Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair

To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy.Abbreviations: DSB, double-strand break; HR, homologous recombination; RER, radiation enhancement ratio; RT, radiation     

二甲双胍抑制胰腺癌干细胞生长的实验研究

摘 要：［目的］ 探讨二甲双胍对胰腺癌干细胞的生长抑制作用。［方法］ 利用无血清培养基的超低粘附培养方法获得干细胞球样胰腺癌PANC1细胞,然后通过定量PCR检测干细胞球样PANC1细胞中CD133及PDX-1的表达情况,验证胰腺癌干细胞;利用干细胞球样PANC1细胞的MTT实验、流式周期检测实验、Annexin-Ⅴ细胞凋亡实验、定量PCR凋亡因子检测实验,研究二甲双胍对干细胞球样PANC1细胞增殖、周期及凋亡的影响;借助干细胞球样PANC1胰腺癌细胞构建鼠移植瘤模型,观察二甲双胍对肿瘤生长的影响。［结果］ 二甲双胍能够明显抑制干细胞球样胰腺癌PANC1细胞增殖,且具有浓度依赖性,最大抑制率为81.3%,半数有效抑制浓度IC50为（11.94±1.43）mmol/L;二甲双胍对干细胞球样PANC1细胞的G0/G1期有阻滞作用;二甲双胍可以诱导干细胞球样PANC1细胞凋亡,与空白对照组比较,二甲双胍组干细胞球样PANC1细胞的Bcl-2 mRNA减少,而Bad、Bax mRNA的表达增加;干细胞球样PANC1细胞接种至裸鼠皮下均能成瘤,且二甲双胍能够明显抑制移植瘤的生长。［结论］ 二甲双胍能够抑制胰腺癌干细胞增殖,阻滞G0/G1期,并诱导胰腺癌干细胞凋亡,发挥抗胰腺癌干细胞生长的作用。     

靶向EGFR基因的shRNA抑制胰腺癌PANC-1细胞增殖的研究

目的探讨EGFR基因对胰腺癌PANC-1细胞的增殖抑制作用。方法构建针对EGFR序列特异性shRNA的表达载体,用脂质体转染胰腺癌PANC-1细胞。采用RT-PCR、Western blot检测EGFR mRNA和蛋白的表达;流式细胞仪检测细胞周期及凋亡;克隆形成实验检测细胞增殖。结果靶向EGFR的序列特异性shRNA明显抑制EGFR mRNA和蛋白的表达,EGFR mRNA和蛋白的抑制率分别为72.1％和67.6％;G1期细胞增多、S期细胞减少(P<0.05);细胞凋亡增加(P<0.05);克隆形成减少(P<0.05)。结论靶向EGFR的序列特异性shRNA能明显抑制胰腺癌细胞增殖、促进凋亡。     

胰腺癌干细胞靶向治疗研究进展

摘 要：胰腺癌干细胞在胰腺癌的发生发展过程中起着很关键的作用。最近不断有报道证实针对肿瘤干细胞的治疗可以明显减弱肿瘤干细胞的信号传导。因此胰腺癌干细胞靶向治疗药物可以明显改善胰腺癌患者的预后。现对胰腺癌干细胞靶向治疗的进展作一综述。     

胃癌干细胞鉴定和治疗的研究进展

胃癌是最常见的消化道恶性肿瘤。近年来,随着对干细胞和肿瘤研究的不断深入,越来越多的证据表明,肿瘤来源于干细胞,肿瘤干细胞在胃癌发病机制中的作用日益受到重视。但是在对胃癌干细胞的筛选方面仍没有非常精准的方法,如何针对胃癌干细胞进行治疗仍是现在研究的热点。本文对胃癌干细胞的研究进展作一综述,将有助于加深对胃癌干细胞的认识,并为从根本上控制胃癌的发生和发展提供线索。      

干细胞、癌症与癌症干细胞

干细胞被认为是一种未分化的细胞,具有永远增殖和自我更新能力.大量研究证明,癌症中存在对化疗更具抵抗性的癌症干细胞.识别癌症干细胞和普通癌症细胞之间的差异,可以发展更有效的癌症分类、诊断和治疗方法.     

Chk1/2对肿瘤细胞凋亡的调节作用

目的:观察顺铂作用下K562细胞及白血病骨髓单个核细胞(BMMNC)的细胞周期变化和转染Chk1/2反义寡核苷酸对顺铂诱导下K562细胞及白血病BMMNC凋亡的影响.方法:用流式细胞仪检测顺铂作用下K562细胞及白血病BMMNC的细胞周期变化.转染Chk1和Chk2反义寡核苷酸于K562细胞及白血病BMMNC,检测顺铂作用下转染细胞的凋亡率.结果:10 μmol/L顺铂作用下K562细胞和白血病BMMNC均出现S期阻滞,转染Chk1/2反义寡核苷酸可明显增加顺铂诱导下K562细胞凋亡,转染Chk1反义寡核苷酸可明显增加顺铂诱导下白血病BMMNC的凋亡率,但转染Chk2反义寡核苷酸未增加白血病BMMNC的凋亡率.结论:Chk1在肿瘤细胞凋亡中有重要调节作用,可作为肿瘤增敏治疗的有效靶点,Chk2在肿瘤细胞凋亡中的作用需进一步研究.     

Nutlin-3-Induced Sensitization of Non-Small Cell Lung Cancer Stem Cells to Axitinib-Induced Apoptosis Through Repression of Akt1/Wnt Signaling

The aim of this study was to investigate the potential biological activities of nutlin-3 in the regulation of growth and proliferation of non-small cell lung cancer (NSCLC) stem cells (CSCs), which may help in sensitizing to axitinib-induced apoptosis. Nutlin-3 induction of p53 expression was used to test its role in controlling the cell division pattern and apoptosis of NSCLC cells. A549 cells and H460 cells were pretreated with nutlin-3 and then treated with either an Akt1 activator or shRNA-GSK3 , to investigate the potential role of p53 sensitization in the biological effects of axitinib. We also determined the expression levels of GSK3 and p-Akt1 in patients with NSCLC and determined their potential association with survival data using Kaplan–Meier plots and CBIOTAL. Increased p53 expression stimulated the induction of apoptosis by axitinib and promoted asymmetric cell division (ACD) of NSCLC CSCs. The repression of Akt phosphorylation induced by nutlin-3 promoted the ACD of lung CSCs, decreasing the proportion of the stem cell population. In addition to the induction of apoptosis by axitinib through inhibition of Wnt signaling, nutlin-3 treatment further enhanced axitinib-induced apoptosis by inhibiting Akt1/GSK3 /Wnt signaling. The low expression of GSK3 and increased expression of p-Akt in patients with NSCLC were closely associated with the development of NSCLC. TP53 stimulates the induction of apoptosis in NSCLC by axitinib and the ACD of lung CSCs through its regulatory effects on the p53/Akt/GSK3 pathways.     

MiR-21 Upregulation Induced by Promoter Zone Histone Acetylation is Associated with Chemoresistance to Gemcitabine and Enhanced Malignancy of Pancreatic Cancer Cells

Background and Aims: MicroRNA-21 (miR-21) is reported to be overexpressed and to contribute toproliferation, apoptosis and gemcitabine resistance in pancreatic ductal adenocarcinomas (PDACs). The aims ofthis study were to explore regulation of miR-21 expression by epigenetic change and its impact on chemoresistanceand malignant properties of of pancreatic cancer. Materials and methods: We retrospectively collected 41 cases ofadvanced pancreatic cancer patients who were sensitive or resistant to gemcitabine and assessed levels of serumcirculating miR-21 for correlation with cytotoxic activity. Histone acetylation in the miR-21 promoter was alsostudied in gemcitabine-sensitive and gemcitabine-resistant PDAC cells. Gemcitabine-resistant HPAC and PANC-1cells were transfected with pre-miR-21 precursors (pre-miR-21) and antisense oligonucleotides (anti-miR-21), andwere treated with TSA. Finally, invasion and metastasis assays were performed and alteration in mir-21, PTEN,AKT and pAKT level was evaluated in these cells. Results: Serum miR-21 levels were increased in gemcitabineresistantPDAC patients compared with gemcitabine-sensitive subjects. The miR-21 levels were increased in 6PDAC cells treated with gemcitabine significantly, associated with 50% inhibitory concentrations (IC50s). Histoneacetylation levels at miR-21 promoter were increased in PDAC cells after treatment with gemcitabine. Enhancedinvasion and metastasis, increased miR-21 expression, decreased PTEN, elevated pAKT level were demonstratedin gemcitabine-resistant HPAC and PANC-1 cells. Pre-miR-21 transfection or TSA treatment further increasedinvasion and metastasis ability, decreased PTEN, and elevated pAKT levels in these two lines. In contrast,anti-miR-21 transfection could reverse invasion and metastasis, and PTEN and pAKT expressions induced bygemcitabine. Conclusions: MiR-21 upregulation induced by histone acetylation in the promoter zone is associatedwith chemoresistance to gemcitabine and enhanced malignant potential in pancreatic cancer cells.     

吉西他滨联合顺铂与吉西他滨单药化疗治疗晚期胰腺癌比较的Meta分析

目的：通过Meta分析.探讨吉西他滨联合顺铂和吉西他滨单药化疗在治疗晚期胰腺癌的意义.方法：通过MEDLLNE,EMBASE,ASCO论文集等数据为检索国内外已发表和未发展的相关文献,选择治疗组为吉西他滨联合铂化疗,对照组为吉西他滨单药化疗的晚期县级腺癌对照试验（randomized controlled trial RCT）.由两位评委者分别按上述检索策略收集资料,按铵入标准入选,主要对生存率进行Meat分析,其次是客观缓解率和毒副反应.结果：共纳入6个RCT,吉西他滨联合顺铂化疗与吉西他滨单药化疗比较,装卸生存率无差别（P=0.25）,临床获益反应率无差别（P=0.58）,客观缓解率提高6%（P=0.08）,血小板减少症增加8%（P=0.17）,恶心/呕吐增加11%（P=0.07）,但均无统计学意义.结论：现有证据不推荐吉西他滨联合铂治疗晚期胰腺癌,吉西他滨单药化疗仍是目前的标准治疗.     

人体胰腺癌细胞株PANC-1中四环素可诱导的周期素D1表达抑制系统的建立

[目的]在人体胰腺癌细胞株PANC-1中建立一个四环素可调控周期素D1表达系统。研究抑制周期素D1对胰腺癌细胞的影响。[方法]反义周期素D1质粒通过两次稳定转染进入胰腺癌细胞株PANC-1细胞中，其表达调控系统采用Tet-Off系统（四环素调控系统）。通过抑制周期素D1表达对PANC-1细胞生长、集落形成能力以及周期素蛋白表达的影响，评价此系统的可调控性和有效性。[结果]通过第一次转染pTet—Off质粒，选择两个最佳表达克隆行第二次转染DTRE-反义周期素D1质粒，并通过免疫印记测定挑选出能在Tet—Off系统中最有效地表达反义周期素D1的克隆。通过Tet—Off系统对反义周期素D1的调控．发现周期素D1的表达抑制可明显地抑制胰腺癌细胞生长和集落能力，并可导致胰腺癌细胞形态学改变。其抑制作用与四环素调控浓度和时间有关。[结论]此研究在PANC-1胰腺癌细胞株中建立了一个高效、可诱导的反义周期素D1的表达系统。通过这个系统的建立，可进一步在体内和体外研究周期素D1的抑制对胰腺癌细胞的影响．并可结合其它治疗手段如化疗来探讨联合治疗在临床的潜在应用价值。     

NID1增强人卵巢癌细胞OVCAR-3干细胞特性及分子机制研究

摘 要：［目的］ 探讨NID1对卵巢癌干细胞表型的潜在影响以及NID1与干细胞标志蛋白联合对预后的潜在价值。［方法］ 采用肿瘤干细胞球形成实验、荧光定量RT-PCR和Western blot分别检测稳定过表达外源NID1的OVCAR-3细胞的自我更新能力和卵巢癌干细胞标志物的表达情况。借助生物信息学分析评估NID1在卵巢癌临床样本中与卵巢癌干细胞标志物的相关性及两者联合对预后预测的价值。［结果］ 与空载细胞相比,稳定过表达NID1的OVCAR-3细胞自我更新能力增强（P=0.006）,同时伴随着干细胞标志物CD44（P=0.000）和ABCG2（P=0.000）表达水平升高。经ERK/MAPK通路的抑制剂U0126下调ERK1/2的磷酸化水平后,其CD44和ABCG2的水平下降。此外,基于两套卵巢癌表达谱芯片数据的生物信息学分析表明,NID1的表达水平与CD44（P=0.006;P=0.036）、ABCG2（P=0.000;P=0.000）表达水平均显著正相关,而NID1高表达并且携带干细胞表型的卵巢癌患者总体存活时间最短（P=0.026;P=0.000）。［结论］ NID1可能通过激活ERK/MAPK通路诱导卵巢癌细胞出现干细胞特性,NID1与干细胞标志物联合可能具有预后预测意义。     

Cancer Stem Cells and Response to Therapy

The cancer stem cell (CSC) model states that cancers are organized in cellular hierarchies, which explains the functional heterogeneity often seen in tumors. Like normal tissue stem cells, CSCs are capable of self-renewal,either by symmetric or asymmetric cell division, and have the exclusive ability to reproduce malignant tumors indefinitely. Current systemic cancer therapies frequently fail to eliminate advanced tumors, which may be dueto their inability to effectively target CSC populations. It has been shown that embryonic pathways such as Wnt, Hedgehog, and Notch control self-renewal and cell fate decisions of stem cells and progenitor cells. These are evolutionary conserved pathways, involved in CSC maintenance. Targeting these pathways may be effective in eradicating CSCs and preventing chemotherapy or radiotherapy resistance.     

Targeting Cancer Stem Cells by Phytochemicals: a Multimodal Approach to Colorectal Cancer

Cancer stem cells (CSCs) exist within a tumor as a rare subpopulation, with the capacity of self-renewal and the ability to differentiate into heterogeneous population of cancer cells. CSCs are increasingly being implicated in tumor recurrence thereby further augmenting the menace of the malignant disease. Characterization of CSCs has unearthed their pivotal role in all the hallmarks of cancer including tumorigenesis, angiogenesis, metastasis and drug resistance, thereby designating cancer as a “stem cell disease.” Here, we discuss the limitations of current therapeutic strategies that spare CSCs thereby failing to achieve complete cure of colorectal cancer, and elucidate the role of multimodal CSC-targeted treatment strategies, using natural phytochemicals and their derivatives. With emerging evidences identifying the molecular targets of phytochemicals in colorectal CSCs, development of better therapeutic strategies uprooting CSCs, the root of all evils, can be envisaged.     

肿瘤干细胞与肿瘤转移

肿瘤干细胞和其微环境住肿瘤形成、浸润性生长和转移灶形成等各步骤均具有关键性作用。阐明其相互作用的分子机制，可为肿瘤转移的诊断、治疗和预后，提供可靠的分子标志和靶点：文章主要就以上进行综述。     

大黄素联合吉西他滨抑制胰腺癌细胞系PANC-1细胞增殖能力的研究

目的:验证大黄素联合吉西他滨(gemcitabine,GEM)抑制胰腺癌细胞系PANC-1细胞增殖的作用,探讨大黄素促PANC-1细胞凋亡的抗肿瘤作用机制.方法:MTT方法检测大黄素单药以及与吉西他滨联合应用对PANC-1细胞增殖能力的影响.ELISA方法测定细胞上清IL-6水平.RT-PCR方法检测大黄素处理后PANC-1细胞内侵袭相关基因MMP-9的表达情况.Western blot方法分别检测大黄素以及吉西他滨处理PANC-1后凋亡相关蛋白Bax和Bcl-2的表达水平.结果:MTT检测结果显示,加药处理细胞72h后,大黄素组(40 μmol/l)PANC-1细胞抑制率为31％,GEM组(20μmol/l)的抑制率为35％,联合用药组的抑制率为49％,与对照组相比差异有统计学意义(P＜0.05).IL-6浓度大黄素组为(22.41±2.27) ng/ml,联合用药组为(15.44±3.91) ng/ml.实验组均显著低于对照组,差异有统计学意义(P＜0.05).大黄素组、联合用药组MMP-9 mRNA表达水平显著低于对照组,GEM组较对照组相比差异无统计学意义(P＞0.05).大黄素组、GEM组、联合用药组Bax表达水平上调,但Bcl-2差异无统计学意义.大黄素组、GEM组、联合用药组的Bax/Bcl-2分别为2.71 ±0.25,4.73---0.17,5.72 ±0.36,均显著高于对照组1.14 ±0.15,结论:体外研究结果显示大黄素能够抑制肿瘤PANC-1细胞增殖,具有促细胞凋亡的作用,与吉西他滨联合应用具有协同效应.     

Induction Gemcitabine Plus Concurrent Gemcitabine and Radiotherapy for Locally Advanced Unresectable or Resected Pancreatic Cancer

AimsTo determine the efficacy of induction gemcitabine followed by biweekly gemcitabine concurrent with radiotherapy for locally advanced pancreatic cancer.Materials and methodsBetween March 2001 and August 2009, 90 patients with unresectable (78) or resected (12) pancreatic cancer were treated with a standard treatment policy of induction gemcitabine (seven doses of weekly gemcitabine at 1000 mg/m²) followed by concurrent radiotherapy (52.5 Gy) and biweekly gemcitabine (40 mg/m²).ResultsAfter induction gemcitabine, 17.8% of patients did not proceed to chemoradiotherapy, due to either disease progression, performance status deterioration or gemcitabine toxicity. Of the patients who received chemoradiotherapy, 68.9% completed the course of 52.5 Gy, whereas 79.7% received more than 45 Gy. Chemoradiotherapy was stopped early due to treatment toxicity in 22.9% of patients. On intention to treat analysis, the median overall survival was 12.7 months in the locally advanced group and 18.2 months in the resected group. On multivariate analysis for the unresectable patients, a larger gross tumour volume was a significant poor prognostic factor for overall survival and local progression-free survival.ConclusionThis large series confirms, in a standard practice setting, similar efficacy and tolerability of treatment as previously reported in our phase I–II study. The benefit to patients with a gross tumour volume >48 cm³ may be limited.     

Phase II Study of Pemetrexed Plus Gemcitabine in Advanced Pancreatic Cancer

Background:Gemcitabine is the standard chemotherapy for the treatment of advanced pancreatic cancer. The novel multitargeted antifolate pemetrexed (Alimta®) has also demonstrated activity in this disease. These two drugs are synergistic in vitro, and a phase I study has demonstrated activity of single-agent pemetrexed in pancreatic cancer. Aim:To evaluate pemetrexed plus gemcitabine in a multicenter phase II study in patients with advanced pancreatic cancer. Patients and methods:Chemonaive patients with advanced pancreatic adenocarcinoma, metastatic or locally advanced, not amenable to curative resection, received intravenous gemcitabine 1250 mg/m² over 30 minutes on days 1 and 8 and intravenous pemetrexed 500 mg/m² over 10 minutes on day 8 every 21 days. The primary endpoint was objective response rate. Patients were evaluated for time-to-event variables including overall survival, time-to progression, time-to-treatment failure, and duration of response. Patients who were symptomatic were evaluated weekly for clinical benefit response. CT scans were obtained every two cycles. Results:Forty-two patients were treated; 41 were evaluable for efficacy. Ninety-five percent of patients had metastatic disease. There were five partial responses (objective response rate 12%), and 44% of patients had stable disease. The 1-year survival rate was 32%; median survival was 6.6 months (95% CI 4.4, 9.9). Of 30 eligible patients, four (13%) had a clinical benefit response. Grade 3 and 4 hematologic toxicities included neutropenia (84%), febrile neutropenia (12%), and thrombocytopenia (33%). Non-hematologic toxicities were minimal. Conclusion:The combination of pemetrexed and gemcitabine is active in advanced pancreatic cancer, and has acceptable toxicity; a 1-year survival rate of 32% is encouraging. A phase III trial comparing pemetrexed plus gemcitabine with gemcitabine has completed accrual. 1 The use of trade names is for product identification purposes only and does not imply endorsement.     

肿瘤干细胞与肿瘤转移

肿瘤干细胞（TSCs）在肿瘤启动作用中的重要性已在造血系统肿瘤中得到证实，最近亦有在实体瘤中的相关报道。TSCs与正常干细胞具相似的生物学特性，这对研究TSCs与肿瘤转移的关系有非常重要意义？文章简要论述TSCs研究现状及TSCs与肿瘤转移两者可能存在的相关性。