Rapamycin delays growth of Wnt-1 tumors in spite of suppression of host immunity

<p>Abstract</p> <p>Background</p> <p>Rapamycin, an inhibitor of mammalian target of Rapamycin (mTOR), is an immunosuppressive agent that has anti-proliferative effects on some tumors. However, the role of Rapamycin-induced immune suppression on tumor progression has not been examined.</p> <p>Methods</p> <p>We developed a transplantation model for generation of mammary tumors in syngeneic recipients that can be used to address the role of the immune system on tumor progression. We examined the effect of Rapamycin on the immune system and growth of MMTV-driven Wnt-1 mammary tumors which were transplanted into irradiated and bone marrow-reconstituted, or naïve mice.</p> <p>Results</p> <p>Rapamycin induced severe immunosuppression and significantly delayed the growth of Wnt-1 tumors. T cell depletion in spleen and thymus and reduction in T cell cytokine secretion were evident within 7 days of therapy. By day 20, splenic but not thymic T cell counts, and cytokine secretion recovered. We determined whether adoptive T cell therapy enhances the anti-cancer effect using <it>ex vivo </it>generated Rapamycin-resistant T cells. However, T cell transfer during Rapamycin therapy did not improve the outcome relative to drug therapy alone. Thus, we could not confirm that suppression of T cell immunity contributes to tumor growth in this model. Consistent with suppression of the mTOR pathway, decreased 4E-BP1, p70 S6-kinase, and S6 protein phosphorylation correlated with a decrease in Wnt-1 tumor cell proliferation.</p> <p>Conclusion</p> <p>Rapamycin has a direct anti-tumor effect on Wnt-1 breast cancer <it>in vivo </it>that involves inhibition of the mTOR pathway at doses that also suppress host immune responses.</p

A Facile Synthetic Approach to Nonracemic Substituted Pyrrolo-allocolchicinoids Starting from Natural Colchicine

A six-step semisynthetic approach towards chiral nonracemic pyrrolo-allocolchicinoids starting from naturally occurring colchicine was developed. The synthetic scheme includes an electrocyclic tropolone ring contraction to afford allocolchicinic acid followed by the Curtius reaction, giving the corresponding aniline. The Sandmeyer reaction and copper-mediated hydrazination gave hydrazine-substituted allocolchicine. This was introduced into the Fischer indole synthesis, affording libraries of regioisomeric indole-based allocolchicine congeners

Cell binding and penetration of quaternized chitosan derivatives

Chitosan (Ch) is an attractive biopolymer with multiple reactive groups. However it is poorly soluble at neutral pH. Quaternization improves its solubility and permits the development of various positively charged drug delivery systems. The aim of this work was to study the solubility, toxicity, cell binding, and penetration of 20 kDa chitosan with 9, 40, 58 and 98% of quaternary ammonium group substitution (ChQ1 to ChQ4 accordingly). We showed that ChQ with substitution degree >40% was soluble in a wide pH range. Unexpectedly ChQ2 and ChQ3 were more toxic to cells than Ch, ChQ1 and ChQ4. Higher toxicity of ChQ was found against macrophage like cell line RAW264.7 than against epithelial cells MiaPaCa-2. All ChQ, in contrast to unmodified Ch, easily bound and penetrated the cells with the highest uptake by ChQ4. Thus, quaternized chitosan derivatives can be used for biomedical applications

Chemokine Homeostasis in Healthy Volunteers and during Pancreatic and Colorectal Tumor Growth in Murine Models

Chemokines are involved in the humoral regulation of body homeostasis. Changes in the blood level of chemokines were found in cancer, atherosclerosis, diabetes, and other systemic diseases. It is essential to distinguish the effects of co-morbid pathologies and cancer on the level of chemokines in the blood. We aimed to analyze, by multiplex cytometry, the levels of chemokines in the blood of healthy young volunteers as well as of intact mice and mice with CT26 colon and Pan02 pancreatic tumors. Two types of chemokines were identified both in human and murine plasmas: homeostatic ones, which were found in high concentrations (&gt;100 pg/mL), and inducible ones, which can be undetectable or determined at very low levels (0&ndash;100 pg/mL). There was a high variability in the chemokine levels, both in healthy humans and mice. To analyze chemokine levels during tumor growth, C57BL/6 and BALB/c were inoculated with Pan02 or CT26 tumor cells, accordingly. The tumors significantly differed in the growth and the mortality of mice. However, the blood chemokine levels did not change in tumor-bearing mice until the very late stages. Taken collectively, blood chemokine level is highly variable and reflects in situ homeostasis. Care should be taken when considering chemokines as prognostic parameters or therapeutic targets in cancer

Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair

(1) Background: We have previously shown that the use of an artificial supramolecular two-component system based on chimeric recombinant proteins 4D5scFv-barnase and barstar-heat shock protein 70 KDa (HSP70) allows targeted delivery of HSP70 to the surface of tumor cells bearing HER2/neu antigen. In this work, we studied the possibility to using DARPin9_29-barnase as the first targeting module recognizing HER2/neu-antigen in the HSP70 delivery system. (2) Methods: The effect of the developed systems for HSP70 delivery to human carcinomas SK-BR-3 and BT474 cells hyperexpressing HER2/neu on the activation of cytotoxic effectors of the immune cells was studied in vitro. (3) Results: The results obtained by confocal microscopy and cytofluorimetric analysis confirmed the binding of HSP70 or its fragment HSP70-16 on the surface of the treated cells. In response to the delivery of HSP70 to tumor cells, we observed an increase in the cytolytic activity of different cytotoxic effector immune cells from human peripheral blood. (4) Conclusions: Targeted modification of the tumor cell surface with molecular structures recognized by cytotoxic effectors of the immune system is among new promising approaches to antitumor immunotherapy

Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death

Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl2 and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions

Susceptibility of mice to invasive aspergillosis correlates with delayed cell influx into the lungs

International audienceP>Ubiquitous fungus Aspergillus fumigatus (A. fumigatus) is involved in invasive pulmonary aspergillosis (IPA), a frequent infection in immunocompromized patients. Genetic differences are likely to play a role predisposing to IPA. This study was aimed to compare six genetically different mouse strains in their susceptibility to IPA and to determine possible mechanisms involved in the pathogenesis of this infection. Immunosuppressed BALB/c and C57BL/6 mice infected with A. fumigatus conidia were more resistant to IPA than DBA/1, DBA/2, CBA, and A/Sn strains. Phagocytosis of A. fumigatus conidia by blood polymorphonuclear neutrophils (PMN) or bone marrow derived dendritic cells showed no difference between strains. All IPA susceptible strains demonstrated decreased PMN influx into the lungs during infection compared with resistant strains. Flow cytometry analysis of the composition of lung infiltrating cells showed that IPA susceptible mice had a decreased number of phagocytes before the infection. After infection the numbers of Gr-1(+)CD11b(+) PMN cells in the lungs of immunosuppressed mice increased from 10-20% to 50-60% while the percentage of CD11(+)F4/80(+) resident macrophages was unchanged. Among susceptible strains DBA/2 and A/Sn have a defect in C5 component of complement. Injection of normal serum into complement deficient but not into complement sufficient CBA or DBA/1 mice significantly improved their survival. We showed that complement replacement significantly increased PMN homing to the lungs of complement deficient mice. Thus, defect in complement system can predispose to IPA. Our results demonstrated that early influx of PMN into the lungs of mice is important for the resistance to IPA

Synthesis of Nonracemic Pyrrolo-allocolchicinoids Exhibiting Potent Cytotoxic Activity

An efficient eight-step semisynthetic approach towards nonracemic pyrrolo-allocolchicinoids starting from natural colchicine was developed by exploiting Pd-catalyzed domino Sonogashira coupling/5-endo-dig cyclization of a 2-iodo-trifluoroacetanilide intermediate to build up the heterocyclic ring system. The N-Me substitution of the pyrrole ring enhanced the antitumor activity of the prepared molecules by 2-3 orders of magnitude. Among the active compounds, the N-methylated colchicinoid exhibited powerful cytotoxic and antiproliferative properties at concentrations < 1 nM

Polyvinylpyrrolidone–Alginate Film Barriers for Abdominal Surgery: Anti-Adhesion Effect in Murine Model

Surgical operations on the peritoneum are often associated with the formation of adhesions, which can interfere with the normal functioning of the internal organs. The effectiveness of existing barrier materials is relatively low. In this work, the effectiveness of soluble alginate–polyvinylpyrrolidone (PVP-Alg) and non-soluble Ca ion cross-linked (PVP-Alg-Ca) films in preventing these adhesions was evaluated. Experiments in vivo were performed on mice via mechanical injury to the adjacent peritoneum wall and the caecum, followed by the application of PVP-Alg or PVP-Alg-Ca films to the injured area. After 7 days, samples from the peritoneal wall and caecum were analyzed using histology and quantitative polymerase chain reaction (qPCR). It was shown that the expression of genes responsible for adhesion formation in the caecum in the PVP-Alg group was comparable to that in the control group, while in the PVP-Alg-Ca group, it increased by 5–10 times. These results were consistent with the histology: in the PVP-Alg group, the adhesions did not form, while in the PVP-Alg-Ca group, the adhesions corresponded to five points on the adhesion scale. Therefore, the formation of intraperitoneal adhesions can be effectively prevented by non-crosslinked, biodegradable PVP-Alg films, whereas cross-linked, not biodegradable PVP-Alg-Ca films cause inflammation and adhesion formation

Design, Synthesis and In Vitro Biological Activity of Novel C-7 Methylene Congeners of Furanoallocolchicinoids

A series of novel heterocyclic colchicine derivatives bearing a C-7 methylene fragment were synthesized via Wittig, Horner–Wadsworth–Emmons and Nenajdenko–Shastin olefination approaches. The in vitro biological activities of the most promising compounds were investigated using MTT assays and cell cycle analyses. Compounds with an electron withdrawing group on the methylene fragment exhibited substantial antiproliferative activity towards COLO-357, BxPC-3, HaCaT, PANC-1 and A549 cell lines. The spatial orientation of the substituent at the double bond significantly influenced its biological activity