FDA Approval Summary: Belantamab Mafodotin for Patients with Relapsed or Refractory Multiple Myeloma.

On August 5, 2020, the FDA granted accelerated approval to belantamab mafodotin-blmf (BLENREP; GlaxoSmithKline) for the treatment of adult patients with relapsed or refractory multiple myeloma who have received at least four prior therapies including an anti-CD38 monoclonal antibody, a proteasome inhibitor, and an immunomodulatory agent. Substantial evidence of effectiveness was obtained from the phase II, multicenter DREAMM-2 trial. Patients received belantamab mafodotin 2.5 or 3.4 mg/kg intravenously once every 3 weeks until disease progression or unacceptable toxicity. The trial demonstrated an overall response rate of 31% in the 2.5 mg/kg cohort and 34% in the 3.4 mg/kg cohort. Keratopathy was the most frequent adverse event, occurring in 71% and 77% of patients, respectively. Other ocular toxicities included changes in visual acuity, blurred vision, and dry eye. The U.S. prescribing information for belantamab mafodotin includes a boxed warning for ocular toxicity, and belantamab mafodotin is available only through a restricted program under a Risk Evaluation and Mitigation Strategy. This article summarizes the data and the FDA review process supporting accelerated approval of belantamab mafodotin 2.5 mg/kg intravenously once every 3 weeks. This approval may be contingent upon verification and description of clinical benefit in confirmatory trial(s).

FDA Approval Summary: Entrectinib for the Treatment of NTRK gene Fusion Solid Tumors.

The FDA-approved entrectinib on August 15, 2019, for the treatment of adult and pediatric patients 12 years of age and older with solid tumors that have a neurotrophic tyrosine receptor kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have progressed following treatment or have no satisfactory alternative therapy. Approval was based on demonstration of a durable overall response rate of 57% (95% confidence interval: 43-71), including a complete response rate of 7%, among 54 entrectinib-treated patients with 10 different tumor types harboring an NTRK fusion enrolled in one of three single-arm clinical trials. The durations of response ranged from 2.8 months to 26.0+ months; 68% of responses lasted ≥ 6 months. The most serious toxicities of entrectinib are congestive heart failure, central nervous system effects, skeletal fractures, hepatotoxicity, hyperuricemia, QT prolongation, and vision disorders. Adverse reactions were manageable through dose interruptions (46%), dose reductions (29%), or discontinuation of entrectinib (9%). This is the third approval of a cancer drug for treatment of a tissue agnostic, biomarker-defined cancer.

Discovery of a JAK1/3 Inhibitor and Use of a Prodrug To Demonstrate Efficacy in a Model of Rheumatoid Arthritis.

The four members of the Janus family of nonreceptor tyrosine kinases play a significant role in immune function. The JAK family kinase inhibitor, tofacitinib 1, has been approved in the United States for use in rheumatoid arthritis (RA) patients. A number of JAK inhibitors with a variety of JAK family selectivity profiles are currently in clinical trials. Our goal was to identify inhibitors that were functionally selective for JAK1 and JAK3. Compound 22 was prepared with the desired functional selectivity profile, but it suffered from poor absorption related to physical properties. Use of the phosphate prodrug 32 enabled progression to a murine collagen induced arthritis (CIA) model. The demonstration of a robust efficacy in the CIA model suggests that use of phosphate prodrugs may resolve issues with progressing this chemotype for the treatment of autoimmune diseases such as RA.

FDA Approval Summary: Daratumumab for Treatment of Multiple Myeloma After One Prior Therapy.

On November 21, 2016, the U.S. Food and Drug Administration granted regular approval to daratumumab in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone, for the treatment of patients with multiple myeloma who have received at least one prior therapy. Approval was based on two randomized, open-label trials in which daratumumab was added to these backbone therapies. The MMY3003 trial demonstrated substantial improvement in progression-free survival (PFS) when daratumumab was added to lenalidomide and dexamethasone compared with lenalidomide and dexamethasone alone. The estimated median PFS had not been reached in the daratumumab arm and was 18.4 months in the control arm (hazard ratio [HR] = 0.37; 95% confidence interval [CI]: 0.27-0.52; p < .0001), representing a 63% reduction in the risk of disease progression or death. Similar results were observed in the MMY3004 trial comparing the combination of daratumumab, bortezomib, and dexamethasone with bortezomib and dexamethasone. The estimated median PFS was not reached in the daratumumab arm and was 7.2 months in the control arm (HR = 0.39; 95% CI: 0.28-0.53; p < .0001), representing a 61% reduction in the risk of disease progression or death. The most frequently reported adverse reactions (greater than or equal to 20%) in MMY3003 were infusion reactions, diarrhea, nausea, fatigue, pyrexia, upper respiratory tract infection, muscle spasm, cough, and dyspnea. The most frequently reported adverse reactions (greater than or equal to 20%) in MMY3004 were infusion reactions, diarrhea, peripheral edema, upper respiratory tract infection, and peripheral sensory neuropathy. Neutropenia and thrombocytopenia have been added to the Warnings and Precautions of the drug label. IMPLICATIONS FOR PRACTICE: Daratumumab, the first monoclonal antibody targeted against CD38, received U.S. Food and Drug Administration accelerated approval in 2015 based on data from single-agent, single-arm trials that provided response rate information. Results of the MMY3003 and MMY3004 trials established that daratumumab can be combined synergistically with some of the most highly active agents used to treat multiple myeloma, leading to daratumumab's regular approval in 2016. Daratumumab added to lenalidomide and dexamethasone, or bortezomib and dexamethasone, provides a substantial improvement in progression-free survival in previously treated patients with multiple myeloma. These combinations will likely improve the survival outlook for patients with multiple myeloma.

Discovery of potent and efficacious pyrrolopyridazines as dual JAK1/3 inhibitors.

A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine model of chronic inflammation. X-ray co-crystal structure determination confirmed the presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will be described.

Discovery of pyrrolo[1,2-b]pyridazine-3-carboxamides as Janus kinase (JAK) inhibitors.

A new class of Janus kinase (JAK) inhibitors was discovered using a rationally designed pyrrolo[1,2-b]pyridazine-3-carboxamide scaffold. Preliminary studies identified (R)-(2,2-dimethylcyclopentyl)amine as a preferred C4 substituent on the pyrrolopyridazine core (3b). Incorporation of amino group to 3-position of the cyclopentane ring resulted in a series of JAK3 inhibitors (4g-4j) that potently inhibited IFNγ production in an IL2-induced whole blood assay and displayed high functional selectivity for JAK3-JAK1 pathway relative to JAK2. Further modifications led to the discovery of an orally bioavailable (2-fluoro-2-methylcyclopentyl)amino analogue 5g which is a nanomolar inhibitor of both JAK3 and TYK2, functionally selective for the JAK3-JAK1 pathway versus JAK2, and active in a human whole blood assay.

Pharmacokinetics and safety of subcutaneous pasireotide and intramuscular pasireotide long-acting release in Chinese male healthy volunteers: a phase I, single-center, open-label, randomized study.

PURPOSE: The purpose of this study was to assess the pharmacokinetic (PK) properties and safety of single and multiple doses of subcutaneous (SC) pasireotide and a single-dose intramuscular (IM) long-acting release (LAR) formulation of pasireotide in Chinese healthy volunteers (HVs) versus the PK properties in Western HVs (pooled from previous PK studies). METHODS: In this phase I, single-center, open-label study, 45 Chinese male HVs were evenly randomized to 1 to 9 treatment sequences: each volunteer received a single dose of 300, 600, or 900 µg of pasireotide SC on day 1, followed by administration of the same dose BID from day 15 to the morning of day 19, and then a single IM dose of 20, 40, or 60 mg of pasireotide LAR on day 33. The PK parameters were assessed with noncompartmental analysis. Statistical comparison of PK parameters, including AUC, Cmax, and CL/F from both formulations, was made for Chinese versus Western male HVs. The safety profile was also assessed. Metabolic parameters, including blood glucose, insulin, and glucagon, and measures that reflect the effects of pasireotide LAR on relatively long-term glucose control, lipid metabolism, and systemic concentrations of pancreatic enzymes and thyrotropin were evaluated. FINDINGS: Of the 45 randomized HVs, 42 completed the study per protocol, 1 withdrew his informed consent for personal reasons, and 2 prematurely discontinued the study because of adverse events (AEs). Concentration-time and safety profiles of both formulations were similar to those reported in Western HVs. Mean geometric mean ratios (GMRs) of Chinese versus Western HVs ranged from 0.79 to 1.42. For most primary PK parameters, 90% CIs for GMRs were within a predefined ethnic insensitivity interval (90% CI, 0.70-1.43). After considering age and weight as covariates in the statistical model, the GMRs and 90% CIs for other PK parameters were within the predefined interval (Cmax in single-dose SC administration) or significantly decreased (Cmin,ss in multiple BID SC doses and first peak Cmax in the single-dose LAR formulation). No serious AEs were reported. Both formulations were well tolerated; pasireotide SC caused transient changes in glucose metabolism. Owing to the differential binding affinity to the somatostatin receptor subtypes, pasireotide LAR elicited a concentration-dependent increase of fasting blood glucose, substantial reduction in triglyceride, and a mild decrease in cholesterol. The most frequently reported AEs after single-dose and multiple-dose pasireotide SC were injection site reaction, nausea, dizziness, and diarrhea; most HVs developed diarrhea with single-dose pasireotide LAR. IMPLICATIONS: The pasireotide formulations had similar PK and safety profiles between Chinese and Western male HVs. Thus, no ethnic sensitivity was found for pasireotide SC or LAR.

Inhibitory effect of ketoconazole on the pharmacokinetics of a multireceptor tyrosine kinase inhibitor BMS-690514 in healthy participants: assessing the mechanism of the interaction with physiologically-based pharmacokinetic simulations.

BMS-690514, a selective inhibitor of the ErbB and vascular endothelial growth factor receptors, has shown antitumor activity in early clinical development. The compound is metabolized by multiple enzymes, with CYP3A4 responsible for the largest fraction (34%) of metabolism. It is also a substrate of P-glycoprotein (P-gp) in vitro. To assess the effect of ketoconazole on BMS-690514 pharmacokinetics, 17 healthy volunteers received 200 mg BMS-690514 alone followed by 100 mg BMS-690514 with ketoconazole (400 mg once daily for 4 days). The AUC(∞) of 100 mg BMS-690514 concomitantly administered with ketoconazole was similar to that of 200 mg BMS-690514 alone. The dose-normalized C(max) and AUC(∞) of BMS-690514 from the 100-mg BMS-690514/400-mg ketoconazole treatment increased by 55% and 127%, respectively, relative to those from 200 mg BMS-690514 alone. Prediction of the drug-drug interaction (DDI) using a population-based simulator (Simcyp) indicated that, in addition to CYP3A4 inhibition, the inhibition of P-gp by ketoconazole in the intestine, liver, and kidneys must be invoked to fully account for the DDI observed. This finding suggests that the inhibition of P-gp by ketoconazole, along with its effect on CYP3A4, needs to be considered when designing a DDI study of ketoconazole with a victim drug that is a dual substrate.

Development of a carbon-14 labeling approach to support disposition studies with a pegylated biologic.

Although it is widely accepted that one can extend the pharmacokinetic half-life of a therapeutic protein by covalent conjugation with polyethylene glycol (PEG), the disposition properties of such biologics have not yet been fully evaluated. Therefore, a novel [¹4C]-labeling method was developed that can be applied to a biologic conjugated with PEG through a maleimide-cysteine reaction. The method was used to study the tissue and tumor distribution of a PEGylated Adnectin, a recombinant protein derived from the 10th type III domain of fibronectin, in nude mice bearing human xenograft tumors. The PEGylated Adnectin contained a 40-kDa branched PEG (P40B) that was labeled with [¹4C] at the linker region between the PEG and Adnectin, without compromising cellular activity and plasma half-life in mice. After a single intravenous or intraperitoneal dose (33 mg/kg, 1.7 µCi per mouse) of [¹4C]-P40B-Adnectin, quantitative whole-body autoradiography analysis revealed that the liver had the highest uptake of the radioactivity among nontumor tissues, followed by the kidneys and lung. The muscle and brain showed the least penetration of the radioactivity among all tissues examined. In addition, the [¹4C]-P40B-EI-tandem penetrated into the tumor tissue, although the extent of accumulation was largely dependent on tumor type. Therefore, it was possible to assess the tissue distribution of a PEGylated biologic after it had been [¹4C] labeled using the novel method described herein.

Cytochrome P450 11A1 bioactivation of a kinase inhibitor in rats: use of radioprofiling, modulation of metabolism, and adrenocortical cell lines to evaluate adrenal toxicity.

A drug candidate, BMS-A ((N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl) 2-oxo-1,2-dihydropyridine- 3-carboxamide)), was associated with dose- and time-dependent vacuolar degeneration and necrosis of the adrenal cortex following oral administration to rats. Pretreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, ameliorated the toxicity. In vivo and in vitro systems, including adrenal cortex-derived cell lines, were used to study the mechanism responsible for the observed toxicity. Following an oral dose of the C-14 labeled compound, two hydroxylated metabolites of the parent (M2 and M3) were identified as prominent species found only in adrenal glands and testes, two steroidogenic organs. In addition, a high level of radioactivity was covalently bound to adrenal tissue proteins, 40% of which was localized in the mitochondrial fraction. ABT pretreatment reduced localization of radioactivity in the adrenal gland. Low levels of radioactivity bound to proteins were also observed in testes. Both M3 and covalent binding to proteins were found in incubations with mitochondrial fraction isolated from adrenal tissue in the presence of NADPH. In vitro formation of M3 and covalent binding to proteins were not affected by addition of GSH or a CYP11B1/2 inhibitor, metyrapone (MTY), but were inhibited by ketoconazole (KTZ) and a CYP11A1 inhibitor, R-(+)-aminoglutethimide (R-AGT). BMS-A induced apoptosis in a mouse adrenocortical cell line (Y-1) but not in a human cell line (H295R). Metabolite M3 and covalent binding to proteins were also produced in Y-1 and to a lesser extent in H295R cells. The cell toxicity, formation of M3, and covalent binding to proteins were all diminished by R-AGT but not by MTY. These results are consistent with a CYP11A1-mediated bioactivation to generate a reactive species, covalent binding to proteins, and subsequently rat adrenal toxicity. The thorough understanding of the metabolism-dependent adrenal toxicity was useful to evaluate cross-species adrenal toxicity potential of this compound and related analogues.

Development and validation of an LC-MS-MS method for the simultaneous determination of sulforaphane and its metabolites in rat plasma and its application in pharmacokinetic studies.

A highly sensitive and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay is developed and validated for the quantification of sulforaphane and its metabolites in rat plasma. Sulforaphane (SFN) and its metabolites, sulforaphane glutathione (SFN-GSH) and sulforaphane N-acetyl cysteine (SFN-NAC) conjugates, are extracted from rat plasma by methanol-formic acid (100:0.1, v/v) and analyzed using a reversed-phase gradient elution on a Develosil 3 µm RP-Aqueous C(30) 140Å column. A 15-min linear gradient with acetonitrile-water (5:95, v/v), containing 10 mM ammonium acetate and 0.2% formic acid, as mobile phase A, and acetonitrile-water (95:5, v/v), containing 10 mM ammonium acetate and 0.2% formic acid as mobile phase B, is used. Sulforaphane and its metabolites are well separated. Sulforaphene is used as the internal standard. The lower limits of quantification are 1 ng/mL for SFN and 10 ng/mL for both SFN-NAC and SFN-GSH. The calibration curves are linear over the concentration range of 25-20,000 ng/mL of plasma for each analyte. This novel LC-MS-MS method shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic studies in rats.

Pharmacokinetics of dietary cancer chemopreventive compound dibenzoylmethane in rats and the impact of nanoemulsion and genetic knockout of Nrf2 on its disposition.

The pharmacokinetic disposition of a dietary cancer chemopreventive compound dibenzoylmethane (DBM) was studied in male Sprague-Dawley rats after intravenous (i.v.) and oral (p.o.) administrations. Following a single i.v. bolus dose, the mean plasma clearance (CL) of DBM was low compared with the hepatic blood flow. DBM displayed a high volume of distribution (Vss). The elimination terminal t1/2 was long. The mean CL, Vss and AUC0-∞/dose were similar between the i.v. 10 and 10 mg/kg doses. After single oral doses (10, 50 and 250 mg/kg), the absolute oral bioavailability (F*) of DBM was 7.4%-13.6%. The increase in AUC was not proportional to the oral doses, suggesting non-linearity. In silico prediction of oral absorption also demonstrated low DBM absorption in vivo. An oil-in-water nanoemulsion containing DBM was formulated to potentially overcome the low F* due to poor water solubility of DBM, with enhanced oral absorption. Finally, to examine the role of Nrf2 on the pharmacokinetics of DBM, since DBM activates the Nrf2-dependent detoxification pathways, Nrf2 wild-type (+/+) mice and Nrf2 knockout (-/-) mice were utilized. There was an increased systemic plasma exposure of DBM in Nrf2 (-/-) mice, suggesting that the Nrf2 genotype could also play a role in the pharmacokinetic disposition of DBM. Taken together, the results show that DBM has low oral bioavailability which could be due in part to poor water solubility and this could be overcome by a nanotechnology-based drug delivery system and furthermore the Nrf2 genotype could also play a role in the pharmacokinetics of DBM.

Site-Selective modification of vitamin D analogue (Deltanoid) through a resin-based version of organoselenium 2,3-sigmatropic rearrangement.

A site-selective modification of a vitamin D analogue (Deltanoid) through a two-step 2,3-sigmatropic rearrangement of organoselenium resin to prepare the key intermediate of calcipotriol has been developed. The polystyrene-supported selenium resins used here not only facilitate separation of product but also assist the crucial 2,3-sigmatropic rearrangement to introduce an important functional group (1α-hydroxyl) with high stereo- and regioselectivity.

Regulation of Nrf2- and AP-1-mediated gene expression by epigallocatechin-3-gallate and sulforaphane in prostate of Nrf2-knockout or C57BL/6J mice and PC-3 AP-1 human prostate cancer cells.

AIM: To examine the regulatory crosstalk between the transcription factors Nrf2 and AP-1 in prostate cancer (PCa) by dietary cancer chemopreventive compounds (-)epigallocatechin-3-gallate (EGCG) from green tea and sulforaphane (SFN) from cruciferous vegetables. METHODS: We performed (i) in vitro studies including luciferase reporter gene assays, MTS cell viability assays, and quantitative real-time PCR (qRT-PCR) in PC-3 AP-1 human PCa cells, (ii) in vivo temporal (3 h and 12 h) microarray studies in the prostate of Nrf2-deficient mice that was validated by qRT-PCR, and (iii) in silico bioinformatic analyses to delineate conserved Transcription Factor Binding Sites (TFBS) in the promoter regions of Nrf2 and AP-1, as well as coregulated genes including ATF-2 and ELK-1. RESULTS: Our study shows that AP-1 activation was attenuated by the combinations of SFN (25 micromol/L) and EGCG (20 or 100 micromol/L) in PC-3 cells. Several key Nrf2-dependent genes were down-regulated (3-fold to 35-fold) after in vivo administration of the combination of EGCG (100 mg/kg) and SFN (45 mg/kg). Conserved TFBS signatures were identified in the promoter regions of Nrf2, AP-1, ATF2, and ELK-1 suggesting a potential regulatory mechanism of crosstalk between them. CONCLUSION: Taken together, our present study of transcriptome profiling the gene expression changes induced by dietary phytochemicals SFN and EGCG in Nrf2-deficient mice and in PC-3 cells in vitro demonstrates that the effects of SFN+EGCG could be mediated via concerted modulation of Nrf2 and AP-1 pathways in the prostate.

Nrf2 plays an important role in coordinated regulation of Phase II drug metabolism enzymes and Phase III drug transporters.

The nuclear transcription factor E2-related factor 2 (Nrf2) has been shown to play pivotal roles in preventing xenobiotic-related toxicity and carcinogen-induced carcinogenesis. These protective roles of Nrf2 have been attributed in part to its involvement in the induction of Phase II drug conjugation/detoxification enzymes as well as antioxidant enzymes through the Nrf2-antioxidant response element (ARE) signaling pathways. This review summarizes the current research status of the identification of Nrf2-regulated drug metabolism enzymes (DMEs), especially Phase II DMEs, and Phase III drug transporters. In addition, the molecular mechanisms underlying the coordinated regulation of Phase II DMEs and Phase III transporters will also be discussed based on findings published in the literature.

Pharmacokinetics and pharmacodynamics of broccoli sprouts on the suppression of prostate cancer in transgenic adenocarcinoma of mouse prostate (TRAMP) mice: implication of induction of Nrf2, HO-1 and apoptosis and the suppression of Akt-dependent kinase pathway.

PURPOSE: In the present study, we have evaluated the pharmacokinetics and the in vivo prostate chemopreventive activity of broccoli sprouts. METHODS: The in vivo pharmacokinetic profiles of sulforaphane (SFN) and SFN- glutathione (GSH) conjugate in rats after oral administration of 200 mg and 500 mg broccoli sprouts were analyzed. Next, 8-week old TRAMP mice were fed with dietary broccoli sprouts at two dosages (60 and 240 mg/mouse/day) for 16 weeks, and the mice were sacrificed to examine the pharmacodynamic response on prostate tumor and some biomarkers. RESULTS: SFN was readily released and conjugated with GSH in the rats after oral administration of broccoli sprouts. TRAMP mice fed with 240 mg broccoli sprouts/mouse/day exhibited a significant retardation of prostate tumor growth. Western blot analysis revealed that the expression levels of Nrf2, HO-1, cleaved-Caspase-3, cleaved-PARP and Bax proteins were increased, but that of Keap1 and Bcl-XL proteins were decreased. In addition, the phosphorylation and/or the expression level of Akt and its downstream kinase and target proteins, e.g. mTOR, 4E-BP1 and cyclin D1, were reduced. CONCLUSIONS: Our findings indicate that broccoli sprouts can serve as a good dietary source of SFN in vivo and that they have significant inhibitory effects on prostate tumorigenesis.

Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily.

Substituted N-(4-(2-aminopyridin-4-yloxy)-3-fluoro-phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamides were identified as potent and selective Met kinase inhibitors. Substitution of the pyridine 3-position gave improved enzyme potency, while substitution of the pyridone 4-position led to improved aqueous solubility and kinase selectivity. Analogue 10 demonstrated complete tumor stasis in a Met-dependent GTL-16 human gastric carcinoma xenograft model following oral administration. Because of its excellent in vivo efficacy and favorable pharmacokinetic and preclinical safety profiles, 10 has been advanced into phase I clinical trials.

Curcumin inhibits Akt/mammalian target of rapamycin signaling through protein phosphatase-dependent mechanism.

Akt/mammalian target of rapamycin (mTOR) signaling plays an important role in tumorigenesis and is dysregulated in many tumors, especially metastatic prostate cancers. Curcumin has been shown to effectively prevent or inhibit prostate cancer in vivo and inhibit Akt/mTOR signaling in vitro, but the mechanism(s) remains unclear. Here, we show that curcumin concentration- and time-dependently inhibited the phosphorylation of Akt, mTOR, and their downstream substrates in human prostate cancer PC-3 cells, and this inhibitory effect acts downstream of phosphatidylinositol 3-kinase and phosphatidylinositol-dependent kinase 1. Overexpression of constitutively activated Akt or disruption of TSC1-TSC2 complex by small interfering RNA or gene knockout only partially restored curcumin-mediated inhibition of mTOR and downstream signaling, indicating that they are not the primary effectors of curcumin-mediated inhibition of Akt/mTOR signaling. Curcumin also activated 5'-AMP-activated protein kinase and mitogen-activated protein kinases; however, inhibition of these kinases failed to rescue the inhibition by curcumin. Finally, it was shown that the inhibition of Akt/mTOR signaling by curcumin is resulted from calyculin A-sensitive protein phosphatase-dependent dephosphorylation. Our study reveals the profound effects of curcumin on the Akt/mTOR signaling network in PC-3 cells and provides new mechanisms for the anticancer effects of curcumin.

Activation of Nrf2-antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis.

Oxidative stress has been implicated in the etiology of neurodegenerative disease, cancer and aging. Indeed, accumulation of reactive oxygen and nitrogen species generated by inflammatory cells that created oxidative stress is thought to be one of the major factor by which chronic inflammation contributes to neoplastic transformation as well as many other diseases. We have recently reported that mice lacking nuclear factor-erythroid 2-related factor 2 (Nrf2) are more susceptible to dextran sulfate sodium (DSS)-induced colitis and colorectal carcinogenesis. Nrf2 is a basic leucine zipper redox-sensitive transcriptional factor that plays a center role in ARE (antioxidant response element)-mediated induction of phase II detoxifying and antioxidant enzymes. We found that increased susceptibility of Nrf2 deficient mice to DSS-induced colitis and colorectal cancer was associated with decreased expression of antioxidant/phase II detoxifying enzymes in parallel with upregulation of pro-inflammatory cytokines/biomarkers. These findings suggest that Nrf2 may play an important role in defense against oxidative stress possibly by activation of cellular antioxidant machinery as well as suppression of pro-inflammatory signaling pathways. In addition, in vivo and in vitro data generated from our laboratory suggest that many dietary compounds can differentially regulate Nrf2-mediated antioxidant/anti-inflammatory signaling pathways as the first line defense or induce apoptosis once the cells have been damaged. In this review, we will summarize our thoughts on the potential cross-talks between Nrf2 and NFkappaB pathways. Although the mechanisms involved in the cross-talk between these signaling pathways are still illusive, targeting Nrf2-antioxidative stress signaling is an ideal strategy to prevent or treat oxidative stress-related diseases.

Synergistic effects of a combination of dietary factors sulforaphane and (-) epigallocatechin-3-gallate in HT-29 AP-1 human colon carcinoma cells.

PURPOSE: The objective of this study was to investigate combinations of two chemopreventive dietary factors: EGCG 20 microM (or 100 microM) and SFN (25 microM) in HT-29 AP-1 human colon carcinoma cells. METHODS: After exposure of HT-29 AP-1 cells to SFN and EGCG, individually or in combination, we performed AP-1 luciferase reporter assays, cell viability assays, isobologram analyses, senescence staining, quantitative real-time PCR (qRT-PCR) assays, Western blotting, and assays for HDAC activity and hydrogen peroxide. In some experiments, we exposed cells to superoxide dismutase (SOD) or Trichostatin A (TSA) in addition to the treatment with dietary factors. RESULTS: The combinations of SFN and EGCG dramatically enhanced transcriptional activation of AP-1 reporter in HT-29 cells (46-fold with 25 microM SFN and 20 microM EGCG; and 175-fold with 25 microM SFN and 100 microM EGCG). Isobologram analysis showed synergistic activation for the combinations with combination index, CI < 1. Interestingly, co-treatment with 20units/ml of SOD, a free radical scavenger, attenuated the synergism elicited by the combinations (2-fold with 25 muM SFN and 20 muM EGCG; and 15-fold with 25 microM SFN and 100 microM EGCG). Cell viability assays showed that the low-dose combination decreased cell viability to 70% whereas the high-dose combination decreased cell viability to 40% at 48 h, with no significant change in cell viability at 24 h as compared to control cells. In addition, 20 microM and 100 microM EGCG, but not 25 microM SFN, showed induction of senescence in the HT-29 AP-1 cells subjected to senescence staining. However, both low- and high-dose combinations of SFN and EGCG attenuated the cellular senescence induced by EGCG alone. There was no significant change in the protein levels of phosphorylated forms of ERK, JNK, p38, and Akt-Ser473 or Akt-Thr308. Besides, qRT-PCR assays corroborated the induction of the luciferase gene seen with the combinations in the reporter assay. Relative expression levels of transcripts of many other genes known to be either under the control of the AP-1 promoter or involved in cell cycle regulation or cellular influx-efflux such as cyclin D1, cMyc, ATF-2, Elk-1, SRF, CREB5, SLCO1B3, MRP1, MRP2 and MRP3 were also quantified by qRT-PCR in the presence and absence of SOD at both 6 and 10 h. In addition, pre-treatment with 100 ng/ml TSA, a potent HDAC inhibitor, potentiated (88-fold) the synergism seen with the low-dose combination on the AP-1 reporter transcriptional activation. Cytoplasmic and nuclear fractions of treated cells were tested for HDAC activity at 2 and 12 h both in the presence and absence of TSA, however, there was no significant change in their HDAC activity. In addition, the H2O2 produced in the cell system was about 2 microM for the low-dose combination which was scavenged to about 1 microM in the presence of SOD. CONCLUSION: Taken together, the synergistic activation of AP-1 by the combination of SFN and EGCG that was potentiated by HDAC inhibitor TSA and attenuated by free radical scavenger SOD point to a possible multifactorial control of colon carcinoma that may involve a role for HDACs, inhibition of cellular senescence, and SOD signaling.