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Proposed Mechanism of Selective LCM Uptake by Tumor Cells
Chapter 14
Proposed Mechanism of Selective LCM Uptake by Tumor Cells: Role of Lipoprotein ReceptorMediated Endocytic Pathways If the same endocytic lipid-coated microbubble (LCM)-uptake mechanism(s) which has been observed and analyzed in detail with C6 and 9L tumor cells in culture (see Section 13.1) were also operative in vivo, it would indicate that a sizable portion of intravenously injected LCM that bypasses the reticuloendothelial system will then become endocytosed directly by tumor cells [531]. The actual endocytic pathways that are likely to be involved in LCM uptake by tumors are not known, at the present time, due to the lack of any detailed receptor-binding studies with LCM to date. However, a few reasonable candidates for such endocytic pathways emerge upon reviewing parts of an extensive research literature describing significantly enhanced, receptor-mediated endocytosis in many different cancerous cells and solid tumors (see below).
14.1 LOW-DENSITY LIPOPROTEIN RECEPTORS, ON TUMOR CELLS, AND LCM Numerous studies have pointed to an important role for cholesterol during proliferation and progression of cancer (e.g., Refs. [612–615]). Rapidly dividing cancer cells have two major routes to fulfill their need for cholesterol to form new cell membrane: endogenous synthesis of cholesterol and/or receptor-mediated uptake of exogenous low-density lipoprotein (LDL) particleassociated cholesterol and cholesterol esters (CEs) [612,613,615]. Each LDL particle contains a CE core surrounded by a polar shell of phospholipids (primarily phosphoglycerides), free cholesterol, and apolipoprotein B [616–618]. Once bound to its cell-surface receptor, LDL is internalized by receptormediated endocytosis and degraded in lysosomes, and the subsequently released cholesterol may be used for membrane synthesis by the tumor [619]. Studies in Interface Science, Vol. 25. DOI: 10.1016/B978-0-444-53798-0.00016-X # 2011 Elsevier B.V. All rights reserved.
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The lipid composition of LCM consists of CEs, free cholesterol, and glycerides (cf. Section 12.1) and is, therefore, similar to the abovementioned lipid composition of the LDL particle. Based upon this molecular similarity, it appears reasonable to expect that injected LCM would readily bind apolipoprotein B (apo B), either alone or already attached to LDL particles, in the bloodstream. (It is useful to note that LDL particles are formed in large part within peripheral capillaries, i.e., along the capillary endothelium, by the progressive removal of triglycerides from the much larger very low-density lipoprotein (VLDL) particles and by their gradual accumulation of CEs [617,620]. The well-documented movement of lipid components between physiologic lipoproteins in the blood suggests that a physical interaction between the injected LCM and LDL particles is feasible). The proposed LCM binding of apo B, either alone or already attached to LDL particles, should influence the subsequent biodistribution of those LCM. This expectation derives from two factors: first, apo B is the LDL component which mediates LDL binding to its cell-surface receptors [608], that is, the LDL receptors, which in turn are involved in receptor-mediated endocytosis [616,620]. The second factor influencing the expected LCM biodistribution involves the frequently reported finding that many different cancer cells and solid tumors take up LDL more effectively than normal tissues (e.g., Refs. [614,615,619,622–625], which many investigators consider is probably a reflection of a higher cholesterol demand of dividing cells as opposed to differentiated cells (e.g., Refs. [612–615]. (This enhanced receptor-mediated active uptake, or endocytosis, of LDL particles by many cancer cells and tumor types can be due to either increased expression or activity, or both, of the LDL receptors on the cell surface (cf. Refs. [613,619,624,626–641]).) Some examples of the reported findings are as follows: leukemic cells isolated from patients with acute myelogenous leukemia have elevated LDL receptor activities compared to normal white blood cells and nucleated bone marrow cells [626]. Gynecologic cancer cells also possess high LDL receptor activity when assayed both in monolayer culture and in membrane preparations from tumor-bearing nude mice [642]. In addition, an enhanced receptor-mediated uptake of LDL by tumor tissue in vivo was demonstrated in animal models and, subsequently, by lung tumors in vivo in humans [619]. Such enhanced LDL receptor-mediated endocytic uptake of LDL particles, and probably also of LCM (by analogy [cf. above]) due to their likely bound apo B and/or LDL, into tumor tissue may therefore explain the extreme rapidity and high selectivity of the LCM accumulation observed within tumors (see below). Specifically, the data reviewed in Chapters 12 and 13 indicate that LCM are rapidly removed from the circulation by the tumor; the maximum accumulation of LCM in the tumor area occurs within the first 30 min after administration [531]. These rapid kinetics for LCM uptake are quite consistent with the well-documented kinetics long-known for the LDL receptor-mediated
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endocytic pathway [616]. For example, Goldstein et al. [643] reported that LDL-ferritin bound to coated pits at 4 C is rapidly internalized when fibroblasts in tissue culture are warmed to 37 C. In this uptake process, the coated pits invaginate to form coated endocytic vesicles. After 5–10 min at 37 C, LDL-ferritin is observed in lysosomes as the result of their fusion with the incoming coated vesicles [643]. The rapid sequence of events visualized in electron micrographs precisely parallels biochemically derived data on the rapid uptake and degradation of radiolabeled LDL [644,645]. An additional, related factor that could be contributing to the rapid removal of LCM from the circulation, by different tumors, is the fact that vascular endothelial cells participate in LDL removal from blood [646]. Moreover, it has been reported by different investigators that growth factors, including tumor-related growth factors, increase LDL receptor expression on vascular endothelial cells (e.g., Refs. [614,647]); this finding raises the possibility that the endothelial cells arising during cancer-associated neovascularization may have high numbers of LDL receptors which could contribute further to the accumulation of LDL-associated substances, viz. LCM, within tumors. Hence, the high expression levels of LDL receptors occurring in many types of tumors (cf. above), and possibly within the tumor capillaries themselves, could lead to the arrest or assemblage of LCM within the tumor’s vascular supply. By this proposed endocytic mechanism of LCM uptake, the initial interaction between LCM and lipoprotein receptors would occur intravascularly, a logical hypothesis for a phenomenon that becomes readily measurable within 2 min following injection of the LCM into the bloodstream (cf. Chapters 12 and 13).
14.2 MULTILIGAND LIPOPROTEIN RECEPTORS As explained in a review by Krieger and Herz [648], LDL receptors and most other mammalian cell-surface receptors, which mediate endocytosis, exhibit two common ligand-binding characteristics: high affinity and narrow specificity. The ligand-binding properties of two more recently characterized lipoprotein receptors, that is, LDL receptor-related protein (LRP) and scavenger receptors, do not conform to a narrow binding specificity. These receptors bind, with high affinity, both lipoprotein and nonlipoprotein ligands and participate in a wide variety of biological processes.
14.2.1 LDL Receptor-Related Protein, on Tumor Cells, and LCM LRP is a member of the “LDL receptor gene family” [649] and, like the LDL receptor, performs an essential role in the removal of certain lipoprotein particles from the bloodstream. As Heeren et al. [650] explain, triglycerides are transported mainly by two distinct classes of lipoproteins, the chylomicrons
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and the VLDL. After assembly in the intestine, chylomicrons are carried via lymph into the bloodstream, where they are transformed at the endothelial surface to remnant lipoproteins through the catalytic action of lipoprotein lipase (for review, see Refs. [651,652]). After lipolysis, the lipoprotein lipase remains associated with the chylomicron remnants and, in conjunction with apolipoprotein E (apo E) [653–655], facilitates their clearance by the liver into hepatocytes [656] via LDL receptors and the LRP [657–660]. (The essential role for both receptors in chylomicron remnant removal in vivo has been demonstrated in gene knockout and gene transfer experiments [661,662] (for review, see Ref. [663].) The remarkable rapidity and specificity of uptake of the chylomicron remnant particles is believed to be highly dependent on their acquisition of apo E in the bloodstream [664,665]; the critical role played by apo E in directing the clearance of chylomicron remnants from the (blood) plasma has been well established by several lines of evidence [664]. Both the LDL receptor (also known as the “apo B,E receptor” [620]) and the LRP have a high affinity for apo E [666], and both receptors play an essential role in the receptormediated endocytosis of chylomicron remnants [650,665]. The chylomicron remnant particles themselves, derived from lipolysis of the larger chylomicrons (cf. above), contain the residual triglyceride and all of the cholesterol and CE from the original chylomicrons. This lipid composition of the chylomicron remnant particles is similar to the above-described lipid composition of both LDL particles (cf. Section 14.1) and LCM (cf. Section 12.1). Based upon this molecular similarity, it appears reasonable to expect that injected LCM could also readily bind apo E (i.e., as an alternative to apo B) in the bloodstream. In this case, the proposed LCM binding of apo E should influence the subsequent biodistribution of those LCM via two endocytic pathways: specifically, one pathway mediated by the LDL receptor (a.k.a. “apo B,E receptor”) (cf. Section 14.1) and the other pathway mediated by the LRP, since both receptor types have a high affinity for apo E (cf. above). As concerns links between apo E and tumors, Section 14.1 included a review of the increased expression and/or activity of LDL receptors on many cancer cells and tumor types. An analogous, but less widespread, pattern of receptor enhancement has been reported by various investigators for the LRP in tumor cells. One series of reports concerns human glioblastomas (a common type of glioma, or brain tumor) and glioblastoma cell lines. Yamamoto et al. [667] investigated immunohistochemical localization of LRP, on sequential frozen sections of human glioblastomas, and showed that the neoplastic glial cells and endothelial cells exhibited intense LRP immunoreactivity. Furthermore, they conclude that LRP is overexpressed in glioblastomas, and that LRP may play a role in facilitating glioblastoma invasiveness and neovascularization within tumor tissues [667]. Similarly, Bu et al. reported that human glioblastoma U87 cells express an abundance of LRP, and determined that LRP at the cell surface and along the cellular processes was
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functional in the binding and endocytosis of its ligands [668]. Finally, in a detailed, recent study by Maletinska et al. [669], the status of both LRP and LDL receptors was evaluated in seven human glioma (i.e., glioblastoma) cell lines. All were found to exhibit LRP, and cell lines SF-539, U-87 MG, and U-343 MG were particularly rich in this receptor. In addition, the presence of specific saturable LDL receptors was proven in six of the cell lines investigated, which had high concentrations of receptors (128,000–950,000 per cell). These authors conclude this finding suggests that LDL receptors on glioblastoma cells could potentially be useful for targeting antitumor agents; they further conclude that LRP, a multifunctional receptor expressed on glioblastoma cells, also has the possibility for serving as a therapeutic target [669]. Another major type of tumor cell examined for the presence of LRP was human breast cancer cells. Li et al. [670] examined six breast cancer cell lines and showed that LRP is expressed at a wide range of levels, that is, approximately 300–6300 sites per cell. Four of the breast cancer cell lines expressed LRP at over 1000 sites per cell, and were markedly invasive in their assay [670].
14.2.2 Scavenger Receptors on Tumor Cells as well as “Activated” Macrophages: LCM Binding, and Its Relation to Certain Disease Sites A second category of multiligand lipoprotein receptor, present in varying amounts in different tissues, consists of the scavenger receptors [671]. Various members of this receptor class were initially identified by their capability for rapid, unregulated uptake (by endocytosis) of chemically “modified” LDL by macrophages, leading to massive cholesterol accumulation [648,671]. The similarity of lipid composition between LDL particles and LCM (cf. Section 12.1) suggests that the LCM could also resemble a chemically “modified” LDL and, hence, act as a ligand for scavenger receptors. The proposed LCM binding with scavenger receptors would provide a third major endocytic pathway which can influence the biodistribution of LCM (subsequent to their binding of cell-surface receptors).
14.2.2.1 Tumor Cell Studies Indirect support for a proposed third available LCM-uptake mechanism (i.e., scavenger receptor-mediated endocytosis) is found in several studies reporting expression of scavenger receptors on certain types of tumor cells, for example, hepatoma [672], renal cell carcinoma [673], human adrenocortical carcinoma [631], human breast carcinoma [615], and histiocytic malignancies [674–676]. Furthermore, when the binding characteristics of these receptors were analyzed in detail in HepG2 (human hepatoma) cells, it was found that in this case the scavenger receptors bound both chemically modified LDL and native LDL [672]. Saturation binding experiments revealed
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moderate-affinity binding sites for both modified and native LDL, and competition binding studies at 4 C showed that both ligands share common binding site(s). Degradation/association ratios for these ligands show that LDLs are very efficiently degraded, and the chemically modified LDL degradation/ association ratio is equivalent to 60% of the LDL degradation ratio. Both lipoprotein ligands were good CE donors to HepG2 hepatoma cells. Finally, hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes [672].
14.2.2.2 Central Nervous System Injury While expression of scavenger receptors has been reported for some tumor cell types (cf. Section 14.2.2.1), there are certain other lesions which far more consistently display increased expression and/or activity of scavenger receptors (see below) on the cell surface. For these particular (noncancerous) disease/injury sites, the above-proposed (cf. Section 14.2.2) third LCM-uptake mechanism (i.e., scavenger receptor-mediated endocytosis) would appear well-suited for targeted drug-delivery therapy, via LCM, of that given disease or injury. An example of this type of lesion is central-nervous-system (CNS) injury, that is, brain injury and/or spinal cord injury (see below). As Bell et al. [677] observed in mouse brain, macrophage scavenger receptor expression on both microglia (the resident macrophages of the CNS) and recruited macrophages was detected 24 h after an intrahippocampal injection of either lipopolysaccharide or kainic acid (i.e., neurotoxic compounds). Macrophage scavenger receptor expression was also detected in microglia 3 days after optic nerve crush both in the nerve segment distal to the crush site and in the superior colliculus. (The monoclonal antibody 2F8 was used to localize the macrophage scavenger receptor by immunohistochemistry. In control adult mice, microglia did not express the receptor. Also, in the aged mouse brain, the pattern of macrophage scavenger receptor expression was no different from that in the young adult brain.) Hence, these studies indicate that the increased macrophage scavenger receptor expression, on microglia and recruited macrophages, arising after brain injury could play a role in the clearance of debris during acute neuronal degeneration [677]. At the same time, this increased receptor expression provides a possible avenue for LCM-targeted drug-delivery treatment of CNS injury sites. Furthermore, passage of LCM across the blood–brain barrier would be facilitated by the fact that scavenger receptors (both types I and II of class A) have been shown, by a reverse transcriptase PCR study of messenger RNA expression, to be present normally in (bovine) brain microvessels [678]. Brain microvessels therefore appear to play an active role in the uptake of native and modified low-density lipoproteins [678]; accordingly, these microvessels would be expected to have a similar role in LCM uptake (cf. Sections 12.1 and 14.2.2 above; see also below).
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Regarding one specific application of LCM to CNS injury, Kureshi et al. [535] reported the affinity of tail vein-injected LCM to injured rat spinal cord (due to a compressive lesion to the upper thoracic region). The accumulation of LCM in the injured spinal cord was analyzed, after labeling it with a lipidsoluble fluorescent dye (diO), by confocal laser scanning microscopy. It was observed that affinity of LCM for spinal cord injury sites appears to be mediated in the early stages after injury by proliferating macrophages in the necrotic center, and then in later stages by (glial fibrillary acidic protein [GFAP]-positive) activated astrocytes in adjacent white matter. These findings suggested a potential for using LCM as a delivery vehicle to concentrate lipid-soluble agents in spinal cord injury sites [535]. As another application of LCM to CNS injury, Ho et al. [533] studied the affinity of LCM to the site of a localized (thermal) brain injury. It had been well documented that in response to injury in the CNS, astrocytes are activated which is accompanied by an increased content of GFAP, hypertrophy, and hyperplasia [679–683]. (This process of gliosis [682] results in scar formation; it has been speculated that the scar may inhibit axonal regeneration [684]) [533]. Ho et al. observed that the influx of LCM began at the time when GFAP-positive cells began to appear. It seemed likely that LCM are initially attracted to the “reactive” astrocytes, but subsequently the LCM were found to be excluded from the area of scar formation. The LCM distribution detected by fluorescent (diO)-labeled LCM implied that the LCM are rapidly (within 10 min after the intravenous injection of LCM) taken up by some cells other than the astrocytes beginning 7–10 days after the injury. The round shape of these cells suggested that they might be blood borne (i.e., microglia) [533]. In follow-up to these findings with brain injury, the experiment program with LCM was expanded to next examine the use of LCM to deliver 7bhydroxycholesterol (7b-OHC) to a radiofrequency (thermal) lesion in the rat brain. With diO-labeled LCM, it was first reaffirmed that LCM target the injury area specifically and not the adjacent normal tissue. With immunohistochemistry and fluorescent immunochemistry, it was then demonstrated that the 7b-OHC, delivered in LCM, exerts an antigliosis effect in the rat brain injury model [534] (see below). 7b-OHC and other oxysterols have been reported, by other investigators, to inhibit astrogliosis as well as tumor cell proliferation both in vitro and in vivo [607–611]. Several groups have proposed different mechanisms for the cytotoxicity of these compounds [607,609–611]. Kupferberg et al. [611] first studied the cytotoxic effects of 7b-OHC and its derivatives on reactive astrocyte proliferation. Their group demonstrated that injection of liposomes containing 7b-OHC decreased the cholesterol biosynthesis by 40% in injured rat brain cortex, with a linear relationship between the cholesterogenesis and astroglial proliferation [607,610,611]. They concluded that oxysterols are potent inhibitors of the endogenous cholesterol biosynthesis, and this
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downregulation accounts for the inhibition of injury-induced astroglial proliferation, thereby reducing astrocytic reaction [534]. The data obtained in this follow-up study, based on immunohistochemical staining of GFAP-positive cells, and with fluorescent confocal microscopy techniques, indicate that both the number of GFAP-expressing astrocytes and the intensity of the staining were reduced when treated with 7b-OHC delivered by the LCM, while not affected by the same dose of intravenously injected 7bOHC in saline. Hence, since regenerating axons appear to be physically blocked by scar formation during healing [684,685], possibly antigliosis agents delivered directly to the injury sites in the optimal concentration (i.e., using targeted drug delivery via LCM) might assist in promoting functional repair [534]. The fact that the 7b-OHC injected alone did not exert the same effect indicates that LCM-bound 7b-OHC might be metabolized through a different pathway, that is, (scavenger) receptor-mediated endocytosis. As observed previously with the drug paclitaxel [532] (see also Section 13.2), delivery of a given drug via LCM appears to greatly magnify the concentration of that drug reaching the target site. Presumably, the mechanism of this enhanced delivery of 7b-OHC to the injury site by LCM shares common features with the receptor-mediated endocytic pathway mechanisms described earlier for the case of tumor cells (cf. Sections 14.1–14.2.2.1). The dissolved free drug, in this case 7b-OHC, would therefore not be expected to reach the desired target sites in as high a concentration compared to when it is carried by the LCM [534] (cf. first three paragraphs of this subsection).
14.2.2.3 Alzheimer’s Disease In Alzheimer’s disease, the characteristic lesions that develop, called senile plaques, are extracellular deposits principally composed of insoluble aggregates of b-amyloid protein (Ab) fibrils, infiltrated by reactive microglia and astrocytes [686]. Ab fibrils exert a cytotoxic effect on neurons and stimulate microglia to produce neurotoxins, such as reactive oxygen species [687]. Mononuclear phagocytes, including microglia, express scavenger receptors that mediate adhesion and/or endocytosis [687,688]; in particular, microglia have been shown to be intimately associated with amyloid deposits [688–690] and have also been implicated as scavengers responsible for clearing Ab fibril deposits of Alzheimer’s disease [688,689]. For example, Paresce et al. [689] reported that microglia in culture rapidly took up fluorescent-labeled Ab microaggregates into discrete vesicles, which were confirmed to be endosomes; at longer incubation times (30–60 min), Ab fluorescence became increasingly concentrated in organelles around the nucleus, consistent with delivery to late endosomes and lysosomes. Moreover, the uptake of Ab microaggregates by the microglia has been shown to be saturable, and is not fluid-phase internalization [689]. Accordingly, El Khoury et al. [687,691] identified class A scavenger receptors as the main cell-surface receptors mediating the interaction of microglia with b-amyloid fibrils. Adhesion of microglia to
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b-amyloid fibrils leads to immobilization of these cells on the fibrils, and induces or “activates” them to produce reactive oxygen species. These authors conclude that microglial scavenger receptors may be novel targets for therapeutic interventions in Alzheimer’s disease [691]. Similarly, a related review by Kalaria [692] provides additional data supporting the belief that microglia play a major role in the cellular response associated with the pathological lesions of Alzheimer’s disease, and concludes that pharmacological agents which suppress microglial activation may prove a useful strategy to slow the progression of Alzheimer’s disease [692] (see also Refs. [693–696]). In view of the above findings and since LCM have already been shown [534] (see also Section 14.2.2.2) to be an effective in vivo targeted drug-delivery vehicle for the antigliosis drug 7b-OHC to experimental injury sites in rat CNS, the LCM/7b-OHC complex may well similarly target the scavenger receptors of Alzheimer’s lesions and, thereby, reach the microglial cell surface and suppress microglial activation. While LCM can be and have been used to carry different lipid-soluble drugs, 7b-OHC is an appealing choice for an antigliosis drug. Specifically, 7b-OHC has a cytotoxic effect on different transformed glial cell lines (and probably also different types of activated, or reactive, glial cells [534]) which is useful since, as noted earlier [686,687,691], the senile plaques of Alzheimer’s disease are infiltrated by both reactive microglia and astrocytes [686]. This added involvement of astrocytes in Alzheimer’s disease is mentioned by various other researchers, such as Malhotra et al. who specifically indicate that the neuritic and amyloid cerebral cortical plaques of Alzheimer’s disease are surrounded by reactive astrocytes [697]. Similarly, El Khoury et al. point out that Alzheimer’s pathological features include neuronal degeneration, astrogliosis, microgliosis, and the extracellular accumulation of neurotoxic altered isoforms of b-amyloid [698] (cf. Refs. [693–696]). Based on these considerations, LCM-directed 7b-OHC delivery to the Alzheimer’s lesions may inhibit amyloid toxicity via gliosis and, potentially, reduce plaque pathology. Passage of the intravenously injected LCM/7b-OHC complex across the blood–brain barrier to Alzheimer’s lesion sites should be readily possible in view of the earlier-mentioned fact that scavenger receptors, both types I and II (of class A), have been shown to be present normally in (bovine) brain microvessels [678]. As stated in Section 14.2.2.2, brain microvessels therefore appear to play an active role in the uptake of native and modified LDL particles [678]; consequently, these microvessels would be expected to have a similar role in uptake of the LCM/7b-OHC complex.
14.2.2.4 Atherosclerotic Lesions The macrophage scavenger receptor family of proteins, including class A (types I and II) receptors and class B receptors, recognizes a wide variety of macromolecules (including modified LDL) which, after binding, can be
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internalized by endocytosis or phagocytosis (or, alternatively, remain at the cell surface and mediate adhesion or lipid transfer through caveolae) (e.g., Ref. [699]). In pathological states, members of this scavenger receptor family mediate the recruitment, activation and transformation of macrophages, and other cells which appear to be related to the development of not only Alzheimer’s disease but also atherosclerosis [699–703]. Lipid accumulation in the blood vessel wall depends on the intracellular uptake by macrophages, which transform into foam cells. Overloaded foam cells finally degenerate, leaving extracellular lipid deposits. The lipid overload of macrophages is brought about by several classes of scavenger receptors that, unlike (native-)LDL receptor, take up modified LDL and are not feedback controlled [701–705]. As one example, a major type of class B scavenger receptor is reported to be upregulated by the proatherogenic, modified LDL particles; hence, binding followed by uptake perpetuates a cycle of lipid accumulation and receptor expression [705]. Both class B [706,707] and class A [700,701] scavenger receptors are expressed in the lipid-laden macrophages in atherosclerotic lesions [704,708]. Furthermore, Nakagama-Toyama et al. [708] have reported on the differential distribution of the scavenger receptor types within human coronary atherosclerotic lesions. In view of the detailed published information available on the presence, functional characteristics, and localization of scavenger receptor populations in atherosclerotic lesions (cf. above) as well as the known structural similarity between modified LDL and LCM (cf. Sections 12.1 and 14.2.2–14.2.2.2), LCM-directed drug delivery to atherosclerotic lesions may offer a means for targeted drug-delivery therapy of atherosclerosis.
Proposed Mechanism of Selective LCM Uptake by Tumor Cells: Role of Lipoprotein ReceptorMediated Endocytic Pathways If the same endocytic lipid-coated microbubble (LCM)-uptake mechanism(s) which has been observed and analyzed in detail with C6 and 9L tumor cells in culture (see Section 13.1) were also operative in vivo, it would indicate that a sizable portion of intravenously injected LCM that bypasses the reticuloendothelial system will then become endocytosed directly by tumor cells [531]. The actual endocytic pathways that are likely to be involved in LCM uptake by tumors are not known, at the present time, due to the lack of any detailed receptor-binding studies with LCM to date. However, a few reasonable candidates for such endocytic pathways emerge upon reviewing parts of an extensive research literature describing significantly enhanced, receptor-mediated endocytosis in many different cancerous cells and solid tumors (see below).
14.1 LOW-DENSITY LIPOPROTEIN RECEPTORS, ON TUMOR CELLS, AND LCM Numerous studies have pointed to an important role for cholesterol during proliferation and progression of cancer (e.g., Refs. [612–615]). Rapidly dividing cancer cells have two major routes to fulfill their need for cholesterol to form new cell membrane: endogenous synthesis of cholesterol and/or receptor-mediated uptake of exogenous low-density lipoprotein (LDL) particleassociated cholesterol and cholesterol esters (CEs) [612,613,615]. Each LDL particle contains a CE core surrounded by a polar shell of phospholipids (primarily phosphoglycerides), free cholesterol, and apolipoprotein B [616–618]. Once bound to its cell-surface receptor, LDL is internalized by receptormediated endocytosis and degraded in lysosomes, and the subsequently released cholesterol may be used for membrane synthesis by the tumor [619]. Studies in Interface Science, Vol. 25. DOI: 10.1016/B978-0-444-53798-0.00016-X # 2011 Elsevier B.V. All rights reserved.
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The lipid composition of LCM consists of CEs, free cholesterol, and glycerides (cf. Section 12.1) and is, therefore, similar to the abovementioned lipid composition of the LDL particle. Based upon this molecular similarity, it appears reasonable to expect that injected LCM would readily bind apolipoprotein B (apo B), either alone or already attached to LDL particles, in the bloodstream. (It is useful to note that LDL particles are formed in large part within peripheral capillaries, i.e., along the capillary endothelium, by the progressive removal of triglycerides from the much larger very low-density lipoprotein (VLDL) particles and by their gradual accumulation of CEs [617,620]. The well-documented movement of lipid components between physiologic lipoproteins in the blood suggests that a physical interaction between the injected LCM and LDL particles is feasible). The proposed LCM binding of apo B, either alone or already attached to LDL particles, should influence the subsequent biodistribution of those LCM. This expectation derives from two factors: first, apo B is the LDL component which mediates LDL binding to its cell-surface receptors [608], that is, the LDL receptors, which in turn are involved in receptor-mediated endocytosis [616,620]. The second factor influencing the expected LCM biodistribution involves the frequently reported finding that many different cancer cells and solid tumors take up LDL more effectively than normal tissues (e.g., Refs. [614,615,619,622–625], which many investigators consider is probably a reflection of a higher cholesterol demand of dividing cells as opposed to differentiated cells (e.g., Refs. [612–615]. (This enhanced receptor-mediated active uptake, or endocytosis, of LDL particles by many cancer cells and tumor types can be due to either increased expression or activity, or both, of the LDL receptors on the cell surface (cf. Refs. [613,619,624,626–641]).) Some examples of the reported findings are as follows: leukemic cells isolated from patients with acute myelogenous leukemia have elevated LDL receptor activities compared to normal white blood cells and nucleated bone marrow cells [626]. Gynecologic cancer cells also possess high LDL receptor activity when assayed both in monolayer culture and in membrane preparations from tumor-bearing nude mice [642]. In addition, an enhanced receptor-mediated uptake of LDL by tumor tissue in vivo was demonstrated in animal models and, subsequently, by lung tumors in vivo in humans [619]. Such enhanced LDL receptor-mediated endocytic uptake of LDL particles, and probably also of LCM (by analogy [cf. above]) due to their likely bound apo B and/or LDL, into tumor tissue may therefore explain the extreme rapidity and high selectivity of the LCM accumulation observed within tumors (see below). Specifically, the data reviewed in Chapters 12 and 13 indicate that LCM are rapidly removed from the circulation by the tumor; the maximum accumulation of LCM in the tumor area occurs within the first 30 min after administration [531]. These rapid kinetics for LCM uptake are quite consistent with the well-documented kinetics long-known for the LDL receptor-mediated
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endocytic pathway [616]. For example, Goldstein et al. [643] reported that LDL-ferritin bound to coated pits at 4 C is rapidly internalized when fibroblasts in tissue culture are warmed to 37 C. In this uptake process, the coated pits invaginate to form coated endocytic vesicles. After 5–10 min at 37 C, LDL-ferritin is observed in lysosomes as the result of their fusion with the incoming coated vesicles [643]. The rapid sequence of events visualized in electron micrographs precisely parallels biochemically derived data on the rapid uptake and degradation of radiolabeled LDL [644,645]. An additional, related factor that could be contributing to the rapid removal of LCM from the circulation, by different tumors, is the fact that vascular endothelial cells participate in LDL removal from blood [646]. Moreover, it has been reported by different investigators that growth factors, including tumor-related growth factors, increase LDL receptor expression on vascular endothelial cells (e.g., Refs. [614,647]); this finding raises the possibility that the endothelial cells arising during cancer-associated neovascularization may have high numbers of LDL receptors which could contribute further to the accumulation of LDL-associated substances, viz. LCM, within tumors. Hence, the high expression levels of LDL receptors occurring in many types of tumors (cf. above), and possibly within the tumor capillaries themselves, could lead to the arrest or assemblage of LCM within the tumor’s vascular supply. By this proposed endocytic mechanism of LCM uptake, the initial interaction between LCM and lipoprotein receptors would occur intravascularly, a logical hypothesis for a phenomenon that becomes readily measurable within 2 min following injection of the LCM into the bloodstream (cf. Chapters 12 and 13).
14.2 MULTILIGAND LIPOPROTEIN RECEPTORS As explained in a review by Krieger and Herz [648], LDL receptors and most other mammalian cell-surface receptors, which mediate endocytosis, exhibit two common ligand-binding characteristics: high affinity and narrow specificity. The ligand-binding properties of two more recently characterized lipoprotein receptors, that is, LDL receptor-related protein (LRP) and scavenger receptors, do not conform to a narrow binding specificity. These receptors bind, with high affinity, both lipoprotein and nonlipoprotein ligands and participate in a wide variety of biological processes.
14.2.1 LDL Receptor-Related Protein, on Tumor Cells, and LCM LRP is a member of the “LDL receptor gene family” [649] and, like the LDL receptor, performs an essential role in the removal of certain lipoprotein particles from the bloodstream. As Heeren et al. [650] explain, triglycerides are transported mainly by two distinct classes of lipoproteins, the chylomicrons
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and the VLDL. After assembly in the intestine, chylomicrons are carried via lymph into the bloodstream, where they are transformed at the endothelial surface to remnant lipoproteins through the catalytic action of lipoprotein lipase (for review, see Refs. [651,652]). After lipolysis, the lipoprotein lipase remains associated with the chylomicron remnants and, in conjunction with apolipoprotein E (apo E) [653–655], facilitates their clearance by the liver into hepatocytes [656] via LDL receptors and the LRP [657–660]. (The essential role for both receptors in chylomicron remnant removal in vivo has been demonstrated in gene knockout and gene transfer experiments [661,662] (for review, see Ref. [663].) The remarkable rapidity and specificity of uptake of the chylomicron remnant particles is believed to be highly dependent on their acquisition of apo E in the bloodstream [664,665]; the critical role played by apo E in directing the clearance of chylomicron remnants from the (blood) plasma has been well established by several lines of evidence [664]. Both the LDL receptor (also known as the “apo B,E receptor” [620]) and the LRP have a high affinity for apo E [666], and both receptors play an essential role in the receptormediated endocytosis of chylomicron remnants [650,665]. The chylomicron remnant particles themselves, derived from lipolysis of the larger chylomicrons (cf. above), contain the residual triglyceride and all of the cholesterol and CE from the original chylomicrons. This lipid composition of the chylomicron remnant particles is similar to the above-described lipid composition of both LDL particles (cf. Section 14.1) and LCM (cf. Section 12.1). Based upon this molecular similarity, it appears reasonable to expect that injected LCM could also readily bind apo E (i.e., as an alternative to apo B) in the bloodstream. In this case, the proposed LCM binding of apo E should influence the subsequent biodistribution of those LCM via two endocytic pathways: specifically, one pathway mediated by the LDL receptor (a.k.a. “apo B,E receptor”) (cf. Section 14.1) and the other pathway mediated by the LRP, since both receptor types have a high affinity for apo E (cf. above). As concerns links between apo E and tumors, Section 14.1 included a review of the increased expression and/or activity of LDL receptors on many cancer cells and tumor types. An analogous, but less widespread, pattern of receptor enhancement has been reported by various investigators for the LRP in tumor cells. One series of reports concerns human glioblastomas (a common type of glioma, or brain tumor) and glioblastoma cell lines. Yamamoto et al. [667] investigated immunohistochemical localization of LRP, on sequential frozen sections of human glioblastomas, and showed that the neoplastic glial cells and endothelial cells exhibited intense LRP immunoreactivity. Furthermore, they conclude that LRP is overexpressed in glioblastomas, and that LRP may play a role in facilitating glioblastoma invasiveness and neovascularization within tumor tissues [667]. Similarly, Bu et al. reported that human glioblastoma U87 cells express an abundance of LRP, and determined that LRP at the cell surface and along the cellular processes was
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functional in the binding and endocytosis of its ligands [668]. Finally, in a detailed, recent study by Maletinska et al. [669], the status of both LRP and LDL receptors was evaluated in seven human glioma (i.e., glioblastoma) cell lines. All were found to exhibit LRP, and cell lines SF-539, U-87 MG, and U-343 MG were particularly rich in this receptor. In addition, the presence of specific saturable LDL receptors was proven in six of the cell lines investigated, which had high concentrations of receptors (128,000–950,000 per cell). These authors conclude this finding suggests that LDL receptors on glioblastoma cells could potentially be useful for targeting antitumor agents; they further conclude that LRP, a multifunctional receptor expressed on glioblastoma cells, also has the possibility for serving as a therapeutic target [669]. Another major type of tumor cell examined for the presence of LRP was human breast cancer cells. Li et al. [670] examined six breast cancer cell lines and showed that LRP is expressed at a wide range of levels, that is, approximately 300–6300 sites per cell. Four of the breast cancer cell lines expressed LRP at over 1000 sites per cell, and were markedly invasive in their assay [670].
14.2.2 Scavenger Receptors on Tumor Cells as well as “Activated” Macrophages: LCM Binding, and Its Relation to Certain Disease Sites A second category of multiligand lipoprotein receptor, present in varying amounts in different tissues, consists of the scavenger receptors [671]. Various members of this receptor class were initially identified by their capability for rapid, unregulated uptake (by endocytosis) of chemically “modified” LDL by macrophages, leading to massive cholesterol accumulation [648,671]. The similarity of lipid composition between LDL particles and LCM (cf. Section 12.1) suggests that the LCM could also resemble a chemically “modified” LDL and, hence, act as a ligand for scavenger receptors. The proposed LCM binding with scavenger receptors would provide a third major endocytic pathway which can influence the biodistribution of LCM (subsequent to their binding of cell-surface receptors).
14.2.2.1 Tumor Cell Studies Indirect support for a proposed third available LCM-uptake mechanism (i.e., scavenger receptor-mediated endocytosis) is found in several studies reporting expression of scavenger receptors on certain types of tumor cells, for example, hepatoma [672], renal cell carcinoma [673], human adrenocortical carcinoma [631], human breast carcinoma [615], and histiocytic malignancies [674–676]. Furthermore, when the binding characteristics of these receptors were analyzed in detail in HepG2 (human hepatoma) cells, it was found that in this case the scavenger receptors bound both chemically modified LDL and native LDL [672]. Saturation binding experiments revealed
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moderate-affinity binding sites for both modified and native LDL, and competition binding studies at 4 C showed that both ligands share common binding site(s). Degradation/association ratios for these ligands show that LDLs are very efficiently degraded, and the chemically modified LDL degradation/ association ratio is equivalent to 60% of the LDL degradation ratio. Both lipoprotein ligands were good CE donors to HepG2 hepatoma cells. Finally, hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes [672].
14.2.2.2 Central Nervous System Injury While expression of scavenger receptors has been reported for some tumor cell types (cf. Section 14.2.2.1), there are certain other lesions which far more consistently display increased expression and/or activity of scavenger receptors (see below) on the cell surface. For these particular (noncancerous) disease/injury sites, the above-proposed (cf. Section 14.2.2) third LCM-uptake mechanism (i.e., scavenger receptor-mediated endocytosis) would appear well-suited for targeted drug-delivery therapy, via LCM, of that given disease or injury. An example of this type of lesion is central-nervous-system (CNS) injury, that is, brain injury and/or spinal cord injury (see below). As Bell et al. [677] observed in mouse brain, macrophage scavenger receptor expression on both microglia (the resident macrophages of the CNS) and recruited macrophages was detected 24 h after an intrahippocampal injection of either lipopolysaccharide or kainic acid (i.e., neurotoxic compounds). Macrophage scavenger receptor expression was also detected in microglia 3 days after optic nerve crush both in the nerve segment distal to the crush site and in the superior colliculus. (The monoclonal antibody 2F8 was used to localize the macrophage scavenger receptor by immunohistochemistry. In control adult mice, microglia did not express the receptor. Also, in the aged mouse brain, the pattern of macrophage scavenger receptor expression was no different from that in the young adult brain.) Hence, these studies indicate that the increased macrophage scavenger receptor expression, on microglia and recruited macrophages, arising after brain injury could play a role in the clearance of debris during acute neuronal degeneration [677]. At the same time, this increased receptor expression provides a possible avenue for LCM-targeted drug-delivery treatment of CNS injury sites. Furthermore, passage of LCM across the blood–brain barrier would be facilitated by the fact that scavenger receptors (both types I and II of class A) have been shown, by a reverse transcriptase PCR study of messenger RNA expression, to be present normally in (bovine) brain microvessels [678]. Brain microvessels therefore appear to play an active role in the uptake of native and modified low-density lipoproteins [678]; accordingly, these microvessels would be expected to have a similar role in LCM uptake (cf. Sections 12.1 and 14.2.2 above; see also below).
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Regarding one specific application of LCM to CNS injury, Kureshi et al. [535] reported the affinity of tail vein-injected LCM to injured rat spinal cord (due to a compressive lesion to the upper thoracic region). The accumulation of LCM in the injured spinal cord was analyzed, after labeling it with a lipidsoluble fluorescent dye (diO), by confocal laser scanning microscopy. It was observed that affinity of LCM for spinal cord injury sites appears to be mediated in the early stages after injury by proliferating macrophages in the necrotic center, and then in later stages by (glial fibrillary acidic protein [GFAP]-positive) activated astrocytes in adjacent white matter. These findings suggested a potential for using LCM as a delivery vehicle to concentrate lipid-soluble agents in spinal cord injury sites [535]. As another application of LCM to CNS injury, Ho et al. [533] studied the affinity of LCM to the site of a localized (thermal) brain injury. It had been well documented that in response to injury in the CNS, astrocytes are activated which is accompanied by an increased content of GFAP, hypertrophy, and hyperplasia [679–683]. (This process of gliosis [682] results in scar formation; it has been speculated that the scar may inhibit axonal regeneration [684]) [533]. Ho et al. observed that the influx of LCM began at the time when GFAP-positive cells began to appear. It seemed likely that LCM are initially attracted to the “reactive” astrocytes, but subsequently the LCM were found to be excluded from the area of scar formation. The LCM distribution detected by fluorescent (diO)-labeled LCM implied that the LCM are rapidly (within 10 min after the intravenous injection of LCM) taken up by some cells other than the astrocytes beginning 7–10 days after the injury. The round shape of these cells suggested that they might be blood borne (i.e., microglia) [533]. In follow-up to these findings with brain injury, the experiment program with LCM was expanded to next examine the use of LCM to deliver 7bhydroxycholesterol (7b-OHC) to a radiofrequency (thermal) lesion in the rat brain. With diO-labeled LCM, it was first reaffirmed that LCM target the injury area specifically and not the adjacent normal tissue. With immunohistochemistry and fluorescent immunochemistry, it was then demonstrated that the 7b-OHC, delivered in LCM, exerts an antigliosis effect in the rat brain injury model [534] (see below). 7b-OHC and other oxysterols have been reported, by other investigators, to inhibit astrogliosis as well as tumor cell proliferation both in vitro and in vivo [607–611]. Several groups have proposed different mechanisms for the cytotoxicity of these compounds [607,609–611]. Kupferberg et al. [611] first studied the cytotoxic effects of 7b-OHC and its derivatives on reactive astrocyte proliferation. Their group demonstrated that injection of liposomes containing 7b-OHC decreased the cholesterol biosynthesis by 40% in injured rat brain cortex, with a linear relationship between the cholesterogenesis and astroglial proliferation [607,610,611]. They concluded that oxysterols are potent inhibitors of the endogenous cholesterol biosynthesis, and this
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downregulation accounts for the inhibition of injury-induced astroglial proliferation, thereby reducing astrocytic reaction [534]. The data obtained in this follow-up study, based on immunohistochemical staining of GFAP-positive cells, and with fluorescent confocal microscopy techniques, indicate that both the number of GFAP-expressing astrocytes and the intensity of the staining were reduced when treated with 7b-OHC delivered by the LCM, while not affected by the same dose of intravenously injected 7bOHC in saline. Hence, since regenerating axons appear to be physically blocked by scar formation during healing [684,685], possibly antigliosis agents delivered directly to the injury sites in the optimal concentration (i.e., using targeted drug delivery via LCM) might assist in promoting functional repair [534]. The fact that the 7b-OHC injected alone did not exert the same effect indicates that LCM-bound 7b-OHC might be metabolized through a different pathway, that is, (scavenger) receptor-mediated endocytosis. As observed previously with the drug paclitaxel [532] (see also Section 13.2), delivery of a given drug via LCM appears to greatly magnify the concentration of that drug reaching the target site. Presumably, the mechanism of this enhanced delivery of 7b-OHC to the injury site by LCM shares common features with the receptor-mediated endocytic pathway mechanisms described earlier for the case of tumor cells (cf. Sections 14.1–14.2.2.1). The dissolved free drug, in this case 7b-OHC, would therefore not be expected to reach the desired target sites in as high a concentration compared to when it is carried by the LCM [534] (cf. first three paragraphs of this subsection).
14.2.2.3 Alzheimer’s Disease In Alzheimer’s disease, the characteristic lesions that develop, called senile plaques, are extracellular deposits principally composed of insoluble aggregates of b-amyloid protein (Ab) fibrils, infiltrated by reactive microglia and astrocytes [686]. Ab fibrils exert a cytotoxic effect on neurons and stimulate microglia to produce neurotoxins, such as reactive oxygen species [687]. Mononuclear phagocytes, including microglia, express scavenger receptors that mediate adhesion and/or endocytosis [687,688]; in particular, microglia have been shown to be intimately associated with amyloid deposits [688–690] and have also been implicated as scavengers responsible for clearing Ab fibril deposits of Alzheimer’s disease [688,689]. For example, Paresce et al. [689] reported that microglia in culture rapidly took up fluorescent-labeled Ab microaggregates into discrete vesicles, which were confirmed to be endosomes; at longer incubation times (30–60 min), Ab fluorescence became increasingly concentrated in organelles around the nucleus, consistent with delivery to late endosomes and lysosomes. Moreover, the uptake of Ab microaggregates by the microglia has been shown to be saturable, and is not fluid-phase internalization [689]. Accordingly, El Khoury et al. [687,691] identified class A scavenger receptors as the main cell-surface receptors mediating the interaction of microglia with b-amyloid fibrils. Adhesion of microglia to
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b-amyloid fibrils leads to immobilization of these cells on the fibrils, and induces or “activates” them to produce reactive oxygen species. These authors conclude that microglial scavenger receptors may be novel targets for therapeutic interventions in Alzheimer’s disease [691]. Similarly, a related review by Kalaria [692] provides additional data supporting the belief that microglia play a major role in the cellular response associated with the pathological lesions of Alzheimer’s disease, and concludes that pharmacological agents which suppress microglial activation may prove a useful strategy to slow the progression of Alzheimer’s disease [692] (see also Refs. [693–696]). In view of the above findings and since LCM have already been shown [534] (see also Section 14.2.2.2) to be an effective in vivo targeted drug-delivery vehicle for the antigliosis drug 7b-OHC to experimental injury sites in rat CNS, the LCM/7b-OHC complex may well similarly target the scavenger receptors of Alzheimer’s lesions and, thereby, reach the microglial cell surface and suppress microglial activation. While LCM can be and have been used to carry different lipid-soluble drugs, 7b-OHC is an appealing choice for an antigliosis drug. Specifically, 7b-OHC has a cytotoxic effect on different transformed glial cell lines (and probably also different types of activated, or reactive, glial cells [534]) which is useful since, as noted earlier [686,687,691], the senile plaques of Alzheimer’s disease are infiltrated by both reactive microglia and astrocytes [686]. This added involvement of astrocytes in Alzheimer’s disease is mentioned by various other researchers, such as Malhotra et al. who specifically indicate that the neuritic and amyloid cerebral cortical plaques of Alzheimer’s disease are surrounded by reactive astrocytes [697]. Similarly, El Khoury et al. point out that Alzheimer’s pathological features include neuronal degeneration, astrogliosis, microgliosis, and the extracellular accumulation of neurotoxic altered isoforms of b-amyloid [698] (cf. Refs. [693–696]). Based on these considerations, LCM-directed 7b-OHC delivery to the Alzheimer’s lesions may inhibit amyloid toxicity via gliosis and, potentially, reduce plaque pathology. Passage of the intravenously injected LCM/7b-OHC complex across the blood–brain barrier to Alzheimer’s lesion sites should be readily possible in view of the earlier-mentioned fact that scavenger receptors, both types I and II (of class A), have been shown to be present normally in (bovine) brain microvessels [678]. As stated in Section 14.2.2.2, brain microvessels therefore appear to play an active role in the uptake of native and modified LDL particles [678]; consequently, these microvessels would be expected to have a similar role in uptake of the LCM/7b-OHC complex.
14.2.2.4 Atherosclerotic Lesions The macrophage scavenger receptor family of proteins, including class A (types I and II) receptors and class B receptors, recognizes a wide variety of macromolecules (including modified LDL) which, after binding, can be
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internalized by endocytosis or phagocytosis (or, alternatively, remain at the cell surface and mediate adhesion or lipid transfer through caveolae) (e.g., Ref. [699]). In pathological states, members of this scavenger receptor family mediate the recruitment, activation and transformation of macrophages, and other cells which appear to be related to the development of not only Alzheimer’s disease but also atherosclerosis [699–703]. Lipid accumulation in the blood vessel wall depends on the intracellular uptake by macrophages, which transform into foam cells. Overloaded foam cells finally degenerate, leaving extracellular lipid deposits. The lipid overload of macrophages is brought about by several classes of scavenger receptors that, unlike (native-)LDL receptor, take up modified LDL and are not feedback controlled [701–705]. As one example, a major type of class B scavenger receptor is reported to be upregulated by the proatherogenic, modified LDL particles; hence, binding followed by uptake perpetuates a cycle of lipid accumulation and receptor expression [705]. Both class B [706,707] and class A [700,701] scavenger receptors are expressed in the lipid-laden macrophages in atherosclerotic lesions [704,708]. Furthermore, Nakagama-Toyama et al. [708] have reported on the differential distribution of the scavenger receptor types within human coronary atherosclerotic lesions. In view of the detailed published information available on the presence, functional characteristics, and localization of scavenger receptor populations in atherosclerotic lesions (cf. above) as well as the known structural similarity between modified LDL and LCM (cf. Sections 12.1 and 14.2.2–14.2.2.2), LCM-directed drug delivery to atherosclerotic lesions may offer a means for targeted drug-delivery therapy of atherosclerosis.