Metabolomic profiling of cancer cells to Aloe vera extract by 1HNMR spectroscopy

Background: The modern life style despite its comforts has made cancer a leading public health problem. As chemotherapeutics has many side effects including resistance, recent attention has focused on plants which may provide a good opportunity for complementary cancer treatment. Aloe vera has been described as a wonder plant due to its many advantages. It has been reported to have anticancer properties. Metabolomics is the measurement and analysis of metabolites in a biological sample by the use of throughput technology like 1HNuclear Magnetic Resonance and Mass Spectrometry. The results are analyzed by multivariate analysis methods. The metabolome reveals the final interaction between the genome of an organism and its environment. We studied the inhibitory effects and metabolomic profile of Aloe Vera extract on Raji cells (cancerous lymphoma cells) using 1HNMR spectroscopy. Methods: The gel was removed from Aloe vera leaves by a sharp knife and chopped by a blender, warmed to 50°C with ascorbic acid as antioxidant and finally cooled to almost 20°C. After filtering, the gel was lyophilized and dissolved in RPMI medium (2mg/ml) and added to Raji cells and 50% inhibitory concentration determined by MTT. Large scale culture of Raji cells were carried out using 50% inhibitory concentration dose of Aloe vera gel. The Raji cells were collected, metabolites extracted using water or chloroform/methanol, lyophilized and sent for 1HNuclear magnetic resonance analysis by NOESY technique. Results: 50% inhibitory concentration of Aloe vera was seen to be 40 μg/ml. The differentiating metabolites were identified and the metabolic cycles were detected. Conclusions: Changes were observed mainly in the amino acid metabolites and the main metabolic cycles involved were protein biosynthesis, mitochondria transport chain, catecholamine biosynthesis and pentose phosphate pathway.


Introduction
Aloe Vera is a plant that belongs to the Aasphodelacea (Liliaceae) family has been used as an herbal medicine for centuries. It is native to Africa and many Mediterranean countries and is used for several healing purposes, like treating various skin disorders such as abcesses, burns and eczema. Its juice may help people with ulcerative colitis, an inflammatory bowel disease. Aloe Vera has been used as a remedy for coughs, wounds, diabetes, gastritis, headaches, arthritis, ulcers, cancer and also immunesystem deficiencies [1]. There are over 100 active biological components found in Aloe Vera, including: anthraquinones, vitamins, minerals, enzymes, amino acids, polysaccharides and plant sterols [2].
Cancer after cardiovascular disease is the second leading cause of death in the world. Approximately 10 million people are diagnosed with it every year and 6 million die of it [3]. Treatment options for cancer include chemotherapy, surgery, radiation and immunotherapy. The use of any of these treatments depends upon the location, grade, tumor stage as well as the general state of a person's health. Chemotherapy and CrossMark ← Click for updates doi: 10.7243/2059-0008-2-1 radiation in addition to cancer cells, destroy normal cells [4] and recently resistance to chemotherapeutic drugs has been reported in some cases and this has renewed an interest in alternative and herbal methods to complement conventional treatment. In fact, medicinal plants are more available, cheaper and possess less toxicity compared to modern(allopathic) drugs. Biological active components of plants can be important sources for new drugs which may lead to new and better treatments for cancer [3].
In this study, Raji cells were used as a model system to study the anti-cancer properties of Aloe vera. Raji cells are a series of cell lines derived from B lymphocyte of a patient with Burkitt's lymphoma (BL) which is an aggressive form of B cell non Hodgkin's lymphoma [5] with reports of chemotherapy resistance.
Metabolomics comprehensively analyzes hundreds of metabolites in a biological sample simultaneously using high throughput technology. Mass spectrometry (MS) and 1 HNuclear Magnetic Resonance spectroscopy ( 1 HNMR) are used in the measurement of metabolites in the sample with chemometrics analysis. Although metabolomics comprises the smallest domain (approximately 5, 000 metabolites) as compared to genomics (30,000 genes) and proteomics (100,000 or more proteins), it is more diverse and involves many different biological molecules, making it chemically and physically more complex than the other two. The importance of metabolomics is in disease diagnosis and drug discovery as the changes in the metabolome are a direct outcome from perturbations in cellular activity [6].
The purpose of this research is to study the effect of Aloe vera gels on Raji cells and to obtain the difference in their metabolic pattern by 1 HNMR spectroscopy so as to identify metabolites and modified metabolic cycles. Earlier work has been done on the effect of ginger on the metabolic pattern of Raji cells and the pathways detected were mainly glucose cycles [6,7].

Preparation of Aloe vera extract
Aloe vera leaves were prepared fresh from herbarium and identified by Tehran University. They were first washed with a solution of sodium hypochloride and rinsed with distilled water at room temperature for 4h. A sharp knife was used for separation of the leaf crusts and the gel collected by a spatula-like knife gel from the lower crust. The gels were chopped by a blender and warmed to 50°C using ascorbic acid as antioxidant and then cooled to 20°C. The Aloe Vera extract was filtered through gauze and the filtrate lyophilized [8].

Cell culture
The Raji cell lines (B-cell lymphoma) were purchased from Pasteur Institute of Iran. The cells were obtained from NCBI code C127 with ATCC number CCL-86, but are DNA typed and karyotyped periodically by the Pasteur Institute of Iran. The culture medium contained 10% fetal calf serum and RPMI-1640, penicillin and streptomycin and incubated at 37°C with 5% CO2 at 95% humidity in a 25 cm 2 flask [9].

50% inhibitory concentration (IC50)
1 mL of culture medium containing 1.6X10 4 Raji cells were added to each well and treated with different concentrations of 0.2, 2, 20, 40, 100 mg/ml Aloe Vera extract with duplicates using controls without extract. The cells were treated for 24, 48, 72 hours.

Viability test
Trypan blue was used to stain dead cells and their viability assessed by counting under an invert microscope using a hemocytometer at 24, 48 and 72 h [10].

MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay: was performed at 24, 48 and 72 h in 96-well plates with the same concentrations of Aloe Vera as viability test. At the end of the time periods, the culture medium supernatant was discarded and the cells were incubated with 50 μg/mL MTT stock solution in PBS for 3 to 4 hours at 37°C. Then, 100 μL formazan was added in dimethyl sulfoxide to the wells and shaken for 30 min and absorbance read at 570 nm using ELISA Reader (Anthos 2020) [11].

Culture of Raji cells for 1 HNMR
After determining IC50 concentration, Raji cells were cultured with IC50 titer of Aloe Vera extract in 75 cm 2 flask for 48 h and then collected by centrifugation at 6,000Xg for 5min and stored in freezing solution containing 90% Fetal Bovine Serum and 10% DMSO at -70˚C.

Cell extraction
The method of extraction using methanol-chloroform-water was done as described previously. The temperature of the extraction procedure was maintained at 4°C by working in a crushed ice bath. Cells were washed in 1X PBS and centrifuged at 6,000g for 5 min and resuspended in 500 μL of ice-cold 2:1 (v/v) methanol-chloroform solution. It was then transferred into a 1.5mL Eppendorf tube, 250 μL of ice-cold H2O 1:1 (v/v) chloroform/H20 was added and vortexed. The cells were sonicated on ice for 10min and centrifuged for 5min at 18000Xg.
The lower lipophilic and the upper hydrophilic extracts were separated and collected in different Eppendorf tubes and lyophilized and stored at −20°C [7].

HNMR spectroscopy
The cell suspensions were placed in 5mm probes for analysis and one dimensional spectroscopy was performed on a 1 HNMR spectrometer (Bruker AV-500) with filed gradient operating at 500.13 MHZ for observation of proton at 298K. One dimensional 1 HNMR spectra were acquired with 6009.6 Hz spectral width, a 10-μs pulse 0.1 s mixing time, 3000 transients and 3.0 s relaxation delay, with standard 1D NOESY (nuclear Overhauser spectroscopy) pulse sequence to suppress the residual water peak [13]. The 1 HNMR spectrum comprising of Fourier Transformed information about metabolites in the control and treated groups(both hydrophilic and lipophilic extracts of each) were imported into MATLAB (v.7.8.0.347) software and first analyzed by ProMetab software (version 1.1) Chemical shifts between 0 and 10 ppm were normalized and spectra binned in 0.004units and the water peak (4.7) removed. The Excel files were then assessed by PLS-Toolbox version 3.0 and Partial Least Square Discriminant Analysis (PLS-DA) was applied [14].

Identification of metabolites
The differentiating metabolites related to these resonances were identified by chemical shift determination using Human Metabolome Database Data Bank (HMDB) (http://www.hmdb. ca/metabolites). The metabolic cycles were determined using Metabo-Analyst software (http://www.metaboanalyst.ca/).

Results
IC50 of Aloe Vera extracton Raji cells is shown to be 40μg/ml at 48 h (Figure 1). Superimposed 1 HNMR spectra of hydrophilic layer and lipophilic layer between experimental and control groups can be seen in (Figures 2A and 2B). The analysis of 1 HNMR spectra are depicted as score plot (Figures 3A and 3B) and loading plot (Figures 4A and 4B) of hydrophilic layer and lipophilic layer between experimental and control groups. Altered metabolites of hydrophilic phase and lipophilic phase are identified in Tables 1 and 2 respectively. Metabolite enrichment analysis is shown in Figure 5 and 6 depicting the main pathways affected by the extract.

Discussion
Aloe vera has many beneficial properties. Anti-cancer properties of Aloe vera have been reported from 2010 when its anti-proliferation effects on some types of cancer cells in vitro such as lung, squamous, glioma, and neuro-ectodermal were described [15,16]. It also has anti tumorigenic properties and increases expression of p53 gene and Bcl2 as demonstrated on human hepatocellular carcinoma cells [16,17]. Ehrlich ascites carcinoma in Swiss albino mice were apparently healed by Aloe vera extract by reversion of their hematological profiles towards normal levels and a decrease of peroxidation enzymes of lipid peroxidation and increased the levels of reduced glutathione and other antioxidant enzymes, like superoxide dismutase, catalase and glutathione peroxidase [16]. The anti-  cancer activity of Aloe vera on Raji cells was exhibited by IC50 of 40µg/ml in vitro confirming the above results.
Metabolomic studies done in this survey also revealed the mechanism of activity of Aloe vera extract on Raji cells. Levels of some amino acids had altered between the two groups (normal and treated) and the metabolic cycles in which they participated were identified by the Metaboanalyst website. These amino acids were involved primarily in protein biosynthesis, a cycle which was affected in both the hydrophilic and hydrophobic extracts. A number of glycoproteins present in Aloe vera gel have been described as antitumor and anti-ulceric and seen to increase proliferation of normal human dermal cells [18]. A substance named Aloin, an anthroquine and the main ingredient of Aloe vera has been shown to possess anticancer potential activities, as it blocks signal transducers and is an activator of transcription 3 activation by inhibiting     tumor angiogenesis and growth demonstrating its potential as a drug candidate for cancer therapy [17]. Investigations carried out in mice fed with fresh leaf pulp extract have shown Aloe vera to be an inducer of phase-II enzyme system which help in detoxification and are associated with carcinogen metabolism. The microsomal and cytosolic proteins were increased in the Aloe vera treated mice indicating the possibility of its involvement in the initiation of protein synthesis [19]. Aloin treatment is shown to inhibit the secretion of vascular endothelial growth factor (VEGF), thus inhibiting proliferation and migration of endothelial cells. VEGF is one of the most important proangiogenic cytokines known and well distinguished as a provoker of tumor neovascularization. VGEF production is shown to be regulated by histamine which is produced from histidine via the enzyme L-histidine decarboxylase [20,21]. Histidine metabolism is one of the cycles detected by our analysis.
L-tyrosine is involved in the catecholamine cycle, another metabolic cycle seen to differ with Aloe vera's action on Raji cells. There have been reports since 1991 that catecholamines and their metabolites are present in single lymphocytes and extracts of T-and B-cell clones as detected by capillary electrophoresis and electrochemical detection. Pharmacological inhibition of tyrosine hydroxylase diminishes perceived catecholamine levels, suggesting catecholamine synthesis by lymphocytes [22]. L-tyrosine is the substrate for tyrosine hydroxylase and is detected in this study.
Succinic acid participates in the mitochondria electron transport chain system. The protective effect of Aloe vera gel on the permeability transition pore in the inner membrane of rat liver mitochondria in vitro has been described. These findings indicate that Aloe vera gel modulates the effect of calcium by moderating the mitochondrial pore opening which  is needed in situations that requires tissue wastage such as in cancer treatment [23]. Degradation of leucine, isoleucine and valine identified in this study has been reported earlier as an important step in cancer metabolism. An investigation carried out on breast cancer cells using microarray has shown this to be the second most important metabolic cycle involved [14,24]. These amino acids are also present in the Aloe vera gel and are likely to be utilized by Raji cells [25].
Gutamate is an antioxidant and is also a source of amino groups for nonessential amino acids like glycine, aspartate, alanine and serine which are required for macromolecular synthesis [26]. The tricarboxylic acid cycle (TCA), the main source of α-ketoglutarate in glutamine consuming cells is glutamate which is also an intermediate for TCA cycle and dioxygenases substrate which can cause protein and DNA modifications. These dioxygenases enzymes include histone demethylases, 5-methylcytosine hydroxylases and prolyl hydroxylases whose requirement for α-ketoglutarate maybe just a fraction of total α-ketoglutarate consumption which makes this metabolite an essential component of cell signaling and epigenetic networks [27]. Aloe vera affects glutamate metabolism by disturbing these crucial networks.
Phenylalanine and tyrosine metabolism was one of the cycles involved and there are reports of its importance in oesphaegal cancer in a serum metabolic study ( [28]). Infact, limitation of pheylalanine and tyrosine amino acids is seen to inhibit prostrate cancer [29].
The pentose phosphate pathway(PPP) is also seen in this study. Energy metabolism is readjusted in many human cancers that bestows many advantages to cancer cells, which include ATP generation, promotion of biosynthesis, detoxification and support of rapid proliferation. Glucose catabolism is carried out by the PPP. It directs the flux of glucose to its oxidative branch and produces a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), an essential reductant in anabolic processes. It is now obvious that PPP has a very important role in regulation of growth of cancer cells by supplying cells with ribose-5-phosphate and also the NADPH required, reductive biosynthesis, detoxification of intracellular reactive oxygen species and ribose biogenesis. It can be concluded that any disturbance in the PPP directly contributes to proliferation of cancer cells, their endurance and weakness. Recent work has proved that regulation oncogenically/or metabolically of PPP is by numerous factors, including proteins such as oncoproteins, tumor suppressors and intracellular metabolites. Cancer cell growth and its survival is due to disturbance of PPP flux. PPP is one of the significant cycles identified by Aloe vera [30].
Earlier work implemented on the metabolic effect of ginger on Raji cells with an IC50 of 1mg/ml. It is interesting that the only common metabolic pathway identified by Aloe vera and ginger is protein biosynthesis which is directly involved in cell proliferation. Ginger seemed to act primarily on the doi: 10.7243/2059-0008-2-1 different glucose cycles, whereas Aloe vera acts mainly on amino acid cycles and the mitochondria. It seems that the two medicinal herbs act on different metabolic pathways to inhibit Raji cell growth [7].

Conclusion
Aloe vera exhibited inhibitory effect on Raji cells with an IC50 of 40 µg/ml. It affects primarily the protein biosynthetic pathway, different amino acid pathways, catecholamine synthesis, mitochondrial transport chain and pentose phosphate pathway exhibiting its unique mechanism of action.