Lih-Jen Su

A study of high‐dose oral silybin‐phytosome followed by prostatectomy in patients with localized prostate cancer

Silibinin is a polyphenolic flavonolignan derived from milk thistle (Silybum marianium) with anti‐oxidant properties. The purpose of the current trial was to determine the tissue and blood effects of high‐dose silybin‐phytosome in prostate cancer patients.

Silibinin synergizes with mitoxantrone to inhibit cell growth and induce apoptosis in human prostate cancer cells

The purpose of these experiments was to assess the synergistic activity of silibinin with chemotherapy agents in clinical use against prostate cancer. Silybin‐phytosome, a commercially available formulation containing silibinin, has recently been studied in a phase I clinical trial. The silibinin doses used in the present study are clinically achievable based on the preliminary phase I data. DU145, PC‐3 and LNCaP prostate cancer cells were seeded in 96‐well plates in triplicate. Twenty‐four hours later, silibinin (10, 20 and 40 μM) and either mitoxantrone or docetaxel were added to the designated wells. Seventy‐two hours post‐treatment, cell viability was determined with a tetrazolium‐based assay. The combination index (CI) for determination of a synergistic effect was calculated, with values of <0.9 indicating synergy and values >1.1 antagonism. Apoptosis was also assessed using a luminescent assay after 72 hr of treatment with media alone, silibinin, mitoxantrone, or silibinin plus mitoxantrone. Silibinin showed a synergistic effect with mitoxantrone, as measured by reduction in cell viability. The CI values ranged from 0.413 to 2.650 for the combination of silibinin and mitoxantrone; in contrast, treatment with docetaxel and silibinin showed little or no synergy, with CI values of 0.898–4.469. In concordance with these findings, the addition of silibinin increased the level of apoptosis compared to mitoxantrone alone, particularly in the PC‐3 cells. The combination of silibinin and mitoxantrone exhibits a pattern of synergy in reducing cell viability with increased apoptosis. These data are important in the planning of future clinical applications of silibinin.

In vitro expression of the diphtheria toxin A-chain gene under the control of human chorionic gonadotropin gene promoters as a means of directing toxicity to ovarian cancer cell lines ☆ ☆☆ ★ ★★

OBJECTIVE Our goal was to determine whether toxicity of the diphtheria toxin A-chain gene regulated by the human chorionic gonadotropin promoter can be directed to malignant ovarian cell lines. STUDY DESIGN Plasmids containing diphtheria toxin A-chain gene linked to the regulatory elements of the metalloergothioneine and human chorionic gonadotropin promoters were transfected into the cell lines. Expression of diphtheria toxin A-chain gene was determined by the inhibition of a cotransfected luciferase reporter gene. RESULTS Cytotoxicity of the diphtheria toxin A-chain gene is shown in a dose-responsive manner. Transfection of a plasmid expressing the diphtheria toxin A-chain gene controlled by a constitutive promoter readily inhibits protein synthesis. Specific inhibition of luciferase protein synthesis occurs in ovarian cancer cells transfected with the diphtheria toxin A-chain gene under the control of the human chorionic gonadotropin promoters when compared with normal ovarian epithelial cells or fibroblasts. CONCLUSIONS These data demonstrate the preferential expression of the diphtheria toxin A-chain gene, regulated by the human chorionic gonadotropin promoter, to ovarian cancer cell lines. This provides an avenue for targeting such cells for suicide, toxin, or cytokine genes.

Diphtheria Toxin–Epidermal Growth Factor Fusion Protein DAB389EGF for the Treatment of Bladder Cancer

Purpose: The novel fusion protein, DAB389EGF, is composed of both the catalytic and the translocation domains of diphtheria toxin that are fused to the human EGF, providing a targeting and a toxicity component. We tested DAB389EGF for antitumor activity in both in vitro and in vivo urinary bladder cancer models. Experimental Design: Human bladder cancer lines were treated with DAB389EGF and assessed for growth inhibition and clonogenic suppression. Using 6- to 8-week-old female athymic nude mice implanted orthotopically with HTB9 cells, DAB389EGF was administered intravesically twice weekly for 2 weeks. The response of the luciferase-expressing HTB9 cells was monitored via bioluminescence as the primary endpoint. Results: Treatment response with DAB389EGF was specific and robust, with an IC50 ranging from 0.5 to 15 ng/mL in eight tested bladder cancer cell lines, but greater than 50 ng/mL in the EGF receptor (EGFR)-negative H520 control cell line. Simulating short-duration intravesical therapy used clinically, a 2-hour treatment exposure of DAB389EGF (10 ng/mL) produced clonogenic suppression in three selected bladder cancer cell lines. In vivo, luciferase activity was suppressed in five of six mice treated with DAB389EGF [70 μL (1 ng/μL) per mouse], as compared with only one of six mice treated with a control diphtheria toxin (DT) fusion protein. Histologic assessment of tumor clearance correlated with the bioluminescent changes observed with DAB389EGF treatment. Immunocompetent mice treated with intravesical DAB389EGF did not show any nonspecific systemic toxicity. Conclusions: The intravesical delivery of targeted toxin fusion proteins is a novel treatment approach for non–muscle-invasive urinary bladder cancer. With appropriate targeting, the treatments are effective and well-tolerated in vivo. Clin Cancer Res; 19(1); 148–57. ©2012 AACR.

Dual epidermal growth factor receptor and vascular endothelial growth factor receptor inhibition with vandetanib sensitizes bladder cancer cells to cisplatin in a dose- and sequence-dependent manner

To investigate the activity of the combination of vandetanib and cytotoxic agents using in vitro models of bladder cancer, as modern chemotherapy regimens are built around cisplatin, with gemcitabine or a taxane such as docetaxel also commonly added in combination for the treatment of advanced bladder cancer.

CUSP/p63 expression in basal cell carcinoma

Abstract: Chronic ulcerative stomatitis protein (CUSP), the most abundant cutaneous isoform of p63, is a p53‐related gene essential for epithelial development. CUSP lacks the N‐terminal transactivation domain found on other p53 family members and has been shown to inhibit p53 function in vitro. In this study, biopsies of normal skin (21 of 21), benign neoplasms [seborrheic keratosis (3 of 3), acrochordon (2 of 3), and verruca plana (3 of 3)], and squamous cell carcinomas (SCC) (4 of 4) displayed strong nuclear CUSP immuno‐reactivity in epidermal cells. In contrast few basal cell carcinomas (BCC) (7 of 27) and sebaceous nevi (1 of 2) displayed this pattern of CUSP immunoreactivity. Thus, biopsies of cutaneous conditions characterized by sonic hedgehog (SHH) pathway dysregulation were more than 86 times as likely to lack CUSP/p63 immunofluorescence as were other cutaneous samples. Adjacent normal‐appearing skin from patients with basal cell nevus syndrome (BCNS) (2 of 3) also lacked CUSP immuno‐staining. Lastly, a BCC arising in a patched heterozygous mouse also lacked CUSP immuno‐staining. Because CUSP mRNA and protein were detected via Northern and Western analysis in BCC samples lacking CUSP immuno‐staining, we sequenced the coding region of CUSP from two non‐staining BCCs but found no mutations. Therefore, CUSP appears to be present, unmutated, and yet frequently undetectable by immunofluorescence in cutaneous lesions in both humans and mice that are associated with SHH pathway dysregulation (BCCs, BCNS, and nevus sebaceous).

miR-125b Regulation of Androgen Receptor Signaling Via Modulation of the Receptor Complex Co-Repressor NCOR2.

Abstract Recognition of micro-RNA function and their contribution to the biology of disease has given a new insight into disease mechanisms, with these discoveries potentially improving clinical diagnostic and therapeutic options. miR-125b has been identified as an important regulator in various cancers, including prostate cancer, but the mechanism of this regulation remains incompletely understood. In these studies, the effect of castration on miR-125b serum expression was evaluated in mice, simulating androgen deprivation. Furthermore, miR-125b expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) in LNCaP prostate cancer cells treated with the antiandrogen bicalutamide. Using LNCaP cells, the effect of miR-125b modulation on apoptotic protein and NCOR2, a co-repressor of androgen receptor (AR), was examined by Western blot. A 3′-untranslated region (UTR) luciferase-binding assay was performed to confirm that miR-125b targets NCOR2. We found that surgical castration induced an initial increase in the expression of circulating miR-125b in mice, while sham surgery did not. In addition, AR blockade via bicalutamide was associated with the rapid release of miR-125b into the cell culture medium of prostate cancer cells. A previously studied target of miR-125b, a regulator in the apoptotic pathway, BAK1, could not completely account for the role of miR-125b in prostate cancer. Thus, we looked for additional targets of miR-125b and found that NCOR2, which is a repressor of AR, is a direct target of miR-125b. We found that NCOR2 protein expression was blocked by mimics of miR-125b, and a luciferase-binding assay confirmed that NCOR2 is a direct target of miR-125b. Our data provide novel evidence that miR-125b is an important regulator of the AR with specific ramification for the effectiveness of antiandrogens and other hormonal therapies in prostate cancer.

Pazopanib synergizes with docetaxel in the treatment of bladder cancer cells

OBJECTIVES To investigate the efficacy of pazopanib, both alone and in combination with docetaxel, in bladder cancer cells. Bladder cancer expresses many potential therapeutic targets of biological agents, including the vascular endothelial growth factor receptor (VEGFR). Pazopanib is a small molecule inhibitor of VEGFR-1, -2-3, platelet-derived growth factor receptor (PDGFR), and c-Kit. MATERIALS AND METHODS Using human bladder cancer cells HTB3, HT1376, J82, RT4, CRL1749, T24, Sup, and HTB9, the treatment effect of pazopanib and cytotoxic chemotherapy was assessed using a tetrazolium-based assay. The combinatorial effect of these agents on clonogenic growth was further examined. Western blotting was used to assess changes in relevant downstream targets, including phospho-AKT, phospho-FAK, total AKT, and total FAK. RESULTS Single-agent pazopanib had modest activity. However, synergy was seen with the combination of docetaxel and pazopanib in these multiple cells lines. J82 and T24 cells were selected for additional clonogenic testing because of their resistance to single-agent docetaxel chemotherapy. 1.25 nM of docetaxel had little effect on clonogenic formation; however, in combination with pazopanib, significant inhibition of colony formation was observed. This combination treatment additionally decreased phospho-AKT, an important mediator of cell survival in all cell lines, whereas phospho-FAK expression was variably affected. CONCLUSIONS The present study demonstrates synergistic efficacy of pazopanib with docetaxel in docetaxel-resistant bladder cancer cells. This work supports future evaluation of pazopanib with docetaxel for the treatment of bladder cancer with the potential of improved efficacy and toxicity.

VEGFR and EGFR inhibition increases epithelial cellular characteristics and chemotherapy sensitivity in mesenchymal bladder cancer cells

The present study investigated the effect of VEGFR and EGFR inhibition via vandetanib (Zactima) on epithelial-mesenchymal transition (EMT) in bladder cancer. Markers of EMT (EGFR, VEGR, E-cadherin and vimentin) were interogated by Western blotting at baseline and after treatment with EGF, VEGF, vandetanib, cisplatin, or their combination using representative epithelial- and mesenchymal-type human bladder cancer cells. Morphological changes induced by these treatments were examined by microscopy over various time courses. The effect of these changes on cisplatin chemotherapy sensitivity was assessed by MTT assay. RT4 and HTB3 cells had epithelial features while CRL1749 and J82 cells had mesenchymal features. After treatment with EGF, the epithelial-type cells demonstrated increased intercellular separation and pseudopodia, with these changes blocked by vandetanib. In contrast, the mesenchymal cells did not exhibit any morphological changes with the EGF treatment but adopted a clustered/epithelial appearance after the administration of vandetanib. Western blotting shows that treatment of epithelial cells with vandetanib increased the expression of E-cadherin. In comparison, mesenchymal cells demonstrated decreased vimentin expression with the treatment of vandetanib in the presence of EGF and VEGF. Improved growth inhibition was seen in the epithelial cells but not in mesenchymal cells with the concurrent treatment of vandetanib and cisplatin. Sequential treatment of mesenchymal cells with vandetanib followed by cisplatin demonstrated synergy with improved cisplatin activity. The findings offer a novel role of vandetanib on the EMT in bladder cancer, providing insight into EMT in bladder cancer.

The CUSP ΔNp63α isoform of human p63 is downregulated by solar-simulated ultraviolet radiation

Background: In normal human keratinocytes, a p53-like protein, ΔNp63α, also known as CUSP, is constitutively and abundantly expressed. The significant constitutive expression of ΔNp63α in stratified epithelium has been proposed to maintain the proliferative capacity of basal cells, blocking the consequences of inappropriate p53 activation. Objective: To determine the response of keratinocyte ΔNp63α to ultraviolet radiation (UVR), a stimulus for p53 activation. Methods: Cultured normal human keratinocytes were exposed to graded doses of solar-simulated UVR. The expression of ΔNp63α protein and mRNA were measured with Western and Northern blotting. Normal mouse skin was exposed to UVR, and ΔNp63α expression assessed with immunohistochemistry. Results: Increasing doses of UVR virtually shut off ΔNp63α protein and mRNA expression in cultured normal human keratinocytes and in normal mouse skin in vivo. Conclusion: This study supports the hypothesis that in situations where p53 activation is desirable, as with DNA-damaging UVR, ΔNp63α downregulation occurs and may possibly allow for better target gene transcription by p53.