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Although interferon (IFN) has been often used as immunotherapy for bladder cancer, its efficacy is rather unsatisfactory, demanding further improvement. Combination therapy is one of viable options, and grape seed proanthocyanidin (GSP) could be such an agent to be used with IFN because it has been shown to have anticancer activity. We thus investigated whether combination of IFN and GSP might enhance the overall antiproliferative effect on bladder cancer cells in vitro. Human bladder cancer T24 cells were employed and treated with the varying concentrations of recombinant IFN-α2b (0-100,000 IU/ml), GSP (0-100 μg/ml), or their combinations. IFN-α2b alone led to a ~50% growth reduction at 20,000 (20K) IU/ml, which further declined to ~67% at ≥50K IU/ml. Similarly, GSP alone induced a ~35% and ~100% growth reduction at 25 and ≥50 μg/ml, respectively. When IFN-α2b and GSP were then combined, combination of 50K IU/ml IFN-α2b and 25 μg/ml GSP resulted in a drastic >95% growth reduction. Cell cycle analysis indicated that such an enhanced growth inhibition was accompanied by a G1 cell cycle arrest. This was further confirmed by Western blot analysis revealing that expressions of G1-specific cell cycle regulators (CDK2, CDK4, cyclin E and p27/Kip1) were distinctly modulated with such IFN-α2b/GSP treatment. Therefore, these findings support the notion that combination of IFN-α2b and GSP is capable of additively enhancing antiproliferative effect on T24 cells with a G1 cell cycle arrest, implying an adjuvant therapeutic modality for superficial bladder cancer.
Bladder cancer is the second most common urologic malignancy next to prostate cancer in the United States, and the majority of bladder cancers present as superficial (80%) with 15% presenting as invasive cancer and 5% as metastatic disease 1. Currently, transitional cell carcinoma (TCC) is the most prevalent primary bladder tumor: 50,000 new cases are diagnosed annually and over 10,000 people die of this disease each year 2. Although endoscopic transurethral resection (TUR) is often performed as a primary therapy, 50%-75% of patients will yet recur in 5 years and about 10% progress to invasive disease 2.
Chemotherapy is another viable option but intravesical administration of bacillus Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis, is currently the most effective immunotherapy for high-grade and recurrent superficial bladder cancer and carcinoma in situ (CIS) 3. In randomized studies, BCG has been shown to be superior to both mitomycin C and adriamycin 4. Intravesical BCG following TUR has also been associated with a significant improvement in progression and survival compared to TUR alone 5. In fact, this protocol has become established therapy for superficial bladder cancers, resulting in a ~40% reduction in cancer recurrence 6. However, side effects of BCG therapy are common and limit its use in clinical practice, demanding a safer, more effective therapeutic modality with fewer side effects.
Interferons (IFNs) have been often used as immunotherapy for a variety of urologic malignancies including prostate, bladder, and renal cell carcinomas 7-9. Especially, IFN-α is used as an intravesical agent for treating superficial bladder cancer because it may cause only minor local and systemic toxicity (compared to BCG) 10. However, since its response rate in patients has been shown to be lower than that of BCG therapy 10, the efficacy of IFN-α combined with BCG was assessed in pilot clinical trials and animal studies 11,12, indicating the better, improved outcomes. Thus, these studies would certainly encourage further exploration into other alternative combination therapies, which may lead to the safer, more effective and satisfactory results.
Proanthocyanidins are naturally occurring plant polyphenolic bioflavonoids in fruits, vegetables, nuts, seeds, flowers and bark 13. They are known as natural antioxidants, having biological, pharmacological and chemoprotective properties against oxidative stress or harmful free radicals 13-15. For example, hydrogen peroxide-induced oxidative stress was significantly reduced by proanthocyanidins in cultured macrophage and neuroactive PC-12 cells 14. They have exhibited antibacterial, antiviral, anti-inflammatory, and vasodilatory actions as well 13. Particularly, a unique grape seed proanthocyanidin (GSP) has been extensively characterized: it is a standardized water-ethanol extract from red grape seeds, consisting of oligomeric proanthocyanidins as active components 15. GSP has also demonstrated its anticancer (cytotoxic) effect on several malignancies including breast, lung and gastric cancers in vitro 16.
Accordingly, we investigated whether IFN-α, GSP or their combination might demonstrate the antiproliferative effect on bladder cancer cells in vitro. We also explored the underlying mechanism - how the cancer cell growth might be inhibited with such agents, focusing on the cell cycle regulation. More detailed studies are described and discussed herein.
The human bladder cancer T24 cells, derived from a patient with TCC, were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml), and were maintained at 37 ºC in a humidified incubator in an atmosphere of 95% air and 5% CO2. For experiments, cells were seeded in 6-well plates (2 ml per well) or T-75 flasks (10 ml per flask) at the initial cell density of 2 x 105 cells/ml and were cultured with recombinant interferon-α2b (IFN-α2b; Schering Corp., Kenilworth, NJ), grape seed proanthocyanidin (GSP; Dry Creek Nutrition, Inc., Modesto, CA) or their combinations. Cell number/viability was then assessed at specified times using the trypan blue exclusion method.
A FACScan flow cytometer (Becton-Dickinson, San Jose, CA), equipped with a double discrimination module, was employed for cell cycle analysis. Control or agents-treated cells (~1 x 106 cells per condition) were first resuspended in 500 μl of propidium iodide solution (20 μg/ml propidium iodide, 0.2 mg/ml RNase, 0.2 mg/ml EDTA, 0.5% Nonidet P-40) and incubated for 1 h at room temperature in the dark. Following incubation, ~10,000 nuclei from each sample were analyzed on a flow cytometer, and CellFit software was used to quantify cell cycle compartments to estimate the % of cells distributed in the different cell cycle phases.
The procedure essentially followed the protocol described previously 17. Briefly, an equal amount of proteins (7 μg) from control and agent-treated cell lysates was resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The blot was first incubated for 90 min with the primary antibodies against CDK2, CDK4, cyclin D1, cyclin E, or p27/Kip1 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with the secondary antibody conjugated with peroxidase for 30 min. The immunoreactive protein bands were detected by chemiluminescence following the manufacturer's protocol (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and quantified using a scan densitometer (Silk Scientific, Oregon, UT).
All data were presented as the mean ± SD (standard deviation), and statistical differences between groups were assessed with either the unpaired Student's t test or one-way analysis of variance (ANOVA). Values of p<0.05 were considered to indicate statistical significance.
To examine possible effects of interferon-α2b (IFN-α2b) and grape seed proanthocyanidin (GSP) on T24 cell growth, cells were cultured with the varying concentrations of IFN-α2b (0-100,000 = 100K IU/ml) or GSP (0-100 μg/ml) for 72 h. IFN-α2b caused a significant (~50%) growth reduction at 20K IU/ml, which further declined to >67% at ≥50K IU/ml (Fig. (Fig.1A).1A). Similarly, a ~35% and >90% growth inhibition were attained with 25 and ≥50 μg/ml of GSP, respectively (Fig. (Fig.1B).1B). A drastic growth reduction (~90%) with 50 μg/ml GSP was actually due to a cytotoxic effect, evidenced by ~30% of cells having been dead. These results thus show that IFN-α2b and GSP are capable of inhibiting T24 cell growth but GSP can also induce cytotoxic cell death at the higher (≥50 μg/ml) concentrations.
It was tempting to examine whether combinations of IFN-α2b and GSP might improve the antiproliferative effects. As 25 μg/ml of GSP appeared to induce a moderate (~35%) growth inhibition (Fig. (Fig.1B),1B), this concentration was used in combination with the varying concentrations of IFN-α2b. Such studies showed that combinations of GSP (25 μg/ml) and IFN-α2b at 10K IU/ml (little effect by itself), 20K IU/ml (a ~50% inhibitory effect), and 50K IU/ml (a ~67% inhibitory effect) led to the improved 43%, 79%, and >95% growth reduction (p<0.05), respectively (Fig. (Fig.2).2). Thus, the IFN-α2b/GSP combinations appear to induce the additive antiproliferative effects on T24 cells.
To explore the mechanism of such additive effects of the IFN-α2b/GSP combinations, cell cycle analysis was performed using the specific concentrations of IFN-α2b (20K IU/ml) and GSP (25 μg/ml), which seemed to be rather suitable for this study. After T24 cells were treated with IFN-α2b (20K IU/ml), GSP (25 μg/ml), or their combination for 72 h, the results of cell cycle analysis were then summarized in Table Table1.1. Compare to cell numbers of the G1 and S phases in controls, significant changes in those numbers (p<0.05) were seen with IFN-α2b treatment, while GSP alone showed only the marginal effects. In contrast, the IFN-α2b/GSP combination induced a 58% increase and 64% decrease in G1 and S phase cell numbers (p<0.05), respectively. This cell accumulation in the G1 phase is known as a G1 cell cycle arrest 18. Thus, the IFN-α2b/GSP combination may primarily target the G1-S phase transition in the cell cycle, subsequently leading to the growth cessation.
To confirm such an IFN-α2b/GSP-induced G1 cell cycle arrest, we also examined its effects on the specific cell cycle regulators for the G1-S phase transition 18. After cells were treated with or without the IFN-α2b (20K IU/ml)/GSP (25 μg/ml) combination for 72 h, the expressions of CDK2, CDK4, cyclin D1, cyclin E, and p27/Kip1 were analyzed on Western blots. Such analysis revealed that the expressions of CDK2, CDK4 and cyclin E were drastically reduced by ~80% with the IFN-α2b/GSP treatment (compared to those in controls), although no change in cyclin D1 was observed (Fig. (Fig.3).3). In contrast, p27/Kip1 protein, a CDK2 inhibitor, was significantly (~2.7 fold) up-regulated in IFN-α2b/GSP-treated cells (Fig. (Fig.3).3). Thus, altered expressions of these regulators would provide the further evidence for a blockage of G1-S phase transition, confirming that the IFN-α2b/GSP-induced growth inhibition is indeed mediated through a G1 cell cycle arrest.
Now, one may raise the question if the IFN-α2b/GSP combination would also demonstrate its enhanced antiproliferative effect on other bladder cancer cells or different cancer cell types. This was tested using another bladder cancer 5637 cells, prostate cancer PC-3 cells, and renal cancer ACHN cells, which had been treated with IFN-α2b (20K IU/ml), GSP (25 μg/ml), or their combination for 72 h. Figure Figure44 shows the effects of these agents on proliferation of three cancer cell lines. Overall, all cancer cells treated with various agents exhibited altered growth patterns, which were significantly different from their respective control cells (p<0.05). IFN-α2b alone was capable of inducing a ~50% growth inhibition in both PC-3 and ACHN cells but a ~30% inhibition in 5637 cells. GSP alone caused a lesser but 22-37% growth reduction in all these cells. Nevertheless, the IFN-α2b/GSP combination resulted in an enhanced 70%, 71%, and 68% growth inhibition in 5637, PC-3, and ACHN cells, respectively. Thus, these results suggest that the IFN-α2b/GSP combination may commonly demonstrate its potentiated antiproliferative effect on not only T24 cells but also a variety of human cancer cells.
Although IFN-α has been often used as immunotherapy for bladder cancer because of its relatively low toxicity (compared to BCG), its less efficacy has been also inquired for a significant improvement. Additionally, IFN-α therapy has several drawbacks, such as high cost and repeated administration. A standard intravesical IFN-α instillation is often carried out with 50-100 million IU of IFN-α 19, but whether this high dosage would be sufficient to induce optimal immunity is uncertain because of its short retention time inside the bladder 19. Accordingly, to improve the efficacy of such IFN-α monotherapy, clinical trials of combination therapy using IFN-α and BCG 11 have been conducted on patients with bladder cancer. Despite some encouraging outcomes, further studies are yet required for establishing the more potent, safer, and cost-effective treatment modalities.
This interesting issue prompted us to explore an alternative approach using grape seed proanthocyanidin (GSP) in combination with IFN-α2b, because GSP has been shown to be a natural, non-toxic antioxidant with anticancer effect 15,16. Our study showed that IFN-α2b or GSP was capable of individually inducing a significant growth reduction in T24 cells. Interestingly, GSP also exhibited the cytotoxic effect (inducing cell death) at its higher concentrations (≥50 μg/ml). When combinations of IFN-α2b and GSP were tested to further improve the overall efficacy, all combinations resulted in the additively enhanced antiproliferative effect, implying that both IFN-α2b and GSP may share the common growth regulatory pathway. In addition, this IFN-α2b/GSP-enhanced growth inhibition in T24 cells was also demonstrated in another bladder cancer (5637), prostate cancer (PC-3), and renal cancer (ACHN) cells (Fig. (Fig.4),4), suggesting its prevalent potency over bladder cancer as well as various cancer cells.
Our next aim was to probe the antiproliferative mechanism of IFN-α2b/GSP combination, focusing on the cell cycle regulation. Such study revealed that the IFN-α2b/GSP-induced growth inhibition was associated with a 64% reduction in the S-phase cell population, due to a blockage of the cells entering from the G1 to the subsequent S phase (i.e. a G1 cell cycle arrest). This finding was also verified by analyzing G1-specific cell cycle regulators: expressions of CDK2, CDK4, and cyclin E were drastically (~80%) down-regulated while p27/Kip1 was greatly (~2.7 fold) up-regulated in IFN-α2b/GSP-treated cells. Specific modulations of these regulators are indicative of a G1 cell cycle arrest, which is the crucial cellular event leading ultimately to a growth cessation. Yet, it is also important to examine the IFN-mediated signaling pathways to further define the mechanism of IFN-α2b/GSP-induced growth inhibition, since activation of specific IFN-inducible genes by signal transduction 20 is well known to dictate biological actions of IFNs (including IFN-α2b). Such study is currently underway in our laboratory.
It would be worthwhile mentioning the possible clinical relevance of IFN-α2b/GSP-enhanced antiproliferative effect. As a high-dose instillation of IFN-α leads to its high cost 19, it would be more practical if such a high dosage could be somehow reduced without losing, or rather, with improving its efficacy. Our study then showed that the relatively low concentrations of IFN-α2b (compared to its monotherapy) were required to be highly effective when combined with GSP. This suggests that the combination of IFN-α2b and GSP may not only help enhance IFN-α2b activity but also help cut its cost down. However, it is yet required to address how the effective concentrations (e.g., 20K or 50K IU/ml) of IFN-α2b and GSP in this in vitro study would be extrapolated to animals or actual patients. Nevertheless, several studies have already reported antitumor activity of GSP in vivo. For example, GSP was found to inhibit prostate tumor growth and angiogenesis 21 as well as breast cancer metastasis in mice 22 or enhance cytotoxic effect of doxorubicin in mice bearing Sarcorma 180 and Hepatoma 22 23. No palpable side effects of GSP have been yet reported in these animal studies, and the LD50 of GSP in the rats has been estimated to be >5,000 mg/kg body weight 24, verifying its low toxicity. Moreover, patients with chronic pancreatitis demonstrated the symptomatic improvements, such as the reduction in both pain index and incidence of vomiting 25, with a daily dose of 200-300 mg of GSP. This also implies that GSP may have few side effects and is safe to be used in clinical practice.
In conclusion, IFN-α2b and GSP can individually demonstrate antiproliferative effect on bladder cancer T24 cells. When they were combined, such inhibitory activity would be additively enhanced, resulting in a nearly complete growth cessation. In addition, this additive potentiation can be seen in other cancer cell types as well. The underlying mechanism of IFN-α2b/GSP-enhanced growth inhibition appears to be more likely attributed to a G1 cell cycle arrest. Therefore, specific IFN-α2b/GSP combination may provide alternative, adjuvant intravesical therapy for superficial bladder cancer.