C26 Cells Research Articles

Anti-inflammatory sesquiterpenes from the fruits of Pittosporum undulatum

Using a bioassay-guided fractionation approach, two unprecedented guaiane-type sesquiterpenes along with five known compounds were isolated from the fruits of Pittosporum undulatum (Pittosporacea), traditionally used in Azores archipelago due to their anti-inflammatory properties. The structures of compounds were established by 1D and 2D NMR spectroscopic techniques and MS analyses. The in vitro anti-inflammatory activity of compounds was evaluated by analyzing their inhibitory effects on chemical mediators released by the LPS activated RAW 264.7 murine macrophages cell line. The new compounds displayed anti-inflammatory activity comparable to that found for the positive control indomethacin and no cytotoxic effect in the mentioned cell line. Furthermore, some compounds were also assessed for their anti-proliferative activity in lung large cell carcinoma COR-L23 and amelanotic melanoma C32 cells.

Blocking Fibroblast Growth Factor Receptor Signaling Inhibits Tumor Growth, Lymphangiogenesis, and Metastasis

Fibroblast Growth Factor receptor (FGFR) activity plays crucial roles in tumor growth and patient survival. However, FGF (Fibroblast Growth Factor) signaling as a target for cancer therapy has been under-investigated compared to other receptor tyrosine kinases. Here, we studied the effect of FGFR signaling inhibition on tumor growth, metastasis and lymphangiogenesis by expressing a dominant negative FGFR (FGFR-2DN) in an orthotopic mouse mammary 66c14 carcinoma model. We show that FGFR-2DN-expressing 66c14 cells proliferate in vitro slower than controls. 66c14 tumor outgrowth and lung metastatic foci are reduced in mice implanted with FGFR-2DN-expressing cells, which also exhibited better overall survival. We found 66c14 cells in the lumen of tumor lymphatic vessels and in lymph nodes. FGFR-2DN-expressing tumors exhibited a decrease in VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) or podoplanin-positive lymphatic vessels, an increase in isolated intratumoral lymphatic endothelial cells and a reduction in VEGF-C (Vascular Endothelial Growth Factor-C) mRNA expression. FGFs may act in an autocrine manner as the inhibition of FGFR signaling in tumor cells suppresses VEGF-C expression in a COX-2 (cyclooxygenase-2) or HIF1-α (hypoxia-inducible factor-1 α) independent manner. FGFs may also act in a paracrine manner on tumor lymphatics by inducing expression of pro-lymphangiogenic molecules such as VEGFR-3, integrin α9, prox1 and netrin-1. Finally, in vitro lymphangiogenesis is impeded in the presence of FGFR-2DN 66c14 cells. These data confirm that both FGF and VEGF signaling are necessary for the maintenance of vascular morphogenesis and provide evidence that targeting FGFR signaling may be an interesting approach to inhibit tumor lymphangiogenesis and metastatic spread.

Abstract 3676: The hypoxia-activated pro-drug tirapazamine suppresses outgrowth of colorectal liver metastases following radiofrequency ablation

Abstract Background Radiofrequency ablation (RFA) is a common procedure for the management of colorectal liver metastases. RFA-generated lesions are surrounded by a rim of hypoxia that is associated with aggressive outgrowth of intrahepatic micrometastases. Hypoxia-activated pro-drugs such as tirapazamine (TPZ) are designed to selectively kill tumour cells under hypoxic conditions. Therefore we hypothesised that TPZ may have therapeutic value in limiting hypoxia-associated tumour outgrowth following RFA. Methods Murine C26 and MC38 colorectal cancer cells were grown under hypoxia and normoxia in vitro and were treated with different concentrations of TPZ. Apoptosis and cell cycle distribution were assessed by western blot and FACS analysis. Proliferative capacity was tested by colony formation assays. Mice harbouring microscopic colorectal liver metastases were treated with RFA, followed by a single injection of TPZ (60 mg/kg) or saline. Tumour load was assessed morphometrically 7 days later. Results TPZ induced apoptosis of colorectal tumour cells under hypoxia in vitro, which completely abolished clone-forming capacity. Under normoxia, TPZ caused a G2 cell cycle arrest from which cells recovered partly. This reduced, but did not abolish, colony-forming capacity. A single dose of TPZ largely prevented accelerated outgrowth of hypoxic micrometastases following RFA. TPZ administration was associated with minimal toxicity. Conclusions TPZ kills colorectal cancer cells in a hypoxia-dependent manner and potently suppresses hypoxia-associated outgrowth of liver metastases with limited toxicity. This warrants further studies to assess the potential value of TPZ, or other hypoxia-activated pro-drugs, as adjuvant therapeutics following RFA treatment of colorectal liver metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3676. doi:1538-7445.AM2012-3676

Effects of tirapazamine on experimental colorectal liver metastases after radiofrequency ablation1

Radiofrequency ablation (RFA) is a common procedure for the management of colorectal liver metastases. RFA-generated lesions are surrounded by a rim of hypoxia that is associated with aggressive outgrowth of intrahepatic micrometastases. Hypoxia-activated prodrugs such as tirapazamine are designed selectively to induce apoptosis in tumour cells under hypoxic conditions. Therefore, it was hypothesized that tirapazamine may have therapeutic value in limiting hypoxia-associated tumour outgrowth following RFA. Murine C26 and MC38 colorectal cancer cells were grown under hypoxia and normal oxygenation in vitro, and treated with different concentrations of tirapazamine. Apoptosis and cell cycle distribution were assessed by western blot and fluorescence-activated cell sorting analysis. Proliferative capacity was tested by means of colony-formation assays. Mice harbouring microscopic colorectal liver metastases were treated with RFA, followed by a single injection of tirapazamine (60 mg/kg) or saline. Tumour load was assessed morphometrically 7 days later. Tirapazamine induced apoptosis of colorectal tumour cells under hypoxia in vitro. Under normal oxygenation, tirapazamine caused a G2 cell cycle arrest from which cells recovered partly. This reduced, but did not abolish, colony-forming capacity. A single dose of tirapazamine largely prevented accelerated outgrowth of hypoxic micrometastases following RFA. Tirapazamine administration was associated with minimal toxicity. Tirapazamine induced apoptosis in colorectal cancer cells in a hypoxia-dependent manner and potently suppressed hypoxia-associated outgrowth of liver metastases with limited toxicity. This warrants further study to assess the potential value of tirapazamine, or other hypoxia-activated prodrugs, as adjuvant therapeutics following RFA treatment of colorectal liver metastases.

Suppression of lamin A/C by short hairpin RNAs promotes adipocyte lineage commitment in mesenchymal progenitor cell line, ROB-C26

Lamin A/C gene encodes a nuclear membrane protein, and mutations in this gene are associated with diverse degenerative diseases that are linked to premature aging. While lamin A/C is involved in the regulation of tissue homeostasis, the distinct expression patterns are poorly understood in the mesenchymal cells differentiating into adipocytes. Here, we examined the expression of lamin A/C in a rat mesenchymal progenitor cell-line, ROB-C26 (C26). Immunocytochemical analysis showed that lamin A/C was transiently down-regulated in immature adipocytes, but its expression increased with terminal differentiation. To elucidate the role of lamin A/C expression on mesenchymal cell differentiation, lamin A/C expression was suppressed using short hairpin RNA (shRNA) molecules in C26 cells. In the absence of adipogenic stimuli, lamin A/C shRNA decreased alkaline phosphatase (ALP) activity, but induced preadipocyte factor -1 (Pref-1) mRNA expression. In the presence of adipogenic stimuli, lamin A/C knockdown promotes adipocytes differentiation, as assessed by the detection of an increase in Oil Red O staining. RT-PCR analysis showed that lamin A/C shRNA resulted in increased mRNA expression of PPARγ2 and aP2 during adipocyte differentiation. These results suggest that decreased lamin A/C expression levels not only suppress osteoblast phenotypes but also promote adipocyte differentiation in C26 cells.

Ultrasound-mediated interferon β gene transfection inhibits growth of malignant melanoma

We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon β (IFN-β) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-β in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-β genes mixed with microbubbles. Successful sonotransfection with IFN-β gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-β gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

In vitro Cytotoxic Activity of Extracts and Isolated Constituents of Salvia leriifoliaBenth. against a Panel of Human Cancer Cell Lines

In the course of recent efforts to identify new potential antiproliferative active principles, Salvia leriifolia extracts and isolated constituents were evaluated for their cytotoxic activity against a panel of human cancer cell lines, including renal adenocarcinoma (ACHN), amelanotic melanoma (C32), colorectal adenocarcinoma (Caco-2), lung large cell carcinoma (COR-L23), malignant melanoma (A375), lung carcinoma (A549), and hepatocellular carcinoma (Huh-7D12) cells. The hexane and CH(2) Cl(2) extracts showed the strongest cytotoxic activity against the C32 cell line with IC(50) values of 11.2 and 13.6 μg/ml, respectively, and the AcOEt extract was the most active extract against the COR-L23 cell line (IC(50) of 20.9 μg/ml). Buchariol, a sesquiterpene obtained by biofractionation of the CH(2) Cl(2) extract, exhibited a higher activity than the positive control vinblastine against the C32 and A549 cell lines (IC(50) values of 2.1 and 12.6 μM, resp.). Interesting results were also obtained for naringenin, a flavonoid isolated from the AcOEt extract, which exhibited a strong cytotoxic activity against the C32, LNCaP, and COR-L23 cell lines (IC(50) values of 2.2, 7.7, and 33.4 μM, resp.), compared to vinblastine (IC(50) values of 3.3, 32.2, 50.0 μM, resp.). None of the tested compounds affected the proliferation of skin fibroblasts (142BR), suggesting a selective activity against tumor cells.

Preparation of Core Cross-Linked PCL-PEG-PCL Micelles for Doxorubicin Delivery In Vitro

Doxorubicin has been widely used in cancer treatment, but its severe side-effects restrict its clinical application greatly. So, we hope to design a novel delivery system to decrease its side-effects. In this paper, we prepared core cross-linked micelle based on poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) (CCPCEC) at about 30 nm in diameter with a narrow distribution. The prepared core cross-linked PCL-PEG-PCL micelles were employed to load doxorubicin by pH-induced self-assembly method. Doxorubicin-loading did not obviously affect the micelle size or size distribution. Furthermore, these micelles exhibited a significantly enhanced thermodynamic stability against dilution with aqueous solvents and showed CMC in the range of 1 x 10(-3) to 2 x 10(-3) mg/mL. Cytotoxicity study confirmed great biocompatibility of the micelles and showed that the encapsulated doxorubicin in CCPCEC micelles enhanced the cytotoxicity of doxorubicin on C26 cell line in vitro. Moreover, in vitro release profile demonstrated a significant difference between rapid release of free doxorubicin and much slower and sustained release of doxorubicin-loaded core cross-linked micelles. In addition, a faster DOX-release from micelles at pH 5.5 than that at pH 7.4 was also observed. These results suggested that this new biodegradable Core Cross-linked PCL-PEG-PCL Micelles might be potential carriers for drug delivery in cancer chemotherapy.

Anti-tumor activity of liposome encapsulated fluoroorotic acid as a single agent and in combination with liposome irinotecan

To test the hypothesis that co-delivery of synergistic drug combinations in the same liposome provides a better anti-tumor effect than the drugs administered in separate liposomes, fluoroorotic acid (FOA) alone and in combination with irinotecan (IRN) were encapsulated in liposomes and evaluated for their anti-tumor activity in the C26 colon carcinoma mouse model. A new chaotropic loading strategy was devised wherein FOA was dissolved in 7 M urea to increase its solubility. This enabled the passive loading of FOA into liposomes at a high concentration. IRN was remote loaded into liposomes that contained the ammonium salt of the multi-valent 1,2,3,4-butanetetracarboxylic acid with a greater than 90% efficiency and at a drug to lipid ratio of 0.2:1. When the two molecules were loaded into the same liposome, FOA was used to remote load IRN. Modulation of the drug/lipid ratio, temperature, and loading time allowed for consistent co-encapsulation of FOA + IRN at various molar ratios. The anti-tumor activity of L-FOA, L-IRN, L-FOA-IRN (5:1), and the L-FOA + L-IRN mixture (5:1) were examined in the C26 mouse model. The maximum tolerated dose of L-FOA was 10 mg/kg given weekly as compared to 100 mg/kg of the non-encapsulated FOA. Delivering two drugs in the same liposome provided a statistically better anti-tumor effect than delivering the drugs in separate liposomes at the same drug ratio. However, the synergistic activity of the 5:1 ratio of free drugs measured on C26 cells in vitro was not observed in the C26 tumor mouse model. These findings point out the challenges to the design of synergistic treatment protocols based upon results from in vitro cytotoxicity studies. L-FOA at 10 mg/kg as a single agent provided the best anti-tumor efficacy which supports previous suggestions that L-FOA has useful properties as a liposome dependent drug.

Abstract 1463: SIX1 induces lymphatic and distant metastasis by upregulating VEGF-C and stimulating lymphangiogenesis in breast cancer

Abstract The homeoprotein Six1 is expressed during embryogenesis and re-expressed in 50% of primary and in 90% of metastatic breast cancer lesions.Using an orthotopic xenograft model of breast cancer, Six1 overexpression results in a 40% increased in lymphatic metastases as compared to controls.Six1 correlates with positive lymph node status in human breast cancer, further suggesting that it influences lymphatic metastasis, perhaps through stimulating lymphangiogenesis.Microarray analysis revealed that the critical lymphangiogenic regulator, VEGF-C, may be a target of Six1.Real-time PCR and VEGF-C promoter-luciferase assays demonstrated that VEGF-C upregulation is mediated transcriptionally by Six1.Chromatin immunoprecipitation analysis demonstrates in vivo binding of Six1 to the VEGF-C promoter.Increased levels of VEGF-C mRNA in response to Six1 overexpression result in increased intracellular and secreted VEGF-C protein.We quantified the number of intra- and peri-tumoral vessels in MCF7-Six1 versus MCF7-Control tumors and the MCF7-Six1 tumors display increased lymphangiogenesis and a 5-fold increase in the frequency of lymphatic invasion compared to the MCF7-Ctrl tumors.To determine whether Six1 depends on VEGF-C to increase lymphangiogenesis and lymphatic metastasis, we knocked down VEGF-C in MCF7-Six1 cells and injected the cells into the mammary gland of nude mice.We observed decreased lymph node metastases in the mice bearing MCF7-Six1/VEGF-C KD tumors compared to the MCF7-Six1 tumors, suggesting Six1-induced lymphatic metastasis is in part through upregulation of VEGF-C.We also used a complementary model, the 66c14 mammary carcinoma cell line which metastasizes primarily through the lymphatic system and expresses high levels of Six1, to study the role of Six1 in lymphatic metastasis.Six1 knockdown (KD) in 66c14 cells resulted in decreased levels of Vegf-c and strikingly, the Six1 KD tumors also contained less lymphatic vessel formation, suggesting a reduction of lymphangiogensis. Finally, Six1 KD dramatically decreased lung metastases in the 66c14 model as seen using bioluminescent imaging. When we rescued Vegf-c expression in 66c14 in the context of Six1 KD, we observed a restoration of lymphatic metastasis, but the rescue failed to restore lung metastasis, suggesting Six1 regulates additional factor/pathways required to accomplish the later stages of metastasis.Regulation of VEGF-C by Six1 is supported by clinical observations that correlation of Six1 and VEGF-C can be extended broadly to breast cancer cell lines (oncomine) and to human breast cancer tissues (tissue array).In conclusion, we demonstrate that Six1 mediates lymphangiogenesis and lymphatic metastasis in multiple mouse models via its ability to regulate VEGF-C, and extend this observation to human tissues where Six1 and VEGF-C overexpression are significantly correlated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1463. doi:10.1158/1538-7445.AM2011-1463

Deltonin Isolated from Dioscorea zingiberensis Inhibits Cancer Cell Growth through Inducing Mitochondrial Apoptosis and Suppressing Akt and Mitogen Activated Protein Kinase Signals

Deltonin is an active component purified from Dioscorea zingiberensis WRIGHT (DZW), and has shown anticancer effects. However, its mechanism of action remains elusive. In the present study, we investigated the effect of Deltonin on a panel of cancer cell lines and analyzed its mechanism in C26 cells, a murine colon carcinoma cell. Our results showed that Deltonin markedly inhibited the growth of all examined cancer cell lines. Deltonin induced dose- and time-dependent apoptosis in C26 cells. The event of apoptosis was accompanied by the release of cytochrome c, depolarization of mitochondrial membrane potential, and dose- and time-dependent reactive oxygen species (ROS) generation. Deltonin also increased the expression of Bax, decreased the expression of B-cell lymphoma/lewkmia-2 (Bcl-2), and induced the activation of caspase 9, caspase 3 and poly(ADP-ribose) polymerase (PARP). Furthermore, Deltonin decreased Akt and extracellular signal-regulated kinase-1/2 (ERK(1/2)) activity. These results demonstrate that Deltonin mediates the growth inhibition of cancer cells through multiple targets, which include the generation of reactive oxygen species (ROS), mitochondrial apoptosis and the inhibition of the mitogen-activated protein kinase (MAPK) and Akt signaling pathways, suggesting Deltonin is a potent cancer preventive and therapeutic agent.

Versican G3 Promotes Mouse Mammary Tumor Cell Growth, Migration, and Metastasis by Influencing EGF Receptor Signaling

Increased versican expression in breast tumors is predictive of relapse and has negative impact on survival rates. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. Here we evaluated the expression of versican in mouse mammary tumor cell lines observing that 4T1 cells expressed highest levels while 66c14 cells expressed low levels. We exogenously expressed a G3 construct in 66c14 cells and analyzed its effects on cell proliferation, migration, cell cycle progression, and EGFR signaling. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and systemic metastasis in vivo. Activation of pERK correlated with high levels of G3 expression. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. G3 expressing cells demonstrated increased CDK2 and GSK-3β (S9P) expression, which were related to cell growth. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling pathway. Taken together, EGFR-signaling appears to be an important pathway in versican G3-mediated breast cancer tumor invasiveness and metastasis.

Preoperative dietary restriction reduces hepatic tumor load by reduced E‐selectin‐mediated adhesion in mice

Inflammatory responses facilitate metastasis by increasing expression of adhesion molecules. Dietary restriction (30% reduction in daily calorie intake) reduces the expression of adhesion molecules and protects against surgically induced inflammation. DR might therefore beneficially interfere with surgery-induced inflammation and subsequent adhesion of circulating tumor cells. BALB/c mice were subjected to 2 weeks dietary restriction prior to inoculation with tumor cells. Intra-splenic injection of 5.0 x 10(4) C26-colon carcinoma cells was followed by splenectomy. Hepatic tumor load was scored after 10 days as a percentage (tumor surface/total liver surface) on H&E stained sections. Liver mRNA expression of adhesion molecules was determined and the effect of serum from dietary restriction mice on in vitro tumor growth and adhesion capacity was assessed. Preoperative dietary restriction significantly reduced mRNA expression levels of E-selectin (P = 0.0087) and hepatic tumor load (P = 0.036). Dietary restriction serum did not affect in vitro cell growth but reduced in vitro adhesion of C26 cells to endothelial cells (P = 0.0043). Preoperative dietary restriction reduces hepatic tumor load after injection with tumor cells. Reduced adhesion to endothelial cells and reduced mRNA expression of E-selectin suggest that dietary restriction reduces tumor load by lowering the adhesion of circulating tumor cells to hepatic vascular endothelium.

HERG K+ channel related chemosensitivity to sparfloxacin in colon cancer cells

Potassium channels are essential for the regulation of cell proliferation. As reported, HERG protein is overexpressed in a wide range of human tumors, including colon carcinoma. The aim of this study was to investigate the effects of antibacterial agents sparfloxacin (SPFX), a blocker of HERG channel, on HERG K+ channel highly expressing colon cancer cells. Expression of HERG and apoptosis correlative proteins was examined by Western blotting. The MTT assay was used to detect the cytotoxicity of drugs and drug combination in vitro. Gene transfection was used to examine the changes in herg-related chemosensitivity. Cell apoptosis was analyzed by flow cytometry. The migration and invasion capacity of tumor cells by SPFX was determined by gelatin zymography assay and Boyden chamber. The in vivo efficacy of SPFX was assessed in murine colon carcinoma C26 in BALB/c mice and human colon carcinoma HCT116 xenografts in nude mice. High expression of HERG protein was detected in colon cancer C26, HCT116 and HT-29 cells. The cell viability of the colon cancer cells was inhibited by SPFX in a dose-dependent manner. SPFX induced apoptosis and inhibited migration and invasion of colon cancer HCT116 cells. The increase in apoptosis was associated with a decrease in procaspase-3 and Bcl-2 protein expression. Study with herg-transfected HEK293 cells and siRNA-knock down HCT116 cells confirmed that the cell viability inhibition by SPFX was correlated with HERG expression. When combined with 5-fluorouracil, SPFX showed synergistic anti-proliferation activity in HCT116 and HT-29 cells. Furthermore, SPFX inhibited the growth of human colon carcinoma HCT116 xenografts and showed synergistic effect with 5-fluorouracil in vivo. Our finding suggested that SPFX could be a biochemical modulator in treatment of colon cancer with chemotherapeutic drugs.

Abstract 1431: Nerve growth factor-expressing stromal cells in the microenvironment of hepatic colorectal carcinoma metastasis: Clinical occurrence and functional implications

Abstract Nerve growth factor (NGF) is increased during hepatic regeneration and carcinogenesis, but its role during hepatic metastasis is unknown. A tissue-array collection of metastases from 24 patients who had undergone hepatic excision of colorectal adenocarcinoma metastases was used to investigate NGF and neurotrophin receptor expression by cancer and stromal cells. NGF immunostaining of cancer cells only occurred in 2 out of 24 patients with hepatic metastases, while around 80% of patients had metastases with NGF-expressing stromal cells. Conversely, high affinity TrkA neurotrophin receptor immunoreactivity was mainly concentrated in cancer cells, with low expression occurring in tumor stroma. However, NGF immunostaining of tumor stroma and cancer cell immunostaining with anti-ki67 antibodies did not correlate, suggesting that NGF was not associated to metastatic cell proliferation. Anti-alpha-smooth muscle actin (ASMA) antobodies revealed that majority of metastasis-associated NGF-expressing cells had a myofibroblast phenotype. Interestingly, NGF immunoreactivity was unequivocally localized to desmin-expressing hepatic stellate cells (HSC), a prototypic myofibroblast precursor, and perimetastatic hepatocytes, located at the invasion front of metastases. NGF-expressing hepatocytes had phenotypic features suggesting epithelial-to-mesenchymal transition (snail, vimentin and ASMA overexpression with E-cadherin expression decrease). Consistent with clinical findings, a similar immunostaining pattern was reproduced in the experimental hepatic colonization of C26 and 51b colorectal cells. Moreover, NGF increased by two-fold in the hepatic blood from metastasis-bearing mice and also significantly increased by 2-3-fold in the supernatant of primary cultured HSC given tumor cell-conditioned medium (CM), and hepatocytes given tumor-activated HSC-CM, but not tumor cell-CM. Both NGF secretion and epithelial-to-mesenchymal transition in hepatocytes were TGFbeta-dependent. Recombinant NGF dose-dependently increased chemotactic migration, but not proliferation and adhesion of neurotrophin receptor-expressing cancer cells in vitro. HSC migration-stimulating activity of VEGF and tumor-activated hepatocytes were also NGF-mediated as shown with anti-NGF antibodies. Our results demonstrate that both hepatocyte- and HSC-derived myofibroblasts secrete NGF in the hepatic metastasis microenvironment of human and experimental colorectal carcinoma. The mechanism is TGFbeta-dependent. The migration-stimulating effect of NGF on both cancer and stromal cells suggests its contribution to hepatic metastasis development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1431.

In vitro Antioxidant and Antiproliferative Activities of Flavonoids from Ailanthus excelsa (Roxb.) (Simaroubaceae) Leaves

The present study aimed to investigate the chemical composition, and the antioxidant and antiproliferative activities of Ailanthus excelsa, a plant used in Egyptian traditional medicine. Chromatographic separation of a methanol extract of A. excelsa leaves yielded four flavones, namely apigenin (1), apigenin 7-O-beta-glucoside (2), luteolin (3), and luteolin 7-O-beta-glucoside (4), and seven flavonols, namely kaempferol (5), kaempferol 3-O-alpha-arabinoside (6), kaempferol 3-O-beta-galactoside (7), quercetin (8), quercetin 3-O-alpha-arabinoside (9), quercetin 3-O-beta-galactoside (10), and quercetin 3-O-rutinoside (11). The A. excelsa extract tested in different in vitro systems (DPPH and FRAP assays) showed significant antioxidant activity. The potential antiproliferative activity of the A. excelsa extract and isolated flavonoids against five human cancer cell lines such as ACHN, COR-L23, A375, C32, and A549 was investigated in vitro by the SRB assay in comparison with one normal cell line, 142BR. The extract exhibited the highest inhibitory activity against C32 cells with an IC50 value of 36.5 microg ml(-1). Interesting activity against COR-L23 was found with 10 (IC50 value of 3.2 microg ml(-1)). Compounds 1 and 3 inhibited cell growth in both amelanotic melanoma and malignant melanoma cells.

A novel approach to deliver anticancer drugs to key cell types in tumors using a PDGF receptor-binding cyclic peptide containing carrier

Tumor stromal cells have been recently recognized to contribute to tumor growth. Therefore, we hypothesized that delivery of anticancer drugs to these cells in addition to the tumor cells might treat cancer more effectively. Stromal cells abundantly expressed Platelet-Derived Growth Factor Receptor-beta (PDGFR-β) in different human tumors as shown with immunohistochemistry. To achieve targeting through PDGFR-β, we developed a carrier by modifying albumin with a PDGFR-β recognizing cyclic peptide (pPB-HSA). pPB-HSA specifically bound to PDGFR-β-expressing 3T3 fibroblasts, C26 and A2780 cancer cells in vitro. Subsequently, doxorubicin was conjugated to pPB-HSA through an acid-sensitive hydrazone linkage. In vitro, Dox-HSA-pPB was taken up by fibroblasts and tumor cells and a short exposure of the conjugate induced cell death in these cells. In vivo, the conjugate rapidly accumulated into PDGFR-β expressing cells in C26 tumors. Treatment with Dox-HSA-pPB significantly reduced the C26 tumor growth in mice while free doxorubicin treated mice had lower response to the therapy. Furthermore, in contrast to free doxorubicin the conjugate did not induce loss in body weight. In conclusion, the present study reveals a novel approach to target key cell types in tumors through PDGFR-β, which can be applied to enhance the therapeutic efficacy of anticancer drugs.

In vitro cytotoxic effects of Senecio stabianus Lacaita (Asteraceae) on human cancer cell lines

This study is aimed to evaluate the in vitro cytotoxicity of Senecio stabianus Lacaita (Asteraceae) against renal adenocarcinoma ACHN, hormone-dependent prostate carcinoma LNCaP, amelanotic melanoma C32 and human breast adenocarcinoma MCF-7 cell lines. The n-hexane extract showed an interesting activity with IC50 values of 62.7 and 71.1 μg mL−1 against C32 and LNCaP, respectively. Two compounds identified in the n-hexane extract, linalool and β-caryophyllene, were found to be active against C32 cells (IC50 values of 23.2 and 20.1 μg mL−1, respectively). Among tested constituents, the highest activity was found when α-humulene was applied to LNCaP cells (IC50 of 11.2 μg mL−1). In order to complete the phytochemical study on S. stabianus, which has not been investigated previously, the most characteristic secondary metabolites of Senecio genus, pyrrolizidine alkaloids (PAs), were analysed. Here we report the isolation, structural elucidation and identification of nine PAs.

Albumin-Binding and Tumor Vasculature Determine the Antitumor Effect of 15-Deoxy-Δ12,14-Prostaglandin-J2in vivo

15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)), a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, induces cell death in tumor cells in vitro; however, no study showed its in vivo effect on tumors. Here, we report that 15d-PGJ(2) shows antitumor effects in vivo in mice. However, its effects correlate with tumor uptake of albumin, to which it reversibly binds. 15d-PGJ(2) induces cell death in B16F10 melanoma and C26 colon carcinoma cells in vitro. These effects were not elicited through PPARgamma-dependent pathways because an irreversible PPARgamma antagonist GW9662 did not inhibit these effects. Caspase- and nuclear factor kappaB- (NF-kappaB) dependent pathways were found to be involved as determined with caspase-3/7 fluorescent assay and NF-kappaB containing plasmid transfection assay, respectively. Noticeably, 15d-PGJ(2) had significantly stronger effects in C26 cells compared with B16 cells in all assays. However, in vivo, there was no effect on C26 tumors, yet it significantly inhibited the B16 tumor growth in mice by 75%. We found that 15d-PGJ(2) rapidly bound to albumin and in vivo albumin greatly distributed to B16 tumors compared with C26 tumors, shown with gamma-camera imaging and immunohistochemical staining. Albumin accumulation can be attributed to the large blood vessel diameter in B16 tumors and an enhanced permeability and retention effect. These findings suggest that 15d-PGJ(2) can be an effective therapeutic agent for cancer, although its effects seem to be limited to the tumors allowing albumin penetration.

Structure–toxicity relationship of phenolic analogs as anti-melanoma agents: An enzyme directed prodrug approach

The aim of this study was to identify a phenolic prodrug compound that is minimally metabolized by rat liver microsomes, but yet could form quinone reactive intermediates in melanoma cells as a result of its bioactivation by tyrosinase. In current work, we investigated 24 phenolic compounds for their metabolism by tyrosinase, rat liver microsomes and their toxicity towards murine B16-F0 and human SK-MEL-28 melanoma cells. A linear correlation was found between toxicities of phenolic analogs towards SK-MEL-28 and B16-F0 melanoma cells, suggesting similar mechanisms of toxicity in both cell lines. 4-HEB was identified as the lead compound. 4-HEB (IC 50 48 h, 75 μM) showed selective toxicity towards five melanocytic melanoma cell lines SK-MEL-28, SK-MEL-5, MeWo, B16-F0 and B16-F10, which express functional tyrosinase, compared to four non-melanoma cells lines SW-620, Saos-2, PC3 and BJ cells and two amelanotic SK-MEL-24, C32 cells, which do not express functional tyrosinase. 4-HEB caused significant intracellular GSH depletion, ROS formation, and showed significantly less toxicity to tyrosinase specific shRNA transfected SK-MEL-28 cells. Our findings suggest that presence of a phenolic group in 4-HEB is critical for its selective toxicity towards melanoma cells.