Curcumin treatment suppresses IKKβ kinase activity of salivary cells of patients with head and neck cancer: a pilot study

Abstract

Purpose: To determine whether curcumin would inhibit IκB kinase β (IKKβ) kinase activity and suppress expression of proinflammatory cytokines in head and neck squamous cell carcinoma cancer (HNSCC) patients.

Experimental design: Saliva was collected before and after subjects chewed curcumin tablets. Protein was extracted and IKKβ kinase activity measured. Interleukin (IL)-6 and IL-8 levels in the salivary supernatants were measured by ELISA. IL-6, IL-8, and other interleukin were also measured independently with ELISA to confirm the inhibitory effect of curcumin on expression and secretion of salivary cytokines.

Results: Curcumin treatment led to a reduction in IKKβ kinase activity in the salivary cells of HNSCC patients (P < 0.05). Treatment of UM-SCC1 cells with curcumin as well as with post-curcumin salivary supernatant showed a reduction of IKKβ kinase activity. Significant reduction of IL-8 levels (P < 0.05) was seen in post-curcumin samples from patients with dental caries. Although there was reduced IL-8 expression in 8 of 21 post-curcumin samples of HNSCC patients, the data did not reach statistical significance. Saliva samples from HNSCC patients were also analyzed in a blinded fashion for expression of cytokines. IL-10, IFN-γ, IL-12p70, and IL-2 clustered together, and granulocyte macrophage colony stimulating factor and TNF-α clustered together. Log₁₀ ratio analysis showed decrease in expression of all nine cytokines in both the salivary supernatant and salivary cells of curcumin-treated samples.

Conclusions: Curcumin inhibited IKKβ kinase activity in the saliva of HNSCC patients, and this inhibition correlated with reduced expression of a number of cytokines. IKKβ kinase could be a useful biomarker for detecting the effect of curcumin in head and neck cancer.

Figure 1

Curcumin inhibits salivary cell IKKβ kinase activity of head and neck cancer patients. A, Dose dependent curve of IKKβ kinase activity was achieved using five IKKβ protein concentrations in increments of 50 pg/mL. The enzyme activity is linear up to 250 pg/mL tested in the present assay. B, Protein was extracted from salivary samples collected from patients with HNSCC before and after a 1-hour curcumin treatment. IKKβ kinase activity was measured using the IKKβ Kinase Assay Kit. Six of 10 samples show a decrease in IKKβ kinase activity, with three samples (#7, #16 and #18) indicating a 50% reduction post curcumin treatment. C, Boxplot showing expression level of IKKβ enzyme activity in pre and post curcumin salivary samples. The results indicate a statistically significant inhibition of IKKβ kinase activity by curcumin with a p value of <0.05. D, The UM-SCC1 cell line was treated with DMSO, curcumin dissolved in DMSO, or saliva samples collected pre and post curcumin treatment. The cells were also pretreated with TNF-α for 15 minutes. As reported earlier, addition of curcumin inhibits IKKβ kinase activity of UM-SCC1 cells. One of the two post curcumin salivary samples (sample # 17) exhibits an inhibitory effect on IKKβ kinase activity, indicating usefulness of this methodology to determine IKKβ kinase levels in the saliva of HNSCC patients. The IKKβ kinase assay performed at four hours in untreated samples (UnTx) shows a reduction in comparison to 1 hour due to loss of growth factors. The 1 hour treatment of UN-SCC1 cells with pre curcumin saliva:medium (50%:50%) shows reduced IKKβ kinase activity in comparison to untreated controls due to the presence of half the level of growth factors in this mixed medium.

Figure 2

Inhibitory effect of curcumin on IL-8 expression in the saliva of patients with dental caries. A, The IL-6 expression is less than 5 pg/mL in four of five normal individuals, which is similar to the expression level reported in the literature. Individual #1 has high level expression that could reflect an inflammatory condition. B, IL-8 expression in three individuals resembles the expression range reported in the literature. Individual #3 has three times the expected expression. Appreciable changes were not observed in IL-6 or IL-8 levels after curcumin treatment. C, Of the thirteen patients with dental caries tested, one sample, #11 shows a 50% reduction and another sample #3 has a four-fold increased expression of IL-6. Appreciable changes are not seen in other samples. D, Eight of the dental caries patients show reduced IL-8 expression and four of them show greater than 25% reduction (#1, 3, 11 and 13). One sample, #8, shows a 20% increase in IL-8 expression post curcumin treatment.

Figure 3

Modest inhibitory effect of curcumin on salivary IL-8 expression in head and neck cancer patients. A, Of the twenty-one head and neck cancer patient samples analyzed, two samples (#10 and 20) show decreased expression and three samples (#11, 13 and 22) show increased expression of IL-6 following curcumin treatment. B, Curcumin treatment results in greater than a 20% decreased expression of IL-8 in five samples (#2, 10, 16, 17 and 19). Higher than 20% expression of IL-8 is seen in two samples, # 11 and # 22, the same samples that have shown increased IL-6 expression after curcumin treatment. The modest inhibitory effect on IL-8 suggests that higher dose of curcumin and longer treatment time periods may be required to induce significant inhibitory effect on cytokine expression in the saliva of different head and neck cancer patients.

Figure 4

Inhibitory effect of curcumin on inflammatory cytokines. Analysis of 14 salivary samples using the Human 9-Plex Ultrasensitive Electrochemiluminescent MULTI-SPOT Assay system followed by the cluster analysis shows decreased expression of all nine cytokines in A, supernatant and in B, cellular pellet. While the inhibition is variable as was seen in figure 3, cluster analysis points to enhanced inhibitory activity on tongue tumors indicating the usefulness of curcumin in the treatment of this tumor type. Overexpression of IL-6 and IL-8 observed in the supernatant samples of #11 and #13 confirmed the results obtained with the individual cytokine ELISA method (Figure 3 above), indicating the usefulness of either of the methods to detect interleukin expression in salivary samples.