Parvin Mahdipoor

Induction of apoptosis by survivin silencing through siRNA delivery in a human breast cancer cell line.

Post-transcriptional silencing of antiapoptotic genes is a promising strategy for cancer therapy, but delivering short interfering RNA (siRNA) molecules against such targets is challenging due to inability of anionic siRNA to cross cellular membranes. Lipid substitution on small molecular weight, nontoxic polyethylenimine (PEI) has been investigated as a promising approach for effective siRNA delivery. In this study, we report on the ability of low molecular weight, lipid-substituted PEI to deliver siRNA against the antiapoptotic protein survivin. Toxicity of a library of lipid-substituted PEIs, as well as their siRNA delivery and survivin silencing efficiency, was evaluated in MDA-MB-231 human breast cancer cells. A significant increase in cellular delivery of siRNA was observed as a result of lipid substitution. Most significant downregulation of survivin was established by caprylic acid-substituted polymers, which resulted in significant levels of apoptosis induction and resultant loss of cell viability. Survivin downregulation prior to anticancer drug treatment decreased the IC(50) of several drugs by 50- to 120-fold. Our experiments indicated an effective downregulation of survivin, a cell protective protein upregulated in tumor cells, by delivering siRNA with hydrophobically modified PEI. This study introduces a promising delivery system for safe and effective siRNA delivery that will be suitable for further investigation in preclinical animal models.

Effective down-regulation of breast cancer resistance protein (BCRP) by siRNA delivery using lipid-substituted aliphatic polymers.

Breast Cancer Resistance Protein (BCRP, ABCG2) is an efflux protein whose aberrant activity has been linked to multidrug resistance in cancer. Although siRNA delivery to down-regulate BCRP expression is promising to sensitize tumor cells against drugs, therapeutic use of siRNA requires effective carriers that can deliver siRNA intracellularly with minimal toxicity on target cells. This study explored the feasibility of special class of cationic polymers, namely lipid-substituted low molecular weight (2kDa) polyethyleneimine (PEI), as a carrier for siRNA-mediated BCRP down-regulation. Structure-function studies methodically evaluated the effect of a range of lipophilic substitutions for siRNA delivery and BCRP down-regulation. Our results showed a significant increase in siRNA delivery as a function of lipid substitution for a range of lipids ranging from C8 to C18. The BCRP silencing was correlated to siRNA delivery efficiency of the polymers, and effectively lasted for ∼5days after a single treatment of siRNA. BCRP down-regulation sensitized the drug-resistant cells to cytotoxic effect of mitoxantrone by a ∼14-fold decrease in the IC(50) value, whose effect was evident even after 14days. This study demonstrated the possibility of functional siRNA delivery by lipid-modified low molecular weight PEI and highlighted the importance of the extent and nature of lipid substitution in effective siRNA delivery.

Insights into the effects of hyperlipoproteinemia on cyclosporine A biodistribution and relationship to renal function

The purpose of this study was to assess the effect of hyperlipoproteinemia on the biodistribution of cyclosporine A (CyA), an extensively lipoprotein bound immunosuppressant, in a rat model and to determine the potential toxicological significance of this effect. Normolipidemic and hyperlipoproteinemic rats were given a single 5 mg/kg dose of CyA as intravenous bolus and at selected times postdose, tissues, blood, and plasma were harvested and assayed for CyA content. Hyperlipoproteinemia was induced by intraperitoneal injection of 1 g/kg poloxamer 407. Compared with normolipidemic animals, hyperlipoproteinemic rats had higher plasma, blood, kidney, and liver CyA concentrations. In contrast, in heart and spleen the concentrations were decreased in hyperlipoproteinemia. The nephrotoxic effect of CyA was also evaluated in normolipidemic and hyperlipoproteinemic rats after 7 days of dosing with 20 mg/kg/day. In both groups of animals, repeated doses of CyA were associated with equivalent decreases in creatinine and urea clearances compared with matching control and predose baseline measures. The concentrations of drug in kidney were equivalent at the conclusion of the study. However, despite these similarities there was microscopic evidence of more severe changes in the kidneys in the hyperlipoproteinemic rats, which also experienced a significant decrease in body weight compared with the normolipedemic animals. In conclusion, the distribution of CyA to kidneys was enhanced in poloxamer 407-induced hyperlipoproteinemic rats after single doses, and with repeated doses there was an apparent greater adverse effect on these animals compared with normolipidemic animals.

Investigating siRNA delivery to chronic myeloid leukemia K562 cells with lipophilic polymers for therapeutic BCR-ABL down-regulation

RNAi represents a new alternative for treatment of chronic myeloid leukemia (CML) to overcome the difficulties of current drug treatments such as the acquired resistance. However, potent carriers that can overcome delivery barriers to RNAi agents and have therapeutic efficacy especially in difficult-to-transfect CML cells are needed. Here, we explored the use of lipid-modified polyethylenimines (PEI) of low molecular weights (0.6, 1.2 and 2.0kDa) in K562 cells and showed that the delivery efficiency was dependent on the type of lipid used for polymer modification, degree of lipid substitution and polymer molecular weight. Among the lipid-substituted polymers investigated, palmitic acid (PA)-substituted 1.2kDa PEI (~2 lipids/PEI) has proven to be highly efficient in delivering siRNA and silencing of the reporter gene green fluorescent protein (GFP). The silencing efficacy achieved with this polymer was found to be higher than the 25kDa PEI and is similar to commercial reagent Lipofectamine™ 2000. Moreover, when BCR-ABL protein was targeted in K562 cells, a reduction in the corresponding mRNA levels was observed, as well as an induction of early and late stage apoptosis. The results of this study demonstrated that PA-substitutions on low MW polymers could be useful for siRNA delivery in CML cells for therapeutic purposes.

Effective response of doxorubicin-sensitive and -resistant breast cancer cells to combinational siRNA therapy.

Chemotherapy is an effective approach to curb uncontrolled proliferation of malignant cells. However, most drugs rapidly lose their efficacy as a result of resistance development. We explored the potential of combinational siRNA silencing to prevent growth of drug-resistant breast cancer cells independent of chemotherapy. Resistance was induced in two breast cancer lines by chronic exposure to doxorubicin. Microarray analysis of apoptosis-related proteins showed Bcl2, survivin, NF B, and Mcl1 to be prominently up-regulated in drug-resistant cells. Human siRNA libraries against apoptosis-related proteins and kinases were screened using lipid-substituted polymers as non-viral carrier, and siRNAs were selected to diminish cell growth without affecting growth of skin fibroblasts. Surprisingly, the selected siRNAs led to similar responses in wild-type and drug-resistant cells, despite their phenotypic differences. Promising kinase siRNAs were co-delivered with anti-apoptotic Mcl-1 siRNA and Ribosomal Protein S6 Kinase (RPS6KA5) was found the most promising candidate for simultaneous silencing with Mcl-1. In both MDA435 wild type (WT) and MDA435 resistant (R) xenografts in nude mice, double silencing of Mcl-1/RPS6KA5 also led to improved inhibition of tumor growth in the absence of chemotherapy. We conclude that combinational silencing of well-selected targets could be a feasible therapeutic strategy in the absence of drug therapy and could provide a new avenue for therapy of drug-resistant breast cancers.

Targeting Cell Cycle Proteins in Breast Cancer Cells with siRNA by Using Lipid-Substituted Polyethylenimines

The cell cycle proteins are key regulators of cell cycle progression whose deregulation is one of the causes of breast cancer. RNA interference (RNAi) is an endogenous mechanism to regulate gene expression and it could serve as the basis of regulating aberrant proteins including cell cycle proteins. Since the delivery of small interfering RNA (siRNA) is a main barrier for implementation of RNAi therapy, we explored the potential of a non-viral delivery system, 2.0 kDa polyethylenimines substituted with linoleic acid and caprylic acid, for this purpose. Using a library of siRNAs against cell cycle proteins, we identified cell division cycle protein 20 (CDC20), a recombinase RAD51, and serine–threonine protein kinase CHEK1 as effective targets for breast cancer therapy, and demonstrated their therapeutic potential in breast cancer MDA-MB-435, MDA-MB-231, and MCF7 cells with respect to another well-studied cell cycle protein, kinesin spindle protein. We also explored the efficacy of dicer-substrate siRNA (DsiRNA) against CDC20, RAD51, and CHEK1, where a particular DsiRNA against CDC20 showed an exceptionally high inhibition of cell growth in vitro. There was no apparent effect of silencing selected cell cycle proteins on the potency of the chemotherapy drug doxorubicin. The efficacy of DsiRNA against CDC20 was subsequently assessed in a xenograft model, which indicated a reduced tumor growth as a result of CDC20 DsiRNA therapy. The presented study highlighted specific cell cycle protein targets critical for breast cancer therapy, and provided a polymeric delivery system for their effective down-regulation.

Effect of siRNA pre-Exposure on Subsequent Response to siRNA Therapy

ABSTRACTPurposeAn alternative cancer therapy based on RNA interference (RNAi) has shown considerable promise but the possibility of resistance development is not known. This study explored the possibility of therapeutic resistance against siRNA nanoparticles in human cancer cells.MethodsTwo approaches to siRNA treatment were undertaken using lipid-modified polyethylenimines, a single high concentration (shock) and repeated increasing concentrations (gradual). The targets were Mcl-1, RPS6KA5 and KSP in MDA-MB-435 cells.ResultsThere was no evidence of resistance development in shock-treated cells, while the decrease in mRNA levels of targeted proteins was not as robust in naïve cells in gradual treatment. However, silencing efficiency was restored after a 7-day recovery period when expression of suppressed proteins returned to normal levels. Cellular uptake of siRNA was not affected by pre-treatments. Other mediators involved in cell survival and proliferation were altered in siRNA-treated cells, but only JUN silencing led to a heightened loss of viability. In vivo experiments demonstrated similar silencing efficiency at mRNA level after repeat doses.ConclusionsHuman cancer cells responded to repeat siRNA nanoparticles in a similar fashion after a temporary initial alteration and little, if any, resistance was evident against repeated siRNA treatments.

Stability of compounded thioguanine oral suspensions

PURPOSE. Updated information on the stability of compounded thioguanine oral suspensions prepared with currently available ingredients, as well as results of testing to determine if the addition of an antioxidant could extend shelf life by inhibiting formation of guanine, are presented. METHODS. Using triturated thioguanine tablets, three compounded suspensions were prepared: (1) a reference formulation containing methylcellulose and simple syrup, (2) an equivalent formulation using Ora-Plus and Ora-Sweet, and (3) an antioxidant-containing formulation prepared by adding ascorbic acid to the equivalent formulation. The compounded batches were stored at room temperature (19-23 °C). The chemical stability of the suspensions was evaluated immediately after compounding and at weekly intervals by a validated liquid chromatography-mass spectrometry (LCMS) assay method; physical stability was evaluated by regular visual checks and weekly pH testing. RESULTS. As demonstrated by serial LCMS testing, mean thioguanine levels in sampled batches of all three suspensions remained above accepted standards and mean guanine formation remained within acceptable limits for up to 63 days. The addition of ascorbic acid appeared to slow guanine formation but did not significantly extend the shelf life of the suspension. CONCLUSION. Compounded oral suspensions of thioguanine 20 mg/mL exhibited acceptable chemical and physical stability for up to nine weeks at 19-23 °C. The addition of ascorbic acid at a concentration of 0.1% to the suspension was not effective in consistently increasing the shelf life of the thioguanine suspensions.

Difatty Acyl-Conjugated Linear and Cyclic Peptides for siRNA Delivery

A number of amphiphilic difatty acyl linear and cyclic R5K2 peptide conjugates were synthesized by solid-phase peptide methods to enhance the interaction with the hydrophobic cellular phospholipid bilayer and to improve siRNA delivery and silencing. Binding to siRNA molecules was significantly less for the cyclic peptide conjugates. A gradual decrease was observed in the particle size of the complexes with increasing peptide/siRNA ratio for most of the synthesized peptides, suggesting the complex formation. Most of the complexes showed a particle size of less than 200 nm, which is considered an appropriate size for in vitro siRNA delivery. A number of fatty acyl-conjugated peptides, such as LP-C16 and LP-C18, displayed near complete protection against serum degradation. Flow cytometry studies demonstrated significantly higher internalization of fluorescence-labeled siRNA (FAM-siRNA) in the presence of LP-C16, LP-C18, and CP-C16 with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) addition. Confocal microscopy confirmed the cellular internalization of fluorescence-labeled siRNA in the presence of LP-C16 and LP-C18 with DOPE when compared with cells exposed to DOPE/FAM-siRNA. While C16- and C18-conjugated peptides (especially linear peptides) showed silencing against kinesin spindle protein (KSP) and janus kinase 2 (JAK2) proteins, the addition of DOPE enhanced the silencing efficiency significantly for all selected peptides, except for CP-C16. In conclusion, C16 and C18 difatty acyl peptide conjugates were found to enhance siRNA delivery and generate silencing of targeted proteins in the presence of DOPE. This study provides insights for the design and potential application of optimized difatty acyl peptide/lipid nanoparticles for effective siRNA delivery.

Abstract B15: In vivo analysis of repeated siRNA silencing on protein expression levels

An ever increasing number of anticancer drugs are under investigation, and enter the clinical setting targeting different tumor survival and/or growth mechanisms; however, development of resistance against the mechanism of action of even the most promising drugs seems to be inevitable and remains a major concern. RNA interference (RNAi) have shown considerable promise in silencing proteins involved in tumor vascularization, as well as intracellular pathways involved in cell proliferation and/or survival. Despite the apparent efficacy of this powerful tool (especially in vitro), our information about the factors affecting siRNA efficiency and the possibility of resistance development against the silencing effect are limited at best. This study focused on determining the possibility of resistance development against siRNA treatment as a result of repeated exposure to siRNA in vivo. Our preliminary experiments in vitro revealed an unaffected siRNA cellular internalization and reproducible silencing efficiency of selected targets. The expression level of other mediators involved in breast cancer cell survival and proliferation (notably survivin, JUN, JAK2, NFkB and STAT3) were, however, altered in siRNA treated cells. In vivo experiments in a xenograft model demonstrated a similar silencing efficiency at the mRNA level after each repeated dose, with little, if any, apparent resistance development to the siRNA therapy. We conclude that cells would respond to repeated siRNA treatments in a similar fashion after a temporary initial alteration. Citation Format: Hamidreza Montazeri Aliabadi, Parvin Mahdipoor, Hasan Uludag. In vivo analysis of repeated siRNA silencing on protein expression levels. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B15.