Low curability of patients diagnosed with acute myeloid leukemia (AML) must be seen as a call for better understanding the disease's mechanisms and improving the treatment strategy. Therapeutic outcome of the crucial anthracycline-based induction therapy often can be compromised by a resistant phenotype associated with overexpression of ABCB1 transporters. Here, we evaluated clinical relevance of ABCB1 in a context of the FMS-like tyrosine kinase 3 (FLT3) inhibitor midostaurin in a set of 28 primary AML samples. ABCB1 gene expression was absolutely quantified, confirming its association with CD34 positivity, adverse cytogenetic risk, and unachieved complete remission (CR). Midostaurin, identified as an ABCB1 inhibitor, increased anthracycline accumulation in peripheral blood mononuclear cells (PBMC) of CD34+ AML patients and those not achieving CR. This effect was independent of FLT3 mutation, indicating even FLT3- AML patients might benefit from midostaurin therapy. In line with these data, midostaurin potentiated proapoptotic processes in ABCB1-overexpressing leukemic cells when combined with anthracyclines. Furthermore, we report a direct linkage of miR-9 to ABCB1 efflux activity in the PBMC and propose miR-9 as a useful prognostic marker in AML. Overall, we highlight the therapeutic value of midostaurin as more than just a FLT3 inhibitor, suggesting its maximal therapeutic outcomes might be very sensitive to proper timing and well-optimized dosage schemes based upon patient's characteristics, such as CD34 positivity and ABCB1 activity. Moreover, we suggest miR-9 as a predictive ABCB1-related biomarker that could be immensely helpful in identifying ABCB1-resistant AML phenotype to enable optimized therapeutic regimen and improved treatment outcome.
- MeSH
- akutní myeloidní leukemie * farmakoterapie genetika metabolismus MeSH
- antracykliny farmakologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- leukocyty mononukleární účinky léků metabolismus MeSH
- lidé MeSH
- mikro RNA * genetika metabolismus MeSH
- mutace MeSH
- P-glykoproteiny * genetika metabolismus MeSH
- staurosporin * analogy a deriváty farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Midostaurin is an FMS-like tyrosine kinase 3 receptor (FLT3) inhibitor that provides renewed hope for treating acute myeloid leukaemia (AML). The limited efficacy of this compound as a monotherapy contrasts with that of its synergistic combination with standard cytarabine and daunorubicin (Dau), suggesting a therapeutic benefit that is not driven only by FLT3 inhibition. In an AML context, the activity of the enzyme aldo-keto reductase 1C3 (AKR1C3) is a crucial factor in chemotherapy resistance, as it mediates the intracellular transformation of anthracyclines to less active hydroxy metabolites. Here, we report that midostaurin is a potent inhibitor of Dau inactivation mediated by AKR1C3 in both its recombinant form as well as during its overexpression in a transfected cell model. Likewise, in the FLT3- AML cell line KG1a, midostaurin was able to increase the cellular accumulation of Dau and significantly decrease its metabolism by AKR1C3 simultaneously. The combination of those mechanisms increased the nuclear localization of Dau, thus synergizing its cytotoxic effects on KG1a cells. Our results provide new in vitro evidence of how the therapeutic activity of midostaurin could operate beyond targeting the FLT3 receptor.
- MeSH
- akutní myeloidní leukemie farmakoterapie enzymologie genetika patologie MeSH
- biotransformace MeSH
- daunomycin metabolismus farmakologie MeSH
- HCT116 buňky MeSH
- inhibitory enzymů farmakologie MeSH
- kolorektální nádory farmakoterapie enzymologie genetika patologie MeSH
- lidé MeSH
- protein AKR1C3 antagonisté a inhibitory genetika metabolismus MeSH
- protokoly antitumorózní kombinované chemoterapie farmakologie MeSH
- staurosporin analogy a deriváty farmakologie MeSH
- synergismus léků MeSH
- tyrosinkinasa 3 podobná fms antagonisté a inhibitory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza účinky léků MeSH
- bisbenzimidazol chemie MeSH
- buněčná smrt účinky léků MeSH
- buněčné jádro účinky léků metabolismus MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- cisplatina farmakologie MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie metody MeSH
- fragmentace DNA účinky léků MeSH
- kamptothecin farmakologie MeSH
- lidé MeSH
- průtoková cytometrie metody MeSH
- reprodukovatelnost výsledků MeSH
- staurosporin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ač je akutní myeloidní leukemie onemocnění s rostoucí incidencí, v posledních desetiletích nedošlo v její léčbě k radikálnímu posunu k lepšímu a prognóza pacientů se zlepšuje pouze pozvolna. V posledních několika letech se však v terapii akutní myeloidní leukemie objevují léčiva, která reprezentují tzv. cílenou léčbu a mají jistou zásluhu na zlepšení. Článek je zaměřen na přehled základních farmakologických vlastností nových léčiv používaných v terapii akutní myeloidní leukemie, zejména na mechanizmus účinku, nejčastější nežádoucí účinky a jejich management či lékové interakce z pohledu klinického farmaceuta.
Although the incidence of acute myeloid leukaemia is on the rise, there has been no radical improvement in its treatment in recent decades and the prognosis of patients is improving only gradually. Drugs for the treatment of acute myeloid leukaemia that have appeared in the past few years and that represent so-called targeted therapy have been responsible for this improvement. This article focuses on an overview of the basic pharmacological properties of new drugs used in the treatment of acute myeloid leukaemia, especially their mechanism of action, their most common adverse reactions and their management or drug interactions from the perspective of the clinical pharmacist.
- Klíčová slova
- midostaurin, gilteritinib, ivosidenib, enasidenib, venetoklax, glasdegib,
- MeSH
- akutní myeloidní leukemie * farmakoterapie MeSH
- antitumorózní látky farmakologie terapeutické užití MeSH
- gemtuzumab farmakologie terapeutické užití MeSH
- inhibitory proteinkinas farmakologie terapeutické užití MeSH
- lékové interakce MeSH
- lidé MeSH
- nežádoucí účinky léčiv MeSH
- staurosporin analogy a deriváty farmakologie terapeutické užití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
The mechanisms of inflammatory pain need to be identified in order to find new superior treatments. Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) are highly co-expressed in dorsal root ganglion neurons and implicated in pain development. Here, we examined the role of spinal PAR2 in hyperalgesia and the modulation of synaptic transmission in carrageenan-induced peripheral inflammation, using intrathecal (i.t.) treatment in the behavioral experiments and recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs and eEPSCs) in spinal cord slices. Intrathecal PAR2-activating peptide (AP) administration aggravated the carrageenan-induced thermal hyperalgesia, and this was prevented by a TRPV1 antagonist (SB 366791) and staurosporine i.t. pretreatment. Additionally, the frequency of the mEPSC and sEPSC and the amplitude of the eEPSC recorded from the superficial dorsal horn neurons were enhanced after acute PAR2 AP application, while prevented with SB 366791 or staurosporine pretreatment. PAR2 antagonist application reduced the thermal hyperalgesia and decreased the frequency of mEPSC and sEPSC and the amplitude of eEPSC. Our findings highlight the contribution of spinal PAR2 activation to carrageenan-induced hyperalgesia and the importance of dorsal horn PAR2 and TRPV1 receptor interactions in the modulation of nociceptive synaptic transmission.
- MeSH
- anilidy farmakologie MeSH
- buňky zadních rohů míšních účinky léků metabolismus fyziologie MeSH
- cinnamáty farmakologie MeSH
- excitační postsynaptické potenciály MeSH
- hyperalgezie etiologie metabolismus patofyziologie MeSH
- karagenan farmakologie toxicita MeSH
- kationtové kanály TRPV antagonisté a inhibitory metabolismus MeSH
- krysa rodu rattus MeSH
- miniaturní postsynaptické potenciály MeSH
- nocicepce MeSH
- potkani Wistar MeSH
- receptor PAR-2 metabolismus MeSH
- staurosporin farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) receptors in the peripheral nerve endings are implicated in the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states. Both PAR2 and TRPV1 receptors are co-expressed in nociceptive dorsal root ganglion (DRG) neurons on their peripheral endings and also on presynaptic endings in the spinal cord dorsal horn. However, the modulation of nociceptive synaptic transmission in the superficial dorsal horn after activation of PAR2 and their functional coupling with TRPV1 is not clear. To investigate the role of spinal PAR2 activation on nociceptive modulation, intrathecal drug application was used in behavioural experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) were performed on superficial dorsal horn neurons in acute rat spinal cord slices. Intrathecal application of PAR2 activating peptide SLIGKV-NH2 induced thermal hyperalgesia, which was prevented by pretreatment with TRPV1 antagonist SB 366791 and was reduced by protein kinases inhibitor staurosporine. Patch-clamp experiments revealed robust decrease of mEPSC frequency (62.8 ± 4.9%), increase of sEPSC frequency (127.0 ± 5.9%) and eEPSC amplitude (126.9 ± 12.0%) in dorsal horn neurons after acute SLIGKV-NH2 application. All these EPSC changes, induced by PAR2 activation, were prevented by SB 366791 and staurosporine pretreatment. Our results demonstrate an important role of spinal PAR2 receptors in modulation of nociceptive transmission in the spinal cord dorsal horn at least partially mediated by activation of presynaptic TRPV1 receptors. The functional coupling between the PAR2 and TRPV1 receptors on the central branches of DRG neurons may be important especially during different pathological states when it may enhance pain perception.
- MeSH
- alergie metabolismus patologie MeSH
- anilidy farmakologie MeSH
- buňky zadních rohů míšních účinky léků fyziologie MeSH
- chování zvířat účinky léků MeSH
- cinnamáty farmakologie MeSH
- excitační postsynaptické potenciály účinky léků MeSH
- hyperalgezie etiologie prevence a kontrola MeSH
- inhibitory proteinkinas farmakologie MeSH
- kationtové kanály TRPV antagonisté a inhibitory metabolismus MeSH
- krysa rodu rattus MeSH
- metoda terčíkového zámku MeSH
- mícha metabolismus MeSH
- nervový přenos fyziologie MeSH
- oligopeptidy farmakologie MeSH
- potkani Wistar MeSH
- receptor PAR-2 agonisté metabolismus MeSH
- staurosporin farmakologie MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.
- MeSH
- ATM protein MeSH
- checkpoint kinasa 2 MeSH
- chromozomální proteiny, nehistonové metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- fosforylace MeSH
- histony metabolismus MeSH
- inhibitory proteinkinas farmakologie MeSH
- jednovláknová DNA metabolismus MeSH
- kofein farmakologie MeSH
- lidé MeSH
- malá interferující RNA farmakologie genetika MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- poškození DNA * fyziologie MeSH
- protein-serin-threoninkinasy fyziologie účinky léků MeSH
- proteinkinasy farmakologie fyziologie genetika MeSH
- proteiny buněčného cyklu fyziologie genetika metabolismus MeSH
- puriny farmakologie MeSH
- replikace DNA fyziologie účinky léků MeSH
- replikační protein A MeSH
- staurosporin * analogy a deriváty farmakologie MeSH
- Check Tag
- lidé MeSH
- MeSH
- apoptóza MeSH
- benzamidy farmakologie MeSH
- chemorezistence MeSH
- fixní kombinace léků farmakologie MeSH
- HL-60 buňky fyziologie účinky léků MeSH
- lidé MeSH
- staurosporin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
- techniky in vitro MeSH
