In Vitro Assessment of Anti-Multiple Myeloma (MM)-Agents On Various MM-Cell Lines (MMCLs) with Use of Bortezomib, Sorafenib, Thalidomide, Lenalidomide and EpiGalloCatechin-3-Gallate (EGCG) Demonstrates to Be a Valuable Tool for the Thorough Analysis and Discovery of Innovative Anti-MM-Agents.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4922-4922
Author(s):  
Josefina Udi ◽  
Dagmar Wider ◽  
Gabriele Ihorst ◽  
Julia Schüler ◽  
Ralph Wäsch ◽  
...  
Keyword(s):  
Cell Growth ◽  
Confocal Microscopy ◽  
Cell Viability ◽  
Cell Lines ◽  
Cytotoxic Effects ◽  
Inhibition Of Proliferation ◽  
Viable Cells ◽  
Morphologic Changes

Abstract Abstract 4922 Introduction Thalidomide and lenalidomide possess antiangiogenic, antiproliferative, proapoptotic and immunomodulatory effects. The proteasome inhibitor bortezomib induces cell death in MMCLs and has demonstrated synergism on various tumor cell lines, when combined with the multikinase inhibitor sorafenib. Sorafenib targets various kinases involved in tumor growth and angiogenesis, which plays a governing role in numerous cancers, including MM. EGCG, the most active catechin in green tea, has been described to induce anti-MM- and anti-amyloid-, but recently also to prevent bortezomib-induced-effects. We therefore tested these compounds individually and in combinations on 3 MMCLs in order to assess their cytotoxicity, cell growth inhibition and phenotype changes. Material and Methods RPMI8226, U266 and L363 were cultured at 1×105 cells/ml, with RPMI1640, 10% FCS and 0.2% pen/strep. On day (d) 0, increasing concentrations of bortezomib, sorafenib, thalidomide, lenalidomide and EGCG were added. On d3 and d6, we determined cytotoxicity and cell viability via trypan blue dye exclusion assay and propidium iodide (PI) staining by flow-cytometry (FACS). Additionally, we analyzed phenotype changes by means of CD138-expression (FACS). To evaluate CD138-expression as well as morphologic changes after sorafenib treatment, we also performed confocal microscopy analyses. Results 100nM bortezomib showed pronounced cytotoxicity on all 3 MMCLs: mean PI-positivity in L363 was 83.9% on d3, remained stable on d6 and was significantly increased as compared to control-L363-cells (p<0.01). In U266, mean PI-positivity on d3 and d6 was 37.5% and 25.2%, respectively, again being significantly higher than in control-U266-cells (p<0.0001). In RPMI, PI-positivity was similarly increased on d3 and d6 with 94.5% and 91.3%, respectively, again substantially higher than in control-RPMI-cells (p<0.001). With 10 and 100μM sorafenib in L363, we observed mean PI+ cells on d3 as high as 61.6% and 94.3% and on d6 of 80.8% and 91.8%, respectively (p<0.0001). A statistically significant dose-dependent decrease in viable cells and CD138-expression with 10 and 100μM sorafenib as compared to L363-control-cells could also be detected. By confocal microscopy, CD138-downregulation was prominent, besides manifest morphologic changes. In U266, mean PI+ cells were 33.1% with 10μM and 78.3% with 100μM, again significantly higher than in control-U266-cells (p<0.001), not substantially increasing on d6. Sorafenib's cytotoxicity was likewise evident in RPMI: PI+ cells on d3 with 10 and 100μM were 89.8% and 95.2% (p<0.001), respectively. In contrast, even with 100μg/ml thalidomide, cytotoxicity in L363 cells was subtle, with mean PI+ cells of 10.0% on d3 and 21.6% on d6 (n.s.), with cell viability only slightly decreasing. Thalidomide did not significantly affect U266 cell growth either. Lenalidomide did not increase PI+ cells in L363 on d3, but induced a noticeable PI+-rise on d6 of 19.2% with 10μM (p<0.01) and 15% with 100μM (p<0.001) as compared to control-L363-cells. No statistically significant decrease in viable cells and CD138-expression was observed. With 10 and 100μM lenalidomide in U266, PI+ cells on d6 were higher with 19.9% and 29.6% (p<0.001), respectively, as compared to control-U266-cells. Exposure of all MMCLs to EGCG, with concentrations ranging from 1 to 500μM, showed potent cytotoxic effects, most evident with concentrations of 250μM or higher in L363 (p<0.01). Conclusions Bortezomib and sorafenib showed impressive cytotoxic effects as single agents and ongoing experiments suggest additive effects between both compounds, which is currently being investigated, both in vitro and in our in vivo NOD/SCID-IL2-receptor-gamma-chain−/− (NSG)-mouse-model. Further investigations will also validate the recently suggested inhibitory effects of EGCG on bortezomib-induced cytotoxicity. Thalidomide and lenalidomide moderately reduced viable cell numbers, confirming that other mechanisms, such as anti-angiogenesis and immunomodulation are of greater relevance on MM cells. In line with earlier work, EGCG induced a pronounced cytotoxic effect and inhibition of proliferation. Our results demonstrate that our in vivo model is valuable for the thorough analysis and discovery of innovative targeted anti-MM-agents. Disclosures No relevant conflicts of interest to declare.

Anti-Myeloma Activity by the Combination of the JAK2 Inhibitor Ruxolitinib with Lenalidomide and Corticosteroids

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2114-2114 ◽  
Author(s):  
Haiming Chen ◽  
Eric Sanchez ◽  
Mingjie Li ◽  
Cathy Wang ◽  
Abby Gillespie ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Scid Mice ◽  
M Protein ◽  
Cytotoxic Effects ◽  
Jak2 Inhibitor

Abstract Introduction: The JAK2 inhibitor ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs (IMiDs) including lenalidomide (LEN) and corticosteroids have shown efficacy for the treatment of multiple myeloma (MM). The JAK-STAT signaling pathway plays key roles in the growth and survival of malignant plasma cells in MM. In this study, we evaluated the preclinical anti-MM effects of RUX in combination with LEN and corticosteroids, both in vitro and in vivo, and in a patient with MM and polycythemia rubra vera (PRV). Methods: The human MM cell lines U266, RPMI8226 and MM1S cells were derived from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow aspirates. The cells were seeded at105 cells/100ul/well in 96-well plates and incubated for 24 h in the presence of vehicle, RUX, LEN or dexamethasone (DEX) alone, RUX + LEN, RUX + DEX, or all three drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. In vitro, synergy between ruxolitinib and lenalidomide or dexamethasone was assessed using the median effect method of Chou and Talalay. For the in vivo studies, the human myeloma tumors (LAGκ-1A or LAGκ-2) were surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. Mice were blindly assigned to one of the experimental groups, and treatment was initiated 7–21 d after tumor implantation. LEN was administered via oral gavage daily (30 mg/kg). RUX (3 mg/kg) was given via intraperitoneal (IP) injection twice daily. Dexamethasone was administered daily (1.5mg/kg) via IP injection. An 88 year old MM patient with PRV who developed MM on RUX alone and then progressed on LEN+DEX was treated with the combination of all three drugs. Results: In vitro, RUX induced concentration-dependent inhibition of viability in all three MM cell lines (U266, RPMI8226 and MM1S) at RUX 50 mM and inhibition of primary MM tumor cells at a higher concentration (100 mM). In contrast, RUX had negligible cytotoxic effects on normal peripheral blood mononuclear cells (PBMCs). We next examined cell viability in the presence of RUX plus LEN or DEX. First, U266 cells were incubated with a fixed concentration of LEN (30 mM) or DEX (40 mM) with increasing concentrations of RUX (0.1–100 mM) for 48 h. At RUX 50 mM, the cytotoxic effects of LEN were enhanced and at RUX 1 mM, the anti-myeloma effect of DEX was increased. Moreover, the cytotoxic effects of RUX, LEN and DEX were greater than RUX in combination with either LEN or DEX in U266 cells. Similar results were obtained using the RPMI8226 and MM1S cell lines as well as primary MM tumor cells. Next, we evaluated RUX in combination with lenalidomide and dexamethasone in vivo using SCID mice bearing either the human LAGκ-1A or LAGκ-2 MM xenografts. RUX (3mg/kg), LEN (15mg/kg) or DEX (1mg/kg) alone did not inhibit tumor growth in either mice bearing LAGκ-1A or LAGκ-2. In contrast, the combination of RUX with DEX but not LEN slightly decreased tumor volume. However, the combination of all three drugs at the same doses showed a marked reduction of tumor size and delay of tumor growth in both human MM xenograft models. In addition, a patient with MM and PRV experienced sustained and ongoing reductions in his serum M-protein, IgG, and 24-urine M-protein with achievement of a partial response on low doses of RUX (2.5 mg twice daily), LEN (2.5 mg daily), and methylprednisolone (20 mg daily) that has been ongoing for more than 12 months after developing MM on RUX alone and then progressing on the combination of LEN and methylprednisolone. Conclusion: This study illustrates that the combination of the JAK2 inhibitor RUX, LEN and corticosteroids shows both preclinical and promising clinical results for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

Striking Anti-Multiple Myeloma (MM) Activity with the In Vitro Use of the Multikinase Inhibitor Sorafenib (S), Cytotoxic Synergy Between Bortezomib (B) and S, but Not Between B and Epigallocatechin-3-Gallate (EGCG; E) Enlarge the Present Knowledge In MM and Promise Novel Insights for Innovative Substance Exploitation In MM-Treatment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5020-5020
Author(s):  
Josefina Udi ◽  
Dagmar Wider ◽  
Julie Catusse ◽  
Gabriele Ihorst ◽  
Dominik Schnerch ◽  
...  
Keyword(s):  
Cell Death ◽  
Confocal Microscopy ◽  
Tumor Cell ◽  
Cell Viability ◽  
Present Knowledge ◽  
Tumor Cell Lines ◽  
Multikinase Inhibitor ◽  
Morphologic Changes

Abstract Abstract 5020 Introduction: As the development of novel anti-MM therapies is pursued worldwide in order to further improve survival in this disease, various innovative agents have to be eagerly tested in in vitro and in vivo models; the former being applied here. Bortezomib (B) has been shown to induce cell death in MMCLs and cytotoxic synergy with sorafenib (S) on various tumor cell lines. S, an oral multikinase inhibitor, targets several cancer-specific pathways and directly affects tumor cell proliferation, cell survival and neovascularization. EGCG (E), one main green tea constituent, causes MM cell toxicity also, but seems to prevent tumor cell death induced by B in vitro and in vivo, this extend not being fully understood as yet. Methods: RPMI8226, U266 and L363 were cultured with RPMI1640/10% FCS. On day (d) 0, cells were treated with increasing concentrations of B, S and/or E. Cell viability and cytotoxicity were assessed on d3 and d6 via trypan blue and PI-staining. CD138 expression and morphologic changes were evaluated via FACS, immunocytochemistry and confocal microscopy. The effect of S on the chemotactic behaviour of L363 in response to conditioned media (CM = supernatant of M210B4 stromal cells) using 96-well chemotaxis chamber plates was also evaluated. Phosphorylation of ERK1/2 was determined by Western blot. The combined effect of S and B was determined using Calcusyn software: the resulting combination index (CI) defines additive effects (CI=1), synergism (CI<1) and antagonism (CI>1). Results: With 10 and 100μM S in L363, we observed increased median PI+ cells (62% and 94% on d3, respectively) as compared to the control (median PI+ d0: 11%), with similar increases on d6 (median 81% and 92%, respectively). In line with PI-observations, viable cells and CD138 expression substantially decreased in a dose- and time-dependent manner. After 3 days pre-incubation with increasing S-concentrations, MM cells were stained with Dapi, Phalloidin-Alexa-549 and CD138-FITC and analyzed by confocal microscopy: L363 cells highly expressed CD138 in the absence of S, whereas impressive CD138 downregulation, morphologic changes and reduction of F-actin content were observed with S-concentrations as low as 1μM. L363 cells exhibited a migratory response to CM, whereas after 3 days of preincubation with 10, 20 and 50μM S, L363 cells showed reduced migratory capacity in response to CM. Western blots showed a decrease in p-ERK1/2 expression levels after 24h inbubation of L363 cells with 10μM S. With 100nM B, PI in L363 increased from 11% on d0 to 84% on d3, albeit not as pronounced with 10nM B as was observed with 10μM S. E induced cytotoxicity in L363, particularly with 50 and 100μM, albeit - different to prior reports - B-induced cell death was preserved when the B-E-combination was tested: of note, however, after addition of increasing E-concentrations, no synergism or additive effect, rather than a plateau cytotoxic effect was observed. Combined B and S use showed synergism with 10nM and 10μM, respectively (CI=0.80). MMCLs stably co-expressing fluorescently labelled cytochrome C and histone H2 will allow the detection of induced apoptosis using live-cell imaging after anti-MM agent treatment. Conclusions: Our MM-based in vitro model revealed that B and S show remarkable therapeutic efficacy as single agents and synergism when combined, which confirms results in other tumor cell lines. E alone induced dose-dependent cell death and decreases in MM cell viability and when combined with B did neither synergize nor abolish B-induced cell death. Our results further enlarge the present knowledge in MM therapy and promise novel insights for innovative substances in the treatment of MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

scholarly journals Long noncoding RNA LINC00941 promotes pancreatic cancer progression by competitively binding miR-335-5p to regulate ROCK1-mediated LIMK1/Cofilin-1 signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Wang ◽  
Zhiwei He ◽  
Jian Xu ◽  
Peng Chen ◽  
Jianxin Jiang
Keyword(s):  
Pancreatic Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Progression ◽  
Noncoding Rna ◽  
Epithelial Mesenchymal Transition ◽  
Loss Of Function ◽  
Mesenchymal Transition

AbstractAn accumulation of evidence indicates that long noncoding RNAs are involved in the tumorigenesis and progression of pancreatic cancer (PC). In this study, we investigated the functions and molecular mechanism of action of LINC00941 in PC. Quantitative PCR was used to examine the expression of LINC00941 and miR-335-5p in PC tissues and cell lines, and to investigate the correlation between LINC00941 expression and clinicopathological features. Plasmid vectors or lentiviruses were used to manipulate the expression of LINC00941, miR-335-5p, and ROCK1 in PC cell lines. Gain or loss-of-function assays and mechanistic assays were employed to verify the roles of LINC00941, miR-335-5p, and ROCK1 in PC cell growth and metastasis, both in vivo and in vitro. LINC00941 and ROCK1 were found to be highly expressed in PC, while miR-335-5p exhibited low expression. High LINC00941 expression was strongly associated with larger tumor size, lymph node metastasis, and poor prognosis. Functional experiments revealed that LINC00941 silencing significantly suppressed PC cell growth, metastasis and epithelial–mesenchymal transition. LINC00941 functioned as a molecular sponge for miR-335-5p, and a competitive endogenous RNA (ceRNA) for ROCK1, promoting ROCK1 upregulation, and LIMK1/Cofilin-1 pathway activation. Our observations lead us to conclude that LINC00941 functions as an oncogene in PC progression, behaving as a ceRNA for miR-335-5p binding. LINC00941 may therefore have potential utility as a diagnostic and treatment target in this disease.

scholarly journals Acetyl-L-Carnitine downregulates invasion (CXCR4/CXCL12, MMP-9) and angiogenesis (VEGF, CXCL8) pathways in prostate cancer cells: rationale for prevention and interception strategies

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Denisa Baci ◽  
Antonino Bruno ◽  
Caterina Cascini ◽  
Matteo Gallazzi ◽  
Lorenzo Mortara ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Growth ◽  
Cancer Cells ◽  
Dietary Supplements ◽  
Cell Lines ◽  
Angiogenic Factors ◽  
Migration And Invasion ◽  
Prostate Cancer Cells

Abstract Background Prostate cancer (PCa) is a leading cause of cancer-related death in males worldwide. Exacerbated inflammation and angiogenesis have been largely demonstrated to contribute to PCa progression. Diverse naturally occurring compounds and dietary supplements are endowed with anti-oxidant, anti-inflammatory and anti-angiogenic activities, representing valid compounds to target the aberrant cytokine/chemokine production governing PCa progression and angiogenesis, in a chemopreventive setting. Using mass spectrometry analysis on serum samples of prostate cancer patients, we have previously found higher levels of carnitines in non-cancer individuals, suggesting a protective role. Here we investigated the ability of Acetyl-L-carnitine (ALCAR) to interfere with key functional properties of prostate cancer progression and angiogenesis in vitro and in vivo and identified target molecules modulated by ALCAR. Methods The chemopreventive/angiopreventive activities ALCAR were investigated in vitro on four different prostate cancer (PCa) cell lines (PC-3, DU-145, LNCaP, 22Rv1) and a benign prostatic hyperplasia (BPH) cell line. The effects of ALCAR on the induction of apoptosis and cell cycle arrest were investigated by flow cytometry (FC). Functional analysis of cell adhesion, migration and invasion (Boyden chambers) were performed. ALCAR modulation of surface antigen receptor (chemokines) and intracellular cytokine production was assessed by FC. The release of pro-angiogenic factors was detected by a multiplex immunoassay. The effects of ALCAR on PCa cell growth in vivo was investigated using tumour xenografts. Results We found that ALCAR reduces cell proliferation, induces apoptosis, hinders the production of pro inflammatory cytokines (TNF-α and IFN-γ) and of chemokines CCL2, CXCL12 and receptor CXCR4 involved in the chemotactic axis and impairs the adhesion, migration and invasion capabilities of PCa and BPH cells in vitro. ALCAR exerts angiopreventive activities on PCa by reducing production/release of pro angiogenic factors (VEGF, CXCL8, CCL2, angiogenin) and metalloprotease MMP-9. Exposure of endothelial cells to conditioned media from PCa cells, pre-treated with ALCAR, inhibited the expression of CXCR4, CXCR1, CXCR2 and CCR2 compared to those from untreated cells. Oral administration (drinking water) of ALCAR to mice xenografted with two different PCa cell lines, resulted in reduced tumour cell growth in vivo. Conclusions Our results highlight the capability of ALCAR to down-modulate growth, adhesion, migration and invasion of prostate cancer cells, by reducing the production of several crucial chemokines, cytokines and MMP9. ALCAR is a widely diffused dietary supplements and our findings provide a rational for studying ALCAR as a possible molecule for chemoprevention approaches in subjects at high risk to develop prostate cancer. We propose ALCAR as a new possible “repurposed agent’ for cancer prevention and interception, similar to aspirin, metformin or beta-blockers.

Cytotoxic Effects of 24/R,25-Dihydroxycholecalciferol on the Proliferation of Various Tumor Cell Lines in vitro and Its Antitumor Effects in vivo

Oncology ◽  
1988 ◽  
Vol 45 (3) ◽  
pp. 206-209 ◽  
Author(s):  
Yuji Maeda ◽  
Tohru Hirai ◽  
Hideyuki Yamato ◽  
Noriko Kobori ◽  
Ken-ichi Matsunaga ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Tumor Cell Lines ◽  
Antitumor Effects ◽  
Cytotoxic Effects

The Kinase Inhibitor Sorafenib Can Disrupt the Survival Support Provided by the Microenvironment and Induce Apoptosis of Chronic Lymphocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 47-47
Author(s):  
Jessie-F Fecteau ◽  
Ila Bharati ◽  
Morgan O'Hayre ◽  
Tracy Handel ◽  
Thomas J. Kipps ◽  
...  
Keyword(s):  
Single Dose ◽  
Cell Viability ◽  
Cell Survival ◽  
Kinase Inhibitor ◽  
Vehicle Control ◽  
Lymphocytic Leukemia ◽  
Viable Cells ◽  
Induced Apoptosis

Abstract Abstract 47 Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of mature monoclonal B cells in the blood, secondary lymphoid tissue, and marrow. Despite their accumulation in vivo, CLL cells undergo spontaneous apoptosis in vitro unless rescued by extrinsic factors derived from the leukemia-cell microenvironment. Monocyte-derived Nurse-Like Cells (NLCs) and Marrow Stromal Cells (MSCs), representing the leukemic microenvironment, have been show to sustain CLL cell survival and more importantly to protect CLL cells from drug-induced apoptosis in vitro and possibly in vivo. Such protective niches are thought to prevent current therapies from achieving complete remission in patients. Investigating the mechanism(s) by which cells from the microenvironment promote CLL cell survival, particularly the signaling pathways triggered, will allow for the identification of new therapeutic targets aiming to disrupt these protective interactions. NLCs and MSCs have been shown to produce the chemokine SDF-1 (CXCL12), which can enhance CLL cell survival. We recently found that ZAP-70+ aggressive CLL cells responded by an increased survival to this chemokine, compared to ZAP-70- indolent CLL cells, and that this response was accompanied by the activation of the ERK pathway. Attempting to abrogate this survival pathway, we found that sorafenib (BAY 43–9006, Nexavar) a multi-kinase inhibitor targeting among others Raf kinases and thereby the RAF/MEK/ERK pathway, strongly reduced CLL cell viability in a time and dose dependent manner. A regimen of one single dose of 10uM of sorafenib significantly reduced CLL cell viability to 18+/−10% cells after 48hrs compared to vehicle control (DMSO; 100%; n=5). The daily addition of 1uM sorafenib also significantly decreased CLL cell viability, leading to 31+/−21% and 11+/−5% viable cells after 6 and 7 days respectively, compared to DMSO (n=5). More importantly, our results show that sorafenib induces CLL cell death in the presence of NLCs and MSCs. A single dose of sorafenib (10uM) rapidly decreased the fraction of viable CLL cells overtime, passing from 40+/−16% after 1 day to 10+/−3% after 4 days (n=4) in the context of NLCs and to 25+/−3% after 2 days and 14+/−3% after 4 days in the presence of MSCs, when compared to vehicle control (>80%; n=4). In the presence of NLCs, the 1uM daily regimen also uncovered an increased sensitivity of ZAP-70+ CLL cells to this drug, reducing in 6 days their viability to 13+/−2% (n=4), which approximately half the fraction of viable cells remaining in the ZAP-70- group (40+/−16%; n=7). We next studied sorafenib-mediated cytotoxicity by investigating its impact on the expression of pro-survival molecules. We found that Mcl-1, Bcl-2 and Bcl-xL protein expression was reduced in CLL cells compared to vehicle control, when stimulated with CXCL12 (n=3). In the presence of NLCs and MSCs, only Mcl-1 expression was downregulated, which was also associated with a reduction of the active form of the transcription factor CREB, involved in Mcl-1 expression. Because Mcl-1 expression can be regulated by ERK and AKT pathways, we next investigated if they were abrogated by sorafenib. We indeed found that MEK, ERK, and AKT activation were reduced by this inhibitor compared to vehicle control (n=3). We therefore propose that the cytotoxic effect of sorafenib on CLL cells is due to its interference with at least these two major survival pathways. Since sorafenib caused apoptosis of CLL cells in context of the microenvironment, we reasoned that it might also cause apoptosis of chemotherapy resistant CLL cells. To test this hypothesis, we studied cells from fludarabine-refractory patients. In the presence of NLCs, a single dose of 10uM sorafenib induced a significant reduction in CLL cell viability after 2 days: only 4+/−2% viable cells remained compared to 78+/−12% for the vehicle control (n=4). A comparable observation was made in the presence of MSCs: sorafenib potently induced apoptosis, leaving 12+/−3% live cells after 2 days, compared to vehicle control (71+/−16%; n=4). These results are very promising as they suggest that sorafenib could be an effective novel therapeutic for CLL, affecting the viability of the leukemic cells even in protective niches. Since sorafenib has been approved by the FDA in 2007 for the treatment of advanced hepatocellular carcinoma, a pilot study is currently being planned at UCSD to evaluate the potential of this drug in CLL in vivo. Disclosures: No relevant conflicts of interest to declare.

A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1841-1841
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Arghya Ray ◽  
Teru Hideshima ◽  
Paul G. Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Advisory Committees ◽  
Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

Preclinical Rationale for the Use of the Combined Treatment with the AKT Inhibitor Perifosine and the Multikinase Inhibitor Sorafenib in Hodgkin Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1653-1653
Author(s):  
Silvia Locatelli ◽  
Arianna Giacomini ◽  
Anna Guidetti ◽  
Loredana Cleris ◽  
Michele Magni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Drug Combination ◽  
Akt Inhibitor ◽  
Sorafenib Treatment ◽  
Responsive Cell

Abstract Abstract 1653 Introduction: A significant proportion of Hodgkin lymphoma (HL) patients refractory to first-line chemotherapy or relapsing after autologous transplantation are not cured with currently available treatments and require new treatments. The PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL. These pathways can be targeted using the AKT inhibitor perifosine (Æterna Zentaris GmBH, Germany, EU), and the RAF/MEK/ERK inhibitor sorafenib (Nexavar®, Bayer, Germany, EU). We hypothesized that perifosine in combination with sorafenib might have a therapeutic activity in HL by overcoming the cytoprotective and anti-apoptotic effects of PI3K/Akt and RAF/MEK/ERK pathways. Since preclinical evidence supporting the anti-lymphoma effects of the perifosine/sorafenib combination are still lacking, the present study aimed at investigating in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS: Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. RESULTS: While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P ≤.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of apoptosis. In responsive cell lines, WB analysis showed that anti-proliferative events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P ≤.0001) as well as mice receiving perifosine alone (49 days, P ≤.03) or sorafenib alone (54 days, P ≤.007). In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P ≤.0001) and necrosis (2- to 8-fold, P ≤.0001), as compared to controls or treatment with single agents. CONCLUSIONS: Perifosine/sorafenib combination resulted in potent anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation in HL patients. Disclosures: No relevant conflicts of interest to declare.

Effect of a novel agent, SL-401, targeting interleukin-3 (IL-3)-receptor on plasmacytoid dendritic cell (pDC)-induced myeloma cell growth and drug resistance.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 8582-8582
Author(s):  
Dharminder Chauhan ◽  
Arghya Ray ◽  
Christopher Brooks ◽  
Eric K. Rowinsky ◽  
Kenneth Carl Anderson
Keyword(s):  
Drug Resistance ◽  
Cell Growth ◽  
Cell Lines ◽  
Plasmacytoid Dendritic Cell ◽  
Myeloma Cell ◽  
Healthy Donors ◽  
Signaling Axis ◽  
Novel Therapeutic Strategies

8582 Background: Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and drug resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Our recent study utilized in vitro and in vivo MM xenograft models to show that plasmacytoid dendritic cells (pDCs) were significantly increased in MM BM and promote MM growth (Chauhan et al., Cancer Cell 2009, 16:309). Importantly, we found increased IL-3 levels upon pDC-MM interaction, which in turn, trigger MM cell growth and pDCs survival. IL-3R is highly expressed on pDCs. We utilized SL-401, a novel biologic conjugate that targets IL-3R, to examine whether abrogation of IL-3–IL-3R signaling axis affects pDC-MM interaction and its tumor promoting sequelae. Methods: MM cell lines, patient MM cells, and pDCs from healthy donors or MM patients were utilized to study the anti-MM activity of SL-401. MM cells and pDCs were cultured alone or together in the presence or absence of SL-401, followed by analysis of cell growth or viability. Results: SL-401 significantly decreased the viability of pDCs at low concentrations (IC50: 0.83 ng/ml; P < 0.005, n = 3). SL-401 also decreased the viability of MM cells at clinically achievable doses. Co-culture of pDCs with MM cells induced growth of MM cell lines; and importantly, low doses (0.8 ng/ml) of SL-401 blocked MM cell growth-promoting activity of pDCs. MM patient-derived pDCs induced growth of MM cell lines and primary MM cells as well; conversely, SL-401 inhibited pDC-triggered MM cell growth (P < 0.005, n= 5). Tumor cells from 3 of the 5 patients were from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. In agreement with these results, SL-401 blocked pDC-induced growth of dexamethasone-resistant MM cell lines. Conclusions: Our study therefore provides the basis for directly targeting pDCs or blocking the pDC-MM interaction, as well as targeting MM, in novel therapeutic strategies with SL-401 to enhance MM cytotoxicity, overcome drug-resistance, and improve patient outcome.

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