Hormetic Dose Response of NaAsO2 on Cell Proliferation of Prostate Cells in Vitro: Implications for Prostate Cancer Initiation and Therapy
Dose-Response: An International Journal April-June 2019:1-6 @ The Author(s) 2019 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/1559325819843374 journals.sagepub.com/home/dos SSAGE
Johan Lundqvist’, Erik Helmersson’, and Agneta Oskarsson
Abstract
Sodium meta-arsenite (NaAsO2) has been suggested to play a role both in initiation/progression of prostate cancer and as a future antiprostate cancer drug. We have studied the effects of NaAsO2 on cell proliferation of prostate cancer and noncancer cells, breast cancer cells, and adrenocortical carcinoma cells in vitro. Further, we have investigated the effect of NaAsO2 on the androgen receptor. We report that NaAsO2 alters the cell proliferation of prostate cells, in a hormetic manner, by increasing cell proliferation at low concentrations and decreasing the cell proliferation at high concentrations. No activation of the androgen receptor was detected. We conclude that NaAsO2 is able to increase cell proliferation of prostate cells in vitro at low con- centrations, while it decreases cell viability at high concentrations. This novel finding has to be further addressed if NaAsO2 should be developed into an antiprostate cancer drug.
Keywords
androgen receptor activation, cell viability, hormetic effects, prostate cancer
Introduction
Prostate cancer is among the most common forms of cancer with 1.3 million new cases worldwide during the year of 2018, accounting for approximately 14% of all cancers diagnosed in men. Prostate cancer is expected to cause more than 350 000 deaths year 2018.1 Growth of prostate cancer is dependent on androgens acting on the androgen receptor. The main treatment of prostate cancer is androgen depravation therapy either by chemical or surgical castration.
The association between inorganic arsenic exposure from drinking water and prostate cancer is well documented from epi- demiological studies.2-5 The role of inorganic arsenic in the devel- opment of prostate cancer and possible mechanisms behind the effect has been reviewed by Benbrahim-Tallaa and Waalkes.6 Due to the high incidence and mortality in prostate cancer, a number of new treatment strategies and drugs are under develop- ment. Sodium meta-arsenite (NaAsO2) has been proposed as a new antiprostate cancer drug.7-10 Sodium meta-arsenite has suc- cessfully been used in the treatment of acute promyelocytic leu- kemia (APL) and has also been used in clinical trials for treatment of solid tumors of various origin, including patients with
metastatic prostate cancer (European Clinical Trials Database number 2006-005607-33). Zhang et al10 have reported that com- binations of NaAsO2 and taxane drugs have synergistic antipro- liferative effects on prostate cancer cells in vitro. Phatak et al9 found that NaAsO2 exerted antiproliferative effects on a wide range of cancer cells in vitro, including prostate cancer cells. Further, they concluded that NaAsO2 is a telomere-targeting agent and that this could be the mechanism for the antiprolifera- tive effect. Kim et al7 reported that NaAsO2 reduced cell viability of both androgen-sensitive and androgen-insensitive prostate cancer cells in vitro. The authors also reported that NaAsO2
Department of Biomedicine and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden
Received 25 September 2018; received revised 11 March 2019; accepted 20 March 2019
Corresponding Author:
Johan Lundqvist, Department of Biomedicine and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7028, SE-750 07 Uppsala, Sweden.
Email: johan.lundqvist@slu.se
CC
BY
NC
induces apoptosis, necrosis, and autophagy in the studied prostate cancer cell lines and suggest that this effect could be via a reactive oxygen species (ROS)-dependent mechanism. Recently, Kim et al8 demonstrated reduced levels and stability as well as trans- location to the nucleus of androgen receptors and reduced levels of prostate-specific antigens (PSAs) after treatment of prostate cancer cells with NaAsO2.
In the present study, we have investigated the effects of NaAsO2 on cell proliferation of cancer and normal cells derived from the prostate, adrenal cortex, and breast. Adreno- cortical and breast cells were included in the study to enable a comparison between cell types and to investigate whether observed effects were specific to prostate cells. We report that NaAsO2 exerts a hormetic effect on cell proliferation of both normal and cancer prostate cells, with a stimulated prolifera- tion at low concentrations of NaAsO2.
Materials and Methods
Chemicals and Cell Culture
Sodium meta-arsenite was purchased from Sigma-Aldrich (Stockholm, Sweden). Human breast cancer MCF-7 cells were cultured in Dulbecco Modified Eagles Medium (DMEM)/F:12 medium (Sigma-Aldrich, Stockholm, Sweden) supplemented with 10% fetal bovine serum (Gibco, Stockholm, Sweden), 2 mM L-glutamine (Gibco, Stockholm, Sweden), 6 ng/ml insulin (Gibco, Stockholm, Sweden), and 100 U/mL penicillin/ 100 µg/mL streptomycin (Gibco, Stockholm, Sweden). Human adrenocortical NCI-H295R cells were cultured in DMEM/F:12 medium (Sigma-Aldrich, Stockholm, Sweden) supplemented with 1% ITS Plus premix (BD Biosciences, Gothenburg, Sweden), 2.5% NuSerum (VWR, Stockholm, Sweden), 2 mM L-glutamine (Gibco, Stockholm, Sweden), and 100 U/mL penicil- lin/100 µg/mL streptomycin (Gibco, Stockholm, Sweden). Human prostate RWPE cells were cultured in Keratinocyte Serum Free Medium (Gibco, Stockholm, Sweden) supplemented with 0.05 mg/mL bovine pituitary extract and 5 ng/ml human recom- binant epidermal growth factor. Human prostate LNCaP cells were cultured in RPMI 1640 medium (Gibco, Stockholm, Sweden) sup- plemented with 2 mM L-glutamine (Gibco, Stockholm, Sweden), 10% fetal bovine serum (Gibco, Stockholm, Sweden), 1 mM sodium pyruvate (Gibco, Stockholm, Sweden), and 100 U/mL penicillin/100 µg/mL streptomycin (Gibco, Stockholm, Sweden). Human prostate VCaP cells were cultured in DMEM/F:12 medium (Sigma-Aldrich, Stockholm, Sweden) supplemented with 2 mM L- glutamine (Gibco, Stockholm, Sweden), 10% fetal bovine serum (Gibco, Stockholm, Sweden), and 100 U/mL penicillin/100 µg/mL streptomycin (Gibco, Stockholm, Sweden). All cells were cultured as monolayers in a humidified environment at 37℃ with 5% CO2 and subcultured weekly.
Cell Proliferation Assay
To assay the effect of NaAsO2 on the cell proliferation in LNCaP, RWPE, VCaP, MCF-7, and NCI-H295R cells, the
capacity of the cells to reduce a tetrazolium compound (MTS) to the colored product formazan was analyzed. The reaction is dependent on mitochondrial function, and the amount of for- mazan formed is directly proportional to the number of living cells in culture. Cells were seeded in 96-well plates with a density of 1 × 104 cells per well and incubated for 48 hours. The cells were then treated with NaAsO2 dissolved in water. Cells were treated with NaAsO2 in concentrations ranging from 0.01 to 300 µM. Cell proliferation was analyzed after 24 hours of exposure, using the CellTiter 96 AQueos One Solution Proliferation Assay System (Promega, Stockholm, Sweden) in accordance with the manufacturer’s recommendations. The absorbance was measured using a Wallac Victor2 1420 micro- plate reader (PerkinElmer, Massachusetts, USA). Relative effects on cell proliferation were compared to the vehicle treated control.
Androgen Receptor Activation Assay
To investigate whether NaAsO2 could activate the androgen receptor, LNCaP, VCaP, and RWPE cells were transfected with a luciferase reporter plasmid, where the expression of luciferase is under the control of a promoter containing andro- gen receptor responsive elements.12 The cells were seeded in 96-well plates with a density of 1 × 104 cells per well and incubated for 48 hours. The cells were then transfected with 0.4 µg luciferase plasmid per well. To standardize for transfec- tion efficiency, the cells were cotransfected with a renilla luci- ferase plasmid. The transfection was performed with 0.3 uL Lipofectamine 2000 per well in accordance with the manufac- turer’s recommendations. Following transfection, the cells were incubated for 24 hours and then treated with various concentrations of NaAsO2 for 24 hours. The luciferase activity was assayed using the Dual-Luciferase Reporter Assay System (Promega, Stockholm, Sweden). The luciferase activity is expressed as percentage compared to the vehicle-treated con- trol group. Testosterone was used as a positive control.
Statistical analysis
Statistical analysis was performed by Kruskal-Wallis test, followed by a Mann-Whitney U test for individual compar- isons against the control. The statistical analysis was performed using Minitab 16. P < . 05 was considered statis- tically significant.
Results
Sodium Meta-Arsenite Alters the Cell Proliferation of Prostate Cells in a Hormetic Manner
We found (Figure 1A-C) that NaAsO2 treatment altered the proliferation of prostate LNCaP, VCaP, and RWPE cells in a hormetic manner, resulting in stimulated cell proliferation with up to approximately 40% in the concentration range 0.1 to 40 µM (depending on the cell line) and reduced cell viability at higher concentrations.
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Effects of NaAsO2 on the Cell Proliferation of Cancer Cell Lines Derived From Breast and Adrenal Cortex
The proliferation of breast cancer MCF-7 cells was increased by treatment with NaAsO2 in the concentration 3 and 10 uM, although the stimulatory effect was only 5% to 7% (Figure 2A). At the concentration 30 µM and above, NaAsO2 was found to significantly decrease the cell viability. The proliferation of
adrenocortical carcinoma NCI-H295R cells was found to be altered by NaAsO2 treatment in a monotonic manner, where increased levels of NaAsO2 resulted in decreased cell prolif- eration (Figure 2B).
Sodium Meta-Arsenite Does Not Activate the Androgen Receptor
The effect of NaAsO2 on androgen receptor activation was assayed at nontoxic concentrations in LNCaP, VCaP, and RWPE cells. We found that NaAsO2 was unable to activate the androgen receptor in LNCaP cells (Figure 3A) and VCaP cells (Figure 3B). The transfected RWPE cells were not respon- sive to the positive control testosterone (data not shown) and excluded from further analysis of receptor activation.
Discussion
Arsenic exposure via drinking water affects millions of people worldwide, and an association with increased incidence of prostate cancer has been reported.2-5 Prostate cancer is a dis- ease with high incidence and mortality. The possibility that arsenite may play a role in the initiation and progression of prostate cancer as well as being a promising candidate com- pound as an antiprostate cancer drug is intriguing. The mole- cular events by which arsenite initiates cancer in normal prostate cells at low concentrations are probably different from the molecular events causing cell deterioration in prostate can- cer cells at high concentrations. NaAsO2, a water soluble and orally bioavailable arsenic compound, has been reported to significantly decrease the proliferation of prostate cancer cells 7-10 with suggested mechanisms at multiple levels, includ- ing that NaAsO2 could act as a telomere-targeting agent, that it could induce apoptosis and necrosis via a ROS-dependent mechanism, or that it could reduce androgen receptor levels, stability, and translocation into the nucleus.
In this article, we report that the effect of NaAsO2 on the proliferation of both cancer and normal prostate cells is in fact hormetic with stimulated cell proliferation in the low- to medium-concentration range and reduced cell proliferation at high concentrations. We have only observed this clear hormetic effect in prostate cells. In cells derived from other organs, NaAsO2 exhibited either a clear monotonic response on the cell proliferation with increasing concentrations of NaAsO2 causing a decrease in cell proliferation or a more unclear dose-response relationship. The hormetic response of NaAsO2 on the proliferation of prostate cells is a novel finding and should be of high relevance to address further whether NaAsO2 is to be developed into an antiprostate cancer drug, as the induced proliferation of prostate cancer cells following low- dose exposure to NaAsO2 could counteract the desired result of the drug therapy (ie, decreased proliferation of prostate cancer cells). We have also investigated whether the stimulated cell proliferation caused by low doses of NaAsO2 could be via an
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androgen receptor-dependent mechanism but found that NaAsO2 is unable to activate the androgen receptor. While androgen receptor signaling is an important regulator of pro- liferation of prostate cells in many cases, this signaling path- way is not the only mechanism by which proliferation is regulated. Our finding that NaAsO2 is affecting the prolifera- tion of prostate cells, but is unable to activate the androgen receptor, indicates that NaAsO2 acts via one of the other proliferation-regulating pathways in prostate cells. The human ether-a-go-go-related gene potassium channel (hERG) has been identified as a novel regulator of growth and death in tumor cells.13 Zhang et al14 recently reported dual effects of arsenic trioxide on tumor cells. They reported stimulated proliferation at low doses, associated with increased expression of hERG and accelerated hERG channel activation, and inhibited proliferation
at high doses, associated with reduced expression of hERG. Further research is needed to clarify the mechanism for the effect of NaAsO2 on prostate cell proliferation.
The effects of arsenite on prostate cells can be compared with the effects of isoflavons, especially genistein and daid- zein, on mammary cells. In estrogen-dependent human breast cancer MCF-7 cells, genistein enhances cell proliferation at low concentrations (0.01 to 10 µM), while higher cell concen- trations inhibit cell growth.15,16 A dose-dependent stimulation of tumor growth has been reported in experimental animals after treatment with isoflavons via the feed.17
Dose-response relationships between arsenic exposure and health effects are debated, and whether there is a dose threshold below which exposure is not harmful.18 Exposure to arsenic in drinking water is associated with increased mortality in various
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forms of cancer, including prostate cancer.2,3,6 Epidemiologi- cal studies with low exposures are sparse. However, a US pro- spective cohort study reported an association between low to moderate exposure to arsenic (measured as urinary levels) and increased mortality in prostate cancer (hazard ratio 3.30; 95% confidence interval 1.28-8.48).4 A dose-dependent association between arsenic in drinking water and prostate cancer was also reported in an ecologic study from Iowa, United states.5
In conclusion, we report that the suggested antiprostate can- cer drug NaAsO2 in fact stimulates the proliferation of prostate cancer cells in vitro at low concentrations and that this is an androgen receptor-independent effect. This novel finding needs
to be further addressed if NaAsO2 should be developed as an antiprostate cancer drug.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research and/or authorship of this article: This work was finan- cially supported by The Swedish Research Council Formas (project 2012-2124).
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