Pifithrin-alpha has a p53-independent cytoprotective effect on docosahexaenoic acid-induced cytotoxicity in human hepatocellular carcinoma HepG2 cells.

Pifithrin-alpha (PFT) is an inhibitor of p53 and is known to protect against a variety of p53-mediated genotoxic agents. In this report, we examined the inhibitory effects of PFT against docosahexaenoic acid (DHA)-induced cytotoxicity in the human hepatocellular carcinoma (HCC) cell line HepG2. PFT significantly abrogated DHA-induced cytotoxicity in wild-type HepG2 cells (normal expression of p53) and after p53-knockdown by siRNA, as well as in Hep3B (p53 null) and Huh7 (p53 mutant) cells. DHA-induced cytotoxicity is mediated by induction of oxidative stress, and PFT inhibited this event, but it does not exert antioxidant effects. PFT significantly suppressed the release of cytochrome c from mitochondria to cytosol, as well as changes in the mitochondrial membrane potential (ΔΨM) by DHA. Therefore, protection of mitochondria by PFT is crucial for its inhibition of DHA-induced cytotoxicity. Although it has been reported that PFT is able to block p53 function, our data suggest that PFT also has a p53-independent inhibition mechanism. This work provided insights into the mechanisms of PFT action on DHA-induced cytotoxicity in HCC.

Albumin modulates docosahexaenoic acid-induced cytotoxicity in human hepatocellular carcinoma cell lines.

Fish oil-containing diets rich in cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) provide protection against tumorigenesis. The mechanisms of the cytotoxic effects of DHA include the production of reactive oxygen species (ROS). Albumin has antioxidant property and binds fatty acids, it may protect the cells against the DHA-induced cytotoxicity. In this study, we compared the susceptibility of three human hepatocellular carcinoma (HCC) cell lines (HepG2, Hep3B, Huh7) to the cytotoxic effects of DHA, and examined the changes in the susceptibility following albumin overexpression using transfection vectors or albumin downregulation using small interfering RNA (siRNA). HepG2 cells were the most susceptible to DHA-induced cytotoxicity and increased oxidative activities by DHA compared to Hep3B and Huh7 cells. The cytotoxic effects of DHA were concentration-dependently abrogated by typical antioxidants, a radical scavenger, an iron chelator and incubation with exogenous albumin. Overexpression of albumin in HepG2 cells markedly attenuated DHA-induced oxidative activities and cytotoxicity. Furthermore, knockdown of albumin in both Hep3B and Huh7 cells significantly enhanced the effects of DHA. The results of our in vitro experiments indicate that the cytotoxic effects of DHA on HCC cell lines are modulated by albumin.

Costunolide-induced apoptosis is caused by receptor-mediated pathway and inhibition of telomerase activity in NALM-6 cells.

Costunolide, isolated from the stem bark of Magnolia sieboldii, is a sesquiterpene lactone that exhibits various biological and immunological actions. We investigated the induction mechanism of apoptosis by costunolide in a human B cell leukemia NALM-6 cell culture system. Costunolide (10 microM)-induced apoptosis time-dependently increased, estimated by nuclear damage observation and flow cytometric analysis. Costunolide did not change Fas-associated factor 1 (FAF1), but the phosphorylation of Fas-associated death domain (FADD) at serine 194 increased from early treatment. The activation of caspase-8 and -9 and degradation of poly-(ADP-ribose) polymerase (PARP) was time-dependently detected by incubation with costunolide. Pretreatment of cells with caspase-3, -8 and broad spectrum caspase inhibitors significantly blocked costunolide-induced apoptosis, but caspase-9 inhibitor failed to block apoptosis. Telomerase activity was significantly suppressed after treatment with costunolide, and human telomerase reverse transcriptase (hTERT), a critical determinant of the enzyme activity of telomerase, decreased the expression of both mRNA and protein levels by costunolide. Costunolide-induced repression of telomerase was prevented by pretreatment of cells with caspase-3, -8 and broad spectrum caspase inhibitors, but caspase-9 inhibitor was no effect. These data suggest that one of the costunolide-induced apoptotic mechanisms is that the receptor-mediated pathway precedes the mitochondria-dependent pathway, caused by the inhibition of telomerase activity via suppression of hTERT in NALM-6 cells.

Trimidox-induced apoptosis is mediated through induction of p53 in NALM-6 cells.

We examined the effect of trimidox-induced apoptosis involvement of p53 in the NALM-6 cell line of acute lymphoblastic leukemia. Trimidox has been shown to increase the induction of p53. Phosphorylation of p53 protein at Ser-15 and Ser-20 residues was activated earlier than the obvious increase in p53 expression. Pifithrin-alpha, a p53 inhibitor, significantly prevented trimidox-induced apoptotic characteristics, as detected by nuclear morphological observation and DNA fragmentation. Trimidox-induced apoptosis was enhanced or attenuated by transfection with wild-type or dominant-negative p53 containing expression vectors, respectively. These results indicate that one of the induction mechanisms of apoptosis by trimidox is the mediated augmentation of p53.

Inhibitory effect of trimidox on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages.

We examined the effect of trimidox (3,4,5-trihydroxybenzamidoxime) on the production of nitric oxide (NO) by lipopolysaccharide (LPS) in mouse RAW 264.7 macrophages. Trimidox (50 - 300 microM) concentration-dependently inhibited NO production by LPS (0.01, 0.1, or 1 microg/ml) after incubation for 24 h. LPS-induced expression of inducible NO synthase (iNOS) and degradation of IkappaBalpha were prevented by trimidox. The protective effect against NO production by LPS was not only observed in prior incubation but also later incubation with trimidox until iNOS was activated by LPS. These results suggest that trimidox has a predominantly protective effect against LPS-induced production of NO via iNOS expression.

Preventive effect of trimidox on oxidative stress in U937 cell line.

Trimidox (3,4,5-trihydroxybenzamidoxime) is one of the most potent ribonucleotide reductase inhibitors, revealing an antitumor effect in several experimental studies. We have examined the effect of trimidox on the induction of cytotoxicity and apoptosis via oxidative stress by typical free radical inducers, hydrogen peroxide (H(2)O(2)), tert-butylhydroperoxide (tBuOOH) or ultraviolet (UV) irradiation in a human diffuse histiocytic lymphoma U937 cell line. Trimidox showed strong radical scavenging activity by the DPPH reduction assay. The 50% rate inhibited the DPPH reduction concentration of trimidox, and its derivates didox, or gallic acid were 8.8 microM, 117.5 microM, or 41.8 microM, respectively. Induction of cytotoxicity by H(2)O(2) (500 microM) or tBuOOH (100 microM) was concentration-dependently attenuated by incubation with Trimidox (10-150 microM). Trimidox also prevented the effect of UV-induced apoptosis estimated by both nuclear morphological change and DNA fragmentation. This effect was due to inhibition of the production of reactive oxygen species. Moreover, the activity and mRNA expression of catalase, an antioxidant enzyme, was significantly increased by trimidox. These results indicate that trimidox has radical scavenging activity and prevents exogenous oxidative stress and increase in catalase; therefore, trimidox is suggested as an anticancer agent exhibiting potent antioxidant properties in this study.