\\MENT OF HEALTH & HUMAN
HHS Public Access Author manuscript Surgery. Author manuscript; available in PMC 2017 January 01.
Published in final edited form as: Surgery. 2016 January ; 159(1): 284-295. doi:10.1016/j.surg.2015.08.023.
sHDL Nanoparticles: A Novel Therapeutic Strategy for Adrenocortical Carcinomas
Chitra Subramanian1, Rui Kuai2,3, Qing Zhu1, Peter White1, James Moon2,3, Anna Schwendeman3,4, and Mark S. Cohen1,5
1Department of Surgery, University of Michigan, Ann Arbor, MI-48109
2Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI-48109
3Biointerfaces Institute, University of Michigan, Ann Arbor, MI-48109
4Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI-48109
5Department of Pharmacology, University of Michigan, Ann Arbor, MI-48109
Abstract
Background-Chemotherapeutic strategies for adrenocortical carcinoma (ACC) carry significant toxicities. Cholesterol is critical for ACC cell growth and steroidogenesis and ACC cells over-express scavenger receptor BI (SR-BI) that uptakes cholesterol from circulating high- density lipoprotein (HDL). We hypothesize that cholesterol-free synthetic-HDL nanoparticles (sHDL) will deplete cholesterol and synergize with chemotherapeutics to achieve enhanced anticancer effects at lower (less toxic) drug levels.
Methods-Anti proliferative efficacy of ACC cells for the combinations of sHDL with chemotherapeutics was tested by cell-Titer Glo. Cortisol levels were measured from the culture media. Effect on steroidogenesis was measured by RT-PCR. Induction of apoptosis was evaluated by flow cytometry.
Results-Combination-Index (CI) for sHDL and either etoposide(E), cisplatin(P) or mitotane(M) demonstrated synergy (CI<1) for anti-proliferation. sHDL alone or in combination with chemo drugs was able to reduce cortisol production by 70-90% compared to cisplatin alone or controls (p<0.01). RT-PCR indicated significant inhibition of steroidogenic enzymes for sHDL (p<0.01 vs. no sHDL). Combination therapy with sHDL increased apoptosis by 30-50% compared to drug or sHDL alone (p<0.03) confirmed by mitochondrial potential decrease.
Conclusion-sHDL can act synergistically and lower the amount of M/E/P needed for anticancer efficacy in ACC in part due to cholesterol starvation. This novel treatment strategy warrants further investigation translationally.
Address correspondence to: Mark S. Cohen MD, FACS, Associate Professor of Surgery and Pharmacology, PI Translational Oncology Program, Director of Endocrine Surgery Research, Department of Surgery, 2920K Taubman Center SPC 5331, University of Michigan Hospital and Health Systems, 1500 E. Medical Center Dr., Ann Arbor, MI 48109-5331, 734-615-4741, cohenmar@med.umich.edu.
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Introduction
Adrenocortical carcinoma is a rare endocrine malignancy (approximately 500 new cases per year in the US) that carries a poor prognosis with advanced disease (1). Unfortunately, a majority of patients will present with advanced disease at the time of diagnosis and once metastatic, the disease has a low 10-20% five-year survival (2). For patients with metastatic disease, the only current FDA approved therapeutic is the adrenolytic agent mitotane, with initial response rates of 20-30% in advanced ACC patients and an improvement in survival rate from 14-50 months (3). Recent years have evaluated mitotane in combination with cytotoxic chemotherapeutics as in the Italian protocol, (etoposide, doxorubicin, cisplatin; EDP) or with streptozotocin (4, 5). EDPM has been shown to carry a higher response rate (23.2% vs. 9.2%) and progression free survival (5.0 months vs. 2.1 months) compared to mitotane with streptozotocin (6-8). Dose-limiting toxicities such as adrenal insufficiency, dizziness, vertigo, central nervous disturbances, hyperlipidemia, and gastrointestinal disorders remain a significant issue with both mitotane and cytotoxic agents given in combination (4). Given this toxicity in combination, development of novel drugs that have the ability to synergize with these agents could allow lower concentrations needed to achieve the same therapeutic effect and potentially mitigate some toxicity.
Normal adrenal and ACC cells require cholesterol for steroidogenesis and are known to express the scavenger receptor class B type I (SR-BI) on their surface to obtain cholesterol esters from circulating HDL (9). This SR-BI receptor is highly over-expressed in ACC cells and several other cancers (breast, prostate, ovarian, lymphoma, nasopharyngeal carcinoma) compared to normal tissues. SR-BI receptors act as bidirectional cholesterol transporters that facilitate uptake of cholesterol into cells and efflux the cholesterol out of cells (10). Since cholesterol transport is an important biologic function of cells including cancer cells, mimetic sHDL nanoparticles that bind to SR-BI recently have come under focus as a novel approach for targeting cancer (11, 12). There are a number of cholesterol-free sHDL products that have been clinically tested for the treatment of atherosclerosis by facilitation of reverse cholesterol transport (RCT) and found to be safe at high doses of 20-40 mg/kg per infusion (13). Many advanced ACC patients will develop steroid over secretion (14). Since these steroids require cholesterol (15), an agent that effluxes cholesterol from cells may have therapeutic benefit in reducing this over secretion functionality. In the current study, we use cholesterol free sHDL nanoparticles and hypothesize that they may be able to generate anti- cancer properties by depleting cholesterol from ACC cells, which could synergize with chemotherapy drugs from the Italian protocol and thereby create novel combination strategies that may lower doses of these cytotoxic drugs needed to achieve the same anticancer benefit.
Methods
Cell lines
Two human ACC cell lines authenticated using genetic finger printing, NCI-H295R (cortisol secretor) and SW13 (non-steroid secretor), were grown in 2D culture in humidified atmosphere of 5% CO2 in air at 37°C. SW13 cells were grown in Dulbecco Modified Eagle’s Medium (DMEM; Life Technologies, Grand Island, NY) supplemented with 10% fetal
Surgery. Author manuscript; available in PMC 2017 January 01.
bovine serum (FBS; Sigma-Aldrich, St. Louis, MO) and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY). NCI-H295R cells were grown in DMEM-Ham’s F12 nutrient medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 1% insulin/transferrin/selenium (ITS) and 1% penicillin/ streptomycin (Life Technologies, Grand Island, NY).
Preparation and characterization of sHDL)
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), and 22A peptide (16) (weight ratio = 1:1:1) were dissolved in glacial acetic acid, which was removed by freeze-drying. Phosphate buffered saline (PBS pH = 7.4) was added to the freeze-dried powder, which then was cycled 3 times between 50°℃ (3min) and 20℃ (3min) with gentle shaking to obtain the sHDL. sHDL purity was analyzed by gel permeation chromatography (GPC). The sHDL was further characterized by transmission electron microscopy (TEM). All images were acquired on JEM 1200EX electron microscope (JEOL USA) equipped with an AMT XR-60 digital camera (Advanced Microscopy Techniques Corp).
Cell viability assay and calculation of combination index
SW13 and NCI-H295R cells were seeded into 96 well plates in triplicate and were treated with varying concentrations of either chemotherapeutic drugs (cisplatin, doxorubicin, etoposide, mitotane or EDPM combination) alone, in simultaneous combinations with sHDL, or sHDL alone for 72 h. A large dose range was initially used to define a more selective dose range for IC50 experiments. Serial dilutions were made from the starting concentrations and viability of cells was then measured based on quantification of the ATP levels after treatment with CellTiter-Glo luminescent assay reagent as per the manufacturer’s instruction (Promega, Madison, WI) with luminescence quantified using a BioTek Synergy Neo plate reader (BioTek, Winooski, VT). Cell viability ratios were calculated using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA) and the combination index (CI) was calculated using Chou-Talalay equation (16) using CompuSyn software (ComboSyn Inc., Paramus, USA). The CI values of less than 1, equal to 1 and greater than 1 represent synergistic, additive and antagonistic effects, respectively. For all cell-based experiments, the experimental control group was either untreated cells or cells treated with single drug alone (when comparing to combination regimens), unless otherwise stated.
Colony formation assay
NCI-H295R and SW13 cells were plated in 6-well plates and allowed to attach. Treatment commenced for 24 h with drug alone or in combination with sHDL (50µg of sHDL in terms of 22A peptide/ml). Untreated or sHDL alone-treated cells were controls. The medium was changed and surviving cells were allowed to grow colonies of 50 cells or more for two weeks, washed, fixed, and stained with coomassie blue and counted. Total colony numbers were normalized to untreated controls.
Analysis of apoptosis by flow cytometry
To analyze combination effect on apoptosis, SW13 and NCI-H295R cells grown in 60 mm plates were treated with either E, P, M alone or in combination with sHDL for 24 h. Following treatment, cells were washed, re-suspended in annexin binding buffer and stained using annexin V-FITC/Propidium iodide as previously described (17). Induction of apoptosis was measured using the CyAn ADP Analyzer (Beckman Coulter, Inc., Indianapolis, IN) at the University of Michigan Flow Cytometry Core.
Mitochondrial membrane potential
SW13 and NCI-H295R were seeded in a 96 well black wall plate. Once attached, they were treated with the drugs as described above. 24 h post drug treatment, 500 nM tetramethylrhodamine, ethyl ester (TMRE) was added, the cells were incubated for 20min at 37°C, and the fluorescent signal was measured after washing using a microplate reader (excitation=549, emission=575). 100nM FCCP (carbonyl cyanide 4- (trifluoromethoxy)phenylhydrazone) was added to cells 10min before the addition of TMRE as a negative control.
Immunoassay for cortisol measurement
The cortisol immunoassay (Alpco, Salem, NH) was used to quantitate cortisol levels in the culture supernatant of steroid producing NCI-H295R cells after treatment with drug combinations (same as in clonogic assay) for 24 h per the manufacturer instructions. Briefly, culture supernatant after treatment was added to the antibody coated-plates containing assay buffer (45min at 25℃). After washing, tetra methyl benzidine (TMB) substrate was incubated at RT and absorbance was measured using a Synergy Neo reader (BioTek, Winooski, VT).
mRNA isolation and real-time (RT)-PCR
RNA from the NCI-H295R cells after drug treatment for 24 h was prepared using Qiagen RNA isolation kit (Qiagen Sciences, Valencia, CA). Approximately 500 ng of RNA was reverse transcribed using superscript RT kit from Life Technologies (Grand Island, NY). qPCR was performed in a step-one RT PCR machine using the gene specific primer sets (Table 1A) as published (18). Relative gene expression levels were calculated after normalization with internal controls. SR-BI expression level in several cancers was confirmed by Western-Blot Analysis(Table 1B).
Three-dimensional Multicellular aggregates (MCAs) treatment
To evaluate the translational potential of the combination therapy, MCAs were developed to mimic the in vivo tumor model as described by Jain et al. (19). Approximately 50,000 SW13 or 100,000 NCI-H295R cells were plated in 24-well ultralow attachment plates (Corning, NY, USA) to generate MCAs. Once MCAs were generated, they were treated with drugs (concentrations specified in our colony formation assay) with or without 50ug/ml sHDL nanoparticle for 24 h. Untreated and sHDL alone treated cells served as controls. The MCAs were photographed before and after treatment and the MCAs were quantified by Image J software (NIH) (20) as described by Jain et al. (19).
Statistical Analysis
All experiments were done in triplicate and the values are presented as mean ± SEM. Comparisons of differences between 2 or more means were determined by the Student unpaired t test (2 means) and the Fisher exact test. More than 2 means were analyzed by 2- way analysis of variance followed by the Duncan multiple range test (2 + means) and Bonferroni post hoc testing via a SPSS version 17.0 (SPSS, Inc, Chicago, IL). Significance was defined as a P value < 0.05.
Results
Our experimental design was to first examine the viability of cells after treatment with combination therapy to determine if synergy of sHDL with EDPM is possible. As such, we first wanted to see if
sHDL nanoparticles enhance the antiproliferative effect of E, P and M in ACC cells
Cell-Titer-GLO viability results showed inhibition of cell proliferation in a dose dependent manner for each of the chemotherapeutics drugs as expected (Fig.1 A [NCI-H295R] and B [SW13]). However, sHDL nanoparticles alone did not induce significant cell death at normal concentrations but only at high concentrations of 100-200ug/ ml of the 22A peptide (Fig.1 A and B left). To determine if combining sHDL nanoparticles and the chemotherapeutic drugs results in synergy or an additive effect, we then calculated the combination index after treating the cells at different combination dosages using the method of Chou-Talalay (21). Doses were chosen based on the higher IC50 value of mitotane in SW13 cells compared to NCI-H295R cells. As indicated in Table 2 and Fig. 1A and B, a true synergistic effect (combination index < 1) was observed at multiple dose ranges by combining very low concentrations of sHDL nanoparticles (25 and 50ug/ml) with cisplatin, etoposide, or mitotane but not with doxorubicin (CI > 1). In each combination, there was a significant reduction in viability compared to either untreated cells or single drug treated cells. We then confirmed this antiproliferative effect by clonogenic assay by testing the combination of sHDL nanoparticles with each chemotherapeutic drug (Fig 2A and B). Combination treatments had a higher reduction in viability for NCI-H295R (A) and SW13 (B) cell lines by 11.8% and 20.4% respectively for cisplatin, 44.6% and 39.52% for etoposide, 39.1% and 22.3% for mitotane compared to single drug alone (p<0.05 for each). HDL treatment alone had minimal effect. Representative images are shown on the right of Fig 2A and B.
With synergy observed in combination with several of the drug compounds in inhibiting cell viability, we next wanted to evaluate if this effect was due to induction of apoptosis or merely a toxic effect of the drug leading to cell necrosis.
sHDL synergizes with chemotherapeutic drugs to induce apoptosis
Next combination dosing was evaluated by flow cytometry for a synergistic effect on apoptotic cell death in both ACC cell lines as determined by analysis of DNA fragmentation using sub-toxic concentrations of E, P, or M alone or in combination with sHDL for 24 h. Given the antagonistic effect of doxorubicin with sHDL on proliferation we did not test this
combination here. Cells undergoing early as well as late apoptosis and necrosis were differentiated based on phosphatidylserine staining on the outer leaflet of the apoptotic cells by Annexin V-FITC / PI staining. Combination treatments with sHDL resulted in a significantly greater increase in apoptotic or necrotic cells compared to each drug alone with negligible cell death noted with sHDL alone or untreated cells. The sHDL nanoparticles in combination with chemo drugs resulted in an increase in the percentage of apoptotic cells (early and late) by 6.16%, 36.46%, and 36.01%) for P, E and M, respectively (p<0.05 vs. drug alone) with minimal changes in necrosis compared to single drug alone for the NCI- H295R cells (Fig. 3A). In the case of non-cortisol secreting SW13 cells, the necrotic cell death increased by 14.71% for P and 21.33% for E (p<0.01) while the apoptotic cell death increased by 11.9% (p<0.05) for M when combined with sHDL vs. drug alone (Fig. 3B).
Given this synergistic effect on cell growth and induction of apoptosis, and since apoptosis and necrosis are mitochondrial dependent pathways, mitochondrial membrane potential was assessed.
Mitochondrial membrane potential is altered by combination therapy with sHDL
To elucidate the role of mitochondrial function in inducing apoptosis, we evaluated the mitochondrial potential (4) using TMRE staining after treatment of cells with sHDL and E, P, or M for 24 h. As a negative control the cells were pretreated with an ionophore FCCP to eliminate mitochondrial membrane potential changes. Treatment of NCI-H295R and SW13 cells with sHDL in combination with each of the chemo drugs resulted in a significant reduction in AY by 13.09% and 6.5% for P, 9.2% and 31.54% for E, and 14.49% and 19.8% for M (p<0.05 vs. chemo drug alone), respectively (Fig. 4A (NCI-H295R) and B (SW13)). This effect was blocked in the presence of mitochondrial depolarizer FCCP.
The effect of combination therapy with sHDL on Cortisol levels
To verify how changes in the steroidogenic pathway are influenced by combination therapy, we measured the concentration of Cortisol in the culture supernatant of hormone-producing NCI-H295R cells after E, P or M treatment alone or in combination with sHDL for 24 h. Treatment of cells with drug alone decreased Cortisol production levels by 89.6% for sHDL, 84.7% for M, and 82.1% for E (p<0.01 each vs. controls while P decreased it only by 8.39% (p=NS) (Fig. 5). In combination with sHDL, this effect was not significantly different for E, or M but decreased 82% with P similar to that seen for the HDL alone.
Next, since Cortisol levels were significantly decreased with sHDL, we wanted to look more at its mechanistic effect on steroidogenesis in these ACC cells. To explore the effect of combination treatment on steroidogenesis, we evaluated the expression of genes involved in steroidogenesis by quantitative RT-PCR after 24 h treatment of hormone producing NCI- H295R cells with either drug alone or in combination with sHDL (primers listed in Table 1A). Relative expression levels of factors of Cortisol biosynthesis by RT-PCR including steroidogenic acute regulatory protein (StAR), the intra mitochondrial cholesterol transporter, CYP11A1 and others were examined. During combination of sHDL with either cisplatin, etoposide, or mitotane, the levels of StAR (0.18-1.88), CYP21A2 (0.05-2.6) and CYP19A1 (0.22-4.9) increased (in terms of fold changes); whereas the levels of CYP11A1
(0.1-0.5), CYP11B1 (0.1-0.64), CYP11B2 (0.04-0.52), CYP17A1 (0.1-0.58) and HSD3B2 (0.1-0.2 for P and M respectively; but increased by 2.1 for E) decreased as fold change. Representative fold changes compared to monotherapy (p<0.05) are shown graphically for StAR, CYP17A1, CYP21A2, CYP11B1, and CYP11B2 (Fig. 6A-E).
Combination therapy with sHDL is effective in targeting in vivo mimicking MCAs
To confirm whether the cytotoxic effect of combination therapy in targeting cells can be translated to tumors in vivo, we have used three-dimensional MCAs as a mimic for tumor model. First, MCAs were developed by seeding the cells in ultralow attachment plates and then treating them with either drug alone or in combination with sHDL. As evident from the results shown in Fig. 7A, we observed approximately 20%, 50% and 30% reduction in NCI- H295R MCAs for cisplatin, etoposide and mitotane, and approximately 50%, 25% and 50% reduction in SW13 MCAs (Fig.7B), respectively, when used in combination with sHDL. These results indicate that sHDL combination is effective in targeting even three- dimensional MCAs.
Combination therapy enhances the efficacy of EDPM
Given the antagonistic effect for doxorubicin, we examined if the complete EDPM regimen with sHDL would still be synergistic in inhibiting ACC cell viability. The viability of the cells was determined as before by CellTiter-Glo after treating both NCI-H295R and SW13 cells with either EDPM (25%, 50%, 75%, or 100% MTD levels) alone or in combination with 25µg/ml or 50ug/ml of sHDL. Untreated cells or sHDL alone treated cells served as controls. Despite the antagonistic effect of doxorubicin, we observed an enhanced dose dependent decrease in viability for both NCI-H295R (Fig.8A) and SW13 (Fig.8B) for sHDL combinations compared to EDPM alone. These results clearly demonstrate that combination therapy with sHDL nanoparticles effectively target ACC cells at lower doses of EDPM, which may lower toxicity profiles.
Discussion
Despite recent insights into the molecular mechanisms underlying the carcinogenesis of ACC and the development of novel targeted therapies for other cancers, advanced ACC remains a deadly disease (22). Patients with ACC rarely present with classic symptoms related to their tumor with only 40-60% of the patients present with symptoms characteristic of hormone excess (22). Hypercortisolism is the most common presentation in 50-80% of the patients with hormone excess and ACC patients diagnosed with hypercortisolism commonly have hypokalemia and hypertension (22). Recently, it was noted that mitotane induces CYP3A4 (23). Since many anti-neoplastic drugs are metabolized by CYP3A4, drug- drug interactions including toxicities could result from such combinations. Therefore, novel treatment options that avoid such drug interactions and have the potential for mitotane dose/ toxicity reduction would be a real advance to the field.
Synthetic HDL nanoparticles have good safety in clinical trials thus far and demonstrate a unique antineoplastic effect in part due to their ability to efflux cholesterol selectively from cancer cells. As such, we have investigated the effect of combination therapy of sHDL with
commonly used chemotherapeutic drugs for ACC. Building on our preliminary findings (Table 1B) indicating that ACC cells express the highest level of SR-BI compared to several other cancers and normal cells, we hypothesized that sHDL nanoparticles can target and inhibit ACC cell viability effectively in combination with suboptimal concentrations of chemotherapeutic drugs.
We first performed a simple cell viability study to determine if combination dosing of sHDL and E, D, P or M would result in a synergistic, additive, or antagonistic effect by calculating combination indices for each combination. Our results clearly demonstrate that cisplatin, etoposide and mitotane act synergistically with sHDL at multiple concentrations whereas doxorubicin acts as an antagonist. This effect was then confirmed using a gold-standard clonogenic assay to demonstrate that combinations of sHDL and low-doses of E, P, or M resulted in improved inhibition of cell viability compared to E, P or M alone at normal therapeutic concentrations. We then demonstrated that combination treatment of E, P or M with sHDL lead to significant increases in ACC apoptosis and decreases in mitochondrial membrane potential compared to monotherapy. As mitochondrial depolarization can be due to either oxidative stress or apoptosis, further studies are needed to fully understand how this combination modulates the mitochondrial respiratory chain activity. Synergistic increases in apoptosis compared to necrosis suggest a mechanism-based pathway synergy, supporting the benefit of combination therapy on tumor biology as opposed to a merely non-targeted toxicity effect observed with necrosis.
Next, we evaluated the role of combination treatment on ACC steroidogenesis and cholesterol production using NCI-H295R human ACC cells that secrete steroid. Drugs like mitotane are known to inhibit CYP11A1, CYP11B1and others (23). Although studies have demonstrated the role of LDL in increasing steroidogenesis (24, 25) very little is known about the role of sHDL in steroidogenesis. Because elevated cortisol levels are a known negative prognostic factor for ACC, we used RT-PCR to examine how our combination treatment modulates key factors involved in the steroidogenesis pathway. Our results indicated that compared to monotherapy, combination therapy with sHDL increased expression levels of StAR, the intra mitochondrial transporter; CYP21A2, the enzyme metabolizing 17-hydroxyprogesterone into 11-deoxycortisol, and CYP19A1 whereas the levels of CYP11B1, CYP11B2, CYP11A1 and CYP21A2 decreased. These results suggest that our combination has multiple effects and blocks both the upstream and the downstream regulators of cortisol. To evaluate if this effect on steroidogenesis leads to down regulation of cortisol production, we measured cortisol production levels in response to treatment. Interestingly, sHDL alone or in combination with P (which does not alter cortisol levels) decreased cortisol production >80%, similar to mitotane. We then showed that the EDPM + sHDL combination treatment retained its synergy despite the antagonism of doxorubicin and that this combination effect is translatable to 3-dimensional cell growth in aggregates. Given the statistically significant reduction in MCAs and viability after EDPM and sHDL treatment, further in vivo evaluation is warranted to confirm if this combinational synergy translates to NCI-H295R xenografts.
In conclusion, our results demonstrate for the first time that sHDL nanoparticles act synergistically with chemotherapy agents used in ACC, allowing lower doses in
combination to generate in vitro efficacy. This synergy may be due in part to targeting of the steroidogenic pathway, similar to mitotane, for potentiating enhanced apoptosis. Since these sHDL nanoparticles have already demonstrated some safety in clinical trials, this combination strategy may be a novel, less toxic approach to improve treatment in combination and avoid dose-limiting toxicities while maintaining the therapeutic benefits of mitotane and the Italian protocol. Further translational evaluation of this combination treatment in vivo will provide additional preclinical evidence to support new treatment strategies for patients with advanced ACC.
Acknowledgments
This work was partly funded by the National Institutes of Health (T32 CA009672, R01 CA173292), The University of Michigan M-TRAC Program, the University of Michigan Comprehensive Cancer Center Support Grant, and the University Of Michigan Department Of Surgery.
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A
1
Cis alone
1
Dos alone DostsHDL 250g/ml
1.25
Mito alone Mito+sHDL 250g/ml
1.25
Cis+sHDL 250g/ml
Ete alone
Cis+sHDL 50ng/ml
Eto+sHDL 25pg/ml
Eto+sHDL 50g/ml
1.5
Y
Dos+sHDL 50pp/ml
1
T
Mito+sHDL 50ng/ml
1
0.75
0.75
Relative Viability
1.25
…
T
Relative Viability
Relative Viability
Relative Viability
Relaive Viability
9,75
0.75
I
0.5
0.5
0,75
0,5
0.5
0.25
0,25
0.5
0.25
0.25
0,25
0
0
0
·
·
.
50
100
150
200
0,625 1.25
2.5
5
7.5
15
6.25
12.5
25
50
100
150
0.3125 0,625
1.25
2.5
5
3.125
6.25
12.5
25
50
HDL Concentration (µg’ml)
Cisplatin Concentration (UM)
Etoposide Concentration (UM)
Dosorubicin Concentration
Mitotame Concentration (M)
(54M)
B
Cis alone
CistsHDL 25µg/ml
Eto alone Eto+sHDL 25gg’ml
Dos alone Dos+sHDL 25,4g/ml
Mito alone Mito+sHDL. 250g/ml
1.25
CistsHDL 50ug/ml
1
Ete+sHDL 50µg’ml
Dos+sHDL 504g/ml
1.25
1.25
Mito+sHDL 500g/ml
1.25
1
T
-
1
.
0.75
1
Relative Viability
Relative Viability
-
Relative Viability
75
Relative Viability
Relative Viability
.75
1.75
3.75
0.5
0.5
0.5
0.5
0,5
0.25
0.25
0.25
0,25
0.25
.
0
50
100
150
200
.
·
0
.
.
HDL Concentration (ng/ml)
0.625
1.25
2.5
5
10
12.5
25
50
75
150
0.3125
0.625
1.25
2.5
12.5
25
50
100
Cisplatin Concentration (JAMI)
Etoposide Concentration (M)
Doxorubicin Concentration
Mitotane Concentration (MI)
(AM)
A
120
sHDL
Fraction of surviving Colonies
100
☐ +SHDL
I
80
*
1
-sHDL
60
40
+sHDL
20
0
Control
Cisplatin
Etoposide
Mitotane
B
120
-SHDL
Fraction of Surviving Colonies
100
☐ +SHDL
T
80
-SHDL
60
40
+sHDL
20
0
Control
Cisplatin
Etoposide
Mitotane
A
B
Coatred
Cisplatin
Ktopuside
Contrel
Cisplatin
Mitatane
Cisplatin+HDL
EtoposidetoHEHE,
Milotane+&HEH
MIDL
Cisplatin +9BDL
ExsposidesHEDL
Mitetane+sHEL
Annexin V-FITC
Annesin V-FITC.
50
Necrotic @ Apoptotic
Necrotic · Apoptocie
-
*
Percent of Cells
Percentage of cells
-
30
A
30
₹
₹
H
20
M
·
Centrul
Cisplatin
Elapoxide
Mitutast
HDL
Caplatis-HDL EtspesidetsHDL, MisotasersHDL
Control
Chplatin
Doposide
Mintast
HDL
A
20
w/o FCCP
% decrease in mitochondrial potential in presence of sHDL
FCCP
15
T
4
10
-
5
0
Cisplatin
Etoposide
Mitotane
B
35
w/o FCCP
% decrease in mitochondrial potential in presence of sHDL
T
30
AFCCP
25
20
15
10
5
0
m
Cisplatin
Etoposide
Mitotane
100
Relative Fold change/Control
☐ -SHDL
80
☐ +sHDL
60
40
20
*
0
Control
Cisplatin
Etoposide
Mitotane
The cortisol production levels were estimated by radioimmunoassay after treatment of cortisol producing NCI-H295R cells with sHDL nanoparticles along with either cisplatin or etoposide or mitotane (at concentrations used in Figure 2) for 24h. Etoposide, mitotane, and sHDL each significantly reduced cortisol production ( *** p<0.05) with treatment while cisplatin alone did not. Addition of sHDL to cisplatin reduced levels to that of sHDL alone and addition of sHDL to Mitotane enhanced this effect (*p<0.05).
A-E
4
StAR
-$HDL
2.5
-SHDL
5
SHDL
☐ +sHDL
CYP17A1
☐ +sHDL
CYP21A2
☐ +sHDL
Fold Change/Actin
3
2
4
Fold Change/Actin
Fold Change/Actin
1.5
3
2
1
2
1
0.5
1
0
0
0
Control
Cisplatin
Etoposide
Mitotane
Control
Cisplatin
Etoposide
Mitotane
Control
Cisplatin
Etoposide
Mitotane
1.5
CYP11B1
-SHDL
+sHDL
1.5
-HDL
CYP11B2
☐ +$HDL
Fold Change/Actin
1
Fold Change/Actin
1
-
5
0.5
0
0
Control
Cisplatin
Etoposide
Mitotane
Control
Cisplatin
Etoposide
Mitotane
Figure 6. A-E mRNA level expressions of key steroidogenesis pathway enzymes in hormone producing H295R cells after treatment with either cisplatin, etoposide or mitotane alone or in combination with sHDL nanoparticles for 24 h Clockwise from top left: enzymes include Panel A = STAR, Panel B = CYP17A1, Panel C = CYP21A2, Panel D=CYP11B1, Panel E =CYP11B2. Expression levels were measured by RT-PCR. Cisplatin consistently decreased enzyme expression levels with treatment, whereas etoposide enhanced them. Mitotane only increased CYP21A2 levels and addition of sHDL to drug enhanced this effect in most of the enzymes. Data were analyzed by relative expression method and the values presented as mean ± SEM. Each experiment was repeated in triplicate. (p<0.05 each *, ** , *** )
Control
Cisplatin
Etoposide
Mitotane
A
900000
☒ -SHDL
Total area occupied by MCAs (in pixels)
800000
☐ +sHDL
-SHDL
700000
600000
500000
400000
300000
+sHDL
200000
100000
0
Control
Cisplatin
Etoposide
Mitotane
B
Control
Cisplatin
Etoposide
Mitotane
900000
☒ -SHDL
Total area occupied by MCAs (in
800000
☐ +sHDL
-SHDL
700000
600000
pixels)
500000
400000
300000
+sHDL
200000
100000
0
Control
Cisplatin
Etoposide
Mitotane
A
1.2
1
T
T
T
I
☒ sHDL untreated
-
舍命南
☐ sHDL 25µg/ml
Relative Viability
0.8
☐ sHDL 50µg/ml
0.6
0.4
0.2
0
Control
EDPM-1
EDPM-2
EDPM-3
EDPM-4
B
1.2
T
☒ sHDL untreated
1
T
T
±
☐ sHDL 25µg/ml
☐ sHDL 50µg/ml
Relative Viability
0.8
E
0.6
T
0.4
0.2
0
Control
EDPM-1
EDPM-2
EDPM-3
EDPM-4
Table 1 A. Primer sequences used for RT-PCR analysis of steroidogenesis pathway enzymes. B. Expression of SR-B1 by western blot analysis
ACC cell lines NCI-H295R, SW13 and RL251, and BHK (baby hamster kidney) cell lines over expressing SR-B1 (positive control) as well as Jurkat cell lines (negative control) were grown in culture in appropriate medium. The cells were lysed and immunoblotted for SR-B1using the method previously described (20). The same blot was reprobed for actin for loading control.
| Gene | Sense | Antisense |
|---|---|---|
| StAR (NM_000349) | TTGCTTTATGGGCTCAAGAATG | GGAGACCCTCTGAGATTCTGCTT |
| CYP11A1 (NM_000781) | CTTCTTCGACCCGGAAAATTT | CCGGAAGTAGGTGATGTTCTTGT |
| HSD3B2 (NM_000198.1) | GCGGCTAATGGGTGGAATCTA | CCTCATTTATACTGGCAGAAAGGAAT |
| CYP11B1 (NM_000497) | TCCCGAGGGCCTCTAGGA | GGGACAAGGTCAGCAAGATCTT |
| CYP11B2 (NM_000498) | TTGTTCAAGCAGCGAGTGTTG | GCATCCTCGGGACCTTCTC |
| CYP17A1 (NM_000102) | GCTGACTCTGGCGCACACT | CCATCCTTGAACAGGGCAAA |
| CYP21A2 (NM_000500) | TCCCAGCACTCAACCAACCT | CAGCTCAGAATTAAGCCTCAATCC |
| CYP19A1 (NM_000103) | ACCAGCATCGTGCCTGAAG | CCAAGAGAAAAAGGCCAGTGA |
BHK-SR-B1
NCI-H295R
SW13
RL251
Jurkat
SR-B1
Actin
Table 2 Combination Index evaluations by drug concentrations
Human ACC cell lines NCI-H295R and SW13 were treated at 25 and 50% IC50 levels of drug [Etoposide (VP16) at 12.5 or 25uM, mitotane at 25 or 50uM, cisplatin at 1.25 or 2.5uM, and doxorubicin at 1.25 or 2.5uM] in combination with either 25µg/ml or 50ug/ml sHDL nanoparticle for 72 h. The viability of the cells was calculated by cell-Titer Glo and the combination index (CI) was calculated using the Chou-Talalay equation (19). CI≤1 is defined as a combination demonstrating synergy (with stronger synergy for CI<0.5), CI=1 means the combination is additive, not synergistic, and CI>1 means an antagonistic effect. While E, P, and M each showed synergy with sHDL in both cell lines, stronger synergy was observed with etoposide and sHDL and in the 1.25uM dose of cisplatin. Each experiment was repeated in triplicate and the viability as a percentage of untreated control was calculated using Graphpad prism.
| sHDL (µg/ml) | Etoposide (µM) | CI for NCI-H295R | CI for SW13 |
|---|---|---|---|
| 50 | 25.0 | 0.694 | 0.158 |
| 50 | 12.5 | 0.602 | 0.392 |
| 25 | 25.0 | 0.641 | 0.172 |
| 25 | 12.5 | 0.521 | 0.196 |
| sHDL (µg/ml) | Mitotane (uM) | CI for NCI-H295R | CI for SW13 |
| 50 | 50 | 0.750 | 0.852 |
| 50 | 25 | 0.709 | 0.652 |
| 25 | 50 | 0.812 | 0.627 |
| 25 | 25 | 0.809 | 0.928 |
| sHDL (ug/ml) | Cisplatin (u.M) | CI for NCI-H295R | CI for SW13 |
| 50 | 2.5 | 0.759 | 0.666 |
| 50 | 1.25 | 0.462 | 0.812 |
| 25 | 2.5 | 0.572 | 0.619 |
| 25 | 1.25 | 0.390 | 0.583 |
| sHDL (µg/ml) | Doxorubicin (uM) | CI for NCI-H295R | CI for SW13 |
| 50 | 2.5 | 5.232 | 1.289 |
| 50 | 1.25 | 3.619 | 1.844 |
| 25 | 2.5 | 5.982 | 1.385 |
| 25 | 1.25 | 4.257 | 1.190 |