A plant alkaloid, veratridine, potentiates cancer chemosensitivity by UBXN2A-dependent inhibition of an oncoprotein, mortalin-2

Abstract

Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53.

Structural studies of UBXN2A and mortalin interaction and the putative role of silenced UBXN2A in preventing response to chemotherapy

“…Yeast two-hybrid, flow cytometry (Annexin V, Caspase-3 and cleaved PARP), immunocytochemistry, and His-tag pull-down assays These four assays were conducted as previously described (Abdullah et al 2015a;Rezvani et al 2009Rezvani et al , 2012Sane et al 2014). …”

“…By binding to mortalin, UBXN2A may antagonize CHIP-dependent destabilization of p53, suggesting a novel regulatory mechanism for CHIP-E3 ubiquitin ligase activity empty or (His) 6 -TYG-UBXN2A vector±(His) 6 -CHIP. Twentyfour hours after transfection, cytoplasmic and nuclear cell lysates (Abdullah et al 2015a) were prepared, followed by WB analysis. Overexpression of CHIP decreases p53 levels in both the cytoplasm and nucleus (Fig.…”

“…These colonies can be untransfected colonies or colonies with low-level expression of exogenous UBXN2A. Cells with high expression of UBXN2A enter into apoptosis 24 h after transfection or induction (Abdullah et al 2015a;Sane et al 2014). In addition, Lu et al reported exogenous stresses, in this case Lipofectamine 2000 (Fiszer-Kierzkowska et al 2011), can increase mortalin-p53 binding, resulting in elevation of cell proliferation (Lu et al 2011).…”

“…By binding to mortalin, UBXN2A reactivates wild-type (WT)-p53 tumor suppressor protein (Sane et al 2014) and induces apoptosis at the cellular level and in tumor xenografts (Abdullah et al 2015b). In addition, we discovered that induction of UBXN2A by its enhancer (Abdullah et al 2015a). Veratridine, can synergistically enhance the effectiveness of the chemotherapeutic agents etoposide and 5-fluorouracil (5-FU), particularly in welldifferentiated colon cancer cells.…”

“…Figure 5b shows efficient silencing of UBXN2A in clones 2 and 3 versus cells expressing scrambled shRNA. The U2OS control group and clones 2 and 3 were treated with DMSO (0.1 % final concentration) or 5-FU (100 μM) (Eby et al 2010) for 48 h, followed by flow cytometry using an early apoptosis marker, Annexin V (Abdullah et al 2015a). We recorded a spontaneous apoptosis (Ehemann et al 2003;Wang et al 1996) in DMSO-treated cells, which was suppressed in the absence of UBXN2A (Fig.…”