Delivery of Toxins and Effectors by Bacterial Membrane Vesicles

Pathogenic bacteria interact with cells of their host via many factors. The surface components, i.e., adhesins, lipoproteins, LPS and glycoconjugates, are particularly important in the initial stages of colonization. They enable adhesion and multiplication, as well as the formation of biofilms. In contrast, virulence factors such as invasins and toxins act quickly to damage host cells, causing tissue destruction and, consequently, organ dysfunction. These proteins must be exported from the bacterium and delivered to the host cell in order to function effectively. Bacteria have developed a number of one- and two-step secretion systems to transport their proteins to target cells. Recently, several authors have postulated the existence of another transport system (sometimes called “secretion system type zero”), which utilizes extracellular structures, namely membrane vesicles (MVs). This review examines the role of MVs as transporters of virulence factors and the interaction of toxin-containing vesicles and other protein effectors with different human cell types. We focus on the unique ability of vesicles to cross the blood–brain barrier and deliver protein effectors from intestinal or oral bacteria to the central nervous system.     

Inhibition of mitochondrial respiration by tigecycline selectively targets thyroid carcinoma and increases chemosensitivity

The role of mitochondria in cancer and mitochondria‐targeted therapy has been gaining attention for its effectiveness and selectivity between cancer and normal cells. In line with this notion, our work demonstrates that inducing mitochondrial dysfunction by tigecycline, a FDA‐approved antibiotic, selectively targets thyroid cancer and enhances chemosensitivity. We found that tigecycline inhibited proliferation and induced apoptosis in a panel of thyroid cancer cell lines. Consistently, tigecycline inhibited thyroid cancer growth in mice without causing significant toxicity. The combination of tigecycline with paclitaxel achieved greater efficacy than paclitaxel alone in vitro and in vivo. Mechanistically, tigecycline inhibited mitochondrial respiration and ATP reduction through decreasing mitochondrial membrane potential and inhibiting mitochondrial translation, leading to oxidative stress and damage. In contrast, tigecycline was ineffective in mitochondrial respiration‐deficient cells, confirming that tigecycline acts on thyroid cancer via inhibiting mitochondrial respiration. Interestingly, although tigecycline inhibited mitochondrial respiration in both thyroid cancer and normal thyroid cells in a similar manner, tigecycline was more effective in thyroid cancer than normal thyroid cells, suggesting that thyroid cancer cells are more dependent on mitochondrial functions than normal thyroid cells. This was supported by our observations that thyroid cancer cells had higher level of mitochondrial biogenesis compared to normal thyroid cells. Our work is the first to demonstrate that the combination of chemotherapy with tigecycline is a potential sensitizing strategy for thyroid cancer treatment. Our findings also highlight the higher dependence of thyroid cancer cells on mitochondrial functions than normal thyroid cells.     

Stimulation of Brain Nicotinic Acetylcholine Receptors (nAChR) and Inhibition of Brain Acetylcholinesterase (AChE) Activity

Introduction: The Fas/FasL system plays a significant role in tumorigenesis. Research has shown that its impairment in cancer cells may lead to apoptosis resistance and contribute to tumor progression. Thus, the development of effective therapies targeting the Fas/FasL system may play an important role in the fight against cancer.

Areas covered: In this review the recent literature on targeting the Fas/FasL system for therapeutic exploitation at different levels is reviewed. Promising pre-clinical approaches and various exceptions are highlighted. The potential of combined therapies is also explored, whereby tumor sensitivity to Fas-mediated apoptosis is restored, before an effective targeted therapy is employed.

Expert opinion: The success of the Fas/FasL system targeting for therapeutics will require a better understanding of the alterations conferring resistance, in order to use the most appropriate sensitizing chemotherapeutic or radiotherapeutic agents in combination with effective targeted therapies.     

No Evidence for the In Vivo Induction of Genomic Instability by Low Doses of CS Gamma Rays in Bone Marrow Cells of BALB/CJ and C57BL/6J Mice

In spite of extensive research, assessment of potential health risks associated with exposure to low-dose (≤ 0.1 Gy) radiation is still challenging. We evaluated the in vivo induction of genomic instability, expressed as late-occurring chromosome aberrations, in bone-marrow cells of two strains of mouse with different genetic background, i.e. the radiosensitive BALB/cJ and the radioresistant C57BL/6J strains following a whole-body exposure to varying doses of (137)Cs gamma rays (0, 0.05, 0.1, and 1.0 Gy). A total of five mice per dose per strain were sacrificed at various times post-irradiation up to 6 months for sample collections. Three-color fluorescence in situ hybridization for mouse chromosomes 1, 2, and 3 was used for the analysis of stable-aberrations in metaphase-cells. All other visible gross structural-abnormalities involving non-painted-chromosomes were also evaluated on the same metaphase-cells used for scoring the stable-aberrations of painted-chromosomes. Our new data demonstrated in bone-marrow cells from both strains that low doses of low LET-radiation (as low as 0.05 Gy) are incapable of inducing genomic instability but are capable of reducing specific aberration-types below the spontaneous rate with time post-irradiation. However, the results showed the induction of genomic instability by 1.0 Gy of (137)Cs gamma rays in the radiosensitive strain only.     

Development and Characterization of Anti-Naja ashei Three-Finger Toxins (3FTxs)-Specific Monoclonal Antibodies and Evaluation of Their In Vitro Inhibition Activity

Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.     

Association of Polygenic Risk Score and Bacterial Toxins at Screening Colonoscopy with Colorectal Cancer Progression: A Multicenter Case-Control Study

Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and its incidence is correlated with infections, chronic inflammation, diet, and genetic factors. An emerging aspect is that microbial dysbiosis and chronic infections triggered by certain bacteria can be risk factors for tumor progression. Recent data suggest that certain bacterial toxins implicated in DNA attack or in proliferation, replication, and death can be risk factors for insurgence and progression of CRC. In this study, we recruited more than 300 biopsy specimens from people undergoing colonoscopy, and we analyzed to determine whether a correlation exists between the presence of bacterial genes coding for toxins possibly involved in CRC onset and progression and the different stages of CRC. We also analyzed to determine whether CRC-predisposing genetic factors could contribute to bacterial toxins response. Our results showed that CIF toxin is associated with polyps or adenomas, whereas pks+ seems to be a predisposing factor for CRC. Toxins from Escherichia coli as a whole have a higher incidence rate in adenocarcinoma patients compared to controls, whereas Bacteroides fragilis toxin does not seem to be associated with pre-cancerous nor with cancerous lesions. These results have been obtained irrespectively of the presence of CRC-risk loci.     

Characterization and Toxicity of Crude Toxins Produced by Cordyceps fumosorosea against Bemisia tabaci (Gennadius) and Aphis craccivora (Koch)

Cordyceps fumosorosea, an insect pathogenic fungus, produces different toxins/secondary metabolites which can act as pest control agents. This study reports the extraction and characterization of crude mycelial extracts of C. fumosorosea isolate SP502 along with their bio-efficacy against Bemisia tabaci and Aphis craccivora. Fourier transform infrared spectroscopy, liquid chromatography, mass spectrometery and nuclear magnetic resonance analysis of C. fumosorosea isolate SP502 extracts showed the presence of five major compounds—Trichodermin, 5-Methylmellein, Brevianamide F, Enniatin and Beauvericin—which all may potentially be involved in insecticidal activity. The HPLC analysis of C. fumosorosea mycelial extracts and Beauvericin standard showed similar chromatographic peaks, with the content of Beauvericin in the crude toxin being calculated as 0.66 mg/ml. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of A. craccivora were 46.35, 54.55, 68.94, and 81.92 µg/mL, respectively. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of B. tabaci were 62.67, 72.84, 77.40, and 94.40 µg/mL, respectively. Our results demonstrate that bioactive compounds produced by C. fumosorosea isolate SP502 have insecticidal properties and could, therefore, be developed into biopesticides for the management of B. tabaci and A. craccivora.     

Application of titanium dioxide (TiO2) nanoparticles in cancer therapies

Abstract

Cancer is one of the most common diseases all over the world; many people suffer from diverse types of cancer. However, currently there is no exact cure or therapy developed for cancer. On the other hand, nanoparticles are defined as microscopic particles that have dimensions less than 100?nm and they are known for their usage in health sciences and medicine, however a few harmful effects on different animal cells. Therefore, researchers began to use nanoparticles for cancer therapies and to develop new methods for much more effective therapies. Nanoparticles in cancer studies are commonly used in photodynamic therapy (PDT) and sonodynamic therapy (SDT) as a sensitising agent, in computed tomography imaging (CT) and radiation therapy as an enhancement agent, in dual-mode image contrast and enhancement therapy as an image contrast agent. Titanium dioxide nanoparticles (TiO₂ NPs) are known as commonly used nanoparticles in medical applications and hence in cancer studies. They are used in PDT, SDT and drug delivery systems. As cancer continues to affect people, new therapeutics and therapies will be developed and nanotechnology for this aim will be an important approach for the researchers.     

Development of an orphanin FQ (OFQ) receptor antagonist for the treatment of chronic pain

Introduction: Hedgehog (Hh) signaling pathway plays key roles in embryonic development, formation and maintenance of cancer stem cells (CSCs) and acquisition of epithelial-to-mesenchymal transition (EMT). Since CSCs and EMT are important biological factors responsible for cancer cell invasion, metastasis, drug resistance and tumor recurrence, the Hh signaling pathway is believed to be an important target for cancer therapy.

Areas covered: In recent years, small-molecule inhibitors of Hh signaling have been synthesized for cancer treatment. Clinical trials using these inhibitors are being conducted to determine their toxicity profiles and efficacies. In addition, nutraceuticals (such as isoflavones, curcumin, vitamin D, etc) have been shown to inhibit cancer growth through downregulation of Hh signaling.

Expert opinion: Inhibition of Hh signaling is important for suppression of cancer growth, invasion, metastasis and recurrence in cancer therapy. However, targeting only one molecule in Hh signaling may not be sufficient to kill cancer cells because cancers show deregulation of multiple signals. Therefore, utilizing new technologies to determine alterations in Hh and other signals for individuals and designing combination strategies with small-molecule Hh inhibitors, nutraceuticals and other chemotherapeutics in targeted personalized therapy could have a significant effect on improving the overall survival of patients with cancers.     

反义DLC-1基因对结直肠癌LoVo细胞增殖和细胞周期的影响

目的 应用核糖核酸干扰(RNAi)技术,通过对大肠癌细胞株LoVo中DLC-1基因mRNA表达的抑制,初步探讨DLC-1基因对大肠癌细胞株的生物学行为影响.方法 构建DCL-1的RNAi重组体pGCsiDLC,转染大肠癌LoVo细胞株.采用RT-PCR观察转染前后细胞株中DLC-1 mRNA以及 Bax、Bcl-2基因表达的改变,MTT比色法检测转染前后细胞增殖,流式细胞仪检测细胞周期.结果 成功构建发卡siRNA真核表达载体pGCsiDLC;pGCsiDLC转染LoVo细胞后,电泳显示DLC-1 mRNA表达水平明显下降;LoVo细胞生长曲线增长幅度亦随时间的增加而逐渐增高,在48h最为明显,RNAi组细胞比脂质体组及空白对照组细胞增殖明显增高(P＜0.05);干扰后的LoVo细胞株G0/G1期发生阻滞,而G2/M 期细胞数量分布减少.而Bax和bcl-2基因表达在干扰前后未见明显改变.结论 成功构建载体介导的DLC-1靶向RNAi重组体可有效抑制LoVo细胞DLC-1 mRNA表达;DLC-1基因可抑制结直肠癌细胞增殖并且对细胞周期重新分布,其可能与结直肠癌的发生发展有一定关系,可能是一个新的抑癌基因.     

2014年美国第50届临床肿瘤年会乳腺癌内科治疗热点撷英

2014年美国临床肿瘤年会（ASCO）在芝加哥召开,大会报告了乳腺癌领域重要的基础研究成果和临床研究数据。本文主要综述了乳腺癌内科治疗领域4项主要临床研究结果：①与他莫西芬+卵巢功能抑制相比,依西美坦+卵巢功能抑制也可以用于激素受体阳性的绝经前早期乳腺癌,但年轻患者要充分做到卵巢功能抑制。②在辅助曲妥珠单抗治疗的基础上联合拉帕替尼没有获得改善HER2阳性乳腺癌的预后。③贝伐珠单抗联合化疗辅助治疗HER2阴性乳腺癌没有获得总生存的改善。④依西美坦+贝伐珠单抗用于转移性乳腺癌维持治疗在治疗周期上有一定延长。本文对乳腺癌内科治疗的进展进行了综述报道。     

Inhibition of Multidrug Resistance-Associated Protein (MRP) Functional Activity with Pluronic Block Copolymers

Purpose. Using monolayers of human pancreatic adenocarcinoma cells (Panc-1) that express multidrug resistance-associated protein (MRP), the present work investigates the effects of Pluronic block copolymers on the functional activity of MRP. Methods. The studies examined the accumulation and efflux of the MRP selective probe fluorescein (FLU) in Panc-1 cell monolayers with and without Pluronic P85 (P85), Pluronic L81 (L81) and Pluronic F108 (F108). Results. Treatment of Panc-1 cells with P85 resulted in concentration-dependent increases in FLU accumulation and elimination of FLU sequestration in vesicular compartments in these cells. The effects of P85 were selective for FLU in the Panc-1 cell monolayers. Inhibition of MRP-mediated transport was dependent on the composition of Pluronic block copolymer: the more hydrophobic copolymer had the greater effect on FLU uptake in Panc-1 monolayers (L81 > P85 > F108). Conclusions. This paper demonstrates for the first time that Pluronic block copolymers inhibit multidrug resistance-associated protein (MRP). The similarities in the effects of Pluronic block copolymers on MRP and P-glycoprotein drug efflux systems suggest that a single unifying mechanism may explain the inhibition observed.     

Suppression of tumor growth in lung cancer xenograft model mice by poly(sorbitol-co-PEI)-mediated delivery of osteopontin siRNA

Small interfering RNA (siRNA)-mediated gene silencing represents a promising strategy for treating diseases such as cancer; however, specific gene silencing requires an effective delivery system to overcome the instability and low transfection efficiency of siRNAs. To address this issue, a polysorbitol-based transporter (PSOT) was prepared by low molecular weight branched polyethylenimine (bPEI) crosslinked with sorbitol diacrylate (SDA). Osteopontin (OPN) gene, which is highly associated with non-small cell lung cancer (NSCLC) was targeted by siRNA therapy using siRNA targeting OPN (siOPN). Characterization study confirmed that PSOT formed compact complexes with siOPN and protected siOPN against degradation by RNase. PSOT/siOPN complexes demonstrated low cytotoxicity and enhanced transfection efficiency in vitro, suggesting that this carrier may be suitable for gene silencing. In the A549 and H460 lung cancer cell lines, PSOT/siOPN complexes demonstrated significant silencing efficiency at both RNA and protein levels. To study in vivo tumor growth suppression, two lung cancer cell-xenograft mouse models were prepared and PSOT/siOPN complexes were delivered into the mice through intravenous injection. The siOPN-treated groups demonstrated significantly reduced OPN expression at both the RNA and protein levels as well as suppression of tumor volume and weight. Taken together, siOPN delivery using PSOT may present an effective and novel therapeutic system for lung cancer treatment.     

Inhibition of a Thrombin-Glycoprotein Ib(GPIb)-GPIIb/IIIa Pathway

Introduction: Caspase-9 is the apoptotic initiator protease of the intrinsic or mitochondrial apoptotic pathway, which is activated at multi-protein activation platforms. Its activation is believed to involve homo-dimerization of the monomeric zymogens. It binds to the apoptosome to retain substantial catalytic activity. Variety of apoptotic stimuli can regulate caspase-9. However, the mechanism of action of various regulators of caspase-9 has not been summarized and compared yet. In this article, we elucidate the regulators of caspase-9 including microRNAs, natural compounds that are related to caspase-9 and ongoing clinical trials with caspase-9 to better understand the caspase-9 in suppressing cancer.

Areas covered: In this study, the basic mechanism of apoptosis pathways, regulators of caspase-9 and the development of drugs to regulate caspase-9 are reviewed. Also, ongoing clinical trials for caspase-9 are discussed.

Expert opinion: Apoptosis has crucial role in cancer, brain disease, aging and heart disease to name a few. Since caspase-9 is an initiator caspase of apoptosis, it is an important therapeutic target of various diseases related to apoptosis. Therefore, a deep understanding on the roles as well as regulators of caspase-9 is required to find more effective ways to conquer apoptosis-related diseases especially cancer.     

A Combined Bioassay and Nanofractionation Approach to Investigate the Anticoagulant Toxins of Mamba and Cobra Venoms and Their Inhibition by Varespladib

Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This study sought to investigate the coagulopathic potential of venoms from medically important elapids of the genera Naja (true cobras), Hemachatus (rinkhals), and Dendroaspis (mambas). Crude venoms were bioassayed for coagulant effects using a plasma coagulation assay before RPLC/MS was used to separate and identify venom toxins in parallel with a nanofractionation module. Subsequently, coagulation bioassays were performed on the nanofractionated toxins, along with in-solution tryptic digestion and proteomics analysis. These experiments were then repeated on both crude venoms and on the nanofractionated venom toxins with the addition of either the phospholipase A₂ (PLA₂) inhibitor varespladib or the snake venom metalloproteinase (SVMP) inhibitor marimastat. Our results demonstrate that various African elapid venoms have an anticoagulant effect, and that this activity is significantly reduced for cobra venoms by the addition of varespladib, though this inhibitor had no effect against anticoagulation caused by mamba venoms. Marimastat showed limited capacity to reduce anticoagulation in elapids, affecting only N. haje and H. haemachatus venom at higher doses. Proteomic analysis of nanofractionated toxins revealed that the anticoagulant toxins in cobra venoms were both acidic and basic PLA₂s, while the causative toxins in mamba venoms remain uncertain. This implies that while PLA₂ inhibitors such as varespladib and metalloproteinase inhibitors such as marimastat are viable candidates for novel snakebite treatments, they are not likely to be effective against mamba envenomings.     

Inhibition of neuronal noradrenaline uptake (uptake1) and desipramine binding by N-ethylmaleimide (NEM)

Summary The inhibition by N-ethylmaleimide (NEM) of uptake₁ and desipramine binding was studied on clonal rat phaeochromocytoma cells (PC12 cells) in different experimental settings: (1) ³H-noradrenaline uptake into intact PC12 cells; (2) ³H-noradrenaline uptake into isolated PC12 plasma membrane vesicles; (3) ³H-desipramine binding to isolated PC12 plasma membrane vesicles.In plasma membrane vesicles, NEM inhibited ³H-desipramine binding and ³H-noradrenaline uptake with similar potency (the IC₅₀'s were 1.36 mmol/l and 1.04 mmol/l, respectively). However, in intact cells, NEM was about 75 times more potent in inhibiting ³H-noradrenaline uptake (IC₅₀ = 0.014 mmol/l). The increased potency of NEM in intact cells is probably due to an inhibition of the Na^{+/K +}-ATPase and not to a direct interaction with the noradrenaline carrier.The inactivation by NEM of ³H-desipramine binding to PC12 plasma membrane vesicles was irreversible. Both an inhibitor (cocaine, 1 mmol/l) and a substrate of uptake₁ (amezinium, 1 mmol/l) protected desipramine binding from inactivation.These results are compatible with the hypothesis of a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.This study was supported by the Deutsche Forschungsgemeinschaft (SFB176) Send offprint requests to E. Schömig at the above address     

Inhibition of human liver microsomal (S)-nicotine oxidation by (-)-menthol and analogues

(-)-Menthol is a widely used flavoring ingredient present in mouthwash, foods, toothpaste, and cigarettes; yet, the pharmacological effects of menthol have not been widely studied. Mentholated cigarette smoking may increase the risk for lung cancer. Many African American smokers smoke mentholated cigarettes, and African Americans have a significantly higher incidence of lung cancer as compared with whites. There may be a relationship between the incidence of lung cancer and the type of cigarette smoked because the use of mentholated cigarettes by white smokers is significantly less and the incidence of lung cancer is less. The mechanism whereby (-)-menthol could increase the health risk of smoking is not known. The results of our in vitro studies herein show that (-)-menthol and synthetic congeners inhibit the microsomal oxidation of nicotine to cotinine and the P450 2A6-mediated 7-hydroxylation of coumarin. Replacement of the alcohol oxygen atom of menthol with other heteroatoms increased the potency of P450 2A6 inhibition. Thus, the K(i) value of (-)-menthol for inhibition of microsomal nicotine oxidation was 69.7 micro M but neomenthyl thiol possesses a K(i) value of 13.8 micro M. Menthylamine inhibited nicotine oxidation with a K(i) value of 49.8 micro M, but its hydroxylamine derivative gave an IC(50) value of 2.2 micro M. A series of 16 menthol derivatives and putative metabolites were procured or chemically synthesized and tested as inhibitors of P450 2A6. While highly potent inhibition of P450 2A6 was not observed for the menthol analogues examined, it is nevertheless possible that smoking mentholated cigarettes leads to inhibition of nicotine metabolism and allows the smoker to achieve a certain elevated dose of nicotine each day. This may be another example of self-medication to obtain the desired effect of nicotine.     

Inhibition of histamine-N-methyltransferase (HNMT) by fragments of 9-amino-1,2,3,4-tetrahydroacridine (tacrine) and by beta-carbolines.

Histamine-N-methyltransferase (HNMT), the major enzyme for the metabolism of histamine in rat brain, is potently inhibited by 9-amino-1,2,3,4-tetrahydroacridine (tacrine). Structural fragments of tacrine were less potent inhibitors of rat brain HNMT than was tacrine itself. Harmaline and a number of other beta-carbolines inhibited HNMT with IC50 values in the range of 1-10 microM. HNMT inhibition by harmaline was competitive with respect to both substrates, S-adenosylmethionine and histamine (Ki = 1.4 microM). These findings are discussed in the context of mechanisms for HNMT inhibition.