Growth suppression of mouse pituitary corticotroph tumor AtT20 cells by curcumin: a model for treating Cushing's disease

Abstract

Background: Pituitary corticotroph tumors secrete excess adrenocorticotrophic hormone (ACTH) resulting in Cushing's disease (CD). Standard treatment includes surgery and, if not successful, radiotherapy, both of which have undesirable side effects and frequent recurrence of the tumor. Pharmacotherapy using PPARgamma agonists, dopamine receptor agonists, retinoic acid or somatostatin analogs is still experimental. Curcumin, a commonly used food additive in South Asian cooking, has potent growth inhibitory effects on cell proliferation. Our laboratory recently demonstrated that curcumin inhibited growth and induced apoptosis in prolactin- and growth hormone-producing tumor cells. Subsequently, Schaaf et.al. confirmed our findings and also showed the in vivo effectiveness of curcumin to suppress pituitary tumorigenesis. However the molecular mechanism that mediate this effect of curcumin are still unknown.

Principal findings: Using the mouse corticotroph tumor cells, AtT20 cells, we report that curcumin had a robust, irreversible inhibitory effect on cell proliferation and clonogenic property. The curcumin-induced growth inhibition was accompanied by decreased NFkappaB activity. Further, curcumin down-regulated the pro-survival protein Bcl-xL, depolarized the mitochondrial membrane, increased PARP cleavage, which led to apoptotic cell death. Finally, curcumin had a concentration-dependent suppressive effect on ACTH secretion from AtT20 cells.

Conclusion: The ability of curcumin to inhibit NFkappaB and induce apoptosis in pituitary corticotroph tumor cells leads us to propose developing it as a novel therapeutic agent for the treatment of CD.

Figure 1. Curcumin suppresses cell proliferation and clonogenic ability of AtT20 cells.

(A) AtT20 cells were treated with the indicated concentrations of curcumin and cell proliferation was determined by MTT assay after 4 days. Data were calculated as % of vehicle control and expressed as mean ±SEM of 3 independent experiments each performed with at least 4 replicates. * indicates significant differences from control, (p<0.05). (B) AtT20 cells were treated with either vehicle or curcumin (5, 10 and 50 µM) for 24 hrs. Cells were trypsinized, washed and re-plated in DMEM medium containing 10% FBS. Cell proliferation was assessed after 4 days. Data were calculated as % of vehicle control and expressed as mean ±SEM of 3 independent experiments each performed with at least 4 replicates. * indicates significant differences from control (p<0.05). (C) AtT20 cells were seeded (1000–3000 cells/well) in a six well plate in complete growth medium containing 10% FBS. Cells were allowed to adhere for 24 hrs, after which medium was replaced with fresh growth medium, containing 10% FBS, together with the indicated concentrations of curcumin. Medium was changed every 3 to 4 days, and colony formation was monitored over a 14–21 day period. Colony formation was detected by crystal violet staining and subsequently photographed. Data shown is from a single experiment, and is a representative of 3 independent experiments yielding similar results. (D) To quantitate colony formation, the number of colonies (each colony consisting of 50 cells or more) in 4 random fields were counted. The average was obtained, and decreases from 0 µM curcumin were calculated and are expressed as % of control. Each value is the mean ±SEM of 3 separate experiments. * indicates significant difference from control, (p<0.05).

Figure 2. Curcumin suppresses constitutively activated NFκB activity, down regulates Bcl-_xl and causes mitochondrial membrane depolarization in AtT20 cells.

(A) AtT20 cells were transiently transfected with an NFκB reporter gene, and after 24 hrs treated with either vehicle or the indicated concentrations of curcumin for 4 hrs. Cells were washed and luciferase activity in cell lysates was determined. Normalized luciferase activity was calculated, and data is presented as fold change over control. Each value is the mean ±SEM of 3 separate experiments each performed in triplicates. * indicates significant difference from control, (p<0.05). (B) AtT20 cells were treated with either vehicle or indicated concentrations of curcumin for 24 hrs. Cell lysates were harvested and equal amount of protein was subjected to western blotting with an anti-Bcl-xL Ab. The filter was stripped and reprobed with anti- β tubulin Ab to confirm equal loading. Data shown is from a single experiment, and is a representative of 2 independent experiments yielding similar results. (C) AtT20 cells were treated with either vehicle or indicated concentrations of curcumin for 24 hrs. Cells were washed and labeled with the dual fluorescence mitochondrial specific dye, JC-1, and analyzed by flowcytometry. The dot plots show that in vehicle and 5 µM curcumin treated cells, the % of cells emitting green fluorescence is low, and is indicative of basal apoptosis. However, treatment with 50 µM curcumin, that caused a decrease in Bcl-xL levels, significantly increased the intensity of green fluorescence. The dot plot shown is from a single experiment that is representative of 3 independent experiments. When data from the light scatter plots were quantitated (D) as % of gated cells, our data show that curcumin in a concentration-dependent manner, lead to increased membrane depolarization. Each value is the mean ±SEM of 3 separate experiments. * indicates significant difference from control, (p<0.05).

Figure 3. Curcumin induced apoptosis in AtT20 cells.

(A) AtT20 cells were treated with the indicated concentration of curcumin for 24 hrs. Cell lysates were harvested and equal amount of protein was subjected to western blotting with an anti-total PARP Ab, to detect apoptosis. Membrane was stripped and reprobed with anti-β tubulin Ab to confirm equal loading. Data presented is from a single experiment, and is a representative of 3 separate experiments yielding similar results. (B) AtT20 cells were treated with the indicated concentration of curcumin for 24 hrs. Cell were washed and labelled with anexinV-FITC and propidium iodide and analyzed by flow cytometry. Data are presented as % of gated cells. Each value is the mean ±SEM of 3 separate determinations from a single experiment, and is a representative of 3 separate experiments yielding similar results. * indicates significant difference from control, (p<0.05).

Figure 4. Curcumin decreases secretion of ACTH.

AtT20 cells were treated with the indicated concentrations of curcumin for 24 hrs, and equal amount of CM were used to detect secreted ACTH. Data were calculated as % of vehicle control and each value is the mean ±SEM of 4 separate experiments. * indicates significant difference from control, (p<0.001).