gx-15-070 and Thyroid-Neoplasms

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Disease Models, Animal; Drug Resistance; Enzyme Inhibitors; Heterografts; Humans; Indoles; Mice, Nude; Phenylurea Compounds; Protein Multimerization; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; Pyrimidines; Pyrroles; Sulfonamides; Thyroid Neoplasms; Treatment Outcome; Vemurafenib

Patients with advanced and metastasized medullary thyroid carcinoma are difficult to treat since tumor cells do not respond to chemotherapeutic treatment and external radiation. Direct induction of cell death is a new therapeutic approach to therapy-resistant tumor cells. In this study we analyzed the effect of the indirubin-derivative 7BIO and the BH3 mimetic drugs ABT-737 and GX15-070 on cell death induction of TT medullary thyroid carcinoma cells.. TT medullary thyroid carcinoma cell line was treated with 7BIO, ABT-737 and GX15-070. Cell viability was analyzed by MTT assay, while cell death was determined by caspase 3/7 activity, measurement of caspase cleavage products and lactate dehydrogenase liberation assay. LC3B cleavage was analyzed by western blot.. Incubation with all 3 drugs efficiently decreased the number of viable TT cells with IC50 values of 4.1 µM (7BIO), 0.19 µM (ABT-737) and 0.23 µM (GX15-070). The BH3 mimetic ABT-737 caused an apoptotic cell death with caspase activation as expected, while 7BIO- and GX15-070-treatment led to a mixed kind of cell death, where caspase activation was detected but had no effect on viability of TT cells. LC3 conversion as a biochemical marker of autophagic cell death was observed after GX15-070 treatment while LDH release pointed to involvement of necrosis after treatment with all 3 drugs.. The BH3 mimetic drugs ABT-737 and GX15-070 efficiently killed TT medullary thyroid carcinoma cells with low IC50 values, while the indirubin-derivative 7BIO was also effective but with a higher IC50 value. Although the exact kind of cell death and target molecules of 7BIO and GX15-070 are not yet defined, direct induction of cell death may be a new therapeutic option in medullary thyroid carcinoma cells.

Topics: BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; Carcinoma, Neuroendocrine; Cell Death; Cell Line, Tumor; Humans; Indoles; Molecular Mimicry; Nitrophenols; Piperazines; Protein Kinase Inhibitors; Pyrroles; Sulfonamides; Thyroid Neoplasms

Poorly differentiated and anaplastic thyroid carcinomas are very aggressive, almost invariably lethal neoplasms for which no effective treatment exists. These tumors are intrinsically resistant to cell death, even when their driver oncogenic signaling pathways are inhibited.We have undertaken a detailed analysis, in mouse and human thyroid cancer cells, of the mechanism through which Obatoclax, a pan-inhibitor of the anti-apoptotic proteins of the BCL2 family, effectively reduces tumor growth in vitro and in vivo.We demonstrate that Obatoclax does not induce apoptosis, but rather necrosis of thyroid cancer cells, and that non-transformed thyroid cells are significantly less affected by this compound. Surprisingly, we show that Obatoclax rapidly localizes to the lysosomes and induces loss of acidification, block of lysosomal fusion with autophagic vacuoles, and subsequent lysosomal permeabilization. Notably, prior lysosome neutralization using different V-ATPase inhibitors partially protects cancer cells from the toxic effects of Obatoclax. Although inhibition of autophagy does not affect Obatoclax-induced cell death, selective down-regulation of ATG7, but not of ATG5, partially impairs Obatoclax effects, suggesting the existence of autophagy-independent functions for ATG7. Strikingly, Obatoclax killing activity depends only on its accumulation in the lysosomes, and not on its interaction with BCL2 family members.Finally, we show that also other lysosome-targeting compounds, Mefloquine and LLOMe, readily induce necrosis in thyroid cancer cells, and that Mefloquine significantly impairs tumor growth in vivo, highlighting a clear vulnerability of these aggressive, apoptosis-resistant tumors that can be therapeutically exploited.

Topics: Animals; Antineoplastic Agents; Apoptosis; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Cell Proliferation; Enzyme Inhibitors; Humans; Indoles; Lysosomes; Mefloquine; Mice; Mice, Knockout; Necrosis; Proto-Oncogene Proteins c-bcl-2; Pyrroles; RNA Interference; RNA, Small Interfering; Spheroids, Cellular; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Tumor Cells, Cultured

The evasion of cell death is one of the hallmarks of cancer, contributing to both tumor progression and resistance to therapy. Dedifferentiated and anaplastic thyroid carcinomas that do not take up radioiodine are resistant to conventional anticancer treatments and patients with these tumors are difficult to treat. BH3 mimetics are a new class of drugs that target anti-apoptotic proteins of the BCL-2 family and promote cell death. The purpose of this study was to analyze the molecular effects of the BH3 mimetic GX15-070 on thyroid carcinoma cell lines and to characterize cell death induced by GX15-070.. A total of 17 cell lines derived from follicular, papillary, and anaplastic thyroid carcinomas were treated with GX15-070. Cell viability was measured with MTT assay while cell cycle phase distribution and subG1 peaks were determined after propidium iodide staining. We assessed cell death via the caspase 3/7 activity, caspase cleavage products, lactate dehydrogenase (LDH) liberation assays, and a LC3 analysis by western blot. Ultrastructural changes were analysed by electron microscopy of GX15-070-treated cells.. After GX15-070 treatment, the number of viable cells was decreased in all cell lines examined, with IC50 values ranging from 48nM to 3.25 μM. We observed biochemical markers of autophagic cell death and necrosis like LC3 conversion and LDH release after the GX15-070 treatment. Electron microscopy revealed several common characteristic ultrastructural changes like swelling of mitochondria, dilatation of rough endoplasmic reticulum, membrane blebbing and formation of vacuoles. GX15-070 treatment induced DNA fragmentation detected by subG1-peak induction and an arrest in G1 phase of the cell cycle. Caspase activation after GX15-070 incubation was detected but had no effect on viability of cells.. With these experiments we demonstrated the efficacy of the BH3 mimetic drug GX15-070 acting against dedifferentiated thyroid carcinoma cells of various histological origins by the induction of cell death. GX15-070-treated cells underwent non-classical cell death with signs of apoptosis, autophagy and necrosis in parallel. GX15-07 and related compounds thus may be a new therapeutic option for dedifferentiated thyroid carcinoma of various histological subtypes.

Topics: Apoptosis; Cell Death; Cell Line, Tumor; Cell Survival; Humans; Indoles; Molecular Targeted Therapy; Pyrroles; Thyroid Neoplasms

Poorly differentiated tumors of the thyroid gland (PDTC) are generally characterized by a poor prognosis due to their resistance to available therapeutic approaches. The relative rarity of these tumors is a major obstacle to our understanding of the molecular mechanisms leading to tumor aggressiveness and drug resistance, and consequently to the development of novel therapies. By simultaneously activating Kras and deleting p53 (Trp53) in thyroid follicular cells, we have generated a novel mouse model that develops papillary thyroid cancer invariably progressing to PDTC. In several cases, tumors further progress to anaplastic carcinomas. The poorly differentiated tumors are morphologically and functionally similar to their human counterparts and depend on MEK/ERK signaling for proliferation. Using primary carcinomas as well as carcinoma-derived cell lines, we also demonstrate that these tumors are intrinsically resistant to apoptosis due to high levels of expression of the Bcl2 family members, Bcl2a1 (Bcl2a1a) and Mcl1, and can be effectively targeted by Obatoclax, a small-molecule pan-inhibitor of the Bcl2 family. Furthermore, we show that Bcl2 family inhibition synergizes with MEK inhibition as well as with doxorubicin in inducing cell death. Thus, our studies in a novel, relevant mouse model have uncovered a promising druggable feature of aggressive thyroid cancers.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Indoles; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinase 2; Mice, Transgenic; Minor Histocompatibility Antigens; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Pyrroles; RNA, Messenger; Thyroid Neoplasms; Tumor Burden