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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Pediatric Research and Electron Microscopy Unit, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
Received February 22, 2007; accepted May 24, 2007.
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). Most patients present in an inoperable stage when chemotherapy is the only alternative. Among the various chemotherapeutics, doxorubicin (Adriamycin hydrochloride; DOX) is broadly prescribed to patients with advanced HCC. DOX belongs to anthracyclines antibiotics, and its antitumor activity is related to the ability of binding DNA and inhibition of nucleic acid synthesis, leading to apoptosis (Gewirtz, 1999). Unfortunately, repetitive DOX treatment induces multidrug resistance of cancer cells, resulting in therapeutic failure (Rittierodt and Harada, 2003; Puhlmann et al., 2005). Novel combination regimens are needed to enhance the efficacy of anthracyclines. Acetaminophen (paracetamol, 4-hydroxyacetanilide; AAP), in high dose (HAAP) rescued by N-acetylcysteine, has been tested for the treatment of advanced HCC and metastatic melanoma (Kobrinsky et al., 1996, 2005; Wolchok et al., 2003). In these trials, HAAP was combined with cisplatin (Kobrinsky et al., 2005) or alkylating agents (Wolchok et al., 2003). It was concluded that chemotherapy combined with HAAP could prolong survival of patients with advanced malignancies.
AAP, a widely used analgesic and antipyretic, is hepatotoxic when given in overdose (Hinson et al., 2004). The mechanisms of AAP hepatotoxicity are well defined, and most steps of its molecular pathogenesis have been elucidated (Hinson et al., 2004; Jaeschke and Bajt, 2006). The predominant event of AAP-induced liver toxicity is its transformation by microsomal enzymes of the P450 family, presumably by CYP2E1, into the reactive metabolite N-acetyl-p-benzoquinone imine, which is detoxified by GSH conjugation. Once GSH is depleted, N-acetyl-p-benzoquinone imine stimulates oxidative stress, causing liver cell necrosis. The role of apoptotic pathway during AAP hepatotoxicity remains controversial (Ray and Jena, 2000; Gujral et al., 2002; Jaeschke and Bajt, 2006). Nevertheless, studies of AAP toxicity in hepatoma cell lines, performed in our laboratory and other laboratories, unequivocally indicate the occurrence of apoptosis (Boulares et al., 2002; Manov et al., 2002, 2004; Neuman, 2002; Macanas-Pirard et al., 2005; Lee et al., 2006b). Because metabolic activation of AAP is reduced or does not occur in tumor cells, unmetabolized AAP contributes directly to the toxicity, causing apoptosis (Boulares and Ren, 2004). Therefore, it was proposed that DOX + HAAP combined chemotherapy may be more effective compared with single-agent treatment. Thus, we investigated the result of DOX and HAAP concomitant treatment using HepG2 cells. These cells, originally derived from HCC (Aden at al., 1979), demonstrate relatively low activities of drug enzymes responsible for AAP metabolic activation (Dai and Cederbaum, 1995). A more recent study revealed a reduced level of CYP2E1 gene expression in HCC, which further decreased during HCC progression (Man et al., 2004). Therefore, use of HepG2 cells to study of chemotherapeutic efficacy of HAAP and HAAP + DOX seems to be justified, and it would reflect a clinical situation in which HAAP is administered to HCC patients as an additional anticancer drug. Contrary to our hypothesis, HAAP markedly reduced the lethal effect of DOX to HepG2 cells. Consequently, we investigated the mechanisms that could be involved in the antagonistic effect of combined DOX + HAAP treatment. Specifically, we studied the roles of the multidrug resistance P-glycoprotein (P-gp) and p44/42-mitogen-activated protein kinase (MAPK) pathways in response to DOX, HAAP, and DOX + HAAP.
P-gp belongs to the family of ATP-binding cassette transporters that carries drugs out of the cells (Hoffmann and Kroemer, 2004). Overexpression of P-gp in tumor cells and its up-regulation during chemotherapy decrease intracellular accumulation of various anticancer drugs, reducing their efficacy (Rittierodt and Harada, 2003; Hoffmann and Kroemer, 2004; Puhlmann et al., 2005). Our previous results indicated that AAP is a P-gp substrate, and in high doses it increases its function in hepatoma-derived cells (Manov et al., 2006). Hence, the modulation of P-gp by HAAP in cancer cells could influence their chemosensitivity. Therefore, we investigated whether HAAP can modulate intracellular DOX concentration and DOX-induced damage in HepG2 cells.
Recent studies have indicated a crucial role of p44/42-MAPK in DNA damage and apoptosis induced by DOX (Tang et al., 2002; Lee et al., 2006a). Scarce information is available regarding the involvement of p44/42-MAPK pathway in response to HAAP (Macanas-Pirard et al., 2005). Subsequently, we explored p44/42-MAPK activation/suppression during the treatments.
Thus, we aimed to investigate the interaction of DOX and HAAP using HepG2 cells and to clarify the involvement of P-gp and p44/42-MAPK pathways in the response to the concomitant exposure. We also studied the effects of the P-gp reversing agent verapamil (verapamil hydrochloride; VRP) and of the inhibitor of p44/42 activation PD98059 on the combined or single-drug treatment.
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-actin monoclonal antibodies were purchased from Sigma-Aldrich Israel Ltd. Anti-p44/42 and anti-phospho-p44/42 antibodies were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., CA). Horseradish peroxidase-conjugated secondary antibody was from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Cell culture reagents were from Biological Industries (Kibbutz Beth HaEmek, Israel). All other chemicals, unless otherwise indicated, were obtained from Sigma-Aldrich Israel Ltd.
Cell Culture and Treatments. The HepG2 cell line was kindly provided by Prof. D. Shouval (Liver Unit, Hadassah University Hospital, Jerusalem, Israel). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, and they were incubated in a humidified incubator at 37°C in 5% CO2. Experiments were initiated when the cells reached 80% confluence. For the experiments, cells were cultured in the fetal calf serum-free medium, and they were treated with 2 and 5 mM HAAP, 2 and 5 µM DOX, and HAAP + DOX for 24 and 48 h. To inhibit P-gp function, 50 µM VRP was simultaneously added to drug-exposed cells. Pretreatment with the p44/42 MAPK inhibitor PD98059 at 30 µM for 2 h was performed to suppress the p44/42 activation in the cells. HAAP was dissolved in cell culture medium, whereas DOX, VRP, and PD98059 were dissolved in DMSO. Final DMSO concentration in culture medium was adjusted to 0.1% in all experiments, including vehicle control. In preliminary experiments, we confirmed that 0.1% DMSO did not affect survival of HepG2 cells. Alamar Blue reduction test (Nakayama et al., 1997) was used for investigation of cell viability as described previously (Manov et al., 2002).
Cell Cycle Analysis. The cell cycle distribution was assessed by flow cytometry of PI-stained nuclei as described previously (Manov et al., 2002). In brief, following drug treatment, cells were harvested by trypsin, combined with medium containing floating cells, washed with PBS, and stained with hypotonic PI solution (50 µg/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100). The PI fluorescence of individual nuclei was recorded by using FL2-A histogram plot (cell cycle analysis) and FL2-H for assessment of nuclear hypodiploidity (FACSCalibur; BD Biosciences, Franklin Lakes, NJ). We acquired 20,000 events, and we corrected for debris and aggregate population.
Detection of Doxorubicin Intracellular Accumulation. Intracellular DOX accumulation, in the presence or absence of HAAP, was evaluated by using fluorescence microscopy and flow cytometry. Cells were grown on glass coverslips in six-well tissue culture plates for 48 h, and then they were treated with 5 µM DOX with or without 2 mM HAAP for 24 h. After treatment, the cells were washed with PBS and fixed in ice-cold methanol. The coverslips were mounted on glass slides, and cells were analyzed with a fluorescent microscope Axioscope2 (Carl Zeiss, Oberkochen, Germany), operated with 546-nm excitation and 590-nm emission filter. For flow cytometry analysis, cells were harvested following the drug treatment (DOX, DOX + HAAP with or without VRP) and washed with PBS. Signals generated from intracellular DOX were determined (FL2, 585-nm emission filter).
Measurement of Rhodamine 123 Retention. Cells were cultured in six-well plates at a density of 5 x 105 cell per well for 48 h. Then, cells were treated with DOX, HAAP, or DOX + HAAP with or without the P-gp inhibitor VRP at 50 µM for 24 h before the addition of 5 µg/ml Rh 123 for 30 min at 37°C. Thereafter, cells were washed with PBS and harvested by 0.05% trypsin with 0.02% EDTA. Intracellular Rh 123 fluorescence was estimated by flow cytometry (FL1, 530-nm emission filter). The mean fluorescence intensity and percentage of Rh 123-positive cells were recorded and combined to provide a fluorescence index (Manov et al., 2006).
Scanning Electron Microscopy. Cells were grown on glass coverslips in six-well tissue culture plates. Then, they were treated with DOX with or without HAAP for 24 h. After treatment, cells were washed with PBS, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h, treated with 2% tannic acid + 2% guanidine HCl in PBS for 1 h, and postfixed in 2% OsO4 for 1 h. Thereafter, specimens were viewed and photographed using a JEOL J-90 scanning electron microscope (JEOL, Tokyo, Japan), as described previously (Levanon et al., 2004).
Western Blot Analysis. Cell lysates, protein determination, electrophoresis, and transfer to polyvinylidene difluoride membrane were performed as described previously (Manov et al., 2006). The proteins were probed with the monoclonal antibody to P-gp (C219; 1:200) or phospho-p44/42 (1:2500), followed by horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescent substrate. The levels of -actin or total p44/42 were measured after stripping of the same membranes and reprobing with anti--actin and anti-total p44/42 (or p42-extracellular signal-regulated kinase-2) antibody. The optical density of the specific protein bands was quantified by densitometry (Vilber Lourmat, Lyon, France).
Statistical Analysis. The data represent the results of at least three separate experiments. Values are the means ± S.D. Statistical differences were analyzed using the Mann-Whitney U test (two-tailed), and p < 0.05 was considered statistically significant.
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). The AAP concentrations of 
2 to 4.5 mM would be achieved in plasma of the patients getting 10 to 30 g/M2 AAP for the treatment of advanced malignancies (Kobrinsky et al., 1996, 2005; Wolchok et al., 2003). In addition, 5 mM AAP has been described previously as the effective dose that caused apoptosis in hepatoma-derived cells (Manov et al., 2002, 2004; Neuman, 2002; Macanas-Pirard et al., 2005). Therefore, the concentrations of 2 and 5 mM HAAP and 2 and 5 µM DOX have been used in the present experiments. The changes of cell viability following exposure to HAAP, DOX, and DOX + HAAP are shown in Fig. 1. After 24 h of exposure to 5 mM HAAP, cell viability decreased (p < 0.05 versus vehicle control and 2 mM HAAP). The 2 and 5 µM concentrations of DOX decreased cell viability as well (p < 0.05 versus vehicle control). Single-drug treatments for 48 h further reduced cell viability. In contrast, combined treatment with DOX + HAAP increased cell viability compared with DOX alone. Marked increase of cell viability was observed, after 48 h, when HAAP was added to 2 µM DOX-exposed cells.
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The cell cycle distributions following the treatment are presented in Fig. 2. DOX at 2 µM induced arrest of proliferation mainly in S phase, whereas DOX at 5 µM significantly increased the frequency of apoptotic cells (sub-G1). Treatment with 2 and 5 mM HAAP produced a moderate increase in apoptotic cells. HAAP added to DOX-exposed cells released cell cycle arrest and decreased apoptosis. Ultrastructural changes in the cells treated with 5 µM DOX, 2 mM HAAP, and DOX + HAAP were investigated by scanning electron microscopy (Fig. 3). Single apoptotic blebs were seen after treatment of HepG2 cells with 2 mM HAAP, whereas prominent apoptotic blebs were present after exposure to 5 µM DOX. These findings disappeared when 5 µM DOX was combined with HAAP.
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Does HAAP Reduce DOX Intracellular Accumulation in HepG2 Cells? Figure 4A presents fluorescence microscopic images of cells treated with 5 µM DOX alone and in combination with 2 mM HAAP for 24 h. DOX accumulation visibly decreased when cells were simultaneously treated with HAAP. In parallel, quantitative flow cytometry analysis revealed a significant decrease of DOX fluorescence indices in cells treated with DOX + HAAP in comparison with single DOX exposure (Fig. 4B). The P-gp inhibitor VRP at 50 µM, when added to DOX + HAAP treatment, restored DOX intracellular accumulation up to the level reached in DOX + VRP exposure (Fig. 4C).
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). To investigate whether P-gp is involved in the effect of HAAP on DOX intracellular accumulation and toxicity, we studied the P-gp activity and expression in cells treated with DOX, HAAP, and DOX + HAAP (Fig. 5). Measurement of the Rh 123 retention was performed to evaluate the P-gp transport activity in the cells. The fluorescent dye Rh 123 is a substrate for P-gp, and its cellular retention has been demonstrated to reflect P-gp function (Pétriz and Garcia-Lopez, 1997). HAAP decreased the Rh 123 retention in HepG2 cells in a concentration-dependent manner that reflects the increasing P-gp transport activity (Fig. 5A). Single treatment with 5 µM DOX markedly decreased Rh 123 fluorescence as well. Combined treatment with DOX + HAAP resulted in the subsequent decrease of Rh 123 retention, and, respectively, increase in P-gp activity. The stimulation of P-gp activity by HAAP, DOX, or DOX + HAAP was fully blocked when the P-gp-reversing agent VRP at 50 µM was added to the cells (Fig. 5B). Immunoblots demonstrated the increase in P-gp expression in cells treated with HAAP for 24 h, 1.2 (2 mM) and 1.7 times (5 mM), in comparison with vehicle control (Fig. 5C). The exposure of cells to DOX alone resulted in a noticeable increase in P-gp content, 2.1 and 3 times at 2 and 5 µM DOX, respectively. Combined treatment with DOX + HAAP did not show a synergistic effect. To evaluate whether VRP can reverse DOX toxicity, we compared cell viability and frequency of hypodiploid (apoptotic) nuclei in HepG2 cells treated with DOX and DOX + HAAP with or without VRP (Fig. 6). VRP significantly decreased cell viability and increased nuclear hypodiploidity of DOX + HAAP-treated cells, fully restoring DOX toxicity.
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Does Inhibition of DOX Toxicity with HAAP Involve a p44/42-Dependent Mechanism? Since recent studies have indicated a critical role for the p44/42-MAPK pathway in the response to DOX (Tang et al., 2002; Lee et al., 2006a), we explored the p44/42-MAPK cascade during the treatment using immunoblotting (Fig. 7). Exposure of HepG2 cells to HAAP decreased the levels of phospho-p44/42 compared with vehicle control. In contrast, DOX significantly activated the p44/42 pathway in a concentration-dependent manner. When HAAP was added to DOX-exposed cells, p44/42 activation was largely diminished compared with single DOX treatment. The changes in phospho-p44/42 levels after the treatment were observed without modifications in total p44/42. To investigate whether inhibition of p44/42 activity could reduce DOX-induced toxicity, we investigated the effect of p44/42 inhibitor PD98059 on DOX exposure (Fig. 8). Pretreatment of HepG2 cells with PD98059 at 30 µM for 2 h inhibited DOX toxicity. Cell viability of DOX-exposed cells pretreated with PD98059 was similar to viability of vehicle control, and it was not different from treatment of PD98059 alone (Fig. 8A). In parallel, DOX-induced nuclear degradation was blocked by PD98059 pretreatment (Fig. 8B). Immunoblot with anti-phospho-p44/42 monoclonal antibody confirmed suppression of p44/42 activation in DOX-exposed or vehicle control cells pretreated with PD98059 (Fig. 8C).
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), and it has been shown to induce apoptosis via activation of caspases and disruption of mitochondrial membrane potential (Gewirtz, 1999). The significant role of MAPK pathways and p53 activation were described as well (Sanchez-Prieto et al., 2000; Tang et al., 2002; Lee et al., 2006a). Several clinical trials demonstrated the effectiveness of HAAP with N-acetylcysteine rescue for the treatment of patients with advanced HCC and metastatic melanoma (Kobrinsky et al., 1996, 2005; Wolchok et al., 2003). Recently, HAAP has been shown to potentiate staurosporine-mediated neuroblastoma cell death (Posadas et al., 2007). Since HAAP causes apoptosis in different tumor cells (Bae at al., 2001; Boulares et al., 2002; Manov et al., 2002, 2004; Macanas-Pirard et al., 2005), we hypothesized that treatment of DOX-exposed HepG2 cells with HAAP would increase tumor cell death. Unpredictably, HAAP strongly reduced the DOX lethal effect to HepG2 cells, increasing cell viability, restoring cell cycle, and decreasing apoptosis (Figs. 1, 2, 3). When tested alone, either DOX or HAAP generated reduced viability, growth inhibition, and increased apoptosis. To determine the molecular mechanisms playing a role in the antagonistic effect during DOX + HAAP exposure, we investigated the involvement of multidrug resistance P-gp and p44/42 MAPK pathways in the response to DOX, HAAP, and cotreatment.
The appearance of drug resistance in tumors is frequently a major obstacle during chemotherapy. Among the transporters, P-gp has been shown to play a central role in the development of multidrug resistance of malignant cells (Hoffmann and Kroemer, 2004). Both P-gp overexpression and its induction by anticancer drugs may generate cancer cell survival and chemotherapeutic inefficiency. DOX has been shown to increase P-gp function in cancer cells (Rittierodt and Harada, 2003; Puhlmann et al., 2005). We previously reported that HAAP can induce P-gp transport activity and expression in hepatoma-derived cells (Manov et al., 2006). Hence, we hypothesized that P-gp stimulation by HAAP may decrease DOX intracellular accumulation, and, consequently, reduce the DOX-induced damage in HepG2 cells. Both DOX and HAAP increased P-gp transport activity and content (Fig. 5, A and C). Combined treatment with DOX + HAAP further stimulated P-gp transport activity, resulting in decreased DOX intracellular accumulation (Fig. 4, A and B) and increased Rh 123 efflux (Fig. 5A). Although P-gp function was induced during combined treatment, no increase of the P-gp expression was observed compared with single DOX treatment (Fig. 5C). Furthermore, reduced P-gp bands were found during coexposure with HAAP and 5 µM DOX. The most likely explanation of this discrepancy is that HAAP, as P-gp substrate, may induce conformational shift in the C219 binding domain, leading to decreased affinity. The finding that P-gp substrates can modulate the P-gp reactivity to bind the specific monoclonal antibody, confirms our suggestion (Druley et al., 2001).
To prove whether stimulation of P-gp contributes to HAAP + DOX antagonism, we studied the effect of the specific inhibitor of P-gp function VRP on DOX accumulation and DOX + HAAP toxicity. If inhibition of DOX toxicity is the result of P-gp activation, VRP would reverse the DOX accumulation and abolish HAAP cytoprotective effect. We have shown that treatment of DOX + HAAP-exposed cells with VRP increased DOX intracellular fluorescence (Fig. 4 C) and fully restored the DOX lethal effect (Fig. 6, A and B).
The mitogen-activated protein kinases, which include extracellular-regulated protein kinases (p44/42), the stress-activated c-Jun NH2-terminal protein kinase, and p38 kinase, play basic roles in survival, proliferation, and apoptosis (Widmann et al., 1999). Little is known about the role of MAPK pathways in HAAP toxicity. It was observed that c-Jun NH2-terminal protein kinase is activated by HAAP in glioma cells (Bae et al., 2001) and HuH7 cells (Macanas-Pirard et al., 2005), but its pharmacological inhibition did not alter HAAP toxicity. A recent report (Macanas-Pirard et al., 2005) showed that inhibition of p38 or p44/42 mitogen-activated protein kinases failed to modulate the ability of HAAP to trigger apoptosis in HuH7 cells; therefore, these authors concluded that these pathways were not involved in the response to HAAP. Our results demonstrated that HAAP suppresses the p44/42 pathway in HepG2 cells, as evidenced by the detection of the corresponding phospho-p44/42 bands (Fig. 7). The p44/42 kinases have been shown to play a central role in the response to DNA-damaging agents such as anthracyclines (Lee et al., 2006a). It was shown that DOX induces the p44/42 pathway in different cell models (Guise et al., 2001; Small et al., 2003; Lee et al., 2006a). Consistent with the previous findings, our results showed that treatment of HepG2 cells with DOX resulted in increased phospho-p44/42 levels (Fig. 7). The role of p44/42 activation in the response to DOX remains controversial. Some investigations suggested an antiapoptotic role of this pathway (Small et al., 2003; Niiya et al., 2004). For example, suppression of p44/42 phosphorylation by the inhibitor U0126 was found to increase the caspase-3 cleavage in DOX-exposed RC-K8 lymphoma cells (Niiya et al., 2004). However, it has not been clearly demonstrated whether the apoptogenic effect is generated via p44/42 inhibition or whether it is due to a direct effect of U0126, which itself markedly enhances the caspase-3 cleavage. Alternatively, suppression of the p44/42 pathway via overexpression of a dominant-negative mutant form (Lee et al., 2006a) or by using antisense strategy (Guise et al., 2001) resulted in decreased DOX toxicity. In our experiments, direct inhibition of p44/42 activity by PD98059 abolished the DOX lethal effect in HepG2 cells (Fig. 8). Similar results were obtained in NIH3T3 fibroblasts and SK-N-SH neuroblastoma cells (Guise et al., 2001; Tang et al., 2002). Taken together, these observations suggest that p44/42 activation is the central event in the response to DOX and induction of apoptosis. Therefore, suppression of the p44/42 pathway could attenuate the DOX lethal effect. Indeed, HAAP was able to diminish the p44/42 activation in DOX-exposed cells. Thus, we suggested that the p44/42 pathway and its inhibition by HAAP could be also involved in the mechanism by which HepG2 cells circumvent the DOX-induced apoptosis.
Our results do not rule out the possibility of other mechanisms playing a role in the DOX + HAAP antagonistic effects. A recent study revealed that AAP stimulates DOX oxidation in human leukemic HL-60 cells that may hinder DOX anticancer activity (Reszka et al., 2004). These authors have shown that GSH protects DOX against its oxidation. When intracellular GSH is depleted by AAP metabolic transformation, DOX undergoes oxidation to a less toxic metabolite. Another possibility is that HAAP, notwithstanding its apoptotic effect to C6 glioma cells, was shown to decrease the level of p53 protein (Lee et al., 2006b). The wild-type p53 is present in HepG2 cells (Lee et al., 2002), and its suppression was found to play a significant role in preventing apoptosis (Sanchez-Prieto et al., 2000; Bai and Cederbaum, 2006). Thus, it may be possible that p53 inhibition by HAAP could also contribute to this effect. Further studies are required to clarify this hypothesis.
In conclusion, HAAP strongly reduces the lethal effect of DOX on HepG2 cells. This phenomenon occurs due to stimulation of P-gp efflux pump activity by HAAP, resulting in decreased DOX intracellular concentration. Furthermore, inhibition of the p44/42 pathway by HAAP could decrease the response to DOX in cancer cells. We presume that coadministration of DOX and HAAP in attempts to improve the chemotherapeutic efficacy may have an opposite effect, resulting in cancer cell survival.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: HCC, hepatocellular carcinoma; DOX, doxorubicin; AAP, acetaminophen; HAAP, high-dose acetaminophen; GSH, glutathione; P-gp, P-glycoprotein; MAPK, mitogen-activated protein kinase; VRP, verapamil; PD98059, 2'-amino-3'-methoxyflavone; PI, propidium iodide; Rh 123, rhodamine 123; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene.
1 Current affiliation: Pediatric Research and Electron Microscopy Unit, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. 
Address correspondence to: Dr. Irena Manov, Pediatric Research and Electron Microscopy Unit, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, Haifa 31096, Israel. E-mail: irmanov{at}tx.technion.ac.il
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