Mammary Gland Tumor Cell Line Research Articles

Establishment of canine mammary gland tumor cell lines harboring PI3K/Akt activation as a therapeutic target

Canine mammary gland tumors (MGT) have a poor prognosis in intact female canines, posing a clinical challenge. This study aimed to establish novel canine mammary cancer cell lines from primary tumors and characterize their cellular and molecular features to find potential therapeutic drugs. The MGT cell lines demonstrated rapid cell proliferation and colony formation in an anchorage-independent manner. Vimentin and α-SMA levels were significantly elevated in MGT cell lines compared to normal canine kidney (MDCK) cells, while CDH1 expression was either significantly lower or not detected at all, based on quantitative real-time PCR (qRT-PCR) analysis. Functional annotation and enrichment analysis revealed that epithelial-mesenchymal transition (EMT) phenotypes and tumor-associated pathways, particularly the PI3K/Akt signaling pathway, were upregulated in MGT cells. BYL719 (Alpelisib), a PI3K inhibitor, was also examined for cytotoxicity on the MGT cell lines. The results show that BYL719 can significantly inhibit the proliferation of MGT cell lines in vitro. Overall, our findings suggest that the MGT cell lines may be valuable for future studies on the development, progression, metastasis, and management of tumors.

Fast Ultrasound Scanning is a Rapid, Sensitive, Precise and Cost-Effective Method to Monitor Tumor Grafts in Mice

In preclinical studies, accurate monitoring of tumor dynamics is crucial for understanding cancer biology and evaluating therapeutic interventions. Traditional methods like caliper measurements and bioluminescence imaging (BLI) have limitations, prompting the need for improved imaging techniques. This study introduces a fast-scan high-frequency ultrasound (HFUS) protocol for the longitudinal assessment of syngeneic breast tumor grafts in mice, comparing its performance with caliper, BLI measurements and with histological analysis. The E0771 mammary gland tumor cell line, engineered to express luciferase, was orthotopically grafted into immunocompetent C57BL/6 mice. Tumor growth was monitored longitudinally at multiple timepoints using caliper measurement, HFUS, and BLI, with the latter two modalities assessed against histopathological standards post-euthanasia. The HFUS protocol was designed for rapid, anesthesia-free scanning, focusing on volume estimation, echogenicity, and necrosis visualization. All mice developed tumors, only 20.6% were palpable at day 4. HFUS detected tumors as small as 2.2 mm in average diameter from day 4 post-implantation, with an average scanning duration of 47 s per mouse. It provided a more accurate volume assessment than caliper, with a lower average bias relative to reference tumor volume. HFUS also revealed tumor necrosis, correlating strongly with BLI in terms of tumor volume and cellularity. Notable discrepancies between HFUS and BLI growth rates were attributed to immune cell infiltration. The fast HFUS protocol enables precise and efficient tumor assessment in preclinical studies, offering significant advantages over traditional methods in terms of speed, accuracy, and animal welfare, aligning with the 3R principle in animal research.

Defucosylated mouse‑dog chimeric anti‑HER2 monoclonal antibody exerts antitumor activities in mouse xenograft models of canine tumors.

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti-HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2-positive breast and gastric cancer. An anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa) was previously developed. In the present study, a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding-affinity [a dissociation constant (KD): 7.5×10−10 M, as determined by flow cytometric analysis] for dog HER2-overexpressed CHO-K1 (CHO/dHER2) cells. H77Bf highly exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for CHO/dHER2 cells by canine mononuclear cells and complement, respectively. Moreover, administration of H77Bf significantly suppressed the development of CHO/dHER2 ×enograft tumor in mice compared with the control dog IgG. H77Bf also possesses a high binding-affinity (KD: 7.2×10−10 M) for a canine mammary gland tumor cell line (SNP), and showed high ADCC and CDC activities for SNP cells. Intraperitoneal administration of H77Bf in mouse xenograft models of SNP significantly suppressed the development of SNP xenograft tumors compared with the control dog IgG. These results indicated that H77Bf exerts antitumor activities against dHER2-positive canine cancers, and could be valuable treatment regimen for canine cancers.

Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Exerted Antitumor Activities in Mouse Xenograft Models of Canine Mammary Gland Tumor.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody, clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed Chinese hamster ovary-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, the reactivity of 134-mG2a-f against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f highly exerted ADCC and CDC for SNP. The administration of 134-mG2a-f significantly suppressed the SNP xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine mammary gland tumors, and could be valuable as part of an antibody treatment regimen for them.

Antitumor Activities in Mouse Xenograft Models of Canine Mammary Gland Tumor by Defucosylated Mouse-Dog Chimeric Anti-Epidermal Growth Factor Receptor Antibody (E134Bf).

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf), and the reactivity of E134Bf against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry. Furthermore, E134Bf highly exerted ADCC and CDC for SNP cells. The administration of E134Bf with canine mononuclear cells significantly suppressed the SNP xenograft growth. These results suggest that E134Bf exerts antitumor effects against dEGFR-expressing canine mammary gland tumors and could be valuable as part of an antibody treatment regimen for them.

In vitro anticancer effects of alpelisib against PIK3CA‑mutated canine hemangiosarcoma cell lines.

Hemangiosarcoma (HSA) is a malignant neoplasm that occurs in humans and canines with a poor prognosis owing to metastatic spread, despite effective treatment. The frequency of spontaneous HSA development is higher in canines than in humans. Therefore, canine HSA is a useful model of intractable human disease, which requires early detection and an effective therapeutic strategy. A high frequency of the p110α phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations is detected in a comprehensive genome-wide analysis of canine cases of HSA. The present cloned the full-length cDNA of canine PIK3CA and identified a mutation in codon 1047 from canine cases of HSA and cell lines that were established from these. The enforced expression of the 1047th histidine residue (H1047)R or L mutants of canine PIK3CA in HeLa cells enhanced epidermal growth factor receptor (EGFR) signaling via Akt phosphorylation. PIK3CA mutant canine HSA cell lines exhibited the hyperphosphorylation of Akt upon EGF stimulation as well. Alpelisib, a molecular targeted drug against PIK3CA activating mutations, exerted a significant antitumor effect in canine PIK3CA-mutated HSA cell lines. By contrast, it had no significant effect on canine mammary gland tumor cell lines harboring PIK3CA mutations. On the whole, the findings of the present study suggest that alpelisib may be highly effective against PIK3CA mutations that occur frequently in canine HSA.

Scorpion Venom Peptide Effects on Inhibiting Proliferation and Inducing Apoptosis in Canine Mammary Gland Tumor Cell Lines

Simple SummaryCanine mammary gland tumors (CMGTs) are quite common in intact female dogs. In diagnosed cases, approximately 50% of mammary gland tumors metastasized. Chemotherapy is a practical treatment to increase the median survival time, but it has severe side effects and impacts on resistance. BmKn-2, an antimicrobial peptide derived from scorpion venom, has displayed anticancer effects in oral and colon human cancer cell lines. Consequently, BmKn-2 may be useful in the molecular treatment of CMGTs. This study investigated the effect of BmKn-2 on the proliferation inhibition, apoptotic induction and the related mechanisms of those actions in CMGT cell lines, metastatic (CHMp-5b) and non-metastatic (CHMp-13a). The experimental results showed that BmKn-2 effectively inhibited proliferation and induced apoptosis in both CMGT cell lines via Bcl-2 (B-cell lymphoma-2) down-regulation and Bax (Bcl2 associated X) up-regulation gene expressions. Therefore, BmKn-2 could be used as a candidate molecular treatment for CMGTs in the future.The most common neoplasms in intact female dogs are CMGTs. BmKn-2, an antimicrobial peptide, is derived from scorpion venom and has published anticancer effects in oral and colon human cancer cell lines. Thus, it is highly likely that BmKn-2 could inhibit CMGT cell lines which has not been previously reported. This study investigated the proliferation and apoptotic properties of BmKn-2 via Bax and Bcl-2 relative gene expression in two CMGT cell lines, metastatic (CHMp-5b) and non-metastatic (CHMp-13a). The results showed that BmKn-2 inhibited proliferation and induced apoptosis in the CMGT cell lines. The cell morphology clearly changed and increased apoptosis in a dose dependent of manner. The half maximum inhibitory concentration (IC50) was 30 µg/mL for CHMp-5b cell line and 54 µg/mL for CHMp-13a cell line. The induction of apoptosis was mediated through Bcl-2 and Bax expression after BmKn-2 treatment. In conclusion, BmKn-2 inhibited proliferation and induced apoptosis in both CHMp-5b and CHMp-13a cell lines via down-regulation of Bcl-2 and up-regulation of Bax relative mRNA expression. Therefore, BmKn-2 could be feasible as candidate treatment for CMGTs.

The inhibitory effect of canine interferon gamma on the growth of canine tumors

Recombinant canine interferon-γ (rc-IFNγ; InterdogⓇ) was exclusively approved as a therapeutic for canine atopic dermatitis. However, it has been used off-label for the treatment of canine cancer. We examined the inhibitory effect of rc-IFNγ on the growth of canine tumor cell lines and analyzed its mechanism of action. Three (CTB-p, CTB-m, and CNM-m) out of seven mammary gland tumor cell lines and two (VIMC and CoMS) out of four mast cell tumor cell lines showed remarkable growth inhibition after treatment with rc-IFNγ. However, one (CLBL-1) out of nine lymphoma cell lines showed a significant amount of cell death. Using CTB-p and CTB-m cell lines, we showed that STAT1 was essential for inducing the growth inhibitory effect of rc-IFNγ. Although rc-IFNγ induced G1 growth arrest in CTB-p cell line, treatment with rc-IFNγ did not alter the expression of cell cycle regulatory proteins. In this study, we observed direct cytotoxicity or cytostatic effects of rc-IFNγ in canine tumor cell lines. However, the detailed mechanisms responsible for these effects need to be elucidated in the future.

Cytotoxic effects of canine nk cells on canine mammary tumor cells

Background & Aim Natural killer (NK) cells play a crucial role in immune responses against various pathogens. NK cells are innate lymphocytes that rapidly provide defenses against tumors and viral infections allowing pathogen elimination or limiting viral spread. Their fast responses mainly rely on the expression of multiple germ-line encoded activating receptors among which natural cytotoxic receptors (NCRs) and NKG2D play the most relevant role in the recognition and killing of infected cells. NK cells have been characterized in a variety of mammals, but canine NK cells remain incompletely characterized. Mammary tumors account for ∼30% of all tumors in the female canine and approximately 50% of the tumors are malignant. Canine mammary gland tumor cell lines are excellent experimental models for identification of cancer-associated genes and understanding cancer biology and progression. In this study, we isolated canine peripheral blood-derived NK cells and defined their immunophenotype and cytotoxic effects on canine mammary tumor cells. In this study, we compared characteristics of canine NK cells against two canine mammary carcinoma cell lines, REM134 and CF41.Mg. Methods, Results & Conclusion Canine NK cells are proliferated and activated in CD5 negative population under specific culture condition. We peripheral blood mononuclear cells (PBMCs) were isolated from female beagle dogs (n=3). Subsequently, CD5 negative cells were isolated by immunomagnetic separation from PBMCs. For activation and expansion of NK cells, CD5 negative cells and co-cultured K562 cells in the presence of IL-2, IL-15, and IL-21 for 21 days. We found that expression markers of activated NK cells such as NKp30, NKp44, NKp46, NKG2D, CD244, perforin, granzyme B, and tumor necrosis factor alpha were highly upregulated. In addition, we found upregulated production of interferon gamma of activated NK cells against target cells such as REM134 and CF41.Mg. Also, we observed that cytotoxicity of activated NK cells against target cells more sensitively reacted to CF41.Mg than REM134. Taken together, we recommend the development of an experimental application of CF41Mg, which has not been reported in canine mammary carcinoma research.

Endogenous Leu332Gln mutation in p53 disrupts the tetramerization ability in a canine mammary gland tumor cell line.

Mutations in the p53 gene are associated with more than half of all human cancers. These mutations often cause a disruption of the tumor-suppressor function of p53 and induce genomic instabilities. Wild‑type p53 requires tetramerization to function as an initiator of cell cycle arrest and apoptosis. Although alterations in p53 tetramerization caused by mutation have been well studied, there are few cell lines containing an endogenous mutation in the tetramerization domain of p53. Here, we report the discovery of a canine mammary gland tumor cell line CTB‑m2, which contains the Leu332Gln (L332Q) mutation corresponding to Leu344 in the tetramerization domain of human p53. Although CTB‑m2 cells are genetically heterozygous for the Leu332Gln mutation, the mutant mRNA was almost exclusively expressed. CTB‑m2 cells showed enhanced cell proliferation compared to wild‑type p53-expressing CTB‑m cells of the same lineage. A p53 tetramerization reporter assay showed that the ability of the p53 mutant to form tetramers was significantly lower than that of wild‑type p53. An immunoblot analysis of cross-linked p53 oligomerized forms demonstrated that the L332Q mutant lacked the ability to form tetramers but retained the ability to form dimers. These data suggest that the p53 mutant cell line CTB‑m2 could be a useful tool for analyzing the precise tetramerization mechanisms of p53 and verifying the effects of therapeutic agents against tumors expressing p53 mutants that lack the ability to tetramerize.

Role of tissue factor expression in thrombin generation by canine tumor cells.

To measure thrombin generation by high and low tissue factor (TF)-expressing canine cancer cell lines. Canine cell lines CMT25 (high TF-expressing mammary gland tumor cell line) and HMPOS (low TF-expressing osteosarcoma cell line). Thrombin generation by cancer cells was measured in pooled normal canine plasma by use of calibrated automated thrombography without added trigger reagents. Results were expressed as lag time, time to peak thrombin concentration, peak thrombin concentration, and total thrombin concentration or thrombin generation potential. Corn trypsin inhibitor, hirudin, and annexin V were used to inhibit contact activation, thrombin formation, and phosphatidylserine activity, respectively. Pooled normal human plasma deficient in coagulation factors VII, VIII, IX, X, XI, or XII was used to assess the role of individual coagulation factors on thrombin generation. CMT25 generated significantly more thrombin than did HMPOS (mean ± SD, 3,555 ± 604 nM thrombin•min and 636 ± 440 nM thrombin•min, respectively). Thrombin generation of CMT25 was dependent on factor VII and phosphatidylserine and was independent of contact activation. In contrast, thrombin generation of HMPOS was attributed to contact activation. High TF-expressing canine mammary cancer cells generated thrombin in a plasma milieu in vitro in a factor VII- and phosphatidylserine-dependent manner. These findings support a role for TF in hypercoagulability detected in dogs with mammary gland tumors and potentially for other tumors that strongly express TF.

Establishment of a canine mammary gland tumor cell line and characterization of its miRNA expression.

Canine mammary gland tumors (CMGTs), which are the most common neoplasms in sexually intact female dogs, have been suggested as a model for studying human breast cancer because of several similarities, including relative age of onset, risk factors, incidence, histological and molecular features, biological behavior, metastatic pattern, and responses to therapy. In the present study, we established a new cell line, the SNP cell line, from a CMGT. A tumor formed in each NOD.CB17-Prkdcscid/J mouse at the site of subcutaneous SNP cell injection. SNP cells are characterized by proliferation in a tubulopapillary pattern and are vimentin positive. Moreover, we examined miRNA expression in the cultured cells and found that the expression values of miRNA-143 and miRNA-138a showed the greatest increase and decrease, respectively, of all miRNAs observed, indicating that these miRNAs might play a significant role in the malignancy of SNP cells. Overall, the results of this study indicate that SNP cells might serve as a model for future genetic analysis and clinical treatments of human breast tumors.

Protective Role of Selenium Compounds on the Proliferation, Apoptosis, and Angiogenesis of a Canine Breast Cancer Cell Line

We herein examined the effects of different doses, forms, and compatibilities of selenium on a canine mammary gland tumor cell line, CTM1211, and explored the related mechanisms. Three selenium compounds, sodium selenite (SSE), methylseleninic acid (MSA), and methylselenocysteine (MSC), were selected for these experiments, and cyclophosphamide (CTX) served as a positive control. In the cell viability assay, the cell viability of each group at 48/72h decreased significantly compared with the control group (p < 0.05), and the cell viability of the CTX + MSA group was lower than that of CTX and MSA groups (p < 0.05). Moreover, the inhibitory effect of selenium on cell proliferation was time-dependent but not concentration-dependent. In the cell apoptosis assay, the apoptosis values of each group increased significantly compared with the control group, and the apoptosis values of the CTX + MSA group increased the most significantly (p < 0.01). The protein and mRNA expression levels of vascular endothelial growth factor-alpha (VEGF-alpha), angiopoietin-2 (Ang-2), and hypoxia inducible factor-1 alpha (HIF-1 alpha) were downregulated in each group, while that of phosphatase and tensin homolog (PTEN) were upregulated (p < 0.05). In conclusion, these three selenium compounds, especially MSA, could significantly inhibit the viability and growth of the CTM1211 cell line, which is partly due to the induction of apoptosis and regulation of tumor angiogenesis.

Transforming growth factor-β transiently induces vimentin expression and invasive capacity in a canine mammary gland tumor cell line

The epithelial–mesenchymal transition (EMT) is a crucial event that occurs during cancer metastasis and can be induced by transforming growth factor-β (TGF-β) in various tumor cells in vitro. However, little is known about the effects of TGF-β in canine mammary gland tumors (CMGTs). Here, we investigated the role of TGF-β in CMGT. We observed that treatment of the CMGT cell line CHMp13a with TGF-β1 leads to transient induction of the mesenchymal marker vimentin. Real-time measurements of cellular electrical impedance also showed that CMGT invasiveness is transiently increased by TGF-β1 treatment, but is reversed after prolonged stimulation. This phenomenon is similar to the mesenchymal–epithelial transition (MET, the reverse phenomenon of EMT), and a process that is implicated in the establishment of secondary metastatic lesions.

Abstract 5228: Multi-modal in vivo imaging of breast cancer cells expressing dual reporters

Abstract We previously engineered tumor cell lines with bioluminescent markers and fluorescent proteins for whole animal imaging. In addition to conventional non-invasive tumor growth detection, these cells when harvested from animals can also be used to analyze cell-cell interactions by immunohistochemistry. Moreover, fluorescent cells can be isolated using fluorescent activated cell sorting (FACS) for gene expression profiling. In this study, we used a mouse mammary gland tumor cell line 4T1 that was labeled with firefly luciferase and tdTomato fluorescent protein (4T1-luc2-tdTomato). The generated cell line was tested for in vitro signal stability with respect to bioluminescence and fluorescence prior to injection into the animals. Tumor cells were implanted subcutaneously and monitored for tumor development by acquisition of bioluminescent and fluorescent images. In a separate study, 4T1-luc2-tdTomato cells were implanted orthotopically into mammary fat pads. Tumor growth was monitored and signals from the implantation sites were reconstructed into three dimensional images. In order to non-invasively detect bone metastases by 4T1-luc2-tdTomato cells, we applied both microCT and optical imaging. For this, an interchangeable imaging platform between an optical imager and microCT machine was developed. 4T1-luc2-tdTomato cells were injected into nu/nu mice by intracardiac injection. Bioluminescent imaging was performed immediately to verify the injection of cells into the left ventricle. Fluorescent signals were not detected, likely due to the small number of cells in the secondary tumor sites. Animals demonstrating substantial whole body bioluminescence were selected for further monitoring until post injection day 9. We found that bioluminescent signals were located in the knee joint regions. For these animals, bioluminescent and X-ray images were also taken without changing the positions of the animals using the interchangeable platform. Bioluminescent images in the knee joints were reconstructed using a diffused luminescence imaging tomography algorithm. These images were then co-registered with microCT images. The results showed that bioluminescent signals were colocalized within the joint area obtained by microCT. Moreover, high resolution images of the joints from these mice revealed bone erosion in the tibia induced by 4T1-luc2-tdTomato cells. These findings demonstrate that multimodal imaging can pinpoint the tumor lesions non-invasively. In addition, low dose microCT imaging can be used to acquire high resolution skeletal images longitudinally without inducing adverse effects on the animals. 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 5228. doi:10.1158/1538-7445.AM2011-5228

Tumor-secreted PGE2 Inhibits CCL5 Production in Activated Macrophages through cAMP/PKA Signaling Pathway

One of the major characteristics of tumors is their ability to evade immunosurveillance through altering the properties and functions of host stromal and/or immune cells. CCL5 has been shown to play important roles in T cell proliferation, IFN-γ, and IL-2 production, which promotes the differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. In this study we found that tumor-bearing mice were more susceptible to bacterial infection and showed reduced CCL5 levels in serum during endotoxic shock. Our data further demonstrated that the soluble factors secreted by mammary gland tumor cells but not normal mammary gland epithelial cells inhibited CCL5 expression in macrophages in response to LPS, but not to TNF-α stimulation. The inhibitory effect of tumor-secreted molecules on LPS-induced CCL5 expression was regulated at the post-transcriptional level. Blocking PGE(2) synthesis by NS398 or through the use of PGE(2) receptor antagonists AH-6809 (EP2 antagonist) and AH-23848 (EP4 antagonist) completely reversed the inhibitory effect of tumor-conditioned medium (TCM) on LPS-induced CCL5 expression. Moreover, PGE(2) and the cAMP analog forskolin could mimic tumor-mediated CCL5 inhibition, and the inhibitory effects of TCM, PGE(2), and cAMP analog on LPS-induced CCL5 expression could be completely reversed by the PKA inhibitor H89. Furthermore, blocking PGE(2) synthesis in vivo led to partial recovery of CCL5 production during endotoxic shock. Taken together, our data indicate that PGE(2) secreted from breast cancer cells suppresses CCL5 secretion in LPS-activated macrophages through a cAMP/PKA signaling pathway, which may result in suppression of host immune responses against subsequent bacterial infection.

Analysis of a cholera toxin B subunit (CTB) and human mucin 1 (MUC1) conjugate protein in a MUC1-tolerant mouse model

Since epithelial mucin 1 (MUC1) is associated with several adenocarcinomas at the mucosal sites, it is pertinent to test the efficacy of a mucosally targeted vaccine formulation. The B subunit of the Vibrio cholerae cholera toxin (CTB) has great potential to act as a mucosal carrier for subunit vaccines. In the present study we evaluated whether a MUC1 tandem repeat (TR) peptide chemically linked to CTB would break self-antigen tolerance in the transgenic MUC1-tolerant mouse model (MUC1.Tg) through oral or parenteral immunizations. We report that oral immunization with the CTB-MUC1 conjugate along with mucosal adjuvant, unmethylated CpG oligodeoxynucleotide (ODN) and interleukin-12 (IL-12) did not break self-antigen tolerance in MUC1.Tg mice, but induced a strong humoral response in wild-type C57BL/6 mice. However, self-antigen tolerance in the MUC1.Tg mouse model was broken after parenteral immunizations with different doses of the CTB-MUC1 conjugate protein and with the adjuvant CpG ODN co-delivered with CTB-MUC1. Importantly, mice immunized systemically with CpG ODN alone and with CTB-MUC1 exhibited decreased tumor burden when challenged with a mammary gland tumor cell line that expresses human MUC1.

Effectiveness of small interfering RNA (siRNA) against the Mcl-1 gene in a canine mammary gland tumor cell line

The effect of down-regulation of Mcl-1 expression by small interfering RNA (siRNA) against the canine Mcl-1 gene on apoptosis was investigated by transfecting CF33 (canine mammary gland tumor cell line) with siRNA using cationic liposomes. The siRNA against canine Mcl-1 increased the rate of apoptotic cells and decreased the numbers of viable cells. Further, sequence-specific down-regulation of Mcl-1 expression was measured by real time-PCR and Western blot analysis. The siRNA directed against the Mcl-1 gene reduced both the mRNA and protein expression in the CF33. Our study suggests the importance of Mcl-1 in canine mammary tumors for inducing apoptosis and reinforces using Mcl-1 as a putative therapeutic target in canine mammary gland tumor.

The effect of small interfering RNA (siRNA) against the Bcl-2 gene on apoptosis and chemosensitivity in a canine mammary gland tumor cell line

The aim of this study was to investigate whether downregulation of Bcl-2 expression by small interfering RNA (siRNA) against the canine Bcl-2 gene would enhance the apoptosis and sensitivity of a canine mammary gland tumor cell line (CF33) to doxorubicin. Transfections of CF33 with siRNA were performed using cationic liposomes. Sequence-specific downregulation of Bcl-2 expression was measured by semiquantitative RT-PCR and Western blot analysis. Total viable cells were determined by MTS assay and apoptotic cell rates were determined by the immunohistochemical analysis on ssDNA. Our data showed the siRNA downregulated Bcl-2 expression which increased apotosis and also increased the sensitivity of CF33 to doxorubicin. This study indicated that downregulation of Bcl-2 expression by siRNA would be useful as a new protocol to increase the effect of doxorubicin on treatment of canine mammary gland tumors, requiring a detailed evaluation of siRNA in vivo.

Genistein, a soy isoflavone, up‐regulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NFκB

We have previously reported that estrogens up-regulate longevity-associated genes. As recent evidence has shown that estrogen replacement therapy is associated with an increased risk of cardiovascular disease, we have studied the effects of genistein, a soy isoflavone with a similar structure to estradiol, on the expression of antioxidant, longevity-related genes. MCF-7 cells (human mammary gland tumor cell line) were incubated for 48 h with 0.5 microM genistein, a concentration found in the plasma of populations consuming diets rich in soy protein. Peroxide levels were determined by fluorimetry, activation of extracellular-signal regulated kinase (ERK1/2), and nuclear factor kappaB (NFkappaB)-signaling pathways by Western blot analysis and ELISA, respectively, and mRNA expression of antioxidant genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Inhibition of basal peroxide levels in MCF-7 cells by genistein was prevented by pretreatment of cells with the estrogen receptor antagonist tamoxifen. Phosphorylation of extracellular regulated kinase (ERK)1/2 led to an activation of NFkappaB, as indicated by increased p50 subunit expression in nuclear extracts, and increased mRNA levels of the antioxidant enzyme manganese-superoxide dismutase (MnSOD). Inhibition of ERK1/2 abrogated genistein-mediated NFkappaB activation and elevated expression of MnSOD. Our molecular studies may provide a basis to determine the effects of genistein and other soy protein-derived products on longevity in both animals and the human population.