The combination of fraction A3 of Cyrtostachys renda Blume with doxorubicin improved cytotoxicity against T47D cell line through cell cycle arrest and apoptotic induction

Experimental design

The T47D breast cancer cell line was used in the in vitro assay. The cells were received from Muhammadiyah University’s Faculty of Medicine and Health Sciences in Yogyakarta, Indonesia, through the in vitro culture laboratory. The cell density is determined using a hemocytometer on confluent developing cells (80%) in a tissue culture dish that is planted in a 96-well microplate with 1 × 10⁴ cells per well. This study has been granted ethical approval (No.006/EC-EXEM-KEPK FKIK UMY/IX/2022). The fractionate of C. renda was dissolved with DMSO and cell culture media until the concentration was less than 0.1% v/v. Doxorubicin was utilized as a chemotherapeutic agent in a combination treatment.

Simplicia preparation

Cyrtostachys renda root was collected in Muara Bungo, Jambi Province, and identified at the Herbarium, Plant Taxonomy Laboratory, Padjadjaran University (No.48/HB/04/2022). It was dried for a week before being dried in a 60°C oven for 1 × 24 hours. The roots were then pulverized with the grinder.

Extraction and fractionation

Cyrtostachys renda root powder (500 g) was macerated in methanol (MeOH) three times for 24 hours. A rotary evaporator was used to evaporate the solvent, yielding 237.7 g dried methanol extract. Liquid–liquid extraction with water was performed to separate the secondary metabolite content based on the level of polarity using the consecutive solvents n-hexane, DCM, ethyl acetate (AcOEt), and water (H₂O). After the solvent was removed, the extracts n-hexane, DCM, AcOEt, and H₂O were formed. Each extract was evaluated the cytotoxicity against T47D cells. The most promising extract was subjected to SiO₂ vacuum liquid chromatography. The fractionates were divided into five fractions (A1–A5) based on the similarity of the stain patterns based on the thin layer chromatography profile. Each fraction was then tested for the cytotoxicity against T47D cell line.

Preparation of cell cultures

T47D cells were grown in RPMI 1,640 media supplemented with 10% FBS and 2% penicillin/streptomycin. Cells were cultivated and propagated in a CO₂ incubator with 5% CO₂ in a humid atmosphere at 37°C. Once the cells have reached confluence, they are ready to be used as test subjects.

Cytotoxicity assay

The cytotoxic fraction A3 of C. renda root, doxorubicin and their combination against T47D breast cancer cells was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid (MTT) assay, as reported in previous studies [22]. Each well of 96–well microplates was filled with 100 μl of T47D cell suspension at a density of 1.5 × 10⁴ cells. The fractionates C. renda with a series of concentration variations ranging from 15.63 to 250 μg/ml and doxorubicin with a series of concentration variations ranging from 0.63 to 10 μg/ml added to the cell cultures. Each treatment group was tested three times. After that, the treated cell cultures were incubated for 24 hours at 37°C with 5% CO₂. The culture medium was discarded at the end of the incubation period, and the cells were washed with phosphate-buffered saline (PBS). A volume of 100 μl MTT solution was then added to each well, followed by 4 hours of incubation at 37°C and 5% CO₂. Following the completion of the incubation, 100 μl of 10% sodium dodecyl sulfate reagent was added as a stopper and stored overnight. An ELISA reader was used to measure cell absorbance at 595 nm. The percentage of living cells was calculated by making a curve between the percentage of cell viability and concentration to obtain the IC₅₀ value of the test compound against the T47D cell line. The significance percent test of cell viability was calculated using the following equation:

$\%\mathrm{cell}\mathrm{viability}=\frac{\mathrm{TC}\mathrm{-}\mathrm{CM}}{\mathrm{CC}\mathrm{-}\mathrm{CM}}\times 100$

where TC is the absorbance of treatment cells, CC is the absorbance of control cells, and CM is the absorbance of control media.

The most cytotoxic fraction A3 was mixed with doxorubicin for the combined cytotoxic test. The combined fraction of A3 and doxorubicin dosage was designed to be less than the A3 and doxorubicin IC₅₀ values, as seen in Table 1. The CI metric was used to quantify the synergistic effect of fraction A3 and doxorubicin.

Cell imaging

Phase-contrast microscopy was used to assess changes in cell morphology after the A3 and doxorubicin were applied in the previous stage. An inverted microscope was used to record images of changes in cell morphology.

Flow cytometry

Flow cytometry (BD FACS Calibur) was used to assess apoptosis and the cell cycle. The T47D cell line was cultured in six-well plates at 5 × 10⁵ cells/well and treated for 24 hours with A3 fraction and doxorubicin. Cells were washed with PBS and centrifuged at 2,000 rpm for 5 minutes. After 3 minutes of separation with 200 μl trypsin-EDTA, 1 ml RPMI was added and centrifuged at 2,000 rpm for 5 minutes. Cell cycle analysis was performed on treated cells using the propidium iodide/RNase staining kit (BD PharmingenTM) according to the kit instructions. Following that, the treated cells were stained with the FITC Annexin V labeling kit for apoptosis assay (BD PharmingenTM) according to the kit protocol. Next, Cell flow cytometry profiles were measured.

Statistical analysis

A two-way analysis of variance (ANOVA) was utilized to assess whether there was an interaction between fraction A3 and the doxorubicin combination. To compare the effects of fraction A3 and doxorubicin on the T47D cell line, a one-way ANOVA test was used.

Bioassay guided fractionation

MeOH extract of C.renda root (237.7 g) was successively partitioned into water-n-hexane, water-DCM, and water-AcOEt to distribute the content of secondary metabolites based on their polarity level to afford n-hexane (1.14g), DCM (36.0 g), AcOEt (4.80 g), and H₂O extract (59.0 g). Based on the cytotoxicity test, only the DCM extract displayed potent cytotoxicity against the T47D cell line (Table 2). Then, the cytotoxic DCM extract (36.0 g) was subjected to SiO₂ vacuum liquid chromatography by using combination eluent of DCM-MeOH-H₂O = (30:3:1), (15:3:1), (7:3:1), (3:3:1), MeOH 100% to give five fractions, A1(5.2 g), A2 (4.4 g), A3 (7.6 g), A4 (8.3 g), and A5 (10.5 g). Each collected fraction was evaluated for the cytotoxicity against T47D cell line to disclose fraction A3 exhibited the most potent with an IC₅₀ value of 43.03 μg/ml (Table 2).

The phytochemical studies on the methanol extract revealed that alkaloids were the major component, along with flavonoids, tannins, and saponins. The phytochemicals study was carried out using conventional methods [23].

Doxorubicin and fraction A3 possess single cytotoxic effect

The cytotoxic activity of fraction A3 and doxorubicin in suppressing the proliferation of the T47D cell line was assessed using the MTT test. The ability of dehydrogenase enzymes in living cells to convert MTT substrate into purple formazan crystals was used as a cytotoxic parameter. The intensity of the purple color is proportional to the number of living cells. The number of living cells increases as the intensity of the purple color increases [24]. In contrast, mitochondria in dead cells produce the enzyme tetrazolium succinate reductase, which can degrade the MTT reagent into formazan crystals, resulting in slightly purple formazan and a pink color change [25,26].

Table 1. Matrix simulation of the combination of fraction A3 and doxorubicin against T47D cell line. [Click here to view]

Table 2. IC50 values and cytotoxicity categories for extracts, fractions, and doxorubicin against the T47D cell line. [Click here to view]

Figure 1. Cytotoxic activity of extracts, fractions and doxorubicin against T47D cell line. The panel showed a decrease in cell viability after treatment for 24 hours. The data were independently carried out for three times. (A) Extracts. (B) Fractions. (C) Doxorubicin. [Click here to view]

Based on the data in Figure 1A and B, an IC₅₀ value of DCM extract; 109.95 ± 2.56 μg/ml and fraction A3; 43.03 ± 0.89 μg/ml were determined to be the most potent among other extracts, and fractions A1–A5. Meanwhile, doxorubicin, used as a positive control, had an IC₅₀ value of 2.22 ± 1.02 μg/ml (Fig. 1C). This was validated by the inhibitory value against T47D cell viability; at the lowest dose (15.63 μg/ml), fraction A3 was able to inhibit cell growth up to 38.75% (Fig. 1B), with an IC₅₀ value of 43.03 μg/ml. Cytotoxic activity was divided into three groups based on the IC₅₀ value: IC₅₀ 100 μg/ml showed potentially cytotoxic, 100 μg/ml IC₅₀ 1000 μg/ml indicated moderate cytotoxic, and IC₅₀ > 1,000 μg/ml indicated nontoxic [27]. In this study, when the cytotoxic effect of fraction A3 was compared to the positive control, doxorubicin exhibited a high cytotoxic effect with an IC₅₀ value of 2.22 μg/ml as a single evaluation after 24 hours of treatment. Meanwhile, fraction A3 had modest cytotoxic activity (Tabel 2). Both samples demonstrated concentration-dependent effects against the T47D cell line. According to the National Cancer Institute, a compound possesses cytotoxic activity if its IC₅₀ value is less than 20 μg/ml. The most potent cytotoxic compounds can be utilized to treat cancer, while moderately cytotoxic compounds can be used to prevent or inhibit cancer cells or as co-chemoprevention agents [27,28]. As a result, the IC₅₀ values of fraction A3 are classified as potentially or strong cytotoxic categories as seen in Table 2. Secondary metabolites, particularly flavonoids, tannins, and saponins, are likely to contribute to fraction A3’s cytotoxic activity. Saponins’ harmful effects are caused by the induction of apoptosis or non-apoptosis [29,30]. Flavonoids have the ability to reduce hydroxyl radicals, superoxide, and anti-lipoperoxidants [31].

The cytotoxic effect of fraction A3 in combination with doxorubicin

The combination treatment’s concentration was determined using the IC₅₀ values of doxorubicin and fraction A3, where the concentrations employed were less than their respective IC₅₀ values. Doxorubicin concentrations of 0.14, 0.28, 0.56, and 1.11 μg/ml were all low, as were fraction A3 values of 2.69, 5.33, 10.75, and 21.50 μg/ml. Fraction A3 demonstrated strong cytotoxicity in reducing the development of T47D cancer cells at the given IC₅₀ value. At the lowest dose of 2.69 μg/ml, cell viability reached 47.82%, meaning that fraction A3 may kill 52.20% of T47D cells. Cell viability was 9.63% at the highest dosage of 21.50 μg/ml, showing that fraction A3 may kill 90.40% more T47D cells (Tabel 3). According to these data, the viability of live T47D cells reduced as the percentage fraction A3 concentration increased. The combined therapy resulted in alterations in shape and decreased cell density. The interaction between fraction A3 and doxorubicin was found to be highly significant (p < 0.05) using a two-way ANOVA. The magnitude of this effect was determined using a one-way ANOVA of cytotoxicity with the Scheffé post-hoc comparison. The cytotoxic mean fraction A3 and doxorubicin concentrations varied significantly among the six concentrations. When evaluated with the T47D cell line, the one-way ANOVA of fraction A3 with the Scheffé post-hoc comparison conclusively demonstrated that fraction A3 and doxorubicin were considerably more active doxorubicin than fraction A3 (p < 0.05). Combination of fraction A3 and doxorubicin were found to be significantly more cytotoxic than fraction A3 and doxorubicin single.

Table 3. Cell viability profile of fraction A3 and doxorubicin combination at IC50 sub concentration. [Click here to view]

Synergistic effect

The IC₅₀ value for doxorubicin was 2.22 μg/ml. These data hint at T47D cell resistance to doxorubicin. The fraction A3 of C. renda root, on the other hand, was insufficient to elicit cytotoxicity in T47D cells. As a result, we employed doxorubicin and the fraction A3 C. renda root at various concentrations based on their single cytotoxic IC₅₀ value. This study found out that increasing the concentration in the combination therapy affects cell viability as shown in Table 3. The largest effect was observed at approximately 0.56 μg/ml of doxorubicin and 21.50 μg/ml of A3, with a 95.63% ± 2.30% decrease in cell viability. When doxorubicin with an IC₅₀ value of 0.14 μg/ml was combined with A3 with combined with A3 with an index combination value of 0.07 and 0.08, a very high synergistic effect as in seen in Table 4. These results suggested that fraction A3 could be used to boost doxorubicin’s cytotoxicity against T47D cells.

Cell morphology by treatment with fraction A3 and doxorubicin

Cell morphology was studied using an inverted microscope in addition to quantitative analysis to determine changes in cell morphology before and after therapy. According to the data presented in Figure 2, T47D cells, which were originally elongated, transformed to a round shape, shrank the cell membrane, and became irregular in shape after being treated with fraction A3 and doxorubicin. These findings could be explained by a relationship between the treatment of the test compounds and cell physiological abnormalities, which are characterized by cell death. There was disharmony between the cell groups when tested with doxorubicin and fraction A3 treatment, indicating a change in cell form to the formation of a dead cell that was spherical/globular, and when compared to the cell control, the dead cells are more noticeable with doxorubicin and fraction A3 treatment than those that did not receive treatment (cell control). Significant cell death was demonstrated by changes in cell morphology to be rounder when compared to the cell control, indicating that the cell was dying, on the morphology of cells administered doxorubicin and fraction A3 treatment. Approximately 38% of cells died at a dose of 1.11 μg/ml doxorubicin and 53% died at a dosage of 2.69 μg/ml, and when fraction A3 concentrations were increased in the combination therapy, more cells died.

Flow cytometry profile

Cell cycle modulation effect of fraction A3 and doxorubicin

Flow cytometry was utilized to investigate the association between cytotoxic activity and cell cycle distribution profile caused by fraction A3, doxorubicin, and their combination. Previous studies have suggested that doxorubicin may cause cell cycle arrest in some cancer cells during the S, G1, and G2/M phases [32]. Simultaneously, the fraction A3 was used to arrest the cell cycle in the G1 and S phases. Fraction A3 and doxorubicin had different cell cycle patterns in the T47D cell line, according to cell cycle analysis. The findings of this study revealed that fraction A3 induced cell accumulation in the G1 phase, whereas doxorubicin promotes cell accumulation in the S phase. Interestingly, treatment with a combination of fraction A3 and doxorubicin contributed to cell accumulation, mainly in the G1 and S phases. In addition to the cell arrest of these two phases, cell accumulation in the G2/M phase increased when fraction A3 and doxorubicin were combined. In contrast, the combination fraction A3 and doxorubicin caused more cells to accumulate in the G2/M phase (22.8 %) than the A3 fraction (15.7 %) and doxorubicin (10.6%) (Fig. 3). These findings point to the importance of fraction A3 concentration in enhancing doxorubicin’s cytotoxic effects through cell cycle arrest.

Table 4. CI analysis of fraction A3 with doxorubicin. [Click here to view]

Figure 2. T47D cell morphology. Untreated (A). Doxorubicin 1.11 μg/ml (B). Fraction A3: 21.50 μg/ml (C). Combination of doxorubicin 0.14 and A3: 2.69 μg/ml (D). Combination of doxorubicin 0.14 and A3: 5.38 μg/ml (E). Combination of doxorubicin 1.11 and A3: 21.50 μg/ml (F). Cells with blue arrows have undergone morphological changes. [Click here to view]

The synergistic effect of the combination of fraction A3 and doxorubicin against T47D cells indicates be correlation to the induced apoptosis and cell cycle arrest, especially in the G1 and S phases. Previous studies have shown that Arecaceae active components cause cell cycle arrest in the G1 phase [33,34], while doxorubicin inhibits topoisomerase II, causing cell cycle arrest in the S phase [35,36]. The combination of these two substances alters cell cycle arrest in both phases (G1 and S), causing many cells to fail to divide and decrease cell viability. This experiment was carried out in 24 hours, which is the normal period for one round of cell division. Based on these findings, the A3 fraction was capable of inducing cell death; however, apoptosis assays were required for more information.

Effect of fraction A3 and doxorubicin on apoptosis

Flow cytometry analysis employing the FITC dye Annexin V confirmed decreased cell viability despite cell cycle arrest and subsequently cell death. The treatment results showed that the single A3 fraction and its combination with doxorubicin increased cell death through inducing apoptosis. However, the number of necrotic cell deaths due to a single fraction of A3 and its combination with doxorubicin was higher than that caused by a single doxorubicin treatment (Fig. 4). Overall, flow cytometry analysis showed no significant difference in the evidence of cell death caused by the single fraction A3 or its combination with doxorubicin through apoptosis induction. However, when compared to a single treatment of doxorubicin, the results were significantly different.

The inhibitory effect of C. renda active components on cell cycle progression appears to be typical for many kinds of cancer cells, which can generally promote apoptosis. The cell cycle arrest effect of the fraction A3 combined with doxorubicin against T47D cells appears to be correlated with apoptosis induction. Flow cytometry analysis (Fig. 4) showed a significant difference in the number of cells that experience mortality between a single treatment of doxorubicin and a combination with fraction A3. The fraction A3 and doxorubicin might act sequentially, despite having been shown that cell population arrest is much reduced in the S phase. This occurrence might be due to the antioxidant properties of the fraction A3, which may counteract the increase in intracellular ROS caused by doxorubicin. However, cells that pass through the S phase have the process inhibited by the fraction A3 and arrest at the G2/M phase. This is consistent with the findings of the cell population arrest at the G2/M phase, which showed an increase. This is interesting to explore further in future studies.

A previous study found that alkaloid-containing fractions promoted apoptosis in T47D cells [37], and that arecoline extracted from Areca cathecu may induce apoptosis in HeLa cells [38]. Alkaloids’ potential to trigger apoptosis by boosting caspase-3 activation [39,40]. Alkaloid compounds can also impact the cell cycle [38], and impede the function of P-glycoprotein [41]. As a result, it was conceivable that compounds from the alkaloid group identified in the A3 fraction promote apoptosis in T47D cells, and the mechanism of apoptosis needs to be investigated further. In the T47D cell line, there were two primary apoptotic mechanisms: intrinsic and extrinsic routes. This apoptotic pathway involves the production of regulatory proteins such as p53, the Bcl-2 family, and caspase activation [42]. Because the T47D cell line lacks p53, apoptosis proceeds via a p53-independent route. Alkaloid compounds block the ERK phosphorylation pathway, which is important in breast cancer cell proliferation [43]. Alkaloid components have also been shown to suppress the proliferation of cancer cells by reducing the production of cyclin D1 and CDK4/6, which are essential regulators in the G1 phase [44,45]. The combination of fraction A3 and doxorubicin may cause an increase in apoptotic cells. However, the precise process needs to be investigated further.

Figure 3. The cell cycle distribution of the T47D cell line was altered by fraction A3. doxorubicin, and their combination. Cells were treated for 24 hours with fraction A3 and doxorubicin, subjected to flow cytometric analysis after stained with propidium iodide/RNase. (A) Flow cytogram of cell cycle profiles; (B) Quantification of cell cycle distribution. [Click here to view]

Figure 4. Fraction A3, doxorubicin and their combination induced apoptosis against T47D cell line. Cells were treated for 24 hours with fraction A3 and doxorubicin and at the appropriate concentrations before being stained with FITC Annexin V for flow cytometric analysis. (A) Flow cytogram profiles of treated cells; (B) Quantification of treated cell death population. [Click here to view]