scholarly journals PNU-74654 Suppresses TNFR1/IKB Alpha/p65 Signaling and Induces Cell Death in Testicular Cancer

2022 ◽  
Vol 44 (1) ◽  
pp. 222-232
Author(s):  
Wen-Jung Chen ◽  
Wen-Wei Sung ◽  
Chia-Ying Yu ◽  
Yu-Ze Luan ◽  
Ya-Chuan Chang ◽  
...  
Keyword(s):  
Cell Death ◽  
Testicular Cancer ◽  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treatment Strategies ◽  
Annexin V ◽  
Western Blots ◽  
Execution Phase ◽  
Induced Apoptosis

Testicular cancer (TC) is a rare malignancy worldwide and is the most common malignancy in males aged 15–44 years. The Wnt/β-catenin signaling pathway mediates numerous essential cellular functions and has potentially important effects on tumorigenesis and cancer progression. The search for drugs to inhibit this pathway has identified a small molecule, PNU-74654, as an inhibitor of the β-catenin/TCF4 interaction. We evaluated the therapeutic role of PNU-74654 in two TC cell lines, NCCIT and NTERA2, by measuring cell viability, cell cycle transition and cell death. Potential pathways were evaluated by protein arrays and Western blots. PNU-74654 decreased cell viability and induced apoptosis of TC cells, with significant increases in the sub G1, Hoechst-stained, Annexin V-PI-positive rates. PNU-74654 treatment of both TC cell lines inhibited the TNFR1/IKB alpha/p65 pathway and the execution phase of apoptosis. Our findings demonstrate that PNU-74654 can induce apoptosis in TC cells through mechanisms involving the execution phase of apoptosis and inhibition of TNFR1/IKB alpha/p65 signaling. Therefore, small molecules such as PNU-74654 may identify potential new treatment strategies for TC.

Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3571-3571
Author(s):  
Matthew F. Clarkson ◽  
Aru Narendran ◽  
Randal N. Johnston
Keyword(s):  
Cell Death ◽  
High Risk ◽  
Cell Viability ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Treatment Strategies ◽  
Leukemia Cells ◽  
Western Blots ◽  
Pediatric Leukemia ◽  
Leukemia Cell Lines

Abstract Abstract 3571 Purpose: Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children. Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design: A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results: Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions: In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures: No relevant conflicts of interest to declare.

In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4459-4459
Author(s):  
Morris Kletzel ◽  
Sarah C. Tallman ◽  
Marie Olszewski ◽  
Wei Huang
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Annexin V ◽  
Resistant Cell ◽  
Primary Tumors ◽  
Induced Apoptosis ◽  
Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

Motexafin Gadolinium Induces Apoptosis in Lymphoid Cell Lines and Demonstrates Enhanced Biological Activity with Akt Kinase Inhibitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3406-3406
Author(s):  
Louie Naumovski ◽  
Jason Ramos ◽  
Jun Chen ◽  
Mint Sirisawad ◽  
David Lucas ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Jurkat Cells ◽  
Motexafin Gadolinium ◽  
Akt Phosphorylation ◽  
Induced Apoptosis

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that catalytically oxidizes intracellular reducing metabolites and produces reactive oxygen species (ROS). In this report, we demonstrate that MGd induces apoptosis or growth inhibition in several hematopoietic tumor-derived cell lines and tumor cells from patients with chronic lymphocytic leukemia. Lymphoma (HF-1, Ramos, DHL-4, DB, Hut78 and Raji) and leukemia (Jurkat, HL-60) cell lines were cultured in RPMI 1640 media with 10% heat inactivated fetal bovine serum with or without 50 uM MGd. MGd inhibited the growth of 6 of the cell lines (HF-1, Ramos, HL-60, DHL-4, Jurkat and DB) and was cytotoxic to HF-1. ROS were implicated in MGd-induced cell death since their presence was detected by dichlorofluorescein diacetate staining and peroxiredoxin oxidation in MGd treated HF-1 cells that undergo apoptosis, but not in Jurkat cells that do not undergo MGd-induced apoptosis. MGd triggered an apoptotic pathway in HF-1 cells as demonstrated by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspases, cleavage of PARP and annexin-V binding. MGd also induced cell death and activated caspases in vitro in primary chronic lymphocytic leukemia cells. Protein lysates from cultured cell lines (HF-1, Ramos) were subjected to immunoblot analysis to determine caspase cleavage patterns, and the phosphorylation status of Akt, a kinase that regulates survival pathways. In MGd treated HF-1, phospho-Akt protein levels initially increased 2–3 fold between 30 min and 1 hr (n=4) and then decreased to 40–50% of control levels by 24–48 hrs (n=4). The drop in phospho-Akt protein coincided with an increase in apoptotic cell death as indicated by morphology, staining with Annexin-V and activation of caspases. Addition of a specific inhibitor of Akt phosphorylation (SH-5) reduced Akt phosphorylation in MGd treated HF-1 cells by 90% and enhanced the cytotoxic effect of MGd. In Ramos cells, which do not undergo apoptosis when treated with MGd, co-treatment with MGd and SH-5 decreased phospho Akt levels by only 15% and did not result in cytotoxicity. These data point to a potential role for Akt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by inhibition of Akt. These data show that the pro-apoptotic effects of MGd involve caspase activation and provide a rationale to evaluate MGd in the treatment of lymphoma and leukemia patients.

The Diterpenoid Lactone Andrographolide (ANDRO) Stimulates Autophagy and Induces Reactive Oxygen Species (ROS)-Dependent Apoptosis in Lymphoma Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4991-4991
Author(s):  
Shuo Yang ◽  
Andrew M. Evens ◽  
Sheila Prachand ◽  
Leo I. Gordon
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Cell Lines ◽  
Redox State ◽  
Annexin V ◽  
Lymphoma Cell ◽  
Time Dependent ◽  
Cellular Redox ◽  
Forkhead Transcription Factors ◽  
Induced Apoptosis

Abstract ANDRO is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an important herbal medicine used in China. It has been reported to have anti-inflammatory, anti-hypertensive, anti-viral and immunostimulant properties. It has also been shown to inhibit cancer cell proliferation and induce apoptosis in HL-60 (leukemia), PC-3 (prostatic adenocarcinoma), MDA-MB-231 (breast cancer), HepG2 (liver cancer), HeLa (cervical cancer) and HCT116 (colorectal cancer) cell lines. The diterpenoids have been found to generate ROS and may increase apoptosis by altering the cellular redox state. We hypothesized that ANDRO would lead to cell death in lymphoma cell lines and that the effect may be related to altered cellular redox state. We studied the Burkitt p53 mutated Ramos cell line, the mantle cell lymphoma line Granta and L428, a resistant EBV-negative Hodgkin lymphoma cell line. We found that after incubation with increasing concentrations of ANDRO, there was dose and time-dependent cell death as measured by MTT. The IC50 (concentration that achieved 50% cell proliferation inhibition) at 48h was 20μM for Ramos, 40μM for Granta, and 50μM for L428. ROS was measured by oxidation of 2’7’dichlorofluorescein diacetate (DCFDA) to dichlorofluorescein (DCF) and analyzed by fluorescence-activated cell sorting (FACS) following incubation at 1hour (h), 2h, 3h, 5h, 38h, and 48h with ANDRO (20–80μM). ANDRO increased ROS production in all lymphoma cell lines, which was abrogated by the antioxidant N-acetyl-L-cysteine (NAC). Maximum ROS generation with ANDRO was seen at 48h for Ramos (1.7 fold), 5h for Granta (1.6 fold), and 38h for L428 (2.4 fold). To determine the mechanism of cell death, we measured apoptosis by Annexin-V/propidium iodide (PI), and detected by flow cytometry (FACS). Cells were treated with ANDRO in the presence or absence of the reduced glutathione (GSH) depleting agent buthionine sulfoximine (BSO) (100μM) for 28h, 48h, and 72h. We found that the AC50 (concentration that achieved 50% apoptosis) was 40μM for Ramos at 72h, 40μM for Granta at 48h and &gt;80μM for L428 at 48h, while in the presence of BSO it was &lt;10μM for Ramos at 72h, between 30–40μM for Granta at 28h and between 30–40μM for L428 at 48h. Apoptosis was completely blocked, by NAC, both in the presence and absence of BSO. Further, ANDRO induced PARP cleavage and activation of caspases 3, 8, and 9 in Granta and Ramos. Next, we explored the relationship of ANDRO and Forkhead transcription factors. ANDRO caused dephosphorylation of FOXO3a or FOXO1, in a dose- and time-dependent manner, and this was reversible by NAC. Downstream proteins of FOXO3a, Bim, p27kip1 and the isoforms of the autophagy-related protein LC3B were upregulated, and this was reversed by NAC. The LC3B isoform-II, which is cleaved from LC3B-I, is a marker of autophagy activation. To determine the role of autophagy in cell death related to ANDRO, we inhibited autophagy with 3-methyladenine (1–2mM) and found significant enhancement of ANDRO-induced apoptosis in Granta and Ramos. Finally, ANDRO induced apoptosis (&gt;60% Annexin-V+/PI+) in malignant B-cells from a patient with chronic lymphocytic leukemia/small lymphocytic lymphoma (trisomy 12, peripheral blood absolute lymphocyte count 95.2 K/uL, bulky adenopathy) very low concentrations (5μM at 18h) in vitro, which was also reversible with NAC. We conclude that ANDRO induces ROS-dependent apoptosis in lymphoma cell lines and in a primary tumor sample, which is enhanced by depletion of GSH and inhibited by the antioxidant NAC. These effects appear to proceed through caspase activation and inhibition of autophagy, and are in part dependent on signaling through forkhead transcription factors and altered cellular redox pathways. Further studies of diterpenoids as single agents or in combination with other anti-lymphoma agents are warranted.

scholarly journals Annona cherimola Seed Extract Activates Extrinsic and Intrinsic Apoptotic Pathways in Leukemic Cells

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 506 ◽  
Author(s):  
Tony Haykal ◽  
Peter Nasr ◽  
Mohammad H. Hodroj ◽  
Robin I. Taleb ◽  
Rita Sarkis ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Seed Extract ◽  
Leukemic Cells ◽  
Western Blots ◽  
Toxic Activity ◽  
Annona Cherimola

Annona cherimola Mill is a large green fruit with black seeds widely known to possess toxic properties due to the presence of Annonaceous acetogenins. The present study investigates the anti-cancer properties of an Annona cherimola Mill ethanolic seed extract on Acute Myeloid Leukemia (AML) cell lines in vitro and elucidates the underlying cellular mechanism. The anti-proliferative effects of the extract on various AML cell lines and normal mesenchymal cells (MSCs) were assessed using WST-1 viability reagent. The pro-apoptotic effect of the extract was evaluated using Annexin V/PI staining and Cell Death ELISA. The underlying mechanism was deciphered by analyzing the expression of various proteins using western blots. Treatment with an A. cherimola seed ethanolic extract promotes a dose- and time-dependent inhibition of the proliferation of various AML cell lines, but not MSCs. Positive Annexin V staining, as well as DNA fragmentation, confirm an increase in apoptotic cell death by upregulating the expression of pro-apoptotic proteins which control both intrinsic and extrinsic pathways of apoptosis. GC/MS analysis revealed the presence of phytosterols, in addition to other bioactive compounds. In conclusion, Annona cherimola Mill seed extract, previously known to possess a potent toxic activity, induces apoptosis in AML cell lines by the activation of both the extrinsic and the intrinsic pathways.

scholarly journals The pro-apoptotic effect of a Terpene-rich Annona cherimola leaf extract on leukemic cell lines

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Carl Ammoury ◽  
Maria Younes ◽  
Marianne El Khoury ◽  
Mohammad H. Hodroj ◽  
Tony Haykal ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Leaf Extract ◽  
Annexin V ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Western Blots ◽  
Annona Cherimola ◽  
Apoptotic Effect ◽  
Ethanolic Leaf Extract

Abstract Background The edible fruit Annona cherimola has previously shown many nutritional and medicinal properties. The current study evaluates the anti-cancer and anti-proliferative properties of Annona cherimola ethanolic leaf extract (AELE) on Acute Myeloid Leukemia (AML) cell lines cultured in vitro (Monomac-1 and KG-1). Methods The anti-proliferative effect of A. cherimola ethanolic leaf extract was evaluated via cell viability assay. Its pro-apoptotic effect was assessed through Cell Death ELISA and dual Annexin V/PI staining. To further investigate the molecular mechanism by which the extract promoted apoptosis and inhibited the proliferation of the AML cells used, apoptotic protein expression was determined through western blots. Extract composition was elucidated by Gas Chromatography-Mass Spectrometry (GC-MS). Results Our results showed that the treatment with A. cherimola ethanolic leaf extract exhibited an inhibitory effect on the proliferation of both cancer cell lines used in a dose- and time-dependent manner, with no toxic effects on normal mononuclear cells (MNCs) isolated from human bone marrow. This effect was mediated by DNA fragmentation and apoptosis, as revealed by Cell Death ELISA and dual Annexin V/PI staining. Western blot analysis revealed a Bax/Bcl2 dependent mechanism of apoptosis, as well as PARP cleavage, confirming the apoptotic results observed previously. These effects may be attributed to the presence of terpenes which constitute a large component of the leafy extract, as revealed via GC-MS. Conclusion All the data presented in our study show that the terpene-rich A. cherimola ethanolic leaf extract exhibits an anti-proliferative and pro-apoptotic effect on the AML cell lines used.

scholarly journals Piper sarmentosum induced apoptosis via mitochondrial pathway in human lung adenocarcinoma cells, A549

2016 ◽  
Vol 15 (1) ◽  
Author(s):  
Azila Sirajudeen ◽  
Aisyah Hanani Mohd Tahir ◽  
Radiah Abdul Ghani
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Death ◽  
Cytochrome C ◽  
Treatment Strategies ◽  
A549 Cells ◽  
Annexin V ◽  
Mitochondrial Pathway ◽  
Cell Cycle Profile ◽  
Induced Apoptosis

Introduction: Lung cancer has been reported as one of the most common types of cancer worldwide. Current cancer treatments like chemotherapy do not result in a complete cure and are known to cause side effects in the patients. Therefore, alternative treatment strategies are being explored, one of which is to investigate the potential of the local herbs in this regard. Piper sarmentosum (daun kaduk) has received much attention due to its anti-cancer properties in A549 cells. In this study, the cell cycle profile and mechanisms of cell death induced by P. sarmentosum were investigated using a flow cytometer. Methods: The cell cycle profile changes were observed using propidium iodide staining while the type of cell death was analyzed using Annexin-V assay. Caspases -3/7,8 and 9 and cytochrome c assays were elucidated using flow cytometry analysis. Results: P.sarmentosum arrested the growth of A549 cells at G0/G1 phase. The Annexin V analysis revealed that P. sarmentosum exhibited significant induction of apoptosis after 24 h exposure. Caspases analysis showed that P. sarmentosum induced apoptosis through mitochondrial pathway, via the activation of caspase 3 and caspase 9. Meanwhile, cytochrome c analysis revealed that P. sarmentosum induced a mitochondrial pathway of cell death through the release of cytochrome c. Conclusions: Based on these preliminary findings, P. sarmentosum has a great potential as a dietary cancer treatment for lung cancer and may perhaps be used for lung cancer pharmacotherapy in the clinical settings in future.

scholarly journals Clinical Impact and Therapeutic Opportunity of Insulin Receptor Substrates 1/2 in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1512-1512
Author(s):  
Juan Luiz Coelho-Silva ◽  
Diego Antonio Pereira-Martins ◽  
Josiane Lilian Schiavinato ◽  
Eduardo Magalhães Rego ◽  
João Agostinho Machado-Neto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Cell Viability ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Annexin V ◽  
Ros Production ◽  
Insulin Receptor Substrates ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Background: The identification of biological and clinical prognostic factors in acute myeloid leukemia (AML) allowed the definition of patient subgroups and the realization of risk-adapted and targeted treatment strategies. Insulin-like growth factor 1 receptor/Insulin Receptor Substrates (IGF1R/IRS) pathway plays an important role in the development of neoplasia. IRS1/2 activates AKT/mTOR and MAPK pathways, through their interaction with PI3K and GRB2, culminating in increasing cell proliferation. NT157 is an allosteric inhibitor of IGF1R-IRS1/2 signaling that showed antineoplastic effects in preclinical studies of solid tumors. However, IRS1/2 clinical function and NT157 effects were not assessed in AML. Aims: To investigate IRS1 and IRS2 mRNA expression in AML patients and their impact in clinical outcomes, and to analyze the effects of the NT157 in AML cell lines. Material and methods: Comparison of IRS1 (probe nº 204686) and IRS2 (probe nº209184_s) expression from 581 AML patients and 8 CD34+ cells from healthy subjects were analyzed using data from Amazonia! Platform. For survival analysis, IRS1 and IRS2 mRNA expression levels from 173 AML patients (92 male - median age 58 years [range: 18-65]) were obtained from TCGA AML study available online on CBioPortal for Cancer Genomics. NB4, NB4-R2, Kasumi-1 and THP1 cell lines were submitted to NT157 (0.5, 1.0, 2.0, 4.0, 8.0 or 16 µM) 72 hours and evaluated for cell viability (MTT assay), apoptosis (Annexin V/PI), cell cycle (PI), ROS production (DCFDA), mitochondria staining (MitoTracker), and protein expression/activation (western blot). Bone marrow mononuclear cells (BMMC) were obtained from 4 AML patients at diagnosis and submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA, Mann-Whitney or Kruskal-Wallis and Spearman correlation tests, as appropriate. For survival analysis, Kaplan-Meyer curves were compared with the log-rank test. Cox regression analysis was also applied. Results: IRS1 expression, but not IRS2, predicted outcomes. Reduced IRS1 expression showed poorer disease-free survival (DFS) (survival median time [MT]: 10.1 months [mos] vs. 28.4 mos, P<0.001; Hazard ratio [HR]: 0.51 [CI95:0.32 - 0.79]) and overall survival (OS) (MT: 14.5 mos vs. 27.4 mos, P=0.009; HR: 0.61 [CI95:0.42 - 0.88]). IRS1 expression independently predicted poorer DFS (HR: 0.59 [CI95: 0.36 - 0.79]; P= 0.03) using cytogenetic risk stratification, age and leukocytes as confounders. Of note, IRS1 level was positively correlated with proapoptotic CD27 (r=0.51; P<0.001) and with IL17RA (r=0.62; P<0.001) related to CD34 cell differentiation. IRS2 expression was upregulated in AML harboring t(15;17) (n=36; P<0.01) and inv(16) (n=37; P=0.01) in comparison to CD34+. In NB4, NB4-R2 and Kasumi-1 cells, NT157≥0.5µM reduced cell viability (P<0.05) and increased apoptosis (P<0.05). The mean percentage of annexin V+ cells for control, NT157 2.5, 5 and 10µM were 11, 47, 73 and 75% for NB4, 11, 41, 69 and 75% for NB4-R2 and 17, 45, 61 and 64% for Kasumi-1, respectively. In TP53-null cell line THP1, NT157 reduced cell viability at doses higher than 2µM (P<0.05) and induced apoptosis at 10µM (9.1 vs. 25%; P<0.05). NT157 induced ROS production in NB4 (fold-increase of mean fluorescence intensity [MFI]: 25.8 and 24.8), NB4-R2 (MFI: 26.7 and 31.4), Kasumi-1 (MFI: 5.8 and 6.6) and THP1 (MFI: 1.8 and 4.1) at 5 and 10µM (all P<0.05) and increased mitochondrial mass in NB4 (MFI: 3.9 and 3.7), NB4-R2 (MFI: 2.6 and 2.9), Kasumi-1 (MFI: 3.2 and 4.7) and THP-1 (MFI: 2.6 and 2.2) (all P<0.05). NT157 also modulated cell cycle progression, as evidenced by G2/M arrest in THP-1 and sub-G0/G1 in other cell lines (P<0.05). The IGF1R-IRS1/2 inhibitor NT157 reduced activation/expression of IGF1R (Tyr1135), IRS1/2 (Tyr612), AKT1/2/3 (Ser473), P70S6K (Thr421/Ser424), 4EBP1 (Thr70), ERK1/2 (Thr185/Tyr187) and induced DNA damage (increased γH2AX). NT157 did not induce autophagy, as demonstrated by non-degradation of p62 and lack of conversion of LC3BI into LC3BII in cell lines tested. NT157≥0.5 µM reduced cell viability and induced apoptosis in BMMC from 4 AML patients in a dose dependent manner (P<0.05). Conclusions: In AML, downregulation of IRS1 predicted dismal prognosis and the IGF1R-IRS1/2 inhibitor NT157 exerted an antineoplastic activity, downregulated PI3K/AKT and MAPK signaling. IRS1/2 arises as a promising therapeutic target for AML patients. Disclosures No relevant conflicts of interest to declare.

Initial Evaluation of Novel Dual PIM/PI3K and Triple PIM/PI3K/mTOR Inhibitors in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5713-5713 ◽  
Author(s):  
Mairead Reidy ◽  
Marianne vanDijk ◽  
Niamh Keane ◽  
Michael O'Neill ◽  
Michael E O'Dwyer
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Mouse Model ◽  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Annexin V ◽  
Mtor Inhibitors ◽  
Pim Kinases

Abstract Background: Despite advances in treatment, Multiple Myeloma (MM) remains incurable. The PI3K/AKT pathway is activated in MM cells in > 50% of cases due to factors such as bone marrow (BM) microenvironmental signaling and hyperactivation following treatment with proteasome inhibitors (PI). Multiple small-molecule inhibitors have been developed to target PI3K/AKT or mTOR kinases, but the efficacy of these drugs is likely to be compromised by the stimulation of compensatory signaling pathways. The redundancy of signaling pathways provides back-up mechanisms allowing escape from targeted inhibition. One such compensatory pathway is that driven by PIM kinases, which produce parallel oncogenic signals to AKT and mTOR and share several downstream molecular targets. As with PI3K/AKT, the BM microenvironment plays a major role in PIM activation and other factors increasing PIM levels include hypoxia and PI treatment. PIM1 and particularly PIM2 are known to be highly expressed in MM and play important roles in regulating MYC-driven transcription, apoptosis, cytokine signaling, cell proliferation and protein translation. Combinations of separate PI3K and PIM inhibitors have shown evidence of synergy in MM cell lines and animal models and a PIM kinase inhibitor has recently shown activity in relapsed/refractory MM. Given this background we wished to evaluate the activity of a novel family of kinase inhibitors capable of inhibiting not only PIM kinases but also PI3K/AKT (dual inhibitors) and PI3K/AKT/mTOR (triple inhibitors). Methods: We evaluated the in-vitro activities of a single pan-PIM (pPIMi), dual PIM/PI3K (IBL-202) and triple PIM/PI3K/mTOR (IBL-301) inhibitor in MM cell lines: MM1.S, NCI-H929, RPMI8226 and KMS11, which is known to be PIM2 dependent, alongside the pan-PI3K inhibitor GDC-0941 and the pan-PIM inhibitor AZD1208. IBL-202 and IBL-301 are optimized lead compounds and are low nanomolar pan-PIM/PI3K and pan-PIM/PI3K/mTOR inhibitors respectively. These dual and triple inhibitors show excellent kinase selectivity profile against a panel of 456 kinases. Cell viability was assessed using the Cell-Titre Glo assay and apoptosis determined by Annexin-V/PI staining. Co-culture experiments were performed with HS-5 stromal cells. Combination treatment was performed with bortezomib and IBL-202 to assess synergy. Results and discussion: IBL-202 and IBL-301 were significantly more potent than pPIMi in all MM cell lines tested (figure 1). IBL-202 and IBL-301 caused a loss in cell viability 50% and 70%, respectively, greater than pPIMi alone. IBL-202 and IBL-301 induced 50-80% and 80-100% cell death, respectively .v. 10% for pPIMi after 48 hrs, p<0.001. The Pim2 dependent MM cell line KMS11 showed a loss in cell viability following treatment with IBL-202 and IBL-301 up to three times greater than either of the PIM kinase inhibitors or GDC-0941. IBL-202 treatment caused a 90% reduction in cell viability at a dose of 5µM and IBL-301 was equally effective at a concentration of just 1µM. GDC-0941(5µM) caused a loss of approximately 30% in cell viability whereas cells remained entirely resistant to pPIMi and AZD1208 at concentrations up to 10µM (p< 0.001). IBL-202 in combination with bortezomib was synergistic in MM cell lines (CI<1). While co-culture with HS-5 cells protected MM cell lines against bortezomib-induced cell death, it promoted the apoptotic effect of both IBL-202 and IBL-301 with an increase in Annexin V positive cells from 15% to 40%. This suggests that micro-environmental stimulation could potentially induce synthetic lethality in the presence of these inhibitors. We observed strong induction of PIM2 in MM1.S cells following co-culture. Mechanistically, cells respond to dual and triple inhibitors with cell cycle arrest, marked apoptosis and strong down-regulation of biomarkers. The dual and triple inhibitors are optimized with respect to their in vitro ADME properties and have excellent oral bioavailability. In-vivo IBL-301 has been well tolerated, with no signs of toxicity even 20 times above the efficacious dose in a transgenic (KRASV12NSCLC) mouse model. Testing of IBL-202 in a relevant MM mouse model is planned in the near future. Conclusions: IBL-201 and IBL-301 show promising activity in MM cellular models with increased potency compared to inhibitors targeting PIM or PI3K alone and warrant further evaluation in this disease. Figure 1. Figure 1. Disclosures O'Neill: Inflection Biosciences: Employment, Equity Ownership. O'Dwyer:Inflection Biosciences: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Smad4 induces cell death in HO-8910 and SKOV3 ovarian carcinoma cell lines via PI3K-mTOR involvement

2020 ◽  
Vol 245 (9) ◽  
pp. 777-784
Author(s):  
Yushuang Yao ◽  
Zhe Zhang ◽  
Fanmao Kong ◽  
Zhuqing Mao ◽  
Zhaoyuan Niu ◽  
...  
Keyword(s):  
Cell Viability ◽  
Ovarian Carcinoma ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Treatment Strategies ◽  
Carcinoma Cells ◽  
Skov3 Cell ◽  
Ovarian Carcinoma Cells ◽  
Skov3 Cells ◽  
Induced Apoptosis

Ovarian carcinoma is one of the most common malignant cancers in women. Previous research has shown that Smad4 participates in the progression of multiple biological reactions as a crucial regulator. Nevertheless, studies on the role of Smad4 in ovarian carcinoma have been extremely limited. The study aim was to explore the mechanism underlying Smad4 regulation of HO-8910 and SKOV3 cell viability and autophagy. We observed that Smad4 gene expression in ovarian carcinoma tissues and cell lines was downregulated, and Smad4 overexpression resulted in decreased proliferation and increased autophagy in HO-8910 and SKOV3 cells (ovarian carcinoma cells). We also found that Smad4 overexpression induced apoptosis of ovarian carcinoma cells. A co-immunoprecipitation assay also revealed that Smad4 interacted with the P85 subunit of PI3K and caused its degradation and dephosphorylation. Subsequently, expression of mTOR was inhibited. Accordingly, these findings showed that further investigation of the biological mechanisms underlying ovarian carcinoma occurrence and progression is warranted, which may lead to new ovarian carcinoma treatment strategies. Impact statement This study investigated the effect and mechanism of Smad4 in ovarian carcinoma (OC) cell viability and demonstrated that Smad4 acted as a tumor suppressor in OC, which may contribute to the understanding of molecular mechanisms underlying OC occurrence and progression. Smad4 expression was decreased in the OC specimens, but Smad4 recovery in the OC cell lines impaired the survival and viability of OC cells by increasing autophagy and apoptosis. Further investigation showed that Smad4 interacted with the P85 subunit of PI3K and caused deactivation of the PI3K/mTOR pathway. Therefore, Smad4 could be considered as a target in cancer therapy due to its regulatory effect in OC carcinogenesis.

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