Identification of angular naphthopyrones from the Philippine echinoderm Comanthus species as inhibitors of the NF-κB signaling pathway

In the past few years heat shock protein 90 (Hsp90) inhibitors have been reported to possess significant antitumor activity. We investigated, for the first time, the antitumor activity of a novel Hsp90 inhibitor 2-(4-acetyloxycyclohexylamino)-4-(3, 6, 6-trimethyl-4-oxo-4, 5, 6, 7-tetrahydro-1H-indazol-1-yl)-benzamide (BJ-B11) and the molecular mechanism underlying the apoptosis it induces in human chronic myeloid leukemia K562 cells. The results revealed that BJ-B11 triggered growth inhibition in K562 cells and other malignant cell lines in vitro with only minor toxicity in a normal human cell line. BJ-B11 inhibited the proliferation of K562 cells in a concentration- and time-dependent manner, with IC(50) values of 1.1 ± 0.2 μM and 0.4 ± 0.1 μM after 48 and 72 h incubations respectively. This most likely results from cell cycle arrest at the G(0)/G(1) phase and the induction of apoptosis. In addition, BJ-B11 degraded the Hsp90 client proteins Bcr-Abl and Akt, induced activation of caspase-9 and caspase-3, and subsequent cleavage of PARP. The caspase signals may originate from mitochondrial dysfunction, which is supported by the finding of cytochrome c release. In addition, inactivation of the Akt signaling pathway may be involved in the process of BJ-B11-induced apoptosis. Taken together, our data provide a putative molecular mechanism for the anticancer effect of BJ-B11 on K562 cells, and suggest a potential application for BJ-B11 in chronic myeloid leukemia therapy.     

Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis

Dietary flavonoids possess a wide spectrum of biochemical and pharmacological actions and are assumed to protect human health. These actions, however, can be antagonistic, and some health claims are mutually exclusive. The antiapoptotic actions of flavonoids may protect against neurodegenerative diseases, whereas their proapoptotic actions could be used for cancer chemotherapy. This study was undertaken to determine whether a cytoprotective dose range of flavonoids could be differentiated from a cytotoxic dose range. Seven structurally related flavonoids were tested for their ability to protect H4IIE rat hepatoma cells against H(2)O(2)-induced damage on the one hand and to induce cellular damage on their own on the other hand. All flavonoids proved to be good antioxidants in a cell-free assay. However, their pharmacologic activity did not correlate with in vitro antioxidant potential but rather with cellular uptake. For quercetin and fisetin, which were readily taken up into the cells, protective effects against H(2)O(2)-induced cytotoxicity, DNA strand breaks, and apoptosis were detected at concentrations as low as 10-25 micromol/L. On the other hand, these flavonoids induced cytotoxicity, DNA strand breaks, oligonucleosomal DNA fragmentation, and caspase activation at concentrations between 50 and 250 micromol/L. Published data on quercetin pharmacokinetics in humans suggest that a dietary supplement of 1-2 g of quercetin may result in plasma concentrations between 10 and 50 micromol/L. Our data suggest that cytoprotective concentrations of some flavonoids are lower by a factor of 5-10 than their DNA-damaging and proapoptotic concentrations.     

The mycotoxin beauvericin induces apoptotic cell death in H4IIE hepatoma cells accompanied by an inhibition of NF-κB-activity and modulation of MAP-kinases

Beauvericin is a world-spread mycotoxin with a high toxicity in mammalian cells. However, its molecular mechanism of action is not fully understood. Using different cancer cell lines (HepG2, C6, Hct116 and H4IIE), we could show that the cyclic peptide is highly toxic (MTT assay) with IC₅₀ values in low micromolar range. As a molecular mechanism of cell death, necrosis was detected in C6 glioma cells (PI staining), but apoptosis prevails in H4IIE hepatoma cells (caspase 3/7 activity, nuclear fragmentation). In H4IIE cells, beauvericin rapidly decreases the phosphorylation of ERK and strongly increases JNK phosphorylation, while p38 phosphorylation was not affected. Furthermore, a strong inhibition of NF-κB signalling was detectable in H4IIE cells. A screening of 21 protein kinases involved in signal transduction pathways (cell proliferation, survival, angiogenesis and metastasis) showed a selective inhibition of src kinase by beauvericin (IC₅₀ = 9.8 μg/ml). We suggest that beauvericin mediates its toxic effects in H4IIE cells, at least in parts, by a distinct modulation of intracellular signalling molecules.     

Dose- and time-dependent effects of doxorubicin on cytotoxicity,cell cycle and apoptotic cell death in human colon cancer cells

The cytostatic drug doxorubicin is a well-known chemotherapeutic agent which is used in treatment of a wide variety of cancers. A key factor in the response of cancer cells to chemotherapeutic drugs is the activation of the apoptotic pathway, a pathway that is often impaired in chemoresistant colon cancer cells. The aim of the present study was to investigate the effects of doxorubicin in Hct-116 human colon carcinoma cells in order to clarify if a time/concentration range for optimal doxorubicin-induced apoptosis exists. We compared a treatment schedule were cells were bolus incubated for 3 h with doxorubicin followed by 24 h in drug-free medium, with a continuous doxorubicin treatment schedule for 24 h. Bolus incubation was carried out to determine effects of doxorubicin accumulated during the first 3 h, whereas continuous incubation allowed further (continuous) exposure to doxorubicin. We found that bolus (3 h) treatment with doxorubicin resulted in a dose-dependent decrease of viable cells and concomitant increase of apoptosis. Additionally, bolus (3 h) doxorubicin incubation led to phosphorylation of p53 at serine 392, induction of p21, G2 arrest and increase of proapoptotic protein Bax. In contrast, continuous (24 h) treatment with doxorubicin reduced the number of living cells with no parallel raise in the amount of dead cells. Continuous (24 h) treatment with 5 μM doxorubicin resulted in cell cycle arrest in G0/G1 phase that was neither accompanied by phosphorylation and activation of p53 nor enhanced expression of p21. These results suggest that doxorubicin is able to induce cell death by apoptosis only at particular dose and treatment conditions and imply a completely different cellular response following bolus or continuous exposure to doxorubicin.     

Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells

Polyphenols are ubiquitous substances in the diet. Their anti-oxidative, anti-inflammatory and anti-viral effects are of interest for human health, and polyphenols such as luteolin are used at high concentrations in food supplements. The aim of this project was to determine the intrinsic effects of luteolin in H4IIE rat hepatoma cells. Luteolin is relatively toxic, cell death was caused via induction of apoptosis as detected by DNA-ladder formation, by nuclear fragmentation and activation of apoptotic enzymes (caspase-2, -3/7, -9 and -8/10). Luteolin (250 microM, 24 h) increased the caspase-3/7 activity four-fold and the caspase-9 activity six-fold. In a time course experiment caspase-9 is activated after 6h, while caspase-2 and -3/7 are activated after 12 h. After 24 h, caspase-8/10 also displays activation. We found a concentration-dependent increase in malondialdehyde release suggesting a prooxidative effect of luteolin. Furthermore, we analysed DNA strand break formation by luteolin and found a distinct increase of DNA strand breaks after incubation for 3h with 100 microM luteolin, a concentration which induces oligonucleosomal DNA cleavage at 24h. In conclusion, the sequence of events is compatible with the assumption that luteolin triggers the mitochondrial pathway of apoptosis, probably by inducing DNA damage.