Study of Binding Thermodynamics in the Optimization of BH3 Mimetics

The use of small molecule B‐cell lymphoma 2 homology domain 3 mimetics to neutralize the B‐cell lymphoma 2 protein is an attractive strategy for cancer treatment due to its ability to cause targeted cell apoptosis. We have previously reported the design and optimization of a series of B‐cell lymphoma 2 homology domain 3‐mimetics, called compounds 1 – 6 . In this study, we evaluated the optimization of B‐cell lymphoma 2 homology domain 3‐mimetics from a thermodynamic perspective. Understanding the thermodynamic parameters of B‐cell lymphoma 2 homology domain 3‐mimetics plays a critical role in the development of B‐cell lymphoma 2 small‐molecule inhibitors. The thermodynamic parameters for the interactions of these compounds with the myeloid cell leukemia sequence 1 protein were obtained using isothermal titration calorimetry. Owing to compounds 1 – 6 overcoming enthalpy–entropy compensation, the affinities of them improved gradually. Toward binding to the myeloid cell leukemia sequence 1 protein, compound 6 was deemed optimal with an obtained K_d value of 238 nm , which is a 10⁴‐fold improvement compared with 1 . Analysis of the enthalpy and ?TΔS efficiencies showed that ligand efficiencies with respect to molecular size are correlated with the enthalpic efficiencies. Notably, an enthalpy gain of 4.65 kcal/mol identified that an additional hydrogen bond is formed by 2 with myeloid cell leukemia sequence 1 compared with compound 1 . For the first time, hydrogen bonding between a small‐molecule inhibitor of B‐cell lymphoma 2 was demonstrated experimentally.     

BH3-only蛋白Noxa在依托泊苷诱导胃腺癌细胞凋亡中的作用

［摘要］目的检测Bcl-2家族BH3-only蛋白Noxa在依托泊苷诱导胃腺癌细胞凋亡中的作用,为临床治疗以及发现新的治疗靶点寻找理论依据。方法噻唑蓝（MTT）法检测依托泊苷处理后细胞存活率;流式细胞仪Annexin Ⅴ单染法检测依托泊苷诱导的细胞凋亡率;流式细胞仪检测广谱caspase抑制药Z VAD fmk对依托泊苷诱导细胞凋亡的抑制率;半定量逆转录 聚合酶链反应（RT PCR）和Western blot分别检测在依托泊苷诱导前后p53、Noxa的mRNA和蛋白的表达水平。结果依托泊苷以剂量、时间依赖的方式诱导胃腺癌细胞凋亡,SGC 7901细胞系比BGC 823更加敏感。广谱caspase抑制药Z VAD fmk能有效抑制依托泊苷诱导的胃腺癌细胞凋亡,抑制率＞50%。在SGC 7901细胞系中依托泊苷作用4 h后就可以观察到p53和其转录目标Noxa的mRNA很快上调,8 h可观察到其蛋白质含量明显增高;而在BGC 823细胞系中依托泊苷作用前后p53和Noxa的mRNA和蛋白质水平变化不大。结论依托泊苷能诱导胃腺癌细胞发生caspase介导的凋亡;依托泊苷主要通过激活p53和其随后转录激活BH3 only蛋白Noxa来诱导胃腺癌细胞凋亡。     

Different contribution of BH3-only proteins and caspases to doxorubicin-induced apoptosis in p53-deficient leukemia cells

Bcl-2 family proteins are key regulators of the intrinsic apoptotic pathway, either facilitating (Bax, Bak, BH3-only) or inhibiting (Bcl-2, Bcl-x_L, Mcl-1, A1) mitochondrial release of apoptogenic factors. The role of caspases in this process is a matter of controversy. We have analyzed the relative contribution of caspases and Bcl-2 family of proteins in the induction phase of apoptosis triggered by doxorubicin in two p53-deficient leukemia cell lines, Jurkat and U937. First, we have found that caspases are dispensable for the induction phase of doxorubicin-induced apoptosis in both cell lines but they are needed to speed up the execution phase in Jurkat cells, not expressing Bax. Thus, down-regulation of Bak expression by siRNA significantly prevented doxorubicin-induced apoptosis in Jurkat but not in U937 cells. Reduction of Mcl-1 protein levels with siRNA increased sensitivity to apoptosis in both cell lines. Moreover, our results indicate that the contribution of BH3-only proteins to apoptosis is cell line specific. In Jurkat cells simultaneous silencing of Bim and PUMA was necessary to reduce doxorubicin-induced apoptosis. In U937 cells silencing of Bim or Noxa reduced sensitivity to doxorubicin. Immunoprecipitation experiments discarded an interaction between Mcl-1 and Bak in both cell lines and underscored the role of Bim and PUMA as mediators of Bax/Bak activation.     

Involvement of BH3-only proapoptotic proteins in mitochondrial-dependent Phenoxodiol-induced apoptosis of human melanoma cells

Phenoxodiol is a chemically modified analogue of the plant hormone isoflavone with antitumour activities. In the present study, we have examined its ability to induce apoptosis in human melanoma cells and the mechanisms involved. Apoptosis was observed in Phenoxodiol-treated cells by using annexin V/propidium iodide staining and determining mitochondrial membrane potential. To determine which caspase pathways were involved in Phenoxodiol-induced apoptosis, studies were performed using specific caspase inhibitors. Western studies were performed to ascertain which proteins of the apoptosis cascade were affected to cause Phenoxodiol-induced apoptosis. We found that induction of apoptosis by Phenoxodiol was maximal at 48 h with a range of apoptosis of 12+/-4 to 48+/-5% in different melanoma lines. This apoptosis was mainly dependent on activation of caspase-3 and caspase-9. Apoptosis was associated with induction of changes in mitochondrial membrane potential and was inhibited by over-expression of Bcl-2. Variation in sensitivity to Phenoxodiol appeared related to events upstream of the mitochondria and the degree of conformational change in Bax. The p53-regulated BH3-only proteins (Bad, PUMA and Noxa) were increased in the sensitive, but not in the resistant lines, whereas Bim was increased in all the lines tested. Bim appeared, however, to be partially involved because reduction of Bim by RNA interference resulted in decreased levels of apoptosis. Together, these studies suggest that Phenoxodiol induces apoptosis of melanoma cells by induction of p53-dependent BH3 proteins (Bad, PUMA and Noxa) and the p53-independent Bim protein, resulting in activation of Bax and its downstream events.     

BH3-only蛋白Bim的移位对紫杉醇诱导的胃腺癌细胞凋亡的影响

目的研究BH3-only蛋白Bim的移位在紫杉醇诱导的胃腺癌细胞凋亡中的作用。方法流式细胞仪AnnexinV／PI双染法检测细胞凋亡率;线粒体分离和Westernblot检测在紫杉醇诱导后BIM的移位情况。结果紫杉醇诱导胃腺癌细胞凋亡具有剂量和时间依赖性,SGC-7901胃腺癌细胞比BGC．823细胞对紫杉醇更具敏感性。紫杉醇能诱导胃腺癌细胞系SGC-7901及BGC-823中Bim移位于线粒体膜,导致其活化形式增加。结论无论对紫杉醇敏感细胞系还是相对不敏感细胞系,Bim的移位都与其诱导的胃腺癌细胞凋亡有关。     

Multidrug Resistance Proteins (MRPs) and Cancer Therapy

The ATP-binding cassette (ABC) transporters are members of a protein superfamily that are known to translocate various substrates across membranes, including metabolic products, lipids and sterols, and xenobiotic drugs. Multidrug resistance proteins (MRPs) belong to the subfamily C in the ABC transporter superfamily. MRPs have been implicated in mediating multidrug resistance by actively extruding chemotherapeutic substrates. Moreover, some MRPs are known to be essential in physiological excretory or regulatory pathways. The importance of MRPs in cancer therapy is also implied by their clinical insights. Modulating the function of MRPs to re-sensitize chemotherapeutic agents in cancer therapy shows great promise in cancer therapy; thus, multiple MRP inhibitors have been developed recently. This review article summarizes the structure, distribution, and physiological as well as pharmacological function of MRP1–MRP9 in cancer chemotherapy. Several novel modulators targeting MRPs in cancer therapy are also discussed.KEY WORDS: ABC transporters, cancer chemotherapy, multidrug resistance, multidrug resistance proteins     

细胞色素P450活性与癌症治疗

本文通过总结影响细胞色素P450活性的因素和抗肿瘤药的生物转化途径,以期探索细胞色素P450同工酶介导的代谢在抗癌药物体内生物转化中的作用,进而探索该酶活性与癌症患者的疗效之间的相关性。发现化疗药物的个体间药代动力学差异会引起治疗的疗效和安全性的不同结果。年龄、性别、单个基因多态性等单一因素并不能阐明个体间对药物反应的差异性,化疗方案中多个药物的相互作用在临床上非常重要。为了更好地评估细胞色素P450酶类对机体代谢的作用,药物的吸收、排泄、活化,以及代谢整个药代动力学途径涉及的酶的多态性都应进行研究。     

伊布替尼联合BH3拟似物ABT737协同抗肿瘤作用及机制

目的 研究伊布替尼联合BH3拟似物ABT737的协同抗实体肿瘤作用及机制。方法 以2种类型实体肿瘤细胞,人非小细胞肺癌细胞株A549、H1299和人脑胶质瘤细胞U251、U87为对象,采用SRB法检测不同浓度伊布替尼(20,15,10,7.5,5 μmol·L^-1)和BH3拟似物ABT737(20,15,10,7.5,5 μmol·L^-1)单独或共同作用24 h后的细胞增殖情况,计算细胞存活率和合用指数;采用克隆形成试验检验10 μmol·L^-1伊布替尼与10 μmol·L^-1ABT737 联合作用120 h后的细胞克隆形成情况;成球试验检测两药联合作用7 d对细胞成球能力的影响;采用PI染色结合流式细胞术检测10 μmol·L^-1伊布替尼与10 μmol·L^-1ABT737联用对U87和U251细胞凋亡的影响;RT-PCR检测两药联合作用24 h后对U87和U251细胞中干细胞标记物Sox2、Nanog和Oct4 mRNA水平影响;Western blotting检测脑胶质瘤干细胞标记物CD44蛋白水平的表达。结果 不同浓度伊布替尼与BH3拟似物ABT737在非小细胞肺癌和脑胶质瘤中均有协同作用,合用组增殖抑制率均高于单用组(P<0.05);两药合用可抑制细胞的克隆形成能力,协同诱导细胞凋亡;同时两者合用可抑制肿瘤干细胞标记物Sox2的mRNA表达水平,使CD44蛋白表达水平降低。结论 伊布替尼联合ABT737可协同抑制实体肿瘤细胞的增殖,促进凋亡发生,其机制可能是通过抑制肿瘤干细胞蛋白的表达。     

Phosphatidylinositol 3-kinase Inhibitor (PIK75) Containing Surface Functionalized Nanoemulsion for Enhanced Drug Delivery, Cytotoxicity and Pro-apoptotic Activity in Ovarian Cancer Cells

Purpose

Ovarian cancer is a debilitating disease, which needs multi-pronged approach of targeted drug delivery and enhanced efficacy with the use of combination therapeutics. In this study, we have examined the anticancer activity of PIK75 incorporated in surface functionalized nanoemulsions for targeted delivery to SKOV-3 cells. A pro-apoptotic molecule C₆-ceramide was also co-delivered to augment therapeutic efficacy.

Methods

EGFR and FR functionalized nanoemulsions incorporating PIK75 and C₆-ceramide were characterized for particle size, surface charge, entrapment efficiency and morphology. Fluorescence and quantitative uptake studies were conducted in SKOV-3 cells to determine intracellular distribution. Cell viability was assessed using MTT assay while mechanism of cytotoxicity was evaluated using capsase-3/7, TUNEL and hROS assay.

Results

Cytotoxicity assay showed 57% decrease in IC₅₀ value of PIK75 following treatment with EGFR targeted nanoemulsion and 40% decrease following treatment with FR targeted nanoemulsion. Combination therapy with PIK75 and ceramide enhanced the cytotoxicity of PIK75 compared to therapy with individual formulations. The increase in cytotoxicity was attributed to increase in cellular apoptosis and hROS activity.

Conclusion

The results of this study showed that the targeted system improved cytotoxicity of PIK75 compared to the non-targeted system. Combination therapy with ceramide augmented PIK75’s therapeutic activity.     

灵芝蛋白质的分离及其免疫活性研究

利用凝胶层析法对灵芝子实体和破壁孢子粉的蛋白初提液进行了分离,通过体外免疫反应初步检测了各蛋白质成分的免疫调节活性.小鼠脾淋巴细胞转化试验表明其中有三种灵芝蛋白体外具有较强的促进淋巴细胞增殖的活性,分别命名为LZP-1,LZP-2和LZP-3,其中来自于灵芝孢子粉的LZP-2和LZP-3用量为50μg/ml时,它们体外促进淋巴细胞的增殖率可达(41.3±3.1)%和(38.7±4.8)%,并且呈现出一定的剂量依赖效应,而来自于灵芝子实体的LZP-1促进淋巴细胞的增殖率较低(18.2±3.9%,使用剂量为50μg/ml).SDS-PAGE测得它们的相对分子质量分别为67,000,43,000和45,700.该蛋白有可能是灵芝中具有免疫调节活性的成分之一,具有潜在的临床应用价值.     

BH3 mimetics to improve cancer therapy; mechanisms and examples

Tumor cell survival is highly dependent on the expression of certain pro-survival Bcl-2 family proteins. An attractive therapeutic approach is to inhibit these proteins using agents that mimic the Bcl-2 homology 3 (BH3) domains of the proapoptotic Bcl-2 family members, which neutralize these proteins by binding to their surface hydrophobic grooves. A number of BH3 mimetic peptides and small molecules have been described, a few of which have advanced into clinical trials. Recent studies have highlighted ABT-737, a bona fide BH3 mimetic and potent inhibitor of antiapoptotic Bcl-2 family members, as a promising anticancer agent. This review summarizes recent advances in understanding the mechanisms of action of BH3 domains and several classes of BH3 mimetics, as well as the prospects of using these agents to improve cancer therapy.     

Heparin Affin Regulatory Peptide Modulates the Endogenous Anticoagulant Activity of Heparin and Heparan Sulphate Mimetics

Pleiotrophin, also known as heparin affin regulatory peptide (HARP), is a growth factor expressed in various tissues and cell lines. In this work, HARP was tested for its capacity to modulate the anticoagulant activity of heparin and heparan sulphate mimetics (OTR4120). We used both in vitro and in vivo assays. HARP was found to be differently effective for neutralization of the anticoagulant activity of the mimetic heparan sulphate (OTR4120) and heparin in purified system and human plasma. HARP was shown to compete with both antithrombin and thrombin for binding to heparin and to OTR4120, respectively. In the presence of OTR4120, the V_max was constant and the calculated maximum velocity was 1.56 U/min; the thrombin Km value (0.011 nM) was affected by HARP concentrations. The Km (HARP) value was 0.085 nM, which is consistent with high affinity of HARP to OTR4120. Under the same conditions, initial velocity patterns for antithrombin–heparin were determined in the presence or in the absence of HARP. The antithrombin value Km (0.022 nM) was affected by HARP (0.077 nM). HARP exhibits efficacy equivalent to or greater than protamine. Interestingly, intraperitoneally administered HARP decreased the anticoagulant activity of heparin and of OTR4120 in mice. Taken together, these data provide the first evidence for a physiological role of HARP in the modulation of anticoagulant activity of heparin and heparin‐like material.     

Pro-apoptotic peptides-based cancer therapies: challenges and strategies to enhance therapeutic efficacy

Cancer is a leading cause of death worldwide. Despite many advances in the field of cancer therapy, an effective cure is yet to be found. As a more potent alternative for the conventional small molecule anti-cancer drugs, pro-apoptotic peptides have emerged as a new class of anticancer agents. By interaction with certain members in the apoptotic pathways, they could effectively kill tumor cells. However, there remain bottleneck challenges for clinical application of these pro-apoptotic peptides in cancer therapy. In this review, we will overview the developed pro-apoptotic peptides and outline the widely adopted molecular-based and nanoparticle-based strategies to enhance their anti-tumor effects.     

Discovery of Novel PTP1B Inhibitors Derived from the BH3 Domain of Proapoptotic Bcl-2 Proteins with Antidiabetic Potency

BH3 peptide analogues are generally believed to exhibit great potency as cancer therapeutics via targeting antiapoptotic Bcl-2 proteins. Here, we describe the synthesis and identification of a new class of palmitoylated peptide BH3 analogues derived from the core region (h1–h4) of BH3 domains of proapoptotic Bcl-2 proteins and as alternative PTP1B inhibitors with antidiabetic potency in vitro and in vivo. PTP1B inhibitors are attractive for treatment of type 2 diabetes. We design the analogues using a simple lipidation approach and discovered novel lead analogues with promising antidiabetic potency in vitro and in vivo. The results presented here expanded the alternative target and function for the BH3 peptide analogues from one member Bim to other members of the proapoptotic Bcl-2 proteins and emphasize their therapeutic potential in T2DM. Furthermore, our findings may provide new proof of the regulatory function of Bcl-2 family proteins in mitochondrial nutrient and energy metabolism.     

肿瘤的生物治疗与分子靶向治疗

在肿瘤的治疗过程中,我们不光要把眼光对准局部异常生长的肿瘤细胞,还要努力纠正存在的全身性缺陷。生物治疗正是从这一角度来寻求自身的定位和价值。     

Colon Targeted Eudragit Coated Beads Loaded with Optimized Fluvastatin-Scorpion Venom Conjugate as a Potential Approach for Colon Cancer Therapy: In Vitro Anticancer Activity and In Vivo Colon Imaging

Preclinical studies suggest that most of statins or 3?hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors possess pleiotropic anticancer activity. The aim of the present work was to investigate the conjugation of the statin fluvastatin (FLV) with scorpion venom (SV), a natural peptide with proven anticancer properties, to enhance FLV cytotoxic activity and prepare colon targeted FLV-SV nanoconjugate beads for management of colon cancer. Response surface design was applied for the optimization of FLV-SV nanoconjugates. FLV-SV particle size and zeta potential were selected as responses. Cytotoxicity of optimized FLV-SV nanoconjugates was carried out on Caco2 cell line. Colon targeted alginate coated Eudragit S100 (ES100) beads for the optimized formula were prepared with the utilization of barium sulfate (BaSO4) as radiopaque contrast substance. Results revealed that optimized FLV-SV nanoconjugates showed a size of 71.21 nm, while the zeta potential was equal to 29.13 mV. Caco2 cells were considerably more sensitive to the FLV-SV formula (half-maximal inhibitory concentration (IC50) = 11.91 µg/mL) compared to SV and FLV used individually, as shown by values of IC50 equal to 30.23 µg/mL and 47.68 µg/mL, respectively. In vivo imaging of colon targeted beads, carried out by employing real-time X-ray radiography, confirmed the efficiency of colon targeted beads. Overall our results indicate that the optimized FLV-SV nanoconjugate loaded alginate coated ES100 beads could represent a promising approach for colon cancer with efficient colon targeting ability.     

Inhibitors of Apoptotic Proteins: New Targets for Anticancer Therapy

Inhibitors of apoptotic proteins (IAPs) can play an important role in inhibiting apoptosis by exerting their negative action on caspases (apoptotic proteins). There are eight proteins in this family: NAIP/BIRC1/NLRB, cellular IAP1 (cIAP1)/human IAP2/BIRC2, cellular IAP2 (cIAP2)/human IAP1/BIRC3, X‐linked IAP (XIAP)/BIRC4, survivin/BIRC5, baculoviral IAP repeat (BIR)‐containing ubiquitin‐conjugating enzyme/apollon/BIRC6, livin/melanoma‐IAP (ML‐IAP)/BIRC7/KIAP, and testis‐specific IAP (Ts‐IAP)/hILP‐2/BIRC8. Deregulation of these inhibitors of apoptotic proteins (IAPs) may push cell toward cancer and neurodegenerative disorders. Inhibitors of apoptotic proteins (IAPs) may provide new target for anticancer therapy. Drugs may be developed that are inhibiting these IAPs to induce apoptosis in cancerous cells.     

Toward Targeted Oral Vaccine Delivery Systems: Selection of Lectin Mimetics from Combinatorial Libraries

Purpose. Various lectins bind specifically to oligosaccharides on intestinal cells. Exploiting this specificity, Ulex europaeus agglutinin I (UEA1) has been used as a ligand for targeted oral vaccine delivery to M cells (antigen-presenting cells) in follicle-associated epithelium. In this study we characterized compounds identified from mixture-based positional scanning synthetic combinatorial libraries, which mimic UEA1 and, thus, may have properties applicable to targeted drug delivery. Methods. Two UEA1 mimetics were synthesized and their activity was verified on live cells. The ability of the lead compound, a tetragalloyl D-Lysine amide construct (4-copy gallic acid construct), to deliver dye-loaded polystyrene particles to M cells was assessed in an in situ mouse gut loop model. Results. The 4-copy gallic acid construct inhibited UEA1 binding to Caco-2 cell membranes with an IC₅₀ of 3 M, a 650- to 5000-fold increase over the natural UEA1 substrate -L-fucose. The biotin-labeled derivative of this construct demonstrated comparable binding activity as verified on live cells by fluorescence-activated cell sorting. Preclinical studies confirmed its ability to mediate M cell-specific delivery of streptavidin-coated particles in vivo. Conclusions. Polyphenolic compounds, D-Lysine scaffolds with multiple galloyl groups, can mimic functional activities of UEA1. Properties of such molecules, including low molecular weight, stability, ease of synthesis and low cost, highlight their potential for application in targeted vaccine delivery.