Effects Of ANXA1 And CYP3A4 On Tumor Multidrug Resistance And Relative Modulating Mechanisms

CYP3A4 is a member of the cytochrome P450 superfamily of enzymes. CYP3A4 is involved in more than 50% of clinical drug metabolism and activation of many typical carcinogens. Its activity can be induced or inhibited by many substances. Understanding of the regulation of CYP3A4 will help guide the clinical use of drugs, to reduce or avoid the toxic side effects of drugs. In this thesis, an adriamycin (ADR)-resistant human erytholeukemia cell line K562/ADR was established by stepwise selection in vitro using ADR. Proteomics techniques were applied to investigate resistance-associated protein and their relation of CYP3A4. These data will be valuable for further study of mechanisms of MDR and may reveal a potential new function of CYP3A4.1. Establishment of adriamycin (ADR)-resistant human erytholeukemia cell line K562/ADR and proteome profiling of K562 and K562/ADR cell linesAdriamycin (ADR)-resistant human erytholeukemia cell line K562/ADR was established by stepwise selection in vitro using ADR. In the selection early stage, the cells were in the proliferation and deaths dynamic process. After about 6 months, the drug concentration was maintained at 1 mg/L, the drug resistant cells were stabilized. The cell morphology turned better; the proliferation became faster:it's no different with the mother cell K562. It was interesting that K562/ADR showed resistance not only to adriamycin but also to other chemicals, about 149-fold,134-fold,63.3-fold, increased resistance to adriamycin, vincristin, and imatinib, respectively, than the parental cell line K562.The adriamycin accumulation results results demonstrated that adriamycin was accumulated in K562 much more than in K562/ADR (p＜0.001) by 2 h incubated with adriamycin, about 2.925 mmol/L·mg (K562) and 1.522 mmol/L·mg (K562/ADR), respectively, suggesting P-gp pump might contribute to the decrease of accumulation of ADR in K562/ADR. But what is more interesting was that after 1 week, drug accumulation in K562/ADR cells increased dramatically without cell dysfunction, whereas all the K562 cells were dead. At this point, it suggested that enhanced efflux of adriamycin by upregulated p-gp was not the only factor in acquired resistance, at least in lone-term resistance to chemotherapeutic drugs. Adriamycin is mainly metabolized by CYP3A4, but CYP3A4 expression changes in K562 and K562/ADR were not the cause in acquired resistance as well, which suggests that there may exists other factors contribute to multidrug resistance.To achieve a global protein profile of these two cell lines, immobilized pH gradient isoelectric-focus and two-dimensional gel electrophoresis were applied. Proteins with a significant differential expression level between the two cell lines were excised, in-gel digested, and analyzed for identification with MALDI-TOF/TOF analysis and subsequent Swiss-Prot database search. These proteins expression changes were caused by cells exposure to chemotherapy drugs. The differentially expressed proteins were classified into groups based on their functions:calcium-binding proteins, chaperones, metabolic enzymes, proteins related to protein synthesis or DNA synthesis, and proteins related to signal transduction.2. Effect of ANXA1 regulation in K562 cells on ADR resistanceThe differential expression levels of spots 1 (ANXA1) were determined by Western blot analysis and immunochemical staining. ANXA1, a well-characterized member of the calcium-and phospholipid-binding protein family, because of its new role in apoptosis of cancer revealed recently. We reasonably hypothesized that ANXA1 may be a contributor to multidrug resistance in K562/ADR cells. For the purpose to evaluate this hypothesis, we knocked down ANXA1 expression using ANXA1-targeting artificial miRNA in K562 cells, and meanwhile, transfected the ANXA1 cDNA into K562/ADR cells. The sensitivity to ADR was decreased by ANXA1 ablation in K562 and vice versa. The functional validation showed that the downregulated ANXA1 expression contributes considerably to drug resistance in K562/ADR cells. Our studies suggest an important role for ANXA1 in contributing to the responses of tumor cells toward chemotherapy and revealed more clues to the study of the mechanisms of MDR.3. Studies on ANXA1 and CYP3A4 modulating mechanismWhile exposed to adriamycin, both K562/ADR/ANXA1 (ANXA1 upregulated K562/ADR cells) and K562/ADR cells were observed typically pronounced DNA ladder, but the DNA fragmentation in ANXA1 transfected K562/ADR cells was much stronger than in K562/ADR cells, which suggested that upregulated ANXA1 expression in K562/ADR cells could resensitize the cells to adriamycin via increased apoptosis. ANXA1 expression is higher in MCF-7/ADR cell lines than MCF-7 cells, which is different from hematological cancer cell lines. Both mRNA and protein levels of ANXA1, IL-6, and CYP3A4 in MCF-7 and MCF-7/ADR showed there might exists a signaling pathway among ANXA1, IL-6, and CYP3A4 in MCF-7 cells, while there is not in K562 cells. The expression levels of ANXA1 regulated the downstream CYP3A4 behavior, influenced drug metabolism, which may also be one of the causes of tumor multi-drug resistance. The multi-drug resistant cell mode established in the present work may be further applied in the studies of ANXAl, IL-6, and CYP3A4 signaling pathway, and also could be used to guide clinical use of drugs.4. Nuclear localization of ANXA1 correlates with advanced disease and peritoneal dissemination in patients with gastric carcinomaA total of 104 paired resected gastric adenocarcinoma and corresponding normal specimens were collected in this study. Expression of ANXA1 was examined by immunohistochemical staining. Both cytoplasmic and nuclear ANXA1 expression levels and their correlation with clinicopathological parameters were assessed. ANXA1 protein expression was positive in 72 of 104 (69.2%) normal tissues and 47 of 104 (45.2%) gastric adenocarcinoma tissues. ANXA1 staining was predominantly localized in the cytoplasm in all 72 ANXA1-positive normal specimens, while 12 ANXAl-positive gastric adenocarcinoma specimens showed positive nuclear staining. The positive nuclear staining correlated well with serosal invasion, peritoneal dissemination and TNM stage. Cases with positive nuclear staining presented more peritoneal dissemination (41.7%,5/12) than those with negative nuclear staining (8.7%,8/92; P= 0.007). A logistic regression model revealed that positive ANXA1 nuclear staining had an independent association with peritoneal dissemination (P=0.039; hazards ratio, 9.499; 95% confidence interval,1.159-77.815). These results indicated that ANXAl is expressed in both gastric adenocarcinoma and normal tissues. In gastric adenocarcinoma tissues ANXA1 is expressed both in cytoplasm and nucleus and its nuclear localization correlates with advanced disease stage and peritoneal dissemination.