The canine prostate cancer cell line CHP‐1 shows over‐expression of the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α

Although androgen therapy resistance and poor clinical outcomes are seen in most canine prostate cancer cases, there are only a few tools for analysing canine prostate cancer by using a cell biological approach. Therefore, to evaluate androgen‐independent neoplastic cell growth, a new canine prostate cancer cell line (CHP‐1) was established in this study. CHP‐1 over‐expressed the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α (SGTA), which is over‐expressed in human androgen‐independent prostate cancer. The CHP‐1 xenograft also showed SGTA over‐expression. Although CHP‐1 shows poor androgen receptor (AR) signalling upon dihydrotestosterone stimulation, forced expression of AR enabled evaluation of AR signalling. Taken together, these results suggest that CHP‐1 will be a useful model for investigating the pathogenesis of androgen‐dependent and androgen‐independent canine prostate cancer.     

Evaluation of the Akt kinase activation in a PTEN deficient canine osteosarcoma cell line

Introduction: Osteosarcoma is a devastating disease in both human and veterinary medicine. The dog has been recognized as an important model system for the study of this disease, with a high incidence of tumor development noted annually. The cure rates in veterinary oncology are between 20 and 30% of patients seen. Akt is a serine/threonine kinase that primarily protects the cell from apoptosis and may prompt proliferation and growth. Akt activation involves sequential phosphorylation of threonine at position 308 (T308) and serine at position 473 (S473). While for the T308 phosphorylation responsible is PDK1 kinase, for S473 phosphorylation no definitive mechanism has yet been identified. The tumor suppressor PTEN, which has been reported to be absent in canine osteosarcoma, negatively regulates Akt. In our study we evaluated the Akt activation sequence in response to serum deprivation. Methods: “Buck,” an immortalized PTEN deficient canine OSA cell line established and characterized in our laboratory was used. Buck cells were grown in the absence of fetal bovine serum for 8, 24, and 48 hours. Activity of Akt was assayed directly by a method described by Kang et al. The phosphorylation status of Akt at T308 and S473, together with serine at position 241 (S241) of PDK1 was assessed by immunoblotting. Results: We noted marked decrease of the Akt kinase activity with the nadir at the 24‐hour time point and subsequent increase of Akt activity at the 48‐hour time point. Decoupling of the two Akt phosphorylation sites at the 24, and 48‐hour time points was observed (p < 0.01). The phosphorylation status of T308 declined steadily throughout the experiment. The phosphorylation status of S473 declined until the 24‐hour time point, and then increased in parallel with the results noted in the kinase activity assay. Phosphorylation of PDK1 S241 was examined and found to decrease at 24 hours, with subsequent increase after 48 hours of serum starvation, in parallel with the phosphorylation pattern of Akt S473 (p < 0.01). Conclusions: We report parallel patterns of PDK1 S241 and Akt S473 phosphorylation in the absence of growth factors. PDK1 activity is not assessed directly, but its phosphorylation status indicates that more PDK1 is in an activated from at the 48‐hour time point than at 24 hours. The increase of Akt activity and S473 phosphorylation at the 48‐hour time point is presumably a response to the apoptogenic conditions of the serum free medium. The same response of PDK1 may indicate the presence of a regulatory autophosphorylation mechanism that members of the AGC kinase family share. Surprisingly, the increased amount of activated PDK1 does not correlate with the phosphorylation pattern of Akt T308, and could be attributed to cytoplasmic sequestration of PDK1.     

PS48 promotes in vitro maturation and developmental competence of porcine oocytes through activating PI3K/Akt signalling pathway

Oocyte maturation plays a vitally important role in porcine reproduction. Regrettably, the quality of oocytes matured in vitro is weaker than that of in vivo matured oocytes. We collected and cultivated porcine cumulus oocyte complexes (COCs) in vitro with phosphoinositide-dependent kinase 1 (PDK1) activator 5-(4-chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48), whose concentrations were 0, 2, 5, 10 and 20 µM to investigate whether the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signalling pathway would impact the oocyte quality. The results showed that 10 µM PS48 increased the oocyte proportion of metaphase II (MII) stage and improved the expansion of cumulus cells (CCs). What's more, the activation of PI3K/Akt signalling pathway could regulate the expression of maturation-related genes and proteins. The results of quantitative real-time PCR showed that 10 µM PS48 increased the mRNA and protein levels of Akt and regulated maturation-related genes, including cyclin B1, MOS, BMP15, GDF9, CDC2, mTOR, BAX, BCL2 and caspase-3. The results of Western blot indicated that 10µM PS48 increased the protein abundance of Akt, phosphorylation of Akt Thr308 (p-Akt^Thr308) and cyclin B1, but decreased the protein abundance of pro-apoptotic BAX. These results suggested that adding 10 µM PS48 to mature culture medium could promote the maturation of porcine oocytes, potentially through activating the PI3K/Akt signalling pathway.     

Dysregulated expression of the key effectors of the mammalian target of rapamycin signalling pathway in cutaneous papillomas of dogs

Cutaneous papillomas (CP) are one of the most common skin neoplasms in dogs. Different murine models have shown that persistent activation of the phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway has a central role in the development and progression of CP. The purpose of this study were to evaluate the immunohistochemical expression pattern of two key molecules involved in the PI3K/Akt/mTOR signalling pathway, pAkt^Ser473, and pS6^Ser235/236, on 36 canine specimens of CP using a tissue microarray. The results show that the PI3K/Akt/mTOR signalling pathway is persistently activated in CP of dogs, pointing to this pathway as a potential therapeutic target.     

In vitro effects of 2-methoxyestradiol on canine tumour cells

The in vitro antiproliferative, apoptotic and cell‐cycle effects of 2‐methoxyestradiol (2ME₂), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME₂. Inhibition of tumour cell proliferation was evaluated using a tetrazolium‐based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V‐fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell‐cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME₂ ranging from 0.88 to 7.67 µM, depending on the cell line tested. Profound G₂/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose‐dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.     

Non‐immunosuppressive FTY720‐derivative OSU‐2S mediates reactive oxygen species‐mediated cytotoxicity in canine B‐cell lymphoma

OSU‐2S is a FTY720 (Fingolimod) derivative that lacks immunosuppressive properties but exhibits strong anti‐tumour activity in several haematological and solid tumour models. We have recently shown OSU‐2S to mediate potent cytotoxicity in human mantle cell lymphoma cell lines and primary cells. We report here the pre‐clinical activity of OSU‐2S in spontaneous B‐cell lymphoma of dogs which shares many characteristics of human lymphoma. OSU‐2S mediated apoptosis in canine B‐cell lines and primary B‐cell lymphoma cells obtained from spontaneous lymphoma bearing dogs. OSU‐2S induced reactive oxygen species (ROS) in canine lymphoma cells and inhibition of ROS partially rescued OSU‐2S‐mediated cell death. These studies provide a rational basis for the use of spontaneous lymphoma in pet dogs as a preclinical large animal model for the development of OSU‐2S as small molecule for treating people and dogs with lymphoma.     

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.     

Wnt5a expression in canine osteosarcoma

Osteosarcoma is the most common primary malignancy of bone in dogs and is associated with poor long‐term outcomes due to its highly metastatic nature. A better understanding of the signalling pathways and proteins involved with osteosarcoma pathogenesis may aid in improved outcomes through the use of targeted therapies. The Wnt5a protein, a ligand for the non‐canonical Wnt signalling pathway, is implicated in mediating the aggressiveness of cancer cell lines, including those of human osteosarcoma origin. Given the close relationship between human and canine osteosarcoma, the primary goal of this study was to characterize Wnt5a expression in canine osteosarcoma. Second, if Wnt5a expression was present in canine osteosarcoma, the study aimed to determine any potential association with clinical outcome and clinical variables in similarly treated osteosarcoma‐bearing dogs. Wnt5a expression was present in 26 of the 48 (54%) cases of canine osteosarcoma. Wnt5a expression was not associated with progression‐free survival (P = 0.4) or overall survival (P = 0.1).     

A novel small molecule Met inhibitor, PF2362376, exhibits biological activity against osteosarcoma

The receptor tyrosine kinase Met is dysregulated in several human cancers including osteosarcoma (OSA) in which overexpression is a negative prognostic indicator and enforced Met expression in normal osteoblasts leads to genomic instability and malignant transformation. Met is also known to be inappropriately expressed in canine OSA tumour samples and cell lines. The purpose of this study was to evaluate the potential utility of an orally bioavailable small molecule Met inhibitor, PF2362376, against canine OSA cell lines as a prelude to future clinical work. PF2362376 inhibited phosphorylation of Met, Gab‐1, Erk and Akt, but not of Src or STAT3. Furthermore, PF2362376 inhibited proliferation of canine OSA cell lines and induced cell death at biologically achievable concentrations. Last, activities associated with Met signalling including migration, invasion, branching morphogenesis and colony formation in soft agar were blocked by PF2362376. These studies support the notion that Met is a relevant target for therapeutic intervention in OSA.     

COX‐2, mPGES‐1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E₂ (PGE₂) tumour‐promoting activity has been confirmed and its production is controlled by Cyclooxygenase‐2 (COX‐2) and microsomal PGE synthase‐1 (mPGES‐1). Evidence suggests that mPGES‐1 and COX‐2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX‐2, mPGES‐1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non‐neoplastic tissues. COX‐2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES‐1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX‐2, EP2 receptor and mPGES‐1 expression was significantly higher in carcinomas than in non‐neoplastic tissues and adenomas. COX‐2, mPGES‐1 and EP2 receptor expression was strongly associated. These findings support a role of the COX‐2/PGE2 pathway in the pathogenesis of these tumours.     

The bisphosphonates alendronate and zoledronate are inhibitors of canine and human osteosarcoma cell growth in vitro

Bisphosphonates (BPs) are a class of non‐hydrolysable analogues of pyrophosphate that have high affinity for bone mineral and are inhibitors of bone resorption. The in vitro effects of two nitrogen‐containing BPs, alendronate (ALE) and zoledronate (ZOL), on growth, induction of apoptosis and effects on cell‐cycle distribution in two canine and two human osteosarcoma (OSA) cell lines are investigated here. Both significantly (P < 0.001) reduced cell growth in all cell lines, as assessed by a colorimetric assay with IC₅₀ values in the range of 7.3–61.4 µM and 7.9–36.3 µM for ALE and ZOL, respectively. Both BPs caused a significant (P < 0.001) dose‐dependent increase in the proportion of cells undergoing apoptosis, as assessed both by cell‐cycle analysis and by annexin‐V binding. Both ALE and ZOL altered the proportion of cells in each phase of the cell cycle, but the extent and proportion was both drug and cell line dependent. These data indicate that the nitrogen‐containing BPs have direct anti‐tumour activity against canine and human OSA cells.     

Neurokinin‐1 receptor expression and antagonism by the NK‐1R antagonist maropitant in canine melanoma cell lines and primary tumour tissues

We interrogated the neurokinin‐1 receptor (NK‐1R)/substance P (SP) pathway in canine melanoma tumour tissues and cell lines. NK‐1R messenger RNA (mRNA) and protein expression were observed in the majority of tumour tissues. Immunohistochemical assessment of archived tissue sections revealed NK‐1R immunoreactivity in 11 of 15 tumours, which may have diagnostic, prognostic and therapeutic utility. However, we were unable to identify a preclinical in vitro cell line or in vivo xenograft model that recapitulates NK‐1R mRNA and protein expression documented in primary tumours. While maropitant inhibited proliferation and enhanced apoptosis in cell lines, in the absence of documented NK‐1R expression, this may represent off‐target effects. Furthermore, maropitant failed to suppress tumour growth in a canine mouse xenograft model derived from a cell line expressing mRNA but not protein. While NK‐1R represents a novel target, in the absence of preclinical models, in‐species clinical trials will be necessary to investigate the therapeutic potential for antagonists such as maropitant.     

Modulation of matrix metalloproteinases by ultraviolet radiation in the canine cornea

Purpose To determine whether ultraviolet (UV) radiation can modulate expression and regulation of matrix metalloproteinases (MMP) in the canine cornea and to examine the expression of MMPs in canine chronic superficial keratitis (CSK). Methods Immunohistochemistry for MMP‐2 and MMP‐9 was performed on samples of CSK. In vitro, canine corneal epithelial cell (CEC) and stromal cell cultures were exposed to UV‐irradiation. Following 2, 8 or 24 h, cells were harvested. MMP expression was examined by zymography, and RT‐PCR was used to examine expression of Slug and Snail. CEC cultures treated with an EGFR inhibitor or a p38 inhibitor were UV‐exposed and harvested 24 h later to examine expression of MMPs, Slug and Snail. Results Canine CSK had increased immunopositivity for both MMP‐2 and MMP‐9 compared to normal canine corneas. In vitro, CEC and stromal cell cultures exposed to UV showed generally increased expression of MMP‐2, ‐9, Slug, and Snail; this response was dose and time dependent. Inhibition of the EGFR pathway did not prevent increased expression of MMP‐2, ‐9, Slug or Snail in UV‐exposed CEC; however, p38 inhibition did attenuate UV induction. Conclusions We have found increased expression of MMPs in clinical samples of CSK compared to normal corneas. In addition, we have shown that there is a temporal association and dose dependency between UV exposure and production of MMPs, Slug, and Snail. These findings suggest that overexpression of MMPs due to UV‐exposure may be linked to changes in the cornea that allow an influx of inflammatory cells and vascularization.     

Effect of porcine glucagon‐like peptides‐2 on tight junction in GLP‐2R + IPEC‐J2 cell through the PI3k/Akt/mTOR/p70S6K signalling pathway

Because of rare glucagon‐like peptide‐2 (GLP‐2) receptor (+) cells within the gut mucosa, the molecular mechanisms transducing the diverse actions of GLP‐2 remain largely obscure. This research identified the naturally occurring intestinal cell lines that endogenously express GLP‐2R and determined the molecular mechanisms of the protective effects of GLP‐2‐mediated tight junctions (TJ) in GLP‐2R (+) cell line. (i) Immunohistochemistry results showed that GLP‐2R is localised to the epithelia, laminae propriae and muscle layers of the small and large bowels of newborn piglets. (ii) GLP‐2R expression was apparent in the cytoplasm of endocrine cells in IPEC‐J2 cell lines. (iii) The protein expressions of ZO‐1, claudin‐1, occludin, p‐PI₃K, p‐Akt, p‐mTOR and p‐p70^S6K significantly (p < 0.05) increased in GLP‐2‐treated IPEC‐J2 cells, and all of them significantly (p < 0.05) decreased when LY‐294002 or rapamycin was added. GLP‐2 improves intestinal TJ expression of GLP‐2R (+) cells through the PI₃k/Akt/mTOR/p70^S6K signalling pathway.     

Annexin A8 (ANXA8) regulates proliferation of porcine endometrial cells via Akt signalling pathway

Annexin A8 (ANXA8) gene, a member of the annexin family, encodes an anticoagulant protein involved in blood coagulation cascade and acts as an indirect inhibitor of the thromboplastin‐specific complex. However, little is known about the function of ANXA8 in porcine endometrial cells so far. Here, ANXA8 mRNA was found to be abundant in porcine endometrium on days 11–13 of pregnancy. Real‐time RT–PCR analysis indicated that the mRNA expression of the leukaemia inhibitory factor (LIF) and the epidermal growth factor (EGF) was upregulated by ANXA8 in porcine endometrial cells. Immunofluorescence technology and cell cycle analysis revealed that ANXA8 promoted the proliferation of endometrial cells, as evidenced by the abundant proliferating cell nuclear antigen (PCNA) expression and an increase in the S phase. Western blot analysis results indicated that ANXA8 activated the phosphorylation of the target protein kinase B (Akt) protein. Immunofluorescence technology results showed that the PCNA protein had no significant change in porcine endometrial cells with both ANXA8 overexpression and the addition of Akt inhibitor. Furthermore, the number of implantation sites was significantly reduced by injection of mus‐siRNA‐ANXA8 into the uterine horn of mice. Collectively, these results suggest that ANXA8 promotes the proliferation of endometrial cells through the Akt signalling pathway.