dTCApFs, a derivative of a novel human hormone peptide, induces apoptosis in cancer cells through a mechanism involving loss of Golgi function.

dTCApFs (Nerofe™) is a 14-amino acid derivative of a longer hormone peptide, tumor-cells apoptosis factor (TCApF), which enters the cells through the T1/ST2 receptor. In the present study, the mechanism of action (MOA) of dTCApFs as an anticancer agent was investigated. Experiments were performed in pancreatic cancer cell lines, and immunofluorescent staining demonstrated that dTCApFs is located in the Golgi apparatus of treated cells. It was also demonstrated in pancreatic, breast and ovarian cell lines that dTCApFs treatment led to Golgi structural changes, loss of Golgi function, and molecular effects associated with endoplasmic reticulum (ER) stress, such as increased levels of C/EBP homologous protein, binding immunoglobulin protein (BiP), phosphorylated inositol-requiring enzyme 1 (pIRE1), and increased phosphorylation of eukaryotic translation initiation factor 2α, and to the generation of reactive oxygen species, which was attenuated by ER stress inhibitors. Moreover, in these cell lines, long-term exposure to dTCApFs led to downregulation of spliced X-box-binding protein 1, which is an ER stress repair mechanism gene, downregulation of the Golgi anti-apoptotic protein, and reduced cell viability. In vivo studies using murine xenograft models of human pancreatic cancer verified the cell culture findings by demonstrating structural changes in the ER/Golgi and increased levels of pIRE1and BiP in dTCApFs-treated mice vs. the controls. Finally, human tissue samples from a patient who received dTCApFs for 11 months in a clinical trial were analyzed, and an increase was observed in the number of cells expressing pIRE1 and BiP post-treatment. In conclusion, we herein report a novel MOA for an anticancer agent involving triggering of apoptosis through induction of opposite effects: ER stress and downregulation of the ER stress repair mechanism. These findings provide the framework for the clinical evaluation of dTCApFs.

NEROFE--a novel human hormone-peptide with anti-cancer activity.

We report the isolation of a novel Tumor-Cells Apoptosis Factor (Nerofe). We found that cDNA of this protein is expressed mainly in the human thymus and partially in the colon and in the frontal lobe of brain. Immunohistochemical studies localize Tumor-Cells Apoptosis Factor (TCApF) to the medulla and Hassal's corpuscles of the thymus gland, which are responsible for negative selection. Treatment of mice with induced AML terminates the cancer development and completely eliminates metastatic cell colonies from the bone marrow and the spleen that reduces probability of the cancer return. We find that TCApF binds to the T1/ST2 receptor and activates caspases 8, 9 and 3 mediated apoptosis, together with activation of JNKinase and p38 MAPKinase. Application of TCApF to cells induced apoptosis in acute myeloid leukemia proliferating cells (U937 premeyloid cells), in human breast carcinoma (MCF7), human glioblastoma, human neuroblastoma, human prostate cancer and human lung cancer proliferating cells. In contrast, TCApF was unable to induce apoptosis in non-proliferating cells. The selectivity of TCApF-induced apoptosis is related to the level of T1/ST2 receptor expression. This is the first report linking the T1/ST2 receptor to apoptosis.

Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions.

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two approximately 2.6 kb DNAs or a monopartite genome of approximately 3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3-20 of TYLCV CP (CP-NLS) or aa 84-106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA-NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin alpha, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin alpha.

AUG sequences are required to sustain nonsense-codon-mediated suppression of splicing.

More than 90% of human genes are rich in intronic latent 5' splice sites whose utilization in pre-mRNA splicing would introduce in-frame stop codons into the resultant mRNAs. We have therefore hypothesized that suppression of splicing (SOS) at latent 5' splice sites regulates alternative 5' splice site selection in a way that prevents the production of toxic nonsense mRNAs and verified this idea by showing that the removal of such in-frame stop codons is sufficient to activate latent splicing. Splicing control by SOS requires recognition of the mRNA reading frame, presumably recognizing the start codon sequence. Here we show that AUG sequences are indeed essential for SOS. Although protein translation does not seem to be required for SOS, the first AUG is shown here to be necessary but not sufficient. We further show that latent splicing can be elicited upon treatment with pactamycin-a drug known to block translation by its ability to recognize an RNA fold-but not by treatment with other drugs that inhibit translation through other mechanisms. The effect of pactamycin on SOS is dependent neither on steady-state translation nor on the pioneer round of translation. This effect is found for both transfected and endogenous genes, indicating that SOS is a natural mechanism.