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Sunitinib can enhance BCG mediated cytotoxicity to transitional cell carcinoma through apoptosis pathway
Urologic Oncology: Seminars and Original Investigations 30 (2012) 652– 659
Original article
Sunitinib can enhance BCG mediated cytotoxicity to transitional cell carcinoma through apoptosis pathway夡 Szu-Yuan Ping, M.S.a,1, Chia-Lun Wu, M.S.b,1, Dah-Shyong Yu, M.D, Ph.D.b,* b
a Graduate Institute of Life Science and Pathology, National Defense Medical Center, Taipei, Taiwan, Republic of China Uro-Oncology Laboratory, Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
Received 12 May 2010; received in revised form 5 July 2010; accepted 6 July 2010
Abstract Bacillus Calmette-Guerin (BCG)-refractory generated a high risk to patients with bladder cancer during treatment. Tyrosine kinase receptor (TKR) and TKR-mediated signal transduction pathways play an important role in tumor initiation, maintenance, angiogenesis, and vascular proliferation. Theoretically, it is helpful in adjuvant treatment for transitional cell carcinoma (TCC). Hence, we proposed that sunitinib, a endothelial growth factor receptor (VEGFR) inhibitor, may have a synergistic effect with BCG in enhancing its cytotoxicity to bladder cancer. The level of VEGF in various TCC cell lines was quantified by real time PCR. High grade TCC-T24 cell line with high level of VEGF expression was selected as representative tumor cells for further study. The single drug and combined inhibitory effects of BCG and sunitinib in T24 cells were determined by MTT method. The drug mediated cell apoptosis in T24 cells was characterized by flow cytometry with PI and annexin V stain. Bcl-2 apoptotic pathway induction by BCG and sunitinib treatment was evaluated by Western blotting method. Inhibitory ability of sunitinib in BCG induced cell migration was verified by cell migration assay. The results shown that expression level of VEGF mRNA in high grade T24 cells was higher than low grade J82, TSGH 8301, and TCC 9202 cell lines. Both BCG and sunitinib treatment presented cytotoxic effect to T24 cells in a dose-dependent manner. Combination of BCG and sunitinib revealed superior cytotoxicity effect than single agent when cells were pretreated with low dosage BCG before sunitinib treatment. By Annexin V analysis it was observed that cell death associated with increased early and late apoptosis process individually. Furthermore, the bcl-2 expression was significant reduced in T24 cells in metachronous BCG and sunitinib combination treatment than single agent. Tumor cell migration activity was also markedly inhibited with BCG and sunitinib combination treatment. In conclusion, these results suggested that during BCG and sunitinib combination treatment both reagents interacted with each other and caused TCC cells apoptosis in addition to direct cytotoxicity. This combination therapeutic model may have the potential for future clinical application to bladder cancer treatment. © 2012 Elsevier Inc. All rights reserved. Keywords: Bacillus Calmette-Guerin (BCG); Sutent; Synergism; Apoptosis; Combined therapy
1. Introduction Transitional cell carcinoma (TCC) of the bladder is the fifth most common solid malignancy in the United States. Over 131,260 new case and over 14,680 deaths of this cancer are estimated for the year 2010 [1]. In Taiwan 1,985 new cases and 681 deaths of bladder cancer were recorded for the year 2006 [2]. Approximately 30% of patients with superficial tumors 夡
This study was supported by grants from the National Science Council (NSC97-2314-B-016-022MY) and Tri-Service General Hospital (TSGH-C98-44). * Corresponding author. Tel.: ⫹886-2-8792-7008; ⫹886-2-8792-7009. E-mail address: [email protected] (D.-S. Yu). 1 These authors contributed equally to this work. 1078-1439/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2010.07.001
will progress to invasive TCC, where radical cystectomy is the standard therapy. Unfortunately, this disease recurs in up to 50% of these patients despite surgery, and is potentially lethal [3]. The treatment goal for superficial bladder cancer is reducing tumor recurrence and preventing tumor progression, which would require additional aggressive therapies. The most common immunotherapeutic agent, BCG (bacillus Calmette-Guérin) is an attenuated mycobacterium developed from the Mycobacterium bovis strain. The anticancer effect of BCG in the treatment of bladder cancer with a complex local immune response, including activated B, T lymphocytes and nature killer cell by inducing multiple cytokines; IL-1, IL-6, IL-8, and GMCSF [4–7]. The critical role of angiogenesis in tumor growth and metastasis has prompted many efforts to develop anti-angiogenic
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therapies, thus providing a rational drug target for using angiogenic inhibitors in bladder cancer tumors [8 –10]. Sunitinib malate is one of several new anti-angiogenic agents undergoing tests of efficacy in the treatment of various types of cancer including gastrointestinal stromal tumors and renal cell carcinoma [11,12]. Several split tyrosine kinase receptor (TKR) domain, namely the vascular endothelial growth factor (VEGF), VEGF receptors, and PDGFR, play prominent roles in tumor angiogenesis [13–15]. Sunitinib malate can block the angiogenesis signaling cascade (VEGF receptors, Flk-1/KDR, and Flt-1) and regulate tumor growth and its angiogenesis. Therefore, inhibiting these targets might be expected to result in broad antitumor efficacy. This study tries to verify the possible synergism effect of BCG and sunitinib malate in treating TCC and its underlying mechanisms.
2. Materials and methods 2.1. Cell culture and reagents The TCC cell lines, T24 (ATCC Rockville, MD), TSGH8301, TSGH8701, TSGH8702, and TCC9202 (established in Uro-Oncology Laboratory, Tri-Service General Hospital) were cultivated in RPMI-1640 medium supplemented with 10% fetal bovine serum, with 100 U/ml penicillin, 50 mg/ml streptomycin, and 2 mM glutamine (Thremo Scientific Hyclone, Logan City, Utah State, UT). Confluent cells were detached with 0.01 M EDTA. BCG (ImmuCyst 81 mg/vial) was purchased from the Sanofi Pasteur Ltd. West Toronto, Ontario, Canada. ImmuCyst 81 mg is freeze-dried preparation made from a culture of the Connaught strain of bacillus Calmette Guérin, which is an attenuated strain of living bovine tubercle bacillus, Mycobacterium bovis. Sunitinib malate (sutent) was kindly provided by Pfizer Inc., Taiwan without financial interest. 2.2. RNA extraction and cDNA synthesis Total RNA was extracted from cultured cells using a commercially available RNA extraction kit (Trizol; Gibco, Carlsbad, CA) according to the manufacturer’s instructions. To avoid contamination by DNA during RNA preparation, RNA was digested with RNase-free DNAse I (Gibco), as recommended by the supplier. cDNA was synthesized in a reaction containing 1 g of total RNA as a template, 0.5 g random hexamers (Promega, Madison, WI), and 200 units of Moloney murine leukemia virus RNase H minus reverse transcriptase (Promega), in a total volume of 25 l according to the manufacturer’s protocol. Duplicate reactions were done both in the presence and absence of the reverse transcriptase enzyme to control for false positive amplification resulting from contaminating chromosomal DNA.
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2.3. Quantitative RT-PCR (qRT-PCR) Detection of VEGFa mRNA was conducted by a twostep procedure using the Light Cycler CK 20 mRNA Quantification Kit (Roche Diagnostics, Indianapolis, IN). First, cDNA was synthesized with a reverse transcription kit (Reverse Transcriptase Master Mix; Roche Diagnostics) using 5 l of RNA and oligo dT serving as primers in a total volume of 20 l. The second step was to amplify 2 l of the cDNA by qRT-PCR using 2 l of the LightCycler PCR Master Mix (CK 20 Detection Mix; Roche Diagnostics). During the qRT-PCR reaction, a 196 bp fragment of VEGFa encoding mRNA was amplified from the cDNA. The amplicon was detected by fluorescence using a specific pair of VEGFa primers: CCC AGG TCA GAC GGA CAG AAA G for VEGFa and AAG CAG GTG AGA GTA AGC GAA GG for VEGFa. The fluorescence emitted after hybridization to the template DNA was measured by the LightCycler 480 instrument. In a separate real time PCR reaction, mRNA encoding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using primers ACC CAC TCC TCC ACC TTT GAC for GAPDH, CAT ACC AGG AAA TGA GCT TGA CAA for GAPDH-rev was processed as when used as a housekeeping gene. Its product served as a control for RNA and relative quantification. All PCR reactions were performed for 40 cycles of 60 seconds at 95°C, 10 seconds at 60°C, and 5 seconds at 72°C. 2.4. Evaluation of the effect of BCG and sunitinib malate on cancer cell viability Cells were exposed to BCG reagent combined with various concentrations of each of the sunitinib for 72 hours. Cellular chemosensitivity was assayed using a modified 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium (MTT; Sigma, St. Louis, MO) assay to determine cell viability in vitro. In brief, T24 cells (4,000 per well) in 100 L culture medium were seeded into 96-well microplates and incubated at 37°C for 24 hours before drug exposure. The plated cell numbers were calculated to keep control cells growing in the exponential phase throughout the 72 hours incubation period. For concurrent treatment, cells were treated with both single BCG reagent and sunitinib (each in 100 l of culture medium) simultaneously and incubated for 72 h. At the point, 50 l of MTT (2 mg/ml in RPMI medium) was added to each well and allowed to react for 2.5 hours. The blue formazan crystals formed were pelleted to the well bottoms by centrifugation, separated from the supernatant, and dissolved in 150 l of DMSO. The optical density was determined by absorbance spectrometry at 492 nm using a microplate reader (MRX-2; Dynex Technologies, Inc., Chantilly, VA). Three separate experiments with triplicate runs in each were performed to obtain mean cell viability. The drug concentrations inhibiting cell growth by 50% (IC50) were determined using a dose– effect analysis model as previously described [16].
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2.5. Median effect analysis of combined cytotoxic effects The combined effects of BCG and sunitinib were subjected to median effect analysis with the mutually nonexclusive model as previously described [17]. Control experiments replaced the active agent with drug-free medium. By combining 2 agents at graded concentrations, numerous combined effects of growth inhibition were obtained and analyzed using the computer software Calcusyn1 (ver, 1.1.1, 1996; Biosoft Inc., Cambridge, UK). For each combined effect of growth inhibition or fraction affected (fa), a combination index (CI) is generated. The effects of the combination are then transformed and displayed as fa–CI plots. CI ⬍ 1, ⫽ 1, and ⬎1 indicate synergism, additivism, and antagonism, respectively. Varied degrees of synergism or antagonism may occur in distinct Fa ranges. We also used the statistical function f(u)1,2 ⫽ f(u)1 ⫹ f(u)2, where f(u)1 ⫽ the fraction unaffected by drug 1, f(u)2 ⫽ the fraction unaffected by drug 2, and f(u)1,2 ⫽ the fraction unaffected by drugs 1 and 2. The expected (presumed to be additive) and observed survival rates of T24 cells were thus obtained from the three independent drug-combined treatments. A statistically significant difference was analyzed by Student’s t-test when P ⬍0.05. 2.6. Annexin V/PI stain for cell apoptotic analysis Cell viability was detected by trypan blue and apoptosis was evaluated by the annexin V/propidium iodide (BD Biosciences, Franklin Lakes, NJ) double staining assay following the manufacturer’s instructions. T24 cells were cultivated with medium alone or medium containing 0.81 mg (1.86 ⫻ 106 CFU) BCG combined with sunitinib (6 M). 106 cells in 10 cm culture dish were harvested and washed with PBS. T24 cells were harvested at the end of treatment, rinsed twice with PBS, and stained with Annexin V-FITC apoptosis detection kit I (BD Biosciences). Analysis was performed on the FACS Calibur using CellQuest. 2.7. Western blotting for bcl-2, bcl-XL, and bim expression 106 exponentially growing T24 cells were co-treated with BCG 0.81 mg (1.86 ⫻ 106 CFU) combined with sunitinib (6 M) at the indicated concentrations for 12, 24, or 48 hours. Then they were pelleted by centrifugation, washed with PBS two times and resuspended in 100 l of lysis buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 2% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 1 mg/ml aprotinin, and sonicated. Protein (50 g) was electrophoresed for 2 hours on 12% SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) by using an electroblotter for 12 h at 4°C. Antibodies rose against bcl-2 (1:2000), bcl-XL (1:2000), bim (1:2000), and -tubulin (1:5000) (Cell Signaling Biotech) was diluted in PBST containing 5% non-fat milk and membranes were incubated for 1 h
with gentle agitation. The blots were washed for three times with PBST and incubated with a sheep anti-mouse antibody conjugated to horseradish peroxidase (1:1000 dilutions in PBST containing 5% non-fat milk; Cell Signaling Biotech, Boston, MA) for 2 hours. After three successive washes with PBST, Western blotting chemiluminescence reagent (Pierce, Thermo Fisher Scientific Inc., Rockford, IL) was used for protein detection. 2.8. Transwell migration assay Disposable 24-well cell culture inserts (no. 140627; Nunc, Thermo Fisher Scientific, Roskilde, Demark) were used for migration studies. The cell suspension was kept positioned on the hydrophilic filter site located directly above the bottom wells. Each filter site is 0.47 m2 square and pores are 3 m in diameter. Wells (“lower chambers”) were filled with 100 l of RPMI 1480 medium with 0.81 mg BCG (1.86 ⫻ 106 CFU) combined with sunitinib (6 M) or 1.2 mg BCG (2.76 ⫻ 106 CFU)], sunitinib (10 M) alone. Polycarbonate filters were positioned on microplates and secured in place with corner pins. T24 cells in 100 l DMEM were placed directly onto the filter sites (“upper chambers”) and incubated for 24 h to 48 h at 37°C in 5% CO2. Non-migrating cells on the top of the filters were removed by gently wiping the filters with cotton swabs. Migrating cells on the bottom side were fixed for 10 min in 3.7% formaldehyde and stained with Gimsa for 3 minutes. Stained cells were then captured using a BX50 microscope (Olympus, Tokyo, Japan) and cells were quantified using morphometry software (Analysis FIVE; Soft Imaging System, Olympus, Tokyo, Japan). Differences between two data points were determined by Student’s t-test where P ⬍ 0.05 was considered significant.
3. Results 3.1. VEGFa mRNA expression in TCC cell lines As demonstrated in Fig. 1, T24 TCC cells with high grade differentiation had the highest expression of VEGF mRNA level among these tested TCC cell lines. It was significantly higher, around 3-fold, compared with TSGH8301 TCC cell line with low grade differentiation (P ⬍ 0.001). This result suggests that T24 cells may have a better pharmaceutical response to sunitinib, which can inhibit the VEGF signaling pathway. 3.2. Combined inhibitory effects of BCG and sunitinib on TCC cells Cells in the exponential growth phases were treated with different doses of BCG in 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, and 1 mg (2.3 ⫻ 102– 8.9 ⫻ 106 CFU) and sunitinib in 0.03, 0.1, 0.3, 1, 3, 10, and 30 M for 72 hours. The results showed that these agents inhibited cell growth in a dose-dependent manner, accounting for 2%–91% and 10%–95% with IC50
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and 48 hour time points. On the contrary, BCG or sunitinib treatment alone showed a weak inhibition of bcl-2 protein in TCC cells (Fig. 4). The change of intracellular bcl-XL pro-
Fig. 1. VEGFa mRNA exhibits differentially expressed patterns in a series of human bladder cancer cell lines; t-test; P 0.05*, P 0.01**, P 0.001***.
around of 0.05 mg and 8.25 M for individual drug (Fig. 2A and B). The combined effects of both reagents were analyzed at high cytotoxic concentrations of cotreatment or pretreatment BCG with sunitinib, which showed a prominent synergistic growth inhibition of T24 cells with CIs bellow 1, respectively (Fig. 2C). In these 2 combinational groups, BCG cotreatment with sunitinib group had a better effect in cell toxicity than metachronous treatment group. To determine whether the BCG cotreatment or pretreatment with sunitinib acted additively or synergistically in killing T24 cells, the predicted and the observed cell survival rates have been compared (Table 1). The average observed cell survival rate was significantly lower than that of the predicted rate after the cotreatment of BCG and sutent (P ⬍ 0.05). Although the group that pretreatment of BCG with sunitinib also showed a lower cell survival rate than expected, the difference of each condition was not significant than BCG and sunitinib cotreatment group. 3.3. Apoptotic change in T24 cells after BCG and sunitinib combination treatment Annexin-V and PI staining results are the indicators of early phase of apoptosis. As shown in Fig. 3, the results indicated that the percentage of early apoptotic cells in T24 cells cotreated with BCG and sunitinib increased markedly compared with those of drug alone (34.9% vs. 15.9% and 26.3%). In Table 2, the percentage of apoptosis (peak of hypodiploid DNA before G1 phase) in T24 cells cotreated with BCG and sunitinib were 65% and 92% at 24 and 72 hours, respectively. The percentage of apoptosis in cotreatment group was higher than BCG or sunitinib treatment alone (65% vs. 16% and 42% vs. 8% individually). 3.4. Bcl family protein reduction by BCG and sunitinib cotreatment in TCC cells The expression of bcl-2 protein was reduced significantly from 11% to 58% by BCG and sunitinib cotreatment at 24
Fig. 2. Dose-dependent cytotoxicity of sunitinib, BCG (A), (B); data are presented as means ⫾ SD (error bar) and median effect analysis of the BCG combination with sunitinib at cotreatment or pretreatment model (C). (CI: combination index, Fa: drug fraction).
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Table 1 Synergism analysis of BCG/sunitinib combination on viability of T24 cells in vitro Combination modality
Co-treatment with BCG BCG (0.3 mg) ⫹ sunitinib (1 M) BCG (0.3 mg) ⫹ sunitinib (3 M) BCG (0.3 mg) ⫹ sunitinib (10 M) Pre-treatment with BCG BCG (0.3 mg) ⫹ sunitinib (1 M) BCG (0.3 mg) ⫹ sunitinib (3 M) BCG (0.3 mg) ⫹ sunitinib (10 M)
Survival rate Expected
Observed
P value
0.36 ⫾ 0.01 0.29 ⫾ 0.01 0.18 ⫾ 0.04
0.30 ⫾ 0.03 0.29 ⫾ 0.01 0.09 ⫾ 0.01
0.003 NS 0.001
0.27 ⫾ 0.06 0.31 ⫾ 0.06 0.22 ⫾ 0.07
0.24 ⫾ 0.07 0.25 ⫾ 0.07 0.13 ⫾ 0.02
NS NS 0.044
NS ⫽ nonspecific.
tein by BCG and sunitinib cotreatment was similar to bcl-2 protein but no bim protein change was seen in TCC cells. 3.5. Effect of BCG and sunitinib in the transmigration of T24 cells T24 cells were treated with BCG/sunitinib (0.3 mg/7 M), BCG (1 mg), or sunitinib (10 M) for 16 hours. Fig. 5 shows that the level of the transmigration of T24 cells was significantly reduced by 75% in the combination treatment group compared with control group. Interestingly, T24 cells treated with BCG alone arising 55% migration ability.
4. Discussion It has previously been shown that VEGF were abnormally expressed in TCC and promotes cancer malignancy [18]. In our study, the VEGF content was parallel correlated to cellular differentiation of TCC cells. T24 cell line was selected for further experiment in this study due to its high content of VEGF and can be similar to clinical scenario in patients with high grade and high stage bladder cancer. As we know, this is the first investigation on the possibility of using BCG and sunitinib combination therapy for bladder cancer [19]. Our rationale is that sunitinib may synergistically enhance the cytotoxicity of BCG to TCC cells through its VEGF inhibition effect. In this in vitro study we have shown that either BCG-cotreatment or metachronous treatment with sunitinib having synergistic cytotoxicity to TCC cells and cotreatment group is better than metachronous group. In our speculation, BCG treatment will conduct direct killing effect to TCC cells and trigger the secretion of various cytokines and chemokines, including VEGF, from tumor cells. Cytokines will enhance the secondary cytotoxicity of BCG. On the contrary, VEGF will increase the ability of tumor cell migration and escape from killing by BCG. Immediate sunitinib addition can suppress the VEGF function secreted by tumor cells and subse-
quently enhance the cytotoxicity of BCG. Therefore, less cytotoxic enhancement will be seen in metachronous addition of sunitinib after BCG treatment. In addition to counteract VEGF activity, sunitinib seems also can increase the apoptotic pathway of tumor cells after BCG treatment as shown in this study. Intracellular bcl proteins marked decreased when T24 cells received BCG and sunitinib combination treatment. It indicates sunitinib also can induce apoptotic effect in addition to inhibition of VEGF activity of tumor cells. Interestingly, in our previous animal studies with orthotopic MBT-2 bladder cancer model, simultaneous instillation of BCG and sunitinib intravesically did not exhibit prominent synergistic cytotoxicity to TCC cells with inhibition of tumor growth as in vitro study (data not shown). In metachronous combination treatment group sunitinib even masked the cytotoxicity of BCG. These results were not comparable to our in vitro study in this study. The possible cause may be related to the time course change of secondary immune responses triggered by BCG in the host. BCG induces local tissue immune responses and attracts serial immune cells aggregation, including white blood cells, neutrophils, B-cells, T-cells, and NK cells several hours after instillation. Among the complex cellular network, various cytokines, chemokines, and interleukins are secreted simultaneously, which further enhances the cellular cytotoxicity to tumor cells. Although in vivo synchronous sunitinib treatment can enhance the early cytotoxicity of BCG in limited degree, the major cytotoxic effect of BCG is dependent on the late immune response. During the host immune response interval, providing sunitinib will restrain the secondary immune response induced by BCG, which will result in immunosuppressive outcome in the late phase. On the contrary, secondary immune response did not exist in in vitro treatment of TCC cells by BCG and sunitinib combination. Therefore, synergistic cytotoxic effect of their combination to TCC cells is prominent in in vitro situation. In this study, we have observed that BCG treatment can enhance the migration activity of TCC cells, which was not reported before. The in vitro migration enhancement of TCC cells should be related to the responsive hypersecretion of VEGF and cyto/chemokines from tumor cells after BCG treatment. Nevertheless, apoptotic activation, direct cytotoxicity, and subsequent immune response should be the major killing effect of BCG to TCC cells. Currently, fewer data are available supporting molecular therapies in bladder cancer [20]. Due to the poor prognosis of advanced bladder carcinoma and the insufficient effects of the chemotherapy agents for this disease, the investigation of the novel genetic and pharmacologic agents, including anti-angiogenic agents that can target pathway-specific molecules, has been the subject of study. Sunitinib malate and sorafenib are the novel Food and Drug Administration (FDA) approved anti-angiogenic agents, which have recently been demonstrated to improve the progression-free survival in patients with metastatic renal cell carcinoma
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Fig. 3. Flow cytometry assay of apoptotic activity in cultured T24 cells under exposure to (B) BCG (3.5 ⫻ 106 CFU) alone, (C) sunitinib (10 M), (D) BCG (1.86 ⫻ 106 CFU) combined sunitinib (6 M) for 48 hours compared with control (A); t-test; P 0.05*, P 0.01**, P 0.001***.
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Table 2 Sub G1 changes of cell cycle in T24 cells after co-treatment by BCG and sunitinib at different time intervals Treatment (concentration)
Control BCG (3.5 ⫻ 106 CFU) Sunitinib (10 M) BCG (1.86 ⫻ 106 CFU) ⫹ sunitinib (6 M)
Sub-G1/G0-G2 phase (%) in cell cycle 24-hour
48-hour
72-hour
Sub G1%
G0/G1-G2/M%
Sub G1%
G0/G1-G2/M%
Sub G1%
G0/G1-G2/M%
5% ⫾ 0.3 16% ⫾ 0.9*** 42% ⫾ 0.7*** 65% ⫾ 1.5***
95% ⫾ 0.5 84% ⫾ 0.5 58% ⫾ 1.2 35% ⫾ 1.6
7% ⫾ 0.2*** 32% ⫾ 0.8*** 51% ⫾ 0.9*** 86% ⫾ 1.2***
93% ⫾ 0.1 68% ⫾ 1.1 49% ⫾ 0.8 24% ⫾ 1.6
8% ⫾ 0.1*** 65% ⫾ 1.5*** 82% ⫾ 1.1*** 92% ⫾ 1***
92% ⫾ 0.2 35% ⫾ 1.3 18% ⫾ 0.7 8% ⫾ 0.6
T-test; P 0.05*, P 0.01**, P 0.001***.
[21]. We have demonstrated that sunitinib can synergistically enhance the cytotoxicity of BCG to TCC cells in in vitro aspect due to its anti-angiogenic and inducing apoptotic effects to tumor cells. In our preliminary results, in vitro is different from in vivo study in using sunitinib and BCG combination therapy for TCC due to the absence of immune response in vitro. Although clinical trials of these agents are
still lacking, the great theoretical potential and the multitude of possible targets and drug combinations support further research into this emergent field of medical treatment of urologic malignancies, especially in the treatment of advanced bladder carcinoma [22,23]. In conclusion, sunitinib can synergistically enhance BCG therapy to TCC cells in vitro through direct anti-VEGF and
Fig. 4. Effect of BCG combined with sunitinib treatment on expression of Bcl-2, bcl-XL and bim in T24 cells. Exponentially growing T24 cells were treated BCG (3.5 ⫻ 106 CFU), sunitinib (10 M) alone, or cotreated with indicated concentrations of BCG (1.86 ⫻ 106 CFU) and sunitinib (6 M) for 24, 48 h. Cell lysates were prepared and protein level of bcl-2 and bcl-XL were determined by immunoblot analysis (A); -tubulin was used for normalization and verification of protein loading, and the results bcl-2 and bcl-XL expression from 3 independent experiments were quantified by densitometry with similar results (B), (C); t-test; P ⬍ 0.05*, P ⬍ 0.01**, P ⬍ 0.001***.
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Fig. 5. T24 cell transmigration after stimulating BCG (3.5 ⫻ 106 CFU), sunitinib (10 M) alone, or cotreating with indicated concentrations of BCG (1.86 ⫻ 106 CFU) and sunitinib (6 M); t-test; P ⬍ 0.05*, P ⬍ 0.01**, P ⬍ 0.001***.
apoptotic activity. The feasibility of this combination regimen for superficial bladder cancer treatment still needs further evaluation.
References [1] Jemal A, Siegel R, Xu J, et al. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277–300. [2] Health Statistics in Taiwan (2006). Part II. Statistics on Causes of Death, Department of Health, Executive Yuan, R.O.C. (Taiwan). [3] Cookson MS, Herr HW, Zhang ZF, et al. The treated natural history of high risk superficial bladder cancer: 15-Year outcome. J Urol 1997;158:62–7. [4] Thanhauser A, Bohle A, Schneider B, et al. The induction of bacillus Calmette-Guerin activated killer cells requires the presence of monocytes and T-helper type-1 cells. Cancer Immunol Immunother 1995; 40:103– 8. [5] Patard JJ, Muscatelli-Groux B, Saint F, et al. Evaluation of local immune response after intravesical bacillus Calmette-Guerin treatment for superficial bladder cancer. Br J Urol 1996;78:709 –14. [6] Luo Y, Chen X, Downs TM, et al. IFN-␣ 2b enhances Th1 cytokine responses in bladder cancer patients receiving Mycobacterium bovis bacillus Calmette-Guerin immunotherapy. J Immunol 1999;162:2399 – 405. [7] Hedges W, Agace M, Svensson H, et al. Uroepithelial cells are part of a mucosal cytokine network. Infect Immunol 1994;62:2315–21.
659
[8] Izawa JI, Sweeney P, Perrotte P, et al. Inhibition of tumorigenicity and metastasis of human bladder cancer growing in athymic mice by interferon-beta gene therapy results partially from various anti-angiogenic effects including endothelial cell apoptosis. Clin Cancer Res 2002;8:1258 –70. [9] Fauconnet S, Lascombe I, Chabannes E, et al. Differential regulation of vascular endothelial growth factor expression by peroxisome proliferator-activated receptors in bladder cancer cells. J Biol Chem 2002;277:23534 – 43. [10] Kausch I, Bohle A. Molecular aspects of bladder cancer III. Prognostic markers of bladder cancer. Eur Urol 2003;41:15–29. [11] Demetri GD, van Oosterom AT, Garret CR, et al. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumor after failure of imatinib: A randomized controlled trial. Lancet 2006; 368:1329 –38. [12] Motzer RJ, Hutson TE, Tomczak P, et al. Sunitinib vs. interferon-␣ in metastatic renal cell carcinoma. N Engl J Med 2007;356:115–24. [13] Cherrington JM, Strawn LM, Shawver LK. New paradigms for the treatment of cancer: The role of anti-angiogenesis agents. In: Klein G, vande Woude GF, editors. Advances in Cancer Research, 1st ed. Vol. LXXIX, San Diego, CA: Academic Press 2000; 1–38. [14] Ferrara N. Molecular and biological properties of vascular endothelial growth factor. J Mol Med 1999;77:527– 43. [15] Laird AD, Christensen JG, Li G, et al. SU6668 inhibits Flk-1/KDR and PDGRFb in vivo, resulting in rapid apoptosis of tumor vasculature and tumor regression in mice. FASEB J 2002;16:681–90. [16] Chou TC. Derivation and properties of Michaelis-Menton type and Hill type equations for reference ligands. J Theor Biol 1976;59:253– 76. [17] Siddiqui IA, Adhami VM, Afaq F, et al. Modulation of phosphatidylinositol-3-kinase/protein kinase B and mitogen-activated protein kinase-pathways by tea polyphenols in human prostate cancer cells. J Cell Biochem 2004;91:232– 42. [18] Liu L, Zhu D, Gao R, Guo H. Expression of vascular endothelial growth factor, receptor KDR, and p53 protein in transitional cell carcinoma of the bladder. Urol Int 2008;81:72– 6. [19] Elfiky AA, Rosenberg JE. Targeting angiogenesis in bladder cancer. Curr Oncol Rep 2009;11:244 –9. [20] Froehner M, Hakenberg OW, Wirth MP. Molecular therapy in urologic oncology. Urol Int 2007;79:1–7. [21] Silay MS, Miroglu C. Sunitinib malate and sorafenib may be beneficial at the treatment of advanced bladder cancer due to their antiangiogenic effects. Hypotheses 2007;69:892–5. [22] Sonpavde G, Jian W, Liu H, et al. Sunitinib malate is active against human urothelial carcinoma and enhances the activity of cisplatin in a preclinical model. Urol Oncol 2009;27:391–9. [23] Bradley DA, Dunn R, Nanus D, et al. Randomized, double-blind, placebo-controlled phase II trial of maintenance sunitinib versus placebo after chemotherapy for patients with advanced urothelial carcinoma: Scientific rationale and study design. Clin Genitourin Cancer 2007;5:460 –3.
Original article
Sunitinib can enhance BCG mediated cytotoxicity to transitional cell carcinoma through apoptosis pathway夡 Szu-Yuan Ping, M.S.a,1, Chia-Lun Wu, M.S.b,1, Dah-Shyong Yu, M.D, Ph.D.b,* b
a Graduate Institute of Life Science and Pathology, National Defense Medical Center, Taipei, Taiwan, Republic of China Uro-Oncology Laboratory, Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
Received 12 May 2010; received in revised form 5 July 2010; accepted 6 July 2010
Abstract Bacillus Calmette-Guerin (BCG)-refractory generated a high risk to patients with bladder cancer during treatment. Tyrosine kinase receptor (TKR) and TKR-mediated signal transduction pathways play an important role in tumor initiation, maintenance, angiogenesis, and vascular proliferation. Theoretically, it is helpful in adjuvant treatment for transitional cell carcinoma (TCC). Hence, we proposed that sunitinib, a endothelial growth factor receptor (VEGFR) inhibitor, may have a synergistic effect with BCG in enhancing its cytotoxicity to bladder cancer. The level of VEGF in various TCC cell lines was quantified by real time PCR. High grade TCC-T24 cell line with high level of VEGF expression was selected as representative tumor cells for further study. The single drug and combined inhibitory effects of BCG and sunitinib in T24 cells were determined by MTT method. The drug mediated cell apoptosis in T24 cells was characterized by flow cytometry with PI and annexin V stain. Bcl-2 apoptotic pathway induction by BCG and sunitinib treatment was evaluated by Western blotting method. Inhibitory ability of sunitinib in BCG induced cell migration was verified by cell migration assay. The results shown that expression level of VEGF mRNA in high grade T24 cells was higher than low grade J82, TSGH 8301, and TCC 9202 cell lines. Both BCG and sunitinib treatment presented cytotoxic effect to T24 cells in a dose-dependent manner. Combination of BCG and sunitinib revealed superior cytotoxicity effect than single agent when cells were pretreated with low dosage BCG before sunitinib treatment. By Annexin V analysis it was observed that cell death associated with increased early and late apoptosis process individually. Furthermore, the bcl-2 expression was significant reduced in T24 cells in metachronous BCG and sunitinib combination treatment than single agent. Tumor cell migration activity was also markedly inhibited with BCG and sunitinib combination treatment. In conclusion, these results suggested that during BCG and sunitinib combination treatment both reagents interacted with each other and caused TCC cells apoptosis in addition to direct cytotoxicity. This combination therapeutic model may have the potential for future clinical application to bladder cancer treatment. © 2012 Elsevier Inc. All rights reserved. Keywords: Bacillus Calmette-Guerin (BCG); Sutent; Synergism; Apoptosis; Combined therapy
1. Introduction Transitional cell carcinoma (TCC) of the bladder is the fifth most common solid malignancy in the United States. Over 131,260 new case and over 14,680 deaths of this cancer are estimated for the year 2010 [1]. In Taiwan 1,985 new cases and 681 deaths of bladder cancer were recorded for the year 2006 [2]. Approximately 30% of patients with superficial tumors 夡
This study was supported by grants from the National Science Council (NSC97-2314-B-016-022MY) and Tri-Service General Hospital (TSGH-C98-44). * Corresponding author. Tel.: ⫹886-2-8792-7008; ⫹886-2-8792-7009. E-mail address: [email protected] (D.-S. Yu). 1 These authors contributed equally to this work. 1078-1439/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2010.07.001
will progress to invasive TCC, where radical cystectomy is the standard therapy. Unfortunately, this disease recurs in up to 50% of these patients despite surgery, and is potentially lethal [3]. The treatment goal for superficial bladder cancer is reducing tumor recurrence and preventing tumor progression, which would require additional aggressive therapies. The most common immunotherapeutic agent, BCG (bacillus Calmette-Guérin) is an attenuated mycobacterium developed from the Mycobacterium bovis strain. The anticancer effect of BCG in the treatment of bladder cancer with a complex local immune response, including activated B, T lymphocytes and nature killer cell by inducing multiple cytokines; IL-1, IL-6, IL-8, and GMCSF [4–7]. The critical role of angiogenesis in tumor growth and metastasis has prompted many efforts to develop anti-angiogenic
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therapies, thus providing a rational drug target for using angiogenic inhibitors in bladder cancer tumors [8 –10]. Sunitinib malate is one of several new anti-angiogenic agents undergoing tests of efficacy in the treatment of various types of cancer including gastrointestinal stromal tumors and renal cell carcinoma [11,12]. Several split tyrosine kinase receptor (TKR) domain, namely the vascular endothelial growth factor (VEGF), VEGF receptors, and PDGFR, play prominent roles in tumor angiogenesis [13–15]. Sunitinib malate can block the angiogenesis signaling cascade (VEGF receptors, Flk-1/KDR, and Flt-1) and regulate tumor growth and its angiogenesis. Therefore, inhibiting these targets might be expected to result in broad antitumor efficacy. This study tries to verify the possible synergism effect of BCG and sunitinib malate in treating TCC and its underlying mechanisms.
2. Materials and methods 2.1. Cell culture and reagents The TCC cell lines, T24 (ATCC Rockville, MD), TSGH8301, TSGH8701, TSGH8702, and TCC9202 (established in Uro-Oncology Laboratory, Tri-Service General Hospital) were cultivated in RPMI-1640 medium supplemented with 10% fetal bovine serum, with 100 U/ml penicillin, 50 mg/ml streptomycin, and 2 mM glutamine (Thremo Scientific Hyclone, Logan City, Utah State, UT). Confluent cells were detached with 0.01 M EDTA. BCG (ImmuCyst 81 mg/vial) was purchased from the Sanofi Pasteur Ltd. West Toronto, Ontario, Canada. ImmuCyst 81 mg is freeze-dried preparation made from a culture of the Connaught strain of bacillus Calmette Guérin, which is an attenuated strain of living bovine tubercle bacillus, Mycobacterium bovis. Sunitinib malate (sutent) was kindly provided by Pfizer Inc., Taiwan without financial interest. 2.2. RNA extraction and cDNA synthesis Total RNA was extracted from cultured cells using a commercially available RNA extraction kit (Trizol; Gibco, Carlsbad, CA) according to the manufacturer’s instructions. To avoid contamination by DNA during RNA preparation, RNA was digested with RNase-free DNAse I (Gibco), as recommended by the supplier. cDNA was synthesized in a reaction containing 1 g of total RNA as a template, 0.5 g random hexamers (Promega, Madison, WI), and 200 units of Moloney murine leukemia virus RNase H minus reverse transcriptase (Promega), in a total volume of 25 l according to the manufacturer’s protocol. Duplicate reactions were done both in the presence and absence of the reverse transcriptase enzyme to control for false positive amplification resulting from contaminating chromosomal DNA.
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2.3. Quantitative RT-PCR (qRT-PCR) Detection of VEGFa mRNA was conducted by a twostep procedure using the Light Cycler CK 20 mRNA Quantification Kit (Roche Diagnostics, Indianapolis, IN). First, cDNA was synthesized with a reverse transcription kit (Reverse Transcriptase Master Mix; Roche Diagnostics) using 5 l of RNA and oligo dT serving as primers in a total volume of 20 l. The second step was to amplify 2 l of the cDNA by qRT-PCR using 2 l of the LightCycler PCR Master Mix (CK 20 Detection Mix; Roche Diagnostics). During the qRT-PCR reaction, a 196 bp fragment of VEGFa encoding mRNA was amplified from the cDNA. The amplicon was detected by fluorescence using a specific pair of VEGFa primers: CCC AGG TCA GAC GGA CAG AAA G for VEGFa and AAG CAG GTG AGA GTA AGC GAA GG for VEGFa. The fluorescence emitted after hybridization to the template DNA was measured by the LightCycler 480 instrument. In a separate real time PCR reaction, mRNA encoding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using primers ACC CAC TCC TCC ACC TTT GAC for GAPDH, CAT ACC AGG AAA TGA GCT TGA CAA for GAPDH-rev was processed as when used as a housekeeping gene. Its product served as a control for RNA and relative quantification. All PCR reactions were performed for 40 cycles of 60 seconds at 95°C, 10 seconds at 60°C, and 5 seconds at 72°C. 2.4. Evaluation of the effect of BCG and sunitinib malate on cancer cell viability Cells were exposed to BCG reagent combined with various concentrations of each of the sunitinib for 72 hours. Cellular chemosensitivity was assayed using a modified 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium (MTT; Sigma, St. Louis, MO) assay to determine cell viability in vitro. In brief, T24 cells (4,000 per well) in 100 L culture medium were seeded into 96-well microplates and incubated at 37°C for 24 hours before drug exposure. The plated cell numbers were calculated to keep control cells growing in the exponential phase throughout the 72 hours incubation period. For concurrent treatment, cells were treated with both single BCG reagent and sunitinib (each in 100 l of culture medium) simultaneously and incubated for 72 h. At the point, 50 l of MTT (2 mg/ml in RPMI medium) was added to each well and allowed to react for 2.5 hours. The blue formazan crystals formed were pelleted to the well bottoms by centrifugation, separated from the supernatant, and dissolved in 150 l of DMSO. The optical density was determined by absorbance spectrometry at 492 nm using a microplate reader (MRX-2; Dynex Technologies, Inc., Chantilly, VA). Three separate experiments with triplicate runs in each were performed to obtain mean cell viability. The drug concentrations inhibiting cell growth by 50% (IC50) were determined using a dose– effect analysis model as previously described [16].
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2.5. Median effect analysis of combined cytotoxic effects The combined effects of BCG and sunitinib were subjected to median effect analysis with the mutually nonexclusive model as previously described [17]. Control experiments replaced the active agent with drug-free medium. By combining 2 agents at graded concentrations, numerous combined effects of growth inhibition were obtained and analyzed using the computer software Calcusyn1 (ver, 1.1.1, 1996; Biosoft Inc., Cambridge, UK). For each combined effect of growth inhibition or fraction affected (fa), a combination index (CI) is generated. The effects of the combination are then transformed and displayed as fa–CI plots. CI ⬍ 1, ⫽ 1, and ⬎1 indicate synergism, additivism, and antagonism, respectively. Varied degrees of synergism or antagonism may occur in distinct Fa ranges. We also used the statistical function f(u)1,2 ⫽ f(u)1 ⫹ f(u)2, where f(u)1 ⫽ the fraction unaffected by drug 1, f(u)2 ⫽ the fraction unaffected by drug 2, and f(u)1,2 ⫽ the fraction unaffected by drugs 1 and 2. The expected (presumed to be additive) and observed survival rates of T24 cells were thus obtained from the three independent drug-combined treatments. A statistically significant difference was analyzed by Student’s t-test when P ⬍0.05. 2.6. Annexin V/PI stain for cell apoptotic analysis Cell viability was detected by trypan blue and apoptosis was evaluated by the annexin V/propidium iodide (BD Biosciences, Franklin Lakes, NJ) double staining assay following the manufacturer’s instructions. T24 cells were cultivated with medium alone or medium containing 0.81 mg (1.86 ⫻ 106 CFU) BCG combined with sunitinib (6 M). 106 cells in 10 cm culture dish were harvested and washed with PBS. T24 cells were harvested at the end of treatment, rinsed twice with PBS, and stained with Annexin V-FITC apoptosis detection kit I (BD Biosciences). Analysis was performed on the FACS Calibur using CellQuest. 2.7. Western blotting for bcl-2, bcl-XL, and bim expression 106 exponentially growing T24 cells were co-treated with BCG 0.81 mg (1.86 ⫻ 106 CFU) combined with sunitinib (6 M) at the indicated concentrations for 12, 24, or 48 hours. Then they were pelleted by centrifugation, washed with PBS two times and resuspended in 100 l of lysis buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 2% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 1 mg/ml aprotinin, and sonicated. Protein (50 g) was electrophoresed for 2 hours on 12% SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) by using an electroblotter for 12 h at 4°C. Antibodies rose against bcl-2 (1:2000), bcl-XL (1:2000), bim (1:2000), and -tubulin (1:5000) (Cell Signaling Biotech) was diluted in PBST containing 5% non-fat milk and membranes were incubated for 1 h
with gentle agitation. The blots were washed for three times with PBST and incubated with a sheep anti-mouse antibody conjugated to horseradish peroxidase (1:1000 dilutions in PBST containing 5% non-fat milk; Cell Signaling Biotech, Boston, MA) for 2 hours. After three successive washes with PBST, Western blotting chemiluminescence reagent (Pierce, Thermo Fisher Scientific Inc., Rockford, IL) was used for protein detection. 2.8. Transwell migration assay Disposable 24-well cell culture inserts (no. 140627; Nunc, Thermo Fisher Scientific, Roskilde, Demark) were used for migration studies. The cell suspension was kept positioned on the hydrophilic filter site located directly above the bottom wells. Each filter site is 0.47 m2 square and pores are 3 m in diameter. Wells (“lower chambers”) were filled with 100 l of RPMI 1480 medium with 0.81 mg BCG (1.86 ⫻ 106 CFU) combined with sunitinib (6 M) or 1.2 mg BCG (2.76 ⫻ 106 CFU)], sunitinib (10 M) alone. Polycarbonate filters were positioned on microplates and secured in place with corner pins. T24 cells in 100 l DMEM were placed directly onto the filter sites (“upper chambers”) and incubated for 24 h to 48 h at 37°C in 5% CO2. Non-migrating cells on the top of the filters were removed by gently wiping the filters with cotton swabs. Migrating cells on the bottom side were fixed for 10 min in 3.7% formaldehyde and stained with Gimsa for 3 minutes. Stained cells were then captured using a BX50 microscope (Olympus, Tokyo, Japan) and cells were quantified using morphometry software (Analysis FIVE; Soft Imaging System, Olympus, Tokyo, Japan). Differences between two data points were determined by Student’s t-test where P ⬍ 0.05 was considered significant.
3. Results 3.1. VEGFa mRNA expression in TCC cell lines As demonstrated in Fig. 1, T24 TCC cells with high grade differentiation had the highest expression of VEGF mRNA level among these tested TCC cell lines. It was significantly higher, around 3-fold, compared with TSGH8301 TCC cell line with low grade differentiation (P ⬍ 0.001). This result suggests that T24 cells may have a better pharmaceutical response to sunitinib, which can inhibit the VEGF signaling pathway. 3.2. Combined inhibitory effects of BCG and sunitinib on TCC cells Cells in the exponential growth phases were treated with different doses of BCG in 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, and 1 mg (2.3 ⫻ 102– 8.9 ⫻ 106 CFU) and sunitinib in 0.03, 0.1, 0.3, 1, 3, 10, and 30 M for 72 hours. The results showed that these agents inhibited cell growth in a dose-dependent manner, accounting for 2%–91% and 10%–95% with IC50
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and 48 hour time points. On the contrary, BCG or sunitinib treatment alone showed a weak inhibition of bcl-2 protein in TCC cells (Fig. 4). The change of intracellular bcl-XL pro-
Fig. 1. VEGFa mRNA exhibits differentially expressed patterns in a series of human bladder cancer cell lines; t-test; P 0.05*, P 0.01**, P 0.001***.
around of 0.05 mg and 8.25 M for individual drug (Fig. 2A and B). The combined effects of both reagents were analyzed at high cytotoxic concentrations of cotreatment or pretreatment BCG with sunitinib, which showed a prominent synergistic growth inhibition of T24 cells with CIs bellow 1, respectively (Fig. 2C). In these 2 combinational groups, BCG cotreatment with sunitinib group had a better effect in cell toxicity than metachronous treatment group. To determine whether the BCG cotreatment or pretreatment with sunitinib acted additively or synergistically in killing T24 cells, the predicted and the observed cell survival rates have been compared (Table 1). The average observed cell survival rate was significantly lower than that of the predicted rate after the cotreatment of BCG and sutent (P ⬍ 0.05). Although the group that pretreatment of BCG with sunitinib also showed a lower cell survival rate than expected, the difference of each condition was not significant than BCG and sunitinib cotreatment group. 3.3. Apoptotic change in T24 cells after BCG and sunitinib combination treatment Annexin-V and PI staining results are the indicators of early phase of apoptosis. As shown in Fig. 3, the results indicated that the percentage of early apoptotic cells in T24 cells cotreated with BCG and sunitinib increased markedly compared with those of drug alone (34.9% vs. 15.9% and 26.3%). In Table 2, the percentage of apoptosis (peak of hypodiploid DNA before G1 phase) in T24 cells cotreated with BCG and sunitinib were 65% and 92% at 24 and 72 hours, respectively. The percentage of apoptosis in cotreatment group was higher than BCG or sunitinib treatment alone (65% vs. 16% and 42% vs. 8% individually). 3.4. Bcl family protein reduction by BCG and sunitinib cotreatment in TCC cells The expression of bcl-2 protein was reduced significantly from 11% to 58% by BCG and sunitinib cotreatment at 24
Fig. 2. Dose-dependent cytotoxicity of sunitinib, BCG (A), (B); data are presented as means ⫾ SD (error bar) and median effect analysis of the BCG combination with sunitinib at cotreatment or pretreatment model (C). (CI: combination index, Fa: drug fraction).
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Table 1 Synergism analysis of BCG/sunitinib combination on viability of T24 cells in vitro Combination modality
Co-treatment with BCG BCG (0.3 mg) ⫹ sunitinib (1 M) BCG (0.3 mg) ⫹ sunitinib (3 M) BCG (0.3 mg) ⫹ sunitinib (10 M) Pre-treatment with BCG BCG (0.3 mg) ⫹ sunitinib (1 M) BCG (0.3 mg) ⫹ sunitinib (3 M) BCG (0.3 mg) ⫹ sunitinib (10 M)
Survival rate Expected
Observed
P value
0.36 ⫾ 0.01 0.29 ⫾ 0.01 0.18 ⫾ 0.04
0.30 ⫾ 0.03 0.29 ⫾ 0.01 0.09 ⫾ 0.01
0.003 NS 0.001
0.27 ⫾ 0.06 0.31 ⫾ 0.06 0.22 ⫾ 0.07
0.24 ⫾ 0.07 0.25 ⫾ 0.07 0.13 ⫾ 0.02
NS NS 0.044
NS ⫽ nonspecific.
tein by BCG and sunitinib cotreatment was similar to bcl-2 protein but no bim protein change was seen in TCC cells. 3.5. Effect of BCG and sunitinib in the transmigration of T24 cells T24 cells were treated with BCG/sunitinib (0.3 mg/7 M), BCG (1 mg), or sunitinib (10 M) for 16 hours. Fig. 5 shows that the level of the transmigration of T24 cells was significantly reduced by 75% in the combination treatment group compared with control group. Interestingly, T24 cells treated with BCG alone arising 55% migration ability.
4. Discussion It has previously been shown that VEGF were abnormally expressed in TCC and promotes cancer malignancy [18]. In our study, the VEGF content was parallel correlated to cellular differentiation of TCC cells. T24 cell line was selected for further experiment in this study due to its high content of VEGF and can be similar to clinical scenario in patients with high grade and high stage bladder cancer. As we know, this is the first investigation on the possibility of using BCG and sunitinib combination therapy for bladder cancer [19]. Our rationale is that sunitinib may synergistically enhance the cytotoxicity of BCG to TCC cells through its VEGF inhibition effect. In this in vitro study we have shown that either BCG-cotreatment or metachronous treatment with sunitinib having synergistic cytotoxicity to TCC cells and cotreatment group is better than metachronous group. In our speculation, BCG treatment will conduct direct killing effect to TCC cells and trigger the secretion of various cytokines and chemokines, including VEGF, from tumor cells. Cytokines will enhance the secondary cytotoxicity of BCG. On the contrary, VEGF will increase the ability of tumor cell migration and escape from killing by BCG. Immediate sunitinib addition can suppress the VEGF function secreted by tumor cells and subse-
quently enhance the cytotoxicity of BCG. Therefore, less cytotoxic enhancement will be seen in metachronous addition of sunitinib after BCG treatment. In addition to counteract VEGF activity, sunitinib seems also can increase the apoptotic pathway of tumor cells after BCG treatment as shown in this study. Intracellular bcl proteins marked decreased when T24 cells received BCG and sunitinib combination treatment. It indicates sunitinib also can induce apoptotic effect in addition to inhibition of VEGF activity of tumor cells. Interestingly, in our previous animal studies with orthotopic MBT-2 bladder cancer model, simultaneous instillation of BCG and sunitinib intravesically did not exhibit prominent synergistic cytotoxicity to TCC cells with inhibition of tumor growth as in vitro study (data not shown). In metachronous combination treatment group sunitinib even masked the cytotoxicity of BCG. These results were not comparable to our in vitro study in this study. The possible cause may be related to the time course change of secondary immune responses triggered by BCG in the host. BCG induces local tissue immune responses and attracts serial immune cells aggregation, including white blood cells, neutrophils, B-cells, T-cells, and NK cells several hours after instillation. Among the complex cellular network, various cytokines, chemokines, and interleukins are secreted simultaneously, which further enhances the cellular cytotoxicity to tumor cells. Although in vivo synchronous sunitinib treatment can enhance the early cytotoxicity of BCG in limited degree, the major cytotoxic effect of BCG is dependent on the late immune response. During the host immune response interval, providing sunitinib will restrain the secondary immune response induced by BCG, which will result in immunosuppressive outcome in the late phase. On the contrary, secondary immune response did not exist in in vitro treatment of TCC cells by BCG and sunitinib combination. Therefore, synergistic cytotoxic effect of their combination to TCC cells is prominent in in vitro situation. In this study, we have observed that BCG treatment can enhance the migration activity of TCC cells, which was not reported before. The in vitro migration enhancement of TCC cells should be related to the responsive hypersecretion of VEGF and cyto/chemokines from tumor cells after BCG treatment. Nevertheless, apoptotic activation, direct cytotoxicity, and subsequent immune response should be the major killing effect of BCG to TCC cells. Currently, fewer data are available supporting molecular therapies in bladder cancer [20]. Due to the poor prognosis of advanced bladder carcinoma and the insufficient effects of the chemotherapy agents for this disease, the investigation of the novel genetic and pharmacologic agents, including anti-angiogenic agents that can target pathway-specific molecules, has been the subject of study. Sunitinib malate and sorafenib are the novel Food and Drug Administration (FDA) approved anti-angiogenic agents, which have recently been demonstrated to improve the progression-free survival in patients with metastatic renal cell carcinoma
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Fig. 3. Flow cytometry assay of apoptotic activity in cultured T24 cells under exposure to (B) BCG (3.5 ⫻ 106 CFU) alone, (C) sunitinib (10 M), (D) BCG (1.86 ⫻ 106 CFU) combined sunitinib (6 M) for 48 hours compared with control (A); t-test; P 0.05*, P 0.01**, P 0.001***.
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Table 2 Sub G1 changes of cell cycle in T24 cells after co-treatment by BCG and sunitinib at different time intervals Treatment (concentration)
Control BCG (3.5 ⫻ 106 CFU) Sunitinib (10 M) BCG (1.86 ⫻ 106 CFU) ⫹ sunitinib (6 M)
Sub-G1/G0-G2 phase (%) in cell cycle 24-hour
48-hour
72-hour
Sub G1%
G0/G1-G2/M%
Sub G1%
G0/G1-G2/M%
Sub G1%
G0/G1-G2/M%
5% ⫾ 0.3 16% ⫾ 0.9*** 42% ⫾ 0.7*** 65% ⫾ 1.5***
95% ⫾ 0.5 84% ⫾ 0.5 58% ⫾ 1.2 35% ⫾ 1.6
7% ⫾ 0.2*** 32% ⫾ 0.8*** 51% ⫾ 0.9*** 86% ⫾ 1.2***
93% ⫾ 0.1 68% ⫾ 1.1 49% ⫾ 0.8 24% ⫾ 1.6
8% ⫾ 0.1*** 65% ⫾ 1.5*** 82% ⫾ 1.1*** 92% ⫾ 1***
92% ⫾ 0.2 35% ⫾ 1.3 18% ⫾ 0.7 8% ⫾ 0.6
T-test; P 0.05*, P 0.01**, P 0.001***.
[21]. We have demonstrated that sunitinib can synergistically enhance the cytotoxicity of BCG to TCC cells in in vitro aspect due to its anti-angiogenic and inducing apoptotic effects to tumor cells. In our preliminary results, in vitro is different from in vivo study in using sunitinib and BCG combination therapy for TCC due to the absence of immune response in vitro. Although clinical trials of these agents are
still lacking, the great theoretical potential and the multitude of possible targets and drug combinations support further research into this emergent field of medical treatment of urologic malignancies, especially in the treatment of advanced bladder carcinoma [22,23]. In conclusion, sunitinib can synergistically enhance BCG therapy to TCC cells in vitro through direct anti-VEGF and
Fig. 4. Effect of BCG combined with sunitinib treatment on expression of Bcl-2, bcl-XL and bim in T24 cells. Exponentially growing T24 cells were treated BCG (3.5 ⫻ 106 CFU), sunitinib (10 M) alone, or cotreated with indicated concentrations of BCG (1.86 ⫻ 106 CFU) and sunitinib (6 M) for 24, 48 h. Cell lysates were prepared and protein level of bcl-2 and bcl-XL were determined by immunoblot analysis (A); -tubulin was used for normalization and verification of protein loading, and the results bcl-2 and bcl-XL expression from 3 independent experiments were quantified by densitometry with similar results (B), (C); t-test; P ⬍ 0.05*, P ⬍ 0.01**, P ⬍ 0.001***.
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Fig. 5. T24 cell transmigration after stimulating BCG (3.5 ⫻ 106 CFU), sunitinib (10 M) alone, or cotreating with indicated concentrations of BCG (1.86 ⫻ 106 CFU) and sunitinib (6 M); t-test; P ⬍ 0.05*, P ⬍ 0.01**, P ⬍ 0.001***.
apoptotic activity. The feasibility of this combination regimen for superficial bladder cancer treatment still needs further evaluation.
References [1] Jemal A, Siegel R, Xu J, et al. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277–300. [2] Health Statistics in Taiwan (2006). Part II. Statistics on Causes of Death, Department of Health, Executive Yuan, R.O.C. (Taiwan). [3] Cookson MS, Herr HW, Zhang ZF, et al. The treated natural history of high risk superficial bladder cancer: 15-Year outcome. J Urol 1997;158:62–7. [4] Thanhauser A, Bohle A, Schneider B, et al. The induction of bacillus Calmette-Guerin activated killer cells requires the presence of monocytes and T-helper type-1 cells. Cancer Immunol Immunother 1995; 40:103– 8. [5] Patard JJ, Muscatelli-Groux B, Saint F, et al. Evaluation of local immune response after intravesical bacillus Calmette-Guerin treatment for superficial bladder cancer. Br J Urol 1996;78:709 –14. [6] Luo Y, Chen X, Downs TM, et al. IFN-␣ 2b enhances Th1 cytokine responses in bladder cancer patients receiving Mycobacterium bovis bacillus Calmette-Guerin immunotherapy. J Immunol 1999;162:2399 – 405. [7] Hedges W, Agace M, Svensson H, et al. Uroepithelial cells are part of a mucosal cytokine network. Infect Immunol 1994;62:2315–21.
659
[8] Izawa JI, Sweeney P, Perrotte P, et al. Inhibition of tumorigenicity and metastasis of human bladder cancer growing in athymic mice by interferon-beta gene therapy results partially from various anti-angiogenic effects including endothelial cell apoptosis. Clin Cancer Res 2002;8:1258 –70. [9] Fauconnet S, Lascombe I, Chabannes E, et al. Differential regulation of vascular endothelial growth factor expression by peroxisome proliferator-activated receptors in bladder cancer cells. J Biol Chem 2002;277:23534 – 43. [10] Kausch I, Bohle A. Molecular aspects of bladder cancer III. Prognostic markers of bladder cancer. Eur Urol 2003;41:15–29. [11] Demetri GD, van Oosterom AT, Garret CR, et al. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumor after failure of imatinib: A randomized controlled trial. Lancet 2006; 368:1329 –38. [12] Motzer RJ, Hutson TE, Tomczak P, et al. Sunitinib vs. interferon-␣ in metastatic renal cell carcinoma. N Engl J Med 2007;356:115–24. [13] Cherrington JM, Strawn LM, Shawver LK. New paradigms for the treatment of cancer: The role of anti-angiogenesis agents. In: Klein G, vande Woude GF, editors. Advances in Cancer Research, 1st ed. Vol. LXXIX, San Diego, CA: Academic Press 2000; 1–38. [14] Ferrara N. Molecular and biological properties of vascular endothelial growth factor. J Mol Med 1999;77:527– 43. [15] Laird AD, Christensen JG, Li G, et al. SU6668 inhibits Flk-1/KDR and PDGRFb in vivo, resulting in rapid apoptosis of tumor vasculature and tumor regression in mice. FASEB J 2002;16:681–90. [16] Chou TC. Derivation and properties of Michaelis-Menton type and Hill type equations for reference ligands. J Theor Biol 1976;59:253– 76. [17] Siddiqui IA, Adhami VM, Afaq F, et al. Modulation of phosphatidylinositol-3-kinase/protein kinase B and mitogen-activated protein kinase-pathways by tea polyphenols in human prostate cancer cells. J Cell Biochem 2004;91:232– 42. [18] Liu L, Zhu D, Gao R, Guo H. Expression of vascular endothelial growth factor, receptor KDR, and p53 protein in transitional cell carcinoma of the bladder. Urol Int 2008;81:72– 6. [19] Elfiky AA, Rosenberg JE. Targeting angiogenesis in bladder cancer. Curr Oncol Rep 2009;11:244 –9. [20] Froehner M, Hakenberg OW, Wirth MP. Molecular therapy in urologic oncology. Urol Int 2007;79:1–7. [21] Silay MS, Miroglu C. Sunitinib malate and sorafenib may be beneficial at the treatment of advanced bladder cancer due to their antiangiogenic effects. Hypotheses 2007;69:892–5. [22] Sonpavde G, Jian W, Liu H, et al. Sunitinib malate is active against human urothelial carcinoma and enhances the activity of cisplatin in a preclinical model. Urol Oncol 2009;27:391–9. [23] Bradley DA, Dunn R, Nanus D, et al. Randomized, double-blind, placebo-controlled phase II trial of maintenance sunitinib versus placebo after chemotherapy for patients with advanced urothelial carcinoma: Scientific rationale and study design. Clin Genitourin Cancer 2007;5:460 –3.