Cancer Investigation, 32:226–235, 2014 ISSN: 0735-7907 print / 1532-4192 online C 2014 Informa Healthcare USA, Inc. Copyright DOI: 10.3109/07357907.2014.905587
ORIGINAL ARTICLE
Apoptosis-Inducing Effects of Melissa officinalis L. Essential Oil in Glioblastoma Multiforme Cells
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Rafaela Muniz de Queiroz,1 Christina Maeda Takiya,1 L´ıvia Paes Tavares Pacheco Guimar˜aes,1 Gleice da Grac¸a Rocha,1 Daniela Sales Alviano,2 Arie Fitzgerald Blank,3 Celuta Sales Alviano,2 and Cerli Rocha Gattass1 Laborat´orio de Imunologia Celular, Instituto de Biof´ısica Carlos Chagas Filho, Centro de Ciˆencias da Sa´ude, Universidade Federal do Rio de Janeiro, Cidade Universit´aria, Rio de Janeiro, RJ, Brazil,1 Laborat´orio de Estruturas de Superf´ıcie de Microorganismos, Instituto de Microbiologia Professor Paulo de Gois, Centro de Ciˆencias da Sa´ude, Universidade Federal do Rio de Janeiro, Cidade Universit´aria, Rio de Janeiro, RJ, Brazil,2 Departamento de Engenharia Agronˆomica, Universidade Federal de Sergipe (UFS), S˜ao Crist´ov˜ao, SE, Brazil3 In general, the cytotoxic effect of many chemotherapeutics is mediated by apoptosis. The apoptotic process is characterized by a series of morphological alterations and biochemical reactions leading to DNA fragmentation and breakdown of the cell into apoptotic bodies. Activation of initiator caspases (caspases 8 and 9), triggered either by activation of death receptors on the plasma membrane (extrinsic pathway) or by stress signals/alterations of the mitochondrial membrane potential (intrinsic pathway), induces the activation of effector caspases (caspases 3, 6, and 7), the main executors of apoptosis (5). It was recently proposed that the generation of reactive oxygen species (ROS) also plays an essential role in the apoptotic cell death induced by cytotoxic drugs (6). Glioblastoma multiforme (GBM) is the most common and aggressive glioma, representing 50% of all gliomas and more than 40% of all central nervous system (CNS) tumors. The standard GBM treatment, which consists of surgical resection, radiation and/or chemotherapy, is rarely curative (7). The location of GBM in the central nervous system and the lack of clear margins prevent complete resection of the tumor. Furthermore, tumor cell resistance to chemoradiation contributes to the poor prognosis of the disease. Thus, despite the significant improvement in early diagnosis, the tumor’s aggressive growth and resistance to available therapies have hindered changes in the outcome of GBM (8). Drug resistance is one of the major obstacles to successful GBM treatment. Among several mechanisms underlying GBM drug resistance, the expression of transporter proteins from the ABC superfamily is the most relevant. These proteins actively remove drugs from cells, decreasing their intracellular concentration and preventing death. Although all transporter proteins are present in GBM cells, members of the MRP family seem to be important for drug resistance,
Current therapies for glioblastoma multiforme (GBM) are not effective. This study investigated the activity of the M. officinalis essential oil (EO) and its major component (citral) in GBM cell lines. Both EO and citral decreased the viability and induced apoptosis of GBM cells as demonstrated by DNA fragmentation and caspase-3 activation. Antioxidant prevented citral-induced death, indicating its dependence on the production of reactive oxygen species. Citral downmodulated the activity and inhibited the expression of multidrug resistance associated protein 1 (MRP1). These results show that EO, through its major component, citral, may be of potential interest for the treatment of GBM. Keywords: Chemotherapy, Gliobastoma multiforme, Melissa officinali essential oil, Citral
INTRODUCTION Lemon balm (M. officinalis L.) is a medicinal plant largely used in Europe and the Mediterranean region as a tea, in aqueous and alcoholic extracts or steeped in wine. Its essential oil (EO) is considered to be responsible for the pharmacological properties of M. officinalis such as antitumoral and antibacterial (1, 2). Interestingly, geranial and neral, two isomers of the monoterpene citral (3,7-dimethyl-2,6octadienal), represent more than 85% of the M. officinalis EO (1). Citral, a key component of essential oils extracted from several herbal plants, is used as a food additive and a fragrance material in cosmetics. It also displays biological activities, including antitumor activity (3, 4). Although the antineoplastic activity of M. officinalis essential oil and citral has already been described, their activity on glioblastoma multiforme (GBM) cell lines has not been investigated.
Correspondence to: Cerli Rocha Gattass, PhD, Laborat´orio de Imunologia Celular, IBCCF, CCS Bloco G, UFRJ, Rio de Janeiro, RJ, Brazil, email: [email protected] Received 26 January 2013; revised 13 January 2014; accepted 13 March 2014.
M. O FFICINALIS EO Induces Apoptosis in Glioblastoma
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and their expression has been correlated with poor prognosis (9–11). Therefore, new therapies, able to overcome mechanisms of drug resistance, should be of interest for GBM treatment. In recent years, several substances isolated from plants were shown to modulate drug resistance in cancer cells (12–14). We have previously shown that the M. officinalis EO was active against several cancer cell lines (1), including a drug-resistant lung cancer cell line. Here, we investigated the in vitro effects of M. officinalis EO and of its major component, citral, on human GBM cell lines. The data obtained demonstrated that EO induces apoptosis of GBM cells and that this effect may be due to its major component, citral. These results support the potential usefulness of M. officinalis EO and citral for the treatment of patients with recurrent GBM.
MATERIALS AND METHODS Reagents The essential oil (EO) from M. officinalis L. (exsiccate #46,008 deposited at the Herbarium of the Federal University of Uberlandia, MG, Brazil) was obtained as described previously (1). Briefly, leaves from a culture of M. officinalis L. established at the Research Station “Campus Rural da UFS,” Federal University of Sergipe, Brazil, were harvested at 09:00 hr and dried at 40◦ C until complete dehydration, and the essential oil was obtained by hydrodistillation in a Clevenger-type apparatus. The composition of the oil has been described elsewhere (1). Citral (MW 152.23) provided by Sigma-Aldrich Chemical Co. (Saint Louis, MO, USA), was dissolved in dimethyl sulfoxide (DMSO, Sigma Chemical Co., Saint Louis, MO, USA), stored at −20◦ C and diluted in culture medium for use. 3-(4,5 dimethylthiozol2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma Chemical Co., Saint Louis, MO, USA), verapamil, penicillin, streptomycin, N-acetyl-L-cysteine (NAC), propidium iodide (PI), and rhodamine 123 (Rho123) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). 5-carboxifluorescein diacetate (5-CFDA) and 2 ,7 -dichlorofluorescein diacetate (H2 –DCFDA) were obtained from Calbiochem (San Diego, CA, USA). MK-571 was provided by Enzo Life Science, Inc. (Farmingdale, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and trypsin-EDTA were from Gibco BRL (Carlsbad, CA, USA). Caspase-3 and -9 assay kits (CaspGlow) were from Biovision (Mountain View, CA, USA). 4 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Apoptag Fluorescein Direct In Situ Apoptosis Detection from Millipore (Billerica, MA, USA). Cell cytotoxicity assay Human glioblastoma multiforme cell lines A172 and U87 were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U penicillin and 100 mg/mL streptomycin in disposable plastic bottles at 37◦ C with 5% CO2 . Cells were subcultured using trypsin-EDTA every 3–4 days. C 2014 Informa Healthcare USA, Inc. Copyright
Cell cytotoxicity was evaluated using the MTT assay. Cells were plated at a density of 1 × 104 cells/well (for 48 hr assay) or 2 × 104 cells/well (for 24 hr assay) in 96-well plate overnight and then treated with medium or various dilutions of the EO or citral (1:200,000; 1:100,000; 1:50,000 or 1:25,000). Four hours before the end of the treatment, cells were incubated with MTT (2.5 mg/mL) and kept in the dark at 37◦ C until the end of the treatment. The formazan produced by the reduction of MTT by viable cells was dissolved in DMSO, and the optical density was measured with an ELISA reader (BenchMark, Bio-Rad, CA) at 570 nm (reference filter 630 nm). Experiments were repeated at least three times. The results were expressed as percentage of the control, considered to be 100%. To determine whether the use of DMSO in first dilution of EO and citral would interfere with cell viability, the same assay was performed with DMSO in the same dilutions used for the OE and citral. No interference on cell viability was observed with the higher concentration (0.2%) of DMSO used. For cotreatment experiments, plated cells were incubated for 48 hr with 2.5 μg/mL cisplatin (CIS), 100 nM vincristine (VCR) or 0.5 μM doxorubicin (DOX) in the presence of absence of different dilutions of citral (1:100,000; 1:50,000, and 1:30,000) and cell viability was assessed by MTT. The dilutions of OE used: 1:200,000; 1:100,000; 1:50,000; and 1:25,000 correspond to 4.6; 9.2; 18.4; and 36.8 μg/mL, respectively, while the dilutions of citral: 1:200,000; 1:100,000; 1:50,000; 30,000; and 1:25,000 correspond to 4.2; 8.4; 16.9; 28.1, and 33.7 μg/mL or 27.7; 55.4; 110.8; 182.8, and 221.6 μM, respectively. This work was preceded by an evaluation of the effect of different concentrations of citral on GBM cell viability. We did not include aldehydes control experiments in this study because an antitumoral effect of citral with concentration higher than that used here was shown previously (3,4). Apoptosis assays Apoptosis was assessed by DNA fragmentation and TUNEL assays. DNA fragmentation was evaluated by cell cycle analysis using flow cytometry. Twenty-four hours after plating, the cells (1 × 104 cells/well - 96-well plate) were treated with medium (control) or various dilutions of citral (1:200,000; 1:100,000; 1:50,000 or 1:25,000 equivalent to 4.2; 8.4; 16.9 and 33.7 μg/mL or 27.7; 55.4; 110.8 and 221.6 μM, respectively) and incubated for different times. Next, cells were harvested, resuspended in a hypotonic fluorescent solution (50 mg/mL PI and 0.1% Triton X-100 in 0.1% Na citrate buffer) for 1 hr in the dark at 4◦ C. The cell cycle was analyzed by flow cytometry (FL-2) (FACSCalibur, Becton Dickinson, San Jose, CA) to determine the sub-G0/G1 DNA content. Subdiploid populations were considered to be apoptotic. Data acquisition and analysis were controlled by CellQuest software, version 3.1f. The results are presented as representative histograms and as the mean ± SD of the percentage of the fragmented DNA. The TUNEL assay was performed using a commercial kit R Fluorescein Direct In Situ Apoptosis Detection (ApopTag Kit, cat. #S7160) according to the instructions of the manufacturer (Millipore, Billerica, MA, USA). An image analysis system composed of a digital camera (Evolution Media
R. Muniz de Queiroz et al.
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Cybernetics Inc., Bethesda, MD) and a computer with the graphical interface software Q-Capture 2.95.0, version 2.0.5, (Silicon Graphic Inc., USA) coupled to an epifluorescence microscope was used to obtain high-quality images (2048 × 1536 pixel buffer) using the 40× objective lens. At least a hundred cells per cover slip were captured, and the percentage of TUNEL-positive nuclei was calculated from the total DAPIpositive nuclei. The results are expressed as the percentage of reactive tissue in the total area (mean ± SD). Caspase activation assay Activation of caspase-3 and -9 were assayed using CaspGlow commercial kits according to the instructions of the manufacturer (Biovision, Mountain View, CA, USA). In brief, cells (5 × 104 /well) were incubated for 24 hr with medium (control) or different dilutions of EO (1:25,000 equivalent to 36.8 μg/mL) or citral (1:50,000 or 1:25,000 equivalent to 16.9 or 33.7 μg/mL or to 110.8 or 221.6 μM, respectively) before being harvested, centrifuged, and suspended in the caspase assay solution. This solution contained a molecule conjugated to FITC that is cell permeable, nontoxic, and binds irreversibly to the activated caspase. After 1 hr of incubation (37◦ C, 5% CO2 ), cells were washed twice with washing buffer, and the percentage of caspase-activated cells was analyzed by flow cytometry (FL-1). The results are presented as representative histograms and as the mean ± SD of the Ratio of Fluorescence Intensity (RIF) compared with the control.
(50 μM verapamil) or MRP1 (50 μM MK-571), or the indicated concentrations of citral (1:50,000; 1:30,000 or 1:25,000 equivalent to 16.9; 28.1 or 33.7 μg/mL, or to 110.8, 182.8 or 221.6 μM, respectively). The cells were washed in PBS, harvested, and kept on ice until flow cytometry analysis. The results are presented as representative histograms or as the mean ± SD of the mean fluorescence intensity (MIF). Expression of MRP1 protein was assessed by western blotting. Cells (5 × 105 ) were platted in 6-wells plates and 24 hr later incubated with medium or citral (1:50,000 or 1:30,000 equivalent to 16.9 or 28.1 μg/mL or to 110.8 or 182.8 μM, respectively) for another 24 hr. Total cell lysates were made by lysing harvested cells in lysis buffer (50 mM Tris pH 7.4, 0.5% Nonidet P-40, 1 mM EDTA, 150 mM NaCl, and proteases inhibitors added at the time of preparation). Samples were fractionated by 8% polyacrylamide gel. The proteins were transferred on to nitrocellulose membrane (Bio-Rad Laboratory Inc., Hercules, CA, USA) which was blocked for 1 hr with Tris-buffered saline containing 0.1% Tween 20–milk 5%. Next, the membrane was incubated overnight at 4◦ C, with anti-MRP1 (A23, Alexis Biochemicals,
Quantification on Reactive Oxygen Species (ROS) ROS was determined by flow cytometry in cells treated with H2 -DCFDA. Cells (2 × 104 cells/well) were plated for 24 hr and then exposed to medium (control) or citral (1:25.000) for different times. After the desired time, cells were harvested, washed with PBS, pH 7.4, and resuspended in 0.16 mL PBS containing 20 μM H2 -DCFDA. After 30-min incubation at 37◦ C, the production of ROS was evaluated by flow cytometry (FL-1). The results are presented as the mean ± SD of Ratio of Fluorescence Intensity (RIF) compared with the control. To assess the role of ROS in citral citotoxicity A172 cells were treated for 24 hr with media, pretreated or not for 2 hr with the ROS inhibitor N-acetyl-L-cysteine (NAC, 10 mM) and then incubated with citral (1:30.000 equivalent to 28.1 μg/mL or 182.8 μM). Cell viability was estimated by the MTT assay and results were presented as percentage of control. Activity and expression of MDR proteins The activity of the MDR proteins was determined by the accumulation of specific substrates as described previously (15). Rho123 and 5-carboxyfluorescein diacetate (CFDA), a nonfluorescent molecule that is converted into the fluorescent carboxy-fluorescein (CF) by intracellular esterases, were used to measure the activity of Pgp/ABCB1 and MRP1/ABCC1, respectively. For each experiment, cells (2 × 104 /well) were plated for 24 hr at 37◦ C/5% CO2 to allow the culture to stabilize. Cells were incubated for 30 min with medium (to measure autofluorescence), 400 nM Rho123 or 3 μM CFDA in the presence of medium, inhibitors of Pgp
Figure 1. Effects of M. officinalis essential oil (EO) and citral on the viability of GBM cells. A172 and U87 cells (104 /well) were incubated with medium or different dilutions of EO or citral [1:200,000 (200), 1:100,000 (100), 1:50,000 (50), 1:25,000 (25)]. After 48 hr, cell viability was assessed by MTT as described in the M&M. Results are expressed as a percentage of the control and represent the mean ± SD of three independent experiments. ∗ , p < .05; ∗∗ , p < .01. OE dilutions correspond to 4.6; 9.2; 18.4; and 36.8 μg/mL respectively, while citral dilutions correspond to 4.2; 8.4; 16.9 and 33.7 μg/mL or 27.7; 55.4; 110.8 and 221.6 μM, respectively. Cancer Investigation
M. O FFICINALIS EO Induces Apoptosis in Glioblastoma for 1 hr at room temperature. After washing the blot was visualized by chemiluminescence detection using the enhanced chemiluminescence (ECL) system (GE Healthcare, Tokyo) according to manufacture’s instructions.
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SanDiego, CA) or anti-β-actina (Sigma Chemical Co., Saint Louis, MO, USA) antibodies. Following a series of washes with Tris-buffered saline - 0.1% Tween 20, a secondary horseradish peroxidase antibody was added and incubated
Figure 2. EO and citral induce apoptosis in GBM cells. Cells (104 /well) were treated with medium or citral [1:200,000 (200), 1:100,000 (100), 1:50,000 (50), 1:25,000 (25)] for the indicated times. DNA fragmentation was assessed by flow cytometry in cells stained with PI, and the sub-G1 peak was considered apoptotic. (a) Representative histograms of A172 cells treated with increasing dilutions of citral after 48 hr. (b and c) Quantitative analysis of DNA fragmentation of A172 and U87 cells treated with the indicated doses of citral for 12, 18, 24, and 48 hr. (d) Quantitative analysis of apoptotic A172 cells treated with EO or citral at 1:25,000 for 24 hr then submitted to the TUNEL assay as described in M&M. The data represent the average of at least three independent experiments ± SD. ∗ , p < .05; ∗∗ , p < .01. Citral dilutions correspond respectively to: 27.7, 55.4, 110.8, and 221.6 μM. C 2014 Informa Healthcare USA, Inc. Copyright
R. Muniz de Queiroz et al.
Statistical analysis All the data reported here are expressed as the mean ± SD from three independent experiments. A significant difference from the respective control for each experimental test condition was assessed by one-way analysis of variance (ANOVA) using GraphPad Prism 4.0 software. Values of p < .05 were considered statistically significant.
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RESULTS M. officinalis essential oil and citral inhibited the viability of GBM cells The cytotoxic effect of M. officinalis EO and citral on GBM cell lines A172 [Figure 1(A)] and U87 [Figure 1(B)] were analyzed by the MTT assay. Cells were treated with media and different dilutions of the oil (1:200,000; 1:100,000; 1:50,000 or 1:25,000 equivalent to 4.6; 9.2; 18.4 or 36.8 μg/mL, respectively) for 48 h, and cell viability was measured by the MTT assay. Treatment with EO reduced the number of viable cells in both cell lines in a dose-dependent manner (Figure 1 - white bars). Because neral and geranial, the two isomers of citral, represent more than 85% of the EO, we investigated whether the cytotoxic effect of EO is due to the action of these components. To do this, the viability of GBM cells treated with commercial citral (1:200,000; 1:100,000; 1:50,000 or 1:25,000 equivalent to 4.2; 8.4; 16.9; 28.1 or 33.7 μg/mL or to 27.7, 55.4, 110.8, or 221.6 μM, respectively) was determined
under the same conditions used for the EO (Figure 1 - black bars). The data showed that the effect of citral on the GBM cells lines U87 and A172 is similar to or even higher than that of the EO, suggesting that the activity of the EO against the tumor cells may be due to the action of citral. Essential oil and citral induce apoptosis in GBM cells Morphological analysis of GBM cells treated with EO or citral (1:50,000 equivalent to 18.4 μg/mL or 110.8 μM) for 48 hr showed a decrease in cell number accompanied by the presence of “blebs” in the plasmatic membrane, a cellular cyto-architecture indicative of apoptosis (data not shown). To further investigate this hypothesis, GBM cells were treated with different concentrations of citral, and DNA fragmentation was evaluated by flow cytometry. The sub-G1 peak of the cell cycle was considered apoptotic. As depicted in Figure 2(a), cell-cycle histograms of A172 showed that treatment with citral for 48 hr led to a significantly dose-dependent increase in the sub-G1 phase [Figure 2(b)]. The DNA fragmentation induced by treatment with citral was also timedependent [Figure 2(c)]. Because similar results were obtained with U87 [Figure 2(c)], only the A172 cell line was used in the next experiments. To confirm the indication of induction of apoptosis by citral and EO, A172 cells were submitted to the TUNEL assay. The data obtained demonstrated that treatment with both EO and citral (1:25,000 equivalent to 36.8 and 33.7 μg/mL for EO and citral, respectively) for
Figure 3. Activation of caspases by EO and citral. Plated A172 cells (5 × 104 /well) were treated for 24 hr with medium, EO [1:25,000 (25)] or citral [1:50,000 (50) and 1:25,000 (25)]. After treatment, cells were harvested, and caspase activity was measured using a commercial kit as described in M&M. The results show representative histograms and quantitative analysis of caspase-9 (a) and -3 (b). Results represent the average of three independent experiments ± SD. ∗ , p < .05; ∗∗ , p < .01. For OE and citral concentrations see legend for Figure 1. Cancer Investigation
M. O FFICINALIS EO Induces Apoptosis in Glioblastoma
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24 hr causes an increase in the number of cells positively labeled for TUNEL, confirming that both compounds induce apoptosis in GBM cells [Figure 2(d)]. To determine whether the induction of apoptosis in GBM cells was dependent on caspase activation, A172 cells were treated with EO (1:25,000 equivalent to 36.8 μg/mL) or citral (1:50,000 or 1:25,000 equivalent to 18.4 or 36.8 μg/mL or to 110.8 or 221.6 μM) for 24 hr, and the levels of activated caspase-9 and -3 were measured by a commercial kit (CaspGlow, Biovision) containing a fluorochrome conjugated to a molecule that irreversibly binds to the activated caspase. As shown in Figure 3(a and b), activation of caspase-9 and -3 were detected in cells treated with either citral or EO. Citral induces a dose-dependent activation of caspases. Apoptosis induced by citral is mediated by ROS To evaluate whether the generation of ROS was involved in citral-induced apoptosis, A172 cells were treated with citral (1:25,000 equivalent to 33.7 μg/mL or 221.6 μM) for different times, and H2 DCF-DA was used to examine ROS generation. As shown in Figure 4(a), treatment with citral led to a rapid increase in ROS generation that peaked at 6 hr and returned to normal levels at 24 hr. Evaluation of cell death, performed in a parallel experiment, revealed that DNA fragmentation begins to increase after 8 hr of citral treatment. Thus, the increase in the ROS levels precedes cell death [Figure 4(a)], suggesting a role for oxidative stress in citral-induced cell death. To confirm this hypothesis, cells were treated for 24 hr with citral (1:30,000 equivalent to 28.1 μg/mL or 182.8 μM) in the presence or absence of the antioxidant Nacetyl-cysteine (NAC), and cell viability was measured by MTT. As shown in Figure 4(b), cellular death induced by citral is completely blocked by the ROS scavenger, indicating that the generation of ROS is an essential process in citralinduced apoptosis. Citral inhibits MRP1 activity and expression Next, we investigated whether citral was able to modulate the activity of MDR transporter proteins expressed by A172 and U87 cells. The activity of Pgp/ABCB1 and MRP1/ABCC1 in these cells was evaluated by their ability to retain substrates of the pumps. To do this, cells were loaded with substrates for Pgp (Rhodamine-123) or MRP1 (5-CFDA), in the presence or absence of specific inhibitors (verapamil or MK571, respectively), and the cell fluorescence, indicative of the pump activity, was measured by flow cytometry [Figure 5(a and b)]. While no increase in fluorescence was observed when cells were incubated with Rho-123 in the presence of verapamil [Figure 5(b)], an increase in fluorescence was observed when cells were incubated with 5-CFDA in the presence of MK571 [Figure 5(a)]. These data demonstrated that MRP1/ABCC1 is active in A172 cells but Pgp/ABCB1 is not. U87 cells showed the same pattern of activity for these pumps [Figure 6(a and b)]. To investigate the effect of citral on MRP1, cells were treated with 5-CFDA and different concentrations of citral (1:50,000, 1:30,000 or 1:25,000 equivalent to 16.9, 28.1 or 33.7 μg/mL or to 110.8, 182.8, 221.6 μM) for 30 min, and the intracellular fluorescence was measured [Figure 5(C)]. The C 2014 Informa Healthcare USA, Inc. Copyright
Figure 4. Involvement of ROS in citral-induced cell death. (a) ROS generation and DNA fragmentation. A172 cells (2 × 104 /well) were treated with medium or citral (1: 25,000) for different times, and ROS generation and DNA fragmentation were measured by flow cytometry. Results are presented as the ratio of the fluorescence of citral over control cells [ROS production (•)] and the percentage of DNA fragmentation (). (b) Effect of NAC. A172 cells were treated with citral (1:30.000) for 24 hr in the presence or absence of NAC (10 mM), and cell viability was estimated by the MTT assay as described in Section 2 of the M&M. Results are expressed as percentage of the control and represent the mean ± SD of three independent experiments. ∗ , p < .05; ∗∗ , p < .01; and ∗∗∗ , p < .001. Citral dilutions correspond respectively to: 221.6 and 182.82 μM.
increase in fluorescence observed in presence of citral [Figure 5(d)], indicates that this substance is able to downmodulate the activity of MRP1. Indeed, the modulatory effect of citral is approximately 50% of that observed with the commercial inhibitor MK571. To probe if the modulation of MRP1 by citral was due to a decrease in protein expression, A172 cells were treated with medium or citral (1:50,000 or 1:30,000 equivalent to 16.9 or 28.1 μg/mL or to 110.8 or 182.8 μM) for 24 hr and protein expression was evaluated by western blotting. As shown in Figure 5(e) citral decrease the expression of MRP1. To confirm the effect of citral on MRP1, U87 cells were treated with 5-CFDA and citral (1:25,000 equivalent to 33.7 μg/mL or 221.6 μM) for 30 min, and the intracellular fluorescence presence of citral [Figure 6(d)] indicates that this substance is able to downmodulate the activity of MRP1. The effect of citral was similar to that observed for A172. Effects of combination of citral with chemotherapy drugs Next, we evaluate if the combination of citral with antineoplastic drugs would decrease the survival of GBM cells. For this, A172 cells were cocultured for 48 hr with different dilutions of citral (1:100,000, 1:50.00, and 1:30,000 corresponding to 8.4, 16.9, and 22.1 μg/mL or to 55.4, 110.8, and 182.8 μM) and 2.5 μg/mL cisplatin (CIS), 100
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R. Muniz de Queiroz et al.
Figure 5. Activity of the efflux pumps. A172 cells (2 × 104 /well) were incubated for 30 min with medium (autofluorescence), 3 μM 5-CFDA or 400 ng/mL Rho-123 in the presence or absence of 50 μM MK751 (MK) or 50 μM verapamil (VRP), respectively. The retention of specific substrates by the cells was evaluated by flow cytometry. (a and b) are representative histograms of the activity of MRP1 (a) and Pgp (b). (c and d) show the effect of citral on the MRP1 pump. Cells were incubated for 30 min with medium (autofluorescence) or 3 μM CFDA in the presence or absence of 50 μM MK751 (MK) or increasing concentrations of citral [1:50,000 (Cit 50); 1:30,000 (Cit 30) or 1:25,000 (Cit 25)], and cell fluorescence was measured by flow cytometry. The results are expressed as representative histograms of cell fluorescence for each condition (c) and by the mean ± SD of the mean fluorescence intensity (MIF) obtained in three different experiments (d). (e) shows the effect of citral on MRP1 expression. Cells were incubated medium or citral 1:50,000 (50) or 1:30,000 (30) for 24 hr and lysates were submitted to western blot as described in M&M. Citral dilutions correspond respectively to: 110.8, 182.82, and 221.6 μM.
nM vincristine (VCR) or 0.5 μg/mL doxorubicin (DOX) and cell viability was assessed by MTT. Results show that cotreatments with citral do not seem to sensitize GBM cells to antineoplastic drugs. DISCUSSION Malignant central nervous system neoplasms, particularly GBM, are among the most lethal and intractable of human tumors. These tumors have defied all current therapeutic modalities, and patient prognosis is poor (8), emphasizing the need to explore new therapeutic strategies for treatment. Thus, the search for new drugs that are able to overcome GBM’s drug resistance mechanisms and prevent tumor recurrence is of great interest for GBM therapy (16). In the last few years, natural products have been recognized as an important source of new antineoplastic drugs (17). Accumulating evidence of the antitumor activity of essential oils (18,19) supports this material as one of these drug sources. In a previous paper (1), we showed that the EO of
M. officinalis was cytotoxic to several cancer cell lines but its effect on GBM cells was not investigated. Although the composition of the M. officinalis EO was described in this paper, the component responsible for the observed antitumor activity was not identified. The data presented in Figure 1 demonstrated that M officinalis EO is also able to cause cell death of established GBM cell lines (U87 and A172) and indicated that this effect is due to its major component citral. The capacity to induce cell death in cancer cell lines is not a property particular to M. officinalis EO. This effect has already been shown for EO from other sources (18, 19) as well as for citral (3, 4). Microscopic observation suggested that the cytotoxic effects of EO and citral on the GBM cell lines (U87 and A172) were due to the induction of apoptosis. Measurement of DNA fragmentation by cell cycle analysis [Figure 2(a–c)] and TUNNEL assays [Figure 2(d)] corroborated this hypothesis. The apoptotic effect of both EO and citral is dosedependent. M. officinalis EO and citral also induced the activation of caspase-9 and -3 [Figure 3 (a and b)], indicating that Cancer Investigation
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M. O FFICINALIS EO Induces Apoptosis in Glioblastoma
Figure 6. Activity of the efflux pumps. U87 cells (2 × 104/well) were incubated for 30 min with medium (autofluorescence), 3 μM 5-CFDA or 400 ng/mL Rho-123 in the presence or absence of 50 μM MK751 (MK) or 50 μM verapamil (VRP), respectively. The retention of specific substrates by the cells was evaluated by flow cytometry. (a and b) are representative histograms of the activity of MRP1 (a) and Pgp (b). (c and d) show the effect of citral on the MRP1 pump. Cells were incubated for 30 min with medium (autofluorescence) or 3 μM CFDA in the presence or absence of 50 μM MK751 (MK) or citral [1:25,000 (Cit 25)], and cell fluorescence was measured by flow cytometry. The results are expressed in (c) as representative histograms of cell fluorescence for each condition and in (d), as mean fluorescence intensity of the experiment shown in c. Results represent two experiments. Citral dilution corresponds, respectively, to 221.6 μM.
their apoptotic effect is caspase-dependent. These data are in agreement with studies showing the activation of caspase-3 by essential oils from other plants (19) and citral (3). Mitochondria play a central role in the apoptotic process induced by several drugs. The activation of caspase-9 [Figure 3(a)] indicated that the effects of M. officinalis EO and citral on GBM cells are mediated by the intrinsic (mitochondrial) pathway of apoptosis. In addition to the release of proapoptotic proteins, the generation of reactive oxygen species (ROS) is used by the intrinsic pathway to induce apoptosis in tumor cells. Due to the high content of ROS, it has been proposed that insults leading to further ROS generation would turn cancer cells very susceptible to ROS damage. Indeed, many cytotoxic agents employed in chemotherapy seem to exert their effects through ROS generation (6, 20, 21). Treatment of GBM cells with citral resulted in a rapid generation of ROS that preceded the induction of cell death [Figure 4(a)]. The addition of the antioxidant NAC prevented citral-induced death, corroborating the dependence of citralinduced death on ROS [Figure 4(b)]. C 2014 Informa Healthcare USA, Inc. Copyright
Expression of transporter proteins from the ABC superfamily is one of the most relevant mechanisms of drug resistance. Drug resistance is a severe limitation of the effect of chemotherapy on various malignancies. A 1998 study (22) showing that GBM patients subjected to chemotherapy have an increase in cells positive for Pgp/ABCB1 and MRP1/ABCC1, suggested a role for these ABC pumps in resistance to treatment. The expression of MRP1 is considered a negative prognostic factor for patients with GBM (10, 11). Indeed, high levels of transporter proteins were also identified in cancer stem cells, a cell population described in recurrent GBM patients (23). In a previous paper (1), we showed that the EO of M. officinalis caused cell death in several cancer cell lines including A549, a lung cancer cell line that expressed several members of the MRP family. Data presented in this paper show that M. officinalis OE and its major component, citral, induced apoptosis of GBM cell lines (A172 and U87) that expressed active MRP1. They also indicated that citral induces production of ROS and inhibits MRP1 expression and activity [Figures 4 and 5 (c, d, e)].
R. Muniz de Queiroz et al. drug resistance in GBM cell points to the potential use of these compounds for the development of new drugs for this neoplasia and as adjuvants for the treatment of glioblastoma multiforme.
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DECLARATION OF INTEREST
Figure 7. Effects of cotreatment of GBM cells with citral and chemetherapics. A172 cells (104 /well) were incubated with medium or different dilutions of citral [1:100,000 (100), 1:50,000 (50), 1:30,000 (30), equivalent to 55.4, 110.8, and 182.82 μM, respectively] in the presence or absence or 2.5 μg/mL cisplatin (CIS), 100 nM vincristine (Vin) or 0.5 μM doxorubicin (DOX). After 48 hr, cell viability was assessed by MTT as described in the M&M. Results are expressed as a percentage of the control and represent the mean ± SD of three independent experiments.
The authors report no declarations of interest. The authors alone are responsible for the content and writing of this article. This study was supported by grants from Fundac¸a˜o de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Instituto Nacional para Pesquisa Translacional em Sa´ude e Ambiente na Regi˜ao Amazˆonica/ Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico/MCT, Brazil (INCT-INPeTAm/CNPq/MCT). The authors thank the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq) for the graduate fellowships awarded to Rafaela Muniz de Queiroz, L´ıvia Paes Tavares Pacheco Guimar˜aes and Gleice da Grac¸a Rocha.
REFERENCES Recent studies showed that temozolomide, the first choice of chemotherapy drug for GBM treatment induces ROS production (21) and that resistance to this drug involved mitochondria dysfunction and a decrease in ROS production (24). Other studies showing that inhibition of MRP1 activity or expression (25–27) lead to an increase in the response of GBM cell lines to chemotherapeutic drugs indicating the importance of this protein in GBM drug resistance. Combination of citral and antineoplastic drugs does not seem to sensitize GBM cells as the resulting decrease in cell viability is equivalent to the added effect of both drugs (Figure 7). However, if a sensitizing effect does exist, it is hard to demonstrate since citral modulation of MRP1 activity was only observed at cytotoxic concentrations. In addition, it should be considered that the modulatory effect of citral on MRP1 activity is quite low [Figure 5 (c, d)] when compared to inhibitors used by other researchers (26,27). However, M. officinalis EO and citral are able to kill resistant cells that express MRP1 (this work) or overexpress Pgp (1) but have no effect on noncancer cells (3,28). Although the relationship between ROS production and inhibition of MRP1 activity by citral was not investigated in this work, data from the literature showed that compounds able to affect ROS or GSH production, altering the cell oxidative stress, may also affect MRP1 expression and/or its activity (29,30). Efficient therapeutic treatments for highly malignant gliomas, such as GBM, are lacking and patient prognosis is poor. Thus, despite the inclusion of targeted drugs in clinical protocols, no significant improvement over the standard protocol has been observed (7, 31). As cancer cells use several mechanisms to evade death, the simultaneous attack to multiple targets is important for a successful cancer therapy. Therefore, the results presented in this paper, showing that M. officinalis OE and citral affect two pathways of
1. de Sousa AC, Alviano DS, Blank AF, Alves PB, Alviano CS, Gattass CR. Melissa officinalis L. essential oil: antitumoral and antioxidant activities. J Pharm Pharmacol 2004;56:677–681. 2. H˘ancianu M, Aprotosoaie AC, Gille E, Poiat˘a A, Tuchilus¸ C, Spac A, St˘anescu U. Chemical composition and in vitro antimicrobial activity of essential oil of Melissa officinalis L. from Romania. Rev Med Chir Soc Med Nat Iasi 2008;112:843–847. 3. Dudai N, Weinstein Y, Krup M, Rabinski T, Ofir R. Citral is a new inducer of caspase-3 in tumor cell lines. Planta Med 2005;71:484–488. 4. Chaouki W, Leger DY, Liagre B, Beneytout JL, Hmamouchi M. Citral inhibits cell proliferation and induces apoptosis and cell cycle arrest in MCF-7 cells. Fundam Clin Pharmacol 2009;23:549–556. 5. Fulda S, Debatin KM. Apoptosis pathways in neuroblastoma therapy. Cancer Lett 2003;197:131–135. 6. Mao X, Yu CR, Li WH, Li WX. Induction of apoptosis by shikonin through a ROS/JNK mediated process in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells. Cell Res 2008;18:879–888. 7. Clarke J, Butowski N, Chang S. Recent advances in therapy for glioblastoma. Arch Neurol 2010;67:279–283. 8. Nishikawa R. Standard therapy for glioblastoma—a review of where we are. Neurol Med Chir (Tokyo) 2010;50:713–719. 9. Decl´eves X, Fajac A, Lehmann-Che J, Tardy M, Mercier C, Hurbain I, Laplanche JL, Bernaudin JF, Scheeemann JM. Molecular and functional MDR1-Ppg and MRPs expression in human glioblastoma multiforme cell lines. Int J Cancer 2002;98:173– 180. 10. Nakagawa T, Ido K, Sakuma T, Takeuchi H, Sato K, Kubota T. Prognostic significance of the immunohistochemical expression of O6-methylguanine-DNA methyltransferase, P-glycoprotein, and multidrug resistance protein-1 in glioblastomas. Neuropathology 2009;29:379–388. 11. Quezada C, Peig˜nan L, Segura R, Riquelme F, Melo R, Rojas DZ, Ayach F, San Mart´ın R, C´arcamo JG. Study of resistance to chemotherapy mediated by ABC transporters in biopsies of glioblastoma multiforme. Rev M´ed Chil 2011;139:415–424. 12. Alvarez AI, Real R, P´erez M, Mendoza G, Prieto JG, Merino G. Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response. J Pharm Sci 2010;99:598–617. Cancer Investigation
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ORIGINAL ARTICLE
Apoptosis-Inducing Effects of Melissa officinalis L. Essential Oil in Glioblastoma Multiforme Cells
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Rafaela Muniz de Queiroz,1 Christina Maeda Takiya,1 L´ıvia Paes Tavares Pacheco Guimar˜aes,1 Gleice da Grac¸a Rocha,1 Daniela Sales Alviano,2 Arie Fitzgerald Blank,3 Celuta Sales Alviano,2 and Cerli Rocha Gattass1 Laborat´orio de Imunologia Celular, Instituto de Biof´ısica Carlos Chagas Filho, Centro de Ciˆencias da Sa´ude, Universidade Federal do Rio de Janeiro, Cidade Universit´aria, Rio de Janeiro, RJ, Brazil,1 Laborat´orio de Estruturas de Superf´ıcie de Microorganismos, Instituto de Microbiologia Professor Paulo de Gois, Centro de Ciˆencias da Sa´ude, Universidade Federal do Rio de Janeiro, Cidade Universit´aria, Rio de Janeiro, RJ, Brazil,2 Departamento de Engenharia Agronˆomica, Universidade Federal de Sergipe (UFS), S˜ao Crist´ov˜ao, SE, Brazil3 In general, the cytotoxic effect of many chemotherapeutics is mediated by apoptosis. The apoptotic process is characterized by a series of morphological alterations and biochemical reactions leading to DNA fragmentation and breakdown of the cell into apoptotic bodies. Activation of initiator caspases (caspases 8 and 9), triggered either by activation of death receptors on the plasma membrane (extrinsic pathway) or by stress signals/alterations of the mitochondrial membrane potential (intrinsic pathway), induces the activation of effector caspases (caspases 3, 6, and 7), the main executors of apoptosis (5). It was recently proposed that the generation of reactive oxygen species (ROS) also plays an essential role in the apoptotic cell death induced by cytotoxic drugs (6). Glioblastoma multiforme (GBM) is the most common and aggressive glioma, representing 50% of all gliomas and more than 40% of all central nervous system (CNS) tumors. The standard GBM treatment, which consists of surgical resection, radiation and/or chemotherapy, is rarely curative (7). The location of GBM in the central nervous system and the lack of clear margins prevent complete resection of the tumor. Furthermore, tumor cell resistance to chemoradiation contributes to the poor prognosis of the disease. Thus, despite the significant improvement in early diagnosis, the tumor’s aggressive growth and resistance to available therapies have hindered changes in the outcome of GBM (8). Drug resistance is one of the major obstacles to successful GBM treatment. Among several mechanisms underlying GBM drug resistance, the expression of transporter proteins from the ABC superfamily is the most relevant. These proteins actively remove drugs from cells, decreasing their intracellular concentration and preventing death. Although all transporter proteins are present in GBM cells, members of the MRP family seem to be important for drug resistance,
Current therapies for glioblastoma multiforme (GBM) are not effective. This study investigated the activity of the M. officinalis essential oil (EO) and its major component (citral) in GBM cell lines. Both EO and citral decreased the viability and induced apoptosis of GBM cells as demonstrated by DNA fragmentation and caspase-3 activation. Antioxidant prevented citral-induced death, indicating its dependence on the production of reactive oxygen species. Citral downmodulated the activity and inhibited the expression of multidrug resistance associated protein 1 (MRP1). These results show that EO, through its major component, citral, may be of potential interest for the treatment of GBM. Keywords: Chemotherapy, Gliobastoma multiforme, Melissa officinali essential oil, Citral
INTRODUCTION Lemon balm (M. officinalis L.) is a medicinal plant largely used in Europe and the Mediterranean region as a tea, in aqueous and alcoholic extracts or steeped in wine. Its essential oil (EO) is considered to be responsible for the pharmacological properties of M. officinalis such as antitumoral and antibacterial (1, 2). Interestingly, geranial and neral, two isomers of the monoterpene citral (3,7-dimethyl-2,6octadienal), represent more than 85% of the M. officinalis EO (1). Citral, a key component of essential oils extracted from several herbal plants, is used as a food additive and a fragrance material in cosmetics. It also displays biological activities, including antitumor activity (3, 4). Although the antineoplastic activity of M. officinalis essential oil and citral has already been described, their activity on glioblastoma multiforme (GBM) cell lines has not been investigated.
Correspondence to: Cerli Rocha Gattass, PhD, Laborat´orio de Imunologia Celular, IBCCF, CCS Bloco G, UFRJ, Rio de Janeiro, RJ, Brazil, email: [email protected] Received 26 January 2013; revised 13 January 2014; accepted 13 March 2014.
M. O FFICINALIS EO Induces Apoptosis in Glioblastoma
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and their expression has been correlated with poor prognosis (9–11). Therefore, new therapies, able to overcome mechanisms of drug resistance, should be of interest for GBM treatment. In recent years, several substances isolated from plants were shown to modulate drug resistance in cancer cells (12–14). We have previously shown that the M. officinalis EO was active against several cancer cell lines (1), including a drug-resistant lung cancer cell line. Here, we investigated the in vitro effects of M. officinalis EO and of its major component, citral, on human GBM cell lines. The data obtained demonstrated that EO induces apoptosis of GBM cells and that this effect may be due to its major component, citral. These results support the potential usefulness of M. officinalis EO and citral for the treatment of patients with recurrent GBM.
MATERIALS AND METHODS Reagents The essential oil (EO) from M. officinalis L. (exsiccate #46,008 deposited at the Herbarium of the Federal University of Uberlandia, MG, Brazil) was obtained as described previously (1). Briefly, leaves from a culture of M. officinalis L. established at the Research Station “Campus Rural da UFS,” Federal University of Sergipe, Brazil, were harvested at 09:00 hr and dried at 40◦ C until complete dehydration, and the essential oil was obtained by hydrodistillation in a Clevenger-type apparatus. The composition of the oil has been described elsewhere (1). Citral (MW 152.23) provided by Sigma-Aldrich Chemical Co. (Saint Louis, MO, USA), was dissolved in dimethyl sulfoxide (DMSO, Sigma Chemical Co., Saint Louis, MO, USA), stored at −20◦ C and diluted in culture medium for use. 3-(4,5 dimethylthiozol2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma Chemical Co., Saint Louis, MO, USA), verapamil, penicillin, streptomycin, N-acetyl-L-cysteine (NAC), propidium iodide (PI), and rhodamine 123 (Rho123) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). 5-carboxifluorescein diacetate (5-CFDA) and 2 ,7 -dichlorofluorescein diacetate (H2 –DCFDA) were obtained from Calbiochem (San Diego, CA, USA). MK-571 was provided by Enzo Life Science, Inc. (Farmingdale, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and trypsin-EDTA were from Gibco BRL (Carlsbad, CA, USA). Caspase-3 and -9 assay kits (CaspGlow) were from Biovision (Mountain View, CA, USA). 4 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Apoptag Fluorescein Direct In Situ Apoptosis Detection from Millipore (Billerica, MA, USA). Cell cytotoxicity assay Human glioblastoma multiforme cell lines A172 and U87 were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U penicillin and 100 mg/mL streptomycin in disposable plastic bottles at 37◦ C with 5% CO2 . Cells were subcultured using trypsin-EDTA every 3–4 days. C 2014 Informa Healthcare USA, Inc. Copyright
Cell cytotoxicity was evaluated using the MTT assay. Cells were plated at a density of 1 × 104 cells/well (for 48 hr assay) or 2 × 104 cells/well (for 24 hr assay) in 96-well plate overnight and then treated with medium or various dilutions of the EO or citral (1:200,000; 1:100,000; 1:50,000 or 1:25,000). Four hours before the end of the treatment, cells were incubated with MTT (2.5 mg/mL) and kept in the dark at 37◦ C until the end of the treatment. The formazan produced by the reduction of MTT by viable cells was dissolved in DMSO, and the optical density was measured with an ELISA reader (BenchMark, Bio-Rad, CA) at 570 nm (reference filter 630 nm). Experiments were repeated at least three times. The results were expressed as percentage of the control, considered to be 100%. To determine whether the use of DMSO in first dilution of EO and citral would interfere with cell viability, the same assay was performed with DMSO in the same dilutions used for the OE and citral. No interference on cell viability was observed with the higher concentration (0.2%) of DMSO used. For cotreatment experiments, plated cells were incubated for 48 hr with 2.5 μg/mL cisplatin (CIS), 100 nM vincristine (VCR) or 0.5 μM doxorubicin (DOX) in the presence of absence of different dilutions of citral (1:100,000; 1:50,000, and 1:30,000) and cell viability was assessed by MTT. The dilutions of OE used: 1:200,000; 1:100,000; 1:50,000; and 1:25,000 correspond to 4.6; 9.2; 18.4; and 36.8 μg/mL, respectively, while the dilutions of citral: 1:200,000; 1:100,000; 1:50,000; 30,000; and 1:25,000 correspond to 4.2; 8.4; 16.9; 28.1, and 33.7 μg/mL or 27.7; 55.4; 110.8; 182.8, and 221.6 μM, respectively. This work was preceded by an evaluation of the effect of different concentrations of citral on GBM cell viability. We did not include aldehydes control experiments in this study because an antitumoral effect of citral with concentration higher than that used here was shown previously (3,4). Apoptosis assays Apoptosis was assessed by DNA fragmentation and TUNEL assays. DNA fragmentation was evaluated by cell cycle analysis using flow cytometry. Twenty-four hours after plating, the cells (1 × 104 cells/well - 96-well plate) were treated with medium (control) or various dilutions of citral (1:200,000; 1:100,000; 1:50,000 or 1:25,000 equivalent to 4.2; 8.4; 16.9 and 33.7 μg/mL or 27.7; 55.4; 110.8 and 221.6 μM, respectively) and incubated for different times. Next, cells were harvested, resuspended in a hypotonic fluorescent solution (50 mg/mL PI and 0.1% Triton X-100 in 0.1% Na citrate buffer) for 1 hr in the dark at 4◦ C. The cell cycle was analyzed by flow cytometry (FL-2) (FACSCalibur, Becton Dickinson, San Jose, CA) to determine the sub-G0/G1 DNA content. Subdiploid populations were considered to be apoptotic. Data acquisition and analysis were controlled by CellQuest software, version 3.1f. The results are presented as representative histograms and as the mean ± SD of the percentage of the fragmented DNA. The TUNEL assay was performed using a commercial kit R Fluorescein Direct In Situ Apoptosis Detection (ApopTag Kit, cat. #S7160) according to the instructions of the manufacturer (Millipore, Billerica, MA, USA). An image analysis system composed of a digital camera (Evolution Media
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Cybernetics Inc., Bethesda, MD) and a computer with the graphical interface software Q-Capture 2.95.0, version 2.0.5, (Silicon Graphic Inc., USA) coupled to an epifluorescence microscope was used to obtain high-quality images (2048 × 1536 pixel buffer) using the 40× objective lens. At least a hundred cells per cover slip were captured, and the percentage of TUNEL-positive nuclei was calculated from the total DAPIpositive nuclei. The results are expressed as the percentage of reactive tissue in the total area (mean ± SD). Caspase activation assay Activation of caspase-3 and -9 were assayed using CaspGlow commercial kits according to the instructions of the manufacturer (Biovision, Mountain View, CA, USA). In brief, cells (5 × 104 /well) were incubated for 24 hr with medium (control) or different dilutions of EO (1:25,000 equivalent to 36.8 μg/mL) or citral (1:50,000 or 1:25,000 equivalent to 16.9 or 33.7 μg/mL or to 110.8 or 221.6 μM, respectively) before being harvested, centrifuged, and suspended in the caspase assay solution. This solution contained a molecule conjugated to FITC that is cell permeable, nontoxic, and binds irreversibly to the activated caspase. After 1 hr of incubation (37◦ C, 5% CO2 ), cells were washed twice with washing buffer, and the percentage of caspase-activated cells was analyzed by flow cytometry (FL-1). The results are presented as representative histograms and as the mean ± SD of the Ratio of Fluorescence Intensity (RIF) compared with the control.
(50 μM verapamil) or MRP1 (50 μM MK-571), or the indicated concentrations of citral (1:50,000; 1:30,000 or 1:25,000 equivalent to 16.9; 28.1 or 33.7 μg/mL, or to 110.8, 182.8 or 221.6 μM, respectively). The cells were washed in PBS, harvested, and kept on ice until flow cytometry analysis. The results are presented as representative histograms or as the mean ± SD of the mean fluorescence intensity (MIF). Expression of MRP1 protein was assessed by western blotting. Cells (5 × 105 ) were platted in 6-wells plates and 24 hr later incubated with medium or citral (1:50,000 or 1:30,000 equivalent to 16.9 or 28.1 μg/mL or to 110.8 or 182.8 μM, respectively) for another 24 hr. Total cell lysates were made by lysing harvested cells in lysis buffer (50 mM Tris pH 7.4, 0.5% Nonidet P-40, 1 mM EDTA, 150 mM NaCl, and proteases inhibitors added at the time of preparation). Samples were fractionated by 8% polyacrylamide gel. The proteins were transferred on to nitrocellulose membrane (Bio-Rad Laboratory Inc., Hercules, CA, USA) which was blocked for 1 hr with Tris-buffered saline containing 0.1% Tween 20–milk 5%. Next, the membrane was incubated overnight at 4◦ C, with anti-MRP1 (A23, Alexis Biochemicals,
Quantification on Reactive Oxygen Species (ROS) ROS was determined by flow cytometry in cells treated with H2 -DCFDA. Cells (2 × 104 cells/well) were plated for 24 hr and then exposed to medium (control) or citral (1:25.000) for different times. After the desired time, cells were harvested, washed with PBS, pH 7.4, and resuspended in 0.16 mL PBS containing 20 μM H2 -DCFDA. After 30-min incubation at 37◦ C, the production of ROS was evaluated by flow cytometry (FL-1). The results are presented as the mean ± SD of Ratio of Fluorescence Intensity (RIF) compared with the control. To assess the role of ROS in citral citotoxicity A172 cells were treated for 24 hr with media, pretreated or not for 2 hr with the ROS inhibitor N-acetyl-L-cysteine (NAC, 10 mM) and then incubated with citral (1:30.000 equivalent to 28.1 μg/mL or 182.8 μM). Cell viability was estimated by the MTT assay and results were presented as percentage of control. Activity and expression of MDR proteins The activity of the MDR proteins was determined by the accumulation of specific substrates as described previously (15). Rho123 and 5-carboxyfluorescein diacetate (CFDA), a nonfluorescent molecule that is converted into the fluorescent carboxy-fluorescein (CF) by intracellular esterases, were used to measure the activity of Pgp/ABCB1 and MRP1/ABCC1, respectively. For each experiment, cells (2 × 104 /well) were plated for 24 hr at 37◦ C/5% CO2 to allow the culture to stabilize. Cells were incubated for 30 min with medium (to measure autofluorescence), 400 nM Rho123 or 3 μM CFDA in the presence of medium, inhibitors of Pgp
Figure 1. Effects of M. officinalis essential oil (EO) and citral on the viability of GBM cells. A172 and U87 cells (104 /well) were incubated with medium or different dilutions of EO or citral [1:200,000 (200), 1:100,000 (100), 1:50,000 (50), 1:25,000 (25)]. After 48 hr, cell viability was assessed by MTT as described in the M&M. Results are expressed as a percentage of the control and represent the mean ± SD of three independent experiments. ∗ , p < .05; ∗∗ , p < .01. OE dilutions correspond to 4.6; 9.2; 18.4; and 36.8 μg/mL respectively, while citral dilutions correspond to 4.2; 8.4; 16.9 and 33.7 μg/mL or 27.7; 55.4; 110.8 and 221.6 μM, respectively. Cancer Investigation
M. O FFICINALIS EO Induces Apoptosis in Glioblastoma for 1 hr at room temperature. After washing the blot was visualized by chemiluminescence detection using the enhanced chemiluminescence (ECL) system (GE Healthcare, Tokyo) according to manufacture’s instructions.
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SanDiego, CA) or anti-β-actina (Sigma Chemical Co., Saint Louis, MO, USA) antibodies. Following a series of washes with Tris-buffered saline - 0.1% Tween 20, a secondary horseradish peroxidase antibody was added and incubated
Figure 2. EO and citral induce apoptosis in GBM cells. Cells (104 /well) were treated with medium or citral [1:200,000 (200), 1:100,000 (100), 1:50,000 (50), 1:25,000 (25)] for the indicated times. DNA fragmentation was assessed by flow cytometry in cells stained with PI, and the sub-G1 peak was considered apoptotic. (a) Representative histograms of A172 cells treated with increasing dilutions of citral after 48 hr. (b and c) Quantitative analysis of DNA fragmentation of A172 and U87 cells treated with the indicated doses of citral for 12, 18, 24, and 48 hr. (d) Quantitative analysis of apoptotic A172 cells treated with EO or citral at 1:25,000 for 24 hr then submitted to the TUNEL assay as described in M&M. The data represent the average of at least three independent experiments ± SD. ∗ , p < .05; ∗∗ , p < .01. Citral dilutions correspond respectively to: 27.7, 55.4, 110.8, and 221.6 μM. C 2014 Informa Healthcare USA, Inc. Copyright
R. Muniz de Queiroz et al.
Statistical analysis All the data reported here are expressed as the mean ± SD from three independent experiments. A significant difference from the respective control for each experimental test condition was assessed by one-way analysis of variance (ANOVA) using GraphPad Prism 4.0 software. Values of p < .05 were considered statistically significant.
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RESULTS M. officinalis essential oil and citral inhibited the viability of GBM cells The cytotoxic effect of M. officinalis EO and citral on GBM cell lines A172 [Figure 1(A)] and U87 [Figure 1(B)] were analyzed by the MTT assay. Cells were treated with media and different dilutions of the oil (1:200,000; 1:100,000; 1:50,000 or 1:25,000 equivalent to 4.6; 9.2; 18.4 or 36.8 μg/mL, respectively) for 48 h, and cell viability was measured by the MTT assay. Treatment with EO reduced the number of viable cells in both cell lines in a dose-dependent manner (Figure 1 - white bars). Because neral and geranial, the two isomers of citral, represent more than 85% of the EO, we investigated whether the cytotoxic effect of EO is due to the action of these components. To do this, the viability of GBM cells treated with commercial citral (1:200,000; 1:100,000; 1:50,000 or 1:25,000 equivalent to 4.2; 8.4; 16.9; 28.1 or 33.7 μg/mL or to 27.7, 55.4, 110.8, or 221.6 μM, respectively) was determined
under the same conditions used for the EO (Figure 1 - black bars). The data showed that the effect of citral on the GBM cells lines U87 and A172 is similar to or even higher than that of the EO, suggesting that the activity of the EO against the tumor cells may be due to the action of citral. Essential oil and citral induce apoptosis in GBM cells Morphological analysis of GBM cells treated with EO or citral (1:50,000 equivalent to 18.4 μg/mL or 110.8 μM) for 48 hr showed a decrease in cell number accompanied by the presence of “blebs” in the plasmatic membrane, a cellular cyto-architecture indicative of apoptosis (data not shown). To further investigate this hypothesis, GBM cells were treated with different concentrations of citral, and DNA fragmentation was evaluated by flow cytometry. The sub-G1 peak of the cell cycle was considered apoptotic. As depicted in Figure 2(a), cell-cycle histograms of A172 showed that treatment with citral for 48 hr led to a significantly dose-dependent increase in the sub-G1 phase [Figure 2(b)]. The DNA fragmentation induced by treatment with citral was also timedependent [Figure 2(c)]. Because similar results were obtained with U87 [Figure 2(c)], only the A172 cell line was used in the next experiments. To confirm the indication of induction of apoptosis by citral and EO, A172 cells were submitted to the TUNEL assay. The data obtained demonstrated that treatment with both EO and citral (1:25,000 equivalent to 36.8 and 33.7 μg/mL for EO and citral, respectively) for
Figure 3. Activation of caspases by EO and citral. Plated A172 cells (5 × 104 /well) were treated for 24 hr with medium, EO [1:25,000 (25)] or citral [1:50,000 (50) and 1:25,000 (25)]. After treatment, cells were harvested, and caspase activity was measured using a commercial kit as described in M&M. The results show representative histograms and quantitative analysis of caspase-9 (a) and -3 (b). Results represent the average of three independent experiments ± SD. ∗ , p < .05; ∗∗ , p < .01. For OE and citral concentrations see legend for Figure 1. Cancer Investigation
M. O FFICINALIS EO Induces Apoptosis in Glioblastoma
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24 hr causes an increase in the number of cells positively labeled for TUNEL, confirming that both compounds induce apoptosis in GBM cells [Figure 2(d)]. To determine whether the induction of apoptosis in GBM cells was dependent on caspase activation, A172 cells were treated with EO (1:25,000 equivalent to 36.8 μg/mL) or citral (1:50,000 or 1:25,000 equivalent to 18.4 or 36.8 μg/mL or to 110.8 or 221.6 μM) for 24 hr, and the levels of activated caspase-9 and -3 were measured by a commercial kit (CaspGlow, Biovision) containing a fluorochrome conjugated to a molecule that irreversibly binds to the activated caspase. As shown in Figure 3(a and b), activation of caspase-9 and -3 were detected in cells treated with either citral or EO. Citral induces a dose-dependent activation of caspases. Apoptosis induced by citral is mediated by ROS To evaluate whether the generation of ROS was involved in citral-induced apoptosis, A172 cells were treated with citral (1:25,000 equivalent to 33.7 μg/mL or 221.6 μM) for different times, and H2 DCF-DA was used to examine ROS generation. As shown in Figure 4(a), treatment with citral led to a rapid increase in ROS generation that peaked at 6 hr and returned to normal levels at 24 hr. Evaluation of cell death, performed in a parallel experiment, revealed that DNA fragmentation begins to increase after 8 hr of citral treatment. Thus, the increase in the ROS levels precedes cell death [Figure 4(a)], suggesting a role for oxidative stress in citral-induced cell death. To confirm this hypothesis, cells were treated for 24 hr with citral (1:30,000 equivalent to 28.1 μg/mL or 182.8 μM) in the presence or absence of the antioxidant Nacetyl-cysteine (NAC), and cell viability was measured by MTT. As shown in Figure 4(b), cellular death induced by citral is completely blocked by the ROS scavenger, indicating that the generation of ROS is an essential process in citralinduced apoptosis. Citral inhibits MRP1 activity and expression Next, we investigated whether citral was able to modulate the activity of MDR transporter proteins expressed by A172 and U87 cells. The activity of Pgp/ABCB1 and MRP1/ABCC1 in these cells was evaluated by their ability to retain substrates of the pumps. To do this, cells were loaded with substrates for Pgp (Rhodamine-123) or MRP1 (5-CFDA), in the presence or absence of specific inhibitors (verapamil or MK571, respectively), and the cell fluorescence, indicative of the pump activity, was measured by flow cytometry [Figure 5(a and b)]. While no increase in fluorescence was observed when cells were incubated with Rho-123 in the presence of verapamil [Figure 5(b)], an increase in fluorescence was observed when cells were incubated with 5-CFDA in the presence of MK571 [Figure 5(a)]. These data demonstrated that MRP1/ABCC1 is active in A172 cells but Pgp/ABCB1 is not. U87 cells showed the same pattern of activity for these pumps [Figure 6(a and b)]. To investigate the effect of citral on MRP1, cells were treated with 5-CFDA and different concentrations of citral (1:50,000, 1:30,000 or 1:25,000 equivalent to 16.9, 28.1 or 33.7 μg/mL or to 110.8, 182.8, 221.6 μM) for 30 min, and the intracellular fluorescence was measured [Figure 5(C)]. The C 2014 Informa Healthcare USA, Inc. Copyright
Figure 4. Involvement of ROS in citral-induced cell death. (a) ROS generation and DNA fragmentation. A172 cells (2 × 104 /well) were treated with medium or citral (1: 25,000) for different times, and ROS generation and DNA fragmentation were measured by flow cytometry. Results are presented as the ratio of the fluorescence of citral over control cells [ROS production (•)] and the percentage of DNA fragmentation (). (b) Effect of NAC. A172 cells were treated with citral (1:30.000) for 24 hr in the presence or absence of NAC (10 mM), and cell viability was estimated by the MTT assay as described in Section 2 of the M&M. Results are expressed as percentage of the control and represent the mean ± SD of three independent experiments. ∗ , p < .05; ∗∗ , p < .01; and ∗∗∗ , p < .001. Citral dilutions correspond respectively to: 221.6 and 182.82 μM.
increase in fluorescence observed in presence of citral [Figure 5(d)], indicates that this substance is able to downmodulate the activity of MRP1. Indeed, the modulatory effect of citral is approximately 50% of that observed with the commercial inhibitor MK571. To probe if the modulation of MRP1 by citral was due to a decrease in protein expression, A172 cells were treated with medium or citral (1:50,000 or 1:30,000 equivalent to 16.9 or 28.1 μg/mL or to 110.8 or 182.8 μM) for 24 hr and protein expression was evaluated by western blotting. As shown in Figure 5(e) citral decrease the expression of MRP1. To confirm the effect of citral on MRP1, U87 cells were treated with 5-CFDA and citral (1:25,000 equivalent to 33.7 μg/mL or 221.6 μM) for 30 min, and the intracellular fluorescence presence of citral [Figure 6(d)] indicates that this substance is able to downmodulate the activity of MRP1. The effect of citral was similar to that observed for A172. Effects of combination of citral with chemotherapy drugs Next, we evaluate if the combination of citral with antineoplastic drugs would decrease the survival of GBM cells. For this, A172 cells were cocultured for 48 hr with different dilutions of citral (1:100,000, 1:50.00, and 1:30,000 corresponding to 8.4, 16.9, and 22.1 μg/mL or to 55.4, 110.8, and 182.8 μM) and 2.5 μg/mL cisplatin (CIS), 100
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R. Muniz de Queiroz et al.
Figure 5. Activity of the efflux pumps. A172 cells (2 × 104 /well) were incubated for 30 min with medium (autofluorescence), 3 μM 5-CFDA or 400 ng/mL Rho-123 in the presence or absence of 50 μM MK751 (MK) or 50 μM verapamil (VRP), respectively. The retention of specific substrates by the cells was evaluated by flow cytometry. (a and b) are representative histograms of the activity of MRP1 (a) and Pgp (b). (c and d) show the effect of citral on the MRP1 pump. Cells were incubated for 30 min with medium (autofluorescence) or 3 μM CFDA in the presence or absence of 50 μM MK751 (MK) or increasing concentrations of citral [1:50,000 (Cit 50); 1:30,000 (Cit 30) or 1:25,000 (Cit 25)], and cell fluorescence was measured by flow cytometry. The results are expressed as representative histograms of cell fluorescence for each condition (c) and by the mean ± SD of the mean fluorescence intensity (MIF) obtained in three different experiments (d). (e) shows the effect of citral on MRP1 expression. Cells were incubated medium or citral 1:50,000 (50) or 1:30,000 (30) for 24 hr and lysates were submitted to western blot as described in M&M. Citral dilutions correspond respectively to: 110.8, 182.82, and 221.6 μM.
nM vincristine (VCR) or 0.5 μg/mL doxorubicin (DOX) and cell viability was assessed by MTT. Results show that cotreatments with citral do not seem to sensitize GBM cells to antineoplastic drugs. DISCUSSION Malignant central nervous system neoplasms, particularly GBM, are among the most lethal and intractable of human tumors. These tumors have defied all current therapeutic modalities, and patient prognosis is poor (8), emphasizing the need to explore new therapeutic strategies for treatment. Thus, the search for new drugs that are able to overcome GBM’s drug resistance mechanisms and prevent tumor recurrence is of great interest for GBM therapy (16). In the last few years, natural products have been recognized as an important source of new antineoplastic drugs (17). Accumulating evidence of the antitumor activity of essential oils (18,19) supports this material as one of these drug sources. In a previous paper (1), we showed that the EO of
M. officinalis was cytotoxic to several cancer cell lines but its effect on GBM cells was not investigated. Although the composition of the M. officinalis EO was described in this paper, the component responsible for the observed antitumor activity was not identified. The data presented in Figure 1 demonstrated that M officinalis EO is also able to cause cell death of established GBM cell lines (U87 and A172) and indicated that this effect is due to its major component citral. The capacity to induce cell death in cancer cell lines is not a property particular to M. officinalis EO. This effect has already been shown for EO from other sources (18, 19) as well as for citral (3, 4). Microscopic observation suggested that the cytotoxic effects of EO and citral on the GBM cell lines (U87 and A172) were due to the induction of apoptosis. Measurement of DNA fragmentation by cell cycle analysis [Figure 2(a–c)] and TUNNEL assays [Figure 2(d)] corroborated this hypothesis. The apoptotic effect of both EO and citral is dosedependent. M. officinalis EO and citral also induced the activation of caspase-9 and -3 [Figure 3 (a and b)], indicating that Cancer Investigation
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M. O FFICINALIS EO Induces Apoptosis in Glioblastoma
Figure 6. Activity of the efflux pumps. U87 cells (2 × 104/well) were incubated for 30 min with medium (autofluorescence), 3 μM 5-CFDA or 400 ng/mL Rho-123 in the presence or absence of 50 μM MK751 (MK) or 50 μM verapamil (VRP), respectively. The retention of specific substrates by the cells was evaluated by flow cytometry. (a and b) are representative histograms of the activity of MRP1 (a) and Pgp (b). (c and d) show the effect of citral on the MRP1 pump. Cells were incubated for 30 min with medium (autofluorescence) or 3 μM CFDA in the presence or absence of 50 μM MK751 (MK) or citral [1:25,000 (Cit 25)], and cell fluorescence was measured by flow cytometry. The results are expressed in (c) as representative histograms of cell fluorescence for each condition and in (d), as mean fluorescence intensity of the experiment shown in c. Results represent two experiments. Citral dilution corresponds, respectively, to 221.6 μM.
their apoptotic effect is caspase-dependent. These data are in agreement with studies showing the activation of caspase-3 by essential oils from other plants (19) and citral (3). Mitochondria play a central role in the apoptotic process induced by several drugs. The activation of caspase-9 [Figure 3(a)] indicated that the effects of M. officinalis EO and citral on GBM cells are mediated by the intrinsic (mitochondrial) pathway of apoptosis. In addition to the release of proapoptotic proteins, the generation of reactive oxygen species (ROS) is used by the intrinsic pathway to induce apoptosis in tumor cells. Due to the high content of ROS, it has been proposed that insults leading to further ROS generation would turn cancer cells very susceptible to ROS damage. Indeed, many cytotoxic agents employed in chemotherapy seem to exert their effects through ROS generation (6, 20, 21). Treatment of GBM cells with citral resulted in a rapid generation of ROS that preceded the induction of cell death [Figure 4(a)]. The addition of the antioxidant NAC prevented citral-induced death, corroborating the dependence of citralinduced death on ROS [Figure 4(b)]. C 2014 Informa Healthcare USA, Inc. Copyright
Expression of transporter proteins from the ABC superfamily is one of the most relevant mechanisms of drug resistance. Drug resistance is a severe limitation of the effect of chemotherapy on various malignancies. A 1998 study (22) showing that GBM patients subjected to chemotherapy have an increase in cells positive for Pgp/ABCB1 and MRP1/ABCC1, suggested a role for these ABC pumps in resistance to treatment. The expression of MRP1 is considered a negative prognostic factor for patients with GBM (10, 11). Indeed, high levels of transporter proteins were also identified in cancer stem cells, a cell population described in recurrent GBM patients (23). In a previous paper (1), we showed that the EO of M. officinalis caused cell death in several cancer cell lines including A549, a lung cancer cell line that expressed several members of the MRP family. Data presented in this paper show that M. officinalis OE and its major component, citral, induced apoptosis of GBM cell lines (A172 and U87) that expressed active MRP1. They also indicated that citral induces production of ROS and inhibits MRP1 expression and activity [Figures 4 and 5 (c, d, e)].
R. Muniz de Queiroz et al. drug resistance in GBM cell points to the potential use of these compounds for the development of new drugs for this neoplasia and as adjuvants for the treatment of glioblastoma multiforme.
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DECLARATION OF INTEREST
Figure 7. Effects of cotreatment of GBM cells with citral and chemetherapics. A172 cells (104 /well) were incubated with medium or different dilutions of citral [1:100,000 (100), 1:50,000 (50), 1:30,000 (30), equivalent to 55.4, 110.8, and 182.82 μM, respectively] in the presence or absence or 2.5 μg/mL cisplatin (CIS), 100 nM vincristine (Vin) or 0.5 μM doxorubicin (DOX). After 48 hr, cell viability was assessed by MTT as described in the M&M. Results are expressed as a percentage of the control and represent the mean ± SD of three independent experiments.
The authors report no declarations of interest. The authors alone are responsible for the content and writing of this article. This study was supported by grants from Fundac¸a˜o de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Instituto Nacional para Pesquisa Translacional em Sa´ude e Ambiente na Regi˜ao Amazˆonica/ Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico/MCT, Brazil (INCT-INPeTAm/CNPq/MCT). The authors thank the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq) for the graduate fellowships awarded to Rafaela Muniz de Queiroz, L´ıvia Paes Tavares Pacheco Guimar˜aes and Gleice da Grac¸a Rocha.
REFERENCES Recent studies showed that temozolomide, the first choice of chemotherapy drug for GBM treatment induces ROS production (21) and that resistance to this drug involved mitochondria dysfunction and a decrease in ROS production (24). Other studies showing that inhibition of MRP1 activity or expression (25–27) lead to an increase in the response of GBM cell lines to chemotherapeutic drugs indicating the importance of this protein in GBM drug resistance. Combination of citral and antineoplastic drugs does not seem to sensitize GBM cells as the resulting decrease in cell viability is equivalent to the added effect of both drugs (Figure 7). However, if a sensitizing effect does exist, it is hard to demonstrate since citral modulation of MRP1 activity was only observed at cytotoxic concentrations. In addition, it should be considered that the modulatory effect of citral on MRP1 activity is quite low [Figure 5 (c, d)] when compared to inhibitors used by other researchers (26,27). However, M. officinalis EO and citral are able to kill resistant cells that express MRP1 (this work) or overexpress Pgp (1) but have no effect on noncancer cells (3,28). Although the relationship between ROS production and inhibition of MRP1 activity by citral was not investigated in this work, data from the literature showed that compounds able to affect ROS or GSH production, altering the cell oxidative stress, may also affect MRP1 expression and/or its activity (29,30). Efficient therapeutic treatments for highly malignant gliomas, such as GBM, are lacking and patient prognosis is poor. Thus, despite the inclusion of targeted drugs in clinical protocols, no significant improvement over the standard protocol has been observed (7, 31). As cancer cells use several mechanisms to evade death, the simultaneous attack to multiple targets is important for a successful cancer therapy. Therefore, the results presented in this paper, showing that M. officinalis OE and citral affect two pathways of
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