Mutual Regulation of FOXM1, NPM and ARF Proteins.

ARF, NPM and FOXM1 proteins interact with each other in mammalian cells. We showed previously that proteasome inhibitors suppress not only FOXM1 expression, but also the expression of ARF and NPM proteins. Using RNA interference we found that the depletion of each of these proteins by RNAi in human cancer HeLa cells leads to down-regulation of the two other partners, suggesting that these proteins stabilize each other in human cancer cells. Since the suppression of FOXM1 is one of hallmarks of proteasome inhibition, suppression of ARF and NPM by proteasome inhibitors may be explained in part as a secondary effect of downregulation of FOXM1 that modulate stability of ARF and NPM1 proteins.

Proteasome inhibitors suppress the protein expression of mutant p53.

Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53.

Structure-activity relationship studies of thalidomide analogs with a taxol-like mode of action.

Anti-microtubule agents such as paclitaxel and docetaxel have played an important role in the treatment of cancer for many years. Recently, a small molecule that has a taxol-like mode of action (5HPP-33) was reported. Herein, the detailed structure-activity relationship (SAR) studies of 5HPP-33 analogs that are substituted at the isoindole and phenyl rings are described. Bulky substitutions (such as di-isopropyl groups) on the phenyl ring result in the isoindole and phenyl rings being perpendicular to each other. It was found that this conformation is critical for anti-microtubule activity. These studies have provided valuable information, which will be helpful in the design of more potent analogs.

Design, synthesis and biological studies of novel tubulin inhibitors.

A series of compounds originally derived from the vascular endothelial growth factor receptor tyrosine kinase inhibitor, SU5416, were synthesized and evaluated. The most potent compound in this series, compound 3, which structurally resembles the potent anti-microtubule agent combretastatin A-4, inhibited tubulin polymerization and showed potent growth inhibitory activities on both prostate and breast cancer lines with IC50 values in the low nanomolar range.

Combination of oxidative stress and FOXM1 inhibitors induces apoptosis in cancer cells and inhibits xenograft tumor growth.

Tumor cells accumulate high level of reactive oxygen species (ROS) because they are metabolically more active than normal cells. Elevated ROS levels increase tumorigenecity but also render cancer cells more vulnerable to oxidative stress than normal cells. The oncogenic transcription factor Forkhead Box M1 (FOXM1), which is overexpressed in a wide range of human cancers, was reported to protect cancer cells from the adverse effects of oxidative stress by up regulating the expression of scavenger enzymes. We therefore hypothesized that the combination of FOXM1 ablation and ROS inducers could selectively eradicate cancer cells. We show that RNA interference-mediated knockdown of FOXM1 further elevates intracellular ROS levels and increases sensitivity of cancer cells to ROS-mediated cell death after treatment with ROS inducers. We also demonstrate that the combination of ROS inducers with FOXM1/proteasome inhibitors induces robust apoptosis in different human cancer cells. In addition, we show evidence that FOXM1/proteasome inhibitor bortezomib in combination with the ROS inducer ß-phenylethyl isothiocyanate efficiently inhibits the growth of breast tumor xenografts in nude mice. We conclude that the combination of ROS inducers and FOXM1 inhibitors could be used as a therapeutic strategy to selectively eliminate cancer cells.

Proteasome inhibitors suppress expression of NPM and ARF proteins.

Proteasome inhibitors stabilize numerous proteins by inhibiting their degradation. Previously we have demonstrated that proteasome inhibitors thiostrepton, MG132 and bortezomib paradoxically inhibit transcriptional activity and mRNA/protein expression of FOXM1. Here we demonstrate that, in addition to FOXM1, the same proteasome inhibitors also decrease mRNA and protein expression of NPM and ARF genes. These data suggest that proteasome inhibitors may suppress gene expression by stabilizing their transcriptional inhibitors.

FoxM1 knockdown sensitizes human cancer cells to proteasome inhibitor-induced apoptosis but not to autophagy.

Apoptosis has been widely accepted as the primary mechanism of drug-induced cell death. Recently, a second type of cell death pathway has been demonstrated: autophagy, also called programmed type II cell death. Autophagy is a highly regulated process, by which selected components of a cell are degraded. It primarily functions as a cell survival mechanism under stress. However, persistent stress can also promote extensive autophagy leading to cell death. Forkhead box M1 (FoxM1), an oncogenic transcription factor that is abundantly expressed in a wide range of human cancers. Here we evaluated the role of FoxM1 in sensitivity of human cancer cells to proteasome inhibitor-induced apoptosis and autophagy. We found that FoxM1 knockdown sensitized the human cancer cells to apoptotic cell death induced by proteasome inhibitors, such as, MG132, bortezomib and thiostrepton, while it did not affect the levels of autophagy following treatment with these drugs.

Thiazole antibiotic thiostrepton synergize with bortezomib to induce apoptosis in cancer cells.

Thiazole antibiotic, thiostrepton was recently identified as proteasome inhibitor. We investigated the therapeutic potential of the combination of thiostrepton and proteasome inhibitor bortezomib (Velcade) on various human tumor cell lines. Combination of sub-lethal concentrations of thiostrepton and bortezomib induced potent apoptosis and inhibition of long-term colony formation in a wide variety of human cancer cell lines. The synergistic relationship between thiostrepton and bortezomib combination was also quantitatively demonstrated by calculating their combination index values that were much lower than 1 in all studied cell lines. The synergy between these drugs was based on their proteasome inhibitory activities, because thiostrepton modification, thiostrepton methyl ester, which did not have intact quinaldic acid ring and did not inhibit proteasome activity failed to demonstrate any synergy in combination with bortezomib.

Proteasome inhibitors induce p53-independent apoptosis in human cancer cells.

Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.

Proteasome inhibitory activity of thiazole antibiotics.

Thiopeptides are sulfur containing highly modified macrocyclic antibiotics with a central pyridine/tetrapyridine/dehydropiperidine ring with up to three thiazole substituents on positions 2, 3 and 6. Thiazole antibiotics with central pyridine nucleus have a macrocyclic loop connecting thiazole rings at position 2 and 3 described as ring A. In addition antibiotics with central tetrahydropyridine nucleus have a quinaldic acid macrocycle also connected to thiazole on position 2 described as ring B. We have demonstrated before that thiazole antibiotics thiostrepton and Siomycin A act as proteasome inhibitors in mammalian tumor cells. Here we decided to test whether other known thiazole antibiotics such as berninamycin, micrococcin P1 and P2, thiocillin and YM-266183 (lacking the quinaldic acid ring B) demonstrate this activity. We found that none of them act as proteasome inhibitors. Moreover, structural modification of thiostrepton to thiostrepton methyl ester (with open B ring) also did not demonstrate this activity. These data suggest that B ring of thiostrepton and Siomycin A that is absent in other thiazole antibiotics determines the proteasome inhibitory activity of these drugs.

ARC synergizes with ABT-737 to induce apoptosis in human cancer cells.

Previously, we reported that the nucleoside analogue/transcriptional inhibitor ARC (4-amino-6-hydrazino-7-beta-D-ribofuranosyl-7H-pyrrolo(2,3-d)-pyrimidine-5-carboxamide) was able to induce p53-independent apoptosis in multiple cancer cell lines of different origins. This occurred, at least in part, by the suppression of short-lived, prosurvival member of the Bcl-2 family, Mcl-1. In contrast, we show here that treatment of human cancer cells with the pan-Bcl-2 inhibitor ABT-737 alone led to upregulation of Mcl-1 protein expression. Combination of subapoptotic concentrations of ABT-737 and ARC induced mitochondrial injury and potent caspase-3/caspase-9-dependent apoptosis in a wide variety of human cancer cell lines. These data suggest that the ABT-737/ARC combination, which simultaneously targets Bcl-2 and Mcl-1, may be efficient against human cancer.

Novel STAT3 phosphorylation inhibitors exhibit potent growth-suppressive activity in pancreatic and breast cancer cells.

The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently detected in most types of human cancer where it plays important roles in survival, drug resistance, angiogenesis, and other functions. Targeting constitutive STAT3 signaling is thus an attractive therapeutic approach for these cancers. We have recently developed novel small-molecule STAT3 inhibitors, known as FLLL31 and FLLL32, which are derived from curcumin (the primary bioactive compound of turmeric). These compounds are designed to bind selectively to Janus kinase 2 and the STAT3 Src homology-2 domain, which serve crucial roles in STAT3 dimerization and signal transduction. Here we show that FLLL31 and FLLL32 are effective inhibitors of STAT3 phosphorylation, DNA-binding activity, and transactivation in vitro, leading to the impediment of multiple oncogenic processes and the induction of apoptosis in pancreatic and breast cancer cell lines. FLLL31 and FLLL32 also inhibit colony formation in soft agar and cell invasion and exhibit synergy with the anticancer drug doxorubicin against breast cancer cells. In addition, we show that FLLL32 can inhibit the induction of STAT3 phosphorylation by IFNalpha and interleukin-6 in breast cancer cells. We also show that administration of FLLL32 can inhibit tumor growth and vascularity in chicken embryo xenografts as well as substantially reduce tumor volumes in mouse xenografts. Our findings highlight the potential of these new compounds and their efficacy in targeting pancreatic and breast cancers that exhibit constitutive STAT3 signaling.

New potential anti-cancer agents synergize with bortezomib and ABT-737 against prostate cancer.

BACKGROUND: We previously described the identification of a transcriptional inhibitor ARC and FoxM1 inhibitors, thiazole antibiotics, Siomycin A and thiostrepton that were able to induce potent p53-independent apoptosis in cancer cell lines of different origin. Here, we report the characterization of these drugs individually or in combination with ABT-737 and bortezomib on a panel of prostate cancer cell lines. METHODS: DU 145, LNCaP and PC-3 prostate cancer cells were treated with ARC, Siomycin A and thiostrepton to evaluate their activity as single agents or in combination with ABT-737 and bortezomib to measure their synergistic potential in anti-proliferative and cell cycle assays. Chou-Talalay method was used to quantitate the synergistic interaction. Western blot method was used to determine Mcl-1 and FoxM1 expression and caspase-3 cleavage. RESULTS: We show that ARC inhibited the viability of prostate cancer cells and induced apoptosis in low nanomolar concentration. It potently downregulated the expression of Mcl-1 and showed synergistic combination effect with Bcl-2 inhibitor ABT-737. Thiazole antibiotics, Siomycin A and thiostrepton inhibited growth, FoxM1 expression and induced cell death in prostate cancer cells in low micromolar concentrations. In addition, thiostrepton and ARC synergistically induced apoptosis in prostate cancer cells following combination treatment with proteasome inhibitor bortezomib. Furthermore, we found that all tested drug combinations were able to induce apoptosis selectively in transformed, but not normal cells of the same origin. CONCLUSIONS: Based on their in vitro activity as single or combination agents, ARC, Siomycin A and thiostrepton represent potential candidates for drug development against prostate cancer.

Biological activity of 1-aryl-3-phenethylamino-1-propanone hydrochlorides and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols on PC-3 cells and DNA topoisomerase I enzyme.

A number of studies reported Mannich bases to manifest antimicrobial, cytotoxic, anticancer, anti-inflammatory, and anticonvulsant activities. A considerable number of therapeutically important cytotoxic compounds are active on DNA topoisomerases that regulate the DNA topology. In the present study we evaluated the biological activity of mono-Mannich bases, 1-aryl-3-phenethylamino-1-propanone hydrochlorides (1a-10a), and semicyclic mono-Mannich bases, 3-aroyl-4-aryl-1-phenethyl-4-piperidinols (1b-9b), synthesized in our laboratory. We employed androgen-independent human prostate cancer cells (PC-3) to assess the cytotoxicity of the compounds and extended the biological activity evaluation to cover supercoil relaxation assays of mammalian type I topoisomerases. Our results showed that the compounds had cytotoxicity within the 8.2-32.1 microM range, while two compounds gave rise to a comparable average value in topo I interference of 42% and 40% for 10a (with a hydroxy substituent on the phenyl ring from mono-Mannich bases) and 5b (with a fluoro substituent on the phenyl ring from the semicyclic mono-Mannich base series, piperidinols), respectively.

p53 negatively regulates expression of FoxM1.

The Forkhead box M1 (FoxM1) oncogenic transcription factor is overexpressed in a majority of human tumors. p53 is a transcription factor and a major tumor suppressor that is mutated in 50% of human cancers. In this study, we compared the levels of FoxM1 in normal BJ human fibroblasts, BJ fibroblasts with p53 knockdown and corresponding BJ immortal/oncogenic cell lines with inactivated p53. We found that partial deletion or inactivation of p53 in these cells leads to upregulation of FoxM1 expression. Similarly, p53 knockdown in several human cancer cell lines with wt-p53 led to upregulation of FoxM1 mRNA and protein expression, while induction of p53 by DNA-damage led to downregulation of FoxM1. These data suggest that p53 negatively regulates FoxM1 expression and therefore inactivation of p53 in tumors could partially explain the phenomenon of FoxM1 overexpression in human cancers.

New curcumin analogues exhibit enhanced growth-suppressive activity and inhibit AKT and signal transducer and activator of transcription 3 phosphorylation in breast and prostate cancer cells.

Curcumin, the active component of turmeric, has been shown to protect against carcinogenesis and prevent tumor development in cancer. To enhance its potency, we tested the efficacy of synthetic curcumin analogues, known as FLLL11 and FLLL12, in cancer cells. We examined the impact of FLLL11 and FLLL12 on cell viability in eight different breast and prostate cancer cell lines. FLLL11 and FLLL12 (IC(50) values 0.3-5.7 and 0.3-3.8 micromol/L, respectively) were substantially more potent than curcumin (IC(50) values between 14.4-50 micromol/L). FLLL11 and FLLL12 were also found to inhibit AKT phosphorylation and downregulate the expression of HER2/neu. In addition, we demonstrate for the first time that FLLL11 and FLLL12 inhibit phosphorylation of signal transducer and activator of transcription (STAT) 3, an oncogene frequently found to be persistently active in many cancer types. The inhibition of STAT3 signaling was confirmed by the inhibition of STAT3 DNA binding and STAT3 transcriptional activity. Furthermore, FLLL11 and FLLL12 were more effective than curcumin in inhibiting cell migration and colony formation in soft agar as well as inducing apoptosis in cancer cells. These results indicate that FLLL11 and FLLL12 exhibit more potent activities than curcumin on the inhibition of STAT3, AKT, and HER-2/neu, as well as inhibit cancer cell growth and migration, and may thus have translational potential as chemopreventive or therapeutic agents for breast and prostate cancers.

Structure-activity relationship studies of curcumin analogues.

Two series of curcumin analogues, a total of twenty-four compounds, were synthesized and evaluated. The most potent compound, compound 23, showed potent growth inhibitory activities on both prostate and breast cancer lines with IC(50) values in sub-micromolar range, fifty times more potent than curcumin. Curcumin analogues might be potential anti-tumor agents for breast and prostate cancers.

Synthesis and cytotoxicity of novel 3-aryl-1-(3'-dibenzylaminomethyl-4'-hydroxyphenyl)-propenones and related compounds.

The reaction of various 4'-hydroxychalcones (1a-e) with paraformaldehyde and dibenzylamine led to the formation of a novel series of 4'-hydroxy-3'-dibenzylaminomethyl chalcones (7a-e) instead of 4'-hydroxy-3',5'-bis-(dibenzylaminomethyl)chalcones 4. In order to rationalise the formation of monoadduct 7, energy minimized model structures of 4a and 7a were compared. The in vitro cytotoxic activities of 7a-e were tested against PC-3 cell lines for the first time in this study and compared with the precursor 4'-hydroxychalcones (1a-e). Except for compound 7a (IC(50): 19.85 microM), insertion of dibenzylaminomethyl function into 4'-hydroxychalcones resulted in complete loss of cytotoxic activity. The results suggested that it is not only the pK(a) but also the shape and size of the amine that is critical in governing the cytotoxic activity.

Design, synthesis, and studies of small molecule STAT3 inhibitors.

A series of small molecule STAT3 inhibitors originally derived from our lead compound STA 21 were synthesized and evaluated. The most potent compound in this series, compound 1, exhibited the same anti-proliferative activities as STA 21 against prostate cancer cell lines that express constitutively active STAT3. Molecular docking showed compound 1 bound to the STAT3beta SH2 domain in a similar manner as STA 21.

Inhibitors of NF-kappaB derived from thalidomide.

A series of compounds originally derived from thalidomide were synthesized and evaluated. The most potent compounds in this series, 5HPP-33 and compound 20, inhibited NF-kappaB activation in HeLa cells. Preliminary study indicated that the mechanism of inhibition of NF-kappaB activation is through inhibition of its translocation from the cytoplasm to the nucleus.