Combinatorial anticancer effects of curcumin and sorafenib towards thyroid cancer cells via PI3K/Akt and ERK pathways

Abstract The objective of this study was to examine the in vitro combinatorial anticancer effects of curcumin and sorafenib towards thyroid cancer cells FTC133 using a MTT cytotoxicity assay, and to test whether the mechanism involves induction of apoptosis. The present results demonstrated that curcumin at 15–25 μM dose-dependently suppressed the proliferation of FTC133. Combined treatment (curcumin (25 μM) and sorafenib (2 μM)) resulted in a reduction in cell colony formation and significantly decreased the invasion and migration of FTC133 cells compared with that treated with individual drugs. Western blot showed that the levels of p-ERK and p-Akt proteins were significantly reduced (p < 0.01) in the medicine-treated FTC133 cells. The curcumin was found to dose-dependently inhibit the apoptosis of FTC133 cells possibly via PI3K/Akt and ERK pathways. There is a synergetic antitumour effect between curcumin and sorafenib. Graphical abstract

anti-inflammatory, anticancer and antidepressant properties. Curcumin has also been reported to enhance wound healing (sharma et al. 2005;Maheshwari et al. 2006;Eshghi et al. 2014;Munigunti et al. 2014;sahebkar 2015a, 2015b. Numerous in vitro and in vivo studies have shown that curcumin possesses anticancer activities via its effect on a variety of biological pathways (Kanitkar et al. 2008;Yao et al. 2013;Liu et al. 2014). Additionally, curcumin affects growth factor receptors and cell adhesion molecules involved in tumour growth, angiogenesis and metastasis, all of which are relevant to cancer. Curcumin has also been shown to inhibit the viability and proliferation of a variety of human cancer cell lines including skin, gastrointestinal, genitourinary, breast, ovarian and lung cancers (Anand et al. 2008). The use of curcumin as a promising new natural chemical for chemoprevention and cancer chemotherapy has been extensively studied over the past several years.
sorafenib is an oral multi-tyrosine kinase inhibitor with reported activity in a variety of tumour types and is approved for the treatment of advanced human hepatocellular carcinoma and gastric cancer (Llovet et al. 2008). sorafenib is able to inhibit cancer growth, either by inhibiting angiogenesis or by directly inducing cancer cell apoptosis (Liu et al. 2006). In a preclinical model, growth of human BTC cells was suppressed by sorafenib alone or in combination (huether et al. 2007;Blechacz et al. 2009).
In this work, we evaluated in vitro combinatorial anticancer effects of curcumin and sorafenib towards thyroid cancer cells.

Results and discussion
In order to estimate the antitumour effect of curcumin, thyroid cancer cells FTC133 was incubated in the presence of curcumin, and tumour cell viability was determined after 48 h by MTT tests. Curcumin reduced tumour cell viability in a dose-dependent manner, with calculated IC 50 values of 19.42 μg/mL. sorafenib could increase antitumour activity of curcumin. A synergetic antitumour effect was displayed between curcumin and sorafenib ( Figure s1).
Annexin V-FITC/PI staining was used to quantify the percentages of apoptotic cells in the total cell population. In FTC133 cells, the percentages of apoptotic cells increased with increasing concentration of curcumin (15, 25 μM). Combined treatment (curcumin (25 μM) and sorafenib (2 μM)) significantly increased the total number of apoptotic cells compared with that treated with individual drugs (p < 0.05). The apoptotic cell populations of FTC133 cells were 10.44 ± 1.23, 18.85 ± 3.84 and 44.17 ± 6.12%, respectively, after treatment with curcumin (15, 25 μM) or their combinations (curcumin (25 μM) and sorafenib (2 μM)). These results indicated that sorafenib could increase the apoptosis inducing effect of curcumin in FTC133 cells (Figure s2).
The long-term suppression effect treated for 14 days with curcumin (15, 25 μM) or curcumin (25 μM) and sorafenib (2 μM) was examined with the clonogenic assay. In this assay, an effective antitumour treatment results in a reduction in cell colony formation. As shown in Figure s3, FTC133 cells colony forming decreased significantly when the concentrations of curcumin increased. Combined treatment (curcumin (25 μM) and sorafenib (2 μM)) showed stronger antitumour effect than curcumin alone (Figure s3).
In vitro invasion and migration assays were used to determine the inhibitory effect of curcumin on the invasive potency of thyroid cancer cells lines FTC133. The invasion and migration of FTC133 cells were inhibited by curcumin in a concentration-dependent manner at 15 and 25 μM (Figure s4). Combined treatment (curcumin (25 μM) and sorafenib (2 μM)) significantly decreased the invasion and migration of FTC133 cells compared with that treated with individual drugs (p < 0.05) (Figure s4).
Aberrant up-regulation of PI3K/AKT and ERK pathways support different aspects of the cancer cell growth, differentiation, survival, transformation, metabolism and cell cycle regulation (Kolch 2000;Chang et al. 2003;Courtney et al. 2010). The role of the ERK signalling pathway in cellular proliferation has been well established. For example, epidermal growth factor stimulates transient ERK activation, which leads to a mitogenic response (Marshall 1995). It has been proposed that the duration of ERK activity is important in determining cellular response and that prolonged activation of the ERK pathway may induce cell growth arrest (Alblas et al. 1998). PI3K/Akt pathway is one of the major cell survival pathways regulating cellular proliferation, transcription and metabolism (Brognard et al. 2001). It is known that PI3K promotes Akt activation through an initial phosphorylation at Thr308 by phosphoinositide-dependent kinase 1 and an additional phosphorylation at ser473 by the mammalian target of rapamycin complex 2 (Martelli et al. 2003). Akt signalling plays a crucial role in carcinogenesis and cancer development. Many reports show that inactivation of Akt by dephosphorylation plays a key role in tumour suppression (Martelli et al. 2003). Western blotting was used to quantitate and compare protein levels of ERK, p-ERK, PI3K, Akt and p-Akt. The results showed that the levels of p-ERK and p-Akt proteins were significantly reduced (p < 0.01) in the medicine-treated FTC133 cells. No statistic significances were found for other proteins (ERK, PI3K and Akt) tested in this experiment (Figures s5 and s6).

Conclusion
In conclusion, the curcumin exhibited in vitro anticancer effects towards thyroid cancer cells FTC133. There is a synergetic anticancer effect between curcumin and sorafenib. Further studies showed that the underlying mechanism involved in the anticancer activity of curcumin is related to PI3K/Akt pathway and ERK pathway.

Disclosure statement
No potential conflict of interest was reported by the authors.