Xichun Liu

Berberine Suppresses Androgen Receptor Signaling in Prostate Cancer

The androgen receptor (AR) is critical in the normal development and function of the prostate, as well as in prostate carcinogenesis. Androgen deprivation therapy is the mainstay in the treatment of advanced prostate cancer; however, after an initial response, the disease inevitably progresses to castration-resistant prostate cancer (CRPC). Recent evidence suggests that continued AR activation, sometimes in a ligand-independent manner, is commonly associated with the development of CRPC. Thus, novel agents targeting the AR are urgently needed as a strategic step in developing new therapies for this disease state. In this study, we investigated the effect of berberine on AR signaling in prostate cancer. We report that berberine decreased the transcriptional activity of AR. Berberine did not affect AR mRNA expression, but induced AR protein degradation. Several ligand-binding, domain-truncated AR splice variants have been identified, and these variants are believed to promote the development of CRPC in patients. Interestingly, we found that these variants were more susceptible to berberine-induced degradation than the full-length AR. Furthermore, although the growth of LNCaP xenografts in nude mice was inhibited by berberine, and AR expression was reduced in the tumors, the morphology and AR expression in normal prostates were not affected. This study is the first to show that berberine suppresses AR signaling and suggests that berberine, or its derivatives, presents a promising agent for the prevention and/or treatment of prostate cancer. Mol Cancer Ther; 10(8); 1346–56. ©2011 AACR.

AKR1C3 overexpression may serve as a promising biomarker for prostate cancer progression

BackgroundAldo-keto reductase family 1 member C3 (AKR1C3) is a key steroidogenic enzyme that is overexpressed in prostate cancer (PCa) and is associated with the development of castration-resistant prostate cancer (CRPC). The aim of this study was to investigate the correlation between the expression level of AKR1C3 and the progression of PCa.MethodsSixty human prostate needle biopsy tissue specimens and ten LNCaP xenografts from intact or castrated male mice were included in the study. The relationship between the level of AKR1C3 expression by immunohistochemistry and evaluation factors for PCa progression, including prostate-specific antigen (PSA), Gleason score (GS) and age, were analyzed.ResultsLow immunoreactivity of AKR1C3 was detected in normal prostate epithelium, benign prostatic hyperplasia (BPH) and prostatic intraepithelial neoplasia (PIN). Positive staining was gradually increased with an elevated GS in PCa epithelium and LNCaP xenografts in mice after castration. The Spearman’s r values (rs) of AKR1C3 to GS and PSA levels were 0.396 (P = 0.025) and -0.377 (P = 0.036), respectively, in PCa biopsies. The rs of AKR1C3 to age was 0.76 (P = 0.011). No statistically significant difference was found with other variables.ConclusionOur study suggests that the level of AKR.1C3 expression is positively correlated with an elevated GS, indicating that AKR1C3 can serve as a promising biomarker for the progression of PCa.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7748245591110149.

Survivin gene silencing sensitizes prostate cancer cells to selenium growth inhibition

BackgroundProstate cancer is a leading cause of cancer-related death in men worldwide. Survivin is a member of the inhibitor of apoptosis (IAP) protein family that is expressed in the majority of human tumors including prostate cancer, but is barely detectable in terminally differentiated normal cells. Downregulation of survivin could sensitize prostate cancer cells to chemotherapeutic agents in vitro and in vivo. Selenium is an essential trace element. Several studies have shown that selenium compounds inhibit the growth of prostate cancer cells. The objective of this study is to investigate whether survivin gene silencing in conjunction with selenium treatment could enhance the therapeutic efficacy for prostate cancer and to elucidate the underlying mechanisms.MethodsExpression of survivin was analyzed in a collection of normal and malignant prostatic tissues by immunohistochemical staining. In vitro studies were conducted in PC-3M, C4-2B, and 22Rv1 prostate cancer cells. The effect of selenium on survivin expression was analyzed by Western blotting and semi-quantitative RT-PCR. Survivin gene knockdown was carried out by transfecting cells with a short hairpin RNA (shRNA) designed against survivin. Cell proliferation was quantitated by the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-Diphenyltetrazolium Bromide (MTT) assay and apoptosis by propidium iodide staining followed by flow cytometry analysis. Finally, in vivo tumor growth assay was performed by establishing PC-3M xenograft in nude mice and monitoring tumor growth following transfection and treatment.ResultsWe found that survivin was undetectable in normal prostatic tissues but was highly expressed in prostate cancers. Survivin knockdown or selenium treatment inhibited the growth of prostate cancer cells, but the selenium effect was modest. In contrast to what have been observed in other cell lines, selenium treatment had little or no effect on survivin expression in several androgen-independent prostate cancer cell lines. Survivin knockdown sensitized these cells to selenium growth inhibition and apoptosis induction. In nude mice bearing PC-3M xenografts, survivin knockdown synergizes with selenium in inhibiting tumor growth.ConclusionsSelenium could inhibit the growth of hormone-refractory prostate cancer cells both in vitro and in vivo, but the effects were modest. The growth inhibition was not mediated by downregulating survivin expression. Survivin silencing greatly enhanced the growth inhibitory effects of selenium.

A Whole Blood Assay for AR-V7 and ARv567es in Patients with Prostate Cancer

PURPOSE Most prostate cancer mortality can be attributed to metastatic castration resistant prostate cancer, an advanced stage that remains incurable despite recent advances. The AR (androgen receptor) signaling axis remains active in castration resistant prostate cancer. Recent studies suggest that expression of the AR-V (AR splice variant) AR-V7 may underlie resistance to abiraterone and enzalutamide. However, controversy exists over the optimal assay. Our objective was to develop a fast and sensitive assay for AR-Vs in patients. MATERIALS AND METHODS Two approaches were assessed in this study. The first approach was based on depletion of leukocytes and the second one used RNA purified directly from whole blood preserved in PAXgene® tubes. Transcript expression was analyzed by quantitative reverse transcription-polymerase chain reaction. RESULTS Through a side-by-side comparison we found that the whole blood approach was suitable to detect AR-Vs. The specificity of the assay was corroborated in a cancer-free cohort. Using the PAXgene assay samples from a cohort of 46 patients with castration resistant prostate cancer were analyzed. Overall, AR-V7 and ARv567es were detected in 67.53% and 29.87% of samples, respectively. Statistical analysis revealed a strong association of AR-V positivity with a history of second line hormonal therapies. CONCLUSIONS To our knowledge this is the first study to demonstrate that PAXgene preserved whole blood can be used to obtain clinically relevant information regarding the expression of 2 AR-Vs. These data on a castration resistant prostate cancer cohort support a role for AR-Vs in resistance to therapies targeting the AR ligand-binding domain.

20(S)-Protopanaxadiol-aglycone Downregulation of the Full-length and Splice Variants of Androgen Receptor

As a public health problem, prostate cancer engenders huge economic and life‐quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high‐grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively active AR splice variants (AR‐Vs) lacking the ligand‐binding domain. Thus, novel agents targeting the receptor, preferentially both the full‐length and AR‐Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)‐protopanaxadiol‐aglycone (PPD) effectively downregulates the expression and activity of both the full‐length AR and AR‐Vs. The effects of PPD on AR and AR‐Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome‐mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full‐length AR and AR‐Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer.

20(S)-Protopanaxadiol Inhibition of Progression and Growth of Castration-Resistant Prostate Cancer

Castration-resistant progression of prostate cancer after androgen deprivation therapies remains the most critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor (AR) activity is an established driver of castration-resistant progression, and upregulation of the full-length AR (AR-FL) and constitutively-active AR splice variants (AR-Vs) has been implicated to contribute to the resurgent AR activity. We reported previously that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) can reduce the abundance of both AR-FL and AR-Vs. In the present study, we further showed that the effect of PPD on AR expression and target genes was independent of androgen. PPD treatment resulted in a suppression of ligand-independent AR transactivation. Moreover, PPD delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation and inhibited the growth of castration-resistant 22Rv1 xenograft tumors with endogenous expression of AR-FL and AR-Vs. This was accompanied by a decline in serum prostate-specific antigen levels as well as a decrease in AR levels and mitoses in the tumors. Notably, the 22Rv1 xenograft tumors were resistant to growth inhibition by the next-generation anti-androgen enzalutamide. The present study represents the first to show the preclinical efficacy of PPD in inhibiting castration-resistant progression and growth of prostate cancer. The findings provide a rationale for further developing PPD or its analogues for prostate cancer therapy.

Methylseleninic acid is a novel suppressor of aromatase expression

Elevated circulating estrogen levels, as a result of increased peripheral aromatization of androgens by aromatase, have been indicated to underlie the association between obesity and a higher risk of breast cancer in postmenopausal women. Although aromatase inhibitors have been used as a first-line therapy for estrogen receptor-positive breast cancer in postmenopausal women, their potential as breast cancer chemopreventive agents has been limited due to toxicities and high costs. It is therefore imperative to develop new aromatase-inhibiting/suppressing agents with lower toxicities and lower costs for breast cancer chemoprevention, especially in obese postmenopausal women. The expression of the aromatase gene, CYP19, is controlled in a tissue-specific manner by the alternate use of different promoters. In obese postmenopausal women, increased peripheral aromatase is primarily attributed to the activity of the glucocorticoid-stimulated promoter, PI.4, and the cAMP-stimulated promoter, PII. In the present study, we show that methylseleninic acid (MSA), a second-generation selenium compound, can effectively suppress aromatase activation by dexamethasone, a synthetic glucocorticoid, and forskolin, a specific activator of adenylate cyclase. Unlike the action of aromatase inhibitors, MSA suppression of aromatase activation is not mediated via direct inhibition of aromatase enzymatic activity. Rather, it is attributable to a marked downregulation of promoters PI.4- and PII-specific aromatase mRNA expression, and thereby a reduction of aromatase protein. Considering the low-cost and low-toxicity nature of MSA, our findings provide a strong rationale for the further development of MSA as a breast cancer chemopreventive agent for obese postmenopausal women.

Plasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth.

Persistent activation of Survivin and its overexpression contribute to the formation, progression and metastasis of several different tumor types. Therefore, Survivin is an ideal target for RNA interference mediated-growth inhibition. Blockade of Survivin using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth. RNA interference does not fully ablate target gene expression, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Survivin-specific shRNA, we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 (GRIM-19). Then, the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium (S. typhimurium) to treat prostate cancer in vitro and in vivo. We found that the co-expressed Survivin-specific shRNA and GRIM-19 synergistically and more effectively inhibited prostate tumor proliferation and survival, when compared with treatment with either single agent alone in vitro and in vivo. This study has provided a novel cancer gene therapeutic approach for prostate cancer.

Methylselenocysteine preventing castration-resistant progression of prostate cancer.

Castration‐resistant progression of prostate cancer after androgen deprivation therapy remains a critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor activity is an established driver of castration‐resistant progression, and upregulation of androgen receptor expression has been implicated to contribute to the resurgent androgen receptor activity. We reported previously that methylselenocysteine can decrease the expression and activity of androgen receptor. Here we investigated the ability of methylselenocysteine to inhibit castration‐resistant progression of prostate cancer.

Abstract 5561: 20(S)-Protopanaxadiol-aglycone downregulates full-length and ligand-independent splice variants of androgen receptor

Prostate carcinogenesis is characterized by a long latency of 20 to 40 years. Chemoprevention to manage the disease at an early stage to prevent it from becoming clinical relevant is increasingly being recognized as an important aspect of prostate cancer control. Androgen signaling plays a vital role in the development and progression of prostate cancer. Finasteride and dutasteride, which inhibit the formation of dihydrotestosterone, are the only chemopreventive agents that have been shown definitively to decrease prostate cancer incidence. However, their effectiveness appears to be limited to Gleason 6 cancers. In addition, cancer cells expressing high level of constitutively-active, ligand-independent splice variants of androgen receptor may not be responsive to treatment with finasteride or dutasteride. Therefore, there is an urgent need to develop new chemopreventive agents that could block androgen signaling through both the full-length and splice variants of androgen receptor. Ginsenosides are the main ingredients responsible for the pharmaceutical functions of ginseng, a commonly used medicinal herb among cancer patients. Several ginsenosides have been implicated to inhibit prostate cancer cell growth. However, the underlying mechanism is largely unknown. Here we provide the first evidence that, in prostate cancer cells, ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of androgen receptor, including both the full-length and the constitutively-active, ligand-independent splice variants. The effect of PPD on androgen receptor is manifested by an immediate drop in protein, followed by a reduction in mRNA. The initial decrease in androgen receptor protein could be attributed to PPD induction of proteasome-mediated degradation, possibly as a result of disrupted androgen receptor N-C interaction. Depressing the inhibitory effect of PPD on androgen receptor by knocking down androgen receptor before PPD treatment weakens significantly the growth-suppressive activity of PPD, indicating the important contribution of androgen receptor downregulation to PPD action in prostate cancer cells. This report is also the first to establish the in vivo preclinical efficacy of PPD against the growth of androgen receptor-expressing prostate cancer cells, which constitute the majority of clinical prostate carcinomas. In addition to tumor growth inhibition, PPD supplementation also leads to in vivo downregulation of androgen receptor and its target gene, prostate-specific antigen. Considering the critical role of androgen receptor signaling in prostate cancer development and progression and the role of the ligand-independent androgen receptor splice variants in disease recurrence, our findings provide strong justification for further development of PPD for prostate cancer prevention and treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5561. doi:10.1158/1538-7445.AM2011-5561