Construction of a bacteriophage-derived vector with potential applications in targeted drug delivery and cell imaging.

There is a strong relationship between the dysregulation of epidermal growth factor receptor (EGFR) and the development of epithelial-derived cancers. Therefore, EGFR has usually been considered the desired target for gene therapy. Here, we propose an approach for targeting EGFR-expressing cells by phage particles capable of displaying EGF and GFP as tumor-targeting and reporting elements, respectively. For this purpose, the superfolder GFP-EGF (sfGFP-EGF) coding sequence was inserted at the N-terminus of the pIII gene in the pIT2 phagemid. The capability of the constructed phage to recognize EGFR-overexpressing cells was monitored by fluorescence microscopy, fluorescence-activated cell sorting (FACS), and cell-based ELISA experiments. FACS analysis showed a significant shift in the mean fluorescence intensity (MFI) of the cells treated with phage displaying sfGFP-EGF compared to phage displaying only sfGFP. The binding of phage displaying sfGFP-EGF to A-431 cells, monitored by fluorescence microscopy, indicated the formation of the sfGFP-EGF-EGFR complex on the surface of the treated cells. Cell-based ELISA experiments showed that phages displaying either EGF or sfGFP-EGF can specifically bind EGFR-expressing cells. The vector constructed in the current study has the potential to be engineered for gene delivery purposes as well as cell-based imaging for tumor detection.

Antitumor Effect of Poplar Propolis on Human Cutaneous Squamous Cell Carcinoma A431 Cells.

Propolis is a gelatinous substance processed by western worker bees from the resin of plant buds and mixed with the secretions of the maxillary glands and beeswax. Propolis has extensive biological activities and antitumor effects. There have been few reports about the antitumor effect of propolis against human cutaneous squamous cell carcinoma (CSCC) A431 cells and its potential mechanism. CCK-8 assays, label-free proteomics, RT-PCR, and a xenograft tumor model were employed to explore this possibility. The results showed that the inhibition rate of A431 cell proliferation by the ethanol extract of propolis (EEP) was dose-dependent, with an IC50 of 39.17 µg/mL. There were 193 differentially expressed proteins in the EEP group compared with the control group (p < 0.05), of which 103 proteins (53.37%) were upregulated, and 90 proteins (46.63%) were downregulated. The main three activated and suppressed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were extracellular matrix (ECM)-receptor interaction, amoebiasis, cell adhesion molecules (CAMs), nonalcoholic fatty liver disease (NAFLD), retrograde endocannabinoid signaling, and Alzheimer's disease. The tumor volume of the 100 mg/kg EEP group was significantly different from that of the control group (p < 0.05). These results provide a theoretical basis for the potential treatment of human CSCC A431 cell tumors using propolis.

Identification of Lilium ledebourii antiproliferative compounds against skin, bone and oral cancer cells.

Objective: This study aimed at the evaluation of anti antiproliferative activity of Lonicera nummularifolia, Lilium ledebourii, Campsis radicans and Parthenocissus quinquefolia extracts. Materials and Methods: The extract was taken from the fresh leaves and bulbs of the plants by maceration method in the dark. After separating the solvent, the remaining dry matter was added to the culture medium containing G292, A431 and KB cancer and HGF-1 normal cells. Cytotoxicity tests, as well as cell cycle and apoptosis tests were performed on cells treated with dry substances and untreated cells. Finally, the most effective extract was separated into fractions by preparative HPLC and the effective fraction was characterized by Triple-Quad LC/MS connected to the UHPLC system. Results: All extracts significantly enhanced cell death rate in the three cancer cell lines more than the HGF-1 line. The Methanolic extract of L. ledebourii bulbs exhibited considerable efficacy on apoptosis induction in the cancer cell lines. It seems that the mode of action for L. ledebourii methanolic extract is mediated through increased BID/MAPK14 expression and decreased MDM2/BCL2/MYC expression, which led to activation of the p53 protein-induced apoptosis. It was also determined that the effective fraction of L. ledebourii methanolic extract consists of substances such as caffeic acid, ferulic acid, coumarin acid, catechin and apigenin. Conclusion: Overall, the findings suggest that L. ledebourii is a promising source of bioactive compounds with anticancer properties.

Cytotoxic Assessment of 3,3-Dichloro-ß-Lactams Prepared through Microwave-Assisted Benzylic C-H Activation from Benzyl-Tethered Trichloroacetamides Catalyzed by RuCl2(PPh3)3.

Natural and synthetic ß-lactam derivatives constitute an interesting class of compounds due to their diverse biological activity. Mostly used as antibiotics, they were also found to have antitubercular, anticancer and antidiabetic activities, among others. In this investigation, six new 3,3-dichloro-ß-lactams prepared in a previous work were evaluated for their hemolytic and cytotoxic properties. The results showed that the proposed compounds have non-hemolytic properties and exhibited an interesting cytotoxic activity toward squamous cell carcinoma (A431 cell line), which was highly dependent on the structure and concentration of these ß-lactams. Among the molecules tested, 2b was the most cytotoxic, with the lowest IC50 values (30-47 µg/mL) and a promising selectivity against the tumor cells compared with non-tumoral cells.

Identification and validation of four photodynamic therapy related genes inhibiting MAPK and inducing cell cycle alteration in squamous cell carcinoma.

Introduction: Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, and photodynamic therapy (PDT) is a promising modality against cSCC. This study investigated the impact of PDT on the MAPK pathway and cell cycle alternation of cSCC as well as the related molecular mechanisms. Method: Expressing mRNA profile data sets GSE98767, GSE45216, and GSE84758 were acquired from the GEO database. The functions of differently expressed genes (DEGs) were enriched by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Least absolute shrinkage and selection operator (Lasso) analysis were used to establish a diagnosis model based on GSE98767. A correlation analysis and a protein-protein interaction (PPI) network were used to evaluate the relationship between cSCC-PDT-related genes and the MAPK pathway. Single-sample gene set enrichment analysis (ssGSEA) was performed on GSE98767 to estimate MAPK activation and cell cycle activity. Finally, the effect of MAPK activation on the cell cycle was explored in vitro. Result: Four cSCC-PDT-related genes, DUSP6, EFNB2, DNAJB1, and CCNL1, were identified as diagnostic markers of cSCC, which were upregulated in cSCC or LC50 PDT-protocol treatment and negatively correlated with the MAPK promoter. Despite having a smaller MAPK activation score, cSCC showed higher cell cycle activity. The PDT treatment suppressed the G1 to G2/M phase in JNK overexpressed A431 cells. Conclusion: CCNL1, DNAJB1, DUSP6, and EFNB2 were identified as potential PDT target genes in cSCC treatment, whose potential therapeutic mechanism was inhibiting the MAPK pathway and inducing cell cycle alternation.

Augmented Therapeutic Efficacy of Naringenin Through Microemulsion-Loaded Sericin Gel Against UVB-Induced Photoaging.

Naringenin (NAR) is a flavonoid found in citrus fruits such as grapes and oranges. Recently, NAR has demonstrated its potential in inhibition of photoaging. The aim of the present study was to investigate the efficacy of sericin (SR) gel loaded with NAR microemulsion (ME) to inhibit UVB-induced photoaging and prevention of epidermoid carcinoma in animal model. NAR -ME was prepared and optimized through Box-Behnken design. The optimized ME was loaded into sericin (SR) gel. The formulations were subjected to various in vitro, in vivo and cytotoxicity studies over A431 cell lines. The optimized ME revealed a globule size of 249.05 ± 3.78 nm, 6.7 ± 0.5 pH and 73.1 ± 2.11% release over a period of 24 h respectively. Cytotoxicity studies revealed a depression in IC50 value in NAR -ME (65.11 ± 1.54 µg/ml) when compared with NAR (118.1 ± 2.09 µg/ml). The NAR-ME-SR gel displayed enhanced therapeutic potential when compared with plain NAR, in terms of augmented antiproliferative activity. Graphical abstract.

ABT-263 induced A431 cell apoptosis and its effect on AKT downstream signaling pathway / 中国职业医学

OBJECTIVE: To investigate the effect of ABT-263,an anti-apoptotic protein inhibitor,on human cutaneous squamous cell carcinoma A431 cells,and to explore its molecular mechanisms. METHODS: i) Total protein was extracted from human immortalized epidermal cells( Ha Ca T cells) and A431 cells in logarithmic growth phase. The protein expression of B-cell lymphoma-2( BCL-2) and BCL2-like 1( BCL-XL) was detected by Western blotting. ii) The A431 cells were treated with ABT-263( inhibitor group) and dimethyl sulfoxide( control group) at a concentration of 50 μmol/L for 4 and 9 hours. The morphological changes of the cells were examined by transmission electron microscopy. iii) The A431 cells were treated with 0,10,25,40,and 50 μmol/L of ABT-263 for 24 hours,and the cell viability was determined by CCK-8 assay. iv) The A431 cells were treated with different doses of ABT-263,and the expression of cleaved Caspase-3, cleaved poly( ADP-ribose) polymerase-1( PARP-1), phosphorylated protein kinase B [p AKT(ser473)],phosphorylated glycogen synthase kinase-3β(p GSK3β) and phosphorylated histone H2 AX(γH2 AX) was detected by Western blot. RESULTS: The relative expression of BCL-2 and BCL-XL in A431 cells were higher than those in Ha Ca T cells( P < 0. 01). Transmission electron microscopy results showed that A431 cells in inhibitor group gradually changed from normal morphology to apoptotic morphology,showing loss of microvilli,increased nuclear chromatin density and aggregation around the nuclear membrane,and nuclear fragmentation. The cell viability of A431 cells in 10,25,40 and 50 μmol/L groups were lower than those in control group( P < 0. 05). The relative expression of cleaved Caspase-3 and cleaved PARP-1 in A431 cells in 10,30 and 50 μmol/L groups were higher than those in control group( P < 0. 05).The relative expression of p AKT( ser473) and p GSK3β in A431 cells in 10,25,40 and 50 μmol/L groups were lower than those of the control group( P < 0. 05) and γH2 AX protein expression was higher than that of the control group( P <0. 05). A431 cell viability and p GSK3β protein expression decreased with the increase of inhibitor dosage( P < 0. 01).The relative expression of cleaved Caspase-3 and γH2 AX protein increased with the increase of inhibitor dosage( P <0. 01),showing dose-effect relationship. CONCLUSION: ABT-263 can induce apoptosis of A431 cells through mitochondria pathway and induce the inactivation of AKT/GSK3β pathway,which can promote the apoptosis of A431 cells with a doseeffect relationship.

Effect of aminolevulinic acid-based photodynamic therapy on the expression of protein kinase D1 and its phosphorylation sites in a cutaneous squamous cell carcinoma cell line A431 / 中华皮肤科杂志

Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression of protein kinase D1 (PKD1) in a cutaneous squamous cell carcinoma cell line A431,and to explore the mechanism underlying ALA-PDT-induced apoptosis of A431 ceils.Methods A431 cells were cultured in vitro,and cell counting kit-8 (CCK-8) assay was performed to select the optimal combination of ALA concentration and PDT dose with the strongest proliferation inhibitory effect.A431 ceils at exponential growth phase were randomly divided into 4 groups:control group receiving no treatment,ALA group treated with ALA solution alone,PDT group treated with PDT alone,and ALA-PDT group treated firstly with ALA solution and then with PDT.After 12-,24-,36-and 48-hour additional culture,CCK-8 assay was conducted to evaluate the cellular proliferation inhibition,and the apoptosis rate at the time point of the strongest proliferation inhibitory effect was measured by flow cytometry.RT-PCR was performed to determine the expression of protein kinase D1 gene (PRKD1) in A431 cells at different time points after the ALA-PDT treatment,and Western blot analysis to measure protein expression of PKD 1 and its phosphorylation at Tyr463 (pTyr463) and Ser916 (pSer916) in A431 cells.Results The combination of ALA at the concentration of 1.5 mmol/L with PDT at an irradiation dose of 2 J/cm2 was optimal due to its strongest proliferation inhibitory effect.After 12-,24-,36-and 48-hour additional culture,there were significant differences in the proliferation inhibition rate among the 4 groups (F =39.56,P ＜ 0.05).At 24 hours after the treatment,the ALA-PDT group showed significantly higher proliferation inhibition rate (46.26％ ± 1.25％) compared with the ALA group (14.65％ ± 0.33％,P ＜ 0.05),PDT group (14.96％ ± 0.68％,P ＜ 0.05) and control group (11.98％ ± 0.32％,P ＜ 0.05),as well as compared with that at 12 hours (P ＜ 0.05).At 24 hours after the treatment,the apoptosis rate significantly differed among the 4 groups (F =16.32,P ＜ 0.05),and the ALA-PDT group showed a significantly higher apoptosis rate (41.92％ ± 3.23％) compared with the control group (4.67％ ± 0.88％,P ＜ 0.05),ALA group (7.02％ ± 1.52％,P ＜ 0.05) and PDT group (8.37％ ± 0.59％,P ＜ 0.05).At 0,6,12,24,36 and 48 hours after the treatment,there were significant differences in the mRNA expression of PRKD 1 among the 4 groups (F =22.24,P ＜ 0.05),and the mRNA expression of PRKD1 at 24 hours was significantly lower than that at 0,6,12 hours (all P ＜ 0.05),but was not significantly different from that at 36 and 48 hours (both P ＞ 0.05).No significant difference in the Ser916-phosphorylated PKD1 expression was found among the 4 groups (F =1.53,P ＞ 0.05),while there were significant differences in the expression of PKD1 and Tyr463-phosphorylated PKD 1 among the 4 groups (F =10.04,8.27,both P ＜ 0.05).Additionally,the ALA-PDT group showed significantly lower expression of PKD 1 and Tyr463-phosphorylated PKD 1 compared with the control group,ALA group and PDT group (all P ＜ 0.05).Conclusion PKD1 may be involved in the photochemical process of A431 cell apoptosis induced by ALA-PDT,and may promote the occurrence of squamous cell carcinoma by Tyr463 phosphorylation.

Effects of resveratrol on the proliferation, apoptosis and telomerase ability of human A431 epidermoid carcinoma cells.

The aim of the present study was to investigate the effect of resveratrol on cell apoptosis, ability of telomerase and the human telomerase reverse transcriptase (hTERT) protein expression in human A431 epidermoid carcinoma cells. A431 cells were treated with different concentrations of resveratrol, and the cell appearance was then observed under a microscope. In addition, the cell proliferation was examined using an MTT assay, and the ability of telomerase was detected using telomeric repeat amplification protocol-polymerase chain reaction-ELISA. Resveratrol significantly inhibited the ability of telomerase and decreased the expression of hTERT protein in a concentration-dependent manner. In conclusion, resveratrol is capable of downregulating the expression of hTERT protein and inhibits the ability of telomerase of A431, which is an important mechanism of action of resveratrol with regard to inhibition of A431 cell proliferation.

Ex vivo localization and permeation of cisplatin from novel topical formulations through excised pig, goat, and mice skin and in vitro characterization for effective management of skin-cited malignancies.

CONTEXT: It would be advantageous to administer cisplatin topically for treatment of cutaneous malignancies. OBJECTIVES: Present work focuses on ex vivo and in vitro characterization of proultraflexible topical formulations. MATERIALS AND METHODS: Permeation of cisplatin through the excised pig, goat, and mice skin was quantitatively determined. RESULTS: Data indicate that protransfersome carbopol gel (pcg) formulation clearly delayed drug permeation through skin. Permeation of cisplatin from protransfersome system (ps) formulation was enhanced by approximately 1.5 fold compared with pcg for pig and goat skin. DISCUSSION: Localization of drug from pcg was higher and showed less permeation. CONCLUSION: Cisplatin-loaded pcg formulation is better to treat cutaneous malignancies.

Effect of berberine on the proliferation and apoptosis of a human skin squamous cell carcinoma cell line A431 / 中华皮肤科杂志

Objective To estimate the effect of berberine on the proliferation of and expressions of apoptosisrelated factors Bax and Bcl-2 in a human skin squamous cell carcinoma cell line A431.Methods A431 cells were cultured in vitro,and classified into various groups to be treated with berberine at different concentrations (12.5,25,5,100 mg/L) or cisplatin at 250 mg/L (positive control group) for different durations (12,24,48 and 72 hours).The A431 cells remaining untreated served as the negative control group.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cell growth,and inverted microscopy to observe cell morphology.Real time quantitative reverse transcription-PCR and an immunofluorescence assay were conducted to measure the mRNA and protein expressions of Bax and Bcl-2 respectively.Statistical analysis was done by multi-way analysis of variance (ANOVA) using the software SPSS 13.0.Results MTr assay showed that berberine inhibited the growth of A431 cells,and the inhibitory effect increased with the increase in concentration (F =1118.312,P ＜ 0.001) and treatment duration (F =510.927,P ＜ 0.001) of berberine.Moreover,there was a significant interaction between the concentration and treatment duration of berberine (F =70.239,P ＜ 0.001).Inverted microscopy revealed that when the concentration of berberine increased,cell density was reduced,and cell morphology changed from polygonal to round with cell body shrinkage.The ratio of bax to Bcl-2 mRNA was elevated with the increase in treatment duration and concentration of berberine,and there were significant differences in the mRNA ratio among cells treated with berberine for different time durations at same concentrations (F =226.231,1300.636,4325.139 for berberine at 25,50 and 100 mg/L respectively,all P＜ 0.001).Immunofluorescence staining indicated that the fluorescence intensity of Bax was enhanced,while that of Bcl-2 was weakened after berberine treatment.Conclusions Berberine inhibits the growth of A431 cells in a dose-and timedependent manner,and may induce the apoptosis of A431 cells via regulating the expressions of Bax and Bcl-2.

Development of novel topical drug delivery system containing cisplatin and imiquimod for dual therapy in cutaneous epithelial malignancy.

CONTEXT: Strategy of dual therapy has been proposed to minimize the amount of each drug and to achieve the synergistic effect for cancer therapies. OBJECTIVE: The aim of this study was to develop an effective drug delivery system for the simultaneous topical delivery of two anti-tumor agents, cisplatin and imiquimod. MATERIAL AND METHODS: The preformulation studies were carried out in terms of tests for identification, solubility profile, determination of partition coefficient and simultaneous estimation of both drugs by UV-Visible spectrophotometer and High Performance Liquid Chromatography (HPLC). Drug-drug and drug-excipients interactions were examined by thin layer chromatography, infrared spectroscopy, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). Provesicular drug delivery system (protransfersome gel formulation) have been prepared and characterized by in vitro and in vivo parameters. RESULTS: The mean size, poly dispersity index (PDI) and zeta potential of transfersomal vesicles formed by protransfersome hydration were 429.5 nm, 0.631 and -68.1 Mv, respectively. The prepared formulation showed toxicity on cutaneous squamous cell carcinoma cell line (A-431) at 200 µg (cisplatin) and 1 mg (imiquimod) concentration of drug in combination against control. The cisplatin- and imiquimod-loaded provesicular dual-drug delivery system achieved an optimal antitumor effect, increase in lifespan, antiviral, and toxicity reduction, revealing the advantage of site specific drug delivery and the modified combination therapy. DISCUSSION: Cisplatin delivery through protransfersome gel in combination with imiquimod may potentiate the activity against solid tumors of epidermal origin. CONCLUSION: Data revealed that combination therapy considerably enhances antitumor efficacy of the drug for skin-cited malignancies.

Effect of baicalin on proliferation and migration activity in human skin SCC cells / 中国药理学通报

Aim To investigate the effect of baicalin on cell proliferation and cell migration in human skin SCC A431 cell line. Methods The A431 cells were incu- bated with 50 mg·L-1 baicalin. The protein level of cofilin-1 was assayed by Western blot. Cofilin-1 specific siRNA fragment was designed , synthesized and trans- fected into A431 cells. The proliferative activity and migration ability of cells were assessed by CCK8 assay and scratch wound healing assay separately. ResultsWestern blot results showed that baicalin treatment in-hibited the cofilin-1 protein expression to 49.3% com-pared with the control group. Single baicalin treatment and cofilin-1 silencing could drease the A431 cell growth and migration. And cofilin-1 silencing signifi- cantly enhanced the efficacy of baicalin. Conclusions Baicalin could significantly inhibit the tumor cell's growth and migration in the A431 cell line. And cofi-lin-1 might become the potential target gene to enhance the effect of anticancer drugs.

Studies on the Radiation Induced Apoptosis by Morphological and Biochemical Analysis in A431 Cells / 대한핵의학회잡지

PURPOSE: We performed this study to evaluate the process of radiation induced apoptosis in A431 skin epithelial cancer cell line. MATERIALS AND METHODS: Low to high dose radiation (0, 2, 5, 10, 25 Gy) was given to A431 cells by Cs-137 cell irradiator. Apoptosis was evaluated by cell morphology, dye exclusion test, and DNA laddering. RESULTS: Cell viability decreased as the radiation dose increased. Number of apoptotic bodies increased as radiation dose increased. It increased most significantly at 12 hours after irradiation. Lactate dehydrogenase activity in culture medium increased according to radiation dose and time after irradiation. CONCLUSION:: Radiation-induced apoptosis which was the main course of cell death in A431 cells could be analyzed quantitatively by counting apoptotic bodies under microscope. Apoptosis increased as radiation dose increased.