The effect of co-treatment with DAPT and oxaliplatin on the biological behavior of human ovarian cancer stem cells

To assess the effects of the combination of DAPT and oxaliplatin on the biological behavior of human ovarian cancer stem cells. In vitro cultured human ovarian cancer stem cells were randomly divided into DAPT, L-OHP, DAPT + L-OHP, and control groups. MTT assays were measured to assess the ability of inhibit proliferation. Inverted microscopy, flow cytometry, and in vitro invasion assays were performed to assess cell morphology, apoptosis, and cell invasion, respectively. Then, western blotting was used to detect Notch-1 and LRP1 expression in the four groups of cells. MTT assay revealed that DAPT and L-OHP monotherapy could inhibit the proliferation of ovarian cancer stem cells in a time- and dose-dependent manner. Inverted microscopy showed that untreated ovarian cancer stem cells had an oval or polygonal morphology, with a plump shape and large nuclei. After treatment with DAPT or L-OHP, cells shrank and cracked, with an irregular shape and increased shedding. Flow cytometry revealed that apoptosis was significantly higher in the DAPT and L-OHP groups compared with the control group; the DAPT + L-OHP group had a significantly higher rate of apoptosis than either the DAPT or L-OHP groups. An in vitro invasion assay revealed that DAPT + L-OHP inhibited cell invasion to a greater extent than either DAPT or L-OHP alone. Western blotting revealed that, compared with control, L-OHP had no effect on Notch-1 protein expression, whereas DAPT and DAPT + L-OHP significantly reduced Notch1 protein levels. In addition, cells treated with DAPT + L-OHP expressed much less Notch than those treated with DAPT alone. In the L-OHP group, LRP protein levels were increased significantly, whereas levels were decreased significantly in the DAPT and DAPT + L-OHP groups. DAPT inhibits the proliferation of ovarian cancer stem cells, promotes their apoptosis, weakens their invasive ability, and functions synergistically with L-OHP.


Background
Ovarian cancer has the highest mortality rate among all gynecological malignancies because early diagnosis is rare and the treatment options available are poor. As such researchers have obtained an in-depth understanding of the biological behavior and mechanisms of ovarian cancer growth in an attempt to identify better treatments. In recent years, progress in cancer stem cytology-based techniques has helped characterize the biological behavior of ovarian cancer from a novel perspective [1]. To further explore the role of ovarian cancer stem cells in the occurrence and development of ovarian cancer, we developed a serum-free suspension culture method with paclitaxel in media containing EGF, bFGF, Noggin and LIF. This method was used both in vitro and in vivo to successfully screen the CDl33+, CD117+ expression characteristics of ovarian cancer stem cells and identify their specific markers and biological characteristics [2].
The current comprehensive treatment program for ovarian cancer is generally surgery supplemented with chemotherapy. Ovarian cancer chemotherapy commonly utilizes platinum drugs, among which oxaliplatin has gained increased clinical emphasis due to its high efficiency and low toxicity [3]. The Notch signaling pathway has been studied extensively in recent years, and was confirmed to be closely associated with the occurrence, development, and drug resistance of several tumors [4]. Among the Notch receptor family members, the Notch-1 receptor is studied most frequently, and it plays a very important role during Notch signaling. Use of the g-secretase inhibitor DAPT, which also inhibits the Notch-1 receptor, has revealed its important function in regulating tumor cell growth, proliferation, and apoptosis [5]. The aim of the current study was to assess the effect of the combination of DAPT and oxaliplatin on the biological behavior of ovarian cancer stem cells to attempt to identify novel strategies for the treatment of ovarian cancer.

Materials
Human ovarian cancer stem cells were screened and stored in the Gynecology Department at the Affiliated Hospital of Xuzhou Medical College [2]. MTT and apoptosis detection kits were purchased from Nanjing KeyGen Biotechnology Development Company. Serum-free medium was purchased from Hangzhou Evergreen Biological Engineering Materials Co.
DAPT and L-OHP was purchased from Wuhan Biochempartner Co. Ltd. Notch-1, and lung resistance protein (LRP) primary and secondary antibodies were purchased from Chemicon (USA). Invasion chambers were purchased from Sigma (USA).

MTT assays
Cells were seeded in 96-well plates at a density of 1.5 × 10 4 cells/well. After 1, 2, 3, 4, 5, and 6 days in culture, 20 μL MTT-working solution was added, and the cells were cultured for an additional 4 h in a CO 2 incubator at 37°C. Dimethyl sulfoxide was then added to stop the reaction, and the optical absorbance (A) at 490 nm of each well was measured using an enzyme-linked immunosorbent detector. The rate of inhibition of proliferation was calculated according to the following formula: inhibition (%) = (1 − OD of the experimental group / OD of the control group) × 100%. The Q method described by Zhang 5 SQ [6]was used to calculate the interaction index of the two drugs: q = E (A + B) / (EA + EB−EA × EB), where EA and EB are the inhibition rate of each drug alone. Q >1.15 defines synergism, q = 0.85-1.15 represents a simple additive effect, and q <0.85 represents antagonistic action. Based on these results, the optimal conditions for DAPT, L-OHP, DAPT + L-OHP, and the control group were selected and used for subsequent analyses.

Inverted microscopy
Proliferating stem cells were harvested, and seeded into 6-well culture plates at a density of 4 × 10 5 cells/ml. Changes in morphology were then observed under an inverted microscope after 24 h in culture.

Flow cytometry
Cells were harvested using 0.25% trypsin, washed with PBS, centrifuged, and diluted to 1.5 × 10 5 cells/ml. They were then stained with 5 µl Annexin V-FITC and 5 µl PI for 10 min in the dark at room temperature, and analyzed by flow cytometry.

In vitro invasion assay
Transwell chambers were used for in vitro invasion assays, with a Matrigel invasion film placed between the upper and lower chambers. A single-celled suspension was plated on the invasion film in 200 μl per well (approximately 10 5 cells), and cells were cultured at 37°C, with 5% CO 2 for 12 h. The cells and Matrigel were then removed from the membrane by wiping, and the invasion film was and fixed ready for staining with hematoxylin and eosin (H&E). Cell number was counted using a microscope, and the rate of invasion inhibition was calculated using the following formula: invasion inhibition (%) = (1 − cell number in experimental group/cell number in the control group) × 100%.

Western blotting
Cells in the exponential growth phase were harvested and lysed in 200 μl of cell lysis buffer on ice. The protein content was quantified using a bicinchoninic acid (BCA) assay.
An equal amount of protein was then electrophoresed on 10% sodium dodecyl sulfonate sodium-polyacrylamide gels (SDS-PAGE), and electrophoretically transferred to nitrocellulose membranes. Membranes were blocked with 5% skimmed milk for 60 min, and then incubated with 1:1,000 anti-Notch-1 or anti-LRP antibodies (rabbit anti-human) at 4°C overnight, followed by horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:10,000) at room temperature for 2 h. Enhanced chemiluminescence (ECL) reagents were then added, and the signal exposed and developed using highly sensitive Xray film in a dark room.

Statistical analysis
SPSS16.0 software (Chicago, IL, USA) was used for all statistical analyses, and experimental data are presented as ± s. Comparisons of mean values between two groups were performed using one-way ANOVA. The difference between two sets of data was compared using t-tests. Correlations were analyzed using Pearson correlation coefficiency. P <0.05 was considered to indicate statistical significance.

Effects of L-OHP and DAPT, alone or in combination, on the proliferation of human ovarian cancer stem cells
Different concentrations of L-OHP inhibited the proliferation of ovarian cancer stem cells to varying degrees. This effect was significant when compared with the control group (P < 0.05). Pearson correlation analysis revealed clear dose-(r = 0.773) and time-dependent effects (r = 0.591; Table 1). Similarly, different concentrations of DAPT also inhibited the proliferation of ovarian cancer stem cells to varying degrees. Compared with control, these effects were statistically significant (P <0.05). Pearson correlation analysis revealed obvious dose-(r = 0.819) and time-dependent effects (r = 0.520; Table 2).
Next, the effect of co-treatment with L-OHP (2.5, 5, and 7.5 µg/ml) and DAPT (20,40,80 and µmol/L) was assessed in ovarian cancer stem cells. Increasing drug concentrations inhibited cell proliferation significantly (P <0.05) compared with the control group and the corresponding monotherapy groups. According to the Q method reported by Zhang SQ [6], co-treatment with 2.5 µg/ml L-OHP and 7.5 µg/ml DAPT had a clear additive effect (q = 0.85-1.15). When 5µg/ml L-OHP was used, synergistic effects occurred after co-treatment with 20, 40, or 80 µmol/L DAPT (q >1.15). Treatment with 5µg/ml L-OHP alone inhibited proliferation by 38.24%; co-treatment with 20, 40, and 80 µmol/L DAPT inhibited the 24 h proliferation rate of ovarian cancer stem cells by 60.13%, 68.17%, and 71.33%, respectively. Therefore, the inhibition rate increased with increasing concentrations of DAPT. This result demonstrates that DAPT synergizes with L-OHP to inhibit the proliferation of ovarian cancer stem cells ( Table 3).

Effects of L-OHP and DAPT, alone or in combination, on the morphology of human ovarian cancer stem cells
Under an inverted microscope, control cells exhibited an oval or polygonal morphology, with a round shape and a large nucleus. However, after 24 h treatment with 5 µg/ml L-OHP, blurred cell membranes were observed, with an enlarged cell gap, and increased apoptosis. Similarly, 24 h treatment with 40 µg/ml DAPT resulted in fusiform, shrunk, and cracked cells with a reduced cell density, enlarged cell gaps, an increased number of nuclear particles, and enhanced apoptosis. After 24 h of treatment with the combination of 40 µg/ml DAPT and 5 µg/ml L-OHP, the cell number was reduced further; in addition, more cells had disintegrated and were undergoing apoptosis (Fig.1).

Increased apoptosis in human ovarian cancer stem cells treated with L-OHT and DAPT alone and in combination
Flow cytometry revealed that the apoptosis rate of cells in the L-OHP (17.7 ± 0.6%) and 8 DAPT groups (15.3 ± 0.2%) was increased significantly compared with the control group (8.1 ± 0.6%; P < 0.05). As expected, the apoptosis rate in the DAPT + L-OHP group (36.5 ± 0.3%) was significantly higher than in the L-OHP or DAPT groups (P <0.05; Fig.2).

Synergistic effects of DAPT and L-OHP on inhibiting the invasion of human ovarian cancer stem cells
The rates of invasion inhibition in the DAPT and L-OHP alone groups were 40.3 ± 4.2% and 50.2 ± 7.8%, respectively. However, co-treatment inhibited invasion by 75.1 ± 2.9%. This suggests that L-OHP could enhance the DAPT-mediated inhibition of cell invasion in ovarian cancer stem cells (Fig.3).
LRP protein levels in the L-OHP group (0.811 ± 0.03) were significantly higher than in the control group (0.686 ± 0.03; P < 0.05). In contrast, levels in the DAPT (0.487 ± 0.01) and DAPT + L-OHP groups (0.318 ± 0.04) were significantly lower than in the control and L-OHP groups (both P < 0.05). Finally, LRP expression was significantly lower in the DAPT + L-OHP group than in the DAPT group (P <0.05; Fig.4).

Discussion
Currently, the standard treatment for ovarian cancer is effective cytoreductive surgery in combination with chemotherapy. Although most patients obtain varying degrees of disease relief after this treatment regimen, most will suffer relapse or become resistant to chemotherapy and consequently die because of the ineffectiveness of the existing antineoplastic therapies.
Ovarian cancer stem cells can cause ovarian cancer; their ability for self-renewal, unlimited proliferation, and differentiation is considered to be a root cause of the occurrence, abnormal proliferation, metastasis, and recurrence of ovarian cancer [7]. Therefore, the search for effective chemotherapy drugs and chemotherapy programs for the ablation of ovarian cancer stem cells has become urgent. Platinum-based drugs, particularly cisplatin and carboplatin, play a very important role in chemotherapy for ovarian cancer; however, the effectiveness of chemotherapy is often unsatisfactory because of drug resistance. Oxaliplatin is a third generation, broad-spectrum platinumbased anticancer drug that produces alkylating conjugates to damage DNA and inhibit DNA synthesis. It has completely different pharmacokinetic characteristics from, and consequently no cross-resistance with, carboplatin and cisplatin; therefore, ovarian cancer patients who are resistant to cisplatin or carboplatin remain sensitive to oxaliplatin [8,9].
Hopfer et al. [10]revealed that the expression of activated Notch-1 was significantly higher in ovarian cancer than in benign ovarian tumors and normal ovarian tissues. In addition, Notch-1 is expressed in the ovarian cancer cell line, suggesting that Notch signaling, and particularly Notch-1, is closely associated with the occurrence, development, and drug resistance of ovarian cancer. Likewise, other investigators demonstrated the importance of the abnormal regulation of Notch signaling in several characteristics of other tumor cells including invasion, metastasis, and drug resistance [11,12].
g-secretase is a multi-molecule complex and a key enzyme during activation of the Notch signaling pathway. As such, g-secretase inhibitors inhibit the activation of Notch signaling [13,14]. As well as directly inhibiting the Notch signaling pathway in tumor cells, g-secretase inhibitors also interfere with Notch signal transduction between tumor cells and tumor vascular epithelial cells, thereby inhibiting angiogenesis [15]. DAPT is a synthetic gsecretase inhibitor that blocks the cleavage of Notch receptors, which is mediated by gsecretase. This inhibits the conversion of Notch receptors into active fragments, therefore suppressing the activation of the Notch signaling pathway [16,17]. Western blotting showed that Notch-1 protein levels were comparable in the L-OHP and control groups. However, levels were reduced significantly in the DAPT and DAPT + L-OHP groups compared with the control and L-OHP groups. Notably, the DAPT + L-OHP group had more reduced expression. The above results suggest that DAPT might inhibit 11 proliferation and induce apoptosis in ovarian cancer stem cells by downregulating Notch-1 expression; these effects are more pronounced after co-treatment with DAPT and L-OHP.
The human LRP gene is located on the short arm of human chromosome 16; the molecular weight of the encoded LRP is 110 kD. LRP plays roles in tumor cell multidrug resistance (MDR) via two mechanisms: (1) pumping out drugs that target nuclei or inhibiting their transition through the nuclear pore into the nucleus; and (2) engulfing drugs into vesicles in the cytoplasm and discharging them to the extracellular compartments via exocytosis.
Therefore, LRP mediates the resistance to DNA-targeting drugs such as platinum-based and DNA-alkylating agents [18][19][20]. In the current study, western blotting revealed that LRP protein levels were increased significantly in the L-OHP group compared with control.
In contrast, levels were reduced significantly in the DAPT and DAPT + L-OHP groups compared with the control and L-OHP groups; the reduction was greater in the DAPT + L-OHP group.
Taken together, these data suggest that the sensitizing effects of DAPT on L-OHP might be related to the downregulation of Notch-1 and LRP protein expression. However, additional experiments are required to verify any potential correlation between Notch signaling and LRP.
Based on the results of the current study, we propose that activation of the Notch signaling pathway might play an important role in the acquisition of chemotherapy resistance in ovarian cancer, as well as the migration and invasion of cancer cells. This suggests that therapeutic interventions targeting the Notch signaling pathway might help improve the chemosensitivity of ovarian cancer stem cells and provide a novel perspective for the treatment of ovarian cancer.

Conflict of Interest
Author Nan Yu declares that she has no conflict of interest. Author Yuchen Yan declares that he has no conflict of interest. Author Lisha Fang declares that she has no conflict of interest. Author Yang Xu declares that she has no conflict of interest. Author Qiang Li declares that he has no conflict of interest. Author Hongchao Yan declares that he has no conflict of interest.

Funding
The work was supported by the General Program of the Natural Science Foundation of Jiangsu Province (No.BK20171182).

Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent
For this type of study, formal consent is not required

Availability of data and material
All data generated or analysed during this study are included in this published article