Isotretinoin and Thalidomide Down-Regulate c-MYC Gene Expression and Modify Proteins Associated with Cancer in Hepatic Cells

Hepatocellular carcinoma (HCC) is the most common form of liver cancer. The number of cases is increasing and the trend for the next few years is not encouraging. HCC is usually detected in the advanced stages of the disease, and pharmacological therapies are not entirely effective. For this reason, it is necessary to search for new therapeutic options. The objective of this work was to evaluate the effect of the drugs isotretinoin and thalidomide on c-MYC expression and cancer-related proteins in an HCC cellular model. The expression of c-MYC was measured using RT-qPCR and western blot assays. In addition, luciferase activity assays were performed for the c-MYC promoters P1 and P2 using recombinant plasmids. Dose-response-time analyses were performed for isotretinoin or thalidomide in cells transfected with the c-MYC promoters. Finally, a proteome profile analysis of cells exposed to these two drugs was performed and the results were validated by western blot. We demonstrated that in HepG2 cells, isotretinoin and thalidomide reduced c-MYC mRNA expression levels, but this decrease in expression was linked to the regulation of P1 and P1-P2 c-MYC promoter activity in isotretinoin only. Thalidomide did not exert any effect on c-MYC promoters. Also, isotretinoin and thalidomide were capable of inducing and repressing proteins associated with cancer. In conclusion, isotretinoin and thalidomide down-regulate c-MYC mRNA expression and this is partially due to P1 or P2 promoter activity, suggesting that these drugs could be promising options for modulating the expression of oncogenes and tumor suppressor genes in HCC.     

p53蛋白与肝细胞癌临床病理特征及预后的关系

目的 探讨p53蛋白表达与肝细胞癌的临床病理特征及预后的关系,为术后综合治疗及判断预后提供依据。方法 采用免疫组织化学方法检测88例肝细胞癌组织中p53蛋白的表达,比较阳性患者和阴性患者的临床病理因素和术后累积复发率、术后累积生存率的差异。结果 p53蛋白表达阳性组1、2、3累积生存率分别为75．0％、54.4%、24．2％,阴性组相应的分别为84．3％、77．8％、56．1％。与阴性组相比,p53蛋白阳性组有较高的术前AFP浓度（P=0．005）、有较大的肿瘤最大直径（P=0．042）、肿瘤包膜不完整或无包膜者多见（P=0．023）。结论 p53蛋白是评价肝细胞癌恶性程度及预后的一个有价值的生物学指标。     

树突状细胞瘤内注射联合放疗对小鼠肾癌细胞因子的影响

目的研究树突状细胞(Dendritic cell,DC)瘤内注射联合放疗对小鼠肾癌治疗后脾细胞分泌细胞因子水平的变化。方法用Renca肾癌细胞(2.5×106/只)制作小鼠皮下移植瘤模型,接受肿瘤局部放射治疗,7Gy 6脉伏电子线/次,并在肿瘤部位直接注射未负载肿瘤抗原的DC,1×106/次,第28 d处死小鼠。检测肿瘤生长速度和瘤体重量。ELISA检测小鼠脾细胞IL-2I、FN-γI、L-4、IL-10和TNF-α分泌水平。结果与肿瘤组比较,DC联合放疗组小鼠的肿瘤生长速度明显缓慢,肿瘤体积明显减小,脾细胞分泌IL-2、IFN-γ和IL-4的能力显著提高。结论瘤内树突状细胞注射联合放疗能够有效地抑制BALB/c小鼠肾癌的生长。     

Hepatoprotective Effect of Millettia dielsiana: In Vitro and In Silico Study

In silico docking studies of 50 selected compounds from Millettia dielsiana Harms ex Diels (family Leguminosae) were docked into the binding pocket of the PI3K/mTOR protein. In there, compounds trans−3−O-p-hydroxycinnamoyl ursolic acid (1) and 5,7,4′−trihydroxyisoflavone 7−O−β−D−apiofuranosyl−(1→6)−β−D−glucopyranoside (2) are predicted to be very promising inhibitors against PI3K/mTOR. They direct their cytotoxic activity against Hepatocellular carcinoma with binding affinity (BA) values, the pulling work spent to the co-crystallized ligand from the binding site of PI3K/mTOR (W and F_max), and the non-equilibrium binding free energy (∆G_neq^Jar) as BA values = −9.237 and −9.083 kcal/mol, W = 83.5 ± 10.6 kcal/mol with F_max = 336.2 ± 45.3 pN and 126.6 ± 21.7 kcal/mol with F_max = 430.3 ± 84.0 pN, and ∆G_neq^Jar = −69.86074 and −101.2317 kcal/mol, respectively. In molecular dynamic simulation, the RMSD value of the PI3K/mTOR complex with compounds (1 and 2) was in the range of 0.3 nm to the end of the simulation. Therefore, the compounds (1 and 2) are predicted to be very promising inhibitors against PI3K/mTOR. The crude extract, ethyl acetate fraction and compounds (1 and 2) from Millettia dielsiana exhibited moderate to potent in vitro cytotoxicity on Hepatocellular carcinoma cell line with IC₅₀ values of 81.2 µg/mL, 60.4 µg/mL, 23.1 μM, and 16.3 μM, respectively, and showed relatively potent to potent in vitro antioxidant activity on mouse hepatocytes with ED₅₀ values of 24.4 µg/mL, 19.3 µg/mL, 30.7 μM, and 20.5 μM, respectively. In conclusion, Millettia dielsiana and compounds (1 and 2) are predicted to have very promising cytotoxic activity against Hepatocellular carcinoma and have a hepatoprotective effect.     

无标记定量蛋白质组学分析AMACR过表达对肝癌细胞生物学行为的影响

研究α-甲酰基辅酶A消旋酶(AMACR)过表达对肝癌细胞生物学行为的影响及其分子机制.首先建立AMACR稳定过表达细胞株,然后提取AMACR过表达细胞株的全蛋白,进行无标记定量蛋白质组学研究,最后对鉴定结果进行生物信息学分析和结果验证,共筛选出138种差异表达蛋白.这些差异表达蛋白主要参与代谢加工、细胞加工等,证明AMACR的过表达对于肝癌细胞的生物学行为影响巨大.IPA分析发现,这些差异表达蛋白主要参与了ERK1/2信号通路和NF-κB信号通路.以上结果说明,AMACR的过表达通过调节ERK1/2和NF-κB信号通路等手段改变肝癌细胞的生物学行为.     

微孔板杂交技术检测乙肝病毒X基因及其临床应用价值

目的 建立微孔板杂交技术检测乙肝病毒X基因的方法,研究其临床应用价值.方法 在微孔板上包被HBV X基因片段的捕获探针;PCR扩增被检标本中目的片段,其引物用Bio-Ⅱ-dUTP修饰,在微孔板上杂交后用链霉亲和素碱性磷酸酶系统显色.结果 该方法检测HBV X基因灵敏,特异;乙肝后肝细胞癌患者X基因阳性率明显高于急、慢性乙肝和肝纤维化组.结论 检测HBV X基因对HCC具有辅助诊断价值.     

From Chemistry to Translational Medicine: The Application of Proteomics to Cancer Biomarker Discovery and Diagnosis

The study of complex protein mixtures and their interactions in cells and tissues has been difficult due to the tedious process involved in their characterization and analysis. The recent emergence of fast‐evolving and state‐of‐the‐art proteomics methodologies has provided a rapid and scalable platform for understanding the comprehensive proteome profiles from complex whole tissues or cells of various biological sources. Therefore, proteomics has been increasingly valuable to examine real‐time changes in protein expression of various tissues or body fluids from patients with various diseases, especially cancer, resulting in the identification of clinically useful biomarkers for diagnosis, prognosis and disease staging. In this review, we focus on potential biomarkers for (1) Helicobacter pylori‐associated gastric cancer, (2) hepatocellular carcinoma (HCC), and (3) renal cell carcinoma (RCC). In addition to the conventional gel‐based proteomics (1‐D or 2‐D gels), we have utilized a more advanced proteomic approach by incorporating stable isotope dimethyl labelling and shotgun proteomics strategy in combination with nanoliquid chromatography and tandem mass spectrometry (nanoLC‐MS/MS) to better characterize the biomarkers in several cancer tissues. By establishing a high‐throughput proteomics platform based on multiple reaction monitoring (MRM), we have successfully detected and analyzed potential protein markers at low concentrations in various normal and tumor tissues. This platform not only highlights the utility of proteomics for biomarker discovery but also can be uniquely applied to disease‐oriented translational medicine for diagnosis of diverse types of cancers and other diseases.     

Phytochemical profiling,molecular docking,and in vitro anti-hepatocellular carcinoid bioactivity of Suaeda vermiculata extracts

The ATP-binding cassette is the major class of transporters responsible for the efflux of chemotherapeutic agents from cancer cells, resulting in treatment failures of cancer’s patients. Suaeda vermiculata Forssk. ex. J. F. Gmel. is traditionally known for its liver protective activity. The LC-MS based chemical profilings of the sequentially partitioned sub-extracts obtained from the alcoholic extract of S. vermiculata using n-hexane, chloroform, ethyl acetate, and n-butanol as fractionating solvents, identified a total of thirty six compounds. These sub-extracts were evaluated for their anti-hepatocarcinoma activity against the sensitive HepG2 and doxorubicin (DOX)-resistant, HepG-2/ADR cell lines. A mixture of doxorubicin and sub-extracts at 20 μg/ml doses were also tested for their anti-hepatocarcinoma activity. The exhibited IC₅₀ values for the chloroform, ethyl acetate, n-hexane, and n-butanol sub-extracts, and the doxorubicin against HepG2, and HepG-2/ADR cell lines were found at 64.5, 66.8, 81.25, 125, 1.3 μg/ml, and 110.1, 91.82, 138.2, 265.7, 4.77 μg/ml levels, respectively. However, the treatment of resistant cells with 20 μg/ml of different sub-extracts in combination with the doxorubicin showed significant improvements in the doxorubicin activity against the resistant cells, and the IC₅₀ values for DOX + chloroform, DOX + ethyl acetate, DOX + n-hexane, and DOX + n-butanol against resistant cells, were at 1.77, 2.05, 2.66, and 2.71 μg/ml levels, respectively. The IC₅₀ values exhibited 2.69x, 2.33x, 1.79x and 1.76x-folds reversal of the sensitivity in the resistant cancer cell lines. The molecular docking studies of the compounds identified in the LC-MS chemical profilings, against three ATP-binding cassette proteins i.e., ABCB1, ABCC1, and ABCG2, showed that flavonoids as the major class of compounds responsible for reversal of the resistant cells sensitivities. The predicted binding affinity for the flavonoids against the above mentioned three ATP-binding cassette proteins’ are in the ranges of ～?8 to ?11 kcal/mol. Our results clearly indicate that the presence of flavonoids, as the major class of compounds in the S. vermiculata is responsible for the chemosensitization of the resistant HCC-cell lines. Moreover, the structures, 21 (5‐O‐methyl visamminol), 22 (N-trans-feruloyl tyramine), 27 (atractylenolide-III), and 32 (ginsenoside-Rh2) were also identified among the potential ATP-binding cassette’s modulators during the current study. These observations put the S. vermiculata in perspective with the traditionally claimed liver protective efficacy of the plant.     

在肝细胞癌诊断中的肿瘤标志物

原发性肝细胞癌(HCC)是最常见的恶性肿瘤之一，其死亡率高居各种恶性肿瘤的第二位，肿瘤标志物检查对本病的早期诊断起着至关重要的作用。文章综述了HCC的一些较为敏感而特异的指标及蛋白质组学技术在确定肿瘤标志物方面的研究进展。     

柴胡逆转肝细胞癌多药耐药作用与相关机制的研究

选用对VCR天然耐药肝癌细胞株Bel-7402为对象来研究柴胡逆转MDR效果与相关的机制,以寻找一种新型的具有多药耐药逆转活性的中药.结果表明,柴胡具有逆转肝细胞癌多药耐药作用,并与抗癌药物VCR有协同作用.柴胡对人肝癌Bel-7402细胞株具有多药耐药逆转作用,提高了耐药细胞内的VCR含量,增加了VCR对细胞G2期阻滞作用,抑制了P-170糖蛋白的表达,抑制MDR1/mRNA的表达,提高了TopoαmRNA水平.