Resveratrol Suppresses Annulus Fibrosus Cell Apoptosis through Regulating Oxidative Stress Reaction in an Inflammatory Environment

During disc degeneration, the increase of inflammatory cytokines and decrease of disc cell density are two prominent features. Enhanced inflammatory reaction contributes to disc annulus fibrosus (AF) cell apoptosis. In this study, we investigated whether resveratrol can suppress AF cell apoptosis in an inflammatory environment. Rat disc AF cells were cultured in medium with or without tumor necrosis factor-α (TNF-α). Resveratrol was added along with the culture medium supplemented with TNF-α. Caspase-3 activity, cell apoptosis ratio, expression of apoptosis-associated molecules (Bcl-2, Bax, caspase-3, cleaved PARP, and cleaved caspase-3), reactive oxygen species (ROS) content, and the total superoxide dismutase (SOD) activity were measured. Our results showed that TNF-α significantly increased caspase-3 activity and AF cell apoptosis ratio and upregulated gene/protein expression of Bax, caspase-3, cleaved caspase-3, and cleaved PARP, whereas it downregulated the expression of Bcl-2. Moreover, TNF-α significantly increased ROS content but decreased the total SOD activity. Further analysis demonstrated that resveratrol partly attenuated the effects of TNF-α on AF cell apoptosis-associated parameters, decreased ROS content, and increased the total SOD activity in the AF cells treated with TNF-α. In conclusion, resveratrol attenuates inflammatory cytokine TNF-α-induced AF cell apoptosis through regulating oxidative stress reaction in vitro. This study sheds a new light on the protective role of resveratrol in alleviating disc degeneration.

Inflammatory Regulation by TNF-α-Activated Adipose-Derived Stem Cells in the Human Bladder Cancer Microenvironment

Mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs), have the most impressive ability to reduce inflammation through paracrine growth factors and cytokines that participate in inflammation. Tumor necrosis factor (TNF)-α bioactivity is a prerequisite in several inflammatory and autoimmune disease models. This study investigated the effects of TNF-α stimulate on ADSCs in the tumor microenvironment. The RNAseq analysis and cytokines assay demonstrated that TNF-α stimulated ADSCs proliferation and pro-inflammatory genes that correlated to leukocytes differentiation were upregulated. We found that upregulation of TLR2 or PTGS2 toward to IRF7 gene-associated with immunomodulatory and antitumor pathway under TNF-α treatment. In TNF-α–treated ADSCs cultured with the bladder cancer (BC) cell medium, the results showed that apoptosis ratio and OCT-4 and TLR2 genes which maintained the self-renewal ability of stem cells were decreased. Furthermore, the cell survival regulation genes including TRAF1, NF-kB, and IRF7 were upregulated in TNF-α–treated ADSCs. Additionally, these genes have not been upregulated in BC cell medium. A parallel study showed that tumor progressing genes were downregulated in TNF-α–treated ADSCs. Hence, the study suggests that TNF-α enhances the immunomodulatory potential of ADSCs during tumorigenesis and provides insight into highly efficacious MSC-based therapeutic options for BC.

Polyphenols from Broussonetia papyrifera Induce Apoptosis of HepG2 Cells via Inactivation of ERK and AKT Signaling Pathways

The extract of Broussonetia papyrifera has been proved to have antitumor activity. However, the underlying mechanism remains unclear. This study aimed to elucidate the mechanism of apoptosis of HepG2 cells induced by polyphenols from Broussonetia papyrifera (PBPs). The results revealed that PBPs inhibited the proliferation of HepG2 cells in a dose-dependent and time-dependent manner. Flow cytometry analysis showed that PBPs increased the apoptosis ratio of HepG2 cells significantly. PBPs increased intracellular reactive oxygen species (ROS) production and decreased intracellular superoxide dismutase (SOD) level of HepG2 cells. PBPs induced cell cycle arrest at G1 phase. Western blotting showed that PBPs upregulated the ratio of Bax/Bcl-2 and the expression level of Caspase-3, and activated p53 in HepG2 cells. The inhibition of proliferative relative signals (protein kinase B, PKB/AKT) and survival relative signals (extracellular signal-regulated kinase, ERK) were also observed in PBP-treated HepG2 cells. Our findings suggest that apoptosis of HepG2 cells induced by PBPs is mitochondria-mediated via inactivation of ERK and AKT signaling pathways.

Aptamer Functionalized Upconversion Nanotheranostic Agent With Nuclear Targeting as the Highly Localized Drug-Delivery System of Doxorubicin

As a widely used anticancer drug, doxorubicin (DOX) could induce cell death mainly via interfering with DNA activity; thus, DOX could perform therapeutic effects mainly in the cell nucleus. However, most of the reported drug delivery systems lacked the well localization in the nucleus and released DOX molecules into the cytoplasm. Due to formidable barriers formed in the nuclear envelope, only around 1% of DOX could reach the nucleus and keep active. Therefore, DOX molecules were inevitably overloaded to achieve the desired therapeutic efficacy, which would induce serious side effects. Herein, we developed a highly localized drug nanocarrier for in situ release of DOX molecules to their action site where they could directly interfere with the DNA activity. In this work, we used cationic polymer-modified upconversion nanoparticles (UCNPs) as the luminescence core and gene carrier, while aptamers served as the DNA nanotrain to load DOX. Finally, the prepared nanotheranostic agent displayed good targetability, high cell apoptosis ratio (93.04%) with quite lower concentration than the LC50 of DOX, and obvious inhibition on tumor growth.

Ginsenoside Rb3 Alleviates the Toxic Effect of Cisplatin on the Kidney during Its Treatment to Oral Cancer via TGF-β-Mediated Mitochondrial Apoptosis

Objective. The research aimed to confirm the role of the transforming growth factor-β (TGF-β) in cisplatin- (CPT-) evoked kidney toxicity and elucidate the mechanism that ginsenoside Rb3 (Rb3) could alleviate the kidney toxicity by CPT during its treatment to oral cancer via TGF-β-mediated mitochondrial apoptosis. Methods. The model of xenograft nude mice bearing oral carcinoma cells ACC83 was established and treated with CPT and/or Rb3, respectively. Bodyweights of the treated mice were weighed, and the kidney tissues were collected; following, the histopathology and the expression of TGF-β were examined using H&E staining and immunohistochemistry. Afterward, the renal cells GP-293 were treated with CPT and/or Rb3. The expression and phosphoration of TGF-β, Smad2, Smad3, Bcl-2, and Bax in GP-293 cells were detected by Western blotting. The cellular apoptosis and mitochondrial membrane potential were analyzed using flow cytometry. Results. The xenograft nude mice exposure to CPT presented the bodyweight loss, necrotic areas, and the increased expression of TGF in kidney tissue, and Rb3 pretreatment relieved these changes evoked by CPT. In GP-293 cells, CPT administration induced the phosphorylation of Smad2 and Smad3, and Rb3 pretreatment suppressed the induced phosphorylation by CPT. Besides, flow cytometry analysis showed that Rb3 inhibited the CPT-evoked cellular apoptosis ratio and mitochondrial membrane depolarization. The Western blotting test indicated that Rb3 alleviated the cleavage of PARP, caspase 3, caspase 8, and caspase 9, the induction of Bax expression, and inhibition of Bcl-2 expression. Additionally, after treating with the TGF inhibitor of disitertide, Rb3 exhibited no alleviation effects on CPT-evoked cellular apoptosis ratio, inhibition of Bax expression, and induction of Bcl-2 expression in GP-293 cells. Conclusion. Rb3 could alleviate CPT-evoked toxic effects on kidney cells during its treatment to oral cancer via TGF-β-mediated mitochondrial apoptosis.

Chemerin enhances the adhesion and migration of human endothelial progenitor cells and increases lipid accumulation in mice with atherosclerosis

Background The role of adipokines in the development of atherosclerosis (AS) has received increasing attention in recent years. This study aimed to explore the effects of chemerin on the functions of human endothelial progenitor cells (EPCs) and to investigate its role in lipid accumulation in ApoE-knockout (ApoE−/−) mice. Methods EPCs were cultured and treated with chemerin together with the specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 in a time- and dose-dependent manner. Changes in migration, adhesion, proliferation and the apoptosis rate of EPCs were detected. ApoE−/− mice with high-fat diet-induced AS were treated with chemerin with or without SB 203580. Weights were recorded, lipid indicators were detected, and tissues sections were stained. Results The data showed that chemerin enhanced the adhesion and migration abilities of EPCs, and reduced the apoptosis ratio and that this effect might be mediated through the p38 MAPK pathway. Additionally, chemerin increased the instability of plaques. Compared with the control group and the inhibitor group, ApoE−/− mice treated with chemerin protein had more serious arterial stenosis, higher lipid contents in plaques and decreased collagen. Lipid accumulation in the liver and kidney and inflammation in the hepatic portal area were enhanced by treatment with chemerin, and the size of adipocytes also increased after chemerin treatment. In conclusion, chemerin can enhance the adhesion and migration abilities of human EPCs and reduce the apoptosis ratio. In animals, chemerin can increase lipid accumulation in atherosclerotic plaques and exacerbate plaques instability. At the same time, chemerin can cause abnormal lipid accumulation in the livers and kidneys of model animals. After specifically blocking the p38 MAPK pathway, the effect of chemerin was reduced. Conclusions In conclusion, this study showed that chemerin enhances the adhesion and migration abilities of EPCs and increases the instability of plaques and abnormal lipid accumulation in ApoE−/− mice. Furthermore, these effects might be mediated through the p38 MAPK pathway.

High-glucose environment accelerates annulus fibrosus cell apoptosis by regulating endoplasmic reticulum stress

Diabetes mellitus (DM) is an important risk factor of intervertebral disc degeneration. However, how DM affects annulus fibrosus (AF) biology remains unclear. The present study was aimed to investigate the effects and mechanism of high glucose on AF cell biology. Rat AF cells were cultured in baseline medium and culture medium with 0.2 M glucose. The inhibitor 4-PBA was added along with the high glucose culture medium to study the role of endoplasmic reticulum (ER) stress in this process. Compared with the control cells, high glucose significantly increased cell apoptosis ratio and caspase-3/9 activity, up-regulated mRNA/protein expression of Bax and caspase-3/cleaved caspase-3, but down-regulated mRNA/protein expression of Bcl-2. Moreover, high glucose increased mRNA and protein expression of CHOP, ATF-6 and GRP78. However, once ER stress was inhibited by the inhibitor 4-PBA in the high glucose group, cell apoptosis ratio and caspase-3/9 activity were decreased, mRNA/protein expression of Bax and caspase-3/cleaved caspase-3 was down-regulated, but mRNA/protein expression of Bcl-2 was up-regulated. In conclusion, high glucose condition can promote AF cell apoptosis through inducing ER stress. The present study helps us understand the mechanism of disc degeneration in DM patients.

Tripartite Motif Containing 11 Interacts with DUSP6 to Promote the Growth of Human Osteosarcoma Cells through Regulating ERK1/2 Pathway

Tripartite Motif Containing 11 (TRIM11), an E3 ubiquitin ligase, is identified as a carcinogen causing certain human cancers. However, the specific role of TRIM11 is still uncovered in human osteosarcoma (OS) cells. To explore the role of TRIM11 in OS cells, TRIM11 was induced by silencing and overexpression in OS cells using RNA interference (RNAi) and lentiviral vector, respectively. qRT-PCR and western blot were used to examine the transcription and translation levels of the target gene. Cell count kit-8 (CCK-8) assays were established to analyze cell proliferation. Cell apoptosis ratio was determined via flow cytometry. In our analyses, TRIM11 was suggested to be upregulated, and it functioned as a pro-proliferation and antiapoptosis factor in OS cells. Moreover, the extracellular-signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 was used to examine the relationship between TRIM11 and ERK1/2 in OS cells. Results demonstrated that the role of TRIM11 was significantly disrupted by the ERK1/2 inhibitor PD98059. Interestingly, we found TRIM11 overexpression did not affect dual-specificity phosphatase 6 (DUSP6) transcription, but improved its translation in OS cells. Co-immunoprecipitation (Co-IP) analyses revealed that TRIM11 interacted with DUSP6. Importantly, overexpression of TRIM11 enhanced DUSP6 ubiquitination in OS cells. Therefore, TRIM11 might suppress the translation of DUSP6 via improving its ubiquitination. Additionally, TRIM11 silencing in OS cells significantly reduced its tumorigenicity in vivo. Overall, our findings firstly revealed that TRIM11 was an oncogene gene in the growth of OS cells and illustrated its potential function as a target in the treatment of OS.

Effects of Aquaporins1, ENAC Induced by Pro-b Cell Clonal Enhancer Factor on Growth, and Expression in Hypoxia/Reoxygenation Treated Human Non-Small Cell Lung Carcinoma Cells

Objective: Lung cancer (LC) remains to be the most frequent cancer among male patients, nonsmall-cell lung cancer (NSCLC) is the most common type of LC. Here we aim to investigate the effect of Pro-b cell clonal enhancer factor (PBEF) on the growth of human NSCLC cells treated with hypoxia/reoxygenation (H/R). Methods : NSCLC A549 cells were divided into four groups: (1) control group; (2) model group; (3) model+ pCAGGS no-load control group; (4) model+ pCAGGS PBEF group. PCAGGS PBEF plasmid was constructed and transfected into the cells, and the expression of PBEF was detected by qPCR and WB, H/R model was constructed, western blot (WB) was used to detect the expressions of AQP1 and ENaC , and cell apoptosis was detected by flow cytometry. Results: Cell apoptosis ratio was higher in H/R model group than that of normal control group; there was no significant difference in apoptosis ratio between the model group and the no-load control group; while the apoptosis ratio in the overexpressed PBEF group was significantly higher than that of the no-load group. AQP1 and ENaC protein levels were significantly lower in the overexpressed PBEF group than the no-load group, while there was no significant difference among normal control group, the model group and the no-load group. Conclusion: Overexpression of PBEF promotes the apoptosis of H/R human NSCLC cells and inhibites the expression of AQP1 and ENaC proteins, indicating that PBEF may play potential adjuvant therapeutic role in the treatment of NSCLC.

mTOR Signal Pathway Inhibition Promote the Cytotoxicity of Chemotherapy on SHI-1 Cells

Acute myeloid leukemia (AML) is a malignant hematologic disorder. Although current chemotherapy could improve the survival rate of AML patients, some patient would suffer from relapse and had bad therapeutic response. Nowadays, many studies have found that the abnormal expression of mTOR signal pathway is closely related to the occurrence and progression of AML, but the roles of mTOR inhibitor in AML is not elucidated. So, in this paper, we would investigate whether rapamycin, a mTOR inhibitor, could enhance the cytotoxicity of chemotherapy. Rapamycin or chemotherapy (adriamycin plus cytarabine) could inhibit the proliferation and induce apoptosis of SHI-1 cells (the proliferation inhibition rate and apoptosis ratio were 35.4%±2.94% and 13.33%±0.74% respectively in rapamycin group; Chemotherapy group: 49.9%±4.11%, 13.2%±1.11%). When rapamycin were combined with chemotherapy, the proliferation inhibition rate and apoptosis ratio were significantly increased to 60.3%±3.36% and 30%±1.74% respectively (p<0.05) (Fig.1A, Fig.1B). Western blotting shown that chemotherapy could enhance the expression of Bax, Beclin-1 and ATG5, and inhibit the expression of Bcl-2, caspase-3, nevertheless chemotherapy didn't influence the expression of proteins in the mTOR signal pathway. When mTOR signal pathway were inhibited by rapamycin, the proteins of mTOR signal pathway such as mTOR, p-mTOR, P70S6K1, p-P70S61, 4EBP1, p-4EBP1, and the apoptosis related proteins Bcl-2 and caspase3 of SHI-1 cells were decreased, otherwise, the expression of autophagy-related proteins LC3B/II, Beclin-1, ATG5 and apoptosis-related proteins Bax increased. Rapamycin combined with chemotherapy could significantly inhibit the expression of proteins in mTOR signal pathway and enhance the expression of autophagy and apoptosis-related proteins of SHI-1 cells than that treated by rapamycin alone (Fig.1C). In order to explore whether mTOR pathway inhibition could enhance the cytotoxicity of chemotherapy in AML animal model. 1×107 SHI-1 cells were inoculated into NPG mice via tail vein to construct systemic leukemia NPG mice. On the 19th day after inoculation, the ratio of human CD45 and 33 double positive cells in peripheral blood of NPG mice was 1.3%, then NPG mice were randomly divided into four groups, control group didn't had treatment, chemotherapy group was treated by adriamycin (1mg/kg) once every other day for three times and cytarabine (100mg/kg) once a day for five days, rapamycin group was treated by rapamycin (10mg/kg) once a day for five days, and combination group was treated by combination chemotherapy and rapamycin. On the first day (24th day)after treatment, the ratio of human CD33 and CD45 double positive cells in peripheral blood, bone marrow and spleen of NPG mice in each treatment group were: control group (20.3%, 23.6%, 15.6%), chemotherapy group (13.6%, 14.1%, 5.50%), rapamycin group (2.03%, 2.68%, 3.31%), combined group (0.308%, 2.49%, 1.10%) (Fig. 1D). The weight of spleen in NPG mice of combination group were significantly lighter than the weight of spleen of NPG mice of other three groups. Pathological immunohistochemistry showed that the infiltration of human CD45 positive cells in spleen of mice in the combination group was significantly lower than that of the other three groups (Fig. 1E). The median survival time of NPG mice in the control group, chemotherapy group, rapamycin group and combination group were 30.5 days, 35.0 days, 34.0 days and 39.0 days, respectively. The survival time of NPG mice in combination group was significantly longer than that in chemotherapy and rapamycin group (P<0.05), while there was no significant difference between chemotherapy group and rapamycin group (P>0.05) (Fig. 1F). When NPG mice were dead, more neoplasms were found in the NPG mice in control group, the treatment of rapamycin or chemotherapy could significantly decrease the degree of extramedullary infiltration of SHI-1 cells in NPG mice, and fewer neoplasms were found in the NPG mice of combination group(Fig.1G). In conclusion, our studies verified that mTOR signal pathway inhibition by rapamycin could enhance the cytotoxicity of chemotherapy via inhibiting mTOR signal pathway, promoting cell autophagy and inducing apoptosis. mTOR signal pathway may be a potential target for the treatment of AML. Figure 1 Disclosures No relevant conflicts of interest to declare.