Horng-Heng Juang

Triiodothyronine Modulates Cell Proliferation of Human Prostatic Carcinoma Cells by Downregulation of the B-Cell Translocation Gene 2

Studies suggest that triiodothyronine (T3) and cognate nuclear receptors (hTR) are involved in regulation of prostatic cell growth and differentiation. To probe mechanisms for T3 effects, we studied prostate carcinoma cells, investigating the effect of T3 on expression of the B‐cell translocation gene 2 (BTG2), which regulates the G1/S transition of the cell cycle.

Curcumin Blocks the Activation of Androgen and Interlukin-6 on Prostate-Specific Antigen Expression in Human Prostatic Carcinoma Cells

Curcumin, a naturally occurring compound, exhibits anticancer chemopreventive effects. We evaluated the effects and mechanisms of curcumin on the gene expression of prostate-specific antigen (PSA) in human androgen-sensitive prostatic carcinoma cells. LNCaP cells were used to determine the effect of curcumin on PSA expression. Quantitative PSA expression was assessed by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunoblot assay. The modulation of androgen, interlukin-6 (IL-6), and prostate-derived Ets factor (PDEF) on the PSA gene was identified by transient gene expression assay with the use of a PSA reporter vector. The effect of curcumin on the activity of androgen receptors was evaluated by electrophoretic mobility shift assay (EMSA). Immunoblot assays, RT-PCR, and ELISA indicated that curcumin treatments blocked the stimulation of methyltrienolone (R1881) and IL-6 on PSA gene expression in LNCaP cells. The effects of curcumin appear to be mediated via the androgen response element of PSA gene. Results from immunoblot assay and EMSA revealed the modulation of curcumin on the expression of androgen receptor and androgen receptor binding activity on androgen response element of PSA gene. Although overexpression of PDEF dramatically enhanced PSA gene expression, the results of immunoblot assays and transient reporter assays indicated that curcumin treatments did not affect the gene expression of PDEF. Curcumin inhibits R1881- and IL-6-mediated PSA gene expression in LNCaP cells through down-regulation of the expression and activity of androgen receptors.

l-Mimosine blocks cell proliferation via upregulation of B-cell translocation gene 2 and N-myc downstream regulated gene 1 in prostate carcinoma cells

L-Mimosine, an iron chelator and a prolyl 4-hydroxylase inhibitor, blocks many cancer cells at the late G1 phase. B-cell translocation gene 2 (Btg2) regulates the G1/S transition phases of the cell cycle. N-myc downstream regulated gene 1 (Ndrg1) is a differentiation-inducing gene upregulated by hypoxia. We evaluated the molecular mechanisms of L-mimosine on cell cycle modulation in PC-3 and LNCaP prostate carcinoma cells. The effect of L-mimosine on cell proliferation of prostate carcinoma cells was determined by the [3H]thymidine incorporation and flow cytometry assays. L-Mimosine arrested the cell cycle at the G1 phase in PC-3 cells and at the S phase in LNCaP cells, thus attenuating cell proliferation. Immunoblot assays indicated that hypoxia and L-mimosine stabilized hypoxia-inducible factor-1α (HIF-1α) and induced Btg2 and Ndrg1 protein expression, but downregulated protein levels of cyclin A in both PC-3 and LNCaP cells. L-Mimosine treatment decreased cyclin D1 protein in PC-3 cells, but not in LNCaP cells. Dimethyloxalylglycine, a pan-prolyl hydroxylase inhibitor, also induced Btg2 and Ndrg1 protein expression in LNCaP cells. The transient gene expression assay revealed that L-mimosine treatment or cotransfection with HIF-1α expression vector enhanced the promoter activities of Btg2 and Ndrg1 genes. Knockdown of HIF-1α attenuated the increasing protein levels of both Btg2 and Ndrg1 by hypoxia or L-mimosine in LNCaP cells. Our results indicated that hypoxia and L-mimosine modulated Btg2 and Ndrg1 at the transcriptional level, which is dependent on HIF-1α. L-Mimosine enhanced expression of Btg2 and Ndrg1, which attenuated cell proliferation of the PC-3 and LNCaP prostate carcinoma cells.

Upregulation of prostate-derived Ets factor by luteolin causes inhibition of cell proliferation and cell invasion in prostate carcinoma cells

Luteolin is a polyphenolic flavone and has antitumor activity for many cancers. The prostate‐derived Ets factor (PDEF), a novel epithelium‐specific Ets transcription factor, acts as an androgen‐independent transcriptional activator of the prostate‐specific antigen (PSA) promoter. We determined the antitumor function of luteolin via upregulation of PDEF gene expression in human prostate carcinoma LNCaP cells. Results from flow cytometry and 3H‐thymidine incorporation assays revealed that luteolin treatments attenuated cell proliferation and arrested the cell cycle at the G1/S phase. High concentration of luteolin (30 μM) induced cell apoptosis. Immunoblot assays and enzyme linked immunosorbent assay revealed that luteolin treatment upregulated PDEF but downregulated androgen receptor (AR) gene expression, which decreased PSA gene expression in LNCaP cells. Results of immunoblot and transient gene expression assays revealed that luteolin treatments at proapoptosis dosage, enhanced gene expression of PDEF, B‐cell translocation gene 2 (BTG2), N‐myc downstream regulated gene 1 (NDRG1) and Maspin. Transient gene expression assays indicated that cotransfection of the PDEF expression vector enhanced the promoter activities of the BTG2, NDRG1 and Maspin genes. Stable overexpression of PDEF significantly induced BTG2, NDRG1 and Maspin gene expression, which markedly attenuated in vitro cell proliferation and invasion of LNCaP cells. The modulatory effect of luteolin on BTG2, NDRG1 and Maspin gene expression were attenuated when PDEF was knocked‐down. These results suggest that luteolin blocks PSA gene expression by downregulation of AR expression. The enhancement of PDEF expression, which induced BTG2, NDRG1 and Maspin gene expression, could account for the function of luteolin for antiproliferation and anti‐invasion in LNCaP cells.

Evaluation of the potential therapeutic role of a new generation of vitamin D analog, MART-10, in human pancreatic cancer cells in vitro and in vivo.

Pancreatic cancer is a lethal disease with no known effective chemotherapy and radiotherapy, and most patients are diagnosed in the late stage, making them unsuitable for surgery. Therefore, new therapeutic strategies are urgently needed. 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is known to possess antitumor actions in many cancer cells in vitro and in vivo models. However, its clinical use is hampered by hypercalcemia. In this study, we investigated the effectiveness and safety of a new generation, less calcemic analog of 1α,25(OH)2D3, 19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (MART-10), in BxPC-3 human pancreatic carcinoma cells in vitro and in vivo. We demonstrate that MART-10 is at least 100-fold more potent than 1α,25(OH)2D3 in inhibiting BxPC-3 cell proliferation in a time- and dose-dependent manner, accompanied by a greater upregulation of cyclin-dependent kinase inhibitors p21 and p27 and a greater downregulation of cyclin D3 and cyclin-dependent kinases 4 and 5, leading to a greater increase in the fraction of cells in G0/G1 phase. No induction of apoptosis and no effect on Cdc25 phosphatases A and C were observed in the presence of either MART-10 or 1α,25(OH)2D3. In a xenograft mouse model, treatment with 0.3 µg/kg body weight of MART-10 twice/week for 3 weeks caused a greater suppression of BxPC-3 tumor growth than the same dose of 1α,25(OH)2D3 without inducing hypercalcemia and weight loss. In conclusion, MART-10 is a promising agent against pancreatic cancer growth. Further clinical trial is warranted.

p53 Downregulates the Gene Expression of Mitochondrial Aconitase in Human Prostate Carcinoma Cells

Mitochondrial aconitase (mACON) is regarded as the key enzyme in citrate oxidation in human prostate epithelial cells, and its abnormal expression has been implicated in tumorigenesis of the prostate. Evidence also supports a broad role for the p53 gene in suppressing prostatic tumorigenesis. We investigated whether p53 regulates mACON expression and explore the potential mechanisms responsible for its effect on prostate cancer cells.

19-Nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (MART-10) is a potent cell growth regulator with enhanced chemotherapeutic potency in liver cancer cells

The discovery that the active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)(2)D] can modulate cellular proliferation and differentiation of cancer cells has led to its potential application as a chemotherapeutic agent to treat a variety of cancers. However, the use of 1α,25(OH)(2)D is limited due to its lethal side effect of hypercalcemia upon systemic administration. To overcome this drawback, numerous analogs have been synthesized. In this report, we examined the anti-proliferative activity of a new analog, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)(2)D(3) (MART-10), in HepG2 liver cancer cells, and studied the potential mechanisms mediating this action. We found that MART-10 exhibited approximately 100-fold greater activity than 1α,25(OH)(2)D(3) in inhibiting HepG2 cell proliferation as determined by cell number counting method. MART-10 was also approximately 100-fold more potent than 1α,25(OH)(2)D(3) in the upregulation of p21 and p27, that in turn arrested HepG2 cells at the G(0)/G(1) phase to a greater extent. Given that no active caspase 3 was detected and treatment with 1α,25(OH)(2)D(3) or MART-10 did not further increase the fractions of apoptotic and necrosis cells over the controls, the growth-inhibitory effect of 1α,25(OH)(2)D(3) and MART-10 on HepG2 cells may not involve apoptosis. Overall, our findings suggest that MART-10 is a good candidate as a novel therapeutic regimen against liver cancer. Further pre-clinical studies using animal models and the subsequent human clinical trials are warranted.

Association of nucleophosmin/B23 with bladder cancer recurrence based on immunohistochemical assessment in clinical samples.

AbstractAim:To investigate the possible correlation of nucleophosmin/B23 expression with bladder carcinoma recurrence.Methods:Surgically-resected bladder tumors staged pTa to pT4 were examined for nucleophosmin/B23 expression by immuno-histochemistry. The study group consisted of 132 consecutive patients surgically treated at Chang Gung Memorial Hospital between December 1998 and November 1999. The mean follow up was 72 months (range: 48-84 months).Results:Nuclear nucleophosmin/B23 staining was detected in 96% of advanced stage and poorly-differentiated tumors. Higher nucleophosmin/B23 levels were linked to more advanced tumor stages, grades, poor prognosis, and likelihood of recurrence (P<0.05). The Cox multivariate analysis indicated the nucleophosmin/B23 expression as an independent indicator for tumor recurrence (P=0.009).Conclusion:The results suggest that nucleophosmin/B23 is a favorable prognostic indicator for bladder cancer. Nucleophosmin/B23 could be a useful molecular tumor marker for predicting bladder cancer recurrence.

The vitamin D analog, MART-10, represses metastasis potential via downregulation of epithelial-mesenchymal transition in pancreatic cancer cells.

Pancreatic cancer (PDA) is a devastating disease and there is no effective treatment available at present. To develop new regiments against PDA is urgently needed. Previously we have shown that vitamin D analog, MART-10 (19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3), exerted potent antiproliferative effect on PDA in vitro and in vivo without causing hypercalcemia. Since metastasis is the major cause of PDA-related death, we therefore investigate the anti-metastasis effect of MART-10 on PDA. Our results showed that both 1α,25(OH)2D3 and MART-10 repressed migration and invasion of BxPC-3 and PANC cells with MART-10 much more potent than 1α,25(OH)2D3. 1α,25(OH)2D3 and MART-10 inhibited epithelial-mesenchymal transition (EMT) in pancreatic cancer cells through downregulation of Snail, Slug, and Vimentin expression in BxPC-3 and PANC cells. MART-10 further blocked cadherin switch (from E-cadherin to N-cadherin) in BxPC-3 cells. The F-actin synthesis in the cytoplasm of BxPC-3 cells was also repressed by 1α,25(OH)2D3 and MART-10 as determined by immunofluorescence stain. Both 1α,25(OH)2D3 and MART-10 decreased MMP-2 and -9 secretion in BxPC-3 cells as determined by western blot and zymography. Collectively, MART-10 should be deemed as a promising regimen against PDA.

Zinc blocks gene expression of mitochondrial aconitase in human prostatic carcinoma cells.

Mitochondrial aconitase (mACON) contains a [4Fe‐4S] cluster as the key enzyme for citrate oxidation in the human prostatic epithelial cell. Although there is accumulating evidence indicating that accumulation of high levels of zinc in prostate epithelial cells causes reduced efficiency of citrate oxidation, zinc regulation on the mACON is still not well understood. From in vitro studies, zinc chloride treatment has been developed using humic acid as the carrier (Zn‐HA) in human prostatic carcinoma cells, PC‐3. Zn‐HA treatment (0.1–10 μM) restricts mACON enzymatic activity, which attenuates citrate utility and decreases intracellular ATP levels in PC‐3 cells, whereas the effect is blocked by adding the zinc chelator, diethylenetriaminepentaacetic acid (DTPA). Immunoblot, ribonuclease‐protection and transient gene‐expression assays indicate that Zn‐HA treatments inhibit mACON gene expression. Mutation of the putative metal response element (MRE) from CTCGCCTTCA to TGATCCTTCA abolishes Zn‐HA inhibition of mACON promoter activity. Our results have demonstrated that zinc possesses a specific regulatory mechanism on the mACON gene, and a biologic function of the putative metal regulatory system in mACON gene transcription has been identified.