Introduction

In 2019, the American Cancer Society estimated ovarian cancer as the leading cause of death in gynecological disease, ranking 5 of the 10 leading cancer types in women1. Ovarian cancer is a rare disease, in which early detection is difficult and surgical strategies are the first step in treatment for this cancer2. However, the extent of surgery depends on how far the ovarian cancer has spread; chemotherapy must be used in the next steps to eradicate any residual cancer cells still present in the body after surgery. The goal of chemotherapy is to destroy the cancer by inhibiting the proliferation of cancer cells. Chemotherapy is a potential treatment for prolonging the cancer patient’s life. There are many kinds of anti-cancer drugs from natural sources, such as plants ( vincristine, irinotecan, and camptothecins) and microorganisms (. doxorubicin, mitomycin, and bleomycin)3.

Doxorubicin (DOX) is the most popular anti-cancer drug, which is currently widely used for treatment of many kinds of human cancers, both solid and hematological4. DOX was shown to induce resistance in 3D spheroids, at a rate higher than that exhibited in standard 2D cell culture5. Additionally, tirapazamine (TPZ; 3-amino-1,2,4-benzotriazine 1,4 dioxide) is a new class of cytotoxic drugs with a focus on treating hypoxic mammalian cells6. When cultured in 2D, the toxicity of DOX with target cells is more sensitive than TPZ. Cell viability of cells with the testing drug was identified by using the Alamar Blue assay. By incorporating a fluorometric/colorimetric growth indicator based on detection of metabolic assay, the Alamar blue assay can quantitatively measure the proliferation of various human and animal cells. Many new drugs and therapies have been under development and are being tested in ongoing clinical trials, with the aim of finding the most effective therapy of ovarian cancer.

Recently, besides modern medicine, ethnomedicine has also become a potential direction for the development of new drugs. Ethnomedicine is a study of the traditional medicine based on the use of plant compounds. Ngai bun () is a ginger species belonging to the family; it is cultivated in tropical countries such as Southeast Asia, India, Sri Lanka and South China. This species has many local names, such as “ngai bun” in Vietnam7, “temu kunci” in Indonesia8, krachai (or krachai-dang) in Thailand, and Chinese key (or fingerroot) in English9. This plant is mainly used as a spice and contains several prenylated chalcones and flavonoids with many biological activities, including antifungal, antibacterial, anti-inflammatory and anti-cancer functions8, 7.

The cancer stem cell (CSC) theory proposes that in the cancer cell population, there exists a small population of stem cells which have the capability of self-renewal and tumor-initiation. Recent research studies had revealed that CSCs in ovarian cancer are not only responsible for primary tumor growth and metastasis but also for chemotherapy resistance10. In studies on ovarian cancer stem cells, CD133 is one of most popular cell surface markers which has been used for the isolation of CSCs from various type of cancers, such as breast, colon, ovarian and prostate cancer11, 10.

In this research study, we aimed to test the toxicity of Ngai bun () extract in different solvents on ovarian cancer OVP-10 cells and CD133 sorted OVP-10 cells (CD133OVP-10 cells) to assess the therapeutic potential of this plant for ovarian cancer treatment.

Materials and Methods

Results

Isolation of human ovarian cancer CD133OVP-10 cells

Human ovarian cancer OVP-10 cells were cultured and expanded in 75-cm flasks. When cells reached approximately 70% confluency (Figure 1B), cells will be trypsinized and subcultured in DMEM/F12 medium supplemented with 10% FBS. After passing two to three times, OVP-10 cells were subjected to bioassays, and/or sorted for CD133 cells using MACS (Figure 1A). CD133OVP-10 cells were labelled with CD133 magnetic beads, isolated and cultured to confluency before treatment with the compounds (Figure 1C). The morphology of OVP-10 cells and CD133OVP-10 cells showed no significant differences; however, in culture, the proliferation time of CD133OVP-10 cells was slightly longer than that of OVP-10 cells.

Testing an ovarian cell model for drug screening with standard drugs

The cell concentration also affected the bioassay for anti-cancer drug screening 12. The growth of OVP-10 cells was tested and the model for screening the different extracts was optimized. As seen in Figure 2A, OVP-10 cells continued to proliferate after 7 days. The bioassay was performed in 3 days: on the first day, cells were plated in wells and incubated for 24 h; next, cells were treated with compounds for 48 h; on the third day, cells were processed for Alamar blue assay and the IC indexes were calculated. During these days, OVP-10 cells were still stable and there was little increase in cell number (Figure 2A). The OVP-10 cells were treated with standard drugs (doxorubicin or tirapazamine) to confirm that the OVP-10 cell model could be used for screening. Doxorubicin is popular standard drug which used as a control in many studies of drug screening. Specifically, doxorubicin only affected the monolayer cell model but not the three-dimensional (3D) cell culture model. In contrast, tirapazamine was only effective in the 3D cell culture model. As shown in Figure 2B and Figure 2C, the IC of doxorubicin in OVP-10 cells (168.9 ± 2.3 nM) and that of CD133OVP-10 cells (567.7 ± 95.7 nM) were highly different (p < 0.01).

Ngai bun extract dissolved in chloroform solvent had a greater effect on both OVP-10 and CD133OVP-10 cancer cells than methanol solvent

The IC index of Ngai bun extract in chloroform solvent (CHCl) had a greater effect on killing ovarian cancer OVP-10 cells when compared with the effect of this extract on adipose-derived stem cells (ADSCs), which were used as the control. The ICof methanol (MeOH) and chloroform (CHCl) solvent were 330.1 ± 16.9 μg/mL and 246.5 ± 21.2 μg/mL, respectively. As indicated inFigure 3A, the MeOH extract had a different effect on ADSCs and OVP-10 cells, as shown by the ICindex of 497.2 ± 32.4 μg/mL and 330.1 ± 16.9 μg/mL, respectively. However, with CD133OVP-10 cells, no significant differences were observed between the two cell lines; the IC of the corresponding cells (ADSCs . CD133OVP-10 cells) with MeOH solvent, respectively, were 497.2 ± 32.4 μg/mL and 411.8 ± 83.7 μg/mL (Figure 3B) (p > 0.05). In Figure 3C, with the same cells in a different solvent (CHCl), the IC index of OVP-10 cells was 246.5 ± 21.2 μg/mL, which was significantly different from the IC of 474.6 ± 18.8 μg/mL for ADSCs (p < 0.05). The results inFigure 3D showed that CD133OVP-10 cells treated with CHCl extract corresponded to an IC index that was significantly lower than that for ADSC cells (307 ± 9.2 μg/mL . 474.6 ± 18.8 μg/mL, respectively) (p < 0.05).

Discussion

Ovarian cancer is the most serious gynecologic cancer, typically diagnosed at an advanced stage14, 13. The current standard treatment for ovarian cancer is surgery and chemotherapy. The chemotherapy strategy is faced with many obstacles including cancer metastasis and resistance of tumor with drugs. This has motivated the development of drug discovery to help find novel potentially therapeutic compounds for anti-cancer treatment. In drug screening, cells must be in the proliferation stage and should be stable in the testing with drugs. In this study, the OVP-10 cells continued to proliferate for 7 days and were suitable for our assay. After 24 h of plating, OVP-10 cells showed a low increase in cell number and after 3 days ( the day of the Alamar blue assay), cells were still in log phase of proliferation.

Despite the disadvantage of the 2D model and the development of the 3D model, the 2D model is still very popular for drug screening. Evaluation of the 2D model using bioassays, such as MTT or Alamar Blue assay, is necessary to assess the efficiency of the 2D model in anti-cancer drug screening12. The cell concentration and drug concentration parameters have a great effect on the success of the bioassays. As shown in this study, the cell concentration for plating in 96-well plates was 1000-2500 cells/well, and 6 parameters of drugs or extract concentrations were at the very least required for calculation of the IC index15.

Use of the traditional extract from plants could kill cancer cells with fewer effects on normal cells. In this study, Ngai bun () extract was demonstrated to be toxic for OVP-10 cells, when compared with adipose derived stem cells as control. When comparing different species (), Jing et al. showed that extract in methanol had the strongest inhibitory effects against CaOV ovarian cancer and different types of cancers, such as breast cancer MDA-MB231 (IC 66.5 ± 2.12 μg/mL), MCF7 (IC51 μg/mL), cervical cancer HeLa (IC66.5 ± 2.12 μg/mL), and colon cancer HT-29 (IC 52 ± 2.12 μg/mL). genera is potentially potent extract for treatment of ovarian cancer. OVP-10 cells is one of the targets for investigation of drug cytotoxicity against ovarian cancer16. Moreover, the synergistic anti-tumor effect would be combined to develop new therapies for ovarian cancer treatment17. From different studies, the extractof genera exhibit robust potency that can be utilized as an potential candidates for the development of new anti-cancer drugs. Advances in drug discovery will require identifying and developing new and innovative marketable pharmaceutical products. Future studies from this research should focus further on the discovery of such compounds.

Besides the toxicity towards ovarian cancer OVP-10 cells in a dose-dependent manner, Ngai bun () extract in chloroform was demonstrated to inhibit the cell viability of CD133OVP-10 cells, representing ovarian cancer stem cells. At the IC of Ngai bun extract which could kill 50% of CD133OVP-10 cells, that concentration could kill more than 50% of OVP-10 cells but less than 50% of ADSC cells. This shows that the dose of drug used for treatment must be chosen carefully.

In this study, two kind of solvents were chosen to evaluate which was the best solvent for dissolving Ngai bun () extract, and still maintain the functions of the extract. As observed, the chloroform-dissolved extract induced better toxicity than the methanol-dissolved extract. Besides the appropriate concentrations, the suitable solvent is also a key factor for determining the success of drug discovery.

Conclusion

Overall, the data obtained from this study shows that Ngai bun () chloroform-dissolved extract is more toxic on OVP-10 cells than on CD133OVP-10 cells. The cytotoxicity of the chloroform extract was also higher that of the methanol extract.

Abbreviations

CSC: Cancer stem cell

DOX: Doxorubicin

IC: the half-maximal inhibitor concentrations

TPZ: Tirapazamine

CONFLICT OF INTEREST

The authors report no conflicts of interest in this work.

AUTHORS’ CONTRIBUTION

All authors equally contributed in this work and approved the final version of manuscript for submission.

Acknowledgement

The authors thank to Vietnam National University, Ho Chi Minh City (VNU-HCM) for funding this project, under grant number A2015-18-01/HD-KHCN.