Curcumin acts as anti-tumorigenic and hormone-suppressive agent in murine and human pituitary tumour cells in vitro and in vivo

  1. U Renner
  1. Neuroendocrinology Group, Max Planck Institute of Psychiatry, Kraepelinstrasse 10, D-80804 Munich, Germany
    1Neurosurgical Clinic, University of Erlangen-Nuremburg, Erlangen, Germany
    2Department of Neurosurgery, Istituto San Raffaele, Milano, Italy
    3Neurosurgical Clinic, Technical University of Munich, Munich, Germany
    4Neurosurgical Clinic, University of Munich, Munich, Germany
    5Department of Internal Medicine 1 and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany
    6Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon II-2 Piso, C1428 Buenos Aires, Argentina
    7IFIBYNE-CONICET, Ciudad Universitaria, Pabellon II-2 Piso, C1428 Buenos Aires, Argentina
  1. (Correspondence should be addressed to U Renner; Email: renner{at}; M J Perone; Email: mperone{at}
  1. Figure 1

    Effect of curcumin on growth of pituitary tumour cell lines. AtT20, MtT/S and GH3 cells were treated with 5–30 μM curcumin for up to 72 h, and effects on [3H]thymidine incorporation and cell numbers were determined. Representative figures show curcumin-induced inhibition of [3H]thymidine incorporation in MtT/S cells ((A) 24 h treatment) and AtT20 cells ((B) 48 h treatment) as well as reduction of cell numbers in GH3 cells ((C) 48 h treatment). For comparative reasons, in figures (A), (B) and (C), values are expressed as percentage of non-treated cells. In (D), an example of a time-course study is shown for AtT20 cells in which the gradual decline of proliferation over time after treatment with 10 μM curcumin is demonstrated. For each time point, the relative decline of cell numbers of curcumin-treated versus non-treated AtT20 cells is shown. *P<0.05; **P<0.01 versus non-treated cells.

  2. Figure 2

    Effect of curcumin on GH3 cell colony formation in soft agar (A) and on GH3 cell tumour growth in nude mice (B and C). (A) GH3 cells were seeded in soft agar in 6-well culture plates and treated with 5–30 μM curcumin or were not treated (0). Curcumin solutions (1 ml) were added on the first day after seeding and were then renewed every 4 days. After 14 days, colony numbers were determined. Since curcumin solutions contained small amounts of DMSO, control cells (CT) were treated with the highest concentration of DMSO applied. Whereas the latter had no effect on colony formation, curcumin dose dependently suppressed the number of GH3 cell colonies. (B and C) Athymic mice bearing GH3 tumour cells were treated with curcumin for 4 weeks (1 mg per animal/ thrice weekly) or vehicle (control; six animals per group) after the tumour size has reached 100 mm3. Suppression of tumour growth by curcumin was evident after 13 days, and the difference in tumour size between curcumin- and vehicle-treated mice gradually increased over treatment time. Representative tumours on day 27 are shown in (B) and, in (C), the development of mean tumour size during the treatment period is documented. *P<0.05; **P<0.01 versus non-treated cells (A) or vehicle controls (B and C) respectively.

  3. Figure 3

    Effect of curcumin on the cell cycle and cell cycle regulating proteins in GH3 cells. After stimulation of GH3 cells with the indicated curcumin concentrations for 24 h, the proportion of cells at different cell cycle phases was determined by FACS analysis (A), and alterations in the expression of cell cycle regulating proteins were measured by western immunoblotting (B). (A) Curcumin dose dependently and significantly enhanced the proportion of GH3 cells in G2/M phase and reduced cell numbers in G1 phase indicating a curcumin-induced growth arrest in G2/M phase. (B) Studies on the effect of curcumin on key cell cycle regulating proteins showed a dose-dependent suppression of cyclin D1 and CDK4 but no effect on p27kip1 in GH3 cells. CT, control with DMSO. *P<0.05 versus non-treated (basal) cells.

  4. Figure 4

    Effect of curcumin on apoptosis and expression of apoptosis-associated proteins in MtT/S cells. (A) Treatment of MtT/S cells with increasing concentrations of curcumin for 24 h induced a dose-dependent, significant increase in DNA fragmentation as measured by cell death ELISA. The application of 30 μM curcumin induced a 9.57±0.71 fold stimulation of apoptosis in comparison to non-treated MtT/S cells. (B) By western immunoblotting, it is shown that 24 h treatment of MtT/S cells with curcumin dose dependently suppressed the expression of anti-apoptotic-acting Bcl-2, whereas pro-apoptotic cleaved caspase-3 expression was enhanced. (C) Detection of normal (grey bars), apoptotic (white bars) and necrotic (black bars) MtT/S cells by FACS analysis after treatment with curcumin (30 μM) for various time periods. Curcumin time dependently and significantly increased the proportion of cells undergoing apoptosis without any sign of induction of necrosis. Non-treated (0) and DMSO-treated (CT) cells were assayed at 24 h and curcumin-treated cells after indicated treatment periods. Positive controls for necrosis (CT 7-AAD) and apoptosis (CT AnnexV) were provided by the manufacturer of the FACS analysis kit. 0, non-treated cells; CT, control with DMSO; *P<0.05; **P<0.01 versus non-treated cells.

  5. Figure 5

    Effect of curcumin on hormone secretion by pituitary tumour cell lines. Corticotroph AtT20 and lactosomatotroph GH3 pituitary tumour cells were stimulated with curcumin (5–30 μM) for 4 and 24 h. At the end of the stimulation period, the cell culture supernatants were cautiously aspirated, centrifuged at 121 g to remove floating apoptotic cells, and then used for hormone measurement by RIA. The cells were then trypsinized and counted with an adapted coulter counter to calculate hormone production per cell. In AtT20 cells, ACTH secretion was significantly reduced by curcumin after 4 (A) and 24 (not shown) h, at 20 and 30 μM curcumin concentrations. In GH3 cells, GH and PRL were significantly reduced after 4 (not shown) and 24 (B and C) h. 0, non-treated cells; CT, controls with DMSO; *P<0.05 versus non-treated cells.

  6. Figure 6

    Effect of curcumin on human pituitary adenoma cells in vitro. Human pituitary adenoma cell cultures were treated with curcumin (0.5–30 μM) and effects on growth, apoptosis and hormone secretion were measured. An overview about all studies in human pituitary tumour cell cultures is documented in Table 1. The figures presented here show dose-dependent curcumin suppression of [3H]thymidine incorporation in a non-functioning ((A) NT3) and a lactosomatotroph ((B) LST1) pituitary adenoma. Curcumin-induced apoptosis measured by cell death ELISA in a non-functioning adenoma (NT3) is shown in (C). Inhibition of GH, PRL and TSH secretion by curcumin in lactosomatotroph (LST1), somatotroph (ST3) and thyreotroph (TT1) adenomas is shown in (D), (E) and (F) respectively. In all experiments, DMSO had no effect on human pituitary adenoma cells. 0, non-treated cells; CT, controls with DMSO; *P<0.05; **P<0.01 versus basal.

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