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The tumor suppressor gene p53, is recognized as the guardian of the genome because it regulates the transcription of numerous genes that code for life and death processes. Wild-type p53 is induced in response to a host of genotoxic and environmental stresses, including chemotherapeutic agents, UV irradiation, hypoxia, heat, or alterations in intracellular nucleotide pools. Many agents used in the treatment of cancer cause DNA damage that is sensed by the p53. Once p53 is induced, a host of target genes are then transcriptionally activated, including p21, bax, and bcl-2. Induction of p21, in turn, leads to cell cycle arrest at both G1 and G2 checkpoints. This function is felt to be essential in preserving the integrity of the cellular genome in response to treatment with cytotoxic agents. In addition to mediating cell cycle arrest, p53 is a potent inducer of the process of apoptosis and the program of cell death. At this time, the specific signals required for p53-mediated cell cycle arrest and/or apoptosis remain complex. However, the final pathway selected seems to depend upon a host of physiologic conditions, the cellular context and environment and the specific cytotoxic and/or cellular stress. In case of a mutation or deletion in the p53 gene the efficiency of the chemotherapy is compromised. It is estimated that more than 50% of all human cancers express either a mutant or altered form of p53. Loss of p53 function has been found to enhance cellular resistance to chemotherapeutic agents. Recently, p53 level is regulated by the rate of its degradation following by two mechanisms: First, which is known to be mediated by the Mdm-2-ubiquitin-proteasome degradation pathway. Second, NQO1 regulates p53 stability and that inhibition of NQO1 activity by dicoumarol induces proteasomal degradation of p53 and inhibits p53-mediated apoptosis. Wild-type NQO1, but not an inactive polymorphic NQO1, stabilizes wild-type p53, and that NQO1 does not inhibit Mdm-2-mediated p53 degradation.
NAD(P)H-quinone oxidoreductase 1 or NQO1 (EC 18.104.22.168), an obligate two-electron reductase that is characterized by its capacity for using either NADH or NADPH as reducing cofactors and by its inhibition by dicumarol. NQO1 has multiple cellular roles, acts as a Phase II detoxification enzyme with the detoxifying step bypassing the formation of free radicals and so protecting tissues against mutagens, carcinogens and cytotoxics. NQO1 can recycle the membrane antioxidants ubiquinone and vitamin E. NQO1 is induced in a "stress response", for example in hypoxic conditions, along with other enzymes, including the glutathione S-transferases which conjugate hydrophobic electrophiles and reactive oxygen species, UDP-transferases which catalyse the conjugation and thus excretion of xenobiotics.
NQO1 may play a role in cancer prevention since it is overexpressed in many cancerous tissues compared to normal tissue, suggesting possible contribution of NQO1 to tumor progression through promotion of proliferation and metastases by which acts to detoxify and protect the cell from toxins and mutagens. NQO1 expression may contribute to resistance to chemotherapeutic agents such as doxorubicin, 5-fluorouracil, cisplatin and gemcitabine in some human cancer. Thus, some studies revealed that suppression of NQO1 activity increased the chemosensitivity of cholangiocarcinoma, urogenital and pancreatic cancer cells. The inhibition of NQO1 with dicoumarol was suggested to stimulate formation of superoxide, oxidative stress and subsequent suppress cell growth and induction of apoptosis.
NQO1 has attracted considerable attention as a potential candidate for targeted anticancer therapy once NQO1 can activate certain xenobiotics, such as MMC, orthonaphthoquinones and aziridinylbenzoquinones. This is of interest since drugs that are effectively bioactivated by NQO1 may allow tumour cytotoxicity without corresponding high levels of toxicity to normal tissues. Reduction of quinones by NQO1 may lead to either production of a reactive alkylating species by rearrangement, or hydroquinones may auto-oxidise leading to the production of reactive oxygen species and toxicity. The reductive cycle can continue along both pathways unless the system becomes anaerobic at which time oxygen radical production decreases and the semiquinone can accumulate. A correlation exists between the cytotoxicity of these bioreductive compounds, NQO1 activity, an increase in the pro-apoptotic protein and the induction of apoptosis. Although, bioreductive quinones are more active in tumour cells that overexpress NQO1, there is still some effect on cells without NQO1, presumably due to single electron enzymatic reduction. Conversely, it would be expected that individuals with the NQO1 polymorphism, and thus no active function, would have a higher susceptibility to developing cancer. Lack of NQO1 is associated with an increased risk of developing adult leukaemia and paediatric leukaemia. In cancer patients, the NQO1 polymorphism may be associated with an increased risk of chemotherapy-related myeloid leukaemia. In addition, individuals with absent NQO1 activity do have increased susceptibility to bone marrow suppression after environmental exposure to benzene and benzene-like compounds. In NQO1 knock-out mice, no detectable change in phenotype is observed and they appear indistinguishable from wild-type mice, however they are more susceptible to quinone toxicity and have increased sensitivity to the development of skin and visceral tumours.
NQO1 is able to physically associate with p53 suggesting that a protein protein interaction may be responsible for the stabilization of p53 by NQO1. In addition to the roles of NQO1 in direct detoxification of quinones and in antioxidant defense, the interaction of NQO1 with p53 may represent an additional mechanism that contributes to the chemosensitivity of NQO1 leading to cell killing in human tumor cells.
Cell Culture and Treatment- The human colon carcinoma cell line HCT116 and human prostate cancer cell lines PC3 were purchased from American Type Culture Collection (Manassas, VA). HCT116 cells were grown in media consisting of modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (Hyclone Inc., Logan, UT), 200 Î¼M L-glutamine (Invitrogen), and 1% penicillin-streptomycin-neomycin (PSN) antibiotic (Invitrogen). PC3 cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin mixture, 1 mmol/ L of sodium pyruvate, 10 mmol/L of HEPES, 1% NEAA mixture (Cambrex), 1% MEM vitamin mixture (Cellgro), and 2 mmol/L of L-glutamine. All Cells were grown in a 5% CO2 atmosphere at 37Â°C.
Drug and radiation treatment- Doxorubicin and 5-Fluorouracil were purchased from Bedford Laboratory (Bedford, OH) and Sigma, respectively. Doxorubicin or Dox (0.1, 0.34, 1 and 5 Î¼M; Sigma Aldrich, St. Louis, MO) was added to complete medium and incubated for various intervals (1, 3, 6, 12, and 24 h). 5-Fluorouracil or 5-FU (25, 50 and 100 Î¼M; Sigma Aldrich, St. Louis, MO) added to complete medium and incubated for 24 h. MG132 or protease inhibitor (0.1, 0.5 and 1 Î¼M; Calbiochem) added to complete medium and pre-incubated for 4 h. A 120 kv X-ray machine (Faxitron Xray Corporation) was used to radiate cells, with a dose rate of 2, 4 and 6 Gy.
Reagents- Unless otherwise stated, all antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal NQO1 antibody was purchased from Abcam (Cambridge, MA). Both control siRNA and p53 siRNA were purchased from Santa Cruz Biotechnology. p53 expression vector (pcDNA/p53) was purchased from Invitrogen.
Transient Transfection- PC3 cells were grown for 24 h in RPMI 1640 medium supplemented with fetal bovine serum, sodium pyruvate, HEPES, NEAA mixture, MEM vitamin mixture, and L-glutamine with no antibiotics. 70-80% confluent cells were transfected with plasmids after a Lipofectamineïƒ’ transfection protocol as directed by the manufacturer. Cells were transfected with 0.1, 1 and 5 Âµg of p53 cDNA construct in pcDNA plasmid vector or pcDNA (-p53) vector alone as control. Twenty-four hours after transfection, the cells were washed twice with PBS and incubated in fresh medium. Cells were grown for another 24 h prior to doxorubicin treatment. One and twenty-four hours post-treatment, the cells were washed with PBS and processed for NQO1 activity.
HCT116 cells were grown for 24 h in modified Eagle's medium supplemented with fetal bovine serum, and L-glutamine with no antibiotics. siRNAs (4 nM) were transfected using Transfectinïƒ’ for 48 h (Santa Cruz Biotechnology) according to the manufacturer's protocol. siRNAs were designed or obtained from commercial sources to interfere with the expression of p53.
Western blot analysis- Total cell extracts were prepared by collecting attached and floating cells in 1x cell lysis buffer with phosphatase inhibitor cocktail and protease inhibitor cocktail kit (Pierce Biotechnology, Rockford, IL). Protein concentration of the cell samples was determined using the Bradfort reagent kit (Bio-Rad) with bovine serum albumin as standard. For Western blot analysis, 50 Î¼g of protein was denatured by heating 95Â°C for 5 min in 10% SDS sample buffer, loaded onto 10% SDS-polyacrylamide gels, and then transferred electrically to a nitrocellulose membrane. The transfer efficiency was assessed by incubation with 0.1% Ponceau solution. The membrane was washed with distilled water until the dye disappeared completely. The membrane was blocked in 5% (wt/vol) nonfat milk 0.1% Tween TBS buffer pH 7.8 (10 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 0.05% (v/v) Tween 20) for at least 1 h at room temperature. After a short wash with TBS-T buffer, the membranes were incubated in the primary antibody for at least 2 h at room temperature or overnight at 4Â°C. The primary antibodies were diluted in TBS-T buffer containing 5% nonfat dried milk at a dilution range of 1000-5000 with the following primary antibodies: anti-MDM2, anti-p53 and anti-Î²-actin (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-NQO1 (Abcam, Cambridge, MA). The membranes were then washed three times each for 10 min with TBS-T. The membranes were incubated with the secondary antibody at a dilution range of 2000-10,000 for 1-2 h at room temperature. The membranes were washed twice with TBS-T buffer for 10 min and once with PBS for 5 min. Anti-Î² actin antibody was used to show equal loading of the protein in the Western blot analyses. Proteins were detected using the ECLïƒ’ system (Amersham Biosciences). The Quantity Image Pro Plus analyzer software program was used for quantitative densitometric analysis.
Immunoprecipitation-Immunoprecipitation studies were performed on HCT116 cells with radioimmune precipitation buffer (9.1mM Na2HPO4, 1.7mM NaH2PO4, 150mM NaCl, pH 7.4, 1% (v/v) Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 Âµg/ml phenylmethylsulfonyl fluoride, 1 Âµg/ml aprotinin). The antibodies used for immunoprecipitation were mouse anti-p53 (FL), rabbit anti-NQO1 and mouse anti-MDM2. One mg of cellular protein was incubated at 4 Â°C overnight with 2 Âµg of corresponding antibodies. After incubation with the antibody, 20 Âµl of protein A/G (Santa Cruz Biotechnology) was added to the reaction mixture of the antibody and nuclear extract and rotated for 2 h at 4 Â°C. Immunoprecipitates were collected by centrifugation at 2500 x g for 5 min, followed by washing three times with radioimmune precipitation buffer. Following the final wash, all of the adhering liquids with the protein A/G beads were removed. Samples were then resuspended in the 1x Laemmli buffer, boiled, and subjected to SDS polyacrylamide gel electrophoresis (10% gel (w/v)), and transferred onto a nitrocellulose membrane; the immunoprecipitated proteins were then detected by Western blotting.
NQO1 activity assay- NQO1 assay is performed essentially according to a previously described method (Buranrat et al, 2009). Cells were seeded at 7.5Ã- 103 cells per well in flat-bottomed 96-well cultured plates overnight. After cells were cultured for the designated time, cells were lysed with 50 Âµl solution containing 0.8% digitonin and agitated on shaker at room temperature for 10 min. 25 Âµl of 0.55% dicoumarol was added into culture wells designated as baseline activity wells while the corresponding paired wells were added with DDW. After that, all wells were added with 200 Âµl of reaction mixture (the following stock solution was prepared for each set of assay: 7.5 ml of 0.5 M Tris-Cl (pH 7.4), 100 mg of bovine serum albumin, 1 ml of 1.5% Tween-20 solution, 0.1 ml of 7.5 mM FAD, 1 ml of 150 mM glucose-6-phosphate, 100 Âµl of 50 mM Î²-NADP, 275 unit of yeast glucose-6-phosphate dehydrogenase, 45 mg MTT, and distilled water to a final volume of 150 ml and menadione (1 Âµl of 50 mM menadione dissolved in acetronitrite per milliliter of reaction mixure) was added just before the mixture is dispensed into the microtiter plates. A blue color developed and the plates were placed into a microplate reader with filter wavelength of 620 nm and readings were made at 0.5 min interval for about 10 min. The slope of the optical readings with times represents the activity of the reaction. NQO1 activity was the activity of sample subtracted with activity of the corresponding baseline activity. Using the extinction coefficient of MTT formazan of 11,300 M-1 cm-1 at 610 nm and correction for the light path of the microplate, activity of NQO1 was expressed as Âµmol/min/mg protein.
Animals- Wild-type (WT) mice are in the C57BL/6 background and were initially generated in the laboratory of Dr.Tyler Jacks at the Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA. Male mice between 10 and 12 weeks old were used in all studies. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Kentucky.
Doxorubicin treatment and tissue collection- Mice were treated with a single dose of 20 mg/kg of Doxorubicin-Adriamycin (Doxorubicin HCl, from Bedford Laboratories, Inc., Bedford, OH) (DOX) or saline via intraperitoneal injection (IP). Three days after treatment, mice were anesthetized using Nembutal\sodium solution (65 mg/kg) (Abbott Laboratories, North Chicago, IL). The colon and liver were excised and immediately processed for ultrastructural studies or frozen in liquid nitrogen for NQO1 activity studies.
Data are expressed as mean ï‚± SE of three separated experiments. An analysis of variance was used to determine significant differences between each experimental group. Statistical analysis was performed using STATA program software. The level of significance was set at * p<0.05.
Chemotherapeutic agents induce p53 inactivate NQO1 activity- To validate the effect of chemotherapeutic agents (Doxo and 5-FU) on NQO1 protein and enzyme activity. The NQO1 activity in HCT116 cells to chemotherapeutic agents were explored using enzymatic assay. HCT116 cells were expose to Doxo (0.01, 0.34, 1 and 5 uM) in time-course for 1, 3, 6, 12, 24 hr and 5-FU (25, 50 and 100 uM) for 24 hr. As shown in Fig 1A, 1B and 1C, the inactivation of NQO1 activity was apparent with in high concentration of chemotherapeutic agents when compared with non treatment group (Doxo: 1 and 5 uM for every time point, 5-FU: 50 and 100 uM). When cells were treated with Doxo and 5-FU, both chemotherapeutic agents show increased p53 and NQO1 protein level in a dose-dependent manner by use Western blot analysis. These results suggest that the chemotherapeutic agents are induced p53 inactivated NQO1 activity in colon cancer cells.
siRNA inhibition of p53 wild-type in HCT116 cells abolishes the inactivate NQO1 activity- To determine the role of p53 mediated inactivation of NQO1 activity in colon cancer cells, p53 siRNA was transfected into p53 wild-type HCT116 cells. The effect of p53 siRNA was confirmed by Western blot analysis (Fig 3a). Inhibition of p53 expression by p53 siRNA showed only a slight increased NQO1 activity (Fig 3b).
Overexpression of p53 wild-type in PC3 cells increases the chemotherapeutic agents effect of inactivate NQO1 activity- To verify the role of of p53 in the inactivate NQO1 activity, we use PC3 cells (prostate cancer cells) that represent null p53 or p53-/- then were treated with Doxo (0.1-5 uM) for 1 hr and 24 hr (Fig 2A) showed NQO1 activity not difference when compared with control. These results demonstrate that p53 is an important for suppress NQO1 activity.
Next, we then tasted whether the expression of p53 was inactivate NQO1 activity when p53 overexpression was combined with Doxo treatment. We transduced p53 cDNA and pcDNA (control vector) in to PC3 cells by various concentration of vector (0.1, 1 and 5 ug). The protein expression of p53 and also NQO1 was verified by Western blot analysis (Fig 2B). Expression of p53 cDNA in PC3 cells conferred the cells more inactivate NQO1 activity (Fig 2b and 2c) and also re-produced the inactivate NQO1 activity effect of Doxo observed in p53 expressing cells (Fig 2C).
It has been shown that the presence of p53 in the cells was due to NQO1 inactivation. To further verify the suppressive effects of p53 on NQO1 activity with the physical interaction between these two proteins. We performed immunoprecipitation studies using antibody to p53 followed by Western blot analysis (Fig 4). The results show that p53 antibody is able to immunoprecipitate NQO1 but not MDM2 in both chemotherapeutic agents (Doxo (Fig 4a, 4b) and 5-FU (Fig 4c)), which is confirmed by Western blot analysis. These results suggest that p53 interacts with endogenously NQO1.
MDM2 dose not play a major role in the NQO1 activity inactivation- Because overexpression of p53 wild-type suppress NQO1 activity, we tested whether the NQO1 activity inactivation is mediated by p53 expression. For this purpose, HCT116 cells were pre-treated with MG132 (a proteasome inhibitor) for 30 min and the co-treated with Doxo 1 uM. The results show that treatment with MG132 also increased MDM2 and p53 protein levels by Western blot analysis (Fig 5a), also suppression of NQO1 activity even presence or absence Doxo.
To further confirm the effect of MDM2 is not mediated NQO1 inactivation, we used immunoprecipitation using antibody to MDM2 followed by Western blot analysis. The results show that MDM2 antibody is not able to immunoprecipitate NQO1 in both treatments; MG132 treated alone and co-treated with Doxo, which is confirmed by Western blot analysis. These results suggest that the presence or absence of MDM2 dose not contribute significantly to the suppression role of NQO1 activity.
The radiosensitization induce p53 but dose not effect of NQO1 inactivation- To evaluate the effect of radiation treatment in p53 wild-type cancer cells, HCT116 cells were cultured and plated in the same RPMI-1640 medium and then cells were exposed to graded doses of x-radiation (0, 2, 4 and 6 Gy). When the radiation shows p53 protein level increased in dose-dependent manner (Fig 6a). By the way, the radiation conferred a not difference NQO1 activity in the both time of treatment 1 and 24 hr (Fig 6b and 6c). These results suggest that the radiation affect on colon cancer cells is enhanced p53 protein but not mediated inactivate NQO1 activity.
Chemotherapeutic agents enhance p53 level inactivation NQO1 activity in vivo- To verify the effect of chemotherapeutic agent on NQO1 activity in vivo. P53 wild-type mice were separated into 2 groups: control and Doxo treated group (20 mg/kg body weight). Three-days after the initiation of the treatment, the liver and colon were collected for Western blot analysis and NQO1 activity. Doxo increased p53 and NQO1 protein levels and then further decrease NQO1 activity in both liver and colon (Fig 7a and 7b). These results confirm that chemotherapeutic agent induce p53 suppress NQO1 activity in vivo.