Effect of apigenin on H₂O₂ -mediated RWPE-1 cell death.

<p>(<b>A</b>) Effect of H₂O₂ on cell death in RWPE-1 cells. The cells were treated with 200 µM H₂O₂ for 6 h. Negative, PI and annexin V only treatments are included as controls. (<b>B</b>) Cells were treated with 10 µM and 20 µM concentration of apigenin for 16 h followed by 200 µM H₂O₂ incubation for 6 h, stained with PI and annexin V for 15 min and analyzed using fluorescence activated cell sorter (FACS). Data shown is representation of FACS graphs analyzed two times in duplicate. Details are described in materials and methods section.</p

Effect of apigenin on reactive oxygen species (ROS) generation and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels with H₂O₂ in transformed human prostate epithelial RWPE-1 cells.

<p>(<b>A</b>) ROS assay with DCF-DA on RWPE-1 cells treated with 10 µM and 20 µM apigenin for 16 h followed by 200 µM H₂O₂ incubation for 6 h. (<b>B</b>) 8-OHdG levels in RWPE-1 cells treated with 10 µM and 20 µM apigenin for 16 h followed by 200 µM H₂O₂ incubation for 6 h. Bars±SD of experiments performed three times. **P<0.001, compared to H₂O₂ treated group. Details are described in materials and methods section.</p

Interaction of apigenin with calf thymus (CT) DNA.

<p>(<b>A</b>) UV-Vis spectra of CT-DNA, apigenin and apigenin+CT-DNA. (<b>B</b>) UV-Vis spectra of CT-DNA with varying concentration of apigenin ranging from 0.025 mM to 0.2 mM in solution. (<b>C</b>) UV-Vis spectra with 0.05 mM apigenin along with varying concentration of CT-DNA ranging from 0.06 mM to 0.1 mM in solution. Absorption spectra of solution were recorded from 230 nm to 500 nm using Nanodrop. The experiment was repeated three times with similar results. Details are described in materials and methods section.</p

Sub-cellular distribution of apigenin in human prostate cancer LNCaP cells.

<p>The cells were incubated with 20 µM apigenin for 48 h with approximately 5×10⁶ cells and processed for different fractions. Bars±SD of experiments performed three times. Distribution is represented as 100% apigenin in all the fractions. Details are described in materials and methods section.</p

Kinetics of apigenin uptake by human prostate cancer LNCaP cells.

<p>(<b>A</b>) Dose-dependent kinetics of apigenin uptake in LNCaP cells incubated for 6 h with apigenin at concentration ranging from 1.25 µM to 40 µM. The rate of apigenin uptake was measured as (cellular apigenin) million cells⁻¹h⁻¹. Points±SD of experiments performed three times. (<b>B</b>) Data evaluated using Michaelis-Menten kinetics by constructing a reciprocal plot between 1/C and 1/V. Details are described in materials and methods section.</p

UV-Vis spectra of H₂O₂ and its quenching by apigenin.

<p>(<b>A</b>) UV-Vis spectra of calf thymus (CT) DNA incubated with varying concentration of H₂O₂ ranging from 1.95 mM to 125 mM. (<b>B</b>) Quenching of UV-Vis spectra by apigenin after H₂O₂ treatment. CT-DNA was incubated with varying concentration of apigenin ranging from 0.2 mM to 0.8 mM followed by H₂O₂ treatment. The experiment was repeated three times with similar results. Details are described in materials and methods section.</p

Chemical structure and stability of apigenin.

<p>(<b>A</b>) 4′, 5, 7-trihydroxyflavone (<b>B</b>) Apigenin stability was determined at 37°C by incubating 20 µM apigenin with or without human prostate cancer PC-3 cells for up to 96 h. The concentration of apigenin at each time point was measured using UV-HPLC. Points±SD, percentage of remaining apigenin performed three times. Details are described in materials and methods section.</p

Uptake of apigenin by various human prostate cell lines.

<p>(<b>A</b>) Time course apigenin uptake by transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells incubated with 20 µM apigenin for up to 16 h. Cellular uptake of apigenin (apigenin/million cells) was determined using UV-HPLC. (<b>B</b>) Bar graph of apignin uptake by various cell lines at 16 h. Bars±SD of experiments performed three times. **P<0.001. Details are described in materials and methods section.</p

Kaposi's sarcoma-associated herpesvirus infection promotes differentiation and polarization of monocytes into tumor-associated macrophages

<p>Tumor associated macrophages (TAMs) promote angiogenesis, tumor invasion and metastasis, and suppression of anti-tumor immunity. These myeloid cells originate from monocytes, which differentiate into TAMs upon exposure to the local tumor microenvironment. We previously reported that Kaposi's sarcoma-associated herpes virus (KSHV) infection of endothelial cells induces the cytokine angiopoietin-2 (Ang-2) to promote migration of monocytes into tumors. Here we report that KSHV infection of endothelial cells induces additional cytokines including interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-13 (IL-13) that drive monocytes to differentiate and polarize into TAMs. The KSHV-induced TAMs not only express TAM-specific markers such as CD-163 and legumain (LGMN) but also display a gene expression profile with characteristic features of viral infection. More importantly, KSHV-induced TAMs enhance tumor growth in nude mice. These results are consistent with the strong presence of TAMs in Kaposi's sarcoma (KS) tumors. Therefore, KSHV infection of endothelial cells generates a local microenvironment that not only promotes the recruitment of monocytes but also induces their differentiation and polarization into TAMs. These findings reveal a new mechanism of KSHV contribution to KS tumor development.</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