Inhibition of Protein Kinase C beta by Enzastaurin (LY 317615) Induces Alterations of Key Regulators of Cell Cycle and Apoptosis in Mantle Cell Lymphoma and Synergizes with Chemotherapeutic Agents in a Sequence Dependent Manner.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2416-2416 ◽  
Author(s):  
Malte Rieken ◽  
Oliver Weigert ◽  
Alessandro Pastore ◽  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Western Blot ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Dependent Manner ◽  
Kinase C ◽  
Mantle Cell

Abstract Mantle Cell Lymphoma (MCL) is a subtype of malignant lymphoma, characterized by a poor clinical outcome, a median survival time of three years and almost no long-term survivors. In previous studies it could be shown that constitutive overexpression of NFκB pathway plays an important role in the pathogenesis of MCL. The protein kinase C (PKC) family is essential in cell signal regulation effecting cellular growth, proliferation and apoptosis. Previous studies in diffuse large B-cell lymphoma suggest that PKCβ overexpression enhances B-cell proliferation and is associated with poor clinical prognosis. The pivotal role of PKCβ in activation of NFκB renders it a promising therapeutic target in the therapy of MCL. Five MCL cell lines (GRANTA 519, HBL-2, Jeko-1, NCEB-1, Rec-1) and two haematological control cell lines (Jurkat, Karpas 422) were treated with Enzastaurin in a proliferation inhibiting dose of 10μM, defined in initial pilot experiments. Real-time PT-PCR and protein expression (Western blot) levels of various regulators of cell cycle and apoptosis were determined at various time points of enzastaurin treatment (0.5 to 12 hours). In addition analysis of cell cycle and apoptosis were performed by flow-cytometry. All, cell lines were also exposed to different doses of enzastaurin in combination with cytarabine, fludarabine or mitoxantrone and analysed for efficacy by evaluating the combination index. Inhibition of proliferation was achieved in all cell lines in a dose and time-dependent manner, however susceptibility varied strongly within the cell lines characterized by IC 50 (24 hours) ranging from 8,0 μM in Jeko-1 to > 50μM in GRANTA 519, Rec-1 and Karpas 422. Induction of apoptosis was detected in all cell lines according to susceptibility, and cell cycle analysis exhibits a moderate accumulation of and cells in the G2/M phase. Significant alterations of RNA expression was detected for BCL-2, cyclin D1 and p14 applying the Kruskal-Wallis-Test. Preliminary Western blot data confirm the altered levels of protein expressions. Synergistic effects of Enzastaurin combinations were sequence dependent, and resulted in a super-additive effect when the exposure of cells to cytostatic agents was followed by Enzastaurin treatment. No synergy effect was observed in combination with fludarabine. Our study revealed alteration of gene expression, critical for the regulation of cell cycle and apoptosis, after Enzastaurin treatment. Even more important, combination with specific cytostatic drugs demonstrated a synergistic efficacy in vitro. Thus, our data suggest that inhibition of PKCβ can overcome chemotherapy resistance by cellular survival mechanisms supporting the evaluation of such combinations in clinical trials.

scholarly journals Prognostic impact of protein kinase C β II expression in R-CHOP-treated diffuse large B-cell lymphoma patients

2010 ◽  
Vol 23 (5) ◽  
pp. 686-693 ◽  
Author(s):  
Sari Riihijärvi ◽  
Satu Koivula ◽  
Heidi Nyman ◽  
Karin Rydström ◽  
Mats Jerkeman ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Prognostic Impact ◽  
Kinase C ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Cisplatin increases B-cell-lymphoma-2 expression via activation of protein kinase C and Akt2 in endometrial cancer cells

2011 ◽  
Vol 130 (8) ◽  
pp. 1755-1767 ◽  
Author(s):  
Alexandre Rouette ◽  
Sophie Parent ◽  
Julie Girouard ◽  
Valérie Leblanc ◽  
Eric Asselin
Keyword(s):  
Endometrial Cancer ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
B Cell ◽  
Cancer Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Kinase C

Protein kinase C-betaII expression in diffuse large B-cell lymphoma predicts for inferior outcome of anthracycline-based chemotherapy with and without rituximab

2009 ◽  
Vol 50 (10) ◽  
pp. 1666-1675 ◽  
Author(s):  
Kritika Chaiwatanatorn ◽  
Georgia Stamaratis ◽  
Ken Opeskin ◽  
Frank Firkin ◽  
Harshal Nandurkar
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Kinase C ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Phase II Study of Enzastaurin, a Protein Kinase C Beta Inhibitor, in Patients With Relapsed or Refractory Diffuse Large B-Cell Lymphoma

2007 ◽  
Vol 25 (13) ◽  
pp. 1741-1746 ◽  
Author(s):  
Michael J. Robertson ◽  
Brad S. Kahl ◽  
Julie M. Vose ◽  
Sven de Vos ◽  
Mary Laughlin ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
B Cell ◽  
Phase Ii Study ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Kinase C ◽  
Large B Cell Lymphoma ◽  
Grade 3 ◽  
Large B Cell

PurposeProtein kinase C beta (PKCβ) was identified by gene-expression profiling, preclinical evaluation, and independent immunohistochemical analysis as a rational therapeutic target in diffuse large B-cell lymphoma (DLBCL). We conducted a multicenter phase II study of a potent inhibitor of PKCβ, enzastaurin, in patients with relapsed or refractory DLBCL.Patients and MethodsEnzastaurin was taken orally once daily until disease progression or unacceptable toxicity occurred. Study end points included freedom from progression (FFP) for ≥ two cycles (one cycle = 28 days), objective response, and toxicity.ResultsFifty-five patients (median age, 68 years) were enrolled. Patients had received a median number of two prior therapies (range, one to five); six patients relapsed after high-dose therapy and autologous stem-cell transplantation. Only one grade 4 toxicity (hypomagnesemia) occurred. Grade 3 toxicities included fatigue (n = 2), edema (n = 1), headache (n = 1), motor neuropathy (n = 1), and thrombocytopenia (n = 1). No grade 3 or 4 neutropenia occurred. No deaths or discontinuations due to toxicity were reported. Fifteen patients completed less than one cycle of therapy. Twelve of 55 patients (22%; 95% CI, 13% to 46%) experienced FFP for ≥ two cycles, and eight patients remained free from progression for ≥ four cycles (15%; 95% CI, 6% to 27%). Four patients (7%; 95% CI, 2% to 18%), including three complete responders and one patient with stable disease, continue to experience FFP 20+ to 50+ months after study entry.ConclusionTreatment with enzastaurin was well-tolerated and associated with prolonged FFP in a small subset of patients with relapsed or refractory DLBCL. Further studies of enzastaurin in DLBCL are warranted.

Constitutive Activation of AKT Contributes to the Pathogenesis and Survival of Blastoid Variants of Mantle Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1908-1908
Author(s):  
Martina Rudelius ◽  
Stefania Pittaluga ◽  
Trinh H.-T. Pham ◽  
Thomas Fountaine ◽  
Leticia Quintanilla-Martinez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Regulatory Proteins ◽  
Akt Pathway ◽  
Akt Inhibitor ◽  
Akt Activation ◽  
Constitutive Activation ◽  
Mantle Cell

Abstract The serine/threonine protein kinase AKT [protein kinase B (PKB)] plays a pivotal role in the tumorigenesis of many human malignancies, including hematopoietic neoplasms. AKT mediates cancer cell survival and cell growth through the phosphorylation of key regulatory proteins such as FRKHL-1, MDM2 and p27. We wished to determine whether the AKT signaling pathway might be involved in the pathogenesis of mantle cell lymphoma (MCL).The activated form of AKT (pAKT) and phosphorylation of several AKT targets, including FRKHL-1, MDM2 and p27 as well as the upstream modulators PTEN and TCL-1 were analyzed by immunoblotting in 31 MCL cases (16 classic MCL and 15 MCL of the blastoid variant) and in four MCL cell lines (Granta 519, NCEB, REC-1 and Z138). PI3KCA mutation analysis at two hotspot regions (Exons 9 and 20) were completed for all cases and cell lines. Subsequently, inhibition of the PIK3/AKT-pathway with LY294002, Wortmannin and AKT-inhibitor was performed in the four cell lines. pAKT was expressed in all 15 blastoid variants of MCL and in the 4 cell lines, with high expression in 13/15 cases and low expression in 2/15 cases. In contrast, only 2/16 typical MCL expressed pAKT, at low levels. Furthermore, pAKT expression in the blastoid variants and cell lines was accompanied by the phosphorylation of multiple downstream targets of activated AKT, including the cell cycle regulatory proteins p27, FRKHL-1, MDM-2 and the apoptosis associated protein bad. 42% of the blastoid MCL cases showed loss of PTEN, while only 2 (12%) of the typical cases showed diminished PTEN expression.TCL-1 expression levels were not correlated with AKT activation and no mutations of PI3KCA were detected. Functional studies of all four MCL lines demonstrated that the phosphorylation of the downstream effectors were regulated by activated AKT:Inhibition of the PI3K/AKT pathway by both 20 μM LY294002 and 8 μM Wortmannin or 40 μM AKT-inhibitor abrogated the phosphorylation of AKT, p27, FRKHL-1, bad and MDM2 after 24 to 48 hours of treatment, and resulted in G1 arrest after 24 hours of treatment and measurable apoptosis by 48 hours. We conclude that constitutive activation of AKT contributes to the pathogenesis of the blastoid variants of MCL. One possible mechanism of AKT activation is the loss of PTEN expression. The inhibition of the PIK3/AKT pathway in MCL cell lines leads to induction of cell cycle arrest and apoptosis, raising the possibility that PI3K/AKT inhibitors may be effective in the treatment of aggressive (blastoid) variants of MCLs.

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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