Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3368-3368 ◽  
Author(s):  
Jessicca M. Rege ◽  
Blaine W. Robinson ◽  
Manish Gupta ◽  
Jeffrey S. Barrett ◽  
Peter C. Adamson ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Family Member ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Single Agent ◽  
Cytotoxic Agents ◽  
Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

Proteasome Inhibitors Can Induce Caspase-3 Activation and Apoptosis in Human CML Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4681-4681
Author(s):  
Byung-Su Kim ◽  
Chang Up Kim ◽  
Young-Ju Kim ◽  
Eun Kyung Bae ◽  
Jinhee Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Proteasome Inhibitor ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Expression Patterns ◽  
Proteasome Inhibitors ◽  
Acridine Orange Staining

Abstract The proteasome is a multi-enzyme complex that provides the ubiquitin-dependent degradation of many cytoplasmic and nuclear proteins involved in cell cycle progression and apoptosis. Inhibition of the proteasome represents a promising approach for the treatment of cancer because it can lead to cell cycle arrest and activation of caspases in tumor cells. There are several proteasome inhibitors that have been reported to induce apoptosis in various tumors. However, the effect of proteasome inhibition in human myeloid leukemia has not been reported so far. In this study, we tested two peptide-aldehyde proteasome inhibitors (MG115, MG132) on two human CML cell lines (K562, KCL22). At first, we treated both cell lines for 24, 48 and 72 hours with different doses of MG115 and MG132 and cell viability was tested by MTT assay. It showed substantial time and dose dependent cytotoxicity in both CML cell lines. Acridine orange staining also revealed DNA fragmentation. We then performed caspase-3 colorimetric assay after treating both cell lines for 6, 12 and 24 hours with 0.78μM of MG115, MG132. K562 showed the continuous rising of caspase-3 activity, while KCL22 exhibited the initial increase and subsequent mild decrease of caspase-3 activity. In addition, western blot analysis showed the reduction of procaspase-3 expression. The expression of Bcl-2 and Bcl-XL was reduced by western blot. p21 expression was slightly increased and that of cyclin D1 was decreased. Additionally, the treatment of proteasome inhibitor in CML cell lines initially induced phosphorylation of Jun kinase. We next examined the expression of heat shock proteins (Hsp70, Hsp90) after treating for 6, 12, 24 hours with the same proteasome inhibitors. Western blot analysis results indicated that expression patterns were different between MG115 and MG132. MG115 induced the slight increase of Hsp70 and Hsp90 in K562, but the reduction of both in KCL22. Meanwhile, MG132 produced the decrease of Hsp70 and Hsp90 in both K562, KCL22. In summary, our work supports that a proteasome inhibitor can induce apoptosis in human CML cell lines. We are currently focusing on the combined effect of proteasome inhibitor and Hsp90 inhibitor on CML. IC50 of Proteasome Inhibitors Cell line Proteasome Inhibitor 24hr 48hr 72hr K562 MG115 3.01 μM 1.14 μM 0.59 μM K562 MG132 μ 2.13 M 1.03 μM 0.57 μM KCL22 MG115 156.92 μM 1.36 μM 0.73 μM KCL22 MG132 1.56 μM 0.93 μM μ 0.75 M

scholarly journals Single Agent and Combinatorial Efficacy of First-in-Class Small Molecule ONC201 in Acute Leukemia and Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2759-2759 ◽  
Author(s):  
Varun V Prabhu ◽  
Amriti Lulla ◽  
Christina L Kline ◽  
Peter J Van den Heuvel ◽  
Mala K. Talekar ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Single Agent ◽  
Employment Equity ◽  
Equity Ownership ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Abstract ONC201 is the founding member of the imipridone class of anti-cancer small molecules that possess a unique core chemical structure. ONC201 is currently being evaluated in several Phase I/II clinical trials for advanced cancers. In the current study, we evaluated the single agent and combinatorial efficacy of ONC201 in preclinical models of acute leukemia and multiple myeloma (MM). In acute leukemia, we evaluated ONC201 anti-cancer effects in acute myeloid leukemia (AML) (Kasumi-1, HL60) and acute lymphoblastic leukemia (ALL) (Reh, Jurkat and MOLT-4) cell lines. We observed a time- and dose-dependent decrease in cell viability for every cell line in the panel (EC50 1-5 µM). Vincristine-resistant cells HL60/VCR were also sensitive to single agent ONC201 with EC50 values on par with corresponding vincristine-sensitive parental cells. Dose- and time-dependent induction of apoptosis was noted in Western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201 for 48 hr. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously reported late-stage signaling effects of ONC201 in solid tumor cells (Allen et al, 2013). Sub-G1 analysis indicated that ONC201 induces apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. Western blot analysis revealed ONC201-mediated apoptosis involves PARP cleavage and caspase-9 activation in ALL cells. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells. We observed inhibition of Akt phosphorylation upon ONC201 treatment of ALL cells. Fresh AML patient cells were also found to be sensitive to ONC201 in cell viability and caspase 3/7 activity assays at 5µM. We observed that independent clones of cancer cells with acquired resistance to ONC201 were more sensitive to cytarabine compared to parental ONC201-sensitive cancer cells. In addition, ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine in AML cell lines. Determination of combination indices (CI) revealed synergy at several concentrations (CI 0.336-0.75 in CMK cells). Also, ONC201 combined additively with midostaurin in CMK cells and vincristine in HL60/VCR cells. Thus, ONC201 is a promising combinatorial partner for AML therapies based on these preclinical sensitization results. In accordance with ONC201-mediated activation of the integrated stress response that B cells are highly sensitive to (Kline et al and Ishizawa et al, 2016), MM was identified as one of the most ONC201-sensitive tumor types in the Genomics of Drug Sensitivity in Cancer collection of cell lines. Three human MM cell lines were used for validation (KMS18, MM.1S and RPMI-8226), which revealed a time- and dose-dependent decrease in cell viability (EC50 1-2.5 µM). Bortezomib-resistant cells MM.1S 33X were sensitive to ONC201 as a single agent with EC50 values comparable to bortezomib-sensitive parental cells. We observed an average of 10-fold induction of ONC201-mediated apoptosis using Sub-G1 analyses in MM cells at 5 µM, 48 hrs post-treatment. Rescue of ONC201-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, Western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Furthermore, ONC201 demonstrated synergistic reduction in cell viability at various concentrations in combination with either ixazomib or dexamethasone, which are used in the clinical treatment of MM, in RPMI8226 cells (CI 0.228-0.75). Also, ONC201 combined additively with bortezomib in RPMI8226 and MM.1S 33X cells. In summary, these preclinical studies support the ongoing ONC201 single agent trials in acute leukemias and MM. Our findings suggest that ONC201 may be an important therapeutic option for patients with hematological malignancies who have developed resistance to approved therapies. Additionally, our results point to specific standard-of-care therapies that may be combined with ONC201 to exert durable responses without adding to the burden of toxicity. Disclosures Prabhu: Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership.

Caspase-Dependent Anti-Tumor Effects of ONC201/TIC10 on Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5224-5224
Author(s):  
Amriti R. Lulla ◽  
Christina Leah B Kline ◽  
Liz J. Hernandez-Borrero ◽  
Varun Vijay Prabhu ◽  
Jessica M Wagner ◽  
...  
Keyword(s):  
Western Blot ◽  
Tumor Cells ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Therapeutic Potential ◽  
Fold Induction ◽  
Dose Dependent Decrease ◽  
Resistant Cells

Abstract PI3K/Akt and Ras/MAPK pathways are attractive therapeutic targets in almost all tumor types, including AML and MM. Apo2L/TRAIL has been deemed a promising therapeutic given its selectivity towards cancer cells although its clinical development has been hampered by various limitations including short half-life and general shortcoming of protein-based therapeutics. ONC201/TIC10 (Oncoceutics, Inc.) is a first-in-class small molecule inducer of TRAIL expression. ONC201/TIC10 has previously been shown to up-regulate TRAIL and its death inducing receptor DR5 in HCT116 colon cancer cells, in part through the inhibition of Foxo3a phosphorylation mediated by dual inhibition of Akt and ERK (Allen JE et al, Sci Transl Med., 2013). Currently, ONC201/TIC10 is set to enter clinical trials for patients with advanced malignancies after the IND was approved by the FDA in March, 2014. We thus investigated the therapeutic potential of ONC201/TIC10 in AML and MM given a major unmet need when conventional therapy fails. We explored the possibility that ONC201/TIC10 induces apoptosis in MM and AML in part through dual inhibition of the PI3K/Akt and Ras/MAPK pathways. We tested a panel of four human MM cell lines (KMS18, MM.1S, MM.1S 33X and RPMI-8226) and three human AML cell lines (Kasumi-1, HL60, HL60/VCR). The Cell-titer Glo assay demonstrated a time and dose-dependent decrease in viability in the entire panel of MM and AML cells. EC50 values ranged from 1-2.5 µM for the MM and 2-5µM for the AML cell lines, respectively. Bortezomib-resistant cells MM.1S 33X and vincristine- resistant cells HL60/VCR were also significantly sensitive to ONC201/ TIC10 as a single agent with EC50s on par with the corresponding parental cell lines. Given the previously reported pro-apoptotic effects of ONC201/TIC10 against solid tumor cells, we assessed apoptosis by performing Sub-G1 analyses and assessing caspase-3 cleavage as two widely used methods to analyze apoptotic cell death. We observed an average of 10-fold induction of ONC201/TIC10–mediated apoptosis in MM cells at 5 mM at 48 hrs post-treatment. Rescue of ONC201/TIC10-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Dose and time dependent induction of apoptosis was noted in western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201/TIC10 for 48 hrs prior to analysis. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously proposed mechanism of ONC201/TIC10 against solid tumor cells. To further investigate the therapeutic potential of ONC201/TIC10 in the context of AML, fresh AML cells were treated with ONC201/TIC10. The primary cells were also found to be sensitive to ONC201/TIC10 (60% decrease in cell viability 72 hrs post 5mM ONC201/TIC10 treatment). Similarly, caspase 3/7 activity was significantly increased as assessed by the Caspase Glo 3/7 assay (~5 fold induction in activity 72 hrs post 5mM ONC201/TIC10 treatment). To explore further the therapeutic potential of ONC201/TIC10, we performed combinatorial experiments with bortezomib and vincristine using the MM.1S 33X MM cells and the HL60/VCR AML cell lines. ONC201/ TIC10 showed an additive effect with both these compounds against the MM and AML lines. Our work demonstrates activity of ONC201/TIC10 against AML and MM cell lines including fresh AML tumor cells. The efficacy data with resistant cells is in par with the applicability of TIC10 in patients with refractory/relapsed hematological malignancies. The long-term goal of this project is to provide a rationale for a phase 1b trial of ONC201/TIC10 for refractory/relapsed MM and AML in combination with existing therapies. Figure 1: Efficacy of ONC201/TIC10 in AML and MM cells Figure 1:. Efficacy of ONC201/TIC10 in AML and MM cells Disclosures Allen: Oncoceutics, Inc.: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.

scholarly journals Comparatively Investigation of Antitumor Activities of Resveratrol and Paclitaxel through Apoptosis and Autophagy in A549 Cells

Proceedings ◽  
2019 ◽  
Vol 40 (1) ◽  
pp. 27
Author(s):  
Imamoglu ◽  
Atalay ◽  
Unsal
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Synergistic Effects ◽  
Biological Effects ◽  
A549 Cells ◽  
Antitumor Effects

Resveratrol, a natural product, has many biological effects including antitumor effects. Paclitaxel, a chemotherapeutic drug, has been widely used in the treatment of lung cancer. Although the antitumor effects of resveratrol and paclitaxel in A549 cells have been studied in separately before, in this study comparatively investigation the anticancer effects of resveratrol and paclitaxel on the apoptosis and autophagy in A549 cells is aimed. The effects on A549 cell viability of resveratrol and paclitaxel (Taxol) were determined by MTT assay. mRNA transcription levels of Bax, Bcl-2 and caspase-3 and protein expression levels of Bax, Bcl-2 and LC3-II were determined by RT-qPCR and western blot analysis, respectively. Our results demonstrated that resveratrol and paclitaxel inhibited the viability of A549 cells. RT-qPCR and western blot analysis showed that paclitaxel stimulated apoptotic cell death in A549 cells by more increasing pro-apoptotic Bax and caspase-3 levels and by more decreasing anti-apoptotic Bcl-2 level in comparison with resveratrol. On the other hand, resveratrol stimulated autophagic cell death by more increasing the level of an autophagic marker LC3-II compared to paclitaxel. In conclusion, we showed that resveratrol exerts its antitumor effects through the induction of autophagy in A549 cells compared to paclitaxel. However, it should be investigated the synergistic effects of resveratrol in combination with paclitaxel on A549 cells. Thus, resveratrol may enhance the effect of paclitaxel on apoptosis by inducing autophagy in A549 cells.

Mer Receptor Tyrosine Kinase Is a Novel Therapeutic Target In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1957-1957
Author(s):  
Sandra Christoph ◽  
Silke Maag ◽  
Deborah DeRyckere ◽  
Douglas K. Graham ◽  
Stephen V Frye ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Blot Analysis ◽  
Colony Formation ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Downstream Signaling ◽  
Tk Inhibitors

Abstract Introduction Although many novel therapeutic agents have been explored, multiple myeloma (MM) remains an incurable hematopoietic malignancy. Mer receptor tyrosine kinase (Mer TK) is a member of the TAM (Tyro3, Axl and Mer) family and is involved in the progression of several human malignancies. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity in vitro, and delays tumor progression in vivo. These observations make Mer TK inhibitors to excellent candidates for targeted therapies. We show here for the first time that Mer TK is ectopically expressed in MM cells and test the response of MM cell lines to our previously described small molecule Mer inhibitor, UNC1062, a substituted pyrazolopyrimidine. Here we demonstrate the biochemical and biologic effects of treatment with UNC1062, which suggests efficacy of Mer inhibitors as a novel strategy for the treatment of MM. Methods: Western blot analysis was used to determine expression of the TAM receptors and to evaluate inhibition of Mer TK activation and downstream signaling by UNC1062 in MM cell lines. UNC1062-mediated anti-myeloma activity with or without bortezomib was determined using short-term (MTT) and long-term (colony-formation) assays. Cleaved PARP and caspase 3 were detected by western blot analysis as indicators of apoptosis. Results: Western blot analysis of protein extract from MM cell lines showed that four (RPMI8226, U226, AMO-1 and KMS-12BM) out of six lines (66.6%) express Mer TK protein at varying levels, with AMO-1 expressing the least and U226 expressing the most. The MM cell lines OPM-2 and NCI-H929 did not express Mer TK. UNC1062 potently inhibits Mer TK activity in vitro (Mer IC50= 1.1 nM, Morrison Ki = 0.33 nM). In cell-based assays, treatment with UNC1062 inhibited accumulation of phospho-Mer and downstream signaling through ERK and Stat5 in U226 cells. UNC1062 also reduced proliferation and/or survival in Mer TK-expressing RPMI8226 cells (IC50 = 0.98 ± 0.14 μM, n=7) and U226 (IC50 = 0.28 ± 0.09 μM, n=3) cells. Treatment with UNC1062 did not lead to a meaningful reduction of metabolically active cells in Mer-negative NCI-H929 and OPM-2 cells. In contrast, UNC1062-treated Mer-positive MM cells exhibited increased levels of cleaved caspase 3 and cleaved PARP confirming apoptosis induction. Treatment with UNC1062 (100 nM) resulted in a statistically significant, dose-dependent decrease in colony-formation in methylcellulose compared to control cultures in Mer-positive RPMI8226 cells (418 ± 23 vs. 291 ± 37 colonies, p = 0.04, n = 3). No significant reduction of colony formation was observed in Mer-negative OPM-2 cells. Treatment of U226 cells with UNC1062 (800 nM) in combination with bortezomib resulted in a statistically significant reduction in relative cell number compared with bortezomib alone (0.61 ± 0.06 vs 0.41 ± 0.06, p = 0.04, n =3). Conclusion: Mer TK is ectopically expressed in the majority of investigated MM cell lines. UNC1062 treatment of Mer TK positive MM cells inhibits Mer TK activity and downstream signaling, mediates anti-neoplastic activity in liquid culture, and decreases colony-formation in methylcellulose. In addition, UNC1062 treatment sensitizes MM cells to bortezomib. Taken together, these data support further development of Mer TK inhibitors as novel MM therapy alone and in combination with bortezomib. Disclosures: No relevant conflicts of interest to declare.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

scholarly journals DIAPH1 Is Upregulated and Inhibits Cell Apoptosis through ATR/p53/Caspase-3 Signaling Pathway in Laryngeal Squamous Cell Carcinoma

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Jiechao Yang ◽  
Liang Zhou ◽  
Yanping Zhang ◽  
Juan Zheng ◽  
Jian Zhou ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Laryngeal Squamous Cell Carcinoma ◽  
Poor Overall Survival

Cancer bioinformatics has been used to screen possible key cancer genes and pathways. Here, through bioinformatics analysis, we found that high expression of diaphanous related formin 1 (DIAPH1) was associated with poor overall survival in head and neck squamous cell carcinoma and laryngeal squamous cell carcinoma (LSCC). The effect of DIAPH1 in LSCC has not been previously investigated. Therefore, we evaluated the expression, function, and molecular mechanisms of DIAPH1 in LSCC. Immunohistochemistry and western blot analysis confirmed the significant upregulation of DIAPH1 in LSCC. We used DIAPH1 RNA interference to construct two DIAPH1-knockdown LSCC cell lines, AMC-HN-8 and FD-LSC-1, and validated the knockdown efficiency. Flow cytometry data showed that DIAPH1 inhibited apoptosis. Further, western blot analysis revealed that DIAPH1 knockdown increased the protein levels of ATR, p-p53, Bax, and cleaved caspase-3, -8, and -9. Thus, DIAPH1 is upregulated in LSCC and may act as an oncogene by inhibiting apoptosis through the ATR/p53/caspase-3 pathway in LSCC cells.

Abstract 255: Cytoprotective Effects of Erythropoietin and Erythropoietin-derivatives in Ischaemic Human Myotubes and the Role of Pi3k/akt Signalling Pathway

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Rebekah Sian Hwee Yu ◽  
Daryll Baker ◽  
David Abraham ◽  
Janice Tsui
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Signalling Pathway ◽  
Critical Limb Ischaemia ◽  
Human Myotubes ◽  
Muscle Biopsies

Objectives Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously demonstrated the presence and interaction of the EpoR and βcR in human skeletal muscle. Here we aim to investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish a potential downstream signalling pathway utilised in protecting cells from apoptosis (including Jak-2, PI3k/Akt, NFkB). Methods Gastrocnemius muscle biopsies were obtained from patients with critical limb ischaemia and control samples were obtained from non-ischaemic patients. Human myoblasts were isolated from muscle biopsies, cultured, and allowed to differentiate into myotubes in order to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitors, LY294002 and wortmannin, were then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Following this, inhibitors against the upstreatm (Jak-2) and downstream (NFkB) molecules were also investigated. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis. Results Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of all inhibitors, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Conclusion The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.

scholarly journals Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

Combination of Farnesyl Transferase Inhibitor (Tipifarnib) with Perifosine Induces Apoptosis through phos-PDK1 in Human Lymphoma and Leukemia Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1488-1488 ◽  
Author(s):  
Ebenezer David ◽  
Rajni Sinha ◽  
Claire Torre ◽  
Jonathan L. Kaufman ◽  
Sagar Lonial
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Targeted Agents ◽  
Leukemia Cell Lines ◽  
Targeted Agent ◽  
The Impact

Abstract Introduction: Novel agents as anti-cancer therapy are used in the setting of specific molecular abnormalities that provide a survival advantage for malignant cells. One such agent, tipifarnib, is theoretically targeted at Ras mutations which are present in a number of different human cancers. Our previous experience with the FTIs (David et al, in press Blood) has demonstrated that they are ideal agents to combine with other targeted agents. We have investigated the combination of the AKT inhibitor perifosine with tipifarnib in human leukemia and lymphoma cell lines with the hypothesis that the combination of 2 targeted agents will disrupt separate survival pathways and ultimately result in synergistic tumor cell death. Methods: In this study we used the human leukemia cell lines HL-60, Jurkat, and the lymphoma cell line HT. Western blot analysis was used to assess for the effect of either single agent perifosine, tipifarnib, or the combination on AKT, p-AKT, PDK-1, and caspase cleavage. Flow cytometry was utilized to assess for Annexin V staining following combination therapy. Results:Dose escalation studies demonstrated that doses of tipifarnib up to 5μm demonstrated a significant cell death in HL-60 and HT cells. Perifosine doses of 1–5uM also induced cell death in both HL-60 and HT cells. When apoptosis was assessed using western blot analysis of caspase 3 activity and cleavage, the combination of perifosine and tipifarnib demonstrated significant apoptosis using low doses of both agents. The apoptosis was associated with downregulation of phos-PDK1, with a resultant downregulation in p-AKT. The level of phos-PDK1 was completely inhibited in less than 24 hrs in both the HL-60 and HT cell lines in combination than when either agent was given alone. Conclusion: The combination of perifosine, and AKT targeted agent, with tipifarnib, a Ras targeted agent, appear to induce significant cell death in lymphoma and leukemia cell lines with rapid downregulation of p-AKT via the PDK-1 pathway. This apoptosis occurs in vitro using concentrations well below those that have been achieved in current clinical trials using these agents. Additional studies are being carried out to further delineate the mechanism of synergy as well as to further explore the impact of sequence of administration using this combination. Further studies are also planned to xplore the impact of the combination on primary human leukemia and lymphoma cells from the blood and bone marrow.

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