Canine Melanoma Cell Research Articles

Abstract 5137: IQGAP1, an ERK1/2 MAPK scaffold, is an oncogenic target in canine melanoma

Abstract BACKGROUND: Human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%). However, human oral melanoma harbors these mutations much less frequently. Similarly, canine oral mucosal melanomas have not been found to have activating Braf or Nras mutations. This makes them an ideal model to study alternative means of ERK1/2 MAPK pathway activation as these tumors are still reliant on MAPK signaling. We have previously studied IQGAP1 as a tumor specific ERK1/2 MAPK scaffold and shown that it is required for cancer progression. METHODS: In this study, we utilized canine oral melanoma cell lines obtained from the University of California-Davis. We examined the expression and localization of IQGAP1, a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human cancer, and validate it as an oncogenic target in the canine melanoma model. Using a CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream pathway activation. Additionally, we assayed proliferation of these cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. RESULTS: We observed that canine IQGAP1 is expressed and localizes to a similar extent in human and canine melanoma by qPCR, Western blot and immunofluorescence. Deletion of IQGAP1 from two canine oral melanoma cell lines using CRISPR-Cas9 genome editing reduces MAPK pathway activation, similar to effects seen in human BrafV600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with the WW blocking peptide in-vitro. We are currently investigating the expression and localization of IQGAP1 in primary canine oral melanoma compared to normal oral mucosa. CONCLUSION: Building on the above findings to explore alternative MAPK signaling in melanoma using the canine melanoma model may yield novel therapeutic strategies and novel means to target canine and human tumors that are resistant to current Braf and MEK targeted therapies. Citation Format: Ashley Zehnder, Becky Lee, Poornima Neela, Paul Khavari. IQGAP1, an ERK1/2 MAPK scaffold, is an oncogenic target in canine melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5137. doi:10.1158/1538-7445.AM2015-5137

Abstract B36: The effects of dasatinib in KIT L579P and NRAS Q61K mutant canine melanoma cells

Abstract Purpose: Malignant melanoma is a common malignant tumor in dog. It is a fatal disease with local invasiveness and frequently metastatic propensity and the poor response has been observed. The aim of this study was to establish the drug-screening platform and used that platform to evaluate small molecular inhibitor, Dasatinib, for therapy of canine melanoma. Experimental Design: Various phenotypes of eight canine melanoma cell lines, CM01, M1, M3, M4, M5, Pu, C1 and KMeC, were examined in this study. Mutation analysis of kit, Nras and Braf was done by PCR/DNA sequencing. Expression of S100, KIT and KIT downstream signaling cascades NRAS, phospho-ERK and phospho-AKT were evaluated by immunocytochemistry. KMeC, M5 and CM01 cell lines were selected for further evaluation of Dasatinib. Proliferation assay and wound healing assay were performed to assess cell viability and motility in the presence of dasatinib. Cell cycle analysis and Annexin V apoptosis assay were also applied to realize the mechanism of cell death caused by Dasatinib. Results: Expressions of S100 and KIT were observed in all of the cell lines. NRAS and activated ERK were identified in several cell lines with different expression levels. However, no activated AKT was identified in any cell lines. kit T1736C mutation causing KIT L579P change was detected in M3, KMeC and C1 cells, while Nras C181A mutation causing NRAS Q61K change was found in M5 cell. ERK activity in KMeC (KIT L579P), M5 (NRAS Q61K) and CM01 (KIT & NRAS wild type) cells was down-regulated when Dasatinib was administered. Dasatinib reduced cell viability with the IC50 values of 0.64 μM in KMeC, 0.24 μM in M5 and 0.37 μM in CM01 cells. In wound healing assay, Dasatinib inhibited cell motility in all of the three cell lines. Cell cycle analysis and Annexin V apoptosis assay revealed that the treatment of Dasatinib causes a G1 cell cycle arrest and induction of apoptosis. The analysis of Dasatinib caused signaling change in canine melanoma cell is ongoing. Conclusions: Various phenotypes of eight canine melanoma cell lines were identified in DNA and protein levels. The NRAS Q61K mutation was first found in canine melanoma. The results showed that KIT L579P mutant type, NRAS Q61K mutant type and wild type canine melanoma cells are sensitive to the treatment of Dasatinib. It caused the reduction of cell viability, and inhibition of motility, moreover, the promotion of G1 cell cycle arrest and cell apoptosis. Dasatinib may potentially be selected as a candidate for therapy of canine melanoma. Citation Format: Lu-Ping Lu, Chen-Si Lin, Albert Taiching Liao. The effects of dasatinib in KIT L579P and NRAS Q61K mutant canine melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B36.

Interferon-β gene transfer induces a strong cytotoxic bystander effect on melanoma cells

A local gene therapy scheme for the delivery of type I interferons could be an alternative for the treatment of melanoma. We evaluated the cytotoxic effects of interferon-β (IFNβ) gene lipofection on tumor cell lines derived from three human cutaneous and four canine mucosal melanomas. The cytotoxicity of human IFNβ gene lipofection resulted higher or equivalent to that of the corresponding addition of the recombinant protein (rhIFNβ) to human cells. IFNβ gene lipofection was not cytotoxic for only one canine melanoma cell line. When cultured as monolayers, three human and three canine IFNβ-lipofected melanoma cell lines displayed a remarkable bystander effect. As spheroids, the same six cell lines were sensitive to IFNβ gene transfer, two displaying a significant multicell resistance phenotype.The effects of conditioned IFNβ-lipofected canine melanoma cell culture media suggested the release of at least one soluble thermolabile cytotoxic factor that could not be detected in human melanoma cells. By using a secretion signal-free truncated human IFNβ, we showed that its intracellular expression was enough to induce cytotoxicity in two human melanoma cell lines. The lower cytoplasmatic levels of reactive oxygen species detected after intracellular IFNβ expression could be related to the resistance displayed by one human melanoma cell line. As IFNβ gene transfer was effective against most of the assayed melanomas in a way not limited by relatively low lipofection efficiencies, the clinical potential of this approach is strongly supported.

Gene Electrotransfer of Canine Interleukin 12 into Canine Melanoma Cell Lines.

A gene electrotransfer (GET) of interleukin 12 (IL-12) had already given good results when treating tumors in human and veterinary clinical trials. So far, plasmids used in veterinary clinical studies encoded a human or a feline IL-12 and an ampicillin resistance gene, which is not recommended by the regulatory agencies to be used in clinical trials. Therefore, the aim of the current study was to construct the plasmid encoding a canine IL-12 with kanamycin antibiotic resistance gene that could be used in veterinary clinical oncology. The validation of the newly constructed plasmid was carried out on canine malignant melanoma cells, which have not been used in GET studies so far, and on human malignant melanoma cells. Canine and human malignant melanoma cell lines were transfected with plasmid encoding enhanced green fluorescence protein at different pulse parameter conditions to determine the transfection efficiency and cell survival. The IL-12 expression of the most suitable conditions for GET of the plasmid encoding canine IL-12 was determined at mRNA level by the qRT-PCR and at protein level with the ELISpot assay. The obtained results showed that the newly constructed plasmid encoding canine IL-12 had similar or even higher expression capacity than the plasmid encoding human IL-12. Therefore, it represents a promising therapeutic plasmid for further IL-12 gene therapy in clinical studies for spontaneous canine tumors. Additionally, it also meets the main regulatory agencies' (FDA and EMA) criteria.

The oncolytic effects of reovirus in canine solid tumor cell lines

Oncolytic virotherapy is a new strategy for cancer treatment for humans and dogs. Reovirus has been proven to be a potent oncolytic virus in human medicine. Our laboratory has previously reported that canine mast cell tumor and canine lymphoma were susceptible to reovirus. In this study, canine solid tumor cell lines (mammary gland tumor, osteosarcoma and malignant melanoma) were tested to determine their susceptibility towards reovirus. We demonstrated that reovirus induces more than 50% cell death in three canine mammary gland tumors and one canine malignant melanoma cell line. The reovirus-induced cell death occurred via the activation of caspase 3. Ras activation has been shown to be one of the important mechanisms of reovirus-susceptibility in human cancers. However, Ras activation was not related to the reovirus-susceptibility in canine solid tumor cell lines, which was similar to reports in canine mast cell tumor and canine lymphoma. The results of this study highly suggest that canine mammary gland tumor and canine malignant melanoma are also potential candidates for reovirus therapy in veterinary oncology.

The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma.

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.

Differentiation-inducing and anti-proliferative activities of lupeol on canine melanoma cells.

Canine melanoma is the most common oral malignant tumor reported in the field of veterinary medicine. We found that lupeol, a lupine triterpene, inhibited mouse melanoma cell growth in vitro and in vivo by inducing cell differentiation. In the present study, we examined the differentiation-inducing activities of lupeol on 4 canine melanoma cells in vitro and in vivo.The induction of canine melanoma cell differentiation by lupeol was confirmed by evaluating some differentiation markers such as tyrosinase with real-time RT-PCR. Furthermore, we transplanted canine melanoma cells into a severe combined immunodeficiency mouse, and studied the anti-progressive effects of lupeol on tumor tissue.The gene expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein-2, which are markers of pigment cell differentiation, was induced in 4 canine oral malignant melanoma cells by lupeol, and the agent markedly inhibited tumor progression in canine melanoma-bearing mice.

Analysis of microRNA-203 function in CREB/MITF/RAB27a pathway: comparison between canine and human melanoma cells.

MicroRNA (miR)-203 is downregulated and acts as an anti-oncomir in melanoma cells. Here, using human and canine melanoma cells, we elucidated the effects of miR-203 on cyclic adenosine monophosphate response element binding protein (CREB)/microphthalmia-associated transcription factor (MITF)/RAB27a pathway, which is known to be important for the development and progression of human melanoma. In this study, we showed that miR-203 directly targeted CREB1 and regulated its downstream targets, MITF and RAB27a. miR-203 significantly suppressed the growth of human and canine melanoma cells and inhibited melanosome transport through the suppression of the signalling pathway. In conclusion, miR-203 was shown to be a common tumour-suppressive miRNA in human and canine melanoma and thus to play a crucial role in the biological mechanisms of melanoma development.

6-Bromoindirubin-3′oxime (BIO) decreases proliferation and migration of canine melanoma cell lines

Despite recent therapeutic advances, malignant melanoma is an aggressive tumor in dogs and is associated with a poor outcome. Novel, targeted agents are necessary to improve survival. In this study, 6-bromoindirubin-3′-oxime (BIO), a serine/threonine kinase inhibitor with reported specificity for glycogen synthase kinase-3 beta (GSK-3β) inhibition, was evaluated in vitro in three canine melanoma cell lines (CML-10C2, UCDK9M2, and UCDK9M3) for β-catenin-mediated transcriptional activity, Axin2 gene and protein expression levels, cell proliferation, chemotoxicity, migration and invasion assays.BIO treatment of canine malignant melanoma cell lines at 5 µM for 72 h enhanced β-catenin-mediated transcriptional activity, suggesting GSK-3β inhibition, and reduced cell proliferation and migration. There were no significant effects on invasion, chemotoxicity, or apoptosis. The results suggest that serine/threonine kinases may be viable therapeutic targets for the treatment of canine malignant melanoma.

Biologic activity of the novel orally bioavailable selective inhibitor of nuclear export (SINE) KPT-335 against canine melanoma cell lines.

BackgroundExportin 1 (XPO1, also known as CRM1), is a chaperone protein responsible for the export of over 200 target proteins out of the nucleus. The expression and activity of XPO1 is upregulated in several human cancers and its expression is also linked to the development of chemotherapy resistance. Recent studies using both human and murine cancer cell lines have demonstrated that XPO1 is a relevant target for therapeutic intervention. The present study sought to characterize the biologic activity of an orally bioavailable selective inhibitor of nuclear export (SINE), KPT-335, against canine melanoma cell lines as a prelude to future clinical trials in dogs with melanoma.ResultsWe evaluated the effects of KPT-335 on 4 canine malignant melanoma cell lines and found that KPT-335 inhibited proliferation, blocked colony formation, and induced apoptosis of treated cells at biologically relevant concentrations of drug. Additionally, KPT-335 downregulated XPO1 protein while inducing a concomitant increase in XPO1 messenger RNA. Lastly, KPT-335 treatment of cell lines upregulated the expression of both protein and mRNA for the tumor suppressor proteins p53 and p21, and promoted their nuclear localization.ConclusionsKPT-335 demonstrates biologic activity against canine melanoma cell lines at physiologically relevant doses, suggesting that KPT-335 may represent a viable treatment option for dogs with malignant melanoma.

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.

Antitumor effects of celecoxib in COX-2 expressing and non-expressing canine melanoma cell lines

Cyclooxygenase-2 (COX-2) is a potential target for chemoprevention and cancer therapy. Celecoxib, a selective COX-2 inhibitor, inhibits cell growth of various types of human cancer including malignant melanoma. In dogs, oral malignant melanoma represents the most common oral tumor and is often a fatal disease. Therefore, there is a desperate need to develop additional therapeutic strategies. The purpose of this study was to investigate the anticancer effects of celecoxib on canine malignant melanoma cell lines that express varying levels of COX-2. Celecoxib induced a significant anti-proliferative effect in both LMeC and CMeC-1 cells. In the CMeC cells, treatment of 50 μM celecoxib caused an increase in cells in the G0/G1 and a decreased proportion of cells in G-2 phase. In the LMeC cells, 50 μM of celecoxib led to an increase in the percentage of cells in the sub-G1 phase and a significant activation of caspase-3 when compared to CMeC-1 cells. In conclusion, these results demonstrate that celecoxib exhibits antitumor effects on canine melanoma LMeC and CMeC-1 cells by induction of G1-S cell cycle arrest and apoptosis. Our data suggest that celecoxib might be effective as a chemotherapeutic agent against canine malignant melanoma.

Binding of VEGF-A to canine cancer cells with preferential expression of VEGFR1

Aim: Despite encouraging results in syngeneic and xenografts cancer models with various inhibitors of vascular endothelial growth factor (VEGF) or its receptors (VEGFRs), beneficial effects have not been consistently translated to the clinic, underscoring the need to develop strategies that go beyond the inhibition of these targets. The purpose of this study was to generate data to support the hypothesis that VEGF may be used as “bait” to selectively deliver therapeutics to VEGFRexpressing cancer cells. Materials and Methods: VEGFR1 and VEGFR2 expression was characterized using real time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) in canine hemangiosarcoma (Grace-HSA, Emma-HSA), melanoma (TLM-1), and thyroid adenocarcinoma (CTAC) cell lines. TLM-1 and Grace-HSA were identified as representative cell lines that selectively expressed high levels of VEGFR1. Flow cytometry was performed to examine binding of a single VEGF molecule (biotinylated VEGFA and avidin conjugated to fluorescein isothiocyanate (FITC)) by these chemoresistant cell

Anti-Tumor Effect of Adipose Tissue Derived-Mesenchymal Stem Cells Expressing Interferon-β and Treatment with Cisplatin in a Xenograft Mouse Model for Canine Melanoma

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are attractive cell-therapy vehicles for the delivery of anti-tumor molecules into the tumor microenvironment. The innate tropism of AT-MSCs for tumors has important implications for effective cellular delivery of anti-tumor molecules, including cytokines, interferon, and pro-drugs. The present study was designed to determine the possibility that the combination of stem cell-based gene therapy with low-dose cisplatin would improve therapeutic efficacy against canine melanoma. The IFN-β transduced canine AT-MSCs (cAT-MSC-IFN-β) inhibited the growth of LMeC canine melanoma cells in direct and indirect in vitro co-culture systems. In animal experiments using BALB/c nude mouse xenografts, which developed by injecting LMeC cells, the combination treatment of cAT-MSC-IFN-β and low-dose cisplatin significantly reduced tumor volume compared with the other treatment groups. Fluorescent microscopic analysis with a TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) assay of tumor section provided evidence for homing of cAT-MSC-IFN-β to the tumor site and revealed that the combination treatment of cAT-MSC-IFN-β with low-dose cisplatin induced high levels of cell apoptosis. These findings may prove useful in further explorations of the application of these combined approaches to the treatment of malignant melanoma and other tumors.

Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma

The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer-related signalling pathways despite differences in activating mutations.

MicroRNAs as tumour suppressors in canine and human melanoma cells and as a prognostic factor in canine melanomas

Malignant melanoma (MM) is one of the most aggressive cancers in dogs and in humans. However, the molecular mechanisms of its development and progression remain unclear. Presently, we examined the expression profile of microRNAs (miRs) in canine oral MM tissues and paired normal oral mucosa tissues by using the microRNA-microarray assay and quantitative RT-PCR. Importantly, a decreased expression of miR-203 was significantly associated with a shorter survival time. Also, miR-203 and -205 were markedly down-regulated in canine and human MM cell lines tested. Furthermore, the ectopic expression of miR-205 had a significant inhibitory effect on the cell growth of canine and human melanoma cells tested by targeting erbb3. Our data suggest that miR-203 is a new prognostic factor in canine oral MMs and that miR-205 functions as a tumour suppressor by targeting erbb3 in both canine and human MM cells.

Identification and evaluation of putative tumour‐initiating cells in canine malignant melanoma cell lines

Tumour-initiating cells (TICs) have been identified in many solid human tumours, including malignant melanoma. In this study, an enriched TIC population was identified in two canine malignant melanoma cell lines (CML1 and CML6M) using cell surface markers and functional assays, including the sphere forming assay, aldehyde dehydrogenase (ALDH) assay, reverse transcriptase-polymerase chain reaction and γH2AX staining for double-stranded DNA (dsDNA)break identification and repair. The CD34(-) population of cells in both cell lines expressed stem cell genes, such as Oct4, Nanog and Ptch1, were more efficient at making spheres in adherence-free media conditions and were able to repair dsDNA breaks faster than the CD34(+) population. A subpopulation of cells with high expression of ALDH was identified in both cell lines by flow cytometry. The findings indicate the presence of TICs in two canine malignant melanoma cell lines.

Radiation up-regulates the expression of VEGF in a canine oral melanoma cell line

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.

Activation of the Canonical Wnt/β-Catenin Signalling Pathway is Rare in Canine Malignant Melanoma Tissue and Cell Lines

Canine malignant melanoma is a highly aggressive tumour associated with a poor overall survival rate due to both local disease recurrence and its highly metastatic nature. Similar to advanced melanoma in man, canine oral melanoma is poorly responsive to conventional anti-cancer therapies. The lack of sustainable disease control warrants investigation of novel therapies, preferably targeting features specific to the tumour and different from normal cells. The Wnt signalling pathway is known to contribute to melanocytic lineage development in vertebrates and perturbation of the Wnt/β-catenin pathway has been implicated in numerous cancer types. Alterations of the Wnt/β-catenin pathway are suggested to occur in a subset of human melanomas, although the precise role of the Wnt/β-catenin pathway in melanoma is yet to be defined. This study investigates the activation status of the canonical Wnt/β-catenin pathway in canine malignant melanoma and its potential as a therapeutic target for treating this disease. The data indicate that canonical Wnt/β-catenin pathway activation is a rare event in canine oral malignant melanoma tissue and canine malignant melanoma cell lines.

Effect of Tolfenamic Acid on Canine Cancer Cell Proliferation, Specificity Protein (Sp) Transcription Factors, and Sp‐Regulated Proteins in Canine Osteosarcoma, Mammary Carcinoma, and Melanoma Cells

Tolfenamic acid (TA) is an NSAID currently under investigation as an anticancer agent in humans. TA induces proteosome-dependent degradation of transcription factors Sp 1, 3, and 4. These proteins are known to be overexpressed in many human cancers. To evaluate the protein expression of Sps in canine tissue, and efficacy of TA against several canine tumor cell lines. Six canine cell lines (2 osteosarcoma, 2 mammary carcinoma, 2 melanoma) were evaluated. Protein levels of Sp 1-4 and their downstream targets were evaluated using Western Blots. Cell survival and TUNEL assays were performed on cell lines, and Sp1 expression was evaluated on histologic samples from archived canine cases. Six immortalized canine cancer cell lines derived from dogs were used. Archived tissue samples were also used. Sps were highly expressed in all 6 cell lines and variably expressed in histologic tissues. TA decreased expression of Sps 1-4 in all cell lines. All of the downstream targets of Sps were inhibited in the cell lines. Variable Sp1 expression was identified in all histologic samples examined. TA significantly inhibited cell survival in all cell lines in a dose dependant fashion. The number of cells undergoing apoptosis was significantly increased (P<.05) in all cell lines after exposure to TA in a dose-dependent fashion. CONCLUSIONS, AND CLINICAL IMPORTANCE: Tolfenamic acid is a potential anticancer NSAID and further investigation is needed to determine its usefulness in a clinical setting.