In this study we showed the efficacy of the indirubin derivative 7BIO in inducing cell death in thyroid carcinoma cell lines. The kind of cell death induced was a non-classical death with cell cycle block, DNA fragmentation as well as signs of non-classical apoptosis and necrosis.
The direct pharmacological induction of cell death is one possibility to overcome the resistance of cancer cells towards apoptosis that is a hallmark of tumour cells [
12]. Dedifferentiated and anaplastic thyroid carcinoma cells that have lost the ability to take up radioiodine are tumour cells that are resistant to cell death induction by external radiation and chemotherapeutic treatment [
23]. One reason for this resistance may be the inability of these cells to undergo apoptotic cell death [
14]. Direct induction of cell death therefore presents one possibility of new treatment strategies for these tumours. 7BIO is an indirubin derivative that has been recently described as inducing cell death in some tumour cell lines of various origins [
3‐
5]. In this study we showed that 7BIO is also active against dedifferentiated thyroid carcinoma cells.
7BIO showed inhibitory effects on all 14 thyroid carcinoma cell lines investigated. IC50 values of our cell lines were in a narrow range (1.54–4.83 µM) with the C643 ATC cell line being the most sensitive cell line (IC50 of 1.54 µM; Table
1). All three papillary cell lines had IC50 values in the higher range (4.68–4.83 µM; Table
1) with BHT101 showing the highest IC50 value of our cell lines. In FTC and ATC cell lines, no correlation was found between the 7BIO sensitivity and the thyroid carcinoma subtype from which the cell lines had been derived (IC50 values in FTC cell lines 1.98–4.62 µM; IC50 in ATC cell lines 1.54–4.32 µM; Table
1). The molecular reasons for these differences have to be elucidated and may be due to specific activation of signaling pathways in these histological thyroid carcinoma subtypes. Moreover, the IC50 values in thyroid carcinoma cells were in the lower range of that reported in 7BIO-treated cell lines of other tumours (2.3–20.0 µM for breast cancer cell lines and approx. 12 µM for SH–SY5Y neuroblastoma cells) [
3,
5] After incubation with 10 µM 7BIO for 48 h, all cell lines except ML1 achieved values of less than 10 % of control pointing to cell death in the majority of cells. A similar effect was already described by Ribas et al. [
4] who showed that a 24 h treatment with 25 µM 7BIO was lethal for the entire cell population of SH-SY5Y neuroblastoma cells and Jurkat leukaemia cells. Our biochemical data in six 7BIO-treated thyroid carcinoma cells argue for an atypical kind of cell death with biochemical signs of necrosis, DNA fragmentation, cell cycle inhibition, and atypical apoptosis. An increased LDH release typical for cell death by necrosis or secondary necrosis was seen in all six cell lines examined after 7BIO stimulation. In parallel, a small but significant increase in activity of caspases seen as direct caspase activity and increase in caspase cleavage products was found in all cells but C643. However, the significance of apoptosis-induction for cell death in 7BIO stimulated cells has to be questioned since the pan-caspase inhibitor Q-VD-OPh did not significantly prevent cell death by 7BIO (Table
3). Similar results have already been described in other cell lines for 7BIO. In neuroblastoma cells and the T cell leukemia cell line Jurkat, cell death by an apoptosis-independent manner was induced by 7BIO [
3,
4]. In IMR-5, IMR-32, Jurkat and HL-60 cells, 7BIO treatment killed cells without activating caspases [
3,
45]. Nicolaou et al. [
5] on the other hand reported on 7BIO-mediated apoptosis induction by caspase-dependent as well as by caspase-independent pathways in breast cancer cell lines with an increase in the uncleaved caspases 3 and 9. From their own data and data in the literature these authors concluded that the response of different tumour cell lines to 7BIO through apoptotic or non-apoptotic mechanisms is a function of cell content [
5]. Our own data suggest a cell death mechanism in which caspase activation occurs but is of little importance. This thesis is supported by the C643 anaplastic thyroid carcinoma cell line that was the most sensitive cell line towards 7BIO incubation but showed no significant activation of caspases by 7BIO. Caspase-independent cell death by apoptosis has already been described in various cell systems [
46]. Physiologically it occurs in cells expressing endogenous caspase inhibitors like XIAP, CrmA or AIF [
47‐
50]. In affected cells, apoptosis-like death is generally caused by serine-protease-mediated processes [
51] that mediate the cleavage of cellular substrates similar to caspase-induction. The lysosomal proteases cathepsin D and B as well as calpains and other serine-proteases and are involved in this caspase-independent, apoptosis-like cell death [
52‐
55]. The findings on the kind of cell death in our 7BIO-stimulated thyroid carcinoma cell lines are in accordance with these findings in cell lines undergoing cell death after various stimuli and also with the recently reported results of Ribas et al. [
4] who reported on the involvement of serine proteases in 7BIO-induced cell death. Further experiments to analyse protease activation and to characterize intracellular targets of 7BIO may elucidate the exact kind of cell death induced by 7BIO.
Regarding increases in subG1 fraction after 7BIO treatment, follicular FTC236 and ML1 cells interestingly showed a large fraction of cell in subG1 peak pointing to massive DNA fragmentation in these cells like already described by others [
5]. In the other cell lines examined, fractions of cells in subG1 fraction were also significantly elevated, but to a lesser extent. Since DNA fragmentation can be mediated by various proteolytic enzymes, a caspase-independent mechanism of DNA fragmentation probably occurred with different degrees of activation in different 7BIO-treated thyroid carcinoma cell lines. In other cell lines, a caspase-independent, serine protease mediated DNA cleavage has already been described [
51,
56,
57]. However, DNA fragmentation resulting in an elevated subG1 fraction may also occur during necrotic cell death [
58] and in our cells may reflect a mixed kind of cell death with non-classical apoptosis and necrotic features. This interpretation is in accordance with data from the literature that cell death in vivo is often characterized not by a single cell death mode but can comprise elements of apoptotic, necrotic, and sometimes autophagic elements. Accordingly, various kinds of cell death can occur independently of each other or simultaneously resulting in a combined cell death phenotype [
59,
60]. Cell cycle analyses of the remaining living cells pointed to different regulation mechanisms in various thyroid carcinoma cell lines treated with 7BIO. All cell lines except C643 showed a significant increase in the portion of cell in G1 fraction after incubation with 7BIO. This is in contrast to some other cell lines described in the literature that exhibited a decrease in G1 fraction following 7BIO treatment [
3,
5] that is also described for other indirubins [
6,
61,
62]. C643 cells showed a similar cell cycle distribution with a small, but not significant decrease in G1 phase (Table
2). The activation of different checkpoints of the cell cycle points to different cellular backgrounds of cells with respect to cell cycle regulatory proteins like p53, pRb as well as CDKs and its regulatory proteins [
63]. Further studies may reveal the correlation of expression pattern of cell cycle regulatory proteins with the cellular effects of 7BIO treatment in different cells.