Promising effect of cisplatin and melatonin combination on the inhibition of cisplatin resistance in ovarian cancer

Materials and sample size

In this study, a laboratory experiment on the combination of cisplatin and melatonin in cisplatin-resistant ovarian cancer cell was conducted using SKOV3 obtained from the American Type Culture Collection (no. HTB-77). This research was conducted at the SCTE (Stem Cell and Tissues Engineering Research Cluster) at the Medical Faculty of Universitas Indonesia. Furthermore, flow cytometry (BD FACS ARIA III) was performed at the Integrated Laboratory of the Faculty of Medicine, Universitas Indonesia, from September 2020 to November 2021. This research has been approved by the ethics committee of Universitas Indonesia with 419/UN2.F1/ETIK/PPM.00.02/2021 on May, 3^rd 2021.

The materials used were IC50 melatonin [Liftmode], IC50 doxorubicin [MBS], IC50 cisplatin [Mybiosource], sodium bicarbonate [Sigma-aldrich], aquabidest, microcarrier beads [Sigma-aldrich], trypsin-EDTA [Gibco], phospate buffer saline (PBS) [Gibco], dimethyl sulfoxide (DMSO) [Gibco], Roswel Park Memorial Institute [RPMI] medium, antibiotic [Penstrep], antimycotic [Gibco], tryphan blue [Gibco], heparin [Gibco], fetal bovine serum (FBS) [Amresco], ethanol 70%, aquadest, whitening [Bayclin], Tris-Cl 0,5 M pH 6,8 [Invitrogen], and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) [Promega]. This research used 8 groups of samples. Mead’s formula was used to calculate sample size. Minimum samples were 16 with three times repetition in each group, so the total was 24 samples. The samples were described in Table 1 and for the protocols can be accessed from dx.doi.org/10.17504/protocols.io.yxmvm2539g3p/v1.

The groupings were as follows: cell control (without any treatment), IC50 doxorubicin, IC50 melatonin, IC50 cisplatin, C1 (combination of 1× IC50 cisplatin and 1x melatonin), C2 (combination of ¾× IC50 cisplatin and ¾× melatonin), C3 (combination of ½× IC50 cisplatin and ½× melatonin), and C4 (combination of ¼× IC50 cisplatin and ¼× melatonin). The dependent variables were the morphology of SKOV3 cells visualized using a microscope with 100x magnification, percentage of cancer cell viability determined via MTS assay, and cell percentage that expressed CTR-1 (as an influx marker) [MBS], p-glycoprotein (Pgp) (as an efflux marker) [FITC], gamma-glutamylcysteinylglycine (GSH) (as a drug inactivation marker) [GSH Kit], excision repair cross-complementation 1 (ERCC 1) (as a damage repair marker) [PE 647], e-cadherin (as a mesenchymal epithelial transition marker) [FITC], and apoptosis (as an annexin V marker) [Mybiosource] using flow cytometry. Positive control was performed with doxorubicin.

SKOV 3 cell culture

Treated cells were harvested by adding 1 mL tripsin-EDTA, centrifuged at 2000 rpm for 5 minutes. The fifth passage of the SKOV3 cell was obtained from a cryopreservation tank and thawed at 37°C for 2 min. Then, the cell was centrifuged for 10 min, and the supernatant was discharged; 1–2 mL of the complete medium was added for cell counting. The cells were cultured and harvested, 2–3 mL of trypsin was added and incubated for 5 min, then the suspension was centrifuged for 10 min and then re-harvested from the 96-well plate for the MTS assay [Promega].

IC50 Determination

IC50 was used to express the 50% decrease in cell viability. To determine the IC50, each concentration of solutions was tested in each cell. The IC50 values of melatonin, cisplatin, and doxorubicin were assessed. Different concentrations were tested to find the IC50 of each variable as follows: melatonin (0.1, 0.5, 1, 2, 5 mM), doxorubicin (25, 50, 100, 150, 200, 300 μM), and cisplatin (10, 20, 40, 50, 80, 100, 200 μM). Spectrophotometry [Shimadzu] was employed with λ = 490 nm for the absorbency. Graphpad software was used to analyze the IC50 values. The IC50 values of the materials were 1.841 mM for melatonin, 117.5 μM for cisplatin, and 14.72 μM for doxorubicin.

Cytotoxic activity

Cells were treated using Roswell Park Memorial Institute (RPMI) medium consisting of 1% penicillin-streptomycine [Penstrep], 1% fungizone [Gibsco], 10% fetal bovine serum [Amresco], and ethanol 70% in a flask incubated at 37°C with CO₂ 5%. SKOV3 cells were seeded onto the 96-well plate with 25x10⁴ cell/well density, then incubated at 37°C for 24 h, then each cell was exposed to each material and incubated for 48 h. To measure the cell viability percentage and cytotoxic activity, MTS assay was employed. The principle is formazan crystal will be produced by the viable cell much more than the nonviable cell. Each of the materials were exposed to the cells and MTS solution was added at each of the wells, then read in λ = 490 nm.

Preparation of cell for flow cytometry

Flow cytometry was performed by adding EDTA into the plate and then centrifuging several times, and cell pellets were washed once with PBS. The cell was put into flow cytometry tube and added 1 mL stain buffer solution, then centrifuged at 2100 rpm for 5 minutes. The supernatant was then separated and the sediment was collected.

Flow cytometry of CTR-1 and Pgp

P-glycoprotein antibody was added into the sediment and incubated for 15 minutes at room temperature, washed with buffer stain, ad centrifuged at 2100 rpm for 5 minutes. 1 mL cytofix was then added and incubated at room temperature for 10 minutes. The cell was centrifuged at 2100 rpm for 5 minutes, the supernatant was discharged and washed with 1 mL of permwash buffer, then recentrifuged at 2100 rpm for 5 minutes. The supernatant was separated, CTR-1 antibody was added, and it was incubated for 20 minutes at room temperature, then washed with permwash buffer and recentrifuged at 2100 rpm for 5 minutes. Samples were analyzed with flow cytometry (BD FACS ARIA III).

Flowcytometry of GSH

5 μL of GSH antibody was added into the sample, which was then incubated at 37°C for 30 minutes, washed with 1 mL buffer, and centrifuged at 2100 rpm for 5 minutes. The sediment was dissolved with stain buffer. Samples were analyzed using flow cytometry (BD FACS ARIA III).

Flowcytometry of ERCC-1 and E-Cadherin

Antibodies of ERCC and E-Cadherin were added into the sample, incubated at room temperature for 20 minutes, then washed with 1 mL permwash buffer for 5 minutes and centrifuged at 2100 rpm for 5 minutes. The sediment was collected and dissolved with stain buffer. Samples were analyzed using flow cytometry (BD FACS ARIA III).

Flowcytometry of apoptosis

0.1 mL annexin V reagent was added into the sediment, incubated at room temperature for 20 minutes, washed with 1 mL stain buffer and recentrifuged at 2100 rpm for 5 minutes. The sediment was collected and stain buffer was added. Samples were analyzed using flowcytometry (BD FACS ARIA III).

Statistical analysis

Mean ± SD was used to express the data. ANOVA with post hoc Bonferroni test was employed for statistical analysis, with significant differences determined as p <0.05. All the statistics were analyzed using SPSS 21.

Influx mechanism using CTR1

CTR1 is a marker of drug influx. An increase in CTR1 expression indicates an increase of drug influx into the cell, which suggests decreased cancer cell resistance to chemotherapy. As can be seen from Table 3 and Figure 3, the control group has the lowest mean expression among the groups. On the contrary, the doxorubicin group has the highest CTR1 expression, indicating that doxorubicin has a higher influx rate than other groups. However, among the combination groups, C1 and C2 had the highest CTR1 expression (P < 0,001), suggesting that these groups have the highest influx rate among the experimental groups. The IC50 melatonin group had lower CTR1 expression than groups C1 and C2, which shows that chemotherapy influx was better in C1 and C2 than the melatonin-only group. If compared with the IC50 of cisplatin, all the combination groups show that the CTR1 expression was higher than the cisplatin alone, which indicated that the combination of cisplatin and melatonin groups were better to increase the influx of the drug than the cisplatin-only group.

Figure 3. CTR 1 expression percentage.

Efflux mechanism using P-glycoprotein (Pgp)

Pgp is an efflux membrane transporter of toxins, cell endogen metabolites, and chemotherapy especially in resistance cells. A high percentage of Pgp indicates a high ability of the cell to inhibit a cytotoxic response, suggesting high chemotherapy resistance of cancer cells. As can be seen from Table 4 and Figure 4, the control group has the highest Pgp expression (44.37%). Among the combination groups, from the mean expression, C1 group has the lowest Pgp expression (6.7%) of all combination groups and C4 has the highest Pgp expression (20.87%). This result indicates that group C1 had the ability to decrease Pgp expression more than the cisplatin-only group (16%). From pair of IC50 melatonin and C1 group, it was shown no difference statistically, but from other pairs shown significantly difference in posthoc analysis. A low rate of drug efflux activity indicates decreased chemotherapy resistance and better outcome. The combination groups had better ability to decrease the efflux mechanism than the cisplatin-only group.

Figure 4. P glycoprotein expression percentage.

Drug inactivation mechanism using GSH

GSH is a peptide, which involved in drug inactivation protein marker. As can be seen from Table 5 and Figure 5, the control group has the highest GSH peptide level (45.23%), whereas the positive control group has the lowest (1.33%), indicating that without giving the materials, the GSH level would high. But if doxorubicin is given, the GSH level would be lowered. Among the combination groups, group C1 has the lowest GSH level (11.73%). When compared with the melatonin-only group (12.57%), group C1 had a lower GSH level. On the other hand, when compared with the cisplatin-only group, all of the combination groups had a higher ability to decrease the GSH level than the cisplatin-only group. But, from pair of IC50 melatonin and C1 in the post hoc analysis, shown no difference statistically. This suggested that the combination groups had decreased drug inactivation activity and chemotherapy resistance as well as a better outcome than the cisplatin-only group. This is presented in Table 5.

Figure 5. GSH level percentage.

Excision repair cross-complementation 1 (ERCC1) mechanism

ERCC1 is a transcription factor used as a DNA repair marker. A high ERCC1 expression indicates high DNA repair mechanism in cancer cells. As can be seen from Table 6 and Figure 6, the control group has the highest ERCC1 expression (48.07%), whereas the positive control group has the lowest (1.57%). Among the combination groups, groups C1, C2, and C3 have the ability to decrease the ERCC1 expression more than the melatonin-only and cisplatin-only groups, but C1 group had the highest ability. The lower the DNA repair activity, the more the chemotherapy resistance decreases, which is shown in combination of melatonin and cisplatin groups.

Figure 6. ERCC1 expression percentage.

E-cadherin examination

E-cadherin is a marker of epithelial mesenchymal transition. A high e-cadherin expression indicates low epithelial mesenchymal transition, which has a low impact on tumor invasion and drug resistance. As can be seen from Table 7 and Figure 7, the positive control group has the highest e-cadherin expression (64.2%), whereas the control group has the lowest (1.7%). When compared with the cisplatin-only group, the combination groups C1-C3 had higher e-cadherin expression than the cisplatin-only group. This suggested that the combination of melatonin and cisplatin might decrease epithelial mesenchymal transition (increased expression of e-cadherin) more than the cisplatin-only group; a decrease in drug resistance could also be observed.

Figure 7. E-Cadherin expression percentage.

Apoptosis mechanism using annexin V

Annexin V was used as an apoptosis marker in this research. A high annexin V activity indicates high apoptosis activity. As can be seen from Table 8 and Figure 8, the positive control group has the highest annexin V activity (70.9%), whereas the control group has the lowest (1.2%). Among the combination groups, group 1 has the highest annexin V activity (53.57%). Compared with the melatonin-only group, all combination groups had a higher ability to decrease annexin V expression. Additionally, compared with the cisplatin-only group, the combination groups all had a better ability to increase apoptosis than the cisplatin-only group. This suggested that the combination of melatonin and cisplatin increases apoptosis to reduce the drug resistance mechanism.

Figure 8. Annexin V expression percentage.

Limitation

This study has shown that cisplatin combine with melatonin may reduce the cisplatin resistance in ovarian cancer, but this study only use one cell line which is SKOV3. In order to see the impact of the combination, study on the several cell lines should be performed. The result of this study may just generally indicative, so another study must be performed and added to see the effect of the melatonin and cisplatin combination in reducing the chemoresistance. The authors also suggest that there should be another research that performed in vivo to see the bioavailability and dose of the combination melatonin and cisplatin, so it can be optimized the cancer treatment.