Evaluation of Anticancer Activity of Neolamarckia cadamba Leaves and its Metabolite Profile

Neolamarckia cadamba (NC) leaf is traditionally used for the treatment of breast cancer, however, this claim is unverified. This study aimed to evaluate the anti-cancer activities of NC leaf ethanol extract on breast cancer cell line (MCF-7 cells) using in vitro cell viability, cytotoxicity, and gene expression assays followed by gas chromatography analysis. Results revealed inhibition concentration (IC 50 ) against MCF-7 at 0.2 mg/mL. The extract exerted a dose and time-dependent inhibitory effect against MCF-7 cells. The cell cycle assay showed that the extract arrested MCF-7 cells in the G0/G1 phase, and apoptosis was observed after 72 hours by Annexin-V assay. The gene expression assay revealed that the cell cycle arrest was associated with the down-regulation of CDK2 and subsequent up-regulation of p21 and cyclin E . The extract induced apoptosis via the mediation of the mitochondrial cell death pathways. Chromatography analysis revealed the contribution of d-pinitol and myo-inositol to the activity observed as the two major bioactive compounds. Overall, the study demonstrated that NC exerts anti-cancer effects on MCF-7 human breast cancer cells through induction of apoptosis and cell cycle arrest thus justifying its traditional use for breast cancer treatment in Malaysia.


Introduction
Breast cancer is the most commonly occurring cancer in women and the second most common cancer overall in terms of fatality after lung cancer 1 . The rate of cases of breast cancer patients is increasing every year, both in developed and developing countries 2 . It is estimated that 26 million new cancer cases and 17 million cancer deaths are likely to occur per year globally by 2030 3 . In Asia alone, the incident in 2008 to 2030 is estimated to rise from 6.1 million to 10.7 million cancer cases and 4.1 million to 7.5 million deaths 4 . Malaysia is one of the Asian countries with an increased prevalence of breast cancer cases 5 affecting both genders but increased exponentially with age in women and thus accounted for the leading cause of cancerrelated death among women in Malaysia 6 . Surgery, radiotherapy, chemotherapy, hormonal therapy, and gene-targeted therapy have been used in treating breast cancer patients. The advances of current treatments and detecting methods have attributed to the increase of the survival rate 7 . Unfortunately, the treatment causes short-term and long-term side effects for the patients. Moreover, studies have demonstrated that the burden of the cost related to cancer treatment is rising 7,8 . Therefore, the use of natural sources as an alternative, effective, and noninvasive entities to treat cancer is warranted.
According to the World Health Organization (WHO), there are many countries including developing countries where people are still using plants and natural source products for therapeutic purposes 9 . About 60% of anticancer agents in the world have been originated from 4 natural sources 10 . The natural-derived compounds are readily available, generally more tolerated, and considered non-toxic to normal human cells 11 .
Neolamarckia cadamba (family: Rubiaceae) is a tree that is used for treating various illnesses. This underexplored evergreen tropical plant has been widely used in the Ayurvedic medicine system of India in treating eye infection, skin diseases, dyspepsia, gum related troubles, stomatitis, cough, fever, anemia, blood disorders, and stomach pain 12,13 . The leaves of this plant have been shown to possess several pharmacological properties including antioxidant 14,15 , antidiabetic 16 , antitumor 17 , anti-inflammatory, antipyretic, analgesic 18 , antimicrobial 19 , and anticancer effect 20 . Furthermore, the N. cadamba leaves have also been used as a topical application for the treatment of breast cancer. However, this traditional application has never been scientifically investigated to verify the claims. Therefore, the purpose of the study was to evaluate the anticancer effects of N. cadamba leaves extract on breast cancer cell line (MCF-7) with emphasis on the mechanism of action, i.e., apoptosis induction and cell cycle arrest. As well, characterization of N. cadamba leaves extract was carried out using gas chromatographymass spectrometry (GCMS) approach to identify bioactive compounds.

Results
Gas Chromatography-Mass Spectrometry (GCMS) analysis of NC. Fig. S1 and Table 1 present the putative compounds present in the NC leaves 80% ethanol extract. The chromatogram showed the highest peak area corresponding to d-pinitol, followed by myoinositol, oleic acid, hexadecanoic acid, and octadecanoic acid. Table 1 presents tentative identified compounds' retention time, % area peak, molecular formula, and similarity index. Breast cancer cells (MCF-7) viability after NC treatment. The anticancer activity of NC leaves 80% ethanol extract was investigated against MCF-7 cells. The cell viability was directly tested using the trypan blue exclusion assay method where the dead cells were stained blue. Fig.   1 (A) shows the anti-proliferative activity of NC leaves 80% ethanol extract on MCF-7 cells after 72 hours of incubation. The finding revealed an IC 50 of 206.0 ± 3.4 µg/mL. The cell viability was found to decrease with the increasing concentration of the extract used. Fig. 1      caspase-9 and caspase-7 were calculated by measuring the fold change with GAPDH and β-actin as a reference gene in untreated cells, whose ratio was equal to 1. Data presented in mean ± 13 standard deviation of triplicates of the experiment. * indicates a significant difference when p < 0.05.

Discussion
Throughout the whole world, breast cancer is affecting women the most compared to other types of cancers with a high incidence and mortality rate 21,22 . Additionally, there are some undocumented and unverified claims for N. cadamba leaf that it is used to treat breast cancer in different parts of Malaysia, however, no scientific study using breast cancer cell lines has ever been carried out to confirm its potential in the management of breast cancer. Hence, this study was performed to verify N. cadamba leaf extract as a potential anticancer agent with minimal toxicities. In this study, NC leaf's 80% ethanol extract was initially characterized with GCMS and then assessed for its in vitro anticancer properties and mechanism of action using MCF- Myo-inositol has been currently used clinically to treat polycystic ovary syndrome (PCOS) 27 .
Hexadecanoic acid was identified in our study as another bioactive compound and identified earlier from methanol (44.88%) and hexane (17.96%) extracts of N. cadamba leaves.
Additionally, octadecanoic acid was also found in methanol, ethyl acetate, chloroform, and hexane extract 28  and down-regulated CDK2 support the G0/G1 cell cycle arrest in the previous data. Interestingly, cyclin E in the present study was found to be up-regulated, which suggested that cyclin E independently binds to CDK2 for halting the cell cycle. It was suggested that inhibition of breast cancer cell MCF-7 proliferation may be linked to the up-regulation of cyclin E and p21 as well as down-regulation of CDK2 upon NC extract treatment. In addition, p21 was found to be associated with apoptosis. A study found the cytochrome c blockage and caspase-3 inactivation in HCT116 p21-/-but not in HCT p53-/-and HCT116 Bax-/-of colon cancer cells after treatment with curcumin. The study also found a reduced expression of Apaf-1 in the HCT116 p21-/-cells whereas it was not expressed in wild type 56 . Cytochrome c release and Apaf-1 are required for caspase-9 activation of the intrinsic apoptosis pathway 57 , which in turn activate executioner caspases, including caspase-3 and caspase-7. Based on the present findings, it is suggested that p21 up-regulated expression may be involved in apoptosis activities by promoting the cytochrome c release to inhibit the proliferation of MCF-7 cells upon treatment.

Conclusions
The present data suggested that NC  to NC and were used as a negative control.
Cell Cycle Analysis. MCF-7 cells collected from the well were prepared in triplicate prior to staining with cell cycle reagent following the manufacturer's instructions. The PI stained cells were observed using a flow cytometer Guava EasyCyte system 58 .
Apoptosis Analysis Using Annexin V. Briefly, MCF-7 cells were exposed to NC for 72 h. The  (Table S1).

Statistical analysis. Statistical and the IC 50 values (half maximal inhibitory concentration)
calculations were carried out using GraphPad Prism software version 6. The flow cytometry analysis and gene expression data results were determined using multiple t-tests (Holm-Sidak method). All data were displayed as means ± SD (standard deviation). * signifies the significant difference when p < 0.05.