TEUCRIUM POLIUM INDUCES APOPTOSIS IN PERIPHERAL BLOOD LYMPHOCYTES ISOLATED FROM HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA

Background/Aim. Chronic lymhocytic leukemia (CLL) is considered more as a disease of cells accumulation due to the defect in apoptosis rather than deregulated cell’s proliferation. The activation of apoptosis is one of the main molecular mechanisms responsible for the anti-cancer activities of most of the currently studied potential anti-cancer agents, including natural compounds. Teucrium polium extracts exhibited strong cytotoxic effects in murine leukemia cell line, RAW 264.7 and human melanoma cell line, C32, but its cytotoxic effects against human leukemia cells were unknown. Methods. The viability of human leukemia cell lines (MOLT-4 and JVM-13), CLL lymphocytes isolated from 28 patients (CLL cells), and peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy subjects treated with Teucrium polium extracts, was determined by MTT assay. Apoptosis of Teucrium polium treated CLL cells was measured by flow cytometry applying Annexin V/7AAD staining. The expressions of active proapoptotic protein Bax, antiapoptotic protein Bcl-2, cytochrome c and the percentage of cells containing cleaved caspase-3 in treated CLL cells was determined by flow cytometry and immunocytochemistry. Results. Teucrium polium methanol extract (TP) decreased the viability of all tested human leukemia cells but it had no effect on the viability of healthy PBMCs. The citotoxic effect of TP was caused by its induction of CLL cells’ apoptosis. TP disarranged the ratio of the expressions of proapoptotic Bax and antiapoptotic Bcl-2 protein in favor of Bax, consequently inducing apoptosis by cytochrome c mitochondrial release and activation of caspase-3 in treated CLL cells. Conclusion. Teucrium polium induced selective apoptosis in CLL cells and it affected the expressions of key proteins involved in the regulation of programmed cell death.


Introduction
Chronic lymhocytic leukemia (CLL) originate from the antigen-stimulated mature B lymphocytes that either avoid death through the intercession of external signals or die by apoptosis, only to be replenished by proliferating precursor cells [1].For that reason, CLL is considered more as a disease of cells accumulation due to the defect in apoptosis rather than deregulated cells proliferation [2].The activation of apoptosis is one of the main molecular mechanisms responsible for the anti-cancer activities of most of the currently studied potential anti-cancer agents, including natural compounds [3,4].Several, novel drugs designed to interfere with the proteins regulating the cell cycle, the apoptotic machinery or leukemic microenvironment, are currently being tested in clinical, in vitro or in vivo trials [5][6][7][8][9].
There are two main cellular death pathways leading to caspase activation and apoptosis: the extrinsic pathway, initiated by "death" receptors and the intrinsic pathway, initiated after cytosolic discharge of mitochondrial derived cytochrome c and the other apoptotic proteins, caused by the mitochondrial outer membrane permeabilization induced by the formation of the proapoptotic proteins oligomerisation pores, such as Bax.Both pathways merge into the activation of caspase 3, the executioner caspase, that ultimately finalizes the apoptosis.The mitochondrial outer membrane permeabilization by pro-apoptotic protein Bax that by following the death signal, translocates from the cytosol to the mitochondrial outer membrane, is suppressed through the actions of cytosolic antiapoptotic proteins such as Bcl-2.Therefore, the changes in cytosolic expressions of Bcl-2 and Bax, play a significant role in the execution of apoptosis [10,11].
Plants of the genus Teucrium were used in traditional medicine because they contained a number of phenolic compounds that exhibit strong biological activity including diterpenoids, monoterpenes, sesquiterpenes, flavonoids and esters of fatty acids [12][13][14].Teucrium polium extracts exhibited strong cytotoxic effects in murine leukemia cell line, RAW 264.7 and human melanoma cell line, C32 [14].However, there has not yet been published data demonstrating both the cytotoxic and proapoptotic effects of Teucrium polium extracts on chronic lymphocytic leukemia cells.
In the present study, for the first time, we investigated the antitumor activity of Teucrium polium methanol extract (TP) against two human leukemic cell lines, MOLT-4 and JVM-13 and peripheral blood lymphocytes isolated from human chronic lymphocytic leukemia patients (CLL lymphocytes).We also determined the cellular pathway responsible for the activation of apoptosis induced by Teucrium polium extracts in CLL lymphocytes.

Chemicals
Unless stated otherwise, all reagents were from Sigma-Aldrich (St. Louis, MO), and all dishes for culturing cells were from Sarstedt (Numbrecht, Germany).

Preparation of drug solution
Leaves of Teucrium polium were collected in the late summer period of 2015.year from the region of Šumarice, Kragujevac, Serbia.The voucher specimen of Teucrium polium was confirmed and deposited in Herbarium at the Department of Biology and Ecology, Faculty of Science, University of Kragujevac.The sampled leaves were air-dried in darkness at room temperature (20ºC).
Air-dried leaves (10g) had been transferred to the dark-coloured flasks.They were mixed with 200ml of methanol and stored at room temperature.After 24h, the extracts were filtered through Whatman No.1 filter paper and residues were again mixed with equal volumes of solvent.After 48h, the process was repeated.Combined supernatants were evaporated to dryness under vacuum at 40ºC using rotary evaporator.The obtained extracts were kept in sterile sample tubes and stored in a refrigerator at 4ºC.
Stock solution of TP was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Germany) at a concentration of 20mM, filtered through a 0.22mm Millipore filter before use, and diluted by a nutrient medium (RPMI 1640) to various working concentrations, so the final concentration of DMSO in culture medium never exceeded 0.5% (v/v).

Patients
The local Ethics Committee accepted the study and prior to the initiation of the study, the written informed consent was obtained from all subjects according to the Declaration of Helsinki.Informed consent was obtained from all individual participants included in the study.CLL was diagnosed by establishing the clinical criteria and it was confirmed by immunophenotypic analysis for the expression of CD5, CD19 and monoclonal immunoglobulin in accordance with updated NCI Working Group Guidelines [15].The control groups were healthy volunteers without known acute and chronic diseases.Peripheral blood samples from 28 CLL patients and 16 healthy control subjects were included in study.

Cell preparation
All blood samples were obtained in the morning and collected in potassium-EDTA coated blood collection tubes (Terumo).Peripheral blood samples (9 ml) were centrifuged at 400xg for 10 minutes to separate plasma and cells.Peripheral blood mononuclear and polymorphonuclear cells were separated by single step continuous density-gradient centrifugation with Histopaque 1077.The separated mononuclear cells were washed three times with culture medium RPMI 1640 and resuspended in RPMI 1640 supplemented with 10% autologous serum.The monocytes were removed by adhesion on plastic Petri dishes [16].

Cell lines
Human leukemia cell lines MOLT-4 (ATCC® CRL-1582™) and JVM 13 (ATCC® CRL-3003™) were acquired as a gift from professor Sonja Dencic, Department of Biochemistry, Belgrade University School of Medicine, Serbia.Both cell lines were maintained in culture medium consisting of RPMI-1640, supplemented with 10% heat-inactivated fetal bovine serum (FBS).

MTT assay
The viability of cultured cells was determined by assaying the reduction of tetrazoliumbromide (MTT) to formazan [17].In brief, cells were treated with different concentrations of TP (10µg/ml, 25µg/ml, 50µg/ml, 100µg/ml, 250µg/ml and 500µg/ml) or cultivated in the cell culture medium containing the appropriate amount of DMSO (control).After 24 and 48 hours of cells incubation at 37°C in the atmosphere containing 5%CO2, the 96 well plates were centrifuged for five minutes at 400xg, the culture medium was removed, and MTT solution (5mg/ml) was added to the cells.After additional 4h of incubation, the microtiter plates were centrifuged again for five minutes at 400xg, the culture medium with MTT solution was removed and DMSO (150µl/well) was added to dissolve the formazan crystals.Absorbance was measured at 595nm with a multiplate reader (Zenyth 3100, Anthos Labtec Instruments, Austria).The results were presented as relative to the control value (untreated cells).

Detection of apoptosis
Apoptosis of CLL lymphocytes was measured using annexin V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD) Apoptosis Kit (BD Biosciences) according to manufacturer's instructions.CLL cells had been treated with TP at earlier specified concentrations and the percentage of apoptotic cells were determined by flow-cytometer FC 500 (Beckman Coulter).Data were presented as density plots of Annexin V-FITC and 7AAD stainings.

Assessment of apoptosis mechanism
In order to understand the mechanism of apoptosis induced by Teucrium polium, we analyzed the expressions of the active proapoptotic protein Bax, antiapoptotic protein Bcl-2, cytochrome c and the percentage of cells containing cleaved caspase-3.Lymphocytes from 28 CLL patients had been incubated for 24 hours with 250µg/ml and 500µg/ml TP or with the culture medium alone (control), washed three times with ice cold PBS, and then resuspended, fixed and permeabilized (Fixation and Permeabilization Kit, eBioscience).Four types of stainings were separately performed afterward.Intended for Bcl-2 staining, the permeabilized cells were then incubated with Bcl-2 fluorescein isothiocyate primary antibody (mhbcl01, Life technologies) for 15 minutes at room temperature.Other three types of staining included incubation of permeabilzed lymfocytes for 30 minutes with primary antibodies against Bax (N20,sc-493; Santa Cruz Biotech.Inc), cytochrome c (G7421, Promega) and caspase-3 (#9661, Cell signaling Тechnology).These cells had been washed and then incubated with the appropriate secondary antibodies for 30 minutes.We used Alexa488 goat anti-mouse IgG (H+L) antibody (A-11001, Life Technologies) for cytochrome c, and goat anti-rabbit IgG FITC (Ab6717-1, Abcam) for Bax and caspase-3 staining.All cells were then washed with PBS and analysed by flow cytometry and/or immunocytochemistry.

Immunocytochemistry
Observation of cells by fluorescent microscope was performed to localize the presence of Bax, Bcl-2, cytochrome c and cleaved caspase 3 in CLL lymphocytes.The images were aquired with a Olympus BX51 at 1000x magnification.

Flow cytometric evaluation
Fluorescence of at least 10000 events per sample had been measured using flow-cytometer FC500 (Beckman Coulter).Fluorescence intensity was standardized using isotype-matched negative control antibodies.The mean fluorescence intensities of Bax and Bcl-2 (MFIs) were calculated as the ratio of raw mean channel fluorescences to isotype control levels, respectively.Cytochrome c and cleaved caspase-3 levels were evaluated as percentage of cells displaying the fluorescence.

Statistical analysis
All values were expressed as mean ± standard deviation (SD).Each experiment was performed in triplicate and conducted on every sample.Commercial SPSS version 20.0 for Windows was used for statistical analysis.The distributions of data were evaluated for normality using the Shapiro-Wilk test.Statistical evaluation was performed by Student's t-test for paired observations, or one-way ANOVA depending on data distribution.P values less than 0.05 were considered significant.

Teucrium polium methanol extract decreased viability of human leukemic cells
After 24 hours of incubation of MOLT-4 leukemia cells with 10 µg/ml, 25 µg/ml and 50 µg/ml of TP, there was no statistically significant decrease in the viability of the examined cells compared to the viability of untreated MOLT-4 leukemia cells (p>0.05).However, at the Teucrium polium extract concentrations of 100µg/ml, 250µg/ml and 500µg/ml, there was a statistically significant reduction in the viability of the MOLT-4 leukemic cells compared to the viability of untreated cells (p<0.05).Specifically, after 24h of incubation of MOLT-4 cells with 100µg/ml, 250µg/ml and 500µg/ml of the TP, the viability of MOLT-4 cells was reduced to 63.56±1.27%,30.32±2.39% and 41.83±1.42%,respectively.

Fig. 1 -Effects of Teucrium polium methanol extract on viability of MOLT-4 cells.
After 24 hours incubation of JVM-13 cells with 10µg/ml, 25µg/ml, 50µg/ml, 100µg/ml and 250µg/ml of TP there was no statistically significant decrease in JVM-13 cells' viability compared to the viability of untreated JVM-13 cells (p>0.05).However, 24h of incubation of JVM-13 cells with 500µg/ml of TP reduced the viability of these cells to 36.24±1.52%compared to the untreated JVM-13 cells.

Fig. 2 -Effects of Teucrium polium methanol extract on viability of JVM-13 cells.
Afterwards, we analyzed the effect of TP on viability of CLL cells and peripheral blood mononuclear cells (PBMCs).After 24 hours, 250µg/ml and 500µg/ml of TP statistically significantly decreased viability of treated CLL cells to 76.85±7.25% for the TP concentration of 250µg/ml and to 50.27±9.25%for 500µg/ml of TP compared to the control group of untreated CLL lymphocytes (p<0.05).
Also, after 48 hours, 250µg/ml and 500µg/ml of TP statistically significantly decreased viability of treated CLL cells to 65.63±8.13%for the TP concentration of 250µg/ml and to 40.39±6.67%for 500 µg/ml of TP compared to the control group of untreated CLL lymphocytes (p<0.05).
After cultivation of healthy PBMCs for 24 and 48 hours with TP at the concentrations ranging from 10µg/ml to 500µg/ml, there were no statistically significant changes in the viability of PBMCs relative to a control group of PBMCs that were not exposed to TPs (p>0.05)(Figure 3).

Teucrium polium methanol extract induced apoptosis of CLL cells
Considering that our previous results showed that TP demonstrated the cytotoxic effect on human leukemia cells, especially on CLL cells, our next goal was to examine the type of cell death induced by TP in CLL cells.The type of cell death was determined by Annexin V/7AAD staining.
Results obtained by Annexin V/7AAD staining after 24 hours of incubation of CLL cells with 500µg/ml of TP, displayed significant increase of percentage of total apoptotic cells of about 50% compared to the untreated cells (p<0.05,Fig. 4).Nevertheless, results obtained after 48 hours of incubation of CLL cells with 250µg/ml and 500µg/ml of TP, also showed significant increase of percentage of total apoptotic cells of 50% and 70%, respectively compared to the untreated cells (p<0.05,Fig. 4).

Teucrium polium methanol extract induced apoptosis in peripheral blood lymphocytes isolated from human CLL patients via mitochondrial apoptotic pathway
To investigate whether TP activated the mitochondrial apoptotic pathway, CLL cells had been either treated with 500µg/ml TP or cultivated in complete medium (control) for 24 hours and the localisation of cytochrome c had been analyzed by fluorescent microscopy.In parallel, we examined the localisation of active Bax proapoptotic protein and the fluorescence intensity representing the total expression of Bcl-2 antiapoptotic protein, along with presence of cleaved (activated) caspase-3 in treated and control cells.Our results showed that Teucrium polium extract induced the activation and translocation of Bax from cytosol to mitochondria, decreased the expression of cellular Bcl-2 protein, and induced the release of cytochrome c from mitochondria to cytosol and caspase-3 cleavage (Fig. 5).
Additionally, in order to quantify these apoptotic changes induced by TP in CLL cells, we analysed the expression levels of Bax, Bcl-2 and cytochrome c by flow cytometry.Furthermore, we identified the amount of cells displaying cleaved caspase-3, to verify that the apoptotic pathway induced by Teucrium polium extract in CLL cells was caspase dependent.The expression of active-Bax and cytosolic concentrations of cytochrome c were significantly increased, while the cytosolic expression of Bcl-2 was significantly decreased in treated CLL lymphocytes compared to the untreated cells.Furthermore, the percentage of cells containing cleaved caspase-3 had been significantly increased (Fig. 6).All these findings showed that TP disarranged the ratio of the expressions of proapoptotic Bax and antiapoptotic Bcl-2 protein in favor of Bax, consequently inducing apoptosis by cytochrome c mitochondrial release and activation of caspase-3.

Discussion
The results of our study showed for the first time that a methanol extract of Teucrium polium reduced the viability of MOLT-4 and JVM-13 cells, and of CLL cells isolated from CLL patients after 24 hours and 48 hours of cells incubation.A very important result of our study is the absence of changes in cells' viability of treated PBMCs compared to the untreated PBMCs, after 24 and 48 hours of cultivation of these cells with methanol extract of Teucrium polium.Although diverse mechanisms of action might contribute to the anti-cancer effects of TP, we have shown that inhibition of Bcl-2 protein expression and activation of Bax are directly involved in Teucrium polium-induced CLL cells' apoptosis.
The results of our research demonstrating the antitumor effects of methanol extract of Teucrium polium in human leukemic cells correlated with the results of previous studies showing the cytotoxic effects of Teucrium polium extracts on cell lines of prostate, colon, lung, and skin tumors [12,14,18,19].In these tumor cell lines, as well as in leukemic cells used in our study, TP decreased the viability of examined cells.Stankovic et al. have demonstrated that the IC50 values of the TP after 72 hours of cells incubation were between 100µg/ml and 200µg/ml that were in correlation with the results of our research.Particularly, the concentration of the TP required to reduce the viability of HeLa cervical adenocarcinoma cells to 50% after 72 hours was 148.02±4.99µg/ml, for Fem-x human melanoma cells was 199.79±0.30µg/ml, and for K562 cells chronic myelogenous leukemia's cells, the concentration was 116.75±24.40µg/ml [20].
Species of plants of the genus Teucrium, are very rich in phenols and flavonoids, which are the carriers of the strong biological activity of various extracts of this plant.Extracts of Teucrium polium had recently been subjected to the increasing number of in vitro studies in which their anticancer potential was tested.The results obtained using the HCT-116 cell line clearly indicated that a methanol extract of Teucrium polium reduced the viability of these cells by the induction of apoptosis.In a study of Stankovic et al., it was shown that after 24 hours of HCT-116 cells' incubation with 250µg/ml of TP, apoptosis occurred in 85% of the total cell population.Our findings were also consistent with the results of a study of Stankovic et al, since we have shown that a TP induced apoptosis in CLL cells [12].Hence, in our research the percentage of apoptotic CLL cells after 48 hours of incubation in the presence of 250µg/ml Teucrium polium extract was about 46%.Therefore, reasults from our study suggest that a TP reduced the viability of CLL cells by inducing apoptosis.
Permeabilization of outer mitochondrial membrane allows apoptotic molecules such as cytochrome c to be released into the cytoplasm that consequently induce the activation of caspases and subsequently execution of apoptosis.Permeabilization of outer mitochondrial membrane is mainly regulated by Bcl-2 family of proteins, such as proapoptotic protein Bax and antiapoptotic protein Bcl-2 [21].The results of our study for the first time demonstrated that a methanol extract of Teucrium polium induced selective mitochondrial apoptosis of CLL cells, by activating proapoptotic protein Bax and reducing the cytosolic expression of antiapoptotic protein Bcl-2, that led to the mitochondrial release of cytochrome c into the cytosol and the activation of caspase-3.The results of various studies that have been published to date were only confirming that a methanol extract of Teucrium polium could induce apoptosis in some cell lines, but to date there have not yet been accurately investigated the potential cellular mechanisms involved in this process [12].The results of some previous studies emphasized the antioxidant capacity of methanol extract of Teucrium polium, but these studies have not yet been performed on tumor cell lines [12,21].It was shown that the methanol extracts of other plants of the genus Teucrium, such as Teucrium chamaedris, Teucrium montanum, Teucrium arduini and Teucrium scordium, could have an antioxidant activity [12].

Conclusions
TP affected key proteins involved in the regulation of programmed cell death of CLL cells.It selectively induced mitochondrial apoptosis in peripheral blood lymphocytes isolated from human CLL and it had no citotoxic and apoptotic effect on peripheral blood mononuclear celles of heathy subjects.In order to precisely define the molecular component, one or more of them, carrying the biological activity of methanol extract of Teucrium polium, it is necessary to perform additional research.The results of our experiments therefore represent a promising start for the future studies in the investigation of the cytotoxic and apoptotic effects of specific components of TP in human leukemia cells.