Britannin, a sesquiterpene lactone induces ROS-dependent apoptosis in NALM-6, REH, and JURKAT cell lines and produces a synergistic effect with vincristine

Britannin, a Sesquiterpene Lactone isolated from Inula aucheriana, has recently gained attraction in the therapeutic fields due to its anti-tumor properties. This study was designed to evaluate the effect of this agent on Acute Lymphoblastic Leukemia (ALL) cell lines, either as a monotherapy or in combination with Vincristine (VCR). To determine the anti-leukemic effects of Britannin on ALL-derived cell lines and suggest a mechanism of action for the agent, we used MTT assay, Annexin-V/PI staining, ROS assay, and real-time PCR analysis. Moreover, by using a combination index (CI), we evaluated the synergistic effect of Britannin on Vincristine. We found that unlike normal Peripheral Blood Mononuclear Cells (PBMCs) and L929 cells, Britannin reduced the viability of NALM-6, REH, and JURKAT cells. Among tested cells, NALM-6 cells had the highest sensitivity to Britannin, and this agent was able to induce p21/p27-mediated G1 cell cycle arrest and Reactive Oxygen Specious (ROS)-mediated apoptotic cell death in this cell line. When NALM-6 cells were treated with Nacetyl-L-Cysteine (NAC), a scavenger of ROS, Britannin could induce neither apoptosis nor reduce the survival of the cells suggesting that the cytotoxic effect of Britannin is induced through ROS-dependent manner. Moreover, we found that a low dose of Britannin enhanced the effect of Vincristine in NALM-6 cells by inducing apoptotic cell death via altering the expression of apoptotic-related genes. Overall, our results proposed a mechanism for the cytotoxic effect of Britannin, either as a single agent or in combination with Vincristine, in NALM-6 cells.


Introduction
As the most common malignancy in pediatrics, acute lymphoblastic leukemia (ALL), with an incidence rate of 25 % of all childhood malignancies, is a heterogeneous disease that mainly affects children and teenagers aged between 1 and 19 years old [1,2]. As the age increases, the prognosis of the disease shifts from a good prognosis with a cure rate of 90 % to a poor prognosis with a cure rate of less than 40 % [3]. According to the affected lineage, ALL could be divided into two main sub-groups; B-cell precursor (BCP) lineage (BCP-ALL), which accounts for 85 % of pediatric ALL, and less common T-cell precursor lineage (T-ALL) [4]. The presence of fundamental genetic abnormalities such as translocations, mutations, and deletions in different types of genes in ALL patients has made the treatment of the disease difficult. Although chemotherapy and CNS radiotherapy have improved the overall survival of the patients, especially in the children group, the intensity of these approaches would bring significant adverse effects for patients.
Despite the increase in survival of patients (94.1 %), the risk of disease relapse is one of the most challenging issues in the treatment of ALL [5]. Currently, the only promising treatment strategy in the cases of relapse or severe disease cases is stem cell transplantation, which the lack of appropriate donor and procedure make it challenging for the physicians [6]. Thereby, the identification of new drugs with higher efficacy has obtained tremendous attention lately. Recently, the identification of new drugs with more efficiency has been considered. The possibilities are to find a new anti-leukemia agent that significantly eliminates the leukemia cell population and has low cytotoxicity in normal cells. The significant anti-cancer properties of vincristine (VCR) and Vinblastine are alkaloids derived from Madagascar periwinkle Catharanthus roseus [7] together with the promising anti-leukemic effects of many other plant-derived agents such as Ergolide [8], have attracted attention to herbal medications in the treatment of ALL.
Asteraceae plants are known for their biologically active sesquiterpene lactones. I. aucheriana, from this family, has been recently investigated for its chemical constituents, and britannin, a sesquiterpene lactone (SL), has been reported from this plant [9,10]. Inula genus has more than 100 species, which could be found in Europe, Asia, and Africa [11]. It has been shown that the SL compounds of this genus have cytotoxic effects due to the presence of α-methyleneγ-lactone [12,13]. Recently, the anti-cancer property of britannin has been reported in several cancer cells [14,15]. It has induced apoptotic cell death through FOXO-dependent up-regulation of BIM in pancreatic cells [13]. Britannin also activated AMP-activated protein kinase (AMPK) and subsequently suppressed mTOR to induce cytotoxicity in liver cancer cells [16]. Despite the compelling number of studies proposing a mechanism for the cytotoxic property of britannin, the mechanism of action of these drugs is elusive in hematologic malignancies. Therefore, we aimed to evaluate the molecular mechanisms of britannin in ALL-derived cell lines.

Isolation of britannin
Britannin was isolated from I. aucheriana DC (voucher herbarium number: TMRC 3173 kept at the Herbarium of TMRC, Shahid Beheshti University of Medical Sciences). The plant material was collected from the West Azerbaijan province of Iran. Britannin was obtained by chromatographic methods, including Vacuum Liquid Chromatography and SPE. NMR spectral data were used to confirm the structure [13]. In the present study, we prepared the working solution of britannin (100 µM) through dissolving 2.8 mg of britannin crystal in 76.7 µL of dimethyl sulfoxide (DMSO). Then, for treating ALL cell lines with concentrations of 0-7 µM, we diluted the working stock several times with the RPMI1640 medium.

Cell culture and drug treatment
ALL-derived NALM-6 cells, REH cells and, JURKAT cells were purchased from the Pasteur Institute (Tehran, Iran) and were cultured in RPMI1640 medium containing 10 % fetal bovine serum, 50 mg/ml Streptomycin, and 30 mg/ml Penicillin at 37 °C in the presence of 5 % CO 2 in a humidified incubator. L929 cells used as control cells were grown in a similar condition as the leukemic cell lines. After reaching the desired confluency, all cells were treated with britannin. In addition, NALM-6 cells were exposed to VCR (Sigma, Germany), autophagy inhibitor chloroquine (CQ) (Sigma, Germany), and N-acetyl-L-cysteine (NAC).

MTT assay
To evaluate the cytotoxic effect of britannin on ALL cell lines, the cells were cultured in 96-well plates and were treated with the agent for up to 48 h. NALM-6 cells were also treated with britannin in the presence of other agents such as CQ, NAC, and VCR for further evaluation. To evaluate the metabolic activity of the cells, after the incubation time, 100 µL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma, Germany) was added to each well, and the plates were kept in the incubator for 4 h. Then, the plates were centrifuged, and the media of each well was replaced with 100 µl of DMSO to dissolve the resulting formazan crystals. The absorbance of each sample was read at 570 nm by an ELISA reader.

Trypan blue staining
Trypan blue was used to evaluate whether britannin could reduce the survival of NALM-6 cells. NALM-6 cells were treated with britannin at concentrations of 1, 3, and 5 µM. After 24 or 48 h, drug-treated cells were harvested and stained by Trypan blue dye. The number of the cells was counted manually using a light microscope.

Cell cycle analysis
PI staining was used to evaluate the effect of britannin on the progression of the cell cycle in NALM-6 cells. The number of NALM-6 cells that were needed at the primary stage of the test was 5 × 10 5 . This number of the cells was exposed to the agent for 48 h and then was harvested using centrifugation. The harvested cells were washed twice with PBS. The cell pellets were fixed with ethanol (70 %) overnight. 1ml of PI MASTER MIX solution containing 40 µL, 10 µL RNase, and 950 µL PBS was added to the cells. After 30 min, the distribution pattern of the cells was examined using flow cytometry.

Annexin-V/PI staining assay
Flow cytometry and Annexin V-FITC staining were performed to examine the effect of britannin, CQ, and VCR, either alone or in combination, on NALM-6 cells. For this analysis, we needed 5 × 10 5 NALM-6 cells at the primary stage. After exposing the cells to the agents, the cells were collected, washed and, stained with Annexin V-FITC and PI (5 µL). After 15 min at dark, BD FACS Calibur (BD biosciences, SanJose, CA, USA) was used to analyze the amount of Phosphatidylserine (PS) externalization on the surface of NALM-6 cells. Flowjo 7.6 software was used for data analysis.

Acridine orange dye method
To investigate whether treatment of NALM-6 cells with britannin is coupled with the induction of autophagy, drugtreated cells were stained with 10 µg /ml of Acridine orange dye. The samples were placed on slides, and the induction of autophagy was evaluated using a fluorescent microscope.

Hoechst 33258 staining
To determine whether the induction of cell death due to britannin was due to the induction of apoptotic cell death, we used Hoechst 33258 staining and a fluorescent microscope. For this staining, 5 × 105 of drug-treated NALM-6 cells were exposed to 1 µl Hoechst 33258 dye. Then, 10 µl of the suspension was placed on a slide, and the staining pattern of the nucleus was analyzed using a fluorescent microscope. As a result, the nucleus of the cells which underwent apoptosis appears as irregular blue fragments; however, the nucleus of the normal cells absorbs the dye uniformly.

RNA extraction, cDNA synthesis and, RT-PCR analysis
Britannin-treated cells were exposed to Trizol reagent for RNA extraction and to TAKARA kit (Japan) for cDNA synthesis. Real-time PCR was performed using SYBR Green PCR. ABL was the housekeeping gene in the present study.

Determination of combination index (CI) and dose reduction index (DRI)
To investigate the efficacy of drug combinations, the reduction of cell survival was examined, and the combination index (CI) was evaluated as described previously [17]. CI values of < 1, =1, and > 1 indicate synergism, additive effect, and antagonism of drugs, respectively (Table 1).

Intracellular ROS measurement
To evaluate the effect of britannin on ROS production in NALM-6 cells, the cells (5 × 10 5 ) were treated with different concentrations of agent and, after 48 h, were subjected to fluorescent reactive oxygen species (ROS) detection kit, which was obtained from Invitrogen detection Technologies (Eugene, Oregon, USA). Flowjo 7.6 software was used for data analysis.

Statistical analysis
One-way ANOVA and post hoc Tukey multiple comparisons were used for data analysis. All analyses were performed using SPSS software version 22. P-value of < 0.05 was considered to be statistically significant.

The cytotoxic effect of britannin on acute lymphoblastic leukemia (ALL) cell lines
To evaluate whether britannin could reduce the survival of different ALL cells with both B lymphocyte and T lymphocyte origin, we treated a panel of ALL-derived cell lines, including NALM-6, REH (pre-B ALL cells), and JURKAT (T-ALL cells), with increasing concentrations of the agent. The results of the MTT assay showed that 48 h treatment with britannin resulted in a reduction of cell viability in  (Fig. 1A).
Given the lower sensitivity of NALM-6 to britannin, we chose this cell line for further investigations. Our results showed that britannin reduced both the metabolic and the viability of NALM-6 cells in a time-dependent manner (Fig. 1A). However, this agent at the same concentrations had a cytotoxic effect neither on normal peripheral blood mononuclear cells (PBMCs) or L929 cells (Fig. 1A).

Effect of britannin on cell cycle progression in NALM-6 cells
NALM-6 cells were exposed to different concentrations of britannin (1, 3 and, 5 µM) for 48 h, and PI staining was used to evaluate the effect of the agent on the cell cycle progression. As shown in Fig. 1B, britannin at the concentrations of 3 and 5 µM increased the percentage of cells in the G1 phase while decreased the percentage of cells in the S phase, but did not affect the proportion of the cells in the G2 phase of the cell cycle as compared to the control group. Also, we used q-RT-PCR to investigate the effect of britannin on the expression of p21 and p27, which are two genes that participate in the regulation of the cell cycle at the S phase.
The results showed that at the time of 48 h, the expression of these genes increased in a dose-dependent manner (Fig. 1C).

Effect of britannin on the induction of apoptosis in NALM-6 cells
Annexin-V/PI staining and flow cytometry analysis was used to evaluate whether britannin at the concentrations of 1, 3, and 5 µM could induce apoptosis in NALM-6 cells. At the time of 48 h, the results showed that britannin increased the percentage of NALM-6 apoptotic cells from 2.33 at the control group to 4.2, 47.58 and, 82.1 at the concentrations of 1 µM, 3 µM and, 5 µM, respectively ( Fig. 2A). To confirm the results of Annexin-V/PI staining and assure that the cell death was due to the induction of apoptosis, Hoechst33258 staining was used. If the induction of apoptotic cell death would be due to induction of apoptosis, then the nucleus of apoptotic cells would have appeared as irregular bright blue dots compared to the blue-stained nucleus of normal cells.
As presented in Fig. 2B, when NALM-6 cells were treated with britannin, their nucleus stained irregularly, which is an indicator of induction of apoptotic cell death. Also, the increase in the expression of Bax (pro-apoptotic gene) and decrease in the expression of XIAP (anti-apoptotic gene) in NALM-6 cells after exposure to increasing concentrations of britannin was another evidence suggesting that this agent could induce apoptotic cell death in ALL-derived cell line (Fig. 2B).

Britannin increased ROS levels in NALM-6 cells
Several anti-cancer drugs induce apoptotic cell death in cancer cells by increasing the production of reactive oxygen specious (ROS) [18,19]. To evaluate whether britannininduced apoptosis is mediated through ROS production, NALM-6 cells were treated with different concentrations of britannin for 48 h. As presented in Fig. 2C, britannin increased the ROS level in NALM-6 cells in a concentrationdependent manner. To ascertain that britannin reduces the survival of NALM-6 cells by inducing ROS-mediated apoptotic cell death, we treated the cells with NAC, a well-known antioxidant and scavenger of ROS [20], either alone or in combination with britannin. We found that when cells were exposed to NAC (80 μm), the ability of britannin to reduce the metabolic activity of the cells was diminished (Fig. 2D), suggesting that probably britannin induced its cytotoxic effects on NALM-6 cells through the production of ROS.

Effect of britannin on the induction of autophagy in NALM-6 cells
There is a compelling body of evidence reporting that some of the anti-cancer agents activate autophagy in cancer cells, but sometimes this happens in favor of the survival of cancer cells [21]. The results showed that britannin elevated the expression of Beclin1, Bnip3, Atg7 and, Atg10, genes involved in regulating autophagy in NALM-6 cells (Fig. 3A). Furthermore, the results of Acridine orange staining showed that while untreated control cells appeared in green fluorescence, britannin-treated cells showed yellowish or reddish-green fluorescence, suggestive of the activation of autophagy in cells (Fig. 3B). To evaluate whether induction of autophagy due to britannin was in favor of NALM-6 cells survival or oppose to it, we treated cells with CQ (20 µM), which is an autophagy inhibitor [22]. The results of the MTT assay showed that inhibition of autophagy could increase the cytotoxic property of britannin, as the metabolic activity of NALM-6 cells diminished more significantly when the cells were treated with britannin and CQ (Fig. 3C). To confirm these results, we also performed Annexin-V/PI staining. As presented in Fig. 3D, flow cytometry results indicated the synergistic effect of concomitant treatment of britannin and CQ on NALM-6 cells. In contrast, britannin (1 µM) and CQ (20 µM) alone increased the percentage of apoptotic cells to 4.2 and 6 %, respectively; the combination of the two agents induced apoptosis in 20.71 % of the cells.

The synergistic effects of britannin and VCR in NALM-6 cells
First, to evaluate the optimum concentration of VCR on NALM-6 cells, the cells were treated with VCR (2-6 nM) for 48 h. The results of the MTT assay showed that after 48 h, the metabolic activity of the cells decreased by 39, 54, 68, 81, 90 % at the concentrations of 2, 3, 4, 5, and 6 nM, respectively (Fig. 4A). Also, the results showed that the IC50 value for VCR in NALM-6 cells at 48 h was 5 nM. Given this, for synergistic experiments, we used VCR at the concentration of 3 nM. Then, NALM-6 cells were treated simultaneously with lower concentrations of britannin (1 and 2 µM) and VCR (3 nM) for 48 and, the viability of the cells was evaluated using MTT assay. Forty-eight hours after treatment, the metabolic activity of the cells treated with the combination of Britannin and VCR decreased more significantly than either agent alone (Fig. 4A), suggesting a synergistic effect between the two agents. The CI and isobologram analysis results also revealed the existence of synergism between britannin and VCR in NALM-6 cells ( Fig. 4B; Table 1). This finding was then confirmed by Annexin-V/PI staining. The flow cytometry results suggested (1 µM) and VCR (3 nM) were capable of inducing apoptosis at 7.7 and 16.2 %, respectively (Fig. 4C). Fig. 3 The effects of britannin on autophagy in NALM-6 cells.
A and B Treatment of NALM-6 cells with britannin (3 and 5 µM ) resulted in the elevation in the expression of Bnip3, Beclin, Atg7 and, Atg1, essential genes in the regulation of the autophagy system. The results of Acridine orange staining also indicated that in the presence of britannin autophagy system was compensatorily activated in NALM-6 cells, as the number of yellowish or reddish cells increased in a concentrationsdependent manner as compared to the untreated control cells. C and D To evaluate whether the inhibition of autophagy could potentiate the antileukemic effects of britannin, we treated NALM-6 cells with the agent together with CQ, an autophagy inhibitor. The results of the MTT assay revealed that inhibition of autophagy could enhance the anti-survival effects of britannin. In agreement with the results of the MTT assay, Annexin-PI staining assays also showed that when NALM-6 cells were treated simultaneously with CQ and britannin, the percentage of apoptotic cells increased more vigorously compared to either agent alone. Values are given as mean ± S.D. of three independent experiments. *P ≤ 0.05 represented significant changes from the control. (Color figure online)

Discussion
Acute lymphoblastic leukemia, a malignancy-affected lymphoid progenitor cell, more frequently occurs in children; however, a proportion of patients suffering from this malignancy are adults whose prognosis, in most cases, is poor. Despite the improvements in understanding the disease and treatment approaches, the risk of disease relapse is still very high in these patients [23]. Some targeted therapies have recently been reached in clinical trials for the treatment of ALL, but even immunotherapies were not successful in preventing the risk of relapse and induction of resistance against chemotherapies [24]. Attempts for the identification of new drugs for the treatment of ALL are still continuing.
In the present study, we found that britannin, a SL derived from I. aucheriana, a native plant of West Azerbaijan (Iran), exerted cytotoxic effects on different ALL-derived cell lines such as NALM-6, REH, and JURKAT. In all cell lines, britannin significantly reduced the survival of the cells and, among them, NALM-6 with the IC 50 value of 3.2 µM was the most sensitive ALL cell line.
Meanwhile, the same concentrations of britannin showed no cytotoxic effects on either normal PBMCs or L929 cells, suggestive of selective behavior of the drug against leukemic Fig. 4 The synergistic effects of britannin and vincristine (VCR). A and B The MTT assay and CI calculation results indicated that britannin could potentiate the sensitivity of NALM-6 cells to VCR, suggestive of the existence of synergism between both agents in ALL-derived NALM-6 cells. C The results of Annexin-PI staining also indicated that the combination of britannin and VCR increased the percentage of apoptotic cells more significantly than either agent alone. Values are given as mean ± S.D. of three independent experiments. *P ≤ 0.05 represented significant changes from the control cells. Also, britannin showed an ability to produce a synergistic effect with VCR, which is one of the chemotherapy drugs used in the treatment of ALL. When NALM-6 cells were treated with britannin and VCR, the percentage of apoptosis induction increased significantly by altering apoptotic-related genes' expression, compared to either agent alone. The previous studies also indicated the potent anti-cancer effects of britannin in several solid tumors, including hepatocarcinoma, breast cancer, and non-small cell lung cancer [9]. Their results in breast cancer cell lines showed that the IC 50 of the agent for MCF-7 and MDA-MB-468 cells were 9.56 and 6.81 µM, respectively [25]. Given these results and based on the previous studies which suggested the cytotoxic activity of britannin on different cancer cells such as breast cancer, liver cancer, and pancreatic cancer [9,13], we evaluate the mechanism of action of agent in NALM-6 cells.
In NALM-6 cells, we found that britannin could induce G1 cell cycle arrest by increasing the expression of p21 and p27, two essential genes that inhibit the cell cycle in the S phase by inhibiting Cyclin D/CDK4/6 [26]. Also, in our previous study, britannin at a concentration lower than 5 µM could reduce the replicative potential of ALL cells by elevating the expression of p21 and p27 [27]. The negative effect of britannin on the progression of the cell cycle was also reported in breast cancer cells which were mediated through Cyclin D1/CDK4-mediated pathway [28]. Another mechanism that was recruited by britannin to reduce the survival of NALM-6 cells was the induction of apoptosis. Apoptosis that is induced in cells through both intracellular and extracellular pathways is one of the significant cell death mechanisms characterized by alteration in the structure of cell membrane and chromatin [29]. Our results showed that after treatment of NALM-6 cells with britannin, while the expression of pro-apoptotic gene Bax increased, the expression of apoptotic inhibitor gene XIAP decreased. These changes in the balance of pro-apoptotic to anti-apoptotic genes subsequently led to the induction of apoptosis in NALM-6 cells. In agreement with our finding, Cui et al. also indicated that britannin induced apoptosis in liver cancer cells by increasing AMPK expression, suppressing mTOR, and activating Caspase-3 [16]. Also, there is some evidence showing that britannin could reduce the expression of BCL-2 protein and increase Bax protein expression in breast cancer cells [25]. One of the best strategies that cytotoxic agents could induce cell death in cancer cells is to produce oxygen-containing entities. Although the production of the small amount of reactive oxygen species (ROS) in the cells could be a help to the development of cancer, if its production surpasses the toxic threshold, ROS could induce apoptosis in cancer cells [30]. Many chemotherapy drugs induce cell death in cancer cells by potentiating ROS production [31,32]. In our study, britannin increased the production of ROS in NALM-6 cells. Also, when NALM-6 cells were treated with NAC, a scavenger of ROS, we found that britannin could induce neither apoptosis nor reduce the survival of the cells suggesting that the cytotoxic effects of britannin are induced through ROS-dependent manner. In parallel to our study, other investigations on the cytotoxic effects of britannin also revealed that ROS production is one of the mechanisms this drug recruited for induction of apoptotic cell death in cancer cells [16,25].
Autophagy has a dual role in the regulation of cancer cell survival. While a group of studies has shown that activation of autophagy can favor cancer cells, especially under metabolic stress caused by anti-cancer stress [33], other studies indicated that activation of autophagy in cancer cells could induce apoptotic cell death [34]. Our results showed that britannin exposure to NALM-6 cells activated autophagy, as revealed by Acridine orange staining and the expression of autophagy-related genes. However, when autophagy was inhibited by using CQ, the sensitivity of NALM-6 cells to the anti-cancer effects of britannin was potentiated. As an interpretation, it is reasonable to assume that the activation of autophagy in response to britannin in NALM-6 cells is a compensatory mechanism through which malignant cells tried to maintain their survival. Although previous studies have reported the positive impact of Britannin on the activation of autophagy in cancer cells [16], no study has so far proposed that this mechanism could be a refractory response of cancer cells to this agent. Overall, our results showed that britannin, a SL, had both anti-proliferative and cytotoxic effects against NALM-6 cells through inducing p21-mediated G1 cell cycle arrest and ROS-mediated apoptotic cell death. Furthermore, it also had no cytotoxic effects against normal cells. Given these, we propose that britannin could be a potent natural compound against leukemic cells; however, efforts are needed to understand the molecular mechanisms of the agent better, and more in vitro and in vivo experiments are required to check the safety of this agent as a monotherapy or in combination.