Lena Lennartsson

Additive/synergistic antitumoral effects on prostate cancer cells in vitro following treatment with a combination of docetaxel and zoledronic acid

Once bone metastasized and androgen independent, prostate cancer is often associated with skeletal morbidity and disability. New treatment modalities that can palliate symptoms from the skeleton and inhibit further progression are warranted. In this study, the antitumoral effects following treatment with a combination of docetaxel and the new generation bisphosphonate, zoledronic acid, were investigated on two hormone-refractory prostate cancer cell lines: PC3 and DU145. The prostate cancer cells were treated with increasing concentrations of zoledronic acid in the absence or presence of docetaxel. Toxicity was measured using fluorometric microculture cytotoxic assay technique. A concentration of 25 µM, zoledronic acid reduced the viable cell number to 68% and 98% for PC3 and DU145 cells respectively. Docetaxel, on the other hand, at a concentration of 0.1 ng/ml, had no effect on the viability. However, a combination of zoledronic acid and docetaxel reduced the cell number to 60% and 81% respectively. Furthermore, zoledronic acid in the concentration range 12.5 µM–50 µM enhanced the antitumoral effects of docetaxel (0.01–1 ng/ml) in an additive and/or synergistic manner for both cell lines. These data support the hypothesis that zoledronic acid, in addition to having bone resorption inhibiting properties, also exhibits anti-tumoral effects. It also appears that combined treatment with docetaxel causes additive and/or synergistic cytostatic effects on prostate cancer cells.

Sorafenib-induced defective autophagy promotes cell death by necroptosis.

Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencing ULK1 and Beclin1 rescues DU145 cells from cell death indicating that, in this setting, autophagy promotes cell death. Re-expression of Atg5 restores the lipidation of LC3 and rescues DU145 and MEF atg5−/− cells from sorafenib-induced cell death. Despite the lack of Atg5 expression and LC3 lipidation, DU145 cells form autophagosomes as demonstrated by transmission and immuno-electron microscopy, and the formation of LC3 positive foci. However, the lack of cellular content in the autophagosomes, the accumulation of long-lived proteins, the presence of GFP-RFP-LC3 positive foci and the accumulated p62 protein levels indicate that these autophagosomes may not be fully functional. DU145 cells treated with sorafenib undergo a caspase-independent cell death that is inhibited by the RIPK1 inhibitor, necrostatin-1. Furthermore, treatment with sorafenib induces the interaction of RIPK1 with p62, as demonstrated by immunoprecipitation and a proximity ligation assay. Silencing of p62 decreases the RIPK1 protein levels and renders necrostatin-1 ineffective in blocking sorafenib-induced cell death. In summary, the formation of Atg5-deficient autophagosomes in response to sorafenib promotes the interaction of p62 with RIPK leading to cell death by necroptosis.

Zoledronic acid induces caspase-dependent apoptosis in renal cancer cell lines

Objective. To study and characterize the potential antitumoral effects of zoledronic acid (ZA) on renal cancer cell lines in vitro. Material and methods. Three different and well-characterized renal cancer cell lines were studied, namely ACHN, A-498 and CAKI-2. The cytotoxic potential of ZA was evaluated using a fluorometric microculture cytotoxic assay. The degree of M30 induction following ZA treatment was measured using an M30 enzyme-linked immunosorbent assay (ELISA), and the blockage of this effect was studied using a pan-caspase inhibitor. Immunofluorescence of the M30 neoepitope was performed to visualize the M30-inducing properties of ZA. Results. A significant reduction in viable cells was seen for all three cell lines following treatment with ZA, compared with untreated controls. This effect was most pronounced for the ACHN cells, as only 4% were viable following incubation with ZA for 72 h. A concomitant increase in the apoptosis significant caspase-dependent M30 antigen was demonstrated. This effect could be blocked by the pan-caspase inhibitor Z-VAD. Conclusions. ZA exerts cytotoxic effects on renal cancer cell lines in vitro. These include caspase-dependent induction of apoptosis, which can be quantified and visualized using M30 ELISA and immunofluorescence, respectively. The clinical relevance of this finding needs to be further investigated, but these results indicate that ZA may be a treatment alternative for selected patients with skeletal metastasized renal cell cancer, particularly for those with impaired performance status without other treatment options.

Prostate cancer cell lines lack amplification : Overexpression of HER2

The potential overexpression of HER2 in prostate cancer cells has attended significant interest during the past few years, both as potential target for HER2 pathway focused therapy and as a mechanism involved in the progression to androgen independence. Conflicting results have been reported concerning HER2 status on clinical material, differences which generally have been attributed to methodological differences. Nevertheless, HER2 has been utilized for targeted therapy of prostate cancer in a number of preclinical studies and is still regarded as an exciting target molecule. In this study, the HER2 status of three widely used prostate cancer cell lines and corresponding xenografts has been analysed. By use of validated and FDA approved analytical staining techniques none of these cell lines or xenografts were shown to overexpress/amplify HER2, as demonstrated by immunohistochemistry and fluorescense in situ hybridization. These findings are important for the interpretation and understanding of the therapeutic effects when developing drugs targeting HER2 in prostate cancer cell lines and also emphasize the importance of using broad and validated analytical techniques.

Identification of mutations, gene expression changes and fusion transcripts by whole transcriptome RNAseq in docetaxel resistant prostate cancer cells

Abstract Docetaxel has been the standard first-line therapy in metastatic castration resistant prostate cancer. The survival benefit is, however, limited by either primary or acquired resistance. In this study, Du145 prostate cancer cells were converted to docetaxel-resistant cells Du145-R and Du145-RB by in vitro culturing. Next generation RNAseq was employed to analyze these cell lines. Forty-two genes were identified to have acquired mutations after the resistance development, of which thirty-four were found to have mutations in published sequencing studies using prostate cancer samples from patients. Fourteen novel and 2 previously known fusion genes were inferred from the RNA-seq data, and 13 of these were validated by RT-PCR and/or re-sequencing. Four in-frame fusion transcripts could be transcribed into fusion proteins in stably transfected HEK293 cells, including MYH9-EIF3D and LDLR-RPL31P11, which were specific identified or up-regulated in the docetaxel resistant DU145 cells. A panel of 615 gene transcripts was identified to have significantly changed expression profile in the docetaxel resistant cells. These transcriptional changes have potential for further study as predictive biomarkers and as targets of docetaxel treatment.

Identification of Mutations, Expression Alterations and Fusion Transcriptsby Next Generation RNAseq in Castration-Resistant Prostate Cancer Celllines with Possible Clinical Relevance

Androgen Deprivation Therapy (ADT) would benefit prostate cancer patients initially but cancer cells can eventually develop castration resistance. In this study, we compared androgen-dependent and androgenindependent cell lines to find potential genes associated with acquired resistance to ADT. Using RNAseq, we found 4397 mutations distributed in 2579 genes, out of which, 1574 mutated genes could also be found in prostate cancer tumor samples collected in Cosmic database (http://cancer.sanger.ac.uk/cosmic). We also discovered 157 and 549 genes which were down and up-regulated respectively in both PC3 and DU145 compared to LNCaP. Network analysis resulted in 3 dominant connection notes: GCR/MCR (NR3C1) and PKA-cat kinase (PRKACB) and PKC family (PRKD1). By ChimeraScan analysis, 48, 117 and 60 chimeric transcripts were discovered in DU145, LNCaP and PC3 respectively. Among them, six predicted fusions expressed specifically in androgen-independent cell lines (DU145 and PC3). Some of these gene mutations and transcription alterations have been reported in tumor samples from prostate cancer patients and may have certain associations with acquired resistance to anti-hormone therapy in prostate cancer. A proportion of mutations are enriched in genes involved in immune response pathways, suggesting new targets to develop effective treatments to overcome castration resistance.