Gambogic acid inhibits angiogenesis and prostate tumor growth by suppressing vascular endothelial growth factor receptor 2 signaling

Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been previously reported to activate apoptosis in many types of cancer cell lines by targeting transferrin receptor and modulating nuclear factor-kappaB signaling pathway. Whether GA inhibits angiogenesis, which is crucial for cancer and other human diseases, remains unknown. Here, we found that GA significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, migration, invasion, tube formation, and microvessel growth at nanomolar concentration. In a xenograft prostate tumor model, we found that GA effectively inhibited tumor angiogenesis and suppressed tumor growth with low side effects using metronomic chemotherapy with GA. GA was more effective in activating apoptosis and inhibiting proliferation and migration in HUVECs than in human prostate cancer cells (PC3), suggesting GA might be a potential drug candidate in cancer therapy through angioprevention with low chemotoxicity. Furthermore, we showed that GA inhibited the activations of vascular endothelial growth factor receptor 2 and its downstream protein kinases, such as c-Src, focal adhesion kinase, and AKT. Together, these data suggest that GA inhibits angiogenesis and may be a viable drug candidate in antiangiogenesis and anticancer therapies.     

All-trans retinoic acid inhibits tumor growth of human osteosarcoma by activating Smad signaling-induced osteogenic differentiation

Osteosarcoma (OS) is one of the most common malignant bone tumors. Despite the advancement of diagnosis and treatment for OS, the prognosis remains poor. We investigated the proliferation inhibitory effect of all-trans retinoic acid (ATRA) for human OS and the possible mechanism underlying this effect. We examined the proliferation inhibition and apoptosis-inducing effects of ATRA in 143B OS cells. We validated this effect by exogenously expressing the retinoic acid receptor alpha (RARα) in 143B OS cells and injecting the cells into nude mice. We explored the possible mechanism for the proliferation inhibitory effect of ATRA on OS cells and multipotential progenitor cells by detecting osteogenic markers. We demonstrated that the endogenous retinoic acid receptor and retinoid X receptor are all detectable in the commercially available OS cell lines and in primary osteosarcoma cells. ATRA inhibits the proliferation of OS cells in a concentration-dependent manner, as well as induces apoptosis in 143B OS cells. The exogenous expression of RARα inhibits the tumor growth and cell proliferation in vivo. The alkaline phosphatase activity, protein levels of osteopontin (OPN) and osteocalcin (OCN) are all promoted by ATRA in OS cells and mouse embryonic fibroblasts (MEFs), at least by activating the Smad signaling pathway. Collectively, our results strongly indicate that ATRA can inhibit the tumor growth of OS by promoting osteogenic differentiation in OS cells, which is mediated in part by activating Smad signaling. Therefore, combination of ATRA with other current chemotherapy agents may be a promising therapy strategy for OS treatment.     

Histone deacetylase inhibitors decrease proliferation and modulate cell cycle gene expression in normal mammary epithelial cells.

Full-term pregnancy early in reproductive life is protective against breast cancer in women. The protective effects of parity have variously been attributed to the differentiation that accompanies pregnancy and lactation, alterations in ovarian hormone receptor levels, and altered sensitivity to ovarian hormones. Butyrate, a short-chain fatty acid, induces differentiation in breast cancer cell lines and decreases hormone receptor expression. Butyrate also inhibits proliferation in breast cancer cell lines and modulates expression of key cell cycle-regulatory proteins including cyclin D1. Given these properties, butyrate could be considered a promising agent for breast cancer prevention. Therefore, this study aimed to determine the effects of butyrate on normal human breast epithelial cells and to compare the effects of two stable butyrate derivatives with more favorable pharmacological properties: phenylacetate and its p.o. active precursor phenylbutyrate. Treatment with each agent resulted in concentration-dependent growth inhibition in a normal breast epithelial cell line and two breast cancer cell lines (MCF-7 and MDA-MB-231). Phenylbutyrate and butyrate inhibited proliferation to a similar extent, but phenylacetate was less effective in all of the cell lines. All three of the agents induced differentiation (accumulation of lipid droplets) in normal as well as in breast cancer cells and caused a decrease in estrogen receptor (ER) mRNA in MCF-7 cells. The butyrates decreased expression of cyclin D1, increased expression of p21(Waf1/Cip1), and hypophosphorylated pRB in the normal mammary epithelial cells. The effects on cyclin D1 expression correlated with the effects on cell proliferation, which suggests that modulation of cyclin D1 expression may underpin the antiproliferative effects of butyrates. We have shown that butyrate and butyrate-like agents are able to decrease proliferation and induce differentiation in normal breast cells as well as in malignant breast cells (ER-positive and ER-negative) and, as such, may be considered as candidate chemopreventative agents for women at high risk of developing breast cancer.     

Dihydromethysticin,a natural molecule from Kava,suppresses the growth of colorectal cancer via the NLRC3/PI3K pathway

Dihydromethysticin (DHM), a natural compound derived from Kava, has been reported to be effective against mental disorders and some malignant tumors. However, little is known about the inhibitory effect of DHM on colorectal cancer (CRC). First, we examined the impact of DHM on human colon cancer cell lines, which demonstrated that DHM inhibits proliferation, migration, and invasion and promotes apoptosis and cell cycle arrest in colon cancer cells in vitro. Using small hairpin RNA, we inhibited nucleotide-oligomerization domain-like receptor subfamily C3 (NLRC3)/phosphoinositide 3-kinase (PI3K) pathway to elucidate the partial signaling of DHM-mediated tumor suppression. Additionally, using an ectopic human CRC model, we verified whether DHM inhibits tumor growth and angiogenesis via the NLRC3/PI3K pathway in vivo. Overall, DHM showed an inhibitory effect on CRC by altering cell proliferation, migration, invasion, apoptosis, cell cycle, and angiogenesis, possibly via the NLRC3/PI3K pathway. Thus, DHM may be a promising candidate for CRC therapy.     

An anti-insulin-like growth factor I receptor antibody that is a potent inhibitor of cancer cell proliferation

An antagonistic monoclonal antibody, designated EM164, has been developed which binds specifically to the human insulin-like growth factor I receptor (IGF-IR) and inhibits the proliferation and survival functions of the receptor in cancer cells. EM164 was initially selected by a rapid cell-based screen of hybridoma supernatants to identify antibodies that bind to IGF-IR but not to the homologous insulin receptor and that show maximal inhibition of IGF-I-stimulated autophosphorylation of IGF-IR. EM164 binds tightly to IGF-IR with a dissociation constant K(d) of 0.1 nM, inhibits binding of IGF-I and antagonizes its effects on cells completely, and has no agonistic activity on its own. EM164 inhibits IGF-I-, IGF-II-, and serum-stimulated proliferation and survival of diverse human cancer cell lines in vitro, including breast, lung, colon, cervical, ovarian, pancreatic, melanoma, prostate, neuroblastoma, rhabdomyosarcoma, and osteosarcoma cancer lines. It also suppresses the autocrine or paracrine proliferation of several cancer cell lines. EM164 was the most potent antagonistic anti-IGF-IR antibody tested when compared with several commercially available antibodies. The in vitro inhibitory effect could be extended to in vivo tumor models, where EM164 caused regression of established BxPC-3 human pancreatic tumor xenografts in SCID mice. The antitumor effect of treatment with EM164 could be enhanced by combining it with the cytotoxic agent gemcitabine. These data support the development of EM164 as a candidate therapeutic agent that targets IGF-IR function in cancer cells.     

The transient receptor potential vanilloid-2 cation channel impairs glioblastoma stem-like cell proliferation and promotes differentiation

Malignant transformation of cells resulting from enhanced proliferation and aberrant differentiation is often accompanied by changes in transient receptor potential vanilloid (TRPV) channels expression. In gliomas, recent evidence indicates that TRPV type 2 (TRPV2) negatively controls glioma cell survival and proliferation. In addition, cannabinoids, the ligands of both cannabinoid and TRPV2 receptors, promote glioblastoma stem-like cells (GSCs) differentiation and inhibit gliomagenesis. Herein, we provide evidence on the expression of TRPV2 in human GSCs and that GSCs differentiation reduces nestin and progressively increases both the glial fibrillary acidic protein (GFAP) and TRPV2 expression. Therefore, we evaluated the role of TRPV2 cation channel in GSC lines differentiation. Treatment of GSC lines with the TRPV antagonist Ruthenium Red, with ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid or knockdown of TRPV2 gene during differentiation, decreases GFAP and class III beta-tubulin (β(III)-tubulin) expression; conversely, phorbol-12-myristate-13-acetate stimulates GSCs proliferation, reduces TRPV2 expression and partially reverts astroglial differentiation. In addition, forced TRPV2 expression in GSC lines by stable TRPV2 transfection increases GFAP and β(III)-tubulin expression and parallelly reduces proliferation. Finally, TRPV2 overexpression inhibits GSCs proliferation in a xenograft mouse model, as shown by reduced tumor diameter and mitotic index, and promotes the differentiation of GSCs toward a more mature glial phenotype. Overall, our results demonstrate that TRPV2 promotes in vitro and in vivo GSCs differentiation and inhibits their proliferation. Better understanding of the molecular mechanisms that regulate the balance between proliferation and differentiation of GSCs would lead to more specific and efficacious pharmacological approaches.     

2-Methoxyestradiol inhibits proliferation of normal and neoplastic glial cells, and induces cell death, in vitro

2-Methoxyestradiol (2ME), a metabolite of estradiol (E), inhibits proliferation of various tumor cells. In this study we determined the effect of 2ME on human glioblastoma cell lines, in vitro. We compared these cells with cultured astrocytes obtained from traumatized adult rat striatum. Exposure to 2ME had a strong antiproliferative effect on human glioblastoma and caused an increase in the population of apoptotic cells, detected by flow cytometry, in some of the investigated cell lines. A significant number of cells were blocked in the G2/M phase of the cell cycle. Concurrently, the population of cells in the G1 phase decreased in all glioblastoma cell lines. Staining with Hoechst 33258 revealed abnormal nuclear morphology in the proliferating cells treated with 2ME. Treatment with 2ME induced upregulation of wild type p53 in one of the human glioblastoma cell lines as well as in proliferating adult rat astrocytes. We conclude that 2ME inhibits the growth of human glioblastoma cell lines and induces apoptosis, in vitro. This compound deserves further investigation as a treatment for gliomas.     

Modulation of differentiation-related responses in human colon carcinoma cells by protein kinase inhibitor H-7.

1-(isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a potent inhibitor of protein kinases, has been used as a tool to examine the role of protein kinases in a variety of cellular functions. Contingent on the cell type, H-7 has been reported either to inhibit or to promote differentiation. The biological effects of H-7 on human colon adenocarcinoma cells have not been reported. In this study we investigated the effects of H-7 on differentiation - related parameters such as cellular morphology, proliferation, the expression of carcinoembryonic antigen (CEA), fibronectin and cytokeratins in human adenocarcinoma cell lines HCT116 and SW480. H-7 induced pronounced morphological alterations in both cell lines. It induced fibronectin expression and down-modulated CEA expression and secretion in the SW480 cells, but not in the HCT116 cells. Expression of acidic keratins was not affected by H-7 treatment in both cell lines. However, the expression of basic keratins were down-modulated in the HCT116 cells and enhanced in the SW480 cells. These studies showed that the protein kinase inhibitor, H-7, modulated phenotypic properties in human colon adenocarcinoma cells. Alterations in phenotypic properties and their significance in regard to the induction of differentiation are discussed.     

Resveratrol pretreatment desensitizes AHTO-7 human osteoblasts to growth stimulation in response to carcinoma cell supernatants.

Resveratrol, a natural phytoestrogen, has been reported to promote differentiation of murine MC3T3-E1 osteoblasts and to inhibit proliferation of prostate cancer cell lines. In the present study we tested the effects of resveratrol on the increased proliferation of human AHTO-7 osteoblastic cell line induced by conditioned media (CM) from a panel of carcinoma cell lines. This compound was found to modulate AHTO-7 proliferation in a tamoxifen-sensitive mechanism at lower concentrations, but failed to induce the osteoblast differentiation marker alkaline phosphatase (ALP) in contrast to vitamin D3. The proliferative response of AHTO-7 cells to conditioned media from carcinoma cell lines was diminished (30-71.4% inhibition) upon pretreatment with 0.5 microM resveratrol. Highest inhibition was demonstrated for pancreas (BxPC3, Panc-1), breast (ZR75-1) and renal (ACHN) carcinoma cell line supernatants whereas the effect on colon carcinoma (SW620, Colo320DM) cell CM and prostate cancer (PC3, DU145 and LNCaP) CM was less pronounced. Direct addition of resveratrol affected only supernatants of cell lines (<25% inhibition) exhibiting growth stimulatory activity for normal WI-38 lung fibroblasts. Resveratrol inhibited proliferation of DU145 and LNCaP cells in concentrations exceeding 5 microM, altered cell cycle distribution of all prostate cancer cell lines in concentrations as low as 0.5 microM, but did not inhibit the production of osteoblastic factors by these lines. In conclusion, resveratrol failed to induce ALP activity as marker of osteoblast differentiation in human osteoblastic AHTO-7 cells, however, inhibited their response to osteoblastic carcinoma-derived growth factors in concentrations significantly lower than those to reduce growth of cancer cells, thus effectively modulating tumor - osteoblast interaction.     

Up-regulation of E-cadherin by an anti-epidermal growth factor receptor monoclonal antibody in lung cancer cell lines.

Many human epithelial carcinomas are characterized by the overexpression and constitutive activation of the epidermal growth factor receptor (EGF-R) via an autocrine signaling loop. We have investigated the effects of a ligand-blocking monoclonal antibody (mAb) against the EGF-R LA1 on selected parameters of human lung cancer cell lines (H322 and H661) and normal human bronchial epithelial (NHBE) cells. Using Western blot analysis, we show that H322 and NHBE cell lines express comparable levels of EGF-R/p170erbB-1. The LA1 mAb against EGF-R inhibits growth, induces differentiation to a more epithelial phenotype, reduces the constitutive activation of EGF-R, and upregulates epithelial cadherin glycoprotein expression in H322 and NHBE cells. In contrast, LA1 had no effect on either growth, differentiation, receptor tyrosine phosphorylation, or the expression of adhesion molecules in H661 cells, which is consistent with our finding that this cell line does not express detectable levels of EGF-R. These studies demonstrate that a blocking anti-EGF-R mAb can regulate proliferation, differentiation, and the expression of cell adhesion molecules in human bronchial epithelial cells. Our findings suggest possible therapeutic avenues for the treatment of invasive carcinomas via the blockade of EGF-R with antibodies.     

Differential effects of 12-O-tetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells

The effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on 10 human lung carcinoma cell lines were compared to those seen on normal human bronchial epithelial (NHBE) cells. TPA (0.1 to 100 nM) did not enhance the clonal growth rate for any of the cell lines. As little as 3 nM TPA induced the NHBE cells to undergo terminal squamous differentiation and thus completely inhibited their proliferation; in contrast, none of the carcinoma cell lines was significantly inhibited at this concentration, and they all continued to proliferate in as much as 100 nM TPA. To determine if this lack of TPA inhibition of clonal growth reflected resistance to TPA induction of terminal squamous differentiation, we measured the ability of TPA to induce cross-linked envelope formation and to increase plasminogen activator activity in four carcinoma cell lines. Cross-linked envelopes were not induced in two lines, and only a small number were induced in the other two lines relative to NHBE cells; plasminogen activator activity was induced in NHBE cells but not in any of the cell lines.     

Stromal–epithelial interactions modulate cross-talk between prolactin receptor and HER2/Neu in breast cancer

Prolactin (PRL) promotes the proliferation and survival of breast cancer cells in part via the transactivation of human epidermal growth factor receptor 2 (HER2), also known as Neu in rodents. A PRL receptor (PRLR) antagonist, G129R, has been developed, which indirectly inhibits the tyrosine phosphorylation of HER2 (p-HER2) in human breast cancer cell lines. In this study, we investigate the effects of cancer-associated fibroblasts (CAFs) upon this molecular cross-talk using tumor cells and CAFs derived from spontaneous mammary tumors of female MMTV-neu transgenic mice. Tumors were resected and cultured as small tumor chunks (~3 mm3) or were cultured in monolayer. G129R reduced tyrosine phosphorylation of Neu (p-Neu) in a dose-dependent manner (IC50~10 μg/ml) in tumor chunks, but had no effect on primary tumor epithelial cells grown in monolayer. Direct co-culture of mouse or human tumor epithelial cell lines with CAFs restored the epithelial cells' response to G129R, similar to that observed in mouse tumor chunks. The addition of PRL, as expected, induced p-Neu in both the tumor chunk and co-culture models. The inhibitory effect of G129R was absent when CAFs were physically separated from mouse tumor epithelial cells using a transwell system, or when CAFs were replaced with normal fibroblasts in direct co-culture with human or mouse tumor epithelial cells. In vivo, G129R reduced p-Neu levels in primary mammary tumors of mice in a time- and dose-dependent manner. In conclusion, CAFs play a critical role in bridging the cross-talk between PRL and HER2/Neu in both mouse and human models of breast cancer. The inhibitory effects of G129R on p-Neu and on tumor growth are dependent upon interactions of tumor epithelial cells with CAFs.     

Human homolog of Drosophila expanded, hEx, functions as a putative tumor suppressor in human cancer cell lines independently of the Hippo pathway

The Hippo signaling network is proving to be an essential regulator within the cell, participating in multiple cellular phenotypes including cell proliferation, apoptosis, cell migration and organ size control. Much of this pathway is conserved from flies to mammals; however, how the upstream components, namely Expanded, affect downstream processes in mammalian systems has remained elusive. Only recently has human Expanded (hEx), also known as FRMD6 or Willin, been identified. However, its functional significance with respect to its putative tumor suppressor function and activation of the Hippo pathway has not been studied. In this study, we show for the first time that hEx possesses several tumor suppressor properties. First, hEx dramatically inhibits cell proliferation in two human cancer cell lines, MDA-MB-231 and MDA-MB-436 cells, and sensitizes these cells to the chemotherapeutic drug Taxol. Furthermore, downregulation of hEx in the immortalized MCF10A breast cell line leads to enhanced proliferation and resistance to Taxol treatment. As evidence for its tumor suppressor function, overexpression of hEx inhibits colony formation, soft agar colony growth in vitro and in vivo tumor growth in nude mice. Although Drosophila expanded (ex) can activate the Hippo pathway, surprisingly no significant alterations were discovered in the phosphorylation status of any of the Hippo pathway components, including downstream tumor suppressor LATS1, upon overexpression of hEx. In addition, knockdown of both LATS1 and LATS2 in hEx-overexpressing cells was unable to rescue the hEx phenotype, suggesting that hEx functions independently of the Hippo pathway in this cell line. Alternatively, we propose a mechanism through which hEx inhibits progression through the S phase of the cell cycle by upregulating p21(Cip1) and downregulating Cyclin A. This is the first study to functionally characterize hEx and show that hEx acts in a distinct manner compared with Drosophila expanded.     

Inhibition of MAPK kinase signaling pathways suppressed renal cell carcinoma growth and angiogenesis in vivo

The mitogen-activated protein kinase (MAPK) signaling pathways play essential roles in cell proliferation and differentiation. Recent studies also show the activation of MAPK signaling pathways in tumorigenesis, metastasis, and angiogenesis of multiple human malignancies, including renal cell carcinoma (RCC). To assess the role of this pathway in regulating the proliferation and survival of RCC cells, we first examined the expression of MAPK kinase (MKK) and MAPK in clear cell RCC and confirmed the overexpression of MKK1 and extracellular signal-regulated kinase 2 (ERK2) in these tumors. We then tested the effects of pharmacologic inhibition of MKK on human RCC cell lines, both in vitro and in vivo, using anthrax lethal toxin (LeTx), which cleaves and inactivates several MKKs. Western blotting showed that the phosphorylation levels of ERK, c-Jun-NH(2) kinase, and p38 MAPK decreased after 72 h of LeTx treatment. Exposure to LeTx for 72 h reduced cell proliferation by 20% without significant effects on cell cycle distribution and apoptosis. Anchorage-independent growth of RCC cells was dramatically inhibited by LeTx. In vivo studies showed that tumor growth of RCC xenografts could be suppressed by LeTx. Extensive necrosis and decreased tumor neovascularization were observed after LeTx treatment. LeTx also showed direct inhibition of proliferation of endothelial cells in vitro. Our results suggest that suppression of one or more MAPK signaling pathways may inhibit RCC growth through the disruption of tumor vasculature.     

Suramin inhibits the proliferation and stimulates the differentiation of tumoral cell lines HT29-D4 and C6]

Suramin is an anti-helminthic drug that has been shown to antagonize the effects of a variety of growth factors including EGF, PDGF and TGF beta. When added to the culture medium, suramin inhibited the proliferation of both human colonic adenocarcinoma cells HT29-D4 and rat glioma cells C6. Suramin also induced the differentiation of both cell lines: appearance of cellular extensions for C6 cells, enterocyte-like epithelial differentiation for HT29-D4-cells. In the latter case, suramin probably acts at the level of glucose metabolism, which is likely to be modulated by autocrine growth factors. The permanent secretion of such factors probably stimulates HT29-D4 proliferation and simultaneously inhibits their differentiation. It is hypothesized that interfering with this autocrine loop, suramin allows HT29-D4 cells to differentiate.