IN VITRO STUDIES ON THE SYNERGISTIC EFFECT OF MORINDA CITRIFOLIA L. (NONI) AND METHOTREXATE ON CYTOTOXICITY OF HELA CELL LINES

Objective: Cancer remains to be one of the leading causes of death around the world. Modern targeted therapies have undeniably improved cancer patients’ survival, but search still continues for safer and more effective drugs. This work is an attempt to assess the effectiveness of cotreatment of aqueous fruit extract of Morinda citrifolia L. (Noni) on methotrexate (MTX)-induced cytotoxicity on HeLa cancer cell line. Methods: HeLa cells were treated with different concentrations of MTX and Noni alone and in combination for 24 and 48 h and studied for the cytotoxic effects by various approaches. Results: There was a dose-dependent inhibition of growth when treated with MTX in a dose range of 0.045–45.4 μg/ml and Noni for a dose range of 0.3–30 mg/ml for 48 h. The IC50 for MTX was 4.45 μg/ml (1 μM/ml), and for Noni, it was 8 mg/ml while the combination exhibited a more intensive growth inhibitory effect. Our results were confirmed with phase-contrast microscopy, whereby the number of viable cells decreased with an increase in the concentration of Noni. Cells were found to have rounded up and detached from the flask indicating apoptosis. Apoptosis in HeLa cell lines was further confirmed by DNA fragmentation and flow cytometry. Conclusion: It can be concluded that Noni may enhance MTX cytotoxicity by inducing apoptosis and cell cycle arrest. Hence, cotreatment with Noni may reduce the dosage of MTX necessary for inhibiting proliferation of malignant cells and hence decrease the toxic effects of MTX on the system.


INTRODUCTION
Many nutraceuticals and plant active compounds are an important historical and ethnobotanical source for developing drugs with broad-spectrum pharmacological activities such as anticancer, antitumor, and antioxidant. An increasing incidence of cancer in developing and developed countries has promoted research toward dietary and medicinal plant extracts as supplements and prophylactics essentially due to the presence of phenolic acids, flavonoids, and phenolic diterpenes [1]. Chemotherapy and radiation on human cells continuously lead to the generation of reactive oxygen radicals, causing noxious effects due to potentially heavy loads of oxidants. Usage of alternative medicine is a necessity of the hour as they can be protective and can possess antioxidant and antitumor agents which can be more effective and reduce toxicity due to the presence of phytochemicals [2,3]. In the present study, we have used methotrexate (MTX) which is an antineoplastic drug widely used in the treatment of several diseases such as autoimmune diseases, lymphoma, and lymphoblastic leukemia but has been associated with mild-to-severe liver and intestinal toxicity. Aqueous fruit extract from Morinda citrifolia L. (Noni), which has a diverse array of active phytoconstituents with multitude of pharmacological activities, has been used for cotreatment in our study. Noni is used as an herbal remedy and has promising antioxidant properties [4].

Bhakti and Thankamani
The supernatant was carefully discarded taking care of not losing the formazan crystals. The crystals were dissolved by adding 100 μl of DMSO, and absorbance was read on a Bio Rad microplate reader (version 680) at 570 nm with a reference wavelength of 655 nm. The experiment was repeated 3 independent times in triplicates and percentage of inhibition was calculated.

Phase-contrast microscopy
Approximately 1×10 4 cells were seeded in a 24 multi-well plate for phase-contrast microscopy. After 24 h, the medium was discarded and cells were supplemented with fresh medium. Cells were treated with MTX at a final concentration of 0.045-45.4 μg/ml alone, 0.3-30 mg/ml of M. citrifolia L. fruit extract, and in combination along with IC 50 (4.54 µg/ml) of MTX with varying concentrations of M. citrifolia L. fruit extract for 48 h. A separate well of control was also maintained. Phase-contrast microscopy study of treated and non-treated cells was performed using Inverted Microscope Nikon ECLIPSE TS100.
DNA laddering assay DNA was extracted by the method of Gong et al. (1994) [7].
HeLa cells (2×10 5 ) were seeded for DNA laddering assay in culture plates. After 24 h, the medium was discarded and cells were washed with ×1 PBS (pH 7.4) and fresh DMEM medium was added. Cells were treated with final concentration IC 50 (4.54 µg/ml) of MTX of alone, IC 50 (8 mg/ml) of Noni alone, and in combination with IC 50 of Noni and MTX. A separate control was also maintained. After 48 h incubation, the supernatant was collected and adhered cells were harvested by trypsin treatment.
Electrophoresis was carried out at 60 V until the loading dye migrated about three-fourth of the gel. DNA molecular marker of 500-1000 bp was run alongside the samples. The DNA was visualized by observing the gel using gel documentation system (Syngene).

Clonogenic assay
Clonogenic assay was performed as per the method of Buch et al. (2012) [8].
Clonogenic assay was performed on exponentially growing cells. A total of 500 cells were seeded in 6 multi-well plates in 1 ml of complete DMEM medium and allowed to adhere overnight. After 16 h, the medium was removed and replaced with medium containing IC 50 (4.54 µg/ml) of MTX alone, IC 50 (8 mg/ml) of Noni alone, and in combination with IC 50 of Noni and MTX. A separate control was also maintained. After 48 h, cells were fed with fresh DMEM medium. After 9 days of treatment, media were carefully removed and then washed with PBS. Cells were then fixed with methanol: acetic acid (3:1) and stained using 0.5% crystal violet in methanol for 15 min. Cells were washed again with PBS and colonies were observed. Treated and non-treated cells were photographed and each experiment was repeated in triplicate.

Flow cytometry-based apoptosis detection
Flow cytometry analysis of apoptotic cells was done as per the method of Riccardi and Nicoletti (2006), Balakrishnan and Bhat (2014), and Da'i et al. (2017) [9][10][11]. Approximately 1×10 5 cells were seeded in multiple 6-well plate for flow cytometry and apoptosis detection. After 16 h, the medium was discarded and cells were supplemented with fresh medium containing IC 50 (4.54 µg/ml) of MTX alone, IC 50 (8 mg/ml) of Noni alone, and in combination with IC 50 of Noni and MTX. A separate control was also maintained. After 24 h of treatment, the supernatant was collected and adhered cells were harvested by trypsin treatment in a tube for each concentration-treated cells. The tubes were centrifuged at 2500 rpm for 10 min to obtain a cell pellet. The supernatant was discarded and cell pellet was washed with 1 ml of PBS (pH 7.4).
The cells were then fixed by adding pre-chilled 2 ml of 70% ethanol dropwise to the pellet. The cells at this stage were stored for a week at −4°C and then processed for analysis. During analysis, cells were first centrifuged at 2500 rpm for 10 min. Ethanol was discarded and cells were washed with PBS (pH 7.4) to remove ethanol completely. 500 μl of PBS was again added to the cell pellet, and the cells were passed gently through 26-gauge needle 3-4 times. 50 μl of RNase A and 5 μl of Triton X-100 were then added to the microfuge tube and incubated at 37°C for 30 min. 12.5 μl of propidium iodide was added per tube at a concentration of 1 mg/ml and incubated in the dark at 37°C for 20 min. The DNA content of the cells was analyzed on BD Accuri Flow Cytometer. C6 analysis software program was used for acquisition and percentage of cells undergoing apoptosis.

Statistical analysis of data
Data are presented as mean ± standard deviation. The statistical significance was assessed using one-way analysis of variance followed by Student's t-test. In figures and tables, symbols represent statistical significance as indicated: *p<0.05, **p<0.01, ***p<0.001 and NS: Nonsignificant.

Cytotoxicity of Noni and MTX
Cytotoxic effect of MTX and Noni alone and in combination on HeLa cell lines was checked first with an exposure time of 24 h, but, since it did not show any significant effect on the HeLa cells, then it was increased to 48 h.

Effect of MTX and Noni on HeLa cell clonogenic assay
Clonogenic assay measures the ability of the cell to retain its normal morphological characteristics, proliferation, and the effectiveness of a cytotoxic agent is added to cancer cells [12]. With increase in concentration of cytotoxic agent there is loss of reproductive integrity and inability to proliferate leading to cell death [13].

Bhakti and Thankamani
Colony formation ability of HeLa cells when treated with MTX and Noni fruit extract was assessed. Our study shows that HeLa cells are sensitive to MTX alone, Noni alone, and combination of Noni and MTX in comparison to control (Plate 5). Since cells number was less than 50, plating efficiency could not be calculated.
As can be observed in Plate 5, cells treated with MTX IC 50 value (4.54 µg/ml), Noni IC 50 value (8 mg/ml), and a combination of 8 mg/ml Noni and 4.54 µg/ml MTX showed distinct fragments that ultimately lead to collapse of the cells, nuclear disintegration, destabilization of nuclear envelope, and lamin proteolysis (Plate 4 -b, c, d). These photographs establish the synergistic effect of Noni fruit juice on MTX-induced cytotoxicity.

Effect of Noni and MTX on DNA fragmentation
Apoptosis is a distinctive feature of cell death due to the release of nuclear DNA from apoptotic cells. HeLa cell lines treated with MTX and Noni were analysed for DNA fragmentation. DNA ladder 500-100bp was used for comparison. (Fig. 4a) DNA of cells treated with MTX IC 50 (Fig. 4c) showed ladder with some smearing. There was no DNA fragmentation or smearing of DNA in control (Fig. 4b), but the combination of Noni + MTX and Noni alone showed smearing which might be due to necrosis of cells (Fig. 4d and e). This also serves as a vital biochemical marker for measurement of drug-mediated cytotoxicity.

Bhakti and Thankamani
Cells treated with MTX IC 50 4.54 µg/ml, however, showed 25.86%±7.78 of apoptotic cells and 74.14% viable cells. Fig. 7a and b shows a number of cells captured with no gate and gate R 2 later. Fig. 7c and d represents the sub G1 phases which determine the number of apoptotic cells in the IC 50 4.54 µg/ml. Cells treated with Noni and MTX, however, showed 21.13%±2.68 of apoptotic cells and 78.87% viable cells. Fig. 8a and b shows a number of cells captured with no gate and gate R 2 later. Fig. 8c and d represents the sub G1 phases which determine the number of apoptotic cells in the Noni and MTX.
Our results indicate that cytotoxicity of Noni or MTX by themselves and in combination as detected by MTT assay and clonogenic assay showed a decrease in survival of cancer cells. As shown in Fig. 9a, combination of Noni and MTX can enhance apoptosis as compared to either Noni alone (p<0.001) or MTX alone (p<0.001). Fig. 9b is a pictorial representation of multiple samples run in gated (P5 in R 2 ). Black peak represents control with 89.41% viable cells and 10% apoptotic cells. Red peak represents Noni extract-treated cells with 75.20% viable cells and 24.8% apoptotic cells. Blue peak represents cells treated with Noni +MTX showing 71.83% viable cells and 28.17% apoptotic cells; similarly, green peak represents cells treated with MTX showing 61.36% viable cells and 38.64% apoptotic cells.
Even though the number of apoptotic cells appears to be more in pictorial graph (Fig. 9b), Fig. 9a shows a greater number of apoptotic cells in Noni and MTX in comparison to that of MTX alone in histogram. This could be due to the scatter of cells obtained during acquisition of data.

DISCUSSION
Natural plant compounds have attracted attention as alternative therapeutics in the fight against diseases, primarily due to their low toxicity and high therapeutic index. Plant-derived drugs such as vinblastine, vincristine, taxol, and camptothecin are already accepted as potent chemotherapeutic agents. Here, we explore the antiproliferative potential of fruit extracts of M. citrifolia alone and in combination with a well-accepted drug, MTX, by in vitro methods.
Our results for viability assay performed on MTX alone, Noni alone, and combination of Noni with MTX-treated cells were found to be highly significant. Singh et al. (2013) have also reported that addition of varying concentration of Noni alone, cisplatin alone, and combination of both reduced the cell survivals, respectively, by 22%, 29%, and 50% in HeLa cell lines [15]. The concentration of cisplatin used in their study was 10 µg/ml and Noni extract used was 10%, v/v for 24 h.
In our study, we have observed that about 50% of HeLa cells showed cytotoxicity even with a concentration of 8 mg/ml of Noni alone and MTX 4.54 µg/ml alone, whereas a minimum dose of MTX 4.54 µg/ml in combination with Noni (8 mg/ml) is sufficient enough to be 52% cytotoxic after 48 h, showing an additive effect and hence proving that small amount of chemotherapeutic drug can be used along with Noni as a chemoadjuvent to boost the cytotoxic potential.
Noni by itself has a cytotoxic effect on HeLa cell lines. Cells rounded up and detached from the plate and their neighbouring cells with subsequent condensation of cytoplasm. Dissolution of nuclear envelope along with separation of the nucleus into distinct fragments leading to collapse of cells into several small intact vesicles were also observed. A similar result was seen when we combined the IC 50 value of MTX with increasing concentration of Noni. Plasma membrane blebbing, collapse of cells, number of viable cells decreased with an increase in concentration, and retraction were observed in treatment with IC 50 value of MTX and increasing concentration of Noni.
Our studies indicate that Noni co-treatment with lower dose of MTX can induce morphological changes such as, necrosis and cell death

Flow cytometry to study the effect of Noni on MTX-induced apoptosis on cell lines
Cells experience a mode of cellular suicide called apoptosis under normal physiological conditions. Cell undergoing apoptosis shows morphological characteristic changes such as formation of round apoptotic bodies appearing like round spherical beads, nuclear condensation, and shrinkage [14].
Flow cytometry and propidium iodide staining was used to investigate antiproliferative effect of Noni and chemotherapeutic drug MTX on the cell cycle distribution of HeLa cells.
Control cell showed a 6.3%±2.56 of apoptotic cells and 93.7% displayed characteristic normal cell cycle distribution. All the analyses were done in triplicates. Fig. 5a and b shows a number of cells captured with no gate and gate R 2 later. Fig. 5c and d represents the subgenomic cells in sub G1 phase peak in red color which determines the number of apoptotic cells in the control. Black peak indicates G0-G1 phase.
Cells treated with Noni IC 50 8 mg/ml, however, showed 9.4%±3.38 of apoptotic cells and 90.60% viable cells. Fig. 6a and b shows a number of cells captured with no gate and gate R 2 later. Fig. 6c and d represents the sub G1 phases which determine the number of apoptotic cells in the Noni IC 50 8 mg/ml.

Bhakti and Thankamani
proving to be very good cytotoxic agent, equivalent to higher doses of MTX alone.
Similar cytotoxicty with extracted chlorophyllin from fresh leaves of M. citrifolia L. has been also reported on Hep G2 and Vero cell lines [16], which suggest higher safety ratio, indicating potential use in cancer treatment as the extract inhibits only the growth of cancer cells but not normal cells.
Clonogenic assay is a conventional and more frequently used in vitro assay to visually witness cloning efficiency. Chemotherapeutic drug leads to remarkable phenotypic changes, [17] and damage to chromosomes leading to apoptosis [18]. This assay detects the ability of all cells that have retained the capacity for producing a large number of progenies after treatment.
The studies on the effect of curcumin-induced apoptosis in human mammary carcinoma cells (MDA-MB-23) reported by Latifah et al. (2006) are similar to our results, which again emphasize Noni fruit juice's synergistic effect on cytotoxicity induced by MTX [19].
Consistent slow growth rate, inability to form clones, no surviving colonies, and only few cells were observed in all the three plates that were treated with Noni and MTX. We observed that the cell number in MTX alone, Noni alone, and combination of Noni and MTX was less than 50 as compared to that of control.
Induction of apoptosis in HeLa cell lines was confirmed by DNA fragmentation. These degraded DNA produces a ladder pattern which is a hallmark of cytotoxicity and drug-induced apoptosis in cancer cells as observed on an agarose gel [20]. Xie et al. (2016) have stated that MTX can be lethal to cancer cells, as well as normal cells, and hence, the use of a combination of Noni and MTX can be more beneficial in the treatment of cancer [21]. Breakdown of cellular DNA caused due to activation of endogenous nuclear endonucleases and proteases, cleave double-stranded DNA at sites situated on linker DNA leading to DNA laddering. The release of these enzymes from cytoplasmic membrane is a sign of inhibition of DNA replication.
Treated or damaged cells can be evaluated by cell cycle analysis. Certain checkpoints are interrupted due to damage that occurs in the cell during cell cycle, hence preventing normal cells from being transformed into cancer cells [9]. G1 phase nuclei of apoptotic cells contain less DNA than healthy cells. Apoptosis is evaluated by quantification of percentage cells in the sub-G1 phase. A sub G0/G1 peak was seen before G1 in a flow cytometry histogram. Cycle cell analysis carried out to check the synergistic effect of Noni and MTX on HeLa cell lines showed an increment in sub G0/G1 population indicating the occurrence of apoptosis which was in concurrence to decrement in the percentage of viable cells in MTT and clonogenic assays. Sharma et al. (2016) reported that cell cycle distribution in MCF -7 and MDA-MB-231 cell when exposed to Noni ethyl acetate extract at IC 50 for 24, 48, and 72 h showed a noticeable increase in the G1/S phase in a time-dependent manner [20]. They have reported 81% apoptotic cells which are comparable to our results for the same time duration [22]. Our results are also in accordance with Gupta et al. (2013) and Alitheen et al. (2016) [2,23]. The latter have used the combination of doxorubicin and damnacanthal, and the population of MCF-7 cells decreased more when treated in a combination of two rather than individual agents alone.
Combination therapy (Noni and MTX) remarkably increased apoptotic index, thereby promising herbal-based anticancer agent. An important homeostatic mechanism can be established by induction of apoptosis which regulates cell division and cell death leading to cytotoxicity that can maintain suitable cell number. Noni has been identified for its antiproliferative potential against tumor cells. In accordance, if plant extracts and their constituents have an ability to inhibit cancer cells without disturbing normal cells, they can have an immense potential in the treatment of cancer [24,25]. The synergistic combinations of Noni with existing anticancer drugs can enhance efficacy, reduce side effects, boost immune modulation, as well as abrogate drug resistance.

CONCLUSION
The results of this study show that Noni extract was able to induce growth inhibition, apoptosis, and cell cycle arrest alone and in  Noni as a chemoadjuvent can be one of the effective methods in reducing MTX dose administered to cancer patients during chemotherapy, thereby reducing toxicity to normal cells as well as side effects caused by chemotherapeutic drugs during the course of treatment.