High Levels Of Cytotoxic Activity Research Articles

Acetylation of Oleanolic Acid Dimers as a Method of Synthesis of Powerful Cytotoxic Agents.

Oleanolic acid, a naturally occurring triterpenoid compound, has garnered significant attention in the scientific community due to its diverse pharmacological properties. Continuing our previous work on the synthesis of oleanolic acid dimers (OADs), a simple, economical, and safe acetylation reaction was performed. The newly obtained derivatives (AcOADs, 3a-3n) were purified using two methods. The structures of all acetylated dimers (3a-3n) were determined based on spectral methods (IR, NMR). For all AcOADs (3a-3n), the relationship between the structure and the expected directions of pharmacological activity was determined using a computational method (QSAR computational analysis). All dimers were also tested for their cytotoxic activity on the SKBR-3, SKOV-3, PC-3, and U-87 cancer cell lines. HDF cell line was applied to evaluate the Selectivity Index of the tested compounds. All cytotoxic tests were performed with the application of the MTT assay. Finally, all dimers of oleanolic acid were subjected to DPPH and CUPRAC tests to evaluate their antioxidant activity. The obtained results indicate a very high level of cytotoxic activity (IC50 for most AcOADs below 5.00 µM) and a fairly high level of antioxidant activity (Trolox equivalent in some cases above 0.04 mg/mL).

Oleanolic Acid Dimers with Potential Application in Medicine-Design, Synthesis, Physico-Chemical Characteristics, Cytotoxic and Antioxidant Activity.

The present work aimed to obtain a set of oleanolic acid derivatives with a high level of cytotoxic and antioxidant activities and a low level of toxicity by applying an economical method. Oleanolic acid was alkylated with α,ω-dihalogenoalkane/α,ω-dihalogenoalkene to obtain 14 derivatives of dimer structure. All of the newly obtained compounds were subjected to QSAR computational analysis to evaluate the probability of the occurrence of different types of pharmacological activities depending on the structure of the analysed compound. All dimers were tested for cytotoxicity activity and antioxidant potential. The cytotoxicity was tested on the SKBR-3, SKOV-3, PC-3, and U-87 cancer cell lines with the application of the MTT assay. The HDF cell line was applied to evaluate the tested compounds' Selectivity Index. The antioxidant test was performed with a DPPH assay. Almost all triterpene dimers showed a high level of cytotoxic activity towards selected cancer cell lines, with an IC50 value below 10 µM. The synthesised derivatives of oleanolic acid exhibited varying degrees of antioxidant activity, surpassing that of the natural compound in several instances. Employing the DPPH assay, compounds 2a, 2b, and 2f emerged as promising candidates, demonstrating significantly higher Trolox equivalents and highlighting their potential for pharmaceutical and nutraceutical applications. Joining two oleanolic acid residues through their C-17 carboxyl group using α,ω-dihalogenoalkanes/α,ω-dihalogenoalkenes resulted in the synthesis of highly potent cytotoxic agents with favourable SIs and high levels of antioxidant activity.

Palladium(II) Pincer Complexes of Functionalized Amides with S-Modified Cysteine and Homocysteine Residues: Cytotoxic Activity and Different Aspects of Their Biological Effect on Living Cells.

In the search for potential new metal-based antitumor agents, two series of nonclassical palladium(II) pincer complexes based on functionalized amides with S-modified cysteine and homocysteine residues have been prepared and fully characterized by 1D and 2D NMR (1H, 13C, COSY, HMQC or HSQC, 1H-13C, and 1H-15N HMBC) and IR spectroscopy and, in some cases, X-ray diffraction. Most of the resulting complexes exhibit a high level of cytotoxic activity against several human cancer cell lines, including colon (HCT116), breast (MCF7), and prostate (PC3) cancers. Some of the compounds under consideration are also efficient in both native and doxorubicin-resistant transformed breast cells HBL100, suggesting the prospects for the creation of therapeutic agents based on the related compounds that would be able to overcome drug resistance. An analysis of different aspects of their biological effects on living cells has revealed a remarkable ability of the S-modified derivatives to induce cell apoptosis and efficient cellular uptake of their fluorescein-conjugated counterpart, confirming the high anticancer potential of Pd(II) pincer complexes derived from functionalized amides with S-donor amino acid pendant arms.

Bioactive Potential of Extracts of Labrenzia aggregata Strain USBA 371, a Halophilic Bacterium Isolated from a Terrestrial Source.

Previous studies revealed the potential of Labrenzia aggregata USBA 371 to produce cytotoxic metabolites. This study explores its metabolic diversity and compounds involved in its cytotoxic activity. Extracts from the extracellular fraction of strain USBA 371 showed high levels of cytotoxic activity associated with the production of diketopiperazines (DKPs). We purified two compounds and a mixture of two other compounds from this fraction. Their structures were characterized by 1D and 2D nuclear magnetic resonance (NMR). The purified compounds were evaluated for additional cytotoxic activities. Compound 1 (cyclo (l-Pro-l-Tyr)) showed cytotoxicity to the following cancer cell lines: breast cancer 4T1 (IC50 57.09 ± 2.11 µM), 4T1H17 (IC50 40.38 ± 1.94), MCF-7 (IC50 87.74 ± 2.32 µM), murine melanoma B16 (IC50 80.87 ± 3.67), human uterus sarcoma MES-SA/Dx5 P-pg (−) (IC50 291.32 ± 5.64) and MES-SA/Dx5 P-pg (+) (IC50 225.28 ± 1.23), and murine colon MCA 38 (IC50 29.85 ± 1.55). In order to elucidate the biosynthetic route of the production of DKPs and other secondary metabolites, we sequenced the genome of L. aggregata USBA 371. We found no evidence for biosynthetic pathways associated with cyclodipeptide synthases (CDPSs) or non-ribosomal peptides (NRPS), but based on proteogenomic analysis we suggest that they are produced by proteolytic enzymes. This is the first report in which the cytotoxic effect of cyclo (l-Pro-l-Tyr) produced by an organism of the genus Labrenzia has been evaluated against several cancer cell lines.

Synthesis and antiproliferative evaluation of novel biheterocycles based on coumarin and 2-aminoselenophene-3-carbonitrile unit

A series of novel coumarins with 2-amino-3-cyanoselenophen-5-yl unit on C-3 have been synthesized. These compounds prepared easily at room temperature, in a short time and in high yield. The importance of biheterocyclic units as dominant structural motif of coumarin derivatives has been well recognized. Anti-cancer activity screening on MCF-7 cell line allowed identification of 2-amino-5-(6-bromo-2-oxo-2H-chromen-3-yl)selenophene-3-carbonitrile with the highest level of cytotoxic activity with mean IC50 and cLogP (partition co-efficient) values 10.84 µM and 3.18, respectively. The most radical scavenging compound was also recognized.

Design, Synthesis and Anticancer Activity of Site Specific Short Chain Cationic Peptide

In spite of current progress in treatment methods, cancer is a major source of morbidity and death rate all over the world. Traditional chemotherapeutic agents aim to divide cancerous cells, are often associated with deleterious side effects to healthy cells and tissues. Host defense peptides Cecropin A and B obtained from insects are capable to lyses various types of human cancer cells at peptide concentrations which are not fatal to normal eukaryotic cells. In the present work we have designed short chain α-helical linear and cyclic peptide from cecropin A having same cationic charge, hydrophobicity and helicity. Synthesis of designed novel short chain linear (10) and cyclic compound (12) was accomplished by using solution phase method. All the coupling reactions were carried out by using dicyclohexylcarbodiimide (DCC) as the coupling reagent at room temperature in the presence of N-methylmorpholine (NMM) as the base. The Structure of newly synthesized peptidse were elucidated by 1H-NMR, 13C-NMR, FT-IR, FABMS and elemental analysis data.Cytotoxicity of synthesized compound was tested against Dalton's Lymphoma Ascites (DLA), Ehrlich's Ascites Carcinoma (EAC) and MCF-7 cell lines by using MTT assay and 5-FU as reference compound. From biological assessment,it was found that short chain cyclicpeptide12 showed high level of cytotoxic activity against DLA and EAC cell lines. By utilizing a structure-based rational approach to anticancer peptide design from cecropin A, we were able to develop short chain linear and cyclic peptides having same charge, hydrophobicity and with improved activity. Systematically removing amino acids, we were able to retaining peptide charge and hydrophobicity/hydrophilicity in linear and cyclic peptide which results to optimize the anticancer activity against DLA and EAC cell lines.

Microbial diversity of saline environments: searching for cytotoxic activities

In order to select halophilic microorganisms as a source of compounds with cytotoxic activities, a total of 135 bacterial strains were isolated from water and sediment samples collected from the Zipaquirá salt mine in the Colombian Andes. We determined the cytotoxic effects of 100 crude extracts from 54 selected organisms on the adherent murine mammary cell carcinoma 4T1 and human mammary adenocarcinoma MCF-7 cell lines. These extracts were obtained from strains of Isoptericola, Ornithinimicrobium, Janibacter, Nesterenkonia, Alkalibacterium, Bacillus, Halomonas, Chromohalobacter, Shewanella, Salipiger, Martellela, Oceanibaculum, Caenispirillum and Labrenzia. The extracts of 23 strains showed an IC50 of less than 100 μg mL−1. They were subsequently analyzed by LC/MS allowing dereplication of 20 compounds. The cytotoxic effect was related to a complex mixture of diketopiperazines present in many of the extracts analyzed. The greatest cytotoxic activity against both of the evaluated cell lines was obtained from the chloroform extract of Labrenzia aggregata USBA 371 which had an IC50 < 6 μg mL−1. Other extracts with high levels of cytotoxic activity were obtained from Bacillus sp. (IC50 < 50 μg mL−1) which contained several compounds such as macrolactin L and A, 7-O-succinoylmacrolactin F and iturin. Shewanella chilikensis USBA 344 also showed high levels of cytotoxic activity against both cell lines in the crude extract: an IC50 < 15 μg mL−1 against the 4T1 cell line and an IC50 < 68 μg mL−1 against the MCF-7 cell line. Nesterenkonia sandarakina CG 35, which has an IC50 of 118 µg mL−1 against 4T1, is a producer of diketopiperazines and 1-acetyl-β-carboline. Also, Ornithinimicrobium kibberense CG 24, which has IC50 < 50 μg mL−1, was a producer of diketopiperazines and lagunamycin. Our study demonstrates that these saline environments are habitats of halophilic and halotolerant bacteria that have previously unreported cytotoxic activity.

Abstract 59: Engineering glypican-3 targeting immunotoxins for the treatment of liver cancer

Abstract The expression of glypican-3 (GPC3) in hepatocellular carcinoma offers a target with high tissue specificity and cell signaling implications. A human single domain antibody discovered by phage display technology, HN3, was fused to domain II and domain III of Pseudomonas exotoxin A. This protein (HN3-PE38) showed a high level of cytotoxic activity with a IC50 of 0.4 nM on Hep3B cells, but a relatively low maximum tolerated dose of 0.8 mg/kg in mice. In order to produce an immunotoxin with reduced toxicity, a new version was constructed that removed domain II and seven B cell epitopes from the Pseudomonas toxin. This deimmunized immunotoxin (HN3-mPE24) was shown to have a similar IC50 of 0.2 nM on Hep3B cells. To determine if further deimmunization was possible, three new versions have been generated with T cell epitopes or a series of both B and T cell epitopes removed. These include the HN3-T20 immunotoxin which had six T cell epitopes removed, HN3-T19 with 4 B cell, 4 T cell and 2 shared epitopes, and HN3-M11 with 5 B cell, 4 T cell and 2 shared epitopes. A comparative analysis of these immunotoxins was made using in vitro cell proliferation assays using Hep3B. Both the HN3-T20 (IC50 = 0.6 nM) and HN3-T19 (IC50 = 0.8 nM) immunotoxins had similar activity to HN3-mPE24 (IC50 = 0.7 nM) in a side by side comparison. The HN3-M11 variant had poor cytotoxicity and was excluded from in vivo examination. A Hep3B subcutaneous xenograft model was generated in athymic nude mice and was followed by nine rounds of intravenous immunotoxin treatments. The mPE24, T20 and T19 immunotoxins all showed an increase in average survival rate 70 days (mPE24), 76 days (T20) and 72 days (T19) when compared to the 55 days for PE38 and the 41 days for PBS alone. Additionally, the T20 and the T19 showed a maximum tolerated dose that was similar to that of the mPE24 with dosages as high as 10 mg/kg being well tolerated. This data would suggest that the HN3-T19 immunotoxins has potential clinical applications because it represents the most deimmunized immunotoxin to date. Citation Format: Bryan D. Fleming, Brittany Nixon, Ira Pastan, Mitchell Ho. Engineering glypican-3 targeting immunotoxins for the treatment of liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 59. doi:10.1158/1538-7445.AM2017-59

Polyamidoamine dendrimers with different surface charge as carriers in anticancer drug delivery

Second-generation (G2) polyamidoamine (PAMAM) dendrimers are branched polymers containing 16 surface primary amine groups. Due to their structural properties, these polymers can be used as universal carriers in various drug delivery systems. Amine-terminated PAMAM dendrimers are characterized by a high positive surface charge, leading to effective but nonspecific interactions with negatively charged cell plasmatic membranes. To reduce the nonspecific internalization of PAMAM dendrimers, their primary amine groups are often modified by acetic or succinic anhydrides, polyethylene glycol derivatives and other compounds. In this work, the role of primary amine groups, which are localized on the surface of doxorubicin-conjugated (Dox) dendrimers, was studied with regard to their intracellular distribution and internalization rates using SKOV3 human ovarian adenocarcinoma cells. It was demonstrated that all Dox-labeled G2-derivatives containing different numbers of acetamide groups synthesized in this work show high rates of cellular uptake at 37°С. As expected, the conjugate carrying the maximum number of primary amine groups demonstrated the highest rates of binding and endocytosis. At the same time, the G2-Dox conjugate containing the maximum number of acetamide groups showed colocalization with LAMP2, a marker of lysosomes and late endosomes, as well as the highest level of cytotoxic activity against SKOV3 cells. We conclude that second-generation PAMAM dendrimers are characterized by varied pathways of internalization and intracellular distribution due to the number of primary amine groups on their surface and, as a consequence, a different surface charge.

In vitro cytotoxic, antibacterial, anti-inflammatory and antioxidant activities and phenolic content in wild-grown flowers of common daisy—A medicinal plant

Bellis perennis L. is a medicinal plant in the family Compositae. It has been used as a remedy for wounds, rheumatism, eczema, eye diseases, inflammation and tonsillitis in folk medicine. In the present study, 19 different extracts and two fractions were obtained from wild-grown flowers, leaves and/or in vitro-grown leaves of common daisy by using different solvents and extraction methods. Biological activities of these extracts and fractions were assessed using selected bioassays: cytotoxic activity, disc diffusion assay, radical scavenging activity (DPPH), total phenolic content, oxygen radical absorbing capacity (ORAC) and 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) cell-based assays. The cytotoxic activity of extracts and fractions was investigated against human lung carcinoma (A-549) and colon adenocarcinoma (DLD-1) cells. In vitro-grown leaf extracts showed the highest cytotoxic activity against selected cell lines. Moreover, n-butanol (n-BuOH) and ethyl acetate (EtOAc) fractions of flowers exerted high levels of cytotoxic activity. The MeOH extract and the EtOAc fraction of flowers exhibited broad-spectrum antibacterial activity against Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, and Enterobacter cloacea. The strongest antioxidant activity was found in the EtOAc fraction of flowers with the highest amount of phenolic content and ORAC value. The MeOH extract of flowers showed strong anti-inflammatory activity on RAW 264.7 macrophages. The amount of the chosen 22 phenolic compounds in dichloromethane (DCM), MeOH extracts, n-BuOH and EtOAc fractions of field-grown flowers was detected using LC–ESI–MS/MS. The results of these studies support the potential use of B. perennis for wounds, rheumatism, inflammation, cancer and eye diseases.

Antiproliferative Activity of Niclosamide Against Melanoma and Colorectal Cancer Cells

Studies of the actions of niclosamide against tumor cells and normal human cells (human embryo kidney cells and pulmonary embryo fibroblast cells) showed higher levels of cytotoxic activity against tumor cells, including melanoma (the Mel-8, Mel-10, MS lines) and colorectal cancer (the Caco-2, COLO 320 HSR, and SW827 cells). Niclosamide was shown to have a high level of cytotoxic activity against cells with the multidrug resistance phenotype due to high levels of expression of MDR1 protein on the plasma membrane (the Mel-8 and MCF-7Adr lines). The proportion of tumor stem cells (TSC) was measured by flow cytometry as the proportion of cells forming side populations (SP). Niclosamide was found to act on TSC cells either more effectively (colorectal cancer line SW 837, (decreases in SP fraction size) or with the same effectiveness as the main population of tumor cells (SP fraction size unaltered).

Total Synthesis and Biological Evaluation of Irciniastatin A (a.k.a. Psymberin) and Irciniastatin B.

Irciniastatin A (a.k.a. psymberin) and irciniastatin B are members of the pederin natural product family, which have potent antitumor activity and structural complexity. Herein, we describe a full account of our total synthesis of (+)-irciniastatin A and (-)-irciniastatin B. Our synthesis features the highly regioselective Eu(OTf)3-catalyzed, DTBMP-assisted epoxide ring opening reaction with MeOH, which enabled a concise synthesis of the C1-C6 fragment, extensive use of AZADO (2-azaadamantane N-oxyl) and its related nitroxyl radical/oxoammonium salt-catalyzed alcohol oxidation throughout the synthesis, and a late-stage assembly of C1-C6, C8-C16, and C17-C25 fragments. In addition, for the synthesis of (-)-irciniastatin B, we achieved the C11-selective control of the oxidation stage via regioselective deprotection and AZADO-catalyzed alcohol oxidation. The synthetic irciniastatins showed high levels of cytotoxic activity against mammalian cells. Furthermore, chemical footprinting experiments using synthetic compounds revealed that the binding site of irciniastatins is the E-site of the ribosome.

Hederagenin as a triterpene template for the development of new antitumor compounds

In this study, a series of novel C-28 esters and amides derivatives of hederagenin (He) were designed and synthesized in attempt to develop potent antitumor agents. Their structures were confirmed by MS, IR, 1H NMR and 13C NMR spectroscopic analyses and their cytotoxic activities were screened in SRB assays using a panel of six human cancer cell lines. Although most of the compounds displayed moderate to high levels of cytotoxic activity they were all more potent than the natural product He. The most active compounds had either an ethylpyrimidinyl (27) or an ethylpyrrolidinyl (28) substituent, with EC50 in the range of 1.1–6.5 μM for six human cancer cell lines. Notably, this corresponds to an approximately 30-fold times greater potency than He.

Dual Targeting of Tumor Angiogenesis and Chemotherapy by Endostatin–Cytosine Deaminase–Uracil Phosphoribosyltransferase

Several antiangiogenic drugs targeting VEGF/VEGF receptor (VEGFR) that were approved by the Food and Drug Administration for many cancer types, including colorectal and lung cancer, can effectively reduce tumor growth. However, targeting the VEGF signaling pathway will probably influence the normal function of endothelial cells in maintaining homeostasis and can cause unwanted adverse effects. Indeed, emerging experimental evidence suggests that VEGF-targeting therapy induced less tumor cell-specific cytotoxicity, allowing residual cells to become more resistant and eventually develop a more malignant phenotype. We report an antitumor therapeutic EndoCD fusion protein developed by linking endostatin (Endo) to cytosine deaminase and uracil phosphoribosyltransferase (CD). Specifically, Endo possesses tumor antiangiogenesis activity that targets tumor endothelial cells, followed by CD, which converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the cytotoxic antitumor drug 5-fluorouracil (5-FU) in the local tumor area. Moreover, selective targeting of tumor sites allows an increasing local intratumoral concentration of 5-FU, thus providing high levels of cytotoxic activity. We showed that treatment with EndoCD plus 5-FC, compared with bevacizumab plus 5-FU treatment, significantly increased the 5-FU concentration around tumor sites and suppressed tumor growth and metastasis in human breast and colorectal orthotropic animal models. In addition, in contrast to treatment with bevacizumab/5-FU, EndoCD/5-FC did not induce cardiotoxicity leading to heart failure in mice after long-term treatment. Our results showed that, compared with currently used antiangiogenic drugs, EndoCD possesses potent anticancer activity with virtually no toxic effects and does not increase tumor invasion or metastasis. Together, these findings suggest that EndoCD/5-FC could become an alternative option for future antiangiogenesis therapy.

Higher Liposomal Membrane Fluidity Enhances the in Vitro Antitumor Activity of Folate-Targeted Liposomal Mitoxantrone

The efficacy of folate-targeted liposomal drug delivery has not been fully achieved in part because of the slow release of the encapsulated drugs following uptake of the liposomes by target cells. Since liposomal mitoxantrone (MXN) composed of lipids with high fluidity was reported to achieve strong anticancer effects in vivo, we hypothesized that folate-targeted liposomal MXN uptake via folate receptor (FR)-mediated endocytosis could effectively release drugs into the endosomal compartment. Folate-targeted liposomal MXN was prepared using two lipids with different fluidities. MXN was released slowly from all types of liposome into PBS, indicating that the cellular uptake of MXN was considered to be in the liposomal form. Folate-targeted liposomes with high fluidity exhibited lower cellular uptake of loaded FITC-labeled dextran into FR (+) KB cells, but, when MXN was loaded, higher cytotoxicity than liposomes with lower fluidity. On the other hand, the cellular uptake of non-folate liposomes was not affected by the membrane fluidity, but higher cytotoxicity was observed in liposomal MXN with high fluidity, which suggested a higher rate of release of the drug from the liposomes. High levels of cytotoxic activity were achieved with folate-targeted liposomal MXN though the cellular uptake rate was restricted by selecting liposomes with higher lipid membrane fluidity. This finding provides a new insight into folate-targeted carrier drug delivery.

Індукція апоптозу та некрозу лейкемічних клітин очищеними препаратами IgG сироватки крові мишей, яким тривалий час згодовували мозок великої рогатої худоби

IgG preparations of mice under study were tested for their ability to induce death of cells of L1210 line of murine leukemia. The highest level of cytotoxic activity was found at affecting cells with IgG of mice that were additionally fed with cattle brain. It was shown that the test cells were dying by both apoptosis (typical signs are destruction of DNA, revealed by the method of DNA-comets, condensation and fragmentation of chromatin, discovered by fluorescent microscopy of cells, stained with Hoechst 33342) and necrosis. The latter was estimated taking into account the level of lactate dehydrogenase activity, which appeared in cultural medium of L1210 cells, and by light microscopy after cell staining according to Romanovsky-Gimza.

Myeloid Derived Dendritic Cells Inhibit CD8+ T Cell Responses by Down-Modulating NKG2D and 4–1BB Expression.

NKG2D is constitutively expressed on naïve human CD8+ T cells (CTLs) and is involved in antitumor and antiviral responses as a costimulatory receptor. Down-modulating NKG2D expression from the surface of CTLs may allow tumors to evade anti-tumor host immune reactions. Thus, stable NKG2D expression on CTLs is a potential clinical strategy to counteract tumor-induced immune evasion in cancer patients. Recently, evidence suggests that tolerogenic myeloid derived dendritic cells (MDCs) recruited to the tumor microenvironment are involved in immune evasion against anti-tumor CTL functions. In this study, we investigated the possibility that tolerogenic MDCs may control CTL activities by modulating NKG2D expression. To test this possibility, we developed MDCs in vitro from CD14+ monocytes isolated from human cord blood through a long term culture with M-CSF and IL-4 (Li et al. J Immunol . 174: 4706, 2005 and Blood in press, 2007) and subsequent activation with LPS. We found that the MDCs generated were able to dramatically inhibit CTL killing activity to the target C1R B lymphoma cell line that expressed MHC class I related chains (MIC), a ligand for NKG2D. Most importantly, the CTLs co-cultured with the allogenic MDCs lost expression of NKG2D, which was necessary to induce 4–1BB, another costimulatory receptor known to be important for effector/memory CTL responses. MDCs generated without IL-4 were less potent in down-regulating NKG2D. In contrast to MDCs, prototype DCs developed with GM-CSF and IL-4 rather increased both NKG2D and 4-1BB expression indicating that capacity for down-modulating NKG2D and 4-1BB on CTLs was unique for MDCs. CTLs co-cultured with MDCs became tolerogenic because they failed to respond to CD3 stimulation. A majority of the cells stayed in a Go cell cycle stage. Neither NKG2D nor cytotoxic activity could be restored in the presence of CD3/CD28 stimulation. We next investigated whether tolerization of CTLs induced by MDCs was correlated with lack of NKG2D and 4-1BB expression. To test this possibility, we generated CTLs of which NKG2D and 4–1BB expression were similarly down-modulated in the absence of MDCs by culturing naïve human cord blood CD8+ T cells with TGF-b1 and IL-4. These CTLs were tolerized similarly as seen in those co-cultured with MDCs, suggesting that lack of both NKG2D and 4–1BB expression on CTLs was a major contributor to the tolerization induced by MDCs. To further verify the importance of NKG2D and 4–1BB expression in CTL functions, we generated CTLs expressing both NKG2D and 4–1BB in vitro from naïve CD8+ T cells by costimulating consecutively with agonistic anti-NKG2D and anti-4–1BB in the presence of anti-CD3. These CTLs were not susceptible to down-modulation of NKG2D by MDCs and exhibited high levels of cytotoxic activity. They expressed CD45RO and CD44 and promptly produced IFN-g in response to IL-12, which are characteristic phenotypes of effector/memory CTL. In conclusion, MDCs developed in vitro from human cord blood CD14+ monocytes showed tolerizing capacity for CTLs by down-modulating NKG2D as well as 4-1BB expression. Because expression of NKG2D and 4–1BB was inter-dependent, CTLs in which NKG2D and 4–1BB expression was down-modulated by MDCs were not able to differentiate into NKG2D+4–1BB+ effector/memory CTLs. Our results suggest that tolerogenic MDCs recruited to the tumor microenvironment might allow evasion of immune tumor responses by down-modulating NKG2D and 4–1BB expression on tumor specific CTLs.

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia

The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.

Expression profiles of TCRβ and CD8α mRNA correlate with virus-specific cell-mediated cytotoxic activity in ginbuna crucian carp

Our previous studies have demonstrated that virus-specific cell-mediated cytotoxicity of sensitized leukocytes can be induced using clonal ginbuna crucian carp and their syngeneic cell lines. In the present study, we attempt to determine if virus-specific cytotoxic cell populations of fish express CD8α and TCRβ genes. Leukocytes from ginbuna crucian carp were separated into four fractions by immunomagnetic separation and density gradient centrifugation: Fraction A, leukocytes with a density of 1.08 g/ml (primarily lymphocytes); Fraction B, sIg-negative leukocytes with density of 1.08 g/ml; Fraction C, sIg-positive cells (primarily B-lymphocytes); Fraction D, leukocytes with a density of 1.08–1.09 g/ml (primarily neutrophils). Leukocytes in all fractions from uninfected fish do not exhibit cytotoxic activity against virus-infected syngeneic cells and weakly express CD8α and TCRβ mRNAs. In contrast, leukocytes in fractions A and B from virus-infected fish exhibit a high level of cytotoxic activity and strongly express CD8α and TCRβ mRNAs. In addition, mRNA expressions of CD8α and TCRβ in effector cells are upregulated by cocultivation with virus-infected target cells but not uninfected ones. The present study suggests that fish possess virus-specific cytotoxic cells with phenotype and gene expression pattern similar to those of CTLs in mammals.

Synthesis and biological evaluation of modified acridines: the effect of N- and O- substituent in the nitrogenated ring on antitumor activity

A series of new acridines has been prepared by cyclodehydration of N-(2,3-dihydro-1,4-benzodioxin-6-yl)anthranilic acid in acidic media following classical procedures. All these compounds have in common a dioxygenated ring fused to the acridine. The tetracyclic system possesses a linear or angular structure formed by intramolecular cyclisation. The last ring and the substituent of the system modify, in an interesting way, the antitumor activity of acridines. Several of the studied compounds displayed significant cytotoxic activity (inhibition of L1210 and HT-29 cell proliferation). The most cytotoxic compound 13a, shows more activity than m-AMSA in inhibiting L1210 and HT-29 cell proliferation and this compound has been selected as a development candidate for further evaluation. The activity results also indicate that the new 11- O-substituted compounds are of considerable interest with high levels of cytotoxic activity. The angular or non-linear dioxinoacridine 10 was equiactive with the linear structure 7. Pentacyclic analogues ( 14 and 15) were more cytotoxic than the tetracyclic compounds (up to twofold).