Cell Apoptosis Rate Research Articles

Therapeutic Effects of Lycopene on Lipopolysaccharide-Induced Acute Lung Injury: Regulation of the PINK1/Parkin Signaling Pathway and Mitochondrial Autophagy

This study investigates the mechanism underlying the therapeutic effects of lycopene (Lyc) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI cell models were established using A549 cells treated with LPS. Cell viability was assessed using cell counting kit-8. ALI cells were treated with Lyc, mitochondrial autophagy inhibitor cyclosporine A (CsA), PTEN-induced kinase 1 (PINK1) activator Valinomycin, and reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC). Apoptosis was evaluated by flow cytometry and JC-1 probe staining. ROS and adenosine triphosphate levels were determined using dichloro-dihydro-fluorescein diacetate staining. Western blot assessed the expression levels of light chain 3 (LC3), Lysosomal-associated membrane protein 1, PINK1, and Parkin. An LPS-induced ALI rat model was treated with different concentrations of Lyc. Lung injury was assessed by hematoxylin and eosin staining. Levels of tumor necrosis factor-α, interleukin-6, and lactate dehydrogenase were measured by ELISA combined with magnetic nanoparticles. In this study, different concentrations of Lyc treatment enhanced cell survival. Additionally, Lyc treatment reduced the cell apoptosis rate, decreased lactate dehydrogenase (LDH) leakage and ROS level in ALI cells, and inhibited the expression of LC3, Beclin-1, PINK1, and Parkin. Consistent effects were seen in Lyc, CsA, Valinomycin, and NAC groups, suggesting similar impacts. In animal experiments, Lyc treatment significantly mitigated edema, inflammation, and autophagy in lung tissues. Furthermore, Lyc exhibited a protective effect on ALI cells by regulating PINK1/Parkin pathway and inhibiting mitochondrial autophagy. Overall, Lyc regulates mitochondrial autophagy in LPS-induced ALI with practical significance for studying its pharmacological mechanism and theoretical implications for understanding autophagy in ALI.

Astragaloside IV enhances the sensitivity of breast cancer stem cells to paclitaxel by inhibiting stemness.

Chemotherapy is one of the common treatments for breast cancer. The induction of cancer stem cells (CSCs) is an important reason for chemotherapy failure and breast cancer recurrence. Astragaloside IV (ASIV) is one of the effective components of the traditional Chinese medicine (TCM) Astragalus membranaceus, which can improve the sensitivity of various tumors to chemotherapy drugs. Here, we explored the sensitization effect of ASIV to chemotherapy drug paclitaxel (PTX) in breast cancer from the perspective of CSCs. The study included both in vitro and in vivo experiments. CSCs from the breast cancer cell line MCF7 with stem cell characteristics were successfully induced in vitro. Cell viability and proliferation were detected using the Cell Counting Kit-8 (CCK-8) and colony formation assays, and flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) methods were performed to detect cell apoptosis. Stemness-related protein expression was determined by western blotting (WB) and immunohistochemistry (IHC). Body weight, histopathology, and visceral organ damage of mice were used to monitor drug toxicity. The expression of stemness markers including Sox2, Nanog, and ALDHA1 was stronger in MCF7-CSCs than in MCF7. PTX treatment inhibited the proliferation of tumor cells by promoting cell apoptosis, whereas the stemness of breast cancer stem cells (BCSCs) resisted the effects of PTX. ASIV decreased the stemness of BCSCs, increased the sensitivity of BCSCs to PTX, and synergistically promoted PTX-induced apoptosis of breast cancer cells. Our results showed that the total cell apoptosis rate increased by about 25% after adding ASIV compared with BCSCs treated with PTX alone. The in vivo experiments demonstrated that ASIV enhanced the ability of PTX to inhibit the growth of breast cancer. WB and IHC showed that ASIV reduced the stemness of CSCs. In this study, the resistance of breast cancer to PTX was attributed to the existence of CSCs; ASIV weakened the resistance of MCF7-CSCs to PTX by significantly attenuating the hallmarks of breast cancer stemness and improved the efficacy of PTX.

Circ0005027 Acting as a ceRNA Affects the Malignant Biological Behavior of Hypopharyngeal Squamous Cell Carcinoma by Modulating miR-548c-3p/CDH1 Axis.

Hypopharyngeal squamous cell carcinoma (HSCC) is a malignant tumor of head and neck. It was verified that circ0005027 was downregulated in HSCC tissues. Here, we aimed to investigate the function and specific regulatory mechanism of circ0005027 in HSCC. Ten pairs tissues of HSCC and adjacent para-cancer were collected. Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) measured circ0005027, miR-548c-3p, and Cadherin 1 (CDH1) mRNA expression. CCK-8 analyzed cell proliferation viability. Flow cytometry assay detected cell cycle and apoptosis rate. Clonal formation assay measured the clonal ability. Transwell detected cell invasion ability. Western blot was performed to detect CDH1, LAST1, p-LAST1, MST1, p-MST1, YAP1, p-YAP1, TAZ and p-TAZ protein level. Dual-luciferase, RIP and RNA pull-down assay identified the target relationship among circ0005027, miR-548c-3p and CDH1. circ0005027 was decreased in tissues and FaDu cells of HSCC. Overexpression of circ0005027 inhibited cell viability, G1-S transition, clonal formation, and invasion and increased cell apoptosis. circ0005027 acted as a ceRNA and decreased circ0005027 enhanced the malignant process of FaDu cells through sponging miR-548c-3p and inhibiting CDH1 expression. Overexpression of CDH1 activated YAP1/TAZ pathway and inhibited the growth of HSCC in vitro. circ0005027 might act as a potential biomarker for the progression and prognosis prediction in HSCC by regulating miR-548c-3p/CDH1/ YAP1/TAZ signaling pathway.

Variable Response of MLL-Rearranged Leukemia Cell Lines to Combinations of Menin, CDK9 and DOT1L Inhibitors

Background: MLL (Mixed Lineage Leukemia) rearrangements occur in approximately 10% of leukemias. Infant leukemia has a higher frequency of MLL rearrangement (MLL-r) than leukemia in other age groups. Despite intensified treatment in infant leukemia, outcomes remain poor. MLL-r leukemia depends on the interaction of MLL fusions with menin, DOT1L, and CDK9. Single-agent treatment with menin, DOT1L, and CDK9 inhibitors has shown promising results in preclinical studies, but their clinical efficacy has been limited by primary and acquired resistance. We hypothesized that combination therapy targeting multiple regulators, including menin, DOT1L, and CDK9, would provide a more effective treatment of MLL-r leukemia. Methods: We performed cell viability experiments on leukemia cell lines (MV4;11, MOLM13, RS4;11, THP1, SEM, KOPN8, NOMO1, HL60, ML2) with single, two and three drug combinations using a menin inhibitor (VTP50469), a DOT1L inhibitor (EPZ5676), and a CDK9 inhibitor (AZD4573). The single drug treatment durations with VTP50469, EPZ5676, and AZD4573 were 10 days, 14 days, and 3 days, respectively. The number of viable cells was determined by trypan blue exclusion staining. After the combination treatment, cells were washed with PBS, placed in fresh media for recovery and counted on day 4 or 6. The effectiveness of the combination treatments was determined using SynergyFinder 3.0 web application. Annexin V/PI and CD11b staining were performed using flow cytometry. To demonstrate MLL-menin binding inhibition, we generated an N-terminal FLAG-tagged MLL expression construct in leukemia cell lines and performed co-immunoprecipitation and western blot analysis. Results:We first determined theIC50 values for each inhibitor separately. Based on the IC50 values of VTP50469 (menin inhibitor), we categorized MLL-r cell lines into three groups: very sensitive (MOLM13 and MV4;11), moderately sensitive (RS4;11 and SEM) and resistant (THP1 and ML2) [Fig. A]. Sensitivity followed a similar pattern (MOLM13 and MV4;11 -sensitive, THP1-resistant) when MLL-r cell lines were treated with EPZ5676 (DOT1L Inhibitor). In contrast, AZD4573 demonstrated consistent efficacy across all MLL-r leukemia cell lines, with THP1 and MOLM13 being particularly sensitive with IC50 values in the low nanomolar (nM) range. After identifying the IC50 values of each inhibitor, we performed viability experiments using two and three drug combinations. The combination of VTP50469 (menin inhibitor) and EPZ5676 (DOT1L inhibitor) was superior in decreasing cell viability in MLL-r leukemia cell lines sensitive to VTP50469 (MV4;11, MOLM13 and RS4;11) but not effective in THP1 cells which are resistant to VTP50469 and EPZ5676 [Fig. B]. Synergy scores analyzed using the ZIP model showed additive effects in MOLM13 and a synergistic response in MV4;11 and RS4;11. After 14-17 days of treatment with EPZ5676 combined with VTP50469, MV4;11, MOLM13 and RS4;11 cells exhibited no recovery from treatment, indicating a profound and persistent antileukemia effect of the combination treatment. Conversely, the combination of VTP5069 (menin inhibitor) and AZD4573 (CDK9 inhibitor) showed a synergistic inhibitory effect on cell viability only in THP1 [Fig. B]. Compared to the single-drug treatments, we observed an increased rate of apoptosis and differentiation in THP1 cells treated with the combination of VTP50469 and AZD4573. The three-drug combination of EPZ5676, VTP50469, and AZD4573 was not superior to the two-drug combinations in any of the MLL-r cell lines examined. To characterize VTP50469 resistance, we analyzed the inhibition of MLL-menin binding using an MLL expression construct in VTP50469 sensitive and resistant cell lines treated with VTP50469 doses ranging between 5-500 nM. Following treatment with VTP50469, the level of expression of the MLL construct correlated with the sensitivity of MLL-r leukemia cell lines to the menin inhibitor. Conclusions: Based on our preclinical in vitro findings, the co-inhibition of menin-DOT1L or menin-CDK9 is a promising approach for the treatment of MLL-r leukemia. The varying responses of different MLL-r leukemia cell lines to drug combination treatment indicate that not all cases of MLL-r leukemia will respond uniformly to treatment combinations. Taken together, our data provide a strategy to determine optimal treatment combinations for personalized treatment of MLL-r leukemia.

Polyethylene microplastics cause apoptosis via the MiR-132/CAPN axis and inflammation in carp ovarian

Microplastics (MPs) are widely distributed pollutants in the environment and accumulate in the aquatic environment due to human activities. Carp, a common edible aquatic organism, has been found to accumulate MPs in body. MicroRNA (miRNAs) is a non-coding short RNA that regulates protein expression by binding to target genes in various physiological processes such as proliferation, differentiation and apoptosis. The ovary is a crucial role in carp reproduction. In this study, we established a model of carp exposed to polyethylene microplastics (PE-MPs) in the aquatic environment to investigate the specific mechanism of PE-MPs causing ovarian injury and the involvement of miR-132/calpain (CAPN) axis. H&E stained sections revealed that PE-PMs induced inflammation in ovarian tissues and impaired oocyte development. TUNEL analysis showed an increased rate of apoptosis in ovarian cells treated with PE-PMs. RT-PCR and Western Blot assays confirmed that exposure to PE-MPs significantly decreased miR-132 expression while increasing CAPN expression at both mRNA and protein levels. The concentration of calcium ions was significantly increased in tissues, leading to CAPN enzyme activity increase. The expression of mitochondrial damage-related genes (bax, AIF, cyt-c, caspase-7, caspase-9, and caspase-3) was higher while the expression of anti-apoptotic genes (bcl-2 and bcl-xl) was lower. Protein levels of bax, AIF, caspase-3, bcl-2 and bcl-xl changed accordingly with the genetic alterations. Additionally, we discovered that PE-MPs can activate the p65 factor through the TRAF6/NF-kB pathway resulting in elevated production of pro-inflammatory factors IL-6, IL-1β and TNF-a which contribute to ovarian inflammation development. This study investigates the impact of PE-MPs on carp ovarian function and provides insights into miRNAs' role and their target genes.

Effect of Nrf2 on brain injury induced by hydraulic shock via regulation of mitophagy and apoptosis.

The specific protective mechanism of mitophagy and Nrf2 in brain injury has not been fully clarified. This study aimed to reveal the effect of Nrf2 on hydraulic shock brain injury in mice, and explore its possible mechanism. Twenty-four Nrf2 knockout (Nrf2-/-) and wild-type mice (WT) of C57BL/6J were randomly divided into two groups: control group (C) and brain injury group (TBI). Hematoxylin-eosin staining (HE) assay was used for the histomorphological observation. The apoptotic state of brain tissue was detected by TUNEL. Mechanical damage in vitro models of glial cells were prepared. The wild-type (WT) and Nrf2 knockout (KO) mice were constructed to investigate the changes of mitophagy and apoptosis-related indicators by Western blotting. The experimental results showed that 24 h after TBI, the tissue structure was highly porous, the cells were highly edema, the neuronal space increased significantly, the neuron degeneration, and the cell vacuolation was obvious. Meanwhile, the number of apoptotic cells and the apoptosis rate of glial cells increased significantly. After injury, the relative expression of Parkin, Pink, Beclin and LC-3II proteins were significantly decreased in all mice. The protein expressions of Caspase3 and Caspase12 were significantly increased. However, in the TBI group, KO mice were more impaired than WT mice. In conclusion, Nrf2 plays a protective role by promoting mitophagy to inhibit apoptosis in the process of brain injury caused by hydraulic shock in mice, which provides a new idea for the effective treatment of brain injury.

Exosomes from adipose-derived stem cells restore fibroblast function and accelerate diabetic wound healing

BackgroundDiabetes is common yet challenging chronic disease, that affects a wide range of people around the world. Complex cellular environments around diabetic wounds tend to damage the function of effector cells, including vascular endothelial cells (VECs), fibroblasts and epithelial cells. This study aims to analyze the differences between diabetic wounds and normal skin as well as whether adipose-derived stem cell (ADSC) exosome could promote healing of diabetic wound. MethodsHuman diabetic wounds and normal skin were collected and stained with HE, Masson, CD31 and 8-hydroxy-2 deoxyguanosine immunohistochemical staining. RNA-seq data were collected for further bioinformatics analysis. ADSC exosomes were isolated and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. The effect of ADSC exosomes on diabetic wound healing was assessed on full thickness wounds in mice. To further verify the regulative impact of ADSCs exosomes in high glucose treated fibroblasts, we isolated fibroblasts from normal skin tissue and measured the cell viability, apoptosis rate, proliferation and migration of fibroblasts. In addition, collagen formation and fibrosis-related molecules were also detected. To further disclose the mechanism of ADSC exosomes on the function of high glucose treated fibroblasts, we detected the expression of apoptosis related molecules including BCL2, Bax, and cleaved caspase-3. ResultsHistological observation indicated that perilesional skin tissues from diabetic patients showed structural disorder, less collagen disposition and increased injury compared with normal skin. Bioinformatics analysis showed that the levels of inflammatory and collagen synthesis related molecules, as well as oxidative stress and apoptosis related molecules, were significantly changed. Furthermore, we found that ADSC exosomes could not only speed up diabetic wound healing, but could also improve healing quality. ADSC exosomes restored high glucose induced damage to cell viability, migration and proliferation activity, as well as fibrosis-related molecules such as SMA, collagen 1 and collagen 3. In addition, we verified that ADSC exosomes downregulated high glucose induced increased apoptosis rate in fibroblast and the protein expression of Bax as well as cleaved caspases 3. ConclusionsThis study indicated that ADSC exosomes alleviated high glucose induced damage to fibroblasts and accelerate diabetic wound healing by inhibiting Bax/caspase 3.

Ropivacaine suppresses the progression of renal cell carcinoma through regulating the lncRNA RMRP/EZH2/CCDC65 axis.

Renal cell carcinoma (RCC) is a common malignancy. Local anesthetics were displayed powerful effects against various cancers. This study aims to probe the functions and molecular mechanism of ropivacaine in RCC. Different concentrations of ropivacaine were performed to administrate RCC cells including 786-O and Caki-1 cells. Cell viability and cell apoptosis were examined using CCK-8 and flow cytometry, respectively. Cell migration and invasion were determined by transwell assay. RMRP and CCDC65 expression was firstly predicted using TCGA dataset and further validated in RCC cells using qRT-PCR and western blot. The interactions among RMRP, EZH2 and CCDC65 were verified by RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assays. Ropivacaine effectively suppressed RCC cell viability, migration and invasion and enhanced cell apoptosis rate. Aberrantly elevated RMRP expression in RCC tissues was predicted by TCGA database. Interestingly, overexpressed RMRP observed in RCC cells could be also blocked upon the administration of ropivacaine. Likewise, RMRP knockdown further strengthened ropivacaine-mediated tumor suppressive effects on RCC cells. In terms of mechanism, RMRP directly interacted with EZH2, thereby modulating the histone methylation of CCDC65 to silence its expression. Moreover, ropivacaine inhibited tumor growth in mice bearing RCC tumor through regulating RMRP/EZH2/CCDC65 axis. In sum up, our work revealed that ropivacaine suppressed capacities of RCC cell viability, migration and invasion through modulating the RMRP/EZH2/CCDC65 axis, which laid the experimental foundation of ropivacaine for clinical application in the future.

Mechanism of the adverse outcome of Chlorella vulgaris exposure to diethyl phthalate: Water environmental health reflected by primary producer toxicity

As a ubiquitous contaminant in aquatic environments, diethyl phthalate (DEP) is a major threat to ecosystems because of its increasing utilization. However, the ecological responses to and toxicity mechanisms of DEP in aquatic organisms remain poorly understood. To address this environmental concern, we selected Chlorella vulgaris (C. vulgaris) as a model organism and investigated the toxicological effects of environmentally relevant DEP concentrations at the individual, physiological, biochemical, and molecular levels. Results showed that the incorporation of DEP significantly inhibited the growth of C. vulgaris, with inhibition rates ranging from 10.3 % to 83.47 %, and disrupted intracellular chloroplast structure at the individual level, while the decrease in photosynthetic pigments, with inhibition rates ranging from 8.95 % to 73.27 %, and the imbalance of redox homeostasis implied an adverse effect of DEP at the physio-biochemical level. Furthermore, DEP significantly reduced the metabolic activity of algal cells and negatively altered the cell membrane integrity and mitochondrial membrane potential. In addition, the apoptosis rate of algal cells presented a significant dose–effect relationship, which was mainly attributed to the fact that DEP pollutants regulated Ca2+ homeostasis and further increased the expression of Caspase-8, Caspase-9, and Caspase-3, which are associated with internal and external pathways. The gene transcriptional expression profile further revealed that DEP-mediated toxicity in C. vulgaris was mainly related to the destruction of the photosynthetic system, terpenoid backbone biosynthesis, and DNA replication. Overall, this study offers constructive understandings for a comprehensive assessment of the toxicity risks posed by DEP to C. vulgaris.

Actinomycin D synergizes with Doxorubicin in triple-negative breast cancer by inducing P53-dependent cell apoptosis.

There are three major subtypes of breast cancer, ER+, HER2+ and triple-negative breast cancer (TNBC), namely ER-, PR-, HER2-. TNBC is the most aggressive breast cancer with poor prognosis and no target drug up to now. Actinomycin D (ActD) is a bioactive metabolite of marine bacteria that has been reported to have antitumor activity. The aim of study is to investigate whether ActD has a synergetic effect on TNBC with Doxorubicin (Dox), the major chemotherapeutic drug for TNBC, and explore the underlying mechanism. TNBC cell lines HCC1937, MDA-MB-436 and nude mice were used in the study. Drug synergy determination, LDH assay, MMP assay, Hoechst 33342 staining, Flow cytometry, Flexible docking and CESTA assay were carried out. The expression of proteins associated with apoptosis was checked by Western blot and siRNA experiments were performed to investigate the role of P53 and PUMA induced by drugs. There was much higher apoptosis rate of cells in the ActD + Dox group than that in ActD group or Dox group. Expression of MDM2 and BCL-2 was reduced while expression of P53, PUMA and BAX were increased in the groups treated with ActD + Dox or Dox compared to the control group. Furthermore, P53 siRNA or PUMA siRNA tremendously abrogated the cell apoptosis in the groups treated by ActD, Dox and ActD + Dox. Flexible docking and CESTA showed that ActD can bind MDM2. ActD had a synergetic effect on TNBC with Dox via P53-dependent apoptosis and it may be a new choice for treatment of TNBC.

Cerebral protective effect of in situ and remote ischemic postconditioning on ischemic stroke rat via the TGFβ1-Smad2/3 signaling pathway

Patients with acute ischemic stroke achieve inadequate benefit due to the short therapeutic window for thrombolysis and the risk of ischemia/reperfusion (IR) injury. Ischemic postconditioning induces endogenous cerebral protection for acute ischemic stroke, although the protective mechanisms associated with ischemic postconditioning haven’t been well clarified. In present study, the rat models of ischemic cerebral stroke with in situ and remote ischemic postconditioning (ISP and RIP) were established successfully. The Zea Longa and the modified neurological severity scoring (mNSS) were carried out to evaluate neurological function in the rats, while the open field test was explored to estimate their autonomic athletic ability. The 2,3,5-riphenyltetrazolium chloride (TTC) staining method was used to measure the size of the infarcts. TUNEL and Nissl's staining were used to detect the apoptosis rate of cells in the ischemic penumbra, with the expression of TGFβ1, Smad2, and Smad3 in the ischemic penumbra and serum detected by immunohistochemical staining, qRT-PCR, Western blots, and ELISA analysis. We showed that application of both types of ischemic postconditioning had cerebral protective effects for the ischemic stroke rats, that included effective reduction in the volume of cerebral infarction, alleviation of apoptosis and inflammation in the ischemic penumbra, and promotion of recovery of neurological function. These effects included significantly enriched gene ontology (GO) terms after RIP intervention that were related to TGFβ1, increased protein levels of TGFβ1 and decreased levels of p-Smad2/3 and smad3 following RIP intervention. We showed that the TGFβ1-Smad2/3 signaling pathway was associated with the cerebral protection of ischemic postconditioning.

Sevoflurane alleviates oxygen-glucose deprivation/reoxygenation-induced damage in HT22 cells by activating the Keap1/Nrf2/ARE pathway to inhibit oxidative stress

Objective: This study aimed to investigate the impact of sevoflurane on oxygen-glucose deprivation/reoxygenation-induced damage in HT22 cells and its associated mechanisms. Methods: HT22 cells were treated with sevoflurane, and an oxygen-glucose deprivation/reoxygenation injury model was established. The HT22 cells were randomly divided into the control group, oxygen-glucose deprivation/reoxygenation group, sevoflurane low-dose group, sevoflurane medium-dose group, and sevoflurane high-dose group. The proliferation of HT22 cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis rate and mitochondrial membrane potential of HT22 cells were determined by flow cytometry. Protein expression levels of B-cell lymphoma-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1) in HT22 cells were examined using Western blot. Reactive oxygen species (ROS) levels were measured with 2',7'-dichlorofluorescin diacetate (DCFH-DA). Malondialdehyde (MDA), glutathione peroxidase (GSH-Px) levels, and superoxide dismutase (SOD) enzyme activity in HT22 cells were determined using assay kits. Results: Compared to controls, OGD/R group had reduced cell viability, mitochondrial potential, Bcl-2, nuclear Nrf2, HO-1, GSH-Px levels, and SOD enzyme activity (p < 0.05), with increased apoptosis, Bax, cytoplasmic Nrf2, ROS, and MDA levels. Sevoflurane groups showed opposite trends (p < 0.05). Conclusion: Sevoflurane can mitigate oxygen-glucose deprivation/reoxygenation-induced damage in HT22 cells, and its mechanism may be related to the activation of the Keap1/Nrf2/ARE pathway to inhibit oxidative stress.

LncRNA ARAP1-AS1 enhances proliferation and impairs apoptosis of lymphoma cells by sponging miR-6867-5p.

Numerous evidence have suggested the vital role of lncRNAs in human tumorigenesis. And lncRNA APAP1-AS1 has been proved to act as an oncogene. Nevertheless, the molecular process underlying ARAP1-AS1 for the lymphoma progression has not been well studied. RT-qPCR was used to ascertain the miR-6867-5p and ARAP1-AS1 in lymphoma cells and tissues. The localization of ARAP1-AS1 was determined via subcellular fractionation analysis. A xenograft model was used to investigate the influence of ARAP1-AS1 in formation of tumor in vivo. In addition, interactions between ARAP-AS1 and miR-6867-5p were tested by bioinformatics analysis, RIP assay, luciferase reporter and Pearson's correlation analysis. Combined with loss-of-function experiments, MTT assays and flow cytometry were performed to evaluate the function of miR-6867-5p and also ARAP-AS1 in proliferation and apoptosis of lymphoma cells, respectively. ARAP1-AS1 was remarkably upregulated in lymphoma cells and tissues, while miR-6867-5p expression was downregulated. Furthermore, high ARAP1-AS1 expression suppressed miR-6867-5p expression in lymphoma cell lines (Raji and CA46), and Pearson's analysis showed negative correlation between ARAP1-AS1 expression and also miR-6867-5p expression. In addition, knockdown of ARAP1-AS1 resulted in weakened cell viability and uplifted apoptosis rate of lymphoma cells (Raji and CA46) as well as a delay in the tumor growth in vivo. Further investigations illustrated that miR-6867-5p inhibitor reversed all above biological activities. LncRNA ARAP1-AS1 served as a tumor-promoter in lymphoma cells by sponging with miR-6867-5p, which may help to provide potential therapeutic target gene for lymphoma patients.

MiR-200a involvement in the biological behavior of hepatoma carcinoma cells by targeting the regulatory expression of mesenchymal-epithelial transition factor

Objective: To study the regulatory effect of miR-200a on mesenchymal-epithelial transition factor (MET) and its impact on the biological behavior of hepatoma carcinoma cells. Method: A luciferase reporter assay was used to determine miR-200a's regulatory impact on MET. Human hepatoma HepG2 cells were divided into a control group, a miR-200a group, a MET overexpression group, and a co-transfection group (miR-200a+MET). After culture, cell proliferation ability, cell migration ability, apoptosis, cell invasion ability, and the expression of MET and apoptosis-related (Bcl-2, Caspase-3, Bax) proteins were detected and observed by cell counting kit-8 (CCK-8), scratch assay, Annexin V-FITC staining, transwell chambers, and western blotting. The two groups were compared using the independent sample t-test. The multiple groups were statistically analyzed using one-way ANOVA. Results: The luciferase experiment showed that miR-200a had target MET. The proliferation rate, number of invasions in cells (55.00 ± 7.21, 85.00 ± 7.94, 164.67 ± 19.22, 104.00± 12.29), scratch healing rate (28.33% ± 5.03%, 61.67% ± 4.04%, 74.67% ± 7.02%, 49.33% ± 9.02%), and expression levels of MET, Bcl-2, and Caspase-3 proteins were lower in the miR-200a group than those in the control group, MET overexpression group, and co-transfection group, while the MET overexpression group had higher indexes than the other three groups, with statistically significant differences between the groups (P <0.05). The apoptosis rate of HepG2 cells and the expression level of Bax protein were higher in the miR-200a group than those in the control group, MET overexpression group, and co-transfection group (19.25% ± 2.98%, 6.80% ± 1.15%, 3.42% ±0.76%, 9.90% ± 2.72%), while the levels of various indexes in the MIF overexpression group were lower than those in the other three groups. The control group and co-transfection group were between the two groups, and the difference between the groups was statistically significant (P <0.05). Conclusion: HepG2 cell proliferation, migration, invasion, and cell apoptosis induction can be inhibited by miR-200a, and the functional mechanism for this may be associated with the miR-200a target's ability to down-regulate MET expression in HepG2 cells.

A novel photosensitizer DTPP-mediated photodynamic therapy induces oxidative stress and apoptosis through mitochondrial pathways in LA795 cells

ObjectiveInvestigation of the effects of 5–5- (4-N, N-diacetoxylphenyl)-10,15,20- tetraphenylporphyrin (DTPP)-mediated photodynamic therapy (PDT) on oxidative stress and mitochondrial apoptosis in LA795 lung cancer cells. MethodsProteomics was used to identify differentially expressed proteins after PDT treatment. The apoptosis rate was determined by flow cytometry. Morphologic observation of apoptosis, reactive oxygen species (ROS) levels, antioxidant indices, nitric oxide (NO) content, mitochondrial membrane potential (MMP), and Caspase- 9 and Caspase-3 were determined by assays; apoptosis-related protein levels of Cytochrome (Cyto) c, Bcl- 2, Bax were determined by Western blot. ResultsTypical apoptosis morphology of LA795 cells was observed after PDT. The cells were mainly in the apoptosis death pathway with high cell apoptosis rates. The proteomics study observed the apoptosis-associated proteins, oxidative stress proteins, antioxidant proteins, the cytoskeletal protein and mitochondrial dysfunction in LA 795 cells. Additional results indicated that PDT could increase levels of ROS, NO; decrease glutathione (GSH) content and MMP; upregulated Bax, Cyto c, and Caspase-3 protein expression, inhibited Bcl-2 protein expression, and further induced cell apoptosis. The effect of DTPP-PDT on lung cancer was: first, mitochondrial Cyto c is released into the cytoplasm, then Caspase- 9 / Caspase-3 was activated, Bcl-2 decreased/Bax increased, initiating cell apoptosis. ConclusionDTPP-PDT could induce oxidative stress and apoptosis via mitochondrial pathways in LA795 cells.

Combined Astragalus, vitamin C, and vitamin E alleviate DEHP-induced oxidative stress and the decreased of insulin synthesis and secretion in INS-1 cells

Di-(2-ethylhexyl)-phthalate (DEHP), a common Phthalic acid ester (PAEs), has been reported to be associated with diabetes mellitus, yet the underlying mechanisms remain unknown. Combined nutrient interventions have been shown to alleviate the diabetic toxicity of DEHP. However, the effects and mechanisms of the combined intervention of Astragalus and vitamins (C and E) are currently unknown. In this study, we investigated the potential mechanisms of DEHP-induced diabetes mellitus through transcriptome analysis and vitro experiments using rat insulinoma cells (INS-1 cells). Furthermore, we explored the protection of the combined Astragalus, vitamin C, and vitamin E on DEHP-induced diabetes mellitus through these mechanisms. INS-1 cells in the logarithmic growth period were exposed to 125 umol/L DEHP followed by high-throughput sequencing analysis. The cell proliferation inhibition rate was determined using MTT assay for each group, and the cell apoptosis rate and intracellular ROS level were measured using flow cytometer. Finally, insulin levels and markers of oxidative stress were detected using ELISA kits in different groups. A total of 372 differentially expressed genes were found between the 125 umol/L DEHP and control groups, subsequent functional enrichment analyses indicated that DEHP induced oxidative stress and disturbed insulin levels. In INS-1 cells, the rate of cell proliferation inhibition, apoptosis, and the degree of oxidative stress increased concentration-dependently with increasing DEHP concentrations, while antioxidant intervention could reverse these changes. Insulin synthesis and secretion decreased after 240 μmol/L DEHP exposure stimulated by 25 mM glucose in INS-1 cells, also could antioxidant intervention alleviate these reductions. Based on these results, the underlying mechanism of DEHP impairing the function of INS-1 cells might be through apoptosis pathways induced by oxidative stress and direct reduction of insulin levels (both synthesis and secretion), while the optimal combination of Astragalus and vitamins (C and E) could exert an alleviating effect.

Norcantharidin-Encapsulated C60-Modified Nanomicelles: A Potential Approach to Mitigate Cytotoxicity in Renal Cells and Simultaneously Enhance Anti-Tumor Activity in Hepatocellular Carcinoma Cells.

The objective of this study was to examine the preparation process of DSPE-PEG-C60/NCTD micelles and assess the impact of fullerenol (C60)-modified micelles on the nephrotoxicity and antitumor activity of NCTD. The micelles containing NCTD were prepared using the ultrasonic method and subsequently optimized and characterized. The cytotoxicity of micelles loaded with NCTD was assessed using the CCK-8 method on human hepatoma cell lines HepG2 and BEL-7402, as well as normal cell lines HK-2 and L02. Acridine orange/ethidium bromide (AO/EB) double staining and flow cytometry were employed to assess the impact of NCTD-loaded micelles on the apoptosis of the HK-2 cells and the HepG2 cells. Additionally, JC-1 fluorescence was utilized to quantify the alterations in mitochondrial membrane potential. The generation of reactive oxygen species (ROS) following micelle treatment was determined through 2',7'-dichlorofluorescein diacetate (DCFDA) staining. The particle size distribution of the DSPE-PEG-C60/NCTD micelles was determined to be 91.57 nm (PDI = 0.231). The zeta potential of the micelles was found to be -13.8 mV. The encapsulation efficiency was measured to be 91.9%. The in vitro release behavior of the micelles followed the Higuchi equation. Cellular experiments demonstrated a notable decrease in the toxicity of the C60-modified micelles against the HK-2 cells, accompanied by an augmented inhibitory effect on cancer cells. Compared to the free NCTD group, the DSPE-PEG-C60 micelles exhibited a decreased apoptosis rate (12%) for the HK-2 cell line, lower than the apoptosis rate observed in the NCTD group (36%) at an NCTD concentration of 75 μM. The rate of apoptosis in the HepG2 cells exhibited a significant increase (49%), surpassing the apoptosis rate observed in the NCTD group (24%) at a concentration of 150 μM NCTD. The HK-2 cells exhibited a reduction in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM) upon exposure to C60-modified micelles compared to the NCTD group. The DSPE-PEG-C60/NCTD micelles, as prepared in this study, demonstrated the ability to decrease cytotoxicity and ROS levels in normal renal cells (HK-2) in vitro. Additionally, these micelles showed an enhanced antitumor activity against human hepatocellular carcinoma cells (HepG2, BEL-7402).

Endogenous ligand of the APJ receptor Apelin-13 inhibits cell apoptosis and oxidative stress of cardiomyocytes.

The present study aimed to investigate the effect of Apelin-13 on nicotine-induced injuries of cardiomyocytes. To establish an H9c2 cell model of nicotine-induced apoptosis, H9c2 cells were divided into the control group, nicotine group, and Apelin-13+nicotine group. The apoptosis rate of H9c2 cells was then detected by flow cytometry. Later, the expressions of indicators related to apoptosis, oxidative stress, and inflammatory responses were measured via Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed that the expression of B-cell lymphoma-2 (Bcl-2) was remarkably down-regulated (P<0.01), while the apoptosis rate and the expressions of apoptosis-related proteins (Bcl-2-associated X protein (Bax) and cysteinyl aspartate specific proteinase-3 (Caspase-3)) were significantly up-regulated (P<0.01) in the nicotine group. However, the variation trends of Bcl-2, Bax, and Caspase-3 in the Apelin-13+nicotine group were contrary to those in the nicotine group (P<0.01). Additionally, the expressions of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) obviously declined (P<0.01), while those of superoxide dismutase 1 (SOD1) and SOD2 dramatically rose in the Apelin-13+nicotine group (P<0.01). Furthermore, Apelin-13 treatment evidently elevated the expressions of phosphorylated protein kinase B (p-AKT) and phosphorylated phosphatidylinositol 3-kinase (PI3K). In conclusion, Apelin-13 inhibits nicotine-induced apoptosis and oxidative stress in H9c2 cells via the PI3K/AKT signaling pathway.

Hematoporphyrin derivative photodynamic therapy induces apoptosis and suppresses the migration of human esophageal squamous cell carcinoma cells by regulating the PI3K/AKT/mTOR signaling pathway.

Esophageal cancer is one of the most common cancer types in humans worldwide. Photodynamic therapy (PDT) is a promising therapeutic strategy for the treatment of cancer. However, its underlying mechanism needs to be studied thoroughly. The present study focused on the antitumor effect and underlying mechanism of the use of hematoporphyrin derivative (HpD)-PDT against human esophageal squamous cell carcinoma cells via regulation of the PI3K/AKT/mTOR signaling pathway. A Cell Counting Kit-8 assay was used to measure cell viability. Migration was evaluated using a wound healing assay. An annexin V-FITC/PI kit was used to determine cell apoptosis rates. Protein expression levels were analyzed via western blotting. Reverse transcription-quantitative PCR was used to detect gene expression levels. A 2',7'-dichlorodihydrofluorescein diacetate kit was chosen to evaluate intracellular reactive oxygen species levels via flow cytometry. Cell viability and migration were decreased in KYSE-150 cells after HpD-PDT treatment. Cellular apoptosis was induced after HpD-PDT treatment, and the same trend was observed for autophagy. Furthermore, the PI3K/AKT/mTOR signaling pathway was inhibited. The viability and migration of KYSE-150 cells were significantly inhibited, and apoptosis was induced more effectively following treatment with a combination of HpD-PDT and the PI3K inhibitor, a final concentration of 20 µM LY294002. In conclusion, HpD-PDT could suppress esophageal cancer cell viability, induce apoptosis and inhibit migration by downregulating the PI3K/AKT/mTOR signaling pathway. Combination of HpD-PDT with PI3K inhibitor (LY294002) could enhance the therapeutic efficacy compared with that demonstrated by HpD-PDT alone. Further studies on combination therapy are required to achieve improved clinical outcomes.

Autophagy and Apoptosis Cross-Talk in Response to Epigallocatechin Gallate in NALM-6 Cell Line

Background: Chemotherapy, the primary treatment for acute lymphoblastic leukemia (ALL), often yields inadequate responses. Epigallocatechin gallate (EGCG) has been shown to significantly affect tumor cells through various mechanisms, including cell cycle arrest, apoptosis, and autophagy. Objectives: The objective of this study is to explore the impact of EGCG on autophagy, apoptosis, and the interplay between them in NALM-6, a pre-B-ALL cell line. Methods: NALM-6 cells were subjected to various concentrations of EGCG for 24 and 48 hours. Additionally, NH4Cl 10 mM was used as an autophagy inhibitor to examine this mechanism. The EGCG effect on cell viability and apoptosis was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), trypan blue exclusion assay, and flow cytometry. Moreover, western blot analysis and real-time PCR were performed to investigate autophagy. Results: Our findings demonstrated that EGCG significantly affected cell proliferation and viability. It reduced cell viability by 55.24 ± 8.43% (P &lt; 0.0001) while inducing apoptosis by 51.04 ± 1.88% (P = 0.006). Furthermore, in the presence of NH4Cl, EGCG led to a 3.92 ± 1.76-fold increase in LC3 protein level (P = 0.001). It also resulted in an approximately 1.34 ± 0.34-fold enhancement in DRAM1 mRNA expression levels (P = 0.013), while reducing of LC3B by 33.3 ± 30.5% (P = 0.008), P62/SQSTM1 by 46.5 ± 28.26% (P &lt; 0.001), and Atg2B by 45.5 ± 16.25% (P &lt; 0.001). However, the inhibition of autophagy did not alter the apoptosis rate in either untreated or EGCG-treated cells. Conclusions: Overall, our findings suggest that EGCG can trigger apoptosis and autophagy in the NALM-6 cell line. However, blockage of autophagy does not appear to impact apoptosis in this cell line.