Polyphenols from the Mediterranean herb rosemary (Rosmarinus officinalis) for prostate cancer

The Mediterranean diet is rich in fruits and vegetables and has been associated with a variety of health benefits including cancer prevention. One aspect of the diet that has not received enough attention is Mediterranean herbs. Specifically, rosemary and its polyphenolic diterpenes (carnosic acid and carnosol) are known to possess anti-oxidant activity that may be beneficial for cancer control. Herein, we describe the in vitro and in vivo studies carried out towards understanding the molecular mechanisms of carnosic acid and carnosol leading to inhibition of prostate cancer. The reported findings suggest that these polyphenols target multiple signaling pathways involved in cell cycle modulation and apoptosis. Further work is required to understand its potential for health promotion and potential drug discovery for prostate cancer chemoprevention

Rosemary (Rosmarinus officinalis) extract modulates CHOP/GADD153 to promote androgen receptor degradation and decreases xenograft tumor growth.

The Mediterranean diet has long been attributed to preventing or delaying the onset of cardiovascular disease, diabetes and various solid organ cancers. In this particular study, a rosemary extract standardized to carnosic acid was evaluated for its potential in disrupting the endoplasmic reticulum machinery to decrease the viability of prostate cancer cells and promote degradation of the androgen receptor. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells procured from two different patients undergoing radical prostatectomy were treated with standardized rosemary extract and evaluated by flow cytometry, MTT, BrdU, Western blot and fluorescent microscopy. A significant modulation of endoplasmic reticulum stress proteins was observed in cancer cells while normal prostate epithelial cells did not undergo endoplasmic reticulum stress. This biphasic response suggests that standardized rosemary extract may preferentially target cancer cells as opposed to "normal" cells. Furthermore, we observed standardized rosemary extract to decrease androgen receptor expression that appears to be regulated by the expression of CHOP/GADD153. Using a xenograft tumor model we observed standardized rosemary extract when given orally to significantly suppress tumor growth by 46% compared to mice not receiving standardized rosemary extract. In the last several years regulatory governing bodies (e.g. European Union) have approved standardized rosemary extracts as food preservatives. These results are especially significant as it is becoming more likely that individuals will be receiving standardized rosemary extracts that are a part of a natural preservative system in various food preparations. Taken a step further, it is possible that the potential benefits that are often associated with a "Mediterranean Diet" in the future may begin to extend beyond the Mediterranean diet as more of the population is consuming standardized rosemary extracts

HPLC of Mangosteen fruit extract (MFE).

<p>[<b>A</b>], Polyphenolic xanthones isolated from the mangosteen fruit. [<b>B</b>], HPLC Chromatogram of mangosteen fruit extract. Sub-panel represents an expansion of the profile to reveal additional constituents in the extract.</p

Selective Modulation of Endoplasmic Reticulum Stress Markers in Prostate Cancer Cells by a Standardized Mangosteen Fruit Extract

<div><p>The increased proliferation of cancer cells is directly dependent on the increased activity of the endoplasmic reticulum (ER) machinery which is responsible for protein folding, assembly, and transport. In fact, it is so critical that perturbations in the endoplasmic reticulum can lead to apoptosis. This carefully regulated organelle represents a unique target of cancer cells while sparing healthy cells. In this study, a standardized mangosteen fruit extract (MFE) was evaluated for modulating ER stress proteins in prostate cancer. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells (PrECs) procured from two patients undergoing radical prostatectomy were treated with MFE. Flow cytometry, MTT, BrdU and Western blot were used to evaluate cell apoptosis, viability, proliferation and ER stress. Next, we evaluated MFE for microsomal stability and anti-cancer activity in nude mice. MFE induced apoptosis, decreased viability and proliferation in prostate cancer cells. MFE increased the expression of ER stress proteins. Interestingly, MFE selectively promotes ER stress in prostate cancer cells while sparing PrECs. MFE suppressed tumor growth in a xenograft tumor model without obvious toxicity. Mangosteen fruit extract selectively promotes endoplasmic reticulum stress in cancer cells while sparing non-tumorigenic prostate epithelial cells. Furthermore, in an in vivo setting mangosteen fruit extract significantly reduces xenograft tumor formation.</p></div

Mangosteen fruit extract (MFE) up-regulated endoplasmic reticulum (ER) stress proteins and chaperones in prostate cancer cells.

<p>22Rv1 and LNCaP cells were treated with increasing doses of MFE or α-Mangostin for 24 hr. Cell lysates were prepared and subjected to western blot for detecting the expression of critical ER stress proteins. [<b>A</b>], Expression of ER stress proteins in increasing doses of MFE-treated 22Rv1 and LNCaP cells. [<b>B</b>], Cleaved caspase-4 levels in increasing doses of MFE-treated 22Rv1 and LNCaP cells. [<b>C</b>], Expression of multiple ER chaperones in increasing doses of MFE-treated 22Rv1 and LNCaP cells. Beta-actin was used as a loading control. These results are representatives from three independent experiments. [<b>D</b>], Upper panel, schematic diagram of XBP-1 splicing during ER stress. Lower panel, agarose gel pictures of RT-PCR products. 22Rv1 and LNCaP cells were treated with 15 µg/ml MFE for 24 hr. Total RNA was extracted and subjected to RT-PCR. XBP-1primers spanning the spliced sequence were used. RT-PCR products were run on a 2% agarose gel. GAPDH was used as a control.</p

The effects of MFE on Prostate epithelial cells (PrECs) from prostate cancer patients.

<p>PrECs were isolated from patients undergoing radical prostatectomy. Samples were then used to prepare cells for treatment with study agents. PrECs from two different prostate cancer patients or 22Rv1 cells were treated with 15 µg/ml of MFE for 24 hr and then subjected to Western blots. Beta-actin was used as a loading control. Minus and plus symbols respectively represent absence and presence of indicated agents. These results are representatives from three independent experiments.</p

Mangosteen fruit extract (MFE) reduced viability and inhibited proliferation <i>in vitro</i> on two different prostate cancer cell lines, 22Rv1 and LNCaP.

<p>[<b>A</b>], Microscopic pictures of MFE-treated and untreated prostate cancer cells, MFE dose was indicated. [<b>B</b>], LNCaP cells were treated with increasing doses of MFE for 24, 48, or 72 hr, cell viability was determined by MTT assay. [<b>C</b>], 22Rv1 cells were treated with increasing doses of MFE for 24, 48, or 72 hr, cell viability was determined by MTT assay. [<b>D</b>], Both 22Rv1 and LNCaP cells were treated with increasing doses of MFE, cell proliferation was evaluated by BrdU assay. Absorbance values at 450 nm (A450) represent proliferating cell numbers. These experiments were performed in triplicate and are represented by the mean along with standard deviation.</p

MFE was incubated with liver microsomes to mimic P450 Phase I metabolism in the mouse or human liver.

<p>Human and mouse liver microsomes were prepared as described in the materials and methods. Black bars represent the respective control for that individual time point. Gray bars represent the microsomal stability of α-Mangostin in Phase I enzyme system in both mouse and human liver microsomes. A ‘no preincubation’ was performed (data not shown) and did not have any impact on the interpretation of this data. [<b>A</b>], Mangosteen Fruit Extract (standardized to α-Mangostin) was incubated with mouse liver microsomes. [<b>B</b>], Mangosteen Fruit Extract (standardized to α-Mangostin) was incubated with human liver microsomes. [<b>C</b>], Twelve animals were subcutaneously injected in each flank of the mouse (i.e. two tumors per mice) with ∼1×10⁶ 22Rv1 cells to initiate tumor growth. Twenty-four hours after cell implantation, the animals in each cohort received by intraperitoneal administration vehicle or mangosteen fruit extract (35 mg/kg). Body weights of athymic nude mice treated with control vehicle or mangosteen fruit extract (35 mg/kg) intraperitoneally twice per week were measured two times weekly. [<b>D</b>], The average tumor volume of control and mangosteen fruit extract treated mice were plotted over days after tumor cell inoculation. Data points represent the mean of 12 tumors from six mice; bars represent standard deviation of the mean, *<i>P</i><0.01. [<b>E</b>], Representatives of tumor bearing mice from control and MFE-treated group.</p

Mangosteen fruit extract (MFE) induced apoptosis and apoptosis-related gene expression in prostate cancer cells.

<p>[<b>A</b>], Percentage of apoptotic cells in MFE-treated and untreated cells were determined by flow cytometry. MFE dose was 15 µg/ml. [<b>B</b>], Cleaved Caspase-3 levels in increasing doses of MFE treated 22Rv1 and LNCaP cells. Cell lysates were prepared from MFE-treated cells and cleaved caspase-3 levels were evaluated from same amounts of cell lysates by ELISA, details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081572#s2" target="_blank"><i>Materials and Methods</i></a>. Relative expression levels were indicated as absorbance values at 450 nm (A450). [<b>C</b>], Bax expression in increasing doses of MFE-treated 22Rv1 and LNCaP. Cell lysates were prepared from MFE-treated cells, Bax expression was detected by western blot, and β-actin was used as a loading control. These results are representatives from three independent experiments.</p