Valproic acid induces Notch1 signaling in small cell lung cancer cells

Abstract

Background: Small cell lung cancer (SCLC) is an aggressive malignancy. Current treatments yield dismal survival rates. We have previously demonstrated that histone deacetylase (HDAC) inhibitors can inhibit neuroendocrine tumor growth. Activation of the Notch1 signaling pathway also impairs SCLC cell viability. In this study, we investigated the ability of the HDAC inhibitor valproic acid (VPA) to activate Notch1 signaling and inhibit proliferation in SCLC cells.

Materials and methods: DMS53 human SCLC cells were treated with VPA (0-10 mM) for 2 d. Light microscopy was used to examine changes in cell morphology. Western analysis was performed using antibodies against various Notch1 pathway proteins to assess Notch1 activation. Additionally, immunoblotting was performed for two neuroendocrine tumor markers, chromogranin A and achaete-scute complex-like 1. Finally, a cell proliferation assay was used to measure the effects of VPA on SCLC growth over 8 d.

Results: After treatment with VPA, DMS53 cells underwent dramatic changes in morphology. VPA induced expression of the full-length and active forms of Notch1 protein. Furthermore, VPA suppressed levels of neuroendocrine tumor markers chromogranin A and ASLC-1. Importantly, VPA treatment led to dose-dependent inhibition of SCLC cell proliferation.

Conclusions: The HDAC inhibitor VPA activates Notch1 signaling in SCLC cells. VPA induces changes in cell morphology and suppresses neuroendocrine tumor markers, indicating a change in phenotype. Additionally, VPA profoundly inhibits SCLC cell growth. These results suggest that VPA has potential as a novel therapeutic agent for SCLC.

FIG 1

(A) VPA Induces Notch1 Signaling in SCLC Cells. Western blots of cell extracts from DMS53 cells treated with control (DMSO 0.1%) or VPA (1 to 10 mM) for 2 d showed increased expression of full-length Notch1 and the cleaved, active Notch intracellular domain (NICD). Levels of Hes-1, a downstream target of Notch1, were also elevated from baseline with VPA treatments. Equal protein loading was confirmed with antibodies against G3PDH. (B) After quantification with image analysis software, there were statistically significant differences between control and treatment groups with full-length Notch, NICD, and Hes-1 expression (p-value <0.001).

FIG 2

VPA Decreases Growth and Induces Morphologic Differentiation in SCLC Cells. DMS53 cells were treated with VPA or control (DMSO 0.1%) for 2 d and examined with light microscopy under 20x magnification. Control plates (A) grew in confluent sheets with ill-defined cellular borders. Cells treated with VPA in 4 mM (B) or 10 mM (C) concentrations progressively assumed a flatter, rounder, and spindle shaped conformation with neuronal-like projections, decreased points of cell-to-cell contact, and more distinct cellular borders.

FIG 3

(A) Induction of Notch1 Signaling by VPA Regulates Levels of the NET Markers ASCL-1 and CgA. Western analysis of cell extracts from DMS53 cells treated with control (DMSO 0.1%) or VPA (1 to 10 mM) for 2 d show decreases in NET markers ASCL-1 and CgA. Equal protein loading was confirmed with G3PDH. (B) After quantification with image analysis software, there were statistically significant differences between control and treatment groups with ASCL-1 and CgA expression (p-value <0.001).

FIG 4

VPA Inhibits Proliferation of SCLC Cells. DMS53 cells were treated with control (DMSO) or varying doses of VPA (1 to 10 mM) for up to 8 d and cell proliferation was measured by the MTT assay. VPA inhibited cell proliferation in a dose-dependent and statistically-significant manner.