Chiu-Ling Chen

Simultaneous induction of autophagy and toll-like receptor signaling pathways by graphene oxide

Graphene oxide (GO) nanosheets have sparked growing interests in biological and medical applications. This study examined how macrophage, the primary immune cell type engaging microbes, responded to GO treatment. We uncovered that incubation of macrophage cell RAW264.7 with GO elicited autophagy in a concentration-dependent manner, as evidenced by the appearance of autophagic vacuoles and activation of autophagic marker proteins. Such GO-induced autophagy was observed in various cell lines and in macrophage treated with GO of different sizes. Strikingly, GO treatment of macrophage provoked the toll-like receptor (TLR) signaling cascades and triggered ensuing cytokine responses. Molecular analysis identified that TLR4 and TLR9 and their downstream signaling mediators MyD88, TRAF6 and NF-κB played pivotal roles in the GO-induced inflammatory responses. By silencing individual genes in the signaling pathway, we further unveiled that the GO-induced autophagy was modulated by TLR4, TLR9 and was dependent on downstream adaptor proteins MyD88, TRIF and TRAF6. Altogether, we demonstrated that GO treatment of cells simultaneously triggers autophagy and TLR4/TLR9-regulated inflammatory responses, and the autophagy was at least partly regulated by the TLRs pathway. This study thus suggests a mechanism by which cells respond to nanomaterials and underscores the importance of future safety evaluation of nanomaterials.

Suppression of hepatocellular carcinoma by baculovirus-mediated expression of long non-coding RNA PTENP1 and MicroRNA regulation.

Long non-coding RNAs (lncRNAs) play regulatory roles in cancers. LncRNA PTENP1 is a pseudogene of the tumor suppressor gene PTEN but its roles in hepatocellular carcinoma (HCC) have yet to be explored. Here we confirmed that PTENP1 and PTEN were downregulated in several HCC cells, thus we constructed Sleeping Beauty (SB)-based hybrid baculovirus (BV) vectors for sustained PTENP1 lncRNA expression. Co-transduction of HCC cells with the SB-BV vector expressing PTENP1 elevated the levels of PTENP1 and PTEN, which suppressed the oncogenic PI3K/AKT pathway, inhibited cell proliferation, migration/invasion as well as induced autophagy and apoptosis. The overexpressed PTENP1 decoyed oncomirs miR-17, miR-19b and miR-20a, which would otherwise target PTEN, PHLPP (a negative AKT regulator) and such autophagy genes as ULK1, ATG7 and p62, indicating that PTENP1 modulated the HCC cell behavior and gene networks by miRNA regulation. Injection of the PTENP1-expressing SB-BV vector into mice bearing HCC tumors effectively mitigated the tumor growth, suppressed intratumoral cell proliferation, elicited apoptosis, autophagy and inhibited angiogenesis. These data collectively unveiled the molecular mechanisms of how PTENP1 repressed the tumorigenic properties of HCC cells and demonstrated the potential of the SB-BV hybrid vector for PTENP1 lncRNA modulation and HCC therapy.

Graphene Oxide Triggers Toll‐Like Receptors/Autophagy Responses In Vitro and Inhibits Tumor Growth In Vivo

Graphene oxide (GO) is a nanomaterial with burgeoning bioapplications, while autophagy is implicated in cancer therapy. Although induction of autophagy by nanomaterials is reported, the underlying signaling mechanism in cancer cells and how this implicates the potential of GO in cancer therapy remain obscure. Here, it is shown that GO itself can induce the toll-like receptors (TLRs) responses and autophagy in cancer cells and confer antitumor effects in mice. GO can be phagocytosed by CT26 colon cancer cells, simultaneously triggering autophagy as well as TLR-4 and TLR-9 signaling cascades. By dissecting the crosstalk between the TLRs and autophagy pathways, it is uncovered that the GO-activated autophagy is regulated through the myeloid differentiation primary response gene 88 (MyD88)- and TNF receptor-associated factor 6 (TRAF6)-associated TLR-4/9 signaling pathways. Injection of GO alone into immunocompetent mice bearing the CT26 colon tumors not only suppresses the tumor progression but also enhances cell death, autophagy, and immune responses within the tumor bed. These data altogether implicate the potential of GO as an effective nanomaterial for autophagy induction and cancer therapy.

Efficient gene delivery into cell lines and stem cells using baculovirus

Baculovirus is a promising vector for transducing numerous types of mammalian cells. We have developed hybrid baculovirus vectors and protocols for the efficient transduction of a variety of cell lines, primary cells and stem cells, including bone marrow–derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs). The hybrid vector enables intracellular minicircle formation and prolongs transgene expression. The advantages of this transduction protocol are that baculovirus supernatant alone needs to be added to cells growing in medium, and transduction occurs after only 4–6 h of incubation at room temperature (25 °C) with gentle shaking. The entire procedure, from virus generation to transduction, can be completed within 4 weeks. Compared with other transduction procedures, this protocol is simple and can confer efficiencies >95% for many cell types. This protocol has potential applications in tissue regeneration, as transduced cells continue to express transgenes after implantation. For example, transduction of rabbit ASCs (rASCs) with growth factor–encoding hybrid baculovirus vectors, as described as an example application in this protocol, enables robust and sustained growth factor expression, stimulates stem cell differentiation and augments tissue regeneration after implantation.

Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study

Baculovirus (BV) is a promising gene vector but mediates transient expression. To prolong the expression, we developed a binary system whereby the transgene in the substrate BV was excised by the recombinase (ΦC31o, Cre or FLPo) expressed by a second BV and recombined into smaller minicircle. The recombination efficiency was lower by ΦC31o (≈40–75%), but approached ≈90–95% by Cre and FLPo in various cell lines and stem cells [e.g. human adipose-derived stem cells (hASCs)]. Compared with FLPo, Cre exerted higher expression level and lower negative effects; thus, we incorporated additional cis-acting element [oriP/Epstein–Barr virus nuclear antigen 1 (EBNA1), scaffold/matrix attached region or human origin of replication (ori)] into the Cre-based BV system. In proliferating cells, only oriP/EBNA1 prolonged the transgene expression and maintained the episomal minicircles for 30 days without inadvertent integration, whereas BV genome was degraded in 10 days. When delivering bmp2 or vegf genes, the efficient recombination/minicircle formation prolonged and enhanced the growth factor expression in hASCs. The prolonged bone morphogenetic protein 2 expression ameliorated the osteogenesis of hASCs, a stem cell with poor osteogenesis potential. Altogether, this BV vector exploiting Cre-mediated recombination and oriP/EBNA1 conferred remarkably high recombination efficiency, which prolonged and enhanced the transgene expression in dividing and non-dividing cells, thereby broadening the applications of BV.

Graphene oxide as a chemosensitizer: Diverted autophagic flux, enhanced nuclear import, elevated necrosis and improved antitumor effects

Graphene oxide (GO) is a nanomaterial that provokes autophagy in CT26 colon cancer cells and confers antitumor effects. Here we demonstrated that both GO and the chemotherapy drug cisplatin (CDDP) induced autophagy but elicited low degrees of CT26 cell death. Strikingly, GO combined with CDDP (GO/CDDP) potentiated the CT26 cell killing via necrosis. GO/CDDP not only elicited autophagy, but induced the nuclear import of CDDP and the autophagy marker LC3. The nuclear LC3 did not co-localize with p62 or Lamp-2, neither did blocking autolysosome formation significantly hinder the nuclear import of LC3/CDDP and necrosis, indicating that autophagosome and autolysosome formation was dispensable. Conversely, suppressing phagophore formation and importin-α/β significantly alleviated the nuclear import of LC3/CDDP and necrosis. These data suggested that GO/CDDP diverted the LC3 flux in the early phase of autophagy, resulting in LC3 trafficking towards the nucleus in an importin-α/β-dependent manner, which concurred with the CDDP nuclear import and necrosis. Intratumoral injection of GO/CDDP into mice bearing CT26 colon tumors potentiated immune cell infiltration and promoted cell death, autophagy and HMGB1 release, thereby synergistically augmenting the antitumor effects. Altogether, we unveiled a mechanism concerning how nanomaterials chemosensitize cancer cells and demonstrated the potentials of GO as a chemosensitizer.

Immune responses during healing of massive segmental femoral bone defects mediated by hybrid baculovirus-engineered ASCs.

Baculovirus holds promise for genetic modification of adipose-derived stem cells (ASCs) and bone engineering. To explore the immune responses during bone healing and the cell fate, ASCs were mock-transduced (Mock group), transduced with the baculovirus transiently expressing growth factors promoting osteogenesis (BMP2) or angiogenesis (VEGF) (S group), or transduced with hybrid baculoviruses persistently expressing BMP2/VEGF (L group). After allotransplantation into massive femoral defects in rabbits, these 3 groups triggered similar degrees of transient inflammatory response (e.g. neutrophil proliferation and immune cell infiltration into the graft site), revealing that baculovirus and transgene products did not exacerbate the inflammation. The cells in all 3 groups underwent apoptosis initially, persisted for at least 4 weeks and were eradicated thereafter. The L group prolonged the in vivo BMP2/VEGF expression (up to 4 weeks), extended the antibody responses, and slightly enhanced the cell-mediated cytotoxicity. Nonetheless, the L group led to remarkably better bone healing and remodeling than the Mock and S groups. These data confirmed that the ASCs engineered with the hybrid BV imparted prolonged expression of BMP2/VEGF which, although stimulated low levels of humoral and cell-mediated immune responses, essentially augmented the healing of massive segmental bone defects.

Baculovirus-Mediated miRNA Regulation to Suppress Hepatocellular Carcinoma Tumorigenicity and Metastasis

MicroRNA 122 (miR-122) is a tumor suppressor for hepatocellular carcinoma (HCC) but is lowly expressed in HCC cells. MiR-151 is aberrantly overexpressed in HCC cells and promotes HCC metastasis yet its roles on HCC tumorigenicity are unknown. To combat HCC tumorigenicity/metastasis, we developed Sleeping Beauty (SB)-based hybrid baculovirus (BV) vectors that expressed (i) miR-122 precursors (pre-miR-122), (ii) miR-151 sponges, or (iii) pre-miR-122 and miR-151 sponges. Transduction of aggressive HCC cells (Mahlavu) with the pre-miR-122-expressing BV tremendously enhanced miR-122 levels for >6 weeks, suppressed the levels of downstream effectors (e.g., ADAM10 and Bcl-w), proliferation, anchorage-independent growth, motility and migration/invasion in vitro. Intratumoral injection of the pre-miR-122-expressing BV attenuated the HCC growth/metastasis. The miR-151 sponges-expressing BV diminished the miR-151 levels for 6 weeks, enhanced RhoGDIA expression, suppressed RhoGTPases, as well as motility and migration/invasion of Mahlavu cells. Intratumoral injection of the miR-151 sponge-expressing BV impeded not only HCC metastasis but also cell proliferation, MMP expression and tumor growth in vivo. The BV co-expressing pre-miR-122 and miR-151 sponges also simultaneously enhanced miR-122 expression and inhibited miR-151, and conferred antitumor/anti-metastasis effects albeit lack of synergism. These data implicate the potentials of the SB-based hybrid BV for persistently modulating miRNA and suppressing HCC tumorigenicity/metastasis.

Defective Antiviral Responses of Induced Pluripotent Stem Cells to Baculoviral Vector Transduction

ABSTRACT Genetic engineering of induced pluripotent stem cells (iPSCs) is important for their clinical applications, and baculovirus (BV) holds promise as a gene delivery vector. To explore the feasibility of using BV for iPSCs transduction, in this study we first examined how iPSCs responded to BV. We determined that BV transduced iPSCs efficiently, without inducing appreciable negative effects on cell proliferation, apoptosis, pluripotency, and differentiation. BV transduction slightly perturbed the transcription of 12 genes involved in the Toll-like receptor (TLR) signaling pathway, but at the protein level BV elicited no well-known cytokines (e.g., interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-α], and beta interferon [IFN-β]) except for IP-10. Molecular analyses revealed that iPSCs expressed no TLR1, -6, -8, or -9 and expressed merely low levels of TLR2, -3, and -4. In spite of evident expression of such RNA/DNA sensors as RIG-I and AIM2, iPSCs barely expressed MDA5 and DAI (DNA-dependent activator of IFN regulatory factor [IRF]). Importantly, BV transduction of iPSCs stimulated none of the aforementioned sensors or their downstream signaling mediators (IRF3 and NF-κB). These data together confirmed that iPSCs responded poorly to BV due to the impaired sensing and signaling system, thereby justifying the transduction of iPSCs with the baculoviral vector.

Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression

MicroRNA (miRNA) plays essential roles in regulating gene expression, but miRNA delivery remains a hurdle, thus entailing a vector system for efficient transfer. Baculovirus emerges as a promising gene delivery vector but its inherent transient expression restricts its applications in some scenarios. Therefore, this study primarily aimed to develop baculovirus as a miRNA expression vector for prolonged gene suppression. We constructed recombinant baculoviruses carrying artificial egfp‐targeting miRNA sequences within the miR155 backbone, which after expression by the cytomegalovirus promoter could knockdown the enhanced green fluorescent protein (EGFP) expression in a sequence‐ and dose‐dependent manner. By swapping the mature miRNA sequences, the baculovirus miRNA shuttle effectively repressed the overexpression of endogenous TNF‐α in arthritic synoviocytes without inducing apoptosis. To prolong the baculovirus‐mediated expression, we further developed a hybrid baculovirus vector that exploited the Sleeping Beauty (SB) transposon for gene integration and sustained miRNA expression. The hybrid baculovirus vector that combined the miR155 scaffold and SB transposon effectively repressed the transgene expression for a prolonged period of time, hence diversifying the applications of baculovirus to indications necessitating prolonged gene regulation such as arthritis. Biotechnol. Bioeng. 2011;108: 2958–2967.