Expression Of CN-â…¡ In Non-Small Cell Lung Cancer And Its Relationship To Gemcitabine Resistance

[Background] Lung cancer is a common malignant carcinoma with the highest morbidity and mortality that is badly endangering the health of human being. Non-small cell lung cancer (NSCLC) makes the majority of lung carcinoma subtypes. Chemical therapy is an important method in the combining treatment of NSCLC. Gemcitabine regimen serves as one of the standard first-line treatment for patients with advanced NSCLC. However, the response rate of this single-agent was only 20% to 30%. And when taken in combination with platinum, the response rate was no more than 54%. Resistance to Gemcitabine has become an obstacle towards its clinical effect. The blocked transportation and the abnormity of metabolism enzymes are considered to be the major cause of Gemcitabine resistance.Cytosolic 5'-nucleotidase II (CN-II) is an important modulation enzyme that regulates the balance of cytosolic nucleotide pools. CN-II behaves both as a nucleotidase and also as a phosphotransferase. The purified enzyme has been shown to use several analogue monophosphates such as dFdCMP as its substrates. And increased CN-II expression may be correlated with Gemcitabine resistance. Further work is needed to identify the exact function of CN-II in nucleotide analogues resistance. The expression of CN-II in NSCLC and its correlation towards response to Gemcitabine and prognosis of patients are still warranted.[ Objective] To detect CN-II expression of both mRNA and protein level and explore the relationship between CN-II and NSCLC as well as Gemcitabine resistance, so as to discuss the potentiality of CN-II to be a latent prognostic factor for Gemcitaine therapy.First, on the basis of investigating the biological characters of parental and Gemcitabine resistant cell lines, both mRNA and protein standard of CN-II expression were assayed and the diversities were compared between different cell lines to elementarily explore the correlationship of CN-II towards Gemcitabine resistance.Second, to study the relation between CN-II and NSCLC, and to retrospectively analyze the role of CN-II in Gemcitabine resistance, we detected CN-II protein level expression in NSCLC tissues so as to further investigate the mechanism of Gemcitabine resistance, and also to offer experimental elements for choosing fit chemotheraputic agents.[Methods] The research work comprises following four parts:Part 1 Biological Characteristics of human NSCLC parental and Gemcitabineresistant cell linesThe risistant index (RI) of A549/Gem and H460/Gem was investigated using the method of MTT assay to validate the resistance .We observed the morphology of NSCLC parental (A549, NCI- H460) and Gemcitabine resistant cells (A549/Gem, H460/Gem). The growth curve and douling time were detected by cell calculating. This part of research was designed to set basis for the later work.Part 2 RT-PCR measurment of CN-IImRNA in NSCLC parental and Gecitabineresistant cell linesTo detect the expression of CN-IImRNA in different NSCLC cell lines, RT-PCR was performed and the relative expression levels of CN-II as toβ-actin were compared between the parental and resistant cells. This part of research was designed to find the wherher CN-II expression mRNA level in Gemcitabine resistant cell line was different from that in parental cells.Part 3 Westernblot and immunocytochemistry measurment of CN-II protein in NSCLC parental and Gecitabine resistant cell lines To investigate the protein level of CN-II in NSCLC cell lines, methods of Westernblot and immunocytochemisry was used to find out whether there was difference of CN-II protein expression between parental and resistant cell lines. The experimental condition of the four cell groups were entirely the same.Part 4 Expression and clinical significance of CN-II protein expression in NSCLCtumor tissuesCN-II protein expression was investigated in 70 cases of NSCLC paraffin-embedded tuomor tissuse by immunohistochemical EnVision technique. The correlation between CN-II expression and clinical parameters as well as pathologic characteristics of NSCLC patients was analyzed using chi-square test. We further chose 21 cases of patients who had ever received Gemcitabine chemotherapy and analyzed the relation between CN-II protein level and patients' response and prognostic to Gemcitabine therapy.[Results ] The results comprise following four parts:Part 1 Biological Characteristics of human NSCLC parental and Gemcitabineresistant cell linesA,IC₅₀ of the four cell lines were as follows:A549 7.58±1.29μmol/L, A549/Gem 852.96±151.52μmol/L; NCI-H460 5.81±1.89μmol/L, H460/Gem 457.53±53.32μmol/L. The resistant index (RI) of the resistant cell lines were as follows: A549/Gem 112.52 and H460/Gem 78.75. The two groups of resistant cell lines had high and steady resistance to Gem.B,The growth curves of the four cell lines were drawn and the doubling time wascalculated according to the curves. The doubling time of the four cell lines were asfollows: A549 30.429h, A549/Gem 39.941h; NCI-H460 39.539h, H460/Gem47.783h. The resistant cell lines had longer doubling time than their parental cells.Part 2 RT-PCR measurment of CN-IImRNA in NSCLC parental and Gecitabineresistant cell linesThe results showed that CN-IImRNA was detectable in four cell lines that had different levels of expression.A,Comparative cotent of CN-IImRNA in A549 was 0.433±0.128, and in A549/Gemwas 0.832±0.139. There was significant variance between two groups (t = 3.655,P<0.05).B,Comparative cotent of CN-IImRNA in NCI-H460 was 0.346±0.127, and inH460/Gem was 0.587±0.101. Resistant cell lines of H460/Gem had hihgerCN-IImRNA expression as to its parental NCI-H460 cells, but there was nosignificant variance between two cells (t =2.583, P>0.05)..C,Comparative cotent of CN-IImRNA in patental cell lines of A549 and NCI-H460was also compared and there was no significant variance between them (t = 0.836,P>0.05).To sum up, Gem resistant cell lines had higher CN-IImRNA expression than their corresponding parental cells. Adenocarcinoma cells had distincter enhancement than the large cell carcinoma cells. The parental cells of A549 and NCI-H460 had close CN-IImRNA comparative cotent as to each other.Part 3 Westernblot and immunocytochemistry measurment of CN-II proteinin NSCLC parental and Gecitabine resistant cell linesThe results showed that CN-II protein was detectable in four cell lines that had different levels of expression.A,CN-II postive expression was mianly sited in cytosol according to theimmunocytochemistry experiment of four cell lines. Gem resistant cell lines hadhigher CN-II protein expression than their corresponding parental cells.B,Positive CN-II band was detected in four groups of cell lines but thebrightness of the four bands was somehow different. SDS-polyacrylamide gelelectrophoresis analyses for CN-II was 56 kDa by Westernblot. The resistant cellsrespectively had higher protein expression than corresponding parental cell lines.C,CN-II protein expression level by Westernblot was identical to the mRNAexpression by RT-PCR. To sum up, resistant cell lines had higher CN-II expression than the parental cells no matter on mRNA or protein levels but the degree of enhancement might be different. Adenocarcinoma cells had significant higher CN-II expression as to its parental cells than the large cell carcinoma cells.Part 4 Expression and clinical significance of CN-II expression in NSCLCtumor tissuesExpression of CN-II was detected in 70 cases of paraffin embedded NSCLC samples by EnVision immunohistochemistry. The results were as follows:A,The positive rate of CN-II in 70 non-small cell lung cancer tissues were 52.9%.CN-II postive expression was mianly sited in cytosol.B,There was no significant relationship between CN-II expression and clinic pathological parameters such as age, gender, TNM stages and tumor differentiation. There was also no significant differenc between the primary and the transferred tumor tissues, as well as between Adenocarcinoma and Squamous carcinoma.C,Among 21 patients who had received gemcitabine chemotherapy, those with tumor progressed (positive rate 83.3%) had higher CN-II expression than the patients with efficacious (positive ratel6.7%)(83.3% VS 16.7%, P<0.05) and disease control chemotherapy (positive rate%)(83.3% VS33.3 %, P<0.05). Meanwhile, patients whose tumor had higher CN-II expression had better prognosis with longer overall survival.To sum up, the results of CN-II expression in NSCLC tissues meets the upper experiments in cell lines. CN-II may be a prognostic factor for gemcitabine chemotherapy to predict patients' response to Gem.[Conclusion] By detecting both CN-II mRNA and protein expression in NSCLC cell lines and tumor tissues, it was definitized that CN-II expression in Gem resistant cell lines was higher than in the corresponding parental cells. The degree of diversity between resistant and parental cells was somehow different with adenocarcinoma higher than large cell carcinoma. CN-II might function in acquired resistance to Gem in NSCLC cell lines. And CN-II may be a prognostic factor for gemcitabine chemotherapy to predict patients' response to Gem.