Combinational treatment of 5-fluorouracil and casticin induces apoptosis in mouse leukemia WEHI-3 cells in vitro.

Leukemia is one of the major diseases causing cancer-related deaths in the young population, and its cure rate is unsatisfying with side effects on patients. Fluorouracil (5-FU) is currently used as an anticancer drug for leukemia patients. Casticin, a natural polymethoxyflavone, exerts anticancer activity against many human cancer cell lines in vitro, but no other reports show 5-FU combined with casticin increased the mouse leukemia cell apoptosis in vitro. Herein, the antileukemia activity of 5-FU combined with casticin in WEHI-3 mouse leukemia cells was investigated in vitro. Treatment of two-drug combination had a higher decrease in cell viability and a higher increase in apoptotic cell death, the level of DNA condensation, and the length of comet tail than that of 5-FU or casticin treatment alone in WEHI-3 cells. In addition, the two-drug combination has a greater production rate of reactive oxygen species but a lower level of Ca2+ release and mitochondrial membrane potential (ΔΨm ) than that of 5-FU alone. Combined drugs also induced higher caspase-3 and caspase-8 activities than that of casticin alone and higher caspase-9 activity than that of 5-FU or casticin alone at 48 hours treatment. Furthermore, 5-FU combined with casticin has a higher expression of Cu/Zn superoxide dismutase (SOD [Cu/Zn]) and lower catalase than that of 5-FU or casticin treatment alone. The combined treatment has higher levels of Bax, Endo G, and cytochrome C of proapoptotic proteins than that of casticin alone and induced lower levels of B-cell lymphoma 2 (BCL-2) and BCL-X of antiapoptotic proteins than that of 5-FU or casticin only. Furthermore, the combined treatment had a higher expression of cleaved poly (ADP-ribose) polymerase (PARP) than that of casticin only. Based on these findings, we may suggest that 5-FU combined with casticin treatment increased apoptotic cell death in WEHI-3 mouse leukemia cells that may undergo mitochondria and caspases signaling pathways in vitro.

Casticin Induces DNA Damage and Affects DNA Repair Associated Protein Expression in Human Lung Cancer A549 Cells (Running Title: Casticin Induces DNA Damage in Lung Cancer Cells).

Casticin was obtained from natural plants, and it has been shown to exert biological functions; however, no report concerns the induction of DNA damage and repair in human lung cancer cells. The objective of this study was to investigate the effects and molecular mechanism of casticin on DNA damage and repair in human lung cancer A549 cells. Cell viability was determined by flow cytometric assay. The DNA damage was evaluated by 4',6-diamidino-2-phenylindole (DAPI) staining and electrophoresis which included comet assay and DNA gel electrophoresis. The protein levels associated with DNA damage and repair were analyzed by western blotting. The expression and translocation of p-H2A.X were observed by confocal laser microscopy. Casticin reduced total viable cell number and induced DNA condensation, fragmentation, and damage in A549 cells. Furthermore, casticin increased p-ATM at 6 h and increased p-ATR and BRCA1 at 6-24 h treatment but decreased p-ATM at 24-48 h, as well as decreased p-ATR and BRCA1 at 48 h. Furthermore, casticin decreased p-p53 at 6-24 h but increased at 48 h. Casticin increased p-H2A.X and MDC1 at 6-48 h treatment. In addition, casticin increased PARP (cleavage) at 6, 24, and 48 h treatment, DNA-PKcs and MGMT at 48 h in A549 cells. Casticin induced the expressions and nuclear translocation of p-H2AX in A549 cells by confocal laser microscopy. Casticin reduced cell number through DNA damage and condensation in human lung cancer A549 cells.

Gefitinib and curcumin-loaded nanoparticles enhance cell apoptosis in human oral cancer SAS cells in vitro and inhibit SAS cell xenografted tumor in vivo.

Curcumin (Cur), a natural product, has been shown to have anti-tumor activities in many human cancer cells. Gefitinib (Gef) is a clinical drug for cancer patients. However, there is no available information to show whether Gef/Cur nanoparticles (NPs) increased cell apoptosis and anti-tumor effects on xenograft mice model in vivo. In this study, γ-polyglutamic acid-coated nanoparticles loaded with Gef and Cur (γ-PGA-Gef/Cur NPs) were developed and its physicochemical properties and antitumor effects were investigated in vitro and in vivo. The γ-PGA-Gef/Cur NPs showed 548.5 ±â€¯93.7 nm in diameter and -40.3 ±â€¯3.87 mV on surface charge. The loading efficiencies of Gef and Cur were 89.5 and 100%, respectively. γ-PGA-Gef/Cur NPs could be internalized into SAS cells and significantly decreased total cell viability of SAS cells. Western blotting results indicated that both free Gef/Cur and γ-PGA-Gef/Cur NPs induced apoptotic cell death via caspase- and mitochondria-dependent pathways. In vivo studies indicated that treatments of PLGA NPs, free Gef/Cur, and γ-PGA-Gef/Cur NPs did not significantly affect appearances and bodyweights of mice. But the γ-PGA-Gef/Cur NPs significantly suppressed tumor size when comparing to free Gef/Cur-treated group. The nanoparticles developed in this study may be used as a potential therapy for oral cancer.

The Ethanol Crude Extraction of Cyperus Rotundus Regulates Apoptosis-associated Gene Expression in HeLa Human Cervical Carcinoma Cells In Vitro.

BACKGROUND/AIM: Cervical cancer is considered poorly chemo-sensitive in women and its treatment remains unsatisfactory. Cyperus rotundus is used in Chinese medicine as a therapeutic agent for women's disease. The effects and molecular mechanisms of the ethanol extraction of C. rotundus (CRE) on cervical cancer remain unclear. We aimed to explore the mechanisms and genetic influence of CRE on cervical cancer. MATERIALS AND METHODS: HeLa, human cervical cancer cells were treated with various doses of CRE and changes in cell morphology and cell viability were assessed using microscopy and flow cytometry. Finally, we performed a microarray analysis to scan related genes. RESULTS: The treatment of CRE on HeLa cells caused morphological changes and induced chromatin condensation. DNA microarray analysis showed that CRE led to up-regulation of 449 genes and down-regulation of 484 genes, which were classified in several interaction pathways. CONCLUSION: CRE changed HeLa cell morphology and induced gene expression which associated with apoptosis and cell-cycle arrest. These results provide important information at the transcription level for targeting treatments of human cervical cancer.

Maslinic Acid Enhances Immune Responses in Leukemic Mice Through Macrophage Phagocytosis and Natural Killer Cell Activities In Vivo.

BACKGROUND/AIM: Maslinic acid (MA), a pentacyclic triterpene extracted from wax-like coatings of olives, has been shown to reduce cancer cell number through induction of autophagy and apoptosis in many human cancer cells including human leukemia HL-60 cells. In the present study, we investigated whether or not MA affects immune responses in a leukemia mouse model. MATERIALS AND METHODS: WEHI-3 cells were intraperitonealIy (i.p.) injected into normal BALB/c mice to develop leukemia. Mice were then treated by i.p. injection with MA at different doses (0, 8, 16 and 32 mg/kg) for 2 weeks. After treatment, all animals were weighed and blood, liver and spleen tissues were weighed. Blood or spleen both were used for determination of cell markers or phagocytosis, natural killer (NK) cell activities and T- and B-cell proliferation, respectively, by using a flow cytometric assay. RESULTS: MA did not significantly affect body, liver, and spleen weights. However, MA increased markers of T-cells (at 16 mg/kg treatment) and monocytes (at 32 mg/kg treatment), but reduced B-cell markers (at 8 mg/kg treatment); MA did not significantly affect cell marker of macrophages. Furthermore, MA increased phagocytosis by macrophages from peripheral blood mononuclear cells and peritoneal cavity at 32 mg/kg treatment and increased NK cell activity at target cell:splenocyte ratio of 25:1 but did not affect B- and T-cell proliferation. CONCLUSION: MA increased immune responses by enhancing macrophage phagocytosis and NK cell activities in leukemic mice.

Phenethyl Isothiocyanate Induces Apoptotic Cell Death Through the Mitochondria-dependent Pathway in Gefitinib-resistant NCI-H460 Human Lung Cancer Cells In Vitro.

BACKGROUND/AIM: Some lung cancer patients treated with gefitinib develop resistance to this drug resulting in unsatisfactory treatment outcomes. Phenethyl isothiocyanate (PEITC), present in our common cruciferous vegetables, exhibits anticancer activities in many human cancer cell lines. Currently, there is no available information on the possible modification of gefitinib resistance of lung cancer in vitro by PEITC. Thus, the effects of PEITC on gefitinib resistant lung cancer NCI-H460 cells were investigated in vitro. MATERIALS AND METHODS: The total cell viability, apoptotic cell death, production of reactive oxygen species (ROS) and Ca2+, levels of mitochondria membrane potential (ΔΨm) and caspase-3, -8 and -9 activities were measured by flow cytometry assay. PEITC induced chromatin condensation was examined by DAPI staining. RESULTS: PEITC-induced cell morphological changes, decreased total viable cell number and induced apoptotic cell death in NCI-H460 and NCI-H460/G cells. PEITC decreased ROS production in NCI-H460 cells, but increased production in NCI-H460/G cells. PEITC increased Ca2+ production, decreased the levels of ΔΨm and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting was used to examine the effect of apoptotic cell death associated protein expression in NCI-H460 NCI-H460/G cells after exposure to PEITC. Results showed that PEITC increased expression of cleaved caspase-3, PARP, GADD153, Endo G and pro-apoptotic protein Bax in NCI-H460/G cells. CONCLUSION: Based on these results, we suggest that PEITC induces apoptotic cell death via the caspase- and mitochondria-dependent pathway in NCI-H460/G cells.

Effects of Chai-Qin-Cheng-Qi decoction () on acute pancreatitis-associated lung injury in mice with acute necrotizing pancreatitis.

OBJECTIVE: To investigate the effects of Chai-Qin-Cheng-Qi Decoction (, CQCQD) on acute pancreatitis-associated lung injury in mice with acute necrotizing pancreatitis (ANP). METHODS: Thirty healthy mice were randomly divided into three groups: an ANP group (ANP+placebo, n=10); a treatment group (ANP+CQCQD, n=10); and a control group (normal mice+placebo, n=10). ANP was induced by intraperitoneal injection with 8% L-arginine (4 µg/kg), and the control group was injected with normal saline. The treatment group received CQCQD (20 mL/kg), and the ANP and control groups received placebo (sucrose and starch) intragastrically at 2 h intervals. After the third intragastric administration, blood, pancreatic tissues and right lung tissues were collected for measurement of serum interleukin-6 (IL-6) and interleukin-10 (IL-10) by enzyme-linked immunosorbent assay, and the expression of heat shock protein 70 (HSP70) in lung tissue was determined by Western blot analysis. Pathological changes of pancreatic tissue and lung tissue were examined. RESULTS: Serum IL-6 was significantly higher in the ANP group compared with the control and the treatment groups (1589.63±377.28 vs. 927.46±210.42 pg/mL, P<0.05, and 1589.63±377.28 vs. 1107.73±351.62 pg/mL, P<0.05, respectively). The IL-10 concentration was significantly lower in the ANP group compared with the treatment group (920.64±101.68 vs. 1177.84±201.72 pg/mL, P<0.05), but no signififi cant difference was found between the ANP and control groups and between the treatment and control groups. The expression level of HSP70 in the ANP and control groups was signififi cantly lower than in the treatment group (0.93±0.03 vs. 1.42±0.21, P<0.01, and 0.81±0.09 vs. 1.42±0.21, P<0.01, respectively). There was no signififi cant difference in HSP70 levels between the ANP and control groups. Histological scores of pancreatic and lung tissue were significantly decreased in the treatment group compared with the ANP groups (4.50±0.54 vs. 6.20±1.65, P<0.05, and 3.00±0.63 vs. 3.87±0.83, P<0.05, respectively). CONCLUSIONS: The incidence of acute pancreatitisassociated lung injury in ANP mice correlates positively with serum IL-6 concentration. CQCQD may inhibit IL-6 induction and increase IL-10 concentration and HSP70 expression, effectively reducing lung injury.

[Effect of chai qin cheng qi decoction on serum CCK-8 and calcium overload of pancreatic acinar in mice with acute pancreatitis].

OBJECTIVE: To investigate the effect of Chai Qin Cheng Qi decoction (CQCQD) on serum cholecystokinin-8 (CCK-8) and calcium overload of pancreatic acinar cells in acute pancreatitis (AP) mice. METHODS: Twenty four mice were randomly divided into control group, AP group, CQCQD group and siRNA group, each comprising 6 mice. AP mouse model was induced by intraperitoneal injection of 8% L-arginine in a dose of 4 g/kg. The AP mice in the CQCQD group were fed with 0.4 mL/100 g of Chai Qin Cheng Qi solution once every two hours. The AP mice in the siRNA group were injected intraperitoneally with CCK-siRNA in a dose of 0.88 mg/kg. The changes of serum CCK-8 and calcium concentrations in the pancreatic acinar cells and pancreatic pathology were observed 6 hours after the interventions. RESULTS: The serum CCK-8 [(3764.3 +/- 369.2) ng/mL], calcium fluorescence intensity (34.8 +/- 27.1) of pancreatic acinar cells and pancreas pathology scores (6.2 +/- 1.1) of the AP mice were significantly greater (P < 0.05) than those in the control group [(1253.5 +/- 39.5) ng/mL, 5.2 +/- 2.3, 2.8 +/- 0.4], CQCQD group [(1230.5 +/- 46.1) ng/mL, 9.6 +/- 1.6, 3.8 +/- 0.8, 4.1 +/- 0.5] and siRNA group[(1702.3 +/- 598.3) ng/mL, 7.6 +/- 2.0]. Serum CCK-8 was positively correlated with intracellular calcium concentrations (r = 0.793, P = 0.021) in pancreatic acinar cells and pancreas pathology scores (r = 0.847, P = 0.000). CONCLUSION: Acute pancreatitis in mice induced by L-arginine is associated with calcium overload in pancreatic acinar cells induced by increased serum CCK-8. CQCQD can reduce serum levels of CCK-8, alleviate calcium overload in pancreatic acinar cells, and reduce pancreas pathological changes in AP mice.

[Histological characteristics of the prostate in men who receive re-TURP for benign prostatic hyperplasia and their clinical significance].

OBJECTIVE: To investigate the pathohistological characteristics of the prostate tissues in patients who receive a second TURP and to evaluate their clinical significance. METHODS: We collected surgical specimens from 50 cases of TURP (the control group) and another 50 cases of re-TURP (the re-TURP group), detected the expressions of CD34, vascular endothelial growth factor (VEGF) and androgen receptor (AR) in the prostate tissues by immunohistochemistry (S-P), and determined microvessel density (MVD) and the expressions of VEGF and AR. We performed statistical analyses on the results obtained from the specimens of the control group as well as from those of the first and second operations of the re-TURP group. RESULTS: VEGF and AR expressed in all the specimens. The expressions of VEGF and AR and MVD were significantly higher in the re-TURP group than in the controls (P < 0.05), but showed no significant differences between the first and second operations in the re-TURP group (P > 0.05). Positive correlations were found between the expressions of AR and VEGF, VEGF and MVD, and AR and MVD (r = 0.650, 0.705 and 0.525, P < 0.05). CONCLUSION: Increased AR, VEGF and MVD in the prostatic tissues may be one of the important causes of recurrence of BPH after TURP, and could be considered as the risk factors for postoperative recurrence and targeted indicators for preventive measures.

[Influence of integrated traditional Chinese and Western medicine therapy on serum resistin levels in patients with severe acute pancreatitis: a randomized controlled trial].

BACKGROUND: Resistin level is high in patients with severe acute pancreatitis (SAP), and resistin is expected to be a new marker for evaluating the severity of acute pancreatitis. OBJECTIVE: To explore the influence of integrated traditional Chinese and Western medicine therapy on serum resistin levels in SAP patients. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: Twenty-eight SAP patients meeting inclusion criteria from Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University were included, and the patients were randomly divided into treatment group and placebo group. There were 13 patients in the treatment group and 15 patients in the placebo group. Patients in the treatment group were given traditional Chinese herbal medicine in addition to the conventional treatment. Patients in the placebo group were given placebo in addition to the conventional treatment. MAIN OUTCOME MEASURES: The serum resistin levels on admission, and days 1, 3, 5, and 7 after the admission were detected. RESULTS: The serum resistin levels on admission in all the patients were higher than normal level, and there was no significant difference between the two groups (P>0.05). On days 1, 3, 5, and 7 after admission, the resistin levels in the treatment group were (3.29 + or - 1.66) microg/L, (3.71 + or - 1.05) microg/L, (3.08 + or - 1.47) microg/L and (3.62 + or - 1.67) microg/L, and in the control group (5.16 + or - 1.93) microg/L, (5.07 + or - 1.53) microg/L, (4.88 + or - 1.47) microg/L and (5.12 + or - 1.48) microg/L, respectively. The resistin levels were lower in the treatment group than in the control group (P<0.05). CONCLUSION: Serum resistin level in SAP patients can be decreased by integrated traditional Chinese medicine and Western medicine therapy.