Aptamer-guided graphene oxide quantum dots for targeted suicide gene therapy in an organoid model of luminal breast cancer.

Breast cancer is one of the most common cancers in women. One of the best therapeutic methods against breast cancer is gene therapy, while having an appropriate gene carrier is the biggest challenge of gene therapy. Hence, developing carriers with low cytotoxicity and high gene transfection efficiency, and preferentially with the selective function of gene delivery is a critical demand for this method. In the present study, we introduce a novel targeted carrier to deliver the inducible caspase-9 suicide gene (pLVSIN-iC9) into breast cancer cells. The carrier is composed of graphene oxide quantum dots decorated with polyethyleneimine, and S2.2; an aptamer with high affinity to MUC1 (GOQD-PEI/S2.2). Due to the overexpression of MUC1 in breast cancer cells, the designed GOQD-PEI/S2.2/pLVSIN-iC9 can selectively target cancer cells. Moreover, to better mimic solid tumor conditions, and to evaluate the selective effect of the GOQD-PEI/S2.2/pLVSIN-iC9, an organoid model derived from human dermal fibroblasts (HDF) and MCF-7 cells (coculture organoid) was generated and characterized. The results demonstrate that the coculture organoid model adapts the tissue structure of luminal breast cancer, as well. Therefore, the organoids were subjected to treatment with targeted gene therapy using GOQD-PEI/S2.2/pLVSIN-iC9. Our evidence supports the targeted killing effect of iC9 on the breast cancer cells of the organoids and suggests the good potential of the newly introduced carriers in targeted gene delivery.

Analysis of the Dynamics of Cognitive Impairments and Expression of Caspase Cascade Genes in Preclinical Stages of Parkinsonism Modeled Using α-Synuclein Oligomers.

The behavioral effects of α-synuclein oligomers were studied at various times after its chronic intranasal administration to 75-day-old C57BL/6J mice in comparison with the dynamics of changes in the transcriptional activity of caspases genes (Casp9, Casp8, and Casp3) in the hippocampus, frontal cortex, and cerebellum. The negative effects of α-synuclein oligomers on exploratory activity and short-term memory in the novel object recognition test were most pronounced after 90 days from the end of administration, while after 1 and 270 days, partial compensation of the studied cognitive functions was observed. Analysis of the expression of caspase genes suggests that early compensatory mechanisms are associated with suppression of the effector caspase-3 gene expression along with increased activity of the genes encoding initiator caspases-9 and -8. Late compensation processes are associated with a decrease in the activity of initiator caspases in the frontal cortex and cerebellum.

The selective proapoptotic impact of the myricetin-loaded alginate-cellulose hybrid nanocrystals (MAC-NCs) on the human AGS gastric cancer cells.

BACKGROUND: Myricetin, a flavanol present in fruits, tea, and vegetables, has the potential to reduce chronic diseases like gastric cancer by promoting cell death and stopping cell growth. However, its limited bioactivity due to its short lifespan and poor solubility in water has been a challenge. The current research focuses on incorporating myricetin into alginate-cellulose hybrid nanocrystals to enhance its selective proapoptotic effects on human AGS gastric cancer cells. METHODS: MAC-NCs, myricetin-loaded alginate-cellulose hybrid nanocrystals, were synthesized using a combined co-precipitation/ultrasonic homogenization method and characterized through Dynamic Light Scattering (DLS), Fourier Transform Infrared Spectroscopy (FTIR), Field Emission Scanning Electron Microscope (FESEM), and Zeta-potential analyses. Their cytotoxic activity was tested on cancerous (AGS) and normal (Huvec) cells, revealing selective toxicity. Apoptotic markers, Caspase 8 and Caspase 9, gene expression was measured, and cell death type was confirmed using DAPI staining and flow cytometry on AGS cells. RESULTS: Synthesized MAC-NCs, measuring 40 nm, showed significant selective toxicity on human gastric cells (IC50 of 31.05 µg/mL) compared to normal endothelial cells (IC50 of 214.26 µg/mL). DAPI and annexin flow cytometry revealed increased apoptotic bodies in gastric cells, indicating apoptosis. However, the apoptosis was found to be independent of Caspase-8 and Caspase-9. CONCLUSION: The current study provides critical insights into the therapeutic potential of MAC-NCs for gastric cancer treatment. Based on the notable induction of apoptosis in the AGS cancer cell line, the synthesized MAC-NCs exhibit promising potential as a selective anti-gastric cancer agent. However, further in-vivo studies are necessary to confirm and quantify the nanoparticle's selective toxicity and pharmaceutical properties in future investigations.

Effects of 630 nm laser on apoptosis, metastasis, invasion and epithelial-to-mesenchymal transition of human lung squamous cell carcinoma H520 cells mediated by hematoporphyrin derivatives.

Photodynamic therapy (PDT) has significant advantages in the treatment of malignant lung tumors. The research on the mechanism of PDT mediated by hematoporphyrin derivatives (HPD) and its cytotoxic effects on lung cancer cells has primarily focused on lung adenocarcinoma cells. However, the impact of HPD-PDT on lung squamous cell carcinoma has not been thoroughly studied. This study aimed to investigate the effects of 630 nm laser on apoptosis, metastasis, invasion, and epithelial-mesenchymal transition (EMT) in human lung squamous cell carcinoma H520 cells mediated by HPD. H520 cells were divided into four groups: control group, photosensitizer group, irradiation group, and HPD-PDT group. Cell proliferation was assessed using CCK8 assay; cell apoptosis was detected by Hoechst 33258 staining and flow cytometry; cell migration and invasion abilities were evaluated using wound-healing and invasion assays; and protein and mRNA expressions were analyzed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively. Results showed that HPD-PDT significantly inhibited cell proliferation, promoted apoptosis (P < 0.05), suppressed cell migration and invasion (P < 0.05), decreased Bcl-2 mRNA expression, and increased Bax and Caspase-9 mRNA expression(P < 0.05). Western blotting analysis indicated increased expression of Bax, Caspase-9, and E-cadherin, and decreased expression of Bcl-2, N-cadherin, and Vimentin (P < 0.05). In conclusion, 630 nm laser mediated by HPD promoted cell apoptosis via upregulation of Bax and caspase-9, and downregulation of Bcl-2, and inhibited cell migration and invasion by regulating EMT in H520 cells.

Targeted biocompatible Zn-metal-organic framework nanocomposites for intelligent chemotherapy of breast cancer cells.

Finding a novel drug delivery system (DDS) represents one of the most challenging endeavors in cancer therapy. Hence, in this study, we developed a new biocompatible and biodegradable zinc-based nanoscale metal-organic framework (Zn-NMOF) coated with folic acid (FA) functionalized chitosan (CS) to facilitate targeted delivery of doxorubicin (D), a standard chemotherapeutic agent, into breast cancer cells. The synthesis of the NMOF-CS-FA-D nanocomposite preceded its comprehensive characterization via FT-IR, DLS, XRD, SEM, and TEM analyses. Subsequent in vitro studies were conducted on MCF-7 breast cancer cells and HFF-1 normal cells, encompassing assessments of cell viability, expression levels of apoptotic and autophagy genes, cell cycle arrest, and apoptotic analyses. The size of the NMOF-CS-FA-D particles was determined to be less than 80 nm, with a drug loading efficiency of 72 ± 5%. The release kinetics of DOX from the nanocomposite were investigated, revealing controlled release behavior at pH 7.4 and accelerated release at pH 5.0, which is conducive to drug delivery into cancer cells. In vitro results indicated a 17.39% ± 6.34 cell viability after 24 h of treatment with a 40 nM concentration of the NMOF-CS-FA-D nanocomposite. Furthermore, the expression levels of Caspase-9 and BAX, key apoptotic genes, along with BECLIN1, an autophagy gene, were found to increase by two-fold, four-fold, and two-fold, respectively, following 5 h of treatment with the nanocomposite. Additionally, analysis of cell cycle distribution revealed 15.4 ± 2% of cells in the sub-G1 phase, indicative of apoptotic cells, and 31.9% of cells undergoing early and late apoptosis in MCF-7 cells. Collectively, these findings underscore the potential of the NMOF-CS-FA-D nanocomposite in inhibiting cancer cell proliferation with low side effects.

Association between NKILA and some apoptotic gene expression in atherosclerosis.

Oxidized light-density lipoprotein (ox-LDL) causes endothelial dysfunction, which is an important determinant of atherogenesis, and subsequently leads to apoptosis. Atherosclerosis is one of the most significant cardiovascular diseases (CVDs) threatening human health and causes death worldwide. Recently, long noncoding RNAs (lncRNAs) have been suggested to involved in vascular biology. Ox-LDL activates nuclear factor kappa-B (NF-κB), and NF-κB interacting lncRNA (NKILA) inhibits NF-κB signaling. In this study, the hypothesis is that NKILA may regulate endothelial cell (EC) apoptosis and, therefore, play a role in the pathogenesis of atherosclerosis. This hypothesis is based on the knowledge that EC apoptosis contributes to atherosclerosis development and that NKILA has become a prominent lncRNA in CVDs. The expression of Bcl-2-associated X protein (BAX), caspase 9 (CASP9), cytochrome c (Cyt c, CYCS), apoptotic protease activating factor 1 (APAF1), and B-cell lymphoma 2 (BCL-2) genes in human umbilical vein endothelial cells (HUVEC) treated with ox-LDL and transfected with NKILA siRNA was analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). BAX, CASP9, CYCS, APAF1, and BCL-2 gene expression was downregulated in ox-LDL and NKILA siRNA-treated HUVEC. In addition, when threshold/quantification cycle (Cq) values of NKILA gene expression increased, Cq values of BAX, CASP9, APAF1, and BCL-2 gene expression increased statistics significantly. The expression detection of all these genes, resulting from NKILA gene silencing, may provide guidance for epigenetic studies on EC apoptosis in atherosclerosis.

[Molecular Mechanism of circVRK1 Regulating the Proliferation and Apoptosis of Acute Lymphoblastic Leukemia KOCL44 Cells by Targeting miR-4428]. / circVRK1靶向miR-4428è°ƒæŽ§æ€¥æ€§æ·‹å·´ç»†èƒžç™½è¡€ç— KOCL44ç»†èƒžå¢žæ®–åŠå‡‹äº¡çš„åˆ†å­æœºåˆ¶ç ”ç©¶.

Objective: To elucidate the role of circVRK1 and its interaction with miR-4428 in regulating proliferation and apoptosis in acute lymphoblastic leukemia (ALL) cells. Methods: KOCL44 ALL cells were cultured in vitro, and experimental groups included pcDNA, pcDNA-circVRK1, anti-miR-NC, anti-miR-4428, si-NC, si-circVRK1, pcDNA-circVRK1+miR-NC, and pcDNA-circVRK1+miR-4428. The expression levels of circVRK1 and miR-4428 were detected using qRT-PCR. CCK-8 assays and flow cytometry were used to assess cell proliferation and apoptosis, respectively. The dual luciferase reporter assays were employed to investigate the interaction between circVRK1 and miR-4428, with groups categorized as WT-circVRK1+miR-NC, WT-circVRK1+miR-4428, MUT-circVRK1+miR-NC, and MUT-circVRK1+ miR-4428. Western blotting was utilized to detect the expression levels of Ki-67, cleaved caspase-3, and cleaved caspase-9 proteins. Results: Compared to the pcDNA group, circVRK1 expression was up-regulated in the pcDNA-circVRK1 group (P<0.05). Compared to transfection with pcDNA or anti-miR-NC, transfection with pcDNA-circVRK1 or anti-miR-4428 led to decreased cell viability and Ki-67 protein levels in KOCL44 cells (P<0.05), and increased apoptosis rates and levels of cleaved caspase-3 and cleaved caspase-9 (P<0.05). circVRK1 was found to negatively regulate miR-4428 expression, with this effect observed only in the WT-circVRK1 group. miR-4428 levels were lower in the pcDNA-circVRK1 group compared to the pcDNA group (P<0.05) and higher in the si-circVRK1 group compared to the si-NC group (P<0.05). Co-transfection with pcDNA-circVRK1+miR-4428 resulted in increased cell viability (P<0.05) and Ki-67 expression (P<0.05), and decreased apoptosis rates and levels of cleaved caspase-3 and cleaved caspase-9 (P<0.05) compared to co-transfection with pcDNA-circVRK1+miR-NC. Conclusion: Overexpression of circVRK1 reduces the proliferation ability of acute ALL cells and induces cell apoptosis by downregulating miR-4428 expression.

Exercise-activated hepatic autophagy combined with silymarin is associated with suppression of apoptosis in rats subjected to dexamethasone induced- fatty liver damage.

AIM: There is a need for effective treatments for non-alcoholic fatty liver disease (NAFLD) that are economically inexpensive, and have few side effects. The present study aimed to investigate exercise training and silymarin on hepatocyte death factors in rats with liver damage. METHODS: Forty-nine male Wistar rats were assigned to seven groups: sedentary control, fatty liver control (DEX), fatty liver + high-intensity interval training (HIIT), fatty liver + HIIT + silymarin (HIIT + SILY), fatty liver + continuous training (CT), fatty liver + CT + silymarin (CT + SILY), and fatty liver + silymarin (SILY). A subcutaneous injection of dexamethasone for 7 days was used to induce fatty liver in rats. Masson's trichrome and hematoxylin-eosin staining were done to evaluate hepatic injury. The hepatocyte apoptosis was determined by TUNEL assay. Real-Time PCR was conducted to evaluate the gene expressions of caspase-9, adenosine monophosphate-activated protein kinase (AMPKα1), mitofusin 2 (Mfn2), and damage-regulated autophagy modulator (DRAM). Liver tissue changes and serum levels of liver enzymes were also evaluated. RESULTS: Liver apoptosis was decreased in the CT, HIIT, HIIT + SILY and CT + SILY groups compared to the DEX group. Both continuous and high-intensity training models produced beneficial alterations in liver morphology and hepatic injuries that were significant in exercise training + silymarin group. This impact was accompanied by increased AMPKα1 and DRAM gene expression and decreased caspase-9 and Mfn2 gene expression. Liver enzyme levels were high in the DEX group and treatment with silymarin significantly reduced it. CONCLUSION: Silymarin supplementation combined with interval or continuous training substantially improves DEX-induced hepatic steatosis and hepatocyte injury mostly through suppressing liver apoptosis and upregulating autophagy, which may provide a novel perspective for NAFLD treatment.

[Ergosterol peroxide inducing apoptosis of human hepatocellular carcinoma by regulating mitochondrial apoptosis pathway].

This study aims to investigate the effect of ergosterol peroxide(EP) on the apoptosis of human hepatocellular carcinoma and its mechanism of action. The cell viability of HepG2 and SK-Hep-1 cells with 0(blank control), 2.5, 5, 10, 20, 40, and 80 µmol·L~(-1) of EP after 24, 48, and 72 h of action was detected by using CCK-8 assay, and the half inhibitory concentrations(IC_(50)) at 24, 48, and 72 h were calculated. Formal experiments were performed to detect the effect of EP on intracellular reactive oxygen species(ROS) using DCFH-DA staining, the effect of EP on intracellular mitochondrial membrane potential using JC-1 staining, the number of apoptotic cells using Annexin V-FITC/PI double-staining after HepG2 cells were co-cultured with 0(blank control), 10, 20, 40 µmol·L~(-1) EP for 48 h. The effects of EP at different concentrations on apoptotic morphology were detected using AO/EB staining. The effects of different concentrations of EP on the protein expression of mitochondrial apoptosis pathway-related proteins B cell lymphoma 2(Bcl-2), cytochrome C(Cyt-C), Bcl-2-related X protein(Bax), caspase-3, cleaved caspase-3, caspase-9, and cleaved caspase-9 were examined by using Western blot. The results showed that different concentrations of EP could inhibit the proliferation of hepatocellular carcinoma with concentration-and time-dependent trends. Compared with the blank control group, the ROS level in the EP-treated group increased significantly(P<0.05). The mitochondrial membrane potential decreased significantly(P<0.05). The total apoptosis rate increased significantly(P<0.05). The expression of Bcl-2 protein was significantly down-regulated, and the expression of Cyt-C, Bax, cleaved caspase-9, and cleaved caspase-3 were significantly up-regulated(P<0.05). In summary, EP may inhibit the proliferation of hepatocellular carcinoma by modulating the mitochondria-mediated apoptosis pathway and induce apoptosis.

Targeted delivery of bee venom to A549 lung cancer cells by PEGylate liposomal formulation: an apoptotic investigation.

This study focused on developing an optimal formulation of liposomes loaded with bee venom (BV) and coated with PEG (BV-Lipo-PEG). The liposomes were characterized using dynamic light scattering, transmission electron microscopy, and Fourier transform infrared spectroscopy. Among the liposomal formulations, F3 exhibited the narrowest size distribution with a low PDI value of 193.72 ± 7.35, indicating minimal agglomeration-related issues and a more uniform size distribution. BV-Lipo-PEG demonstrated remarkable stability over 3 months when stored at 4 °C. Furthermore, the release of the drug from the liposomal formulations was found to be pH-dependent. Moreover, BV-Lipo-PEG exhibited favorable entrapment efficiencies, with values reaching 96.74 ± 1.49. The anticancer potential of the liposomal nanocarriers was evaluated through MTT assay, flow cytometry, cell cycle analysis, and real-time experiments. The functionalization of the liposomal system enhanced endocytosis. The IC50 value of BV-Lipo-PEG showed a notable decrease compared to both the free drug and BV-Lipo alone, signifying that BV-Lipo-PEG is more effective in inducing cell death in A549 cell lines. BV-Lipo-PEG exhibited a higher apoptotic rate in A549 cell lines compared to other samples. In A549 cell lines treated with BV-Lipo-PEG, the expression levels of MMP-2, MMP-9, and Cyclin E genes decreased, whereas the expression levels of Caspase3 and Caspase9 increased. These findings suggest that delivering BV via PEGylated liposomes holds significant promise for the treatment of lung cancer.

Thymoquinone Increases the Sensitivity of SW-480 Colon Cancer Cells to 5-Fluorouracil.

Background: Studies have concentrated on the therapeutic potential of thymoquinone (TQ), a natural polyphenol, in diverse malignancies, such as colorectal cancer. Nevertheless, the precise mechanisms of TQ-mediated anticancer properties are not yet fully elucidated. Objective: The present study has been designed to scrutinize the impact of TQ on 5-fluorouracil (5-FU)-mediated apoptosis in SW-480 cells. Materials and Methods: SW-480 cells were treated with TQ, 5-FU, and a combination of TQ + 5-FU. MTT assay was employed to assess cell viability. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to evaluate apoptotic markers comprising Bcl-2, Bax, and caspase-9 expression levels. The γ-H2AX protein expression was assessed by western blotting, and Annexin V flow cytometry was implemented to determine the apoptosis rate. Results: 5-FU significantly reversed the cell proliferation in a dose-dependent circumstance. The concurrent administration of TQ and 5-FU led to a substantial inhibition of cell growth in comparison to single treatments (p < 0.05). TQ also facilitated apoptosis via upregulating Bax and caspase-9 proapoptotic markers and suppressing antiapoptotic mediators, like Bcl-2. In addition, TQ augmented 5-FU-induced apoptosis in SW-480 cells. 5-FU, combined with TQ, increased the protein expression of γ-H2AX in SW-480 cells compared with groups treated with TQ and 5-FU alone. Conclusion: The present study's findings unveil the significance of TQ as a potential therapeutic substance in colorectal cancer, particularly through enhancing 5-FU-induced apoptosis.

Potential use of silver nanoparticles green synthesized using Astragalus spinosus extract for treating cystic echinococcosis.

The present investigation aims to develop and evaluate silver nanoparticles (AgNP) synthesized through environmentally friendly methods and to assess their effectiveness against hydatid cysts through in vitro, ex vivo, and in vivo experiments. The green synthesis of ANP was accomplished using the precipitation technique with Astragalus spinosus extract. The in vitro protoscolicidal effects of ANP were evaluated on hydatid cyst protoscoleces (PTS) through eosin exclusion test. The study also investigated the effect of ANP on the gene expression levels of caspase-3 and 9, as well as the external morphology of PTS. The in vivo efficacy was assessed by analyzing the quantity, dimensions, and weight of hydatid cysts in infected mice. Real-time PCR was used to analyze the gene expression levels of antioxidant and inflammatory cytokines. ANP exhibited significant (p < 0.001) in vitro protoscolicidal activity in a dose- and time-dependent manner. Treatment with ANP resulted in creases and protrusions on the plasma membrane, indicating bleb formation and an increase in the expression of caspase-3 and caspase-9 genes. Notably, there was a significant (p < 0.001) reduction in the number, size, and weight of hydatid cysts following ANP treatment. Administration of ANP resulted in a significant increase in the expression of antioxidant genes (glutathione peroxidase and superoxide dismutase) and a notable decrease in oxidative stress markers, as well as in the expression levels of Interleukin-4 (IL-4) and IL-10. Due to its antioxidant and anti-inflammatory properties, ANP shows potential as a scolicidal agent and holds promise in managing hydatid cysts in a mouse model. Nevertheless, further clinical trials are imperative to validate the efficacy of ANP in treating hydatidosis.

Application of Repetitive Sequences in Fish Cell Depletion as a Target for the CRISPR/Cas9 System.

Specific cell depletion is a common means to study the physiological function of cell lineages and tissue regeneration. However, 100% depletion is difficult to achieve with existing cell depletion strategies. With the increasing maturity of CRISPR/Cas9 technology, it is increasingly used for the depletion of various cells. However, even with this technology, it is difficult to complete the depletion of specific gene knockout cells. For this reason, cell depletion with the use of repetitive sequences as the target of CRISPR/Cas9 was explored using zebrafish. All cells were used as the target cells for the first set of experiments. The results showed that injection of a mixture of DANA-gRNA and Cas9 mRNA into zygotes resulted in substantial cell apoptosis. Cells are almost invisible in the embryonic animal pole during the dome stage. The activities of the caspase-3 and caspase-9 proteins and the mRNA level of the P53 gene were significantly increased. Then, primordial germ cells (PGCs) in embryos were used as the target cells in subsequent experiments. To specifically knock out PGCs, we injected the mix of DANA-gRNA, pkop: Cas9 plasmid (the kop promotor allows Cas9 expression only in PGCs), and eGFP-nos3'UTR mRNA into zebrafish fertilized eggs. The results revealed that the activity of the caspase-3 protein was significantly increased, and the mRNA levels of P53, ku70, and ku80 were significantly upregulated, while the number of PGCs decreased gradually. Few PGCs labeled with GFP could be seen 20 h post-fertilization (hpf), and no PGCs could be seen at the germinal ridge 24 hpf. Therefore, the combination of CRISPR/Cas9 technology and repetitive sequences can achieve efficient cell depletion regardless of whether there is generalized expression or expression in specific cells. These results indicate that it is feasible to eliminate cells by using repeat sequences as CRISPR/Cas9 system target sites.

Chimeric Antigen Receptor T Cell with an Inducible Caspase-9 Suicide Gene Eradicates Uveal Melanoma Liver Metastases via B7-H3 Targeting.

PURPOSE: Uveal melanoma (UM) is the most common intraocular malignant tumor. Despite successful treatment of the primary tumor, about 50% of patients will recur with systemic diseases for which there are no effective treatment strategies. Here we investigated the preclinical efficacy of a chimeric antigen receptor (CAR) T-cell-based immunotherapy targeting B7-H3. EXPERIMENTAL DESIGN: B7-H3 expression on primary and metastatic human UM samples and cell lines was assessed by RNA sequencing, flow cytometry, and immunohistochemistry. Antitumor activity of CAR T cells targeting B7-H3 was tested in vitro with UM cell lines, patient-derived organotypic tumor spheroids from patients with metastatic UM, and in immunodeficient and humanized murine models. RESULTS: B7-H3 is expressed at high levels in >95% UM tumor cells in vitro and in vivo. We generated a B7-H3 CAR with an inducible caspase-9 (iCas9) suicide gene controlled by the chemical inducer of dimerization AP1903, which effectively kills UM cells in vitro and eradicates UM liver metastases in murine models. Delivery of iCas9.B7-H3 CAR T cells in experimental models of UM liver metastases demonstrates a durable antitumor response, even upon tumor rechallenge or in the presence of a significant metastatic disease burden. We demonstrate effective iCas9.B7-H3 CAR T-cell elimination in vitro and in vivo in response to AP1903. Our studies demonstrate more effective tumor suppression with iCas9.B7-H3 CAR T cells as compared to a B7-H3-targeted humanized monoclonal antibody. CONCLUSIONS: These studies support a phase I clinical trial with iCas9.B7-H3 CAR T cells to treat patients with metastatic UM.

A Non-Apoptotic Pattern of Caspase-9/Caspase-3 Activation During Differentiation of Human Embryonic Stem Cells into Cardiomyocytes.

In vitro studies have demonstrated that the differentiation of embryonic stem cells (ESCs) into cardiomyocytes requires activation of caspases through the mitochondrial pathway. These studies have relied on synthetic substrates for activity measurements, which can be misleading due to potential none-specific hydrolysis of these substrates by proteases other than caspases. Hence, caspase-9 and caspase-3 activation are investigated during the differentiation of human ESCs (hESCs) by directly assessing caspase-9 and -3 cleavage. Western blot reveals the presence of the cleaved caspase-9 prior to and during the differentiation of human ESCs (hESCs) into cardiomyocytes at early stages, which diminishes as the differentiation progresses, without cleavage and activation of endogenous procaspase-3. Activation of exogenous procaspase-3 by endogenous caspase-9 and subsequent cleavage of chromogenic caspase-3 substrate i.e. DEVD-pNA during the course of differentiation confirmes that endogenous caspase-9 has the potency to recognize and activate procaspase-3, but for reasons that are unknown to us fails to do so. These observations suggest the existence of distinct mechanisms of caspase regulation in differentiation as compared to apoptosis. Bioinformatics analysis suggests the presence of caspase-9 regulators, which may influence proteolytic function under specific conditions.

In Vitro Inhibition of Colorectal Cancer Gene Targets by Withania somnifera L. Methanolic Extracts: A Focus on Specific Genome Regulation.

An approach that shows promise for quickening the evolution of innovative anticancer drugs is the assessment of natural biomass sources. Our study sought to assess the effect of W. somnifera L. (WS) methanolic root and stem extracts on the expression of five targeted genes (cyclooxygenase-2, caspase-9, 5-Lipoxygenase, B-cell lymphoma-extra-large, and B-cell lymphoma 2) in colon cancer cell lines (Caco-2 cell lines). Plant extracts were prepared for bioassay by dissolving them in dimethyl sulfoxide. Caco-2 cell lines were exposed to various concentrations of plant extracts, followed by RNA extraction for analysis. By explicitly relating phytoconstituents of WS to the dose-dependent overexpression of caspase-9 genes and the inhibition of cyclooxygenase-2, 5-Lipoxygenase, B-cell lymphoma-extra-large, and B-cell lymphoma 2 genes, our novel findings characterize WS as a promising natural inhibitor of colorectal cancer (CRC) growth. Nonetheless, we recommend additional in vitro research to verify the current findings. With significant clinical benefits hypothesized, we offer WS methanolic root and stem extracts as potential organic antagonists for colorectal carcinogenesis and suggest further in vivo and clinical investigations, following successful in vitro trials. We recommend more investigation into the specific phytoconstituents in WS that contribute to the regulatory mechanisms that inhibit the growth of colon cancer cells.

Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics.

Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.

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.

Synergetic effects and inhibition mechanisms of the polysaccharide-selenium nanoparticle complex in human hepatocarcinoma cell proliferation.

BACKGROUND: Active components from natural fungal products have shown promising potential as anti-tumor therapeutic agents. In the search for anti-tumor agents, research to overcome the drawbacks of high molecular weight and low bioavailability of pure polysaccharides, polysaccharide-conjugated selenium nanoparticles (SeNPs) has attracted much attention. RESULTS: A novel polysaccharide-selenium nanoparticle complex was produced, in which SeNPs were decorated with polysaccharide obtained from fermented mycelia broth of Lactarius deliciosus (FLDP). Transmission electron microscope, dynamic light scattering, and X-ray photoelectron spectroscopy were utilized to characterize the FLDP-SeNPs; and human hepatocarcinoma cell line (HepG2) was used to assess growth inhibition efficacy. The FLDP-SeNPs that were prepared had a spherical shape with the smallest mean diameter of 32 nm. The FLDP-SeNPs showed satisfactory dispersibility and stability after combination, demonstrating that a reliable consolidated structure had formed. The results revealed that FLDP-SeNPs had notable growth inhibition effects on HepG2 cells. They reduced the membrane potential of mitochondria significantly, increased the generation of reactive oxygen species, enhanced levels of both Caspase-3 and Caspase-9, and led to the nucleus in a wrinkled form. CONCLUSION: The FLDP-SeNPs could exert a synergetic toxicity reduction and inhibition enhancement effect on HepG2 cells by inducing early apoptosis, through mitochondria-mediated cytochrome C-Caspases and reactive oxygen species-induced DNA damage pathways. These results indicate that FLDP-SeNP treatment of HepG2 cells induced early apoptosis with synergetic efficacy, showing that FLDP-SeNPs can be useful as natural anti-tumor agents. © 2024 Society of Chemical Industry.

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.