Fenretinide (4HPR) Inhibits Growth of Myeloma Cells in Their Microenvironment and Is a Potent Inhibitor of Angiogenesis and Osteoclastogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3480-3480
Author(s):  
Xin Li ◽  
Wen Ling ◽  
Rinku Saha ◽  
Paul Perkins ◽  
Angela Pennisi ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Mtt Assay ◽  
Cell Apoptosis ◽  
Mononuclear Cells ◽  
Annexin V ◽  
Dependent Manner ◽  
Angiogenic Potential ◽  
Inhibition Of Angiogenesis ◽  
Induced Apoptosis

Abstract Fenretinide (4HPR) is a relatively safe neoclassical retinoid analog that inhibits growth of various tumors through increased intracellular ceramide and ROS, induction of tumor cell apoptosis and inhibition of angiogenesis. 4HPR has been successfully tested as a chemopreventive and chemotherapeutic agent in clinical trials on various malignancies. In contrast to retinoic acid, 4HPR induces cell apoptosis rather than differentiation and shows synergistic responses with chemotherapeutic drugs in different tumor cell types. The biological effect and therapeutic value in multiple myeloma (MM) has not been investigated. The aim of this study was to investigate the anti-MM effect and mechanism of action of 4HPR using 3 stroma-dependent and 2 stroma-independent MM cell lines established in our laboratory, CD138-selected primary MM cells and co-culture systems of these cells with human osteoclasts and mesenchymal stem cells (MSCs) as previously described (Yaccoby et al., Cancer Res 2004). MM cell apoptosis detected by annexin V flow cytometry and TUNNEL, tumor growth by MTT assay, changes in caspase 3, 8 and 9 activity using Western blotting and ROS production by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay. 4HPR inhibits growth of all tested MM cells in a dose- and time-dependent manner. The IC50 after 48 hrs in serum-containing media was 10 μM using MTT assay. 4HPR (3 μM) increased percent of apoptotic MM cells by 2.5±0.4 folds (p<0.01). Co-culture of these cell lines with osteoclasts only partially protected MM cells from the proapoptotic effect of this drug. Furthermore, 4HPR also induced apoptosis of primary CD138-selected MM cells co-cultured with osteoclasts or MSCs, and inhibited growth of bortezomib-resistant MM cell lines. In contrast, 4HPR had only minimal cytotoxic effect on blood mononuclear cells and MSCs. The proapoptotic effect of 4HPR involved increased level of ROS by 2.55±0.67 folds in MM cells (p<0.01). We also detected reduced levels of procaspase and increased cleaved caspase 8, 9 and 3 within 24 hrs of incubation with this drug. Sphingosine-1 phosphate (S1P) partially protected MM cells from 4HPR-induced apoptosis suggesting that, as reported for other tumors, anti-MM mechanism of this drug involved increased intracellular ceramide. 4HPR significantly inhibited tube formation by HUVEC in a matrigel assay (p<0.0001), confirming its anti-angiogenic potential. This drug also effectively prevented formation of multinucleated osteoclasts in culture of human osteoclast precursors with RANKL and M-CSF (p<0.0001). Furthermore, mature osteoclasts viability as assessed by MTT assays was reduced following incubation with 3 μM 4HPR (p<0.0001). We conclude that 4HPR is a potent anti-MM agent, affecting growth of MM cells in their microenvironment directly through induction of apoptosis in mechanisms involving ROS, caspase and possibly ceramide, and indirectly through inhibition of angiogenesis and osteoclastogenesis. Our data also suggests that S1P, which is highly produced by activated platelets, is an important survival factor for MM cells. Study is underway to test anti-MM efficacy of 4HPR in the SCID-hu model for primary myeloma.

scholarly journals Dihydrochalcone Derivative Induces Breast Cancer Cell Apoptosis via Intrinsic, Extrinsic, and ER Stress Pathways but Abolishes EGFR/MAPK Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-18 ◽  
Author(s):  
Wasitta Rachakhom ◽  
Patompong Khaw-on ◽  
Wilart Pompimon ◽  
Ratana Banjerdpongchai
Keyword(s):  
Breast Cancer ◽  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Mapk Pathway ◽  
Annexin V ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Mcf 7

Dihydrochalcone derivatives are active compounds that have been purified from the Thai medicinal plant Cyathostemma argenteum. The objectives of this study were to investigate the effects of two dihydrochalcone derivatives on human breast cancer MDA-MB-231 and MCF-7 cell proliferation and to study the relevant mechanisms involved. The two dihydrochalcone derivatives are 4′,6′-dihydroxy-2′,4-dimethoxy-5′-(2″-hydroxybenzyl)dihydrochalcone (compound 1) and calomelanone (2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, compound 2), both of which induced cytotoxicity toward both cell lines in a dose-dependent manner by using MTT assay. Treatment with both derivatives induced apoptosis as determined by annexin V-FITC/propidium iodide employing flow cytometry. The reduction of mitochondrial transmembrane potential (staining with 3,3′-dihexyloxacarbocyanine iodide, DiOC6, employing a flow cytometer) was established in the compound 1-treated cells. Compound 1 induced caspase-3, caspase-8, and caspase-9 activities in both cell lines, as has been determined by specific colorimetric substrates and a spectrophotometric microplate reader which indicated the involvement of both the extrinsic and intrinsic pathways. Calcium ion levels in mitochondrial and cytosolic compartments increased in compound 1-treated cells as detected by Rhod-2AM and Fluo-3AM intensity, respectively, indicating the involvement of the endoplasmic reticulum (ER) stress pathway. Compound 1 induced cell cycle arrest via enhanced atm and atr expressions and by upregulating proapoptotic proteins, namely, Bim, Bad, and tBid. Moreover, compound 1 significantly inhibited the EGFR/MAPK signaling pathway. In conclusion, compound 1 induced MDA-MB-231 and MCF-7 cell apoptosis via intrinsic, extrinsic, and ER stress pathways, whereas it ameliorated the EGFR/MAPK pathway in the MCF-7 cell line. Consequently, it is believed that compound 1 could be effectively developed for cancer treatments.

Cyclin-Dependent Kinase 4/6 Inhibitor Abemaciclib Exerts Dose-Dependent Cytostatic and Cytocidal Effects on Multiple Myeloma Cells Via Autophagy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4478-4478 ◽  
Author(s):  
Noriyoshi Iriyama ◽  
Hirotsugu Hino ◽  
Shota Moriya ◽  
Masaki Hiramoto ◽  
Yoshihiro Hatta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Annexin V ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Dose Dependent

Abstract Background:Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of abnormal plasma cells in the bone marrow. D-type cyclins (CCNDs), an important family of cell cycle regulators, are thought to be implicated in multiple myeloma (MM) development because CCNDs are commonly expressed in myeloma cells. CCND is known to positively regulate the cell cycle from G1 to S-phase initiation by binding to cyclin-dependent kinase (CDK) 4/6, resulting in potentiation of myeloma cell growth. These findings suggest a possible role for CDK4/6-targeting therapy in MM, yet the details remain incompletely understood. In this regard, we investigated the biological activity of abemaciclib, a potent, highly selective CDK4/6 inhibitor, in myeloma cell lines, to elucidate the mechanisms underlying the involvement of the CCND-CDK4/6 complex in cell cycle regulation and survival. Methods:The effects of abemaciclib on myeloma cells were investigated using three myeloma cell lines, KMS12-PE (CCND1-positive and CCND2-negative), RPMI8226 (CCND1-negative and CCND2-positive), and IM-9 (both CCND1- and CCND2-positive). Cell growth was assessed by trypan blue exclusion assay. Cell cycle analysis was performed using propidium iodide (PI) and apoptosis was measured using annexin V/PI staining via flow cytometry. Cell cycle regulated proteins, including p21 and p27, and phosphorylated proteins, including STAT1, STAT3, ERK, JNK, p38, and AKT, were evaluated using a phospho-flow method. Autophagy was assessed using CYTO-ID via flow cytometry. PARP cleavage was investigated via western blotting. Clarithromycin, an antibiotic agent belonging to the macrolide class, was used as an autophagy inhibitor. Results:Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all the cell lines evaluated, with significant differences seen at a concentration of 320 nM. Annexin V/PI staining revealed that 1 μM abemaciclib showed little or no effect on apoptosis, but 3.2 μM abemaciclib induced apparent myeloma cell apoptosis, with an increase in both the early and late apoptotic fractions. Therefore, 1 and 3.2 μM of abemaciclib were used in subsequent experiments for the assessment of cell growth and apoptosis, respectively. Cell cycle analyses revealed that 1 μM abemaciclib increased the fraction of cells in G0/G1 phase and decreased the fraction in S-G2/M phase. Furthermore, this effect was associated with the upregulation of p21 and p27 in the evaluated myeloma cells. PARP cleavage was observed in KMS12-PE cells treated with 3.2 μM abemaciclib, but not 1 μM, suggesting a close connection between the degree of PARP cleavage and apoptosis in myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner. However, no apparent inhibitory effect on the autophagy-related phosphorylated proteins STAT1 (Y701), STAT3 (Y705), ERK (T202/Y204), JNK (T183/Y185), p38 (T180/Y182), or AKT (Y315) was observed in myeloma cells treated with 3.2 μM abemaciclib. To investigate the role of abemaciclib-induced autophagy on myeloma cell apoptosis, we further assessed the apoptotic effect of 3.2 μM abemaciclib or 50 μg/mL clarithromycin, alone or in combination. Clarithromycin did not induce apoptosis of myeloma cells. Importantly, clarithromycin treatment in combination with abemaciclib attenuated the apoptotic effect of abemaciclib. Discussion & Conclusions: Although the underlying mechanisms conferring the level of CCND expression are known to differ greatly (e.g., CCND translocation, hyperdiploidy, or activation of upstream pathways of CCND transcription), the results of the current study indicate that the CCND-CDK4/6 complex is closely involved in myeloma cell growth and survival regardless of the CCND family member present. In addition, we demonstrate that abemaciclib exerts multiple effects, such as myeloma cell apoptosis, via the PARP pathway or autophagy, as well as cell cycle regulation. Because abemaciclib in combination with clarithromycin inhibits myeloma cell apoptosis, the autophagy induced by abemaciclib is considered to have a critical role in the induction of apoptosis, so-called "autophagic cell death." These results provide novel insights into a possible therapeutic approach using abemaciclib to target CDK4/6 in patients with MM, and offer new possibilities for combination therapy with CDK4/6 inhibitors and autophagy regulators. Disclosures Iriyama: Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Hatta:Novartis Pharma: Honoraria.

Identification of the TAK1-NF-κB Axis As Critical Regulator of AML Stem and Progenitor Cell Survival.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2982-2982
Author(s):  
Matthieu C.J. Bosman ◽  
Jan J. Schuringa ◽  
Wim J. Quax ◽  
Edo Vellenga
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Cell Survival ◽  
Cell Lines ◽  
Combined Treatment ◽  
Annexin V ◽  
Fold Increase ◽  
Dependent Manner ◽  
Leukemic Stem Cells ◽  
Induced Apoptosis

Abstract Abstract 2982 A small population of leukemic stem cells is resistant to chemotherapy and is responsible for the leukemic out-growth and relapse in acute myeloid leukemia (AML) patients. Evasion of apoptosis might be one of the essential mechanisms involved in this process. In order to gain more insight into the differences in the apoptotic programming between normal and leukemic (stem) cells, we recently performed gene array analysis by comparing CD34+ AML cells versus CD34+ normal bone marrow (NBM) cells. Gene ontology (GO) analysis of the differentially expressed genes between AML and NBM cells revealed differences in GO terms metabolic processes and apoptosis. In order to characterize differences in apoptotic programming in more detail 429 apoptotic related genes were selected and cluster analysis showed that CD34+ AML and CD34+ NBM cells could be separated into two distinct groups. In particular TGF-β activated kinase 1 (TAK1)/MAP3K7 was one of the apoptosis-related genes that was significantly higher expressed in CD34+ AML cells compared to CD34+ NBM cells (p = 1.8e−7). This increased expression of TAK1 could be confirmed by Q-PCR, showing an increase of on average 5.8 fold in TAK1 expression in the studied CD34+ AML cells. In mice it has been demonstrated that TAK1 is required for the survival of hematopoietic cells which is largely dependent on TNFR1 and TNFR2. In accordance with these data, we showed that TAK1 is also necessary in human hematopoiesis. Colony-forming cell (CFC) assays showed that inhibition of TAK1 in human cord blood CD34+cells, either by shRNAs targeting TAK1 or the TAK1 inhibitor 5z–7-oxozeaenol, resulted in a 2 fold reduction in CFU-GM and BFU-E frequencies compared to control cells. The efficacy was strongly further enhanced by the addition of TNFα, which resulted in a 9.4 fold decrease in CFC colonies upon TAK1 inhibition. Subsequently, we questioned whether TAK1 inhibition would affect CD34+ AML cell survival. Treatment of the AML cell lines MOLM13, OCI-M3 and HL60 with the TAK1 inhibitor 5z–7-oxozeaenol alone only induced modest effects, but in combination with TNFα for 24 hrs a strong induction of apoptosis was observed (IC50 respectively = 23nM, 215nM and 60 nM). Comparable results were observed in HL60 cells transduced with shRNAs targeting TAK1 whereby a downmodulation of TAK1 resulted in a 5.4 fold increase in Annexin V+ cells upon TNFα addition. In accordance with previous data, Western blot analysis showed that TAK1 inhibition reduced the levels of p-IκBα, p-p38, p-ERK and p-C-JUN. To test which of these pathways would be important for cell survival, AML cell lines were treated with either the p38 inhibitor SB203580, MEK/ERK inhibitor U0126, JNK inhibitor SP600125 and the NF-κB inhibitor BMS-345541, alone or in combination with TNFα. Addition of the NF-κB inhibitor BMS-345541 induced apoptosis in OCI-M3 and MOLM13 which was significantly increased in combination with TNFα (2.4 fold, p = 0.02). In contrast, inhibition of p38, MEK/ERK and JNK, either alone or in combination with TNFα, did not induce cell death in the AML cell lines. These data suggest that cell death induced by TAK1 inhibition is mainly due to inhibition of the NF-κB pathway. To determine the effects of TAK1 inhibition on primary AML cells, long-term expansion of the leukemic stem cell enriched CD34+ AML cell fraction was evaluated in MS5 stromal co-cultures in the absence or presence of TAK1 inhibitor and/or TNFα. Combined treatment for a period of 2 weeks completely abrogated the out-growth of CD34+ AML cells, indicating that both leukemic progenitors as well as leukemic stem cells were targeted. In contrast, addition of the single agents did not efficiently reduce cell growth. Similarly, downmodulation of TAK1 using shRNAs strongly sensitized primary CD34+ AML cells for TNFα-induced apoptosis, showing a 6 fold increase in Annexin V+ cells compared to control cells. Results on the in vivo efficacy of TAK1 inhibition on primary AML cells are in progress. In conclusion, our results show that TAK1 is frequently overexpressed in CD34+ AML cells, and that inhibition of TAK1 in combination with TNFα is highly efficient in inducing apoptosis of leukemic stem/progenitor cells in a NF-κB-dependent manner. Disclosures: No relevant conflicts of interest to declare.

Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 603-603
Author(s):  
Hisato Kawakami ◽  
Shengbing Huang ◽  
Krishnendu Pal ◽  
Debabrata Mukhopadhyay ◽  
Frank A. Sinicrope
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Annexin V ◽  
Treatment Resistance ◽  
Dependent Manner ◽  
Apoptosis Resistance ◽  
Caspase Cleavage ◽  
Control Shrna ◽  
Induced Apoptosis

603 Background: Oncogenic BRAFV600E mutations activate MAP kinase signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer (CRC). In BRAFV600E mutant CRCs, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. Methods: BRAF isogenic RKO CRC cells and RKO, HT29, WiDr cell lines were treated with cobimetinib ± the small molecule MCL-1 inhibitor, A-1210477. Apoptosis was measured by Annexin V staining and caspase cleavage. Gene knockdown or overexpression was achieved by lentivirus; ERK siRNA was utilized. Competitive RT-PCR was performed for MCL-1 mRNA expression. HT-29 cells with control or MCL-1 shRNA were xenografted into SCID mice, treated with cobimetinib or vehicle, and tumor volume was measured. Results: We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using CRC cell lines isogenic for BRAF. BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by ERK siRNA that suppressed MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively.MEK /ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced pro-apoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown vs control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. A-1210477 also enhanced cobimetinib-induced apoptosis in vitrothat was due to disruption of the interaction of MCL-1 with pro-apoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. Conclusions: BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism.

Carfilzomib Inhibits Constitutive NF-κB Activation in Mantle Cell Lymphoma B Cells and Leads to the Induction of Apoptosis

2017 ◽  
Vol 137 (2) ◽  
pp. 106-112 ◽  
Author(s):  
Yong-Li Zhang ◽  
Matthew Ho Zhi Guang ◽  
Hui-Qin Zhuo ◽  
Xiang-Hui Min ◽  
Qin Yao ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Cell Lymphoma ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Mantle Cell ◽  
Induced Apoptosis

Mantle cell lymphoma (MCL) remains incurable and new treatments are needed, especially in the relapsed/refractory setting. We therefore investigated the effects of carfilzomib, a novel, long-acting, second-generation proteasome inhibitor, in MCL cells. Eight established MCL cell lines and freshly isolated primary MCL cells were treated with carfilzomib. Cell proliferation was assessed by a 3H-thymidine incorporation assay. Cell apoptosis was evaluated by flow cytometry with annexin V and propidium iodide. Electrophoresis mobility shift (EMSA), Western blot, and luciferase assays were used to analyze NF-κB activation and related signaling proteins. Carfilzomib inhibited growth and induced apoptosis in both established MCL cell lines and freshly isolated primary MCL cells in a dose-dependent manner. In contrast, carfilzomib was less toxic to normal peripheral blood mononuclear cells from healthy individuals. The carfilzomib-induced apoptosis of MCL cells occurred in a caspase-dependent manner through both intrinsic and extrinsic caspase pathways. In addition, carfilzomib inhibited constitutive activation of the NF-κB signaling cascade, both in MCL cell lines and primary MCL cells, by completely blocking the phosphorylation of IκBα. Our results demonstrate that carfilzomib can induce growth arrest and apoptosis in MCL cells and that the mechanism may involve the NF-κB signaling pathway.

Poly-L-arginine: Enhancing Cytotoxicity and Cellular Uptake of Doxorubicin and Necrotic Cell Death

2019 ◽  
Vol 18 (10) ◽  
pp. 1448-1456 ◽  
Author(s):  
Bahareh Movafegh ◽  
Razieh Jalal ◽  
Zobeideh Mohammadi ◽  
Seyyede A. Aldaghi
Keyword(s):  
Cell Death ◽  
Cellular Uptake ◽  
Combined Treatment ◽  
Annexin V ◽  
Cell Penetrating Peptides ◽  
Short Exposure ◽  
Dependent Manner ◽  
Du145 Cells ◽  
Induced Apoptosis ◽  
Resistance To Doxorubicin

Objective: Cell resistance to doxorubicin and its toxicity to healthy tissue reduce its efficiency. The use of cell-penetrating peptides as drug delivery system along with doxorubicin is a strategy to reduce its side effects. In this study, the influence of poly-L-arginine on doxorubicin cytotoxicity, its cellular uptake and doxorubicin-induced apoptosis on human prostate cancer DU145 cells are assessed. Methods: The cytotoxicity of doxorubicin and poly-L-arginine, alone and in combination, in DU145 cells was evaluated at different exposure times using MTT assay. The influence of poly-L-arginine on doxorubicin delivery into cells was evaluated by fluorescence microscopy and ultraviolet spectroscopy. DAPI and ethidium bromide- acridine orange stainings, flow cytometry using annexin V/propidium iodide, western blot analysis with anti-p21 antibody and caspase-3 activity were used to examine the influence of poly-L-arginine on doxorubicininduced cell death. Results: Poly-L-arginine had no cytotoxicity at low concentrations and short exposure times. Poly-L-arginine increased the cytotoxic effect of doxorubicin in DU145 cells in a time-dependent manner. But no significant reduction was found in HFF cell viability. Poly-L-arginine seems to facilitate doxorubicin uptake and increase its intracellular concentration. 24h combined treatment of cells with doxorubicin (0.5 µM) and poly-L-arginine (1 µg ml-1) caused a small increase in doxorubicin-induced apoptosis and significantly elevated necrosis in DU145 cells as compared to each agent alone. Conclusion: Our results indicate that poly-L-arginine at lowest and highest concentrations act as proliferationinducing and antiproliferative agents, respectively. Between these concentrations, poly-L-arginine increases the cellular uptake of doxorubicin and its cytotoxicity through induction of necrosis.

Cytotoxic Effects of Ferula Latisecta on Human Glioma U87 Cells

Drug Research ◽  
2019 ◽  
Vol 69 (12) ◽  
pp. 665-670 ◽  
Author(s):  
Mohammad Jalili-Nik ◽  
Hamed Sabri ◽  
Ehsan Zamiri ◽  
Mohammad Soukhtanloo ◽  
Mostafa Karimi Roshan ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Gelatin Zymography ◽  
Annexin V ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Induced Apoptosis ◽  
Natural Herb ◽  
First Time ◽  
U87 Cells

AbstractGlioblastoma multiforme (GBM) is the fatal type of astrocytic tumors with a survival rate of 12 months. The present study, for the first time, evaluated the cytotoxic impacts of Ferula latisecta (F. latisecta) hydroalcoholic extract on U87 GBM cell line. The MTT assay measured the cellular toxicity following 24- and 48 h treatment with various doses of F. latisecta (0–800 μg/mL). Apoptosis was evaluated by an Annexin V/propidium iodide (PI) staining 24 h after treatment by F. latisecta. Moreover, to determine the cellular metastasis of U87 cells, we used a gelatin zymography assay (matrix metalloproteinase [MMP]-2/-9 enzymatic activity). The outcomes showed that F. latisecta mitigated the viability of U87 cells in a concentration- and time-dependent manner with IC50 values of 145.3 and 192.3 μg/mL obtained for 24- and 48 h treatments, respectively. F. latisecta induced apoptosis in a concentration-dependent manner after 24 h. Also, MMP-9 activity was significantly decreased following 24 h after treatment concentration-dependently with no change in MMP-2 enzymatic activity. This study showed that F. latisecta induced cytotoxicity and apoptosis, and mitigated metastasis of U87 GBM cells. Hence, F. latisecta could be beneficial as a promising natural herb against GBM after further studies.

scholarly journals Impact of GADD34 on Apoptosis of Tonsillar Mononuclear Cells from IgA Nephropathy Patients by Regulating Eif2α Phosphorylation

2018 ◽  
Vol 50 (6) ◽  
pp. 2203-2215 ◽  
Author(s):  
Xiaofei Peng ◽  
Tao Yang ◽  
Liyu He ◽  
Xian Chen ◽  
Yafeng Jiang ◽  
...  
Keyword(s):  
Western Blotting ◽  
Cell Apoptosis ◽  
Mononuclear Cells ◽  
Gradient Centrifugation ◽  
Annexin V ◽  
Enzyme Linked Immunosorbent Assay ◽  
Eif2α Phosphorylation ◽  
Significant Difference ◽  
Mrna And Protein Expression ◽  
Sirna Interference

Background/Aims: Tonsillectomy may be an important method to achieve a long-term remission of IgAN, but patients’ physical status may limit their access to this surgery. We proposed an encouraging solution through inhibiting GADD34 expression in order to promote tonsillar mononuclear cells (TMCs) apoptosis and reduce nephropathic IgA secretion. Methods: A total of 12 IgAN and 9 non-IgAN patients were involved from March 2015 to May 2016. After TMCs were extracted by density gradient centrifugation and stimulated by inactivated hemolytic streptococcus, the mRNA and protein expression of GADD34, GRP78, CHOP, Bcl-2, Bcl-XL, AID, Iα-Cα, and cleaved caspase-3 were examined by fluorescent RT-PCR and Western blotting. Guanabenz treatment and siRNA interference were applied to downregulate GADD34 in tonsillar mononuclear cells from IgAN patients, and P-eIF2α expression was examined by Western Blotting. Cell apoptosis was evaluated by Annexin V FITC/PI flowcytometry, and IgA secretion in cultural supernatant was inspected by enzyme linked immunosorbent assay. Results: After stimulation, the expression of GADD34 was significantly increased in IgAN patients (P< 0.05). Cell apoptosis was mitigated and IgA secretion level was elevated (P< 0.05). To be noticed, CHOP expression had no significant difference between two groups. After guanabenz treatment and siRNA interference, a prolonged elevation of P-eIF2α expression was observed. Cell apoptosis was reinforced and IgA secretion level was decreased (P< 0.05). Conclusion: GADD34 may be a potential therapeutic target for IgAN treatment due to its effect on cell apoptosis.

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