Melatonin Increases the Anticancer Potential of Doxorubicin in Caco-2 Colorectal Cancer Cells

Colorectal cancer (CC) is an important human malignancy with high cancer related death worldwide. The chemotherapy using doxorubicin hydrochloride is one of the most common cancer therapeutic methods. However, drug resistance lowers the treatment ecacy in CC patients. The combination therapies seem to be more promising by taking the advantage of synergistic effects. The present study aimed to evaluate a new strategy to enhance the anticancer activity of doxorubicin in Caco-2 CC cell line by co-administration of melatonin. The effects of doxorubicin, melatonin, and their combinations (Dox-Mel) were investigated on the proliferation and viability, morphological alterations, and tumor spheroid formation. Flow cytometry was employed to compare the apoptotic situation of the cells in study groups. Changes in metastatic potential of the cells were assessed by wound healing assay and trans-well migration assays. Moreover, expression of BAX, SMAC, BCL-2, SURVIVIN, MMP-2, and MMP-9 genes were evaluated by quantitative real time PCR (qRT-PCR) and western blotting. Our study showed that doxorubicin, melatonin, and Dox-Mel signicantly decreased the proliferation and viability, tumor spheroid formation, invasion and migration. Furthermore, the changes were in a concentration and time dependent manner. There was an increase in apoptosis rate in the treatment groups. Expression of genes involved in apoptosis and cell motility were altered signicantly. It was observed that anticancer activity of Dox-Mel combination was signicantly more than doxorubicin and melatonin treatments alone. We showed an enhanced apoptotic and anticancer activity of doxorubicin and melatonin combination chemotherapy on CC cell line than doxorubicin or melatonin treatments alone. This combination could promote the treatment eciency and alleviate the un-intended side effects by lowering the dose of doxorubicin prescription.


Introduction
Colorectal cancer (CC) is a highly prevalent malignancy and causative of cancer related death in the world. The incidence of CC is much lower in the developing countries of Africa and Asia compared with the United States and developed European countries (Haggar and Boushey, 2009). Nowadays, surgery and chemotherapy are the most common methods for treatment of CC. However, due to development of drug-resistant CC cells the e cacy of chemotherapy is limited (Wang et al., 2017). Therefore, application of combinational methods seems to be more effective and result in prolonged survival of CC patients Therefore, therapeutic e cacy of doxorubicin on cancer cells were decreased through overexpression of e ux transporters and inhibition of apoptosis (Hu et al.,206). Combination of doxorubicin with different compounds and molecules to overcome the drug resistance has been found to be promising in various cancer cells (Menéndez et al., 2019;Fic et al., 2007).
Melatonin is a natural hormone secreted by the pineal gland as well as testes, retina, ovary, skin, and intestine (Chen et al., 2016). Previous studies reported that, melatonin plays an important role in regulation of several biological pathways such as secretion and modulation of hormones, reproduction, and circadian rhythms (Kim et al., 2012;Wang et al., 2012;. In addition, melatonin has shown an appropriate anticancer activity without side effects on several cancer cells (Wang et  So far, several studies have been conducted to assess the anticancer effects of various natural compounds in combination with doxorubicin against cancer cells. However, this is the rst study on the anticancer effects of doxorubicin-melatonin combination and underlying mechanisms in CC cell line which can help making decisions for clinical applications in the future.

Cancer cell culture
The CC cell line Caco-2 was obtained from Immunology Research Center, Tabriz University of Medical Sciences. The cell culture was performed using Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum (FBS) and appropriate concentrations of penicillin (5,000 units/mL)and streptomycin (5,000 mg/mL). The cells were then incubated in standard conditions (5% CO 2 , 95% humidity and 37 °C).

Assessment of the morphological alterations
The Caco-2 cancer cells were seeded in a 6 well culture plates (1.5 × 10 5 cells/well) in 2000 µL culture medium. After 24 hours of incubation, the cells were then treated with doxorubicin (1 µM), melatonin (2 mM), and Dox-Mel (0.8 µM-mM), and then incubated for 24-72 hours. Finally, morphological alterations of cancer cells were monitored by an inverted phase contrast microscope.

Spheroid formation assay
The Caco-2cells were seeded in a6well culture plates (1.5 × 10 5 cells/well) in 2000 µL culture mediumcontaining10 µM basic broblast growth factor (bFGF) and 10 µM epidermal growth factor (EGF), and then incubated for 24 hours. Subsequently, the cancer cells were treated with doxorubicin (1 µM), melatonin (2 mM), and Dox-Mel (0.8 µM-mM), and maintained in an incubator under standard conditions. The culture medium was substituted every 72 hours. Finally, formation of tumor sphere-like cells was monitored by an inverted phase contrast microscope, after 7-12 days Wound healing assay The Caco-2 cancer cells were seeded in a 6 well culture plates (1.5 × 10 5 cells/well) in 2000 µL culture medium, and then incubated. After reaching to more than 90% con uence, the cancer cell monolayers in each well were scratched with a sterile pipette tip. Then, cancer cells were treated with doxorubicin (1 µM), melatonin (2 mM), and Dox-Mel (0.8 µM-mM), and incubated for 0-20 hours. Finally, migration of cancer cells was monitored by an inverted phase contrast microscope.

Trans-well migration assay
The Caco-2 cancer cells were seeded in the upper trans-well chamber in serum free culture medium and treated by doxorubicin (1 µM), melatonin (2 mM), and Dox-Mel (0.8 µM-mM) for 10-20 hours. In parallel, supplemented culture medium was added to the bottom trans-well chamber, and incubated for 20 hours. Then, non-migrated cancer cells in the upper chamber were removed, and the migrated cancer cells in the bottom chamber were xed by 4% paraformaldehyde and stained by 0.1% crystal violet. Finally, migration of cancer cells in 3 random elds was monitored by an inverted phase contrast microscope.
After 24 hours of incubation, the cells were treated with doxorubicin (1 µM), melatonin (2 mM), and Dox-Mel (0.8 µM-mM), and maintained for 48 hours. The cancer cells were detached, washed, and resuspended in binding buffer containing Annexin V-FITC (5 mL) and PI (10 mL), and incubated at room temperature in dark for 15 minutes. Finally, apoptosis rate in cancer cells was measured by a ow cytometry instrument (Becton Dickinson Bioscience).

Gene expression assay by real time PCR
The Caco-2 cancer cells were seeded in a 6 well culture plates (1.5 × 10 5 cells/well) in 2000 µL culture medium, and then incubated overnight. Then, the cancer cells were treated with doxorubicin (1 µM), melatonin (2 mM), and Dox-Mel (0.8 µM-mM), and incubated for 48 hours. The cancer cells were detached, washed, and re-suspended in TRIzol reagent, and total RNA extraction was performed according to the manufacturer's instructions. Then, random hexamer primer was used for cDNA synthesis. The mRNA expression levels of BAX, BCL-2, SMAC, SURVIVIN, MMP-2, and MMP-9 genes were evaluated using quantitative Real Time PCR (qRT-PCR). The used primers sequences were presented in Table 1. The Real Time PCR was performed in 20 µl total volume comprising: 1 µl cDNA, 0.5 µl forward and reverse primers (5 µM), and 10 µl of PCR pre-Mix (Takara, RR820L). The cycling program included: 1 cycle of 1 minutes at 94 °C) followed by 45 cycles of denaturation at 94 °C for 10 seconds, annealing at 59 °C for 30 seconds, and extension at 72 °C for 20 seconds. The ACTB gene (β-actin) was considered as endogenous control, and calculations were conducted by 2 −ΔΔCt (Livak) formula.

Gene expression assay by western blotting
The Caco-2 cancer cells were seeded in a 6 well culture plates (1.5 × 10 5 cells/well) in 2000 µL culture medium, and then incubated for 24 hours. Then, the cancer cells were treated with doxorubicin (1 µM), melatonin (2 mM), and Dox-Mel (0.8 µM-mM), and incubated for 48 hours. The cancer cells were detached, washed, and lysed inice-cold RIPA lysis buffer containing protease inhibitor for 30 minutes. The cell lysates centrifuged and supernatant collected. The protein expression levels of Bax, Bcl-2, Smac, Survivin, MMP-2, and MMP-9 were evaluated using western blot analysis. Approximately 100 µg of cells protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred on to polyvinylidene di uoride (PVDF) membrane. The membrane was blocked using non-fat dry milk (5%) and then incubated with primary antibodies at 4 °C for 24 hours. The horseradish peroxidase (HRP) conjugated secondary antibody was used as secondary antibody at room temperature for 60 minutes. The protein detection was performed using enhanced chemiluminescent (ECL) detection system.

Statistical analysis
The all performed experiments were repeated for three times, and obtained data were presented as mean ± standard deviation (SD). The Student's ttest, Tukey (posthoc), and oneway analysis of variance (ANOVA) were used to statistical analysis using the Graph Pad Prism software. The p-value < 0.05 was considered as signi cant.

Cancer cell viability
According to the results, both doxorubicin and melatonin demonstrated a time and concentration dependent anticancer activity. The viability of the cancer cells was signi cantly decreased in the high concentrations of doxorubicin and melatonin. In addition, the anticancer activity of Dox-Mel was signi cantly more than doxorubicin and melatonin. The half maximal inhibitory concentration (IC 50 ) of doxorubicin, melatonin, and Dox-Mel on CC cells after 48 hours were: 1 µM, 2 mM, and 0.8 µM-mM, respectively (Fig. 1).

Morphological alterations
The cancer cells treated with doxorubicin, melatonin, and Dox-Mel showed several morphological alterations including shrinkage, decreased cell size, fragmented nuclei, and membrane damages, which can result in programmed cell death. The cells treated with Dox-Mel revealed signi cantly more morphological alterations than the cells with doxorubicin and melatonin monotherapy (Fig. 2).

Tumor spheroid
The treatment of cancer cells with doxorubicin, melatonin, and Dox-Mell showed signi cant decrease in the number and size of the spheroids compared to the untreated cancer cells. The size and number of tumor spheroids in the treated cancer cells with Dox-Mel were signi cantly less than in Caco-2 cells treated with doxorubicin and melatonin alone (Fig. 3).

Cancer cell migration and invasion
The trans-well assay showed that the migration and invasion of cancer cells treated with doxorubicin, melatonin, and Dox-Mel was signi cantly decreased in a time dependent manner (Fig. 4A). The wound healing showed a signi cant decrease in the migration and invasion of cancer cells treated with doxorubicin, melatonin, and Dox-Mel in a time dependent manner (Fig. 5A). However, inhibition of migration and invasion in the cancer cells treated with Dox-Mel was signi cantly more than doxorubicin and melatonin groups ( Fig. 4B and Fig. 5B).

Cancer cell apoptosis
The Annexin V-FITC/PI assay showed that early and late apoptosis rate were signi cantly increased in cancer cells treated with doxorubicin, melatonin and Dox-Mel compared to untreated cells. Total apoptosis rate in the cancer cells treated with doxorubicin and melatonin were 34% and 29%, respectively; whereas the total apoptosis rate was 42% in cancer cells treated with Dox-Mel combination (Fig. 6).

Transcript quanti cation of migration and invasion related genes
The mRNA expression levels of MMP-2 and MMP-9 genes were signi cantly decreased in treated Caco-2 cells with doxorubicin, melatonin, and Dox-Mel. However, regulation of migration and invasion related genes expression in the treated cancer cells with Dox-Mel was signi cantly more profound than cells treated with doxorubicin and melatonin (Fig. 7A, B).

Expression analysis of migration and invasion related genes in protein level
The migration and invasion related MMP-2 and MMP-9 protein levels signi cantly decreased in Caco-2 cells treated with doxorubicin, melatonin, and Dox-Mel. However, regulation of migration and invasion related proteins levels in the treated cancer cells with Dox-Mel was signi cantly more profound than treated cancer cells with doxorubicin and melatonin (Fig. 8).
Transcript quanti cation of apoptosis related genes mRNA expression of the apoptosis related genes including BCL-2 and SURVIVIN signi cantly decreased in treated cancer cells with doxorubicin, melatonin, and Dox-Mel (Fig. 7C, D). Moreover, apoptosis related BAX and SMAC genes mRNA expression signi cantly increased treated cancer cells with doxorubicin, melatonin, and Dox-Mel (Fig. 7E, F). However, regulation of apoptosis related genes expression in the treated cancer cells with Dox-Mel was signi cantly more profound than treated cancer cells with doxorubicin and melatonin.

Expression analysis of apoptosis related genes in protein level
The apoptosis related Bcl-2 and Survivin proteins levels signi cantly decreased in treated cancer cells with doxorubicin, melatonin, and Dox-Mel. Moreover, apoptosis related Bax and Smac proteins levels signi cantly increased in treated cancer cells with doxorubicin, melatonin, and Dox-Mel. However, regulation of apoptosis related proteins levels in the treated cancer cells with Dox-Mel was signi cantly more profound than treated cancer cells with doxorubicin and melatonin (Fig. 9). In the present study, we assessed the anticancer effects of doxorubicin, melatonin, and Dox-Mel on CC cells and underlying mechanisms of action. We demonstrated that, melatonin increases the anticancer effects of doxorubicin against CC cells through regulation of several mechanisms involved in cell viability.

Discussion
We evaluated the morphological alterations and tumor spheroid formation in CC cells treated with doxorubicin, melatonin, and Dox-Mel in comparison with untreated CC cells. According to the results, various morphological alterations such as cell shrinkage, chromatin condensation, and apoptotic body formation as indicators of apoptosis induction were increased in CC cells treated with doxorubicin, melatonin, and Dox-Mel. However, morphological alterations of CC cells treated with Dox-Mel signi cantly more increased, as compared to doxorubicin and melatonin monotherapy. Moreover, tumor spheroid formation was signi cantly decreased in CC cells treated with Dox-Mel, as compared with doxorubicin and melatonin monotherapy.
The high lethality in patients with CC is due to metastasis and tumor growth at distant tissues (Bakhshaiesh et al., 2015). In this study, wound healing and trans-well migration assays demonstrated that invasion and migration of CC cells treated with Dox-Mel decreased more signi cantly, as compared doxorubicin or melatonin monotherapy. Moreover, we evaluated them RNA and protein levels of MMP-2 and MMP-9, which is related to CC cells metastasis and invasion. Our results showed that doxorubicin, The current study represents a novel combination chemotherapeutic approach to treatment of patients with doxorubicin-resistant CC. It was shown that, melatonin enhanced the anticancer activity of doxorubicin through induction of intrinsic apoptotic pathway and suppressed the invasiveness of CC cells. Therefore, the Dox-Mel can be used in the future to control and even treatment of patients with CC.
However, further studies are required to identify the enhanced anticancer mechanisms of Dox-Mel to better management of patients with CC and prescription of lower doses of chemotherapeutic agents.
The current study represents a novel combination chemotherapeutic approach to treatment of patients with doxorubicin-resistant CC. It was shown that, melatonin enhanced the anticancer activity of doxorubicin through induction of intrinsic apoptotic pathway and suppressed the invasiveness of CC cells. Therefore, the Dox-Mel can be used in the future to control and even treatment of patients with CC. However, further studies are required to identify the enhanced anticancer mechanisms of Dox-Mel to better management of patients with CC and prescription of lower doses of chemotherapeutic agents. Figure 1 The  The morphological alteration of CC cells treated with doxorubicin (1 μM), melatonin (2 mM), and Dox-Mel (0.8 μM-mM) in 24, 48, and 72 hours. Treatment with Dox-Mel caused more profound morphological alterations.

Figure 3
The    The apoptosis rate of CC cells treated with doxorubicin (1 μM), melatonin (2 mM), and Dox-Mel (0.8 μM-mM). The induction of apoptosis in the CC cells treated with Dox-Mel was signi cantly more than doxorubicin and melatonin alone.  The western blot analysis (A) of MMP-2 (B) and MMP-9 (C) proteins following the treatment with doxorubicin (1 μM), melatonin (2 mM), and Dox-Mel (0.8 μM-mM). The regulation of migration and invasion related genes expression in the CC cells treated with Dox-Mel was signi cantly more than doxorubicin and melatonin alone.