Suberoyl bishydroxamic acid activates notch1 signaling and suppresses tumor progression in an animal model of medullary thyroid carcinoma

Abstract

Background: Medullary thyroid carcinoma (MTC) is a neuroendocrine malignancy that frequently metastasizes and has few treatments. This study was aimed at assessing the antitumor effects of suberoyl bishydroxamic acid (SBHA) in an in vivo model of MTC.

Methods: Nude mice were injected with human MTC cells, and the groups were treated with SBHA (200 mg/kg) or vehicle (dimethyl sulfoxide) in saline injection every other day for 12 days. Tumors were measured every 4 days and collected at 12 days for Western blot analysis.

Results: Treatment with SBHA resulted in an average 55% inhibition of tumor growth in the treatment group (P < .05). Analysis of SBHA-treated MTC tumors revealed a marked increase in the active form of Notch1 (NICD) with a concomitant decrease in achaete-scute complex-like 1 (ASCL1), a downstream target of Notch1 signaling, as well as the neuroendocrine tumor marker chromogranin A. Importantly, SBHA treatment resulted in an increase in protein levels of p21(CIP1/WAF1), p27(KIP1), cleaved caspase-9, cleaved caspase-3, and cleaved poly ADP-ribose polymerase and concomitant with a decrease in cyclin D1 and cyclin B1, indicating that the growth inhibition was due to both cell cycle arrest and apoptosis. Moreover, SBHA downregulated cell survival proteins Bcl-2 and Bcl-X(L), but upregulated apoptotic proteins Bax, Bad, and Bmf.

Conclusion: These results demonstrate that SBHA inhibits MTC growth in vivo. SBHA is a promising candidate for further preclinical and clinical studies in MTC.

Figure 1

SBHA inhibits growth of human medullary thyroid carcinoma cells in vivo. Nude mice were injected with TT cells (10⁶). Tumor bearing animals were treated with SBHA (200 mg/kg) or DMSO by intraperitoneal (i.p.) injections every other day for 12 days. Growth of tumors during course of experiment was measured by increases in calculated tumor volumes. P < 0.05 between control tumors and SAHA-treated tumors by Bonferroni post hoc test.

Figure 2

SBHA induces accumulation of acetyl-histone H₄ (Lys12) (AH₄). Total tumor tissue extracts were isolated from SBHA-treated or DMSO-treated mice and analyzed for the levels of AH₄. Western blot analysis showed that AH₄ is significantly increased in SBHA-treated samples compared to those in the control group. G3PDH was used to confirm equal protein loading. The quantification of the bands was generated by comparison with G3PDH.

Figure 3

SBHA activates the Notch1 signaling pathway and decreases levels of chromogranin A (CgA). Total tumor tissue extracts were isolated from SBHA-treated or DMSO-treated mice and analyzed by Western blotting for expression of Notch1 intracellular domain (NICD), achaete-scute complex-like 1 (ASCL1) and CgA. SBHA treatment induced NICD protein, which led to a concomitant decrease in ASCL1, a downstream target of Notch1 signaling, as well as NE tumor marker CgA. G3PDH was used to confirm equal protein loading. The quantification of the bands was generated by comparison with G3PDH.

Figure 4

SBHA induces cell cycle arrest in MTC xenografts. Tumor tissue lysates from SBHA- or DMSO-treated mice were analyzed by Western blotting for levels of cell cycle regulators. Treatment of animals with SBHA resulted in significant increases in protein levels of p21^CIP1/WAF1and p27^KIP1 and significant decreases in levels of cyclin B1 and cyclin D1, indicating cell cycle arrest. G3PDH was used to confirm equal protein loading. The quantification of the bands was generated by comparison with G3PDH.

Figure 5

SBHA regulates the expression of apoptotic mediators and induces caspase-dependent apoptosis in a mouse model of MTC. Western blot analysis was performed for the levels of apoptotic mediators utilizing tumor tissue lysates. (A) SBHA treatment resulted in increased caspase-9, caspase-3 and PARP cleavage, indicating activation of the apoptotic pathway. (B) After SBHA treatment, the antiaopototic proteins Bcl-2 and Bcl-X_L were markedly down-regulated. In contrast, the expression levels of proapoptotic proteins Bad, Bmf and Bax were up-regulated. G3PDH was used to confirm equal protein loading.