Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jul 3;13(7):e0198627.
doi: 10.1371/journal.pone.0198627. eCollection 2018.

Combined acetyl-11-keto-β-boswellic Acid and Radiation Treatment Inhibited Glioblastoma Tumor Cells

Affiliations
Free PMC article

Combined acetyl-11-keto-β-boswellic Acid and Radiation Treatment Inhibited Glioblastoma Tumor Cells

Sefora Conti et al. PLoS One. .
Free PMC article

Abstract

Glioblastoma multiforme (GBM) is the most common and most aggressive subtype of malignant gliomas. The current standard of care for newly diagnosed GBM patients involves maximal surgical debulking, followed by radiation therapy and temozolomide chemotherapy. Despite the advances in GBM therapy, its outcome remains poor with a median survival of less than two years. This poor outcome is partly due to the ability of GBM tumors to acquire adaptive resistance to therapy and in particular to radiation. One of the mechanisms contributing to GBM tumor progression and resistance is an aberrant activation of NF-ĸB, a family of inducible transcription factors that play a pivotal role in regulation of many immune, inflammatory and carcinogenic responses. Acetyl-11-keto-β-boswellic acid (AKBA) is a pentacyclic terpenoid extracted from the gum Ayurvedic therapeutic plant Boswellia serrata. AKBA is anti-inflammatory agent that exhibits potent cytotoxic activities against various types of tumors including GBM. One of the mechanisms underlying AKBA anti-tumor activity is its ability to modulate the NF-ĸB signaling pathway. The present study investigated in vitro and in vivo the effect of combining AKBA with ionizing radiation in the treatment of GBM and assessed AKBA anti-tumor activity and radio-enhancing potential. The effect of AKBA and/or radiation on the survival of cultured glioblastoma cancer cells was evaluated by XTT assay. The mode of interaction of treatments tested was calculated using CalcuSyn software. Inducing of apoptosis following AKBA treatment was evaluated using flow cytometry. The effect of combined treatment on the expression of PARP protein was analysed by Western blot assay. Ectopic (subcutaneous) GBM model in nude mice was used for the evaluation of the effect of combined treatment on tumor growth. Immunohistochemical analysis of formalin-fixed paraffin-embedded tumor sections was used to assess treatment-related changes in Ki-67, CD31, p53, Bcl-2 and NF-ĸB-inhibitor IĸB-α. AKBA treatment was found to inhibit the survival of all four tested cell lines in a dose dependent manner. The combined treatment resulted in a more significant inhibitory effect compared to the effect of treatment with radiation alone. A synergistic effect was detected in some of the tested cell lines. Flow cytometric analysis with Annexin V-FITC/PI double staining of AKBA treated cells indicated induction of apoptosis. AKBA apoptotic activity was also confirmed by PARP cleavage detected by Western blot analysis. The combined treatment suppressed tumor growth in vivo compared to no treatment and each treatment alone. Immunohistochemical analysis showed anti-angiogenic and anti-proliferative activity of AKBA in vivo. It also demonstrated a decrease in p53 nuclear staining and in Bcl-2 staining and an increase in IĸB-α staining following AKBA treatment both alone and in combination with radiotherapy. In this study, we demonstrated that AKBA exerts potent anti-proliferative and apoptotic activity, and significantly inhibits both the survival of glioblastoma cells in vitro and the growth of tumors generated by these cells. Combination of AKBA with radiotherapy was found to inhibit factors which involved in cell death regulation, tumor progression and radioresistence, therefore it may serve as a novel approach for GBM patients.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Combined effect of AKBA and ionizing radiation on the survival of glioblastoma cells.
Cells were irradiated with a single dose and then exposed for 72 hrs to different concentrations of AKBA. The survival was evaluated by XTT colorimetric assay. The graph represents the average survival of treated cells relative to the survival of untreated control cells. The data are mean ± SE values from three individual experiments, each performed in triplicates. Statistical significance was determined by one way ANOVA test (*P < 0.05; **P < 0.01; ***P < 0.001). Normalized isobolograms indicated the mode of interaction between AKBA and ionizing radiation. Combination index for each variant of the combined treatment was calculated using Calcusyn software. The line is the line of additivity: interactions below the line are synergistic while interactions above the line are sub-additive.
Fig 2
Fig 2. Effect of AKBA on induction of apoptosis in A172 cells.
(A) A172 cells were treated with AKBA for 72 hrs and apoptosis was assessed using Annexin-V-FITC/PI double staining and flow cytometry. Double negative cells indicated the live cell population, Annexin-V-FITC positive cells indicated the early apoptotic population, double positive cells indicated late apoptotic population and PI positive cells indicated the necrotic population. (B) Cells were treated with 30 μM AKBA for 24 and 48 hrs. Cell lysates from A172 monolayers were subjected to Western blot analysis with mouse anti-human PARP and β-actin antibodies.
Fig 3
Fig 3. Effect of AKBA, radiation and combined treatment on tumor growth in an ectopic GBM model.
(A)Tumor growth monitored twice weekly for 7 weeks. Data are mean ± SE values from 6 individual mice in each group. (B) The tumor volumes measured at the end of the study, before the mice were sacrificed. The data are averages relative to control with SE (error bars) from 6 individual mice in each group. Differences in tumor volume after exposure to various treatments were determined using the one-way ANOVA test. *P < 0.05; **P< 0.01 vs. controls. (C) Representative tumors from corresponding treatments groups.
Fig 4
Fig 4. Effect of AKBA treatment on proliferation and intra-tumoral microvessel density in an ectopic GBM model.
Formalin-fixed paraffin-embedded sections were stained immunohistochemically for Ki-67 (proliferation marker) and CD31 (endothelial marker). Representative microphotographs from each treatment group are shown (original magnification x400 –Ki-67, x200- CD31).
Fig 5
Fig 5. Effect of AKBA treatment on p53 and Bcl-2 expression in an ectopic GBM model.
Formalin-fixed paraffin-embedded sections were stained immunohistochemically for p53 and Bcl-2. Representative microphotographs from each treatment group are shown (original magnification x200).
Fig 6
Fig 6. Effect of AKBA treatment on NF-ĸB-related protein expression in an ectopic GBM model.
Formalin-fixed paraffin-embedded sections were stained immuno-histochemically for IĸB-α. Representative microphotographs from each treatment group are shown (original magnification x200).

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007. WHO classification of tumours of the central nervous system. Acta Neuropathol. [Internet]. 2007 [cited 2014 Jul 11];114:97–109. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1929165&tool=pmcentrez&rendertype=abstract doi: 10.1007/s00401-007-0243-4 - DOI - PMC - PubMed
    1. Barani IJ, Larson DA. Radiation therapy of glioblastoma. Cancer Treat. Res. [Internet]. 2015. [cited 2015 Sep 12];163:49–73. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25468225 doi: 10.1007/978-3-319-12048-5_4 - DOI - PubMed
    1. Mealey J, Norrell HA, Ransohoff J, Wilson CB, Gehan EA, Strike TA. Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas. 1978;49:333–43. - PubMed
    1. Ammon HP,Salai Guggal—Boswellia serrata: from a herbal medicine to a non-redox inhibitor of leukotriene biosynthesis. Eur. J. Med. Res. [Internet]. 1996. [cited 2015 Oct 8];1:369–70. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9360935 - PubMed
    1. Gupta I, Parihar A, Malhotra P, Singh GB, Lüdtke R, Safayhi H, et al. Effects of Boswellia serrata gum resin in patients with ulcerative colitis. Eur. J. Med. Res. [Internet]. 1997. [cited 2015 Oct 8];2:37–43. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9049593 - PubMed

Publication types

MeSH terms

Grant support

The authors received no specific funding for this work.
Feedback