Anticancer effects of mifepristone on human uveal melanoma cells

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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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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Fisetin Induced Cell Death in Human Ovarian Cancer Cell Lines via ZBP1 Mediated Necroptosis.

10.21203/rs.3.rs-520727/v1 ◽

2021 ◽

Author(s):

Yaxian Liu ◽

Wenhong Cao ◽

Yanhui Zhao ◽

Lijuan Shan ◽

Shuhai Lan

Keyword(s):

Ovarian Cancer ◽

Cell Death ◽

Cell Growth ◽

Cell Lines ◽

Annexin V ◽

Dependent Manner ◽

Apoptotic Process ◽

Ovarian Cancer Cell Lines ◽

And Migration

Abstract Background: Ovarian cancer leads to severe female mortality among all reproductive cancers. Fisetin, a natural flavonoid, exerts pharmacological characteristics on inhibiting cancer growth from various origins. Although multiple mechanisms involving in regulating cell death, there is still unclear if and how fisetin exhibits anti-cancer effect on ovarian cancer. The presented study aimed to evaluate cell apoptotic and necroptotic processes occurring in ovarian carcinoma (OC) cell lines induced by fisetin Methods: Cell growth was evaluated by MTT assay in both OC cell lines treated with or without fisetin. Annexin V/Propidium iodide staining followed by flow cytometry were used to characterize fisetin induced cell death. The apoptotic process was suppressed by z-VAD intervention then cell necroptosis was assessed by introducing ZBP1 knockdown OC cell lines coupled with fisetin intervention. The expression of necroptosis-related mediators and migration capability of respective cells were evaluated by western blotting and in vitro cell invasion assay. Result: Fisetin successfully reduced cell growth on both OC cell lines in a dose-dependent manner. Both apoptosis and necroptosis were induced by fisetin. Suppression on cell apoptotic process failed to enhance proliferation of fisetin treated cells. The induced cell death as well as robust expression of necroptotic markers RIP3 and MLKL were alleviated by knocking down the expression of ZBP1 protein in both OC cell lines.Conclusion: The present study demonstrated in vitro evidence supporting that both apoptosis and necroptosis were involved in fisetin induced OC cell death, while ZBP1 regulates necroptotic process via RIP3/MLKL pathway.

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Cytoprotective Activity ofGlycyrrhizae radixExtract against Arsenite-Induced Cytotoxicity

Evidence-based Complementary and Alternative Medicine ◽

10.1093/ecam/nem014 ◽

2008 ◽

Vol 5 (2) ◽

pp. 165-171 ◽

Cited By ~ 14

Author(s):

Sang Chan Kim ◽

Sook Jahr Park ◽

Jong Rok Lee ◽

Jung Cheol Seo ◽

Chae Ha Yang ◽

...

Keyword(s):

Cell Death ◽

Caspase 3 ◽

Apoptotic Cell ◽

Herbal Medicines ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cytoprotective Activity ◽

Flow Cytometric ◽

H4iie Cells ◽

Cell Line Cell

Licorice,Glycyrrhizae radix, is one of the herbal medicines in East Asia that has been commonly used for treating various diseases, including stomach disorders. This study investigated the effect of licorice on arsenite (As)-induced cytotoxicity in H4IIE cells, a rat hepatocyte-derived cell line. Cell viability was significantly diminished in As-treated H4IIE cells in a time and concentration-dependent manner. Furthermore, results from flow cytometric assay and DNA laddering in H4IIE cells showed that As treatment induced apoptotic cell death by activating caspase-3. Licorice (0.1 and 1.0 mg ml−1) treatment significantly inhibited cell death and the activity of caspase-3 in response to As exposure. These results demonstrate that licorice induced a cytoprotective effect against As-induced cell death by inhibition of caspase-3.

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Marine Actinomycetes-Derived Secondary Metabolites Overcome TRAIL-Resistance via the Intrinsic Pathway through Downregulation of Survivin and XIAP

Cells ◽

10.3390/cells9081760 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1760 ◽

Cited By ~ 1

Author(s):

Mohammed I. Y. Elmallah ◽

Sheron Cogo ◽

Andrei A. Constantinescu ◽

Selene Elifio-Esposito ◽

Mohammed S. Abdelfattah ◽

...

Keyword(s):

Cell Death ◽

Secondary Metabolites ◽

Cell Lines ◽

Hct116 Cell ◽

Cancer Cell Line ◽

Cancer Drug ◽

Dependent Manner ◽

Marine Actinomycetes ◽

Intrinsic Pathway ◽

Concentration Dependent Manner

Resistance of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis represents the major hurdle to the clinical use of TRAIL or its derivatives. The discovery and development of lead compounds able to sensitize tumor cells to TRAIL-induced cell death is thus likely to overcome this limitation. We recently reported that marine actinomycetes’ crude extracts could restore TRAIL sensitivity of the MDA-MB-231 resistant triple negative breast cancer cell line. We demonstrate in this study, that purified secondary metabolites originating from distinct marine actinomycetes (sharkquinone (1), resistomycin (2), undecylprodigiosin (3), butylcyclopentylprodigiosin (4), elloxizanone A (5) and B (6), carboxyexfoliazone (7), and exfoliazone (8)), alone, and in a concentration-dependent manner, induce killing in both MDA-MB-231 and HCT116 cell lines. Combined with TRAIL, these compounds displayed additive to synergistic apoptotic activity in the Jurkat, HCT116 and MDA-MB-231 cell lines. Mechanistically, these secondary metabolites induced and enhanced procaspase-10, -8, -9 and -3 activation leading to an increase in PARP and lamin A/C cleavage. Apoptosis induced by these compounds was blocked by the pan-caspase inhibitor QvD, but not by a deficiency in caspase-8, FADD or TRAIL agonist receptors. Activation of the intrinsic pathway, on the other hand, is likely to explain both their ability to trigger cell death and to restore sensitivity to TRAIL, as it was evidenced that these compounds could induce the downregulation of XIAP and survivin. Our data further highlight that compounds derived from marine sources may lead to novel anti-cancer drug discovery.

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Inhibition of ERK1/2 Activity by the MEK1/2 Inhibitor AZD6244 (ARRY-142886) Induces Human Multiple Myeloma Cell Apoptosis in the Bone Marrow Microenvironment: A New Therapeutic Strategy for MM.

Blood ◽

10.1182/blood.v108.11.3460.3460 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3460-3460 ◽

Cited By ~ 1

Author(s):

Yu-Tzu Tai ◽

Xian-Feng Li ◽

Iris Breitkreutz ◽

Weihua Song ◽

Peter Burger ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Growth ◽

Cell Lines ◽

Caspase 3 ◽

Parp Cleavage ◽

Dependent Manner ◽

Growth And Survival ◽

Cyclin E1 ◽

Human Multiple Myeloma

Abstract Activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) signaling pathway mediates tumor cell growth in many cancers, including human multiple myeloma (MM). Specifically, this pathway mediates MM cell growth and survival induced by cytokines/growth factors (i.e. IL-6, IGF-1, CD40, BAFF) and adhesion to bone marrow stromal cells (BMSCs), thereby conferring resistance to apoptosis in the bone marrow (BM) milieu. In this study, we therefore examined the effect of the MEK1/2 inhibitor AZD6244 (ARRY-142886), on human MM cell lines, freshly isolated patient MM cells and MM cells adhered to BMSCs. AZD6244, inhibits constitutive and cytokine (IL-6, IGF-1, CD40)-stimulated ERK1/2, but not AKT phosphorylation. Importantly, AZD6244 inhibits the proliferation and survival of human MM cell lines, regardless of sensitivity to conventional chemotherapy, as well as freshly isolated patient MM cells. AZD6244 induces apoptosis in patient MM cells even in the presence of BMSCs, as evidenced by caspase 3 activity and PARP cleavage at concentrations as low as 20 nM. AZD6244 overcomes resistance to apoptosis in MM cells conferred by IL-6 and BMSCs, and inhibits IL-6 secretion induced by MM adhesion to BMSCs. AZD6244 suppresses MM cell survival/growth signaling pathways (i.e., STAT3, Bcl-2, cyclin E1, CDK1, CDK3, CDK7, p21/Cdc42/Rac1-activated kinase 1, casein kinase 1e, IRS1, c-maf) and up-regulates proapoptotic cascades (i.e., BAX, BINP3, BIM, BAG1, caspase 3, 8, 6). AZD6244 also upregulates proteins triggering cell cycle arrest (i.e. p16INK4A, p18INK4C, p21/WAF1 [Cdkn1a], p27 [kip1], p57). In addition, AZD6244 inhibits adhesion molecule expression in MM cells (i.e. integrin a4 [VLA-4], integrin b7, ICAM-1, ICAM-2, ICAM-3, catenin a1, c-maf) associated with decreased MM adhesion to BMSCs. These pleiotropic proapoptotic, anti-survival, anti-adhesion and -cytokine secretion effects of AZD6244 abrogate BMSC-derived protection of MM cells, thereby sensitizing them to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. In contrast, AZD6244 has minimal cytotoxicity in BMSCs and does not inhibit DNA synthesis in CD40 ligand-stimulated CD19 expressing B-cells derived from normal donors at concentrations toxic to MM cells (between 0.02–2 mM). Furthermore, AZD6244 inhibits the expression/secretion of osteoclast (OC)-activating factors (i.e., macrophage inflammatory protein (MIP)-1a, MIP-1b, IL-1b, VEGF) from MM cells. It also downregulates MM growth and survival factors (IL-6, BAFF, APRIL) in OC cultures derived from MM patient peripheral blood mononuclear cells (PBMCs). Significantly, AZD6244 inhibits OC differentiation from MM PBMCs (n=10) in a dose-dependent manner. Together these results provide the preclinical basis for clinical trials with AZD6244 (ARRY-142886) in MM.

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KRN5500, a Spicamycin Derivative, Exerts Anti-Myeloma Effects through Impairing Both Myeloma Cells and Osteoclasts.

Blood ◽

10.1182/blood.v112.11.1669.1669 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1669-1669

Author(s):

Hirokazu Miki ◽

Shuji Ozaki ◽

Osamu Tanaka ◽

Shingen Nakamura ◽

Ayako Nakano ◽

...

Keyword(s):

Bone Marrow ◽

Drug Resistance ◽

Cell Death ◽

Cell Growth ◽

Cell Lines ◽

G1 Arrest ◽

Dependent Manner ◽

Osteolytic Bone Disease ◽

Rpmi 8226

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by devastating bone destruction due to enhanced bone resorption and suppressed bone formation. Although high-dose chemotherapy and new agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains incurable due to drug resistance mediated by interactions with osteoclasts or stroma cells. Moreover, osteolytic bone disease continues to be a major problem for many patients. Therefore, alternative approaches are necessary to overcome drug resistance and inhibit osteoclasts activity in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan), which potently inhibits protein synthesis and induces cell death in human tumor cell lines. Phase I studies of KRN5500 in patients with solid tumors such as colon cancer and gastric cancer showed acceptable toxicity with Cmax values of 1000––3000 nM. In this study, we investigated the effects of KRN5500 against MM cells and osteoclasts in vitro and in vivo. MM cell lines such as RPMI 8226, MM.1S, INA-6, KMS12-BM, UTMC-2, TSPC-1, and OPC were incubated with various concentrations of KRN5500 for 3 days. Cell proliferation assay showed marked inhibition of cell growth with G1 arrest in these MM cells (IC50: 4–100 nM). KRN5500 (100 nM) also induced 30–90% of cell death in primary MM cells (n=7). Annexin V/propidium iodide staining showed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Western blot analysis confirmed activation of caspase-8, -9, and −3, cleavage of poly (ADP-ribose) polymerase (PARP), and down-regulation of Mcl-1. We next examined the effect of KRN5500 against MM cell lines and primary MM cells in the presence of bone marrow stroma cells and osteoclasts. Co-culture of these cells enhanced viability of MM cells; however, KRN5500 still induced strong cytotoxicity to MM cells. Of interest, KRN5500 specifically mediated apoptosis in osteoclasts but not stroma cells as assessed by TUNEL staining. More than 90% of osteoclasts were killed even at a low concentration of KRN5500 (20 nM). Finally, we evaluated the effect of KRN5500 against MM cells and osteoclasts in vivo. Two xenograft models were established in SCID mice by either subcutaneous injection of RPMI 8226 cells or intra-bone injection of INA-6 cells into subcutaneously implanted rabbit bones (SCID-rab model). These mice were treated with intraperitoneal injection of KRN5500 (5 mg/kg/dose) or saline thrice a week for 3 weeks after tumor development. In a subcutaneous tumor model, KRN5500 inhibited the tumor growth compared with control mice (increased tumor size, 232 ± 54% vs 950 ± 422%, p<0.001, n=6 per group). In a SCID-rab model, KRN5500 also inhibited MM cell growth in the bone marrow (increase of serum human sIL6-R derived from INA-6, 134 ± 19% vs 1112 ± 101%, p<0.001, n=5 per group). Notably, the destruction of the rabbit bones was also prevented in the KRN5500-treated mice as evaluated by radiography. Therefore, these results suggest that KRN5500 exerts anti-MM effects through impairing both MM cells and osteoclasts and that this unique mechanism of action provides a valuable therapeutic option to improve the prognosis in patients with MM.

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

Blood ◽

10.1182/blood.v128.22.4478.4478 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4478-4478 ◽

Cited By ~ 1

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.

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SAR650984 (SAR) Directly Promotes Homotypic Adhesion-Related Multiple Myeloma (MM) Cell Death and SAR-Induced Anti-MM Activities Are Enhanced By Pomalidomide, More Potently Than Lenalidomide

Blood ◽

10.1182/blood.v124.21.2124.2124 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2124-2124 ◽

Cited By ~ 1

Author(s):

Hua Jiang ◽

Gang An ◽

Chirag Acharya ◽

Mike Y Zhong ◽

Ti Cai ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Caspase 3 ◽

Single Agent ◽

Annexin V ◽

Cell Killing ◽

Individual Agent ◽

Direct Cell ◽

Direct Killing

Abstract SAR650984 (SAR) is a naked humanized IgG1 monoclonal antibody (mAb) selectively targeting the membrane protein CD38 in early clinical development to treat multiple myeloma (MM) and other CD38+ hematological malignancies. SAR has demonstrated encouraging single agent activity in relapsed/refractory (R/R) MM patients (ASCO abstract #8532) and even better efficacy when combined with Dexamethasone and Lenalidomide (Len), without reaching a maximum tolerated dose in patients with heavily pretreated MM (ASCO Abstract #8512). It functions through multiple mechanisms including antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and direct killing against CD38-positive tumor cells including MM. Although SAR induces lysis of all CD38-expressing MM cell lines via ADCC, it only significantly induces direct killing of MOLP8 cells that express the highest CD38 surface density (~580,000/cell) among > 17 MM cell lines. We first sought to determine whether direct cell death induced by SAR depends on CD38 levels on MM cell membrane by generating RPMI8226 cells overexpressing CD38 (R-CD38) (Abstract #67338). R-CD38 cells express > 6-fold higher CD38 mRNA and surface protein levels than parental RPMI8226 cells (577,304/cell vs. 128,713/cell). Direct MM cell killing by SAR was determined using caspase 3/7 activity and CellTiter-Glo luminescent cell viability assays without goat anti-human IgG crosslinking, in the presence or absence of IL-6 or bone marrow stromal cells (BMSCs). Following overnight incubation, SAR significantly induced homotypic aggregation (HA) of R-CD38, but not control RPMI8226 cells, associated with dose-dependent activation of pro-apoptotic caspase 3/7 in R-CD38, but not control cells. Importantly, SAR decreased the viability of R-CD38, but not control cells, regardless of the presence of IL-6 or BMSCs. Direct cell death induced by SAR depends on SAR-induced HA in MM cells since SAR only blocked survival of R-CD38 and MOLP8 MM cells that show significant HA. Thus, direct apoptosis induced by SAR depends on the level of CD38 surface expression, which may contribute to clinical responses in R/R MM expressing higher CD38 levels. Next, we evaluated the combination effect of Len or Pomalidomide (Pom) with SAR on MM cells. BM mononuclear cells from MM patients were incubated with SAR (10 mg/ml) with or without 10 mM of Len or Pom overnight, followed by flow cytometric analysis to determine % Annexin V/PI staining of CD138+/BCMA+ MM cells. As expected, Pom alone induced slightly higher % of Annexin V+/PI+ MM cells than Len (41 + 1.8 % vs 49 + 1.5 %). Either combination further increased the % of double positive MM patient cells when compared with individual agent alone (from 40 + 2.1% to 70 + 3.1% combined with Len; from 40 + 2.1% to 86 + 3.4% combined with Pom). In addition, PBMC effectors from normal donors (n=4) were pretreated with Len or Pom (5 mM) for 3-7 days and used for SAR-mediated ADCC assays against MM cells (MM1S, MM1R, RPMI8226, R-38, MOLP8), with or without HS-5 or BMSCs from patients. Pom, more potently than Len, further increased SAR-induced MM cell lysis regardless of the presence of BMSCs. Moreover, additional pretreatment of MM cells with Pom overnight further enhanced SAR-induced ADCC by Pom-pretreated PBMC effectors. Both MOLP8 and R-CD38 are relative resistant to direct cytotoxicities induced by Len or Pom. Significantly, Pom, also more potently than Len, augmented direct toxicities induced by SAR in MOLP8 and R-CD38 MM cells. Taken together, we here demonstrate that SAR directly induces apoptosis of MM cells with higher CD38 levels; and that Pom, more effectively than Len, increases SAR-induced MM cell killing via apoptosis and ADCC. These data strongly support SAR as a monotherapy or in combination treatment to improve the outcome of MM patients. Disclosures Cai: Sanofi: Employment. Song:Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Munshi:Celgene: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Sanofi-Aventis: Consultancy; Ocopep: Consultancy, Equity Ownership, Patents & Royalties. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

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ID: 1085 Sodium citrate induces apoptosis in HepG2 cell lines

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.359 ◽

2017 ◽

Vol 4 (S) ◽

pp. 174

Author(s):

Sinh Truong Nguyen ◽

Phuc Hong Vo ◽

Oanh Thi-Kieu Nguyen ◽

Nghia Minh Do ◽

Phuc Van Pham

Keyword(s):

Hepatocellular Carcinoma ◽

Cancer Cells ◽

Dna Fragmentation ◽

Hepg2 Cell ◽

Cell Lines ◽

Hepg2 Cells ◽

Caspase 3 ◽

Sodium Citrate ◽

Dependent Manner ◽

Hepg2 Cell Lines

PURPOSES: Cancer cells were observed to increase glucose uptake and fermentation of glucose to lactate to to synthesis rapidly ATP for cell growth, survival and proliferation. Thus, inhibition of glycolysis might be useful in antitumor treatment. This phenomenon occurred even with fully functioning mitochondria, and known as Warberg effect. Sodium citrate, an inhibitor of Warberg effect, was reported to antiproliferate many cancer cells line. However, sodium citrate has not been studied in Hepatocellular Carcinoma cells line yet. Here we aimed to investigate the effect of sodium citrate in HepG2 cells line. MATERIAL AND METHODS: HepG2 cell lines was treated with sodium citrate at different concentrations. Viable cells were determined by Alamar Blue. The apoptosis induced-cells was detected by Annexin V with FCM technique. Disintegrated nuclei and DNA fragmentation was analyzed. The activity of caspase-3 was also tested. RESULTS: We observed that the IC50 value of sodium citrate on HepG2 is at 10mM. FCM analysis showed that sodium citrate induced apoptosis in HepG2 cell line in dose-dependent manner. At 10mM sodium citrate, the caspase-3/7 was observed to be activated in time-dependent manner. Sodium citrate also induced nuclei disintergated in HepG2. DNA fragmentation was observed when HepG2 cells were treated with 10mM sodium citrate. CONCLUSIONS: We have shown that sodium citrate possesses the antiproliferative ability on HepG2 at IC50 10mM. Sodidum citrate induces apoptosis cells in hepatocellular carcinoma HepG2 by capases-3 activation. More investigation of glycolysis inhibition of sodium citrate on HepG2 should be performed in animals

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Tetraspanin Overexpression Induces Multiple Myeloma Cell Death.

Blood ◽

10.1182/blood.v106.11.5067.5067 ◽

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.

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