Sensitization of human malignant lymphoid cells to etoposide by fucoidan, a brown seaweed polysaccharide

The search for the substances sensitizing cancer cells to apoptosis induction by chemotherapeutic agents is a task of high importance in the modern strategy of anticancer therapy. The aim of the study was to investigate the apoptogenic and apoptosis-modulating activities of fucoidan (sulfated polysaccharide) isolated from far-eastern brown seaweeds Fucus evanescens in two human malignant lymphoid cell lines, MT-4 and Namalwa. Methods: Apoptosis was assessed morphologically and quantified by flow cytometry analysis of cells stained with propidium iodide. Caspase-3 activation was assayed by flow cytometry with the aid of labeled monoclonal antibodies. Results: The fucoidan at 500 µg/ml was not cytotoxic in MT-4 or Namalwa cells even in the setting of long-term presence in culture medium up to 14 days. Nevertheless, pretreatment of MT-4 but not Namalwa cells with fucoidan followed by the exposure to DNA topoisomerase II inhibitor etoposide led to about two-fold increase in the relative apoptotic index as compared with etoposide alone. Apoptosis enhancement of MT-4 cells by fucoidan was not accompanied by further increase in the number of the cells with active form of caspase-3. Conclusion: The present findings demonstrate for the first time that fucoidan enhances etoposide induced caspase-dependent cell death pathway in MT-4 but not Namalwa cell line. The mechanisms of such enhancement do not seem to be related directly to caspase-3 activation.Одной из важных задач современной стратегии противоопухолевой терапии является поиск веществ, повышающих чувствительность опухолевых клеток к индукции апоптоза под действием химиопрепаратов. Цель: изучение апоптогенной и апоптозмодулирующей активности фукоидана — сульфатированного полисахарида, выделенного из дальневосточной бурой водоросли Fucus evanescens, на двух линиях злокачественных лимфоидных клеток человека MT-4 и Namalwa. Методы: апоптоз выявляли морфологически и количественно проточной цитометрией клеток, окрашенных йодистым пропидием. Активацию каспазы-3 изучали методом проточной цитометрии клеток после реакции с конъюгированными моноклональными антителами. Результаты: фукоидан в дозе 500 мкг/мл не проявлял токсичности в клетках MT-4 или Namalwa даже при длительном присутствии препарата в культурах до 14 сут. Предварительная инкубация клеток MT-4 с фукоиданом в указанной дозе приводила к двукратному повышению относительного апоптотического индекса при действии ингибитора ДНК топоизомеразы II этопозида, причем такого эффекта в клетках Namalwa не отмечено. Повышение апоптотического индекса в клетках MT-4 под влиянием фукоидана при индукции апоптоза этопозидом не сопровождалось приростом процентного содержания клеток с активной формой каспазы-3, в сравнении с таковым при действии одного лишь индуктора апоптоза этопозида. Выводы: впервые продемонстрирована способность фукоидана усиливать этопозидиндуцированный каспазозависимый апоптоз в клетках MT-4. Подобный эффект отсутствовал в клетках Namalwa. Механизм повышения чувствительности злокачественных лимфоидных клеток человека к этопозиду при действии фукоидана, по-видимому, напрямую не связан с активацией каспазы-3

Jurkat/A4 cells with multidrug resistance exhibit reduced sensitivity to quercetin

Background: While multidrug resistance of cancer cells is a well-known phenomenon, little is known on the cross resistance between cytotoxic chemotherapeutical agents and unrelated substances such as natural flavonoids. Aim: To compare the effects of cytotoxic drug, vepeside and natural flavonoid, quercetin in Jurkat cells and their multidrug-resistant subline Jurkat/A4, in particular to analyze the effector mechanisms of apoptosis and the profiles of several pro- and antiapoptotic proteins in these cells upon exposure to vepeside or quercetin. Methods: Apoptosis and poly (ADP-ribose) polymerase cleavage were assessed by flow cytometry. Expression of apoptosisrelated proteins was analyzed by Western blotting. Results: Jurkat/A4 cells are less sensitive to antiproliferative effects of quercetin as compared with the parental Jurkat cell line. While vepeside as well as quercetin initially induces apoptosis in both cell lines, the following survival of the exposed cells is essentially different. In resistant Jurkat/A4 cells, vepeside or quercetin treatment activates significantly less caspase-9 and -3 as compared with that in the parental cells. The expression of Bad and BNip1 proteins in Jurkat/A4 cells is lower than in the parental cell line. At the same time, XIAP and CAS levels in Jurkat/A4 cells increase. Upon apoptosis induction, XIAP and CAS levels in Jurkat cells decrease, this effect being negligible in resistant cells. Conclusion: Multidrug-resistant Jurkat/A4 cells exhibit reduced sensitivity to cytotoxic effects of quercetin. The expression profile of Jurkat/A4 cells is characterized by the increased levels of XIAP and CAS representing the endogenous inhibitors of apoptosis

Molecular markers of apoptosis in cancer patients exposed to ionizing radiation: the post-chornobyl view

During the past three decades, the deleterious consequences of Chornobyl accident including carcinogenic effects in the people who were accidentally exposed to radiation have been intensively studied. In particular, recent studies provided increased knowledge of the molecular pathogenesis of thyroid tumors in children exposed to Chornobyl fallout. The risk of several forms of leukemia including myelodysplastic syndromes is elevated in Chornobyl liquidators. Furthermore, the upward trends of increases in a variety of other tumors including breast cancer, cancers of central nervous system and renal cancer have been reported in the persons exposed to Chornobyl fallout. There is growing evidence that insufficient apoptosis allows irradiated cells to survive and thereby contributes to carcinogenesis. The purpose of the present survey is to summarize the recent findings related to apoptotic biomarkers among cancer patients from the different populations affected by the Chornobyl catastrophe. Among the particularly radiosensitive cancer sites, we focused on thyroid cancer and leukemia. Several genes and/or proteins controlling apoptosis directly or indirectly have been incorporated into the analysis. The data reviewed here provide a mechanistic link between the apoptosis alterations and development of radiation-related cancer in the 30-year post-Chornobyl period. We suggest that the type of mutations arising from misrepair of DNA double strand breaks (gene fusion and amplification) is the initial signature event in radiation-induced thyroid cancer. Much work has to be done over the next years to elucidate central questions related to the nature of human radiation carcinogenesis. This article is part of a Special Issue entitled “The Chornobyl Nuclear Accident: Thirty Years After”

Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide

The apoptotic index, cell cycle progression and caspase-3 activation in K-562 cells induced to differentiate by DMSO or quercetin have been studied. Quercetin treatment of K-562 cells was accompanied by cell cycle arrest in G2 /M and apoptosis with caspase-3 activation. In contrast, DMSO-induced differentiation was accompanied by the complete cell cycle arrest in G1 /G0 with negligible caspase-3 activation. In spite of the appearance of benzidine-positive cells and the decreased CD71 level in K-562 cells after exposure to quercetin, the analysis of 1H NMR spectra revealed the overall balance in favor of apoptosis, namely the increase in the content of NMR-visible mobile lipid domains and the decreased intensity of choline-containing metabolites.Визначали апоптотичний індекс, проходження клітинного циклу та активацію каспази-3 в клітинах K-562 в разі диференціювання, індукованого кверцетином або диметилсульфоксидом (ДМСО). Обробка клітин кверцетином супроводжувалась зупинкою клітинного циклу в фазі G2/M та призводила до апоптозу і активації каспази-3. Навпаки, в разі диференціювання, індукованого ДМСО, спостерігали повну зупинку клітинного циклу у фазі G1/G0 без активації каспази-3. Незважаючи на появу бензидин-позитивних клітин та зниження вмісту CD71 після інкубації з кверцетином, аналіз спектрів 1H ЯМР виявив баланс змін у бік апоптозу, а саме збільшення вмісту ЯМР-візуалізованих мобільних ліпідних доменів та зниження інтенсивності сигналу холін-вмісних метаболітів.Определяли апоптотический индекс, прохождение клеточного цикла и активацию каспазы-3 в клетках K-562 при индукции дифференцировки кверцетином или диметилсульфоксидом (ДМСО). Обработка клеток кверцетином сопровождалась остановкой клеточного цикла в фазе G2 /M и активацией каспазы-3. В противоположность этому при дифференцировке, индуцированной ДМСО, отмечали полную остановку клеточного цикла в фазе G1 /G0 без активации каспазы-3. Несмотря на появление бензидин-положительных клеток и снижение содержания CD71 после инкубации с кверцетином, анализ спектров 1H ЯМР выявил баланс изменений в сторону апоптоза, а именно, увеличение содержания ЯМР-визуализируемых мобильных липидных доменов и снижение интенсивности сигнала холин-содержащих метаболитов

Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide

The apoptotic index, cell cycle progression and caspase-3 activation in K-562 cells induced to differentiate by DMSO or quercetin have been studied. Quercetin treatment of K-562 cells was accompanied by cell cycle arrest in G2 /M and apoptosis with caspase-3 activation. In contrast, DMSO-induced differentiation was accompanied by the complete cell cycle arrest in G1 /G0 with negligible caspase-3 activation. In spite of the appearance of benzidine-positive cells and the decreased CD71 level in K-562 cells after exposure to quercetin, the analysis of 1H NMR spectra revealed the overall balance in favor of apoptosis, namely the increase in the content of NMR-visible mobile lipid domains and the decreased intensity of choline-containing metabolites.Визначали апоптотичний індекс, проходження клітинного циклу та активацію каспази-3 в клітинах K-562 в разі диференціювання, індукованого кверцетином або диметилсульфоксидом (ДМСО). Обробка клітин кверцетином супроводжувалась зупинкою клітинного циклу в фазі G2/M та призводила до апоптозу і активації каспази-3. Навпаки, в разі диференціювання, індукованого ДМСО, спостерігали повну зупинку клітинного циклу у фазі G1/G0 без активації каспази-3. Незважаючи на появу бензидин-позитивних клітин та зниження вмісту CD71 після інкубації з кверцетином, аналіз спектрів 1H ЯМР виявив баланс змін у бік апоптозу, а саме збільшення вмісту ЯМР-візуалізованих мобільних ліпідних доменів та зниження інтенсивності сигналу холін-вмісних метаболітів.Определяли апоптотический индекс, прохождение клеточного цикла и активацию каспазы-3 в клетках K-562 при индукции дифференцировки кверцетином или диметилсульфоксидом (ДМСО). Обработка клеток кверцетином сопровождалась остановкой клеточного цикла в фазе G2 /M и активацией каспазы-3. В противоположность этому при дифференцировке, индуцированной ДМСО, отмечали полную остановку клеточного цикла в фазе G1 /G0 без активации каспазы-3. Несмотря на появление бензидин-положительных клеток и снижение содержания CD71 после инкубации с кверцетином, анализ спектров 1H ЯМР выявил баланс изменений в сторону апоптоза, а именно, увеличение содержания ЯМР-визуализируемых мобильных липидных доменов и снижение интенсивности сигнала холин-содержащих метаболитов

Homeostasis

Homeostasi

Photodynamic responsiveness of human leukemia Jurkat/A4 cells with multidrug resistant phenotype

Photodynamic therapy (PDT) is considered as a possible alternative approach to overcoming multidrug resistance (MDR). Analysis of cross-resistance to PDT in cells with different MDR pathways and resistance levels seems to be advantageous for elucidating the general mechanisms of cancer cell resistance to various treatment modalities. Aim: The aim of the study was to clarify whether the Jurkat/A4 leukemia cells with MDR phenotype are cross-resistant to PDT. Methods: Human T-cell acute lymphoblastic leukemia line Jurkat and Jurkat/A4 subline with MDR phenotype were used. 5-Aminolevulinic acid (ALA) and Photolon (a complex of chlorine-e6 and polyvinylpyrrolidone; PL) or gold nanocomposite of PL were applied as photosensitizers. The cells were pretreated with photosensitizers and exposed to laser radiation at corresponding wavelengths. The phototoxicity was assessed in trypan blue exclusion test. The hypodiploid cell fraction was analyzed by flow cytometry of propidium iodide-stained cells. Expression of genes related to PDT resistance was analyzed by microarray technique with Affymetrix U133A chips. Results: ALA-mediated PDT resulted in dose-dependent cell death in both lines, the relative photodynamic efficacy in Jurkat/A4 cells being inferior to that in the parental Jurkat cells. There was no correlation between phototoxicity and apoptosis induction both in Jurkat and Jurkat/A4 cells. PL-mediated general phototoxicity in Jurkat cells amounted up to 75% at the maximal photosensitizer dose with about 40% of apoptotic death fraction. PL-phototoxicity in Jurkat/A4 cells was considerably lower. In contrast to Jurkat cells, PL-gold composite did not increase the efficacy of photosensitization as compared to free PL in Jurkat/A4 cells. Conclusions: Multidrug-resistant Jurkat/A4 cells exhibit reduced sensitivity to phototoxic effect in comparison with parental Jurkat cells independently of nature of the photosensitizer being assayed. Key Words: photodynamic therapy, leukemia cells, multidrug resistance, apoptosis

Squamocin modulates histone H3 phosphorylation levels and induces G1 phase arrest and apoptosis in cancer cells

<p>Abstract</p> <p>Background</p> <p>Histone modifications in tumorigenesis are increasingly recognized as important epigenetic factors leading to cancer. Increased phosphorylation levels of histone H3 as a result of aurora B and pMSK1 overexpression were observed in various tumors. We selected <it>aurora B </it>and <it>MSK1 </it>as representatives for testing various compounds and drugs, and found that squamocin, a bis-tetrahydrofuran annonaceous acetogenin, exerted a potent effect on histone H3 phosphorylation.</p> <p>Methods</p> <p>GBM8401, Huh-7, and SW620 cells were incubated with 15, 30, and 60 μM squamocin for 24 h. The expressions of mRNA and proteins were analyzed by qRT-PCR and Western blotting, respectively. The cell viability was determined by an MTT assay. Cell cycle distribution and apoptotic cells were analyzed by flow cytometry.</p> <p>Results</p> <p>Our results showed that squamocin inhibited the proliferation of GBM8401, Huh-7, and SW620 cells, arrested the cell cycle at the G₁phase, and activated both intrinsic and extrinsic pathways to apoptosis. In addition, we demonstrated that squamocin had the ability to modulate the phosphorylation levels of H3S10 (H3S10p) and H3S28 (H3S28p) in association with the downregulation of aurora B and pMSK1 expressions.</p> <p>Conclusions</p> <p>This study is the first to show that squamocin affects epigenetic alterations by modulating histone H3 phosphorylation at S10 and S28, providing a novel view of the antitumor mechanism of squamocin.</p

Down-Regulation of AP-4 Inhibits Proliferation, Induces Cell Cycle Arrest and Promotes Apoptosis in Human Gastric Cancer Cells

BACKGROUND: AP-4 belongs to the basic helix-loop-helix leucine-zipper subgroup; it controls target gene expression, regulates growth, development and cell apoptosis and has been implicated in tumorigenesis. Our previous studies indicated that AP-4 was frequently overexpressed in gastric cancers and may be associated with the poor prognosis. The purpose of this study is to examine whether silencing of AP-4 can alter biological characteristics of gastric cancer cells. METHODS: Two specific siRNAs targeting AP-4 were designed, synthesized, and transfected into gastric cancer cell lines and human normal mucosa cells. AP-4 expression was measured with real-time quantitative PCR and Western blot. Cell proliferation and chemo-sensitivity were detected by CCK-8 assay. Cell cycle assay and apoptosis assay were performed by flow cytometer, and relative expression of cell cycle regulators were detected by real-time quantitative PCR and Western blot, expression of the factors involved in the apoptosis pathway were examined in mRNA and protein level. RESULTS: The expression of AP-4 was silenced by the siRNAs transfection and the effects of AP-4 knockdown lasted 24 to 96 hrs. The siRNA-mediated silencing of AP-4 suppressed the cellular proliferation, induced apoptosis and sensitized cancer cells to anticancer drugs. In addition, the expression level of p21, p53 and Caspase-9 were increased when AP-4 was knockdown, but the expression of cyclin D1, Bcl-2 and Bcl-x(L) was inhibited. It didn't induce cell cycle arrest when AP-4 was knockdown in p53 defect gastric cancer cell line Kato-III. CONCLUSIONS: These results illustrated that gene silencing of AP-4 can efficiently inhibited cell proliferation, triggered apoptosis and sensitized cancer cells to anticancer drugs in vitro, suggesting that AP-4 siRNAs mediated silencing has a potential value in the treatment of human gastric cancer

Gene expression profile of cervical and skin tissues from human papillomavirus type 16 E6 transgenic mice

<p>Abstract</p> <p>Background</p> <p>Although K14E6 transgenic mice develop spontaneous tumors of the skin epithelium, no spontaneous reproductive tract malignancies arise, unless the transgenic mice were treated chronically with 17β-estradiol. These findings suggest that E6 performs critical functions in normal adult cervix and skin, highlighting the need to define E6-controlled transcriptional programs in these tissues.</p> <p>Methods</p> <p>We evaluated the expression profile of 14,000 genes in skin or cervix from young K14E6 transgenic mice compared with nontransgenic. To identify differentially expressed genes a linear model was implemented using R and the LIMMA package. Two criteria were used to select the set of relevant genes. First a set of genes with a Log-odds ≥ 3 were selected. Then, a hierarchical search of genes was based on Log Fold Changes.</p> <p>Results</p> <p>Microarray analysis identified a total of 676 and 1154 genes that were significantly up and down-regulated, respectively, in skin from K14E6 transgenic mice. On the other hand, in the cervix from K14E6 transgenic mice we found that only 97 and 252 genes were significantly up and down-regulated, respectively. One of the most affected processes in the skin from K14E6 transgenic mice was the cell cycle. We also found that skin from transgenic mice showed down-regulation of pro-apoptotic genes and genes related to the immune response. In the cervix of K14E6 transgenic mice, we could not find affected any gene related to the cell cycle and apoptosis pathways but did observe alterations in the expression of immune response genes. Pathways such as angiogenesis, cell junction and epidermis development, also were altered in their gene expression profiles in both tissues.</p> <p>Conclusion</p> <p>Expression of the HPV16 E6 oncoprotein in our model alters expression of genes that fell into several functional groups providing insights into pathways by which E6 deregulate cell cycle progression, apoptosis, the host resistance to infection and immune function, providing new opportunities for early diagnostic markers and therapeutic drug targets.</p