TNF-alpha pathway mediated leukemic cell death

Accepted Manuscript Self assembled nano fibers of betulinic acid: A selective inducer for ROS/TNFalpha pathway mediated leukemic cell death Sandeep Kumar Dash, Sourav Chattopadhyay, Shib Shankar Dash, Satyajit Tripathy, Balaram Das, Santanu Kar Mahapatra, Braja Gopal Bag, Parimal Karmakar, Somenath Roy PII: DOI: Reference:

S0045-2068(15)30021-3 http://dx.doi.org/10.1016/j.bioorg.2015.09.006 YBIOO 1844

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

29 June 2015 11 September 2015 26 September 2015

Please cite this article as: S.K. Dash, S. Chattopadhyay, S.S. Dash, S. Tripathy, B. Das, S.K. Mahapatra, B.G. Bag, P. Karmakar, S. Roy, Self assembled nano fibers of betulinic acid: A selective inducer for ROS/TNF-alpha pathway mediated leukemic cell death, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.09.006

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Self assembled nano fibers of betulinic acid: A selective inducer for ROS/TNF-alpha pathway mediated leukemic cell death Sandeep Kumar Dasha, Sourav Chattopadhyaya, Shib Shankar Dashb, Satyajit Tripathya, Balaram Dasa, Santanu Kar Mahapatrac, Braja Gopal Bagb, Parimal Karmakard, Somenath Roya * a

Immunology and Microbiology Laboratory, Department of Human Physiology with Community

Health, Vidyasagar University, Midnapore-721 102, West Bengal, India. b

Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore, 721

102, West Bengal, India c

School of Chemical & Biotechnology, SASTRA University, Thanjavur – 613 401, Tamil Nadu,

India. d

Department of Life Science and Biotechnology, Jadavpur University, Kolkata, India.

Running title: Anti-cancer activity of SA-BA

*Address of Correspondence Immunology and Microbiology Laboratory Department of Human Physiology with Community Health Vidyasagar University Midnapore-721 102, West Bengal, India.

E-mail: [email protected]

1

Abstract The main complication in betulinic acid (BA) based drug delivery has been observed due to its bulk structure. The present study demonstrates the potential effects of self assembled nano size betulinic acid (SA-BA) by treating human leukemic cell lines (KG-1A and K562) and its normal counterpart. Self assembly property of BA was investigated using SEM and DLS study which showed that the BA forms fibrous structure having few nanometers in diameter. Selective antileukemic efficacy study of SA-BA was investigated by cell viability assay. Mode of leukemic cell death and probable pathway of apoptosis was monitored by measuring ROS level, pro and anti-inflammatory cytokine

status

and

expression of caspase-8

and

caspage-3

by

immunocytochemistry. Higher efficacy of SA-BA over non-assemble BA was monitored towards cancer cells, with no relevant toxicity to normal blood cells. SA-BA facilitated reactive oxygen species (ROS) mediated leukemic cell death, confirmed by pre-treatment of N-acetyl- Lcysteine. Induction of apoptosis by SA-BA treatment increased pro-inflammatory cytokines, specifically potentiated TNF-α mediated cell death, confirmed by expression of caspase-8 and caspage-3 by immunocytochemistry. This study explored the better anti-leukemic efficacy of SA-BA over BA and this modification will enrich the use of BA in cancer therapy.

Key words: betulinic acid, self assembled, reactive oxygen species, TNF-α, mitochondrial membrane potential.

2

1.Introduction

Cancer is one of the leading causes of human death worldwide. World health organization (WHO) reported that it is the second foremost cause of human death globally. In spite of substantial advancement in biomedical researches on cancer biology, identifications of cancer biomarkers, different surgical procedures, antibody therapy, radiotherapy, and chemotherapy, the overall survival rate of cancer patients has not significantly improved in the last few decades [1]. Leukemia, the cancer of white blood cells originates from bone marrow and discretely spreads throughout the body using the blood stream. Acute myeloid leukemia (AML) is one of the fetal form of leukemia, occurs mainly in children. The abnormal, immature white blood cells (called ‘blast’) spreads quickly and ultimately cause death in a very short period. Chronic myeloid leukemia (CML) is a cancer of myeloid cell, found in the bone marrow. It occurs by the chromosomal translocation (Philadelphia chromosome). In CML, huge number of granulocytes is produced and spreads throughout the body, disturbing the normal functions of the blood cells. Natural resources have been used for thousands of years to prevent human diseases. The therapeutic benefits of different plant products have become one of the major areas of interest in cancer therapy. Naturally occurring substances show an escalating role in different bio-medical applications. So far most of the anticancer drugs are discovered from plant sources [2]. Betulinic acid (BA) is a member of pentacyclic triterpene, discovered in 1995 from the stem bark of the plant Zizyphus mauritiana, and it is widely distributed among plant kingdom. Previous study reported that BA showed potent anti inflammatory, anti HIV and antitumor property against melanoma cells [3]. The anti-leukemic activity BA was found by triggering apoptosis pathway, 3

which is not only restricted to human melanoma cells, but also several other types of cells [4]. Hata et al. [5] reported that BA showed potent anti-proliferative activity on HL-60, U937 and K562 cell lines. Gradual development in BA based research proved the diversive anti-tumor activity to vast areas of cancer, including lung carcinomas, ovarian and cervical carcinomas [6]. Treatment of BA on human neuroblastoma (SHEP) cell line have revealed that BA acts on the mitochondria without affecting cell surface receptor and induced apoptosis in cancer cells, but not in lymphoid cell lines [7]. Later on BA was isolated from the heavy wood powder of Ziziphus jujube tree and purified by column chromatography [8]. This group first reported the self-assembly properties of BA in different organic liquids mixture and alcohol mixtures along with their morphological characteristics. Self-assembly is one of the unique techniques for nanofabrication which involves designing new structured molecules with supramolecular entities.

Self-assembly possesses various advantages in biological applications including

modification of drugs complexity and functional structures which facilitates the potency of drugs compared to its native form [9]. Along with the different organic solvents they reported that in ethanol-water mixture, crystal structure of BA converted to a fibrillar network having fibers nano to micrometer cross sections and >100 micrometer lengths. This study also revealed that BA has a 1.31 nm long, rigid, 6-6-6-6-5 pentacyclic backbone, with the hydroxyl and the carboxy groups separated by 0.97 nm. In our previous study we observed that non-self assembled BA showed good anti-leukemic activity against KG-1A and K562 cells at 25µg/ml dose for 48 hr treatment. But in this study the percentage of leukemic cell death was below 65% in both cells [10]. The best of our knowledge, studies regarding the anti leukemic effect of self-assembled nano-sized betulinic acid (SA-BA) on acute and chronic myeloid leukemia have not been carried out till now. So for the first time, the present work has been carried out to understand the anticancer 4

activity of the SA-BA against acute (KG-1A) and chronic (K562) myeloid leukemia cell lines in comparison to doxorubicin (DOX) as a control drug. Human peripheral lymphocytes were compared in this study to find out any cytotoxicity and selectivity of said drug in vitro. The results obtained from the previous study [10] were compared with the present study to identify the efficacy of SA-BA over BA.

2. Materials and methods

2.1. Culture media and chemicals

Histopaque 1077, and Rhodamine B, RPMI 1640, penicillin, streptomycin, pentoxifylline (POF), N-acetyl-L-cysteine (NAC), doxorubicin were procured from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from GIBCO/Invitrogen. MTT was purchased from Himedia, India. Zinc perchlorate hexahydrate, Tris–HCl, Tris buffer, Titron X-100, Sodium dodecyl sulphate (SDS), phenol, chloroform, iso-amyl alcohol, ethidium bromide (EtBr), 2vinylpyridine were procured from Merck Ltd., SRL Pvt. Ltd., Mumbai, India. Commercially available dimethyl sulfoxide (DMSO) was procured from Hi-media, India, and was purified by vacuum distillation over KOH. All other chemicals were from Merck Ltd and SRL Pvt. Ltd. Mumbai, were of the highest purity grade available.

2.2. Synthesis and purification of Betulinic acid

Synthesis and purification of BA were previously performed and reported elsewhere [11]. 5

2.3. Physical measurements

Purified BA was characterized X-Ray Diffraction (XRD) study, Fourier transform infrared spectrum, 1H NMR, and Reversed-phase HPLC analysis. Self assembled property of BA was examined by Optical polarized microscopic images (OPM) and Scanning electron microscopy (SEM) studies were previously reported [11]. In the present study, we have examined the self assembled property of BA dissolved in ethanol: water mixture (16:4) using the following methods

2.3.1. Dynamic light scattering (DLS) and Zeta potential analysis DLS and Zeta potential of SA-BA was done by Zetasizer Nano ZS (Malvern Instruments) [12]. The SA-BA was (200 μg/ml in ethanol: water mixture 16:4 ratio) sonicated for 10 min and hydro-dynamic particle sizes were measured by suspending two drops of aqueous suspension of SA-BA in 1 ml of Millipore water. When particle was completely dispersed in water, then particles were analyzed with a dynamic light scattering analyzer. The obtained z-average value was noted as average size of the conjugate. Zeta potential was also performed in the same instrument by universal Zeta dip cell using the same solution used for DLS study.

2.3.2. Optical polarized microscopy (OPM) and Scanning electron microscopy (SEM) study for self assembly property The self-assembled property of BA dissolved in ethanol: water mixture (16:4) was studied using OPM and SEM image analysis. For this, 2 mg of BA was dissolved in 1 ml ethanol-water mixture (16:4) and subsequently used for OPM and SEM analysis. For OPM analysis the 6

prepared sample (SA-BA) was dried slightly and spread on to cover slip and subjected to examine under polarized light using NIKON ECLIPSE LV100POL (Japan) microscope.

The size of materials and nano-structure were studied by scanning electron microscopy (ZEISS EVO-MA 10, India). In brief, sample was dissolved in ethanol: water mixture (16:4) at a concentration of 2 mg/ml and then the sample were syndicated using a sonicator bath (REMI) until the sample formed a homogeneous suspension. For size measurement, the syndicated stock solution of sample (0.5 mg/ml) was diluted 20 times using the same solvent mixture. The sample was coated with a thin layer of gold before SEM examination [12]. The self assembly property of BA was also investigated in presence of RPMI-1640 complete media. For this purpose 1 mg of BA was dissolved in ethanol: water mixture (16:4, 0.5% w/v). Like this way 1 mg of BA was dissolved in DMSO for structural identification of the compound in non-self assembly form. In both cases the dissolved BA was added to RPMI-1640 culture media at 25µg/ml dose and incubated for 24 hrs. After incubation a small amount of media was taken for SEM analysis.

2.4. Cell lines culture and maintenance

KG-1A (AML) and K562 (CML) cell lines were obtained from NCCS, Pune (India). The cells were cultivated and maintained in RPMI-1640 complete media supplemented with 10% heatinactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin under 5% CO2 and 95% humidified atmosphere at 37°C in CO2 incubator. Cells

7

were cultured and maintained in logarithmic growth phase until number of cells reaches at 1.0 X 106 cells/ml.

2.5. Selection of human subjects for collection of lymphocytes

Six healthy subjects were chosen at random to collect the blood sample for separation of lymphocytes. The subjects enrolled in this study were all asymptomatic and none of them was physically abnormal confirmed by routine laboratory tests. The subjects were from same geographical area and same economic status, non-smokers and non-alcoholic, and having same food habit. These subjects received no medication, including vitamin-E and vitamin C. All subjects gave informed consent. The selection excluded not only individuals with acute infections or chronic diseases, but also excluded healthy individuals undergoing supplementation with antioxidants. The study protocol was in accordance with the declaration of Helsinki, and was approved by the ethical committee of Vidyasagar University [10].

2.5.1. Isolation of peripheral blood lymphocytes Blood samples were collected from these six healthy human volunteers by vein-puncture in 5 ml heparin coated Vacutainers satisfying the method of Hudson and Hay [14]. Five milliliters of blood were diluted 1:1 with phosphate buffered saline (PBS) and layered onto Histopaque 1077 (Sigma) by using a Pasteur pipette and centrifuged at 400 x g (1500 rpm) for 40 min at room temperature. The upper monolayer of buffy coat i.e lymphocytes was transferred using a clean Pasteur pipette to a clean centrifuge tube and washed three times in balanced salt solution. The peripheral blood lymphocytes (PBL) were re-suspended in RPMI complete media supplemented 8

with 10% FBS and incubated for a day at 37°C in a 95% air/5% CO2 atmosphere in CO2 incubator. As KG-1A cells were already in the proliferative state, so PBL was stimulated with 1µg/ml LPS for 1 hour, followed by washing prior to most of the experiments [15].

2.6. Drug preparation

A 10 mg/ml stock of BA was prepared by dissolving 10 mg of BA in ethanol-water mixture (16:4) which forms self assembled BA (SA-BA). Non self-assembled BA was prepared by dissolving 10 mg of BA in DMSO. Stock concentrations of BA were then serially diluted with RPMI media to prepare working concentrations. The amount of ethanol and DMSO for each concentration, was never exceeded >0.75%.

2.7. Experimental design

Each type of cells was divided into 10 groups. Each group contained 6 petri dishes (2 X 105 cells in each). The following groups were considered for the experiment and cultured for 24 hrs: Group I: Control i.e., Cells + culture media, Group II: Cells + 5 μg/ml Doxorubicin in culture media, Group III: Cells + 5 μg/ml BA in culture media, Group IV: Cells + 5 μg/ml SA-BA in culture media, Group V: Cells + 10 μg/ml Doxorubicin in culture media, Group VI: Cells + 10 μg/ml BA in culture media, Group VII: Cells + 10 μg/ml SA-BA in culture media, Group VIII: Cells + 25 μg/ml Doxorubicin in culture media, Group IX: Cells + 25 μg/ml BA in culture media, Group X: Cells + 25 μg/ml SA-BA in culture media.

9

After the treatment schedule the cells were collected from the petri dishes separately and centrifuged at 2,200 rpm for 10 min at 4°C to separate cells and supernatants [10]. The cells were washed twice with 50 mM PBS, pH 7.4. A required amount of cells were lysed using hypotonic lysis buffer (10 mM TRIS, 1 mM EDTA and Titron X-100, pH 8.0) for 45 min at 37ºC and then processed for the biochemical estimation. Intact cells were used for mitochondrial membrane potential, ROS, and different microscopic observations.

2.8. In vitro cell viability assay

The dose and duration dependent cytotoxicity of DOX, BA and SA-BA on PBL, KG-1A and K562 cells were quantitatively estimated by a non-radioactive, colorimetric assay system using tetrazolium salt, 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenil-tetrazolium bromide (MTT) [16]. The percentage of proliferation was calculated by using the following equation: % Proliferation = [OD sample – OD control] X 100/OD control The concentration required for a 50% inhibition of viability (IC 50) was determined graphically. Multiple linear regressions were used to compare data using Statistica version 5.0 (Statsoft, India) software package.

2.9. Hemolysis assay

EDTA-stabilized human blood samples were freshly obtained from healthy subjects according to the previous mentioned protocol. First, 5 ml of blood sample was added to 10 ml of phosphatebuffered saline (PBS), and then red blood cells (RBCs) were isolated from serum by 10

centrifugation at 2,200 rpm for 10 min. The RBCs were further washed five times with 10 ml of PBS solution. The purified blood was diluted to 50 ml with PBS. Prior to SA-BA exposure, the absorption spectrum of the positive control supernatant was checked and used only if the observance was in the range from 0.50 to 0.55 optical density units to reduce differences in samples from different donors. RBC incubation with deionized water and with PBS was used as the positive and negative controls, respectively. Then, 0.2 ml of diluted RBC suspension was added to 0.8 ml of SA-BA solutions at systematically varied concentrations and mixed gently. The SA-BA suspended in PBS solutions with different concentrations was prepared immediately before RBC incubation by serial dilution. All the sample tubes were kept in the static condition at room temperature for 3 h. Finally, the mixtures were centrifuged at 3000 rpm for 3 min, and 100 µl of supernatant from all samples was transferred to a 96-well plate. The absorbance of the supernatants at 570 nm was determined by using a microplate reader with the absorbance at 655 nm as a reference [17]. The percent hemolysis of RBCs was calculated using the following formula: Hemolysis (%) = (Asample − Anegative control) / (Apositive control − Anegative control) × 100, Where Asample, Anegitive

control

and Apositive

control

are denoted as absorbencies of the sample, and

negative and positive controls, respectively. All hemolysis experiments were carried out in triplicate.

2.10. In vitro drug uptake assay

To find out the internalization of SA-BA on PBL and leukemic cells (KG-1A and K562), we performed in vitro drug uptake assay using fluorescence microscopic imaging [10]. Briefly, Rh11

B labeled SA-BA was prepared through the following process. Ten (10) µg/ml SA-BA was conjugated with 50 μl (2 mg/ml) of Rh-B. This mixture was stirred for 24 hrs at 37°C using magnetic starrier (REMI, India). Then, these fluoro labelled SA-BA were separated by centrifugation at 4ºC. The obtained sediment was washed with de-ionized water and redispersed. This process was repeated three times to remove the un-reacted Rh-B. To determine successful tagging of Rh-B on SA-BA, we examined this fluoro labeled compound under fluorescence-polarized microscopy. Finally, the obtained Rh-B labeled SA-BA was dispersed in culture medium for in vitro experiment. Cells were plated at a density of 2× 104cells/Petridis (35 mm) for 24 h. Rh-B tagged SABA at 25 µg/ml conc. were incubated for 6 hr at 37°C in a 95% air/5% CO2 atmosphere in CO2 incubator. After defined time, the cover slips were removed; the cells were washed 2 times with PBS and immediately observed in green light under the fluorescence microscope (NIKON ECLIPSE LV100POL) for uptake assessment. Images were acquired at 50X optical zoom and analysis was done using ImageJ software v.r. 1.43 (NIH).

2.11. Determination of reduced glutathione (GSH)

Reduced glutathione estimation in cell lysate was performed by the method of Kar Mahapatra et al [16]. The required amount of the sample was mixed with 25% of TCA and centrifuged at 2,000×g for 15 min to settle down the precipitated proteins. The supernatant was aspirated and diluted to 1 ml with 0.2 M sodium phosphate buffer (pH 8.0). After that, 2 ml of 0.6 mM DTNB was added. After 10 minutes the optical density of the yellow-colored complex formed by the reaction of GSH and DTNB (Ellman’s reagent) was measured at 405 nm. A standard curve was 12

obtained with standard reduced glutathione. The levels of GSH were expressed as μg of GSH mg / protein.

2.12. Determination of oxidized glutathione (GSSG)

The oxidized glutathione level was measured after derevatization of GSH with 2- vinyl pyidine according to the method of Kar Mahapatra et al [16]. In brief, with 0.5 ml sample, 2 μl of 2vinylpyidine was added and incubates for 1 hr at 37°C. Then the mixture was de-protenized with 4% sulfosalicylic acid and centrifuged at 1,000×g for 10 min to settle the precipitated proteins. The supernatant was aspirated and GSSG level was estimated with the reaction of DTNB at 412 nm in spectrophotometer and calculated with standard GSSG curves. The levels of GSSG were expressed as μg of GSSG mg/protein.

2.13. Intracellular ROS measurement

ROS measurement was performed using H2DCFDA according to our previously reported method [18]. In brief, normal PBL, KG-1A and K562 cell lines (2X 105cells per milliliter) were treated with SA-BA at 25 µg/ml for 24 hrs. As a positive control, those cells were incubated with H2O2 (100 µM) for 30 min prior to the analysis [19]. After treatment schedule cells were washed with culture media followed by incubation with 1 µg/ml H2DCFDA for 30 min at 37ºC. Then the cells were washed three times with fresh culture media. DCF fluorescence was determined at 485 nm excitation and 520 nm emission using a Hitachi F-7000 Fluorescence Spectrophotometer and

13

was also observed by fluorescence microscopy (NIKON ECLIPSE LV100POL).

All

measurements were done in triplicate.

2.14. Measurement of mitochondrial membrane potential (ΔΨm)

The alteration of mitochondrial membrane potential by spectro-fluorometric method was done according to our previous reported method [20]. In brief, PBL, KG-1A and K562 cell lines (2X 105cells per milliliter) were treated with DOX and SA-BA at 25 µg/ml dose for 24 hrs. It was evident from the previous study that DOX can decrease mitochondrial membrane potential significantly [21]. After treatment schedule cells were washed with culture media followed by incubation with 1.5 µM Rh-123 for 10 min at 37ºC in a humidified incubator. Then the cells were washed three times with culture media. The cellular fluorescence intensity of Rh-123 was monitored for 2 min using Hitachi F-7000 Fluorescence Spectrophotometer. A required amount of cells was also used for microscopic observations (NIKON ECLIPSE LV100POL). The cellular mitochondrial membrane potential was expressed as a percentage of control cells at an excitation wave length of 493 nm and an emission wavelength of 522 nm. Both excitation and emission slit width were set to 5.0.

2.15. DNA fragmentation study by alkaline comet assay

The alkaline comet assay was performed according to the previously reported method [18]. Comet tail length was calculated as the distance between the end of nuclei heads and end of each tail. Tail moments were defined as the product of the percentage of DNA in each tail, and the 14

distance between the mean of the head and tail distributions and presented as: %DNA (tail) = TA X TAI X 100/ [(TA X TAI) + (HA X HAI)]; where TA = tail area, TAI = tail area intensity, HA = head area and HAI = head area intensity. Tail DNA % was calculated using image J 1.44 with comet macros function (NIH).

2.16. Measurement of apoptosis by FACS staining

Cellular apoptosis was measured by flow cytometric analysis. In brief, after DOX and SA-BA exposure (25 µg/ml) for 24 hrs, the cells were collected and centrifuged with the supernatant medium at 1400 rpm for 5 min. Following washes, cells were re-suspended in PBS and fixed in 70 % ethanol for 1 hr on ice. Fixed cells were washed with PBS and stained with Annexin VFITC and propidium iodide solution as per the manufacture’s instruction (E-bioscience, India). Then, cells were analyzed on a Becton Dickinson FACS Calibur flow cytometer. The cell populations were analyzed by CellQuest software [22].

2.17. Examination of apoptosis by DAPI staining

All the test cells were seeded into six well plates. A number of 2X105 cells/ml were treated with or without SA-BA (0, and 25 µg/ml) for 24 hrs and were then isolated for DAPI staining according to the method of Lin et al [23] with some modification. After treatment, the cells were fixed with 2.5% glutaraldehyde for 15 min, permeabilized with 0.1% Triton X-100 and stained with 1 µg/ml DAPI for 5 min at 37 C. The cells were then washed with PBS and examined by fluorescence microscopy (NIKON ECLIPSE LV100POL). 15

2.18. Measurement of pro-inflammatory and anti-inflammatory cytokine level by sandwich ELISA

The level of TNF- α, IL-12p70, IL-10 and TGF-β in serum free culture supernatants were measured using an enzyme-linked immunosorbent assay (ELISA) kit with pre-coated plates (Human TNF alpha ELISA Ready-SET-Go, E-bioscience, India) according to manufacturer’s instruction. In brief, PBL and KG-1A and K562 cells were treated with SA-BA (25 µg/ml) and 1 µg/ml LPS for 24 hrs. It was found from the study that, LPS is one of potential stimulator for cytokines releases from various types of cells [24]. After treatment culture supernatants were separated by centrifugation (2200 rpm for 10 min). Hundred micro liters of each supernatant were taken as a sample of cytokine release level. O.D was recorded using an ELISA micro plate reader, Bio Rad, India. The sensitivity limits were 4.0, 4.0, 2.0 and 8.0 pg/ ml for TNF- α, IL12p70, IL-10 and TGF-β cytokines. Cytokines concentration was expressed as pg/ml/10 6 cells to correct for the number of total PBL, KG-1A and K562 cells.

2.19. Pretreatment with N-acetyl- L-cysteine and incubation with pentoxifylline

To determine the role of ROS in SA-BA induced cell death, KG-1A and K562 cells were seeded in a 96-well plate at 0.2 ml per well at a concentration of 2 X 105cells per milliliter. A stock solution of N-acetyl- L-cysteine (NAC; Sigma- Aldrich) was made with sterile water and added to cells at 5 and 10 mM for 1 h. After NAC pretreatment, cells were cultured with SA-BA (2 µg/ml) for 24 hrs. Viability was determined by the MTT method [16].

16

To understand the potential contribution of TNF-α in SA-BA induced cell death, PBL, KG-1 and K562 cells were co-cultured with 1mM and 2 mM Pentoxifylline (a potent TNF-α inhibitor) with 25 µg/ml dose of SA-BA for 24 hrr. The doses of pentoxifylline were selected from the previous study of Marques et al [25]. After the treatment schedule cells were washed two times with culture medium and cell viability was estimated by MTT assay [16]. 2.20. Immunofluorescent staining

Both the KG-1A and K562 cells were treated with 25μg/ml of SA-BA for 24 h. At the end of treatment(s), the cells were washed with PBS, pH 7.2, and were fixed in 4% paraformaldehyde solution for 15 min at 4°C. Next, the cells were washed three times with the blocking buffer (5% bovine serum albumin in PBS), followed by permeabilization with 0.5% Triton X-100 in PBS for 20 min at 25°C. After three washes with the blocking buffer, the cells were incubated for 1 h at 25°C with human antibodies specific for caspase-8, 3 (e-bioscience, India). The antibodies were used at dilutions of 1:100, in blocking buffer. The cells were then rinsed three times with the blocking buffer and probed in the dark with Rh-B conjugated compatible secondary antibodies for 1 h at 25°C. Following three washes with the blocking buffer, the coverslips were mounted on glass slides using a Fluoromount G (Electron Microscopy Sciences, Fort Washington, Pa., USA). The slides were viewed using a fluorescence microscope (NIKON ECLIPSE LV100POL) equipped with the suitable wavelength filters [26].

2.21. Protein estimation

17

Protein was determined according to Lowry et al., 1951 using bovine serum albumin as Standard [27].

2.22. Statistical analysis

All the parameters were repeated at least three times. The data were expressed as mean ± SEM, n = 06. Comparisons between the means of control and treated group were made by one-way ANOVA test (using a statistical package, Origin 6.1, Northampton, MA 01060 USA) with multiple comparison t tests, p<0.05 as a limit of significance.

3. Results

3.1. Self assembly study of betulinic acid

3.1.1. Dynamic light scattering (DLS) and Zeta potential The size distribution of self assembled betulinic acid (SA-BA) suspended in aqueous medium was estimated by DLS. The hydrodynamic size (Z-average) of the material was 67.42 nm with a polydispersity index (PDI) of 0.639 (Fig. 1A). The zeta-potential measurement showed that SABA had a negative zeta potential (-17.3 mV), indicated that the material possess a negatively charged surface (Fig. 1B).

18

A 40

B

Number (%)

30

20

10

0 40

60

80

100

Size (r.nm)

Figure 1

3.1.2. Optical polarized microscopy and scanning electron microscopy Self-assembly property of BA was observed using OPM and SEM analysis (Fig 2 A and 2B respectively). OPM image showed that, in ethanol-water mixtures (16:4), BA produced several fibrillar networks. SEM imaging confirmed that the fibers of BA were appeared in the nanometer to micrometer cross sections and micrometer lengths. The single fiber’s cross section was noted to be 15-25 nm with 100 nm to 3 µm lengths (Fig. 2). It was also found that fibers were aggregated together and formed a network like arch structure. A

B

19

Figure 2

SEM imaging showed that, in culture medium, the BA (dissolved in DMSO) appeared as highly aggregated bulk structure (Supplementary Fig 1A). This suggested the non assembled configuration of BA. But on the other set of experiment the BA (dissolved in ethanol+water mixture) showed appearance of several fibrillar networks suggested the formation self assembled BA (SA-BA) (Supplementary Fig 1B)

3.2. In vitro cell viability assay

Cell viability was measured by MTT experiments. Fig. 3A shows the viability of PBL, KG-1A and K-562 cells after DOX exposure at 1, 5, 10 and 25 µg/ml doses after 24 hr of incubation time. It was found that, doxorubicin showed potent ant proliferative effects on both types of leukemic cells significantly (p<0.05) at all concentrations. At 25µg/ml of DOX treatment, cell viability of KG-1A and K562 were significantly (p<0.05) decreased by 90.82% and 92.89% respectively when compared with the control group. Side-by-side DOX showed potential toxic effects on PBL. From Fig 3A it was observed that, cell viability of DOX treated PBL significantly (p<0.05) decreased by 52.87%, 61.17% and 65.32% in 5, 10 and 25 µg/ml doses respectively when compared to control group. Cell viability of BA and SA-BA treated PBL, KG1A and K562 cells were examined by same manner to identify the applicability of those drugs over DOX. It was manifest that the viability of BA treated leukemic cells were decreased with the increasing concentration of BA, which was also dependent on the period of incubation (Fig 3B and Fig 3D). BA exposure for 24 hrs reduced the viability of KG-1A and K562 cells significantly by 47.37%, 70.93% and 38.08%, 63.67% at 10 and 25 µg/ml doses, respectively (Fig 3B) where as SA-BA decreased the viability of those leukemic cells by 54.66%, 59.86%, 20

83.96% (KG-1A cells) and by 40.84%, 57.08%, 69.73% in 5, 10 and 25 µg/ml doses respectively when compared to control group. Surprisingly, it was observed that both BA and SA-BA did not produce any significant anti-proliferative effects on normal PBL at all concentrations (Fig 3C). Thus, it can be stated that SA-BA showed more potent anti-proliferative activity than BA on both leukemic cells. From Fig 3D it was found that the cytotoxicity of SA-BA depends on concentration of drug and time of exposure. Time dependent killing kinetic assay showed that 25 µg/ml dose of SA-BA for 24 hour incubation played maximal anti-leukemic effects on KG-1A and K562 cells. The IC50 value of BA and SA-BA against PBL, KG-1A and K562 cells were found to be 159.62, 14.87, 17.59 µg/ml, respectively, for the BA and 124, 10.16, 13.93 µg/ml respectively for SA-BA, after interaction for 24 hours (Supplementary Fig 2). From the cell viability experiment it was found that SA-BA showed higher cell killing ability in compare to BA towards both leukemic cells. So this 25 µg/ml dose of only SA-BA with 24 hour incubation time was selected for further experiments.

21

B

A 110

PBL+DOX

KG-1A+DOX

K562+DOX

110

100

PBL+BA

KG-1A+BA

K562+BA

100 Cell viability (%of control)

Cell viability (% of control)

90 80 70 60 50 40 30 20

90 80 70 60 50 40 30

10 Control

5

1

10

20

25

Control

10

25

D

C PBL+SA-BA

5

Concentration of BA (g/ml)

Concentration of DOX (g/ml)

110

1

KG-1A+SA-BA

K562+SA-BA

KG-1A+SA-BA 25 g/ml

100

K562+SA-BA 25 g/ml

100 Cell viability (% of control)

Cell viability (%of control)

90 80 70 60 50 40 30 20

80 60 40 20

10 Control

1

5

10

Concentration of SA-BA (g/ml)

0

25

O hr 2 hr 4 hr 8 hr 12 hr 16 hr 18 hr 24 hr

Figure 3

22

Incubation Time (hr)

3.3. Dose-dependent hemolytic activity of SA-BA

The hemolysis assay was used to evaluate the cytotoxic effect of SA-BA on human normal RBCs. The RBCs were exposed to SA-BA at concentrations from 1 to 100 µg/ml doses for 3 hr. It was observed that no significant hemolytic activity of SA-BA was noted at any doses used (Fig 4).

% of Hemolysis

100

80

--

µg /m l 5 µg /m l 10 µg /m 25 l µg /m l 50 µg /m l 10 0µ g/ m l

C

1

-v e

+

ve

C

0

Concentration of SA-BA (µg/ml)

Figure 4 3.4. Intracellular localization of drug by fluorescence microscopy

Cellular internalization of SA-BA was performed by fluorescence microscopy. Successful tagging of SA-BA with Rh-B was confirmed by fluorescence images (Fig 5A-C). The fluorescence imaging revealed that Rh-B labeled SA-BA was successfully taken up by KG-1A (Fig 5c-d) and K562 cells (Fig 5e-f). A very little amount of fluoro labelled SA-BA was 23

internalized in PBL (Fig 5a-b). It was observed that SA-BA distributed throughout the cells maintaining its self assembly configuration. A

C

B

Figure 5 24

3.5. Examination of cellular redox status (GSH and GSSG level)

From Fig 6 and Fig 7 it was found that PBL, DOX (25 µg/ml) treatment significantly (p<0.05) decreased GSH level by 62.58% and significantly (p<0.05) elevated GSSG level by 122% as compared to control. But treatment with SA-BA (25 µg/ml) in PBL showed completely reverse action. Here, SA-BA treatment significantly (p<0.05) increased GSH level by 77.67% and significantly (p<0.05) decreased GSSG level by 76.56% as compared with control. Alteration of GSH and GSSG level in leukemic cells was observed to be same manner. DOX treatment significantly (p<0.05) diminished the level of GSH by 90.21% and increased GSSG level by 247.10% in KG-1A cells (Fig. 6 and Fig. 7 respectively). The same type of changes was also noted for K562 cells. Here the DOX treatment also significantly (p<0.05) decreased GSH level by 88.00% and significantly (p<0.05) increased GSSG level by 158.59% when compared with the control group (Fig. 6 and Fig. 7 respectively). SA-BA exposure significantly (p<0.05) diminished GSH level by 86.42% in KG-1A cells and by 65.04% in K562 cells. GSSG levels of SA-BA treated KG-1A cells was increased by 99.65% and also increased by 36.67% in K562 cells at the effective dose compared with the control group. 25

GSH (µg/mg protein)

20 15

Figure 6

10 5

PB L Co PB + D ntr L+ O ol P SA X 2 BL -B 5 µ A g/m 25 l µg /m K G C l K -1A ontr G o + -1 l A DO KG + SA X 2 -1A -B 5 µ A g/m 25 l µg /m K l 56 C K 2 + ontr 56 2 DO ol K + SA X 2 562 -B 5 µ A g/m 25 l µg /m l

0

25

X Axis Title

25

GSSG (µg/mg protein)

20 15 10 5

nt

SA

L+

PB

L+

D

O

Co PB

X

ro

lP 25 BL -B µ A g/m 25 l µg /m K G C l K -1A ontr G o + -1 l A DO KG + X SA 2 -1A -B 5 µ A g/m 25 l µg /m K l 56 C K 2 + ontr 56 2 DO ol K + SA X 2 562 -B 5 µ A g/m 25 l µg /m l

0

X Axis Title

Figure 7

3.6. Estimation of cellular ROS level

From Fig 8. It was found that treatment with DOX (25 µg/ml) elevated cellular ROS level by 443.34%, 523.39% and 531.021% in PBL, KG-1A and K562 cells respectively. SA-BA treatment selectively increased ROS level in both leukemic cells and significantly (p<0.05) decreased in PBL. SA-BA treatment was able to significantly (p<0.05) increased ROS level by 449.30% in KG-1A cells and by 241.866% in K562 cells as compared with the control group. From fluorescent microscopic images this alteration of cellular ROS level was clearly observed (Fig 8a-6f).

26

PB L Co PB + D ntr L+ O ol P SA X 2 BL -B 5 µ A g/m 25 l µg /m K G C l K -1A ontr G -1 + D ol K A + OX G-1 SA 2 A -B 5 µ g A 25 /ml µg /m K l 56 C K 2 + ontr 56 2 DO ol K + SA X 2 562 -B 5 µ A g/m 25 l µg /m l

DCF fluorescence intensity (% of control)

700

A 600

500

400

300

200

100

0 b

X Axis Title

27 Figure 8

3.7. Determination of mitochondrial membrane potential

Alteration of mitochondrial membrane potential is one of the effective mechanisms for initiation of apoptosis. Thus we examine cellular mitochondrial membrane potential (MMP) in term of Rh123 fluorescence intensity after H2O2 and SA-BA exposure for defining dose and time period. From Fig. 9, it was observed that treatment with SA-BA significantly (p<0.05) reduced MMP by 58.442% in KG-1A cells and by 41.95% in K562 cells compared with the control group. Potent ROS generating substance, H2O2 also decreased MMP significantly (p<0.05) by 73.24% and 70.75% in KG-1A and K562 cells respectively cells compared with the control group.. 120 100 90 80 70 60 50 40 30 20 10

K

56

2

+

2 56 K

X Axis Title

Figure 9 28

25

µg /m l

µg /m l -B A

+

SA

D

O

Co

X

nt

ro

25

lK

56

2

µg /m l

25 -B A

K

G

G

-1 A

-1 A

+

+

SA

D

Co

O

nt

X

ro

25

lK

G

-1 A

µg /m l

0

K

Rhodamin 123 fluorescence intensity (% of control)

110

3.8. DNA fragmentation estimation by alkaline comet assay

Genotoxicity of SA-BA was determined by alkaline comet assay. After electrophoresis, SA-BA treated leukemic cells were examined under fluorescence microscope and images were captured and analyzed with ImageJ (NIH) software (Fig. 10A-10B). The percentage of DNA (tail) was observed in leukemic cells treated with 25 µg/ml of SA-BA. Percentage of tail DNA intensity reveled that DNA damage was significantly increased in both leukemic cell lines.

A

B

70 60

a

b

c

d

Tail DNA %

50 40 30 20 10

25 + 2 56 K

K

G

-1 A

+

SA

SA

-B A

K

-B A

56 2

25

Co nt

µg /m l

l ro

l ro nt Co -1 A K

G

µg /m l

0

X Axis Title

Figure 10

3.9. Apoptotic cell population by FACS study

The induction of cell death by SA-BA was estimated by FACS using Annexin V-FITC and PI staining. Percentages of only Annexin V-FITC positive cells (LR) were denoted as early 29

apoptotic cells, Annexin-FITC+PI positive cells were considered as late apoptotic cells and only PI positive cells were marked as necrotic or dead cells. The LL quadrant showed viable cells. The result showed that SA-BA treatment significantly (p<0.05) elevated early apoptotic cells in both leukemic cells. A lower number of late apoptotic cells were also noted. But, in case of PBL no significant apoptotic cell population was found due to SA-BA treatment (Fig. 11a-f and 11B).

A

B

a

b

Q1 LL (Viable cells) Q1 UR (Late apoptosis)

Q1 LR (Early apoptosis) Q1 UL (Necrosis)

100

% of cells

Propidium iodide (PI)

80

c

60 40

d 20 0 KG

e

f

Annexin V+FITC

Figure 11

30

1A

C

KG

1

SA A+

A -B

6 K5

2C

A -B

K5

A +S 62

L PB

C S L+ PB

A-

BA

3.10. Nuclear morphological changes using DAPI staining

DAPI preferentially stains the nucleus by binding strongly to A-T rich regions in DNA, which is observed as blue fluorescence when excited under a fluorescence microscope (excitation 330– 380 nm, emission 430–460 nm). In our present study, DAPI staining exposed the changes associated with apoptosis in KG-1A and K562 cells treated with the SA-BA (Fig. 12). The morphological changes coupled with apoptosis such as chromatin condensation, nuclear fragmentation, and margination of nucleus (marked by arrows in Fig. 12b) were manifest in leukemic cells upon treatment with SA-BA. A very small number of apoptotic cells were noted for PBL.

Figure 12 31

3.11. Determination of pro and anti inflammatory cytokine release by ELISA

To find out whether inflammation plays any role in the antileukemic activity of SA-BA we performed pro inflammatory (TNF-α and IL-12) and anti inflammatory (TGF-β and IL-10) cytokine levels by ELISA (Fig 13A-13D).

It was found that LPS treatment in KG-1A cells

significantly (p<0.05) increased TNF-α level by 136.02%, IL-12 level by 89.78% (Fig 13A and 13B) and significantly (p<0.05) reduced TGF-β level by 78.40%, IL-10 level by 68.75% (Fig 13C and 13D) compared with the control group. In case of K562 cells, LPS treatment significantly (p<0.05) increased TNF-α level by 170.33%, IL-12 level by 81.15% and significantly (p<0.05) reduced TGF-β level by 78.77%, IL-10 level by 45.68% compared with the control group. SA-BA also pro inflammatory cytokine balance and reduced antiinflammatory cytokines balance. When KG-1A cells were treated with SA-BA for defining dose and time period TNF-α level was significantly elevated by 74.02% and IL-12 level was also increased by 63.94%. SA-BA treatment in KG-1A cells decreased TGF-β level significantly by 65.79% and the IL-10 level by 59.27% compared with the control group. In case of K562 cells, treatment with SA-BA significantly (p<0.05) increased TNF-α level by 78.61%, IL-12 level by 52.01% and significantly (p<0.05) reduced TGF-β level by 45.68%, IL-10 level by 54.44% compared with the control group.

32

K

X Axis Title

Figure 13

33

X Axis Title

+

SA

1

25

LP S

-B A

+

µg /m l

µg /m l

2

ro lK 56

µg /m l

2

K 56

+

LP S SA -B A

+

25

1

µg /m l

µg /m l

25

20

µg /m l

µg /m l

lK 56 2

Co nt ro

-B A

SA

56 2

K

+

270

56 2

K

K 56 2

25

µg /m l

C

Co nt

-B A

SA

0

+

90

1

30 -1 A

0

LP S

30

G -1 A

60

K G

90

+

210

Release of IL-12p70 (pg/ml)

210

LP S

X Axis Title

K

tro l

µg /m l

µg /m l

2

120

G -1 A

K

Co n

1

56

lK

25

LP S

-B A

SA

+

ro

µg /m l

µg /m l

-1 A

240

+

60

Release of IL-10 (pg/ml)

+

2

1

25

nt

Co

-B A

SA

G

150

-1 A

µg /m l

µg /m l

2

56

K

+

56

K

-1 A

+L PS

lK

ro

180

G

25

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lK

nt ro

Co

SA -B A

LP S

2

56

K

G

K

-1 A

G

Release of TNF- (pg/ml)

240

-1 A

G

+

+

ro l

nt

µg /m l

µg /m l

-1 A

K

nt

Co

A

-1 A

G

K

2

K 56

2

Co

1

25

LP S

-B A

SA

56

+

+

G

lK

tro

Release of TGF- (pg/ml)

120

K

-1 A

G

K

-1 A

G

K

Co n

270

B

180

150

120 90

60

30 0

X Axis Title

D

120

90

60

30

0

3.12. Involvement of TNF-α and ROS in SA-BA induced cytotoxicity in leukemia cells

To observe the role of TNF-α on SA-BA induced cancer cell killing, we co-culture the both leukemia cells with 1 and 2mM POF (a potent TNF-α inhibitor) and SA-BA (25 µg/ml) (Fig 14A-14B). The viability was estimated by MTT method. It was observed that POF played a great role in eliminating toxic effects induced by SA-BA in both leukemic cells. In each case cell viability was >89%. Studies showed that TNF-α is thought to generate reactive oxygen species (ROS) and activate the downstream apoptosis signals. To search out whether ROS played pivotal role in SA-BA induced leukemic cells, we pre-treated KG-1A and K562 cells by 2 and 5mM of NAC, a potent ROS inhibitor, for 4-6 hr before SA-BA exposure and cell viability was estimated by MTT method. It was found that pre-treatment with 5mM NAC effectively protected the cells from SA-BA induced cytotoxicity. Cell viability of KG-1A cells was reached to 89.49% and to 91.099% in K562 cells compared with the control group.

A

B

KG-1A cells

K562 cells

KG-1A cells

K562 cells

Cell viability (% of control)

100

80 60 40 20

80 60 40 20 0

Doses of Pentoxifylline (POF)

Doses of N-acetyl cysteine (NAC) and Pentoxifylline (POF) Doses of NAC

Figure 14 34

M 2m

M 1m

Co n

M 5m

M 2m

tro

l

tro

l

0 Co n

Cell viability (% of control)

100

3.13. Immunoflouroscence staining of apoptotic marker proteins

Higher expression of caspage 8 and 3 protein in SA-BA treated leukemic cells was observed from their respective fluorescence images (Fig. 15). Elevated expression of those pro-apototic markers proteins in SA-BA treated KG-1A and K562 cells suggested the activation of apoptosis

Caspase 8

Caspase 3

phenomenon.

Figure 15

35

4. Discussion Severe side effects of anticancer drugs produce an urgent need to develop new classes of anticancer agents with great potency as well as selectivity. Plant derived secondary metabolites plays new therapeutic benefits as well as the crucial role in anticancer drug development. The capability of some natural products or naturally occurring substances in inhibition of cancer cell’s growth was investigated through modulation of the immune system and provides a greatest alternative in solving toxic side effects towards healthy cells. Triterpenoids shows a highly promising class of natural compounds in bio-medical applications. BA, a pentacyclic triterpenoid compound has been identified as potentially effective agent against a wide variety of cancer cells, also those derived from therapy resistant and refractory tumors, whereas it has been found relatively non-toxic to healthy cells. First report on self assembly of BA in twenty two organic liquids and alcohol–water mixtures was studied earlier by Bag et al [8]. The present study report showed that in alcohol-water mixture BA revealed fibrillar networks having fibers of nano to micro-meter cross sections and micrometer lengths (Supplementary Fig 1 and Fig 1B). The nano archistructure of SA-BA was investigated by DLS and zeta potential study (Fig. 1A and Fig 1B respectively). From the SEM image, it was found that the fibers having cross-sectional diameter within 10-25 nm (Fig 2B), but in DLS analysis the size was obtained 67.42 nm (Fig 1A). This because, the images obtained from SEM described the size in the dried state of the sample, whereas DLS measured the size in the hydrated state of the sample, so that the size measured by DLS was a hydrodynamic diameter and larger in size. However, one has to bear in mind that by SEM we image single particles, while DLS gives an average size estimation, which is biased toward the larger-size end of the population distribution [17]. The obtained zeta-potential indicated that the fibers were considerably stable in aqueous medium (Fig. 1B). Best of our 36

knowledge, no bio-activity study of this self assembled BA was done earlier. Not only that, no relevant study regarding anti-leukemic effects of BA against KG-1A cell line has been reported till now. This study demonstrated that both BA and SA-BA could inhibit proliferation of KG-1A and K562 cells selectively in a dose-dependent manner, with no toxic effects on normal PBL cells (Fig. 3). From this study it was found that anti-proliferative activity of SA-BA against leukemic cell lines were more than non-self assembled BA which was corroborate with our previous result [10]. In this previous observation BA mediated leukemic cell (KG-1A and K562 cells) death was under 65% in 25 µg/ml dose at 48 hr treatment period [10]. In this report the percentage of dead leukemic cells was drastically elevated in SA-BA (25 µg/ml) treatment for 24 hr (Fig 3C and D). The higher effectiveness of SA-BA over BA was may be due to the formation of several micros and nano diameter fibrillar network, which may facilitated the entry of the compound leukemic cells easily compared to bulk materials. Side by side, both types of BA did not produce any toxic effects towards the normal cells. The biocompatibility of SA-BA was also noted for human normal RBCs. It was noted that at all doses of SA-BA had no significant hemolytic activity (Fig. 4). The non-hemolytic nature of SA-BA is also an important parameter for evaluation of optimum potency of anticancer drugs. The IC50 value of SA-BA against K562 cells was much lower than other reported studies [28-29] (Supplementary Fig 2). It was also observed that the anti-proliferative activity of the SA-BA on KG-1A was higher than K562 cells which may be due to the maturation status of the cells. KG-1A cells are comparatively less mature than K562 cells. According to Graham-Evans et al [30], cytotoxicity has been defined as the cell killing property of a chemical compound independent from the mechanism of death. In vitro assessment of a compound’s toxicity to various cell types is one of 37

the primary and essential test which can easily estimate and widely used [31]. It was also found that the direct effect of various reactive compounds at sub toxic concentrations shows direct interaction with DNA that will result in various types of damage, including pro-mutagenic lesions [32]. Intracellular uptake is noted as important parameter for localization of the drug in the cells. Substantial uptake of the bio-active compound is proportional to the relative bio-activity. Fluorescence labeled SA-BA was examined under fluorescence microscopy to understand the successful entrapment of Rh-B within the fibrillar network (Fig. 5A-5C). This Rh-B labeled SABA was used in vitro for examination of SA-BA internalization in PBL and leukemic cells. The result showed that SA-BA was found to be distributed in the cytoplasm. This indicated that cellular uptake was associated instead of adhering to the surface and the SA-BA was preferentially targeted the cancer cells and were interned. In PBL a very little internalization of SA-BA were observed (Fig. 5g-5h). Having minimal internalization of SA-BA in PBL, no toxic effects were found and on the other side, it maintained cellular redox balance which was confirmed by further experiments. The cellular uptake study clearly revealed that Rh-B conjugated SA-BA slightly attached to cellular surfaces and maximally entered into the leukemic cells. It was also found that during internalization process SA-BA maintained its self assembly structure (Fig.5c-5h). This internalization might be due to endocytosis in which passive targeting of SA-BA in both leukemic cells were occurred [33-34]. Dysregulated cholesterol metabolism (cholesterol is a part of the cell membrane responsible for plasma membrane integrity, and regulates the uptake process) in leukemic cells [35] may enhance the uptake of SA-BA into cancer cells. In addition, tumor cell membranes contain a higher amount of phospholipids than normal cells [36-37]. The presence of a high amount of phospholipids may influence the 38

attachment of SA-BA to the cancer cell membrane, entered the cell and allows apoptosis followed by necrosis by activation of different biochemical processes. This selective uptake of SA-BA was responsible for selective cytotoxic effects which were found from cell viability study. Glutathione, the crucial component in the cellular anti-oxidant defense system, offers protections against free radicals, peroxide and toxic compounds [20, 38]. Many pathological conditions markedly reduce intra cellular GSH load by non-enzymatic oxidation of GSH to glutathione disulfide (GSSG). This conversion of GSH to GSSG is proceeding by oxidative stress [39]. This anti-oxidant molecule not only protects the cell from oxidative damage but also found to be crucial for the regulation of cell proliferation, cell cycle progression and apoptosis. The ability of GSH was found to neutralize numerous oxidizing compounds, including reactive oxygen species (ROS). It has been proposed that efflux of GSSG from the cell, and then occurs in order to preserve the cellular normal redox state so that a depletion of oxidized glutathione levels is regularly observed [40]. The present study showed that SA-BA produced antiproliferative effect on leukemic cells by altering the intracellular redox status (Fig. 6 and Fig. 7). Depleted level of GSH and higher level of GSSG in leukemic cells were may be due to the effective conversion of GSH to GSSG by free radicals (ROS and NO). In PBL, SA-BA treatment, significantly elevated cellular GSH level and diminished GSSG level. Thus, it can be understood that SA-BA killed the both leukemic cells by generating oxidative stress, side by side; it maintains the cellular redox status in normal cells by maintaining GSH level and decreasing GSSG level. The same type of result was also found in our previous report [10]. ROS are generated as byproducts of aerobic respiration and various other catabolic and anabolic processes. Many researchers have found that elevation of cellular ROS level is one of 39

the major causes of cell death [41-42]. Mitochondria are the major producer of ROS in cells during aerobic respiration during electron transport system. Electrons leak from the electron transport chain directed towards oxygen and thereby produces short-lived free radicals such as superoxide anion (O2−). This O2

−

can be easily converted to non-radical derivatives such as

hydrogen peroxide (H2O2) either spontaneously or it can be catalyzed by superoxide dismutase (SOD). H2O2 can be easily comes to cytosole due to its relative stability and higher permeability. Most of the anti-oxidants systems are present in cytosolic environment. Those anti-oxidants (such as catalase, glutathione peroxidase etc.) removes the free radicals from the cells [10]. In our study SA-BA effectively diminished cellular reduced glutathione level and increased the level of ROS in leukemic cells (Fig. 8A and 8c-f). Depletion of cellular anti-oxidant system was not able to eliminate ROS from those cells. On the other side in PBL, due no little SA-BA internalization an anti-oxidative role of SA-BA was noticed (Fig. 8a-8b). So higher level of GSH in PBL was may be the contributor mechanism for lowering cellular ROS level [10]. Previous studies indicated that mitochondria are the major target of betulinic acid [4344]. So, we investigated the mitochondrial metabolic status in term of membrane potential measurement after SA-BA exposure. Mitochondrial membrane potential of DOX and BA treated PBL, KG-1A and K562 cells were estimated using mitochondrial membrane specific fluorescence dye Rh-123 (Fig. 9). We found that treatment with SA-BA significantly decreases MMP in both leukemic cells, whereas it did not show any loss of MMP in PBL. Treatment with DOX showed significant loss of MMP in all types of cells. Studies showed that mitochondria are caught up commonly in programmed cell death due to its contribution in release of mitochondrial proteins into the cytosol, triggers several relevant pathways [45-46]. The release of such proteins requires specific changes in the mitochondrial membrane that allow the passage of pro-apoptotic 40

proteins. That alteration can be easily detected using Rh-123. Rh-123 specifically binds to the mitochondrial membrane which has higher polarization. In our study a gradual depolarization of mitochondrial membrane potential in leukemia cells due to SA-BA treatment suggested that the cells undergo programmed cell death (Fig. 10). In our study, comet assay confirmed the potent genotoxic effects of SA-BA on leukemic cells. The increased tail DNA (%) may be due to direct induction of DNA strand breaks or disruption of DNA backbones by SA-BA or its byproducts, like free radicals. In our study, elevated levels of ROS may contribute to severe genotoxic effects in leukemic cell lines. Thus, significant DNA damage in SA-BA treated leukemic cells suggests the involvement of the apoptosis phenomenon which was also observed in BA treatment [10] (Fig. 10A-10B). To confirm the SA-BA induced leukemic cell death was surely due to apoptosis, we examined the nuclear morphological changes of the SA-BA treated cells using DAPI staining and analyzed the apoptotic cell percentage by FACS analysis using Annexin V-FITC+PI dual staining (Fig. 11 A-B). The initiation of apoptosis associated with SA-BA treatment and estimation of exert population of viable cells, apoptotic cells and necrotic cells were confirmed by dual staining analysis of Annexin V+FITC. Annexin V+FITC fluorescence (FL1) and PI fluorescence (FL3) gave different cell populations. In our study SA-BA treatment significantly (p<0.05) increased the number of apoptotic cells in both leukemic cells. Induction of apoptosis was slightly higher for KG-1A cells than K562 cells (Fig 11). On the other no such apoptotic cell population was observed in case of PBL. DAPI staining denoted the visual events of apoptosis by detecting nuclear fragmentation which is one of the characteristic features of apoptotic mode of cell death (Fig. 12). Significant numbers of KG-1A and K562 cells with fragmented nuclei were observed upon treatment with SA-BA, suggested the induction of apoptosis in both 41

leukemic cells [49]. The result suggested that normal cells are highly resistant to SA-BA, where as leukemic cell are highly susceptible. Maes et al [48] reported that, the ratio of Th1/Th2 responders has been shown to be more indicative of immune function. In our present study, it is well documented that SA-BA can trigger the pro-inflammatory cytokines (TNF-α and IL-12), and diminish the anti-inflammatory (TGF-β and IL-10) cytokine release from both leukemic cells, indicated that, SA-BA may have at least short term damaging effect on the normal immune balance (Fig. 13). The significant deficit of anti-inflammatory cytokine response to counter the increased pro-inflammatory cytokine response in SA-BA exposed KG-1A and K562 cells suggested that, SA-BA, disrupts the normal balance of the Th1/Th2 cytokine levels. Elevation of pro-inflammatory cytokines level suggested the possible underlying etiology for SA-BA induced anti-leukemic effects may be due to severe inflammation. In this study LPS was also incorporated using same fashion as it is a very well known inducer of pro-inflammatory cytokines and inhibitor of anti-inflammatory cytokines level [23]. It was found that pre-treatment with NAC significantly protected the KG-1A and K562 cells from SA-BA induced toxicity (Fig. 14A). Restoration of leukemic cell viability >89% suggested that ROS is the main agent which played a crucial anti-leukemic potential caused by SA-BA. Association of ROS in SA-BA induced leukemic cell death supports the previously published reports of many researchers [41-42]. Studies showed that TNF-α initiates many downstream signaling pathways, including activation of NF-κB, MAP kinase and the induction of both apoptosis and necrosis [49]. TNF-α has been found to escort to ROS generation through the activation of NADPH oxidase by mitochondrial pathways, or other enzymes. ROS produced by TNF-α have an important function in cell death by activating c-Jun N-terminal kinase 42

pathway [50]. So in our study, we confirmed the major contribution of TNF-α and ROS in SABA induced leukemic cell death. To establish a connection between SA-BA induced leukemic cell death with TNF-α we co-treated the both leukemic cells with POF and SA-BA and cell viability was estimated. Pentoxifylline (POF), a methylxanthine derivative, suppressed TNFalpha gene transcription and downregulates the expression of TNF-alpha mRNA and the secretion of TNF-alpha protein in macrophages and monocytes [51]. It was found that administration of 2 mM POF protected the both leukemic cells from SA-BA induced cytotoxicity suggested the potential contribution of TNF-α underlying the etiology of cell death (Fig. 14B). On the other hand it was established that higher expression of inflammatory cytokines mainly TNF-α stimulates the caspase pathway. The obtained result indicated that SA-BA activated caspases (8 and 3) in KG-1A and K562 cells (Fig. 15). Involvement of caspase in triggering apoptosis was demonstrated as an apical initiator in receptor and stress-induced apoptosis [52]. Binding of TNF-α with TNF receptor 1, induce apoptosis through activation of caspase 8 [17, 52]. Our findings are in accordance with the effect of SA-BA on KG-1A and K562 cells with increasing TNF-α expression and their induction of TNF-α mediated cell death through apoptosis (Scheme 1).

5. Conclusion In this study, we found that self assembled form of betulinic acid showed more potent antileukemic activity towards KG-1A and K562 cell lines. On the other hand it did not exert any toxic effects on PBL. Selective cytotoxicity of SA-BA on both leukemic cells occurred by the alteration of cellular redox balance, disruption of mitochondrial outer membrane potential and 43

thereby induction of apoptosis phenomenon. Induction of apoptosis in both leukemic cell lines through activation of upstream caspases (Caspase 3 and 8) was observed. It was also revealed from the study that involvement of ROS followed by TNF-α is the major contributors of SA-BA induced anti leukemic activity through apoptosis mechanism. Hence, this study elicited the probable mechanism of action of SA-BA towards KG-1A and K562 cells, thus this molecule with its self assembled configuration can be used as a lead molecule for generation of drugs in leukemia treatment and management.

Scheme 1: Self-assembled betulinic acid mediated selective leukemic cell death through activation of ROS/TNF-α pathway.

Abbreviations

44

BA:

Betulinic acid

DMSO:

Dimethyl sulfoxide

DTNB:

5,5’-Dithiobis-(2-Nitrobenzoic Acid)

GSH:

Reduced Glutathione

GSSG:

Glutathione disulfide

H2DCFDA:

2’,7’-Dichlorodihydrofluorescein diacetate

HEPES:

N-(2-hydroxyethyl)-piperazine-N- (2-ethanesulfonic acid)

MDA:

Malondialdehyde

MMP:

Mitochondrial membrane potential

NAC:

N-acetyl-l-cysteine

PI:

Propidium iodide

Rh-B

Rhodamine B

Rh-123

Rhodamine 123

ROS:

Reactive oxygen species

SA-BA:

Self assembled betulinic acid

TNF-α:

Tumor necrosis factor alpha

Competing interests

The authors declare there are no conflicts of interest.

Acknowledgement

45

The authors express gratefulness to the Vidyasagar University, Midnapore and CRNN, University of Calcutta for providing the facilities to execute these studies. We are heartily thankful to Dr. Ashok Maiti, MD Pathologist, Medipath Diagnostic and Consultation Centre, West Midnapore, West Bengal for providing us healthy human volunteer’s blood samples.

References

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Figure legends

Figure 1: Dynamic light scattering (DLS) (A) and Zeta potential (B) measurement of SA-BA. Figure S1: Self assembly property and non self assembly study of BA in culture medium by SEM (B) imaging. One milligram of BA was dissolved in either 1 ml of ethanol-water mixture (16:4, 0.5%, w/v) or 1ml DMSO. A thin layer of the mixture was layered on the slide and subjected to SEM analysis. Here: A: BA dissolved in DMSO, B: dissolved in ethanol-water mixture. Figure 2: Self assembly property study of betulinic acid by OPM (A) and SEM (B) imaging. Two milligram BA was dissolved in 1 ml of ethanol-water mixture (16:4). A thin layer of the mixture was layered on the slide and subjected to SEM analysis. Figure 3: In vitro cell viability assay of DOX (A), BA (B) and SA-BA (C) treated PBL, KG-1A and K562 cell lines. Cells were treated with DOX, BA and SA-BA for 24 h at 37 °C. Cell viability was measured by the MTT method as described in materials and methods. D: In vitro killing kinetic assay of SA-BA on KG-1A and K562 cells.Values are expressed as mean ± SEM of three experiments; superscripts indicate significant differences (p < 0.05) compared with the control group. Figure S2: Estimation of IC50 value of DOX (A), BA (B) and SA-BA (C) on PBL, KG-1A and K562 cells. The dose response graph was prepared and IC50 values of those drugs were calculated using Statistica software.

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Figure 4: Estimation of haemolytic activity of SA-BA. Human RBCs were treated with varying concentration of SA-BA (1-100 µg/ml). The absorbance of the supernatants at 570 nm was determined by using a microplate reader with the absorbance at 655 nm as a reference. The result was expressed as percentage of hemolysis as compared to control groups. Figure 5A-C: Fluorescence microscopic images of Rh-B tagged SA-BA. Here, A: Gray scale image of Rh-B tagged SA-BA, B: Mixed light source image of Rh-B tagged SA-BA and C: Fluorescence image of Rh-B tagged SA-BA. Images were taken at 100X magnification. d-i: Intracellular uptake of SA-BA on PBL, KG-1A and K562 cells by fluorescence imaging. A required amount of cells was treated with Rhodamin B labeled SA-BA (25 µg ml-1) for 6 h. Intracellular uptake was examined using fluorescence microscope at 100X magnification. Here, d: KG-1A control, e: SA-BA treated KG-1A cells, f: K562 control, g: SA-BA treated K562 cells, h: PBL control, i: SA-BA treated PBL. Figure 6: Intracellular reduced glutathione (GSH) levels of DOX treated and SA-BAtreated PBL, KG-1A and K562 cell lines. The levels of GSH were expressed as μg of GSH/mg protein. Values are expressed as mean ± SEM of three experiments; superscripts indicate significant difference (p < 0.05) compared with the control group. Figure 7: Intracellular oxidized glutathione (GSSG) levels of DOX treated and SA-BAtreated PBL, KG-1A and K562 cell lines. The levels of GSSG were expressed in term of μg of GSSG/mg protein. Values are expressed as mean ± SEM of three experiments;

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superscripts indicate significant differences (p < 0.05) compared with the control group. Figure 8: Effects of DOX and SA-BA on ROS induction in PBL, KG-1A and K562 cell lines. DCF fluorescence intensity was expressed in term of ROS production. Results represent the means of three separate experiments, and error bars represent the standard error of the mean. Values are expressed as mean ± SEM of three experiments; superscripts indicate significant difference (p < 0.05) compared with the control group. Intensity of control cells were set to 100. Data is represented as the percentage of the ROS level in the control group. Fig. (a-f). Qualitative characterization of reactive oxygen species formation by DCFH2-DA staining using fluorescence microscopy. After the said treatment schedule leukemic were incubated with DCFH2-DA. At the end of DCFH2-DA exposure, cells were washed with PBS and they were visualized by fluorescence microscopy at an excitation wavelength of 485 nm and an emission wavelength of 520 nm (scale bar = 10 µm). Here, a: PBL control, b: SA-BA treated PBL, c: KG-1A control, d: SA-BA treated KG-1A cells, e: K562 control and f: SA-BA treated K562. Figure 9: Measurement of mitochondrial membrane potential (MMP) of DOX and SA-BA treated PBL, KG-1A and K562 cell lines. The normal and leukemic cells were treated with 25 µg/ml DOX and SA-BA for 24 hrs and after a treatment schedule MMP was determined by measuring Rhodamin 123 fluorescence intensity using fluorescence spectrophotometer as described in materials and methods section. Values are

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expressed as mean ± SEM of three experiments; superscripts indicate significant difference (p < 0.05) compared with the control group. Figure 10: Determination of genotoxic effects of SA-BA (25 µg/ml) treated KG-1A and K562 cell lines by Comet assay (A and a- d). A: Estimation of percentage of tail DNA intensity of SA-BA treated KG-1A and K562 cells. Values are expressed as mean ± SEM of three experiments; superscripts indicate significant differences (p < 0.05) compared with the control group. Here: a: KG-1A control, b: SA-BA treated KG-1A cells, c: K562 control and d: SA-BA treated K562. Figure 11: A: Cellular apoptosis measurements by flow cytometric analysis. Here, a: KG-1A control, b: SA-BA treated KG-1A cells, c: K562 control and d: SA-BA treated K562, e: PBL control, f: SA-BA treated PBL. B: Graphical representation of percentage of cells in viable (LL), early apoptosis (LR), late apoptosis (UR) and necrosis or dead (UL) phases. Figure 12: Qualitative characterization nuclear morphology by DAPI staining using fluorescence microscopy. After the treatment schedule PBL and leukemic cells were incubated with DAPI. At the end of DAPI exposure, cells were washed with PBS and they were visualized by fluorescence microscopy at excitation 330–380 nm and emission 430–460 nm (scale bar = 10 µm). Here, A: PBL control, B: SA-BA treated PBL, C: KG-1A control, D: SA-BA treated KG-1A cells, E: K562 control and F: SABA treated K562.

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Figure 13: Measurement of TNF-α (A), IL-12p70 (B), TGF-β (C) and IL-10 (D) cytokines level in serum free culture supernatant. KG-1A and K562 cells were treated with LPS and SA-BA at 1 µg/ml and 25 µg/ml doses respectively for 24 hrs. Cytokines release levels were measured by ELISA. Values are expressed as mean ± SEM of three experiments; superscripts indicate significant differences (p < 0.05) compared with the control group. Figure 14: A. Quenching of ROS rescues KG-1A and K562 cells from SA-BA induced cytotoxicity. KG-1A and K562 cells were pre-treated with 2 and 5mM NAC for 4-6 hrs and then subsequently exposed to SA-BA 25 µg/ml dose. Cell viability was estimated by MTT assay. B. Inhibition of TNF-α production protects the KG-1A and K562 cells from SA-BA induced cytotoxicity. KG-1A and K562 cells were pre-treated with 1mM Pentoxifylline (POF), a potent TNF- α inhibitor for 24 hrs and then subsequently exposed to SA-BA 25 µg/ml dose. Cell viability was estimated by MTT assay. Figure 15: Detection of pro-apoptotic protein expression by immunoflourescence staining. KG1A and K562 cells were treated with SA-BA (25 µg/ml) for 24 hr. After the treatment schedule cells were incubated with anti caspase 3 and 8 antibodies and subsequently exposed to rhodamine-conjugated compatible secondary antibodies for 1 h at 25 0C. Cells were visualized by fluorescence microscopy. Here: A&E: KG-1A control, B&F: SA-BA treated KG-1A cells, C&G: K562 control and D&H: SA-BA treated K562.

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Graphical abstract

57

Highlights



SA-BA significantly decreased the viability of leukemic cells.



SA-BA did not produce any toxic effects on normal cells.



ROS and TNF-α played main role in the etiology of leukemic cell death.



Leukemic cell death occurs due to induction of caspase mediated apoptosis pathway.

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