Low Vitamin D Might Be Linked to Prostate Cancer

Adequate levels may help keep cell growth in check, but researchers say more study needed

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By Brenda Goodman

HealthDay Reporter

THURSDAY, May 1, 2014 (HealthDay News) -- Low blood levels of vitamin D may be linked to more aggressive and advanced cases of prostate cancer in men, a new study suggests.

And black men with low vitamin D levels were more likely than those with normal levels to test positive for cancer after a prostate biopsy.

The study, published May 1 in the journal Clinical Cancer Research, suggests that vitamin D may play an important role in how prostate cancer starts and spreads, although it does not prove a cause-and-effect relationship. Researchers aren't yet sure exactly how it comes into play or even if taking extra vitamin D might keep prostate cancer in check.

"There are still many questions about this relationship that have to be answered," said Dr. Len Lichtenfeld, deputy chief medical officer at the American Cancer Society. He was not involved in the research.

"We really don't know, for certain, what role vitamin D plays in cancer -- either the genesis or beginning of cancer -- or in defining how aggressive the cancer may be," he said. "Further research has to be done."

What is known is that vitamin D plays several critical roles in how cells develop and grow.

"It seems to regulate normal differentiation of cells as they change from stem cells to adult cells. And it regulates the growth rate of normal cells and cancer cells," said study author Dr. Adam Murphy, an assistant professor of urology at Northwestern University's Feinberg School of Medicine, in Chicago.

Vitamin D is also known as the "sunshine vitamin" because skin makes it when exposed to sunlight. Vitamin D levels tend to drop with advancing age, and deficiency is more common in seasons and regions that get less sunlight and in people with darker skin, which naturally blocks the sun.

What about the vitamin's possible relationship to cancer?

"When you squirt vitamin D on prostate cells in a petri dish, their rate of growth slows down," Murphy said.

The idea is that too little of this critical vitamin in the body may cause cell growth to go awry, leading to cancer.

To test that idea, researchers checked vitamin D levels in 667 Chicago men between the ages of 40 and 79 who were having prostate biopsies because they'd recently had an abnormal prostate specific antigen (PSA) test or because a doctor felt changes to the prostate during a physical exam.

Normal vitamin D levels are in the range of 30 to 80 nanograms per milliliter (ng/ml).

Vitamin D deficiency, or a level under 20 ng/ml, was relatively common among all the men tested.

About 44 percent of the men with positive biopsies and 38 percent of those who tested negative for cancer had low vitamin D levels.

Among men who tested positive for cancer after their biopsies, those who also had very low levels of vitamin D -- under 12 ng/ml -- had greater odds of more advanced and aggressive cancers than those with normal levels.

The connection between vitamin D and cancer seemed to be even stronger in black men.

Black men with vitamin D levels under 12 ng/ml were far more likely than those with normal levels to test positive for prostate cancer in the first place.

In general, black men are also more likely to be diagnosed with prostate cancer. On average, men have about a one-in-seven lifetime risk of getting prostate cancer. That risk rises to one in five for black men, according to the U.S. Centers for Disease Control and Prevention.

Researchers aren't sure whether lower vitamin D levels may help to explain why black men are at higher risk for prostate cancer.

They say longer and larger studies are needed to sort out the connection.

http://www.webmd.com/prostate-cancer/news/20140501/low-vitamin-d-linked-to-aggressive-advanced-prostate-cancers-study

E-cigarettes 'contain higher levels of certain toxins than regular cigarettes

• U.S. researchers found vapour from e-cigarettes contains toxic element chromium and four times more nickel than found in normal cigarettes
• They were also found to contain lead, zinc and other toxic metals
•  World Health Organisation has called for e-cigarettes to be banned indoors and advertising stopped until industry provides evidence for claims
• British Medical Association has called for tighter regulation of the gadgets so they can't be aggressively marketed to minors
• BBC investigation found toxic chemical dialectyl in one brand of e-cigarettes, which when inhaled can lead to serious lung disease

E-cigarettes can contain higher amounts of certain toxins than regular cigarettes, according to U.S. scientists.

Researchers at the University of South California found vapour emitted by the gadgets contains the toxic element chromium, which is not found in traditional cigarettes, as well as four times more nickel than found in traditional tobacco.

The electronic replacements also contain lead, zinc and other toxic metals, although in lower levels than cigarettes.

Professor Constantinos Sioutas, from the University of South California said the raised levels of toxic metals found in electronic cigarettes are concerning, although overall his research found e-cigarettes are less harmful than traditional cigarettes.

He said: ‘Our results demonstrate that overall electronic cigarettes seem to be less harmful than regular cigarettes, but their elevated content of toxic metals such as nickel and chromium do raise concerns.’

Lead author of the study, Arian Saffari, added: 'The metal particles likely come from the cartridge of the e-cigarette devices themselves – which opens up the possibility that better manufacturing standards for the devices could reduce the quantity of metals in the smoke.

'Studies of this kind are necessary for implementing effective regulatory measures. E-cigarettes are so new, there just isn’t much research available on them yet.'

He said: ‘Our results demonstrate that overall electronic cigarettes seem to be less harmful than regular cigarettes, but their elevated content of toxic metals such as nickel and chromium do raise concerns.’

Lead author of the study, Arian Saffari, added: 'The metal particles likely come from the cartridge of the e-cigarette devices themselves – which opens up the possibility that better manufacturing standards for the devices could reduce the quantity of metals in the smoke.

'Studies of this kind are necessary for implementing effective regulatory measures. E-cigarettes are so new, there just isn’t much research available on them yet.'

REVEALED: LIQUID FOUND IN E-CIGARETTES LINKED TO SERIOUS LUNG DISEASE

E-cigarettes sold in the north east of England were found to contain a chemical called diacetyl, which can cause serious lung disease when inhaled

E-cigarettes sold in the north east of England were found to contain a chemical called diacetyl, which can cause serious lung disease when inhaled.

Diacetyl- used to add butterscotch flavour to liquid tobacco- is harmless to eat, but when inhaled is known to cause a serious condition called ‘popcorn lung’, or bronchiolitis obliterans. 

‘Popcorn lung’, which struck workers in popcorn factories who inhaled large amounts of diacetyl, is an irreversible disease which scars the lung and makes it impossible to breathe properly.

The e-cigarettes were being made by one of the country’s biggest e-cigarettes firms VIP, and have been withdrawn following an investigation by the BBC.

VIP have withdrawn the product, and Lynne White, head of retail for the company said VIP was investigating how this happened.

She told the BBC: ‘This is our first issue in five years. We sell millions of bottles a year. We are very sorry it has happened, we are investigating how it has happened.’

Dr Graham Burns, consultant physician in Respiratory Medicine at Newcastle upon Tyne Hospitals NHS Foundation Trust told the BBC that popcorn lung can sometimes be so severe it leads to a lung transplantation.

He said: ‘That particular chemical - diacetyl - is associated with an unusual but well-established lung condition called popcorn worker’s lung.’

‘It has been inhaled in significant quantities in people who worked in popcorn manufacturing factories.’

‘In those individuals it has caused a very serious lung condition, serious enough to warrant lung transplantation.’ 

http://www.dailymail.co.uk/health/article-2739610/E-cigarettes-contain-higher-levels-certain-toxins-regular-cigarettes.html

The Health Claims Of E-Cigarettes Are Going Up In Smoke

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The sales pitch of electronic cigarette manufacturers seemed too good to be true. Could nicotine addicts around the world really get their fix whilst dodging the health risks of puffing away on cancer sticks?
Sadly for smokers and “vapers”, the answer is far from clear. Over the past week, new evidence has emerged which suggests that E-Cigarettes can be dangerous too – in some cases carrying higher amounts of certain toxins than the blazing tobacco of old.
Researchers at the University of South California have found that although E-Cigarettes are less harmful than ordinary smokes, the vapour emitted by the gadgets contains the toxic element chromium, which is not found in traditional cigarettes, as well as levels of nickel four times higher than in real tobacco. The electronic replacements also contain lead, zinc and other toxic metals, although in lower levels than cigarettes.

Two electronic cigarette models. (Photo credit: Wikipedia)

“Our results demonstrate that overall electronic cigarettes seem to be less harmful than regular cigarettes, but their elevated content of toxic metals such as nickel and chromium do raise concerns,” said Constantinos Sioutas, professor at the USC Viterbi School of Engineering.
There is a bit of good news for reforming smokers, as E-Cigarettes contain virtually no detectable polycyclic aromatic hydrocarbons, which are known to cause cancer. The researchers also said the metal particles were likely to come from the cartridge of the E-Cigarette devices, opening up the possibility that a change in the production process could eliminate the dangerous chemicals.
But this isn’t the only bit of bad news for vape inhalers. In the UK, the BBC reported that E-Cigarette liquid sold in the north east of England was found to contain a chemical called diacetyl, which is used to add butterscotch flavor to liquid tobacco.
Whilst this substance is harmless to eat, it is extremely dangerous to inhale. The chemical is known to cause a serious condition called popcorn lung, or bronchiolitis obliterans, an irreversible disease which scars the lung and makes it impossible to breathe properly. This illness has struck workers in popcorn factories, who are known to breathe in vast quantities of diacetyl, as well as ordinary people who eat a lot of popcorn.
Commenting on the report, Dr Graham Burns, a respiratory expert at Newcastle’s Royal Victoria Infirmary, said the illness is often “serious enough to warrant lung transplantation”.
VP, the firm which manufactured the cigarettes, immediately withdrew the liquid from sale, and Lynne White, head of retail distribution, admitted there were concerns about inhaling the liquid on a long-term basis.
“We are very sorry it has happened, we are investigating how it has happened,” she added.
“Because of the small amount the vaper would actually consume it was deemed in the short term there would be no health concerns.
“Long-term yes there could well be, however we decided it was a withdrawal rather than a recall of the product and that was based on Ecita’s (Electronic Cigarette Industry Trade Association) guidelines.”
But for many health-conscious people, the damage is likely to have been done. Once upon a time, the world didn’t know about the many terrible effects of cigarettes. It was only after a concerted campaign by scientists, doctors and activists that the risks began to be publicized and governments began to act on them. The long term implications of switching to E-Cigarettes have not yet been tested, so anyone using the devices has to ask themselves if they are willing to take the risk of becoming a guinea pig.

Marlboro Cigarettes (Photo credit: Wikipedia)

The World Health Organisation has issued a report advising that use of E-Cigarettes should be banned indoors and and all advertising stopped until the emerging industry produces “convincing supporting scientific evidence and obtains regulatory approval”.

Backing this call, the British Medical Association board of science deputy chair Ram Moorth said “tighter controls are needed to ensure their use does not undermine current tobacco control measures and reinforces the normalcy of smoking behaviour”.
‘There is a need for research to understand the health impacts of E-Cigarettes on both the user and bystanders, and it is vital that the sale of e-cigarettes is appropriately regulated to ensure they are not sold to minors, and are not aggressively marketed to young people as tobacco was in the past,” he continued.
“Any health claims must be substantiated by robust independent scientific evidence to ensure that the consumer is fully informed regarding potential benefits and risks of E-Cigarettes.”
Are you willing to take the chance and keep on vaping?

http://www.forbes.com/sites/jasperhamill/2014/08/31/the-health-claims-of-e-cigarettes-are-going-up-in-smoke/

Vitamin E succinate fights prostate cancer

 Int J Cancer. 2006 May 15;118(10):2441-7.

Vitamin E succinate suppresses prostate tumor growth by inducing apoptosis.

Malafa MP¹, Fokum FD, Andoh J, Neitzel LT, Bandyopadhyay S, Zhan R, Iiizumi M, Furuta E, Horvath E, Watabe K.

Author information

Abstract

Prostate cancer is a major cause of cancer death and morbidity in western countries. However, because of its intrinsic nature of chemoresistance, there is only limited systemic therapy available for the patients. Vitamin E (VE) has been under intensive study as a chemopreventive agent for various types of cancers. Preclinical studies suggest that vitamin E succinate (VES) is the most effective antitumor analogue of VE, yet there are scarce studies of VES in prostate cancer. In this study, we investigated the effects of VES on a panel of prostate cancer cells, and a xenograft model of prostate cancer. Our results indicate that VES significantly inhibited proliferation and induced apoptosis of prostate cancer cell lines in a dose and time dependent manner. The results of microarray analysis followed by real-time RT-PCR and inhibitor analyses indicated that the VES-induced apoptosis is mediated by caspase-4 in prostate tumor cells. In our animal model of prostate cancer in SCID mouse, daily injection of VES significantly suppressed tumor growth as well as lung metastases. These results suggest a potential therapeutic utility of VES for patients with prostate cancer.

http://www.ncbi.nlm.nih.gov/pubmed/16380976

Nutr Cancer. 2000;36(1):90-100.

Vitamin E succinate induces apoptosis in human prostate cancer cells: role for Fas in vitamin E succinate-triggered apoptosis.

Israel K¹, Yu W, Sanders BG, Kline K.

Author information

Abstract

The apoptosis-triggering properties of vitamin E succinate (VES, RRR-alpha-tocopheryl succinate) for human LNCaP and PC-3 prostate carcinoma cells and normal PrEC human prostate epithelial cells were investigated. LNCaP and PC-3 cells were sensitive to VES-induced apoptosis, with 100% and 60% of cells undergoing apoptosis after three days of treatment with 10 micrograms of VES/ml, respectively. PrEC cells were resistant to VES-induced apoptosis. Treatment of prostate cells with agonistic anti-Fas antibody triggered apoptosis in approximately 50% of PC-3 cells within 48 hours, whereas LNCaP and PrEC cells were resistant. Prostate cells simultaneously treated with VES and agonistic anti-Fas antibodies revealed 1) no effect on PrEC cells, 2) an additive effect on Fas-sensitive PC-3 cells, and 3) a synergistic effect on LNCaP cells. VES treatment of LNCaP cells caused depletion of cytosolic 43-kDa Fas, enhanced membrane levels of 43-kDa Fas, and induced Fas sensitivity. PC-3 cells expressed high levels of membrane 43-kDa Fas that were enhanced by VES treatments. Fas ligand expression by LNCaP cells was enhanced by VES treatments. In summary, VES triggers apoptosis in human prostate carcinoma cells but not normal prostate cells in vitro, and VES modulates Fas signaling.

Proc Natl Acad Sci U S A. May 28, 2002; 99(11): 7408–7413.

doi:  10.1073/pnas.102014399

PMCID: PMC124244

Biochemistry

http://www.ncbi.nlm.nih.gov/pubmed/10798221

Biochem Biophys Res Commun. 2003 Jan 10;300(2):357-63.

Vitamin E succinate inhibits human prostate cancer cell growth via modulating cell cycle regulatory machinery.

Ni J¹, Chen M, Zhang Y, Li R, Huang J, Yeh S.

Author information

Abstract

Several epidemiological studies have demonstrated that vitamin E is a chemopreventative agent for prostate cancer. alpha-Tocopheryl succinate (VES), a derivative of vitamin E, effectively modulates prostate cancer cell growth. However, little is known about the mechanisms regarding this action. Here we show that VES causes human prostate cancer cell LNCaP arrest at G1 phase. This effect is accomplished through VES significantly decreasing expression of the cell cycle regulatory proteins cyclin D1, D3, and E, cdk2 and 4, but not cdk6. Furthermore, VES reduces cdk4 kinase activity, Rb phosphorylation, and cyclin E mRNA expression. Recently there is increasing interest in the protective effect of the VES and selenium combination on prostate cancer. Here we show that VES and selenium work through different mechanisms to exert their inhibitory effects on prostate cancer cells. Taken together, our studies suggest that VES-mediated prostate cancer cell G1/S arrest is a consequence of the regulation of multiple molecules of the cell cycle regulatory machinery.

 http://www.ncbi.nlm.nih.gov/pubmed/12504091

Vitamin E succinate inhibits the function of androgen receptor and the expression of prostate-specific antigen in prostate cancer cells

Yu Zhang,^*† Jing Ni,^*† Edward M. Messing,^* Eugene Chang,^* Chin-Rang Yang,^‡ and Shuyuan Yeh^*†§

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

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Abstract

Although epidemiological evidence indicates that a daily supplement of vitamin E may reduce the risk of prostate cancer, the detailed mechanism underlying this effect remains unclear. Here we demonstrate that α-tocopheryl succinate (VES) can suppress the expression of prostate-specific antigen (PSA), a marker for the progression of prostate cancer. VES can also suppress androgen receptor (AR) expression by means of transcriptional and posttranscriptional modulation, but not ligand binding, nuclear translocation, or AR dimerization. This VES-mediated inhibition of AR is selective because VES does not repress the expression of other nuclear receptors. Cell growth studies further show that VES inhibits the growth of prostate cancer LNCaP cells. In contrast, hydroxyflutamide (HF), an antiandrogen currently used to treat prostate cancer patients, only slightly inhibits LNCaP cell growth. Interestingly, simultaneous addition of HF and VES results in a more significant inhibition of LNCaP cell growth. Moreover, selenomethionine (SM), a prostate cancer treatment adjuvant, shows an inhibitory effect on LNCaP cell growth, yet has no effect on the AR/PSA pathway. Together, our data indicate that VES may suppress androgen/AR-mediated cell growth and PSA expression by inhibiting AR expression at both the transcription and translation levels. This previously undescribed mechanism may explain how VES inhibits the growth of prostate cancer cells and help us to establish new therapeutic concepts for the prevention and treatment of prostate cancer.

Keywords: α-tocopheryl succinate‖selenium‖hydroxyflutamide‖vitamin D receptor

Although vitamin E was identified as an essential nutrient for many decades, the detailed mechanisms of its physiological functions remain unclear (1). Early reports showed that a daily supplement of α-tocopherol (vitamin E) decreased the incidence of prostate cancer from 17.8% to 11.7% in male smokers, whereas β-carotene (another antioxidant) had no effect (2). This was the first large-scale epidemiological study showing that vitamin E may play an important role in the prevention of prostate cancer.

Among vitamin E derivatives used to study the inhibition of cancer cell growth, α-vitamin E succinate (VES) effectively inhibits the growth of several cancer cells (3–6). Previous studies suggested that VES could inhibit the proliferation of prostate cancer cells by arresting DNA synthesis, or by stimulating transforming growth factor beta (TGF-β) (7). The detailed mechanisms by which VES prevents prostate cancer cell proliferation, however, remain largely unknown.

Prostate cancer is the most common noncutaneous cancer and second leading cause of cancer death in American men (8). The androgen receptor (AR) is required for the development of both the normal prostate gland and prostate cancer. In the early stages of prostate cancer, almost all cancer cells are androgen-dependent and highly sensitive to anti-androgens. However, prostate cancer usually recurs after a few years of androgen ablative treatment, and most cancer cells become androgen-independent, rendering antiandrogen therapy useless (9). Reports suggest that mutations in the AR ligand-binding domain, AR coregulators, or receptor phosphorylation may enable the AR to respond to nonandrogen agonists (10–13). Furthermore, the activation of the AR by these factors during androgen ablation therapy may facilitate androgen-independent prostate cancer growth. As androgen-independent prostate tumors are incurable, the prevention of such aberrant AR activation is an attractive therapeutic target. Prostate-specific antigen (PSA) is a key androgen-regulated gene, and is a sensitive and selective marker for prostate cancer screening and assessment (14). Consequently, PSA is used as an indicator of disease progression and response for prostate cancer therapies.

Here we use the androgen-dependent LNCaP human prostate cancer cell line (15) as a cell model to study the potential mechanisms of VES to prevent prostate cancer development and progression. We demonstrate that VES decreases intracellular and secreted levels of PSA in LNCaP cells, which have been cultured either in normal serum or in androgen-stimulated conditions. Furthermore, our results indicate that inhibition of PSA is concomitant with VES-mediated down-regulation of AR protein levels. We have also found that the inhibition of AR protein is not only because of regulation of AR mRNA level but also because VES affects the efficiency of AR protein translation.

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Materials and Methods

Chemicals and Reagents.

VES, succinic acid (Suc), selenomethionine (SM), and 5α-dihydrotestosterone (DHT) were purchased from Sigma. HF was a gift from Schering. Antibodies to vitamin D receptor (VDR), peroxisome-proliferator activated receptor α (PPARα), and retinoid X receptor α (RXRα) and β-actin were from Santa Cruz Biotechnology. PSA (clone ER-PR8) antibody was purchased from Dako.

Cell Culture and VES Treatment.

The LNCaP and COS-1 cells were purchased from the American Type Culture Collection (Manassas, VA). Fibroblast cells were primarily cultured from normal prostate tissue. LNCaP cells were grown in phenol red-free RPMI medium 1640 with 8% fetal bovine serum (FBS). The fibroblast cells and COS-1 cells were cultured in phenol red-free Dulbecco's modified Eagle's medium (DMEM) with 10% FBS. The cells were treated with Suc as a control, VES, HF, SM, or DHT at designated concentrations. During the treatment, the medium was changed every 4 days and fresh compounds were added every 2 days.

Cell Counting and Thiazolyl Blue (MTT) Assay.

LNCaP cells (5 × 10⁴) were seeded in each well of 12-well plates. After 36–48 h, the medium was changed to phenol red-free RPMI 1640 medium with 8% FBS or charcoal-stripped FBS (CS-FBS) for another 2, 4, and 6 days, with different treatments. For cell counting, cells were trypsinized, neutralized by medium, and counted on hemocytometers. The MTT assay is a quantitative colorimetric assay for mammalian cell survival and proliferation (13, 16). Fibroblast cells were seeded at 2 × 10⁵ per well in 6-well plates, and cell growth assays were conducted by using the same MTT assay used for LNCaP cells.

Northern Blot Analysis.

Total RNA was extracted by using Trizol according to the manufacturer's instructions (GIBCO), and 20 μg of total RNA was electrophoresed and transferred to the membrane (17). The fragments of the human AR, PSA, or β-actin cDNAs were labeled with [³²P]dCTP. Membranes were prehybridized, hybridized, and washed. The mRNA signals were visualized by using a PhosphorImager (Molecular Dynamics).

In Vivo AR Radioligand Competition Binding Assay.

LNCaP cells were plated into 60-mm dishes and grown to ≈60% confluence. Cells were pretreated with ethanol or 10 μM VES (0.1% vol/vol) for 24 h. Then medium was changed to RPMI 1640 with 8% CS-FBS, and competition ligand binding was performed by using 2.5 nM [³H]R1881, with or without 100-fold excess of unlabeled R1881 (250 nM) (18). After 1-h incubation, cells were harvested by lysis buffer (PBS with 1% Triton X-100). Equal protein amounts of cell extract were subjected to binding assays, which were terminated by adding hydroxylapatite. Each sample was filtered by using a sampling manifold (Millipore) and unbound ligand was removed by washing. Filter papers that contained bound ligand were transferred to counting vials containing 5 ml of liquid scintillation fluid and counted with a multipurpose scintillation counter (Beckman).

[³⁵S]Methionine Labeling of AR.

LNCaP cells were plated into 60-mm dishes and grown to ≈75% confluence. Cells were pretreated with 10 μM VES or ethanol (0.1% vol/vol) for 24 h. Then medium was changed to methionine-free DMEM + 5% dialyzed FCS with or without 10 μM VES at 37°C for 2 h. After 2 h, cells were labeled by incubation with 37°C pulse medium for either (i) 2 h followed by 2-, 6-, and 12-h incubation with chase medium (no [³⁵S]methionine) for protein-stability assay, or (ii) 0.5-, 2-, 6-, and 12-h incubation with no chase period for protein-translation assay. Pulse medium consisted of 100 μCi/ml [³⁵S]methionine (1 Ci = 37 GBq) and 5 μM unlabeled methionine in methionine-free DMEM with 5% dialyzed FBS. To lyse cells, precooled RIPA buffer (1% Nonidet P-40/0.1% SDS/0.5% sodium deoxycholate/1× PBS) plus 1 mM PMSF was added to each dish.

Immunoprecipitation of [³⁵S]Methionine-Labeled Cell Lysate.

Three hundred micrograms of total cellular protein was transferred to new microcentrifuge tubes, and then 3 μl of rabbit anti-AR polyclonal antibody-NH27 (19) and 500 μl of reaction buffer (0.15 M NaCl/0% Triton X-100/20 mM Tris⋅HCl, pH 8.0) was added (20), and incubated for 2 h at 4°C with constant rocking. Twenty-five microliters of protein A/G beads, was added to the solution and incubated for 2 h at 4°C with constant rocking. Samples were centrifuged at 2,500 × g for 3 min at 4°C to collect the beads and then washed three times using ice-cold reaction buffer. Fifty microliters of 1.5× SDS gel-loading buffer was added and boiled for 4 min. Aliquots (25 μl)were subjected to gel electrophoresis, followed by autoradiographic signal quantitation using iqmac software (Molecular Dynamics).

Cell Transfection and Reporter Gene Assay.

For PSA promoter luciferase assay, LNCaP cells were plated in 60-mm dishes until ≈60–70% confluence, and then transfected with 6-kb PSA promoter-linked luciferase reporter (PSA6.0-Luc) by using Superfect (Qiagen, Valencia, CA). Twenty-four hours after transfection, the cells were treated with various compounds for an additional 24 h. For AR N-terminal/C-terminal (N-C) interaction assay, COS-1 cells (1 × 10⁵) were plated on 12-well plates 12 h before being transfected with 0.5 μg of pG5-Luc reporter and other expression vectors depicted in the figure legends. After 24 h transfection, 10 nM DHT and/or 10 μM VES was added for another 24 h. For each transfection, simian virus 40 promoter driven Renilla luciferase (SV40RL) was used as an internal control.

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Results

VES Represses the Growth of LNCaP Cells, but Not Prostate Fibroblasts.

Many prostate tumors progress to a hormone-refractory stage concomitant with the flutamide withdrawal syndrome (21), enabling the tumor to grow in the presence of antiandrogens, such as HF. It is necessary, therefore, to search for more effective antiproliferative reagents to manage prostate cancer. Here, we compare the inhibitory effect of VES with HF in LNCaP cells. Using the MTT assay, Fig. ​Fig.11A demonstrates that 5 nM DHT can stimulate LNCaP cell growth, and the addition of 5 μM HF fails to repress this DHT-induced cell growth in medium with 8% CS-FBS. In contrast, the addition of 10 μM VES effectively represses DHT-mediated cell growth. Interestingly, addition of both 5 μM HF and 10 μM VES can further repress DHT-mediated cell growth. In addition, when we replace 8% CS-FBS with 8% FBS without DHT, 5 μM HF induces LNCaP cell growth at day 2, with the induction gradually diminishing after day 4 (Fig. ​(Fig.11B). Again, 10 μM VES inhibits LNCaP cell growth and the combination of both 10 μM VES and 5 μM HF could further repress LNCaP cell growth after day 4. Together, results from Fig. ​Fig.11 A and B demonstrate that 10 μM VES can effectively inhibit LNCaP cell growth, either in FBS or in CS-FBS in the presence of 5 nM DHT. The combination of 10 μM VES and 5 μM HF further represses LNCaP cell growth. At the same time, we also observed a morphologic change in the LNCaP cells during the treatment period with most of the cells dying after VES treatment for 4 days (Fig. ​(Fig.11C).

Figure 1

VES inhibits the growth of LNCaP cells, but not prostate fibroblast cells. (A) LNCaP cells were cultured in 8% CS-FBS RPMI and treated with DHT (5 nM), Suc (10 μM), HF (5 μM), VES (1 μM or 10 μM), or VES (10 μM) ...

Surprisingly, when we replaced tumor cells with primary cultured fibroblasts from normal prostate tissue, 10 μM VES had only a marginal inhibitory effect on cell growth (Fig. ​(Fig.11D), suggesting that VES may have selective inhibitory effects on tumor cells that are androgen sensitive. Direct cell-number counting by using a hemocytometer (data not shown) further confirmed these cell growth results.

VES Inhibits the Expression of PSA.

Knowing that VES can inhibit the growth of prostate cancer cells, we were interested in determining whether VES also affects the expression of PSA, a marker used to monitor the progression of prostate cancer (14). As shown in Fig. ​Fig.22 A and B, using Western blotting and Northern blotting analyses, we found that both mRNA and protein expression of PSA were induced by 5 nM DHT, and the addition of 10 μM VES effectively repressed PSA expression at both the mRNA and protein levels in LNCaP cells cultured under the same conditions as described for Fig. ​Fig.11 A and B. To further study whether VES-repressed PSA expression occurred at the transcription level, we applied a luciferase reporter linked with the 6.0-kb PSA promoter (PSA6.0-Luc) to assay the VES effect. As shown in Fig. ​Fig.22C, 5 nM DHT induced PSA6.0-Luc activity, and the addition of 10 μM VES, but not Suc, repressed DHT induced-PSA6.0-Luc activity. To test whether the VES-mediated inhibition of PSA promoter is specific, we examined the effect of VES on the transactivation of SP1 by testing GAL4 DNA-binding domain (DBD) fused SP1, which can bind to and activate GAL4 binding site-linked luciferase reporter, pG5-Luc. Our results indicate that 10 μM VES did not significantly inhibit GAL4-SP1 transcription activity (Fig. ​(Fig.22D). Together, our data show that 10 μM VES not only represses DHT-mediated cell growth, but also selectively represses DHT-induced PSA expression in LNCaP cells.

Figure 2

VES inhibits the expression of PSA. (A) VES inhibits PSA expression at the protein level. LNCaP cells were cultured in 8% FBS RPMI or 8% CS-FBS RPMI plus 5 nM DHT and treated with ethanol, 10 μM Suc, or 10 μM VES (0.1% ...

VES Affects AR mRNA and Protein Expression.

As AR has been demonstrated to play essential roles for the induction of PSA expression (22), we were interested in determining the potential influence of VES on AR functions. As shown in Fig. ​Fig.33A, Northern blotting data indicate that VES inhibits AR mRNA and protein expression; however, PSA mRNA and protein levels begin to decrease at earlier times.

Figure 3

VES differentially regulates the protein level of AR, VDR, PPARα, and RXRα. (A) VES down-regulates AR at the transcription and posttranscription level. LNCaP cells were cultured in 8% FBS RPMI and treated with 10 μM VES, ...

As androgen/AR play major roles in the induction of PSA and the down-regulation of AR mRNA is a later event than the down-regulation of PSA activity (Fig. ​(Fig.3),3), we were interested in knowing whether the VES mediated-suppression of PSA occurs by other mechanisms to influence the androgen/AR function rather than regulating AR protein and mRNA level.

VES Does Not Affect the Ligand-Binding, N-C Dimerization, or Nuclear Translocation of AR.

After binding to androgen(s), AR will form a dimer (23), translocate from the cytoplasm to the nucleus (24), and activate its target genes by recognition of androgen-response elements (25). First, we used competition radioligand-binding assay to examine whether VES would affect AR-ligand-binding ability. Results show that unlabeled R1881 can compete for 95% of the specific binding, and VES treatment has little influence on AR ligand binding (Fig. ​(Fig.44A). Next, we examined whether VES affects the N-C interaction of AR, which has been suggested to play an important role in AR transactivation (26). We used a mammalian two-hybrid system, which included the hinge and ligand-binding domain of AR fused with the GAL4-DBD (GAL-ARHLBD), the N terminus of AR fused with VP16 (VP16-ARN), and a pG5-Luc reporter (23). Our results show that 10 nM DHT triggers the AR N-C interaction and addition of 10 μM VES has little influence on the AR N-C interaction (Fig. ​(Fig.44B, lane 3 vs. 4). We also examined whether VES could influence translocation of AR. Although VES has little influence on the AR distribution between cytosol and nucleus, the total AR-staining intensity is reduced, suggesting that VES may affect AR protein expression (data not shown). These immunostaining results not only confirm our earlier Northern and Western blotting assays, but also indicate that VES may function via a posttranscription pathway to down-regulate AR protein function.

Figure 4

VES cannot affect the ligand binding and N-C dimerization of AR. (A) LNCaP cells cultured in 8% CS-FBS RPMI were treated with 2.5 nM [³H]R1881, with or without 100-fold excess of unlabeled R1881. Cells were harvested and washed, ...

Together, our data suggest that VES cannot influence the ligand-binding, N-C dimerization, and nuclear translocation of AR. Instead, VES reduces the overall AR-staining intensity, suggesting that VES may affect AR expression at the transcriptional or translational level.

VES Inhibits AR Protein Translation in LNCaP Cells.

To determine the possible mechanism involved in the regulation of AR expression at the posttranscriptional level, a pulse-chase labeling was applied to characterize whether VES affects AR-protein-translation efficiency or stability (27). Although the intensity of signal is different at the starting point, the degradation rates of the AR are similar in the absence or presence of VES. Our data indicate that VES has little effect on AR-protein stability (Fig. ​(Fig.55A). On the other hand, after treatment with VES, AR-protein synthesis is much slower compared with that of the control group (Fig. ​(Fig.55B). These results suggest that VES may regulate AR protein level through inhibition of protein translation rather than influencing stability.

Figure 5

VES has no effect on AR protein stability, but reduces AR translation. (A) For the stability assay, after pretreatment with ethanol or 10 μM VES (0.1% vol/vol) for 24 h, LNCaP cells were labeled with [³⁵S]methionine. After ...

VES Differentially Regulates the Expression of AR, VDR, PPARα, and RXRα.

To test whether the VES-mediated down-regulation of AR function is specific, we examined the expression level of other nuclear receptors under the same conditions. When antibodies for AR, VDR, PPARα, and RXRα were used, our results indicated that 10 μM VES, but not 10 μM Suc, could suppress AR protein level. This VES-mediated AR repression is selective as 10 μM VES showed little effect on the PPARα and RXRα expression (Fig. ​(Fig.33B) and, in contrast, increased the expression of VDR (Fig. ​(Fig.33B).

VES, but Not Selenium, Affects AR and PSA Expression.

In previous research, selenium has been combined with vitamin E to study their antitumor activity, especially in epidemiological studies (4, 28). Therefore, we were interested in testing whether selenium could function similarly to vitamin E, which inhibits AR expression in LNCaP cells. SM is known to be the major source of selenium in the diet. In the current study, we used 10 μM SM, which has been reported to inhibit LNCaP cell growth (29). Although we observed the SM-mediated growth inhibition in LNCaP cells after 4 days treatment (data not shown), our Western blot data suggest that SM has no effect on AR and PSA expression (Fig. ​(Fig.6).6). Together, our results suggest that VES, but not selenium, down-regulates the expression of AR and PSA. The VES-mediated growth inhibition of prostate cancer cells may be partly due to down-regulated AR expression, and SM may function through other mechanisms to inhibit the growth of prostate cancer cells.

Figure 6

SM has no effect on AR and PSA expression. LNCaP cells were cultured in 8% FBS RPMI and treated with 10 μM SM, 10 μM VES, or ethanol (0.1% vol/vol). Protein harvested from cells without treatment on the first day (day 0) ...

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Discussion

VES Differentially Inhibits the Growth of Cancer Cells and Primary Fibroblast Cultures.

The LNCaP cell line is derived from lymph node prostate cancer metastasis (15), and is one of the best in vitro models for human prostate cancer studies, as it represents a hormone-refractory prostate carcinoma, and its growth is responsive to androgen. In addition, LNCaP cells express a functional mutant AR, and produce PSA, which is a sensitive and specific tumor marker for prostate cancer screening and assessment (22, 30–32). Whereas both the wild-type AR and the LNCaP mutant respond to androgen, estrogenic compounds and some androgens bind to the LNCaP mutant AR with higher affinity, and more effectively stimulate AR-transcriptional activity and PSA expression (12, 33). Our growth assay results indicated that VES could effectively inhibit the growth of LNCaP cells. HF, a popular antiandrogen, cannot effectively suppress the growth of LNCaP cells, which is consistent with the results from previous reports. However, the combination of VES and HF more effectively inhibits LNCaP cell growth than VES alone (Fig. ​(Fig.1).1). This combination may be a potential application for clinical treatment and could warrant further study.

VES Regulates the Expression of AR and PSA in Prostate Cancer.

AR is a critical factor in the development and differentiation of the prostate gland and prostate cancer. In the later stages of prostate cancer, more than 80% of prostate cancer tissues remain positive for AR staining (34). Overall, these observations indicate the importance of the AR in the initiation and progression of prostate cancer. In the present study, our results indicate that VES could effectively down-regulate the protein level of AR, which could be one of the major reasons accounting for VES-mediated growth inhibition in the prostate cancer cells.

Many factors may influence AR function, including interrupting production of the AR at the mRNA or protein levels, ligand binding, dimerization, nuclear translocation, the presence of AR-associated proteins, etc. Our data indicate that VES has a delayed and marginal inhibiting effect on the transcription of AR (Fig. ​(Fig.3),3), with no obvious effects on ligand binding, nuclear translocation, and the interaction between the N terminus and the C terminus of AR (Fig. ​(Fig.4).4). VES, however, influences AR function by down-regulating the efficiency of its translation in LNCaP cells (Fig. ​(Fig.5).5). This could represent a previously unreported mechanism for regulating AR function.

The Antioxidant Effects of Vitamin E and Other Possible Factors for VES-Mediated Functions in Prostate Cells.

One possible mechanism for VES to suppress prostate tumor growth and AR expression may be through its antioxidant effects. Although this is possible, our unpublished data suggest that ascorbic acid, which has antioxidant activity, has little effect on the LNCaP growth and AR-protein expression (S.Y., Y.Z., and J.N., unpublished observation). In addition, VES, an esterified vitamin E analog, has little antioxidant activity. Therefore, it is insufficient to hypothesize that the antioxidant activity is the major factor contributing to VES-mediated suppression of AR and PSA expression. Moreover, the minimal effect VES had on cultured prostate fibroblasts (Fig. ​(Fig.11D) and on other non-androgen-stimulated prostate cancer cell (data not shown) would support the concept that the major inhibition of prostate cancer cell growth by VES may be achieved through its effects on AR expression and function.

Recently, a 46-kDa tocopherol-associated protein (TAP) has been identified from the cytosol of bovine liver (35). In the followup study, a human TAP (hTAP) was isolated, and the recombinant hTAP was capable of binding to vitamin E with a K_d of 460 nM. Since Northern blotting assays indicate that higher levels of hTAP mRNA are found in the liver, brain, and prostate, this molecule may be important for and predictive of functional roles of vitamin E in prostate cancer management (36).

In addition, reports indicated that LNCaP and PC-3 cells were sensitive to VES-induced apoptosis, with 100%, and 60% of cells, respectively, undergoing apoptosis after 3 days of treatment with 20 μM of VES, respectively. However, prostate epithelial cells were resistant to VES-induced apoptosis (37). Whether VES interruption of AR function has any correlation with VES-mediated cell apoptosis, however, remains interesting yet unclear. Further experiments, using microarrays to characterize the downstream biological targets of VES, may help obtain better insight into the functions of VES in prostate cancer cells.

VES, VDR, and Prostate Cancer.

It is well documented that 10 nM 1α,25-dihydroxyvitamin D₃ (vitamin D₃) can inhibit prostate cancer growth (38). Recently, data from several clinical trials using vitamin D₃ to treat prostate cancer patients suggested that vitamin D₃ could decrease the PSA levels in patients' sera (39). We found that 10 μM VES could induce VDR expression (Fig. ​(Fig.33B). Whether increased VDR expression contributes to vitamin D₃-mediated suppression of prostate cancer growth, however, is not known. Nevertheless, this provides another possible explanation for VES' suppression of prostate cancer cell growth, and the possible molecular mechanisms for VES-regulated VDR expression could be an interesting project for future study.

Vitamin E, Selenium, and Prostate Cancer.

In addition to vitamin E, a growing body of evidence suggests that a higher serum level of dietary supplemental selenium substantially reduces the incidence of lung, colon, or prostate cancer (40, 41). Other studies also reported that the selenium compounds inhibit the growth of prostate cancer cells through the induction of apoptosis. However, it is unclear whether vitamin E and selenium function through similar mechanisms to inhibit LNCaP-cell growth.

Because SM is the major component of the selenium in our daily diet (42), we have applied SM to compare the vitamin E-mediated down-regulation of AR and PSA. Although SM can inhibit LNCaP cell growth, we found no significant effect of SM on AR/PSA protein expression. Therefore, it is likely that SM and vitamin E function via different pathways to inhibit LNCaP cell growth.

Various Compounds Inhibit the AR at Different Transcription or Translation Steps.

AR is a key player in the initiation and progression of prostate cancer. If the combination of different compounds can elicit more profound effects on AR-mediated prostate cancer growth, a greater potential for cancer prevention and chemotherapy is possible.

Our data indicate that, unlike other natural products that also showed an inhibitory effect on AR expression at either the transcription (43) or nuclear translocation level (44), vitamin E could be a natural product that inhibits prostate cancer growth by influencing the translation of AR. This newly discovered mechanism could provide an opportunity for the combination of vitamin E with other natural products to coordinately suppress AR function and prevent prostate tumor progression.

In sum, our results may contribute new knowledge to understand the vitamin E-mediated suppression of prostate tumor growth, which may help to design a better therapeutic treatment for prostate cancer patients.

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Acknowledgments

We thank Dr. Chawnshang Chang for helpful discussion and plasmid support, and Karen Wolf, Erik Sampson, and Brenna Simons for assistance in manuscript preparation. This research was partially supported by National Institutes of Health Grant DK60912.

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Abbreviations

AR

androgen receptor

PSA

prostate-specific antigen

DHT

5α-dihydrotestosterone

Suc

succinic acid

VES

α-tocopheryl succinate (vitamin E succinate)

HF

hydroxyflutamide

N-C

N-terminal/C-terminal

VDR

vitamin D receptor

PPAR

peroxisome proliferator-activated receptor

RXR

retinoid X receptor

Luc

luciferase

MTT

thiazolyl blue [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]

CS-FBS

charcoal-stripped fetal bovine serum

SM

selenomethionine

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Footnotes

This paper was submitted directly (Track II) to the PNAS office.

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