The curcumin analogue PAC has potent anti-anaplastic thyroid cancer effects

Abstract

Anaplastic thyroid carcinoma (ATC) is the rarest type of thyroid cancer, but is the common cause of death from these tumors. The aggressive behavior of ATC makes it resistant to the conventional therapeutic approaches. Thus, the present study was designed to evaluate the anti-ATC efficacy of the piperidone analogue of curcumin (PAC). We have shown that PAC induces apoptosis in thyroid cancer cells in a time-dependent fashion through the mitochondrial pathway. Immunoblotting analysis revealed that PAC suppressed the epithelial-to-mesenchymal transition (EMT) process in ATC cells by upregulating the epithelial marker E-cadherin and reducing the level of the mesenchymal markers N-cadherin, Snail, and Twist1. This anti-EMT effect was confirmed by showing PAC-dependent inhibition of the proliferation and migration abilities of ATC cells. Furthermore, PAC inhibited the AKT/mTOR pathway in ATC cells. Indeed, PAC downregulated mTOR and its downstream effectors p70S6K and 4E-BP1 more efficiently than the well-known mTOR inhibitor rapamycin. In addition to the promising in vitro anticancer efficacy, PAC significantly suppressed the growth of humanized thyroid tumor xenografts in mice. Together, these findings indicate that PAC could be considered as promising therapeutic agent for anaplastic thyroid carcinomas.

Figure 1

PAC induces apoptosis in anaplastic thyroid cancer cells. (A) Thyroid cancer cells were either sham-treated (DMSO ≤ 1%) or challenged with the indicated doses of PAC for 72 h, and then the proportion of apoptosis was analyzed by Annexin V/PI-flow cytometry. Flow charts. (B) CAL-62 cells were challenged with PAC for the indicated periods of time, and then the proportion of apoptotic cells was analyzed by Annexin V/PI-flowCytometry. (C, D) CAL-62 cells were treated with PAC (10 µM) for the indicated periods of time, and then cell lysates were prepared and used for immunoblotting analysis using specific antibodies for the indicated proteins. Histograms show quantifications of the immunoblots performed by densitometry relative to GAPDH and presented as fold change relative to the control. Error bars represent mean ± SEM (n = 3). *P ≤ 0.05, **P ≤ 0.01, ns: not significant.

Figure 2

PAC suppresses the epithelial-to-mesenchymal transition processe in thyroid cancer cells. (A, B) CAL-62 cells were either sham-treated (DMSO ≤ 1%) or challenged with PAC (3, 6 or 10 µM) for the indicated periods of time. Exponentially growing cells (10⁴) were seeded independently in the E-plate (proliferation) or CIM plate (migration), and then the assessments were performed using the RTCA-DP xCELLigence System. Data are representative of different experiments performed in triplicate. (C) CAL-62 cells were treated with PAC (10 µM) for the indicated periods of time, and then cell lysates were prepared and were used for immunobloting analysis utilizing specific antibodies against the indicated proteins. (D) Histograms show quantifications of the immunoblots performed by densitometry relative to GAPDH and β-actin, and presented as fold change relative to the control. Error bars represent mean ± SEM (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns: not significant.

Figure 3

PAC suppresses the AKT/mTOR pathway in thyroid cancer cells. (A) CAL-62 cells were treated with PAC (10 µM) for the indicated periods of time, and then cell lysates were prepared and were used for immunebloting analysis utilizing specific antibodies against the indicated proteins. (B) CAL-62 cells were treated with PAC (10 µM) or rapamycin (50 nM) for 48 h, and then cell lysates were prepared and were used for immunobloting analysis utilizing specific antibodies against the indicated proteins. Histograms show quantifications of the immunoblots performed by densitometry relative to GAPDH, and presented as fold change relative to the control. The levels of phosphorylated proteins were normalized against the total amount of their relative non-phosphorylated forms. Error bars represent mean ± SEM (n = 2). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns: not significant.

Figure 4

PAC inhibits the growth of human thyroid tumor xenografts. Thyroid cancer xenografts were created by subcutaneous injection of CAL-62 cells (5.10⁶) into nude mice (n = 6). Subsequent to the growth of the tumors, animals were randomized into two sub-groups. One group was PAC-treated intraperitonially at a dose of 100 µg/kg daily. The control group was treated with DMSO. The volumes of the tumors were measured at the indicated periods of time. The error bars represent means ± SEM, **P = 0.0085.