Enhanced Antiproliferative and Apoptotic Response of HT-29 Adenocarcinoma Cells to Combination of Photoactivated Hypericin and Farnesyltransferase Inhibitor Manumycin A

Several photodynamically-active substances and farnesyltransferase inhibitors are currently being investigated as promising anticancer drugs. In this study, the combined effect of hypericin (the photodynamically-active pigment from Hypericum perforatum) and selective farnesyltransferase inhibitor manumycin (manumycin A; the selective farnesyltransferase inhibitor from Streptomyces parvulus) on HT-29 adenocarcinoma cells was examined. We found that the combination treatment of cells with photoactivated hypericin and manumycin resulted in enhanced antiproliferative and apoptotic response compared to the effect of single treatments. This was associated with increased suppression of clonogenic growth, S phase cell cycle arrest, elevated caspase-3/7 activity and time-dependent total cleavage of procaspase-3 and lamin B, cleavage of p21Bax into p18Bax and massive PARP cleavage. Moreover, we found that the apoptosis-inducing factor is implicated in signaling events triggered by photoactivated hypericin. Our results showed the relocalization of apoptosis-inducing factor (AIF) to the nuclei after hypericin treatment. In addition, we discovered that not only manumycin but also photoactivated hypericin induced the reduction of total Ras protein level. Manumycin decreased the amount of farnesylated Ras, and the combination treatment decreased the amount of both farnesylated and non-farnesylated Ras protein more dramatically. The present findings indicate that the inhibition of Ras processing may be the determining factor for enhancing the antiproliferative and apoptotic effects of combination treatment on HT-29 cells

Se-methylselenocysteine inhibits phosphatidylinositol 3-kinase activity of mouse mammary epithelial tumor cells in vitro

INTRODUCTION: Se-methylselenocysteine (MSC), a naturally occurring selenium compound, is a promising chemopreventive agent against in vivo and in vitro models of carcinogen-induced mouse and rat mammary tumorigenesis. We have demonstrated previously that MSC induces apoptosis after a cell growth arrest in S phase in a mouse mammary epithelial tumor cell model (TM6 cells) in vitro. The present study was designed to examine the involvement of the phosphatidylinositol 3-kinase (PI3-K) pathway in TM6 tumor model in vitro after treatment with MSC. METHODS: Synchronized TM6 cells treated with MSC and collected at different time points were examined for PI3-K activity and Akt phosphorylation along with phosphorylations of Raf, MAP kinase/ERK kinase (MEK), extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The growth inhibition was determined with a [(3)H]thymidine incorporation assay. Immunoblotting and a kinase assay were used to examine the molecules of the survival pathway. RESULTS: PI3-K activity was inhibited by MSC followed by dephosphorylation of Akt. The phosphorylation of p38 MAPK was also downregulated after these cells were treated with MSC. In parallel experiments MSC inhibited the Raf–MEK–ERK signaling pathway. CONCLUSION: These studies suggest that MSC blocks multiple signaling pathways in mouse mammary tumor cells. MSC inhibits cell growth by inhibiting the activity of PI3-K and its downstream effector molecules in mouse mammary tumor cells in vitro

Gradient Descent Optimization in Gene Regulatory Pathways

BACKGROUND: Gene Regulatory Networks (GRNs) have become a major focus of interest in recent years. Elucidating the architecture and dynamics of large scale gene regulatory networks is an important goal in systems biology. The knowledge of the gene regulatory networks further gives insights about gene regulatory pathways. This information leads to many potential applications in medicine and molecular biology, examples of which are identification of metabolic pathways, complex genetic diseases, drug discovery and toxicology analysis. High-throughput technologies allow studying various aspects of gene regulatory networks on a genome-wide scale and we will discuss recent advances as well as limitations and future challenges for gene network modeling. Novel approaches are needed to both infer the causal genes and generate hypothesis on the underlying regulatory mechanisms. METHODOLOGY: In the present article, we introduce a new method for identifying a set of optimal gene regulatory pathways by using structural equations as a tool for modeling gene regulatory networks. The method, first of all, generates data on reaction flows in a pathway. A set of constraints is formulated incorporating weighting coefficients. Finally the gene regulatory pathways are obtained through optimization of an objective function with respect to these weighting coefficients. The effectiveness of the present method is successfully tested on ten gene regulatory networks existing in the literature. A comparative study with the existing extreme pathway analysis also forms a part of this investigation. The results compare favorably with earlier experimental results. The validated pathways point to a combination of previously documented and novel findings. CONCLUSIONS: We show that our method can correctly identify the causal genes and effectively output experimentally verified pathways. The present method has been successful in deriving the optimal regulatory pathways for all the regulatory networks considered. The biological significance and applicability of the optimal pathways has also been discussed. Finally the usefulness of the present method on genetic engineering is depicted with an example

Involvement of FKHR-Dependent TRADD Expression in Chemotherapeutic Drug-Induced Apoptosis

Chemotherapeutic drugs exhibit their cytotoxic effect by inducing apoptosis in tumor cells. Because the serine/threonine kinase Akt is involved in apoptosis suppression, we investigated the relationship between Akt activity and drug sensitivity. We discovered that certain chemotherapeutic drugs induced apoptosis with caspase activation only when Akt was inactivated after drug treatment, while inactivation of Akt was not observed when tumor cells showed resistance to the drug-induced caspase activation. So, turn-off of the Akt-mediated survival signal is correlated with the sensitivity of the cells to chemotherapy. With a cDNA microarray, we revealed that tumor necrosis factor receptor-associated death domain (tradd) gene expression was elevated in response to Akt inactivation. Reportedly, Forkhead family transcription factors are phosphorylated by Akt, which results in their nuclear exit and inactivation. Analysis of the tradd promoter revealed that it contains at least one potential Forkhead family transcription factor-responsive element, and we confirmed that this element was involved in chemotherapeutic drug-induced TRADD expression. Overexpression of mutant TRADD proteins to block its apoptosis-inducing capability attenuated chemotherapeutic drug-induced apoptosis. Thus, chemotherapeutic drugs exhibited their cytotoxic effects in part by down-regulating Akt signaling following TRADD expression. These results indicate that Akt kinase activity after drug treatment is a hallmark of sensitivity of the cells to chemotherapeutic drugs