Damage Effects on Bacille Calmette-Guérin by Low-Frequency, Low-Intensity Ultrasound: A Pilot Study.

OBJECTIVES: To perform an in vitro experimental study of the possible damage effects on Bacille Calmette-Guérin (BCG) by low-frequency (42-kHz) ultrasound (US) irradiation at low spatially and temporally averaged intensities and different exposure times. METHODS: A 2-mL BCG suspension was added to the wells of a 24-well cell culture plate. Then the samples were randomly divided into 4 groups, each group including 3 wells, with group 1 as a control group and groups 2, 3, and 4, as US treatment groups. The samples for groups 2, 3, and 4 were irradiated with US at 0.13 W/cm(2) for 5 minutes, 0.13 W/cm(2) for 15 minutes, and 1.53 W/cm(2) for 15 minutes, respectively. After irradiation, the temperature, ratio of damage, and structure of the bacteria were examined. The cavitation effect of the device was detected by the passive cavitation detection method. RESULTS: After US irradiation at the different doses (intensity and exposure time), no significant temperature change was found in all sample suspensions. The ratio of bacterial damage tested by flow cytometry and the optical density of the suspensions as assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric method showed that the US-irradiated groups were significantly different from the control group. The BCG damage ratio reached 28% at the intensity of 1.53 W/cm(2). Transmission electron microscopic results showed that the bacterial structure of BCG could be destroyed by low-frequency, low-intensity US. CONCLUSIONS: Low-frequency, low-intensity US can cause acute injury to BCG, and the degree of injury is closely correlated with the US dose applied.

[Manufacture and application of laparoscopic extraperitoneal sigmoid colostomy].

OBJECTIVE: To investigate the safety and feasibility of laparoscopic extraperitoneal sigmoid colostomy. METHODS: Thirty-six patients with low rectal cancer undergoing laproscopic abdominoperineal resection from July 2011 to July 2012 were prospectively enrolled in the study and randomly divided into extraperitoneal colostomy group(EPC, n=18) and internal peritoneal colostomy group(IPC, n=18). Follow-up period was 4-16 (median, 7) months and postoperative complications were compared between two groups. RESULTS: One case in EPC group was converted to IPC because of poor blood supply of the proximal sigmoid, who was eliminated from the subsequent analysis. Compared with the IPC group, the surgery time was longer in EPC group [(25.3±8.5) min vs. (14.7±6.4) min], while the difference was not statistically significant(P>0.05). Each group had 1 case of stoma ischemia, who both received the colostomy reconstructive surgery. The incidence of stoma edema was significantly higher in EPC group[35.3%(6/17) vs. 0, P<0.05). The early postoperative complications rate did not significantly different between the two groups[58.8%(10/17) vs. 27.8%(5/18), P>0.05]. The late postoperative complications rate was 22.2%(4/18) in IPC group, including 1 case of stoma prolapse, 1 case of stoma stenosis and 2 cases of parastomal hernia. No later postoperative complication occurred in EPC group. CONCLUSION: Extraperitoneal sigmoid colostomy is an easy and safe procedure with lower late complications as compared to internal peritoneal sigmoid colostomy.

Focused-ultrasound termination of an early pregnancy in rhesus macaques (Macaca mulatta): a pilot study.

We explored the effectiveness, safety, and feasibility of focused ultrasound in terminating undesired pregnancy. A high-intensity focused ultrasound therapeutic unit was employed to terminate early pregnancies in rhesus macaques. B-mode ultrasound incorporated within the system was used to locate and study the gestational sacs of 6 rhesus macaques with gestation ages of 37 to 66 days, and varying modes of ultrasound exposure were adopted in the termination of the early pregnancies of the rhesus macaques. After focused ultrasound exposure, B-mode ultrasound of the gestational sacs showed significant lethal changes. Of the 6 rhesus macaques, 5 underwent complete abortions whereas 1 rhesus macaque underwent an incomplete abortion. The rhesus macaques resumed their menstrual cycles 50 days after focused-ultrasound treatment. The results suggested that focused ultrasound could be safe, feasible, and effective in terminating early pregnancies in rhesus macaques. As a novel physical method, it may be a promising ablation for a potentially clinical application.

[Changes of blood pressure and pulse rate and the microstructure of celiac ganglion in rabbits with damaged celiac ganglion induced by high intensity focused ultrasound and alcohol].

OBJECTIVE: To observe the changes of blood pressure (BP), pulse rate (PR) and the microstructure of celiac ganglion (CG) in rabbits with damaged CGs induced by alcohol and high intensity focused ultrasound. METHODS: Fourteen rabbits were randomly divided into two groups. The CGs of the rabbits in group A and group B were damaged by alcohol and high intensity focused ultrasound respectively. The changes of BP and PR 0, 1, 3, 5, and 10 minutes after the damage were recorded and compared. The microstructure changes of the damaged CGs were examined under optics microscope and electron microscope. RESULTS: Ganglionic morphology changes were obvious in both groups, with moved and concentrated karyons. In the CGs damaged by alcohol, the nucleolus still existed; some organelles could be identified; the myelination nerve fibre lost its myelin sheath or delaminated while the unmyelination nerve fibre exhibited vacuole formation. In the CGs damaged by high intensity focused ultrasound, all nucleolus disappeared, vacuole formed, intracellular membrane disappeared, axone locally necrotized. The BPs of the rabbits started to decrease three minutes after the alcohol treatment (P < 0.01), one minute after the high intensity focused ultrasound (P < 0.01). Significant differences of BP decline were observed between the two groups one minute after the CG damages. (P < 0.01). The PRs of the rabbits increased 5 minutes and 10 minutes after the high intensity focused ultrasound (P < 0.05, P < 0.01). CONCLUSION: Using high intensity focused ultrasound to damage CGs has more significant impacts on BPs and PRs than alcohol.

Effects of mifepristone on proliferation of human gastric adenocarcinoma cell line SGC-7901 in vitro.

AIM: To explore the effects of mifepristone, a progesterone receptor (PR) antagonist, on the proliferation of human gastric adenocarcinoma cell line SGC-7 901 in vitro and the possible mechanisms involved. METHODS: In situ hybridization was used to detect the expression of PR mRNA in SGC-7901 cells. After treatment with various concentrations of mifepristone (2.5, 5, 10, 20 micromol/L) at various time intervals, the ultrastructural changes, cell proliferation, cell-cycle phase distribution, and the expression of caspase-3 and Bcl-XL were analyzed using transmission electron microscopy (TEM), tetrazolium blue(MTT) assay, 3H-TdR incorporation, flow cytometry, and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Mifepristone markedly induced apoptosis and inhibited cell proliferation of PR- positive SGC-7901 cells revealed by TEM, MTT assay and 3H-TdR incorporation, in a dose- and time-dependent manner. The inhibitory rate was increased from 8.98% to 51.29%. Flow cytometric analysis showed mifepristone dose-dependently decreased cells in S and G2/M phases, increased cells in G0/G1 phase, reduced the proliferative index from 57.75% to 22.83%. In addition, mifepristone up-regulated the expression of caspase-3, and down- regulated the Bcl-XL expression, dose-dependently. CONCLUSION: Mifepristone effectively inhibited the proliferation of PR-positive human gastric adenocarcinoma cell line SGC-7901 in vitro through multiple mechanisms, and may be a beneficial agent against human adenocarcinoma.

Reversal of multidrug resistance in drug-resistant human gastric cancer cell line SGC7901/VCR by antiprogestin drug mifepristone.

AIM: To explore the reversal effect of mifepristone on multidrug resistance (MDR) in drug-resistant human gastric cancer cell line SGC7901/VCR and its mechanisms. METHODS: Expression of multidrug resistance-associated protein(MRP) was detected using reverse transcription-polymerase chain reaction(RT-PCR). Flow cytometry was used to assay the expression of P-glycoprotein(P-gp), Bcl-2, Bax, and the mean fluorescent intensity of intracellular rhodamine 123 in the cells. Meanwhile, the protein levels of Bcl-2 and Bax were also detected by Western blotting analysis. The sensitivity of cells to the anticancer agent, vincrimycin(VCR), and the intracellular [(3) H]VCR accumulation were determined by tetrazolium blue (MTT) assay and a liquid scintillation counter, respectively. RESULTS: Expression of MRP and P-gp in SGC7901/VCR cells was 6.04-and 8.37-fold higher as compared with its parental SGC7901 cells, respectively. After treatment with 1, 5, 10, and 20 micromol/L mifepristone, SGC7901/VCR cells showed a 1.34-, 2.29-, 3.11-, and 3.71-fold increase in the accumulation of intracellular VCR, a known substrate of MRP, and a 1.03-, 2.04-, 3.08-, and 3.68-fold increase in the retention of rhodamine 123, an indicator of P-gp function, respectively. MTT assay revealed that the resistance of SGC7901/VCR cells to VCR was 11.96-fold higher than that of its parental cells. The chemosensitivity of SGC7901/VCR cells to VCR was enhanced by 1.02-, 7.19-, 12.84-, and 21.17-fold after treatment with mifepristone at above-mentioned dose. After 96 h of incubation with mifepristone 10 micromol/L, a concentration close to plasma concentrations achievable in human, the expression of Bcl-2 protein was decreased to (9.21+/-0.65)% from (25.32+/-1.44)%, whereas the expression of Bax protein was increased to (19.69+/-1.13)% from (1.24+/-0.78)% (P<0.01). Additionally, the effects of mifepristone on the expression of Bcl-2 and Bax proteins in SGC7901/VCR cells were further demonstrated by Western blotting analysis. CONCLUSION: Mifepristone has potent reversal effect on MDR in SGC7901/VCR via inhibiting the function of MRP and P-gp, modulating the expression of Bcl-2 and Bax proteins, and enhancing the sensitivity to anticancer agent VCR.

Effects of mifepristone on invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 in vitro and in vivo.

AIM: To investigate the effects of mifepristone on the invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 and its mechanisms. METHODS: After incubation with various concentrations of mifepristone (5, 10, 20 micromol/L), the adhesion to artificial basement membrane, Matrigel, and the migration of MKN-45 cells were assayed using MTT assay and Transwell cell culture chambers, respectively. Enzyme- linked immunoabsorbent assay (ELISA) and flow cytometry were used to determine the expression of vascular endothelial growth factor (VEGF) and integrin beta3 in the cells. After subcutaneous transplantation of MKN-45 cells in nude mice, mifepristone (50 mg/kg.d) was administrated subcutaneously for 8 wk to assess its effects on tumor metastasis. Immunohistochemical analysis was used to detect the expression of VEGF and microvascular density (MVD) in xenografted tumors. RESULTS: Mifepristone dose-dependently inhibited the heterotypic adhesion to Matrigel of MKN-45 cells. The inhibition was accompanied by a significant down-regulation of integrin beta3 expression in the cells. After incubation with 5, 10, 20 micromol/L mifepristone, the number of migrated MKN-45 cells was 72+/-8, 50+/-6, 41+/-5 in experiment group, and 94+/-16 in control group (P<0.01). Meanwhile, secreted VEGF protein of MKN-45 cells in mifepristone-treated group (14.2+/-2.9, 8.9+/-3.1, 5.4+/-2.1 ng/g per liter) was significantly lower than that in control group (22.7+/-4.3 ng/g per liter, P<0.01). In vivo, mifepristone decreased the number of metastatic foci in lungs of nude mice and down-regulated the expression of VEGF and MVD in the xenograted tumors. CONCLUSION: Mifepristone can effectively inhibit the invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 in vitro and in vivo through inhibition of heterotypic adhesion to basement membrane, cell migration and angiogenesis.