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.

[A clinical analysis of liver disease patients with abnormal glucose metabolism].

OBJECTIVE: To study the clinical features of liver disease patients with abnormal glucose metabolism. METHODS: Liver functions and levels of FPG, PPG, FINS, PINS, FCP, and PCP in 91 chronic hepatitis B patients with abnormal glucose metabolism (62 had liver cirrhosis) were analyzed. RESULTS: (1) The incidence of hepatogenic impaired glucose tolerance (IGT) and of diabetes mellitus (DM) in hepatitis B patients with liver cirrhosis (20.53%; 24.11%) were higher than those without cirrhosis (3.82%; 1.64%; P<0.05, P<0.01). (2) There were no diabetic symptoms among any of the hepatogenic IGT and DM patients. 12 of 19 chronic hepatitis B patients with primary DM and 6 of 12 hepatitis B associated liver cirrhosis patients with primary DM had diabetic symptoms. (3) The levels of FPG and PPG in chronic hepatitis B patients with hepatogenic IGT and DM were lower than those in the patients with primary DM (P<0.05), but the levels of PINS and PCP in chronic hepatitis B patients with hepatogenic IGT and DM were higher than those in the patients with primary DM (P<0.05). (4) There were no differences in the levels of FPG and PPG between the hepatitis B associated liver cirrhosis patients with hepatogenic DM and those with primary DM (P<0.05). The levels of FINS, PINS, FCP, and PCP were higher in the hepatitis B associated liver cirrhosis patients with hepatogenic DM than those in the hepatitis B associated liver cirrhosis patients with primary DM (P<0.05). The levels of FPG and PPG in the hepatogenic DM patients were higher than those in the hepatogenic IGT patients (P<0.05), but their levels of FINS, PINS, FCP and PCP were lower than those in the hepatogenic IGT patients (P<0.05, P<0.01). CONCLUSION: Hepatogenic IGT and DM are always secondary in severe liver cirrhosis patients, who always showed no diabetic symptoms. The chronic hepatitis B patients with hepatogenic DM had increased insulin secretion, while the hepatitis B associated liver cirrhosis patients with hepatogenic DM had decreased insulin secretion.

[The changes of ROS and mitochondria membrane potential in HepG2 cells on the pressure of cisplatin].

OBJECTIVE: To explore the changes and significance of the level of reactive oxygen species (ROS) and mitochondria membrane potential (Delta Psi) in HepG2 cells under the stress of cisplatin (CDDP). METHODS: HepG2 cells were incubated with CDDP. The changes in the level of ROS were determined by a probe (2,7-dichloro fluorescein-ciactate, DCFH-DA) and the changes of Delta Psi were reflected as changes of intensities of fluorescence seen under a laser scan microscope using a probe (rhodamine-123). All these changes in cells at 0 h, 24 h, 48 h, 72 h, 120 h, 168 h were dynamically observed. RESULTS: The level of ROS was much higher after the CDDP treatment than the non-treated, and the increase lasted for 24 h and 48 h. Then it started to decrease at 72 h, gradually returning to normal level at 120 h. Under the selective pressure of CDDP, the fluorescence intensity of rhodamine-123 in HepG2 cells was decreasing at 24 h and 48 h, then gradually started to increase at 72 h. There were no such changes in the cells of the controls. CONCLUSION: The changes of ROS and Delta Psi in HepG2 cells under the pressure of CDDP suggest that the cells change themselves adapting to such pressures.

[Evaluation of the effects of dense endoscopic ligation for bleeding esophageal varices].

OBJECTIVE: To evaluate the short-term and long-term effects of dense endoscopic variceal ligation (DEVL) for bleeding esophageal varices. METHODS: Patients with acute or with a history of esophageal variceal bleeding underwent regular DEVLs with a 2-3 week interval between 2 sessions until their varices disappeared at the lower 5-6 cm part of the esophagus. Follow-up study and gastroscopy were performed at 3, 6 and 12 months after the final DEVL in all patients. The results at 3 months were classified as short-term effects and those after 6 months as long-term ones. RESULTS: 126 patients underwent DEVLs with 403 sessions and 3641 ligations; each patient was ligated with a mean of 3.2 sessions and at 28.9 points. The cure rate of acute variceal bleeding was 100.0%; short-term rate of variceal eradication was 94.4% and variceal rebleeding occurred in 3.9% patients. After a mean of 22.3 months follow-up period, the recurrence of esophageal varices was observed in 11.9% patients, but the variceal rebleeding rate was only 3.2% and no patients died from it. CONCLUSION: DEVL was very useful and effective in both short-term and long-term variceal eradication and prevention of variceal rebleeding.

Dynamical influence of Cordyceps sinensis on the activity of hepatic insulinase of experimental liver cirrhosis.

BACKGROUND: Cordeceps sinensis (CS) is a herb which can inhibit the liver fibrosis. Hyperinsulinemia is common in liver cirrhosis patients. The activity of insulin degrading enzyme could reflect the metabolism of insulin. This study was to detect the dynamical effects and mechanisms of CS on the activity of hepatic insulinase in CCl4 induced liver cirrhosis in rats. METHODS: Rats were randomly allocated into three groups: normal group, model group and CS group. The rats in the normal group were sacrificed at the beginning of experiment, and the other two groups were sacrificed randomly at the end of the third, sixth and ninth weeks. Blood and tissue specimens were taken. Biochemical assays were used to determine the changes of alanine transaminase (ALT), albumin levels in serum. And radioimmunological assays were used to determine the changes of hyaluronic acid (HA), insulin levels in serum and the activity of hepatic insulinase. RESULTS: No significant differences were seen in the serum levels of ALT, albumin, HA between the CS group and the model group at the third and sixth weeks (P>0.05). The serum levels of ALT, HA in the CS group were lower than those in the model group at the ninth week (P<0.05), but the serum level of albumin in the CS group was higher than that in the model group at the ninth week (P<0.05). No significant differences were observed in the serum levels of insulin and the activity of hepatic insulinase between the CS and model groups at the third week and the normal group (P>0.05). The serum levels of insulin in the CS and model groups at the sixth and ninth weeks were higher than those in the normal group (P<0.05). But the activity of hepatic insulinase was lower than that in the normal group (P<0.05 or P<0.01). No significant differences were found in the serum levels of insulin and the activity of hepatic insulinase between the CS and model groups at the third, sixth and ninth weeks (P>0.05). CONCLUSIONS: CS may decrease the damage to hepatocyte by CCl4, and inhibit hepatic fibrogenesis. Six weeks after CCl4 administration, the activity of hepatic insulinase began decreasing. CS could not inhibit the decrease of the activity of hepatic insulinase.

[Influence of Cordyceps sinensis on pancreatic islet beta cells in rats with experimental liver fibrogenesis].

OBJECTIVE: To investigate the effect of Cordyceps sinensis (CS) on pancreatic islet B cells of experimental hepatic fibrogenesis rats. METHODS: Rats were randomly allocated into three groups: normal group, model group and CS group. The rats in the latter two groups were administered with CCl(4) solution to induce liver fibrosis, the CS group was also treated with CS 10 days after the beginning of CCl(4) administration. Rats in normal group were sacrificed at the beginning of the experiment, while the rats in the other two groups were sacrificed randomly at the end of the third and sixth weeks. The rats' islets were isolated and cultured in vitro, then the basal insulin level of the islets and the serum level of insulin were determined by radioimmunological assay. RESULTS: It seemed no change that the levels of serum insulin and basal insulin between the model group and the normal group at the third week. But at the sixth week, both insulin levels in the model group were higher than those in the normal group (52.6 mU/L2.5 mU/L vs 23.7 mU/L 2.3 mU/L, q=13.01, p<0.05; 52.94muU/ml 13.12muU/ml vs 35.16muU/ml 5.64muU/ml, q=10.06, p<0.01). No significant change could be seen in the serum levels of insulin between the CS group and the model group at the third and sixth weeks. But the basal insulin levels in the CS group were apparently higher than those in the model group at the third and sixth weeks (156.63muU/ml 6.57muU/ml vs 39.64muU/ml 3.95muU/ml, q=66.94, p<0.001; 140.44muU/ml 38.53muU/ml vs 52.94muU/ml 13.12muU/ml, q=12.98, p<0.01). CONCLUSION: Cordyceps sinensis can increase the basal insulin level of the islets in CCl(4)-induced liver fibrosis rats.

[Effect of Rhein on the development of hepatic fibrosis in rats].

OBJECTIVE: To investigate the effect of rhein on the development of hepatic fibrosis. METHODS: The animal models were made with carbon tetrachloride (CCl(4)) mixed with vegetable oil (3/2, v/v), which was injected subcutaneously twice a week for 6 weeks, and with 5% ethanol for free drinking water. At the same time, Rhein was administrated at the dose of 25 mg/kg or 100 mg/kg once a day for 6 weeks. The changes of both biochemical markers, such as the levels of alanine aminotransferase (ALT), hyaluronic acid (HA), procollagen type III (PCIII) in serum and SOD, malondialdehyde (MDA) in liver, and related histopathological parametres were determined. RESULTS: Compared with the model group, there were three kinds of changes in the larger quantity of rhein treated group. (1) The levels of ALT, HA, PCIII in serum and MDA in liver homogenate were decreased significantly (from 150 U/L +/- 16 U/L to 78 U/L +/- 18 U/L, 321 microg/L +/- 97 microg/L to 217 microg/L +/- 75 microg/L, 31 microg/L +/- 14 microg/L to 16 microg/L +/- 6 microg/L and 3.67 nmol/mg +/- 0.68 nmol/mg to 1.88 nmol/mg +/- 0.34 nmol/mg, respectively, t > or 2.977, P<0.01). However the level of SOD in liver was increased (from 62.45 NU/mg +/- 8.74 NU/mg to 91.26 NU/mg +/- 14.04 NU/mg, t=4.453, P<0.01). (2) The expressions of transforming growth factor beta 1 (TGF-beta 1) and alpha-smooth muscle actin (alpha-SMA) in liver were markedly reduced (P<0.05 and P<0.01). (3) The collagen staining positive area was decreased and the grade of fibrosis was reduced significantly in liver (P<0.05 and P<0.01). CONCLUSION: Rhein can protect hepatocyte from injury and prevent the progress of hepatic fibrosis in rats, which may associate with that rhein plays a role in antioxidation, anti-inflammation, inhibiting the expression of TGF-beta1 and suppressing the activation of hepatic stellate cells (HSCs).