, PgLPS induced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), extracellular signal-regulated kinase (ERK), and protein kinase B/Akt. Melittin also inhibited the expression of proinflammatory cytokines by suppressing the activation of the NF-κB signaling pathway, ERK, and Akt. Melittin attenuates the PgLPS-induced inflammatory response and could therefore be applied in the treatment of periodontitis for anti-inflammatory effects.

P68 prompts the epithelial-mesenchymal transition in cervical cancer cells by transcriptionally activating the TGF-β1 signaling pathway.

Overexpression of p68 has been reported in various types of cancer. However, little study has been conducted on the expression and role of p68 in cervical cancer. Therefore, the present study focuses on the role of p68 in cervical cancer cells, which may elucidate its potential mechanism of cervical cancer progression and shed light on the precision medical treatment of cervical cancer. Firstly, the expression of p68 was analyzed using reverse transcription-quantitative polymerase chain reaction and western blot analysis. The changes to cell morphology were observed using an inverted microscope (XDS-500D; Shanghai Caikon Optical Instrument Co., Ltd., Shanghai, China). Cell migration was determined using an in vitro scratch assay. The present study demonstrated that the mRNA and protein levels of p68 were significantly enhanced in cervical cancer CaSki, HeLa [human papillomavirus (HPV)-18-positive], SiHa (HPV-16-positive) and C-33A (HPV-negative) cell lines compared with the human keratinocyte HaCaT cell line. Overexpression of p68 induced an elongated and spindle-shaped morphology in CaSki cells. Upregulation of p68 increased the expression of α-smooth muscle actin, vimentin and fibronectin however, epithelial marker E-cadherin was significantly decreased. Furthermore, the in vitro scratch assay demonstrated that overexpression of p68 markedly enhanced CaSki cell migration capacity at 24 and 48 h. Knockdown of p68 partially reversed transforming growth factor-β1 (TGF-β1)-induced changes in epithelial-mesenchymal transition (EMT) markers and cell morphological changes. In summary, the present study demonstrated that p68 transcriptionally activated the expression of TGF-β1, thereby prompting EMT in cervical cancer cells.

Potential inhibitory effects of the traditional herbal prescription Hyangso-san against skin inflammation via inhibition of chemokine production and inactivation of STAT1 in HaCaT keratinocytes.

Inflammatory skin disease are caused by multiple factors, including susceptibility genes, and immunologic and environmental factors, and are characterized by an increase in epidermal thickness and the infiltration of macrophages, keratinocytes, mast cells, eosinophils and other inflammatory cells. Keratinocytes may serve an important role in the pathogenesis of inflammatory skin diseases. The traditional herbal decoction Hyangso‑san (HSS) has been used to treat symptoms of the common cold, including headache, pantalgia, fever and chills. However, to the best of our knowledge, there is no evidence regarding whether HSS has an effect on inflammatory skin diseases. The present study investigated the anti‑skin inflammation activity of HSS using the HaCaT human keratinocyte cell line. The mRNA expression and production of inflammatory chemokines, including C‑C motif chemokine ligand22 (CCL22), CCL5, CCL17, and interleukin(IL)‑8, was measured using reverse transcription polymerase chain reaction and ELISA analyses. Moreover, we evaluated the effect of HSS on signal transducer and activator of transcription1 (STAT1) pathway in HaCaT cells. The cells were stimulated with tumor necrosis factor‑α (TNF‑α) and interferon‑γ (IFN‑γ) to induce an inflammatory reaction. In the TNF‑α‑and IFN‑γ‑stimulated cells, the production and expression of inflammatory chemokines were observed, including CCL22, CCL5, CCL17 andIL‑8. In addition, stimulation with TNF‑α and IFN‑γ increased the phosphorylation and nuclear translocation of STAT1 in HaCaT cells. By contrast, HSS extract treatment inhibited TNF‑α‑andIFN‑γ‑induced STAT1 activation. Results from the present study indicated that HSS exhibited inhibitory effects on TNF‑α‑and IFN‑γ‑mediated chemokine production and expression by targeting STAT1 in keratinocytes. Overall, the results indicated that HSS may be a potential candidate therapeutic drug for inflammatory skin diseases such as atopic dermatitis.

A comparative study of digital PCR and real-time qPCR for the detection and quantification of HPV mRNA in sentinel lymph nodes of cervical cancer patients

BackgroundQualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR.MethodsSerial dilutions of 5 ng–5 pg RNA (≙ 500–0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template.ResultsFor the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis.ConclusionsCompared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.

Effect of Green and Brown Propolis Extracts on the Expression Levels of microRNAs, mRNAs and Proteins, Related to Oxidative Stress and Inflammation

A large body of evidence highlights that propolis exerts many biological functions that can be ascribed to its antioxidant and anti-inflammatory components, including different polyphenol classes. Nevertheless, the molecular mechanisms are yet unknown. The aim of this study is to investigate the mechanisms at the basis of propolis anti-inflammatory and antioxidant activities. The effects of two brown and green propolis extracts—chemically characterized by RP-HPLC-PDA-ESI-MSn—on the expression levels of miRNAs associated with inflammatory responses (miR-19a-3p and miR-203a-3p) and oxidative stress (miR-27a-3p and miR-17-3p), were determined in human keratinocyte HaCat cell lines, treated with non-cytotoxic concentrations. The results showed that brown propolis, whose major polyphenolic components are flavonoids, induced changes in the expression levels of all miRNAs, and was more active than green propolis (whose main polyphenolic components are hydroxycinnamic acid derivatives) which caused changes only in the expression levels of miR-19a-3p and miR-27a-3p. In addition, only brown propolis was able to modify (1) the expression levels of mRNAs, the target of the reported miRNAs, which code for Tumor Necrosis Factor-α (TNF-α), Nuclear Factor, Erythroid 2 Like 2 (NFE2L2) and Glutathione Peroxidase 2 (GPX2), and (2) the protein levels of TNF-α and NFE2L2. In conclusion, brown and green propolis, which showed different metabolite profiles, exert their biological functions through different mechanisms of action.

Pro-oxidant status and Nrf2 levels in psoriasis vulgaris skin tissues and dimethyl fumarate-treated HaCaT cells.

Reactive oxygen species (ROS) contribute to pathogenesis of many inflammatory skin diseases, including psoriasis. The aim of this study is to compare antioxidant protein expression in psoriasis vulgaris (PV) skin tissues with that in normal skin tissues in vivo and to evaluate the effects of dimethyl fumarate (DMF), used for the treatment of psoriasis, on ROS generation and apoptosis in a human keratinocyte cell line HaCaT. Compared with normal skin tissues, PV skin tissues showed increased protein oxidation as well as down-regulation of Nrf2 and its regulatory proteins such as HO-1 and AKR1C3. Using HaCaT cells to model DMF-induced pro-oxidant effects in the skin cells, we found that DMF treatment induced increased ROS levels and apoptotic cell death, as signified by increased proportion of cells with Annexin V-PE(+) staining and a sub-G0/G1 peak in the cell cycle. Preceding these changes, DMF treatment resulted in up-regulation of Nrf2, HO-1, and AKR1C3 proteins in these cells. Collectively, increased oxidative stress and impaired cellular anti-oxidant enzyme systems may participate in the pathogenesis of PV. DMF may exert an additive therapeutic efficacy in PV by attenuating the redox burden and subsequent oxidative damage to normal keratinocytes through activation of Nrf2 pathway relative to PV.

Bortezomib, a proteasome inhibitor, alleviates atopic dermatitis by increasing claudin 1 protein expression

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Many studies investigating AD pathogenesis and its therapy have been conducted but none have been successful. One of the causes of AD is dysfunction of tight junctions through reduction of claudin 1 expression in the epidermal barrier of the skin. In the present study, we investigated the role of bortezomib (BTZ) in the restoration of the reduced expression of claudin 1. Immunoblot and immunofluorescence analyses revealed that BTZ increased the protein expression level of claudin 1 in the human keratinocyte cell line HaCaT, thereby forming paracellular barriers. Furthermore, repeated application of BTZ alleviated atopic symptoms on the backs and ears of 2, 4-dinitrochlorobenzene (DNCB)-induced AD mice, and led to the formation of normal tight junctions in the epidermal barrier of DNCB-induced mice skin. Taken together, these results demonstrate that BTZ-induced claudin 1 expression may be a valuable therapeutic approach for AD.

Coordination-Driven Self-Assembly of Ionic Irregular Hexagonal Metallamacrocycles via an Organometallic Clip and Their Cytotoxicity Potency.

Two new irregular hexagons (6 and 7) were synthesized from a pyrazine motif containing an organometallic acceptor clip [bearing platinum(II) centers] and different neutral donor ligands (4,4'-bipyridine or pyrazine) using a coordination-driven self-assembly protocol. The two-dimensional supramolecules were characterized by multinuclear NMR, mass spectrometry, and elemental analyses. Additionally, one of the macrocycles (6) was characterized by single-crystal X-ray analyses. Macrocycles are unique examples of [2 + 2] self-assembled ensembles that are hexagonal but irregular in shape. These hexagon frameworks require the assembly of only four tectons/subunits. The cytotoxicity of platinum(II)-based macrocycles was studied using various cell lines such as A549 (human lung carcinoma), KB (human oral cancer), MCF7 (human breast cancer), and HaCaT (human skin keratinocyte) cell lines, and the results were compared with those of cisplatin. The smaller macrocycle (7) exhibited a higher cytotoxic effect against all cell types, and its sensitivity was found to be comparable with that of cisplatin for A549 and MCF7 cells. Cell cycle analysis and live propidium iodide staining suggest that the macrocycles 6 and 7 induced a loss of membrane integrity that ultimately might lead to necrotic cell death.

Alteration of miRNA expression in a sulfur mustard resistant cell line

BackgroundMicroRNAs (miRNAs) are responsible for post-transcriptional control of protein expression. Numerous miRNAs have been identified to be responsible for the resistance of tumor cells to cytostatic drugs. Possibly, the same miRNAs also play a role in the sulfur mustard (SM)-resistance of the keratinocyte cell line HaCaT/SM as alkylating cytostatics exhibit similar cytotoxic effects as SM. MethodsBasal expression levels of 1920 miRNAs in total were analyzed in HaCaT/SM compared to the origin human keratinocyte cell line HaCaT. The effect for selected miRNAs on cell survival was analyzed using antagomirs for ectopic miRNA level decrease or miRNA mimics for increase. Cell survival was calculated as SM dose-dependent-curves. ResultsOut of 1920 miRNAs analyzed, 49 were significantly up- and 29 were significantly downregulated in HaCaT/SM when compared to HaCaT controls. Out of these, 36 could be grouped in miRNA families. Most of the 15 miRNA family members showed either a common increase or decrease. Only the members of miR-10, miR-154, miR-430 and miR-548 family showed an inconsistent picture. The ectopic increase of miR-181 in HaCaT/SM had a positive effect on cell survival in the presence of SM. ConclusionIn summary, the extensive differences in miRNA expression pattern between these cell lines indicate that specific miRNAs may play a role in the resistance mechanism against sulfur mustard. The miR-125b-2 and miR-181b alone are not responsible for the resistance development against SM, but an ectopic increase of miR-181 even enhances the SM resistance of HaCaT/SM. Improving the resistance in normal keratinocytes by treatment with either both miRNAs together or a different combination might be used as an initial step in development of an innovative new drug or prophylactic agent against SM.

Skin renewal activity of non-thermal plasma through the activation of \u03b2-catenin in keratinocytes

For recent years, devices that generate non-thermal plasma (NTP) have been introduced into the field of dermatology. Since NTP has demonstrated strong anti-pathogenic activity with safety of use, NTP was first applied to sterilize the skin surface to aid in the healing of various kinds of skin diseases. However, the effect of NTP on skin regeneration has not yet been fully explored. In this study, the effect of NTP on the growth of keratinocytes was tested using the HaCaT human keratinocyte cell line and HRM2 hairless mice. Treatment with NTP allowed confluent keratinocytes to escape from G1 cell cycle arrest and increased the proportion of cells in S and G2 phases. In particular, NTP treatment immediately dispersed E-cadherin-mediated cell-to-cell interactions, resulting in the translocation of β-catenin to the nucleus and leading to the enhanced transcription of target genes including c-MYC and cyclin D1. Moreover, repeated treatment of the mice with NTP also stimulated epidermal expansion by activating β-catenin in the epidermal cells. The symptoms of cellular DNA damage were not detected after NTP treatment. Taken together, these results demonstrate that NTP may be employed as a new type of skin regenerating device.

Influence of chronic low-dose/dose-rate high-LET irradiation from radium-226 in a human colorectal carcinoma cell line

PurposeTo evaluate potential damages of chronic environmentally relevant low-dose/dose-rate high-LET irradiation from a naturally occurring alpha-emitting radionuclide (radium-226, 226Ra) on a human colorectal carcinoma HCT116 p53+/+ cell line. MethodsClonogenic survival assays and mitochondrial membrane potential (MMP) measurement with a sensitive fluorescent MMP probe JC-1 were performed in HCT116 p53+/+ cells chronically exposure to low doses/dose rates of 226Ra with high-LET. Comparisons were made with the human non-transformed keratinocyte HaCaT cell line and acute low-dose direct low-LET gamma radiation. Results and conclusionThe chronic low-dose/dose-rate alpha radiation (CLD/DRAR) did not reduce the clonogenic survival of HCT116 p53+/+ cells over the period of 70 days of exposure. Only one significant reduction in the HCT116 p53+/+ cells’ clonogenic survival was when cells were grown with 10,000mBq/mL 226Ra for 40 days and progeny cells were clonogenically assessed in the presence of 10,000mBq/mL 226Ra. The cumulative doses that cells received during this period ranged from 0.05 to 46.2mGy. The mitochondrial membrane potential (MMP) dropped initially in both HCT116 p53+/+ and HaCaT cells in response to CLD/DRAR. The MMP in HCT116 p53+/+ cells recovered more quickly at all dose points than and that in HaCaT cells until the end of the exposure period. The highest dose rate of 0.66mGy/day depolarized the HaCaT's mitochondria more consistently during the exposure period. The faster recovery status of the MMP in HCT116 p53+/+ cells than that in HaCaT cells was also observed after exposure to acute low-dose gamma rays. Overall, it was found that CLD/DRAR had little impact on the MMP of human colorectal cancer and keratinocyte cell lines.

Protective effects of Scutellaria baicalensis Georgi against hydrogen peroxide-induced DNA damage and apoptosis in HaCaT human skin keratinocytes.

Oxidative stress due to excessive accumulation of reactive oxygen species (ROS) is one of the risk factors for the development of several chronic diseases. In this study, we investigated the protective effects of Scutellaria baicalensis rhizome ethanol extract (SBRE) against oxidative stress-induced cellular damage and elucidated the underlying mechanisms in the HaCaT human skin keratinocyte cell line. Our results revealed that treatment with SBRE prior to hydrogen peroxide (H2O2) exposure significantly increased viability of HaCaT cells. SBRE also effectively attenuated H2O2-induced comet tail formation and inhibited the H2O2-induced phosphorylation levels of the histone γH2AX, as well as the number of apoptotic bodies and Annexin V-positive cells. In addition, SBRE exhibited scavenging activity against intracellular ROS generation and restored the mitochondrial membrane potential loss by H2O2. Moreover, H2O2 enhanced the cleavage of caspase-3 and degradation of poly (ADP-ribose)-polymerase, a typical substrate protein of activated caspase-3, as well as DNA fragmentation; however, these events were almost totally reversed by pretreatment with SBRE. Furthermore, SBRE increased the levels of heme oxygenase-1 (HO-1), which is a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). According to our data, SBRE is able to protect HaCaT cells from H2O2-induced DNA damage and apoptosis through blocking cellular damage related to oxidative stress through a mechanism that would affect ROS elimination and activating the Nrf2/HO-1 signaling pathway.

Attenuation of UV-B exposure-induced inflammation by abalone hypobranchial gland and gill extracts

Exposure to solar ultraviolet B (UV-B) is a known causative factor for many skin complications such as wrinkles, black spots, shedding and inflammation. Within the wavelengths 280–320 nm, UV-B can penetrate to the epidermal level. This investigation aimed to test whether extracts from the tropical abalone [Haliotis asinina (H. asinina)] mucus-secreting tissues, the hypobranchial gland (HBG) and gills, were able to attenuate the inflammatory process, using the human keratinocyte HaCaT cell line. Cytotoxicity of abalone tissue extracts was determined using an AlamarBlue viability assay. Results showed that HaCaT cells could survive when incubated in crude HBG and gill extracts at concentrations between <11.8 and <16.9 μg/ml, respectively. Subsequently, cell viability was compared between cultured HaCaT cells exposed to serial doses of UV-B from 1 to 11 (x10) mJ/cm2 and containing 4 different concentrations of abalone extract from both the HBG and gill (0, 0.1, 2.5, 5 μg/ml). A significant increase in cell viability was observed (P<0.001) following treatment with 2.5 and 5 μg/ml extract. Without extract, cell viability was significantly reduced upon exposure to UV-B at 4 mJ/cm2. Three morphological changes were observed in HaCaT cells following UV-B exposure, including i) condensation of cytoplasm; ii) shrunken cells and plasma membrane bubbling; and iii) condensation of chromatin material. A calcein AM-propidium iodide live-dead assay showed that cells could survive cytoplasmic condensation, yet cell death occurred when damage also included membrane bubbling and chromatin changes. Western blot analysis of HaCaT cell COX-2, p38, phospho-p38, SPK/JNK and phospho-SPK/JNK following exposure to >2.5 μg/ml extract showed a significant decrease in intensity for COX-2, phospho-p38 and phospho-SPK/JNK. The present study demonstrated that abalone extracts from the HGB and gill can attenuate inflammatory proteins triggered by UV-B. Hence, the contents of abalone extract, including cellmetabolites and peptides, may provide new agents for skin anti-inflammation, preventing damage due to UV-B.