Introduction
Ovarian cancer is one of the most common malignant tumors of gynecology, with the highest mortality compared with other gynecologic cancer because of its acute onset, rapid progress and high metastasis rate [
1,
2]. Epithelial ovarian cancer (EOC) accounts for 85–90% of total ovarian carcinoma and is the most aggressive one. In early stage, surgical resection combined with chemotherapy is an effective therapy method [
3]. Unfortunately, most of the patients reach advanced stage at the time of diagnosis [
4,
5]. For patients with advanced EOC, platinum-based chemotherapy is the standard of care. More than 80% of ovarian tumors response to first-line platinum-based therapy [
6], however, the majority of patients acquire resistance to cisplatin (CDDP) treatment and ultimately result in relapse and poor prognosis [
7,
8]. Therefore, it is necessary to develop appropriate combined reagents to solve drug resistance and enhance the sensitivity of EOC to cisplatin treatment.
Chemotherapy resistance is a key factor that limits the cure rate of ovarian cancer. The mechanisms underlying cancer cells resistance to cisplatin are not fully understood. It is acknowledged that various mechanisms are responsible for drug-resistance, including the decrease of the effective concentration of drugs in cells, the abnormalities of drug targets, and the abnormal regulation of cell apoptosis [
9]. Currently, there are some ways to overcome the chemo-resistance, such as maintenance therapy, novel cytotoxic agents, modulation of apoptosis and combination therapy [
10]. Natural medicine, with its small side effects and significant therapeutic effect, attracts a lot attention as a potential combination agent for cisplatin treatment.
Luteolin is one of the most common flavonoid compound that is widely existed in various plants including peppermint, rosemary, thyme, pinophyte, and pteridophyta [
11]. Numerous studies suggested that luteolin possesses a variety of pharmacological properties including anti-inflammatory, antiallergic, antioxidant, antimicrobial, immune regulation and anticancer activities [
11,
12]. Among all these properties, anti-tumor effect has attracted a lot of attention. Researchers have found that luteolin exerts anti-tumor activities via several mechanisms, including cell cycle arrest, apoptosis induction, angiogenesis and metastasis inhibition [
13‐
16]. A previous study has demonstrated that luteolin can sensitize oxaliplatin-resistant colorectal cancer cells to chemotherapeutic drugs through the inhibition of the Nrf2 pathway [
17]. Another study reported that luteolin can be used as a chemosensitizer to improve the therapeutic effect of tamoxifen in drug-resistant human breast cancer cells via the inhibition of cyclin E2 expression [
18]. These results suggest that luteolin exhibits potential chemosensitivity property for various cancers. However, whether luteolin can increase the chemotherapy sensitivity of cisplatin-resistant ovarian cancer and the underlying mechanisms is rarely reported, which needs to be further studied.
In the current study, we investigated the synergistic effects of luteolin combined with cisplatin in drug-resistant ovarian cancer cell line CAOV3/DDP both in vitro and in vivo, and tried to explore associated molecular mechanisms.
Materials and methods
Reagents and cell lines
Luteolin was bought from Jin Sui Biological Technology (Shanghai, China). It was dissolved in DMSO as a stock of 500 mM and stored at − 20 °C. Cisplatin was purchased from QILU Pharmaceutical (Shandong, China). Human drug-resistant ovarian cancer cell line, CAOV3/DDP were obtained from the Shanghai Sixin Biotechnology company (Shanghai, China) and maintained in RPMI1640 (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2.
Cell proliferation assay
Cell proliferation was measured using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto,Japan). Briefly, CAOV3/DDP cells (5 × 103) were seeded into 96-well plates and allowed for adhesion overnight. Then the cells were administrated with eight treatments as follows: control (culture medium); low-dose of luteolin (10 μM); medial-dose of luteolin (50 μM); high-dose of luteolin (100 μM); CDDP (2 μg/ml); CDDP (2 μg/ml) + low-dose of luteolin (10 μM); CDDP (2 μg/ml) + medial-dose of luteolin (50 μM); CDDP (2 μg/ml) + high-dose of luteolin (100 μM). After 48 h treatment, the culture medium was removed and CCK-8 was added according to the manufacturer’s instruction. Then the cells were incubated for 1–4 h at 37 °C and the absorbance was detected at 450 nm using a microplate reader. Cell proliferation was calculated as follows:
Cell proliferation (%) = [(OD of experiment group – OD of blank) / (OD of control group – OD of blank)] × 100%.
Apoptosis analysis
Cell apoptosis was detected using Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, Franklin Lakes, NJ, USA). Cells (2 × 104) were seeded into 6-well plates and treated with various concentration of luteolin (0, 10, 50, 100 μM) or CDDP alone or in combination for 48 h. Then both the adherent and floating cells were harvested and stained according to the manufacturer’s protocol. The apoptosis rate was analyzed by flow cytometry.
Wound-healing assay
Cell migration ability was measured by wound-healing assay. Briefly, cells were seeded into 6-well plates and allowed to grow to a monolayer. Subsequently, a straight scratch was generated across the plate using a 200 μl pipet tip. The cells were washed with PBS and incubated with various concentration of luteolin (0, 10, 50, 100 μM) and CDDP alone or in combination (dissolve the chemicals in serum-free culture medium). Wound healing was observed and photographed at 0 and 48 h.
Matrigel invasion assay
The Matrigel was diluted in serum-free RPMI-1640 (RPMI-1640: Matrigel = 8:1) and added into the upper chamber. After treatment with various concentrations of luteolin (0, 10, 50, 100 μM) and CDDP alone or in combination for 48 h, the cells (5 × 104) were trypsinized and collected. 5 × 104 cells in 200 μl serum-free medium were seeded into the upper chamber. The lower chamber was filled with 600 μl complete medium containing 10% FBS. After incubation for 48 h, the invaded cells were stained with crystal violet and pictured under a microscope at x100 magnification.
qRT-PCR
After treatment, the medium was removed and the cells were washed with PBS. The total RNA of each group was extracted using TRIzol (Invitrogen, California, USA). Then the RNA was reversely transcribed to cDNA using the PrimeScript™ RT Reagent kit (Takara, Dalian, China) according to the manufacturer’s instruction. The qPCR was performed using a SYBR Premix Ex Taq (Tli RNaseH Plus) in Applied Biosystem 7300 (Applied Biosystems, Foster city, CA, USA). The BCL-2 mRNA expression was analyzed using the 2-ΔΔCq method taking β-Actin as reference. The gene primer sequences were shown in Table
1.
Table 1
Primer sequences for genes
BCL-2 | F: 5’-AACATCGCCCTGTGGATGAC-3’ |
R: 5’-AGAGTCTTCAGAGACAGCCAGGAG-3’ |
β-Actin | F: 5’-CATTGCCGACAGGATGCAG-3’ |
R: 5’-CTCGTCATACTCCTGCTTGCTG-3’ |
Western blot
CAOV3/DDP cells were seeded into 6-well plates (2 × 105/well),and treated with increasing doses of luteolin (0, 10, 50, 100 μM) or cisplatin (2 μg/ml) or both for 48 h. Then, the cells were harvested, and total proteins were extracted using cell lysis buffer (1 mM PMSF, 50 mM Tris (pH 8.1), 1% SDS, sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, sodium fluoride, EDTA, leupeptin and other inhibitors) (Beyotime Biotechnology, Shanghai, China. No. P0013G). The protein concentration was detected using BCA assay (Mai Bio Co., Ltd.). 20 μg proteins of each group were separated on SDS-PAGE, and then transferred onto PVDF membranes (Millipore Corp., Bedford, MA, USA). Membranes were blocked with 5% non-fat dry milk, and probed with primary antibodies against Bax (1:4000, Cell Signaling Technology, USA), Bcl-2 (1:4000, Cell Signaling Technology, USA), and β-Actin (1:5000, ProteinTech Group, Inc., USA) at 4 °C overnight. Then the membrane was washed with PBS and incubated with HRP-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Finally, the blots were imaged with ECL (EMD Millipore).
In vivo xenograft experiment
Female BALB/c nude mice (5–6 weeks old) were obtained from the Shanghai Experimental Animal Center. Animals were raised in pathogen-free conditions at 22 °C, 50% humidity. Animal experiments were approved by the Institutional of Animal Care and Use Committee of Jinshan Hospital, Fudan University. The cisplatin resistant cell line CAOV3/DDP (5 × 106 cells) in a volume of 100 μl of PBS were inoculated in the subcutaneous tissue of the nude mice. Two weeks after implantation, the tumors were visible and the mice were randomly allocated into 8 groups (6 mice per group): (1) control group (normal saline); (2) luteolin low-dose (10 mg·kg− 1·d− 1) group; (3) luteolin medial-dose (20 mg·kg− 1·d− 1) group; (4) luteolin high-dose (40 mg·kg− 1·d− 1) group; (5) CDDP (3 mg·kg− 1·d− 1) group; (6) CDDP (3 mg·kg− 1·d− 1) plus luteolin low-dose (10 mg·kg− 1·d− 1) group; (7) CDDP (3 mg·kg− 1·d− 1) plus luteolin medial-dose (20 mg·kg− 1·d− 1) group; (8) CDDP (3 mg·kg− 1·d− 1) plus luteolin high-dose (40 mg·kg− 1·d− 1) group. The CDDP were intraperitoneal injected once daily, and luteolin were given by gavage once daily for 5 days. The tumor volume was measured three times a week. Three weeks after treatment, the mice were sacrificed, and the tumor volume and weight were measured. The tumor tissues were used for histopathologic examination.
TUNEL
Tumor paraffin tissue sections were processed with TUNEL assay to analyze apoptosis. The procedure was performed according to instructions of the TUNEL kit (KeyGen, Nanjing, China). The samples were observed under a microscope at × 100 magnification. The apoptotic cells were counted in three random fields for each sample, and the apoptosis percentage was calculated as follows: (Number of TUNEL-positive cells/Total number of cells in the field) × 100%.
Drug combination effect analysis
Combination effect between the luteolin and cisplatin was analyzed by the Zheng-Jun Jin method [
19‐
21]. In this method, the combination rate was evaluated by the inhibition rate via the Q value. The formula for the Q value is: Q = Ea + b / (Ea + Eb - Ea × Eb), where Ea + b, Ea, and Eb are the inhibition rate of the combination group, drug a and drug b, respectively. Q = 1 would mean simple addition; Q > l, synergism or potentiation, Q < 1, antagonism.
Statistical analysis
All the experiments were repeated three times. The data were presented as mean ± SD. The difference between indicated groups were analyzed using Student’s t-test. P < 0.05 was considered be statistically significant.
Discussion
Cisplatin is one of the most effective therapeutic agents widely used in clinic for the treatment of EOC. However, drug resistance is a major problem that limits its clinical application. Therefore, combination treatment with new sensitizing agents is an effective strategy to overcome cisplatin resistance [
10]. Luteolin, a flavonoid that has been identified in many plants, has demonstrated in numbers studies to exhibit chemopreventive or chemosensitising properties against various human cancers. In the current study, we provide experimental evidence both in vivo and in vitro that luteolin is able to enhance the therapeutic potential of cisplatin in ovarian cancer.
In the current study, firstly, we evaluated the effect of luteolin or cisplatin or the combination of both on the cell proliferation in human cisplatin-resistant ovarian cancer CAOV3/DDP cells. We found that luteolin alone inhibited the cell proliferation in a dose-dependent manner, and co-treatment with both agents could further decrease cell proliferation. These results suggested that luteolin could exert synergistic anti-proliferation effect with cisplatin in CAOV3/DDP cells.
Apoptosis inhibition is one of the main mechanisms responsible for the resistance of chemotherapy [
22]. Cisplatin is one of the most effective drugs for the treatment of ovarian cancer, and the mechanism involved in the process of its cytotoxicity include survival inhibition and apoptosis induction. Once the apoptotic pathway is blocked, tumor cells acquire resistance to pro-apoptotic effect of cisplatin, which reduces the antitumor effect of cisplatin [
23]. Therefore, inhibition of apoptosis is an effective strategy to overcome the drug resistance and promote the anti-tumor effect of cisplatin [
24]. Luteolin has been reported to induce apoptosis in various cancer cells such as human cervical cancer cells [
13], esophageal carcinoma cells [
25] and colorectal cancer cells [
26]. Our study found that the single treatment with luteolin could dose-dependently induce apoptosis in CAOV3/DDP cells, when combined with cisplatin, luteolin could significantly enhance cisplatin-induced cell apoptosis, indicating that luteolin enhanced the sensitivity of cisplatin, in part, through apoptosis induction.
The BCL-2 protein family plays a key role in the regulation of cell apoptosis. The BCL-2 protein family can be divided into three different subfamilies, including pro-survival factions such as BCL-2, MCL1 and BCL-XL, which inhibit the apoptosis process, and two pro-apoptotic subfamilies, the death effectors BAX and BAK and the BH3-only proteins such as BID, BIM and PUMA, which contribute to cell apoptosis [
27‐
29]. Consequently, the ratio of Bcl-2/Bax is an essential factor to determine whether a tumor cell commits apoptosis or not. Overexpression of Bcl-2 can inhibit cell apoptosis, lead to resistance to cisplatin, and result in poor prognosis of cancer patients. Recent study has demonstrated that Bcl-2 is overexpressed in ovarian cancer [
30,
31] and has a significant positive correlation with sensitivity to cisplatin in ovarian cancer cells [
32]. Therefore, targeting Bcl-2 may provide an effective therapeutic method to solve drug resistance in ovarian cancer. It was previously reported that luteolin could decrease Bcl-2 expression in various cancer cells [
33]. In the current study, results from qRT-PCR showed that luteolin at high concentration (100 μM) could significantly decrease the Bcl-2 mRNA level, and the combination of luteolin with cisplatin could evidently inhibit Bcl-2 expression compared with cisplatin alone. This suggests that the combined treatment induced cell apoptosis through the inhibition of Bcl-2 expression. The BCL-2 family proteins control the permeability of mitochondria and the release of cytochrome c to the cytoplasm, following the activation of a group of caspases, which proceeds apoptosis [
27]. This suggests that mitochondrial apoptosis pathway may be involved, and further study should be focused on the pathway.
Our data also revealed the potent antitumor effect of luteolin with cisplatin in ovarian cancer in vivo. Single treatment with increasing doses of luteolin showed growth inhibition in xenograft tumor. In addition, tumor volume and weight were significantly decreased in mice of combination treatment group compared with cisplatin alone. What’s more, the combination therapy synergistically induced more apoptosis than cisplatin, which is in consistent with in vitro study. These results further demonstrate that the inhibition of tumor growth was induced, in part, by the enhancement of cisplatin induced apoptosis.
Ovarian cancer is highly susceptible to occur metastasis in late stage. In most patients, though appearance of the lesion is still localized in the ovary, subclinical metastasis may already exist in many parts of the peritoneal or omentum [
34]. In addition, chemotherapy resistance leads to the decrease of chemotherapy sensitivity in ovarian cancer cells, and also enhance its malignant degree. It suggests that the occurrence of chemotherapy resistance is closely related to the promotion of invasion and metastasis in cancer cells [
35,
36]. Cancer metastasis involves several processes including adhesion, migration, and invasion. Targeting these processes provides effective strategy to enhance the chemosensitivity of cisplatin [
37]. Luteolin has been proven to inhibit metastasis in various caner types such as breast cancer [
38] and prostate cancer [
39]. In our experiment, wound-healing assay and Matrigel invasion assay showed that luteolin exhibited a dose-dependent suppression on migration as well as invasion in CAOV3/DDP cells. Additionally, the inhibition effect became stronger when treated the cells with both increasing concentrations of luteolin and cisplatin than single agent treatment. These results indicate that the improved anticancer effect of cisplatin in CAOV3/DDP cells by luteolin is partially mediated through inhibition in cell migration and invasion.
In conclusion, our study shows that luteolin, a natural flavonoid, significantly enhances the anti-tumor effect of cisplatin in ovarian cancer both in vivo and in vitro. Combination of luteolin and cisplatin is more effective in suppressing CAOV3/DDP cell growth and metastasis. Luteolin could enhance cisplatin induced apoptosis in cisplatin-resistant ovarian cancer CAOV3/DDP cells via decreasing Bcl-2 expression. Our preliminary data provide experimental evidence for potential clinical application of luteolin as a novel chemosensitizer in the chemotherapy in ovarian cancer.
Acknowledgements
We thank Longxiang Zhou, from Department of Science and Education, Jinshan branch of Shanghai Sixth People’s Hospital, Shanghai Jiaotong University, and Guiping Gan, from Department of Obstetrics and Gynecology, Jinshan branch of Shanghai Sixth People’s Hospital, Shanghai Jiaotong University, for their guidance and help in our work.