Rajiv Nayar

Retention of vital dyes correlates inversely with the multidrug-resistant phenotype of adriamycin-selected murine fibrosarcoma variants

Retention of the vital dyes rhodamine 123 (R-123) and hydroethidine (HET) correlates inversely with the multidrug resistant phenotypes of the adriamycin (ADM)-selected variants of a uv-induced murine fibrosarcoma cell line (UV-2237M). The differential affinity of these dyes for specific cellular organelles makes them unique compounds for studies of cellular transport. HET enters viable cells freely, is dehydrogenated to ethidium bromide (EtBr), and is subsequently accumulated in the nucleus. Viable cells are impermeable to extracellular EtBr, facilitating kinetic analysis of the efflux of intracellular EtBr. We found that the metabolite EtBr was rapidly cleared by ADM-resistant but not by ADM-sensitive cells. R-123 has a high affinity to mitochondria. Our results show that ADM-sensitive cells retain R-123 whereas the ADM-resistant cells do not. The clearance of both R-123 and EtBr from these cells was inhibited by verapamil. Therefore, R-123 and HET may be considered MDR-associated compounds useful in studying the MDR phenotype of cancer cells. Previously we reported a direct correlation between the level of activity of the calcium- and phospholipid-dependent protein kinase (protein kinases C) and ADM resistance in UV-2237M variant lines. In this report, we demonstrate a direct correlation between cellular calcium and MDR in these cells. Although chelation of extracellular calcium by EDTA did not alter the fluorescence profile of R-123 of the various cell lines, treating the ADM-resistant variants with verapamil restored cellular calcium to the same level as that of the parental cells and, at the same time, retarded the facilitated efflux of R-123 and EtBr and partially reversed cancer cell resistance to ADM.

The Systemic Activation of Macrophages by Liposomes Containing Immunomodulators

ConclusionsThe demonstration that appropriately activated macrophages can destroy microorganisms and cancer cells has prompted an intense search to identify agents which can render these cells active in vivo. Several natural products, e. g., lymphokines or synthetic molecules, e. g., MDP can produce the tumoricidal state in macrophages. The in vivo use of these agents has been limited, since they have a very short half life.Liposomes offer a most useful carrier system to transport agents to phagocytic cells in vivo. Once in the circulation, liposomes are cleared by phagocytic cells and this passive localization provides an effective mechanism for targeting liposome-entrapped materials to macrophages. In this review we have described the exploitation of this mechanism to deliver lymphokines or other synthetic molecules to macrophages in situ. Since not all liposomes home equally to macrophages, there is still a great need to identify vesicles with ideal properties for this task. The potential application of liposome encapsulated agents to activate macrophages is tremendous. Only future studies will determine the effectiveness and limitations for activated macrophages in enhancing host defense against infections and cancer.

In vitro activation of tumoricidal properties in mouse macrophages using the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) incorporated in liposomes

SummaryWe investigated the ability of free or liposome-incorporated synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) to generate tumoricidal properties in mouse macrophages. As FMLP contains three hydrophobic amino acid residues, it can readily be incorporated into multilamellar vesicles (MLV) consisting of phosphatidylcholine (PC) and phosphatidylserine (PS). The incorporation of FMLP into MLV with a PC:PS ratio of 7:3 mol at FMLP concentrations of up to 10−4M did not affect the phagocytosis of liposomes by mouse peritoneal macrophages. Studies with radioactive FMLP revealed that higher levels of FMLP can be delivered to macrophages by liposomes than in the free, nonencapsulated form. Treatment of mouse macrophages with liposome-encapsulated FMLP, but not with free FMLP, generated tumoricidal properties in the macrophages. The mechanism appears to involve an intracellular site since 100-fold concentrations of free FMLP or free N-acetyl-methionyl-leucyl-phenylalanine, the FMLP agonist, failed to competitively inhibit the macrophages tumoricidal properties generated by liposome-encapsulated FMLP.

Liposome Encapsulation of Muramyl Peptides for Activation of Macrophage Cytotoxic Properties

Publisher Summary This chapter describes a microcytoxicity assay that measures the tumoricidal activity of macrophages activated by liposome-encapsulated muramyl peptides against tumor target cells prelabeled with [ 125 I]iododeoxyuridine ([ 125 I]IUdR). It discusses the advantages and disadvantages of hydrophilic and lipophilic muramyl peptide derivatives for the activation of macrophage cytotoxic properties and the techniques employed to generate appropriate phospholipid vesicles for achieving macrophage activation. The procedure to assess macrophage activation is also presented in the chapter. The activation of macrophages by liposomes containing muramyl peptides can be achieved using three different types of liposomes—namely, (1) multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), and large unilamellar/oligolamellar vesicles produced by reverse-phase evaporation (REVs). The standard method for generating MLVs involves hydrating a dry lipid film on a glass test tube or a lyophilized phospholipid mixture by vortex mixing above the gel-to-liquid crystalline transition temperature of the lipid. An extensive sonication of MLVs results in the formation of smaller unilamellar vesicles (SUVs). The reverse-phase evaporation vesicles (REVs) are formed from water-in-oil emulsions of phospholipids and buffer in an excess organic phase, followed by removal of the organic phase under reduced pressure.

Role of Macrophages in Recognition and Destruction of Metastatic Cells

The most devastating aspect of cancer is the emergence of metastases in organs distant from the primary tumor. Metastasis has already taken place by the time many cancers are diagnosed, and therefore, despite significant advances in surgical technique and general patient care, most deaths from cancer are still due to the uncontrolled growth of metastases. There are several reasons for the failure to treat metastasis. First, in the majority of patients, by the time of diagnosis of primary malignant neoplasms, excluding skin cancers, metastasis may well have occurred and eluded detection (22). Second, metastases are typically located in different organs and may also be in different locations within the same organ. This limits the delivery of chemotherapeutic agents and effective radiation therapy to the lesions without damaging normal tissues. Third, malignant neoplasms contain multiple cell populations exhibiting tremendous biological heterogeneity. This results in the rapid emergence of metastases that are resistant to conventional therapy (26, 85).