Optimization of the chemoradiotherapy treatment schedule increases anticancer efficacy
In order to optimize the treatment schedule of MTA plus irradiation, the concentration of MTA and level of exposure to irradiation that induced an intermediate level of growth inhibition was determined. Exposure to 5 Gy IR or treatment with 1 µM MTA resulted in an intermediate reduction of cell numbers (Additional file 1: Figure S1; Fig. 1b) and these conditions were therefore applied to the combination therapy. Three different treatment regimens were compared to determine whether the effectiveness of the MTA-IR combination therapy is dependent on the treatment schedule, including continuous MTA (1 μM) treatment for 72 h combined with exposure to 5 Gy IR at different time points (Fig. 1a). In detail, cells were irradiated 1, 48 or 71 h after the initiation of the 72 h MTA treatment (treatment #1, #2 or #3, respectively). The doubling time (day 0➙3) of untreated A549 cells was found to be approximately 22 h (Tièche et al., manuscript in preparation), which is in agreement with the information provided by the American Type Culture Collection. Compared to the untreated control, cell growth slightly decreased after 24 h MTA treatment alone (treatments #2 and #3, day 1) and further decreased after either 48 or 71 h MTA alone (treatment #3, day 2 or 3 respectively) (Fig. 1b). When the IR treatment was applied 1 h after the start of the MTA treatment (treatment #1), cell numbers did not increase considerably during the subsequent 24 h. In contrast, after pretreatment with MTA-for 48 h, cell numbers steadily increased during the 24 h following irradiation (treatment #2, day 2➙3). At the end of the treatment phase (day 4), the absolute cell counts were significantly lower after treatment #1 compared to treatment #2 and #3. However, during the extended recovery phase (day 6➙9), cell numbers steadily increased after treatment #1 whereas cell growth was significantly decreased after treatment #2 compared to treatment #1 and were further reduced after treatment #3 (Fig. 1b). In detail, during the recovery phase (day 6➙9) the doubling time of recovering cells after treatment #1 was 83 h whereas treatment #3 significantly prolonged the doubling time of the recovering cells (176 h) and with treatment #2 inducing an intermediate level of growth delay (116 h). To evaluate the growth capacity of the remaining cells, the residual cells were harvested at day 9 of the recovery phase and reseeded at low density. At day 13 of the recovery phase, cell numbers compared to treatment #1 were 1.9 and 2.9 times lower after treatments #2 and #3, respectively (Fig. 1c). Thus, treatment #3 reduced overall survival by a factor of ~7 compared to treatment #1 (fold difference in cell number at day 9 multiplied by day 13, i.e. 2.4 × 2.9). In other words, long-term cell growth was significantly reduced after extended pretreatment when compared to concomitant treatment.
Prolonged MTA pretreatment and subsequent irradiation induces senescence
Prolonged cell cycle arrest after DNA damage induction results at the molecular level in DNA double strand break formation and at the cellular level in a terminal proliferation halt, i.e. senescence [11]. By permanently arresting proliferation of damaged cells, senescence serves as a barrier to cancer development.
Visual examination of the recovered cells after treatment #1 revealed that clones formed by small, cuboid cells could be distinguished from surrounding cells, which displayed morphologic changes that are associated with senescence, namely increased cell size and flattened shape (Fig. 2a; reviewed in [12]). The fraction of cells featuring a senescence phenotype was increased after treatment #2 and maximized after treatment #3 (Fig. 2a). Cells were stained to detect senescence-associated β-galactosidase (SA-β-Gal) activity. At day 6, the fraction of SA-β-Gal-positive cells (indicated by blue staining in Fig. 2a) was 4.5-fold higher after treatment #3 compared to treatment #1 (Fig. 2b, P > 0.05). Detectable by flow cytometry, increased forward (cell size) and side (cellular granularity) scatter intensity (F/S-high) is an additional feature associated with senescence (reviewed in [13]). Flow cytometric analysis at day 9 of the recovery phase revealed that the highest frequency of cells with a F/S-high phenotype was observed after treatment #3 (Fig. 2c). In detail, compared to the untreated control (13.3 %), the frequency of F/S-high cells was 1.5 and 3.3-fold higher after treatment #1 and treatment #3, respectively (19.8 and 43.2 %, P > 0.05), with treatment 2 inducing an intermediate level of senescence. Nevertheless, flow cytometric analysis at the end of the treatment phase (day 3) and during the early recovery phase (day 6) confirmed that for all three treatments, a fraction of the cells maintained a normal forward and side scatter intensity (F/S-low), indicating the presence of cells resistant to the tested treatment regimens (Additional file 2: Figure S2). However, compared to treatment #1, treatment #3 significantly decreased the fraction of resistant cells, e.g. F/S-low cells, indicating that extended MTA pretreatment can augment the anticancer activity of radiation therapy.
Prolonged MTA pretreatment enhances S-phase accumulation prior to irradiation
Terminal cell cycle arrest is a classic hallmark of senescence, and has been observed after treatment with chemotherapy (reviewed in [12]). Therefore, we monitored the cell cycle distribution of A549 cells during and after combined MTA-IR treatment (Fig. 3). MTA-alone treatment for 24 h did not result in a significantly changed cell cycle distribution (treatment #2 or 3, day 1). MTA-alone treatment for 48 h increased the fraction of cells in S-phase (treatment #3, day 2), which was even more pronounced after 72 h (treatment #3, day 3). Thus, at the start of the irradiation during treatment #1 (day 0), the cells mainly resided in the G1-phase of the cell cycle. In contrast, at the start of irradiation during treatment #2 (day 2), a significant fraction of the cells (37 %) was arrested in S-phase, which was further increased at the start of the irradiation during treatment #3 (day 3, 47 %).
Interestingly, when irradiation was preceded by MTA pretreatment for 48 h, the fraction of cells in S-phase decreased during the 24 h after irradiation (treatment #2, day 2 compared to day 3). An adverse effect of irradiation on the subsequent MTA-induced S-phase accumulation was also detectable after concurrent therapy. In detail, concurrent irradiation (treatment #1, day 0) completely abolished the MTA-induced S-phase accumulation at the end of the treatment phase (day 3, treatment #1 compared to treatment #3).
The cell cycle distribution of the recovering culture was normal during the early recovery phase (day 3➙6) after treatment #1. Since treatment #1 did not result in pronounced growth retardation, the cell culture became confluent during the later recovery phase (day 9) as also indicated by the accumulation in the G1-phase. Reseeding at low density revealed that the cell culture acquired a normal cell cycle distribution during the extended recovery phase, which was also detectable after treatment #2 and #3 (day 13). A sub-G1 DNA content is a hallmark of cells undergoing apoptosis. A very small fraction of sub-G1 cells (1.4 %) was observed 24 h after irradiation during treatment #1 (day 2) but not after treatment #2 (day 3) and also not 3 days after treatment #3 (day 6) indicating that the tested treatment regimen did not result in significant induction of apoptosis. In summary, pretreatment with MTA for a prolonged period enhanced S-phase accumulation prior to irradiation. However, this effect was transitory in the remaining, therapy-resistant fraction of the cells and cell growth returned to normal during the extended recovery phase.
Prolonged MTA pretreatment results in persistent DNA damage accumulation
Induction of DNA breaks or DNA replication stress leads to the activation of the DNA damage response (DDR) (reviewed in [14]). During DDR, phosphorylation of histone variant H2AX (γH2AX) serves as a key mediator for the assembly of DNA repair proteins at the sites of DNA damage as well as for the activation of checkpoint proteins. Consequently, analysis of γH2AX is frequently used as a surrogate marker for DDR activation.
We have previously demonstrated that accumulation of persistent DNA damage leads to a cell cycle arrest and induction of senescence in A549 cells [15]. Thus, we determined the effect of the different treatment regimens on H2AX phosphorylation. Analysis of the total population revealed that irradiation with 5 Gy IR resulted in phosphorylation of H2AX in more than 90 % of the cells irrespective if MTA was administered concomitantly or as pretreatment (Fig. 4a). MTA-alone treatment for 24 h only marginally increased γH2AX levels in the total population (treatments #2 and 3, day 0➙1) (Fig. 4a). However, analysis of the cell cycle specific subpopulations revealed that MTA-alone treatment slightly increased γH2AX levels in S-phase and G2/M-phase cells whereas cells in the G1-phase were not affected (Fig. 4b). MTA treatment for 48 h (treatment #3, day 2) resulted in robust H2AX phosphorylation in a fraction of S-phase cells (34 %), which was also observed in the majority of cells in the G2/M-phase (66 %). After MTA-IR co-treatment, H2AX was rapidly phosphorylated in the majority of cells in all cell cycle phases (93 % of total population) (treatment #1, day 1, Fig. 4a). 24 h after irradiation, γH2AX phosphorylation returned to basal levels even in the presence of MTA co-treatment (treatment #1, day 0➙1). Similarly, H2AX phosphorylation was increased to nearly maximal levels in all phases of the cell cycle when irradiation was preceded by 48 or 71 h MTA pretreatment (91 and 97 % of total population, respectively) (treatment #2, day 2 and treatment #3, day 3, respectively). H2AX phosphorylation also returned to basal levels 24 h after irradiation during treatment #2 (treatment #2, day 2➙3). However, H2AX phosphorylation levels were still increased 3 and 6 days after termination of treatment #3 (day 6 and 9, respectively) compared to treatment #1 (25.7 and 20.5 % for treatment #3 compared to 16.5 and 6.0 % for treatment #1, respectively, P < 0.05) (Fig. 4a). During the recovery phase after treatment #3 (day 9), only a small fraction of cells in the G1-phase contained high levels of H2AX phosphorylation whereas persistent H2AX phosphorylation was detectable in the majority of cells in the G2/M-phase, which was absent after treatment #1 (Fig. 4b). In summary, prolonged MTA pretreatment (treatment #3) increased H2AX phosphorylation levels during the extended recovery phase.
In summary, the inhibitory effect of MTA-IR combination therapy on lung cancer cell growth can be further augmented by the optimization of the treatment schedule. Prolonged MTA pretreatment preceding irradiation reduces cell growth compared to concomitant treatment and increases the fraction of senescent cells. In addition, extended MTA-pretreatment induces a pronounced S-phase accumulation, which is abrogated by concomitant IR treatment. Finally, our investigations reveal that prolonged MTA pretreatment significantly delays recovery after DNA damage induction.