RESEARCH ARTICLE
Promising effect of cisplatin and melatonin combination on the inhibition of cisplatin resistance in ovarian cancer
[version 1; peer review: awaiting peer review]
Cut Adeya Adella
1, M Fidel Ganis Siregar
2, Imam B Putra
3, Poppy Anjelisa Hasibuan
4, Andrijono Andrijono
5, Adang Bachtiar
6, Sarma N Lumbanraja
7, Iqbal P Nasution
81Obstetric and Gynecology Department, Gynecology Oncology Division, Medical Faculty, Universitas Sumatera Utara, Medan, Sumatera Utara, 20155, Indonesia
2Obstetric and Gynecology Department, Fertility Endocrinology Reproduction Division, Medical Faculty, Universitas Sumatera Utara, Medan, Sumatera Utara, 20155, Indonesia
3Dermatology and Venerology Department, Medical Faculty, Universitas Sumatera Utara, Medan, Sumatera Utara, 20155, Indonesia 4Faculty of Pharmacy, Universitas Sumatera Utara, Medan, Sumatera Utara, 20155, Indonesia
5Obstetric and Gynecology Department, Gynecology Oncology Division, Universitas Indonesia, Jakarta, DKI Jakarta, 10430, Indonesia 6Public Health Faculty, Universitas Indonesia, Jakarta, DKI Jakarta, 10430, Indonesia
7Obstetric and Gynecology Department, Feto Maternal Division, Medical Faculty, Universitas Sumatera Utara, Medan, Sumatera Utara, 20155, Indonesia
8Surgery Department, Medical Faculty, Universitas Sumatera Utara, Medan, Sumatera Utara, 20155, Indonesia
First published: 21 Mar 2023, 12:313
https://doi.org/10.12688/f1000research.130172.1 Latest published: 21 Mar 2023, 12:313
https://doi.org/10.12688/f1000research.130172.1
v1
Abstract
Background: Ovarian cancer management has not yet given a satisfactory result, and the recurrence rate is still high. One of the reasons for this is resistance to chemotherapy. Melatonin and cisplatin may be involved in the chemotherapy resistance of ovarian cancer.
Methods: A laboratory experiment was performed using melatonin and cisplatin in the SKOV3 cell, from September 2020 to November 2021 at the SCTE and Integrated Laboratory & Research Center Universitas Indonesia. Several variables were used, such as doxorubicin, melatonin, cisplatin, and combination of cisplatin and melatonin at several concentrations (1×, 3/4×, 1/2×, and 1/4×). A total of 24 samples were included and divided into 8 groups. The IC50 values of melatonin, doxorubicin, and cisplatin as well as cell viability was calculated via MTS assay. Subsequently, flow cytometry was performed to assess the effect of cisplatin and melatonin on the mechanisms of CTR1, p-glycoprotein, GSH, ERCC1, e-cadherin, and apoptosis. Analysis of variance and Bonferroni test were employed for the study.
Results: The IC50 values of melatonin, cisplatin, and doxorubicin were 1.841 mM, 117.5 mM, and 14.72 mM, respectively. The combination
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Corresponding author: Cut Adeya Adella (adeya.adella@gmail.com)
Author roles: Adeya Adella C: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Resources, Supervision, Writing – Original Draft Preparation; Siregar MFG: Formal Analysis, Supervision, Validation, Visualization, Writing – Review & Editing; Putra IB: Formal Analysis, Project Administration, Supervision, Validation, Writing – Review & Editing;
Hasibuan PA: Formal Analysis, Resources, Supervision, Visualization, Writing – Review & Editing; Andrijono A: Conceptualization, Supervision, Validation, Writing – Review & Editing; Bachtiar A: Formal Analysis, Supervision, Validation, Writing – Review & Editing;
Lumbanraja SN: Supervision, Visualization, Writing – Review & Editing; Nasution IP: Supervision, Validation, Writing – Review & Editing Competing interests: No competing interests were disclosed.
Grant information: The author(s) declared that no grants were involved in supporting this work.
Copyright: © 2023 Adeya Adella C et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite this article: Adeya Adella C, Siregar MFG, Putra IB et al. Promising effect of cisplatin and melatonin combination on the inhibition of cisplatin resistance in ovarian cancer [version 1; peer review: awaiting peer review] F1000Research 2023, 12:313 https://doi.org/10.12688/f1000research.130172.1
First published: 21 Mar 2023, 12:313 https://doi.org/10.12688/f1000research.130172.1 groups of cisplatin and melatonin reduced cell viability; decreased the CTR1 mean (19.73), Pgp (6.7), GSH (11.73), and ERCC1 (4.27) in the combination 1 (C1) group; and increased e-cadherin (32.2) and annexin V (53.57) also in the combination 1 (C1) group.
Conclusions: The combination of melatonin and cisplatin might have an impact on drug resistance via several mechanisms in ovarian cancer.
Keywords
Cisplatin, melatonin, doxorubicin, SKOV3
This article is included in the Oncology gateway.
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Introduction
To date, the management of ovarian cancer, especially epithelial ovarian cancer, has not yet given satisfactory results (Raziet al., 2016). Ovarian cancer is known to have high morbidity and mortality rates as well as poor prognosis (Coburn et al., 2017). It ranks third in the world after cervical and uterine cancer (Brayet al., 2018). According to the Global Burden Cancer 2020 database, the number of ovarian cancer cases worldwide is 313,959, with the number of deaths being 207,252 (Sunget al., 2021). In Indonesia, the rate of ovarian cancer is still high at around 3,398 from 2016 to 2020 (INASGO, 2021).
At present, ovarian cancer recurrence rate is still high and influences the morbidity and mortality rates. One of the reasons why the recurrence rate remains high is chemotherapy resistance, especially by cisplatin at cancer stem cell (CSC), because evasion from apoptosis caused the cell to redevelop after therapy has been performed (Wooet al., 2012). There are multiple factors in cisplatin resistance, and the mechanism is still unclear from the perspective of cellular mechanism (Sousa,et al.,2014).
There are many intracellular mechanisms that affect the evasion of a cell from the cytotoxic effect of chemotherapy, such as the process that regulates drug bioavailability, mesenchymal epithelial transition, and oncogenic signals producing a phenotype that causes resistance to chemotherapy (Yeldag,et al.,2018).
Melatonin has been considered an alternative for managing chemotherapy resistance, especially in ovarian cancer (Chuffa,et al.,2017). It is synthesized at the pineal gland. Melatonin exerts antioxidant and antiapoptotic effects on normal cells but exerts pro-oxidative, antiproliferative, antiangiogenic, and immunomodulatory effects on cancer cells, especially hormone-dependent cancer. Melatonin has anticancer effects, e.g., it potentiates antimetastasis, enhances drug sensitivity, induces apoptosis, inhibits cancer growth, and exhibits antiangiogenic and antiinvasive activities (Su,et al., 2017). In addition, melatonin has a good effect on clinical outcomes in several cancers such as colon, breast, lung, and ovarian cancers. However, to date, the study of melatonin in cisplatin-resistant cancer is scarce. Therefore, this study was conducted to find new alternatives for ovarian resistant cancer cell by using a combination of cisplatin and melatonin.
Methods
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, 3rd2021.
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], and3-(4,5-dimethylthiazol-2-yl)-5-(3-carbox- ymethoxyphenyl)-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 inTable 1and for the protocols c] an be accessed fromdx.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 1IC50 cisplatin and 1x melatonin), C2 (combination of ¾IC50 cisplatin and ¾melatonin), C3 (combination of ½IC50 cisplatin and ½melatonin), and C4 (combination of ¼IC50 cisplatin and ¼melatonin).
The dependent variables were the morphology of SKOV3 cells visualized using a microscope with 100x magnification, percentage of cancer cell viability determinedviaMTS 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, 300μM), and cisplatin (10, 20, 40, 50, 80, 100, 200μM). 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, 117.5μM for cisplatin, and 14.72μM 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 37oC with CO25%. SKOV3 cells were seeded onto the 96-well plate with 25x104cell/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 inλ= 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
5μL of GSH antibody was added into the sample, which was then incubated at 37oC 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).
Table 1.Sample size in each group.
Study groups Well Size
Cell Control 3
IC50 Doxorubicin 3
IC50 Melatonin 3
IC50 Cisplatin 3
Group 1 1 x IC50 (Melatonin-Cisplatin) 3
Group 2¾x IC50 (Melatonin-Cisplatin) 3
Group 3½x IC50 (Melatonin-Cisplatin) 3
Group 4¼x IC50 (Melatonin-Cisplatin) 3
TOTAL 24
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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
MeanSD was used to express the data. All the statistics were analyzed using SPSS 21.
Results
In this study, SKOV3 cells were used. SKOV3 cell culture was observed under a microscope, as presented inFigure 1.
After calculating the IC50 values of the materials (melatonin, 1.841 mM; cisplatin, 117.5μM; and doxorubicin, 14.72 μM), cell viability was calculatedviaMTS assay and observed under microscope as inFigure 2. The highest rate of cell viability decrease was in group C1 (IC50 melatonin and cisplatin) at 37.57%, as shown inTable 2(Adella 2023a;Adella 2023b;Adella 2023c).
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 fromTable 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 decrease the influx of the drug than the cisplatin-only group.
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 fromTable 4, the control group has the highest Pgp expression (44.37%). Among the combination groups, group C1 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%). 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.
Drug inactivation mechanism using GSH
GSH is a drug inactivation protein marker. As can be seen fromTable 5, the control group has the highest GSH expression (45.23%), whereas the positive control group has the lowest (1.33%), indicating that without giving the materials, the
Figure 1.SKOV 3 cell culture under microscope with 100×magnification.
Figure 2.Microscopic feature of SKOV3 after exposure of MTS for 48 hours with magnification 100×.
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Table 2.Viability cell study.
Study groups Percentage of viability cell
Cell control 100%
Medium control 0%
IC50 Doxorubicin 38.31%
IC50 Melatonin 47.68%
IC50 Cisplatin 55.16%
Group 1 (1x IC50 Melatonin + IC50 Cisplatin) 37.57%
Group 2 (3/4x IC50 Melatonin + IC50 Cisplatin) 40.63%
Group 3 (1/2x IC50 Melatonin + IC50 Cisplatin) 42.78%
Group 4 (1/4x IC50 Melatonin + IC50 Cisplatin) 48.43%
Table 3.CTR 1 expression in study groups.
Groups Mean (SD) pa Posthocb
Dox Mel Cis C1 C2 C3 C4
Control 2.17 (0.21) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 IC50
Doxorubicin
30.33 (0.4) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
IC50 Melatonin
14.8 (0.1) <0.001 <0.001 <0.001 1.000 0.001
IC50 Cisplatin
7.37 (0.7) <0.001 <0.001 <0.001 <0.001
C1 19.73 (0.49) 1.000 <0.001 <0.001
C2 18.73 (0.84) <0.001 <0.001
C3 14.53 (1.14) 0.002
C4 11.77 (0.55)
aANOVA.
bBonferroni.
Table 4.Pgp expression in study groups.
Groups Mean (SD) pa Posthocb
Dox Mel Cis C1 C2 C3 C4
Control 44.37 (1.55) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 IC50
Doxorubicin
0 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
IC50 Melatonin
7.37 (0.21) <0.001 1.000 0.986 <0.001 <0.001
IC50 Cisplatin
16 (1.59) <0.001 <0.001 1.000 <0.001
C1 6.7 (0.17) 0.161 <0.001 <0.001
C2 9.1 (0.1) <0.001 <0.001
C3 15.43 (1.25) <0.001
C4 20.87 (0.49)
aANOVA.
bBonferroni.
GSH expression would high. But if doxorubicin is given, the GSH expression would be lowered. Among the combination groups, group C1 has the lowest GSH expression (11.73%). When compared with the melatonin-only group (12.57%), group C1 had a lower GSH expression. On the other hand, when compared with the cisplatin-only group, all of the combination groups had a higher ability to decrease the GSH expression than the cisplatin-only group. 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 inTable 5.
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 fromTable 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.
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 fromTable 7, the Table 5.GSH expression in study groups.
Groups Mean (SD) pa Posthocb
Dox Mel Cis C1 C2 C3 C4
Control 45.23 (0.5) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 IC50
Doxorubicin
1.33 (0.06) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
IC50 Melatonin
12.57 (0.12) <0.001 1.000 <0.001 <0.001 <0.001
IC50 Cisplatin
33.2 (0.87) <0.001 <0.001 <0.001 <0.001
C1 11.73 (0.67) <0.001 <0.001 <0.001
C2 20.5 (1.42) 0.008 <0.001
C3 23.07 (0.23) <0.001
C4 28.93 (0.38)
aANOVA.
bBonferroni.
Table 6.ERCC1 expression in study groups.
Groups Mean (SD) pa Posthocb
Dox Mel Cis C1 C2 C3 C4
Control 48.07 (0.45) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 IC50
Doxorubicin
1.57 (0.06) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
IC50 Melatonin
14.63 (0.49) <0.001 <0.001 <0.001 <0.001 <0.001
IC50 Cisplatin
20.2 (0.17) <0.001 <0.001 <0.001 0.001
C1 4.27 (0.21) <0.001 <0.001 <0.001
C2 9.53 (0.6) 0.001 <0.001
C3 11.63 (0.6) <0.001
C4 22.8 (0.52)
aANOVA.
bBonferroni.
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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 mesen- chymal transition (increased expression of e-cadherin) more than the cisplatin-only group; a decrease in drug resistance could also be observed.
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 fromTable 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.
Discussion
The IC50 of doxorubicin was used as a positive control in this study as doxorubicin has much evidence as an anticancer agent and has been demonstrated to be effective when cells are resistant to cisplatin (Yi S,et al.,2020). Doxorubicin has a DNA intercalation chain mechanism, inhibition of topoisomerase II to destroy DNA and increase apoptosis (Johnson and Dubey 2021). The combination groups had higher cell viability decrease than the cisplatin group. These groups may have the capability to increase the cytotoxic effect of chemotherapy more than that of the single-use therapy. Melatonin Table 7.E-Cadherin expression in study groups.
Groups E_Chaderin p-valuea P-valueb
MeanSD Control IC50
Doxorubicin IC50 Melatonin
IC50 Cisplatin
C1 C2 C3 C4
Control 1.700.10 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
IC50 Doxorubicin 64.21.76 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
IC50 Melatonin 19.20.36 <0.001 <0.001 <0.001 <0.001 <0.001
IC50 Cisplatin 7.200.59 <0.001 <0.001 <0.001 1.00
C1 32.21.60 <0.001 <0.001 <0.001
C2 15.530.85 0.588 <0.001
C3 14.300.46 <0.001
C4 6.930.65
aANOVA.
bBonferroni.
Table 8.Annexin V expression in study groups.
Groups Mean (SD) pa Posthocb
Dox Mel Cis C1 C2 C3 C4
Control 1.2 (0) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 IC50
Doxorubicin
70.9 (0.1) <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
IC50 Melatonin
15.77 (0.21) 0.005 <0.001 <0.001 <0.001 <0.001
IC50 Cisplatin
10.87 (0.91) <0.001 <0.001 <0.001 <0.001
C1 53.57 (1.33) <0.001 <0.001 <0.001
C2 46.77 (0.06) <0.001 <0.001
C3 32.43 (2.83) <0.001
C4 23.2 (1.31)
aANOVA.
bBonferroni.
exhibits anti-inflammatory, anti-tumor, and anti-proliferative activities, with minimal side effects. In addition, it was found to reduce cell proliferation and angiogenesis and act as a pro-apoptosis and immunomodulator agent in ovarian cancer (Chuffa, 2017). Melatonin has no issues regarding bioavailability compared with other drugs because it has faster solubility through the cell and nuclear membranes (Tamura,et al.,2020).
CTR1 and Pgp were found to be associated with cell resistance. Low influx and high efflux may contribute to drug resistance (Zhanget al.,2019). CTR1 plays a role in the absorption of platinum drugs, such as cisplatin and oxaliplatin, and decreases transporter expression, thus influencing tumor resistance (Chen and Chang,2019). Pgp may pump the drug outside of the cell to reduce cytotoxic activity. A high Pgp indicates a high ability of the cell to inhibit the cytotoxic agent (Amiri-Kordestani,et al.,2012). Melatonin is capable of reducing Pgp in diffuse large B-cell lymphoma and activating the NF-kB pathway. In a study using the combination of epirubicin and melatonin, melatonin made the lymphoma cell sensitive to epirubicin from the inhibition of Pgp expression through the pathway of NF-kB. Melatonin decreases P65 in the nucleus and inhibits Pgp expression (Liu,et al.,2021).
GSH has the ability to detoxify intracellular toxins, but the GSH in cell cancer is capable of inhibiting and inactivating chemotherapy (Tapia,et al.,2013). GSH binds to cisplatin to inhibit cisplatin binding with DNA, reduce reactive oxygen species, and decrease cell sensitivity to apoptosis (Galluzzi,et al.,2012). Melatonin can decrease the GSH level and increase the GSH peroxidase activity (Medina-Leendertz,et al.,2018). Furthermore, it induces antioxidant synthesis by inducing gamma glutamylcysteine synthetase (Meng,et al.,2017). In addition, melatonin increases antioxidant activity that increases glutathione level and induces glutathione peroxidase (Harderland, 2017). Melatonin increases the apoptosis activity of cisplatin, reduces oxidative stress by decreasing the GSH level, and increases glutathione synthesis (Fernandezet al.,2019).
Melatonin has the ability to inhibit mTOR and ERCC1 expressions and increase the activity of intracellular autophago- somes. It is used as a cancer therapy adjuvant to repair the sensitivity of chemotherapy and managing the side effects of cisplatin (Bennukulet al.,2014). ERCC1 overexpression has poor prognosis in patients with osteosarcoma or lung cancer who received cisplatin.Li et al. (2017)demonstrated that low ERCC1 expression increases the sensitivity of platinum chemotherapy in ovarian cancer. The low repair activity of the DNA decreases chemotherapy resistance (Li,et al.,2017).
E-cadherin has an impact in cell adhesion and influences cell growth. A decrease in e-cadherin expression increases activity of epithelial mesenchymal transition, thus reducing the cell adhesion strength (Rosso,et al., 2017). High e-cadherin was associated with high activities of invasive cancer and metastasis (Loh, et al., 2019). Melatonin administration increased e-cadherin expression, decreased N-cadherin and vimentin expressions in CSC from CMT- U229. Melatonin may decrease the migration and invasion activities of cancer cells (Goncalves,et al.,2016). It also decreases proteins associated with inflammation, oxidative stress, cell-cycle, proliferation, and apoptosis (Zare,et al., 2019)
Apoptosis indicates low chemotherapy resistance. In this study, we used annexin V as the marker. High annexin V expression indicates high apoptosis activity in the cell (Wang,et al.,2012). Melatonin increases p53 expression and activates it, increasing apoptosis in several cancers such as colon and uterine cancer. Melatonin is involved in the BAX gene expression, decreases the expression of BCL-2 as an antiapoptotic gene, and regulates the Bax/Bcl-2 ratio (Chuffa, et al.,2017). Melatonin and cisplatin potentiate apoptosis by increasing the depolarization of mitochondrial membrane, activating caspase-3/7, and inducing cell-cycle arrest, if compared with the cisplatin-only group (Plaimee,et al.,2015).
Conclusion
This study has shown that group C1 (combination of 1x IC50 melatonin and 1x IC50 cisplatin) may reduce the cisplatin resistance by decreasing cell viability, increase influx activity by increasing CTR-1 expression, reduce efflux activity by reducing p-glycoprotein expression, decrease drug inactivity by reducing GSH expression, decrease DNA repair mechanism by reducing ERCC-1 expression, increase epithelial mesenchymal transition by increasing e-cadherin expression, and increase apoptosis mechanism by increase of annexin V expression.
Data availability Underlying data
Mendeley: Raw Data of Melatonin-Cisplatin.https://doi.org/10.17632/ybpbjbkfb4.1(Adella, 2023a)
Mendeley: Combination of Cisplatin and Melatonin on the Inhibition of Cisplatin Resistance in SKOV3 Cells.https://
www.doi.org/10.17632/ftdpsfwrtv.1(Adella, 2023b)
Page 10 of 12
Mendeley: File for Combination of Melatonin and Cisplatin on the Inhibition of Ovarian Cancer Cell. https://doi.
org/10.17632/475dpkhk3w.2(Adella, 2023c)
Data are available under the terms of theCreative Commons Attribution 4.0 International license(CC-BY 4.0).
Acknowledgments
All of the authors give regard to the people who participated in this research.
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