The second-generation curcumin analogue RL71 elicits G2/M cell cycle arrest and apoptosis in canine osteosarcoma cells

Canine osteosarcoma is an aggressive cancer, comprising 85% of canine bone neoplasms. Current treatment practices of surgery and chemotherapy increase 1-year survival by only 45%. The curcumin analogue RL71, has demonstrated potent in vitro and in vivo efficacy in several models of human breast cancer through increased apoptosis and cell cycle arrest. Thus, the present study aimed to investigate efficacy of curcumin analogues in two canine osteosarcoma cell lines. Osteosarcoma cell viability was assessed using the sulforhodamine B assay and mechanisms of action were determined by analysing the levels of cell cycle and apoptotic regulatory proteins via Western blotting. Further evidence was obtained using flow cytometry to detect cell cycle distribution and the number of apoptotic cells. RL71 was the most potent curcumin analogue with EC₅₀ values of 0.64 ± 0.04 and 0.38 ± 0.009 μM (n = 3) in D-17 (commercial) and Gracie canine osteosarcoma cells, respectively. RL71 significantly increased the ratio of cleaved-caspase 3 to pro-caspase 3 and the level of apoptotic cells at the 2× and 5× EC₅₀ concentration (p < 0.001, n = 3). Furthermore, at the same concentration, RL71 significantly increased the number of cells in the G2/M phase. In conclusion, RL71 has potent cytotoxic activity in canine osteosarcoma cells triggering G2/M arrest and apoptosis at concentrations achievable in vivo. Future research should further investigate molecular mechanisms for these changes in other canine osteosarcoma cell lines prior to in vivo investigation.     

Effect of tetrandrine on radiosensitivity of nasopharyngeal carcinoma cells

AIM: To investigate the mechanism of the radiosensitizing effect of maximum non-cytotoxic doses of tetrandrine (Tet) on nasopharyngeal carcinoma cell lines CNE1 and CNE2.METHODS: The cells were treated with ma-ximum non-cytotoxic doses of Tet (for CNE1 cells at 1.5 μmol/L and for CNE2 cells at 1.8 μmol/L), irradiation at 4 Gy, or combination of irradiation and maximum non-cytotoxic doses of Tet. The cell cycle distribution was analyzed by flow cytometry. The protein levels of γ-H2AX, cleaved caspase-3, p-CDC25C, CDK1, p-CDK1, cyclin B1, ERK and p-ERK were determined by Western blot.RESULTS: The expression of γ-H2AX was increased in CNE1 cells and CNE2 cells after combined treatment with irradiation and maximum non-cytotoxic doses of Tet. The percentages of CNE1 cells and CNE2 cells at G₂/M phase in irradiation group were (18.09±0.42)% and (18.48±1.32)%, respectively, which were decreased to (15.88±1.04)% and (13.80±0.82)% in combined treatment group, respectively (P<0.05). Combined treatment enhanced the increase in the protein level of cleaved caspase-3 caused by irradiation. The protein levels of p-CDC25C and p-CDK1 were increased in a dose-dependent manner by Tet treatment (P<0.05), while the expression of CDK1 showed no difference among different doses of Tet treatments. The protein levels of p-CDC25C, p-CDK1 and CDK1 showed no difference after the treatment with maximum non-cytotoxic doses of Tet. The combined treatment with irradiation and the maximum non-cytotoxic doses of Tet decreased the protein levels of p-CDC25C and p-CDK1 (P<0.05), increased the expression of cyclin B1, and had no influence on the expression of CDK1 (P<0.05). The combined treatment resulted in an increase in the protein level of p-ERK1 (P<0.05).CONCLUSION: The maximum non-cytotoxic doses of Tet enhance the DNA damage and apoptosis in CNE1 cells and CNE2 cells caused by irradiation, and the mechanism might be associated with ending of G₂/M arrest via activation of ERK/CDC25C/CDK1/cyclin B1 pathways.     

Successful cardiopulmonary resuscitation in an adult horse following cardiovascular collapse on recovery from general anaesthesia in the Trendelenburg position

Equine anaesthesia is associated with a high risk of perioperative morbidity and mortality, particularly in the recovery period. Cardiovascular collapse and cardiopulmonary arrest (CPA) have been reported to account for one-third of anaesthesia-related perioperative deaths in the horse. This case report describes the successful cardiopulmonary resuscitation of a healthy adult Thoroughbred mare that developed cardiovascular collapse and subsequent CPA following positioning in the recovery box after general anaesthesia in the Trendelenburg position. Cardiopulmonary resuscitation (CPR) was instigated and included thoracic compressions, intermittent positive pressure ventilation and adrenaline administered via the intravenous and intra-tracheal routes. Return of spontaneous circulation (ROSC) was detected 5 min and 50 s after the start of CPR. A stable stance was achieved approximately 100 min after ROSC. The mare was discharged from the hospital 3 days later with no known complications. To the authors’ knowledge, this is the first case report documenting CPA following general anaesthesia in the Trendelenburg position in a horse, with subsequent successful CPR. The precise cause of the CPA is unknown but a number of plausible hypothesis are discussed including hypoventilation and a Bezold-Jarisch reflex.     

Expression of hypoxia-inducible factor 1 and neuroglobin in piglet cortex during deep hypothermic circulatory arrest

AIM: To observe the expression of hypoxia-inducible factor 1 (HIF-1) and neuroglobin (NGB) in piglet cortex during deep hypothermic circulatory arrest. METHODS: Wuzhishan piglets were randomly assigned to cardiopulmonary bypass group (CPB group), 40 min of circulatory arrest (CA) at 18 ℃ without cerebral perfusion (DHCA group) or with selective antegrade cerebral perfusion (SACP group). After 180 min of reperfusion, cortical tissue was harvested for determining HIF-1α and NGB expression by HE staining, Western blot and real-time PCR. RESULTS: Severer cerebral injury was observed in DHCA group than that in SACP group. After 180 min of reperfusion, HIF-1α protein and mRNA levels were significantly higher in DHCA group than those in CPB group (P<0.05). Accordingly, SACP animal had higher levels of HIF-1α protein and mRNA than those in DHCA group (P<0.05). Simultaneously, higher NGB protein and mRNA levels were found in DHCA group than those in CPB group after 180 min of reperfusion (P<0.05). The SACP animal had higher levels of NGB protein and mRNA than those in DHCA group (P<0.05). CONCLUSION: Up-regulation of HIF-1 and NGB are involved in the mechanism against cerebral injury resulting from DHCA in the cortex and possibly a part of cerebral protective effect of SACP.     

华蟾毒精对胃腺癌细胞MGC-803作用的研究

研究华蟾毒精（cinobufagin,CBG）体外对人胃腺癌细胞MGC-803的抑制作用,并初步探讨其抗肿瘤的作用机制。MTT法测定CBG对胃腺癌MGC-803细胞的生长抑制情况;采用光镜观察CBG对人胃腺癌细胞MGC-803形态的影响;流式细胞术检测CBG对MGC-803细胞的抑制作用、细胞周期及细胞早期凋亡的影响。一定浓度范围内CBG对MGC-803细胞的增殖有剂量、时间依赖性抑制作用;光镜下可见细胞形态明显发生变化,细胞变圆,分泌颗粒增多,细胞折光性下降,大部分细胞脱壁;流式细胞仪检测MGC-803细胞,G1期细胞减少,大量细胞停滞在G2/M期,凋亡细胞增多。CBG对MGC-803细胞的抑制作用与诱导其细胞阻滞及凋亡密切相关。