The human colon cancer cell line HCT116, kindly provided by Dr G. Marra (Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland), was maintained in McCoy’s 5A medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% heat-inactivated (56°C, 30 min) fetal calf serum (FCS, Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), and 50 μg/ml gentamicin (Euroclone, Milan, Italy).

The human colon cancer cell line HT-29, obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), was routinely grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich), supplemented with 2 mM L-glutamine, 60 μM gentamicin and 10% heat-inactivated FCS (Sigma-Aldrich), hereafter referred to as complete medium D (CMD). Both HCT116 and HT-29 cells growing as plastic adherent cells were removed using a solution of 0.05% trypsin and 0.02% EDTA in phosphate-buffered saline (PBS) without calcium and magnesium.

The human Jurkat CD4⁺ T cell leukemia cell line and the human promyelocytic leukemia cell line HL-60 were obtained from the ATCC and were cultured at 37°C in a 5% CO₂ humidified atmosphere in RPMI-1640 (Sigma-Aldrich), supplemented with 10% heat-inactivated FCS (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), and 50 μg/ml gentamicin (Euroclone) hereafter referred to as complete medium (CM).

The human melanoma cell line M10, kindly donated by Dr D. Del Bufalo (Regina Elena Cancer Institute, Rome, Italy), was originally established from a cutaneous metastasis of advanced melanoma. The line, regularly cultured in CM in our laboratory, shows fairly high levels of MGMT activity (14). M10 cells were removed from continuous culture as described for the colon cancer cell lines.

Peripheral blood mononuclear cells were obtained by centrifugation on a Ficoll-Hypaque (Lymphoprep™, Axis-Shield, Oslo, Norway) gradient of buffy coats from healthy blood donors, and washed twice in RPMI-1640 medium. Non adherent mononuclear cells (NAMNC) were separated by plastic adherence at 37°C for 1 h in CM. Informed consent was obtained from blood donors according to our institutional guidelines.

Orlistat was purchased from Sigma-Aldrich, dissolved in sterile DMSO (Sigma-Aldrich) at the concentration of 20 mM, aliquoted and stored at −70°C until use. Lomeguatrib (LM) was a generous gift from Professor G. Margison (Centre for Occupational and Environmental Health, University of Manchester, Manchester, UK). The compound was dissolved in DMSO at the concentration of 10 mM, aliquoted and stored at −70°C until use. Temozolomide (TMZ) was supplied by Schering-Plough Co. (Milan, Italy) and was always prepared freshly in culture medium and added immediately to cell suspensions, because the drug readily decomposes in aqueous solution.

MGMT activity was determined by measuring the transfer of ³H-methyl groups from a DNA substrate to the MGMT protein (15). Briefly, cell pellets (1×10⁶ cells) were re-suspended in 1 ml of lysis buffer (0.5% CHAPS, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 3 mM dithiothreitol, 100 mM NaCl, 10% glycerol) supplemented with a cocktail of protease inhibitors (Roche, Mannheim, Germany) and incubated for 30 min at 4°C. Cell lysates were then centrifuged at 18,000 × g for 10 min at 4°C. Aliquots of supernatants were then diluted in 50 mM Tris-HCl buffer, pH 8.3, containing 1 mM EDTA, and 3 mM dithiothreitol, and incubated with 10 μg of ³H-methylated-DNA at 37°C for 1 h. DNA was then hydrolyzed by heating samples at 75°C for 45 min, in the presence of 1 N perchloric acid, and protein precipitated using 1 mg bovine serum albumin as carrier. Pellets were washed with 1 N perchloric acid, re-suspended in 0.01 N NaOH, and radioactivity measured in a liquid scintillation counter (TRI-CARB 1900, Packard Instruments Co., Meriden, CT, USA), after addition of scintillation liquid (Ultima Gold, Packard Instruments Chemical Operation, Groningen, The Netherlands). Protein concentration in supernatants was evaluated according to the method of Bradford using the Bio-Rad Protein Assay Dye reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA) and bovine serum albumin as standard. MGMT activity was expressed in terms of fmoles of ³H-methyl groups transferred per mg of protein in cell extract.

Cells were washed with PBS. The cell pellet was suspended in 100 μl extraction buffer [(12.5 mM Na₂HPO₄, pH 7.2; 94 mM NaCl; 50 mM NaF; 1% di-Triton X-100; 2 mM EGTA, 1% protease inhibitor cocktail (Sigma-Aldrich), 0.5% saponin (Sigma-Aldrich) and 1 mM Na₃VO₄: (Sigma-Aldrich)] kept on ice for 30 min, and centrifuged for 30 min at 15,000 × g at 4°C in an Eppendorf microcentrifuge. The protein concentrations were determined using Bio-Rad Protein Assay Dye. The samples were stored at −80°C until use.

The proteins were heated in a boiling water bath for 2 min and separated by running cell extracts on 10% polyacrylamide pre-cast gels (NuPAGE^® Novex Bis-Tris, Invitrogen, Life Technologies, Grand Island, NY, USA), using XCell SureLock™ Mini-Cell apparatus (Invitrogen) following the instructions of the producer. At the end of the electrophoretic separation, proteins were transferred to Hybond-ECL nitrocellulose filters (GE Healthcare Life Sciences, Pittsburg, PA, USA) by electrotransfer with miniblot apparatus (Invitrogen). The transfer was carried out at 25 V overnight. Thereafter, the membranes were incubated with 3% non-fat dry milk (Bio-Rad) in Tris-buffered saline (TBS) at pH 7.5, for 60 min at room temperature and then incubated with an anti-actin rabbit polyclonal antibody (Sigma-Aldrich) diluted 1:3,000 in TBS containing 0.05% Tween-20 (TBST) and mouse monoclonal antibody against MGMT (Millipore, Billerica, MA, USA) diluted 1:500 in TBST for 60 min. The membranes were washed twice with TBST and incubated for 45 min with the secondary alkaline phosphate-conjugated antibody. Bands were developed using westernBreeze chemiluminescent immunodetection kit (Invitrogen), according to the manufacturer’s instructions. The film was scanned on a GS-710 Calibrated Imaging Densitometer and analyzed by means of Quantity One software, version 4.1.1 (Bio-Rad Laboratories). The optical density (OD) of MGMT and actin bands was expressed as arbitrary units. MGMT expression was evaluated on the basis MGMT:actin ratio (OD-R).

Total RNA was extracted, from 2×10⁶ viable cells using 1 ml of TRI Reagent solution (Ambion, Life Technologies, Monza, Italy) according to the manufacturer’s instructions. RNA purity and concentration were checked with a NanoQuant Infinite M200 instrument (Tecan Group Ltd., Mannedorf, Switzerland). Two microliters of RNA was purified by clearance of DNA traces using Turbo DNA-free kit (Applied Biosystems, Life Technologies, Monza, Italy). cDNA was synthesized from 2 μg of RNA by reverse transcription using TaqMan RT kit (Applied Biosystems), according to the manufacturer’s instructions. Five microliters (i.e., 2 μg) of cDNA/sample was amplified, according to the manufacturer’s instructions, by the Stratagene Mx3005P qPCR System (La Jolla, CA, USA) using a TaqMan gene expression assay kit (Applied Biosystems, Assay-On-Demand: code no. Hs.00172470_m1 for MGMT). Levels of MGMT mRNA were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene expression (Applied Biosystems, code no. 4326317E). All RT-qPCR reactions were performed in triplicate.

The relative quantification of MGMT was performed using the comparative threshold cycle (C_T) method (as described by Applied Biosystems protocol) that uses an arithmetic formula (2^−ΔΔCT). ΔΔC_T is the difference between the ΔC_T of the sample under investigation and the ΔC_T of the calibrator sample. The RNA obtained from Jurkat cells was chosen as the calibrator sample.

Statistical significance among different mean values was assessed using two-tailed Student’s t-test analysis.

In order to determine the concentration range of orlistat to be utilized in the experiments illustrated in the present report, a concentration/effect study was performed using Jurkat target cells. Leukemic cells were cultured in the presence of graded concentrations of orlistat for two days. Control cultures were exposed to DMSO alone at the concentration corresponding to that utilized for orlistat 40 μM. At the end of the in vitro treatment, leukemic cells were lysed and subjected to western blot (WB) analysis. The results, illustrated in Fig. 1A, indicate that orlistat, at the concentration of 40 μM, was able to reduce by >50% the MGMT level, whereas little or no effect was found when lower concentrations were used. These findings were qualitatively confirmed by four additional experiments conducted with Jurkat cells (data not shown), although downregulation of MGMT protein expression appears to be noticeably variable (i.e., inhibition among different experiments ranging from ~30 to 70%). Based on these results, we decided to utilize the concentration of 40 μM as the standard concentration of the agent in the majority of the experimental procedures herein described.

Time-course studies were performed on Jurkat cells in order to explore the effect of orlistat on MGMT expression when the leukemic blasts were exposed to the drug for a total of 72 h. Incubation of target cells with orlistat (40 μM) started on day 0, and MGMT levels were tested by WB analysis on days 1, 2 and 3. The results (Fig. 1B) show that downregulation of MGMT by orlistat is not detectable on day 1, starts on day 2 and persists up to day 3.

Similar concentration/effect and time-course studies were conducted with the epithelial colon cancer HCT116 cells. The results, illustrated in Fig. 1C, confirm that downregulation of MGMT expression is produced by orlistat in these cells at the concentration of 40 μM. This effect was even more pronounced after 4-day exposure to the agent, showing a decline of MGMT protein concentration >70%.

Analysis of the effect of continuous exposure to orlistat was extended to normal NAMNC and to other tumor cell types, such as melanoma M10, promyelocytic leukemia HL-60, and colon cancer HT-29 cell lines. After 2-day incubation with the agent, the results, illustrated in Fig. 1D, confirm that the drug provoked an ~50% reduction of MGMT level in all target cells except for melanoma M10 cells that showed no downregulation of the protein.

Among well established MGMT inhibitors, LM represents one of the best molecule able to rapidly and potently inhibit MGMT functional activity (16). This agent behaves as a false substrate that binds MGMT irreversibly and promotes its degradation through the ubiquitin-proteasome pathway (17). This mechanism implies that MGMT function is almost immediately suppressed by LM and that the inactive protein undergoes a fairly rapid degradation within the cell. Thereafter, the cell starts to synthesize de novo the MGMT protein in order to restore its DNA repair functional activity. These observations prompted us to compare the effect of orlistat with that of LM on the kinetics of MGMT protein level decline in a short-term assay. Previous studies pointed out that 10 μM LM was able to downregulate markedly the MGMT function in target cells (18). Therefore, we employed this inhibitor concentration in all experiments described in the present report. Jurkat cells were incubated with orlistat (40 μM) alone, or with LM alone. At different time intervals (i.e., 1, 3, 6 and 24 h) the cells were collected and tested for MGMT protein expression by WB analysis. The results, illustrated in Fig. 2A, confirmed that no noticeable downregulation of MGMT protein level was found at all time intervals explored when target cells were treated with orlistat. In contrast, a manifest decline of MGMT protein was detected in LM-treated cells as early as 3 h after start of treatment, followed by a further reduction of ~70% at 24 h.

Additional experiments were conducted with the intent to establish whether orlistat could be able to influence the functional activity of MGMT with a mechanism conceivably similar to that of LM. Therefore, HCT116 and Jurkat cells were treated with 40 μM orlistat or 10 μM LM, and MGMT activity was tested as early as 2 h after start of treatment. As expected, and in line with previous reports (16,18), LM provoked a drastic decrease of MGMT function in both cell lines, whereas minimal or no significant downregulation of the enzymatic activity was detected in Jurkat and HCT116 samples, respectively, exposed to orlistat (Table I).

Further studies were performed to evaluate the possible influence of orlistat and LM on MGMT gene transcription. RT-qPCR performed on Jurkat cells at different times after exposure to orlistat (40 μM) or to LM (10 μM) pointed out that no remarkable changes of MGMT transcription respect to DMSO-treated controls were detectable in orlistat-treated cells, for ≤24 h after start of treatment (Fig. 2B). However, a transient increase of MGMT mRNA was found at 48 and 72 h. Limited downregulation of MGMT expression was found instead, shortly after exposure to LM. In this case, mRNA values returned to normal levels at 72 h of incubation with the MGMT inhibitor.

The results illustrated in the previous section favor the hypothesis that orlistat inhibits MGMT expression with a mechanism distinct from that involved in LM activity. Therefore it was of interest to explore the combined effects of the two agents on MGMT protein levels of normal or leukemic cells.

Normal NAMNC were exposed to 40 μM orlistat alone, to 10 μM LM alone, or to orlistat + LM for 6 consecutive days. WB analysis was performed on cells collected shortly (i.e., on day 1), or late (i.e., on day 6) from the onset of drug treatment. The results, shown in Fig. 3A, demonstrate that, in line with the previous results, orlistat alone had marginal effect on MGMT protein levels on day 1, whereas LM reduced MGMT expression by >50%. Combination of the two agents was slightly additive, as confirmed by other two experiments that gave similar results. When the WB assay was performed on day 6, both orlistat and LM showed remarkable inhibitory effects, that were higher when the two drugs were used in combination.

In a second set of experiments Jurkat cells were incubated with either 40 μM orlistat or 10 μM LM or with the combination of the two drugs. In this case, however, target cells were incubated with the agents for 24 h only. Thereafter, the cells were washed carefully and all cultures were reconstitute in culture medium alone. MGMT protein levels were determined on days 2, 3 and 6 of total culture time. The results, illustrated in Fig. 3B, show that progressive decline of MGMT levels occurred during the time-course study in all groups treated with LM, alone or in combination with orlistat, with the combination being particularly active on day 2. However, since this type of experiment was conducted with target cells exposed to drugs for 24 h only, it was possible to detect a complete recovery of MGMT levels in the group treated with orlistat alone. On the contrary, the effect of LM, alone or in combination was fully detectable up to 6 days, as confirmed by other two experiments (data not shown).

It is known that methylation of oxygen 6 of DNA guanine by endogenous or exogenous DNA methylating agents is an important step in chemical carcinogenesis leading to mutation-mediated malignant transformation (19,20). MGMT is crucially involved in the physiological defenses against carcinogenesis consequent to methylation of O⁶-guanine and undergoes inactivation and proteasome-mediated degradation after removing, in a stoichiometric reaction, methyl adducts from O⁶-guanine (21). We therefore decided to investigate the effects of a combined treatment with low concentrations of a mono-methylating agent followed by orlistat on MGMT protein levels.

We directed our attention to TMZ, a triazene compound that is activated in vitro and is capable of inducing DNA O⁶-guanine methyl adducts (20). This drug has been found recently to increase and accelerate tumorigenesis in intestinal cells of mice in a murine model of Lynch syndrome (22). Removal of TMZ-induced methyl adducts operated by MGMT is necessarily followed by depletion of the enzyme. Therefore, it is not surprising that TMZ downregulates the enzymatic activity (23) and the cellular levels of MGMT as a result of the DNA repair process.

In the first set of experiments, the ability of the triazene compound to downregulate the level of MGMT protein in a short-term assay was tested using HCT116 target cells that were incubated with graded concentrations of TMZ (40–320 μM) for 24 h. No influence of TMZ was detectable on MGMT levels when drug concentrations were lower than 160 μM (data not shown). At the concentration of 160 or 320 μM the drug provoked a marginal or a strong (close to 70%) downregulation of MGMT, respectively (Fig. 4A), thus confirming that TMZ was able to produce the expected enzymatic depletion in our model.

Drug combination experiments were performed on HCT116 cells that were exposed to TMZ (40 μM) for 24 h. Thereafter, the drug was removed by thorough washing and the cells were incubated for additional three days with 40 μM orlistat. The results confirm that MGMT protein levels were not affected by 40 μM TMZ, either when the protein amounts were evaluate at the end of drug treatment (Fig. 4B, day 1) or after three additional days of culture in the absence of the drug (Fig. 4B, day 4). Conversely, 3-day incubation with orlistat alone (i.e., not preceded by TMZ treatment) reduced MGMT by ~50% respect to untreated controls. Of particular interest is the finding that the group treated in sequence with TMZ and orlistat showed an overall reduction of MGMT protein level of approximately 75% with respect to untreated control. A similar set of experiments were performed with the colon cancer cell line HT-29 (Fig. 4C). In these studies, cancer cells were exposed to TMZ (40 μM) for 24 h, washed and incubated with orlistat (40 μM) for four days (i.e., from days 1–5). The results of WB analysis performed on day 1 show that TMZ, at the concentration of 40 μM, did not influence MGMT level. On day 5 of culture, HT-29 cells treated with 40 μM TMZ for 24 h, followed by 4-day culture without drugs, did not display changes of MGMT protein levels. Orlistat alone, at the concentration of 40 μM produced marginal (~8%) inhibition, probably due to lower susceptibility to orlistat of HT-29 cell line with respect to that of HCT116 after 4-day incubation with the agent. However, most importantly, orlistat combined with TMZ at the same molar concentration (i.e., 40 μM), reduced MGMT levels by >60% respect to control, suggesting that the combined effects of the two agents on MGMT could be synergistic.