Kun Cheng

The role of HER2 in cancer therapy and targeted drug delivery

HER2 is highly expressed in a significant proportion of breast cancer, ovarian cancer, and gastric cancer. Since the discovery of its role in tumorigenesis, HER2 has received great attention in cancer research during the past two decades. Successful development of the humanized monoclonal anti-HER2 antibody (Trastuzumab) for the treatment of breast cancer further spurred scientists to develop various HER2 specific antibodies, dimerization inhibitors and kinase inhibitors for cancer therapy. On the other hand, the high expression of HER2 and the accessibility of its extracellular domain make HER2 an ideal target for the targeted delivery of anti-tumor drugs as well as imaging agents. Although there is no natural ligand for HER2, artificial ligands targeting HER2 have been developed and applied in various targeted drug delivery systems. The emphasis of this review is to elucidate the roles of HER2 in cancer therapy and targeted drug delivery. The structure and signal pathway of HER2 will be briefly described. The role of HER2 in tumorigenesis and its relationship with other tumor markers will be discussed. For the HER2 targeted cancer therapy, numerous strategies including the blockage of receptor dimerization, inhibition of the tyrosine kinase activity, and interruption of the downstream signal pathway will be summarized. For the targeted drug delivery to HER2 positive tumor cells, various targeting ligands and their delivery systems will be described in details.

Prodrugs for improving tumor targetability and efficiency.

As the mainstay in the treatment of various cancers for several decades, chemotherapy is successful but still faces challenges including non-selectivity and high toxicity. Improving the selectivity is therefore a critical step to improve the therapeutic efficacy of chemotherapy. Prodrug is one of the most promising approaches to increase the selectivity and efficacy of a chemotherapy drug. The classical prodrug approach is to improve the pharmaceutical properties (solubility, stability, permeability, irritation, distribution, etc.) via a simple chemical modification. This review will focus on various targeted prodrug designs that have been developed to increase the selectivity of chemotherapy drugs. Various tumor-targeting ligands, transporter-associated ligands, and polymers can be incorporated in a prodrug to enhance the tumor uptake. Prodrugs can also be activated by enzymes that are specifically expressed at a higher level in tumors, leading to a selective anti-tumor effect. This can be achieved by conjugating the enzyme to a tumor-specific antibody, or delivering a vector expressing the enzyme into tumor cells.

Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA.

Antisense oligodeoxynucleotides, triplex-forming oligodeoxynucleotides and double-stranded small interfering RNAs have great potential for the treatment of many severe and debilitating diseases. Concerted efforts from both industry and academia have made significant progress in turning these nucleic acid drugs into therapeutics, and there is already one FDA-approved antisense drug in the clinic. Despite the success of one product and several other ongoing clinical trials, challenges still exist in their stability, cellular uptake, disposition, site-specific delivery and therapeutic efficacy. The principles, strategies and delivery consideration of these nucleic acids are reviewed. Furthermore, the ways to overcome the biological barriers are also discussed so that therapeutic concentrations at their target sites can be maintained for a desired period.

Vascular endothelial growth factor gene delivery for revascularization in transplanted human islets.

AbstractPurpose. Islet transplantation is limited by islet graft failure because of poor revascularization, host immune rejection, and nonspecific inflammatory response. Human vascular endothelial growth factor (hVEGF) gene delivery is likely to promote islet revascularization and survival. Methods. We evaluated gene expression from a bicistronic plasmid encoding hVEGF and enhanced green fluorescent protein (EGFP) (pCMS-EGFP-hVEGF). Glucose responsiveness of islets was evaluated both in vitro and in vivo, and revascularization in islet graft was evaluated by immunohistochemistry. Results. After transfection, hVEGF and EGFP expression levels were comparable with original monocistronic plasmids in Jurkat cells but higher and prolonged hVEGF expression in islets transfected with the bicistronic plasmid was observed, possibly as the result of differences in promoter strength and hypoxia response. The 3:1 w/w complexes showed little toxicity to islets at a dose of 5 μg DNA per 2000 islets. On glucose challenge, insulin release from transfected islets as well as secretion from islets after transplantation under the mouse kidney capsules in response to glucose stimulation, increased with time. Immunohistochemical staining of transplanted islets using mouse anti-human insulin, mouse anti-human von Willebrand factor, and rat anti-mouse CD31 antibodies suggests that islets are functional and there is new blood vessel formation. Conclusions. These findings suggest that transient hVEGF gene expression by the islets may promote islet revascularization and prolong islet survival after transplantation.

TGF-β1 Gene Silencing for Treating Liver Fibrosis

Small interfering RNA (siRNA) and short hairpin RNA (shRNA) targeting different regions of transforming growth factor beta1 (TGF-beta1) mRNA were designed and the silencing effect was determined after transfection into immortalized rat liver stellate cells (HSC-T6). There was not only significant decrease in TGF-beta1, tissue inhibitor of metalloproteinase 1 (TIMP-1), alpha-smooth muscle actin (alpha-SMA) and type I collagen after transfection with TGF-beta1 siRNAs, but also synergism in gene silencing when siRNAs targeting two different start sites were used as a pool for transfection. The two siRNA sequences which efficiently inhibited TGF-beta1 gene expression were converted to shRNAs via cloning into the pSilencer1.0. There was significant decrease in TGF-beta1 and TIMP-1 when HSC-T6 cells were transfected with pshRNA targeting the same regions of TGF-beta1 mRNA as siRNAs. Furthermore, TGF-beta1 gene silencing in HSC-T6 cells significantly decreased the levels of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta). In conclusion, both siRNA and shRNA showed sequence-specific and dose dependent TGF-beta1 gene silencing and have the potential to treat liver fibrosis.

Enhanced Hepatic Uptake and Bioactivity of Type α1(I) Collagen Gene Promoter-Specific Triplex-Forming Oligonucleotides after Conjugation with Cholesterol

A triplex-forming oligonucleotide (TFO) specific for type α1(I) collagen promoter is a promising candidate for treating liver fibrosis. Earlier, we determined the pharmacokinetics and biodistribution of TFO after systemic administration into normal and fibrotic rats. In this study, we conjugated cholesterol to the 3′ end of the TFO via a disulfide bond and determined its cellular and nuclear uptake and bioactivity using HSC-T6 cell lines in vitro, followed by biodistribution at whole-body, organ (liver), and subcellular levels. Conjugation with cholesterol had little effect on the triplex-forming ability of the TFO with target duplex DNA, and the cellular uptake of 33P-TFO-cholesterol (Chol) increased by 2- to approximately 4-fold. Real-time reverse transcriptase-polymerase chain reaction analysis after transfection of HSC-T6 cells with TFO-Chol or TFO indicated that TFO-Chol had higher inhibition on type α1(I) collagen primary transcript than naked TFO at low concentration (200 nM) but showed similar inhibition at higher concentration (500 and 1000 nM). There was increase in the inhibition on primary transcript with transfection time. The hepatic uptake of 33P-TFO-Chol after systemic administration was 72.22% of the dose compared with 45.8% of 33P-TFO. There was significant increase in the uptake of 33P-TFO-Chol by hepatic stellate cells and hepatocytes. More importantly, the nuclear uptake of TFO-Chol was higher than TFO in cell culture system and in vivo studies. In conclusion, TFO-Chol is a potential antifibrotic agent.

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.

Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets

Islet transplantation is limited by islet graft failure due to poor revascularization, host immune rejection and nonspecific inflammatory response. Delivery of human vascular endothelial growth factor (hVEGF) gene to the islets is likely to promote islet revascularization and survival. We used a bicistronic adenoviral vector encoding hVEGF and CpG-free allele of green fluorescent protein (Adv-GFP-hVEGF) and introduced into human pancreatic islets by transfection. We found that transfection efficiency and apoptosis were dependent on the multiplicity of infection (MOI). Compared to Adv-GFP transfected and nontransfected islets, the levels of hVEGF secreted from Adv-GFP-hVEGF transfected islets were higher and exhibit a linear relationship between hVEGF expression and MOI (10–5000). Persistent, but low level expression of hVEGF from nontransfected islets was also observed. This may be due to expression of the endogenous hVEGF gene under hypoxic conditions. The levels of DNA fragmentation determined by ELISA of islet lysates were dependent on the MOI of Adv-GFP-hVEGF. On glucose challenge, insulin release from transfected islets was comparable to nontransfected islets. Immunohistochemical staining for hVEGF was very high in Adv-GFP-hVEGF transfected islets. Weak staining was also observed for hCD31 in both transfected and nontransfected islets. These findings suggest that Adv-GFP-hVEGF is a potential candidate for promoting islet revascularization.

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.