SEL120

Effect of Imipramine on radiosensitivity of Prostate Cancer: An In Vitro Study

Songul Barlaz Us, Fatma Sogut, Metin Yildirim, Derya Yetkin, Serap Yalin, Sakir Necat Yilmaz & Ulku Comelekoglu

KEYWORDS
Imipramine; prostate cancer; radiotherapy; cell index; oxidative stress; EAG1 channel

Introduction

Prostate cancer is the most common cancer and major cause of cancer death for males in the world (1,2). RAD is one of the treatment methods and widely used as a curative treatment for local pros- tate cancer (3–5). However, prostate cancer cells are resistant to RAD in a number of patients. The combination of radiosensitizer agent and RAD in prostate cancer may increase the effectiveness of treatment with minimal side effects (6).The voltage sensitive ether `a-go-go potassium (EAG 1) channel is the voltage-gated potassium channel that plays a role in
oncogenesis. In recent studies, it has explored the role of EAG1 in the control of cell proliferation (7–10). EAG1 protein expression has been detected in several cell lines derived from human malignant tumors, such as neuroblastoma, melanoma, breast, cervical, and prostate carcinoma. Specific inhibition of EAG1 expression by or by nonspecific blockers lead to a reduction of tumor cell proliferation in vitro (11). Reactive oxygen species (ROS) are generated during metabolic processes in a normal cell. Increased ROS generation has been associated with tissue injury or DNA, protein and lipid damage (12). The term oxidative stress is used to mean to the imbalance between levels of ROS and the antioxidant Defense system. Organism is protected against the harmful ROS activity by antioxidant system. Some clinical studies have indicated that increased ROS is related to pros- tate cancer and that some antioxidants have the potential to protect from prostate cancer (13).

Several studies have identified depression as a risk factor for cancer, but some studies have reported no association between cancer and depression (14–18). IMI, a chemical name 10,11- Dihydro-N, N-dimethyl-5H-dibenz [b, f] azepine- 5-propanamine hydrochloride (C19H24N2.HCl), is a tricyclic synthetic antidepressant. It is used in the treatment of major depressive disorders and neuropathic pain (19). Besides their antidepres- sant effects, tricyclic antidepressant drugs have been shown to have antineoplastic effects (20). They cause the apoptotic cell death and inhibit proliferation of malignant tumor cells (21,22). Additionally, it was shown that when present in the growth medium of EAG1-expressing tumor cells, IMI slows cell proliferation (23). There are few studies investigating the antineo- plastic effect of IMI in the literature (24–26). But there is no study to investigate effect of IMI, RAD and IMI plus RAD on prostate cancer cells. The aim of this study was to investigate the radiosensi- tizing effect of IMI, a tricyclic antidepressant, on the DU145 prostate cancer cell line.

Material and methods

Cell culture

The human DU-145 prostate cancer cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and pre- served in our laboratory. Cells were routinely maintained in phenol red RPMI, supplemented with 10% FBS and 1% penicillin-streptomycin complete RPMI) at 37 ◦C in a water-saturated atmosphere of 5% CO2 incubator. The medium was refreshed every 3 days and, up to approxi- mately 80–90% confluence, cells were detached from the petri dish with 0.25% trypsin-EDTA solution for subculture at a ratio of 1:3.

Doses of RAD and IMI

DU145 cells were exposed to 6 Gy radiation dose with 8 MeV electron energy at a dose rate of 300 MU/min as a single fraction (Siemens, PRIMUSTM, Germany) (27–29). Compared with the photon energy, the effect of build-up in elec- tron is less. Therefore, electron energy was used in the irradiation. A 5 mm bolus was placed on the petri dish to obtain a uniform dose distribution. IMI (IMI hydrochloride ≥99%; CAS Number: 113-52-0; EC Number: 204-030-7) was purchasedfrom Sigma Aldrich Chemical Co (St Louis, MO, USA). Dose of IMI was determined using data obtained preliminary experiments. Different doses of IMI (10 nM, 100 nM, 1 mM, 20 mM, 50 mM and 75 mM) were incubated DU145 prostate cancer cells. Then cell index values were determined at 72 h by using xCELLigence DP system (ACEA Biosciences). IC50 value (the concentration of IMI that inhibits cell proliferation by 50%) was calcu- lated from dose-response curve by using the GNUPLOT package program (http://www.gnuplot. info). IC50 value was found to be 2.15 mM for DU145 prostate cancer cells. In this study, we used 1 mM dose of IMI. The cells were divided into 4 groups. Group 1 was the control group. In this group cells were left untreated. Group 2 was the IMI group, in which 1 mM IMI was given to the prostate cells in this group. Group 3 was the RAD group, in which the prostate cells were radiated. In group 4 both IMI and RAD were given together (IMI + RAD). All cells were incubated 24 hours after RAD with IMI.

Cell proliferation assay
The growth curves of DU-145 prostate cancer cells were (adhesion, proliferation and stationary phase) were established according to the cellular density at seeding using cell impedance measure- ments with the xCELLigence DP real time cell analysis system (ACEA Biosciences, San Diego, CA, USA). The xCELLigence DP system is a Real-Time Cell Analyzer based on the assessment of cell-impedance variations. First, 100 ll of cell culture medium (RPMI supplemented with 10% FBS) was added to each well of E-plate 16. The E-plate 16 was then connected to the system to check for proper electrical contacts and to obtain background impedance readings in the absence of cells. Meanwhile, the DU-145 cells were resus- pended in the appropriate cell culture medium, then, 100 ll of every cellular density was added to the wells containing 90 ll of culture medium in order to determine the optimum cell concen- tration. After 30 min incubation at room tem- perature, the E-plate 16 was placed onto the RTCA SP Station located inside the incubator (5% CO2; at 37 ◦C) for continuous impedance recording. The viability and proliferation of the cells were monitored every 15 min in the first 24 h and monitored every 60 min for up to 100 h. Measured electrical impedance were translated as a dimensionless parameter, the Cell Index (CI).

Analysis of cell cycle
Cell cycle and apoptosis analysis kit (BD 558662 and BioLegend 640932) was used according to the manufacturer’s instructions. Briefly, cells were plated in 6-well plates and treated with different objective compounds in experimental RPMI for 72 h. After incubation, both the suspension and the adherent cells were collected into flow cytome- try tube and centrifuged at 1000 g for 5 min to obtain cell pellets. And then, cells were washed with precooling PBS, and incubated with propi- dium iodide (PI) staining solution (0.5 mL staining buff, 25 lL PI staining solution, and 10 lL RNAase A) for 30 min at 37 ◦C in the dark. Analysis was performed on a FACS ARIA III ana-
lyzer (BD Biosciences, Franklin Lakes, NJ, USA). The tests were performed in four independent experiments. The percentage of cells distributed in different phases, sub-G0, G0/G1, S and G2/M, were calculated using FACS Diva software.

Apoptosis assessment
Cell apoptosis ratio was assessed using Annexin V-FITC apoptosis detection kit (BioLegend 640932) according to the manufacturer’s instruc- tions. After treated with objective compounds the same as cell cycle analysis, both the adherent and floating cells were harvested with trypsinization free of EDTA and washed with precooling PBS. Then the cells were incubated with staining solu- tion (195 lL Annexin V-APC binding buffer, 5 lL Annexin V- APC and 10 lL PI) for 20 min at room temperature in the dark. The cell apop- tosis was analyzed with a BD FACS ARIA III within 1 h and calculated by dot plot analysis with the built-in software. Generally, Annexin V- APC positive cells were considered to be under- going apoptosis including early apoptotic cells (Annexin V+/PI—) and necrotic or late apoptotic cells (Annexin V+/PI+), and those negative for APC were considered to be alive. Recordings of EAG1 channel currents using whole cell patch- clamp technique Whole-cell recordings were acquired from DU145 prostate cancer cells with the patch-clamp technique (30) using an Multiclamp 700 B amplifier (Axon Instrument, CA, USA) and data analyzed with Clampfit 9.2 (Axon Instruments, CA, USA) software. Borosilicate glass pipettes (WPI, USA, model 1B150F-4) were pulled by a horizontally puller (Sutter Instruments Co. P-97). Electrode resistance ranging between 2–5 MX were used in the records. Pipettes and cells were visualized by an inverted microscope (BM-37XB, U-Therm, International (H.K.) Limited). External solution contained 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2) and internal solution con- tained 150 mM KCl, 1 mM MgCl2 and 5 mM ethylene glycol tetraacetic acid (EGTA) and 10 mM HEPES (pH 7.2). Experiments were performed at room temperature (23 ◦C to 25 ◦C). The current amplitudes were determined by depolarizing pulses from a holding potential of —40 mV to voltage ranging between -100 mV and +100 mV in 20 mV steps.

Evaluation of lipid peroxidation and antioxidant activity

The cells in all groups were dissolved in RIPA lysis and extraction buffer (Sigma Aldrich, Chemical Co, St Louis, MO, USA), then cell lysates were homo- genized with IKA homogenizer (T 25 Ultra-Turrax, IKA Werke GmbH, Staufen, Germany) finally cen- trifuged (Hettich Mikro 22 R, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany) for 10 min at 10000 rpm at 4 ◦C. The supernatant was used for the measurement of antioxidant enzyme activities and lipid peroxidation determination. Malondialdehyde (MDA) levels were deter- mined by thiobarbituric acid reaction. A Carry 50 spectrophotometer (Varian, Inc., Palo Alto, CA, USA) was used, at a wavelength of 532 nm. The principle of the method depended on the meas- urement of the pink color produced by the inter- action of barbituric acid with MDA elaborated as a result of lipid peroxidation. The colored reac- tion with 1,1,3,3- tetraethoxy propane was used as the primary standard (Sigma-Aldrich Chemical Co, MO, USA). The determination of MDA lev- els was performed by the method of Yagi (31) as a nanomol per milligram of protein (nmol/mg protein). Catalase (CAT) activity was measured according to procedure of Aebi (32) Catalase- mediated decomposition of H2O2 was measured in spectrophotometer at 240 nm. The principle of measurement of SOD enzyme activity is based on the measurement of the color absorbed at 560 nm by nitroblue tetrazolium (NBT) of the superoxide radicals produced by xanthine oxidase in the pres- ence of xanthine (33). Activity of glutathione perox- idase (GSH-Px) was measured spectrophotometrically at 340 nm. The method was based on the changes in absorbance resulting from the conversion of NADPH into NADP. Protein level in cells was deter- mined based on the procedure described by Lowry et al. (34). Enzyme activities were expressed in U/mg protein.

Statistical analysis

Data were analyzed using SPSS 17.0 statistical package program (SPSS Inc., Chicago IL, USA). The checks of normality of variables were tested with Kolmogorov-Smirnov test. Statistics analysis for comparisons of groups were evaluated by using ANOVA test. Repeated measures analysis were used for comparison of the data within the groups. Data was expressed as mean ± standard deviation. p < .05 was considered statistically significant. of ion channels that lead to oxidation, nitrosylation and/or nitration of specific amino acid residues, or indirectly modulate channel function by affecting gene transcription, traffic, and signal paths (43). CAT, SOD, and GSH-Px enzymes are considered primary endogenous antioxidants. These antioxi- dants protect cells against ROS induced damage during metabolism in living organisms. While SOD metabolizes the superoxide radical to H2O2 and molecular O2, GSH-Px catalyzes the reaction of reduction of organic hydroperoxides or hydro- gen peroxide by the GSH and CAT provides decomposition of H2O2 to H2O and O2 (13). In this study, we measured CAT, SOD and GSH-Px activities in all groups and we observed that both IMI and RAD inhibited antioxidant enzyme activ- ities. But inhibitor effect of IMI was higher than RAD and combined IMI and RAD. The previous studies have shown that IMI is a potent antioxi- dant (44). Some cancer chemopreventive com- pounds having antioxidant properties have been documented to potentiate RAD–induced cytotoxic effects on cancer cells. In this regard, Raffoul et al. showed that phytochemical soy isoflavones, via their antioxidant activities, could be used as potent radiosensitizers to enhance the efficacy of RAD- mediated suppression of the proliferation and metastatic ability of cancers, including prostate cancer (45). In another study by Raffoul et al. It was demonstrated that both soy and genistein inhibited the proliferation of human prostate can- cer cells and these effects were enhanced when soy or genistein was combined with RAD (46). Contrary to Raffoul et.al’s findings, our results suggest that antioxidant IMI is not radiosensitizer, but it is an potent anticarcinogenic agent. The lipid peroxidation with the formation of reactive compounds can lead to changes in the permeability and fluidity of the membrane lipid bilayer and can dramatically alter cell integrity. Lipid peroxidation modifies the environment of not only membrane proteins including ion chan- nels and may influence their functional efficiency. In our study MDA levels in IMI, RAD and IMI + RAD groups were significantly higher than control group. The reduction in EAG1 channel current may be also associated with increased lipid peroxidation as well as an increase in oxida- tive stress. This suggestion is consistent with Starks’s report (2005), who reported that the pre- dominant effect of increased MDA levels is inhib- ition of membrane functions (47). Conclusion Our findings showed that co-treatment with IMI and RAD did not enhance radiosensitivity of DU145 prostate cancer cell but interestingly, treatment of IMI alone was more effective in reducing prostate cancer cell proliferation. In addition, in this study, the effect of RAD on EAG1 channel currents was first shown and it was found that tricyclic antidepressant IMI inhib- ited EAG1 channel activity in DU145 prostate cancer cells more than RAD and combined IMI and RAD. This increased inhibition was thought to be associated with high oxidative stress and apoptosis induced by IMI in cancer cells. Results from this study suggested that IMI may be an alternative to RAD in the treatment of prostate cancer. However, further research is needed to thoroughly determine molecular mechanism. 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