Spatiotemporally-controlled hydrophobic drug delivery via photosensitizer-driven assembly-disassembly for enhanced triple-negative breast cancer treatment.

Therapeutic approaches for triple-negative breast cancer (TNBC) have been continuously advancing, but inadequate control over release behavior, insufficient tumor selectivity, and limited drug availability continue to impede therapeutic outcomes in nanodrug systems. In this study, we propose a general hydrophobic antineoplastic delivery system, termed spatiotemporally-controlled hydrophobic antineoplastic delivery system (SCHADS) for enhanced TNBC treatment. The key feature of SCHADS is the formation of metastable photosensitive-antineoplastic complexes (PACs) through the self-assembly of hydrophobic drugs driven by photosensitive molecules. With the further decoration of tumor-targeting peptides coupled with the EPR effect, the PACs tend to accumulate in the tumor site tremendously, promoting drug delivery efficiency. Meanwhile, the disassembly behavior of the metastable PACs could be driven by light on demand to achieve in situ drug release, thus promoting chemotherapeutics availability. Furthermore, the abundant ROS generated by the photosensitizer could effectively kill tumor cells, ultimately realizing an effective combination of photodynamic and chemotherapeutic therapy. As an exemplary presentation, chlorin e6 has been chosen to drive the formation of PACs with the system xc- inhibitor sorafenib. Compared with pure drug treatment, the PACs with the above-described preponderances exhibit superior therapeutic effects both in vitro and in vivo and circumvent the side effects due to off-target. By manipulating the laser irradiation, the PACs-treated cell death mechanism could be dynamically regulated, thus providing the potential to remedy intrinsic/acquired resistance of tumor. Collectively, this SCHADS achieves spatio-temporal control of the drug that greatly enhances the availability of anticarcinogen and realizes synergistic antitumor effect in TNBC treatment, even ultimately being extended to the treatment of other types of tumors.

Synthetic Biohybrids of Red Blood Cells and Cascaded-Enzymes@ Metal-Organic Frameworks for Hyperuricemia Treatment.

Hyperuricemia, caused by an imbalance between the rates of production and excretion of uric acid (UA), may greatly increase the mortality rates in patients with cardiovascular and cerebrovascular diseases. Herein, for fast-acting and long-lasting hyperuricemia treatment, armored red blood cell (RBC) biohybrids, integrated RBCs with proximal, cascaded-enzymes of urate oxidase (UOX) and catalase (CAT) encapsulated within ZIF-8 framework-based nanoparticles, have been fabricated based on a super-assembly approach. Each component is crucial for hyperuricemia treatment: 1) RBCs significantly increase the circulation time of nanoparticles; 2) ZIF-8 nanoparticles-based superstructure greatly enhances RBCs resistance against external stressors while preserving native RBC properties (such as oxygen carrying capability); 3) the ZIF-8 scaffold protects the encapsulated enzymes from enzymatic degradation; 4) no physical barrier exists for urate diffusion, and thus allow fast degradation of UA in blood and neutralizes the toxic by-product H2 O2 . In vivo results demonstrate that the biohybrids can effectively normalize the UA level of an acute hyperuricemia mouse model within 2 h and possess a longer elimination half-life (49.7 ± 4.9 h). They anticipate that their simple and general method that combines functional nanomaterials with living cell carriers will be a starting point for the development of innovative drug delivery systems.

Protocol to assemble metal-phenolic framework nanoparticles based on polyphenol-mediated biomimetic mineralization for wound healing in rats.

Here, we present a protocol for controllable biomimetic mineralization at the nano-scale, simulating natural ion-enriched sedimentary mineralization. We describe steps for treatment of metal-organic frameworks with polyphenol-mediated stabilized mineralized precursor solution. We then detail their use as templates to assemble metal-phenolic frameworks (MPFs) with mineralized layers. Furthermore, we demonstrate the therapeutic benefits of MPF delivery by hydrogel to the full-thickness skin defect model in rats. For complete details on the use and execution of this protocol, please refer to Zhan et al. (2022).1.

Dual-Stage Irradiation of Size-Switchable Albumin Nanocluster for Cascaded Tumor Enhanced Penetration and Photothermal Therapy.

The triple-negative breast cancer (TNBC) microenvironment makes a feature of aberrant vasculature, high interstitial pressure, and compact extracellular matrix, which combine to reduce the delivery and penetration of therapeutic agents, bringing about incomplete elimination of cancer cells. Herein, employing the tumor penetration strategy of size-shrinkage combined with ligand modification, we constructed a photothermal nanocluster for cascaded deep penetration in tumor parenchyma and efficient eradication of TNBC cells. In our approach, the photothermal agent indocyanine green (ICG) is laded in human serum albumin (HSA), which is cross-linked by a thermally labile azo linker (VA057) and then further modified with a tumor homing/penetrating tLyP-1 peptide (HP), resulting in a TNBC-targeting photothermal-responsive size-switchable albumin nanocluster (ICG@HSA-Azo-HP). Aided by the enhanced permeability and retention effect and guidance of HP, the ca. 149 nm nanoclusters selectively accumulate in the tumor site and then, upon mild irradiation with the 808 nm laser, disintegrate into 11 nm albumin fractions that possess enhanced intratumoral diffusion ability. Meanwhile, HP initiates the CendR pathway among the nutrient-deficient tumor cells and facilitates the transcellular delivery of the nanocluster and its disintegrated fractions for subsequent therapy. By employing this size-shrinkage and peptide-initiated transcytosis strategy, ICG@HSA-Azo-HP possesses excellent penetration capabilities and shows extensive penetration depth in three-dimensional multicellular tumor spheroids after irradiation. Moreover, with a superior photothermal conversion effect, the tumor-penetrating nanocluster can implement effective photothermal therapy throughout the tumor tissue under a second robust irradiation. Both in vivo orthotopic and ectopic TNBC therapy confirmed the efficient tumor inhibition of ICG@HSA-Azo-HP after dual-stage irradiation. The synergistic penetration strategy of on-demanded size-shrinkage and ligand guidance accompanied by clinically feasible NIR irradiation provides a promising approach for deep-penetrating TNBC therapy.

Glutathione-Depleting Organic Metal Adjuvants for Effective NIR-II Photothermal Immunotherapy.

Although photothermal immunotherapy (PTI) is a compelling strategy for tumor therapy, the development of promising photothermal agents to overcome the insufficient immunogenicity of tumor cells and the poor immune response encountered in PTI is still challenging. Herein, commercial small-molecule-based organic metal adjuvants (OMAs) are presented, with second near-infrared photoacoustic and photothermal properties as well as the ability to perturb redox homeostasis to potentiate immunogenicity and immune responsiveness. OMAs, assembled from charge-transfer complexes and characterized by a broad substrate scope, high accessibility, and flexibly tuned optical properties, demonstrate strong phototherapeutic and adjuvant abilities via the depletion of glutathione and cysteine, and subsequently elicit systemic immunity by evoking immunogenic cell death, promoting dendritic cell maturation, and increasing T cell infiltration. Furthermore, programmed cell death protein 1 antibody can be employed to synergize with OMAs to suppress tumor immune evasion and ultimately improve the treatment outcomes. This study unlocks new paradigms to provide a versatile OMA-based scaffold for future practical applications.

Approach for evaluating inundation risks in urban drainage systems.

Urban inundation is a serious challenge that increasingly confronts the residents of many cities, as well as policymakers. Hence, inundation evaluation is becoming increasingly important around the world. This comprehensive assessment involves numerous indices in urban catchments, but the high-dimensional and non-linear relationship between the indices and the risk presents an enormous challenge for accurate evaluation. Therefore, an approach is hereby proposed to qualitatively and quantitatively evaluate inundation risks in urban drainage systems based on a storm water management model, the projection pursuit method, the ordinary kriging method and the K-means clustering method. This approach is tested using a residential district in Guangzhou, China. Seven evaluation indices were selected and twenty rainfall-runoff events were used to calibrate and validate the parameters of the rainfall-runoff model. The inundation risks in the study area drainage system were evaluated under different rainfall scenarios. The following conclusions are reached. (1) The proposed approach, without subjective factors, can identify the main driving factors, i.e., inundation duration, largest water flow and total flood amount in this study area. (2) The inundation risk of each manhole can be qualitatively analyzed and quantitatively calculated. There are 1, 8, 11, 14, 21, and 21 manholes at risk under the return periods of 1-year, 5-years, 10-years, 20-years, 50-years and 100-years, respectively. (3) The areas of levels III, IV and V increase with increasing rainfall return period based on analyzing the inundation risks for a variety of characteristics. (4) The relationships between rainfall intensity and inundation-affected areas are revealed by a logarithmic model. This study proposes a novel and successful approach to assessing risk in urban drainage systems and provides guidance for improving urban drainage systems and inundation preparedness.

[Corrigendum] MicroRNA-320a inhibits tumor invasion by targeting neuropilin 1 and is associated with liver metastasis in colorectal cancer.

After carefully checking the original data of migration and invasion experiment, we find we had selected the wrong picture to show the effect of microRNA320a on the migration of LoVo cells. The corrected version of Fig. 6A shown below contains appropriate representative images. We apologize for the error and appreciate the opportunity to correct the scientific record. All authors agree with this correction. [the original article was published in the Oncology Reports 27: 685-694, 2012 DOI: 10.3892/or.2011.1561]

Identification of two new HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from colorectal carcinoma-associated antigen PLAC1/CP1.

BACKGROUND: To explore the potential application of placenta-specific PLAC1/Cancer Placenta (CP) 1 antigen for immunotherapy in CRC patients, further identification of the cytotoxic T lymphocyte epitopes from this antigen is necessary. METHODS: We assessed the protein expression of PLAC1/CP1 using a tissue chip and immunochemistry staining in CRC samples. Simultaneously, we predicted four PLAC1/CP1-derived HLA-A*0201-restricted peptides by using reverse immunology methods. Peptide-specific CD8(+) T cell responses were assessed by an IFN-γ release ELISPOT assay. Effector CD8(+) T cells lyse HLA-A*0201 CRC cell line SW620 was detected in a granzyme-B release ELISPOT cytotoxicity assay. RESULTS: Our results indicated that PLAC1/CP1 was highly expressed in 56.7 % (55/97) of adenocarcinomas. PLAC1/CP1 protein expression was associated with CRC tumor differentiation, the tumor/node/metastasis stage, and lymph node metastasis. Two of four peptides showed high affinities in an HLA-A2 binding assay. In 66.7 % (6/9) of peripheral blood mononuclear cells of CRC samples with PLAC1/CP1 protein-positive expression, these two peptides, PLAC1/CP1 p41-50 (FMLNNDVCV) and PLAC1/CP1 p69-77 (HAYQFTYRV), were immunogenic in the induction of peptide-specific CD8(+) T cell responses as assessed by an IFN-γ release ELISPOT assay. Furthermore, the generated effector CD8(+) T cells could specifically lyse the PLAC1/CP1 HLA-A*0201 CRC cell line SW620 in a granzyme-B release ELISPOT cytotoxicity assay. CONCLUSIONS: These results show that the PLAC1/CP1 antigen is a possible prognostic marker of CRC and that PLAC1/CP1 p41-50 and PLAC1/CP1 p69-77 are novel HLA-A*0201-restricted CD8(+) T cell epitopes and potential targets for peptide-based immunotherapy in CRC patients.

Effects of chemokine-like factor 1 on vascular smooth muscle cell migration and proliferation in vascular inflammation.

OBJECTIVE: Vascular smooth muscle cell (VSMC) migration and proliferation are key components of vascular inflammation that may lead to atherosclerosis and restenosis, in which cytokines are considered as pivotal factors regarding recruitment of VSMC. A member of recently described family of chemokines, chemokine-like factor 1 (CKLF1), displays a wide spectrum of chemotaxis. This study investigated the role of CKLF1 in VSMC migration and proliferation during the process of vascular inflammation. METHODS AND RESULTS: : The effects of CKLF1 on migration, proliferation and neointimal formation were investigated in cultured VSMCs, rat balloon injured arteries and human atherosclerotic plaques. CKLF1 overexpression greatly enhanced, whereas shRNA knockdown markedly retarded, VSMC migration and proliferation in vitro. In addition, CKLF1 protein accumulated preferentially in neointima of the injured rat arteries in vivo. CKLF1 overexpression resulted in a 2.5-fold increase in intimal thickness. In contrast, shRNA-mediated CKLF1 knockdown significantly suppressed neointima formation by 70% compared that in control group. Intriguingly, besides animal model, higher level of CKLF1 was observed in human atherosclerotic plaques than that in normal arteries. CONCLUSION: CKLF1 plays an essential role in migration and proliferation of VSMCs, which in turn facilitated neointimal hyperplasia and atherosclerosis. Inhibition of CKLF1 activity provides a potential target for the prevention of atherosclerosis and restenosis.

Effect of kappa elastin on melanogenesis in A375 human melanoma cells and its related mechanism.

BACKGROUND: Elastin derived peptides can regulate melanocyte precursor development. Ultraviolet irradiation, infrared radiation and heat can increase the synthesis of tropoelastin in human skin epidermis. The aim of this study was to investigate whether the over expressed tropoelastin in epidermis has some role in melanogenesis of melanocytes. METHODS: A375 human melanoma cells were treated with different concentrations of kappa elastin for 24 hours. A375 human melanoma cells were randomly assigned to control, kappa elastin, and lactose pre-incubated groups. The cell viabilities were detected by the methyl thiazoleterazolium assay. Melanin content and tyrosinase activity in A375 melanoma cells were measured. The expressions of endothelin B receptor (ET(B)R) mRNA and c-kit mRNA in A375 melanoma cells were measured by quantative reverse transcription polymerase chain reaction. RESULTS: Fifty µg/ml of kappa elastin significantly increased the melanin content by 56.64% compared with the control (P < 0.05). Kappa elastin increased cellular tyrosinase activity by 46.73% compared with the control at 24 hours (P < 0.05). Kappa elastin increased the expressions of ET(B)R and c-kit mRNA levels by 2.13-fold and 2.47-fold compared with the controls, respectively. When pre-incubating cells with a lactose solution (10 mmol/L), the inhibition on melanin production was 34.96% compared with the kappa elastin group (P < 0.05), tyrosinase activity was inhibited by 29.93% compared with kappa elastin group (P < 0.05), and the expressions of ET(B)R mRNA and c-kit mRNA were decreased by 1.56-fold and 0.82-fold compared with kappa elastin group, respectively. CONCLUSION: Kappa elastin increased the melanogenesis in A375 melanoma cells via the stimulation of tyrosinase activity and the expression of ET(B)R and c-kit. The over expressed tropoelastin produced by keratinocytes might play a role in melanogenesis of epidermal melanocytes.

Genistein inhibit cytokines or growth factor-induced proliferation and transformation phenotype in fibroblast-like synoviocytes of rheumatoid arthritis.

The purpose of this research is to study the effect of genistein on cytokines or growth factor-induced proliferation and transformation phenotype of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). RA-FLS were primarily cultured. With respective stimulation of IL-1ß, TNF-α, and EGF, genistein was applied to elucidate its effect on synoviocytes' growth number, cell proliferation assay, cell cycle using cell counts, (3)H-TdR incorporation and flow cytometry, the colony numbers under anchorage-independent condition, and the expression of MMP-2 and MMP-9 in synovial fibroblasts. EGF, IL-1ß, and TNF-α increased (3)H incorporation in RA-FLS, respectively. EGF augmented clone numbers of RA-FLS under anchorage-independent condition and not IL-1ß and TNF-α. Genistein had an inhibitory role on cell number and (3)H-TdR incorporation of RA-FLS stimulated with IL-1ß, TNF-α and EGF; genistein arrested the cell cycle at G(1) restriction point; genistein decreased colony numbers under anchorage-independent condition stimulated by EGF in serum condition. IL-1ß or TNF-α increased expression of MMP-9 and MMP-2 in rheumatoid synoviocytes; EGF stimulated expression of MMP-9 but not of MMP-2; genistein suppressed production of MMP-9 more than MMP-2 induced by IL-1ß or TNF-α; rMMP-9, rMMP-2, or their inhibitors had no effect on the (3)H-TdR incorporation of synovial cells. Erk1/2 inhibitor (PD098 059) had obvious inhibitory effect on the (3)H incorporation induced by TNF-α or IL-1ß; inhibitors of JNK (SP600 125) had no significant effect on the (3)H incorporation. While pretreatment with PD098059 had no marked inhibitory effect on MMP-9 expression induced by TNF-α or IL-1ß, SP600125 decreased significantly the MMP-9 expression induced by TNF-α or IL-1ß. Neither PD098059 nor SP600 125 could inhibit the MMP-2 expression induced by TNF-α or IL-1ß. Genistein inhibited IL-1ß, TNF-α or EGF-induced proliferation and MMP-9 expression in fibroblast-like synoviocytes of rheumatoid arthritis; the proliferation of RA-FLS was mediated by Erk1/2 but not JNK activation, while JNK activation was involved in the signal transduction pathway leading to MMP-9 expression in rheumatoid synoviocytes.

microRNA-320a inhibits tumor invasion by targeting neuropilin 1 and is associated with liver metastasis in colorectal cancer.

MicroRNAs (miRNAs) have been implicated in regulating diverse cellular pathways. Although there is emerging evidence that various miRNAs function as oncogenes or tumor suppressors in colorectal cancer (CRC), the role of miRNAs in mediating liver metastasis remains unexplored. The expression profile of miRNAs in liver metastasis and primary CRC tissues was analyzed by miRNA microarrays and verified by real-time polymerase chain reaction (PCR). In 62 CRC patients, the expression levels of miR-320a were determined by real-time PCR, and the effects on migration and invasion of miR-320a were determined using a transwell assay. miR-320a target genes were confirmed by luciferase assay, real-time PCR and western blot analysis. A set of miRNAs was found to be dysregulated in the liver metastasis tissues compared to matched primary CRC tissues, and the expression levels of miR-320a were significantly decreased in the liver metastasis tissues examined. miR-320a was correlated with tumor progression in CRC. miR-320a was downregulated in liver metastatic colon cancer cells and inhibited liver metastatic colon cancer cell migration and invasion. miR-320a directly binds to the 3'UTR of neuropilin 1 (NRP-1), a protein that functions as a co-receptor of vascular epithelial growth factor. miR-320a downregulated the expression of NRP-1 at both the mRNA and protein levels. These data demonstrated that miR-320a may be useful for identifying CRC patients that are at an elevated risk for developing liver metastasis. Our findings suggest that miR-320a may be a novel therapeutic candidate for the treatment of colorectal cancer.

[Effect of integrin-linked kinase on the growth of prostate cancer in nude mice].

OBJECTIVE: To investigate the effect of the integrin-linked kinase (ILK) small interfering RNA (siRNA) on prostate cancer in nude mice by orthotopic injection of human cell line DU145. METHODS: The cultured human cell line DU145 was knocked down for ILK using a siRNA .Cellular ILK expression was quantified by RT-PCR and Western blot analysis. Moreover, cell attachment, invasiveness and microfilament dynamics assays were performed. Furthermore, the impact of the ILK siRNA on the prostate cancer was tested using a nude mice model in which prostate cancer was induced by orthotopic injection of human prostate cancer cell line DU145.Gross tumor volume of prostate in nude mice,cell differentiation,the state of apoptosis and proliferation were tested after 5 weeks of injection. RESULTS: The expression of ILK was suppressed significantly by siRNA, cellular mRNA and protein of ILK decreased 87% and 81% separately. The knockdown of ILK also induced the attachment and invasiveness of DU145 cell growing down. The tumor volume, cell differentiation, apoptosis index and proliferation index of prostate in nude mice of ILK siRNA orthotopic injection model were significantly smaller, better, increased and decreased separately than those in control group. CONCLUSION: Targeting inhibition of ILK not only decreases attachment and invasiveness of human DU145 cells, but also suppresses the growth and development of prostate cancer of orthotopic injection human DU145 cell line model in nude mice.

Quercetin in combating H2O2 induced early cell apoptosis and mitochondrial damage to normal human keratinocytes.

BACKGROUND: Oxidative stress plays an important role in the pathogenesis of epidermal diseases. This study aimed to investigate the effects of quercetin on the anti-oxidative response and on mitochondrial protection in cultured normal human keratinocytes. METHODS: Cultured HaCaT cells were treated with different concentrations of H2O2 (0, 50, 100, 250, 500 micromol/L) for different periods of time (0.5, 1, 2, 4 hours) to establish an oxidative stress model. The cultured HaCaT cells were randomly assigned to control, H2O2, and quercetin + H2O2 groups. For the quercetin groups, the cells were treated with different concentrations of quercetin (0, 10, 25, 50 micromol/L) before exposure to H2O2. Morphological changes of the cells were observed under an inverted microscope and an electron microscope. The cell viability was detected by the MTT method. The cell apoptosis (AnnexinV/propidium iodide double stain) and mitochondrial membrane potential (DeltaPsim) changes were detected by flow cytometry. RESULTS: An oxidative stress model of HaCaT cells was established under a suitable concentration (250 micromol/L) and treated time of H2O2 (2 hours). The cell viability and DeltaPsim decreased in a concentration-dependent and time-dependent manner while the percentage of apoptotic cells significantly increased in the H2O2 groups compared with the control group (P < 0.05). The cell viability and DeltaPsim of the quercetin treated group increased (P < 0.05) and the percentage of apoptotic cells decreased at concentrations of 1-50 micromol/L quercetin (P < 0.01) compared with H2O2 treated group. CONCLUSION: Quercetin can relieve the cell damage and apoptosis from H2O2 induced injury to HaCaT cells by anti-oxidation and mitochondrial protection.

Targeting of integrin-linked kinase with a small interfering RNA inhibits endothelial cell migration, proliferation and tube formation in vitro.

PURPOSE: To investigate the role of integrin-linked kinase (ILK) in endothelial cell migration, proliferation and tube formation in vitro. MATERIALS AND METHODS: Cultured RF/6A cells were knocked down for ILK using small interfering RNA (siRNA). Cellular ILK expression was quantified by real-time quantitative PCR and Western blot analysis. Cell migration was measured by cell counting in modified Boyden chambers, while microfilament dynamics were assessed by immunofluorescence analysis. Cell cycling was determined using a FACS Calibur flow cytometer, and the expression of cyclin D(1) was also measured by the Western blot assay. The secretion of vascular endothelial growth factor (VEGF) in culture medium samples was assessed by ELISA, and capillary/tube-like network formation assays were performed using matrigel. RESULTS: Both ILK mRNA and protein levels were virtually undetectable after transfection with ILK siRNA. In addition, there was a significant accumulation of cells in the G(0)-G(1) phase and a marked decrease in cell numbers in the S phase in ILK-specific siRNA-transfected cells, and the expression of cyclin D(1) was decreased after transfection. The knockdown of ILK significantly inhibited cell migration ability by disturbing F actin assembly, and VEGF secretion in conditioned medium was also reduced by 33%. Furthermore, treatment with ILK siRNA suppressed tube formation in RF/6A cells and significantly reduced the overall length of the tubes. CONCLUSIONS: Knockdown of ILK with siRNA effectively inhibits endothelial cell migration, proliferation and tube formation in vitro.

[Chemoattractive effects of chemokine-like factor 1 on human arterial smooth muscle cells].

OBJECTIVE: To investigate the chemotactic effect of chemokine-like factor 1 (CKLF1) on human arterial smooth muscle cells (ASMCs). METHODS: The recombinant eukaryotic expression vectors pEGFP-N1-CKLF1 (test group) and pEGFP-N1 (control group) were transiently transfected into 293T cells. The supernatants were harvested 72 h after transfection for bioactivity study. The chemotactic effect of CKLF1 on ASMCs were assayed by cellular chemotactic experiments. RESULTS: ASMCs number migrated from the test and control groups diluted by none and 10-fold supernatants had statistical significance (114+/-4 vs 41+/-4, P<0.05; 74+/-4 vs 34+/-3, P<0.01), but have no statistical significance (28+/-4 vs 25+/-5, P>0.05; 26+/-5 vs 23+/-5, P>0.05) between the two groups diluted by 100-fold and 1 000-fold supernatants. When ASMCs were treated at different concentrations of 0 and 2 microg/L of pertussis toxin (PTX), the cell number migrated from the test and control groups diluted by 10-fold supernatants, they had statistical significance (74+/-4 vs 34+/-3, P<0.01; 45+/-3 vs 34+/-3, P<0.01). And when ASMCs were treated at 10 microg/L of PTX, the cell number migrated from both groups diluted by 10-fold supernatants, they had no statistical significance (37+/-4 vs 34+/-3, P>0.05). CONCLUSION: CKLF1 has significant chemotactic effects on ASMCs and such a CKLF1-induced chemotaxis could be inhibited by PTX at concentration of 10 microg/L.

Targeting of integrin-linked kinase with a small interfering RNA suppresses progression of experimental proliferative vitreoretinopathy.

Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts through its COOH terminus with beta1 and beta3 integrins, which mediates a diversity of cell functions by coupling integrins and growth factors to cascades of downstream signaling events. The purpose of this work was to investigate the effects of ILK on development of experimental proliferative vitreoretinopathy (PVR). Cultured human RPE cell line D407 was knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by MTT assay. Moreover, cell attachment, spreading, migration, microfilament dynamics, and cell cycling assays were performed. Furthermore, the impact of the ILK-specific siRNA on PVR was tested using a rabbit model in which PVR was induced by the injection of human RPE cells. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection. The results showed that blocking the expression of ILK by siRNA significantly inhibited human RPE cell attachment, spreading, migration and proliferation. The knockdown of ILK also disturbed F-actin assembly and induced a cellular arrest in the G1 phase of the cell cycle. Though the eyes injected with ILK-specific siRNA also developed features of PVR, the severities of day 28 post-injection were significantly lower than those in the control eyes (P<0.01). We conclude that targeting of ILK with a small interfering RNA not only inhibits human RPE cell attachment, spreading, migration and proliferation in vitro, but also effectively suppresses development of proliferative vitreoretinopathy in a rabbit model. This may be a potential therapeutic usefulness in treating PVR.

[Phosphodiesterase type 5 siRNA increases cGMP in the smooth muscle cells of human corpus cavernosum].

OBJECTIVE: To investigate the effect of phosphodiesterase type 5 (PDE5) small interfering RNA (siRNA) on the cGMP in the smooth muscle cells of human corpus cavernosum, and to provide an experimental groundwork for the gene therapy of erectile dysfunction (ED). METHODS: Small interfering RNAs targeting PDE5 gene were synthesized by using web design software provided by Ambion, three siRNAs and a control siRNA were synthesized by Ambion. siRNAs were transfected into the smooth muscle cells of human corpus cavernosum by using siPORT Lipid reagent. cGMP was detected by ELISA at different times (24, 48, 72 and 96 h) after transfection. RESULTS: The cGMP levels of the siRNA1, siRNA2 and siRNA3 groups were significantly higher than those of the siRNA control and blank control groups (P < 0.05), and so was it in the siRNA1 group than the siRNA2 and siRNA3 groups (P < 0.05), with significant difference between the siRNA control and the blank control group (P > 0.05). CONCLUSION: The synthesized siRNAs in vitro are capable of increasing the level of cGMP in the smooth muscle cells of human corpus cavernosum, different siRNAs with different capabilities. The siRNA technique could provide not only an extremely powerful tool for the functional analysis of genome but also a new approach to ED gene therapy.

Antisense oligonucleotide targeting c-fos mRNA limits retinal pigment epithelial cell proliferation: a key step in the progression of proliferative vitreoretinopathy.

The purpose of this work was to investigate the effect of c-fos antisense oligonucleotide (c-fos-AS-ON) on proliferative vitreoretinopathy (PVR). Cultures of human retinal pigment epithelial (hRPE) cells were established from adult human corneal donors. These cells were positively stained for cytokeratins. C-fos-AS-ON effect on serum-stimulated cell proliferation was estimated by evaluating the incorporation of 5-bromo-2'-deoxy-uridine (BrdU) into cellular DNA. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were respectively performed to quantify the serum-stimulated c-fos gene mRNA and protein expression in hRPE cells. Eight rabbits (16 eyes) were divided into c-fos-AS-ON treatment group and control group. 2.5 x 10(5) cultured hRPE cells were injected into the vitreous cavity of eyes to establish a PVR model. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection and by pathological study on days 28 post-injection. The results showed that blocking the expression of c-fos by the addition of c-fos-AS-ON to the culture medium significantly inhibited the hRPE cells proliferation. This effect of c-fos-AS-ON was found to be sequence specific (the use of a sense or a mismatch sense oligonucleotide had no such an effect) and dose-dependent (0.375 microM was the lowest effective dose tested). Growth inhibition by c-fos-AS-ON remained for at least 72 h. By using RT-PCR and Western blotting, we found that the c-fos-AS-ON could specifically inhibit c-fos mRNA and protein synthesis in cultured hRPE cells. Though the eyes injected with c-fos-AS-ON also developed features of PVR, the severities of days 14, 21 and 28 post-injection were significantly lower than those in the control eyes (P<0.05). We conclude that c-fos-AS-ON can inhibit cultured hRPE cell proliferation, which mechanism may relate to blocking the expression of c-fos and can reduce the prevalence of experimental PVR. These findings establish a rationale for investigating the potential use of a c-fos-AS-ON as a novel therapeutical tool in the treatment of PVR.

Apoptin induces chromatin condensation in normal cells.

Apoptin, a chicken anemia virus protein, was reported to induce tumor specific apoptosis, which was correlated with the nuclear localization of the protein in tumor cells. While in normal human cells, Apoptin was detected mainly in the cytoplasm and did not induce apoptosis. Using a recombinant adenovirus expressing Apoptin, we have found that Apoptin induced G(2)-M cell cycle arrest and chromatin condensation in cancer cells. Here we report that adenovirus mediated Apoptin expression also induces G(2)-M arrest in normal cells. In normal cells Apoptin is localized mainly in the cytoplasm but is also found in the nucleus of a subset of cells. Apoptin induces chromatin condensation not only when it is expressed in the nucleus but also when it is expressed in the cytoplasm. Our results indicate that Apoptin-induced chromatin condensation in the normal cells may not correlate with its nuclear localization and the mechanism of regulating the G(2)-M transition might be a target for Apoptin.