Examination of PDK1/AKT/mTOR transcription and exosomal mRNA levels in human glioblastoma cell line treated with a combination of temozolomide and hesperidin

Abstract

The most malignant type of tumor in the brain is high-grade gliomas. Glioblastoma (GB), a grade 4 glioma, has the lowest 5-year survival rate and is associated with poor prognosis. An important signaling pathway involved in the pathogenesis of GB is the mammalian target of rapamycin (mTOR). Therefore, our study aimed to investigate how exosomes obtained from GB cells applied with different doses of hesperidin (HSP) affect miR- 9 and change the PDK1/AKT/mTOR pathway. For this purpose, T98G cells were treated with different doses (5, 10, 25, and 50 µg/mL) of HSP in combination with temozolomide (TMZ- 10 µg/mL). At the end of 24 h, cell viability, flow cytometry, and biochemical tests were performed. Additionally, exosomes were isolated from cells belonging to the control, TMZ, and high-concentration TMZ-HSP groups. miR- 9, PDK1, PTEN, AKT- 1, Bax, Bcl- 2, and Caspase 3 genes were expressed in both application groups and exosomes belonging to these groups. HSP was found to reduce the viability of GB cells significantly. The viability was significantly reduced, especially in the TMZ-HSP 50 µg/mL group. Depending on the dose, there was a significant increase in the LDH level and oxidative stress level. The apoptosis level was approximately 26% in the TMZ-HSP 50 µg/mL group. Along with all this, gene expressions changed at the exosomal level, and miR- 9 and miR- 146 levels increased. Similarly, it changed the expression of proteins related to the PDK1/AKT/mTOR signaling pathway at the exosomal level (p < 0.05). In conclusion, the TMZ-HSP combination showed anticancer effects in T98G cells, influenced exosome profiles, and appeared non-toxic and potentially beneficial to healthy cells, highlighting its potential therapeutic value.

Keywords: AKT; Glioblastoma; T98G; mTOR; miR- 9.

Fig. 1

SEM examined exosomes. The white arrow shows particle size. Pa: particle size in nm, P: particle, Pa R: particle radius, and Pb: particle angle (The Carl Zeiss Evo 40 SEM (Jena, Germany) was used to capture the images)

Fig. 2

A The MTT assay was utilized to determine cell viability (n = 12)—the consequences of TMZ, HSP, and TMZ combined on the viability of T98G cells. B Impact of TMZ and TMZ + HSP on LDH Activity in T98G Cells (n = 6). LDH activity has been determined in cells that were treated with TMZ (10 g/mL), HSP (5,10,25, and 50 g/mL), and combinations of TMZ + HSP for 24 h in 96-well plates. The average of three different experiments is used to represent the results. Meaningful statistically: *p < 0.05; **p < 0.01

Fig. 3

Live, early, late apoptotic, and necrotic cell populations were distributed in T98G cells after treatment with TMZ and HSP for 24 h. Apoptotic cells positive for Annexin V can be seen in the lower right quadrant, and dead cells positive for both annexin and PI can be seen in the upper right quadrant. Both stains show negative results in healthy cells (lower left quadrant). a Control, b TMZ 10 µg/mL, c HSP 50 µg/mL, d TMZ- HSP 10 µg/mL, e TMZ—HSP 25 µg/mL, and f TMZ- HSP 50 µg/mL. The symbols for statistical significance are *p < 0.05 and **p < 0.01

Fig. 4

A Effect of the combination of TMZ and HSP on T98G cell TAC (n = 6). B Effect of the combination of TMZ and HSP on T98G cell TOS (n = 6). C Effect of the combination of TMZ and HSP on T98G cell 8-OhDG (n = 6). Meaningful statistically: *p < 0.05; **p < 0.01. The symbols for statistical significance are *p < 0.05 and **p < 0.01

Fig. 5

Effect of the combination of TMZ and HSP on T98G cell mTOR (n = 6). Meaningful statistically: *p < 0.05; **p < 0.01. The symbols for statistical significance are *p < 0.05 and **p < 0.01

Fig. 6

A miRNA9 gene level of treatment groups. B miRNA- 9 gene level of exosomes obtained from treatment groups. C PDK- 1 gene level of treatment groups. D miRNA9 gene level of exosomes obtained from treatment groups. E AKT gene level of treatment groups. F AKT gene level of exosomes obtained from treatment groups. G PTEN gene level of treatment groups. H PTEN gene level of exosomes obtained from treatment groups. Experiments were performed in triplicate, and the results are given as average. Meaningful statistically: *p < 0.05; **p < 0.01

Fig. 6

A miRNA9 gene level of treatment groups. B miRNA- 9 gene level of exosomes obtained from treatment groups. C PDK- 1 gene level of treatment groups. D miRNA9 gene level of exosomes obtained from treatment groups. E AKT gene level of treatment groups. F AKT gene level of exosomes obtained from treatment groups. G PTEN gene level of treatment groups. H PTEN gene level of exosomes obtained from treatment groups. Experiments were performed in triplicate, and the results are given as average. Meaningful statistically: *p < 0.05; **p < 0.01

Fig. 7

A miRNA 146 - 5p gene level of treatment groups. B miRNA 146 - 5p gene level of exosomes obtained from treatment groups. Experiments were performed in triplicate, and the results are given as the average. Meaningful statistically: *p < 0.05; **p < 0.01

Fig. 8

A BAX gene level of treatment groups. B Bcl- 2 gene level of treatment groups. C Caspase 3 gene level of treatment groups. Experiments were performed in triplicate, and the results are given as average. Meaningful statistically: *p < 0.05; **p < 0.01

Fig. 9

A MTT, LDH, TAC, and TOS results of human dermal fibroblast cells. Cell viability decreased, and cytotoxicity increased in the Wound group compared to the control group. Cell viability increased in the groups treated with HSP compared to the wound group. B Wound healing figures of the experiment groups. Experiments were performed in triplicate, and the results are given as the average. Meaningful statistically: *p < 0.05; **p < 0.01; p < 0.05; ^##p < 0.01