| BACKGROUNDMultidrug resistance (MDR) is a major obstacle to successful chemotherapy. One of the well-known mechanisms of MDR is over-expression of P-glycoprotein (P-gp). Multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) are also involved in MDR development. They are plasma membrane proteins, which belong to a larger family of ATP-binding cassette proteins, and confer resistance to tumor cells by extruding many structurally and functionally unrelated hydrophobic anticancer drugs using the energy of ATP hydrolysis. Furthermore, alterations of the genes and the proteins related to the control of apoptosis, such as especially Bax and Bcl-2, contribute to MDR. Up to now, many strategies and methods have been investigated for their roles in reversing tumor MDR. As severe toxicity, side effects and lack of organ specificity, their clinical applications are extremely limited. In our previous studies we found that ultrasound could reverse MDR in tumor cells in vitro by downregulating the expression of MDR proteins on the cellular membrane and anti-apoptosis protein Bcl-2, and inducing apoptosis of MDR tumor cells. However, as many host factors can influence tumor MDR development, it is still unclear whether ultrasound could reverse tumor MDR in vivo. OBJECTIVEThe objectives of this study are described as follows: ⑴ To establish the subclone of adriamycin-resistant human hepatic carcinoma HepG2/ADM in vivo using nude mice. Morphological and MDR characteristics were evaluated. ⑵ To evaluate the effects of ultrasound on the biological characteristics of HepG2/ADM tumor in nude mice, and the possibility of ultrasound in the reversal of tumor MDR in vivo.⑶ To investigate the mechanisms of ultrasound in reversing MDR HepG2/ADM tumor in nude mice. Using molecular biological techniques, the gene expressions of MDR, Bcl-2 and Bax were assessed, MDR proteins and apoptosis proteins were measures, and activities of adenosinetriphosphatase (ATPase) were evaluated in vivo in nude mice respectively.METHODS⑴ A total of 20 athymic nude mice were used to establish human adriamycin-resistant HepG/ADM model. They were randomized to two groups: HepG2 group, in which 10 nude mice received a subcutaneous injection of 5 x 106/ml adriamycin-sensitive HepG2 cells; HepG2/ADM group, in which 10 received a subcutaneous injection of 5 x 106/ml adriamycin-resistant subclone HepG2/ADM. Biological characteristics, morphologic and histological changes were observed in both groups. MTT assay was performed to assess drug sensitivities, and Western blot was used to detect the expression of MDR proteins and apoptosis proteins in nude mice bearing either HepG2 or HepG2/ADM tumors respectively. SAS 8.0 statistical software was employed for statistical analysis. ⑵ A total of 40 nude mice bearing HepG2/ADM tumor were randomized to four groups as follows: control group (without any intervention); ADM group, in which 5 mg/kg ADM was injected via the tail vein; ultrasound (US) group, in which HepG2/ADM tumor was exposed to ultrasound (0.4302W/cm2, 0.8MHz, 5 minutes); ADM combined with US group, in which ADM and US were simultaneously performed. There were 10 nude mice in each group. Temperature distribution at the US-treated region was measured. Histological changes were examined in nude mice using light and electronic microscope. Tumor growth and the evaluation of reversal effects on MDR were performed in four groups. ANOVA test and student t test were used for the statistical analysis. ⑶ A total of 40 nude mice bearing HepG2/ADM tumor were randomized to control group (n=10), ADM group (n=20), US group (n=20), and ADM plus US group (n=20). The tumor specimens were harvested from the mice in each group 28 days after the interventions. P-gp, MRP, LRP, and apoptosis proteins including Bcl-2 and Bax were assessed by Western blot analysis and immunohistochemical staining respectively. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNNEL)...
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