Franky L. Chan

Up-regulation of TWIST in prostate cancer and its implication as a therapeutic target

Androgen-independent metastatic prostate cancer is the main obstacle in the treatment of this cancer. Unlike a majority of solid cancers, prostate cancer usually shows poor response to chemotherapeutic drugs. In this study, we have shown a potential novel target, TWIST, a highly conserved bHLH transcription factor, in the treatment of prostate cancer. Using malignant and nonmalignant prostate tissues, we found that TWIST expression was highly expressed in the majority (90%) of prostate cancer tissues but only in a small percentage (6.7%) of benign prostate hyperplasia. In addition, the TWIST expression levels were positively correlated with Gleason grading and metastasis, indicating its role in the development and progression of prostate cancer. Furthermore, down-regulation of TWIST through small interfering RNA in androgen-independent prostate cancer cell lines, DU145 and PC3, resulted in increased sensitivity to the anticancer drug taxol-induced cell death which was associated with decreased Bcl/Bax ratio, leading to activation of the apoptosis pathway. More importantly, inactivation of TWIST suppressed migration and invasion abilities of androgen-independent prostate cancer cells, which was correlated with induction of E-cadherin expression as well as morphologic and molecular changes associated with mesenchymal to epithelial transition. These results were further confirmed on the androgen-dependent LNCaP cells ectopically expressing the TWIST protein. Our results have identified TWIST as a critical regulator of prostate cancer cell growth and suggest a potential therapeutic approach to inhibit the growth and metastasis of androgen-independent prostate cancer through inactivation of the TWIST gene.

Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin.

The flavonoid baicalin (baicalein 7-D-beta-glucuronate), isolated from the dried root of Scutellaria baicalensis Georgi (Huang Qin), is widely used in the traditional Chinese herbal medicine for its anti-inflammatory, anti-pyretic and anti-hypersensitivity effects. In the present study, we investigated the in vitro effects of baicalin on the growth, viability, and induction of apoptosis in several human prostate cancer cell lines, including DU145, PC-3, LNCaP and CA-HPV-10. The cell viability after treating with baicalin for 2-4 days was quantified by a colorimetric 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium (MTS) assay. The results showed that baicalin could inhibit the proliferation of prostate cancer cells. The responses to baicalin were different among different cell lines, with DU145 cells being the most sensitive and LNCaP cells the most resistant. Baicalin caused a 50% inhibition of DU145 cells at concentrations of 150 microM or above. The inhibition of proliferation of prostate cancer cells after a short period of exposure to baicalin was associated with induction by apoptosis, as evidenced by the typical nuclear fragmentation using Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling, DNA fragmentation, activation of caspase-3 and cleavage of poly-ADP-ribose polymerase (PARP). The results indicate that baicalin has direct anti-tumor effects on human prostate cancer cells.

Urocortin‐induced endothelium‐dependent relaxation of rat coronary artery: role of nitric oxide and K+ channels

The mechanisms underlying the vasodilator response to urocortin are incompletely understood. The present study was designed to examine the role of endothelial nitric oxide and Ba2+‐sensitive K+ channels in the endothelium‐dependent component of urocortin‐induced relaxation in the rat left anterior descending coronary artery. Urocortin induced both endothelium‐dependent and ‐independent relaxation with respective pD2 of 8.64±0.03 and 7.90±0.10. Removal of endothelium reduced the relaxing potency of urocortin. In rings pretreated with 10−4 M NG‐nitro‐L‐arginine methyl ester, 10−5 M methylene blue or 10−5 M ODQ, the urocortin‐induced relaxation was similar to that observed in endothelium‐denuded rings. L‐Arginine (5×10−4 M) antagonized the effect of NG‐nitro‐L‐arginine methyl ester. The relaxant response to urocortin was reduced in endothelium‐intact rings preconstricted by 3.5×10−2 M K+ and abolished when extracellular K+ was raised to 5×10−2 M. Pretreatment with 10−4 M BaCl2 significantly inhibited urocortin‐induced relaxation. Combined treatment with 10−4 M BaCl2 plus 10−4 M NG‐nitro‐L‐arginine methyl ester did not cause further inhibition. In urocortin (10−8 M)‐relaxed rings, BaCl2 induced concentration‐dependent reversal in vessel tone. Tertiapin‐Q (10−6 M) also attenuated urocortin‐induced relaxation. In contrast, BaCl2 did not alter urocortin‐induced relaxation in endothelium‐denuded rings. In endothelium‐denuded rings, hydroxylamine‐ and nitroprusside‐induced relaxation was inhibited by 10−4 M BaCl2, but not by 10−6 M tertiapin‐Q. The endothelium of the coronary artery was moderately stained with the antiserum against urocortin. Taken together, the present results indicate that the urocortin‐induced endothelium‐dependent relaxation of rat coronary arteries is likely attributable to endothelial nitric oxide and subsequent activation of Ba2+‐ or tertiapin‐Q‐sensitive K+ channels. The urocortin‐induced endothelium‐dependent relaxation appears to be mediated by cyclic GMP‐dependent mechanisms.

FTY720, a fungus metabolite, inhibits in vivo growth of androgen‐independent prostate cancer

FTY720, a derivative of fungus, has demonstrated dramatic anticancer effect in several malignancies recently. Our study evaluates the therapeutic potential of FTY720 in the treatment of androgen‐independent prostate cancer using a human prostate cancer xenograft in nude mice. CWR22R, an androgen‐independent human prostate tumor xenograft was inoculated into castrated nude mice and the animals were administrated with either normal saline or FTY720 (10 mg/kg) through intraperitoneal (i.p.) injection for 20 days. Body weight and tumor volume were recorded every 2 days, and serum prostate specific antigen (PSA) levels were also measured before and after the treatment. The effect of FTY720 on tumor cell proliferation was examined using antibodies against PCNA and Ki‐67 by immunohistochemical staining, MTT assay and colony forming assay, whereas apoptotic effect of FTY720 was evaluated by TUNEL assay and immunostaining using antibodies against cleaved caspase 3 and Bcl‐2. In addition, the potential inhibitory effect of FTY720 on prostate cancer angiogenesis and metastasis was investigated by immunostaining of CD31, VEGF, E‐cadherin and β‐catenin. Our results showed that FTY720 treatment led to suppression of CWR22R tumor growth without causing any detectable side effects in nude mice. The FTY720‐induced tumor suppression was correlated with decreased serum PSA level as well as reduced proliferation rate, suppression of angiogenic factors, and restoration of E‐cadherin and β‐catenin expression. In addition, the FTY720‐treated tumors showed increased apoptosis rate demonstrated by increased TUNEL‐ and cleaved caspase 3‐positive cells, and decreased Bcl‐2 expression. Our results suggest a potential novel agent in the suppression of androgen‐independent prostate cancer.

ERRγ Suppresses Cell Proliferation and Tumor Growth of Androgen-Sensitive and Androgen-Insensitive Prostate Cancer Cells and Its Implication as a Therapeutic Target for Prostate Cancer

Estrogen receptor-related receptors (ERR) are orphan nuclear receptors, which are constitutively activated without estrogen binding. Recent evidence indicates that the ligand-independent ERRs may be involved in similar ER-mediated regulatory pathways and modulate estrogen responsiveness in certain target cells. We recently showed that an ERR subtype, ERRgamma, is coexpressed with ERbeta in normal human prostatic epithelial cells and exhibits reduced expression in many prostate cancer cell lines and clinical neoplastic prostate tissues. Based on this, we hypothesize that ERRgamma may have growth regulatory roles in prostate and prostate cancer. We showed in this study that ERRgamma was expressed in epithelial cell nuclei in fetal and pubertal human prostates, whereas its nuclear expression became reduced in advanced prostate cancer lesions. Stable ERRgamma expression by retroviral transduction suppressed significantly both in vitro cell growth and in vivo tumorigenicity of two prostate cancer cell lines, LNCaP and DU145, as evidenced by a cell-cycle arrest at G(1)-S transition and also induction of two cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1). We further showed by reporter assay that induction of p21 and p27 by ERRgamma was mediated through direct transactivation of their gene promoters. Moreover, we also showed that a selective ERRgamma-agonist, DY131, could potentiate the ERRgamma-induced growth inhibition in LNCaP-ERRgamma and DU145-ERRgamma cells in a dose-dependent manner compared with respective parental cells. Taken together, our results show that ERRgamma may perform an antiproliferative or tumor-suppressing function in prostate cancer cells. More importantly, our results suggest that ERRgamma could be a novel therapeutic target for prostate cancer treatment.

Difference in flavonoid and isoflavone profile between soybean and soy leaf

The present study was to compare the flavonoid profile between soybean and soy leaves. Soybean was most abundant in malonyl-genistin followed by malonyl-daidzin, genistin, daidzin, genistein and daidzein in a decreasing order. In contrast, soy leaves contained only trace amounts of malonyl-genistin and genistin, but they had the six unknown flavonoids that were absent in soybean. The six unknown compounds were isolated by using various chromatographic techniques and the structures were identified by studying their varying spectra of ultraviolet (UV), infrared (IR), Mass, 1H-NMR and 13C-NMR. It was found that the six unknown compounds were all kaempferol glycosides namely kaempferol-3-O-alpha-L-rhamnopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->6)-beta-D-galactopyranoside, kaempferol-3-O-(2,6-di-O-alpha-rhamnopyranosyl)-beta-galactopyranoside, kaempferol-3-O-alpha-L-rhamnopyranosyl (1-->6)-beta-D-galactopyranoside, kaempferol-3-O-digalactopyranoside, kaempferol-3-O-diglucopyranoside and kaempferol-3-O-rutinoside. It was concluded that the flavonoids in soy leaves were mainly kaempferol glycosides, whereas those in soybean were mainly isoflavone glycosides and derivatives.

Silencing of the retinoid response gene TIG1 by promoter hypermethylation in nasopharyngeal carcinoma

Tazarotene‐induced gene 1 (TIG1) and Tazarotene‐induced gene 3 (TIG3) are retinoid acid (RA) target genes as well as candidate tumor suppressor genes in human cancers. In our study, we have investigated the expression of TIG1 and TIG3 in nasopharyngeal carcinoma (NPC). Loss of TIG1 expression was found in 80% of NPC cell lines and 33% of xenografts, whereas TIG3 was expressed in all NPC samples and immortalized nasopharyngeal epithelial cells. In order to elucidate the epigenetic silencing of TIG1 in NPC, the methylation status of TIG1 promoter was examined by genomic bisulfite sequencing and methylation‐specific PCR (MSP). We have detected dense methylation of TIG1 5′CpG island in the 5 TIG1‐negative NPC cell lines and xenograft (C666‐1, CNE1, CNE2, HONE1 and X666). Partial methylation was observed in 1 NPC cell line HK1 showing dramatic decreased in TIG1 expression. Promoter methylation was absent in 2 TIG1‐expressed NPC xenografts and the normal epithelial cells. Restoration of TIG1 expression and unmethylated alleles were observed in NPC cell lines after 5‐aza‐2′‐deoxycytidine treatment. Moreover, the methylated TIG1 sequence was detected in 39 of 43 (90.7%) primary NPC tumors by MSP. In conclusion, our results showed that TIG1 expression is lost in the majority of NPC cell lines and xenografts, while promoter hypermethylation is the major mechanism for TIG1 silencing. Furthermore, the frequent epigenetic inactivation of TIG1 in primary NPC tumors implied that it may play an important role in NPC tumorigenesis.

A protein kinase G-sensitive channel mediates flow-induced Ca2+ entry into vascular endothelial cells

The hemodynamic force generated by blood flow is considered to be the physiologically most important stimulus for the release of nitric oxide (NO) and prostacyclin (PGI2) from vascular endothelial cells (1). NO and PGI2 then act on the underlying smooth muscle cells, causing vasodilation and thus lowering blood pressure (2, 3). One critical early event occurring in this flow‐induced regulation of vascular tone is that blood flow induces Ca2+ entry into vascular endothelial cells, which in turn leads to the formation of NO (4, 5). Here we report a mechanosensitive Ca2+‐permeable channel in vascular endothelial cells. The activity of the channel was inhibited by 8‐Br‐cGMP, a membrane‐permeant activator of protein kinase G (PKG), in cell‐attached membrane patches. The inhibition could be reversed by PKG inhibitor KT5823 or H‐8. A direct application of active PKG in inside‐out patches blocked the channel activity. Gd3+, Ni2+, or SK&F‐96365 also inhibited the channel activity. A study of fluorescent Ca2+ entry revealed a striking pharmacological similarity between the Ca2+ entry elicited by flow and the mechanosensitive Ca2+‐permeable channel we identified, suggesting that this channel is the primary pathway mediating flow‐induced Ca2+ entry into vascular endothelial cells.—Yao, X., Kwan, H. Y., Chan, F. L., Chan, N. W. K., Huang, Y. A protein kinase G‐sensitive channel mediates flow‐induced Ca2+ entry into vascular endothelial cells. FASEB J. 14, 932–938 (2000)

Transient receptor potential channel TRPC5 is essential for P-glycoprotein induction in drug-resistant cancer cells

An attractive strategy to overcome multidrug resistance in cancer chemotherapy is to suppress P-glycoprotein (P-gp), which is a pump overproduced in cancer cells to remove cytotoxic drugs from cells. In the present study, a Ca2+-permeable channel TRPC5 was found to be overproduced together with P-gp in adriamycin-resistant breast cancer cell line MCF-7/ADM. Suppressing TRPC5 activity/expression reduced the P-gp induction and caused a remarkable reversal of adriamycin resistance in MCF-7/ADM. In an athymic nude mouse model of adriamycin-resistant human breast tumor, suppressing TRPC5 decreased the growth of tumor xenografts. Nuclear factor of activated T cells isoform c3 (NFATc3) was the transcriptional factor that links the TRPC5 activity to P-gp production. Together, we demonstrated an essential role of TRPC5–NFATc3–P-gp signaling cascade in P-gp induction in drug-resistant cancer cells.

Orphan nuclear receptor estrogen-related receptor-β suppresses in vitro and in vivo growth of prostate cancer cells via p21WAF1/CIP1 induction and as a potential therapeutic target in prostate cancer

Recent studies indicate that estrogen-related receptors (ERRs) are involved in similar estrogen receptor (ER) regulatory pathways and play roles in energy and lipid metabolism. Here, we analysed the functional role of ERRβ in prostate cancer cell growth regulation in an androgen-sensitive and androgen-insensitive prostate cancer cell lines. ERRβ was expressed in normal human prostates, but exhibited a reduced expression in prostate cancer lesions. Stable ERRβ expression suppressed significantly cell proliferation and tumorigenicity of LNCaP and DU145 cells, accompanied by an S-phase suppression and increased p21 expression. Reporter and chromatin immunoprecipitation assays showed that ERRβ could directly transactivate p21 gene promoter, which could be further enhanced by peroxisome proliferator-activated receptor-γ coactivator-1α. Truncation analysis showed that ERRβ-mediated p21 transactivation and prostate cancer cell growth inhibition required intact DNA-binding domain and AF2 domains in ERRβ. Interestingly, ERRβ displayed a cell cycle associated downregulated expression pattern in ERRβ-transduced and non-transduced cells. Finally, we showed that ERRβ-mediated growth inhibition could be potentiated by an ERRβ/γ agonist DY131. Knockdown of ERRβ by RNA interference could reduce the DY131-induced growth inhibition in prostate cancer cells. Taken together, our findings indicate that ERRβ performs a tumor suppressing function in prostate cancer cells, and targeting ERRβ could be a potential therapeutic strategy for prostate cancer.