BACKGROUND: Resistance to chemotherapy is a major problem in the treatment of patients with triple-negative breast cancer (TNBC). Preclinical data suggest that TNBC is dependent on proteasomes; however, clinical observations indicate that the efficacy of proteasome inhibitors in TNBC may be limited, suggesting the need for combination therapies. METHODS: We compared bortezomib and carfilzomib and their combinations with nelfinavir and lopinavir in TNBC cell lines and primary cells with regard to their cytotoxic activity, functional proteasome inhibition, and induction of the unfolded protein response (UPR). Furthermore, we evaluated the involvement of sXBP1, ABCB1, and ABCG2 in the cytotoxic activity of drug combinations. RESULTS: Carfilzomib, via proteasome β5 + β2 inhibition, is more cytotoxic in TNBC than bortezomib, which inhibits β5 + β1 proteasome subunits. The cytotoxicity of carfilzomib was significantly potentiated by nelfinavir or lopinavir. Carfilzomib with lopinavir induced endoplasmic reticulum stress and pro-apoptotic UPR through the accumulation of excess proteasomal substrate protein in TNBC in vitro. Moreover, lopinavir increased the intracellular availability of carfilzomib by inhibiting carfilzomib export from cells that express high levels and activity of ABCB1, but not ABCG2. CONCLUSION: Proteasome inhibition by carfilzomib combined with nelfinavir/lopinavir represents a potential treatment option for TNBC, warranting further investigation.
- MeSH
- ABC transportér z rodiny G, člen 2 * metabolismus antagonisté a inhibitory MeSH
- apoptóza účinky léků MeSH
- bortezomib * farmakologie MeSH
- inhibitory HIV-proteasy * farmakologie MeSH
- inhibitory proteasomu farmakologie MeSH
- lidé MeSH
- Lopinavir * farmakologie MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny antagonisté a inhibitory metabolismus MeSH
- nelfinavir * farmakologie MeSH
- oligopeptidy * farmakologie MeSH
- P-glykoproteiny metabolismus MeSH
- protokoly protinádorové kombinované chemoterapie farmakologie MeSH
- signální dráha UPR * účinky léků MeSH
- stres endoplazmatického retikula účinky léků MeSH
- synergismus léků * MeSH
- triple-negativní karcinom prsu * farmakoterapie patologie MeSH
- XBP1 metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- bortezomib farmakologie MeSH
- chemorezistence * MeSH
- homeostáza MeSH
- inhibitory proteasomu farmakologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipidy chemie MeSH
- metabolomika MeSH
- mitochondrie metabolismus MeSH
- mnohočetný myelom farmakoterapie MeSH
- nádorové buněčné linie MeSH
- oligopeptidy farmakologie MeSH
- peroxid vodíku chemie MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- protinádorové látky farmakologie MeSH
- protokoly protinádorové kombinované chemoterapie farmakologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- sbalování proteinů MeSH
- senioři MeSH
- sfingomyeliny biosyntéza MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
OBJECTIVES: To assess the potential of second-generation proteasome inhibition by carfilzomib and its combination with the human immunodeficiency virus (HIV) protease inhibitors (HIV-PIs) lopinavir and nelfinavir in vitro for improved treatment of clear cell renal cell cancer (ccRCC). MATERIALS AND METHODS: Cytotoxicity, reactive oxygen species (ROS) production, and unfolded protein response (UPR) activation of proteasome inhibitors, HIV-PIs, and their combination were assessed in three cell lines and primary cells derived from three ccRCC tumours by MTS assay, flow cytometry, quantitative reverse transcriptase-polymerase chain reaction and western blot, respectively. Proteasome activity was determined by activity based probes. Flow cytometry was used to assess apoptosis by annexin V/propidium iodide assay and ATP-binding cassette sub-family B member 1 (ABCB1) activity by MitoTrackerTM Green FM efflux assay (Thermo Fisher Scientific, MA, USA). RESULTS: Lopinavir and nelfinavir significantly increased the cytotoxic effect of carfilzomib in all cell lines and primary cells. ABCB1 efflux pump inhibition, induction of ROS production, and UPR pre-activation by lopinavir were identified as underlying mechanisms of this strong synergistic effect. Combined treatment led to unresolved protein stress, increased activation of pro-apoptotic UPR pathway, and a significant increase in apoptosis. CONCLUSION: The combination of the proteasome inhibitor carfilzomib and the HIV-PIs lopinavir and nelfinavir has a strong synergistic cytotoxic activity against ccRCCin vitro at therapeutically relevant drug concentrations. This effect is most likely explained by synergistic UPR triggering and ABCB1-modulation caused by HIV-PIs. Our findings suggest that combined treatment of second-generation proteasome inhibitors and HIV-PIs should be investigated in patients with metastatic RCC within a clinical trial.
- MeSH
- chemorezistence MeSH
- inhibitory HIV-proteasy terapeutické užití MeSH
- inhibitory proteasomu terapeutické užití MeSH
- karcinom z renálních buněk farmakoterapie MeSH
- lidé MeSH
- Lopinavir terapeutické užití MeSH
- nádorové buněčné linie MeSH
- nádory ledvin farmakoterapie MeSH
- nelfinavir terapeutické užití MeSH
- protinádorové látky terapeutické užití MeSH
- stres endoplazmatického retikula účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Glioblastoma stem-like cells (GSCs) are critical for the aggressiveness and progression of glioblastoma (GBM) and contribute to its resistance to adjuvant treatment. MicroRNAs (miRNAs) are small, non-coding RNAs controlling gene expression at the post-transcriptional level, which are known to be important regulators of the stem-like features. Moreover, miRNAs have been previously proved to be promising diagnostic biomarkers in several cancers including GBM. Using global expression analysis of miRNAs in 10 paired in-vitro as well as in-vivo characterized primary GSC and non-stem glioblastoma cultures, we identified a miRNA signature associated with the stem-like phenotype in GBM. 51 most deregulated miRNAs classified the cell cultures into GSC and non-stem cell clusters and identified a subgroup of GSC cultures with more pronounced stem-cell characteristics. The importance of the identified miRNA signature was further supported by demonstrating that a Risk Score based on the expression of seven miRNAs overexpressed in GSC predicted overall survival in GBM patients in the TCGA dataset independently of the IDH1 status. In summary, we identified miRNAs differentially expressed in GSCs and described their association with GBM patient survival. We propose that these miRNAs participate on GSC features and could represent helpful prognostic markers and potential therapeutic targets in GBM.
- MeSH
- analýza přežití MeSH
- glioblastom genetika MeSH
- isocitrátdehydrogenasa genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikro RNA genetika MeSH
- mutace MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádorové buňky kultivované MeSH
- nádorové kmenové buňky chemie MeSH
- nádory mozku genetika MeSH
- nestin MeSH
- regulace genové exprese u nádorů MeSH
- senioři MeSH
- stanovení celkové genové exprese metody MeSH
- transkripční faktory SOXB1 genetika MeSH
- transplantace nádorů MeSH
- zvířata MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Glioblastoma multiforme (GBM) is the most aggressive intracranial tumor characterized with infaust prognosis. Despite advances in neurosurgical and radiotherapeutic techniques and chemotherapy, the median overall survival ranges between 12-15 months from diagnosis. The main cause of treatment failure is considered the presence of tumor cells resistant to conventional therapy, mainly radiotherapy. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and have been repeatedly proven to play important roles in pathogenesis and biological features of many cancers, including GBM and its radioresistant phenotype. In our study, we established radioresistant cells from the commonly used human GBM cell lines T98G, U87MG and U251. Consequently, we performed global miRNA expression profiling in both radioresistant and parental cell lines and identified 113 miRNAs with significantly different expression (p<0.05) between these two groups (73 miRNAs were up-regulated, 40 miRNAs were down-regulated). Some of these miRNAs have been previously described in relation to ionizing radiation, and others were herein identified for the first time. We believe that after deeper functional investigation of identified miRNAs in relation to radioresistance, these miRNAs present potential predictive biomarkers or therapeutic targets in GBM.
- MeSH
- apoptóza MeSH
- fenotyp MeSH
- glioblastom genetika patologie MeSH
- lidé MeSH
- mikro RNA genetika MeSH
- nádorové biomarkery metabolismus MeSH
- nádorové buněčné linie MeSH
- nádory mozku genetika patologie MeSH
- prognóza MeSH
- proliferace buněk MeSH
- regulace genové exprese u nádorů * MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- stanovení celkové genové exprese MeSH
- tolerance záření genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with an extremely low median survival rate of 12-15 months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to the treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain-specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and miR-338-5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased cell proliferation, increased cell cycle arrest, and apoptosis in comparison to irradiation-only cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, and ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation, and cell cycle regulation. To our knowledge, this is the first study to describe the role of miR-338-5p in GBM and its potential to improve the sensitivity of GBM to radiation.
- MeSH
- buněčné dělení účinky léků účinky záření MeSH
- glioblastom genetika patologie MeSH
- kontrolní body buněčného cyklu účinky léků účinky záření MeSH
- lidé středního věku MeSH
- lidé MeSH
- membránové proteiny biosyntéza genetika MeSH
- mikro RNA genetika MeSH
- monomerní proteiny vázající GTP biosyntéza genetika MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny biosyntéza genetika MeSH
- nádory mozku genetika patologie MeSH
- neuropeptidy biosyntéza genetika MeSH
- poškození DNA genetika MeSH
- proteinfosfatasa 2 biosyntéza genetika MeSH
- regulace genové exprese u nádorů genetika MeSH
- RNA nádorová genetika MeSH
- stanovení celkové genové exprese MeSH
- tolerance záření genetika MeSH
- transportní proteiny biosyntéza genetika MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
