Protein kinase D1 attenuates tumorigenesis in colon cancer by modulating ß-catenin/T cell factor activity.

Over 80% of colon cancer development and progression is a result of the dysregulation of ß-catenin signaling pathway. Herein, for the first time, we demonstrate that a serine-threonine kinase, Protein Kinase D1 (PKD1), modulates the functions of ß-catenin to suppress colon cancer growth. Analysis of normal and colon cancer tissues reveals downregulation of PKD1 expression in advanced stages of colon cancer and its co-localization with ß-catenin in the colon crypts. This PKD1 downregulation corresponds with the aberrant expression and nuclear localization of ß-catenin. In-vitro investigation of the PKD1-ß-catenin interaction in colon cancer cells reveal that PKD1 overexpression suppresses cell proliferation and clonogenic potential and enhances cell-cell aggregation. We demonstrate that PKD1 directly interacts with ß-catenin and attenuates ß-catenin transcriptional activity by decreasing nuclear ß-catenin levels. Additionally, we show that inhibition of nuclear ß-catenin transcriptional activity is predominantly influenced by nucleus targeted PKD1. This subcellular modulation of ß-catenin results in enhanced membrane localization of ß-catenin and thereby increases cell-cell adhesion. Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia. Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of ß-catenin functions in cells.

Anti-cancer activity of curcumin loaded nanoparticles in prostate cancer.

Prostate cancer is the most commonly diagnosed cancer disease in men in the Unites States and its management remains a challenge in everyday oncology practice. Thus, advanced therapeutic strategies are required to treat prostate cancer patients. Curcumin (CUR) is a promising anticancer agent for various cancer types. The objective of this study was to evaluate therapeutic potential of novel poly(lactic-co-glycolic acid)- CUR nanoparticles (PLGA-CUR NPs) for prostate cancer treatment. Our results indicate that PLGA-CUR NPs efficiently internalize in prostate cancer cells and release biologically active CUR in cytosolic compartment of cells for effective therapeutic activity. Cell proliferation (MTS), clonogenic, and Western blot analyses reveal that PLGA-CUR NPs can effectively inhibit proliferation and colony formation ability of prostate cancer cells than free CUR. PLGA-CUR NPs showed superior tumor regression compared to CUR in xenograft mice. Further investigations reveal that PLGA-CUR NPs inhibit nuclear ß-catenin and AR expression in cells and in tumor xenograft tissues. It also suppresses STAT3 and AKT phosphorylation and leads to apoptosis via inhibition of key anti-apoptotic proteins, Mcl-1, Bcl-xL and caused induction of PARP cleavage. Additionally, significant downregulation of oncogenic miR21 and up-regulation of miR-205 was observed with PLGA-CUR NPs treatment as determined by RT-PCR and in situ hybridization analyses. A superior anti-cancer potential was attained with PSMA antibody conjugated PLGA-CUR NPs in prostate cancer cells and a significant tumor targeting of (131)I labeled PSMA antibody was achieved with PLGA-CUR NPs in prostate cancer xenograft mice model. In conclusion, PLGA-CUR NPs can significantly accumulate and exhibit superior anticancer activity in prostate cancer.

Anti-cancer potential of a novel SERM ormeloxifene.

Ormeloxifene is a non-steroidal Selective Estrogen Receptor Modulator (SERM) that is used as an oral contraceptive. Recent studies have shown its potent anti-cancer activities in breast, head and neck, and chronic myeloid leukemia cells. Several in vivo and clinical studies have reported that ormeloxifene possesses an excellent therapeutic index and has been well-tolerated, without any haematological, biochemical or histopathological toxicity, even with chronic administration. A reasonably long period of time and an enormous financial commitment are required to develop a lead compound into a clinically approved anti-cancer drug. For these reasons and to circumvent these obstacles, ormeloxifene is a promising candidate on a fast track for the development or repurposing established drugs as anti-cancer agents for cancer treatment. The current review summarizes recent findings on ormeloxifene as an anti-cancer agent and future prospects of this clinically safe pharmacophore.

Novel curcumin-loaded magnetic nanoparticles for pancreatic cancer treatment.

Curcumin (CUR), a naturally occurring polyphenol derived from the root of Curcuma longa, has showed potent anticancer and cancer prevention activity in a variety of cancers. However, the clinical translation of CUR has been significantly hampered due to its extensive degradation, suboptimal pharmacokinetics, and poor bioavailability. To address these clinically relevant issues, we have developed a novel CUR-loaded magnetic nanoparticle (MNP-CUR) formulation. Herein, we have evaluated the in vitro and in vivo therapeutic efficacy of this novel MNP-CUR formulation in pancreatic cancer. Human pancreatic cancer cells (HPAF-II and Panc-1) exhibited efficient internalization of the MNP-CUR formulation in a dose-dependent manner. As a result, the MNP-CUR formulation effectively inhibited growth of HPAF-II and Panc-1 cells in cell proliferation and colony formation assays. The MNP-CUR formulation suppressed pancreatic tumor growth in an HPAF-II xenograft mouse model and improved the survival of mice by delaying tumor growth. The growth-inhibitory effect of MNP-CUR formulation correlated with the suppression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-extra large (Bcl-xL), induced myeloid leukemia cell differentiation protein (Mcl-1), cell surface-associated Mucin 1 (MUC1), collagen I, and enhanced membrane ß-catenin expression. MNP-CUR formulation did not show any sign of hemotoxicity and was stable after incubation with human serum proteins. In addition, the MNP-CUR formulation improved serum bioavailability of CUR in mice up to 2.5-fold as compared with free CUR. Biodistribution studies show that a significant amount of MNP-CUR formulation was able to reach the pancreatic xenograft tumor(s), which suggests its clinical translational potential. In conclusion, this study suggests that our novel MNP-CUR formulation can be valuable for the treatment of pancreatic cancer.

Increased expression and aberrant localization of mucin 13 in metastatic colon cancer.

MUC13 is a newly identified transmembrane mucin. Although MUC13 is known to be overexpressed in ovarian and gastric cancers, limited information is available regarding the expression of MUC13 in metastatic colon cancer. Herein, we investigated the expression profile of MUC13 in colon cancer using a novel anti-MUC13 monoclonal antibody (MAb, clone ppz0020) by immunohistochemical (IHC) analysis. A cohort of colon cancer samples and tissue microarrays containing adjacent normal, non-metastatic colon cancer, metastatic colon cancer, and liver metastasis tissues was used in this study to investigate the expression pattern of MUC13. IHC analysis revealed significantly higher (p<0.001) MUC13 expression in non-metastatic colon cancer samples compared with faint or very low expression in adjacent normal tissues. Interestingly, metastatic colon cancer and liver metastasis tissue samples demonstrated significantly (p<0.05) higher cytoplasmic and nuclear MUC13 expression compared with non-metastatic colon cancer and adjacent normal colon samples. Moreover, cytoplasmic and nuclear MUC13 expression correlated with larger and poorly differentiated tumors. Four of six tested colon cancer cell lines also expressed MUC13 at RNA and protein levels. These studies demonstrate a significant increase in MUC13 expression in metastatic colon cancer and suggest a correlation between aberrant MUC13 localization (cytoplasmic and nuclear expression) and metastatic colon cancer.

Cellular requirements for LARK in the Drosophila circadian system.

RNA-binding proteins mediate posttranscriptional functions in the circadian systems of multiple species. A conserved RNA recognition motif (RRM) protein encoded by the lark gene is postulated to serve circadian output and molecular oscillator functions in Drosophila and mammals, respectively. In no species, however, has LARK been eliminated, in vivo, to determine the consequences for circadian timing. The present study utilized RNA interference (RNAi) techniques in Drosophila to decrease LARK levels in clock neurons and other cell types in order to evaluate the circadian functions of the protein. Knockdown of LARK in timeless (TIM)- or pigment dispersing factor (PDF)-containing clock cells caused a significant number of flies to exhibit arrhythmic locomotor activity, demonstrating a requirement for the protein in pacemaker cells. There was no obvious effect on PER protein cycling in lark interference (RNAi) flies, but a knockdown within the PDF neurons was associated with increased PDF immunoreactivity at the dorsal termini of the small ventral lateral neuronal (s-LNv) projections, suggesting an effect on neuropeptide release. The expression of lark RNAi in multiple neurosecretory cell populations demonstrated that LARK is required within pacemaker and nonpacemaker cells for the manifestation of normal locomotor activity rhythms. Interestingly, decreased LARK function in the prothoracic gland (PG), a peripheral organ containing a clock required for the circadian control of eclosion, was associated with weak population eclosion rhythms or arrhythmicity.

Curcumin attenuates ß-catenin signaling in prostate cancer cells through activation of protein kinase D1.

Prostate cancer is the most commonly diagnosed cancer affecting 1 in 6 males in the US. Understanding the molecular basis of prostate cancer progression can serve as a tool for early diagnosis and development of novel treatment strategies for this disease. Protein Kinase D1 (PKD1) is a multifunctional kinase that is highly expressed in normal prostate. The decreased expression of PKD1 has been associated with the progression of prostate cancer. Therefore, synthetic or natural products that regulate this signaling pathway can serve as novel therapeutic modalities for prostate cancer prevention and treatment. Curcumin, the active ingredient of turmeric, has shown anti-cancer properties via modulation of a number of different molecular pathways. Herein, we have demonstrated that curcumin activates PKD1, resulting in changes in ß-catenin signaling by inhibiting nuclear ß-catenin transcription activity and enhancing the levels of membrane ß-catenin in prostate cancer cells. Modulation of these cellular events by curcumin correlated with decreased cell proliferation, colony formation and cell motility and enhanced cell-cell aggregation in prostate cancer cells. In addition, we have also revealed that inhibition of cell motility by curcumin is mediated by decreasing the levels of active cofilin, a downstream target of PKD1. The potent anti-cancer effects of curcumin in vitro were also reflected in a prostate cancer xenograft mouse model. The in vivo inhibition of tumor growth also correlated with enhanced membrane localization of ß-catenin. Overall, our findings herein have revealed a novel molecular mechanism of curcumin action via the activation of PKD1 in prostate cancer cells.

Emerging roles of protein kinase D1 in cancer.

Protein kinase D1 (PKD1) is a serine-threonine kinase that regulates various functions within the cell, including cell proliferation, apoptosis, adhesion, and cell motility. In normal cells, this protein plays key roles in multiple signaling pathways by relaying information from the extracellular environment and/or upstream kinases and converting them into a regulated intracellular response. The aberrant expression of PKD1 is associated with enhanced cancer phenotypes, such as deregulated cell proliferation, survival, motility, and epithelial mesenchymal transition. In this review, we summarize the structural and functional aspects of PKD1 and highlight the pathobiological roles of this kinase in cancer.

Curcumin induces chemo/radio-sensitization in ovarian cancer cells and curcumin nanoparticles inhibit ovarian cancer cell growth.

BACKGROUND: Chemo/radio-resistance is a major obstacle in treating advanced ovarian cancer. The efficacy of current treatments may be improved by increasing the sensitivity of cancer cells to chemo/radiation therapies. Curcumin is a naturally occurring compound with anti-cancer activity in multiple cancers; however, its chemo/radio-sensitizing potential is not well studied in ovarian cancer. Herein, we demonstrate the effectiveness of a curcumin pre-treatment strategy for chemo/radio-sensitizing cisplatin resistant ovarian cancer cells. To improve the efficacy and specificity of curcumin induced chemo/radio sensitization, we developed a curcumin nanoparticle formulation conjugated with a monoclonal antibody specific for cancer cells. METHODS: Cisplatin resistant A2780CP ovarian cancer cells were pre-treated with curcumin followed by exposure to cisplatin or radiation and the effect on cell growth was determined by MTS and colony formation assays. The effect of curcumin pre-treatment on the expression of apoptosis related proteins and beta-catenin was determined by Western blotting or Flow Cytometry. A luciferase reporter assay was used to determine the effect of curcumin on beta-catenin transcription activity. The poly(lactic acid-co-glycolic acid) (PLGA) nanoparticle formulation of curcumin (Nano-CUR) was developed by a modified nano-precipitation method and physico-chemical characterization was performed by transmission electron microscopy and dynamic light scattering methods. RESULTS: Curcumin pre-treatment considerably reduced the dose of cisplatin and radiation required to inhibit the growth of cisplatin resistant ovarian cancer cells. During the 6 hr pre-treatment, curcumin down regulated the expression of Bcl-XL and Mcl-1 pro-survival proteins. Curcumin pre-treatment followed by exposure to low doses of cisplatin increased apoptosis as indicated by annexin V staining and cleavage of caspase 9 and PARP. Additionally, curcumin pre-treatment lowered beta-catenin expression and transcriptional activity. Nano-CUR was successfully generated and physico-chemical characterization of Nano-CUR indicated an average particle size of ~70 nm, steady and prolonged release of curcumin, antibody conjugation capability and effective inhibition of ovarian cancer cell growth. CONCLUSION: Curcumin pre-treatment enhances chemo/radio-sensitization in A2780CP ovarian cancer cells through multiple molecular mechanisms. Therefore, curcumin pre-treatment may effectively improve ovarian cancer therapeutics. A targeted PLGA nanoparticle formulation of curcumin is feasible and may improve the in vivo therapeutic efficacy of curcumin.

The Drosophila FMRP and LARK RNA-binding proteins function together to regulate eye development and circadian behavior.

Fragile X syndrome (FXS) is the most common form of hereditary mental retardation. FXS patients have a deficit for the fragile X mental retardation protein (FMRP) that results in abnormal neuronal dendritic spine morphology and behavioral phenotypes, including sleep abnormalities. In a Drosophila model of FXS, flies lacking the dfmr1 protein (dFMRP) have abnormal circadian rhythms apparently as a result of altered clock output. In this study, we present biochemical and genetic evidence that dFMRP interacts with a known clock output component, the LARK RNA-binding protein. Our studies demonstrate physical interactions between dFMRP and LARK, that the two proteins are present in a complex in vivo, and that LARK promotes the stability of dFMRP. Furthermore, we show genetic interactions between the corresponding genes indicating that dFMRP and LARK function together to regulate eye development and circadian behavior.

Genetic and biochemical strategies for identifying Drosophila genes that function in circadian control.

Explicit biochemical models have been elaborated for the circadian oscillators of cyanobacterial, fungal, insect, and mammalian species. In contrast, much remains to be learned about how such circadian oscillators regulate rhythmic physiological processes. This article summarizes contemporary genetic and biochemical strategies that are useful for identifying gene products that have a role in circadian control.

Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover.

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (alpha, beta, and gamma), and also their two-domain combinations, viz. alphabeta and betagamma in Escherichia coli. After purification, alpha and beta showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs alphabeta and betagamma showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK.HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK.HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels.

Involvement of a nine-residue loop of streptokinase in the generation of macromolecular substrate specificity by the activator complex through interaction with substrate kringle domains.

The selective deletion of a discrete surface-exposed epitope (residues 254-262; 250-loop) in the beta domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SK(del254-262)). A kinetic analysis of SK(del254-262) revealed that its low HPG activator activity arose from a 5-6-fold increase in K(m) for HPG as substrate, with little alteration in k(cat) rates. This increase in the K(m) for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SK(del254-262). The involvement of kringles was further indicated by a hypersusceptibility of the SK(del254-262).plasmin activator complex to epsilon-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK.plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SK(del254-262).HPG was reduced by about 3-fold in comparison with that of nSK.HPG . Overall, these observations identify the 250 loop in the beta domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK.HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process.