Reversal of Stathmin-Mediated Microtubule Destabilization Sensitizes Retinoblastoma Cells to A Low Dose of Antimicrotubule Agents: A Novel Synergistic Therapeutic Intervention

PURPOSE. To explore the possibility of stathmin as an effective therapeutic target and to evaluate the synergistic combination of stathmin RNAi and the antimicrotubule agents paclitaxel and vincristine to retinoblastoma Y79 cells. METHODS. RNAi-mediated specific inhibition of stathmin expression in Y79 cells was shown by real-time quantitative RT-PCR (RT-Q-PCR), its effect on cell proliferation by MTT assay, cell invasion using matrigel, microtubule polymerization by immunohistochemistry, apoptosis, cell cycle analysis by flow cytometry analysis, and the changes in FOXM1 protein expression were studied by Western blot. The effect of combination treatment of stathmin siRNA and paclitaxel/ vincristine was studied by assessing cell viability and apoptosis. RESULTS. Short interfering RNA-mediated transient stathmin downregulation resulted in a marked inhibition of retinoblastoma cell proliferation and cell invasion in vitro. Stathmin inhibition promoted Y79 cells to G2/M phase, and ultimately there were increased apoptotic events as evidenced by higher caspase-3 activation and cleaved poly-(ADP-ribose) polymerase expression. Cells transfected with stathmin siRNA showed long and bundled microtubule polymers and sensitized the Y79 cells significantly to paclitaxel and vincristine. CONCLUSIONS. Stathmin may be a pivotal determinant for retinoblastoma tumorigenesis and chemosensitivity. Strategies to inhibit stathmin will help to enhance the cytotoxic effect of paclitaxel while reducing toxicity (or side effects) to normal cells caused by high doses. (Invest Ophthalmol Vis Sci. 2011; 52:5441-5448

Human surfactant protein D alters oxidative stress and HMGA1 expression to induce p53 apoptotic pathway in eosinophil leukemic cell line

This article is made available through the Brunel Open Access Publishing Fund. Copyright: © 2013 Mahajan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and timedependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SPD in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host’s immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.Department of Biotechnology, Indi

Proposed mechanism for rhSP-D mediated apoptosis in AML14.3D10 eosinophilic leukemic cells.

<p> rhSP-D leads to oxidative stress and reduction in the levels of HMGA1. This possibly results in phosphorylation of p53 (Ser15) and Cdc2 (Tyr15). p21 levels were also found to be upregulated. p21 elevation and Cdc2 phosphorylation contribute to Cdc2 inhibition leading to G2/M arrest. Taken together, these overall changes lead to G2/M arrest followed by apoptosis. The superscripts (1-9) are the changes observed during the study.</p

rhSP-D treatment results in downregulation of HMGA1 levels and activates p53 and the apoptotic pathway.

<p>(<b>i</b>) AML14.3D10 cells treated with rhSP-D (5 and 10µg/ml) for 48h, were analyzed for HMGA1 expression by western blot. The cells incubated with rhSP-D (10µg/ml) for 24h were also analyzed for the levels of (ii) phospho-p53 (Ser15), (iii) cleaved caspase 9, (iv) cleaved PARP, (<b>v</b>) cleaved caspase 7, (vi) p21, (vii) cyclin B and (viii) phospho-cdc2 by immunoblotting. NS points to a non-specific band that displays equal protein loading in different lanes. Cleavage of caspase 9 (iii) produces two bands of molecular mass 37 kDa (upper panel; black arrow) and 17 kDa (lower panel; black arrow). Hollow arrow points to a non-specific band that displays equal protein loading. On the right is shown the quantification of the normalized values, *p<0.05, n=3 independent experiments.</p

rhSP-D leads to G2/M phase arrest, while its removal post-24h reversed cell cycle arrest.

<p><b>A</b>. AML14.3D10 cells were incubated in the presence of rhSP-D (10µg/ml) (Right panel) or in culture medium alone (control) (Left panel) for 24 or 48h, Top and Bottom panel, respectively. <b>B</b>. AML14.3D10 cells were incubated continuously with or without rhSP-D (10µg/ml) for 72h (rhSP-D-72h, Top right) and (Control-72h, Top left) respectively. Further, the cells were cultured in the presence of rhSP-D (10µg/ml) for 24h initially, followed by washing and removal of rhSP-D. The cells were then cultured in culture media for another 48h to complete an overall incubation of 72h (rhSP-D 24h-Release-72h, Bottom right); while the control cells were cultured without rhSP-D for 24h and then given same treatment as test (Control 24h-Release-72h, Bottom left).The cell cycle distribution was determined by flow cytometry of propidium iodide-stained DNA. The inset shows the percentage of total cells that are present in different phases. The flow cytometry data was acquired on Becton Dickinson FACS Calibur machine using Cell Quest Pro software. The figure shows representative histograms from one of the three independent experiments. The total population of cells has been divided in the G1, S and G2 phases based on the Dean/Jett/Fox method of FlowJo software. </p

rhSP-D decreases viability of AML14.3D10 cells in a dose and time dependent manner.

<p><b>A</b>. AML14.3D10 cells were incubated with varying concentrations of rhSP-D (1, 5, 10 or 20µg/ml) for the indicated time intervals. The viability of AML14.3D10 cells was assessed using a modified MTT assay. Results expressed as % viability ± SEM of cells, from 5 independent experiments, with 3 parallel samples in each experiment, in comparison to that of control (taken as 100%) at that time interval and compared by one-way ANOVA and Tukey’s test, * p<0.05 versus control. <b>B</b>. The cells were treated with rhSP-D (10 or 20µg/ml) for 48h and the apoptotic cells were detected by annexin-V-FITC/PI staining (<b>i</b>) Untreated, (ii) rhSP-D (10µg/ml), (iii) rhSP-D (20µg/ml), (iv) FSC and SSC. The increase in PI positive cells in rhSP-D treated cells represents late apoptotic cells. The figure shows representative scatter plots from one of the three independent experiments. <b>C</b>. Representative histograms for rhSP-D induced DNA fragmentation in AML14.3D10 cells studied by hypotonic propidium iodide staining. AML14.3D10 cells were incubated with rhSP-D (10 and 20µg/ml) for 72h at 37°C. IL-5 (25ng/ml) incubated cells with intact DNA were used to define marker (M1). The marker (M1) represents percentage apoptotic cells indicating the sub-diploid DNA. The figure shows representative histograms from one of the three independent experiments.</p

Involvement of CRD region of SP-D in its interaction with AML14.3D10 cells.

<p><b>A</b>. <b>rhSP-D shows dose and calcium dependent binding to AML14.3D10 cells</b>. (<b>i</b>) <b>AML14.3D10</b> cells were incubated with increasing concentrations of the FITC-labeled rhSP-D in the presence of 2mM Ca²⁺ (Ca²⁺) or 2mM Ca²⁺ and 10mM EDTA (EDTA). Data are Mean ± SE of the mean fluorescence of rhSP-D binding to the AML14.3D10 cells; n=3 independent experiments. Significant difference from Ca²⁺ alone: * p<0.05 by Student’s t-test. (ii) A representative FACS histogram of AML14.3D10 cells with FITC-labeled rhSP-D (5µg/ml) showing decreased binding in presence of EDTA. <b>B</b>. <b>Engagement of CRD with cellular debris inhibits the interaction with AML14.3D10 cells</b>. AML14.3D10 cells were incubated with rhSP-D (10µg/ml) (<b>i</b>) or rhSP-D (10µg/ml) pre-incubated with monoclonal antibody against CRD domain of SP-D (ii) or rhSP-D (10µg/ml) in the presence of cellular debris (10 debris : 1 AML 14.3D10 cells) (iii) or rhSP-D (10µg/ml) in the presence of 100 mM Maltose (iv). The cells were then washed and probed with anti-native human SP-D raised in rabbit followed by incubation with anti-rabbit IgG-FITC. The cells were washed, and fixed and analyzed by FACS. % indicates the percentage of fluorescent AML14.3D10 cells. The figure shows representative histograms from one of the three independent experiments.</p

2-DE gel images of proteins extracted from AML14.3D10 cells.

<p>Proteins were extracted from AML14.3D10 cells <b>A</b>. untreated and <b>B</b>. treated with rhSP-D (10µg/ml) for 48h. Proteins (400µg) were separated by iso-electric focusing on IPG strips, 17cm, pI range 5-8 and SDS-PAGE on 12% Laemmli gels (n=3). Differentially expressed proteins (PDQuest Image Analysis Software version 7.2) showing a change of ≥ 3 were identified by MALDI-TOF-MS and MALDI-TOF-MS-MS (75 proteins, marked with arrows) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085046#pone.0085046.s002" target="_blank">Table S1</a>). The proteins marked in box are identified as HMGA1 proteins, which were downregulated on rhSP-D treatment.</p

rhSP-D induces apoptosis in leukemia cell lines having different origins.

<p> Human PBMCs, AML14.3D10, Jurkat, Raji, THP-1 or MCF-7 cells were treated with rhSP-D (10 or 20µg/ml) for 48h. The viability of rhSP-D treated or untreated cells was assessed by a modified MTT assay. Results are expressed as % viability ± SEM in comparison to control from 3 independent experiments, and evaluated by Student’s t-tests, * p<0.05.</p