Advances in mechanism and regulatory factors of corneal stromal repair / 国际眼科杂志(Guoji Yanke Zazhi)

Corneal stromal is an important structure to maintain corneal transparency. The corneal stroma can be injured by trauma, infection and surgery. Therefore, corneal stromal wound starts repair with phenotype changes in stromal cell, extracellular matrix remodeling and immune cells migration. The corneal scar was the leading cause of blindness worldwide, which can be caused by corneal stromal fibrosis from increased myofibroblasts and deposited extracellular matrix after sever damage. At present, corneal transplantation is the main treatment for corneal scar, which has limited therapeutic effect because of corneal donor shortage, surgical requirements and the risk of postoperative immune rejection. Recently, great progress has been made in the study of control mechanism of corneal stromal wound healing with various molecules, cells and tissues. This paper reviews the repair mechanism of corneal stromal injury and the regulation mechanism of cause of corneal injury, corneal structure and molecule factors towards corneal stromal injury. It aims at providing new ideas for exploring the mechanism of corneal stromal repair and regeneration, which is supposed to help prevent corneal scar clinically.

Macrophage Polarization and Anti-tumor Mechanism of Chinese Medicine：A Review / 中国实验方剂学杂志

Macrophages are natural immune cells with strong plasticity. The polarization of macrophages mainly responds to stimuli in the microenvironment by changing their phenotype and related functions. In recent years， studies have found that the polarization of macrophages is involved in the occurrence and development of various diseases such as bone arthritis， skin diseases， diabetes， coronary heart disease， breast cancer， colorectal cancer， and lung cancer， especially the metastasis of malignancies and drug resistance， through multiple signaling pathways， including nuclear factor kappa-B（NF-κB）， c-Jun N-terminal kinase（JNK）， protein kinase B （Akt）， mitogen-activated protein kinase （MAPK）， signal transducer and activator of transcription 6 （STAT6）， Wnt/β-catenin， and mammalian target of rapamycin （mTOR） and regulatory factors， such as granulocyte-macrophage colony-stimulating factor （GM-CSF）， interleukin （IL）-4， IL-10， transforming growth factor （TGF）-β， tumor necrosis factor（TNF）-α， and interferon-γ（IFN-γ）. Chinese medicine is also pivotal in the prevention and treatment of malignancies. In recent years， therefore， the specific anti-tumor mechanism of Chinese medicine and its active ingredients has become a research hotspot. The tumor microenvironment is crucial to the occurrence and development of tumors. The polarization of tumor-associated macrophages is involved in the proliferation， apoptosis， and metastasis of tumor cells. Therefore， targeted regulation of the polarization of tumor-associated macrophages is a potential target for clinical treatment of malignancies. Based on the research articles published in the past three years， this article reviewed macrophage polarization and the anti-tumor mechanism of Chinese medicine from four perspectives， i.e.， macrophage polarization， related pathways and regulatory factors of macrophage polarization， macrophage polarization and breast cancer， colorectal cancer， and lung cancer， and macrophage polarization and anti-tumor effects of Chinese medicine， active ingredients of Chinese medicine， and self-formulated prescriptions/classic prescriptions. This study is expected to provide certain ideas for the clinical treatment， basic research， and development of new Chinese medicine in the treatment of tumors.

Differential expression patterns of myogenic regulatory factors in the postnatal longissimus dorsi muscle of Jeju Native Pig and Berkshire breeds along with their co-expression with Pax7

BACKGROUND: Myogenic regulatory factors (MRFs) such as MyoD, Myf6 and Myf5 play a vital role in the growth and development of muscles. Jeju Native Pig (JNP) is the top ranker in Korea amongst the indigenous livestock reared for meat purpose. Few studies covering transcript abundance of the MRFs and related to their co-expression with Pax7 in JNP have been conducted. Despite having better quality pork, JNP does not have a comparative growth rate with respect to western breeds. Therefore, the present study was designed with the objective to study the relative transcript levels of MRFs in the postnatal myogenesis of longissimus dorsi muscles in JNP and Berkshire breeds. RESULTS: Relative transcript levels were analyzed by qRT-PCR and blot expression analysis through Western blotting. Immunocytochemistry was performed to analyze their expressions at cellular levels. ToppCluster aided in the analysis of gene ontology of biological processes. The quantitative transcript levels of MyoD and Pax7 were significantly (P < 0.05) higher in Berkshire than in JNP. Myotube formation was observed under the co-expression of MyoD and Pax7. ToppCluster helped in the understanding of the linking of biological processes of the MRFs with the different signaling pathways. MyBPH had significantly (P < 0.05) high transcript levels during the chosen age groups in JNP than Berkshire. CONCLUSIONS: The current study can be helpful in understanding the genetic basis for myogenesis in postnatal stage. Moreover, it can act as stepping stone for the identification of marker genes related to body growth and meat quality in JNP.

Effect of HBsAg on the production of interferon-α in peripheral blood plasmacytoid dendritic cells induced by the stimulator of interferon genes signaling pathway / 临床肝胆病杂志

ObjectiveTo investigate the effect of HBsAg on the expression of interferon-α (IFN-α) in peripheral blood plasmacytoid dendritic cells (pDCs) induced by the stimulator of interferon genes (STING) signaling pathway activated by cyclic GMP-AMP (cGAMP). MethodPeripheral venous blood was collected from healthy adults and the patients with chronic hepatitis B virus (HBV) infection who attended the outpatient service or were hospitalized in Department of Infectious Diseases, The Affiliated Hospital of Xuzhou Medical University, from February to December 2016, and peripheral blood mononuclear cells (PBMCs) were isolated and extracted. After the STING agonist cGAMP was added to PBMCs, ELISA was used to measure the levels of IFN-α, interferon-β, and tumor necrosis factor-α in supernatant. PBMCs from healthy adults were pre-incubated with HBsAg and then stimulated by cGAMP, and supernatant was collected to measure IFN-α. The magnetic-activated cell sorting method was used to remove pDCs from PBMCs, and after culture with cGAMP, ELISA was used to measure the level of IFN-α in supernatant. PBMCs from healthy adults were stimulated by HBsAg and/or cGAMP, and then flow cytometry was used to measure the frequency of pDCs. The independent samples t-test was used for comparison of continuous data between two groups. ResultsPBMCs from the patients with chronic HBV infection stimulated by cGAMP in vitro had a significantly lower level of IFN-α than healthy controls (469.72±18.95 vs 599.90±84.06, t=4.868, P=0.001). PBMCs from healthy adults co-cultured with HBsAg and stimulated by cGAMP had a significantly lower level of IFN-α than those in the non-HBsAg group (448.5±52.0 vs 571.0±30.8, t=4.500, P=0.011). Compared with PBMCs containing pDCs, PBMCs without pDCs stimulated by cGAMP had a significant reduction in the level of IFN-α (164.50±40.73 vs 339.50±35.33, t=6.482, P=0.001). Compared with PBMCs from healthy adults stimulated by cGAMP, PBMCs pre-incubated with HBsAg and then stimulated by cGAMP had a significant reduction in the frequency of pDCs (0.12%±0.04% vs 0.24%±0.04%, t=5.176, P=0.014). ConclusionHBsAg can inhibit the expression of IFN-α induced by the STING pathway in pDCs activated by cGAMP.

Relationship between the severity of iron overload in peripheral blood CD4+T cells and the key transcription factors of RORrt, IRF8, STAT3 for cell proliferation and differentiation in elderly patients with myelodysplastic syndrome / 中华老年医学杂志

Objective To investigate the effects RORrt (RORrt),IRF8 (IRF8) and STAT3 (STAT3) in peripheral blood CD4+T cells on the cell proliferation and differentiation in elderly patients with iron-overload myelodysplastic syndrome(MDS).Methods A prospective case-control study was conducted.Twenty-two elderly hospitalized patients(12 males and 10 females)aged 60-78 years with iron-overload MDS from Jan.2017 to Dec.2018 were enrolled and considered as the observation group.Twenty MDS patients without iron overload hospitalized in the same period were selected as the non-iron overload group,and 26 healthy elderly people were considered as the healthy control group.Peripheral blood monocytes(PBM)were prepared and resident CD4+T cells were sorted by flow cytometry.The mRNA and protein expression levels of transcription factors of RORrt,p-STAT3 and IRF8 were detected by quantitative real time polymerase chain reaction(qRT-PCR)and Western blotting.Results In peripheral blood CD4+T cells,the mRNA expression level of RORrt and p-STAT3 were higher and that of IRF8 was lower in the iron-overload group than in the non-iron overload group and the healthy control group(42.634± 18.613 vs.21.289 ± 15.158 and 22.520 ± 9.896;29.710±9.689 vs.12.355±4.681 and 9.818±3.845;19.293±8.258 vs.23.785±12.498 and 69.619±23.768,P＜0.01).In peripheral blood CD4+T cells,the protein expression level of RORrt and p-STAT3 was higher,and that of IRF8 was lower in the iron overload group than in the non-iron overload group and healthy control group(P＜0.01).Conclusions The abnormalities of the mRNA and protein expression levels of transcription factors of RORrt,IRF8 and p-STAT3 in CD4+ T cells play a fundamental role in the pathogenesis of iron overload MDS in elderly patients.

Construction of yeast one-hybrid library and screening of transcription factors regulating LS expression in Ganoderma lucidum / 中国中药杂志

Lanosterol synthase( LS) is a key enzyme involving in the mevalonate pathway( MVA pathway) to produce lanosterol,which is a precursor of ganoderma triterpenoid. And the transcriptional regulation of LS gene directly affects the content of triterpenes in Ganoderma lucidum. In order to study the transcriptional regulation mechanism of LS gene,yeast one-hybrid technique was used to screen the transcription regulators which interact withthe promoter of LS. The bait vector was constructed by LS promoter,then the vector was transformed yeast cells to construct bait yeast strain. One-hybrid c DNA library was constructed via SMART technology. Then the c DNA and p GADT7-Rec vector were co-transformed into the bait yeast strain to screen the upstream regulatory factors of the promoter region of LS by homologous recombination. Total of 23 positive clones were screened. After sequencing,blast was performed against the whole-genome sequence of G. lucidum. As a result,8 regulatory factors were screened out including the transcription initiation TFIIB,the alpha/beta hydrolase super family,ALDH-SF superfamily,60 S ribosomal protein L21,ATP synthase β-subunit,microtubule associated protein Cript,prote asome subunit β-1,and transaldolase. Until now,the regulation effect of these 8 regulatory factors in G.lucidum has not been reported. This study provides candidate proteins for in-depth study on the expression regulation of LS.

Research progress in applications used for guided tissue regeneration in endodontic surgery / 口腔疾病防治

@#Conventional root canal therapy can not completely treat all periradicular lesions, especially combined endodontic-periodontic diseases, large periapical lesions and bone defect lesions. Guided tissue regeneration greatly improves the success rate of endodontic surgery in treating these lesions. This article focuses on the application of various grafting materials, guided bone regeneration membranes and regulatory factors in endodontic surgery.

Evaluation of skeletal muscle regeneration in experimental model after malnutrition / Avaliação da regeneração do músculo esquelético em modelo experimental após desnutrição

Abstract The aim of this study was to analyze muscle regeneration after cryoinjury in the tibialis anterior muscle of young rats that were malnourished and then recovered. Forty Wistar rats were divided into a nourished group that received a normal protein diet (14% casein) for 90 days and a malnourished and recovered rats group (MR) that was submitted to 45 days of malnutrition with a hypoproteic diet (6% casein) followed by 45 days of a normal protein diet (14% casein). After the recovery period, all of the animals underwent cryoinjury in the right tibialis anterior muscle and euthanasia after 7, 14 and 21 days. The amount of connective tissue and the inflammation area was higher in the malnutrition recovered injury MR group (MRI) at 14 days post-injury (p < 0.05). Additionally, the cross-sectional area (CSA) of the regenerated fibers was decreased in the MRI (p < 0.05). The MyoD and myogenin protein levels were higher in the nourished injury group. Similar levels of TGF-β1 were found between groups. The proposed malnutrition protocol was effective in showing delayed changes in the regeneration process of the tibialis anterior muscle of young rats. Furthermore, we observed a delay in muscle repair even after nutritional recovery.

Resumo O objetivo do presente estudo foi analisar a regeneração muscular após criolesão no músculo tibial anterior de ratos jovens desnutridos e recuperados. Foram utilizados 40 ratos da linhagem Wistar, divididos em 2 grupos: ratos nutridos receberam dieta normoproteica (14% de caseína) por 90 dias; e ratos desnutridos e recuperado submetidos a duas fases nutricionais pós-desmame, correspondendo a 45 dias de desnutrição com dieta hipoproteica (6% caseína), seguida por 45 dias de dieta normoproteica (14% caseína). Ao completar a fase de recuperação, todos os animais foram submetidos à criolesão no músculo tibial anterior direito e a eutanasia ocorreu 7, 14 e 21 dias após a lesão. A quantidade de tecido conjuntivo e a área de inflamação 14 dias pós-lesão foi maior no grupo desnutrido, recuperado e lesado (MRI – malnourished, recovered and injured group) (p < 0,05). A área de secção transversa (AST) das fibras regeneradas do grupo MRI foi menor (p < 0,05). O conteúdo das proteínas MyoD e Miogenina foi maior no grupo nutridos e lesados. A citocina TGF-β1 não apresentou diferença entre os grupos. O protocolo proposto foi eficaz para demonstrar alterações no processo de regeneração do músculo tibial anterior de ratos jovens, atrasando o reparo muscular mesmo após a recuperação nutricional.

Effects of pulsed radiofrequency application to dorsal root ganglia on expression of IRF8 in spinal cord and BDNF in nucleus accumbens of rats with neuropathic pain / 中华麻醉学杂志

Objective To evaluate the effects of pulsed radiofrequency (PRF) application to dorsal root ganglia on the expression of interferon regulatory factor 8 (IRF8) in the spinal cord and brain-derived neurotrophic factor (BDNF) protein in the nucleus accumbens of rats with neuropathic pain (NP).Methods Forty healthy pathogen-free male Wistar rats,weighing 180-200 g,aged 2 months,were divided into 4 groups (n=10 each) using a random number table:sham operation group (group Sham),group NP,sham PRF group (group SPRF) and PRF group.NP was induced by chronic constriction injury (CCI) to the left sciatic nerve of anesthetized rats.The mechanical paw withdrawal threshold (MWT) was measured before CCI and at 3,7,10,14,21,28,35 and 42 days after CCI.Sucrose preference test and forced-swim test were performed at 42 days after CCI for determination of the expression of IRF8 in the spinal cord and BDNF in the nucleus accumbens by Western blot.Results Compared with group Sham,the MWT at each time point after CCI and rate of preference for sucrose were significantly decreased,the duration of immobility in forced-swim test was prolonged,and the expression of IRF8 and BDNF was up-regulated in NP,SPRF and PRF groups (P＜0.05).Compared with group NP,the MWT at 10-42 days after CCI and rate of preference for sucrose were significantly increased,the duration of immobility in forced-swim test was shortened,and the expression of IRF8 and BDNF was down-regulated in group PRF (P＜0.05),and no significant changes were found in the parameters mentioned above in group SPRF (P＞O.05).Conclusion The mechanism by which PRF application to dorsal root ganglia alleviates NP and depressive-like behaviors is probably related to down-regulation of the expression of IRF8 in the spinal cord and BDNF in the nucleus accumbens of rats.

Envejecimiento y metabolismo: cambios y regulación

Los estudios sobre los efectos del envejecimiento en la fisiología y el metabolismo cada vez son más, uno de sus objetivos es contribuir a instrumentar programas para mejorar la calidad de vida y prevenir discapacidades en la vejez. Es de gran importancia mencionar que durante el envejecimiento se presenta una desaceleración natural del metabolismo, se produce una serie de cambios en la regulación de la energía, lo que contribuye a la pérdida de peso y grasa; estos cambios en la regulación de la ingesta calórica contribuyen en un aumento de la susceptibilidad al desequilibrio energético tanto positivo como negativo, lo cual va asociado a un deterioro en la salud. Sin embargo, el llegar a la vejez, no es una sentencia de muerte para el metabolismo, por el contrario, éste puede ser controlado mediante el mantenimiento de un estilo de vida activo, aunado a esto investigaciones han demostrado que el metabolismo puede ser regulado mediante el papel que desempeña un sistema de reloj sincronizado (ritmos biológicos), el cual a su vez es modulado por varias proteínas reguladoras; esta relación garantiza que las células funcionen correctamente y por tanto el mantenerse saludables. El objetivo de esta revisión es aportar información actualizada sobre la regulación metabolismo-energía y su relación con la gran variedad de componentes involucrados en el gasto energético que acompañan al envejecimiento; analizar la regulación de este sistema para mejorar la calidad de vida y mantener la salud en la vejez.

Aging and metabolism: changes and regulation. Studies about the effects of aging in the physiology and metabolism are increasingly, one of its objectives is to help implement programs to improve the quality of life and prevent disability in elderly. It is relevant to mention that, during aging, there is a natural metabolic deceleration, a series of changes in the regulation of energy are produced, which contributes to loss of weight and fat; the changes in the regulation of caloric intake contribute to increase the susceptibility to energy imbalance both positive and negative, which is associated with a deterioration in health. However, to grow old, is not a death sentence for metabolism, on the other hand, it can be controlled by maintaining an active lifestyle, coupled with this, research has shown that the metabolism can be regulated by a synchronized clock (circadian rhythms), which is mediated by regulatory proteins, this relationship ensures the proper functioning of the cells and therefore good health. The aim of this review is to provide updated information on the energy- metabolism-regulation and its relationship with the great variety of components involved in energy expenditure that accompany aging, to analyze the regulation of this system to improve the quality of life and maintenance of health in old age.

Effects of isoflurane anenthesia on myocyte enhancer factor 2 signaling pathway in neonatal rat hippocampus / 中华麻醉学杂志

Objective To investigate the effects of isoflurane anenthesia on myocyte enhancer factor 2(MEF2) signaling pathway in neonatal rat hippocampus. Methods Twenty-four 5-day-old SD rats of both sexes,weighing 10-13 g, were randomly divided into 2 groups ( n = 12 each): control group (group C) and isoflurane group (group I). In group I, 1.5% isoflurane in 100% O2 was inhaled for 6 h. Group C received no treatment.Three rata in each group were sacrificed at 2, 4, 6 h of isoflurane anenthesia and 24 h after isoflurane anenthesia (T1-4), and the hippocampi removed for determination of MEF2 mRNA, synGAP Ⅰ mRNA, Arc mRNA and synapsinⅠ mRNA expression (by PT-PCR) and synapsin Ⅰ protein expression (by Western blot).Results Compared with group C, the expression of MEF2 mRNA, synGAP Ⅰ mRNA, Arc mRNA and synapsin Ⅰ mRNA at T1-3 and synapsin Ⅰ protein at T2-4 was up-regulated in group I ( P ＜ 0.05). Conclusion Inhalation of anaesthetic concentration of isoflurane may affect synapse formation during the development of central nervous system by actirating hippocampal MEF2 signaling pathways in neonatal rats.

Functional expression of oligopeptide transporter PepT1 in the gastrointestinal tract and its regulatory factors / 中华临床营养杂志

Many peptide transporters have been identified in mammals, among which PepT1 has been widely studied. PepT1, a member of proton-coupled oligopeptide transporter (POT) superfamily, is a peptide transporter of low affinity and high capacity and is mainly expressed in the brush border membrane of intestinal epithelial cells. PepT1 plays an important role in the absorption of di/tri-peptide (the degradation products of protein in intestinal tract). Meanwhile, it mediates the transport of peptide-like drugs and the bacterial products. Therefore,the changes of the functional expression of PepT1 in the gastrointestinal tract may dramatically affect the internal and external environmental stability and drug absorption. This paper reviews the structural features and function,distribution, transport mechanisms, and regulatory factors of PepT1.

Effect of Shugan Jianpi Formula on Proliferation/Apoptosis of Hyperplastic Tissues of Mammary Gland in Model Rats / 中国药房

OBJECTIVE:To explore the effect of Shugan jianpi formula on the proliferation and apoptosis of the hyperplastic tissues of mammary gland of model rats.METHODS:The rats were injected with benzestrofol and progesterone to establish mammoplasia model,meanwhile which were treated intragastrically with Shugan jianpi formula for 30 days.The degree of mammoplasia,as well as the PCNA(proliferating cell nuclear antigen),the apoptotic related regulating factors such as Bcl-2 and Bax and the apoptotic index(AI)of the mammary gland were detected.RESULTS:Shugan jianpi formula inhibited the mammoplasia,down-regulated the expression levels of PCNA and Bcl-2,up-regulated the expression level of Bax and AI,inhibited the proliferation of the mammary tissues and promoted apoptosis.CONCLUSION:Shugan jianpi formula blocks or reverses the hyperplasia of mammary gland by regulating the cell proliferation/apoptosis of the hyperplastic tissues of mammary gland.

Progress in Pre-mRNA Alternative Splicing in Gliomas / 生物化学与生物物理进展

Gliomas constitute the most common type of primary brain tumor. Alternative pre-mRNA splicing may play roles in the carcinogenesis, cell growth and invasion in various types of gliomas. Factors regulating the alternative spicing in gliomas include cis-regulatory element (for example, ESE, ISS and ESS) and trans-regulatory factors (such as SRp55, SC35, SF2/ASF and PTB). Recent progresses demonstrate that many genes associated with gliomas, including those encoding tumor suppressors or promoters, enzymes, receptors and ion channels are subject to regulation by alternative pre-mRNA splicing. Therefore, studying the alternative pre-mRNA splicing in gliomas will be of benefit to understanding the molecular basis of gliomas and identifying new targets potentially useful for early diagnose as well as therapy of gliomas.

The role of negative-regulatory factors of toll-like receptors signal pathways in the immunological pathogenesis of Kawasaki disease / 中华风湿病学杂志

Objective To investigate the role of negative-regulatory factors of toll-like receptors (TLRs)signal pathways in immunological pathogenesis of Kawasaki disease(KD).Methods Thirty-two chil- dren with Kawasaki disease and 16 age-matched healthy children were studied.Reverse-transcription PCR (RT-PCR)and real-time PCR were used to evaluate the mRNA expression levels of toll-like receptor 4(TLR4), MD-2,MyD88,IRAK-4,TRAF6,T1/ST2,IRAK-M,Triad 3A,and proinflammatory factors such as IL-1?, IL-6,IL-8 and TNF-?,in peripheral blood monocytes/macrophages(MC).The expression of TLR4 protein in MC was analyzed by flow cytometry.Results①Compared with the control group,the mRNA levels of TLR4, MD-2,MyD88,IRAK-4 and TRAF6 in KD group were up-regulated significantly(P＜0.01),and the expression level of TLR4 protein was also found to be up-regulated in KD group during acute phase.It was detected that expression levels of TLR4 protein in KD with coronary artery lesion(KD-CAL~+)was significantly higher than that of KD without coronary artery lesion(KD-CAL-)[flow cytometry:(6.5?1.7)% vs(11.9_+2.4)%,P＜0.01].②The expression level of negative-regulatory factors such as IRAK-M and Triad3A were significantly up-regulat- ed in acute phase of Kawasaki disease,while the mRNA levels of IRAK-M and Triad3A in KD-CAL~+ group was found to be significantly lower than those of KD-CAL~- group(P＜0.01).No difference of T1/ST2 mRNA expres sion level was detected among all groups(P＞0.05).③The expressions of proinflammatory eytokines such as IL-1?, IL-6,IL-8 and TNF-?in monoeytes/macrophages during acute phase of Kawasaki disease were higher than those of the control group(P＜0.01),and expression of proinflammatory cytokines in KD-CAL~+ group was significantly higher than that of KD-CAL~- group.Conclusion Relative insufficient expression of negative-regulatory factors, such as IRAK-M and Triad3A,maybe correlate with immunological pathogenesis of Kawasaki disease.

EFFECTS OF MRF4 TRANSFECTION ON DIFFERENTIATION AND EXPRESSION OF MYOGENIC REGULATORY FACTORS IN CULTERED HUMAN RHABDOMYOSARCOMA RD CELLS / 解放军医学杂志

To study the effects of MRF4 transfection on differentiation and expression of myogenic regulatory factors of human rhabdomyosarcoma RD cells, the plasmid-MRF4 cDNA was transfected into cultured rhabdomyosarcoma RD cells with lipofectin method. The myogenic regulatory factors MRF4 and MyoD mRNA were measured with in situ hybridization and the expressions of myosin heavy chain(MHC) and a-actin in the cells were assayed with immunocytochemical method. The cell growth and morphology were observed at the same time. It was found that the morphology of differentiation increased and the growth was suppressed in RD cells after transfection. The expression of MHC and a-actin were significantly increased in RD cells after transfection, while the expressions of MRF4 and MyoD mRNA were up-regulated. It is suggested that transfection of MRF4 can induce differentiation of RD cells and up-regulate the expression of MyoD.

Effects of carcinogenicity and growth ragulatory factors in human epithelial cells exposed with tobacco-specific N-nitrosamine

Since NNK is one of the most abundant tobacco-specific alkaloids and a strong carcinogenic nitrosamine, it has been used for evaluating a potential of carcinogenicity in the animal models. The present study has attempted to examine the potential of carcinogenicity of NNK in human epithelial cells, from which the cell type the most of cancers including oral cancer and nasal cavity cancer are originated. The cellular model used for the study is a human keratinocyte cell system immortalized by Ad12-SV40 hybrid virus. The cellular system has successfully been used for the carcinogenicity studies because of its limitless life span, epithelial morphology and non-tumorigenicity. When cells were treated with a variety of NNK concentrations, levels of saturation density and soft agar colony formation were increased in a dose-dependent fashion. Colonies of large cell aggregates were above 5 at the higher doses. The results indicate that exposure of human cells with NNK induced loss of contact inhibition and increases of anchorage independence and cellular adhesion, which are typical characteristics of the neoplatically transformed cells. When cells were exposed with 100uM NNK for 2hr, mRNA levels of IL-1 and PAI-2 were increased in a dose-dependent manner, but expression of TGF- 1 was not affected. While expression of growth regulatory factors were altered with a short-term exposure, there was no alteration of these factors in the NNK-transformed cells. However, mRNA levels of fibronectin were increased both in the short-term treatment and in the transformation. The results suggest that altered expression of extracellular matrix such as fibronectin following short-term exposure might be fixed in the genome and these altered properties be continuously transfered throughout the cell division. Western blot analysis showed a translocation of PKC- from cytosolic fraction to the particulate fraction, indicating a possible role of NNK in the signal transduction pathway. The present study provided an evidence that NNK in the smoking may be associated with epithelial origin cancer such as oral and nasal cavity cancers. In addition, this study suggested that altered expression of extracellular matrix and PKC may play an important role in the carcinogenic mechanism of NNK.