|Year : 2016 | Volume
| Issue : 1 | Page : 14-19
Anti-leukemic activity of betulinic acid from bulk to self-assembled structure
Sandeep Kumar Dash1, Sourav Chattopadhyay1, Parimal Karmakar2, Somenath Roy1
1 Department of Human Physiology with Community Health, Immunology and Microbiology Laboratory, Vidyasagar University, Midnapore, India
2 Department of Life Science and Biotechnology, Jadavpur University, Kolkata, West Bengal, India
|Date of Submission||22-Apr-2016|
|Date of Acceptance||04-May-2016|
|Date of Web Publication||2-Jun-2016|
Department of Human Physiology with Community Health, Immunology and Microbiology Laboratory, Vidyasagar University, Midnapore - 721 102, West Bengal
Source of Support: None, Conflict of Interest: None
The Ziziphus jujuba tree is one of the major sources of betulinic acid (BA). After isolation, the bulk structure of the compound was converted to a self-assembled nanofibers (SA-BA) configuration which showed better anti-leukemic efficacy than its bulk form. After internalization in leukemic cells, SA-BA elevated reactive oxygen species (ROS) and pro-inflammatory cytokine secretion which ultimately activated apoptosis pathway. The SA-BA showed potent ameliorative role against acute chemotherapeutic toxicity induced by doxorubicin in human peripheral blood lymphocytes through the mechanism totally opposite to said pathway. Thus, SA-BA showed cell specific distinct effects. It was also revealed that the SA-BA had potent immunomodulatory affected on T cells and macrophages by polarizing the cytokine balance toward Th1 at a slightly higher dose. SA-BA arrested the growth of in vivo cancer by increasing the CD4 + cells in associated with increased cytotoxic T-cell response. SA-BA was also selectively internalized in folate receptor overexpressing leukemic cells. For this purpose, folic acid (FA) and polyethylene glycol (PEG) were conjugated on the nanostructured of SA-BA. After internalization, the conjugate (FA-PEG-SA-BA) diminished the cellular redox system and generated an excess amount of ROS which induced tumor necrosis factor-alpha-mediated cell death through activation of caspase 8 and 3 cascade system. Throughout all these studies, no toxic effects of the conjugates toward normal cells were observed. Thus, the whole study enlightens the multifunctional role of SA-BA in different aspects of anti-leukemic therapy which may be useful in future treatment policies.
Keywords: Anti-leukemic, betulinic acid, drug delivery, folate receptor, immunomodulation, self-assembled
|How to cite this article:|
Dash SK, Chattopadhyay S, Karmakar P, Roy S. Anti-leukemic activity of betulinic acid from bulk to self-assembled structure. BLDE Univ J Health Sci 2016;1:14-9
|How to cite this URL:|
Dash SK, Chattopadhyay S, Karmakar P, Roy S. Anti-leukemic activity of betulinic acid from bulk to self-assembled structure. BLDE Univ J Health Sci [serial online] 2016 [cited 2019 May 21];1:14-9. Available from: http://www.bldeujournalhs.in/text.asp?2016/1/1/14/183269
The use of plant in disease control and prevention is believed to date back to prehistoric medicine. Bioactive phytochemicals are used in treatment of cancer from the "Vedic" period. Nowadays bio-active ingredients from a natural source and naturally occurring process have become a wide area of anticancer based research. Secondary metabolites of various plants have been used to combat human diseases for several years, as they exhibit a wide range of biological properties that can be exploited for medical application.  Among various bio-active ingredients, triterpenoids received significant attention in cancer research owing to their cancer cell specific activity as well as low or considerable cytotoxicity toward healthy cells. The terpene group consists of more than 30,000 known members with more than 4000 compounds has been classified as cyclic triterpenoid-like substances,  among them the free triterpenoids, saponins (triterpenoid glycosides), and phytosterols are presents. Among the various secondary metabolites of plants, pentacyclic triterpenoids receive significant attention in the field of selective cancer therapy. Pentacyclic triterpenoids are widely distributed in plant kingdom; possess strong protective effects against drug-induced toxicities especially by chemotherapeutics. They can interact with multiple biological molecules to exert potent bio-efficacy side-by-side do not act as conservative cytotoxic agents or monotargated drugs that inimitably triggered the single pathway.  Easy availability, less toxicity, and routine consumption across a large number of populations produce their attention toward their safety. One of the most well-studied lupane-type triterpenes is betulinic acid (BA), widely distributed throughout the plant kingdom. The most structurally related precursor of BA is betulin which also widely distributed among plants but comparatively less available than BA.  Betulin, the reduced form of BA was first isolated from plants in 1788 by Johann Tobias Lowitz and found to be a prominent constituent of the outer bark of white-barked birch trees. Birch tree (Betula spp., Betulaceae), is one of the most widely reported sources of BA. ,,] Other substantial plant sources of BA include Ziziphus spp. (Rhamnaceae), Syzygium spp. (Myrtaceae), Diospyros spp. (Ebenaceae), and Paeonia spp. (Paeoniaceae).  Pisha et al. first reported the anticancer activity of this compound toward human melanoma.  Gradual developing studies showed that this compound is not only melanoma-specific but also hold the ability to kill many other cancer cell types holds the significant anti-human immunodeficiency virus type-1, antibacterial, antimalarial, anti-inflammatory, and anthelmintic activities. 
| Molecular Mechanism of Betulinic Acid in Cancer Therapy|| |
In 1995, BA was first reported as a highly selective antiproliferative agent against human melanoma, neuroectodermal, and malignant tumor cells and the mode of action underlying the mechanism of cell death was reported to induce apoptosis in these cells.  Till now thousands of studies has been carried out to test the ability of BA as selective anticancer agent. Most interesting fact is that it did not affect normal cell status, and its lack of cytotoxic activity has been demonstrated in human astrocytes, human dermal fibroblasts, peripheral blood lymphoblasts, and animal studies. The antitumor effects and the cytotoxicity of BA and its different derivatives have been widely examined using a large variety of malignant tumor cell lines as well as applying xenograft mouse models and primary tumor samples.  Recently, BA was found to be significantly effective against human acute and chronic myeloid leukemic cells. ,
Contradictory results are reported in some studies regarding the alteration of p53 status in BA-treated malignant cells. Some studies reported that the apoptosis induced by BA was independent of the cellular p53 status. ,, On the other hand, another study reported the up-regulation p53 status in metastatic melanoma cells.  Treatment with BA in melanoma cells in vitro resulted in the appearance of typical characteristics of apoptosis including surface blebbing and cytoplasmic shrinking.  Mitochondria has been recognized as major target of BA, induces apoptosis through the induction of changes in mitochondrial transmembrane potential, production of excessive reactive oxygen species (ROS), and permeability transition pore (PT pore) openings.  In isolated mitochondria, i.e., in cell-free systems, BA-induced loss of mitochondrial transmembrane potential in a manner that was not affected by the caspase inhibitor Z-VAD-FMK. , This effect was also noted in intact cells.  This phenomenon helps to release of mitochondrial apogenic factors which activate caspases, and DNA fragmentation and ultimately induces apoptosis. ,,
BA treatment was found to induce cleavage of both caspase-8 and caspase-3 in cytosolic extracts. Cytochrome c, released was also manifest with BA-mediated PT, activated caspase-3 but not caspase-8 in a cell-free system. The activation of caspase system and disturbance of mitochondrial membrane potential was largely depends on the generation of ROS.  BA also found to suppress STAT3 (Sigma, USA) activation along with inhibition of nuclear factor-kappa B (NF-κB) activity. Constitutive STAT3 activation has been found in various types of carcinoma, sarcoma, lymphoma, and leukemia.  The previous study showed that erythropoietin activates NF-κB through JAK2 kinase pathway.  Thus, it assumed that the suppression of JAK activation was highly correlated with NF-κB and STAT3 activation by BA treatment. 
The synergistic effects of BA with other anticancer agents were found to be highly effective for reduction of tumor cells compare to the independent application. The combined effects drastically induced loss of mitochondrial membrane potential and the release of cytochrome c and Smac from mitochondria which resulted activation of caspases and apoptosis.  The same type of result was noted for human cervical adenocarcinoma cell (HeLa), human lung cancer A549, and human hepatoma HepG2 cells when BA was treated in combination with Ginsenoside Rh2, isolated from the root of Panax ginseng. In this report, additive effects of BA with Ginsenoside Rh2 drastically up-regulated the apoptosis phenomenon on the said cancer cell types.  Another combinational study using α-mangostin enhanced BA's cytotoxicity against HCT 116 human colorectal carcinoma cells showed synergistic elevation of BA's cytotoxicity. The BA and α-mangostin targets different signaling pathways of apoptosis and the compounds could act synergistically when used in combination.  Differential molecular targets of the two natural compounds may provide helpful advantages including decreasing the likelihood of developing drug resistance and the anti-angiogenic effect of BA. , One of the very recent studies showed that the combined treatment of 5-fluorouracil (5-FU) and BA on ovarian carcinoma cells was found to increase the loss of mitochondrial membrane potential and Sub-G1 cell population resulted significant growth rate inhibition. The proteomic analysis showed a high concentration of cytochrome c in the cell cytosol after 24 h of 5-FU and BA combination treatment. 
| Self-assembly: An Overview|| |
The process of "self-assembly" is designated as the impulsive organization of individual components into a well-organized structure without external directions.  The main characteristics of the self-assembled (SA) structured components is largely depends on the mass concentration of the molecule, solvent system, incubation temperature, etc. The mechanism of molecular self-assembly is naturally included by hydrogen bonding, ionic interactions, water-mediated hydrogen bonds, hydrophobic, and van-der-Waals interactions.  The contribution of individual factors in self-assembly process is quite low, whereas their summative effects play strong contributory effects in this process. Nowadays molecular self-assembly based research has become one of the most important tools for fabrication of micro to nano ranged materials which enable them to have a strong platform in biomedical applications in the coming decades.  Numerous self-assembling systems have already been developed, ranging from block copolymers and surfactant-like materials to scaffolds for three-dimensional cell culture, DNA-based structures, and models to study protein folding and protein conformational disease. The first self-assembly property of BA was observed by Bag and Dash.  In this study, they found that BA showed self-assembly in 22 organic liquids and alcohol-water mixtures. Optical and electron microscopy and atomic force microscopy studies clearly demonstrated the fibrillar networks having fibers of nano- to micro-meter cross sections and micrometer lengths. The anti-leukemic efficacy study of the SA-BA as well as in drug targeting were reported for the first time by Dash et al. ,
| Application of Self-assembled Betulinic Acid in Anti-leukemic Therapy|| |
Anti-leukemic efficacy of self-assembled betulinic acid
Ziziphus jujuba tree is one of the richest sources of BA. The compound was isolated from heavy wood bark of that tree and purified by column chromatography. , The compound was then characterized by reverse-phase high-performance liquid chromatography analysis, Fourier transform infrared spectroscopy, X-ray powder diffraction and proton nuclear magnetic resonance spectral analysis which confirmed the purity of BA and revealed 6-6-6-6-5 pentacyclic triterpenic acid structure.  The toxicity of the compound against normal cells as well as anti-leukemic activity of BA was examined to test its usefulness in cancer therapy.  The outcome showed that BA drastically reduced the viability of human acute and chronic myeloid leukemia cells (KG-1A and K562 cells, respectively) in dose and dose-dependent fashion while showing no significant toxicity toward normal counterparts. After internalization into leukemic cells, it downregulated the cellular redox status by elevating ROS levels, which caused DNA damage and ultimately induced apoptosis, confirmed by Annexin V-FITC + propidium iodide dual staining using flow cytometry technique (FACS). Involvement of caspase-3 was also noted for BA-mediated apoptosis in both leukemic cells.  The anti-leukemic efficacy of BA was upgraded by converting its bulk structure into well-organized self-assembly configuration (SA-BA) using ethanol + water (16:4, 0.5% w/v) as solvent system.  SEM images showed the presence of several fibrous networks having single's fiber diameter of 15-25 nm with 1-3 micrometer length. Dynamic light scattering and zeta potential estimation confirmed that the fibers were in nanoscale with negatively charged surface. Higher efficacy of SA-BA over nonassemble BA was monitored toward leukemic cells with no relevant toxicity to normal blood cells. Here also the compound elevated ROS level and pro-inflammatory cytokines, primarily tumor necrosis factor-alpha (TNF-α) mediated activation of caspase-8 and caspage-3 was confirmed by immunocytochemistry. 
Protective role of self-assembled betulinic acid
Besides the potent antilukemic property, SA-BA served as an effective protective agent against acute chemotherapeutic toxicity in human peripheral blood lymphocytes (PBLs) induced by doxorubicin (DOX). DOX, is a well-known and widely used drug for treatment of different types of cancers including solid tumors (breast and ovarian cancer) as well as hematological malignancies (leukemia and lymphomas). ,, However, the clinical use of DOX is now restricted due to its severe side effects in blood and organs. Cardiotoxicity is the major obstacle of DOX treatment associated with severe cardiac attack and massive myocardial injury leading to left ventricular systolic dysfunction and congestive heart failure.  DOX therapy actually impact lymphocytes. Loss of lymphocyte viability leads to unmanageable condition during chemotherapy. , DOX treatment destroyed the antioxidant system in PBLs leading to significant cell death through apoptosis.  Pretreatment with SA-BA followed by DOX exposure for 24 h protected the PBLs from DOX-induced oxidative stress and associated apoptosis activity confirmed by FACS analysis and Western blot assay.  Excess amount of ROS in PBL by DOX treatment was the main cause of severe inflammation which also reduced by pretreatment with SA-BA [Figure 1]. Thus, this finding indicates that SA-BA can be applied to ameliorate the cytotoxic effects of DOX which can be a helpful strategy in overcoming complications during DOX based chemotherapy in cancer patients.
|Figure 1: Protective role of self-assembled-betulinic acid against doxorubicin induced toxicity|
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Immunomodulatory role of self-assembled betulinic acid
Conventional chemotherapeutic agents lead to lethal toxic effects in the body during and after the treatment of cancer. Multi-organ toxicity has been widely reported in the patients attending chemotherapy schedule.  To minimize the cytotoxic effects of chemotherapeutic drugs organo-protective adjuvants has been prescribed to the patients. However, till to date very limited types of organo-protective adjuvants have been discovered. Current advancement on immunomodulatory approaches is largely based on the use of pentacyclic triterpenoids as effective adjuvants in cancer treatment. ,, Immunomodulation is the safest way to treat malignant cells. The use of immunotherapy for the treatment of cancer promises to be helpful to overcome the resistance of tumors to the immune system.  Study showed that oral administration of BA (0.25-1 mg/kg BW) for 14 days significantly elevated the thymic (CD4 + ) and spleenic (CD19 + ) cell populations in dose-dependent fashion. The percentage elevation of the ratios of CD4(+)/CD8(+) in spleen were also confirmed by flow cytometric assay. It was also found that BA did not show any toxic effects up to 0.5 mg/kg BW dose in mice for 14 days treatment period.  Furthermore, it was observed that activation of TNF-α from mice peritoneal macrophages treated with BA. The association of TNF-α and interleukin-1 beta were found to modulate the immunomodulatory properties of BA.  Slight elevation of nitric oxide generation with downregulation of cyclooxygenase-2 denoted the potential role of TNF-mediated immune stimulation through activation of macrophages. Activated macrophages which serve as both antigen-presenting cells and effectors cell, represents a promising immunotherapeutic approach against cancer. , It was reported that SA-BA can be considered as an adjuvant to provoke immunostimulatory activity against cancer. The concept of immunostimulatory activity of SA-BA is based on the activation of immune system against cancer antigen.  It was found that SA-BA pulsed human macrophages significantly arrested the KG-1A and K562 cell growth in vitro setup at 1:10 ratio for 48 h. SA-BA significantly induced pro-inflammatory cytokines especially IFN-γ and TNF-α followed by activation of cell-mediated immunity based on CD4 + T cell response. SA-BA pulsed macrophages displayed substantial T cell allostimulatory capacity and promoted the generation of cytotoxic T lymphocytes. The adjuvanticity of SA-BA was proved by the increased CD4 + response and IgG response in vivo [Figure 2]. Collectively, these findings may enrich the biomedical applications of SA-BA as a potent immune stimulating agent which might be an alternative way in the cancer immunotherapy.
|Figure 2: Immunomodulatory activity of self-assembled-betulinic acid. Scheme showing a balanced Th1/Th2 response|
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Efficacy of self-assembled betulinic acid in receptor-mediated delivery
Folate receptor (FR) targeting is one of the best choices for drug delivery adopted by a vast number of researchers. Folic acid (FA, folate or Vitamin B9), is an essential vitamin requires by all living cells for nucleotide biosynthesis and for the proper metabolic maintenance of 1-carbon pathways.  Aside from its cofactor role for intracellular enzymes, FA also displays high affinity for the FR, a glycosylphosphatidylinositol-linked protein that captures its ligands from the extracellular milieu and transports them inside the cell via a nondestructive, recycling endosomal pathway. , There are four isoforms of this receptor family have been identified and are classified as FR-alpha (FRα), FR beta (FRβ), delta, and gamma, respectively. FRα, FRβ isoforms are both glycosylphosphatidylinositol-anchored proteins contain two N-glycosylation sites and comprise high affinity for FA/Vitamin B9. , It was found that expression of FRα is frequently augmented in various types of epithelial cancers, whereas FRβ expression is noted in myeloid leukemia and chronic inflammatory diseases.  The FR is also easily recognized by several tumor antigens and biomarkers. Because of this, diagnostic and therapeutic methods which exploit the FR's function are being developed for cancer. Certain cancer types found to overexpress FR which includes the cancers of ovary, lung, breast, kidney, brain, endometrium, colon, and hematopoietic cells of myelogenous origin.  Normal healthy cells have minimum expression of FR. It was found that FR is overexpressed approximately 500 times more in cancer cells than normal cells/tissue. The differential regulation of FR between cancer cells and normal cells provides the unique tool for folate-mediated drug delivery.  From a mechanistic perspective, the FR functions to concentrate exogenous ligands (e.g., folates and folate-drug conjugates) into the cell cytosol by endocytosis. The term endocytosis refers to the process whereby the plasma membrane invaginates and eventually forms a distinct intracellular compartment. The endocytic vesicles (endosomes) rapidly become acidified to allow the FR to release its ligand.  Afterward, the empty FR returns to the cell surface where is can participate in another round of ligand-mediated endocytosis. 
SA-BA was also found significantly effective in targeted drug delivery system. For this purpose, polyethylene glycol (PEG) stabilized SA-BA followed by conjugated with FA (FA-PEG-SA-BA) was prepared by simple physical reaction and were characterized by a series of physical measurements techniques.  Dynamic functional theory and its computation were adopted to analyze the conjugation chemistry of SA-BA with PEG and SA-BA-PEG with FA. It was found that H-bonding between the components played the substantial role in physical conjugation. The FA-PEG-SA-BA showed maximum stability at acidic pH which facilitated the internalization of the conjugate maximally through FR in FR overexpressing K562 cells and comparatively lower in FR lower expressing KG-1A cells. Here also the intracellular trafficking of FA-PEG-SA-BA triggered ROS-TNF-α-caspase mediated leukemic cell death. The conjugate showed good compatibility with normal cells. 
| Conclusion|| |
The present review largely focused on the anti-leukemic efficacy of BA and its mechanism of action. It was established that change of structure, i.e., from bulk to SA nano-sized fibers formation elevated the reactivity of the compounds towards myeloid leukemia cells. Along with the better anti-leukemic efficacy the SA-BA showed protective role against DOX-induced lymphocyte toxicity. It was observed that SA-BA restored the cellular antioxidant level to ameliorate the PBLs from DOX-induced toxicity. The compound was also capable of activating immune system which was very much essential for cancer therapy. A balanced Th1/Th2 response was noted in SA-BA treatment. In receptor-mediated drug delivery, FA-PEG-SA-BA internalized maximally in FR overexpressing leukemic cells. In every cases of SA-BA treatment, the leukemic cell death was manifested by disruption of cellular antioxidant level which ultimately activated caspase-mediated cell death.
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[Figure 1], [Figure 2]