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

Abstract

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.

Novel nucleolar localization of clusterin and its associated functions in human oral cancers: An in vitro and in silico analysis

“…Immunofluorescence assay was carried out as described previously 17 . For nuclease treatment related experiments, cells were fixed and permeabilized with methanol for 5 min at −20°C and then incubated with 10 μg/mL DNase I or 100 μg/mL RNase A for 30 min at 37°C.…”

“…RNA extraction, cDNA synthesis and quantitative real‐time PCR (qRT‐PCR) were carried out as described previously 17 . The primers used for CLU and rRNA transcript detection are listed in Table 3 in Data S1.…”

Targeting Autophagy for Cancer Treatment and Tumor Chemosensitization

“…Its orally available enantiomeric form (-)-gossypol (AT-101) has shown to induce ACD in malignant glioma [56], but the induced autophagy has also been accompanied by apoptosis in head and neck squamous cell carcinoma [53], malignant mesothelioma [54], and colon cancer cells [55]. Obatoclax (GX15-070) is another BH3 mimetic that has shown autophagic-mediated necroptosis in oral squamous cell carcinoma [57], rhabdomyosarcoma cells [58], and acute lymphoblastic leukemia cells [59]. Moreover, obatoclax induced autophagy in adenoid cystic carcinoma [87] and Beclin-1 independent autophagy inhibition in colorectal cancer cells [88].…”

RIPK1 and RIPK3 - emerging targets in cancer?

“…These include, for example, disruption of the activity of the members of HOX family of transcription factors in AML (97); inhibition of Aurora kinase A in pancreatic carcinoma cells (98); PPARγ-induced Annexin A1 expression in triple negative breast cancer cells, promoting cIAP1-degradation and, thus, activation of RIPK1-dependent cell death (99); activation of MLKL by ceramide liposomes in ovarian carcinoma cells (100); induction of ROS-dependent TNFα synthesis and necroptosis by selenium nanoparticles in PC-3 human prostate cancer cells (101); and activation of mitochondrial stress and autophagy-dependent necroptosis by BH3 mimetic drug Obatoclax (GX15-070) in human oral cancer cells (102). These and other recent findings indicate that exciting new strategies to therapeutically activate RIPK1/ RIPK3-dependent pathways in human cancers may be possible in the near future, but further effort to translate these initial findings into clinically relevant approaches is required.…”