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GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca²⁺ nanoparticles underlies distinct gene therapy effect in ovarian cancer.

ABSTRACT: Practice of tumor-targeted suicide gene therapy is hampered by unsafe and low efficient delivery of plasmid DNA (pDNA). Using HIV-Tat-derived peptide (Tat) to non-covalently form Tat/pDNA complexes advances the delivery performance. However, this innovative approach is still limited by intracellular delivery efficiency and cell-cycle status. In this study, Tat/pDNA complexes were further condensed into smaller, nontoxic nanoparticles by Ca²⁺ addition. Formulated Tat/pDNA-Ca²⁺ nanoparticles mainly use macropinocytosis for intercellular delivery, and their macropinocytic uptake was persisted in mitosis (M-) phase and highly activated in DNA synthesis (S-) phase of cell-cycle. Over-expression or phosphorylation of a mitochondrial chaperone, 75-kDa glucose-regulated protein (GRP75), promoted monopolar spindle kinase 1 (MPS1)-controlled centrosome duplication and cell-cycle progress, but also driven cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca²⁺ nanoparticles. Further in vivo molecular imaging based on DF (Fluc-eGFP)-TF (RFP-Rluc-HSV-ttk) system showed that Tat/pDNA-Ca²⁺ nanoparticles exhibited highly suicide gene therapy efficiency in mouse model xenografted with human ovarian cancer. Furthermore, arresting cell-cycle at S-phase markedly enhanced delivery performance of Tat/pDNA-Ca²⁺ nanoparticles, whereas targeting GRP75 reduced their macropinocytic delivery. More importantly, in vivo targeting GRP75 combined with cell-cycle or macropinocytosis inhibitors exhibited distinct suicide gene therapy efficiency. In summary, our data highlight that mitochondrial chaperone GRP75 moonlights as a biphasic driver underlying cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca²⁺ nanoparticles in ovarian cancer.

SUBMITTER: Su L

PROVIDER: S-EPMC9301890 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles underlies distinct gene therapy effect in ovarian cancer.

Su Linjia L Sun Zhe Z Qi Fangzheng F Su Huishan H Qian Luomeng L Li Jing J Zuo Liang L Huang Jinhai J Yu Zhilin Z Li Jinping J Chen Zhinan Z Zhang Sihe S

Journal of nanobiotechnology 20220720 1

Practice of tumor-targeted suicide gene therapy is hampered by unsafe and low efficient delivery of plasmid DNA (pDNA). Using HIV-Tat-derived peptide (Tat) to non-covalently form Tat/pDNA complexes advances the delivery performance. However, this innovative approach is still limited by intracellular delivery efficiency and cell-cycle status. In this study, Tat/pDNA complexes were further condensed into smaller, nontoxic nanoparticles by Ca2+ addition. Formulated Tat/pDNA-Ca2+</su ...[more]

PMID: 35858873

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Project description:BackgroundSuccessful implementation of gene therapy heavily relies on efficiently delivering genetic materials and specific targeting into cells. Oncogene-driven endocytosis stimulates nutrient uptake and also develops an endocytosis-mediated defense against therapeutic agents. Cell-penetrating peptides, typically HIV-Tat, are well known for efficient delivery of nucleic acid drugs but lack targeting specificity. Various passive targeting strategies were pursued to enhance the tumor targeting efficiency; however, they are still limited by complicated cellular endocytosis routes and the heterogeneity of cancer types.MethodsTat/pDNA complexes were noncovalently compacted and their physiochemical properties were determined. The siRNA pool and pLV-RNAi-GFP lentivirus were used to knock down dbl oncogene (originally isolated from diffuse B-cell lymphoma) expression, and its overexpression was performed by plasmid transient transfection. The cellular uptake of fluorescent ligands was quantified by confocal imaging and flow cytometry analysis. The transgene efficiency was determined by the Luciferase expression assay. Rho GTPase activation was checked by the GST-Rho GTPase-binding domain pull-down assay.ResultspGL3 plasmid DNA was noncovalently compacted with the Tat peptide into nano-size complexes at high N/P ratios. Macropinocytosis, a clathrin- and caveolin-independent endocytosis process, was shown to contribute to the uptake of middle-sized (∼600 nm) Tat/pGL3 complexes. Cell-type-specific variation in macropinocytosis was essentially controlled by the action of the Dbl oncogene. Onco-Dbl presentation constantly induced a high level of macropinocytosis activity in ovarian cancer cells. Onco-Dbl overexpression hyperstimulated macropinocytosis enhancement in cells mainly through actin cytoskeleton reorganization mediated by the PH domain and Rac1 activation. The Dbl-driven Rho GTPase signaling collectively determined the cell-type-specific macropinocytosis phenotype.ConclusionSuch an aspect can be exploited to selectively confer targeted delivery of Tat/pDNA nano-complexes into ovarian cancer cells. Our work provides a novel alternative for targeted delivery of cell-penetrating peptide-based nucleic acid drugs into certain tumor types if specific endocytosis pathways are used.

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GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca²⁺ nanoparticles underlies distinct gene therapy effect in ovarian cancer.

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GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca<sup>2+</sup> nanoparticles underlies distinct gene therapy effect in ovarian cancer.

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GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles underlies distinct gene therapy effect in ovarian cancer.

Publications

GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca&lt;sup&gt;2+&lt;/sup&gt; nanoparticles underlies distinct gene therapy effect in ovarian cancer.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca²⁺ nanoparticles underlies distinct gene therapy effect in ovarian cancer.

GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca<sup>2+</sup> nanoparticles underlies distinct gene therapy effect in ovarian cancer.