BACKGROUND: Cystathionine β-synthase (CBS), one of three enzymes that endogenously produce hydrogen sulfide, is extensively studied for its relevance in the cells of various tumors. In our previous work, we observed that the immunofluorescence pattern of CBS is very similar to that of tubulin and actin. Therefore, we focused on the potential interaction of CBS with cytoskeletal proteins β-actin and β-tubulin and the functional relevance of the potential interaction of these proteins in colorectal carcinoma cell lines. METHODS: To study the potential interaction of CBS with cytoskeletal proteins and its functional consequences, a CBS-knockout DLD1 (DLDx) cell line was established by using the CRISPR/Cas9 gene editing method. The interaction of the selected cytoskeletal protein with CBS was studied by immunoprecipitation, Western blot analysis, immunofluorescence, and proximity ligation assay. The functional consequences were studied by proliferation and migration assays and by generation of xenografts in SCID/bg mice. RESULTS: We have found that CBS, an enzyme that endogenously produces H2S, binds to cytoskeletal β-tubulin and, to a lesser extent, also to β-actin in colorectal carcinoma-derived cells. When CBS was knocked out by the CRISPR/Cas9 technique (DLDx), we observed a de-arranged cytoskeleton compared to the unmodified DLD1 cell line. Treatment of these cells with a slow sulfide donor GYY4137 resulted in normal organization of the cytoskeleton, thus pointing to the role of CBS in microtubule dynamics. To evaluate the physiological importance of this observation, both DLD1 and DLDx cells were injected into SCID/bg mice, and the size and mass of the developed xenografts were evaluated. Significantly larger tumors developed from DLDx compared to the DLD1 cells, which correlated with the increased proliferation of these cells. CONCLUSIONS: Taken together, in colorectal cancer DLD1 cells, CBS binds to the cytoskeleton, modulates microtubule dynamics, and thus affects the proliferation and migration in the colorectal carcinoma stable cell line.

• Klíčová slova
• colorectal carcinoma cells, cystathionine beta-synthase, cytoskeleton, xenografts, β-tubulin,
• Publikační typ
• časopisecké články MeSH

Intensive investigation for novel antiproliferative and cytotoxic effective chemical compounds is currently concentrated on structurally modified agents of natural or synthetic source. The selenium derivative of triorganotin compound, triphenyltin isoselenocyanate (TPT-NCSe) caused higher cytotoxicity in hormone sensitive MCF 7 (IC 50-250 nM) in comparison with triple-negative MDA-MB-231 breast carcinoma cell line (IC 50-450 nM) as determined by MTT assay. Measurement of DNA damage showed presence of crosslinks in both cell lines treated by increasing TPT-NCSe concentrations. This compound decreased mitochondrial membrane potential shown by JC-1 staining in a concentration-dependent manner in both cell lines. Activation of caspases-3/7 was observed in MDA-MB-231 cells and was significant only by concentrations causing significant level of crosslinks. On the other hand, migration assay revealed inhibitory effect of viability keeping 100 nM concentration of TPT-NCSe on migration of MDA-MB-231 cells. Our data has shown that this selenium containing triorganotin molecule exerts DNA damage-linked antineoplastic activity in breast carcinoma cell lines studied.

• Klíčová slova
• Apoptosis, Breast cancer, Cytotoxicity, DNA crosslinks, Migration, Triorganotin isoselenocyanates,
• MeSH
• antitumorózní látky * chemie   farmakologie   MeSH
• apoptóza MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádorové buněčné linie MeSH
• nádory prsu * farmakoterapie   MeSH
• organocínové sloučeniny MeSH
• organoselenové sloučeniny MeSH
• proliferace buněk MeSH
• selen * farmakologie   MeSH
• triple-negativní karcinom prsu * metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antitumorózní látky * MeSH
• isoselenocyanate MeSH Prohlížeč
• organocínové sloučeniny MeSH
• organoselenové sloučeniny MeSH
• selen * MeSH
• triphenyltin MeSH Prohlížeč

Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance. A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively. Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.

• Klíčová slova
• 5-fluorouracil, Cancer therapy, Chemotherapeutics, High resolution mass spectrometry, Metabolites,
• MeSH
• chromatografie kapalinová MeSH
• cytosindeaminasa MeSH
• flucytosin * MeSH
• fluorouracil MeSH
• hmotnostní spektrometrie MeSH
• prekurzory léčiv * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytosindeaminasa MeSH
• flucytosin * MeSH
• fluorouracil MeSH
• prekurzory léčiv * MeSH

Several papers have reported that calcium channel blocking drugs were associated with increased breast cancer risk and worsened prognosis. One of the most common signs of breast tumors is the presence of small deposits of calcium, known as microcalcifications. Therefore, we studied the effect of dihydropyridine nifedipine on selected calcium transport systems in MDA-MB-231 cells, originating from triple negative breast tumor and JIMT1 cells that represent a model of HER2-positive breast cancer, which possesses amplification of HER2 receptor, but cells do not response to HER2 inhibition treatment with trastuzumab. Also, we compared the effect of nifedipine on colorectal DLD1 and ovarian A2780 cancer cells. Both, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and type 1 sodium calcium exchanger (NCX1) were upregulated due to nifedipine in DLD1 and A2780 cells, but not in breast cancer MDA-MB-231 and JIMT1 cells. On contrary to MDA-MB-231 and JIMT1 cells, in DLD1 and A2780 cells nifedipine induced apoptosis in a concentration-dependent manner. After NCX1 silencing and subsequent treatment with nifedipine, proliferation was decreased in MDA-MB-231, increased in DLD1 cells, and not changed in JIMT1 cells. Silencing of IP3R1 revealed increase in proliferation in DLD1 and JIMT1 cells, but caused decrease in proliferation in MDA-MB-231 cell line after nifedipine treatment. Interestingly, after nifedipine treatment migration was not significantly affected in any of tested cell lines after NCX1 silencing. Due to IP3R1 silencing, significant decrease in migration occurred in MDA-MB-231 cells after nifedipine treatment, but not in other tested cells. These results support different function of the NCX1 and IP3R1 in the invasiveness of various cancer cells due to nifedipine treatment.

• Klíčová slova
• Apoptosis, Breast cancer, Inositol 1,4,5-trisphosphate receptor, Migration, Sodium calcium exchanger 1,
• MeSH
• apoptóza účinky léků   genetika   MeSH
• blokátory kalciových kanálů farmakologie   MeSH
• inositol-1,4,5-trisfosfát - receptory genetika   metabolismus   MeSH
• kolorektální nádory genetika   metabolismus   patologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prsu genetika   metabolismus   patologie   MeSH
• nádory vaječníků genetika   metabolismus   patologie   MeSH
• nifedipin farmakologie   MeSH
• pohyb buněk účinky léků   genetika   MeSH
• proliferace buněk účinky léků   genetika   MeSH
• protinádorové látky imunologicky aktivní farmakologie   MeSH
• pumpa pro výměnu sodíku a vápníku genetika   metabolismus   MeSH
• receptor erbB-2 genetika   metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• RNA interference MeSH
• trastuzumab farmakologie   MeSH
• triple-negativní karcinom prsu genetika   metabolismus   patologie   MeSH
• vápníková signalizace účinky léků   genetika   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• blokátory kalciových kanálů MeSH
• inositol-1,4,5-trisfosfát - receptory MeSH
• nifedipin MeSH
• protinádorové látky imunologicky aktivní MeSH
• pumpa pro výměnu sodíku a vápníku MeSH
• receptor erbB-2 MeSH
• sodium-calcium exchanger 1 MeSH Prohlížeč
• trastuzumab MeSH

We explored possibility that sodium/calcium exchanger 1 (NCX1) is involved in pH modulation and apoptosis induction in GYY4137 treated cells. We have shown that although 10 days treatment with GYY4137 did not significantly decreased volume of tumors induced by colorectal cancer DLD1 cells in nude mice, it already induced apoptosis in these tumors. Treatment of DLD1 and ovarian cancer A2780 cells with GYY4137 resulted in intracellular acidification in a concentration-dependent manner. We observed increased mRNA and protein expression of both, NCX1 and sodium/hydrogen exchanger 1 (NHE1) in DLD1-induced tumors from GYY4137-treated mice. NCX1 was coupled with NHE1 in A2780 and DLD1 cells and this complex partially disintegrated after GYY4137 treatment. We proposed that intracellular acidification is due to uncoupling of NCX1/NHE1 complex rather than blocking of the reverse mode of NCX1, probably due to internalization of NHE1. Results might contribute to understanding molecular mechanism of H2S-induced apoptosis in tumor cells.

• Klíčová slova
• Apoptosis, Hydrogen sulfide, Intracellular acidification, Sodium/calcium exchanger, Sodium/hydrogen exchanger,
• MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza účinky léků   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• morfoliny farmakologie   MeSH
• myši nahé MeSH
• nádorové buněčné linie MeSH
• organothiofosforové sloučeniny farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• pumpa pro výměnu sodíku a vápníku metabolismus   MeSH
• sodíko-vodíkový výměnný transportér 1 metabolismus   MeSH
• sulfan metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antitumorózní látky MeSH
• GYY 4137 MeSH Prohlížeč
• morfoliny MeSH
• NCX1 protein, mouse MeSH Prohlížeč
• organothiofosforové sloučeniny MeSH
• pumpa pro výměnu sodíku a vápníku MeSH
• SLC9A1 protein, human MeSH Prohlížeč
• Slc9a1 protein, mouse MeSH Prohlížeč
• sodíko-vodíkový výměnný transportér 1 MeSH
• sodium-calcium exchanger 1 MeSH Prohlížeč
• sulfan 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