Synthesis and in vitro cytotoxic evaluation of new 1H-benzo[d]imidazole derivatives of dehydroabietic acid

A series of new 1H-benzo[d]imidazole derivatives of dehydroabietic acid were designed and synthesized as potent antitumor agents. Structures of the target molecules were characterized using MS, IR, ¹H NMR, ¹³C NMR and elemental analyses. In the in vitro cytotoxic assay, most compounds showed significant cytotoxic activities against two hepatocarcinoma cells (SMMC-7721 and HepG2) and reduced cytotoxicity against noncancerous human hepatocyte (LO2). Among them, compound 7b exhibited the best cytotoxicity against SMMC-7721 cells (IC₅₀: 0.36 ± 0.13 μM), while 7e was most potent to HepG2 cells (IC₅₀: 0.12 ± 0.03 μM). The cell cycle analysis indicated that compound 7b caused cell cycle arrest of SMMC-7721 cells at G2/M phase. Further, compound 7b also induced the apoptosis of SMMC-7721 cells in Annexin V-APC/7-AAD binding assay.     

SMMC-7721肝癌细胞67kD层粘连蛋白受体的分离纯化

分离纯化肝癌细胞的 6 7kD层粘连蛋白受体 (6 7LR) ,以便进一步研究 6 7LR的结构、功能及其在肝癌浸润、转移过程中的作用 .以SMMC 772 1肝癌细胞和L 0 2正常肝细胞为材料 ,采用13 1I标记的层粘连蛋白测定其与细胞的结合能力 ;亲和层析法分离纯化层粘连蛋白受体 ,用SDS PAGE、放射自显影及体外竞争结合实验进行鉴定 .在相同条件下SMMC 772 1肝癌细胞与层粘连蛋白特异结合量为 17 5 4± 0 4 9ng 10 5细胞 ,而L 0 2正常肝细胞与层粘连蛋白的特异结合量为 8 36± 0 4 8ng 10 5细胞 .经过亲和层析 ,从SMMC 772 1肝癌细胞和L 0 2正常肝细胞均可获得纯化受体 ,SDS PAGE显示为单一条带 ,分子量为 6 7kD ,放射自显影及体外竞争结合实验表明其具有较强的与层粘连蛋白结合的活性 .体外竞争结合实验表明 ,SMMC 772 1肝癌细胞层粘连蛋白受体 (772 1LnR)的抑制率可达到 96 2 7± 2 2 9% ,而L 0 2正常肝细胞层粘连蛋白受体 (L 0 2LnR)的抑制率为 4 8 71± 3 79% ,这说明 772 1LnR与层粘连蛋白的亲和力明显高于L 0 2LnR(P <0 0 0 1) .结果表明 ,与L 0 2肝细胞比较 ,SMMC 772 1肝癌细胞具有与层粘连蛋白较强结合能力的特异受体 ,并从肝癌细胞膜上分离纯化到与层粘连蛋白有较强亲和力的 6 7LR     

Cortactin and Exo70 mediated invasion of hepatoma carcinoma cells by MMP-9 secretion

This study was aimed to evaluate the regulation mechanism of cortactin (CTTN) on matrix metalloproteinases 9 (MMP-9) and its relations with Exo70 in invasion of hepatoma carcinoma (HCC) cells. The expression levels of CTTN, Exo70 and MMP-9 were detected in normal hepatocytes and various HCC cells by real-time PCR. Then the migration and invasion ability of these cells was revealed by scratch and invasion assay. The effects of CTTN on MMP-9 and the ability of migration and invasion were evaluated by silence and overexpress CTTN. During this process, the expression of CTTN was detected by Western blot, the activity and concentration of MMP-9 in supernatant of culture medium was detected by zymography and ELISA assay. Besides, Exo70 was also inhibited to reveal its effects on MMP-9 and the migration and invasion ability of LM3. Increased expression of CTTN, MMP-9, Exo70, reduced scratch area and increased puncture cell numbers were found in HCC cells (p < 0.05). The expression of CTTN was significantly correlated with Exo70 and the migration and invasion ability of HCC (p < 0.05). In addition, the activity and concentration of MMP-9 were significantly affected by the expression level of CTTN, while the expression of MMP-9 was not influenced. Besides, Exo70-si also exhibited significantly inhibition effects on the activity and concentration of MMP-9 and puncture cell numbers (p < 0.05). A synergistic reaction may exhibited on CTTN and Exo70, which could mediate the secretion of MMPs thereby regulate tumor invasion.     

OIP5对肝癌细胞SMMC-7721增殖和侵袭迁移能力的影响

目的:探讨OIP5对肝癌细胞SMMC-7721增殖和侵袭迁移能力的影响。方法:采用RNA干扰技术沉默肝癌细胞中OIP5的表达后,通过qRT-PCR和Western-blot技术检测OIP5的下调效率,CCK-8和平板克隆法检测肝癌细胞的增殖能力,Transwell法检测肝癌细胞的侵袭和迁移能力。结果:转染OIP5-siRNA后,肝癌细胞SMMC-7721中OIP5 mRNA和蛋白的表达水平均明显降低(P0.05);同时,与对照组相比,OIP5-siRNA组肝癌细胞SMMC-7721的CCK-8实验的OD值、平板克隆法测得的克隆球个数、Transwell法测得的迁移细胞数与侵袭细胞数均明显低于对照组(P0.05)。结论:OIP5能够促进肝癌细胞的增殖和侵袭迁移,可能作为肝癌治疗的潜在靶点。     

Downregulation of NIN/RPN12 binding protein inhibit the growth of human hepatocellular carcinoma cells

The ribosome assembly factor NIN/RPN12 binding protein (Nob1) has been suggested to be essential for processing of the 20S pre-rRNA to the mature 18S rRNA, and is also reported to participate in proteasome biogenesis. However, it is unclear whether Nob1 is involved in tumor cells growth. The aim of this study was to determine whether the suppression of Nob1 by short hairpin RNA (shRNA) inhibits the growth of human hepatocellular carcinoma (HCC) cells. Recombinant lentiviral shRNA expression vector carrying Nob1 was constructed and then infected into human HCC cell line SMMC-7721. The growth properties of SMMC-7721/pGCSIL-GFP-shNC and pGCSIL-GFP-shNob1 cells were determined by MTT, BrdU incorporation assay, and flow cytometric analysis. In addition, the colony formation and tumor growth ability in nude mice were detected to define the function of Nob1 in cell transformation and tumorigenesis. Our data showed that the growth and proliferation of SMMC-7721/pGCSIL-GFP-shNob1 cells were significantly reduced compared with the SMMC-7721/pGCSIL-GFP-shNC. In addition, the colony formation was impaired after the suppression of Nob1 in SMMC-7721 cells. And in vivo, the tumor formation ability of the SMMC-7721/pGCSIL-GFP-shNob1 cells was significantly reduced compared with the control cells. Our data support that Nob1 is an important regulator of the tumorigenic properties of human HCC and could be used as a candidate therapeutic target in human HCC.     

Longikaurin A,a natural ent-kaurane,induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/c-Jun pathway in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, and is also highly resistant to conventional chemotherapy treatments. In this study, we report that Longikaurin A (LK-A), an ent-kaurane diterpenoid isolated from the plant Isodon ternifolius, induced cell cycle arrest and apoptosis in human HCC cell lines. LK-A also suppressed tumor growth in SMMC-7721 xenograft models, without inducing any notable major organ-related toxicity. LK-A treatment led to reduced expression of the proto-oncogene S phase kinase-associated protein 2 (Skp2) in SMMC-7721 cells. Lower Skp2 levels correlated with increased expression of p21 and p-cdc2 (Try15), and a corresponding decrease in protein levels of Cyclin B1 and cdc2. Overexpression of Skp2 significantly inhibited LK-A-induced cell cycle arrest in SMMC-7721 cells, suggesting that LK-A may target Skp2 to arrest cells at the G2/M phase. LK-A also induced reactive oxygen species (ROS) production and apoptosis in SMMC-7721 cells. LK-A induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase and P38 MAP kinase. Treatment with, the JNK inhibitor SP600125 prevented LK-A-induced apoptosis in SMMC-7721 cells. Moreover, the antioxidant N-acetylcysteine prevented phosphorylation of both JNK and c-Jun. Taken together, these data indicate that LK-A induces cell cycle arrest and apoptosis in cancer cells by dampening Skp2 expression, and thereby activating the ROS/JNK/c-Jun signaling pathways. LK-A is therefore a potential lead compound for development of antitumor drugs targeting HCC.     

FOXQ1在肝癌中的临床意义及其对SMMC-7721细胞体外血管形成的影响

探讨叉头框蛋白Q1(forkhead box Q1, FOXQ1)基因在肝癌中的临床意义及对肝癌细胞体外血管生成作用.利用 qRT-PCR法及Western印迹法,检测24例肝癌、癌旁组织、正常肝细胞L02及肝癌细胞SMMC-7721中FOXQ1的mRNA和蛋白质的表达;利用免疫组织化学法检测68例肝癌及癌旁组织中FOXQ1的蛋白质表达.合成shRNA-FOXQ1及shRNA-NC慢病毒,转染到SMMC-7721细胞.用体外血管生成实验检测转染shRNA-FOXQ1的肝癌细胞血管生成能力. 用qRT-PCR和Western印迹法检测细胞间FOXQ1、VEGF基因和蛋白质的表达.结果显示,癌组织和SMMC-7721细胞中FOXQ1 mRNA和蛋白质的表达均高于癌旁组织和正常肝细胞(P<0.05),FOXQ1蛋白的表达与TNM分期、肿瘤分化程度、肿瘤数目、肿瘤大小等参数差异显著(P<0.05).shRNA-FOXQ1组血管生成能力明显低于shRNA-NC组和空白组(P<0.05),FOXQ1、VEGF基因和蛋白质的表达也明显低于shRNA-NC组和空白组(P<0.05).研究结果证实,FOXQ1在肝癌中高表达,如果沉默FOXQ1的表达可抑制肝癌细胞血管生成,与肝癌的临床病理特征密切相关.     

Roles of cortactin, an actin polymerization mediator, in cell endocytosis

Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS 1 oncogene. Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents. Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 （SH3）-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation. Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.     

Microfilament regulatory protein MENA increases activity of RhoA and promotes metastasis of hepatocellular carcinoma

Mammalian enabled (MENA), usually known as a direct regulator of microfilament polymerization and bundling, promotes metastasis in various cancers. Here we focus on the role of MENA in hepatocellular carcinoma (HCC) metastasis and the relevant mechanism from the view of RhoA activity regulation. By HCC tissue microarray analysis, we found that MENA expression was positively associated with satellite lesions (P<0.01) and vascular invasion (P<0.01). Cases with membrane reinforcement of MENA staining in HCC tissues had significantly higher rates of early recurrence in the intermediate MENA expression group. Knockdown of MENA significantly suppressed HCC cell migration and invasion in vitro, as well as their intrahepatic and distant metastasis in vivo. Knockdown of MENA also decreased filopodia and stress fibers in SMMC-7721 cells. Furthermore, a decrease of RhoA activity was detected by a pull-down assay in SMMC-7721-shMENA cells. The ROCK inhibitor, Y-27632, suppressed migration of both MENA knockdown SMMC-7721 cells and control cells, but diminished their difference. Thus, our findings suggest that MENA promotes HCC cell motility by activating RhoA.     

Regulation of human hepatocellular carcinoma cells by Spred2 and correlative studies on its mechanism

Members of the Spred gene family are negative regulators of the Ras/Raf-1/ERK pathway, which has been associated with several features of the tumor malignancy. However, the effect of Spred genes on hepatocellular carcinoma (HCC) remains uninvestigated. In the present work, we analyzed the in vitro and in vivo effects of Spred2 expression on the hepatic carcinoma cell line, SMMC-7721. In addition to attenuated ERK activation, which inhibited the proliferation and migration of unstimulated and HGF-stimulated SMMC-7721 cells. Adenovirus-mediated Spred2 overexpression induced the activation of caspase-3 and apoptosis, as well as reduced the expression level of Mcl-1. Most importantly, the knockdown of Spred2 markedly enhanced tumor growth in vivo. In conclusion, these results suggest that Spred2 could qualify as a potential therapeutic target in HCC.     

Compound Kushen Injection intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating β-catenin/c-Myc signaling

BackgroundHepatocellular carcinoma (HCC) is one of the most extensive and most deadly cancers worldwide. The invasion and metastasis characteristics of HCC dramatically affect the prognosis and survival of HCC patients. Compound Kushen Injection (CKI) is a GMP produced, proverbially applied traditional Chinese medicine formula in China to treat cancer-associated pains, and used as an adjunctive therapy for HCC. Until so far, whether CKI could suppress the metastasis of HCC through regulation of epithelial-mesenchymal transition or metabolic reprogramming is still ambiguous.PurposeIn this study, the anti-metastasis effects of CKI were clarified and its pharmacological mechanisms were systematically explored.MethodsCell invasion and cell adhesion assay were performed in SMMC-7721 cells to assess the anti-metastasis role of CKI, and the histopathological evaluation and biochemical detection were utilized in DEN-induced HCC rats to verify the anti-HCC effect of CKI. Serum and liver samples were analyzed with ¹H NMR metabolomics approach to screen the differential metabolites and further target quantification the content of key metabolites. Finally, western blotting and immunofluorescence assay were applied to verify the crucial signaling pathway involved in metabolites.ResultsCKI markedly repressed the invasion and adhesion in SMMC-7721 cells and significantly improved the liver function of DEN-induced HCC rats. CKI significantly regulated the expression of epithelial-mesenchymal transition (EMT) markers (Vimentin and E-cadherin). Metabolomics results showed that CKI regulated the metabolic reprogramming of HCC by inhibiting the key metabolites (citrate and lactate) and enzymes (HK and PK) in glycolysis process. Importantly, we found that c-Myc mediates the inhibitory effect of CKI on glycolysis. We further demonstrated that CKI inhibits c-Myc expression through modulating Wnt/β-catenin pathway in SMMC-7721 cells and DEN-induced HCC rats. Furthermore, through activating Wnt/β-catenin pathway with LiCl, the inhibitory effects of CKI on HCC were diminished.ConclusionTogether, this study reveals that CKI intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating β-catenin/c-Myc signaling pathway. Our research provides a new understanding of the mechanism of CKI against invasion and metastasis of HCC from the perspective of metabolic reprogramming.     

Identification and expression profiles of genes and protens in SMMC-7721 cells

In the study presented here, we first evaluated effect of CDDP on liver cancer cells SMMC-7721 apoptosis and motility capacity. Then, we evaluate inhibitory effect of CDDP on tumour growth and its possible molecular mechanism in liver cancer mice model. Results showed that the apoptosis rate of cells decreased with increasing CDDP. Analysis of the effect of the CDDP on cell cycle was performed by flow cytometry and results show a dose-dependent increase in the percentage of cells in the S-phase of the cell cycle, with a decrease in the percentage of cells in the G1 and G2/M phases. CDDP did not close the wound even after 48 h, as opposed to untreated cells (0 mg/l). Similarly, the migratory and invasion capacity of SMMC-7721 cells was also reduced after treatment with CDDP, as evaluated by a transwell assay. Animal experiment indicated that CDDP administration could increase blood WBC, total protein, albumin and A/G, decrease blood alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in hepatocellular carcinomas mice. Immunohistochemistry analysis showed that positive expression of Fas and Bax proteins in the medicine-treated (II, III) group was significantly higher, whereas the expression of NF-κB, P53, Bcl-2 proteins was significantly lower than those of the control group. Gene expression analysis using Real time PCR methods revealed a significant up-regulation in the expression levels of Bax mRNA in the medicne-treated (II, III) group when compared to untreated control. In contrast, CDDP-treated group showed a significant down regulation in the expression levels of Bcl-2 mRNA as compared to untreated control group. These results are in agreement with immunohistochemistry data. Our observations indicate that CDDP has damaged effects on liver tumour cells SMMC-7721 including apoptosis, motility and cell cycle under in vitro. CDDP can enhance pro-apoptosis gene Fas, Bax expression, decrease anti-apoptosis genes Bcl-2 expression, and mutant genes P53, NF-κB proteins expression.     

In vitro and in vivo anti-metastatic effect of the alkaliod matrine from Sophora flavecens on hepatocellular carcinoma and its mechanisms

BackgroundsHepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer with high metastasis and recurrence rates. Hypoxia-induced miRNAs and HIF-1α are demonstrated to play essential roles in tumor metastasis. Matrine (C₁₅H₂₄N₂O), an alkaloid extracted from Sophora flavescens Aiton, has been used as adjuvant therapy for liver cancer in China. The anti-metastasis effects of matrine on HCC and the underlying mechanisms remain poorly understood.PurposeWe aimed to investigate the effects of matrine on metastasis of HCC both in vitro and in vivo, and explored whether miR-199a-5p and HIF-1α are involved in the action of matrine.MethodsMTT method, colony formation, wound healing and matrigel transwell assays were performed to evaluate the effects of matrine on cell proliferation, migration and invasion. Nude mice xenograft model and immunohistochemistry (IHC) assay were employed to investigate the anti-metastatic action of matrine in vivo. Quantitative real-time PCR, western blot and dual luciferase reporter assay were conducted to determine the underlying mechanisms of matrine.ResultsMatrine exerted stronger anti-proliferative action on Bel7402 and SMMC-7721 cells under hypoxia than that in normoxia. Both matrine and miR-199a-5p exhibited significant inhibitory effects on migration, invasion and EMT in Bel7402 and SMMC-7721 cells under hypoxia. Further study showed that miR-199a-5p was downregulated in HCC cell lines, and this microRNA was identified to directly target HIF-1α, resulting in decreased HIF-1α expression. Matrine induced miR-199a-5p expression, decreased HIF-1α expression and inhibited metastasis of Bel7402 and SMMC-7721 cells, while miR-199a-5p knockdown reversed the inhibitory effects of matrine on cell migration, invasion, EMT and HIF-1α expression. In vivo, matrine showed significant anti-metastatic activity in the nude mouse xenograft model. H&E and IHC analysis indicated that lung and liver metastasis nodules were reduced, and the protein expression of HIF-1α and Vimentin were significantly decreased by i.p injection of matrine.ConclusionsMatrine exhibits significant anti-metastatic effect on HCC, which is attributed to enhanced miR-199a-5p expression and subsequently impaired HIF-1α signaling and EMT. These findings suggest that miR-199a-5p is a potential therapeutic target of HCC, and matrine may represent a promising anti-metastatic medication for HCC therapy.     

Stealth siRNA against CD147 inhibits hepatocellular carcinoma cell metastatic properties

CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to secrete matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this research was to investigate the inhibitory effects of stealth small interfering RNA (siRNA) against CD147 on HCC cell line (SMMC-7721) metastatic properties including invasion, adhesion to ECM, gelatinase production, focal adhesion kinase (FAK) and vinculin expression. Flow cytometry (FCM) and western blot assays were employed to detect the transfection efficiency of the stealth siRNA against CD147. Invasion assays and gelatin zymography were also used to detect the effects of stealth siRNA against CD147 on SMMC-7721 cells’ invasion and gelatinase production. The effects of stealth siRNA against CD147 on FAK and vinculiln expression in SMMC-7721 cells were also detected by western blot. The results showed that stealth siRNA against CD147 inhibited SMMC-7721 invasion, adhesion to ECM proteins, MMP-2 production, and FAK and vinculin expression. These findings indicate that CD147 is required for tumor cell invasion and adhesion. Perturbation of CD147 expression may have potential therapeutic uses in the prevention of MMP-2-dependent tumor invasion.     

Design,synthesis and in vitro anticancer activity of novel quinoline and oxadiazole derivatives of ursolic acid

A series of new quinoline derivatives of ursolic acid were designed and synthesized in an attempt to develop potential anticancer agents. The structures of these compounds were identified by ¹H NMR, ¹³C NMR, IR and ESI-MS spectra analysis. The target compounds were evaluated for their in vitro cytotoxicity against three human cancer cell lines (MDA-MB-231, Hela and SMMC-7721). From the results, compounds 3a–d displayed significant antitumor activity against three cancer cell lines. Especially, compound 3b was found to be the most potent derivative with IC₅₀ values of 0.61 ± 0.07, 0.36 ± 0.05, 12.49 ± 0.08 μM against MDA-MB-231, HeLa and SMMC-7721 cells, respectively, stronger than positive control etoposide. Furthermore, the Annexin V-FITC/PI dual staining assay revealed that compound 3b could significantly induce the apoptosis of MDA-MB-231 cells in a dose-dependent manner. The cell cycle analysis also indicated that compound 3b could cause cell cycle arrest of MDA-MB-231 cells at G0/G1 phase.     

Lentivirus mediated silencing of Ubiquitin Specific Peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro

Background

Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells.

Results

In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G₂/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells.

Conclusions

All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0006-y) contains supplementary material, which is available to authorized users.     

The expression and significance of NET-1 and Contactin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. In this study, the significance of NET and Contactin on the pathogenesis and prognosis of HCC was investigated, and further to explore their functions in vitro and in vivo by down regulation with siRNA. The expression of NET-1 and Contactin in HCC and in adjacent non-tumor tissues (ANT) were evaluated by immunohistochemistry, and the correlations of the expression of NET-1 and Contactin with the clinicopathological characteristics and survival of HCC patients were also analyzed. After inhibited by single-target siRNA or dual-target siRNA, the expressions of NET-1 and Contactin mRNA and protein in SMMC-7221 cells were determined by RT-PCR, Western blot and immunofluorescence stain. The cell proliferation and apoptosis were assessed by CCK-8 assays and flow cytometry (FCM). The ability of cell migration and invasion were evaluated by wound-healing migrating assay and transwell chamber assays, respectively. Subsequently, transmission system encapsulated cationic liposome was used to deliver dual-siRNA into HCC xenografts in mice. The expressions of NET-1, Cortactin, Ki67, Bax, Bcl2 and Survivin in xenograft tumor were detected by immunohistochemical staining, respectively. The positive rates of NET-1 and Cortactin in HCC tissues were significantly higher than those in ANT. In HCC, the expression of NET-1 was related to Edmondson''s grade (P<0.05), cirrhosis background (P<0.001) and TNM stage (P<0.05). The expression of Cortactin was related to tumor infiltration (P<0.05), vascular invasion (P <0.001) and TNM stage (P<0.001). The expressions of NET-1 and Cortactin were positively correlated (r=0.280, P=0.004). Significant differences in the 5-year survival rates were seen between the NET-1 negative group and the positive group (P<0.05), and between the Contactin negative group (50%) and Contactin positive group (28.0%, P<0.01). The 5-year overall survival rate (OS) in NET-1 and Contactin co-expression cases (27.78%) were remarkably lower than that in both NET-1 and Contactin negative cases (54.54%) and in NET-1 positive while Contactin negative cases (47.06%, P<0.05). Univariate and multivariate Cox regression analysis revealed that NET-1 and Contactin over-expression were independent indicators for OS in HCC patients (P<0.01). There were higher expressions of NET-1 and Contactin in SMMC-7721 cells than that in other HCC cells. Dual-siRNA was demonstrated to be more effective on inhibiting cancer cell proliferation, migration and inducing apoptosis than individual siRNAs used alone in vitro and in vivo (P<0.05). The results suggest that dual-siRNA may be a great potential in siRNA-based therapeutic applications.     

蛋白激酶PRKX对人肝癌细胞粘附和迁移能力的影响

目的观察蛋白激酶PRKX对人肝癌细胞SMMC-7721粘附和迁移能力的影响。方法采用脂质体转染的方法,将PRKX表达质粒转染到SMMC-7721细胞中,蛋白印迹方法鉴定转染前后PRKX蛋白的表达。细胞-基质粘附实验测定对照组和PRKX转染组SMMC-7721细胞的粘附能力。细胞迁移实验测定对照组和PRKX转染组SMMC-7721细胞的迁移能力。结果 SMMC-7721细胞转染组PRKX蛋白的表达增加,SMMC-7721细胞转染组的粘附能力和迁移能力均较对照组增加。结论 PRKX可增加人肝癌细胞SMMC-7721的粘附和迁移能力。     

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone,from buds of Cleistocalyx operculatus,induces apoptosis in human hepatoma SMMC-7721 cells through a reactive oxygen species-dependent mechanism

Nowadays, much effort is being devoted to detect new substances that not only significantly induce the death of tumor cells, but also have little side effect on normal cells. Our previous study showed that 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) exhibited significant cytotoxic potential with an IC₅₀ value of 32.3 ± 1.13 μM against SMMC-7721 cells and could induce SMMC-7721 cells apoptosis. In the present study, we found that DMC was almost nontoxic to human normal liver L-02 and human normal fetal lung fibroblast HFL-1 cells as their IC₅₀ values (111.0 ± 4.57 and 152.0 ± 4.83 µM for L-02 and HFL-1 cells, respectively) were much higher. To further explore the apoptotic mechanism of DMC, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by DMC in SMMC-7721 cells. Our results suggested that the cytotoxicity and the generation of intracellular ROS were inhibited by N-acetylcysteine (NAC). Reversal of apoptosis in NAC pretreated cells indicated the involvement of ROS in DMC-induced apoptosis. The loss of mitochondrial membrane potential (ΔΨm) induced by DMC was significantly blocked by NAC. NAC also prevented the decrease of Caspase-3 and -9 activities, the increase of Bcl-2 protein expression and the decrease of p53 and PUMA protein expressions. Together, these results indicated that ROS played a key role in the apoptosis induced by DMC in human hepatoma SMMC-7721 cells.