Silica nanoparticles induce male reproductive toxicity via Crem hypermethylation mediated spermatocyte apoptosis and sperm flagella damage.

Exposure to silica nanoparticles (SiNPs) could causally contribute to malfunctioning of the spermatogenesis, but the underlying mechanism is rarely known. This study was designed to explore the mechanism of Crem hypermethylation in SiNP-induced reproductive toxicity. The male mice were exposure to SiNPs (0 and 20 mg/kg·bw) once every 5 days via intratracheal instillation for 35 days. After exposure stopped, half of each group was killed, and the rest were sacrificed after another 15-day feeding. GC-2 cells were treated with 0 and 20 µg/mL SiNPs. The results showed that SiNPs led to structure damage of spermatocyte and sperm, caused spermatocyte apoptosis, and decreased sperm quantity and quality. After 15 days of the withdrawal, the testicular tissue damage gradually recovered. Mechanistic study showed that SiNPs induced hypermethylation of the gene of cAMP responsive element modulator (Crem) in the promoter region. Downregulation of Crem inhibited the expression of outer dense fiber 1 (Odf1), resulting in abnormal sperm flagella structure; at the same time, Crem inhibited the expression of Bcl-xl, causing upregulation of cytochrome-C, cleaved-caspase-9/caspase-9, cleaved-caspase-3/caspase-3, resulting in mitochondrial dependent apoptotic pathway. However, 5-aza, DNA methylation inhibitor, could reverse the SiNP-induced downregulation of Crem and reverse the Crem/Bcl-xl-mediated mitochondrial dependent apoptotic pathway. These results suggested SiNPs could disrupt spermatogenesis by causing Crem hypermethylation to regulate the Odf1 and Bcl-xl in spermatocytes resulting in the sperm flagella structure and spermatocyte apoptosis. Our study provided new insights into the male reproductive toxicity mechanism of SiNPs; Crem demethylation may be a potential way to prevent reproductive dysfunction from SiNP exposure.

Lentivirus-based shRNA of Caspase-3 gene silencing inhibits chondrocyte apoptosis and delays the progression of surgically induced osteoarthritis.

Chondrocyte apoptosis is an important pathological feature of osteoarthritis (OA). Excessive apoptosis of chondrocytes disrupts the dynamic balance of cell proliferation and apoptosis, with a marked reduction in chondrocytes and cartilage matrix disintegration, which represents the main pathology of OA. Caspases, especially Caspase-3, play a central role in cell apoptosis. In this study, a lentiviral vector was used to transduce caspase-3 short hairpin RNA (shRNA) into rat chondrocytes (RCs), and the apoptotic and phenotypic genes of RCs were analyzed using real-time PCR and western blotting in vitro. In addition, in vivo intra-articular injection of Caspase-3 shRNA lentivirus was performed in a surgically induced OA rat model. Our results showed that Caspase-3 gene silencing could down-regulate the TNF-α-mediated inflammatory gene expression of TNFR1, FADD, and IL-1ß, apoptotic gene expression of APAF1, Caspase-3, and Caspase-9, thereby attenuating the apoptotic pathway in vitro. Caspase-3 gene silencing also attenuated TNF-α-mediated decreased gene expression of ACAN, Col1-a1, and Col2-a1. Furthermore, Caspase-3 gene silencing could effectively reduce the OARSI score, and gene expression of Caspase-3, Caspase-9, MMP13, and TNF-α in a surgically induced OA rat model. Caspase-3 gene silencing may serve as a novel therapeutic strategy for cartilage injury and OA.

Roles of Cyt-c/Caspase-9/Caspase-3/Bax/Bcl-2 pathway in Cd-induced testicular injury in rats and the protective effect of quercetin.

Cadmium (Cd) exposure causes oxidative damage to mitochondria, which would adversely affect rat testicular tissue. Quercetin (Que) is a natural antioxidant with anti-inflammatory, antioxidant and anti-apoptotic effects. However, the mechanism by which Que inhibits Cd-induced apoptosis of testicular cells remains unclear. The purpose of this study was to investigate the role of mitochondrial apoptosis pathway (Cyt-c/Caspase-9/Caspase-3/Bax/Bcl-2 pathway) in inhibiting Cd-induced apoptosis of testicular cells by Que. We used SD rats to simulate Cd chloride exposure by treating all sides of the rats with CdCl2 and/or Que. The levels of GSH and MDA in rat testis were detected using reagent kits. The effects of CdCl2 and/or Que on tissue damage, apoptosis, and gene and protein expression of the Cyt-c/Caspase-9/Caspase-3/Bax/Bcl-2 pathway in rat testis were examined by HE, TUNEL, RNA extraction and reverse-transcriptase polymerase chain reaction (RT-PCR), and Western blot (Wb). The results show that Cd significantly increased the contents of GSH and MDA in rat testis (P < 0.01); conversely, Que significantly reduced the contents of GSH and MDA (P < 0.01). Cd inflicted damage to testicular tissue, and Que addition significantly reduced the damage. Cd increased the number of apoptosis of testicle cells, and Que inhibited testicle-cell apoptosis. In addition, the results of reverse transcription PCR and Wb assays confirmed that, as expected, Cd increased the expression levels of Cyt-c, Caspase-9, Caspase-3, and Bax mRNAs as well as proteins. And at the same time decreased the expression of the anti-apoptotic factor Bcl-2 in the cells. Surprisingly, these effects were reversed when Que was added. Therefore, Que can play an antioxidant and anti-apoptotic role in reducing the testicular tissue damage caused by Cd exposure. This provides a conceptual basis for the later development and utilization of Que as well as the prevention and treatment of tissue damage caused by Cd exposure.

Caspase-9 inhibition triggers Hsp90-based chemotherapy-mediated tumor intrinsic innate sensing and enhances antitumor immunity.

BACKGROUND: Antineoplastic chemotherapies are dramatically efficient when they provoke immunogenic cell death (ICD), thus inducing an antitumor immune response and even tumor elimination. However, activated caspases, the hallmark of most cancer chemotherapeutic agents, render apoptosis immunologically silent. Whether they are dispensable for chemotherapy-induced cell death and the apoptotic clearance of cells in vivo is still elusive. METHODS: A rational cell-based anticancer drug library screening was performed to explore the immunogenic apoptosis pathway and therapeutic targets under apoptotic caspase inhibition. Based on this screening, the potential of caspase inhibition in enhancing chemotherapy-induced antitumor immunity and the mechanism of actions was investigated by various cells and mouse models. RESULTS: Heat shock protein 90 (Hsp90) inhibition activates caspases in tumor cells to produce abundant genomic and mitochondrial DNA fragments and results in cell apoptosis. Meanwhile, it hijacks Caspase-9 signaling to suppress intrinsic DNA sensing. Pharmacological blockade or genetic deletion of Caspase-9 causes tumor cells to secrete interferon (IFN)-ß via tumor intrinsic mitochondrial DNA/the second messenger cyclic GMP-AMP (cGAS) /stimulator of interferon genes (STING) pathway without impairing Hsp90 inhibition-induced cell death. Importantly, both Caspase-9 and Hsp90 inhibition triggers an ICD, leading to the release of numerous damage-associated molecular patterns such as high-mobility group box protein 1, ATP and type I IFNs in vitro and remarkable antitumor effects in vivo. Moreover, the combination treatment also induces adaptive resistance by upregulating programmed death-ligand 1 (PD-L1). Additional PD-L1 blockade can further overcome this acquired immune resistance and achieve complete tumor regression. CONCLUSIONS: Blockade of Caspase-9 signaling selectively provokes Hsp90-based chemotherapy-mediated tumor innate sensing, leading to CD8+ T cell-dependent tumor control. Our findings implicate that pharmacological modulation of caspase pathway increases the tumor-intrinsic innate sensing and immunogenicity of chemotherapy-induced apoptosis, and synergizes with immunotherapy to overcome adaptive resistance.

Baicalin inhibits apoptosis and enhances chondrocyte proliferation in thiram-induced tibial dyschondroplasia in chickens by regulating Bcl-2/Caspase-9 and Sox-9/Collagen-II expressions.

Avian tibial dyschondroplasia (TD) is a skeletal disease affecting fast growing chickens, resulting in non-mineralized avascular cartilage. This metabolic disorder is characterized by lameness and reduced growth performance causing economic losses. The aim of this study was to investigate the protective effects of baicalin against TD caused by thiram exposure. A total of two hundred and forty (n = 240) one day-old broiler chickens were uniformly and randomly allocated into three different groups (n = 80) viz. control, TD, and baicalin groups. All chickens received standard feed, however, to induce TD, the TD and baicalin groups received thiram (tetramethylthiuram disulfide) at a rate of 50 mg/kg feed from days 4-7. The thiram induction in TD and baicalin groups resulted in lameness, high mortality, and enlarged growth-plate, poor production performance, reduction in ALP, GSH-Px, SOD, and T-AOC levels, and increased AST and ALT, and MDA levels. Furthermore, histopathological results showed less vascularization, and mRNA and protein expression levels of Sox-9, Col-II, and Bcl-2 showed significant downward trend, while caspase-9 displayed significant up-regulation in TD-affected chickens. After the TD induction, the baicalin group was orally administered with baicalin at a rate of 200 mg/kg from days 8-18. Baicalin administration increased the vascularization, and chondrocytes with intact nuclei, alleviated lameness, decreased GP size, increased productive capacity, and restored the liver antioxidant enzymes and serum biochemical levels. Furthermore, baicalin significantly up-regulated the gene and protein expressions of Sox-9, Col-II, and Bcl-2, and significantly down-regulated the expression of caspase-9 (p < 0.05). Therefore, the obtained results suggest that baicalin could be a possible choice in thiram toxicity alleviation by regulating apoptosis and chondrocyte proliferation in thiram-induced tibial dyschondroplasia.

Inhibition of mitochondria induces apoptosis and reduces telomere length and activity in acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) is a highly lethal hematological malignancy in adults and children. Abnormal proliferation of leukemia stem cells (LSC) with CD34+ and CD38- phenotypes are the main clinical features of AML. Patients with AML face drug resistance and treatment failure due to a default in stem and progenitor cells. Therefore, defining LSC properties is necessary for targeting leukemia-initiating cells. Mitochondrial mass and activity increase in AML initiating cells compared with normal stem cells. This idea has offered the inhibition of the mitochondrial translation machinery to reduce the number of leukemia-initiating cells in patients with AML Tigecycline is an FDA-approved microbial antibiotic that inhibits oxidative phosphorylation in mitochondria, resulting in the suppression of leukemia cell proliferation with little toxicity to normal cells. Thus, the present study was conducted to evaluate whether LSC is influenced by mitochondrial inhibition. We measured the IC50 of tigecycline in KG-1a AML cell lines. KG-1a AML cell lines were separated into CD34+ and CD34- cells by MACS. In the following, these cells were treated with 20 µM (IC50) tigecycline. The expression of Annexin/PI, Caspase 3, apoptotic genes (BCL2, BCLX, BAX, BAD, and P53) and proteins (P53, BAX, BCL2 and Caspase 9) was evaluated in CD34+ , CD34- and KG-1a AML cells. In addition, the telomere length and expression of hTERT were evaluated in this study. The results indicated that BCl2 (gene and protein) and BCLX gene dramatically decreased. In addition, BAD, BAX, and P53 gene and protein expression significantly increased in CD34+ AML cells compared to CD34- AML cells. The results also suggested that tigecycline induced intrinsic (Cleaved-caspase 9/Pro-Caspase 9 ratio) and p53-mediated apoptosis. Furthermore, hTERT gene expression and telomere length decreased in the tigecycline-treated groups. Taken together, our findings indicate that inhibition of mitochondrial activity with tigecycline can induce apoptosis in cancer stem cells and can be used as a novel method for cancer therapy.

[Mechanism of Proliferation and Apoptosis of Acute Promyelocytic Leukemia Cell Line NB4 Induced by TPA].

OBJECTIVE: To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism. METHODS: The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT. RESULTS: Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G1 phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells. CONCLUSION: TPA induces NB4 cell cycle arrest in G1 phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.

[Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy].

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

Combined oncolytic virotherapy gold nanoparticles as synergistic immunotherapy agent in breast cancer control.

Combining viruses and nanoparticles may be a way to successfully treat cancer and minimize adverse effects. The current work aimed to evaluate the efficacy of a specific combination of gold nanoparticles (GNPs) and Newcastle disease virus (NDV) to enhance the antitumor effect of breast cancer in both in vitro and in vivo models. Two human breast cancer cell lines (MCF-7 and AMJ-13) and a normal epithelial cell line (HBL-100) were used and treated with NDV and/or GNPs. The MTT assay was used to study the anticancer potentials of NDV and GNP. The colony formation assay and apoptosis markers were used to confirm the killing mechanisms of NDV and GNP against breast cancer cell lines. p53 and caspase-9 expression tested by the qRT-PCR technique. Our results showed that combination therapy had a significant killing effect against breast cancer cells. The findings demonstrated that NDV and GNPs induced apoptosis in cancer cells by activating caspase-9, the p53 protein, and other proteins related to apoptosis, which holds promise as a combination therapy for breast cancer.

Rhein suppresses African swine fever virus replication in vitro via activating the caspase-dependent mitochondrial apoptosis pathway.

African swine fever (ASF) is a virulent infectious diseases of pigs caused by the African swine fever virus (ASFV) that can spread widely and cause high fatality rates. Currently, there is no effective way to treat the disease, and there is no effective vaccine to prevent it. Rhein, an anthraquinone compound extracted from many traditional Chinese medicines, exhibits anti-inflammatory, anti-tumor, and anti-viral activities. However, the anti-viral effects of rhein on ASFV remain unclear. Therefore, this study aimed to investigate the anti-ASFV activity of rhein in porcine alveolar macrophages (PAMs) and the underlying mechanisms. In this study, we confirmed that rhein inhibits ASFV replication significantly in a dose-dependent manner in vitro. Moreover, rhein could alter the susceptibility of PAMs to ASFV and promoted the production of superoxide in the mitochondria, which induced the loss of mitochondrial membrane potential, leading to the activation of caspase-9, caspase-3, and apoptosis. Mito-TEMPO, a mitochondria-targeted antioxidant, blocked rhein-induced mitochondrial superoxide generation and loss of mitochondrial membrane potential, prevented caspase-9 and caspase-3 activation, alleviated apoptosis, and suppressed the anti-ASFV activity of rhein. Altogether, our results suggested that rhein could play an anti-ASFV role by inducing apoptosis through the activation of the caspase-dependent mitochondrial apoptotic pathway and may provide a novel compound for developing anti-ASFV drugs.

Variants of the CASP9 gene as candidate markers for primary response to anti-TNF therapy in Crohn's disease patients.

Anti-tumor necrosis factor (TNF) therapy is used to induce and maintain remission in Crohn's disease (CD) patients. However, primary non-responders to initial treatment constitute 20-40% of cases. The causes of this phenomenon are still unknown. We aim to investigate the impact of the caspase 9 (CASP9) gene variants on the variable reactions of CD patients to anti-TNF therapy. The study group included 196 diagnosed and clinically characterized CD Polish patients following anti-TNF therapy. The sequence of the CASP9 gene was analyzed using next-generation and Sanger sequencing and was analyzed with the response to biological treatment. Using the RT-qPCR analysis, we estimated the CASP9 gene mRNA level in colon biopsies material from inflamed and non-inflamed tissue (21 CD patients: 14 responders and seven non-responders to anti-TNF therapy and six controls), as well as in vitro in a peripheral blood mononuclear cells (PBMCs) from CD patients (seven responders and seven non-responders to anti-TNF therapy) and eight controls. Our findings indicated association of variants rs1052571 and rs4645978 with response to anti-TNF monoclonal antibodies (mAbs). Moreover, we observed tendency for reduced expression after incubation with anti-TNF in the group of CD patients, in contrast to the control group. Our results suggest that response to anti-TNF therapy in CD patients may be an effect of variants of the CASP9 gene as a key effector of the internal pathway of apoptosis; however, further population and functional research are necessary.

L-asparaginase from human breast milk Lactobacillus reuteri induces apoptosis using therapeutic targets Caspase-8 and Caspase-9 in breast cancer cell line.

BACKGROUND: Breast cancer is the most fatal type of cancer in women worldwide. Many chemotherapeutics targeted breast cancer however, they have frightening side effects. One method of controlling cancer cell growth is targeting apoptosis. OBJECTIVE: This study aimed to induce apoptosis in breast cancer cells by purifying L-asparaginase from human breast milk Lactobacillus reuteri isolates via inhibition of Caspases 8 and 9. METHODS: The best L. reuteri isolates producing L-asparagine with the highest enzyme activity were identified from human breast milk and chosen for L-asparaginase purification. The MTT cell viability assay used for measure the toxicity of the enzyme. Breast cancer cell line was used to study the effect of the enzyme on the caspase 8 and caspase 9 gene expression. RESULTS: The MTT cell viability assay showed the inhibition rates ranged between 30% and 80%, of cell death, occurred when 3.125, 6.25, 12.5, 25, 50, and 100 µg/ml of the enzyme used and IC50 was 4.305 µg/ml. The breast cell lines were treated with the enzyme at a concentration of IC50 value. The Cas8 and Cas9 genes expression in L-asparagine treated breast cancer cell line at a concentration of IC50 value were upregulated (the fold of gene expression are 2.071 and 1.197 respectively). CONCLUSIONS: Breast milk L. reuteri L-asparaginase induces apoptosis via Cas8 and Cas9 upregulation in the breast cancer cell line. L. reuteri L-asparaginase treatment may be the hopeful approach for the management of breast cancer. Furthermore, the results may highlight the fact that the presence of L-asparaginase-producing L. reuteri isolates in human breast milk may aid in breast cancer improvement or even prevention.

[Effect of autophagy in cadmium chloride induced apoptosis of mouse spermatogenic cells].

OBJECTIVE: To study the effect of autophagy in cadmium chloride(CdCl_2)-induced apoptosis of mouse spermatocytes(GC-2 spd) cells and explore the underlying molecular mechanisms. METHODS: The cells were treated with different concentrations of CdCl_2(0, 5 and 10 µmol/L) for 24 h. Hoechst33342 staining and monodansylcadaverine(MDC) were performed to explore the formation of autophagosomes and apoptotic bodies. The apoptosis of cadmium-treated cells was examined by TUNEL staining. Autophagy inhibitor 3-methyladenine(3-MA)(60 µmol/L), apoptotic inhibitorCaspase inhibitor Z-VAD-FMK( zVAD-FMK)(50 nmol/L), autophagy inducer rapamycin(RAPA)(50 nmol/L) and lysosomal inhibitor chloroquine(CQ)(10 µmol/L) were added to cell culture in the presence/absence of CdCl_2(10 µmol/L) to treat GC-2 spd cells for 24 h. The expression levels of autophagy-related proteins LC3, P62, and pro-apoptotic proteins cleaved Caspase-3 and cleaved Caspase-9 were examined by Western blot. RESULTS: Autophagosomes aggregated and the number of apoptotic cells increased after exposure to CdCl_2 for 24 h. Western blot result showed that in the 5 and 10 µmol/L CdCl_2 exposure groups, the protein expression levels of LC3II/LC3I increased to 9.23±0.81 and 12.15±0.80 compared with the control group(5.50±0.56)(P<0.05), LC3II protein expression level increased to 3.35±0.14 and 3.47±0.32 compared with the control group(2.35±0.34)(P<0.05), P62 protein expression level increased to 1.48±0.12 and 1.80±0.22 compared with the control group(0.83±0.09)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of LC3II/LC3I, LC3II, P62, cleaved Caspase-9 and cleaved Caspase-3 after 3-MA treatment decreased to 0.90±0.07(CdCl_2 group: 1.47±0.06), 1.57±0.14(CdCl_2 group: 2.45±0.29), 0.82±0.05(CdCl_2 group: 1.44±0.18), 0.18±0.01(CdCl_2 group: 0.28±0.01) and 0.61±0.84(CdCl_2 group: 1.15±0.04)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of cleaved Caspase-9 and cleaved Caspase-3 after zVAD-FMK treatment decreased to 0.12±0.01(CdCl_2 group: 0.28±0.01) and 0.34±0.01(CdCl_2 group: 1.15±0.04)(P<0.05), while those of LC3II/LC3I, LC3II and P62 had no significant change(P>0.05). Compared with the CdCl_2-treated group, RAPA enhanced cadmium-induced LC3II/LC3I, LC3II and P62 protein expressions to 2.22±0.21(CdCl_2 group: 1.56±0.06), 3.72±0.21(CdCl_2 group: 2.97±0.15) and 2.41±0.19(CdCl_2 group: 1.52±0.35)(P<0.05). Western blot result showed that compared with the CdCl_2 group, the protein expressions of LC3II/LC3I, LC3II, P62 and cleaved Caspase-3 in the CdCl_2 and CQ treatment groups increased to 3.21±0.31(CdCl_2 group: 2.09±0.25), 4.49±0.43(CdCl_2 group: 2.72±0.26), 2.59±0.19(CdCl_2 group: 1.84±0.19) and 2.43±0.23(CdCl_2 group: 1.50±0.27)(P<0.05). CONCLUSION: Cadmium chloride induces apoptosis of mouse spermatocyte cells by inhibiting autophagosome-lysosomal fusion and prompting abnormal aggregation of autophagosomes.

[Stellera chamaejasme extract against multidrug resistance of breast cancer cell line MCF-7].

This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.

Fatal dermatophytic pseudomycetoma in a patient with non-HIV CD4 lymphocytopenia.

Dermatophytic pseudomycetoma is a rare invasive infection, involving both immunocompetent and immunocompromised individuals. Since the discovery of inherited immune disorders such as the impairment of CARD9 gene, extended dermatophyte infections are mostly ascribed to any of these host factors. This study is to present and explore the potential causes in a fatal dermatophytic pseudomycetoma patient. We present a chronic and deep pseudomycetoma caused by the common dermatophyte Microsporum canis which ultimately led to the death of the patient. Mycological examination, genetic studies and host immune responses against fungi were performed to explore the potential factors. The patient had decreased lymphocyte counts with significantly reduced CD4+ T cells, although all currently known genetic parameters proved to be normal. Through functional studies, we demonstrated that peripheral blood mononuclear cells from the patient showed severe impairment of adaptive cytokine production upon fungus-specific stimulation, whereas innate immune responses were partially defective. This is, to our knowledge, the first report of fatal dermatophytic pseudomycetoma in a patient with non-HIV CD4 lymphocytopenia, which highlights the importance of screening for immune deficiencies in patients with deep dermatophytosis.

Modulatory role of BV6 and chloroquine on the regulation of apoptosis and autophagy in non-small cell lung cancer cells.

Aims: Non-small cell lung cancer (NSCLC) is one of the aggressive tumors mostly diagnosed in the advanced stage. Therapeutic failure and drug resistance pose a major problem in NSCLC treatment primarily due to alterations in autophagy and loss of apoptosis. Therefore, the present study aimed to investigate the importance of the second mitochondria-derived activator of caspase mimetic BV6 and autophagy inhibitor chloroquine (CQ) on the regulation of apoptosis and autophagy, respectively. Subjects and Methods: Study was conducted on NCI-H23 and NCI-H522 cell lines to evaluate the effect of BV6 and CQ on the transcription and translation level of LC3-II, caspase-3, and caspase-9 genes by quantitative real-time-polymerase chain reaction and western blotting techniques. Results: In NCI-H23 cell line, BV6 and CQ treatments showed increased mRNA and protein expression of caspase-3, and caspase-9 compared to its untreated counterpart. BV6 and CQ treatments also caused downregulation of LC3-II protein expression compared to its counterpart. In NCI-H522 cell line, BV6 treatment showed a significantly increased expression of caspase-3 and caspase-9 mRNA and protein expression levels whereas BV6 treatment downregulated the expression level of LC3-II protein. A similar pattern was also observed in CQ treatment when compared with the respective controls. Both BV6 and CQ modulated in vitro expression of caspases and LC3-II which have critical regulatory roles in apoptosis and autophagy, respectively. Conclusions: Our findings suggest that BV6 and CQ could be promising candidates in NSCLC treatment and there is a need to explore them in vivo and in clinical applications.

RapaCaspase-9-based suicide gene applied to the safety of IL-1RAP CAR-T cells.

Even if adoptive cell transfer (ACT) has already shown great clinical efficiency in different types of disease, such as cancer, some adverse events consistently occur, and suicide genes are an interesting system to manage these events. Our team developed a new medical drug candidate, a chimeric antigen receptor (CAR) targeting interleukin-1 receptor accessory protein (IL-1RAP), which needs to be evaluated in clinical trials with a clinically applicable suicide gene system. To prevent side effects and ensure the safety of our candidate, we devised two constructs carrying an inducible suicide gene, RapaCasp9-G or RapaCasp9-A, containing a single-nucleotide polymorphism (rs1052576) affecting the efficiency of endogenous caspase 9. These suicide genes are activated by rapamycin and based on the fusion of human caspase 9 with a modified human FK-binding protein, allowing conditional dimerization. RapaCasp9-G- and RapaCasp9-A-expressing gene-modified T cells (GMTCs) were produced from healthy donors (HDs) and acute myeloid leukemia (AML) donors. The RapaCasp9-G suicide gene demonstrated better efficiency, and we showed its in vitro functionality in different clinically relevant culture conditions. Moreover, as rapamycin is not pharmacologically inert, we also demonstrated its safe use as part of our therapy.

Japanese encephalitis virus induces apoptosis by activating the RIG-1 signaling pathway.

Japanese encephalitis virus (JEV) infection can cause brain tissue lesions characterized by neuronal death, and apoptosis is involved in JEV-induced neuronopathy. In the present study, mouse microglia were infected with JEV, and pyknosis with dark-staining nuclei of infected cells was detected using Hoechst 33342 staining. TUNEL staining showed that JEV infection promoted the apoptosis of BV2 cells, and the apoptosis rate was significantly increased at 24-60 hours postinfection (hpi) (P < 0.01) and was the highest at 36 h (P < 0.0001). Western blot results showed that the expression of the Bcl-2 protein in JEV-infected cells was downregulated significantly at 60 hpi (P < 0.001), whereas that of the Bax protein was observably upregulated at 60 hpi (P < 0.001). At the same time, the level of cytochrome c (Cyt c) was significantly increased (P < 0.001), and the expression levels of two apoptosis-related proteins, namely, cleaved caspase-3 (P < 0.01) and caspase-9 (P < 0.001), were elevated significantly. Immunofluorescence staining showed that the amount of Cyt c increased with time after infection. After BV2 cells were infected with JEV, the expression of RIG-1 increased significantly from 24 hpi to 60 h (P < 0.001). The expression of MAVS increased significantly at 24 h (P < 0.001) and decreased gradually from 24 h to 60 hpi. The expression of TBK1 and NF-κB (p65) was not significantly changed. The expression of p-TBK1 and p-NF-κB (p-p65) increased significantly within 24 h (P < 0.001) and decreased from 24 to 60 hpi. The expression levels of IRF3 and p-IRF3 peaked at 24 hpi (P < 0.001) and decreased gradually from 24 to 60 hpi. However, the expression levels of JEV proteins showed no significant change at 24 and 36 hpi but were markedly elevated at 48 and 60 hpi. Interference with the expression of the RIG-1 protein in BV2 cells resulted in a dramatic increase in the expression of the anti-apoptotic protein Bcl-2 (P < 0.05), whereas the pro-apoptotic protein Bax, cleaved caspase-9, and especially cleaved caspase-3 were downregulated (P < 0.05), and viral protein expression was notably reduced (P < 0.05). These results indicate that JEV induces apoptosis through mitochondrial-dependent apoptosis pathways, interfering with the expression of RIG-1 in BV2 cells can inhibit viral replication and inhibit apoptosis.

GD2-CART01 for Relapsed or Refractory High-Risk Neuroblastoma.

BACKGROUND: Immunotherapy with chimeric antigen receptor (CAR)-expressing T cells that target the disialoganglioside GD2 expressed on tumor cells may be a therapeutic option for patients with high-risk neuroblastoma. METHODS: In an academic, phase 1-2 clinical trial, we enrolled patients (1 to 25 years of age) with relapsed or refractory, high-risk neuroblastoma in order to test autologous, third-generation GD2-CAR T cells expressing the inducible caspase 9 suicide gene (GD2-CART01). RESULTS: A total of 27 children with heavily pretreated neuroblastoma (12 with refractory disease, 14 with relapsed disease, and 1 with a complete response at the end of first-line therapy) were enrolled and received GD2-CART01. No failure to generate GD2-CART01 was observed. Three dose levels were tested (3-, 6-, and 10×106 CAR-positive T cells per kilogram of body weight) in the phase 1 portion of the trial, and no dose-limiting toxic effects were recorded; the recommended dose for the phase 2 portion of the trial was 10×106 CAR-positive T cells per kilogram. Cytokine release syndrome occurred in 20 of 27 patients (74%) and was mild in 19 of 20 (95%). In 1 patient, the suicide gene was activated, with rapid elimination of GD2-CART01. GD2-targeted CAR T cells expanded in vivo and were detectable in peripheral blood in 26 of 27 patients up to 30 months after infusion (median persistence, 3 months; range, 1 to 30). Seventeen children had a response to the treatment (overall response, 63%); 9 patients had a complete response, and 8 had a partial response. Among the patients who received the recommended dose, the 3-year overall survival and event-free survival were 60% and 36%, respectively. CONCLUSIONS: The use of GD2-CART01 was feasible and safe in treating high-risk neuroblastoma. Treatment-related toxic effects developed, and the activation of the suicide gene controlled side effects. GD2-CART01 may have a sustained antitumor effect. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT03373097.).

Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process.

Introduction: B-cell lymphoma-2 (Bcl-2) is the first identified member of the Bcl-2 family that performs an anti-apoptotic function in mammals. However, its role in teleosts is not fully understood. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned, and its role in apoptosis was investigated. Methods: In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect its mRNA expression level in healthy condition and after LPS stimulation. Subcellular localization was performed by transfecting the pTroBcl2-N3 plasmid into golden pompano snout (GPS) cells and observed under an inverted fluorescence microscope DMi8 and further verified by immunoblotting. In vivo overexpression and RNAi knockdown method were performed to evaluate the role of TroBcl2 in apoptosis. The anti-apoptotic activity of TroBcl2 was detected by flow cytometry. The effect of TroBcl2 on the mitochondrial membrane potential (MMP) was measured by an enhanced mitochondrial membrane potential assay kit with JC-1. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was performed to evaluate the role of TroBcl2 in the DNA fragmentation. Immunoblotting was used to verify whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm. The Caspase 3 and Caspase 9 Activity Assay Kits were used to investigate the effect of TroBcl2 on caspase 3 and caspase 9 activities. The effects of TroBcl2 on the expression of apoptosis-related and nuclear factor- κB (NF-κB) signaling pathway-related genes in vitro were evaluated by qRT-PCR and Enzyme linked immunosorbent assay (ELISA). Luciferase reporter assay was used to evaluate the activity in NF-κB signaling pathway. Results and discussion: The full-length coding sequence of TroBcl2 contains 687 bp and encodes a protein containing 228 amino acids. Four conserved Bcl-2 homology (BH) domains and one invariant "NWGR" motif located in BH1 were identified in TroBcl2. In healthy T. ovatus, TroBcl2 was widely distributed in the eleven tested tissues, and higher expression levels were found in immune-related tissues, such as spleen and head kidney tissues. After stimulation with lipopolysaccharide (LPS), the expression of TroBcl2 in the head kidney, spleen, and liver was significantly upregulated. In addition, subcellular localization analysis revealed that TroBcl2 was localized in both the cytoplasm and nucleus. Functional experiments showed that TroBcl2 inhibited apoptosis, possibly by reducing mitochondrial membrane potential loss, decreasing DNA fragmentation, preventing cytochrome c release into cytoplasm, and reducing the caspase 3 and caspase 9 activations. Moreover, upon LPS stimulation, overexpression of TroBcl2 suppressed the activation of several apoptosis-related genes, such as BOK, caspase-9, caspase-7, caspase-3, cytochrome c, and p53. Furthermore, knockdown of TroBcl2 significantly increased the expression of those apoptosis-related genes. In addition, TroBcl2 overexpression or knockdown induced or inhibited, respectively, the transcription of NF-κB and regulated the expression of genes (such as NF-κB1 and c-Rel) in the NF-κB signaling pathway as well as the expression of the downstream inflammatory cytokine IL-1ß. Overall, our study suggested that TroBcl2 performs its conserved anti-apoptotic function via the mitochondrial pathway and may serve as an anti-apoptotic regulator in T. ovatus.