The effect of administration of A2a receptor antagonist on the activity of A2a R/CD37 pathway and induction of apoptosis in glioblastoma tumor cells in BALB/c mice

doi:10.21203/rs.3.rs-2120555/v1

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The effect of administration of A2a receptor antagonist on the activity of A2a R/CD37 pathway and induction of apoptosis in glioblastoma tumor cells in BALB/c mice

https://doi.org/10.21203/rs.3.rs-2120555/v1

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Glioblastoma is the most prevalent primary brain tumor with survival rate of about 20.5 months. Hence, investigation on the involved signaling pathways to treat the disease is urgently needed. CD73 and adenosine play key roles in tumor invasion, proliferation, angiogenesis, and chemical resistance of cancer cells. Theophylline are known as adenosine A2a receptor antagonists. Current study investigates the plausible impacts of theophylline in the mice model of glioblastoma.

Materials and Methods: Initially, induction of glioblastoma tumor was performed within 10 days in male Balb/ C mice. Next, the mice were treated with theophylline for 14 days. Garcia's behavioral test was performed. Moreover, histopathological changes in brain tissue were examined using hematoxylin and eosin staining. The expression of A2a and CD73 proteins was assessed by western blotting. Additionally, the expression of VEGF was indicated by immunohistochemistry. Apoptosis was examined by the TUNEL method. Finally, serum levels of MDA and SOD were investigated using the ELISA method.

Results: Garcia's behavioral test indicated a reduction in behavioral and cognitive impairments in theophylline-treated mice. Pathological results demonstrated decreased tumor size and angiogenesis in treatment group. The presence of A2a and CD73 proteins were lower in the treated mice. Moreover, immunohistochemistry investigations showed a decline in VEGF expression in brain tissue and angiogenesis in the theophylline-treated group compared to the control group. Finally, serum levels of MDA and SOD were reduced in theophylline-treated mice.

Conclusion: Theophylline seem to ameliorate glioblastoma cancer by improving CD73 or adenosine signaling.

Glioblastoma

Methylxanthine

A2aR receptor

CD73

Theophylline

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary malignant brain tumor [1], which can be arised from astrocyte, oligodendrocyte, and neural stem cells (NSCs) [2]. The GBM is well-known for its heterogeneous entity and its high capacity for cell proliferation. Common treatments for glioblastoma include surgery, radiation therapy, chemotherapy, and new treatments such as immunotherapy [3]. Despite of the multiple treatments, the frequency of tumor recurrence is very significant [4] with approximately 7% of 5-year survival [5]. It is notable that even after the most eradicative treatments average survival for glioblastoma patients is about 12–19 months [3] concerning a therapeutic gap in GBM that has yet to be addressed. Hence, finding and affective treatment against GBM is urgently needed.

Purinergic molecules such as adenosine are highly conserved among different species, which has a great impact on regulating cell survival and proliferation progression, especially the relationship between glial cells and central nervous system cells [6, 7]. Numerous studies have indicated that adenosine plays a vital role in the development and progression of cancer by reducing the immune system responses [8, 9]. Moreover, induction of the adenosine signaling pathway via the A2a receptor reduces the secretion of inflammatory mediators, including interferon (IFN), interleukin 12 (IL-12), tumor necrosis factor (TNF). It also increases anti-inflammatory mediators such as IL-10, Transforming growth factor (TGF), and vascular endothelial growth factor (VEGF) that has a great effect on the cancer progression [10].

On the other hand, in cancerous tissues, adenosine triphosphate (ATP) is released into the extracellular space. Extracellular adenosine is involved in many cellular functions, including adhesion, cell proliferation, tumor invasion and cell angiogenesis [11, 12]. CD73, an ecto-5′-nucleotidase, is a main enzyme in extracellular ATP metabolism that resulting in converting ATP to adenosine monophosphate (AMP). Extracellular adenosine production through CD73 activity plays an important role in cancer progression, including involvement in immune suppression, angiogenesis, and cell proliferation. In addition to enzymatic function, CD73 is involved in the extracellular matrix interactions of the tumor, which is also involved in tumor invasion and metastasis [13].

Based on a study by Jung et al. on whether combined antitumor activity with CD73 and A2aR inhibition is stronger than their anti-cancer therapeutic alone, the results demonstrated that targeting these dependent pathways in combination with each other significantly suppressed tumor proliferation, growth and metastasis in the mouse model of fibrosarcoma [14]. Therefore, targeting CD73 and adenosine signaling via A2aR should be considered as a new therapeutic potential for glioblastoma patients. Methylxanthines are natural compounds with anti-cancer properties, including apoptosis induction, reducing cell migration, and inhibition of cell cycle, all of which are therapeutic targets against glioblastoma. Methylxanthines are currently used for several non-neoplastic diseases, with Pharmacodynamical profiles with high safety are proposed [15]. It is noteworthy that methylxanthines have the ability to bind to adenosine receptors. Also, methylxanthines are known as A2a receptor antagonists [15].

Theophylline, a member of the methylxanthine family, has a significant effect on reducing metastasis, inflammation, of cancer cells via inhibition of the phosphoinositide 3-kinase (PI3K), an active pathway in cancer that causes metastasis and resistance to tumor cell therapy [16]. In a study by Moon et al., theophylline was shown to reduce the survival of A172 and U87MG cells. In fact, the use of theophylline increases the basal level of cell death and inhibits the cell cycle in the G2/M stage [17].

Considering the vital role of adenosine pathway in glioblastoma progression through CD73 and A2aR signaling, the impact of theophylline in reducing the invasion of cancer cells cancers and the fact that theophylline acts as A2aR antagonist. In the current study, the effect of theophylline on glioblastoma process through CD73 and A2aR pathways was investigated.

Animals

Eighteen male BALB/c mice, weighing 20 ± 2 g and 6 weeks’ old were purchased from Razi Vaccine and Serum Research Institute (Karaj, Iran). The animals in groups of 6 were placed in cages covered with woodchip under standard laboratory conditions in a temperature and humidity-controlled environment with 12 h light/dark schedule. The animals had access to adequate pellet food and water. All the experimental procedures were performed in compliance with the Animal Research Ethics Committee of Islamic Azad University Science and Research Branch (Code of Ethics: IR.IAU.SRB.REC.1400.58).

Tumor Cell Line

CT-2A cell line has been widely used in glioblastoma tumor assessment. Preserved CT-2A cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco-Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco-Invitrogen, USA) and 1% penicillin along with streptomycin (Gibco-Invitrogen, USA). The cells were incubated at 37°C in a humidified atmosphere with 5% carbon dioxide (CO2). The medium was changed every 2–3 days, until achieving approximately 75% confluency. Then, cells were washed with phosphate-buffered saline (PBS) (Gibco-Invitrogen, USA) and detached by 0.04% trypsin-EDTA (Gibco-Invitrogen, USA). Next, the cells were centrifuged (500 g for 5 min) and resuspended in DMEM.

Glioblastoma induction

The experiment design consists of three groups as follow: healthy group (negative control), glioblastoma-induced group (positive control), and theophylline-recipient group (experimental). Mice were randomly allocated in these groups, and they were coded and housed in group of 6 in cages before starting the experiments.

To induce glioblastoma tumor, mice were anesthetized by intraperitoneal (ip) injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). The top of the mice’s head hair was removed and the animals were fixed to stereotaxic apparatus (Stoelting®, model 51700). The implantation position was determined at the following coordinates: Anterior-posterior (AP): 2.0 mm; Later-lateral (LL) 2.0 mm, and Abdominal depth (AD) of 2.5 mm. Mice received approximately 1 ×10⁶ CT-2A cells slowly over 10 minutes’ period. The healthy group was only injected by PBS as the negative control group.

Treatment with theophylline

After induction of glioblastoma tumor in 10 days, mice in theophylline-recipient group were administered theophylline (Darou Pakhsh, Iran) (200 µl/kg) orally by gavage daily during the 14 days of treatment period. There was no intervention in healthy group and glioblastoma-induced group. At the 15th day, mice were euthanized by carbon dioxide (CO2) narcosis. Serum and brain samples were collected for further investigation. Schematic presentation of in vivo experiments are shown in Fig. 1.

Garcia behavioral assessment

Various methods are used to assess neurological disorders such as motor, sensory, cognitive and behavioral in laboratory animals, which is in fact similar to what is used in stroke patients. On the 7th day of treatment, Garcia test was performed to evaluate motor and sensory functions on mice. The animals were scored as follows: spontaneous activity (0–4), symmetry of limb movement (0–4), forelimb outstretching (0–4), climbing and grip strength (0–4), symmetry of body proprioception (0–4), and sensory function of vibrissae (0–4). In this test, the animal's movement disorders were given a score of zero to four on a conventional basis for which a lower score illustrated more severe stroke outcome.

Histopathological and immunohistochemistry assessments

Brain tissues were immediately sectioned after euthanization and were paraffin-embedded that divided into two serial sections. One part of the divided sections was stained with hematoxylin and eosin (H&E) to evaluate the alternate brain histological features. The other part of sections was processed for immunohistochemistry (IHC) according to the standard protocols to evaluate the VEGF protein expression [18]. The sections were incubated overnight at 4°C with primary antibodies against VEGF (Abcam 69479, UK). Peroxidase-conjugated secondary antibodies were applied for chromogenic detection by oxidizing 3, 3′-Diaminobenzidin (1:200, ab205718). The nuclei were stained by Hematoxylin. CD73, and A2aR staining was quantified for the percentage of the positive area using Image J software.

Detection of apoptosis by TUNEL assessment

Apoptosis was determined by terminal deoxynucleotidyl transferase–mediated dUTP end labeling (TUNEL) assay kit (Roche, Germany) based on the manufacturer’s instructions. Briefly, brain tissue samples were immersed in xylol for 10 minutes and then washed with 90, 80 and 70% alcohols, respectively. Next, the samples were washed with PBS and then incubated with proteinase k (Thermo Fisher Scientific, UK) for 20 min at 37 ° C. They were then washed again with PBS. In the next step, 50 µl of TUNEL dye solution was poured on each tissue sample and incubated for one hour at 37 ° C. After final washing with PBS, the dead cells were observed under a fluorescent microscope. A minimum of 5 fields were randomly selected, and all dead cells were counted in each field. A surface equivalent to 1 mm² in each group was considered for counting. In this method, both TUNEL-Enzyme solution and TUNEL-Label solution in a ratio of 1 to 9 were used to stain positive control samples. While for negative control samples only TUNEL-Label solution was used.

Western blotting

The protein content of brain tissues was extracted using lysis buffer (Invitrogen; Thermo Fisher Scientific, UK) based on the manufacturer's instruction. The total protein concentrations in sample lysate were measured using Bradford assay employing bovine serum albumin (BSA). Proteins (40 µg/lane) were electrophoretically separated on the 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring onto poly-vinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membrane was then blocked with Tris-buffered saline (TBS) using 3% skim milk powder (Sigma-Aldrich, Germany) incubated at 4°C overnight and then probed with the primary antibodies of rabbit monoclonal antibody against CD73 (1:1000 dilution) (BS-4834R, Bioss Inc, USA), A2aR (1:1000 dilution) (BS-1456R, Bioss Inc, USA) and mouse monoclonal antibody against GAPDH (Abcam 181602, UK), applying as a loading control (1:5000 dilution). For the appearance of the protein bands, the membranes were incubated with suitable HRP-coupled secondary antibodies (Abcam 9572, UK), for 1 h at room temperature. Protein signals were detected by using enhanced electrochemiluminescence (ECL) reagents following autoradiography. Protein bands quantification was assessed by Image J software.

Oxidative stress markers investigation

After anesthesia, blood samples were allowed to clot, and serum was separated using centrifugation at 3000 rpm for 10 minutes at 4°C. The serum was used for investigation on oxidative stress markers, including the malondialdehyde (MDA) level (Zellbio, Germany) and the activity of the antioxidant enzyme superoxide dismutase (SOD) (Zellbio, Germany) based on the commercial assay kits. The content of MDA was measured by reacting with thiobarbituric acid to form a chromophoric product at 532 nm wavelength. SOD activity was determined by the conversion of superoxide anion to hydrogen peroxide under enzymatic conditions at 420 nm wavelength.

Statistics

The analysis of the data was carried out by Prism software version 5 (GraphPad Software, 224 USA). All values were expressed as Mean ± S.E.M. Statistical significance of differences between groups were analyzed using one-way analysis of variance and Tukey's test with a significance level set at p value ≤ 0.05.

Evaluation of sensory and motor disorders using Garcia behavioral assessment

To address motor and sensory functions on mice, on the 7th day of treatment, Garcia behavioral test was examined in all three groups of healthy, Glioblastoma- induced, and theophylline-recipient group and scored according to Garcia's conventional criteria. According to Garcia Behavioral, there was a statistically significant difference between healthy, Glioblastoma- induced, and theophylline-recipient groups (p < 0.001). The theophylline-recipient group showed a significant decrease in Garcia behavioral test scores, compared to the Glioblastoma- induced group (p < 0.01). On the other hand, there was not any significant difference between healthy control group and theophylline-recipient group (Fig. 2). The results showed the effectiveness of theophylline in improving motor and cognitive disorders caused by brain tumor induction.

Histopathological assessment verified the ameliorative potential of theophylline against glioblastoma

At the end of the treatment period, the brain excised for histopathological assessment. The H&E staining was used to evaluate the impact of theophylline on the progression of brain tumor. The healthy control group represented no tumor formation. Also, infiltration of inflammatory cells was not observed, and tumor formation was confirmed in the Glioblastoma- induced group. moreover, the accumulation of inflammatory cells into the tissue was observed to a large extent. Slightly necrotic areas were observed inside the tumor tissue, which may be due to a lack of oxygen supply to the tumor center. Cells were detected in the mitotic phase, which indicates the high activity of cells in the tumor area. A significant reduction of the tumor size was observed in theophylline-recipient group compared to the Glioblastoma- induced group and tumor cells have been detected in apoptosis phase. Infiltration of inflammatory cells into the brain tissue was also reduced compared to the Glioblastoma- induced group (Fig. 3).

Evaluation of the of VEGF protein content in brain tissues

The immunohistochemistry (IHC DAB staining) images revealed a significant reduction of VEGF protein in 14 days of the treatment period in the theophylline-recipient group compared to the Glioblastoma- induced group (p < 0.01). According to the results obtained from the studied groups, the expression of VEGF protein in the theophylline-recipient group was significantly higher in comparison with healthy group (p < 0.05) (Fig. 4). The results proved the therapeutic impact of theophylline in reducing the expression of VEGF protein and thus in reducing angiogenesis.

Evaluation of apoptosis in brain tissues by TUNEL assay

In order to evaluate apoptosis, TUNEL method was used. The results indicated that the positive TUNEL cells in the healthy, theophylline-recipient, and the Glioblastoma-induced groups have been detected. Statistical analysis showed a significant increase in the level of positive TUNEL cells in theophylline-recipient group compared to the healthy control group (p < 0.001). On the other hand, the expression level of positive TUNEL cells in the theophylline-recipient group was higher than the Glioblastoma- induced group, which was not statistically significant (Fig. 5). The results showed the effect of theophylline in increasing the rate of apoptosis in the tumor area.

MDA level and SOD activity measurement

MDA level and SOD activity were measured in the three groups of healthy, Glioblastoma-induced, and theophylline-recipient. The results are illustrated in Fig. 6. The SOD activity in mice treated with theophylline was higher compared to the Glioblastoma-induced group but this increase was no statistically significance. The results indicate the effectiveness of theophylline in reducing lipid peroxidation and increasing SOD activity. On the other hand, Data analysis showed that theophylline-recipient mice had lower levels of MDA than the Glioblastoma-induced group and the differences was statistically significant (p < 0.01).

The GBM is the most common primary malignant brain tumor known for its high cell proliferative capacity [19]. The treatment strategies against glioblastoma include surgery and then chemotherapy with radiotherapy. Currently, the average survival for glioblastoma patients is within 20 months, even with existing treatments [20]. Hence, the therapeutical approaches have focused on inhibiting intracellular signaling pathways.

During cancer progression, tumor cells increase adenosine production and decrease its consumption, which is achieved by increasing CD73 expression [21, 22]. Specific conditions of cancerous microenvironment, including hypoxia, high TGF expression, results in CD73 overexpression [23]. Moreover, up-regulation of A2aR and the further signaling pathways suppresses anti-tumor immune responses. Numerous studies demonstrated that CD73 and adenosine play a vital role in tumor adhesion, invasion, proliferation, angiogenesis, and chemical resistance [13, 21, 24].

In the current study, the therapeutical impact of theophylline against glioblastoma progression was investigated by focusing on inhibiting CD73 and A2aR pathways. For this purpose, the glioblastoma tumor was induced in male BALB/C mice by CT-2A cells injection within 10 days. Then, mice were treated with theophylline for 14 days.

To evaluate motor and sensory disorders caused by brain tumor Garcia behavioral test was performed. The results showed a difference in the Garcia score between the Glioblastoma- induced and theophylline recipient group. On the other hand, there was not a statistically significant difference between theophylline-treated group in comparison with the healthy group. This data suggested the effectiveness of theophylline in improving motor and sensory disorders caused by brain tumor induction.

Next, based on the histopathological assessment and H&E staining, multiple angiogenesis, the presence of inflammatory cells and an increase of cancer cells in the mitotic phase were observed in Glioblastoma- induced group. However, after theophylline treatment, the apoptosis rate among tumor cells increased and cell divisions, angiogenesis and penetration into the brain tissue were notably reduced, indicating the impact of theophylline in reducing brain tumors size and also preventing the progression of glioma by diminishing angiogenesis.

In the next step, IHC DAB staining results illustrated the significant reduction of VEGF protein in the theophylline- recipient group, which indicates the effectiveness of theophylline administration in reducing the expression of VEGF protein and thus in reducing angiogenesis.

To investigate the cause of VEGF protein depletion by theophylline, a study by Adir et al. indicates that under hypoxia, HIF-1-induced transcriptional factor rearrangement increased production of CD73 and extracellular adenosine. Further adenosine signaling via A2aR caused release of VEGF, which promotes angiogenesis and tumor invasion [25]. Consistently, data from Allard et al. study indicated that tumor-derived CD73 increases the production of VEGF, which is required for in vivo angiogenic responses. They showed that inhibition of CD73 with monoclonal antibodies significantly reduced tumor VEGF levels and suppressed tumor angiogenesis in mice breast cancer model [26].

After treatment with theophylline, protein levels of A2aR and CD73 were assessed by Western blotting. According to the findings, theophylline therapy results in reducing the expression of CD73 protein and A2aR protein in tumor tissue, which ultimately reduces angiogenesis in the tumor area. In consistence with our study, a study by Qalamfarsa et al. showed that CD73 regulates tumor angiogenesis via the VEGF signaling pathway [27]. Therefore, inhibition of CD73 and A2aR pathways by applying theophylline as A2aR antagonist results in downregulation of VEGF protein and further reducing angiogenesis and hence preventing tumor invasion.

Tunnel method was used to evaluate apoptosis. The results showed a significant increase in the level of positive TUNEL cells in the theophylline-recipient group in comparison with Glioblastoma- induced group. In line with the present study, investigation of Moon et al. showed that theophylline reduced the survival rate of A172 and U87MG cells. In fact, the use of theophylline increases the basal level of cell death and inhibits the cell cycle in the G2/M phase [17].

Based on Bortez et al. investigation to evaluate the concentration of MDA and SOD activity in the serum of patients with primary brain tumor namely glioblastoma, the results showed a statistically higher concentration of MDA with lower SOD activity in serum of patients with brain tumors compared to the healthy control group [28]. In the present study, it was shown that theophylline-recipient mice had lower levels of MDA than the Glioblastoma-induced ones. Also SOD activity level of theophylline-recipient group was higher. According to the results, the administration of theophylline has a great potential in amelioration of glioblastoma.

Overall, indication of theophylline administration as A2aR antagonist in induced glioblastoma model showed the alleviation of glioma characteristics through downregulation CD73 and A2aR pathway and subsequently downregulation of the VEGF level in these models. Hence, this study provides a pathway analysis of theophylline as A2aR antagonist concerning the crucial role of adenosine pathway in glioblastoma. Meanwhile, theophylline may exert its anti-tumor activity via inhibition of CD73 and adenosine pathways that make it a promising approach in treatment of glioblastoma.

Acknowledgments The authors would like to acknowledge the financial support of the Research Council of the Islamic Azad University, Science and Research Branch.

Authors' Contributions Design of experiments by HJ and MGh. FB performed experiments. All authors read and approved the final manuscript.

Data Availability The data are available from the corresponding author upon reseanable contact.

Conflict of Interest The authors declare no financial or non-financial conflict of interest.

Ethical Approval Animal experiments were conducted with approval from the Animal Research Ethics Committee of Islamic Azad University Science and Research Branch.

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The effect of administration of A2a receptor antagonist on the activity of A2a R/CD37 pathway and induction of apoptosis in glioblastoma tumor cells in BALB/c mice

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Animals

Tumor Cell Line

Glioblastoma induction

Treatment with theophylline

Garcia behavioral assessment

Histopathological and immunohistochemistry assessments

Detection of apoptosis by TUNEL assessment

Western blotting

Oxidative stress markers investigation

Statistics

Results

Evaluation of sensory and motor disorders using Garcia behavioral assessment

Histopathological assessment verified the ameliorative potential of theophylline against glioblastoma

Evaluation of the of VEGF protein content in brain tissues

Evaluation of apoptosis in brain tissues by TUNEL assay

MDA level and SOD activity measurement

Discussion

Declarations

References

Status:

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