Significant Killing Effect Research Articles

Efficacy of a chlorocresol-based disinfectant product on Toxocara canis eggs.

Toxocara canis is a common parasite of dogs and can cause zoonotic toxocariasis in humans. As a part of control programs for this agent, optimized hygiene including chemical disinfection is considered essential in the prevention and control of zoonotic toxocariasis in humans. However, commonly used disinfectants at present mostly fail to inhibit the embryogenesis and viability of T. canis eggs. To this effect, the present study was designed to evaluate the effect of a chlorocresol-based disinfectant product Neopredisan®135-1 (NP) on embryonic development of T. canis eggs in vitro and to investigate the infectivity of exposed eggs by assessing larval establishment in a mouse model. Under in vitro conditions, NP at a final concentration of 0.25, 0.50, 1, 2, or 4% all exhibited significant killing effect on T. canis embryogenesis compared with the control eggs (P < 0.05), regardless of contact times (30, 60, 90, or 120min). Such killing activity increased in a concentration- and time-dependent manner, with a maximum killing efficacy of 95.81% at 4% concentration and 120min exposure time. Comparisons between low and high concentrations and between short and long contact times concluded that a protocol using the 1% concentration of NP with a 90-min contact could be the most suitable for practical application. Additionally, the lower larval recovery in mice inoculated with eggs treated by either 0.25 or 0.5% NP than that from their corresponding controls (P < 0.05) verified once again that NP had an adverse impact on the larval development of T. canis eggs even at a low concentration. To the best of our knowledge, this is the first study to report the effect of the chlorocresol-based disinfectant NP on the embryonation and larval development of T. canis eggs, and the results presented here would contribute to environmental clearance and control of toxocariasis by providing an alternative disinfectant resource. However, it is highlighted that the clearance of the novel and existing sources of infection including larvated eggs in places treated with NP is not guaranteed and therefore continuous monitoring and additional disinfection are still required.

Dihydroartemisinin-Loaded Magnetic Nanoparticles for Enhanced Chemodynamic Therapy.

Recently, chemodynamic therapy (CDT) has represented a new approach for cancer treatment with low toxicity and side effects. Nonetheless, it has been a challenge to improve the therapeutic effect through increasing the amount of reactive oxygen species (ROS). Herein, we increased the amount of ROS agents in the Fenton-like reaction by loading dihydroartemisinin (DHA) which was an artemisinin (ART) derivative containing peroxide groups, into magnetic nanoparticles (MNP), thereby improving the therapeutic effect of CDT. Blank MNP were almost non-cytotoxic, whereas three MNP loading ART-based drugs, MNP-ART, MNP-DHA, and MNP-artesunate (MNP-AS), all showed significant killing effect on breast cancer cells (MCF-7 cells), in which MNP-DHA were the most potent. What’s more, the MNP-DHA showed high toxicity to drug-resistant breast cancer cells (MCF-7/ADR cells), demonstrating its ability to overcome multidrug resistance (MDR). The study revealed that MNP could produce ferrous ions under the acidic condition of tumor microenvironment, which catalyzed DHA to produce large amounts of ROS, leading to cell death. Further experiments also showed that the MNP-DHA had significant inhibitory effect on another two aggressive breast cancer cell lines (MDA-MB-231 and MDA-MB-453 cells), which indicated that the great potential of MNP-DHA for the treatment of intractable breast cancers.

Chemovirotherapy of Lung Squamous Cell Carcinoma by Combining Oncolytic Adenovirus With Gemcitabine.

Oncolytic virotherapy is emerging as an important agent in cancer treatment. In a previous study, we designed and constructed Ad-Apoptin-hTERTp-E1a (Ad-VT), a dual cancer-selective anti-tumor recombinant adenovirus. In this study, crystal violet staining and WST-1 assays showed that Ad-VT has a significant tumor killing effect in a time and dose dependent manner. The combination of Ad-VT (10 MOI) and gemcitabine (10 nM) significantly inhibited NCI-H226 cells, but did not increase the killing effect of gemcitabine on human normal bronchial epithelial cells BEAS-2B. Hoechst, JC-1 and Annexin V experiments demonstrated that the combination of Ad-VT and gemcitabine mainly inhibited NCI-H226 cell proliferation by inducing apoptosis (mitochondrial pathway). The combination also significantly inhibited the migration and invasion abilities of NCI-H226 cells. In vivo, Ad-VT in combination with low-dose gemcitabine could effectively inhibit tumor growth and prolong survival of mice. Ad-VT has the characteristics of tumor-selective replication and killing, in vitro and in vivo. The combined application of Ad-VT and gemcitabine has a synergistic effect, which can increase the anti-tumor effect and reduce the toxicity of chemotherapy drugs, indicating that Ad-VT has a potential clinical value in the treatment of lung squamous cell carcinoma.

Candida albicans Biofilm Heterogeneity and Tolerance of Clinical Isolates: Implications for Secondary Endodontic Infections.

Aim: Endodontic infections are caused by the invasion of various microorganisms into the root canal system. Candida albicans is a biofilm forming yeast and the most prevalent eukaryotic microorganism in endodontic infections. In this study we investigated the ability of C. albicans to tolerate treatment with standard endodontic irrigants NaOCl (sodium hypochlorite), ethylenediaminetetraacetic acid (EDTA) and a combination thereof. We hypothesized that biofilm formed from a panel of clinical isolates differentially tolerate disinfectant regimens, and this may have implications for secondary endodontic infections. Methodology: Mature C. albicans biofilms were formed from 30 laboratory and oral clinical isolates and treated with either 3% NaOCl, 17% EDTA or a sequential treatment of 3% NaOCl followed by 17% EDTA for 5 min. Biofilms were then washed, media replenished and cells reincubated for an additional 24, 48 and 72 h at 37 °C. Regrowth was quantified using metabolic reduction, electrical impedance, biofilm biomass and microscopy at 0, 24, 48 and 72 h. Results: Microscopic analysis and viability readings revealed a significant initial killing effect by NaOCl, followed by a time dependent significant regrowth of C. albicans, but with inter-strain variability. In contrast to NaOCl, there was a continuous reduction in viability after EDTA treatment. Moreover, EDTA significantly inhibited regrowth after NaOCl treatment, though viable cells were still observed. Conclusions: Our results indicate that different C. albicans biofilm phenotypes grown in a non-complex surface topography have the potential to differentially tolerate standard endodontic irrigation protocols. This is the first study to report a strain dependent impact on efficacy of endodontic irrigants. Its suggested that within the complex topography of the root canal, a more difficult antimicrobial challenge, that existing endodontic irrigant regimens permit cells to regrow and drive secondary infections.

Characterization of synergistic antibacterial effect of silver nanoparticles and ebselen

The emerging and spreading of multi-drug resistant (MDR) bacteria have been becoming one of the most severe threats to human health. Enhancing oxidative stress as mimicking immune system was considered as a potential strategy to fight against infection of MDR bacteria. In this study, we investigated the antibacterial efficiency of such a strategy which combines silver nanoparticles (AgNPs) with ebselen. The results showed that AgNPs and ebselen combination had significant synergistic killing effects both on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in vitro, including model strains of China Veterinary Culture Collection and MDR clinical isolates, which is similar as the combination of silver ion and ebselen. AgNPs exhibited to be a strong inhibitor of bacterial thioredoxin reductase, same as a free silver ion. Ebselen mitigated the cytotoxicity of AgNPs to HeLa cells. However, in a bacteria-cell coexistence condition, the synergistic bactericidal effect was only observed on S. aureus (p<.05), while the temporary synergistic inhibitory effect on E. coli within 4 hours treatment (p<.01). In mice infection model, a combination of AgNPs and ebselen did not increase protection against the challenge of clinical E. coli CQ10 strain. Our data demonstrated that AgNPs and ebselen combination may be a promising strategy to fight against the increasingly MDR bacteria targeting bacterial thiol redox system.

3D printed zirconia ceramic hip joint with precise structure and broad-spectrum antibacterial properties.

Background: Nowadays, zirconia ceramic implants are widely used as a kind of hip prosthesis material because of their excellent biocompatibility and long-term wear resistance. However, the hip joint is one of the major joints with complex 3D morphological structure and greatly individual differences, which usually causes great material waste during the process of surgical selection of prosthesis.Methods: In this paper, by combining ceramic 3D printing technology with antibacterial nano-modification, zirconia ceramic implant material was obtained with precise 3D structure and effective antibacterial properties. Among which, two technical problems (fragile and sintering induced irregular shrinkage) of 3D printed ceramics were effectively minimized by optimizing the reaction conditions and selective area inversing compensation. Through in vivo and in vitro experiments, it was confirmed that the as prepared hip prosthesis could precisely matched the corresponding parts, which also exhibited good biocompatibility and impressive antibacterial activities.Results: 1) Two inherent technical problems (fragile and sintering induced irregular shrinkage) of 3D printed ceramics were effectively minimized by optimizing the reaction conditions and selective area inversing compensation. 2) It could be seen that the surface of the ZrO2 material was covered with a layer of ZnO nano-particles. A universal testing machine was used to measure the tensile, bending and compression experiments of ceramic samples. It could be found that the proposed ZnO modification had no significant effect on the mechanical properties of ZrO2 ceramics. 3) According to the plate counting results, ceramics modified with ZnO exhibited significantly higher antibacterial efficiency than pure ZrO2 ceramics, the ZrO2-ZnO ceramics had a significant killing effect 8 hours. 4) The removed implants and the tissue surrounding the implant were subjected to HE staining. For ZrO2-ZnO ceramics, inflammation was slight, while for pure ZrO2 ceramics, the inflammatory response could be seen that the antibacterial rate of the ZrO2-ZnO ceramics was significantly better than that of the pure ZrO2 ceramics group. 5) It could be seen that the cytotoxicity did not increase proportionally with the increase of concentration, all of viability were still above 80%. This suggested that our materials were safe and could be applied as a type of potential biomaterial in the future. 6) Further animal studies demonstrated that the implant was in good position without dislocation. This resulted implied that the proposed method can achieve accurate 3D printing preparation of ceramic joints. In addition, the femurs and surrounding muscles around the implant were then sectioned and HE stained. Results of muscle tissue sections further showed no significant tissue abnormalities, and the growth of new bone tissue was observed in the sections of bone tissue.Conclusion: 1) The ceramic 3D printing technology combined with antibacterial nano-modification can quickly customize the ideal implant material with precise structure, wear-resistant and effective antibacterial properties. 2) Two inherent technical problems (fragile and sintering induced irregular shrinkage) of 3D printed ceramics were effectively minimized by optimizing the reaction conditions and selective area inversing compensation. 3) ZnO nano-materials were modified on the ceramic surface, which could effectively killing pathogenic bacteria.

Abstract 305: Combination analysis for the potential chemomodulatory effects of mansorin-A and its naphthoquinone derivative (mansonone-G) to 5-fluorouracil against liver cancer cells

Abstract Mansorin-A and its naphthoquinone derivative, mansonone-G are naturally occurring compounds isolated from the trunk of Mansonia gagei, family Sterculariaceae. In a previous work, they showed potential anticancer effects besides their antioxidant properties. Herein, we investigated their potential chemomodulatory effects to 5-fluorouracil (5FU) against two liver cancer cell lines (Huh7 and HepG2). Despite the significant cell killing effect of 5FU to Huh7 and HepG2 cells (IC50’s of 2.6±0.2 µM and 0.82 ± 0.2 µM, respectively), the resistance fractions of both cell lines were considerably high (53.2±1.7% and 39.4±2.7%, respectively). Mansorin-A and mansonone-G induced moderate cytotoxicity against both liver cancer cell lines with IC50’s ranging from 25.8±3.2 µM to 36.3±2.7 µM. In Huh7, mansorin-A and mansonone-G synergized cell killing effect of 5FU and decreased its IC50’s to 0.9±0.1 and 1.0±2.1 µM, respectively. On the other hand, mansorin-A and mansonone-G showed apparent antagonism by combination with 5FU (CI values of 6.2 and 9.4, respectively) against HepG2 cells. However, both compounds significantly abolished the resistant fraction of HepG2 to 5FU (3.3±0.3% and 8.4 ± 2.6%, respectively). Further investigation showed that mansonone-G alone showed antiproliferative effect and arrested cells in G0/G1-phase. Both mansorin-A and mansonone-G enhanced 5FU induced cell cycle arrest at G0/G1-phase with reciprocal decrease in cells entering S-phase and G2/M-phase. In addition, apoptosis due to 5FU treatment alone and in combination with mansorin-A and mansonone-G was assessed using annexin-V/FITC staining coupled with flowcytometry. Yet, both mansorin-A and mansonone-G enhanced 5FU induced apoptotic effect against liver cancer cells. Mansonone-G alone induced significant apoptosis in both HepG2 and Huh7 cells. Besides apoptosis, autophagy induction properties of mansorin-A and mansonone-G were assessed using flowcytometry after acridine orange staining. Mansonone-G aborted autophagy in both HepG2 and Huh7 cell lines and indirectly forced liver cancer cells to undergo apoptotic cell death. On the other hand, both mansorin-A and mansonone-G significantly induced the production of reactive oxygen species within both HepG2 and Huh-7 cells which might directly induce apoptosis in both cell lines. In conclusion, mansonone-G and to a lesser extent mansorin-A, potentiated the anticancer properties of 5FU against liver cancer cells via potentiating its cell cycle arrest and potentiating its apoptotic influence due to ROS production. Citation Format: Hanadi G. Aljohani, Gehan A. Hegazy, Ali M. El-Halawany, Aliaa A. Alamoudi, Ghada M.A. Agabnoor, Ahmed M. Al-Abd. Combination analysis for the potential chemomodulatory effects of mansorin-A and its naphthoquinone derivative (mansonone-G) to 5-fluorouracil against liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 305.

Nitric oxide releasing two-part creams containing S-nitrosoglutathione and zinc oxide for potential topical antimicrobial applications

Currently, most antimicrobial topical treatments utilize antibiotics to prevent or treat infection at a wound site. However, with the ongoing evolution of multi-drug resistant bacterial strains, there is a high demand for alternative antimicrobial treatments. Nitric oxide (NO) is an endogenous gas molecule with potent antimicrobial activity, which is effective against a wide variety of bacterial strains. In this study, the potential for creating NO releasing creams containing the naturally occurring NO carrier, S-nitrosoglutathione (GSNO), are characterized and evaluated. GSNO is shown to have prolonged stability (>300 days) when mixed and stored within Vaseline at 24 °C. Further, enhanced proliferation of NO from GSNO using zinc oxide nanoparticles (ZnO) is demonstrated. Triggering NO release from the GSNO/Vaseline mixture using a commercial zinc oxide-containing cream exhibits first-order NO release kinetics with the highest %NO release over the first 6 h. Significant killing effects against S. aureus, S. epidermidis, and P. aeruginosa are demonstrated for the GSNO/Vaseline/ZnO cream mixtures in a proportional manner dependent upon the concentration of GSNO in the final mixture.

Enhancement Effect of Shark Cartilage Extract on Treatment of Leishmania infantum with Artemisinin and Glucantime and Evaluation of killing Factors and Apoptosis in-vitro Condition.

In this study we examined enhancement effects of Artemisinin plus Glucantime and shark cartilage extract on promastigotes and amastigotes of L.infantum in in-vitro condition.The toxicity of artemisinin, glucantime, and shark cartilage extract on the L. infantum promastigotes and amastigote-infected macrophages was evaluated using MTT assay. The role of these drugs inducing apoptosis in promastigotes, un- infected, and parasite- infected macrophages was also studied. Using promastigote assay, IC50 values of artemisinin and glucantime as standalone drugs as well as in combination were obtained to be 50, 400, and 100µg/mL respectively. The flow cytometry analysis of apoptotic promastigotes stained with Annexin-V FITC staining showed that artemisinin, glucantime, artemisinin plus glucantime, artemisinin plus shark cartilage extract, and shark cartilage extract alone applied at their IC50 concentrations resulted in 53.5%, 73.92%, 64.46%, 49.9%, and 47.34% apoptosis respectively. The results of MTT assay indicated that cytotoxicity of artemisinin, glucantime, artemisinin plus glucantime, shark cartilage plus artemisinin, and shark cartilage in infected macrophages after 72h was 75%, 84%, 82%, 30%, and 3% respectively. In un- infected macrophages, cytotoxicity of Artemisinin, Glucantime, Artemisinin plus Glucantime and shark cartilage was 15%, 31%, 21%, 2%, and 0% respectively.This study suggests that artemisinin, glucantime, artemisinin plus glucantime, and shark cartilage extract have significant killing effects on promastigotes and amastigotes. Also, it proved that artimisinin alone and in combination with glucantime and shark cartilage extract has little toxic effect on macrophages, but could induce apoptosis in L.infantum promastigotes and amastigote-infected macrophages. Thus, these chemicals can be used as alternative drugs for in-vivo studies.

Enhanced efficacy of chemo-photodynamic therapy of rhabdomyosarcoma cells by using vitamin K3 as a neoadjuvant agent

Nowadays combination of various therapeutic modalities is an attractive strategy in cancer treatment. In order to develop more efficient combinational therapy, we investigated the effect of Vitamin K3 (menadione) to enhance the cytotoxic action of chemo-photodynamic therapy against Rhabdomyosarcoma (RD) cell line, using Photosense as a photosensitizer and different chemo therapeutic agents. RD cell line was cultured for the evaluation of efficacy in the presence and absence of vitamin K3 with other chemotherapeutic agents. Diode laser (λ = 635 nm) was used for initializing the photodynamic action of Photosense mediated photodynamic therapy (PDT). Cytotoxicity was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Pre-treatment for 24 h to RD cells with K3 significantly potentiates sensitivity of chemotherapy. Chemotherapeutic agents and K3 showed significant killing effect when combined with PDT, at very low doses. These results suggest that this procedure could be the basis for an improved chemo-PDT protocol for cancer control.

Targeting Oncoprotein Translation with Rocaglates in MYC-Driven Lymphoma

Background: c-MYC (MYC) is commonly dysregulated in aggressive B cell lymphomas. MYC associated lymphoma, especially Double Hit lymphoma (DHL) and Double-Expression Lymphoma (DEL) which are characterized by MYC and BCL2 dual overexpression usually present with the inferior outcome as rapid disease progression and poor response to standard chemotherapy regimen. Nevertheless, MYC is considered as an “undruggable” target and targeting strategies such as suppressing MYC transcription by bromodomain (BRD)-4 inhibitors have been widely investigated in both preclinical models and clinical trials. However, increasing evidence has shown that lymphoma cells displayed a wide range of resistance to BRD-4 inhibition, due to transcription adaptation or kinome reprogramming. Hence, alternative approaches for suppressing MYC or its function are urgently needed. Strategies directed against oncoprotein translation may efficiently repress key oncoproteins regardless of the abundant MYC mRNA due to genetic aberrations and secondary transcription up-regulated by MYC. Rocaglate is a class of natural products derived from plants of the Aglaia genus that have been demonstrated to potently inhibit protein translation initiation via eIF4A. The use of rocaglates for anti-cancer treatment was limited due to the scarcity and instability of these natural products e.g. Silvestrol. Recent chemical modification and screening studies have unveiled a few synthetic rocaglates that are more potent than Silvestrol such as SDS-1-021-(−), which unlocked the potential use of rocaglates for clinical applications.Methods and Results: To probe effective reagents for MYC-driven lymphoma, a screening of 50 drugs targeting common oncogenic pathways and tumorigenic machinery was performed in two isogenic B-lymphoma lines. Several protein translation inhibitors, such as mTOR kinase inhibitors (TORKi), and eIF4A inhibitor Silvestrol were identified as the most potent drugs in all of the tested cells. Further, we found that Silvestrol but not TORKi efficiently repressed MYC protein translation in MYC-driven B lymphoma cells, whereas neither of them inhibited BCL2 expression. Moreover, we demonstrated that eIF4E knockdown or eIF4E/eIF4G disruptor Briciclib did not significantly affect MYC expression, whereas eIF4A inhibitor hippuristanol and rocaglates derivate SDS-1-021-(−) diminished MYC expression similar to that observed in Silvestrol treated cells. By using dual luciferase assay, we demonstrated that rocaglates had stronger cap-dependent and IRES translation inhibition than TORKi in lymphoma cells. Next, by native RNA immunoprecipitation and siRNA knocking down, we found that rocaglates repressed MYC translation not only via eIF4A1 but also via eIF4A2, however, with different underlying mechanisms. Furthermore, to explore the molecular targets of rocaglates treatment in B cell lymphoma, we performed TMT-Mass Spectrometry which identified multiple oncoproteins including NEK2, MYC, MCL1, TCF3, BCL6, PLK1, AURKA, and WEE1 were significantly down-regulated by SDS-1-021-(−) treatment. Finally, we demonstrated that SDS-1-021-(−) is highly potent as a single agent and synergized with ABT199 at a low dose (0.2mg/kg) in PDX models with DHL/DEL.Brief summary: Our pre-clinical study provided strong evidence that rocaglates but not TORKi efficiently suppress MYC protein translation because 1). Rocaglates exhibit strong inhibition on both Cap- and IRES-dependent translation, 2). Rocaglates decrease PLK1 and AURKA/B thus destabilizing MYC protein. The synthetic rocaglate SDS-1-021-(−) is a potent agent that exhibits significant synergistic killing effect with ABT199 on DHL/DEL cells in the pre-clinical animal study. [Display omitted] DisclosuresLunning:TG Therapeutics: Consultancy; AbbVie: Consultancy; Genentech: Consultancy; Astra-Zeneca: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Gilead: Consultancy; Spectrum: Consultancy; Genentech: Consultancy; Seattle Genetics: Consultancy; Portola: Consultancy; Kite: Consultancy; Juno: Consultancy; Janssen: Consultancy; Verastem: Consultancy. Vose:Novartis: Honoraria, Research Funding; Epizyme: Honoraria; Incyte Corp.: Research Funding; Bristol Myers Squibb: Research Funding; Kite Pharma: Research Funding; Merck Sharp & Dohme Corp.: Research Funding; Abbvie: Honoraria; Legend Pharmaceuticals: Honoraria; Seattle Genetics, Inc.: Research Funding; Acerta Pharma: Research Funding; Celgene: Research Funding; Roche: Honoraria.

PEGylated liposome-encapsulated rhenium-188 radiopharmaceutical inhibits proliferation and epithelial\u2013mesenchymal transition of human head and neck cancer cells in vivo with repeated therapy

Human head and neck squamous cell carcinoma (HNSCC) is usually treated with chemoradiotherapy, but the therapeutic efficacy could be hampered by intrinsic radioresistance and early relapse. Repeated administrations of rhenium-188 (188Re)-conjugated radiopharmaceutical has been reported to escalate the radiation doses for better control of advanced human cancers. Here we found that high dosage of 188Re-liposome, the liposome-encapsulated 188Re nanoparticles exhibited significant killing effects on HNSCC FaDu cells and SAS cells but not on OECM-1 cells. To investigate the biological and pharmaceutical responses of high 188Re-liposomal dosage in vivo, repeated doses of 188Re-liposome was injected into the orthotopic tumor model. FaDu cells harboring luciferase reporter genes were implanted in the buccal positions of nude mice followed by intravenous injection of 188Re-liposome. The Cerenkov luminescence imaging (CLI) was performed to demonstrate an increased accumulation of 188Re-liposome in the tumor lesion of nude mice with repeated doses compared to a single dose. Repeated doses also enhanced tumor growth delay and elongated the survival of tumor-bearing mice. These observations were associated with significant loss of Ki-67 proliferative marker and epithelial–mesenchymal transition (EMT) markers in excised tumor cells. The body weights of mice were not significantly changed using different doses of 188Re-liposome, yet repeated doses led to lower blood counts than a single dose. Furthermore, the pharmacokinetic analysis showed that the internal circulation of repeated 188Re-liposomal therapy was elongated. The biodistribution analysis also demonstrated that accumulations of 188Re-liposome in tumor lesions and bone marrow were increased using repeated doses. The absorbed dose of repeated doses over a single dose was about twofold estimated for a 1 g tumor. Together, these data suggest that the radiopharmacotherapy of 188Re-liposome can enhance tumor suppression, survival extension, and internal circulation without acute toxicity using repeated administrations.

Regulation of Enterococcus faecalis Biofilm Formation and Quorum Sensing Related Virulence Factors with Ultra-low Dose Reactive Species Produced by Plasma Activated Water

Enterococcus faecalis (E. faecalis) is the species which can cause life-threatening infections in human beings and exhibit highly antibacterial resistance in the nosocomial environment. The purpose of this study was to evaluate the inhibition effect and the mechanisms of ultra-low dose reactive oxygen species produced by plasma activated water (PAW) to the E. faecalis biofilm. The bactericidal effect of E. faecalis planktonic growth was evaluated to find out the maximum processing time. The level of intracellular reactive oxygen species (ROS) and extracellular hydrogen peroxide (H2O2) produced by the PAW without significant killing effect were defined as ultra-low. Then the pre-treated bacteria suspension by the PAW were incubated with brain heart infusion (BHI) broth for 6 h, 12 h, 24 h and 48 h. The biofilm development was evaluated with 96-well polystyrene method (OD570) and confocal laser scanning microscopy (CLSM). Meanwhile, total organic carbon (TOC) was measured with a Shimadzu TOC-L analyzer. The quorum sensing related virulence genes, cylR1, cylA, gelE and sprE, were evaluated with real-time PCR. The PAW did not have significant killing effect within 30 s and the maximum corresponding fluroscence intensity of ROS is under 57.33 ± 0.58 and the concentration of H2O2 is 19.87 ± 2.01 μmol/L. The OD570, CLSM results and TOC results indicated that the biofilm development was effectively inhibited with PAW treatment. Four quorum sensing related virulence genes were all down-regulated. In conclusion, the PAW treatment has potential application to control E. faecalis biofilm development and reduce quorum sensing related virulence genes expression in vitro.

Tumor hypoxia directed multimodal nanotherapy for overcoming drug resistance in renal cell carcinoma and reprogramming macrophages

Drug resistance is one of the significant clinical burden in renal cell carcinoma (RCC). The development of drug resistance is attributed to many factors, including impairment of apoptosis, elevation of carbonic anhydrase IX (CA IX, a marker of tumor hypoxia), and infiltration of tumorigenic immune cells. To alleviate the drug resistance, we have used Sorafenib (Sor) in combination with tumor hypoxia directed nanoparticle (NP) loaded with a new class of apoptosis inducer, CFM 4.16 (C4.16), namely CA IX-C4.16. The NP is designed to selectively deliver the payload to the hypoxic tumor (core), provoke superior cell death in parental (WT) and Everolimus-resistant (Evr-res) RCC and selectively downmodulate tumorigenic M2-macrophage. Copper-free ‘click’ chemistry was utilized for conjugating SMA-TPGS with Acetazolamide (ATZ, a CA IX-specific targeting ligand). The NP was further tagged with a clinically approved NIR dye (S0456) for evaluating hypoxic tumor core penetration and organ distribution. Imaging of tumor spheroid treated with NIR dye-labeled CA IX-SMA-TPGS revealed remarkable tumor core penetration that was modulated by CA IX-mediated targeting in hypoxic-A498 RCC cells. The significant cell killing effect with synergistic combination index (CI) of CA IX-C4.16 and Sor treatment suggests efficient reversal of Evr-resistance in A498 cells. The CA IX directed nanoplatform in combination with Sor has shown multiple benefits in overcoming drug resistance through (i) inhibition of p-AKT, (ii) upregulation of tumoricidal M1 macrophages resulting in induction of caspase 3/7 mediated apoptosis of Evr-res A498 cells in macrophage-RCC co-culturing condition, (iii) significant in vitro and in vivo Evr-res A498 tumor growth inhibition as compared to individual therapy, and (iv) untraceable liver and kidney toxicity in mice. Near-infrared (NIR) imaging of CA IX-SMA-TPGS-S0456 in Evr-res A498 RCC model exhibited significant accumulation of CA IX-oligomer in tumor core with >3-fold higher tumor uptake as compared to control. In conclusion, this proof-of-concept study demonstrates versatile tumor hypoxia directed nanoplatform that can work in synergy with existing drugs for reversing drug-resistance in RCC accompanied with re-education of tumor-associated macrophages, that could be applied universally for several hypoxic tumors.

In vitro expansion of γδT cells and their killing effect on different tumor cells

Objective To study the killing effect of γδT cells on different tumor cells by inducing and expanding γδT cells in vitro. Methods Collected the peripheral venous blood of 20 healthy volunteers recruited at Xinqiao Hospital of Third Military Medical University from January to March 2018. Peripheral blood mononuclear cells (PBMCs) were induced and expanded using zoledronic acid combined with interleukin-2 (IL-2) to obtain γδT cells. The cell purity was detected on day 0, 7, 10, 14 and 16 using flow cytometry. The killing activity was detected by CCK-8 kit. Cells cultured on the 14th day were used as effector cells, and breast cancer cell BCap-37 and liver cancer cell Bel-7402 were used as target cells, and the cell killing activity was detected at the effective target ratio (E∶T) of 5∶1, 10∶1 and 20∶1 respectively. Results The purity of γδT cells were respectively (3.35±1.32)%, (50.76±5.76)%, (80.43±4.53)%, (90.56±3.34)%, (89.54±4.42)% on day 0, 7, 10, 14 , 16, with significant difference (F=18.431, P=0.012). The efficiency of γδT cells killing BCap-37 tumor cells were (31.3±2.0)% (E∶T=5∶1), (48.7±1.6)% (E∶T=10∶1), (71.3±2.4)% (E∶T=20∶1) respectively, with significant difference (F=16.724, P=0.016), further comparison between each two groups: 10∶1 vs. 5∶1 (P=0.013), 20∶1 vs. 5∶1 (P=0.017), 20∶1 vs. 10∶1 (P=0.011). The killing rate increased with the increase of target ratio. The efficiency of γδT cells killing Bel-7402 tumor cells were (34.5±2.0)% (E∶T=5∶1), (52.4±1.9)% (E∶T=10∶1), (74.5±1.6)% (E∶T=20∶1) respectively, with significant difference (F=18.253, P=0.013), further comparison between each two groups: 10∶1 vs. 5∶1 (P=0.015), 20∶1 vs. 5∶1 (P=0.012), 20∶1 vs. 10∶1 (P=0.015). The killing rate increased with the increase of target ratio. Conclusion We can obtain high purity γδT cells using zoledronic acid combined with IL-2 induced PBMCs, and the amplified γδT cells have significant killing effect on BCap-37 and Bel-7402 tumor cells. Key words: γδT cells; Zoledronic acid; Killing function

Improving the specific antitumor efficacy of ONC by fusion with N-terminal domain of transferrin.

Onconase (ONC) as a novel anti-tumor drug has a significant killing effect on a variety of tumor cells. Drug delivery system mediated by transferrin (TF) and TF receptor (TfR), which can significantly increase the amount of drug uptake in the tumor cells, enhance the initiative target efficiency of drugs and reduce its toxic side effects. It has been widely used in drug delivery and clinical trials. In this study, the rONC-TFn was expressed in Escherichia coli by linking ONC with the N-terminal domain of TF (TFn). ELISA and competitive binding analysis demonstrated that rONC-TFn can bind to TfR. The rONC-TFn protein showed much higher cytotoxicity to the cultured HepG2 and Hela cells than rONC. These results suggested that the N-terminal domain protein of TF promoted the tumor targeting of ONC, and thus the rONC-TFn fusion protein may be further developed as a potential targeted anti-tumor drug.

Reduction/temperature/pH multi-stimuli responsive core cross-linked polypeptide hybrid micelles for triggered and intracellular drug release

The high toxicity, poor stability, premature drug release, and lack of intracellular stimuli responsibility of current polymeric micelles still hinder them for potential clinical applications. To address these challenges, a novel type of multi-stimuli responsive, core cross-linked polypeptide hybrid micelles (CCMs) was developed for triggered anticancer drug delivery in tumor microenvironment. The CCMs was prepared via free radical copolymerization by using N,N'-methylene-bis-acylamide (BACy) as the cross-linking agent, 2,2-azobisisobutyronitrile (AIBN) as the initiator, where poly (γ-benzyl-L-glutamate) (PBLG) and N-isopropylacrylamide (NIPPAM) as comonomers. The doxorubicin (DOX) was then introduced into the CCMs by hydrazone bond to prepare the drug-incorporated core cross-linked micelles (CCMs-DOX). By the experimental results, the CCMs showed reduction responsibility due to the degradable disulfide bond in the polymer network. The hydrazone bond can be broken under acidic condition causing a controllable drug release for CCMs-DOX. Compared to only 7.7% DOX release under pH 7.4 at 37°C, a much higher DOX release rate up to 85.3% was observed under 10 mM GSH (pH 5.0, 42°C). In vitro cell assays showed that the blank CCMs showed almost no toxicity against HUVEC cells while the CCMS-DOX exhibited significant cancer cell killing effect. These experimental results suggested that the prepared multi-stimuli responsive polymeric micelles could serve as a smart and promising drug delivery candidate for anti-cancer therapy.

DC-CIK cells derived from ovarian cancer patient menstrual blood activate the TNFR1-ASK1-AIP1 pathway to kill autologous ovarian cancer stem cells.

Ovarian cancer stem cells (OCSCs) are highly carcinogenic and have very strong resistance to traditional chemotherapeutic drugs; therefore, they are an important factor in ovarian cancer metastasis and recurrence. It has been reported that dendritic cell (DC)‐cytokine‐induced killer (CIK) cells have significant killing effects on all cancer cells across many systems including the blood, digestive, respiratory, urinary and reproductive systems. However, whether DC‐CIK cells can selectively kill OCSCs is currently unclear. In this study, we collected ovarian cancer patient menstrual blood (OCPMB) samples to acquire mononuclear cells and isolated DC‐CIK cells in vitro. In addition, autologous CD44+/CD133+ OCSCs were isolated and used as target cells. The experimental results showed that when DC‐CIK cells and OCSCs were mixed and cultured in vitro at ratios of 5:1, 10:1 and 50:1, the DC‐CIK cells killed significant amounts of OCSCs, inhibited their invasion in vitro and promoted their apoptosis. The qPCR and Western blot results showed that DC‐CIK cells stimulated high expression levels and phosphorylation of TNFR1, ASK1, AIP1 and JNK in OCSCs through the release of TNF‐α. After the endogenous TNFR1 gene was knocked out in OCSCs using the CRISPR/Cas9 technology, the killing function of DC‐CIK cells on target OCSCs was significantly attenuated. The results of the analyses of clinical samples suggested that the TNFR1 expression level was negatively correlated with ovarian cancer stage and prognosis. Therefore, we innovatively confirmed that DC‐CIK cells derived from OCPMB could secret TNF‐α to activate the expression of the TNFR1‐ASK1‐AIP1‐JNK pathway in OCSCs and kill autologous OCSCs.

Antimicrobial Effects of β-Lactams on Imipenem-Resistant Ceftazidime-Susceptible Pseudomonas aeruginosa.

We studied the resistance mechanism and antimicrobial effects of β-lactams on imipenem-resistant Pseudomonas aeruginosa isolates that were susceptible to ceftazidime as detected by time-kill curve methods. Among 215 P. aeruginosa isolates from hospitalized patients in eight hospitals in the Republic of Korea, 18 isolates (23.4% of 77 imipenem-resistant isolates) were imipenem resistant and ceftazidime susceptible. Multilocus sequence typing revealed diverse genotypes, which indicated independent emergence. These 18 isolates were negative for carbapenemase genes. All 18 imipenem-resistant ceftazidime-susceptible isolates showed decreased mRNA expression of oprD, and overexpression of mexB was observed in 13 isolates. In contrast, overexpression of ampC, mexD, mexF, or mexY was rarely found. Time-kill curve methods were applied to three selected imipenem-resistant ceftazidime-susceptible isolates at a standard inoculum (5 × 105 CFU/ml) or at a high inoculum (5 × 107 CFU/ml) to evaluate the antimicrobial effects of β-lactams. Inoculum effects were detected for all three β-lactam antibiotics, ceftazidime, cefepime, and piperacillin-tazobactam, against all three isolates. The antibiotics had significant killing effects in the standard inoculum, but no effects in the high inoculum were observed. Our results suggest that β-lactam antibiotics should be used with caution in patients with imipenem-resistant ceftazidime-susceptible P. aeruginosa infection, especially in high-inoculum infections such as endocarditis and osteomyelitis.

Identification of a fluorescent small-molecule enhancer for therapeutic autophagy in colorectal cancer by targeting mitochondrial protein translocase TIM44

ObjectiveAs the modulation of autophagic processes can be therapeutically beneficial to cancer treatment, the identification of novel autophagic enhancers is highly anticipated. However, current autophagy-inducing anticancer agents exert undesired side...