Hsp90 is a ubiquitous cellular protein and its functions as a molecular chaperone are vital for cell survival [4
]. Hsp90 mediates the proper folding and prevention of aggregation of numerous proteins in the cell [6
]. It has a wide substrate specificity ranging from steroid hormone receptors, transcription factors, kinases and polymerases [3
]. Hsp90 is also essential for the activation of many regulatory and signaling proteins [3
]. The conversion of extracellular stimuli into appropriate intracellular responses is mediated through the coordinated actions of proteins involved in the signal transduction pathway. Proteins involved in major signal transduction pathways also include the mitogen-activated protein kinases, MAPK [12
]. Hsp90 has been shown to stabilize cell signaling proteins and the binding of HSP90 to these proteins helps in the folding, conformational maturation, translocation and formation of immobilized signal transduction complexes [8
]. p38 MAPK appears to be stimulated primarily through environmental stress and cytokines and is inhibited by the anti-inflammatory drug SB203580 which has been used extensively to delineate this signaling pathway [12
GA, an inhibitor of Hsp90 is a commonly used anti-tumor drug which is effective due to the essential role of Hsp90 in the proliferation and survival of cancer cells [9
]. The chaperone functions of Hsp90 depend on its ATP binding and hydrolyzing ability and GA binds at its ATP-binding site, thus acting as a competitive inhibitor and affecting Hsp90 binding affinity to substrates and co-chaperones [9
]. Several studies have shown that GA disrupts the complex formation of Hsp90 with signal transduction proteins and thus interferes with pathways involving these proteins [36
]. CYP2E1 is a major source of oxidative stress in cells and previously, we have shown that GA potentiates CYP2E1 toxicity in HepG2 cells [22
]. GA also disrupts the binding of Hsp90 to kinases involved in signal transduction pathways [30
]. However, the possible role of MAP kinases involved in signal transduction pathways in GA mediated regulation of CYP2E1 toxicity in HepG2 cells has not been studied.
In the present study GA was toxic to E47 cells and SB203580 increased the viability and prevented the aberrations in morphology in E47 cells due to GA treatment. These results suggest a role of the p38MAPK pathway in GA mediated toxicity in E47 cells. The protective effects of SB203580 were observed only when the E47 cells were pretreated with the p38 MAPK inhibitor before subsequent addition of GA. When SB203580 was added after GA addition, the decline in viability of E47 cells was not prevented. Even addition of SB203580 as early as 5 min after GA failed to protect suggesting that an early activation of p38 MAPK is a prerequisite for GA toxicity in the HepG2 cells and the inhibition of p38 MAPK does not protect the cells from CYP2E1 mediated toxicity once GA initiated its effects.
Since the inhibition of p38MAPK resulted in prevention of the toxicity of GA in the E47 cells, it was important to examine whether inhibition of Hsp90 with GA leads to the activation of p38MAPK, as measured by the increase in the phosphorylated to the non-phosphorylated ratio. Indeed, the pp38 MAPK levels were increased from 15min-4h after GA addition and decreased to the basal levels at 36h in the E47 cells, indicating that the activation of p38 MAPK is an early event which eventually leads to cell toxicity during further incubation. The GA treated E47 cells showed elevated and especially more sustained activation of pp38 MAPK when compared to the C34 cells, suggesting that potentiation of CYP2E1 toxicity in the E47 cells is due, in part to a loss of the protective functions of Hsp90 by GA treatment coupled to p38 MAPK activation.
Hsp90 has been shown to play an anti-apoptotic role [39
] and our previous study has shown that inhibition of Hsp90 with GA increases apoptosis in E47 cells [22
]. In the present study, inhibition of p38 MAPK with SB203580 decreased the percentage of apoptotic E47 cells significantly, as assessed by Annexin V staining or DNA analysis, consistent with other studies where the p38 MAPK inhibitor SB203580 reversed apoptosis [43
We also investigated the involvement of other kinase pathways in mediating the toxic effects of GA in E47 cells using LY294002, a PI3 kinase inhibitor, PD98059, an ERK MAPK inhibitor and SP600125, a JNK inhibitor. These inhibitors were found to be ineffective in preventing GA mediated toxicity in E47 cells, suggesting that PI3 kinase, ERK MAPK and JNK pathways are not involved in the GA-mediated toxicity.
We have previously shown that GA increases ROS levels and lipid peroxidation and decreases GSH content in E47 cells [22
]. We hypothesized that GA inhibition of Hsp90 function lowered cellular protection against CYP2E1 resulting in an increase in CYP2E1- generated oxidant stress by GA. Indeed, the 2-fold elevation in Hsp90 content in E47 compared to C34 cells like the increase in GSH and other antioxidants [29
] reflect metabolic adaptations by the E47 cells to remove the CYP2E1 oxidant stress. Activation of p38MAPK by GA may be mediated by the increased oxidative stress produced by GA, or the activation of MAPK by GA may be important in the developing oxidative stress. The inhibition of p38 MAPK with SB203580 lowered ROS and malondialdehyde accumulation and increased the levels of GSH in GA treated E47 cells. A recent report demonstrated that SB203580 protects dopaminergic neurons against interferon-LPS injury by the suppression of nitric oxide generation [45
]. In a recent study using HeLa cells, the addition of H2O2 did not affect activation of p38 MAPK, suggesting that p38 MAPK functions upstream of ROS [46
]. The reduction in ROS and lipid peroxidation and the partial restoration of GSH in SB203580 treated E47 cells may indicate that the elevated ROS functions downstream of MAPK pathways and the GA-mediated inhibition of Hsp90 in CYP2E1 over-expressing E47 cells leads to oxidative stress. The suppression of activated p38 MAPK with SB203580 downregulates the increased oxidant stress due to GA treatment and protects against loss of cell viability.
Another possible explanation for the lowering of the GA-induced elevated oxidant stress by SB203580 may reflect the effects of SB203580 on CYP2E1 or Hsp90 levels. However, SB203580 had no effect on the levels or activity of CYP2E1 in the E47 cells. Hsp90 levels were elevated in the E47 cells compared to the C34 cells. GA caused an insignificant decrease in Hsp90 levels in the E47 cells, and while a decrease in Hsp90 would promote an increase in oxidative stress, the extent of decrease does not appear to be sufficient to account for the elevated oxidant stress. SB203580 did not alter basal Hsp90 levels significantly in the E47 or C34 cells. The SB203580 mediated prevention of the GA-induced increase in toxicity is not at the levels of CYP2E1 or Hsp90.
Damage to the mitochondria and consequent decline in mitochondrial membrane potential plays an important role in apoptosis [47
] and CYP2E1-mediated toxicity [48
]. GA caused mitochondrial damage in E47 cells, and to a lesser extent in C34 cells, as assessed by loss in mitochondrial membrane potential. SB203580 partially reversed the mitochondrial damage in the E47 cells. Mitochondrial dysfunction has been shown to activate MAPK pathways [49
] but activated p38 MAPK may cause mitochondrial dysfunction [34
]. The decrease in percentage of cells with low mitochondrial membrane potential after SB203580 treatment suggests that GA toxicity in CYP2E1 over-expressing cells through a p38 MAPK pathway and perhaps increased oxidant stress may cause damage to the mitochondria. Alternatively, mitochondrial damage due to CYP2E1 plus GA toxicity may activate p38 MAPK. Further studies will be needed to evaluate whether activated p38 MAPK is upstream of the mitochondrial damage or is a result of the mitochondrial damage.
In summary, the p38 MAPK inhibitor, SB203580, prevents GA toxicity in E47 cells indicating that the potentiation of toxicity due to Hsp90 inhibition by GA in HepG2 cells over-expressing CYP2E1 is mediated through the activation of a p38 MAPK pathway. The addition of SB203580 prior to GA treatment was critical for its protective effects. Short-term treatment with GA caused activation of p38 MAPK in the E47 cells. SB203580 decreased the GA-induced elevation of ROS levels and lipid peroxidation, and increased GSH levels in the E47 cells. The increase in number of GA treated cells showing an apoptotic mode of cell death and low mitochondrial membrane potential were decreased significantly with SB203580 treatment. The results suggest that the Hsp90 inhibitor GA potentiates CYP2E1 toxicity through a p38 MAPK pathway.