scholarly journals Divergent Actions of Pyruvate and ATP in Glucose-mediated Irinotecan Chemoresistance in Colorectal Cancer

2021 ◽  
Author(s):  
Chung-Yen Huang ◽  
Yu-Chen Pai ◽  
Linda Chia-Hui Yu
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
High Glucose ◽  
Structural Damage ◽  
Topoisomerase I ◽  
Molecular Mechanisms ◽  
Flow Cytometric Analysis ◽  
Quiescent State ◽  
Altered Glucose ◽  
Induced Apoptosis

Abstract Background: Altered glucose metabolism is associated with chemoresistance in colorectal cancer (CRC). The aim of this study was to illustrate the molecular mechanisms of glucose-mediated chemoresistance against irinotecan, a topoisomerase I inhibitor, focusing on the distinct roles of metabolites such as pyruvate and ATP in modulating cell death and proliferation. Methods: Four human CRC cell lines, tumorspheres, and mouse xenograft models were treated with various doses of irinotecan in the presence of high concentrations of glucose, pyruvate or ATP-encapsulated liposomes. Cell apoptosis was measured by DNA fragmentation and caspase activities, and necroptosis was evaluated by immunoprecipitation of receptor-interacting protein kinase (RIP) 1/3 complex. Cell cycles were assessed by flow cytometric analysis.Results: Human CRC cell lines treated with irinotecan in the presence of high glucose displayed increased cell viability and larger xenograft tumor sizes in mouse models compared to those treated in the presence of normal glucose. Irinotecan induced apoptosis and necroptosis, both of which were mitigated by high glucose. Liposomal ATP prevented irinotecan-induced apoptosis, while it had no effect on necroptosis. In contrast, pyruvate attenuated the RIP1/3-dependent necroptosis via free radical scavenging, without modulating apoptotic levels. Regarding the cell cycle, liposomal ATP aggravated irinotecan-induced G0/G1 shift whereas pyruvate diminished the G0/G1 shift, showing opposite effects on proliferation. Last, tumorsphere structural damage, an index of solid tumor responsiveness to chemotherapy, was determined. Liposomal ATP increased tumorsphere sizes while pyruvate prevented the deformation of spheroid mass. Conclusions: Glucose metabolites confer tumor chemoresistance via multiple modes of action. Glycolytic pyruvate attenuated irinotecan-induced necroptosis and potentiated drug insensitivity by shifting cells from a proliferative to quiescent state. On the other hand, ATP decreased irinotecan-induced apoptosis and promoted active cell proliferation, which might contribute to tumor recurrence.

scholarly journals Antiproliferative Efficacy of N-(3-chloro-4-fluorophenyl)-6,7-dimethoxyquinazolin-4-amine, DW-8, in Colon Cancer Cells Is Mediated by Intrinsic Apoptosis

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4417
Author(s):  
Rabin Neupane ◽  
Saloni Malla ◽  
Mariam Sami Abou-Dahech ◽  
Swapnaa Balaji ◽  
Shikha Kumari ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Colon Cancer Cells ◽  
Apoptotic Pathway ◽  
Anticancer Efficacy ◽  
Colon Cell ◽  
Ic50 Values ◽  
Induced Apoptosis

A novel series of 4-anilinoquinazoline analogues, DW (1–10), were evaluated for anticancer efficacy in human breast cancer (BT-20) and human colorectal cancer (CRC) cell lines (HCT116, HT29, and SW620). The compound, DW-8, had the highest anticancer efficacy and selectivity in the colorectal cancer cell lines, HCT116, HT29, and SW620, with IC50 values of 8.50 ± 2.53 µM, 5.80 ± 0.92 µM, and 6.15 ± 0.37 µM, respectively, compared to the non-cancerous colon cell line, CRL1459, with an IC50 of 14.05 ± 0.37 µM. The selectivity index of DW-8 was >2-fold in colon cancer cells incubated with vehicle. We further determined the mechanisms of cell death induced by DW-8 in SW620 CRC cancer cells. DW-8 (10 and 30 µM) induced apoptosis by (1) producing cell cycle arrest at the G2 phase; (2) activating the intrinsic apoptotic pathway, as indicated by the activation of caspase-9 and the executioner caspases-3 and 7; (3) nuclear fragmentation and (4) increasing the levels of reactive oxygen species (ROS). Overall, our results suggest that DW-8 may represent a suitable lead for developing novel compounds to treat CRC.

scholarly journals Molecular subtype-specific responses of colon cancer cells to the SMAC mimetic Birinapant

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Michael Fichtner ◽  
Emir Bozkurt ◽  
Manuela Salvucci ◽  
Christopher McCann ◽  
Katherine A. McAllister ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Caspase 8 ◽  
Colon Cancer Cells ◽  
Apoptosis Pathway ◽  
Iap Antagonist

AbstractColorectal cancer is a molecularly heterogeneous disease. Responses to genotoxic chemotherapy in the adjuvant or palliative setting vary greatly between patients, and colorectal cancer cells often resist chemotherapy by evading apoptosis. Antagonists of an inhibitor of apoptosis proteins (IAPs) can restore defective apoptosis signaling by degrading cIAP1 and cIAP2 proteins and by inhibition of XIAP. Due to the multiple molecular mechanisms-of-action of these targets, responses to IAP antagonist may differ between molecularly distinct colon cancer cells. In this study, responses to the IAP antagonist Birinapant and oxaliplatin/5-fluorouracil (5-FU) were investigated in 14 colon cancer cell lines, representing the consensus molecular subtypes (CMS). Treatment with Birinapant alone did not result in a substantial increase in apoptotic cells in this cell line panel. Annexin-V/PI assays quantified by flow cytometry and high-content screening showed that Birinapant increased responses of CMS1 and partially CMS3 cell lines to oxaliplatin/5-FU, whereas CMS2 cells were not effectively sensitized. FRET-based imaging of caspase-8 and -3 activation validated these differences at the single-cell level, with CMS1 cells displaying sustained activation of caspase-8-like activity during Birinapant and oxaliplatin/5-FU co-treatment, ultimately activating the intrinsic mitochondrial apoptosis pathway. In CMS2 cell lines, Birinapant exhibited synergistic effects in combination with TNFα, suggesting that Birinapant can restore extrinsic apoptosis signaling in the context of inflammatory signals in this subtype. To explore this further, we co-cultured CMS2 and CMS1 colon cancer cells with peripheral blood mononuclear cells. We observed increased cell death during Birinapant single treatment in these co-cultures, which was abrogated by anti-TNFα-neutralizing antibodies. Collectively, our study demonstrates that IAP inhibition is a promising modulator of response to oxaliplatin/5-FU in colorectal cancers of the CMS1 subtype, and may show promise as in the CMS2 subtype, suggesting that molecular subtyping may aid as a patient stratification tool for IAP antagonists in this disease.

scholarly journals miR-128 Targets the SIRT1/ROS/DR5 Pathway to Sensitize Colorectal Cancer to TRAIL-Induced Apoptosis

2018 ◽  
Vol 49 (6) ◽  
pp. 2151-2162 ◽  
Author(s):  
Bo Lian ◽  
Dongxiang Yang ◽  
Yanlong Liu ◽  
Gang Shi ◽  
Jibin Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cancer Cells ◽  
Cell Lines ◽  
Sirtuin 1 ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Reporter Assays ◽  
Induced Apoptosis

Background/Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an ideal anti-tumor drug because it exhibits selective cytotoxicity against cancer cells. However, certain cancer cells are resistant to TRAIL, and the potential mechanisms are still unclear. The aim of this study was to reduce the resistance of colorectal cancer (CRC) cells to TRAIL. Methods: Quantitative real-time PCR analysis was performed to detect the expression of microRNA-128 (miR-128) in tissues from patients with CRC and CRC cell lines. MTT assays were used to evaluate the effect of miR-128 on TRAIL-induced cytotoxicity against CRC cell lines. The distribution of death receptor 5 (DR5) and the production of reactive oxygen species (ROS) were detected by flow cytometry analysis. Western blot, flow cytometry, and luciferase reporter assays were performed to evaluate the potential mechanism and pathway of miR-128-promoted apoptosis in TRAIL-treated CRC cells. Results: MiR-128 expression was downregulated in tumor tissues from patients with CRC as well as in CRC cell lines in vitro. The enforced expression of miR-128 sensitized CRC cells to TRAIL-induced cytotoxicity by inducing apoptosis. Mechanistically, bioinformatics, western blot analysis, and luciferase reporter assays showed that miR-128 directly targeted sirtuin 1 (SIRT1) in CRC cells. miR-128 overexpression suppressed SIRT1 expression, which promoted the production of ROS in TRAIL-treated CRC cells. This increase of ROS subsequently induced DR5 expression, and thus increased TRAIL-induced apoptosis in CRC cells. Conclusion: The combination of miR-128 with TRAIL may represent a novel approach for the treatment of CRC.

scholarly journals SENP1-Mediated Desumoylation of DBC1 Inhibits Apoptosis Induced by High Glucose in Bovine Retinal Pericytes

2016 ◽  
Vol 2016 ◽  
pp. 1-11
Author(s):  
Jian Gao ◽  
Xia Chen ◽  
Qing Gu ◽  
Xiaoxiao Liu ◽  
Xun Xu
Keyword(s):  
Diabetic Retinopathy ◽  
High Glucose ◽  
Molecular Mechanisms ◽  
Characteristic Change ◽  
High Concentration ◽  
Retinal Pericytes ◽  
Specific Protease ◽  
Induced Apoptosis ◽  
Pericyte Loss

Pericyte loss is an early characteristic change in diabetic retinopathy, but its precise molecular mechanisms have not been elucidated. This study investigated the role of SENP1 in pericyte loss in diabetic retinopathy. We demonstrated that a high concentration of glucose inhibited the expression of the Sentrin/SUMO-specific protease 1 (SENP1), which resulted in an increase in DBC1 sumoylation in bovine retinal pericytes (BRPCs). Furthermore, SENP1 overexpression attenuated hyperemia-induced apoptosis of BPRCs, and SENP1 knockdown aggravated this effect. We also provide evidence that DBC1 sumoylation/desumoylation is involved in the SENP1-regulated apoptosis of BRPCs under high glucose conditions. Understanding the role of SENP1 in the pathogenesis of high glucose induced pericyte loss could help elucidate important targets for future pharmacological interventions.

Combination of a Spicamycin Analogue KRN5500 and Chemotherapeutic Agents Synergistically Enhances Their Cytotoxicity in Human Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4783-4783
Author(s):  
Hirokazu Miki ◽  
Shuji Ozaki ◽  
Osamu Tanaka ◽  
Shiro Fujii ◽  
Shingen Nakamura ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Growth ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Flow Cytometric Analysis ◽  
Human Tumor ◽  
Chemotherapeutic Agents ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. Although new classes of agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains an incurable disease due to increased resistance to these agents. Therefore, alternative approaches are necessary to overcome drug resistance in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan). This drug potently decreases protein synthesis and inhibits cell growth in human tumor cell lines both in vitro and in vivo. Several phase I studies of KRN5500 were conducted in patients with solid tumors, which showed Cmax values of 1000–3000 nM at the maximum tolerated doses. However, no objective anti-tumor response to KRN5500 alone was observed in these patients. In this study, we examined the anti-tumor activity of KRN5500 against MM cells and evaluated its therapeutic potential in combination with other anti-MM agents. MM cell lines and freshly-isolated MM cells were incubated with various concentrations of KRN5500 for 24 hours. Cell proliferation assay showed marked inhibition of cell growth in MM cells such as RPMI 8226, KMS12-BM, and UTMC-2 (IC50 = 10–40 nM), and U266, MM.1S, and primary MM cells (IC50 = 500–1000 nM). Importantly, a chemotherapy-resistant subclone of RPMI 8226 had a similar sensitivity to KRN5500. Annexin V/propidium iodide staining confirmed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Moreover, cleavage of poly (ADP-ribose) polymerase (PARP) was detected after 24 hours with only modest activation of caspase-8, -9, and -3 by immunoblotting. Flow cytometric analysis of anti-apoptotic proteins revealed that apoptosis induced by KRN5500 was associated with down-regulation of Mcl-1 and Bcl-2 expression. To determine the effect of KRN5500 on the unfolded protein response (UPR), splicing of XBP-1 mRNA was analyzed by reverse transcription-polymerase chain reaction. In response to stimulation with KRN5500, splicing of XBP-1 mRNA occurred after 24 hours in RPMI 8226 cells, suggesting that KRN5500-induced apoptosis is mediated in part by the inhibition of UPR. Furthermore, synergistic effects on MM cells were observed when KRN5500 was combined with anti-MM agents including melphalan, dexamethasone, and bortezomib. These results suggest that KRN5500 induces apoptosis in MM cells mainly by the caspase-independent pathway and that its unique mechanism of action provides a valuable therapeutic option to overcome drug resistance in patients with MM.

Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1772-1772 ◽  
Author(s):  
Jahangir Abdi ◽  
Yijun Yang ◽  
Patrick Meyer-Erlach ◽  
Hong Chang
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Molecular Mechanisms ◽  
Marrow Stromal Cells ◽  
Survivin Gene ◽  
Induced Apoptosis

Abstract INTRODUCTION It is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known. METHODS Human myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia). RESULTS BMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05). CONCLUSION Our results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines

Metabolomics ◽  
2021 ◽  
Vol 17 (12) ◽  
Author(s):  
Dorna Varshavi ◽  
Dorsa Varshavi ◽  
Nicola McCarthy ◽  
Kirill Veselkov ◽  
Hector C. Keun ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Single Point ◽  
Hct116 Cell ◽  
Cellular Transformation ◽  
Single Copy ◽  
Driver Mutations ◽  
Glutathione Metabolism ◽  
Single Point Mutation

Abstract Introduction KRAS was one of the earliest human oncogenes to be described and is one of the most commonly mutated genes in different human cancers, including colorectal cancer. Despite KRAS mutants being known driver mutations, KRAS has proved difficult to target therapeutically, necessitating a comprehensive understanding of the molecular mechanisms underlying KRAS-driven cellular transformation. Objectives To investigate the metabolic signatures associated with single copy mutant KRAS in isogenic human colorectal cancer cells and to determine what metabolic pathways are affected. Methods Using NMR-based metabonomics, we compared wildtype (WT)-KRAS and mutant KRAS effects on cancer cell metabolism using metabolic profiling of the parental KRASG13D/+ HCT116 cell line and its isogenic, derivative cell lines KRAS+/– and KRASG13D/–. Results Mutation in the KRAS oncogene leads to a general metabolic remodelling to sustain growth and counter stress, including alterations in the metabolism of amino acids and enhanced glutathione biosynthesis. Additionally, we show that KRASG13D/+ and KRASG13D/− cells have a distinct metabolic profile characterized by dysregulation of TCA cycle, up-regulation of glycolysis and glutathione metabolism pathway as well as increased glutamine uptake and acetate utilization. Conclusions Our study showed the effect of a single point mutation in one KRAS allele and KRAS allele loss in an isogenic genetic background, hence avoiding confounding genetic factors. Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS. Graphical abstract

scholarly journals Anti-EGFR-Coated Gold Nanoparticles In Vitro Carry 5-Fluorouracil to Colorectal Cancer Cells

Materials ◽  
2020 ◽  
Vol 13 (2) ◽  
pp. 375 ◽  
Author(s):  
Raquel B. Liszbinski ◽  
Graziela G. Romagnoli ◽  
Carolina M. Gorgulho ◽  
Caroline R. Basso ◽  
Valber A. Pedrosa ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Gold Nanoparticles ◽  
Cancer Cells ◽  
Cell Lines ◽  
Antineoplastic Agent ◽  
Physicochemical Characterization ◽  
Colorectal Cancer Cells ◽  
Induced Apoptosis ◽  
20 Nm

The aim of the current study is to present a strategy to improve the efficiency of 5-fluorouracil (5-FU), which is widely used as antineoplastic agent against solid tumors-based on the use of gold nanocarriers to overcome the resistance of colorectal cancer cells. 5-FU was loaded on gold nanoparticles (AuNP) coated with anti-EGFR antibodies in order to target them towards colorectal cancer cells that overexpress epidermal growth factor receptors (EGFR). Physicochemical characterization has shown that AuNP size was approximately 20 nm and that AuNP functionalization led to spherical nanoparticles. Flow cytometry allowed observing that some compounds synthesized by our research group have induced apoptosis/necrosis and impaired the proliferation of colon cancer cell lines ‘HCT-116′ and ‘HT-29′. The antibody/drug combination in AuNP (AuNP 5FU EGFR) has improved the apoptosis rate and impaired cell proliferation in both cell lines, regardless of the exposure time. Overall, these results have shown that AuNP functionalization with monoclonal antibodies focused on delivering 5-FU to tumor cells is an exciting strategy against colorectal cancer.

Dual AKT inhibition with perifosine and GCP enhances apoptosis in prostate cancer: Clinical implications

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 14096-14096
Author(s):  
R. L. Vinall ◽  
K. Hwa ◽  
C. Pan ◽  
P. Ghosh ◽  
P. N. Lara ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Western Blot ◽  
Cell Lines ◽  
Combination Treatment ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
Parp Cleavage ◽  
Clonogenic Potential ◽  
Ic50 Values ◽  
Induced Apoptosis

14096 Background: Genistein combined polysaccharide (GCP) has been shown to exhibit anti-cancer properties in both experimental and clinical models of prostate cancer (CaP). Perifosine is an alkylphospholipid with clinical anti-neoplastic activity. Both agents inhibit the AKT signaling pathway. Methods: Prostate cancer cell lines (LNCaP, LNCaP stably transfected with R273H or P151S p53 mutant allele, cds1, and PC3) were treated with GCP, perifosine, or both. IC50 values were established using the MTT assay. Apoptosis was assessed by flow cytometry, Western blot of PARP cleavage, and caspase activity. Clonogenic potential was assessed by colony assay. Status of AKT, p53, p21, AR and PSA was determined by Western blot. All experiments were performed in triplicate. Results: Perifosine inhibited AKT activity in all the cell lines. GCP had little or no effect on AKT activity but reduced AR and PSA levels. The combination of GCP and perifosine further increased the level of AKT inhibition and maintained inhibition for longer when compared to treatment with perifosine alone. Flow cytometric analysis of LNCaP revealed that combination treatment dramatically increased SubG1 levels (23-fold increase versus a 4.4-fold increase for GCP alone and a 6.5% increase for perifosine alone). Apoptosis was confirmed by PARP and caspase analysis. As single agents, the main effect of GCP or perifosine was to induce growth arrest as shown by a decrease in S-phase and increased p21. The cds1 cell line responded similarly to LNCaP, however, cell lines that expressed mutant p53 or were p53 null were not susceptible to GCP/perifosine-induced apoptosis. Combination treatment further decreased the clonogenic potential in all of the cell lines assessed when compared to treatment with the single agents alone. Conclusions: Treatment with a combination of the AKT inhibitors GCP and perifosine dramatically increases apoptosis and/or inhibits clonogenic potential in several CaP cell lines. The effect of the combination treatment on apoptosis appears to be dependent on p53 status. Clinical validation of these findings is warranted. A clinical trial of hormone therapy with or without GCP/perifosine is presently in development. No significant financial relationships to disclose.

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