Studies suggest that survivin contributes to chemo-resistance and enhances cell survival through two potential mechanisms: 1) the inhibition of apoptosis by blocking activated caspases [18, 22], and 2) by stabilizing the microtubule network to avert cell catastrophe . These two mechanisms exert different weights in the contribution to cancer cell survival, depending on the therapeutic agent used and possibly the cancer type. For example, in lung cancer, Normura et al. demonstrated that inhibition of apoptosis mediated by survivin contributed to cisplatin-resistance . In another study, Zhang et al. showed that that adenovirus-mediated inhibition of survivin expression actually sensitized human prostate cancer cells to paclitaxel . Their studies also suggested that survivin mediates resistance to paclitaxel through suppression of caspase-mediated apoptosis . However, another study revealed that survivin is required for stable checkpoint activation in paclitaxel-treated HeLa cells . Down-regulation of survivin by siRNA abrogated the ability of cells to sustain paclitaxel-induced mitotic arrest . In addition, down-regulation of survivin alone did not induce massive death in HeLa cells . Therefore, question remains as to whether survivin plays a dominant role in processing cytokinesis or inhibiting caspases activity in cells treated with anti-mitotic compounds. Interestingly, survivin-induced resistant to microtubule de-stabilizers such as Vinca alkalodis, cochicine and Combretastatin A-4 related compounds in cancers was rarely demonstrated in the past . In our study, we found that survivin counteracts the therapeutic effect of microtubule de-stabilizer, BPR0L075, by stabilizing tubulin polymers.
BPR0L075 is a novel anti-mitotic compound and its mechanisms of action have been published elsewhere [11, 12]. In brief, BPR0L075 is a heterocyclic Combretastatin A-4 (CA-4) analog that inhibits tubulin polymerization by binding to tubulin at the colchicine-binding site [11, 12]. In the current study, up-regulation of survivin in response to BPR0L075 was observed in both p53-wildtype KB and p53-mutated HONE-1 cancer cells. In addition, the baseline expression of survivin was also increased in KB-derived BPR0L075-resistant KB-L30, as compared to its drug-sensitive parental cells. Therefore, one important aim of our study was to investigate whether survivin could be potentially important in sensitivity to the microtubule-destabilizing compound, BPR0L075, especially in BPR0L075-resistant cancer cells. Here, we demonstrate that down-regulation of survivin by siRNA induced hyper-sensitivity to BPR0L075 in KB and partially restored sensitivity to the drug in BPR0L075-resistant KB-L30 cells. These results suggested for the first time that survivin plays an important role in the sensitivity and resistant to the microtubule de-stabilizer, BPR0L075. Furthermore, p53 is widely believed to be a negative regulator of survivin expression [25, 36–41]. However, our current results indicate that whether p53 plays a definite role in survivin regulation under various chemo-stresses remains agurable. In our study, dose-dependent co-expression of p53 and survivin in response to BPR0L075 was shown in p53-wildtype KB cells. Interestingly, co-incubation of a p53-inhibitor, pifithrin-α, was unable to increase the level of survivin expression in BPR0L075-treated cells. In addition, over-expression of survivin in response to BPR0L075 was also observed in p53-mutated HONE-1 cells. Taken together, our study suggested that BPR0L075 induces overexpression of survivin through a p53-independent pathway.
It has been reported that VEGF-induced expression of survivin preserved the microtubule integrity and stabilization of the microtubule networks subsequently induced drug-resistance to both CDDP and paclitaxel in HUVEC cells . A role for altered microtubule polymer levels in vincristine resistance of acute lymphoblastic leukemia has also been demonstrated in vivo . In the present study, increased tubulin polymer levels were observed in the BPR0L075-resistant KB-L30 cells as compared to its drug-sensitive parental cells. We also demostrated that down-regulation of survivin de-stabilizes tubulin polymers in both KB and KB-L30 cancer cells. In addition, the level of acetylated α-tubulin was synergistically reduced in the survivin/BPR0L075 combination therapy as compared to the monotherapy in KB cells. Thus, the over-expression of survivin in response to the microtubule de-stabilizer BPR0L075 reflects one of the possible cell survival mechanisms by stabilizing microtubule. It is also worth noting that over-expression of survivin was observed in both colchicine- and vincristine-treated KB cells (data not shown). In addition, KB-L30 cells are relatively more sensitive to paclitaxel (microtubule stabilizer) and at the same time more resistant to both colchicine and vincristine (microtubule de-stabilizers), as compare to their parental cells (data not shown). Interestingly, Ong et al's study also revealed that a vincristine-resistant xenograft with high levels of polymerized tubulin was relatively sensitive to the microtubule-polymerizing drug paclitaxel . Indeed, further investigations are needed to determine the relationships between the survivin-interfered microtubule dynamics and the resistance to various microtubule de-stabilizers in depth.
Although various studies reveal that survivin interferes with cytokinesis and inhibits caspases during apoptosis, the question remains as to whether survivin plays a dominant role in regulating cell cycle or inhibiting cell death. In fact, down-regulation of survivin did not induce massive cell death in HeLa cells . However, the process of cytokinesis was heavily impaired by the same treatment . In the present study, down-regulation of survivin did not induce cell death in both KB and KB-L30 cells under BPR0L075-free conditions. In accord with the results from cell viability study, down-regulation of survivin did not induce caspase-3/-7 activities in both KB and KB-L30 cells cultured under drug-free conditions. It is widely believed that anti-mitotic compounds induce mitotic arrest by activating the spindle checkpoint and these molecular changes subsequently activate caspase-dependent apoptosis. Our previous study agreed with that hypothesis as the microtubule de-stabilizer, BPR0L075, induced activation of caspase-3 in various cancer cell lines . Interestingly, down-regulation of survivin did not enhance caspase-3/-7 activities in the survivin/BPR0L075 co-treated KB cells, given that the microtubule dynamics were heavily impaired in the same treatment. These results suggested that survivin functions predominantly in microtubule stabilization or mitotic progression, as opposed to inhibition of caspase-dependent cell death in KB cells under BPR0L075-free conditions. However, down-regulation of survivin induced caspase-3/-7 activities in the survivin/BPR0L075 co-treated KB-L30 cells. Therefore, whether survivin interferes with the caspase-3/-7 during BPR0L075 treatment seems to be cell type dependent. Furthermore, incomplete restoration to the drug sensitivity in survivin down-regulated KB-L30 cells indicates that other important drug resistance mechanisms may co-exist.
As we mentioned earlier, it is believed that anti-mitotic compounds induce mitotic arrest by activating the spindle checkpoint and these molecular changes subsequently activate caspase-dependent apoptosis. However, our current study reveals that down-regulation of survivin synergistically affects the microtubule dynamics and cell viability without significant induction in caspases activity. In addition, increased DNA fragmentation and nucleus degradation were observed in the survivin/BPR0L075 combination treatments, as compared to the BPR0L075 monotherapy. Therefore, down-regulation of survivin together with BPR0L075 may initiate caspase-independent DNA fragmentation and cell death in KB cells. During one form of caspase-independent apoptosis, apoptosis-inducing factor (AIF) translocates from the mitochondria to the nucleus [42–44]. AIF is a flavoprotein that is normally confined to the mitochondrial intermembrane space, but induces chromatin condensation and fragmentation of DNA into high molecular weight forms of >50 kb when it translocates to the nucleus [31, 32]. It has been suggested that survivin interferes with the translocation of AIF. In fact, translocation of AIF from the mitochondria to the nucleus was shown in YUSAC2 melanoma cells treated with a cell-permeable dominant-negative survivin protein . Furthermore, involvement of AIF in nocodazole (microtubule de-stabilizer)-induced caspase-independent mitotic cell death was also demostrated perviosuly . In Niikura et al.'s study, treatment with nocodazole or paclitaxel induced kinetochore-microtubule detachement, mitotic arrest and subsequent AIF-involved caspase-independent cell death in cancer cells . In our study, translocation of AIF from the cytoplasm to the nucleus following the down-regulation of survivin indicates that survivin inhibits caspase-independent apoptosis in KB cells. Thus, both caspase-dependent and caspase-independent DNA fragmentation could be co-operated in KB cells during survivin-targeted BPR0L075 combination treatments. These results suggested that down-regulation of survivn together with microtubule de-stabilizers may induces caspase-independent mitotic cell death, possible through AIF activation. However, our study could not determine the rate of AIF activity between survivin monotherapy and survivin/BPR0L075 combination. Further investigation is needed to determine the level of AIF activities between different treatments.