|Year : 2013 | Volume
| Issue : 1 | Page : 54-59
M-ds-P21 induces cell apoptosis in bladder cancer T24 cells through P53 independent pathway
Haifeng Wang, Wujiang Liu, Jie Jin, Liqun Zhou, Lili Liang, Yinglu Guo
Department of Urology, Peking University First Hospital and Institute of Urology, Peking University, National Research Center for Genitourinary Oncology, Beijing, China
|Date of Web Publication||10-Apr-2013|
Department of Urology, Peking University First Hospital and Institute of Urology, Peking University, National Research Center for Genitourinary Oncology, Beijing
Source of Support: None, Conflict of Interest: None
Objectives: To investigate the effect of M-ds-P21 on the apoptosis of bladder cancer T24 cells and its potential mechanism.
Materials and Methods: Effect of M-ds-P21 on T24 cells were assessed by cell morphology and Western blot. Apoptosis was quantified by Annexin-V flow-cytometry analysis. To uncover the role of P53 in M-ds-P21-mediated apoptosis of T24 cells, we knocked down P53 before treating cells with M-ds-P21, and then assayed P21 and apoptosis-related protein by Western blot. To uncover the mechanism by which M-ds-P21 played stronger effect than ds-P21, we performed confocal microscope analyses.
Results: Both M-ds-P21 and ds-P21 treatment changed the cell morphology, leading to cell apoptosis after 3 days. Apoptosis induced by M-ds-P21 and ds-P21 treatment is not P53-dependent but caspase-dependent. Compared with ds-P21, M-ds-P21 significantly increased the bioavailability of ds-RNA in T24 cells.
Conclusions: M-ds-P21 treatment induces more apoptotic population than ds-P21 does. The mechanism for stronger effect of M-ds-P21 is partly due to the enhanced bioavailability of ds-RNA in human bladder cancer T24 cells, and not P53-dependent but caspase-dependent.
Keywords: 2′- Fluoride modification of ds-P21, apoptosis, bladder cancer cells, ds-P21, T24
|How to cite this article:|
Wang H, Liu W, Jin J, Zhou L, Liang L, Guo Y. M-ds-P21 induces cell apoptosis in bladder cancer T24 cells through P53 independent pathway. J Can Res Ther 2013;9:54-9
|How to cite this URL:|
Wang H, Liu W, Jin J, Zhou L, Liang L, Guo Y. M-ds-P21 induces cell apoptosis in bladder cancer T24 cells through P53 independent pathway. J Can Res Ther [serial online] 2013 [cited 2020 Aug 3];9:54-9. Available from: http://www.cancerjournal.net/text.asp?2013/9/1/54/110367
| > Introduction|| |
The major bottlenecks of siRNA (small interfering RNA) in the clinical application are the stability, poor transportation, bioavailability and ineffective targeting strategy. Scientists around the world have adopted a variety of chemical modification for siRNA to improve its bioavailability and bioactivity. Up to date, 2'- hydroxy (2'-OH) modified by fluoride was supposed to improve siRNA stability and enhance the binding of mRNA with its complementary mRNA. 
Ds-P21 is a synthesized 21-nucleotide double strand RNA (dsRNA) targeting the p21WAF1/CIP1 gene promoter at position -322 related to the transcription start site.  The P21 protein level could be significantly up-regulated by ds-P21 transfection, although the exact molecular mechanism is still mysterious. p21WAF1/CIP1 gene encodes a potent cycling-dependent kinase inhibitor, which binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle progression at G1. The expression of p21WAF1/CIP1 gene is tightly controlled by the tumor suppressor protein p53,  and this protein mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of stress stimuli. P21 was supposed to be specifically cleaved by caspase-3-like caspases, which thus leads to a dramatic activation of CDK2, and may be instrumental in the execution of apoptosis following caspase activation.  The dramatically enhanced apoptotic cell population was observed in T24 bladder cancer cell after ds-P21 transfection,  thus ds-P21 is a good inducer of apoptosis. Cell model derived from this phenomenon is perfect to investigate the apoptosis mechanism and the effect of chemical modification of double strand RNA on cell apoptosis.
The most important modification of the siRNA is nucleotide pentose modification at 2'-OH site. 2'-OH is the main difference between RNA and DNA. Catalyzed by the RNA enzyme, 2'-OH attack on the phosphate first, disconnect phosphate ester bonds, at the same time form cyclic phosphate ester, and then re-form the hydrolyzed products by alkaline hydrolysis. At the ribose 2'-OH position, the introduction of certain substituents such as methyl and fluorine made the siRNA a greater resistance to nuclease hydrolysis.  2′- fluoride modification is that the 2'-OH of 2, 4-dihydroxypyrimidine in double stranded RNA was substituted by 2'- fluoride (2'-F). This modification makes the ds-RNA difficult to be identified by the RNA enzymes, thus increasing the stability of ds-RNA.  Furthermore, this modification can increase the binding ability to the targeted mRNA, and may contribute to the increased bioactivity. 
In this study, we synthesized the 2'-fluoride-modified-ds-P21, named M-ds-P21. The bioavailability, the protein induction and the apoptosis mechanism were investigated compared with its unmodified partner, ds-P21.
| > Materials and Methods|| |
ds-RNA and 2'- fluoride modification
ds-P21 was designed and synthesized as previously described.  Simultaneously, ds-Control was a randomly selected sequence without significant homology to all known human gene sequences. The 2'-OH of 2, 4-dihydroxypyrimidine in uracil of ds-RNA was replaced by 2′- fluoride [Figure 1]. The modified ds-P21 was named M-ds-P21. 2′- fluoride modification of ds-Control was used for M-ds-P21 control. siP53 was ordered from Sigma Co. Ltd (origin, USA).
|Figure 1: Schematic diagram of 2'-fluoride modification of ds-P21. ds-P21 targeted the P21 gene promoter -322 nucleotide and covered 20 bp length (a). The 2'-OH of uracil base in ds-RNA was replaced by 2′-fluoride (b). Five uracil bases in guide strand and six uracil bases in following strands were modified (c)|
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Cell culture and RNA transfection
Bladder cancer cell lines T24 was purchased from American Type Culture Collection (ATCC), cultured in regular RPMI containing 10% FBS and 1% penicillin-streptomycin. Double-stranded P21 (ds-P21), negative control RNA (ds-Cont), 2'-F-ds-P21 (M-ds-P21) and 2'-F-ds-control RNA (M-ds-Cont) labeled with FAM were synthesized from Shanghai Genepharma (Shanghai, SH, China). Transfection reagent-RNAiMAX was purchased from Invitrogen (Carlsbad, CA, USA). Before transfection, cells were plated in 3.5 cm dishes to reach about 30% confluent. 100 pmol ds-RNA and 5 ul transfection reagent in 100 ul regular RPMI (without FBS) were used for each transfection. Photographs of cell were captured with a cell culture by microscope (Olympus, Japan). siRNA of P53 was transfected to the cells 2 days before ds-P21 or M-ds-P21 treatment.
Cells were harvested at 48 h after transfection to assay P53 and P21 protein levels. Antibodies against P53 or P21 and GAPDH were purchased from cell signaling (Billerica, MA, USA). Cell pellets were lysed in Triton X-100/glycerol lysis buffer plus 1% sodium deoxycholate and 0.1% SDS, and then electrophoresed in 15% SDS-PAGE for P21 detection, and then transferred onto PVDF membranes for 4 hours. After blocking with 5% non-fat dry milk, the membranes were incubated with the primary antibodies at 1:1000 dilution for P21 and 1:3000 dilution for GAPDH at 4˚C during overnight, followed by the secondary antibodies conjugated with horseradish peroxidase at 1:8000 dilution for 2 hours in room temperature. Protein bands were visualized on X-ray film and data were scanned into personal computer.
The proteins related to the apoptosis including caspase-1, caspase-3 and caspase-7, and BCLAF1 were also assayed on the same experiment. The corresponding primary antibodies came from Santa Cruz (California, USA). All primary antibodies are of rabbit origin. The second antibody is goat anti-rabbit. The other is nearly the same to keep the minimal variation.
Apoptosis assay by flow cytometry
Annexin-V FITC Apoptosis Detection Kit was purchased from Roche (South San Francisco, CA, USA). The cells were trypsinized (including floating cells), washed twice with PBS, and then adjusted to 5 × 10 5 cells/500 ul in the binding buffer prior to incubation with Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI). The apoptotic population was investigated by flow-cytometry assay (BD FACSAria, Becton Dickinson, MD, USA).
T24 cells were grown on cover slips, transfected with M-ds-P21 and control. After an 8-hour transfection, cells were collected and washed twice with PBS, and fluorescence was analyzed using co-focal microscope (FV1000 Laser Scanning, Olympus). The intensity of fluorescence was divided into two categories: bright or dim. Randomly selected 10 high-power fields calculated the total bright fluorescence cells in each transfection group. Standard deviation was expressed, and statistical analyses were performed.
All experiments were repeated at least 3 times. Each indicator was quantified as the average value, expressed as X±M. The paired test was used for statistical analysis. P value was calculated by the SPSS 15.0 version. If P < 0.05, it was considered as statistically significant.
| > Results|| |
M-ds-P21 transfection induce morphology change and increase P21 protein expression
To evaluate if the activity of ds-P21 was affected by 2'-fluoride modification, we transfected the human bladder cancer cell line-T24 with mock, ds-Control, ds-P21, M-ds-Control and M-ds-P21. Three days later, morphology changes were observed in the ds-P21 and M-ds-P21 treated groups [Figure 2]a and M-ds-P21 induces more severe cell morphology changes. Most cells died, floated and clustered in the complete medium and M-ds-P21 transfected cells showed more cell death than ds-P21. Given that the cell death is the consequence of increased P21 protein expression, we speculated that M-ds-P21 has more P21 protein expression. Then, cells were collected for real-time PCR and Western blot analysis for P21. Surprisingly, the mRNA of P21 did not have any change among mock, ds-control, M-ds-control, ds-P21 and M-ds-P21 treatments (data not shown), but the protein level of P21 increased in both ds-P21 and M-ds-P21 treated groups, compared with the mock, ds-control and M-ds-control treatments (P < 0.05). Compared with ds-P21 treated group, further increase was observed in M-ds-P21 treated group (P < 0.01) [Figure 2]b and c. The protein level of P21 among mock, ds-control and M-ds-control treated groups showed no statistical difference (P > 0.05). The increased morphology changes and P21 protein levels are not due to the 2'-fluoride modification in M-ds-P21 treatment compared with ds-P21 treatment since there was no difference observed between M-ds-control treatment and ds-control treatment (P > 0.05) [Figure 2]b and c.
|Figure 2: M-ds-P21 and ds-P21 induced cell morphology change and P21 protein expression. Compared with ds-P21, M-ds-P21 induced severe morphology change in T24 cells (a). P21 protein level was dramatically increased by the ds-P21 transfection, especially further increased by the M-ds-P21 transfection (b). Statistical analysis of the P21 protein expression in each group: Mock, ds-Control, M-ds-Control, ds-P21 and M-ds-P21. Experiments were performed three times. The value was expressed as average ± standard deviation. '*' represents significant statistical difference (P < 0.05). '**' represents very significant statistical difference (P < 0.01)|
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2-Fluoride modification facilitates cell apoptosis
To quantify the apoptosis, we use the Annexin-V to stain the apoptotic cells transfected with ds-P21 and M-ds-P21. [Figure 3] showed that the apoptosis cells increased in ds-P21 or M-ds-P21 transfected cells compared with mock, ds-control and M-ds-control treated groups. Especially in M-ds-P21 transfected cells, the ratio of apoptotic cells to total cells reached to 56.5% compared with ds-P21 transfection, 40.4% (P < 0.05). The apoptotic cell increment parallels with the P21 protein increment showed in [Figure 2] and the increment is not due to the direct effect of 2'-fluoride modification since there was not much differences between M-ds-Control transfection and ds-control transfection [Figure 3]a and b.
|Figure 3: Flow-cytometry analysis of the cell apoptosis in each group. Annexin-V positive cells were dramatically increased after ds-P21 treatment, especially further increased after M-ds-P21 treatment (a). Statistical analysis of the apoptotic cells in each group: Mock, ds-Control, M-ds-Control, ds-P21 and M-ds-P21. Experiments were performed four times. The value was expressed as average ± standard deviation. '*' represents significant statistical difference (P < 0.05)|
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Apoptosis induced by M-ds-P21 and ds-P21 is not P53-dependent
P21 is a downstream gene of P53. To understand the role of P53 in the ds-P21 response, bladder cancer cells were transfected with siP53 two days before ds-P21 transfection. As showed in [Figure 4]a, P53 protein levels maintained the same level before and after ds-P21 transfection [Figure 4]a and b. This manifested that the up-regulation of P21 is not due to P53 change. In other words, although P53-mediated cell cycle arrest and apoptosis are the main patterns of cell death, the ds-P21 induced apoptosis is not mediated by P53. When the cells were pretreated with siP53 two days followed by ds-P21 transfection, siP53 down-regulated the P21 protein expression in mock or control groups [Figure 3]c and d but the P21 protein expression was recovered in cells treated with M-ds-p21 or ds-P21 [Figure 4]c and d.
|Figure 4: Role of P53 in the M-ds-P21 and ds-P21 induced apoptosis. P53 protein expression was not changed by the M-ds-P21 treatment or ds-P21 treatment (a). Statistical analysis of the P53 expression in each group: Mock, ds-Control, M-ds-Control, ds-P21 and M-ds-P21 (b). Experiments were repeated three times. The value was expressed as average ± standard deviation. siP53 reduced the P21 protein expression, but P21 protein level still could be up-regulated by the ds-P21 treatment or M-ds-P21 treatment(s). Statistical analysis of the P21 protein expression in each group: Mock, ds-Control, M-ds-Control, ds-P21 and M-ds-P21. Experiments were performed four times. The value was expressed as average ± standard deviation. '*' represents significant statistical difference (P < 0.05)|
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M-ds-P21 and ds-P21 induced apoptosis are caspase-1, 3, 7-dependent
As P21 protein is mainly degraded by caspase-3,  we first screened the apoptosis-related genes by RT-PCR. RT-PCR showed that caspase-1 and caspase-7 were unregulated, while caspase-3 and BCLAF1 were down-regulated with ds-p21 and M-ds-p21 treatment (data not shown). Then, we performed Western blot to confirm the results. Western blot showed that both ds-P21 and M-ds-P21 transfection up-regulated caspase-1 and caspase-7, as well down-regulated caspase-3 and BCLAF1 compared with mock, ds-Control and M-ds-Control treated group (P < 0.01) [Figure 5]. Interestingly, when compared with ds-P21, M-ds-P21 showed more increase of caspase-1 and caspase-7, as well as more decrease of caspase-3 (P < 0.05). Although there were no obvious differences of BLCAF1 between ds-P21 treatment and M-ds-P21 treatment, it is still significant when compared them with mock, ds-control and M-ds-control treatment (P < 0.05) [Figure 5]a and e. Results show that in [Figure 5] are the same samples of P53 study [Figure 4] to make the target protein comparable.
|Figure 5: Caspase-dependent apoptosis-related signal pathway analysis. Compared to ds-P21, M-ds-P21 significantly up-regulated caspase-1 and caspase-7, together with down-regulating caspase-3 and BCLAF1 (a). Statistically analysis of caspase-1 (b), caspase-7 (c), caspase-3 (d) and BCLAF1 (e) protein expression in each group: Mock, ds-Control, M-ds-Control, ds-P21 and M-ds-P21. Experiments were performed three times. The value was expressed as average ± standard deviation. '*' represents significant statistical difference in M-ds-P21 treatment compared to ds-P21 treatment (P < 0.05). '**'represents very significant statistical difference (P < 0.01). RE = relative expression = target gene/actin|
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2'-fluoride modification increases the bioavailability of ds-P21
To investigate the mechanism of the increased effect of M-ds-P21 compared with ds-P21, we labeled all ds-RNA with FAM fluorescence. Cells were collected and observed under confocal microscopy. The results clearly showed that there is more fluorescence molecule in the cells treated by M-ds-P21 and M-ds-Control than other treatments [Figure 6]a. Moreover, we noted that 2'-fluoride modified ds-control treatment had more molecular penetration than its partner ¾ ds-control. This indicates that 2'-fluoride modification in ds-RNA has a positive effect on penetration capability. The statistical analysis revealed that the cellular concentration of M-ds-P21 was nearly three times as ds-P21 [Figure 6]b.
|Figure 6: Bio-availability of M-ds-P21. Under confocal microscope, more fluorescence positive cells were observed in M-ds-P21 treatment (a). Statistical analysis of the fluorescence positive cells in each group: Mock, ds-Control, M-ds-Control, ds-P21 and M-ds-P21. Experiments were performed four times. The value was expressed as average ± standard deviation. '*' represents significant statistical difference in M-ds-Control treatment compared to ds-Control treatment (P < 0.05)|
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| > Discussion|| |
We evaluated the effect of M-ds-P21 on the apoptosis of bladder cancer T24 cells, and observed that M-ds-P21 is more powerful on cell apoptosis induction than on its counterpart ds-P21. Further study revealed that both M-ds-P21 and ds-P21 induced apoptosis is not P53-dependent, however, caspase-1, 3, 7 were involved in this process. Moreover, M-ds-P21 has more capability to enter into the cells than its counterpart, ds-P21, thus it may interpret the increased activity of the M-ds-P21.
The first observation of this study is that 2'-fluoride modification of double-stranded RNA improves the penetration capability. Although RNA activation (RNAa) is a newly defined phenomenon found in applying ds-P21, which targets the promoter region of P21 gene for siRNA investigation,  the mechanism for this phenomenon is largely unknown. This study takes this phenomenon as a good cell model for investigating the effect of chemical modification of double-stranded RNA on tumor cell apoptosis. It at least has two advantages: first, the effect could be directly observed by cell morphology under microscope; second, the apoptotic cells could be calculated by flow cytometry. In this study, we observed that the apoptotic cell population is dramatically increased after M-ds-P21 transfection. Further study revealed that this effect is partly because M-ds-P21 has more penetration capability than its counterpart ¾ ds-P21. As the same molecular structure of siRNA and ds-P21 (all contains A, U, G, C base), it is rational inferred that this modification may have the same application for siRNA. So this study provides a new method for chemical modification of siRNA/saRNA, it may have the advantage for the therapeutic use of the double strand RNA. Although our modification has some minimal difference from other 2'-F-RNA modifications investigated in siRNA study ,  we could infer that M-ds-P21 might enhance the bioactivity through below pathways based on other studies: 1 small size and high electronegativity of fluorine  2 increased target affinity  3 increased stabilization of the modified strand against nucleases  4 compatibility with the RNA-induced silencing complex (RISC)  5 decreased degradation and prolonged half-life of oligonucleotides  and 6 reduction in immunostimulatory effects.  For our study, we added the information that 2'-F-RNA enhanced the bioactivity from the beginning when it encountered the cells, it showed more molecules in the cells than its counterpart, ds-P21.
The second observation of this study is that both M-ds-P21 and ds-P21 induced T24 cells apoptosis is related to P21 protein increment. Previous studies showed that ds-P21 induced P21 protein expression occurred at transcript level, ,, but in our study, we did not find any P21 change in mRNA level. It does not rule out the rapid degradation of P21 mRNA, but a more rational explanation is that the increase of P21 protein may be due to the decreased degradation of P21 protein. One reason for this explanation is that we found caspase-3, the main molecular responsible for P21 protein degradation,  was dramatically decreased after ds-P21 treatment [Figure 5]a. Apoptosis induced by ds-P21 can lead to caspase-3 decrease,  thus then leading to decreased P21 protein degradation, and ultimately P21 protein may accumulate in cells. Whether apoptosis induces P21 protein increase, or contrarily, increased P21 protein induces apoptosis, needed to be profoundly investigated. The compromise conclusion is that both mechanisms exist in the ds-P21 treatment, and may form a vicious circle to induce the T24 cell apoptosis. No matter which mechanism, both M-ds-P21 and ds-P21 can induce apoptosis and increase the P21 protein expression. Both apoptosis and P21 increase is beneficial to combat the bladder cancer cells.
The third observation of this study is that both M-ds-P21 and ds-P21 induced apoptosis is not P53-dependent, but caspase-1, 3, 7-dependent. RNA activation is still in debate because of exact mechanism. Many people doubt that it is just off-target effect or by-product of other genes' down-regulation such as P53.  Our results show that P53 protein level was not affected by M-ds-P21 or ds-P21 treatment. M-ds-P21 and ds-P21 still induced the P21 protein increase after P53 was knocked down by siP53 treatment [Figure 4]. First, it manifests that the reason M-ds-P21 (ds-P21) induced apoptosis is not P53-dependent. Second, it also shows that M-ds-P21 (ds-P21) induced P21 protein increase is not through the P53-P21 signal pathway. Other novel signal pathways may involve this process. Moreover, we found M-ds-P21 induced apoptosis involves caspase-1 and caspase-7 increase while caspase-3 and BCLAF1 decrease. Caspase-1 is a apoptosis initiator, and also an enzyme that proteolytically cleaves other proteins, such as the precursor forms of the inflammatory cytokines interleukin 1-β and interleukin 18, into active mature peptides.  From this point, we inferred that apoptosis induced by ds-P21 treatment is mediated by caspase-1, and the intracell immunity observed in ds-P21 treatment need more caspase-1 to activate them. Caspase-7 is a member of the caspase (cysteine aspartate protease) family of proteins and has been shown to be an executioner protein of apoptosis.  Caspase-7 increase in this study potentially is a good indicator of T24 apoptosis induced by M-ds-P21 (or ds-P21) treatment. Just as mentioned above, whether caspase-3 decrease is the cause of P21 increase or the result of P21 increase is still under investigation, many studies had showed that caspase-3 and BCLAF1 decrease are promotional factors of cell apoptosis process.  Collectively, this study reveal the multifactorial role of M-ds-P21 (ds-P21) on apoptosis in bladder cancer T24 cells.
| > Acknowledgment|| |
We thank Dr. Yu Qi and Dr. Minghui Zhao for providing good advice and for editorial review of this paper.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
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