Clustering of anthracycline-containing lysosomes in drug-resistant MCF-7 cells in response to tesmilifene

3223 A clinical trial by the National Cancer Institute of Canada Clinical Trials Group has shown that N,N-Diethyl-2-[4-(Phenylmethyl)Phenoxy] Ethanamine (tesmilifene) in combination with doxorubicin was superior to doxorubicin alone in patients with metastatic breast cancer in terms of overall survival (p=0.021. J. Clin. Oncol.22: 269-276). To address the mechanism for tesmilifene-induced clinical potentiation of drug cytotoxicity, doxorubicin-resistant (MCF-7DOX-2) and paclitaxel-resistant (MCF-7TAX-2) variants of MCF-7 cells were established in our laboratory. Clonogenic assays revealed that tesmilifene could effectively augment the cytotoxicity of a variety of chemotherapy agents specifically in these drug-resistant cell lines and this potentiation was independent of P-glycoprotein status. To better understand the mechanism of action of tesmilifene, confocal fluorescence microscopy was used to examine the distribution and localization of doxorubicin and epirubicin in drug-resistant cells in the absence or presence of tesmilifene. In wildtype MCF-7 cells, lysosomes were well distributed throughout the cytoplasm, while doxorubicin and epirubicin were consistently observed within the nucleus. In contrast, both drugs were located in the cytoplasm of some or all of MCF-7TAX-2 and MCF-7DOX-2 cells, respectively, where they co-localized with lysosomes (as visualized by LysoTracker staining). Addition of tesmilifene to the drug-resistant cells induced a very striking clustering of doxorubicin- or epirubicin-containing lysosomes to the perinuclear region. Clustering of lysosomes around the nucleus is a hallmark feature of necrosis, suggesting that tesmilifene may induce necrotic death in drug-resistant tumour cells. To identify genes which may be related to this phenomenon, a survey of gene expression using Operon Human Genome arrays (AROS V4) containing 35035 oligonucleotide probes was conducted in order to identify tesmilifene-induced changes in gene expression in the drug resistant cell lines in the presence of either doxorubicin or paclitaxel. Sixty-two genes were upregulated and 29 genes down-regulated by tesmilifene in MCF-7DOX-2 cells in the presence of doxorubicin. Tesmilifene upregulated 9 genes in MCF-7TAX-2 cells in the presence of paclitaxel. Two of the tesmilifene-dependent genes in MCF-7DOX-2 cells were found to be related to lysosome function and may play a role in the above-described phenomenon. These include the overexpression of cathepsin C (CTSC) and Lysozyme G-like Protein Precursor (LYG2). In MCF-7TAX-2 cells, Bcl-2 like 13 protein (BCL2L13) was induced by tesmilifene and is known to promote paclitaxel-induced cell death. Further studies are underway to identify the precise mechanism by which tesmilifene induces lysosomal clustering and the above changes in gene expression.