John T. Piper

Mechanisms of anticarcinogenic properties of curcumin: the effect of curcumin on glutathione linked detoxification enzymes in rat liver.

Curcumin, an antioxidant isolated from turmeric (curcuma longa), has been shown to attenuate chemical carcinogenesis in rodents. Previous studies have shown that curcumin causes an increase in glutathione S-transferase (GST) activity in rodent liver which may contribute to its anti-cancer and anti-inflammatory activities. Since the effects of curcumin on specific GST isozymes and other glutathione (GSH)-linked enzymes are incompletely defined, we have examined in the present studies the effect of curcumin on hepatic non-protein sulfhydryls and GSH-linked enzymes in male Sprague-Dawley rats. When rats were fed curcumin at doses from 1 to 500 mg kg-1 body weight daily for 14 days, the induction of hepatic GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was found to be biphasic, with maximal induction of about 1.5 fold at the 25 to 50 mg kg-1 body weight dosage. At higher doses, a decrease was observed in the activity and in the rats treated with 500 mg kg-1 curcumin this activity was below the levels observed in controls. In contrast, GST activity towards 4-hydroxynonenal (4-HNE) increased in a saturable, dose dependent manner. Western-blot analyses of liver cytosols revealed that curcumin caused a dose dependent induction of rGST 8-8, an isozyme which is known to display the highest activity towards 4-HNE, a highly toxic product of lipid peroxidation. Glutathione peroxidase (GPx) activity towards cumene hydroperoxide in liver homogenate was also found to be increased in a saturable manner with respect to curcumin dose. Our results suggest that induction of enzymes involved in the detoxification of the electrophilic products of lipid peroxidation may contribute to the anti-inflammatory and anti-cancer activities of curcumin.

Characterization of a chlorambucil-resistant human ovarian carcinoma cell line overexpressing glutathione S-transferase μ

Ovarian carcinoma cells 10-fold resistant to the alkylating agent chlorambucil (CBL) were isolated after repeated exposure of the parent cells to gradually escalating concentrations of the drug. The resistant variant, A2780(100), was highly cross-resistant (9-fold) to melphalan and showed lower-level resistance to other cross-linking agents. The resistant A2780(100) cells had almost 5-fold higher glutathione S-transferase (GST) activity than the parental A2780 cells with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The pi-class GST(s) was the major isoform(s) in both cell lines. However, the resistant A2780(100) cells had at least 11-fold higher GST mu as compared with the parental cells, in which this isoform was barely detectable. A significant induction of GST mu was observed in A2780 cells, but not in the resistant cells, 18 hr after a single exposure to 100 microM CBL. The induction of GST mu by CBL was both time- and concentration-dependent. Assays of the conjugation of CBL with GSH showed that the human mu-class GST had 3.6- and 5.2-fold higher catalytic efficiency relative to the pi- and alpha-class GSTs, respectively. This difference was reflected in the relatively higher (about 6-fold) efficiency of CBL conjugation in A2780(100) cells as compared with the parental cells. These results have demonstrated for the first time a near-linear correlation between CBL resistance and overexpression of mu-class GSTs and suggest that this overexpression maybe responsible, at least in part, for the acquired resistance of ovarian carcinoma cells to CBL, and possibly the other bifunctional alkylating agents. Consistent with this hypothesis, we found evidence for decreased formation of DNA lesions in A2780(100) compared with the drug-sensitive A2780 cells after exposure to CBL.

The effect of curcumin on glutathione-linked enzymes in K562 human leukemia cells

Curcumin, an antioxidant present in the spice turmeric (Curcuma longa), has been shown to inhibit chemical carcinogenesis in animal models and has been shown to be an anti-inflammatory agent. While mechanisms of its biological activities are not understood, previous studies have shown that it modulates glutathione (GSH)-linked detoxification mechanisms in rats. In the present studies, we have examined the effects of curcumin on GSH-linked enzymes in K562 human leukemia cells. One micromolar curcumin in medium (16 h) did not cause any noticeable change in glutathione peroxidase (GPx), glutathione reductase, and glucose-6-phosphate dehydrogenase activities. Gamma-glutamyl-cysteinyl synthetase activity was induced 1.6-fold accompanied by a 1.2-fold increase in GSH levels. GSH S-transferase (GST) activities towards 1-chloro-2,4-dinitrobenzene, and 4-hydroxynonenal (4HNE) were increased in curcumin-treated cells 1.3- and 1.6-fold, respectively (P = 0.05). The GST isozyme composition of K562 cells was determined as follows: 66% of GST Pl-1, 31% of Mu class GST(s), and 3% of an anionic Alpha-class isozyme hGST 5.8, which was immunologically similar to mouse GSTA4-4 and displayed substrate preference for 4HNE. The isozyme hGST 5.8 appeared to be preferentially induced by curcumin, as indicated by a relatively greater increase in activity toward 4HNE. Immunoprecipitation showed that GPx activity expressed by GST 5.8 contributed significantly (approximately 50%) to the total cytosolic GPx activity of K562 cells to lipid hydroperoxides. Taken together, these results suggest that GSTs play a major role in detoxification of lipid peroxidation products in K562 cells, and that these enzymes are modulated by curcumin.

Role of oxidative stress and MAPK signaling in reference moist smokeless tobacco-induced HOK-16B cell death.

The use of smokeless tobacco products is often associated with an oral injury at the site of repeated use. To further our understanding of this injury process, the effect of reference moist smokeless tobacco extract (STE) on cell death, oxidative stress, and MAPK signaling in a human oral keratinocyte cell line, HOK-16B, was investigated. STE caused dose-dependent cell death and reactive oxygen species (ROS) production within 30 min to 3h of exposure. This same insult enhanced the activity of ERK1/2, JNK1/2, p38 MAPK and ASK1, an upstream activator of JNK1/2 and p38 MAPK. Inhibition of JNK1/2 and to a lesser extent p38 MAPK, but not ERK1/2, suppressed STE-induced cell death. Pretreatment with antioxidants and an iron chelator, deferoxamine suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Interestingly, extracellular free iron levels in STE (29.4+/-0.5 microM) were significantly elevated as compared with cell culture medium (4.9+/-0.6 microM) and the addition of extracellular free iron (14, 30 or 70 microM) to HOK-16B cultures (without STE) caused dose-dependent cell death after 3h. Thus, acute exposure to STE leads to HOK-16B cell death in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways.

Attenuation of galactose cataract by low levels of dietary curcumin

Abstract Our previous studies have shown that curcumin, a dietary antioxidant present in turmeric, attenuates 4-hydroxynonenal induced lens opacification in organ culture (Awasthi et al , Amer. J. Clin. Nutr. 1996;64:761–766). Oxidative stress has been implicated in the mechanism of galactosemic cataract formation. Therefore, the present studies were designed to examine whether dietary curcumin can attenuate galactosemic cataract in rats in-vivo . 8 week old Sprague-Dawley rats were randomized into four groups and fed with either AIN-76 diet or that containing 30% galactose with or without curcumin 0.0025% (ww) in the diet. Progression of cataract formation was monitored by slit lamp biomicroscopy at days 1, 22 and 29. The eyes were removed out at day 29 and the lenses were dissected out. Images of the isolated lenses were acquired using a digital imager. The lens epithelium was dissected and lens epithelial cells were examined for apoptosis. Lenses of the animals fed a diet containing neither galactose nor curcumin and those fed only a curcumin containing diet remained transparent. The transparency of lenses from rats without or with curcumin was identical as assessed by measuring the average intensity of transmitted light (AITL) (241 ± 4, n=12). Lenses from galactose fed animals without curcumin were partially opacified with an AITL value of 77 ± 9 % of the controls, (n=10, p

Purification and characterization of glutathione S-transferases of rat uterus

Glutathione S-transferases (GSTs) provide protection to cells against electrophilic xenobiotics as well as lipid hydroperoxides and 4-hydroxynonenal generated during lipid peroxidation. The catalytic properties of the alpha class GSTs are well suited for detoxification of electrophilic products of lipid peroxidation. An immunologically distinct subgroup of the alpha class GST isozymes having high activity towards 4-hydroxynonenal has been recently identified in several mammalian tissues [Zimniak et al. (1994) J. Biol. Chem. 269, 992-1000]. Since oxidative stress can exert deleterious effects during gestation, the present studies were performed to determine whether the rat homolog of this group of GSTs, rGST 8-8, is expressed in gravid rat uterus, where it may function as a defense mechanism against oxidative stress. GSTs were purified by GSH-affinity chromatography. Individual GST isozymes were separated by column isoelectric focusing and their immunologic identities were established using highly specific polyclonal antibodies in Western blot analysis. Their expression was quantified and kinetic properties were characterized. Rat uterus contained an alpha class GST (pI 9.8), a pi class GST (pI 8.1), two mu class GSTs (pI 6.7 and 6.2) and rGST 8-8. This result indicated that rGST subunits 1, 2, 3, 4, 7 and 8 are present in rat uterus. The relative abundance of rGST 8-8 in the gravid rat uterus was found to be about three-fold higher as compared with that previously seen in rat liver. Higher relative abundance of rGST8-8 in gravid rat uterus suggests that it may play a protective role against the deleterious effects of lipid peroxidation during gestation.

Rat GST 8-8 is expressed predominantly in myeloid origin cells infiltrating the gravid uterus

Previous studies from our laboratory have shown a relatively high expression of rGST8-8 in uterine tissues. This GST isozyme displays relatively high glutathione-peroxidase activity towards lipid-hydroperoxides and towards toxic 4-hydroxyalkenals generated from lipid peroxidation. Since the uterus is a unique organ, subject to oxidative stress due to infiltration by immune effector cells during gestation and because this infiltration is readily identifiable histologically, the studies reported herein were performed to localize the cell specific expression of rGST8-8 to determine whether immune effector cells infiltrating the pregnant rat uterus specifically expressed rGST8-8. A 75 bp end-radiolabeled cRNA probe was prepared from the full length mGSTA4-4 cDNA from the region which is highly homologous with rGST8-8. This cRNA probe was used for in situ hybridization studies to localize rGST8-8 in specific cell types of gravid rat uterus. Results of these studies indicate that this GST isozyme is selectively expressed in myeloid origin cells such as monocytes/macrophages, and neutrophils infiltrating the uterine endometrium and in vascular walls. Selective expression of rGST8-8 in the myeloid origin cells, which are known to generate higher levels of reactive oxygen species, suggests that this GST isozyme plays an important role in the protection mechanisms against lipid peroxidation.