The aim of the present study was to determine the structural requirements for dibenzocyclooctadiene lignans essential for P-glycoprotein inhibition. Altogether 15 structurally related lignans isolated from Schisandra chinensis or prepared by modification of their backbone were investigated, including three pairs of enantiomers. P-Glycoprotein inhibition was quantified using a doxorubicin accumulation assay in human promyelotic leukemia HL60/MDR cells overexpressing P-glycoprotein. A preliminary quantitative structure-activity relationship analysis revealed three main structural features involved in P-glycoprotein inhibition: a 1,2,3-trimethoxy moiety, a 6-acyloxy group, and the absence of a 7-hydroxy group. The most effective inhibitors, (-)-gomisin N (1) and (+)-deoxyschizandrin [(+)-2], were selected for further evaluation of their effects. Both these lignans restored the cytotoxic effect of doxorubicin in HL60/MDR cells and when combined with a subtoxic concentration of this compound increased the proportion of G2/M cells significantly, which is a usual response to treatment with this anticancer drug.
- MeSH
- cyklooktany * chemie izolace a purifikace farmakologie MeSH
- doxorubicin farmakologie MeSH
- kontrolní body fáze G2 buněčného cyklu účinky léků MeSH
- kvantitativní vztahy mezi strukturou a aktivitou MeSH
- lidé MeSH
- lignany * chemie izolace a purifikace farmakologie MeSH
- molekulární struktura MeSH
- P-glykoproteiny antagonisté a inhibitory MeSH
- polycyklické sloučeniny * chemie izolace a purifikace farmakologie MeSH
- Schisandra chemie MeSH
- semena rostlinná chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Rusko MeSH
The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the antiproliferative effect of interferon gamma in relation to SOCS3 expression in a panel of breast cancer cell lines and normal mammary epithelial cells. Compared to normal cells most breast cancer lines (7/8) were highly resistant to IFN-gamma. Using Northern blot and real time RT-PCR we investigated transcription of SOCS3 genes. All normal epithelial cells (4/4) showed SOCS3 induction (2-14 fold) while most breast cancer lines did not or weakly activated SOCS3 after the interferon gamma treatment. Among the cancer lines, the MDA-MB-468 cells showed increased sensitivity to IFN-gamma and relatively high level of SOCS3 induction (2-3 fold). Together, there was a good correlation
- MeSH
- chemorezistence MeSH
- fosforylace MeSH
- interferon gama farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory prsu farmakoterapie metabolismus patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteiny SOCS genetika MeSH
- prsy metabolismus účinky léků MeSH
- transkripční faktor STAT1 metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Interferon-alpha (IFN-alpha) is an important drug used in anti-melanoma therapy. However, metastases eventually reappear in almost 60% of melanoma patients, who have received adjuvant cytokine therapy suggesting that IFN-alpha can paradoxically promote disease progression in some cases, at least. In this study, we have investigated the possibility that a growth-promoting STAT3 protein might be activated by interferon-alpha in melanoma cells. We examined 24 primary cultures established from node metastases of melanoma patients who were monitored in a 5-year clinical follow-up. The patients differed in the course of disease and survival end-points. Using Western blot analyses, we show that interferon-alpha stimulated STAT3 phosphorylation at tyrosine (Y705) residue in 17% of cases. These over-reactive cell populations originated from patients who had the shortest disease-free intervals. A significant correlation was obtained between the length of survival end-points and a lack of STAT3 activation by IFN-alpha. No STAT3 induction was observed in normal melanocytes. The STAT1 activation at tyrosine (Y701) occurred at a similar frequency as that of STAT3 (17%) albeit in different patients, no clear correlation with the clinical status could be made. The interferon-alpha/beta receptors (IRFARs) were expressed irrespective to the signal transducers and activators of transcription (STATs) inducibility suggesting that signalling defects occur downstream from IRFAR. We propose that in some cases the application of IFN-alpha could increase the probability of disease progression via overactive STAT3. The tests for STAT3 inducibility prior to cytokine immunotherapy in the clinic are therefore warranted.
- MeSH
- dospělí MeSH
- financování organizované MeSH
- fosforylace MeSH
- imunohistochemie MeSH
- imunologické faktory škodlivé účinky MeSH
- interferon alfa škodlivé účinky MeSH
- Kaplanův-Meierův odhad MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfatické metastázy MeSH
- melanom farmakoterapie metabolismus MeSH
- messenger RNA analýza MeSH
- nádorové buňky kultivované MeSH
- nádory kůže farmakoterapie metabolismus MeSH
- přežití bez známek nemoci MeSH
- progrese nemoci MeSH
- proliferace buněk účinky léků MeSH
- receptor interferonu alfa-beta genetika MeSH
- senioři MeSH
- transkripční faktor STAT3 analýza metabolismus MeSH
- upregulace MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- western blotting metody MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
Přeruš. str. : il., tab. ; 30 cm
Studie hodlá analyzovat molekulární a funkční abnormality v signálních proteinech STAT/SOCS a v expresi jejich cílových genů, které jsou specifické pro nádorová onemocnění, a mohly by představovat nové nádorové markery a být nápomocné při imunoterapii. Výsledky by mohly rozšířit naše znalosti o tom, jak výše zmíněné systémy fungují a interagují v přenosu signálu interferonů a jaké jsou důsledky defektní aktivity v signálních mediátorech. Western a Northern hybridizace, technika tkáňových kultur a DNA makroarray expresní profily zastupují dominantní metody.; The study intends to analyze molecular and functional cancer-specific abnormalities in STAT/SOCS signaling proteins and in the expression of their target genes which might represent new tumor markers and be of help in immunotherapeutic strategies. The results are also expected to extend our understanding how the above signaling systems operate and interact in interferon-induced signaling and what are the consequences of defective activity within signaling mediators. Western and Northern hybridization, tissue cultures and DNA macroarray expression profiling exemplify the dominant methods.
- MeSH
- DNA sondy MeSH
- exprese genu MeSH
- interferony MeSH
- messenger RNA analýza MeSH
- nádory diagnóza MeSH
- proteiny SOCS analýza diagnostické užití MeSH
- sondy nukleových kyselin MeSH
- transkripční faktor STAT1 analýza diagnostické užití MeSH
- transkripční faktor STAT3 analýza diagnostické užití MeSH
- transkripční faktor STAT5 analýza diagnostické užití MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- onkologie
- biologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
Signal transducer and activator of transcription 3 (STAT3) protein has been documented as a significant mediator of interferon (IFN) signaling. Physiological STAT3 phosphorylation involves tyrosine (Y705) and serine (S727) activation. Impairment of STAT3 protein levels and/or of STAT3 phosphorylation after IFN treatment has been found in many pathological conditions such as cancer, immunopathy and inflammatory disease. To analyze tumor-associated defective STAT3 response to IFNs, the induction of S727 and Y705 STAT3 activation after IFN exposure was evaluated in 18 human malignant melanoma cell lines and 68 primary cell cultures established from the lymph node metastases of melanoma patients. STAT3 expression and STAT3 phosphorylated forms were assayed by Western blot analysis employing specific STAT3 antibodies. All melanoma cell lines as well as samples derived from metastatic melanoma patients expressed STAT3 with variable signal intensities depending on the appropriate cell type. Significantly altered IFNγ-induced S727 STAT3 activation was found in both experimental models, with on average 94.1% of patients detected to be non-responders in lymph node cell cultures and 83.3% in melanoma cell lines. Moreover, a deficiency in IFNα-induced S727 induction was detected in 88.9% of melanoma cell lines. Defects in Y705 STAT3 phosphorylation were determined in clinical material (61.8% after IFNγ exposure) as well as in melanoma cell lines (absence of response to IFNα/γ in 83.3 and 55.5%, respectively). Our data clearly confirm STAT3 pathophysiological perturbances in human malignant melanoma cells. Depending on the induction of STAT3-activated phosphoforms by IFNs, three categories of melanoma cells were identified: a) phosphorylation on both the S727 and Y705 amino acid residues; b) STAT3 activation on Y705 only; c) phosphorylation at neither S727 nor Y705. The significance of in vitro STAT3 activation for predicting patient response to immunotherapy will be examined in a prospective clinical study by our group.
The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the evolution of an IFN-resistant state in vitro using melanoma cell lines. We found that the cells became less sensitive to antiproliferative effect of IFN-gamma after prolonged cultivation enabling us to isolate sensitive and resistant subclones of the parental line. We investigated transcription of signal transducer and activator of transcription (STAT) 1-6 and suppressor of cytokine signalling (SOCS) 1-3 genes, and phosphorylation of STAT 1 protein. The resistant subline (termed WM 1158R) differed from the sensitive subline (WM 1158S) by a constitutive expression of SOCS 3, lack or weak SOCS 1-3 activation following IFN-gamma, and short duration of cytokine activatory signal. Similar correlations were observed in additional melanoma lines differing in IFN sensitivities. At the protein level, IFN-gamma induced strong and prolonged STAT 1 activation at serine 727 (S727) in WM 1158R while in WM 1158S cells phosphorylation of this amino acid was much less pronounced. On the other hand, phosphorylation of tyrosine 701 (Y701) was stimulated regardless of the sensitivity phenotype. In conclusion, constitutive expression of SOCS 3 is correlated with attenuation of its induction following IFN treatment. These results suggest that progression of melanoma cells from IFN sensitivity to IFN insensitivity associates with changes in SOCS expression.
- MeSH
- chemorezistence genetika MeSH
- exprese genu MeSH
- financování organizované MeSH
- fosforylace MeSH
- interferon gama farmakologie terapeutické užití MeSH
- lidé MeSH
- melanom farmakoterapie genetika MeSH
- nádorové buněčné linie MeSH
- nádory kůže farmakoterapie genetika MeSH
- proliferace buněk účinky léků MeSH
- proteiny SOCS genetika MeSH
- regulace genové exprese u nádorů MeSH
- stanovení celkové genové exprese MeSH
- transkripční faktor STAT1 metabolismus MeSH
- Check Tag
- lidé MeSH
