1-Chromonyl-5-Imidazolylpentadienone Demonstrates Anti-Cancer Action against TNBC and Exhibits Synergism with Paclitaxel.

Curcumin has been well studied for its anti-oxidant, anti-inflammatory, and anti-cancer action. Its potential as a therapy is limited due to its low bioavailability and rapid metabolism. To overcome these challenges, investigators are developing curcumin analogs, nanoparticle formulations, and combining curcumin with other compounds or dietary components. In the present study, we used a 1-chromonyl-5-imidazolylpentadienone named KY-20-22 that contains both the pharmacophore of curcumin and 1,4 benzopyrone (chromone) moiety typical for flavonoids, and also included specific moieties to enhance the bioavailability. When we tested the in vitro effect of KY-20-22 in triple-negative breast cancer (TNBC) cell lines, we found that it decreased the cell survival and colony formation of MDA-MB-231 and MDA-MB-468 cells. An increase in mitochondrial reactive oxygen species was also observed in TNBC cells exposed to KY-20-22. Furthermore, KY-20-22 decreased epithelial-mesenchymal formation (EMT) as evidenced by the modulation of the EMT markers E-cadherin and N-cadherin. Based on the fact that KY-20-22 regulates interleukin-6, a cytokine involved in chemotherapy resistance, we combined it with paclitaxel and found that it synergistically induced anti-proliferative action in TNBC cells. The results from this study suggested that 1-chromonyl-5-imidazolylpentadienone KY-20-22 exhibited anti-cancer action in MDA-MB-231 and MDA-MB-468 cells. Future studies are required to evaluate the anti-cancer ability and bioavailability of KY-20-22 in the TNBC animal model.

Synergistic anticancer action of quercetin and curcumin against triple-negative breast cancer cell lines.

Women with the breast cancer type 1 susceptibility protein (BRCA1) mutation and loss of BRCA1 expression are reported to have an increased risk of triple-negative breast cancer (TNBC). Targeting BRCA1 modulation might offer a therapeutic option to treat TNBC patients. Our studies detected that BRCA1 is poorly expressed in TNBC cell lines and highly expressed in ER+ breast cancer cell lines. To modulate BRCA1 expression, we tested two different dietary components to find out if any would induce tumor suppressor genes. We detected that quercetin and curcumin dose-dependently enhanced the BRCA1 expression. Further, a synergistic action of quercetin and curcumin was observed in modulating the BRCA1 level and in inhibiting the cell survival and migration of TNBC cell lines. Quercetin and curcumin appeared to induce BRCA1 promoter histone acetylation. Furthermore, BRCA1 knockdown induced cell survival and cell migration in ER + cells were significantly decreased by the combined treatment of quercetin and curcumin. Our present study concluded that the combination treatment of quercetin and curcumin acts synergistically to induce anticancer activity against TNBC cells by modulating tumor suppressor genes.

Quercetin regulates ß-catenin signaling and reduces the migration of triple negative breast cancer.

Triple negative breast cancer (TNBC) is characterized by a lack in estrogen, progesterone, and epidermal growth factor 2 receptors. TNBC exhibits most of the characteristics of basal-like and claudin-low breast cancer subtypes. The main contributor in the mortality of TNBC is due to the higher invasive and migratory ability of these tumor cells. Some plant flavonoids inhibit the epithelial mesenchymal transition (EMT) of tumor cells and suppress cancer metastasis. In this study, we aimed to determine whether the flavonoid quercetin is effective in modulating the molecular signaling associated with EMT in TNBC. Our data indicated that quercetin can induce the expression of E-cadherin and also downregulate vimentin levels in TNBC. The ability of quercetin to modulate these EMT markers resulted in a mesenchymal-to-epithelial transition (MET). Quercetin-induced MET was linked with the alteration of nuclear localization of ß-catenin and modulation of ß-catenin target genes such as cyclin D1 and c-Myc. Furthermore, we observed that quercetin induced the anti-tumor activity of doxorubicin by inhibiting the migratory ability of TNBC cells. These results suggested that quercetin may inhibit TNBC metastasis and also improve the therapeutic efficacy of existing chemotherapeutic drugs.

Novel targeted siRNA-loaded hybrid nanoparticles: preparation, characterization and in vitro evaluation.

BACKGROUND: siRNAs have a high potential for silencing critical molecular pathways that are pathogenic. Nevertheless, their clinical application has been limited by a lack of effective and safe nanotechnology-based delivery system that allows a controlled and safe transfection to cytosol of targeted cells without the associated adverse effects. Our group recently reported a very effective and safe hybrid nanoparticle delivery system composing human IgG and poloxamer-188 for siRNA delivery to cancer cells. However, these nanoparticles need to be optimized in terms of particle size, loading capacity and encapsulation efficiency. In the present study, we explored the effects of certain production parameters on particle size, loading capacity and encapsulation efficiency. Further, to make these nanoparticles more specific in their delivery of siRNA, we conjugated anti-NTSR1-mAb to the surface of these nanoparticles to target NTSR1-overexpressing cancer cells. The mechanism of siRNA release from these antiNTSR1-mAb functionalized nanoparticles was also elucidated. RESULTS: It was demonstrated that the concentration of human IgG in the starting nanoprecipitation medium and the rotation speed of the magnetic stirrer influenced the encapsulation efficiency, loading capacity and the size of the nanoparticles produced. We also successfully transformed these nanoparticles into actively targeted nanoparticles by functionalizing with anti-NTSR1-mAb to specifically target NTSR1-overexpressing cancer cells, hence able to avoid undesired accumulation in normal cells. The mechanism of siRNA release from these nanoparticles was elucidated to be by Fickian diffusion. Using flow cytometry and fluorescence microscopy, we were able to confirm the active involvement of NTSR1 in the uptake of these anti-NTSR1-mAb functionalized hybrid nanoparticles by lung adenocarcinoma cells. CONCLUSIONS: This hybrid nanoparticle delivery system can be used as a platform technology for intracellular delivery of siRNAs to NTSR1-overexpressing tumor cells.

Investigation of the stability and cellular uptake of self-associated monoclonal antibody (MAb) nanoparticles by non-small lung cancer cells.

The inability to deliver MAbs to intracellular targets still remains a limitation to their application in cancer therapy and diagnosis. Selective targeting of MAbs to oncoproteins in cancer cells while avoiding their accumulation in normal cells may reduce some of the well-documented adverse effects accompanying antibody therapy. One of the remarkable characteristics of malignant cells is the alteration in the biological properties of the cellular plasma membrane. Taking advantage of this alteration, we hope to selectively deliver self-associated MAb nanoparticles to cancer cells while reducing their accumulation in normal cells. We hypothesized that self-associated MAb nanoparticles can be preferentially taken up by non-small lung cancer cells in comparison to normal cells due to the absence or dysfunction of tight junctions (TJ) in confluent cancer cells and increased permeability of the cancer cell membrane. Self-associated bevacizumab nanoparticles were prepared and characterized for particle size and biochemical stability. Fluorescence microscopy, TEM, and flow cytometry revealed that these bevacizumab nanoparticles were internalized by A549 cells three times more than MRC-5 cells. Macropinocytosis and energy-dependent pathways were elucidated to be involved in their uptake by A549 cells. Further, uptake was by nonspecific interaction with cell membrane. Results obtained from this study suggest that self-associated MAb nanoparticles can be selectively delivered to cancer cells.

Effect of probiotics and gut microbiota on anti-cancer drugs: Mechanistic perspectives.

Bacteria present in probiotics, particularly the common Lactobacillus and Bifidobacterium microbes, have been found to induce anti-cancer action by enhancing cancer cell apoptosis and protecting against oxidative stress. Probiotics supplements also decrease the cancer-producing microorganism Fusobacterium. Studies have demonstrated that gut microbiota modifies the effect of chemo/radiation therapy. Gut microbes not only enhance the action of chemotherapy drugs but also reduce the side effects of these medications. Additionally, gut microbes reduce immunotherapy toxicity, in particular, the presence of Bacteroidetes or Bifidobacterium decreases the development of colitis by ipilimumab therapy. Probiotics supplements containing Bifidobacterium also reduce chemotherapy-induced mucositis and radiation-induced diarrhea. This review focused on elucidating the mechanism behind the anti-cancer action of Bifidobacterium species. Available studies have revealed Bifidobacterium species decrease cancer cell proliferation via the inhibition of growth factor signaling as well as inducing mitochondrial-mediated apoptosis. Moreover, Bifidobacterium species reduce the adverse effects of chemo/immuno/radiation therapy by inhibiting proinflammatory cytokines. Further clinical studies are needed to identify the powerful and suitable Bifidobacterium strain for the development of adjuvant therapy to support chemo/immuno/radiation therapy.

Myristoyltransferase and calcineurin: novel molecular therapeutic target for epilepsy.

N-myristoylation is a co-translational, irreversible addition of a fatty acyl moiety to the amino terminus of many eukaryotic cellular proteins. These myristoylated proteins in the cell have diverse biological functions such as signal transduction, cellular transformation and oncogensis. Known myristoylated proteins [Src family kinases, the catalytic subunit of cAMP-dependent protein kinase and calcineurin (CaN)] are either protein kinases or a protein phosphatases which modulate various cellular metabolic processes. Myristoylation is catalyzed by N-myristoyltransferase (NMT) and is recognized to be a widespread and functionally important modification of proteins. The main objective of this review is to focus on the potential role of NMT and CaN in epileptic brain and its involvement in neuronal apoptosis. The findings on the interaction of NMT and CaN with various signaling molecules in epileptic chickens adds to our understanding of the mechanism of CaN signaling in neuronal apoptosis. Understanding the regulation of NMT by specific inhibitors may help us to control the action of this enzyme on its specific substrates and may lead to improvements in the management of various neurological disorders like Alzheimer's disease, ischemia and epilepsy.

Flavonoids and Other Polyphenols Act as Epigenetic Modifiers in Breast Cancer.

Breast cancer is a common cancer that occurs due to different epigenetic alterations and genetic mutations. Various epidemiological studies have demonstrated an inverse correlation between breast cancer incidence and flavonoid intake. The anti-cancer action of flavonoids, a class of polyphenolic compounds that are present in plants, as secondary metabolites has been a major topic of research for many years. Our review analysis demonstrates that flavonoids exhibit anti-cancer activity against breast cancer occurring in different ethnic populations. Breast cancer subtype and menopausal status are the key factors in inducing the flavonoid's anti-cancer action in breast cancer. The dose is another key factor, with research showing that approximately 10 mg/day of isoflavones is required to inhibit breast cancer occurrence. In addition, flavonoids also influence the epigenetic machinery in breast cancer, with research demonstrating that epigallocatechin, genistein, and resveratrol all inhibited DNA methyltransferase and altered chromatin modification in breast cancer. These flavonoids can induce the expression of different tumor suppressor genes that may contribute to decreasing breast cancer progression and metastasis. Additional studies are required to confirm the contribution of epigenetic modifications by flavonoids to breast cancer prevention.

NC2213: a novel methionine aminopeptidase 2 inhibitor in human colon cancer HT29 cells.

Methionine aminopeptidase 2 (MetAP2) is a bifunctional protein that plays a critical role in the regulation of post-translational processing and protein synthesis. MetAP2 is overexpressed in human colon cancer. In this report we screened various MetAP2 inhibitors and treated HT29 cells with various concentrations of compounds. We evaluated the expression of MetAP2 and pp60c-src expressions in HT29 cells. In addition we also carried out the cell proliferation and cell cycle analysis in the MetAP2 inhibitor-treated HT29 cells. The cell cycle analysis of HT29 treated with 1.0 microM of NC2213 showed an arrest in the G2 phase followed by an induction in the percentage of cells undergoing apoptosis in the sub-G1 phase. Western blot analysis revealed that the MetAP2 expression was dose-dependently decreased when the HT29 cells were treated with the 3,5-bis(benzylidene)-4-piperidone derivative (NC2213). In addition, phosphorylation of Src, a myristoylated oncoprotein was significantly decreased by 1.0 microM of NC2213 as revealed by Western blot analysis. Furthermore, NC2213 also inhibits the expression of pp60c-src in HT29 cells. Interestingly, this compound also inhibits the phosphorylation at Tyr416 of pp60c-src while increasing the phosphorylation at Tyr527 of pp60c-src. NC2213 inhibits the growth of HT29 cells by inducing apoptosis and might be useful for the treatment of human colon cancer.

Biochemical characterization of bovine brain myristoyl-CoA:protein N-myristoyltransferase type 2.

Protein N-myristoylation is a lipidic modification which refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral, and fungal proteins. In this paper, we have cloned the gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) from Bos tarus brain. The open reading frame codes for a 410-amino-acid protein and overexpressed in Escherichia coli. Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities. The metal ion Ca(2+) had stimulatory effects on NMT2 activity while Mn(2+) and Zn(2+) inhibited the enzyme activity. In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect. Biochemical characterization suggested that both forms of NMT have unique characteristics. Further analysis towards functional role NMT2 will lead the development of therapeutic target for the progression of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases.

Potential role of N-myristoyltransferase in cancer.

Colorectal cancer is the second leading cause of malignant death, and better preventive strategies are needed. The treatment of colonic cancer remains difficult because of the lack of effective chemotherapeutic agents; therefore it is important to continue to search for cellular functions that can be disrupted by chemotherapeutic drugs resulting in the inhibition of the development and progression of cancer. The current knowledge of the modification of proteins by myristoylation involving myristoyl-CoA: protein N-myristoyltransferase (NMT) is in its infancy. This process is involved in the pathogenesis of cancer. We have reported for the first time that NMT activity and protein expression were higher in human colorectal cancer, gallbladder carcinoma and brain tumors. In addition, an increase in NMT activity appeared at an early stage in colonic carcinogenesis. It is conceivable therefore that NMT can be used as a potential marker for the early detection of cancer. These observations lead to the possibility of developing NMT specific inhibitors, which may be therapeutically useful. We proposed that HSC70 and/or enolase could be used as an anticancer therapeutic target. This review summarized the status of NMT in cancer which has been carried in our laboratory.

Cardiac high molecular weight calmodulin-binding protein is homologous to calpastatin I and calpastatin II.

Calpastatin is an endogenous inhibitor of calpain, which has been implicated in various physiological and pathological processes. In the present study we determined the molecular and inhibitory properties of HMWCaMBP, calpastatin I, and calpastatin II. Western blot analysis with antibodies raised against either full length HMWCaMBP or internal peptides that are common to all isoforms showed that all three homologs have common antigenic epitopes. However, additional Western blot analysis with N-terminal specific antibodies showed that all three proteins are different at the N-terminal end. HMWCaMBP is clearly different from two other homologues, calpastatin I and II, at the N-terminal end. In addition, HMWCaMBP also showed the same affinities for m-calpain as calpastatin I and calpastatin II. Our findings suggest that HMWCaMBP is the homolog of calpastatin and may be a CaM-binding form of calpastatin.

Methionine aminopeptidase 2 and cancer.

Methionine aminopeptidase (MetAP) is a bifunctional protein that plays a critical role in the regulation of post-translational processing and protein synthesis. In yeasts and humans, two proteins are known to possess MetAP activity, which are known as MetAP1 and MetAP2. MetAP2 has attracted much more attention than MetAP1 due to the discovery of MetAP2 as a target molecule of the anti-angiogenic compounds, fumallin and ovalicin. MetAP2 plays an important role in the development of different types of cancer. Recently, we observed a high expression of MetAP2 in human colorectal cancer tissues and colon cancer cell lines. In addition, pp60(c-src) expression was correlated with the expression of MetAP2 and N-myristoyltransferase. In this review, we discuss the recent developments of MetAP2 and its inhibitors. Future detailed studies related to MetAP2 and apoptosis will shed light on the involvement of this enzyme in the regulation of various apoptotic factors.

Interaction between heat shock protein 70 kDa and calcineurin in cardiovascular systems (Review).

Cells have the capability of defending themselves from various stressors by activating a genetic program with the production of substances known as heat shock proteins (Hsps) and their regulatory partners, the heat shock transcription factors. Hsps play a major role in systemic hypertension, coronary artery disease, carotid atherosclerosis, myocardial infarction and myocardial ischemia. In this review we discuss the interaction between Hsp70 and CaN which was carried out in our laboratory. We demonstrated that the cardiac Hsp70 stimulated a 2-fold increase in calcineurin (CaN) activity. In addition, the pull-down assay revealed that Hsp70 directly interacts with CaN. Furthermore, expressed cardiac specific Hsp70 was phosphorylated in vitro by cAMP-dependent protein kinase. The phosphorylated Hsp70 was unable to activate the phosphatase activity of CaN. For the first time we demonstrated that Hsp70 is phosphorylated by cAMP-dependent protein kinase and provides an on/off switch for the regulation of CaN signaling by Hsp70. This will lead to therapeutic benefit in human diseases such as atherosclerosis, cardiomyopathy, congestive heart failure, and ischemia.

Regulation of calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1): review.

The response of living cells to change in cell environment depends on the action of second messenger molecules. The two second messenger molecules cAMP and Ca2+ regulate a large number of eukaryotic cellular events. Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1) is one of the key enzymes involved in the complex interaction between cAMP and Ca2+ second messenger systems. Some PDE1 isozymes have similar kinetic and immunological properties but are differentially regulated by Ca2+ and calmodulin. Accumulating evidence suggests that the activity of PDE1 is selectively regulated by cross-talk between Ca2+ and cAMP signalling pathways. These isozymes are also further distinguished by various pharmacological agents. We have demonstrated a potentially novel regulation of PDE1 by calpain. This study suggests that limited proteolysis by calpain could be an alternative mechanism for the activation of PDE1. We have also shown PDE1 activity, expression and effect of calpain in the rat model in vitro of cardiac ischemia-reperfusion.

Aptamer-hybrid nanoparticle bioconjugate efficiently delivers miRNA-29b to non-small-cell lung cancer cells and inhibits growth by downregulating essential oncoproteins.

MicroRNAs (miRNAs) are potentially attractive candidates for cancer therapy. However, their therapeutic application is limited by lack of availability of an efficient delivery system to stably deliver these potent molecules intracellularly to cancer cells while avoiding healthy cells. We developed a novel aptamer-hybrid nanoparticle bioconjugate delivery system to selectively deliver miRNA-29b to MUC1-expressing cancer cells. Significant downregulation of oncoproteins DNMT3b and MCL1 was demonstrated by these MUC1 aptamer-functionalized hybrid nanoparticles in A549 cells. Furthermore, downregulation of these oncoproteins led to antiproliferative effect and induction of apoptosis in a superior version when compared with Lipofectamine 2000. This novel aptamer-hybrid nanoparticle bioconjugate delivery system could potentially serve as a platform for intracellular delivery of miRNAs to cancer cells, hence improving the therapeutic outcome of lung cancer.

Molecular cloning, expression, purification and characterization of calcineurin from bovine cardiac muscle.

Calcineurin (CaN), also known as calmodulin-dependent phosphatase, was cloned from bovine cardiac muscle and the deduced amino acid sequences of CaN A revealed that it had an open reading frame of 511 amino acid residues. As compared to bovine brain CaN A, the cardiac enzyme contains a 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory region. A deletion analysis of the catalytic domain revealed a 20% decrease in phosphatase activity when the N-terminal 200 amino acids were removed from CaN A as compared to the wild type enzyme. The C-terminal deletions of CaN A revealed that in addition to the autoinhibitory domain (residues 457-480), additional adjacent residues (407-456) also inhibited CaN activity. These results point to either a second autoinhibitory region within CaN A or an extension of the previously noted autoinhibitory region within the cardiac CaN A enzyme.

High expression of methionine aminopeptidase 2 in human colorectal adenocarcinomas.

PURPOSE: Several viral and eukaryotic proteins required for signal transduction and regulatory functions undergo lipophilic modification by the enzyme N-myristoyltransferase. Previously we reported that N-myristoyltransferase activity is higher in colon and gallbladder carcinoma than in the corresponding normal tissues. Methionine aminopeptidase 2 (MetAP2) is a bifunctional protein that plays a critical role in the regulation of post-translational processing and protein synthesis. To investigate whether MetAP2 contributes to the pathogenesis of colon carcinoma, we investigated the expression of MetAP2 in both normal and invasive tumor components of human samples. EXPERIMENTAL DESIGN: We evaluated 50 cases of colon carcinoma for this study. In this report we analyzed 15 cases for MetAP2 activity and 13 cases for the expression of MetAP2 by Western blot in both the normal and in invasive tumor components of human samples. In addition, immunohistochemistry analysis was also carried out on samples from all patients. RESULTS: MetAP activity was elevated in all cancerous tissues compared with normal tissues. Western blot analysis also showed the higher expression of MetAP2 in all cases of cancerous tissues. In addition, immunohistochemistry analysis revealed that all cases of colorectal adenocarcinoma showed moderate to strong cytoplasmic positivity for MetAP2 with increased intensity in the invasive component. CONCLUSIONS: Elevated MetAP protein expression is associated with metastatic tumor progression and appears to be a strong molecular marker for clinical prognosis. MetAP2 inhibition may represent a potential target for therapeutic intervention in colorectal carcinoma.

Overexpression of m-calpain in human colorectal adenocarcinomas.

BACKGROUND: Calpains represent a well-conserved family of Ca2+ -dependent proteolytic enzymes. Recently, the importance of calpain in the metastatic process has received great attention. To investigate whether m-calpain contributes to the pathogenesis of colorectal cancer, we investigated the expression of m-calpain and its inhibitors, calpastatin and high-molecular-weight calmodulin-binding protein (HMWCaMBP), in human colorectal surgical specimens. METHODS: Fifty cases of colon carcinoma were evaluated for this study. Of 50 cases evaluated, we presented in this report six cases for m-calpain, calpastatin and HMWCaMBP protein expression by Western blot analyses was done in both normal and invasive tumor components of human samples. In addition, immunohistochemistry analysis was also carried out in all patients. RESULTS: The activity and protein expression of m-calpain was significantly higher in colorectal adenocarcinoma than in normal colonic mucosa. This finding was corroborated by immunohistochemical studies that showed strong cytoplasmic staining in the colon tumors with m-calpain antibody. The decreased expression of these calpain inhibitors (calpastatin and HMWCaMBP) paralleled increased activity and expression of calpain in colorectal adenocarcinoma and the well-documented involvement of this Ca2+ -dependent protease in colon tumor. CONCLUSION: Increased activity and moderate staining of m-calpain in polyps show the usage of this enzyme as a marker for the early detection of colorectal adenocarcinoma using immunologic approaches. These findings represent the first description of calpain overexpression in colorectal cancer. This has implications with regard to the design of chemotherapeutic drugs as well as in monitoring colorectal cancer in early stages of the metastatic process.

Epigenetic silencing of Na,K-ATPase ß 1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma.

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na(+) and uptake of K(+) across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-ß 1 (NaK-ß) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-ß in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-ß gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients' tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-ß. Furthermore, treatment with 5-Aza-2'-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-ß protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-ß expression, which might contribute to RCC initiation and/or disease progression.