We report results showing that the silencing of carbonic anhydrase I (siCA1) in prostatic (PC3) tumour cells has a significant impact on exosome formation. An increased diameter, concentration and diversity of the produced exosomes were noticed as a consequence of this knock-down. The protein composition of the exosomes' cargo was also altered. Liquid chromatography and mass spectrometry analyses identified 42 proteins significantly altered in PC3 siCA1 exosomes compared with controls. The affected proteins are mainly involved in metabolic processes, biogenesis, cell component organization and defense/immunity. Interestingly, almost all of them have been described as 'enhancers' of tumour development through the promotion of cell proliferation, migration and invasion. Thus, our results indicate that the reduced expression of the CA1 protein enhances the malignant potential of PC3 cells.
- Klíčová slova
- LC-MS, PC3 cells, carbonic anhydrase I, exosomes, malignant potential, siCA1, siMock,
- MeSH
- buňky PC-3 MeSH
- energetický metabolismus genetika MeSH
- exozómy genetika metabolismus MeSH
- karboanhydrasa I genetika metabolismus MeSH
- lidé MeSH
- nádory prostaty genetika metabolismus patologie MeSH
- pohyb buněk genetika MeSH
- proliferace buněk genetika MeSH
- regulace genové exprese enzymů * MeSH
- regulace genové exprese u nádorů * MeSH
- RNA interference * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- karboanhydrasa I MeSH
Gene-directed enzyme/prodrug therapy (GDEPT) mediated by mesenchymal stromal cells (MSC) was already approved for clinical study on a progressive disease refractory to standard therapy. In this work, we examined the effect of several GDEPT approaches on chemoresistant cells. First, we derived 5-fluorouracil (5-FU)-resistant variant of human colorectal adenocarcinoma cells HT-29 designated HT-29/EGFP/FUR. Our data show that the upregulation of thymidylate synthase (TS) and downregulation of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT) and dihydropyrimidine dehydrogenase (DPD) contributed to the 5-FU resistance in cancer cells. Next, we combined the MSC expressing either yeast cytosine deaminase (CD-MSC) or fusion yeast CD::uracil phosphoribosyl transferase (CD::UPRT-MSC) and prodrug 5-fluorocytosine (5-FC) in a cell-mediated GDEPT approach. Bystander cytotoxic effect in the direct co-cultures of the tumor and therapeutic cells mixed in a 5:1 ratio resulted in 55% and 70% inhibition of proliferation, respectively. However, the acquired chemoresistance to 5-FU can be overcome by introducing the prodrug-converting transgene into the tumor cells. When the transgene CD::UPRT was expressed in the chemoresistant cells (CD::UPRT-FUR), substantial suicide effect and a 90% decrease in viability was observed using non-toxic concentration of 62.5 µg/ml 5-FC. In summary, we demonstrate here that the transgene introduction circumvented 5-FU resistance in the tumor cells.
- MeSH
- fluorouracil farmakologie terapeutické užití MeSH
- genetická terapie metody MeSH
- lidé MeSH
- myši SCID MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- prekurzory léčiv farmakologie terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fluorouracil MeSH
- prekurzory léčiv MeSH
- Publikační typ
- tisková chyba MeSH
BACKGROUND: Cells of the tumor microenvironment are recognized as important determinants of the tumor biology. The adjacent non-malignant cells can regulate drug responses of the cancer cells by secreted paracrine factors and direct interactions with tumor cells. RESULTS: Human mesenchymal stromal cells (MSC) actively contribute to tumor microenvironment. Here we focused on their response to chemotherapy as during the treatment these cells become affected. We have shown that the secretory phenotype and behavior of mesenchymal stromal cells influenced by cisplatin differs from the naïve MSC. MSC were more resistant to the concentrations of cisplatin, which was cytotoxic for tumor cells. They did not undergo apoptosis, but a part of MSC population underwent senescence. However, MSC pretreatment with cisplatin led to changes in phosphorylation profiles of many kinases and also increased secretion of IL-6 and IL-8 cytokines. These changes in cytokine and phosphorylation profile of MSC led to increased chemoresistance and stemness of breast cancer cells. CONCLUSION: Taken together here we suggest that the exposure of the chemoresistant cells in the tumor microenvironment leads to substantial alterations and might lead to promotion of acquired microenvironment-mediated chemoresistance and stemness.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza účinky léků MeSH
- chemorezistence MeSH
- cisplatina farmakologie MeSH
- interleukin-6 imunologie MeSH
- interleukin-8 imunologie MeSH
- lidé MeSH
- mezenchymální kmenové buňky cytologie účinky léků imunologie MeSH
- nádorové buněčné linie MeSH
- nádorové mikroprostředí účinky léků MeSH
- nádory prsu farmakoterapie imunologie MeSH
- prsy účinky léků imunologie MeSH
- stárnutí buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antitumorózní látky MeSH
- cisplatina MeSH
- interleukin-6 MeSH
- interleukin-8 MeSH