Grape Polyphenols
Phytochemicals

Inhibition of mammary tumor growth and metastases to bone and liver by grape polyphenols

Introduction

Cancer is one of the main reasons of death in both men and women, claiming over 6 million people
each year worldwide.. Conventional cancer therapies, result in serious side effects and, at best, merely extend the patient's lifespan by a few years.
Chemoprevention, a relatively new and promising strategy to prevent cancer, is defined as the use of either natural or synthetic substances or their combinations to block, reverse or retard the process of carcinogenesis Searching for effective prevention is of high priority in both basic and clinical sciences.
In recent decades natural products have been considered to be an important source of cancer chemopreventive agents. A diet rich in vegetable and fruit reduces the risk of cancer. Red wine polyphenols, which consisted of various powerful antioxidants such as flavonoids and stilbenes, have been implicated in cancer prevention and that promote human health without recognizable side effects.
Polyphenols in foods and beverages are thought to be among the constituents responsible for the reduced cancer risk because have been shown to block carcinogenesis and to inhibit the growth of tumors in whole animals or in cell culture.
Grapes and red wine are an abundant source of polyphenols and represent an important dietary component for some populations. In recent years, a large body of literature has been devoted to studies describing the potential cancer chemopreventive activities of red wine polyphenols.

Resveratrol, a major component of red wine polyphenols, has been studied and reviewed extensively for the chemopreventive activity to interfere with the multistage carcinogenesis. However, resveratrol was not believed to be the only phytochemical that contribute to the chemopreventive activity of red wine. Resveratrol, quercetin, and catechin, represent about 70% of the total polyphenols in red wine and have been shown to be the most effective anticancer compounds.
Much of the studies on cancer preventive mechanisms of grape polyphenols have been conducted with individual compounds at concentrations too high to be achieved via dietary consumption.

Breast cancer is the most commonly diagnosed form of cancer and the second major cause of death from cancer in women. Recent clinical advances have remarkably increased the survival rates from primary breast cancer; however, the prognosis of breast cancer patients is still limited by metastases that can occur years after initial diagnosis and potential cure. Malignant breast cancers often overexpress epidermal growth factor receptor (EGFR) isoforms such as Her-2 that further confound effective treatment of metastatic breast cancer
It’s been recently reported that combined grape polyphenols at physiologically relevant concentrations are more effective than individual compounds at inhibition of mammary tumor and site-specific metastasis.
Resveratrol, quercetin, and catechin, are grape polyphenols selected for this study.
Resveratrol is found in low, but significant amounts in red wine and comprises about 1% of total polyphenols. In breast cancer, resveratrol has been implicated in prevention of multistage carcinogenesis.
Quercetin comprises about 6% of total polyphenols in red wine and has been reported to decrease Her-2 expression. Her-2 is often overexpressed in metastatic cancers including the MDA-MB-435 cell line that was used for this study.
The monomeric form of catechin constitutes up to 65–70% of total red wine polyphenols and has been shown to delay tumor initiation.
Resveratrol, quercetin, and catechin are all viable chemopreventives because they are absorbed and metabolized rapidly in vivo and can be detected in plasma and urine samples in the intact form in humans and rodent models

Most breast cancers preferentially metastasize to bone and liver, where ~80% of patients with advanced breast cancer develop bone cancer, causing severe morbidity and mortality.
Mammary fat pad tumors were established in nude mice and we show that dietary grape polyphenols inhibit both primary tumor growth and metastatic cancer progression from the breast to bone and liver.

Cell culture, proliferation and cycle progression
Human metastatic cancer cell lines MDA-MB-231 (ERα−, ERβ+) (American Type Culture Collection, Manassas, VA, USA) and a bone metastatic variant of MDA-MB-435 (ER−) stably expressing GFP were used for the study (kind gift of Dr. Danny Welch, The University of Alabama at Birmingham, AL, USA) to test the effect of grape polyphenols. Cells were cultured in DMEM with 10% heat-inactivated FBS
MDA-MB-435 (2 × 105) cells were treated every 48 h for 96 h with vehicle (0.2–0.5% DMSO), 0.5, 5, or 20 μM resveratrol, quercetin, or catechin or a combination RQC at 0.5, 5, or 20 μM each. Cells were fixed, nuclei stained with PI and cell proliferation quantified as the number of cells with intact nuclei. Cell cycle stage of MDA-MB-435 cells was determined by flow cytometry of PI-stained cells, following treatment with 5 μM resveratrol, quercetin, or catechin or 5 μM RQC every 48 h for 96 h.

Results and discussion
we demonstrated that a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 μM reduced cell number significantly from control and was more efficient than individual compounds in the MDA-MB-231 ERα (−) ERβ (+) human low metastatic breast cancer cell line. However, due to the low metastatic nature of this cell line, we did not observe adequate metastases in a nude mouse model to enable a statistical analysis of the role of grape polyphenols on metastasis
Therefore, we tested the effect of dietary RQC on a highly metastatic ER (−) cancer cell line, MDA-MB-435.

As shown, 0.5 or 5 μM treatment with resveratrol, quercetin, or catechin alone did not decrease MDA-MB-435 cell proliferation. Resveratrol or quercetin at high concentrations (20 μM) significantly inhibited cell proliferation by 80 and 60% while catechin alone increases cell proliferation significantly. Therefore, the effects on cell proliferation at 20 μM RQC appear to be an additive effect of resveratrol and quercetin. The combined RQC treatment significantly inhibited MDA-MB-435 cell proliferation by ~50, 80, and 90% at 0.5, 5, or 20 μM of each polyphenol (Fig. 1a). These compounds were more effective in the MDA-MB-231 cell line where both resveratrol and quercetin inhibited cell proliferation at 5 and 20 μM by ~60 and ~95%; while combined resveratrol, quercetin, and catechin (RQC) treatment at 0.5, 5, and 20 μM each inhibited cell proliferation by ~60, 85, and 98%, respectively, compared to vehicle controls. Since combined RQC treatment induced a significant reduction on MDA-MB-435 cell proliferation, we then tested the cell cycle stage of MDA-MB-435 cells following 5 μM RQC treatment and found that these compounds arrested MDA-MB-435 cells at S phase.

Therefore, we investigated the effect of RQC treatment on apoptosis of MDA-MB-435 cells by caspase 3 activity. Resveratrol and quercetin at high concentrations have been implicated in apoptosis of cancer cell lines by inducing caspase activity and inhibition of cell survival via PI3K/Akt pathways. In MDA-MB-231 cells, by western blotting with total Akt and phospho-Aktser-473 antibodies, 5 μM RQC (15 min) was found to decrease Akt activity by ~50% compared to vehicle. Therefore, the observed decrease in breast cancer cell numbers in response to RQC treatment is thought to be due to a block in cell cycle progression, increased apoptosis, and reduced cell survival signaling.

Since directed cell migration has been implicated with metastatic efficiency, we tested the effect of individual and combined grape polyphenols on cell migration.
The effect of resveratrol is similar to our previous results that reported low concentrations of resveratrol to act comparable to estrogen and increase cell migration. None of the other grape polyphenols significantly changed breast cancer cell migration. At 0.5 and 5 μM, combined RQC treatment significantly reduced MDA-MB-231 cell migration by ~60% when compared to vehicle controls; whereas, 0.5 μM combined RQC treatment reduced MDA-MB-435 cell migration by ~20%, and 5 μM RQC significantly reduced cell migration by 40%.

The lower response of the inhibitory effect of RQC treatment in the ER (−) MDA-MB-435 cells compared to the ERβ (+) MDA-MB-231 cells may be due to grape polyphenols acting as antiestrogenic compounds in the MDA-MB-231 cells. Also, since MDA-MB-435 cells are Her2++ it is possible that combined grape polyphenols are not as efficient at inhibiting the increased Her-2 signaling in this highly aggressive cancer cell line.

In vivo
To test the effect of resveratrol, quercetin, and catechin on metastatic breast cancer progression in vivo, we established mammary fat pad tumors from GFP-tagged highly meta-static MDA-MB-435 cancer cells. Tumor growth remained linear and similar for both vehicle and RQC treated mice for 60 days. After 60 days, the RQC-treated mice demonstrated reduced tumor growth compared to vehicle. At the end of the study on day 77, the mice following RQC diet demonstrated smaller tumors that were reduced by ~37% in a statistically significant manner.
This data indicates that combined grape polyphenols can be safe and effective therapeutics and preventives of primary tumor growth of ER (−) breast cancer.

A molecular analysis of the effect of grape polyphenols on breast cancer analyzes changes in expression of PI3-K pathway genes because this pathway is a central regulator of cancer cell survival and invasion. Interestingly, real-time PCR arrays for PI3-K pathway genes from tumor extracts revealed that expression of FOXO1 transcription factor was upregulated significantly in mouse mammary tumors following RQC treatment. FOXO factors have been shown to function as tumor suppressors in a variety of cancers. They are actively involved in promoting apoptosis in a mitochondria-independent and -dependent manner by inducing the expression of death receptor ligands and pro-apoptotic Bcl-2 family members. Forkhead proteins have been shown to be important for the anticancer activities of resveratrol. This is the first time that elevation of death promoting genes have been implicated in vivo for a combination of dietary grape polyphenols in reducing mammary tumor growth. RQC treatment of breast cancer cells significantly reduces active phospho Akt1 (p-Akt ser473) levels in 15 min without affecting total Akt1 levels

NfκB transcription factor that regulates tumorigenic and immunomodulatory signaling is a potential target for the chemopreventive activity of grape polyphenols, resveratrol, and quercetin. NFKBIA, which codes for IκB, the subunit that sequesters NfκB in an inactive state, was also upregulated significantly in mammary tumors following RQC treatment. This data indicates that inhibition of NfκB signaling may contribute to the observed reduced mammary tumor growth and metastasis by grape polyphenols.

Effect on metastasis
Since breast cancers preferentially metastasize to the bone, this trend was simulated by inoculating the MDA-MB-435 bone metastatic variant into the mammary fat pad. The ability of the GFP-MDA-MB-435 mammary tumors to invade bone was investigated by fluorescent image analysis of excised, cleaned femurs from mice on vehicle control or RQC diets. In vehicle controls, 5/8 mice presented with bone metastases while only 2/8 mice following RQC treatment demonstrated fluorescent metastatic foci in femurs. Of the mice with bone metastases, the number of metastatic lesions were higher for vehicle treated mice compared to RQC treatments in a statistically significant manner. Similarly, the number of mice with liver metastases and the number of metastatic lesions/liver were also significantly lower in RQC treated mice compared to vehicle controls.

Red Wine Polyphenols for Cancer Prevention, 2008

Inhibition of mammary tumor growth and metastases to bone and liver by dietary grape polyphenols, 2009

Daniela Di Giacomo