New curcumin analogues exhibit enhanced growth-suppressive activity and inhibit AKT and signal transducer and activator of transcription 3 phosphorylation in breast and prostate cancer cells

Abstract

Curcumin, the active component of turmeric, has been shown to protect against carcinogenesis and prevent tumor development in cancer. To enhance its potency, we tested the efficacy of synthetic curcumin analogues, known as FLLL11 and FLLL12, in cancer cells. We examined the impact of FLLL11 and FLLL12 on cell viability in eight different breast and prostate cancer cell lines. FLLL11 and FLLL12 (IC(50) values 0.3-5.7 and 0.3-3.8 micromol/L, respectively) were substantially more potent than curcumin (IC(50) values between 14.4-50 micromol/L). FLLL11 and FLLL12 were also found to inhibit AKT phosphorylation and downregulate the expression of HER2/neu. In addition, we demonstrate for the first time that FLLL11 and FLLL12 inhibit phosphorylation of signal transducer and activator of transcription (STAT) 3, an oncogene frequently found to be persistently active in many cancer types. The inhibition of STAT3 signaling was confirmed by the inhibition of STAT3 DNA binding and STAT3 transcriptional activity. Furthermore, FLLL11 and FLLL12 were more effective than curcumin in inhibiting cell migration and colony formation in soft agar as well as inducing apoptosis in cancer cells. These results indicate that FLLL11 and FLLL12 exhibit more potent activities than curcumin on the inhibition of STAT3, AKT, and HER-2/neu, as well as inhibit cancer cell growth and migration, and may thus have translational potential as chemopreventive or therapeutic agents for breast and prostate cancers.

Figure 1

(a) The chemical structures of curcumin, FLLL11, and FLLL12. (b) The general synthesis scheme for FLLL11 and FLLL12.

Figure 2

The inhibitory effects of AKT phosphorylation and HER‐2/neu protein expression by FLLL11, FLLL12, and curcumin in (a) BT‐474, (b) SK‐BR‐3, (c) MDA‐MB‐453, and (d) PC‐3 human cancer cell lines. Cancer cells were treated with 5–10 µmol/L FLLL11 and FLLL12, or 5–10 µmol/L curcumin for 24 h. Membranes were blotted with HER‐2/neu, phospho‐specific AKT (S473), cleaved poly (ADP‐ribose) polymerase (PARP), cleaved caspase‐3, and GAPDH antibodies.

Figure 3

FLLL11 and FLLL12 inhibit STAT 3 phosphorylation in (a) MDA‐MB‐231, (b) SK‐BR‐3, and (c) DU145 breast and prostate cancer cells. Cells were treated for 24 h. Membranes were blotted with phospho‐specific STAT3, phospho‐independent STAT3, phospho‐specific ERK1/2, cleaved poly (ADP‐ribose) polymerase (PARP), cleaved caspase‐3, and GAPDH antibodies. (d) FLLL11 and FLLL12 (10 µmol/L) induce cleaved PARP and cleaved caspase‐3 in breast cancer cells but not in human mammary epithelial cells (HMEC) and WI‐38 normal human lung fibroblasts. The inhibitory effects of FLLL11 and FLLL12 on STAT3 DNA binding activity in (e) MDA‐MB‐231 (f), SK‐BR‐3, and (g) DU145 cancer cells. The nuclear extracts were analyzed for STAT3 DNA binding activity using STAT3‐specific TransFactor kit. Statistical significance (P < 0.01) relative to the DMSO vehicle control is designated by an asterisk. (h) The inhibitory effect of FLLL11 and FLLL12 on STAT3‐dependent transcriptional activity was analyzed in MDA‐MB‐231 breast cancer cells that stably integrate the STAT3‐dependent luciferase reporter construct. The data were subsequently normalized and presented here as percentages of the DMSO vehicle control. Asterisks denote statistically significant (P < 0.01) decreases in luciferase activity.

Figure 3

FLLL11 and FLLL12 inhibit STAT 3 phosphorylation in (a) MDA‐MB‐231, (b) SK‐BR‐3, and (c) DU145 breast and prostate cancer cells. Cells were treated for 24 h. Membranes were blotted with phospho‐specific STAT3, phospho‐independent STAT3, phospho‐specific ERK1/2, cleaved poly (ADP‐ribose) polymerase (PARP), cleaved caspase‐3, and GAPDH antibodies. (d) FLLL11 and FLLL12 (10 µmol/L) induce cleaved PARP and cleaved caspase‐3 in breast cancer cells but not in human mammary epithelial cells (HMEC) and WI‐38 normal human lung fibroblasts. The inhibitory effects of FLLL11 and FLLL12 on STAT3 DNA binding activity in (e) MDA‐MB‐231 (f), SK‐BR‐3, and (g) DU145 cancer cells. The nuclear extracts were analyzed for STAT3 DNA binding activity using STAT3‐specific TransFactor kit. Statistical significance (P < 0.01) relative to the DMSO vehicle control is designated by an asterisk. (h) The inhibitory effect of FLLL11 and FLLL12 on STAT3‐dependent transcriptional activity was analyzed in MDA‐MB‐231 breast cancer cells that stably integrate the STAT3‐dependent luciferase reporter construct. The data were subsequently normalized and presented here as percentages of the DMSO vehicle control. Asterisks denote statistically significant (P < 0.01) decreases in luciferase activity.

Figure 4

The ability of MDA‐MB‐231 breast cancer cells to form colonies in soft agar is decreased following treatment with 5 µmol/L curcumin or 1–5 µmol/L FLLL11 and FLLL12 in (a) MDA‐MB‐231 and (b) SK‐BR‐3 breast cancer cells. (c) Migration of MDA‐MB‐231 into a scratch wound is impeded after 4 h of treatment with curcumin, FLLL11, or FLLL12. (d) MTT assays of MDA‐MB‐231 cells reveal that the dosages of these agents used in the migration assay have minimal impact on viability over 4 h of drug treatment and an additional 20 h without treatment.

Figure 5

The inhibitory effects of cell viability by FLLL11 and FLLL12 in combination with doxorubicin in the MDA‐MB‐231 human breast cancer cell line. MDA‐MB‐231 breast cancer cells were treated with (a) FLLL11, (b) FLLL12, and doxorubicin for 72 h. The cell viability was determined by MTT assay. The untreated cells were set at 100% and the viability of doxorubicin‐, FLLL11‐, and FLLL12‐treated cells was determined relative to the untreated cells. Asterisks represent synergism (a combinational index less than 1).