scholarly journals Sensitization of breast cancer cells to doxorubicin via stable cell line generation and overexpression of DFF40

2015 ◽  
Vol 93 (6) ◽  
pp. 604-610 ◽  
Author(s):  
Fatemeh Bagheri ◽  
Shahrokh Safarian ◽  
Mohamadreza Baghaban Eslaminejad ◽  
Nader Sheibani
Keyword(s):  
Cell Cycle ◽  
Annexin V ◽  
Eukaryotic Expression ◽  
Stable Cell Line ◽  
Cell Cycle Distribution ◽  
Distribution Analysis ◽  
Transfected Cells ◽  
Dna Laddering ◽  
Stable Cell Line Generation ◽  
Cell Line Generation

There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.

Silencing of the DEK gene induces apoptosis and senescence in CaSki cervical carcinoma cells via the up-regulation of NF-κB p65

2012 ◽  
Vol 32 (3) ◽  
pp. 323-332 ◽  
Author(s):  
Kuiran Liu ◽  
Tianda Feng ◽  
Jie Liu ◽  
Ming Zhong ◽  
Shulan Zhang
Keyword(s):  
Cell Cycle ◽  
Nuclear Translocation ◽  
Annexin V ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Cell Senescence ◽  
Stable Cell Line ◽  
Reverse Transcription Pcr ◽  
Dna Binding Activity ◽  
Nuclear Extracts

The human DEK proto-oncogene has been found to play an important role in autoimmune disease, viral infection and human carcinogenesis. Although it is transcriptionally up-regulated in cervical cancer, its intracellular function and regulation is still unexplored. In the present study, DEK and IκBα [inhibitor of NF-κB (nuclear factor κB) α] shRNAs (short hairpin RNAs) were constructed and transfected into CaSki cells using Lipofectamine™. The stable cell line CaSki–DEK was obtained after G418 selection. CaSki–IκB cells were observed at 48 h after psiRNA-IκB transfection. The inhibitory efficiency of shRNAs were detected by RT (reverse transcription)–PCR and Western blot analysis. The proliferation activity of cells were measured using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, cell apoptosis was measured using an Annexin V/PI (propidium iodide) kit, the cell cycle was analysed by flow cytometry and cell senescence was detected using senescence β-galactosidase staining. The intracellular expression of NF-κB p65 protein was studied by cytochemistry. The expression levels of NF-κB p65, p50, c-Rel, IκBα and phospho-IκBα protein were analysed by immunoblotting in whole-cell lysates, cytosolic fractions and nuclear extracts. The protein expression and activity of p38 and JNK (c-Jun N-terminal kinase) were also assayed. In addition, the NF-κB p65 DNA-binding activity was measured by ELISA. Following the silencing of DEK and IκBα, cell proliferation was inhibited, apoptosis was increased, the cell cycle was blocked in the G0/G1-phase with a corresponding decrease in the G2/M-phase, and cell senescence was induced. All of these effects may be related to the up-regulation of NF-κB p65 expression and its nuclear translocation.

Tetraspanin Overexpression Induces Multiple Myeloma Cell Death.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5067-5067
Author(s):  
Tali Tohami ◽  
Liat Drucker ◽  
Judith Radnay ◽  
Hava Shapiro ◽  
Michael Lishner
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Annexin V ◽  
Myeloma Cell ◽  
Transfected Cells ◽  
Over Expression ◽  
Rpmi 8226

Abstract Background: Medullary and extra-medullary dissemination of multiple myeloma (MM) cells involves cell-cell and cell-extracellular matrix (ECM) interactions. Proteins coordinating these intricate networks regulate the signaling cascades in a spatial and time dependent manner. Tetraspanins facilitate multiprotein complexing in defined membranal microdomains and select family members have been identified as metastasis suppressors. In preliminary studies, we observed that tetraspanins CD82, frequently down regulated or lost at the advanced clinical stages of various cancers, was absent in MM (8 BM samples, 5 cell lines) and CD81, characteristically expressed in leukocytes plasma membranes, was under-expressed (4/8 BM samples, 4/5 cell lines). We aimed to investigate the consequences of CD81 and CD82 over-expression in myeloma cell lines. Methods: CAG and RPMI 8226 were transfected with pEGFP-N1/C1 fusion vectors of CD81 and CD82. Transfected cells were assessed for - cell morphology (light and fluorescent microscope); cell survival (eGFP+/PI- cells); cell death (Annexin V/7AAD, pre-G1, activated caspase-3 (IC), caspase dependence with pan caspase inhibitor z-VAD-fmk); cell cycle (PI staining). Results: CD82 induced cell death was determined by morphologic characteristics in stably transfected CAG cells (50%) compared to their mock-transfected counterparts (8%) (p<0.05). Activated caspase-3 was also detected (40% of the CD82 transfected cells) (p<0.05). In CD82 transiently transfected MM cell lines a reduced fraction of surviving cells was observed compared to mocks (~60%) (p<0.05) yet, no increases in pre-G1 or Annexin V+/7AAD- subgroups were observed. Moreover, CD82 induced cell death could not be inhibited by the use of z-VAD-fmk. CD82 transfection did not affect the cell cycle of CAG and RPMI 8226 lines. CD81 stably transfected cell lines (CAG and RPMI 8226) could not be established. Indeed, in transiently transfected cells we determined a massive rate of CD81 induced cell death. This is demonstrated in a surviving fraction of only 10% CAG cells and 30% RPMI 8226 (compared to mock) (p<0.05). The CD81 transfected cells were negative for PS exposure, pre-G1 sub-population, or inhibition of death with z-VAD-fmk. The death inducing effect of both tetraspanins in the two cell lines was evident with the pEGFP-N1 orientation vector only. Conclusions: CD81 and CD82 over-expression in MM cell lines causes cell death. Based on the restriction of the killing effect to the pEGFP-N1 clone it may be speculated that its implementation is either dependent on the interactions of the N1 tetraspanin terminus or the proteins’ conformation. It is of interest that CD81 though normally expressed in RPMI 8226 still induced cell death when over-expressed, possibly indicative of ’negative signaling’. Tetraspanins’ suppressive effects on adhesion, motility, and metastasis in solid tumors combined with its capacity to induce myeloma cell death underscore the significance of its absence in MM cell lines and patients. We suspect that a better understanding of CD81/82 mediated signaling pathways will promote future treatment of myeloma cell in their microenvironment. Current studies designed to assess the involvement of oxidative stress in CD81/CD82 induced death are underway.

scholarly journals The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Andreia Ascenso ◽  
Tiago Pedrosa ◽  
Sónia Pinho ◽  
Francisco Pinho ◽  
José Miguel P. Ferreira de Oliveira ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Annexin V ◽  
Human Keratinocytes ◽  
Protective Role ◽  
Cell Cycle Distribution ◽  
Skin Cells ◽  
A Cell ◽  
Previously Treated ◽  
Annexin V Assay ◽  
And Oxidative Stress

Lycopene has been reported as the antioxidant most quickly depleted in skin upon UV irradiation, and thus it might play a protective role. Our goal was to investigate the effects of preexposure to lycopene on UV-B-irradiated skin cells. Cells were exposed for 24 h to 10 M lycopene, and subsequently irradiated and left to recover for another 24 h period. Thereafter, several parameters were analyzed by FCM and RT-PCR: genotoxicity/clastogenicity by assessing the cell cycle distribution; apoptosis by performing the Annexin-V assay and analyzing gene expression of apoptosis biomarkers; and oxidative stress by ROS quantification. Lycopene did not significantly affect the profile of apoptotic, necrotic and viable cells in nonirradiated cells neither showed cytostatic effects. However, irradiated cells previously treated with lycopene showed an increase in both dead and viable subpopulations compared to nonexposed irradiated cells. In irradiated cells, lycopene preexposure resulted in overexpression ofBAXgene compared to nonexposed irradiated cells. This was accompanied by a cell cycle delay at S-phase transition and consequent decrease of cells in G0/G1 phase. Thus, lycopene seems to play a corrective role in irradiated cells depending on the level of photodamage. Thus, our findings may have implications for the management of skin cancer.

Cell cycle distribution, cellular viability and mRNA expression of hGCase-gene-transfected cells in dairy goat

2010 ◽  
Vol 34 (6) ◽  
pp. 679-685 ◽  
Author(s):  
Yan‑Li Zhang ◽  
Yong‑Jie Wan ◽  
Zi‑Yu Wang ◽  
Wei‑Wei Qi ◽  
Zheng‑Rong Zhou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mrna Expression ◽  
Dairy Goat ◽  
Cell Cycle Distribution ◽  
Cellular Viability ◽  
Transfected Cells

scholarly journals Farrerol Induces Cancer Cell Death via ERK Activation in SKOV3 Cells and Attenuates TNF-α-Mediated Lipolysis

2021 ◽  
Vol 22 (17) ◽  
pp. 9400
Author(s):  
Jongbeom Chae ◽  
Jin Soo Kim ◽  
seok tae Choi ◽  
Seul Gi Lee ◽  
Oyindamola Vivian Ojulari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mapk Pathway ◽  
Adipogenic Differentiation ◽  
Cell Cycle Distribution ◽  
Dependent Manner ◽  
Distribution Analysis ◽  
M Cell ◽  
Cancer Cell Death ◽  
Skov3 Cells ◽  
Tnf Α

Farrerol (FA) is a flavanone isolated from the Chinese herbal medicine “Man-shan-hong” (Rhododendron dauricum L.). In the present study, FA decreased the viability of SKOV3 cells in a dose- and time-dependent manner, and it induced G2/M cell cycle arrest and cell apoptosis. Cell cycle distribution analysis via flow cytometry showed that FA decreased G1 populations and increased G2/M populations in SKOV3 cells. Additionally, Western blotting confirmed an increase in the expression level of proteins involved in the cell cycle, e.g., CDK and cyclins. FA-induced apoptosis in SKOV3 cells was also investigated using a TUNEL assay, and increased expression levels of proapoptotic factors, including Caspase-3 and poly ADP ribose polymerase (PARP), through the Extracellular signal-regulated kinase (ERK)/MAPK pathway were investigated. Proinflammatory cytokines (e.g., IL-6, TNF-α, and IL-1) have been identified as a driver of the pathological mechanisms underlying involuntary weight loss and impaired physical function, i.e., cachexia, during cancer; in the present study, we showed that farrerol attenuates TNF-α-induced lipolysis and increases adipogenic differentiation in 3T3-L1 cells. Thus, farrerol could potentially be used as an anticancer agent or anticachetic drug.

scholarly journals Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

PLoS ONE ◽  
2018 ◽  
Vol 13 (3) ◽  
pp. e0194887 ◽  
Author(s):  
Roman J. Szczesny ◽  
Katarzyna Kowalska ◽  
Kamila Klosowska-Kosicka ◽  
Aleksander Chlebowski ◽  
Ewelina P. Owczarek ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Cell Line ◽  
Stable Cell Line ◽  
Recombination System ◽  
Stable Cell Line Generation ◽  
Human Proteins ◽  
Cell Line Generation ◽  
Line Generation

scholarly journals Distinct Modulatory Effects of Fever-Range Hyperthermia on the Response of Breast Cancer Cells and Macrophages to Mistletoe (Viscum album L.) Extract

2021 ◽  
Vol 14 (6) ◽  
pp. 551
Author(s):  
Henryk M. Kozłowski ◽  
Małgorzata Pawlikowska ◽  
Justyna Sobocińska ◽  
Tomasz Jędrzejewski ◽  
Artur Dzialuk ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Cell Lines ◽  
Breast Cancer Cells ◽  
Inflammatory Factors ◽  
Cell Cycle Distribution ◽  
Distribution Analysis ◽  
Treated Breast ◽  
Treated Breast Cancer

Heat utility as a critical component of fever is often ignored, although the symptom is observed in many medical conditions. Mistletoe extract (ME) is an adjunctive medication prescribed to cancer patients. The increase in body temperature is frequently observed in patients following ME administration. Nevertheless, the impact of this fever on the effectiveness of therapy is unknown. Therefore, we aimed to investigate the effect of fever-range temperatures on ME-treated breast cancer cells and macrophages. The cells were simultaneously stimulated with ME and subjected to fever-range hyperthermia (FRH; 39 °C or 41 °C). After co-treatment, the cell viability, generation of reactive oxygen species (ROS), cell cycle distribution, and production of pro-inflammatory factors (interleukin (IL)-1β, IL-6, and cyclooxygenase (COX)-2) were evaluated. The results showed that the exposure of ME-treated breast cancer cells to FRH at 39 °C resulted in a slight decrease in their viability, whereas FRH of 41 °C enhanced this effect. Only FRH of 41 °C induced minor changes in ROS level in ME-treated breast cancer cell lines. In ME-treated macrophages, FRH stimulated cell proliferation. The cell cycle distribution analysis showed a difference between cells cultured at 39 °C and 41 °C in all examined cell lines. Moreover, hyperthermia at 41 °C completely inhibited the ME-induced increase in IL-1β and IL-6 expression in MCF-7 breast cancer cells, whereas this effect was not observed in 4T1 breast cancer cells. In contrast, in ME-treated macrophages, FRH of 41 °C strongly up-regulated expression of the pro-inflammatory factors. We conclude that fever is an important component of ME therapy that differentially affects cancer and immune cells.

scholarly journals Antibacterial and Cytotoxic Activity of Compounds Isolated fromFlourensia oolepis

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Mariana Belén Joray ◽  
Lucas Daniel Trucco ◽  
María Laura González ◽  
Georgina Natalia Díaz Napal ◽  
Sara María Palacios ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cytotoxic Activity ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Multidrug Resistant ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Cycle Arrest ◽  
M Phase ◽  
Lower Effect

The antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract ofFlourensia oolepiswere evaluated. Bioguided fractionation led to five flavonoids, identified as 2′,4′-dihydroxychalcone (1), isoliquiritigenin (2), pinocembrin (3), 7-hydroxyflavanone (4), and 7,4′-dihydroxy-3′-methoxyflavanone (5). Compound1showed the highest antibacterial effect, with minimum inhibitory concentration (MIC) values ranging from 31 to 62 and 62 to 250 μg/mL, against Gram-positive and Gram-negative bacteria, respectively. On further assays, the cytotoxic effect of compounds1–5was determined by MTT assay on acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) cell lines including their multidrug resistant (MDR) phenotypes. Compound1induced a remarkable cytotoxic activity toward ALL cells (IC50= 6.6–9.9 μM) and a lower effect against CML cells (IC50= 27.5–30.0 μM). Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and by Annexin V/PI staining, respectively. Upon treatment,1induced cell cycle arrest in the G2/M phase accompanied by a strong induction of apoptosis. These results describe for the first time the antibacterial metabolites ofF. oolepisextract, with1being the most effective. This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells, highlighting its potential as a lead compound.

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