Cimicifuga racemosa extract BNO 1055 inhibits proliferation of the human prostate cancer cell line LNCaP

Extracts from black cohosh (Cimicifuga racemosa, CR) exert an anti-proliferative action in human breast cancer cell cultures, which has been attributed to an anti-estrogenic effect. However, CR constituents do not bind to either of the known estrogen receptors. Thus, the anti-tumor effect of CR me be mediated by mechanisms not involving these receptors. Polycyclic aromatic hydrocarbons are toxic environmental pollutants, which indirectly act as anti-estrogens by activating the aryl hydrocarbon receptor (AhR). The AhR is widely expressed in mammalian tissues and tumors. A recent screening study demonstrated activation of the AhR by a variety of herbal extracts, among others, CR. Since activation of the AhR causes inhibition of growth of prostate cancer cells, we addressed the question, whether CR may not only inhibit growth of breast cancer--but also of prostate cancer cells. In the AhR ligand assay, the CR extract BNO 1055 reduced tracer binding to 71% of the control demonstrating interaction of constituents of this extract with the receptor. Under basal as well as under estradiol- and dihydrotestosterone stimulated conditions, the CR extract dose dependently inhibited proliferation of LNCaP cells. A significant reduction of cell growth was observed at a concentration as low as 50 ng/ml. Thus, it is demonstrated for the first time that CR compounds potently inhibit the growth of human prostate cancer cells in vitro. This anti-proliferative effect may be mediated via the AhR.     

Activation of two distinct anti-proliferative pathways, apoptosis and p38 MAP kinase-dependent cell cycle arrest, by tumor necrosis factor in human melanoma cell line A375

The proliferation of human melanoma cell line A375-6 cells is inhibited by several cytokines, including interleukin-1 (IL-1). A375-R8 cells, a subclone of A375-6, are resistant to IL-1-induced growth inhibition. The proliferation of both cell lines is inhibitable by tumor necrosis factor (TNF). In this study, we characterized the mechanisms of TNF-induced growth inhibition. TNF-induced growth inhibition in both cell lines was partially suppressed by a selective p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), whereas a combination of SB203580 and Z-VAD-fmk, an inhibitor for a wide range of caspases, completely blocked TNF-induced growth inhibition, indicating that TNF-induced growth inhibition is mediated by both p38 MAPK and caspases. However, Z-VAD-fmk alone suppressed TNF-induced growth inhibition in A375-R8, but not A375-6, cells, suggesting that there may exist a TNF-induced anti-apoptotic mechanism in A375-6 cells which is lost or mutated in A375-R8 cells. Evidence in support of this notion includes (1) TNF-induced apoptosis only in A375-R8, but not A375-6 cells; (2) cycloheximide enabled TNF to induce apoptosis even in A375-6 cells; and (3) somatic hybrid cells between A375-6 and A375-R8 cells are resistant to TNF-induced apoptosis. Since TNF-induced NF-kappa B activation, cell cycle arrest, RB dephosphorylation, and E2F downregulation are indistinguishable in both cell lines, none of these factors is likely to be involved in the TNF-induced anti-apoptotic mechanism in A375-6 cells. Our results indicate that TNF activates two distinct anti-proliferative pathways including p38 MAPK-dependent cell cycle arrest and caspase-mediated apoptosis, as well as an anti-apoptotic mechanism in melanoma cells.     

Leflunomide Reduces Proliferation and Induces Apoptosis in Neuroblastoma Cells In Vitro and In Vivo

Leflunomide as an immunosuppressive drug is generally used in the treatment of rheumatoid arthritis. It inhibits DHODH (dihydroorotate dehydrogenase ), which is one of the essential enzymes in the de novo pyrimidine biosynthetic pathway. Here we showed that leflunomide significantly reduced cell proliferation and self-renewal activity. Annexin V-FITC/PI staining assay revealed that leflunomide induced S-phase cell cycle arrest, and promoted cell apoptosis. In vivo xenograft study in SCID mice showed that leflunomide inhibited tumor growth and development. We also observed that DHODH was commonly expressed in neuroblastoma. When treated with leflunomide, the neuroblastoma cell lines BE(2)-C, SK-N-DZ, and SK-N-F1 showed dramatic inhibition of DHODH at mRNA and protein levels. Considering the favorable toxicity profile and the successful clinical experience with leflunomide in rheumatoid arthritis, this drug represents a potential new candidate for targeted therapy in neuroblastoma.     

Lidocaine inhibits melanoma cell proliferation by regulating ERK phosphorylation

The melanoma is responsible for the majority of all skin cancer–related deaths worldwide. Evidence suggests that local anesthetics provide some benefit in the treatment of cancer via inhibition of cellular proliferation, invasion and migration. However, the potential antiproliferative effects of local anesthetics in the treatment of melanoma remain to be elucidated. In this study, we investigated the antiproliferative effects and underlying mechanism of the commonly used local anesthetic (lidocaine) on melanoma cells. A375 melanoma cells were treated by lidocaine or vemurafenib. Cell Counting Kit-8, histological staining, flow cytometric analysis, immunohistochemical staining, and Western blot analyses were carried out to test the effects of lidocaine and vemurafenib on A375 cells. BALB/C-nu/nu mice intraperitoneally injected with A375 cells were treated by lidocaine, and then tumor volume and weight were calculated. Lidocaine exhibited vemurafenib-like effects totally. Lidocaine inhibited A375 melanoma cell proliferation in a dose- and time-dependent manner and colony formation also showed a dose-dependent inhibition. Lidocaine treatment resulted in the arrest of cell-cycle progression in the G1 phase and inhibited Ki-67 expression in a dose-dependent manner. This effect was associated with inhibited extracellular signal–regulated kinase (ERK) phosphorylation. In vivo experiments revealed that intravenous injections of lidocaine suppressed tumor volume and weight. Lidocaine inhibits melanoma cell proliferation in a dose- and time-dependent manner via a mechanism that may involve inhibition of the ERK signaling pathway. Thus, lidocaine may provide some benefit for the treatment of melanoma.     

Accelerative effect of leflunomide on recovery from hepatic fibrosis involves TRAIL-mediated hepatic stellate cell apoptosis

AimsHepatic fibrosis is reversible, associated with apoptosis of activated hepatic stellate cells (HSCs) as injury subsides, thus providing potential targets for therapy. Little is known, however, about the course of this condition. The objective of this study was to elucidate the mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis and the effect of leflunomide on this process.Main methodsWe harvested Kupffer cells from rats during spontaneous recovery from liver fibrosis induced by carbon tetrachloride (CCl₄) and prepared recovery Kupffer cell conditioned medium (KCCM). Culture-activated HSCs were pretreated in the absence or presence of A771726, the active metabolite of leflunomide, and then stimulated with recovery KCCM.Key findingsFollowing stimulation with recovery KCCM, HSCs showed a decrease in proliferation and an increase in apoptosis by a caspase-dependent mechanism. Furthermore, pretreatment with A771726 markedly enhanced these effects. Real-time quantitative PCR (Q-PCR) analysis showed increased expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Kupffer cells during the spontaneous recovery phase. The pro-apoptotic function of KCCM prepared from TRAIL siRNA-treated Kupffer cells was obviously decreased, suggesting that TRAIL played an important role in recovery from hepatic fibrosis. Moreover, A771726 enhanced recovery KCCM-induced apoptosis of HSCs by a mechanism involving the inhibition of nuclear factor-kappa B (NF-κB) activation.SignificanceOur results showed the role of TRAIL in the apoptosis of activated HSCs that is induced by Kupffer cells prepared from livers recovering from CCI₄-induced fibrosis and provided insights into the resolution of fibrosis and the mechanisms by which leflunomide might act upon liver fibrosis.     

Gomisin A ameliorates metastatic melanoma by inhibiting AMPK and ERK/JNK-mediated cell survival and metastatic phenotypes

BackgroundGomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods.PurposeThe objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma.Study designThe anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo.MethodsWST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays.ResultsG.A (25–100 μM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5–20 μM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2–50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells.ConclusionThese results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.     

阿霉素与青蒿素半乳糖苷联合用药对乳腺癌细胞的体外抑制作用及机制

目的：青蒿素及其衍生物具有抗癌活性,本研究旨在探讨阿霉素（DOX）与青蒿素半乳糖苷（AG）联合用药对乳腺癌细胞的体外抑制作用及机制。方法：DOX与AG联合对乳腺癌MCF-7细胞株进行干预,MTT法评价二者对癌细胞增殖的影响;流式细胞计量术检测细胞凋亡;免疫印迹法检测凋亡相关蛋白表达情况。结果：DOX与AG联合作用对MCF-7细胞增殖抑制率最高可达91．6％,均显著高于同浓度DOX或AG抑制效果（P〈0．01）,且浓度分别为10IzM和20μM量效比最佳。10斗MDOX＋20μMAG联合干预纽癌细胞凋亡率为19．8％,显著高于对照组及单用10μMDOX或20／zMAG干预组。DOX与AG联合给药比单独应用其中一种均更加显著激活caspase级联信号通路,进而更加有效的促进癌细胞凋亡。结论：DOX和AG联合用药对人乳腺癌MCF-7细胞具有协同抑制效应,其机制可能与caspase家族介导的蛋白酶级联反应以及PARP裂解失活有关。这项研究为提高DoX治疗乳腺癌的有效性提供了新的思路。     

新霉胺通过抑制血管生成素活性抑制人黑色素瘤细胞增殖、迁移和侵润

新霉胺（neamine）是一种无毒的新霉素（neomycin）降解产物;已有研究证明,其可抑制血管生成素（angiogenin,ANG）诱导的内皮细胞血管生成作用,阻滞异种移植的人结肠腺癌在裸鼠的生长.本研究证明,新霉胺对人黑色瘤细胞株A375的细胞增殖、迁移和侵润作用.MTT法及软琼脂培养显示,新霉胺可明显抑制A375细胞的增殖和集落形成能力. Transwell试验证明,新霉胺可阻滞A375细胞,乃至血管生成素诱导的A375细胞的迁移和侵润能力.此外,免疫荧光揭示新霉胺可阻断血管生成素的核转位,从而抑制血管生成素诱导的A375细胞增殖.上述结果提示,新霉胺可通过抑制血管生成素的核转位,从而抑制肿瘤细胞增殖、迁移和侵润.鉴于与新霉素比较,新霉胺毒性小,因此新霉胺可望作为黑色素瘤治疗的先导药物,颇具开发前景和潜力.     

Involvement of autophagy in recombinant human arginase-induced cell apoptosis and growth inhibition of malignant melanoma cells

Recombinant human arginase (rhArg) has been developed for arginine derivation therapy of cancer and is currently in clinical trials for a variety of malignant solid tumors. In this study, we reported for the first time that rhArg could induce obvious autophagy in human melanoma cells; inhibition of autophagy by chloroquine (CQ) significantly increased rhArg-induced cell apoptosis and growth inhibition of A375 cells. A significant increase in mitochondrial membrane potential loss and elevated intracellular reactive oxygen species (ROS) levels were detected in A375 cells after rhArg treatment when compared with control. Membrane transition inhibitor cyclosporine A blocked autophagy and accelerated cell death induced by rhArg, indicating that rhArg induced autophagy via mitochondria pathway. Furthermore, antioxidant N-acetyl-l-cysteine suppressed rhArg-induced autophagy and rescued cells from cell growth inhibition, suggesting that ROS played an important role in rhArg-induced A375 cell growth inhibition and autophagy. Akt/mTOR signaling pathway was involved in autophagy induced by rhArg in a time-dependent manner. Moreover, rhArg could induce ERK1/2 activation in a dose- and time-dependent manner and rhArg-induced autophagy was attenuated when p-ERK1/2 was inhibited by MEK 1/2 inhibitor, U0126. Taken together, this study provides new insight into the molecular mechanism of autophagy involved in rhArg-induced cell apoptosis and growth inhibition, which facilitates the development of rhArg in combination with CQ as a potential therapy for malignant melanoma.     

Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents

BACKGROUND: Dihydroorotate dehydrogenase (DHODH) catalyzes the fourth committed step in the de novo biosynthesis of pyrimidines. As rapidly proliferating human T cells have an exceptional requirement for de novo pyrimidine biosynthesis, small molecule DHODH inhibitors constitute an attractive therapeutic approach to autoimmune diseases, immunosuppression, and cancer. Neither the structure of human DHODH nor any member of its family was known. RESULTS: The high-resolution crystal structures of human DHODH in complex with two different inhibitors have been solved. The initial set of phases was obtained using multiwavelength anomalous diffraction phasing with selenomethionine-containing DHODH. The structures have been refined to crystallographic R factors of 16.8% and 16.2% at resolutions of 1. 6 A and 1.8 A for inhibitors related to brequinar and leflunomide, respectively. CONCLUSIONS: Human DHODH has two domains: an alpha/beta-barrel domain containing the active site and an alpha-helical domain that forms the opening of a tunnel leading to the active site. Both inhibitors share a common binding site in this tunnel, and differences in the binding region govern drug sensitivity or resistance. The active site of human DHODH is generally similar to that of the previously reported bacterial active site. The greatest differences are that the catalytic base removing the proton from dihydroorotate is a serine rather than a cysteine, and that packing of the flavin mononucleotide in its binding site is tighter.     

Novel Small Molecule XPO1/CRM1 Inhibitors Induce Nuclear Accumulation of TP53, Phosphorylated MAPK and Apoptosis in Human Melanoma Cells

XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE). In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI)], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E) cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p<0.0001) and CHL-1 (p = 0.0087) human melanoma cell lines in vivo at well tolerated doses. Inhibition of XPO1 using SINE represents a potential therapeutic approach for melanoma across cells with diverse molecular phenotypes by promoting growth inhibition and apoptosis.     

Multiple inhibitor analysis of the brequinar and leflunomide binding sites on human dihydroorotate dehydrogenase

Brequinar and the active metabolite of leflunomide, A77 1726, have been clearly shown to inhibit human dihydroorotate dehydrogenase (DHODH), but conflicting mechanisms for their inhibition have been reported. DHODH catalyses the conversion of dihydroorotate (DHO) to orotate concurrent with the reduction of ubiquinone. This study presents data that indicates brequinar is a competitive inhibitor versus ubiquinone; A77 1726 is noncompetitive versus ubiquinone and both are uncompetitive versus DHO. 2-Phenyl 5-quinolinecarboxylic acid (PQC), the core moiety of brequinar also shows competitive inhibition versus ubiquinone. Multiple inhibition experiments indicate that PQC (and thus brequinar) and A77 1726 have overlapping binding sites. Both PQC and A77 1726 are also mutually exclusive with barbituric acid (a competitive inhibitor versus DHO). In addition, we failed to observe brequinar binding to E.orotate by isothermal titration calorimetry (ITC). These results indicate that the E.DHO.inhibitor and E.orotate.inhibitor ternary complexes do not form. The absence of these complexes is consistent with the two-site ping-pong mechanism reported for DHODH. This kinetic data suggests that recent crystal structures of human DHODH complexed with orotate and A77 1726 or brequinar may not represent the relevant physiological binding sites for these inhibitors [Liu, S., Neidhardt, E. A., Grossman, T. H., Ocain, T., and Clardy J. (2000) Structure 8, 25-33].     

Identification of vibsanin A analog as a novel HSP90 inhibitor

Vibsanin A is the first natural product isolated from Viburnum awabuki and has several biological activities. We have reported that a vibsanin A analog, obtained from process of total synthesis of vibsanin A, has anti-proliferative activity against human cancer cell lines. In this study, we evaluated anti-proliferative effect of the vibsanin A analogs against various human cancer cell lines and examined molecular target of the analog in human cells. Among the vibsanin A analogs, vibsanin A analog C (VAC) showed anti-proliferative effect against various cancer cell lines, and the anti-proliferative activity was strongest among the vibsanin A analogs. Additionally, VAC fluctuated amounts of HSP90-related proteins in cells and inhibited HSP90-mediated protein refolding of luciferase in vitro. These results suggest that the anti-proliferative activity of VAC is due to HSP90 inhibition, and VAC has a potential as novel anti-cancer drug as HSP90 inhibitor.     

Mechanism of leflunomide-induced proliferation of mitochondria in mammalian cells

Leflunomide (LFM) is an inhibitor of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) that catalyzes the conversion of dihydroorotate to orotate coupled with the generation of reactive oxygen species (ROS) from mitochondria. We demonstrate here that LFM causes an unrestrained proliferation of mitochondria both in human osteosarcoma cell line 143B cells and rat liver derived RL-34 cells. Increases in the total mass of mitochondria per cell in LFM-treated cells were evidenced by the application of Green FM or 10-n-nonyl acridine orange to flow cytometry, an enhanced replication of mtDNA and electron microscopy. Externally added uridine improved the disturbance in cell cycle progression in LFM-treated cells, but failed to suppress such unrestrained mitochondrial proliferation. On the contrary, lapacol and 5-fluoroorotate, inhibitors of DHODH besides LFM, suppressed the biogenesis of mitochondria during the cell cycle progression. LFM, but not lapacol or 5-fluoroorotate, caused increases of the intracellular level of acetylated alpha-tubulin. These data suggest that the inhibition of DHODH may not be at least primarily related to the LFM-induced abnormal proliferation of mitochondria, and support our recent published observation that changes in the physicochemical properties of microtubules may be in someway concerned with the biogenesis of mitochondria.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

MicroRNA-330 inhibits growth and migration of melanoma A375 cells: In vitro study

Melanoma skin cancer is one of the main causes of male cancer-related deaths worldwide. It has been suggested that miR-330-5p can act as a tumor suppressor in various types of cancers. So, in this study, we replaced miR-330 in melanoma cancer cells by vector-based miR-330 to evaluate the effects of this microRNA on the growth and migration inhibition of melanoma cancer cells, and to determine the molecular mechanisms underlying its action. By using the MTT assay, the IC₅₀ of Geneticin antibiotic was obtained as 460 µg/mL. The results of the qRT-PCR showed the increased expression level of miR-330 and decreased expression levels of MMP-9, CXCR4, Vimentin, melanoma cell adhesion molecule, AKT1, and E2F1 messenger RNA in A375 transfected cells. The cytotoxicity assay results demonstrated the inhibition of cancer cells proliferation. Furthermore, the wound healing test results showed a migration reduction of transfected cells with miR-330 compared with nontransfected ones. In addition, 4′,6-diamidino-2-phenylindoleLB: Luria-Bertani (DAPI) staining revealed the significant nucleus fragmentation in miR-330 replaced cells, which correspond to apoptosis induction in replaced cells. The results showed that increase in miR-330 expression level could significantly inhibit the tumor cell growth and the migration of melanoma cancer cells.