Cytotoxic Potential of Arthrospira platensis Extract on Cervical Cancer Cells Line Hela: Study on Antiproliferative, Cell Cycle, Apoptosis Induction and Anti Metastasis

Indonesian J Pharm 31(1), 2020, 19-26 | DOI: 10.14499/indonesianjpharm31iss1pp19 indonesianjpharm.farmasi.ugm.ac.id Copyright © 2019 THE AUTHOR(S). This article is distributed under a Creative Commons Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) Cytotoxic Potential of Arthrospira platensis Extract on Cervical Cancer Cells Line Hela: Study on Antiproliferative, Cell Cycle, Apoptosis Induction and Anti Metastasis


INTRODUCTION
Cervical cancer can occur due to an infection by the Human Papilloma Virus (HPV) that cause mutations in the p53 gene and increased expression of the protein Bcl-2 which resulted in an imbalance between proliferation and cell (Sari and Syahrul, 2014). One of the methods chosen to treat cervical cancer is by giving chemotherapy agents. However, chemotherapy agents have side effects and complications in the form of normal cell damage (Huang et al., 2018). Therefore, it is necessary to develop alternative anticancer agents which are selective towards cancer cells without affecting normal cells.
Previous research showed that A. platensis contains antioxidant compounds such as βcarotene, flavonoids, terpenoids, and saponin compounds (Muszynska et al., 2018). Antioxidant compounds are able to regulate antiapoptotic proteins in the mitochondria to increase the induction of apoptosis (Muszynska et al., 2018). In addition, antioxidant compounds also have the ability to repair DNA or RNA damage in normal cells which will trigger the activation of the p53 gene so that there is a cessation of cell cycle and inhibition of cancer cell proliferation (Nugraheni et al., 2013). Based on this, it is necessary to investigate A. platensis cytotoxicity against HeLa cervical cancer cells. HeLa cells are cervical cell cultures infected with HPV and express 2 oncogens, namely E6 and E7 so that they can multiply in culture media and have cancer properties (Lucey et al., 2009). The selectivity test for A. platensis is done using Human Dermal Fibroblast Adult (HDFa) cells, HDFa cells are primary cell cultures that can present responses to stress and other biological approaches that approach in vivo situations in humans (Özen et al., 2005). The cytotoxic mechanism of an anticancer agent is related to its ability to inhibit proliferation and induce apoptosis (Moningka, 2019). Therefore, an antiproliferation test was performed to determine the doubling time value to show the ability of A. platensis to inhibit HeLa cells proliferation, flow cytometry analysis to observe inhibition patterns in the cell cycle and the magnitude of apoptosis induction in HeLa cells due to A. platensis treatment, and cell migration inhibition test with wound healing assay to determine the effect of A. platensis on the process of metastatic HeLa cancer cells (Lee et al., 2014). 20 Volume 31 Issue 1 (2020)

Plant material and cell culture
A. platensis obtained from the Serba Alami Store, Bantul, Yogyakarta in November 2018. The sample has been identified at the Faculty of Biology, Gadjah Mada University. The test subjects used were HeLa cells and HDFa cells which are collections from the in Vitro Cell Culture Laboratory at Yogyakarta Muhammadiyah University. HeLa and HDFa cells were maintained in the RPMI medium with supplemented with FBS and cultured under 5% CO2 and 37ᵒC.

Cytotoxicity tests on HeLa cells and HFDa cells
Cells were distributed into wells in 96 well plates (each well containing 2x10 4 cells) and incubated at 37ᵒC, 5% CO2 overnight. Following incubation, the various concentration series (62.5-2000µg/mL) of A.platensis extract were added into each well and incubated for 24h. At the end of the incubation, 10μL 0.5% MTT in PBS was added to each well. The reaction was stopped by adding 10% SDS, and stand for 4h before measuring the intensity. The intensity of the purple color formed was measured by the ELISA reader at a wavelength of 595nm (Sulistyani and Nurkhasanah, 2017).

Doubling time test
Cells were starved in culture media containing 0.5% FBS for 24h in 96 well plate (each well containing 2x10 4 cells), then cells were incubated with ethanol extract of A. platensis with a concentrations 156.575μg/mL and 78.288μg/mL. Cell population in each series of experiments was calculated at 24, 48, and 72h incubation time with ELISA readers at a wavelength of 595nm, then a curve of cell number vs. incubation time ware made. The doubling time is calculated from the slop after a straight line is drawn on the curve (CCRC, 2017).

Cell cycle analysis by flow cytometry
Cells were distributed into 6 well plates (each containing 5x10 5 cells) and incubated with test samples at a concentrations of 156.575 μg/mL and 78.288μg/mL for 24h. At the end of the treatment, the media in each well was put in a different conical. Cells were washed with 500μL PBS and added with 200μL trypsin-EDTA 0.25% to the cell, then incubated for 3min to detach the cells and then added with 500μL of media. Cell suspension was then centrifuged at 600 rpm for 5 minutes. The precipitate was fixed with cold 70% ethanol, and let it for 30min at room temperature and followed by centrifugation at 2000 rpm for 3 minutes and washed with PBS. Following washing, 400μL PI reagent (propidium iodide) 1mg/mL (Sigma), 10mg/mL RNAse (Sigma) and 0.1% (v/v) Triton-X 100 (Sigma) were added. Cells were resuspended and incubated for 5min in a dark room, then read with a flow cytometer with a light beam of 488nm and a medium speed of 500cells/s (Mahfudh and Pihie, 2008).

Annexin V apoptosis detection assay
For apoptotic observations, Following the treatment, incubation and washing, the cells was then resuspended with 100μL binding buffer, 2μL Anexin-V and 2μL PI, then incubated in a dark room for 10 minutes. Cell suspension is read with a flow cytometer with a light beam of 488nm and a medium speed of 500cells/s (Mahfudh and Pihie, 2008).

Scratch wound healing assay
Cells were starved in culture media containing 0.5% FBS for 24h into 6 well plates (each containing 7.5x10 4 cells). Then a scratch was made on the surface of the well using sterile yellow tips and given a test sample with a concentration of 156.575μg/mL and 78.288μg/mL. Cell condition was observed and documented after incubation of 0, 18, 24 and 48h. Then the stroke distance between cell control and treatment was compared using ImageJ Software (Jonkman et al, 2014).

Data analysis
Data obtained from ELISA in the form of absorbance is converted as a percentage of cell death. After the percentage (%) of cell death was obtained, it can be calculated that the extract concentration can inhibit the growth of 50% of the cell population (IC50 value) by probit analysis according to SPSS version 20. Selectivity is calculated by comparing the IC50 value of HDFa cells with IC50 cells of HeLa cells. The data of doubling time, % apoptosis, and % cell migration were analyzed by One Way Anova followed by Post Hoc Tukey's Test at p<0.05. The cell cycle was analyzed by comparing the largest cell accumulation between treatment and control.

Phytochemical screening
The maceration process produced 2.235 gram of ethanol extract of A. platensis with an extraction rate of 2.233%. Based on TLC results, ethanol extract of A. platensis contains β-carotene, terpenoids, and flavonoid compounds ( Table I).
The previous research showed the potency of these compound to increase apoptosis and inhibit proliferation of cancer cells (Wati et al., 2016).

Cytotoxicity tests on HeLa cells and HFDa cells
The treatment of ethanol extract of A. platensis causes morphological changes in HeLa cells and HDFa cells. Some of HeLa cells were shrink and detach to each other (contact inhibition) and decrease in number, as well as HDFa cells ( Figure  1). After observing cell morphology, cells are given an MTT solution. The more formazan crystals formed, the absorbance measured in ELISA Reader is higher and more cells are still alive (Suzery and Cahyono, 2014). The percentage of deaths due to ethanol extract of A. platensis in HeLa cells increases when the concentration increases. The same thing happened with HDFa cells, an increase in the concentration of ethanol extract A. platensis also resulted in an increase in the percentage of cell death. However, the dose needed to cause hindrance to HDFa cell viability is higher than the

Figure 2. The cytotoxic effect of the ethanol extract of A. platensis on HeLa cells and HDFa cells
The extract has a strong cytotoxic activity if it has IC50 <100μg/mL or moderate if 100μg/mL <IC50<1000μg/mL against cancer cells, while the extract is toxic to normal cells if it has an IC50 value <1000μg/mL and said not toxic if the IC50 value >1000μg/mL (Benedetti et al., 2010). The present study found that, the IC50 values of A.platensis extract against Hela cells and HDFa cells were 260.444μg/mL and 2065.662 ug/mL respectively. Therefore, it can be concluded that the ethanol extract of A. platensis has moderate cytotoxic activity against HeLa cells and is not toxic to normal HDFa cells. The cytotoxic activity of ethanol extract of A. platensis could be caused by its chemical content including β-carotene, terpenoid, and flavonoid. The previous studies suggested the potency of compounds as cytotoxic agent (Wati et al., 2016). The study of cytotoxicity mechanism of the extract was carried out in concentration under IC50 including 156.575ug/mL and 78.288ug/mL to allow the cells grow and could be observed.

The selectivity of ethanol extract of A. platensis
Ethanol extract of A. platensis has a selectivity index (SI) of 7.9313, so it can be concluded that ethanol extract A. platensis is selective against HeLa cancer cells. The mechanism of ethanol extract of A. platensis to distinguish cancer cells from normal cells is based on the cells need for Adenosine Triphosphate (ATP). Because cancer cells move, grow and multiply faster and are more active than normal cells, cancer cells need more ATP energy. The previous studies showed the potency of flavonoids as anticancer agent (Abotaleb et al., 2019) through inducing apoptosis. Another mechanism of flavonoid in inhibiting of cancer progression is through its ability to target molecular pathways involved in glucose metabolism and mitochondrial function (Reyes-Fariaz and Carrasco-Pozo, 2019).

Doubling time test
Based on Post Hoc Tukey's Test stated that ethanol extract of A. platensis caused a significant increasing of HeLa cells doubling time compared to the control group (Figure 3). The higher the concentration of the extract given, the longer the doubling time obtained (Table II). This result showed that treatment of A.platensis extract could inhibit the cancer cells proliferation. In the control group, it was found the doubling time is shorter. It showed that without treatment, Hela cells proliferate faster. Inhibition of proliferation on HeLa cells due to the administration of ethanol extract of A. platensis occurs through the mechanism of inhibiting the action of oxidation enzymes that play a role in the production of ATP by flavonoids so that the energy used for cell division is less available (Sahid et al., 2013). After that, β-carotene can increase p53 which gives effect to cell cycle termination (Nugraheni et al., 2013). When the cell cycle is stopped, it is possible for cells to make DNA repairs, which are maximized by terpenoids. Terpenoids have the ability to inhibit cancer cell proliferation by triggering DNA repair mechanisms and inhibiting DNA fragmentation (Thoppil and Bishayee, 2011).

Cell cycle analysis by flow cytometry
Ethanol extract of A. platensis can cause accumulation of HeLa cells in the S phase which can cause the inhibition of HeLa cells to enter the G2/M phase (Figure 4). In the G2/M phase the cells are ready to divide, in this phase the process of DNA replication and the biosynthesis of proteins and RNA needed for cell division have been perfected. Barriers to cell cycles in the G2/M phase produce regulatory errors that cause delayed or disrupted cell division (Lacroix and Maddox, 2014).
Inhibition of the cell cycle in HeLa cells is caused by the content of β-carotene, flavonoid, and terpenoids compounds contained in the ethanol extract of A. platensis. In general, β-carotene compounds have the ability to increase p53 which is able to recognize cells with damaged DNA and stimulate transcription of genes such as p21 or Bax so that the cell cycle stops and DNA has the opportunity to repair itself before entering the next division stage (Nugraheni et al., 2013). While the mechanism of action of flavonoid compounds is to prevent the formation of active CDK-Cyclin complexes thereby suppressing Maturation Promoting Factor (MPF), this results in disruption of checkpoints in the S and G2/M phases so that cells cannot complete their cell cycle (Wati et al., 2016). The terpenoid compounds have a mechanism of action in blocking the cell cycle in the G2/M phase by stabilizing the spindle threads in the mitotic phase, thus causing the process of mitosis to be inhibited (Setiawati et al., 2007).

Annexin V apoptosis detection assay
Based on flowytometry analysis, ethanol extract of A. platensis has the ability to induce cell death through the necrosis pathway, this is reflected in the number of cells undergoing necrosis more than the number of cells undergoing apoptosis in both treatment groups ( Figure 5). However, it should be noted that in both treatment groups the range of cells undergoing necrosis and apoptosis is not too far away, so it is necessary to optimize the concentration ethanol extract of A. platensis so that the cell population undergoing apoptosis is more optimal. Necrosis can also be caused by an incubation period that is too long (24h) so that the apoptotic phase has passed. Apoptosis induction in HeLa cells is caused by the content of β-carotene, flavonoid, and terpenoid compounds contained in ethanol extract of A. platensis. Flavonoid compounds have the ability to increase the expression of p53. The p53 gene will stimulate the mitochondria to release cytochrome C to the cytosol and activate the exclusionary caspase so that it can cause apoptotic cell death (Setiawan et al., 2017). The terpenoid compounds will make the bond between the topoisomerase enzyme and the DNA of the cancer cell held longer so that it results in fragmentation or damage to the cancer cell DNA. DNA damage can increase the expression of proapoptotic proteins such as Bax and Bak and decrease the expression of antiapoptotic proteins namely Bcl-2 and Bcl-XL (Susianti, 2016). The mechanism of action is then strengthened by β-carotene compounds which  have the ability to reduce the expression of Bcl-2 and induce the release of cytochrome c from mitochondria in HeLa cells which results in increased induction of apoptosis (Palozza, 2005).

Scratch wound healing assay
The scratch wound healing assay was carried out to observed the effect of extract in inhibiting migration of the cancerous cells. The capability of extract to inhibit the cell migration could inhibit the progression of cells, which is the important part of cancerous cells to spread and metastatic.
Based on the Post Hoc Tukey's test stated that ethanol extract of A. platensis caused significant decreasing the migration rate of HeLa cells when compared to the control group ( Figure  6). Decreased rate of cell migration in HeLa cells is caused by suppression of MMP-9 expression due to inhibition of NF-κB activity by antioxidant compounds such as β-carotene, flavonoid, and terpenoid compounds, which are contained in the ethanol extract of A. platensis (Sung et al., 2012). Decreased rate of cell migration can illustrate a decrease in the ability of cancer cells to separate from primary tumors into more distant tissues and colonize other organs to form secondary tumors (Zhang et al., 2007). Barriers to cell spreading signify a delay in the metastatic process from cancer (Jiang et al., 2015).

CONCLUSION
Ethanol extract of A. platensis has a cytotoxic effect on HeLa cells with an IC50 value of 260.444 μg/mL and selective against normal HDFa with SI 7.9313. These cytotoxic properties are related to delays in doubling time, triggers apoptosis, inhibition of the cell cycle in the G2/M phase, and inhibition of cell migration. Cytotoxic effects are caused by β-carotene, terpenoid, and flavonoid compounds contained in the ethanol extract of A. platensis.