Ovarian cancer, the most lethal gynecological cancer, is the second most common malignancy after breast cancer in women over the age of 40 [1
]. About 80% of all ovarian cancers are of the high-grade serous type, arising from the serous epithelial layer in the abdominopelvic cavity [2
]. Since most ovarian cancers are diagnosed at advanced stages, they have a poor prognosis, and a low overall survival rate [3
]. Moreover, the survival rate of patients with ovarian cancer has barely changed since platinum-based treatment was introduced more than 30 years ago [4
]. Therefore, there is an important need for new strategies to treat ovarian cancers and a better understanding of the molecular events leading to the resistance of treatment.
Forkhead box M1 (FOXM1), a member of the Forkhead box transcription factor family, is an oncogenic transcription factor, and its overexpression is associated with poor prognosis in several types of human cancers, such as pancreatic cancer, breast cancer, and lung cancer [5
]. FOXM1 is involved in cell cycle progression through regulation of gene expression in the G1/S and G2/M phases and by inducing the proper execution of the mitotic program [8
]. Moreover, FOXM1 plays an important role in the early stage of metastasis, by stimulating the expression of genes associated with the invasion and migration of cancer cells [11
]. FOXM1 also increases the population, proliferation, and motility of cancer stem cells, which make cancers tolerant to drugs like cisplatin [14
]. Hence, targeting FOXM1 could be effective for treating several cancers and sensitizing drug-resistant cancer cells.
Rosmarinic acid methyl ester (RAME), a derivative of rosmarinic acid (RA), has several biological effects, such as anti-microbial, anti-inflammatory, and anti-allergic effects [16
]. RAME also exhibits anti-oxidant and anti-melanogenesis in vivo [19
]. Recently, the anti-cancer effect of RAME via the inhibition of mTOR-S6K1 signaling in cervical cancer was reported [20
]. It was also reported that RAME induced the apoptosis of cervical cancer cells and enhanced the anti-tumor effect of cisplatin in cervical cancer. However, the regulation of gene expression by RAME treatment and the mechanisms through which gene expression is regulated, are unclear.
Here, we performed RNA-sequencing in RAME-treated ovarian cancer cells. Differentially Expressed Gene (DEG) analysis and Gene Ontology (GO) analysis suggest that the expression of mitosis-associated genes known to be regulated by FOXM1 were downregulated. Treatment of ovarian cancer cells with RAME effectively inhibited the binding of FOXM1 to its target gene promoters by decreasing the FOXM1 expression. We also observed that RAME repressed the migration and invasion of ovarian cancer cells. Moreover, co-treatment with RAME and cisplatin sensitized a cisplatin-resistant ovarian cancer cell line and induced the expression of apoptosis-associated genes. Collectively, our studies suggested that RAME, a derivative of rosmarinic acid, has the potential for use as a therapeutic substance for patients with ovarian cancer, especially for those with cisplatin-resistant tumors.
Several studies revealed that FOXM1 is overexpressed in various cancer cells, such as ovarian, breast, lung, and cervical cancer cells [12
]. Upregulation of FOXM1 enhances cell proliferation and inhibits apoptosis [14
]. Moreover, upregulated FOXM1 is related to poor prognosis in several cancers [12
]. Therefore, FOXM1 is a promising therapeutic target for inducing anti-cancer effects in various cancer cells [36
]. In this study, we hypothesized that RAME could exert anti-cancer effects since it downregulated the expression of FOXM1 target genes, according to our RNA sequencing data. Based on this hypothesis, we investigated the expression of FOXM1 target genes and the recruitment of FOXM1 onto the promoters of its target genes. We identified that RAME suppressed the expression of FOXM1 target genes by inhibiting the enrichment of FOXM1 on their promoters, as shown in Figure 1
C,D and Figure 2
. Moreover, by suppressing the expression of the FOXM1 and its target genes, the migration and invasion abilities of ovarian cancer cells were suppressed (Figure 3
Ovarian cancer is commonly diagnosed in the advanced stages; however, ovarian cancer is more curable at the early stages than at the later stages [38
]. This is due to the lack of warning signs or obvious symptoms in early-stage ovarian cancer; hence, ovarian cancer is usually called “the silent killer” [38
]. Unfortunately, the diagnosis of ovarian cancer at advanced stages leads to the acquisition of resistance to conventional chemotherapy and poor prognosis [41
]. For these reasons, there is an increasing need for an effective treatment against chemotherapy-resistant ovarian cancer.
Cisplatin is one of the conventional agents used to treat ovarian cancer. Its anti-cancer effect is a result of DNA damage-induced apoptosis [43
]. Ovarian cancer often develops cisplatin resistance after cisplatin chemotherapy [44
]. Furthermore, recurrence of ovarian cancer is up to 75% and the recurrent cancer can result in the acquisition of cisplatin resistance, as well [43
]. Thus, cisplatin resistance is a limitation of cisplatin chemotherapy, since there are several molecular mechanisms leading to cisplatin resistance [45
]. Among the mechanical reasons, previous studies showed that upregulation of FOXM1 induced cisplatin resistance in several cancer cells [48
]. From these previous studies, we hypothesized that RAME could overcome cisplatin resistance in ovarian cancer cells. Our data revealed that co-treatment with RAME and cisplatin was effective against cisplatin-resistant cancer cells by inhibiting the expression of FOXM1 and its target genes, ultimately inducing apoptosis in these cells (Figure 5
4. Materials and Methods
4.1. Antibodies and Reagents
Anti-FOXM1 (GeneTex, Irvine, CA, USA; GTX102170) and anti-β-Actin (Millipore, Temecula, CA, USA; mab1501) antibodies were utilized for the immunoblotting assay in this study. In addition, for the Chromatin Immunoprecipitation assay, anti-FOXM1 (GeneTex, Irvine, CA, USA; GTX102170) and anti-H3Ac (Millipore, Temecula, CA, USA; 06-599) antibodies were used. Rosmarinic acid methyl ester (RAME) was purchased from Chemfaces (Wuhan, China; No. CFN97567). RAME was extracted from the herbs of Rosmarinus officinalis L. and its purity was ≥98%.
4.2. Cell Culture and Establishment of Cisplatin-Resistant TOV-21G (TOV/CisR) Cells
SKOV-3, a human ovarian cancer cell line, was maintained in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S) (100×). Additionally, another ovarian cancer cell line, TOV-21G, was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 1% P/S (100×). These cell types were obtained from the American Type Culture Collection (ATCC) and maintained in a humidified atmosphere (37 °C, 5% CO2
). Cisplatin-resistant TOV-21G (TOV/CisR) cells were established by culturing the cells with gradually increasing concentrations of cisplatin [51
4.3. RNA Isolation, Library Preparation, and RNA-Sequencing
The total RNA of SKOV-3 cells was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). For the gene expression profiling, RNA-sequencing was performed at Ebiogen Inc. (Seoul, Korea). RNA quality was assessed with an Agilent 2100 Bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using an ND-2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For the RNAs of DMSO- and RAME-treated SKOV-3 cells, a library was constructed using the QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen Inc., Vienna, Austria), according to the manufacturer’s instructions. In brief, 500 ng of total RNA was prepared and an oligo-dT primer containing an Illumina-compatible sequence at its 5′ end was hybridized to the RNA; reverse transcription was then performed. After degradation of the RNA template, second strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5′ end. The double-stranded library was purified using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The finished library was purified from the PCR components. High-throughput sequencing was performed as single-end 75 sequencing using NextSeq 500 (Illumina Inc., San Diego, CA, USA). QuantSeq 3′ mRNA-Seq reads were aligned using Bowtie2 [52
]. Bowtie2 indices were either generated from the genome assembly sequence or the representative transcript sequences for alignment to the genome and transcriptome, respectively. The alignment file was used for assembling transcripts, estimating their abundance, and detecting the differential expression of genes. Analysis of the relationship between differentially expressed genes was performed with the Excel-based Differentially Expressed Gene Analysis (ExDEGA v.2.0.0) software by eBiogen (ebiogen.com). Differentially expressed genes with fold changes >2 and p
-values < 0.05 were further identified by GO analysis using the Enrichr tool [53
4.4. RNA Extraction and Quantative Real-Time PCR (qPCR)
Total RNA was isolated from the cells using an Easy-Blue reagent (iNtRON Biotechnology, Seongnam, Korea). Next, 1 μg of RNA was reverse-transcribed using a Maxim RT-PreMix Kit (iNtRON Biotechnology, Seongnam, Korea). Quantitative real-time PCR (qPCR) was performed using a KAPATM SYBR® FAST qPCR Master Mix (Kapa Biosystems, Wilmington, MA, USA) and CFX96 TouchTM real-time PCR detector (Bio-Rad, Hercules, CA, USA). The relative mRNA expression levels of the target genes were normalized to the mRNA levels of GAPDH for each reaction. The primer sequences used for RT-qPCR were as follows: FOXM1 forward, 5′-AACCGCTACTTGACATTG G-3′; FOXM1 reverse, 5′-GCAGTGGCTTCATCTTCC-3′; CCNB1 forward, 5′-CCAGTGCCAGTGTCTGAGC-3′; CCNB1 reverse, 5′-TGGAGAGGCAGTATCAACCA-3′; CENPF forward, 5′-CAAGAATATGCACAACGTCCTGC-3′; CENPF reverse, 5′ GAACGCCTGTTCAGCTCTG-3′; TOP2A forward, 5′-CTGCGGACAACAAACAAAGG-3′; TOP2A reverse, 5′-ACACAATTTGGCTCCATAGC-3′; UBE2C forward, 5′-GGATTTCTGCCTTCCCTGAA-3′; UBE2C reverse, 5′-GATAGCAGGGCGTGAGGAAC-3′; GAPDH forward, 5′-ACGGATTTGGTCGTATTGGGCG-3′; GAPDH reverse, 5′-CTCCTGGAAGATGGTGATGG-3′; P53 forward: 5′-TCTTCCTCTGAGGCGAGCT-3′, P53 reverse: 5′-AGGTGTGTGTGTCTGAGCCC-3′, 14-3-3A forward: 5′-: GGCCATGGACATCAGCAAGAA-3′, 14-3-3A reverse: 5′-CGAAAGTGGTCTTGGCCAGAG-3′, NOXA forward: 5′-AGAGCTGGAAGTCGAGTGT-3′, NOXA reverse: 5′-GCACCTTCACATTCCTCTC-3′.
4.5. Protein Extraction and Immunoblotting
To extract the total protein, cells were lysed using a Pro-Prep reagent (iNtRON Biotechnology, Seongnam, Korea). Each sample contained the same amounts of protein, as determined by quantifying the protein contents in the lysates. The samples were loaded and separated via SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred onto polyvinylidene difluoride (PVDF, Millipore, Temecula, CA, USA) membranes using the wet transfer method. The membranes were blocked using 5% skim milk for 1 h; then, they were incubated overnight with primary antibodies at 4 °C. After incubation with the primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h (Millipore, Temecula, CA, USA). The signals were detected using chemiluminescence reagents (AbClon, Seoul, Korea) and quantified using ImageJ software.
4.6. Chromatin Immnoprecipitation (ChIP)-qPCR
Cells were crosslinked with 1% formaldehyde in PBS. Then, the crosslinked samples were sheared by sonification to obtain chromatin fragments that were 200–500 bp in size. The sheared chromatin fragments were incubated overnight at 4 °C with antibodies and magnetic beads, except for 2% input DNA. After immunoprecipitation, the chromatins were de-crosslinked at 65 °C, followed by the addition of RNase A and Proteinase K. DNA was purified from the samples and subjected to PCR as a template. The result was expressed as IP/Input (2%). The primers for the Chromatin Immunoprecipitation (ChIP) assay were the targets of the promoters of each gene, and the sequences were as follows: CCNB1 forward, 5′-CGCGATCGCCCTGGAAACGCA-3′; CCNB1 reverse, 5′-CGCGATCGCCCTGGAAACGCA-3′; UBE2C forward, 5′-CATTGGCTGGATCAAACCCA-3′; UBE2C reverse, 5′-GGAGAACACGACTGCAACTG-3′.
4.7. Wound Healing Assay
Cells were grown on 6 well plates until 100% confluence, followed by scratch with a 200 µL pipette tip. After washing with PBS to remove the floating cells, the cells were incubated in a medium with RAME, at concentrations of 0, 20 and 40 µM for 24 h. Gap width between the migrated cells was estimated in 4 random fields, under microscope, and averaged.
4.8. Transwell Migration Assay
A cell migration assay was performed using the Transwell chamber inserts (24-well; Corning Incorporated, Corning, NY, USA) in a 24-well plate. The upper membranes were coated with Geltrex (Life Technologies, Carlsbad, CA, USA) as a barrier (100 µL/well) for the invasion assay. Then, 5 × 104 cells suspended in 100 µL of culture medium containing 1% FBS were added to the upper chamber. Culture medium containing 20% FBS was placed in the bottom chamber. The cells were incubated for 24 h at 37 °C. After incubation, the inserts were removed from the plate and fixed with 100% methanol for 2 min, followed by staining with crystal violet for 25 min. The cells on the upper surface of the insert were wiped off with a cotton swab. The cells that migrated to the lower layer of the microporous membrane were counted under a microscope. Five fields in each sample were randomly selected and the mean values were calculated.
4.9. Knockdown of FOXM1
SKOV-3 cells were transfected with siRNA using Lipofectamine 2000 reagent (Life Technologies, Carlsbad, CA), according to the manufacturer’s protocol. The siRNA sequences targeting FOXM1 were as follows:
FOXM1 sense: 5′-CCCUGCCCAACAGGAGUCUAAUCAA-3′,
FOXM1 antisense: 5′-UUGAUUAGACUCCUGUUGGGCAGGG-3′
4.10. Cell Viability Assay
A cell viability assay was performed using PrestoBlue™ (Invitrogen, Carlsbad, CA, USA; A13261) in a 96-well plate. The cancer cell lines were cultured at a density of 1 × 104 cells per well. After incubation for 24 h at 37 °C, the cells were treated with 0–100 µM of cisplatin for 24 h, and the wavelength absorbance test was performed using Cytation™ 5 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA).
4.11. Statistical Analysis
Statistical significance was analyzed using Student’s t-test (two-tailed) and Pearson’s correlation test. Statistical differences were assessed based on the following criteria for P-values: * p < 0.05, ** p < 0.01, and *** p < 0.001.
In conclusion, our study showed that RAME suppressed the expression of a novel molecular target, FOXM1, leading to anti-cancerous effects. By inhibiting the expression of the transcription factor FOXM1, the expression of its target genes was also downregulated. In fact, many transcription factors, such as Twist, CREB, and CTCF, are known to regulate the expression of FOXM1 by binding to its promoter [55
]. Moreover, FOXM1 can be served as a transcription factor and can auto-regulate the expression of FOXM1 [56
]. Therefore, further studies are needed to identify how RAME treatment regulates these various transcription factors, hence resulting in FOXM1 suppression. Our results also showed an effective decrease in the migration and invasion abilities of the ovarian cancer cells. Moreover, FOXM1 upregulation in cisplatin-resistant ovarian cancer cells was inhibited by RAME treatment. Consistently, co-treatment with RAME and cisplatin resulted in an increased sensitivity to chemotherapy and enhanced the apoptosis of cancer cells. These results showed that RAME could be an effective therapeutic agent to treat ovarian cancer patients, who also later develop chemoresistance. Additionally, further studies should be performed to elucidate the mechanism underlying the RAME-induced suppression of FOXM1 expression not only in ovarian cancer cells, but also in other cancer cells.