Rosuvastatin inhibit spheroid formation and epithelial-mesenchymal transition (EMT) in prostate cancer PC-3 cell line.

There is a growing body of evidence suggesting antitumor activity of statins. In metastasis and invasion of cancer the Epithelial-Mesenchymal Transition (EMT) of cancerous cells is an important process. Our goal was to understand the effect of Rosuvastatin on the EMT process in human prostate cancer cell line PC-3 cells in adherent 2 dimensional (2D) and spheroid 3 dimensional (3D) culture. PC-3 cells were cultured in adherence and/or spheroid culture system. The cells were treated with different concentrations of Rosuvastatin. After 96 h, the cell proliferation, viability, type and number of spheroids, the expression of E-Cadherin, Vimentin and Zeb-1 were analyzed. The results show that Rosuvastatin inhibit cell proliferation without significant cytotoxicity. The spheroid formation and spheroid sizes were inhibited by Rousavastatin in a dose dependent manner. In 2D culture, expression of the E-Cadherin was increased up to 2.0 fold in a dose dependent linear manner (R2 = 0.89). Vimentin and Zeb-1 expressions were decreased up to 40 and 20% of untreated control cells expression level respectively, (R2 = 0.99 and 0.92). In 3D system, the expression of E-Cadherin did not show a significant change, but Vimentin and Zeb-1 expressions were decreased up to 70 and 40% of untreated control cells expression level respectively in a dose dependent linear manner in comparison to 2D system (R2 = 0.36 and 0.90). Our finding indicates that Rousavastatin inhibit cell proliferation and spheroid formation of PC-3 cells. This inhibition accompanies by inhibition of EMT markers. Therefor, this cholesterol lowering agent could probably have potential in the prevention and suppression of cancer in androgen dependent prostate cancer.

Prostaglandin F-2α Stimulates The Secretion of Vascular Endothelial Growth Factor and Induces Cell Proliferation and Migration of Adipose Tissue Derived Mesenchymal Stem Cells.

OBJECTIVES: Tissue engineering today uses factors that can induce differentiation of mesenchymal stem cells (MSCs) into other cell types. However, the problem of angiogenesis in this differentiated tissue remains an unresolved area of research interest. The aim of this study was to investigate the effects of prostaglandin F-2α (PGF-2α) on the expression of vascular endothelial growth factor (VEGF) in human adipose tissue derived MSCs. MATERIALS AND METHODS: In this experimental research, human adipose tissue was digested using collagenase. The isolated MSCs cells were treated with PGF-2α (up to 5 µg/ml) and incubated for 96 hours. Cell proliferation, secretion of VEGF and cell migration were spontaneously assayed by MTT, BrdU, ELISA, RT-PCR and scratching methods. RESULTS: Cell growth at 1.0, 2.5, 5 µg/ml of PGF-2α was not significantly reduced compared to control cells, suggesting that these concentrations of PGF-2α are not toxic to cell growth. The results of the BrdU incorporation assay indicated that, in comparison to untreated cells, BrdU incorporation was respectively 1.08, 1.96, 2.0 and 1.8 fold among cells treated with 0.1, 1.0, 2.5 and 5.0 µg/ml of PGF-2α. The scratching test also demonstrated a positive influence on cell proliferation and migration. Cells treated with 1.0 µg/ml of PGF-2α for 12 hours showed the highest relative migration and coverage in comparison to untreated cells. Quantitative VEGF ELISA and RTPCR results indicated an increase in VEGF expression and secretion in the presence of PGF-2α. The amount of VEGF produced in response to 0.1, 1.0, 2.5 and 5.0 µg/ml of PGF-2α was 62.4 ± 3.2 , 66.3 ± 3.7, 53.1 ± 2.6 and 49.0 ± 2.3 pg/ml, respectively, compared to the 35.2 ± 2.1 pg/ml produced by untreated cells. CONCLUSIONS: Stimulation of VEGF secretion by PGF-2α treated MSCs could be useful for the induction of angiogenesis in tissue engineering in vitro.

The Effects of Purified Artemia Extract Proteins on Proliferation, Differentiation and Apoptosis of Human Leukemic HL-60 Cells

There has been an increment in the number of studies focused on marine bioactive materials. Many peptides and other biomaterials with anticancer potential have been extracted from various marine animals. Artemia extracts have found uses in sun-light protection cosmetics and anti-aging products. However, contents of biochemical compounds in Artemia spp. and molecular mechanisms of have not been clearly studied in leukemic cells in vitro. In this work, we isolated and purified proteins of Artemia Urmiana. Six clear fractions (A-F) observed on DEAE-cellulose chromatography were assayed for effects on cell growth, differentiation and apoptosis using the human leukemic HL-60 cell line. Cell proliferation analysis by MTT and BrdU assays indicated that did not affect cells, growth. Cells treated with crude extract and fractions A, B and C, but not E and F (up to 100 µg/mL), exhibited increase of cell growth in a dose dependent manner. Stimulatory effects of fraction D were observed at concentrations of 10 µg/ml and above. In nitro blue tetrazolium (NBT) reduction assays, treatment with 100 µg/mL of fraction E or F for 96 hr increased the fraction of differentiated cells up to 14.8 ± 3.56% and 16.5 ± 2.08% respectively. Combination of those fractions with retinoic acid had significant synergistic effects on the differentiation of cells (56.8 ± 3.7% and 67.4 ± 4.2%, p≤0.01). Annexin-V FITC staining for apoptosis and flow cytometric assays indicated induction of apoptosis by fractions E and F up to 23.8 and 31.8% of cells

Differential sensitivity of telomerase from human hematopoietic stem cells and leukemic cell lines to mild hyperthermia.

We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0-4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed.

Studying the enucleation process, DNA breakdown and telomerase activity of the K562 cell lines during erythroid differentiation in vitro.

During erythropoiesis, some organelles such as mitochondria and nucleus are lost by autophagy and enucleation processes in the presence of macrophages in vivo. In vitro production of erythrocytes has raised many questions about the mechanism of enucleation. The aim of this work was to study the DNA breakdown, enucleation, hemoglobin synthesis and telomerase activity of K562 cells during erythroid differentiation. For these purposes, K562 cells were induced to differentiate by erythropoietin + rhGM-CSF, DMSO, and sodium butyrate separately up to 14 d. In different time intervals, hemoglobin synthesis was evaluated by benzidine staining and RT-PCR for γ-globin gene expression. DNA breakdown was analyzed by 4',6-diamidino-2-phenylindole (DAPI) staining, DNA ladder electrophoresis and comet assay. The telomerase activity was evaluated by TRAP assay. Our result indicated that, sodium butyrate and DMSO inhibited K562 cell growth about 50-60% in comparison to untreated control cells. The percentage of benzidine-positive cells was about 45% in the presence of sodium butyrate after 10 d. Densitometric analysis of RT-PCR and calculated data indicated a 1.5-fold increase in relative γ-globin gene expression at 96 h, in the presence of 1 mM sodium butyrate in comparison with untreated cells. DAPI staining did not reveal any evidence of internal lysis of the nucleus during erythroid differentiation at first wk, but this was obvious in the second wk. DNA laddering pattern was not observed in differentiated cells during 14 d. In comet assay, the percentage of DNA in tail, tail length, and tail moment were significantly different between untreated and treated cells (p < 0.05). Telomerase activity was inhibited up to 90.3% during erythroid differentiation of these cells.

Schwann cell apoptosis and p75(NTR) siRNA.

The p75 pan-neurotrophin receptor (p75(NTR)) plays a pivotal role in linking the immune system with the nervous system. p75(NTR) is required for the development of several characteristic features of allergic asthma. Also p75(NTR) upregulated by reactive Schwann cells after peripheral nerve injury. Moreover p75(NTR) and RhoA play a critical role in the regulation of apoptosis. To determine whether the designed siRNA for p75(NTR) can downregulates both p75(NTR) and Rho-A at RNA level in rats and, if so, at what magnitude, Schwann cell apoptosis occurs. Isolation and purification of neonate Schwann cells were prepared from rat sciatic nerve. Specific siRNA duplex was designed for p75(NTR) . To investigate the role of siRNA-mediated knockdown of p75(NTR) , the gene expression in p75(NTR) was examined with reverse transcription-polymerase chain reaction (RT-PCR) and Real-Time RT-PCR. Schwann cell apoptosis was performed by Annexin and TUNEL assays after 24 hours. Following p75(NTR) Transfection siRNA, p75(NTR) gene, compared with control, was downregulated by 73%. Without using siRNA for Rho-A, Rho-A gene was downregulated by 89% at the same time. Based on Annexin assay, apoptosis of Schwann cells occurred in siRNA+NGF and control+NGF by 16.76%±2.27 and 92.39%±1.82, respectively. TUNEL data showed that apoptosis of Schwann cells occurred in siRNA and control by 12.91%±6.39 and 78.55%±11.85, respectively. Thus, p75-siRNA downregulated both p75(NTR) and Rho-A at RNA level in rats and showed a role on decreased cell apoptosis compared to the controls.

Evaluation of the biological activity of human growth hormone by enzyme linked immuno-receptor assay (ELIRA) method.

The aim of this research was to study the application and effectiveness of Enzyme Linked Immuno Receptor Assay (ELIRA) method for understanding the bioactivity of human Growth Hormone (hGH) in micro-titer plates. For this purpose, rabbit hepatocyte microsomes which contained hGH receptors were used for coating of ELISA micro-titer plates. Then hGH was interacted with coated receptors. Fractions of bounded complexes were identified by antibodies in an Enzyme-based substrate detection system. Different assay conditions such as: buffers, blocking agents, temperatures and times of incubation were analyzed. Our result indicated that, the carbonate coating buffer was not effective in receptor coating in ELIRA. Overnight incubation of hGH and hGH receptors in HEPES assay buffer and BSA blocking resulted in the lower linearity and correlations (R(2) = 0.46 to 0.85). However, 3 h incubation in Tris-HCl assay buffer at 30°C resulted in higher linearity and correlations (R(2) = 0.95 to 0.97). Finally, the coating of microwells by 250 µg/ml of microsome membranes in Tris buffer at 30°C for 3 hr and blocking by skim milk resulted to the best linearity and higher correlation, (R(2) = 0.985) and lower detection limit about 2 ng/ml of bioactive hGH.

Apoptosis and reduced cell proliferation of HL-60 cell line caused by human telomerase reverse transcriptase inhibition by siRNA.

BACKGROUND: The close correlation between telomerase activity and human telomerase reverse transcriptase (hTERT) expression has made hTERT to be considered as a selective molecular target for human cancer therapy. In this study, the ability of short-interfering RNA (siRNA) to downregulate hTERT expression and its correlation with cell growth and apoptosis in the promyelocytic cell line HL-60 was evaluated. MATERIALS AND METHODS: hTERT siRNA was designed and transfected to HL-60. hTERT mRNA expression, cell proliferation and apoptotic cells were measured. RESULTS AND CONCLUSION: The results indicated that hTERT siRNA resulted in 97.2 ± 0.6% downregulation of the hTERT mRNA content; inhibition of the cell proliferation rate was about 52.8 ± 2.3% and the apoptotic index of cells was 30.5 ± 1.5%. hTERT plays an essential role in cell proliferation and control of the viability of leukemic cells, thus promising the development of drugs for leukemia.

Slug/SNAI2 regulates cell proliferation and invasiveness of metastatic prostate cancer cell lines.

Many metastatic cancers recapitulate the epithelial-to-mesenchymal transition (EMT) resulting in enhanced cell motility and invasiveness. The EMT is regulated by several transcription factors, including the zinc finger protein SNAI2, also named Slug, which appears to exert additional functions during development and cancer progression. We have studied the function of SNAI2 in prostate cancer cells. Quantitative RT-PCR analysis showed strong SNAI2 expression particularly in the PC-3 and PC3-16 prostate carcinoma cell lines. Knockdown of SNAI2 by specific siRNA induced changes in EMT markers and inhibited invasion of both cell lines into a matrigel matrix. SNAI2 siRNA-treated cells did not tolerate detachment from the culture plates, likely at least in part due to downregulation of integrin alpha6beta4. SNAI2 knockdown disturbed the microtubular and actin cytoskeletons, especially severely in PC-3 cells, resulting in grossly enlarged, flattened, and sometimes multinuclear cells. Knockdown also decreased cell proliferation, with a prominent G0/G1 arrest in PC3-16. Together, our data imply that SNAI2 exerts strong effects on the cytoskeleton and adhesion of those prostate cancer cells that express it and is necessary for their proliferation and invasiveness.

Induced apoptosis by mild hyperthermia occurs via telomerase inhibition on the three human myeloid leukemia cell lines: TF-1, K562, and HL-60.

The purpose of this research was to understand the effect of hyperthermia on the telomerase activity in human leukemic cell lines (HL-60, K562, and TF-1). The cells were treated by hyperthermia at the range of 41-44 degrees C for 120 min and incubated for 96 h. Then telomerase activity, cell proliferation, and apoptosis were assessed. The results indicated that hyperthermia significantly induced apoptosis on the cells. The cells exhibited pre-apoptotic pattern at 41 and 42 degrees C at 60-120 min and apoptotic pattern at 43 and 44 degrees C over 30 min after hyperthermia. Telomerase activity (that was assayed immediately after hyperthermia) was stable at 41-42 degrees C for 60 min but decreased to 35-40% at 120 min. However, at severe hyperthermia (43-44 degrees C) telomerase activity was decreased in a time- and dose-dependent manner. Following hyperthermia (41-44 degrees C up to 120 min), the cells were incubated for 96 h. In these conditions, the telomerase activity was decreased by about 60-80% in comparison with that untreated control cells.

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast cell-mediated allergic reactions.