Mouse Cancer Cell Lines Research Articles

The Anti-Tumor Effect of Motesanib in Pre-Clinical Models of Human Basal-Like Breast Cancer.

Abstract Background: Growth of breast cancer is dependent upon sustained angiogenesis, of which vascular endothelial growth factor (VEGF) is a major stimulant. Increased expression of VEGF in breast cancers is associated with poor prognosis and disease recurrence, and anti-angiogenic agents are being developed as potential new therapies. Motesanib is an oral inhibitor of VEGF receptors 1, 2 and 3, platelet-derived growth factor receptor, and stem cell factor receptor (c-Kit). This study determined the effects of motesanib on the growth of human basal-like breast cancer cell lines in immunodeficient mice, alone and in combination with paclitaxel.Materials and Methods: Breast cancer cells BT-549 and GILM2 were injected into the mammary fatpad (MFP) of mice; HCC1187 cells were injected on the subcutaneous flank. Mice with established tumors (150-200 mm3) received motesanib orally BID, at 7.5, 25 or 75 mg/kg for three weeks. Tumors were collected for analysis of blood vessels (CD-31 staining), viable tumor area, and for markers of proliferation (Ki67) and apoptosis (cleaved caspase-3). GILM2 cells were injected into the tibia of mice to model growth in bone; mice with GILM2 MFP or tibia tumors received motesanib (25 mg/kg BID) or paclitaxel (24 mg/kg Q4Dx5), or the combination. Intratibial GILM2 tumors expressing luciferase were monitored by bioluminescence and radiography to evaluate osteolysis. Analyses of tumor size were assessed by ANOVA, and immunohistochemical tests by Student's t tests.Results: Motesanib produced dose-dependent inhibition of growth of BT-549, GILM2 and HCC1187 tumors. All doses of motesanib significantly inhibited the growth of BT-549 tumors (>55% inhibition, P<0.01 vs. control). For GILM2 tumors, 25 and 75 mg/kg motesanib gave 47% and 58% growth inhibition, respectively (P<0.008), and for HCC1187, these doses produced 81% and 91% growth inhibition, respectively (P<0.001). GILM2 tumors from mice receiving >25mg/kg had smaller viable tissue areas (P<0.001), and fewer CD-31 positive blood vessels (P<0.001) than tumors from control mice. Immunostaining showed that 25mg/kg motesanib reduced proliferation of GILM2 tumors (P<0.001 vs. control), with no change in numbers of apoptotic cells. Treatment with 25 mg/kg motesanib or paclitaxel (24mg/kg Q4Dx5) significantly reduced GILM2 tumor burden in the MFP (46% and 49% inhibition, respectively) and in the bone (77% and 68% inhibition, respectively) (P<0.05 vs. control). Combining the agents did not enhance the anti-tumor effects of paclitaxel. Bioluminescent measurements and X-rays showed that motesanib alone was as effective as paclitaxel at reducing GILM2 growth and osteolytic destruction in bone (P<0.05, vs. control).Conclusion: Oral administration of motesanib significantly inhibited the growth of three ER-, PR- HER2-negative basal-like breast cancer cell lines in vivo. Motesanib was as effective as paclitaxel at controlling the growth of GILM2 in the MFP, and reduced osteolysis in bone tumors. These data suggest that motesanib has anti-angiogenic action in diverse tumor microenvironments. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5057.

Methyl Dehydro-Jasmonate Has Anti-Inflammatory Effect Cells and Its Molecular Targets Mir-155 and NF-Kb Pathway against LPS Stimulation On RAW264.7.

Abstract 1357Poster Board I-380Jasmonic acid and methyl jasmonate are fatty acid-derived cyclopentanones, found in the plants, and play major roles in a defense against insects and disease. Methyl jasmonate suppresses cellular proliferation and induces apoptosis in human and mouse cancer cell lines. Methyl jasmonate increased the life span of EL-4 lymphoma-bearing mice with selective cytotoxicity against lymphoma cells while sparing normal blood lymphocytes.Inflammation is one of the defence mechanisms against pathogens, caused by diverse microbial products. Microbial products are detected by the Toll-like receptors (TLRs) that are expressed at high levels on macrophages and dendritic cells. The complex of the TLRs and their ligand initiates a wide spectrum of responses from phagocytosis to production of a variety of cytokines, which enhance the inflammatory and adaptive immune responses. Of these TLRs, TLR4 recognizes the product of gram-negative bacteria, LPS. LPS stimulated-cells produce inflammatory cytokines (TNF-α and IL-6), and inducible enzymes of iNOS and COX-2. Recent evidence reveal that some microRNAs (miRNAs) play important roles in inflammation; miRNA-146a plays central roles in the negative feedback regulation of IL-1b-induced inflammation and miR-155 enhances the release of inflammatory mediators during the innate immune responses. Our structural analysis shows that methyl jasmonate contains enone group which is a common functional moiety in anti-inflammatory drugs. Our previous works found that methyl jasmonate has anti-inflammatory effects and the related compound methyl dehydro-jasmonate (J2) had the highest anti-inflammatory effect in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophage cells of all synthesized methyl jasmonate analogues. In this study, we wanted to elucidate molecular targets of J2 action in its anti-inflammatory properties. We observed that a LPS stimulation of RAW264.7 cell line also induced known inflammatory markers; TNF-α, IL-6, iNOS and COX-2. Our analysis of miRNAs changes revealed an increase of miR-155 (>8 fold) and miR-146a (>3 fold), but not miR-125b. In a J2 toxicity test on the LPS stimulated RAW264.7 cells, the J2 treatment protected LPS treated RAW264.7 cells starting at 6.25 μM. We then tested J2 effects on various mediators of inflammation. We found that J2 suppressed inductions of TNF-α, IL-6, iNOS and COX-2 at a transcript level in a dose-dependent manner (IC50 = 18∼25 μM) and confirmed it also at a protein level for iNOS and COX-2. We then found that miR-155 induction was inhibited by J2 dose-dependent manner, J2 suppressed miR-146a only at 50 μM. The NF-kB pathway and MAPK pathway are thought to be important mediators of LPS induced inflammation and we show that J2 had significant effects on NF-kB, p65, and IkB while no or minimal effect on JNK, p38, and ERK.In conclusion, the present study demonstrates that J2 supressed LPS stimulation in RAW264.7 cells and it targets miR-155, and NF-kB pathway. In addition, our results also suggest that MAPK pathway may not contribute to the induction of inflammatory markers (i.e. TNF-α, IL-6, iNOS, and COX-2). DisclosuresNo relevant conflicts of interest to declare.

Synthesis, structure, electrochemistry and spectral characterization of ( d-glucopyranose)-4-phenylthiosemicarbazide metal complexes and their antitumor activity against Ehrlich Ascites Carcinoma in Swiss albino mice

The novel glycosyl saccharide derivative, ( d-glucopyranose)-4-phenylthiosemicarbazide (LH) and its complexes, with cobalt(II), nickel(II), copper(II) and zinc(II) were synthesized, characterised and tested for cytotoxic effects. The copper complex, [CuLCl] inhibited Ehrlich Ascites Carcinoma (EAC) induced cancer cell lines in Swiss albino mice at LC 50 = 1.94 × 10 −8 (LC 50 = 2.76 × 10 −8 for cisplatin) and so distinctly better than free ligand and other complexes.

Association of nuclear-intermediate filament lamin B1 with necrotic- and apoptotic-morphologies in cell death Induced by 5-fluoro-2'-deoxyuridine

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analyses. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Our present finding provides an interesting possibility that lamin-B1 may have an important role in regulating cell-death morphology.

HPMA copolymer conjugates with reduced anti-CD20 antibody for cell-specific drug targeting. I. Synthesis and in vitro evaluation of binding efficacy and cytostatic activity

Synthesis, physicochemical and biological properties and preliminary anticancer activity of new star-shaped polymer–doxorubicin (DOX) conjugates targeted with anti-CD20 monoclonal antibody were investigated. Mild reduction of antibody (Ab) with dithiothreitol (DTT) resulted in introduction of thiol groups into Ab. Polymer precursors used for the synthesis of the conjugates were based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with a functional group at the polymer chain end. The copolymers were linked to the thiol groups of the reduced Ab via one-point attachment forming a star-shaped structure with central antibody surrounded by hydrophilic polymer chains. Neither reduction nor polymer modification of Ab influenced binding activity of the Ab to its specific cancer cell membrane antigen as it was confirmed in vitro by standard flow cytometry. The anticancer drug DOX was attached to the HPMA copolymer chain in an Ab–polymer system via a pH–labile hydrazone linkage or via an oligopeptide sequence degradable by lysosomal enzymes. Such Ab–polymer–DOX conjugates were fairly stable in aqueous solution at pH 7.4 and the drug was readily released in mildly acid environment at pH 5–5.5 by hydrolysis of hydrazone bond or more slowly by enzymolysis with lysosomal enzymes. The cytostatic activity of the anti-CD20 monoclonal Ab-targeted conjugates tested on several CD20-positive or negative human and mouse cancer cell lines confirmed considerable targeting capacity of the monoclonal Ab after its binding to the polymer carrier. New method of synthesis of star antibody-targeted polymer–drug conjugates with pH-controlled drug release described in this paper opens new perspectives for development of new therapeutics intended for cancer therapy.

Imidazole metalloporphyrins as photosensitizers for photodynamic therapy: Role of molecular charge, central metal and hydroxyl radical production

The in vitro photodynamic therapy activity of four imidazole-substituted metalloporphyrins has been studied using human (HeLa) and mouse (CT26) cancer cell lines: an anionic Zn porphyrin and a homologous series of three cationic Zn, Pd or InCl porphyrins. A dramatic difference in phototoxicity was found: Pd cationic > InCl cationic > Zn cationic > Zn anionic. HeLa cells were more susceptible than CT26 cells. Induction of apoptosis was demonstrated using a fluorescent caspase assay. The anionic Zn porphyrin localized in lysosomes while the cationic Zn porphyrin localized in lysosomes and mitochondria, as assessed by fluorescence microscopy. Studies using fluorescent probes suggested that the cationic Pd porphyrin produced more hydroxyl radicals as the reactive oxygen species. Thus, the cationic Pd porphyrin has high potential as a photosensitizer and gives insights into characteristics for improved molecular designs.

Nuclear receptor dependent chromatin remodeling

The necessary compaction of DNA with histones to form chromatin provides a multifaceted opportunity to regulate gene expression in response to environmental and developmental ques. Nuclear receptors represent a particularly useful class of transcription factors have played a profound role in the investigation of the underlying mechanisms of chromatin biology. The physical and functional interaction between receptors and the ATP dependent chromatin remodeling complexes reveal the importance of a diverse group of associated or co‐activator proteins. We have made use of specific human and mouse cancer cell line models to manipulate the expression of these varied molecules to understand the specific roles they play in the activation of gene transcription by steroid hormones. The focus of our attention has been the human BRG1 remodeling complex ATPase and its associated factors (BAFs). Our studies revel that chromatin remodeling is a necessary but not sufficient condition for hormone mediated transcription. Further, mutational analysis the remodeling complex enzymatic core reveals critical requirements for specific subunits of the complex. Our studies contribute to an evolving view of gene activation that places chromatin remodeling at the centre of this vital biological process.

Snake venom inhibits the growth of mouse mammary tumor cells in vitro and in vivo

Crude preparations as well as purified components of venoms isolated from American, Asian and European snakes have been shown to inhibit the growth of some mouse and human cancer cell lines. However, it is not known if Cerastes cerastes venom (CCV) obtained from the Egyptian desert possesses any anti-tumor activity. In the present study we examined in vitro the effect of CCV on the growth rate and morphology of a mouse mammary tumor virus-induced mammary tumor cell line (RIII/Sa-MT). In addition, the effect of the venom on the growth of RIII/Sa-MT cells transplanted into syngeneic mice was investigated. Our results show that CCV at a concentration of 7 μg/ml kills in vitro a significant number of cells (≈55%) within a period of 48 h. CCV (1 μg/mouse), administered once per week directly into growing tumors for a period of 4 weeks, was found to reduce tumor load by 54%, and as a consequence the CCV-treated mice lived for more than 35 days longer than untreated mice. Histological and ultrastructural examination of the cells and tumors, as well as nuclear staining of the cells and DNA fragmentation studies, led us to conclude that necrosis is most likely the underlying mechanism by which CCV inhibited the growth of mouse mammary tumor cells both in vitro and in vivo.

Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death

Dying tumour cells can elicit a potent anticancer immune response by exposing the calreticulin (CRT)/ERp57 complex on the cell surface before the cells manifest any signs of apoptosis. Here, we enumerate elements of the pathway that mediates pre-apoptotic CRT/ERp57 exposure in response to several immunogenic anticancer agents. Early activation of the endoplasmic reticulum (ER)-sessile kinase PERK leads to phosphorylation of the translation initiation factor eIF2alpha, followed by partial activation of caspase-8 (but not caspase-3), caspase-8-mediated cleavage of the ER protein BAP31 and conformational activation of Bax and Bak. Finally, a pool of CRT that has transited the Golgi apparatus is secreted by SNARE-dependent exocytosis. Knock-in mutation of eIF2alpha (to make it non-phosphorylatable) or BAP31 (to render it uncleavable), depletion of PERK, caspase-8, BAP31, Bax, Bak or SNAREs abolished CRT/ERp57 exposure induced by anthracyclines, oxaliplatin and ultraviolet C light. Depletion of PERK, caspase-8 or SNAREs had no effect on cell death induced by anthracyclines, yet abolished the immunogenicity of cell death, which could be restored by absorbing recombinant CRT to the cell surface.

PIM-1–specific mAb suppresses human and mouse tumor growth by decreasing PIM-1 levels, reducing Akt phosphorylation, and activating apoptosis

Provirus integration site for Moloney murine leukemia virus (PIM1) is a proto-oncogene that encodes a serine/threonine kinase with multiple cellular functions. Overexpression of PIM-1 plays a critical role in progression of prostatic and hematopoietic malignancies. Here we describe the generation of a mAb specific for GST-PIM-1, which reacted strongly with most human and mouse cancer tissues and cell lines of prostate, breast, and colon origin but only weakly (if at all) with normal tissues. The mAb binds to PIM-1 in the cytosol and nucleus as well as to PIM-1 on the surface of human and murine cancer cells. Treatment of human and mouse prostate cancer cell lines with the PIM-1-specific mAb resulted in disruption of PIM-1/Hsp90 complexes, decreased PIM-1 and Hsp90 levels, reduced Akt phosphorylation at Ser473, reduced phosphorylation of Bad at Ser112 and Ser136, and increased cleavage of caspase-9, an indicator of activation of the mitochondrial cell death pathway. The mAb induced cancer cell apoptosis and synergistically enhanced antitumor activity when used in combination with cisplatin and epirubicin. In tumor models, the PIM-1-specific mAb substantially inhibited growth of the human prostate cancer cell line DU145 in SCID mice and the mouse prostate cancer cell TRAMP-C1 in C57BL/6 mice. These findings are important because they provide what we believe to be the first in vivo evidence that treatment of prostate cancer may be possible by targeting PIM-1 using an Ab-based therapy.

Blockade of Notch Signaling in Tumor-Bearing Mice May Lead to Tumor Regression, Progression, or Metastasis, Depending on Tumor Cell Types

It has been reported that blocking Notch signaling in tumor-bearing mice results in abortive angiogenesis and tumor regression. However, given that Notch signaling influences numerous cellular processes in vivo, a comprehensive evaluation of the effect of Notch inactivation on tumor growth would be favorable. In this study, we inoculated four cancer cell lines in mice with the conditional inactivation of recombination signal-binding protein-Jkappa (RBP-J), which mediates signaling from all four mammalian Notch receptors. We found that whereas three tumors including hepatocarcinoma, lung cancer, and osteogenic sarcoma grew slower in the RBP-J-deficient mice, at least a melanoma, B16, grew significantly faster in the RBP-J-deficient mice than in the controls, suggesting that the RBP-J-deficient hosts could provide permissive cues for tumor growth. All these tumors showed increased microvessels and up-regulated hypoxia-inducible factor 1alpha, suggesting that whereas defective angiogenesis resulted in hypoxia, different tumors might grow differentially in the RBP-J-deleted mice. Similarly, increased infiltration of Gr1(+)/Mac1(+) cells were noticed in tumors grown in the RBP-J-inactivated mice. Moreover, we found that when inoculated in the RBP-J knockout hosts, the H22 hepatoma cells had a high frequency of metastasis and lethality, suggesting that at least for H22, deficiency of environmental Notch signaling favored tumor metastasis. Our findings suggested that the general blockade of Notch signaling in tumor-bearing mice could lead to defective angiogenesis in tumors, but depending on tumor cell types, general inhibition of Notch signaling might result in tumor regression, progression, or metastasis.

Differential effects of anticoagulants on tumor development of mouse cancer cell lines B16, K1735 and CT26 in lung

Cancer progression is facilitated by blood coagulation. Anticoagulants, such as Hirudin and low molecular weight heparins (LMWHs), reduce metastasis mainly by inhibition of thrombin formation and L- and P-selectin-mediated cell-cell adhesion. It is unknown whether the effects are dependent on cancer cell type. The effects of anticoagulants on tumor development of K1735 and B16 melanoma cells and CT26 colon cancer cells were investigated in mouse lung. Tumor load was determined noninvasively each week up to day 21 in all experiments using bioluminescence imaging. Effects of anticoagulants on tumor development of the three cell lines were correlated with the fibrin/fibrinogen content in the tumors, expression of tissue factor (TF), protease activated receptor (PAR)-1 and -4 and CD24, a ligand of L- and P-selectins. Hirudin inhibited tumor development of B16 cells in lungs completely but did not affect tumor growth of K1735 and CT26 cells. Low molecular weight heparin did not have an effect on K1735 melanoma tumor growth either. TF and PAR-4 expression was similar in the three cell lines. PAR-1 and CD24 were hardly expressed by K1735, whereas CT26 cells expressed low levels and B16 high levels of PAR-1 and CD24. Fibrin content of the tumors was not affected by LMWH. It is concluded that effects of anticoagulants are dependent on cancer cell type and are correlated with their CD24 and PAR-1 expression.

Cytotoxic activities of diarylheptanoids from Alnus japonica

The diarylheptanoids (1-10) 1,7-bis-(3,4-dihydroxyphenyl)-heptane-3-O-beta-D-glucopyranosyl(1-->3)-beta-D-xylopyranoside (1), 1,7-bis-(3,4-dihydroxyphenyl)-heptane-3-O-beta-D-apiofuranosyl(1-->6)-beta-D-glucopyranoside (2), 1,7-bis-(3,4-dihydroxyphenyl)-heptane-5-O-beta-D-glucopyranoside (3), 1,7-bis-(3,4-dihydroxyphenyl)-5-hydroxyheptane (4), 1,7-bis-(3,4-dihydroxyphenyl)-heptane-3-one-5-O-beta-D-glucopyranoside (5), oregonin (6), hirsutanonol (7), hirsutenone (8), 1,7-bis-(3,4-dihydroxyphenyl)-5-hydroxyheptane-3-O-beta-D-xylopyranoside (9), and platyphylloside (10), isolated from the bark of Alnus japonica, were analyzed for their cytotoxic activities on various human and mouse cancer cell lines. The cytotoxic activities of these ten compounds were evaluated against murine B16 melanoma, human SNU-1 gastric cancer, human SNU-354 hepatoma cancer and human SNU-C4 colorectal cell lines. The diarylheptanoids showed potent cytotoxic activities against murine B16 melanoma cells and human SNU-C1 gastric cancer cell when the cell viability was analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay.

Molecular mechanisms in two cell death-types, necrosis and apoptosis, induced 5-fluoro-2'-deoxyuridine

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analysis. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FudR-treated original F28-7. This finding suggests a new role for lamin B1 as a regulator in the cell death.

Roles of a metastasis-associated molecule, RTVP-1, in cancer immunosurveillance

Objective To elucidate distinct functions of a recently identified cancer metastasis-associated molecule, related to testes-specific, vespid, and pathogenesis protein-1 (RTVP-1) in the mammalian immune system. Methods Immunohistochemical assays and functional analysis on the immune system were performed on RTVP –/– mice and RTVP-1 +/+ mice. Protein-protein interaction (PPI) was used to predict the functions of RTVP-1. Results Abnormal lymphocyte growth kinetics and reduced numbers of CD8 + T cells were revealed in lymph nodes of RTVP-1 –/– mice. Expression of phenotypic markers of maturation in bone marrow-derived dendritic cells (DC) was impaired in RTVP-1 –/– DC following antigenic stimulation in vitro and RTVP-1 –/– DC failed to provide normal CD4 + T cell stimulatory activities in vivo. RTVP-1 –/– mice failed to generate normal CTL or antibody responses in vivo after vaccination. In vivo tumor challenge experiments using a mouse cancer cell line demonstrated that the growth rate of subcutaneous tumors in RTVP-1 –/– mice was significantly increased and CD8 + T cell infiltration significantly reduced compared to RTVP-1 +/+ mice. PPI showed that RTVP-1 protein closely interacted with molecules associated with immune response and cancer metastasis. Conclusion RTVP-1 might function as a tumor metastasis suppressor and immunosurveillance molecule in cancer.

Association of Nuclear Membrane Protein Lamin B1 with Necrosis and Apoptosis in Cell Death Induced by 5-Fluoro-2′-Deoxyuridine

We report that anticancer 5-fluoro-2 ′-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells was performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Thus, the swelling feature for the necrosis was no longer observable, and instead cell shrinkage typical of apoptosis took place in almost all the cells examined. This finding suggests a new role for lamin B1 as a regulator in cell death.

Targeting CD24 for Treatment of Colorectal and Pancreatic Cancer by Monoclonal Antibodies or Small Interfering RNA

CD24 is a potential oncogene reported to be overexpressed in a large variety of human malignancies. We have shown that CD24 is overexpressed in 90% of colorectal tumors at a fairly early stage in the multistep process of carcinogenesis. Anti-CD24 monoclonal antibodies (mAb) induce a significant growth inhibition in colorectal and pancreatic cancer cell lines that express the protein. This study is designed to investigate further the effects of CD24 down-regulation using mAb or small interfering RNA in vitro and in vivo. Western blot analysis showed that anti-CD24 mAb induced CD24 protein down-regulation through lysosomal degradation. mAb augmented growth inhibition in combination with five classic chemotherapies. Xenograft models in vivo showed that tumor growth was significantly reduced in mAb-treated mice. Similarly, stable growth inhibition of cancer cell lines was achieved by down-regulation of CD24 expression using short hairpin RNA (shRNA). The produced clones proliferated more slowly, reached lower saturation densities, and showed impaired motility. Most importantly, down-regulation of CD24 retarded tumorigenicity of human cancer cell lines in nude mice. Microarray analysis revealed a similar pattern of gene expression alterations when cells were subjected to anti-CD24 mAb or shRNA. Genes in the Ras pathway, mitogen-activated protein kinase, or BCL-2 family and others of oncogenic association were frequently down-regulated. As a putative new oncogene that is overexpressed in gastrointestinal malignancies early in the carcinogenesis process, CD24 is a potential target for early intervention in the prevention and treatment of cancer.

Of Mice and (Wo)Men: Is This Any Way to Test a New Drug?

Of Mice and (Wo)Men: Is This Any Way to Test a New Drug?

Isolation and Characterization of Two Korean Mistletoe Lectins

Two isolectins (KML-IIU and the KML-IIL) were individually isolated from the previously reported Korean mistletoe lectin, KML-C, by using an immunoaffinity column. Molecular weights of the KML-IIU and the KML-IIL were 64 kDa and 60 kDa respectively. Both of the lectins were composed of heterogeneous A and B subunits linked with a disulfide bond, and showed the same carbohydrate-binding specificities for Gal and GalNAc. However, they are different not only in biophysical properties (glycosylation and amino acid compositions) but also bioactivities (cell killing and cytokine induction). The KML-IIL showed 17-145 times stronger in cytotoxicities to various human and mouse cancer cell lines than the KML-IIU. The KML-IIL also induced TNF-alpha secretion from mouse peritoneal macrophages 4.5 times better than the KML-IIU. The results demonstrated isolectins in Korean mistletoe were varied in bioactivities and the KML-IIL may be developed as an anti-cancer agent.

Targeting Thioredoxin Reductase 1 Reduction in Cancer Cells Inhibits Self-Sufficient Growth and DNA Replication

Thioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1's role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase α, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy.