| Objective:Growing evidences have suggested that cellular senescence is not only associated with aging, but also with multiple pathological processes including tumorigenesis and tumor cells reaction to chemotherapy and radiation. In response to cytotoxic agents, tumor cells often manage to survive through the chemotherapy treatment and gradually transform themselves into a state called premature cellular senescence. Cancer cells in this state can maintain cytostasis for weeks or months with lower potential of proliferation and invasiveness, exhibit typical senescence phenotype characterized by positive SA-beta-gal staining. Despite differences in pharmacological mechanism and dosage regimen, the majority of anti-neoplastic genotoxicants are capable of premature senescence induction in different cellular contexts, from normal fibroblasts to malignant cancer cells. In experimental treatment of glioma cells, Temozolomide (TMZ), the alkylating agent applied as standard adjuvant chemotherapy for gliomablastoma in recent years, has been shown to elicits typical senescence response as well as apoptosis. In fact, it is the same06-MeG lesion brought by TMZ alkylating that triggered both apoptosis and senescence in glioma cells. And whether to go apoptosis or senescence is largely a collective outcome of multiple effector mechanisms in cancer cells following the TMZ treatment. Along with apoptosis and other kinds of cell death, senescence contributes to the overall therapeutic effects of TMZ in glioma cells, especially when glioma cells are considered to be more resistant to apoptosis. However, on the other hand, other than apoptosis to completely destroy cancer cell, TMZ-induced senescence can also be viewed as a survival strategy of glioma cells to maintain viability and to avoid further damage that cause apoptosis and lethal mitosis. Moreover, senescence is not the end state of cancer cells that under genomic stress, so the cell fate in therapy-induced senescence is great important to cancer treatment outcome. There are accumulating evidences that suggested senescence cells can bypass therapy-induced senescence and reentry the proliferative cycle, leading to recurrence of the tumor and generating resistance to former therapy.Due to the reversibility of senescence, it is assumable that the treatment outcome may improve either by reducing senescence escape or by abrogating senescence response to induce cell death. Although the actual molecular basis of cancer cells to initiate and maintain senescence is still largely obscure. Signaling pathways that conduct senescence may overlap with that of apoptosis and proliferation. The common signal elements for these pathways can serve different function to each pathway to maintain cell balance or coordinate responses to various stresses. For example, it’s shown that suppressing apoptosis by expression of the anti-apoptotic gene BCL-2during chemotherapy is able to induce senescence. Here, we focus on survivin, a multifunction member of the inhibitor of apoptosis protein (IAP) family, mediator of apoptosis resistance and cell cycle progression, and is highly expressed in cancer cells. Survivin has been shown to protect cancer cell against apoptosis during chemotherapy and promote cell proliferation in various kinds of cancer. Given to these observations, survivin is likely to play a role to resist apoptosis or to re-activate cell cycle progress during senescence. In our study, we examined the influences of survivin interfering on fate of glioma cells exposed to TMZ and sought to explore the effects of survivin in TMZ treatment to glioma cells.Methods: Cell lines and culture conditions:The U87and U251glioma cells were maintained in DMEM supplemented with10%fetal bovine serum at37℃and5%CO2humidified incubator. Cells were treated with TMZ at indicted concentrations at30%cell density for24hours, rinsed with PBS, and harvested or allowed to recover for indicted time in full medium before harvest.Small interefering RNA transfections:SiRNAs against survivin (si-survivin) and p53(si-p53) were designed and synthesized by GenePharma Co.(Shanghai, China). Briefly, cells were seeded at70%confluence and cultured in non-serum medium. Gene-specific siRNAs and the control siRNAs (20-40nM) were addd to media using lipophilic transfection reagent (Lipofectamine2000, Invitrgen Inc) and incubated overnight.Drugs and drug treatment:TMZ was dissolved in DMSO and diluted in sterile water to the indicated concentration before used. All experiments were repeated at least three times and data were evaluated statistically using the t-test (*p<0.05).Determination of apoptosis and senescence:The apoptotic frequency was determined by Annexin V-FITC/propidium iodide (PI) double-staining and quantified by flow cytometry with FACSCanto (Becton Dickinson).Quantitative in situ SA-beta-gal staining was done on indicated cells after three PBS washes and fixation in4%paraformaldehyde with1.7mg/ml of5-bromo-4-chloro-3-indolyl-beta-galactoside at PH6.0.Cell cycle analyses:At each time point, the cells attached to the culture dish were trypsinized and collected together with the cell floating in the media. These cells were washed in PBS, fixed in70%(v/v) ethanol, and stored at-20%for up to2weeks. Before analyses of FACS, the cells were washed with PBS, followed incubation in PBS containing40μg/ml propidium iodine and200μg/ml RNase A for lh at room temperature in the dark. Stained nuclei were then analyzed using FACScan flow cytometer (Becton Dickinson). CellQuest (BD Bio-sciences) software was used to assess cell cycle distribution.Colony escape assay:The senescent U251and U87cells were plated at a known cell density after exposed to TMZ at indicated concentrations. The cells were recovered in fresh media and characterized for senescence and escape colony formation. Colonies typically emerge15days following chemotherapy and are scored from triplicate plates. In experiments involving inhibition of survivin, transfection of Survivin SiRNA and control RNA was performed2days after cell seeded.Preparation of protein extracts and Western-blot analysisThe cultured cells were washed with ice-cold PBS, scraped form the culture dish, and incubated in the lysis buffer containing lOmM KCL,1M sucrose,2mM MgCL2,0.5%Igepal CA-630,1mM EDTA,1mM DTT, lOmM beta-glycerophosephate,1mM Na3VO4, lOmM NAF,100μg/ml PMSF, and10μg/ml aprotinin for30min on ice. The cell lysate was centrifuged at12000g for15min at4℃, and the supernatant was were collected as total proteins of the cultured cells and stroed at-80℃until use. The protein concentrations were measured using the BCA assay kit. Prontein (40μg) was subjected to SDS-PAGE and electroblotted on PVDF membranes (PVDF, Millipore). The membrane was blocked in5%nonfat skim milk at4℃overnight and probed with mouse monoclonal antibodies human survivin, p53, and p21for1hour at room temperature. Secondary antibody was detected with goat antimouse IgG using an enhanced chemiluminescence system (ECL, CST) on Kodak2000MM. Actin protein was detected as a control.Statistics:All values are reported as mean±SD values and analyzed with SPSS13.0version.Results:Silencing survivin in U251cell leads to increase of early apoptosis and decrease of senescence in TMZ therapy TMZ is the genotoxic agent that is capable to activate both apoptosis and senescence. It has been shown that glioma cells underwent apoptosis and senescence after transient exposed to TMZ in clinical relevant dose (100μM). In our study, unsynchronized U251cells were treated with100μM of TMZ for24hours, and then the cells were gently washed and followed for at least6days. Protein levels of survivin in U251cells were stable expressed after TMZ treatment. SiRNA interfering to determine the role of survivin in mediating chemoresistance of TMZ in U251cell was carried out a day before TMZ treatment, the siRNA targeting against survivin mRNA (SiSurvivin) and a control with a scrambled sequence (SC) were transfected into U251cells and down-regulation of survivin expression was comfirmed by Western blot analysis2days after the transfection.Cells were harvested each day after TMZ treatment and subjected to a variety of analyses. As indicated by trypan blue exclusion assay, viable U251cells that transfected with control SiRNA still rised in number for the first2days before reaching a plateau from day3to day5, then they decreased in the next4days and the cell numbers remain largely constant afterward. The decrease of viable cells was due to the onset of apoptosis in these cells as shown by Annexin V staining. And the reasons of cytostasis are because TMZ further arrested control U251cell cycle in G2/M phase and elicited senenscence response in these cells. which is revealed by cell cycle analyses and Sa-beta-gal staining. Compared with the control cells, survivin interfering cells are much more sensitive to TMZ, silencing survivin has accelerated the apoptosis process in U251cells and the rise of apoptotic cells appeared2days ahead of the controls’time. This caused a prominent decrease of their cell number in the first5days. In addition, the production of senescent cells was also decreased by survivin interfering, as indicated by SA-beta-gal staining, relatively small numbers of SiSurvivin transfected cells transform into senescent cell compared to the survivin interfering cell after5days of TMZ treatment.Together, these findings suggested that survivin is responsible for chemoresistance to TMZ and down-regulation of survivin sensitized U251cell by switching the response to TMZ treatment from senescence to apoptosis. Establishment of TMZ resistant subclones from senescent U251cellAlthough cell senescence is originally defined as an irreversible cellular progress in response to various stresses, recent studies have discovered that a small subset of this senescent cells are able to infrequently escape senescence and regenerate themselves into colonies.0To further investigate the cell properties of these senescence escape cell, U251cell were exposed to intermittent treatment of TMZ at low concentration (10μM) repeatedly until most of these cells (80%) assumed senescence phenotype. After that, proliferating colonies in these senescent cells were selected, allowed to recovered from TMZ treatment and resumed the growth rates ofnon-senescence U251cells. We analyzed2subclones (SE3-U251/SE5-U251) of these senescence escape cell and increase percentage of cells in G0/G1and S phase has confirmed the re-entry of cell cycle in these cells. Cell viabilities assessed by trypan blue dye-exclusion assays have shown that both of these cells are more refractory to TMZ treatment compared to untreated U251cells. To determine apoptosis and senescence response to standard TMZ treatment (100μM) in these cells, we also performed apoptosis and senescence analyses in day4and day6after TMZ exposure. For both of the subclones, there’re only small percentage of cells underwent apoptosis, but the abilities to senescence transformation are still partly retained. Moreover, high protein expression of survivin compared to senescent cells is also found in these senescence escape cells, which may indicate a further relationship between survivin and senescence escape.Survivin is required for U251subclones to escape from senescenceSurvivin is a dual mediator of apoptosis resistance and cell cycle progression. It’s possible that the high protein level of survivin expression in U251senescence escape cells is the reason of these cells to proliferate in TMZ-induced senescence. In order to clarify the effect of survivin in U251senescent cells, the same schedule of TMZ treatment we mentioned earlier to induce a large majority of senescence transformation was performed in U251cells. SiSurvivin was transfected into these cells and cells were followed by analyses of proliferation potential. The proliferation potential of these senescent U251cells is assessed by colony escape assay to quantify sub colonies derived from their senescent parent cells.10%densities of the cells were plated as separated cell and we scored up the colonies generated from the single cell after15days. The formation of escape colony in SiSurvivin transfected cells was significantly reduced compared to those cells transfected scramble control. Cell cycle analyses also demonstrated a smaller portion of S phase cells in survivin down-regulated cells.The decline of the escape of the senescent cell indicates that survivin is essential to help senescent cell to overcome cell cycle arrest and re-populate themselves into new colonies.Effects of survivin interfering in U87cells are associated with p53activationP53is one of the most well studied tumor suppressor protein in cancer cells. Contrary to survivin’s effects, p53blocks cell cycle progression and/or induces apoptosis by transactivation of specific target gene. Previous studies also have suggested that survivin is negatively regulated by p53. To determine whether effects of survivin interfering in glioma cells are in related with p53expression, we used the wild-type p53expressing U87cells for further studies. U87cells were treated with TMZ in the same dosage regimen and activation of p53in response to TMZ was confirmed by induction of p21by Western blotting. Expression of p53in U87cells was knockdown by transfection of p53SiRNA and survivin expression was analyzed4days followed the TMZ treatment. Compared to U87cells transfected with control SiRNA, no significant promotion of survivin expression was observed in p53down-regulated U87cells. Survivin interfering was also performed in U87cells combined with treatment of TMZ. In accordance with survivin interfering in U251cells, knock-down of survivin reduce cell viability and sensitize U87cells to apoptosis. Interestingly, combination treatments of p53and survivin SiRNA can partially rescued cell viability and decreased apoptosis within these cells. This suggested that increase of apoptosis by survivin interfering in U87cells is mediated by p53 activation. In summary, our findings suggested that in wild-type p53expressing U87cells, survivin blocked the process of TMZ-induced apoptosis and inhibition of survivin may allow for re-activation of this p53dependent apoptosis which overcome the resistance to TMZ in these cells.Conclusion:1. Cellular senescence but not apoptosis is the major response of glioma cell in TMZ therapy.2. Survivin interfering increase the sensitivity of glioma cell to TMZ therapy, mainly through the early onset of apoptosis and reduction of senescence3. Glioma cell under senescence can maintain long term viability and recover its proliferative capacity.4. Survivin is required for senescence escape in glioma cell.5. Interfering of survivin increase the p53-dependent apoptosis in TMZ treatment. |
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