膀胱癌细胞中舞茸D-fraction与干扰素的协同增效作用.docx

声明：本文摘自BJU International （2010年4月，第105卷第7期），即British Journal of Urology International《英国国际泌尿学杂志》。是国际泌尿学领域的主要杂志，发表关于成人及小儿泌尿学的原创论文、综述、评论等，为泌尿科、肾病科、肿瘤科、放射科、妇科、男科、外科及儿科的临床医生提供最新的技术、治疗方法等信息。为方便广大读者阅读，我们对论文摘要进行了中文翻译，译文仅供参考，如有不足之处，敬请指正。
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Synergistic potentiation of interferon activity with maitake mushroom D-fraction on bladder cancer cells
                               膀胱癌细胞中舞茸D-fraction与干扰素的协同增效作用
目的：检验在膀胱癌T24细胞的体外试验中，一种具有生物活性的菇类提取物舞茸D-fraction (PDF)与IFN-α合用时，是否能提高IFN-α的的抗癌活性。
材料与方法：评估72小时后不同浓度IFN-α2b（0-50000IU/ml），PDF(0-700μg/ml)，以及两者的组合对T24细胞的作用。进行细胞周期分析以及双链DNA依赖蛋白激酶试验来发现这些药物可能的抗增殖机制。
结果：浓度为20000IU/ml的IFN-α2b能显著降低细胞的生长（≈50%），当IFN-α2b达到50000IU/m时，生长下降达到66%。PDF浓度小于200μg/ml时，对癌细胞生长没有影响，但当其浓度为400和700μg/ml时，其生长减少率分别达到≈20%和≈53%。当不同浓度的IFN-α2b和PDF组合时，10000IU/ml的IFN-α2b和200μg/ml的PDF联合使用时，细胞生长减少率约为75%。细胞周期分析发现，与此同时出现了G1细胞周期停滞现象。同时，经IFN-α2b和PDF联合处理后的细胞内的DNA依赖蛋白激酶活性比对照组几乎高3倍。
结论：T24细胞中10000 IU/ml的IFN-α2b和200μg/ml的PDF联合使用能导致约75%的细胞生长减少。这表明这两者具有协同作用，并能激活DNA依赖蛋白激酶活性并诱导G1细胞周期停滞。所以，IFN-α2b和PDF联合使用能触发DNA依赖蛋白激酶活性，它可能作用于细胞周期使癌细胞停止生长。
关键词：干扰素，D-fraction，联合疗法，协同作用，膀胱癌

                              BJU International, Volume 105, Issue 7, April 2010, Pages 1011-1015
Synergistic potentiation of interferon activity with maitake mushroom D-fraction on bladder cancer cells
             Brandon Louie, Srinivas Rajamahanty, John Won, Muhammad Choudhury and Sensuke Konno
                          Department of Urology, New York Medical College, Valhalla, NY, USA

                                               Accepted for publication 9 June 2009
OBJECTIVE
To examine whether the combination of interferon (IFN)-αand maitake mushroom D-fraction (PDF), a bioactive mushroom extract, might potentiate the anticancer activity of IFN-α in bladder cancer T24 cells in vitro.
MATERIALS AND METHODS
Effects of recombinant IFN-α2b (0–50 000 IU/mL), PDF (0–700μg/mL), or their combinations were assessed on T24 cell growth at 72 h. Cell cycle analysis and assays for double-stranded DNA-dependent protein kinase (DNA-PK) were performed to explore possible antiproliferative mechanism of these agents.
RESULTS
IFN-α2b was able to induce a significant (≈50%) growth reduction at 20 000 IU/mL, which further declined to ≈66% at 50 000 IU/mL. PDF had no effects up to 200μg/mL, but there was an ≈20% and ≈53% growth reduction at 400 and 700μg/mL, respectively. When the varying concentrations of IFN-α2b and PDF were combined, 10 000 IU/mL of IFN-α2b combined with 200μg/mL of PDF resulted in an ≈75% growth reduction. This was accompanied by a G1 cell cycle arrest, shown by cell cycle analysis. Concurrently, DNA-PK activity in IFN-α2b/PDF-treated
cells was almost three-fold higher than controls.
CONCLUSIONS
The combination of IFN-α2b(10 000 IU/mL) and PDF (200μg/mL) reduced growth by ≈75% in T24 cells. This appears to be due to a synergistic potentiation of these two agents, inducing a G1 arrest with DNA-PK activation. Therefore, the IFN-α2b /PDF combination could trigger DNA-PK activation that may act on the cell cycle to cease cancer cell growth.
KEYWORDS
interferon, D-fraction, combined therapy, synergism, bladder cancer
.
INTRODUCTION
The bladder is the most common site of cancer in the urinary system and ≈90% of bladder cancers are TCCs. Of these TCCs, ≈80% are diagnosed as superficial bladder tumours [1]. Transurethral resection is the primary method for removal of those superficial bladder tumours; however, nearly 65% of patients will have tumour recurrence in 5 years while 10–20% will have progression to muscle invasion [2,3]. It is thus conceivable that the primary therapeutic aim is to prevent multiple recurrences and progression to a more advanced, invasive disease.
Several cytotoxic and immune modifying agents have been used intravesically for therapeutic purpose. Among them, intravesical administration of BCG is highly effective in reducing the recurrence rate and altering the progression rate of the disease with an increased survival rate [4]. In fact, adjuvant intravesical BCG therapy after surgical resection has become established therapy for superficial bladder cancers, resulting in ≈40% reduction in cancer recurrence [4,5]. However, its benefits are sometimes outweighed by its severe side-effects: cystitis occurs in 90% of patients and other potential adverse effects (fever, allergic reactions, sepsis, etc.) cannot be excluded [6,7]. These drawbacks thus limit its use in clinical practice and request a safer and effective treatment method with few side-effects, promoting the use of unconventional therapies with other various immunomodulators.
Interferons (IFNs) have been widely used as immunotherapy for various human malignancies including prostate, bladder, and RCCs [8–10]. Particularly, IFN-α has been used as an intravesical agent for treating superficial bladder cancer, resulting in an ≈40% response rate in patients [11]. Although this response rate is lower than that of BCG therapy, IFN-α causes only minimal local and systemic toxicity (compared with BCG) [11,12]. To improve the efficacy of IFN-α therapy, combined therapy (e.g. IFN-α/BCG) has been proposed and is being assessed in pilot clinical trials and animal studies, which are showing better and encouraging outcomes [13,14]. This suggests that further exploration of effective treatment methods such as an alternative and/or combined therapy is warranted.
D-fraction (PDF) is a bioactive proteoglucan extracted from maitake mushrooms (Grifola frondosa) [15]. The standardized PDF has been commercially available for medical and scientific research. Several published and unpublished studies have to date suggested the immunomodulatory and antitumour activities of PDF [16,17]. It was shown in an animal model that PDF was capable of activating immune-competent cells such as natural killer cells and cytotoxic T-cells with a concomitant increase in interleukin-1 production [16,17], indicating stimulation of immune responses. Meanwhile, the safety of PDF is supported by the fact that the USA Food and Drug Administration (FDA) has exempted a phase I study of toxicology tests. Additionally, the FDA has approved PDF for the Investigational New Drug application for a phase II pilot study on patients with advanced breast and prostate cancer [18].
Accordingly, we investigated whether IFN-α, PDF or their combination might have a growth inhibitory effect on bladder cancer T24 cells in vitro, and the underlying mechanism of such activity was also explored.
MATERIALS AND METHODS
The human bladder cancer T24 cells, derived from a patient with TCC, were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in McCoy’s 5a medium containing 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). Routinely, culture medium was changed every 3–4 days and the passage of cells was performed weekly. For experiments, cells were seeded in T-75 flasks or six-well culture plates at the initial cell density of 2×105 cells/mL and were cultured with recombinant IFN-α2b (Schering Corp., Kenilworth, NJ, USA), PDF (Maitake Products, Inc., Paramus, NJ, USA) or their combinations. Cell numbers were then assessed at specified times using the Trypan blue exclusion method.
Cell cycle analysis was performed using a FACScan flow cytometer (Becton-Dickinson), equipped with a double discrimination module. About 1×106 cells were resuspended in 500μL of propidium iodide solution (20μg/mL propidium iodide, 0.2 mg/mL RNase, 0.2 mg/mL EDTA, 0.5% NP-40) and incubated at room temperature for 1 h. In all, 10 000 nuclei were analysed for each sample, and CellFit software was used to quantify cell cycle compartments and estimate cell cycle phase fractions.
In vitro phosphorylation assay was performed as previously described [19]. Cell lysates were first prepared from control and IFN-α2b/PDF-treated cells by three cycles of freeze-thaw in liquid nitrogen. A 5μg aliquot of cell lysate preparation was added to the phosphorylation cocktail containing 55.5 kBq of [γ-32P]-ATP (specific activity: 166.5 TBq/mmol) and incubated at 37°C for 15 min. To activate endogenous double-stranded DNA-dependent protein kinase (DNA-PK), 2μg/mL of fragmented calf thymus DNA was also included in the reaction mixture. Phosphoproteins were then separated by 10% SDS-PAGE and analysed by autoradiography. Intensities of specific DNA-PK bands were then quantified using a scan densitometer (Silk Scientific, Oregon, UT, USA).
For statistical analysis, all data were presented as the mean (SD), and statistical differences between groups were assessed with the unpaired Student’s t-test; P<005 was considered to indicate statistical significance.
RESULTS
EFFECTS OF IFN-α2b AND PDF ON T24 CELL GROWTH
To examine possible effects of IFN-α2b and PDF on T24 cell proliferation, cells were cultured with the varying concentrations of IFN-α2b (0–50 000 IU/mL) or PDF (0–700μg/mL) for 72 h. IFN-α2b caused an ≈50% reduction in cell number at 20 000 IU/mL, which further declined to ≈66% at 50 000 IU/mL (Fig. 1A). PDF had no effects up to 200μg/mL, but there was an ≈20% and ≈ 53% growth reduction at 400 and 700 μg/mL, respectively (Fig. 1B). Thus, these results show that 20000 IU/mL of IFN-α2b or 700 μg/mL of PDF is required to significantly inhibit the growth of T24 cells.
SYNERGISTIC GROWTH INHIBITORY EFFECTS OF IFN-α2b AND PDF
We next examined whether the combination of IFN-α2b and PDF might have a better growth inhibitory effect. Cells were cultured with combinations of IFN-α2b and PDF at the varying concentrations and cell growth was assessed at 72 h. The combination of 10 000 IU/mL IFN-α2b and 200 μg/mL PDF resulted in an ≈75% reduction in cell growth (Fig. 2). This suggests that the IFN-α2b/PDF-induced growth reduction is due to a synergistic effect, because the effect of the two agents was greater than the effect of each agent individually (Fig. 1A,B).

FIG. 1. Effects of IFN-α2b or PDF on T24 cell growth. T24 cells were cultured with varying concentrations of either IFN-α2b(0–50 000 IU/mL) or PDF (0–700μg/mL), and cell numbers in IFN-α2b-treated (A) or PDF-treated (B) were determined at 72 h. All data are the mean±SD from three separate experiments; *P<0.03; **P<0.05.

 FIG. 2. Effects of combinations of IFN-α2b and PDF on cell growth. Cells were cultured with varying concentrations of IFN-α2b/PDF combination for 72 h, and cell growth was assessed by the percentage relative to the cell number in control (100%). Cell growth in control, IFN-α2b(10 000 IU/mL)-treated, PDF (200μg/mL)-treated, or IFN-α2b(10 000 IU/mL)/PDF (200μg/mL)-treated cells is shown. The data are mean±SD from three independent experiments; *P<0.02.
EFFECTS OF COMBINED IFN-α2b AND PDF ON THE CELL CYCLE
To explore the underlying mechanism of such a synergistic growth inhibition induced by the IFN-α2b/PDF combination, cells were treated with IFN-α2b (10 000 IU/mL), PDF (200 μg/mL), or their combination for 72 h and subjected to cell cycle analysis. As shown in Table 1, IFN-α2b or PDF alone had little effects similar to cell cycle phase distribution in control cells; in contrast, the IFN-α2b/PDF combination caused an ≈63% decrease in cell number in the S phase with a concomitant 55% increase in the G1-phase cell population, compared with those in controls. These results thus indicate that the IFN-α2b/PDF combination may cause a blockage of cells entering from the G1 to the S phase, known as a G1 cell cycle arrest.

INVOLVEMENT OF DNA-DEPENDENT PROTEIN KINASE (DNA-PK) IN GROWTH INHIBITION
As many proteins and enzymes are known to be modulated by IFNs [20,21], it is feasible that the growth-inhibitory activity of IFN-α2b with PDF may involve specific PK(s) acting on the signal transduction pathway for cell proliferation. Particularly, we are interested in a PK, namely double-stranded DNA-PK, whose activity relies essentially on small double-stranded DNA [22]. Accordingly, cell extracts obtained from control and IFN-α2b (10 000 IU/mL)/PDF (200 μg/mL)-treated cells at 72 h were subjected to in vitro phosphorylation assay in the presence or absence of exogenous DNA (served as a DNA-PK activator). The basal phosphorylation state of DNA-PK in IFN-α2b/PDF-treated cells was ≈three-fold higher than controls (compare lanes 1 with 3 in Fig. 3), and such phosphorylation was increased (autophosphorylated) by additional three-fold
with DNA (compare lanes 3 with 4 in Fig. 3). Therefore, these studies show that the IFN-α2b/PDF-induced growth inhibition is accompanied by activation of DNA-PK.

 FIG. 3. In vitro phosphorylation assays. Cell lysates from control (lanes 1 and 2) and IFN-α2b/PDF-treated cells (lanes 3 and 4) for 72 h were subjected to in vitro phosphorylation assays with (lanes 2 and 4) or without (lanes 1 and 3) DNA, as described in the Methods. Autophosphorylated DNA-PK detected on autoradiogram is shown, and the relative intensities
of DNA-PK bands were quantified (by a scan densitometer) and expressed by arbitrary values.
DISCUSSION
In an attempt to establish an improved method for bladder cancer therapy, pilot clinical trials using combinations of IFN-α and BCG has been conducted. These trials showed that such combined therapy could lower BCG toxicity against tumours [13]. However, the exact mechanism by which IFN-α potentiates BCG-mediated anti-bladder cancer immunity has not been fully understood. In addition, IFN-α therapy has several drawbacks, such as high cost and repeated administration. A standard intravesical IFN-α instillation (combined with BCG) is carried out with 50–100×106 IU of IFN-α [23], although such a high dosage appears to be in excess of the actual amount needed for an effective antitumour immunity. Moreover, even a high dose of IFN-α may not be sufficient to induce optimal immunity because of its short retention time inside the bladder [23].
Consequently, we explored an alternative approach for bladder cancer immunotherapy by combining IFN-α2b and PDF, a bioactive proteoglucan of maitake mushroom. We found that IFN-α2b at concentrations of ≥20 000 IU/mL were able to induce a significant (≈50%) growth reduction in T24 cells. PDF could be also effective at a relatively high concentration of 700 μg/mL, leading to an ≈53% growth inhibition. This PDF concentration seems to be yet higher than a physiologically achievable concentration, although such a concentration has not been established at this time.
Nevertheless, the possibility that PDF might be able to enhance or potentiate antiproliferative activity of IFN-α2b was then tested. The combination of 10 000 IU/mL IFN-α2b and 200 μg/mL PDF was capable of inducing an ≈75% growth reduction (Fig. 2). This enhanced growth inhibition probably resulted from a synergistic potentiation of the two agents, because neither IFN-α2b (10 000 IU/mL) nor PDF (200 μg/mL) alone had such growth-inhibitory activity (Fig. 1A,B). It also show