Despite considerable research efforts aimed at developing novel therapeutic approaches against NSCLC, including molecularly targeted therapy and immunotherapy, the frequency of MDR severely restricts the effectiveness of various anticancer drugs [3, 4]. Currently, apoptosis tolerance-mediated MDR has been considered the most common and complex drug-resistant mechanism [5, 33]. However, in addition to apoptosis, anticancer drugs can also trigger other anticancer mechanisms, such as autophagy, MC, ferroptosis, pyroptosis, necrosis and senescence, to inhibit the proliferation of cancer cells and kill them by avoiding the apoptosis-resistant pathway [11, 17, 34, 35]. In our previous studies, we confirmed that BZML has potent anticancer activity and overcomes MDR by inducing MC in A549/Taxol cells, an MDR cell line [24]. However, a detailed and clear explanation of the mechanisms underlying MC occurrence and development in BZML-treated A549/Taxol cells was lacking.
Cellular senescence is a permanent state of cell cycle arrest and has been considered a novel anticancer mechanism [14, 17]. In our study, BZML treatment induced the cellular senescence in A549/Taxol cells, showing a significantly increased proportion of SA-β-gal-positive cells and some other typical features of cellular senescence, such as flattened morphology, increased size and granularity and an elevated lysosomal mass in the cells. To further investigate whether these senescent cancer cells were dormant cells that can contribute to cancer redevelopment with the decrease in the concentrations of therapeutic drugs, the BZML-containing medium was replaced with drug-free medium at 48 h post-BZML treatment, and the results from the SA-β-gal staining and microscopy assays of the size and shape of the cells demonstrated that the A549/Taxol cells underwent senescence even after BZML withdrawal. Importantly, the senescent cancer cells neither reentered the normal cell cycle nor divided; thus, they lost normal proliferative potential, and the increase in cell death was significant after the withdrawal of the BZML treatment. Therefore, given the important role of senescence in cancer treatment, our findings support the possibility that BZML-induced senescence might act as an anticancer mechanism against MDR in NSCLC.
MC is a newly discovered form of tumor suppression that differs from other cell death modes and is characterized by unique nuclear alterations, such as multi and/or micronucleation [24, 36, 37]. Interestingly, a number of studies reported that senescence is closely related to MC and that cells undergoing senescence may undergo polyploidization and/or become multinucleated [37, 38]. In contrast, some studies showed that C85 cells senescence induced by methotrexate did not undergo polyploidization or multinucleation [39]. Therefore, to elaborate the relationship between BZML-induced MC and the senescence-like phenotype of A549/Taxol cells, we first performed a time kinetic study to assess the dynamic changes of key parameters during MC and senescence. Notably, BZML-induced MC, indicated by the appearance of polyploid and/or multinucleated cells, occurred as early as 12 h, while an increase in SA-β-gal-positive cells was detected 48 h after BZML treatment. Analysis of these 2 events over the experimental time course indicated that BZML-induced MC occurred relatively early in the A549/Taxol cells. Moreover, SA-β-gal and Hoechst 33342 double staining revealed that almost all the SA-β-gal-positive cells were multi- and/or micronucleated, but not all the polyploid cells were stained positive for SA-β-gal activity. Interestingly, our previous study proved that BZML-induced MC was independent of p53 [24]. As expected, regardless of p53 status, BZML did not induce MC in A549, H1299 or MDA-MB-231 cells, which are p53-wild-type, p53-null and p53-mutant cells, respectively. Importantly, these BZML-treated cells did not undergo senescence. In addition, in contrast to traditional cellular senescence, which is often accompanied by G0/G1 phase arrest [37], in our study, a substantial level of polyploidy was detected in the senescent A549/Taxol cells after BZML treatment. Together, these data strongly support the supposition that BZML-induced senescence is downstream of MC and acts as an important phenotype associated with MC occurrence and development in A549/Taxol cells.
Interestingly, elevated ROS levels have been widely accepted as a major trigger of cellular senescence [37, 40], and BZML was confirmed in our previous study to cause ROS generation in a time-dependent manner, but ROS are not inducers of MC occurrence or development in A549/Taxol cells [25]. In addition, in this study, we also found that NAC did not reverse the BZML-induced senescence-like phenotype of the A549/Taxol cells (Supplementary Fig. S8a and b), suggesting that the senescence-like phenotype is secondary to the BZML-induced MC in A549/Taxol cells and may be attributed to mechanisms other than ROS. BZML-induced MC is independent of p53, but the expression of p53 was increased in BZML-treated A549/Taxol cells in a time-dependent manner. Given these considerations, p53-p21 pathway activation represents the trigger of senescence, and the p16INK4α-Rb pathway is involved in maintaining senescence [37, 41]. As expected, the expression of p21 was gradually increased, followed by an increase in Rb expression and a decrease in Rb phosphorylation in the BZML-treated A549/Taxol cells. Moreover, our study also demonstrated that pifithrin-α and p53-siRNAs can reverse the BZML-induced senescence-like phenotype, indicating that p53 might play a decisive role at the beginning of senescence in BZML-treated A549/Taxol cells.
Under normal conditions, p53 is a short-lived protein that shuttles between the nucleus and the cytoplasm in a cell cycle-specific manner [31]. Relocation of p53 to the nucleus in response to cellular stress is a contributor to the inhibition of the growth of cancer cells [42]. In this study, the expression of p53 was significantly increased in the BZML-treated A549/Taxol cells at both the mRNA and protein levels. Importantly, the results from the western blot analysis and fluorescence microscopy assay all indicated that BZML treatment increased p53 levels, mainly in the nucleus. In addition, increasing attention has been focused on MDM2 because it acts as an oncogene that negatively regulates the functions of the tumor suppressor p53 by inhibiting transcriptional activity and accelerating the degradation of p53 [43]. Interestingly, several reports have demonstrated that some CBSIs, such as combretastatin A-4 and SQ, exhibit a potential MDM2 inhibitory effect in breast cancer cells [44]. Furthermore, in this study, BZML also significantly decreased the expression of MDM2 in the A549/Taxol cells. Additionally, it cannot be ignored that the ubiquitin-proteasome system (UPS) functions as the primary route of degradation for thousands of short-lived proteins [45, 46]. Here, its important components, proteasome 20S core subunits and PMSA6, were significantly downregulated in the BZML-treated A549/Taxol cells, indicating that BZML might cause the destruction of the UPS. In fact, the half-life of p53 was significantly prolonged by BZML in the A549/Taxol cells. Therefore, these results suggest that BZML can activate p53 via multiple mechanisms in the A549/Taxol cells and that the destruction of the UPS also contributes to the increase in p53 protein, at least in part.
In cancer cells, survivin located in the cytoplasm plays indispensable roles in cell proliferation and apoptosis inhibition [32]. Interestingly, in this study, BZML treatment increased survivin mainly in the nucleus of the A549/Taxol cells. Given these considerations, survivin is also a short-lived protein and can be polyubiquitylated and undergo proteasomal destruction [47]. Furthermore, nuclear survivin is significantly more unstable [20]. Interestingly, survivin was expressed at low levels under unstressed conditions; however, in response to BZML-induced MC, its stability in the nucleus was quickly increased, and its half-life was significantly prolonged at the same time. This outcome suggests that the increase in survivin in the nucleus may also be attributed to the destruction of the UPS in BZML-treated A549/Taxol cells. Notably, chemotherapy and radiation therapy can increase survivin expression, which is attributed to mitotic arrest at the G2/M phase and to the augmented stability of the survivin protein through its phosphorylation at Thr34 by a cdc2/cyclin B1 complex [21, 22, 48]. In the present study, the expression of survivin was also slightly increased at the mRNA level. In addition, our previous study showed that BZML treatment caused a transient decrease in cyclin B1 within 24 h, followed by a significant and stable increase in cyclin B1 expression during BZML-induced MC [24]. Remarkably, in this study, the expression of survivin and cyclin B1 changed in parallel during this process. Importantly, the overexpression of survivin neither mediated the apoptosis resistance against BZML nor promoted BZML to induce MC in the A549 cells, suggesting that survivin is not an inducer to the BZML induction of MC in A549/Taxol cells, at least in some settings. Therefore, we speculated that the increase in survivin in the nucleus may result from the increase in cyclin B1 and the destruction of the UPS, and the nuclear expression of survivin is an important biological phenotype associated with MC occurrence and development.
Additionally, some studies have demonstrated that MC and senescence play important and paradoxical roles in the process of cancer treatment [36, 37]. In some circumstances, senescent cells cannot be cleared at the time of treatment in time, which may result in the resumed division and apoptosis of drug-resistant cancer cells, and eventually potentiate cancer progression [49, 50]. Therefore, the development new strategies to remove cells undergoing MC and senescence in a timely manner after the initial anticancer response are urgent. Our data showed that survivin was not required for a cell to undergo MC, but once cellular senescence, secondary to MC, was induced, the upregulation of survivin may provide additional vulnerability to and critical opportunities for sequentially applied therapies. Interestingly, in this study, there was no synergistic effect of YM155 treatment in response to BZML-induced MC during short periods, but the inhibition of survivin by YM155 significantly enhanced the efficiency of BZML in overcoming the MDR of the A549/Taxol cells after 72 h of treatment. Importantly, after BZML-induced MC associated with senescence, dose-sequence-dependent combination therapy with YM155 exhibited a synergistic lethal effect. This suggests that the synergistic effectiveness of BZML and YM155 occurs in the context of the MC-associated senescence induced by BZML. Combinational treatment is typically employed to achieve a better response rate than monotherapy and is based on the use of drugs with different cytotoxicity-inducing mechanisms [22, 23]. Herein, sequential monotherapy not only retained the synergistic effect but also further reduced the toxicity of the anticancer drugs in clinical application. Due to the short half-life of YM155 in the human body [23], we proposed a ‘‘one-two punch’’ approach to cancer treatment based on our findings. That is, at the beginning of cancer treatment, MC-associated senescence is selectively induced in cancer cells; subsequently, in consecutive therapy, these senescent cells are killed by dose-sequence-dependent molecularly targeted drugs. Thus, MC-associated irreversible senescence induced by BZML may provide a treatment window for the opportunistic cell elimination using synergistic YM155. In addition, considering that the aberrant activation of an oncogene can cause cellular senescence, survivin, as an oncogene, may also promote the phosphorylation of Rb to maintain the senescence phenotype [17, 20, 37]. Subsequently, we tried to clarify whether the nuclear accumulation of survivin was critical for the senescence caused by BZML-induced MC. Notably, YM155 significantly decreased the percentage of SA-β-gal-positive cells among the BZML-treated A549/Taxol cells, suggesting that the nuclear accumulation of survivin may mediate self-protection by inducing a senescence-like phenotype during MC. Our data provide strong evidence for targeting survivin as a strategy for enhancing the efficiency of BZML because it induces MC to overcome the MDR of A549/Taxol cells.