MATERIALS AND METHODS

Study area: The study was carried out at Microbiology and Phytochemistry Lab, Department of Pharmacy, Ma Chung University from April-July, 2020.

Chemicals: Methanol, n-hexane, chloroform, ethyl acetate, n-butanol was purchased from Merck, Germany. 4T1 cells (American Type Culture Collection, Manassas, VA), were cultured in RPMI-1640 medium containing 10% (v/v) Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin (Life Technologies, USA) at 37°C in 5% CO₂ humidified incubator. Vero cells were courtesy from Parasitology Laboratory, Medical Faculty, Gadjah Mada University cultured in Dulbecco’s Modified Eagle Media (DMEM). Doxorubicin (DOX) and 3-(4,5-dimethylthiazol-zyl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma, USA. The 4T1 cells (ATCC^® CRL-2539^™) were incubated with mouse monoclonal anti-Bcl-2 (Dako, Carpinteria, USA) or rabbit polyclonal anti-Bax (Dako) antibody, biotinylated universal secondary antibody (Biocare, USA) and streptavidin HRP label (Biocare). Finally, the cells were added with the DAB substrate (Dako).

Plant collections and extractions: Pterygota alata collected from a specific area of Kupang City, East Nusa Tenggara, Indonesia. Plant identification using the library Flora of Java and identified by Taxonomist, namely Dr. Budi Sumarta. The voucher specimens were deposited in the Laboratory of Pharmacognosy, Ma Chung University Malang, Indonesia with specimen numbers as followed: Pterygota alata (Roxb.) R.Br (MCH/002/X). Extraction using the maceration method, 3.0 kg of dry bark soaked using 80% methanol for 24 hrs. The supernatant and pulp are separated, the supernatant is evaporated until a thick extract is obtained.

Liquid-liquid partitions: About 60.0 g of the extract is placed into the separating funnel and 600 mL of 80% methanol is added. Total 600 mL of n-hexane was added into the solution and vigorously shaken until dual-phase being formed. The hexane phase was collected and then the 80% methanol phase was added and was re-shaken with the chloroform in the same volume ratio until a separate phase was formed. The obtained n-hexane and chloroform phases were then evaporated under reduced pressure to collect n-hexane and chloroform fractions. The 80% methanol phase was added and re-shaken with an equal volume of ethyl acetate until two separate phases were formed. The ethyl acetate phase and 80% methanol phase were dried until ethyl acetate and 80% methanol fractions were obtained. The 80% dry methanol partition was added with n-butanol and stirred for 2 hrs to form an n-butanol partition and an insoluble n-butanol partition. The two partitions were evaporated to obtain n-butanol and insoluble n-butanol fractions. All fractions were used for in vitro cytotoxic tests.

In vitro cytotoxicity assay: Determine the cytotoxicity of on 4T1 and Vero cells; the MTT assay was employed. 4T1 is a triple-negative breast cancer cell line from Mus musculus¹⁷ which contains PEX9 and Vero cell is a non-tumorigenic cell from the kidney tissue of African green monkey¹⁸. The assay detects the reduction of MTT and reflects the normal functioning of mitochondria and hence cytotoxicity. Fraction solutions were prepared in the following concentrations: 10, 20, 40, 80, 160, 320, 640 and 1280 μg mL^–1. Cisplatin was added to the cultures at the following concentrations: 0, 2,5, 5, 10, 20 and 40 μg in a final volume of 100 μL as a positive control using DMSO 0.1%. Cells (1×10⁴/well) were seeded in 96-well at-bottomed plates and incubated with each extract at various concentrations for 24 hrs. Total 30 μL of MTT solution (5 mg mL^–1 in PBS) was added to each well and the plate was incubated at 37°C for another 4 hrs. Then, the medium was discarded and 150 μL of DMSO was added to dissolve the formazan crystals. The test was carried out for 5 replications. The absorbance of each sample was read at 595 nm using a microplate reader. Results were expressed as a percentage of cell viability to untreated control cells (as 100%). Microsoft excel prepared the data calculation and the drug-dose dependent curve.

Immunocytochemistry: T41 cells were planted at a density of 5×10⁴ cells/well. Upon reaching 90% confluent, the cells were treated with ¼IC₅₀, ½IC₅₀ and IC₅₀ of the IFB for 24 hrs. After that, the cells were washed and fixed with methanol, then treated with 3.7% H₂O₂ in methanol for 10 min. Then, the cells were washed and treated with blocking serum for 15 min. The cells were added with a monoclonal anti-Bcl-2 mouse or rabbit polyclonal anti-Bax antibody for 60 min, a biotinylated universal secondary antibody for 20 min and streptavidin HRP label for 10 min. At the end of the process, the cells were added with DAB substrate (Dako) to visualize the antigen-antibody bound and counterstained with hematoxylin. The control group is treated with 1% ethanol as vehicle control. Expressions of Bcl-2 and Bax were observed as the brown color at the cytoplasm and membranes of 4T1 cells under the microscope. The test was carried out five times of repetition. The expressions were then semi quantified using ImageJ version 1.51w software.

GC-MS analysis metabolomic-based assay: The structural identification of the most active fraction was carried on GC-MS. For this 1 mg of the fraction was dissolved in 1 mL of dichloromethane. Rtx^®-50 column (30×250×0.25 μm) was used as the stationary phase with helium as a carrier gas at a flow rate of 1 mL min^–1 and a pressure of 8.80 psi. The GC oven temperature was kept at 70 and 300°C for initial and final temperatures, respectively. The temperature rate increase was set at 10°C min^–1 for 15 min. Splitless injections were implemented with the liquid injection method in this experiment. A library search for the structural identification was carried out using Wiley 7 Nist05 library.

Data analysis: Statistical analysis was performed using a one-way analysis of variance (ANOVA method) followed by post hoc Tukey’s multiple comparisons test in SPSS 20 software. Differences were considered significant at the p<0.05 level.

RESULTS

In vitro cytotoxicity assay: The purpose of this test is to determine the cytotoxic effect of each fraction resulting from the separation. Besides, this test is used to evaluate the selectivity index of the fraction resulting from separation against normal cells. The in vitro cytotoxic test was carried out using the MTT method. The principle of the MTT method is the reduction of the yellow salt of tetrazolium MTT (3-(4.5-dimethylthiazol-2-il)-2,5-diphenyltetrazolium bromide) by the reductase system. First, a test of each fraction resulted from the separation of 4T1 and Vero cells was conducted. We used 4T1 cells as a model of highly metastatic triple-negative breast cancer and Vero as a model for normal cells. Fractions treatment showed a dose-dependent phenomenon in inducing 4T1 cell death. The percentage of viable 4T1 cells was decreased along with the increase of fractions concentration (Fig. 1). The cytotoxic test results showed that IFB was the most active fraction in inhibiting 4T1 cells with an IC₅₀ (Table 1). IFB had no significant difference with cisplatin as a positive control (p>0.05). Another fraction showed weak activity against 4T1 cells with a value range of 88.24-748.24 μg mL^–1.

Fig. 1:

Drug dose-dependent curves of five fractions against 4T1 cells

Fig. 2(a-f):

4T1 cell morphologies after and before treatment with IC50 concentration on each fraction for 24 hrs as followed, (a) Untreated cell, (b) n-Hexane, (c) Chloroform, (d) n-Butanol, (e) IFB and (f) Cisplatin 1: Viable cell, 2: Dead cell. Bar: 100 μm

The results of the cytotoxic test fraction of another test against Cisplatin as positive control showed a significant difference (p<0.05). The potential of a compound is classified as a strong cytotoxic agent if the IC₅₀ value <20 μg mL^–1, moderate if the IC₅₀ value <50 μg mL^–1 and weak if the IC₅₀ value >50 μg mL^–1.

The selectivity index of chemo preventive agents is a test parameter to determine that only cells identified as cancer cells are inhibited growth. The higher the selectivity index value means that the test compound can inhibit cancer cells and has a low cytotoxic effect on normal cells. A small selectivity index indicates that the compound is less toxic to normal cells rather than to cancer cells. Compounds with high SI values offer the potential for safer and more effective therapy in cancer therapy. The test results showed that IFB was selective against 4T1 cells with SI values>3, while the other fractions were less selective against 4T1 cells with SI<3.

Morphology fraction-treated 4T1 cells: Morphological changes in 4T1 cells after administration of each fraction and positive control with a concentration of IC₅₀ for 24 hrs are shown in Fig. 2. Living cells appeared as leaf-shape and had black spots, while dead cells as round and white. Cells show morphological changes that are possible because the cytoskeleton is cut and proteins that play a role in cell adhesions do not undergo polymerization so that the cell bonds are released and the lipid membrane will become round (Fig. 2a-f).

Fig. 3(a-e):

Bcl-2 expression of IFB on 4T1 cells. 4T1 cells were treated with (a) Control, (b) 3.8, (c) 7.71, (d) 15.14 μg mL–1 IFB and (e) Graphical representation of Bcl-2 expression Cells were processed with immunohistochemistry to detect Bcl-2 as described in Methods. Semiquantified Bcl-2 expression with ImageJ. Bar: 150 μm. Values area means±SD, n = 5 Replicates, *p-value<0.05 then indicate statistically significant differences in comparison to control

Bcl-2 expressions of IFB-treated 4T1 cells: The expression of Bcl-2 can be seen in Fig. 3. Expressions of Bcl-2 were indicated by the presence of Bcl-2 monoclonal antibodies bonds, detected in brown color in the cytoplasm and membrane of 4T1 cells (Fig. 3a-d).

Fig. 4(a-e):

Bax expression of IFB on 4T1 cells 4T1 cells were treated with (a) Control, (b) 3.8, (c) 7.71, (d) 15.14 μg mL–1 IFB and (e) Graphical representation of Bax expression Cells were processed with Immunohistochemistry to detect Bax as described in Methods. Semiquantified Bcl-2 expression with ImageJ. Bar: 150 μm. Values area means±SD, n = 5 Replicates, *p-value<0.05 then indicate statistically significant differences in comparison to control

The arrows point to the positive-staining cells. The Bcl-2 expression semi quantification of control (1% ethanol), 3.8 (¼ of IC₅₀), 7.71 (½ of IC₅₀) and 15.14 (IC₅₀) μg mL^–1 IFB-treated 4T1 cells were 9.72, 6.67, 2.83 and 1.31% (Fig. 3e). These results showed that the treatment of 3.8, 7.71 and 15.14 μg mL^–1 IFB decreased the expression of Bcl-2 until 22, 65 and 91%, respectively. The test results at each test concentration of all fractions showed a significant difference with the control group (p<0.05).

Bax expressions of IFB-treated 4T1 cells: The expression of Bax can be seen in Fig. 4. Expressions of Bax were indicated by the presence of Bax monoclonal antibodies bonds, detected in brown color in the cytoplasm and membrane of 4T1 cells (Fig. 4a-d). The arrows point to the positive-staining cells.

Fig. 5:

GC chromatogram of IFB of P. alata bark

The Bax expression semi quantification of control (1% ethanol), 3.8 (¼ of IC₅₀), 7.71 (½ of IC₅₀) and 15.14 (IC₅₀) μg mL^–1 IFB-treated 4T1 cells were 1.13, 3.17, 6.38 and 8.5% (Fig. 4e). These results showed that the treatment of 3.8, 7.71 and 15.14 μg mL^–1 IFB increased the expression of Bax until 16, 45 and 65%, respectively. The test results at each test concentration of all fractions showed a significant difference with the control group (p<0.05).

GC-MS analysis of IFB: The pharmacological activity of a plant will be determined by its metabolites. Therefore we analyzed the compounds contained in IFB using GC-MS to determine the metabolite profile. There are a total of 12 compounds identified in the IFB, representing about 100% of the total compound. The major components of IFB are beta-caryophyllene (26.12%), beta-bisabolene (9.65), hexadecanoic acid (9.33) and 1,3-dimethylbutyl acetate (8.20%) (Fig. 5 and Table 2). The results of the literature search show that almost all chemical compounds contained in IFB have cytotoxic activity. Likewise, we studied that there are taraxerol compounds that have antioxidant and cytotoxic activity on MCF-7 cells and T47D cell lines. As a result, the IFB on cancer and normal cells in concern with the metabolic profiles needed to be explored further.

DISCUSSION

Although P. alata has been widely known to have pharmacological activity, in anticancer tests, it has not been widely studied. This is a critical point to prove P. alata has an activity to maintain health and has anticancer activity, so our focus is to evaluate the evidence-based cytotoxic properties of metastatic breast cells, namely 4T1 cells and normal cells, namely Vero cells. The results of the study prove that cisplatin can inhibit the growth of 4T1 cells, cisplatin is reported to be able to induce Bcl-2 downregulation in T47D breast cancer cells³¹. It can affect DNA replication and transcription, resulting in stopping the cell cycle in the S phase and triggering apoptosis³². Downregulation of Bcl-2 (anti-apoptotic protein) will decrease cell survival and increase sensitivity to chemotherapy agents³³. From the statistical test results, Cisplatin and IFB did not have a significant difference. The study indicated that IFB has almost equal power in inhibiting the growth of 4T1 cells so that IFB is used for the next test phase, especially in its role in apoptosis.

Apoptosis plays a massive role in tumor development and progression by eliminating the cancerous and preneoplastic cells form. Morphological analysis of cells demonstrating IFB induces typical apoptotic characteristics such as nuclear shrinkage and fragmentation³⁴. As apoptosis is modulated by a group of anti-apoptotic and pro-apoptotic regulators, we examined the expression of these key regulatory proteins in apoptosis in the tumor sections. Bax promotes the release of cytochrome C into the cytosol from the mitochondria³⁵. The antiapoptotic proteins, such as Bcl-2 preserve the integrity of the mitochondria. This blocks the release of cytochrome C that activates the effectors of apoptosis³⁶. Since Bcl-2 functions by forming a heterodimer with its pro-apoptotic partner Bax, the Bcl-2/Bax ratio is proportional to the relative sensitivity or resistance of the cells to various apoptotic stimuli³⁷. By comparing with vehicle control, our work indicated that IFB specifically and significantly increased the number of cells that expressed Bax and decreased the number of cells that expressed Bcl-2. These data show that increased numbers of cells underwent apoptosis after IFB treatment, suggesting that IFB inhibited breast tumor growth by induction of apoptosis. Besides, these data indicate that the molecular mechanism occurs through IFB induced mitochondria-mediated apoptosis. Apoptosis occurs by inhibiting the expression of the anti-apoptotic factor that releases cytochrome C from the mitochondria and activation of caspase-3. Besides, the results strongly supported the hypothesis of the mitochondrial apoptotic pathway involvement.

The chemical compounds in IFB are responsible for cytotoxic activity (Table 2), lowering BCL-2 levels and increasing Bax levels. The class of compounds detected from GC-MS in IFB is a polyketide compound. Many polyketide compounds are known to have cytotoxic activity^37-42. Germacrene has cytotoxic activity on LNCaP cancer cells¹⁹, besides that according to the study of Govindam et al.⁴³ reported that germacrene isolated from the soft coral Lobophytum pauciflorum has an activity to inhibit human epidermoid carcinoma A431 cells by inhibiting the activation of NF-κB activation by IKKa/b. NF-κB has an essential role as transcription factors that regulate the expression of anti-apoptotic genes⁴⁴. Beta-caryophyllene is found as a major compound (26.12%) in IFB. Major compounds have a considerable role in pharmacological activity⁴⁵. Park et al.⁴⁶ reported that Beta-caryophyllene isolated from Psidium guajava leaves could induce apoptosis in PC-3 and MCF-7 cells by inducing Bax. Bax induces the release of Cytochrome C from mitochondria explicitly, Cytochrome C will form a complex with Protease Activating Factor-1 (Apaf1) and Procaspase-9 apoptotic which are called apoptosome complexes⁴⁷. The apoptosome will activate caspase-9 and there will be activation of caspase 6 and caspase 7 and induce apoptosis⁴⁸.

Besides apoptosis, cytotoxic effects can also occur due to the induction of cell cycle⁴⁹. Abdel Bar et al.⁵⁰ reported that methyl eugenol has a cytotoxic effect on human cervical cancer cells (HeLa, C4-1, MEK-180, C33A) by inducing G2/M cell cycle arrest. Beta-bisabolene has also been reported to be induced G2/M cell cycle arrest and apoptosis, elevated levels of cell cycle regulatory proteins p53 and p21 and lowered levels of G2/M checkpoint proteins Cdc2 and Cyclin B1 in melanoma cells⁵¹. The cytotoxic effect can occur due to the various chemical compounds possessed by IFB; the cytotoxic effect can occur due to the synergistic effect of the compounds it has. Either through the process of apoptosis or induction of the cell cycle. However, all these things need to be proven more deeply to ensure the cytotoxic mechanism against 4T1 cells.

CONCLUSION

Pterygota alata has empirically beneficial as traditional medicine, especially on the bark. The bark of P. alata was fractionated using five types of solvents. The insoluble fraction of n-butanol (IFB) had the highest cytotoxic activity against 4T1 cells and was less toxic to normal cells. IFB induces apoptosis in 4T1 cells through the intrinsic apoptotic pathway by decreasing Bcl-2 expression and increasing Bax expression. The cytotoxic IC₅₀ of IFB on 4T1 cells was 15.14±2.81 μg mL^–1. GC-MS spectrum analysis showed that IFB had five main compounds, namely beta-caryophyllene (26.12%), beta-bisabolene (9.65), hexadecanoic acid (9.33) and 1,3-dimethylbutyl acetate (8.20%), which were proven to have cytotoxic activity.

SIGNIFICANCE STATEMENT

This research was conducted to investigate the active compounds in P. alata bark which have cytotoxic activity with apoptotic mechanisms. This study will provide new evidence and theory to researchers about the use of P. alata medicinal plants for the treatment of breast cancer.

ACKNOWLEDGMENT

This research article is part of the doctoral thesis and this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.