High Expression of NEK2 Induces Drug Resistance by Inhibiting Premature Senescence Through Activation of the PI3K/Akt Pathway in Myeloma and Other Cancers,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3923-3923
Author(s):  
Fenghuang Zhan ◽  
Fang Xiao ◽  
Maurizio Zangari ◽  
Hongwei Xu ◽  
Guido J. Tricot
Keyword(s):  
Drug Resistance ◽  
Mouse Model ◽  
Cancer Cells ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Autologous Transplantation ◽  
Akt Pathway ◽  
Proliferative Potential ◽  
Premature Senescence ◽  
Akt Inhibitor

Abstract Abstract 3923 Background: Autologous transplantation in combination with the newer drugs has significantly prolonged survival of patients with myeloma (MM). However, the majority of patients still relapses. Our previous study has shown that NEK2, a chromosomal instability (CIN) gene, is up-regulated in many types of cancer cells and is highly related to drug resistance. Recently, premature senescence has been determined to be an indicator of the efficacy of anti-cancer drugs. In this study, we investigate the correlation between the inhibition of premature senescence and NEK2 induced drug resistance. Materials and Methods: Four MM cell lines (Δ47, KMS28PE, ARP1 and U266) and 2 other cancer cell lines (MCF7, H1299) over- and under-expressing NEK2 using a lentiviral delivery system were studied. NEK2 induced drug resistance was evaluated by colonogenic and standard apoptotic assays. The phenotypic changes of premature senescence were determined by using SA β-galactosidase (SA β-gal) staining. The mechanisms of NEK2-induced anti-senescence activity were studied in vitro and in the 5TGM1 murine MM mouse model. Results: Over-expression of NEK2 stimulated cancer cells into increased S and G2/M phase, resulting in polyploidy. All NEK2 over-expressing cancer cells were able to override doxorubicin-induced premature senescence and to maintain their proliferative potential. Mechanistic studies revealed that NEK2 activated the PI3K/Akt pathway. This was confirmed by the use of a specific PI3K/Akt inhibitor, KP372-1, which abrogated NEK2-induced drug resistance by decreasing clonogenicity of NEK2 over-expressing cells and by decreasing tumor burden and extending mouse survival in the 5TGM1 myeloma mouse model even better than bortezomib. In contrast, knockdown NEK2 by shRNA also induced premature senescence. We determined that NEK2 activates PI3K/Akt pathway by directly interacting with pp1. And the Akt down-stream targets GSK-3, MDM2, NF-kB and the tumor suppressor genes p53, p21, p27, the pro-apoptotic and pro-survival genes of the Bcl-2 family as well as the ABC transporters that related to drug resistance were identified to contribute to the inhibition of premature senescence resulting in drug resistance. Conclusion: NEK2 inhibits doxorubicin-induced premature senescence, resulting in increased drug resistance by activating the PI3K/Akt pathway. Alternatively, down regulation of NEK2 by shRNA inhibits cell proliferation and decreases drug resistance. Thus, targeting PI3K/Akt pathway should eliminate NEK2 induced drug resistance by re-establishing drug-induced premature senescence. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Negative Regulation of PTEN by MicroRNA-221 and Its Association with Drug Resistance and Cellular Senescence in Lung Cancer Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Ning Wang ◽  
Chen Zhu ◽  
Ye Xu ◽  
Wenliang Qian ◽  
Min Zheng
Keyword(s):  
Lung Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Term Treatment ◽  
Lung Cancer Cells ◽  
Akt Pathway ◽  
Long Term Treatment ◽  
Important Regulator

Objective.Chemotherapy is the routine method for treating many cancers, but long-term treatment may result in developing resistance to the drugs. The aim of this study was to identify whether noncoding RNAs play a role in drug resistance and how they affect drug resistance.Materials and Methods.The expression levels of miR-221 in different lung cancer cell lines H226, H1299, and A549 were measured. H1299 and A549 cell lines were transfected to overexpress and downexpress miR-221, and cell viability and cell senescence were determined. The PTEN/Akt pathway was then examined by real-time polymerase chain reaction and Western blot analysis.Results. MiR-221 together with proteins MDR1 and ABCG2 was upregulated in Cisplatin-resistant A549 lung cancer cells. Anti-miR-221 inhibits proliferation and induces senescence in lung cancer cells. PTEN/Akt pathway axis was identified as a target of drug resistance induced by miR-221.Conclusion. Our results revealed that miR-221 is an important regulator for chemotherapy sensitivity and showed miR-221 as a potential target for drug sensitization.

M2 Macrophages, but Not M1 Macrophages, Support Megakaryopoiesis Via up-Regulating PI3K-AKT Pathway

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-1
Author(s):  
Hong-Yan Zhao ◽  
Qi Wen ◽  
Zhong-Shi Lyu ◽  
Shu-Qian Tang ◽  
Yuan-Yuan Zhang ◽  
...  
Keyword(s):  
Platelet Count ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Polarization State ◽  
Graft Function ◽  
Underlying Mechanism ◽  
Akt Pathway ◽  
Monocyte Subsets ◽  
Akt Inhibitor ◽  
Cd34 Cells

Background Megakaryopoiesis and platelets production intensely depend on bone marrow(BM) microenvironment. Our previous studies found that impaired BM microenvironment and dysfunctional megakaryopoiesis are responsible for the occurrence of prolonged isolated thrombocytopenia (PT), which is defined as the engraftment of all peripheral blood cell lines other than a platelet count less than 20×109/L or a dependence on platelet transfusions for more than 60 days following allo-HSCT(BBMT 2014; BBMT 2017; Brit J Haematol 2018; Am J Hematol 2018). As an important component of the BM microenvironment, macrophages (MՓs) are heterogeneous and polarized into classically activated (M1) MՓs and alternatively activated (M2) MՓs with distinct phenotypes and function. Although inconsistent effect of BM MՓs was reported on megakaryopoiesis, the functional role of M1 and M2 MՓs and related pathway in regulating megakaryopoiesis and its effect on PT patients post-allotransplant remain to be elucidated. Aims To address the roles of M1 MФs and M2 MФs in regulating megakaryopoiesis as well as PI3K-AKT pathway in the process. Moreover, polarization status and the function of BM MФs in regulating megakaryopoiesis were evaluated in PT patients. Methods This prospective nested case-control study enrolled 12 patients with PT, 24 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 12 healthy donors (HD). BM standard monocyte subsets and M1/M2 MՓs polarization state were analyzed by flow cytometry. To generate M1 and M2 MՓs, both primary BM MՓs and THP1 cell lines were treated with LPS and IFN-γ or with IL-4 and IL-13. The functions of BM MՓs were evaluated by migration, phagocytosis and cytokine secretion assay. The sorted CD34+ cells from HD were co-cultured with BM MՓs from PT and GGF patients or M1 and M2 MՓs respectively for megakaryopoiesis. The quantification of the megakaryocytes(MKs), MKs apoptosis, MKs polyploidy distribution, colony-forming unit MK(CFU-MK) efficiency, and platelet production were analyzed in the coculture system. To understand the underlying mechanism of MՓs polarization in regulating MKs, RNA-seq analyses were performed in BM MՓs from PT and GGF patients. In addition, M1 and M2 MՓs were treated with the chemical inhibitors and lentivirus for PI3K-AKT pathway. Results Elevated intermediate and non-classical monocyte subsets were found in PT patients when compared with those in GGF patients. Moreover, PT patients displayed increased M1 and reduced M2 MՓs, resulting an unbalanced M1/M2 polarization, compared with GGF and HD. BM MՓs from PT patients, with high TNF-α levels and low TGF-β levels, showed decreased megakaryopoiesis-supporting ability. No significant differences in migration and phagocytosis function of MՓs among the three groups. RNA sequencing of BM MՓs showed down-regulated PI3K-AKT pathway in MՓs of PT patients compared with GGF. Consistently, the phosphorylation levels of AKT decreased significantly in MՓs of PT patients, suggesting that PI3K-AKT pathway may functionally regulate megakaryopoiesis-supporting ability of MՓs. Subsequently, BM-M2 and THP1-M2 showed superior effect on megakaryopoiesis-supporting ability compared with BM-M1 and THP1-M1. Specifically, the BM CD34+ cells cocultured with M2 MՓs demonstrated significant increased percentages of MKs and MK polyploidy, CFU-MK efficiency, and platelet count compared with those cocultured with M1 MՓs. Preventing PI3K-AKT pathway by PI3K inhibitor or Akt inhibitor significantly reduced the megakaryopoiesis-supporting ability of M2 MՓs. Moreover, knockdown of AKT1 induced the impairment of megakaryopoiesis-supporting ability via suppressing M2 MՓs polarization, which could be attenuated by AKT1 overexpression complementarily. Summary/Conclusion The current study demonstrated the polarization status of MՓs modulates their ability to support megakaryopoiesis. M2 MՓs, but not M1 MՓs, support megakaryopoiesis via up-regulating PI3K-AKT pathway. Defective M2 MՓs polarization via down-regulating PI3K-AKT pathway may be responsible for the pathogenesis of PT post-allotransplant, which provides a promising therapeutic target for PT patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Assessment of PI3K/mTOR/AKT Pathway Elements to Serve as Biomarkers and Therapeutic Targets in Penile Cancer

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2323
Author(s):  
Anita Thomas ◽  
Sascha Reetz ◽  
Philipp Stenzel ◽  
Katrin Tagscherer ◽  
Wilfried Roth ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Penile Cancer ◽  
Multivariate Regression Analysis ◽  
Clinicopathological Characteristics ◽  
Akt Pathway ◽  
Akt Inhibitor ◽  
Free Survival ◽  
Kaplan Meier ◽  
Treatment Naïve

The PI3K/mTOR/AKT pathway might represent an intriguing option for treatment of penile cancer (PeCa). We aimed to assess whether members of this pathway might serve as biomarkers and targets for systemic therapy. Tissue of primary cancer from treatment-naïve PeCa patients was used for tissue microarray analysis. Immunohistochemical staining was performed with antibodies against AKT, pAKT, mTOR, pmTOR, pS6, pPRAS, p4EBP1, S6K1 and pp70S6K. Protein expression was correlated with clinicopathological characteristics as well as overall survival (OS), disease-specific survival (DSS), recurrence-free survival (RFS) and metastasis-free survival (MFS). AKT inhibition was tested in two primarily established, treatment-naïve PeCa cell lines by treatment with capivasertib and analysis of cell viability and chemotaxis. A total of 76 patients surgically treated for invasive PeCa were included. Higher expression of AKT was significantly more prevalent in high-grade tumors and predictive of DSS and OS in the Kaplan–Meier analysis, and an independent predictor of worse OS and DSS in the multivariate regression analysis. Treatment with pan-AKT inhibitor capivasertib in PeCa cell lines induced a significant downregulation of both total AKT and pAKT as well as decreased cell viability and chemotaxis. Selected protein candidates of the mTOR/AKT signaling pathway demonstrate association with histological and survival parameters of PeCa patients, whereas AKT appears to be the most promising one.

scholarly journals Drug-Like Small Molecule HSP27 Functional Inhibitor Sensitizes Lung Cancer Cells to Gefitinib or Cisplatin by Inducing Altered Cross-Linked Hsp27 Dimers

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 630
Author(s):  
Hawon Yoo ◽  
Seul-Ki Choi ◽  
Jaeok Lee ◽  
So Hyeon Park ◽  
You Na Park ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Small Molecule ◽  
Anticancer Agents ◽  
Tumor Growth Inhibition ◽  
Dependent Manner ◽  
Hsp27 Expression ◽  
Cancer Aggressiveness

Relationships between heat shock protein 27 (HSP27) and cancer aggressiveness, metastasis, drug resistance, and poor patient outcomes in various cancer types including non-small cell lung cancer (NSCLC) were reported, and inhibition of HSP27 expression is suggested to be a possible strategy for cancer therapy. Unlike HSP90 or HSP70, HSP27 does not have an ATP-binding pocket, and no effective HSP27 inhibitors have been identified. Previously, NSCLC cancer cells were sensitized to radiation and chemotherapy when co-treated with small molecule HSP27 functional inhibitors such as zerumbone (ZER), SW15, and J2 that can induce abnormal cross-linked HSP27 dimer. In this study, cancer inhibition effects of NA49, a chromenone compound with better solubility, longer circulation time, and less toxicity than J2, were examined in combination with anticancer drugs such as cisplatin and gefitinib in NSCLC cell lines. When the cytotoxic drug cisplatin was treated in combination with NA49 in epidermal growth factor receptors (EGFRs) WT cell lines, sensitization was induced in an HSP27 expression-dependent manner. With gefitinib treatment, NA49 showed increased combination effects in both EGFR WT and Mut cell lines, also with HSP27 expression-dependent patterns. Moreover, NA49 induced sensitization in EGFR Mut cells with a secondary mutation of T790M when combined with gefitinib. Augmented tumor growth inhibition was shown with the combination of cisplatin or gefitinib and NA49 in nude mouse xenograft models. These results suggest the combination of HSP27 inhibitor NA49 and anticancer agents as a candidate for overcoming HSP27-mediated drug resistance in NSCLC patients.

scholarly journals Intra-Tumoral Expression of SLC7A11 Is Associated with Immune Microenvironment, Drug Resistance, and Prognosis in Cancers: A Pan-Cancer Analysis

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiajun He ◽  
Hongjian Ding ◽  
Huaqing Li ◽  
Zhiyu Pan ◽  
Qian Chen
Keyword(s):  
Pancreatic Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Pancreatic Cancer Cell ◽  
Mononuclear Cells ◽  
Cancer Cell Lines ◽  
Clinical Practices ◽  
Pan Cancer

While many anti-cancer modalities have shown potent efficacy in clinical practices, cancer prevention, timely detection, and effective treatment are still challenging. As a newly recognized iron-dependent cell death mechanism characterized by excessive generation of lipid peroxidation, ferroptosis is regarded as a potent weapon in clearing cancer cells. The cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11) is the core target for ferroptosis regulation, the overexpression of which dictates downregulated sensitivity to ferroptosis in cancer cells. Hence, we elaborated the pan-cancer level bioinformatic study and systematically elucidated the role of intra-tumoral expression of SLC7A11 in the survival of cancer patients and potential immunotherapeutic response. Specifically, 25/27 (92.6%) cancers were featured with upregulated SLC7A11 expression, where SLC7A11 overexpression is a risk factor for worse overall survival in 8 cancers. We also validated SLC7A11 expression in multiple pancreatic cancer cell lines in vitro and found that it was upregulated in most pancreatic cancer cell lines (p < 0.05). Single-cell sequencing method revealed the SLC7A11 was majorly expressed in cancer cells and mononuclear cells. To further explore the function of SLC7A11 in cancer progression, we analyzed the influence on cell proliferation after the knockdown or knockout of SLC7A11 by either CRISPR or RNAi methods. Besides, the association between SLC7A11 and drug resistance was characterized using bioinformatic approaches as well. We also analyzed the association between the expression of SLC7A11 in multi-omics level and the intra-tumoral infiltration of immune cells based on cell annotation algorithms. Moreover, the relationship between SLC7A11 and the expression of MHC, immune stimulators, immune inhibitors as well as the response to immunotherapy was investigated. In addition, the SLC7A11 expression in colon adenocarcinoma, uterine corpus endometrial carcinoma, and stomach adenocarcinoma (STAD) is also positively associated with microsatellite instability and that in head and neck squamous cell carcinoma, STAD, and prostate adenocarcinoma is positively associated with neoantigen level, which further revealed the potential relationship between SLC7A11 and immunotherapeutic response.

scholarly journals Mutual Expression of ALDH1A1, LOX, and Collagens in Ovarian Cancer Cell Lines as Combined CSCs- and ECM-Related Models of Drug Resistance Development

2018 ◽  
Vol 20 (1) ◽  
pp. 54 ◽  
Author(s):  
Karolina Sterzyńska ◽  
Andrzej Klejewski ◽  
Karolina Wojtowicz ◽  
Monika Świerczewska ◽  
Marta Nowacka ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Extracellular Matrix ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Lysyl Oxidase ◽  
Resistant Cell ◽  
Pcr Analysis ◽  
Ovarian Cancer Cells

A major contributor leading to treatment failure of ovarian cancer patients is the drug resistance of cancer cell. CSCs- (cancer stem cells) and ECM (extracellular matrix)-related models of drug resistance are described as independently occurring in cancer cells. Lysyl oxidase (LOX) is another extracellular protein involved in collagen cross-linking and remodeling of extracellular matrix and has been correlated with tumor progression. The expression of LOX, COL1A2, COL3A1, and ALDH1A1 was performed in sensitive (A2780, W1) and resistant to paclitaxel (PAC) (A2780PR1 and W1PR2) and topotecan (TOP) (W1TR) cell lines at the mRNA (real-time PCR analysis) and protein level (Western blot and immunofluorescence analysis). The ALDH1A1 activity was measured with the ALDEFLUOR test and flow cytometry analysis. The protein expression in ovarian cancer tissues was determined by immunohistochemistry. We observed an increased expression of LOX and collagens in PAC and TOP resistant cell lines. Subpopulations of ALDH1A1 positive and negative cells were also noted for examined cell lines. Additionally, the coexpression of LOX with ALDH1A1 and COL1A2 with ALDH1A1 was observed. The expression of LOX, collagens, and ALDH1A1 was also detected in ovarian cancer lesions. In our study LOX, ALDH1A1 and collagens were found to be coordinately expressed by cells resistant to PAC (LOX, ALDH1A1, and COL1A2) or to TOP (LOX and ALDH1A1). This represents the study where molecules related with CSCs (ALDH1A1) and ECM (LOX, collagens) models of drug resistance are described as occurring simultaneously in ovarian cancer cells treated with PAC and TOP.

scholarly journals The Effect of Compound Sophora on Fluorouracil and Oxaliplatin Resistance in Colorectal Cancer Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
WeiHua Yin ◽  
GuPing Zhong ◽  
HuiZhen Fan ◽  
HongMei Xia
Keyword(s):  
Colorectal Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Sw480 Cell ◽  
Gastric Cancer Cells ◽  
Glycoprotein P ◽  
Chemotherapy Drugs ◽  
Protein Levels ◽  
Enhanced Expression

Fluorouracil (5-FU) and oxaliplatin (L-OHP) are the most commonly used chemotherapy drugs for colorectal cancer, though resistance is common. Compound Sophora injection is a traditional Chinese medicine that can protect the liver against oxidation, improve immunity, and enhance sensitivity to chemotherapy; it may have an effect of reversing resistance in 5-FU- and L-OHP-resistant gastric cancer cells (5-FU/SW480 and L-OHP/SW480, respectively). A concentration gradient experiment was performed to identify a nontoxic dose of compound Sophora injection. 5-FU/SW480 and L-OHP/SW480 cells were treated with the nontoxic dose of compound radix Sophorae injection for 48 h, and changes in drug resistance to 5-FU and L-OHP were detected. Alterations in apoptosis and the cell cycle were assessed, as were the mRNA and protein levels of permeability glycoprotein (P-gp), annexin A1 (ANXA1), and ATP-binding cassette superfamily G member 2 (ABCG2). Flow cytometry showed a reduction in the number of cells in the G1 phase and an increase of cells in the S phase (P<0.05). mRNA and protein expression of P-gp and ABCG2 was significantly higher in 5-FU/SW480 and L-OHP/SW480 cell lines, and ANXA1 expression decreased significantly (P<0.05). Compound Sophora injection can reverse the drug resistance of 5-FU/SW480 and L-OHP/SW480 cell lines to 5-FU and L-OHP, respectively, possibly through a mechanism involving reduced expression of P-gp and ABCG2 but enhanced expression of ANXA1, which is the basis for the identification of clinical drug resistance in colorectal cancer.

scholarly journals GLUL Ablation Can Confer Drug Resistance to Cancer Cells via a Malate-Aspartate Shuttle-Mediated Mechanism

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1945
Author(s):  
Magesh Muthu ◽  
Ranjeet Kumar ◽  
Azharuddin Sajid Syed Khaja ◽  
Jonathan D. Gilthorpe ◽  
Jenny L. Persson ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Small Cell Lung Carcinoma ◽  
A549 Cells ◽  
Cancer Drug ◽  
Aminooxyacetic Acid ◽  
Cell Lung Carcinoma ◽  
Metabolic Fitness ◽  
Malate Aspartate Shuttle

Glutamate-ammonia ligase (GLUL) is important for acid-base homeostasis, ammonia detoxification, cell signaling, and proliferation. Here, we reported that GLUL ablation conferred resistance to several anticancer drugs in specific cancer cell lines while leaving other cell lines non-resistant to the same drugs. To understand the biochemical mechanics supporting this drug resistance, we compared drug-resistant GLUL knockout (KO) A549 non-small-cell lung carcinoma (NSCLC) cells with non-resistant GLUL KO H1299 NSCLC cells and found that the resistant A549 cells, to a larger extent, depended on exogenous glucose for proliferation. As GLUL activity is linked to the tricarboxylic acid (TCA) cycle via reversed glutaminolysis, we probed carbon flux through both glycolysis and TCA pathways by means of 13C5 glutamine, 13C5 glutamate, and 13C6 glucose tracing. We observed increased labeling of malate and aspartate in A549 GLUL KO cells, whereas the non-resistant GLUL KO H1299 cells displayed decreased 13C-labeling. The malate and aspartate shuttle supported cellular NADH production and was associated with cellular metabolic fitness. Inhibition of the malate-aspartate shuttle with aminooxyacetic acid significantly impacted upon cell viability with an IC50 of 11.5 μM in resistant GLUL KO A549 cells compared to 28 μM in control A549 cells, linking resistance to the malate-aspartate shuttle. Additionally, rescuing GLUL expression in A549 KO cells increased drug sensitivity. We proposed a novel metabolic mechanism in cancer drug resistance where the increased capacity of the malate-aspartate shuttle increased metabolic fitness, thereby facilitating cancer cells to escape drug pressure.

Rituximab-Mediated Inhibition of the PI3K-Akt Survival Signaling Pathway in B-NHL Cell Lines Leading to Downregulation of Bclxl Expression and Chemosensitization.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2830-2830
Author(s):  
Eriko Suzuki ◽  
Ali R. Jazirehi ◽  
Benjamin Bonavida
Keyword(s):  
Cell Survival ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Akt Pathway ◽  
Akt Inhibitor ◽  
Drug Induced ◽  
Similar Time ◽  
Cell Lysates ◽  
Induced Apoptosis

Abstract Rituximab (chimeric anti-CD20 monoclonal antibody) has been used in the treatment of B-NHL. We have reported in vitro that rituximab treatment signals B-NHL cell lines Ramos and Daudi and inhibits both the ERK 1/2 MAPK and NF-κB signaling pathways leading to selective inhibition of Bclxl expression and sensitization to drug-induced apoptosis. The inhibition of the NF-κB signaling pathway by rituximab was shown to be due, in part, to the induction of the Raf Kinase Inhibitor Protein (RKIP) (Jazirehi, et al., 2005 Cancer Research 65:264–276). The PI3K-Akt signaling pathway is a key regulator of cell survival and aberrant activation of the PI3K-Akt pathway has been implicated in both drug resistance and resistance to apoptosis-inducing stimuli. Akt can promote cell survival by indirectly activating the proximal transcription factor NF-κB through the phosphorylation of I-kappa B kinase (I-κB) (Ozes et. al. Nature401:82–85, 1999). This study investigated whether NF-κB inhibition by rituximab and downregulation of Bclxl expression was also the result of rituximab-mediated inhibition of the PI3K-Akt pathway. Ramos and Daudi B-NHL cell lines were treated with rituximab (20 ug/ml) and cell lysates were prepared and both Akt and phospho-Akt (p-Akt) expression were examined by western blot. The findings demonstrate that both cell lines show constitutively activated p-Akt and treatment with rituximab significantly inhibited p-Akt but not Akt. Time kinetics analysis demonstrated that inhibition of p-Akt was first detected at 3–6 hours following rituximab treatment and inhibition was maintained up to 24 hours. Concomitantlly, a similar time kinetics revealed inhibition of NF-κB activity as assessed by EMSA. Since the inhibition of NF-kB activity resulted in significant downregulation of Bclxl expression, we also examined the role of the Akt pathway in the regulation of Bclxl expression. Tumor cells were treated with the Akt inhibitor LY294002 and analysis of cell lysates showed significant downregulation of Bclxl expression. Rituximab was previously shown to sensitize B-NHL cells to drug-induced apoptosis via inhibition of NF-κB activity and Bclxl expression. We examined if inhibition of the Akt pathway also chemosensitized the cells. Treatment of Ramos cells with the Akt inhibitor LY294002 significantly sensitized the cells to CDDP-induced apoptosis and synergy was achieved. Altogether, these findings demonstrate, for the first time, that rituximab inhibits the Akt pathway and that this pathway is involved in the regulation of tumor- cell resistance to chemotherapeutic drugs. This study also proposes that the Akt pathway is a potential targeting pathway for therapeutic intervention in the treatment of rituximab and drug-resistant B-NHL.

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