Evaluation of the antiproliferative effect of Iso-mukaadial acetate on breast and ovarian cancer cells

Natural compounds derived from various medicinal plants may activate several physiological pathways which can be valuable to diseases such as cancer. Isomukaadial acetate has previously been shown to possess antimalarial and antidiabetic properties. The purpose of this study was to evaluate the antiproliferative effects of isomukaadial acetate on breast and ovarian cancer cell lines. Cell viability assays were conducted using AlamarBlue assay and xCELLigence system. Cell apoptosis and cell cycle arrest were determined and analyzed by flow cytometer. Effector caspase (3/7) activation was evaluated by caspase Glo®-3/7 reagent and gene expression was analyzed by Real-Time Polymerase Chain Reaction. The Alamar blue assay and xCELLigence showed that Iso-mukaadial acetate exhibited anti-proliferative effects on MDA-MB 231, RMG-1, and HEK 293 cell lines in a concentration-dependent manner. Iso-mukaadial acetate induced apoptosis in both cancer cell lines caused cell cycle arrest at the S phase (RMG-1) and early G2 phase (MDA-MB 231) and expressed caspase 3/7 activity in MDA-MB 231 and RMG-1 cells. BAX and p21 were upregulated in MDA-MB 231 and RMG-1 cells after treatment. IMA significantly inhibited cancer growth and induced cell apoptosis with cell cycle modulation. IMA may be considered a promising candidate for the development of anticancer drugs either for its cytotoxic or cytostatic effect Furthermore, IMA requires to be further studied more to clearly understand its mechanism of action on cancer cells.


Introduction
Breast cancer is one of the most occurring cancer in women around the world, with high occurrence rates in well-established countries. It is documented to be the second leading cause of death rate in women worldwide (Luo et al. 2017). Breast cancer does not affect women only, but it can occur in men including transgender (Iqbal et al. 2018). Ovarian cancer is a lethal disease that affects 1-2% of females and about 20,000 new cases are identified yearly with 130,000 deaths globally. It is documented that the occurrence rate of ovarian cancer has increased over time affecting most people in developed countries. Ovarian cancer is the 6th most occurring cancer affecting females all over the world and the 5th cause of death (Sak 2015). Apoptosis is a programmed cell death 1 3 that is caused by the expression of explicit genes to remove damaged cells, for instance during cell development. Apoptosis is induced by DNA damage in precancerous lesions and other factors, which removes the harmful cells thereby blocking the development or growth of a tumour. Since cancer cells are reported to be resistant to apoptosis, better therapeutic approaches need to be implemented to reestablish cancer cell's sensitivity to apoptosis (Pistritto et al. 2016).
Medicinal plants have continued to be substantial hope for drug discovery and development in various diseases. Natural plants have been and still are explored as vital resources for cancer therapies including therapies for other diseases such as diabetes, malaria. Medicinal plants are used by approximately 70% of the world as a source for potential drug agents Pandiella 2018a, b, 2019) and about 70-95% of people in the developing countries use traditional medicinal plants for health care services (Majolo et al. 2019). Different cultures across the world have been acquiring knowledge, skills, and practice of using medicinal plants in diagnosis, prevention, and treatment of infections/diseases before the development of western medicine/pharmaceutical industries (Barata et al. 2016). Natural compounds derived from various medicinal plants may activate several physiological pathways which can be valuable to diseases such as cancer. Previous studies have reported that natural compounds have a positive impact on the health benefits of people by targeting specific genes or metabolic pathways. They can effectively reduce side effects such as nausea, fatigue, and anemia coming from chemotherapy or other cancer treatments and advance the existing and survival rates of patients (Liao et al. 2020) and have minimal toxicity (Mitra and Dash 2018).
Previous studies have shown that iso-mukaadial acetate, a drimane sesquiterpenoid possesses antimalarial and antidiabetic properties (Msomi et al. 2019;Nyaba et al. 2018;Opoku et al. 2019. This compound was isolated from a South African plant (Warburgia salutaris) that is extensively used for the treatment of bronchitis, ulcers, and oral thrush (Maroyi 2014;Kotina et al. 2014). The plant is also recommended by traditional healers in treating and managing cancer (Soyingbe et al. 2018;Kotina et al. 2014). In this study, isomukaadial acetate (IMA) was evaluated for its antiproliferative activities on breast and ovarian cancer cell lines, which triggered cell apoptosis by the expression of cell apoptotic genes and cell cycle arrest. Note, this is the first findings of the antiproliferative effect of is-mukaadial acetate on cancer cell lines.

Materials and method
Reagents Iso-mukaadial acetate was previously isolated from the University of KwaZulu Natal by Simelane and colleagues.
Breast cancer (MDA-MB 231) cell lines and normal embryonic kidney (HEK293) cell lines were donated by Dr. Engelbrecht Z (University of Witwatersrand). The RMG-1 cell lines were donated by Prof Motadi LR (University of Johannesburg). Dulbecco's Modified Eagle's medium (DMEM) and trypsin were manufactured by LONZA (RSA). Dulbecco's phosphate-buffered saline (DPBS) was manufactured by HyClone, GE Healthcare. AlamarBlue cell viability reagent was manufactured by Invitrogen, Thermo Fischer Scientific. Trypan blue (0.4%) was manufactured by LONZA (RSA). Staurosporine was obtained from Prof Motadi (University of Johannesburg) and etoposide from Sigma. Fetal bovine serum (FBS) was manufactured by HyClone, GE Healthcare. Caspase-Glo® 3/7 assay was manufactured by Promega (USA). Relia-Prep RNA cell miniprep kit was manufactured by Promega (USA). Propidium Iodide was manufactured by Sigma (USA) and Annexin V/FITC+PI was manufactured by R&D System (USA).

Preparation of the compound
The Iso-mukaadial acetate (IMA) was weighed separately and dissolved in DMSO to a stock concentration of 10 mM. The compound was stored at 4 °C until use.

Cell culture
The HEK 293, MDA-MB 321, and RMG-1 cell lines were maintained in 75 cm 2 and 25 cm 2 cell culture flasks under the following conditions: 37 °C, 5% CO 2 , and 95% humidity atmosphere in an incubator. The human cell lines were grown until they reached a confluency of about 70-80% using DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. When the cancer cell lines reached 70-80% confluency, the cells were washed with 1X DPBS and exposed to 1X trypsin-EDTA for less than 1 min (depend on the type of cell line). After incubation with trypsin, a 10% FBS media was added into a flask to deactivate trypsin, then the mixture was transferred into a 50 ml falcon tube and centrifuged for 4 min at 2200×g. The cell viability concentration was determined by using 0.4% of trypan blue. From the 50 ml falcon tube containing cells with media, 20 µl of cells were mixed with 20 µl of 0.4% of trypan blue. The mixture was aspirated gently, then 10 µl of the mixture was spread on the cell counter slide using a micropipette and the number of viable cells was counted using the TC20 Automated Cell Counter.

Cell proliferation assay using AlamarBlue
Firstly, the cell lines were grown in an incubator (with the same conditions mentioned in 2.3) until 80% of cell confluence was reached. After the cell lines were sub-cultured and seeded in 96 well plates. Trypan blue was used to check the viability of the cells. The desired concentration of cells used was 5.0 × 104 for the HEK293 cell line, 1.0 × 104 for MDA-MB 321 and RMG-1, then a total of 100 µl of cells/ ml was seeded on the 96 well plates for 24 h. After 24 h of incubation, the cell lines were treated with different concentrations of the compounds namely, 5 µM, 2.5 µM, 1.25 µM, and 0.65 µM for IMA for 24 h. Positive control was etoposide at a concentration of 100 µM. Negative control was DMSO with cells (0.1%) cells with media only. After 24 h, 10 µl of AlamarBlue was added to each well in the dark, then the plates were wrapped with aluminum foil as the dye is sensitive to light and incubated for 2 h. After 2 h of incubation, the plates were read by a fluorescence microplate reader (Synergy HT).

Real-time cell viability analysis using xCELLigence system
The xCELLigence RTCA was placed in 37 °C, 5% CO 2 , and a 95% humidified incubator and was calibrated before use. Different cell concentrations for MDA-MB 321, RMG-1 and HEK 293 cell lines (1 × 104 cell/ml, 5 × 104 cell/ml and 1.5 × 105 cell/ml) were seeded on the E16 well plates for 22 h. The cell lines were left for 30 min to allow cell attachment before running the RTCA system. After 22 h, the cell lines were treated with different compound concentrations: IMA (10, 5, 2.5, 1.3, 0.6 µM) for 3 days. A 0.1% was used as negative control and 100 µM etoposides were used as a positive control which was run in duplicates including the different compound concentrations. Etoposides, MA, and DMSO were dissolved in DMEM media before treating the cells. The RTCA was supervised every 15 min and the results were recorded by the RTCA software.

Apoptosis assay using Annexin V + FITC and PI
The cell lines were seeded into Petri dishes MDA-MB 231 (2 × 105) and RMG-1 (2 × 105) for 24 h. The cell lines were then treated with the IC50 of iso-mukaadial acetate for 24 h. Then the treated cells plus media were transferred into 2 ml Eppendorf tubes, washed with DPBS, and the remaining attached cells in the Petri dishes were trypsinized. The control cells were resuspended in fresh media after trypsinization to recover in an incubator. The trypsinized cells were transferred in the same Eppendorf tubes. The mixtures were micro-centrifuged at 500×g for 2-5 min. Following the manufacturer's protocol, the supernatants were removed, and the pellets were washed twice with icecold DPBS at 500×g for 2-5 min depending on the cell line. The binding buffer (1x) was prepared with autoclaved distilled water and 90 µl of it was added into different Eppendorf's containing the cell including 5 µl of Annexin V/FITC and 5 µl PI to make a final volume of 100 µl. The mixtures were vortexed and incubated in the dark on ice for 15 min. About 300 µl of the binding buffer was added into the flow tubes containing the cells and the dyes before reading the results. A flow cytometer (BD FACS AriaTm ll) was then used to measure apoptosis.

Cell division cycle using Propidium Iodide
In this study, the cells were counted using a T20 cell counter of about 1 × 105 to 1 × 106 depending on the cell line. The cell lines were seeded in small Petri dishes respectively and incubated for 24 h. The plated cells were treated with both compounds respectively using the IC50 obtained. The media was decanted into the Eppendorf tubes. Trypsin was added into the Petri dishes to detach the remaining cells and poured off into the same Eppendorf tubes. The mixture in the Eppendorf tubes was centrifuged at 300×g for 3 min and decanted the supernatant. Cells were washed with ice-cold 1 × DPBS (500 ul), before pouring off, they were centrifuged at 300×g for 3 min and carefully poured off the supernatant. The cells were fixed in cold 0.5 ml (500 µl) 70% absolute ethanol at 4 °C and incubated overnight or stored at − 20 °C for several days until use. After incubation, the cell samples were centrifuged at 500×g for 5 min, washed cells with cold 1X PBS twice, and centrifuged at 500×g for 3 min. Cells have treated the cells with RNase A by adding 5 µl of 10 mg/ml solution followed by 300 µl of PI (10 mg/ml) and incubated for 30 min on ice in the dark until read by flow cytometer (BD FACS AriaTm ll).

Caspase-Glo® 3/7 activity
Following the manufacture's protocol from Promega, caspase-Glo® 3/7 assay was used to measure the activation of caspase 3/7 in MDA-MB 231 and RMG-1 cell lines after treatment with IC50 of iso-mukaadial acetate. MDA-MB 231 (2 × 105) and RMG-1 (2 × 105) cell lines were seeded in the 96 well microplates for 24 h. The cell lines were treated with iso-mukaadial acetate IC50 values for 24 h. The caspase-Glo® reagent was then added to the wells and incubated on the shaker for 30 min. The caspase activities were measured by a luminescence microplate reader (Synergy HT) and the results were represented as Relative Luminescence Units.

RNA extraction using Relia-Prep™ RNA-cell miniprep kit
Following manufacturers protocol from Promega, RNA was extracted from MDA-MB 231 and RMG-1 cell lines using Relia-Prep™ RNA-cell miniprep kit after treatment with IMA IC50. The three cell lines were harvested before cell lysis, then collected cells in an Eppendorf tube by microcentrifugation at 300×g for 5 min. The cell pellets were washed with ice-cold 1X DPBS and microcentrifuge at 300×g for 5 min. The supernatant was discarded. The BL-TG Buffer mixture was prepared and about 100 µl was added to the pellets respectively, then vortexed. About 35 µl of isopropanol was added to the mixture and vortexed for 5 s. Aseptically, the cell lysates were transferred into a Minicolumn in a collection tube and were microcentrifuge at 13,000×g for 30 s. About 500 µl of RNA wash solution was added to the Minicolumn and microcentrifuge at 13,000×g for 30 s. DNase I incubation mixture was prepared and 30 µl of the mixture was added into each Minicolumn tubes and incubated at room temperature for 15 min. Column wash solution (200 µl) was added after the incubation period and microcentrifuge at 13,000 for 15 s. The RNA wash solution was added and microcentrifuge at a higher speed for 2 min. The Minicolumn was transferred into an elution tube and nuclease-free water was added, microcentrifuge at 13,000×g for 1 min. The extracted RNA was quantified by determining its purity using Nano-Drop and an A260/A280 ratio of 2.0 was considered as pure. Then the RNA was stored at − 80 °C until further use.

Real-time polymerase chain reaction using SYBR green
After mixing the following components (RNA template, primers, random primers), the mixture was incubated at 70 °C using a master-cycler gradient for 5 min. Then the mixture was chilled on ice immediately for 5 min, vortexed for 10 s, and chilled on the ice again until the reverse transcription mixture is added. The reverse transcription mixture was combined with the 5 µl of RNA and the primers. Then the cDNA synthesis conditions were applied, and the synthesized cDNA was stored at − 80 °C until further use. The amplification of cDNA was done using GoTaq G2 Green master mix 2X. The cycle conditions were repeated 40 times and the samples were held at 4 °C for several hours before the amplicons were visualized on agarose gel electrophoresis. The iTaq™ Universal SYBR® Green Supermix, primers, and cDNA were used. The components above were mixed by vortexing for few seconds and stored on ice in the dark until further use. A CFX96-connect machine was used to run real-time PCR and the following thermal cycling was programmed.

Statistical analysis
Results were analyzed statistically by using Graph Pad Prism, ModFit LT and REST 2007 software. The data were expressed as mean ± Standard Deviation (*P < 0.05, **P < 0.01 ***P < 0.001). A P-value less than 0.05 (P < 0.05) was considered as significant.

Results
The results below represent the antiproliferative effects of Iso-mukaadial acetate (IMA) at various concentrations on breast cancer, ovarian cancer, and embryonic kidney normal cell lines. As shown in Fig. 1, IMA exhibited antiproliferative effects on MDAMB 231, RMG-1, and HEK 293 cell lines stained/treated with the AlamarBlue reagent (endpoint assay). Real-time cell analysis (xCELLigence) has also shown the inhibitory effects of IMA on the cells (Fig. 1). IMA induced apoptosis, cell cycle arrest (Fig. 2a, b), and the activation of caspase 3/7 (Fig. 3) was also observed. The expression of selected genes was determined as shown in Fig. 4. IMA may be a promising anticancer agent in the future on cancer cell lines.

Anti-proliferative effect of IMA using Alamarblue and xCELLigence system on MDA-MB 231, RMG-1 and HEK 293 cell lines
The antiproliferative effect of IMA was measured using Alamarblue reagent, which is an assay reagent based on the metabolic reduction of resazurin compound to a form known as resorufin by mitochondrial enzymes. MDA-MB 231, RMG-1 and HEK 293 were exposed to different concentrations of IMA (0.6-10 µM) for 24 h. IMA decreased the viability of the cell lines in a concentration-dependent manner in an endpoint assay (figure A, C and E). xCELLigence system was used to track down the effectiveness or potency of IMA over 48 h treatment. The IC 50 values of IMA were automatically calculated by the xCELLigence system which were used in subsequent experiments to determine the anticancer activities it may possess on breast and ovarian cell lines. IMA, the compound of interest exhibited anti-proliferative effects on MDA-MB 231, RMG-1 and HEK 293 cell lines as shown in Fig. 1. At higher concentrations (10 and 5 µM), IMA showed to be highly toxic in comparison with etoposide observed in all cell lines. Then, in Fig. 1b, d, low concentrations showed that IMA was moderately cytotoxic within 24 h of treatment. DMSO (0.1%) showed to promote cell proliferation in comparison to untreated control (see Fig. 1b). DMSO (0.1%) in figure D, showed to slow down cell proliferation in comparison to untreated control. Higher concentrations of IMA were toxic to the HEK 293 cells in comparison to low concentrations (figure E and F). The cytotoxic effect of etoposide was also observed. DMSO did not have any effect on the HEK 293 cells in comparison to untreated cells and IMA response.

Apoptotic induction and cell cycle arrest using Flow cytometer on MDA-MB 231 and RMG-1 cell lines
To determine whether IMA triggered apoptosis, cell apoptotic assay was conducted using Annexin-V FITC-PI staining. Early apoptosis was expressed as a percentage of cells which are positive for Annexin V-FITC but negative for PI, and late apoptosis was expressed as a percentage of cells which are positive for both Annexin V-FITC and PI. The percentage of apoptosis was calculated as the sum of early and late apoptosis. IMA was shown to significantly induce apoptosis on MDA-MB 231 and RMG-1 cell lines. The data were expressed as mean ± Standard Deviation (*P < 0.05, **P < 0.01 ***P < 0.001, ****P < 0.0001) from three biological repeats obtained were read and analyzed by flow cytometer and ModFit LT 5.0 software. The data were expressed as mean ± standard deviation (**P < 0.01; ***P < 0.001; ****P < 0.0001) from three biological repeats Higher percentage of apoptotic cells in a treated sample was observed in comparison to untreated cells in both cell lines. The overall percentages of cell apoptosis induced by IMA was low in comparison to etoposide as a positive control in both cell lines. Cell cycle arrest is an assay that measures the DNA content in each cell cycle phases (G1, G2, M phases). PI was used since it can intercalate and stain DNA in the nuclease. Cell cycle results on MDA-MB 231 cells exhibited that etoposide caused G2/M phase (99%) arrest while IMA caused S phase (72%) and early G2 phase (25%) cell cycle arrest in comparison to untreated cells. Etoposide caused S phase (76%) arrest and IMA caused S phase (81%) cell cycle arrest with a decrease in cell population in other phases on RMG-1 cells in comparison to untreated cells. Fig. 3 Caspase 3/7 activation modulated by the biological activity of IMA on the MDAMB 231, and RMG-1 cell lines. The cells were treated with IMA IC 50 (MDA-MB 231 (2.7 µM)) and (RMG-1 (2.3 µM)) for 24 h. Caspase 3/7 activity was analyzed by luminescence. The data were expressed as mean ± standard deviation (*P < 0.05, ***P < 0.001) from three biological repeats tive expression of normalization of the selected genes. GAPDH was used as a reference gene. The data were expressed as mean ± standard deviation from two biological repeats (*P < 0.05, **P < 0.01, ****P < 0.0001) The apoptotic induction cell results may be supported by the cell cycle results observed after 24-h treatment with IMA in both cell lines.

Activation of caspase 3/7 using caspase 3/7 Glo reagent on MDA-MB 231 and RMG-1 cell lines
Caspases play a major role in the process of apoptotic induction. The activation of caspase 3/7 is important for the induction of the downstream processes that lead to cell death. To determine caspase 3/7 activation, caspase 3/7 reagent was used and measured using luminescence after 24 h of treatment with IMA on both cell lines. IMA expressed a low level of caspase 3/7 activation in comparison to etoposide which expressed a high level of caspase activity (A). In Fig. 4b, IMA expressed high level of caspase 3/7 activity in comparison to etoposide as a positive control (B). The activation of caspase 3/7 indicates that apoptosis was induced during treatment with IMA, and this may further proof that IMA may possess properties that elicit apoptosis response.

Gene expression using real-time PCR
To examine if IMA induced apoptosis by influencing cellular processes responsible for eliciting cell death, Real-Time PCR was conducted. In MDA-MB 231 cells, the rtPCR data showed that p53 was downregulated in comparison to control by a factor of 0.380 where S.E rage was 0.287-0.517 (A, C). In RMG-1 cells, the rtPCR data showed that p53 was significantly downregulated in comparison to control by a factor of 0.002 where S.E range is 0.001-0.004. Bax gene was statistically highly upregulated in both cell lines and Bcl2 as an anti-apoptotic gene was slightly upregulated in MDA-MB cells by a factor of 1.557. No significant changes were observed in the expression of Bcl2 in RMG-1 cells before and after treatment with IMA. In MDA-MB 231 cells, p21, RBBP6 and CDK2 showed no significant changes in comparison to control. Their expression was the same before and after treatment. While p21 expression in RMG-1 was the same before and after treatment. CDK2 was not expressed at all on RMG-1 (see Fig. 4b, d).

Discussion
Natural compounds derived from medicinal plants are promising to advance the development and treatment efficacy in human cancer cells (Choudhari et al. 2017(Choudhari et al. , 2020. Researchers are moving towards natural compounds due to the gene mutations and cancer-causing properties of the synthesized compounds (Moodley et al. 2014). The anti-proliferative effect of IMA was evaluated using Alamarblue (endpoint assay) and xCELLigence system (real-time cell analyzer).
We found that IMA significantly exhibited an antiproliferative effect on MDA-MB 231 and RMG-1 cells in a concentration-dependent manner. The cytotoxic effect of IMA on cancer cell lines in comparison with non-cancer cells (HEK 293) exhibits that IMA is significantly selective based on the type of cell line. Etoposide (positive control) in both endpoint assay and real-time cell analysis exhibited a cytotoxic effect on the MDA-MB 231, RMG-1, and HEK 293 cells at higher concentrations (100 µM) and it was not selective on the cell lines in comparison to IMA.
The endpoint assay (Alamarblue, Fig. 1) showed the cytotoxic effect of IMA on the cancer cells in a concentrationdependent manner after 24 h of treatment. In Realtime cell viability analysis (Fig. 1b), IMA exhibited an inhibitory effect on the MDA-MB 231 cell lines over 48 h of treatment. The real-time cell analyzer results, exhibit that MDA-MB 231 was resistant to treatment within the first 24 h of treatment. This may be an interesting point because at 10 and 5 µM concentrations, the cells lost their viability, hence a decline in cell proliferation was observed. This exhibits the toxicity of IMA at higher concentrations with the possibility of apoptosis induction. At lower concentrations (2.5, 1.3, and 0.6 µM), Fig. 1b, showed that IMA may have slowed down/ inhibited cell proliferation and growth within the first few hours after treatment. This revealed that IMA may not be toxic at lower concentrations but suggesting that IMA may act as a cytostatic compound that inhibits cellular proliferation at low concentrations (Peña-Morán et al. 2016). Nonetheless, after the first 24 h (after treatment), a rise in recovery and cell proliferation of MDA-MB 231 cells was observed. It may be that MDA-MB 231 formed a resistance mechanism towards the compound (Li et al. 2018), after a certain period of exposure which is what cancer cells do to defend themselves. This result reveals the understanding and importance of shorter and longer drug agent exposure to cells. Even so, the activity of IMA based on the results obtained is highly efficient as compared to the activity of etoposide. By analyzing the real-time cell proliferation results, IMA cytotoxic/cytostatic effect on MDA-MB 231 requires further evaluation to understand its effect on breast cancer cells. IMA exhibited anti-proliferative effects on RMG-1 cell lines (see Fig. 1c, d). In real-time cell viability analysis, IMA exhibited anticancer activity, as inhibition of cell proliferation was observed over 48-h treatments (see Fig. 1d). The same trend from MDA-MB 231 cells results was observed on RMG-1.
IMA reduced the cell viability of non-cancer HEK 293 cell lines in a concentration-dependent manner (see Fig. 1e, f). At 10, 5, and 2.5 µM, there was a decline in cell viability which may show the toxicity of IMA on non-cancer cells. At lower concentrations, IMA did not eradicate or affect the viability of non-cancer cells, but this shows that IMA may be toxic at higher concentrations to both cancer and non-cancer cells. Therefore, optimization of the exact activity of IMA on cancer and non-cancer is required to be investigated in the future. DMSO (0.1%) was used as a negative control. The activity of DMSO may depend on the type of cell lines and it may either slow down cell proliferation or promote cell proliferation. Therefore, based on the cell antiproliferative results, the decreasing concentrations of DMSO did not affect the biological activity of IMA on the cells or contributed to IMA activity.
Furthermore, the results in Fig. 2a, exhibited that IMA induced cell apoptosis on MDA-MB 231 cell lines at IC 50 (2.7 µM). Etoposide showed a higher apoptotic rate than IMA as shown in Fig. 2a, i. This observation may correlate with results obtained in Fig. 1b. Again, the caspase 3/7 results attest to this as IMA expressed caspase 3/7 in low proportions as compared to etoposide. The upregulation of BAX exhibit that some percentages of cells were damaged beyond repair and were signaled to undergo apoptosis hence Fig. 2a, i, iii were observed. It was documented that the p53-independent pathways result in apoptotic response using other alternative death pathways, upregulating BAX and p21 expressions (Shankar et al. 2017). In a previous study, the p53-independent apoptotic pathway was also observed in MCF-7 and T47D cell lines as the p53 response was found to be the same after treatment (Tiwari et al. 2014).
Using flow cytometric analysis of propidium iodide stain (Fig. 2a ii and iv), IMA was assessed its effect on cell cycle progression on MDA-MB 231 cells. Cell cycle arrest is the response to DNA damage or cellular stress. After 24 h treatment, the number of cells in the GO/G1 phase was very low and a significant increase in the number of cells in the S phase was observed, with reductions of cell population in the G2/M phase. The cell cycle results indicated that IMA encouraged cell cycle arrest in the S-phase and early G2 phases. The arrest in the S phase may relate to the induction of p21 expression observed in Fig. 4. This observation suggested that IMA may have prevented the DNA replication process and arrested cells in the S phase. Nonetheless, low expression of CDK2 was observed and this may have slightly delayed the S phase to G2 phase progression (Bačević et al. 2017) as about 25% of G2/M phase cell populations were observed. IMA may possess the ability to cause cell cycle arrest by inhibiting DNA synthesis and cause DNA damage leading to G2 arrest (Badmus et al. 2019).
The results in Fig. 2b i and iii, exhibited that IMA induced cell apoptosis on RMG-1 cell lines at IC 50 concentration (2.3 µM). It can be supported by the upregulation of the BAX gene and the downregulation of anti-Bcl2. Caspase 3/7 activity (Fig. 3b) after treatment with IMA was highly expressed in comparison with etoposide. This correlates with the apoptosis and cell cycle regulation results. Cell cycle arrest was observed in the S phase with a high number of cells as compared to the other phases (Fig. 2b ii and iv). The cell cycle arrest in the S phase only may reveal that IMA has the properties to inhibit damaged DNA to replicate, hence cells were inhibited from progression within 24 h of treatment. Therefore, IMA may possess anticancer effects on RMG-1 cancer cell lines, and it may be a promising agent to develop cancer therapies targeting the synthesis phase. P21 was slightly upregulated on RMG-1 cells and highly upregulated on MDA-MB 231 cells (Fig. 4). P21 is known to enhance apoptosis in the absence of p53 through other complex mechanisms (Manu et al. 2019). The biological activity of IMA may differ based on the type of cell lines and period of exposure.

Conclusion
This study has reported the first findings of the antiproliferative effect of IMA in cancer cell lines. IMA has been shown to inhibit cell proliferation of breast and ovarian cancer cells, modulate the cell cycle arrest, and induced cell death through the intrinsic apoptotic pathway. With the activation of the apoptotic genes such as BAX, and expression of caspase 3/7 pathway. IMA may be considered a promising candidate for the development of anticancer drugs either for its cytotoxic or cytostatic effect with proper regulation on the non-cancer cells. Furthermore, IMA requires to be further studied more to clearly understand its mechanism of action. It will also serve the purpose to evaluate IMA's optimum cytotoxic effect within various time points. In the future, protein expression will be evaluated including other genes involved in the cell cycle arrest and apoptosis such as pRB, p16.

Declarations
Ethical statement This article does not contain any studies involving animals performed by any of the authors. This article does not contain any studies involving human participants performed by any of the authors.

Conflict of interest
Portia P. Raphela-Choma has no conflict of interest. Mthokozisi B. C. Simelane has no conflict of interest. Mpho S. Choene has no conflict of interest.