Bcl-2 Antagonist Obatoclax Reactivates Latent HIV-1 via the NF-κB Pathway and Induces Latent Reservoir Cell Apoptosis in Latently Infected Cells.

The implementation of combined antiretroviral therapy (cART) has rendered HIV-1 infection clinically manageable and efficiently improves the quality of life for patients with AIDS. However, the persistence of a latent HIV-1 reservoir is a major obstacle to achieving a cure for AIDS. A "shock and kill" strategy aims to reactivate latent HIV and then kill it by the immune system or cART drugs. To date, none of the LRA candidates has yet demonstrated effectiveness in achieving a promising functional cure. Interestingly, the phosphorylation and activation of antiapoptotic Bcl-2 protein induce resistance to apoptosis during HIV-1 infection and the reactivation of HIV-1 latency in central memory CD4+ T cells from HIV-1-positive patients. Therefore, a Bcl-2 antagonist might be an effective LRA candidate for HIV-1 cure. In this study, we reported that a pan-Bcl-2 antagonist obatoclax induces HIV-1 reactivation in latently infected cell lines in vitro and in PBMCs/CD4+ T cells of HIV-infected individuals ex vivo. Obatoclax promotes HIV-1 transcriptional initiation and elongation by regulating the NF-κB pathway. Obatoclax activates caspase 8 and does not induce the phosphorylation of the antiapoptotic protein Bcl-2 in latent HIV-1 infected cell lines. More importantly, it preferentially induces apoptosis in latently infected cells. In addition, obatoclax exhibited potent anti-HIV-1 activity on target cells. The abilities to reactivate latent HIV-1 reservoirs, inhibit HIV-1 infection, and induce HIV-1 latent cell apoptosis make obatoclax worth investigating for development as an ideal LRA for use in the "shock and kill" approach.

Obatoclax inhibits SARS-CoV-2 entry by altered endosomal acidification and impaired cathepsin and furin activity in vitro.

Coronavirus disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has set off a global pandemic. There is an urgent unmet need for safe, affordable, and effective therapeutics against COVID-19. In this regard, drug repurposing is considered as a promising approach. We assessed the compounds that affect the endosomal acidic environment by applying human angiotensin-converting enzyme 2 (hACE2)- expressing cells infected with a SARS-CoV-2 spike (S) protein-pseudotyped HIV reporter virus and identified that obatoclax resulted in the strongest inhibition of S protein-mediated virus entry. The potent antiviral activity of obatoclax at nanomolar concentrations was confirmed in different human lung and intestinal cells infected with the SARS-CoV-2 pseudotype system as well as clinical virus isolates. Furthermore, we uncovered that obatoclax executes a double-strike against SARS-CoV-2. It prevented SARS-CoV-2 entry by blocking endocytosis of virions through diminished endosomal acidification and the corresponding inhibition of the enzymatic activity of the endosomal cysteine protease cathepsin L. Additionally, obatoclax impaired the SARS-CoV-2 S-mediated membrane fusion by targeting the MCL-1 protein and reducing furin protease activity. In accordance with these overarching mechanisms, obatoclax blocked the virus entry mediated by different S proteins derived from several SARS-CoV-2 variants of concern such as, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Taken together, our results identified obatoclax as a novel effective antiviral compound that keeps SARS-CoV-2 at bay by blocking both endocytosis and membrane fusion. Our data suggested that obatoclax should be further explored as a clinical drug for the treatment of COVID-19.

Antimetastatic Properties of Prodigiosin and the BH3-Mimetic Obatoclax (GX15-070) in Melanoma.

Metastasis is the primary cause of death in cancer patients. Many current chemotherapeutic agents only show cytotoxic, but not antimetastatic properties. This leads to a reduction in tumor size, but allows cancer cells to disseminate, which ultimately causes patient death. Therefore, novel anticancer compounds with both effects need to be developed. In this work, we analyze the antimetastatic properties of prodigiosin and obatoclax (GX15-070), anticancer drugs of the Prodiginines (PGs) family. We studied PGs' effects on cellular adhesion and morphology in the human primary and metastatic melanoma cell lines, SK-MEL-28 and SK-MEL-5, and in the murine melanoma cell line, B16F10A. Cell adhesion sharply decreased in the treated cells, and this was accompanied by a reduction in filopodia protrusions and a significant decrease in the number of focal-adhesion structures. Moreover, cell migration was assessed through the wound-healing assay and cell motility was severely inhibited after 24 h of treatment. To elucidate the molecular mechanisms involved, changes in metastasis-related genes were analyzed through a gene-expression array. Key genes related to cellular invasion, migration and chemoresistance were significantly down-regulated. Finally, an in vivo model of melanoma-induced lung metastasis was established and significant differences in lung tumors were observed in the obatoclax-treated mice. Altogether, these results describe, in depth, PGs' cellular antimetastatic effects and identify in vivo antimetastatic properties of Obatoclax.

Tambjamines and Prodiginines: Biocidal Activity against Trypanosoma cruzi.

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 Trypanosoma cruzi 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 IC50 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 IC50), 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.

Obatoclax, the pan-Bcl-2 inhibitor sensitizes hepatocellular carcinoma cells to promote the anti-tumor efficacy in combination with immune checkpoint blockade.

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.

Combinations of proteasome inhibitors with obatoclax are effective for small cell lung cancer.

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.

[Effects of obatoclax combined with gemcitabine on breast cancer cells under hypoxia condition].

Objective: 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.Methods: 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% CO2 for 24 h and 48 h; Hypoxia group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2 for 24 h and 48 h; GEM group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2, adding 10 µmol/L GEM for 24 h and 48 h; OBX + GEM group: Cells were cultured at 37℃, 1% O2, 5% CO2, 94% N2, 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. Results: 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. Conclusion: 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.

Biomimetic nanoparticle loading obatoclax mesylate for the treatment of non-small-cell lung cancer (NSCLC) through suppressing Bcl-2 signaling.

Lung cancer still remains a leading cause of cancer mortality in the world. Obatoclax mesylate (OM), a B cell chronic lymphocytic leukemia/lymphoma 2 (Bcl-2) family antagonist, is a potential antitumor drug. However, its poor aqueous solubility restricts its clinical application. Although these inherent defects, nanotechnology can be used to improve the solubility and tumor target of OM, promoting its antitumor efficiency. In the present study, the poly(lactic-coglycolic acid) (PLGA) was used and combined with red blood-cell membrane (RBCm) to explore if OM-loaded RBCm nanoparticles could improve the antitumor efficacy of OM for the treatment of lung cancer with relatively lower side effects compared with the free OM. The good physicochemical stability of the prepared RBCm-OM/PLGA nanoparticles was confirmed, and the optimal size of 153 nm was screened out, along with sustained drug release behavior. We found that RBCm-OM/PLGA nanoparticles effectively reduced the proliferation of lung cancer cells. Additionally, RBCm-OM/PLGA nanoparticles considerably induced apoptosis in lung cancer cells by reducing Bcl-2 expression levels, accompanied with the improved Cyto-c releases in cytoplasm and Caspase-3 activation. Mitochondrial membrane potential was also obviously impaired in lung cancer cells incubated with RBCm-OM/PLGA nanoparticles. Compared with free OM, RBCm-OM/PLGA nanoparticles could greatly prolong the drug circulation time in vivo and upgraded the drug concentration accumulated in tumor tissue. Furthermore, RBCm-OM/PLGA nanoparticles exerted stronger antitumor efficacy in vivo against lung cancer progression with superior safety. Therefore, RBCm-OM/PLGA nanoparticles provided new potential for lung cancer therapy with the improved safety and therapeutic effect.

The pan-Bcl-2 inhibitor obatoclax promotes differentiation and apoptosis of acute myeloid leukemia cells.

One of the key features of acute myeloid leukemia (AML) is the arrest of differentiation at the early progenitor stage of myelopoiesis. Therefore, the identification of new agents that could overcome this differentiation block and force leukemic cells to enter the apoptotic pathway is essential for the development of new treatment strategies in AML. Regarding this, herein we report the pro-differentiation activity of the pan-Bcl-2 inhibitor, obatoclax. Obatoclax promoted differentiation of human AML HL-60 cells and triggered their apoptosis in a dose- and time-dependent manner. Importantly, obatoclax-induced apoptosis was associated with leukemic cell differentiation. Moreover, decreased expression of Bcl-2 protein was observed in obatoclax-treated HL-60 cells. Furthermore, differentiation of these cells was accompanied by the loss of their proliferative capacity, as shown by G0/G1 cell cycle arrest. Taken together, these findings indicate that the anti-AML effects of obatoclax involve not only the induction of apoptosis but also differentiation of leukemic cells. Therefore, obatoclax represents a promising treatment for AML that warrants further exploration.

Obatoclax, a Pan-BCL-2 Inhibitor, Downregulates Survivin to Induce Apoptosis in Human Colorectal Carcinoma Cells Via Suppressing WNT/ß-catenin Signaling.

Colorectal cancer (CRC) is a highly prevailing cancer and the fourth leading cause of cancer mortality worldwide. Aberrant expression of antiapoptotic BCL-2 family proteins is closely linked to neoplastic progression and chemoresistance. Obatoclax is a clinically developed drug, which binds antiapoptotic BCL-2, BCL-xL, and MCL-1 for inhibition to elicit apoptosis. Survivin is an antiapoptotic protein, whose upregulation correlates with pathogenesis, therapeutic resistance, and poor prognosis in CRC. Herein, we provide the first evidence delineating the functional linkage between Obatoclax and survivin in the context of human CRC cells. In detail, Obatoclax was found to markedly downregulate survivin. This downregulation was mainly achieved via transcriptional repression, as Obatoclax lowered the levels of both survivin mRNA and promoter activity, while blocking proteasomal degradation failed to prevent survivin from downregulation by Obatoclax. Notably, ectopic survivin expression curtailed Obatoclax-induced apoptosis and cytotoxicity, confirming an essential role of survivin downregulation in Obatoclax-elicited anti-CRC effect. Moreover, Obatoclax was found to repress hyperactive WNT/ß-catenin signaling activity commonly present in human CRC cells, and, markedly, ectopic expression of dominant-active ß-catenin mutant rescued the levels of survivin along with elevated cell viability. We further revealed that, depending on the cell context, Obatoclax suppresses WNT/ß-catenin signaling in HCT 116 cells likely via inducing ß-catenin destabilization, or by downregulating LEF1 in DLD-1 cells. Collectively, we for the first time define survivin downregulation as a novel, pro-apoptotic mechanism of Obatoclax as a consequence of Obatocalx acting as an antagonist to WNT/ß-catenin signaling.

Dual-stimuli responsive nanotheranostics for mild hyperthermia enhanced inhibition of Wnt/ß-catenin signaling.

Wnt/ß-catenin signaling cascade is highly associated with tumorigenesis and progression of various cancers. Targeting Wnt/ß-catenin signaling exhibits a promising way for cancer treatment. Herein, dual-stimuli responsive nanotheranostics was synthesized, which was composed of melanin coated magnetic nanoparticles (MMNs) and Wnt signaling inhibitor obatoclax (OBX) for multimodality imaging guided mild hyperthermia-enhanced chemotherapy. The MMNs could be used as contrast agents for magnetic resonance imaging (MRI) and photoacoustic imaging (PAI) guided photothermal therapy. In addition, OBX-loaded MMNs (OBX-MMNs) were specific response to both pH changes and near-infrared (NIR) light illumination, which could trigger OBX release. Most intriguingly, tumor tissue accumulation and cellular internalization of this nanotheranostics could be dramatically enhanced through mild hyperthermia generated by laser-irradiated MMNs. Laser irradiation exhibited efficient chemotherapeutic outcome through enhancing OBX-mediated inhibition of the Wnt/ß-catenin signaling. Our results indicated the as-prepared OBX-MMNs hold great potential for MR/PA dual-modal imaging guided mild hyperthermia-enhanced chemotherapy.

[Bortezomib and obatoclax for dual blockade of protein degradation pathways show 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.

Targeting sphingosine kinase 2 suppresses cell growth and synergizes with BCL2/BCL-XL inhibitors through NOXA-mediated MCL1 degradation in cholangiocarcinoma.

Sphingosine kinase 2 (SPHK2) is a key factor within sphingolipid metabolism, responsible for the conversion of pro-apoptotic sphingosine to the pro-survival sphingosine-1-phosphate. We have previously shown that ABC294640, a first-in-class SPHK2 inhibitor, inhibits growth of cholangiocarcinoma cells. In a Phase I study of ABC294640 in tumors, the best response was achieved in a cholangiocarcinoma patient. These data suggest SPHK2 as a novel therapeutic target of cholangiocarcinoma. However, the antitumor mechanism of ABC294640 in cholangiocarcinoma remains not clear. In the current study, we found that ABC294640 upregulated expression of pro-apoptotic NOXA. In cholangiocarcinoma patients, high NOXA mRNA expression was associated with better overall survival. Also, SPHK2 mRNA expression was negatively correlated with NOXA mRNA expression. NOXA is known to degrade MCL1, an anti-apoptotic BCL2 protein. We showed that ABC294640 directed MCL1 for proteasome degradation. Knockdown of NOXA prevented ABC294640-induced MCL1 degradation and apoptosis. In addition, ABC294640 had a synergistic effect with BCL2/BCL-XL inhibitors ABT-263 and Obatoclax in inhibiting cell growth. Combined treatment with ABC294640 and BCL2/BCL-XL inhibitors induced potent apoptosis. Silencing of MCL1 also potentiated ABT-263-induced cytotoxicity. Furthermore, we found that both SPHK2 and MCL1 protein expression were significantly higher in cholangiocarcinoma than that in nontumoral bile ducts. SPHK2 expression correlated significantly with MCL1 expression. Our study reveals that ABC294640 inhibits cholangiocarcinoma cell growth and sensitizes the antitumor effect of BCL2/BCL-XL inhibitors through NOXA-mediated MCL1 degradation. Combinations of ABC294640 with BCL2/BCL-XL inhibitors may provide novel strategies for the treatment of cholangiocarcinoma.

Obatoclax, a BH3 Mimetic, Enhances Cisplatin-Induced Apoptosis and Decreases the Clonogenicity of Muscle Invasive Bladder Cancer Cells via Mechanisms That Involve the Inhibition of Pro-Survival Molecules as Well as Cell Cycle Regulators.

Several studies by our group and others have determined that expression levels of Bcl-2 and/or Bcl-xL, pro-survival molecules which are associated with chemoresistance, are elevated in patients with muscle invasive bladder cancer (MI-BC). The goal of this study was to determine whether combining Obatoclax, a BH3 mimetic which inhibits pro-survival Bcl-2 family members, can improve responses to cisplatin chemotherapy, the standard of care treatment for MI-BC. Three MI-BC cell lines (T24, TCCSuP, 5637) were treated with Obatoclax alone or in combination with cisplatin and/or pre-miR-34a, a molecule which we have previously shown to inhibit MI-BC cell proliferation via decreasing Cdk6 expression. Proliferation, clonogenic, and apoptosis assays confirmed that Obatoclax can decrease cell proliferation and promote apoptosis in a dose-dependent manner. Combination treatment experiments identified Obatoclax + cisplatin as the most effective treatment. Immunoprecipitation and Western analyses indicate that, in addition to being able to inhibit Bcl-2 and Bcl-xL, Obatoclax can also decrease cyclin D1 and Cdk4/6 expression levels. This has not previously been reported. The combined data demonstrate that Obatoclax can inhibit cell proliferation, promote apoptosis, and significantly enhance the effectiveness of cisplatin in MI-BC cells via mechanisms that likely involve the inhibition of both pro-survival molecules and cell cycle regulators.

Obatoclax and Paclitaxel Synergistically Induce Apoptosis and Overcome Paclitaxel Resistance in Urothelial Cancer Cells.

Paclitaxel is a treatment option for advanced or metastatic bladder cancer after the failure of first-line cisplatin and gemcitabine, although resistance limits its clinical benefits. Mcl-1 is an anti-apoptotic protein that promotes resistance to paclitaxel in different tumors. Obatoclax, a BH3 mimetic of the Bcl-2 family of proteins, antagonizes Mcl-1 and hence may reverse paclitaxel resistance in Mcl-1-overexpressing tumors. In this study, paclitaxel-sensitive 5637 and -resistant HT1197 bladder cancer cells were treated with paclitaxel, obatoclax, or combinations of both. Apoptosis, cell cycle, and autophagy were measured by Western blot, flow cytometry, and fluorescence microscopy. Moreover, Mcl-1 expression was analyzed by immunohistochemistry in bladder carcinoma tissues. Our results confirmed that paclitaxel alone induced Mcl-1 downregulation and apoptosis in 5637, but not in HT1197 cells; however, combinations of obatoclax and paclitaxel sensitized HT1197 cells to the treatment. In obatoclax-treated 5637 and obatoclax + paclitaxel-treated HT1197 cells, the blockade of the autophagic flux correlated with apoptosis and was associated with caspase-dependent cleavage of beclin-1. Obatoclax alone delayed the cell cycle in 5637, but not in HT1197 cells, whereas combinations of both retarded the cell cycle and reduced mitotic slippage. In conclusion, obatoclax sensitizes HT1197 cells to paclitaxel-induced apoptosis through the blockade of the autophagic flux and effects on the cell cycle. Furthermore, Mcl-1 is overexpressed in many invasive bladder carcinomas, and it is related to tumor progression, so Mcl-1 expression may be of predictive value in bladder cancer.

Decoding the antineoplastic efficacy of Aplysin targeting Bcl-2: A de novo perspective.

The B-cell lymphoma-2 (Bcl-2) family proteins have been attributed to be the key regulators in programmed cell death and apoptosis with a prominent role in human cancer. Understanding the fundamental principles of cell survival and death have been the main cornerstone in cancer drug discovery for identification of novel anticancer agents. In this context the Bcl-2 family of anti-and pro-apoptotic proteins provide an excellent opportunity for development of anticancer agents, as blocking the Bcl-2 or Bcl-XL functionally promotes apoptosis in tumor cells and also sensitize them to chemo- and radiotherapies. The present study reports the identification of novel Aplysin analogs as BCL-2 inhibitors from a sequential virtual screening approach using drug-like, ADMET, docking, pharmacophore filters and molecular dynamics simulation. We identified promising Aplysin analogs that have a potential to be Bcl-2 inhibitors just like the standard drug Obatoclax. One of the compound analog 11 was identified to be a promising inhibitor of Bcl-2 in the docking, pharmacophore and simulation based models.The molecular modeling information provided here can be vital in designing of the novel Bcl-2 inhibitors.

Ionophores: Potential Use as Anticancer Drugs and Chemosensitizers.

Ion homeostasis is extremely important for the survival of both normal as well as neoplastic cells. The altered ion homeostasis found in cancer cells prompted the investigation of several ionophores as potential anticancer agents. Few ionophores, such as Salinomycin, Nigericin and Obatoclax, have demonstrated potent anticancer activities against cancer stem-like cells that are considered highly resistant to chemotherapy and responsible for tumor relapse. The preclinical success of these compounds in in vitro and in vivo models have not been translated into clinical trials. At present, phase I/II clinical trials demonstrated limited benefit of Obatoclax alone or in combination with other anticancer drugs. However, future development in targeted drug delivery may be useful to improve the efficacy of these compounds. Alternatively, these compounds may be used as leading molecules for the development of less toxic derivatives.

BH3 mimetic Obatoclax (GX15-070) mediates mitochondrial stress predominantly via MCL-1 inhibition and induces autophagy-dependent necroptosis in human oral cancer cells.

We have previously reported overexpression of antiapoptotic MCL-1 protein in human oral cancers and its association with therapy resistance and poor prognosis, implying it to be a potential therapeutic target. Hence, we investigated the efficacy and mechanism of action of Obatoclax, a BH3 mimetic pan BCL-2 inhibitor in human oral cancer cell lines. All cell lines exhibited high sensitivity to Obatoclax with complete clonogenic inhibition at 200-400 nM concentration which correlated with their MCL-1 expression. Mechanistic insights revealed that Obatoclax induced a caspase-independent cell death primarily by induction of a defective autophagy. Suppression of autophagy by ATG5 downregulation significantly blocked Obatoclax-induced cell death. Further, Obatoclax induced interaction of p62 with key components of the necrosome RIP1K and RIP3K. Necrostatin-1 mediated inhibition of RIP1K significantly protected the cells from Obatoclax induced cell death. Moreover, Obatoclax caused extensive mitochondrial stress leading to their dysfunction. Interestingly, MCL-1 downregulation alone caused mitochondrial stress, highlighting its importance for mitochondrial homeostasis. We also demonstrated in vivo efficacy of Obatoclax against oral cancer xenografts and its synergism with ionizing radiation in vitro. Our studies thus suggest that Obatoclax induces autophagy-dependent necroptosis in oral cancer cells and holds a great promise in the improved management of oral cancer patients.

Breast cancer cell obatoclax response characterization using passivated-electrode insulator-based dielectrophoresis.

Inherent electrical properties of cells can be beneficial to characterize different cell lines and their response to experimental drugs. This paper presents a novel method to characterize the response of breast cancer cells to drug stimuli through use of off-chip passivated-electrode insulator-based dielectrophoresis (OπDEP) and the application of AC electric fields. This work is the first to demonstrate the ability of OπDEP to differentiate between two closely related breast cancer cell lines, LCC1 and LCC9 while assessing their drug sensitivity to an experimental anti-cancer agent, Obatoclax. Although both cell lines are derivatives of estrogen-responsive MCF-7 breast cancer cells, growth of LCC1 is estrogen independent and anti-estrogen responsive, while LCC9 is both estrogen-independent and anti-estrogen resistant. Under the same operating conditions, LCC1 and LCC9 had different DEP profiles. LCC1 cells had a trapping onset (crossover) frequency of 700 kHz and trapping efficiencies between 30-40%, while LCC9 cells had a lower crossover frequency (100 kHz) and showed higher trapping efficiencies of 40-60%. When exposed to the Obatoclax, both cell lines exhibited dose-dependent shifts in DEP crossover frequency and trapping efficiency. Here, DEP results supplemented with cell morphology and proliferation assays help us to understand the response of these breast cancer cells to Obatoclax.

Obatoclax and LY3009120 Efficiently Overcome Vemurafenib Resistance in Differentiated Thyroid Cancer.

Although the prognosis of differentiated thyroid cancer (DTC) is relatively good, 30-40% of patients with distant metastases develop resistance to radioactive iodine therapy due to tumor dedifferentiation. For DTC patients harboring BRAFV600E mutation, Vemurafenib, a BRAF kinase inhibitor, has dramatically changed the therapeutic landscape, but side effects and drug resistance often lead to termination of the single agent treatment. In the present study, we showed that either LY3009120 or Obatoclax (GX15-070) efficiently inhibited cell cycle progression and induced massive death of DTC cells. We established that BRAF/CRAF dimerization was an underlying mechanism for Vemurafenib resistance. LY3009120, the newly discovered pan-RAF inhibitor, successfully overcame Vemurafenib resistance and suppressed the growth of DTC cells in vitro and in vivo. We also observed that expression of anti-apoptotic Bcl-2 increased substantially following BRAF inhibitor treatment in Vemurafenib-resistant K1 cells, and both Obatoclax and LY3009120 efficiently induced apoptosis of these resistant cells. Specifically, Obatoclax exerted its anti-cancer activity by inducing loss of mitochondrial membrane potential (ΔΨm), dysfunction of mitochondrial respiration, reduction of cellular glycolysis, autophagy, neutralization of lysosomes, and caspase-related apoptosis. Furthermore, the cancer killing effects of LY3009120 and Obatoclax extended to two more Vemurafenib-resistant DTC cell lines, KTC-1 and BCPAP. Taken together, our results highlighted the potential value of LY3009120 for both Vemurafenib-sensitive and -resistant DTC and provided evidence for the combination therapy using Vemurafenib and Obatoclax for radioiodine-refractory DTC.