Antileukemic Potential of Momordica charantia Seed Extracts on Human Myeloid Leukemic HL60 Cells

Momordica charantia (bitter gourd) has been used in the traditional system of medicine for the treatment of various diseases. Anticancer activity of M. charantia extracts has been demonstrated by numerous in vitro and in vivo studies. In the present study, we investigated the differentiation inducing potential of fractionated M. charantia seed extracts in human myeloid HL60 cells. We found that the HL60 cells treated with the fractionated seed extracts differentiated into granulocytic lineage as characterized by NBT staining, CD11b expression, and specific esterase activity. The differentiation inducing principle was found to be heat-stable, and organic in nature. The differentiation was accompanied by a downregulation of c-myc transcript, indicating the involvement of c-myc pathway, at least in part, in differentiation. Taken together these results indicate that fractionated extracts of M. charantia seeds possess differentiation inducing activity and therefore can be evaluated for their potential use in differentiation therapy for leukemia in combination with other inducers of differentiation.


Introduction
Acute myeloid leukemia (AML) is a complex disease, characterized by abnormal differentiation and unlimited cellular proliferation that develops due to the accumulation of genetic and epigenetic alterations. Current treatments including chemotherapeutic drugs, radiation, and stem cell transplantation are associated with incomplete remission and side effects [1]. Therefore, there is a need to discover novel anticancer drugs that are effective and have minimal side-effects associated with the treatment. In traditional system of medicine and folklores, plant-based formulations with potential anticancer properties have been described [2,3]. Investigating dietary compounds with potential chemopreventive and anticancer activity has provided important leads for the development of clinically relevant anticancer drugs [3,4] However, systematic scientific studies have been lacking to understand the mechanism (s) of their anticancer activity.
In the present study, we have focused our attention on a tropical plant, Momordica charantia L, belonging to Cucurbitaceae family that has been used in the traditional health care world over and the ethnobotanical use of this medicinal plant is well documented [5,6]. M. charantia has been reported to possess a number of diverse medicinal properties such as antimicrobial, antidiabetic, antifertility and abortifacient activity [7][8][9][10]. Antigrowth properties of fractionated M. charantia whole plant extracts were first reported by West et al. [11]. Subsequently, a number of growth inhibitors have been isolated from M. charantia seeds and its antiproliferative activity has been demonstrated in a variety of tumor cell lines [8,[12][13][14][15]. M. charantia fruit extract and its components have also been shown to be cytotoxic to leukemic lymphocytes and induce antitumor activity in vivo [8,16,17].
While antiproliferative and antitumor activity of M. charantia has been demonstrated, its differentiation inducing potential in leukemic cell lines has not been investigated. We have used human myeloid leukemic HL60 cells as an in vitro system to test the differentiation inducing potential of M. charantia and to elucidate the molecular mechanisms involved in the differentiation process. HL60 cells have been used for screening of compounds for their 2 Evidence-Based Complementary and Alternative Medicine antileukemic potential and to elucidate the molecular mechanisms involved in the differentiation process. Different compounds have been reported to induce HL60 cells towards granulocytic, macrophagic, and monocytic lineage [18][19][20][21]. In the present study, we report for the first time that the fractionated M. charantia seed extract induced differentiation of HL60 to granulocytes and thus has potential to be developed as a therapeutic supplement.

Cells and Reagents.
The human promyelocytic leukemia HL60 cells (ATCC#CCL240) were maintained in 1 × RPMI 1640 medium supplemented with fetal calf serum (FCS, 20%), penicillin (100 U/mL), and streptomycin (100 μg/mL) and 2 mM L-glutamine in a 5% CO 2 humidified atmosphere. The chemicals required for cell culture were procured from Biological Industries, Israel. Other chemicals were from standard commercial suppliers.

Extraction and Partial Purification of M. charantia Seed
Extract. Seeds of M. charantia L. (Pusa Vishesh variety, developed, verified, and released by Indian Agriculture Research Institute (IARI), Pusa, New Delhi [22]) were obtained from IARI, Pusa, New Delhi. Decorticated seeds (100 g) were extracted in 75% ethanol containing 0.2 N HCl and 1 mM PMSF. The crude extract was centrifuged at 20,000 ×g for 1 hr at 4 • C. The supernatant was evaporated to 1/4 volume under reduced pressure followed by neutralization using NH 4 OH. Differential fractionation of the extract (Mc-1, dry weight ∼16.5 g) thus obtained using 0.05 M (NH 4 ) 2 CO 3 followed by centrifugation at 20,000 ×g for 45 min at 4 • C resulted in the pellet (Mc-2, dry weight ∼2.3 g) and supernatant (Mc-3, dry weight ∼14.2 g) fractions. All the fractions were evaporated and resuspended in Milli-Q water prior to use. The fraction Mc-2 was further fractionated using acetone extraction (Mc-2Ac, dry weight ∼400 mg). Protein was estimated using Lowry's method [23].

Cell Viability and Cell Proliferation
Assay. Cell viability of HL60 cells upon treatment with M. charantia fractions, namely, Mc-1, Mc-2, Mc-3, and Mc-2Ac was assayed using MTT essentially as described by Yedjou et al. [24]. HL60 cells were seeded in 96-well plate at an initial density of 10 5 cells/mL. Different concentrations M. charantia test fractions (diluted in the medium) were added to the cells. The blank sample contained medium only. The cells were placed in humidified 5% CO 2 incubator at 37 • C. After 72 h of incubation, the cells were centrifuged at 800 rpm for 5 min and the supernatant culture medium carefully aspirated. The cells were given 2 washes with PBS, followed by addition of 200 μL fresh medium containing MTT (0.5 mg/mL). The cells were further incubated at 37 • C for 2 h, followed by centrifugation at 800 rpm for 5 min. The medium containing MTT was carefully removed followed by addition of 100 μL DMSO to each well. The plate was incubated at room temperature with shaking for 10 min to solubilize formazan crystals. The absorbance at 570 nm was measured in a microplate reader (Tecan, Switzerland). The amount of color produced is directly proportional to the number of viable cells. All assays were done in triplicates for each concentration and cell viability was estimated by using means + SD values for each treatment. Cell viability was calculated using MTT absorbance of the control and treated cells: % survival = (mean value treated sample/mean value of untreated sample) * 100.
Effect of M. charantia fractions on cell proliferation rate was determined by viable cell count using a hemocytometer as described by Lee et al. [20]. Briefly, HL60 cells (10 5 cells/mL) were plated in a 24-well plate and incubated with different fractions of M. charantia at a final concentration of 20 μg/mL for 5 days. Viable cell counts were determined on each day post treatment using trypan blue dye exclusion assay using a light microscope (Leica, Germany).

Differentiation of HL60
Cells. HL60 cells (2 × 10 5 /mL) were incubated with the test fractions at the indicated concentrations for different time intervals. Cell differentiation was assayed by NBT (Nitroblue tetrazolium) reduction, Wright-Giemsa staining, specific and nonspecific esterase activities, and by FACS analysis.

Nitroblue Tetrazolium (NBT) Reduction.
NBT staining was used to assay for HL60 cell differentiation essentially as described earlier [25]. Approximately 200 cells in triplicates were scored under a light microscope (Leica, Germany) for each test sample. Extent of differentiation is expressed as number of % NBT positive cells. The data corresponding to the number of total NBT positive cells out of the total cells counted for different experiments are given in Table 1 in supplementary material available online at doi: 10.1155/2012/732404. The data were analyzed using one-way ANOVA and Tukey post hoc test to compare the difference between the treated and the untreated control. An alpha value of <0.05 was considered to be significant.

Wright-Giemsa Staining.
Morphological changes in the HL60 cells induced by the test samples were evaluated using Wright-Giemsa staining as per the manufacturer's instructions (Sigma-Aldrich, USA). Differential cell count was performed under a light microscope (Leica, Germany) from at least 3 independent glass slides for each experimental time point in triplicates.

Specific and Nonspecific Esterase
Activity. Specific and nonspecific esterase activities in the HL60 cell lysates of the control and treated cells were estimated using cytochemical kits as per manufacturer's instruction (Sigma 91-A and 91-C, Sigma-Aldrich, USA). The presence of granulation (reddish and blackish granulation indicating the specific and nonspecific esterase, respectively) indicated the areas of enzyme activity, which was scored in a scale of 0 to +4, 0 indicating no staining, hence no enzyme activity and +4 Evidence-Based Complementary and Alternative Medicine 3 indicating brilliant staining, and hence maximal enzyme activity.

Analysis for CD11b
Expression. Control and treated HL60 cells (5 × 10 5 cells) washed with PBS containing 1% FCS and 0.01% sodium azide were incubated for 30 min in fetal calf serum at 4 • C. Subsequently, FITC-conjugated anti-human CD11b antibody (1 μg/mL, eBiosciences, USA) was added to the cells and incubated at 25 • C for 45 min followed by washing with PBS. The cells were then fixed in 1% paraformaldehyde and analyzed on Becton Dickinson Flow cytometer. Isotypic control (Rat IgG2b) was also used to check for nonspecific binding. The results were plotted using WinMdi software (Flow Core Facility, Scripps Research Institute; http://facs.scripps.edu/software.html).

Northern Blot Analysis of c-myc Transcript. HL60 cells (2 × 10 5 cells/mL) were treated with fractions Mc-2 and
Mc-2Ac (20 μg/mL each). Total RNA from the control and treated cells was isolated on day 4 of the treatment using TRIzol reagent (Life Technologies, USA) as per the manufacturer's instructions. Briefly, total RNA (10 μg each) isolated from the control and treated HL60 cells resolved on 1% formaldehyde agarose gel was transferred onto nitrocellulose membrane and subjected to Northern blotting using α-P 32 -dCTPlabeled c-myc gene specific probe [26] essentially as described earlier [27].  (Figure 1(a)).

Acid Ethanol Extractable Fractions of M. charantia Seeds
In order to assess if the observed increase in NBT positive cells is due to death of immature cells caused by the test fractions, cytotoxicity analysis was carried out with the fractions Mc-1, Mc-2, and Mc-3 of M. charantia. No effect on the cell viability was observed with any of the fractions upto 100 μg/mL (the maximum concentration tested; Figure  1(b)). These data indicate that the three fractions were noncytotoxic even at 5× concentrations than those used for differentiation studies (Figure 1(a)). Similar results were also obtained using Alamar blue reduction assay for cell survival. Percentage reduction of Alamar blue in HL60 cells treated with the three factions was comparable to the control cells confirming that the M. charantia fractions were noncytotoxic (data not shown).

Time Course of Induction of Differentiation.
To investigate the time kinetics of the differentiation induced by fraction Mc-2, the HL60 cells were treated with Mc-2 at a final concentration of 20 μg/mL and scored for NBT-positive cells from day 1 to day 5 of the treatment. As evident from Figure 1(c), differentiation was observed on day 3, which increased further on day 4 after which no significant change in the percentage of differentiated cells was observed. The fraction Mc-2Ac showed higher differentiation inducing potential. As evident from the data, fraction Mc-2 showed between 50 and 53% differentiation at 10 and 20 μg/mL, whereas fraction Mc-2Ac was able to bring about 59% differentiation even at 10 μg/mL concentration (cf. Figures  1(a) and 2(a)). Like the parent fraction Mc-2, fraction Mc-2Ac also did not show any cytotoxic effect on HL60 cells even at 5× concentration than that used for differentiation ( Figure  2(c)).  All the three fractions resulted in significantly increased NBT staining, relative to untreated control. The data in all the panels represent mean ± SD of three independent experiments performed in triplicates. * P < 0.05, * * P < 0.01, and * * * P < 0.001 relative to untreated control.

Minimum Time Required for Fraction
known to cause both differentiation and inhibit proliferation [28][29][30], effect of different fractions of M. charantia was also evaluated on proliferation of HL60 cells. As evident from Figure 3, no change in rate of proliferation was noted till day 3 of the treatment, after which a slight decline in the cell count was observed on day 4 and day 5 in the treated samples, when compared to the untreated control.  Lineage of differentiation was further confirmed by determination of the specific/nonspecific esterase activity in HL60 cells. The HL60 cells treated with either Mc-2 or Mc-2Ac were found to be positive for specific esterase activity (+2 to +3) and negative (0) for nonspecific esterase activity confirming to the granulocytic differentiation of the HL60 cells by M. charantia seed fractions.

Effect of Fraction Mc-2Ac on c-myc Expression in HL60
Cells. Since c-myc expression has been reported to be downregulated in leukemic cells that were induced to differentiate [31][32][33], c-myc transcript levels were measured in HL60 cells treated with fractions Mc-2 and Mc-2Ac on day 4 after treatment. Northern blot analysis performed with total RNA isolated from the control and treated cells revealed a significant decrease in the levels of c-myc transcript

Discussion
In the present study, we for the first time demonstrate the differentiation induction potential of the M. charantia seeds in HL60 cells. It is clearly demonstrated that fraction Mc-1 and its subsequent fractions Mc-2 and Mc-2Ac are able to differentiate HL60 cells. As Mc-1 is crude extract, a decrease in the differentiated cells in Mc-1-treated cells at higher concentration (i.e., 20 μg/mL) in comparison with that observed with 10 μg/mL could possibly be due to the presence of some compounds that could inhibit differentiation and which gain their inhibitory effect when concentration is increased to 20 μg/mL. Upon further fractionation into Mc-2 and Mc-3, these inhibitors are fractionated away and that is why no decrease in differentiation is seen in fractions Mc-2 at 20 μg/mL. The test fractions of M. charantia capable of inducing differentiation did not exhibit any cytotoxic effect on HL60 cells. These data suggest that the increase in NBTpositive differentiated cells upon treatment is indeed due to differentiation brought about by the M. charnatia fractions and not caused by cell death of immature undifferentiated cells. Our results are in agreement with the reports put forth by Sharma et al., who also did not observe any cytotoxic effect of ethanolic extract of M. charantia on a number of cancer cells [34].
Unlike many other plant-derived compounds which induce the HL60 cells towards monocytic or macrophagic pathway [35,36], fractionated M. charantia extracts induced HL60 cells into granulocytic pathway, evident by the morphological characteristics of granulocytic lineage, presence of specific esterase activity, and expression of CD11b in the Mc-2-and Mc-2Ac-treated HL60 cells. Granulocytic differentiation of HL60 cells by fractionated M. charantia seed extract is comparable to that observed with retinoic acid [25].
Induction of differentiation of HL60 cells by fractionated M. charantia seed extracts in a time-and concentration-dependent manner supports the "all or none" response similar to that of DMSO and sodium butyrate [37] as opposed to the graded response seen by another inducer of HL60 differentiation such as retinoic acid [25]. It is known that different inducers require different lengthes of time to trigger differentiation in different cell types [38][39][40]. During this period, the cells become committed to differentiation and this has been proposed to be a rate limiting step for the cells to synthesize cellular constituents above a threshold value [41]. While difluoromethylornithine (DFMO) is reported to trigger differentiation of MEL cells when present in the medium for only 24 h, vitamin D3 is required to be present for 48 h in the medium to induce differentiation of MEL cells [40]. On the other hand, vitamin D3 was able to induce differentiation of HL60 cells on day 1 itself [38]. In the present study, it was established that presence of Mc-2Ac for a minimum time of ∼72 h is required to observe any significant differentiation, though some differentiation could be observed on day 2 also. Since the fraction Mc-2A did not show any cytotoxic effect even at 2.5× concentration that induced maximum differentiation, it is advantageous over other inducers such as DFMO or DMSO, that exhibit cytotoxic effect at higher concentration.
The nature of the differentiation inducing compound(s), as established by the retention of the differentiation inducing activity even after heat and protease treatment, is in line with other studies wherein a large proportion of the inducers of HL60 differentiation are nonproteinaceous in nature [35,36]. The extent of differentiation induced with the Mc-2 and Mc-2Ac fractions is comparable with the differentiation induced by many other plant-derived compounds [42].
M. charantia seed fractions did not exhibit any antiproliferative activity. Till day 3, there was no change in the growth profile of the treated cells when compared to the untreated cells. Similar results have been obtained by induction of differentiation of human K562 cells with hemin [43]. A slight decline in the cell count in Mc-1, Mc-2, and Mc-2Ac fractions on day 4 and day 5 of the treatment is due to induction of differentiation as the differentiated cell population does not proliferate further. These data suggest that the test fractions do not possess antiproliferative activity. Although M. charantia extracts have been reported to have antitumor and antiproliferative activities ( [13][14][15][16], for review please see [10,44]), most of such activities have been attributed to a number of proteins identified as lectins-ribosome inactivating proteins such as alpha momorcharin, momordin [7,15,45], and MAP30 ( [8,46], for review, please see [10,44]). Unlike these, the differentiation inducing fractions did not exhibit any antiproliferative activity and the differentiation inducing principle(s) was found to be nonproteinaceous in nature, suggesting that the differentiation inducing factors of M. charantia are different from its antitumor factors.
Since HL60 cells are induced to differentiate by retinoic acid, it was imperative to rule out the possibility that the differentiation by M. charantia extract is not due to retinoic acid present in the extracts, if any. For this purpose, differentiation inducing potential of the fraction Mc-1 was assessed in K562 cells, prototype human myeloid leukemia cell type that is resistant to retinoic acid induced differentiation [28,47] by morphological examination as described earlier [48]. While treatment with positive inducer sodium butyrate resulted in upto 85-90% cells to be differentiated, fraction Mc-1 resulted in differentiation of ∼40-45% cells (Ramani Soundararajan, Madhu Geeta, and Aparna Dixit, unpublished data). Vitamin A levels have been reported to be undetectable in methanolic extracts of M. charantia [49].
In the present study, seed extraction was performed with acid ethanol, and therefore, it was unlikely to have retinoic acid. The transcription factor c-Myc plays a role in the control of cell proliferation and differentiation [50]. A significant decrease in the c-myc expression in the Mc-2-and Mc-2Ac-treated cells is in line with previous reports wherein a modulation in c-myc expression in HL60 cells induced to differentiate has been established [31,32]. Murine erythroleukemic (MEL) cells induced to differentiate by DMSO also showed a biphasic change in the c-myc levels suggesting that the changes in c-myc may be important for irreversible commitment of MEL cells towards terminal erythroid lineage [33]. The reduced expression of c-myc can be related to the loss of proliferative potential of the differentiated cells [32]. A slight decrease in c-myc transcript in the Mc-2Ac-treated cells relates well with the decrease in number of cells from day 3 onwards, relating to the loss of proliferating potential of the differentiated cells as c-Myc has been shown to be functionally involved in DNA synthesis [51].
Thus, the present study for the first time demonstrates differentiation inducing activity of the fractionated M. charantia seed extracts which can be further assessed as inducer of differentiation for the treatment of APL, either alone or in combination with the suboptimal concentrations of other known inducers of differentiation. Studies are in progress to understand the mechanism by which the compounds present in fraction Mc-2Ac are inducing differentiation.