Role of CXCR4 and SDF-1 in mammary tumor metastasis in the cat

It has recently been suggested that the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12) promote metastasis of various cancers in humans. Since feline mammary tumors also metastasize to distant organs frequently, we used real-time quantitative PCR to examine the expression of feline CXCR4 (fCXCR4) in ten feline mammary tumor cell lines and seven feline mammary tumor tissues, and also the expression of feline SDF-1 (fSDF-1) in various organs. Cell lines derived from metastatic regions expressed more fCXCR4 than those derived from primary tumors. Mammary tumor tissues overexpressed more fCXCR4 than normal mammary tissues. Organs with high levels of fSDF-1 expression represent common sites of metastasis. Migration assays using the feline mammary tumor cell line NAC were also performed to test the activity of TN14003 and TC14012, antagonists of human CXCR4, to antagonize fCXCR4 expressed on NAC cells. TN14003 and TC14012 inhibited migration of NAC cells. We conclude that fCXCR4 may be a therapeutic target for feline mammary tumors.     

Tumour necrosis factor‐related apoptosis‐inducing ligand induces apoptosis in canine hemangiosarcoma cells in vitro

Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is an apoptosis‐inducing cytokine that shows potential therapeutic value for human neoplasms, and is effective in some canine tumours; however, its potential for killing canine hemangiosarcoma (HSA) cells is unknown. Thus, we evaluated the proapoptotic effect of TRAIL in nine canine HSA cell lines. Cells (JuA1, JuB2, JuB2‐1, JuB4, Re11, Re12, Re21, Ud2 and Ud6) were cultured with three recombinant human TRAILs (rhTRAILs): TRAIL‐TEC derived from Escherichia coli, TRAIL‐TL derived from mammalian cells and isoleucine zipper recombinant human TRAIL (izTRAIL) containing an isoleucine‐zippered structure that facilitates trimerization. TRAIL‐TEC did not decrease the cell viability in any of the cell lines tested, whereas the other two rhTRAILs effectively decreased the viability of all cell lines as assessed by the WST‐1 assay. In canine HSA cells, izTRAIL induced apoptosis more effectively than TRAIL‐TL. In JuB4, Re12, and Ud6 cells, izTRAIL increased the activation of caspase‐3 and caspase‐8 and caused poly (ADP‐ribose) polymerase degradation. Moreover, izTRAIL treatment increased the proportion of Annexin V+/ Propidium iodide (PI)? apoptotic cells and nuclear fragmentation in izTRAIL‐sensitive cells. These results show that rhTRAIL can induce apoptosis in canine HSA cells, but the sensitivity of TRAIL was different depending on the cell lines. Therefore, TRAIL could be an effective therapeutic agent against canine HSA, but the specific mechanism of resistance should be determined to clarify under what conditions this treatment would be most effective.     

CXCL8 attenuates chemoattractant-induced equine neutrophil migration

The chemokine, CXCL8, is a potent chemoattractant but it has also been shown to attenuate the migratory response of human neutrophils to the bacterial peptide, FMLP; this could lead to retention of cells in infected tissue and, potentially, to enhanced clearance of bacteria. This study has examined the effect of CXCL8 on equine neutrophil migration and adherence in response to PAF and LTB(4), chemoattractants that may play a role in non-infectious inflammatory conditions of the horse associated with neutrophil recruitment to the target tissue. The effects of CXCL8 on PAF- and LTB(4)-induced responses were determined using a ChemoTx plate migration assay and by measuring adhesion to protein-coated plastic. The CXCR1/2 antagonist, SB225002, was used to investigate whether the observed effects were receptor mediated and the role of cAMP was examined by measuring intracellular cAMP following exposure to agonists alone and in combination and by establishing the effect of dibutyryl cAMP on neutrophil migration. CXCL8, LTB(4) and PAF each induced migration and adhesion. Exposure of neutrophils to a combination of CXCL8 and PAF reduced the magnitude of the responses to that of unstimulated cells. In contrast, although the effect was less than additive, the response to co-stimulation with CXCL8 and LTB(4) were not nearly as pronounced. CXCL8 acted in a receptor mediated manner, the attenuation of PAF-induced responses being reversed by SB225002 at a concentration that blocks CXCR2. CXCL8, PAF and LTB(4) alone increased intracellular cAMP. In co-incubation studies, combination of CXCL8 with PAF led to an additive increase in cAMP whereas no increase above that obtained in response to LTB(4) alone was seen. Dibutyryl cAMP significantly reduced neutrophil migration in response to either CXCL8 or PAF alone. These results demonstrate that CXCL8, in addition to being a potent chemoattractant and pro-adhesive molecule for equine neutrophils, is able to attenuate responses to PAF and, to a much lesser extent, LTB(4). This effect, which appears to be CXCR2-mediated and cAMP dependent, could lead in vivo to trapping of cells at sites of inflammation resulting potentially in either enhanced clearance of injurious stimuli or increased local tissue damage by activated cells.     

Anticancer effects of resveratrol in canine hemangiosarcoma cell lines

Hemangiosarcoma (HSA) is a highly malignant tumour with aggressive biological behaviour. HSAs are more common in dogs than other domestic animals. The median survival time of dogs with HSA remains short, even with chemotherapy and surgery. Therefore, there is a critical need to improve the adjuvant chemotherapeutic regimens to improve clinical outcomes in dogs with HSA. Resveratrol has been shown to possess strong anti‐proliferative and/or pro‐apoptotic properties in human cancer cell lines. Nevertheless, the potential anticancer effects of resveratrol have not been reported in canine HSAs. The objective of this study is to determine the growth inhibitory effects of resveratrol in HSA cells when used alone or in combination with doxorubicin, a commonly used chemotherapeutic agent. Frog and DD‐1 canine HSA cell lines were treated with varying concentrations of resveratrol with and without doxorubicin. Cell viability was measured by the MTT assay. The expression of apoptotic proteins, activation of p38 mitogen‐activated protein kinase (MAPK), AMP‐activated protein kinase (AMPK) and extracellular signal‐regulated kinase 1/2 (ERK1/2) were assessed by western blotting. Similar to human cancer cell lines, resveratrol markedly inhibited the growth and induced apoptosis in both HSA cell lines. Mechanistically, resveratrol activated p38 MAPK, but did not affect the AMPK or the ERK1/2 pathways. Additional experiments showed that resveratrol augmented the growth‐inhibitory and apoptotic effects of doxorubicin in both HSA cell lines. These findings suggest that resveratrol has pro‐apoptotic effects in canine HSA cells; therefore, its use as a potential adjunct therapy in canine HSA patients warrants further investigation.     

血小板凝血酶蛋白1对犬乳腺肿瘤细胞体外生物学特性的影响分析

本研究旨在深入探讨血小板凝血酶蛋白1(THBS1)对犬乳腺肿瘤细胞CHMp的影响,并阐明其影响犬乳腺肿瘤发生发展的作用机制。通过构建THBS1慢病毒稳定过表达和THBS1沉默表达的犬乳腺肿瘤细胞CHMp细胞系,采用CCK-8试验、划痕试验、Transwell试验、原位荧光检测和流式细胞术检测THBS1对CHMp细胞增殖、迁移、侵袭、细胞凋亡和细胞周期情况的影响;通过qRT-PCR和Western blot检测THBS1对CHMp细胞凋亡相关因子(p53、Bcl-2、Bax)表达情况的影响,验证THBS1对CHMp细胞凋亡通路的影响。结果显示,过表达THBS1能有效增强犬乳腺肿瘤细胞CHMp的增殖、迁移和侵袭能力,并且减少CHMp细胞的凋亡数量,而沉默THBS1后结果与之相反;流式细胞术得出THBS1能够影响CHMp细胞的细胞周期分布;经qRT-PCR和Western blot检测发现,在THBS1过表达后,p53和Bcl-2的表达量均明显增高,Bax的表达量明显减少,在沉默THBS1后结果与之相反。结果表明,THBS1的异常表达能够影响犬乳腺肿瘤细胞CHMp的增殖、迁移、侵袭以及细胞周期;此外,THBS1能够通过影响细胞凋亡因子p53、Bcl-2和Bax的表达来影响CHMp细胞凋亡相关通路,进而影响犬乳腺肿瘤的发生发展。     

Transcriptomic profile reveals molecular events associated to focal adhesion and invasion in canine mammary gland tumour cell lines

The prevalence of cancer in animals has increased significantly over the years. Mammary tumours are the most common neoplasia in dogs, in which around 50% are presented in the malignant form. Hence, the development and characterization of in vitro models for the study of canine tumours are important for the improvement of cancer diagnosis and treatment. Thus, the aim of this study was to characterize cell lines derived from canine mammary gland neoplasias which could be further used for basic and applied oncology research. Samples of canine mammary carcinomas were taken for cell culture and 2 cell lines were established and characterized in terms of cell morphology, tumourigenicity and global gene expression. Both cell lines presented spindle‐shape morphology and shown common malignant features as in vitro invasion potential and expression of epithelial and mesenchymal proteins. Also, we found gene expression patterns between the 2 cell cultures in comparison to the normal mammary gland tissue. Cells from M25 culture showed a higher invasion and in vivo tumourigenic potential, associated to the overexpression of genes involved in focal adhesion and extracellular matrix communication, such as FN1, ITGA8 and THBS2. The phenotypic characterization of these cells along with their global gene expression profile potentially determine new therapeutic targets for mammary tumours.     

Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma

Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival.     

Interferon stimulated genes, CXCR4 and immune cell responses in peripheral blood mononuclear cells infected with bovine viral diarrhea virus

Non-cytopathic bovine viral diarrhea virus (ncpBVDV) induces immune responses mediated by chemokines and interferon (IFN) stimulated genes (ISGs). Cultured bovine peripheral blood mononuclear cells (PBMC) from ncpBVDV-naïve cattle were used herein to demonstrate that BVDV infection modulates chemokine receptor 4 (CXCR4), CXCL12, IFN-I, ISGs and selected immune cell marker (CD4, CD8, CD14) mRNAs, and that these acute responses to viral infection are reflected in PBMC cultured with serum from heifers carrying fetuses persistently infected (PI) with ncpBVDV. Infection of PBMC with ncpBVDV increased IFN-β, ISG15, RIG-I, CXCR4, CXCL12, and CD8 mRNA concentrations after 32 h. Culture of PBMC with uterine vein serum from acutely infected heifers, inoculated with ncpBVDV during early gestation to generate PI fetuses, also increased the concentration of CXCR4, RIG-I and ISG15 mRNAs. In vitro PBMC treatment with ncpBVDV or uterine vein serum from acutely infected pregnant heifers activates chemokine, ISG and immune cell responses.     

Canine osteosarcoma cell lines from patients with differing serum alkaline phosphatase concentrations display no behavioural differences in vitro

Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumour size, presence of metastatic disease and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behaviour of osteosarcoma cells differ based on serum ALP concentration. Here, we report on the generation of six canine osteosarcoma cell lines from osteosarcoma‐bearing dogs with differences in serum ALP concentration. To determine whether in vitro behaviour differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP, assays were performed to evaluate proliferation, migration, invasion and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion or chemosensitivity between cell lines associated with normal or increased serum ALP concentration.     

Tumour necrosis factor stimulated gene 6 intrinsically regulates PD-L1 expressions in breast cancer cells,leading to modulation of tumour microenvironment

Recent studies have shown that tumour cells express tumour necrosis factor-inducible gene 6 (TSG-6) and its protein, which is known to play a key role in regulating excessive immune responses and proliferation and growth of mesenchymal stem cells (MSCs). It has not been confirmed whether the inhibition of TSG-6 for tumour cells can suppress tumour cell growth and regulate the activation of immune cells in the tumour microenvironment (TME). TSG-6-specific small interfering RNA was transfected into canine and human breast cancer cells (CIPp, CIPm and BT-20). TSG-6-down-regulated (siTSG-6) cells showed decreased cell proliferation, migration, and invasion abilities. Decreased mRNA expressions of NF-κB, STAT3 and Sox2, confirming that TSG-6 is an upper factor governing tumour growth and metastasis. Notably, siTSG-6 cells showed significantly decreased expression levels of CD44 and PD-L1. Direct and indirect co-culture of canine peripheral blood mononuclear cells (cPBMCs) and the siTSG-6 cells showed significant activation in M1 type macrophages and cytotoxic T cells. They also showed a tendency to decrease in the expression of CTLA-4 and increase in the expression of PD-1. In conclusion, this study suggests that the down-regulation of TSG-6 in breast cancer cells could not only suppress tumour growth and metastasis, and but also regulate TME. Since modulation of immune checkpoint proteins occurs in both tumour cells and immune cells, inhibiting TSG-6 and its protein within the TME could be novel therapeutic target for anticancer treatment.     

Characterization, expression and function of c-Met in canine spontaneous cancers

Aberrant expression of the proto‐oncogene c‐Met has been noted in a variety of human cancers. To better define the potential role of Met dysregulation in canine cancer, the canine Met, hepatocyte growth factor (HGF) and HGF activator were cloned. Inappropriate expression of Met was present in canine tumour cell lines derived from a wide variety of cancers. Furthermore, both HGF and HGF activator were also expressed in several of these cell lines, providing evidence of a possible autocrine loop of Met activation. Stimulation of tumour cell lines with recombinant human HGF induced Met autophosphorylation, as well as activation of the downstream signalling elements Gab‐1, Akt and Erk1/2. Scattering of tumour cells and migration across a defect occurred in response to HGF stimulation. The Met inhibitor PHA665752 blocked both HGF‐induced phosphorylation of canine Met and HGF‐mediated cell cycling, scattering and migration. These studies provide evidence that Met dysregulation may play a role in the biology of canine cancer and lay the groundwork for future studies employing Met inhibitors.     

Molecular cloning of canine membrane-anchored inhibitor of matrix metalloproteinase, RECK

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5% and 91.9% homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation, migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.     

Role of monocyte recruitment in hemangiosarcoma metastasis in dogs

Canine hemangiosarcoma (HSA) is a highly malignant tumour associated with short survival times because of early and widespread metastasis. In humans and rodents, monocytes play key roles in promoting tumour metastasis through stimulating tumour cell extravasation, seeding, growth and angiogenesis. Therefore, we investigated the potential association between monocyte infiltration and tumour metastasis in HSA and other common canine tumours. Immunohistochemistry was used to quantify CD18⁺ monocytes within metastases. We found that HSA metastases had significantly greater numbers of CD18⁺ monocytes compared with metastases from other tumour types. HSA cells were the highest producers of the monocyte chemokine CCL2, and stimulated canine monocyte migration in a CCL2 dependent manner. These results are consistent with the hypothesis that overexpression of CCL2 and recruitment of large numbers of monocytes may explain in part the aggressive metastatic nature of canine HSA. Thus, therapies designed to block monocyte recruitment may be an effective adjuvant strategy for suppressing HSA metastasis in dogs.     

Comparative study of anti-oncogenic microRNA-145 in canine and human malignant melanoma

MicroRNA-145 (miRNA-145; miR-145) is aberrantly expressed in most of human cancers and plays a significant role in carcinogenesis and cancer progression. In the current study, we focused on how miR-145 plays a role in canine and human malignant melanomas. MiR-145 was significantly downregulated in canine malignant melanoma tissues and canine melanoma cell lines, as well as human melanoma cell lines tested. The ectopic expression of miR-145 showed a significant growth inhibition in both canine and human melanoma cells tested, and the effect was achieved partly by suppressing c-MYC in canine melanoma LMeC and in human melanoma A2058 and Mewo cells. At the same time, a suppressive tendency on cell migration in canine melanoma KMeC cells and significant suppression of cell migration in human melanoma A2058 cells by suppressing FASCIN1 were also found. These findings suggest that miR-145 acts as a tumor suppressor in both canine and human malignant melanomas.     

Tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine‐derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine‐derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8). CDV replication‐induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV‐induced cancer therapy.     

In vitro effects of the tyrosine kinase inhibitor, masitinib mesylate, on canine hemangiosarcoma cell lines

This study evaluated the in vitro activity of masitinib mesylate against canine hemangiosarcoma (HSA) cell lines after treatment with increasing concentrations of masitinib mesylate (0.01-100?μM) for 24, 48 and 72 h. Results indicated that masitinib mesylate caused a dose- and time-dependent decrease in HSA cell proliferation. The 50% inhibitory concentration (IC(50) ) at 72 h for three HSA cell lines (DEN, Fitz and SB) was found to be 8.56, 9.41 and 10.65?μM, respectively. Further investigation demonstrated that masitinib mesylate induced apoptosis in all HSA cell lines, including activation of caspase-3/7. Measurement of VEGF levels in cell supernatant found a statistically significant increased VEGF in close proximity to the IC(50) of each cell line followed by a decline back towards baseline. These findings indicate that masitinib mesylate causes dose-dependent HSA cell death in vitro and supports future clinical trials of masitinib for canine HSA.