scholarly journals Bacterial Ribonuclease MazF-Mediated Apoptosis as Potential Cancer Therapy

2021 ◽  
Author(s):  
Maryam Saffarian Abbas Zadeh ◽  
Rebecca Anne MacPherson ◽  
Guohui Huang ◽  
Hui Ding ◽  
Rhonda Reigers Powell ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Synthesis ◽  
Cell Lines ◽  
Mrna Translation ◽  
Protein Translation ◽  
Programmed Death ◽  
Inhibition Of Protein Synthesis ◽  
Cancer Agent ◽  
Cell Suicide ◽  
The Impact

Abstract Programmed cell death is a dynamic and critical mechanism of cell suicide in eukaryotes and prokaryotes. MazF is a ribonuclease protein involved in bacterial intracellular programmed death. This protein cleaves mRNAs at ACA sequences, leading to inhibition of protein synthesis and triggering cell death. Given that cancer is heterogenic and has varied susceptibility to treatment, we examined the impact of MazF proteins on the growth and viability of three cancer cell lines: MCF7, HT29, and AGS. These cell lines were transfected with ACA-less mazF mRNAs and evaluated for MazF-mediated cell death. The data illustrated that efficient MazF translation leads to a significant reduction in cell viability and is modulated by structural elements of ACA-less mazF mRNAs. In the presence of MazF, the levels of activated caspase-3 and -7 were significantly elevated in transfected cells, confirming the occurrence of apoptosis. We also quantified mRNA translation on a single-cell basis in MCF7 and AGS cell lines to examine MazF-mediated inhibition of protein synthesis. MazF expression significant decreases the levels of protein translation in the examined cell lines. This is the first report of MazF as a potential anti-cancer agent via induction of apoptosis in MCF7, AGS, and HT-29 cell lines.

Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth

1997 ◽  
Vol 273 (5) ◽  
pp. L941-L949 ◽  
Author(s):  
Barbara Driscoll ◽  
Lingtao Wu ◽  
Susan Buckley ◽  
Frederick L. Hall ◽  
Kathryn D. Anderson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cyclin D ◽  
Lung Cancer Cell ◽  
Cancer Cell Growth ◽  
Transfected Cells ◽  
The Impact

To investigate the role of cyclin D1 in the regulation of lung cancer cell growth, we created five stably transfected cell lines carrying a cyclin D1 antisense construct. The transfected cells exhibited a marked decrease in the rate of cell growth, in contrast to the original lines (A549 and NCI-H441). The expression of several cell cycle-regulating proteins, including cyclin A, the cyclin-dependent kinases (cdk) 2 and cdk4, in addition to cyclin D1 itself, was markedly decreased. The expression of one cdk inhibitor, p21WAF1/CIP1, increased in the A549-derived cell lines. A specific target of cyclin D1 activity, the growth-suppressing product of the retinoblastoma gene, pRb, exhibited decreased expression and a decreased level of phosphorylation in the transfected cells. Decreased expression of pRb due to a significant increase in its turnover rate suggested that the stability of the protein may depend on phosphorylation by cyclin D1-dependent cdk activity. In addition to the impact on pRb stability, decreased expression of cyclin D1 induced susceptibility to cell death after withdrawal of exogenous growth factors in the antisense transfected cell lines, a response that was not observed in the original cancer cell lines. We conclude that abrogation of cyclin D1 overexpression in lung cancer cells disrupts several key pathways that are required for uncontrolled cell growth and induces those that lead to cell death after growth factor deprivation. Therefore, we speculate that use of antisense cyclin D1 expression in appropriate gene vectors could be a useful method for retarding lung cancer cell growth in accessible tumors such as those of the lung epithelium.

Inhibition of protein synthesis and heat protection: Histidinol-resistant mutant cell lines

1991 ◽  
Vol 149 (3) ◽  
pp. 396-402 ◽  
Author(s):  
Yong J. Lee ◽  
Zi-Zheng Hou ◽  
Dooha Kim ◽  
Abdul Al-Saadi ◽  
Peter M. Corry
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Mutant Cell ◽  
Resistant Mutant ◽  
Heat Protection ◽  
Inhibition Of Protein Synthesis

Combination of Farnesyl Transferase Inhibitor (Tipifarnib) with Perifosine Induces Apoptosis through phos-PDK1 in Human Lymphoma and Leukemia Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1488-1488 ◽  
Author(s):  
Ebenezer David ◽  
Rajni Sinha ◽  
Claire Torre ◽  
Jonathan L. Kaufman ◽  
Sagar Lonial
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Targeted Agents ◽  
Leukemia Cell Lines ◽  
Targeted Agent ◽  
The Impact

Abstract Introduction: Novel agents as anti-cancer therapy are used in the setting of specific molecular abnormalities that provide a survival advantage for malignant cells. One such agent, tipifarnib, is theoretically targeted at Ras mutations which are present in a number of different human cancers. Our previous experience with the FTIs (David et al, in press Blood) has demonstrated that they are ideal agents to combine with other targeted agents. We have investigated the combination of the AKT inhibitor perifosine with tipifarnib in human leukemia and lymphoma cell lines with the hypothesis that the combination of 2 targeted agents will disrupt separate survival pathways and ultimately result in synergistic tumor cell death. Methods: In this study we used the human leukemia cell lines HL-60, Jurkat, and the lymphoma cell line HT. Western blot analysis was used to assess for the effect of either single agent perifosine, tipifarnib, or the combination on AKT, p-AKT, PDK-1, and caspase cleavage. Flow cytometry was utilized to assess for Annexin V staining following combination therapy. Results:Dose escalation studies demonstrated that doses of tipifarnib up to 5μm demonstrated a significant cell death in HL-60 and HT cells. Perifosine doses of 1–5uM also induced cell death in both HL-60 and HT cells. When apoptosis was assessed using western blot analysis of caspase 3 activity and cleavage, the combination of perifosine and tipifarnib demonstrated significant apoptosis using low doses of both agents. The apoptosis was associated with downregulation of phos-PDK1, with a resultant downregulation in p-AKT. The level of phos-PDK1 was completely inhibited in less than 24 hrs in both the HL-60 and HT cell lines in combination than when either agent was given alone. Conclusion: The combination of perifosine, and AKT targeted agent, with tipifarnib, a Ras targeted agent, appear to induce significant cell death in lymphoma and leukemia cell lines with rapid downregulation of p-AKT via the PDK-1 pathway. This apoptosis occurs in vitro using concentrations well below those that have been achieved in current clinical trials using these agents. Additional studies are being carried out to further delineate the mechanism of synergy as well as to further explore the impact of sequence of administration using this combination. Further studies are also planned to xplore the impact of the combination on primary human leukemia and lymphoma cells from the blood and bone marrow.

The Lipid Addiction of Diffuse Large B-Cell Lymphoma (DLBCL) and Potential Treatment Strategies with Novel Fatty Acid Synthase (FASN) Small Molecule Inhibitors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4490-4490 ◽  
Author(s):  
Ravi Dashnamoorthy ◽  
Nassera Abermil ◽  
Afshin Behesti ◽  
Paige Kozlowski ◽  
Frederick Lansigan ◽  
...  
Keyword(s):  
Cell Death ◽  
Fatty Acid ◽  
Lipid Metabolism ◽  
Cell Survival ◽  
Binding Affinity ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Fatty Acid Synthase ◽  
The Impact

Abstract Background: Fatty acid (FA) metabolism is altered in several cancers through increased de novo synthesis of lipids via up-regulation fatty acid synthase (FASN) and increased utilization of lipids via β-oxidation. We investigated the dependence of DLBCL survival on FA metabolism. In addition, we examined novel FASN inhibitors TVB3567 and TVB3166 in comparison with cerulenin for the effects on cell survival and PI3K and MAPK-related biological pathways associated with tumor-related FA metabolism in DLBCL. Methods: FASN inhibitors, TVB3567 and TVB3166 (3V Biosciences, CA), cerulenin (FASN inhibitor), orlistat (anti-lipoprotein lipase (LPL) and FASN), PI3K/mTOR, and MEK small molecule inhibitors were studied in OCI-LY3, OCI-LY19, SUDHL4, SUDHL6, and SUDHL10 DLBCL cell lines for the effects of FA inhibition on lipid metabolism, cell signaling, and cell death. The effects of FASN inhibition on global gene expression profile (GEP) were also determined with Affymetrix Human 2.0 ST Genechip with Gene set enrichment analysis (GSEA). We also utilized short hairpin RNA interference (shRNA) to study interactions between FASN and PI3K/MAPK signaling. Finally, AutoDock Vina software (autodock.scripps.edu) was utilized to analyze drug target (FASN enzyme) binding affinity and assist in the design of FASN inhibitors with higher target binding affinity. Results: DLBCL cell lines OCI-LY3, SUDHL4, and SUDHL6 grown in the presence of lipoprotein-depleted serum showed exquisite sensitivity to lipid deprivation resulting in near complete cytotoxicity by MTT. Lipid deprivation-induced apoptotic cell death, detected as cleaved caspase 3 and PARP and Annexin-V/PI positivity, in these cells. Further, these effects were completely rescued by Very Low Density Lipoprotein (VLDL) supplementation to growth medium in SUDHL4 confirming the high lipid-dependency on cell survival in DLBCL. Treatment with pharmacological inhibitors of FASN (ie, TVB3567, TVB3166, cerulenin, or orlistat) resulted in a dose- and time-dependent reduction in cell viability in all DLBCL cell lines. Ingenuity Pathway Analysis (IPA) from GEP with cerulenin-treated OCI-LY3 showed prominent suppression of CD40, TNF, and NFκB dependent inflammatory responses as well as activation of apoptosis as predominant biological activities including significant down-regulation of genes involved in Krebs cycle and p38 MAPK pathways. Interestingly, upstream regulation by IPA predicted activation of MEK/ERK and MYC-dependent functions; and in OCI-LY3 with shRNA knock down of FASN, we observed constitutive activation of ERK as detected with increased phosphorylation by western blot. Activation of MEK/ERK and MYC is expected in part owing to metabolic stress induced by FASN inhibition. Considering the impact of MEK/ERK pathways on lipid metabolism, we next investigated the impact of MEK/ERK on FA metabolism. FASN was significantly decreased following MEK or ERK shRNA in OCILY-3 and SUDHL10 cells. Similarly, pharmacological inhibition of MEK or PI3K/mTOR (using novel small molecule agents AZD6244 (selumetinib) or BEZ235, respectively) resulted in marked down-regulation of FASN expression. Based on these results, FASN inhibition appears to a promising therapeutic target for the treatment of DLBCL, however attaining clinical efficacy with existing compounds require the effective drug concentration to be within the nanomolar range. Thus, we utilized AutoDock to determine drug docking enzyme inhibition constant (ki). We identified high ki values of 33μM and 180μM for Cerulenin and Orilstat, respectively. Therefore, we have developed/constructed modified novel and potent anti-FA compounds with ki <1μM that are currently being investigated. Conclusions: Collectively, we demonstrated that DLBCL cell survival is highly dependent on FA metabolism and that targeting lipid metabolism may be harnessed as a potential therapeutic strategy. We also showed that MEK/ERK-dependent mechanisms are intimately involved in promoting lipid addiction in DLBCL cells. Further investigation is warranted to delineate the mechanisms through which MEK/ERK regulate FASN expression and to determine in vivo implications of FASN inhibition on DLBCL tumor growth. In addition, continued development, design, and enhancement are needed to construct the most optimal anti-FA therapeutic agents. Disclosures Lansigan: Teva Pharmaceuticals: Research Funding; Spectrum Pharmaceuticals: Research Funding.

scholarly journals Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells

1993 ◽  
Vol 289 (1) ◽  
pp. 71-79 ◽  
Author(s):  
W L Wong ◽  
M A Brostrom ◽  
G Kuznetsov ◽  
D Gmitter-Yellen ◽  
C O Brostrom
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Extracellular Fluid ◽  
Mrna Translation ◽  
Protein Processing ◽  
Pituitary Cells ◽  
Translational Initiation ◽  
Early Protein ◽  
Inhibition Of Protein Synthesis

Thapsigargin, a tumour-promoting sesquiterpene lactone, selectively inhibits the Ca(2+)-ATPase responsible for Ca2+ accumulation by the endoplasmic reticulum (ER). Mobilization of ER-sequestered Ca2+ to the cytosol and to the extracellular fluid subsequently ensues, with concomitant alteration of cellular functions. Thapsigargin was found to serve as a rapid, potent and efficacious inhibitor of amino acid incorporation in cultured mammalian cells. At concentrations mobilizing cell-associated Ca2+ to the extracellular fluid, thapsigargin provoked extensive inhibition of protein synthesis within 10 min. The inhibition in GH3 pituitary cells involved the synthesis of almost all polypeptides, was not associated with increased cytosolic free Ca2+ concentration ([Ca2+]i), and was not reversed at high extracellular Ca2+. The transient rise in [Ca2+]i triggered by ionomycin was diminished by thapsigargin. Polysomes failed to accumulate in the presence of the drug, indicative of impaired translational initiation. With longer (1-3 h) exposures to thapsigargin, recovery of translational activity was observed accompanied by increased synthesis of the ER protein glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein (‘GRP78/BiP’) and its mRNA. Such inductions were comparable with those observed previously with Ca2+ ionophores which mobilize the cation from all intracellular sequestered sites. Actin mRNA concentrations declined significantly during such treatments. In HepG2 cells processing and secretion of the glycoprotein alpha 1-antitrypsin were rapidly suppressed by thapsigargin. Ca2+ sequestered specifically by the ER is concluded to be essential for optimal protein synthesis and processing. These rapid effects of thapsigargin on mRNA translation, protein processing and gene expression should be considered when evaluating potential mechanisms by which this tumour promoter influences cellular events.

scholarly journals Human Retrovirus Codon Usage from tRNA Point of View: Therapeutic Insights

2013 ◽  
Vol 7 ◽  
pp. BBI.S12093 ◽  
Author(s):  
Diego Frias ◽  
Joana P. Monteiro-Cunha ◽  
Aline C. Mota-Miranda ◽  
Vagner S. Fonseca ◽  
Tulio De Oliveira ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Codon Usage ◽  
Supply And Demand ◽  
Mrna Translation ◽  
Protein Translation ◽  
Point Of View ◽  
T Score ◽  
Synonymous Codons ◽  
Trna Species ◽  
Gene Data

The purpose of this study was to investigate the balance between transfer ribonucleic acid (tRNA) supply and demand in retrovirus-infected cells, seeking the best targets for antiretroviral therapy based on the hypothetical tRNA Inhibition Therapy (TRIT). Codon usage and tRNA gene data were retrieved from public databases. Based on logistic principles, a therapeutic score (T-score) was calculated for all sense codons, in each retrovirus-host system. Codons that are critical for viral protein translation, but not as critical for the host, have the highest T-score values. Theoretically, inactivating the cognate tRNA species should imply a severe reduction of the elongation rate during viral mRNA translation. We developed a method to predict tRNA species critical for retroviral protein synthesis. Four of the best TRIT targets in HIV-1 and HIV-2 encode Large Hydrophobic Residues (LHR), which have a central role in protein folding. One of them, codon CUA, is also a TRIT target in both HTLV-1 and HTLV-2. Therefore, a drug designed for inactivating or reducing the cytoplasmatic concentration of tRNA species with anticodon TAG could attenuate significantly both HIV and HTLV protein synthesis rates. Inversely, replacing codons ending in UA by synonymous codons should increase the expression, which is relevant for DNA vaccine design.

Stable isotope-labelling analysis of the impact of inhibition of the mammalian target of rapamycin on protein synthesis

2012 ◽  
Vol 444 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Yilin Huo ◽  
Valentina Iadevaia ◽  
Zhong Yao ◽  
Isabelle Kelly ◽  
Sabina Cosulich ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Stable Isotope ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Target Of Rapamycin ◽  
Stable Isotope Labelling ◽  
Isotope Labelling ◽  
Cell Functions ◽  
Specific Mrnas ◽  
The Impact

mTORC1 [mTOR (mammalian target of rapamycin) complex 1] regulates diverse cell functions. mTORC1 controls the phosphorylation of several proteins involved in mRNA translation and the translation of specific mRNAs, including those containing a 5′-TOP (5′-terminal oligopyrimidine). To date, most of the proteins encoded by known 5′-TOP mRNAs are proteins involved in mRNA translation, such as ribosomal proteins and elongation factors. Rapamycin inhibits some mTORC1 functions, whereas mTOR-KIs (mTOR kinase inhibitors) interfere with all of them. mTOR-KIs inhibit overall protein synthesis more strongly than rapamycin. To study the effects of rapamycin or mTOR-KIs on synthesis of specific proteins, we applied pSILAC [pulsed SILAC (stable isotope-labelling with amino acids in cell culture)]. Our results reveal, first, that mTOR-KIs and rapamycin differentially affect the synthesis of many proteins. Secondly, mTOR-KIs inhibit the synthesis of proteins encoded by 5′-TOP mRNAs much more strongly than rapamycin does, revealing that these mRNAs are controlled by rapamycin-insensitive outputs from mTOR. Thirdly, the synthesis of certain other proteins shows a similar pattern of inhibition. Some of them appear to be encoded by ‘novel’ 5′-TOP mRNAs; they include proteins which, like known 5′-TOP mRNA-encoded proteins, are involved in protein synthesis, whereas others are enzymes involved in intermediary or anabolic metabolism. These results indicate that mTOR signalling may promote diverse biosynthetic processes through the translational up-regulation of specific mRNAs. Lastly, a SILAC-based approach revealed that, although rapamycin and mTOR-KIs have little effect on general protein stability, they stabilize proteins encoded by 5′-TOP mRNAs.

scholarly journals A chemical screen identifies a link between lipid metabolism and mRNA translation

2020 ◽  
Author(s):  
Alba Corman ◽  
Dimitris C. Kanellis ◽  
Maria Häggblad ◽  
Vanesa Lafarga ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Mrna Translation ◽  
Protein Translation ◽  
Sphingosine Kinases ◽  
Chemical Screen ◽  
Approved Drugs ◽  
The Impact

ABSTRACTmRNA translation is one of the most energy-demanding processes for living cells, alterations of which have been frequently documented in human disease. Using recently developed technologies that enable image-based quantitation of overall translation levels, we here conducted a chemical screen to evaluate how medically approved drugs, as well as drugs that are currently under development, influence overall translation levels. Consistent with current knowledge, inhibitors of the mTOR signaling pathway were the most represented class among translation suppresors. In addition, we identified that inhibitors of sphingosine kinases (SPHKs) also reduce mRNA translation levels independently of mTOR. Mechanistically this is explained by an effect of the compounds on the membranes of the endoplasmic reticulum, which activates the integrated stress response (ISR). Accordingly, the impact of SPHK inhibitors on translation is alleviated by the concomitant inhibition of ISR kinases. On the other hand, and despite the large number of molecules tested, our study failed to identify chemicals capable of substantially increasing mRNA translation, raising doubts on to what extent translation can be supra-physiologically stimulated in mammalian cells. In summary, our study provides the first comprehensive characterization of the effect of known drugs on protein translation and has helped to unravel a new link between lipid metabolism and mRNA translation in human cells.

Investigating the Potential Use of L-Asparaginase in Myeloid Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3641-3641
Author(s):  
Sung Ah Jun ◽  
Lusia Sepiashvili ◽  
Thomas Kislinger ◽  
Mark D. Minden
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Acute Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Nuclear Antigen ◽  
Intracellular Enzyme ◽  
Inhibition Of Protein Synthesis ◽  
Factor C ◽  
Resistant Cells

Abstract Abstract 3641 Introduction: L-asparaginase (LA) in combination with other drugs has been one of the standard components of acute lymphocytic leukemia (ALL) therapy for decades. Its antineoplastic effects are likely caused by its depletion of extracellular asparagine and glutamine creating a state of amino acid deficiency and subsequent inhibition of protein synthesis. Despite its efficacy in ALL, LA has been used only occasionally in the treatment of other leukemias and solid tumors. Previous in vitro studies have observed varied response to LA in acute myeloid leukemia (AML) across the French-American-British subtypes. We wanted to elucidate the possible resistance mechanisms of myeloid leukemic cells during LA treatment to increase the efficacy of LA for AML treatment. One of the candidate proteins identified in multiple studies was asparagine synthetase (ASNS), an intracellular enzyme catalyzing the reverse reaction of LA whose expression is up-regulated during nutrient stress. The aim of this study was to investigate the potential of repositioning LA for AML treatment by identifying key components of the cellular response to LA in myeloid leukemic cell lines and primary AML samples. Results: In all the cell lines treated with LA, we observed an inhibition of growth rate and colony formation. Furthermore, we detected apoptotic death by annexin V and propidium iodide staining in most cell lines except for K562. We also observed a leftward shift towards monosomes in polysome profiles of LA sensitive but not insensitive cells, indicating a role for global inhibition of protein synthesis in the effect of LA. To further understand the differences in the responses between resistant and sensitive cell lines at the protein level, we utilized MudPIT (multidimensional protein identification technology) that combines 2-dimensional liquid chromatography coupled to mass spectrometry to separate and identify proteins. Using DAVID, an online program that identifies statistically significant enriched biological themes in gene lists, we compared the MudPIT identified proteomes in LA treated and untreated HL-60 (LA sensitive) and K562 (LA resistant) cells. In HL60, up-regulated proteins in the treated sample were enriched for carbohydrate metabolism (aldolase A, lactate dehydrogenase, 6-phosphogluconolactonase). We also observed decreased expression of proteins involved in cell division (replication factor C, proliferating cell nuclear antigen, minichromosome maintenance complex component 3). The data from K562 is currently being analyzed. A reported predictor of sensitivity to LA is the level of ASNS. To see if this was involved in the resistance of K562 to LA we used shRNA to knockdown ASNS in these cells. While there was some increase in the sensitivity of the cells to LA, the degree of killing did not approach that of other cell lines. Finally, AML primary samples treated with LA were inhibited in their ability to form colonies compared to untreated controls. Interestingly there was no correlation between the level of ASNS and sensitivity of the primary cells. Taken together these studies suggest that other factors are important in mediating the response of cells to LA. Conclusions: Our study shows that LA is effective in killing some forms of AML by inhibiting growth, blocking protein synthesis and inducing apoptosis. Increased sensitivity to LA in ASNS knockdown cell lines indicate a role for ASNS in LA resistance but the absence of strong correlation between ASNS expression and LA resistance in primary samples suggest that ASNS is not solely responsible. The availability of sensitive and resistant myeloid cells provides us with the opportunity to identify mechanisms of resistance. The identification of differentially expressed proteins in the sensitive and resistant cells using MudPIT will help to identify targets that if blocked can synergize with LA and render a resistant cell sensitive. Disclosures: Off Label Use: L-asparaginase is a drug used to treat acute lymphocytic leukemia.

Sign in / Sign up

Export Citation Format

Share Document