High-throughput Drug Library Screening Identifies Colchicine As A Thyroid Cancer Inhibitor

Objection: Papillary thyroid carcinoma is the most frequent thyroid cancer accounting for 85–90% of all thyroid malignancies. BRAFV600 E mutation is the most common mutation identified in the BRAF gene,BRAF mutations are known to alter the biological status ofcancer cells, promoting tumor progression and represents a novel indicator of poorer prognosis in a broad range of human cancers including thyroid cancer, colon cancer and melanoma carcinoma, especially in thyroid cancers. BRAFV600 E mutation was found exclusively in papillary thyroid carcinoma and in papillary thyroid carcinoma-derived anaplastic thyroid carcinoma. Aggressive thyroid cancers remain resistant to traditional therapeutic ways. Novel targeted BRAF V600 E compounds are being tested in the preclinical and clinical applications. Inhibition of the BRAFV600 E oncoprotein by small-molecule drugs such as PLX4032(vemurafenib) is highly effective in the treatment of melanoma. However, thyroid cancer patients who are radioiodine refractory and harbor the same BRAFV600 E oncogenic mutation, show limited response to this class of inhibitors. It was reported that BRAF(V600E) inhibition causes a rapid feedback activation of EGFR, and blockade of the epidermal growth factor receptor(EGFR) shows strong synergy with BRAF(V600E) inhibition. Although such drug combinations may show more effective action, the potential for increased toxicity and economic implications can limit clinical feasibility. Prompted by these limitations, we set out to identify existing compounds active in thyroid cancer cells and explore their mechanism of cytotoxicity.Methods: To identify small molecules that can inhibit the viability of thyroid cancer cells,we developed a screening platform using 8505 C and KTC-1 cells that harbor the BRAFV600 E mutation and, as a reference for a non-thyroidal BRAF mutant phenotype, human melanoma Malme-3M cells. The screening libraries included approximately 5200 small molecules selected to facilitate drug reposition and mechanistic studies. cell viability was detected by Alamar Blue assay, and cell growth was also monitored by directly cell count. To examine the mechanisms underlying growth inhibition of colchicine, we monitored cell cycle phase progression by flow cytometry. Impact of colchicine on thyroid cancer cell apoptosis was detected by western blot and Annexin V-FITC and PI staining using flow cytometry. Mechanisms in colchicine-induced thyroid cancer cell apoptosis were determined by western blot and development of colchicine-resistant sub-lines. To confirm the activity of colchicine in vivo and the toxicity in mice, we establishment the thyroid cancer mice models.Results: Primary screens yielded a number of hits differentially active between thyroid and melanoma cells. In particular, amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Validation testing demonstrated the ability of colchicine to decrease 8505C、KTC-1、WRO、TPC-1 thyroid cancer cell viability and cell density by Alamar Blue assay and direct cell count. The impact of colchicine on increasing the proportion of 8505 C and WRO cells in G2/M phase, and shows a markedly diminished entry of cells into the G1 phase by flow cytometry. Externalization of phosphatidylserine, an early marker of apoptosis detected by Annexin V, and late marker of apoptosis detected by PI, were observed by flow cytometry in live cells treated with variable concentrations of colchicine(0.01-1.0 μM) across different time points(24-72 hrs). Further, we observed that the effect of colchicine correlated with PARP cleavage in a time and dose-dependent manner, as displayed by Western blotting.Importantly, the pro-apoptotic action of colchicine was accompanied by the activation of multiple signaling pathways. In 8505 C cells, we noted increased phosphorylation of the MAP kinases MEK/ERK, p38, and JNK. Further, despite anearly inhibitory impact detected after 24 hrs treatment, AKT phosphorylation was increased by 72 hrs in both cell types. In WRO cells, we also noted increased MEK, p38, and JNK phosphorylation in response to colchicine treatment, while elevated p ERK levels remained unaffected.To study the significance of these pathway alterations in relation to colchicine mode-of-action, we co-incubated colchicine with a variety of chemicals known to impact specific cell signaling routes. Inhibition of MEK1/2 with U0126 virtually abolished the apoptotic effect of colchicine, as shown by absence of PARP and Caspase 3 degradation, reduced apoptotic cell fraction, and rescue of cell viability. Similarly, we noted the ability of the JNK inhibitor SP600125 to diminish colchicine-induced apoptosis and rescue cell viability. In contrast, the effect of the p38 inhibitor SB203580 was less evident on PARP and Caspase 3 degradation in WRO cells, and it only partially rescued these cells from apoptosis with minimal impact on cell viability in response to colchicine treatment. Additionally, SB203580 failed to rescue colchicine-induced PARP and Caspase 3 degradation, apoptosis, or viability of 8505 C cells. Moreover, the AKT inhibitor LY294002 had no measureable impact on colchicine action in either cell type including apoptosis and cell viability.To further distinguish between the roles of different MAP kinases on colchicine action, we generated colchicine-resistant 8505 C cells. Compared with their sensitive parental cells, colchicine-resistant 8505 C cells continued to exhibit measureable p38 phosphorylation responses, but failed to stimulate MEK, ERK1/2, or JNK. To determine whether the effects of colchicine observed in vitro had an impact on tumor growth in vivo, we tested this drug in thyroid 8505 C and WRO cancer cell mouse xenografts. These studies revealed the ability of colchicine to significantly reduce 8505 C tumor volume and tumor weight in a dose-dependent manner. Comparable results were also recapitulated in the WRO cell xenografts. Further, PHH3 staining demonstrated the ability of colchicine to reduce the proportion of proliferating malignant cells in both cell line xenografts. TUNEL staining also identified asignificant impact on apoptosis in both animal models. Furthermore, we observed that colchicine administration was well-tolerated in mice with no adverse impact on body weight, and we found no gross morphological changes in the H& E stained tissues obtained from mice.Conclusion: 1. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer. 2. The colchicine activity involves with BRAF-mutant and BRAF-WT thyroid cancer cells. 3. Colchicine exhibits cytotoxicity against thyroid cancer cell lines through arresting of thyroid cancer cells at G2/M phase, as well as apoptosis in a time-and dose-dependent manner. 4. Colchicine-induced activation of MEK1/2 and JNK pathways mediates apoptosis over thyroid cancer cell. 5. Systemic colchicine effectively arrested thyroid cancer progression in xenografted mice. In vivo studies revealed that colchicine was well tolerated in mice with no adverse impact on body weight and overall health.