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Nan Fang Yi Ke Da Xue Xue Bao = Journal... Apr 2016To explore whether MG-132 could enhance the anti-tumor activity of obatoclax against esophageal cancer cell line CaES-17.
OBJECTIVE
To explore whether MG-132 could enhance the anti-tumor activity of obatoclax against esophageal cancer cell line CaES-17.
METHODS
MTT assay was used to determine the cytotoxicity of obatoclax and MG-132 in CaES-17 cells. The IC(50) of obatoclax and MG-132 were used to determine the molar ratio (1:2.4) of the two drugs for combined treatment of the cells. The concentrations of obatoclax and MG-132 ranged from 1/8 IC(50) to 4 IC(50) after serial dilution, and their combination index (CI) was calculated using CompuSyn software. The expression of ubiquitin and the cleavage of PARP, caspase-9, phospho-histone H3 and phospho-aurora A/B/C in the exposed cells were examined with Western blotting; the cell apoptosis was measured by flow cytometry with Annexin V staining, and the percentage of cells in each cell cycle phase was also determined by flow cytometry.
RESULTS
The CI of obatoclax and MG-132 was 0.296 for a 50% inhibition of Caes-17 cells and was 0.104 for a 95% inhibition. The cells treated with obatoclax or MG-132 alone showed increased expression of ubiquitin and cleavage of PARP and caspase-9. Compared with the cells treated with obatoclax or MG-132 alone, the cells with a combined treatment exhibited significantly increased expression of ubiquitin, cleavage of PARP and caspase-9, and expression of phospho-Histone H3 (P<0.05). The combined treatment of the cells also resulted in significantly increased expression of phospho-Aurora A/B/C compared with obatoclax treatment alone. The cells with the combined treatment showed significantly higher percentages of apoptotic cells and cells in sub-G(1) and G(2)/M phases compared with the cells treated with either of the drugs (P<0.05).
CONCLUSION
Obatoclax combined with MG-132 shows a significant synergistic anti-tumor effect against esophageal cancer CaES-17 cells by inducing apoptosis and cell cycle arrest.
Topics: Apoptosis; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Esophageal Neoplasms; Histones; Humans; Indoles; Leupeptins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Pyrroles
PubMed: 27113178
DOI: No ID Found -
Zhongguo Ying Yong Sheng Li Xue Za Zhi... May 2020To explore the effects of obatoclax(OBX) combined with gemcitabine(GEM) on breast cancer cells MCF-7 and BT-20 cell activity, migration, invasion and apoptosis under...
To explore the effects of obatoclax(OBX) combined with gemcitabine(GEM) on breast cancer cells MCF-7 and BT-20 cell activity, migration, invasion and apoptosis under hypoxia condition. Breast cancer cells MCF-7 and BT-20 were divided into normal group, hypoxia group, GEM group, OBX+GEM group. Normal group: Cells were cultured at 37℃, 5% CO for 24 h and 48 h; Hypoxia group: Cells were cultured at 37℃, 1% O, 5% CO, 94% N for 24 h and 48 h; GEM group: Cells were cultured at 37℃, 1% O, 5% CO, 94% N, adding 10 μmol/L GEM for 24 h and 48 h; OBX + GEM group: Cells were cultured at 37℃, 1% O, 5% CO, 94% N, adding 10 μmol/L GEM and 50 nmol/L OBX for 24 h and 48 h. Western blot method was used to detect the expressions of HIF-1α in MCF-7 and BT-20 cells under normal oxygen and hypoxia condition. CCK-8 method was used to detect cancer cell activity, each group was provided with 15 compound holes. Scratch experiment was used to detect cells migration ability, each group was provided with 6 compound holes. Western blot method was used to detect the expressions of vimentin, E-Cadherin and p53 protein in cells of each group. Under hypoxia condition, the expression of HIF-1α in MCF-7 and BT-20 cells was much higher than that under normal oxygen(P<0.05). Compared with hypoxia group, GEM could reduce MCF-7 and BT-20 cells migration ability(P<0.01)and cell activity(P<0.05), while decrease the expression of vimentin protein(P<0.01)and promote the expressions of E-Cadherin (P<0.01)and p53 protein(P<0.01) in tumor cells under hypoxia condition. In OBX combined with GEM group, the cell activity and the migration ability of MCF-7 and BT-20 were reduced significantly(P<0.01). The expression of vimentin in cells was further reduced(P<0.01). The expressions of E-Cadherin(P<0.01)and p53(P<0.01) protein were increased significantly compared with GEM group. Under hypoxia condition, OBX combined with a low-dose of GEM can significantly inhibit the growth, migration and invasion of breast cancer cells, and enhance the pro-apoptotic effect of GEM, but the specific mechanism needs further study.
Topics: Antineoplastic Agents; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Deoxycytidine; Gene Expression Regulation, Neoplastic; Humans; Indoles; Pyrroles; Gemcitabine
PubMed: 32981284
DOI: 10.12047/j.cjap.5955.2020.059 -
Oral Oncology Sep 2022The purpose of this review was to summarise available literature concerning the anticancer effects of both putative and validated BH3-mimetics in head and neck squamous... (Review)
Review
The purpose of this review was to summarise available literature concerning the anticancer effects of both putative and validated BH3-mimetics in head and neck squamous cell carcinomas. A literature search was performed and studies assessing malignant cell lines, xenograft models, and/or humans were considered eligible. A total of 501 studies were identified, of which 40 were included. One phase-II clinical trial assessing gossypol (combined with docetaxel) was found. The remaining 39 preclinical studies investigated cell lines and/or xenograft models involving the use of six validated BH3-mimetics (A-1210477, A-1331852, ABT-737, navitoclax, S63845, venetoclax) and six putative BH3-mimetics (ApoG2, gossypol, obatoclax, sabutoclax, TW-37, and YC137). In preclinical settings, most validated BH3-mimetics were capable of inducing apoptosis (in-vitro) and tumour growth inhibition (in-vivo). The majority of putative BH3-mimetics were also capable of inducing cell death, although important off-target effects, such as autophagy induction, were also described. Combinations with conventional anticancer drugs, ionising radiation, or multiple BH3-mimetics generally resulted in enhanced anticancer effects, such as increased sensitivity to apoptotic stimuli, especially considering some cell lines that showed resistance to either treatment alone. In conclusion, although clinical data are still insufficient to evaluate the anticancer effects of BH3-mimetics in head and neck squamous cell carcinomas, promising results in preclinical settings were observed concerning induction of cell death and inhibition of tumour growth. Therefore, further clinical trials are highly encouraged.
Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Gossypol; Head and Neck Neoplasms; Humans; Proto-Oncogene Proteins c-bcl-2; Squamous Cell Carcinoma of Head and Neck
PubMed: 35816876
DOI: 10.1016/j.oraloncology.2022.105979 -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... Apr 2019To explore whether bortezomib and a Bcl-2 inhibitor exhibit synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells.
OBJECTIVE
To explore whether bortezomib and a Bcl-2 inhibitor exhibit synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells.
METHODS
MTT assay was used to determine the cytotoxicity of bortezomib in the absence or presence of Bcl-2 inhibitors (obatoclax, AT-101 and ABT-199) in Jurkat cells. The effects of drug treatment on the expression of Bcl-2 family proteins, LC3B, p62, ubiquitin, BiP/Grp78, p-JNK, p-p38 and CHOP proteins were examined by Western blotting. Flow cytometry was used to determine the effects of bortezomib and Bcl-2 inhibitors (obatoclax, AT-101 and ABT-199) on cell apoptosis. Quantitative real-time PCR was used to measure the mRNA expression levels of the key regulatory factors of unfolded protein reaction (UPR). A zebrafish xenograft model was used to study the anti-tumor effect of bortezomib, obatoclax and their combination in vivo.
RESULTS
Bortezomib or Bcl-2 inhibitors alone inhibited the cell viability of Jurkat cells, but only obatoclax and bortezomib showed synergistic cytotoxicity and pro-apoptotic effect. Obatoclax, rather than AT-101 and ABT- 199, blocked autophagic flux in the cells evidenced by concomitant accumulation of LC3B-Ⅱ and p62. Both bortezomib and obatoclax alone caused accumulation of polyubiquinated proteins, and their combination showed a synergistic effect, which was consistent with their synergistic cytotoxicity. The dual blockade of proteasome and autophagy by the combination of bortezomib and obatoclax triggered unfolded protein response followed by cell apoptosis. Preventing UPS dysfunction by tauroursodeoxycholic acid (TUDCA) significantly attenuated the cytotoxicity and pro-apoptotic effect of bortezomib in combination with obatoclax. In zebrafish xenograft models, bortezomib combined with obatoclax significantly decreased tumor foci formation.
CONCLUSIONS
Bortezomib and obatoclax for dual blockade of protein degradation pathways show synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells.
Topics: Antineoplastic Agents; Apoptosis; Bortezomib; Cell Line, Tumor; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Humans; Indoles; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Pyrroles
PubMed: 31068282
DOI: 10.12122/j.issn.1673-4254.2019.04.04 -
Pharmaceutics May 2021The aim of this work was to explore new therapeutic options against Chagas disease by the in vitro analysis of the biocidal activities of several tambjamine and...
The aim of this work was to explore new therapeutic options against Chagas disease by the in vitro analysis of the biocidal activities of several tambjamine and prodiginine derivatives, against the CLB strain (DTU TcVI). The compounds were initially screened against epimastigotes. The five more active compounds were assayed in intracellular forms. The tambjamine MM3 and both synthetic and natural prodigiosins displayed the highest trypanocidal profiles, with IC values of 4.52, 0.46, and 0.54 µM for epimastigotes and 1.9, 0.57, and 0.1 µM for trypomastigotes/amastigotes, respectively. Moreover, the combination treatment of these molecules with benznidazole showed no synergism. Finally, oxygen consumption inhibition determinations performed using high-resolution respirometry, revealed a potent effect of prodigiosin on parasite respiration (73% of inhibition at ½ IC), suggesting that its mode of action involves the mitochondria. Moreover, its promising selectivity index (50) pointed out an interesting trypanocidal potential and highlighted the value of prodigiosin as a new candidate to fight Chagas disease.
PubMed: 34065993
DOI: 10.3390/pharmaceutics13050705 -
Scientific Reports Jun 2021Extracellular vesicles (EVs) are secreted from most cells and play important roles in cell-cell communication by transporting proteins, lipids, and nucleic acids. As the...
Extracellular vesicles (EVs) are secreted from most cells and play important roles in cell-cell communication by transporting proteins, lipids, and nucleic acids. As the involvement of EVs in diseases has become apparent, druggable regulators of EV secretion are required. However, the lack of a highly sensitive EV detection system has made the development of EV regulators difficult. We developed an ELISA system using a high-affinity phosphatidylserine-binder TIM4 to capture EVs and screened a 1567-compound library. Consequently, we identified one inhibitor and three activators of EV secretion in a variety of cells. The inhibitor, apoptosis activator 2, suppressed EV secretion via a different mechanism and had a broader cellular specificity than GW4869. Moreover, the three activators, namely cucurbitacin B, gossypol, and obatoclax, had broad cellular specificity, including HEK293T cells and human mesenchymal stem cells (hMSCs). In vitro bioactivity assays revealed that some regulators control EV secretion from glioblastoma and hMSCs, which induces angiogenesis and protects cardiomyocytes against apoptosis, respectively. In conclusion, we developed a high-throughput method to detect EVs with high sensitivity and versatility, and identified four compounds that can regulate the bioactivity of EVs.
Topics: Animals; Apoptosis; Drug Evaluation, Preclinical; Extracellular Vesicles; HCT116 Cells; HEK293 Cells; Humans; Jurkat Cells; K562 Cells; Mesenchymal Stem Cells; Mice; NIH 3T3 Cells; Small Molecule Libraries; THP-1 Cells
PubMed: 34188113
DOI: 10.1038/s41598-021-92860-2 -
Acta Pharmacologica Sinica Aug 2021Proteasome inhibitors, bortezomib (BTZ), and carfilzomib (CFZ) are approved drugs for hematological malignancies, but lack anticancer activities against most solid...
Proteasome inhibitors, bortezomib (BTZ), and carfilzomib (CFZ) are approved drugs for hematological malignancies, but lack anticancer activities against most solid tumors. Small cell lung cancer (SCLC) is a very aggressive neuroendocrine carcinoma of the lungs demanding effective therapy. In this study we investigated whether BTZ or CFZ combined with obatoclax (OBX), an antagonist for MCL-1 and a pan-BCL family inhibitor, could cause synergistic growth inhibition of SCLC cells. We showed that combined application of BTZ or CFZ with OBX caused synergistic growth inhibition of human SCLC cell lines (H82, H526, DMS79, H196, H1963, and H69) than single agent alone. Both BTZ-OBX and CFZ-OBX combinations displayed marked synergism on inducing apoptosis (~50% increase vs BTZ or CFZ alone). A comprehensive proteomics analysis revealed that BTZ preferentially induced the expression of MCL-1, an antiapoptotic protein, in SCLC cells. Thus, proteasome inhibitor-OBX combinations could specifically induce massive growth inhibition and apoptosis in SCLC cells. Subsequent proteome-wide profiling analysis of activated transcription factors suggested that BTZ- or CFZ-induced MCL-1 upregulation was transcriptionally driven by FOXM1. In nude mice bearing in SCLC H82 xenografts, both BTZ-OBX, and CFZ-OBX combinations exhibited remarkable antitumor activities against SCLC tumors evidenced by significant reduction of tumor size and the proliferation marker Ki-67 signals in tumor tissues as compared with single agent alone. Thus, proteasome inhibitor-OBX combinations are worth immediate assessments for SCLC in clinical settings.
Topics: Animals; Antineoplastic Agents; Apoptosis; Bortezomib; Cell Line, Tumor; Drug Synergism; Forkhead Box Protein M1; HEK293 Cells; Humans; Indoles; Lung Neoplasms; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Oligopeptides; Proteasome Inhibitors; Pyrroles; Small Cell Lung Carcinoma; Up-Regulation; Xenograft Model Antitumor Assays; Mice
PubMed: 33139838
DOI: 10.1038/s41401-020-00544-w -
Translational Oncology Aug 2021Bcl-2 family proteins play critical roles in regulating lymphocyte development and maintain homeostasis, and have also been proved to be involved in various cancer types...
Bcl-2 family proteins play critical roles in regulating lymphocyte development and maintain homeostasis, and have also been proved to be involved in various cancer types development. However, the role of Bcl-2 in hepatocellular carcinoma (HCC) development has not been clearly studied. Here, we reported the pan-Bcl-2 inhibitor, obatoclax could directly inhibit HCC growth in vitro. We further demonstrated in murine HCC model that obatoclax also suppressed HCC development in vivo. We also proved that although obatoclax inhibited T cells expansion, it had no influence on T cells activation in vivo. Mechanism study revealed that obatoclax sensitized HCC cells to T cell-mediated killing. Combination therapy of obatoclax with anti-PD-1 antibody synergistically suppressed HCC development and prolonged the survival rate of tumor-bearing mice. The combination therapy promoted T cells activation and effector cytokines expression both in spleen and tumor. In summary, our results proved that obatoclax sensitized HCC cells to T cell -mediated killing. Combination of obatoclax with immune checkpoint blockade served as a promising therapeutic strategy for HCC treatment.
PubMed: 33975180
DOI: 10.1016/j.tranon.2021.101116 -
International Journal of Molecular... Dec 2016Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to...
Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to tumorigenesis and poor prognosis in colorectal cancer. Obatoclax is an inhibitor targeting all antiapoptotic BCL-2 proteins. A previous study has described the antiproliferative action of obatoclax in one human colorectal cancer cell line without elucidating the underlying mechanisms. We herein reported that, in a panel of human colorectal cancer cell lines, obatoclax inhibits cell proliferation, suppresses clonogenicity, and induces G₁-phase cell cycle arrest, along with cyclin D1 downregulation. Notably, ectopic cyclin D1 overexpression abrogated clonogenicity suppression but also G₁-phase arrest elicited by obatoclax. Mechanistically, pre-treatment with the proteasome inhibitor MG-132 restored cyclin D1 levels in all obatoclax-treated cell lines. Cycloheximide chase analyses further revealed an evident reduction in the half-life of cyclin D1 protein by obatoclax, confirming that obatoclax downregulates cyclin D1 through induction of cyclin D1 proteasomal degradation. Lastly, threonine 286 phosphorylation of cyclin D1, which is essential for initiating cyclin D1 proteasomal degradation, was induced by obatoclax in one cell line but not others. Collectively, we reveal a novel anticancer mechanism of obatoclax by validating that obatoclax targets cyclin D1 for proteasomal degradation to downregulate cyclin D1 for inducing antiproliferation.
Topics: Carcinoma; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; HCT116 Cells; HT29 Cells; Humans; Indoles; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Pyrroles
PubMed: 28035994
DOI: 10.3390/ijms18010044 -
The Journal of Pharmacy and Pharmacology Nov 2015Obatoclax is a pan-Bcl-2 inhibitor with promising efficacy, especially when combined with other antineoplastic agents. Pharmacokinetic drug-drug interactions can occur...
OBJECTIVES
Obatoclax is a pan-Bcl-2 inhibitor with promising efficacy, especially when combined with other antineoplastic agents. Pharmacokinetic drug-drug interactions can occur systemically and at the level of the tumour cell. Thus, this study scrutinised the interaction potential of obatoclax in vitro.
METHODS
Obatoclax was screened for P-gp inhibition by calcein assay, for breast cancer resistance protein (BCRP) inhibition by pheophorbide A assay and for inhibition of cytochrome P450 isoenzymes (CYPs) by commercial kits. Induction of mRNA of drug-metabolising enzymes and drug transporters was quantified in LS180 cells via real-time polymerase chain reaction and involvement of nuclear receptors was assessed by reporter gene assays. Proliferation assays were used to assess whether obatoclax retains its efficacy in cell lines overexpressing BCRP, P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (MRP2).
KEY FINDINGS
Obatoclax induced the mRNA expression of several genes (e.g. CYP1A1, CYP1A2 and ABCG2 (five to seven-fold) through activation of the aryl hydrocarbon receptor in the nanomolar range. Obatoclax inhibits P-gp, BCRP and some CYPs at concentrations exceeding plasma levels. P-gp, MPR2 or BCRP overexpression did not influence the efficacy of obatoclax.
CONCLUSIONS
Obatoclax retains its efficacy in cells overexpressing P-gp, MRP2 or BCRP and might act as a perpetrator drug in interactions with drugs, for example being substrates of CYP1A2 or BCRP.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Animals; Cell Line; Cell Line, Tumor; Cytochrome P-450 Enzyme System; Drug Interactions; Drug Resistance, Multiple; Gene Expression Regulation; Humans; Indoles; LLC-PK1 Cells; Mice; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Pyrroles; RNA, Messenger; Real-Time Polymerase Chain Reaction; Swine
PubMed: 26255619
DOI: 10.1111/jphp.12455