肾细胞癌的新治疗方法维生素K3与舞茸Dfraction相结合诱导癌细胞凋亡.docx

1、肾细胞癌的新治疗方法维生素K3与舞茸Dfraction相结合诱导癌细胞凋亡声明：本文摘自美国Journal of Endourology，即内泌尿学杂志，是微创泌尿外科学领域的主要杂志，为临床外科医生提供泌尿生殖道的良性和恶性疾病的最新治疗方法。为方便广大读者阅读，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。 -本论文由杭州正树保健食品有限公司整理编辑【参考译文】Alternative Therapeutic Approach to Renal-Cell Carcinoma:Induction of Apoptosis with Combinationof Vitamin

2、K3 and D-fraction肾细胞癌的替代治疗方法：维生素K3与舞茸D-fraction相结合诱导癌细胞凋亡ABSTRACT 翻译参考摘 要 目的：由于晚期肾细胞癌（RCC）的预测性差，因此我们研究了一种替代治疗方法，即联合使用维生素K3(VK3)和舞茸D-fraction(DF)。VK3是一种人造维生素K的衍生物，而DF是一种菇类的生物活性提取物，这两者都被证实具有抗肿瘤活性。因此，我们进行了体外实验来研究VK3与舞茸D-fraction联合对抗晚期肾细胞癌的效果。 材料与方法：将ACHN细胞株（RCC）分别用不同浓度的VK3和D-fraction以及两者的混合物处理，72小时后用MT

3、T法测定细胞生存率。为了考察其可能存在的抗癌机制，我们对细胞周期，染色质修饰以及细胞凋亡等部分进行研究。 结果：当VK3浓度为4M时 ，可导致癌细胞生存率减少约20 %，而当舞茸D-fraction浓度大于等于500g/mL时，癌细胞生存率减少20%至45 %。然而，当VK3（4M）与舞茸D-fraction（300g/ml）联合使用时，癌细胞生存率降低了90 %以上。细胞周期分析表明，VK3/DF 疗法会引起G1细胞周期停滞，同时伴随着p21WAF1 和p27Kip1增长。在实验过程中，组蛋白脱乙酰基酶(HDAC)也明显被灭活(约60%)，这指示染色质修饰的情况。另外，蛋白质印迹分析表明，经

4、VK3/DF处理的细胞中出现Bax上升以及（ADP-核糖）聚合酶（PARP）活化现象，这些都会诱发癌细胞的凋亡。 结论：VK3和DF联合导致ACHN细胞生存率显著降低，并通过p21WAF1 介导的G1细胞周期停滞最终诱导细胞凋亡。因此，VK3/DF的联合应用可能具有临床意义，它能作为一种替代疗法用于改善晚期RCC的治疗模式。论文原文Journal of Endourology Volume 27, Number 12, December 2013, pp 1499-1503Alternative Therapeutic Approach to Renal-Cell Carcinoma:Indu

5、ction of Apoptosis with Combinationof Vitamin K3and D-fractionMichael Degen, MD, Bobby Alexander, MD, Muhammad Choudhury, MD,Majid Eshghi, MD, and Sensuke Konno, PhDAbstract Purpose: Because of a dismal prognosis for advanced renal-cell carcinoma (RCC), an alternative therapeutic approach, using vit

6、amin K3(VK3) and D-fraction (DF) was investigated. VK3 is a synthetic VK derivative and DF is a bioactive mushroom extract, and they have been shown to have antitumor activity. We examined if the combination of VK3 and DF would exhibit the improved anticancer effect on RCC in vitro. Materials and Me

7、thods: Human RCC, ACHN cell line, were treated with varying concentrations of VK3, DF, or a combination of the two. Cell viability was assessed at 72 hours by MTT assay. To explore the possible anticancer mechanism, studies on cell cycle, chromatin modifications, and apoptosis were conducted. Result

8、s: VK3 alone led to a 20% reduction in cell viability at 4M, while DF alone induced a 20% to 45% viability reduction at 500g/mL. A combination of VK3(4M) and DF (300g/mL) led to a drastic 90% viability reduction, however. Cell cycle analysis indicated that VK3/DF treatment induced a G1 cell cycle ar

9、rest, accompanied by the up-regulation of p21WAF1and p27Kip1. Histone deacetylase (HDAC) was also significantly (60%) inactivated, indicating chromatin modifications. In addition, Western blot analysis revealed that the up-regulation of Bax and activation of poly-(ADP-ribose)-polymerase (PARP) were

10、seen in VK3/DF-treated cells, indicating induction of apoptosis. Conclusions: The combination of VK3 and DF can lead to a profound reduction in ACHN cell viability, through a p21WAF1-mediated G1 cell cycle arrest, and ultimately induces apoptosis. Therefore, the combination of VK3/DF may have clinic

11、al implications as an alternative, improved therapeutic modality for advanced RCC.Introduction Renal-cell carcinoma (RCC) is the third most common genitourinary tumor in the United States,1and nearly 30% of patients with RCC will present with metastatic disease.2,3Standard treatment for nonmetastati

12、c RCC is complete resection of the tumor by either a radical or partialnephrectomy4,5; however, 20% to 30% of these patients will progress to metastatic disease. While a majority of these patients in whom metastsis develops after surgery presented with larger tumors necessitating radical nephrectomi

13、es, a recent study suggested that distant metastasis developed in 4% of patients who had positive surgical margins after nephron-sparing surgery, commonly performed laparoscopically or robotically.6 With a 5-year survival rate of 10% for metastatic RCC,7 these patients are left with a dismal prognos

14、is, making RCC one of the most fatal genitourinary cancers. Unfortunately, conventional therapeutic modalities for metastatic RCC, including cytoreductive surgery, radiotherapy, chemotherapy, or immunotherapy, were not very effective.3,8 Newer, specifically targeted treatment options for distant met

15、astasis, including tyrosine kinase inhibitors (sunitinib, etc.), mammalian target of rapamycin inhibitors (temsirolimus, etc.), and monoclonal antibodies (G250, etc.), are promising, but the survival results are still poor.9 These reasons thus prompted us to explore a novel treatment for patients wi

16、th metastatic RCC. We have been working with vitamins and natural agents/substances as an alternative approach to such metastatic RCC cases. Particularly, we were interested in vitamin K3 (menadione; VK3) as a potential therapeutic agent. VK3 is a synthetic derivative of naturally occurring fat-solu

17、ble vitamin K (VK)10 and has been shown to have an antitumor effect on several cancer cells.11-13 Because VK3 requires a relatively high dose to be effective,11 combinations of VK3 and other agents have been postulated to improve its efficacy at a lower dose. At the same time, we have also been stud

18、ying the bioactive extract from maitake (Grifola frondosa), known as D-fraction (DF). DF has been extensively studied for the past 30 years and has been shown to have immunomodulatory and antitumor activities.1416 For instance, antitumor activity of DF has been demonstrated in tumor-bearing mice thr

19、ough activation of various immune effectors.14,15 In addition,induction of apoptosis by DF was also reported in breast cancer cells.16 DF thus appears to be an interesting and promising natural agent that could be used in cancer treatment. The purpose of this study was to investigate whether a combi

20、nation of VK3 and DF would exhibit the improved and enhanced anticancer effect on RCC in vitro, as an alternative therapeutic approach. These studies were performed focusing mainly on cell viability, cell cycle, chromatin modifications, and apoptosis.Materials and MethodsCell culture The human renal

21、 carcinoma ACHN cells (with aggressive grade IV property) were maintained in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100g/mL). For experiments, ACHN cells were seeded at the initial cell density of 2105 cells/mL in six-well plates or T-75 flas

22、ks and cultured with varying concentrations of VK3, DF, or their combinations. Cell viability was then assessed at 72 hours by 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay.Cell viability assay (MTT assay) Cell viability was determined by MTT(3-4,5-dimethylthiazol-2-yl-2

23、,5-diphenyl-tetrazolium bromide) assay following the protocol of the vendor (Sigma-Aldrich,St.Louis,MO).Briefly, at the harvest time, MTT reagent (0.5mg/mL) was added to each well in the six-well plate, followed by 3-hour incubation at 37.After removing MTT reagent, dimethyl sulfoxide was added to e

24、ach well and absorbance of formazan solution was read on a microplate reader. Cell viability was then expressed as percentage (%) of viable cells relative to the control reading(100%).Cell cycle analysis A BD FACscan flow cytometer (Franklin Lakes, NJ), equipped with a double discrimination module,

25、was used for cell cycle analysis. Approximately 1106 cells were re-suspended in 500L of propidium iodide solution and incubated at room temperature for 1 hour. Ten thousand nucleiwere analyzed for each sample, quantified in cell cycle compartments, and estimated as cell cycle phase fractions.Histone

26、 deacetylase (HDAC) assay HDAC activity was measured using the Epigenase HDAC Assay Kit following the protocol of the manufacturer (Epigentek, Farmingdale, NY). Briefly, 10g of nuclear extracts from each sample were added to the coated microplate wells. After 90-minute incubation at 37, all wells in

27、 the plate were treated with 1st antibody for 60 minutes, followed by 30-minute incubation with 2nd antibody. The plate was then treated with reaction solution for 10 minutes, and the reaction was terminated with stop solution. Absorbance of samples was read on a microplate reader, and HDAC activity

28、 was calculated and expressed by the % relative to controls (100%).Western blot analysis The detailed procedures are described elsewhere.17 An equal amount of cell lysates (7g), obtained from control and agent-treated cells, was first subjected to 10% SDS gel electrophoresisand transferred to a nitr

29、ocellulose membrane (blot). The blot was incubated for 90 minutes with the primary antibodies against p21WAF1, p27Kip1, Bax, or poly-(ADP-ribose)-polymerase (PARP) (Santa Cruz Biotechnology, Santa Cruz, CA), followed by 30-minute incubation with the secondary antibody conjugates. The specific immuno

30、reactive protein bands were then detected by chemiluminescence following the protocol of the manufacturer (KPL, Gaithersburg, MD).Statistical analysis All data were presented as mean+standard deviation, and statistical differences between groups were assessed with either one-way analysis of variance

31、 or the unpaired Student t test. Values of P0.05 were considered to indicate statistical significance.ResultsEffects of VK3, DF, or their combinations on ACHN cell growth ACHN cells were cultured separately with varying concentrations of VK3(04M) or DF (0700g/mL). After 72 hours, MTT assay showed th

32、at VK3 alone had little effects but led to a 20% reduction in cell viability at 4M (Fig. 1A), while DF induced a 20% to 45% viability reduction at500g/mL (Fig. 1B). Whether the combination of VK3 and DF may potentiate the anticancer effect was also examined. Such study showed that a profound cell viability reduction, from severe (90%) cell death, was attained with the specific combination of 4M VK3 and 300g/mL DF (Fig. 2).Effects of VK3/DF combination on cell cycle To explore how such VK3/DF combination would induce a viability reduction or cell