Goniothalamus lanceolatus extract inhibits the growth of human ovarian cancer cells through migration suppression and apoptosis induction

Nasibah Razali1,2, Norodiyah Othman3,4, Nur Hilwani Ismail1*, Nur Vicky Bihud1,2, Nor Hadiani Ismail1,2, Farida Zuraina Mohd Yusof1,5 1School of Biological Sciences, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), Shah Alam, Malaysia. 2Atta-ur-Rahman Institute for Natural Product Discovery (AuRIns), Universiti Teknologi MARA (UiTM), Puncak Alam, Malaysia. 3Haematology Unit, Cancer Research Centre, Institute for Medical Research (IMR), National Institutes of Health (NIH) Complex, Shah Alam, Malaysia. 4Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam, Malaysia. 5Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM), Puncak Alam, Malaysia.


INTRODUCTION
Ovarian cancer is known to be one of the most aggressive gynecological cancers causing death in women. An estimated 240,000 new cases have been recorded in 2018, leaving ovarian cancer at ranking number seven as the most common cancer among women worldwide (Henderson et al., 2018). Ovarian cancer cases have been mostly identified in the advanced stages due to their asymptomatic characteristics (Jayson et al., 2014). Nonetheless, some patients may experience bloating, pelvic pain, frequent urination, and changes in bowel movements (Jayson et al., 2014). Cisplatin is a standard drug used for ovarian cancer treatment; however, the cell resistance has been shown to cause relapse in some patients following initial treatment (Noviyani et al., 2019). This substantially reduced the effectiveness and outcome of chemotherapy and resulted in less than 30% for a 5-year survival rate (Cornelison et al., 2017). Hence, it is extremely crucial to discover alternative approaches to treat ovarian cancer, especially in overcoming cisplatin resistance with minimal side effects on patients.
Goniothalamus is one of the largest paleotropical genera of a plant in the Annonaceae family, comprising over 130 species and is mainly distributed in the Malesian floristic region, comprising Malaysia, Borneo, New Guinea, Sumatra, and Philippines (Saunders, 2003;Yang et al., 2020). Goniothalamin (GTN) as the main bioactive compounds has been reported to exert a cytotoxic effect and anticancer properties in human promyelocytic leukemia (HL-60), leukemia monocytic (U937) (Petsophonsakul et al., 2013), squamous cell carcinoma (H400 cells) (Li et al., 2016), and cervical cancer (HeLa) cell lines (Sophonnithiprasert et al., 2017). The compound is capable of limiting the development of cancer cells by increasing the number of dead cancer cells without producing inflammatory reactions on the healthy cells (Seyed et al., 2014;Umar-Tsafe et al., 2004).
Goniothalamus lanceolatus (GL) is native to the rainforest in Sarawak, Malaysia, locally known as "Getimang," and is believed by the Iban people to repel mosquitoes due to the pungent scent and thick smoke when burned. It has also been used to treat cold, fever, and skin diseases by the indigenous people as alternative medicine (Wiart, 2007). The isolation of the pure compounds and the cytotoxicity chemical classification of the active compounds are currently ongoing. The newly discovered alkaloids of goniolanceolactam and 2-acetyl-3-amino-1,4naphthoquinone isolated from the dichloromethane (DCM) root extract were reported to have a cytotoxic effect on human colon and lung cancer cells (Rasol et al., 2018a). Moreover, eight new bis-styryllactones and goniolanceolatins A−H, and four known styryllactones with a rare (6S)-styrylpyrone and (1S)pyranopyrone moieties were also isolated from the GL (Bihud et al., 2019). These compounds have been reported to be associated with antimalarial properties against chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum (Kaharudin et al., 2020). Apart from these findings, antiproliferative and anticancer activities of GL on ovarian cancer cells have not yet been reported. Therefore, this study aims to investigate the cell viability, migration, and apoptotic effects of GL extracts in three different solvents on chemosensitive and chemoresistant ovarian cancer cells.

Preparation of plant extracts
GL leaves and roots were collected from Sematan Sarawak, Malaysia, in June 2012, described as FBAUMS 108 by a botanist Professor Dr. Kamaruddin Mat Salleh (Universiti Kebangsaan Malaysia). The leaves and the roots of the plants were oven-dried before being subjected to extraction using DCM, hexane, and methanol solvents. The solvents were removed under a vacuum condition using a rotary evaporator resulting in a total of six extracts. The extracts were purified using high-performance liquid chromatography (HPLC) and recycling HPLC leaving the isolation of a series of compounds (Rasol et al., 2018b). The extracts were reconstituted in dimethyl sulfoxide (DMSO) to form a 20 mg/ml stock solution and preserved for further use at -20°C.

Cell culture
Two ovarian cancer cell lines, namely, PEO1 (chemosensitive) and PEO4 (chemoresistant), were provided by Dr. Normala Abd. Latip of the Atta-ur-Rahman Institute for Natural Product Discovery, which was previously purchased from the European Cell Culture Collection (ECACC, UK). The ovarian cancer cell lines were derived from ascites of the same patient who received cisplatin, 5-fluorouracil, and chlorambucil treatment. PEO1 was sensitive to cisplatin taken when the patient was able to respond to the treatments. Meanwhile, PEO4 was taken 10 months later after the patients developed resistance toward cisplatin chemotherapy. RPMI-1640 complete media containing 10% fetal bovine serum and 1% penicillin-streptomycin were used for cell culture and incubated in a humidified atmosphere containing 5% CO 2 at 37°C. Seeding of monolayer cell cultures was performed in a 75 cm 2 tissue culture flask and maintained up to 70% confluency prior to treatment.

MTT assay
Cell viability and inhibition were determined using the MTT assay. Briefly, each cell line was plated in a 96-well plate seeded with 2.0 × 10 4 cells/well followed by 24 hours cell treatment with different extract concentrations at two-fold serial dilutions (0-1,000 μg/ml). Cell viability was determined by adding 5 mg/ ml MTT solution to each well. After 4 hours of incubation, 50 μl of DMSO was added to each well as a stop solution and incubated for 10 minutes at room temperature on a shaker. The absorbance was measured at 570 nm wavelength using a microplate reader (BMG Labtech, Offenburg, Germany). A graph of cell viability percentage versus extract concentration was plotted and the inhibitory concentration (IC 50 ) was determined from the concentration-response inhibition curves. Cells without treatment (untreated cells) were used as negative control (NC) while cells treated with cisplatin were used as a positive control. The extracts with the lowest IC 50 value were selected for subsequent testing.

Scratch assay
The migration rate of both cell lines was determined using a scratch assay. Briefly, the cells were cultured in a six-well plate until it reached 70% confluency. The cells were then treated with the extracts and cisplatin (positive control). A scratch was introduced using a sterile pipette tip followed by 16, 24, 48, and 72 hours incubation period. The closure of the scratched wounds was considered as the completion of the migration process. The migration rate of both cells was determined by comparing the migration rate of the treated cells with the untreated cells (negative control). The following formula was used to calculate the migration rate (Warden et al., 2020): Rate of migration (%) = Area of scratch at 0 hour − area of scratch at (n) hour Area of scratch at 0 hour ×100 where n = time of incubation.

Cell apoptosis assay by flow cytometry
Cell apoptosis was assessed by the BD Pharmigen TM FITC Annexin V (AV) Apoptosis Detection Kit as per the manufacturer's protocol. A cell density of 2 × 10 6 cells/well was seeded in a six-well plate, followed by cell treatment and incubation for 24 and 48 hours. The cells were harvested by trypsinization, washed three times with cold phosphate-buffered saline (PBS), pelleted, and resuspend pellet in a 1× binding buffer. The suspension was then stained with 5 µl FITC-conjugated AV and 5 µl propidium iodide (PI) and incubated for 15 minutes at room temperature in a dark space, followed by the addition of 400 µl binding buffer. Finally, the samples were analyzed by a flow cytometer (FACSCalibur, Becton-Dickinson, San Jose, CA). The cell distribution was analyzed using CellQuest™ software (Becton-Dickinson) within 1 hour of staining in which 10,000 cells were collected for each sample. Samples with different cell groups were separated in either early or late apoptosis, marked by AV while necrosis was marked by PI and presented in a separate plot group.

Statistical analysis
The statistical analysis test was carried out using GraphPad Prism Software version 6.0. Data are presented as mean ± standard mean error (SEM). The statistical comparative analysis was performed using one-way analysis of variance, followed by Tukey's post-hoc test. A p-value of less than 0.05 was considered to be significant. Figure 1 shows the cell viability in PEO1 after 24 hours of treatment with different concentrations of the extracts. DCM and methanol leaves extract significantly reduced the Figure 1. Cell viability of the PEO1 cell line treated with GL leaf and root extracts. Each leaf and root extract was dissolved in DCM, hexane, and methanol and treated at different concentrations for 24 hours in triplicate. All data are expressed as mean ± SEM. *p < 0.05; **p < 0.01,; ****p < 0.0001 versus negative control (NC). cell viability (p < 0.05) at the highest concentration (1,000 μg/ ml) after 24 hours of treatment. A significantly decreased cell viability to below 20% was observed in DCM roots extract at 250 μg/ml and above concentration while methanol extract decreased the cell viability at around 50% at a concentration of 62.5-1,000 μg/ml. Figure 2 shows the cell viability in PEO4 after 24 hours of treatment with different concentrations of leaf and root extracts of GL. The viability of the cells decreased significantly by the DCM leaves extract at a concentration of 1,000 μg/ml compared to the hexane leaves extract at 500 μg/ml. In addition, the DCM root extracts significantly decreased the cell viability (p < 0.0001) to less than 20% at 125-1,000 μg/ml concentration. The methanol root extract showed a significant reduction of less than 50% cell viability with a concentration of 7.81-1,000 μg/ml compared to the control group. Figure 3 shows the cell viability in both cell lines after 24 hours of cisplatin treatment. In the PEO1 cell line, the viability decreased significantly at 1.56 μM and almost 90% inhibition was observed at 6.25 μM as shown in Figure 3a. A nonsignificant cell viability decrease was seen in PEO4 after cisplatin treatment except at 3.125 μM as shown in Figure 3b.

Effect of GL on cell migration
The extract with the lowest IC 50 value from the cell viability analysis for each cell line was used to assess cell migration. PEO1 cells were treated with methanolic roots extract at IC 20, IC 50 , and IC 80 concentrations at 16, 24, 48, and 72 hours time points, respectively ( Fig. 4). At 72 hours, cell migration decreased from 21.37% at IC 20 to 18.65% at IC 80. In comparison, untreated cells showed a 76.63% cell migration. Cisplatin was able to fully inhibit PEO1 cell migration (-2.49%).
PEO4 cells were treated with DCM leaf extracts at IC 20, IC 50 , and IC 80 concentrations and incubated at four different time points (Fig. 5). At 72 hours, cell migration was suppressed to 16.73% at IC 80 compared to 31.99% in the untreated cells. Meanwhile, the migration was almost the same in the untreated cells when treated with cisplatin (30.3%).

Effects of GL in inducing apoptosis
The effects of the extracts on inducing apoptosis using flow cytometry were examined at 24 and 48 hours. In PEO1, cells treated with methanol root extracts at IC 20 and IC 80 showed increased early apoptosis to 6.76% and 13.52%, respectively, after 24 hours. The cells at IC 20 and IC 80 shifted to late apoptosis at 4.48% and 6.64%, respectively. Early apoptosis significantly decreased while the cells shift toward late apoptosis after 48 hours of treatment as shown in Figure 6A-C.
In PEO4 cells, DCM leaves extract significantly increased early apoptosis at IC 20 to 42.94% but dropped after 24 hours to 24.63% at IC 80 . However, early apoptosis at IC 20 and IC 80 increased to 16.28% and 26.5%, respectively, after 48 hours. Late apoptosis increased significantly at both dose-dependent times as shown in Figure 7A-C.

DISCUSSION
Cell viability assay following the administration of GL methanol root extract gave the lowest IC 50 value at 31.40 ± 0.77 µg/ml in PEO1. However, a higher concentration at 72.51 ± 0.27 µg/ml was needed to inhibit PEO4. The results showed that methanol root extract exhibited a higher cytotoxic effect against chemosensitive cells compared to the chemoresistant cells. However, DCM leaves extract was found to be the most cytotoxic to the chemoresistant cells, PEO4 (IC 50 : 22.02 ± 0.52 µg/ml) compared to the chemosensitive cells and PEO1 (IC 50 : 166.10 ± 0.15 µg/ml). The selection of chemosensitive and chemoresistant cells was done to compare the effects of the plant extracts on cells toward different chemosensitivity profiles.
Chemoresistance in the ovarian cancer cells was initiated by the cell adaptation using different energy pathways such as glycolysis or oxidative phosphorylation (Dar et al., 2017). Chemosensitive ovarian cancer cell lines (A2780 and PEO1) appeared to exhibit a glycolytic phenotype and could not tolerate  glucose deprivation. However, the chemoresistant counterparts (C200 and PEO4) showed a high metabolically active phenotype and the ability to switch between oxidative phosphorylation and glycolysis (Dar et al., 2017). Cell proliferation can be suppressed by apoptosis through inhibition of signal transducers and transcription activator 3 and activation of Janus kinase 2 (Jo et al., 2012). Alonezi et al. (2016) demonstrated that chemoresistant cells (A2780CR) were highly sensitive to melittin and had a slightly lower IC 50 value of 4.5 μg/ml compared to 6.8 μg/ml in the chemosensitive cells (A2780). The GL leaves and roots were extracted using different solvents (DCM, hexane, and methanol) of different polarities to obtain different groups of active compounds. DCM is a moderate polar organochloride solvent that is widely used to dissolve most organic compounds. Hexane is a nonpolar solvent commonly used in the extraction of nonpolar compounds and methanol is a polar and universal solvent. Due to these differences, the presence and quantity of the compounds will also affect the activity of the cells. The phytochemical analysis indicates the presents of goniodiol, 8-epi-9-deoxygoniopypyrone 9-deoxygoniopypyrone, digoniodiol, and GTN in the GL extract (Zohdi et al., 2017). Two new alkaloids, goniolanceolactam and 2-acetyl-3-amino-1,4naphthoquinone, were isolated from the DCM root extract of the GL (Rasol et al., 2018a). Goniolanceolactam showed cytotoxic activity on human colon and lung cancer cell lines with IC 50 values ranging from 5.32 to 9.91 μM. A new styryl lactone, 5R,6R-5hydroxy-6-styryltetrahydropyrane-2-one, was isolated from the GL roots with cytotoxic activity of IC 50 : 2.38-7.59 µM against human colon and lung cancer cell lines (Rasol et al., 2018b).
PEO1 and PEO4 cells are primarily derived from patients with poorly differentiated serous adenocarcinoma at different stages of ovarian cancer. The cells were found as single adhesive cells or small clusters in vitro with approximately 37 hours of doubling times (Langdon and Lawrie, 2001). Cancer cell migration is generally associated with the alteration of the cellmatrix interface on the cell surface (Kim et al., 2012). Inhibition of cell migration prevents cancer metastasis and enhances patient survival in vivo (Helbig et al., 2003). Therefore, it was hypothesized that GL extracts could modulate cell migration while controlling disease progression. The scratch assay revealed that the GL extracts inhibited cell migration in a concentrationdependent manner compared to the negative control. Cells treated with a higher concentration of GL extracts inhibited cell migration more than with a lower concentration. Interestingly, suppression of cell migration by the GL extract was found to be dependent on the type of cell resistance, whereby it inhibited more chemoresistant cells than chemosensitive cells compared to the negative control cells.
A previous study showed that GTN inhibits human lung cancer cell line migration (H1299) at a concentration of less than 10 μg/ml. The inhibition of migration is associated with decreased levels of metalloproteinase matrix (MMP-2 and MMP-9) activity (Chiu et al., 2011). Another study found that Goniolactone-C from G. cheliensis strongly inhibited PDGF-BBinduced vascular smooth muscle cell (VSMC) migration by the suppression of adhesion molecule expression (Sun et al., 2014). Similarly, auraptene and Kaempferia parviflora extract suppressed cell migration and invasion of ovarian cancer in vitro by inhibiting the MMP-2 and MMP-9 activities (Jamialahmadi et al., 2018;Paramee et al., 2018).
Apoptosis is essential in a multicellular organism and is a dominant tumor-suppressive pathway, which can potentially deplete cancer cells (Ghante and Jamkhande, 2019). Cancer cells have the ability to a range of modifications and responses. Mutation is one of the modifications that can cause dysfunction to the apoptotic machinery pathway, thus rendering the cancer cells to be resistant to the drug (Das et al., 2016). Apoptosis has been shown by the externalization of phosphatidylserine indicating early apoptosis in cell death with intact membrane integrity. Necrotic cells are the late apoptotic cells with damaged membranes (Foo et al., 2014). This study showed that GL extracts induced apoptosis in both cell lines; however, GL has a higher ability to induce apoptosis in chemoresistant cells compared to the chemosensitive cells.

CONCLUSION
Goniothalamus lanceolatus extracts exhibited a significant inhibitory effect on cell viability, cell migration, and apoptosis initiation in ovarian cancer cells (PEO1 and PEO4). Nonetheless, careful attention is needed to better understand these promising findings and their interaction as natural extract products containing a complex mixture of natural compounds and may involve multiple molecular targets and pathways.