Sensitization of head and neck cancer to cisplatin through the use of a novel curcumin analog

Abstract

Objective: To determine whether a novel small molecule inhibitor derived from curcumin (FLLL32) that targets signal transducer and activator of transcription (STAT) 3 would induce cytotoxic effects in STAT3-dependent head and neck squamous cell cancer (HNSCC) cells and would sensitize tumors to cisplatin.

Design: Basic science. Two HNSCC cell lines, UM-SCC-29 and UM-SCC-74B, were characterized for cisplatin [cis-diammineplatinum(II) dichloride] sensitivity. Baseline expression of STAT3 and other apoptosis proteins was determined. The FLLL32 50% inhibitory concentration (IC(50)) dose was determined for each cell line, and the effect of FLLL32 treatment on the expression of phosphorylated STAT3 and other key proteins was elucidated. The antitumor efficacy of cisplatin, FLLL32, and combination treatment was measured. The proportion of apoptotic cells after cisplatin, FLLL32, or combination therapy was determined.

Results: The UM-SCC-29 cell line is cisplatin resistant, and the UM-SCC-74B cell line is cisplatin sensitive. Both cell lines express STAT3, phosphorylated STAT3 (pSTAT3), and key apoptotic proteins. FLLL32 downregulates the active form of STAT3, pSTAT3, in HNSCC cells and induces a potent antitumor effect. FLLL32, alone or with cisplatin, increases the proportion of apoptotic cells. FLLL32 sensitized cisplatin-resistant cancer cells, achieving an equivalent tumor kill with a 4-fold lower dose of cisplatin.

Conclusions: FLLL32 monotherapy induces a potent antitumor effect and sensitizes cancer cells to cisplatin, permitting an equivalent or improved antitumor effect at lower doses of cisplatin. Our results suggest that FLLL32 acts by inhibiting STAT3 phosphorylation, reduced survival signaling, increased susceptibility to apoptosis, and sensitization to cisplatin.

Figure 1

MTT chemosensitivity assays were performed for a range of cell lines using a wide range of cisplatin doses. The UM-SCC-29 cell line demonstrates cisplatin resistance (IC50=12.5μM) relative to the cisplatin sensitive UM-SCC-74B cell line (IC50=4.8μM).

Figure 2

The IC50 for FLLL32 was determined in the UM-SCC-29 and UM-SCC-74B through an MTT chemosensitivity assay. The IC50 for the UM-SCC-74B and UM-SCC-29 cell lines was 1.4 and 0.85 μM, respectively.

Figure 3

UM-SCC-74B and UM-SCC-29 cells were characterized for STAT3 and p-STAT3 expression, as well as anti-apoptotic proteins (AKT1, p-AKT1, Bcl-xL, survivin) and pro-apoptotic proteins (SMAC, caspase-3) involved in the JAK-STAT pathway. Expression of EGFR, an upstream STAT3 activator, and p53 was determined.

Figure 4

Western blot demonstrates a down-regulation of phosphorylated STAT3 in the UM-SCC-29 and -74B cell lines following administration of FLLL32, with or without cisplatin. The expression level of non-phosphorylated, inactive STAT3 remains unchanged in both cell lines after the addition of FLLL32 or cisplatin, alone or in combination. JAK2 and non-phosphorylated STAT3 expression was stable across cell lines and treatment groups. Phosphorylated JAK2 protein (not shown) could not be isolated. FLLL32, with or without cisplatin, did not alter the expression of the pro-apoptotic proteins Bcl-xL or AKT or of the upstream signaling molecule EGFR. GAPDH was used as a positive loading control.

Figure 5

In UM-SCC-74B cells, which are relatively cisplatin sensitive, combination therapy with FLLL32 and cisplatin (1.5625μM) induced an equivalent cytotoxic effect to cisplatin monotherapy at a dose four times higher (6.25μM) and enhanced cytotoxicity versus cisplatin monotherapy at doses of 1.5625 – 3.25 μM (p<0.001). The same effect was observed in cisplatin resistant UM-SCC-29 cells. This suggests that FLLL32 can induce sensitivity in HNSCC cells and that this effect is not compromised by intrinsic resistance to cisplatin.

Figure 6

FLLL32 alone induced an increase in apoptosis in UM-SCC-74B and -29. The combination of FLLL32 and cisplatin induces greater apoptosis than either agent alone. Interestingly, the cisplatin resistant UM-SCC-29 cell line exhibited greater increases in apoptosis than UM-SCC-74B in all treatment groups. Data is shown as apoptosis flow cytometry plots (y-axis: logarithmic quantification of annexin V-FITC positive cells; x-axis: logarithmic quantification of propidium iodide positive cells), and as bar graphs with data tables.