Info were presented as the percentage relative to the drug-untreated control. 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. Keywords: obatoclax, BH3 (BCL-2 homology 3) mimetics, cyclin D1, proteasomal degradation, G1-phase arrest, antiproliferation, colorectal cancer == 1 . Introduction == Colorectal cancer was the third most commonly diagnosed cancer and the fourth leading cause of cancer-related death globally in 2012 [1]. Accumulation of inactivating mutations in tumor suppressor genes such asadenomatous polyposis coli(APC), SMAD4, orTP53and oncogenic mutations inKRAS, PI3KCA, orBRAFare critical for the development and progression of colorectal cancer. In particular, loss of APC and consequent increase in -catenin facilitates the formation of adenoma, thereby initiating the adenoma-carcinoma sequence for colorectal tumorigenesis [2]. Colorectal cancer is often first diagnosed at an advanced stage [3]. Surgical resection followed by systemic chemotherapy is recommended for the treatment of patients with advanced colorectal cancer; however , clinical relapse frequently occurs and accounts for most colorectal cancer-related mortality [4]. Identification of novel chemotherapeutics with better efficacy for colorectal cancer therapy is therefore in urgent demand. Apoptosis constitutes a fundamental intrinsic mechanism of tumor suppression, as the resistance of apoptosis is a well-established hallmark of cancer [5]. Apoptosis is predominantly regulated by members of the BCL-2 (B-cell lymphoma 2) family [6]. The antiapoptotic members, including BCL-2, BCL-xL (B-cell lymphoma-extra large), and MCL-1 (Myeloid cell leukemia 1), suppress apoptosis by binding to proapoptotic BAK (BCL-2 homologous antagonist/killer) and BAX (BCL-2-associated X protein) to prevent their activation for initiating the mitochondrial apoptotic signaling. Furthermore, proapoptotic BH3-only proteins function either as BAX/BAK activators through direct binding to BAX/BAK, or act as sensitizers of proapoptotic stimuli by fitting into the BH3-binding groove of antiapoptotic members to release the activator BH3-only proteins for BAX/BAK activation [7]. As Atosiban Acetate to colorectal cancer, it has been proven that aberrant overexpression of antiapoptotic BCL-2 family proteins is closely linked to colorectal tumorigenesis, poor prognosis, and drug resistance, thus highlighting the potential of drugs targeting antiapoptotic BCL-2 proteins for colorectal cancer therapy [8, 9, 10]. To this end, a novel class of cancer therapeutics coined as BH3 mimetics, including ABT-737, ABT-263, and obatoclax, were developed to function as inhibitors of antiapoptotic BCL-2 members by mimicking the modes of action of BH3-only proteins [11, 12, 13, 14, 15]. These BH3 mimetics are potent inducers of apoptosis in vitro and have been under intensive clinical trials [11, 15]. Obatoclax is classified as a BH3 mimetic by its ability to bind to the BH3-bidning groove of BCL-2, BCL-xL, and ALK2-IN-2 MCL-1, leading to inhibition of these antiapoptotic BCL-2 proteins and consequent BAX/BAK-dependent apoptosis [11, 16, 17]. Obatoclax as a single agent induces apoptosis in ALK2-IN-2 cells derived from hematological cancers and solid tumors, but also potentiates the cytotoxicity of conventional chemotherapeutics or targeted therapy drugs [17, 18, 19]. Of note, obatoclax is unique by its inhibitory action on MCL-1 compared to ABT-737 and its orally available derivative ABT-263 (navitoclax), which bind to BCL-2 and BCL-xL, but not MCL-1, thus allowing obatoclax to conquer MCL-1-mediated ABT-737 resistance [20]. Phase III clinical trials of obatoclax in combination therapies are currently ongoing [11, 15]. Intriguingly, evidence that obatoclax can also evoke BAX- and BAK-independent cell death [17] highlights additional mechanisms of action that account for the anticancer effect of obatoclax. Indeed, obatoclax has been documented to induce autophagic cell death [21], likely due to ALK2-IN-2 blocking the end stage of autophagy through inhibition of lysosomal activity [22, 23]. Furthermore, the obatoclax-mediated death of rhabdomyosarcoma.