Survivin and p53 Modulate Quercetin-induced Cell Growth Inhibition and Apoptosis in Human Lung Carcinoma Cells*

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells. However, the regulation of survivin and p53 on the quercetin-induced cell growth inhibition and apoptosis in cancer cells remains unclear. In this study, we investigated the roles of survivin and p53 in the quercetin-treated human lung carcinoma cells. Quercetin (20-80 μm for 24 h) induced the cytotoxicity and apoptosis in both A549 and H1299 lung carcinoma cells in a concentration-dependent manner. Additionally, quercetin inhibited the cell growth, increased the fractions of G2/M phase, and raised the levels of cyclin B1 and phospho-cdc2 (threonine 161) proteins. Moreover, quercetin induced abnormal chromosome segregation in H1299 cells. The survivin proteins were highly expressed in mitotic phase and were located on the midbody of cytokinesis; however, the survivin proteins were increased and concentrated on the nuclei following quercetin treatment in the lung carcinoma cells. Transfection of a survivin antisense oligodeoxynucleotide enhanced the quercetin-induced cell growth inhibition and cytotoxicity. Subsequently, quercetin increased the levels of total p53 (DO-1), phospho-p53 (serine 15), and p21 proteins, which were translocated to the nuclei in A549 cells. Treatment with a specific p53 inhibitor, pifithrin-α, or transfection of a p53 antisense oligodeoxynucleotide enhanced the cytotoxicity of the quercetin-treated cells. Furthermore, transfection of a small interfering RNA of p21 enhanced the quercetin-induced cell death in A549 cells. Together, our results suggest that survivin can reduce the cell growth inhibition and apoptosis, and p53 elevates the p21 level, which may attenuate the cell death in the quercetin-treated human lung carcinoma cells.

Survivin is expressed in human cancer cells, but it is undetectable in most normal adult cells (1)(2)(3). It has been shown that survivin may exhibit anti-apoptotic effect and inhibit the activity of caspases in cancer cells (3)(4)(5)(6). The survivin activity resulted from the phosphorylation of threonine 34 by the mitotic kinase complex cdc2/cyclin B1 (7,8). Survivin has been demonstrated to promote mitotic progression (2,7,8) and cytokinesis (9 -11) in cancer cells. Moreover, it has been proposed that survivin may serve as a radio-and chemoresistance factor (8,12). Treatments with adriamycin and taxol increase survivin expression in cancer cells (8). However, a semisynthetic flavonoid, flavopiridol, suppresses the survivin phosphorylation on threonine 34 and enhances cancer cell apoptosis induced by anticancer agents, e.g. adriamycin and UVB irradiation (8).
p53 is a cellular gatekeeper for the cell growth and division (13)(14)(15). It has been shown that p53 can regulate cell cycle arrest, apoptosis, and DNA repair in a variety of cells (14,15). The p53 downstream effectors include p21, which participates in cell cycle arrest, and bax, which triggers apoptosis (13,14). Inhibition of p53 function may cause the decrease of DNA repair and the increase of genomic instability (16,17). Also, the p53 protein mediates the G 1 and G 2 /M checkpoints, which trigger the p21 to participate in cell cycle arrests (18). In addition, p21 can play a protective role on survival signal against apoptosis (19 -21). The diverse phosphorylation sites of p53 have been indicated to play important roles in the regulation of many cellular responses (22)(23)(24). Moreover, it has been proposed that the phosphorylation of p53 at serine 15 is an important target for p53 activation (25) and stabilization (22,26).
The antitumor effects of plant flavonoids have been reported to induce cell growth inhibition and apoptosis in a variety of cancer cells (27)(28)(29). Quercetin, a ubiquitous bioactive flavonoid, can inhibit the proliferation of cancer cells (30 -32). It has been shown that quercetin treatment caused cell cycle arrests such as G 2 /M arrest or G 1 arrest in different cell types (31)(32)(33)(34). Moreover, quercetin-mediated apoptosis may result from the induction of stress proteins, disruption of microtubules and mitochondrial, release of cytochrome c, and activation of caspases (32,(35)(36)(37). However, the roles of survivin and p53 proteins in the quercetin-induced cell growth inhibition and apoptosis in cancer cells are still not clear.
In this study, we investigated the expression of survivin, p53, and p21 proteins in quercetin-induced cell growth inhibition and apoptosis of the human lung cancer cells. We further studied the effects of survivin, p53, and p21 gene knockdowns by using the antisense oligodeoxynucleotides of survivin, p53, and a small interfering RNA (siRNA) 1 of p21, respectively, in the quercetin-treated lung carcinoma cells. The enhanced survivin expression may act to reduce cell growth inhibition and apoptosis, and p53-mediated elevation of the p21 level may moderate cell death in the quercetin-treated human lung carcinoma cells.

EXPERIMENTAL PROCEDURES
Chemicals and Antibodies-Hoechst 33258, pifithrin-␣, propidium iodide, quercetin, and 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazo-* This work was supported by Grant NSC 93-2320-B-320-014 from the National Science Council, Taiwan and Tzu Chi University Grant TCMRC 92009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 2. Effects of quercetin on cell growth and cell cycle progression in human lung carcinoma cells. A, the cells were plated at a density of 1 ϫ 10 6 cells/p100 Petri dish for 18 h. Then the cells were treated with 0 -80 M quercetin for 24 h. After drug treatment, the cells were washed twice with PBS and incubated for various times before they were counted by a hemocytometer. Results were obtained from 5-6 experiments, and the bars represent Ϯ S.E. p Ͻ 0.05 (*) and p Ͻ 0.01 (**) indicate the difference between untreated and quercetin-treated samples. B, the cells were treated with 0-60 M quercetin for 24 h. After treatment, the cells were trypsinized and then subjected to flow cytometry analyses. The data represented the average values from three experiments. C, the cells were exposed to 0 -60 M quercetin for 24 h. The total protein extracts were prepared for Western blot analysis using anti-cdc2, anti-cyclin B1, anti-cyclin D1, anti-phospho-cdc2 (threonine 161), and anti-ERK-2 antibodies.
were incubated with rabbit anti-apoptosis inducing factor antibody and then incubated with goat anti-rabbit Cy5. The apoptosis inducing factor protein displayed red fluorescence with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. Arrows indicate the apoptotic nuclei. D, the H1299 cells were treated with 60 M quercetin for 24 h. The F-actin was stained with BODIPY FL phallacidin, which displayed green fluorescence. Arrows indicate the apoptotic nuclei. E, the percentage of apoptosis was scored from the apoptotic nuclei. Results were obtained from 4 -5 experiments, and the bars represent mean Ϯ S.E. p Ͻ 0.05 (*) and p Ͻ 0.01 (**) indicate the difference between untreated and quercetin-treated samples. Cell Culture-The p53-contained A549 cell line was derived from lung carcinoma of a 58-year-old Caucasian male (38). The H1299 cell line was p53-null and was derived from a non-small cell lung adenocarcinoma tumor (39,40). These lung carcinoma cell lines were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, L-glutamine (0.03%, w/v), and sodium bicarbonate (2.2%, w/v). The cells were cultured at 37°C and 5% CO 2 in a humidified incubator (310/Thermo, Forma Scientific, Inc., Marietta, OH).
Cytotoxicity Assay-The cells were plated in 96-well plates at a density of 1 ϫ 10 4 cells/well for 16 -20 h. Then the cells were treated with 0 -80 M quercetin for 24 h in serum-free RPMI 1640 medium. After drug treatment, the cells were washed twice with phosphatebuffered saline (PBS) and were re-cultured in complete RPMI 1640 medium (containing 10% fetal bovine serum) for 2 days. Subsequently, the medium was replaced, and the cells were incubated with 0.5 mg/ml of MTT in complete RPMI 1640 medium for 4 h. The surviving cells converted MTT to formazan that generates a blue-purple color when dissolved in dimethyl sulfoxide. The intensity was measured at 545 nm using a plate reader (OPTImax, Molecular Dynamics) for enzymelinked immunosorbent assays. The relative percentage of survival was calculated by dividing the absorbance of treated cells by that of the control in each experiment.
Apoptosis Assay-The adherent cells were cultured on coverslips. After exposure to quercetin, the cells were washed twice with isotonic PBS (pH 7.4). The cells were stained with annexin V-FITC (Strong Biotech, Taipei, Taiwan) in binding buffer containing propidium iodide for 30 min in the dark. Then the cells were washed three times with isotonic PBS and analyzed using a fluorescence microscope.
To score the percentage of apoptosis, the adherent cells were cultured on coverslips. After drug treatment, the cells were washed with isotonic PBS (pH 7.4) and then fixed in 4% paraformaldehyde solution in PBS for 1 h at 37°C. Then the nuclei were stained with 2.5 g/ml Hoechst 33258 for 10 min. The number of apoptotic nuclei was counted by a hemocytometer under a fluorescence microscope. A total of 200 cells were examined for the calculation of apoptotic percentage in each treatment.
Cell Cycle Analysis-The cells were plated at a density of 1 ϫ 10 6 cells per p60 Petri dish in complete RPMI 1640 medium for 16 -20 h. Then the cells were treated with 0 -60 M quercetin for 24 h in serumfree RPMI 1640 medium. At the end of treatment, the cells were collected and fixed with ice-cold 70% ethanol overnight at Ϫ20°C. After centrifugation, the cell pellets were treated with 4 g/ml propidium iodide solution containing 100 g/ml RNase and 1% Triton X-100 for 30 min. Subsequently, the samples were analyzed in a FACScalibur system (BD Biosciences) using CellQuest software. The percentage of cell cycle phases was analysis by a ModFit LT software (Version 2.0, BD Biosciences).
Cell Growth Assay-The cells were plated at a density of 1 ϫ 10 6 cells per p100 Petri dish in complete RPMI 1640 medium for 16 -18 h. Then the cells were treated with 0 -80 M quercetin for 24 h in serum-free RPMI 1640 medium. After drug treatment, the cells were washed twice with PBS and re-cultured in complete RPMI 1640 medium. Subsequently, the cells were incubated for various times before they were counted by a hemocytometer.
Indirect Immunofluorescence and Confocal Microscopy-The cells were cultured on coverslips, which were kept in a p60 Petri dish for 16 -20 h before treatment. After treatment with or without 60 M quercetin for 24 h, the cells were washed with isotonic PBS (pH 7.4) and then fixed in 4% paraformaldehyde solution in PBS for 1 h at 37°C. Then the coverslips were washed three times with PBS, and nonspecific binding sites were blocked in PBS containing 10% normal goat serum, 0.3% Triton X-100 for 1 h. The cells were incubated with rabbit antisurvivin antibody (1:250) in PBS containing 0.3% Triton X-100 and 10% normal goat serum overnight at 4°C and washed three times with 0.3% Triton X-100 in PBS. Then the cells were incubated with goat antirabbit Cy5 (1:250) in PBS containing 0.3% Triton X-100 and 10% normal goat serum for 2-3 h at 37°C and washed three times with 0.3% Triton X-100 in PBS. The nuclei were stained with 2.5 g/ml Hoechst 33258 for 10 min. The actin filament (F-actin) was stained with 20 units/ml BODIPY FL phallacidin for 30 min. Finally, the samples were stored in the dark at 4°C until examined under a Leica confocal laser scanning microscope (Mannheim, Germany) that was equipped with a UV laser (351/364 nm), an argon laser (457/488/514 nm), and a HeNe laser (543 nm/633 nm). The emitted fluorescence was displayed through the frame store in the computer, and the images were processed for presentation by Adobe Photoshop software (Version 6.0, Adobe Systems, San Jose, CA).

FIG. 3. The expression and location of survivin proteins in a variety of human cancer cells.
A, the total protein extracts were prepared for Western blot analysis using anti-survivin and anti-ERK-2 antibodies. B, the cells were incubated with rabbit anti-survivin antibody and then incubated with goat anti-rabbit Cy5. The survivin protein displayed red fluorescence with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. The F-actin was stained with BODIPY FL phallacidin, which displayed green fluorescence. Arrows indicate the location of survivin proteins in the midbody of cytokinesis.
Transfection-The antisense oligodeoxynucleotides were synthesized by Medclub Scientific Co. (Taipei, Taiwan) in the form of phosphorothioate oligodeoxynucleotides. The sequence of survivin antisense oligodeoxynucleotide was 5Ј-CCCAGCCTTCCAGCTCCTTG-3Ј (41), p53 antisense oligodeoxynucleotide was 5Ј-CCCTGCTCCCCCCTGGCTCC-3Ј, and the control oligodeoxynucleotide was 5Ј-GGAGCCAGGGGG-GAGCAGGG-3Ј. These oligodeoxynucleotides were employed for transfections in A549 cells. The cells (5 ϫ 10 5 cells/p35 dish) were transfected with 100 nM of control or antisense oligodeoxynucleotides by using LipofectAMINE TM 2000 (Invitrogen) in 1 ml of serum-free medium for 5 h at 37°C in a CO 2 incubator according to the manufacturer's recommendations. Then, 1 ml of Dulbecco's modified Eagle's medium with 20% fetal bovine serum was added without removing the transfection mixture, and incubation proceeded for an additional 24 h. In addition, a p21 siRNA (Santa Cruz Biotechnology, Inc.) was utilized for the p21 gene knockdown in A549 cells according to the manufacturer's recommendations. After transfection, the cells were subjected to cell growth or MTT assay as described above.
Statistical Analysis-Data were analyzed using Student's t test, and significant differences were found between values obtained in the population of cells treated with different conditions. A p value of Ͻ 0.05 was considered to be statistically significant in the experiments.

Quercetin Induces Cytotoxicity and Apoptosis in Human
Lung Carcinoma Cells-The human lung cancer cell lines were treated with 0 -80 M quercetin for 24 h. As shown in Fig. 1A, quercetin induced cell death in a concentration-dependent manner, and ϳ70 and 30% of A549 and H1299 cells, respec- tively, survived after exposure to 40 M quercetin for 24 h. However, the LC 50 values of quercetin toward cultured human normal lung fibroblasts were Ͼ 120 M (results not shown). We further assessed apoptosis from the cells that had been exposed to quercetin. Quercetin-treated cells were heavily stained with annexin V-FITC (early apoptotic cells) that displayed green color (Fig. 1B, lower right panel, stars) and increased the nuclear fragmentation and apoptotic bodies (Fig. 1C and Fig. 1D, arrows) in both A549 and H1299 cells. Quercetin partially induced the translocation of apoptosis inducing factor into the nuclei in A549 cells (Fig. 1C). Moreover, quercetin caused a significantly higher level of apoptosis inducing factor translocation into the nuclei of H1299 cells (data not shown). The late apoptotic cells were stained with annexin V-FITC and propidium iodide (Fig. 1B, lower right panel, arrows). In contrast, the control cells were not significantly stained with fluorochrome (Fig. 1B, upper right panel). The percentage of apopto-sis was counted under a fluorescence microscope. As shown in Fig. 1E, the percentage of apoptosis was increased by quercetin (40 -60 M) in A549 cells in a concentration-dependent manner. Quercetin at 60 M induced about 25% of apoptosis in H1299 cells (Fig. 1E).

Quercetin Induces Cell Growth Inhibition and Increases the Fractions of G 2 /M Phase in Human Lung Carcinoma Cells-To
examine the effect of quercetin on tumor cell growth, A549 cells were plated at a density of 1 ϫ 10 6 cells per p100 dish and were treated with 0 -80 M quercetin for 24 h. Cell numbers were counted using a hemocytometer. As shown in Fig. 2A fraction while increasing the G 2 /M fraction in both A549 and H1299 cells (Fig. 2B). Moreover, immunoblot analysis showed that quercetin increased the levels of cyclin B1 and phospho-cdc2 (threonine 161) proteins in both A549 and H1299 cells (Fig. 2C). However, quercetin did not significantly change the levels of cyclin D1 and cdc2 proteins in these cells (Fig. 2C). ERK-2 protein was used as an internal control in this study.
The Expression and Location of Survivin Protein in a Variety of Human Cancer Cells-To examine the expression of survivin in cancer cells, total cellular proteins were extracted and subjected to immunoblot analysis. As shown in Fig. 3A, the human cancer cell lines including lung cancer (A549 and H1299), colon cancer (RKO and SW480), cervical cancer (HeLa), and breast cancer (MCF-7) expressed the high levels of survivin proteins. To further study the location of survivin protein, the cancer cells were subjected to immunofluorescence staining and confocal microscopy. As shown in Fig. 3B, the intensity of red fluorescence (Cy5) exhibited by survivin expressed in a variety of cancer cells. The survivin proteins were highly expressed in mitotic phase and concentrated on the midbody of cytokinesis of cancer cells (Fig. 3B and Fig. 4A, arrows).

Quercetin Induces Abnormal Chromosome Segregation and Increases the Level of Survivin Proteins in Human Lung
Carcinoma Cells-To investigate the effect of quercetin on the survivin protein expression, cells were treated with quercetin and subjected to immunofluorescence staining and immunoblot analysis. The survivin proteins were highly expressed in mitotic phase and concentrated on the midbody of cytokinesis in both A549 and H1299 cells (Fig. 4A and Fig. 5, arrows). Moreover, quercetin increased the level of survivin proteins in A549 cells (Fig. 4B) in a concentration-dependent manner. The red fluorescence (Cy5) intensity exhibited by survivin was significantly increased when exposed to 60 M quercetin for 24 h in both A549 and H1299 cells (Fig. 4C and Fig. 5A). The increased survivin proteins were concentrated on the nuclei of these cells ( Fig. 4C and Fig. 5A). In addition, quercetin induced abnormal chromosome segregation in H1299 cells (Fig. 5, B and C, lower panels). In this respect, quercetin induced a significantly higher degree of abnormal chromosome segregation in H1299 than in A549 cells (data not shown). The increased survivin proteins were co-localized with abnormal chromosomes in the quercetin-treated H1299 cells (Fig. 5, B and C, lower panels).
Survivin Antisense Oligodeoxynucleotide Enhances Quercetin-induced Cell Growth Inhibition and Cytotoxicity-A survivin antisense oligodeoxynucleotide was used for transfection in A549 cells to examine the effect of survivin on quercetininduced cell growth inhibition and cell death. As shown in Fig.  6A, treatment with quercetin (60 M for 24 h) or a transfection of survivin antisense oligodeoxynucleotide inhibited the cell growth in A549 cells. Moreover, the inhibition of cell growth caused by quercetin (60 M for 24 h) was significantly enhanced by transfection with the survivin antisense oligodeoxynucleotides (Fig. 6A). To further investigate the role of survivin in quercetin-induced cytotoxicity, the survivin antisense oligodeoxynucleotide-transfected cells were treated with or without quercetin (60 M for 24 h), and the percentage of cell survival was estimated by MTT assay. As shown in Fig. 6B, treatment with quercetin or transfection of a survivin antisense oligodeoxynucleotide induced cell death in A549 cells. Subsequently, the cytotoxicity caused by quercetin was significantly enhanced by transfection with the survivin antisense oligodeoxynucleotides (Fig. 6B).
Quercetin Increases the Levels of Total p53 (DO-1), Phospho-p53 (Serine 15), and p21 Proteins in A549 Cells-We have investigated the possible roles of p53 and p21 in the quercetin-FIG. 6. Effects of a survivin antisense oligodeoxynucleotide on the cell growth inhibition and cytotoxicity induced by quercetin. A, the cells were plated at a density of 5 ϫ 10 5 cells/p60 Petri dish for 18 h. The survivin antisense oligodeoxynucleotide transfected-cells were exposed to 60 M quercetin for 24 h in serum-free medium. Then the cells were washed twice with PBS and incubated for various times before they were counted by a hemocytometer. B, the cell survival was measured by MTT assay. Results were obtained from four experiments, and the bars represent Ϯ S.E. p Ͻ 0.05 ( # ) and p Ͻ 0.01 ( ## ) indicate the difference between the control and the survivin antisense oligodeoxynucleotides-transfected cells without quercetin treatment. p Ͻ 0.05 (*) and p Ͻ 0.01 (**) indicate the difference between the control and the survivin antisense oligodeoxynucleotides transfected-cells after quercetin treatment. mediated cytotoxicity of lung carcinoma cells. The green fluorescence (FITC) intensity exhibited by p53 (DO-1) was significantly increased following exposure to 60 M quercetin for 24 h in A549 cells (Fig. 7A, lower right panel). A549 cells were treated with quercetin, and the protein level of p53 (DO-1) was assayed by the immunoblot. As shown in Fig. 7B, treatment with quercetin at 20 -60 M for 24 h or at 40 M for 4 -16 h significantly increased the levels of p53 (DO-1) proteins. In addition, quercetin (60 M for 24 h) increased the level of phospho-p53 (serine 15) proteins, which were concentrated on the nuclei in A549 cells (Fig. 7C, arrows). Also, the p53 downstream p21 protein was increased (Fig. 7D) and concentrated on the nuclei in the quercetin-treated A549 cells (Fig. 7E, arrows).
Pifithrin-␣, p53 Antisense Oligodeoxynucleotide, and p21 siRNA Enhance Quercetin-induced Cytotoxicity in A549 Cells-A specific p53 inhibitor, pifithrin-␣ (42), or a p53 antisense oligodeoxynucleotide was used to examine the effects of p53 on the levels of cell death and p21 protein in quercetintreated A549 cells. As shown in Fig. 8A, pifithrin-␣ (20 M for 24 h), which alone was without effect on cell survival, significantly enhanced the cytotoxicity mediated by quercetin. Similarly, transfection of a p53 antisense oligodeoxynucleotide enhanced the quercetin-induced cell death in A549 cells (Fig. 8B). Interestingly, transfection of a p53 antisense oligodeoxynucleotide reduced the elevation of p21 protein expression in the quercetin-treated A549 cells (Fig. 8C). To further investigate  8. Effects of a p53 inhibitor or a p53 antisense oligodeoxynucleotide on the cytotoxicity and the level of p21 proteins in quercetin-treated A549 cells. A, the A549 cells were treated with 60 M quercetin alone or in combination with 20 M pifithrin-␣ for 24 h. The cell survival was measured by MTT assay. Results were obtained from 4 -6 experiments, and the bars represent Ϯ S.E. **, p Ͻ 0.01 indicates the difference between quercetin alone and quercetin in combination with pifithrin-␣. B, the control or p53 antisense oligodeoxynucleotide-transfected cells were exposed to 60 M quercetin for 24 h. The cell survival was measured by MTT assay. C, the total protein extracts were prepared for Western blot analysis using anti-p21, anti-p53, and anti-ERK-2 antibodies. D, the control or p21 siRNA transfected-cells were exposed to 40 M quercetin for 24 h. The cell survival was measured by MTT assay. Results were obtained from three experiments, and the bars represent Ϯ S.E. *, p Ͻ 0.05 indicates the difference between the control and the transfection with p53 antisense oligodeoxynucleotides or p21 siRNA after quercetin treatment.
the role of p21 in quercetin-induced cytotoxicity, the p21 siRNA-transfected cells were treated with or without quercetin (40 M for 24 h), and the percentage of cell survival was estimated by MTT assay. As shown in Fig. 8D, p21 siRNA alone did not affect the cell survival. However, cells transfected with p21 siRNA were more sensitive to quercetin-induced cytotoxicity than the control cells (Fig. 8D).

DISCUSSION
The balance between apoptosis and anti-apoptosis signal pathways plays a role in the pathogenesis of a variety of cancers (43,44). Survivin has been demonstrated to inhibit apoptosis and promotes the mitotic progression in cancer cells (4,5,7). The inhibition of survivin would block the anti-apoptosis and mitotic progression in tumor cells, providing an important strategy in cancer therapy (8). However, treatment with anticancer drugs, such as adriamycin and taxol, increased survivin expression in cancer cells (8). It has been proposed that survivin may serve as a radio-and chemoresistance factor (8,12). In the present study, we found that quercetin induced the cell growth inhibition and apoptosis in human lung carcinoma cells. However, quercetin increased the expression of survivin proteins. The survivin activity resulted from the phosphorylation of threonine 34 by the mitotic kinase complex cdc2/cyclin B1 (7,8). We found that quercetin increased the levels of cyclin B1 and phopho-cdc2 (threonine 161) proteins in the lung carcinoma cells. The activation of cdc2 is through phosphorylation of threonine 161 by cdc2 activating kinase (45). Our results indicate that the increases of cyclin B1 proteins and cdc2 kinase activity may involve in the enhancement of survivin protein expression in the quercetin-treated cells (Fig. 9). Moreover, the inhibition of survivin by transfection with a survivin antisense oligodeoxynucleotide enhanced the quercetin-induced cell growth inhibition and cell death in the lung carcinoma cells. Therefore, we suggest that survivin expression may act to reduce the cell growth inhibition and apoptosis in the quercetin-treated human lung carcinoma cells.
Subsequently, we found that quercetin increased the expression of survivin proteins, which were concentrated on the nuclei in both A549 (p53-contained) and H1299 (p53-null) lung carcinoma cells. The data indicate that quercetin-mediated increase in survivin expression may result from a p53-independent pathway. Moreover, the H1299 cells were more susceptible to quercetin-induced cytotoxicity, apoptosis, and abnormal chromosome segregation than A549 cells after treatment. The inhibition of p53 by transfection with a p53 antisense oligodeoxynucleotide or a specific p53 inhibitor, pifithrin-␣, enhanced quercetin-induced cell death in A549 cells. It has also been reported that disruption of normal p53 function may decrease DNA repair and increase genome instability (16,17). Thus, we suggest that p53 protein appears to reduce the cell death and to maintain the genome stability in quercetin-treated lung carcinoma cells.
The protective role of p53 is dependent on its downstream gene, p21 (20). p21 can participate in survival signal against apoptosis (19,46). We observed that quercetin increased the levels of total p53, phospho-p53 (serine 15), and p21 proteins in A549 cells. Moreover, the phospho-p53 (serine 15) and p21 proteins were found to translocate to the nuclei of quercetin-treated cells. Phosphorylation of serine 15 was a key target site of p53 for its activation and stabilization (25,26). The phosphorylation of p53 (serine 15) may transmit a survival signal and may suppress apoptosis in response to several stimuli (47). Additionally, the p21 may rescue cells from an apoptotic state to survival condition (19,21). It has been shown that phosphorylation of p53 (serine 15) increases the level of p21 proteins and induces the G 2 /M cell cycle checkpoint (47). In this study, a specific p53 inhibitor, pifithrin-␣, or a p53 antisense oligodeoxynucleotide significantly enhanced the cytotoxicity concomitant with the decrease of p21 protein expression in the quercetin-treated A549 cells. Furthermore, transfection of p21 siRNA enhanced the cell death in the quercetin-treated A549 cells. Accordingly, our results suggest that p53 elevates the p21 level, which may attenuate the quercetin-induced cell death in lung carcinoma cells. However, the bax protein has been shown to be a downstream protein of p53 for the induction of apoptosis (14,48). Thus, the role of p53 regulates the bax expression in the quercetin-induced apoptosis that need further investigation.
In summary, we propose that the survivin expression can reduce the cell growth inhibition and apoptosis, and the p53regulated p21 protein expression appears the reduced cell death and aberrant mitosis in lung carcinoma cells following quercetin treatment (Fig. 9). Survivin may support p21/procaspase 3 complex formation resulting in the suppression of cell death signaling (49). Nevertheless, quercetin can mediate the differential pathways for aberrant mitosis and apoptosis via the induction of stress proteins, disruption of microtubules and mitochondrial, release of cytochrome c, and activation of caspases (32,(35)(36)(37). Understanding the mechanisms by which survivin, p53, and p21 pathways modulate the quercetin-induced cell growth inhibition and apoptosis of cancer cells may contribute to the development of novel therapeutic strategies that can control the apoptotic and anti-apoptotic balance in such disease states.