Induction of pulmonary precancerous lesions by tobacco-specific NNK in Wistar rats / 中国肺癌杂志

<p><b>BACKGROUND</b>Tobacco-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.</p><p><b>METHODS</b>Fifteen 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>Transbronchial 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.</p>

Different expressions of p53 gene family members and their clinical significance in human non-small cell lung cancer / 中国肺癌杂志

<p><b>BACKGROUND</b>To investigate the different expressions of p53 gene family members p53, p63 and p73, and their clinical significance in non small cell lung cancer (NSCLC).</p><p><b>METHODS</b>p53, p63 and p73 protein expressions were detected in 60 NSCLC tissues and 7 normal lung tissues by immunohistochemistry.</p><p><b>RESULTS</b>In NSCLC, positive rate of p53, p63 and p73 protein was 61.67%(37/60), 80.00%(48/60), 73.33% (44/60) respectively. There were significant differences in positive rate of three proteins as compared to normal lung tissue ( P < 0.05). p53 protein expression was closely associated with tumor cell differentiation degree ( P =0.023), but was not associated with histological classification, lymph node metastasis and clinical stages ( P > 0.05). Expression of p63 protein was closely related to lymph node metastasis ( P =0.028) and histological classification ( P =0.001), but not to cell differentiation degree and clinical stages ( P > 0.05). There was no significant relationship between p73 protein expression and clinical characteristics of NSCLC ( P > 0.05). A positive correlation was present between p63 and p73 protein expressions ( P =0.000 1). No statistical correlation was found between p53 and p73 ( P > 0.05).</p><p><b>CONCLUSIONS</b>p53 gene family may be related to the oncogenesis and development of NSCLC. p63 and p73 proteins may have different biological function from p53 protein, and both might play oncogenic roles.</p>

Cyclooxygenase-2, inducible nitric oxide synthase protein expression and vasculature during experimental rat lung carcinogenesis / 中国肺癌杂志

<p><b>BACKGROUND</b>To investigate the expression of cyclooxygenase-2 (COX-2) protein and inducible nitric oxide synthase (iNOS) protein during the experimental lung carcinogenesis in rats, as well as their association with microvessel density (MVD).</p><p><b>METHODS</b>Diethylinitrosamine and 3-methylcholanthrene were instilled into the left lobar bronchus to induce lung squamous cell carcinoma in 88 Wistar rats, and 10 nomal rats as controls. COX-2, iNOS expression and MVD count of the specimens obtained from the rats were examined by immunohistochemistry.</p><p><b>RESULTS</b>A total of 155 specimens of various pathological phase during the carcinogenesis were obtained: 14 hyperplasia, 25 squamous metaplasia, 33 dysplasia, 12 carcinoma in situ, 54 infiltration carcinoma, and 17 metastasis. The immunohistochemical score (IHS) of COX-2 significantly increased in dysplasia, carcinoma in situ and metastasis (P < 0.01,P < 0.05,P < 0.01). IHS of iNOS significantly increased in hyperplasia and metastasis (P < 0.05,P < 0.01 ). Remarkably increased MVD was found in carcinoma in situ, infiltration carcinoma and metastasis (P < 0.01, P < 0.01, P < 0.01). There was a positive correlation between COX-2 and iNOS (r=0.601 6,P < 0.001) expression. Expression of COX-2 or iNOS were remarkably related to MVD count (P < 0.01,P < 0.01)</p><p><b>CONCLUSIONS</b>COX-2 and iNOS may play important roles in the carcinogenesis of experimental rat lung squamous cell carcinoma as well as its progress, and it may be associated with stimulating angiogenesis.</p>

Angiogenesis and blood supply during the course of pulmonary carcinogenesis in experimental rat / 中国肺癌杂志

<p><b>BACKGROUND</b>To 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.</p><p><b>METHODS</b>One 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.</p><p><b>RESULTS</b>In 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).</p><p><b>CONCLUSIONS</b>Angiogenesis 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.</p>

Expression of survivin gene and its significance in the carcinogenesis and development of non-small cell lung cancer / 中国肺癌杂志

<p><b>BACKGROUND</b>To study the expression of survivin mRNA in non-small cell lung cancer (NSCLC), and to explore its relationship with carcinogenesis, development invasion and metastasis of NSCLC.</p><p><b>METHODS</b>In situ hybridization was applied to detect survivin mRNA expression in 12 normal bronchial epithelium, 9 dysplasia, 34 NSCLC and 12 metastatic lymph nodes. The relationship between survivin expression and clinicopathological characteristics was analyzed.</p><p><b>RESULTS</b>In normal bronchial epithelium, dysplasia, NSCLC and metastatic lymph nodes, the positive rate of survivin mRNA expression were 16.67% (2/12), 33.33% (3/9), 61.76% (21/34), and 91.67% (11/12), respectively. There were significant differences in survivin mRNA expression between lung cancer and normal bronchial epithelium ( P < 0.01), as well as between metastatic lymph nodes and normal bronchial epithelium ( P < 0.001). There were remarkably higher survivin mRNA expressions in poor- and moderate-differentiated groups than that in well-differentiated group ( P =0.003, P =0.004). The expression of survivin mRNA was not related to histologic classification and lymph node status ( P > 0.05, P > 0.05).</p><p><b>CONCLUSIONS</b>Survivin mRNA expression may play an important role in the carcinogenesis and development of NSCLC. It may be a new target in gene therapy of lung cancer through blocking or down-regulating survivin mRNA expression to recover the normal regulation mechanism of apoptosis.</p>

Dynamic study on expression of gelatinase A and its natural inhibitor during invasion and metastasis of induced lung cancer in Wistar rats / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Squamous 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.</p><p><b>RESULTS</b>Along 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).</p><p><b>CONCLUSION</b>The 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.</p>

Expression of inflammation related enzymes during experimental rat lung carcinogenesis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of two inflammation related enzymes - cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) during the experimental rat lung carcinogenesis.</p><p><b>METHODS</b>Eighty 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.</p><p><b>RESULTS</b>155 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).</p><p><b>CONCLUSION</b>COX-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.</p>

Study on the specific expressions of p53, bcl-2 and c-myc in non-small cell lung cancer with neuroendocrine differentiation / 中国肺癌杂志

<p><b>BACKGROUND</b>To 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).</p><p><b>METHODS</b>The 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).</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>A 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.</p>

Microdissection and PCR-SSCP detected mutation and expression of p53 and K-ras gene in carcinogenesis and development of induced rat lung carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Microdissection 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.</p><p><b>RESULTS</b>No 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).</p><p><b>CONCLUSIONS</b>p53 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.</p>

The relationship between caspase-3 expression and cell proliferation in rat lung squamous cell carcinoma / 中国肺癌杂志

<p><b>BACKGROUND</b>To 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.</p><p><b>METHODS</b>The 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).</p><p><b>RESULTS</b>Caspase-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).</p><p><b>CONCLUSIONS</b>Loss 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.</p>

Effects of triptolide on the proliferation and apoptosis of cell line A549 in human lung adenocarcinoma / 中国药理学通报

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