Androgen receptor (AR) degradation enhancer ASC-J9® in an FDA-approved formulated solution suppresses castration resistant prostate cancer cell growth

doi:10.1016/j.canlet.2017.11.038

Cancer Letters

Volume 417, 28 March 2018, Pages 182-191

https://doi.org/10.1016/j.canlet.2017.11.038 Get rights and content

Highlights

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ASCJ9^® targeting prostate cancer through both AR-dependent and AR-independentmechanistic pathways.
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Lower IC₅₀ values in AR-positive cells than in AR-negative cells suggest ASC-J9^®'s importance as an AR-degradation enhancer.
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ASC-J9^® could also function via an AR-independent apoptotic pathway.
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ASC-J9^® has similar AR degradation effects when dissolved in various FDA-approved solvents in vivo.
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ASC-J9^®suppresses prostate cancer at the castration resistant stage to support futurehuman clinical trials.

Abstract

ASC-J9^® is a recently-developed androgen receptor (AR)-degradation enhancer that effectively suppresses castration resistant prostate cancer (PCa) cell proliferation and invasion. The optimal half maximum inhibitory concentrations (IC₅₀) of ASC-J9^® at various PCa cell confluences (20%, 50%, and 100%) were assessed via both short-term MTT growth assays and long-term clonogenic proliferation assays. Our results indicate that the IC₅₀ values for ASC-J9^® increased with increasing cell confluency. The IC₅₀ values were significantly decreased in PCa AR-positive cells compared to PCa AR-negative cells or in normal prostate cells. This suggests that ASC-J9^® may function mainly via targeting the AR-positive PCa cells with limited unwanted side-effects to suppress the surrounding normal prostate cells. Mechanism dissection indicated that ASC-J9^® might function via altering the apoptosis signals to suppress the PCa AR-negative PC-3 cells. Preclinical studies using multiple in vitro PCa cell lines and an in vivo mouse model with xenografted castration-resistant PCa CWR22Rv1 cells demonstrated that ASC-J9^® has similar AR degradation effects when dissolved in FDA-approved solvents, including DMSO, PEG-400:Tween-80 (95:5), DMA:Labrasol:Tween-80 (10:45:45), and DMA:Labrasol:Tween-20 (10:45:45). Together, results from preclinical studies suggest a potential new therapy with AR-degradation enhancer ASC-J9^® may potentially be ready to be used in human clinical trials in order to better suppress PCa at later castration resistant stages.

Introduction

An estimated 2.9 million men (as reported by the America Cancer Society) are currently affected by prostate cancer (PCa) in the United States. Approximately 15.3% of males will be diagnosed with PCa at some point during their lifetime [1]. PCa is one of the most commonly-diagnosed cancers with one of the highest cancer-related mortality rates [2]. In recent years, both androgen deprivation therapy (ADT) and chemotherapy have been the standard treatments for PCa, yet they may eventually be less commonly used due to the many unwanted side-effects [3].

Previous studies have shown that PCa development is closely-linked to the androgen receptor (AR) [4], [5], [6]. In males, the AR plays a major role in phenotypic development and is a crucial component in the progression of several androgen-related tumors [4]. The novel AR degradation enhancer ASC-J9^® (5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one) has been shown to effectively inhibit the growth of AR-positive (AR+) PCa cells with low toxicity, minimal side-effects and drug resistance [7], [8].

The IC₅₀ values of ASC-J9^® in different cell lines at varying densities have numerous effects on cell survival inhibition. The cell densities of PCa cell lines were defined as how much surface area of the culture wells was covered at the time of drug treatment. One of the experiments performed showed that a higher cell density would increase the contact between cells to an extent that they may develop contact inhibition, as is the case for the 100% confluent cells that results in discontinued growth [9]. Since the same dosage of ASC-J9^® was distributed to every cell within the culture wells, the same drug concentration was distributed over increasing numbers of cells. This resulted in diminished ASC-J9^®-elicited cytotoxicity received by each cell, hence higher half maximal inhibitory concentration (IC₅₀) values.

The goal of this study was to test the IC₅₀ of ASC-J9^® dissolved in the FDA-approved formulated solutions allowed in human clinical trials. Three AR+ PCa cell lines, LNCaP, C4-2, and CWR22Rv1, one AR negative (AR-) PCa cell line, PC-3, a normal prostate stromal myofibroblast cell line (WPMY-1), and a stromal fibroblast human embryonic kidney (HEK) cell line (HEK-293T), were treated with different concentrations of ASC-J9^® at different PCa cell confluencies to determine the IC₅₀ values using short-term MTT growth assays and long-term clonogenic proliferation (colony formation) assays. In addition, a preclinical study using an in vivo mouse model xenografted with castration-resistant CWR22Rv1 cells also demonstrated that ASC-J9^® has similar AR degradation effects when dissolved in FDA-approved solvents.

Section snippets

Cell lines and cell cultures

All cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured at 5% CO₂ and 37 °C. LNCaP is an androgen-responsive and androgen-dependent human PCa cell line with a mutant AR (T877A). C4-2 and CWR22Rv1 are also androgen-responsive, but also androgen-independent human PCa cell lines, which express endogenous AR. PC-3 is an androgen-independent human PCa cell line that lacks AR expression. LNCaP, C4-2, and CWR22Rv1 cell lines were maintained in

ASC-J9^® IC₅₀ values gradually increased with increasing cell confluency from 20% to 50% to 100% in AR+ PCa CRPC cell lines

The IC₅₀ values of ASC-J9^® in different PCa cell lines were determined through survival curves produced from MTT cytotoxicity assays. We first determined the ASC-J9^® IC₅₀ values in 100% confluent cells to mimic a malignant tumor morphology. We also determined the ASC-J9^® IC₅₀ values in 20% confluent cells to mimic cancer cells circulating in the bloodstream, and then in 50% confluent cells to confirm a trend between low and high cell density conditions.

The results revealed that in 20% confluent

Discussion

This study demonstrated cell-specific and time-dependent growth effects of ASC-J9^® on PCa cell line viabilities and in mice using multiple FDA-approved solvents. The IC₅₀ values of ASC-J9^® in different cell lines were determined through various cell densities to see the effect that confluency has on cell survival inhibition. The cell densities of PCa cell lines were defined as how much surface area of the culture well/dish was covered at the time of drug treatment [14], [15]. The 100% confluent

Authors contributions

Max A. Cheng, Fu-Ju Chou, Keliang Wang, Rachel Yang: designed and carried out most experiments; Max A. Cheng and Fu-Ju Chou: wrote the manuscript; Jie Ding: helped assay and maintain cell lines; Qiaoxia Zhang, Gonghui Li, and Defeng Xu: designed and carried out all in vivo mouse experiments; and Shuyuan Yeh and Chawnshang Chang: supervised the entire project.

Disclosure summary

ASC-J9^® was patented by the University of Rochester, the University of North Carolina, and AndroScience, and then licensed to AndroScience. Both the University of Rochester and C.C. own royalties and equity in AndroScience.

Conflicts of interest statement

We have no Conflicts of Interest other than those mentioned in the Disclosure Statement.

Acknowledgments

We thank Karen Wolf very much for her assistance in preparing the manuscript. This work was supported by the George Whipple Professor Endowment and NIH Grants (CA127300 and CA156700), and Taiwan Department of Health Clinical Trial and Research Center of Excellence Grant DOH99-TD-B-111-004 (China Medical University, Taichung, Taiwan) and Shenzhen Municipal Government of China (ZD201111080117A).

References (26)

K.-P. Lai
New therapeutic approach to suppress castration-resistant prostate cancer using ASC-J9 via targeting androgen receptor in selective prostate cells
Am. J. pathology
(2013)
L. Cui
Effects of salts on oxidative stability of lipids in Tween-20 stabilized oil-in-water emulsions
Food Chem.
(2016)
M.H. Park
A novel aqueous parenteral formulation of docetaxel using prodrugs
Int. J. Pharm.
(2014)
D. Xu
Cryptotanshinone suppresses androgen receptor-mediated growth in androgen dependent and castration resistant prostate cancer cells
Cancer Lett.
(2012)
K.P. Lai
New therapeutic approach to suppress castration-resistant prostate cancer using ASC-J9 via targeting androgen receptor in selective prostate cells
Am. J. Pathol.
(2013)
K. McDavid
Prostate cancer incidence and mortality rates and trends in the United States and Canada
Public Health Rep.
(2004)
A. Ahmed et al.
Advances in androgen receptor targeted therapy for prostate cancer
J. Cell Physiol.
(2014)
C. Massard et al.
Targeting continued androgen receptor signaling in prostate cancer
Clin. Cancer Res.
(2011)
L. Li
Infiltrating mast cells enhance prostate cancer invasion via altering LncRNA-HOTAIR/PRC2-androgen receptor (AR)-MMP9 signals and increased stem/progenitor cell population
Oncotarget
(2015)
C.A. Heinlein et al.
Androgen receptor in prostate cancer
Endocr. Rev.
(2004)

T. Visakorpi

In vivo amplification of the androgen receptor gene and progression of human prostate cancer

Nat. Genet.

(1995)

K. Izumi et al.

Targeting inflammatory cytokines-androgen receptor (AR) signaling with ASC-J9 to better battle prostate cancer progression

Oncoimmunology

(2013)

Z. Yang

ASC-J9 ameliorates spinal and bulbar muscular atrophy phenotype via degradation of androgen receptor

Nat. Med.

(2007)

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¹: Contributed equally.

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Original ArticlesAndrogen receptor (AR) degradation enhancer ASC-J9® in an FDA-approved formulated solution suppresses castration resistant prostate cancer cell growth

Highlights

Abstract

Introduction

Section snippets

Cell lines and cell cultures

ASC-J9® IC50 values gradually increased with increasing cell confluency from 20% to 50% to 100% in AR+ PCa CRPC cell lines

Discussion

Authors contributions

Disclosure summary

Conflicts of interest statement

Acknowledgments

Am. J. pathology

Food Chem.

Int. J. Pharm.

Cancer Lett.

Am. J. Pathol.

Prostate cancer incidence and mortality rates and trends in the United States and Canada

Public Health Rep.

Advances in androgen receptor targeted therapy for prostate cancer

J. Cell Physiol.

Targeting continued androgen receptor signaling in prostate cancer

Clin. Cancer Res.

Infiltrating mast cells enhance prostate cancer invasion via altering LncRNA-HOTAIR/PRC2-androgen receptor (AR)-MMP9 signals and increased stem/progenitor cell population

Oncotarget

Androgen receptor in prostate cancer

Endocr. Rev.