Curcumin: a novel Stat3 pathway inhibitor for chemoprevention of lung cancer

Abstract

Multiple studies from independent groups find evidence for signal transducer and activator of transcription 3 (Stat3) activation in nearly 50% of lung cancers, suggesting a functional role for this target in subsets of lung cancer. On the basis of the existing evidence, we hypothesized that bioavailable curcuminoid complex may modulate lung carcinogenesis, primarily by inhibiting Stat3 activation. With the safety of this being botanically well established, the objective of these studies was to test our hypothesis in vitro and in vivo in an effort to inform the design of a phase II chemoprevention trial in former smokers. We treated non-tumor-derived, normal (but immortalized) human bronchial epithelial cells (AALE) (Lundberg et al., 2002; Pillai et al., 2011) and lung adenocarcinoma-derived cells (H441) with bioactive curcumin C3 complex. Asynchronous cells in each case were treated with curcumin for 24 h, followed by immunoblotting for Stat3 and activated Stat3-P, prior signal of which was used for normalization. We also completed a preclinical trial in which 12 mice were randomly divided into three groups and subjected to 3 days or 9 days of curcumin intraperitoneal injections, followed by analysis of lung tissues for Stat3-P changes and growth suppressive effects of the curcumin. The growth suppressive effects were measured using Cyclin D1 and the replicative helicase subunit, Mcm2, as surrogates for the proliferative capacity of the tissues. In-vitro studies with curcuminoid complex demonstrated that the activity of Stat3 in both normal bronchoepithelial cells and lung cancer-derived cells is sensitive to curcumin exposure. In a dose-dependent manner, curcumin treatment resulted in significant suppression of Stat3 phosphorylation and reduction in the proliferative capacity of both cell types. In the preclinical trial with rodent models, curcumin reduced Stat3-P and the proliferative markers CycD1 and Mcm2 in mice lung tissues in vivo. These culture and preclinical studies indicate that the activity of the Stat3 pathway can be suppressed by curcumin treatment, concomitant with a reduction in cell proliferation, supporting our hypothesis that inhibition of the Stat3 pathway represents at least one important mechanism by which curcumin elicits its effects on the bronchoepithelium. These data provide a rationale for the use of curcumin as a promising chemopreventive agent in high-risk populations such as former smokers.

Figure 1. Curcumin Inhibits the Stat3 Pathway in Normal and Lung Cancer Derived Human Bronchoepithelial Cells

Asynchronous AALE (A) or H441 (B) cells were treated with curcumin at the doses indicated for 24 hrs, following which immunoblots were performed on total protein extracts for Ptyr705-Stat3 and total Stat3. Densitometric readings were used to normalize P-Stat3:Stat3, and resulting data are shown +/- 1 S.D. Untreated samples were arbitrarily set at 100% Stat3-P level (first column in each set). Statistically significant differences are indicated with P-values.

Figure 2. Curcumin Inhibits the Proliferation of Human Normal and Lung Cancer Cells

Asynchronous AALE (A) or H441 (B) cells were treated with curcumin at the doses indicated for 24 hrs, following which cell viability (GLO) assays were performed to determine growth suppressive effects of curcumin on each cell type. Triplicate samples were tested, and average results are shown +/- 1 S.D. Samples were normalized to untreated samples shown in first column in each graph, arbitrarily set to 100% viability.

Figure 3. Curcumin Suppresses the Proliferative Capacity of Normal Lung Tissue in vivo in Mice

Curcumin (cur) in DMSO (or DMSO as a control) was given to mice at 50mg/2.5ml/kg daily by i.p. injection, for 3 or 9 days. 12 mice were randomly divided into 3 groups: (9d DMSO, 3d cur, 9d cur), and results from two mice for each condition are shown. Immunoblots were performed on total protein extracts using antibodies indicated, from mice sacrificed to obtain whole lung tissue for protein samples. Similar results were obtained in the remaining mice for each condition.