The novel platinum(IV) complex LA-12 induces p53 and p53/47 responses that differ from the related drug, cisplatin

The platinum(II)-based complex cisplatin is one of the most frequently used antitumour agents; however, a high incidence of harmful side effects and the frequent emergence of acquired resistance are the major clinical problems. The novel platinum(IV)-based complex LA-12 exhibits a high efficacy against cancer cell lines, including cisplatin-insensitive cells, but the mechanisms by which LA-12 perturbs cell growth are unclear. We tested the effects of LA-12 on the p53 response and demonstrate that LA-12 induces unique changes in the profile of gene expression compared with cisplatin and doxorubicin. Furthermore, the ability of LA-12 to disrupt cellular proliferation is greatly enhanced by the expression of p53 and p53/47 indicating both p53-dependent and p53-independent effects of LA-12. Exposure of the human cancer cell lines H1299, A2780, BT549 and BT474 to LA-12 alters the expression of p53 and p53/47 in both a time-dependent and dose-dependent manner. Treatment of cells with a low concentration of the drug results in accumulation of p53 and p53/47 concomitant with their posttranslational modification, whereas a high dose results in the disappearance of both the forms of p53. The distinct p53 activation profile of LA-12 compared with cisplatin and doxorubicin provides a molecular explanation for the ability of this drug to treat cisplatin-resistant cells and indicates its potential usefulness as an alternative antitumour agent in first-line therapy or as a second-line therapy in patients with acquired cisplatin resistance.     

Chemotherapy compounds in cervical cancer cells primed by reconstitution of p53 function after short interfering RNA-mediated degradation of human papillomavirus 18 E6 mRNA: opposite effect of siRNA in combination with different drugs

Constant expression of E6 and E7 mRNA by high-risk human papillomaviruses (HPV) abrogates p53 and retinoblastoma protein function, respectively, and is essential for the development of cervical cancer. Despite E6, some chemotherapy drugs can stabilize p53 in cervical cancer cells. It is not known how chemotherapy-induced p53 activation and cytotoxicity are affected when the amount of E6 mRNA is decreased before the drug treatment. In this study, HPV18-positive HeLa cervical cancer cells were transfected with short interfering RNA (siRNA) molecules targeting HPV18 E6 mRNA before treatment with carboplatin, cisplatin, doxorubicin, etoposide, gemcitabine, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, and topotecan. Transfection with siRNA was followed by nuclear accumulation of p53, but the effect was transient despite continuously suppressed HPV mRNA levels. When treatment with E6 siRNA was coupled with chemotherapy, the p53 activity after treatment with carboplatin and paclitaxel was additively increased, whereas the p53 activation induced by the rest of the drugs was synergistically increased. Treatment with E6 siRNA alone moderately inhibited HeLa cell proliferation but did not induce detectable apoptosis. The combined cytotoxic effect of E6 siRNA and chemotherapy ranged from subadditive to synergistic, depending on the drug. The decrease of E6 mRNA sensitized HeLa cells, for example, to doxorubicin and gemcitabine but counteracted the cytotoxicity of cisplatin and etoposide. In conclusion, activating p53 by degrading E6 mRNA may either increase or decrease the chemosensitivity of cervical cancer cells, depending on the chemotherapy compound.     

p53基因联合化疗治疗喉鳞癌的实验研究

目的：研究喉癌细胞系Hep-2中腺病毒介导的p53与顺铂及紫杉醇联合应用在体内及体外的协同效应,指导进一步临床应用。方法：将以腺病毒为载体的p53基因与顺铂及紫杉醇等药物联合应用于喉癌细胞系Hep-2,通过细胞增殖分析,流式细胞仪细胞周期分析,集落形成实验,观察其对喉癌细胞系的作用机制。结果：p53基因联合化疗治疗组肿瘤生长明显受到抑制,肿瘤生长速度显著慢于单纯基因治疗组及单纯化疗治疗组。结论：腺病毒介导的p53基因治疗联合顺铂及紫杉醇的化疗方案可显著提高喉鳞癌的治疗效果。     

多聚二磷酸腺苷核糖聚合酶抑制剂对顺铂在肺癌治疗中增敏的作用研究

目的探讨多聚二磷酸腺苷核糖聚合酶(PARP-1)抑制剂4-氨基-1,8-萘二胺(4-AN)对顺铂在肺腺癌治疗中的增敏作用及相关机制。方法应用MTT法和克隆形成试验检测4-AN与顺铂联合作用对A549细胞的细胞毒性作用;应用单细胞凝胶电泳和微核试验检测4-AN与顺铂联合作用对A549细胞的遗传毒性作用。结果 4-AN可以增加顺铂对A549细胞的杀伤作用,且杀伤作用随4-AN浓度增加而增强;4-AN可以增加顺铂导致的DNA单双链断裂和染色体损伤,而且随药物浓度的增加,损伤作用增强。结论抑制PARP-1可以有效的增加A549对顺铂的敏感性,其作用机制可能是通过抑制A549细胞DNA单双链损伤修复,继而引起染色体损伤,导致细胞的生长和克隆受到抑制。     

肺癌耐顺铂细胞A549/CIS产生肿瘤干细胞特性的分子机制研究

目的 研究人肺癌耐顺铂细胞A549/CIS获得肿瘤干细胞特性的分子机制。方法 微球形成实验比较A549和A549/CIS细胞肿瘤干细胞特性的差别,以及磷脂酰肌醇3-激酶/丝氨酸-苏氨酸激酶(PI3K/AKT)通路抑制剂LY294002对A549/CIS细胞微球形成能力的影响。Western blotting检测A549和A549/CIS细胞中AKT、p65以及MAPK-p38信号通路的活化情况。CCK8实验检测A549和A549/CIS细胞对顺铂的敏感性,以及PI3K/AKT通路抑制剂LY294002对A549/CIS细胞对顺铂敏感性的影响。结果 A549/CIS细胞对顺铂的敏感性以及微球形成能力相对于A549明显增强,Western blotting结果表明:A549/CIS细胞中AKT通路明显被激活,而p65和MAPK-p38通路没有明显变化。LY294002可以抑制A549/CIS细胞的肿瘤干细胞特性,并提高A549/CIS细胞对顺铂的敏感性。结论 相对于敏感株细胞A549,耐药株A549/CIS细胞的肿瘤干细胞特性明显增强,而其机制涉及PI3K/AKT信号通路的激活。     

Wild type p53 increased chemosensitivity of drug-resistant human hepatocellular carcinoma Be17402 ／ 5-FU cells

AIM: To study the effect of wild type (wt) p53 gene transfection on drug resistant human hepatocellular carcinoma (HCC) cells induced by 5-Fluorouracil (5-FU). METHODS: The cytotoxicity of anticancer drugs on Bel7402 and Bel7402/5-FU cells was assessed using SRB assay. p53 expression was detected at its mRNA level by RT-PCR assay and at its protein level Western blot or immunocytochemistry assay in Bel7402/5-FU cells transfected with either control vector or wt p53. AnnexinV-FITC/PI double labeled assay was performed to detect apoptosis. The chemosensitivity of Bel7402/5-FU cells transfected with wt p53 was assessed using SRB assay. RESULTS: Bel7402/ 5-FU cells exhibited cross-resistance to vincristine, doxorubicin, paclitaxel, and so on. wt p53 gene transfection upregulated the expression of p53 in Bel7402/5-FU cells. wt p53 was able to greatly inhibit cell     

Potential role of short hairpin RNA targeting epidermal growth factor receptor in growth and sensitivity to drugs of human lung adenocarcinoma cells

Upregulation of expression and activation of epidermal growth factor receptor (EGFR) is involved in the development and progression of a wide range of human cancers. The present study aims at determining gene-silencing effects of vector-based short hairpin RNA (shRNA) targeting EGFR on receptor expression and cell growth and evaluating its modulation of responsiveness to drugs in human lung adenocarcinoma cells (HLAC). A vector-based polymerase 3-promotor system was used to express shRNA targeting EGFR in HLAC lines (A549 and SPC-A1). EGFR was detected by immunofluorescence staining and quantified by Western blot. The effect of shRNA targeting EGFR on tumor cell growth was assessed by colony formation assay, cell cycle and apoptosis by flow cytometry, and the responsiveness of HLAC lines to cytotoxic drugs by 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay. Vectors expressing shRNA against EGFR significantly downregulated receptor expression by 74 and 85% and the colony number by 63 and 69% in A549 and SPC-A1, respectively. Vector-based shRNA against EGFR caused G1 arrest, induced apoptosis, and subsequently increased the sensitivity to cisplatin, doxorubicin and paclitaxel by about four- to seven-fold in both HLAC lines. Our data suggest that vector-based shRNA could be considered as an alternative to effectively inhibit EGFR expression in HLACs, probably with the higher efficacy in combination therapies with conventional chemotherapeutic drugs.     

靶向调控AQP4对非小细胞肺癌细胞化疗敏感性的调控作用

摘要：目的 探讨靶向沉默水通道蛋白4（AQP4）基因对非小细胞肺癌细胞化疗敏感性影响,并研究其分子作用 机制。方法 实时荧光定量 PCR（qRT-PCR）和 Western blot 分别检测非小细胞肺癌 A549 细胞以及顺铂耐药菌株 A549/DDP中AQP4 mRNA和蛋白的表达。设计靶向AQP4的siRNA-1和siRNA-2,采用siRNA抑制A549/DDP细胞 中AQP4的表达,筛选出最优siRNA,将其转染A549/DDP细胞,设立siRNA-NC组和空白对照组。CCK-8法检测细胞 增殖能力并计算各组细胞的半数抑制浓度（IC50）;流式细胞术检测细胞凋亡。Western blot检测P53和Bcl-2蛋白的 表达。结果 AQP4 mRNA和蛋白在A549/DDP细胞中的表达水平显著高于A549细胞,AQP4表达沉默后A549/DDP 细胞增殖抑制,细胞凋亡增加,对顺铂的敏感性增加,凋亡相关蛋白P53表达增加,而Bcl-2蛋白表达降低。结论 通 过靶向调控AQP4的表达可以增加非小细胞肺癌细胞对化疗药物的敏感性。     

OTU deubiquitinase 5 inhibits the progression of non‐small cell lung cancer via regulating p53 and PDCD5

Non‐small cell lung cancer (NSCLC) has the highest morbidity and mortality worldwide. OTU deubiquitinase 5 (OTUD5), a deubiquitinating enzyme, can enhance the stability of p53 and programmed cell death 5 (PDCD5), a protein related to the apoptosis, by deubiquitination. This study aimed to explore the biological function and underlying mechanism of OTUD5 in NSCLC. Western blot and qRT‐PCR were used to detect the expression of OTUD5 protein and mRNA in NSCLC tissues and cells, respectively. RNAi was adopted to construct an OTUD5 low‐expression model while the plasmids overexpressing p53 and PDCD5 were used to establish the overexpression models, respectively. CCK‐8 assay, transwell assay, and apoptosis assay were carried out to analyze the changes in the proliferation, migration, and chemoresistance of A549 and HCC827 cells. The mechanism of OTUD5 in NSCLC was studied by Western blot. Down‐regulated OTUD5 in NSCLC tissues was significantly correlated to a poor prognosis. The knockdown of OTUD5 inactivated p53 and PDCD5, promoting the proliferation and metastasis of NSCLC cells while inhibiting their apoptosis. OTUD5 knockdown also enhanced the resistance of NSCLC cells to doxorubicin and cisplatin. OTUD5 acted as a tumor suppressor in NSCLC by regulating the p53 and PDCD5 pathways.     

缓激肽介导的治疗心血管和糖尿病药物致血管性水肿的研究进展

目的 本研究旨在调查慢性射血分数降低的心力衰竭(HFrEF)患者神经内分泌抑制剂-血管紧张素转换酶抑制剂(ACEI)或血管紧张素II受体阻滞剂(ARB)、β受体阻滞剂(BB)和醛固酮受体拮抗剂(MRA)使用现状及其与指南的差距。方法 搜集并分析2018年01月到12月HFrEF住院患者基本资料和三类神经内分泌抑制剂使用情况。结果 共纳入301例慢性HFrEF患者,平均年龄为71.3±11岁,男性占56.5%,平均EF值32.1±7.2%,51.8%合并有高血压,心衰最常见的病因是心脏瓣膜病。ACEI/ARB、BB和MRA使用率分别为77.4%,60.5%和94.0%,剂量达标率分别为44.2%、22.5%和100%。48.8%患者采用指南推荐的联合用药,包括1.3%两药联合(ACEI/ARB+BB)和47.4%三药联合(ACEI/ARB+ BB+MRA)。结论 该院慢性HFrEF患者神经内分泌抑制剂应用现状与指南仍有差异,ACEI/ARB和BB使用率和剂量达标率不足,而MRA使用过度,需进一步提高医生对指南依从性。     

Isokotomolide A, a new butanolide extracted from the leaves of Cinnamomum kotoense, arrests cell cycle progression and induces apoptosis through the induction of p53/p21 and the initiation of mitochondrial system in human non-small cell lung cancer A549 cells

This study is the first to investigate isokotomolide A (IKA), a butanolide compound isolated from the leaves of Cinnamomum kotoense Kanehira & Sasaki (Lauraceaee), which exhibits an anti-proliferative activity in human non-small cell lung cancer A549 cells. The results show that IKA inhibits the proliferation of A549 by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. Blockade of cell cycle was associated with increased p21/WAF1 levels and reduced amounts of cyclin D1, cyclin E, Cdk2, Cdk4, and Cdk6 in a p53-mediated manner. IKA treatment also increased p53 phosphorylation (Ser15) and decreased the interaction of p53-MDM2. IKA treatment triggered the mitochondrial apoptotic pathway, indicated by changing Bax/Bcl-2 ratios, cytochrome c release and caspase-9 activation. In addition, pre-treatment of cells with caspase-9 inhibitor inhibited IKA-induced apoptosis, indicating that caspase-9 activation was involved in A549 cells' apoptosis induced by IKA. Our study reports here for the first time that the induction of p53/p21 and the initiation of the mitochondrial apoptotic system may participate in the anti-proliferative activity of IKA in human non-small cell lung cancer cells.     

The association between p53 protein phosphorylation at serine 15, serine 20 and sensitivity of cells isolated from patients with ovarian cancer and cell lines to chemotherapy in in vitro study

Background

The association between p53 protein phosphorylated at serine 15 (Ser15), serine 20 (Ser20) and ovarian tumor cell sensitivity after chemotherapy was analyzed in order to define the influence of p53 activation on tumor cell sensitivity to chemotherapy.

Chemosensitization by fibroblast growth factor-2 is not dependent upon proliferation,S-phase accumulation,or p53 status

Fibroblast growth factor-2 (bFGF/FGF-2) is a pleiotropic growth factor that functions as a survival factor and directs apoptosis during embryogenesis and development. As a survival factor, FGF-2 would be expected to protect cells against drug toxicities. Such protection has been reported in some cells treated with some chemotherapeutic drugs. However, we recently demonstrated that FGF-2 can sensitize NIH 3T3 mouse fibroblasts to the cytotoxic and apoptotic effects of cisplatin. Sensitization requires prolonged incubation of cells with FGF-2 before the addition of cisplatin, and it requires an FGF-2 concentration (5-10 ng/mL) that is higher than that needed for its mitogenic effects (0.5 ng/mL). We now report that FGF-2 can also sensitize MCF7 human breast cancer cells and A2780 human ovarian cancer cells, as well as NIH 3T3 cells, to cisplatin. FGF-2 did not affect the cisplatin sensitivity of SKOV3 ovarian cancer cells or a panel of seven pancreatic cancer cell lines. We have demonstrated that the sensitizing effect is not simply a function of the mitogenic activity of FGF-2 on cells, as we did not observe sensitization with other growth-stimulatory factors (FGF-1 and epidermal growth factor); the sensitizing effect of FGF-2 was observed even with cell lines that were not growth-stimulated by FGF-2; and sensitization was not restricted to cells in S-phase of the cell cycle. These results indicate that cell proliferation is neither necessary nor sufficient for sensitization by FGF-2. Moreover, sensitization to cisplatin appears to be p53-independent, as p53-null 3T3 10-1 cells were equally sensitized by FGF-2. Finally, FGF-2 also sensitized NIH 3T3 and MCF7 cells to carboplatin, and had smaller effects on the sensitivity of these cell lines to doxorubicin and docetaxel. FGF-2 had no effect on sensitivity to etoposide in any cell line tested. Therefore, sensitization by FGF-2 was most effective with the platinum compounds, suggesting that this activity may be specific to particular mechanisms of drug action.     

siRNA 靶向沉默 Lipocalin-2基因增加人肺癌 A549细胞对顺铂的敏感性

目的：利用针对 Lipocalin-2的小干扰 RNA（siRNA）沉默人肺癌 A549细胞中 Lipocalin-2基因表达,观察其对顺铂化疗敏感性的影响。方法分别采用免疫组织化学 SP 法和 Western blot 检测 Lipocalin-2在肺癌组织和3种肺癌细胞株（A549、NCI-H661、NCI-H446）中的表达情况。设计并构建靶向 Lipocalin-2的 siRNA 转染 A549细胞,采用Real-time PCR 和 Western blot 检测转染效率。MTT 法检测顺铂处理后细胞存活率及半数抑制浓度（IC50）。流式细胞术检测顺铂处理后细胞凋亡率。结果 Lipocalin-2蛋白在肺癌组织和肺癌 A549、NCI-H661、NCI-H446细胞中均异常高表达（ P ＜0.05）。siRNA-Lipocalin-2转染 A549细胞能在 mRNA 和蛋白水平显著抑制 Lipocalin-2表达,同时顺铂诱导的细胞存活率显著降低,凋亡率显著增高（ P ＜0.05）。结论利用特异性 siRNA 能有效抑制人肺癌 A549细胞中Lipocalin-2 mRNA 和蛋白表达,增强细胞对顺铂的敏感性。     

基于前药构建混合胶束实现紫杉醇/阿霉素共递送

为了达到靶向递送,实现肿瘤的联合治疗,制备两亲性紫杉醇-聚乙二醇前药以及小分子阿霉素前药,两者共同构成混合胶束实现共递送.合成还原敏感性的聚乙二醇-紫杉醇前体药物(mPEG-SS-PTX)和靶向性叶酸修饰的聚乙二醇-紫杉醇前体药物(FA-PEG-SS-PTX).同时合成pH敏感阿霉素-乌头酸酐(CAD)小分子前药,采用...     

通窍止咳液增强顺铂对肿瘤细胞内p38活性的研究

目的:研究通窍止咳液增强顺铂对肿瘤细胞内p38的活性。方法:通过Western-Blot检测通窍止咳液对人鼻咽癌细胞CNE2、人肺癌细胞A549内p38活性的变化;通过MTT比色法检测通窍止咳液单用以及联合顺铂对CNE2、A549细胞生长的影响。结果:Western-Blot检测结果显示,通窍止咳液能够激活CNE2和A549细胞内p38,增高细胞内p38的磷酸化水平;MTT检测结果显示高浓度的通窍止咳液可以抑制CNE2、A549细胞的生长,通窍止咳液能增强顺铂对CNE2细胞的细胞毒作用,而对A549细胞则无此作用。结论:通窍止咳液增强顺铂的细胞毒作用可能与其激活p38有关,这一作用为逆转顺铂耐药提供了新的治疗方案。     

shRNA对A549细胞survivin基因表达及紫杉醇敏感性的影响

目的观察靶向survivin基因的短发夹RNA(shorthairpin RNA,shRNA)对人肺腺癌细胞A549生物学行为及紫杉醇敏感性的影响。方法将靶向survivin的基因片段插入载体后构建重组质粒,用转染试剂FuGENE(HD将其导入A549细胞,RT-PCR及Western blot分析转染前后survivin基因的表达情况,TUNEL法检测细胞凋亡情况,MTT法检测转染后A549细胞对紫杉醇的敏感性变化。结果成功构建重组质粒。与对照组相比,转染重组质粒后,survivin基因的表达明显降低,细胞凋亡率增加。转染前紫杉醇抑制A549细胞的IC50为转染后的11.9倍,两者比较,差异有显著性(P<0.05)。结论靶向survivin的shRNA能下调survivin基因表达,诱导细胞凋亡,增强A549细胞对紫杉醇的敏感性。     

KIF4A knockdown inhibits tumor progression and promotes chemo-sensitivity via induction of P21 in lung cancer cells

KIF4A has been demonstrated to play a crucial function in the pathogenesis of a broad number of tumors and have close association with PI3K/AKT pathway. The aim of this study was to explore the potential function of KIF4A in lung cancer progression by targeting PI3K/AKT pathway and P21 combination with doxorubicin. A549 cell lines were transfected with siRNA against KIF4A and negative control siRNA (si-NC). MTT assay and trypan blue staining were used to evaluate the effect of si-KIF4A on the doxorubicin cytotoxicity. The mRNA and protein expression levels of KIF4A and p21 were assessed by qRT-PCR and Western blotting. Apoptosis was measured by cell death ELISA kit. Our result revealed that KIF4A silencing decreased cellular proliferation in A549 lung cancer cells. Doxorubicin in combination with si-KIF4A led to significant reduction in the survival rate of A549 cell. KIF4A silencing upregulated p21. In conclusion, our results demonstrate that KIF4A inhibition sensitizes A549 cells to doxorubicin by targeting p21 and PI3K/AKT pathway, indicating a significant role for KIF4A in lung cancer chemotherapy.