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

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.

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

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180412114750 ◽

2019 ◽

Vol 18 (10) ◽

pp. 1448-1456 ◽

Cited By ~ 3

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.

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Anticancer effects of mifepristone on human uveal melanoma cells

Cancer Cell International ◽

10.1186/s12935-021-02306-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Prisca Bustamante Alvarez ◽

Alexander Laskaris ◽

Alicia A. Goyeneche ◽

Yunxi Chen ◽

Carlos M. Telleria ◽

...

Keyword(s):

Cell Death ◽

Cell Growth ◽

Progesterone Receptor ◽

Dna Fragmentation ◽

Cell Lines ◽

Caspase 3 ◽

Annexin V ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Free Dna

Abstract Background Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. Methods The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. Results MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

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

BioMed Research International ◽

10.1155/2019/7298539 ◽

2019 ◽

Vol 2019 ◽

pp. 1-18 ◽

Cited By ~ 3

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.

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

Blood ◽

10.1182/blood.v108.11.3480.3480 ◽

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.

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Combined Pharmacologic Inhibition of Bcl-Xl/Bcl-2 and mTORC1/2 Survival Signals Trigger Apoptosis in BCR-ABL1+in Vitro Models of Blast Crisis Chronic Myelogenous Leukemia (CML-BC), and Primary CD34+/CD38− Stem and CD34+ progenitor Cells From CML-BC Patients

Blood ◽

10.1182/blood.v118.21.2738.2738 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2738-2738

Author(s):

Jason G Harb ◽

Paolo Neviani ◽

Claudia S Huettner ◽

Guido Marcucci ◽

Danilo Perrotti

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Colony Formation ◽

Mitochondrial Membrane ◽

Kinase Inhibitors ◽

Blast Crisis ◽

Combined Treatment ◽

Annexin V ◽

Clonogenic Potential ◽

Induced Apoptosis

Abstract Abstract 2738 Tyrosine kinase inhibitors (TKIs) have become frontline therapy for CML; however, alternative therapies are required, as TKIs do not induce long-term response in CML patients undergoing blastic transformation and are ineffective against Philadelphia-positive (Ph+) quiescent stem cells, which show innate resistance to BCR-ABL1 kinase inhibitors. Therapeutic targets of interest are survival factors conferring resistance to TKI-induced apoptosis and/or those increasing proliferation of leukemic progenitors. We previously reported (Harb JG et al., ASH 2007) that genetic inactivation of Bcl-x did not inhibit BCR-ABL1 leukemogenesis in an inducible mouse model of CML. Thus, we hypothesize that BCR/ABL mediated post-translational modification and inactivation of pro-apoptotic BAD negates the requirement for the anti-apoptotic function of Bcl-xL in stem/progenitor cells from SCLtTA-BCR/ABL1/Bcl-x−/− mice. Following this rationale, we tested if simultaneous pharmacologic BAD activation and Bcl-xL inhibition may be an efficient way of killing CML stem/progenitor cells. To test this, loss of Bcl-xL function with increased levels of active BAD was achieved by expressing Bcl-x shRNA in 32D-BCR/ABL mouse myeloid precursors that were then treated with LY294002 (LY), which suppresses the inhibitory effects of PI-3K/Akt activation on BAD. Flow cytometric analysis of Annexin V+ cells revealed that levels of apoptosis were three times higher in BCR-ABL1+ cells expressing the Bcl-x shRNA when compared with vector-transduced BCR-ABL1+ cells. As expected, increased levels of dephosphorylated (active) BAD at the mitochondrial membrane were found in LY-treated BCR-ABL+ cells. Interestingly, co-treatment of Bcl-x shRNA-expressing BCR-ABL1+ cells with LY and the Bcl-xL/Bcl-2 antagonist ABT-263 (ABT) did not further promote apoptosis, suggesting that decreased survival of BCR-ABL1+ cells was dependent on downregulation of Bcl-xL and not Bcl-2. To determine efficacy of combined pharmacologic Bcl-xL inhibition and BAD activation, 32D-BCR/ABL and K562 cells were treated with compounds expected to activate BAD upon inhibition of PI-3K/Akt/mTOR-generated signals, used alone or in combination with ABT. Individually, at suboptimal doses, LY, Rapamycin (RAP), mTORC1/2 inhibitor PP242, and ABT were tolerated with apoptosis levels lower than 20%. Notably, when combined with ABT, all three efficiently induced apoptosis (∼90% Annexin V+) of BCR-ABL1+ cells. As with LY, increased levels of active BAD were found at the mitochondrial membrane of RAP- and PP242-treated BCR-ABL1+ cells. We found that PP242 downregulated p-Akt (92%), Mcl-1 (67%) and Bcl-xL (51%) more efficiently than RAP or LY. It has been shown that PP242 impairs the clonogenic potential of TKI-resistant mononuclear BM CML-BC cells; however, its effects when used alone or in combination with ABT on survival of normal and leukemic hematopoietic stem (HSCs) and progenitor cells is still unknown. Thus, HSC-enriched (CD34+/CD38-) and progenitor (CD34+) CML-BC cell fractions were isolated from bone marrow and peripheral blood and used in colony forming (CFC) assays with ABT, PP242 or ABT/PP242. ABT alone did not suppress colony formation of Ph+ CD34+/CD38− cells, while PP242 reduced it by nearly 50%. Conversely, ABT/PP242 combination decreased Ph+ stem and progenitor colony formation by ∼80%. Furthermore, the self-renewal of Ph+ CD34+/CD38− cells was markedly impaired by ABT/PP242 as demonstrated by the 80% decrease in replating efficiency. To assess if non-leukemic stem cells would tolerate ABT/PP242, colony assays were performed with LSK from wild type mice treated with ABT, PP242, RAP and ABT/PP242. We did not find a significant effect of ABT or PP242 on clonogenic potential when given as single agents. More importantly, combined treatment decreased CFC output by only 35% while RAP, which has an acceptable toxicity profile as it has been used in clinical trials for patients unresponsive to TKIs, decreased LSK colony forming potential by 50%. In summary, our data showing that combined treatment with the mTORC1/2 inhibitor/BAD activator PP242 and the BCl-xL/Bcl-2 antagonist ABT-263 markedly induces apoptosis of BCR-ABL+ cell lines, in HSCs and in progenitors from CML-BC patients. This approach warrants further pre-clinical investigation aimed at inclusion in clinical protocols for treating blast crisis CML. Disclosures: No relevant conflicts of interest to declare.

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Dihydroartemisinin-Transferrin Adducts Enhance TRAIL-Induced Apoptosis in Triple-Negative Breast Cancer in a P53-Independent and ROS-Dependent Manner

Frontiers in Oncology ◽

10.3389/fonc.2021.789336 ◽

2022 ◽

Vol 11 ◽

Author(s):

Xinyu Zhou ◽

Abel Soto-Gamez ◽

Fleur Nijdam ◽

Rita Setroikromo ◽

Wim J. Quax

Keyword(s):

Breast Cancer ◽

Cell Death ◽

Cell Lines ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Breast Cancer Subtype ◽

Death Receptor ◽

Dependent Manner ◽

Tnbc Cell ◽

Induced Apoptosis

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype independent of estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2. It has a poor prognosis and high recurrence. Due to its limited treatment options in the clinic, novel therapies are urgently needed. Single treatment with the death receptor ligand TRAIL was shown to be poorly effective. Recently, we have shown that artemisinin derivatives enhance TRAIL-induced apoptosis in colon cancer cells. Here, we utilized transferrin (TF) to enhance the effectiveness of dihydroartemisinin (DHA) in inducing cell death in TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and BT549). We found that the combination of DHA-TF and the death receptor 5-specific TRAIL variant DHER leads to an increase in DR5 expression in all four TNBC cell lines, while higher cytotoxicity was observed in MDA-MB-231, and MDA-MB-436. All the data point to the finding that DHA-TF stimulates cell death in TNBC cells, while the combination of DHA-TF with TRAIL variants will trigger more cell death in TRAIL-sensitive cells. Overall, DHA-TF in combination with TRAIL variants represents a potential novel combination therapy for triple-negative breast cancer.

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Anticancer Effects of Mifepristone on Human Uveal Melanoma Cells

10.21203/rs.3.rs-794697/v1 ◽

2021 ◽

Author(s):

Alexander Laskaris ◽

Prisca Bustamante ◽

Alicia A. Goyeneche ◽

Carlos M. Telleria ◽

Julia V Burnier

Keyword(s):

Cell Death ◽

Cell Growth ◽

Progesterone Receptor ◽

Dna Fragmentation ◽

Cell Lines ◽

Uveal Melanoma ◽

Annexin V ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Free Dna

Abstract Background: Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant cancer. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytostatic and cytotoxic effects of MF in human UM cell lines of different genetic backgrounds.Methods: The effects of incremental concentrations of MF (0, 5, 10, 20, 30 or 40 mM) on a panel of human UM primary (MP46, 92.1, MP41, MEL270) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 hours before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or propidium iodide (PI), caspases 3/7 activities, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by ddPCR, while the expression of progesterone and glucocorticoid receptors was determined by qPCR. Results: MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI-double positive cells, caspase 3/7 activities, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion: This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

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Motexafin Gadolinium Induces Apoptosis in Lymphoid Cell Lines and Demonstrates Enhanced Biological Activity with Akt Kinase Inhibitors.

Blood ◽

10.1182/blood.v104.11.3406.3406 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3406-3406

Author(s):

Louie Naumovski ◽

Jason Ramos ◽

Jun Chen ◽

Mint Sirisawad ◽

David Lucas ◽

...

Keyword(s):

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Specific Inhibitor ◽

Annexin V ◽

Lymphocytic Leukemia ◽

Jurkat Cells ◽

Motexafin Gadolinium ◽

Akt Phosphorylation ◽

Induced Apoptosis

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that catalytically oxidizes intracellular reducing metabolites and produces reactive oxygen species (ROS). In this report, we demonstrate that MGd induces apoptosis or growth inhibition in several hematopoietic tumor-derived cell lines and tumor cells from patients with chronic lymphocytic leukemia. Lymphoma (HF-1, Ramos, DHL-4, DB, Hut78 and Raji) and leukemia (Jurkat, HL-60) cell lines were cultured in RPMI 1640 media with 10% heat inactivated fetal bovine serum with or without 50 uM MGd. MGd inhibited the growth of 6 of the cell lines (HF-1, Ramos, HL-60, DHL-4, Jurkat and DB) and was cytotoxic to HF-1. ROS were implicated in MGd-induced cell death since their presence was detected by dichlorofluorescein diacetate staining and peroxiredoxin oxidation in MGd treated HF-1 cells that undergo apoptosis, but not in Jurkat cells that do not undergo MGd-induced apoptosis. MGd triggered an apoptotic pathway in HF-1 cells as demonstrated by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspases, cleavage of PARP and annexin-V binding. MGd also induced cell death and activated caspases in vitro in primary chronic lymphocytic leukemia cells. Protein lysates from cultured cell lines (HF-1, Ramos) were subjected to immunoblot analysis to determine caspase cleavage patterns, and the phosphorylation status of Akt, a kinase that regulates survival pathways. In MGd treated HF-1, phospho-Akt protein levels initially increased 2–3 fold between 30 min and 1 hr (n=4) and then decreased to 40–50% of control levels by 24–48 hrs (n=4). The drop in phospho-Akt protein coincided with an increase in apoptotic cell death as indicated by morphology, staining with Annexin-V and activation of caspases. Addition of a specific inhibitor of Akt phosphorylation (SH-5) reduced Akt phosphorylation in MGd treated HF-1 cells by 90% and enhanced the cytotoxic effect of MGd. In Ramos cells, which do not undergo apoptosis when treated with MGd, co-treatment with MGd and SH-5 decreased phospho Akt levels by only 15% and did not result in cytotoxicity. These data point to a potential role for Akt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by inhibition of Akt. These data show that the pro-apoptotic effects of MGd involve caspase activation and provide a rationale to evaluate MGd in the treatment of lymphoma and leukemia patients.

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In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽

10.1182/blood.v106.11.4459.4459 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4459-4459

Author(s):

Morris Kletzel ◽

Sarah C. Tallman ◽

Marie Olszewski ◽

Wei Huang

Keyword(s):

Cell Death ◽

Cell Viability ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Annexin V ◽

Resistant Cell ◽

Primary Tumors ◽

Induced Apoptosis ◽

Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

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The Diterpenoid Lactone Andrographolide (ANDRO) Stimulates Autophagy and Induces Reactive Oxygen Species (ROS)-Dependent Apoptosis in Lymphoma Cell Lines

Blood ◽

10.1182/blood.v112.11.4991.4991 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4991-4991

Author(s):

Shuo Yang ◽

Andrew M. Evens ◽

Sheila Prachand ◽

Leo I. Gordon

Keyword(s):

Cell Proliferation ◽

Cell Death ◽

Cell Lines ◽

Redox State ◽

Annexin V ◽

Lymphoma Cell ◽

Time Dependent ◽

Cellular Redox ◽

Forkhead Transcription Factors ◽

Induced Apoptosis

Abstract ANDRO is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an important herbal medicine used in China. It has been reported to have anti-inflammatory, anti-hypertensive, anti-viral and immunostimulant properties. It has also been shown to inhibit cancer cell proliferation and induce apoptosis in HL-60 (leukemia), PC-3 (prostatic adenocarcinoma), MDA-MB-231 (breast cancer), HepG2 (liver cancer), HeLa (cervical cancer) and HCT116 (colorectal cancer) cell lines. The diterpenoids have been found to generate ROS and may increase apoptosis by altering the cellular redox state. We hypothesized that ANDRO would lead to cell death in lymphoma cell lines and that the effect may be related to altered cellular redox state. We studied the Burkitt p53 mutated Ramos cell line, the mantle cell lymphoma line Granta and L428, a resistant EBV-negative Hodgkin lymphoma cell line. We found that after incubation with increasing concentrations of ANDRO, there was dose and time-dependent cell death as measured by MTT. The IC50 (concentration that achieved 50% cell proliferation inhibition) at 48h was 20μM for Ramos, 40μM for Granta, and 50μM for L428. ROS was measured by oxidation of 2’7’dichlorofluorescein diacetate (DCFDA) to dichlorofluorescein (DCF) and analyzed by fluorescence-activated cell sorting (FACS) following incubation at 1hour (h), 2h, 3h, 5h, 38h, and 48h with ANDRO (20–80μM). ANDRO increased ROS production in all lymphoma cell lines, which was abrogated by the antioxidant N-acetyl-L-cysteine (NAC). Maximum ROS generation with ANDRO was seen at 48h for Ramos (1.7 fold), 5h for Granta (1.6 fold), and 38h for L428 (2.4 fold). To determine the mechanism of cell death, we measured apoptosis by Annexin-V/propidium iodide (PI), and detected by flow cytometry (FACS). Cells were treated with ANDRO in the presence or absence of the reduced glutathione (GSH) depleting agent buthionine sulfoximine (BSO) (100μM) for 28h, 48h, and 72h. We found that the AC50 (concentration that achieved 50% apoptosis) was 40μM for Ramos at 72h, 40μM for Granta at 48h and >80μM for L428 at 48h, while in the presence of BSO it was <10μM for Ramos at 72h, between 30–40μM for Granta at 28h and between 30–40μM for L428 at 48h. Apoptosis was completely blocked, by NAC, both in the presence and absence of BSO. Further, ANDRO induced PARP cleavage and activation of caspases 3, 8, and 9 in Granta and Ramos. Next, we explored the relationship of ANDRO and Forkhead transcription factors. ANDRO caused dephosphorylation of FOXO3a or FOXO1, in a dose- and time-dependent manner, and this was reversible by NAC. Downstream proteins of FOXO3a, Bim, p27kip1 and the isoforms of the autophagy-related protein LC3B were upregulated, and this was reversed by NAC. The LC3B isoform-II, which is cleaved from LC3B-I, is a marker of autophagy activation. To determine the role of autophagy in cell death related to ANDRO, we inhibited autophagy with 3-methyladenine (1–2mM) and found significant enhancement of ANDRO-induced apoptosis in Granta and Ramos. Finally, ANDRO induced apoptosis (>60% Annexin-V+/PI+) in malignant B-cells from a patient with chronic lymphocytic leukemia/small lymphocytic lymphoma (trisomy 12, peripheral blood absolute lymphocyte count 95.2 K/uL, bulky adenopathy) very low concentrations (5μM at 18h) in vitro, which was also reversible with NAC. We conclude that ANDRO induces ROS-dependent apoptosis in lymphoma cell lines and in a primary tumor sample, which is enhanced by depletion of GSH and inhibited by the antioxidant NAC. These effects appear to proceed through caspase activation and inhibition of autophagy, and are in part dependent on signaling through forkhead transcription factors and altered cellular redox pathways. Further studies of diterpenoids as single agents or in combination with other anti-lymphoma agents are warranted.

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