Green tea polyphenols induce apoptosis irrespective of p53 status in prostate cancer LNCaPshV and LNCaPshp53 cells.

<p>[<b>A</b>] Cells were treated with 20–80 µg/ml concentration of GTP for 24 h and Western blotting was performed for p53, Akt, p-Akt (Ser473), BAD, p-BAD (Ser136), p21/waf1, PUMA and Bax proteins. [<b>B</b>] Cells were treated with 40 µg/ml concentration of GTP for 3, 6, 9, 12, 24 and 48 h and Western blotting was performed for procaspase-9, cleaved caspase-9 and cleaved caspase-3 proteins. A typical actin blot demonstrates internal loading control. Cytochrome c release from mitochondria to cytosol was determined by Western blotting in GTP treated cells. A typical actin blot demonstrates internal loading control for cytosol whereas VDAC as internal loading control for mitochondria. [<b>D</b>] Cells were treated with 20 µM concentration of PI3K-Akt inhibitor LY294002 for 8 h and with 40 µg/ml GTP for 16 h alone, or LY294002 for 8 h followed by GTP treatment in combination followed by Western blotting for p-Akt, Akt, p-BAD and BAD proteins. The expressions of native proteins were considered as loading controls. [<b>D</b>] Cell death measurement was performed by photometric enzyme immunoassay Cell Death Detection ELISA kit. The bars represent mean±SD of at least two independent experiments each performed in duplicate, **<i>p</i><0.001 represents significant differences as compared to control group. [<b>E</b>] Light microscopic images of LNCaPshV and LNCaPshp53 cells treated with LY294002 and GTP alone or in combination. The details are described in the materials and methods section.</p

Camptothecin induces differential apoptosis in LNCaPshV and LNCaPshp53 cells.

<p>Isogenic cells with lenti-virus vector background were generated by permanent knockdown of p53 in LNCaP cells [LNCaPshp53] and transfected with control vector, LNCaPshV cells. [<b>A</b>] Cell treated with 50 and 100 ng/ml camptothecin for 24 h were harvested, stained with PI and analyzed by flow cytometry to measure sub-G1 and G1 population. [<b>B</b>] Cells were treated with 50, 100 and 200 ng/ml of camptothecin for 24 h and stained with methylene blue. The intensity of methylene blue taken up by live cells was measured spectrophotometerically after eluting the dye in 0.1N HCl and compared with untreated cells. [<b>C</b>] Knockdown of p53 upregulated Akt/BAD signaling in prostate cancer cells. LNCaPshV and LNCaPshp53 cells were lysed and Western blotting was performed for p53, Akt, p-Akt (Ser473), BAD, p-BAD (Ser136) proteins. Actin was used as internal loading control. [<b>D</b>] Relative intensities of p-Akt and p-Bad protein in LNCaPshV and LNCaPshp53RNA cells where bands were normalized to actin and expressed in relative values compared to the native protein. The details are described in the materials and methods section.</p

Green tea polyphenols-mediated apoptosis induced by death receptor pathway in prostate cancer LNCaPshV and LNCaPshp53 cells.

<p>[<b>A</b>] Cells were treated with 40 µg/ml concentration of GTP for 1, 5, 10, 15, 30 and 60 min and Western blotting was performed for SAPK/JNK, p-JNK, FAS, FADD, p-FADD, BID and t-BID proteins. A typical actin blot demonstrates internal loading control. [<b>B</b>] Cells were treated with 40 µg/ml concentration of GTP for 3, 6, 9, 12, 24 and 48 h and Western blotting was performed for cleaved PARP, procaspase-8, cleaved caspase-8, c-IAP and XIAP proteins. A typical actin blot demonstrates internal loading control. [<b>C</b>] Cells were treated with 20 µM concentration of JNK inhibitor SP600125 for 8 h and with 40 µg/ml GTP for 16 h alone, or SP600125 for 8 h followed by GTP treatment in combination followed by Western blotting for JNK, p-JNK, FAS and p-FADD proteins. The expression of native JNK protein was considered as loading control. [<b>D</b>] Cell death measurement was performed by photometric enzyme immunoassay Cell Death Detection ELISA kit. The bars represent mean±SD of at least two independent experiments each performed in duplicate, **<i>p</i><0.001 represents significant differences as compared to control group. [<b>E</b>] Light microscopic images of LNCaPshV and LNCaPshp53 cells treated with SP600125 and GTP alone or in combination. The details are described in the materials and methods section.</p

Green tea polyphenols decrease cell viability and induces apoptosis in LNCaPshV and LNCaPshp53 cells.

<p>[<b>A</b>] Cells were exposed to 20–80 µg/ml concentration of GTP for 24 h, and viability of the cells was determined by the MTT assay. Cell viabilities are depicted as percentages; vehicle-treated cells were regarded as 100% viable. The bars represent mean±SD of at least two independent experiments each performed in duplicate, **<i>p</i><0.001 represents significant differences as compared to control group without GTP treatment. [<b>B</b>] Cell were treated with 40 µg/ml GTP for 24, 48, 72 and 96 h and distribution of cells were recorded in different stages of cell cycle analyzed using FACS analysis. [<b>C</b>] Cells were treated with 40 and 80 µg/ml GTP for 96 h and the number of cells undergoing apoptosis were determined by measuring cell population in sub G1 phase of the cell cycle. The bars represent mean±SD of at least two independent experiments each performed in duplicate, **<i>p</i><0.001 represents significant differences as compared to control group without GTP treatment. [<b>D</b>] Light microscopic images of LNCaPshV and LNCaPshp53 cells treated with 40 and 80 µg/ml GTP for 96 h. GTP treatment exhibits morphological changes consistent with apoptosis in both these cells. [<b>E</b>] DNA fragmentation assay. The cells were treated with 40 µg/ml concentration of GTP for 48 h, collected for DNA isolation and subjected to agarose gel electrophoresis, followed by visualization of bands under UV light. The details are described in the materials and methods section.</p