• Users Online: 307
  • Print this page
  • Email this page


 
 
Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 3  |  Issue : 1  |  Page : 16-23

Evaluation of combination gene therapy with SLC22A18 upregulation and sequence binding protein 1 downregulation for glioma U251 cells in vitro and in vivo


Department of Neurosurgery, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

Date of Submission18-Sep-2019
Date of Acceptance06-Mar-2020
Date of Web Publication13-Apr-2020

Correspondence Address:
Dr. Shenghua Chu
Department of Neurosurgery, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 280 Mohe Road, Baoshan District, Shanghai 201999
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/glioma.glioma_19_19

Rights and Permissions
  Abstract 

Background and Aim: Our previous study demonstrated that SLC22A18 downregulation through promoter methylation and Sequence binding protein 1 (SATB1) upregulation are associated with the development and progression of glioma. This study aimed to examine the effect of combined SLC22A18 and short hairpin RNA (shRNA) targeting SATB1 gene therapy on glioma growth and invasion. Materials and Methods: Here, a combined gene therapy to upregulate SLC22A18 and downregulate SATB1 in malignant glioma was evaluated both in vitro and in vivo. Glioma U251 cells overexpressing SLC22A18 and underexpressing SATB1 were generated to investigate the effects of these changes on cell survival, as measured by the methyl thiazol tetrazolium assay, and on cell invasion, as measured by cell invasion and migration assays. In addition, analysis of the cell cycle was performed using flow cytometry in vitro. The animal experiments were approved by the Ethics Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Zhongnan Hospital of Wuhan University Ethics Committees (approval No. ZNHWHU0389, NTPHSHJTUSM046). Results: The upregulation of SLC22A18 and downregulation of SATB1 significantly inhibited growth and invasion in vitro and reduced in vivo tumor growth of human glioma U251 cells. Furthermore, we revealed that U251 cells were arrested at the G0/G1 phase. Similar data for apoptotic glioma cell death were obtained in tumor cells from an in vivo glioma xenograft model after transfection. At the same total dose, the therapeutic effect was markedly better in the glioma xenografts transfected with both SLC22A18 gene and SATB1 short hairpin RNA (shRNA) expression vectors compared with tumors transfected with either agent alone. The levels of SLC22A18 and SATB1 protein expression were respectively increased and decreased in the glioma cells.Conclusion: These results demonstrate that combination treatment with SLC22A18 gene and SATB1 shRNA expression vectors effectively inhibits the growth of human malignant glioma cells both in vitro and in glioma xenografts in vivo, suggesting a promising novel strategy for glioma therapy that warrants further study.

Keywords: Gene therapy, glioma, sequence binding protein 1, SLC22A18


How to cite this article:
Chu S, Ma Y. Evaluation of combination gene therapy with SLC22A18 upregulation and sequence binding protein 1 downregulation for glioma U251 cells in vitro and in vivo. Glioma 2020;3:16-23

How to cite this URL:
Chu S, Ma Y. Evaluation of combination gene therapy with SLC22A18 upregulation and sequence binding protein 1 downregulation for glioma U251 cells in vitro and in vivo. Glioma [serial online] 2020 [cited 2022 Dec 8];3:16-23. Available from: http://www.jglioma.com/text.asp?2020/3/1/16/282425


  Introduction Top


Gliomas include well-differentiated low-grade astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme.[1] Resection remains the most effective therapy for glioma, but postoperative recurrence is common and leads to poor outcomes.[2] Consequently, a top priority in glioma research is the development of new therapeutic strategies. It has been reported thatSLC22A18 expression is markedly decreased in gliomas; SLC22A18 expression was much higher in gliomas that did not recur within 6 months after resection than in those that recurred within 6 months. In addition, promoter methylation of SLC22A18 was detected in 50% of tumor tissues, but not in the adjacent normal tissues in cases of brain glioma. Compared with gliomas without SLC22A18 promoter methylation, SLC22A18 expression was markedly downregulated in the gliomas that had SLC22A18 promoter methylation. The SLC22A18 promoter is methylated in U251 cells, which are a glioma cell line; thus, upregulation of SLC22A18 protein expression using the demethylating agent 5-aza-2-deoxycytidine may reduce U251 cell proliferation. The development and progression of human glioma is associated with low expression of SLC22A18 via promoter methylation, suggesting that SLC22A18 is a very important tumor suppressor in glioma.[3],[4],[5],[6],[7] We have previously demonstrated that 62.9% of gliomas are positive for special AT-rich sequence binding protein 1 (SATB1) expression. In addition, SATB1 expression is related to histological tumor grade and poor survival in glioma. In glioma, expression of SATB1 protein has also been reported to be positively correlated with Ki67 and negatively correlated with O6-methylguanine-DNA methyltransferase promoter methylation. Therefore, SATB1 might have an important role as a positive regulator of human glioma development and progression, and SATB1 may be a very useful molecular biomarker for predicting glioma prognosis.[8],[9],[10],[11]

During the last decade, a large number of gene therapy methods have been developed and used for the treatment of glioma in animal models, but therapeutic effects remain unsatisfactory because it is difficult to choose effective target genes.[12],[13],[14],[15] There are numerous abnormal genes that are associated with malignancy in gliomas, and single-gene therapy has a limited impact. Combined gene therapy, with more than one target gene, may thus increase the treatment effect. Our previous studies have revealed that SLC22A18 and SATB1 may have critical roles in glioma pathogenesis,[3],[4],[8],[9],[10] thus indicating that they might be effective target genes for the clinical therapy of gliomas. In the present study, we examined the effect of combined SLC22A18 and short hairpin RNA (shRNA) targeting SATB1 gene therapy on glioma growth and invasion. First, U251 cells overexpressing SLC22A18 were generated and SATB1-specific shRNA sequences were synthesized, and SLC22A18 overexpressing U251 cells were then transfected with SATB1 RNA interference plasmids. We demonstrated that combination treatment with SLC22A18 gene and SATB1 shRNA expression vectors effectively inhibited the growth and invasion of human malignant glioma cells in vitro and in glioma xenografts in vivo.


  Materials and Methods Top


Cell culture and transfection of tumor cells with pcDNA3.1-SLC22A18

Human glioma U251 cells were purchased from China Center for Type Culture Collection (Wuhan, China). Tumor cells were cultured in Roswell Park Memorial Institute (RPMI-1640 (Gibco Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and penicillin-streptomycin (10,000 IU penicillin/mL, 10,000 μg streptomycin/mL, Invitrogen). As previously described by our group,[3],[6] for SLC22A18 transfection, tumor cells (1 × 10[6]) were plated in six-well plates 24 h before transfection. Lipofectamine 2000 was used to mediate transfection using 5.0 μg of pcDNA3.1-SLC22A18 vector or 5.0 μg of empty pcDNA 3.1, according to the manufacturer's instructions (Invitrogen). After 48 h of transfection, the cells were selected in media supplemented with 150 μg/mL G418. The medium was changed every 48 h, and non-transfected U251 cells died within 2 weeks. G418-resistant cells transfected with pcDNA3.1 were referred to as U251-EV cells, while G418-resistant cells transfected with pcDNA3.1–SLC22A18 were referred to as U251–SLC22A18 cells.

Knockdown of sequence binding protein 1 by RNA interference in U251 cells

As previously described by our group,[8],[11]SATB1-specific shRNA sequences were synthesized and inserted into the pGCsi-H1/Neo/green fluorescent protein (GFP)/siNEGative vector (Genscript, Piscataway, NJ, USA), which co-expresses GFP to allow identification of transfection efficiency. The SATB1 shRNA sequence and the non-specific shRNA sequence were as follows: SATB1-shRNA 5′-GTC CAC CTT GTC TTC TCT C-3′ and control-shRNA-GFP 5′-ACG TGA CAC GTT CGG AGA A-3′, respectively. U251 cells were transiently transfected with SATB1 RNA interference plasmids using an electroporator (BTX, San Diego, CA, USA).

Cell survival assay

The effect of SLC22A18 and/ or SATB1-shRNA on cell survival in U251 cells was measured using the methyl thiazole tetrazolium (MTT) assay.[3],[16],[17] Cells were seeded in 24-well plates at a density of 1 × 10[4] cells/well for 24 h, and 200 μL of 5 mg/μL MTT (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) was then added to each well and incubated at 37°C for 4 h. The precipitate was solubilized in 100 μL of 100% dimethyl sulfoxide (Sigma-Aldrich) and shaken for 15 min. Absorbance values were determined using an enzyme-linked immunosorbent assay reader (Model 318; Thermo, Shanghai, China) at 540 nm. Each assay was performed at least three times.

Apoptosis and proliferation assays

As previously described by our group,[18] apoptosis was quantified using the Guava Nexin assay (Guava Technologies, Hayward, CA, USA) following the manufacturer's instructions. Briefly, 3.0 × 10[4] cells (50 μL) were added to 150 μL of staining solution containing 135 μL of 1× apoptosis buffer, 10 μL of Annexin V-PE, and 5 μL of 7-AAD. The cells were incubated at room temperature in the dark for 20 min. Samples with 2000 cells/well were analyzed on a Guava EasyCyte flow cytometer (Merck Millipore, Darmstadt, Germany).

Immunochemistry for Ki-67 was used as a marker of proliferation activity in glioma cells. Briefly, cells were adhered to glass slides and nonspecific binding sites and endogenous peroxidases were blocked with goat serum and H2O2, respectively. The cell smears were incubated with Ki-67 antibody (1:100; Dako Corporation, Via Real, CA, USA) overnight at 4°C, and the slides were then incubated with a biotinylated secondary antibody combined with an avidin-biotin-peroxidase complex. The slides were then incubated in a diaminobenzidine peroxidase substrate solution to visualize the Ki-67-positive staining.

Cell invasion and migration assay

Chambers were covered with 60 μL Matrigel diluted in RPMI-1640 to a certain dilution (1–5 mg/mL) and incubated at 37°C for 2–4 h. Next, 4 × 10[4] U251 cells were suspended in 200 μL RPMI-1640 and seeded in the upper chambers, and 500 μL RPMI-1640 with 10% fetal bovine serum was added to the lower chamber. The cells that invaded the lower chamber were fixed and stained for 30 min in a 0.1% cresyl violet solution after being incubated at 37°C.

Flow cytometry analysis of the cell cycle

A minimum of 15,000 U251 cells at log phase were harvested and washed twice with PBS for cell cycle phase distribution analysis. Cell pellets were fixed in 80% ethanol treated with 0.1 mg/mL RNase A (Boehringer Mannheim, Indianapolis, IN, USA) and stained with 40 μg/mL propidium iodide (Sigma-Aldrich). DNA content was determined using a flow cytometer (Becton Dickinson, San Jose, CA, USA).

Western blot analysis

U251 cells were washed in ice-cold PBS and lysed in buffer using standard methods.[19] The tumor tissue samples were homogenized in RIPA lysis buffer. Lysates were cleared by centrifuging (14,000 r/min) at 4°C for 30 min. Protein samples (approximately 40 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (with a 15% gel), transferred to a polyvinylidene difluoride membrane, and the non-specific binding sites were blocked by incubation in 5% non-fat milk for 60 min. Membranes were then incubated overnight at 4°C with polyclonal anti-SLC22A18 antibody (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or anti-SATB1 antibody (1:200 dilution; Sigma-Aldrich). The membrane was then washed three times with Tris-buffered saline with Tween-20 for 10 min before being probed with horseradish peroxidase-conjugated secondary antibody (1:2000 dilution; Dako, Glostrap, Denmark) at room temperature for 30 min. After being washed three times, the membrane was developed using an enhanced chemiluminescence system (Pierce Chemicals, Rockford, IL, USA).

Subcutaneous tumor model

Male 4–6-week-old BALB/c athymic nude mice (n = 15/group) were acquired from the National Cancer Institute. They were inoculated subcutaneously into the left abdominal quadrant with 3 × 10[7] U251 (U251 group), U251-EV and control-shRNA-GFP (vector group), U251-SLC22A18 (SLC22A18 group), SATB1-shRNA (SATB1 group), or U251-SLC22A18 combined with SATB1-shRNA (combined group) cells. The tumor diameters were measured at regular intervals using digital calipers, and the tumor volume was calculated using the following formula in mm[3]: volume = (width) 2 × length × 0.52. After 30 days, the mice were killed humanely. Tumors were resected to determine Ki-67, SLC22A18, and SATB1 protein expressions by western blot analysis, and apoptosis was quantified using the Guava Nexin assay. The animal experiments were approved by the Ethics Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Zhongnan Hospital of Wuhan University Ethics Committees (approval No. ZNHWHU0389, NTPHSHJTUSM046). During the study, all steps were taken to minimize the pain and suffering of all animals. The anesthesia used during tumor inoculation was isoflurane/O2. During the course of the experiments, all animals were given access to food and water ad libitum. Euthanasia was carried out using CO2 asphyxiation followed by cervical dislocation.

Orthotopic brain tumor model

Male 4–6-week-old BALB/c athymic nude mice (n = 10/group) were anesthetized and stereotactically inoculated with tumor cells (1 × 10[4] cells in 2 μL PBS) into the left forebrain (1 mm anterior and 2 mm lateral to bregma, at a 3 mm depth from the skull surface).[7],[18] These tumor cells were U251 (U251 group), U251-EV and control-shRNA-GFP (vector group), U251-SLC22A18 (SLC22A18 group), SATB1-shRNA (SATB1 group), or U251-SLC22A18 combined with SATB1-shRNA (combined group) cells. On day 14, tumor imaging in animal models was performed using a small animal coil on a High-Field GE Signa 3 Tesla clinical MR scanner (Signa; GE Healthcare, Waukesha, WI, USA), and the images were gained using a standard T1 protocol following intraperitoneal injection of gadolinium (Gd-DTPA, 100 μL/20 g; Magnevist, Berlex Laboratories, Wayne, NJ, USA) 10 min before examination. In enhanced scanning, the scanning parameters were: AxT1 FSE series; scan plane: oblique; phase FOV: 0.60; FOV: 5.0; spacing: 0.0 mm; slice thickness: 1.0 mm; freq DIR: R/L; minimum TR: 60; and auto-TR: 600. Tumor sizes were measured, and the tumor volumes in mm[3] were calculated using the formula: volume = (width) 2 × length/2 using function analysis software.[7],[18] Moribund mice or mice with severe neurological symptoms were euthanized.

Statistical analysis

Statistical analyses and graphing were performed using SPSS (version 12.0, for Windows; SPSS, Chicago, IL, USA). Quantitative values were expressed as the mean ± standard deviation. Statistical differences between groups were examined using Student's t-test. P < 0.05 was considered statistically significant.


  Results Top


Effects of SLC22A18 upregulation and sequence binding protein 1 downregulation on U251 cell survival, apoptosis, and proliferation

After U251 cells were transfected with SLC22A18 gene and SATB1 shRNA expression vectors, survival rates were evaluated using an MTT assay. Transfection with SLC22A18 gene and/or SATB1 shRNA expression vectors reduced cell survival significantly [Figure 1]A. The survival rate on day 5 was 45.25%, and the lowest rate of survival on day 6 was 34.75% in the combined group compared with the U251 group (P < 0.01). In addition, the number of apoptotic U251 cells was significantly higher in the U251 cells transfected with both SLC22A18 gene and SATB1 shRNA expression vectors compared with the U251 group (P < 0.05). On day 3, the percentages of apoptotic U251 cells were 6.63%, 10.01%, and 13.30% in the SLC22A18, SATB1, and combined groups, respectively. Similar data for apoptotic glioma cell death were obtained in the tumor cells from an in vivo glioma xenograft model after transfection [Figure 1]B. Because Ki67 is a signature gene for tumor cell proliferation, we determined the effect of SLC22A18 upregulation and SATB1 downregulation on cell proliferation by observing Ki-67 immunocytochemical staining. [Figure 1]C shows that U251 cell proliferation was markedly suppressed following transfection of the SLC22A18 gene and SATB1 shRNA expression vectors. On day 3, the Ki67-positive rates were 57.25%, 51.83%, and 40.12% in the SLC22A18, SATB1, and combined groups, respectively; these values were significantly lower than those of the U251 group (P < 0.05 or P < 0.01), in which U251 cells were not transfected. Similar Ki-67-positive rates were also observed in tumor cells from the in vivo glioma mouse model [Figure 1]C, demonstrating the inhibitory effect of SLC22A18 upregulation and SATB1 downregulation on the growth and proliferation of glioma U251 cells both in vitro and in vivo.
Figure 1: Effects of SLC22A18 upregulation and SATB1 downregulation on U251 cell survival, apoptosis, and proliferation in vitro and in vivo. (A) U251 cell survival in vitro. Cell survival was assessed by the MTT assay. Cell survival rates are expressed relative to those of the control U251 cells (control value, 100%). Each assay was performed at least three times. (B) U251 cell apoptosis in vitro and in vivo. Apoptosis of the glioma cells, in vitro and in vivo, was detected and quantified using the Guava Nexin assay at 3 days. (C) U251 cell proliferation in vitro and in vivo. Ki67 was determined as a parameter of cell proliferation activity. Tumor cells were stained with Ki67 antibody (1:100) using immunocytochemistry. Data are expressed as the mean ± standard deviation, and analyzed using the Student's t-test. *P < 0.05, **P < 0.01, vs. U251 group

Click here to view


Effects of SLC22A18 upregulation and sequence binding protein 1downregulation on U251 cell invasion and migration

As shown in [Figure 2], the invasion and migration of U251 cells was inhibited following transfection of SLC22A18 gene and SATB1 shRNA expression vectors; the numbers of these invading and migrating cells were significantly lower than in the U251 group (P < 0.05 or P < 0.01), indicating a suppressing effect of SLC22A18 upregulation and SATB1 downregulation on the invasion and migration of glioma U251 cells.
Figure 2: Effects of SLC22A18 upregulation and SATB1 downregulation on U251 cell invasion (A) and migration (B). Cells were U251 (U251 group), U251-EV and control- short hairpin RNA -GFP (vector group), U251-SLC22A18 (SLC22A18 group), SATB1- short hairpin RNA (SATB1 group), or U251-SLC22A18 combined with SATB1- short hairpin RNA (combined group). The tumor cells were stained with 0.1% cresyl violet (upper), and the cell numbers of the controls (U251 group) were expressed as 100% (lower). The data are representative of three separate experiments. Data are expressed as the mean ± standard deviation, and were analyzed using the Student's t-test. *P < 0.05, **P < 0.01, vs. U251 group

Click here to view


Effects of SLC22A18 upregulation and sequence binding protein 1downregulation on the U251 cell cycle

We next examined whether the suppressing effect of SLC22A18 upregulation and SATB1 downregulation on cell proliferation was mediated through control of the cell cycle, at least in part. After transfecting U251 cells with the SLC22A18 gene and SATB1 shRNA expression vectors, the cell cycle was analyzed using flow cytometry, and the cells were arrested at the G0/G1 phase [Figure 3]. The proliferative index values were 52.48, 45.64, and 27.52, respectively, in the SLC22A18, SATB1, and combined groups, and in the S phase fractions were 36.34, 34.54, and 22.85, respectively. Thus, the proliferation rate was decreased by SLC22A18 upregulation and SATB1 downregulation in glioma U251 cells, and the maximum effect was found in U251 cells transfected with both the SLC22A18 gene and SATB1 shRNA expression vectors.
Figure 3: Effects of SLC22A18 upregulation and SATB1 downregulation on the U251 cell cycle. The cells were harvested and stained, and the cell cycle distribution of propidium iodide-labeled cells was analyzed using flow cytometry. The data are representative of three separate experiments. U251: U251 group, vector: U251-EV and control-short hairpin RNA-GFP group, SLC22A18: U251-SLC22A18 group, SATB1: SATB1-short hairpin RNA group, combined: U251-SLC22A18 combined with SATB1-short hairpin RNA group

Click here to view


SLC22A18 and sequence binding protein 1 protein expression in U251 cells

After transfecting U251 cells with SLC22A18 gene, SATB1 shRNA, or both expression vectors, we used western blotting to examine the levels of SLC22A18 and SATB1 protein expression in these cells. As shown in [Figure 4], the level of SLC22A18 protein was significantly upregulated in U251 cells transfected with the SLC22A18 gene expression vector alone or with both the SLC22A18 gene and SATB1 shRNA expression vectors (P < 0.05 or P < 0.01). In contrast, the SATB1 protein level was significantly lower in U251 cells transfected with the SATB1 shRNA expression vector alone or with both the SLC22A18 gene and SATB1 shRNA expression vectors, compared with the level in the U251 group (P < 0.05).
Figure 4: SLC22A18 and SATB1 protein expression in U251 cells in vitro using western blot analysis. (A) Representative images of SLC22A18 and SATB1 western blots. Cellular protein was extracted from the U251 cells, and the protein levels of SLC22A18 and SATB1 were analyzed using western blot assay. (B) The ratio of SLC22A18 and SATB1 protein expression to β-actin. Western blot data are shown that are representative of the data obtained from three separate experiments. Data are expressed as the mean ± standard deviation and analyzed using Student's t-test. *P < 0.05, **P < 0.01 , vs. U251 group

Click here to view


Effects of SLC22A18 upregulation and sequence binding protein 1downregulation on tumor growth in vivo

Because the survival and proliferation rates were reduced in glioma U251 cells in vitro after transfection of the SLC22A18 gene and SATB1 shRNA expression vectors, we next determined the effects of SLC22A18 upregulation and SATB1 downregulation on tumor growth in a subcutaneous tumor model. The tumors in the SLC22A18 and SATB1 groups grew slowly in vivo, with dimensions that were markedly smaller than in the U251 group. There was a significant difference in tumor size between the SLC22A18 and SATB1 groups and the U251 groups, even on day 14; at the end of the study, there were large differences in tumor volumes among the SLC22A18, SATB1, and combined groups, and the antitumor effect in the combined group was markedly better than in the other groups (P < 0.001). As shown in [Figure 5], on day 30, the average tumor volumes were 3.64, 2.76, and 1.78 cm[3] in the SLC22A18, SATB1, and combined groups, respectively; in contrast, the average glioma volume in the U251 group was 7.28 cm[3]. The effects of SLC22A18 upregulation and SATB1 downregulation on tumor growth in the orthotopic brain tumor model were observed using magnetic resonance imaging. By day 14, both SLC22A18 upregulation and SATB1 downregulation monotherapies had significantly inhibited U251 tumor growth by 45.2% and 52.6% compared with the U251 group [Figure 6]A and [Figure 6]B, which was further reduced by the combination of gene therapies, to 80.4%.
Figure 5: Effects of SLC22A18 upregulation and SATB1 downregulation on the growth of human glioma U251 cells in a subcutaneous tumor model. (A) Representative images of the subcutaneous tumor model. The tumors are indicated by arrows. (B) Tumor growth curves of each group over 30 days. Data are expressed as the mean ± standard deviation and analyzed using Student's t-test.###P < 0.001, vs. combined group. U251: U251 group, vector: U251-EV and control-short hairpin RNA-GFP group, SLC22A18: U251-SLC22A18 group, SATB1: SATB1-short hairpin RNA group, combined: U251-SLC22A18 combined with SATB1- short hairpin RNA group

Click here to view
Figure 6: Effects of upregulation of SLC22A18 and downregulation of SATB1 on the growth of human glioma U251 cells in an orthotophic brain tumor model. (A) The representative MRI images from the U251, vector, SLC22A18, SATB1 or combined group at 14 days. (B) Tumor volumes estimated from magnetic resonance imaging. Data were expressed as the mean ± standard deviation, and analyzed by Student's t-test. *P < 0.05, **P < 0.01, vs. U251 group. U251: U251 group, vector: U251-EV and control-short hairpin RNA-GFP group, SLC22A18: U251-SLC22A18 group, SATB1: SATB1-short hairpin RNA group, combined: U251-SLC22A18 combined with SATB1-short hairpin RNA group

Click here to view



  Discussion Top


In the present study, we constructed SLC22A18 gene and SATB1 shRNA expression vectors and evaluated their effects on tumor growth and invasion in a human glioma U251 cell line both in vitro and in vivo. We demonstrated that transfection with SLC22A18 gene and SATB1 shRNA expression vectors significantly inhibited growth and invasion in vitro and reduced in vivo tumor growth of U251 cells in a time-dependent manner. Furthermore, we demonstrated that the protein expression level of SLC22A18 was significantly reduced in these cells, whereas the level of SATB1 protein expression was significantly increased. Finally, we revealed that the number of apoptotic cells was significantly increased in human glioma U251 cells transfected with SLC22A18 gene and SATB1 shRNA expression vectors, but that cell proliferation was markedly reduced, as observed using flow cytometry. The results demonstrated cell cycle arrest at G1/G0, and there were substantially decreased proliferative index and S-phase fraction values in the U251 cells co-transfected with SLC22A18 gene and SATB1 shRNA expression vectors. These findings imply that the antiproliferative effect and anticancer activity of the combination SLC22A18 and SATB1 shRNA therapy in glioma are mediated by SLC22A18 upregulation and SATB1 downregulation. These observations also indicate a framework and rationale for developing the combination of SLC22A18 and SATB1 shRNA as a potentially safe and effective targeted gene treatment for patients with gliomas and other brain tumors. We have previously reported that RNA interference of SATB1 successfully inhibits the expression of SATB1 protein and mRNA, as well as tumor growth, adhesion, invasion, metastasis, and angiogenesis in vitro, and tumor growth and angiogenesis in vivo. These results might be caused by reduced SATB1, c-Met, and Bcl-2 protein expression and increased SLC22A18 and caspase-3 expression.[8] The mechanism underlying the combination anticancer gene therapy with SLC22A18 gene and SATB1 shRNA is currently not very clearly defined, but these findings are potentially relevant for our understanding of the effects of the SLC22A18 gene and SATB1 shRNA on proliferation in human tumor cells.

In conclusion, our study demonstrates that gene transfer might be a potentially optimal method for the treatment of glioma. We have provided evidence that transfecting SLC22A18 or SATB1 shRNA into U251 cells leads to the effective inhibition of invasive growth in vitro and in vivo. In addition, at the same total viral dose, the treatment efficacy and xenografts transfected with both SLC22A18 and SATB1 shRNA was significantly higher than in tumor cells transfected with either agent alone. In addition, our findings revealed that the inhibitory effect of the combination gene therapy on the growth and survival of glioma occurred through G1 cell cycle arrest, as well as through the upregulation of SLC22A18 and the downregulation of SATB1. There is a lack of effective treatment strategies for the invasive growth of glioma, with surgery, chemotherapy, and radiotherapy being the management mainstays. We therefore advocate the combination treatment with SLC22A18 and SATB1 shRNA as an attractive, novel strategy for the treatment of gliomas that are not cured by conventional treatment.

Financial support and sponsorship

The study was sponsored by the Natural Science Foundation of Shanghai, China (No. 19ZR1429800), Shanghai Jiao Tong University Medicine-Engineering Cross Research Foundation, China (No. YG2015MS25) and the Research Foundation of Shanghai No. 3 People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, China (No. syz2015-015).

Institutional review board statement

The animal experiments were approved by the Ethics Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Zhongnan Hospital of Wuhan University Ethics Committees (approval No. ZNHWHU0389, NTPHSHJTUSM046).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Li D, Chen Y, Guo C, Zhang X, Sai K, Ke C, et al. Real-world management and survival outcomes of patients with newly diagnosed gliomas from a single institution in China: A retrospective cohort study. Glioma 2019;2:96-104.  Back to cited text no. 1
  [Full text]  
2.
Jansen JA, Spliet WG, de Leng W, Robe PA. Histologic characterization of the immune infiltrate in isocitrate dehydrogenase wild-type and mutant World Health Organization Grade II and III gliomas. Glioma 2018;1:196-200.  Back to cited text no. 2
  [Full text]  
3.
Chu SH, Feng DF, Ma YB, Zhang H, Zhu ZA, Li ZQ, et al. Promoter methylation and downregulation of SLC22A18 are associated with the development and progression of human glioma. J Transl Med 2011;9:156.  Back to cited text no. 3
    
4.
Chu SH, Ma YB, Feng DF, Zhang H, Zhu ZA, Li ZQ, et al. Correlation of low SLC22A18 expression with poor prognosis in patients with glioma. J Clin Neurosci 2012;19:95-8.  Back to cited text no. 4
    
5.
Chu SH, Ma YB, Feng DF, Zhang H, Qiu JH, Zhu ZA. Effect of 5-Aza-2'-deoxycytidine on SLC22A18 in glioma U251 cells. Mol Med Rep 2012;5:138-41.  Back to cited text no. 5
    
6.
Chu SH, Ma YB, Feng DF, Zhang H, Qiu JH, Zhu ZA. Elevated expression of solute carrier family 22 member 18 increases the sensitivity of U251 glioma cells to BCNU. Oncol Lett 2011;2:1139-42.  Back to cited text no. 6
    
7.
Chu SH, Zhou ZM, Karri S, Li ZQ, Zhao JM.In vitro and in vivo radiosensitization of human glioma U251 cells induced by upregulated expression of SLC22A18. Cancer Gene Ther 2014;21:103-9.  Back to cited text no. 7
    
8.
Chu SH, Ma YB, Feng DF, Zhang H, Zhu ZA, Li ZQ, et al. Upregulation of SATB1 is associated with the development and progression of glioma. J Transl Med 2012;10:149.  Back to cited text no. 8
    
9.
Chu SH, Ma YB, Feng DF, Zhang H, Qiu JH, Zhu ZA, et al. Relationship between SATB1 expression and prognosis in astrocytoma. J Clin Neurosci 2013;20:543-7.  Back to cited text no. 9
    
10.
Chu SH, Ma YB, Feng DF, Li ZQ, Jiang PC. Correlation between SATB1 and Bcl-2 expression in human glioblastoma multiforme. Mol Med Rep 2013;7:139-43.  Back to cited text no. 10
    
11.
Chu SH, Zhou ZM, Feng DF, Ma YB. Inhibition of human glioma U251 cells growth in vitro and in vivo by hydroxyapatite nanoparticle-assisted delivery of short hairpin RNAs against SATB1. Mol Biol Rep 2014;41:977-86.  Back to cited text no. 11
    
12.
Barone A, Sengupta R, Warrington NM, Smith E, Wen PY, Brekken RA, et al. Combined VEGF and CXCR4 antagonism targets the GBM stem cell population and synergistically improves survival in an intracranial mouse model of glioblastoma. Oncotarget 2014;5:9811-22.  Back to cited text no. 12
    
13.
Daniele S, Costa B, Zappelli E, Da Pozzo E, Sestito S, Nesi G, et al. Combined inhibition of AKT/mTOR and MDM2 enhances Glioblastoma Multiforme cell apoptosis and differentiation of cancer stem cells. Sci Rep 2015;5:9956.  Back to cited text no. 13
    
14.
Singh MM, Johnson B, Venkatarayan A, Flores ER, Zhang J, Su X, et al. Preclinical activity of combined HDAC and KDM1A inhibition in glioblastoma. Neuro Oncol 2015;17:1463-73.  Back to cited text no. 14
    
15.
Chu SH, Zhang H, Ma YB, Feng DF, Zhu ZA, Yuan XH, et al. c-Met antisense oligodeoxynucleotides as a novel therapeutic agent for glioma:In vitro and in vivo studies of uptake, effects, and toxicity. J Surg Res 2007;141:284-8.  Back to cited text no. 15
    
16.
Chu SH, Feng DF, Zhang H, Chen ET, Duan ZX, Li XY, et al. c-Met-targeted RNA interference inhibits growth and metastasis of glioma U251 cells in vitro. J Neurooncol 2009;93:183-9.  Back to cited text no. 16
    
17.
Chu SH, Feng DF, Ma YB, Li ZQ. Hydroxyapatite nanoparticles inhibit the growth of human glioma cells in vitro and in vivo. Int J Nanomedicine 2012;7:3659-66.  Back to cited text no. 17
    
18.
Chu SH, Karri S, Ma YB, Feng DF, Li ZQ.In vitro and in vivo radiosensitization induced by hydroxyapatite nanoparticles. Neuro Oncol 2013;15:880-90.  Back to cited text no. 18
    
19.
Chu SH, Ma YB, Feng DF, Zhang H, Qiu JH, Zhu ZA. c-Met antisense oligodeoxynucleotides increase sensitivity of human glioma cells to paclitaxel. Oncol Rep 2010;24:189-94.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


This article has been cited by
1 Epigenetics of Glioblastoma Multiforme: From Molecular Mechanisms to Therapeutic Approaches
Md. Sahab Uddin,Abdullah Al Mamun,Badrah S. Alghamdi,Devesh Tewari,Philippe Jeandet,Md. Shahid Sarwar,Ghulam Md. Ashraf
Seminars in Cancer Biology. 2020;
[Pubmed] | [DOI]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
References
Article Figures

 Article Access Statistics
    Viewed2366    
    Printed125    
    Emailed0    
    PDF Downloaded203    
    Comments [Add]    
    Cited by others 1    

Recommend this journal