EPIC-1042 as a potent PTRF/Cavin1-caveolin-1 interaction inhibitor to induce PARP1 autophagic degradation and suppress temozolomide efflux for glioblastoma.

BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma is determined by various mechanisms such as TMZ efflux, autophagy, base excision repair (BER) pathway, and the level of O6-methylguanine-DNA methyltransferase (MGMT). Here, we reported a novel small-molecular inhibitor (SMI) EPIC-1042 (C20H28N6) with the potential to decrease TMZ efflux and promote PARP1 degradation via autolysosomes in the early stage. METHODS: EPIC-1042 was obtained from receptor-based virtual screening. Co-immunoprecipitation and pull-down assays were applied to verify the blocking effect of EPIC-1042. Western blotting, co-immunoprecipitation, and immunofluorescence were used to elucidate the underlying mechanisms of EPIC-1042. In vivo experiments were performed to verify the efficacy of EPIC-1042 in sensitizing glioblastoma cells to TMZ. RESULTS: EPIC-1042 physically interrupted the interaction of PTRF/Cavin1 and caveolin-1, leading to reduced secretion of small extracellular vesicles (sEVs) to decrease TMZ efflux. It also induced PARP1 autophagic degradation via increased p62 expression that more p62 bound to PARP1 and specially promoted PARP1 translocation into autolysosomes for degradation in the early stage. Moreover, EPIC-1042 inhibited autophagy flux at last. The application of EPIC-1042 enhanced TMZ efficacy in glioblastoma in vivo. CONCLUSION: EPIC-1042 reinforced the effect of TMZ by preventing TMZ efflux, inducing PARP1 degradation via autolysosomes to perturb the BER pathway and recruitment of MGMT, and inhibiting autophagy flux in the later stage. Therefore, this study provided a novel therapeutic strategy using the combination of TMZ with EPIC-1042 for glioblastoma treatment.

HSPA8 Activates Wnt/ß-Catenin Signaling to Facilitate BRAF V600E Colorectal Cancer Progression by CMA-Mediated CAV1 Degradation.

BRAF V600E attracts wide attention in the treatment of colorectal cancer (CRC) as stratifying and predicting a refractory classification of CRC. Recent evidence indicates that Wnt/ß-catenin signaling is broadly activated and participates in the refractoriness of BRAF V600E CRC, but the underlying molecular mechanism needs to be elucidated. Here, heat shock 70 kDa protein 8 (HSPA8), an essential regulator in chaperone-mediated autophagy (CMA), is identified as a potential therapeutic target for advanced BRAF V600E CRC. These results show that HSPA8 is transcriptionally upregulated in BRAF V600E CRC, which promotes CMA-dependent degradation of caveolin-1 (CAV1) to release ß-catenin into the nucleus and thus activates the Wnt/ß-catenin pathway, contributing to metastasis and progression of BRAF V600E CRC. Of note, HSPA8 directly interacts with the KIFSN motif on CAV1, the interaction can be enhanced by p38 MAPK-mediated CAV1 S168 phosphorylation. Furthermore, pharmacological targeting HSPA8 by VER155008 exhibits synergistic effects with BRAF inhibitors on CRC mouse models. In summary, these findings discover the important role of the HSPA8/CAV1/ß-catenin axis in the development of refractory BRAF V600E CRC and highlight HSPA8 as a predictive biomarker and therapeutic target in clinical practice.

Caveolin-mediated cytosolic delivery of spike nanoparticle enhances antitumor immunity of neoantigen vaccine for hepatocellular carcinoma.

Rationale: Although neoantigen-based cancer vaccines have shown promise in various solid tumors, limited immune responses and clinical outcomes have been reported in patients with advanced disease. Cytosolic transport of neoantigen and adjuvant is required for the activation of intracellular Toll-like receptors (TLRs) and cross-presentation to prime neoantigen-specific CD8+T cells but remains a significant challenge. Methods: In this study, we aimed to develop a virus-like silicon vaccine (V-scVLPs) with a unique spike topological structure, capable of efficiently co-delivering a hepatocellular carcinoma (HCC)-specific neoantigen and a TLR9 agonist to dendritic cells (DCs) to induce a robust CD8+T cell response to prevent orthotopic tumor growth. We evaluated the antitumor efficacy of V-scVLPs by examining tumor growth and survival time in animal models, as well as analyzing tumor-infiltrating CD8+T cells and cytokine responses in the tumor microenvironment (TME). To evaluate the synergistic efficacy of V-scVLPs in combination with α-TIM-3 in HCC, we used an orthotopic HCC mouse model, a lung metastasis model, and a tumor rechallenge model after hepatectomy. Results: We found that V-scVLPs can efficiently co-deliver the hepatocellular carcinoma (HCC)-specific neoantigen and the TLR9 agonist to DCs via caveolin-mediated endocytosis. This advanced delivery strategy results in efficient lymph node draining of V-scVLPs to activate lymphoid DC maturation for promoting robust CD8+T cells and central memory T cells responses, which effectively prevents orthotopic HCC tumor growth. However, in the established orthotopic liver tumor models, the inhibitory receptor of TIM-3 was significantly upregulated in tumor-infiltrating CD8+T cells after immunization with V-scVLPs. Blocking the TIM-3 signaling further restored the antitumor activity of V-scVLPs-induced CD8+T cells, reduced the proportion of regulatory T cells, and increased the levels of cytokines to alter the tumor microenvironment to efficiently suppress established orthotopic HCC tumor growth, and inhibit lung metastasis as well as recurrence after hepatectomy. Conclusion: Overall, the developed novel spike nanoparticles with efficient neoantigen and adjuvant intracellular delivery capability holds great promise for future clinical translation to improve HCC immunotherapy.

Cardioprotective and anti-inflammatory effects of Caveolin 1 in experimental diabetic cardiomyopathy.

Previous studies of the Caveolin 1 (Cav1) protein and caveolae, which are lipid raft structures found on the plasma membranes of certain cells, are associated with fat metabolism disorders, inflammation, diabetes, and cardiovascular disease. However, there have been no reports linking Cav1 to diabetic cardiomyopathy (DCM). In the present study, we established a relationship between Cav1 and the development of DCM. We found that compared with Cav1+/+ mice, Cav1-/- diabetic mice exhibited more severe cardiac injury, increased activation of NF-κB signaling, and up-regulation of downstream genes, including hypertrophic factors and inflammatory fibrosis factors in heart tissues. Additionally, in vitro results showed that knocking down Cav1 further activated HG-induced NF-κB signaling, increased the expression of downstream target genes, and decreased the expression of inhibitor α of NF-κB (iκBα), all of which have been linked to DCM pathogenesis. In contrast, Cav1 overexpression resulted in the opposite effects. Our study suggests that Cav1 knockdown promotes cardiac injury in DCM by activating the NF-κB signaling pathway, and targeting Cav1 may lead to the development of novel treatments for DCM.

Adipose tissue-derived mesenchymal stem cells attenuate lung inflammation and fibrosis in the bleomycin-induced pulmonary fibrosis rat model via caveolin-1/NF-kB signaling axis.

Stem cells have emerged as promising therapeutic options for several human diseases, including pulmonary fibrosis (PF). In this study, we investigated the therapeutic effects of adipose tissue-derived mesenchymal stem cells (ADMSCs) in the bleomycin-induced PF model rats and the underlying mechanisms. The PF model rats were generated by intratracheal injections of 5 mg/kg bleomycin sulfate. The ADMSC group rats were generated by injecting 2×10(6) ADMSCs via the tail vein at 0, 12, and 24 h after bleomycin injection. The control, PF, and ADMSC group rats were sacrificed on day 21 after bleomycin injections and the changes in lung histology and the levels of pro-inflammatory cytokines, collagen I, and caveolin-1 (Cav-1), and the activity of the NF-kappaB signaling pathway in the lung tissues was assessed by hematoxylin-eosin staining, ELISA, and western blotting assays. The lung tissues of the PF model rats showed significant infiltration of neutrophils, tissue destruction, and collagen deposition, but these effects were abrogated by the ADMSCs. The levels of pro-inflammatory cytokines such as IL-6, IL-1beta, and TGF-beta1 were elevated in the lung tissues and the bronchoalveolar lavage fluid (BALF) of the bleomycin-induced PF model rats, but these effects were reversed by the ADMSCs. The lung tissues of the PF model rats showed significant downregulation of Cav-1 and significantly higher activation of the pro-inflammatory NF-kappaB pathway. However, administration of the ADMSCs restored the expression levels of Cav-1 and suppressed the NF-kappaB signaling pathway in the lungs of the bleomycin-induced PF model rats. In conclusion, this study demonstrated that the ADMSCs protected against bleomycin-induced PF in the rat model by modulating the Cav-1/NF-kappaB axis.

Subpial delivery of adeno-associated virus 9-synapsin-caveolin-1 (AAV9-SynCav1) preserves motor neuron and neuromuscular junction morphology, motor function, delays disease onset, and extends survival in hSOD1G93A mice.

Elevating neuroprotective proteins using adeno-associated virus (AAV)-mediated gene delivery shows great promise in combating devastating neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is one such disease resulting from loss of upper and lower motor neurons (MNs) with 90-95% of cases sporadic (SALS) in nature. Due to the unknown etiology of SALS, interventions that afford neuronal protection and preservation are urgently needed. Caveolin-1 (Cav-1), a membrane/lipid rafts (MLRs) scaffolding and neuroprotective protein, and MLR-associated signaling components are decreased in degenerating neurons in postmortem human brains. We previously showed that, when crossing our SynCav1 transgenic mouse (TG) with the mutant human superoxide dismutase 1 (hSOD1G93A) mouse model of ALS, the double transgenic mouse (SynCav1 TG/hSOD1G93A) exhibited better motor function and longer survival. The objective of the current study was to test whether neuron-targeted Cav-1 upregulation in the spinal cord using AAV9-SynCav1 could improve motor function and extend longevity in mutant humanized mouse and rat (hSOD1G93A) models of familial (F)ALS. Methods: Motor function was assessed by voluntary running wheel (RW) in mice and forelimb grip strength (GS) and motor evoked potentials (MEP) in rats. Immunofluorescence (IF) microscopy for choline acetyltransferase (ChAT) was used to assess MN morphology. Neuromuscular junctions (NMJs) were measured by bungarotoxin-a (Btx-a) and synaptophysin IF. Body weight (BW) was measured weekly, and the survival curve was determined by Kaplan-Meier analysis. Results: Following subpial gene delivery to the lumbar spinal cord, male and female hSOD1G93A mice treated with SynCav1 exhibited delayed disease onset, greater running-wheel performance, preserved spinal alpha-motor neuron morphology and NMJ integrity, and 10% increased longevity, independent of affecting expression of the mutant hSOD1G93A protein. Cervical subpial SynCav1 delivery to hSOD1G93A rats preserved forelimb GS and MEPs in the brachial and gastrocnemius muscles. Conclusion: In summary, subpial delivery of SynCav1 protects and preserves spinal motor neurons, and extends longevity in a familial mouse model of ALS without reducing the toxic monogenic component. Furthermore, subpial SynCav1 delivery preserved neuromuscular function in a rat model of FALS. The latter findings strongly indicate the therapeutic applicability of SynCav1 to treat ALS attributed to monogenic (FALS) and potentially in sporadic cases (i.e., SALS).

Melanosomal targeting via caveolin-1 dependent endocytosis mediates ZN(II) phthalocyanine phototoxic action in melanoma cells.

Melanosomes have been considered crucial targets in melanoma treatments. In this study we explored the role of melanosomes in photodynamic therapy (PDT), employing the synthetic Zn(II) phthalocyanine Pc13, a potent photosensitizer that promotes melanoma cell death after irradiation. Phototoxic action is mediated by reactive oxygen species increase. The internalization mechanism of Pc13 and its consequent subcellular localization were evaluated in melanotic B16-F0 cells. Pharmacological inhibitors of dynamin or caveolae, but not of clathrin, decreased Pc13 cellular uptake and phototoxicity. Similar results were obtained when cells over-expressed dominant negative mutants of dynamin-2 and caveolin-1, indicating that Pc13 is internalized by caveolae-mediated endocytosis. Confocal microscopy analysis revealed that Pc13 targets melanosomes and damage of these structures after irradiation was demonstrated by transmission electron microscopy. Treatment of pigmented B16-F0 and WM35 melanoma cells with the melanin synthesis inhibitor phenylthiourea for 48 h led to cell depigmentation and enhanced cell death after irradiation, whereas a 3-h period of inhibition did not modify melanin content but produced a marked reduction of Pc13 phototoxicity, together with a decrease of oxidative melanin synthesis intermediates. In contrast, the effect of Pc13 in amelanotic A375 cells was not altered by phenylthiourea treatment. These results provide evidence that melanosomes have a dual role in the efficacy of PDT. While melanin antagonizes the phototoxic action of Pc13, the release of cytotoxic synthetic intermediates to cytosol after irradiation and melanosome damage is conducive to the phototoxic response. Based on these findings, we demonstrate that melanosome-targeted PDT could be an effective approach for melanoma treatment.

Role of IL-26+CD26+CD4 T Cells in Pulmonary Chronic Graft-Versus-Host Disease and Treatment with Caveolin-1-Ig Fc Conjugate.

Obliterative bronchiolitis is the primary noninfectious pulmonary complication after allogeneic hematopoietic cell transplantation and the only pathognomonic manifestation of pulmonary chronic graft-versus-host disease (cGVHD). In our recent study, we identified a novel effect of IL-26, which is absent in rodents, on transplant related-obliterative bronchiolitis. Sublethally irradiated NOD/Shi-scidIL2rγnull mice transplanted with human umbilical cord blood gradually exhibited obliterative bronchiolitis with increased collagen deposition and predominant infiltration with human IL-26+CD26+CD4 T cells. Moreover, we showed that IL-26 increased collagen synthesis in fibroblasts in vitro and that collagen contents were increased in a murine GVHD model using IL26 transgenic mice. In vitro analysis demonstrated a significant increase in IL-26 production by CD4 T cells following CD26 costimulation, while immunoglobulin Fc domain fused with the N-terminal of caveolin-1, the ligand for CD26, (Cav-Ig) effectively inhibited production of IL-26. Administration of Cav-Ig before or after onset of GVHD impeded the development of clinical and histologic features of GVHD without interrupting engraftment of donor-derived human cells, with preservation of the graft-versus-leukemia effect. We concluded that cGVHD of the lungs is caused in part by IL-26+CD26+CD4 T cells, and that treatment with Cav-Ig could be beneficial for cGVHD prevention and therapy.

Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system.

Alveolar type II (ATII) cell apoptosis and depressed fibrinolysis that promotes alveolar fibrin deposition are associated with acute lung injury (ALI) and the development of pulmonary fibrosis (PF). We therefore sought to determine whether p53-mediated inhibition of urokinase-type plasminogen activator (uPA) and induction of plasminogen activator inhibitor-1 (PAI-1) contribute to ATII cell apoptosis that precedes the development of PF. We also sought to determine whether caveolin-1 scaffolding domain peptide (CSP) reverses these changes to protect against ALI and PF. Tissues as well as isolated ATII cells from the lungs of wild-type (WT) mice with BLM injury show increased apoptosis, p53, and PAI-1, and reciprocal suppression of uPA and uPA receptor (uPAR) protein expression. Treatment of WT mice with CSP reverses these effects and protects ATII cells against bleomycin (BLM)-induced apoptosis whereas CSP fails to attenuate ATII cell apoptosis or decrease p53 or PAI-1 in uPA-deficient mice. These mice demonstrate more severe PF. Thus p53 is increased and inhibits expression of uPA and uPAR while increasing PAI-1, changes that promote ATII cell apoptosis in mice with BLM-induced ALI. We show that CSP, an intervention targeting this pathway, protects the lung epithelium from apoptosis and prevents PF in BLM-induced lung injury via uPA-mediated inhibition of p53 and PAI-1.

Caveolin-1 enhances resveratrol-mediated cytotoxicity and transport in a hepatocellular carcinoma model.

BACKGROUND: Resveratrol (RES), an estrogen analog, is considered as a potential cancer chemo-preventive agent. However, it remains unclear how RES is transported into cells. In this study, we observed that Caveolin-1(CAV1) expression can increase the cytotoxic and pro-apoptotic activity of RES in a dose- and time-dependent manner both in vitro and in vivo in a Hepatocellular Carcinoma animal model. METHODS: High performance liquid chromatography (HPLC) demonstrated that RES intra-cellular concentration is increased about 2-fold in cells stably expressing CAV1 or CAVM1 (a scaffolding domain (81-101AA)-defective CAV1 mutant) compared to the untransduced human Hepatoblastoma cell line (HepG2) or after transduction with the green fluorescent protein (GFP) control vector. The increased intra-cellular transport of RES was abolished in cells stably expressing CAVM2 (a cholesterol shuttle domain (143-156AA)-defective CAV1 mutant) or CAVRNAi. In order to further characterize CAV1-dependent RES transport, we synthesized RES-dansyl chloride derivatives as fluorescent probes to visualize the transport process, which demonstrated a distribution consistent with that of CAV1 in HepG2 cells. RESULTS: In addition, RES endocytosis was not mediated by estrogen receptor (ER) alpha and beta, as suggested by lack of competitive inhibition by estrogen or Tamoxifen. Pathway analysis showed that RES can up-regulate the expression of endogenous CAV1; this activates further the MAPK pathway and caspase-3 expression. DISCUSSION: This study provides novel insights about the role played by CAV1 in modulating cellular sensitivity to RES through enhancement of its internalization and trafficking.

Short-term administration of a cell-permeable caveolin-1 peptide prevents the development of monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy.

BACKGROUND: Caveolins (Cavs), the principal structural proteins of caveolar microdomains, have been implicated in the development of pulmonary hypertension (PH). Mice with homozygous deletion of the Cav-1 gene develop PH and right ventricular hypertrophy (RVH). Reductions in pulmonary Cav-1 expression have been shown in several animal models of PH and in patients with severe PH. Whether in vivo modulation of Cav-1 expression could affect the development of PH and RVH remains unknown. Therefore, we investigated the effect of in vivo administration of a Cav-1 mimetic peptide on the development of monocrotaline (MCT)-induced PH. METHODS AND RESULTS: Thirty minutes after injection of saline or 60 mg/kg MCT, rats were assigned to receive a daily injection of saline, a peptide corresponding to the homeodomain of the Drosophila transcription factor antennapedia (AP; 2.5 mg x kg(-1) x d(-1)), or a peptide consisting of the Cav-1-scaffolding domain coupled to AP (AP-Cav; 2.5 mg x kg(-1) x d(-1)) for 2 weeks. MCT and MCT+AP rats developed PH with respective right ventricular systolic pressures of 40.2 +/- 1.5 and 39.6 +/- 1.5 mm Hg. Administration of AP-Cav to MCT rats significantly reduced the right ventricular systolic pressure to 30.1 +/- 1.3 mm Hg. MCT and MCT+AP rats also developed pulmonary artery medial hypertrophy and RVH, which was normalized by administration of AP-Cav. Mechanistically, the development of PH was associated with reduced expression of pulmonary Cav-1 and Cav-2, hyperactivation of the STAT3 signaling cascade, and upregulation of cyclin D1 and D3 protein levels, all of which were prevented by administration of AP-Cav. CONCLUSIONS: Short-term administration of a Cav-based cell-permeable peptide to MCT rats prevents the development of pulmonary artery medial hypertrophy, PH, and RVH.