[Abstract] Objective detected 113 cases of non-small cell lung cancer (NSCLC) enzyme glutathione S-transferase π (GST  π), lung resistance-related protein (LRP), DNA topoisomerase II (Topo Ⅱ a.), Multi-drug resistant drug-associated protein (MRP) and multidrug resistance (MDR1) gene expression observed correlation between the expression of the above indicators with clinical pathological factors and indicators. The methods using immunohistochemical SP assay GST  π, LRP, Topo Ⅱ a protein; application situ hybridization to detect MRP and MDR1 mRNA expression. The results (1) GST  π, of LRP, Topo Ⅱ a protein and MRP, in 113 patients with NSCLC of MDR1 mRNA positive expression rate was 75.2%, 80.5%, 60.2%, 79.6% and 51.3%, respectively. Which expression of LRP highest MDR1 expression lowest. (2) of LRP, Topo Ⅱ a and MRP expression were associated with tumor histological type. (3) GST  π and MRP (P <0.05), LRP and MRP (P <0.01), the correlation of MDR1 and MRP (P <0.01) expression. Conclusion (1) over-expression of a variety of drug-related genes and their interactions may NSCLC produce a primary MDR important reasons. (2) LRP and MRP overexpression, a high expression of Topo Ⅱ MRP low expression may be associated with lung adenocarcinoma, squamous cell carcinoma chemosensitivity.

Key words】 non-small cell lung cancer; multidrug resistance; immunohistochemistry; situ hybridization

    Multi-drug resistant tumor to a chemotherapeutic drug resistance while many other different structure, a different mechanism of action of chemotherapy drugs also produce cross-resistance phenomenon. Compared with small cell lung cancer, non-small cell lung cancer (NSCLC) combination chemotherapy less efficient, their resistance is more prominent. This study, immunohistochemistry and in situ hybridization to detect NSCLC tissues multidrug resistance-associated protein or mRNA expression, trying to in-depth understanding of multidrug resistance in NSCLC situation, the relationship between different resistance mechanisms, provide a theoretical basis for the development of rational chemotherapy regimens for NSCLC cases.

    1 Data and methods

    1.1 cases of sources collected Wuhan University Zhongnan Hospital Pathology Archive and the five years between 1996 to 2000 cases of a total of 113 cases of pathologically confirmed NSCLC surgical resection. All cases are without put chemotherapy, 91 cases were male and 22 females; aged 34 to 77 years old, the average age of 57 years old; 61 cases of 39 cases of squamous cell carcinoma, adenocarcinoma (including bronchioloalveolar carcinoma), adenosquamous cancer, 13 cases; 43 cases of well-differentiated carcinoma, moderately differentiated carcinoma and 47 cases, 23 cases of poorly differentiated carcinoma. 113 cases, lymph node submission of 90 cases (37 cases with lymph node metastasis and without lymph node metastasis, 53 patients), 23 patients without lymph node censorship record; according to the TNM staging Ⅰ 52 cases, Ⅱ 26 cases, 11 cases of stage III, IV, 1 case, 23 cases recorded. The specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, thick of 4 ~~ 5 μm serial sections.

    1.2 Experimental Methods

    1.2.1 slice production

    The specimens were 10% neutral Formalin solution fixed, dehydrated, embedded in paraffin, 4μm thick slices. Each case slices 6. 1 HE staining for tumor routine pathological diagnosis and classification. 3 Immunohistochemical staining for GST  π tumors was observed, LRP and Topo Ⅱ a protein; the two situ hybridization for the detection of tumor MRP and MDR1 mRNA expression.

    1.2.2 Immunohistochemical staining and in situ hybridization

    By immunohistochemical SP method. Used mouse anti-human GST  π, LRP and of of Topo Ⅱ a monoclonal antibody was purchased from Fuzhou Maixin biotechnology company, the Shanghai Instrument Tao Bioinstrumentation Co., Ltd., are that type. Known positive sections as a positive control, PBS replaced the primary antibody as a negative control.

    In situ hybridization with digoxigenin mark of MDR1, MRP oligonucleotide probes and detection kit was purchased from Wuhan Boster Biological Engineering Co., Ltd.. The detection method is carried out according to kit instructions. With known positive piece as positive control, the prehybridization solution instead of primary antibody as a negative control.

    1.2.3 The results of judgment

    GST  π protein of LRP, MRP and MDR1 mRNA positive expression are in addition to protein expression of Topo Ⅱ a cell nucleus brownish yellow the cytoplasm brown particles. Each case randomly selected 10 high power field (× 400), and counting the percentage of positive cells. Positive cell rate> 5% as positive, ≤ 5% considered negative [1].

    1.2.4 Statistical

    SPSS 11.5 statistical software, to the difference between the bidirectional unordered R × C table χ2 test and Fisher’s exact test for indicators and correlation analysis. P <0.05 for significance test standards.

    2 Results

    2.1 5 multidrug resistance-associated protein or mRNA expression in NSCLC

    The ① LRP positive expression of the protein in NSCLC highest rate, shown in Figure 1 to 5. MDR1 mRNA expression in the lowest rate; and ② of LRP, of Topo Ⅱ a protein and MRP mRNA expression with NSCLC histological type. LRP protein expression for adenocarcinoma> of adenosquamous carcinoma> squamous cell carcinoma, adenocarcinoma was significantly higher than adenosquamous carcinoma, squamous cell carcinoma (P <0.05); highest of Topo Ⅱ a protein expression in squamous cell carcinoma, significantly higher than the gland carcinoma and adenosquamous carcinoma (P <0.05); expression of MRP mRNA in squamous cell carcinoma was significantly lower than the glandular squamous cell carcinoma and adenocarcinoma (P <0.05); ③ the five kinds of multidrug resistance-associated protein or mRNA expression and tumor the degree of differentiation, lymph node metastasis status and clinical stage were not correlated (P> 0.05), are shown in Table 1.

    2.2 5 multidrug resistance-associated protein or mRNA expression

    The statistical analysis shows that have a positive correlation between the GST  π protein MRP mRNA (P <0.05), the expression of LRP protein MRP mRNA (P <0.01), MDR1 mRNA and MRP mRNA (P <0.01). No correlation between the rest of the multidrug resistance protein or mRNA expression. Table 1113 patients with NSCLC multidrug resistance-associated protein or mRNA expression

3 Discussion

    The multidrug resistance gene (MDR1) gene family belong to the human MDR. P-glycoprotein (P  gp) encoded product of the MDR1 gene, MDR1 gene can lead to its overexpression. Located in the membrane of the endoplasmic reticulum surrounding P  gp is a transmembrane protein, will enter the anti-cancer drugs within the cancer cells to pump out the cancer cells and lead to cancer drug resistance [2]. MDR1 expression in almost all tumor types, but its expression level and lung resistance and prognosis still remains controversial. Most scholars believe that the MDR1 lung cancer multidrug resistance has nothing to do and even scholars independent view of MDR1 mRNA and protein expression in NSCLC chemotherapy sensitivity or intracellular / intranuclear cisplatin accumulation [3]. There are scholars think that the existence of a significant relationship between the two [4]. In this study, MDR1 positive expression in untreated NSCLC was 51.3%, that they may be one of the factors that lead to tumor multidrug resistance; MDR1 in this study, five kinds of multidrug resistance-associated protein or mRNA expression of the lowest rates, and its expression with clinicopathological factors has nothing to do of MDR1 with NSCLC biological phenotype relationship needs further study.

    Numerous studies show that the MRP gene expression is an important participant in NSCLC primary multidrug resistance factors, especially vincristine, relying on the process of formation of primary resistance to etoposide and doxorubicin drug involved in tumor [5]. The group of 113 patients with NSCLC, MRP positive expression rate was 79.6%, suggesting that NSCLC MRP gene primary resistance, and its expression is related to histological type in squamous cell carcinomas of the positive expression rate lower than glandular squamous cell carcinoma and adenocarcinoma (P <0.05), which is consistent with Wright et al [6] reported. Recently Doubre etc. [7], the percentage of positive cells MRP1 DNA aneuploid cells was significantly higher than DNA diploid cells, finds its expression can be used to predict tumor response to chemotherapy, advocates of DNA aneuploid tumors should be adopted Unlike the method of treatment of the diploid tumors.

    1998, Ikeda et al [8] found that the LRP were expressed in 14 lung cancer cell lines, and its mRNA levels with a variety of cross-resistance of anticancer drugs. Berger et al [9] observed LRP expression in 16 NSCLC cell lines and found that each cell systems of different levels of LRP expression and significantly associated with cisplatin resistance. , Half of the cells in the LRP expression was significantly elevated in the medium daunorubicin and bleomycin. The above studies have shown that LRP expression in NSCLC frequency is very high, and the tumor is closely related to both intrinsic and acquired MDR. Observed in this study, five kinds of multidrug resistance-associated protein or mRNA expression, LRP expression level of the highest (80.5%), and and especially expression in lung adenocarcinoma was significantly higher than adenosquamous carcinoma and squamous cell carcinoma. Our observations not only consistent with the literature, and further LRP is the main lead NSCLC multidrug resistance [10].

    Generally, GST  π in many tumors have gene amplification and expression levels, is currently considered the most closely related to a relationship with tumor resistance GST. Nakanishi et al [11] observed 54 cases with immunohistochemical method untreated NSCLC, results of GST  π-positive expression rate was 69%, but the positive expression group and negative expression rate of response to chemotherapy group were 16% and 47 %. Unsal, etc. [12] proposed GST  π expression does not always predict the reactivity of NSCLC to cisplatin-containing combination chemotherapy drugs. The group of NSCLC cases, GST  π-positive expression rate was 75.2%, but its expression with clinicopathological factors were not correlated. Therefore, GST  π in NSCLC expression meaning still need to continue to explore.

    Topo Ⅱ a, a can catalyze DNA supercoiling local conformational change gene nucleic acid, especially Topo Ⅱ a important target for cancer chemotherapy. Topo Ⅱ inhibitor application by forming drug – enzyme-DNA complexes interfere with the activity of the enzyme, thus affecting DNA metabolism of tumor cells to exert its cytotoxic effect, while the sensitivity of tumor cells Topo Ⅱ inhibitors mainly depends on the intracellular Topo Ⅱ level. Kreisholt found in untreated NSCLC, Topo Ⅱ a was significantly lower than the expression of SCLC, Topo Ⅱ a reduction in the number and / or activity decreased significantly correlated with tumor drug resistance, is also one of the shaping factors of NSCLC primary drug resistance [13 ]. The group of NSCLC, Topo Ⅱ a positive expression rate was 60.2%, suggesting that it is also the formation of primary drug resistance factor in tumor. Topo Ⅱ a positive expression rate was significantly higher than in the squamous cell carcinoma of the lung adenocarcinoma and adenosquamous carcinoma (P <0.05), and clinical practice consistent with lung squamous cell carcinoma is more sensitive to chemotherapeutic drugs. Chemotherapy in NSCLC, planned way Topo Ⅱ a high expression of lung squamous cell applications Topo Ⅱ inhibitors may achieve better efficacy.

    The results of this study show that, GST  π the MRP of LRP and the MRP of MDR1 and MRP in NSCLC expression exist obvious or significant with correlation, indicating that the formation of NSCLC original hair MDR’s is a multi-factor, multi-step complex process, which include multi- kinds of participation of the resistance-related genes and synergy with each other. Therefore, in-depth study the the tumor MDR phenomenon and its mechanism and targeted individualized treatment plan for the development, improve the effect of chemotherapy for NSCLC patients is of great significance.