With our experiments we showed the efficacy of the BH3 mimetic drug ABT-737 in reducing the number of viable cells and in inducing apoptotic cell death in thyroid carcinoma cells of various histological origins.
Patients with dedifferentiated and anaplastic thyroid carcinomas are difficult to treat since during dedifferentiation thyroid carcinoma cells lose their ability to take up radioiodine. Furthermore, thyroid carcinoma cells are resistant to external radiation and chemotherapeutic treatment [
4]. The inability of these cells to undergo apoptotic cell death is one reason for this resistance [
12,
13] and may be influenced by a new class of pharmacological compounds termed BH3 mimetics [
23,
35]. BH3 mimetic drugs, such as ABT-737, are due to counter apoptotic blocks by binding to anti-apoptotic proteins like BCL-2, BCL-xL and BCL-w [
23,
24]. In preclinical studies, ABT-737 showed single agent efficacy against some tumor types like small cell lung cancer, follicular lymphoma and chronic lymphocytic leukemia [
36‐
38]. Some recently completed phase 1 trials with ABT-263, the orally available analogue drug of ABT-737, also showed clinical responses in 30–50 % of patients with chronic lymphocytic leukemia (CLL) [
26,
27] but not in small cell lung cancer (SCLC) patients [
28].
A better sensitivity towards ABT-737 was, with the exception of FTC cell lines FTC238 and RO82W, found in FTC and PTC cell lines compared to more dedifferentiated ATC cell lines reflecting the aggressiveness and treatment resistance of ATC cells. Regarding the sensitivity towards ABT-737, it was reported that ABT-737 is effective in cells with overexpressed BCL-2, BCL-xL or BCL-w but does not bind to the anti-apoptotic proteins MCL1 or A1 [
23]. Accordingly, it has been shown that an elevated expression of MCL1 or BFL-1/A1 may mediate the resistance of tumor cells to ABT-737-mediated apoptosis [
36,
43]. Furthermore, the effect of ABT-737 is dependent on the expression of the pro-apoptotic proteins BAK and/or BAX which execute apoptosis when liberated from BCL-2 after ABT-737 treatment [
39,
44]. In thyroid tissues, an elevated expression of BAX was demonstrated in thyroid carcinoma compared to adenoma [
45]. Furthermore it was shown in different studies that BCL-2 expression is strong in most differentiated thyroid carcinoma but decreases in less differentiated subtypes [
46‐
49]. BCL-xL expression on the other hand was shown to be stronger in high-risk subtypes of thyroid carcinoma [
50], while little is known about MCL1 expression in thyroid carcinoma tissues. Expression profiles of BCL-2 family proteins in our thyroid carcinoma cells was already analyzed in a recent study by our group [
51]: While expression of BAK was relatively constant as a prerequisite for ABT-737 action, BAX was not expressed in FTC238 and C643 and showed a weak expression in sensitive BHT101 and insensitive 8305 and 8505 cells [
51]. Expression of BCL-2, BCL-xL and MCL-1 was variable [
51] but not related to ABT-737 sensitivity (Table
1): The most sensitive cell line B-CPAP (IC50: 0.73 μM) showed a weak expression of both BCL-2 and BCL-xL, while MCL-1 was moderately expressed [
51]. The four most sensitive cell lines besides B-CPAP (BHT101, ML1, FTC133 and TT2609) on the other hand showed a strong expression of either BCL-2 or BCL-xL and moderate expression of MCL-1 [
51]. The most insensitive cell line RO82W expressed BCL-xL strongly and BCL-2 moderately [
51]. In sensitive BHT101 (IC50: 1.20 μM) as well as in insensitive 8305 cells (IC50: 10.9 μM), a very low expression of both BCL-2 and BCL-xL proteins was seen [
51]. Furthermore, in contrast to recent data of Iacovelli et al. [
52] who showed that in five acute lymphocytic leukemia (ALL) cell lines the ratio of MCL-1/BCL-2 plus BCL-xL protein ratio was correlated with sensitivity to ABT-737, in the 16 thyroid carcinoma cell lines analyzed in this study, no such correlation was found [
51]. In mouse cell lines derived from thyroids of mouse strains with deleted
p53 and activated
Kras that develop PTC and PDTC, high expression of
Bcl-
2 and
Mcl1 was reported that mediate resistance to apoptosis [
53]. These cell lines can be targeted by GX15-070 (obatoclax), a pan-inhibitor of the BCL-2 family, while ABT-263 was modestly effective [
53] which generally showed the suitability of BH3 mimetics for treatment of thyroid carcinoma cells. In one early study, Mitsiades and coworkers [
54] also showed the efficacy of the BH3 inhibitors BH3I-1 and BH3I-2 in some thyroid carcinoma cell lines as well as sensitization to other anti-tumor substances [
54]. In own experiments, we have recently shown the potency of GX15-070 against dedifferentiated thyroid carcinoma cells of various histological origins [
51]. Treatment with GX15-070 resulted in a non-classical cell death with signs of apoptosis, autophagy and necrosis in parallel [
51] that was also seen in other cell systems [
55‐
57]. While GX15-070 and its cellular targets besides the proteins of the BCL-2 family are not known yet, ABT-737 was shown to act as a real BH3 mimetic [
23]. However, our data indicate that the expression of pro- and anti-apoptotic proteins alone does not predict sensitivity to ABT-737. These results are underlined by several other recent papers: In ovarian carcinomas, it was shown that phospho-ERK1/2 as well as a low expression of BIM are biomarkers for absence of response to ABT-737 [
58]. Phosphorylation of MCL-1 and BCL-2 are found to be further determinants of sensitivity to ABT-737 [
59,
60]. Phosphorylation of MCL-1 at various threonine and serine residues by Cyclin E/Cdk2 kinase, ERK (extracellular-signal regulated kinase), JNK (c-jun N-terminal kinase), p38 MAPK (mitogen-activated kinase) and GSK-3β (glycogen synthase kinase-3β) can lead to stabilization as well as destabilization of MCL-1 [
59,
61‐
64], while phosphorylation of BCL-2 leads to a structural alteration in the BH3-binding groove and resistance to ABT-737 [
60]. Furthermore, treatment of cells with ABT-737 can lead to altered expression of proteins of the BCL-2 family [
65,
66]. Thus, prediction of sensitivity of a cell line to ABT-737 treatment is a topic under investigation in many cell systems and also needs further investigation in thyroid carcinoma cells. However, with the availability of ABT-737 and its orally active derivative ABT-263, our data on the potency of BH3 mimetics become a current topic.