Annexin V-propidium Iodide Staining Research Articles

Piperlongumine induces apoptosis and autophagy via the PI3K/Akt/mTOR pathway in KB human cervical cancer cells.

Natural products are continuously being researched to develop safe and effective treatment options for cervical cancer, the fourth most common cancer in women. Piperlongumine (PL), an amide alkaloid mainly present in long pepper, exhibits neuroprotective and anti-cancer properties. However, the specific effect of PL in cervical cancer and the relationship between the anti-cancer pathway and autophagy remain unclear. Therefore, we aimed to investigate PL-induced apoptosis in KB human cervical cancer cells and the relationship between apoptosis and autophagy therein. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and wound-healing assays showed that PL treatment suppressed KB cell viability and proliferation. Apoptosis was identified through 4',6-diamidino-2-phenylindole and annexin V-propidium iodide staining, increased cleaved-poly (ADP-ribose) polymerase and Bcl-2 associated X levels, and decreased B cell lymphoma 2 levels. Acridine orange staining and increased microtubule-associated protein 1A/1B-light chain 3-II and Beclin-1 levels confirmed autophagy. We determined that KB cell-related autophagy exerted cytoprotective effects using the autophagy inhibitors 3-methyladenine and hydroxychloroquine. PL treatment promoted apoptosis by inhibiting the phosphatidylinositol-3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin pathway in KB cells; inhibiting the pathway using PI3K inhibitors increased autophagy. We suggest that PL is a potential natural anticancer agent for cervical cancer treatment.

Plumbagin downregulates UHRF1, p-Akt, MMP-2 and suppresses survival, growth and migration of cervical cancer CaSki cells

Plumbagin is a natural compound known to impede growth of cancerous cells. However, anti-cervical cancer effects of plumbagin and its underlying molecular mechanism still remains elusive. In this study, plumbagin reduced the viability of CaSki cells in a concentration dependent manner and suppressed their colony formation potential. It led to G2/M phase arrest with downregulation of E2F1 and upregulation of p21. Plumbagin reduced mitochondrial membrane potential and concomitantly increased the percentage of apoptotic cells as revealed by annexin V-propidium iodide staining. Real Time PCR and western blotting confirmed that plumbagin induced apoptosis by reducing the expression of pAkt, procaspase 9 and full-length PARP. Furthermore, scratch assay showed that plumbagin suppressed migratory potential of CaSki cells which could be due to the reduced expression and activity of MMP-2 and upregulation of TIMP2. Interestingly, plumbagin also downregulated UHRF1 expression. Transient silencing of UHRF1 like plumbagin, induced G2/M phase arrest, enhanced apoptosis and suppressed metastasis of CaSki cells suggesting the role of UHRF1 in mediating anti-cancer activities of plumbagin. Plumbagin at IC20 (1 μM) interacted synergistically with cisplatin and reduced its IC50 value by 13.23 fold with improved effectivity as revealed by augmented apoptosis in CaSki cells.

Extract of Codiaeum luzonicum Merr. overcomes multidrug resistance in human colon cancer cells by modulating P-glycoprotein

Objective: To investigate anti-multidrug resistance (MDR) activity and safety of the bioactive fraction (CL11) from Codiaeum luzonicum crude leaf extract. Methods: Cytotoxic activity of CL11 against MDR and non- resistant colon cancer cells was assessed using MTT assay. Mode of cell death was investigated by annexin V-propidium iodide staining, TUNEL, and JC-1 assays. To examine mechanism of action, the effect on the expression and function of the MDR-implicated protein P-glycoprotein was tested using Western blotting and calcein assay, respectively. Results: CL11 had an EC50 of 0.18, 1.03 and 38.52 μg/mL against HCT-15, HCT-15/Dox and HCT116, respectively. Cytotoxicity was mediated by inhibition of P-glycoprotein function and expression. The mode of cell death involved mitochondrial membrane depolarization and was mostly non-apoptotic at EC50 concentrations against HCT-15 and HCT-15/Dox. Conclusions: Fraction CL11 of Codiaeum luzonicum induces non- apoptotic cell death in MDR cancer cells by overcoming MDR through inhibition of P-glycoprotein expression and function.

Resveratrol Protects Against Nicotine-Induced Apoptosis by Enhancing Autophagy in BEAS-2B Lung Epithelial Cells

Background: Nicotine (Nic), the major component of tobacco products, can induce apoptosis in lung epithelial cells, and the resulting damage contributes to chronic obstructive pulmonary disease. Apoptosis is closely related to autophagy. Resveratrol (Res) can induce autophagy and inhibit apoptosis. Therefore, the present study investigated whether Nic induces apoptosis of lung epithelial cells by regulating autophagy and the effect of Res on apoptosis of Nic-exposed lung epithelial cells. Methods: The BEAS-2B lung epithelial cell line was used to study the harmful effects of Nic and the potential benefits of Res as well as the underlying mechanisms. Viability and apoptosis were examined using the Cell Counting Kit-8 and annexin V-propidium iodide staining, respectively. The expression of levels of apoptosis-related proteins, autophagy-related proteins, and members of the PI3K/Akt/mTOR pathway was measured by western blotting. Autophagic flux was detected via mRFP-GFP-LC3 double-labeled adenovirus transfection and transmission electron microscopy. Results: Nic significantly reduce the viability and increased the apoptosis of BEAS-2B cells in a concentration-dependent manner. Nic treatment also increased the numbers of autophagosomes in BEAS-2B cells and upregulated LC3II and p62 expression. Moreover, Res at concentration of 2, 10, and 50 μM protected BEAS-2B cells from Nic apoptosis, and the expression of LC3II increased further and p62 decreased in Res pretreatment group. Apart from this, Res reduced Akt and mTOR phosphorylation. Subsequently, upon inhibiting PI3K phosphorylation by PI3K inhibitors, BEAS-2B cell autophagy induced by Res was obviously abolished. Conclusions: Nic-induced BEAS-2B cell apoptosis by inhibiting the late-stage autophagic flux, but Res could protect BEAS-2B cells from the detrimental effects of nicotine by enhancing autophagy via the PI3K/Akt/mTOR pathway. These results will provide an experimental basis for the prevention and treatment of COPD.

In vitro anticancer activity of ethanolic extract of Stoechospermum marginatum against HT-29 human colon adenocarcinoma cells

Colorectal cancer is a one of the leading causes of death globally and its clinical management of cancer involves chemotherapy. Increase in the development of resistance to the drugs used in the cancer treatment and serious side effects associated with chemotherapeutic drugs are the major limitations in cancer therapy. Hence, there exists a huge need to develop safer natural therapeutic products for cancer therapy. In this study, ethanolic extract of Stoechospermum marginatum was evaluated for its anticancer activity. The cytotoxicity of S. marginatum extract was evaluated on HT-29 cells by MTT assay. Trypan blue cell viability was also carried out to evaluate cytotoxicity and antiproliferative effect. The apoptosis-inducing potential of the extract was analyzed by acridine orange and ethidium bromide dual staining method, mitochondrial membrane potential assay and FITC Annexin V-Propidium iodide staining method. The ethanolic extract of S. marginatum showed significant dose-dependent cytotoxicity in HT-29 cells Treatment with S. marginatum extract increased number of apoptotic cells in HT-29 cells and caused damage to mitochondrial membrane potential. The findings of the present study confirmed in vitro anticancer activity of ethanolic extract S. marginatum

Combination of Cytochalasin H and zinc oxide nanoparticles in human breast cancer: an insight into apoptosis study

Nanoparticle-mediated combination therapies have significant impact on improving the therapeutic efficacy of cancer. The aim of the current work was to investigate the apoptotic efficacy of Zinc Oxid nanoparticles (ZnONPs) in combination with Cytochalasin H (CytoH) in human MCF-7 breast cancer cells. The anti cancer activity of combined treatment of CytoH and ZnONPs was performed using MTT assay, Annexin V-propidium iodide staining, Caspase assay, and Hoechst 33342 staining. Quantitative real-time polymerase chain reaction was conducted to detect of p53 as a tumor suppressor gene expression in cancerous cells. Also, measurement of ROS generation was performed using flow cytometry. According to the MTT assay results, the combination of CytoH and ZnONPs significantly decreased the viability of cancer MCF-7 cells than either CytoH or ZnONPs alone as compared with normal HEK293 cells. The experiments, such as caspase 8 and 9 assay and Hoechst 33342 staining, showed that the combined treatment of CytoH and ZnONPs significantly increased the level of cell apoptosis pathway as compared with breast cancer cells treated with CytoH or ZnONPs alone. As demonstrated by annexin V/PI detection assay, the percentage of apoptotic MCF-7 cells was significantly increased, following combined treatment with CytoH and ZnONPs as compared with CytoH or ZnONPs alone. Moreover, by comparing the combination of CytoH and ZnONPs to CytoH and ZnONPs alone the ability of combined treatment to up-regulate p53 gene expression was observed. The results of the present study showed that combination of CytoH and ZnONPs could have a powerful synergistic cytotoxic effect on breast cancer cell line. Moreover, this is the first report indicating a new insight into how CytoH and ZnONPs can enhance apoptosis and cell growth in breast cancer cells.

Kevetrin induces apoptosis in TP53 wild‑type and mutant acute myeloid leukemia cells.

Tumor protein p53 is a key regulator of several cellular pathways, including DNA repair, cell cycle and angiogenesis. Kevetrin exhibits p53-dependent as well as-independent activity in solid tumors, while its effects on leukemic cells remain unknown. The aim of the present study was to analyze the response of acute myeloid leukemia (AML) cell lines (TP53 wild-type: OCI-AML3 and MOLM-13; and TP53-mutant: KASUMI-1 and NOMO-1) to kevetrin at a concentration range of 85–340 µM. The cellular and molecular effects of the treatment were analyzed in terms of cell growth, viability [Annexin V-propidium iodide (PI) staining] and cell cycle alterations (PI staining). Gene expression profiling, western blotting and immunofluorescence were performed to elucidate the pathways underlying kevetrin activity. Pulsed exposure exerted no effect on the wild-type cells, but was effective on mutant cells. After continuous treatment, significant cell growth arrest and apoptosis were observed in all cell lines, with TP53-mutant models displaying a higher sensitivity and p53 induction. Kevetrin also displayed efficacy against TP53 wild-type and mutant primary AML, with a preferential cytotoxic activity against blast cells. Gene expression profiling revealed a common core transcriptional program altered by drug exposure and the downregulation of glycolysis, DNA repair and unfolded protein response signatures. These findings suggest that kevetrin may be a promising therapeutic option for patients with both wild-type and TP53-mutant AML.

15-hydroxy-6α,12-epoxy-7β,10αH,11βH-spiroax-4-ene-12-one exerts anti-tumor effects against osteosarcoma through apoptosis induction.

Osteosarcoma is the most common type of malignant bone tumor, which has an overall survival rate of only 15-30%. The present study aimed to investigate the effects of 15-hydroxy-6α,12-epoxy-7β,10αH,11βH-spiroax-4-ene-12-one (HESEO), a compound extracted from the endophytic fungus Penicillium sp. FJ-1 isolated from Avicennia marina, on the proliferation of osteosarcoma cells and to explore its underlying mechanisms of action. Cell number was counted to measure the cell proliferation. JC-1 reagent was used to measure mitochondrial membrane potential. ELISA was used to measure the cytochrome c level and caspase activities. Apoptosis was detected by Annexin V-Propidium Iodide staining. Gene and protein expression were measured by reverse-transcription-PCR and western blot analysis, respectively. Additionally, the anti-tumor effects of HESEO were explored within a syngeneic osteosarcoma tumor model. The results suggested that HESEO significantly inhibited the proliferation of osteosarcoma cells and induced apoptosis of MG-63 cells, evidenced by their decreased mitochondrial membrane potential, and increased cytochrome c release, caspase activities and percentage of apoptotic cells. In addition, HESEO increased the expression of pro-apoptotic genes and proteins compared with control cells. The results indicated that HESEO may act through increasing p53 upregulated modulator of apoptosis expression. Furthermore, HESEO treatment significantly increased the survival time and decreased the tumor burden of osteosarcoma tumor-bearing mice compared with vehicle treatment. Furthermore, combined treatment with HESEO enhanced the effects of the chemotherapeutic agent methotrexate on a lung metastasis osteosarcoma model. These data suggested that HESEO could be developed as a potential anti-tumor agent against osteosarcoma.

Exosomes derived from mesenchymal stem cells improved function and survival of neutrophils from severe congenital neutropenia patients in vitro

Severe congenital neutropenia (SCN) is described by the absolute neutrophil counts less than 500 cells/mm3, bacterial infections, and an arrest of neutrophil differentiation. So, effective strategies for improving the function and lifespan of the existing neutrophils in these patients are necessary. Mesenchymal stem cells (MSCs) have supportive effects on neutrophils. Recently, it was determined that MSCs exert their effects, mostly by secreting soluble factors and exosomes. So, in this study, neutrophils were isolated from the bloodstream of healthy donors and SCN patients and cultured with medium, MSC-exosomes or MSC-conditioned media (MSC-CM). Then, the effects of the two treatments on neutrophil respiratory burst, apoptosis and phagocytosis percentage were assessed using nitro blue tetrazolium (NBT) assay, annexin V-propidium iodide (PI) and Giemsa staining, respectively. Both treatments could significantly augment respiratory burst of neutrophils from SCN patients and healthy donors. But, only CM could significantly enhance phagocytosis index. About the lifespan of neutrophils, only exosomes could significantly enhance it in both groups. Based on these results, both exosomes and CM derived from MSCs could be attractive candidates for rescuing SCN patients from serious infections.

High Glucose Affects the Cytotoxic Potential of Rapamycin, Metformin and Hydrogen Peroxide in Cultured Human Mesenchymal Stem Cells.

Oxidative stress and chronic hyperglycemia are two major side effects of type 2 diabetes affecting all cell types including mesenchymal stem cells (MSCs). As a cell therapy choice, understanding the behavior of MSCs will provide crucial information for efficient treatment. Placental mesenchymal stem cells were treated with various concentrations of glucose, metformin, rapamycin, and hydrogen peroxide to monitor their viability and cell cycle distribution. Cellular viability was examined via the MTT assay. Cell cycle distribution was studied by propidium iodide staining and apoptosis was determined using Annexin Vpropidium iodide staining and flow cytometry. Involvement of potential signaling pathways was evaluated by Western blotting for activation of Akt, P70S6K, and AMPK. The results indicated that high glucose augmented cell viability and reduced metformin toxic potential. However, the hydrogen peroxide and rapamycin toxicities were exacerbated. Our findings suggest that high glucose concentration has a major effect on placental mesenchymal stem cell viability in the presence of rapamycin, metformin and hydrogen peroxide in culture.

Porphyromonas gingivalis degrades integrin β1 and induces AIF-mediated apoptosis of epithelial cells

Background: Porphyromonas gingivalis, a major pathogen of chronic periodontitis, adheres to and invades epithelial cells via an interaction between fimbriae and integrin. P. gingivalis proliferation and infection may affect the survival of cells. In this study, we further examined alternative signaling pathways mediating epithelial-cell death induced by P. gingivalis and the role of the cell-adhesion molecule integrin.Methods: Human epithelial KB cells interacted with P. gingivalis to evaluate cell death by Annexin V-propidium iodide (PI) staining. JC-1 staining was used to measure mitochondrial membrane potential (MMP). The mRNA and protein of integrin β1, apoptosis-inducing factor (AIF) and caspase-3 were detected by real-time PCR and western blot. Caspase-3 activity was analyzed by spectrophotometry.Results: P. gingivalis infection downregulated integrin β1 and led to cell detachment in a dose and time-dependent manner. Large amount of P. gingivalis induced MMP depolarization and apoptosis in KB cells. Moreover, P. gingivalis up-regulated AIF, but not activate caspase-3 during apoptosis. In addition, AIF inhibitor N-Phenylmaleimide almost inhibited the P. gingivalis-induced apoptosis.Conclusions: P. gingivalis disrupts epithelial-cell adhesion by degrading integrin β1 and induces caspase-independent, AIF-mediated mitochondrial apoptosis, which may promote the damage of oral tissue.

MYC as a therapeutic target for the treatment of triple-negative breast cancer.

e12550 Background: Despite intensive efforts, a validated targeted therapy for triple-negative breast cancer (TNBC) remains elusive. One of the most frequent genetic alterations identified in TNBC is amplification of the MYC gene which is found in > 60% of samples. The aim of this study was therefore to investigate the potential value of targeting MYC activation with the recently identified selective and high-affinity low molecular weight MYC inhibitor MYCMI-6, for the treatment of TNBC. Methods: Cell viability was determined using the MTT assay and apoptosis by flow cytometry using annexin V-propidium iodide staining. Results: Based on a panel of 13 breast cancer cell lines, IC50 values for inhibition of proliferation by MYCMI-6 ranged from 1.2 to 96.7 μM. Significantly lower IC50 values were found for TNBC than for non-TNBC cell lines (p = 0.04), i.e., TN cell lines were more sensitive to MYCMI-6 than non-TN cell lines. IC50 values for MYCMI-6 were compared with 2 older MYC inhibitors, i.e., 10058-F4 and KJ Pyr 9. Of these 3 inhibitors, the lowest IC50 values were found for MYCMI-6, i.e., MYCMI-6 was the most sensitive of the 3 inhibitors investigated (p < 0.0001 for MYCMI-6 versus both 10058-F4 and KJ Pyr 9). In addition to inhibiting cell proliferation, MYCMI-6 was found to induce apoptosis in a time and concentration-dependent manner in multiple breast cancer cell lines. A significant correlation was found between MYCMI-6 IC50 and induction of apoptosis ((p = 0.0499, r 2= 0.9026, n = 4), i.e., the lower the IC50 value, the greater the number of apoptotic cells present. This finding suggests that MYCMI-6 decreases cell proliferation by inducing apoptosis. Conclusions: We conclude that the MYC targeting compound, MYCMI-6 is a potential new treatment for TNBC.

Mechanisms of mixed umbilical cord blood-derived mesenchymal stem cells inhibiting proliferation and inducing apoptosis of C6 glioma cells in vitro

Objective To study the mechanisms of the mixed umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) inhibiting proliferation and inducing apoptosis of C6 glioma cells in vitro. Methods Cysteinyl aspartate-specific protease (Caspase)-8, B cell lymphoma/leukemia-2 associated X protein (bax), B cell lymphoma/leukemia-2 (bcl-2) and Caspase-3 protein expression in C6 cells was analyzed by immunohistochemistry and Western blotting, respectively. Caspase-8, bax, bcl-2 and Caspase-3 mRNA levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Cell cycle was detected by flow cytometry. Apoptosis of C6 cells was examined by Annexin V-propidium iodide (PI) staining and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) methods, respectively. Results The mixed UCB-MSCs had a tendency to increase the expression of Caspase-8, bax, Caspase-3 mRNA, and decrease the expression of bcl-2 mRNA. The expression of apoptosis protein and the number of apoptosis cells increased with the increase of the ratio of E/T (E: effect cells namely UCB-MSCs; T: target cells namely C6 cells), showing a proportional dependence relationship; E∶T=(5+ 5)∶1. The relative expression of Caspase-8, bax, bcl-2, and Caspase-3 mRNA in C6 cells was 0.78±0.13, 0.28±0.03, 0.70±0.08, and 0.76±0.12, respectively. When E/T=0∶1, 5∶1, (2.5+ 2.5)∶1, 10∶1, (5+ 5)∶1, the number of apoptosis C6 cells was (2.9±0.6), (3.1±0.5), (14.5±1.8), (14.3±2.2), and (22.8±4.5) cells, respectively. Conclusion After phytohemagglutinin (PHA) stimulation, the mixed UCB-MSCs could inhibit proliferation of C6 cells in vitro, which was related to induction of apoptosis of glioma cells. Key words: Glioma; Umbilical cord blood; Mesenchymal stem cells; Apoptosis; Proliferation

Comparative Research on Anti-Inflammatory Effects of Different Dietary Antioxidants on Alcohol-Induced Damage in Gastric Cells

Objective: Antioxidants prevent ill-effects of free radicals through regulation of different proand anti-inflammatory enzymes of the cell which have association with the pathophysiology of alcohol-induced gastropathy. This study was aimed to explore the mechanism in gastric adenocarcinoma (AGS) cell line and protection by dietary antioxidants e.g. catechin, resveratrol, quercetin, curcumin and 6-gingerol. Methods: AGS cells were exposed to 2.5% ethanol for varied time span in presence and absence of antioxidants. Cytotoxicity was assessed by MTT assay and annexin V-propidium iodide staining. The damages in cellular morphology were observed by DAPI and EtBr-acridine orange staining. Changes in ROS generation were examined through confocal microscopy. Western blots were performed using cell extracts to investigate the changes in expression level of COX-1, -2, catalase, superoxide dismutase (SOD), iNOS, MMP-9 and TIMP-1. Immunofluorescence study was done to cross check the expression of COX-2. Results: A concentration-dependent cytotoxicity was observed in the cell viability on ethanol exposure which was reduced by antioxidant treatment. Antioxidants reduced the inflammation by downregulating the expression of COX-2, iNOS and MMP-9 and by upregulating the expressions of catalase, SOD and TIMP-1 significantly. Catechin and quercetin demonstrated most prominent cytoprotection amongst the five, followed by resveratrol. These results were corroborated with MTT assay, DAPI and EtBr-acridine orange staining. Curcumin and 6-gingerol did not show any significant effect. Conclusion: Dietary antioxidants protect AGS cells from oxidative stress by maintaining the homeostasis between oxidant-antioxidant and protease-antiprotease ratio.

Manilkara zapota (L.) P. Royen leaf water extract triggered apoptosis and activated caspase-dependent pathway in HT-29 human colorectal cancer cell line

Manilkara zapota (L.) P. Royen (Family: Sapotaceae), commonly called as sapodilla, has been applied as traditional folk medicine for diarrhea and pulmonary infections. Conventional therapy in colorectal cancer is not likely effective due to undesirable outcomes. The anti-colon cancer properties of Manilkara zapota leaf water extract have yet to be investigated thus far. Therefore, our present study aimed to evaluate the ability to induce apoptosis and the underlying mechanisms of Manilkara zapota leaf water extract against human colorectal cancer (HT-29) cells. The cytotoxicity of Manilkara zapota leaf water extract was screened in different cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) analyses. The morphological changes in HT-29 cell lines after exposure to Manilkara zapota leaf water extract were viewed under fluorescence and inverted light microscope. The apoptotic cell was measured by Annexin V-propidium iodide staining. The caspase-3 and -8 activities were assessed by colorimetric assay. Overall analyses revealed that treatment with Manilkara zapota leaf water extract for 72 h can inhibit the viability of HT-29 cells. Incubation with Manilkara zapota leaf water extract for 24, 48, and 72 h significantly increased (p < 0.05) the total apoptotic cells compared to the control. Treatment with 21, 42, and 84 μg/mL of Manilkara zapota leaf water extract for 72 h triggered both caspase-3 and -8 activities in a concentration-dependent pattern. We also found that the catalase level in the two treatment groups (21 and 42 μg/mL) was significantly elevated after 24 h incubation. Incubation with Manilkara zapota leaf water extract for 72 h triggered the transcriptional elevation of the adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β), AXIN1, and casein kinase 1 (CK1). The β-catenin mRNA levels were reduced accordingly when the concentration of the Manilkara zapota leaf water extract was increased. Our results suggested that Manilkara zapota leaf water extract offer great potential against colorectal cancer through modulation of Wnt/β-catenin signaling pathway, caspase-dependent pathway, and antioxidant enzyme.

Deguelin induces apoptosis in colorectal cancer cells by activating the p38 MAPK pathway.

ObjectivesDeguelin, a rotenoid extracted from Mundulea sericea (Leguminosae), exhibits antitumor effects on several types of human cancers. Due to the limited studies of deguelin on colorectal cancer (CRC), the present study was designed to investigate the antitumor effect of deguelin and to explore the underlying mechanism in CRC.Materials and methodsCell viability was assessed by the cell counting kit-8 (CCK-8) assay, and cell apoptosis was determined by the annexin v-propidium iodide staining using flow cytometry and Western blot in CRC cell lines after incubation with deguelin. The antitumor effect of deguelin was further evaluated in tumor xenograft models. Moreover, SB203580, a specific inhibitor of p38 MAPK, was used to confirm the involvement of p38 MAPK pathway in deguelin-induced apoptosis.ResultsDeguelin significantly inhibited cell proliferation and induced apoptosis in CRC cell lines (SW620 and RKO) in a time-dependent and dose-dependent manner. Western blot analysis also showed that the expression of proapoptotic proteins (cleaved caspase 3 and cleaved PARP) was upregulated, while that of antiapoptotic proteins (Bcl-2 and survivin) was downregulated after deguelin treatment in CRC cell lines. Moreover, oral administration of deguelin significantly suppressed tumor growth and induced apoptosis in subcutaneous xenograft mouse models without obvious toxicity. Additionally, Western blot revealed that deguelin-induced apoptosis might be regulated by the p38 MAPK pathway and inhibition of p38 MAPK could attenuate deguelin-induced proliferative inhibition and apoptosis in CRC cells.ConclusionCollectively, these results demonstrated that deguelin inhibited CRC cell growth by inducing apoptosis via activation of p38 MAPK pathway.

High Mir-17-92 Expression Is Associated with in Vitro chemoresistance in Burkitt Lymphoma

Background: Copy number gain and/or MYC induction leads to increased expression of miRs in the miR-17~92 cluster. Development of Burkitt lymphoma (BL) is associated with translocation of MYC resulting in MYC expression and frequent recurrent copy number gains of chromosome 13q containing the miR-17~92 cluster of miRs. MiRs in the miR-17-92 cluster, including miR19 and miR17, have been associated with B-cell lymphomagenesis. Copy number gain of 13q and high expression of miR-17 have also been associated with possible inferior outcome in children with BL. MiR-17~92 miRs can decrease expression of the PI3K inhibiting protein PTEN leading to activation of the PI3K/AKT pathway, a pathway implicated in BL lymphomagenesis. These miRs also target the pro-apoptotic protein BIM, suppression of which has also been associated with chemoresistance in BL.Objectives: Investigate the in vitro effect of altered miR-17-92 expression on chemoresponsiveness in BL cells.Methods: MiR expression was determined by qPCR. The miR-17-92 locus was deleted in Raji cells using custom CRISPR-Cas9 lentiviral vectors. Single cell-derived clones, R5 and R24, were established and locus deletion determined by genomic DNA PCR. Protein expression and Caspase cleavage was assessed by western blotting. Apoptosis induction was determined by flow cytometry for Annexin V-propidium iodide staining. Cell viability following exposure to chemotherapy was determined by AlamarBlue assay. SCID mice were inoculated with Raji or R24 cells by tail vein injection and survival determined by Kaplan-Meier analysis. Hind limb paralysis was used as an end-point for survival. Antagomir targeting miR17 was transduced into Raji cells by electroporation.Results: Chemotherapy resistant Raji 4RH cells exhibit increased miR17 and miR19 expression and increased PI3K/AKT pathway activation compared to parental Raji. R5 and R24 Raji KO lines exhibit decreased miR17 and miR19 expression and increased expression of miR targets PTEN and BIM. When xenografted into SCID mice, R24 cells demonstrated a significant prolongation in survival compared to Raji (Raji vs R24: median 29 days vs not reached at 150 days, P<0.05) (Figure 1B). A significant decrease in [IC50] in R24 cells compared to Raji was observed following 48h exposure to doxorubicin (Raji vs. R24: 65±23nM vs 12±2nM), dexamethasone (1579±10e5nM vs 49±57nM), etoposide (571±106nM vs 106±22nM) or cisplatin (2.5±0.7uM vs 0.96±0.3uM) (Figure 1A). Raji R24 cells also demonstrate an increase in apoptosis induction following exposure to doxorubicin or etoposide. To investigate the effect of direct miR targeting, Raji cells were transfected with an antagomir targeting miR17 resulting in decreased viability following exposure to doxorubicin or cisplatin compared to Raji cells transfected with a control oligomer (Figure 1C). Antagomir transfected Raji cells express higher levels of PTEN and BIM compared to control Raji cells.Conclusion: High expression of miR17-92 cluster miRs is associated with in vitro chemotherapy resistance. Knockout of the MIR17HG gene in BL cells results in impaired proliferation, impaired in vivo engraftement with prolonged survival in SCID mice and increased in vitro chemoresponsiveness associated with an increase in expression of PTEN and BIM. Targeting of miR17 using an antagomir approach resulted in increased PTEN and BIM and increased chemoresponsiveness. These findings highlight the role of MYC-associated miRs in in vitro chemoresistance in BL cells warranting continued investigation as a possible therapeutic target for relapsed/refractory BL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PARP inhibitor re‑sensitizes Adriamycin resistant leukemia cells through DNA damage and apoptosis.

Resistance to Adriamycin (ADR) is an increasing problem in the treatment of leukemia and the development of novel therapeutic strategies is becoming increasingly important. Olaparib is a poly (adenosine diphosphate-ribose) polymerase (PARP) 1 inhibitor, which has promising antitumor activity in patients with metastatic breast cancer and germline BRCA mutations. Previously published studies have indicated that Olaparib is able to overcome drug resistance in cancer; however, its underlying mechanism of action is yet to be elucidated. The aim of the present study was to explore the mechanism underlying re-sensitization. Annexin V-propidium iodide staining indicated that the percentage of apoptotic ADR resistant cells was markedly increased and the cell cycle was blocked at the G2/M-phase following treatment with ADR combined with Olaparib, when compared with the control group. The alkaline comet assay demonstrated that ADR combined with Olaparib significantly upregulated the induction of the DNA damage response in ADR-resistant cells. Western blot analysis revealed that the protein expression of γ-H2A histone family member X, cleaved PARP, caspase 3 and cleaved caspase 3 was markedly enhanced, while the cell cycle-associated protein cyclin B1 was downregulated in K562/ADR cells following treatment with a combination of ADR and Olaparib. Similar synergistic cytotoxicity was observed in blood mononuclear cells, which were isolated from patients with chemotherapy-resistant leukemia. As Olaparib is available for clinical use, the results of the present study provide a rationale for the development of Olaparib combinational therapies for cases of ADR resistant leukemia.

Retinoic acid receptor α expression exerts an anti-apoptosis effect on PC12 cells following oxygen-glucose deprivation.

It has been established that the primary form of neuron death following hypoxic ischemic brain damage is apoptosis. Imbalances in the expression of genes in the B-cell lymphoma 2 (Bcl-2) family located in the mitochondrion, and in the expression of their encoded proteins, are key events in the mitochondrial apoptotic pathway, which lead to damage of cellular structure and function. The present study aimed to explore the regulatory effect of retinoic acid receptor α (RAR-α) on the apoptosis of PC12 cells induced by oxygen-glucose deprivation (OGD) in the retinoic acid signaling pathway. Recombinant adenovirus RAR-α small interfering RNA (Ad-siRAR-α) was used to transduce PC12 cells, and the efficiency of RAR-α expression inhibition was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). An empty adenovirus vector was transfected in PC12 cells, which were used as the control. Flow cytometry with Annexin V-propidium iodide (PI) and fluorescence probe JC-1 staining was used to detect the apoptosis rate and mitochondrial transmembrane potential (MMP), respectively, of PC12 cells after transduction with Ad-siRAR-α. Furthermore, the expression levels of key genes in the RAR-α and mitochondrial apoptotic pathway, Bcl-2 and Bcl-2-associated protein (Bax) were analyzed by RT-quantitative (q)PCR and western blot analysis. RAR-α mRNA expression was observed to be decreased in PC12 cells following OGD-induced injury, and this decrease can be reversed by 4 µmol/l ATRA treatment. After 36 h transfection with Ad-siRAR-α, RAR-α gene expression was significantly inhibited compared with the control (P<0.05). The results of Annexin V-PI, fluorescence probe JC-1 staining and flow cytometry demonstrated that the apoptosis rate significantly increased and MMP significantly decreased in OGD-induced PC12 cells following transduction with Ad-siRAR-α compared with the control (both P<0.05). RT-qPCR and western blot analysis indicated that Bax expression was significantly increased and Bcl-2 expression was significantly decreased in PC12 cells transduced with Ad-siRAR-α after OGD-induced injury at the mRNA and protein level (P<0.05). In conclusion, Ad-siRAR-α transduction could promote apoptosis in OGD-induced PC12 cells. This suggests that the expression of Bax and Bcl-2 in the mitochondrial apoptosis signaling pathway is, at least in part, mediated by RAR-α expression, thereby indicating that RAR-α expression exerts an anti-apoptotic effect on OGD-damaged PC12 cells.

ROS-mediated lysosomal membrane permeabilization is involved in bupivacaine-induced death of rabbit intervertebral disc cells

Bupivacaine is frequently administered for diagnosing and controlling spine-related pain in interventional spine procedures. However, the potential cytotoxic effects of bupivacaine on intervertebral disc (IVD) cells and the underlying molecular mechanisms have not yet been fully established. Here, we showed that bupivacaine decreased the viability of rabbit IVD cells in a dose- and time-dependent manner. Moreover, the short-term cytotoxicity of bupivacaine in IVD cells was primarily due to cell necrosis, as assessed by Annexin V-propidium iodide staining and live/dead cell staining. Necrosis was verified by observations of swollen organelles, plasma membrane rupture, and cellular lysis under transmission electronic microscopy. Interestingly, our data indicated that bupivacaine-induced primary necrosis might involve the necroptosis pathway. The key finding of this study was that bupivacaine was able to induce lysosomal membrane permeabilization (LMP) with the release of cathepsins into the cytosol, as evidenced by LysoTracker Red staining, acridine orange staining, and cathepsin D immunofluorescence staining. Consistently, inhibitors of lysosomal cathepsins, CA074-Me and pepstatin A, significantly reduced bupivacaine-induced cell death. Finally, we found that bupivacaine resulted in an increase in intracellular reactive oxygen species (ROS) and that inhibition of ROS by N-acetyl-L-cysteine effectively blocked bupivacaine-induced LMP and cell death. In summary, the results of this in vitro study reveal a novel mechanism underlying bupivacaine-induced cell death involving ROS-mediated LMP. Our findings establish a basis for the further investigation of bupivacaine cytotoxicity in an in vivo system.