Figure 1
(A,B) Clonogenic survival assays of LN229 and LN18 glioblastoma cell lines after treatment with photon irradiation. (C,D) Clonogenic survival assays after comparing LN229 and LN18 cell lines after combined treatment with TMZ (C) or metformin (D). Error bars represent standard deviations. * shows a statistical significance (P <0.05). In LN229 enhanced cell kill was observed by TMZ and in LN18 cells lines metformin treatment resulted in increased cell toxicity compared to the control, as well as the combination of both agents itself. Combined bimodal and trimodal treatment results in superior cell toxicity.
Table 1
Sensitizer enhancement ratio (SER 20%) for LN229 and LN18 cells after treatment with metformin, temozolomide (TMZ) and combined treatment with 2Gy irradiation
| SER (20% survival) / 2 Gy | ||
|---|---|---|
| Cell line | LN18 | LN229 |
| 50 µM TMZ | 1.11 | 2.15 |
| 1mM Metformin | 0.67 | 0.74 |
| 20 mM Metformin | 3.13 | 0.61 |
| 50 µM TMZ + 20mM Metformin | 2.57 | 2.69 |
[i] SER values (ranging from 0.67–3.13) depending on cell line, chemotherapeutic agent and dose
Table 2
Cell cycle distribution into G1, S, and G2/M phase of LN18 and LN229 cells after various treatments. Measurements were performed after 72h. * shows a statistical significance (P <0.05) of the treatment compared to the control
| Ln18 24h | 72h | G1 (%) ± sth.dev. | S (%) ± sth.dev. | G2/M (%) ± sth.dev. |
|---|---|---|---|---|
| Ctrl | 74.5 ± 8 | 5.6 ± 0 | 19.9 ± 1 | |
| 50µM TMZ | 66.0 ± 8 | 9.5 ± 1 | 24.6 ± 3 | |
| 1mM Metformin | 71.0 ± 5 | 6.3 ± 1 | 22.7 ± 0 | |
| 20mM Metformin | 46.9 ± 3 | 9.3 ± 2 | 43.8 ± 2* | |
| 50µM TMZ + 1mM Metformin | 64.5 ± 0 | 8.6 ± 4 | 26.9 ± 4 | |
| 50µM TMZ + 20mM Metformin | 41.2 ± 0 | 19.4 ± 7 | 39.4 ± 2 | |
| 2Gy | 71.7 ± 1 | 7.5 ± 1 | 20.8 ± 4 | |
| 6Gy | 56.2 ± 5 | 8.0 ± 1 | 35.7 ± 1 | |
| 2Gy + 50µM TMZ | 62.0 ± 5 | 6.6 ± 5 | 31.4 ± 4 | |
| 2Gy + 1mM Metformin | 68.2 ± 7 | 10.3 ± 1 | 21.5 ± 2 | |
| 2 Gy + 20mM Metformin | 41.3 ± 0 | 16.1 ± 1 | 42.6 ± 4* | |
| 2Gy + 50µM TMZ + 1mM Metformin | 60.1 ± 7 | 8.6 ± 1 | 31.3 ± 0 | |
| 2Gy + 50µM TMZ + 20mM Metformin | 33.5 ± 2 | 20.0 ± 3 | 46.5 ± 4* | |
| 6Gy + 50µM TMZ | 47.1 ± 7 | 11.6 ± 3 | 41.3 ± 3* | |
| 6Gy + 1mM Metformin | 51.3 ± 2 | 10.4 ± 0 | 38.2 ± 3 | |
| 6Gy + 20mM Metformin | 36.1 ± 3 | 14.2 ± 0 | 49.7 ± 3* | |
| 6Gy + 50µM TMZ + 1mM Metformin | 46.3 ± 4 | 10.7 ± 1 | 42.9 ± 2* | |
| 6Gy + 50µM TMZ + 20mM Metformin | 27.9 ± 1 | 14.1 ± 3 | 58.0 ± 2* | |
| Ln229 24h | 72h | G1 (%) | S (%) | G2/M (%) |
| Ctrl | 86.0 ± 2 | 3.6 ± 2 | 10.4 ± 2 | |
| 50µM TMZ | 37.4 ± 3 | 8.1 ± 2 | 54.6 ± 5* | |
| 1mM Metformin | 87.4 ± 3 | 3.3 ± 1 | 9.3 ± 0 | |
| 20mM Metformin | 63.9 ± 10 | 14.9 ± 1 | 21.2 ± 9 | |
| 50µM TMZ + 1mM Metformin | 38.9 ± 0 | 6.1 ± 2 | 55.0 ± 4* | |
| 50µM TMZ + 20mM Metformin | 25.2 ± 1 | 25.4 ± 2 | 49.4 ± 2* | |
| 2Gy | 83.5 ± 5 | 3.3 ± 2 | 13.2 ± 5 | |
| 6Gy | 67.1 ± 2 | 6.2 ± 1 | 26.7 ± 1 | |
| 2Gy + 50µM TMZ | 44.4 ± 1 | 6.9 ± 1 | 48.7 ± 4* | |
| 2Gy + 1mM Metformin | 82.3 ± 1 | 3.5 ± 1 | 14.2 ± 2 | |
| 2 Gy + 20mM Metformin | 55.6 ± 2 | 11.8 ± 2 | 32.6 ± 1 * | |
| 2Gy + 50µM TMZ + 1mM Metformin | 46.0 ± 1 | 7.9 ± 1 | 46.1 ± 0* | |
| 2Gy + 50µM TMZ + 20mM Metformin | 32.3 ± 3 | 28.9 ± 7 | 38.8 ± 5* | |
| 6Gy + 50µM TMZ | 41.6 ± 2 | 8.9 ± 3 | 49.5 ± 3 | |
| 6Gy + 1mM Metformin | 55.5 ± 11 | 8.8 ± 4 | 35.7 ± 23 | |
| 6Gy + 20mM Metformin | 56.5 ± 18 | 7.8 ± 6 | 35.7 ± 16 | |
| 6Gy + 50µM TMZ + 1mM Metformin | 41.9 ± 2 | 5.9 ± 2 | 52.2 ± 2* | |
| 6Gy + 50µM TMZ + 20mM Metformin | 18.4 ± 1 | 16.2 ± 3 | 65.4 ± 3* | |
Figure 2
(A) In LN229 pAMPK measurements were significantly increased after treatment with Metformin, 2 Gy and temozolomide and 6 Gy combined with Metformin compared to the control if not otherwise specified (* p<0.05). (B) No significant increase of pAMPK levels were observed in cell line LN18. Error bars represent standard deviation.