Evaluation of Decay Kinetics of Black Elderberry Antioxidants from Fruits and Flowers.

The health-promoting properties of black elderberry are related to its high content of polyphenols (natural antioxidants), which eliminate free radicals and prevent the formation of oxidative stress responsible for many diseases. The aim of this work was to determine, the anti-radical effect of Sambucus nigra infusions based on the reaction with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and galvinoxyl (Glv) radicals and to determine the function describing the disappearance curves of these radicals. The antioxidant properties of infusions obtained from the flowers and fruits of this plant were tested using the modified Brand-Williams method using DPPH and Glv radicals. Higher antioxidant activity towards both the DPPH and Glv radicals was found in flowers compared to fruits. In addition, it was found that the process of quenching radicals in the reaction with Sambucus nigra infusions proceeds in accordance with the assumptions of second-order reaction kinetics. The infusion obtained from flowers quenched radicals faster than fruit infusions. The applied second-order kinetics equation may enable estimation of antioxidants levels in natural sources of radicals.

DNA-damaging imidazoacridinone C-1311 induces autophagy followed by irreversible growth arrest and senescence in human lung cancer cells.

Imidazoacridinone 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor inhibitor of topoisomerase II and FMS-like tyrosine kinase 3 receptor. In this study, we describe the unique sequence of cellular responses to C-1311 in human non-small cell lung cancer (NSCLC) cell lines, A549 and H460. In A549 cells, C-1311 (IC80 = 0.08 µM) induced G1 and G2/M arrests, whereas H460 cells (IC80 = 0.051 µM) accumulated predominantly in the G1 phase. In both cell lines, cell cycle arrest was initiated by overexpression of p53 but was sustained for an extended time by elevated levels of p21. Despite prolonged drug exposure (up to 192 hours), no apoptotic response was detected in either cell line. Instead, cells developed a senescent phenotype and did not resume proliferation even after 2 weeks of post-treatment, indicating that C-1311-triggered senescence was permanent. When cell cycle arrest was evident but there were no signs of senescence, C-1311 significantly induced autophagic cells. Pharmacological inhibition of autophagy by 3-methyladenine profoundly reduced the senescent phenotype and slightly sensitized cancer cells to C-1311 by increasing cell death, suggesting a link between both autophagy and senescence. However, a small interfering RNA-mediated knockdown of the autophagy-associated Beclin 1 and ATG5 genes attenuated but failed to block development of senescence. Taken together, our studies suggest that in NSCLC, a C-1311-induced senescence program is preceded and corroborated but not exclusively determined by the induction of autophagy.

Modulation of CYP3A4 activity and induction of apoptosis, necrosis and senescence by the anti-tumour imidazoacridinone C-1311 in human hepatoma cells.

There is increasing evidence that the expression level of drug metabolic enzymes affects the final cellular response following drug treatment. Moreover, anti-tumour agents may modulate enzymatic activity and/or cellular expression of metabolic enzymes in tumour cells. We have investigated the influence of CYP3A4 overexpression on the cellular response induced by the anti-tumour agent C-1311 in hepatoma cells. C-1311-mediated CYP3A4 activity modulation and the effect of CYP3A4 overexpression on C-1311 metabolism have also been examined. With the HepG2 cell line and its CYP3A4-overexpressing variant, Hep3A4, experiments involving DAPI staining, cell cycle analysis, phosphatidylserine externalisation and senescence-associated (SA)-ß-galactosidase expression, were used to monitor the effects of C-1311 exposure. C-1311 cellular metabolism and CYP3A4 activity were investigated by high-performance liquid chromatography. C-1311 metabolism was very low in both hepatoma cell lines and slightly influenced by CYP3A4 expression. Interestingly, in HepG2 cells, C-1311 was an effective modulator of CYP3A4 enzymatic activity, being the inhibitor of this isoenzyme in Hep3A4 cells. Cell cycle analysis showed that HepG2 cells underwent a rather stable G(2) /M arrest following C-1311 exposure, whereas CYP3A4-overexpressing cells accumulated only slightly in this compartment. C-1311-treated cells died by apoptosis and necrosis, whereas surviving cells underwent senescence; however, these effects occurred faster and more intensely in Hep3A4 cells. Although CYP3A4 did not influence C-1311 metabolism, changes in CYP3A4 levels affected the C-1311-induced response in hepatoma cells. Therefore, inter-patient differences in CYP3A4 levels should be considered when assessing the potential therapeutic effects of C-1311.

[Autophagy--molecular mechanism, apoptosis and cancer]. / Autofagia--mechanizm molekularny, apoptoza i nowotwory.

Autophagy is a catabolic process involving the degradation of long-lived proteins and organelles through the lysosomal machinery. In eukaryotic cells, among the three types of autophagy the most extensively studied is macroautophagy. Macroautophagy (hereafter referred to as autophagy) is characterized by sequestration of bulk cytoplasm in double-membrane vesicles, called autophagosomes, which ultimately fuse with lysosomes, resulting in degradation of their contents. Autophagy is responsible for the maintenance of intracellular homeostasis and enables cell survival under stress conditions. However, this process is also involved in the pathogenesis of diverse diseases, including cancers. In the cancer cell, autophagy plays a dual role, as a mechanism responsible for protecting or killing the cell. In most cases chemotherapy-induced autophagy in tumor cells is a prosurvival response which potentially leads to development of drug resistance. However, autophagy can also lead to cell death, thus enhancing treatment efficacy. It is important for the anticancer therapy to find the type of cancer cells which are susceptible to autophagy and to determine whether the autophagy induced by the applied therapy leads to cells' death or their survival and subsequently to therapy resistance. In this review, the molecular mechanism of macroautophagy and the most important signaling transduction pathways involved in regulation of this process in cancer cells are presented. The dual function of autophagy in tumorigenesis and the implications of autophagy modulation for cancer therapy are also discussed.

Targeting of FLT3-ITD kinase contributes to high selectivity of imidazoacridinone C-1311 against FLT3-activated leukemia cells.

Drugs targeting receptor tyrosine kinase FLT3 are of particular interest since activating FLT3-internal tandem duplication (ITD) mutations abundantly occur in fatal acute myeloid leukemias (AMLs). Imidazoacridinone C-1311, a DNA-reactive inhibitor of topoisomerase II, has been previously shown to be a potent and selective inhibitor of recombinant FLT3. Here, we expand those findings by studying its effect on leukemia cells with wild-type FLT3, FLT3-ITD mutant and no FLT3 receptor. While brief C-1311 exposure blocked wild-type and FLT3-ITD activity, profound and sustained inhibition was achieved only for FLT3-ITD mutants. C-1311 inhibited FLT3 downstream pathways (MAPK and AKT) independent of FLT3 status, yet translation to decreased viability was significant in FLT3-ITD cells. RNA interference against FLT3-ITD reduced cytotoxic effect and apoptosis induced by C-1311, indicating selective inhibition of FLT3-ITD crucial for high efficacy of drug against activated leukemia cells. Cellular responses in treated FLT3-ITD mutants included G1 and G2/M phase arrest, moderate inhibition of Bcl-2, caspase-3 activation, PARP cleavage, and depolarization of mitochondria. Consistent with selective decrease in FLT3-ITD activity, C-1311 remarkably reduced antiapoptotic survivin mRNA and protein expression, correlating well with enhanced apoptosis of FLT3-ITD cells. No survivin decrease and respectively lower level of apoptosis was found in wild-type and null-FLT3 cells. Combination of C-1311 with cytarabine or doxorubicin again showed distinct synergistic activity in FLT3-ITD-positive cells. The ability of C-1311 to selectively target constitutively active FLT3, suggests a favorable therapeutic index for AML carrying FLT3-ITD mutations. Thus further preclinical and clinical studies addressing its potency against FLT3-ITD kinase is well justified.