Anat Cell Biol.  2024 Mar;57(1):85-96. 10.5115/acb.23.159.

Inhibitory effect of temozolomide on apoptosis induction of cinnamaldehyde in human glioblastoma multiforme T98G cell line

• Abband H
• Dabirian S
• Jafari A
• Nasiri M
• Nasiri E

• KMID: 2554243
• DOI: http://doi.org/10.5115/acb.23.159

Abstract

Glioblastoma is the most common primary malignant brain tumor in adults. Temozolomide (TMZ) is an FDAapproved drug used to treat this type of cancer. Cinnamaldehyde (CIN) is a derivative of cinnamon extract and makes up 99% of it. The aim of this study was to investigate the in vitro combined effect of CIN and TMZ on human glioblastoma multiforme T98G cell line viability. In this study, we used 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tertazolium bromide (MTT) method to evaluate the extent of IC₅₀ , acridine orange, Giemsa and Hoechst staining to evaluate the manner of apoptosis and the Western blotting method to examine the expression change of apoptotic proteins. Our results show that TMZ has an inhibitory effect on CIN when both used in combination at concentrations of 300 and 100 μM (P<0.05) and has a cytotoxic effect when used alone at the same concentrations (P<0.05). The western blotting result showed that TMZ at concentrations of 2,000 and 1,000 μM significantly increased Bax expression and decreased Bcl2 expression (P<0.05), indicating that TMZ induced apoptosis through the mitochondrial pathway. However, CIN had no effect on Bax and Bcl2 expressions, thus causing apoptosis from another pathway. Also, the Bax:Bcl2 expression ratio at concentrations combined was lower than that for TMZ 1,000 μM and higher than that for CIN 150 and 100 μM (P<0.05), which confirms the inhibitory effect of TMZ on CIN. From the present study, we conclude that TMZ in combination with CIN has an inhibitory effect on increasing the cytotoxicity rate.

Keyword

Glioblastoma; T98G cell line; Cinnamaldehyde; Temozolomide; Apoptosis

Figure

• 

  Fig. 1 (A) Chemical structure of trans-cinnamaldehyde. (B) Chemical structure of temozolomide.

• 

  Fig. 2 Cell viability and IC50. 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tertazolium bromide (MTT) assay show the viability of T98G cells after exposure to increasing concentrations of cinnamaldehyde for 48 hours. Analysis of MTT assay results using the GraphPad Prism v.8 software (GraphPad). In these graphs, the x-axis shows the logarithm of the cinnamaldehyde concentration and the y-axis shows the cell viability rate in percentage. The mean of IC50 for cinnamaldehyde was estimated at 211.96±14.82 μM.

• 

  Fig. 3 (A) Comparison of cell cytotoxicity rate (mean±SD) in the combined groups of temozolomide (TMZ) and cinnamaldehyde (CIN) 300 μM (T+C300) with the sum of cytotoxicity at concentrations of TMZ and CIN 300 μM considered individually. ****P≤0.0001, ***P=0.004 and P=0.001 at concentrations of 50 and 25 μM, respectively. **P=0.01. (B) Comparison of cell viability rate (mean±SD) in the combined groups of TMZ with CIN 300 μM (T+C300) with that of the two groups of TMZ and CIN 300 μM. In this diagram, * shows the significance of the TMZ groups compared to the combined groups and # shows the significance of CIN 300 μM compared to the combined groups. ****P≤0.0001. ###P=0.008 and P=0.002, respectively, at concentrations of 50 and 25 μM. ##P=0.02, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

• 

  Fig. 4 (A) Comparison of cell cytotoxicity rate (mean±SD) in the combined groups of temozolomide (TMZ) and cinnamaldehyde (CIN) 150 μM (T+C150) with the sum of cytotoxicity at concentrations of TMZ and CIN 150 μM considered individually. (B) Comparison of cell viability rate (mean±SD) in the combined groups of TMZ and CIN 150 μM (T+C150) with that of the two groups of TMZ and CIN 150 μM. In this diagram, * shows the significance of the TMZ groups compared to the combined groups and # shows the significance of CIN 150 μM compared to the combined groups. ****P<0.0001 and ####P<0.0001.

• 

  Fig. 5 (A) Comparison of cell cytotoxicity rate (mean±SD) in the combined groups of temozolomide (TMZ) and cinnamaldehyde (CIN) 100 μM (T+C100) with the sum of cytotoxicity at concentrations of TMZ and CIN 100 μM considered individually. ****P≤0.0001 and ***at concentrations of TMZ 50 μM and 25 μM at P=0.002 and P=0.001, respectively. (B) Comparison of cell viability rate (mean±SD) in the combined groups of TMZ with CIN 100 μM (T+C100) with that in the two groups of TMZ and CIN 100 μM. In this diagram, * shows the significance of the TMZ groups compared to that of the combined groups and # shows the significance of CIN 100 μM compared to that of the combined groups. ****P≤0.0001 and ***at concentrations of TMZ 2,000, 50, and 25 μM at P=0.001, P=0.004, and P=0.008, respectively. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

• 

  Fig. 6 Acridine orange, Giemsa, and Hoechst staining; the scale bar is 100 μm. (A) Acridine orange staining to identify acidic vesicular organelles under the red filter. (B) Giemsa staining to evaluate the morphology of apoptotic cells. The arrow indicates apoptotic cells. In the group T1000+C150, the star represents the pyknotic cell and a number of apoptotic bodies around it. (C) Hoechst staining to investigate nuclear fragmentation in apoptotic cells. The arrow indicates apoptotic bodies.

• 

  Fig. 7 Altered Bax and Bcl2 expression and Bax:Bcl2 expression ratio in TG98 cell line in different groups. (A) Bax expression (21 kDa) in different groups. Bcl2 expression (26 kDa) in different groups. β-Actin was used as a loading control (45 kDa). (B, C) Densitometric comparison of the average expression of Bax and Bcl2. (B) ANOVA, tukey-hoc test. ****P≤0.0001, ***P=0.001, **P=0.01. Data are represented as mean±SD, n=4. (C) ANOVA, tukey-hoc test. ****P≤0.0001, ***P=0.005, **P=0.01. Data are represented as mean±SD, n=4. (D) Bax:Bcl2 expression ratio in treatment groups. ANOVA, tukey-hoc test. ****P≤0.0001, ***P=0.001, **P=0.003. Data are represented as mean±SD, n=4.

Reference

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