Curcumin glucuronides: assessing the proliferative activity against human cell lines.

A gram scale synthesis of the glucuronide metabolites of curcumin were completed in four steps. The newly synthesized curcumin glucuronide compounds 2 and 3 along with curcumin 1 were tested and their anti-proliferative effects against KBM-5, Jurkat cell, U266, and A549 cell lines were reported. Biological data revealed that as much as 1 µM curcumin 1 exhibited anticancer activity and almost 100% cell kill was noted at 10 µM on two out of four cell lines; while curcumin mono-glucuronide 2 as well as di-glucuronide 3 displayed no suppression of cell proliferation.

Imatinib analogs as potential agents for PET imaging of Bcr-Abl and c-KIT expression at a kinase level.

We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [(18)F]-labeled STI-571 was prepared with high specific activity (75 GBq/µmol) by nucleophilic displacement and an average radiochemical yield of 12%. [(131)I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [(18)F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [(18)F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.

Degrasyn-like symmetrical compounds: possible therapeutic agents for multiple myeloma (MM-I).

A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents.

Imaging epigenetic regulation by histone deacetylases in the brain using PET/MRI with ¹8F-FAHA.

Epigenetic modifications mediated by histone deacetylases (HDACs) play important roles in the mechanisms of different neurologic diseases and HDAC inhibitors (HDACIs) have shown promise in therapy. However, pharmacodynamic profiles of many HDACIs in the brain remain largely unknown due to the lack of validated methods for noninvasive imaging of HDAC expression-activity. In this study, dynamic PET/CT imaging was performed in 4 rhesus macaques using [(18)F]FAHA, a novel HDAC substrate, and [(18)F]fluoroacetate, the major radio-metabolite of [(18)F]FAHA, and fused with corresponding MR images of the brain. Quantification of [(18)F]FAHA accumulation in the brain was performed using a customized dual-tracer pharmacokinetic model. Immunohistochemical analyses of brain tissue revealed the heterogeneity of expression of individual HDACs in different brain structures and cell types and confirmed that PET/CT/MRI with [(18)F]FAHA reflects the level of expression-activity of HDAC class IIa enzymes. Furthermore, PET/CT/MRI with [(18)F]FAHA enabled non-invasive, quantitative assessment of pharmacodynamics of HDAC inhibitor SAHA in the brain.

Asymmetric Synthesis of the C1-C6 Portion of the Psymberin Using an Evans Chiral Auxiliary.

The C1-C6 region of the potent cytotoxic agent psymberin has been synthesized. The key transformations of the synthesis are an auxiliary-controlled addition of a Sn(II)-glycolate enolate to an aldehyde to yield the anti aldol product and transforming the primary alcohol into a terminal olefin utilizing organoselenium chemistry.

Kit inhibitor APcK110 extends survival in an AML xenograft mouse model.

BACKGROUND: Constitutive activation of kit contributes to pathogenesis of acute myeloid leukemia (AML) and targeting Kit may be of therapeutic benefit. APcK110, a novel inhibitor of Kit, has potent proapoptotic and antiproliferative activity in AML cell lines and primary AML samples. Here we extend our studies to the activity of APcK110 in a xenograft mouse model. METHODS: After sub-lethal whole body radiation, OCI/AML3 cells were injected intravenously in NOD-SCID mice. Ten days later, either APcK110 or phosphate buffered saline (PBS) was injected intraperitoneally every other day. Kaplan-Meier estimates were used to calculate survival. RESULTS: We show that 1) all mice injected with OCI/AML3 cells developed a clinical and histological picture consistent with myelomonocytic AML; and 2) survival of APcK110-treated mice was significantly longer compared with mice injected with PBS (p = .02). CONCLUSIONS: APcK110 is a novel kit kinase inhibitor with anti-AML activity in vitro and in vivo. Further evaluation in toxicology and clinical studies is warranted.

Tyrphostin-like compounds with ubiquitin modulatory activity as possible therapeutic agents for multiple myeloma.

With the goal of developing small molecules as novel regulators of signal transduction and apoptosis, a series of tyrphostin-like compounds were synthesized and screened for their activity against MM-1 (multiple myeloma) cells and other cell lines representing this malignancy. Synthesis was completed in solution-phase initially and then adopted to solid-phase for generating a more diverse set of compounds. A positive correlation was noted between compounds capable of inducing apoptosis and their modulation of protein ubiquitination. Further analysis suggested that ubiquitin modulation occurs through inhibition of cellular deubiquitinase activity. Bulky groups on the sidechain near the α,ß-unsaturated ketone caused a complete loss of activity, whereas cyclization on the opposite side was tolerated. Theoretical calculations at the B3LYP/LACV3P(∗∗) level were completed on each molecule, and the resulting molecular orbitals and Fukui reactivity values for C(ß) carbon were utilized in developing a model to explain the compound activity.

Mechanism of activation and inhibition of the HER4/ErbB4 kinase.

HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.

Activity of lapatinib is independent of EGFR expression level in HER2-overexpressing breast cancer cells.

Epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2/neu), members of the ErbB receptor tyrosine kinase family, are frequently overexpressed in breast cancer and are known to drive tumor growth and progression, making them promising targets for cancer therapy. Lapatinib is a selective competitive inhibitor of both the HER2 and EGFR tyrosine kinases. Although lapatinib showed significant activity in patients with HER2-positive breast cancer, the role of EGFR in the response of breast cancer to lapatinib has not been defined. Here, we examined the role of EGFR expression levels in the sensitivity of HER2-overexpressing breast cancer cells to lapatinib. Depletion of EGFR by EGFR small-interfering RNA knockdown did not affect lapatinib sensitivity in these cells, whereas treated HER2 siRNA knockdown cells became more resistant to lapatinib. We conclude that the in vitro activity of lapatinib is not dependent on EGFR expression level in HER2-overexpressing breast cancer cells.

Interferon-inducible protein IFIXalpha inhibits cell invasion by upregulating the metastasis suppressor maspin.

IFIXalpha, a member of the interferon-inducible HIN-200 family, has been identified as a putative tumor suppressor. However, the molecular mechanisms underlying IFIXalpha-mediated tumor suppression are poorly understood. In the present study, we demonstrated that the metastasis suppressor maspin acts as the downstream target of IFIXalpha. IFIXalpha suppressed the invasion activity of MDA-MB-468 breast cancer cells, and its inhibitory effect was reversed by the knockdown of maspin. Both Maspin mRNA and protein were upregulated by IFIXalpha. Histone deacetylase (HDAC) inhibitors, but not DNA methyltransferase inhibitor upregulated maspin, and HDAC1 inhibited the transactivation of maspin promoter. Although the HDAC1 protein was downregulated in IFIXalpha-expressing cells, IFIXalpha did not affect HDAC1 mRNA levels. Conversely, a proteasome inhibitor restored the level of HDAC1 protein in IFIXalpha-expressing cells, and the polyubiqutination of HDAC1 was promoted by IFIXalpha, suggesting that HDAC1 is regulated by IFIXalpha through a ubiquitin-proteasome pathway. Together, these data provide novel insights into the tumor-suppressive function of IFIXalpha.

Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells.

The proteasome inhibitor bortezomib (formerly known as PS-341) recently received Food and Drug Administration approval for the treatment of multiple myeloma, and its activity is currently being evaluated in solid tumors. Bortezomib triggers apoptosis in pancreatic cancer cells, but the mechanisms involved have not been fully elucidated. Here, we show that pancreatic cancer cells exposed to bortezomib formed aggregates of ubiquitin-conjugated proteins ("aggresomes") in vitro and in vivo. Bortezomib-induced aggresome formation was determined to be cytoprotective and could be disrupted using histone deacetylase (HDAC) 6 small interfering RNA or chemical HDAC inhibitors, which resulted in endoplasmic reticulum stress and synergistic levels of apoptosis in vitro and in an orthotopic pancreatic cancer xenograft model in vivo. Interestingly, bortezomib did not induce aggresome formation in immortalized normal human pancreatic epithelial cells in vitro or in murine pancreatic epithelial cells in vivo. In addition, these cells did not undergo apoptosis following treatment with bortezomib, suberoylanilide hydroxamic acid, or the combination, showing tumor selectivity. Taken together, our study shows that inhibition of aggresome formation can strongly potentiate the efficacy of bortezomib and provides the foundation for clinical trials of bortezomib in combination with HDAC inhibitors for the treatment of pancreatic cancer.

Radiosynthesis of 2'-deoxy-2'-[18F]-fluoro-5-methyl-1-beta-L-arabinofuranosyluracil ([18F]-L-FMAU) for PET.

Radiosynthesis of 2'-deoxy-2'-[(18)F]-fluoro-5-methyl-1-beta-L-arabinofuranosyluracil ([(18)F]-L-FMAU) is reported. Compound 1 was synthesized and converted to 2-triflate 2. Compound 3 was prepared from 2 using tetrabutylammonium[(18)F]fluoride, converted to 4, and then coupled with 5. The crude product was hydrolyzed, and purified by HPLC to obtain 7a. The radiochemical yield of [(18)F]-L-FMAU was 26% decay corrected (d.c.) in four runs with radiochemical purity >99% and specific activity 2200 mCi/micromol. The synthesis time was 3.3-3.5h from the end of bombardment (EOB).

Detection of histone deacetylase inhibition by noninvasive magnetic resonance spectroscopy.

Histone deacetylase (HDAC) inhibitors are new and promising antineoplastic agents. Current methods for monitoring early response rely on invasive biopsies or indirect blood-derived markers. Our goal was to develop a magnetic resonance spectroscopy (MRS)-based method to detect HDAC inhibition. The fluorinated lysine derivative Boc-Lys-(Tfa)-OH (BLT) was investigated as a (19)F MRS molecular marker of HDAC activity together with (31)P MRS of endogenous metabolites. In silico modeling of the BLT-HDAC interaction and in vitro MRS studies of BLT cleavage by HDAC confirmed BLT as a HDAC substrate. BLT did not affect cell viability or HDAC activity in PC3 prostate cancer cells. PC3 cells were treated, in the presence of BLT, with the HDAC inhibitor p-fluoro-suberoylanilide hydroxamic acid (FSAHA) over the range of 0 to 10 micromol/L, and HDAC activity and MRS spectra were monitored. Following FSAHA treatment, HDAC activity dropped, reaching 53% of control at 10 micromol/L FSAHA. In parallel, a steady increase in intracellular BLT from 14 to 32 fmol/cell was observed. BLT levels negatively correlated with HDAC activity consistent with higher levels of uncleaved BLT in cells with inhibited HDAC. Phosphocholine, detected by (31)P MRS, increased from 7 to 16 fmol/cell following treatment with FSAHA and also negatively correlated with HDAC activity. Increased phosphocholine is probably due to heat shock protein 90 inhibition as indicated by depletion of client proteins. In summary, (19)F MRS of BLT, combined with (31)P MRS, can be used to monitor HDAC activity in cells. In principle, this could be applied in vivo to noninvasively monitor HDAC activity.

Sporadic and familial myoclonic dystonia: Report of three cases and review of literature.

Myoclonic dystonia refers to a clinical syndrome characterized by rapid jerky movements along with dystonic posturing of the limbs. Clinically, it is characterized by sudden, brief, electric shock-like movements, mostly involving the upper extremities, shoulders, neck and trunk. Characteristically, the movements wane with consumption of small dose of alcohol in about 50% of cases. Additionally, dystonic contractions are observed in most of the patients in the affected body parts and some patients may exhibit cervical dystonia or graphospasm as well. It may manifest as an autosomal dominant condition or sometimes, as a sporadic entity, though there are doubts whether these represent cases with reduced penetrance. The condition is usually treated with a combination of an anticholinergic agent like, benztropine, pimozide and tetrabenazine. We report one sporadic case and one familial case where the father and the son are affected. The cases were collected from the Movement Disorders Clinic of Bangur Institute of Neurosciences, Kolkata, West Bengal in a period of ten months. Myoclonic dystonia is a rare condition and to the best of our knowledge, this series is the first one reported from our country. Videos of the patients are also provided with the article.

Designing and developing S100P inhibitor 5-methyl cromolyn for pancreatic cancer therapy.

We have previously shown that the antiallergic drug cromolyn blocks S100P interaction with its receptor receptor for advanced glycation end product (RAGE) and improves gemcitabine effectiveness in pancreatic ductal adenocarcinoma (PDAC). However, the concentration required to achieve its effectiveness was high (100 µmol/L). In this study, we designed and synthesized analogs of cromolyn and analyzed their effectiveness compared with the parent molecule. An ELISA was used to confirm the binding of S100P with RAGE and to test the effectiveness of the different analogs. Analog 5-methyl cromolyn (C5OH) blocked S100P binding as well as the increases in NF-κB activity, cell growth, and apoptosis normally caused by S100P. In vivo C5OH systemic delivery reduced NF-κB activity to a greater extent than cromolyn and at 10 times lesser dose (50 mg vs. 5 mg). Treatment of mice-bearing syngeneic PDAC tumors showed that C5OH treatment reduced both tumor growth and metastasis. C5OH treatment of nude mice bearing orthotopic highly aggressive pancreatic Mpanc96 cells increased the overall animal survival. Therefore, the cromolyn analog, C5OH, was found to be more efficient and potent than cromolyn as a therapeutic for PDAC.

Radiosynthesis and initial in vitro evaluation of [18F]F-PEG6-IPQA--a novel PET radiotracer for imaging EGFR expression-activity in lung carcinomas.

INTRODUCTION: Epidermal growth factor receptor (EGFR)-targeted therapies with antibodies and small molecular EGFR kinase inhibitors have shown poor efficacy in unselected populations of patients with advanced non-small cell lung carcinomas (NSCLC). In contrast, patients with overexpression of EGFR and activating mutations in EGFR kinase domain demonstrated improved responses to EGFR kinase inhibitors. Therefore, we have developed a novel radiotracer, [(18)F]F-PEG(6)-IPQA for PET imaging of EGFR expression-activity in NSCLC, and have described its radiosynthesis and in vitro evaluation in two NSCLC cell lines with wild-type and L858R active mutant EGFR. METHODS: A mesylate precursor was synthesized in multiple steps and radiofluorinated using K(18)F/Kryptofix. The fluorinated intermediate compound was reduced to an amino derivative then treated with acryloyl isobutyl carbonate, followed by purification by HPLC to obtain the desired product. RESULTS: Decay-corrected radiochemical yields of [(18)F]F-PEG(6)-IPQA were 3.9-17.6%, with an average of 9.0% (n = 11). Radiochemical purity was >97% with specific activity of 34 GBq/µmol (mean value, n = 10) at the end of synthesis. The accumulation of [(18)F]F-PEG(6)-IPQA in H3255 cells was ten-fold higher than in H441 cells, despite a two-fold lower level of activated phospho-EGFR expression in H3255 cells compared with H441 cells. The accumulation of [(18)F]F-PEG(6)-IPQA in both cell lines was significantly decreased in the presence of a small molecular EGFR kinase inhibitor, Iressa, at 100 µM concentration in culture medium. CONCLUSION: We have synthesized [(18)F]F-PEG(6)-IPQA and demonstrated its highly selective accumulation in active mutant L858R EGFR-expressing NSCLC cells in vitro. Further in vivo studies are warranted to assess the ability of PET imaging with [(18)F]F-PEG(6)-IPQA to discriminate the active mutant L858R EGFR-expressing NSCLC that are sensitive to therapy with EGFR kinase inhibitors vs NSCLC that express wild-type EGFR.

Synthesis and ex vivo autoradiographic evaluation of ethyl-ß-D-galactopyranosyl-(1,4')-2'-deoxy-2'-[18F]fluoro-ß-D-glucopyranoside--a novel radioligand for lactose-binding protein: implications for early detection of pancreatic carcinomas with PET.

INTRODUCTION: Previous studies demonstrated that the lactose-binding protein (hepatocellular carcinoma-intestine-pancreas and pancreatitis-associated proteins (HIP/PAP)) is upregulated >130 times in peritumoral pancreatic tissue as compared to normal pancreatic tissue. Therefore, we developed a new radiolabeled ligand of HIP/PAP, the ethyl-ß-D-galactopyranosyl-(1,4')-2'-deoxy-2'-[¹8F]fluoro-ß-D-glucopyranoside (Et-[¹8F]FDL) for noninvasive imaging of pancreatic carcinoma using positron emission tomography and computerized tomography (PET/CT). METHODS: The novel precursor and radiolabeling methods for synthesis of Et-[¹8F]FDL produced no isomers; the average decay-corrected radiochemical yield was 68%, radiochemical purity >99%, and specific activity >74 GBq/µmol. The radioligand properties of Et-[¹8F]FDL were evaluated using an ex vivo autoradiography and immunohistochemistry in pancreatic tissue sections obtained from mice-bearing orthotopic pancreatic tumor xenografts. RESULTS AND DISCUSSION: Et-[¹8F]FDL binding to peritumoral pancreatic tissue sections strongly correlated with HIP/PAP expression (r = 0.81) and could be completely blocked by treatment with 1 mM lactose. CONCLUSION: These results suggest that Et-[¹8F]FDL is a promising agent which should be evaluated for detection of early pancreatic carcinomas by PET/CT imaging.

Suberoylanilide hydroxamic acid blocks self-renewal and homotypic aggregation of inflammatory breast cancer spheroids.

BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC commonly present with skin metastasis, which are observed microscopically as tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly overexpress E-cadherin and exhibit the ability to undergo self-renewal and are highly invasive. There are no therapeutics yet identified that target the structure and functions of IBC tumor emboli. The present studies evaluated the effects of the pan-histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) using IBC tumor spheroids derived from established IBC cell lines and tumor spheroids derived from pleural effusion (PE) aspirates of patients with IBC and LABC, designated as PE-IBC and PE-LABC. METHODS: Methods used are as follows: culture of IBC cells from clonal density single cells in low adherence culture conditions that promote formation of IBC tumor spheroids; clonogenic assays; cell fractionation and Western blotting; confocal microscropy; and modified Boyden chamber invasion assays. RESULTS: SAHA inhibited self-renewal of IBC tumor spheroids from established IBC cell lines and PE-IBC and PE-LABC, as assessed by decreased clonogenic growth. SAHA blocked homotypic aggregation of the cells that comprised the IBC tumor spheroids leading to loss of their 3-dimensional (3D) structure, which was associated with a change in location of E-cadherin protein from the plasma membrane in untreated IBC tumor spheroids to the cytoplasm of cells within IBC tumor spheroids with SAHA treatment. In addition, SAHA blocked the robust invasion exhibited by IBC tumor spheroids of established cell lines as well as by tumor spheroids derived from PE-IBC and PE-LABC. CONCLUSIONS: SAHA targets the integrity and biological activities of IBC tumor spheroids and may be a promising agent to evaluate for its effectiveness in treatment of IBC.