Design, synthesis and biological evaluation of 3-fluoroalkenyloxindole ring-fused 3-trifluoromethyloxindoles obtained from indoline-2,3-diones and difluoromethylene phosphabetaine

<p></p> <p>A wide variety of multi-substituted 3-fluoroalkenyloxindole ring-fused 3-trifluoromethyloxindoles were obtained in good yields by the reaction of indoline-2,3-diones with difluoromethylene phosphabetaine. Their biological activities against human prostate cancer cells <b>PC-3</b> and human Breast cancer cells <b>MCF-7</b> have been preliminarily demonstrated, using MTT-based assays with the commercially available standard drug Paclitaxel as a positive control. Several compounds exhibited comparable <i>in vitro</i> inhibitory activities against human prostate cancer cells <b>PC-3</b> and human Breast cancer cells <b>MCF-7</b> to Paclitaxel. These results indicate that 3-fluoroalkenyloxindole ring-fused 3-trifluoromethyloxindoles may be potential lead compounds for further biological screening.</p

Effects of TPP1 overexpression on localization of TRF2 with telomeres, telomere length and telomerase activity.

<div><p>(A) Mean TRF lengths at different PDs were detected by southern blot. PD, population doubling. The position of MWs (kb) is indicated to the left.</p> <p>(B) TRAP PCR ELISA assay was used in the analysis of telomerase activity at different PDs.</p> <p>(C) Western blot analysis revealed that TPP1 overexpression had no significant influence on the expression of hTERT.</p> <p>(D) Telomere-ChIP assays were performed using a TRF2 antibody to examine the telomeric DNA bound to by TRF2. Input, supernatant before immunoprecipitation; ppt, protein-DNA immunoprecipitate complex. Specific (telomeric) and nonspecific (Alu) probes were used.</p> <p>Telomeric DNA in ChIP (%) =Telomeric DNA signals of ppt / Telomeric DNA signals of input* 100%.</p></div

TPP1 production, radiosensitivity (SF2) and telomere length (TRF) in human colorectal cancer cell lines.

<div><p>(A) TPP1 production was detected by western blotting.. </p> <p>(B) Telomere length was examined by Southern blot analysis. </p> <p>(C) Relative TPP1 production, radiosensitivity (SF2) and telomere length (TRF) in human colorectal cancer cell lines.</p> <p>(D) Correlation between TPP1 production and radiosensitivity (SF2) in colorectal cancer cells was examined.</p> <p>(E) Correlation between TPP1 production and the TRF length in colorectal cancer cells was examined.</p></div

TPP1 overexpression increased <b>ATM</b>/<b>ATR</b> expression and induced prolonged Chk1 (p³⁴⁵) phosphorylation.

<div><p>(A) Western blot analysis revealed that TPP1 overexpression increased the expression of ATM and ATR.</p> <p>(B) HCT116-Mock and-TPP1 cells were irradiated with 6 Gy X-ray and incubated for indicated times. Western blots were preformed to detect the expression of Chk1 and p-Ser³⁴⁵-Chk1.</p></div

hTERT interactors identified in Y2H library screen.

<p>hTERT interactors identified in Y2H library screen.</p

The detection of protein(UBE2D3, hTERT, cyclin D1, β-actin) expressions were illustrated.

<p>(<b>A</b>) Western blotting analysis showing the effect of overexpression and knockdown of UBE2D3 on UBE2D3 and hTERT levels in MCF-7 cells. Control cells were transfected with negative control shRNA. (<b>B</b>) Western blotting analysis showing the effect of knockdown of UBE2D3 on cyclin D1 levels in MCF-7 cells. (<b>C</b>) Western blotting analysis showing the effect of overexpression of hTERT on UBE2D3 and hTERT levels in MCF-7 cells. Experiments were repeated 3 times with similar results.</p

Effects of TPP1 overexpression on the radiosensitivity and cell cycle in HCT116 cells.

<div><p>(A)Verification of TPP1 overexpression by western blotting.</p> <p>(B) HCT116-Mock and-TPP1 cells were irradiated with X-rays and then cell survival was determined using clonogenic assay.</p> <p>(C) HCT116-Mock and-TPP1 cells were irradiated with 6 Gy X-ray and recovered for indicated times. Cell cycle was analyzed by FACS.</p> <p>(D) The population of cells in G2/M phases over time in HCT116- Mock and -TPP1 cells.</p></div

The MCF-7 cells radiosensitivity detection were illustrated when exposed to irradiation, depending on doses in GY, MCF-7 cells transfected with pshRNA-UBE2D3 showed reductions of clonogenic survival compared to negative control.

<p>Each group of cells were irradiated at the dose point of 0, 1, 2, 4, 6, 8, 10 GY respectively. After 14 days of incubation, the colonies were fixed and stained. Those colonies containing >50 cells were scored as viable colonies. The data were fit into the linear-quadratic model, and survival curve of each group were demonstrated by Graphpad prism 5.0 software. Each experiment was done at least three times in triplicate wells.</p

The total RNA, isolated from Human laryngeal squamous cell carcinoma radioresistant cell Hep2R, was used to synthesized the first-strand cDNA and double-strand cDNA by SMART method (Clontech).

<p>The cDNA fragments were inserted into the pGADT7 vector, and the recombinant phage were packaged in vitro. A small portion packaged phage was used to infected DH10B Competent Cells. Titration and the positive clones were assayed by PCR. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064660#pone-0064660-g001" target="_blank">Fig. 1</a> shows the inserted fragment of Hep2R cell full length cDNA library detected through construction electrophoresis. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064660#pone-0064660-t001" target="_blank">Table 1</a> shows the proteins found through Y2H from Hep2R cell cDNA library.</p

TPP1 overexpression inhibits IR induced apoptosis.

<div><p>HCT116-Mock and-TPP1 cells were irradiated with 5 Gy X-ray and incubated for 24h. The percentage of apoptotic cells was measured by flow cytometry.</p> <p>(A) Representative results of diffrerent groups are shown.</p> <p>(B) Data shown are means±SEM from three independent experiments.</p> <p>*, P < 0.05.</p></div