Browsing by Author "Panchoo, Marlyn"
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Item Cholesterol Metabolism in High Risk Neuroblastoma. Contributions by the SR-B1 Receptor and Cholesterol Ester Accumulation(2017-03-14) Spolitu, Stefano; Deligia, Stefania; Batetta, Barbara; Lacko, Andras G.; Panchoo, MarlynBackground: Cancer cells promote their survival by reprogramming metabolic pathways. Alterations in cholesterol metabolism have been observed as one of these mechanisms, including reduced levels of high-density lipoprotein (HDL) cholesterol in cancer patients. In high-risk neuroblastoma (HRNB), an extra cranial pediatric cancer, activation of genes associated with cholesterol synthesis has been reported. However, it is unclear whether modification in exogenous sources of cholesterol also occur in HRNB. An external source of cholesterol is from lipoproteins via the scavenger receptor class B type 1 (SR-B1) lipoprotein receptor that mediates the selective uptake of cholesteryl esters (CE) into the cell. In neuroblastoma high expression of SR-B1 correlates with poor prognosis. Objective: Our goal was to examine whether the accumulation of cholesteryl ester occurs in HRNB cells and whether this process is correlated with SR-B1 expression. Methods and Results: Human neuroblastoma cell lines expressing wild type p53; SH-SY-5Y, SMS-KCNR, and mutated p53; SK-N-BE (2), and BE (2) C were cultured. Western blot analysis confirmed the presence of the HDL receptor, SR-B1, and the multidrug resistant protein 1 (MDR1). Fluorescence staining of lipid droplets was performed using Nile Red. The neutral lipid content in SH-SY-5Y and SMS-KCNR, was higher at 24 hours than 72 hours suggesting that at 24 hours either increased accumulation or synthesis of neutral lipids occurred. To examine cholesterol esterification, cells were incubated with 14C-oleate. Cells were collected, lipids extracted with cold acetone and neutral lipids separated by thin layer chromatography. The results indicated notable reduction in cholesterol esterification in p53-mutated cells as compared to the wild type cells. However, triglyceride synthesis seems unaffected by p53 mutation when compared with the wild type. Conclusions: We confirm that there is accumulation of cholesteryl esters and high expression of SR-B1 in HRNB. These findings present a more in depth understanding of molecular mechanisms that drive progression of HRNB and may provide unique molecular targets to combat HRNB and other cancers. Future plans will include investigating cholesteryl ester accumulation and SR-B1 expression as a function of cell proliferation in HRNB. Additionally, human neuroblastoma tissue samples will be used to investigate the association between overexpression of SR-B1 and tumor aggressiveness.Item Overcoming Chemoresistance in Neuroblastoma Cells(2015-03) Panchoo, Marlyn; Sabnis, Nirupama; Lacko, Andras G.Background: The development of multi-drug resistance (MDR) in tumor cells continues to be a major challenge to effective cancer therapeutics. Regarding neuroblastoma (NB), a large proportion of the cases (~40%), designated as high risk NB (HRNB), present with a poor prognosis, with death occurring within 2-3 years of diagnosis. Most of these HRNB tumors become resistant and thus unresponsive during extended treatment, leading to the exceedingly poor prognosis. MDR is often due to the efflux (pumping out) of the drugs from the malignant cells via the ATP Binding Cassette (ABC) transporters. Hypothesis: The design of this study is based on the concept that the incorporation of the drug doxorubicin (Dox) into reconstituted high density lipoprotein (rHDL) nanoparticles will overcome MDR during treatment. This hypothesis also proposes a mechanism where delivery of Dox via the scavenger receptor type B1 (SR-B1), directly into the cytosol, will allow the drug to bypass the membrane MDR efflux pump and thus limit or eliminate drug resistance. Methods and Results: Drug resistance was induced in SMS-KCNR neuroblastoma cells by incubating the cells with 50 ng/ml of interleukin-6 (IL- 6) for five days. The IL-6 treated cells showed a 5 fold increase in resistance compared to the untreated cells. To evaluate the Dox encapsulated in rHDL (rHDL-Dox nanoparticles) for its ability to overcome MDR, the cytotoxicity of the free Dox vs. the encapsulated Dox was compared against resistant cells. The results showed that the rHDL-Dox formulation was more effective in killing the drug resistant cells than the free Dox (IC50 = 0.08 µg/ml vs. 0.52 µg/ml). These findings show that the rHDL-Dox formulation is indeed effective in limiting drug resistance. Conclusion: We anticipate that the increased sensitivity of MDR cells to rHDL-Dox nanoparticles, shown by these studies, could be extended to other drugs. Consequently, we also anticipate that these rHDL nanoparticle formulations could provide a safe and effective treatment for HRNB patients that otherwise would be resistant to therapy. Future plans will include screening of drug resistant NB cells for the expression of the SR-B1 receptor, monitoring of downstream events such as apoptosis, cell migration, and localization of the drug to document cytosolic delivery, in addition to studies with tumor carrying mice.Item Role of Scavenger Receptor Class B Type 1 in High-Risk Neuroblastoma(2017-12-01) Panchoo, Marlyn; Lacko, Andras G.; Jones, Harlan P.; Nair, Maya P.Neuroblastoma (NB), the most common extra-cranial childhood cancer in the United States is responsible for 15% of all pediatric cancer deaths. Due to advancements in treatment approaches, survival in low- and intermediate-risk NB patients now exceeds 90%. However, patient outcome for high-risk NB still remains poor with an overall survival of less than 50%. Nearly all high-risk NB patients present with metastatic disease at diagnosis and are unresponsive to intense chemotherapy, radiotherapy or aggressive surgery. Recently, the high-density lipoprotein (HDL) receptor, scavenger receptor class B type 1 (SR-B1), has emerged as an important indicator of cancer progression and patient outcome. Moreover, cancerous cells exhibit a higher expression of SR-B1 than normal non-malignant cells. SR-B1 is mainly responsible for the selective uptake of cholesteryl ester (CE) from HDL but also mediates reverse cholesterol transport. In this study, the expression of SR-B1 was identified on high-risk NB cells. Blocking of SR-B1 diminished cell proliferation, migration and invasion and induced apoptosis. Additionally, inhibition of SR-B1 reduced CE content in high-risk NB cells. Finally, high expression of SR-B1 in NB biopsy samples correlated with poor patient outcome. Taken together, this study identified SR-B1 expression as a potential regulator of high-risk NB progression linked to changes in cellular cholesterol metabolism. These findings also identify SR-B1 as a potential target for treatment of high-risk NB.Item The Therapeutic Role of rHDL Nanoparticles with Saquinavir in Fighting High Risk Neuroblastoma(2017-03-14) Panchoo, Marlyn; Sabnis, Nirupama; Lacko, Andras G.; Maharaj, AnshumaanHypothesis: Our hypothesis is to determine how effective rHDL nanoparticles with saquinavir are in killing neuroblastoma cells. Specifically, we are looking to see how they effect specific cell lines that do express the SR-B1 receptor versus cell lines that do not express the SR-B1 receptor. We are also measuring the effect of rHDL with saquinavir versus free saquinavir in killing neuroblastoma cells. Materials and Methods: The rHDL-saquinavir nanoparticles were prepared by cholate dialysis method, and their biochemical composition was determined using standard assay kits for the different components of the nanoparticle. The average size of the particles was measured using DeLsa Nano particle size analyzer. The cytotoxic effect of the rHDL-saquinavir combination versus free saquinavir was measured against a HRNB cell line, SMS-KCNR, using a CCK-8 kit. Results: The most pertinent result came from the cytotoxicity assay, which showed that free saquinavir was more effective than rHDL with saquinavir in killing neuroblastoma cells from the SMS-KCNR cell line, which has low expression of SR-B1. This is contrary to the previous experiment (from last years data on this same project) which showed that rHDL wth saquinavir was more effective than free saquinavir in killing neuroblastoma cells from the IMR-5 and SJNKP cell lines, which have high expression of SR-B1. Conclusions: From this project, we were able to conclude several things. First was that saquinavir was successfully incorporated into rHDL capsules to form a viable nanoparticle. The particle was also small enough to be incorporated into the cells. The biggest and main conclusion was that the SR-B1 receptor plays a key role in regulating uptake of the rHDL nanoparticles. Cells lines that had high expression of SR-B1 showed more uptake of the nanoparticles, and therefore more cell death. Cell lines that had low levels of expression of SR-B1 showed less uptake of the nanoparticles, and therefore less cell death. Free saquinavir showed more effective killing of cancer cells than encapsulated saquinavir when SR-B1 levels were low. Ergo, cancers that express high levels of SR-B1 can be targeted with encapsulated chemotherapeutic agents such as rHDL. Overall, rHDL nanoparticles are a novel therapeutic treatment strategy that can potentially be used in patients with high risk neuroblastoma as well as other forms of cancer.