scholarly journals Propranolol Sensitizes Vascular Sarcoma Cells to Doxorubicin by Altering Lysosomal Drug Sequestration and Drug Efflux

2021 ◽  
Vol 10 ◽  
Author(s):  
Jhuma Saha ◽  
Jong Hyuk Kim ◽  
Clarissa N. Amaya ◽  
Caleb Witcher ◽  
Ali Khammanivong ◽  
...  
Keyword(s):  
Cell Lines ◽  
Therapeutic Potential ◽  
Treatment Options ◽  
Resistant Cell ◽  
Weak Base ◽  
Cellular Targets ◽  
Downstream Signaling ◽  
Lysosomal Accumulation ◽  
Sarcoma Cells

Angiosarcoma is a rare cancer of blood vessel–forming cells with a high patient mortality and few treatment options. Although chemotherapy often produces initial clinical responses, outcomes remain poor, largely due to the development of drug resistance. We previously identified a subset of doxorubicin-resistant cells in human angiosarcoma and canine hemangiosarcoma cell lines that exhibit high lysosomal accumulation of doxorubicin. Hydrophobic, weak base chemotherapeutics, like doxorubicin, are known to sequester within lysosomes, promoting resistance by limiting drug accessibility to cellular targets. Drug synergy between the beta adrenergic receptor (β-AR) antagonist, propranolol, and multiple chemotherapeutics has been documented in vitro, and clinical data have corroborated the increased therapeutic potential of propranolol with chemotherapy in angiosarcoma patients. Because propranolol is also a weak base and accumulates in lysosomes, we sought to determine whether propranolol enhanced doxorubicin cytotoxicity via antagonism of β-ARs or by preventing the lysosomal accumulation of doxorubicin. β-AR-like immunoreactivities were confirmed in primary tumor tissues and cell lines; receptor function was verified by monitoring downstream signaling pathways of β-ARs in response to receptor agonists and antagonists. Mechanistically, propranolol increased cytoplasmic doxorubicin concentrations in sarcoma cells by decreasing the lysosomal accumulation and cellular efflux of this chemotherapeutic agent. Equivalent concentrations of the receptor-active S-(−) and -inactive R-(+) enantiomers of propranolol produced similar effects, supporting a β-AR-independent mechanism. Long-term exposure of hemangiosarcoma cells to propranolol expanded both lysosomal size and number, yet cells remained sensitive to doxorubicin in the presence of propranolol. In contrast, removal of propranolol increased cellular resistance to doxorubicin, underscoring lysosomal doxorubicin sequestration as a key mechanism of resistance. Our results support the repurposing of the R-(+) enantiomer of propranolol with weak base chemotherapeutics to increase cytotoxicity and reduce the development of drug-resistant cell populations without the cardiovascular and other side effects associated with antagonism of β-ARs.

scholarly journals GSK1838705a, an IGF-1R/ALK Inhibitor, Overcomes Resistance to Crizotinib in ALK-Positive ALCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4069-4069
Author(s):  
Wenyu Shi ◽  
Jian-Yong Li
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Large Cell ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Expression Level ◽  
Downstream Signaling ◽  
Single Drug

Anaplastic large cell lymphoma (ALCL) is a type of CD30-expressing non-Hodgkin's lymphoma (NHL), which accounts for 2% to 3% of adult non-Hodgkin's lymphoma,accounting for 15% to 30% of children with large cell lymphoma. Anaplastic lymphoma kinase (ALK) positive ALCL is highly invasive, and currently it is generally based on CHOP combined with chemotherapy. The proportion of patients with complete relief of symptoms is as high as 90%, but the proportion of recurrence is also as high as 40%. Crizotinib is the first generation of ALK inhibitors that have been approved for the treatment of ALK+ ALCL. Unfortunately, most patients treated with crizotinib relapse after a significant initial response. The median progression-free survival of clinical trials was 10.5 months. Various mutations in the ALK kinase domain and amplification of the ALK gene copy number, activation of the alternative pathway, and tumor heterogeneity are major causes of crizotinib resistance. Studies have shown that IGF-1R interacts with NPM-ALK to promote ALK+ALCL transformation, proliferation and migration. GSK is a small molecule kinase inhibitor that inhibits both IGF-IR and ALK. Therefore, GSK with simultaneous inhibition of the bidirectional potential of IGF-IR and ALK has a promising prospect in the targeted therapy of NPM-ALK+ALCL. This study explored the inhibitory effects of GSK on NPM-ALK+ALCL and crizotinib-resistant NPM-ALK+ALCL by in vivo and in vitro experiments. In vitro experiments: The sensitivity of ALCL cell line to GSK1838705a was detected by CCK8 and flow cytometry. The expression of phosphorylation of IGF-1R and NPM-ALK signaling pathway in Karpas299 and SR786 cell lines stimulated by GSK was detected by WB method. In order to study the crizotinib resistance mutation, we established ALK+ALCL crizotinib-resistant cell lines Karpas299-R and SR786-R, and identified the resistance of Karpas299-R and SR786-R cell lines by CCK8 and flow cytometry. The drug-resistant and non-resistant strains were stimulated with gradient concentrations of crizotinib and gradient GSK, and the IC50 of the two were compared by CCK8. The WB method was used to compare the phosphorylation levels of downstream signaling pathways in drug-resistant and non-resistant strains. In vivo experiment: The ALK+ALCL and resistant-ALK+ALCL mouse model was established, and three groups of mice treated with control, GSK single drug 30 mg/kg, GSK single drug 60 mg/kg, were established. The tumor volume and body weight of the four groups were compared. Immunohistochemistry was used to compare the expression levels of key signaling molecules and apoptotic proteins in each group. SPSS statistical software draws survival curves. As the concentration of GSK gradually increases, the survival rate of ALCL cells gradually decreases. The expression of pIGF-1R, pNPM-ALK, pSTAT3, pAKT, casepase3 and other molecules decreased in the downstream signaling pathway, and the expression level of cleaved-casepase3 increased.In the crizotinib-resistant cell line, with the increase of the concentration of GSK, the apoptosis rate of the cells increased and the phosphorylation level of the downstream molecules gradually decreased. Tumor volume of three groups of mouse models: control>GSK single drug 30 mg/kg>GSK single drug 60 mg/kg. Immunohistochemistry results showed that the expression level of key signaling molecules in GSK-treated CHOP-treated mice decreased, and the expression level of apoptotic proteins increased. In this research, we explored the effects of GSK1838705A on proliferation, apoptosis, and clonogenesis of ALCL cell lines. Subsequently, we established a crizotinib-resistant cell line and noticed that GSK1838705A can effectively reduce the viability of resistant ALCL cells and significantly restrain the transmission of downstream survival signaling pathways induced by IGF1R/IR phosphorylation. Besides, we discovered that GSK1838705A inhibited the development of both crizotinib-sensitive and crizotinib-resistant ALCL tumors in the ALCL mouse model established by subcutaneous tumorigenesis. Based on the results of previous clinical trials, we put forward to use GSK1838705A as an alternative treatment strategy to overcome crizotinib-resistant ALCL. Disclosures No relevant conflicts of interest to declare.

Stew in its Own Juice: Protein Homeostasis Machinery Inhibition Reduces Cell Viability in Multiple Myeloma Cell Lines

2019 ◽  
Vol 19 (2) ◽  
pp. 112-119 ◽  
Author(s):  
Mariana B. de Oliveira ◽  
Luiz F.G. Sanson ◽  
Angela I.P. Eugenio ◽  
Rebecca S.S. Barbosa-Dantas ◽  
Gisele W.B. Colleoni
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Treatment Options ◽  
Compensatory Mechanism ◽  
Protein Homeostasis ◽  
Protein Response ◽  
Rpmi 8226

Introduction:Multiple myeloma (MM) cells accumulate in the bone marrow and produce enormous quantities of immunoglobulins, causing endoplasmatic reticulum stress and activation of protein handling machinery, such as heat shock protein response, autophagy and unfolded protein response (UPR).Methods:We evaluated cell lines viability after treatment with bortezomib (B) in combination with HSP70 (VER-15508) and autophagy (SBI-0206965) or UPR (STF- 083010) inhibitors.Results:For RPMI-8226, after 72 hours of treatment with B+VER+STF or B+VER+SBI, we observed 15% of viable cells, but treatment with B alone was better (90% of cell death). For U266, treatment with B+VER+STF or with B+VER+SBI for 72 hours resulted in 20% of cell viability and both treatments were better than treatment with B alone (40% of cell death). After both triplet combinations, RPMI-8226 and U266 presented the overexpression of XBP-1 UPR protein, suggesting that it is acting as a compensatory mechanism, in an attempt of the cell to handle the otherwise lethal large amount of immunoglobulin overload.Conclusion:Our in vitro results provide additional evidence that combinations of protein homeostasis inhibitors might be explored as treatment options for MM.

scholarly journals Lomeguatrib Increases the Radiosensitivity of MGMT Unmethylated Human Glioblastoma Multiforme Cell Lines

2021 ◽  
Vol 22 (13) ◽  
pp. 6781
Author(s):  
Anna Kirstein ◽  
Daniela Schilling ◽  
Stephanie E. Combs ◽  
Thomas E. Schmid
Keyword(s):  
Glioblastoma Multiforme ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Treatment Options ◽  
Treatment Resistance ◽  
Cell Cycle Distribution ◽  
Human Glioblastoma ◽  
Human Glioblastoma Multiforme ◽  
Mgmt Protein

Background: Treatment resistance of glioblastoma multiforme to chemo- and radiotherapy remains a challenge yet to overcome. In particular, the O6-methylguanine-DNA-methyltransferase (MGMT) promoter unmethylated patients have only little benefit from chemotherapy treatment using temozolomide since MGMT counteracts its therapeutic efficacy. Therefore, new treatment options in radiotherapy need to be developed to inhibit MGMT and increase radiotherapy response. Methods: Lomeguatrib, a highly specific MGMT inhibitor, was used to inactivate MGMT protein in vitro. Radiosensitivity of established human glioblastoma multiforme cell lines in combination with lomeguatrib was investigated using the clonogenic survival assay. Inhibition of MGMT was analyzed using Western Blot. Cell cycle distribution and apoptosis were investigated to determine the effects of lomeguatrib alone as well as in combination with ionizing radiation. Results: Lomeguatrib significantly decreased MGMT protein and reduced radiation-induced G2/M arrest. A radiosensitizing effect of lomeguatrib was observed when administered at 1 µM and increased radioresistance at 20 µM. Conclusion: Low concentrations of lomeguatrib elicit radiosensitization, while high concentrations mediate a radioprotective effect.

scholarly journals Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell Lines

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2012
Author(s):  
Kathryn M. Appleton ◽  
Charuta C. Palsuledesai ◽  
Sean A. Misek ◽  
Maja Blake ◽  
Joseph Zagorski ◽  
...  
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Therapeutic Potential ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Intrinsic Resistance ◽  
Primary Resistance ◽  
Induced Apoptosis ◽  
Melanoma Cell Lines

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (≈50% BRAFV600 mutations and ≈30% NRAS mutations). While drugs targeting the MAPK pathway have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in patients with NRAS mutant melanomas in part due to their cytostatic effects and primary resistance. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. A combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells, which are highly resistant to trametinib. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlight the therapeutic potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

scholarly journals ZN148 Is a Modular Synthetic Metallo-β-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Ørjan Samuelsen ◽  
Ove Alexander Høgmoen Åstrand ◽  
Christopher Fröhlich ◽  
Adam Heikal ◽  
Susann Skagseth ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Potential ◽  
Treatment Options ◽  
Carbapenem Resistance ◽  
Functional Space ◽  
Bactericidal Effect ◽  
Gram Negative ◽  
Content Type

ABSTRACT Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2797-2805 ◽  
Author(s):  
Feng-Ting Liu ◽  
Samir G. Agrawal ◽  
John G. Gribben ◽  
Hongtao Ye ◽  
Ming-Qing Du ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Resistant Cell ◽  
Protein Levels ◽  
Bax Protein ◽  
Degradation Activity ◽  
Potent Drug ◽  
Induced Apoptosis

Proapoptotic Bcl-2 family member Bax is a crucial protein in the induction of apoptosis, and its activation is required for this process. Here we report that Bax is a short-lived protein in malignant B cells and Bax protein levels decreased rapidly when protein synthesis was blocked. Malignant B cells were relatively resistant to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)–induced apoptosis, and this correlated with low basal Bax protein levels. Furthermore, during treatment with TRAIL, the resistant cell lines showed prominent Bax degradation activity. This degradation activity was localized to mitochondrial Bax and could be prevented by truncated Bid, a BH3-only protein; in contrast, cytosolic Bax was relatively stable. The proteasome inhibitor bortezomib is a potent drug in inducing apoptosis in vitro in malignant B-cell lines and primary chronic lymphocytic leukemic (CLL) cells. In CLL cells, bortezomib induced Bax accumulation, translocation to mitochondria, conformational change, and oligomerization. Accumulation and stabilization of Bax protein by bortezomib-sensitized malignant B cells to TRAIL-induced apoptosis. This study reveals that Bax instability confers resistance to TRAIL, which can be reversed by Bax stabilization with a proteasome inhibitor.

cis-Dichloroplatinum(II) complexes tethered to 9-aminoacridine-4-carboxamides: synthesis and action in resistant cell lines in vitro

2001 ◽  
Vol 85 (2-3) ◽  
pp. 209-217 ◽  
Author(s):  
Rodney J Holmes ◽  
Mark J McKeage ◽  
Vincent Murray ◽  
William A Denny ◽  
W.David McFadyen
Keyword(s):  
Cell Lines ◽  
Resistant Cell

scholarly journals Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

2021 ◽  
Vol 12 ◽  
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Javier Martin Broto ◽  
Katia Scotlandi ◽  
Michele Cavo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Effector T Cells ◽  
Sarcoma Cell ◽  
Ifn Γ ◽  
Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1

Blood ◽  
2021 ◽  
Author(s):  
Roger Belizaire ◽  
Sebastian Hassan John Koochaki ◽  
Namrata D. Udeshi ◽  
Alexis Vedder ◽  
Lei Sun ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Therapeutic Potential ◽  
Mutant Cell ◽  
E3 Ubiquitin Ligase ◽  
Akt Signaling ◽  
Allelic Series ◽  
Genetic Ablation

CBL encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and non-receptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10-20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, PIK3R1 recruitment, and downstream PI3K/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine-kinase binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

scholarly journals High LET Radiation Overcomes In Vitro Resistance to X-Rays of Chondrosarcoma Cell Lines

2019 ◽  
Vol 18 ◽  
pp. 153303381987130
Author(s):  
Francois Chevalier ◽  
Dounia Houria Hamdi ◽  
Charlotte Lepleux ◽  
Mihaela Temelie ◽  
Anaïs Nicol ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Relative Biological Effectiveness ◽  
Radiation Treatment ◽  
Malignant Tumors ◽  
Treatment Options ◽  
X Rays ◽  
Chondrosarcoma Cell ◽  
Biological Effectiveness

Chondrosarcomas are malignant tumors of the cartilage that are chemoresistant and radioresistant to X-rays. This restricts the treatment options essential to surgery. In this study, we investigated the sensitivity of chondrosarcoma to X-rays and C-ions in vitro. The sensitivity of 4 chondrosarcoma cell lines (SW1353, CH2879, OUMS27, and L835) was determined by clonogenic survival assays and cell cycle progression. In addition, biomarkers of DNA damage responses were analyzed in the SW1353 cell line. Chondrosarcoma cells showed a heterogeneous sensitivity toward irradiation. Chondrosarcoma cell lines were more sensitive to C-ions exposure compared to X-rays. Using D10 values, the relative biological effectiveness of C-ions was higher (relative biological effectiveness = 5.5) with cells resistant to X-rays (CH2879) and lower (relative biological effectiveness = 3.7) with sensitive cells (L835). C-ions induced more G2 phase blockage and micronuclei in SW1353 cells as compared to X-rays with the same doses. Persistent unrepaired DNA damage was also higher following C-ions irradiation. These results indicate that chondrosarcoma cell lines displayed a heterogeneous response to conventional radiation treatment; however, treatment with C-ions irradiation was more efficient in killing chondrosarcoma cells, compared to X-rays.

Sign in / Sign up

Export Citation Format

Share Document