Characterization of receptor tyrosine kinase activation and biological activity of toceranib phosphate in canine urothelial carcinoma cell lines.

BACKGROUND: Urothelial carcinoma (UC) accounts for > 90% of canine tumors occurring in the urinary bladder. Toceranib phosphate (TOC) is a multi-target receptor tyrosine kinase (RTK) inhibitor that exhibits activity against members of the split kinase family of RTKs. The purpose of this study was to evaluate primary UC tumors and UC cell lines for the expression and activation of VEGFR2, PDGFRα, PDGFRß, and KIT to assess whether dysregulation of these RTKs may contribute to the observed biological activity of TOC. RESULTS: Transcript for VEGFR2, PDGFRα, PDGFRß, and KIT was detected in all UC tissue samples and UC cell lines. The Proteome Profiler™ Human Phospho-RTK Array Kit (R & D Systems) provided a platform to assess phosphorylation of 42 different RTKs in primary UC tumors and UC cell lines. Evidence of PDGFRα and PDGFRß phosphorylation was present in only 11% or 33% of UC tumors, respectively, and 25% of UC cell lines. Treatment of UC cell lines with TOC had no significant impact on cell proliferation, including UC cell lines with evidence of PDGFRß phosphorylation. CONCLUSIONS: Phosphorylation of several key RTKs targeted by TOC is present in a small subset of primary UC tumors and UC cell lines, suggesting that these RTKs do not exist in a state of continuous activation. These data suggest that activation of RTKs targeted by TOC is present in a small subset of UC tumors and UC cell lines and that treatment with TOC at physiologically relevant concentrations has no direct anti-proliferative effect on UC cells.

The selective inhibitor of nuclear export (SINE) verdinexor exhibits biologic activity against canine osteosarcoma cell lines.

Verdinexor (KPT-335) is a novel orally bioavailable selective inhibitor of nuclear export (SINE) compound that inhibits the function of the nuclear export protein Exportin 1 (XPO1/CRM1). In the present study, we sought to characterize the expression of XPO1 in primary canine osteosarcoma (OS) tumour samples, OS cell lines and normal osteoblasts and evaluate the in vitro activity of verdinexor alone or in combination with doxorubicin. Canine OS cell lines and a subset of primary OS tumours showed increased XPO1 transcript and protein expression as compared with normal canine osteoblast cells. All canine OS cell lines exhibited dose-dependent growth inhibition and increased caspase 3,7 activity in response to low nanomolar concentrations of verdinexor (IC50 concentrations ranging from 21 to 74 nM). Notably, growth inhibition of normal canine osteoblast cell lines treated with verdinexor was observed at high micromolar concentrations (IC50  = 21 µM). The combination of verdinexor and doxorubicin resulted in potent inhibition of cell viability and demonstrated synergetic activity in three canine OS cell lines. Concordantly, OS cell lines showed increased γH2A.X foci following treatment with doxorubicin and recovery in verdinexor compared with cells treated with doxorubicin and recovered in normal media for 24 hours. These findings demonstrate that verdinexor has biologic activity against canine OS cell lines at physiologically relevant doses and suggest that XPO1 inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in canine OS.

Plasma Cytokeratin 18 and fecal Alpha-1 Antitrypsin concentrations in dogs with osteosarcoma receiving carboplatin chemotherapy.

Gastrointestinal (GI) toxicosis is a common side effect of cytotoxic chemotherapy treatment in humans and dogs. Measurement of cytokeratin 18 (CK18), an intracellular structural protein released during epithelial apoptosis, and Alpha1-Antitrypsin (A1AT) in faeces provides a mechanism for evaluating damage to the intestinal mucosa secondary to cytotoxic chemotherapy. Our goal was to evaluate the clinical utility of plasma CK18 and faecal A1-AT levels as non-invasive biomarkers of cytotoxic chemotherapy induced GI toxicity. We conducted a prospective cohort study in dogs (N = 10) with osteosarcoma undergoing amputation followed by carboplatin chemotherapy. We hypothesized that plasma CK18 and faecal A1-AT levels would increase following carboplatin administration due to drug-induced GI epithelial damage/apoptosis, and that plasma CK18 and faecal A1-AT levels would correlate with severity of GI toxicity. Mean baseline plasma CK18 concentration was variable amongst patients; however, CK18 concentration prior to carboplatin chemotherapy treatment was not significantly different from CK18 levels after treatment. There was significant intra and inter-patient variability in mean faecal A1-AT levels at baseline. Mean A1-AT concentration did not change significantly from day 0 to day 21. Gastrointestinal toxicity was minimal; therefore, we were unable to determine the association of plasma CK18 and faecal A1-AT concentrations with development of GI toxicosis. In this study population, plasma CK18 and faecal A1-AT concentration were not clinically useful biomarkers for the detection of GI toxicosis secondary to carboplatin administration. Further prospective evaluation of CK18 and A1-AT as biomarkers of drug-induced GI toxicity is warranted in a larger cohort of dogs receiving cytotoxic chemotherapy. AVMA clinical trial registration number: AAHSD004827.