Administration Of Phenethyl Isothiocyanate Research Articles

Synthesis of PEITC-loaded gold nanoparticles and evaluation of the hepatoprotective effect on CCl4-induced damage through Nrf2 pathway

Abstract Objectives The purpose of the study was to prepare an effective and new drug delivery system for enhancing the stability of Phenethyl isothiocyanate (PEITC), and its hepatoprotective effect in the carbon tetrachloride (CCl4)-induced damage in hepatocellular carcinoma G2 (HepG2) cell line via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. Methods Gold nanoparticles were synthesized and then characterized by XRD, SEM, SEM-EDX analysis, hydrodynamic diameter and zeta potential measurements. 1.0024 mM PEITC, a naturally occurring isothiocyanate, an active ingredient was loaded onto the characterized AuNPs. The cytotoxicity test of PEITC-AuNP and effects on ALT, AST, Nrf2 levels and total antioxidant capacity (TAC) of CCI4-induced HepG2 cells were investigated. Results PEITC-AuNPs and PEITC decreased ALT and AST levels (p<0.05). This reduction was greater with PEITC-AuNPs. PEITC-AuNPs increased Nrf2 level but it was nonsignificantly (p>0.05). PEITC didn’t increase the Nrf2 level in CCI4-induced HepG2 cells. TAC of both PEITC-AuNPs and PEITC administration increased significantly compared with CCl4 group (p<0.05). But PEITC-AuNPs enhanced the TAC level higher than PEITC significantly (p<0.05). Conclusions PEITC-AuNPs were more effective than PEITC which resulted in more hepatoprotective and antioxidant effects via Nrf2 activation against CCl4-induced liver injury in HepG2 cells.

Phenethyl isothiocyanate alleviates renal injury in STZ‐induced diabetic rats: Effect on oxidative stress, glycative stress and inflammation

Background Diabetic nephropathy (DN) is a serious microvascular complication of diabetes mellitus (DM) and the major cause of end-stage renal disease. The pathogenesis of DN is multifactorial. Indeed, advanced glycation end products, inflammation and oxidative stress were shown to significantly contribute to DN development/progression. Aim of the work Given the reported beneficial biological effects of phenethyl isothiocyanate (PEITC), the present study aims to evaluate the potential protective effects of PEITC in streptozotocin (STZ)-induced diabetic Sprague Dawley rats. Methods PEITC was administrated at 3, 10, 30 mg/kg/day for 8 weeks after confirming experimental induction of DM. Renal function (creatinine, urea, proteinuria), hypertrophy index, inflammation (NLRP3/TXNIP/Caspase-1 and pro-inflammatory cytokines IL-1β,TNF-α, IL-6), redox homeostasis (Nrf2/Keap1/HO1/γGCS, lipid peroxidation, total antioxidant capacity, and antioxidants GSH, SOD) and protein glycation (AGE/RAGE/GLO1) were assessed by spectrophotometry, ELISA and immunohistochemistry. Moreover, renal structural and ultra-structural alterations were evaluated. Results According to our results, PEITC administration improved renal function, restored oxidant/antioxidant balance, diminished TXNIP/NLRP3-dependent inflammation and suppressed glycative stress in a dose dependent manner. Furthermore, results from histological and electron microscopy studies revealed preservation of renal structure by PEITC treatment. Conclusion These findings underline for the first time that PEITC confers a considerable protection against diabetes-induced kidney injury, suggesting potential use of PEITC as early therapeutic intervene that might slow down the progression of DN via multifactorial pathways targeting.

PEITC triggers multiple forms of cell death by GSH-iron-ROS regulation in K7M2 murine osteosarcoma cells

Phenethyl isothiocyanate (PEITC) is an isothiocyanate that largely exists in cruciferous vegetables and exhibits chemopreventive and chemotherapeutic potential against various cancers. However, it is little known about the molecular mechanisms of its antitumor action against osteosarcoma, which is the second highest cause of cancer-related death in children and adolescents. In this study, we investigated the effects of PEITC on K7M2 murine osteosarcoma both in vitro and in vivo. We found that treatment with PEITC dose-dependently inhibited the viability of K7M2 murine osteosarcoma cells with an IC50 value of 33.49 μM at 24 h. PEITC (1, 15, 30 μM) dose-dependently inhibited the cell proliferation, caused G2/M cell cycle arrest, depleted glutathione (GSH), generated reactive oxygen species (ROS), altered iron metabolism, and triggered multiple forms of cell death, namely ferroptosis, apoptosis, and autophagy in K7M2 cells. We further revealed that PEITC treatment activated MAPK signaling pathway, and ROS generation was a major cause of PEITC-induced cell death. In a syngeneic orthotopic osteosarcoma mouse model, administration of PEITC (30, 60 mg/kg every day, ig, for 24 days) significantly inhibited the tumor growth, but higher dose of PEITC (90 mg/kg every day) compromised its anti-osteosarcoma effect. Histological examination showed that multiple cell death processes were initiated, iron metabolism was altered and MAPK signaling pathway was activated in the tumor tissues. In conclusion, we demonstrate that PEITC induces ferroptosis, autophagy, and apoptosis in K7M2 osteosarcoma cells by activating the ROS-related MAPK signaling pathway. PEITC has promising anti-osteosarcoma activity. This study sheds light on the redox signaling-based chemotherapeutics for cancers.

Cooking has the potential to decrease the antitumor effect of fresh Betong watercress.

Betong watercress (Nasturtium officinale R.Br.) contains phenethyl isothiocyanate (PEITC), derived from myrosinase-mediated hydrolysis of glucosinolates. Effects of fresh and cooked Betong watercress (FBW & CBW) on N-demethylation and C-8-hydroxylation of caffeine (CF) in rats were investigated. Wistar rats received a single dose of CF before and after pretreatments with a single or five-day administration of PEITC, FBW, and CBW dry powders. Plasma CF metabolic ratios (CMRs) were compared between before and after pretreatments. Single pretreatment with PEITC, FBW, but not CBW, significantly decreased CMRs. Five-day pretreatment with PEITC, FBW, and CBW significantly decreased CMRs. The decreases in CMRs after multiple doses of PEITC, FBW, and CBW were significantly higher than after a single dose. The decrease in CMRs caused by CBW was significantly lower than those by FBW, both single- and multiple doses. Cooking decreases the activity of FBW in inhibition of CYP1A2 mediating CF metabolism. PRACTICAL APPLICATIONS: PEITC and fresh watercress possess chemoprotective effects due to the inhibitory activity of PEITC on cytochrome P450s mediated bioactivation of carcinogens. Several clinical trials of the therapeutic uses of PEITC for cancer and other diseases are still in the pipeline. Betong watercress is a common ingredient in hot soup and stir-fried Thai recipes. Cooking heat inactivates plant myrosinase involving the production of PEITC. Consumption of watercress in cooked form may contribute less chemoprotective benefit. More appropriate preparation to deliver PEITC is needed to be evaluated.

Phenethyl isothiocyanate activates leptin signaling and decreases food intake.

Obesity, a principal risk factor for the development of diabetes mellitus, heart disease, and hypertension, is a growing and serious health problem all over the world. Leptin is a weight-reducing hormone produced by adipose tissue, which decreases food intake via hypothalamic leptin receptors (Ob-Rb) and the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway. Protein tyrosine phosphatase 1B (PTP1B) negatively regulates leptin signaling by dephosphorylating JAK2, and the increased activity of PTP1B is implicated in the pathogenesis of obesity. Hence, inhibition of PTP1B may help prevent and reduce obesity. In this study, we revealed that phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate in certain cruciferous vegetables, potently inhibits recombinant PTP1B by binding to the reactive cysteinyl thiol. Moreover, we found that PEITC causes the ligand-independent phosphorylation of Ob-Rb, JAK2, and STAT3 by inhibiting cellular PTP1B in differentiated human SH-SY5Y neuronal cells. PEITC treatment also induced nuclear accumulation of phosphorylated STAT3, resulting in enhanced anorexigenic POMC expression and suppressed orexigenic NPY/AGRP expression. We demonstrated that oral administration of PEITC to mice significantly reduces food intake, and stimulates hypothalamic leptin signaling. Our results suggest that PEITC might help prevent and improve obesity.

Fatty Acid Synthesis Intermediates Represent Novel Noninvasive Biomarkers of Prostate Cancer Chemoprevention by Phenethyl Isothiocyanate.

Increased de novo synthesis of fatty acids is a distinctive feature of prostate cancer, which continues to be a leading cause of cancer-related deaths among American men. Therefore, inhibition of de novo fatty acid synthesis represents an attractive strategy for chemoprevention of prostate cancer. We have shown previously that dietary feeding of phenethyl isothiocyanate (PEITC), a phytochemical derived from edible cruciferous vegetables such as watercress, inhibits incidence and burden of poorly differentiated prostate cancer in transgenic adenocarcinoma of mouse prostate (TRAMP) model. The current study was designed to test the hypothesis of whether fatty acid intermediate(s) can serve as noninvasive biomarker(s) of prostate cancer chemoprevention by PEITC using archived plasma and tumor specimens from the TRAMP study as well as cellular models of prostate cancer. Exposure of prostate cancer cells (LNCaP and 22Rv1) to pharmacologic concentrations of PEITC resulted in downregulation of key fatty acid metabolism proteins, including acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and carnitine palmitoyltransferase 1A (CPT1A). The mRNA expression of FASN and CPT1A as well as acetyl-CoA levels were decreased by PEITC treatment in both cell lines. PEITC administration to TRAMP mice also resulted in a significant decrease in tumor expression of FASN protein. Consistent with these findings, the levels of total free fatty acids, total phospholipids, triglyceride, and ATP were significantly lower in the plasma and/or prostate tumors of PEITC-treated TRAMP mice compared with controls. The current study is the first to implicate inhibition of fatty acid synthesis in prostate cancer chemoprevention by PEITC. Cancer Prev Res; 10(5); 279-89. ©2017 AACR.

PEITC treatment suppresses myeloid derived tumor suppressor cells to inhibit breast tumor growth

Breast tumors are heterogeneous with a complex etiology. The immune system plays a crucial role in the development of tumors and can facilitate tumor growth pleiotropically. Myeloid derived suppressor cells (MDSCs) generate reactive oxygen species (ROS) and cytokines to suppress T cells, dendritic cells and natural killer (NK) cells. Hence, the inhibition of MDSCs could be an important strategy for anticancer therapeutics. Phenethyl isothiocyanate (PEITC), a bioactive compound present in cruciferous vegetables, is known to have anticancer properties. However, the effects of PEITC administration on the immune system have not been previously reported. In the current study, we evaluated the effects of administering PEITC to immunocompromised NOD-SCID IL2Rγ−/− (SCID/NSG) host mice bearing MDA-MB-231 xenografts on MDSCs in the peripheral blood. Our results reveal that oral administration of 12 μmol PEITC attenuated tumor growth by 76%. This was marked tumor-inhibitory phenotype was associated with a significant reduction in the levels of MDSCs bearing the surface markers CD33, CD34 and CD11b in PEITC treated mice, indicating that overall tumor growth suppression by PEITC correlates with inhibition of MDSCs. To the best of our knowledge, this is the first study showing effects of PEITC on MDSCs.

Phenethyl Isothiocyanate Inhibits Proliferation and Induces Apoptosis in Pancreatic Cancer Cells In Vitro and in a MIAPaca2 Xenograft Animal Model

Pancreatic cancer is often diagnosed at an advanced stage and it has a poor prognosis that points to an increased need to develop effective chemoprevention strategies for this disease. We examined the ability of phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate found in cruciferous vegetables, to inhibit the growth of pancreatic cancer cells in vitro and in a MIAPaca2 xenograft animal model. Exposure to PEITC inhibited pancreatic cancer cell growth in a dose-dependent manner, with an IC50 of approximately 7 μmol/L. PEITC treatment induced G2/M phase cell cycle arrest, downregulated the antiapoptotic proteins Bcl-2 and Bcl-XL, upregulated the proapoptotic protein Bak, and suppressed Notch 1 and 2 levels. In addition, treatment with PEITC induced cleavage of poly-(ADP-ribose) polymerase and led to increased cytoplasmic histone-associated DNA fragmentation and subdiploid (apoptotic) fraction in pancreatic cancer cells. Oral administration of PEITC suppressed the growth of pancreatic cancer cells in a MIAPaca2 xenograft animal model. Our data show that PEITC exerts its inhibitory effect on pancreatic cancer cells through several mechanisms, including G2/M phase cell cycle arrest and induction of apoptosis, and supports further investigation of PEITC as a chemopreventive agent for pancreatic cancer.

Biomarkers of Phenethyl Isothiocyanate-Mediated Mammary Cancer Chemoprevention in a Clinically Relevant Mouse Model

Phenethyl isothiocyanate (PEITC) is a natural plant compound with chemopreventative potential against some cancers and the ability to induce apoptosis in breast cancer cells. Female mouse mammary tumor virus-neu mice were fed a control AIN-76A diet (n = 35) or the same diet supplemented with 3 µmol PEITC/g diet (n = 33) for 29 weeks, at which time they were killed. Breast tissue sections were stained with hematoxylin and eosin for histopathological assessments, and incidence and size of macroscopic mammary tumors were assessed. Cell proliferation (Ki-67 staining), apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-labeling), and neoangiogenesis (CD31 staining) were determined in tumor sections. Plasma levels of transthyretin were measured in treated and control mice. Expression of proteins in mammary tumor sections was determined by immunohistochemistry. Proteomic profiling was performed by two-dimensional gel electrophoresis followed by mass spectrometry. All statistical tests were two-sided. Administration of PEITC for 29 weeks was associated with 53.13% decreased incidence of macroscopic mammary tumors (mean tumor incidence, PEITC-supplemented diet vs control diet, 18.75% vs 40.00%, difference = -21.25%, 95% confidence interval [CI] = -43.19% to 0.69%, P = .07) and with a 56.25% reduction in microscopic mammary carcinoma lesions greater than 2 mm(2) (mean incidence, PEITC-supplemented diet vs control diet, 18.75% vs 42.86%, difference = -24.11%, 95% CI = -46.35% to -1.86%, P = .04). PEITC-mediated mammary cancer growth inhibition was not because of suppression of human epidermal growth factor receptor-2 expression but was associated with reduced cellular proliferation and neoangiogenesis, increased apoptosis, and altered expression of several proteins, including decreased ATP synthase in the tumor and increased plasma levels of transthyretin. PEITC inhibits the growth of mammary cancers in a mouse model with similarities to human breast cancer progression. ATP synthase and transthyretin appear to be novel biomarkers associated with PEITC exposure.

Abstract 568: Biomarkers of phenethyl isothiocyanate-mediated mammary cancer prevention in MMTV-neu mouse model

Abstract Biomarker(s) predictive of efficacy is necessary for clinical development of promising cancer chemopreventive agents because cancer incidence is too rigorous of an end point for malignancies with long latency such as breast cancer. The present study was undertaken to identify biomarker(s) associated with phenethyl isothiocyanate (PEITC)-mediated mammary cancer prevention in MMTV-neu transgenic mouse model. The PEITC is a promising cancer chemopreventive constituent of cruciferous vegetables (e.g., watercress). Two-dimensional gel electrophoresis-mass spectrometry of tumor specimens from control and PEITC-fed mice (n= 3) revealed alterations in expression of several proteins, including hexokinase-1, isoform CRA_f (1.36-fold increase by PEITC administration), L-lactate dehydrogenase A chain isoform 1 (1.57-fold increase by PEITC administration), pyruvate kinase isozymes M1/M2 (1.33-fold decrease by PEITC administration), and mitochondrial ATP synthase, H+ transporting F1 complex beta subunit (1.38-fold decrease by PEITC administration). Interestingly, the PEITC-induced apoptotic cell death in cultured cancer cells is associated with inhibition of oxidative phosphorylation leading to production of reactive oxygen species. Down-regulation of mitochondrial ATP synthase protein in tumors of PEITC-fed mice was confirmed by immunohistochemistry. Expression of mitochondrial ATP synthase was reduced by about 66% in the tumors from PEITC-fed mice in comparison with control (P= 0.05 by two-sided Student's t-test, n= 6-7). In addition, plasma level of transthyretin was significantly higher in the PEITC treatment group compared with control (P= 0.04 by two-sided Student's t-test, n= 5). In conclusion, the present study not only underscores the power of proteomics in cancer chemoprevention research but also suggests that mitochondrial ATP synthase, and possibly hexokinase-1, pyruvate kinase isozymes M1/M2, and/or L-lactate dehydrogenase A chain isoform 1, may be useful biomarkers to assess the biological activity of PEITC in future clinical trials. This study was supported in part by the US PHS grants CA101753 and CA129347, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 568. doi:1538-7445.AM2012-568

Abstract 563: Chemoprevention of mammary cancer in MMTV-neu transgenic mice by dietary administration of cruciferous vegetable constituent phenethyl isothiocyanate

Abstract We have shown previously that phenethyl isothiocyanate (PEITC), which is a highly promising cancer chemopreventive constituent of edible cruciferous vegetables (e.g., watercress), inhibits growth of cultured breast cancer cells in association with apoptosis induction. The present study was undertaken to determine the efficacy of PEITC for prevention of breast cancer using MMTV-neu mouse model. PEITC administration in the diet (3 mmol PEITC/kg AIN76A diet) for 29 weeks caused an approximate 27% decrease in incidence of mammary hyperplasia compared with control, but the difference between control and PEITC treatment groups did not reach statistical significance. On the other hand, incidence of palpable tumors was reduced by about 53% by PEITC administration (P= 0.07 by two-sided Fisher's exact test). Moreover, size of microscopic carcinoma lesions was significantly lower in mice fed PEITC-supplemented diet compared with those fed basal diet (P= 0.05 by two-sided Fisher's exact test). Average body weight of the PEITC-fed mice was modestly but significantly higher than that of control mice at earlier time points (weeks 1-7) but this differential was not observed at later time points (weeks 8-29). Diet consumption was comparable for mice placed on basal diet and PEITC-supplemented diet. Mean PEITC concentration in the plasma and tumor tissue was 901 nM (95% confidence interval- 741-1060 nM) and 87 nmol/kg (95% confidence interval- 67-107 nmol/kg), respectively. Expression of HER-2 oncoprotein as well as number of CD31-positive blood vessels in the tumor did not differ between control and PEITC treatment groups. On the other hand, tumors from PEITC treatment group exhibited about 46% decrease in Ki-67 expression in comparison with control (P= 0.01). Consistent with cellular observations, number of TUNEL-positive apoptotic bodies was about 4.4-fold higher in the tumors of PEITC-fed mice as compared with controls (P= 0.01). Pulmonary metastasis was evident in 3/35 control mice but only 1/32 mice placed on PEITC-supplemented diet exhibited microscopic evidence of metastasis. Size of pulmonary metastasis was also smaller in the PEITC treatment group compared with control. The results indicate that PEITC administration in the diet inhibits carcinoma development in MMTV-neu mice without any signs of overt toxicity. This investigation was supported in part by the US PHS grants CA101753 and CA129347 awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 563. doi:1538-7445.AM2012-563

Abstract 2584: Notch1 and Notch2 activation by phenethyl isothiocyanate hinders its inhibitory effect on prostate cancer cell migration

Abstract Practical and safe modalities for chemoprevention of prostate cancer are clinically attractive because of high mortality associated with this malignancy in American men. Plant products, including constituents of fruits and vegetables, continue to attract attention for the discovery of novel cancer chemopreventive agents. Phenethyl isothiocyanate (PEITC) is one such promising cancer chemopreventive agent abundant in edible cruciferous vegetables such as watercress. Evidence for protective effect of cruciferous vegetables and their components, including PEITC, against prostate cancer derives from population-based observational studies as well as laboratory investigations. For example, we have shown previously that PEITC administration in the diet (3 µmol/g diet) significantly inhibits incidence as well as burden of poorly-differentiated prostate cancer in the dorsolateral prostate of Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) transgenic mice. Cancer chemopreventive response to PEITC is attributed to its ability to inhibit multiple oncogenic signaling pathways, including nuclear factor-βB, Akt, and androgen receptor. The present study demonstrates, for the first time, that PEITC treatment activates Notch1 and Notch2 in malignant as well as normal human prostate cells. The Notch1 and Notch2 belong to a family of transmembrane receptors implicated in prostate cancer development and metastasis. Exposure of human prostate cancer cells (LNCaP, PC-3, and DU145) and a normal human prostate epithelial cell line (PrEC) to PEITC resulted in cleavage (active form) of Notch1 and Notch2, which was associated with an increase in transcriptional activity of Notch. PEITC treatment caused induction of Notch ligands Jagged1 and Jagged2, overexpression of γ-secretase complex components Presenilin1 and Nicastrin, nuclear enrichment of cleaved Notch2, and/or up-regulation of Notch1, Notch2, Jagged1, and/or Jagged2 mRNA in cancer cells. PEITC-induced apoptosis in LNCaP and PC-3 cells was significantly attenuated by RNA interference of Notch2, but not by pharmacological inhibition of Notch1. Inhibition of PC-3 and LNCaP cell migration resulting from PEITC exposure was significantly augmented by RNA interference of Notch2 as well as pharmacological inhibition of Notch1 activation. Nuclear expression of cleaved Notch2 protein was significantly higher in PC-3 xenografts from PEITC-treated athymic mice and dorsolateral prostates from PEITC-fed TRAMP mice compared with the respective control. Because Notch signaling is implicated in epithelial-mesenchymal transition and metastasis, the present study suggests that anti-metastatic effect of PEITC may be augmented by a combination regimen involving PEITC and a Notch inhibitor. This investigation was supported by the US PHS grant RO1 CA101753-08. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2584. doi:1538-7445.AM2012-2584

Abstract 587: Phenethyl isothiocyanate suppresses inhibitor of apoptosis family protein expression in prostate cancer cells in culture and in vivo

Abstract Bioactive food components continue to gain traction for possible chemoprevention of prostate cancer, which is a leading cause of cancer-related death among American men. Phenethyl isothiocyanate (PEITC) is one such compound that is under active investigation for chemoprevention of prostate cancer as well as other types of malignancies, including colon, lung, and breast cancers. PEITC occurs naturally as a thioglucoside conjugate (gluconasturtiin) in a variety of edible cruciferous vegetables (e.g., watercress), and is generated upon cutting or chewing of cruciferous vegetables due to myrosinase-catalyzed breakdown of the gluconasturtiin. Initial evidence supporting prostate cancer protective effect of cruciferous vegetables emerged from population-based case control studies. In vivo chemopreventive efficacy of PEITC against prostate cancer has now been demonstrated in a transgenic mouse model (Transgenic Adenocarcinoma of Mouse Prostate mice; hereafter abbreviated as TRAMP mice). Post-initiation cancer chemopreventive response to PEITC is associated with apoptosis induction but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of inhibitor of apoptosis (IAP) family proteins in regulation of PEITC-induced apoptosis. The IAP family proteins play critical roles in apoptosis regulation by inhibiting caspases. Elevated expression of IAP proteins, including X-linked inhibitor of apoptosis (XIAP) and Survivin has been reported in human prostate cancers. Exposure of PC-3 and LNCaP human prostate cancer cells to PEITC resulted in downregulation of XIAP and Survivin proteins and suppression of Survivin mRNA expression. PEITC administration to Transgenic Adenocarcinoma of Mouse Prostate mice caused modest but significant downregulation of XIAP and Survivin proteins in the dorsolateral prostate. Proapoptotic response to PEITC was significantly attenuated by ectopic expression of XIAP and Survivin proteins. In addition, Survivin overexpression conferred modest but significant protection against PEITC-mediated inhibition of PC-3 cell migration. In conclusion, these results indicate that cellular responses to PEITC, including apoptosis induction and inhibition of cell migration, in prostate cancer cells are mediated by downregulation of XIAP and/or Survivin, which may serve as valid biomarkers to assess PEITC response in future clinical investigations. This investigation was supported by the USPHS grant RO1 CA101753-08. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 587. doi:1538-7445.AM2012-587

Inhibition of androgen-responsive LNCaP prostate cancer cell tumor xenograft growth by dietary phenethyl isothiocyanate correlates with decreased angiogenesis and inhibition of cell attachment

Phenethyl isothiocyanate (PEITC) is a candidate anticancer compound found in certain cruciferous vegetables. In our tumor cell xenograft model, dietary administration of PEITC (100–150 mg/kg body weight/d) inhibited androgen-responsive LNCaP human prostate cancer cell tumor growth. We found that dietary treatment with PEITC significantly inhibited tumor platelet/endothelial cell adhesion molecule (PECAM-1/CD31) expression, a marker of angiogenesis. By contrast, we did not find the inhibitory effects of PEITC on tumor growth to be associated with alteration of specific markers for apoptosis, cell proliferation or androgen receptor-mediated pathways. Consistent with in vivo results, PEITC exerted little effects on cell proliferation, cell cycle and androgen-dependent pathways. Interestingly, PEITC significantly attenuated LNCaP cell plating efficiency that correlated with inhibition of integrin family proteins integrin β1, α2 and α6 mRNA expression. Thus, PEITC may be a dietary factor that inhibits androgen-responsive prostate tumor growth indirectly by selectively targeting factors involved in the tumor microenvironment.

Phenethyl isothiocyanate suppresses inhibitor of apoptosis family protein expression in prostate cancer cells in culture and in vivo

Cruciferous vegetable constituent phenethyl isothiocyanate (PEITC) causes apoptosis in prostate cancer cells through mechanisms not fully understood. The present study was designed to determine the role of inhibitor of apoptosis (IAP) family proteins in PEITC-induced apoptosis induction. Effect of PEITC treatment on protein and mRNA expression of IAP in cells was determined by Western blotting and reverse transcription PCR, respectively. Immunohistochemistry was performed to determine the in vivo effect of PEITC administration on X-linked IAP (XIAP) and Survivin protein expression. Overexpression of desired protein was achieved by transient transfection. Cell viability was determined by trypan blue dye exclusion assay, whereas apoptosis was quantified by measurement of histone-associated DNA fragment release into the cytosol. Transwell chamber assay was used to determine cell migration. Exposure of PC-3 and LNCaP human prostate cancer cells to PEITC resulted in downregulation of XIAP and Survivin proteins and Survivin mRNA. PEITC administration to transgenic adenocarcinoma of mouse prostate mice caused modest but significant downregulation of XIAP and Survivin proteins in the dorsolateral prostate. Proapoptotic response to PEITC was significantly attenuated by ectopic expression of XIAP and Survivin proteins. Survivin overexpression also conferred modest but significant protection against PEITC-mediated inhibition of PC-3 cell migration. The present study demonstrates that cellular responses to PEITC, including apoptosis induction and inhibition of cell migration, in prostate cancer cells are mediated by downregulation of XIAP and/or Survivin, which may serve as valid biomarkers of PEITC response in future clinical investigations.

Inhibition of Premature Death by Isothiocyanates through Immune Restoration in LP-BM5 Leukemia Retrovirus-Infected C57BL/6 Mice

The purpose of this study was to determine the effect of isothiocyanates (ITCs) in delaying the progression of the murine immunodeficiency virus to murine AIDS, resulting in increased life span. Furthermore, we investigated the role of ITCs in modulating immune dysfunction caused by LP-BM5 retrovirus infection. Among the tested ITCs, oral administration of sulforaphane (SUL), benzyl isothiocyante (BITC), and phenethyl isothiocyanate (PEITC) showed the inhibition of premature death caused by LP-BM5 retrovirus infection, while indolo[3,2-b] carbazole (ICZ) and indole-3-carbinol (I3C) did not delay the progress of the LP-BM5 retrovirus to murine AIDS. Inhibition of premature death by BITC, PEITC, and SUL could be explained by restoration of the immune system and down regulation of free radicals. Dysfunction of T and B cell mitogenesis caused by retrovirus infection in primary cultured splenocytes has been partially recovered with administration of BITC, PEITC, and SUL. There was a shift from imbalanced cytokine production (increased Th2 and decreased Th1 cell cytokine production) into balanced Th1/Th2 cell secretion of cytokines under administration of these ITCs during the development of murine AIDS. Hepatic vitamin E level was significantly restored by administration of these ITCs, in accordance with reduced hepatic lipid peroxidation levels. This study suggests that certain types of ITCs have beneficial effects in preventing premature death during progression to murine AIDS by restoration of immune dysfunction and removal of excessive free radicals, implying that selective usage of ITCs would be helpful in retarding the progression from HIV infection to AIDS.

Abstract 5563: Dietary phenethyl isothiocyanate administration inhibits prostate cancer development in transgenic adenocarcinoma of mouse prostate mice in association with induction of autophagy and suppression of plasma clusterin

Abstract We have shown previously that phenethyl isothiocyanate (PEITC), a constituent of edible cruciferous vegetables (e.g., watercress), suppresses growth of cultured and xenografted human prostate cancer cells in association with apoptotic and autophagic cell death. The goals of the present study were to: (a) determine effect of dietary administration of PEITC on prostate cancer development using a transgenic mouse model (Transgenic Adenocarcinoma of Mouse Prostate; TRAMP); (b) elucidate whether PEITC utilizes the same mechanisms in vivo as it does in cultured prostate cancer cells; and (c) identify biomarker(s) predictive of PEITC response for future clinical applications. Administration of PEITC in the diet (3 μmol PEITC/g diet for 19 weeks) significantly inhibited burden (affected area) of poorly-differentiated cancer in the dorsolateral prostate (DLP) of TRAMP mice without causing weight loss or any other side effects. PEITC was detectable in plasma (∼1451 nM) as well as in the prostate (∼1138 nmol/kg) of non-transgenic littermates placed on PEITC-supplemented diet. The PEITC-mediated inhibition of prostate cancer progression correlated with a decrease in the expression of cell proliferation marker Ki-67. To our surprise, number of apoptotic bodies was comparable in the DLP of Control and PEITC-fed mice. On the other hand, the DLP from mice placed on PEITC-supplemented diet exhibited 2-3 fold increase in number of autophagosomes (judged by transmission electron microscopy) and elevated expression of autophagy regulator microtubule-associated protein 1 light chain 3 (judged by immunohistochemistry), as compared with that of Control group. Consistent with these observations, level of selective autophagy substrate p62 (also known as sequestosome 1) was reduced in the DLP of PEITC-fed mice in comparison with Control. The PEITC-mediated inhibition of prostate cancer development was accompanied by a marked increase in expression of E-cadherin. Additionally, 2-dimensional gel electrophoresis coupled with mass spectrometry revealed a marked decrease in plasma clusterin in PEITC-treated mice compared with Controls. Together these results indicate that PEITC administration inhibits prostate cancer progression in TRAMP mice in association with reduced cell proliferation and induction of autophagy. Furthermore, the present study discovers plasma clusterin as a potential biomarker of PEITC response. This investigation was supported by the USPHS grant 2 RO1 CA101753-07, awarded by the National Cancer Institute. 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 5563. doi:10.1158/1538-7445.AM2011-5563

Abstract 4596: Phenethyl isothiocyanate inhibits growth of MIAPaca2 human pancreatic cancer xenograft in vivo in association with apoptosis induction

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in the U.S. with a 5-year survival rate of less than 5%. Low survival rate for patients with pancreatic cancer points towards an increased need for novel chemoprevention, early detection, and therapeutic strategies for this disease. This study was undertaken to determine the efficacy of phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate found in cruciferous vegetables, against pancreatic cancer cells in vitro and in vivo. Exposure of human pancreatic cancer cells (MIAPaca2, PL-45, and BxPC3) to PEITC resulted in growth inhibition, which was accompanied by apoptotic cell death regardless of the K-ras status. Apoptotic cell death by PEITC treatment was characterized by an increase in cytoplasmic histone-associated DNA fragmentation and enrichment of sub-diploid fraction. PEITC-induced apoptosis correlated with induction of pro-apoptotic protein Bak, down-regulation of anti-apoptotic proteins Bcl-2 and Bcl-xL, and cleavage of caspase-3 and PARP. Oral administration of 12 µmol PEITC/mouse (five times per week) significantly retarded the growth of MIAPaca2 xenografts in male nude mice without causing weight loss or any other signs of toxicity. PEITC administration significantly reduced proliferation as determined by PCNA and Ki-67 stainings and increased apoptosis in tumors as shown by TUNEL assay. In conclusion, the present study demonstrates, for the first time, that PEITC administration retards the growth of human pancreatic cancer xenografts by inducing apoptotic cell death. The present study provides pre-clinical evidence for development of PEITC as a chemopreventive agent for pancreatic cancer. This investigation was supported in part by the Wayne Fusaro Pancreatic Cancer Research Fund (DCW), the Shirley Hobbs Martin Memorial Fund (REB), and the NCI grants CA101753 and CA113363 (SVS). 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 4596. doi:10.1158/1538-7445.AM2011-4596

Phenethyl isothiocyanate exhibits antileukemic activity in vitro and in vivo by inactivation of Akt and activation of JNK pathways

Effects of phenethyl isothiocyanate (PEITC) have been investigated in human leukemia cells (U937, Jurkat, and HL-60) as well as in primary human acute myeloid leukemia (AML) cells in relation to apoptosis and cell signaling events. Exposure of cells to PEITC resulted in pronounced increase in the activation of caspase-3, -8, -9, cleavage/degradation of PARP, and apoptosis in dose- and time-dependent manners. These events were accompanied by the caspase-independent downregulation of Mcl-1, inactivation of Akt, as well as activation of Jun N-terminal kinase (JNK). Inhibition of PI3K/Akt by LY294002 significantly enhanced PEITC-induced apoptosis. Conversely, enforced activation of Akt by a constitutively active Akt construct markedly abrogated PEITC-mediated JNK activation, Mcl-1 downregulation, caspase activation, and apoptosis, and also interruption of the JNK pathway by pharmacological or genetically (e.g., siRNA) attenuated PEITC-induced apoptosis. Finally, administration of PEITC markedly inhibited tumor growth and induced apoptosis in U937 xenograft model in association with inactivation of Akt, activation of JNK, as well as downregulation of Mcl-1. Taken together, these findings represent a novel mechanism by which agents targeting Akt/JNK/Mcl-1 pathway potentiate PEITC lethality in transformed and primary human leukemia cells and inhibitory activity of tumor growth of U937 xenograft model.

Chemopreventative Potential of the Cruciferous Vegetable Constituent Phenethyl Isothiocyanate in a Mouse Model of Prostate Cancer

This study was undertaken to determine the chemopreventative efficacy of phenethyl isothiocyanate (PEITC), a bioactive constituent of many edible cruciferous vegetables, in a mouse model of prostate cancer, and to identify potential biomarker(s) associated with PEITC response. The chemopreventative activity of dietary PEITC was investigated in Transgenic Adenocarcinoma of Mouse Prostate mice that were fed a control diet or one containing 3 μmol PEITC/g (n = 21 mice per group) for 19 weeks. Dorsolateral prostate tissue sections were stained with hematoxylin and eosin for histopathologic evaluations and subjected to immunohistochemistry for analysis of cell proliferation (Ki-67 expression), autophagy (p62 and LC3 protein expression), and E-cadherin expression. Autophagosomes were visualized by transmission electron microscopy. Apoptotic bodies were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Plasma proteomics was performed by two-dimensional gel electrophoresis followed by mass spectrometry to identify potential biomarkers of PEITC activity. All statistical tests were two-sided. Administration of PEITC (3 μmol/g diet) decreased incidence (PEITC diet vs control diet, mean = 21.65 vs 57.58%, difference = -35.93%, 95% confidence interval = -45.48% to -13.10%, P = .04) as well as burden (affected area) (PEITC diet vs control diet, mean = 18.53% vs 45.01%, difference = -26.48%, 95% confidence interval = -49.78% to -3.19%, P = .02) of poorly differentiated tumors in the dorsolateral prostate of transgenic mice compared with control mice, with no toxic effects. PEITC-mediated inhibition of prostate carcinogenesis was associated with induction of autophagy and overexpression of E-cadherin in the dorsolateral prostate. However, PEITC treatment was not associated with a decrease in cellular proliferation, apoptosis induction, or inhibition of neoangiogenesis. Plasma proteomics revealed distinct changes in the expression of several proteins (eg, suppression of clusterin protein) in the PEITC-treated mice compared with control mice. In this transgenic model, dietary PEITC suppressed prostate cancer progression by induction of autophagic cell death. Potential biomarkers to assess the response to PEITC treatment in plasma were identified.