scholarly journals Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells Via JAK2/STAT3 Pathway Inhibition

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1343-1343 ◽  
Author(s):  
Ly Quoc Trung ◽  
Luis Jorge Espinoza ◽  
Akiyoshi Takami ◽  
Akiyo Yoshida ◽  
Shinji Nakao
Keyword(s):  
Cell Cycle ◽  
Nk Cells ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Nk Cell ◽  
Therapeutic Potential ◽  
Dependent Manner ◽  
Stat3 Pathway ◽  
Cycle Arrest

Abstract Abstract 1343 Natural killer (NK)-cell malignancies, particularly aggressive NK-cell leukemias/lymphomas, have poor prognoses. Although recent regimens that include L-asparaginase substantially improve outcomes, novel therapeutic approaches are needed to improve clinical responses. Recent reports have shown that the signal transducer and activator of transcription 3 (STAT3) pathway is critical for proliferation and survival of malignant NK cells. Resveratrol is a naturally-occurring polyphenol that has been extensively studied for its anti-inflammatory, cardioprotective, and anti-cancer activities. In this study, we investigated the potential anti-tumor activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92, and NK-YS. Resveratrol significantly suppressed cell proliferation in a dose- and time-dependent manner in these four cell lines. Flow cytometry analysis with annexin V/propidium iodide staining showed a variable but consistent induction of apoptosis in the four cell lines treated with resveratrol for 48 hours, ranging from 57.1±6.9% of apoptosis in the L-asparaginase resistant cell line KHYG-1 to 53.4±9.7%, 28.8±3.3%, and 51.7±6.7% in NKL, NK-92, and NK-YS cells respectively (Fig. 1a). Notably, the anti-tumor activity of resveratrol against NK cell lines was p53 independent as demonstrated by equal efficacy of resveratrol against NK cell lines pretreated with the p53 inhibitor Pifithrin-α. Immunoblot analysis to study intracellular signaling in resveratrol-treated cells showed suppression of constitutively active STAT3 in all four cell lines 24 hours after treatment. Remarkably, resveratrol inhibited JAK2 phosphorylation, but had no effect on other known upstream mediators of STAT3 activation such as PTEN and Tyk2 (Fig. 1b). Resveratrol induced robust G1 cell cycle arrest and down-regulation of two anti-apoptotic proteins, Mcl-1 and survivin, both of which are downstream effectors of the STAT-3 pathway. Furthermore, resveratrol enhanced the pro-apoptotic and anti-proliferative activities of L-asparaginase against NKL and NK-92 cells by 32% and 126% respectively. These data indicate that resveratrol possesses a potent anti-tumor effect via inactivation of the STAT3 pathway in malignant NK cells. These mechanistic findings suggest that resveratrol may have therapeutic potential against NK cell malignancies. Our finding that resveratrol is a bonafide JAK2 inhibitor extends its therapeutic potential to other diseases with deregulated JAK2 signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Glioblastoma Multiforme Stem Cell Cycle Arrest by Alkylaminophenol through the Modulation of EGFR and CSC Signaling Pathways

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 681 ◽  
Author(s):  
Phuong Doan ◽  
Aliyu Musa ◽  
Akshaya Murugesan ◽  
Vili Sipilä ◽  
Nuno R. Candeias ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Glioblastoma Multiforme ◽  
Cell Cycle Arrest ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Cell Population ◽  
Significant Role ◽  
Dependent Manner ◽  
Cycle Arrest

Cancer stem cells (CSCs), a small subpopulation of cells existing in the tumor microenvironment promoting cell proliferation and growth. Targeting the stemness of the CSC population would offer a vital therapeutic opportunity. 3,4-Dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), a small synthetic phenol compound, is proposed to play a significant role in controlling the CSC proliferation and survival. We assessed the potential therapeutic effects of THTMP on glioblastoma multiforme (GBM) and its underlying mechanism in various signaling pathways. To fully comprehend the effect of THTMP on the CSCs, CD133+ GBM stem cell (GSC) and CD133- GBM Non-stem cancer cells (NSCC) population from LN229 and SNB19 cell lines was used. Cell cycle arrest, apoptosis assay and transcriptome analysis were performed for individual cell population. THTMP strongly inhibited NSCC and in a subtle way for GSC in a time-dependent manner and inhibit the resistance variants better than that of temozolomide (TMZ). THTMP arrest the CSC cell population at both G1/S and G2/M phase and induce ROS-mediated apoptosis. Gene expression profiling characterize THTMP as an inhibitor of the p53 signaling pathway causing DNA damage and cell cycle arrest in CSC population. We show that the THTMP majorly affects the EGFR and CSC signaling pathways. Specifically, modulation of key genes involved in Wnt, Notch and Hedgehog, revealed the significant role of THTMP in disrupting the CSCs’ stemness and functions. Moreover, THTMP inhibited cell growth, proliferation and metastasis of multiple mesenchymal patient-tissue derived GBM-cell lines. THTMP arrests GBM stem cell cycle through the modulation of EGFR and CSC signaling pathways.

scholarly journals Degalactotigonin, a Steroidal Glycoside from Solanum nigrum, Induces Apoptosis and Cell Cycle Arrest via Inhibiting the EGFR Signaling Pathways in Pancreatic Cancer Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Hoang Le Tuan Anh ◽  
Phuong Thao Tran ◽  
Do Thi Thao ◽  
Duong Thu Trang ◽  
Nguyen Hai Dang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Pancreatic Cancer ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Solanum Nigrum ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cycle Arrest

Degalactotigonin (1) and three other steroidal compounds solasodine (2), O-acetyl solasodine (3), and soladulcoside A (4) were isolated from the methanolic extract of Solanum nigrum, and their chemical structures were elucidated by spectroscopic analyses. The isolated compounds were evaluated for cytotoxic activity against human pancreatic cancer cell lines (PANC1 and MIA-PaCa2) and lung cancer cell lines (A549, NCI-H1975, and NCI-H1299). Only degalactotigonin (1) showed potent cytotoxicity against these cancer cell lines. Compound 1 induced apoptosis in PANC1 and A549 cells. Further study on its mechanism of action in PANC1 cells demonstrated that 1 significantly inhibited EGF-induced proliferation and migration in a concentration-dependent manner. Treatment of PANC1 cells with degalactotigonin induced cell cycle arrest at G0/G1 phase. Compound 1 induced downregulation of cyclin D1 and upregulation of p21 in a time- and concentration-dependent manner and inhibited EGF-induced phosphorylation of EGFR, as well as activation of EGFR downstream signaling molecules such as Akt and ERK.

scholarly journals Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

PLoS ONE ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. e55183 ◽  
Author(s):  
Ly Quoc Trung ◽  
J. Luis Espinoza ◽  
Akiyoshi Takami ◽  
Shinji Nakao
Keyword(s):  
Cell Cycle ◽  
Nk Cells ◽  
Cell Cycle Arrest ◽  
Stat3 Pathway ◽  
Cycle Arrest ◽  
Pathway Inhibition

PI3K a Selective Inhibitor Induces Growth Inhibition In Mantle Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1812-1812
Author(s):  
Yixin Zhou ◽  
Linhua Jin ◽  
Stefania Pittaluga ◽  
Mark Raffeld ◽  
Takashi Miida ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Inhibition ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Pi3k Inhibitor ◽  
Dependent Manner ◽  
Pi3k Inhibitor Ly294002 ◽  
Pi3k Inhibitors ◽  
Cycle Arrest

Abstract Abstract 1812 Deregulation of the phosphatidylinositol 3-kinase (PI3K)-mediated signaling plays an important role in the development of cell proliferation of mantle cell lymphoma (MCL). The PI3K pathway activation in MCL has been shown to result from constitutive B cell receptor (BCR) activation which is directly mediated by the Class IA PI3K p110 isoforms (a, β, and d). However, their relative contribution in MCL is not fully understood. In this study, the activity and molecular mechanisms of isoform-selective PI3K inhibitors which target different isoforms of the p110-kDa subunit has been investigated. We utilized the isoform-selective PI3K inhibitors; PI3-Ka inhibitor IV (p110a), TGX115 (p110b), IC87114 (p110d) and the non-specific PI3K inhibitor LY294002 (all inhibitors were purchased commercially). The p110a and p110d but not p110b isoform protein expression was detected in all tested MCL cell lines (Granta 519, JVM-2, Z138, Jeko-1, MINO). PI3-Ka inhibitor IV as well as non-specific PI3K inhibitor LY294002 induced cell growth inhibition with dose-dependent manner (IC50 at 48 hrs; PI3-Ka inhibitor IV: 17.5 μM for Granta 519, 14.3 μM for Jeko-1, 16.5 μM for Z138, LY294002: 14.8 μM for Granta 519, 19.4 μM for Jeko-1, 15.0 μM for Z138, MTT test). However, neither IC87114 nor TGX115 showed significant cell growth inhibition up to 40mM. Low dose of PI3-Ka inhibitor IV (5 μM) or LY294002 (5 μM) induced G0/G1 cell cycle arrest (increase of G0/G1 phase: PI3-Ka inhibitor IV 17.9 % for Granta 519, 28.2 % for Jeko-1, LY294002 19.3 % for Granta 519, 14.5 % for Jeko-1), and the higher dose (10 μM) increased apoptosis(specific apoptosis: PI3-Ka inhibitor IV 10.8 % for Granta 519, 15.3 % for Jeko-1, LY294002 13.6 % for Granta 519, 19.6 % for Jeko-1). No induction of cell cycle arrest/apoptosis by IC87114 or TGX115 treatment was observed. We then tried to assess the inhibition of PI3K/Akt signaling activation by p110a and p110d inhibitors. PI3-Ka inhibitor IV (10 μM) completely diminished phosphorylated (p-) Akt in all cell lines analyzed. Further investigation with 1–10 μM PI3-Ka inhibitor IV or IC87114 in Granta 519 and Jeko-1 cells declared that 1 μM PI3-Ka inhibitor IV almost diminished p-Akt and p-S6rp in both cells. The phosphorylation level of other PI3K/Akt signaling downstream substrates, GSK3-b and 4E-BP1, were down-regulated in dose dependent manner. Recently, GSK3-b kinase has been shown to negatively regulate cell cycle progression through Cyclin D1 repression in MCL. We observed that PI3-Ka inhibitor IV decreased Cyclin D1 expression and active pRb which are responsible for G0/G1 cell cycle arrest. The treatment with IC87114 (10 μM) performed moderate decrease of p-Akt, p-S6rp, and p-4E-BP, while no change in the levels of p-GSK3-b, Cyclin D1, or p-pRb was observed in both Granta 519 and Jeko-1 cells. We also tested whether the combination of PI3-Ka inhibitor IV or IC87114 with the proteasome inhibitor bortezomib induces synergistic cytotoxicity in MCL. No synergistic anti-proliferative effect was observed in any of the MCL cell lines analyzed. These findings demonstrate that p110a may be the responsible Class IA PI3K isoform for the development of MCL cell proliferation, and p110a isoform-selective PI3K inhibitor but not p110d or p110b inhibitors may provide a better therapeutic index relative to pan-PI3K inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MDM4 is an essential disease driver targeted by 1q gain in Burkitt lymphoma

2018 ◽  
Author(s):  
Jennifer Hüllein ◽  
Mikołaj Słabicki ◽  
Maciej Rosolowski ◽  
Alexander Jethwa ◽  
Stefan Habringer ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
Mutant Cell ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
1Q Gain

AbstractOncogenic MYC activation promotes cellular proliferation in Burkitt lymphoma (BL), but also induces cell cycle arrest and apoptosis mediated by TP53, a tumor suppressor gene that is mutated in 40% of BL cases. To identify therapeutic targets in BL, we investigated molecular dependencies in BL cell lines using RNAi-based, loss-of-function screening. By integrating genotypic and RNAi data, we identified a number of genotype-specific dependencies including the dependence of TCF3/ID3 mutant cell lines on TCF3 and of MYD88 mutant cell lines on TLR signaling. TP53 wild-type (TP53wt) BL were dependent on MDM4, a negative regulator of TP53. In BL cell lines, MDM4 knockdown induced cell cycle arrest and decreased tumor growth in a xenograft model in a p53-dependent manner, while small molecule inhibition of the MDM4-p53 interaction restored p53 activity resulting in cell cycle arrest. Consistent with the pathogenic effect of MDM4 upregulation in BL, we found that TP53wt BL samples were enriched for gain of chromosome 1q which includes the MDM4 locus. 1q gain was also enriched across non-BL cancer cell lines (n=789) without TP53 mutation (23% in TP53wt and 12% in TP53mut, p<0.001). In a set of 216 cell lines representing 19 cancer entities from the Achilles project, MDM4 was the strongest genetic dependency in TP53wt cell lines (p<0.001).Our findings show that in TP53wt BL, MDM4-mediated inhibition of p53 is a mechanism to evade cell cycle arrest. The data highlight the critical role of p53 as a tumor suppressor in BL, and identifies MDM4 as a key functional target of 1q gain in a wide range of cancers, which is therapeutically targetable.

Effects of the multikinase inhibitor regorafenib in neuroblastoma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 10553-10553
Author(s):  
Peter E. Zage ◽  
Divya Subramonian ◽  
Qianxing Mo ◽  
Shixia Huang
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Viability ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Therapeutic Potential ◽  
Neuroblastoma Cells ◽  
Dependent Manner ◽  
Western Blots

10553 Background: Neuroblastoma (NB) is the most common extracranial solid pediatric tumor, and children with high-risk NB have poor survival rates and need novel treatment strategies. Regorafenib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for treating adult solid tumors such as advanced metastatic colorectal cancer and gastrointestinal stromal tumors, inhibits many RTKs, including PDGFR-β, VEGFR1-3, RET, c-Kit and FGFR family members. Based on the potential roles for these targets in neuroblastoma pathogenesis, we explored the therapeutic potential of Regorafenib alone and in combination with 13-cis-retinoic acid against neuroblastoma cells. Methods: We treated NB cell lines with increasing concentrations of Regorafenib and measured cell viability using MTT assays. We further measured the occupied percent confluence over time using continuous live cell imaging. We performed Western blots for caspase cleavage to measure apoptosis and flow cytometry to determine cell cycle expression. We performed Reverse Phase Protein Array (RPPA) analysis of neuroblastoma cells before and after treatment with regorafenib combined with 13- cis-retinoic acid. Results: IC50values for the tested cell lines ranged between 2.5mcM and 12.5mcM after 72 hours of exposure to Regorafenib, and decreased viability was due to a combination of apoptosis and cell cycle arrest. RPPA analysis identified alterations in multiple proteins and pathways after Regorafenib with retinoic acid treatment, including the PI3K/Akt/mTOR and Jak/Stat pathways. Phosphorylation of Erk1/2, S6, Akt, and c-Jun were decreased, while protein expression of GATA3 was increased in a dose-dependent manner. Conclusions: Regorafenib treatment results in reduced neuroblastoma cell viability and increased apoptosis via effects on several signaling pathways. Effects on intracellular signaling pathways associated with responses to the combination of regorafenib plus retinoic acid represent opportunities to develop novel combination therapies, representing potential new therapeutic strategies for children with neuroblastoma.

PI3KCA and PIM Inhibitors in Peripheral T-Cell Lymphomas

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4917-4917
Author(s):  
Esperanza Martin-Sanchez ◽  
Socorro M. Rodriguez-Pinilla ◽  
Luis Lombardia ◽  
Margarita Sanchez-Beato ◽  
Beatriz Dominguez-Gonzalez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
T Cell ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Gene Expression Signature ◽  
Dependent Manner ◽  
Expression Signature ◽  
Cycle Arrest ◽  
T Cell Lymphomas

Abstract Abstract 4917 T-cell lymphomas (TCL) are a heterogeneous group of aggressive malignancies lacking specific and efficient therapy. Unfortunately, there are neither animal models nor representative cell lines for most TCL types, making functional and pharmacogenomics studies even more difficult. PI3K and PIM are kinases involved in cell proliferation, frequently altered in human cancer that seems to play a critical role in T-cell development and activation. Genomic studies have identified PIK3CD subunit to be significantly associated with in activation of CD40, NF-kB and TCR-pathways. The aim of this project is to determine the efficiency of PI3K inhibitors (PI3Ki) and PIM inhibitors (PIMi) in TCL, looking for biomarkers of their mechanism of action and to identify markers that could identify responders from non-responders. Twenty PTCL and seven reactive lymph nodes were studied using gene expression microarrays. We performed an in silico analysis using the Connectivity Map program to identify drugs that could potentially reverse PTCL gene expression signature. Among them, several PI3K/mTOR inhibitors were found. A panel of 6 TCL cell lines belonging to different TCL subgroups were treated with 3 PI3Ki (LY294002, ETP-45658, GDC-0941) and one PIMi (ETP-39010). Functional studies were also done to establish the role of each of the targeted genes. In vitro studies showed that PI3Ki induced G1 cell cycle arrest in all cell lines, and apoptosis in a portion of them, in a time/dose-dependent manner. We also observed a decrease in the levels of pAKT(S473), pGSK3B(S9) and p-p70S6K(T389) after treatment. In addition, both the analysis of the PTCL gene expression signature as well as western blot studies on TCL cell lines has shown overexpression of PIM family genes, A decrease in cell viability, and a strong induction of apoptosis in all cell lines was seen after PIM inhibition, without cell cycle arrest. Several diagnostic and pharmacodynamic biomarkers of PIMi have been identified at the mRNA and protein level in both cell lines In conclusion, our results indicate that PI3Ki and PIMi are effective therapeutic approaches for TCLs, identifying potential markers for patient's stratification and pharmacodynamic assessment. Disclosures: No relevant conflicts of interest to declare.

Optimized anti–tumor effects of anthracyclines plus Vinca alkaloids using a novel, mechanism-based application schedule

Blood ◽  
2011 ◽  
Vol 118 (23) ◽  
pp. 6123-6131 ◽  
Author(s):  
Harald Ehrhardt ◽  
David Schrembs ◽  
Christian Moritz ◽  
Franziska Wachter ◽  
Subrata Haldar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Childhood Leukemia ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Vinca Alkaloids ◽  
Cycle Arrest ◽  
Induced Apoptosis

Abstract Application of anthracyclines and Vinca alkaloids on the same day represents a hallmark of polychemotherapy protocols for hematopoietic malignancies. Here we show, for the first time, that both drugs might act most efficiently if they are applied on different days. Proof-of-concept studies in 18 cell lines revealed that anthracyclines inhibited cell death by Vinca alkaloids in 83% of cell lines. Importantly, in a preclinical mouse model, doxorubicin reduced the anti–tumor effect of vincristine. Both drugs acted in a sequence-dependent manner and the strongest anti–tumor effect was obtained if both drugs were applied on different days. Most notably for clinical relevance, in 34% of 35 fresh primary childhood leukemia cells tested in vitro, doxorubicin reduced the anti–tumor effect of vincristine. As underlying mechanism, doxorubicin activated p53, p53 induced cell-cycle arrest, and cell-cycle arrest disabled inactivation of antiapoptotic Bcl-2 family members by vincristine; therefore, vincristine was unable to activate downstream apoptosis signaling. As molecular proof, antagonism was rescued by knockdown of p53, whereas knockdown of cyclin A inhibited vincristine-induced apoptosis. Our data suggest evaluating anthracyclines and Vinca alkaloids on different days in future trials. Selecting drug combinations based on mechanistic understanding represents a novel conceptional strategy for potent polychemotherapy protocols.

scholarly journals Sulforaphane Causes Cell Cycle Arrest and Apoptosis in Human Glioblastoma U87MG and U373MG Cell Lines under Hypoxic Conditions

2021 ◽  
Vol 22 (20) ◽  
pp. 11201
Author(s):  
Giulia Sita ◽  
Agnese Graziosi ◽  
Patrizia Hrelia ◽  
Fabiana Morroni
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Hypoxic Condition ◽  
Primary Brain Tumor ◽  
Dependent Manner ◽  
Hypoxic Conditions ◽  
Cycle Arrest ◽  
Extracellular Signal ◽  
Dose Dependent

Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary brain tumor. The median survival rate from diagnosis ranges from 15 to 17 months because the tumor is resistant to most therapeutic strategies. GBM exhibits microvascular hyperplasia and pronounced necrosis triggered by hypoxia. Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, has already demonstrated the ability to inhibit cell proliferation, by provoking cell cycle arrest, and leading to apoptosis in many cell lines. In this study, we investigated the antineoplastic effects of SFN [20–80 μM for 48 h] in GBM cells under normoxic and hypoxic conditions. Cell viability assays, flow cytometry, and Western blot results revealed that SFN could induce apoptosis of GBM cells in a dose-dependent manner, under both conditions. In particular, SFN significantly induced caspase 3/7 activation and DNA fragmentation. Moreover, our results demonstrated that SFN suppressed GBM cells proliferation by arresting the cell cycle at the S-phase, also under hypoxic condition, and that these effects may be due in part to its ability to induce oxidative stress by reducing glutathione levels and to increase the phosphorylation of extracellular signal-regulated kinases (ERKs). Overall, we hypothesized that SFN treatment might serve as a potential therapeutic strategy, alone or in combination, against GBM.

Molecular Mechanisms of the mTOR Inhibitor Temsirolimus (CCI-779) Antiproliferative Effects in Mantle Cell Lymphoma: Induction of Cell Cycle Arrest, Autophagy, and Synergy with Vorinostat (SAHA).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2493-2493 ◽  
Author(s):  
Victor Y. Yazbeck ◽  
Georgios V. Georgakis ◽  
Yang Li ◽  
Eiji Iwado ◽  
Seiji Kondo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Cell Lymphoma ◽  
Time Dependent ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Aberrant activation of the PI3-Kinase/Akt/mTOR survival pathway has been implicated in promoting the growth and survival of a variety of cancers, including lymphoma, and is currently being explored for cancer therapy. Importantly, the small molecule mTOR inhibitor temsirolimus (CCI-779) recently demonstrated significant clinical activity in patients with relapsed mantle cell lymphoma (MCL). However, the mechanism of action of temsirolimus in MCL cells is unknown. In this study, we demonstrated that temsirolimus induced cell growth inhibition in three MCL cell lines in a time-dependent and dose-dependent manner. The activity of temsirolimus was determined in 3 mantle cell lymphoma cell lines (Jeko-1, Mino, SP53). Temsirolimus upregulated p27 without altering cyclin D1 levels, resulting in cell cycle arrest in the G0/G1 phase. The Akt/mTOR pathway has been implicated in regulating cellular autophagy in yeasts and in mammalian cells. Thus, we examined whether temsirolimus may also induce autophagy in MCL cells, which is identified by the sequestering of cytoplasmic proteins into the lytic autophagosomes and autolysosome, and the formation of acidic vesicular organelles (AVOs). Temsirolimus induced AVOs formation indicative of autophagy in all MCL cell lines at doses ranging between 1 and 1000 nM in a time-dependent manner, with the highest activity observed between 72 and 96 hours of incubation. LC3 is essential for amino acid starvation-induced autophagy in yeasts. LC3-I is the cytoplasmic form, which is processed into the LC3-II form that is associated with the autophagosome membrane. Incubation of the SP53 cells with temsirolimus (1,000 nM) for 96 hours, resulted in processing LC3-I into LC3-II, indicative of autophagy induction. To further confirm induction of autophagy, SP53 cells expressing LC3-fused green fluorescent protein (GFP-LC3) were treated with temsirolimus and the pattern of LC3 distribution was compared with untreated cells using fluorescence microscopy. Untreated control cells showed a diffuse cytoplasmic distribution of LC3, whereas temsirolimus -treated cells showed a punctate pattern of green fluorescence, indicative of its association with autophagosomes. Furthermore, temsirolimus increased acidic vesicular organelles and microtubule-associated protein 1 light chain 3 (LC3) processing as determined by Western blot, which are characteristic of autophagy. In contrast, temsirolimus had minimal induction of apoptosis. Moreover, temsirolimus inhibited ribosomal S6 phosphorylation, an mTOR downstream target. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) demonstrated antiproliferative activity in a dose and time dependent manner in all three MCL cell lines. SAHA enhanced the activity of temsirolimus, which was associated with ERK dephosphorylation and caspase 3 activation. In contrast, temsirolimus did not potentiate the antitumor effects of bortezomib, doxorubicin, or gemcitabine. Our results demonstrate that in short-term culture, temsirolimus is primarily a cytostatic drug, and suggest that SAHA may potentiate the clinical efficacy of temsirolimus patients with MCL.

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