BACKGROUND & AIMS: :Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Unfortunately, chemo-resistance is a huge obstacle in the treatment of OS. However, the underlying molecular mechanisms of OS chemo-resistance still remain unknown. Here we reported that the resistance to camptothecin (cpt) therapy was driven by degradation of DDX5. DDX5 knockdown decreased cell death and DNA damage and recovered cell proliferation in cpt treated 143B cells. Furthermore, we found that DDX5 bound to NONO, a kind of DNA repairing protein, and regulated NONO functions. Our data verified that cpt-induced degradation of DDX5 following by breaking down the protein bound of NONO, which participated in the resistance of cpt. In the summary, according to our results, DDX5 might be a potential therapeutic target for improving clinical outcomes of cpt in OS.

背景与目标： ：骨肉瘤（OS）是儿童和青少年中最常见的原发性恶性骨肿瘤。不幸的是，耐化学性是OS治疗的巨大障碍。但是，OS化学抗性的潜在分子机制仍然是未知的。在这里，我们报道了喜树碱（cpt）治疗的耐药性是由DDX5降解引起的。 DDX5组合物降低了cpt处理的143B细胞的细胞死亡和DNA损伤，并恢复了细胞增殖。此外，我们发现DDX5与NONO（一种DNA修复蛋白）结合，并调节NONO功能。我们的数据证实了cpt诱导的DDX5降解是通过破坏NONO的蛋白质结合而引起的，NONO参与了cpt的抗性。总之，根据我们的结果，DDX5可能是改善OS中cpt临床结果的潜在治疗靶标。

BACKGROUND & AIMS: :ST1968, a novel hydrophilic camptothecin analogue of the 7-oxyiminomethyl series, is characterised by the formation of stable DNA-topoisomerase I cleavable complex and by a promising profile of antitumour activity. The present study was designed to extend preclinical evaluation of the novel camptothecin in human squamous cell carcinoma (SCC) models. ST1968 exhibited an impressive activity with a high cure rate in SCC models. ST1968 produced 100% of complete response without evidence of regrowth in tumours characterised by susceptibility to drug-induced apoptosis (FaDu, A431 and A2780). In contrast to irinotecan, ST1968 still showed an excellent, persisting activity in models less susceptible to apoptosis induction (KB, Caski and SiHa), in which drug treatment elicited a persistent DNA damage response, as documented by phosphorylation of p53, RPA-2 and histone H2AX, resulting in delayed apoptosis and senescence. This behaviour was associated with a marked cellular/tumour drug accumulation. In conclusion, ST1968 exhibited an outstanding antitumour activity superior to that of irinotecan against SCC. A high intracellular accumulation, resulting in fast apoptosis or DNA damage persistence, appeared to be a critical determinant of SCC sensitivity to ST1968.

背景与目标： ：ST1968是7-氧亚氨基甲基系列的新型亲水喜树碱类似物，其特征在于形成了稳定的DNA-拓扑异构酶I可裂解复合物，并具有令人信服的抗肿瘤活性。本研究旨在扩展新型喜树碱在人鳞状细胞癌（SCC）模型中的临床前评价。在SCC模型中，ST1968表现出令人印象深刻的活性和高治愈率。 ST1968产生了100％的完全应答，而没有以药物诱导的凋亡（FaDu，A431和A2780）为特征的肿瘤再生长的证据。与伊立替康相反，ST1968在较不易发生凋亡诱导的模型（KB，Caski和SiHa）中仍表现出优异的持久性，其中药物治疗引起持久性DNA损伤反应，如p53，RPA-2和P53的磷酸化所证明。组蛋白H2AX，导致延迟的细胞凋亡和衰老。这种行为与明显的细胞/肿瘤药物蓄积有关。总之，ST1968具有优异的抗肿瘤活性，优于伊立替康对SCC的抗肿瘤活性。大量的细胞内积累导致快速的细胞凋亡或DNA损伤持久性，似乎是SCC对ST1968敏感性的关键决定因素。

BACKGROUND & AIMS: :Cutaneous malignant melanoma is a life-threatening cancer with poor prognosis due to a high metastasis potential. The main obstacle in treatment of metastatic melanoma is the resistance to chemotherapy. Recent studies indicated that apoptosis is a common mechanism of action for various cytotoxic agents. As p53 plays an important part in apoptosis, we investigated the role of p53 in chemosensitivity of melanoma cells. Previously, we found that melanoma cell lines containing wild-type p53 have significantly higher response rates to chemotherapy than cell lines with a mutant p53 gene. To confirm the role of p53 in melanoma chemosensitivity further, we transfected an expression vector, pED1, which carries a mutant p53 gene, into a wild-type p53 melanoma cell line, MMAN. We examined the effect of mutant p53 on camptothecin-induced apoptosis and the expression of genes which are known to be involved in apoptosis or drug resistance, such as bcl-2, bax, bak, p21waf1, and P-glycoprotein. Our results indicate that overexpression of the mutant p53 increased the growth rate of MMAN cells, reduced the sensitivity to camptothecin, and lowered drug-induced apoptosis by 2-3-fold. Flow cytometry indicated that the camptothecin-induced apoptosis is not associated with G1 arrest. Furthermore, camptothecin treatment reduced bcl-2 and P-glycoprotein expression in wild-type p53 MMAN cells, but not cells overexpressing mutant p53. These results demonstrate that p53 mutational status is a determinant of melanoma chemosensitivity. p53 may downregulate bcl-2 and P-glycoprotein to induce apoptosis in melanoma cells after chemotherapy.

背景与目标： ：恶性黑色素瘤是一种威胁生命的癌症，因其高转移潜力而预后较差。治疗转移性黑色素瘤的主要障碍是对化学疗法的抵抗力。最近的研究表明，凋亡是各种细胞毒剂的共同作用机制。由于p53在凋亡中起重要作用，因此我们研究了p53在黑色素瘤细胞化学敏感性中的作用。以前，我们发现含有野生型p53的黑素瘤细胞系对化疗的反应率明显高于具有突变p53基因的细胞系。为了进一步证实p53在黑色素瘤化学敏感性中的作用，我们将携带突变p53基因的表达载体pED1转染到野生型p53黑色素瘤细胞系MMAN中。我们检查了突变体p53对喜树碱诱导的细胞凋亡的作用以及已知与细胞凋亡或耐药有关的基因的表达，例如bcl-2，bax，bak，p21waf1和P-糖蛋白。我们的结果表明，突变体p53的过表达提高了MMAN细胞的生长速率，降低了对喜树碱的敏感性，并使药物诱导的细胞凋亡降低了2至3倍。流式细胞仪表明喜树碱诱导的细胞凋亡与G1阻滞无关。此外，喜树碱处理可降低野生型p53 MMAN细胞中bcl-2和P-糖蛋白的表达，但不会过度表达突变型p53的细胞。这些结果表明，p53突变状态是黑色素瘤化学敏感性的决定因素。在化疗后，p53可能下调bcl-2和P-糖蛋白以诱导黑色素瘤细胞凋亡。

BACKGROUND & AIMS: To achieve a sequence-specific DNA cleavage by topoisomerase I, derivatives of the antitumor drug camptothecin have been covalently linked to triple helix-forming oligonucleotides that bind in a sequence-specific manner to the major groove of double-helical DNA. Triplex formation at the target sequence positions the drug selectively at the triplex site, thereby stimulating topoisomerase I-mediated DNA cleavage at this site. In a continuous effort to optimize this strategy, a broad set of conjugates consisting of (i) 16-20-base-long oligonucleotides, (ii) alkyl linkers of variable length, and (iii) camptothecin derivatives substituted on the A or B quinoline ring were designed and synthesized. Analysis of the cleavage sites at nucleotide resolution reveals that the specificity and efficacy of cleavage depends markedly on the length of both the triple-helical structure and the linker between the oligonucleotide and the poison. The optimized hybrid molecules induced strong and highly specific cleavage at a site adjacent to the triplex. Furthermore, the drug-stabilized DNA-topoisomerase I cleavage complexes were shown to be more resistant to salt-induced reversal than the complexes induced by camptothecin alone. Such rationally designed camptothecin conjugates could provide useful antitumor drugs directed selectively against genes bearing the targeted triplex binding site. In addition, they represent a powerful tool to probe the molecular interactions in the DNA-topoisomerase I complex.

背景与目标： 为了实现拓扑异构酶I的序列特异性DNA切割，抗肿瘤药物喜树碱的衍生物已与三螺旋形成寡核苷酸共价连接，该寡核苷酸以序列特异性方式结合到双螺旋DNA的主沟上。在靶序列上的三链体形成将药物选择性地定位在三链体位点，从而刺激拓扑异构酶I介导的在该位点的DNA切割。在不断努力优化此策略的过程中，广泛的共轭物由（i）16-20个碱基长的寡核苷酸，（ii）可变长度的烷基接头和（iii）在A或B喹啉上取代的喜树碱衍生物组成设计并合成了环。以核苷酸分辨率对切割位点的分析表明，切割的特异性和效力显着取决于三螺旋结构的长度以及寡核苷酸与毒物之间的接头。优化的杂合分子在邻近三链体的位点诱导强烈且高度特异性的切割。此外，与单独由喜树碱诱导的复合物相比，药物稳定的DNA-拓扑异构酶I裂解复合物显示出对盐诱导的逆转更具抗性。这种经过合理设计的喜树碱偶联物可以提供有用的抗肿瘤药物，选择性地针对带有目标三链结合位点的基因。此外，它们是探测DNA-拓扑异构酶I复合物中分子相互作用的有力工具。

BACKGROUND & AIMS: By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin-treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti-nucleolar antibodies seen in various pathological disorders.

背景与目标： 通过免疫荧光和免疫电子显微镜，我们研究了喜树碱处理的HL60细胞凋亡过程中不同核仁成分的命运。我们已经发现，RNA聚合酶I消失了，而UBF与先前描述的纤维状螺纹体相关。相反，在晚期凋亡细胞的微核中存在的颗粒状物质中，仍可检测到原纤维蛋白，C23 /核仁蛋白和B23 /核磷脂。双重免疫标记实验显示C23和B23与原纤维蛋白共定位。免疫印迹分析表明，UBF可以被蛋白水解降解，而原纤维蛋白，C23 /核仁蛋白和B23 /核磷蛋白则没有。这些结果可能有助于解释在各种病理性疾病中发现的抗核仁抗体的存在。

BACKGROUND & AIMS: :Stimuli-responsive systems for controlled drug release have been extensively explored in recent years. In this work, we developed a reduction-responsive camptothecin (CPT) nanocapsule (CPT-NC) by combining nanoprecipitation and in situ polymerization using a polymerized surface ligand and a disulfide bond-containing crosslinker. Dissolution rate studies proved that the CPT-NCs have robust drug-release profiles in the presence of glutathione (GSH) owing to the division of the disulfide bond crosslinker which triggers the collapse of the polymer layer. Furthermore, the in vitro investigations demonstrated that the CPT-NCs exhibited a high-cellular uptake efficiency and cytotoxicity for cancer cells of squamous cell carcinoma (SCC-15). Our approach thus presents an effective intracellular drug delivery strategy for anticancer therapy.

背景与目标： 近年来，已广泛探索了用于控制药物释放的刺激反应系统。在这项工作中，我们通过使用聚合的表面配体和含二硫键的交联剂将纳米沉淀和原位聚合相结合，开发了一种还原反应性喜树碱（CPT）纳米胶囊（CPT-NC）。溶出度研究证明，由于二硫键交联剂的分裂触发了聚合物层的塌陷，因此在存在谷胱甘肽（GSH）的情况下，CPT-NC具有强大的药物释放特性。此外，体外研究表明，CPT-NC对鳞状细胞癌（SCC-15）的癌细胞表现出高细胞摄取效率和细胞毒性。因此，我们的方法为抗癌治疗提出了有效的细胞内药物递送策略。

BACKGROUND & AIMS: :The interleukin-23/interleukin 17A (IL-23/IL-17A) cytokine axis plays a critical role in the pathogenesis of psoriasis. In this study, we report the effects of topical calcipotriol, camptothecin, clobetasol and tazarotene on the treatment of imiquimod (IMQ)-induced psoriasis-like inflammation, the development of which is dependent on the IL-23/IL-17A axis. IMQ-induced epidermal hyperplasia and inflammation in the BALB/c mouse ear were significantly inhibited following clobetasol treatment but not calcipotriol, camptothecin or tazarotene treatments. Real-time polymerase chain reaction showed that the mRNA levels of IL-17A, IL-17F, IL-22, IL-1β, IL-6 and TNF-α in ear skin were significantly decreased by clobetasol. In addition, we observed that calcipotriol, camptothecin and tazarotene failed to show any inhibitory effects on the IL-23/IL-17A/IL-22 axis. We also found that clobetasol treatment inhibited the proliferation of γδ T cells and C-C chemokine receptor type 6 (CCR6) expression induced by IMQ. Calcipotriol, camptothecin and tazarotene not only failed to inhibit this proliferation but also enhanced retinoic acid-related orphan receptor γ (RORγ) expression in IMQ-induced psoriasis-like inflammation. In conclusion, we suggest that clobetasol induces the relief of IMQ-induced psoriasis-like inflammation in a mouse model but that calcipotriol, camptothecin and tazarotene cannot. Therefore, we suggest that more in-depth studies on pharmacological effects of tazarotene, camptothecin and calcipotriol should be carried out.

背景与目标： 白细胞介素-23 /白介素17A（IL-23 / IL-17A）细胞因子轴在牛皮癣的发病机理中起关键作用。在这项研究中，我们报道了局部卡泊三醇，喜树碱，氯倍他索和他扎罗汀对咪喹莫特（IMQ）诱导的牛皮癣样炎症的治疗效果，其发展取决于IL-23 / IL-17A轴。 IMQ诱导的BALB / c小鼠耳朵中的表皮增生和炎症在氯倍他索治疗后得到显着抑制，而卡泊三醇，喜树碱或他扎罗汀治疗则无明显抑制作用。实时聚合酶链反应显示，氯倍他索可显着降低耳部皮肤中IL-17A，IL-17F，IL-22，IL-1β，IL-6和TNF-α的mRNA水平。此外，我们观察到卡泊三醇，喜树碱和他扎罗汀未能对IL-23 / IL-17A / IL-22轴显示任何抑制作用。我们还发现氯倍他索治疗可抑制IMQ诱导的γδT细胞增殖和C-C趋化因子受体6型（CCR6）表达。 Calcipotriol，喜树碱和他扎罗汀不仅不能抑制这种增殖，而且在IMQ诱导的牛皮癣样炎症中，维甲酸相关的孤儿受体γ（RORγ）的表达也增加了。总之，我们建议氯倍他索在小鼠模型中诱导IMQ诱导的牛皮癣样炎症缓解，但卡泊三醇，喜树碱和他扎罗汀不能。因此，我们建议对他扎罗汀，喜树碱和卡泊三醇的药理作用进行更深入的研究。

BACKGROUND & AIMS: :Previous research has shown synergistic growth inhibition between UCN-01 and camptothecin (CPT) in tumor cells with mutant p53 versus tumor cells with wild-type p53. To determine the possible role of p53 in this drug combination, we tested the hypothesis that the synergistic growth inhibition is due to the absence of p53, and can result from the induction of DNA double-strand breaks (DSBs). Experiments were performed with the use of normal human mammary epithelial cells (HMEC); HMEC transfected with HPV16 E6 protein which inactivates p53 (HE6), or p53-mutant MDA-MB-231 tumor cells. CPT, UCN-01, or a 1:1 combination of both, in either HMEC or HE6 cells did not induce DSBs. In contrast, simultaneous treatment of MDA-MB-231 cells with both UCN-01 and CPT induced significant levels of DSBs while treatment with either drug alone did not. While UCN-01 was surprisingly potent against HMEC, the growth inhibition was only additive between UCN-01 and CPT against these cells. HE6 cells were much less sensitive than HMEC to UCN-01 and slightly less sensitive to the combined treatment with UCN-01 and CPT. The drug combination was synergistic against HE6 cells, due to their lower sensitivity to UCN-01. Unlike what was observed previously in MDA-MB-231 cells, UCN-01 did not abrogate CPT-induced inhibition of DNA synthesis in either HMEC or HE6 cells. These data indicate that synergistic growth inhibition by UCN-01 and CPT against p53 mutant MDA-MB-231 tumor cells may be due to induction of DSBs however the loss of p53 function alone does not sensitize normal cells to the combination of both drugs.

背景与目标： ：先前的研究表明，在具有突变型p53的肿瘤细胞与具有野生型p53的肿瘤细胞中，UCN-01和喜树碱（CPT）之间具有协同生长抑制作用。为了确定p53在这种药物组合中的可能作用，我们测试了以下假设：协同生长抑制是由于p53的缺失，并且可能是由DNA双链断裂（DSBs）引起的。使用正常人乳腺上皮细胞（HMEC）进行实验；用HPV16 E6蛋白转染的HMEC，该蛋白可灭活p53（HE6）或p53突变的MDA-MB-231肿瘤细胞。 HMEC或HE6细胞中的CPT，UCN-01或两者的1：1组合均不会诱导DSB。相反，用UCN-01和CPT同时处理MDA-MB-231细胞可诱导显着水平的DSB，而单独使用任何一种药物均不能。尽管UCN-01令人惊讶地对HMEC有效，但其生长抑制作用仅是UCN-01和CPT之间对这些细胞的加和作用。 HE6细胞对UCN-01的敏感性比HMEC低得多，而对UCN-01和CPT的联合治疗敏感性稍低。该药物组合对HE6细胞具有协同作用，因为它们对UCN-01的敏感性较低。与以前在MDA-MB-231细胞中观察到的不同，UCN-01在HMEC或HE6细胞中都没有消除CPT诱导的DNA合成抑制作用。这些数据表明，UCN-01和CPT对p53突变MDA-MB-231肿瘤细胞产生的协同生长抑制作用可能是由于DSB的诱导所致，但是仅p53功能的丧失并不能使正常细胞对两种药物的组合敏感。

BACKGROUND & AIMS: :Fas ligand (L) is a membrane protein from the tumor necrosis factor (TNF) family. It induces apoptosis upon contact with its Fas/CD95/APO1 receptor. Trimerization of FasL on the surface of effector cells is essential in the binding of the Fas trimer of the target cells. The receptor then recruits an adaptor and caspase-like proteins which lead apoptosis. This paper reports on the fate of FasL in HEp-2 cells committed to apoptosis by induction with campthotecin. Our main results demonstrated that in non-apoptotic cells, FasL aggregates in the cytoplasm forming trimers of 120 kDa. Apoptosis increases the trimeric FasL species, but also induces its dissociation into monomers of 35 kDa. In conclusion, camptothecin appears to perturb the Fas and FasL segregation in the cytoplasm by promoting the transit of FasL to the cell surface, thus fostering a process of autocrine or paracrine apoptosis. FasL is trimerized prior to Fas/FasL complex formation, and after apoptosis, FasL undergoes an intense turnover.

背景与目标： ：Fas配体（L）是一种来自肿瘤坏死因子（TNF）家族的膜蛋白。与Fas / CD95 / APO1受体接触后，它会诱导凋亡。 FasL在效应细胞表面的三聚化对于靶细胞Fas三聚体的结合至关重要。然后，受体募集衔接子和胱天蛋白酶样蛋白，从而导致细胞凋亡。这篇论文报道了通过喜树碱诱导的凋亡导致Hep-2细胞中FasL的命运。我们的主要结果表明，在非凋亡细胞中，FasL在细胞质中聚集，形成120 kDa的三聚体。凋亡增加了三聚体FasL种类，但也诱导其解离成35kDa的单体。总之，喜树碱通过促进FasL向细胞表面的转移，似乎干扰了Fas和FasL在细胞质中的分离，从而促进了自分泌或旁分泌细胞凋亡的过程。 FasL在Fas / FasL复合物形成之前被三聚，并且在细胞凋亡后，FasL经历强烈的更新。

BACKGROUND & AIMS: :A folate targeted camptothecin small molecule drug conjugate (SMDC) was synthesized using a monodisperse PEG spacer linked to folate via a releasable disulfide carbonate linker. Cell cytotoxicity in human KB cells exhibited an IC50 of 6nM. Importantly, activity of the prodrug was blocked by excess folate, demonstrating receptor-mediated celluar uptake of the PEG conjugate.

背景与目标： 叶酸靶向喜树碱小分子药物偶联物（SMDC）的合成是通过可释放的二硫化碳碳酸酯连接子与叶酸连接的单分散PEG间隔基进行的。人KB细胞中的细胞毒性显示出6nM的IC50。重要的是，前药的活性被过量的叶酸所阻断，表明受体介导的细胞对PEG缀合物的摄取。

BACKGROUND & AIMS: :Camptothecins represent an established class of effective agents that selectively target topoisomerase I by trapping the catalytic intermediate of the topoisomerase I-DNA reaction, the cleavage complex. The water-soluble salt camptothecin-sodium - introduced in early trials in the 1960s - was highly toxic in animals, whereas the semisynthetic derivatives irinotecan and topotecan did not cause haemorrhagic cystitis because of their higher physicochemical stability and solubility at lower pH values. Myelosuppression, neutropenia and, to a lesser extent, thrombocytopenia are dose-limiting toxic effects of topotecan. In contrast to the structurally-related topotecan, irinotecan is a prodrug which has to be converted to SN-38, its active form. SN-38 is inactivated by conjugation, thus patients with Gilbert's syndrome and other forms of genetic glucuronidation deficiency are at an increased risk of irinotecan-induced adverse effects, such as neutropenia and diarrhoea. The cytotoxic mechanism of podophyllotoxin is the inhibition of topoisomerase II. Common adverse effects of etoposide include dose-limiting myelosuppression. Hypersensitivity reactions are more common with etoposide and teniposide than with etoposide phosphate because the formulations of the former contain sensitising solubilisers. Leukopenia and thrombocytopenia occur in 65% and 80%, respectively, of patients after administration of conventional doses of teniposide. Anorexia, vomiting and diarrhoea are generally of mild severity after administration of conventional doses of topoisomerase II inhibitors. Clinical pharmacokinetic studies have revealed substantial interindividual variabilities regarding the area under the concentration-time curve values and steady-state concentrations for all drugs reviewed in this article. Irinotecan, etoposide and teniposide are degraded via complex metabolic pathways. In contrast, topotecan primarily undergoes renal excretion. Regarding etoposide and teniposide, the extent of catechol formation over time during drug metabolism may be associated with a higher risk for secondary malignancies.

背景与目标： 喜树碱代表一类已建立的有效药物，通过捕获拓扑异构酶I-DNA反应的催化中间体，裂解复合物，选择性地靶向拓扑异构酶I。 1960年代早期试验中引入的水溶性喜树碱钠盐对动物具有高毒性，而半合成衍生物伊立替康和托泊替康因其较高的理化稳定性和在较低pH值下的溶解度而不会引起出血性膀胱炎。骨髓抑制，中性粒细胞减少和血小板减少是托泊替​​康的剂量限制性毒性作用。与结构相关的拓扑替康相反，伊立替康是一种前药，必须将其转变为其活性形式SN-38。 SN-38会因结合而失活，因此患有吉尔伯特综合症和其他形式的遗传性葡萄糖醛酸缺乏症的患者罹患伊立替康引起的不良反应（如中性粒细胞减少和腹泻）的风险增加。鬼臼毒素的细胞毒性机制是对拓扑异构酶II的抑制。依托泊苷的常见不良反应包括限制剂量的骨髓抑制。依托泊苷和替尼泊苷的过敏反应比依托泊苷磷酸酯的过敏反应更为常见，因为前者的配方中含有增敏剂。常规剂量的替尼泊苷给药后，白细胞减少症和血小板减少症分别发生在65％和80％的患者中。常规剂量的拓扑异构酶II抑制剂给药后，厌食，呕吐和腹泻的严重程度通常较轻。临床药代动力学研究表明，本文中综述的所有药物的浓度-时间曲线值和稳态浓度下的面积之间存在很大的个体差异。伊立替康，依托泊苷和替尼泊苷通过复杂的代谢途径降解。相反，拓扑替康主要经历肾脏排泄。关于依托泊苷和替尼泊苷，药物代谢期间儿茶酚形成的程度可能与继发性恶性肿瘤的较高风险有关。

BACKGROUND & AIMS: :The plant Ophiorrhiza pumila produces camptothecin (CPT), a kind of terpene indole alkaloid (TIAs) that has been widely used in treatment of cancer. Tryptophan-arginine-lysine-tyrosine (WRKY) transcription factors have been reported to play important roles in plant metabolism and development. In this study, a novel WRKY transcription factor named OpWRKY3 was isolated from O. pumila, with full-length open reading frame (ORF) of 1128 bp, encoding 375 amino acids. Phylogenetic tree analysis revealed that OpWRKY3 shared the highest homology with VvWRKY30, and it is a significant feature belonging to group III. OpWRKY3 was responsive to various treatments, including gibberellin (GA3), methyl jasmonate (MJ), acetylsalicylic acid (ASA), salicylic acid (SA), and abscisic acid (ABA). Besides, OpWRKY3 is expressed predominantly in stems. Subcellular localization analysis showed that OpWRKY3 localized in the nucleus. The biomass of OpWRKY3-SRDX transgenic hairy roots (S line) was visibly suppressed, while there were slight changes between overexpression of the OpWRKY3 line (OE line) and the control. In addition, the concentration and total production of camptothecin precursors including loganin and secologanin were significantly changed in both OE and S lines while total production of CPT was significantly changed in most transgenic lines. Thus, the present work revealed that OpWRKY3 may act as a regulator in the growth and development of O. pumila, and in production of camptothecin and its precursors.

背景与目标： ：Ophiorrhiza pumila植物产生喜树碱（CPT），这是一种被广泛用于治疗癌症的萜烯吲哚生物碱（TIA）。色氨酸-精氨酸-赖氨酸-酪氨酸（WRKY）转录因子据报道在植物代谢和发育中起重要作用。在这项研究中，从O. pumila中分离出了一种名为OpWRKY3的新型WRKY转录因子，其全长开放阅读框（ORF）为1128 bp，编码375个氨基酸。系统进化树分析表明，OpWRKY3与VvWRKY30具有最高的同源性，这是第三类的重要特征。 OpWRKY3对多种处理有响应，包括赤霉素（GA3），茉莉酸甲酯（MJ），乙酰水杨酸（ASA），水杨酸（SA）和脱落酸（ABA）。此外，OpWRKY3主要在茎中表达。亚细胞定位分析表明OpWRKY3定位在细胞核中。 OpWRKY3-SRDX转基因毛状根（S系）的生物量受到明显抑制，而OpWRKY3系（OE系）的过表达与对照之间略有变化。另外，喜树碱前体包括loganin和secologanin的浓度和总产量在OE和S系中均发生了显着变化，而CPT的总产量在大多数转基因系中均发生了显着变化。因此，目前的工作表明，OpWRKY3可能在O. pumila的生长和发育以及喜树碱及其前体的生产中起调节作用。

BACKGROUND & AIMS: :Camptothecin (CPT) is an anticancer drug that promotes DNA breakage at replication forks and the formation of lesions that activate the processes of homologous recombination (HR) and nonhomologous end joining. We have taken advantage of the CPT-induced damage response by coupling it to gene repair directed by synthetic oligonucleotides, a process in which a mutant base pair is converted into a wild-type one. Here, we show that pretreating DLD-1 cells with CPT leads to a significant stimulation in the frequency of correction of an integrated mutant enhanced green fluorescent protein gene. The stimulation is dose-dependent and coincident with the formation of double-strand DNA breaks. Caffeine, but not vanillin, blocks the enhancement of gene repair suggesting that, in this system, HR is the pathway most responsible for elevating the frequency of correction. The involvement of HR is further proven by studies in which wortmannin was seen to inhibit gene repair at high concentrations but not at lower levels that are known to inhibit DNA-PK activity. Taken together, our results suggest that DNA damage induced by CPT activates a cellular response that stimulates gene repair in mammalian cells.

背景与目标： 喜树碱（CPT）是一种抗癌药，可促进复制叉处的DNA断裂和损伤的形成，从而激活同源重组（HR）和非同源末端连接的过程。我们通过将CPT诱导的损伤反应与合成寡核苷酸指导的基因修复相结合来利用CPT诱导的损伤反应，在该过程中，突变碱基对被转化为野生型。在这里，我们显示用CPT对DLD-1细胞进行预处理可显着刺激整合突变增强型绿色荧光蛋白基因的校正频率。刺激是剂量依赖性的，并且与双链DNA断裂的形成同时发生。咖啡因而不是香草醛阻止了基因修复的增强，这表明在该系统中，HR是最主要的途径来提高校正频率。研究表明，渥曼青霉素在抑制DNA-PK活性的高浓度下抑制基因修复，但在较低水平上却不抑制基因修复，这进一步证实了HR的参与。综上所述，我们的结果表明，CPT诱导的DNA损伤会激活细胞反应，从而刺激哺乳动物细胞中的基因修复。

BACKGROUND & AIMS: PURPOSE:The purpose of this study was to investigate the pro-apoptotic effect of camptothecin (CPT) on Y79 retinoblastoma cells and the role of Forkhead box, class O (FOXO1) in CPT-induced apoptosis. METHODS:CPT-induced apoptosis was determined by flow cytometry with annexin V-FITC positive cells and Western blot of PARP expression, respectively. The expressions of FOXO1 were detected by Western blot. The transcriptional activity of FOXO1 was determined by luciferase reporter assay. siRNAs specifically inhibiting FOXO1 were used, and flow cytometry and Western blot were executed to test the role of FOXO1 in CPT-induced apoptosis. RESULTS:CPT was extremely effective in inducing apoptosis of Y79 retinoblastoma cells. FOXO1 was highly expressed in Y79 cells. CPT not only elevated the FOXO1 dephosphorylation level but also promoted its transcriptional activity, suggesting that the activation of FOXO1 was, at least in part, triggered by CPT. The decreased annexin V positive cells and less PARP cleavage demonstrated that siRNAs-mediated inhibition of FOXO1 significantly abrogated CPT-induced apoptosis, indicating that FOXO1 plays an important role in CPT-induced apoptosis. Moreover, the expression of Bim was also elevated with the treatment of CPT, which is in accordance with the activation of FOXO1. CONCLUSIONS:Our study provides the evidence that a high level of endogenous FOXO1 expression in retinoblastoma cells contributes, at least in part, to CPT-induced apoptosis, which may help broad application of CPT in retinoblastoma therapy in the future.

背景与目标： 目的：本研究旨在探讨喜树碱（CPT）对Y79视网膜母细胞瘤细胞的促凋亡作用以及O型叉头盒（FOXO1）在CPT诱导的细胞凋亡中的作用。
方法：用膜联蛋白V-FITC阳性细胞通过流式细胞术和PARP表达的Western印迹分别测定CPT诱导的细胞凋亡。 Western blot检测FOXO1的表达。 FOXO1的转录活性通过荧光素酶报告基因测定。使用特异性抑制FOXO1的siRNA，并进行流式细胞仪和Western印迹法测试FOXO1在CPT诱导的细胞凋亡中的作用。
结果：CPT在诱导Y79视网膜母细胞瘤细胞凋亡中非常有效。 FOXO1在Y79细胞中高表达。 CPT不仅提高了FOXO1的去磷酸化水平，而且还促进了其转录活性，这表明FOXO1的激活至少部分是由CPT触发的。膜联蛋白V阳性细胞的减少和PARP裂解的减少表明siRNA介导的FOXO1抑制显着废除了CPT诱导的凋亡，表明FOXO1在CPT诱导的凋亡中起重要作用。此外，随着CPT的处理，Bim的表达也升高，这与FOXO1的激活有关。
结论：我们的研究提供了证据，视网膜母细胞瘤细胞中高水平的内源性FOXO1表达至少部分地促进了CPT诱导的细胞凋亡，这可能有助于CPT在将来的视网膜母细胞瘤治疗中广泛应用。

BACKGROUND & AIMS: :DE-310, a new macromolecular prodrug, was designed to enhance the pharmacological profiles of a novel camptothecin analog (DX-8951f), and a single treatment with DE-310 exhibits a similar or greater therapeutic effect than do optimally scheduled multiple administrations of DX-8951f in several types of tumors. In this study, the drug-release mechanism by which DE-310 excites antitumor activity was investigated in Meth A cells, a malignant ascites model of murine fibrosarcoma. A single i.v. injection of DE-310 at the maximum tolerated dose (MTD) prolonged survival of Meth A-bearing mice by 300%. DX-8951 and glycyl-8951 (G-DX-8951), enzymatic cleavage products of DE-310, were detected in serum and ascites fluid, and also in the culture medium of Meth A ascites cells incubated in vitro with DE-310. The total amounts of DX-8951, G-DX-8951, and conjugated DX-8951 in Meth A tumor cells were three times higher than that in macrophages. Furthermore, DX-8951-related fluorescence was observed in Meth A ascites cells obtained from Meth A-bearing mice that had received DE-310 or CM-Dex-PA-DX-8951 that does not release free DX-8951. DX-8951-related fluorescence was also observed at the site of lysosomes in cells incubated in vitro with DE-310 at 37 degrees C, but not in those incubated at 4 degrees C. Drugs were released from DE-310 by cysteine proteinase prepared from Meth A tumor tissue. These results suggest that the mechanism by which DX-8951 is released from DE-310 in vivo is involved in the process of uptake of DE-310 into tumor or macrophages, digestion by intracellular lysosomal cysteine proteinase, and subsequent secretion of the drugs.

背景与目标： ：DE-310是一种新的大分子前药，旨在增强新型喜树碱类似物（DX-8951f）的药理作用，并且与最佳预定多次给药相比，DE-310的单次治疗具有相似或更高的治疗效果。 DX-8951f在几种类型的肿瘤中。在这项研究中，研究了在小鼠纤维肉瘤的恶性腹水模型Meth A细胞中DE-310激发抗肿瘤活性的药物释放机制。单个i.v.以最大耐受剂量（MTD）注射DE-310可将含Meth A的小鼠的生存期延长300％。在血清和腹水液中以及在与DE-310体外孵育的Meth A腹水细胞的培养基中检测到DX-8951和glycyl-8951（G-DX-8951）（DE-310的酶促裂解产物）。 Meth A肿瘤细胞中DX-8951，G-DX-8951和结合DX-8951的总量是巨噬细胞的三倍。此外，在得自接受DE-310或未释放游离DX-8951的CM-Dex-PA-DX-8951的携带Meth A的小鼠的Meth A腹水细胞中，观察到了DX-8951相关的荧光。在与DE-310于37°C体外温育的细胞中，在溶酶体的位点也观察到了DX-8951相关的荧光，而在4°C的温育细胞中则未观察到。方法：肿瘤组织。这些结果表明DX-8951在体内从DE-310释放的机制涉及DE-310被摄取到肿瘤或巨噬细胞中，被细胞内溶酶体半胱氨酸蛋白酶消化以及随后的药物分泌过程。