Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways - PubMed (original) (raw)

Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways

Suksanti Prakobwong et al. Carcinogenesis. 2011 Sep.

Abstract

Cholangiocarcinoma (CCA) is a tumor with poor prognosis that is resistant to all currently available treatments. Whether curcumin, a nutraceutical derived from turmeric (Curcuma longa), has potential therapeutic activity against human CCA was investigated using three CCA cell lines (KKU100, KKU-M156 and KKU-M213). Examination of mitochondrial dehydrogenase activity, phosphatidylserine externalization, esterase staining, caspase activation and poly-adenosine diphosphate ribose polymerase cleavage demonstrated that curcumin inhibited proliferation of and induced apoptosis in these biliary cancer cells. Colony-formation assay confirmed the growth-inhibitory effect of curcumin on CCA cells. When examined for the mechanism, curcumin was found to activate multiple cell signaling pathways in these cells. First, all CCA cells exhibited constitutively active nuclear factor (NF)-κB, and treatment with curcumin abolished this activation as indicated by DNA binding, nuclear translocation and p65 phosphorylation. Second, curcumin suppressed activation of signal transducer and activator of transcription-3 as indicated by decreased phosphorylation at both tyrosine(705) and serine(727) and inhibition of janus kinase-1 phosphorylation. Third, curcumin induced expression of peroxisome proliferator-activated receptor gamma. Fourth, curcumin upregulated death receptors, DR4 and DR5. Fifth, curcumin suppressed the Akt activation pathway. Sixth, curcumin inhibited expression of cell survival proteins such as B-cell lymphoma-2, B-cell leukemia protein xL, X-linked inhibitor of apoptosis protein, c-FLIP, cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2 and survivin and proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Seventh, the growth inhibitory effect of curcumin was enhanced in the IκB kinase-deficient cells, the enzyme required for nuclear factor-kappaB activation. Overall, our results indicate that curcumin mediates its antiproliferative and apoptotic effects through activation of multiple cell signaling pathways, and thus, its activity against CCA should be further investigated.

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Figures

Fig. 1.

Curcumin induces apoptosis and inhibits proliferation of CCA cells. (A) The chemical structure of curcumin. (B) Cells were treated with indicated concentrations of curcumin, and cell viability was determined by measuring mitochondrial dehydrogenase activity on days 0, 2, 4 and 6. (C) Curcumin suppressed long-term colony formation in CCA cells. Cells treated with indicated concentrations of curcumin were allowed to form colonies for 6 days. The values given are the means ± standard errors of the mean of three replicates. One of three independent experiments is shown. (D) Curcumin enhanced apoptosis in CCA cells as determined by the Live/Dead assay reagent. Cells were treated with the indicated concentrations of curcumin for 24 h, stained with a Live/Dead assay reagent for 30 min and then analyzed under a fluorescence microscope. Values below each photomicrograph represent percentage of apoptotic cells. (E) Curcumin induced early apoptosis in CCA cells, as determined by the annexin V assay. Cells were treated with indicated concentrations of curcumin for 24 h. The cells were then incubated with a fluorescein isothiocyanate-conjugated annexin V antibody, and early apoptotic cells were analyzed by flow cytometry. Asterisk indicates significance of the difference compared with control; P < 0.05.

Fig. 2.

Curcumin induces caspase activation and PARP cleavage and downregulates expression of antiapoptotic and proliferative gene products in KKU-M156 cells**.** Cells were treated with 50 μM curcumin for the indicated times, and whole cell extracts were prepared and analyzed using indicated antibodies. β-Actin was used as an internal control.

Fig. 3.

Curcumin inhibits constitutive NF-κB activation in CCA cells. (A, left) Nuclear extracts prepared from KKU100, KKU-M156 and KKU-M214 cells were analyzed for NF-κB activation by EMSA. (A, middle) Effect of curcumin on constitutive NF-κB activation. KKU-M156 cells were incubated with curcumin at the indicated concentrations for 4 h. Nuclear extracts were prepared and analyzed for NF-κB activation by EMSA. (A, right) The binding of NF-κB to the DNA is specific and consists of p50 and p65 subunits. Nuclear extracts were prepared from KKU-M156 cells, incubated for 30 min with indicated antibodies, preimmune serum and mutant or unlabeled NF-κB oligonucleotide probe and then assayed for NF-κB by EMSA. (B) Curcumin induced redistribution of p65. KKU-M156 cells were incubated without or with curcumin (50 μM) for 4 h and then analyzed for the distribution of p65 by immunocytochemistry. Red stain indicates p65, and blue stain indicates nucleus. (C, D) Curcumin inhibited phosphorylation of p65 and expression of TRAF1 and TRAF2. KKU-M156 cells were treated with 50 μM curcumin for the indicated times, and whole cell extracts were prepared and analyzed by western blotting using antibodies against phospho-p65 (Ser536), TRAF1 and TRAF2. β-Actin was used as an internal control. Figures are representative of three independent experiments.

Fig. 4.

Curcumin inhibits STAT-3 and JAK-1 phosphorylation in KKU-M156 cells. (A) Cells (1 × 106) were treated with indicated concentrations of curcumin for 6 h, whole cell extracts were prepared and analyzed for STAT-3 phosphorylation using p-STAT-3 (Tyr705, Ser 727) antibody. (B and C) KKU-M156 cells were treated with 50 μM curcumin for the indicated times, and whole cell extracts were prepared and analyzed for STAT-3 phosphorylation (at Tyr705 and Ser727) and JAK-1 phosphorylation. Data shown are representative of three independent experiments.

Fig. 5.

Curcumin upregulates PPAR-γ and death receptors and downregulates p-Akt in KKU-M156 cells. (A) Cells were treated with indicated concentrations of curcumin for 6 h or (B–D) with 50 μM curcumin for indicated times. Whole cell extracts were prepared and analyzed using indicated antibodies. Data shown are representative of two independent experiments.

Fig. 6.

IKK-deficient cells are more sensitive to growth inhibition by curcumin: (A) The whole cell extract prepared from wild-type and knockout cells were analyzed by western blotting using indicated antibodies. β-Actin was used as an internal control. (B) Wild-type and deficient cells were treated with indicated concentrations of curcumin for 3 days. Cell growth was examined by measuring mitochondrial dehydrogenase activity as described in Material and Methods. (C) A schematic diagram showing curcumin’s mechanism of action in CCA cells.

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Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways - PubMed (original) (raw)