Induction of thyroid cancer cell apoptosis by a novel nuclear factor k B inhibitor, dehydroxymethylepoxyquinomicin

長崎大学学位論文 学位記番号:博(医)甲第1,182号 学位授与年月日:平成17年3月18

Differential effects of low and high doses of taxol in anaplastic thyroid cancer cells: possible implication of the PIN1 prolyl isomerase

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 р27KIP1 and p21WAF1, 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 G1/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 G2/M cell cycle arrest.Цель: изучить молекулярный механизм дозозависимых эффектов противоопухолевого препарата таксола на клеточный цикл и ассоциированные с апоптозом белки в клетках линий ARO и KTC-2 анапластического рака щитовидной железы. Материалы и методы: для количественного анализа содержания различных белков и их фосфорилированных форм использовали Вестерн блоттинг. Результаты: при действии таксола в дозе 25 нмоль/л, которая вызывает апоптоз клеток ARO и KTC-2, отмечается усиление фосфорилирования белка Rb, снижение экспрессии ингибиторов циклинзависимых киназ р27KIP1 и p21WAF1, а также накопление фосфорилированного р53 и пролил-изомеразы Pin1. Высокие дозы таксола (100 и 1000 нмоль/л), вызывающие некрозоподобную гибель клеток, заметно снижают уровень Pin1 в обеих клеточных линиях. Выводы: низкие дозы таксола способствуют переходу из G1 в S-фазу клеточного цикла, что свидетельствует о митогенподобном действии препарата. Индуцированное таксолом накопление изомеразы Pin1, возможно, облегчает этот переход и параллельно участвует в апоптотических процессах через p53/p73-зависимый механизм. При более высоких дозах препарата отмечают существенное снижение уровня Pin1, что может быть одной из причин задержки клеточного цикла на стадии G2 /M

Down-regulation of ABCC11 protein (MRP8) in human breast cancer

Aim of this article is to investigate the expression of ABCC11 (MRP8) protein in normal breast tissue, and examine the difference in ABCC11 mRNA and protein expression between normal breast and breast cancer tissues taking into account ABCC11 genotype (a functional SNP, rs17822931) and estrogen receptor (ER) status

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

Aim of this article is 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

Nuclear Factor-Kappa B Inhibition Can Enhance Apoptosis of Differentiated Thyroid Cancer Cells Induced by 131I

Objective: To evaluate changes of nuclear factor-kappa B (NF-kB) during radioiodine 131 ( 131 I) therapy and whether NF-kB inhibition could enhance 131 I-induced apoptosis in differentiated thyroid cancer (DTC) cells in a synergistic manner. Methods: Three human DTC cell lines were used. NF-kB inhibition was achieved by using a NF-kB inhibitor (Bay 11-7082) or by p65 siRNA transfection. Methyl-thiazolyl-tetrazolium assay was performed for cell viability assessment. DNA-binding assay, luciferase reporter assay, and Western blot were adopted to determine function and expression changes of NF-kB. Then NF-kB regulated anti-apoptotic factors XIAP, cIAP1, and Bcl-xL were measured. Apoptosis was analyzed by Western blot for caspase 3 and PARP, and by flow cytometry as well. An iodide uptake assay was performed to determine whether NF-kB inhibition could influence radioactive iodide uptake. Results: The methyl-thiazolyl-tetrazolium assay showed significant decrease of viable cells by combination therapy than by mono-therapies. The DNA-binding assay and luciferase reporter assay showed enhanced NF-kB function and reporter gene activities due to 131 I, yet significant suppression was achieved by NF-kB inhibition. Western blot proved 131 I could increase nuclear NF-kB concentration, while NF-kB inhibition reduced NF-kB concentration. Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after 131 I therapy. And inhibition of NF-kB could significantly downregulate these factors. Finally, synergism induced by combined therapy was displayed by significant enhancements o

Evidence for Association of the rs17822931-A Allele in ABCC11 with a Decreased Risk of Estrogen Receptor-negative Breast Cancer

The rs17822931 SNP of the human ABCC11 gene determines earwax types, and is also associated with some functions of apocrine glands, including the mammary gland. Nevertheless, whether the ABCC11 polymorphism is correlated with estrogen receptor (ER) status of breast cancer (BC) remains unclear. To investigate the correlation between rs17822931 and BC, we screened the genotypes in a total of 276 and 295 histological BC samples collected from Japanese and Ukrainian BC patients, and 269 and 264 ethnically-matched healthy individuals, respectively, using TaqManTM PCR. Genotype frequencies at rs178229131 in Japanese and Ukrainian BC patients were not significantly different from those in their respective control populations. Consistently, no correlation between rs178229131 and the susceptibility to BC was found. The AA genotype, which corresponds to dry earwax, occurred less frequently in ER -negative BC in Japanese [odds ratio, 0.48; 95% confidential interval, 0.29-0.80] but not in Ukrainian patients although a similar correlation was weakly observed. Our results indicate that the rs178229131-A allele may be important in reducing the risk of ER -negative BC development

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

Disruption of Microtubules Sensitizes the DNA Damage-induced Apoptosis Through Inhibiting Nuclear Factor κB (NF-κB) DNA-binding Activity

The massive reorganization of microtubule network involves in transcriptional regulation of several genes by controlling transcriptional factor, nuclear factor-kappa B (NF-κB) activity. The exact molecular mechanism by which microtubule rearrangement leads to NF-κB activation largely remains to be identified. However microtubule disrupting agents may possibly act in synergy or antagonism against apoptotic cell death in response to conventional chemotherapy targeting DNA damage such as adriamycin or comptothecin in cancer cells. Interestingly pretreatment of microtubule disrupting agents (colchicine, vinblastine and nocodazole) was observed to lead to paradoxical suppression of DNA damage-induced NF-κB binding activity, even though these could enhance NF-κB signaling in the absence of other stimuli. Moreover this suppressed NF-κB binding activity subsequently resulted in synergic apoptotic response, as evident by the combination with Adr and low doses of microtubule disrupting agents was able to potentiate the cytotoxic action through caspase-dependent pathway. Taken together, these results suggested that inhibition of microtubule network chemosensitizes the cancer cells to die by apoptosis through suppressing NF-κB DNA binding activity. Therefore, our study provided a possible anti-cancer mechanism of microtubule disrupting agent to overcome resistance against to chemotherapy such as DNA damaging agent

Selective Mitochondrial Uptake of MKT-077 Can Suppress Medullary Thyroid Carcinoma Cell Survival and

BackgroundMedullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the rearranged during transfection (RET) proto-oncogene. Not all patients with progressive MTC respond to current therapy inhibiting RET, demanding additional therapeutic strategies. We recently demonstrated that disrupting mitochondrial metabolism using a mitochondria-targeted agent or by depleting a mitochondrial chaperone effectively suppressed human MTC cells in culture and in mouse xenografts by inducing apoptosis and RET downregulation. These observations led us to hypothesize that mitochondria are potential therapeutic targets for MTC. This study further tests this hypothesis using1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride (MKT-077), a water-soluble rhodocyanine dye analogue, which can selectively accumulate in mitochondria.MethodsThe effects of MKT-077 on cell proliferation, survival, expression of RET and tumor protein 53 (TP53), and mitochondrial activity were determined in the human MTC lines in culture and in mouse xenografts.ResultsMKT-077 induced cell cycle arrest in TT and MZ-CRC-1. Intriguingly, MKT-077 also induced RET downregulation and strong cell death responses in TT cells, but not in MZ-CRC-1 cells. This discrepancy was mainly due to the difference between the capacities of these cell lines to retain MKT-077 in mitochondria. The cytotoxicity of MKT-077 in TT cells was mainly attributed to oxidative stress while being independent of TP53. MKT-077 also effectively suppressed tumor growth of TT xenografts.ConclusionMKT-077 can suppress cell survival of certain MTC subtypes by accumulating in mitochondria and interfering with mitochondrial activity although it can also suppress cell proliferation via other mechanisms. These results consistently support the hypothesis that mitochondrial targeting has therapeutic potential for MTC