Aim To investigate the effects of triptolide on the proliferation and apoptosis of cell line A549 in human Lung Adenocarcinoma. Methods MTT, DNA fragmentation assay, fluorescence stain and flow cytometric analysis were carried out.Results Triptolide notably reduced the survival rate of A549 cells, and inhibited the proliferation of A549 cells. The 50 % inhibitory concentration (IC50) for 72 h was 75 nmol?L-1 and it arrested the cell cycle at S phase at the concentration of 14 nmol?L-1 (P

BACKGROUND:The living standard of farmers and herdsmen in Xinjiang Uygur Autonomous Region, China is significantly lower than that of urban residents in general. Meanwhile, the shortage of doctors and medicines and lack of medical knowledge are the main reasons for local farmers and herdsmen to suffer from knee osteoarthritis. Most of the farmers and herdsmen are not aware of or prevent knee osteoarthritis in the early days to remove pathogenic factors, which results in a serious condition at the time of their medical treatment. OBJECTIVE: To investigate the main pathogenic factors of knee osteoarthritis in Uygur, Kazakh and Han populations in pastoral areas of northern Xinjiang Uygur Autonomous Region, China.METHODS:The permanent residents of Uygur, Kazakh and Han ethnic groups in northern Xinjiang, China who met the criteria were selected by the method of stratified, multistage and cluster random sampling during June 2012 to October 2014. The investigation of knee osteoarthritis was conducted among al the residents who meet the inclusion criteria using the method of home scene closed questionnaire. X-ray lateral plain film examination of the knee joint was performed among the patients presenting with the symptoms of knee osteoarthritis. The database was established. The multi-factor and unconditional Logistic regression analysis was conducted among the 40 variables using SPSS 20.0 software. RESULTS AND CONCLUSION:The 3 402 of 3 540 questionnaires were valid. The Logistic regression analysis suggest that the common pathogenic factors in Han, Uygur and Kazakh ethnic groups are associated with older ages, female gender, high body mass and drink alcohol. Smoking, history of internal disease, high education level, standing position, climbing, trauma, family history, fried food, housing conditions and amenorrhea, different nationalities, churchgoing of ethnic minorities are also the major risk factors. Among the three ethnic groups, the prevalence of female patients in Uygur and Kazak ethnic groups is significantly higher than that of Chinese Han nationality. The morbidity is different among these three nationalities. The dietary preferences cannot be determined as the related influencing factor of knee osteoarthritis. Whether the means of transportation, residence climate and environment, the number of pregnancies and deliveries, drinking tea or not, the type of tea are related to knee osteoarthritis remains unclear.

BACKGROUNDTo investigate the expressions of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) and their correlation with metastasis and prognosis in human lung cancer.

METHODSImmunohistochemical S-P method was used to detect the expression of MMP-9 and TIMP-1 in 65 lung cancer tissues, 35 hyperplastic and dysplastic epithelium from patients with non-cancerous pulmonary diseases, and 30 normal epithelial tissues of the lung.

RESULTSThe positive expression rates of MMP-9 in normal tissue, hyperplastic or dysplastic epithelium, and lung cancer tissue were 16.7%(5/30), 42.9%(15/35) and 72.3%(47/65) respectively, whereas the positive rates of TIMP-1 expression in normal tissue, hyperplastic or dysplastic epithelium, and lung cancer tissue were 6.7%(2/30), 28.6%(10/35) and 50.8%(33/65) respectively. Significant differences of the expression rates of MMP-9 and TIMP-1 were found between lung cancer and normal groups, between lung cancer and hyperplasia groups, and between hyperplasia and normal groups (P < 0.05). Small cell carcinoma and adenocarcinoma had higher MMP-9 expression than squamous cell carcinoma (P < 0.025). Expression rate of MMP-9 had negative relation with cell differentiation of lung cancer (P < 0.05), and positive relation with TNM stage (P < 0.025). Between the survival time < 2 years group and ≥2 years groups, both the expressions of MMP-9 and TIMP-1 had significant difference (P < 0.05 ). The expression of MMP-9 was closely related to metastasis of lung cancer (P < 0.005), but the expression of TIMP-1 was not related to metastasis.

CONCLUSIONSOverexpression of MMP-9 may appear in precancerous lesion and at the early stage of lung cancer. Activation of MMP-9 gene may be an important factor for oncogenesis of the lung. MMP-9 and TIMP-1 may play important roles in lung cancer invasion and metastasis, their overexpression could act as a reference to evaluate metastasis and unfavourable prognosis of lung cancer.

BACKGROUNDTo study the specific expression of tumor-related genes (p53, bcl-2 and c-myc) in non small cell lung cancer with neuroendocrine differentiation (NSCLC-NE).

METHODSThe expression of neuron-specific enolase (NSE), chromogranin A (CgA), synaptophysin(Syn), c-myc, bcl-2 and p53 was detected in 60 surgically resected and paraffin-embedded non-small cell lung cancer (NSCLC) specimens by immunohistochemistry (S-P method).

RESULTSThe positive rates of NSE, CgA, Syn expressed in 60 cases of NSCLC were 45.00%(27/60), 13.33%(8/60), 31.67% (19/60) respectively. According to the results of these three markers, 41.67%(25/60) of 60 specimens was proved to be as NE differentiation cancer. The NE differentiation in NSCLC was remarkably related to differentiation of tumor cells (P < 0.05). NSCLC-NE had a higher metastatic rate (P < 0.05) and a higher clinical staging (P < 0.05) than NSCLC without NE differentiation. The positive rates of bcl-2, p53 and c-myc expression in NSCLC-NE were 68.00% (17/25), 80.00% (20/25), 68.00% (17/25) respectively, and the expression of bcl-2 and p53 was closely related to NE differentiation (P < 0.05).

CONCLUSIONSA certain part of NSCLC have NE differentiation, which has different biological features from NSCLC without NE differentiation. High expression of bcl-2 and mutant p53 can be observed in NSCLC-NE, and bcl-2/Bax unbalance associated with p53 mutation may play an important role in oncogenesis and development of NSCLC-NE.

OBJECTIVETo investigate the dynamic expression and its relation of gelatinase A (MMP-2), its natural inhibitor (TIMP-2) and DNA index (DI) changes during carcinogenesis, invasion and metastasis in Wistar rats.

METHODSSquamous cell carcinoma of lung was induced with 3-methylcholanthrene (MCA) and diethyinitrosamine (DEN) in iodized oil by left intra-bronchial instillation in 80 Wistar rats. Immrno histochemistay (IHC) and in situ hybridigation were used in the monitor of MMP-2, TIMP-2 proteins and mRNA expression during invasion and metastasis of lung cancer in these rats, DNA index (DI) value was measured by guantitatove image analysis on feulgen stained sections.

RESULTSAlong with the carcinogenis, the average poritive MNP-2 and TIMP-2 expressions increased, with positive rates of 8.5% - 85.7% and 6.4% - 35.7%. DI value also underwent the same changes (1.47 +/- 0.54) - (2.87 +/- 0.55). The difference of MMP-2 expression in carcinoma in situ versus early carcinoma and early carcinoma versus metastatic carcinoma are statistically significant (P < 0.05). Companing lung carcinome, the contrel group and non-cancerous lesions, the elevation of MNP-2 and TIMP-2 expressions were also sigmificant (P < 0.01). The DI elevation in carcinoma in situ and dysplasia were obviously significant (P < 0.05). Meanwhile a negative relation was noted in TINP-2 and MMP-2 expressions during carcinogenesis. There was a positive relation between MMP-2 expression and DNA poikiloidy (P < 0.01), which was related to the close relationship between MMP-2 and metastasis in advanced rat lung carcinoma (P < 0.05).

CONCLUSIONThe excess degradation and disruption of basement membranes by activated MMP-2 may be a key step in inducing lung cancer invasion and metastasis. The imbalance between MMP and TIMP may be a critical factor which affects biologic behavior of lung carcinogenesis, invasion and metastasis.

Alkylating Agents ; toxicity ; Animals ; Carcinoma, Squamous Cell ; chemically induced ; genetics ; pathology ; Diethylnitrosamine ; toxicity ; Female ; Gene Expression Regulation, Neoplastic ; Lung ; drug effects ; metabolism ; pathology ; Lung Neoplasms ; chemically induced ; genetics ; pathology ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Methylcholanthrene ; toxicity ; Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; metabolism

BACKGROUNDTo investigate the origin of tumor blood vessel and blood supply during pulmonary carcinogenesis, and the relationship between vascular endothelial growth factor (VEGF), its receptor Flk-1 and angiogenesis.

METHODSOne hundred Wistar rats were instilled with 3-methylcholanthrene (MCA) and diethylinitrosamine (DEN) to induce pulmonary squamous cell carcinoma through left lower lobe bronchus. To acquire different pathological phase during the carcinogenesis, rats were killed in 15, 35, 55, 65, 75 days after instillation. Yellow and green silastics were respectively injected into the bronchial and pulmonary arteries of 30 rats in 55, 65, 75 days after instillation. Intertumor microvessel density (MVD) was marked by anti-von Willebrand factor monoantibody. VEGF and Flk-1 expression were examined by immunohistochemistry.

RESULTSIn the tumor area the tumor blood vessels were yellow and connected with distorted bronchial artery and very few green incomplete branches of pulmonary artery were seen. Silastic particles could be seen in the disordered tumor blood vessels by microscope after bronchial artery perfusion. There was no silastic particles in the carcinoma interstitial blood vessels after pulmonary artery perfusion. MVD count significantly increased in carcinoma in situ (39.50±12.60) and infiltrative carcinoma (61.05±19.92) as compared to atypical hyperplasia (8.92±3.80)(both P < 0.01), and the increased vessels originated from bronchial artery, but not pulmonary artery. The expression of VEGF and Flk-1 increased during pulmonary carcinogenesis. The positive coefficients of VEGF and FLK-1 expressions became higher and higher from epithelial proliferation to squamous metaplasia, to atypical hyperplasia, to carcinoma in situ and finally to infiltrative carcinoma. There was significant correlation between MVD and VEGF expression (r=0.979 8, P < 0.005), as well as between MVD and Flk-1 expression (r=0.907 8, P < 0.05).

CONCLUSIONSAngiogenesis is the important phenomenon of the rat pulmonary carcinogenesis and the newly formed blood vessels in tumor connect with the branches of bronchial artery, but not pulmonary artery. This confirms that the blood supply of pulmonary carcinoma is from bronchial artery, not from pulmonary artery. VEGF and Flk-1 are closely related to angiogenesis of tumor.

BACKGROUNDTo analyse the relationship between caspase-3 expression and cell proliferation, and to find molecular-biology markers to adjust canceration during rat lung squamous cell carcinogenesis.

METHODSThe 3-methylcholanthrene(MCA) and diethyinitrosamine (DEN) were used to induce lung squamous cell carcinoma by intra-left lobar-bronchial instillation in 50 Wistar rats, and 10 normal rats as controls. Expression of caspase-3 and PCNA were evaluated by immunohistochemistry (IHC).

RESULTSCaspase-3 protein positive rate was 44.12% in 34 rat lung squamous cell carcinomas, and positive coefficient value was 1.38±0.95, which were significantly lower than that of normal bronchial epithelium (P=0.007, P < 0.01) and premalignant lesions (P < 0.05, P < 0.05). The mean PCNA-labeling indexes (PCNA-LI) of normal rat bronchial epithelium, premalignant lesions and lung cancer were 14.10±5.02, 28.13±8.72 and 41.88±14.24 (P < 0.05), respectively. There was a negative correlation between caspase 3 and PCNA LI (r=-0.730 6, P < 0.01).

CONCLUSIONSLoss expression of caspase-3 may promote tumor cell growth, and it may be important in rat lung squamous cell carcinogenesis. Detection of caspase-3 and PCNA proteins can be regarded as major markers in the diagnosis of lung canceration.

OBJECTIVETo investigate the roles of p53 and K-ras gene in carcinogenesis and development of the lung carcinoma induced by 3-methylcholanthrene (MCA) and diethylinitrosamine (DEN) in Wistar rats, and to elucidate the relationships between the protein expression and gene mutation of p53 and K-ras.

METHODSMicrodissection was used to obtain pure cell populations of each phase in the carcinogenesis and development of lung carcinoma induced by MCA and DEN. DNA of the microdissected cell populations was extracted and used to analyze the mutations of p53 exons 5 approximately 8 and K-ras exons 1 approximately 2 by PCR-SSCP. The expressions of p53 and K-ras protein in each phase were detected by immunohistochemistry.

RESULTSNo mutation and protein expression of p53 and K-ras was found in the 30 cases with normal bronchial epithelium. Mutation of p53 was detected in 3.1% of 18 hyperplasia and 14 squamous metaplasia cases, 28.6% of 21 dysplasia, 30.0% of 12 carcinomas in situ, 51.2% of 43 infiltration carcinomas, 52.9% of 17 metastases. The positive immunostaining rate of p53 protein was 0, 42.9%, 50.0%, 60.5% and 64.7% respectively. K-ras mutation rate was 0, 4.8%, 8.3%, 9.3%, 11.8% respectively, while the overexpression rate of K-ras protein was 15.6%, 19.0%, 25.0%, 41.9%, 52.9% respectively. p53 protein expression was closely related with p53 mutation (P < 0.005, Pearson's R = 0.599 6). There was no relationship between the protein expression and gene mutation of K-ras (P > 0.500).

CONCLUSIONSp53 gene mutation and K-ras overexpression were early events in the carcinogenesis and development of rat lung carcinoma induced by MCA and DEN, while K-ras mutation does not play any important role.

Animals ; Genes, p53 ; Genes, ras ; Humans ; Immunohistochemistry ; Lung Neoplasms ; chemically induced ; chemistry ; genetics ; Mice ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Rats ; Tumor Suppressor Protein p53 ; analysis ; ras Proteins ; analysis

OBJECTIVETo investigate the expression of two inflammation related enzymes - cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) during the experimental rat lung carcinogenesis.

METHODSEighty Wistar rats were instilled with 3-methylcholanthrene (MCA) and diethylinitrosamine (DEN) into the left lobar branchus to induce lung squamous cell carcinoma. To obtain specimen in every pathological phase during the carcinogenesis, these rats were sacrificed at different intervals. The expression of COX-2 and iNOS in every pathological phase during the carcinogenesis were examined by immunohistochemical method. The immunohistochemical scores (IHS) were calculated by combining an estimate of the percentage of immunoreactive cells with that of the stain intensity.

RESULTS155 specimens of every pathological phase during the carcinogenesis showed: hyperplasia 14, squamous metaplasia 25, dysplasia 33, carcinoma in situ 12, infiltrating carcinoma 54 and metastasis 17. Inflammation and elevated expressions of COX-2 and iNOS were shown in the precancerous lesions. The COX-2 IHS was significantly increased in dysplasia, carcinoma in situ and metastasis (P < 0.01, P < 0.05, P < 0.01 respectively). The iNOS IHS significantly increased in hyperplasia and metastasis (P < 0.05, P < 0.01 respectively). There was a positive correlation between the expression of COX-2 and iNOS (gamma = 0.601 6, P < 0.001).

CONCLUSIONCOX-2 and iNOS, two inflammation related enzymes, playing important roles in the carcinogenesis of MCA and DEN, induce rat lung squamous cell carcinoma as well as its metastasis. The relation between inflammation and carcinogenesis may partly be explained by the elevated expression of these two enzymes. Nonsteroidal antiinflammatory drug (COX-2 inhibitors) and iNOS inhibitors may possess antitumor activities because of their prevention of bronchial dysplasia, carcinogenesis and metastasis.

Animals ; Carcinogens ; adverse effects ; Carcinoma, Squamous Cell ; chemically induced ; enzymology ; pathology ; Cyclooxygenase 2 ; Female ; Immunohistochemistry ; methods ; Isoenzymes ; biosynthesis ; Lung Neoplasms ; chemically induced ; enzymology ; pathology ; Male ; Methylcholanthrene ; adverse effects ; Neoplasms, Experimental ; chemically induced ; enzymology ; pathology ; Nitric Oxide Synthase ; biosynthesis ; Nitric Oxide Synthase Type II ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; Rats ; Rats, Wistar

BACKGROUNDTobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most important carcinogen in cigarette. Models induced by NNK are widely used in investigations about the mechanisms of pulmonary neoplasia and chemoprevention studies. The aim of this study is to explore the pulmonary precancerous lesions induced by NNK and its possible mechanisms.

METHODSFifteen Wistar rats were divided into two trial groups, in which the high-dose group was instilled with iodized oil including 10 mg (50 mg/kg) NNK into the left lower lobar bronchus, and the low-dose group received 5mg ( 25mg/kg) NNK. Another 15 Wistar rats were instilled only with iodized oil as control group. All rats were examined immediately after instillation and followed up periodically by pulmogram. The pulmonary tissues of rats were pathologically examined, and the expression of AE1/AE3, PCNA and p53 was detected by immunohistochemical method.

RESULTSThe pulmograms showed that the iodized oil localized at the bottom of left lobe and disappeared 107 days later. In trial group, 10 of 15 rats (67%) had nodus at the bottom of left lobe. All of rats in trial group (15/15) displayed atypical hyperplasia in alveolar region, showing single or multiple layers of proliferative epithelial cells along intact alveolar septa with irregular and non-discrete margins of lesion, but continuous alveolar spaces were not obliterated by proliferative epithelial cells. Ten of 15 rats in trial group showed severe atypical hyperplasia of glandular epithelium with occasional infiltrating to muscular layer. All of those atypical hyperplasia cells showed positive AE1/AE3 expression. The positive rate of PCNA was 90% (9/10) and 100% (5/5) in low-dose group and high-dose group respectively, which was significantly higher than that in control group (13%, 2/15) (P=0.000, P=0.001). The positive rate of p53 expression was 50% (5/10) and 60% (3/5) in low-dose group and high-dose group respectively, which was significantly higher than that in control group (0) (P=0.005, P=0.009). However, there was no remarkable difference in PCNA and p53 expression between low-dose group and high-dose group (P > 0.05).

CONCLUSIONSTransbronchial instillation of iodized oil including tobacco-specific NNK can induce pulmonary lesions as atypical hyperplasia of alveolar cell and glandular epithelium in Wistar rats. This model can be used in experimental studies about tobacco-related lung cancer.