FKBP12 mediates necroptosis by initiating RIPK1-RIPK3-MLKL signal transduction in response to TNF receptor 1 ligation.

Necroptosis is a regulated form of necrotic cell death that is mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and mixed-lineage kinase domain-like protein (MLKL), which mediates necroptotic signal transduction induced by tumor necrosis factor (TNF). Although many target proteins for necroptosis have been identified, no report had indicated that FK506-binding protein 12 (FKBP12, also known as FKBP1A), an endogenous protein that regulates protein folding and conformation alteration, is involved in mediating necroptosis. In this study, we found that FKBP12 acts as a novel target protein in mediating necroptosis and the related systemic inflammatory response syndrome triggered by TNF. The mechanistic study discovered that FKBP12 is essential for initiating necrosome formation and RIPK1-RIPK3-MLKL signaling pathway activation in response to TNF receptor 1 ligation. In addition, FKBP12 is indispensable for RIPK1 and RIPK3 expression and subsequent spontaneous phosphorylation, which are essential processes for initial necrosome formation and necroptotic signal transduction; therefore, FKBP12 may target RIPK1 and RIPK3 to mediate necroptosis in vitro and in vivo Collectively, our data demonstrate that FKBP12 could be a potential therapeutic target for the clinical treatment of necroptosis-associated diseases.

JS-K induces reactive oxygen species-dependent anti-cancer effects by targeting mitochondria respiratory chain complexes in gastric cancer.

As a nitric oxide (NO) donor prodrug, JS-K inhibits cancer cell proliferation, induces the differentiation of human leukaemia cells, and triggers apoptotic cell death in various cancer models. However, the anti-cancer effect of JS-K in gastric cancer has not been reported. In this study, we found that JS-K inhibited the proliferation of gastric cancer cells in vitro and in vivo and triggered mitochondrial apoptosis. Moreover, JS-K induced a significant accumulation of reactive oxygen species (ROS), and the clearance of ROS by antioxidant reagents reversed JS-K-induced toxicity in gastric cancer cells and subcutaneous xenografts. Although JS-K triggered significant NO release, NO scavenging had no effect on JS-K-induced toxicity in vivo and in vitro. Therefore, ROS, but not NO, mediated the anti-cancer effects of JS-K in gastric cancer. We also explored the potential mechanism of JS-K-induced ROS accumulation and found that JS-K significantly down-regulated the core proteins of mitochondria respiratory chain (MRC) complex I and IV, resulting in the reduction of MRC complex I and IV activity and the subsequent ROS production. Moreover, JS-K inhibited the expression of antioxidant enzymes, including copper-zinc-containing superoxide dismutase (SOD1) and catalase, which contributed to the decrease of antioxidant enzymes activity and the subsequent inhibition of ROS clearance. Therefore, JS-K may target MRC complex I and IV and antioxidant enzymes to exert ROS-dependent anti-cancer function, leading to the potential usage of JS-K in the prevention and treatment of gastric cancer.

Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis.

To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with CAVA, a SFV replicon DNA vaccine targeting survivin and ß-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, ß-hCG and VEGFR2 simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic.

Intraarticular gene transfer of SPRY2 suppresses adjuvant-induced arthritis in rats.

AKT and ERK pathways have been implicated as therapeutic targets for human rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) inhibition, and thus RA treatment. Sprouty2 (SPRY2) has been known as a tumor suppressor by blocking both ERK and AKT signaling cascades. Whether SPRY2 can function as a suppressor of tumor-like inflammatory FLS and RA through negatively regulating AKT and ERK activation has not been reported. The purpose of this study was to determine whether SPRY2 might have antiarthritic effects in experimental animal model of RA. We first determined that expression of SPRY2 mRNA was decreased in FLS from patients with RA compared with patients with osteoarthritis (OA). Further studies demonstrated that intraarticular gene transfer with AdSPRY2, the recombinant adenovirus containing SPRY2 complementary DNA, resulted in a significant suppression of rat adjuvant-induced arthritis (AIA) compared with the control AdGFP, the adenoviral vector encoding green fluorescent protein, as reflected in both clinical and histological observations. AdSPRY2 suppressed the production of proinflammatory cytokines and matrix metalloproteinases (MMPs), and the activation of ERK and AKT signals in AIA ankle joints. These results suggest that using SPRY2 to block the AKT and ERK pathways effectively reduces the inflammatory responses and arthritic progression in AIA. Thus, the development of an immunoregulatory strategy based on SPRY2 may therefore have therapeutic potential in the treatment of RA.

Combination therapy with TNFR-Fc and CTLA4-FasL using the recombinant adeno-associated virus potently suppresses adjuvant-induced arthritis in rats.

The complexity of rheumatoid arthritis (RA) pathogenesis makes combined blockade of key pathogenic factors an attractive therapeutic strategy. We have previously reported a novel recombinant adeno-associated virus (AAV) vector, AAV.TFCF, which mediates separate coexpression of TNFα antagonist TNFR-Fc and T cell antagonist CTLA4-FasL both in vitro and in vivo (the injected joints). The purpose of this study was to examine the efficacy of TNFR-Fc/CTLA4-FasL combination therapy mediated by AAV.TFCF in experimental model of RA. Adjuvant-induced arthritis (AIA) was induced in Lewis rats, and the recombinant AAV.TFCF was injected into rat ankle joints. AAV vector encoding CTLA4-FasL (AAV.CTFA) or TNFR-Fc (AAV.TRFC) was used as the monotherapy control, and an AAV vector mediating the expression of enhanced green fluorescent protein (AAV.EGFP) was used as the negative control. The combination treatment mediated by AAV.TFCF demonstrated a more effective suppression of AIA compared with those monotherapy controls, as reflected in the clinical and histological observations. The synergistic anti-inflammatory effect of TNFR-Fc combining with CTLA4-FasL was proved to be associated with the greater reductions of inflammatory CD4+ T cell infiltration and proinflammatory cytokine TNFα level in the arthritic joints. In addition, the combination therapy was found to be able to increase the frequency of CD4 + CD25 + FoxP3+ regulatory T cell population in rat draining lymph nodes and suppress splenic inflammatory responses. These results suggest that combination treatment with TNFR-Fc and CTLA4-FasL may achieve superior efficacy in suppressing RA, and using this novel recombinant AAV.TFCF to obtain the combined counteraction of both pathogenic T cells and the key proinflammatory cytokine TNFα may provide a more effective and desirable strategy for treatment of RA.

Intraarticular gene delivery of CTLA4-FasL suppresses experimental arthritis.

T lymphocytes are key inflammatory cells contributing significantly to the pathogenesis of Rheumatoid arthritis (RA). Biological treatments targeting T lymphocytes may provide an efficient approach for treatment of RA. CTLA4-FasL, a fusion product of extracellular domains of CTLA4 and FasL, integrating two inhibitory elements against T cells into one molecule, might be a desirable derivative of engineered soluble FasL or CTLA4 and have therapeutic potential in RA. The aim of this study was to investigate whether simultaneous induction of Fas-mediated apoptosis and blockade of co-stimulation signal by CTLA4-FasL gene delivery has a suppressive effect on adjuvant-induced arthritis (AIA) in Lewis rats. Recombinant adeno-associated virus (rAAV) vectors encoding rat CTLA4-FasL fusion gene (rAAV.CTLA4-FasL) or enhanced green fluorescent protein (rAAV.EGFP) were injected intraarticularly into both ankle joints after immunization. The ankles were monitored by measures of clinical, histological and inflammatory cytokines' changes. Treatment using rAAV.CTLA4-FasL resulted in a significant suppression of AIA compared with rAAV.EGFP control, as reflected in the mainly clinical signs including articular index, ankle joint thickness and paw swelling and typically histological characters of arthritic joints including synovial hyperplasia, inflammatory cells infiltration and cartilage degradation. Treatment with rAAV.CTLA4-FasL also significantly decreased the levels of key proinflammatory cytokines in AIA joints. Moreover, local productions of transgene mRNA and protein of CTLA4-FasL were found in injected joints without systemic distribution. Our results indicate that rAAV.CTLA4-FasL profoundly suppressed experimental model of RA, implicating the potential therapeutic applications for suppression of RA by local joint delivery of CTLA4-FasL.

Design, expression and characterization of a novel coexpression system of two antiarthritic molecules.

The complexity of rheumatoid arthritis (RA) pathogenesis makes combined blockade of multiple targets an attractive therapeutic strategy. The combination therapy with anti-TNF plus anti-T-cell has been mostly reported to provide greater efficacy than anti-TNF alone. TNFR (p75)-Fc fusion protein, which has been proven effective in clinics, is chosen as the TNF antagonist in this study. CTLA4-FasL fusion molecule, which has been well characterized in our previous studies for its suppressive effect in rat arthritis model, is chosen as the T-cell antagonist. In this study, furin cleavage site and 2A self-processing sequence were introduced to link upstream TNFR-Fc and downstream CTLA4-FasL and mediate separate coexpression of the two fusion proteins in a single recombinant adeno-associated virus (rAAV) vector. Using this expression system, we generated two fusion proteins with same size as their individual counterparts in vitro and in vivo, and the proteins desirably retained their parent biological activities. In vivo results demonstrated that furin-2A technology is able to regulate separate coexpression of these proteins under arthritic inflammatory conditions. This study describes a single rAAV vector for production of two antiarthritic molecules antagonizing both TNF and T cells, which may serve as an attractive expression system for RA gene therapy.

[Transformation activity and antigenicity of the human papillomavirus type 58 E6E7 fusion gene mutant].

OBJECTIVE: To develop a prophylactic and therapeutic vaccine against human papillomavirus (HPV) type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity. METHODS: The E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7. The recombinant HPV58 mE6E7 gene was inserted into pIRES-neo vector to generate plasmid pIRES-neo-HPV58 mE6E7. Then NIH/3T3 cell line was transfected with plasmid pIRES-neo-HPV58 mE6E7. The pIRES-neo-HPV58 mE6E7-transfected cells were the experimental group, pIRES-neo-HPV58 E6E7-transfected cells were the positive control group, and pIRES-neo empty vector-transfected cells were the negative control group. The expression of HPV58 mE6E7 protein in the experimental cells was detected by flow cytometry, immunofluorescence and Western blot. The transformation activity of HPV58 mE6E7 was tested by soft agar colony formation assay and subcutaneously tumors in nude mice. Finally, DNA vaccine was constructed with HPV58 mE6E7 fusion antigen and used to immunize C57BL/6 mice with the vaccine plasmids. The specific serum antibodies were detected by EIISA, and the number of splenic specific CD8(+) T cells secreting IFN-γ of the immunized mice was detected by ELISPOT assay. RESULTS: Sequencing confirmed the expected mutation and a 100% homogeneity of the HPV58 E6E7 fusion gene. Stable transfected NIH/3T3 cells expressing HPV58 mE6E7 and HPV58 E6E7 gene were 70.3% and 84.1%, respectively. The relative expressions of HPV58 mE6E7 and HPV58 E6E7 fusion protein in 3T3-HPV58 mE6E7 experimental cells and 3T3-HPV58 E6E7 positive control cells were 2.1 ± 1.7 and 3.8 ± 1.4, respectively, and were negative in the negative control group. No colony formation was found in the experimental and 3T3-neo negative control cell groups, and 31 colonies were found in the positive control cell group, among them 10 colonies were consisted of more than 50 cells. No tumor mass was formed within 4 weeks in the nude mice of experimental and negative control groups, but among the 10 mice of positive control group tumor was formed in 6 mice. Using HPV58 mE6E7 fusion gene as target antigen of DNA vaccine, the antibody titer was 25 600, and specific immunity spots were 218.8 ± 34.4, significantly higher than that in the control group. CONCLUSIONS: The fused and modified HPV58 E6E7 amino acid codons can abolish the transformation activity but preserve its antigenicity. HPV58 mE6E7 is a potential target gene for the development of therapeutic DNA vaccine against HPV58-associated cervical cancer.

[Preliminary observation on antitumor effect of HPV58 composite DNA vaccine].

OBJECTIVE: To initially observe the antitumor immune of PVAX1-HPV58mE6E7FcGB composite DNA vaccine. METHODS: Before detecting immune effect of the vaccine, the B16-HPV58E6E7 tumor cell line was built which could steadily express HPV58E6E7 fusion gene. Then, HPV58E6E7-GST fusion protein as an antigen was expressed and purified. Before or after immunized with the vaccine, the C57BL/6 mice were challenged by B16-HPV58E6E7 cells. Anti-tumor transplantation and tumor growth inhibition experiment were performed to observe prevention and treatment effects on the vaccine. Specific humoral and cellular immune responses in the immunized mice were detected by ELISA, enzyme linked immunospot assay (ELISPOT) and cytotoxic T lymphocyte (CTL) method. RESULTS: In the anti-tumor transplantation experiment, tumor formation rate was only 9/15 in the mice which were immunized by PVAX1-HPV58mE6E7FcGB vaccine, time before tumor formation was the longest [(13.6 ± 1.7) days] and tumor growth was the slowest in the vaccine group. In tumor growth inhibition experiment, inhibition rate reached 81.4% in the vaccine group. Except tumor formation rate, all data in the vaccine group was superior to the pure antigen PVAX1-HPV58mE6E7Fc group (P < 0.05). Humoral immune effect showed that both the vaccine and the pure antigen could induce specific antibody in the immunized mice, and the highest titer were 1: 25 600 and 1: 12 800, respectively. Although there was not significant difference of antibody titer between the vaccine and the pure antigen group (P > 0.05), the number of activated T cells in the vaccine group was almost four times as that in the pure antigen group [(219 ± 34)/4×10(5) spleen lymphocytes versus (55 ± 25)/4×10(5) spleen lymphocytes, P < 0.05], and the highest specific CTL that vaccine induced was significantly higher than that of pure antigen (43.3% versus 31.3%, P < 0.05). CONCLUSIONS: Humoral and cellular immune response could be effectively stimulated by PVAX1-HPV58mE6E7FcGB composite DNA vaccine. Growth of B16-HPV58E6E7 cells was significantly inhibited in the immunized mice. The cellular immune effect on the vaccine was superior to the pure antigen. Therefore, PVAX1-HPV58mE6E7FcGB could be used as a candidate vaccine for immune therapy to the HPV58 positive tumors and precancerous lesions.

[Construction and eukaryotic expression of PVAX1-hPV58mE6E7fcGB composite gene vaccine].

To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.

Recent Progress of Lipid Nanoparticles-Based Lipophilic Drug Delivery: Focus on Surface Modifications.

Numerous drugs have emerged to treat various diseases, such as COVID-19, cancer, and protect human health. Approximately 40% of them are lipophilic and are used for treating diseases through various delivery routes, including skin absorption, oral administration, and injection. However, as lipophilic drugs have a low solubility in the human body, drug delivery systems (DDSs) are being actively developed to increase drug bioavailability. Liposomes, micro-sponges, and polymer-based nanoparticles have been proposed as DDS carriers for lipophilic drugs. However, their instability, cytotoxicity, and lack of targeting ability limit their commercialization. Lipid nanoparticles (LNPs) have fewer side effects, excellent biocompatibility, and high physical stability. LNPs are considered efficient vehicles of lipophilic drugs owing to their lipid-based internal structure. In addition, recent LNP studies suggest that the bioavailability of LNP can be increased through surface modifications, such as PEGylation, chitosan, and surfactant protein coating. Thus, their combinations have an abundant utilization potential in the fields of DDSs for carrying lipophilic drugs. In this review, the functions and efficiencies of various types of LNPs and surface modifications developed to optimize lipophilic drug delivery are discussed.

Recent Progress in Diatom Biosilica: A Natural Nanoporous Silica Material as Sustained Release Carrier.

A drug delivery system (DDS) is a useful technology that efficiently delivers a target drug to a patient's specific diseased tissue with minimal side effects. DDS is a convergence of several areas of study, comprising pharmacy, medicine, biotechnology, and chemistry fields. In the traditional pharmacological concept, developing drugs for disease treatment has been the primary research field of pharmacology. The significance of DDS in delivering drugs with optimal formulation to target areas to increase bioavailability and minimize side effects has been recently highlighted. In addition, since the burst release found in various DDS platforms can reduce drug delivery efficiency due to unpredictable drug loss, many recent DDS studies have focused on developing carriers with a sustained release. Among various drug carriers, mesoporous silica DDS (MS-DDS) is applied to various drug administration routes, based on its sustained releases, nanosized porous structures, and excellent solubility for poorly soluble drugs. However, the synthesized MS-DDS has caused complications such as toxicity in the body, long-term accumulation, and poor excretion ability owing to acid treatment-centered manufacturing methods. Therefore, biosilica obtained from diatoms, as a natural MS-DDS, has recently emerged as an alternative to synthesized MS-DDS. This natural silica carrier is an optimal DDS platform because culturing diatoms is easy, and the silica can be separated from diatoms using a simple treatment. In this review, we discuss the manufacturing methods and applications to various disease models based on the advantages of biosilica.

Adjuvant DNA vaccine pNMM promotes enhanced specific immunity and anti-tumor effects.

DNA vaccines containing only antigenic components have limited efficacy and may fail to induce effective immune responses. Consequently, adjuvant molecules are often added to enhance immunogenicity. In this study, we generated a tumor vaccine using a plasmid encoding NMM (NY-ESO-1/MAGE-A3/MUC1) target antigens and immune-associated molecules. The products of the vaccine were analyzed in 293 T cells by western blotting, flow cytometry, and meso-scale discovery electrochemiluminescence. To assess the immunogenicity obtained, C57BL/6 mice were immunized using the DNA vaccine. The results revealed that following immunization, this DNA vaccine induced cellular immune responses in C57BL/6 mice, as evaluated by the release of IFN-γ, and we also detected increases in the percentages of nonspecific lymphocytes, as well as those of antigen-specific T cells. Furthermore, immunization with the pNMM vaccine was found to significantly inhibit tumor growth and prolonged the survival of mice with B16-NMM+-tumors. Our data revealed that pNMM DNA vaccines not only confer enhanced immunity against tumors but also provide a potentially novel approach for vaccine design. Moreover, our findings provide a basis for further studies on vaccine pharmacodynamics and pharmacology, and lay a solid foundation for clinical application.

Puerarin: a novel antagonist to inward rectifier potassium channel (IK1).

Puerarin, isolated from the root of pueraria, had been widely used to prevent and treat arrhythmia. We show that puerarin effectively prevents and reverses aconitine-induced arrhythmias in perfused heart in vitro and in rats in vivo. To study the mechanisms of antiarrhythmic action of puerarin, we investigated the electrophysiological actions of puerarin using whole-cell clamp in isolated rodent ventricular myocytes and two electrode voltage-clamp (TEV) in I(K1)-expressing Xenopus oocytes. Puerarin had no prominent effect on action potentials of ventricular myocytes from guinea pig. However, puerarin (1.2 mM) significantly inhibited the I(K1) current in rat ventricular cells. Consistently, puerarin blocked I(K1) expressed in Xenopus oocytes in a dose-dependent manner. Puerarin competed with barium, an open-channel blocker of I(K1), to inhibit I(K1) currents. Thus, our data demonstrated that puerarin is a novel open-channel blocker of I(K1), which may underlie the antiarrhythmic action of puerarin.

TRADD Mediates RIPK1-Independent Necroptosis Induced by Tumor Necrosis Factor.

As a programmed necrotic cell death, necroptosis has the intrinsic initiators, including receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and mixed-lineage kinase domain-like protein (MLKL), which combine to form necroptotic signaling pathway and mediate necroptosis induced by various necroptotic stimuli, such as tumor necrosis factor (TNF). Although chemical inhibition of RIPK1 blocks TNF-induced necroptosis, genetic elimination of RIPK1 does not suppress but facilitate necroptosis triggered by TNF. Moreover, RIPK3 has been reported to mediate the RIPK1-independent necroptosis, but the involved mechanism is unclear. In this study, we found that TRADD was essential for TNF-induced necroptosis in RIPK1-knockdown L929 and HT-22 cells. Mechanistic study demonstrated that TRADD bound RIPK3 to form new protein complex, which then promoted RIPK3 phosphorylation via facilitating RIPK3 oligomerization, leading to RIPK3-MLKL signaling pathway activation. Therefore, TRADD acted as a partner of RIPK3 to initiate necroptosis in RIPK1-knockdown L929 and HT-22 cells in response to TNF stimulation. In addition, TRADD was critical for the accumulation of reactive oxygen species (ROS), which contributed to RIPK1-independent necroptosis triggered by TNF. Collectively, our data demonstrate that TRADD acts as the new target protein for TNF-induced RIPK3 activation and the subsequent necroptosis in a RIPK1-independent manner.

Fabry disease and immunoglobulin A nephropathy presenting with Alport syndrome-like findings: A case report.

RATIONALE: Fabry's disease is an X-linked inherited syndrome. Herein, we presented an unusual case of Fabry disease coexisting with immunoglobulin A nephropathy (IgAN) presenting with Alport syndrome-like pathological findings. PATIENT CONCERNS: We report a 30-year-old male who presented with proteinuria and elevated serum creatinine and for whom the initial pathologic diagnosis supported Alport syndrome. DIAGNOSES: A diagnosis of Fabry disease with immunoglobulin A nephropathy (IgAN) was finally made after further examination. INTERVENTIONS: After the initial diagnosis the patient was treated with herbal medications and mecobalamin. OUTCOMES: The patient was discharged 1 week later. He was maintained on these treatments and received regular follow-up in our hospital. LESSONS SUBSECTIONS AS PER STYLE: FD coexisting with IgAN is rare and may have nonspecific clinical presentations. Laboratory examination and genetic diagnosis is needed for confirmation. Timely diagnosis and reproductive intervention is needed for therapy.

Evaluation of MC3T3 Cells Proliferation and Drug Release Study from Sodium Hyaluronate-1,4-butanediol Diglycidyl Ether Patterned Gel.

A pattern gel has been fabricated using sodium hyaluronate (HA) and 1,4-butanediol diglycidyl ether (BDDGE) through the micro-molding technique. The cellular behavior of osteoblast cells (MC3T3) in the presence and absence of dimethyloxalylglycine (DMOG) and sodium borate (NaB) in the pattern gel (HA-BDDGE) has been evaluated for its potential application in bone regeneration. The Fourier transform infrared spectroscopy (FTIR), 13C-nuclear magnetic resonance spectroscopy (13C NMR), and thermogravimetric analysis (TGA) results implied the crosslinking reaction between HA and BDDGE. The scanning electron microscopy (SEM) analysis confirmed the formation of pattern on the surface of HA-BDDGE. The gel property of the crosslinked HA-BDDGE has been investigated by swelling study in distilled water at 37 °C. The HA-BDDGE gel releases DMOG in a controlled way for up to seven days in water at 37 °C. The synthesized gel is biocompatible and the bolus drug delivery results indicated that the DMOG containing patterned gel demonstrates a better cell migration ability on the surface than NaB. For local delivery, the pattern gel with 300 µM NaB or 300 µM DMOG induced cell clusters formation, and the gel with 150 µM NaB/DMOG showed high cell proliferation capability only. The vital role of NaB for bone regeneration has been endorsed from the formation of cell clusters in presence of NaB in the media. The in vitro results indicated that the pattern gel showed angiogenic and osteogenic responses with good ALP activity and enhanced HIF-1α, and Runx2 levels in the presence of DMOG and NaB in MC3T3 cells. Hence, the HA-BDDGE gel could be used in bone regeneration application.

TRADD mediates the tumor necrosis factor-induced apoptosis of L929 cells in the absence of RIP3.

Receptor-interacting protein kinase 3 (RIP3) is a critical initiator in mediating necroptosis induced by tumor necrosis factor alpha (TNFα) in L929 cells, so knockdown of RIP3 inhibits TNFα-induced L929 cell necroptosis. However, RIP3 knockdown was shown to switch TNFα-induced necroptosis to apoptosis in L929 cells in other studies. Therefore, whether RIP3 knockdown blocks the TNFα-induced death of L929 cells is controversial. In this study, TNFα activated caspase pathway and induced cell death in RIP3 knockdown L929 cells, and the RIP3-independent cell death had been blocked by Z-VAD-FMK (pan-caspase inhibitor) or caspase 8 knockdown, demonstrating that RIP3 knockdown switched TNFα-induced necroptosis to caspase-dependent apoptosis. Although both TNF receptor type 1-associated death domain protein (TRADD) and RIP1 have been reported to mediate TNFα-induced apoptosis, the knockdown of TRADD, but not RIP1, suppressed TNFα-induced activation of the caspase pathway and subsequent apoptosis in RIP3 knockdown L929 cells. In addition, TRADD bound and activated caspase 8 during the RIP3-independent apoptosis process, indicating that TRADD initiates RIP3-independent apoptosis by activating the caspase pathway. Collectively, we identified the target and mechanism underlying RIP3-independent apoptosis and elucidated the coordinated roles of RIP3 and TRADD in mediating the programmed cell death of L929 cells following TNFα stimulation.

[Establishment of a human bladder cancer cell line stably co-expressing hSPRY2 and luciferase genes and its subcutaneous tumor xenograft model in nude mice].

Objective To establish a human bladder cancer cell line stably co-expressing human sprouty2 (hSPRY2) and luciferase (Luc) genes simultaneously, and develop its subcutaneous tumor xenograft model in nude mice. Methods The hSPRY2 and Luc gene segments were amplified by PCR, and were cloned into lentiviral vector pCDH and pLVX respectively to produce corresponding lentivirus particles. The J82 human bladder cancer cells were infected with these two kinds of lentivirus particles, and then further screened by puromycin and G418. The expressions of hSPRY2 and Luc genes were detected by bioluminescence, immunofluorescence and Western blot analysis. The screened J82-hSPRY2/Luc cells were injected subcutaneously into BALB/c nude mice, and the growth of tumor was monitored dynamically using in vivo fluorescence imaging system. Results J82-hSPRY2/Luc cell line stably expressing hSPRY2 and Luc genes was established successfully. Bioluminescence, immunofluorescence and Western blot analysis validated the expressions of hSPRY2 and Luc genes. The in vivo fluorescence imaging system showed obvious fluorescence in subcutaneous tumor xenograft in nude mice. Conclusion The J82-hSPRY2/Luc bladder cancer cell line and its subcutaneous tumor xenograft model in nude mice have been established successfully.

Shikonin induces mitochondria-mediated apoptosis and enhances chemotherapeutic sensitivity of gastric cancer through reactive oxygen species.

The prognosis of gastric cancer remains poor due to clinical drug resistance. Novel drugs are urgently needed. Shikonin (SHK), a natural naphthoquinone, has been reported to trigger cell death and overcome drug resistance in anti-tumour therapy. In this study, we investigated the effectiveness and molecular mechanisms of SHK in treatment with gastric cancer. In vitro, SHK suppresses proliferation and triggers cell death of gastric cancer cells but leads minor damage to gastric epithelial cells. SHK induces the generation of intracellular reactive oxygen species (ROS), depolarizes the mitochondrial membrane potential (MMP) and ultimately triggers mitochondria-mediated apoptosis. We confirmed that SHK induces apoptosis of gastric cancer cells not only in a caspase-dependent manner which releases Cytochrome C and triggers the caspase cascade, but also in a caspase-independent manner which mediates the nuclear translocation of apoptosis-inducing factor and Endonuclease G. Furthermore, we demonstrated that SHK enhanced the chemotherapeutic sensitivity of 5-fluorouracil and oxaliplatin in vitro and in vivo. Taken together, our data show that SHK may be a novel therapeutic agent in the clinical treatment of gastric cancer.