Etoposide-induced Apoptosis Research Articles

Developing targeted therapies for neuroblastoma by dissecting the effects of metabolic reprogramming on tumor microenvironments and progression.

Rationale: Synergic reprogramming of metabolic dominates neuroblastoma (NB) progression. It is of great clinical implications to develop an individualized risk prognostication approach with stratification-guided therapeutic options for NB based on elucidating molecular mechanisms of metabolic reprogramming. Methods: With a machine learning-based multi-step program, the synergic mechanisms of metabolic reprogramming-driven malignant progression of NB were elucidated at single-cell and metabolite flux dimensions. Subsequently, a promising metabolic reprogramming-associated prognostic signature (MPS) and individualized therapeutic approaches based on MPS-stratification were developed and further validated independently using pre-clinical models. Results: MPS-identified MPS-I NB showed significantly higher activity of metabolic reprogramming than MPS-II counterparts. MPS demonstrated improved accuracy compared to current clinical characteristics [AUC: 0.915 vs. 0.657 (MYCN), 0.713 (INSS-stage), and 0.808 (INRG-stratification)] in predicting prognosis. AZD7762 and etoposide were identified as potent therapeutics against MPS-I and II NB, respectively. Subsequent biological tests revealed AZD7762 substantially inhibited growth, migration, and invasion of MPS-I NB cells, more effectively than that of MPS-II cells. Conversely, etoposide had better therapeutic effects on MPS-II NB cells. More encouragingly, AZD7762 and etoposide significantly inhibited in-vivo subcutaneous tumorigenesis, proliferation, and pulmonary metastasis in MPS-I and MPS-II samples, respectively; thereby prolonging survival of tumor-bearing mice. Mechanistically, AZD7762 and etoposide-induced apoptosis of the MPS-I and MPS-II cells, respectively, through mitochondria-dependent pathways; and MPS-I NB resisted etoposide-induced apoptosis by addiction of glutamate metabolism and acetyl coenzyme A. MPS-I NB progression was fueled by multiple metabolic reprogramming-driven factors including multidrug resistance, immunosuppressive and tumor-promoting inflammatory microenvironments. Immunologically, MPS-I NB suppressed immune cells via MIF and THBS signaling pathways. Metabolically, the malignant proliferation of MPS-I NB cells was remarkably supported by reprogrammed glutamate metabolism, tricarboxylic acid cycle, urea cycle, etc. Furthermore, MPS-I NB cells manifested a distinct tumor-promoting developmental lineage and self-communication patterns, as evidenced by enhanced oncogenic signaling pathways activated with development and self-communications. Conclusions: This study provides deep insights into the molecular mechanisms underlying metabolic reprogramming-mediated malignant progression of NB. It also sheds light on developing targeted medications guided by the novel precise risk prognostication approaches, which could contribute to a significantly improved therapeutic strategy for NB.

Amino Acid Composition of a Chum Salmon (Oncorhynchus keta) Skin Gelatin Hydrolysate and Its Antiapoptotic Effects on Etoposide-Induced Osteoblasts.

A gelatin hydrolysate with a hydrolysis degree of 13.7% was generated using the skin gelatin of chum salmon (Oncorhynchus keta) and papain-catalyzed enzymatic hydrolysis. The results of analysis demonstrated that four amino acids, namely Ala, Gly, Pro, and 4-Hyp, were the most abundant in the obtained gelatin hydrolysate with measured molar percentages ranging from 7.2% to 35.4%; more importantly, the four amino acids accounted for 2/3 of the total measured amino acids. However, two amino acids, Cys and Tyr, were not detected in the generated gelatin hydrolysate. The experimental results indicated that the gelatin hydrolysate at a dose of 50 µg/mL could combat etoposide-induced apoptosis in human fetal osteoblasts (hFOB 1.19 cells), causing a decrease in the total apoptotic cells from 31.6% to 13.6% (via apoptotic prevention) or 13.3% to 11.8% (via apoptotic reversal). Meanwhile, the osteoblasts exposed to the gelatin hydrolysate showed expression changes for 157 genes (expression folds > 1.5-fold), among which JNKK, JNK1, and JNK3 were from the JNK family with a 1.5-2.7-fold downregulated expression. Furthermore, the protein expressions of JNKK, JNK1, JNK3, and Bax in the treated osteoblasts showed a 1.25-1.41 fold down-regulation, whereas JNK2 expression was not detected in the osteoblasts. It is thus suggested that gelatin hydrolysate is rich in the four amino acids and has an in vitro antiapoptotic effect on etoposide-stimulated osteoblasts via mitochondrial-mediated JNKK/JNK(1,3)/Bax downregulation.

Wip1 phosphatase inhibition enhances etoposide mediated apoptosis by increasing p53 phosphorylation in Rhabdoid tumor cells

Protein phosphatase magnesium-dependent 1 delta (PPM1D/Wip1) is a genotoxic stress-activated serine/threonine phosphatase playing a central role in the timely termination of DNA damage response (DDR). Wip1 is frequently mutated, amplified, and overexpressed in common human cancers and is recognized as an oncogene. Rhabdoid tumors (RT) are a type of soft tissue sarcoma being quite aggressive and resistant to chemotherapy. In this study, the role of Wip1 in the regulation of genotoxic stress-induced apoptosis was studied in RT cell line A204. First PPM1D mRNA levels were analyzed in RT compared to normal tissues by in silico analysis utilizing RNA-seq data generated by TARGET from TCGA Pan Cancer. Second, MCF-7, BJ, and A204 cells were used to test the gene copy number and mRNA levels of Wip1 by qRT-PCR. Small molecule inhibitor GSK2830371 and etoposide were used to target Wip1 and to trigger genotoxic stress, respectively. MTT and AnexinV/7AAD analyzes were used to determine the viability and apoptotic response of A204 cells. Inhibition of Wip1 and total and phosphorylated levels of p53 was demonstrated by WB analysis. Thus, here we showed that Wip1 is overexpressed and amplified in the A204 RT cell line. Wip1 overexpressing A204 cells display resistance to etoposide-induced apoptosis due to decreased phosphorylation of p53. Targeting of Wip1 by GSK2830371 increased the phosphorylation of p53 in response to etoposide treatment. Additionally combined use of GSK2830371 and etoposide enhances p53-mediated apoptosis in A204 cells.

LncRNA PRR34-AS1 knockdown represses neuroinflammation and neuronal death in traumatic brain injury by inhibiting microRNA-498 expression

ABSTRACT Objective Traumatic brain injury (TBI) can result in motor and cognitive dysfunction and is a possible risk factor for the subsequent development of dementia. However, the pathogenesis of TBI remains largely unclear. This study investigated the roles of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in inflammation and neuronal apoptosis following TBI. Methods The lncRNA expression profiles in the cerebral cortices of TBI model mice and sham-operated mice were analyzed using microarray. We focused on an upregulated lncRNA, PRR34-AS1, because of its known modulatory role in apoptosis and inflammation. Results Our findings indicated that the knockdown of PRR34-AS1 inhibited inflammation and neuronal apoptosis and improved long-term neurological function. Using an in vitro, cell-based model of etoposide-induced primary cortical neuronal injury, we demonstrated that PRR34-AS1 levels were higher in injured model cells than in untreated control cells. Silencing of PRR34-AS1 suppressed etoposide-induced apoptosis and the production of inflammatory mediators in primary cortical neurons. PRR34-AS1 directly targets microRNA-498 (miR-498) in primary cortical neurons. Importantly, the inhibition of miR-498 expression counteracted the effects of PRR34-AS1 silencing on neuronal apoptosis and inflammation. Conclusions These findings indicate that PRR34-AS1 may be a useful therapeutic target for TBI.

Mucin 21 confers resistance to apoptosis in an O-glycosylation-dependent manner

Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.

Novel two-photon absorption compounds with different organic cations: facile synthesis, photophysical properties, detection of viscosity, and selective imaging of the endoplasmic reticulum in living cells.

Three novel imidazole-based two-photon absorption compounds bearing different organic cations (1PIPy, 2PIQu, and 3PIIm) were facilely synthesized and fully characterized by 1H NMR, 13C NMR, FT-IR, and HRMS. The linear and nonlinear photophysical properties of the target compounds were systematically investigated in various solvents, supplemented with the density functional theory calculations to shed light on their structure-property relationships. The maximum two-photon action cross-sections (Φ × δmax) were determined to be 22.4-98.2 (CH2Cl2), 9.6-41.3 (DMF), and 3.9-11.8 (H2O) GM. It is found that 3PIIm shows significant viscosity sensitivity with a sharp 27-fold increase in fluorescence intensity. Its fluorescence intensity also exhibits a linear relationship with the viscosity of the media in a logarithmic plot. The Φ × δmax value of 3PIIm in highly viscous glycerol was found to be 107.5 GM. Cytotoxicity tests indicate that these compounds have relatively low cytotoxicity. All the target compounds were successfully characterized by one- and two-photon fluorescence imaging in living cells. The colocalization experiments reveal that 1PIPy and 3PIIm are specially located in the endoplasmic reticulum (ER) with the Pearson's coefficients above 0.90. 3PIIm can also monitor the fluctuation of ER viscosity during etoposide-induced apoptosis.

The Classical Apoptotic Adaptor FADD Regulates Glycolytic Capacity in Acute Lymphoblastic Leukemia.

The Fas-associated death domain (FADD) has long been regarded as a crucial adaptor protein in the extrinsic apoptotic pathway. Despite the non-apoptotic function of FADD is gradually being discovered and confirmed, its corresponding physiological and pathological significance is still unclear. Based on the database of GWAS catalog and GTEx Portal, 17 SNPs associated with leukemia susceptibility were found to be linked to FADD expression. We then investigated a regulatory role of FADD in T-acute lymphoblastic leukemia (T-ALL) using Jurkat cells as a model. Jurkat cells stably depleted of FADD (FADD-/- Jurkat) expression exhibited dampened proliferation, hypersensitivity to Etoposide-induced intrinsic apoptosis whereas near total resistance to TRAIL-induced extrinsic apoptosis. Comparison between wild type and FADD-/- Jurkat cells using iTRAQ-based proteomics revealed considerably altered expression spectrum of genes, and led us to focus on metabolic pathways. Investigation of glycolytic and mitochondrial pathways and relevant enzymes revealed that FADD knockout triggered a metabolic shift from glycolysis to mitochondrial respiration in Jurkat cells. Re-expression of FADD in FADD-/- Jurkat cells partially rescued glycolytic capacity. FADD loss triggers global metabolic reprogramming in Jurkat cells and therefore remains as a potential druggable target for ALL treatment.

LncRNA DDGC participates in premature ovarian insufficiency through regulating RAD51 and WT1

The list of long non-coding RNAs (lncRNAs) that participate in the function of ovarian granulosa cells (GCs) is rapidly expanding, but the mechanisms through which lncRNAs regulate GC function are not yet fully understood. Here, we recognized a minimally expressed lncRNA RP4-545C24.1 (which we named DDGC) in GCs from patients with biochemical premature ovarian insufficiency (bPOI). We further explored the role of lncRNA DDGC in GC function and its contribution to the development of bPOI. Mechanistically, silencing DDGC downregulated RAD51 by competitively binding with miR-589-5p, and this resulted in significant inhibition of DNA damage repair capacity. In addition, decreased expression of DDGC promoted ubiquitin-mediated degradation of Wilms tumor 1 (WT1) protein through interactions with heat shock protein 90 (HSP90), which led to aberrant differentiation of GCs. Moreover, DDGC was able to ameliorate the etoposide-induced DNA damage and apoptosis in vivo. Taken together, these findings provide new insights into the contribution of lncRNAs in POI pathogenesis.

Phospholipase D2 is a positive regulator of sirtuin 1 and modulates p53-mediated apoptosis via sirtuin 1

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent histone deacetylase that plays diverse physiological roles. However, little is known about the regulation of SIRT1 activity. Here, we show that phospholipase D2 (PLD2), but not PLD1, selectively interacts with SIRT1 and increases the deacetylase activity of SIRT1. PLD2 does not interact with the other isozymes of SIRT (SIRT2–7). Two leucine residues in the LXXLL motif (L173 and L174) in the phox domain of PLD2 interact with the region essential for SIRT1 activity. PLD2 stimulates the SIRT1-mediated deacetylation of p53 independent of its lipase activity. In our study, mutagenesis of the LXXLL motif suppressed the interaction of PLD2 with SIRT1 and inhibited SIRT1-mediated p53 deacetylation and p53-induced transactivation of proapoptotic genes. Ultimately, overexpression of wild-type PLD2 but not that of LXXLL-mutant PLD2 protected cells against etoposide-induced apoptosis. Moreover, PLD2 did not protect against apoptosis induced by SIRT1 depletion under genotoxic stress. Collectively, our results suggest that PLD2 is a positive regulator of SIRT1 and modulates p53-mediated apoptosis via SIRT1.

Synergistic anti-cancer effects of silibinin-etoposide combination against human breast carcinoma MCF-7 and MDA-MB-231 cell lines.

Objective(s):Recently, there is a significant focus on combination chemotherapy for cancer using a cytotoxic drug and a phytochemical compound. We investigated the effect of silibinin on etoposide-induced apoptosis in MCF-7 and MDA-MB-231 breast carcinoma cell lines.Materials and Methods:The cytotoxic effects of silibinin and etoposide were determined using MTT assay after 24 and 48 hr incubation with these drugs individually and combined. The mRNA expression of Bax and Bcl2, and protein levels of P53, phosphorylated p53 (P-P53), and P21 were determined using real-time PCR and western blot analysis, respectively. The caspase 9 activity was measured using an ELISA kit. Results:Silibinin and etoposide alone and combined significantly inhibit cell growth in a dose and time-dependent manner in both cell lines. The strongest synergistic effects in terms of MCF-7 cell growth inhibition [combination index (CI) = 0.066] were evident. The silibinin-etoposide combinations cause a much powerful apoptotic death (47% and 40%) compared with each compound individually in MCF-7 and MDA-MB 231 cells, respectively. Additionally, the silibinin-etoposide combinations significantly increased the expression of P53, P-P53, and P21 in MCF-7 cells. Neither silibinin nor etoposide individually increased the level of P53 and P-P53 in MDA-MB-231 cells, but both of them individually and combined increased the level of P21.Conclusion:Since the silibinin-etoposide combination induces apoptosis in both cell lines with and without expression of p53, thus, it is suggested that this combination may be a successful therapeutic strategy for breast cancer regardless of P53 status.

The inhibition of GHR enhanced cytotoxic effects of etoposide on neuroblastoma

Etoposide, a DNA damage-inducing agent, is widely used to treat neuroblastoma. Etoposide binds to and inhibits topoisomerase II, thereby inducing the DNA damage response. However, the underlying mechanism of etoposide resistance in neuroblastoma remains unclear. The results of the present study revealed that etoposide upregulated growth hormone receptor (GHR) expression levels in etoposide-resistant neuroblastoma cells, suggesting that GHR upregulation may be involved in the underlying mechanism of etoposide resistance. Thus, the combined effect of GHR knockdown and etoposide treatment on cell viability, apoptosis and migration in vitro, as well as tumor growth in mouse xenograft models in vivo, was subsequently analyzed. The results of cell viability and colony formation assays demonstrated that GHR knockdown enhanced the inhibitory effects of etoposide on cell viability and sensitized cells to etoposide. The enhanced cell viability was discovered to be, at least in part, due to the increase in etoposide-induced apoptosis following GHR knockdown. Moreover, the knockdown of GHR enhanced the inhibitory effect of etoposide on cell migration. Mouse xenograft studies confirmed the effects of GHR silencing in etoposide-resistant neuroblastoma progression in vivo. Furthermore, the effects of GHR knockdown in etoposide resistance were hypothesized to occur via the inactivation of the MEK/ERK signaling pathway. In conclusion, the results of the present study provided novel insight into the underlying mechanism of etoposide resistance and a potential target for the treatment of etoposide-resistant neuroblastoma.

Glyceraldehyde‐3‐phosphate dehydrogenase protects smooth muscle cells against oxidative/genotoxic stress by activation of the apurinic/apyrimidinic endonuclease I pathway

The role of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a potential regulator of cell death was initially interrogated by our laboratory as it pertains to the protection and stimulation of DNA repair that comes from the direct binding of nuclear GAPDH to apurinic/apyrimidinic endonuclease 1 (Ape1). To further elucidate this mechanism, we set to perturbate the GAPDH system in vascular smooth muscle cells (SMC) using a lentiviral GAPDH-overexpressing vector and cells were treated with etoposide (10-240uM), a pro-oxidant and genotoxic compound. Basal GAPDH and Ape1 protein levels were measured via Western Blotting. Etoposide (20 uM) decreased GAPDH and Ape1 protein levels at 54±7% and 42±7%, respectively. Etoposide increased phospho-histone H2A.X (S139) (double-strand DNA damage marker), and cleaved caspase-3 (Asp 175) (apoptosis marker) levels by 170±17% and 230±15%, respectively. Etoposide induced cell apoptosis in dose-dependent manner (Cell Death Elisa). Etoposide induced appearance of oxidized GAPDH in cell nuclear and cytosolic compartments (immunofluorescence with oxidized GAPDH antibody, 4A1 clone) and Etoposide also markedly suppressed GAPDH-Ape1 binding in the cell nucleus (proximity ligation assay). Cell transfection with GAPDH-overexpressing virus increased GAPDH protein, decreased Etoposide-induced cytotoxicity (live cell assay with Incucyte Sx5/Cytotox Red) and suppressed Etoposide-elicited caspase3/7 activation (Incucyte Caspase 3/7 Red dye). GAPDH overexpression reduced Etoposide-induced apoptosis and decreased phospho-histone H2A.X levels. In summary, we have created an efficient oxidative/genotoxic stress model in vascular SMC to analyze DNA damage and its mechanistic role in cell death and apoptosis-associated pathologies. We show that Etoposide induced a potent cytotoxic/genotoxic stress in SMC including DNA damage, and induction of cell apoptosis and these effects were associated with oxidation of GAPDH, reduction in GAPDH-Ape1 binding and marked Ape1 endonuclease downregulation. GAPDH overexpression prevented Etoposide-induced cytotoxicity and apoptosis potentially via stimulation of Ape1/DNA repair pathway.

Inhibition of protein PMP22 enhances etoposide-induced cell apoptosis by p53 signaling pathway in Gastric Cancer.

Gastric Cancer (GC) is one of the main causes leading to death. PMP22, as a member of the GAS3 family of tetraspan proteins, it is associated with a variety of neurological diseases. Recently, more and more studies have shown that PMP22 play a great role in the physiological processes such as cells adhesion, migration, proliferation and tumorigenesis, but the involvement and functional mechanisms of PMP22 in Gastric carcinoma are not investigated clearly. In this study, we found that the PMP22 was overexpressed in the GC cells and tissue. Knockdown of PMP22 inhibits cell growth. Over-expressed PMP22 inhibits the etoposide-induced apoptosis, meanwhile knockdown of PMP22 promotes the etoposide-induced proliferation suppression, and increases cell apoptosis in GC cells. Furthermore, PMP22 enhanced the inhibition of the p53 transcriptional activities and down-regulated the p53 targeting genes, including p21, BAX and PUMA with or without treatment of etoposide. Finally, our results showed that PMP22 reduced the etoposide-induced tumor growth suppression in nude mice. Taken together, our research provided an anti-apoptotic properties alternative mechanism for PMP22 in gastric carcinoma and suggested PMP22 can be a potential target for the treatment of gastric cancer.

Fluorescent Visualization of Nucleolar G-Quadruplex RNA and Dynamics of Cytoplasm and Intranuclear Viscosity

The nucleolus, the locus of ribosome biogenesis, was found to be the predominant intracellular target of a new fluorescent probe, V-P1. In solution, the probe demonstrated both a selectivity to RNA...

The Parotoid Gland Secretion from Peruvian Toad Rhinella horribilis (Wiegmann, 1833): Chemical Composition and Effect on the Proliferation and Migration of Lung Cancer Cells.

Since Rhinella sp. toads produce bioactive substances, some species have been used in traditional medicine and magical practices by ancient cultures in Peru. During several decades, the Rhinella horribilis toad was confused with the invasive toad Rhinella marina, a species documented with extensive toxinological studies. In contrast, the chemical composition and biological effects of the parotoid gland secretions (PGS) remain still unknown for R. horribilis. In this work, we determine for the first time 55 compounds from the PGS of R. horribilis, which were identified using HPLC-MS/MS. The crude extract inhibited the proliferation of A549 cancer cells with IC50 values of 0.031 ± 0.007 and 0.015 ± 0.001 µg/mL at 24 and 48 h of exposure, respectively. Moreover, it inhibited the clonogenic capacity, increased ROS levels, and prevented the etoposide-induced apoptosis, suggesting that the effect of R. horribilis poison secretion was by cell cycle blocking before of G2/M-phase checkpoint. Fraction B was the most active and strongly inhibited cancer cell migration. Our results indicate that the PGS of R. horribilis are composed of alkaloids, bufadienolides, and argininyl diacids derivatives, inhibiting the proliferation and migration of A549 cells.

Prostate cancer cells modulate the differentiation of THP-1 cells in response to etoposide and TLR agonists treatments.

The aim of this study was to determine the polarization of macrophages in the tumor microenvironment, as well as the effect of soluble factors secreted from these polarized macrophages on etoposide-induced cancer cell apoptosis. We investigated the effect of soluble factors secreted from the supernatant of PC3 cells treated with TLR4 and TLR8 agonists, and etoposide on macrophage polarization at the protein level through flow cytometry and enzyme-linked immunosorbent assay. We further explored the cell cycle distribution and phagocytic activity of THP-1 cells by flow cytometry. To imitate the relationship between cancer cells and tumor-associated macrophages (TAMs), we cocultured macrophages with etoposide-treated PC3 cells. After the incubation, the apoptosis in cancer cells was assessed through FACS analysis and by annexin V and PI staining. Our results demonstrate that protein expression of M1 and M2 markers confirmed the upregulation of M1 markers upon etoposide treatment, and mixed M1/M2 phenotype upon treatment with TLR agonists-treated PC3 supernatant. In coculture methods, our results demonstrate that the apoptosis of etoposide-treated cancer cells increases in the presence of M0 macrophages and THP-1 cells incubated with the supernatant of TLR4 agonists-treated PC3 cells. These results indicate clear protective effects of M0 macrophages and THP-1 cells incubated with the supernatant of PC3 cells treated with TLR4 agonists (THP-1 + SUP + TLR4a) on etoposide-induced cancer cell apoptosis.

Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells.

The apoptotic protease-activating factor 1 (Apaf-1) split luciferase biosensor has been used as a biological tool for the detection of early stage of apoptosis. The effect of doxorubicin in a cell-based assay and the addition of cytochrome c and ATP in a cell-free system have been used to test the functionality of the reporter for the detection of apoptosome formation. Here, our data established a drug- and cytochrome c/ATP-independent way of apoptosis induction relying on the expression of the biosensor itself to induce formation of apoptosome. Overexpression of Apaf-1 constructs led to increased split luciferase activity and caspase-3 activity in the absence of any drug treatment. Caspase-3 activity was significantly inhibited when caspase-9DN was co-overexpressed, while the activity of the Apaf1 biosensor was significantly increased. Our results show that the Apaf-1 biosensor does not detect etoposide-induced apoptosis.

GARP is a key molecule for mesenchymal stromal cell responses to TGF-β and fundamental to control mitochondrial ROS levels.

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenerative medicine and for autoimmune/inflammatory diseases. However, a main hurdle for MSCs‐based therapies is the loss of their proliferative potential in vitro. Here we report that glycoprotein A repetitions predominant (GARP) is required for the proliferation and survival of adipose‐derived MSCs (ASCs) via its regulation of transforming growth factor‐β (TGF‐β) activation. Silencing of GARP in human ASCs increased their activation of TGF‐β which augmented the levels of mitochondrial reactive oxygen species (mtROS), resulting in DNA damage, a block in proliferation and apoptosis. Inhibition of TGF‐β signaling reduced the levels of mtROS and DNA damage and restored the ability of GARP−/lowASCs to proliferate. In contrast, overexpression of GARP in ASCs increased their proliferative capacity and rendered them more resistant to etoposide‐induced DNA damage and apoptosis, in a TGF‐β‐dependent manner. In summary, our data show that the presence or absence of GARP on ASCs gives rise to distinct TGF‐β responses with diametrically opposing effects on ASC proliferation and survival.

MiR-129-5p sensitization of lung cancer cells to etoposide-induced apoptosis by reducing YWHAB.

Background: Lung cancer is the most common cause of death from cancer worldwide and recent studies have revealed that microRNAs play critical roles to regulate lung carcinogenesis. microRNA-129-5p (miR-129-5p) has been reported to regulate cell proliferation and invasion in lung cancer, but its role in lung cancer apoptosis remains unknown.Methods: The expression of miR-129-5p and YWHAB in lung cancer tissues were analyzed from data downloaded from the NCBI Gene Expression Omnibus (GEO) database. Luciferase reporter assay, Western blot and qRT-PCR were used to determine the regulatory effect of miR-129-5p on YWHAB. Cell apoptosis was detected by using the PI/Annexin V Cell Apoptosis Kit. The effect of miR-129-5p and YWHAB on the survival of lung cancer patients was also explored.Results: In this study, by combining the data derived from six GEO database, our results showed that miR-129-5p was downregulated in lung cancer tissues and YWHAB was upregulated in lung cancer patient' serum. A significant negative correlation between miR-129-5p and YWHAB was found in lung cancer tissues. Both the expression of YWHAB and miR-129-5p were associated significantly with prognosis (overall survival) in patients with lung cancer. Overexpression of miR-129-5p promotes VP16-induced lung cancer cell apoptosis and YWHAB was shown to be a direct downstream target of miR-129-5p.Conclusion: Overexpression of expression miR-129-5p contributes to etoposide-induced lung cancer apoptosis by modulating YWHAB.

Isobaric tags for relative and absolute quantitation-based quantitative proteomic analysis of X-linked inhibitor of apoptosis and H2AX in etoposide-induced renal cell carcinoma apoptosis.

Background:X-linked inhibitor of apoptosis (XIAP) is a vital factor in the anti-apoptosis mechanism of tumors and is highly expressed in renal cell carcinoma (RCC). However, the mechanism through which XIAP regulates DNA damage repair is unknown. This study investigated the regulatory mechanism of XIAP in etoposide-induced apoptosis in two Caki-1 cell lines with high or low XIAP expression.Methods:The two cell lines were established using RNA interference technology. The differentially expressed proteins in the two cell lines were globally analyzed through an isobaric tags for relative and absolute quantitation-based quantitative proteomics approach. Proteomic analysis revealed 255, 375, 362, and 5 differentially expressed proteins after 0, 0.5, 3, and 12 h of drug stimulation, respectively, between the two cell lines. The identified differentially expressed proteins were involved in numerous biological processes. In addition, the expression of histone proteins (H1.4, H2AX, H3.1, H3.2, and H3.3) was drastically altered, and the effects of XIAP silencing were accompanied by the marked downregulation of H2AX. Protein-protein interactions were assessed and confirmed through immunofluorescence and Western blot analyses.Results:The results suggested that XIAP may act as a vital cell signal regulator that regulates the expression of DNA repair-related proteins, such as H2AX, and influences the DNA repair process.Conclusions:Given these functions, XIAP may be the decisive factor in determining the sensitivity of RCC cell apoptosis induction in response to chemotherapeutic agents.