scholarly journals Diclofenac Induces Apoptosis and Suppresses Diffuse Large B-Cell Lymphoma Proliferation Independent of P53 Status

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5485-5485
Author(s):  
Hesham Hassan ◽  
Michelle Varney ◽  
Bhavana J Dave ◽  
Rakesh K Singh
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Flow Cytometric Analysis ◽  
B Cell Lymphoma ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Flow Cytometric ◽  
Large B Cell Lymphoma ◽  
P53 Status

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL).Despite long-term remission achieved with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone), relapse occurs in almost one third of the patients. Therefore, there is a need for novel therapeutic targets that are relevant to DLBCL pathogenesis. TP73 gene is a member of the p53 tumor suppressor gene family, which is critical in the regulation of cell cycle and apoptosis. TP73 is located in distal 1p36 chromosomal region that is commonly disrupted in DLBCL. Our previous studies had shown that the differential expression of p73 isoforms correlates with proliferation and apoptosis in DLBCL patient specimens. Furthermore, the experimental modulation of p73 isoforms using expression vectors or siRNA modulates the behavior and regulate the chemotherapeutic response of DLBCL cell line models. Diclofenac is NSAID that has been shown to increase p73 activity, substitute p53 activity and suppress the growth of neuroblastoma. In the present study, we investigated whether diclofenac modulates DLBCL apoptosis and cell cycle progression independent of p53 status. We used cell line models of the GCB-DLBCL (DHL-16 and OCI-Ly7) and the ABC-DLBCL (OCI-Ly3 and Pfeiffer). Because OCI-Ly7 and Pfeiffer have a mutant p53, these cells can model the activity of diclofenac in the presence of mutant p53. We used MTT assay to study the response of the DLBCL cells to various concentrations of diclofenac (25, 50, 100, 150, 200, 250 µM) and at different time points (24, 48, and 72 hours). To decipher the biological effects of diclofenac treatment on DLBCL cells Hema-3 staining was done to visualize morphologic evidence of cell death; propidium iodide-based flow cytometric analysis for cell cycle progression; BrdU incorporation for proliferation; and Annexin-V-Flous flow cytometric analysis for apoptosis. Molecularly, Caspase-GLO assay was used for evaluation of Caspase-3, 7, 8 activity and qRT-PCR was used to estimate the effect of diclofenac treatment on p73 and the p53 family transcriptional target regulating cell cycle (p21) and apoptosis (PUMA, NOXA, BIM, and CD95). Mann-Whitney (for two groups) or ANOVA (for more than two groups) analyses were used to determine the statistical significance for comparisons between different treatment groups. Diclofenac treatment displayed a concentration and duration-dependent suppressive cell proliferative activity against a panel of DLBCL cells independent of p53 status including experimental therapy-resistant models. Diclofenac treatment resulted in cell cycle arrest mainly at the G2/M phase, decreased proliferation, and caused profound cell death (mainly apoptosis and possibly necroptosis). Molecularly, diclofenac treatment was associated with increased activity of caspases- 3, -7 and -8. Increased p53 pathway activity as suggested by induction of expression of a panel of p53 transcriptional targets including the cell cycle regulatory molecule p21 and the pro-apoptotic molecules, PUMA, NOXA, BIM, and CD95, was detected in diclofenac treated DLBCL cells. More importantly diclofenac treatment was associated with enhanced expression of the pro-apoptotic isoforms of the p53 homologue, TAp73. Together, our data demonstrate that clinically non-toxic doses of diclofenac treatment, induces apoptosis and cell cycle arrest of both GCB and ABC-DLBCL cells independent of p53 status and is associated with increased expression of the p73 homologue TAp73. These data highlight the potential of diclofenac as a novel adjuvant therapy in DLBCL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-Tumor Effect of Rutin on Human Neuroblastoma Cell Lines through Inducing G2/M Cell Cycle Arrest and Promoting Apoptosis

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Hongyan Chen ◽  
Qing Miao ◽  
Miao Geng ◽  
Jing Liu ◽  
Yazhuo Hu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Apoptosis ◽  
Cell Cycle Progression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Human Neuroblastoma ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Flow Cytometric

Aims. To further investigate the antineuroblastoma effect of rutin which is a type of flavonoid.Methods. The antiproliferation of rutin in human neuroblastoma cells LAN-5 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Chemotaxis of LAN-5 cells was assessed using transwell migration chambers and scratch wound migration assay. The cell cycle arrest and apoptosis in a dose-dependent manner was measured by flow cytometric and fluorescent microscopy analyses. The apoptosis-related proteins BAX and BCL2 as well as MYCN mRNA express were determined by RT-PCR analysis. Secreted TNF-αlevel were determined using specific enzyme-linked immunosorbent assay kits.Results. Rutin significantly inhibited the growth of LAN-5 cells and chemotactic ability. Flow cytometric analysis revealed that rutin induced G2/M arrest in the cell cycle progression and induced cell apoptosis. The RT-PCR showed that rutin could decrease BCL2 expression and BCL2/BAX ratio. In the meantime, the MYCN mRNA level and the secretion of TNF-αwere inhibited.Conclusion. These results suggest that rutin produces obvious antineuroblastoma effects via induced G2/M arrest in the cell cycle progression and induced cell apoptosis as well as regulating the expression of gene related to apoptosis and so on. It supports the viability of developing rutin as a novel therapeutic prodrug for neuroblastoma treatment, as well as providing a new path on anticancer effect of Chinese traditional drug.

scholarly journals SYK inhibition and response prediction in diffuse large B-cell lymphoma

Blood ◽  
2011 ◽  
Vol 118 (24) ◽  
pp. 6342-6352 ◽  
Author(s):  
Shuhua Cheng ◽  
Greg Coffey ◽  
X. Hannah Zhang ◽  
Rita Shaknovich ◽  
Zibo Song ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Cycle Arrest ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G1-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.

scholarly journals Coix lacryma-jobi var. ma-yuen Stapf sprout extract induces cell cycle arrest and apoptosis in human cervical carcinoma cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Eun Suk Son ◽  
Se-Hee Kim ◽  
Young Ock Kim ◽  
Young Eun Lee ◽  
Sun Young Kyung ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Hela Cells ◽  
Flow Cytometric Analysis ◽  
B Cell Lymphoma ◽  
Cell Counting ◽  
Cycle Arrest ◽  
Flow Cytometric

Abstract Background Cervical cancer is the second-leading cause of cancer-related mortality in females. Coix lacryma-jobi L. var. ma-yuen (Rom.Caill.) Stapf ex Hook. f. is the most widely recognized medicinal herb for its remedial effects against inflammation, endocrine system dysfunctions, warts, chapped skin, rheumatism, and neuralgia and is also a nourishing food. Methods To investigate the activity of Coix lacryma-jobi sprout extract (CLSE) on cell proliferation in human cervical cancer HeLa cells, we conducted a Cell Counting Kit-8 (CCK-8) assay. Flow-cytometric analysis and western blot analysis were performed to verify the effect of CLSE on the regulation of the cell cycle and apoptosis in HeLa cells. Results We observed that CLSE significantly inhibited cell proliferation. Furthermore, CLSE dose-dependently promoted cell cycle arrest at the sub-G1/ S phase in HeLa cells, as detected by bromodeoxyuridine (BrdU) staining. The cell-cycle-arrest effects of CLSE in HeLa cells were associated with downregulation of cyclin D1 and cyclin-dependent kinases (CDKs) 2, 4, and 6. Moreover, CLSE induced apoptosis, as determined by flow-cytometric analysis and nuclear DNA fragmentation with Annexin V/propidium iodide (PI) and 4′6′-diamidino-2-phenylindole (DAPI) staining. Induction of apoptosis by CLSE was involved in inhibition of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) and upregulation of the apoptotic proteins p53, cleaved poly (ADP-ribose) polymerase (PARP), cleaved caspase-3, and cleaved caspase-8. Finally, we observed that CLSE inactivated the phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) pathways. Conclusions CLSE causes cell cycle arrest and apoptotic cell death through inactivation of the PI3K/AKT pathway in HeLa cells, suggesting it is a viable therapeutic agent for cervical cancer owing to its anticancer effects.

STAT3 Regulates the Growth and Immunoglobulin Production of BCL1 B Cell Lymphoma through Control of Cell Cycle Progression

2000 ◽  
Vol 202 (2) ◽  
pp. 124-135 ◽  
Author(s):  
James G. Karras ◽  
Robert A. McKay ◽  
Tao Lu ◽  
Jaclyn Pych ◽  
David A. Frank ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Immunoglobulin Production ◽  
Cycle Progression

The Small Molecule CHK1/CHK2 Inhibitor PF-0477736 (Pfizer) Demonstrates Single Agent Activity and Synergizes with Chemotherapy in Diffuse Large B-Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2732-2732
Author(s):  
Enrico Derenzini ◽  
Ilaria Iacobucci ◽  
Elisa Brighenti ◽  
Federica Cattina ◽  
Richard Eric Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Cycle Arrest ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 2732 The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the signal transduction mechanims of the DNA damage response pathway. Once activated by upstream kinases [Ataxia-Telangiectasia mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases] following DNA damage, they phosphorylate downstream targets such as CDC25 phosphatases and p53, promoting G2/M cell cycle arrest, in order to facilitate DNA repair. Furthermore is now clear that the efficacy of conventional DNA-damaging anticancer drugs is limited by the activity of these protective cell cycle checkpoints. The tumor suppressor p53 is activated in normal cells following extensive DNA damage and promotes G1 cell cycle arrest and apoptosis. Cells lacking p53 activity are more resistant to genotoxic agents. It has been shown that CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, by inhibiting the response to DNA damage, preferentially in p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL harboring p53 mutations and/or CDKN2A loss have been recently shown to have a dismal outcome, being refractory to conventional antracyclin-based chemotherapy. Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a large panel of B cell lymphoma cell lines, and explore its mechanisms of action. Nine cell lines were used for in vitro viability assays: 3 Germinal center (GCB) Diffuse Large B-cell lymphoma (DLBCL) derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2. All the cell lines were screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but an homozygous deletion of CDKN2A was detected. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were first incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs of incubation, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with PF-0477736 10nM for 24 hours. The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 250–500 nM induced cell death by apoptosis in a time and dose dependent manner after 24 and 48 hours of incubation. Lower concentrations of PF-0477736 (25–50 nM) promoted a statistically significant increase in cell death only in the BJAB cells. For functional studies we characterized the two most sensitive cell lines (BJAB and U2932) and the two resistant cell lines (SUDHL-4 and KM-H2). Inhibition of cdc25c ser216 phosphorylation was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25–250 nM). A marked increase in levels of the DNA damage marker γH2AX, was detected in the BJAB, U2932, SUDHL-4 cell lines after 24 hrs. KM-H2 did not show any increase of γH2AX following treatment. All the cell lines demonstrated baseline CHK1 activation but there was no correlation with outcome. Interestingly levels of baseline pcdc25c ser216 were higher in the sensitive BJAB and U2932 cells. PF-0477736 at the fixed dose of 50 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) in the BJAB and U2932 cells at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The integrity of the p53 axis seems to be the major determinant of efficacy of PF-0477736. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome. Our study provides the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Disclosures: No relevant conflicts of interest to declare.

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