In Vitro Bioassay-Guided Identification of Anticancer Properties

pharmaceuticals Article In Vitro Bioassay-Guided Identification of Anticancer Properties from Moringa oleifera Lam. Leaf against the MDA-MB-231 Cell Line Prapakorn Wisitpongpun 1, Nungruthai Suphrom 2, Pachuen Potup 1, Nitra Nuengchamnong 3, Philip C. Calder 4 and Kanchana Usuwanthim 1,* 1 Cellular and Molecular Immunology Research Unit (CMIRU), Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand; [email protected] (P.W.); [email protected] (P.P.) 2 Department of Chemistry, Faculty of Science and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 65000, Thailand; [email protected] 3 Science Laboratory Centre, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand; [email protected] 4 School of Human Development and Health, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK; [email protected] * Correspondence: [email protected]; Tel.: +66-89-780-3878 Received: 12 October 2020; Accepted: 4 December 2020; Published: 15 December 2020 Abstract: Moringa oleifera Lam. (MO) is a medicinal plant distributed across the Middle East, Asia, and Africa. MO has been used in the traditional treatment of various diseases including cancer. This study aimed to perform bioassay-guided fractionation and identification of bioactive compounds from MO leaf against MDA-MB-231 breast cancer cells. MO leaf was sequentially extracted with hexane, ethyl acetate (EtOAc), and ethanol. The most effective extract was subjected to fractionation. MO extract and its derived fractions were continuously screened for anti-cancer activities. The strongest fraction was selected for re-fractionation and identification of bioactive compounds using LC-ESI-QTOF-MS/MS analysis. The best anticancer activities were related to the fraction no. 7-derived crude EtOAc extract. This fraction significantly reduced cell viability and clonogenic growth and increased cells apoptosis. Moreover, sub-fraction no. 7.7-derived fraction no. 7 was selected for the identification of bioactive compounds. There were 10 candidate compounds tentatively identified by LC-ESI-QTOF-MS. Three of identified compounds (7-octenoic acid, oleamide, and 1-phenyl-2-pentanol) showed anticancer activities by inducing cell cycle arrest and triggering apoptosis through suppressed Bcl-2 expression which subsequently promotes activation of caspase 3, indicators for the apoptosis pathway. This study identified 10 candidate compounds that may have potential in the field of anticancer substances. Keywords: Moringa oleifera; triple negative breast cancer; MDA-MB-231; oleamide; 7-octenoic acid; 1-phenyl-2-pentanol; LC-ESI-QTOF-MS/MS 1. Introduction Breast cancer is the most frequently diagnosed cancer in women and is one of the leading causes of cancer death for women. Worldwide, over 1.3 million cases of invasive breast cancer are diagnosed and more than 450,000 women die from breast cancer annually [1,2]. Breast cancer is a heterogeneous disease with distinct pathological entities and therefore needs diverse therapeutic interventions. Approximately 10–20% of invasive breast cancers are triple-negative breast cancer (TNBC) which is defined as the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor (HER) 2 [3]. TNBC is associated with poor prognosis, and the survival after metastasis is Pharmaceuticals 2020, 13, 464; doi:10.3390/ph13120464 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 464 2 of 20 worse compared to other subtypes [3,4]. Unfortunately, due to lack of targeted therapy, radiotherapy and chemotherapy remain the only recommended option for TNBC [3,5,6]. Therefore, it is urgent to develop new alternative therapies for TNBC. Moringa oleifera Lam. (MO) is a highly valued medicinal plant native to India and now distributed widely across the Middle East, Africa, and Asia, including Thailand. It belongs to the family Moringaceae and is commonly referred to as the “Drumstick tree” [7–9]. All parts of the MO possess medicinal properties, and the leaf has the highest nutritional value [10]. MO leaves (MOL) contain high levels of vitamins C and A, potassium, calcium, iron, and proteins. Additionally, the leaves contain phytochemicals like carotenoids, alkaloids, flavonoids, and amino acids, such as cystine, lysine, methionine, and tryptophan [10–12]. In vitro and in vivo studies have demonstrated that MOL extract has various biological activities and therapeutic effects, including cardioprotective [13], hypocholesterolaemic [14], neuroprotective [15], anti-inflammatory [16], antioxidant [17–19], anti-hypertensive [20,21], antidiabetic [22,23], antibacterial [24,25], immunomodulatory [26,27], and anticancer properties [28–30]. With regard to the anticancer properties, MOL extracts have been shown to disrupt the proliferation of different cancer cell lines, for example the hot aqueous MOL extract induced apoptosis in human lung cancer A549 cells by affecting mitochondrial viability in a ROS-dependent manner [29]. It also can induced cell cycle arrest in murine B16F10 melanoma cells by increasing of p53, p21WAF1/Cip1 and p27Kip1 proteins [30]. The methanolic MOL extract induced apoptosis in human cervical cancer HeLa cells by promoting DNA fragmentation [31]. Moreover, oral administration of cold aqueous MOL extract induced apoptosis of human hepatocellular carcinoma HepG2 cells by affecting the apoptosis-related proteins Bcl-2 and caspase-3 [32]. In breast cancer, most of the previous studies of MOL extracts have used the MCF-7 cell line, a hormone receptor-positive breast cancer model [32,33]. Only one study has investigated the effect of MOL on the TNBC cell line, MDA-MB-231: the authors found that ethanol MOL extract arrested these cells at the G2/M phase and effectively induced apoptosis [32]. However, the underlying mechanism and the bioactive compounds involved have not yet been fully elucidated. In this study, we investigated the in vitro anticancer effect of MOL extract against MDA-MB-231 cells by bioassay-guided fractionation, and identification of potential bioactive compounds responsible for the observed effects. We found that MOL extracts and derived fractions showed a remarkable anticancer activity with a significant decrease of cell viability, striking reduction of colony formation, and induction of apoptosis and cell cycle arrest at the G2/M phase. Additionally, we tentatively identified 10 bioactive compounds by LC-ESI-QTOF-MS analysis. Three of them (7-octenoic acid, oleamide, and 1-phenyl-2-pentanol) can arrest the cell cycle and induce apoptosis of MDA-MB-231 cells. We also demonstrated the anticancer properties of oleamide on human myelogenous leukemia cell K562 and human squamous cell carcinoma SCC-15. 2. Results 2.1. Screening for Cytotoxic Effects of Crude Hexane, EtOAc, and EtOH Extracts of MOL To compare the cytotoxic effects of crude MOL extracts, MDA-MB-231 cells were plated into 96-well plates and incubated with serial concentrations of the crude hexane, crude EtOAc, and crude ethanolic (EtOH) extracts for 24 h. Cell viability was assessed using the MTT assay. Crude EtOAc extract exhibited the lowest IC50 value (233.5 µg/mL) followed by crude EtOH extract (241.1 µg/mL), and crude hexane extract (342.6 µg/mL), respectively (Figure1A,B). The crude EtOAc MOL extract was subjected to further fractionation. Pharmaceuticals 2020, 13, 464 3 of 20 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 3 of 20 (A) (B) FigureFigure 1. 1.E Effectsffects ofof crudecrude hexane,hexane, EtOAc,EtOAc, and EtOH extracts of MOL MOL on on the the viability viability of of MDA MDA-MB-231-MB-231 cells.cells. ( A(A)) Cells Cells were were plated plated into into 96-well 96-well plates plates and and incubated incubated with with each each extract extract for 24for h. 24 IC h.50 ICvalues50 values were calculatedwere calculated using GraphPad using GraphPad Prism 6.0 Prism software. 6.0 software Each dot. representsEach dot represents mean SEM mean of three ± SEM independent of three ± experiments.independent ( experimentsB) Cell viability. (B) after Cell treatmentviability after with treatment each extract with at each IC50 concentrationsextract at IC50 concentrations for 24 h. One-way for ANOVA24 h. One was-way performed ANOVA with was multiple performed comparison with multiple correction comparison (Dunnett correction test). Data (Dunnett represent test). the Data mean representSEM of threethe mean independent ± SEM experiments.of three independent IC50, the experiments. half-maximal IC50, inhibitory the half concentration;-maximal inhibitory EtOAc, ± ethylconcentration; acetate; EtOH, EtOAc, ethanol. ethyl acetate; EtOH, ethanol. 2.2.2.2. Cytotoxic Cytotoxic EEffffectsects ofof EtOAcEtOAc ExtractExtract ofof MOLMOL andand Itsits D Derivederived F Fractionsractions on on MDA MDA-MB-231-MB-231 Cells Cells ToTo examine examine the the inhibitory inhibitory eeffectsffects ofof crudecrude EtOAcEtOAc extract and its its derived derived fractions, fractions, MDA MDA-MB-231-MB-231 cellscells were were incubatedincubated with crude crude EtOAc EtOAc extract extract and and fractions fractions no. no. 1–11 1–11 at concentrations at concentrations of 75, of 100, 75, and 100, and150 150μg/mLµg/mL for 24 for h. 24 Cell h. Cellviability viability was wasdetermined determined using using the MTT the MTTassay assay (Figure (Figure 2A). 2CellA). viabil Cell viabilityity was wassignificantly significantly decreased decreased after after treatment treatment with with crude crude EtOAc

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