Stefan A. Cohen

Selective effects of adriamycin on murine host defense systems.

The clinical value of cancer chemotherapy is limited by the fact that the , anticancer agents available to date are not sufficiently selective in their antitumor action. Because of this, it may not be possible to overcome even relatively moderate degrees of tumor resistance and still avoid unacceptable toxicity. As discussed elsewhere (Mihich 1978), current approaches in cancer chemotherapy are aimed at reducing these limitations through the development of new selective treatments. Measurable differences in antigenicity have been found in tumor cells in animals (see Mihich 1978) and in man (Reinherz & Schlossman 1981, Ritzet al. 1981). Although in humans the differences seem to be primarily quantitative in nature, their existence has led to the hope that responses of the host to tumorassociated antigens might be exploited in designing more selective treatments of neoplastic disease. Difficulties that may have to be overcome in order to exploit tumor immunity therapeuticaily include those related to the fact that anticancer drugs may suppress host defenses through the very antiproliferative action which is at the basis of their antitumor effects (Mihich 1971, 1975). Furthermore, augmented tumor growth may be a consequence of modifications of immunoregulation possibly caused by tumor, drug or immunomanipulations. Thus, an understanding of the interactions between chemotherapeutic treatments and host

Natural antitumor defense system of the murine liver.

Murine nonparenchymal liver cells from various genetic strains isolated by collagenase digestion and differential sedimentation contain both lymphocytes and macrophages. Nonparenchymal liver cells as well as spleen cells, mononuclear blood cells, and peritoneal exudate cells from C3HeB/FeJ mice were tested for natural cytotoxicity against YAC‐1 (sensitive to NK cells) and P815 (resistant to NK cells) tumor cell lines. Resident peritoneal exudate cells exerted no cytotoxicity against either tumor cell, whereas spleen and mononuclear blood cells lysed only YAC‐1. In contrast, nonparenchymal liver cells lysed both YAC‐1 (4 h) and P815 (18 h) tumor cells. Treatment of nonparenchymal liver cells with anti‐asialo GM1 and complement abolished the antitumor activity against both tumor cell lines but not the phagocytic activity. Nonadherent nonparenchymal liver cells exerted greater cytotoxicity against YAC‐1 tumor cells but little cytotoxicity against P815 tumor cells when compared with unfractionated cells. Adherent nonparenchymal liver cells (macrophages) from untreated mice exerted no antitumor activity against either tumor cell. In contrast, adherent nonparenchymal liver cells from Coryn‐ ebacterium parvum treated mice were directly cytotoxic to P815 tumor cells. Spleen cells that are normally not cytotoxic to P815 tumor cells (18 h) became cytotoxic when mixed with adherent nonparenchymal liver cells from untreated mice. These results indicate that the tumoricidal effector cell in nonparenchymal liver cells from untreated mice appears to be the NK cell. Apparently, murine liver marophages from untreated mice do not have tumoricidal activity per se but can “activate” NK cells to kill tumor cells normally resistant to NK cells.

Low density lipoproteins and Lovastatin modulate the organ-specific transendothelial migration of primary and metastatic human colon adenocarcinoma cell lines in vitro

Tumor cell arrest and tumor migration are two of the critical steps in the metastatic cascade. We hypothesized that these steps may be facilitated by the low density lipoprotein (LDL)-induced activation of microvessel endothelial cells (MVEC). The purpose of our study was to investigate the biological effects of an LDL-enriched milieu and the effects of the anticholesterol drug Lovastatin on metastatic behavior. The SW480 and SW620 are primary and metastatic human colonic adenocarcinoma cell lines derived from the same patient. We investigated the effect of LDL on adhesion and migration of the two tumor cell lines across human brain, lung, liver and dermal endothelial monolayers. Adhesion and migration assays were done before and after pretreat-ment of the MVEC or tumor cells with LDL (100 mg/ml) for 24 h. Although metastatic SW620 cells were more adherent to MVEC compared with primary SW480 cells, LDL pretreatment of SW480 and SW620 cells did not affect tumor cell adhesion to MVEC. In contrast, tumor cell migration was significantly increased across endothelial monolayers when MVEC were pretreated with LDL. Transendothelial cell migration was not sig-nificantly affected by pretreatment of the tumor cells with LDL. Lovastatin is an inhibitor of HMG-CoA reduc-tase, the rate-limiting enzyme in cholesterol biosynthesis. It has been shown to have anti-tumor activity in vitro. We investigated the effect of Lovastatin on tumor cell kinetics and tumor cell migration across MVEC. Growth curves and migration assays were done before and after pretreatment of the tumor cells with Lovastatin (30 mg/ml). Migration assays were also done after treatment of unstimulated or LDL-stimulated MVEC (100 mg/ml) for 24 h with Lovastatin. Lovastatin inhibited the in vitro growth of the metastatic SW620 cell line to a greater extent than the invasive SW480E cell line. On the other hand, pretreatment of tumor cells with Lovastatin (30 mg/ml) did not suppress transendothelial tumor cell migration of tumor cells. Finally, Lovastatin given to mice effectively suppressed the number of MCA-26 tumor colonies in the liver of Balb/c mice com-pared with untreated mice. ©Lippincott Williams & Wilkins

Adriamycin induced immunomodulation: Dependence upon time of administration

The immunomodulating capabilities of the anti-neoplastic agent, Adriamycin, were investigated. The day of Adriamycin administration to mice was varied from -15 to -1, day 0 being when mice were either immunized or sacrificed and their spleen cells sensitized in culture. Humoral and cellular immune responses against allogeneic or xenogeneic cellular antigens in mice and in culture, as well as phagocytic and ADCC activities were evaluated using spleen cell populations. The cellular responses and phagocytic activities were affected in a cyclical manner with time after Adriamycins administration. Peaks of increased activity were seen subsequent to day -5 and day -11, administration and low activities following day -1, -3 and day -7, -9 administration. The humoral responses were not affected in a biphasic manner but single peaks of increased activity were seen which corresponded to the times of low cellular cytolytic and phagocytic activities. The ADCC was independent of time of Adriamycin administration. The significance of these findings to the design of therapeutic protocols is discussed.

Adriamycin-induced activation of NK activity may initially involve LAF production

SummaryC3HeB/FeJ spleen cells (unseparated or passaged over nylon wool columns) were cultured overnight (1∓2×106 cells/microwell) in the presence and absence of resident or ADM-induced PEC and anti-YAC-1 (4 h) NK activity was determined. The addition of resident PEC to spleen cells had little effect on NK activity. However, the addition of ADM-elicited PEC (10 mg/kg, IP, day −1 and day −5) to spleen cells prior to culture significantly augmented NK activity. If ADM-induced PEC were treated with carbonyl iron prior to coculture with spleen cells, augmentation of anti-YAC-1 activity was not observed. This suggested that ADM-activated macrophages augmented cultured splenic NK activity. Supernatants from overnight-cultured resident or ADM-induced adherent PEC were then prepared, dialyzed (to remove ADM), and tested for mitogenic activity or cocultured with spleen cells overnight. ADM-induced adherent PEC supernatants stimulated the proliferation of murine thymocytes (both LAF and IL-2 also stimulate) but not cultured CTL (only IL-2 stimulates). ADM-induced adherent PEC supernatants (as well as LAF, IL-2, and IFN) augmented overnight-cultured C3HeB/FeJ splenic NK activity. However, only IL-2 and IFN could augment overnight-cultured athymic BALB/c · nu/nu splenic NK activity. This suggested that ADM-elicited macrophages produce LAF which may act directly on NK cells or, more likely, may induce T cells to produce IL-2, IFN, or both.

Endogeneous interferon α/β produced by murine Kupffer cells augments liver-associated natural killing activity

SummaryNonparenchymal liver cells from untreated C3HeB/FeJ mice, when incubated in medium containing-10% fetal bovine serum or portal serum, produced significant amounts of interferon alpha/beta (IFNα/β). In contrast, other cell populations (spleen, mononuclear blood cells and peritoneal cells) from C3HeB/FeJ mice or nonparenchymal liver cells from other strains of mice (C3H/HeJ, germ-free C3H/HeN and C57Bl/6J) produced little or no detectable IFN in fetal bovine serum under the same culture conditions. The cells in the nonparenchymal liver cell population responsible for IFNα/β production were adherent, phagocytic, silica-sensitive, carbonyl-iron-sensitive, and Thy1.2−, presumably Kupffer cells or resident liver macrophages. IFNα/β production by cultured Kupffer cells was not observed if medium containing fetal bovine serum or portal serum was treated with polymyxin B or if Kupffer cells were cultured in serum-free medium. This suggested that small amounts of endotoxin in fetal bovine or portal serum stimulated Kupffer cells to produce IFNα/β. Possibly, Kupffer cells are in a different state of activation/maturation than peritoneal and splenic macrophages since the sensitivity of resident Kupffer cells from C3HeB/FeJ mice to the stimulatory effects of endotoxin. The endogenous production of IFNα/β by Kupffer cells from C3HeB/FeJ mice can augment liver-associated natural killer (NK) activity against YAC-1 cells (4 h) and induce liver-associated cytotoxic activity, not restricted by the major histocompatibility complex, against NK resistant P815 mastocytoma cells (18 h).

Cellular effects of hypercholesterolemia in modulation of cancer growth and metastasis: a review of the evidence

Hypercholesterolemia and increased cancer risk have been associated, particularly with the high fat diets characteristic of Western societies. We were interested in the possible association between preexisting hypercholesterolemia and the rapidity and extent of tumor metastases in these patients. To date there has been only a few studies that have suggested and explored this determinant of cancer metastases although it may play a role in a subset of patients who develop cancers. This article will review the literature on the effects of LDL-cholesterol on cell proliferation and differentiation and speculate on mechanisms of involvement of a hypercholesterolemic milieu on cancer progression and enhancement of metastatic potential.

Indomethacin modulation of Adriamycin-induced effects on multiple cytolytic effector functions*

SummaryThe anticancer agent, Adriamycin (ADM), in addition to being a potent cytotoxic drug has been shown to be an effective immunomodulator. This study was undertaken to determine whether ADM-induced changes in the production of prostaglandins (particularly PGE2) are involved in ADM-associated modifications of selected host defenses. Spleen cells from normal or ADM-treated (5 mg/kg; day −5) C57BL/6 mice were assessed for the following activities: fresh (day 0) and cultured natural killer (NK), cytotoxic T lymphocyte, lymphokine-activated killer (LAK), Fc-dependent phagocytosis and tumoricidal macrophage. All activities were assessed with and without the addition of indomethacin, an inhibitor of the first step of the cyclo-oxygenase pathway of prostaglandin synthesis. Depending on culture conditions, the cytotoxic T lymphocyte and splenic tumoricidal macrophage activities were either unaffected or were augmented by ADM treatment of the spleen donor mice or by addition of indomethacin to the culture, and these effects were apparently independent of one another. In contrast, ADM treatment generally resulted in reduced NK and LAK activities relative to control and elevated Fc-dependent phagocytosis. The addition of indomethacin to the culture effectively reversed these effects. Furthermore, spleen cells from ADM-treated mice were found to produce twice the amount of PGE2 in culture compared to cells from untreated mice. Finally, the direct addition of PGE2 to NK cultures resulted in a dose-dependent inhibition of NK activity and the dose required was comparable to the amount of PGE2 produced by cultured spleen cells from ADM-treated mice. Taken together, these results indicate that at least some of the immunomodulatory effects of ADM are an indirect result of ADM-induced changes in PGE2 production.

Immunoradiometric assay of lipid A: a test for detecting and quantitating endotoxins of various origins

The ability to measure circulating endotoxin in various disease states has been hampered by the lack of a specific and quantitative assay. The test most commonly used has been the Limulus gelation assay, which measures an enzymatic effect of endotoxin rather than the substance itself. Based on a solid-phase immunoradiometric assay previously developed to detect the specific lipopolysaccharide from Escherichia coli 026, a similar assay has been developed for the lipid A moiety of endotoxins. The assay uses rabbit antibodies to lipid A which do not react with ketodeoxyoctonate, myristic or beta-hydroxymyristic acids, and detects lipid A obtained from endotoxins of various origins after acid hydrolysis of lipopolysaccharide. Experiments in rats given exogenous endotoxin suggest that this assay can be useful for quantitation of bacterial endotoxins in serum and for studying the pathophysiology of experimental endotoxemia.

Endogenous interferon α/β produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor α production and interleukin-2-induced activation of nonparenchymal liver cells

SummaryWe have previously reported liver-specific interferon (IFN) α/β production by murine Kupffer cells that was not observed with other tissue macrophages incubated in the absence of stimulators such as IFNγ or lipopolysaccharide (LPS). Consequently, while interleukin-2 (IL-2) alone induced pronounced lymphokine-activated killer (LAK) activity from splenocytes, combination of anti-IFNα/β antibody with IL-2 was required to generate significant LAK activity from nonparenchymal liver cells. This endogenous IFNα/β production by Kupffer cells was not induced by LPS because (a) addition of polymyxin B did not abolish the positive effects of anti-IFNα/β antibody on nonparenchymal liver cells, and (b) similar results were obtained when comparing the responses of LPS-responsive C3HeB/FeJ and LPS-hyporesponsive C3H/HeJ mice. The possibility of hepatotropic infection was also ruled out in that anti-IFNα/β antibody enhanced hepatic but not splenic LAK cell induction in vitro in both conventional and germfree C3H/HeN mice. IFNα/β played an autoregulatory role by down-regulating the production of IL-1 and tumor necrosis factor α by Kupffer cells. However, the augmenting effect of anti-IFNα/β antibody on LAK induction from non-parenchymal liver cells was not mediated through an increase in the level of either IL-1 or TNFα, as specific antisera against either cytokine did not abrogate this positive effect. Finally, flow-cytometry analysis showed that IFNα/β significantly diminished the expression of IL-2 receptor α chain, indicating an inhibition of LAK cell generation at a relatively early stage of induction.