RETRACTED ARTICLE: Genistein inhibits stemness of SKOV3 cells induced by macrophages co-cultured with ovarian cancer stem-like cells through IL-8/STAT3 axis

Human ovarian cancer SKOV3 and OVCAR-3 cells and human monocyte THP-1 cells were obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China).

SKOV3 and OVCAR-3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Gibco, Grand Island, NY, USA), containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin, and then were incubated at 37 °C in an atmosphere with 5% CO₂. The second generation spheres of SKOV3 cells were obtained using sphere-forming assay, and then were considered as OCSLCs [15].

THP-1 cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 100 IU/ml penicillin G, and 100 μg/ml streptomycin, and then were incubated in a humidified atmosphere of 5% CO₂ at 37 °C. THP-1 macrophages were induced by phorbol-12-myristate-13-acetate (PMA; final concentration: 100 ng/ml) for 24 h. Activated THP-1 macrophages were obtained as previously described [15]. In brief, the THP-1 macrophages (2 × 10⁶) were plated in the lower chamber and cultured for 12 h. Then the SKOV3- and OVCAR-3-derived OCSLCs (2 × 10⁶) were seeded in the upper chamber and co-cultured for 24 h in transwell system (BD Biosciences, San Jose, CA, USA). After that, the THP-1 macrophages activated OCSLCs in the lower chamber and the supernatant of co-culture (Co-CM) were collected.

SKOV3 and OVCAR-3 cells (2 × 10³) were suspended in serum-free DMEM/F12 mixture containing 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml hrEGF, 20 ng/ml hbFGF, 0.2% B27, 0.4% BSA, and 4 μg/ml insulin (cancer stem cell conditioned medium, CSC-CM) as well as Co-CM (v/v: 1:1). The cells were then seeded in an ultra low attachment 6-well plate (Corning Inc., Coring, NY, USA). The total number of tumor spheres was counted after culturing for 8 days. The efficiency of sphere formation was calculated as previously described [15]. Three independent experiments were performed.

DMEM medium containing 0.7% agarose was added into a 6-well plate. Then, 10⁴ SKOV3 and OVACR-3 cells were seeded per well in CSC-CM as well as Co-CM (v/v: 1:1) containing 0.4% agarose (top layer), and incubated for 3 weeks. Colony count was carried out by using an inverted microscope (Olympus IX53, Japan). Three independent experiments were performed.

Co-CM was collected, and was incubated with IL-8 neutralizing antibody (50 nM) (PeproTechInc, USA) for overnight at 4 °C. Then the medium was centrifuged at 12,000 g for 10 min, and the supernatants were collected for use as conditioned medium for IL-8 immunodepletion. Collected Co-CM was mixed with sterile 10 ng/ml of recombinant human IL-8 (R&D Systems, USA), and this was considered as the conditioned medium for adding IL-8.

THP-1 macrophages (1 × 10⁵) were seeded into 6-well culture plates (Corning Inc.) and incubated overnight until they reach 50% confluence, and infected with adenoviral particles loaded with pHBad-MCMV-EGFP-STAT3, pHBad-MCMV-EGFP, pHBad-U6-GFP-shSTAT3, and pHBad-U6-GFP plasmids (Hanbio Biotechnology Co. Ltd., Shanghai, China), respectively. The cells were cultured with Opti-MEM containing 50.0 μL adenoviral particles (Han Heng Biotech Corp) using Enhanced Infection Solution (Jikai gene Co., Ltd., Shanghai, China) for 2 h, and after which, the medium was replaced with DMEM containing 10% FBS. Infection efficiency was calculated by counting GFP-positive cells and live cells using the same high power field under a fluorescent microscope (Olympus IX53, Japan).

Co-CM was collected from SKOV3- or OVCAR-3-derived OCSLC/THP-1 macrophage co-cultures, centrifuged at 1000 g for 5 min to obtain the supernatants, and then assessed for IL-10, IL-12, and IL-8 levels by ELISA with specific kits (Neobioscience, Shenzhen, Guangdong, China) according to the manufacturer’s instructions. Absorbance was immediately read at 450 nm on a microplate reader (BioTek, Winooski, Vermont, USA).

To examine the effects of GEN on co-cultures, THP-1 macrophages co-cultured with SKOV3- or OVCAR-3-derived OCSLCs were treated with or without different concentrations of GEN (10, 20, and 40 μmol/L) for 24 h. For determining the induced effects of GEN combined with depletion or addition of Co-CM on macrophage polarization and SKOV3 cell stemness, the THP-1 macrophages and SKOV3 cells were treated with or without the conditioned medium from Co-CM depleted IL-8 by neutralizing antibody or added recombinant human IL-8 in the presence or absence of GEN (10 μM). To evaluate the linkage between STAT3 activation in THP-1 macrophages and GEN treatment in co-cultured THP-1 macrophages with SKOV3-derived OCSLCs, the co-culture of THP-1 macrophages expressing STAT3 or STAT3 shRNA or both with SKOV3-derived OCSLCs were treated with or without GEN (10 μM).

The cells were harvested and lysed using ice-cold RIPA lysis buffer (Beyotime Biotechnology, CN). Bradford assay (Bio-Rad Laboratories, Hercules, USA) was used to determine the protein concentration. Equal amounts of protein (40 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a polyvinylidenedifluoride membrane (Millipore, Billerica, USA). The membranes were then blocked with TBST supplemented with 5% BSA for 2 h at room temperature, and incubated with primary antibodies against CD133, CD44, CD163 and STAT3 (Abcam, Burlingame, USA, dilution of 1:2000), Nanog and Oct4 (Cell Signaling Technology; Danvers, MA, USA), p-STAT3 (Tyr 705) and β-actin (Santa Cruz, USA, dilution of 1:2000) for overnight at 4 °C. The membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody (Beyotime Institute of Biotechnology, Shanghai, China) for 1 h. The protein bands were detected using enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, USA).

Female BALB/c-nude mice (4–5 weeks of age, body weight 12–14 g) were purchased from Nanjing Institute of Biomedical Research in Nanjing University. The experimental procedure was performed in accordance with the standard protocols and approved by the Ethics Committee of Hunan Normal University (No. 2015–055) and the Committee of Experimental Animal Feeding and Management (ID: 201607119). Mice were acclimated to their new environment for 1 week prior to undergoing the experiment.

To determine that effects of interaction between SKOV3-derived OCSLCs and THP-1 macrophages on the growth of tumors in nude mice in vivo, the mice were injected with SKOV3-derived OCSLCs (1 × 10⁵ cells) in the left flank, and co-injected OCSLCs (1 × 10⁵ cells)/THP-1 macrophages (2 × 10⁵ cells) in the right flank, respectively.

For GEN therapeutic experiments, SKOV3-derived OCSLCs (1 × 10⁵) and THP-1 macrophages (2 × 10⁵) were mixed with matrigel (1:1), and then 100 μL mixture was injected subcutaneously into each Balb/c-nu mouse. After the xenograft volume achieved about 100mm³, the mice were randomly divided into 4 groups, with 4 mice in each group. Group 1 mice were given olive oil by gavage and was considered as control group; Group 2 mice were orally given Genistein dissolved in olive oil (50 mg/kg), once on alternate days, for a total of 10 times; Group 3 mice were intratumorally injected with 20 μL per mouse of adenovirus loaded with pHBad-U6-GFP-shSTAT3 (Hanbio Biotechnology Co. Ltd), once a week, for a total of 3 times; and Group 4 mice were orally given Genistein (50 mg/kg) plus injected the adenovirus expressing STAT3 shRNA. Then, the longest (L) and shortest (W) diameters of the subcutaneous xenografts were measured with a Vernier caliper for volume assessment, according to the following formula: V (transplanted tumor volume, mm³) = L × (W)² × 0.5. At the end of the experiment, the mice were euthanized and xenografts were weighed after extraction. Xenograft specimens were fixed in 10% neutral formalin. Tissue sections were submitted to H&E staining, and the histopathological morphology was evaluated by optical microscopy.

Data were analyzed using SPSS 20.0 for Windows (SPSS Inc., Chicago, USA). All the experiments were repeated three times and the data were presented as means±SD. Comparisons between the groups for statistical significance were conducted using a two-tailed Student’s t-test. The differences between multiple groups were analyzed by one-way analysis of variance. First, the homogeneity of variance was determined, and all the pairwise comparisons between the groups were analyzed using least significant difference (LSD) method. Tukey’s test was performed in the event of incomplete variance of both the control and the experimental groups. Significance was determined as p < 0.05.

To determine the effects of GEN on M2 phenotype of THP-1 macrophages co-cultured with OCSLCs, the co-culture system of SKOV3-derived OCSLCs/THP-1 macrophages was used. Figure 1a and b indicated that GEN down-regulated CD163 and p-STAT3 expression of THP-1 macrophage, although the expression of STAT3 showed no significant change. In addition, GEN also decreased the levels of IL-10 (Fig. 1c), increased the levels of IL-12 (Fig. 1c) and nitric oxide (NO) (Fig. 1d) in a dose-dependent manner in Co-CM. Furthermore, we found that the levels of IL-8 in Co-CM were reduced by GEN treatment (Fig. 1e). The similarity findings were observed in OVCAR-3-derived OCSLCs/THP-1 macrophages co-culture (Additional file 1: Figure S1). These results suggested that GEN inhibition of M2 polarization might be involved in decreasing IL-8 secretion and inhibiting STAT3 activation in THP-1 macrophages co-cultured with OCSLCs.

To assess the inhibitory effects of GEN on ovarian cancer cell stemness induced by co-culture, the Co-CM from the co-culture system of OCSLCs/THP-1 macrophages treated with or without GEN was obtained. The sphere and colony formation assay revealed that GEN could suppress self-renewal ability (Fig. 2a) and in vitro tumorigenic capabilities (Fig. 2b) in SKOV3 cells induced by Co-CM. Furthermore, compared to vehicle (0.1% DMSO), Co-CM containing GEN from the co-culture system significantly decreased the protein expression levels of the cancer stem cell surface markers CD44, CD133 (Fig. 2c) and the multipotent transcription factors Nanog and OCT4 (Fig. 2d) in SKOV3 cells in a dose-dependent manner. The similarity findings were observed in OVCAR-3 cells induced by Co-CM. (Additional file 2: Figure S2). These results suggested that GEN could also inhibit the stemness of ovarian cancer cells induced by Co-CM.

Given that the GEN inhibits macrophage M2 polarization co-cultured with OCSLCs and this might be involved in regulating IL-8 secretion, THP-1 macrophages treated with depletion or addition of IL-8 Co-CM in the presence or absence of GEN was prepared. We found that depletion of IL-8 and GEN together suppressed CD163 and p-STAT3 expression (Fig. 3a and b), but not STAT3 expression in THP-1 macrophages, and reduced IL-10 (Fig. 3c) as well as increased IL-12 (Fig. 3c) and NO (Fig. 3d) in the conditioned medium obtained from THP-1 macrophages treated with IL-8 depletion of Co-CM.

In contrast, addition of IL-8 significantly abolished the inhibitory effects of GEN on CD163 and p-STAT3 expression of THP-1 macrophages (Fig. 3e and f). ELISA analyses revealed the addition of IL-8 addition exhibited antagonistic activity against GEN on IL-10 and IL-12 secretion (Fig. 3g) as well as NO (Fig. 3h) in the conditioned medium obtained from THP-1 macrophages treated by IL-8 addition to Co-CM. Together, these findings demonstrated that the inhibitory effect of GEN on M2 polarization of THP-1 macrophages required inhibition of IL-8 secretion caused by co-culture.

Since GEN could inhibit the secretion of IL-8 through co-culture system, we sought to investigate whether secretion of IL-8 was involved in the effects of GEN on stemness of SKOV3 cells. The results demonstrated that co-treatment of depletion of IL-8 in Co-CM and GEN in co-culture system together attenuated the self-renewal ability (Fig. 4a) and in vitro tumorigenic capabilities (Fig. 4b) in SKOV3 cells. Furthermore, co-treatment significantly decreased the expression levels of CD44 and CD133 in SKOV3 cells (Fig. 4c). Conversely, the addition of IL-8 significantly neutralized GEN decreased the expression levels of CD44 and CD133 in SKOV3 cells induced by Co-CM (Fig. 4d). Addition of IL-8 effectively opposed the GEN attenuated self-renewal ability (Fig. 4e) and in vitro tumorigenic capabilities (Fig. 4f) in SKOV3 cells induced by Co-CM. Together, these findings suggested that the inhibitory effects of GEN on stemness of SKOV3 cells are necessary for the inhibition of IL-8 secretion in co-culture system.

To explore the role of STAT3 activation in GEN inhibition of M2 polarization of THP-1 macrophages, the THP-1 macrophages expressing STAT3 shRNA were initially used in the co-culture system. STAT3 knockdown and GEN treatment alone down-regulated CD163, STAT3 and p-STAT3 expression in THP-1 macrophages, suggesting the suppression of the above by their combined activity (Fig. 5a and b). In addition, STAT3 knockdown and GEN together reduced IL-10 secretion (Fig. 5c) as well as increased IL-12 secretion (Fig. 5c) and NO (Fig. 5d) in Co-CM. Furthermore, we found that the IL-8 levels (Fig. 5e) in Co-CM in response to GEN treatment was further reduced by STAT3 knockdown.

To further identify the role of STAT3 activation in GEN inhibition of M2 polarization of THP-1 macrophages, THP-1 macrophages expressing STAT3 were used in the co-culture system. Our data showed that overexpression of STAT3 attenuated GEN suppressed CD163 and p-STAT3 expression in THP-1 macrophages (Fig. 5f and g). In addition, overexpression of STAT3 abrogated GEN reduced IL-10 secretion (Fig. 5h) as well as increased IL-12 secretion (Fig. 5h) and NO (Fig. 5i) in THP-1 macrophages. In addition, overexpression of STAT3 also abrogated GEN reduced IL-8 levels in Co-CM (Fig. 5j).

To ascertain the role of STAT3 activation in GEN inhibition of M2 polarization of THP-1 macrophage induced by co-culture, THP-1 macrophages expressing STAT3 in those expressing STAT3 shRNA were established. Additional file 3: Figure S3A and B depicted that overexpression of STAT3 attenuated STAT3 shRNA combined with GEN suppressed CD163 and p-STAT3 expression in THP-1 macrophages. In addition, overexpression of STAT3 abrogated co-treatment of STAT3 shRNA and GEN reduced IL-10 secretion (Additional file 3: Figure S3C) as well as increased IL-12 secretion (Additional file 3: Figure S3C) and NO product (Additional file 3: Figure S3D) in THP-1 macrophages. Importantly, overexpression of STAT3 significantly antagonized the inhibitory effects of STAT3 shRNA and GEN co-treatment on IL-8 secretion in Co-CM (Additional file 3: Figure S3E). Collectively, these findings demonstrated that the effects of GEN on M2 polarization of THP-1 macrophages are dependent on the inhibition of STAT3 activation of THP-1 macrophages in the co-culture system.

To investigate the role of STAT3 activation in GEN inhibition of stemness of SKOV3 cells, Co-CM from the co-culture of OCSLCs with THP-1 macrophages expressing STAT3 shRNA was obtained. The results showed that combination of STAT3 knockdown and GEN together attenuated self-renewal ability (Fig. 6a) and in vitro tumorigenic capabilities (Fig. 6b) in SKOV3 cells induced by Co-CM. As indicated in Fig. 6c, combination of STAT3 knockdown and GEN significantly decreased the expression levels of CD44 and CD133 in SKOV3 cells induced by Co-CM.

To further examine the role of STAT3 activation in GEN inhibition of stemness of SKOV3 cells, Co-CM from co-culture of OCSLCs with THP-1 macrophages expressing STAT3 was obtained. Figure 6d and e indicated that overexpression of STAT3 reduced GEN, which inhibited the self-renewability and in vitro tumorigenic capabilities in SKOV3 cells induced by Co-CM. Overexpression of STAT3 abrogated GEN, decreasing the expression levels of CD44 and CD133 in SKOV3 cells induced by Co-CM (Fig. 6f).

To corroborate the role of STAT3 activation in GEN inhibition of stemness of SKOV3 cells induced by Co-CM, Co-CM from co-culture of OCSLCs with THP-1 macrophages expressing STAT3 in those expressing STAT3 shRNA THP-1 macrophages was prepared. Overexpression of STAT3 reduced STAT3 shRNA combined with GEN, which inhibited the self-renewal ability (Additional file 4: Figure S4A) and in vitro tumorigenic capabilities (Additional file 4: Figure S4B) in SKOV3 cells induced by Co-CM. As indicated in Additional file 4: Figure S4A, overexpression of STAT3 abrogated STAT3 shRNA combined with GEN decreased the expression levels of CD44 and CD133 in SKOV3 cells induced by co-culture. Collectively, these findings demonstrated that the effects of GEN on stemness of SKOV3 cells are dependent on the inhibition of STAT3 activation of THP-1 macrophages in the co-culture system.

To clarify whether IL-8/STAT3 axis was involved in GEN inhibition of M2 polarization of THP-1 macrophages, depletion of IL-8 of Co-CM in THP-1 macrophage expressing STAT3 was treated with or without GEN. Figure 7a and b showed that overexpression of STAT3 attenuated depletion of IL-8 combined with GEN, suppressing the expression of CD163 and p-STAT3 in THP-1 macrophages. In addition, overexpression of STAT3 abrogated the depletion of IL-8 combined with GEN reduced IL-10 secretion (Fig. 7c) as well as increased IL-12 secretion (Fig. 7c) and NO product (Fig. 7d) in Co-CM. In addition, overexpression of STAT3 partly attenuated the depletion of IL-8 combined with GEN decreased IL-8 levels in Co-CM (Fig. 7e). These finding suggested that the effects of GEN on M2 polarization of THP-1 macrophage involved IL-8/STAT3 axis in the co-culture system.

To determine the role of IL-8/STAT3 axis in GEN inhibition of stemness of SKOV3 cells induced by Co-CM, depletion of IL-8 of Co-CM in THP-1 macrophages expressing STAT3 with or without GEN treatment was prepared. As indicated in Fig. 8a, overexpression of STAT3 abrogated the depletion of IL-8 combined with GEN decreased the expression levels of CD44 and CD133 in SKOV3 cells induced by Co-CM. Figure 8b and c showed that overexpression of STAT3 reduced the depletion of IL-8 combined with GEN inhibited the self-renewal ability and in vitro tumorigenic capabilities in SKOV3 cells induced by Co-CM. These findings demonstrated that the effects of GEN on stemness of SKOV3 cells required modulation of IL-8/STAT3 axis in the co-culture system.

The results from the nude mouse xenograft model showed that injection with SKOV3-derived OCSLCs alone and co-injection with THP-1 macrophages could form subcutaneous tumors in 30 days; however, the tumor growth by co-injection with OCSLCs/THP-1 macrophages was significantly accelerated than that of injection with OCSLCs alone (Additional file 5: Figure S5A, B and C). Immunohistochemisty revealed elevated human CD68 antigen, IL-8 and p-STAT3 expressions in the co-injected xenografts, compared to OCSLC injection alone (Additional file 3: Figure S3D). These results suggested that the interaction between OCSLCs and THP-1 macrophages promoted the growth of tumors in nude mice in vivo and may be involved in the activation of IL-8/STAT3 signaling pathway.

We also found that GEN plus Ad-STAT3 shRNA reduced the size and weight of xenografts in nude mice co-injected with OCSLCs/THP-1 macrophages (Fig. 9a, b and c). The immunohistochemical staining showed that GEN plus Ad-STAT3 shRNA decreased the expression levels of human CD68, IL-8 and p-STAT3 in tumors of nude mice co-injected with OCSLCs/THP-1 macrophages than OCSLCs alone (Fig. 9d). These results demonstrated that GEN inhibits the growth of tumors co-inoculated with OCSLCs/THP-1 macrophages in nude mice in vivo through blocking IL-8/STAT3 signaling.