Demethoxycurcumin inhibited human epithelia ovarian cancer cells’ growth via up-regulating miR-551a

Abstract

Curcumin is a natural agent that has ability to dampen tumor cells’ growth. However, the natural form of curcumin is prone to degrade and unstable in vitro. Here, we demonstrated that demethoxycurcumin (a curcumin-related demethoxy compound) could inhibit cell proliferation and induce apoptosis of ovarian cancer cells. Moreover, IRS2/PI3K/Akt axis was inactivated in cells treated with demethoxycurcumin. Quantitative real-time reverse transcription polymerase chain reaction demonstrated that miR-551a was down-regulated in ovarian cancer tissues and ovarian cancer cell lines. Over-expression of miR-551a inhibited cell proliferation and induced apoptosis of ovarian cancer cells, whereas down-regulation of miR-551a exerted the opposite function. Luciferase assays confirmed that there was a binding site of miR-551a in IRS2, and we found that miR-551a exerted tumor-suppressive function by targeting IRS2 in ovarian cancer cells. Remarkably, miR-551a was up-regulated in the cells treated with demethoxycurcumin, and demethoxycurcumin suppressed IRS2 by restoration of miR-551a. In conclusion, demethoxycurcumin hindered ovarian cancer cells’ malignant progress via up-regulating miR-551a.

Keywords

Ovarian cancer demethoxycurcumin miR-551a IRS2 gene therapy

Introduction

Epithelial ovarian cancer (EOC) is one of the most frequent gynecologic tumors in women worldwide. It is the fifth common reason for death by cancer in women.¹ EOC is characterized by difficult early diagnosis and extremely poor prognosis. Despite debulking surgery and chemotherapy, EOC cells are prone to metastasize and recur, and the patients experience poor survival.² Therefore, it is necessary to explore an efficient way in EOC therapy.

Curcumin (CUR) is a member of curcuminoids which can be extracted from the rhizomes of Curcuma longa linn. CUR acts as inhibitor in tumor initiation in vivo and possesses anti-proliferative activities against cancer cells in vitro.^3–5 However, CUR is prone to degrade and unstable both in vitro and in vivo. Demethoxycurcumin (DMC) is a CUR-related demethoxy compound that can restrain tumor cells’ malignant behavior, including prostate cancer PC-3 cells, human glioblastoma 8410 cells, and human renal carcinoma caki cells.^6–8 Moreover, accumulating evidence proved that DMC was a more efficient and stable agent than CUR in cancer therapy. However, the effects and mechanism of DMC on ovarian cancer still remain poorly defined.

MicroRNAs (miRNAs) are a subgroup of non-coding RNAs with various expressions in normal tissues and tumors.⁹ Accumulating evidence showed that miRNAs involved in cancer cells’ biological process by targeting 3′ untranslated region (UTR) of their downstream genes. MiR-186 was found to inhibit lung adenocarcinoma cell proliferation by inducing G1–S checkpoint arrest and predicted better prognosis in human lung adenocarcinoma.¹⁰ In contrary, miR-148a acted as an oncogene in glioblastoma by targeting BIM and MIG6, and inversely correlates with patient survival.¹¹ Also, miRNAs were capable to sensitize tumor cells to chemotherapeutics agents. MiR-566 sensitized glioblastoma cells to nimotuzumab by activating epidermal growth factor receptor (EGFR) signaling pathway.¹² Meanwhile, over-expression of miR-508-5p efficiency reversed gastric cancer cell resistance to multidrug in vitro and sensitize tumors to chemotherapeutics in vivo.¹³ In addition, miR-497 attenuated cisplatin resistant in ovarian cancer by inactivating mTOR/P70S6K1 pathway.¹⁴ miR-551a was characterized as a tumor suppressor in various tumors such as human colorectal cancer and gastric cancer. Also, over-expression of miR-551a restrained tumor cells’ malignant progression.^15,16 However, the expression and function of miR-551a in ovarian cancer cells still need to be investigated.

In this study, we aimed to investigate the effect of DMC and miR-551a on ovarian cancer cells and the expression of miR-551a in ovarian cancer tissues (OCT). Moreover, IRS2/PI3K/Akt axis was detected to investigate the mechanism of DMC moderate ovarian cancer cells’ biology characteristics by up-regulating miR-551a.

Materials and methods

Human tissue samples and cell culture

In total, 15 pairs of OCT and normal ovarian tissues (NOT) were gathered from patients undergoing surgery at the Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University. The information and the subtype of ovarian cancer samples used are listed in Table 1. Parts of the fresh OCT were sent for routine pathology of tumor evaluation after surgery resection, and the rest were put in liquid nitrogen or used for the ensuing experiments. Informed consents were obtained from all patients, and the study was approved by the Ethics Committee of Shengjing Hospital of China Medical University. Human fallopian tube epithelial cell line FTE187, human ovarian cancer cell line ES2, HO8640, HO8640PM, SKOV3, and human embryonic kidney (HEK)-293T cells were purchased from the Cell Bank of Chinese Academy of Sciences. FTE187 cells were cultured in M199 medium (Gibco, USA) with 10% fetal bovine serum (FBS; Corning, NY, USA); ES2 cells were cultured in McCoy’s 5A (American Type Culture Collection (ATCC), Manassas, VA, USA) with 10% FBS; HO8640, HO8640PM, and SKOV3 were maintained in RPMI 1640 (Corning) supplemented with 10% FBS; and HEK-293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM)/high glucose supplemented (Gibco, Carlsbad, CA, USA) containing 10% FBS—all cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

Table 1.

Clinicopathological information of 15 ovarian cancer samples.

Number	Age (years)	FIGO stage	Histotype	Grade	Chemotherapy
1	53	III	Serous cystadenocarcinoma	Moderate	NA
2	57	II	Serous cystadenocarcinoma	Moderate	NA
3	51	III	Serous cystadenocarcinoma	Moderate	NA
4	44	I	Endometrioid carcinoma	Well	NA
5	63	III	Serous cystadenocarcinoma	Moderate	NA
6	61	IV	Endometrioid carcinoma	Poor	NA
7	59	II	Mucinous cystadenocarcinoma	Moderate	NA
8	46	III	Serous cystadenocarcinoma	Poor	NA
9	49	IV	Serous cystadenocarcinoma	Moderate	NA
10	50	II	Serous cystadenocarcinoma	Moderate	NA
11	58	III	Mucinous cystadenocarcinoma	Moderate	NA
12	60	II	Serous cystadenocarcinoma	Moderate	NA
13	49	III	Endometrioid carcinoma	Moderate	NA
14	52	III	Serous cystadenocarcinoma	Moderate	NA
15	56	I	Serous cystadenocarcinoma	Well	NA

FIGO: Federation of Gynecology and Obstetrics; NA: not applicable.

Drug preparation

DMC was obtained from Chengdu Mansite Pharmaceutical Co., China (>98% purity), dissolved in dimethyl sulfoxide (DMSO) (Sigma, USA) to 160 mM for storage and diluted in RPMI 1640 medium to different final concentrations in the following experiments.

Cell viability assay

Cell Counting Kit-8 (CCK-8) assay (Dojin, Kumanoto, Japan) was used to determine cell viability and proliferation. SKOV3 cells were seeded in a 96-well suspension culture plate at 8 × 10³ cells/well. We selected the various concentration and time referred to the previous research. After treatments with DMC (5, 10, 20, 40, 80, and 160 µM) for 12, 24, 36, and 48 h, and 0.9% sodium chloride as control, and the cells were incubated at the indicating time at 37.5°C. Afterward, 10 µL of CCK-8 were added to each well and the plates incubated for 2 h at 37.0°C. Absorbance was measured at OD 450 nm with a Thermo Varioskan Flash reader (Theromo Fisher, Carlsbad, CA, USA).

Quantitative real-time reverse transcription polymerase chain reaction

According to the manufacturer’s instructions, total RNA was extracted from the tissues or cells with TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA). Taqman MicroRNA Reverse Transcription Kit and TaqMan Universal Master Mix II with the TaqMan MicroRNA Assay of miR-551a and U6 (Applied Biosystems, Foster City, CA, USA) were used to measure the miR-551a expression. U6 was used as endogenous controls. Relative expression of the tested genes was calculated using relative quantification (2^−ΔΔ Ct) method.

Cell transfections

Lipofectamine 3000 reagent was used (Life Technologies Corporation) for cell transfections according to the manufacturer’s instructions. SKOV3 cells were transfected with miR-551a agomir (pre-miR-551a), miR-551a antagomir (anti-miR-551a), or their respective negative control (NC, non-targeting sequence) which were synthesized from GenePharma (Shanghai, China). At 48 h post-transfection, the medium was replaced with fresh medium. The expression levels of miR-551a in the transfected cells were detected by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Since the highest transfection efficiency occurred at 48 h, 72 h post-transfection was considered as the harvest time in the subsequent experiments. SKOV3 cells transfected with miRNAs were divided into five groups as follows: Control group (cells given no miRNAs), pre-NC group, pre-miR-551a group, anti-NC group, and anti-miR-551a group. Those stable expressing cells treated with DMC in different concentrations were divided into seven groups as follows: Control group, DMC (20 µM) + pre-miR-551a, DMC (20 µM) + anti-miR-551a, DMC (40 µM) + pre-miR-551a, DMC (40 µM) + anti-miR-551a, DMC (80 µM) + pre-miR-551a, and DMC (80 µM) + anti-miR-551a.

Western blot analysis

Using radioimmunoprecipitation assay (RIPA) buffer to extract total proteins form the cells with protease inhibitors (Beyotime Institute of Biotechnology, Beijing, China) on ice, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated in 5% milk dissolved in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 3 h at room temperature and then incubated with primary antibodies as follows: IRS2 (1:1000, Proteintech, Chicago, IL, USA), PI3K, p-PI3K, Akt, p-Akt (1:1000, Cell Signaling Technology, Beverly, MA, USA), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase, 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with appropriate correlated HRP-conjugated secondary antibody. The membranes were incubated with secondary antibodies (Santa Cruz Biotechnology) at room temperature for 2 h. Immunoblots were visualized by enhanced chemiluminescence (ECL Kit; Santa Cruz Biotechnology) and scanned using ChemImager 5500 V2.03 software. The relative integrated density values (IDVs) were calculated based on GAPDH as an internal control.

Quantization of apoptosis by flow cytometry

Cell apoptosis was measured by Annexin V-FITC/PI staining (BD, Birmingham, AL, USA). After washing with phosphate-buffered saline (PBS) twice, according to the manufacturer’s instructions, cells were resuspended with Annexin V-FITC/PE. Then, the cells were analyzed by flow cytometry (FACScan; BD Biosciences, San Jose, CA) and apoptotic fractions were acquired.

Reporter vectors construction and luciferase assays

MiR-551a-3′UTR and IRS2 3′UTR sequences were amplified by PCR and cloned into a pmirGlo Dualluciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to construct 3′UTR-luciferase reporter vector (miR-551a-WT and IRS2-WT; GenePharma). The sequence of putative binding site was replaced as indicated (miR-551a-Mut and IRS2-Mut) to mutate the putative binding site of miR-551a or IRS2 in the 3′UTR-containing vector. HEK-293T cells were seeded in 96-well plates and the cells were co-transfected with miR-551a-WT (or miR-551a-Mut) or IRS2-WT (or IRS2-Mut) plasmids when they reached 50%–70% density. The luciferase activities were measured at 48 h after transfection by Dual-Luciferase reporter assay kit (Promega).

Statistical analysis

Data were presented as means ± standard deviation (SD). Differences were analyzed using SPSS 18.0 statistical software with the Student’s t-test or one-way analysis of variance (ANOVA). If p < 0.05, differences were considered significant.

Results

DMC inhibited SKOV3 cells’ proliferation and induced apoptosis

To investigate the effect of DMC on SKOV3 cells, we first used CCK-8 assay to determine the relative viability of SKOV3 cells treated with different concentration of DMC at the indicated time. As shown in Figure 1(a), DMC reduced cell viability in both dose- and time-dependent manner. 20, 40, and 80 µM of DMC treatment for 48 h caused significant growth inhibition compared with the control group. Therefore, we chose 48 h as the optimal dose to be used in the subsequent experiments. Then, flow cytometry was employed to measure the effect of DMC on SKOV3 cells; as shown in Figure 1(b), apoptosis rate of cells treated with DMC at concentration 20, 40, and 80 µM for 48 h increased as the concentration of DMC increased. These results inferred that DMC could inhibit ovarian cancer cells’ progress.

Figure 1.

Effect of DMC on ovarian cancer cells. (a) DMC reduced cell viability in a dose- and time-dependent manner in SKOV3 cells. (b) DMC induced cell apoptosis in SKOV3 cells. Data are presented as the mean ± SD (n = 5, each group).

DMC modulated IRS2/PI3K/Akt axis

Previous study showed that IRS2 was up-regulated in ovarian cancer and activated PI3K/Akt pathway.¹⁷ To determine the underlying mechanism of DMC modulating SKOV3 cells biological behavior, the expression of IRS2, p-PI3K, PI3K, p-Akt, and Akt proteins were determined. As shown in Figure 2, SKOV3 cells treated with DMC (20, 40, and 80 µM) exhibited lower expression of IRS2, p-PI3K, and p-Akt compared to control group. This implied IRS2/PI3K/Akt pathway might be the underlying mechanism of DMC affecting SKOV3 cells’ biological behaviors.

Figure 2.

DMC blocked IRS2/PI3K/Akt axis. Western blot analysis for DMC regulated IDVs of IRS2, p-PI3K, PI3K, p-Akt, and Akt; they are shown using GAPDH as endogenous control. Data are presented as the mean ± SD (n = 5, each group).

MiR-551a was down-regulated in EOC and ovarian cancer cell lines, and retarded SKOV3 cells’ growth and induced cell apoptosis

MiR-551a exerts tumor-suppressive role in various cancers. qRT-PCR was conducted to detect the relative expression of miR-551a in EOC tissues (cancer tissues) and normal epithelial ovarian tissues (normal tissues). Figure 3(a) showed that the expression of miR-551a was obviously down-regulated in cancer tissues than in normal tissues (p < 0.01). As shown in Figure 3(b), abundance of miR-551a was higher than that in several ovarian cancer cell lines, especially in SKOV3 cells. These results implied that miR-551a may act as a tumor-suppressor gene in ovarian cancer cells. CCK-8 assay and flow cytometry were used to measure the miR-551a-mediated effect on SKOV3 cells. As shown in Figure 3(c), over-expression of miR-551a resulted in decreased proliferation of SKOV3 cells compared to control group. Similarly, as shown in Figure 3(d), miR-551a deletion led to a significant inhibition of apoptosis, while over-expression of miR-551a led to a significant induction of apoptosis in SKOV3 cells. These results gave a solid evidence that miR-551a exerted tumor suppressor in ovarian cancer cells.

Figure 3.

MiR-551a expression in ovarian cancer tissues and ovarian cancer cell lines, over-expression of miR-384 inhibited the malignant progression of SKOV3 cells. (a) Expression levels of miR-551a in normal tissues and cancer tissues. Data are presented as the mean ± SD (n = 15, each group). **p < 0.01 versus normal tissues group. (b) Expression levels of miR-384 in human fallopian tube epithelial cell line FTE187 cells and ovarian cancer cell lines. Data are presented as the mean ± SD (n = 5, each group). *p < 0.05 versus FTE187 group. (c) CCK-8 assay was applied to evaluate the proliferation effect of miR-551a on SKOV3 cells. Data are presented as the mean ± SD (n = 5, each group). *p < 0.05 versus pre-NC group; ^#p < 0.05 versus anti-NC group. (d) Flow cytometry analysis of SKOV3 cells with the expression of miR-551a changed. Data are presented as the mean ± SD (n = 5, each group). *p < 0.05 versus pre-NC group; ^#p < 0.05 versus anti-NC group.

MiR-551a inactivating IRS2/PI3K/Akt axis by targeting IRS2

According to the bioinformatics databases (Starbase and miRanda), we scanned the downstream genes of miR-551a. IRS2 was identified as a downstream gene of miR-551a. As previously reported, IRS2 acted as an oncogene in ovarian cancer and was involved in diverse processes of ovarian cancer progress.^17,18 Since we chose IRS2 as the putative gene that miR-551a may targeted. To determine whether IRS2 mRNA 3′UTR is a direct target of miR-551a, pre-miR-551a and IRS2 3′UTR reporter construct (IRS2-3′UTR-Wt) were co-transfected into HEK-293T cells and luciferase activity was measured. The results showed that up-regulated miR-551a prominently decreased the luciferase activities, whereas the pre-miR-551a-NC transfected did not alter the luciferase activities. However, to determine whether the predicted region is the direct binding site (Figure 4(a)), pre-miR-551a and the IRS2 3′UTR mutated reporter construct (IRS2-3’UTR-Mut) were co-transfected into HEK-293T cells. As shown in Figure 4(b), the luciferase activity in the IRS2-Wt + miR-551a group was significantly attenuated than that in the control group, while the luciferase activity in the IRS2-Mut group was not affected. These results proved that IRS2 was a direct target of miR-551a with a specific binding site.

Figure 4.

MiR-551a inactivated IRS2/PI3K/Akt axis through targeting IRS2 3′UTR. (a) The predicted miR-551a binding sites in the 3′UTR of IRS2 (IRS2-Wt) or and the designed mutant sequence (IRS2-Mut) were indicated. (b) Luciferase reporter assay of HEK-293T cells transfected with PIWIL4-3′UTR-Wt (IRS2-Wt) (or the IRS2-3′UTR-Mut (IRS2-Mut)) and the indicated miRNAs. Data are presented as the mean ± SD (n = 5, each group). *p < 0.05 versus IRS2-Wt + miR-551a-NC group. (c) Western blot analysis for DMC regulated IDVs of IRS2, p-PI3K, PI3K, p-Akt, and Akt; they are shown using GAPDH as endogenous control. Data are presented as the mean ± SD (n = 5, each group). *p < 0.05 versus pre-NC group; ^#p < 0.05 versus anti-NC group.

To investigate whether miR-551a could dampen the activation of PI3K/Akt axis by targeting IRS2, the expression of IRS2, p-PI3K, PI3K, p-Akt, and Akt proteins were measured by western blot. As Figure 4(c) showed, over-expression of miR-551a inhibits the expression of IRS2, p-PI3K, and p-Akt than in pre-miR-551a-NC group. Similarly, miR-551a deletion significantly increases the expression of IRS2, p-PI3K, and p-Akt than in anti-miR-551a-NC group. This implies that miR-551a could inhibit the activation of IRS2/PI3K/Akt pathway by directly targeting IRS2.

MiR-551a was positive correlated with DMC

The above results showed that both DMC and miR-551a could inhibit SKOV3 cell proliferation and induce apoptosis via down-regulation of IRS2. We hypothesized whether miR-551a was positively correlated with DMC. qRT-PCR was conducted to measure the expression of miR-551a in the cells treated with different concentrations of DMC. As we expected, Figure 5(a) showed that compared to control group, the level of miR-551a was obviously elevated in DMC (80 µM) group. Furthermore, the expression of miR-551a was higher in the cells with 40 µM than the cells with 20 µM. These results indicated that miR-551a was positively correlated with DMC treatment.

Figure 5.

DMC modulated SKOV3 cells’ progression via up-regulation of miR-551a. (a) Expression levels of miR-551a in SKOV3 cells treated with different concentrate of DMC. Data are presented as the mean ± SD (n = 15, each group). *p < 0.05 versus control group; **p < 0.01 versus control group. (b) CCK-8 assay was employed to evaluate the relative viability of DMC and miR-551a on SKOV3 cells. Data are presented as the mean ± SD (n = 5, each group). *p < 0.05 versus control group; ^#p < 0.05 versus DMC (20 µM) + anti-miR-551a group; ^&p < 0.05 versus DMC (40 µM) + anti-miR-551a group; ^▲p < 0.05 versus DMC (80 µM) + anti-miR-551a group. (c) Flow cytometry analysis of DMC and miR-551a on SKOV3 cells. Data are presented as the mean ± SD (n = 5, each group). *p < 0.05 versus control group; ^#p < 0.05 versus DMC (20 µM) + anti-miR-551a group; ^&p < 0.05 versus DMC (40 µM) + anti-miR-551a group; ^▲p < 0.05 versus DMC (80 µM) + anti-miR-551a group. (d) Western blot analysis for DMC regulated IDVs of IRS2, p-PI3K, PI3K, p-Akt, and Akt via up-regulated miR-551a; they are shown using GAPDH as endogenous control. Data are presented as the mean ± SD (n = 5, each group). *p < 0.05 versus control group; ^#p < 0.05 versus DMC (20 µM) + anti-miR-551a group; ^&p < 0.05 versus DMC (40 µM) + anti-miR-551a group; ^▲p < 0.05 versus DMC (80 µM) + anti-miR-551a group.

MiR-551a sensitized SKOV3 cells to DMC by targeting IRS2

Either miR-551a or DMC could affect the biological behavior of SKOV3 cells, and miR-551a was positively correlated with DMC, and miR-551a expression was positively correlated with DMC treatment. We hypothesized that DMC might restrain SKOV3 cells’ progress via up-regulating miR-551a. To investigate the underlying mechanism, SKOV3 cells were treated with both DMC and pre-miR-551a/anti-miR-551a. CCK-8 assays showed the relative viability of cells treated with DMC, and pre-miR-551a were lower than cells treated with DMC and anti-miR-551a (Figure 5(b)). Moreover, SKOV3 cells’ apoptosis ratio was higher in DMC and miR-551a group (Figure 5(c)). The expression of IRS2/PI3K/Akt was measured by western blot to ascertain underlying mechanism. As Figure 5(d) showed, the expression of IRS2/PI3K/Akt was down-regulated in DMC + pre-miR-551a group. This gave us a clear evidence that DMC attenuated ovarian cancer cells’ malignancy via up-regulated miR-551a.

Discussion

EOC is the most common and malignant tumor in women worldwide.¹⁹ Finding new chemotherapeutic agents and fighting against chemotherapy resistance is the urgent mission in EOC treatment. In this study, we demonstrated that DMC could inhibit SKOV3 cell proliferation and induce apoptosis. MiR-551a had a low expression in OCT than in normal tissues. Over-expression of miR-551a inhibited SKOV3 cell proliferation and induced cell apoptosis. Furthermore, miR-551a was up-regulated when SKOV3 cells were treated with DMC. In addition, miR-551a could down-regulate PI3K/Akt axis by targeting IRS2 which could inactivate PI3K/Akt pathway.

Accumulating evidence showed that DMC is a more effective and stable natural agent than CUR or bisdemethoxycurcumin (BDMC, 3%−5%) in cancer treatment. DMC inhibited prostate cancer PC-3 cells’ proliferation via mitogen-activated protein kinase (MAPK) pathway and displayed the most effective among the three compounds in prostate cancer PC-3 cells.²⁰ Similarly, DMC led to G2/M arrest and induced apoptosis in human glioblastoma U87 cells.²¹ Moreover, DMC induced apoptosis by increasing the level of reactive oxygen species and the activating of caspase-3 in human renal carcinoma caki cells.⁸ Consistent with the previous reports, we showed that DMC suppressed human EOC SKOV3 cells’ growth and induced apoptosis. However, the mechanism of DMC involved in the cellular process in SKOV3 cells needs to be elucidated.

MiRNAs are involved in many cellular biological processes such as proliferation, migration, invasion, and apoptosis. MiR-551a was first found to act as a suppressor gene in colorectal cancer and gastric cancer.^15,16 MiR-551a also exerted the function of inhibiting migration and invasion in gastric cancer cells.¹⁶ Consistent with the previous reports, our results showed that miR-551a was down-regulated in OCT and cells, and over-expression of miR-551a inhibited proliferation and induced apoptosis in SKOV3 cells. However, the underlying mechanism needed to be elucidated.

IRS2 was considered as the target gene of miR-551a in our study. IRS2 is a member of insulin receptor substrate (IRS) family, linking upstream activator to diverse multiple downstream effectors involved in cellular growth, metabolism, and differentiation. IRS2 is dysregulated and acts as an oncogene in many solid tumors such as colorectal cancer, gastric cancer, and breast cancer.^22–25 Moreover, IRS2 is remarkably up-regulated and involved in the malignant progression of ovarian cancer.¹⁷ The activation of PI3K/Akt pathway involved in many tumor pathological processes in tumor cells such as proliferation, apoptosis, and chemotherapeutics resistant.²⁶ Remarkably, previous study found that IRS2-deleted blocked PI3K/Akt pathway.^27,28 To investigate the underlying mechanism of DMC and miR-551a effect on SKOV3 cells, we validated the expression of IRS2/PI3K/Akt axis. Our results showed that IRS2/PI3K/Akt axis was blocked in the SKOV3 cells with DMC than in the SKOV3 cells with no extra treatment. We also found that over-expression of miR-551a decreased the expression of IRS2 protein, and reporter vectors’ construction and luciferase assays’ results showed that miR-551a could target IRS2 3′UTR, indicating that IRS2 was the downstream gene of miR-551a. Furthermore, PI3K/Akt pathway was blocked in the stable expressing pre-miR-551a SKOV3 cells. Thus, over-expression of miR-551a attenuated PI3K/Akt axis in ovarian cancer cells by targeting IRS2. However, whether miR-551a could sensitize ovarian cancer cells to DMC remains unclear.

Previous studies described that miRNAs are involved in resisting tumor cells to chemotherapeutics by targeting diverse pathway.^29–31 Liu et al. demonstrated miR-451a sensitized breast cancer to Tamoxifen and inhibited growth of breast cancer cells. In addition, Mussnich et al.³² illustrated miR-199a-5p and miR-375 reduced colon cancer sensitive to CTX. Our results showed the expression of miR-551a was up-regulated in the SKOV3 cells treated with DMC. This inferred miR-551a involved in the functional effect of DMC on ovarian cancer cells. Furthermore, the cells treated with DMC and pre-miR-551a displayed the lowest expression of IRS2/PI3K/Akt. Moreover, in DMC + anti-miR-551a group, activity of IRS2/PI3K/Akt pathway was rescued. However, deeper insight into the mechanism of DMC affects the expression of miR-551a remains to further explore. The mechanism underlying the suppression of ovarian cancer cells by DMC is schematically presented in Figure 6.

Figure 6.

The schematic cartoon of the mechanism of DMC-hindered cell growth and induced cell apoptosis via negative regulation of miR-551a in ovarian cancer cells.

Collectively, our study showed that miR-551a was down-regulated in EOT. MiR-551a sensitized ovarian cancer SKOV3 cells to DMC. Mechanistically, miR-551a reduced the expression of IRS2 and dampened the activation of PI3K/Akt pathway. More importantly, DMC/miR-551a might represent a potential therapeutic target for the treatment of human EOC.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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