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 . 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 . 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 .
Some forms of BC are easy to treat, whereas others are very difficult 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, 28–31]. Moreover, most of the commercial chemotherapeutic agents are not safe since they can kill normal healthy cells in the body especially at high doses . 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 inflicting mankind [15, 33]. In two previous studies, we investigated the beneficial 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 find 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 findings 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 .
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 efficacious 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 significant 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 water-soluble 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 . The results of this study have shown that either the MC ethanol extract or K-J had no significant 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 significant 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 finding 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 . The main objective of this study was to find 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) amplification were used in this study.
The results presented in Fig. 4 are highly significant 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 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, 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 [41–45]. 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.