Ultrasound reverses multidrug resistance in human cancer cells by altering gene expression of ABC transporter proteins and Bax protein.

Multidrug resistance (MDR) is the major obstacle to successful chemotherapy of human malignancies and strategies for overcoming MDR phenomena are still unavailable to clinical use. Previous results showed that ultrasound (US) exposure could make MDR cancer cells become more sensitive to anticancer drugs, and the physical parameters of US exposure could adjust the uptake and retention of rhodamine 123 in MDR cells. In this study, we investigated the mechanisms of therapeutic ultrasound as a physical approach to overcoming MDR in a multidrug resistant human hepatocarcinoma cell line (HepG2/ADM). Our results demonstrated that the percentage of P-glycoprotein(+) (P-gp), multidrug resistance-associated protein(+) (MRP) and lung resistance-related protein(+) (LRP) cells was 96.97% ± 2.41%, 20.84% ± 3.12% and 1.16% ± 0.59% in HepG2/ADM cells, and 62.84% ± 3.42%, 10.26% ± 1.18% and 3.05% ± 0.37% in US-exposed HepG2/ADM cells, respectively. A significant decrease in the number of P-gp(+) and MRP(+) cells was observed between US-exposed HepG2/ADM and HepG2/ADM cells (p < 0.05). Using RT-PCR technique, we found that US could significantly downregulate the expression of P-glycoprotein (P-gp) and (MRP) at the mRNA level in HepG2/ADM cells. Compared with the control, the percentage of apoptotic cell death was significantly increased in HepG2/ADM after ultrasound exposure. Using immunocytochemistry, the percentage of Bcl-2(+) and Bax(+) cells was 21.7% and 4.1% in the control, and 18.46% and 8.1% in the US-exposed cells, respectively. The percentage of Bax(+) cells was significantly higher in US-exposed HepG2/ADM cells (p < 0.05), suggesting that US exposure could lead to cellular apoptosis in HepG2/ADM cells. It is concluded that US exposure could reverse MDR in HepG2/ADM cells via decreasing P-gp and MRP levels and their mRNA expressions and increasing expression of Bax protein. It may lead to the development of a novel strategy of using a targeted, noninvasive physical approach for the induction of MDR reversal in cancer cells.

Optimization of ultrasound-mediated in vitro reversal of multidrug resistance in human hepatocarcinoma cell line HepG2.

Previous studies have shown that ultrasound (US) could enhance cellular uptake and cytotoxicity of chemotherapeutic agents in drug-resistant cancer cells. The goal of this study was to investigate the optimization of physical parameters of US exposure for in vitro reversal of multidrug resistance (MDR) in human hepatocarcinoma cell line (HepG2). Using a constant total energy density (3.87 J/cm(2)) that could maintain cell viability at the 90% level, we exposed parent (HepG2) and MDR variant (HepG2/ADM) tumor cells to US in vitro to a variety of US frequency, exposure intensity and duty cycle. After US exposure, flow cytometry was performed to measure retention of rhodamine 123 (Rh123) in both HepG2 and HepG2/MDR cells. The results showed that US frequency and duty cycle (DC) could influence the intracellular retention of Rh123 in HepG2/ADM tumor cells; intensity and exposure duration appeared to be of little importance. At a constant total energy density of 3.87 J/cm(2), the optimal US parameters for in vitro reversal of MDR in HepG2/ADM tumor cells appear to be 0.8 MHz, 0.43 W/cm(2) and 60% DC, respectively. These findings support our hypothesis that varying the physical parameters would have an effect on efficiency of US-mediated reversal of MDR in cancer cells.

Cytotoxic effects and in vitro reversal of multidrug resistance by therapeutic ultrasound in human hepatocarcinoma cell line (HepG2).

Multidrug resistance (MDR) is one of the major obstacles to successful chemotherapy of human malignancies. Although many strategies have been explored to overcome MDR, none of them have been proven to be clinically useful until now. The aim of this study was to investigate whether a novel therapeutic ultrasound (US) approach would have useful effects on the reversal of MDR in cancer cells. Wild-type and MDR phenotype (HepG2/ADM) cells of the human hepatocarcinoma cell line HepG2 were exposed to 0.8 MHz US at an intensity of 0.43 W/cm(2) for a 9s exposure (total energy density: 3.87 J/cm(2)). After US exposure, cell number and viability were counted immediately, and flow cytometry was performed to measure retention of rhodamine 123 and adriamycin in HepG2 and HepG2/MDR cells. Both cell lines were then incubated in suspension with adriamycin, vincristine, etoposide, cisplatin and 5-fluorouracil, respectively, and the MTT assay was used to determine cytotoxicity. The results showed that US exposure could significantly increase the uptake of Rh123 and ADM by HepG2/ADM tumor cells. The resistant index for the chemotherapeutic drugs was significantly lower in the US-exposed HepG2/ADM cells than in those not exposed to US. It was therefore concluded that US exposure could enhance the sensitivity of HepG2/ADM tumor cells to these chemotherapeutic agents, and the functional and structural changes induced by previous US exposure in MDR tumor cells may be responsible for it. However, further study is needed to investigate the mechanism behind US-mediated reversal of MDR.

Development and characterization of multidrug resistant human hepatocarcinoma cell line in nude mice.

AIM: To establish a multidrug resistant (MDR) cell sub-line from the human hepatocarcinoma cell line (HepG2) in nude mice. METHODS: HepG2 cell cultures were incubated with increasing concentrations of adriamycin (ADM) to develop an ADM-resistant cell subline (HepG2/ADM) with cross-resistance to other chemotherapeutic agents. Twenty male athymic BALB/c-nu/nu mice were randomized into HepG2/nude and HepG2/ADM/nude groups (10 in each group). A cell suspension (either HepG2 or HepG2/ADM) was injected subcutaneously into mice in each group. Tumor growth was recorded, and animals were sacrificed 4-5 wk after cell implantation. Tumors were prepared for histology, and viable tumor was dispersed into a single-cell suspension. The IC50 values for a number of chemotherapeutic agents were determined by 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (MTT) assay. Rhodamine-123 retention/efflux and the level of resistance-associated proteins were determined by flow cytometry. The mRNA expression of mdr1, mrp and lrp genes was detected using reverse transcriptase polymerase chain reaction (RT-PCR) in HepG2/nude and HepG2/ADM/nude groups. RESULTS: The appearances of HepG2/nude cells were slightly different from those of HepG2/ADM/nude cells. Similar tumor growth curves were determined in both groups. A cross-resistance to ADM, vincristine, cisplatin and 5-fluorouracil was seen in HepG2/ADM/nude group. The levels of P-glycoprotein and multidrug resistance-associated proteins were significantly increased. The mRNA expression levels of mdr1, mrp and lrp were higher in HepG2/ADM/nude cells. CONCLUSION: ADM-resistant HepG2 subline in nude mice has a cross resistance to chemotherapeutic drugs. It may be used as an in vivo model to investigate the mechanisms of MDR, and explore the targeted approaches to overcoming MDR.

[Establishment of in vivo adriamycin-induced multidrug resistance models of subcutaneous and hepatic transplanted human liver cancer in nude mice].

BACKGROUND & OBJECTIVE: Multidrug resistance (MDR) phenotype is the obstacle of chemotherapy in tumors and inspired research interesting. This study was to establish multidrug resistance (MDR) models induced with adriamycin in vivo of subcutaneous or in situ hepatic transplanted human liver cancer in nude mice (BALB/C nu/nu), and explore the biological characteristics and mechanism of multidrug resistance, which can provide an ideal animal model for the basic,and clinical study of MDR. METHODS: After successful performing either subcutaneous or hepatic transplantation in nude mice with liver cancer cell line HepG2, adriamycin (ADM) was injected into abdominal cavity to induce multidrug resistance. The chemosensitivity of HepG2 and ADM-cultured HepG2 cells was determined by MTT assay. P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), and cell pump-efflux to Rhodamine 123 (R123) were determined by flow cytometry. The mRNA expression of P-gp, MRP, and LRP was assessed with reverse transcriptase polymerase chain reaction(RT-PCR). RESULTS: Most biological characteristics of either subcutaneous or hepatic transplanted HepG2 liver cancer in nude mice like the parent HepG2 liver cancer. Both of them showed significant multidrug resistance. Among them, the resistance index of ADM was 26.4 times in hepatic transplanted group, and 24.6 times in subcutaneous transplanted group higher than that of parent cell line HepG2. In subcutaneous,and hepatic transplanted ADM-cultured groups, P-gp [(77.3+/-2.1)%, and (78.1+/-1.9)%], MRP [(72.1+/-4.3)%, and (72.7+/-5.0)%], and LRP [(31.1+/-1.0)%, and (32.2+/-1.4)%] positive cells increased significantly, their mRNA positive expression increased also, and the cell pump-efflux to R123 strengthened. However, no significant difference of above data was observed between subcutaneous,and hepatic transplanted tumor group (P >0.05). CONCLUSION: Both MDR subcutaneous and hepatic HepG2 transplanted models induced by in vivo injection of adriamycin in nude mice have the same biological characteristics as parent HepG2 liver cancer. These 2 animal models could be used as in vivo MDR models to explore the mechanism of MDR and reversing therapeutic methods.

[Establishment of human multidrug-resistant hepatocellular carcinoma cell line (HepG2/Adm) and biological characteristics evaluation].

BACKGROUND & OBJECTIVE: Multidrug resistance (MDR) is considered to be the major obstacle for chemotherapy. In order to reverse tumor MDR in vitro, we designed this study to establish human multidrug-resistant hepatocellular carcinoma cell line (HepG2/Adm) and to investigate its biological characteristics. METHODS: An adriamycin-resistant human hepatocellular carcinoma cell subline (HepG2/Adm) was established in vitro using gradually increased concentration of adriamycin (ADM) in culture. Cell growth was measured and multidrug resistance to multianticancer agents was evaluated by MTT assay. Flow cytometry (FCM) was performed to determine cell cycle,and to assess the expression of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP),lung-related protein (LRP),and glutathione S-transferase (GST), respectively. MDR mRNA expression related to these proteins were determined with reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Compared to HepG2, HepG2/Adm had a prolonged doubling time of 30.1 hours. The number of cells in S-phase was significantly decreased (5.6+/-0.03)% in HepG2/Adm while those in G1 and G2-phase increased [(4.2+/-0.09)% and (1.5+/-0.08)%, respectively]. Furthermore, HepG2/Adm was resistant to many anti-tumor agents, and its IC(50) of ADM was 26 times higher than that of parent cell line HepG2. Significant overexpression of P-gp, MRP, and GST were detected. RT-PCR showed that mRNA expression of P-gp, MRP, LRP, and GST were significantly increased in HepG2/Adm. CONCLUSION: HepG2/Adm is human multidrug-resistant, and its resistance may be closely related to the overexpression of P-gp, MRP, and GST.

[Establishment of human hepatocellular carcinoma multidrug-resistance cell line (HepG2/Adm) and study apoptosis induced by low-frequency pulse ultrasound exposure].

OBJECTIVE: To establish human hepatocellular carcinoma multidrug-resistance cell line (HepG2/ADM) and to determine the effect of low-frequency pulse ultrasound (US) on MDR cells and investigate its mechanism. METHODS: Using gradual increase of adriamycin (ADM) concentrations in culture, an adriamycin-resistant human hepatocellular carcinoma cell sub line (HepG2/ADM) was established in vitro. HepG2/ADM cells were cultured in vitro and randomly divided into 4 groups: the control group (HepG2/ADM only), the group ADM by 1.0 mug/ml adriamycin for 1 h, the group US by low-frequency pulse ultrasound for 10 min, and the group US by low-frequency pulse ultrasound and treated with adriamycin simultaneously at same time. A sonication at a frequency of 0.8 MHz, was delivered with an intensity level of 0.5W/cm2, with continuous exposure of 10 min was applied. The ability of US to induce the apoptosis of MDR was evaluated by analyses of fluorescence microscopy, DNA fragmentation assay and flow cytometry assay. RESULTS: HepG2/Adm was resistant to many anti-tumor agents, and its IC50 of ADM was 26 times higher than that of parent cell line HepG2. Significant over expressions of P-gp, MRP, LRP and GSTs were detected. HepG2/ADM cells radiated by US had the typical characteristics of apoptosis. Compared with the control group (HepG2/ADM, 3.47%); the apoptosis rates were higher in US (12.23%). The therapeutic alliance of US with ADM for MDR cells, have a significant change in the ratio of apoptosis (18.81%, t=1.46 to 5.36, P<0.01). CONCLUSION: HepG2/ADM could have the biological characteristics of human multidrug-resistance cell line. The US sonication of 0.8 MHz could induce apoptosis of HepG2/ADM cell in vitro, and could act synergistically with Adriamycin.

[Reversal of multidrug resistance of human hepatocarcinoma HepG2/Adm cells by high intensity focused ultrasound].

BACKGROUND & OBJECTIVE: The effects of ultrasound have been shown on increasing cell membrane permeability and thereby might promote cellular uptake of cytotoxic drugs and enhance chemotoxicity. The aim of this study was to examine the reversal effects of high intensity focused ultrasound (HIFU) and co-administration with Adriamycin (ADM) on multidrug resistance (MDR) in HepG2/Adm cells, and to investigate its mechanism. METHODS: HepG2 cells and HepG2/Adm cells were divided into 4 groups: HepG2 (control group),HepG2 (treated with HIFU 5s),HepG2/Adm (non HIFU), and HepG2/Adm (treated with HIFU 5s). MTT assay was used to examine the chemosensitivity to ADM in HIFU-treated HepG2 and HepG2/Adm cell lines. Before and after the treatment with HIFU 5s, MDR1 gene expression was determined by immunocytochemistry assay. Flow cytometry was used to determine intracellular ADM concentration and the expression of P-gp on cell membranes. RESULTS: HIFU (0.8MHz, 460W/cm2) partly overcame the MDR of cell line HepG2/Adm and decreased the drug resistance. The relative reverse efficiencies to ADM, cisplatin (DDP), mytomycin (MMC), 5-fluorouracil (5-FU), and methotrexate (MTX) were 66.4%, 63.4%, 89.4%, 72.4%, and 75.0%, respectively. While, the concentration of ADM in HepG2/Adm cells was increased by 88%. The expression of P-glycoprotein (P-gp) protein in HepG2/Adm was down-regulated by 83.1%. CONCLUSION: HIFU can not only increase the ADM concentration in HepG2/Adm cells and decrease P-gp expression on cellular and nuclear membrane, but also partly overcome MDR of tumor cells.