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. 2010 Jan;9(1):202-10.
doi: 10.1158/1535-7163.MCT-09-0771. Epub 2010 Jan 6.

Notch-1 inhibition by Withaferin-A: a therapeutic target against colon carcinogenesis

Srinivas Koduru  1 , Raj Kumar, Sowmyalakshmi Srinivasan, Mark B Evers, Chendil Damodaran
Affiliations

Notch-1 inhibition by Withaferin-A: a therapeutic target against colon carcinogenesis

Srinivas Koduru et al. Mol Cancer Ther. 2010 Jan.

Abstract

Notch signaling plays a crucial role in the development of colon cancer; targeting the Notch pathway may sensitize colon cancers to various adjuvant agents. The focus of our current study is to identify natural compounds that target Notch signaling and that might be beneficial for the prevention and treatment of colon cancer. Withaferin-A (WA) is a bioactive compound derived from Withania somnifera, which inhibits Notch-1 signaling and downregulates prosurvival pathways, such as Akt/NF-kappaB/Bcl-2, in three colon cancer cell lines (HCT-116, SW-480, and SW-620). In addition, WA downregulated the expression of mammalian target of rapamycin signaling components, pS6K and p4E-BP1, and activated c-Jun-NH(2)-kinase-mediated apoptosis in colon cancer cells. We also established the molecular link between Notch/Akt/mammalian target of rapamycin signaling by complementary approaches (i.e., overexpression of Notch-1 or inhibition of Notch-1 by small interfering RNA). Our results suggest that WA inhibits Notch-mediated prosurvival signaling, which facilitates c-Jun-NH(2)-kinase-mediated apoptosis in colon cancer cell lines. These results underscore the anticancer activity of WA, which exhibits potential for further development for targeted chemotherapy and/or chemoprevention strategies in the context of colon cancer.

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Figures

Figure 1
Figure 1. Withaferin-A inhibits Notch signaling in colon cancer cells
HCT-116, SW-480, and SW-620 cells were treated with either vehicle control (DMSO) or WA for varying time intervals. Cell lysates were subjected to western blot analysis using (A) Notch-1 (cleaved), Hey-1 and Hes-1 antibodies, and (B) gamma-secretase components. GAPDH was used as the internal loading control.
Figure 2
Figure 2. Withaferin-A Inhibits Akt/ NF-κB/Bcl-2 signaling in colon cancer cells
(A, B and C) HCT-116, SW-480, and SW-620 cells were treated with either vehicle control (DMSO) or WA for varying time intervals, and cell lysates were subjected to western blot analysis using the indicated antibodies. GAPDH was used as the internal loading control.
Figure 3
Figure 3. Withaferin-A regulates mTOR, ERK and JNK pathways in colon cancer cells, leading to the induction of apoptosis
(A, B, and C) HCT-116, SW-480 and SW-620 cells were treated with either vehicle control (DMSO) or WA for varying time intervals, and cell lysates were subjected to western blot analysis using the indicated antibodies. GAPDH was used as loading control.
Figure 4
Figure 4. Knockdown or overexpression of Notch-1 significantly modulates Akt/mTOR signaling in colon cancer cells
(A) Full-length Notch-1 was overexpressed in HCT-116 and SW-620 cells followed by treatment with vehicle or DMSO, and cell lysates were subjected to western blot analysis using the indicated antibodies. (B) HCT-116 and SW-620 cells were transfected with siRNA and cell lysates were subjected to western blot analysis; GAPDH was used as a loading control.
Figure 5
Figure 5. Withaferin-A inhibits cell viability and induces apoptosis in colon cancer cells
(A) HCT-116, SW-480, SW-620, and FHC cells were treated with varying concentrations of WA for 24h. Trypan Blue Exclusion assay was performed. Each data point represents the mean of four wells from three independent experiments (mean ± SE). (B) Apoptotic assays were performed using Annexin V-FITC staining. The data shown are representative of the combined means from three independent experiments, and the error bars represent the standard error.
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