Jen-Wei Chiao

J W Chiao; What causes prostate cancer-learning from a tragedy, Carcinogenesis, , bgab078, https://doi.org/10.1093/carcin/bgab078

// <![CDATA[ $('.header-date').hide();$('#titleAuthors').hide();$('#abstractHeader').hide(); // ]]> Yamei Chen1, 4, *, Shundong Cang1, 2, *, Liying Han3, Christina Liu1, Patrick Yang1, Zeeshan Solangi1, Quanyi Lu4, Delong Liu1, 5, J.W. Chiao1 1Department of Medicine, New York Medical College, Valhalla, NY 10595, USA 2Department of Oncology, Henan Province People’s Hospital, Zhengzhou University, Zhengzhou, China 3Department of Pathology, New York Medical College, Valhalla, NY 10595, USA 4Department of Hematology, Zhongshan Hospital, Xiamen University, Xiamen, Fujian, China 5Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China *These authors have contributed equally to this work Correspondence to: J.W. Chiao, email: Jen-Wei_Chiao@NYMC.edu Delong Liu, email: delong_liu@nymc.edu Quanyi Lu, email: quanyilu@hotmail.com Keywords: prostate cancer, stem cells, sphere, epigenome, androgen Received: September 24, 2015 Accepted: March 06, 2016 Published: March 28, 2016 ABSTRACT Prostate cancer can transform from androgen-responsive to an androgen-independent phenotype. The mechanism responsible for the transformation remains unclear. We studied the effects of an epigenetic modulator, phenethyl isothiocyanate (PEITC), on the androgen-responsive LNCaP cells. After treatment with PEITC, floating spheres were formed with characteristics of prostate cancer stem cells (PCSC). These spheres were capable of self-renewal in media with and without androgen. They have been maintained in both types of media as long term cultures. Upon androgen deprivation, the adherent spheres differentiated to neuroendocrine cells (NEC) with decreased proliferation, expression of androgen receptor, and PSA. NEC reverse differentiated to spheres when androgen was replenished. The sphere cells expressed surface marker CD44 and had enhanced histone H3K4 acetylation, DNMT1 down-regulation and GSTP1 activation. We hypothesize that PEITC-mediated alteration in epigenomics of LNCaP cells may give rise to sphere cells, whereas reversible androgenomic alterations govern the shuttling between sphere PCSC and progeny NEC. Our findings identify unrecognized properties of prostate cancer sphere cells with multi-potential plasticity. This system will facilitate development of novel therapeutic agents and allow further exploration into epigenomics and androgenomics governing the transformation to hormone refractory prostate cancer.

Hypoacetylation on histone H3 of human prostate cancer cells has been described. Little is known about the modifications of other histones from prostate cancer cells. Histones were isolated from the prostate cancer cell line DU-145 and the non-malignant prostatic cell line RC170N/h. Post-translational modifications of histone H2B were determined by liquid chromatography-mass spectrometry (LC-MS)/MS. The histone H2B of the prostate cancer cell line DU-145 was found to have hypoacetylation, hypomethylation, and dephosphorylation as compared to the non-malignant prostatic cell line RC170N/h. H2B regained acetylation on multiple lysine residues, phosphorylation on Thr19, and methylation on Lys23 and Lys43 in the DU-145 cells after sodium butyrate treatment. The histone H2B of DU-145 prostate cancer cells are hypoacetylated, hypomethylated, and dephosphorylated. Histone deacetylase inhibitor reversed this phenotype. Epigenetic agent may therefore be useful for prostate cancer therapy and worth further investigation.

Phenethyl isothiocyanate (PEITC), present naturally in cruciferous vegetables, is a chemopreventive agent. It blocks initiation and post-initiation progression of carcinogenesis. Mechanism study in human prostate cancer cells revealed that PEITC is a dual inhibitor of aberrant DNA hypermethylation and histone deacetylases, reactivating silenced genes and regulating the androgen-mediated growth of tumor cells. The identity of the cellular organelle that initially interacts with PEITC has not been fully described. Human prostate cancer LNCaP cells were exposed to PEITC and the effects on cellular fine structure examined by transmission electron microscopic studies. Alteration of mitochondrial membrane potential and cytochrome c release were evaluated as early events of apoptosis, and the TUNEL method for quantifying apoptotic cells. Mitochondria were isolated for determining their protein expression. Ultrastructural analyses have revealed condensed mitochondria and a perturbed mitochondrial cristae structure, which assumed a rounded and dilated shape within 4-hours of PEITC contact, and became more pronounced with longer PEITC exposure. They presented as the most prominent intracellular alterations in the early hours. Mitochondria structure alterations were demonstrated, for the first time, with the isothiocyanates. An increase in the number of smooth endoplasmic reticulum and vacuoles were also noted that is consistent with the presence of autophagy. Early events of apoptosis were detected, with cytochrome c released along with the appearance of mitochondrial alteration. Mitochondrial membrane potential was disrupted within 18 hours of PEITC exposure, preceding the appearance of apoptotic cells with DNA strand breaks. In parallel, the expression of the mitochondrial class III ß-tubulin in the outer membrane, which associates with the permeability transition pore, was significantly reduced as examined with isolated mitochondria. Mitochondria may represent the organelle target of the isothiocyanates, indicating that the isothiocyanates may be mitochondria-interacting agents to inhibit carcinogenesis.

Combination of phenethyl isothiocyanate (PEITC) and paclitaxel (taxol) has been shown to work synergistically to increase apoptosis and cell cycle arrest in breast cancer cells. In this report, we further explored the mechanisms for the synergistic activity of PEITC and taxol in MCF7 and MDA-MB-231 (MB) breast cancer cell lines. By Western blotting analysis, treatment of MCF7 cells with both PEITC and taxol led to a 10.4-fold and 5.96-fold increase in specific acetylation of alpha-tubulin over single agent PEITC and taxol, respectively. This synergistic effect on acetylation of alpha-tubulin was also seen in MB cells. The combination of PEITC and taxol also reduced expressions of cell cycle regulator Cdk1, and anti-apoptotic protein bcl-2, enhanced expression of Bax and cleavage of PARP proteins. In conclusion, this study provided biochemical evidence for the mechanism of synergistic effect between the epigenetic agent PEITC and the chemotherapeutic agent taxol.

This study examined whether combining paclitaxel (taxol) with a novel epigenetic agent phenethyl isothiocyanate (PEITC) will yield a synergistic effect on inhibiting breast cancer cells. Two drug-resistant breast cancer cell lines, MCF7 and MDA-MB-231, were treated with PEITC and taxol. Cell growth, cell cycle, and apoptosis were examined. The combination of PEITC and taxol significantly decreased the IC₅₀ of PEITC and taxol over each agent alone. The combination also increased apoptosis by more than two fold over each single agent in both cell lines. A significant increase of cells in the G2/M phases was detected. In conclusion, the combination of PEITC and taxol exhibits a synergistic effect on growth inhibition in breast cancer cells. This combination deserves further study in vivo.

Background: We have previously studied the histone acetylation in primary human leukemia cells. However, histone H3 methylation in these cells has not been characterized. Methods: This study examined the methylation status at histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) in primary acute leukemia cells obtained from patients and compared with those in the non-leukemia and healthy cells. We further characterized the effect of phenylhexyl isothiocyanate (PHI), Trichostatin A (TSA), and 5-aza-2’-deoxycytidine (5-Aza) on the cells. Results: We found that methylation of histone H3K4 was virtually undetectable, while methylation at H3K9 was significantly higher in primary human leukemia cells. The histone H3K9 hypermethylation and histone H3K4 hypomethylation were observed in both myeloid and lymphoid leukemia cells. PHI was found to be able to normalize the methylation level in the primary leukemia cells. We further showed that PHI was able to enhance the methyltransferase activity of H3K4 and decrease the activity of H3K9 methyltransferase. 5-Aza had similar effect on H3K4, but minimal effect on H3K9, whereas TSA had no effect on H3K4 and H3K9 methyltransferases. Conclusions: This study revealed opposite methylation level of H3K4 and H3K9 in primary human leukemia cells and demonstrated for the first time that PHI has different effects on the methyltransferases for H3K4 and H3K9.

The regimen of cytarabine, aclarubicin and G-CSF (CAG) has been widely used in China and Japan for treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We searched literature on CAG between 1995 and 2010 and performed a meta-analysis to determine its overall efficacy using a random-effects or fixed-effects model. Thirty five trials with a total of 1029 AML (n = 814) and MDS (n = 215) patients were included for analysis. The CR rate of AML (57.9%) was significantly higher than that of MDS (45.7%) (p < 0.01). No difference in CR was noted between the new (56.7%) and relapsed/refractory AML (60.1%) (p > 0.05). The CR rate was also significantly higher in patients with favorable (64.5%) and intermediate (69.6%) karyotypes than those with unfavorable one (29.5%) (p < 0.05). Remarkably, the CR rate of CAG was significantly higher than those of non-CAG regimens (odds ratio 2.43). CAG regimen was well tolerated, with cardiotoxicity in 2.3% and early death in 5.2% of the cases. In conclusion, CAG regimen was an effective and safe regimen for the treatment of AML, and may be more effective than non-CAG regimens. Randomized controlled trials are strongly recommended to evaluate its efficacy and safety in comparison with the current standard treatment.

Background: We have previously demonstrated that phenylhexyl isothiocyanate (PHI), a synthetic isothiocyanate, inhibits histone deacetylases and remodels chromatins to induce growth arrest in HL-60 myeloid leukemia cells in a concentration-dependent manner.Methods: To investigate the effect of PHI, a novel histone deacetylases inhibitor (HDACi), on demethylation and activation of transcription ofp15in acute lymphoid leukemia cell line Molt-4, and to further decipher the potential mechanism of demethylation, DNA sequencing and modified methylation specific PCR (MSP) were used to screenp15-M andp15-U mRNA after Molt-4 cells were treated with PHI, 5-Aza and TSA. DNA methyltransferase 1 (DNMT1), 3A (DNMT3A), 3B (DNMT3B) andp15mRNA were measured by RT-PCR. P15 protein, acetylated histone H3 and histone H4 were detected by Western Blot.Results: The genep15in Molt-4 cells was hypermethylated and inactive. Hypermethylation of genep15was attenuated andp15gene was activated de novo after 5 days exposure to PHI in a concentration-dependent manner. DNMT1 and DNMT3B were inhibited by PHI (P < 0.05). Alteration of DNMT3A was not significant at those concentrations. Acetylated histone H3 and histone H4 were accumulated markedly after exposure to PHI.Conclusion: PHI could induce both DNA demethylation and acetylated H3 and H4 accumulation in Molt-4 cells. Hypermethylation of genep15was reversed andp15transcription could be reactivated de novo by PHI.

Histone deacetylase (HDAC) inhibitors are a new class of chemotherapeutic agents. Our laboratory has recently reported that phenylhexyl isothiocyanate (PHI), a synthetic isothiocyanate, is an inhibitor of HDAC. In this study we examined whether PHI is a hypomethylating agent and its effects on myeloma cells. RPMI8226, a myeloma cell line, was treated with PHI. PHI inhibited the proliferation of the myeloma cells and induced apoptosis in a concentration as low as 0.5 μM. Cell proliferation was reduced to 50% of control with PHI concentration of 0.5 μM. Cell cycle analysis revealed that PHI caused G1-phase arrest of RPMI8226 cells. PHI induced p16 hypomethylation in a concentration- dependent manner. PHI was further shown to induce histone H3 hyperacetylation in a concentration-dependent manner. It was also demonstrated that PHI inhibited IL-6 receptor expression and VEGF production in the RPMI8226 cells, and reactivated p21 expression. It was found that PHI induced apoptosis through disruption of mitochondrial membrane potential. For the first time we show that PHI can induce both p16 hypomethylation and histone H3 hyperacetylation. We conclude that PHI has dual epigenetic effects on p16 hypomethylation and histone hyperacetylation in myeloma cells and targets several critical processes of myeloma proliferation.