Transplanted Tumor Cells Research Articles

The Effect of Coumarin Derivate from the Stem Bark of Calophyllum biflorum on the In Vivo Growth of Transplantable C3H Mammary Tumor Cells

Calanone, is a coumarin derivate which was isolated from the stem bark of Calophyllum biflorum.To know the effect of calanone on the in vivo growth of transplantable C3H mammary tumor cells, C3H mice were used which were divided into : one group of untreated control, one group of solvent control (injected with 0,1 mL PEG 400) and four treated groups, each of which were injected subcutaneously near the tumor with 0,1 mL of 1 mg/mL, 2 mg/mL, 4 mg/mL, and 8 mg/mL of calanone in PEG 400 solvent respectively. The injections were given three times a week, for four weeks. By using Friedman test, for non parametric statistical analysis of the weekly observed tumor volume, it was shown that there was a significant decrease in the tumor growth of the group treated with calanone solution of 4 mg/mL dosage, compared to the control or other groups.  

The effect of monotherapy and combined therapy with NSC-631570 (Ukrain) on growth of low- and high-metastasizing B16 melanoma in mice

NSC-631570 (ukrain) is a semisynthetic derivative of the Chelidonium majus alcaloids and the alkylans thiotepa. It exerts a selective cytotoxic effect on tumor cells in vitro and in vivo and shows the ability to modulate immunocyte functions. Purpose. The aim of our work was to carry out a comparative investigation of the effects of NSC-631570 alone or in combination with pathogen-associated molecules (PAM) on the growth of low- and high-metastasizing melanoma B16 in mice. NSC-631570 was administered intravenously and PAM intramuscularly to tumor-bearing mice seven times every third day, starting from the second day after the transplantation of tumor cells. The effect of monotherapy and combined therapy on tumor growth was evaluated by the indices of tumor growth inhibition in experimental animals. Cell cycle distribution of cancer cells was determined by flow cytometry. TAP1 and TAP2 expression was evaluated by RT-PCR. The metabolic activity of phagocytes was determined by NBT-test, phagocytosis was tested by flow cytometry, and arginase activity was estimated by colorimetric determination of urea. Combined therapy and monotherapy with NSC-631570 resulted in significant inhibition of tumor growth in melanoma-bearing mice. Monotherapy with Ukrain was more effective in mice with high-metastasizing tumors. The therapeutic efficacy of NSC-631570 used in combination with PAM was more expressed in mice with low-metastasizing melanoma. The effectiveness of monotherapy and combined therapy with NSC-631570 in the treatment of melanoma B16 depends on the biological properties of the tumor and the immune state of the organism.

Critical Review of Preclinical Approaches to Evaluate the Potential of Immunosuppressive Drugs to Influence Human Neoplasia

Many immunosuppressive drugs are associated with an increased risk of B-cell lymphoma, squamous cell carcinoma, and Kaposi sarcoma. Thirteen immunosuppressive drugs have been tested in 2-year carcinogenicity studies (abatacept; azathioprine; busulfan; cyclophosphamide; cyclosporine; dexamethasone; everolimus; leflunomide; methotrexate; mycophenolate mofetil; prednisone; sirolimus; and tacrolimus) and in additional models including neonatal and genetically modified mice; chemical, viral, ultraviolet, and ionizing radiation co-carcinogenesis, and in models with transplanted tumor cells. The purpose of this review is to outline the mechanisms by which immunosuppressive drugs can influence neoplasia, to summarize the available preclinical data on the 13 drugs, and to critically review the performance of the models. A combination of primary tumor and metastasis assays conducted with transplanted cells may provide the highest value for hazard identification and can be applied on a case-by-case basis. However, for both small molecules and therapeutic proteins, determining the relative risk to patients from preclinical data remains problematic. Classifying immunosuppressive drugs based on their mechanism of action and hazard identification from preclinical studies and a prospective pharmacovigilance program to monitor carcinogenic risk may be a feasible way to manage patient safety during the clinical development program and postmarketing.

Effect of silencing heparanase on the biological behavior of gastric carcinoma in nude mice

To investigate the effects of silencing heparanase (HPA) on growth, angiogenesis and metastasis of human gastric carcinoma transplanted in nude mice. Human gastric carcinoma SGC-7901 cells and those cells with silenced HPA (gastric carcinoma SGC-7901-HPA(-)) were separately transplanted subcutaneously in 6 nude mice. The time, size and speed of tumor growth were recorded. RT-PCR and Western-blot were used to detect the expression of HPA mRNA and protein in the subcutaneous tumors of the two groups. Immunohistochemical staining was used to detect microvessel density (MVD) in the subcutaneous tumors of the two groups. Cells of the subcutaneous transplanted tumors of the two groups were separately injected into the peritoneal cavity of nude mice, 6 mice each. The growth of metastatic tumors in nude mice was observed. Human gastric carcinoma SGC-7901 cells and SGC-7901-HPA(-) cells were subcutaneously inoculated in nude mice, and tumors appeared at 4 days and 7 days after inoculation, respectively. The MVD was (20.69 ± 1.20)/HP and (11.35 ± 1.94)/HP, respectively (P < 0.05). The expressions of HPA mRNA and protein of the subcutaneously transplanted SGC-7901-HPA(-) tumor were decreased. Four voluminous metastatic tumors caused by SGC-7901 cells occurred in 3 mice in the liver, right kidney, omentum and intestine. Two smaller abdominal metastatic tumors of SGC-7901-HPA(-) cells were found in the liver and right kidney. Silencing HPA can inhibit the tumor growth, angiogenesis and metastasis of human gastric cancer in nude mice. It suggests that HPA might become a new target for prevention and treatment of gastric cancer.

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-γ) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 × 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

Organ microenvironment plays significant roles through Fas ligand in vaccine‐induced CD4+ T cell dependent suppression of tumor growth at the orthotopic site

Growth of colon carcinoma cells transfected with mucin 1 (MUC1) was effectively suppressed by vaccination with MUC1 cDNA. The suppression was dependent on the presence of Fas ligand (FasL) in the cecum, whereas it was independent of FasL in the spleen and in the liver, as revealed by the use of gld/gld mice as the recipients of vaccination, and transplantation of tumor cells expressing MUC1. CD4(+) T cells were transferred from mice immunized with MUC1 cDNA to naive gld/gld or C57BL/6 mice, and the suppression of colon carcinoma growth in the cecum was tested. The results clearly showed that FasL in the recipient played a significant role. In the cecum, FasL was associated with intratumoral CD11b(+) cells, which are likely to be responsible for vaccine-induced tumor suppression. The T cell response to MUC1 was not influenced by the gld/gld status.

Quantitative relationship between guanine O6-alkyl lesions produced by Onrigin™ and tumor resistance by O6-alkylguanine-DNA alkyltransferase

O 6-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O 6-chloroethyl (Onrigin™ and carmustine) and O 6-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O 6-methylguanines/cell and ∼300 O 6-chloroethylguanines/cell, respectively. AGT repaired O 6-methylguanines until the AGT pool was exhausted, while its repair of O 6-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O 6-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O 6-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.

Metal Complexes as Protein Kinase Inhibitors

Angiogenesis inhibition: Metal complexes are often overlooked in the development of new drugs. However, rationally designed iridium complexes with bidentate pyridocarbazole ligands have been found to act as selective protein kinase inhibitors. Experiments in zebrafish embryos revealed impressive anti-angiogenic effects for this type of metallodrug. (Picture: tumor-induced angiogenesis in a zebrafish embryo; red: transplanted tumor cells, green: blood vessels).

INDUCTION OF ANTICANCER RESISTANCE IN MICE TREATED WITH LECTIN OF BACTERIAL ORIGIN

The preventive antitumor effect of cytotoxic lectin isolated from culture me- from culture medium Васillus subtilis B-7025 on the models of experimental tumours sarcoma-37, Ehrlich carcinoma and metastatic Lewis lung carcinoma was firstly shown. The injection of lectin to intact mice leads to stimulation of anticancer resistance to transplanted tumour cells, tumour growth inhibition and increasing of animal survival. Presurgical injection of lectin to Lewis lung carcinoma bearing mice was found to result in the substantial antimetastatic effect in surgical treated mice. Our results prove that cytotoxic lectin has mediate activating influence on antitumour immunity of animals.

Establishment of a nude mouse model of highly metastatic gastric lymphoma constructed with orthotopic transplantation of surgical specimen

To construct a mouse model of highly metastatic gastric lymphoma with orthotopic transplantation of human primary gastric lymphoma specimen. A fresh surgical specimen of primary gastric lymphoma was obtained intraoperatively and implanted into the submucosa of stomach in nude mice. Tumor formation, invasion, metastasis, morphological characteristics under light microscopy and electron microscopy, immunohistochemistry,and the karyotype of orthotopically transplanted tumor cells were studied. An orthotopic highly metastatic model of human primary gastric lymphoma in nude mice(HGBL-0305) was successfully established. Histopathology of transplanted tumors showed primary gastric diffuse large B cell lymphoma. CD19, CD20, CD22 and CD79alpha were positive, while CD3 and CD7 were negative. The number of chromosome ranged from 56 to 69. DNA index(DI) was 1.47+/-0.12(i.e. heteroploid). Until now, HGBL-0305 model has been maintained for 45 generations by orthotopic passage for almost 4 years in nude mice. A total of 156 nude mice were used for transplantation. The growth rate and resuscitation rate of liquid nitrogen cryopreservation of transplanted tumor cells were both 100%. The autonomic growth of the transplanted tumor cells invaded and destructed all the layers of the nude mice stomach. The metastasis rates of liver, spleen, lymph node, and peritoneal seeding were 69.5%, 55.6%, 45.7%, and 30.5%, respectively. An orthotopic highly metastatic model of human primary gastric lymphoma in nude mice is successfully established. HGBL-0305 model may simulate the natural course of primary gastric lymphoma in human and provides an ideal animal model for studies on pathogenesis, metastasis biology and anti-metastatic therapies of primary gastric lymphoma.

Lung metastasis targeting by a tumor-killing Salmonella typhimurium mutant.

e13631 Background: We have previously developed a tumor-targeting double auxotroph of Salmonella typhimurium termed A1-R (Cancer Research 66, 7647-7652, 2006; Proc. Natl. Acad. Sci. USA 104, 10170-10174, 2007). A1-R requires arginine and leucine, which disable these bacteria from growing in normal tissue but allow the bacteria to have high antitumor virulence in nude mouse models. In the present study, we have observed the efficacy of A1-R bacteria on Lewis lung cancer-bearing C57 immunocompetent mice. Methods: Lung tumors were obtained after injection of 2×106 Lewis lung cancer cells, expressing red fluorescent protein (RFP), into the tail vein of C57 B16 immunocompetent mice. Three days after tumor cell transplantation, the animals were treated with A1-R bacteria via tail-vein injection. Results: Lung tumor growth was inhibited in the treated group, and the mice had much fewer lung metastases tumors compared to the untreated group. A1-R was also injected into the thoracic cavity of the Lewis lung cancer-bearing C57 mice weekly for three weeks. All animals were sacrificed at the end of the experiment. The excised lungs were weighed and imaged by RFP fluorescence. The lung tumors in the treated group were significantly smaller than in the untreated group. Intrathoracic injection of bacteria allowed 108 bacteria to be administered weekly to the host with no obvious toxicity. Various i.v. doses of A1-R were tested, including bolus treatment with a single i.v. injection, medium dose using weekly i.v. administration, and metronomic treatment using a small dose twice a week, i.v. In contrast to intrathoracic administration of A1-R, bolus treatment was toxic to the host. However, the medium i.v. dose and the i.v. metronomic dosing of A1-R were well tolerated. Conclusions: These results suggest that S. typhimurium A1-R is effective against metastatic tumors in immunocompetent mice which is important for future clinical development of bacterial cancer therapy. No significant financial relationships to disclose.

Abstract 5323: Distinct roles of IL-17A in inflammation-induced tumor development and tumor immunosurveillance

Abstract While chronic inflammation is considered to be a critical component of tumorigenesis, the immune system can also prevent tumor development and/or growth. IL-17A and IL-17A secreting T cells, termed Th17 mediate inflammation via activation of a wide range of downstream inflammatory mediators that contribute to chronic inflammatory diseases. It also has been suggested that Th17 can be a constituent in tumor immunosurveillance since tumor-specific Th17 has been shown to eradicate established tumor. In the present study, we addressed the effects of inflammation/immunity driven by IL-17A on a primary tumor development model using a chemical carcinogen 3′-methylcholanthrene (MCA). We found that IL-17A deficient (IL-17A−/−) mice were protected from the primary tumor development by MCA. Paradoxically, the growth of primary tumors developed in IL-17A−/− mice was faster than that of tumors in WT (C57BL/6) mice. Although tumor cell lines derived from WT and IL-17A−/− mice grew equally in immunocompromised (T, B cell deficient C. B-17 scid) mice, those from IL-17−/− mice were easily rejected in C57BL/6 mice as compared to those from WT mice. Consistent with this, tumor growth of B16 melanoma, a non-immunogenic transplantable tumor cell line, was enhanced in IL-17A−/− mice. The apparently contrasting effects of IL-17A on tumor growth and development may reflect difference in cells producing IL-17A, namely cells mediate innate or acquired immunity. We transferred CD4+ T cells from WT mice into IL-17A−/−. While the transfer with CD4+ T cells from WT mice did not affect the primary tumor development in IL-17A−/− mice, the growth of primary tumors in IL-17A−/− mice transferred with WT CD4+ T cells was significantly suppressed. These results indicate that IL-17A promotes tumorigenesis possibly by enhancing inflammation and IL-17A produced by CD4+ T cells contributes to the immune surveillance to the emerging tumors as well. Overall, these results not only demonstrate that a single cytokine, IL-17A, plays contrasting role in tumor development and growth, but also indicate that immune inflammation-driven tumorigenesis and tumor immunosurveillance coexist in the same host. 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 5323.

Abstract 1719: Leptin, an obesity-related adipocytokine, promotes accelerated growth of Wnt-1 mammary tumors and modulates basal-like and luminal tumor markers

Abstract Over 2/3 of the US population is obese or overweight, a condition associated with increased risk and decreased survival of various cancers including breast cancer. There is a large unmet medical need to identify mechanisms underlying the link between obesity and cancer. The purpose of these studies is to elucidate the obesity-dependent mechanisms promoting aggressive mammary tumor growth. Circulating levels of leptin, an adipocyte-secreted cytokine that regulates body weight, are elevated in obese and overweight individuals. We orthotopically transplanted tumor cells derived from primary spontaneous MMTV-Wnt-1 tumors into morbidly obese leptin-deficient (ob/ob; lack wild-type leptin) and leptin receptor-deficient (db/db; hyperleptinemic) mice. The MMTV-Wnt-1 tumors, like many human breast tumors, are mixed lineage giving rise to tumors expressing both basal and luminal tumor markers. We determined that orthotopic transplant of MMTV-Wnt-1 tumor cells into obese db/db mice, with high serum leptin, leads to accelerated tumor growth. In contrast, transplant into obese ob/ob mice leads to small, disorganized tumors. More importantly, tumors from leptin-deficient ob/ob mice are predominantly comprised of differentiated epithelial (E-cadherin) and myoepithelial (α—SMA and keratin 14) tumors cells and surprisingly lack basal (keratin 5 and 6) and luminal (keratin 8) lineage tumor cells. Indeed, we determined that AKT cell survival/proliferation pathway was suppressed in the tumors from leptin-deficient mice. The phospho-AKT (p-AKT) immunopositive cells were primarily also expressing keratin 6, and not αSMA or keratin 8, indicating that AKT is activated in the keratin 6 basal-like tumor cells. These studies are highly significant because they indicate leptin, elevated in obese patients, promotes aggressive tumor growth by targeting survival, proliferation, and/or differentiation of subpopulations of mammary tumor cells. 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 1719.

Abstract 2370: A novel Src/Ezrin pathway regulates local invasion and metastasis of breast

Abstract The membrane cytoskeletal crosslinker, ezrin, is frequently over-expressed in human breast cancer, and is required for motility and invasion of epithelial cells. Our group recently showed that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in transformation of epithelial cells, requiring phosphorylation of specific tyrosines on ezrin by Src (Srivistava et al. MBC, 16:1481-90, 2005; Naba et al. EMBO J. 27:38-50, 2008). We showed that Src binds to ezrin Y190 via its SH2 domain and that ezrin Y145 and Y477 are phosphorylated by Src. Expression of Y145F or Y477F ezrin mutants, which cannot be phosphorylated by Src, delays cell spreading of epithelial cells on fibronectin substratum. Furthermore, ezrin is required for metastasis in a breast tumor xenograft model (Elliott et al. BCR 7:R365-73, 2005). We therefore examined whether Src/ezrin interaction regulates invasion and metastasis of breast cancer, using a mouse breast tumor (AC2M2) xenograft model. Transplanted tumor cells expressing vector alone rapidly infiltrated into surrounding stroma, underlying abdominal muscle, visceral organs and metastasized to lung and other sites. Interestingly, both Y145F and Y477F ezrin mutants had little effect on primary tumor growth rate. However, Y145F ezrin showed a marked attenuating effect on pulmonary metastasis. Furthermore, transplanted mouse breast carcinoma cells over-expressing Y477F ezrin formed cohesive tumors that were circumscribed by normal stroma with no detectable infiltration into underlying muscle. Marked lymphovascular involvement, a prognostic indicator of relapse in clinically advanced human breast cancers, was evident in control tumors but not in tumors expressing Y477F ezrin. In a preliminary IHC analysis, total ezrin showed strong cytoplasmic and weak membranous staining in the cortical region of control primary tumors, but showed strong membranous and weak cytoplasmic staining in Y477F ezrin-expressing primary tumors. The cohesive phenotype of Y477F ezrin is likely a consequence of a defect in cell adhesion as described by Naba et al. Thus, the precise state of ezrin phosphorylation may elicit different effects on local invasion and metastasis. Our study implicates a role of the Src/ezrin pathway in regulating local invasion and metastasis of breast carcinoma cells, and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic marker and treatment target for invasive breast cancer. (Funded by CBCRA 17374 and ARC 4823) 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 2370.

Therapy for minimal residual tumor disease: β‐Galactosylceramide inhibits the growth of recurrent HPV16‐associated neoplasms after surgery and chemotherapy

Natural killer T (NKT) cells are potent modulators of antitumor immunity. Their protective effects can be achieved upon their activation by glycolipid ligands presented in the context of the CD1d molecule. These CD1d-binding glycolipid antigens have been described as potent therapeutic agents against tumors, infections, as well as autoimmune diseases. Immunoregulatory and therapeutic effects of glycolipid ligands depend on their structure and modes of administration. Therefore, more studies are needed for optimization of the particular therapeutic settings. This study was focused on the tumor-inhibitory effects of 12 carbon acyl chain beta-galactosyl ceramide (C12 beta-D-Galactosyl Ceramide; beta-GalCer(C12)) on the growth of human papillomavirus type 16 (HPV16)-associated neoplasms transplanted in syngeneic mice. Treatment of tumor-bearing mice with beta-GalCer(C12) 3-14 days after tumor cell transplantation significantly inhibited the growth of the major histocompatibility complex (MHC) Class I-positive (TC-1), as well as MHC Class I-deficient (TC-1/A9) HPV16-associated tumors. Moreover, administration of beta-GalCer(C12) after surgical removal of TC-1 tumors inhibited the growth of tumor recurrences. Similar results were obtained in the treatment of tumors after chemotherapy. beta-GalCer(C12) treatment turned out to be also synergistic with immunotherapy based on administration of IL-12-producing cellular vaccines. These results suggest that beta-GalCer(C12), whose antitumor effects have so far not been studied in detail, can be effective for the treatment of minimal residual tumor disease as well as an adjuvant for cancer immunotherapy.

Tellurite-induced oxidative stress leads to cell death of murine hepatocarcinoma cells

Data regarding tellurium (Te) toxicity are scarce. Studies on its metabolism, performed mainly in bacteria, underline a major role of reactive oxygen species (ROS). We investigated whether tellurite undergoes redox cycling leading to ROS formation and cancer cell death. The murine hepatocarcinoma Transplantable Liver Tumor (TLT) cells were challenged with tellurite either in the presence or in the absence of different compounds as N-acetylcysteine (NAC), 3-methyladenine, BAPTA-AM, and catalase. NAC inhibition of tellurite-mediated toxicity suggested a major role of oxidative stress. Tellurite also decreased both glutathione (GSH) and ATP content by 57 and 80%, respectively. In the presence of NAC however, the levels of such markers were almost fully restored. Tellurite-mediated ROS generation was assessed both by using the fluorescent, oxidation-sensitive probe dichlorodihydrofluorescein diacetate (DCHF-DA) and electron spin resonance (ESR) spectroscopy to detect hydroxyl radical formation. Cell death occurs by a caspase-independent mechanism, as shown by the lack of caspase-3 activity and no cleavage of poly(ADP-ribose)polymerase (PARP). The presence of gamma-H2AX suggests tellurite-induced DNA strand breaking, NAC being unable to counteract it. Although the calcium chelator BAPTA-AM did show no effect, the rapid phosphorylation of eIF2alpha suggests that, in addition to oxidative stress, an endoplasmic reticulum (ER) stress may be involved in the mechanisms leading to cell death by tellurite.

Synthesis, antineoplastic and cytotoxic activities of some mononuclear Ru(II) complexes

A series of mononuclear Ru(II) complexes of the type [Ru(S)2(K)]2+, where S = 1,10-phenanthroline/2,2′-bipyridine and K = 4-OH-btsz, 4-CH3-btsz, 3,4-di-OCH3-btsz, 4-OH-binh, 4-CH3-binh, 3,4-di-OCH3-binh, were prepared and characterized by elemental analysis, FTIR, 1H-NMR, and mass spectroscopy. The complexes displayed metal–ligand charge transfer (MLCT) transitions in the visible region. These ligands formed bidentate octahedral ruthenium complexes. The title complexes were evaluated for their in vivo anticancer activity against a transplantable murine tumor cell line, Ehrlisch’s ascites carcinoma (EAC), and in vitro cytotoxic activity against human cancer cell lines Molt 4/C8 and CEM and murine tumor cell line L1210. The ruthenium complexes showed promising biological activity especially in decreasing tumor volume and viable ascites cell counts. Treatment with these complexes prolonged the life span of mice bearing EAC tumors by 10–52%. In vitro evaluation of these ruthenium complexes revealed cytotoxic activity from 0.21 to 24 μM against Molt 4/C8, 0.16 to 19 μM aginst CEM, and 0.75 to 32 μM against L1210.

Growth Factor Independence 1 (Gfi1) Is Required for Initiation, Maintenance, Progression, and Transplantability of Lymphoma.

Abstract 447One of the transcription factor genes, which are aberrantly expressed in human T-cell lymphoma, is Growth Factor Independence 1 (Gfi1). Since over expression of Gfi1 accelerates experimentally induced T-cell tumors on mice, it is likely that Gfi1 plays a crucial role in establishing or maintaining lymphoid neoplasms. To test this we have used, N-ethyl-N-nitrosourea (ENU) to induce T-cell tumors in WT and Gfi1-deficient mice. Nearly all WT mice (37/43) developed T-cell lymphoma with a median disease-free survival time of 106 days. In contrast, only 3/19 Gfi1-/- mice developed hematopoietic neoplasms with a prolonged median latency period of 131 days (p≤0.02 for latency and p≤0.00001 for tumor incidence). Other approaches using infection of newborn mice with Moloney Murine leukemia virus (MoMuLV) showed a similar dependency of tumor formation on the expression of Gfi1. To test whether Gfi1 is also required for the maintenance of T-cell leukemia, we used conditional Gfi1 deficient mice, in which exon 4 and exon 5 of Gfi1 encoding N-terminal zinc fingers critical for DNA binding are flanked by loxp sites and can be eliminated by Cre recombinase. Mx-Cre-Gfi1fl/fl and, as a control, Gfi1fl/fl mice, were treated with ENU to induce T-cell lymphoma. About 7 weeks after ENU injection, Cre expression was induced by poly(I:C). At this time a tumor has already been initiated but is not visible phenotypically. Nearly all (19/22) of ENU and poly(I:C) treated Gfi1fl/fl mice succumbed to lymphoma (median survival time 98 days) whereas only 5/19 poly(I:C) treated Mx-Cre-Gfi1fl/fl mice developed T-cell lymphoma (median survival time 132 days, p≤0.01 for survival and p≤0.0001 for incidence). Interestingly, all tumors arising in poly(I:C) treated Mx-Cre-Gfi1fl/fl mice showed only partial excision of the Gfi1 locus, further demonstrating the requirement of Gfi1 for tumor maintenance. We also treated a cohort of Mx-Cre-Gfi1fl/fl and Gfi1fl/fl mice with ENU and observed them for development of tumors by ultrasound. After the appearance of tumors in these mice, we treated Mx-Cre-Gfi1fl/fl and Gfi1fl/fl mice with poly(I:C) and observed a noticeable tumor regression only when the Gfi1 gene was deleted (average reduction of tumor size 0.4). In contrast, the tumors progressed in poly(I:C) treated Gfi1fl/fl mice (average increase 4.5 times, p≤0.01 between Mx-Cre-Gfi1fl/fl and Gfi1fl/fl mice). Next we transplanted tumor cells from the different experiments and could show that loss of Gfi1 abrogated completely the engraftment of tumors in the recipient mice whereas tumors expressing Gfi1 engrafted perfectly well (n = 12 for Gfi1fl/fl and n=6 for Mx-Cre-Gfi1fl/fl mice p≤0.001 for incidence and survival between both cohorts). Inhibition of apoptosis is one of the hallmarks of cancer. Thus we investigated whether Gfi1 regulates apoptosis in the hematopoietic system. In-vitro, we demonstrated that loss of Gfi1 led to an enhanced apoptosis in explanted thymocytes and hematopoietic stem cells (defined as Lin-, Sca1+, c-kit+: LSK) upon DNA damage induction either by gamma-radiation or cytotoxic substances. In-vivo, over 60 % of Gfi1 deficient mice but only 10% of WT mice succumbed to an exposure of a non-lethal dose of gamma irradiation (6 Gy) (p≤0.001 for survival) with a higher degree of cell death within the LSK fraction of Gfi1-/- mice. On the molecular level we found that Gfi1 inhibits the DNA damage induced upregulation of the proapoptotic factors (such as Bax) and is required for up regulation of the antiapoptotic factor p21. Our findings suggest that Gfi1 is required for initiation, maintenance, progression and transplantability of lymphoid neoplasm and that it exerts this function by inhibiting apoptosis. Gfi1 might be a new target to treat lymphoid leukemia in humans. Disclosures:No relevant conflicts of interest to declare.

Roles of polysaccharide from Branchiostoma belcheri in anti-DNA oxidation and anti-tumor activity in S180 mice

In this study, we isolated a polysaccharide from Branchiostoma belcheri (PBB) by enzymatic protein hydrolysis and alcohol precipitation. We investigated the effects of PBB supplementation on DNA oxidation and growth of the transplanted tumor cells Sarcoma (S180) in mice. Sixty healthy Kunming mice weighing between 18 and 25 g were randomly assigned to 6 groups, each consisting of 10 animals. All the mice, except for the blank control group, were inoculated with S180 sarcoma cells into the axilla of the left foreleg. PBB was given to mice by gavage at doses of 0 (model control), 25, 50, or 100 mg/kg b.w. in 0.2 ml saline for 30 days. The fifth group of S180-mice was given cytoxan (50 mg/kg) by peritoneal injection as a positive control group. The animals had free access to food and water. The mice were sacrificed after the final treatment and blood was quickly collected. Spontaneous and oxidized DNA damage of peripheral lymphocytes induced by H2O2 were analyzed by SCGE. O6-methyl-guanine (O6-MeG) was measured by high-performance capillary zone electrophoresis. The average tumor weights (0.856–1.118 g) of the three PBB groups were significantly lower than that of the model control group (1.836 g) (p<0.05). The tumor inhibition ratios of the PBB groups were 39.1%–53.4% and similar to the cytoxan positive group (57.5%). There were no significant differences in spontaneous DNA damage in peripheral lymphocytes among the groups. The oxidative DNA damage induced by 10 µmol/L H2O2 in the 50 and 100 mg/kg b.w. groups were 246.1 AU and 221.7 AU, respectively, both of which were significantly lower than that in the model group (289.0 AU; p<0.05). The plasma concentrations of O6-MeG in the 25, 50, and 100 mg/kg supplemented groups were 2.09 µmol/L, 1.86 µmol/L, and 1.63 µmol/L, respectively, all of which were significantly lower than that of the model group (2.67 µmol/L; p<0.05). These results indicated that PBB may have antioxidative activity and thus reduce oxidation-induced DNA damage.

Extensive conservation of genomic imbalances in canine transmissible venereal tumors (CTVT) detected by microarray-based CGH analysis

Canine transmissible venereal tumor (CTVT) is an intriguing cancer that is transmitted naturally as an allograft by transplantation of viable tumor cells from affected to susceptible dogs. At least initially, the tumor is able to evade the host's immune response; thus, CTVT has potential to provide novel insights into tumor immunobiology. The nature of CTVT as a "contagious" cancer, originating from a common ancestral source of infection, has been demonstrated previously by a series of studies comparing geographically distinct tumors at the molecular level. While these studies have revealed that apparently unrelated tumors share a striking degree of karyotypic conservation, technological restraints have limited the ability to investigate the chromosome composition of CTVTs in any detail. We present characterization of a strategically selected panel of CTVT cases using microarray-based comparative genomic hybridization analysis at ~one-megabase resolution. These data show for the first time that the tumor presents with an extensive range of non-random chromosome copy number aberrations that are distributed widely throughout the dog genome. The majority of abnormalities detected were imbalances of small subchromosomal regions, often involving centromeric and telomeric sequences. All cases also showed the sex chromosome complement XO. There was remarkable conservation in the cytogenetic profiles of the tumors analyzed, with only minor variation observed between different cases. These data suggest that the CTVT genome demonstrates a vast degree of both structural and numerical reorganization that is maintained during transmission among the domestic dog population.