Expression Of ABCG2 In Lung Cancer Tissues And Effect Of EGF, IL-4 To Regulate ABCG2 Expression In Human Lung Adencarcinoma Cell Lines

ObjectiveLung cancer is one of the commonest malignant tumors. Its morbidity and mortality are increasing in the world. The situation of lung cancer's prevention and treatment is serious. At present, lung cancer lacks effective treatment, so to improve therapeutic effect of lung cancer has become very urgent in the world. Lung cancer includes small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). In the combined treatments, chemotherapy is the basic and the most important one. But there are two drawbacks in chemotherapy. One is the side effect of the chemical drugs. The other is drug resistance, especially multi-drug resistance. The multi-drug resistance will affect the clinical effects. This is one of the important factors that induce failures in chemical therapy.The multi-drug resistance is correlated with overexpression of multi-drug resistance genes and proteins. The ATP-binding cassette (ABC) transporter family contains membrane proteins that are involved in trafficking of a wide variety of substrates (including metabolic products, drugs, toxins, endogenous lipids, peptides, nucleotides and sterols) across extra- and intracellular membranes. To date, there have been 49 human ABC transporters classified, localized and functionally evaluated in a variety of mammalian tissues. Several of these proteins have been associated with the phenomenon of multidrug resistance. The following three seem to account for most of multidrug resistance—P-glycoprotein (ABCB1, MDR1), multidrug resistance associated protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2, MXR, ABCP). BCRP gene is the latest of MDR gene discovered. Based on structural and sequence homology, BCRP belongs to the ABCG subfamily. The Human Genome Nomenclature Committee named the transporter ABCG2. Unlike most of the other ABC transporters, BCRP contains only one NBD, so called a half-transporter. ABCG2 limits intracellular concentration of substrate agents by pumping them out of cell through an active, energy dependent mechanism. Recent studies have shown that ABCG2 is involved in MDR in human tumor cells. But the relationship of ABCG2 and lung cancer was less investigated; furthermore, the regulation of ABCG2 is poorly understood. Based on others'work, we have observed the expression of ABCG2 in lung cancer tissues and the relationship of its expression and clinical pathological factors. Moreover, we investigated the expression of ABCG2 and the effect of EGF, IL-4 to regulate ABCG2 expression in human lung adencarcinoma cell lines. This will help understand the mechanism of drug resistance in lung adencarcinoma and may be also relevant for the treatment of NSCLC, especially lung adenocarcinoma.Materials and methodsLung tumor tissues, malignant pleural mesothelioma tissues and lung adenocarcinoma cell lines were observed in our study. The relationship of ABCG2 and lung cancer was investigated in organ, cell and molecule levels. The possible mechanisms were further studied.1. The ABCG2 expression of 83 patients was determined by immunohistochemistry to investigate the expression and signification of ABCG2 in lung cancer tissues.2. The ABCG2 expression of 26 malignant pleural mesothelioma and 29 lung adencarcinoma tissues which embed in paraffin and preserved by formalin was determined by immunohistochemistry.3. The ABCG2 mRNA expression was determined by semi-quantitative reverse transcription-PCR, while the ABCG2 protein expression was measured by flow cytometric assay in lung adenocarcinoma cell lines.4. The ABCG2 mRNA and EGFR mRNA expression in 34 lung adenocarcinoma tissues were examined by real-time PCR.5. The collocation of ABCG2 and EGFR in A549 and SPC-A-1 cell lines was explored using flow cytometry.6. ABCG2 mRNA and protein expression induced by EGF ligand in A549 and SPC-A-1 cell lines was measured by real-time PCR and western-blot. Viability assays after treatment with topotecan was measured by MTT.7. ABCG2 mRNA and protein expression induced by IL-4 in SPC-A-1 cell lines was measured by semi-quantitative reverse transcription-PCR and western-blot.1. The ABCG2 positive rate in the tissues of NSCLC patients determined by immunohistochemistry was 71.88% (46/64), higher than it in the SCLC patients 10.53% (2/19), it was significantly difference (P<0.05).2. The expression levels of ABCG2 are not associated with gender, tumor size, histology and in NSCLC (P>0.05). The expression levels of ABCG2 in lymph node metastasis group were higher than them in non-lymph node metastasis group (P<0.05). It was also significant in different differentiation group of NSCLC (P<0.05).3. The ABCG2 positive rate in the tissues of 26 malignant pleural mesothelioma patients determined by immunohistochemistry was 7.69% (2/26).4. The ABCG2 mRNA expression in the tissues of lung adencarcinoma was higher than it in paracancerous tissues and in normal lung tissues. The ABCG2 mRNA was readily detected in lung adencarcinoma cell lines A549 and SPC-A-1. The ABCG2 expression level in A549 was slightly higher than that in SPC-A-15. The mRNA expression was found in 34 tumors (100%) for both of ABCG2 and EGFR. The median relative target gene expression levels in the tumor samples were as follows: ABCG2 0.959 (range: 0.005-3.721), EGFR 1.299 (range: 0.009-7.224; Table 1). The levels of the ABCG2 mRNA expression were correlated significantly with those of the EGFR mRNA expression (r=0.609, P <0.001).6. The proportion of anti-ABCG2 positive cells of A549 and SPC-A-1 cells were 10.21±1.34% and 6.32±0.81%, while the proportion of double-staining cells was 7.05±1.21% and 5.49±0.85% respectively.7. The result of real-time PCR and western-blot showed a significant increase of ABCG2 expression by EGF in A549 and SPC-A-1cells.8. The result of MTT showed that EGF-treated A549 cells exhibit a statistically significant increase in viability after 24 h of incubation with topotecan in concentrations of 100nM compared with control cells, while EGF-treated SPC-A-1 cells in concentrations of 100nM and 1μM compared with control cells.9. There is not a significant change of ABCG2 expression by IL-4 in SPC-A-1 cells on both mRNA and protein levels.1. The expression of ABCG2 in SCLC is less.2. The expression of ABCG2 found in NSCLC tissues may closely related to oncogenesis and progression of NSCLC.3. The expression levels of ABCG2 are associated with lymph node metastasis and differentiation in NSCLC. 4. The expression of ABCG2 was determined by immunohistochemistry is helpful in differential diagnosis of malignant pleural mesothelioma and lung adencarcinoma. In addition, the lack of ABCG2 expression in malignant pleural mesothelioma may have relationship with irinotecan having anti- mesothelioma activity.5. There is the expression of ABCG2 in lung adencarcinoma cell lines.6. EGF regulates ABCG2 expression in human lung adencarcinoma cell line A549 and SPC-A-1. In view of EGF signal transduction as a target for drugs (e.g., gefitinib), which are in turn substrates and/or inhibitors of ABCG2, this regulation has therapeutic consequences.7. IL-4 does not regulate ABCG2 expression in human lung adencarcinoma cell line SPC-A-1.