Increase In Apoptotic Population Research Articles

The anticancer potential of tetrahydrocurcumin-phytosomes against oral carcinoma progression

BackgroundHerbal medicine combined with nanotechnology offers an alternative to the increasing burden of surgery and/or chemotherapy, the main therapeutics of oral carcinoma. Phytosomes are nano-vesicular systems formed by the interaction between phospholipids and phyto-active components via hydrogen bonding, exhibiting superior efficacy over pure phytocomponents in drug delivery.MethodsTetrahydrocurcumin (THC)-phytosomes were prepared by thin film hydration method. After characterization, in vitro cytotoxicity, antiproliferative capacity, antioxidant potential and full apoptotic workup were paneled on oral squamous cell carcinoma (SCC4) in comparison with native THC-solution and cisplatin (3.58 µg/mL intravenous injection), as positive controls. In addition, we tested the three medications on normal oral keratinocytes and gingival fibroblasts to attest to their tissue-selectivity.ResultsSuccessful preparation of THC-phytosomes using 1:1 molar ratio of THC to phospholipid exhibited significantly increased aqueous solubility, good colloidal properties, and complete drug release after one hour. On SCC4 cells, THC-phytosomes, at their dose-/time-dependency at ~ 60.06 µg/mL escalated cell percentages in the S-phase with 32.5 ± 6.22% increase, as well as a startling 29.69 ± 2.3% increase in apoptotic population. Depletion of the cell colonies survival to 0.29 ± 0.1% together with restraining the migratory rate by -6.4 ± 6.8% validated THC-phytosomes’ antiproliferative capacity. Comparatively, the corresponding results of THC-solution and cisplatin revealed 12.9 ± 0.9% and 25.8 ± 1.1% for apoptosis and 0.9 ± 0.1% and 0.7 ± 0.08% for colony survival fraction, respectively. Furthermore, the nanoformulation exhibited the strongest immuno-positivity to caspase-3, which positively correlated with intense mitochondrial fluorescence by Mitotracker Red, suggesting its implication in the mitochondrial pathway of apoptosis, a finding further explained by the enormously high Bax and caspase-8 expression by RT-qPCR. Finally, the THC groups showed the lowest oxidative stress index, marking their highest free radical-scavenging potential among the test groups.ConclusionsTHC-phytosomes are depicted to be an efficient nanoformulation that enhanced the anticancer efficacy over the free drug counterpart and the conventional chemotherapeutic. Additionally, being selective to cancer cells and less cytotoxic to normal cells makes THC-phytosomes a potential candidate for tissue-targeted therapy.

Abstract 5503: Targeting CDK12-mediated DNA damage response to overcome cisplatin resistance in human urothelial carcinoma

Abstract Urothelial carcinoma (UC) accounts for more than 90% of bladder cancer. According to the 2019 Surveillance, Epidemiology and End Results (SEER) Program database, the 5-year survival rates of regional and metastatic bladder cancer are 36.3% and 4.6%, respectively. Patients who received radical cystectomy suffer from a high recurrence rate of up to 30%. As for metastatic UC, only half of the patients respond to standard platinum-based chemotherapy, with 20% of 5-year overall survival. CDK12 shows transcription regulatory effects via regulating RNA polymerase II CTD phosphorylation. In our research, we used CDK12 selective inhibitor SR-4835 to treat the UC cell lines including T24, BFTC905, RT4, and cisplatin-resistant T24/R. MTT assay result has demonstrated the cytotoxicity of SR-4835 against UC cells. Using q-RT-PCR and immunoblotting, we have confirmed the reduction of ATM mRNA and protein expression after the treatment of SR-4835. The expression of DNA damage marker gamma-H2AX was also elevated. Flow cytometry data indicated an increase in apoptotic population after treatment. According to these findings, we presume SR-4835 can lead to a DNA damage-induced apoptosis process. Furthermore, we also observed the inhibition of AKT pathway, which is related to cell survival and proliferation. Due to the effects of DNA-repair inhibition, we aim to combine SR-4835 to enhance the anti-cancer effects of cisplatin. The results of UC mice xenograft have confirmed that SR-4835 and cisplatin display a synergistic effect against UC tumor growth in vivo. We will further dissect the mechanisms of the in vitro synergistic effects between SR-4835 and cisplatin using immunoblotting, RNA-sequencing, comet assay, and various functional assays. Conclusively, our study aims to explore the role of CDK12 in urothelial carcinoma and the therapeutic effects of SR-4835, especially its potential to overcome cisplatin resistance and manage metastatic disease. Citation Format: Po-Ming Chow, Jun-Ren Dong, Chung-Sheng Shi, Kuo-How Huang. Targeting CDK12-mediated DNA damage response to overcome cisplatin resistance in human urothelial carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5503.

Identification of second generation benzylidene chromanone analogues as novel, potent DHODH inhibitors in acute myeloid leukemia cells

Dihydroorotate dehydrogenase (DHODH) remains as an active target at the preclinical level against acute myeloid leukemia (AML). Herein we report potent second generation benzylidene chromanone (SBL-105) analogues to inhibit DHODH in AML cells. Virtual docking and molecular dynamic simulations were performed. Human-recombinant (rh)DHODH, THP-1, TF-1 and HL-60 cell lines were used. MTT assay was used for cell viability. Flow cytometry was used for differentiation analysis. Computational modeling and simulations predict, SBL-105 analogs bind efficiently to DHODH with improved binding energies. While all tested analogues of SBL-105 inhibited rh DHODH enzyme, SBL-105-4 and SBL-105-6 more effectively inhibited rh DHODH with an IC50 value of 3.62 and 13.61 nM respectively. SBL-105-4 exhibited excellent anti proliferative effects against THP-1, TF-1 and HL-60 cells with GI50 values of 18.78, 38.11 and 63.83 nM respectively. A similar effect was also observed in SBL-105-6 treated AML cells with respective GI50 values of 34.56, 44.40 and 38.65 nM in THP-1, TF-1 and HL-60 cells. An increase in apoptotic populations were enumerated in all three AML cells. Both these compounds also increased the differentiation marker CD11b positive populations in all the three AML cells tested. In conclusion, SBL-105-4 and SBL-105-6 were identified as potent second generation DHODH inhibitors, which drives attention for further preclinical developments. Communicated by Ramaswamy H. Sarma

Computational and in vitro characterization of ICY-5: A potential candidate promoting mitochondrial apoptosis via the c-MET andSTAT3 pathways.

Targeted chemotherapy remains the primary choice in controlling various forms of breast cancer (BC) due to its heterogenous gene expressions in various subtypes. In silico and in vitro evaluation of ICY-5, a novel arylidene analogue against c-MET, was performed. ICY-5 exhibited a docking score of -9.6 kcal/mol in inactive conformation and, - 8.6 kcal/mol in active conformation for c-MET. ICY-5 inhibited c-MET enzyme with an IC50 of 34.34 nM. The compound effectively inhibited MDA-MB 231 and MCF-7 cell proliferation, with GI50 values of 62.61 and 75.31 nM, respectively, and hepatocyte growth factor (HGF)/R c-MET phosphorylation with IC50 s of 71.41 and 83.77 nM, respectively. ICY-5 dose-dependently inhibited HGF-induced transmigration, cell scattering, invasion and altered cell cycle. An increase in apoptotic populations of these cells, with a dose-dependent decease in phosphorylation of STAT3 protein was observed. Furthermore, ICY-5 upregulated the caspase-3, caspase-9, Bcl-2-associated X andsurvivin, and downregulated Bcl-2, vascular endothelial growth factor, matrix metalloproteinase-2 (MMP-2), and MMP-9 in both BC cell lines. In summary, ICY-5 exhibited excellent efficacy in BC cells, targeting c-MET/SAT-3-mediated mitochondrial apoptosis. Further research will be required to ascertain ICY-5 suitability as a targeted chemotherapeutic against multiple forms of BC.

25-Hydroxycholesterol Induces Death Receptor-mediated Extrinsic and Mitochondria-dependent Intrinsic Apoptosis in Head and Neck Squamous Cell Carcinoma Cells.

Oxysterol plays important physiological roles in diverse biological processes including apoptosis. However, the mechanisms underlying oxysterol-induced apoptosis remain unknown. 25-hydroxycholesterol (25-HC) is an oxysterol synthesized by cholesterol 25-hydroxylase from cholesterol during sterol metabolism. The aim of present study was to investigate 25-HC-induced apoptosis and associated signalling pathways in FaDu cells, which is originated form human head and neck squamous cell carcinoma cells. 25-HC-induced apoptosis was investigated by cell cytotoxicity assay using MTT, cell viability assay using cell LIVE/DEAD cell viability assay, haematoxylin & eosin staining, nuclear staining, fluorescence-activated cell sorting, western blotting using specific antibodies associated with extrinsic and intrinsic apoptosis pathways, and caspase-3/-7 activity assay in FaDu cells. 25-HC dose-dependently decreased the viability of FaDu cells and up-regulated apoptotic events, such as alteration in morphology, and nuclear condensation. Flow cytometric analysis showed an increase in apoptotic population upon 25-HC treatment, suggesting that 25-HC induces apoptosis in FaDu cells. Moreover, 25-HC-induced apoptosis in FaDu cells was dependent on the activation of caspases by Fas antigen ligand-triggered death receptor-mediated extrinsic pathway and mitochondria-dependent intrinsic pathway via mitogen activated protein kinases. Cholesterol-derived oxysterol, 25-HC has potential anti-cancer function in FaDu cells and may have potential properties for the discovery of anti-cancer agents.

High Glucose Represses the Anti-Proliferative and Pro-Apoptotic Effect of Metformin in Triple Negative Breast Cancer Cells.

Metformin, the most widely prescribed anti-diabetic drug, is shown to possess anti-cancer potential in treatment of cancers, including breast cancer; decreases breast cancer risk; and improves overall survival. However, reports suggest that higher glucose concentrations may negatively impact the anti-cancer efficacy of metformin. Therefore, we examined the anti-cancer potential of metformin in triple-negative breast cancer cells (TNBCs) exposed to different glucose (25 mM, 5.5 mM and zero glucose/glucose-starved) conditions. Our data indicates that a high glucose (25 mM) concentration (mimicking diabetes) significantly abrogated the effect of metformin on cell proliferation, cell death and cell cycle arrest in addition to loss of efficacy in inhibition of the mTOR pathway, a key metabolic pathway in TNBC cells. The mTOR pathway is activated in TNBCs compared to other subtypes of breast cancer, regulates the synthesis of proteins that are critical for the growth and survival of cancer cells and its activation is correlated to poor outcomes among TNBC patients, while also contributing to metastatic progression and development of resistance to chemotherapy/radiotherapy. Our studies were performed in two different types of TNBCs, MDA-MB-231 cells (mesenchymal stem cell-like (MSL)) and MDA-MB-468 (basal like-1 (BL-1)). Interestingly, lower concentrations of metformin (50, 100, 250, and 500 μM) significantly increased cell proliferation in 25 mM glucose exposed MDA-MB-231 cells, an effect which was not observed in MDA-MB-468 cells, indicating that the effective concentration of metformin when used as anti-cancer drug in TNBCs may have to be determined based on cell type and blood glucose concentration. Our data indicates that metformin treatment was most effective under zero glucose/glucose-starved conditions in MDA-MB-468 with a significant increase in the apoptotic population (62.3 ± 1.5%; p-value < 0.01). Under 5.5 mM glucose conditions in both MDA-MB-231 and MDA-MB-468 cells our data showed reduced viability of 73.56 ± 2.53%; p-value < 0.05 and 70.49 ± 1.68%; p-value < 0.001, respectively, along with a significant increase in apoptotic populations of both cell types. Furthermore, metformin (2 mM) inhibited the mTOR pathway and its downstream components under zero glucose/glucose-starved conditions indicating that using metformin in combination with agents that inhibit the glycolytic pathway should be more beneficial for the treatment of triple-negative breast cancers in diabetic individuals.

Gold nanoparticles–conjugated quercetin induces apoptosis via inhibition of EGFR/PI3K/Akt–mediated pathway in breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

Epidermal growth factor plays a major role in breast cancer cell proliferation, survival, and metastasis. Quercetin, a bioactive flavonoid, is shown to exhibit anticarcinogenic effects against various cancers including breast cancer. Hence, the present study was designed to evaluate the effects of gold nanoparticles-conjugated quercetin (AuNPs-Qu-5) in MCF-7 and MDA-MB-231 breast cancer cell lines. Borohydride reduced AuNPs were synthesized and conjugated with quercetin to yield AuNPs-Qu-5. Both were thoroughly characterized by several physicochemical techniques, and their cytotoxic effects were assessed by MTT assay. Apoptotic studies such as DAPI, AO/EtBr dual staining, and annexin V-FITC staining were performed. AuNPs and AuNPs-Qu-5 were spherical with crystalline nature, and the size of particles range from 3.0 to 4.5 nm. AuNPs-Qu-5 exhibited lower IC50 value compared to free Qu. There was a considerable increase in apoptotic population with increased nuclear condensation seen upon treatment with AuNPs-Qu-5. To delineate the molecular mechanism behind its apoptotic role, we analysed the proteins involved in apoptosis and epidermal growth factor receptor (EGFR)-mediated PI3K/Akt/GSK-3β signalling by immunoblotting and immunocytochemistry. The pro-apoptotic proteins (Bax, Caspase-3) were found to be up regulated and anti-apoptotic protein (Bcl-2) was down regulated on treatment with AuNPs-Qu-5. Additionally, AuNPs-Qu-5 treatment inhibited the EGFR and its downstream signalling molecules PI3K/Akt/mTOR/GSK-3β. In conclusion, administration of AuNPs-Qu-5 in breast cancer cell lines curtails cell proliferation through induction of apoptosis and also suppresses EGFR signalling. AuNPs-Qu-5 is more potent than free quercetin in causing cancer cell death, and hence, this could be a potential drug delivery system in breast cancer therapy.

Folate Receptor-Targeted Polymeric Micellar Nanocarriers for Delivery of Orlistat as a Repurposed Drug against Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a recalcitrant malignancy with no available targeted therapy. Off-target effects and poor bioavailability of the FDA-approved antiobesity drug orlistat hinder its clinical translation as a repurposed new drug against TNBC. Here, we demonstrate a newly engineered drug formulation for packaging orlistat tailored to TNBC treatment. We synthesized TNBC-specific folate receptor-targeted micellar nanoparticles (NP) carrying orlistat, which improved the solubility (70-80 μg/mL) of this water-insoluble drug. The targeted NPs also improved the delivery and bioavailability of orlistat to MDA-MB-231 cells in culture and to tumor xenografts in a nude mouse model. We prepared HEA-EHA copolymer micellar NPs by copolymerization of 2-hydroxyethylacrylate (HEA) and 2-ethylhexylacrylate (EHA), and functionalized them with folic acid and an imaging dye. Fluorescence-activated cell sorting (FACS) analysis of TNBC cells indicated a dose-dependent increase in apoptotic populations in cells treated with free orlistat, orlistat NPs, and folate-receptor-targeted Fol-HEA-EHA-orlistat NPs in which Fol-HEA-EHA-orlistat NPs showed significantly higher cytotoxicity than free orlistat. In vitro analysis data demonstrated significant apoptosis at nanomolar concentrations in cells activated through caspase-3 and PARP inhibition. In vivo analysis demonstrated significant antitumor effects in living mice after targeted treatment of tumors, and confirmed by fluorescence imaging. Moreover, folate receptor-targeted Fol-DyLight747-orlistat NP-treated mice exhibited significantly higher reduction in tumor volume compared to control group. Taken together, these results indicate that orlistat packaged in HEA-b-EHA micellar NPs is a highly promising new drug formulation for TNBC therapy. Mol Cancer Ther; 15(2); 221-31. ©2015 AACR.

Combined 2-deoxy glucose and metformin improves therapeutic efficacy of sodium-iodide symporter-mediated targeted radioiodine therapy in breast cancer cells.

Radiosensitization using either metformin or 2-deoxy-d-glucose (2-DG) in various cancer cells has been reported. The present study reveals novel information on combining these drugs to enhance radiosensitization effect in breast cancer (BC) cells. Responses to low-dose Cobalt60 radiation, as well as a newly emerged radioiodine therapy target for BC, that is, sodium-iodide symporter (NIS or SLC5A5) protein, are tested. As therapeutic potential of NIS in BC is often limited due to low uptake and fast efflux rate of iodine, the scope of these two radiosensitizers to further improve NIS-mediated 131I therapeutic efficacy is explored. Two BC cell lines, MCF-7, and MDA MB231 are tested to optimize minimal drug doses required for radiosensitization. A combination of 2 mM metformin and 20 mM 2-DG with 2 grey (Gy) Cobalt60 radiation shows significant radiosensitization effect (P=0.0002). In cells treated with the combination therapy, increased γH2A.X foci formation was noted. Further, MCF-7 BC cells overexpressing NIS (MCF-7 NIS) was established, and using the optimized drug concentrations, significant radiosensitization (P=0.0019) by 50 μ Ci 131I usage was found to be the case as well. Apoptosis data corroborates with the result of clonogenic assay showing significant increase in apoptotic population upon dual drug-mediated radiosensitization. In case of metformin treatment, lowered adenosine triphosphate (ATP) content of the cell has been observed. The encouraging radiosensitization effect observed using combined 2-DG and metformin may aid in reducing Cobalt60 radiation exposure or for targeted radioiodine therapy in BC cells with NIS expression. This study indicates high potential of this drug combination in sensitizing BC cells for NIS-mediated-targeted radioiodine therapy, which otherwise may have lacked efficacy.

Effect of FSH Receptor‐Binding Inhibitor‐8 on FSH‐Mediated Granulosa Cell Signaling and Proliferation

Follicle-stimulating hormone is important for mammalian reproduction. It acts through specific receptors located on the plasma membrane of granulosa cells in ovaries and Sertoli cells in testes. The binding of follicle-stimulating hormone to its receptor activates intracytoplasmic signaling pathways leading to steroidogenesis. These steroids in turn regulate the follicle-stimulating hormone action from the anterior pituitary through exerting negative feedback effect. In addition to steroids, non-steroidal factors secreted by the ovaries are believed to modulate follicle-stimulating hormone action through autocrine/paracrine mode. One such low molecular weight peptide referred to as follicle-stimulating hormone receptor-binding inhibitor-8 purified from human follicular fluid has been extensively studied. Follicle-stimulating hormone receptor-binding inhibitor-8 has been shown to inhibit binding of follicle-stimulating hormone to its receptor. The present article describes the effect of follicle-stimulating hormone receptor-binding inhibitor-8 on follicle-stimulating hormone-induced signaling in rat granulosa cells. Follicle-stimulating hormone receptor-binding inhibitor-8 inhibited the follicle-stimulating hormone-induced cAMP, and the effect was observed to be mediated through the protein kinase A. Further, an inhibitory effect of follicle-stimulating hormone receptor-binding inhibitor-8 on the granulosa cell proliferation was evaluated using COV434 cell line which is derived from the human granulosa cell tumor. The effect of the peptide on the cell cycle analysis showed an increase in apoptotic population and the arrest of G1 phase. These findings suggest that follicle-stimulating hormone receptor-binding inhibitor-8 acts as a follicle-stimulating hormone antagonist and affects the follicle-stimulating hormone-mediated signaling and proliferation in the granulosa cells.

OncogeneGAEC1regulatesCAPN10expression which predicts survival in esophageal squamous cell carcinoma

To identify the downstream regulated genes of GAEC1 oncogene in esophageal squamous cell carcinoma and their clinicopathological significance. The anti-proliferative effect of knocking down the expression of GAEC1 oncogene was studied by using the RNA interference (RNAi) approach through transfecting the GAEC1-overexpressed esophageal carcinoma cell line KYSE150 with the pSilencer vector cloned with a GAEC1-targeted sequence, followed by MTS cell proliferation assay and cell cycle analysis using flow cytometry. RNA was then extracted from the parental, pSilencer-GAEC1-targeted sequence transfected and pSilencer negative control vector transfected KYSE150 cells for further analysis of different patterns in gene expression. Genes differentially expressed with suppressed GAEC1 expression were then determined using Human Genome U133 Plus 2.0 cDNA microarray analysis by comparing with the parental cells and normalized with the pSilencer negative control vector transfected cells. The most prominently regulated genes were then studied by immunohistochemical staining using tissue microarrays to determine their clinicopathological correlations in esophageal squamous cell carcinoma by statistical analyses. The RNAi approach of knocking down gene expression showed the effective suppression of GAEC1 expression in esophageal squamous cell carcinoma cell line KYSE150 that resulted in the inhibition of cell proliferation and increase of apoptotic population. cDNA microarray analysis for identifying differentially expressed genes detected the greatest levels of downregulation of calpain 10 (CAPN10) and upregulation of trinucleotide repeat containing 6C (TNRC6C) transcripts when GAEC1 expression was suppressed. At the tissue level, the high level expression of calpain 10 protein was significantly associated with longer patient survival (month) of esophageal squamous cell carcinoma compared to the patients with low level of calpain 10 expression (37.73 ± 16.33 vs 12.62 ± 12.44, P = 0.032). No significant correction was observed among the TNRC6C protein expression level and the clinocopathologcial features of esophageal squamous cell carcinoma. GAEC1 regulates the expression of CAPN10 and TNRC6C downstream. Calpain 10 expression is a potential prognostic marker in patients with esophageal squamous cell carcinoma.

The PI3K Inhibitor GS-1101 (CAL-101) Synergistically Potentiates HDAC-Induced Proliferation Inhibition and Apoptosis Through the Activation of JNK in Lymphoma Cells

AbstractAbstract 3714Pre-clinical studies demonstrated the potential therapeutic value of targeting the PI3K Delta (PI3Kδ) pathway in lymphoma. A selective PI3Kδ inhibitor, GS-1101, shows promising single agent response rates in patients with a variety of lymphoid malignancies. Previously, we showed that inhibition of PKCβ/AKT pathways (pathways that augments survival and proliferation signals) augments histone deacetylase (HDAC) inhibitor-induced stress response, and the irreversible engagement of the apoptotic cascade in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by GS-1101 in combination with HDAC inhibitors panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA).Cytotoxicity was determined by WST-1 proliferation assay. Apoptosis was measured by flow cytometry, caspase activation and PARP cleavage. Cell signaling transduction pathways were analyzed by Western blot and by phosphoproteome array.Six DLBCL, two MCL and two Burkitt lymphoma cell lines were treated with different concentration of GS-1101. To determine the molecular mechanisms by which GS-1101 decreases the viability in various cell lines differently, three cell models (SU-DHL-6 as the most sensitive cells, SU-DHL-16 with moderate sensitivity, and Raji the most resistant cells) were selected for phosphoproteome profiling. SU-DHL-6 cells with IC50 only 0.2 μM had constitutively active AKT pathway and highly active ERK pathway. GS-1101 treatment significantly decreased the level p-AKT in this cell line, but more interestingly it also resulted in a decrease in the level of p-ERK. Significant dephosphorylation of ERK was observed also in SU-DHL-16 cells, however with a minimal effect on p-AKT levels. In contrast, the analysis of Raji cells highlighted a very low activity of AKT pathway and no changes in p-ERK levels after GS-1101 treatment.To enhance the effect of GS-1101, SU-DHL-6/16 cells and NHL primary cells were simultaneously treated with the HDAC inhibitor LBH589. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation in all tested cells and induced a significant increase of caspase-dependent apoptosis in SU-DHL-6/16 cells. For example, in SU-DHL-6 cells co-treatment of 0.1 μM GS-1101 with 2.5 nM LBH589 induced 62% increase of apoptotic population in comparison to the untreated sample, while GS-1101 alone induced 13% increase and LBH589 induced 11% increase. Moreover, substituting SAHA for LBH589 suggests that the synergy between GS-1101 and LBH589 is a class effect between HDAC inhibitors and GS-1101, rather than specific to LBH589.Mechanistically, in SU-DHL-6 cells, LBH589 single treatment increased the level of many phosphoproteins; such as p-ERK, p-AKT, p-CREB, p-AMPKα1/2, p-GSK3α/β, p-HSP27, p-MEK1/2, p-MSK1/2, p-P38a, and p-STAT2. Phosphoproteome analysis of cells treated simultaneously with both agents mostly recapitulated the profile of cells treated with GS-1101 only. Exceptions are p-Chk2, p-c-Jun, p-JNK, and p-STAT5, where levels were significantly higher than in either GS-1101 or LBH589 single treatments. This suggests that GS-1101 and LBH589 may enhance stress-related responses through JNK pathway.These data suggest that PI3Kδ inhibition in combination with HDAC inhibitors might have potential therapeutic value in lymphoma treatment, and provide pre-clinical rationale for combination therapy in patients with lymphoid malignancies. [Display omitted] Disclosures:Lannutti:Gilead Sciences Inc: Employment.

Bax Translocation Mediated Mitochondrial Apoptosis and Caspase Dependent Photosensitizing Effect of Ficus religiosa on Cancer Cells

The main aim of the present work was to investigate the potential effect of acetone extract of Ficus religosa leaf (FAE) in multiple apoptosis signalling in human breast cancer cells. FAE treatment significantly induced dose and time dependent, irreversible inhibition of breast cancer cell growth with moderate toxicity to normal breast epithelial cells. This observation was validated using Sulforhodamine B assay. Cell cycle analysis by Flow cytometry showed cell cycle arrest in G1 phase and induction of sub-G0 peak. FAE induced chromatin condensation and displayed an increase in apoptotic population in Annexin V-FITC/PI (Fluorescein isothiocyanate/Propidium iodide) double staining. FAE stimulated the loss of mitochondrial membrane potential in multiple breast cancer cell lines when compared to normal diploid cells. To understand the role of Bax in FAE induced apoptosis, we employed a sensitive cell based platform of MCF-7 cells expressing Bax-EGFP. Bax translocation to mitochondria was accompanied by the disruption of mitochondrial membrane potential and marked elevation in LEHDase activity (Caspase 9). Consistent with this data, FAE induced Caspase activation as evidenced by ratio change in FRET Caspase sensor expressing MCF-7 cell line and cleavage of prominent Caspases and PARP. Interestingly, FAE accelerated cell death in a mitochondrial dependent manner in continuous live cell imaging mode indicating its possible photosensitizing effect. Intracellular generation of reactive oxygen species (ROS) by FAE played a critical role in mediating apoptotic cell death and photosensitizing activity. FAE induced dose and time dependent inhibition of cancer cell growth which was associated with Bax translocation and mitochondria mediated apoptosis with the activation of Caspase 9 dependent Caspase cascade. FAE also possessed strong photosensitizing effect on cancer cell line that was mediated through rapid mitochondrial transmembrane potential loss and partial Caspase activation involving generation of intracellular ROS.