Isovitexin Suppresses Cancer Stemness Property And Induces Apoptosis Of Osteosarcoma Cells By Disruption Of The DNMT1/miR-34a/Bcl-2 Axis.

BACKGROUND: Isovitexin (apigenin-6-C-glucoside, ISOV) is a natural flavonoid that exhibits tumor suppressive activity on various types of cancer. However, it is unknown whether the mechanism of its action in osteosarcoma (OS) is associated with epigenetic regulation and whether it involves DNA methyltransferase 1 (DNMT1), microRNAs and their targets.
MATERIALS AND METHODS: The present study investigated the effects of ISOV on DNMT1 activation and miR-34a and Bcl-2 expression levels in order to explain the mechanism underlying ISOV-mediated repression of proliferation and stemness. In addition, the induction of apoptosis in the spheres derived from OS cells was investigated.
RESULTS: The results indicated that ISOV significantly repressed survival, induced apoptosis and decreased the level of CD133, CD44, ABCG2 and ALDH1 mRNA in the spheres derived from U2OS (U2OS-SC) and MG63 cells (MG63-SC). ISOV further reduced the sphere formation rate of U2OS-SC and MG63-SC. It is important to noted that, ISOV inhibited tumor growth and reduced tumor size of U2OS-SC xenografts in nude mice, which was accompanied by decreased CD133 protein levels, elevated apoptotic index, downregulation of proliferating cell nuclear antigen (PCNA) expression, reduced DNMT1 activity and expression, increased miR-34a and decreased Bcl-2 levels. We identified that Bcl-2 as a direct functional target of miR-34a. Furthermore, ISOV exhibited a synergistic effect with 5-aza-2'-deoxycytidine, the miR-34a mimic or ABT-263 in order to repress cell survival, induce apoptosis, downregulate CD133, CD44, ABCG2 and ALDH1 mRNA expression levels and reduce sphere formation rates of U2OS-SC and MG63-SC cells.
CONCLUSION: The findings suggested that ISOV-mediated epigenetic regulation involved the DNMT1/miR-34a/Bcl-2 axis and caused the suppression of stemness and induction of apoptosis in the spheres derived from OS cells. The data indicated that ISOV exhibited a novel efficient potential for the treatment of OS.

Introduction

Osteosarcoma (OS) is a common bone derived malignant cancer represents a disease with high incidence rates in children and young adults.1,2 No significant improvements with regard to the long-term survival of the patients with OS have been reported due to metastases at initial diagnosis and due to the lack of sensitivity to chemotherapy with current therapeutic regimens.3 Therefore, the development of effective therapeutic agents that can increase drug-response rates is a promising strategy to avoid chemoresistance and improve clinical outcomes of subjects with OS.4

Increasing evidence supports the notion that a small population of cancer cells with stem cell characteristics, termed as cancer stem-like cells (CSLC) are the primary cause for chemoresistance owing to their resistance to apoptosis induced by chemotherapy and to the reduction of the stem-like features, including sphere formation ability and expression of stem cell specific markers (CD133, CD44, ABCG2 and ALDH1).5 Therefore, the investigation of the underlying mechanism by which inhibition of cell survival and induction of apoptotic cell death is mediated in human osteosarcoma stem-like cells (OSLCs) may aid the development of novel treatment strategies for patients with OS.

Emerging evidence has indicated that epigenetic regulation acts as a driver of tumorigenesis by regulating stem-like features of CSLCs.6,7 Aberrant DNA methylation can promote progression of OS.8,9 DNA methyltransferase 1 (DNMT1) is a key enzyme that is involved in DNA methylation and contributes to the maintenance of stem-like features in multiple cancer types, including OS.7,10–13 MiRNAs play a crucial role in self-renewal of CSLCs. Muhammad et al14 reported that anti-miR-203 significantly inhibited the stemness of breast cancer cells. More importantly, tumor suppressive miRNAs, such as miR-34a can be silenced by DNA hypermethylation in its promoter region using DNMT1 catalysis.15,16 Although Zou et al5 reported that the stem-like feature of the spheres from the U2OS cell line with OSLCs characteristics were regulated by miR-34a, the role of aberrant DNMT1 activation in silencing miR-34a expression was not fully discovered in these phenotypes.

Several agents derived from natural products have been used for clinical treatment, suggesting multiple biological activities, such as anti-inflammation and anti-tumor properties. Isovitexin (apigenin-6-C-glucoside, ISOV, also known as homovitexin17 or saponaretin18) is one of the major active components of the herbs Cucurbitaceae, Vigna radiate, Passiflora mucronata, Cannabis sativa and Vitex trifolia L.19–23 ISOV has demonstrated anti-cancer effects in cervical cancer, hepatic carcinoma and prostate cancer cells by inducing apoptotic cell death.24–26 Our recent study indicated that ISOV could suppress self-renewal capacity in spheres from human hepatocellular carcinoma MHCC97H cells.27 However, it is currently unknown whether and how ISOV represses proliferation and stem-like features. Furthermore, the exact mechanism by which ISOV promotes apoptotic cell death in OSLCs has not been clarified. Considering that Bcl-2, a mitochondrion-associated anti-apoptotic protein, was directly regulated by miR-34a,28–30 the purpose of the present study was to examine the in vivo and in vitro multiplication inhibitory activities and the corresponding apoptosis inducing effects of ISOV in OSLCs. Furthermore, we aimed to investigate the potential mechanisms associated with these processes, notably with regard to the involvement of epigenetic regulations in OS cells.

Materials And Methods

Reagents And Sphere Culture Of OS Cell Lines

Isovitexin (ISOV) and 5-aza-2′-deoxycytidine (Aza-dC) were purchased from Sigma–Aldrich (St. Louis, MO, USA). ABT-263 (Navitoclax) was purchased from Medchemexpress (Monmouth Junction, NJ, USA). The human OS U2OS and MG63 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

For sphere culture, U2OS and MG63 cells (5,000 cells/wells) were suspended in CSC-conditioned medium (CSC-CM) composed of serum-free DMEM/F12 (Invitrogen, Carlsbad, CA, USA) with 100 IU/mL penicillin, 100 µg/mL streptomycin, 20 ng/mL hrEGF (Invitrogen), 20 ng/mL hbFGF (Invitrogen), 2% B27 (Invitrogen), 0.4% BSA (Invitrogen) and 4 µg/mL insulin (Sigma-Aldrich). The cells were plated into ultra-low attachment 6-well plates (Corning Inc., Corning, NY, USA) for two weeks. The spheres that were obtained from U2OS and MG63 cells (U2OS-SC and MG63-SC) and exhibited OSLC characteristics, as demonstrated previously by Zou et al5 and Liu et al,31 were used in the present study.

Preparation Of PBMCs

Normal human peripheral blood mononuclear cells (PBMCs) were collected from the healthy people’s medical examination specimen at the Second Xiangya Hospital of Central South University. The use of the primary cells was approved by the ethics committee of the Second Xiangya Hospital of Central South University (Changsha, China). PBMCs were isolated by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density-gradient centrifugation and cultured in RPMI 1640 medium supplemented with 20% FBS.

Determination Of Cell Viability

The cell counting kit-8 (Dojindo, Kumamoto, Japan) was used to measure the cell viability according to the manufacturer’s instructions. Briefly, the single cell suspensions were cultured at a density of 1,000 cells/well (CSC-CM of U2OS-SC or MG63-SC or PBMCs). The cells were plated into 96-well plates and treated with or without various concentrations of ISOV (1.0, 3.0 and 10.0 μM), Aza-dC (2.5 μM) or ABT-263 (1.0 μM) alone or in combination for 72 h. A total of 10 μl of CCK-8 solution was added in each well for 2 h. The absorbance of each sample was detected at 450 nm by a microplate reader (Bio-Rad, Hercules, CA, USA).

Determination Of Apoptotic Cell Death

The sub-G1 cell population was analyzed by flow cytometry using propidium iodide (PI) staining, as described by Zhao et al.32 Briefly, the single cell suspensions were adjusted at a density of 1×105 cells/well (CSC-CM of U2OS-SC and MG6-SC) and were plated into 6-well plates. The cells were treated with or without the aforementioned agents for 72 h. PI staining was performed for DNA analysis using a FACScan flow cytometer and the FlowJo software, ver. 7.1.0 (Tree Star, USA).

Determination Of The Histone/DNA Fragment

The internuclear shear of apoptotic cell was estimated using the cell apoptosis ELISA detection kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s protocol. Briefly, the single cell suspensions (1×104 cells/well in CSC-CM of U2OS-SC and MG63-SC) were plated into 96-well plates and treated with or without the aforementioned compounds for 24 h. Following 24 h of incubation, the cytoplasmic fraction of the cells was transferred to the 96-well plates and incubated with streptavidin and the biotinylated histone antibody containing the peroxidase-tag mouse anti-human DNA for 2 h at room temperature. The absorbance was measured at 405 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).

Quantitative Real-Time PCR

For mRNA detection, U2OS-SC (1×105 cells), MG63-SC (1×105 cells), or xenograft tissues (50 mg) were used to extract the total RNA using the TRIzol universal reagent (Cat. DP424, Tiangen biotech, Beijing, China). The SureScript™ first-strand cDNA synthesis kit (Cat. QP057, GeneCopoeia Inc., Maryland, USA) was used to transcribe 2 μg total RNA into cDNA. The BlazeTaq™ One-Step SYBR Green qRT-PCR kit (Cat. QP047, GeneCopoeia Inc., Maryland, USA) was employed for cDNA amplification in a CFX Connect fluorescent quantitative PCR analyzer (Bio-Rad Laboratories). The primers used are listed in the . The amplifiable procedures were set as follows: 95°C for 10 min, followed by 35 cycles of 95°C (30 sec), 55°C (30 sec) and 70°C (30 sec). Human β-actin mRNA was used as an internal control for RNA normalization.

For miRNA detection, the miRcute miRNA isolation kit (Cat. DP501, Tiangen biotech, Beijing, China) was used to prepare the sample miRNA following the manufacturer’s protocol. MiRNA (2 μg) was transcribed into cDNA using All-in-One™ miRNA qRT-PCR detection kit (Cat. QP016, GeneCopoeia Inc., Maryland, USA), which included the All-in-One miRNA first-strand cDNA synthesis kit and the All-in-One miRNA qRT-PCR detection kit. The primers used are depicted in the . The results were analyzed by the method of 2−ΔΔCt. U6 RNA was used as an internal control.

Determination Of Sphere Formation Rates

The first-forming spheres as described in the above sphere culture section were treated with or without the aforementioned agents for 14 days. The second-forming spheres were cultured in the absence of an agent for an additional 14 days. In the second sphere culture, the single cell suspensions of U2OS-SC and MG63-SC (1,000 cells/well) were plated into ultra-low attachment 24-well plates (Corning Inc., Corning, NY, USA). The sphere formation rate was measured as follows: total number of spheres obtained/viable cells. The result was multiplied by a factor of 100, derived from the second-forming spheres.

In Vivo Xenograft Experiments

Male BALB/c-nu mice (old, 32 days; body weight:12–14 g) provided by the Hunan Silaike Jingda Laboratory Animal Co., Ltd. (Changsha, China) were used in this study (certificate No: 430047 00050992, Changsha, China) and the procedures were approved by the Ethics Committee of the Hunan Normal University (Permit No: Hunnu-2019012). Animal welfare and experimental procedures were carried out strictly in accordance with the care and use of laboratory animals (National Research Council, 1996) and was performed in compliance with the Animal Protection Law of the People’s Republic of China (2009) for experimental animals.

To estimate the effects of ISOV on the tumor growth of nude mouse xenografts, U2OS-SC (2×106/mL) suspended in CSC-CM was obtained from U2OS cells stably expressing red fluorescein protein. A total of 100 μl mixture [the cell suspensions mixed with matrigel (1:1; BD Biosciences, San Jose, CA, USA)] was injected subcutaneously into the left and right flanks of each mouse respectively. The mice were orally administered 200 μl of 2% DMSO (control group) and isovitexin (10, 20 and 40 mg/kg body weight, respectively) daily for a total of 14 times (experiment groups). The tumor diameters were estimated from the beginning of the treatment and the tumor volume (length × width2/2) and the body weight of mice was monitored twice weekly during the experiment period. The fluorescence intensity was recorded and analyzed by in vivo imaging using specific software (PerkinElmer Inc., NY, USA) following one week after the end of treatment. The xenografts were extracted, weighed, and frozen or fixed for further analysis.

Immunohistochemical Staining And TUNEL Assay

Immunohistochemical staining and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay were conducted following standard procedures. The tissue slides were incubated at 4°C overnight with an anti-DNMT1 antibody (1:200; DNMT1, Catalog No. 3598S, Cell Signaling Technology), an anti-CD133 antibody (1:100; CD133, Catalog No. ab16518), or an anti-PCNA antibody [EPR3822] (1:100; PCNA, Catalog No. ab92729). Phosphate-buffered saline instead of primary antibody was used as the negative controls. The apoptotic and proliferative indices were quantified using a digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) and the AxioVision Rel.4.6 computerized image analysis system (Carl Zeiss Co Ltd., Jena, Germany) as described by Wu et al33 in each group. A total of 6 tumor tissue samples were used.

Western Blot Analysis

The lysates were prepared from U2OS-SC or MG63-SC (1×106 cells) or from xenograft tissues (50 mg). Immunoblotting with primary antibodies against β-actin (1:5,000; Catalog No. A5441; Sigma-Aldrich), Bcl-2(C-2) (1:1,000; Catalog No. sc-7382; Santa Cruz Biotechnology) and DNMT1 and PARP (1:2,000; Catalog No. 3598S and #9542, Cell Signaling Technology) was performed as described previously.33

Detection Of DNMT1 Activity

The EpiQuik™ Nuclear Extraction Kit (Epigentek Group, USA) was used to prepare the nuclear extracts. DNMT1 activity was measured by a non-radioactive protocol using the DNA Methyltransferase Activity/Inhibition Assay Kit (Epigentek) as instructed by the manufacturer, based on the following formula: DNMT activity (OD/h/mg) = (No inhibitor OD-Blank OD) 1000/Protein amount (µg) × hour. The relative DNMT1 activity was normalized to the activity of the control group.

miRNA Transfection

MicrON™ miR-34a mimic (miR-34a) was provided by RiboBio (Guangzhou, China) and was transfected into U2OS-SC and MG63-SC cells using the iboFECT™ CP reagent (RiboBio Co., Ltd, Guangzhou, China) at a final concentration of 50 nM following the manufacturer’s instructions. The transfection protocol for the miR-34a mimic negative control (miR-NC) was the same as that for the miR-34a sample.

Luciferase Reporter Assay

U2OS-SC and MG63-SC (1×105 cells) were co-transfected with miR-34a or miR-NC and pLUC vector containing the firefly luciferase reporter and the wild-type or mutated 3′-UTR sequence of the Bcl-2 gene. Following 48 h of transfection, luciferase activity was detected using a luciferase assay kit (Promega, Madison, WI, USA) and the assays were conducted in triplicate.

Statistical Analysis

Statistical analysis was performed using the SPSS 20.0 software (IBM, Armonk, NY, USA) and presented as mean ± standard deviation (SD). The comparisons with the control groups were performed using a two-tailed Student’s t-test. All the pairwise comparisons between the groups were analyzed by the Tukey’s post-hoc test using one-way ANOVA. The significance was determined by a P value lower than 0.05 (p<0.05).

Results

ISOV Represses Proliferation, Promotes Apoptosis And Reduces Stemness Of U2OS-SC And MG63-SC In Vitro

Given that U2OS-SC and MG63-SC have shown the characteristics of OSLCs,5,31 we initially measured the effects of ISOV on cell viability using the cell counting kit-8 (CCK-8). Although no significant cytotoxicity was noted in PBMCs () at the concentration range of 1 μM to 100 μM, ISOV preferentially suppressed the proliferation of U2OS-SC and MG63-SC cells in a dose-dependent manner with an IC50 of approximately 10 μM following 72 h of treatment (Figure 1A and B) compared with U2OS and MG63 cells ( and ). Furthermore, ISOV induced apoptotic cell death of U2OS-SC and MG63-SC cells in a dose-dependent manner, as indicated by the increased sub-G1 population, the DNA/Histone fragment detection and the PARP cleavage products (Figure 1C–F and and ). It is important to note that ISOV downregulated the mRNA expression levels of CSC markers and reduced tumor sphere formation rates in both spheres (Figure 1G–J).

Figure 1

ISOV represses proliferation, promotes apoptosis and reduces stemness of U2OS-SC and MG63-SC in vitro. ISOV decreased cell viabilities of U2OS-SC (A) and MG63-SC (B); increased sub-G1 population of U2OS-SC (C) and MG63-SC (D) and increased the amount of the DNA/Histone fragment in U2OS-SC (E) and MG63-SC (F); ISOV reduced the mRNA levels of CD133, CD44, ABCG2 and ALDH1 in U2OS-SC (G) and MG63-SC (H) and the sphere formation rates of U2OS-SC(I, Scale bar=200μm) and MG63-SC (J, Scale bar=100μm). *p<0.05 vs control; #p<0.05 vs 1.0 μM ISOV treatment (n=3).

ISOV Inhibits Tumor Growth Of U2OS-SC Xenografts In A Nude Mouse Model

We further examined ISOV-associated suppression in vivo with nude mouse models bearing subcutaneous U2OS-SC xenografts. ISOV significantly decreased tumor size, volume and weight in the ISOV-treated groups compared with those noted in the model control (Figure 2A–C). Moreover, ISOV reduced the amount of the CD133 protein and increased the apoptotic index of U2OS-SC tumor xenografts in nude mouse models as determined by immunohistochemical and TUNEL staining (Figure 2D and E). Concomitantly, it decreased the proliferative index, as determined by immunohistochemical analysis (Figure 2F). The data further indicated that ISOV significantly decreased the expression levels of the Bcl-2 and DNMT1 proteins, as determined by immunoblotting (Figure 2G and J). In addition, no significant differences were noted in the average body weight of the mice between the vehicle and the ISOV groups (). The expressions of miR-34a were upregulated and the DNMT1 activity was reduced by ISOV treatment in U2OS-SC tumor xenografts (Figure 2H and I).

Figure 2

ISOV restrains tumor growth in U2OS-SC xenograft nude mouse models. Living images of the nude mice bearing tumors (A); the tumor volume (B), tumor weight (C); H&E staining and immunohistochemichal results following incubation with the CD133 antibody (D); TUNEL assay (E); immunohistochemical assay using PCNA antibody (F); immunoblotting using a Bcl-2 antibody (G); miR-34a-5p expression by qRT-PCR analysis (H); DNMT activity detected ELISA (I) and immunoblotting using a DNMT1 antibody (J). The samples were derived from ISOV-treated mice (0, 10, 20, and 40 mg/kg/day). Magnification: 400; *p<0.05 vs control; #p<0.05 vs 10.0 mg/kg ISOV treatment (n=6).

Identification Of Bcl-2 As A Direct Target Of miR-34a

Previous studies reported that miR-34a re-expression could repress proliferation and promote apoptosis by regulation of Bcl-2.29,30 Since the aforementioned results revealed that ISOV reduced the expression of Bcl-2 protein in vivo, we examined if the effects of ISOV on proliferation, apoptosis and stem-like features of OSLCs were involved in the regulation of Bcl-2 by miR-34a. Consistent with the results reported by Zhao et al,29 we found that Bcl-2 may be a putative target of miR-34a, as predicted by TargetScan software (Figure 3A). To confirm this hypothesis, the miR-34a binding sequences present at the 3′-UTR of Bcl-2 mRNA (3′-UTR-wt) or its mutant (3′-UTR-mut) form were subcloned into the downstream of the firely luciferase reporter gene in the pLUC vector. These two vectors were labeled pLUC/Bcl-2-3′-UTR-wt and pLUC/Bcl-2-3′-UTR-mut, respectively (Figure 3A). pLUC/Bcl-2-3′-UTR-wt or pLUC/Bcl-2-3′-UTR-mut, and miR-34a or miR-NC were co-transfected into U2OS-SC and MG63-SC and the luciferase activity was determined. The luciferase activity was inhibited by treatment of the cells with the miR-34 mimic, when the wildtype 3′-UTR sequence of Bcl-2 was used. However, in the presence of the mutated 3′-UTR of Bcl-2, this effect was not noted (Figure 3B and C). Furthermore, miR-34a mimic and ISOV cooperatively decreased the amount of Bcl-2 protein and upregulated the expression of miR-34a, without affecting the inhibition of DNMT1 activity and the inhibition in its expression levels caused by ISOV in U2OS-SC and MG63-SC cells (Figure 3D–K).

Figure 3

MiR-34a binds to the 3ʹ-UTR of human Bcl-2 mRNA. The mutation was generated on the Bcl-2 3′-UTR sequence at the complementary site for the seed region of miR-34a-5p, as shown (A). Relative luciferase activity was analyzed in U2OS-SC (B) and MG63-SC (C) co-transfected with wildtype or mutant 3′-UTR reporter plasmids and miR-34a or miR-NC. Immunoblotting with Bcl-2 antibody in U2OS-SC (D) and MG63-SC (E); miR-34a-5p expression by qRT-PCR analysis in U2OS-SC (F) and MG63-SC (G); DNMT activity by ELISA in U2OS-SC (H) and MG63-SC (I); immunoblotting using a DNMT1 antibody in U2OS-SC (J)and MG63-SC (K) following treatment with miR-NC or miR-NC in the presence of ISOV (3 μM) or miR-34a or both. *p<0.05 vs miR-NC; #p<0.05 vs miR-NC plus 3 μM ISOV treatment (n=3).

The Effects Of The Combination Treatment Of ISOV With Aza-dC On Proliferation, Apoptosis And Stemness Of U2OS-SC And MG63-SC In Vitro

To determine the combined effects of DNMT1 inhibition and ISOV on proliferation, apoptosis and stemness of U2OS-SC and MG63-SC cells in vitro, we used the DNMT1 inhibitor Aza-dC and demonstrated that ISOV and Aza-dC cooperatively repressed proliferation of U2OS-SC and MG63-SC cells (Figure 4A and B). In addition, ISOV and Aza-dC cooperatively promoted apoptotic cell death of U2OS-SC and MG63-SC, as determined by increased sub-G1 population, DNA/Histone fragment detection and PARP cleavage (Figure 4C–F and and ). Furthermore, ISOV and Aza-dC cooperatively decreased the mRNA levels of CD133, CD44, ABCG2 and ALDH1 and tumor sphere formation rates in these cells (Figure 4G–J).

Figure 4

ISOV cooperates with Aza-dC to repress proliferation, promote apoptosis and reduce stemness of U2OS-SC and MG63-SC in vitro. ISOV(3 μM) cooperated with Aza-dC(2.5 μM) to decrease cell viability of U2OS-SC (A) and MG63-SC (B); ISOV increased sub-G1 population of U2OS-SC (C) and MG63-SC (D) as well as amount of the DNA/Histone fragments of U2OS-SC (E) and MG63-SC (F); ISOV reduced the mRNA levels of CD133, CD44, ABCG2 and ALDH1 in U2OS-SC (G) and MG63-SC (H) as well as sphere formation rates of U2OS-SC(I, Scale bar=200μm) and MG63-SC (J, Scale bar=100μm). *p<0.05 vs control; #p<0.05 vs 3 μM ISOV treatment (n=3).

Combination Effects Of ISOV With miR-34a Mimic On Proliferation, Apoptosis And The Stemness Of U2OS-SC And MG63-SC In Vitro

To examine the effects of miR-34a expression on the mechanism of action of ISOV, we measured the effects of ISOV in the presence of the miR-34 mimic (miR-34a) on cell viability, and demonstrated that ISOV and miR-34a cooperatively suppressed the proliferation of U2OS-SC and MG63-SC cells (Figure 5A and B). In addition, ISOV and miR-34a cooperatively promoted apoptotic cell death of U2OS-SC and MG63-SC, as demonstrated by increased sub-G1 population and DNA/Histone fragment detection (Figure 5C–F). In addition, ISOV and miR-34a cooperatively decreased the mRNA amounts of CD133, CD44, ABCG2 and ALDH1 and tumor sphere formation rates in these cells (Figure 5G–J).

Figure 5

ISOV cooperates with miR-34a mimic to repress proliferation, promote apoptosis and reduce stemness of U2OS-SC and MG63-SC in vitro. ISOV (3 μM) synergized with miR-34a mimic to decrease cell viability of U2OS-SC (A) and MG63-SC (B); ISOV increased sub-G1 population of U2OS-SC (C) and MG63-SC (D) as well as amount of DNA/Histone fragments of U2OS-SC (E) and MG63-SC (F); ISOV reduced the mRNA levels of CD133, CD44, ABCG2 and ALDH1 in U2OS-SC (G) and MG63-SC (H) as well as sphere formation rates of U2OS-SC(I, Scale bar=200 μm) and MG63-SC (J, Scale bar=100 μm). *p<0.05 vs control; #p<0.05 vs 3 μM ISOV treatment (n=3).

Effects Of The ISOV Combination With ABT-263 On Proliferation, Apoptosis And Stemness Of U2OS-SC And MG63-SC In Vitro

To confirm the effects of Bcl-2 downregulation on the action of ISOV, we measured the effects of ISOV combined with the Bcl-2 inhibitor ABT-263 on cell viability and demonstrated that ISOV and ABT-263 cooperatively suppressed the proliferation of U2OS-SC and MG63-SC cells (Figure 6A and B). In addition, ISOV and ABT-263 cooperatively promoted apoptotic cell death of U2OS-SC and MG63-SC, as determined by the increased sub-G1 population and the DNA/Histone fragment detection (Figure 6C–F). It is important to note that ISOV and ABT-263 cooperatively decreased the mRNA levels of CD133, CD44, ABCG2 and ALDH1 and the tumor sphere formation rates of these cells (Figure 6G–J).

Figure 6

ISOV cooperates with ABT-263 to repress proliferation, promote apoptosis and reduce stemness of U2OS-SC and MG63-SC in vitro. ISOV(3 μM) acted in a synergistic mode with ABT-263 (1 μM) to decrease cell viability of U2OS-SC (A) and MG63-SC (B); increase sub-G1 population of U2OS-SC (C) and MG63-SC (D) as well increase the amount of the DNA/Histone fragments of U2OS-SC (E) and MG63-SC (F); ISOV reduced the mRNA levels of CD133, CD44, ABCG2 and ALDH1 in U2OS-SC (G) and MG63-SC (H) as well as sphere formation rates of U2OS-SC(I, Scale bar=200μm) and MG63-SC (J, Scale bar=100μm). *p<0.05 vs control; #p<0.05 vs 3 μM ISOV treatment (n=3).

Discussion

In the current study, we initially demonstrated that ISOV could preferentially repress proliferation, induce apoptotic cell death and decrease stemness of OS spheres at least partly via interruption of the DNMT1/miR-34a/Bcl-2 axis in vitro and in vivo. ISOV thus exhibited therapeutic potential for OS by targeting OSLCs. The mechanism of action involved the epigenetic regulation on DNMT1, miR-34a and its target Bcl-2.

Natural products play a critical role in the discovery and the development of numerous drugs for the treatment of various types of diseases, including cancer.34–36 ISOV is present in several edible or medicinal plants and possesses various biological activities, including anti-inflammatory and neuroprotective properties.17–23,37–39 ISOV also induces apoptosis in various cancer cell types by the regulation of the expression of anti-apoptotic proteins.24–26 However, it is still unknown whether the regulation of anti-apoptotic proteins (e.g. Bcl-2) is involved in the alteration of the microRNA levels by the modification of DNMT1 activity caused by ISOV treatment. It has been shown that apigenin, which is an analogue of ISOV, could inhibition of DNMT1 activity.40 The data of the present study demonstrated for the first time that ISOV effectively promoted apoptotic cell death of U2OS-SC and MG63-SC via downregulation of Bcl-2 protein levels and upregulation of miR-34a levels. In addition, ISOV was capable of inhibiting the expression and activity of DNMT1. This notion is supported by the following evidence: (i) ISOV reduced DNMT1 activity and protein expression accompanied by an increase in miR-34a levels and a decrease in Bcl-2 levels in U2OS-SC tumor xenografts; (ii) miR-34a mimic and ISOV cooperatively caused an increase of miR-34a and a decrease of the Bcl-2 protein without affecting the inhibition on DNMT1 activity and the reduction in its expression levels caused by ISOV treatment alone in U2OS-SC and MG63-SC; (iii) Bcl-2 was a direct functional target of miR-34a; (iv) ISOV and Aza-dC, miR-34a mimic or ABT-263 cooperatively suppressed the proliferation and promoted apoptotic cell death in U2OS-SC and MG63-SC. These results increase our understanding on the epigenetic mechanism by which ISOV suppresses cell proliferation and induces apoptotic cell death in spheres from OS cells that contain an enriched OSLC population. These effects were regulated by the DNMT1/miR-34a/Bcl-2 signaling axis.

Increasing evidence has demonstrated the presence of CSCs in OS, which exert substantial impact on the design and development of novel targeted treatments for OS.41 Therefore, agents that can directly target OSLCs or sensitize OSLCs to current chemotherapy regimens are required for the treatment of patients with OS.31 Previous studies have reported the use of sphere culture in the isolation and identification of OSLCs in U2OS and MG63 cells.5,31 Our recent study has demonstrated that ISOV inhibits carcinogenicity and stemness in hepatic carcinoma stem-like cells.27 In the current study, ISOV repressed sphere formation, induced apoptotic cell death, and reduced mRNA levels of CSC markers in U2OS-SC and MG63-SC cells. Moreover, it significantly decreased the CSC population with CD133 positive expression in nude mouse tumor xenografts, which demonstrated that ISOV may be an effective agent for OS therapy by targeting OSLCs. Therefore, ISOV may have the potential to improve the clinical outcomes of patients with OS. MiR-34a has been reported to have multiple direct targets, such as the CSC marker CD44,42 the carcinogenic transcription factor FoxM143 and the CSC signaling pathways, including Wnt44 and Notch.45 Additionaly, Muhammad et al46 demonstrated that c-Fos overexpression enhanced the epithelial-mesenchymal transition (EMT) state and the expression of CSC markers (Nanog, c-Myc, Sox2, and Notch1). Therefore, further studies are required to establish whether these pathways are all regulated by elevated levels of miR-34a in CSLCs treated with ISOV.

Conclusions

In summary, the present study provides comprehensive evidence for ISOV with regard to the suppression of OSLC features, the induction of apoptotic cell death and the inhibition of cell survival and tumor growth by the epigenetic regulation of DNMT1, miR-34a and its target Bcl-2 (Figure 7). We suggest that ISOV is a promising therapeutic candidate for patients with OS. The current study further identified the DNMT1/miR-34a/Bcl-2 signaling axis as a novel regulatory mechanism of OSLCs. These findings can be used for the development of promising therapeutic agents for patient with OS.

Figure 7

Schematic diagram of the mechanism underlying isovitexin inhibits the stemness and induces apoptotic of OSLC via the DNMT1/miR-34a/Bcl-2 axis. Constitutive activation of DNMT1, epigenetic silencing of miR-34a expression, leading to up-regulation of Bcl-2 expression by disinhibition has an important role in the promotion and maintenance of OSLC characteristics. ISOV inhibits the stemness and induces apoptotic of OSLC by disruption of the DNMT1/miR-34a/Bcl-2 axis.