INHIBITOR OF THE TRANSCRIPTION FACTOR NF-κB, DHMEQ, ENHANCES THE EFFECT OF PACLITAXEL ON CELLS OF ANAPLASTIC THYROID CARCINOMA IN VITRO AND IN VIVO.

Anticancer drug paclitaxel (Ptx) effect on biochemical mechanisms, regulating apoptosis in anaplas- tic thyroid carcinoma cells, was studied. It was shown that in addition to apoptotic cell death, Ptx induces signaling cascades that ensure cell survival. Paclitaxel-induced activation of nuclear factor kappa B (NF-κB) leads to an increase of some antiapoptotic proteins expression such as survivin, cIAP, XIAP. A novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), was found to enhance cytotoxic effect of Ptx in anaplastic thyroid carcinoma cells. An enhancement of caspase-3 and -9 activation and PARP cleavage as well as the decreased levels of proteins-inhibitors of apoptosis were observed when cells were treated with a combination of both drugs. Mitochondria transmembrane potential (Δψ (m)) loss was observed at higher concentrations of Ptx and DHMEQ. NF-κB inhibition also potentiates paclitaxel effect at tumors formed by xenotransplantation of FRO cells into mice. Tumor mass reduction, significantly different from the effects of each of the compounds alone, was observed in animals, treated with paclitaxel and NF-κB inhibitor. Thus, the combined use of paclitaxel and NF-κB inhibitor inhibits biochemical processes that contribute to the resistance of anaplastic thyroid carcinoma cells to paclitaxel action.

Effects of Paclitaxel and combination of the drug with radiation therapy in an in vivo model of anaplastic thyroid carcinoma.

AIM: To study the effects of Paclitaxel (Ptx), γ-irradiation (IR) and their combination on the growth of xenografted tumors derived from undifferentiated thyroid cancer cells. MATERIALS AND METHODS: Experiments were performed in nude mice with tumors developing from implanted undifferentiated thyroid carcinoma cells (FRO). Animals were treated with Ptx i.p. and exposed locally to a single dose of 5 Gy of IR. Apoptosis in situ was detected using ApopTag Peroxidase Kit. RESULTS: In the in vivo experiments, IR significantly inhibited but did not abrogate tumor growth. Ptx effect was stronger, and the combination therapy with Ptx and IR led to the decrease of tumor volume to 0-0.3% of the control (P < 0.01). The systemic administration of Ptx to the animals with advanced tumors resulted in a profound tumor growth suppression and in apoptosis in tumor tissues in time-dependent manner. CONCLUSION: The combination of Ptx and IR is a promising strategy for further preclinical and clinical trials aimed at the development of new therapeutic approaches to the treatment of undifferentiated thyroid cancer.

Effects of low and high concentrations of antitumour drug taxol in anaplastic thyroid cancer cells.

AIM: To study the changes of cell cycle, mitochondrial membrane potential and caspase activation in response to an antitumour drug Taxol in ARO and KTC-2 cell lines of anaplastic thyroid carcinoma. METHODS: Experiments were done on thyroid anaplastic cancer cell lines ARO and KTC-2 using Western blotting, flow cytometry, light and fluorescent microscopy. RESULTS: Taxol significantly activated caspases in ARO cells starting from drug concentration of 5 nM. Maximum activation was observed at 25 nM and further increase of Taxol concentration to 100 nM resulted in a reduction of caspase activation. Concomitant to caspase activation, a loss of mitochondrial membrane potential was observed. At Taxol concentration of 100 nM, most cells lost their mitochondrial membrane potential. Low Taxol concentrations (10 nM) caused changes in the cell cycle that are typical for apoptosis without cell cycle arrest. Higher drug doses starting from 50 nM arrested cell cycle in G2/M phase. In KTC-2 cell line Taxol concentration as low as 1 nM induced apoptosis. 6-15 nM of the drug caused massive (75-83%) cell death. Upon Taxol action, the increase in the number of cells displaying manifestations of accelerated senescence was insignificant. CONCLUSION: Taxol induces bona fide apoptosis in thyroid cancer cell cultures at low (1-25 nM) concentrations. Higher drug doses cause the loss of mitochondrial membrane potential and possibly lead to other types of cell death. No accelerated senescence at different Taxol concentrations was observed. The significance of subG1 and G2/M cell populations at low and high doses of Taxol is discussed.

Differential effects of low and high doses of Taxol in anaplastic thyroid cancer cells: possible implication of the Pin1 prolyl isomerase.

AIM: To study the molecular mechanisms of dose-dependent effects of an anticancer drug, Taxol, on the cell cycle machinery and apoptosis-related proteins in thyroid anaplastic cancer cell lines ARO and KTC-2. MATERIALS AND METHODS: Western blot analysis was used for the detection of various proteins and of their phosphorylated forms. RESULTS: Low dose of Taxol that cause apoptosis (25 nM) enhanced Rb protein phosphorylation, decreased the expression of cyclin-dependent kinase inhibitors p27(KIP1) and p21(WAF1) , and potentiated the accumulation of phosphorylated p53 and of the prolyl isomerase Pin1. High Taxol doses (100 and 1000 nM) that cause necrosis-like cell death drastically decreased Pin1 level in both cell lines. CONCLUSION: Low doses of Taxol promoted G(1)/S transition, thus exhibiting mitogen-like effect. Drug-induced Pin1 accumulation could probably facilitate this transition and in parallel contribute to apoptosis via the p53/p73-dependent mechanism. At higher doses of Taxol, there was a dramatic decrease of Pin1 levels which may be a reason for G(2)/M cell cycle arrest.

Molecular mechanisms of the effects of low concentrations of taxol in anaplastic thyroid cancer cells.

Understanding the detailed mechanisms of a chemotherapeutic agent action on cancer cells is essential for planning the clinical applications because drug effects are often tissue and cell type specific. This study set out to elucidate the molecular pathways of Taxol effects in human anaplastic thyroid cancer cells using as an experimental model four cell lines, ARO, KTC-2, KTC-3 (anaplastic thyroid cancer), and FRO (undifferentiated follicular cancer), and primary thyrocytes. All cell lines were sensitive to Taxol, although to different extent. In primary thyrocytes the drug displayed substantially lower cytotoxicity. In thyroid cancer cells, Taxol-induced changes characteristic to apoptosis such as poly (ADP-ribose) polymerase and procaspase cleavage and alteration of membrane asymmetry only within a narrow concentration range, from 6 to 50 nm. At higher concentration, other form(s) of cell death perhaps associated with mitochondrial collapse was observed. Low doses of Taxol enhanced Bcl2 phosphorylation and led to its degradation observed on the background of a sustained or increasing Bax level and accumulation of survivin and X-chromosome-linked inhibitor of apoptosis. c-jun-NH(2) terminal kinase activation was essential for the apoptosis in anaplastic thyroid cancer cells, whereas Raf/MAPK kinase/ERK and phosphatidylinositol-3-OH kinase/Akt were likely to comprise main survival mechanisms. Our results suggest an importance of cautious interpreting of biological effects of Taxol in laboratory studies and for determining optimal doses of Taxol to achieve the desired therapeutic effect in anaplastic thyroid cancers.