Abstract
This study was designed to adjust effective chemotherapy doses of cabazitaxel (CBZ) on cognitive behaviors, inflammatory cytokines and oxidative stress parameters, and survival rate in C6-induced GBM of rats. Male Sprague–Dawley rats bearing intra-caudate nucleus (CN) C6 inoculation were randomly divided into nine groups as follows: sham, tumor, Temozolomide (TMZ) vehicle, TMZ, CBZ vehicle, CBZ at doses of 0.5, 1, 2 and 4 mg/kg. Behavioral tests survival rate, histopathology, immunohistochemistry, oxidative stress, and inflammatory cytokines were evaluated. All drug treatments reduced the volume and number of tumor cells dose-dependently and CBZ4 was able to cause the greatest reduction. The %Survival rate of animals using CBZ1 significantly increased compared to other treatment groups. CBZ1 reduced anxiety-like behaviors and increased the balance of the animal with GBM. CBZ1 and CBZ2 groups improved C6-induced learning disabilities. Treatments could ameliorate tumor-induced dysregulation of oxidative stress. TNF-α/IL-10 decreased in the CBZ1 group compared to other treatment groups, which may indicate an improvement in inflammatory balance. Our findings demonstrate that the administration of CBZ at a dosage of 1 mg/kg exerts advantageous impacts on both the survival rate and neurocognitive performance of rats within the GBM model. However, our results showed that CBZ may have toxic effects, especially in a dose of 4 mg/kg.
Keywords: glioblastoma, Temozolomide (TMZ), Cabazitaxel (CBZ), cognitive behavior, inflammatory factors, oxidative stress
Graphical Abstract
Graphical Abstract.
Introduction
Cancer of the central nervous system (CNS) continues to be a prominent contributor to global mortality, resulting in approximately 308,102 newly diagnosed cases and 251,329 fatalities in 2020.1 It is estimated that in 2023, CNS cancer is expected to become the tenth leading cause of cancer-related mortality across all age groups for both genders.2 Glioblastoma (GBM) is a prevailing tumor of the CNS, constituting a significant quandary in the realm of cancer therapy owing to its remarkable aggressiveness and poor prognosis of a 9% 5 yr survival rate.3,4 Despite various therapeutic approaches such as surgery, chemotherapy, and radiation therapy, the average survival after diagnosis of GBMs is only 14–16 mo.3 Increased intracranial pressure, impaired cognitive function, and seizures are the main clinical manifestations of glioma patients.3 Among these, behavior disorders plays an important role in predicting the prognosis of glioma patients and are even considered as the first indicator of tumor recurrence after treatment.5
For patients with glioma who cannot undergo neurosurgery, chemotherapy is the first-line treatment.6 In either systemic or intra-CSF administration, chemotherapy can adversely affect the central and peripheral nervous systems.7 Neurological complications of chemotherapy drugs can be serious, such as acute encephalopathy, dementia, neuropathy, vasculopathy, and stroke.7 These complications can worsen the patient’s quality of life and restrict the dosage of the medication employed. Direct toxic effects or metabolic disturbances of the chemotherapy agent may cause these complications.8,9 Even for drugs such as taxanes, the dose-limiting toxicity of chemotherapy is defined as neurotoxicity.10 Therefore, the most important thing is to adjust the dose of chemotherapy because there is no specific antidote for most chemotherapy-induced neurotoxicity.10
Cabazitaxel (CBZ) is a potent anticancer drug from the second-generation taxanes. It is approved by the US FDA for the treatment of prostate cancer.11 The anti-neoplastic impact of CBZ is characterized by microtubule inhibition, cell cycle disruption, and induction of apoptosis in tumor cells.12,13 Additionally, this drug exhibits other effects such as the reduction of angiogenesis, cell migration, and tumor volume in both in vitro and ex vivo glioma models.14 In preclinical studies, CBZ has been documented to possess a heightened capacity for traversing the blood–brain barrier (BBB) in contrast to first-generation taxanes.15,16
Currently, the prevailing chemotherapeutic agent employed in the treatment of patients diagnosed with GBM is temozolomide (TMZ), which induces apoptosis in tumor cells by elevating oxygen-free radicals.17,18 Studies indicate that treatment with TMZ is associated with cognitive impairment and an even higher incidence of subsequent cancers.19,20 Therefore, given that the current treatments do not provide sufficient protection against mortality and tumor progression, nor do they improve survival and quality of life.21 The present study evaluates CBZ ‘s impact on cognitive function, tumor volume, histopathology, oxidative stress, and inflammatory cytokines in tumors caused by C6 as a reliable model for assessing treatment efficacy.22 In addition, the safe and effective dose of CBZ as chemotherapy for GBM was studied due to the importance of adjusting chemotherapy doses.
Materials and methods
The timetable and flow chart of the experimental groups have been demonstrated in Fig. 1.
Fig 1.
Time-table of the research study.
Cell culture
The rat C6 cell line was purchased from Pasteur Institute (Tehran, Iran) and was cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, USA), enriched by 10% fetal bovine serum, 1% penicillin/ streptomycin, at the 5% carbon dioxide (CO2) and 37 °C. Cells have been tested each day and passaged serially once they arrived at a minimum 70% confluence.23 Afterward, cells were suspended in DMEM at the concentration of 1.0 × 106 cells per 10 μL in a tube for tumor implantation.
Drugs
TMZ and CBZ were obtained from Razavi Pharmaceutical Service Institute and NanoAlvand Pharmaceutical Co, respectively.
Animals
In this experiment, 108 male Sprague Dawley rats that matured at 8 wk and weighed 200–250 g were purchased from the Pasteur Institute (Tehran, Iran). The animals were kept in a temperature-controlled room (22 ± 2 °C) on a 12/12 h light/dark cycle at the Kerman University of Medical Sciences Animal house and they were provided with food through a stainless-steel grid and water ad-libitum. All experiments and behavioral tests followed the protocols approved by the Ethical Committee for Animal Care and the Code of Ethics approval IR.KMU.AH.REC.1399.136. We used 12 animals in each group (seven rats for evaluating behavioral and molecular tests, and five rats for survival rate). However, we missed some animals in various groups for glioblastoma or side effects of treatments.
Experimental procedure
Rats were randomly partitioned into nine groups: Sham group, C6 glioma cells (1 × 106 cells/30 μL/rats), C6 glioma cells + TMZ vehicle, C6 glioma cells + TMZ, C6 glioma cells + CBZ vehicle, C6 glioma cells + CBZ 0.5 mg/kg, C6 glioma cells + CBZ 1 mg/kg, C6 glioma cells + CBZ 2 mg/kg, C6 glioma cells+ CBZ4 mg/kg. It should be noted that researchers evaluated CBZ doses based on a pilot study and investigation by Karavelioglu et al (2016).9
Sham group
This group of animals underwent surgery and received 30 μL of culture medium.23
Tumor group
These animals underwent surgery to receive tumor cells in the form of 1 × 106 cells/30 μL/rats.24
TMZ vehicle group
Tumor-induced animals received DMSO10% + normal saline 90% at a rate of 1 mL/kg intraperitoneally from the fifth day after tumor induction for five days in a row.23,25
TMZ group
Tumor-induced animals received TMZ at a dose of 7.5 mg /kg intraperitoneally from the fifth day after tumor induction for five days.26
CBZ vehicle
Tumor-induced animals received ethanol 13% distilled water 87% at a rate of 1 mL/kg intraperitoneally from the fifth day after tumor induction for four days.
CBZ0.5 group
Tumor-induced animals received CBZ at a dose of 0.5 mg/kg intraperitoneally from the fifth day after tumor induction for four days.
CBZ1 group
Tumor-induced animals received CBZ at a dose of 1 mg/kg intraperitoneally from the fifth day after tumor induction for four days.
CBZ2 group
Tumor-induced animals received CBZ at a dose of 2 mg/kg intraperitoneally from the fifth day after tumor induction for four days.
CBZ4 group
Tumor-induced animals received CBZ at a dose of 4 mg/kg intraperitoneally from the fifth day after tumor induction for four days.
Stereotaxic surgery for C6 implantation into rat’s brain
The method was the same as described earlier.27 All rats were anesthetized with intraperitoneal injections of ketamine (55 mg/kg) and xylazine (10 mg/kg). First, the hair wherever the surgery was performed was shaved. They were fixed on a rodent stereotactic frame, and the pericranium was moved laterally to expose the bregma and a midline incision was made on the dorsal part of the head. A tiny hole with a diameter of 1 mm and a depth of 5 mm was bored at the spot designated for the guide-screw entry in the right 3 mm and anterior 1 mm of the bregma. The caudate nucleus is regarded by the Paxinos and Watson atlas as a very reliable intracranial site for the development of tumors. The culture medium or an add up to 1*106 single C6 glioma cells in 30 μL of culture medium was infused with a Hamilton syringe for 10 minutes. The needle is cleared out within the infusion location for 2 min to prevent the injected cells from dismissing, and after that, the needle is tenderly pulled out. The cranium bone gap was repaired utilizing bone wax and the cranium skin was sutured with 4–0 nylon strings.
Open field test (OFT)
The instrument utilized in this check could be a Plexiglas box with a length and dimension of 90 × 90 × 30 cm3, the ground of which is split into sixteen little squares by hypothetic lines. Each rat was located in the central zone and allowed five minutes of unfastened exploration. All periods have been recorded after which analyzed the use of the Ethovision software (version 7), a video monitoring machine for automation of behavioral tests. The following behavioral indices have been then evaluated: overall distance moved (m); overall period of mobility (s) and immobility (s); and the wide variety of rearing and grooming. The equipment becomes wiped clean after trying out every rat.27,28
Elevated plus-maze (EPM)
The apparatus was the same as described earlier.27 EPM equipment consisted of 2 open and 2 closed arms of the same size (50 × 50 cm2). 40 cm-high walls surrounded the 2 closed arms. The four arms were joined by a central sq. platform. The maze was elevated 50 cm on top of the ground. All experiments were done in a test room set alight by a 100w light bulb placed on top of the center of the EPM. The percentage of open arm entries (%OAE) open arm times (%OAT) and locomotor activity as the standard anxiety indices were assessed as follows: (a) %OAE (the ratio of entries into open arms to total entries × 100); (b) %OAT (the ratio of times spent in the open arms to total times spent in any arms × 100); and (c) locomotor activity (total arm entries).29
Rotarod test
The rotarod device consists of an associate fast-rotating rod, which is accepted equipment for evaluating activity parameters indicating motor coordination and balance in rats. The experiment began with a minimum speed of ten revolutions per minute (rpm) to a maximum speed of sixty rpm. Animals were placed on the rod, and 3 min trials with a thirty min interval between trials, were devoted to every rat. The latency to fall as a motor coordination index was recorded.27
Passive avoidance test
The basis of this test, which was performed 15–16 days after tumor induction to assess associative learning and memory, is the intrinsic inclination of rodents to dark and closed environments and learning to avoid negative stimuli.27 The gadget for testing is a shuttle box comprising two dark and light compartments and a controllable entryway between them. First, the rat was habituated to the apparatus for 5 min. The day after habituation, the rat was returned to the light part, the door opened and the animal was allowed to move to the dark chamber. The delay time for taking off the light area and entering the dark area is recorded. 1 h later, the rat was placed into the light part, the door was opened, and the rat received an electric shock as soon as it entered the dark part (50 Hz, 3 s, 1 mA intensity; via wires embedded in the dark part floor. This was done up to five times at 1-hour intervals until the rat learned not to enter the dark area (stays in the light area for at least 300 seconds) and recorded the number of shocks needed to learn. 24 h after the learning phase, the rat was placed into the light area to assess its memory, and after 30 seconds, the door was opened and the time required for the animal to enter the dark chamber was recorded.27,30
Weight recording and tumor volume measurement
From the day of operation, the weights of the rats were monitored every day. Data have not been provided since there was no statistically significant difference between the groups. Additionally, each animal underwent thorough anesthesia when the behavioral testing was finished, after which the skull was swiftly opened and the brain was extracted from each animal. When evaluating tumor size, the researchers were not aware of the test settings. The tumor volume was measured using caliper measurements. The formula L*H*W*π/6, where L is the length, W is the width, and H is the height of the tumor in millimeters, was used to estimate tumor volumes (mm3).27
Histopathological and Immunohistochemical evaluation
Histopathology
On the 17th day after tumor induction, the brain tissue was removed after induction of deep anesthesia and placed in a 10% formalin container. Then, 5 μm sections of the blocks were prepared by microtome stained with Hematoxylin and Eosin (HE) for histopathological evaluation.27,31 The histologic findings including tumor necrosis, mitotic counts in ten high-power fields, and infiltration of inflammatory cells (mild, moderate, severe) were evaluated by a blind pathologist.
Immunohistochemistry
Histologic sections of 3 mm thick paraffin blocks were deparaffinized and rehydrated with xylene and ethanol, respectively, for the IHC investigation. Tris-EDTA (PH = 9) buffer was used for antigen retrieval, which was then microwaved for 10 min. Methanol 0.5% and H2O2 inhibited tissue peroxidase activity. Following that, the primary monoclonal antibody, which included Ki67, was incubated for 30 min at room temperature (Dako MIB1, df 1:50, Denmark). Using a biotin and streptavidin peroxidase method, the secondary antibody was applied. Diaminobenzidine tetrahydrochloride (DAB) chromogen was used to demonstrate antigen–antibody bands. The background was stained with hematoxylin, and cells with positive nuclear staining were assessed according to Sebastian CJ Steiniger et al’s study.27
Preparation of tissue homogenates
Following the procedure, the tissue samples were separated, cleaned with 0.9% NaCl, and then refrigerated at 80 °C. To provide homogenates, a homogenizer (Polytron PT 2100, KINEMATIC AG, Switzerland) was employed. The homogenates used for the SOD, GPx, MDA, TAC, IL-10, and TNF- α measurements contained 100 mg of tissue in 1 ml of phosphate buffer (50 mmol/L; pH 7.5), along with 1 mm of EDTA. After centrifuging at 20,000 g for 15 min. at 4 °C, the supernatants were collected for biochemical analysis.27
Assessment of superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities
Tissue samples kept at −80 ° C are used to assess these parameters. According to the manufacturer’s instructions, a Randox assay kit (London, England) was used to measure these parameters in the brain tissue. U/μg protein for SOD, as well as U/mg protein for GPX, were used to allocate findings.27
Measurement of total antioxidant capacity (TAC) level
The combination of 140 μL of the working reagent and 10 μL of the standard sample was added to the 96-well plate. The plate was incubated for 15 min at 37 ° C. Then the absorption was read at 593 nm wavelength.27
Measurement of malondialdehyde (MDA) activity level
200 μL of the working reagent mixed with 100 μL of the standard sample incubated at 90 ° C for 1 h and then cooled to 4 ° C. After cooling, the mixture was centrifuged at 3000 RPM. Then, 150 μL of the supernatant solution was removed and poured into the 96-well kit. Its light absorption at 550 nm wavelength was read by a spectrophotometer. We draw a standard diagram and obtain the MDA of unknown samples using statistical software.27
Evaluation of interleukin 10 (IL-10) and tumor necrosis factor (TNF-α) level
Brain tissue cytokines, including IL-10 and TNF-α, were measured based on the instructions of ELISA kits purchased from (Karmania Pars Gene, Iran). Values are displayed as pg/mg tissue in the brain tissue.27
Statistical analysis
Normality and homogeneity of variances assumptions were used by Shapiro–Wilk and Leven tests, respectively. All acquired data except survival rate were analyzed by one-way ANOVA and when the F-value was significant, Tukey’s post-hoc analysis test was performed for evaluating specific inter-groups. Furthermore, the survival rate evaluation and significant survival differences were done using the Kaplan–Meier method and the Wilcoxon test, respectively. The significance level was less than 0.05. GraphPad Prism software version 8 (GraphPad Software, San Diego, USA) analyzed the data and plotted the diagram. Results were reported as mean ± SEM.
Results
The effect of TMZ and various doses of CBZ on tumor volume in GBM
One-way ANOVA indicates a significant difference among treatment groups for tumor volume [F (7, 32) = 12.96, P˂0.001]. Supplementary evaluation using the Tukey test showed that treatment groups reduced tumor volume as compared to vehicles (P < 0.001). Besides, injection of 4 mg/kg CBZ could have a greater effect on lowering tumor volume than different remedy groups (P < 0.05) (Fig. 2).
Fig 2.
The effect of TMZ and various doses of CBZ on tumor volume in C6-induced GBM in rats. Each bar represents mean ± SEM. Significant differences: +++P < 0.001 compared with sham group; & P < 0.05 compared with TMZ, CBZ 0.5, 1, 2 groups (n = 5 in each group).
The impact of TMZ and different doses of CBZ on histopathological and immunohistochemical parameters in C6-induced GBM in rats
Histopathology
The impact of intraperitoneal administration of TMZ (1 mg/kg) on the 5th to 9th days after tumor induction and CBZ at doses of 0.5, 1, 2, and 4 mg /kg on the 5th to 8th days after induction on histopathological parameters in C6-induced glioblastoma in the male rat is shown in Fig. 3. No histopathological lesions had been determined in the sham group (Aa). In the tumor group, microscopic examinations confirmed tumoral mass composed of neoplastic cells with spherical nuclei containing one or two distinct nuclei is consistent with C6 GBM. A high mitotic index was observed (Ba, Bb). In the TMZ vehicle and CBZ vehicle, the tumor mass was hypercellular and contained active tumor cells. No findings of growth regression were discovered (Bc, Bd). In the TMZ group, the tumor mass was significantly smaller than in the tumor group, and some tumor cells showed necrotic changes. There was space at many points in the tumor matrix due to the destruction of tumor cells (Ab). Histopathologically, in CBZ groups, regression of tumor mass was observed—the severity of those changes expanded with raising the dose of the drug inside the treatment groups. The size of the tumor mass decreased and had an exact and restrained border. A noticeable reduction in tumor cells was seen. There was also a large number of empty spaces in the tumor matrix. Unlike the control group, where the cells had an active and specific nucleus, the nuclei of the cells were very dense and dark in the treatment group. Also, the penetration rate of inflammatory cells increased compared to the tumor group (Ac-Af) (Fig. 3A and B).
Fig 3.
The effect of TMZ and different doses of CBZ on histopathological parameters in C6-induced GBM in rats. (A) Histopathological staining: a) sham group: no changes in histopathological examination was seen, b) TMZ group: Decreased number of tumor cells (circle) and cells with contraction and dense and dark nuclei, c) CBZ0.5 group: Low cell tumor mass (circle) and decreased cytoplasm/nucleus ratio, d) CBZ1 group: Tumoral cells are scattered and specific necrotic areas (hollow star) are seen with the infiltration of inflammatory cells around it (circle), e) CBZ2 group: Tumor mass size is significantly reduced and has a limited and clear border (arrow), f) CBZ4 group: The number of tumor cells is reduced and these cells have pyknotic nuclei (dark and dense). There are several empty spaces in the matrix (circle) (HE, X200). B) Histopathological staining: a &b) tumor group: Hypercellular tumor mass consisting of pleomorphic cells often have a round nucleus with distinct nuclei (circle). There is a high mitotic index as well as a high cytoplasm/nucleus ratio(arrow), c &d) TMZ and CBZ-vehicle groups: Tumor mass is similar to tumor group and contains active tumor cells. (HE, X200). (C) Immunohistochemical staining: Immunohistochemistry of tumor tissue for Ki67 expression in different groups. Ki67 positive cells were more numerous (arrows) in tumor group (a), TMZ-vehicle group (b), and CBZ group (c) compared with treatment groups, TMZ group (d), CBZ 0.5 mg (e), CBZ 1 mg (f), CBZ 2 mg (g) and CBZ 4 mg (f). IHC, bar = 20 μm.
Immunohistochemistry
The results of Ki-67 immunohistochemical staining are shown in Table 1 and Fig. 3C). High Ki-67 expression in the tumor and vehicle groups was observed. The Ki67 level significantly decreased in TMZ and CBZ 0.5 mg, 1 mg, 2 mg, and 4 mg, respectively.
Table 1.
Comparison of histopathological and immunohistochemical findings among treatment groups.
| Groups Parameter | Tumor | TMZ | CBZ 0.5 mg | CBZ 1 mg | CBZ 2 mg | CBZ 4 mg |
|---|---|---|---|---|---|---|
| Tumor necrosis (%) | ˂5% | 20% | 20% | 25% | 30% | 30% |
| Mitotic counts | 35/10hpf | 14/10hpf | 12/10hpf | 10/10hpf | 7/10hpf | 6/10hpf |
| Infiltration of Inflammatory cells | Mild | Moderate | Moderate | Moderate | Severe | Severe |
| Ki-67 (Score) | 4 | 3 | 2 | 2 | 1 | 1 |
| ˃15% | (10%–15%) | (5%–10%) | (5%–10%) | ˂5% | ˂5% |
The effect of TMZ and various doses of CBZ on survival rate in GBM
Kaplan–Meier estimator shows a significant difference among treatment groups for survival rate (P < 0.001) as shown in Fig. 4. The Wilcoxon test showed a considerable difference among tumor and sham groups (P < 0.001). In addition, the administration of CBZ 1 mg/kg significantly increased the survival rate in comparison to the vehicle (P < 0.01), TMZ, and other CBZ groups (P < 0.001). In the 4-mo follow-up, the mean survival time for the sham group, tumor group, TMZ Vehicle, TMZ, CBZ Vehicle, CBZ 0.5, 1, 2, 4 groups were 120, 25, 27, 31, 24, 44, 77, 38, and 13 days, respectively.
Fig 4.
The effect of TMZ and various doses of CBZ on survival rate in C6-induced GBM in rats. Each bar represents mean ± SEM. Significant differences: ***P < 0.001 compared with tumor group; ++P < 0.01 compared with vehicle group; ^^^P < 0.001 compared with TMZ, CBZ 0.5, 2 and 4 groups (n = 5 in each group).
Effects of TMZ and different doses of CBZ on the OFT indices in C6-induced GBM in rats
There was a statistically significant difference among treatment groups by one-way ANOVA for time spent in center part [F (7, 40) = 5.15, P = 0.0003] and grooming [F (7, 40) = 4.29, P = 0.0013], but not for distance moved [F (7, 40) = 1.08, P = 0.38] and rearing [F (7, 40) = 1.37, P = 0.24]. A complementary analysis using the Tukey test indicated that receiving a 2 mg/kg dose of CBZ significantly increased the time spent in the central part compared to the sham group (P < 0.01) (Fig. 5B). No significant difference was observed among treatment groups in the parameter of grooming (Fig. 5C) following Tukey’s posthoc test (Fig. 5).
Fig 5.
The effect of TMZ and various doses of CBZ on distance moved (A), time spent in the central part (B), grooming (C), and rearing (D) in the OFT in C6-induced GBM in rats. Each bar represents mean ± SEM. Significant differences: ++P < 0.001 compared with the vehicle group (n = 6 in each group).
The effect of TMZ and different doses of CBZ on anxiety-like behavior parameters in the EPM test in GBM
One-way ANOVA showed a significant difference among treatment groups for %OAT [F (7, 40) = 5.15, P = 0.003]. Complementary analysis using the Tukey test showed a significant reduction in the %OAT in the tumor group compared to the sham group, suggesting an increase of anxiety-like behaviors after C6 injection (P < 0.05) (Fig. 6A). In addition, in the CBZ1 group, compared to the sham group, % OAT increased (P < 0.05), indicating a decrease in anxiety-like behaviors. One-way ANOVA of %OAE showed that there was a significant difference among the treatment groups [F (7, 40) = 5.39, P = 0.0002]. A significantly increased %OAE was observed in the TMZ-receiving rats compared to the sham group in the Tukey’s test (Fig. 6B) (P < 0.01). No significant difference in locomotor activity was observed among the treatment groups [F (7, 40) = 1.25, P = 0.29].
Fig 6.
The effect of TMZ and various doses of CBZ on anxiety-like behavior parameters of the EPM test including %OAT (A), %OAE (B), and locomotor activity (C) in C6-induced GBM in rats. Each bar represents mean ± SEM. Significant differences: *P < 0.01 compared with the sham group; +P < 0.05 compared with the CBZ vehicle group; ++P < 0.01 compared with the TMZ vehicle group (n = 6 in each group).
The effect of TMZ and different doses of CBZ on time on the rod as an index of motor coordination in rotarod test in C6-induced GBM in rats
One-way ANOVA revealed a significant difference among treatment groups for the duration of stay on the rod [F (7, 40) = 5.13 P = 0.0003]. Tukey’s test showed that C6 injection shortened the stay on the rod compared to the sham group (P < 0.01), indicating motor disturbances. The duration of time spent on the rod dramatically increased in the CBZ1 group compared to the sham group, suggesting improvement of motor symptoms and balance (P < 0.05) (Fig. 7).
Fig 7.
The effect of TMZ and various doses of CBZ on time on the rod as an index of motor coordination in the rotarod test in C6-induced GBM in rats. Each bar represents mean ± SEM. Significant differences: **P < 0.01 compared with the sham group; +P < 0.05 compared with the CBZ vehicle group (n = 6 in each group).
The effect of TMZ and different doses of CBZ on avoidance memory in the shuttle box in C6-induced GBM in rats
One-way analysis of variance showed a significant difference among treatment groups for the avoidance memory index obtained from the time of the delay to entering the shock zone chamber [F (7, 40) = 6.91, P < 0.001]. Further analysis using Tukey’s test indicated that after induction of the tumor, step-through latency (s) was significantly decreased as compared to the sham group (P < 0.01), indicating memory impairment in the tumor group. In addition, post-hoc analysis revealed that administration of TMZ, CBZ1, and CBZ2 significantly increased step-through latency (s) compared to their vehicle (P < 0.01, P < 0.05, P < 0.05, respectively), suggesting an ameliorating effect of these treatments on the memory of GBM rats (Fig. 8).
Fig 8.
The effect of TMZ and different doses of CBZ on step-through latency in the passive avoidance test in C6-induced GBM in rats. Each bar represents mean ± SEM. Significant differences: **P < 0.01 compared with sham group; ++P < 0.05 compared with TMZ vehicle group; +P < 0.05 compared with CBZ vehicle group (n = 6 in each group).
The effects of TMZ and different doses of CBZ on antioxidant indices including GPx, TAC, and SOD in C6-induced GBM in rats
One-way ANOVA showed a significant difference among treatment groups for GPx activity [F (8, 36) = 73.69, P < 0.001]. Additional analysis using Tukey’s test showed that C6 injection significantly decreased GPx activity compared to the sham group (P < 0.001), indicating a decrease in antioxidant activity. GPx activity increased in rats that received TMZ compared to its vehicle group (P < 0.05). Also, The CBZ1 and CBZ4 groups showed an increase in GPx activity compared to their vehicle group (P < 0.01 and P < 0.001, respectively), which indicates an improvement in antioxidant status. CBZ0.5 group showed a decrease in GPx activity (P < 0.05) compared to CBZ vehicles. This antioxidant index in the TMZ group increased compared to CBZ0.5 and 2 groups and decreased compared to the CBZ 4 group (P < 0.001). In addition, GPx was significantly decreased in CBZ0.5, 2 groups compared to the CBZ1 group (P < 0.001). Also, this index in the CBZ4 group showed a considerable increase compared to the CBZ1 group (P < 0.01) and CBZ 0.5 and 2 groups (P < 0.001) (Fig. 9A).
Fig 9.
The effects of TMZ and different doses of CBZ on antioxidant indices including GPx (A), TAC (B), and SOD (C) in C6-induced GBM in rats. Each bar represents mean ± SEM. A) Significant differences: ***P < 0.001 compared with sham group; +P < 0.05, ++P < 0.01, +++P < 0.001 compared with vehicle group; ###P < 0.001 compared with CBZ 0.5, 2 and 4 groups; ^^^P < 0.001 compared with CBZ 0.5 and 2 groups; &&P < 0.01 compared with CBZ1 group; &&&P < 0.001 compared with CBZ 0.5 and 2 groups (n = 6 in each group). B) Significant differences: ***P < 0.001 compared with sham group; +P < 0.05, ++P < 0.01 compared with vehicle group; ###P < 0.001 compared with CBZ 0.5, 1, 2 groups; &&&P < 0.001 compared with CBZ 0.5, 1, 2 groups (n = 6 in each group). C) Significant differences: ***P < 0.001 compared with sham group; ++P < 0.01 compared with vehicle group; ###P < 0.001 compared with CBZ 0.5 groups; &&P < 0.01 compared with CBZ 0.5 group (n = 6 in each group).
Evaluation of TAC using one-way ANOVA showed that there was a significant difference among the treatment groups [F (8, 36) = 87.91, P < 0.001]. Further analysis using Tukey’s test indicated that C6 injection reduced TAC in rats compared to the sham group (P < 0.001). TMZ increased the TAC levels compared to the TMZ vehicle group (P < 0.05). CBZ 0.5, 1, and 2 groups decreased TAC levels compared to their vehicle group (P < 0.01, P < 0.01, P < 0.05, respectively). TAC levels in CBZ 0.5, 1, and 2 groups showed a significant decrease compared to TMZ and CBZ4 groups (P < 0.001) (Fig. 9B).
One-way ANOVA showed a significant difference among treatment groups for SOD [F (8, 36) = 17.60, P < 0.001]. Further analysis using Tukey’s test showed that C6 injection reduced SOD in comparison to the sham group (P < 0.001). The CBZ0.5 group also reduced SOD compared to the CBZ vehicle, TMZ, and CBZ4 groups (P < 0.01) (Fig. 9C).
The effect of TMZ and different doses of CBZ on MDA oxidative index in GBM
One-way ANOVA showed a significant difference among treatment groups for MDA [F (8, 36) = 21.88, P < 0.001]. Additional analysis using Tukey’s test showed that C6 injection increased MDA levels in rats compared to the sham group (P < 0.001). In addition, CBZ 0.5, 1, 2 mg/kg reduced the MDA level compared to their vehicle, which indicates a decrease in oxidative activity after injection of these drugs (P < 0.001). Our results also demonstrated that MDA level decreased in the CBZ 0.5 and CBZ 2 groups compared to TMZ and CBZ4 groups (P < 0.01, P < 0.001, respectively (Fig. 10).
Fig 10.
The effect of TMZ and different doses of CBZ on MDA oxidative index in C6-induced GBM in rats. Each bar represents mean ± SEM. Significant differences: ***P < 0.001 compared with sham group; +++P < 0.001 compared with CBZ vehicle group; ##P < 0.01 compared with CBZ 0.5 group; ###P < 0.001 compared with CBZ 2 group; &&P < 0.01 compared with CB Z0.5 group; &&&P < 0.001 compared with CBZ 2 group (n = 6 in each group).
The effect of TMZ and different doses of CBZ on TNF-α, IL-10, and TNF-α /IL-10 in a rat model of GBM
One-way ANOVA showed a significant difference between treatment groups for TNF-α [F (8, 36) = 12.85, P < 0.001]. Supplementary analysis using Tukey’s test showed that C6 injection caused a significant increase in TNF-α (P < 0.001). The CBZ 0.5 group decreases TNF-α compared to the CBZ 2 group (P < 0.05). In addition, in the CBZ4 group, the TNF-α levels increased significantly compared to CBZ vehicle, TMZ, and CBZ 0.5 groups (P < 0.001), as well as CBZ1 (P < 0.01) and CBZ2 groups (P < 0.05) (Fig. 11A).
Fig 11.
The effect of TMZ and different doses of CBZ on TNF-α (A), IL-10 (B), and TNF-α / IL-10 (C) in C6-induced GBM in rats. Each bar represents mean ± SEM. Significant differences: ***P < 0.001 compared with sham group; +++P < 0.001 compared with vehicle group; @P < 0.05 compared with CBZ 2 group; &P < 0.05 compared with CBZ 2 group; &&P < 0.01 compared with CBZ1 group; &&&P < 0.001 compared with CBZ0.5 and TMZ group (n = 6 in each group). B) Significant differences: ***P < 0.001 compared with sham group; +++P < 0.001 compared with vehicle group; ^^^P < 0.001 compared with CBZ 0.5, 2 groups and TMZ group; ^^P < 0.01 compared with CBZ4 group (n = 6 in each group). C) Significant differences: @P < 0.05 compared with TMZ group; ^P < 0.05 compared with CBZ 2, 4 groups; ^^P < 0.01 compared with TMZ group (n = 6 in each group).
One-way ANOVA showed a significant difference among the treatment groups for IL-10 [F (8, 36) = 11.13, p < 0.001]. Supplementary analysis using Tukey’s test showed that tumor induction caused a significant increase in IL-10 (P < 0.001). In addition, CBZ1 increased the level of IL-10 compared to CBZ vehicle, TMZ, CBZ0.5 CBZ2 groups (P < 0.001), and also the CBZ4 group (P < 0.01) (Fig. 11B).
One-way ANOVA showed considerable differences among treatment groups for TNF-α/IL-10 ratio [F (8, 36) = 4.38, P < 0.001]. Supplementary analysis using Tukey’s test showed a decline in the CBZ 0.5 group compared to the TMZ group (P < 0.05). In addition, the CBZ1 group showed a significant decrease in the TNF-α/IL-10 compared to the TMZ (P < 0.01), CBZ 2, and CBZ4 groups (P < 0.05) (Fig. 11C).
Discussion
In the present study, we first investigated the effect of CBZ therapy on tumor volume, histopathology, survival rate, anxiety-like behaviors, balance, memory, oxidative, and inflammatory status in brain tissue of rats with C6-induced GBM. In the second step, according to the side effects of CBZ and the importance of determining the safe dose in chemotherapy drugs, the effective and safe dose of CBZ in the treatment of GBM was determined. CBZ is a highly efficacious chemotherapeutic agent specifically crafted with the intention of inhibiting microtubule function in tumors. Previous experimental studies have reported that CBZ is useful in a variety of in vitro and preclinical models of glioma.15,32–34
In the histopathological examination, treatment with TMZ and CBZ caused tumor regression, and this reduction was greater and dose-dependent in the CBZ groups. These results are confirmed by a previous study in which CBZ reduced tumor size and increased apoptotic cells in mice receiving orthotopic glioma.35
Our results showed that the tumor group had high levels of Ki-67 expression, which was considerably reduced by CBZ dose-dependently. In cancer cells, as expected, the percentage of Ki-67 expression as a non-histone nuclear protein expressed during the proliferative cell cycle is high.27 It is well-recognized that rising Ki-67 labeling index values correspond to increased malignancy grades.36 Our previous study showed that the tumor induced by C6 cells increases the expression of Ki-67 in the tumor group, and treatment with TMZ decreases its expression.27 Previous studies have shown the effectiveness of CBZ in reducing the expression of Ki-67 in various tumors.37
An interesting finding in our study was a significant increase in survival rate in the CBZ1 group. Also, in Wu et al’.s study, the CBZ and nanocrystal CBZ groups increased the survival rate in the GBM mouse model compared to the PBS group.32 In the study of Bteich et al., CBZ nanoparticle groups increased the survival of the Orthotopic LN-229 GBM xenograft model of mice compared to the vehicle group.38
To evaluate the effect of drugs on the cognitive deficit of tumor growth, OFT, EPM, Rotarod, and passive avoidance learning tests were performed. TMZ and CBZ 1 could in part decrease GBM-induced anxiety-like behaviors in the EPM test. The imbalance in tumor animals which was determined by reducing the duration of stay on the rod in the rotarod test ameliorated with administration of CBZ1. In the shuttle box test, TMZ, CBZ1, and 2 were able to improve the memory impairment caused by the tumor. In line with these data, we found in our previous study that rats with C6-induced GBM showed anxiety-like behavior, imbalance, and memory impairment which in part ameliorated by TMZ.27 Some investigations have shown that TMZ can increase anxiety-like behaviors, depression, and hippocampal-dependent memory impairment.39 Inconsistency in results may be due to a lack of tumor induction and focus on behavioral effects in previous studies on TMZ. The duration and dosage of drug administration varied in these studies. Lee et al. found that the increase in inflammatory proteins in GBM leads to anxiety-like behavior and cognitive impairment, neuronal damage, and synaptic function impairment.40
Tumor formation in the brain can occur in association with oxidative stress caused by an imbalance between free radicals and antioxidants.41 We investigated the effect of TMZ and different doses of CBZ on GPx, SOD, and TAC as antioxidant indices, and MDA as an oxidant factor in the GBM. The results revealed a decrease in antioxidant indices and an increase in MDA in brain tissue of tumor groups. Tanriordi et al. reported a relationship between decreased GPx activity and high protein oxidation in human glioma tissue samples.42 Our findings indicated that all treatment groups, except the CBZ2 group, experienced an increase in GPx activity. Also, about SOD, except CBZ4, the other groups had a decrease in SOD. Kabza et al’.s experiment on rats with C6 cell-induced GBM showed that serum SOD level in the tumor group increased significantly compared to the control group and serum GPx level decreased.43 The results described by other researchers in human patients show great heterogeneity. In some studies, a lower level of SOD has been described in patients with brain tumors compared to controls.44,45 We also reported that TAC increased in the TMZ group and CBZ4 group and decreased in CBZ0.5, 1, and 2 groups. In an investigation on cancer, Tuzgen et al. reported that DNA oxidative damage increased in correlation with TAC reduction.46 Recent research has given insight into the formation of reactive oxygen species (ROS) by anticancer medications in cancer cells, which may affect cell death or treatment resistance.47,48 According to Kosaka et al., CBZ produces ROS by reducing Sestrin-3 expression, which is a preventing gene of ROS accumulation and oxidative DNA damage.49 MDA levels also decreased with CBZ 0.5, 1, and 2 groups. In other experiments Similar to the results of this study, it has been shown that treatment with higher doses of CBZ significantly increases MDA.9
In the current study, an increase in the levels of both IL-10 and TNF-α in GBM rats was seen. The CBZ4 group increased TNF-α level. The CBZ1 group increased the IL-10 level and decreased the TNF-α/IL-10 ratio, both of which may play a role in curing. IL-10 is a dual-function cytokine in many types of tumors. In a study by Moore et al, findings showing that mice with a genetic defect in TNF were immune to skin cancer provided evidence of TNF-dependent inflammation and cancer.50 The production of TNF-α by tumors is associated with a worse prognosis51 as TNF helps tumor progression by increasing tumor growth and invasion, the presence of leukocytes, angiogenesis, and facilitating epithelial to mesenchymal conversion.52 Also, Huettner et al. in the study of IL-10 protein expression, Found that expression levels were significantly increased by glioma malignancy.53
High systemic toxicity substantially restricts the use of CBZ in therapeutic settings54 Patients who were given CBZ experienced febrile neutropenia, neutropenic infection, diarrhea, and haematuria more frequently.54 Overdose with CBZ causes bone marrow suppression and gastrointestinal complications. In the present study, severe diarrhea and dehydration were probably one of the causes of death in the CBZ4 group, while this group dramatically decreased tumor volume and histopathological lesions. To prevent side effects, novel drug delivery systems such as liposomes can be used. CBZ nanocrystal-loaded liposome was used in the treatment of glioma, and in addition to reducing side effects, it exhibited a substantial suppressive impact on tumor proliferation.32
Conclusion
In conclusion, this report demonstrates the effectiveness of CBZ as a viable treatment option for GBM with protective effects such as increasing GPx activity, TAC level, and IL-10 level and reducing the inflammatory balance of TNFα/IL-10. In the current study, we have concluded that CBZ at a dose of 1 mg/kg exhibits a notable enhancement in the survival rate compared to other doses and even TMZ (as a positive control). This dose also had the best effect in improving the animal’s anxiety-like behaviors, coordination, and memory compared to other therapeutic doses of CBZ. However, higher doses (4 mg/kg) may reduce the survival rate to a great extent, which can be attributed to an increase in inflammation and gastrointestinal complications. These findings may provide a basis for clinical trials to find a new therapeutic approach for the management of GBM.
Study limitations
Due to financial constraints, we could not evaluate the signaling pathways. In addition, it was not possible to perform an MRI test in the center where the tests were performed.
Future perspective
The demonstrated activity of CBZ in GBM with innate or acquired resistance to TMZ, may make this agent a candidate for further clinical evaluation in a broader range of patient populations. In addition, by conducting these basic studies, it is possible to achieve a better understanding of the pathophysiology of the disease.
Supplementary Material
Acknowledgment
The authors would like to acknowledge the Kerman Neuroscience Research Center for their support of the study—also, many thanks to 2 anonymous reviewers whose insightful comments greatly improved the manuscript.
Contributor Information
Zahra Mohammadzadeh, Department of Physiology and Pharmacology, Afzalipour School of Medicine, Kerman University of Medical Sciences, Haft-Bagh Highway, P. O. Box 76169 13555, Kerman, Iran.
Mohammad Khaksari, Endocrinology and Metabolism Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Haft-Bagh Highway, P. O. Box 76169 13555, Kerman, Iran.
Mohammad Hadi Nematollahi, Physiology Research Center, Afzalipour School of Medicine, Kerman University of Medical Sciences, Haft-Bagh Highway, P. O. Box 76169 13555, Kerman, Iran.
Reza Kheirandish, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Imam Khomeni Highway, P. O. Box 76169 13439, Kerman, Iran.
Amirhossein Moslemizadeh, Department of Immunology, Tehran University of Medical Sciences, Dameshgh St., Vali-e Asr Ave., P. O. Box 14167 53955, Tehran, Iran.
Sina Delshad, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Imam Khomeni Highway, P. O. Box 76169 13439, Kerman, Iran.
Sanaz Faramarz, Department of Biochemistry, Kerman University of Medical Sciences, Haft-Bagh Highway, P. O. Box 76169 13555, Kerman, Iran.
Sara Sheibani Tezerji, Department of Behavioural and Molecular Neurobiology, Regensburg Center for Neuroscience, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Deutschland, Germany.
Mohammad Torkashvand, College of Engineering, University of Tehran, Dameshgh St., Vali-e Asr Ave., P. O. Box 14167 53955, Tehran, Iran.
Samira Shahba, Department of Biotechnology, School of Medicine, Semnan University of Medical Sciences, Bassij Blvd, P. O. Box 35147-99442, Semnan, Iran.
Hamideh Bashiri, Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Haft-Bagh Highway, P. O. Box 76169 13555, Kerman, Iran.
Author contributions
Hamideh Bashiri, and Mohammad Khaksari: Conceptualization; Zahra Mohammadzadeh, Amirhossein Moslemizadeh, Sanaz Faramarz, Samira Shahba, and Mohammad Torkashvand: Investigation, Methodology, Project administration; Hamideh Bashiri: Software; Reza Kheirandish: Visualization, performed the histopathological examination of the brain; Hamideh Bashiri and Mohammad Hadi Nematollahi: Data curation; Hamideh Bashiri, Writing – original draft – review and editing; Sina Delshad: review and editing; All authors read and approved the final manuscript.
Funding
Funding for this study was obtained from the Neuroscience Research Center of Kerman University of Medical Sciences (Code: 99000458).
Conflict of interest statement
There is no conflict of interest with other people or organizations that could inappropriately influence or bias the content of the paper.
Data availability
Data will be made available on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Data will be made available on reasonable request.