The Cytotoxic Effect of Ethanol Extract of Momordica Charantia, Kuguacin-J and Cisplatin on Healthy MCF10A and MCF-7 and MDAMB-231 Breast Cancer Cell Lines in Vitro

Traditional medicines, derived from plants, could present alternative treatment strategy for cancer therapy. One such plant is Momordica charantia (MC) which possesses anti-carcinogenic properties. This study investigated the anticancer effect of an ethanol extract of MC fruit, Kuguacin-J (K-J), an isolated compound from the leaves of MC and cisplatin, either alone or combination on healthy MCF-10A mammary cells and compared with breast cancer MCF-7 and MDAMB-231 cell lines. Cell viability was tested using 8 μg/mL and 80 μg/mL doses of MC extract, K-J and cisplatin individually or combined for 24 and 48 hours. Caspase-3- activity was measured in MCF-7 and MDA-MB-231 cells using established methods. The results revealed that MC extract and K-J had no effect on healthy MCF-10A cell viability as compared to cisplatin which induced dose and time-dependent cell death. Similarly, treatment of MCF-7 cells with cisplatin induced cell death at high concentration at both the time points, while MC extract and K-J only induce MCF-7 cell death at high dose after 48 hours only. During combination therapy, both doses of cisplatin enhanced MCF-7 cell death when combined with MC extract or K-J after 24 and 48 hours. In MDAMB-231 cells, the three drugs, either alone or combined, evoked signicant cell death at both the doses and time points. All three drugs, at high dose, elicited signicant increase in caspase-3- activity in MCF-7 and MDA-MB-231 cells as compared to untreated cells. The results revealed that either MC extract or K-J alone or combined with cisplatin, can elicit signicant increase in cell death and caspase–3-activity in MCF-7 and MDA-MB-231cells as compared to untreated cells. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. Figure 1 shows light microscope morphological images of MCF-10A cells (C) without any drug treatment (control) and following treatment with 8 µg/mL of either (D) the alcohol soluble extract of M. charantia, (E) K-J or (F) cisplatin. These initial morphological studies have shown that the alcohol soluble extract and K-J, using 8 µg/mL, had little or no effect on the viability of healthy MCF-10A cell line as compared to cisplatin which reduced cell viability causing cell death.


Introduction
At present, the primary methods of treatment of cancer include surgery, radiation therapy, and systemic treatment, including chemotherapy, targeted therapy, hormonal therapy, and immunotherapy. Throughout the last half-century, cancer treatment has been characterized by highs and lows, due to the side effects, the ineffectiveness of treatment and the failed hope of full remittal and cure. Among the therapeutic options, in conjunction with surgery in the case of solid tumors, antitumor drugs and radiation have been the treatment of choice in many cases [11]. Amongst many, chemotherapeutic agents are extensively utilised for cancer, cisplatin is one of the most compelling ones. It was the rst FDA-approved platinum compound for cancer treatment in 1978 [11]. Platinum effectiveness is high primarily, but many cancer patients eventually deteriorate with cisplatin-resistant disease. Hence, drug resistance has been detected in many patients who have relapsed from cisplatin therapy and also the drug caused various side effects [12].
In contrast to classic alkylating agents, cisplatin binds with DNA to form intra-strand crosslinks and products that leads to changes in the structure of the DNA and eventually disturbs the DNA replication. Other mechanisms of cisplatin cytotoxicity comprise decreased ATPase activity, mitochondrial impairment and transformed cellular transport mechanisms. Previous studies have identi ed the DNA as a critical target for cisplatin cytotoxicity [12,13].
With the aforementioned cytotoxic side effects of this and other commercial antitumor drugs, it is of paramount importance to develop alternative therapies which can be applied either alone or in combination with other components to treat BC. Recently, immunotherapy has become an essential therapeutic alternative, and even nanotechnology has recently arrived on the scene, offering nanostructures as new therapeutic alternatives. However, side effects and remission persist [14].
One such alternative that has been used for many years to treat cancer and other diseases is plant-based extracts. Research has shown that the plant, Momordica charantia (M.charantia or MC) (Family: Cucurbitaceae) has been used as traditional medicine since ancient times for its various medicinal properties [15]. Several studies have conducted extensive research and published research and review papers on the extract of this plant and its components obtained from its fruits, seeds, leaves and stem [16][17][18][19][20].
The medicinal bene ts of MC come from the bioactive phytochemical components. These components are responsible for the physiological effects on the human immune system, protecting it from various diseases. The main groups that were identi ed and isolated from MC are proteins and triterpenes, previously published review details the MC isolated bioactive compounds [18], amongst one compound which have revealed strong biological activities is cucurbitacins, a group of cucurbitane-type triterpenoids. Currently, more than 50 triterpenoids have been isolated from MC including Kuguacin J (K-J), a triterpenoid isolated from the leaves of Momordica charantia which have demonstrated apoptosis in androgen-dependent human prostate cancer [20,21]. The unripe green fruits of M. charantia were obtained from the local market and subsequently cleaned of internal tissues and cut into small pieces [22]. Approximately one kilogram of chopped green fruit was liquidized in 70% ethanol for 5-10 min using a blender at room temperature. The ethanol sample was poured into a 2L bottle and capped. The bottle was then kept at room temperature for 6-8 weeks and mixed daily by shaking periodically. It was then ltered through Whatman lter paper (No: 4 Whatman, UK) to remove any residue. The ltered brown ethanol sample was then transferred to the 1000 mL round bottom rotating ask (Fig. 1A). The ask was then connected to the Rota-evaporator machine through a clamp. The rotating ask was then heated by partial emersion in a hot water bath at a temperature of 40°C. A typical 120 rpm speed was used for the ask rotation. The rotary-evaporated sample was then scrapped using spatula and dried overnight in an oven at 43°C. This crude ethanol soluble extract was stored at 2°C for further use (Fig. 1B).
Measurement of cytotoxicity effect of MC alcohol-soluble extract, K-J and cisplatin on the MCF-7 and MDAMB 231 breast cancer and MCF-10A healthy control cell lines using Presto-Blue Assay An amount of 30 mg of the crude alcohol-soluble extract of M. charantia was initially dissolved in 100 µl DMSO by continuous stirring and with the brief use of a sonicator water bath. This was made up to 5 mL by adding 4.5 mL of the cell medium. The alcohol-soluble crude extract stock solution was ltered using 0.22 µm sterile lters. These stock solutions were stored in a sealed tube in the fridge until required. Once removed from the fridge, the prepared alcohol-soluble extract of M. charantia solutions were gently warmed in a water bath at 37°C in order to ensure that the MC ethanol soluble extract was mixed complete in solution, before aliquoting into the concentrations of 8 µg/mL and 80 µg/mL. Similarly, either K-J or cisplatin was weighed and dissolved in 100 µl DMSO and then diluted in the incubating medium at concentrations of 8 µl/mL and 80 µl/mL. The two different concentrations of the MC alcohol soluble extract in cell medium were transferred in triplicate using a Gilson pipette to 96 well plates to give a nal volume of 200 µl to the treated cell wells. An equivalent volume of 200 µl of the medium was added to the control (untreated) well with cells. For combination treatment, cisplatin (either 8 µg/mL or 80 µg/mL) was combined with either the alcohol soluble extract or K-J using either 8 µl/mL or 80 µl/mL. In this study, both time and dose-dependent experiments were performed either with each drug individually or during combination compared to control experiments without either drug for comparison. The time-dependent experiments were done initially over a period of 24 and 48 hours to ascertain the time that produced maximal cell death. Once the times of cell death were established, both time-and dose-dependent experiments were performed during the incubation period of 24 and 48 hours either alone or when combined. At the end of the treatment with either the MC alcohol soluble extract, K-J or cisplatin, the uorescence was determined using Tecan-microtiter plate reader at excitation 570 nm and emission 610 nm [22]. Control experiments were also done using DMSO alone in the media which contained cells. The results showed that DMSO had no effect on cell death. The percentage cell viability (cell death) was calculated compared to the control subtracting the background reference employing the following equation: Measurements of caspase-3 activity in untreated and treated MCF-7 and MDAMB-231 breast cancer cell lines with either the MC ethanol extract, K-J or cisplatin The two different breast cancer cells lines were incubated with 80 µg/mL of either crude water-soluble extract of M. charantia or K-J. The induced apoptosis cell suspension contained roughly 10 7 cells. A sample of noninduced cells for a zero-time (control) also contained 10 7 cells. The induced and non-induced cells were incubated for 48 h at 37°C in 5% CO 2 atmosphere. The induced cells and the control cells were transferred to 15 ml centrifuge tube and centrifugation at 600 x g for 5 min at 4°C. The supernatant was removed by gentle aspiration for both induced and control cell suspensions. The cells were then pelleted and washed once with 1 ml of phosphate buffer solution and centrifuged at 5000 rpm for 5 min and the supernatant was completely removed by gentle aspiration. The centrifuged cell pellets were then treated with 1 X lysis buffer at a concentration of 100 µl per 10 7 cells, and the cells were incubated on ice for 15-20 min. The lysed cells were centrifuged at 16,000 to 20,000 x g for 10 to 15 min at 4°C and the supernatants were transferred to new 1 ml tube and subsequently frozen in liquid nitrogen and stored in aliquots at -70°C for further use (All the values were expressed as µmoL/min/mL.

Analysis of M. charantia for some of its contents
The green fruit of M. charantia was dried and pulverized into a powder and a known amount was dissolved in either deionized water or 70% ethanol for chemical analysis. These soluble samples were used to measure cation concentrations using ICPMS technique while antioxidant activity, vitamin C, total protein, phenolic and antioxidant contents were measured by well-established biochemical methods [23,24]. Statistical Analysis All control and test data collected from the different experiments were analyzed using Student's t -test and ANOVA test via PRISM Graph-Pad software for comparison. Data obtained were expressed as mean ± standard deviation (S.D). All experiments were performed in triplicate (n = 3). A value of (p < 0.05) was taken as a signi cant result.

Results
Cytotoxicity of MC extract, K-J and cisplatin either alone or in combination The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. Figure 1 shows light microscope morphological images of MCF-10A cells (C) without any drug treatment (control) and following treatment with 8 µg/mL of either (D) the alcohol soluble extract of M. charantia, (E) K-J or (F) cisplatin. These initial morphological studies have shown that the alcohol soluble extract and K-J, using 8 µg/mL, had little or no effect on the viability of healthy MCF-10A cell line as compared to cisplatin which reduced cell viability causing cell death.
The data from three different experiments in Fig. 1, using two different doses, were quanti ed and presented graphically as bar charts in Fig. 2 for comparison. The results show that at low and high doses (8 µg/mL and 80 µg/mL), MC ethanol extract either alone or when combined with cisplatin had no signi cant (p > 0.05) effect on the viability of healthy MCF-10A cell line at 24 and 48 hours of incubation ( Fig. 2A-D). On the other hand, K-J, a puri ed single anticancer compound from M.charantia, also had no effect on MCF-10A cell line at low dose of 8 ug/mL either alone or in combination with high and low doses of cisplatin after 24 hours of incubation ( Fig. 2A/B). When the dose of K-J was increased to 80 ug/mL, it killed MCF-7 cells signi cantly (P < 0.05) compared to untreated cells (Fig. 2C). On the other hand, cisplatin, at both low and high doses (8 µg/mL and 80 µg/mL), killed MCF-10A cells signi cantly (p < 0.05) as compared to untreated (control) cells at both 24 and 48 hours of incubation ( Fig. 2A/C). Surprisingly, when K-J at a high dose was combined with either a low and a high doses of cisplatin, it had no signi cant (p > 0.05) effect on cell viability compared to untreated (control) cells ( Fig. 1E/F). These ndings indicate that MC ethanol extract is safe to use in breast cancer therapy either alone or when combined with cisplatin. On the other hand, K-J alone at a high dose seems to be less safe just like cisplatin since both can kill healthy breast cells. Figure 3 shows the concentration-dependent cytotoxicity effect of either MC alcohol extract, K-J or cisplatin on MCF-7 breast cancer cells employing a separate (either 8 µg/mL or high 80 µg/mL) dose and combination treatment with either low or high dose (8 µg/mL or high 80 µg/mL) of cisplatin with MC ethanol extract or K-J for 24 hours and 48 hours of treatment. The results show that at a low dose, all three compounds had no effect on cell viability after 24 hours of treatment. However, when the cells were treated with a higher dose (80 µg/mL) of either of the three compounds for 24 hours, only cisplatin killed the cells signi cantly (p < 05) compared to untreated (control) cells ( Fig. 2A). During combination treatment for 24 hours, employing a low dose (8 µg/mL) of cisplatin with a high dose (80 µg/mL) of either MC ethanol extract or K-J, cell viability reduced by around 25%. However, when the dose of cisplatin was increased to 80 µg/mL and combined with 8 µg/mL of either the ethanol extract or K-J, cell death increased by about 25% after 24 hours of treatment. In contrast, when the dose of all the three compounds was increased to 80 µg/mL, there was signi cant (p < 0.05) increase in cell death (decreased cell viability) for both K-J and MC ethanol extract after 24 hours which was more or less similar to the effect of cisplatin alone (Fig. 1E).
Incubation of MCF-7 breast cancer cell line for 48 hours with 8 µg/mL with either compound individually resulted is a small, but signi cant (p < 0.05) increase in cell death as compared to untreated (control) cells. When the concentration was increase to 80 µg/mL, both the MC ethanol extract and cisplatin killed MCF-7 cells signi cantly (p < 0.05) compared to untreated cells (Fig. 2C). During combination treatments, cisplatin at either 8 µg/mL or 80 µg/mL was able to kill MCF-7 cells signi cantly (p < 0.05) when incubated with either low (8 µg/mL) or high (80 µg/mL) of the MC ethanol extract or K-J (Fig. 2D). These nding support the hypothesis that combined application is a safer option for breast cancer therapy using a low dose of cisplatin with a high dose of MC ethanol extract or K-J for 48 hours of treatment.

Measurement of caspase − 3 activity
There are a number of subcellular, cellular, molecular and structural mechanisms whereby anti-cancer drugs can induce cell death including apoptosis, autophagy, necrosis, microtubule disassembling, DNA damage, mitochondrial cytochrome release and calcium over-loading as well as others. In some cases, more than one processes occur, and however it is well known that apoptosis is the dominant type involving the formation of multi-protein complexes that provide the molecular scaffolding for the activation of the initiator caspases including caspase − 8 (extrinsic pathway), caspase − 9 (intrinsic pathway) and caspase − 3 (executioner caspase caspase-3 pathway) [25,26].
Since the MC ethanol extract, K-J and cisplatin can induce cell death of both MCF-7 and MDA-MB-231-triple negative cell lines, it was relevant to investigate the cellular mechanism(s) or pathway(s) involved in cell death. Caspase-3 activity was investigated on using caspase-3 calorimetric kit reading the absorbance using Tecan plate reader. Caspase activity was observed after treating the cells with a dose of 80 µg/mL for 48 hours. The amount of protein released during the assay was quanti ed and used to determine the caspase activity. The results are presented in Fig. 5  Chemical contents of M. charantia Following chemical analysis, the results show that the fruit is rich in proteins (2.86 ± 0.07 %), vitamin C (125 ± 1.75mg/100 g), carotenoid (1.09 ± 0.06 mg/100 g), phenolic content (2.42 ± 0.25 mg/100 g), anti-oxidant activity dry weight (44.5 ± 2.46%), including the presence of caffeic (221 ng/g) and celuic acid (623.6 ng/g) and a number of cations including sodium (7.76 ± 0.51 mg/g), potassium (0.667 ± 0.044 mg/g), calcium 28.95 ± 2.21 mg/g), magnesium (0.443 ± 0.0028 mg/g), manganese (0.42 ± 0.03 mg/g), copper (0.26 ± 0.031 mg/g) and zinc (0.99 ± 0.03 mg/g). Among the cations, the fruit had signi cantly (p < 0.05) more sodium and calcium compared to the other cations present.

Discussion
Globally, breast cancer (BC) is the most common cancer in women with the most recent published statistics indicating 2,088,849 new cases in 2018, second only to lung cancer, which corresponds to 11.6% of all new cancer cases. Research has shown that breast cancer was responsible for 626,679 deaths in 2018, representing 6.6% of cancer deaths [1]. Sadly, it is estimated that these values will continue to increase as it is estimated that by 2050, the yearly incidence of BC will reach around 3.2 million [27]. BC is caused by many risk factors including age, obesity, radiation, genetic disposition, family history of the disease which may link to inherited genes, excess alcohol intake, lack of exercise, hormone replacement therapy (HRT), personal history of BC, race/ethnicity, menstrual, breast-feeding and pregnancy history, dense breast, exposure to environmental factors, chemicals, drugs and many others [28,29]. BC can be easily prevented by eating a balanced diet, exercising regularly, and reducing alcohol intake and avoid smoking. Moreover, knowledge of the disease and regular mammogram tests can help in early diagnosis and treatment of the disease successfully resulting in a better quality of life for the victim [28].
Some forms of BC are easy to treat, whereas others are very di cult to treat, and occurrence is very prevalent. Currently, the primary forms of treatments include surgery followed by radiotherapy, chemotherapy and others such as palliative care for living well [11,12]. Most commercial drugs are given, depending on the type of the BC that is being treated, where the cancer is located in the breast and types of breast cells affected [18,[28][29][30][31]. Moreover, most of the commercial chemotherapeutic agents are not safe since they can kill normal healthy cells in the body especially at high doses [32]. As such, it is of paramount importance to look for new and safe forms of therapy. This is where the current research project comes in as an alternative form of safe treatment for BC using natural plant-based medicine either alone or in combination with a low dose of a commercial therapeutic anti-cancer agent combined with a high dose of the natural medicine or extract [18,21].
Nature is a rich source of different types of natural medicinal and phytochemical products and for years scientists have been utilizing plant-based extracts and medicine to treat different types of cancers [21,32].
Second, if single drug therapy is unable to treat the cancer, then it is important to employ combined therapy. Momordica charantia (MC) or bitter melon is a well-known plant which has been used for thousands of years as a natural medicine to treat several diseases including diabetes and cancer in icting mankind [15,33]. In two previous studies, we investigated the bene cial anticancer effects of a water-soluble extract of MC and α, β momorcharin, an isolated compound of MC, on several different cancer cell lines including 1321N1, Gos-3, U87-MG, Sk Mel, Corl − 23, Weri Rb-1 [16,17]. In light of these two previous studies, the current investigation was designed to ascertain a cost-effective and safe approach to treat BC using an ethanol soluble extract from M. charantia and compare with the effect of K-J, an isolated anticancer compound from the leaves of M. charantia and cisplatin, a commercial anticancer drug. The rationale of this study was to nd a safe dose of BC ethanol soluble extract or K-J either alone or in combination with a low dose cisplatin to treat BC without killing healthy cells and where possible, the mechanism involved in BC cell death. The discussion will now focus on the main ndings of this study and their relevance in BC therapy, especially for people in low-income countries in the world where they have to pay for healthcare [34].
The results of this study have demonstrated that the active anticancer ingredient(s) from the green succulent fruit in M. charantia is ethanol soluble and several studies have employed the same procedure in preparing the extracts which are highly e cacious in treating other diseases successfully including diabetes and cancer [15,19,35]. In the present study, both low and high doses of the MC ethanol extract and K-J had no effect on the viability of healthy MCF-10A breast cell lines compared to cisplatin which killed over 60% of the cells after 24 hours of treatment. However, during 48 hours of treatment both doses of MC ethanol extract seemed to enhance cell viability slightly compared to K-J and cisplatin which killed MCF-10A cells with cisplatin being more toxic at both low and high doses compared to K-J at the higher concentration. Surprisingly, when cisplatin was combined with either MC ethanol extract or K-J, employing both doses, they had little or no signi cant effect on MCF-10A cell at both 24 and 48 hours of treatment. The results have clearly demonstrated that MC ethanol extract is safe to use in breast cancer therapy either alone or when combined with cisplatin since it does not kill the healthy cells even during 48 hours of treatment and rather reduce the toxic effects of cisplatin. In contrast, K-J alone, especially at high dose and longer treatment time, can kill the healthy cells. Interestingly, both MC ethanol extract and K-J seem to exert a protecting effect on the healthy cells against the adverse effect of cisplatin at both low and high doses at either 24 or 48 hours of treatment (( Fig. 2B/D). The current results are in agreement with two previous studies which revealed that that the watersoluble extract of MC had no cytotoxic effect on healthy cell lines [16,17]. MCF-7 is a human breast cancer cell line was isolated in 1970 from a Caucasian woman with metastasis and it has estrogen, progesterone and glucocorticoid receptors and as such it is a well-established in vitro as a cell line model to study the effects of drugs on cell viability [36]. The results of this study have shown that either the MC ethanol extract or K-J had no signi cant effect at a low dose on MCF-7 cell viability at 24 hours of treatment. However, at a higher dose of 80 µg/mL and 24 hours of treatment, MC ethanol extract seemed to enhance MCF-7 cell proliferation compared to K-J which reduced cell viability slightly (Fig. 3A). When a high dose of cisplatin was combined with a high dose of either MC ethanol extract or K-J, they elicited more than 80% of cell death compared to untreated (control) cell at 24 hours (Fig. 3B). This effect was more or less the same as with cisplatin alone. However, during 48 hours of treatment with a low dose, either MC ethanol extract, K-J or cisplatin reduced cell viability by about 25%. At a high dose and during 48 hours of treatment, K-J evoked only a small effect on cell death compared to the MC ethanol which induced about 50% of cell and cisplatin which killed more than 90% of the cancer cells (Fig. 3C). Interestingly, when either a low or a high dose of cisplatin was combined with a high dose of either MC ethanol extract or K-J for 48 hours, they evoked signi cant increases in MCF-7 cell death. Together, the main results from this part of the study revealed that a low dose of cisplatin combined with a high dose of either MC ethanol extract or K-J could elicit a synergistic effect on MCF-7 cell death after 48 hours of treatment. As such, the nding supports the hypothesis that combined application is a safer option for breast cancer therapy using a low dose of cisplatin and a high dose of MC ethanol extract or K-J. These results are in total agreement with related studies which show that bitter melon extract or juice alone can reduced the viability of MCF-7 and other cancer cell lines especially at a longer duration of treatment [19,37].
Triple negative breast cancer (TNBC) represents almost 10-20% of all breast cancers globally. This is due to the absence of estrogen and progesterone receptors and HER2 proteins. TNBC cells do not respond to hormone therapy and as such, new and novel medicines are required to prevent their growth and subsequent death, but at the same time leaving healthy cells intact [38]. The main objective of this study was to nd a natural product approach using plant-based medicine to treat TNBC. MDA-MB-231 is a highly aggressive, invasive and poorly differentiated triple-negative breast cancer (TNBC) cell line as it lacks ER and PR expression, as well as HER2 (human epidermal growth factor receptor 2) ampli cation were used in this study.
The results presented in Fig. 4 are highly signi cant and demonstrated that either the MC ethanol soluble extract, K-J or cisplatin either alone or in combination at low and high doses could kill MDAMB-231 TNBC cells by more than 90% after both 24 and 48 hours of treatment, making the MC ethanol extract and possible K-J at low concentration as very safe options in TNBC therapy. The present results are in agreement with a related study which employed 2% of bitter melon fruit juice (v/v) alone on both MCF-7 and MDAMB-231using 12, 24 and 48 hours of treatment [19,37,39]. In addition to individual application of either MC ethanol extract, K-J or cisplatin like other studies, the present study also investigated combined therapy employing cisplatin with either MC ethanol extract or K-J.
It is now known that different anticancer drugs exert their killing effect via different cellular, subcellular and molecular mechanisms involving autophagy, cellular calcium overloading, necrosis, microtubule disassembling, DNA damage, cell cycle arrest, suppression of c-Met signaling, mitochondrial cytochrome release and apoptosis [25,26]. Programmed cell death or apoptosis occurs naturally in cells and many drugs kill cancer cells by this process. However, the underlying mechanisms of the growth inhibitory effects of M charantia extract and K-J in breast cancer cell remains poorly understood. In some cases, more than one processes occur, however, it is known that apoptosis is the dominant type involving the formation of multiprotein complexes that provide the molecular scaffolding for the activation of the initiator caspases including caspase − 8 (extrinsic pathway), caspase − 9 (intrinsic pathway) and caspase − 3 (executioner caspase caspase-3 pathway [25,26,40,41]. The results presented in Fig. 5 show that in both MDAMB-231 and MCF-7 cell lines, MC ethanol extract, K-J and cisplatin were able to increase caspase-3 activity by 82%, 75% and 76%, respectively, indicating that all three agents are inducing breast cancer cell death through the activation of caspase-3 pathway. In two previous studies [17,22], we had shown that a water soluble of M. charantia and α, β momorcharin, an isolated compound from bitter melon, were able to kill several cancer cell lines via apoptosis and cellular calcium overloading [16,17]. It is tempting to suggest that other intracellular signally pathways may be involved in MDAMB-231 and MCF-7 cell death but this warrants further experiments. The results of this study have revealed that M. charantia is rich in a number of cations, especially sodium and calcium, proteins, phenols, antioxidants and other compounds. Other studies have reported more than 250 bioactive compounds with medicinal and nutritious characteristics in M. charantia [41][42][43][44][45]. The question which now arises is: how MC ethanol extract and K-J exerts their lethal effect when applied extracellularly to cells in vitro. Control experiments have shown that the dose of DMSO employed to dissolve either K-J, cisplatin or the MC extract had no effect on all three cell lines employed in this study. It is possible that either K-J or the active agent (s) in MC extract is lipid soluble and as such they can enter the cells very easily to activate different signaling pathways to elicit cell death via apoptosis [19,21]. Currently, there is no evidence of receptor -mediated effect of extract of M. charantia or its isolated compounds.

Conclusion
The results of this study have clearly demonstrated that MC alcohol soluble extract had no effect on healthy human mammary epithelial MCF-10A cells as compared to cisplatin at all concentrations and K-J at a high