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ORIGINAL ARTICLE
Year : 2022 | Volume
: 18
| Issue : 7 | Page : 2033-2040
Bone marrow-derived mesenchymal stem cells expressing BMP2 suppress glioma stem cell growth and stemness through Bcl-2/Bax signaling
Jizhen Feng1, Zhigang Yao2, Hongan Yang3, Jiwei Ma2, Xiuming Zhong2, Yejun Qin2, Jiamei Li2
1 Department of Radiology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
2 Department of Pathology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
3 Department of Neurosurgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
Correspondence Address:
Jiamei Li
Department of Pathology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, No. 324 Jingwu Road, Jinan Shandong - 250 021
China
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/jcrt.jcrt_1983_21
Objectives: To find an effective molecule that controls glioma stem cell (GSC) proliferation and differentiation for the development of future therapeutic interventions against glioblastoma.
Material and Methods: Bone marrow-derived mesenchymal stem cells (BMSCs) were infected with a lentiviral vector to express BMP2. Cell viability, cell counting, and tumor sphere formation assays, as well as flow cytometry, immunofluorescence staining, and Western blotting were used to investigate the effects of BMSC-BMP2 on GSCs.
Results: The results of flow cytometry and the CKK-8 assay showed that BMSC-BMP2 induced GSC apoptosis while inhibiting proliferation. BMSC-BMP2 decreased GSC neurosphere formation and neurospheres' transverse and vertical diameter. Meanwhile, BMSC-BMP2 downregulated GSC Nanog and OCT4 expression levels, suggesting stemness inhibition. Western blotting showed that BMSC-BMP2 increased Bax protein expression and significantly decreased Bcl-2 protein expression. Accordingly, the Bcl-2/Bax ratio increased.
Conclusion: BMSC-BMP2 could effectively inhibit GSC proliferation, induce GSC apoptosis, and decrease GSC stemness, thereby providing a novel strategy for treating malignant glioma.
Keywords: Apoptosis, Bcl-2/Bax ratio, bone marrow-derived mesenchymal stem cells, bone morphogenetic proteins 2, glioma stem cells, glioma, proliferation
Malignant glioma is the most common and aggressive brain tumor, as there are limited therapeutic options. Available treatments, including surgical excision, radiation, and chemotherapy, do not improve the prognosis of malignant glioma.[1] The median survival of patients with high-grade glioma is <14 months,[2],[3] and the 5-year survival rate of patients with glioblastoma (GBM) <3%.[4]
Recently, it was reported that gliomas include glioma stem cells (GSCs) with unlimited self-renewal capacity and potent tumorigenicity.[5] These cells are resistant to various chemotherapeutic agents.[6] Therefore, strategies targeting GSCs or restraining their invasive capacity might provide effective therapies. Recent studies have shown that GSCs can be induced to differentiate into mature glial cells, thus eliminating GSCs.[7] Accordingly, we attempted to find an effective molecule to regulate GSC proliferation and differentiation with potential for GBM treatment.
Bone morphogenetic proteins (BMPs) are a family of cytokines with extensive biological activity. BMPs have been found to play an important role in nervous system development, regulating neural stem cell (NSC) proliferation and differentiation.[8] In vitro, BMP2 is an important switch for transforming neural progenitor cells to glial cells, changing the direction of NSC differentiation toward astrocytes. Further, BMP2 can inhibit brain tumor stem cell proliferation and glioma growth, prolonging survival in mice.[9] In a mouse model of transplanted tumors, simvastatin-induced BMP2 expression promoted differentiation of colorectal CSCs, inhibiting metastasis.[10]
Mesenchymal stem cells (MSCs) have been suggested as a promising cell population for cell-based therapeutic strategies.[11] MSCs have the intrinsic capability to migrate to solid tumor tissues and have been used to deliver anti-cancer agents. This method reduces systemic adverse effects and promotes effective local anti-tumor activity.[12],[13],[14] In addition, MSCs' immune modulatory capacity is crucial in anti-tumoral strategies.[15],[16],[17],[18],[19] We have previously used bone marrow-derived MSCs (BMSCs) as a safe alternative for clinical intervention in patients with central nervous system (CNS) disorders.[20] In the present study, we used BMSCs as a vehicle for delivering BMP2 to GBM as a novel and effective means of eliminating GSCs. Our results show that BMP2-BMSC carriers (BMSC-BMP2) can inhibit GSC proliferation, stemness, and induce GSC apoptosis.
> Material and MethodsBMSC isolation and expansion
We used 3-week-old Wistar rats for all experiments. Rat bone marrow aspirates were selected by the plastic adherence method.[21] The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) and incubated at 37°C and 5% CO2. Flow cytometry was used to analyze the expression of MSC surface markers. MSC phenotypic markers, namely CD29, CD90, and CD45 (all from Becton Dickinson Biosciences, USA), were used to confirm the stem cell-like features of the isolated stromal cells. The cells were subsequently expanded through several passages; we used passage four cells for further experiments. The approval from the ethics committee has been obtained before. The data of the approval is March 3, 2015. The number is CN0.2015-019.
Vector construction and cell transduction
BMSCs were infected with a lentiviral vector constructed to express BMP2. The lentivirus was constructed according to manufacturer's instructions (Thermo Fisher Scientific, USA). A human complementary DNA (cDNA) BMP2 fragment was directionally cloned into the pDC316 plasmid, which was subsequently recombined with the pLenti6.3 vector. Then, the pLenti6.3-BMP2 plasmid was transfected with pLP1, pLP2, and pLP/VSVG plasmids into BMSCs. After 48 h, the virus was harvested from the culture medium and filtered. Lentivirus with the BMP2 sequence was obtained according to manufacturer's instructions and used to infect BMSCs (multiplicity of infection = 10 virus particles/mL) and selected after 2 weeks using G418. Transfection was accomplished using Lipofectamine 2000 (Thermo Fisher Scientific, USA).
BMSC-BMP2 proliferation was detected by a cell cycle assay, and BMSCs' differentiation ability was detected by a BMSC adipogenic differentiation kit.
GSC isolation and expansion
Human glioma U251 cells were inoculated in NSC medium containing DMEM/F12 (Invitrogen, Carlsbad, CA, USA), leukemia inhibitory factor (10 ng/mL), B27 (1×, Thermo Fisher Scientific, USA), recombinant murine epidermal growth factor (20 ng/mL, Thermo Fisher Scientific, USA), and basic fibroblast growth factor (20 ng/mL, Thermo Fisher Scientific, USA) at 37°C in a humidified 5% CO2 atmosphere. Half of the medium was replaced once every 3–4 days. Neurospheres were collected after they had grown in numerous proliferations and digested by 0.25% trypsin for 2 min; digestion was terminated using 10% FBS. The cell suspension was filtered through a 200-mesh sieve and centrifuged at 1000 rpm. After disposing of the supernatant, the primary GSCs were washed again with serum-free medium. Then, CD133+ cells were separated according to the instructions of a CD133 cell isolation kit (Miltenyi Biotec, USA), and collected and inoculated into the NSC medium.
Apoptosis assay
The apoptosis rate was calculated by flow cytometry. Experiments were randomly divided into control (normal saline), negative control (NC), and low- (0.5 × 104 cells) and high-dose (1 × 104 cells) BMSC-BMP2 groups. After collection, cells were rinsed and resuspended in phosphate-buffered saline (PBS). Then, the cells were stained using an annexin V–FITC (fluorescein isothiocyanate)/PI (propidium iodide) Apoptosis Detection Kit (BD Biosciences, USA) as per manufacturer's instructions. FACS Calibur was used to determine the apoptosis rate of the stained cells; data were measured by FACS Diva software. The experiments were repeated three times.
Cell counting kit-8 assay
Cell proliferation was examined using the cell counting kit-8 (CCK-8) and cell cycle assays. GSCs were plated in 96-well plates at approximately 1 × 106 cells per well and co-cultured with normal saline, BMSC-NC, or low- and high-dose BMSC-BMP2 for 48 and 72 h. Then, the inhibitory effect of BMSC-BMP2 on GSCs was confirmed using the CCK-8 assay. A CCK-8 reagent was added to the cells and incubated for 2 h. Next, the optical density at 450 nm was measured by a microplate reader according to manufacturer's instructions (Thermo Fisher Scientific, USA). All results correspond to the means of three experiments, each performed in triplicate.
Quantitative real-time PCR
RNA was isolated from cells using an RNA extraction kit. cDNA was synthesized using a SuperScript RNAse Reverse Transcriptase (Invitrogen, USA). Quantitative real-time PCR was run in triplicate using SYBR Green master mix (Toyobo, Japan). Fluorescence emission was recorded in real time (Applied Biosystems, CA). Beta-actin was used as endogenous control gene to normalize mRNA expression. The relative mRNA expression was calculated by the comparative threshold cycle (2-ΔΔCt) method.
Immunofluorescent staining
BMSC-BMP2-treated GSCs were washed three times with ice-cold PBS and fixed with 4% paraformaldehyde-PBS. After a 15-min incubation with 0.1% Triton-PBS, the cells were blocked with 1% bovine serum albumin-PBS. Then, the GSC spheres were incubated with antibodies against CD133, Nanog, and OCT4 (all from Abcam, UK). The samples were incubated with the primary antibodies overnight at 4°C. After a PBS wash, cells were incubated with goat anti-rabbit Alexa Fluor 594 for 1 h at 37°C. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) for 30 min. Images were obtained with a confocal microscopy system.
Western blotting
Total protein was extracted using a NP-40 lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, USA). The lysates were centrifuged at 12,000 rpm for 10 min, and the supernatant was collected for further use. Protein lysates (40 mg) were resolved on 4–20% denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad Laboratories, USA). The membranes were probed with the following antibodies: mouse anti-CD133, anti-Nanog, anti-OCT4, anti-Bax, and anti-Bcl-2 (all from Abcam, UK). Then, horseradish peroxidase-conjugated secondary antibody was added for 1 h at room temperature and the blot visualized using an enhanced chemiluminescent reagent kit (GE Healthcare, USA).
Neurosphere formation assay
Sphere-forming GSCs were dissociated into single-cell suspensions. The cells were diluted to 1000–5000 cells/mL, and 100 μL of cell solution was added to each well of a 96-well plate. The cells were allowed to grow for 6–8 days; 50 μL fresh medium was added at day 3 or 4. Then, the number and diameter of neurospheres in each well were detected under a microscope.
Statistical analyses
The values are presented as mean ± standard error of the mean. Each experiment was repeated ≥3 times and analyzed by Student's t-test or two-way analysis of variance. P < 0.05 was considered to indicate statistical significance.
> ResultsBMSC isolation and characterization
The BMSCs used in this study were positive for surface antigens CD29 and CD90, and negative for CD45, displaying the capacity for differentiation into adipocytes (data not shown).
BMSC-BMP2 proliferation and differentiation
There was an increased frequency of BMSC-BMP2 cells in the G2/M phase and a decreased frequency of cells in the S phase [p < 0.05, [Figure 1]a]. The proliferation index of BMSC-BMP2 increased [p < 0.05, [Figure 1]b] compared to the control and NC groups. The effect of transduction on BMSC-BMP2 differentiation was detected using a BMSC adipogenic differentiation kit. Oil Red O staining demonstrated numerous intracellular lipid droplets in the BMSC-BMP2 after 2 weeks of culture [Figure 1]c and [Figure 1]d.
Figure 1: Characterization of BMSCs overexpressing BMP2. (a) BMSCs-BMP2 cell cycle showing increased frequency of cells in the G2/M phase and decreased frequency of cells in the S phase compared to BMSCs. (b) Upregulation of BMSC-BMP2's proliferation index. (c and d) Numerous intracellular lipid droplets in BMSCs-BMP2. BMSCs = bone marrow-derived MSCs, BMP2 = bone morphogenetic proteins 2, Con = Control group, NC = Negative control group, BMP2-OE = BMSC-BMP2 group. *p < 0.05GSC isolation and characterization
Stem-like cells derived from U251 cells were cultured in NSC medium. The cells grew together and formed cell spheres after 3–5 days [Figure 2]a. The cells in the spheres were CD133-positive [Figure 2]b and expressed CD133 protein [Figure 2]c and [Figure 2]d. Next, stem-like cell spheres were cultured in DMEM/F12 plus 10% FBS medium. After three days, stem-like cell spheres adhered to the bottom of the well; thick dendrite-like pseudopodia grew after one week and showed typical morphological differentiation toward astrocytic lineages, identified as they were positive for glial fibrillary acid protein (data not shown).
Figure 2: GSC characterization. (a) GSC spheres from U251 cells. (b) Immunofluorescent staining showing CD133-positive GSC spheres. (c) Western blot showing higher CD133 protein expression in GSC spheres than in U251 cells. (d) Relative CD133 protein expression in GSC spheres and U251 cells. GSC = glioma stem cell. *p < 0.05BMSC-BMP2-induced GSC apoptosis and inhibited GSC proliferation
To determine whether BMSCs-BMP2 induce GSC apoptosis, U251 GSCs were analyzed by flow cytometry. The apoptotic rate of GSCs increased significantly from 5.84% to 7.06% after 48 h of treatment with low-dose BMSC-BMP2 and from 6.97% to 20.47% after 72 h. Furthermore, 48 and 72 h treatment with high-dose BMSC-BMP2 significantly increased the GSC apoptosis rate from 5.84% to 8.64% and from 6.97% to 24.57%, respectively [Figure 3]a and [Figure 3]b.
Figure 3: BMSC-BMP2 induces GSC apoptosis and inhibited GSC proliferation. (a and b) Cell apoptosis in the BMSC-BMP2 group compared to the control and NC group. (c) BMSC-BMP2 decreases GSC viability. BMSCs = bone marrow-derived MSCs, BMP2 = bone morphogenetic proteins 2, GSC = glioma stem cell, Con = Control group, NC = Negative control group, Low = Low-dose BMSC-BMP2 group, High = High-dose BMSC-BMP2 group. *p < 0.05To determine whether BMSCs-BMP2 had inhibitory effects on GSC proliferation, GSCs were co-cultured with low- and high-dose BMSC-BMP2 for 48 and 72 h, and cell activity was detected using CCK-8. Treated GSCs exhibited reduced proliferation levels in response to BMSC-BMP2 in a dose-dependent manner. There was a significant shift in the proliferation inhibition curve of BMSC-BMP2 groups compared with the control and NC groups [Figure 3]c.
BMSC-BMP2 inhibited neurosphere formation
We investigated if BMSCs-BMP2 would inhibit neurosphere formation. The number of neurospheres decreased significantly from 131.33 (control group) and 110 (NC group) to 43 and 24 after a 72 h treatment with low- and high-dose BMSC-BMP2, respectively; moreover, there was an obvious decrease in neurosphere size. The transverse and vertical diameter of neurospheres were 72.45 μm and 61.97 μm in the control group, 72.45 μm and 61.97 μm in the BMSC-NC group, 52.02 μm and 54.43 μm in the low-dose BMSC-BMP2 group, and 58.42 μm and 50.98 μm in the high-dose BMSC-BMP2 group, respectively [Figure 4]. Accordingly, BMSCs-BMP2 inhibit GSC neurosphere formation.
Figure 4: BMSC-BMP2 inhibits GSC neurosphere formation. The number of GSC neurospheres decreased significantly, as did their TD and VD. BMSCs = bone marrow-derived MSCs, BMP2 = bone morphogenetic proteins 2, GSC = glioma stem cell, Con = Control group, NC = Negative control group, Low = Low-dose BMSC-BMP2 group, High = High-dose BMSC-BMP2 group, TD = transverse diameter, VD = vertical diameter. *p < 0.05BMSC-BMP2 decreased GSC stemness
We explored whether BMSC-BMP2 regulate GSC stemness. To that end, we investigated the levels of OCT4 and Nanog, two stemness markers. Immunofluorescence staining and Western blotting showed lower Nanog [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d, [Figure 5]i and [Figure 5]j and OCT 4 [Figure 5]e, [Figure 5]f, [Figure 5]g, [Figure 5]h, [Figure 5]i and [Figure 5]k expression levels in the BMSC-BMP2 groups compared to the control and NC groups (p < 0.05). These data suggest that BMSC-BMP2 affects GSC proliferation and stemness.
Figure 5: BMSC-BMP2 decreased expression of the GSC stem cell markers Nanog and OCT4. Immunofluorescent staining showing strong and diffuse Nanog and OCT4 expression in the control (a and e) and NC groups (b and f). GSCs treated with low- (c and g) and high-dose (d and h) BMSC-BMP2 showing low Nanog and OCT4 expression levels, respectively. (i) Nanog and OCT4 expression levels by Western blotting. (j and k) Relative Nanog and OCT4 expression. BMSCs = Bone marrow-derived MSCs, BMP2 = Bone morphogenetic proteins 2, GSC = Glioma stem cell, Con = Control group, NC = Negative control group, Low = Low-dose BMSC-BMP2 group, High = High-dose BMSC-BMP2 group. *p < 0.05Bax and Bcl-2 expression
After a 72 h treatment with low- and high-dose BMSC-BMP2, Bax and Bcl-2 protein expression in GSC sphere cells was detected by Western blotting. As shown in [Figure 6], Bax expression was significantly upregulated in BMSC-BMP2 groups (p < 0.01); however, Bcl-2 protein expression was significantly downregulated (p < 0.01). Accordingly, the Bcl-2/Bax ratio significantly increased (p < 0.01).
Figure 6: Bax and Bcl 2 protein expression. (a and b) BMSC BMP2 decreases Bcl 2 protein expression. (a and c) BMSC BMP2 increases Bax protein expression. (d) significantly increased Bcl-2/Bax ratio. BMSCs = Bone marrow-derived MSCs, BMP2 = Bone morphogenetic proteins 2, Con = Control group, NC = Negative control group, Low = Low-dose BMSC-BMP2 group, High = High-dose BMSC-BMP2 group. *p < 0.05 > Discussion In the present study, we attempted to explore a new practical clinical application against GSCs. BMP2 can sensitize GSCs to temozolomide by affecting hypoxia-inducible factor 1 alpha (HIF-1α) stability and MGMT expression.[22] Valproic acid-induced BMP2 mRNA expression induces apoptosis in glioma-initiating cells.[23] However, the delivery pathway restricts the inhibitory effects of BMP2 on glioma cells. Since BMP2 cannot cross the blood–brain barrier when administered intravenously, and direct intracerebral injection cannot achieve long-term efficacy, we used BMSCs as a vehicle for delivering BMP2 as a novel and effective means of eliminating GSCs. First, we observed the effect of the transfection on BMSC proliferation and stemness. BMP2-transfected BMSCs could differentiate into adipocytes. Moreover, BMP2 increased BMSC proliferation and self-renewal but not BMSC stemness.
Sustained proliferative signaling and resistance to apoptosis are two hallmarks of cancer. In the present study, BMSC-BMP2-treated GSCs underwent G0/G1 arrest and showed inhibited proliferation and decreased stemness; BMSC-BMP2 also induced GSC apoptosis. Morphologically, there was an obvious decrease in the number and size of gliospheres formed in vitro. Accordingly, BMSC-BMP2 can inhibit GSC growth by inhibiting cell proliferation and promoting apoptosis.
Differentiation status significantly affects the properties of malignant glioma cells; non-stem cells induce tumor enlargement and stem-like cells drive tumor initiation and treatment resistance to reduce GSC stemness. Triggering GSC differentiation reduces their tumor-propagating potential[7] while reprogramming GSCs to induced pluripotent stem cells alters their capacity to propagate tumors.[24] In the present study, stem cell markers were detected to observe the differentiation status of BMSC-BMP2-treated GSCs. Here, BMSC-BMP2-treated GSCs showed significantly decreased Nanog and OCT4 expression. Taken together, our data demonstrate that BMSC-BMP2 can effectively attenuate GSC-related pluripotency gene expression and reduce cancer stemness, greatly enhancing the GSC targeting effect.
Moreover, we detected significant Bax upregulation in the BMSC-BMP2 groups; however, Bcl-2 was downregulated. BMSCs-BMP2 inhibited cell cycle progression in the G2/M phase and induced apoptosis by regulating the Bcl-2/Bax signaling pathways in vitro. Combined with the neurosphere number and size and the formation of in vitro colonies, our results show that BMSC-BMP2 exhibited robust GSC-eliminating properties by regulating Bcl-2/Bax signaling in vitro. Cell proliferation and apoptosis analyses showed the increased anti-cancer effects of BMSC-BMP2 on GSCs in vitro.
Our study investigated the potential of BMSC-based therapy for glioma treatment. The results demonstrate the outstanding performance of BMSC-BMP2 in inhibiting GSC proliferation, inducing GSC apoptosis, and decreasing GSC stemness. BMSC-mediated BMP2 may be a promising alternative for treating GMB.
Financial support and sponsorship
The present research was supported by grants from the National Natural Science Foundation of China (81502568) and Natural Science Foundation of Shandong Province (ZR2019BH063).
Conflicts of interest
There are no conflicts of interest.
> References
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