Inhibition of growth in medullary thyroid cancer cells with histone deacetylase inhibitors and lithium chloride

Abstract

Background: While representing only 3% of thyroid malignancies, medullary thyroid cancer (MTC) accounts for 14% of thyroid cancer deaths. MTC has a high rate of recurrence and lacks effective treatments. The histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) activate the Notch1 signaling pathway, while lithium chloride inhibits the glycogen synthase kinase-3ss (GSK-3ss) pathway. These compounds have been shown to limit growth and suppress hormonal secretion; thus, targeting different signaling pathways may be an effective treatment.

Methods: MTC cells were treated with varying combinations of up to 20 mM lithium chloride with either 3 mM VPA or 20 muM SBHA for 48 h. Western analysis was used to measure the effects on Notch1, GSK-3ss, and neuroendocrine (NE) markers. Growth was assessed by a methylthiazolyldiphenyl-tetrazolium (MTT) bromide cellular proliferation assay. Western analysis was used to determine the mechanism of growth regulation.

Results: Combination therapy increased active Notch1, inhibited the GSK-3ss pathway, and decreased NE markers. Additive inhibition of growth was observed with combination therapy. Lower-dose combination therapy achieved greater decreases on NE markers and growth than treatment with any of the drugs alone. Moreover, an increase in the cleavage of the apoptotic markers caspase-3 and PARP was observed.

Conclusions: Combination therapy with lithium chloride and HDAC inhibitors suppresses NE markers and decreases growth via apoptosis of MTC cells in vitro. With the possibility of increased efficacy and decreased toxicity, combination therapy may represent a new strategy to treat MTC.

Figure 1

Combination therapy limits growth of MTC cells. MTC cells were treated with the indicated combinations of VPA, SBHA, and lithium chloride for up to 6 days, and cell viability was determined by an MTT colorimetric growth assay. Points represent mean +/- SE. All treatments are significantly different from control after two days of treatment, and this inhibition became more significant over time (* = P < 0.01, ** = P < 0.001, one-way ANOVA). MTC, medullary thyroid cancer, VPA, valproic acid, SBHA, suberoyl bis-hydroxamic acid, Li, lithium chloride, MTT, methylthiazolyldiphenyl-tetrazolium.

Figure 2

The mechanism of growth inhibition by combination therapy is apoptosis. MTC cells were treated with the indicated concentrations of VPA, SBHA, and lithium chloride for 2 days, and total cell lysates were prepared. An increase in the cleavage of PARP and caspase-3 suggests that the mechanism of inhibition is apoptosis. GAPDH was used as a loading control. MTC, medullary thyroid cancer, VPA, valproic acid, SBHA, suberoyl bis-hydroxamic acid, Li, lithium chloride, GAPDH, glyceraldehyde 3-phosphate dehydrogenase, PARP, poly-ADP ribose polymerase.

Figure 3

Combination therapy upregulates Notch1 and inhibits GSK-3ß in MTC cells. At baseline, MTC cells express minimal active Notch1. Treatment for 2 days with the HDAC inhibitors VPA (lane 3) or SBHA (lane 5) increases the amount of cleaved Notch1 protein (NICD). Additionally, treatment with lithium chloride phosphorylates GSK-3ß, demonstrating inhibition of the pathway (lane 2). Combination therapy with either VPA or SBHA and lithium chloride (lanes 4 and 6) affects both pathways simultaneously. GAPDH is shown as a loading control. MTC, medullary thyroid cancer. HDAC, histone deacetylase, VPA, valproic acid, SBHA, suberoyl bis-hydroxamic acid, Li, lithium chloride, GSK-3ß, glycogen synthase kinase-3ß, GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

Treatment with the combination of lithium chloride and either VPA or SBHA reduces neuroendocrine markers more than treatment with higher doses of the drugs alone in MTC cells. Western blot analysis showed a decrease in levels of ASCL1, a pro-neuroendocrine gene, and CgA, a marker of hormonal secretion. Importantly, lower-dose combination therapy was more effective than treatment with higher doses of the drugs alone. MTC, medullary thyroid cancer, VPA, valproic acid, SBHA, suberoyl bis-hydroxamic acid, Li, lithium chloride, GAPDH, glyceraldehyde 3-phosphate dehydrogenase, CgA, chromogranin A, ASCL1, achaete scute complex-like 1.