Samples from the culture medium of BHK-21 cells infected with MHV-68 were fractionated using conventional chemical, chromatographic and separation techniques. Obtained fractions were tested for biological activity. Subsequently, the MHGF-68 fractions with antiproliferative activity were tested in vivo to see their inhibitory activity on tumor growth in mice. In this study were used four weeks old athymicnudemice CD1, subcutaneously infected with the suspension of Hepa 1c1c7 cells. Within 8 days post administration of cells, tumor proliferation was observed. MHGF-68 fractions 1B1 and 1D2 with the antiproliferative effect were intravenously administered to mice and they were tested for inhibition of progression of tumor growth. Tumors in experimental mice treated with MHGF-68 fractions exhibited decreased growth of tumors in comparison to control mice. These data show that tumor xenografts were suppressed after the treatment with 1B1 and 1D2 fractions of MHGF-68. Histological findings did not confirm substantial differences in the tumor tissue between control and experimental mice. Cytologically, there were tumor formations/masses consisting of a little differentiated population of cells with cellular and nuclear pleiomorphia with atypia, mitoses. In further studies tumor tissues collected from mice after administration of MHGF-68 will be studied in more detail by immuno-histochemical methods. Taken together, the promising potential of MHGF-68 in the modulation of tumor growth could be used as a foundation for development of novel cancer treatment measures with antiproliferative effects.
A highly significant, but unanswered, question in the pathogenesis of psoriasis relates to how normal appearing and diseased skin can coexist, undergo spontaneous flares and remissions, and yet appear to be genetically acquired. A plausible explanation for these disparate observations is that there is a basic defect in epidermal proliferation of skin of subjects with psoriasis and that disease expression is governed by other host factors. To address this question, we compared epidermal proliferation of skin involved and uninvolved with psoriasis with normal skin before and after transplantation to congenitally athymic (nude) mice, a biologic milieu free of humoral factors unique to the donor host. Results demonstrated that (a) before transplant, synthesis of DNA by the epidermal cells from skin uninvolved and involved with psoriasis is significantly higher than normal, 1.6 and 3.6 times, respectively; (b) 6 wk after transplantation, synthesis of DNA by epidermal cells is
In this study, we show that Py-induced mammary tumor cell lines established from two independent mammary adenocarci- nomas, which do not contain free viral genomes and are still tumorigenic in athymicnudemice, exhibit alterations of the c-myc gene structure and/or expression. Recent studies with woodchucks, an animal model for hepadnavirus-induced pri- mary hepatocellular carcinomas, have shown that in some tu- mors, the viral DNA integrates at or close to the cellular c-myc proto-oncogene and disturbs its expression (25, 32). On the other hand, Couturier et al. (14) identified in genital carcino- mas papillomavirus DNA integration next to the c-myc gene which also modifies the transcription of the gene.
Melanoma is the most deadly form of cutaneous malignancy, and its incidence rates are rising worldwide. In melanoma, constitutive activation of the BRAF/MEK/ ERK (MAPK) and PI3K/AKT/mTOR (PI3K) signaling pathways plays a pivotal role in cell proliferation, survival and tumorigenesis. A combination of compounds that lead to an optimal blockade of these critical signaling pathways may provide an effective strategy for prevention and treatment of melanoma. The phytochemical fisetin is known to possess anti-proliferative and pro-apoptotic activities. We found that fisetin treatment inhibited PI3K signaling pathway in melanoma cells. Therefore, we investigated the effect of fisetin and sorafenib (an RAF inhibitor) alone and in combination on cell proliferation, apoptosis and tumor growth. Combination treatment (fisetin + sorafenib) more effectively reduced the growth of BRAF-mutated human melanoma cells at lower doses when compared to individual agents. In addition, combination treatment resulted in enhanced (i) apoptosis, (ii) cleavage of caspase-3 and PARP, (iii) expression of Bax and Bak, (iv) inhibition of Bcl2 and Mcl-1, and (v) inhibition of expression of PI3K, phosphorylation of MEK1/2, ERK1/2, AKT and mTOR. In athymicnudemice subcutaneously implanted with melanoma cells (A375 and SK-MEL-28), we found that combination therapy resulted in greater reduction of tumor growth when compared to individual agents. Furthermore, combination therapy was more effective than monotherapy in: (i) inhibition of proliferation and angiogenesis, (ii) induction of apoptosis, and (iii) inhibition of the MAPK and PI3K pathways in xenograft tumors. These data suggest that simultaneous inhibition of both these signaling pathways using combination of fisetin and sorafenib may serve as a therapeutic option for the management of melanoma.
CDIM9 exhibited remarkable growth inhibitory effects on basal-like breast cancer in our animal model, with 87% tumor growth inhibition after daily treatment for 35 days. The dose used was twofold below the toxic dose of CDIM9; we there- fore estimate a therapeutic index of at least 2, and this may be significantly higher because we did not investigate lower doses of CDIM9. In addition, six out of 13 (46%) of the tumor- bearing animals had complete tumor regression with no re- growth of tumors by day 50 and absence of basal-like breast tumor cells by histologic examination of necropsy specimens. This anticancer efficacy compares favorably with results obtained in similar subcutaneous MDA-MB231 xenografts in athymicnudemice treated with gamma radiation (94% tumor growth inhibition), doxorubicin (63% tumor growth inhibition),
Angiogenesis is essential for tumor development. Accumulating evidence suggests that adenosine monophosphate-activated protein kinase (AMPK), an energy sensor and redox modulator, is associated with cancer development. However, the effect of AMPK on tumor development is controversial, and whether AMPK affects tumor angiogenesis has not been resolved. We show that deletion of AMPKa1, but not AMPKa2, upregulates non-canonical nuclear factor kappa B2 (NF-kB2)/p52- mediated cyclin-dependent kinase 2 (CDK2), which is responsible for the anchorage- independent cell growth of immortalized mouse embryo fibroblasts (MEFs). Co-culture with AMPKa1 knockout MEFs (or their conditioned medium) enhances the migration and network formation of human microvascular endothelial cells, which is dependent on p52-upregulated erythropoietin (Epo). AMPKa1 deletion stimulates cellular proliferation of allograft MEFs, angiogenesis, and tumor development in athymic nu/nu mice, which is partly ameliorated by antibody-mediated Epo neutralization. Therefore, the AMPKa1-p52-Epo pathway may be involved in stromal fibroblast- mediated angiogenesis and tumorigenesis.
Finally, we tested F1 effect in mice harboring PDXs of TNBC . First, quantification by sandwich ELISA in whole cytosolic extracts of five representative TNBC PDXs showed that cath-D concentration varied from 18 to 77 pmol/mg of total protein (Fig. 6a). These values were in the same range as those detected in whole cytosolic extracts prepared from MDA-MB-231 tumor xenografts (Fig. 6a), and from 40 TNBC samples (Fig. 6b). Immunostaining of the B1995 and B3977 primary tumors with an anti-cath-D antibody confirmed that cath-D was detected in tumor cells and microenvironment (Fig. 6c), as previously observed with the TNBC TMA (Fig. 1c and e). These results indicate these PDX models are representative of the disease, at least concerning cath-D expression. We then engrafted athymicnudemice with PDX B1995 or PDX B3977, the two PDXs showing the fastest growth in nudemice (average passage duration for the first three passages: 46 days for B1995 and 42 days for B3977) (Fig. 6d). Tumor volume increase was significantly slowed down in mice treated with F1 com- pared with control (Fig. 6e).
Because cetuximab in combination with rIL-1α showed some anti-tumor activity in athymicnudemice where NK cells (and not T cells) are present (Fig. 4A), and cetuximab along with IL-1 ligands have been previously reported to activate NK cell activity, we initially proposed that NK cells may be involved in the anti-tumor immune response to CTX + IL-1α-NP. However, immune cells isolated from spleens of CTX + IL-1α-NP-treated mice demonstrated no differences in the frequency of NK cells or activated NK cells in the spleen (Additional file 6: Figure S5) or tumors (Additional file 7: Figure S6) compared to the other treat- ment groups. However, spleens from mice administered CTX + IL-1α-NP showed significantly decreased percent- ages of PD-1 + CD4 + T cells (Fig. 5G), significantly increased percentages of CD8 + T cells (Fig. 5H), and significantly de- creased CD25 + CD8 + T cells compared to IgG + EMP-NP (Fig. 5I). Percentages of CD69 + CD4 + and IFNγ + CD8 + T cells were elevated in tumors from CTX + IL-1α-NP-- treated mice but did not reach statistical significance com- pared to the other treatment groups (Additional file 8: Figure S7). To further interrogate the role of T cell-dependent immune response, female BALB/c mice bearing TUBO-EGFR tumors were treated with CTX + IL-1α-NP (Fig. 6A,B) as already described in Fig. 5 with or without anti-CD4 (100 μg (clone GK1.5)) (Fig. 6A,C) or anti-CD8 (300 μg (clone 53–6.7)) (Fig. 6A,D) 1 and 3 days prior to tumor inoculation, and every 3–4 days after tumor inoculation. Specific depletion of CD4+ (Fig. 6E) and CD8+ T cells (Fig. 6F) from tumors was confirmed by flow cytom- etry. Both CD4 + and CD8 + T cell depletion significantly re- versed the anti-tumor effect of CTX + IL-1α-NP, and CD8 +
Multiple human tumor xenograft models were used to investigate the tumor preferential accumulation of NIR-27 in vivo. Athymic BALB/c nudemice with human MKN-45, A549, and GFP-labelled HL-60 tumor xenografts in the subcutaneous spaces were established and subjected to NIR imaging after a single dose administration of NIR-27 at 0.3 mg/kg through intravenous injection. Figure 3A shows that NIR florescence signals associated with the implanted MKN-45, A549, (see Figure S4) and GFP-labelled HL-60 tumor xenograft sites can be clearly visualized with low background interfering signals after injection with NIR-27. Furthermore, fluorescence of NIR and GFP can be observed to be co-localized in the area of the tumor. The NIR fluorescence signals of the dissected organs further confirmed the cancer-targeting ability of NIR-27 in vivo (Figure 3B). Moreover, the cytosol accumulation of NIR-27 in GFP-labelled HL-60 tumor cells was identified by histopatho- logic analysis of the tumor specimens (Figure 3D). In addition, we also observed that the fluorescent signals continued for 14 days after a single injection of NIR-27 in the athymicnudemice bearing HL-60 tumor xenograft (see Figure S5). These results suggest NIR-27 can not only be preferentially accumu- lated in multiple tumors, but also maintain stable retention for a long time. The subcellular localization of the dye in tumor
A2780 ovarian cancer xenograft model: Female athymicnudemice (6–8 weeks) were subcutaneously inoculated in the right flank with 8 × 10 6 human A2780 ovarian cancer cells (Sigma Aldrich) resuspended in 50% growth medium and 50% Matrigel. Two sets of experiments were performed: early stage tumor treatment starting after tumor sizes reached ca. 100–200 mm 3 ; or late stage tumor treatment when tumor sizes reached ca. 400 mm 3 . Animals were randomized into groups of seven mice such that the mean tumor volumes were similar between groups and then administered with following formulations: 1) Normal saline; 2) Taxol (20 mg/kg PTX at determined MTD dose); 3) Abraxane (45 and 90 mg/kg PTX at determined ½MTD and MTD doses); 4) PTX loaded micelles (75 and 150 mg/kg at determined ½MTD and MTD doses). The formulations were administered via tail vein following q4d × 4 regimen (on the days 0, 4, 8, 12). Tumor growth was monitored twice weekly for 15 weeks or earlier end-points defined by tumor volume (> 2000 mm 3 ), animal weight loss (> 15%), or animals becoming moribund. Tumor length (L), width (W) were measured and tumor volume (TV) was calculated as TV =1/2 × L × W 2 . Survival and body
To generate tumor xenografts, we injected the HCT-116 colorectal cancer cells in athymicnudemice and, after seven days, treatments with bevacizumab, anti- human PlGF mAb 16D3, iVR1 and CP peptides started (Figure 2C). Surprisingly, tumor growth curves in iVR1 and bevacizumab treated mice were fully superimposable, resulting in a significant tumor growth delay starting from four days of treatment, compared to vehicle and CP. The mAb 16D3, able to block only PlGF produced by human cells, also determined a significant inhibition compared to vehicle and CP. Bevacizumab and iVR1 tumor growth inhibitions were also significantly higher compared to Figure 1: Anti-angiogenic activity of iVR1 in vivo. (A) Chemical structure of iVR1 tetrameric tripeptide that has a calculated molecular mass of 2362.02 g/mol. (B) Schematic representation of the iVR1. L-Cys(Bzl): L-cysteine(S-benzyl); L-Cha, L-cyclohexylalanine. (C) iVR1 inhibited laser-induced CNV in a dose-dependent manner, whereas CP was ineffective. CNV volumes were measured by confocal evaluation of Isolectin B4 staining of RPE-choroid flat mounts. Data are represented as the mean ± SEM (N = 8). *p < 0.0005 and § p < 0.01
Fastidious mycobacteria usually infect immunocompromised hosts (human immunodeficiency virus-infected or otherwise immunosuppressed patients). We here describe severe lymphadenitis, caused by a fastidious mycobacterium closely related to Mycobacterium genavense, in an apparently immunocompetent woman, whose brother had died from an unidentified mycobacterial infection in 1969. A variety of techniques, including inoculation of nudemice, histopathology, electron microscopy, lipid analysis, ATP measurements, and molec- ular biology, were used to characterize this mycobacterium. All attempts to culture the etiological agent on many different media failed. The organism multiplied only in congenitally athymicnudemice. Although phenotypically similar to M. genavense, the mycobacterium differs from M. genavense by three nucleotides of the 16S rRNA gene sequence. Various antimycobacterial drugs were administered, including gamma interferon, but multiple relapses occurred. Finally, therapy with a combined regimen of clarithromycin, clofazimine, rifabutin, and ethambutol was curative. To our knowledge, this is the first report of lymphadenitis in an apparently immunocompetent patient, caused by a noncultivable Mycobacterium sp. closely related to M. genavense. This study emphasizes the importance of employing a variety of diagnostic approaches such as the inoculation of laboratory animals, histopathology, electron microscopy, lipid analysis, ATP measurements, and molecular biology to characterize novel microorganisms that cannot be cultured in vitro.
vein injection. Five animals were sacrificed by sodium pentobarbital overdose (~200 mg/kg) at 0.5, 1, 4, 24 and 72 h postinjection (p.i.). Blood samples were withdrawn from the heart of tumor-bearing mice. The tumor and normal organs (brain, eyes, heart, spleen, lungs, liver, kidneys, muscle and intestine) were har- vested, washed with saline, dried with absorbent tis- sue, weighed, and counted on a Perkin Elmer Wizard – 1480 -counter (Shelton, CT). The organ uptake was calculated as the percentage of injected dose per gram of organ mass (%ID/g) or the percentage of injected dose per organ (%ID/organ). For the blocking ex- periment, five tumor-bearing athymicnudemice (20 – 25 g) were used, and each animal was administered with ~3 Ci of 111 In(DOTA-6P-RGD 4 ) along with ~350
Results: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymicnudemice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke ’ s type A-stage HCT-116 and Duke ’ s type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN- γ , IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF- γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls.
H5.110CMVhIFN- β treatment inhibits growth of human colon cancer cells in a dose-dependent fashion in an intrahepatic microscopic dis- ease athymicnude mouse liver metastases model and improves survival in an intrasplenic microscopic disease athymicnude mouse liver metas- tases model. (a) KM12L4 cells were directly injected into the livers of athymicnudemice. On day 5, mice were randomized to receive PBS, H5.010CMV β -gal (Ad β -gal), or H5.110CMVhIFN- β (AdhIFN- β ) via tail vein injection. On day 19, animals were sacrificed and livers were har- vested for tumor volume measurement. Each point represents a single animal. Mean tumor volume is indicated by the large cross. (P = 0.0001.) One animal in the PBS group died on day 17 of diffuse metastasis and was not included in the final average. (b) KM12L4 cells were injected as described above. On day 5, mice were randomized to receive PBS or H5.110CMVhIFN. Animals were sacrificed on day 19 and the livers harvested for tumor volume measurement. (P < 0.0001.) (c) KM12L4 cells were directly injected in the spleens of athymicnudemice followed by splenectomy. On day 5, mice were randomized to receive PBS, H5.010CMV β -gal, or H5.110CMVhIFN- β via tail vein injection. Mice were sacrificed when they became moribund by preestablished criteria, and their survival curves were plotted.
primary lesions or distant metastases (Albelda, S. M., S. A. Mette, D. E. Elder, R. Stewart, L. Damjanovich, M. Herlyn, and C. A. Buck. 1990. Cancer Res. 50:6757-6764), suggesting a role for this adhesion receptor in the malignant growth of human melanoma tumors. A cell model was established to analyze the role of alpha v integrins on the tumorigenicity of human melanoma. From M21 human melanoma cells, stable variants were selected that lack alpha v gene expression and thus fail to express integrin alpha v beta 3 (M21-L cells). These cells not only lost the ability to attach to vitronectin but showed a dramatic reduction in tumorigenicity when transplanted into athymicnudemice, compared with M21 cells, even though both cell types showed identical beta 1 integrin expression and growth properties in vitro. M21-L cells were stably transfected with a cDNA-encoding alpha v. This resulted in the functional expression of integrin alpha v beta 3 on these cells and completely restored their tumorigenicity. Thus, integrin alpha v gene expression and the resulting adhesive phenotype are directly involved in the […]
The effect of dmrFABP5 on levels of phosphorylated PPARγ (p-PPARγ1 and p-PPARγ2, the presumed biologically active forms of PPARγ) was shown in Figure 5. Western blots using anti-p-PPARγ detected 2 bands representing the isoforms of p-PPARγ1 and p-PPARγ2 at 54 and 57kDa, respectively (Figure 5A). If the levels of p-PPARγ1 and p-PPARγ2 in PNT2 were both set at 1 and 1, relative levels in weakly malignant LNCaP, moderately malignant 22RV1, and highly malignant DU145, PC3 and PC3-M cells were 9.54 ± 1.81 and 9.5 ± 0.5; 25.4 ± 1.8 and 47.0 ± 1.7; 26.99 ± 1.72 and 85.5 ± 14.5; 12.08 ± 1.8 and 30 ± 5; 21.99 ± 2.63 and 80 ± 5, respectively (Figure 5B). Thus the levels of p-PPARγ, particularly p-PPARγ2, were significantly increased in all the malignant cell lines studied (Student’s t test, p < 0.001). To investigate the effect of dmrFABP5 on p-PPARγ, PC3-M cells were treated for 24 hours with the PPARγ antagonist GW9662, with the PPARγ agonist Rosiglitazone plus dmrFABP5 and with the combination of dmrFABP5 and SB-FI-26 (Figure 5C). If levels of p-PPARγ1 and p-PPARγ2 in untreated cells were set at 1 and 1 (Figure 5D), the levels in cells treated with GW9662 and dmrFABP5 were reduced significantly by 52% and 51%; 50% and 65%, respectively (Student’s t test, p < 0.001). Interestingly, cells treated with a combination of dmrFABP5 and SB- FI-26 dramatically reduced the level of p-PPARγ2 by 90%, and that of p-PPARγ1 by 52%. However, those mice treated with rosiglitazone significantly increased the levels of both p-PPARγ isoforms (Student’s t test, p < 0.01) (Figure 5D). When LNCaP cells, which expressed very low levels of p-PPARγ1 and 2, were treated with wtrFABP5, the levels of both isoforms were increased by 1- and 0.9- fold, respectively. However, these increases were reversed completely by adding dmrFABP5 to the cells. Furthermore, addition of dmrFABP5 to LNCaP cells reduced the level of p-PPARγ2 by 5-fold (Student’s t test, p < 0.001) to a level lower than that obtained in the pretreatment group (Figure 5E, 5F). When androgen- sensitive 22Rv1 cells were treated with wtrFABP5, with dmrFABP5 or with a combination of both (Figure 5G), wtrFABP5 significantly increased the levels of both p-PPARγ1 and 2 (Student’s t test, P < 0.01) (Figure 5H). However, treatments with dmrFABP5 significantly suppressed the level of both PPARγ isoforms. Furthermore, the promoting effect of wtrFABP5 on levels of both p-PPARγ isoforms was completed blocked by treatment of mice with dmrFABP5.
Mammary adipose tissue (1 ml) was obtained from patients undergoing plastic surgery and implanted subcutaneously into non-obese diabetic/severe com- bined immunodeficient (NOD/SCID) mice to form a fat pat. MDA-MB-231(negative control or shPAI-1) cells (1 × 10 6 ) were then either injected into the fat pad or injected alone, while untreated fat pads were also used as a separate control. Each group included six mice. After 7 days, the mice injected both adi- pose and MDA-MB-231 negative control cells were assigned to the negative control group and minoxidil group (8 mice/group, tail vein injection of 0.9% NaCl and 2.5 mg/kg minoxidil at a frequency of once every 2 days, respectively). After 6 weeks, tumor metastasis was assessed by bioluminescent imaging on the Xenogen In Vivo Imaging System (IVIS, Caliper Life Science, Hopkinton, MA). Mice were then sacrificed and lungs were formalin-fixed and paraffin-embedded for hematoxylin and eosin staining. Lung metastases were quantified by human genomic DNA extraction from mouse lungs.
Male inbred Buffalo rat weights from 280 ~ 350 g were applied as donors and recipients for rat liver transplantation model. Male nudemice around 4~8 weeks old and weights from 20 ~ 25 g were applied for orthotopic and subcutaneous xenograft nudemice models. These buffalo rat and nudemice were housed in a standard laboratory environment with sufficient water, chow and free activity. They were kept under constant environment with a 12-hour light/dark cycle. They were fasted 12 hours before operation. All the operations were performed under sterilized condition. All the animal studies were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR), The University of Hong Kong. Rat liver transplantation model