Cell Apoptosis Rate Research Articles

Silibinin Induces Both Apoptosis and Necroptosis with Potential Anti-tumor Efficacy in Lung Cancer.

Lung cancer incidence is steadily on the rise, posing a growing threat to human health. The search for therapeutic drugs from natural active substance and elucidating their mechanism have been the focus of anti-tumor research. In our work, Silibinin (SiL) was chosen as a possible substance that could inhibit lung cancer. and its effects on inducing tumor cell death have been studied. CCK-8 analysis and morphological observation were used to assess the cytotoxic impacts of SiL on lung cancer cells in vitro. The alterations in mitochondrial membrane potential (MMP) and apoptosis rate of cells were detected by flow cytometry. The level of lactate dehydrogenase (LDH) release out of cells was measured. The expression changes of apoptosis or necroptosis-related proteins were detected using western blotting. Protein interactions among RIPK1, RIPK3 and MLKL were analyzed using the co-immunoprecipitation technique. In vivo, SiL was evaluated for its antitumor effects using LLC tumor-bearing mice with mouse lung cancer. With an increased dose of SiL, the proliferation ability of A549 cells was considerably inhibited, and the accompanying cell morphology changed. The results of flow cytometry showed that after SiL treatment, MMP levels decreased, and the proportion of cells undergoing apoptosis increased. The proteins associated with apoptosis were upregulated and activated. The amount of LDH released from the cells increased following SiL treatment, accompanied by augmented expression and phosphorylation levels of necroptosis-related proteins. The co-IP assay further confirmed necrosome formation induced by SiL. Furthermore, Necrosulfonamide (an MLKL inhibitor) increased the apoptotic rate of SiL-treated cells and aggravated the cytotoxic effect of SiL, indicating that necroptosis blockade could switch cell death to apoptosis and increase the inhibitory effect of SiL on A549 cells. In LLC-bearing mice, gastric administration of SiL significantly inhibited tumor growth. This study helped clarify the anti-tumor mechanism of SiL against lung cancer, elucidating its role in dual induction of apoptosis and necroptosis. In particular, necroptosis blockade could switch cell death to apoptosis and increase the inhibitory effect of SiL. Our work provided an experimental basis for the research on cell death induced by SiL and revealed its possible applications for improving the management of lung cancer.</p>.

A small-molecule carrier for the intracellular delivery of a membrane-impermeable protein with retained bioactivity.

Intracellular protein delivery has the potential to revolutionize cell-biological research and medicinal therapy, with broad applications in bioimaging, disease treatment, and genome editing. Herein, we demonstrate successful delivery of a functional protein, cytochrome c (CYC), by using a boron cluster anion as molecular carrier of the superchaotropic anion type (B12Br11OPr2-). CYC was delivered into lipid bilayer vesicles as well as living cells, with a cellular uptake ratio approaching 90%. Mechanistic studies showed that CYC was internalized into cells through a permeation pathway directly into the cytoplasm, bypassing endosomal entrapment. Upon carrier-assisted internalization, CYC retained its bioactivity, as reflected by an induced cell apoptosis rate of 25% at low dose (1 µM). This study furbishes a direct protein delivery method by a molecular carrier with high efficiency, confirming the potential of inorganic cluster ions as protein transport vehicles with an extensive range of future cell-biological or biomedical applications.

Effects of PTPN6 Gene Knockdown in SKM-1 Cells on Apoptosis, Erythroid Differentiation and Inflammations

Objective: Protein tyrosine phosphatase non-receptor type 6 (PTPN6) is a cytoplasmic phosphatase that acts as a key regulatory protein in cell signaling to control inflammation and cell death. In order to investigate the role of PTPN6 in hematologic tumor myelodysplastic syndrome (MDS), this study infected SKM-1 cell line (MDS cell line) with packaged H_PTPN6-shRNA lentivirus to obtain H_PTPN6-shRNA SKM-1 stable strain. The effect of PTPN6 knockdown on apoptosis, erythroid differentiation, and inflammations in SKM-1 cell line was examined. Methods: The stable knockdown SKM-1 cell line was validated using qPCR and Western blot assays. The proliferation activity, apoptosi, erythroid differentiation, and inflammatory cytokines in SKM-1 cells were assessed before and after transfection. Results: qPCR confirmed that the expression level of H_PTPN6-shRNA in SKM-1 cells was significantly reduced, and Western blot showed that the protein expression level of H_PTPN6-shRNA in SKM-1 cells was also significantly reduced. The CCK-8 cell viability assay confirmed that stable gene knockdown did not affect cell viability. Flow cytometry revealed that the apoptosis rate of cells in the PTPN6 knockdown group was 0.8%, lower than the 2.7% observed in the empty plasmid group; the expression rate of the erythroid differentiation marker CD235a was 13.2%, lower than the 25.0% observed in the empty plasmid group. The expression levels of the proinflammatory factors IL-6 and IL-8 increased, and the expression levels of the inhibitor factor IL-4 decreased. Conclusions: The PTPN6 gene was successfully knocked down using lentivirus-mediated transduction, and the constructed cell line was validated using PCR and Western blot. The CCK-8 cell viability assay confirmed that stable gene knockdown did not affect cell proliferation viability. Flow cytometry analysis of apoptosis and erythroid differentiation indicated that PTPN6 knockdown inhibits apoptosis and erythroid differentiation in SKM-1 cells and also alters the level of inflammations in the bone marrow microenvironment. It suggests that the PTPN6 gene acts as a tumor suppressor in myelodysplastic syndrome cells, influencing hematopoietic cell apoptosis, erythroid differentiation, and inflammations. This provides a reliable experimental basis for further in-depth studies on the mechanism of PTPN6 in MDS and related pharmacological research.

Assessment of the Role of miR-30a-5p on the Proliferation and Apoptosis of Hair Follicle Stem Cells.

To investigate the role of miR-30a-5p on the proliferation and apoptosis of hair follicle stem cells (HFSCs) and whether the Wnt/β-catenin signaling pathway is involved. HFSCs derived from the vibrissa of mammary rats were obtained by enzymatic digestion, and subsequently the obtained HFSCs were treated with Lipofectamine 2000 cell transfection and divided into normal cell culture group (control), miR-30a-5p overexpression group (miR-30a-5p mimic), miR-30a-5p empty vector group (miR-NC), miR-30a-5p inhibitor group (in-miR-30a-5p), and in-miR-30a-5p empty vector group (in-miR-NC). After transfection, the cell proliferation and apoptosis rates were examined separately. In addition, the mRNA expression of β-catenin, proliferating cell nuclear antigen (PCNA) and apoptosis-related genes (Bax and Bcl-2) were examined. The results of cell proliferation ability showed that in-miR-30a-5p group promoted cell proliferation of HFSCs relative to other groups, along with significant upregulation of gene levels of PCNA. Apoptosis analysis indicated that apoptosis rate was reduced in the in-miR-30a-5p group, and the expression of Bax was suppressed, while that of Bcl-2 was promoted. Wnt/β-catenin signaling pathway investigation revealed a significant increase in the levels of β-catenin in HFSCs in the in-miR-30a-5p group. Downregulation of miR-30a-5p levels inhibited HFSCs apoptosis and simultaneously promoted proliferation, furthermore, the increased expression of β-catenin indirectly confirmed the activation of the Wnt/β-catenin signaling pathway.

Effects of Ovarian Stimulation with Gonadotropins in the Conditions of Chronic Psychosocial Stress on the Quality of Murine Oocyte

Chronic psychosocial stress may negatively affect the female reproductive system. Meanwhile, the effect of ovarian stimulation with gonadotropins during stress on the quality of oocytes remains poorly studied. The purpose of this work was to investigate the effects of chronic psychosocial stress on the quality of murine cumulus-oocyte complexes during natural estrus cycle, as well as during ovarian stimulation with exogenous gonadotropins; the latter is an important part of modern assisted reproductive technologies. The results of the study demonstrate that psychosocial stress does not affect the number of ovulating oocytes, but worsens their quality, i. e. reduces the percentage of mature oocytes. In addition, stressed mice exhibited the increased accumulation of reactive oxygen species in oocytes, which is accompanied by the enhanced rate of apoptosis in cumulus cells. Hormonal stimulation of the ovaries with gonadotropins alleviates the negative changes associated with the psychosocial stress, normalizing the level of reactive oxygen species in oocytes and reducing the rate of apoptosis in cumulus cells.

FHOD3 Promotes the Progression of Lung Cancer by Regulating the Caspase-3-Mediated Signaling Pathway.

Lung cancer causes hundreds of thousands of deaths each year worldwide. FHOD3 was reported to accelerate the progression of brain cancer. However, its role in lung cancer is not clear. This study aimed to investigate the role of FHOD3 in lung cancer. The clinical significance of FHOD3 in lung cancer was analyzed based on the data from the TCGA database. The expression level of FHOD3 was detected by qPCR technology. Cell proliferation was detected by CCK-8 assay, and cell invasion was detected by transwell assay. The activity of caspase-3 was determined by the ELISA method, cell apoptosis was iden-tified by TUNEL assay, and protein expression was measured by western blotting technology. Based on the TCGA data, FHOD3 was overexpressed in tumor tissues compared to the normal tissues. Patients with higher FHOD3 expression exhibited a worse survival rate. The expression levels of FHOD3 in lung cancer cell lines were much higher than that in normal cells. When FHOD3 was knocked down, the ability of cell proliferation and invasion was sig-nificantly inhibited. Cell apoptosis rate was increased reversely. The activity of caspase-3 was increased significantly. In addition, the expression level of cleaved caspase-3 was increased. The expression levels of Bax, caspase-8, and ICAD were also increased significantly. However, the expression of antiapoptotic molecule Bcl-2 was decreased reversely. This suggests that the caspase-3-mediated apoptosis signaling pathway was activated by FHOD3 knockdown. FHOD3 was overexpressed and negatively associated with survival rate in lung cancer patients. FHOD3 regulates cell proliferation, invasion, and apoptosis through the caspase-3-mediated signaling pathway. This study indicates that FHOD3 is an important gene contributing to the progression of lung cancer and might be a new drug target for the therapy of lung cancer.

Comparative hepatoprotective effects of flavonoids-rich fractions from flowers and leaves of Penthorum chinense Pursh in vitro

Ethnopharmacological relevancePenthorum chinense Pursh is a traditional Miao ethnomedicine rich in bioactive components, widely recognized for its hepatoprotective properties. However, the hepatoprotective effects of its flowers and leaves have not been individually elucidated. Aims of the studyThe objective of this study was to isolate and purify flavonoids-rich fractions from the flowers (PFF) and leaves (PLF) of P. chinense, and to assess their potential protective effects against oxidative, alcohol-induced, and free fatty acid (FFA) induced injury in hepatic cells. Materials and methodsThe P. chinense flowers and leaves flavonoids-rich fractions were extracted by the method optimized by response surface methodology, and the extracts were subsequently purified using petroleum ether and microporous column. The physical characteristics and component composition of PFF and PLF were analyzed by FT-IR and UPLC-MS/MS. The hepatoprotective activities of PFF and PLF were evaluated by the alcohol, H2O2, and FFA-induced hepatocyte injury cellular model in vitro. The protective effects of PFF and PLF on the hepatic cells were evaluated by assessing cell apoptosis rate, enzymes activities, mitochondrial membrane potential, and mRNA expression in relevant signaling pathways. ResultsThe results revealed that PFF was mainly composed of pinocembrin, quercitrin and quercetin, while PLF was predominantly composed of quercetin, pinocembrin, and kaempferol and their derivatives. PFF and PLF exhibited distinct effects on increasing the cell proliferation rate, regulating the MDA, GOT and GPT levels, and modulating the mRNA expression in apoptosis and antioxidant pathways in alcohol damaged LO2 cells. PFF exhibited superior efficacy in reducing cell apoptosis in alcohol-damage cells compared to PLF. Both PFF and PLF alleviated mitochondrial stress in H2O2-induced LO2 cells. Additionally, the PFF and PLF attenuated lipid accumulation and activated mRNA expressions in PPARα/ACOX1/CPT-1 lipid metabolism pathways, as well as Nrf2/ARE oxidative stress pathways. ConclusionThis study compared the hepatoprotective activities of flavonoids-rich fractions purified from the flowers and leaves of P. chinense. The results contribute to the enhanced development and utilization of various parts of P. chinense aimed at medical and health food applications.

The Inhibition of Reactive Oxygen Species Modulator 1 Attenuates Sevoflurane-Induced Neural Injury via Reducing Apoptosis and Oxidative Stress.

Sevoflurane causes neural injury by promoting apoptosis and oxidative stress. Reactive oxygen species modulator 1 (ROMO1) regulates apoptosis and oxidative stress, while its role in sevoflurane-induced neural injury remains unclear. This study intended to investigate the effect of ROMO1 knockdown on viability, apoptosis, and oxidative stress in sevoflurane-treated HT22 cells and its downstream pathway. HT22 cells were untreated (blank control), or treated with 1%, 2%, and 4% sevoflurane, respectively. Moreover, HT22 cells were transfected with siROMO1 small interfering RNA (siROMO1) or negative control siRNA (siNC) and then stimulated with 4% sevoflurane for further assays. Sevoflurane dose-dependently decreased cell viability and increased apoptosis rate versus blank control in HT22 cells. Sevoflurane elevated reactive oxygen species (ROS) fluorescence intensity, malondialdehyde (MDA), and lactate dehydrogenase (LDH) release, while reducing superoxide dismutase (SOD) activity in a dose-dependent manner versus blank control in HT22 cells. It also dose-dependently increased the relative mRNA and protein expressions of ROMO1 versus blank treatment in HT22 cells. Moreover, siROMO1 plus 4% sevoflurane increased cell viability, while decreasing apoptosis rate, ROS fluorescence intensity, MDA, and LDH release versus siNC plus 4% sevoflurane in HT22 cells. siROMO1 plus 4% sevoflurane elevated the phosphorylation of protein kinase B (AKT) versus siNC plus 4% sevoflurane in HT22 cells. ROMO1 inhibition reverses sevoflurane-induced neural injury by reducing apoptosis and oxidative stress in HT22 cells. The results indicate that ROMO1 may be a potential target for the management of sevoflurane-induced neural injury.

Co-delivery of Doxorubicin and VEGF siRNA using carcinoembryonic antigen-targeted chitosan nanoparticles for colorectal cancer

Developing a targeted nanocarrier-based therapeutic approach is highly effective in delivering therapeutics to specific cancer cells. Combining two or more therapeutic approaches with different mechanisms of action can effectively maintain their unique benefits while considerably improving cancer treatment. Doxorubicin (DOX) binds to DNA by intercalation and triggers a cascade of biochemical processes in tumor cells leading to death. Anti-VEGF medicines may sensitize cancer cells to chemotherapeutics by limiting blood supply and increasing penetration by disrupting tumor vasculature. In this study, DOX and vascular endothelial growth factor siRNA (VEGF siRNA) loaded chitosan nanoparticles (ChNPs) were prepared by using the ionic gelation method. The DOX-siRNA-ChNPs surface was then functionalized with carcinoembryonic antigen (CEA) targeted monoclonal antibody (DOX-siRNA-ChNPs-CMAb), thereby delivering therapeutic moieties into the cancer milieu. The cytotoxicity and cellular uptake of the developed nanocarrier were tested in-vitro on HT-29, which revealed that DOX-siRNA-ChNPs-CMAb possessed the highest cytotoxicity when compared with other prepared formulations and free DOX. The cellular uptake using fluorescence microscopy displayed a significantly higher uptake of DOX-siRNA-ChNPs-CMAb (targeted) than free DOX and DOX-siRNA-ChNPs (non-targeted). Apoptosis analysis in flow cytometry using annexin-V/PI staining demonstrated a substantially higher apoptosis rate of HT-29 cells using DOX-siRNA-ChNPs-CMAb than DOX-siRNA-ChNPs. Moreover, DOX-siRNA-ChNPs-CMAb were studied in CRC induced animal models, showing that DOX-siRNA-ChNPs-CMAb specifically targets CRC cells without showing organ toxicity. Thus, the novel approach of simultaneous delivery of DOX and siRNA using CMAb conjugated ChNPs demonstrated significant targeting in both in-vitro and in-vivo with minimal organ toxicity.

DNA fragmentation factor 40-based therapeutic approaches for cancer: a review article.

DNA Fragmentation Factor (DFF) is a heterodimer protein involved in DNA fragmentation during apoptosis, which acts as a trigger downstream of caspase-3 activation. DFF40 catalytically active homo-oligomers break down chromosomal DNA. Previous scientific investigations have revealed a link between DFF40 expression changes and various cancers. DFF40 deletion or down-regulation has been observed in some cancers. Consequently, therapeutic strategies involving the DFF40 molecule compensating led to an increased rate of cancer cell apoptosis. In this review article, we aimed to introduce cancers with low expression of this protein first. The second part of this paper focuses on studies that utilized exogenous DFF40 protein produced by recombinant DNA technology and surveyed during in vitro and in vivo tests. Finally, compensation for diminished expression of the mentioned protein via gene therapy-based techniques to make up for this apoptotic molecule's low expression is the topic of thelast part of this review article.

6615 Clinical features and genetic etiology of primary ovarian insufficiency in 46,XX children

Abstract Disclosure: F. Wang: None. X. Yin: None. X. Lan: None. P. Li: None. Purpose: The clinical features of primary ovarian insufficiency in 46,XX children were analyzed, the pathogenic genes of POI were screened, and the genetic etiology of primary ovarian insufficiency in children 46,XX was clarified. Method: The clinical data of 46,XX POI patients were collected, including uterine and ovarian ultrasound, serum follicle-stimulating hormone (FSH), estrogen, anti-mullerian hormone (AMH), chromosomal karyotype analysis, adrenal and thyroid function, and immune function. Whole exome sequencing (WES) and CNV were used to screen POI candidate genes. Novel mutations such as MCM8 gene were used to verify cell function. HE staining, immunohistochemistry, cell fluorescence, and Tunnel method were used to observe the changes in ovarian tissue structure. Construction overexpression plasmids of Mutant MCM8 gene in Hela cells. Result: 1. Clinical features of 46,XX POI: The age of onset of the children was 11.9∼16.1 years old. In addition to POI, the clinical phenotype showed height growth retardation. Among them, 4 cases were short stature, 3 cases were growth hormone deficiency, and 5 cases were backward in bone age. All of them showed elevated FSH (&amp;gt;25 IU/L), and 7 cases had low serum AMH (&amp;lt;0.5 ng/ml). 2.The Screening of POI candidate genes and pathogenicity analysis: 5 of the 10 children carried the mutations of the pathogenic genes with POI, that is, 2 cases (sisters of the same family) carried two novel heterozygous mutations of MCM8 gene, one infant carried two novel heterozygous mutations of CYP19A1 gene, and one case with delayed puberty carried one homozygous mutation of ZSW1M7 gene. 3.The MCM8 mutant were functionally validated: The sisters carried two novel mutations, namely p.C242R and p.S445*. The elder sister (the proband) showed POI, short stature and tumor susceptibility, while the younger sister showed a trend of gradual increase in FSH level and ovarian decline. The follicles of the proband disappeared and fibrous tissue proliferation, the MCM8 expression was lower, and the positive rate of cell apoptosis was higher than normal control. The downregulation of Bax and Capase3 expression in mutant MCM8 of Hela cell was delayed compared with wild type, indicating the persistence of apoptosis. Conclusion: 1. The clinical characteristics of 46,XX POI in children are ovarian hypoplasia, often combined with height growth retardation, biochemical manifestations of hypergonadotropic hypogonadism, decreased serum AMH, and karyotype 46,XX. 2.46,XX POI genetic etiology analysis found that five known POI-related pathogenic genes, namely CYP19A1, MCM8, and ZSW1M7, in which five novel mutations were identified.3.Mutant MCM8 promotes ovarian apoptosis by activating the Bcl-2/Bax/Caspase 3 pathway. Keywords: Primary ovarian insufficiency, short stature, apoptosis Presentation: 6/3/2024

AEBP1 restores osteoblastic differentiation under dexamethasone treatment by activating PI3K/AKT signalling.

Adipocyte enhancer-binding protein 1 (AEBP1) is closely implicated in osteoblastic differentiation and bone fracture; this research aimed to investigate the effect of AEBP1 on restoring osteoblastic differentiation under dexamethasone (Dex) treatment, and its interaction with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Pre-osteoblastic MC3T3-E1 cells were cultured in osteogenic medium and treated by Dex to mimic steroid-induced osteonecrosis cellular model. They were then further transfected with control or AEBP1-overexpressed lentiviral vectors. Finally, cells were treated with the PI3K inhibitor LY294002, with or without AEBP1-overexpressed lentiviral vectors. AEBP1 expression showed a downward trend in MC3T3-E1 cells under Dex treatment in a dose-dependent manner. AEBP1-overexpressed lentiviral vectors increased relative cell viability, alkaline phosphatase (ALP) staining, Alizarin red staining and osteoblastic differentiation markers including osteocalcin (OCN), osteopontin (OPN), collagen type I alpha 1 (COL1A1), runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), but decreased cell apoptosis rate in MC3T3-E1 cells under Dex treatment; besides, AEBP1-overexpressed lentiviral vectors positively regulated p-PI3K and p-AKT expressions. Furthermore, LY294002 treatment decreased relative cell viability, Alizarin red staining, osteoblastic differentiation markers including OCN, OPN, RUNX2 and BMP, increased cell apoptosis rate and did not affect ALP staining in MC3T3-E1 cells under Dex treatment; meanwhile, LY294002 treatment weakened the effect of AEBP1 overexpression vectors on the above cell functions. AEBP1 restores osteoblastic differentiation under Dex treatment by activating the PI3K/AKT pathway.

Magnetic lipid-poly(lactic-co-glycolic acid) nanoparticles conjugated with epidermal growth factor receptor antibody for dual-targeted delivery of CPT-11

To entrap sparingly water-soluble drugs like CPT-11 (irinotecan), the poly(lactic-co-glycolic acid) (PLGA) nanoparticle (NP) is highly favored due to its low cytotoxicity and approval for clinical use. On the other hand, entrapping hydrophobic oleic acid-coated iron oxide magnetic nanoparticles (OMNP) in PLGA NP can provide a nanovehicle for magnetically targeted drug delivery. Our goal in this study is to develop a new dual-targeted magnetic lipid-polymer NP for the delivery of CPT-11. We first co-entrap OMNP and CPT-11 in self-assembled lipid-PLGA NP to prepare OLNP@CPT-11. The OLNP@CPT-11 surface was modified with an epidermal growth factor receptor (EGFR) antibody Cetuximab (CET), which can actively target the overexpressed EGFR on the U87 glioblastoma cell surface. The OLNP-CET@CPT-11 enables dual targeting through both external magnetic guidance and CET-mediated active targeting. The NP was characterized for physicochemical properties using various analytical techniques. In vitro study confirms ligand-receptor interaction results in enhanced endocytosis of OLNP-CET@CPT-11 by U87 cells, which offers increased cytotoxicity and elevated cell apoptosis rates. Furthermore, magnetic guidance of OLNP-CET@CPT-11 to U87 cells can induce cell death exclusively in the magnetically targeted zone. The dual-targeted strategy also provides the best therapeutic efficacy against subcutaneously implanted U87 tumors in nude mice with intravenously delivered OLNP-CET@CPT-11.

Cephalomannine reduces radiotherapy resistance in non-small cell lung cancer cells by blocking the β-catenin-BMP2 signaling pathway

BackgroundThe management of non-small cell lung cancer (NSCLC) often includes the use of radiotherapy, with individual outcomes being impacted by the tumor's response to this treatment modality. Cephalomannine (CPM), a taxane diterpenoid found in Taxus spp, has been found to have anti-tumor activity. This study was aim to the explore the role and mechanism by which CPM affects radiotherapy resistance in NSCLC. MethodsH460 cells were pretreated with different doses of CPM. H460 cells were transfected with β-catenin overexpression plasmids. The cell viability, colony-forming ability, migration ability, and sphere-forming ability and apoptosis of the cells were measured by using CCK-8, colony-forming, transwell, and sphere-forming assay and flow cytometry. Western blot assay was employed to detect the expression of β-catenin and BMP2. ResultsThe cell viability, proliferation, migration and sphere-forming ability of cells in the radiotherapy-resistant (RR) group were significantly higher than those in the radiotherapy-sensitivity (RS) group. Conversely, the apoptosis rate of cells in the RR group was lower than that in the RS group. However, after CPM pretreatment of RR group cells, the above phenomena were reversed in a CPM dose-dependent manner. Subsequently, pretreatment with CPM resulted in a decrease in the expression levels of β-catenin and BMP2 in the RR group. In addition, overexpression of β-catenin mitigated the inhibitory effects of CPM on radiotherapy-resistant NSCLC cells. ConclusionCPM has the potential to decrease radiotherapy resistance in NSCLC cells by inhibiting the β-catenin-BMP2 signaling pathway, promoting apoptosis, and ultimately impeding cell growth.

Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells

Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group (P＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group (P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group (P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group (P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group (P＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group (P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group (P＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

Study on the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells and the intervention effect of N-acetylcysteine

Objective: To investigate the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells (BEAS-2B cells), and to study the intervention effect and mechanism of N-acetylcysteine (NAC) on ultrafine carbon black-induced oxidative damage in BEAS-2B cells. Methods: In March 2023, BEAS-2B cells were used as research object, an in vitro airway model exposed to ultrafine carbon black was constructed. A control group and three carbon black exposure groups (50, 100, 200 μg/ml) were set up, and the cells were treated with corresponding concentrations of ultrafine carbon black for 24 hours. In addition, the experiment was divided into control group, NAC+ control group, 100 μg/ml carbon black exposure group and NAC+ exposure group. The corresponding groups were treated with 2 mmol/L NAC for 1 h and 100 μg/ml ultrafine carbon black for 24 h, respectively. Cell viability was measured by CCK-8 assay. Intracellular reactive oxygen species (ROS) level was detected by chemical fluorescence method. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), as well as the content of malondialdehyde (MDA) were detected by colorimetry. The mRNA and protein expressions of autophagy-related genes[Atg5, Atg7, Beclin1, microtubule-associated protein light chain 3B (LC3B), p62 and lysosome-associated membrane protein 2 (LAMP2) ] and apoptosis-related genes [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase3, Caspase9 and poly (ADP-ribose) polymerase 1 (PARP1) ] were determined by fluorescence quantitative PCR and Western blot. Cell apoptosis was determined by flow cytometry. Results: Compared with the control group, the relative survival rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly decreased, the levels of ROS and MDA were significantly increased, and the activities of SOD, GSH-Px and CAT were significantly decreased (P<0.05). The relative survival rate, ROS and MDA levels, SOD, GSH-Px and CAT activities were significantly correlated with the exposure dose of ultrafine carbon black (r(s)=-0.755, 0.826, 0.934, -0.810, -0.880, -0.840, P<0.05). Compared with the control group, the relative expression levels of Atg5, Atg7, Beclin1, LC3B, p62, LAMP2, Bax, Caspase3, Caspase9, PARP1 mRNA and Atg5, Atg7, Beclin1, LC3BⅡ, p62, LAMP2, Bax, cleaved Caspase3 (C-Caspase3), cleaved Caspase9 (C-Caspase9), cleaved PARP1 (C-PARP1) protein and the ratio of LC3BⅡ/LC3BⅠ in 50, 100 and 200 μg/ml carbon black exposure groups were significantly increased, while the relative expression levels of Bcl-2 mRNA and protein were significantly decreased (P<0.05). The changes of the above indexes were significantly correlated with the exposure dose of carbon black (r(s)=0.892, 0.879, 0.944, 0.892, 0.828, 0.880, 0.814, 0.794, 0.931, 0.918, 0.813, 0.866, 0.774, 0.695, 0.918, 0.761, 0.794, 0.944, 0.833, 0.866, 0.905, -0.886, -0.748, P<0.05). Compared with 100 μg/ml carbon black exposure group, the relative survival rate, the activities of SOD, GSH-Px and CAT in NAC+exposure group were significantly increased, while the levels of ROS and MDA were significantly decreased, and the relative expression levels of LC3B, p62 and Caspase3 mRNA and protein as well as the ratio of LC3BⅡ/LC3BⅠ were significantly decreased, and the differences were statistically significant (P<0.05). Compared with the control group, the apoptosis rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly increased (P<0.05), and there was a significant positive correlation between ultrafine carbon black exposure dose and cell apoptosis rate (r(s)=0.944, P<0.05). While compared with 100 μg/ml carbon black exposure group, the apoptosis rate of NAC+exposure group was significantly decreased, and the difference was statistically significant (P<0.05) . Conclusion: Cell autophagy and apoptosis may be important pathophysiological mechanisms of ultrafine carbon black-induced oxidative damage in BEAS-2B cells. NAC can alleviate the occurrence of BEAS-2B cell damage caused by ultrafine carbon black by regulating oxidative stress and the cascading autophagy and apoptosis pathways.

Astaxanthin attenuates glucose-induced liver injury in largemouth bass: role of p38MAPK and PI3K/Akt signaling pathways

BackgroundAstaxanthin (ASX) has been documented to exert beneficial influence on various processes in fish. Largemouth bass (Micropterus salmoides) serves as a common model for studying glucose-induced liver disease, making it imperative to investigate the regulatory mechanisms underlying its liver health.MethodsLargemouth bass were fed with a control diet (CON), a high carbohydrate diet (HC), or a HC diet supplemented astaxanthin (HCA) for 8-weeks, followed by the glucose tolerance test (GTT). Primary hepatocytes were treated with low glucose and high glucose combined with different concentrations of astaxanthin for 48 h. The histopathology, enzymology, transcriptomics, molecular biology and cell biology were combined to investigate the mechanism of liver injury.ResultsThis study provides evidence for the protective effects of ASX against growth performance reduction and hepatic liver injure in largemouth bass fed HC diet. In GTT, HCA diet exhibited an improvement in glucose tolerance following glucose loading. Although HCA diet did not restore the expression of insulin resistance-related genes in livers at different time during the GTT, the addition of ASX in the long-term HC diet did improve the insulin resistance pathway by regulating the PTP1B/PI3K/Akt signaling pathway. Hepatic transcriptome analyses showed that ASX plays an essential role in the modulation of glucose homeostasis in response to treated with HC diet. In in vitro study, ASX treatment resulted in an exaltation in cell viability and a reduction in the rate of cell apoptosis and reactive oxygen species (ROS). Additionally, astaxanthin was observed to improve apoptosis induced by high-glucose via p38MAPK/bcl-2/caspase-3 signaling pathway.ConclusionsAstaxanthin exhibited a protective effect against apoptosis by regulating p38MAPK/bcl-2/caspase-3 pathway, and ameliorated insulin resistance by activating the PTP1B/PI3K/Akt pathway. This study elucidated the mechanism of astaxanthin in the liver injury of largemouth bass from a new perspective and provided a new target for the treatment of insulin resistance.Graphical

Epicatechin ameliorates palmitate-induced insulin resistance in C2C12 myogenic cells by alleviating oxidative stress and activating the AMPK/ACC pathway

ABSTRACT Epicatechin (EC) is a water-soluble natural organic compound belonging to the flavanol class of compounds. It exhibits various physiological activities such as anti-inflammatory, antioxidant, treatment of mitochondrial diseases, and prevention of cardiovascular diseases.To examineEC’s impact and mechanism on adult myoblasts’ insulin resistance. The effects of various EC concentrations on the viability of C2C12 adult myoblasts were measured, as were glucose consumption and glycogen content; in vitro antioxidant activity of EC was measured; cellular oxidative marker content and apoptosis were measured; and protein expression was detected by Western blot. A model of insulin resistance in C2C12 myogenic cells was established using palmitic acid. EC significantly increased the glucose consumption and glycogen content; EC increased the levels of CAT, SOD and GSH and decreased the levels of NO, MDA and ROS; EC decreased the apoptosis rate of cells; EC treatment upregulated the levels of GLUT4, p-AMPK, p-ACC, NRF2, and HO-1 proteins. These results suggest that EC can regulate the NRF2/HO-1 signaling pathway, enhance GLUT4 transport, activate AMPK/ACC pathway, enhance cellular uptake of glucose, thus effectively improving insulin resistance in C2C12 cells.

Integration of Transcriptomics and Metabolomics Reveals the Antitumor Mechanism of Protopanaxadiol Triphenylphosphate Derivative in Non-Small-Cell Lung Cancer.

The objective of this study was to enhance the membrane permeability and anticancer effectiveness of (20S)-protopanaxadiol (PPD) by introducing triphenylphosphonium into the OH group at the C-3 site. This study shows that the anti-proliferation activity of CTPPPPD, with an IC50 value of 1.65 ± 0.10 μmol/L, was 33-times better than that of PPD (with an IC50 value of 54.56 ± 4.56 μmol/L) and superior to that of cisplatin (with an IC50 value of 1.82 ± 0.25 μmol/L) against A549 cells. Biological examinations suggested that CTPPPPD treatment reduced the growth rate of A549 cells, increased the permeability of cell membranes, and changed the structure of chromosomal DNA in a concentration-dependent manner. Annexin V/PI assay and flow cytometry were employed to detect the effect of CTPPPPD on the apoptosis of A549 cells. The results showed that CTPPPPD could induce the apoptosis of A549 cells, and the apoptosis rate of A549 cells treated with 0, 1.0, 2.0, and 4.0 μM of CTPPPPD for 24 h was 0%, 4.9%, 12.7%, and 31.0%, respectively. The integration of transcriptomics and metabolomics provided a systematic and detailed perspective on the induced antitumor mechanisms. A combined analysis of DEGs and DAMs suggested that they were primarily involved in the central carbon metabolism pathway in cancer, as well as the metabolism of aminoacyl-tRNA biosynthesis, alanine, aspartate, and glutamate. Central carbon metabolism in cancer-related genes, i.e., SLC16A3, FGFR3, LDHA, PGAM1, and SLC2A1, significantly reduced after treatment with CTPPPPD. In particular, the dominant mechanism responsible for total antitumor activity may be attributed to perturbations in the PI3K-AKT, MAPK, and P53 pathways. The findings derived from transcriptomics and metabolomics were empirically confirmed through q-PCR and molecular docking. Further analyses revealed that CTPPPPD could be a promising lead for the development of protopanaxadiol for non-small-cell lung cancer (NSCLC) drugs.

Metformin-loaded chitosan nanoparticles augment silver nanoparticle-induced radiosensitization in breast cancer cells during radiation therapy

Recent research has focused on enhancing tumor response to radiation therapy using radiosensitizers to increase radiation absorption by cancerous tissues. This study utilized silver nanoparticles (AgNPs) as radiation sensitizers and chitosan as a nanocarrier to deliver metformin to breast cancer cells. Metformin-loaded chitosan nanoparticles (Met NPs) and AgNPs were synthesized and characterized. MCF-7 breast cancer cells were pretreated with Met NPs, followed by treatment with AgNPs and irradiation with X-rays at 2, 4, and 8 Gy doses. Cellular cytotoxicity, apoptosis, DNA damage, and 3D spheroid formation were evaluated. The synthesized Met NPs and AgNPs had average diameters of 51.5 ± 9.4 nm and 3.02 ± 0.03 nm, respectively. Cellular cytotoxicity assessment revealed the highest cytotoxicity in MCF-7 cells pretreated with Met NPs, treated with AgNPs, and irradiated with 8 Gy. Flow cytometry analysis demonstrated a 67.58 % apoptosis rate in cells pretreated with Met NPs, compared to 30.42 % in cells pretreated with plain metformin. DAPI staining revealed a 1.8-fold increase in DNA damage in cells pretreated with Met NPs and Ag NPs upon exposure to radiation. The 3D spheroid culture model confirmed a 60 % enhancement in the radiosensitivity of breast cancer cells in the presence of Met NPs and Ag NPs. The combination of Met NPs and Ag NPs represents a promising strategy to improve the therapeutic efficacy of radiation therapy for breast cancer treatment. The delivery of metformin can potentiate the radiosensitizing effects of Ag NPs, offering a novel approach to enhance cancer cells' response to radiation.