ABSTRACT
The anti-tumour mechanisms following Bacillus Calmette-Guérin (BCG) treatment of bladder-cancer remain largely unknown. Previous studies have shown involvement of nitric-oxide (NO) formation in the BCG-mediated effect. We analyzed the effects of macrophage secreted factors (MSFs) from BCG-stimulated RAW264.7 cells on the bladder-cancer cell line MBT2. Direct treatment with BCG did not induce NO in MBT2-cells whereas supernatant from BCG-stimulated macrophages increased NOS2 mRNA and protein expression, NO concentrations and cell-death. Blocking NO-synthesis with the NOS-inhibitor L-NAME did not affect levels of cell-death suggesting cytotoxic pathways involving other signalling molecules than NO. Several such candidate genes were identified in a microarray.
Subject(s)
Antineoplastic Agents/therapeutic use , BCG Vaccine/therapeutic use , Macrophages/drug effects , Nitric Oxide/metabolism , Paracrine Communication/drug effects , Urinary Bladder Neoplasms/drug therapy , Animals , Cell Death/drug effects , Cell Line, Tumor , Culture Media, Conditioned/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Gene Expression Profiling/methods , Humans , Macrophages/immunology , Macrophages/metabolism , Mice , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Signal Transduction/drug effects , Treatment Outcome , Up-Regulation , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/immunology , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
Endocannabinoids have been implicated in cancer development and cause heterogenous effects in tumor cells, by inducing apoptosis, reducing migration, causing anti-angiogenic activity and alterations in the cell cycle resulting in growth arrest. Recently, several novel amides of fatty acids that are structurally related to endocannabinoids have been isolated from mammalian sources, although the functions of these fatty amides are not well studied. One group of these novel fatty acid amides are the N-acyl taurines (fatty acids conjugated to the amino acid taurine). This study examined if N-acyl taurines, specifically N-arachidonoyl taurine and N-oleoyl taurine could function in a similar way to endocannabinoids and result in cell cycle alterations or growth arrest in the human prostate adenocarcinoma cell line PC-3. PC-3 cells were treated with various concentrations of N-arachidonoyl taurine and N-oleoyl taurine and cell proliferation and viability was measured using resazurin and colony formation assays. Effects of N-acyl taurines on the cell cycle was measured using FACS analysis. Treatment with N-arachidonoyl taurine and N-oleoyl taurine resulted in a significant reduction in proliferation of PC-3 cells, even at concentrations as low as 1 µM. Treatment with N-oleoyl taurine resulted in an increased number of cells in the subG1 population, suggesting apoptosis, and a lower number of cells in S-phase of the cell cycle. In summary, our results show that novel biologically active lipids, the N-acyl taurines, result in reduced proliferation in PC-3 cells.
Subject(s)
Adenocarcinoma/drug therapy , Arachidonic Acids/pharmacology , Prostate/drug effects , Prostatic Neoplasms/drug therapy , Taurine/analogs & derivatives , Adenocarcinoma/pathology , Apoptosis/drug effects , Arachidonic Acids/chemistry , Cannabinoid Receptor Modulators/chemistry , Cannabinoid Receptor Modulators/pharmacology , Cell Count , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Male , Oxazines , Prostate/pathology , Prostatic Neoplasms/pathology , Taurine/chemistry , Taurine/pharmacology , XanthenesABSTRACT
Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.
