Verteporfin disrupts multiple steps of autophagy and regulates p53 to sensitize osteosarcoma cells

Abstract

Background Osteosarcoma (OS) is a malignant tumor of the bone mostly observed in children and adolescents. The current treatment approach includes neoadjuvant and adjuvant chemotherapy; however, drug resistance often hinders therapy in OS patients. Also, the post-relapse survival of OS patients is as low as 20%. We therefore planned to understand the molecular cause for its poor prognosis and design an appropriate therapeutic strategy to combat the disease. Methods We analyzed OS patient dataset from Gene Expression Omnibus (GEO) and identified the differentially expressed genes and the top deregulated pathways in OS. Subsequently, drugs targeting the major de-regulated pathways were selected and the following assays were conducted- MTT assay to assess cytotoxicity of drugs in OS cells; immunoblotting and immunostaining to analyze key protein expression and localization after drug treatment; LysoTracker staining to monitor lysosomes; Acridine Orange to label acidic vesicles; and DCFDA to measure Reactive Oxygen Species (ROS). Results The differential gene expression analysis from OS patient dataset implicated the striking involvement of cellular processes linked to autophagy and protein processing in the development of OS. We therefore selected the FDA approved drugs, chloroquine (CQ) and verteporfin (VP) known for autophagy inhibitory and proteotoxic functions to explore against OS. Importantly, VP, but not CQ, showed an extensive dose-dependent cytotoxicity. It resulted in autophagy disruption at multiple steps extending from perturbation of early autophagic processes, inhibition of autophagic flux to induction of lysosomal instability. Interestingly, VP treated protein lysates showed a ROS-dependent high molecular weight (HMW) band when probed for P62 and P53 protein. Further, VP triggered accumulation of ubiquitinated proteins as well. Since VP had a pronounced disruptive effect on cellular protein homeostasis, we explored the possibility of simultaneous inhibition of the ubiquitin-proteasomal system (UPS) by MG-132 (MG). Addition of a proteasomal inhibitor significantly aggravated VP induced cytotoxicity. MG co-treatment also led to selective targeting of P53 to the lysosomes. Conclusion Herein, we propose VP and MG induce regulation of autophagy and protein homeostasis which can be exploited as an effective therapeutic strategy against osteosarcoma.

Interleukin-6 Induced Proliferation Is Attenuated by Transforming Growth Factor-β-Induced Signaling in Human Hepatocellular Carcinoma Cells

“…Post multiple PBS washes, cells were blocked with 2.5% bovine serum albumin (BSA) blocking solution for 1 h. The cells were then incubated overnight with primary antibody (1:500 dilution in 2.5% BSA) at 4°C, followed by washing twice with PBS, and then incubated with TRITC-conjugated secondary antibody (1:1,000 in 2.5% BSA) for 1 h. Coverslips were mounted on slides using antifade DAPI and imaged under a fluorescent microscope (Zeiss, Axio Scope A1, or Axio Observer.Z1/7). Images were analyzed using the Zen 3.2 (blue edition) software ( 48 ).…”

p53 Modulation of Autophagy Signaling in Cancer Therapies: Perspectives Mechanism and Therapeutic Targets

“…Furthermore, metformin inhibited matrix metalloproteinase-9 activation, decreased endogenous insulin resistance, suppressed HER2 (erbB-2) oncoprotein overexpression, improved cancer patient's survival in type 2 diabetes, and blocked migration as well as invasion of cancer cells (Sui et al, 2011). Recently, Saini et al found that verteporfin, known as autophagy inhibitory and proteotoxic functions, disrupts multiple steps of autophagy in addition to regulate p53 to sensitize osteosarcoma of human osteosarcoma cells-HOS (R156P mutant P53) (Saini et al, 2021). microRNA, miR-26b, improves the sensitivity of hepatocellular carcinoma to doxorubicin by USP9X-dependent degradation of p53 as well as autophagy regulation (Chen et al, 2021).…”

Constitutive Activation of p62/Sequestosome-1-Mediated Proteaphagy Regulates Proteolysis and Impairs Cell Death in Bortezomib-Resistant Mantle Cell Lymphoma

“…To explore the role of p62 in proteaphagy, verteporfin (VTP, a clinical-grade small molecule inhibitor of this autophagy receptor) was employed [ 30 , 31 ]. Unlike BafA, VTP inhibits autophagy at an early stage by aggregation of p62 and reduces autophagosome accumulation such that it can be assessed by a reduction of the autophagy marker LC3B [ 32 , 33 ]. Whereas the levels of LC3B and its lipidated form decreased, high-molecular weight aggregates of p62 accumulated after VTP treatment in both Z-138 and ZBR cells.…”