白藜芦醇对CNE-1鼻咽癌细胞放射增敏效应的研究

目的:探讨白藜芦醇(RV)对鼻咽癌细胞CNE-1的放射增敏作用及其机制.方法:将实验分为空白对照组、单纯用药组(10、20和50 μmol/L RV)、单纯照射组(1、2、3、4、5和7 Gy)以及实验组(药物+照射).利用多靶单击模型拟合放射剂量-细胞生存曲线,检测RV对鼻咽癌细胞CNE-1的放射增敏效应.用流式细胞仪观察RV对细胞周期分布和细胞凋亡的影响,并观察细胞的形态学变化.结果:RV作用48 h后,10、20、50 μmol/L RV的放射增敏比分别为1.07、1.32和1.66,呈药物浓度依赖性.流式细胞仪检测显示,RV和照射均可使G2/M期细胞阻滞,凋亡值(AI)增加,单纯用药组(10、20和50 μmol/L RV)、单纯照射组(1、2、3、4、5和7 Gy)和实验组(药物+照射)的G2/M及AI值均随药物浓度的增加而增加,P值均＜0.01.形态学检测可见凋亡小体,且两者具有协同作用.结论:RV对人鼻咽癌细胞CNE-1具有放射增敏效应,其机制可能与RV抑制细胞修复、导致G2/M期细胞阻滞和诱导细胞凋亡有关.     

肿瘤放射增敏剂的研究进展

放射增敏剂的研究包括亲电子类化合物、生物还原活性物、靶点药物等方面,部分药物已取得了不同程度的临床疗效。替拉加明(TPZ)对于头颈癌、非小细胞肺癌和宫颈癌,尼莫唑(nimorazole)对于头颈癌和宫颈癌,爱必妥(C225)对于表皮生长因子受体过表达和(或)突变程度高的肿瘤,希美钠(CMNa)对于头颈癌、食管癌和肺癌等表现出明确的放射增敏作用,有很好的临床应用价值。而高效、低毒、广谱的放射增敏剂将是本领域的研究方向。     

肿瘤放射增敏剂的研究进展

放射增敏剂的研究包括亲电子类化合物、生物还原活性物、靶点药物等方面,部分药物已取得了不同程度的临床疗效。替拉加明（TPZ）对于头颈癌、非小细胞肺癌和官颈癌,尼莫唑（nimorazole）对于头颈癌和官颈癌,爱必妥（C225）对于表皮生长因子受体过表达和（或）突变程度高的肿瘤,希美钠（CMNa）对于头颈癌、食管癌和肺癌等表现出明确的放射增敏作用,有很好的临床应用价值。而高效、低毒、广谱的放射增敏剂将是本领域的研究方向。     

青蒿素对人宫颈癌细胞放射增敏作用及其机制的研究

目的:研究青蒿素(artemisinin)对人宫颈癌HeLa细胞放射敏感性增强的作用机制.方法:MTT法检测青蒿素对正常人皮肤成纤维细胞GM0639、宫颈鳞癌SiHa细胞和宫颈腺癌HeLa细胞的生长抑制作用.克隆形成实验检测青蒿素对GM0639、SiHa和HeLa细胞的放射增敏作用.Western 印迹法检测CyclinB1和Wee1蛋白在HeLa细胞中表达的变化.结果:MTT法检测结果显示,不同浓度的青嵩素(27.67～442.75 nmol/L)对GM0639、SiHa和HeLa细胞作用24和48 h时均有细胞生长抑制作用;而青蒿素(110.69 nmol/mL)分别作用GM0639、SiHa和HeLa细胞6 h后,对3株细胞的生长抑制率分别为0.93±0.25、0.84±0.10和0.77±0.03,对照组(未加药组)3株细胞的生长抑制率分别为0.92±0.13、0.83±0.09和0.75±0.21,加药组和对照组之间差异无统计学意义(P>0.05).青蒿素对GM0639和SiHa细胞无放射增敏作用,对HeLa细胞有放射增敏作用,SER=1.12;青嵩素和照射联合作用HeLa细胞后,细胞中CyclinB1蛋白表达比照射组增加,Wee1蛋白表达比照射组减少.结论:青蒿素能增加HeLa细胞的放射敏感性,其作用机制可能与上调CylinB1及下调Wee1的蛋白表达有关.     

天然活性产物的放射增敏作用

 天然产物中存在大量具有放射增敏作用的活性物质,这些物质能够通过不同的作用机制,提高肿瘤的放射敏感性,达到增强放疗效果之目的。探讨放射增敏作用的机制,对具有发展成为放射增敏药物潜力的天然化合物进行分类,可为天然放射增敏剂的进一步研发提供一定思路。     

放疗增敏剂研究趋势

利用放射增敏剂提高肿瘤中乏氧细胞对放疗的敏感性,能提高放射线对肿瘤细胞的杀伤率,增强放疗效果。理想的放射增敏剂应可以显著增加放疗的疗效,对正常组织没有或很少有毒副反应。国内外学者进行了大量的研究,在基因和分子靶向药物等方面取得了新进展,显示了未来放疗增敏剂发展的趋势。     

放疗增敏剂研究趋势

利用放射增敏剂提高肿瘤中乏氧细胞对放疗的敏感性,能提高放射线对肿瘤细胞的杀伤率,增强放疗效果。理想的放射增敏剂应可以显著增加放疗的疗效,对正常组织没有或很少有毒副反应。国内外学者进行了大量的研究,在基因和分子靶向药物等方面取得了新进展,显示了未来放疗增敏剂发展的趋势。     

化疗药物的放射增敏作用研究进展

放射增敏剂一直是放射肿瘤学研究的课题，虽然已发现许多药物有放射增敏作用，但多数实用价值不大，细胞毒性化疗药物在临床上与放疗同时应用显示出协同和放射增敏作用，化疗药物作为一种放疗增敏剂已得到了广泛的研究和应用。     

化疗药物的放射增敏作用研究进展

放射增敏剂一直是放射肿瘤学研究的课题,虽然已发现许多药物有放射增敏作用,但多数实用价值不大,细胞毒性化疗药物在临床上与放疗同时应用显示出协同和放射增敏作用,化疗药物作为一种放疗增敏剂已得到了广泛的研究和应用.     

UCN-01增加鼻咽癌细胞放射敏感性的相关机制

目的 在前期研究基础上，进一步探讨UCN-01增加鼻咽癌细胞放射敏感性的相关机制。方法 应用流式细胞仪和凋亡试剂盒测定不同条件下CNE-1细胞的凋亡比例。应用彗星分析法测定不同实验组肿瘤细胞在照射后不同时间的DNA损伤程度，用以观察UCN-01对CNE-1细胞修复DNA损伤能力的影响。结果 照射＋UCN-01组与单纯照射组相比，细胞总凋亡比例仅有轻微增加（14．9％：13．6％），其中主要表现为早期凋亡比例轻度增加（5．0％：3．9％），两组细胞晚期凋亡比例相仿（9．9％：9．7％），细胞凋亡不是UCN-01对CNE-1细胞放射增敏的主要机制。照射后加入UCN-01组细胞的尾力矩下降速度较单纯照射组明显降低，即DNA的修复速度降低，两组的半修复时间分别为3．1、0．4h，说明UCN-01使CNE-1细胞修复放射性DNA损伤的能力下降。结论 UCN-01未明显增加放射后CNE-1细胞的凋亡比例；UCN-01使细胞修复放射性DNA损伤的能力降低可能是该药放射增敏的重要机制之一。     

吉西他滨对非小细胞肺癌细胞放射增敏的实验研究

目的　研究低浓度吉西他滨对非小细胞肺癌细胞系A5 4 9(wtp5 3)的放射增敏作用、并探讨用药后最佳放射时间。方法　①以不同浓度的吉西他滨与A5 4 9细胞作用 2 4h ,MTT法选择生长抑制率≤ 10 %的药物浓度 (IC10 )。②将细胞分为对照组 (Ct)、单纯用药组 (Ch)、单纯照射组 (R1、R2 )、未弃药照射组 (CR)及弃药后照射组 (DCR1、DCR2 )。R1组行单纯60 Co2Gy照射 ;Ch组单纯用药 ;CR组及DCR1组均用IC10 浓度作用 2 4h ,CR组于 2 4h末、DCR1分别于弃药后 30min、1、3、6、12及 2 4h予60 Co2Gy照射。比较细胞存活分数 ,选择最佳弃药时间 (T)。③用IC10 药物浓度作用DCR2 2 4h ,于弃药后T时间经60 Co0 .5、1.0、2 .0、4 .0、6 .0、8.0Gy剂量照射 ,绘制细胞存活曲线 ;与R2 组 (同剂量单纯照射 )比较 ,计算增敏比 (D0 值比和Dq 值比 )。结果　IC10 浓度吉西他滨的放射增敏作用在弃药后 3h最为明显 [增敏比分别为 1.88(D0 值比 )、1.90 (Dq 值比 ) ];弃药 6h后与对照组比较差异无显著性意义 (P >0 .0 5 ) ;这种增敏作用在照射剂量较低时 ( 0 .5、1.0Gy)即已明显表现出来。结论　低浓度吉西他滨对离体非小细胞肺癌A5 4 9(wtp5 3)细胞系具有较好的放射增敏作用 ,且最佳照射时间为弃药后 3h。     

干扰素α-2a对人鼻咽癌细胞株CNE-I放射敏感性的影响

目的探讨干扰素α2a对人鼻咽癌细胞株CNEI放射敏感性及其细胞周期的影响。方法用不同浓度的干扰素α2a处理CNEI细胞后用6MVX线照射,计算细胞存活率。流式细胞仪分析CNEI细胞周期动力学改变。结果0.5×106、1.0×106、1.5×106IU/L干扰素α2a干预CNEI细胞后细胞存活分数分别为0.62、0.43、0.20,放射增敏比为1.16、1.57、1.93;1×106IU/L干扰素α2a干预CNEI细胞24h后,与对照组比较细胞增殖周期G1和G2+M细胞数增多,S期细胞数减少(P<0.01)。结论干扰素α2a能抑制人鼻咽癌细胞株CNEI的生长,并具有放射增敏效应,放射增敏机制可能与其调节CNEI细胞增殖周期有关。     

环氧合酶-2抑制剂NS-398对食管癌细胞放射增敏作用及机制初探

目的探讨COX-2选择性抑制剂NS-398对食管癌细胞株EC706放射敏感性的影响及作用机制。方法选用高表达Cox-2的人食管癌细胞株EC706为靶细胞，加入不同浓度(10、20、50、100μmol／L)的NS-398，分别作用24、48h，用直线加速器分别给予0、2、4、6、8、10Gy剂量照射，采用成克隆实验分析增敏效应，用DNA片段分析法及流式细胞术检测细胞凋亡情况。结果用10、20、50、100μmol／L的NS-398作用24、48h均能检测到放射增敏作用，其放射增敏比(Dq比)分别为1．11、1．24、1．40、1．54和1．11、1．27、1．58、1．67，呈剂量和时间依赖性；同时使细胞凋亡率增加。结论NS-398能增强人食管癌细胞株EC706对放射的敏感性，其增敏机制与抑制亚致死性损伤修复和诱导细胞凋亡增加有关。     

受缺氧反应元件调控的荧光素酶报告基因在缺氧肿瘤细胞内的表达活性研究

 目的 研究缺氧反应元件调控的荧光素酶报告基因在肿瘤细胞内的缺氧反应性。方法 将pGL3-6HRE-TATA-luciferase-SV40pa报告载体以Lipofectamine 2000介导瞬时转染Hela S3和HepG2细胞，分别于缺氧（1 % O2）和常氧（21 % O2）培养后检测相对荧光素酶活性。结果 pGL3-6HRE- TATA- luciferase-SV40pa报告载体转染细胞后，缺氧培养条件下相对荧光素酶的活性较常氧培养条件下显著上调，差异具有统计学意义（P＜0.01），并且该缺氧诱导性具有一定的时间依赖性，即Hela S3细胞中，缺氧培养12～24 h，相对荧光素酶的活性上升到了最高值；而HepG2细胞中，缺氧培养36 h，相对荧光素酶的活性上升到了最高值。结论 6HRE人工启动子在肿瘤细胞内具有显著的缺氧诱导性；6HRE人工启动子缺氧诱导性具有一定的时间效应，并且在不同肿瘤细胞中其诱导效应和时间效应可存在着差异。研究结果为实体肿瘤基因治疗奠定了理论和实验基础。     

Recombinant protein TRAIL-Mu3 enhances the antitumor effects in pancreatic cancer cells by strengthening the apoptotic signaling pathway

Pancreatic cancer is a highly malignant type of cancer and its treatment remains a major challenge. The novel recombinant protein TNF-related apoptosis-inducing ligand (TRAIL)-Mu3 has been shown to exert stronger tumor inhibitory effects in colon cancer in vitro and in vivo compared with TRAIL. The present study investigated the antitumor effects of TRAIL-Mu3 on pancreatic cancer cells, and the possible mechanisms were further examined. Compared with TRAIL, TRAIL-Mu3 exhibited significantly higher cytotoxic effects on pancreatic cancer cell lines. The inhibitory effect of TRAIL-Mu3 on the viability of PANC-1 cells was shown to be a caspase-dependent process. The affinity of TRAIL-Mu3 to PANC-1 cell membranes was significantly enhanced compared with TRAIL. In addition, TRAIL-Mu3 upregulated death receptor (DR) expression in PANC-1 cells and promoted the redistribution of DR5 in lipid rafts. Western blotting results demonstrated that TRAIL-Mu3 activated the caspase cascade in a faster and more efficient manner compared with TRAIL in PANC-1 cells. Therefore, TRAIL-Mu3 enhanced the antitumor effects in pancreatic cancer cells by strengthening the apoptotic signaling pathway. The present study indicated the potential of TRAIL-Mu3 for the treatment of pancreatic cancer.     

PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells

Background:

The PIAS4 protein belongs to the family of protein inhibitors of activated STAT, but has since been implicated in various biological activities including the post-translational modification known as sumoylation. In this study, we explored the roles of PIAS4 in pancreatic tumourigenesis.

Methods:

The expression levels of PIAS4 in pancreatic cancer cells were examined. Cell proliferation and invasion was studied after overexpression and gene silencing of PIAS4. The effect of PIAS4 on hypoxia signalling was investigated.

Results:

The protein was overexpressed in pancreatic cancer cells compared with the normal pancreas. Gene silencing by PIAS4 small interfering RNA (siRNA) suppressed pancreatic cancer cell growth and overexpression of PIAS4 induced expression of genes related to cell growth. The overexpression of PIAS4 is essential for the regulation of the hypoxia signalling pathway. PIAS4 interacts with the tumour suppressor von Hippel-Lindau (VHL) and leads to VHL sumoylation, oligomerization, and impaired function. Pancreatic cancer cells (Panc0327, MiaPaCa2) treated with PIAS4 siRNA suppressed expression of the hypoxia-inducible factor hypoxia-inducible factor 1 alpha and its target genes JMJD1A, VEGF, and STAT3.

Conclusion:

Our study elucidates the role of PIAS4 in the regulation of pancreatic cancer cell growth, where the suppression of its activity represents a novel therapeutic target for pancreatic cancers.     

紫杉醇联合放射线照射对肺腺癌细胞放射增敏作用的研究

目的 探讨化疗药紫杉醇是否对肺腺癌A973细胞有放射增敏作用.方法 取指数生长期的人肺腺癌细胞A973,采用成克隆分析法检测紫杉醇毒性,确定IC10、IC50和IC90剂量作为药物浓度.分析照射前后紫杉醇IC10、IC50和IC90浓度下作用24 h,照射剂最分别为0、1、2、4、6、8、10 Gy时的细胞存活分数,并用多靶单击模型拟合细胞存活曲线.采用流式细胞术分析不同浓度紫杉醇作用0、2、4、6、10、18、24 h,A973细胞周期分布变化.结果 紫杉醇对A973细胞的IC10、IC50和IC90剂量分别为0.5、2.6和8.7 nmol/L.IC10剂量紫杉醇照前给药增敏比为0.97(D0值比)、1.01(D0值比)、1.00(SF2值比),照后给药为0.97、1.02、1.02;IC50剂量照前给药为1.06、129.00、2.61,照后给药为0.94、129.00、2.14;IC90剂量照前给药为1.00、120.00、2.09,照后给药为0.98、120.00、2.09.IC10剂量紫杉醇对A973细胞无明显的G2+M期阻滞作用,而IC50和IC90剂量紫杉醇分别于2和18 h将A973细胞阻滞在G2+M期.结论 紫杉醇对肺腺癌细胞A973产生明显的放射增敏作用,且照射前后给药均有相似的增敏作用,中高浓度剂量联合小剂量X线照射增敏效果最好.     

Circulating tumor cells in pancreatic cancer patients: Methods of detection and clinical implications

The poor prognosis of pancreatic cancer patients is associated with the frequent and early dissemination of the disease, as well as late detection due to unspecific and late symptoms from the primary tumor. Pancreatic cancers frequently spread to the liver, lung and skeletal system, suggesting that pancreatic tumor cells must be able to intravasate and travel through the circulation to distant organs. Circulating tumor cells (CTCs) are tumor cells that have acquired the ability to enter the circulatory system; this cell population is ultimately responsible for the development of metastases in distant organs. Clinical studies have revealed that the presence of CTCs in blood is correlated with disease progression for other cancers, such as breast, colorectal and prostate cancer. However, as CTCs are extremely rare, both enrichment and sensitive methods of detection are required for their enumeration. This review highlights various enrichment procedures and methods for the detection of CTCs. Furthermore, we systematically review previously reported studies of the clinical relevance of CTC detection in pancreatic cancer patients. There is evidence that the presence of CTCs also correlates with an unfavorable outcome in pancreatic cancer patients. However, technical/methodological issues may explain why some studies only show a trend toward an association between CTC detection and disease progression. Larger studies, as well as characterization of the CTC population, are required to achieve further insight into the clinical implications of CTC detection in pancreatic cancer patients.     

Synergistic interaction of novel lactate dehydrogenase inhibitors with gemcitabine against pancreatic cancer cells in hypoxia

Background:

Hypoxia is a driving force in pancreatic-ductal-adenocarcinoma (PDAC) growth, metastasis and chemoresistance. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis, ensuring supply of energy and anabolites in hypoxic-environments. Therefore, we investigated the molecular mechanisms underlying the pharmacological interaction of novel LDH-A inhibitors in combination with gemcitabine in PDAC cells.

Methods:

Lactate dehydrogenase A levels were studied by quantitative RT–PCR, western blot, immunofluorescence and activity assays in 14 PDAC cells, including primary-cell-cultures and spheroids, in normoxic and hypoxic conditions. Cell proliferation, migration and key determinants of drug activity were evaluated by sulforhodamine-B-assay, wound-healing assay, PCR and LC-MS/MS.

Results:

Lactate dehydrogenase A was significantly increased under hypoxic conditions (1% O₂), where the novel LDH-A inhibitors proved to be particularly effective (e.g., with IC₅₀ values of 0.9 vs 16.3 μM for NHI-1 in LPC006 in hypoxia vs normoxia, respectively). These compounds induced apoptosis, affected invasiveness and spheroid-growth, reducing expression of metalloproteinases and cancer-stem-like-cells markers (CD133+). Their synergistic interaction with gemcitabine, with combination index values <0.4 in hypoxia, might also be attributed to modulation of gemcitabine metabolism, overcoming the reduced synthesis of phosphorylated metabolites.

Conclusion:

Lactate dehydrogenase A is a viable target in PDAC, and novel LDH-A inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours.