The Bispidinone Derivative 3,7-Bis-[2-(S)-amino-3-(1H-indol-3-yl)-propionyl]-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one Dihydrochloride Induces an Apoptosis-Mediated Cytotoxic Effect on Pancreatic Cancer Cells In Vitro

Pancreatic cancer (PC) is a complex, heterogeneous disease with a dismal prognosis. Current therapies have failed to improve survival outcomes, urging the need for discovery of novel targeted treatments. Bispidinone derivatives have yet to be investigated as cytotoxic agents against PC cells. The cytotoxic effect of four bispidinone derivatives (BisP1: 1,5-diphenyl-3,7-bis(2-hydroxyethyl)-3,7-diazabicyclo[3.3.1]nonan-9-one; BisP2: 3,7-bis-(2-(S)-amino-4-methylsulfanylbutyryl)-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride; BisP3: [2-{7-[2-(S)-tert-butoxycarbonylamino-3-(1H-indol-3-yl)-propionyl]-9-oxo-1,5-diphenyl-3,7-diazabicyclo[3.3.1]non-3-yl}-1-(S)-(1H-indol-3-ylmethyl)-2-oxoethyl]-carbamic acid tertbutyl ester; BisP4: 3,7-bis-[2-(S)-amino-3-(1H-indol-3-yl)-propionyl]-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride) was assessed against PC cell lines (MiaPaca-2, CFPAC-1 and BxPC-3). Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) colorimetric assay, while apoptotic cell death was confirmed using fluorescence microscopy and flow cytometry. Initial viability screening revealed significant cytotoxic activity from BisP4 treatment (1 µM–100 µM) on all three cell lines, with IC50 values for MiaPaca-2, BxPC-3, and CFPAC-1 16.9 µM, 23.7 µM, and 36.3 µM, respectively. Cytotoxic treatment time-response (4 h, 24 h, and 48 h) revealed a 24 h treatment time was sufficient to produce a cytotoxic effect on all cell lines. Light microscopy evaluation (DAPI staining) of BisP4 treated MiaPaca-2 PC cells revealed dose-dependent characteristic apoptotic morphological changes. In addition, flow cytometry confirmed BisP4 induced apoptotic cell death induction of activated caspase-3/-7. The bispidinone derivative BisP4 induced an apoptosis-mediated cytotoxic effect on MiaPaca-2 cell lines and significant cytotoxicity on CFPAC-1 and BxPC-3 cell lines. Further investigations into the precise cellular mechanisms of action of this class of compounds are necessary for potential development into pre-clinical trials.

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Effect of hyperthermia combined with gemcitabine on apoptotic cell death in cultured human pancreatic cancer cell lines

International Journal of Hyperthermia ◽

10.1080/02656730802657036 ◽

2009 ◽

Vol 25 (3) ◽

pp. 210-219 ◽

Cited By ~ 46

Author(s):

Satoko Adachi ◽

Satoshi Kokura ◽

Tetsuya Okayama ◽

Takeshi Ishikawa ◽

Tomohisa Takagi ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cell Death ◽

Cell Lines ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Apoptotic Cell ◽

Cancer Cell Lines ◽

Apoptotic Cell Death ◽

Human Pancreatic Cancer ◽

Human Pancreatic Cancer Cell

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Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines

International Journal of Oncology ◽

10.3892/ijo.2015.3237 ◽

2015 ◽

Vol 48 (1) ◽

pp. 45-54 ◽

Cited By ~ 19

Author(s):

SHUNTARO MUKAI ◽

SHOTA MORIYA ◽

MASAKI HIRAMOTO ◽

HIROMI KAZAMA ◽

HIROKO KOKUBA ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cell Death ◽

Cell Lines ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Apoptotic Cell ◽

Cancer Cell Lines ◽

Apoptotic Cell Death ◽

Autophagy Flux ◽

Egfr Tki

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Britannin, a Sesquiterpene Lactone Induces ROS-Dependent Apoptosis in NALM-6, REH, and JURKAT Cell Lines And Produces a Synergistic Effect With Vincristine

10.21203/rs.3.rs-369748/v1 ◽

2021 ◽

Author(s):

Hassan Mohammadlou ◽

Maryam Hamzeloo-Moghadam ◽

Mohammad Hossein Mohammadi ◽

Amir Yami ◽

Ahmad Gharehbaghian

Keyword(s):

Cell Death ◽

Sesquiterpene Lactone ◽

Cell Lines ◽

Cytotoxic Effect ◽

Apoptotic Cell ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Apoptotic Cell Death ◽

Dependent Manner ◽

Normal Peripheral Blood

Abstract Britannin, a Sesquiterpene Lactone isolated from Inula aucheriana, has recently gained attractions in the therapeutic fields due to its vast cytotoxic properties in different types of cancers. This study was designed to evaluate the cytotoxic effect of this agent on Acute Lymphoblastic Leukemia (ALL) cell lines, either as a monotherapy or in combination with Vincristine (VCR). The results obtained in this study showed that while Britannin reduced the viability of ALL cell lines such as NALM-6, REH, and JURKAT cells, it did not exert cytotoxicity against normal Peripheral Blood Mononuclear Cells (PBMCs) and L929 cells. Among tested cells, pre-B ALL-derived NALM-6 cells had the highest sensitivity to Britannin. Moreover, we found that Britannin induced p21/p27-mediated G1 cell cycle arrest cells and Reactive Oxygen Specious (ROS)-mediated apoptotic cell death in NALM-6 cells. When NALM-6 cells were treated with N-acetyl-L-Cysteine (NAC), a scavenger of ROS, we found that Britannin could induce neither apoptosis nor reduce the survival of the cells, suggesting that the cytotoxic effect of Britannin is induced through ROS-dependent manner. The cytotoxic effect of Britannin also was potentiated by autophagy suppression using Chloroquine (CQ). Moreover, we found that a low dose of Britannin enhanced the effect of Vincristine in NALM-6 cells by inducing apoptotic cell death via altering the expression of apoptotic-related genes. Overall, our results proposed a mechanism for the cytotoxic effect of Britannin, either as a single agent or in combination with Vincristine, in NALM-6 cells.

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Inhibition of Replication Induces Non-Apoptotic Cell Death in Fibroblast Cell Lines Derived from LEC Rats

Journal of Veterinary Medical Science ◽

10.1292/jvms.65.249 ◽

2003 ◽

Vol 65 (2) ◽

pp. 249-254 ◽

Cited By ~ 1

Author(s):

Masanobu HAYASHI ◽

Taku HAMASU ◽

Daiji ENDOH ◽

Reiko SHIMOJIMA ◽

Toyo OKUI

Keyword(s):

Cell Death ◽

Cell Lines ◽

Apoptotic Cell ◽

Fibroblast Cell ◽

Apoptotic Cell Death ◽

Lec Rats ◽

Fibroblast Cell Lines

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ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

Infection and Immunity ◽

10.1128/iai.00651-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4600-4608 ◽

Cited By ~ 40

Author(s):

Karin Heine ◽

Sascha Pust ◽

Stefanie Enzenmüller ◽

Holger Barth

Keyword(s):

Cell Death ◽

Cell Lines ◽

Mammalian Cells ◽

Clostridium Botulinum ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Adp Ribosylation ◽

Mammalian Cell Lines ◽

Adp Ribosyltransferase ◽

C2 Toxin

ABSTRACT The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.

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Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase.

Journal of Experimental Medicine ◽

10.1084/jem.180.4.1547 ◽

1994 ◽

Vol 180 (4) ◽

pp. 1547-1552 ◽

Cited By ~ 373

Author(s):

M G Cifone ◽

R De Maria ◽

P Roncaioli ◽

M R Rippo ◽

M Azuma ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Intracellular Signaling ◽

Apoptotic Cell ◽

Human Tumor ◽

Apoptotic Cell Death ◽

Cell Surface Receptor ◽

Cross Linking ◽

Cell Extracts

Intracellular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monoclonal antibody was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase (SMase) in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death.

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Flow Cytometry and FTIR Spectroscopy for Detection of Early Apoptosis in Human T Cells

Proceedings ◽

10.3390/proceedings2251558 ◽

2018 ◽

Vol 2 (25) ◽

pp. 1558 ◽

Cited By ~ 1

Author(s):

Günnur Güler ◽

Ayten Nalbant

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Cell Death ◽

Ftir Spectroscopy ◽

Apoptotic Cell ◽

Annexin V ◽

Apoptotic Cell Death ◽

Biochemical Changes ◽

Human T Cells ◽

Early Apoptosis

Apoptosis, a programmed cell death, has a vital role in various cellular processes. Apoptotic cells exhibit morphological and biochemical changes, detected by a variety of assays (caspases, mitochondrial dyes, DNA laddering). Flow cytometry is a powerful tool for detection of apoptotic cell death and allows information about the cell size and molecules associated with cell-bound antibodies. Recently, Fourier transform infrared (FTIR) spectroscopy as rapid and low-cost tool has been extensively used for cellular studies, providing information on cellular structures. The aim of this study was to detect early apoptosis and obtain further insights into the capability of FTIR spectroscopy, comparing the results with flow cytometry. In this study, apoptotic cell death was induced in human Jurkat T cells with Camptothecin (CPT), a DNA topoisomerase I inhibitor. Cells were cultured with 4µM CPT in RPMI (with 5% FCS) for 24 h. Immunoflourescence labeling for multicolor flow cytometry was accomplished with Annexin V concomitantly with 7-AAD. The same cells were also analyzed with ATR-FTIR spectroscopy. Flow cytometry data represents that the cells are Annexin V positive but 7AAD negative. This indicates that cells are in the early apoptotic stage, only externalization of phosphatidylserine exists on the plasma membrane. FTIR data reveals that membrane phospholipids and proteins undergo changes; fatty acid acyl chains are disordered and increased in mobility after treatment, which result from the early apoptosis process after CPT-treatment, confirmed by the flow cytometry. A combined study of flow cytometry and FTIR spectroscopy for analysis of apoptosis in human T cells exhibited compatible and complementary results. Existence of biophysical and biochemical changes in T cells after treatment were also demonstrated.

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