Synthesis and biological evaluation of furoxan-based nitric oxide-releasing derivatives of glycyrrhetinic acid as anti-hepatocellular carcinoma agents

A series of novel furoxan-based nitric oxide (NO)-releasing derivatives of glycyrrhetinic acid (GA) were designed, synthesized, and evaluated for their in vitro cytotoxicity against human hepatocellular carcinoma (HCC) and non-tumor liver cells. Five furoxan/GA hybrids, 7b-d, 7f, and 7g, displayed potent cytotoxicity against HCC cells (IC(50): 0.25-1.10 μM against BEL-7402 cells and 1.32-6.78 μM against HepG2 cells), but had a little effect on the growth of LO2 cells, indicating that these compounds had selective cytotoxicity against HCC cells. Furthermore, these compounds produced high concentrations of NO in HCC cells, but low in LO2 cells and treatment with hemoglobin partially reduced the cytotoxicity of the hybrid in HCC cells. Apparently, the high concentrations of NO produced by NO donor moieties and the bioactivity of GA synergistically contribute to the cytotoxicity, but the NO is a major player against HCC cells in vitro. Potentially, our findings may aid in the design of new chemotherapeutic reagents for the intervention of human HCC at clinic.     

百里香酚的抗肿瘤作用

目的：观察百里香酚对体外培养的肝癌细胞的抑制作用。方法：体外培养人肝癌细胞（Bel-7402）,采用MTT法、AO/EB荧光染色法观察百里香酚对人肝癌细胞Bel-7402的作用。结果：百里香酚可显著抑制Bel-7402细胞的生长;经百里香酚作用后,肝癌细胞在显微镜形态明显改变。结论：百里香酚能抑制肝癌Bel-7402细胞生长。     

Anticancer drugs are synergistic with freezing in induction of apoptosis in HCC cells

Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from −15 to −80 °C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.     

Effects of Gekko sulfated polysaccharide on the proliferation and differentiation of hepatic cancer cell line

Gekko swinhonis Gūenther has been used as an anti-cancer drug in traditional Chinese medicine. Gekko sulfated polysaccharide (Gepsin) was investigated for its activity in hepatocellular carcinoma. Hepatocarcinoma cell line (Bel-7402) and liver cell line (L-02) were exposed to Gepsin (100 microg/ml and 10 microg/ml). Gepsin did not suppress the proliferation and viability of normal liver L-02 cells, but strongly inhibited the proliferation of hepatocarcinoma Bel-7402 cells. Gepsin did not induce the apoptosis of Bel-7402 cells, but blocked cells in G2/M phase. Treated with Gepsin, Bel-7402 cells showed ultrastructural features of differentiation. AFP secretion decreased while ALB secretion increased markedly on Gepsin-treated cells. The data show that Gepsin suppressed the proliferation and induced differentiation of hepatocarcinoma, but the toxicity to normal liver cells was negligible.     

ONTD induces apoptosis of human hepatoma Bel-7402 cells via a MAPK-dependent mitochondrial pathway and the depletion of intracellular glutathione

3-Oxo-29-noroleana-1,9(11),12-trien-2,20-dicarbonitrile (ONTD) is a novel synthetic derivative of glycyrrhetinic acid (GA), which has the ability to inhibit the proliferation of human hepatocellular carcinoma (HCC) cells. However, the mechanisms by which ONTD exerts its inhibitory effects remain elusive. The present study was conducted to investigate the cytotoxicity of ONTD in Bel-7402 cells and its molecular mechanisms. We found that ONTD depleted intracellular GSH, increased the level of ROS, and consequently induced mitochondrial permeability transition (MPT) leading to the release of apoptosis-inducing factor (AIF) and cytochrome c (Cyt c) to the cytosol. Mitochondrial alteration and subsequent apoptotic cell death in ONTD-treated Bel-7402 cells could be blocked by addition of exogenous antioxidants N-acetylcystein (NAC), GSH and the MTP inhibitor cyclosporin A (CsA). In addition, ONTD activated the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPK) but not extracellular signal-regulated protein kinases (ERK 1/2). When the cells were exposed to SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), the deregulation of the expression of apoptotic proteins was attenuated. Furthermore, 40 mg/kg ONTD significantly reduced tumor weight (?70.62%, p < 0.01) in the H22 tumor-bearing mouse model in vivo. Taken together, these findings provide the first experimental evidence supporting that ONTD could induce apoptosis of Bel-7402 cells via MAPK-mediated mitochondrial pathway and ONTD has the potential to be developed as a therapeutic agent for the treatment of HCC.     

Antiproliferative activity and apoptosis inducing effects of nitric oxide donating derivatives of evodiamine

The first series of nitric oxide donating derivatives of evodiamine were designed and prepared. NO releasing ability of all target derivatives was evaluated in BGC-823, Bel-7402 and L-02 cells. The cytotoxicity was evaluated against three human tumor cell lines (Bel-7402, A549 and BGC-823) and normal human liver cells L-02. The nitrate derivatives 11a and 11b only exhibited moderate activity and furoxan-based derivatives 13a–c, 14a and 14b showed promising activity. 13c showed good cytotoxic selectivity between tumor and normal liver cells and was further investigated for its apoptotic properties on human hepatocarcinoma Bel-7402 cells. The molecular mode of action revealed that 13c caused cell-cycle arrest at S phase and induced apoptosis in Bel-7402 cells through mitochondria-related caspase-dependent pathways.     

13,28-Epoxy triterpenoid saponins from Ardisia japonica selectively inhibit proliferation of liver cancer cells without affecting normal liver cells

Twenty 13,28-epoxy and related triterpenoid saponins from Ardisia japonica were evaluated for their anti-proliferative activity on human liver cancer cells and normal liver cells. Eight saponins selectively inhibited the growth of liver cancer Bel-7402 and HepG-2 cells without affecting the survival of normal liver HL-7702 cells. The structure-activity relationship analyses indicated that the 13,28-epoxy, 16α-hydroxy, and C-30 methyl moieties in the sapogenin parts and the glycosyl moiety consisting from tetra- to hepta-saccharide units are important for this activity. Among the active saponins, ardisianoside B (2) and 3β-O-β-d-glucopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)]-α-l-arabinopyranosyl-13β,28-epoxy-16α-hydroxyoleanane (3) showed the most potent anti-proliferative activity against Bel-7402 cells in a dose- and time-dependent manner. The selective anti-proliferative activity is attributed to the different cellular responses (CDKs and cyclins levels, cell cycle arrest and apoptosis) between tumor and normal liver cells. Exposure to 2 and 3 selectively led to cell cycle arrest and apoptosis in Bel-7402 cells together with the increased pro-apoptotic caspase-8 and the decreased anti-apoptotic Cdc25A levels.     

rhddADAM15抑制肝癌细胞Bel-7402增殖的作用研究

ADAMl5属于跨膜蛋白ADAM家族中的一员,在乳腺癌、宫颈癌、卵巢癌等多种实体瘤中均发现其表达量提高。ADAMl5在降解细胞外基质、介导细胞的黏附、细胞间信号转导及在肿瘤发展进程中起到重要作用。因其去整合素区域含有RGD序列,ADAMl5可与多种整合素相互作用。在前期工作中,实验组利用大肠杆菌表达系统表达了重组人ADAMl5去整合素区域蛋白,记作rhddADAMl5。该研究将进一步针对rhddADAMl5抑制肝癌细胞Bel-7402增殖的机理进行分析及探讨。SRB法显示,rhddADAMl5可抑制Bel-7402细胞增殖并呈剂量依赖性,IC50为1．14gmol／L;利用DAPI核染发现,rhddADAMl5可显著诱导Bel-7402细胞凋亡;流式细胞仪分析发现,rhddAD—AMl5浓度为6gmol／L时,（87．44±7．25）％的细胞发生凋亡;PI单染分析细胞周期表明,rhddADAMl5作用后,部分Bel-7402细胞周期被阻滞于S期及G2／M期,并呈剂量依赖性,4gmol／LrhddADAMl5处理后G0／Gl期含量下降约14％;Westernblot分析显示,rhddADAMl5可下调Bel-7402细胞周期蛋白CDC26的表达,抑制CDC2-TyrM的去磷酸化,引起G2／M期的阻滞。     

Hydrogen sulfide releasing enmein-type diterpenoid derivatives as apoptosis inducers through mitochondria-related pathways

In this study, we combined two enemin-type diterpenoid derivatives with two well-established hydrogen sulfide moieties via ester or different anhydride linkers, to search apoptosis inducing drug candidate capable of hydrogen sulfide generating. Therefore, a series of hybrids were synthesized and superior antiproliferative efficacy accompanied with enhanced selectivity was observed under extensive pharmacological evaluations. A standard methylene blue (MB⁺) method was applied to measure the capacity for the hydrogen sulfide generation of all the target derivatives. One particular molecule A1, which contained α-thioctic acid moiety for hydrogen sulfide donating, manifested more potent antiproliferative activity. It exerted inhibitory effects against Bel-7402, SGC-7901 and A549 cell lines with IC₅₀ values of 2.16, 5.07 and 6.98 μM respectively. While it exacted relatively low effects over human normal cell lines L-02 and PBMC with IC₅₀ values of 15.81 μM for the prior and 14.15 μM for the latter, and displayed better selectivity index (SI) than parent diterpenoids. A high dosage of H₂S release was also recorded. Hence, A1 was most suitable for mechanistic exploration on account of both safety and efficacy. The ensuing biological assays revealed central role of apoptosis in A1’s mode of action for antiproliferative efficacy, which led to further confirmation of G1 phase cell cycle arrest, mitochondria membrane potential collapse and apoptotic activation in Bel-7402 cells. Further western blot assay on intrinsic mitochondria pathway unlocked intricate interplay among a series of apoptotic related proteins in which Bax, caspase-3 and cytochrome c went through up-regulation, while Bcl-2, Bcl-xL and procaspase-3 undergone down-regulation. In a nutshell, a hydrogen sulfide releasing hybrid A1 was synthesized and antiproliferative evaluation identified it to be a worthy drug candidate for future in depth study.     

Tertiary sulphonamide derivatives as dual acting small molecules that inhibit LSD1 and suppress tubulin polymerisation against liver cancer

A series of tertiary sulphonamide derivatives were synthesised and evaluated for their antiproliferative activity against liver cancer cell lines (SNU-475, HepG-2, and Bel-7402). Among these tertiary sulphonamides, compound 17a displayed the best anti-liver cancer activity against Bel-7402 cells with an IC₅₀ value of 0.32 μM. Compound 17a could effectively inhibit tubulin polymerisation with an IC₅₀ value of 1.27 μM. Meanwhile, it selectively suppressed LSD1 with an IC₅₀ value of 63 nM. It also concentration-dependently inhibited migration against Bel-7402 cells. Importantly, tertiary sulphonamide 17a exhibited the potent antitumor activity in vivo. All these findings revealed that compound 17a might be a tertiary sulphonamide-based dual inhibitor of tubulin polymerisation and LSD1 to treat liver cancer.     

细胞内F-actin的聚合与解聚对肝癌Bel-7402细胞的影响

目的：为探讨癌细胞内F—actin的解聚与聚合对癌细胞的形态、迁移、侵入的影响。方法：利用激光共聚焦显微镜对贴附培养的人肝癌：Bel-7402细胞形态及其细胞内F-actin进行观察;使用流式细胞仪对贴附的Bel一7402细胞及其脱落细胞与Cyt—B处理后Bel-7402细胞内F-actin的含量进行分析。结果：Bel-7402细胞在培养的过程中,癌细胞形态伸展,出现侵入性生长,细胞内F-actin聚合形成粗大的贯通细胞内的F-actin束,F-acfin含量增高;癌细胞在生长过程中,常出现重叠生长,细胞变圆,F_actin解聚变短,F-acfin小体增高,细胞有脱落的趋势,其脱落细胞内的F-actin含量低于贴附细胞。结论：人肝癌：Bel-7402细胞内F-actin的聚合可增加癌细胞的贴附和侵入性：细胞内F-actin的解聚,及Gactin重新聚合形成F-aefin小体可影响到癌细胞脱落及迁移。     

Perifosine induces cell cycle arrest and apoptosis in human hepatocellular carcinoma cell lines by blockade of Akt phosphorylation

Hepatocellular carcinoma (HCC) is one of the most common solid cancers, representing the third cause of cancer-related death among cirrhotic patients. Treatment of advanced HCC has become a very active area of research. Perifosine, a new synthetic alkylphospholipid Akt inhibitor, has shown anti-tumor activity by inhibition of Akt phosphorylation. In this study, the effect of perifosine on the cell proliferation and apoptosis in hepatoma cells has been investigated. Cell growth inhibition was detected by MTT assay, cell cycle was analyzed by flow cytometry, AnnexinV-FITC apoptosis detection kit was used to detect cell apoptosis, and protein expression was examined by Western blotting analysis. Our present studies showed that Akt phosphorylation was inhibited by perifosine in HepG2 and Bel-7402 human hepatocellular carcinoma cells. Perifosine inhibited the growth of HepG2 cells and Bel-7402 cells in a dose-dependent manner, and arrested cell cycle progression at the G₂ phase. Apoptosis induction became more effective with increasing perifosine concentration. The caspase cascade and its downstream effectors, Poly (ADP-ribose) polymerase (PARP), were also activated simultaneously upon perifosine treatment. The proapoptotic effect of perifosine was in part depending on regulation of the phosphorylation level of ERK and JNK. Perifosine cotreatment substantially increased cytotoxic effects of cisplatin in HepG2 cells. Down-regulating the expression of Bcl-2 and up-regulating the level of Bax may be the potential mechanism for this synergistic effect. Our findings suggest that the small molecule Akt inhibitor perifosine shows substantial anti-tumor activity in human hepatoma cancer cell lines, and is a good candidate for treatment combinations with classical cytostatic compounds in hepatocellular carcinoma.     

基于肝癌细胞线粒体功能受损和caspase-3信号通路探讨罗哌卡因促进肝癌细胞凋亡的作用机制研究

摘要 目的：基于肝癌细胞线粒体功能受损和天冬氨酸蛋白水解酶3（caspase-3）信号通路探讨罗哌卡因促进肝癌细胞凋亡的作用机制。方法：选用细胞株人肝癌细胞BEL-7402进行实验研究。用不同浓度罗哌卡因处理BEL-7402细胞后,采用溴化噻唑蓝四氮唑（MTT）法检测肝癌细胞的增殖情况,光镜及4,6-二苯胺-2-苯吲哚二盐酸盐（DAPI）溶液染色观察细胞形态,台盼蓝染色法测定细胞活力,流式细胞术分析BEL-7402细胞的凋亡情况,电子显微镜下观察细胞线粒体,激光共聚焦显微镜观察caspase-3在BEL-7402细胞中的细胞核迁移情况,蛋白免疫印迹试验评价罗哌卡因对细胞质凋亡相关蛋白、线粒体凋亡相关蛋白、BEL-7402细胞和线粒体凋亡相关蛋白表达的影响。结果：罗哌卡因能够抑制肝癌细胞的生长,并呈剂量依赖性和时间依赖性。罗哌卡因可诱导BEL-7402细胞发生凋亡,显著增加BEL-7402细胞的凋亡率。罗哌卡因能够损伤肝癌细胞线粒体功能。激光共聚焦显微镜观察显示caspase-3分子迁移到细胞核。罗哌卡因与caspase-3相互作用,促进caspase-3向细胞核内迁移,刺激caspase-3和聚腺苷二磷酸核糖聚合酶（PARP-1）、天冬氨酸蛋白水解酶9（caspase-9）蛋白的表达,抑制B细胞淋巴瘤-2基因（Bcl-2）的表达,促进凋亡酶激活因子（Apaf-1）的表达,促进线粒体释放细胞色素C（Cytochrome C）,激活caspase-3活性。结论：罗哌卡因具有促进肝癌细胞凋亡的作用,其作用机制可能与破坏肝癌细胞线粒体功能和激活caspase-3信号通路有关。     

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.     

Synthesis, characterization and biological activity of lanthanum(III) complexes containing 2-methylene-1,10-phenanthroline units bridged by aliphatic diamines

Two novel lanthanum(III) complexes containing 2-methylene-1,10-phenanthroline units bridged by aliphatic diamines were synthesized and characterized by elemental analysis, IR, NMR, thermal analysis and conductance measurements. They have been assayed for anticancer activity in vitro against HL-60 (human leukocytoma) cells, PC-3MIE8 (human prostate carcinoma) cells, BGC-823 (human stomach carcinoma) cells, MDA-MB-435 (human galactophore carcinoma) cells, Bel-7402 (human liver carcinoma) cells, and Hela (human cervix carcinoma) cells. The results show that the two complexes exhibit good cytotoxic activities against different cell lines in general, especially more effective than cisplatin against Bel-7402, BGC-823 and MDA-MB-435 cell lines. DNA-binding studies indicate that, besides the intercalation, the complexes bind to DNA by the other interaction(s), which might be responsible for the production of more compact DNA, coinciding with more A-like feature of DNA as suggested by CD spectra.     

Nitrogen-containing flavonoids as CDK1/Cyclin B inhibitors: design, synthesis, and biological evaluation

A novel series of nitrogen-containing flavonoids 5a-l, 6a,b, and 7a,b were designed and synthesized as cyclin-dependent kinases (CDKs) inhibitors. The representative compounds 5a, 5b, 5e, and 5g showed potent CDK1/Cyclin B inhibitory activities. All compounds displayed a significant growth inhibitory action in vitro against Bel-7402, PC-3, ECA-109, A-549, HL-60, and MCF-7 cancer cell lines. Flow cytometry analysis showed that 5b induced apoptosis in PC-3 cells.     

细胞内微丝骨架的变化对人肝癌Bel-7402细胞形态的影响

癌细胞的形态及癌细胞的生物学行为(如粘附、迁移和浸润)与细胞内骨架系统密切相关,本研究利用Phalloidin-FTTC标记人肝癌Bel-7402细胞内F-肌动蛋白(F-aetin),使用激光扫描共聚焦显微镜观测细胞微丝骨架与癌细胞形态的关系,及Cyt-B处理Bel-7402细胞后,癌细胞内微丝骨架的变化。结果显示：癌细胞内F-aedn解聚,F-aetin小体的聚集重组是癌细胞的形态变化的主要因素之一。     

Natural static magnetic field-induced apoptosis in liver cancer cell

Purpose: To observe the apoptotic effects of NSMF on human hepatoma cells and to investigate the mechanisms. Materials and methods: Human hepatoma cell line Bel-7402 and Hep G-2 were treated by 0.2?T rotary NSMF (30?min/d) with 250?Hz, 400?Hz and 500?Hz for 3?d and 6?d, respectively. Apoptosis was analyzed with flow cytometry. Cell proliferation was measured with XTT assay. Expression of Bcl-2, caspase3/8/9 was analyzed with ELISA. Results: After 6?d treatment, significant apoptosis was induced by 400?Hz in Bel-7402 cells. Slight cell apoptosis was observed at 250?Hz, while Hep G-2 cells exhibited slight apoptosis at 250?Hz and 400?Hz. After 3?d treatment, no apoptosis exhibited in both cell types. Compared with control group, expression of Bcl-2 and Caspase 8 in treated Bel-7402 cells were significantly reduced (p?p?Conclusions: NSMF upregulates caspase 9 and downregulates Bel-2 expression, which results in higher level of active caspase 3 to trigger apoptosis in cells. Different cell types require different NSMF factors like rotary frequency and treatment time to induce apoptosis.