Giganteaside D induces ROS-mediated apoptosis in human hepatocellular carcinoma cells through the MAPK pathway

Purpose

Every year, almost one million individuals are diagnosed with hepatocellular carcinoma (HCC) worldwide and more than 690,000 patients die of it. At present, most therapeutic anti-HCC agents are not effective, which is due to the appearance of chemo-resistance and/or toxic side effects. Therefore, it is imperative to find novel more effective anti-HCC agents. Here, we evaluated the effect of giganteaside D (GD), an oleanolic acid saponin from P. scabiosaefolia, on the growth and apoptosis of HCC cells.

Methods and results

Using MTT and clonogenic assays, we found that GD exhibited a significant growth inhibitory effect on the HCC-derived cell lines HepG2 and Bel-7402. In addition, we found that GD induced mitochondria-mediated apoptosis in these HCC-derived cells, as indicated by a decreased mitochondrial potential, activation of Caspase-9 and Caspase-3, cleavage of PARP and release of Cytochrome C from the mitochondria. Besides, we found that GD stimulated the generation of reactive oxygen species (ROS) and that blockage of ROS attenuated the GD-induced mitochondria-mediated apoptosis. Additionally, we found that GD treatment led to a decrease in phosphorylated Erk (p-Erk) and triggered the generation of p-JNK, both components of the mitogen-activated protein kinase (MAPK) signaling pathway. Inhibition of Erk or JNK by specific inhibitors or siRNAs augmented or attenuated the cytotoxic and apoptotic effects of GD.

Conclusions

From our results we conclude that GD can induce ROS-mediated apoptosis in HCC-derived cells through the MAPK pathway. This observation may open up avenues to explore the future use of GD as a HCC chemotherapeutic agent.

     

维生素C对顺铂杀伤卵巢癌细胞的影响

目的:探讨维生素C 是否能影响顺铂杀伤卵巢癌细胞株OVCAR5、SKOV3、CAOV3,并对其作用机制进行初探。方法:使用梯度浓度维生素C及顺铂处理卵巢癌细胞株,显微镜下观测细胞的形态学变化;使用ATP-TCA方法检测细胞生长抑制情况。选择对维生素C敏感的卵巢癌细胞株。氧化敏感探针DCFH-DA标记细胞 ,检测不同浓度维生素C处理不同时间后细胞内过氧化氢(H2O2 )水平。结果:三株卵巢癌细胞维生素C处理的敏感度不同,但总体呈现出随着维生素C浓度的增加,细胞受到抑制作用增加,维生素C对于顺铂杀伤细胞的作用具有辅助加强效应,该作用呈现剂量依赖效应。其中较低浓度维生素C处理OVCAR5细胞15min各组胞内H2O2 水平相比对照组稍高,当处理浓度为5 000μmol/L时细胞H2O2 水平明显升高,达到164%。培养30min后,荧光强度显示细胞内H2O2 水平比15min时升高更明显。不同浓度组分别达到对照组的94%、106%、122%、188%,培养60min后检测荧光强度发现各给药组H2O2 水平均已低于对照组。12h后检测到细胞内的H2O2 水平处于更低水平,各组均未达到对照组的50%。结论:较高浓度维生素C能增强顺铂杀伤卵巢癌细胞株的能力,并呈现一定的剂量依赖性。该结果可能由于维生素 C通过自氧化机制产生过氧化氢促进卵巢癌细胞凋亡。     

Two BRM promoter polymorphisms predict poor survival in patients with hepatocellular carcinoma

Polymorphisms in the promoter of the BRM gene, a critical subunit of the chromatin remodeling SWI/SNF complex, have previously been implicated in risk and prognosis in Caucasian‐predominant lung, head and neck, esophageal, and pancreatic cancers, and in hepatocellular cancers in Asians. We investigated the role of these polymorphisms in hepatocellular carcinoma (HCC) risk and prognosis. HCC cases were recruited in a comprehensive cancer center while the matched controls were recruited from family practice units from the same catchment area. For risk analyses, unconditional logistic regression analyses were performed in HCC patients and matched healthy controls. Overall survival analyses were performed using Cox proportional hazard models, Kaplan‐Meier curves, and log‐rank tests. In 266 HCC cases and 536 controls, no association between either BRM promoter polymorphism (BRM‐741 or BRM‐1321) and risk of HCC was identified (P > 0.10 for all comparisons). There was significant worsening of overall survival as the number of variant alleles increased: BRM‐741 per variant allele adjusted hazards ratio (aHR) 5.77, 95% confidence interval (CI) 2.89‐11.54 and BRM‐1321 per variant allele aHR 4.09, 95%CI 2.22‐7.51. The effects of these two polymorphisms were at least additive, where individuals who were double homozygotes for the variant alleles had a 45‐fold increase in risk of death when compared to those who were double wild‐type for the two polymorphisms. Two BRM promoter polymorphisms were strongly associated with HCC prognosis but were not associated with increased HCC susceptibility. The association was strongest in double homozygotes for the allele variants.     

血常规相关指标与食管癌患者治疗效果和预后的关系

炎症在恶性肿瘤的发生、进展、转移中扮演着重要的角色,在食管癌病程进展中的作用也逐渐引起人们重视。近年来食管癌患者血常规检查中相关指标与其治疗疗效和预后的关系逐步成为研究热点。现就EC患者血常规检查中一些指标与其治疗疗效和预后的关系进行概述,并主要介绍血常规中炎症复合标记物与EC患者治疗疗效和预后关系的研究进展。     

生物反应调节剂（BRM－102）冲剂诱变和抗诱变作用的研究

采用微核法对生物反应调节剂冲剂（ＢＲＭ－１０２）的诱变和抑制诱变作用进行了研究。结果显示ＢＲＭ－１０２提取液各种浓度组与阴性对照组统计学处理无意义，表明该冲剂尚未有诱发正常人淋巴细胞微核率升高的作用，不致引起体细胞遗传损伤；对诱变剂丝裂霉素Ｃ诱发正常人淋已细胞微核率有明显的抑制作用。     

Normalizing GDNF expression in the spinal cord alleviates cutaneous hyperalgesia but not ongoing pain in a rat model of bone cancer pain

Bone cancer pain (BCP) is the most common complication in patients with bone cancer. Glial cell line‐derived neurotrophic factor (GDNF) is believed to be involved in chronic pain conditions. In this article, the expression and roles of GDNF were studied in a rat model of BCP induced by tibia injection of Walker 256 rat mammary gland carcinoma cells. Significant mechanical and thermal hyperalgesia and ongoing pain were observed beginning as early as day 5 post injection. The expression level of GDNF protein examined on day 16 after tibia injection was decreased in the L3 dorsal root ganglion (DRG) and lumbar spinal cord, but not in other spinal levels or the anterior cingulate cortex. Phosphorylation of Ret, the receptor for GDNF family ligands, was also decreased. Furthermore, normalizing GDNF expression with lentiviral vector constructs in the spinal cord significantly reduced mechanical and thermal hyperalgesia, spinal glial activation, and pERK induction induced by tibia injection, but did not affect ongoing pain. Together these findings provide new evidence for the use of GDNF as a therapeutic treatment for bone cancer pain states.     

miR-221/222及其靶基因在妊娠期肝内胆汁淤积症发病机制中作用的研究

目的：研究微小核糖核酸（MicroRNA,miRNA）221/222及其靶基因在正常孕妇和妊娠期肝内胆汁淤积症（intrahepatic cholestasis of pregnancy,ICP）孕妇胎盘中表达的差异,并通过细胞实验过表达方法研究miR-221/222对靶基因表达影响,探讨其在ICP发病机制中的作用。方法：搜集2015年9月到2016年3月在重庆医科大学附属第二医院妇产科行剖宫产分娩的正常孕妇和ICP孕妇的胎盘组织各20例,直接提取miRNA,采用实时荧光定量逆转录PCR（qRT-PCR）检测miR-221/222在2组胎盘组织中的表达情况;预测miR-221/222的靶基因是顶端钠依赖性胆酸转运体（apical sodium-dependent bile acid transporter,ASBT,又称SLC10A2）;Western blot检测ASBT在ICP和正常胎盘组织中的表达情况;用Lipofectaine2000脂质体包裹合成的miR-221/222 mimic转染正常滋养细胞HTR-8;qRT-PCR检测miR-221/222表达水平;蛋白质印迹法检测转染后ASBT蛋白表达。结果：①经过内参U6的校正,miR-221在ICP胎盘表达量为1.066±0.044,正常胎盘表达量为0.053±0.009;miR-222在ICP胎盘表达量为13.724±4.355,正常胎盘表达量为0.833±0.189。可见miR-221/222在ICP胎盘中的表达明显上调（P<0.05）。②SLC10A2在ICP组中的表达量为0.328±0.102,在正常组中的表达量为0.604±0.119,其在ICP组的表达较正常组的表达下调（P=0.000）。③经过内参ACTIN校正,ASBT在转染miR-221组细胞内表达量为0.338±0.064,对照组表达量为0.583±0.040;ASBT在转染miR-222组细胞内表达量为0.371±0.024,对照组表达量为0.624±0.031,可见ASBT在转染组中较对照组表达下调。结论：miR-221/222在ICP胎盘组织中表达升高,而其预测靶基因ASBT的表达则下降;细胞实验中,miR-221/222转染组靶基因ASBT表达下降。两者之间可能存在负调控关系。可能由此影响了孕妇胆汁酸在肠道的重吸收及在肝脏的转运而导致妊娠期肝内胆汁淤积。     

Intravenous injection of oncolytic picornavirus SVV-001 prolongs animal survival in a panel of primary tumor–based orthotopic xenograft mouse models of pediatric glioma

Background

Seneca Valley virus (SVV-001) is a nonpathogenic oncolytic virus that can be systemically administered and can pass through the blood–brain barrier. We examined its therapeutic efficacy and the mechanism of tumor cell infection in pediatric malignant gliomas.

Methods

In vitro antitumor activities were examined in primary cultures, preformed neurospheres, and self-renewing glioma cells derived from 6 patient tumor orthotopic xenograft mouse models (1 anaplastic astrocytoma and 5 GBM). In vivo therapeutic efficacy was examined by systemic treatment of preformed xenografts in 3 permissive and 2 resistant models. The functional role of sialic acid in mediating SVV-001 infection was investigated using neuraminidase and lectins that cleave or competitively bind to linkage-specific sialic acids.

Results

SVV-001 at a multiplicity of infection of 0.5 to 25 replicated in and effectively killed primary cultures, preformed neurospheres, and self-renewing stemlike single glioma cells derived from 4 of the 6 glioma models in vitro. A single i.v. injection of SVV-001 (5 × 10¹² viral particles/kg) led to the infection of orthotopic xenografts without harming normal mouse brain cells, resulting in significantly prolonged survival in all 3 permissive and 1 resistant mouse models (P < .05). Treatment with neuraminidase and competitive binding using lectins specific for α2,3-linked and/or α2,6-linked sialic acid significantly suppressed SVV-001 infectivity (P < .01).

Conclusion

SVV-001 possesses strong antitumor activity against pediatric malignant gliomas and utilizes α2,3-linked and α2,6-linked sialic acids as mediators of tumor cell infection. Our findings support the consideration of SVV-001 for clinical trials in children with malignant glioma.     

p78/MCRS1 forms a complex with centrosomal protein Nde1 and is essential for cell viability

The centrosome, an organelle that functions as the major microtubule-organizing center, plays an essential role in the formation of the mitotic spindle and guiding accurate chromosome segregation. Centrosome aberrations are frequently associated with various forms of human cancers and it is thought that defects in this organelle contribute to genomic instability and malignant transformation. We recently identified and characterized a centrosome-localized protein complex that is comprised of Su48 and Nde1. Disruption of the normal function of these proteins leads to abnormal cell division. To extend our understanding of how this protein complex operates, we sought to identify Nde1-interacting molecules by the yeast two-hybrid screening method. Here, we demonstrate that both Nde1 and Su48 can associate with p78/MCRS1, a protein implicated in cancer development. We found that, whereas the majority of p78 localizes to the nucleus as reported in earlier studies, a fraction of the p78 protein can be detected in the centrosome. Moreover, we determined that a region containing the forkhead-associated domain of p78 is involved in association with Nde1 and Su48, as well as in centrosomal localization. We also provide evidence that the association between p78 and Nde1 is regulated by phosphorylation on Nde1. Furthermore, abrogation of the endogenous p78 function by small interfering RNA knockdown causes cell death and a modest delay in mitosis. These results indicate that a subset of the p78 proteins comprises a component of the centrosome and that p78 is essential for cell viability.