Methanolic extract of Moringa oleifera leaves induces Cell cycle arrest and downregulates Mitochondrial membrane potential in Dalton's Lymphoma cells.


 Cancer is a group of diseases characterised by abnormal and undifferentiated cell growth that has the potential to spread to other parts of the body. It is the world's second leading cause of death and morbidity. According to the GLOBOCAN 2020 report, out of 19.3 million new cancer cases and 10 million deaths reported, 544352 new cases and 259793 deaths occurred by non-Hodgkin lymphoma (NHLs). Although, numerous therapeutic approaches like, surgery, radiotherapy, chemotherapy and immunotherapy have been developed to treat cancer, limited success has been achieved, possibly due to severe side effects associated with the drugs used during chemotherapy. Therefore, deciphering the novel compound with least side effects and highly potent against cancer is urgently required. In the present study we used leaf extract of M. oleifera, well-known for its anti-cancer efficacy against different cancer cells, however, its effect on Dalton’s lymphoma, a type of spontaneously occurring T cell lymphoma originated in the thymus of DBA mice is seriously lacking. Therefore, present study was aimed to analyze the therapeutic efficacy of M. oleifera against DL cells. Our results show that leaf extract of M. oleifera (MOML) significantly induces morphological changes in DL cells followed by chromatin condensation, nuclear fragmentation, and ROS generation. We also found significant changes in mitochondrial membrane potential (ΔΨm) in a dose dependent manner. Furthermore, apoptosis of DL cells induced by cell cycle arrest at G2/M and S phase suggested that MOML could be used to treat NHL effectively


Introduction
Cancer is a group of disease occur in multi-cellular organism, characterized by an uncontrolled proliferation and irregular growth of abnormal cells. It is the second leading cause of mortality and morbidity worldwide [1,2]. According to the GLOBOCAN 2020 report, about 19.3 million new cancer cases and 10 million deaths were reported in 2020.
Report further suggested that out of the 19.3 million new cancer cases 10 million deaths in 2020, 83087 new cases and 23376 deaths by Hodgkin Lymhoma and 544352 new cases and 259793 deaths by non-Hodgkin lymphoma (NHLs) occurred worldwide. Non-Hodgkin's lymphoma is a group of heterogeneous malignant cells of the lymphatic system, characterized by an uncontrolled growth and proliferation of T cells, B cells or both [3].
Dalton's lymphoma (DL) is one of the most common NHL reported in the mice. DL is a spontaneously occurring transplantable T-cell lymphoma arises in the thymus of murine mice, and mimics human T cell lymphoma [4]. Although, numerous therapeutic approaches like, surgery, radiotherapy, chemotherapy and immunotherapy have been developed, limited success achieved in the treatment of cancers. The drugs used during chemotherapy, a most frequently and widely used approach to cure cancer, causes mild to severe side effects in the patients. Therefore, deciphering the novel compound with least side effects and highly potent against cancer is urgently required.
Moringa oleifera is the most prevalent cultivated angiospermic deciduous perennial tree of the family Moringaceae. It is widely distributed in the tropics and subtropics of Asia including India, Philippines, Cambodia, Central America, North and South America, Saudi Arabia and the Caribbean Islands [5]. The plant has numerous common names across regions where it is cultivated, like drumstick tree, horse radish tree, kelor tree or simply moringa in English [6]. It has high nutritional and medicinal values, and therefore, frequently used in India both as nutritional and medicinal purposes [7]. The medicinal properties of moringa lies in their different parts like, leaves, bark, seed and root help in the treatment of cardiovascular diseases, diabetes, and rheumatoid arthritis [8]. The methanolic extract of the leaves contains several potent bioactive compounds such as flavonoid pigments (kaempferol, rhamnetin, isoquercitrin and kaempferitrin), polyphenols, phenolic acids, minerals, and vitamins [9] that shows antimicrobial, antioxidant, antihypertensive, anti-inflammatory, and anticancer properties [10,11]. Furthermore, M. oleifera leaves are rich source of glycoside compounds, isothiocynates, beta-sitosterol, glucosinolates, gitoxigenin and betulic acid that shows significant anti-cancer properties [12,13,14].
Various reports suggested the anti-cancer efficacy of M. oleifera against different cancer cells [15], however, there is no any study suggesting the anti cancer activity of leaf extract of M. oleifera in Daltons lymphoma. Therefore, present study was aimed to analysis the therapeutic efficacy of M. oleifera against DL cells. Our results showed that leaf extract of M. oleifera significantly induces apoptosis and arrest cell cycle progression at G1 phase in DL cells.

Plant material and preparation of methanolic leaf extract
Greenish fresh leaves of M. oleifera were collected from Botanical garden, BHU, Varanasi (Uttar Pradesh), India. The collected leaves were washed gently 2-3 times with autoclaved double distilled water, and carefully air dried. Leaves were grinded into a fine powder; added 10 gm leaves powder in 100 ml methanol in a 200 ml round bottom flask and mixed for 10-12 hr until all components were dissolved. Filtered the extract with the help of Whatman filter paper and evaporated in a rotatory vacuum evaporator at 50°C. The filtrate obtained was stored at -20°C for further experiment.

Maintenances of Animal
The 8-10 week old inbred population of pathogen-free adult BALB/c (H 2 d) mice of either sex were used in the study. Mice were kept in a conventional polystyrene cage with an utmost pathogen free condition; sterilized food and water ad libitum were given to the mice.

GC-HRMS analysis of methanolic leaf extract of M. oleifera
Analysis of phytochemicals present in the methanolic extract of Moringa leaves was done by gas chromatography combined to mass spectrometer (GC-HRMS) (AccuTOF GCV) at SAIF laboratory, IIT Bombay, Powai, Mumbai, India. [16]. This technique is the combination of gas chromatography and mass spectrometry used to find out different biologically active phytochemicals present in the methanolic leaf extracts of M. oleifera (MOML).

Reactive oxygen species (ROS) estimation
The secretion of intracellular oxidative ROS by cells after treatment was analyzes with the help of DCFH-DA staining [18]. Briefly, 1 × 10 5

Ao/Etbr staining
Acridine orange/ethidium bromide is a fluorescence dye used to observe the changes occurred in the nucleus of apoptotic cells [19]. To identify the morphological changes in DL

Mitochondrial membrane potential (MMP)
A change in the mitochondrial membrane potential is an important factor leads to apoptosis of the cell. Mitochondrial membrane potential (ΔΨm) are typically assessed using Rhodamine-123, cationic fluorescence dyes [21]. Briefly, 1 × 10 5

DNA Fragmentation Assay
Apoptosis is a natural phenomenon of cell death recognised by cytoplasmic condensation, membrane blebbing, and nuclear pycnosis followed by nuclear DNA fragmentation [22].

GC-MS analysis of MOML
The chromatogram of GC-MS analysis showing the peaks of different compounds present in the methanolic extract of M. oleifera (Fig. 1)

M. oleifera leaf extract reduced cell viability
Cell viability and cytotoxic effects of MOML was evaluated by standard MTT assay. Results show that MOML reduced viability of DL cells in concentration dependent manner as compare to control, and IC50 value was found to be 300 μg/ml (Fig. 2). In further experiments, 150, 300, 450 µg/ml doses of MOML were used for cell treatment.

MOML induces intracellular ROS generation in DL cells
Generation of Reactive Oxygen species (ROS) play an important roles in apoptosis induction [23]. The effect of MOML on ROS generation in DL cells was assessed by fluorescence microscope and flow cytometer. Results show that generation of ROS in treated cells increases in a concentration dependent manner with respect to the control. Further, secretion of ROS was examine by flow cytometer and found that groups of treated cells exhibit significantly increased ROS level, 46.44%, 56.11% and 60.57% respectively in concentration dependent manner suggested 450 µg/ml as a most effective dose as compared to 6.44% in control (Fig.). concentrations. However, morphological changes induce by MOML in the nucleus of DL cells at any concentration was always higher than control or untreated cells (Fig. 3).

MOML induces apoptosis in DL cells
Apoptosis is an energy-dependent biochemical mechanism of programmed cell death that play critical role in maintaining cellular homeostasis in tissues [24]. Flow cytometry analysis of cells after AO/EtBr staining shows that cells treated with different doses of MOML display high proportion of yellowish green fluorescence with damaged cell membrane indicating early stage of apoptosis. However, the remaining cells show orange/red fluorescence with condensed nucleus that indicates late stage of apoptosis. Apoptosis induced by MOML was further confirmed by Annexin V/PI staining, and found that percentage of apoptotic cells increases by 13%, 16% and 70.9% respectively as compared to control group in dose dependent manner ( Fig. 9a and 9d). These observations suggested that MOML treatment effectively induce apoptosis in DL cells to maintain cellular homeostasis.

MOML arrest G2/M and S phase of cell cycle of DL cells
After measuring apoptotic potential of MOML, an experiment was performed to study its effect on cell cycle. were arrested at S phase respectively ( Fig. 8a and 8b). It is evident from the result that higher dose of MOML (450 µg/ml) is more effective in arresting cells at G2 and S phase.
Although, difference between the percentage of cells arrested at S phase by 300 µg/ml and 450 µg/ml MOML was not significant, it was always higher when compared with control.
Based on these observations, we concluded that MOML has ability to inhibit cell cycle progression at different stages, and down regulate DL progression.

MOML down-regulated mitochondrial membrane potential (ΔΨm) in DL cells
Any changes in the normal function of mitochondria causes depolarization of mitochondrial membrane potential, as a result apoptosis occur [25]. Therefore, to measure the effect of

MOML induce DNA Fragmentation of DL cells
To validate the apoptotic effect of MOML on DL cells, DNA fragmentation assay was performed. During apoptosis intracellular endonucleases are activated and cleave the internucleosomal linker DNA, as a result genomic DNA fragmentation occur [26].

Discussion
It has been reported that more than 60% medicinal plants exhibits cytotoxic properties and effective directly or indirectly against different types of cancer [27]. The GC-MS analysis shows that 14 major biologically active compound are present in MOML, where some of them having well reported anticancer properties [28]. Over past decades, M. oleifera leaves or its extract have been using effectively as antibacterial, anti-inflammatory, antioxidant, anti-parasitic, and anticancer agent [29,30,31]. Several studies have shown that M. oleifera exert significant anti-cancer activity against MCF-7, MDA-MB 231, EAC, HCT-8, Kasumi-1, A549 cancer cells by interfering with signalling cascade that promote development and proliferation of cancer cells [32]. However, the antitumor and anti-proliferative efficacy of MOML against NHLs, preferably T cell lymphoma have not explored yet.
Reactive oxygen species (ROS) is an effective stress mediated cellular intermediate metabolite acts as a secondary messenger in cell signalling, and play crucial role in various biochemical process with pro-survival activity in healthy cells [33]. However, due to high metabolic and peroxisome activities, cancer cells produces large amount of reactive oxygen species (ROS) that kill cells [34,35]. Keeping this fact in view, we performed a comprehensive study using DCFH-DA staining to find out the effect of MOML on ROS generation. We found that MOML treatment effectively induces ROS generation in DL cells.
Results also suggested that secretion of ROS is dose dependent and therefore, highest cytotoxity was observed at highest dose of MOML. However, at any dose secretion of ROS was always higher when compared to the control or untreated cells.
First time we reported putative effect of MOML on the growth and proliferation of T cell lymphoma in vitro and found remarkable anti-proliferative activities against DL cells. We also found that MOML was effectively reducing cell viability of DL cells in concentration dependent manner, where highest cell death occurs at 400 mg/ml MOML. Although, it was earlier reported that leaves extract of M. oleifera cell decreases viability in several cancer [36], their effect on apoptosis was not deciphered in T cell lymphoma. Therefore, we further analyzed the effect of MOML on DL cells apoptosis and found that MOML effectively induces membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies at higher dose, which are directly correlated with apoptosis. Results from DAPI, AO/EtBr staining analysis suggested that MOML not only down regulate DL cell proliferation, but also induces apoptosis to maintain normal cellular homeostasis of the tissue in dose dependent manner [37,38]. Further Annexin V/PI staining and Flow cytometer analysis of MOML treated DL cells show that most of the cells undergo apoptosis due to their cell cycle arrest at early and late phase, suggest significant anti-proliferative potential of MOML. However, these effects were dose dependent and maximum effects observed when the cells were treated with highest dose.
We next perform an experiment to study the effect of MOML on mitochondrial membrane potential. Studies have reported that mitochondria is a highly polarised organelle play important roles in several biochemical processes like oxidative phosphorylation, electron transport chain, and apoptosis [39]; however, any change in mitochondrial membrane potential results in the signals transduction that triggers apoptosis [40].  Figure 1 The chromatogram was obtained from the GC-HRMS with the methanolic extract of Moringa oleifera leaves.

Figure 2
DL cells were incubated with indicated concentrations of MOML for 24hrs and cytotoxic activity of MOML was measured by standard MTT assay. The results were denoted as a percentage of control±SD and statistically analyzed via using Bonferroni posttests as well as unpaired T-test (two-way ANOVA) for at least three independent experiments.

Figure 3
MOML treated and control cells were stained with the nuclear stain DAPI. Observation of Nuclear morphology was done by uorescent microscope(40x).

Figure 4
Composite images of DL cells stained with AO/EtBr after 24hrs treatment with various concentrations of MOML.

Figure 5
MOMLtreated and control cells analyzed by BD FACSCalibur ow cytometer. Bar graph showing increase in uorescent intensity of H2DCFDA dose-dependent manner in MOML treated DL cells.

Figure 6
An increase in H2DCFDA uorescence intensity was also visualized under a uorescence microscope (EVOS) showing that ROS production has enhanced.  Decrease of the Rh-123 uorescence intensity was also visualized under the uorescence microscope (EVOS) representing that MMP has disrupted.  The Cell cycle analysis of DL cells stained with PI dye after 24hrs of treatment with various concentrations of MOML. Figure 11 1.8 % Agarose gel electrophoresis was run to separate DNA fragments after incubation with various concentrations of MOML for 24hrs.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.