Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

Aims:

Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy.

Methods:

An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses.

Results:

Several specific APE1 inhibitors were isolated by this approach. The IC₅₀ for APE1 inhibition ranged between 30 n and 50 μ. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines.

Conclusions:

Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.     

siRNA重组腺病毒增强贝伐单抗对移植骨肉瘤的治疗作用

目的：探讨以DNA损伤修复基因脱嘌呤/脱嘧啶核酸内切酶基因（apurinic/aprimidinic endonuclease1,APE1）为靶点的siRNA在骨肉瘤治疗中的作用及其与贝伐单抗（bevacizumab,Avastin）的协同效应。方法：建立人骨肉瘤9901细胞荷瘤裸鼠模型,16只荷瘤鼠随机分为4组：EGFP对照组,APE1 siRNA治疗组,Avastin治疗组和联合治疗组（Avastin+APE1 siRNA）。观察移植瘤生长情况并计算抑瘤率,免疫组织化学法检测肿瘤组织微血管密度和Ki67表达,TUNEL法检测肿瘤细胞凋亡,激光共聚焦检测肿瘤组织的缺氧状态,Western blotting检测肿瘤组织内VEGF蛋白的表达。结果：与APE1 siRNA治疗组和Avastin治疗组相比,Avastin+APE1 siRNA治疗组的抑瘤率显著增加（P<0.01）。各治疗组的微血管密度及Ki67表达明显低于对照组,且Avastin+APE1 siRNA治疗组微血管密度和Ki67表达显著低于单独治疗组（P<0.01）。各治疗组的凋亡指数明显高于对照组,且Avastin+APE1 siRNA治疗组明显高于单独治疗组（P<0.01）。APE1siRNA或Avastin治疗均可引起肿瘤组织缺氧,抑制肿瘤组织中VEGF的表达,Avastin+APE1 siRNA治疗效果更加明显。结论：以APE1为靶点的siRNA能显著抑制裸鼠移植骨肉瘤的血管生成和瘤体生长,并诱导肿瘤细胞凋亡,且与Avastin具有协同作用。     

PD-L1和APE1在三阴性乳腺癌组织中的表达及其临床意义

目的:检测三阴性乳腺癌（triple negative breast cancer,TNBC）组织中程序性死亡配体1（programmed death ligand 1,PD-L1）与脱嘌呤/脱嘧啶内切核酸酶1（apurinic/apyrimidinic endonuclease 1,APE1）的表达水平,分析探究其相关性及临床意义。方法:研究选取60例三阴性乳腺浸润性导管癌女性患者,使用免疫组织化学SP法检测组织标本中PD-L1和APE1表达水平;使用Spearman等级法、Kaplan-Meier生存曲线分析PD-L1与APE-1表达相关性及与患者预后关系;建立Cox回归模型进行多因素预后分析。结果:60例患者组织标本中PD-L1阳性表达率为56.67%,与患者是否有淋巴结转移有相关性（P=0.021）;APE1阳性表达率为58.33%,与肿瘤组织分化程度有相关性（P=0.011）。PD-L1与APE1表达呈正相关（r2=0.383,P=0.003）。PD-L1、APE1共阳性表达患者中位生存时间显著差于共阴性表达患者（P=0.018）。Cox回归模型分析显示:PD-L1阳性表达是影响乳腺癌患者预后的一项独立危险因素（P=0.025）。结论:PD-L1与APE1可能共同参与了三阴性乳腺癌发生、发展和转移,临床上检测其表达对预测乳腺癌进展及判断预后具有重要的参考价值。     

TGF‐β inactivation and TGF‐α overexpression cooperate in an in vivo mouse model to induce hepatocellular carcinoma that recapitulates molecular features of human liver cancer

Hepatocellular carcinoma (HCC) results from the cumulative effects of deregulated tumor suppressor genes and oncogenes. The tumor suppressor and oncogenes commonly affected include growth factors, receptors and their downstream signaling pathway components. The overexpression of transforming growth factor alpha (TGF‐α) and the inhibition of TGF‐β signaling are especially common in human liver cancer. Thus, we assessed whether TGF‐α overexpression and TGF‐β signaling inactivation cooperate in hepatocarcinogenesis using an in vivo mouse model, MT1/TGFa;AlbCre/Tgfbr2^flx/flx mice (“TGFa;Tgfbr2^hepko”), which overexpresses TGF‐α and lacks a TGF‐β receptor in the liver. TGF‐β signaling inactivation did not alter the frequency or number of cancers in mice with overexpression of TGF‐α. However, the tumors in the TGFa;Tgfbr2^hepko mice displayed increased proliferation and increased cdk2, cyclin E and cyclin A expression as well as decreased Cdkn1a/p21 expression compared to normal liver and compared to the cancers arising in the TGF‐α overexpressing mice with intact TGF‐β receptors. Increased phosphorylated ERK1/2 expression was also present in the tumors from the TGFa;Tgfbr2^hepko mice and correlated with downregulated Raf kinase inhibitor protein expression, which is a common molecular event in human HCC. Thus, TGF‐β signaling inactivation appears to cooperate with TGF‐α in vivo to promote the formation of liver cancer that recapitulates molecular features of human HCC.     

Synthetic lethal targeting of DNA double-strand break repair deficient cells by human apurinic/apyrimidinic endonuclease inhibitors

An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In this study, we have investigated the ability of APE1 inhibitors to induce synthetic lethality (SL) in a panel of DNA double-strand break (DSB) repair deficient and proficient cells; i) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant [V-C8(Rev1)]. ii) Human cancer cells: BRCA1 deficient (MDA-MB-436), BRCA1 proficient (MCF-7), BRCA2 deficient (CAPAN-1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested SL in CH ovary cells expressing a dominant-negative form of APE1 (E8 cells) using ATM inhibitors and DNA-PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. SL was also demonstrated in CH cells expressing a dominant-negative form of APE1 treated with ATM or DNA-PKcs inhibitors. We conclude that APE1 is a promising SL target in cancer.     

Establishment and characterization of TSGH9201, a human gastric carcinoma cell line that is growth inhibited by epidermal growth factor

A human signet ring gastric carcinoma cell line TSGH9201 was established in vitro. The cells grew in vitro as a monolayer with polygonal morphology and had a population doubling time of 34 hours. The cells secreted tumor markers CEA and CA 125. They were, however, not tumorigenic in athymic nude mice. Karyotypic analysis demonstrated a near tetraploidy with a modal chromosome number of 98. Northern blotting and immunocytochemical analysis revealed the expression of both transforming growth factor α and high levels of epidermal growth factor receptor. Cell growth was inhibited by the epidermal growth factor in vitro. The cell line may be a useful tool to study autocrine growth regulation through the epidermal growth factor receptor. © 1995 Wiley-Liss, Inc.     

Changes in protein expression during multistage mouse skin carcinogenesis

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12-dimethylbenz[a]anthracene–initiated, 12-O-tetradecanoylphorbol-13-acetate–promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor-β1 (TGFβ1) were strongly expressed in the suprabasal layers, whereas integrin α6β4 was expressed only in basal cells and only moderate staining for transforming growth factor-α (TGFα) was seen. In hyperplastic skin, TGFα expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin-binding EGF-like growth factor (HB-EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap-junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of α6β4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20–30 wk), focal areas of γ-glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFβ1 expression began to decrease. Cx26 and TGFα staining became patchier in some late-stage papillomas (30–40 wk), whereas suprabasal α6β4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFα, HB-EGF, TGFβ1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for α6β4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity. Mol. Carcinog. 20:125–136, 1997. © 1997 Wiley-Liss, Inc.     

TGFB1 and TGFBR1 polymorphic variants in relationship to bladder cancer risk and prognosis

The transforming growth factor‐beta (TGF‐β) signalling pathway plays an important role in tumor development and progression. We aimed at analyzing whether 7 different common variants in genes coding for 2 key members of the TGF‐β signalling pathway (TGFB1 and TGFBR1) are associated with bladder cancer risk and prognosis. A total of 1,157 cases with urothelial cell carcinoma of the bladder and 1,157 matched controls where genotyped for 3 single nucleotide polymorphisms (SNPs) in TGFB1 (rs1982073, rs1800472, rs1800471) and an additional 3 SNPs and 1 indel polymorphism in TGFBR1 (rs868, rs928180, rs334358 and rs11466445, respectively). In the case‐control study, we estimated odds ratios and 95% confidence intervals for each individual genetic variant using unconditional logistic regression adjusting for age, gender, study area and smoking status. Survival analysis was performed using the Kaplan‐Meier method and Cox models. The endpoints of interest were tumor relapse, progression and death from bladder cancer. All the SNPs analyzed showed a similar distribution among cases and controls. The distribution of the TGFBR1*6A allele (rs11466445) was also similar among cases and controls, indicating no association with bladder cancer risk. Similarly, none of the haplotypes was significantly associated with bladder cancer risk. Among patients with muscle‐invasive tumors, we found a significant association between TGFBR1‐rs868 and disease‐specific mortality with an allele dosage effect (p‐trend = 0.003). In conclusion, the genetic variants analyzed were not associated with an increased risk of bladder cancer. The association of TGFBR1‐rs868 with outcome should be validated in independent patient series. © 2008 Wiley‐Liss, Inc.     

APE1与P-gp在晚期非小细胞肺癌中的表达及其临床意义

目的 探讨脱嘌呤脱嘧啶核酸内切酶1（APE1／ref-1）和P糖蛋白（P-gP）在晚期非小细胞肺癌（NSCLC）中的表达及其临床意义。方法采用免疫组化方法,检测晚期NSCLC患者肿瘤标本中的APE1和P—gP的表达,探讨APE1和P-gp与晚期NSCLC患者化疗疗效的相关性。结果58例非小细胞肺癌中45例APE1呈高表达,其表达与患者年龄、性别、肿瘤病理类型及临床分期无关;P—gp高表达28例,腺癌中其高表达率高于鳞癌,但与患者年龄、性别及临床分期无关。APEI表达与P-gP表达呈正相关。APE1高表达组、APE1低表达组、P—gP高表达组及P-gP低表达组化疗有效率依次为26．7％、61．5％、21．4％和46．7％,差异有统计学意义。结论APE1和P—gP表达与晚期NSCLC化疗疗效密切相关,对晚期NSCLC耐药机制的研究及其化疗疗效的评价具有重要意义。     

Significance of transforming growth factor β1 as a new tumor marker for colorectal cancer

Transforming growth factor β1 (TGF‐β1) is thought to be involved in cancer growth and progression. TGF‐β1 changes to its active form after being secreted in its latent form. Our aim was to clarify the significance of plasma concentrations of active and total TGF‐β1 of patients with colorectal cancer. Plasma concentrations of active and total TGF‐β1 in 45 patients with colorectal cancer and 23 healthy volunteers were measured using ELISA and the activation rate (ratio of active to total TGF‐β1) was determined. Plasma concentrations of active TGF‐β1 (21.9 ± 12.8 pg/ml) were significantly higher in patients with colorectal cancer than in healthy volunteers (9.9 ± 5.9 pg/ml; p < 0.001, Welch's t‐test). Concentration of total TGF‐β1 was also significantly higher for patients with colorectal cancer (18.0 ± 13.0 ng/ml vs. 11.1 ± 6.4 ng/ml; p < 0.01, Welch's t‐test). However, there was no significant difference in the TGF‐β1 activation rate between the 2 groups. There was a correlation between Dukes' stage and plasma concentration of active or total TGF‐β1 (p < 0.01, Spearman's rank correlation test) and on day 7 the active TGF‐β1 levels for patients recovering from curative resection were similar to those of the control group of healthy volunteers. These results suggest that active TGF‐β1 might be used as a tumor marker for colorectal cancer. © 2001 Wiley‐Liss, Inc.     

Aryl hydrocarbon nuclear translocator (hypoxia inducible factor 1β) activity is required more during early than late tumor growth

c4 is a derivative of the mouse hepatoma cell line, Hepa‐1, that harbors a mutation in the aryl hydrocarbon receptor nuclear translocator gene (Arnt, or hypoxia inducible factor 1β [HIF‐1β]) leading to loss of activity. Clone 3 cells were generated by introducing a doxycycline‐repressible Arnt expression vector into c4 cells. Clone 3 cells were injected subcutaneously into immunosuppressed mice, which were treated with doxycyline (a) throughout the growth of the subsequent tumor xenografts, or (b) from day 7 through to the end of the experiment (day 30), or not treated (c). Tumors in all groups grew exponentially between days 14 and 30, and at rates that were indistinguishable from each other. However, tumors in group a were smaller than those of the other two groups throughout the measurable growth period, while tumor volumes in groups b and c were not significantly different from each other. The degrees of vascularity and apoptosis did not correlate with the differences in degrees of growth between the different groups. Thus, Arnt is required during the early stages of growth of the tumors but less in later stages. Since Arnt does not detectably effect the growth kinetics of Hepa‐1 cells either during hypoxia or normoxia, this requirement is unlikely to reflect a direct effect of Arnt on cell proliferation, and is therefore probably a consequence of altered interaction(s) between the tumor cells and the host. These studies suggest that Arnt (and HIF‐1α/HIF‐2α) inhibitors will be particularly effective against smaller tumors, including micrometastases. © 2009 Wiley‐Liss, Inc.     

Measurement of apoptosis, proliferation and three cytokines in 46 patients with myelodysplastic syndromes

Extensive apoptosis or programmed cell death (PCD) of both hematopoietic (erythroid, myeloid, megakaryocytic) and stromal cells in myelodysplastic syndromes (MDS) cancels the high birth-rate resulting in ineffective hematopoiesis and has been demonstrated as the probable basis for peripheral cytopenias in MDS by our group. It is proposed that factors present in the microenvironment are inducing apoptosis in all the cells whether stromal or parenchymal. To investigate this hypothesis further, bone marrow biopsies from 46 MDS patients and eight normal individuals were examined for the presence of three cytokines, tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β) and granulocyte macrophage-colony stimulating factor (GM-CSF) and one cellular component, macrophages, by the use of monoclonal antibodies immunohistochemically. Results showed the presence of TNF-α and TGF-β in 41/46 and 40/46 cases of MDS respectively, while only 15 cases showed the presence of GM-CSF. Further a significant direct relationship was found between the degree of TNF-α and the incidence of PCD (p = 0.0015). Patients who showed high PCD also had an elevated TNF-α level. Thus, the expression of high amounts of TNF-α and TGF-β and low amounts of the viability factor GM-CSF may be responsible for the high incidence of PCD leading to ineffective hematopoiesis in MDS. Future studies will be directed at attempting to reverse the lesion in MDS by using anti-TNF-α drugs such as pentoxifylline.     

Differential expression of cytokines in rat osteosarcoma and malignant fibrous histiocytoma cell lines induced by 4-(hydroxyamino)quinoline-1-oxide

Cytokines are considered to play an important role in tumor pathogenesis and progression, and recent studies have demonstrated that a variety of forms, including interleukins (ILs) and transforming growth factor-beta(s) (TGF-beta(s)), may regulate tumors. In the present study, the expression of TGF-beta isoforms and ILs was investigated in cell lines from a rat osteosarcoma and a malignant fibrous histiocytoma (MFH), both established from transplantable tumors induced by 4-(hydroxyamino) quinoline 1-oxide (4-HAQO) in syngeneic F344 male rats. The results of a multiprobe RNase protection assay showed TGF-beta1 expression to be remarkably elevated, with no TGF-beta2 and beta3 detectable in MFH cells, while TGF-beta1 and -beta2 were found to be moderately and TGF-beta3 weakly expressed in osteosarcoma lines. All cell lines of osteosarcomas and MFHs expressed macrophage migration inhibitory factor at similar levels. In contrast to the lack of ILs in the MFH cells, moderate IL-6 and very weak IL-1beta expression was detected in the osteosarcoma cells. These results suggest that variation in expression pattern of these cytokines in osteosarcomas and MFHs might be involved in differences in histological appearance and biological behavior, including metastatic ability, between these two mesenchyme-derived tumor types.