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Despite the widespread use of nanotechnology in radio-imaging applications, lipoprotein based delivery systems have received only limited attention so far. These studies involve the synthesis of a novel hydrophobic radio-imaging tracer consisting of a hydrazinonicotinic acid (HYNIC)-N-dodecylamide and 99mTc conjugate that can be encapsulated into rHDL nanoparticles (NPs). These rHDL NPs can selectively target the Scavenger Receptor type B1 (SR-B1) that is overexpressed on most cancer cells due to excess demand for cholesterol for membrane biogenesis and thus can target tumors in vivo. We provide details of the tracer synthesis, characterization of the rHDL/tracer complex, in vitro uptake, stability studies and in vivo application of this new radio-imaging approach.

Gynecologic cancers (GC) affect millions of women of all ages globally each year. Apart from a crucial economic impact of these cancers on a society, it affects the emotional, physical, material, and social well-being of survivors and their kinfolks as well. GC diagnostics is often at advanced stages, restricting the options of therapeutics. In addition, patients with GCs undergoing chemotherapy often show resistance to chemotherapeutic drugs and recurrence. In fact, the largest hurdle in GC therapeutics is multidrug (chemo) resistance (MDR). Comprehending the key mechanisms involved in chemoresistance, innovative research in the area of targeted drug discoveries and identifying unique and effective drug targets/inhibitorsare necessary for the clinical management of GC patients efficiently. In this chapter, classical as well as novel molecular mechanisms of drug resistance in GCs, particularly ovarian, cervical, and endometrial cancers from relevant peer-reviewed literature, have been investigated. Finally, targeted drug deliveries to combat MDR in GCs are discussed in detail. The knowledge gained could assist design meaningful preclinical and translational studies to overcome MDR in GCs.

The tumor microenvironment (TME) plays a key role in enhancing the growth of malignant tumors and thus contributing to "aggressive phenotypes," supporting sustained tumor growth and metastasis. The precise interplay between the numerous components of the TME that contribute to the emergence of these aggressive phenotypes is yet to be elucidated and currently under intense investigation. The purpose of this article is to identify specific role(s) for lipoproteins as part of these processes that facilitate (or oppose) malignant growth as they interact with specific components of the TME during tumor development and treatment. Because of the scarcity of literature reports regarding the interaction of lipoproteins with the components of the tumor microenvironment, we were compelled to explore topics that were only tangentially related to this topic, to ensure that we have not missed any important concepts.

Reconstituted high-density lipoprotein (HDL) containing apolipoprotein A-I (Apo A-I) mimics the structure and function of endogenous (human plasma) HDL due to its function and potential therapeutic utility in atherosclerosis, cancer, neurodegenerative diseases, and inflammatory diseases. Recently, a new class of HDL mimetics has emerged, involving peptides with amino acid sequences that simulate the the primary structure of the amphipathic alpha helices within the Apo A-I protein. The findings reported in this communication were obtained using a similar amphiphilic peptide (modified via conjugation of a myristic acid residue at the amino terminal aspartic acid) that self-assembles (by itself) into nanoparticles while retaining the key features of endogenous HDL. The studies presented here involve the macromolecular assembly of the myristic acid conjugated peptide (MYR-5A) into nanomicellar structures and its characterization via steady-state and time-resolved fluorescence spectroscopy. The structural differences between the free peptide (5A) and MYR-5A conjugate were also probed, using tryptophan fluorescence, Fӧrster resonance energy transfer (FRET), dynamic light scattering, and gel exclusion chromatography. To our knowledge, this is the first report of a lipoprotein assembly generated from a single ingredient and without a separate lipid component. The therapeutic utility of these nanoparticles (due to their capablity to incorporate a wide range of drugs into their core region for targeted delivery) was also investigated by probing the role of the scavenger receptor type B1 in this process. SIGNIFICANCE STATEMENT: Although lipoproteins have been considered as effective drug delivery agents, none of these nanoformulations has entered clinical trials to date. A major challenge to advancing lipoprotein-based formulations to the clinic has been the availability of a cost-effective protein or peptide constituent, needed for the assembly of the drug/lipoprotein nanocomplexes. This report of a robust, spontaneously assembling drug transport system from a single component could provide the template for a superior, targeted drug delivery strategy for therapeutics of cancer and other diseases (Counsell and Pohland, 1982).

The tumor microenvironment (TME) plays a key role in enhancing the growth of malignant tumors and thus contributing to "aggressive phenotypes," supporting sustained tumor growth and metastasis. The precise interplay between the numerous components of the TME that contribute to the emergence of these aggressive phenotypes is yet to be elucidated and currently under intense investigation. The purpose of this article is to identify specific role(s) for lipoproteins as part of these processes that facilitate (or oppose) malignant growth as they interact with specific components of the TME during tumor development and treatment. Because of the scarcity of literature reports regarding the interaction of lipoproteins with the components of the tumor microenvironment, we were compelled to explore topics that were only tangentially related to this topic, to ensure that we have not missed any important concepts.

The physiological role(s) of mammalian plasma lipoproteins is to transport hydrophobic molecules (primarily cholesterol and triacylglycerols) to their respective destinations. Lipoproteins have also been studied as drug-delivery agents due to their advantageous payload capacity, long residence time in the circulation and biocompatibility. The purpose of this review is to briefly discuss current findings with the focus on each type of formulation's potential for clinical applications. Regarding utilizing lipoprotein type formulation for cancer therapeutics, their potential for tumor-selective delivery is also discussed.

Neuroblastoma (NB) is an extra cranial pediatric embryonal tumor most prevalent in children less than 1 year of age. NB accounts for 7% of all pediatric cancers but accounts for 15% of all childhood cancer deaths. Scavenger receptor class B type 1 (SR-B1), a mediator of cellular cholesterol uptake, is overexpressed in and have been linked to the aggressiveness of many cancers. Nevertheless, no studies have so far investigated the relationship between SR-B1 and NB. Elucidation of receptors that promote NB may pave the way for discovery of new therapeutic targets. Here we show that inhibition of SR-B1 reduced cell survival, migration and invasion, and cholesterol content in NB cell lines. Additionally analysis of SR-B1 levels in NB patient biopsies using the R2: Genomics Analysis and Visualization Platform showed that high SR-B1 expression correlated with decreased overall and event-free survival.

Based on data from the World Health Organization, breast cancer is the most common type of cancer among women, accounting for about 15% of all cancer-related deaths. Thus, new treatment options are urgently needed to decrease this mortality rate. In recent years, mesoionic compounds have shown promising potential as anti-cancer agents due to their unique structure and reaction properties. We recently reported that a 1,3-thiazolium-5-thiolate mesoionic compound (MIH 2.4Bl) inhibited oxidative phosphorylation in the MCF-7 breast cancer cell line compared to normal human mammary epithelial cells. Furthermore, MIH 2.4Bl induced cytotoxicity by activating autography-related proteins (Beclin-1 and ATG5) and cell cycle arrest at the G2/M phase. Based on our previous findings, MIH 2.4BI is a promising candidate for treating breast cancer. However, a major challenge facing cancer therapeutics is tumor delivery in vivo for the selective destruction of malignant cells while sparing normal cells to preserve tissue integrity. The development and use of drug delivery systems is a recognized approach to improve the efficacy of chemotherapy agents. Nonetheless, drug delivery systems have been largely unexplored in the context of mesoionic compounds. Lipoproteins are ideal for carrying transporting lipophilic anti-cancer drugs and imaging agents as they circulate in the bloodstream for an extended period. In addition, the hydrophobic core of lipoprotein particles allows the incorporation of lipophilic components (including a number of anti-cancer agents). Reconstituted high-density lipoprotein (rHDL) mimics the structure and function of endogenous (i.e., human plasma) HDL and thus presents a potentially markedly improved therapeutic strategy for cancer drug delivery. Previous studies from our group have shown that a stable reconstituted synthetic rHDL-drug complex could be prepared by combining paclitaxel and other chemotherapy drugs using the natural lipid and protein components of circulating HDL via a novel procedure. In this preliminary work, we present an improved strategy of using a newly developed formulation of MIH 2.4BI compound with rHDL nanoparticles as the delivery agent. Also, physico-chemical characterization of the nanoparticles and cytotoxicity analyses using a panel of breast cancer cell lines were performed. These studies support the potential therapeutic use of MIH 2.4Bl in treating breast cancer. (D. Debnath and R.M. Petty contributed equally to this work) Citation Format: Dipti Debnath, R. Max Petty, Nirupama Sabnis, Jinmin Zhang, Andras G. Lacko, Helivaldo Diógenes Souza, Petrônio Filgueiras Filho, J. Michael Mathis, Rafal Fudala. An improved strategy for delivering the mesoionic compound MIH 2.4Bl utilizing reconstituted high density nanoparticles (rHDL) in treating breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 300.

Patients with triple-negative breast cancer (TNBC) have a considerably less favorable prognosis than those with hormone-positive breast cancers. TNBC patients do not respond to current endocrine treatment and have a 5-year survival prognosis of, Video abstract

Current anti-angiogenic therapy for cancer is based mainly on inhibition of the vascular endothelial growth factor pathway. However, due to the transient and only modest benefit from such therapy, additional approaches are needed. Deregulation of microRNAs (miRNAs) has been demonstrated to be involved in tumor angiogenesis and offers opportunities for a new therapeutic approach. However, effective miRNA-delivery systems are needed for such approaches to be successful. In this study, miRNA profiling of patient data sets, along with in vitro and in vivo experiments, revealed that miR-204-5p could promote angiogenesis in ovarian tumors through THBS1. By binding with scavenger receptor class B type 1 (SCARB1), reconstituted high-density lipoprotein-nanoparticles (rHDL-NPs) were effective in delivering miR-204-5p inhibitor (miR-204-5p-inh) to tumor sites to suppress tumor growth. These results offer a new understanding of miR-204-5p in regulating tumor angiogenesis.

Purpose. The goal of these studies was to provide proof of concept for a novel targeted therapy for Glioblastoma Multiforme (GBM). Methods. These studies involve the evaluation of reconstituted high density lipoprotein (rHDL) nanoparticles (NPs) as delivery agents for the drug, mammalian Target of Rapamycin (mTOR) inhibitor Everolimus (EVR) to GBM cells. Cytotoxicity studies and assessment of downstream effects, including apoptosis, migration, and cell cycle events, were probed, in relation to the expression of scavenger receptor B type 1 (SR-B1) by GBM cells. Results. Findings from cytotoxicity studies indicate that the rHDL/EVR formulation was 185 times more potent than free EVR against high SR-B1 expressing cell line (LN 229). Cell cycle analysis revealed that rHDL/EVR treated LN229 cells had a 5.8 times higher apoptotic cell population than those treated with EVR. The sensitivity of GBM cells to EVR treatment was strongly correlated with SR-B1 expression. Conclusions. These studies present strong proof of concept regarding the efficacy of delivering EVR and likely other agents, via a biocompatible transport system, targeted to the SR-B1 receptor that is upregulated in most cancers, including GBM. Targeting the SR-B1 receptor could thus lead to effective personalized therapy of GBM.

Drug delivery to malignant tumors is limited by several factors, including off-target toxicities and suboptimal benefits to cancer patient. Major research efforts have been directed toward developing novel technologies involving nanoparticles (NPs) to overcome these challenges. Major obstacles, however, including, opsonization, transport across cancer cell membranes, multidrug-resistant proteins, and endosomal sequestration of the therapeutic agent continue to limit the efficiency of cancer chemotherapy. Lipoprotein-based drug delivery technology, “nature’s drug delivery system,” while exhibits highly desirable characteristics, it still needs substantial investment from private/government stakeholders to promote its eventual advance to the bedside. Consequently, this review focuses specifically on the synthetic (reconstituted) high-density lipoprotein rHDL NPs, evaluating their potential to overcome specific biological barriers and the challenges of translation toward clinical utilization and commercialization. This highly robust drug transport system provides site-specific, tumor-selective delivery of anti-cancer agents while reducing harmful off-target effects. Utilizing rHDL NPs for anti-cancer therapeutics and tumor imaging revolutionizes the future strategy for the management of a broad range of cancers and other diseases.

In cancer treatment, there is a need to improve chemotherapeutic drug uptake at the local tumor site to both enhance dose efficacy and minimize any toxic side effects. It is well known now that stably cavitating microbubbles (MBs) in an ultrasound (US) field can increase the permeability of tumor blood vessel walls. This consequently can enhance the extravasation and accumulation of a circulating anticancer drug. Reconstituted high-density lipoprotein (rHDL) nanoparticles (NPs) allow selective delivery of anticancer agents to tumor cells via the overexpressed scavenger receptor class B type 1 (SR-B1). The goal of this study was to evaluate the potential of US-stimulated drug delivery for improving tumor uptake of rHDL NPs that were preloaded with IR-780 dye (i.e., drug surrogate). Mice implanted with human breast cancer cells were divided into five groups: control, and low- or high-dose rHDL NPs ± US therapy. In vivo fluorescence optical imaging was used to quantify intratumoral NP accumulation at baseline and again at 1, 30, 60 min, 24 and 48 h after dosing. At 48 hours after treatment, tumors were resected and subjected to a fluorescent dye extraction study. Results of in vivo optical imaging revealed that the US therapy group animals exhibited stronger rHDL dye localization in the tumor area comparing to non-US groups. Moreover, in the dye extraction study, higher dye levels were detected in the US therapy groups compared to the non-US exposed animals given the same rHDL doses. Meanwhile, administration of a higher dose of rHDL NPs promoted dye accumulation in tumor sites. Overall, US-stimulated drug delivery can noninvasively promote the improved local tumor uptake of rHDL NP vectors.

We studied steady-state and time-resolved fluorescence properties of an anticancer drug Doxorubicin in a saline buffer and poly-vinyl alcohol (PVA) film. Absorption of Doxorubicin, located at blue-green spectral region, allows a convenient excitation with visible light emitting diodes or laser diodes. Emission of Doxorubicin with maximum near 600nm can be easily detected with photomultipliers and CCD cameras. Both, absorption and fluorescence spectra in polymeric matrix show more pronounced vibronic structures than in solution. Also, the steady-state anisotropy in the polymer film is significantly higher than in the saline solution. In PVA film the fluorescence anisotropy is about 0.30 whereas in the saline buffer only 0.07. Quantum efficiencies of Doxorubicin were compared to a known standard Rhodamine 101 which has fluorescence emission in a similar spectral region. The quantum yield of Doxorubicin in PVA film is more than 10% and about twice higher than in the saline solution. Similarly, the lifetime of doxorubicin in PVA film is about 2 ns whereas in the saline solution only about 1 ns. The fluorescence anisotropy decays show that Doxorubicin molecules are freely rotating in the saline buffer with a correlation time of about 290 ps, and are almost completely immobilized in the PVA film. The spectroscopic investigations presented in this manuscript are important, as they provide answers to changes in molecular properties of Doxorubicin depending changes in the local environment, which is useful when synthesizing nano-particles for Doxorubicin entrapment.

Sarika Sabnis,1 Nirupama A Sabnis,1 Sangram Raut,2 Andras G Lacko1,3 1Institute of Cardiovascular and Metabolic Diseases, University of North Texas Health Science Center, 2Department of Physics, Texas Christian University, 3Department of Pediatrics, University of North Texas Health Science Center, Fort Worth, TX, USA Abstract: Current cancer chemotherapy is frequently associated with short- and long-term side effects, affecting the quality of life of cancer survivors. Because malignant cells are known to overexpress specific surface antigens, including receptors, targeted drug delivery is often utilized to reduce or overcome side effects. The current study involves a novel targeting approach using specifically designed nanoparticles, including encapsulation of the anti-cancer drug valrubicin into superparamagnetic iron oxide nanoparticle (SPION) containing reconstituted high-density lipoprotein (rHDL) nanoparticles. Specifically, rHDL–SPION–valrubicin hybrid nanoparticles were assembled and characterized with respect to their physical and chemical properties, drug entrapment efficiency and receptor-mediated release of the drug valrubicin from the nanoparticles to prostate cancer (PC-3) cells. Prussian blue staining was used to assess nanoparticle movement in a magnetic field. Measurements of cytotoxicity toward PC-3 cells showed that rHDL–SPION–valrubicin nanoparticles were up to 4.6 and 31 times more effective at the respective valrubicin concentrations of 42.4µg/mL and 85µg/mL than the drug valrubicin alone. These studies showed, for the first time, that lipoprotein drug delivery enhanced via magnetic targeting could be an effective chemotherapeutic strategy for prostate cancer. Keywords: drug delivery, nanoparticles, rHDL, magnetic nanoparticles, SPION

Malignant tumors display remarkable heterogeneity to the extent that even at the same tissue site different types of cells with varying genetic background may be found. In contrast, a relatively consistent marker the scavenger receptor type B1 (SR-B1) has been found to be consistently overexpressed by most tumor cells. Scavenger Receptor Class B Type I (SR-BI) is a high density lipoprotein (HDL) receptor that facilitates the uptake of cholesterol esters from circulating lipoproteins. Additional findings suggest a critical role for SR-BI in cholesterol metabolism, signaling, motility, and proliferation of cancer cells and thus a potential major impact in carcinogenesis and metastasis. Recent findings indicate that the level of SR-BI expression correlate with aggressiveness and poor survival in breast and prostate cancer. Moreover, genomic data show that depending on the type of cancer, high or low SR-BI expression may promote poor survival. This review discusses the importance of SR-BI as a diagnostic as well as prognostic indicator of cancer to help elucidate the contributions of this protein to cancer development, progression, and survival. In addition, the SR-B1 receptor has been shown to serve as a potential gateway for the delivery of therapeutic agents when reconstituted high density lipoprotein nanoparticles are used for their transport to cancer cells and tumors. Opportunities for the development of new technologies, particularly in the areas of cancer therapy and tumor imaging are discussed.

Nirupama Sabnis1, Maya Nair1, Mervyn Israel2, Walter J McConathy3, Andras G Lacko11University of North Texas Health Science Center, Fort Worth, TX, 2University of Tennessee Health Science Center, Memphis, TN, 3Texas Tech University Health Sciences Center–Permian Basin, Odessa, TX, USAAbstract: Among numerous drug-delivery approaches, reconstituted high-density lipoprotein (rHDL) nanocarriers have proven particularly applicable for delivering highly hydrophobic drugs. In this study, we have investigated the enhancement of the therapeutic impact of valrubicin (AD-32), an antineoplastic agent that has been limited to intravesicular application against bladder cancer, despite the encouraging original preclinical data. Earlier studies validated the superior therapeutic efficacy of AD-32 over doxorubicin. In the present study, rHDL/AD-32 nanoparticles were formulated and characterized with regard to encapsulation efficiency, physicochemical properties, selective toxicity, and receptor-mediated uptake. The half maximal inhibitory concentration values (IC50) for rHDL/AD-32 nanoparticles were 1.8 and 2.6 times lower than the free AD-32 for prostate (PC-3) and ovarian (SKOV-3) cancer cell lines, respectively, whereas nonmalignant cell lines demonstrated 5 and 1.48 times higher IC50 doses with rHDL/AD-32 formulations. The data obtained demonstrated effective receptor-mediated uptake of AD-32 from the rHDL nanocarriers by PC-3 and SKOV-3 cancer cells via a targeted drug-delivery process. The rHDL/AD-32 formulation was stable for 6 months when stored at 4°C or at –20°C, as 92% of the AD-32 was retained in the nanoparticles. The findings from this study show that the rHDL/AD-32 formulation can overcome the solubility barriers of AD-32 and thus serve as an effective systemically administered chemotherapeutic agent.Keywords: AD-32, rHDL, nanoparticles, targeted drug delivery, selective drug delivery

Nanoparticles are target-specific drug delivery agents that are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Valrubicin is an anti-cancer drug, currently approved only for vesicular bladder cancer treatment because of its poor water solubility. On the other hand, valrubicin carrying reconstituted high density lipoprotein (rHDL) nanoparticles appear ideally suited for extended applications, including systemic cancer chemotherapy. We determined selected fluorescence properties of the free (unencapsulated) drug vs. valrubicin incorporated into rHDL nanoparticles. We have found that upon encapsulation into rHDL nanoparticles the quantum yield of valrubicin fluorescence increased six fold while its fluorescence lifetime increased about 2 fold. Accordingly, these and potassium iodide (KI) quenching data suggest that upon incorporation, valrubicin is localized deep in the interior of the nanoparticle, inside the lipid matrix. Fluorescence anisotropy of the rHDL valrubicin nanoparticles was also found to be high along with extended rotational correlation time. The fluorescence of valrubicin could also be utilized to assess its distribution upon delivery to prostate cancer (PC3) cells. Overall the fluorescence properties of the rHDL: valrubicin complex reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy.

This review is intended to evaluate the research findings and potential clinical applications of drug transport systems, developed based on the concepts of the structure/function and physiological role(s) of high density lipoprotein type nanoparticles. These macromolecules provide targeted transport of cholesteryl esters (a highly lipophilic payload) in their natural/physiological environment. The ability to accommodate highly water insoluble constituents in their core regions enables High density lipoproteins (HDL) type nanoparticles to effectively transport hydrophobic drugs subsequent to systemic administration. Even though the application of reconstituted HDL in the treatment of a number of diseases is reviewed, the primary focus is on the application of HDL type drug delivery agents in cancer chemotherapy. The use of both native and synthetic HDL as drug delivery agents is compared to evaluate their respective potentials for commercial and clinical development. The current status and future perspectives for HDL type nanoparticles are discussed, including current obstacles and future applications in therapeutics.

Objective. Assess the prevalence of abnormal glucose metabolism among Hispanic parents of children with acanthosis nigricans (AN). Methods. Hispanic families (n=258) were evaluated for metabolic and anthropometric parameters including fasting glucose levels and AN status. Results. Mothers with AN+ children had IFG (17.3%) and 4% had glucose levels ≥126 mg/dL (P=0.028) compared to 7.1% and 1.8% of mothers with AN− children, respectively. Mothers of AN+ children also had greater odds of having impaired fasting glucose levels (OR: 3.917, 95% CI: 1.475–10.404; P<0.004) but this was not the case for fathers (OR: 1.125, 95% CI: 0.489–2.586; P=0.781). Mothers of AN+ children were also more likely to be AN+ (OR: 5.76, 95% CI: 2.98–11.13, P<0.001). Screening discovered glucose levels >126 mg/dL in 9% of fathers with AN+ children. Conclusions. Hispanic mothers of AN+ children are at higher risk of carbohydrate metabolism abnormalities. AN in children can be a marker for prevention and delay programs aimed at identifying adults at risk for diabetes.

The purpose of these studies was to determine the mechanism(s) whereby paclitaxel (PTX), is taken up by cancer cells, once encapsulated into synthetic/reconstituted high density lipoprotein (rHDL). The uptake of PTX was found to be facilitated by the scavenger receptor type B-1 (SR-B1) when drug-loaded rHDL particles were incubated with cells that express the SRB1 receptor. Studies with double-labeled, PTX containing rHDL nanoparticles showed that prostate cancer (PC-3) cells incorporated PTX primarily via a selective (SR-B1 type) uptake mechanism. In the presence of a 10-fold excess of plasma HDL, PTX uptake decreased to 30% of the control. These findings suggest that the incorporation of lipophilic drugs by cancer cells from rHDL nanoparticles is facilitated by a receptor mediated (SR-B1) mechanism.

The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 microM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 microM(-1) and for UV excitation at 295 nm was 3.2 microM(-1). In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.

Reconstituted (synthetic) high-density lipoprotein particles carrying paclitaxel (rHDL/PTX) were prepared with substantially higher PTX content than reported earlier. The rHDL/PTX complexes seemed to be primarily spherical nanoparticles when examined via electron microscopy, with a constant composition, molecular weight and exceptional stability even after ultracentrifugation and storage for up to 6 months. The rHDL/PTX nanoparticles had superior cytotoxicity against several cancer cell lines (MCF7, DU145, OV1063 and OVCAR-3), the half maximal inhibitory concentration (IC50) having been found to be 5-20 times lower than that of the free drug. Studies with mice showed that the rHDL/PTX nanoparticles were substantially better tolerated than the corresponding dosages of either Taxol or Abraxane.

Fluorescence properties of a novel homodimeric BODIPY dye rotor for Fluorescence Lifetime Imaging Microscopy (FLIM) are reported. Steady state and time resolved fluorescence measurements established the viscosity dependent behaviour in vitro. Homodimeric BODIPY embedded in different membrane mimicking lipid vesicles (DPPC, POPC and POPC plus cholesterol) is demonstrated to be a viable sensor for fluorescence lifetime based viscosity measurements. Moreover, SKOV3 cells readily endocytosed the dye, which accumulated in membranous structures inside the cytoplasm thereby allowing viscosity mapping of internal cell components.

To assess risk factors for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) with the hypothesis that risk for T2DM in children would be associated with an increase in risk factors for CVD.Subjects from a group of Mexican-American school children (aged 10-12 years) identified to be at risk for T2DM, and their siblings, were selected for this study. There were 68 children with acanthosis nigricans (AN+), and 71 without AN (AN-). Both AN+ and AN- children were assessed for T2DM and CVD risk factors. Probands and siblings were evaluated by physical examination, family history, and fasting serum parameters: glucose, insulin, body mass index (BMI), serum lipoproteins, and oxidized lipids. Data were analyzed by descriptive, univariate, and multivariate procedures.BMI, waist/hip ratio, systolic and diastolic blood pressure were all significantly higher (p.002) in AN+, whereas Tanner stages were similar in both groups. Fasting serum glucose was in the normal range, whereas insulin was elevated in AN+ compared with AN- (30.0 +/- 1.9 microU/mL vs. 14.8 +/- 1.0 p.0001). Insulin resistance as assessed by the homeostasis assessment model (HOMA-IR) was elevated in both groups, although higher among AN+ (p.0001). High-density lipoprotein-cholesterol (HDL-C) was lower (6.2 mg/dL) in the AN+ group (p.003). The lower HDL-C in AN+ was associated with elevated triglycerides and a higher serum total cholesterol TC/HDL-C ratio when contrasted with the AN- values (145.9 +/- 7.6 mg/dL vs. 97.1 +/- 0.07, p.0001; 4.1 +/- 0.2 vs. 3.4 +/- 0.1, p.0001, respectively). In addition to the high prevalence of overweight/obesity (BMI85th percentile) in this population (76.3%, 106/139), elevated insulin (59.7%15 microU/mL), low HDL-C (27.3%40 mg/dL), and elevated low-density lipoprotein cholesterol (LDL-C) (41.0%100 mg/dL) were also detected.The altered metabolic pattern observed in this group of Mexican-American children is characteristic of metabolic syndrome, a condition associated with obesity and increased risk for both T2DM and CVD in adults. This study points to the value of BMI and acanthosis nigricans as easily accessible markers for children and nuclear families at increased risk for developing T2DM and CVD.

Ewing sarcoma (ES) is one of the most frequent primary bone and soft tissue tumors that occur in pediatric and adolescent population. ES is often diagnosed after the disease has already metastasized. With available treatment options, the prognosis of metastatic ES patients is poor and the 5-year survival rate is less than 20%. Therefore, identifying precise targeted therapies to induce therapeutic response in ES patients is urgently needed. An oncogenic fusion protein and transcription factor EWS-FLI1 is associated with more than 85% of ES tumors. Mithramycin has been identified as an effective agent to target EWS-FLI1 and is currently in clinical trials. In this study, we investigated the efficacy of Mithramycin to induce the anti-proliferative activity of chemotherapeutic agents commonly used for the treatment of this malignancy using human ES cell lines. TC71, TC32, CHLA32, CHLA10 and TC205 cells were treated with increasing concentrations of Mithramycin or Vincristine or Doxorubicin or Etoposide. Cell growth inhibition was evaluated at 24 and 48 h post-treatment using CellTiter Glo kit. Results showed a dose/time-dependent anti-proliferative effect for all agents. To confirm the effect of Mithramycin on selected cell lines, mRNA expression of downstream targets of EWS-FLI1 (ID2, LDB2, NROB1 and RCOR1 genes) was determined by quantitative PCR following treatment with Mithramycin. Mithramycin significantly down-regulated all tested genes and these results are in agreement with published work. In order to determine the combination response, cell growth inhibition of the chemotherapeutic drugs was assessed in the presence of added Mithramycin. The combination Index was evaluated by Chou-Talalay method. Further studies were performed for the combination of Mithramycin and Vincristine to investigate its effect on apoptosis and cell cycle arrest using CHLA10 and TC205 cells. Apoptotic markers such as the expression of cleaved Poly (ADP-ribose) polymerase (c-PARP) and survivin were determined by Western blot analysis. The apoptotic cell population was measured by Annexin-V staining using flow cytometry. When compared to individual agents, the combination of Mithramycin and Vincristine showed greater effect on apoptosis in both cell lines as evinced by an increase in Annexin-V positive cells, decrease in survivin expression and up-regulation of c-PARP. Overall, these results indicate the potential clinical advantage of combination treatment involving Mithramycin along with other chemotherapeutic agents for inhibiting the growth of ES cells. Molecular profiling analysis is underway to elucidate the candidate pathways associated with beneficial effect of this combination treatment. Note: This abstract was not presented at the meeting. Citation Format: Anish Ray, Bhavani Nagarajan, Umesh T. Sankpal, Sagar Shelake, W. Paul Bowman, Andras Lacko, Riyaz M. Basha. Mithramycin induces the antiproliferative activity of chemotherapeutic agents in Ewing sarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1532. doi:10.1158/1538-7445.AM2017-1532

The two major forms of leukemia, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) account for about one third of the malignancies diagnosed in children. Despite the marked successes in ALL and AML treatment, concerns remain regarding the occurrence of resistant disease in subsets of patients the residual effects of therapy that often persist for decades beyond the cessation of treatment. Therefore, new approaches are needed to reduce or to avoid off target toxicities, associated with chemotherapy and their long term residual effects. Recently, nanotechnology has been employed to enhance cancer therapy, via improving the bioavailability and therapeutic efficacy of anti-cancer agents. While in the last several years, numerous review articles appeared detailing the size, composition, assembly and performance evaluation of different types of drug carrying nanoparticles, the description and evaluation of lipoprotein based drug carriers have been conspicuously absent from most of these major reviews. The current review focuses on such information regarding nanoparticles with an emphasis on high density lipoprotein (HDL)-based drug delivery systems to examine their potential role(s) in the enhanced treatment of children with leukemia.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of plasma lipoprotein cholesterol in mammals as part of the reverse cholesterol transport pathway. Studies of the natural mutations of LCAT revealed a region that is highly sensitive to mutations (residues 121–136) and it is highly conserved in six animal species. The purpose of these studies was to investigate the reactivity of wild type and several mutated forms of LCAT, with a series polyclonal antibodies to further characterize this specific domain (residues 121–136). Two polyclonal antibodies directed against the whole enzyme, one against human plasma LCAT and the other against purified recombinant LCAT, and one site specific polyclonal antibody, directed against the 121–136 region of LCAT, were employed. All three antibodies reacted with a recombinant form of purified LCAT; however, only the polyclonal antibodies directed against the whole enzyme were able to recognize the LCAT when it was adsorbed to a hydrophobic surface in a solid phase immunoassay, or when bound to HDL in a sink immunoassay. These findings indicate that the epitope(s) of the 121–136 region are not accessible to antibodies under these conditions. Three mutant forms of LCAT, representing alterations in the 121–136 region, were also examined for their immunoreactivity with the same panel of antibodies and compared to the wild-type enzyme. These studies demonstrate that in its native configuration the 121–136 region of LCAT is likely to reside on a surface of LCAT. Furthermore, mutations within this region appear to markedly impact the exposure of epitopes at additional sites. These findings suggest that the 121–136 region could play an important role in enzyme interaction with its hydrophobic lipoprotein substrates as mutations within this region appear to alter enzyme conformation, catalytic activity, and the specificity of LCAT.

We examined the effects of dietary fats with specific fatty acid compositions, on serum paraoxonase (PON1) activity in rats. Male adult Sprague-Dawley rats were divided randomly into four dietary groups. One group received the control diet [AIN 93M with soybean oil (5 g/100 g diet)], whereas the remaining three groups received the modified control diet supplemented with (15 g/100 g diet) triolein, tripalmitin or fish oil, respectively. After 20 d, blood was obtained after overnight food deprivation and PON1 activity was determined. Serum lipids and lipid components of lipoproteins were also determined. Serum PON1 activity [micromol/(L.min)] was significantly (P:0.05) higher in triolein (98 +/- 6) and lower in fish oil (41 +/- 4), compared with tripalmitin-fed rats (63 +/- 11). Serum PON1 activity in tripalmitin-fed rats was comparable to that of controls (67 +/- 9). Serum PON1 activity correlated significantly with serum lecithin:cholesterol acyltransferase (LCAT) activity (r = 0.77, P:0.001) and was transported in blood principally in association with the denser subfraction of HDL, very high density lipoprotein (VHDL; d1.15 kg/L). Serum PON1 activity correlated strongly with serum lipids as well as lipids of VLDL, HDL and its subfractions. Multiple linear regression analysis, however, showed a significant relationship of serum PON1 activity, principally with the phospholipids of VHDL (r = 0.47, P:0.002). These data suggest that the modulation of serum PON1 activity by dietary fat may be mediated via the effect of the specific fatty acids on the synthesis and secretion of VHDL, the subfraction of HDL that transports the majority of PON1 in the blood.

Abstract —We studied the effect of hyperbaric oxygen (HBO) treatment on the extent of diet-induced accumulation of lipid oxidation products in rabbit plasma and tissues, on plasma paraoxonase activity, and on the extent of progression and regression of atherosclerotic lesions in the rabbit aorta. HBO treatment of cholesterol-fed rabbits dramatically reduces the development of arterial lesions despite having little or no effect on plasma or individual lipoprotein cholesterol concentrations. Compared with no treatment in cholesterol-fed animals, HBO treatment also substantially reduces the accumulation of lipid oxidation products (conjugated dienes, trienes, and thiobarbituric acid–reactive substances) in plasma, in the low density lipoprotein and high density lipoprotein fractions of plasma, in the liver, and in the aortic tissues. In addition, HBO treatment prevents the decrease in plasma paraoxonase activity observed in rabbits fed cholesterol-rich diets. Similarly, in regression studies, HBO treatment has no effect on the rate of plasma (or lipoprotein) cholesterol decline but significantly accelerates aortic lesion regression compared with no treatment. Direct measures of aortic cholesterol content support these morphological observations. On the basis of these results, we conclude that repeated, but relatively short, exposure to HBO induces an antioxidant defense mechanism(s) that is responsible for retarding the development or accelerating the regression of atherosclerotic lesions.

Apolipoprotein D (apoD) levels were examined in the temporal cortex as well as an assessment of the location of apoD positive cells within the brain by immunohistochemical and biochemical methods in young control (YC), aged control (AC), and Alzheimer's demented (AD) probands. Scattered apoD positive astrocytes and oligodendrocytes were found throughout the white matter by immunohistochemistry. ApoD immunoreactivity was also observed in the cerebellar oligodendrocytes of the YC group. There was faint positive apoD staining in scattered cortical astrocytes and a few neurons in the same group. In contrast, some of the AC and all of the AD probands had intense and frequent apoD immunostained cortical astrocytes and pyramidal neurons. The cortical senile plaques and neurofibrillary tangles were apoD immunonegative. No quantitative differences were found between the cortical apoD levels in the AC and AD groups, determined by immunoblotting. ApoD detected in the brain tissue was different in molecular weight (29 kDal) from that seen in CSF or in the serum (32 kDal). Our results indicate apoD is present in the human brain, especially in glial cells, and has increased abundance in the elderly and AD subjects.

Several immunoassay models, including sandwich ELISA, solid phase ELISA and sink immunoassay and antibody combinations were investigated to develop an ELISA assay for LCAT with an appropriate linear range and sensitivity. Solid phase immunoassays were found to be most suitable for measuring LCAT from cell culture medium and in partially purified preparations. The immunoassays were analyzed for matrix interference, recovery studies, intra-run precision and inter-run precision. These studies have identified a reliable method for measuring LCAT in purified preparations and cell culture media, and provide the foundation for further development of immunoassays for clinical application.

There is now sufficient evidence to suggest that cardiovascular pathology and altered lipid metabolism contribute to the development of late-onset Alzheimer's disease (AD). In the present study, 24 AD patients and 15 controls were assessed for cardiovascular risk based on serum lipid and lipid oxidation parameters. The AD patients appeared to have a more favorable cardiovascular risk profile than the controls based on high-density lipoprotein cholesterol (HDL-C) values. The levels of thiobarbituric-acid-reactive substances and the activity of the enzyme paraoxonase (PON) following copper oxidation indicate that female patients may have better protection against serum and perhaps tissue oxidants than males with AD. While the higher HDL-C values indicate lower cardiovascular risk, additional data on oxidized lipid parameters suggest a lower level of protection against serum oxidants in male AD probands. CopyrightCopyright 1999S.KargerAG,Basel

This report represents the continuation of our studies on the effects of gemfibrozil therapy on high density lipoprotein cholesterol levels. Previously, we reported that despite an impressive mean increase in high density lipoprotein cholesterol (20 %), the response to 12 weeks of gemfibrozil therapy was highly variable. Accordingly, out of the 27 subjects studied, five actually had lower high density lipoprotein cholesterol at the conclusion of therapy compared to baseline values. The changes observed in plasma lipids, combined with correlational relationships suggest that the conversion of triglyceride rich lipoprotein components into high density lipoprotein may be impaired in those subjects that respond poorly or negatively to gemfibrozil therapy.

The major N-linked carbohydrate structures were determined for recombinant human plasma lecithin:cholesterol acyltransferase (LCAT). The analysis of the structure of oligosaccharides by fast atom bombardment mass spectrometry (FAB-MS) and linkage analysis was preceded by reduction and carboxymethylation of the intact glycoproteins and digestion with trypsin and proline specific endopeptidase. The N-glycans were subsequently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak® cartridge. The data from the combination of FAB spectrometry and linkage analysis show that the N-linked glycans present on recombinant LCAT (rLCAT) were composed primarily of triantennary and tetraantennary structures with and without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or more antennae. The LCAT activities of both recombinant and circulating forms of plasma LCAT were determined using low molecular weight and lipoprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical. These findings validate the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundation for subsequent structural studies.—Lacko, A. G., A. J. Reason, C. Nuckolls, B. J. Kudchodkar, M. P. Nair, G. Sundarrajan, P. H. Pritchard, H. R. Morris, and A. Dell. Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties. J. Lipid Res. 1998. 39: 807–820.

Production and purification of recombinant human lecithin:cholesterol acyltransferase (LCAT), secreted by baby hamster kidney (BHK) cells, has been improved by limiting the harvesting times for the conditioned medium and introducing an additional purification step. The recombinant BHK cells were grown until nearly confluent on multilayered flasks in a fetal-calf-serum-enriched medium. Subsequently, the cells were washed and supplied with serum free medium for 24-h periods. The conditioned medium, containing recombinant LCAT, was harvested at 24 and 48 h and subjected to chromatography on phenyl-Sepharose and ACA-44 agarose to isolate the recombinant enzyme. The second chromatography step revealed the presence of a low-molecular-weight contaminant that exhibited a carbohydrate/protein composition similar to proteoglycans. The major purified component contained LCAT activity and was homogeneous by acrylamide gel electrophoresis.