Protein Kinase C α-Responsive Gene Carrier for Cancer Therapy
Protein Kinase C α-Responsive Gene Carrier for Cancer-Specific Transgene Expression and Cancer Therapy
The presence of intracellular signal transduction and its abnormal activities in many cancers has potential for medical and pharmaceutical applications. They recently developed a protein kinase C α (PKCα)-responsive gene carrier for cancer-specific gene delivery. Here, they demonstrate an in-depth analysis of cellular signal-responsive gene carrier and the impact of its selective transgene expression in response to malfunctioning intracellular signaling in cancer cells. They prepared a novel gene carrier consisting of a linear polyethylenimine (LPEI) main chain grafted to a cationic PKCα-specific substrate (FKKQGSFAKKK-NH2). The LPEI–peptide conjugate formed a nanosized polyplex with pDNA and mediated efficient cellular uptake and endosomal escape. This polyplex also led to successful transgene expression which responded to the target PKCα in various cancer cells and exhibited a 10–100-fold higher efficiency compared to the control group. In xenograft tumor models, the LPEI–peptide conjugate promoted transgene expression showing a clear-cut response to PKCα. Furthermore, when a plasmid containing a therapeutic gene, human caspase-8 (pcDNA-hcasp8), was used, the LPEI–peptide conjugate had significant cancer-suppressive effects and extended animal survival. Collectively, these results reveal that their method has great potential for cancer-specific gene delivery and therapy.
An important consideration for gene-delivery systems is target cell- or tissue-specific delivery for efficient transgene expression. Various approaches have been combined to develop an efficient gene-delivery system, in which transgenes can be specifically expressed at pathological sites. These approaches, consisting of “passive targeting” and “active targeting”, are increasingly used for cancer therapy; however, significant obstacles need to be overcome before therapeutic genes can be effectively delivered to target sites. Furthermore, to achieve the practical usage of a gene-delivery system in clinical applications, more precise and accurate approaches have to be considered to ensure the therapeutic payload to target cells without side effects. Abnormal intracellular signaling, especially protein kinases (PKs), is an important factor in cancer and other diseases.
Intracellular images of polyplexes. (a) Cellular uptakes of LPEI polyplex, LPEI–pentyne polyplex, and LPEI(S) polyplex. (b) Quantitative analysis of mean fluorescence intensity for polyplexes (n = 3 per group). *P < 0.05. (c) Time-course distribution of the LPEI(S) polyplex in A549 cells. (d) Higher magnification of the yellow boxed areas in (c). Confocal images were taken at 1, 3, 6, and 24 h after a 6 h transfection of polyplexes. pDNA was labeled with Cy5 (red). Nuclei were stained with Hoechst 33342 (blue) and late endosomes/lysosomes were stained with LysoTracker Green (green) and Hoechst 33342 (blue), respectively. Scale bar: 20 μm.
For instance, PKs such as c-SRC, phosphotidylinositol-3-kinase (PI3K)/Akt, protein kinase A, and protein kinase C α (PKCα) involved in many aspects of cancer proliferation, progression, and metastasis, wherein PKs are known to be commonly activated in various types of cancer cells. In particular, abnormal protein kinase C α (PKCα) activity is closely related to cancer malignancy, bad prognosis, and metastatic activity. Thus, PKs are considered potential therapeutic targets for the treatment of cancer formation and progression in humans. Approaches to use these abnormal activities have been merged into advances in nano/biotechnologies toward cell- and tissue-specific targeting strategies.
In vivo transgene expression responding to PKCα and therapeutic effects of the LPEI–peptide conjugate. (a) Transgene expression in A549 and U87-MG cancers. Luciferase expression was monitored using an IVIS imaging system 24 h after the injection of LPEI–peptide conjugate/pCMV-luc2 polyplexes (60 μL) (pCMV-luc2, 10 μg). Total numbers of positive-expressing mice are as follows: 5/5 [LPEI(S)] and 0/4 [LPEI(A)] in A549 cancer tissues and 8/8 [LPEI(S)] and 1/5 [LPEI(A)] in U87-MG cancer tissues. (b) Change in cancer growth after the transfection of various polyplex formations containing pcDNA-hcasp8 (10 μg) (n = 5–7). ***P < 0.001. (c) Survival curve of mice treated by the direct injection of various polyplex formations. (d) Body weight of mice in response to treatment with various polyplex formations.
Recently, they developed a cellular signal-responsive gene carrier that can be selectively responsive to malfunctioning intracellular signaling in cancer cells.This novel gene carrier consisting of a linear polyethylenimine (LPEI) main chain grafted to a cationic PKCα-specific substrate (FKKQGSFAKKK-NH2). The LPEI–peptide conjugate produced successful transgene expression in vitro and in vivo responding to target PKCα in several cancer cells and tissues.
PKCα is overactivated in many cancer cells and tissues and shows negligible activity in normal cells and tissues. The cationic LPEI–peptide conjugate forms a complex with an anionic gene through an electrostatic interaction that inhibits gene expression. However, when the complex is transferred into target cancer cells, the phosphorylation of the side-chain PKCα-specific peptide by activated PKCα results in dissociation of the complex enabling the gene to bind transcription factors. On the other hand, the negligible activity of PKCα in normal cells leads to little or no effect on the phosphorylation of the side-chain peptide1.
Protein Kinase C α-Responsive Gene Carrier for Cancer-Specific Transgene Expression and Cancer Therapy Chan Woo Kim, Riki Toita, Jeong-Hun Kang, Takeshi Mori, Akihiro Kishimura, and Yoshiki Katayama ACS Biomaterials Science & Engineering Article ASAP DOI: 10.1021/acsbiomaterials.1c00213