Bin Qin

Co-administration strategy to enhance brain accumulation of vandetanib by modulating P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) mediated efflux with m-TOR inhibitors

The objectives of this study were (i) to characterize the interaction of vandetanib with P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) in vitro and in vivo (ii) to study the modulation of P-gp and BCRP mediated efflux of vandetanib with specific transport inhibitors and m-TOR inhibitors, everolimus and temsirolimus. Cellular accumulation and bi-directional transport studies in MDCKII cell monolayers were conducted to delineate the role of efflux transporters on disposition of vandetanib. Brain distribution studies were conducted in male FVB wild-type mice with vandetanib administered intravenously either alone or in the presence of specific inhibitors and m-TOR inhibitors. In vitro studies suggested that vandetanib is a high affinity substrate of Bcrp1 but is not transported by P-gp. Interestingly, in vivo brain distribution studies in FVB wild type mice indicated that vandetanib penetration into the brain is restricted by both Bcrp1 and P-gp mediated active efflux at the blood brain barrier (BBB). Co-administration of elacridar, a dual P-gp/BCRP inhibitor increased the brain to plasma concentration ratio of vandetanib upto 5 fold. Of the two m-TOR pathway inhibitors examined; everolimus showed potent effect on modulating vandetanib brain penetration whereas no significant affect on vandetanib brain uptake was observed following temsirolimus co-administration. This finding could be clinically relevant as everolimus can provide synergistic pharmacological effect in addition to primary role of vandetanib efflux modulation at BBB for the treatment of brain tumors.

Development of a Peptide–Drug Conjugate for Prostate Cancer Therapy

TGX-221 is a highly potent phosphoinositide 3-kinase β (PI3Kβ) inhibitor that holds great promise as a novel chemotherapeutic agent to treat prostate cancer. However, poor solubility and lack of targetability limit its therapeutic applications. The objective of this present study is to develop a peptide-drug conjugate to specifically deliver TGX-221 to HER2 overexpressing prostate cancer cells. Four TGX-221 derivatives with added hydroxyl groups were synthesized for peptide conjugation. Among them, TGX-D1 exhibited a similar bioactivity to TGX-221, and it was selected for conjugation with a peptide promoiety containing a HER2-targeting ligand and a prostate specific antigen (PSA) substrate linkage. From this selection, the peptide-drug conjugate was proven to be gradually cleaved by PSA to release TGX-D1. Cellular uptake of the peptide-drug conjugate was significantly higher in prostate cancer cells compared to the parent drug. Moreover, both the peptide-drug conjugate and its cleaved products demonstrated comparable activities as the parent drug TGX-D1. Our results suggest that this peptide-drug conjugate may provide a promising chemotherapy for prostate cancer patients.

Enhanced brain accumulation of pazopanib by modulating P-gp and Bcrp1 mediated efflux with canertinib or erlotinib.

Primary objective of this investigation was to delineate the differential impact of efflux transporters P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) on brain disposition and plasma pharmacokinetics of pazopanib. In addition, this research investigated whether inhibition of these efflux transporters with clinically relevant efflux modulators canertinib or erlotinib could be a viable strategy for improving pazopanib brain delivery. In vitro assays with MDCKII cell monolayers suggested that pazopanib is a high affinity substrate for Bcrp1 and a moderate substrate for P-gp. Co-incubation with specific transport inhibitors restored cell accumulation and completely abolished the directionality of pazopanib flux. Brain and plasma pharmacokinetic studies were conducted in FVB wild type mice in the absence and presence of specific transport inhibitors. Drug levels in plasma and brain were determined using a validated high performance liquid chromatography method using vandetanib as an internal standard. In vivo studies indicated that specific inhibition of either P-gp (by zosuquidar or LY335979) or Bcrp1 (by Ko143) alone did not significantly alter pazopanib brain accumulation. However, dual P-gp/Bcrp1 inhibition by elacridar (GF120918), significantly enhanced pazopanib brain penetration by ~5-fold without altering its plasma concentrations. Thus, even though Bcrp1 showed higher affinity towards pazopanib in vitro, in vivo at the mouse BBB both P-gp and Bcrp1 act in concert to limit brain accumulation of pazopanib. Furthermore, erlotinib and canertinib as clinically relevant efflux modulators efficiently abrogated directionality in pazopanib efflux in vitro and their co-administration resulted in 2-2.5-fold increase in pazopanib brain accumulation in vivo. Further pre-clinical and clinical investigations are warranted as erlotinib or canertinib may have a synergistic pharmacological effect in addition to their primary role of pazopanib efflux modulation as a combination regimen for the treatment of recurrent brain tumors.

Silencing of the IKKε gene by siRNA inhibits invasiveness and growth of breast cancer cells

IntroductionIκB kinase ε (IKKε) is a member of the IKK family that plays an important role in the activation of NF-κB. Overexpressed in more than 30% of breast cancers, IKKε has been recently identified as a potential breast cancer oncogene. The purpose of the present study is to examine the therapeutic potential of IKKε siRNA on human breast cancer cells.MethodsEight siRNAs targeting different regions of the IKKε mRNA were designed, and the silencing effect was screened by quantitative real-time RT-PCR. The biological effects of synthetic siRNAs on human breast cancer cells were investigated by examining the cell proliferation, migration, invasion, focus formation, anchorage-independent growth (via soft agar assay), cell cycle arrest, apoptosis (via annexing binding), NF-κB basal level, and NF-κB-related gene expressions upon the IKKε silencing.ResultsSilencing of IKKε in human breast cancer cells resulted in a decrease of focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of IKKε suppressed cell proliferation. Cell cycle assay showed that the anti-proliferation effect of IKKε siRNA was mediated by arresting cells in the G0/G1 phase, which was caused by downregulation of cyclin D1. Furthermore, we demonstrated that silencing of IKKε inhibited the NF-κB basal activity as well as the Bcl-2 expression. Significant apoptosis was not observed in breast cancer cells upon the silencing of IKKε. The present study provided the first evidence that silencing IKKε using synthetic siRNA can inhibit the invasiveness properties and proliferation of breast cancer cells.ConclusionsOur results suggested that silencing IKKε using synthetic siRNA may offer a novel therapeutic strategy for breast cancer.

Inhibition of breast cancer cell growth and invasiveness by dual silencing of HER-2 and VEGF.

Overexpression of HER-2 accounts for approximately 25% of all breast cancer cases, while 87.7% of HER-2 positive breast cancers are associated with upregulated VEGF. The objective of this study is to explore the combination therapy of blocking HER-2 and VEGF expressions simultaneously using siRNA. This is the first report to examine the effect of dual silencing of HER-2 and VEGF genes on tumor growth and invasiveness. We have designed nine HER-2 siRNAs and ten VEGF siRNAs, and identified potent siRNA which can silence the target gene up to 75-83.5%. The most potent HER-2 and VEGF siRNAs were used to conduct functional studies in HER-2 positive breast cancer cells. Tumor invasiveness properties including cell morphology change, in vitro migration, cell spreading, and adhesion to ECM were evaluated. In addition, cell proliferation and apoptosis were examined after the siRNA treatment. Our data demonstrated for the first time that HER-2 siRNA could inhibit cell migration and invasion abilities. Combination of HER-2 and VEGF siRNAs exhibited synergistic silencing effect on VEGF. Both HER-2 siRNA and VEGF siRNA showed significant inhibition on cell migration and proliferation. HER-2 siRNA also demonstrated dramatic suppression on cell spreading and adhesion to ECM, as well as induction of apoptosis. Dual silencing of HER-2 and VEGF exhibited significant cell morphology change, and substantial suppression on migration, spreading, cell adhesion, and proliferation. Our observations suggested that HER-2 positive breast cancer may be more effectively treated by dual inhibition of HER-2 and VEGF gene expressions using siRNA.

Peptides Used in the Delivery of Small Noncoding RNA

RNA interference (RNAi) is an endogenous process in which small noncoding RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), post-transcriptionally regulate gene expressions. In general, siRNA and miRNA/miRNA mimics are similar in nature and activity except their origin and specificity. Although both siRNAs and miRNAs have been extensively studied as novel therapeutics for a wide range of diseases, the large molecular weight, anionic surface charges, instability in blood circulation, and intracellular trafficking to the RISC after cellular uptake have hindered the translation of these RNAs from bench to clinic. As a result, a great variety of delivery systems have been investigated for safe and effective delivery of small noncoding RNAs. Among these systems, peptides, especially cationic peptides, have emerged as a promising type of carrier due to their inherent ability to condense negatively charged RNAs, ease of synthesis, controllable size, and tunable structure. In this review, we will focus on three major types of cationic peptides, including poly(l-lysine) (PLL), protamine, and cell penetrating peptides (CPP), as well as peptide targeting ligands that have been extensively used in RNA delivery. The delivery strategies, applications, and limitations of these cationic peptides in siRNA/miRNA delivery will be discussed.

Identification of a LNCaP-Specific Binding Peptide Using Phage Display

ABSTRACTPurposeTo identify a LNCaP-specific peptide using a phage display library and evaluate its potential applications in targeted drug delivery.MethodsBinding abilities of selected phages were evaluated by cell phage ELISA. The KYL peptide encoded by the most specific phage clone was synthesized, labeled with fluorescein, and assayed in various cell lines. A fusion peptide composed of the KYL peptide and a proapoptotic peptide D(KLAKLAK)2 was synthesized, and the cell death effect was evaluated on different cells. Moreover, the KYL peptide was conjugated to a cationic protein, protamine, to explore its potential application in siRNA delivery.ResultsOne phage clone with a high binding affinity to LNCaP cells was identified. Cell phage ELISA and immunostaining demonstrated high specificity of this phage to LNCaP cells. The fluorescein-labeled KYL peptide exhibited higher binding to LNCaP cells in comparison to other cells. The fusion peptide composed of the KYL peptide and the proapoptotic peptide induced cell death in LNCaP cells, but not in PC-3 cells. The KYL peptide-protamine conjugate also efficiently delivered a fluorescein-labeled siRNA into LNCaP cells.ConclusionWe identified a LNCaP-specific peptide and demonstrated its potential applications in targeted drug delivery to LNCaP cells.

Blocking IKKα expression inhibits prostate cancer invasiveness.

ABSTRACTPurposeIKKα has been recently identified as a key mediator of the inflammation and metastasis in prostate cancer. In the present study, we intend to silence the IKKα expression in prostate cancer cells using synthetic siRNAs and examine their biological effects on tumor cell invasiveness and growth.MethodsThree synthetic siRNAs targeting different regions of the IKKα mRNA were designed, and the silencing effect was determined in PC-3 and DU145 cells. Numerous studies, including wound-healing assay, migration assay, invasion assay, cell attachment assay, cell proliferation, and cell cycle analysis, were conducted to investigate the biological effects of the IKKα siRNAs on prostate cancer cells.ResultsWe have identified potent siRNAs that can silence the IKKα up to 74%. Inhibition of IKKα reduced the wound healing, migration, invasion and cell attachment capabilities of prostate cancer cells. Similar anti-invasive effects were also observed in the presence of RANKL. However, silencing of IKKα only showed a negligible effect on cell proliferation and cell cycle distribution.ConclusionThis study presents compelling evidence that IKKα plays a major role in prostate cancer invasion and metastasis, but not in cell proliferation. Silencing of IKKα with siRNA may therefore provide a promising therapeutic strategy for prostate cancer patients.

Development of cholesteryl peptide micelles for siRNA delivery

Despite the rapid progress in the siRNA field, developing a safe and efficient delivery system of siRNA remains to be an obstacle in the therapeutical application of siRNA. The purpose of this study is to develop an efficient peptide-based siRNA delivery system for cancer therapy. To this end, cholesterol was conjugated to a series of peptides composed of lysine and histidine residues. The resultant cholesteryl peptides were characterized, and their potential for siRNA delivery was evaluated. Our results indicate that short peptides (11-21 mer) composed of various numbers of lysine and histidine residues alone are not sufficient to mediate efficient siRNA delivery. However, the amphiphilic cholesteryl peptides can self-assemble to form a micelle-like structure in aqueous solutions, which significantly promotes the siRNA condensation capability of the peptides. The cholesteryl peptides form stable complex with siRNA and effectively protect siRNA from degradation in rat serum up to three days. Furthermore, the cholesteryl peptides efficiently transfect siRNA into different cancer cells and trigger potent gene silencing effect, whereas peptides without cholesterol modification cannot deliver siRNA into the cells. In addition, one of the cholesteryl peptides Chol-H3K2s displays comparable cellular uptake and gene silencing effect but less cytotoxicity compared with branched polyethylenimine (bPEI) and Lipofectamine-2000. Our results reveal that the cholesteryl peptides possess great potential as an efficient siRNA delivery system.

PCBP2 siRNA Reverses the Alcohol-induced Pro-fibrogenic Effects in Hepatic Stellate Cells

ABSTRACTPurposeType I collagen accumulates during liver fibrosis primarily because α-complex protein-2 (αCP2), encoded by the poly(rC) binding protein 2 (PCBP2) gene, binds to the 3′ end of the collagen mRNA and increases its half-life. This study aimed to reverse the pro-fibrogenic effect of alcohol on hepatic stellate cells (HSCs) by silencing the PCBP2 gene with siRNA.MethodsThe silencing effects of a series of predesigned PCBP2 siRNAs were evaluated in the rat hepatic stellate cell line, HSC-T6. The pro-fibrogenic effects of alcohol on the expression levels of PCBP2 and type-I collagen were examined by several methods. The effect of PCBP2 siRNA on the stability of type I collagen α1(I) mRNA was investigated by an in vitro mRNA decay assay.ResultsWe identified one potent PCBP2 siRNA that reversed the alcohol-induced expression of PCBP2 in HSCs. The decay rate of the collagen α1(I) mRNA increased significantly in HSCs treated with the PCBP2 siRNA.ConclusionThis study provides the first evidence that alcohol up-regulates the expression of PCBP2, which subsequently increases the half-life of collagen α1(I) mRNA. Silencing of PCBP2 using siRNA may provide a promising strategy to reverse the alcohol-induced pro-fibrogenic effects in HSCs.