Combination hyperbaric oxygen and temozolomide therapy in c6 rat glioma model

Terapia combinada de oxigênio hiperbárico e temozomida no modelo C6 de glioma em ratos

Yaşar Dagıstan Ismail Karaca Erol Ruştu Bozkurt Engin Ozar Kaan Yagmurlu Akin Toklu Ayhan Bilir About the authors

Abstracts

PURPOSE: Temozolomide (TMZ) has anti-tumor activity in patients with malignant glioma. Hyperbaric oxygen (HBO) may enhance the efficacy of certain therapies that are limited because of the hypoxic tumor microenvironment. We examined the combined effects of TMZ-HBO in a rat glioma model. METHODS: After stereotactic injection of C6/LacZ rat glioma cells into the Wistar rats brain, the rats were randomly assigned to three treatment groups [group 1, control treatment; group 2, TMZ alone; group 3, a combination of TMZ and HBO]. Rats were sacrificed 18 days after treatment, and number of intra-/peri-tumoral vessels, microendothelial proliferations, immunohistochemistry and necrotic area were evaluated. RESULTS: Tumoral tissue was stained only sparsely with GFAP. Temozolomide treatment was significantly decreased in tumor tissue intratumoral vessel number / total tumor area level. The level of Ki67 was significantly decreased in the tumor tissue of the group 3. Additionally, the total necrotic area / total tumor volume (%) was decreased significantly in tumor tissue of the group 3 rats compared to group1 and 2. CONCLUSION: The combination of hyperbaric oxygen with temozolomide produced an important reduction in glioma growth and effective approach to the treatment of glioblastoma.

Glioma; Glioblastoma; Temozolomide; Hyperbaric Oxygenation; Rats


OBJETIVO: A temozolomida (TMZ) tem atividade anti-tumoral em pacientes com glioma maligno. Oxigênio hiperbárico (HBO) pode aumentar a eficácia de terapias que são limitadas devido a um microambiente do tumor hipóxico. Foram examinados os efeitos combinados de TMZ-HBO em um modelo de glioma em rato. MÉTODOS: Após a injeção estereotáxica de células de glioma de rato C6/LacZ no cérebro de ratos Wistar, os ratos foram distribuídos aleatoriamente em três grupos de tratamento: Grupo 1: tratamento de controle. Grupo 2: TMZ sozinho. Grupo 3: uma combinação de TMZ e HBO. Os ratos foram sacrificados 18 dias após o tratamento. Foram avaliados o número de vasos intra-/peri-tumoral, proliferação microendotelial, imunohistoquímica e área necrótica . RESULTADOS: O tecido tumoral foi marcado apenas esparsamente com GFAP. O tratamento com temozolomida diminuiu significativamente o tecido intratumoral e a área total do tumor. O nível de Ki67 foi significativamente diminuído no tecido do tumor do grupo 3. Além disso, a superfície necrótica total / volume total do tumor (%) diminuiu significativamente no tecido do tumor do grupo 3 em comparação com grupo 1 e 2. CONCLUSÃO: A combinação de oxigênio hiperbárico com temozolomida produziu uma redução importante no crescimento do glioma podendo ser abordagem eficaz para o tratamento do glioblastoma.

Glioma; Glioblastoma; Temozolomida.Oxigenação Hiperbárica; Ratos


5 – ORIGINAL ARTICLE

EXPERIMENTAL NEUROLOGY

Combination hyperbaric oxygen and temozolomide therapy in c6 rat glioma model1 1 Research performed at Postgraduate Program in Neurosurgery, First Neurosurgery Clinic in Bakirkoy Mental Diseases Hospital, Bakirkoy, Istanbul.

Terapia combinada de oxigênio hiperbárico e temozomida no modelo C6 de glioma em ratos

Yaşar DagıstanI; Ismail KaracaII; Erol Ruştu BozkurtIII; Engin OzarII; Kaan YagmurluII; Akin TokluIV; Ayhan BilirV

IMD, Specialist in Neurosurgery, Department of Neurosurgery, Izzet Baysal Hospital, Bolu, Turkey. Main author. Conception, design, intellectual and scientific content of the study; acquisition, analysis and interpretation of data; manuscript writing, critical revision

IIMD, Specialist in Neurosurgery, 1st Neurosurgery Clinic in Bakirkoy Mental Diseases Hospital, Bakirkoy, Istanbul, Turkey. Acquisition and interpretation of data, involved in technical procedures

IIIAssociate Professor, Department of Pathology, Istanbul Education and Research Hospital, Abdurrahman Nafiz Gurman Street Kocamustafapasa Fatih, Istanbul, Turkey. Scientific and intellectual content of the study and interpretation of data

IVMD, Professor, Department of Underwater and Hyperbaric Medicine, Istanbul University Istanbul, Turkey. Scientific and intellectual content of the study

VProfessor, Department of Histology and Embryology, Institute of Oncology, Faculty of Medicine, Istanbul University, Turkey. Supervised all phases of the study and interpretation of data

Correspondence

ABSTRACT

PURPOSE: Temozolomide (TMZ) has anti-tumor activity in patients with malignant glioma. Hyperbaric oxygen (HBO) may enhance the efficacy of certain therapies that are limited because of the hypoxic tumor microenvironment. We examined the combined effects of TMZ–HBO in a rat glioma model.

METHODS: After stereotactic injection of C6/LacZ rat glioma cells into the Wistar rats brain, the rats were randomly assigned to three treatment groups [group 1, control treatment; group 2, TMZ alone; group 3, a combination of TMZ and HBO]. Rats were sacrificed 18 days after treatment, and number of intra-/peri-tumoral vessels, microendothelial proliferations, immunohistochemistry and necrotic area were evaluated.

RESULTS: Tumoral tissue was stained only sparsely with GFAP. Temozolomide treatment was significantly decreased in tumor tissue intratumoral vessel number / total tumor area level. The level of Ki67 was significantly decreased in the tumor tissue of the group 3. Additionally, the total necrotic area / total tumor volume (%) was decreased significantly in tumor tissue of the group 3 rats compared to group1 and 2.

CONCLUSION: The combination of hyperbaric oxygen with temozolomide produced an important reduction in glioma growth and effective approach to the treatment of glioblastoma.

Key words: Glioma. Glioblastoma. Temozolomide. Hyperbaric Oxygenation. Rats.

RESUMO

OBJETIVO: A temozolomida (TMZ) tem atividade anti-tumoral em pacientes com glioma maligno. Oxigênio hiperbárico (HBO) pode aumentar a eficácia de terapias que são limitadas devido a um microambiente do tumor hipóxico. Foram examinados os efeitos combinados de TMZ-HBO em um modelo de glioma em rato.

MÉTODOS: Após a injeção estereotáxica de células de glioma de rato C6/LacZ no cérebro de ratos Wistar, os ratos foram distribuídos aleatoriamente em três grupos de tratamento: Grupo 1: tratamento de controle. Grupo 2: TMZ sozinho. Grupo 3: uma combinação de TMZ e HBO. Os ratos foram sacrificados 18 dias após o tratamento. Foram avaliados o número de vasos intra-/peri-tumoral, proliferação microendotelial, imunohistoquímica e área necrótica .

RESULTADOS: O tecido tumoral foi marcado apenas esparsamente com GFAP. O tratamento com temozolomida diminuiu significativamente o tecido intratumoral e a área total do tumor. O nível de Ki67 foi significativamente diminuído no tecido do tumor do grupo 3. Além disso, a superfície necrótica total / volume total do tumor (%) diminuiu significativamente no tecido do tumor do grupo 3 em comparação com grupo 1 e 2.

CONCLUSÃO: A combinação de oxigênio hiperbárico com temozolomida produziu uma redução importante no crescimento do glioma podendo ser abordagem eficaz para o tratamento do glioblastoma.

Descritores: Glioma. Glioblastoma. Temozolomida.Oxigenação Hiperbárica. Ratos.

Introduction

Glioblastomas are the most frequent types of intracranial tumors, and locally infiltrating, aggressive and hypervascularized tumors with a median survival rate of less than one year. The current therapy is cytoreductive surgery followed by radiotherapy, with a more limited role for adjuvant chemotherapy. Glioblastomas are dependent on angiogenesis, as proliferation of microvascular endothelial cells1–4.

Temozolomide (TMZ), in many cancers including gliomas, has broad-spectrum anti-tumor activity5. Temozolomide penetrated excellent into the brain tissue is agent an oral imidazotetrazinone methylating. Also, inhibition of protein kinase C has been implicated as a possible mechanism of action6.

In high-grade gliomas have been found to be areas hypoxic. In particular, the C6 line has demonstrated tumor hypoxia when used in experimental models. Hypoxia may decrease the effectiveness of adjuvant therapy7,8. Hyperbaric oxygen treatment (HBO), which in turn will improve the radiation response in solid tumors, including gliomas, will enhance the oxygen tension in tumors9-11.

In the present study, we evaluated the effects of the combination treatment with temozolomide and hyperbaric oxygen in a rat brain tumor model using C6 glioma cell line.

Methods

Glioma cell lines and culture condition

The rat glioblastoma cell line C6 was obtained from ATCC (Rockville, USA) and serial passages were made at the Histology and Embryology Department of Istanbul Medical School in modified Eagle medium of Dulbecco, which contained 15% heat inactivated fetal calf serum, 0.2 mM glutamine, 50 mg/ml neomycin, and 100 mg/ml streptomycin. Culture flasks were kept in electronic incubator (Sanyo) under humidified atmosphere with 5% CO2 at 37°C.

Animals and implantation procedure

Cells were harvested via 2 ml trypsin- EDTA solution C (Biological Industries, Israel) and centrifuged after the addition of 1.5 cc of F12 medium and fetal calf serum mixture at 1x103 rpm for three minutes. After removing the supernatant, pellet was resuspended with 2 cc of medium. Cell suspension was concentrated; so that 5 microliters of any injection volume would contain 5x105 cells, and then placed in a microcentrifuge tube, and kept in a water-ice mixture environment during the whole implantation procedure, which always lasted less than 2 hours.

Male Wistar rats, weighing between 260 and 300 grams, were anesthetized by intramuscular injection of a solution containing 42.8 mg/ml of 10% ketamine and 8.6 mg/ml of 2% xylazine (this dosage did not exceed 0.7 mg/kg of body weight in total). The rats were secured into a stereotaxis apparatus (Trent Welles Inc., South Gate, Ca, USA). After antiseptic preparation of the scalp with betadine, the skin was incised on the mid-sagittal line for 1 cm. A burrhole (1.5 mm wide) was made on the right side 3 mm lateral to midline, and 2 mm proximal to the bregma using a dental drill. Injection of the C6 glioma cell suspension was made using a Hamilton syringe with a 27 gauge needle, which was fixed to the manipulation arm of the stereotaxis apparatus and advanced to the center of the right caudate nucleus which was 5 mm deep to the surface of the brain. A total of 5 ml solution was injected over a 5 minute of time in 0.5 μl aliquo. The needle was kept in same place for an additional 3 minutes and then withdrawn very gently over two minutes. The operation area was irrigated with saline and the burrhole was covered with bone-wax. The scalp was sutured using 5-0 vicryl.

Therapy for established C6 rat glioma growing in the brain of rats

All animals underwent implantation of guide screws and after one week they were divided into three groups. For all animals (21 rats), 5x105 C6/LacZ glioma cells were injected and the animals were sacrificed 18 days later following specific treatment. Animals in group 1 (Control, seven rats) were treated by saline (23 ml/kg/day) p.o. daily from 11th to 15th day. Animals in group 2 (TMZ treated, n=7) were treated by temozolomide, which was administered at a dose of 150 mg/m2/day by gastric catheter (6°F ) doses of temozolomide (Temodar, Scherling-Plough) on days 11–15 after tumor implantation. Animals in group 3 (TMZ and HBO combination treated n=7) were treated by the combination of hyperbaric oxygen (2.0 atm, 7–12 exposures, seven days) and temozolomide.

Histopathological evaluation

Brains fixed in 10% buffered formalin were dissected in 10 mm coronal slices via taking inoculation hole as origin. All samples were photographed. Glass slides of 4 microns thickness were prepared. Histology sections from these were stained with conventional haematoxylin-eosin technique for routine analysis. Number of intra-/ peri-tumoral vessels and glomeruloid microendothelial proliferations were counted in five high power field (HPF) area (0.238 mm2); according to these data, two indexes were calculated, i1and i2 respectively. i1 = Intratumoral vessel number / total tumor area, i2 = Peritumoral vessel number / intratumoral vessel number.

For immunohistochemistry

Primary antibodies to glial fibrillary acidic protein (GFAP), and Ki-67 (clone SP6, Lab Vision, Fremont, CA, USA), were used. Immunoreactivity was visualized by a biotin-streptavidin-peroxidase kit (Nichirei, Tokyo, Japan) and 3,3′-diaminobenzidine solution. To assess Ki-67 labeling index, more than 1000 neoplastic cells from each histologic type of brain tumor were counted.

Necrotic area quantification

Hematoxylin and eosin (H&E) sections (4 micron thick, paraffin embedded) of each tumor were analyzed by a pathologist. For tumor necrotic area quantification (μm²), images were captured using a digital camera (Olympus Camedia 7070) and analyzed using SPOT version 3.2.4 software. (Diagnostic Instruments, Inc.). Total necrotic area / total tumor volume was given as a percentage (%).

Statistics

Data are expressed as mean ± S.D. and analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by post-hoc for multiple comparisons (Tukey's test) using an Instat software package (GraphPad Software, San Diego, CA, USA). Differences between mean values were considered significant when p<0.05.

Results

The i1, i2, Ki67 and necrotic area values for the different groups are shown in Table 1.

Histopathologically, all tumors contained pleomorphic cellular elements, mostly with vesicular nuclei and moderate amounts of eosinophilic cytoplasm; mitotic figures and atypical nuclei were medium, as well as necrosis with pseudopalisading. Tumoral tissue was stained with GFAP (Figure 1a).

The i1 levels in the group 1 and 3 were increased (Figure 1b); however, Temozolomide treatment was significantly decreased in tumor tissue i1 level (p<0.05) (Figure 1c). For i2 level in group 2 and 3 was found to be not different than that of the control group.

The level of Ki67 was significantly increased in the tumor tissue of the group 1 (p<0.001) compared to the group 2 and 3 (respectively p<0.05, p<0.05) (Figure 1d). Additionally, the level of Ki67 was significantly decreased in the tumor tissue of the group 3 (p<0.001) compared to the group 2 (p<0.05) (Figure 1e).

The total necrotic area / total tumor volume (%) was decreased significantly in tumor tissue of the group 3 rats (Figure 1f) compared to group1 and 2 (Figure 1g) (p<0.05, p<0.05, respectively).

Discussion

Data obtained from the rat glioma model of this study indicate that the combination of TMZ and HBO significantly inhibits cell proliferation, angiogenesis and induce the stimulation of apoptosis compared with the effect of TMZ alone.

TMZ suggests capability as an effective chemotherapeutic agent for the treatment of patients with malignant glioma12. TMZ is quickly absorbed after oral administration and is cleaved in vivo to monoethyl triazenoimidazole carboxamide, a reactive DNA methylating species13. TMZ has excellent central nervous system entry, and reaches the brain in therapeutical concentrations14. Son et al.15 found that treatment with TMZ alone significantly decreased the number of proliferating cell nuclear antigen-positive proliferating cells and increased the number of TUNEL-positive apoptotic cells. Kim et al.16 demonstrated that temozolomide inhibited the growth of tumors and was accompanied by a reduction in tumor cell proliferation and induction of apoptosis. In the present study, TMZ treatment was significantly decreased in tumor tissue intratumoral vessel number. This result is similar to the reports by other authors that temozolomide inhibits angiogenesis.

Hypoxia in tumor tissue restricts the efficacy of treatment. Malignant cells in hypoxic fields are exposed to lower drug concentrations because of the limited access of parenterally administered drugs17. Additionally, these cells in hypoxic regions are less susceptible to radiotherapy, which target rapidly proliferating cells. Furthermore, hypoxia directly influences the expression of numerous gene products that are involved in angiogenesis, apoptosis, and glycolysis17. Tumor hypoxia in human malignant glioma has been studied by direct and indirect measurements. In direct pO2 measurement, Kayama et al.18 found that the intratumoural pO2 value was significantly lower than that of the surrounding brain. Hypoxia is suggested to contribute to the resistance of gliomas to radiosurgery, radiation therapy, and chemotherapeutic agents such as temozolomide19. Kohshi et al.20 in study multivariate analysis a small series revealed that combination with HBO was a good predictive prognostic factor for survival. Radiotherapy after HBO exposure is one of the initial treatment options for patients with malignant gliomas. Stuhr et al.21 shown that repeated HBO induces an anti-angiogenic effect in the dimethylbenzanthracene induced tumors. The Ki-67 protein is a cellular marker for proliferation22. Ki-67 immunostaining of a brain tumor associated with a high proliferative rate. In our study, the level of Ki67 was significantly decreased in the tumor tissue of the group 3 compared to the group 2. Additionally, the total necrotic area / total tumor volume (%) was decreased significantly in tumor tissue of the group 3 rats compared to group1 and 2.

Conclusions

The combination of hyperbaric oxygen with temozolomide produced an important reduction in glioma growth and effective approach to the treatment of glioblastoma. The treatment demonstrated an inhibition to rat glioma tumors, a significant decrease in cell proliferation and an increase in apoptosis.

Received: January 11, 2012

Review: March 14, 2012

Accepted: April 12, 2012

Conflict of interest: none

Financial source: none

  • 1. Benítez JA, Domínguez-Monzón G, Segovia J. Conventional and gene therapy strategies for the treatment of brain tumors. Curr Med Chem. 2008;15(8):729-42.
  • 2. Attenello FJ, Mukherjee D, Datoo G, McGirt MJ, Bohan E, Weingart JD, Olivi A, Quinones-Hinojosa A, Brem H. Use of Gliadel (BCNU) wafer in the surgical treatment of malignant glioma: a 10-year institutional experience. Ann Surg Oncol. 2008;15(10):2887-93.
  • 3. Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med. 2008;359(5):492-507.
  • 4. Combs SE, Wagner J, Bischof M, Welzel T, Edler L, Rausch R, Wagner F, Zabel-du Bois A, Debus J, Schulz-Ertner D. Radiochemotherapy in patients with primary glioblastoma comparing two temozolomide dose regimens. Int J Radiat Oncol Biol Phys. 2008;71(4):999-1005.
  • 5. Chang SM, Lamborn KR, Malec M, Larson D, Wara W, Sneed P, Rabbitt J, Page M, Nicholas MK, Prados MD. Phase II study of temozolomide and thalidomide with radiation therapy for newly diagnosed glioblastoma multiforme. Int J Radiat Oncol Biol Phys. 2004;60(2):353-7.
  • 6. Tentori L, Leonetti C, Aquino A. Temozolomide reduces the metastatic potential of Lewis lung carcinoma (3LL) in mice: role of alpha-6 integrin phosphorylation. Eur J Cancer. 1995;31A(5):746-54.
  • 7. Khan N, Li H, Hou H, Lariviere JP, Gladstone DJ, Demidenko E, Swartz HM. Tissue pO2 of orthotopic 9L and C6 gliomas and tumor-specific response to radiotherapy and hyperoxygenation. Int J Radiat Oncol Biol Phys. 2009;73(3):878-85.
  • 8. Sheehan J, Sherman J, Cifarelli C, Jagannathan J, Dassoulas K, Olson C, Rainey J, Han S. Effect of trans sodium crocetinate on brain tumor oxygenation. Laboratory investigation. J Neurosurg. 2009;111(2):226-9.
  • 9. Chang CH. Hyperbaric oxygen and radiation therapy in the management of glioblastoma. Natl Cancer Inst Monogr. 1977;46:163-9.
  • 10. Dowling S, Fischer JJ, Rockwell S. Fluosol and hyperbaric oxygen as an adjunct to radiation therapy in the treatment of malignant gliomas: a pilot study. Biomater Artif Cells Immobilization Biotechnol. 1992;20(2-4):903-5.
  • 11. Al-Waili NS, Butler GJ, Beale J, Hamilton RW, Lee BY, Lucas P. Hyperbaric oxygen and malignancies: a potential role in radiotherapy, chemotherapy, tumor surgery and phototherapy. Med Sci Monit. 2005;11(9):RA279-89.
  • 12. Chang SM, Lamborn KR, Malec M, Larson D, Wara W, Sneed P, Rabbitt J, Page M, Nicholas MK, Prados MD. Phase II study of temozolomide and thalidomide with radiation therapy for newly diagnosed glioblastoma multiforme. Int J Radiat Oncol Biol Phys. 2004;60(2):353-7.
  • 13. Payne MJ, Pratap SE, Middleton MR. Temozolamide in the treatment of solid tumours: current results and rationale for dosing/scheduling. Crit Rev Oncol Hematol. 2005;53(3):24152.
  • 14. Plowman J, Waud WR, Koutsoukos AD, Rubinstein LV, Moore TD, Grever MR. Preclinical antitumor activity of temozolomide in mice: efficacy againsthuman brain tumor xenografts and synergism with 1, 3-bis (2-chloroethyl)-1-nitrosourea. Cancer Res. 1994;54(14):3793-9.
  • 15. Son MJ, Kim JS, Kim MH, Song HS, Kim JT, Kim H, Shin T, Jeon HJ, Lee DS, Park SY, Kim YJ, Kim JH, Nam DH. Combination treatment with temozolomide and thalidomide inhibits tumor growth and angiogenesis in an orthotopic glioma model. Int J Oncol. 2006;28(1):53-9.
  • 16. Kang SG, Kim JS, Park K, Kim JS, Groves MD, Nam DH. Combination celecoxib and temozolomide in C6 rat glioma orthotopic model. Oncol Rep. 2006;15(1):7-13.
  • 17. Daruwalla J, Christophi C. Hyperbaric oxygen therapy for malignancy: a review.  World J Surg. 2006;30(12):2112-31.
  • 18. Kayama T, Yoshimoto T, Fujimoto S, Sakurai Y. Intratumoral oxygen pressure in malignant brain tumor. J Neurosurg. 1991;74(1):55-9.
  • 19. Spence AM, Muzi M, Swanson KR, O'Sullivan F, Rockhill JK, Rajendran JG, Adamsen TC, Link JM, Swanson PE, Yagle KJ, Rostomily RC, Silbergeld DL, Krohn KA. Regional hypoxia in glioblastoma multiforme quantified with [18F] fluoromisonidazole positron emission tomography before radiotherapy: correlation with time to progression and survival. Clin Cancer Res. 2008;14(9):2623-30.
  • 20. Kohshi K, Kinoshita Y, Imada H, Kunugita N, Abe H, Terashima H, Tokui N, Uemura S. Effects of radiotherapy after hyperbaric oxygenation on malignant gliomas. Br J Cancer. 1999;80(1-2):236-41.
  • 21. Stuhr LB, Raa A, Řyan AM, Kalland KH, Sakariassen PO, Petersen K, Bjerkvig R, Reed RK. Hyperoxia retards growth and induces apoptosis, changes in vascular density and gene expression in transplanted gliomas in nude rats. J Neurooncology. 2007;85(2):191-202.
  • 22. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182 (3):31122.

  • Correspondence:
    Yasar Dagıstan
    Department of Neurosurgery, Izzet Baysal Hospital, Bolu, Turkey
    Phone: (+90)374 2703879
  • 1
    Research performed at Postgraduate Program in Neurosurgery, First Neurosurgery Clinic in Bakirkoy Mental Diseases Hospital, Bakirkoy, Istanbul.

Publication Dates

  • Publication in this collection
    04 June 2012
  • Date of issue
    June 2012

History

  • Received
    11 Jan 2012
  • Accepted
    12 Apr 2012
  • Reviewed
    14 Mar 2012
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