Effects of Akt-, PI3K-, and mTOR-inhibition on the migration of GBM cells
We recently reported that the migration activity of SNB19 cells is much less responsive to PI3K/mTOR inhibition than that of DK-MG cells [25]. In the present study, we tested whether Akt inhibition can reduce the motility of SNB19 cells. First, we examined the migration of individual DK-MG and SNB19 cells (Fig. 1 and Additional file 3: Movie S1) by time-lapse video-microscopy [25].
As evident from the motility diagrams shown in Fig. 1a and Additional file 3: Movie S1, which are statistically summarized in Fig. 1b, treatment with MK-2206 decreased the migration speed of DK-MG cells by ~ 30% with respect to controls cells. In contrast, MK-2206 did not affect the migration activity of SNB19 cells (Fig. 1b). Data on the effects of PI-103 have been previously reported [25]. The effects of both inhibitors on the migration of irradiated cells were similar to their effects on non-irradiated samples from both cell lines (data not shown).
Additionally to individual cell tracking, we analyzed cell movement by wound healing (scratch) assay. Representative data of the wound healing tests on both cell lines shown in Fig. 1d are statistically summarized in Fig. 1c. Similarly to the results of the single-cell tracking assay, drug-treated DK-MG cells closed the wounded area much slower (by ~ 65–70%) than control cell samples (Fig. 1c, d).
A further interesting finding of the wound healing test was that the Akt-inhibitor was less effective in SNB19 cells (Fig. 1c, d). Thus, treatment with MK-2206 alone only moderately (~ 20%) affected the wound closure rate of SNB19 cells. Besides this, compared to drug-free controls, treatment of SNB19 cells with PI-103 alone and with both drugs caused, respectively, an ~ 35 and ~ 45% decrease in the wound healing rate compared with the control cells (Fig. 1c), which is much less than the almost 70% inhibition observed in drug-treated DK-MG cells. Qualitatively similar data were obtained on irradiated cells (data not shown).
Effect of MK-2206 alone and in combination with PI-103 on colony-forming ability after irradiation
We further analyzed whether the Akt inhibition alone or in combination affected the radiation sensitivity of tumor cells. Figure 2 shows the normalized colony-forming curves of untreated and drug-treated cells plotted against the radiation dose, along with the best fit curves of the LQ model (Eq. 1) to the survival data. The plating efficiencies (PE) of control cell samples, as well as the values obtained with the LQ model, including the colony-forming fraction at 2 Gy (CF2), the radiation exposure dose required to reduce colony-forming ability by 10% (D10) and the growth inhibition factor (IF10) are summarized in Additional file 2: Table S3.
As seen in Fig. 2 and in accordance with our previous results [24], PI-103 alone acts as a potent radiosensitizer in both cell lines (Fig. 2, curves 2 in parts A and B). In contrast, MK-2206 given alone had little - if any - effect on the radiation sensitivity of DK-MG cells (curve 3, Fig. 2a) and, unexpectedly, it significantly increased the radiation resistance of SNB19 cells (curve 3, Fig. 2b). Upon combined drug treatment, MK-2206 only slightly enhanced the radiosensitizing effect of PI-103 in DK-MG cells (Fig. 2a, curve 4), without affecting the PI-103-mediated radiosensitization in SNB19 cells (Fig. 2b, curve 4).
Effects of MK-2206, PI-103 and/or irradiation on cell cycle
To find the molecular basis and to identify the mechanisms underlying the absence of the radiosensitizing effect of the Akt inhibitor MK-2206 in DK-MG cells (Fig. 2a) or the radioresistance in respective SNB19 cells (Fig. 2b) the attempts were focused on the inhibitor’s possible impact on cell cycle progression. The data for both cell lines are summarized in Fig. 3. The large fractions of cells in the S- and G2/M-phases (> 70%) in control non-irradiated probes (Fig. 3) show that the cells were growing exponentially at the start of experiments. As expected, 30 min post-IR, non-irradiated and irradiated cells (within the respective cell line) exhibited similar cell cycle phase distribution (Fig. 3). However, as shown previously [24], prolonged incubation with PI-103 caused an increase of G1 phase cells from about 40% to ~ 60%. In contrast, upon a long-term incubation with MK-2206, the cell cycle phase distributions in both cell lines were nearly the same as in respective non-treated controls (Fig. 3). Combined prolonged drug treatment caused a strong G2/M arrest in irradiated samples of both cell lines similar to that observed after treatment with PI-103 alone and IR.
The cell cycle impairment caused by PI-103 alone and in combination shown above (Fig. 3) led us to determine the expression levels of several cell-cycle regulating factors such as cyclin dependent kinases (Cdk1, Cdk4), and pRb by Western blotting. As shown in the Additional file 4: Figure S1, the expressions of Cdk1 and pRb decreased although to a different extent in both GBM lines after prolonged exposure to PI-103 alone or in combination, especially in non-irradiated cells. In contrast, the levels of cell cycle-related proteins were mostly unchanged in non-irradiated cells treated with MK-2206 alone.
To sum up, long-term incubation with PI-103 caused a significant G1 arrest in both cell lines, but combined with IR it produced a strong G2/M arrest. In contrast, treatment with MK-2206 alone had no influence on the cell cycle. In both cell lines, combined treatment with both drugs and IR caused a profound G2/M arrest 24 h post-IR, similar to that induced by PI-103 alone and IR.
Effects of MK-2206 and PI-103 and/or radiation on late-stage apoptosis and autophagy
To further dissect the mechanisms underlying the radiation survival of GBM cells after single Akt- and combined PI3K/Akt/mTOR-inhibition illustrated in Fig. 2, we also analyzed cleaved PARP, a well-known marker of apoptosis. As seen in Additional file 4: Figure S2, DK-MG cells treated with PI-103 alone or in combination with MK-2206 exhibited negligible levels of cleaved PARP, independent of IR, whereas no any cleaved PARP was detected in SNB19 samples. Likewise, the Akt-inhibitor did not induce any measurable amounts of cleaved PARP. In addition, the apoptosis rate was evaluated by the sub-G1 fraction. As seen in Fig. 4, inhibitor-treated and irradiated DK-MG samples contained much larger fractions of apoptotic cells and debris than corresponding SNB19 cell samples, especially 24 h after IR.
Because the Akt/mTOR pathway is recognized as a major pathway regulating autophagy [16], we also studied the role of autophagy in the development of radiation resistance in MK-2206-treated cells, especially in case of the SNB19 cell line. To this end, we detected the autophagosomal membrane-bound LC3B protein along with the expression of the p62/sequestome protein, a pleiotropic protein that is consumed during autophagy [31]. As seen in the Additional file 4: Figure S3, IR increased the expression of LC3B-I and LC3B-II proteins in both cell lines. This result is corroborated by earlier findings that radiation enhances autophagy in tumor cells (for review, see [32]). We also found that the dual PI3K- and mTOR-inhibitor PI-103 alone and especially in combination with MK-2206 strongly induced autophagy, as evident from the increased levels of LC3B-II protein (Additional file 4: Figure S3). However, the ratio between LC3B-II and LC3B-I was lower in irradiated cells compared to non-irradiated. Furthermore, the enhanced autophagy in drug-treated samples, detected by LC3B-II expression, was also corroborated by the strong depletion of p62 protein, another marker of autophagy (Additional file 4: Figure S3). These findings agree well with the results of Fan et al. (2017) who found that dual inhibition of PI3K and mTOR promotes survival of glioma cells by inducing cytoprotective autophagy [33]. Regarding MK-2206, the extent of drug-induced autophagy measured by LC3B-II expression was lower than that induced by PI-103. Likewise, when assessed by p62 expression, MK-2206 alone induced autophagy to a much lesser extent than PI-103. The highest extent of autophagy was observed in samples treated with both inhibitors simultaneously. Interestingly, the background expression of p62 in DK-MG cell line was much lower than in SNB19 cells. The difference may reside in the wild type status of p53 in DK-MG cells because functional p53 is known to inhibit autophagy [34].
Effects of MK-2206 and PI-103 and/or radiation on the expression of marker proteins
To further investigated the molecular basis for the observed effects in MK-2206-treated tumor cells with or without addition of PI-103, or IR exposure (Figs. 1, 2), we studied the expression of two groups of proteins. The first group (Fig. 5 and Additional file 4: Figure S4) includes several marker proteins of the PI3K-pathway, i.e. p-Akt and mTOR, along with p-4E-BP1 and p-S6. The second group includes two proteins of the MAPK-pathway, i.e. MEK1/2 and Erk1/2 (Additional file 4: Figure S5). Figure 5 shows exemplarily Western blot data of control and drug-treated cells probed for p-Akt, p-mTOR, p-S6 and p-4E-BP1 proteins 30 min and 24 h after IR with 8 Gy. Samples shown on the left- and right-hand sides (LHS, RHS) of Fig. 5 were obtained from DK-MG and SNB19 cells, respectively.
Complete loss of PTEN in PTEN-mutated SNB19 cells, commonly leads to an over-activation of the PI3K pathway. As seen in Fig. 5 (RHS column), the expression of p-Akt in SNB19 cells was much higher than in DK-MG cells containing wild type PTEN. Addition of MK-2206 or PI-103 either alone or in combination strongly reduced the p-Akt level after a 3-h drug treatment, with and without IR. However, in accordance with our previous findings [24], prolonged treatment with PI-103 caused reactivation of the Akt function in both cell lines. Thus, p-Akt expression (1.1 a.u.) in SNB19 cells almost recovered to the background level of 1.4 a.u. (Fig. 5, RHS). In contrast, both cell lines remained depleted of p-Akt after prolonged treatment with MK-2206 alone or in combination with PI-103, independent of IR exposure.
In addition to p-Akt, we analyzed the expression of p-mTOR and its downstreams, ribosomal S6 and translational repressor 4E-BP1 proteins, which are known to influence cell-cycle progression and cell growth [35, 36]. The expression of p-mTOR decreased after short incubation with PI-103 alone or in combination with MK-2206 (Fig. 5) in both cell lines. Upon long-term exposure to both drugs, p-mTOR expression almost returned to the control level in SNB19 cells, but not in DK-MG cells. As a result of mTOR inhibition by PI-103, both p-4E-BP1 and p-S6 were also strongly suppressed 30 min or even depleted (p-S6) 24 h post-IR (Fig. 5) in cells treated with PI-103 alone and especially in combination with MK-2206.
Contrary to the inhibition of p-mTOR by PI-103, MK-2206 moderately increased the p-mTOR level in SNB19 cells (but not in DK-MG cells) 30 min post-IR (1.0–1.1 a.u.), as compared to control (0.84–0.89 a.u.) and especially to PI-103-treated cells (0.69–0.71 a.u.; Fig. 5). As a result, p-4E-BP1 and p-S6 proteins were also increased after addition of MK-2206 alone to SNB19 cells, whereas they were strongly reduced or even vanished after prolonged PI-103 treatment. At the same time, in DK-MG cells the expressions of p-mTOR and p-4E-BP1 remained mostly unchanged (30 min and 24 h post-IR) in the samples treated with MK-2206 alone or in combination with PI-103. The corresponding bands of β-actin are shown in Additional file 4: Figure S4.
Because of the mutual dependence of the PI3K and MAPK pathways [37], we also analyzed two kinases of the MAPK signaling pathway, p-MEK1/2 and p-Erk1/2 (Additional file 4: Figure S5). The MAPK pathway, which is frequently mutated in cancer cells [38, 39], transmits signals from surface receptors to stimulate cell survival, proliferation and migration [40]. We found that a short incubation with MK-2206 alone or in combination with PI-103 slightly increased the expressions of p-MEK1/2 and p-Erk1/2 (Additional file 4: Figure S5). However, after prolonged incubation with MK-2206 alone or in combination with PI-103 the effect was less evident.
Next we studied Rheb protein, which was shown to activate mTORC1 in vitro [41]. As seen in Additional file 4: Figure S6, after a short incubation with MK-2206 the Rheb expression was increased in SNB19 but not in DK-MG cells. At 24 h post-IR the expression of Rheb was higher in all drug-treated and irradiated probes of SNB19 cells compared to non-treated controls. At the same time, the expression of Rheb in DK-MG cells remained mostly unchanged by drug application.
Effects of MK-2206 and PI-103 and/or radiation on DNA damage
To elucidate the cause for the radioprotective effect of the Akt inhibitor MK-2206 in colony-forming tests, especially in SNB19 cells (Fig. 2), we evaluated DNA damage in control and drug-treated cells after IR. The results are statistically summarized in Fig. 6 as the mean (± SE) values of the amount of DNA damage detected 30 min and 24 h after IR in all cell probes. As seen in Fig. 6a, irradiated drug-free DK-MG cells showed increased residual damage 24 h post-IR. However, no differences in the induction and repair of DNA damage were found between irradiated DK-MG cells independent of drug pretreatment. On the contrary, in irradiated SNB19 cells (Fig. 6b) the residual (24 h post-IR) damage to DNA after addition of PI-103 alone or in combination with MK-2206 was much higher than in drug-free irradiated cells. Interestingly, MK-2206 alone did not influence the degree of induced or residual DNA damage in both cell lines.
Driven by the finding that both inhibitors added alone differently affect the repair of DNA damage in irradiated SNB19 cells (Fig. 6b), without influencing the repair process in DK-MG cells (Fig. 6a), we analyzed the expression of several DNA repair proteins. Figure 7 shows representative Western blot detections of several DNA repair proteins in both cell lines treated with drugs and IR. Thirty minutes after IR, the expression levels of DNA-PK, ATM and ATR proteins were very similar in control and drug-treated samples of DK-MG cells (Fig. 7, LHS). However, 24 h post-IR the expression of DNA-PK and ATM proteins was higher in drug-treated than in drug-free DK-MG cell samples. On the contrary, drug treatment did not cause any changes in the expression of DNA-PK and ATM in SNB19 cells (Fig. 7, RHS). The expression of ATR was mostly unchanged throughout the experiment in both cell lines, except within irradiated SNB19 cells treated for more than 24 h with PI-103 alone or in a combination.
Furthermore, we studied the expression of DNA repair proteins Rad50, Rad51, Ku70 and Ku80 (Additional file 4: Figure S7). We found no differences in the expression of Rad50, Ku70 and Ku80 in both cell lines throughout the whole study. In contrast, the expression of Rad51 was strongly reduced in non-irradiated samples of both cell lines treated with PI-103 alone or in combination with MK-2206 (Additional file 4: Figure S7). Given that Rad51 operates mostly in the G2 phase of the cell cycle [42], the decrease in Rad51 can be explained by the G1-arrest after addition of PI-103. However, despite the strong reduction of Rad51 in both cell lines, DK-MG cells showed normal repair of DNA damage, whereas SNB19 cells showed protracted DNA repair. This means that the impaired DNA repair capacity revealed by the high residual histone γ-H2AX levels in SNB19 cells treated with PI-103 alone or in combination with MK-2206 (Fig. 6b) cannot be explained by the reduction of Rad51 (Additional file 4: Figure S7, RHS). The corresponding blots for β-actin expression are shown in Additional file 4: Figure S8.
Effects of MK-2206 and PI-103 on the plasma membrane properties probed by electrorotation (ROT)
In a series of studies, we have shown recently that the two glioblastoma cell lines (DK-MG and SNB19) studied here are very different not only in their invasiveness and migratory behavior [25, 43], but also in their morphological appearance including membrane surface area and folding [44], analyzed by electron microscopy and electrorotation. Although derived from the same tumor entity, the two cell lines differ markedly in the mutational status of the most prominent tumor suppressors PTEN and p53 [43, 44]. Both genes are wild type in DK-MG cells, whereas in the SNB19 cell line both genes are mutated, which leads to a marked difference between the two cell lines regarding their phospholipid and membrane synthesis (for detail, see Fig. 7 in [44]). Furthermore, the two cell lines differ greatly from each other in their response to the pharmacological inhibition of PI3K and mTOR in terms of migration [25]. Taken together, the above mentioned findings prompted us to analyze in this study the effects of the PI3K-Akt-mTOR inhibition not only on GBM cell migration but also on the plasma membrane morphology, which is closely related to cellular motility and invasion [43].
As shown above (Fig. 5), a 3-h incubation with MK-2206 had different effects on the mTOR expression in tested cell lines, i.e. an up-regulation in SNB19 cells and a down-regulation in DK-MG cells. In view of the essential role of mTOR in cellular metabolism, including protein and phospholipid synthesis [45], it can be expected that the plasma membrane properties will be differently affected by MK-2206 in the two tested GBM cell lines. To prove this assumption, we examined the impact of MK-2206 and PI-103 on the plasma membrane folding and area by means of the contra-rotating field (CRF) technique [30]. Using this technique, we measured the area-specific plasma membrane capacitance Cm [μF/cm2] and the whole-cell capacitance CC [pF]. Cm reflects the morphological complexity of the cell surface, such as membrane folds, protrusions and microvilli [44], whereas the parameter CC accounts for the total electrically accessible cell membrane, including both smooth and folded membrane regions. The results of electrorotation experiments are shown in the Additional file 4: Figures S9, S10 and summarized in Additional file 2: Table S4.
As seen in Additional file 2: Table S4, the Cm value of control DK-MG cells (~ 2.5 μF/cm2) is much lower than that of SNB19 cells (~ 3.9 μF/cm2), which can be explained by a lower degree of membrane folding in DK-MG cells [44]. Treatment with the Akt inhibitor MK-2206 for 3 h significantly reduced both Cm and CC of DK-MG cells by ~ 25% (2.5 → 1.86 μF/cm2) and ~ 33% (19.5 → 13.2 pF), respectively, indicating a marked decline of the plasma membrane area. In contrast and as expected, in SNB19 cells, MK-2206 reduced the Cm and CC by ~ 15% (3.9 → 3.3 μF/cm2) and 25% (30.3 → 23.0 pF), respectively, to a lesser extent than in DK-MG cells.
Interestingly, PI-103 also reduced both Cm and CC of DK-MG, but not as strong as MK-2206. Combined MK-2206 and PI-103 treatment caused the strongest observed reduction of Cm (− 30%) and CC (− 40%) in DK-MG cells compared to controls, while no additional reduction of Cm was observed in SNB19 cells treated with both drugs. In the light of the recent finding that the plasma membrane turnover actively contributes to cell migration [46], the drug-induced reduction of the membrane surface area (probed by CC) in both cell lines (Additional file 2: Table S4), may at least partly be responsible for the observed drug-mediated inhibition of cell migration revealed by wound-healing assay (Fig. 1c, d).