Effects of lycopene and tomato paste extracts on DNA and lipid oxidation in LNCaP human prostate cancer cells

Animal and epidemiological studies point to a cancer preventive/therapeutic role for tomato products and its antioxidant, lycopene. It is hypothesized that lycopene will behave as an antioxidant at low concentrations and as a prooxidant at high concentrations in LNCaP human prostate cancer cell culture systems. We characterized the antioxidant, and prooxidant effects of a hexane extract of tomato paste (TP) and water solubilized lycopene at different concentrations using a prostate cancer cell line. Placebo (5% triglyceride, Roche Inc.) was used as a control. After 6, 24 hr and 48 hr incubation, LNCaP cells were harvested and used for each measurement. Cellular proliferation was determined using the MTT colorimetric assay. Lycopene and TP hexane extract inhibited cell growth in a dose-dependent (0.1-50 microM lycopene) manner and growth inhibition was 55% and 35% at 1 microM lycopene and TP hexane extract, respectively after 48 hr incubation. The levels of 8-hydroxydeoxyguanosine/deoxyguanosine (an oxidative DNA damage product) was significantly increased starting at 5 microM lycopene from both TP hexane extract and pure lycopene after 24 and 48 hr incubation with no protection at the lower concentrations. Malondialdehyde formation (a lipid peroxidation product measured by HPLC separation of the MDA-TBA adduct) was significantly reduced at low concentrations (0.1-1 microM) of lycopene in all treatments. Clinically relevant concentrations of lycopene and the tomato fraction containing lycopene significantly reduced LNCaP cancer cell survival which can only be partially explained by increased DNA damage at high lycopene concentrations (> 5 microM). Low concentrations of lycopene acted as a lipid antioxidant but did not protect DNA.     

Effect of the standardized Cimicifuga foetida extract on Hsp 27 expression in the MCF-7 cell line

Cimicifuga foetida, an Asian Cimicifuga species, has been employed as a cooling and detoxification agent in traditional Chinese medicine since ancient times. For this herb, two cycloartane triterpene glycosides isolated from the rhizomes have demonstrated cytotoxicity on rat tumor and human cancer cell lines. Since human Hsp27 is increased in various human cancers and exhibits cytoprotective activity that affects tumorigenesis and the susceptibility of tumours to cancer treatment, the purpose of this research was to study the expression of this protein in MCF-7 breast cancer cells. To accomplish this aim, MCF-7 cells were exposed to different concentrations of Cimicifuga foetida extract showing a reduction in cell number measured by the sulforhodamine assay. In addition, the expression of Hsp-27 mRNA detected by RT-PCR and Hsp-27 protein detected by immnofluorescence was present in all conditions, except when using the highest concentration of Cimicifuga foetida extract (2,000 jig /L). We conclude that Hsp 27 expression at 2,000 jig /L Cimicifuga foetida extract is diminished. This is the first report showing the Hsp-27 expression after exposure to Cimicifuga foetida extract in MCF-7 cells.     

The Structure of Remsamabilinin B

A new cucurbitacin glycoside, named hemsamabilinin B (2), was isolat-ed from Hemsleya amabilis Diels. 2 was hydrolyzed by enzyme on acid to produce 23,24-dihydrocucurbitacin F (3) or D-glucose respectively. On the basis of the spectrosco-pic data of 2 and its acetate (4) (1H NMR, 13CNMR and MS), the structure of 2 wasassigned as 2-0-β-D-glucopyranoside of 3.     

Suppression of Glial Tumor Growth by Expression of Glial Fibrillary Acidic Protein

We examined the effect of expression of glial fibrillary acidic protein (GFAP) on the tumor growth of astrocytoma in vivo. When rat astrocytoma C6 cells were injected subcutaneously in athymic mice, the cells produced tumors that grew rapidly. The tumor growth of C6 cells transfected with GFAP cDNA was significantly reduced compared to that of control NeoC6 cells transfected only with the neomycin resistant gene. After implantation of C6 cells transfected with mutated GFAP cDNA at the phosphorylation sites, the tumor growth was suppressed similar to that of the wild GFAP transfectants. To determine whether the cell growth suppression by GFAP is specific for astroglial cells, we assessed the effect of GFAP on the cell growth of an L cell of fibroblast origin in vitro. By GFAP cDNA transfection, L cells showed morphological changes, but the cell growth was not reduced. These results suggest that GFAP is a critical regulator of the tumor growth of astrocytoma.     

MiR-181b-5p Downregulates NOVA1 to Suppress Proliferation,Migration and Invasion and Promote Apoptosis in Astrocytoma

MicroRNAs (miRNAs) are small, short noncoding RNAs that modulate the expression of numerous genes by targeting their mRNA. Numerous abnormal miRNA expression patterns are observed in various human malignancies, and certain miRNAs can act as oncogenes or tumor suppressors. Astrocytoma, the most common neuroepithelial cancer, represents the majority of malignant brain tumors in humans. In our previous studies, we found that the downregulation of miR-181b-5p in astrocytomas is associated with a poor prognosis. The aim of the present study was to investigate the functional role of miR-181b-5p and its possible target genes. miR-181b-5p was significantly downregulated in astrocytoma specimens, and the reduced expression of miR-181b-5p was inversely correlated with the clinical stage. The ectopic expression of miR-181b-5p inhibited proliferation, migration and invasion and induced apoptosis in astrocytoma cancer cells in vitro. The NOVA1 (neuro-oncological ventral antigen 1) gene was further identified as a novel direct target of miR-181b-5p. Specifically, miR-181b-5p bound directly to the 3''-untranslated region (UTR) of NOVA1 and suppressed its expression. In clinical specimens, NOVA1 was overexpressed, and its protein levels were inversely correlated with miR-181b-5p expression. Furthermore, the changing level of NOVA1 was significantly associated with a poor survival outcome. Similar to restoring miR-181b-5p expression, downregulating NOVA1 inhibited cell growth, migration and invasion. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. Our results indicate that miR-181b-5p is a tumor suppressor in astrocytoma that inhibits tumor progression by targeting NOVA1. These findings suggest that miR-181b-5p may serve as a novel therapeutic target for astrocytoma.     

Chemopreventive effects of sage oil on skin papillomas in mice.

Salvia libanotica (sage) extract is a popular plant remedy used by Middle Eastern people to treat common complaints such as colds and abdominal pain. In this study, the chemopreventive effects of sage oil on 7,12 dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin papillomas was investigated. Furthermore, its growth inhibitory and cytotoxic effects on a mouse papilloma-derived cell line (SP-1) were studied using 3H-thymidine incorporation, cell count and trypan blue dye exclusion assays. Sage oil was either applied topically to mouse skin at concentrations of 5, 50 and 100% in acetone, injected intraperitoneally at concentrations of 4 (37 mg/ml) and 8% (75 mg/ml) in saline or given by gavage at 100% twice per week for 20 weeks, 20 minutes prior to each promotion treatment with TPA. The topically applied 100% oil extract delayed tumor appearance by 4 weeks and inhibited tumor incidence and yield by 19 and 61%, respectively, at week 20. Topical application of 50% and 5% sage oil inhibited tumor yield by 41% at week 20. Tumor weight was decreased by 75% and 80% following treatment of mouse skin with 50% and 100% oil, respectively. Intraperitoneal injections and gavage treatments failed to inhibit the promotion of tumors in mouse skin, but significantly decreased tumor weight and volume. Sage oil displayed strong growth inhibitory effects on the SP-1 papilloma derived cell line following 24 hrs of treatment with estimated IC50 of 50 microg/ml. This observed growth inhibition was due to cytostatic and not cytotoxic effects. Our results suggest that the oil extract of the sage plant has potent suppressive activities against tumor promotion in mouse skin and thus could be an effective chemopreventive agent against skin cancer.     

掌叶半夏有效提取物对宫颈癌细胞株增殖的抑制作用

目的既往的临床研究证实中药掌叶半夏可有效治疗宫颈癌。在此基础上,课题组观察了中药掌叶半夏有效提取物(Pinellia Extract,PE)对宫颈癌细胞株CaSki和HeLa细胞增殖的作用,先前采用MTT法检测细胞活性以及倒置相差显微镜观察细胞形态的变化,初步显示了PE对CaSki和HeLa细胞的增殖有抑制作用;本文将对PE的增殖抑制作用进行进一步的研究。方法采用扫描电镜和透射电镜观察PE作用后CaSki和HeLa细胞表面及内部超微结构的变化,In-cell Western blot检测不同浓度PE作用15min后CaSki和HeLa细胞磷酸化ERK的表达。结果0.15mg/mlPE作用24h后CaSki和HeLa细胞表面结构及内部结构均发生了显著的变化,提示细胞增殖受到抑制;不同浓度PE作用15min后CaSki和HeLa细胞磷酸化ERK的表达随PE浓度的增加逐渐下降。结论PE对CaSki和HeLa宫颈癌细胞株的增殖有明显抑制作用,表现在细胞表面及内部超微结构的变化;增殖抑制作用之一是阻断ERK的磷酸化。实验结果为进一步研究PE的抗宫颈癌作用机制以及新药的研制开发奠定了一定的理论依据和实验基础。     

Mechanism of differential effect of low dose adaptogens on the functional activity of normal and transformed cellular elements in vitro]

Influence of water solutions of chemically pure adaptogen--synthetic analog of Rhodiola Rosea extract phenol composition (SAR) on functional activity of hemopoietic and tumor cells of mice with Ehrlich ascite cancer was studied in vitro. The periodical character of SAR effects was shown to be different for both types of cells, and at 1 x 10(-2) and 1 x 10(-26) M concentrations simultaneous stimulation of blood marrow cells colony-forming activity and inhibition of the latter in tumor elements was revealed. Essential changes of reactions of both cell types after adding the DNA-dependent RNA polymerase blocker Actinomycin D permit to suggest SAR effects to be connected with drug influence on the membrane RNA of the target cells.     

Role of myosin II activity and the regulation of myosin light chain phosphorylation in astrocytomas

The generation of contractile force mediated by actin-myosin interactions is essential for cell motility. Myosin activity is promoted by phosphorylation of myosin light chain (MLC). MLC phosphorylation in large part is controlled by kinases that are effectors of Rho family GTPases. Accordingly, in this study we examined the effects of ROCK and Rac1 inhibition on MLC phosphorylation in astrocytoma cells. We found that low concentrations of the ROCK inhibitor Y27632 increased the phosphorylation state of the Triton X-100 soluble fraction of MLC, whereas higher concentrations of Y27632 decreased soluble phospho-MLC. These effects of Y27632 were dependent on Rac1. The soluble form of phospho-MLC comprises about 10% of total phospho-MLC in control cells. Interestingly, ROCK inhibition led to a decrease in the phosphorylation state of total MLC, whereas Rac1 inhibition had little effect. Thus, the soluble form of MLC is differentially regulated by ROCK and Rac1 compared with MLC examined in a total cell extract. We also observed that astrocytoma migration is stimulated by low concentrations of the myosin II inhibitor blebbistatin. However, higher concentrations of blebbistatin inhibit migration leading us to believe that migration has a biphasic dependence on myosin II activity. Taken together, our data show that modulation of myosin II activity is important in determining optimal astrocytoma migration. In addition, these findings suggest that there are at least two populations of MLC that are differentially regulated.     

Berberis libanotica Ehrenb Extract Shows Anti-Neoplastic Effects on Prostate Cancer Stem/Progenitor Cells

Cancer stem cells (CSCs), including those of advanced prostate cancer, are a suggested reason for tumor resistance toward conventional tumor therapy. Therefore, new therapeutic agents are urgently needed for targeting CSCs. Despite the minimal understanding of their modes of action, natural products and herbal therapies have been commonly used in the prevention and treatment of many cancers. Berberis libanotica Ehrenb (BLE) is a plant rich in alkaloids which may possess anti-cancer activity and a high potential for eliminating CSCs. We tested the effect of BLE on prostate cancer cells and our data indicated that this extract induced significant reduction in cell viability and inhibited the proliferation of human prostate cancer cell lines (DU145, PC3 and 22Rv1) in a dose- and time-dependent manner. BLE extract induced a perturbation of the cell cycle, leading to a G0-G1 arrest. Furthermore, we noted 50% cell death, characterized by the production of high levels of reactive oxidative species (ROS). Inhibition of cellular migration and invasion was also achieved upon treatment with BLE extract, suggesting a role in inhibiting metastasis. Interestingly, BLE extract had a major effect on CSCs. Cells were grown in a 3D sphere-formation assay to enrich for a population of cancer stem/progenitor cells. Our results showed a significant reduction in sphere formation ability. Three rounds of treatment with BLE extract were sufficient to eradicate the self-renewal ability of highly resistant CSCs. In conclusion, our results suggest a high therapeutic potential of BLE extract in targeting prostate cancer and its CSCs.     

Induction of apoptosis and differentiation in neuroblastoma and astrocytoma cells by the overexpression of Bin1, a novel Myc interacting protein.

Bin1 is a novel protein that specifically binds Myc and inhibits, at least in part, Myc transactivation. Bin1 seems to play a role in cell cycle control, acting as a tumor suppressor gene. Since MYC family genes play a regulatory role in the proliferation, differentiation, and apoptosis of the nervous system, we studied the effects of the overexpression of the Myc-interacting protein, Bin1, in neuroblastoma and astrocytoma cell lines, which were chosen as neural cell system models. The major effects of BIN1 overexpression observed in undifferentiated neuroblastoma and astrocytoma cells were a significant reduction of cell growth, an increase in the G(0)/G(1) cell population and the induction of apoptosis. The trigger of programmed cell death by Bin1 is described for the first time. Bin1 overexpression in undifferentiated cells did not induce any maturation process as neither neuronal nor astrocyte differentiation markers were upregulated in neuroblastoma and astrocytoma cells, respectively. On the other side, the effects of Bin1 overproduction in neuroblastoma and astrocytoma cells committed towards neuronal and astrocyte differentiation, respectively, were different from those observed in undifferentiated cells. Although we did not evidence any triggering of programmed cell death, we did notice a further induction towards more differentiated phenotypes. Our studies suggest that Bin1 overexpression in neuroblastoma and astrocytoma cells can result in one of the following pathways: (1) suppressed cell proliferation, (2) induced differentiation, or (3) apoptosis. Thus, it appears that Bin1 operates through different pathways that involve activation of different genes: the chosen pathway however will depend on the proliferating or differentiated state of the cell.     

Interleukin-2 inhibits proliferation of HPV-associated tumor cells and halts tumor growth in vivo

Previous studies have shown inhibition of cervical cancer cell growth by treatment with high concentrations of IL-2. In the present study, we evaluated the in vitro and in vivo effects of recombinant human IL-2 on HPV-associated tumor cells (3T3-16). Treatment of 3T3-16 cells with rhIL-2 for 72 h inhibited cell growth in a dose-dependent manner and this effect was evidenced at nanomolar concentrations. These tumor cells expressed mRNA for beta and gamma subunits of the IL-2 receptor, which are required for signal transduction. In experiments to explore the effect of IL-2 on the growth of the HPV-associated tumor, mice received rhIL-2 through different routes: (i) intraperitoneal; (ii) subcutaneous, at the tumor inoculation site; or (iii) subcutaneous, distant from the tumor inoculation site. An effective antitumor response was observed only in those animals that received IL-2 at the tumor site (P<0.01). These results indicate the potential adequacy of therapeutic strategies based on local administration of rhIL-2 for cervical carcinoma, not only based on the ability of this cytokine to stimulate cellular-mediated immunity but also because of its direct effects on tumor cells.     

Characterization of a Novel Anti-Cancer Compound for Astrocytomas

The standard chemotherapy for brain tumors is temozolomide (TMZ), however, as many as 50% of brain tumors are reportedly TMZ resistant leaving patients without a chemotherapeutic option. We performed serial screening of TMZ resistant astrocytoma cell lines, and identified compounds that are cytotoxic to these cells. The most cytotoxic compound was an analog of thiobarbituric acid that we refer to as CC-I. There is a dose-dependent cytotoxic effect of CC-I in TMZ resistant astrocytoma cells. Cell death appears to occur via apoptosis. Following CC-I exposure, there was an increase in astrocytoma cells in the S and G2/M phases. In in vivo athymic (nu/nu) nude mice subcutaneous and intracranial tumor models, CC-I completely inhibited tumor growth without liver or kidney toxicity. Molecular modeling and enzyme activity assays indicate that CC-I selectively inhibits topoisomerase IIα similar to other drugs in its class, but its cytotoxic effects on astrocytoma cells are stronger than these compounds. The cytotoxic effect of CC-I is stronger in cells expressing unmethylated O⁶-methylguanine methyltransferase (MGMT) but is still toxic to cells with methylated MGMT. CC-I can also enhance the toxic effect of TMZ on astrocytoma when the two compounds are combined. In conclusion, we have identified a compound that is effective against astrocytomas including TMZ resistant astrocytomas in both cell culture and in vivo brain tumor models. The enhanced cytotoxicity of CC-I and the safety profile of this family of drugs could provide an interesting tool for broader evaluation against brain tumors.     

山奈酚对胃癌细胞PARP1以及P53基因表达的影响研究

胃癌(GC)是最常见的恶性肿瘤之一,是人类健康的主要威胁,其发病机制是一个单基因或多基因逐步突变的过程,与细胞的侵袭、增殖和转移有关,包括癌基因遗传和表观遗传的突变、肿瘤抑制基因、DNA修复途径基因、细胞周期途径基因和幽门螺杆菌感染等。而山奈酚具有多种生物学活性,能够抑制多种肿瘤细胞的细胞周期,诱导肿瘤细胞凋亡从而抑制肿瘤细胞/组织的侵袭及转移。因此本研究用不同浓度的山奈酚处理胃癌细胞,并检测了胃癌细胞的形态变化情况、癌细胞凋亡相关因子P53和PARP1基因的表达水平和其对应的蛋白质表达变化。结果表明大于100μmol/L山奈酚处理后的胃癌细胞中P53基因和P53蛋白的表达水平被显著提高,而相反的PARP1基因和蛋白的表达则被显著抑制,且山奈酚处理后胃癌细胞的凋亡数目也明显增加,因此本实验结果表明,山奈酚能够有效的促进胃癌细胞凋亡的发生,以此来达到抑制癌细胞恶性增殖的作用。这一结果可以为后续针对胃癌新疗法的研究提供一些思路和理论支持。     

姜半夏乙醇提取物对人胃癌SGC7901细胞增殖和凋亡的影响

目的：观察姜半夏乙醇提取物对人胃癌SGC7901细胞增殖和凋亡的影响。方法：不同浓度姜半夏乙醇提取物（终浓度为1mg／ml,0．5mg／ml,0．25mg／ml,0．125mg／ml）处理SGC7901细胞后,倒置相差显微镜下观察细胞的形态学变化;通过甲基噻唑基四唑法检测细胞增殖状况、描绘生长曲线,使用紫外分光光度法观察药物干预后细胞ATP酶活力;AnnexinV．异硫氰酸荧光素（fluorescein isothiocyanate,FITC）／碘化丙啶（propidium iodide,PI）双标记法流式细胞术检测姜半夏乙醇提取物对SGC7901细胞诱导凋亡的情况。结果：不同浓度姜半夏乙醇提取物均能不同程度地抑制人胃癌SGC7901细胞的增殖;在姜半夏乙醇提取物诱导细胞后细胞发生了边缘毛刺、体积缩小等形态学变化,同时可见细胞折光度和贴壁能力下降;Annexin V-F／TC／PI双标记法检测显示姜半夏乙醇提取物可诱导细胞发生凋亡;细胞总ATP酶活力在药物干预72小时后出现明显下降;并且随着药物浓度增加细胞凋亡率、细胞形态异常改变以及ATP酶活力抑制作用均呈上升趋势。结论：姜半夏乙醇提取物可抑制人胃癌SGC7901细胞的增殖,促进其凋亡,抑制细胞ATP酶活力。     

Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28

Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI₅₀) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p < 0.0001, T-test). At 8 μg/ml (GI₇₀), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p < 0.05, T-test, n = 8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI₅₀ of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p < 0.05, T-test, n = 9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.     

蓝舌病毒HbC3对几株人和动物肿瘤细胞的感染特性研究

用BTV-HbC3感染人肺癌SPC—A-1细胞,人宫颈癌HeLa细胞,人星型胶质瘤U251细胞,小鼠星形胶质瘤C6细胞及人胚肺HEL细胞后,观察细胞病变效应(CPE);运用透射电镜技术及琼脂双扩散试验检测BTV-HbC3对各种不同肿瘤细胞及人胚肺HEL细胞的感染性;并用RT-PCR技术检测蓝舌病毒的增殖情况。结果显示,BTV-HbC3对正常HEL不感染,但能在不同来源的某些肿瘤细胞中选择性增殖,产生不同程度的细胞病变效应(CPE)及调亡现象,终致肿瘤细胞死亡。其中以人肺癌SPC-A-1细胞对其最为敏感。因此,初步认为BTV-HbC3株能靶向性杀死某些肿瘤细胞,从而为深入开展BTV-HbC3靶向性抗肿瘤的研究提供了第一手实验室依据。     

植物提取十八碳二烯酸对人胃癌组织细胞生态毒理学作用机制研究

为了探索植物提取十八碳二烯酸对人胃癌组织细胞生态毒理学作用机制,取胃癌患者肿瘤组织移植到重症联合免疫缺陷(SCID)小鼠皮下,腹腔注射100、300、900 mg·kg-1植物提取物十八碳二烯酸,分析对移植瘤生长抑制作用;用酶联免疫吸附法(ELISA)检测瘤组织cAMP、P53、PI3K水平.将瘤组织经酶解消化分离出细...     

Diindolilmethane (DIM) selectively inhibits cancer stem cells

Epidemiologic studies repeatedly have shown chemopreventive effects of cruciferous vegetables. Indole-3-carbinol (I3C) and its metabolite diindolylmethane (DIM) were identified in these plants as active ingredients and theirs anti-tumor activities were confirmed in multiple in vitro and in vivo experiments. Here, we demonstrate that DIM is a selective and potent inhibitor of cancer stem cells (CSCs). In several cancer cell lines, DIM inhibited tumor sphere formation at the concentrations 30-300 times lower than concentrations required for growth inhibition of parental cells cultured as adherent culture. We also found that treatment with DIM overcomes chemoresistance of CSCs to cytotoxics, such as paclitaxel, doxorubicin, and SN-38. Pre-treatment of tumor spheres with DIM before implantation to mice significantly retarded the growth of primary tumors compared to tumors formed by untreated tumor spheres. The concentrations of DIM required to suppress CSCs formation are in the close range to those achievable in human plasma after oral dosing of the compound. Therefore, DIM can potentially be used in cancer patients, either alone, or in combinations with existing drugs.