Hyperthermia and cisplatin combination therapy promotes caspase-8 accumulation and activation to enhance apoptosis and pyroptosis in cancer cells.

BACKGROUND: Hyperthermia can play a synergistic role with chemotherapy in combination therapy. Although the association between caspase activation, apoptosis, and pyroptosis have been published for both cisplatin (CDDP) and hyperthermia therapies independently, the interactions between these molecular pathways in combination therapy are unknown. The present study aimed to investigate the possible interactions between caspase 8 activation, apoptosis, and pyroptosis in combination therapy. METHODS: Cells were treated with CDDP (15 µg/ml), followed by hyperthermia at optimized temperature (42.5 °C) in water-bath. After combination therapy, cell viability was analyzed by CCK-8, and cell death was analyzed by Annexin-V-FITC/PI and caspases activation. Immuno-staining and co-immuno-precipitation were used to examine the interaction between p62 and caspase-8. Pyroptosis was investigated by western blotting and transmission electron microscopy. E3 ligase Cullin 3 was knockdown by siRNA. In addition, caspase-8 activation was modulated by CRISPR-Cas9 gene-editing or pharmacological inhibition. RESULTS: Combination therapy promoted K63-linked polyubiquitination of caspase-8 and cellular accumulation of caspase-8. In turn, polyubiquitinated caspase-8 interacted with p62 and led to the activation of caspase-3. Knockdown of the E3 ligase Cullin 3 by siRNA reduced caspase-8 polyubiquitination and activation. In addition, combination therapy induced release of the pore-forming N-terminus from gasdermins and promoted pyroptosis along with caspase-8 accumulation and activation. Knockdown of caspase-8 by CRISPR/Cas9 based gene editing reduced the sensitivity of tumor cells to apoptosis and pyroptosis. CONCLUSIONS: Our study presented a novel mechanism in which hyperthermia synergized with chemotherapy in promoting apoptosis and pyroptosis in a caspase-8 dependent manner.

Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis.

Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.

Zn2 SiO4 Bioceramic Attenuates Cardiac Remodeling after Myocardial Infarction.

In the pursuit of therapeutic strategies for myocardial infarction (MI), a pivotal objective lies in the concurrent restoration of blood perfusion and reduction of cardiomyocyte apoptosis. However, achieving these dual goals simultaneously presents a considerable challenge. In this study, a Zn2 SiO4 bioceramic capable of concurrently sustaining the release of bioactive SiO3 2- and Zn2+ ions, which exhibit a synergistic impact on endothelial cell angiogenesis promotion, cardiomyocyte apoptosis inhibition, and myocardial mitochondrial protection against oxygen-free radical (reactive oxygen species) induced injury is developed. Furthermore, in vivo outcomes from a murine MI model demonstrate that either systemic administration via tail vein injection of Zn2 SiO4 extract or local application through intramyocardial injection of a Zn2 SiO4 composite hydrogel promotes cardiac function and reduces cardiac fibrosis, thus aiding myocardial repair. This research is the first to elucidate the advantageous effects of dual bioactive ions in myocardial protection and may offer a novel therapeutic avenue for ischemic heart disease based on meticulously engineered bioceramics.

Dissolvable hyaluronic acid microneedles loaded with ß-Elemene for the treatment of psoriasis.

The pathology of psoriasis involves the over-proliferation of keratinocytes, exaggerated inflammation of keratinocytes, and infiltration of inflammatory cells such as macrophages (Mø), etc. The therapeutic outcomes of current treatment targeting one single pathological process are less than satisfactory. Based on their diverse biological activities, natural products offer a potential solution to this problem. In this study, we investigated the effects of ß-Elemene (ELE) on both psoriatic keratinocytes and M1-type Mø (M1-Mø) in vitro. Hyaluronic acid (HA) microneedles loaded with ELE (HA-ELE-MN) were also fabricated and tested for the treatment of psoriasis in vivo using an imiquimod (IMQ)-induced psoriatic mice model. Our data suggest that ELE induces apoptosis and inhibits inflammation of psoriatic keratinocytes. In addition, ELE attenuates the expression of inflammatory cytokines secreted from M1-Mø, thus indirectly inhibiting the inflammation of keratinocytes. Furthermore, HA-ELE-MN has been found to significantly alleviate symptoms in an IMQ-induced psoriatic mice model by inducing keratinocytes apoptosis, suppressing keratinocytes proliferation, and inhibiting M1-Mø infiltration. Taken together, this study demonstrates that ELE can be used for the treatment of psoriasis by targeting both keratinocytes and M1-Mø, which provides a potential novel reagent for psoriasis treatment.

Surface Ligand Valency and Immunoliposome Binding: when More Is Not Always Better.

PURPOSE: Nano-drug delivery systems are designed to contain surface ligands including antibodies for "active targeting". The number of ligands on each nanoparticle, known as the valency, is considered a critical determinant of the "targeting" property. We sought to understand the correlation between valency and binding properties using antibody conjugated liposomes, i.e. immunoliposomes (ILs), as the model. METHODS: Anti-CD3 Fab containing a terminal cysteine residue were conjugated to DSPE-PEG-maleimide and incubated with preformed liposomes at 60°C. The un-incorporated antibodies were removed and the obtained ILs were characterized to contain in average 2-22 copies of anti-CD3 Fabs per liposome. The Biolayer Interferometry (BLI) probe surface was coated with various densities of CD3 epsilon&delta heterodimer (CD3D/E) to imitate different CD3 expression levels on target cells. The inference wavelength shifts upon anti-CD3 liposome binding were monitored and analyzed. RESULTS: The data indicated ILs may bind either monovalently or multivalently, determined mainly by the surface ligand density rather than the ILs antibody valency. The ILs valency indeed correlated with the dissociation rate constant (Koff), but not with the association rate constant (Kon). Their binding capabilities also did not necessarily increase with the surface anti-CD3 valency. CONCLUSION: We proposed a model for understanding the binding properties of ILs with different ligand valencies. The binding mode may change when the targeted surfaces had different antigen densities. The model should be important for the designing and optimization of active targeting drug delivery systems to fit different applications.

Size-Dependent Absorption through Stratum Corneum by Drug-Loaded Liposomes.

PURPOSE: Topical treatment of various skin disorders requires drug absorption and penetration through the stratum corneum (SC) into the epidermis and dermis tissues. The use of nano-drug delivery systems including liposomes and lipid nanoparticles (SLNs) have been shown to facilitate SC penetration. The goal of this work was to study the impact of liposome sizes and the resulted drug distribution inside various skin tissue. METHODS: All trans retinoic acid (ATRA) was used as the model drug and loaded into gel phase HSPC/CHOL/DSPE-PEG liposomes (lipo-ATRA) with sizes ranging from 80 nm to more than 300 nm. The percutaneous drug absorption process was monitored and analyzed. RESULTS: There were significant differences in percutaneous absorption and tissue distribution resulted from liposomes smaller than 100 nm and those bigger than 200 nm. Lipo-ATRA with a mean diameter of 83 nm can deliver the content to epidermis and dermis. But for 200 nm - 300 nm liposomes, the resulted epidermis and dermis ATRA levels were less than about one third, suggesting bigger liposomes had poor penetration through the brick and mortar structure of SC. CONCLUSIONS: Gel phase liposomes with sizes under 100 nm improved encapsulated drug absorption and distribution into the epidermis and dermis tissues. A size dependent mechanism for liposome penetration of the stratum corneum was proposed.

Combination therapy with BH3 mimetic and hyperthermia tends to be more effective on anti-melanoma treatment.

Malignant melanoma has shown increased incidence and high mortality rate in the last three decades. In this study, we investigated whether combination therapy with ch282-5 (a novel BH3 mimetic) and microwave hyperthermia could display synergistic antitumor effects against melanoma. Our results indicated that combination therapy reduced the viability and proliferation of melanoma cells. Through inhibiting the expression of anti-apoptotic proteins of Bcl-2 and IAP family and activating MAPK proteins, combined hyperthermia enhanced ch282-5-induced apoptosis. Combination therapy also synergistically disturbed the mTOR/p70S6k signaling pathway, which is important for cell survival and migration. Moreover, our results showed that combination therapy remarkably suppressed melanoma cell migration in vitro and significantly reduced experimental pulmonary metastasis in vivo. In conclusion, our results indicate that combination therapy with ch282-5 and hyperthermia has synergistic antitumor effects and provides a possible therapeutic strategy for advanced melanoma.

Functional role of asparaginyl endopeptidase ubiquitination by TRAF6 in tumor invasion and metastasis.

BACKGROUND: Asparaginyl endopeptidase (AEP) has been implicated in human cancer development. However, the molecular mechanisms underlying AEP regulation, including the role of pro-AEP activation, remain elusive. METHODS: We investigated the regulation of AEP by TRAF6 and its effects on tumor progression and metastasis in cancer cell lines, murine models, and specimens from patients using biochemical analyses, confocal microscopy, immunoelectron microscopy, and migration-invasion assays. The sera of healthy donors and breast cancer patients were examined by enzyme-linked immunosorbent assay, and a tissue array of 314 breast cancer specimens was assessed for AEP and TRAF6 by immunohistochemistry. Furthermore, the effects of AEP inhibitors or monoclonal antibodies on pulmonary metastasis were evaluated in murine models. The statistical significance between groups was determined using two-tailed Student t tests. RESULTS: We demonstrate that TRAF6 ubiquitinates the proform of AEP through K63-linked polyubiquitin, reversible by USP17, and forms a complex with HSP90α to subsequently promote pro-AEP intracellular stability as well as secretion. Disrupting the interaction between pro-AEP and TRAF6 or inhibiting HSP90α reduced pro-AEP secretion and consequently reduced tumor metastasis. Higher circulating AEP levels were detected in the sera of breast cancer patients, and AEP inhibitors or neutralizing antibodies remarkably decreased tumor metastasis in murine models. Notably, TRAF6 and AEP were overexpressed in human breast neoplasms and correlated with poor prognosis. Patients with low AEP/TRAF6 expression survived for a mean of 111 months (95% confidence interval [CI] = 108 to 115 months), whereas those with high AEP/TRAF6 expression survived for a mean of only 61 months (95% CI = 42 to 79 months; P < .001). CONCLUSIONS: Our study elucidates a novel mechanism of AEP regulation and an alternative oncogenic pathway for TRAF6 in breast cancer, which suggests that AEP and TRAF6 protein levels may have prognostic implications in breast cancer patients. Thus, AEP may serve as a biomarker as well as new therapeutic target.

Effects of legumain as a potential prognostic factor on gastric cancers.

Although legumain has been found to be a prognostic factor in both breast cancer and colorectal cancer, its effects on gastric cancer are unknown. In this study, we investigated effects of legumain on gastric cancer and the correlation between legumain expression and prognosis of gastric cancer patients. SGC7901 cells were transduced with legumain cDNA (SGC7901-hLeg) for overexpression of legumain or with legumain shRNA to knock down legumain. In vitro tumor migration was examined by wound healing assay. Furthermore, a tumorigenicity and metastasis mouse model was used to examine legumain function in vivo; asparaginyl endopeptidase inhibitor (AEPI, an inhibitor of legumain) was injected to the mice (i.p.) to evaluate its therapeutic effect. Tissue microarray analysis from 112 gastric cancer patients was performed to evaluate the association between legumain expression and the cumulative survival time. Legumain was highly expressed in gastric cancer patients and some gastric cancer cell lines. Legumain promoted gastric cell migration in vitro and promoted gastric tumor growth and metastasis in vivo, and these effects were reversed by knockdown of legumain with shRNA or treated with AEPI. In gastric cancer clinical samples, legumain expression in tumor was significantly higher than in non-tumor and was negatively associated with the cumulative survival rate. In conclusion, legumain was highly expressed in gastric adenocarcinoma; legumain promoted gastric cancer tumorigenesis and metastasis in vitro and in vivo. Legumain expression in tumor was a poor prognostic factor for gastric cancer patients, and legumain could be a potential target molecule for gastric cancer therapy in clinic.

Anti-CD3 antibodies modulate anti-factor VIII immune responses in hemophilia A mice after factor VIII plasmid-mediated gene therapy.

One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells, whereas increased transforming growth factor-beta levels in plasma and the frequency of both CD4+CD25+FoxP3+ and CD4+CD25-Foxp3+ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+ cells did not abrogate tolerance induction, adoptive transfer of CD4+ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3-treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-beta levels and the generation of adaptive FVIII-specific CD4+Foxp3+ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

CD4+FOXP3+ regulatory T cells confer long-term regulation of factor VIII-specific immune responses in plasmid-mediated gene therapy-treated hemophilia mice.

Gene transfer of a factor VIII (FVIII) plasmid into hemophilia A (HemA) mice achieved supraphysiologic FVIII expression, but triggered production of high-titer FVIII-specific antibodies and loss of functional FVIII activity. To test whether FVIII-specific regulatory T cells (Tregs) can modulate immune responses against FVIII, we developed a HemA mouse model in which all T cells overexpressed Foxp3 (HemA/Foxp3-Tg). FVIII plasmid therapy did not induce antibody production in HemA/Foxp3-Tg mice. CD4(+)Foxp3(+) T cells isolated from plasmid-treated HemA/Foxp3-Tg mice significantly suppressed proliferation of FVIII-stimulated CD4(+) effector T cells. The percentage of CD4(+) T cells expressing CD25, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4 increased significantly in spleen and peripheral blood for 9 weeks. Mice receiving adoptively transferred Tregs from FVIII-exposed HemA/Foxp3-Tg mice produced significantly reduced antibody titers compared with controls after initial challenge with FVIII plasmid and second challenge 16 weeks after first plasmid treatment. Adoptively transferred Tregs engrafted and distributed at 2% to 4% in the Treg compartment of blood, lymph nodes, and spleens of the recipient mice and induced activation of endogenous Tregs; the engraftment decreased to negligible levels over 8 to 12 weeks. Antigen-specific Tregs can provide long-lasting protection against immune responses in vivo and limit recall responses induced by a second challenge via infectious tolerance.

Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice.

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid-treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS(+)CD4(+) T cells and activation of CD25(+)Foxp3(+) Tregs in the peripheral blood, spleen, and lymph nodes. CD4(+) T cells from anti-ICOS-treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-beta. Moreover, CD4(+)CD25(+) Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS-treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Phix 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

[Application of quantum dots fluorescent nanoprobes in following the migration of inflammatory cells from local tissue to draining lymph node].

Quantum dots (QDs) have favorable physical and photochemical properties. In this work, we used QDs fluorescent nanoprobes to follow the migration of inflammatory cells from local tissue to draining lymph node in inflammation resolution. Electric pulse stimulation was used to establish inflammation model in mouse tibialis anterior. QDs injected to inflammatory tissue were found to aggregate and endocytosed by inflammatory cells. While in the draining lymph node, QDs mainly distributed in the T cell area. TEM and confocal observation showed that most of QDs in the draining lymph node were located in the endosomes of monocytes/macrophages. Our work shows that QDs can be used as fluorescent probes to follow migration of cells especially phagocytic cells. Thus QDs may be explored in application in immunology research.

Electric pulses applied prior to intramuscular DNA vaccination greatly improve the vaccine immunogenicity.

Electroporation can improve intramuscular DNA vaccination efficacy but the exact antigen presentation mechanism remains unclear. We reported here that a similar immuno-potentiation effect was also observed by stimulating the skeletal muscles with electric pulses (EP) a few days prior to DNA inoculation (EP + n days + DNA). The application of EP by itself activated proinflammatory chemokine genes and stress genes. It also triggered an influx of inflammatory monocytes/macrophages (MPs). After DNA inoculation, the plasmids were seen taken up by these inflammatory MPs, which migrated to the draining LNs subsequently. The antibody responses results were fast and strong. Furthermore, MPs isolated from the draining LNs of EP + n days + DNA treated mice were capable of stimulating Ag specific CD4+ T cell proliferation in vitro. Based on these observations, we proposed that the local inflammation resulted from EP treatment played an important role in facilitating antigen presentation of the DNA vaccines.

Anti-HBV immune responses in rhesus macaques elicited by electroporation mediated DNA vaccination.

Electroporation has been shown to be an effective method to improve the efficiency of gene expression and the immunogenicity of DNA vaccines. In order to optimize the procedure and test for its efficacy in more clinically relevant large animal models, we examined the detailed immune responses in rhesus macaques after vaccination intramuscularly with electroporation using the plasmid encoding for HBV preS(2)-S antigen and an adjuvant plasmid encoding for hIL-2 and hIFN-gamma. Several important factors were examined, including the dose response relationships, the effect of various prime and boost regimens, and different combinations of electro-pulse parameters. The immune responses were closely followed for more than a year. The results showed that in rhesus macaques, electroporation can significantly enhance the immunogenicity of the DNA vaccines, resulting in greatly improved antibody responses as well as peptide-stimulated IFN-gamma producing T cell responses. In addition, we also reported the different antibody response behaviors resulted from different electro-pulse parameters. The detailed data would be useful to suggest possible optimization strategies for better DNA vaccine efficacy.

In vivo plasmid DNA electroporation resulted in transfection of satellite cells and lasting transgene expression in regenerated muscle fibers.

In vivo plasmid DNA electroporation resulted in elevated and lasting transgene expression in skeletal muscles. But the nature of the cells that contributed to sustained gene expression remains unknown. We followed the fate of plasmid DNA delivered with electroporation and systematically investigated the time course and location of transgene expression in muscle tissues both with GFP and luciferase. Furthermore, satellite cell activation after electroporation was confirmed by RT-PCR and immunohistochemistry analysis. The activated satellite cells were shown to be able to uptake the injected plasmid DNA and express transgene products as regenerated myocytes. We found that cells with longer gene expression durations were mostly regenerated muscle fibers. In contrast, expression in pre-existing muscle fibers was rather transient. We also presented in this study that immune response to transgene products might hamper the lasting gene expression. Based on these observations, we proposed that the underlying mechanism for prolonged transgene expression in the muscles after electroporation is related to the activation and transfection of myogenic satellite cells which subsequently developed into regenerated muscle fibers.

Visualization and characterization of drug carrier transportation and distribution in vivo.

Micro/nano-particle drug delivery systems are being developed for improving therapeutic effects. However, most studies can only evaluate the delivery properties by indirectly measuring the pharmacokinetic changes of the drugs. It had been difficult to directly assay the drug carrier status and distribution in vivo. We described here a method using intravital microscopy to visualize individual drug carriers traveling through blood vessels. The transportation and extravasation process of these particles were monitored. The in vivo distribution patterns were also confirmed by fluorescence microscopy studies of the various tissue samples.