C. elegans model of ALS. In this kinome-wide survey, we identified homologs of the tau tubulin kinases 1 and 2 (TTBK1 and TTBK2), which were also identified in a prior screen for kinase modifiers of TDP-43 behavioral phenotypes. Using refined methodology, we demonstrate TTBK1 and TTBK2 directly phosphorylate TDP-43 in vitro and promote TDP-43 phosphorylation in mammalian cultured cells. TTBK1/2 overexpression drives phosphorylation and relocalization of TDP-43 from the nucleus to cytoplasmic inclusions reminiscent of neuropathologic changes in disease states. Furthermore, protein levels of TTBK1 and TTBK2 are increased in frontal cortex of FTLD-TDP patients, and TTBK1 and TTBK2 co-localize with TDP-43 inclusions in ALS spinal cord. These kinases may represent attractive targets for therapeutic intervention for TDP-43 proteinopathies such as ALS and FTLD-TDP.Pathological aggregates of phosphorylated TDP-43 characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), two devastating groups of neurodegenerative disease. Kinase hyperactivity may be a consistent feature of ALS and FTLD-TDP, as phosphorylated TDP-43 is not observed in the absence of neurodegeneration. By examining changes in TDP-43 phosphorylation state, we have identified kinases controlling TDP-43 phosphorylation in a  in vitro, and control TDP-43 phosphorylation in cellular and simple animal models of ALS. This has uncovered a molecular mechanism by which pathological phosphorylated TDP-43 can occur in disease. To determine whether changes in TTBK1/2 protein are contributing to TDP-43 pathology, we examined diseased brain and spinal cord tissue from patients with ALS or FTLD-TDP. We observed changes in the abundance of TTBK1 and TTBK2 in disease-affected neurons, and the coexistence of TTBK1/2 with phosphorylated TDP-43 aggregates in both FTLD-TDP and ALS. Therefore, increased abundance or activity of TTBK1 or TTBK2 may contribute to the neurodegeneration observed in ALS and FTLD-TDP.Aggregated proteins are a hallmark of many neurodegenerative diseases. In ALS and FTLD-TDP, these aggregates contain abnormal TDP-43 modified by phosphorylation. Protein phosphorylation normally controls protein activity, stability, or location, but in some neurodegenerative diseases the phosphorylated proteins accumulate in excess. Kinases are the enzymes responsible for protein phosphorylation. We have identified two TDP-43 kinases, TTBK1 and TTBK2, using a novel approach combining reverse genetics and biochemical screening to identify the kinases responsible for changes in TDP-43 phosphorylation. We show TTBK1 and TTBK2 directly phosphorylate TDP-43  TARDBP, were found to cause ALS in a subset of affected families, supporting a causal role for TDP-43 in disease initiation C. elegans, Drosophila, zebrafish, mice, and rats have demonstrated neurotoxicity resulting from mutant TDP-43 Ubiquitinated, hyperphosphorylated inclusions of the protein TDP-43 characterize disease-affected neurons in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD-TDP) TDP-43 undergoes a number of pathological modifications in disease-affected neurons including ubiquitination, phosphorylation, and proteolytic processing. These modifications may promote aggregation and the formation of detergent-insoluble inclusions. The precise molecular cause underlying neurotoxicity in most TDP-43 proteinopathies remains unclear, although the toxicity of mutant TDP-43 expressed in multiple model systems indicates it may be acting through a gain-of-function mechanism via aberrant interactions with proteins and/or nucleic acids C. elegans model of TDP-43 proteinopathy exhibiting TDP-43 phosphorylation dependent neurodegeneration and neurotoxicity; in C. elegans, phosphorylation of TDP-43 at serines 409 and 410 suffices to promote TDP-43 mediated neurotoxicity C. elegans, because detectable phosphorylated TDP-43 remains in the absence of CDC7. Inhibition of the kinases CDC7 or CK1 has also been shown to reduce but not eliminate TDP-43 phosphorylation in cultured cells C. elegans. We have identified homologs of the tau tubulin kinases TTBK1 and TTBK2 and characterized their function as regulators of TDP-43 phosphorylation. TTBK1/2 may be attractive drug targets for therapeutic interventions in TDP-43 proteinopathies such as FTLD-TDP and ALS.In order to investigate the causes driving pathological TDP-43 phosphorylation, we have developed a C. elegans. We have assembled an RNAi library targeting 451 predicted kinase genes in C. elegans . This library has been previously employed to identify kinase modifiers of TDP-43 dependent behavioral phenotypes, and identified CDC7 as a direct TDP-43 kinase responsible for promoting TDP-43 neurotoxicity C. elegans model as detectable phosphorylation at S409/410 is still observed in a cdc-7(−/−) null mutant background. Thus other kinases play conserved roles phosphorylating TDP-43, and previous behavior-based screening may have failed to uncover kinases with multiple roles in vivo, or kinases whose loss of function could adversely impact motor function or viability independent of TDP-43. To identify additional TDP-43 kinases, a direct biochemical assay of TDP-43 phosphorylation in TDP-43 transgenic C. elegans was used to screen for alterations in pS409/410 TDP-43 phosphorylation. Populations of transgenic C. elegans expressing ALS-mutant M337V TDP-43 were grown on bacteria producing double stranded RNA targeting each kinase, then harvested and tested by immunoblot for changes in TDP-43 phosphorylation  family members C. elegans suggests a diversification of functional roles for the TTBK1/2 like kinases in the nematode.To identify TDP-43 kinases, we undertook a comprehensive survey utilizing kinase-targeting RNAi coupled with direct immunoblot detection of changes in TDP-43 phosphorylation in rylation . Transgecdc-7(−/−), H05L14.1(−/−), and dkf-2(−/−), dramatically reduce TDP-43 phosphorylation with only moderate or no changes in total levels of TDP-43, consistent with the results from the initial RNAi screen (dkf-2(−/−) exhibited significant differences in total TDP-43 levels. cdc-7(−/−) has been previously characterized as a TDP-43 kinase dkf-2(−/−).RNAi can inactivate multiple genes simultaneously depending on their sequence similarity, potentially confounding the identification of any single gene responsible for TDP-43 phosphorylation. To unambiguously determine the effects of single kinase gene loss of function on TDP-43 phosphorylation, we generated TDP-43 transgenic animals with viable deletion mutants eliminating the kinase active domain of each candidate gene of interest . Each ofi screen . We obsei screen , but thecdc-7(−/−);TDP-43, H05L14.1(−/−);TDP-43 or dkf-2(−/−);TDP-43 had a more natural and vigorous movement profile relative to the TDP-43 transgene alone. We assessed motor function by measuring the average dispersal velocity of the animals, and found significant improvements compared to TDP-43  C. elegans and other ecdysozoa. The H05L14.1 kinase domain has 40% sequence identity to the highly homologous tau tubulin kinases TTBK1 and TTBK2 at the amino acid level   . PRKD2/3immunity  [29], [3activity . Our prein vitro  to decrease levels of TTBK1 gene expression in mammalian cultured cells. We have modeled pathological TDP-43 phosphorylation in the mouse motor neuron-like NSC-34 cell line using the chemical trigger ethacrynic acid (EA). EA acts by depleting cytosolic and mitochondrial glutathione, resulting in robust TDP-43 phosphorylation Both TTBK1 and TTBK2 are expressed in the brain, although TTBK2 is expressed in other tissues as well Of the six ALS cases examined, only two had phospho-TDP-43 aggregates in the frontal cortex and hippocampus, while all six demonstrated phospho-TDP-43 aggregates within spinal cord. ALS spinal cord motor neurons immunoreactive for phospho-TDP-43 pathology also co-labeled with TTBK1 and TTBK2 . Of the C. elegans demonstrated a causal relationship between neurodegeneration and S409/410 phosphorylation of TDP-43 C. elegans behavior-based screening tool to identify TDP-43 kinases C. elegans kinome by RNAi knockdown for modifiers of TDP-43 phosphorylation. For this survey we employed sensitive and specific S409/410 phosphorylation dependent TDP-43 antibodies C. elegans was conducted by testing deletion mutations within the kinase genes of interest. Three identified candidate kinases, cdc-7, dkf-2, and H05L14.1, reduced TDP-43 S409/410 phosphorylation and improved TDP-43 dependent behavioral phenotypes in C. elegans. TDP-43 is a known substrate of CDC7, as it was previously uncovered in a reverse genetic screen to identify modifiers of TDP-43 behavioral phenotypes dkf-2 and H05L14.1. Interestingly, H05L14.1 was a homolog of the mammalian tau tubulin kinases TTBK1 and TTBK2. Our previous behavior-based screen for TDP-43 kinases identified a different C. elegans homolog of TTBK1/2 as a TDP-43 kinase, although at the time we were unable to demonstrate a direct relationship between human TTBK1/2 and TDP-43 in vitro kinase reaction conditions, we demonstrate here that human TTBK1/2 are able to directly phosphorylate TDP-43. We then overexpressed TTBK1/2 in cultured human cells. TTBK1/2 overexpression in the absence of other stressors promoted robust phosphorylation of endogenous TDP-43. Furthermore, this phosphorylated TDP-43 localized to the cytoplasm in inclusion-like aggregates. We also found that reduction of TTBK1 mRNA levels attenuated TDP-43 phosphorylation in a chemically induced model of pathological phospho-TDP-43 accumulation. Finally, to explore whether TDP-43 kinase hyperactivity could underlie the etiology of TDP-43 proteinopathies, we immunostained tissue from FTLD-TDP and ALS for TTBK1/2. We observe increased TTBK1/2 in FTLD-TDP frontal cortex, and co-localization with TDP-43 positive aggregates in FTLD frontal cortex and ALS spinal cord. One possible explanation for these data is the observed differences in TTBK1/2 expression drives neurodegeneration in TDP-43 proteinopathies. Taken together these data support a pivotal role for TTBK1/2 hyperactivity in TDP-43 proteinopathy.Tandem phosphorylation at TDP-43 serines 409 and 410 (pS409/410) is a consistent and robust feature of TDP-43 pathology in ALS and FTLD-TDP. Our previous work in TDP-43 transgenic in vitro and in vivo. The kinases CK1 C. elegans, mutant TDP-43 still exhibits varying but detectable degrees of phosphorylation  in TTBK1 are associated with decreased Alzheimer's disease risk in studies of Spanish and Han Chinese populations Kinases regulating TDP-43 phosphorylation present an attractive target for therapeutic intervention in both ALS and FTLD-TDP. No specific small molecule inhibitors targeting TTBK1 or TTBK2 has been reported to date, despite their potential roles contributing to tauopathies by hyperphosphorylating tau. Development of brain penetrant TTBK1 and TTBK2 inhibitors may also provide a viable strategy for intervening in TDP-43 proteinopathy disorders including ALS and FTLD-TDP.C. elegans and maintained as previously described C. elegans were performed at room temperature unless otherwise noted. CK423 (TDP-43 M337V) and eri-1(mg366);lin-15b(n744);TDP-43 M337V transgenic strains were generated previously cdc-7(tm4391);TDP-43, CK602 H05L14.1(tm4720);TDP-43, CK600 dkf-2(tm4076);TDP-43, CK597 nsy-1(ok593);TDP-43, CK613 kin-20(ok505);TDP-43, CK574 mlk-1(ok2471);TDP-43, CK623 F39F10.3(tm4396);TDP-43.N2 (Bristol) was used as wild type C. elegans was derived from the C. elegans kinome project eri-1(−/−);lin-15(−/−) RNAi enhancing mutant background st generation gravid adults with 2nd generation L2–L3 animals were harvested by washing with M9 buffer into 96 well plates and frozen at −80°C, for subsequent immunoblot analysis. Each RNAi treated population was evaluated semi-quantitatively for reduction in phospho-TDP-43 relative to control treated animals. Positives candidates were retested for effects on TDP-43 phosphorylation by independent RNAi treatment and immunoblot, and the RNAi gene target for each plasmid was confirmed by sequencing.The list of predicted kinase genes in C. elegans β-tubulin levels were measured using monoclonal antibody E7 as a loading control as previously described Equivalent mixed-stage worm lysate fractions were loaded and resolved on precast 4–15% gradient SDS-PAGE gels and transferred to PVDF membrane as recommended by the manufacturer (Bio-Rad). On immunoblots, human TDP-43 was detected with a commercially available monoclonal antibody ab57105 (Abcam) directed against human TDP-43 amino acids 1–261. TDP-43 phosphorylated at S409/S410 was detected by a monoclonal antibody called anti phospho TDP-43 (pS409/410) available from Cosmobio . GST-TDP-43 (WT) and GST-TDP-43 (M337V) fusion proteins were purified from BL21 (DE3) expression host cells as previously described HEK 293 cells (ATCC) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% defined fetal bovine serum (FBS) and penicillin (50 IU/ml)–streptomycin (50 mg/ml). NSC-34 cells (Cedarlane Labs) and SHSY-5Y cells (ATCC) were cultured in DMEM/HAM's F12 (50/50) with 10% FBS and penicillin (50 IU/ml)–streptomycin (50 mg/ml).2+/Mg2+, then blocked in antibody buffer +10% normal goat serum. Primary antibody was applied and incubated for 1 hour at room temperature . Cells were washed 3×5 min in PBS/Ca2+/Mg2+, then re-blocked for 10 min. Appropriate secondary antibody was applied and incubated for 20 min at room temperature. Cells were again washed 3×5 min in PBS/Ca2+/Mg2+, counterstained with 300 nM DAPI and mounted with ProLong Gold antifade. Microscopy was performed on a Delta Vision microscope  using a 60× oil immersion objective, a sCMOS camera, and 2×2 binning. Image analysis was performed using softWoRx 6.0 Beta software.Cells were seeded onto poly-D-Lysine coated (Sigma Aldrich) 12 mm round glass cover slips in 24-well plates. Cells were transfected with the plasmid encoding TTBK2-GFP with GenePorter 2 (Genlantis) using the manufacturer's protocol. Cells were fixed for imaging in 4% formaldehyde 96 hours after transfection. Cells were washed 3×5 min in PBS/CaHEK 293 cells were treated with 150 µM ethacrynic acid (EA) for 5 hours to induce endogenous TDP-43 phosphorylation Transfection of plasmids containing full-length TTBK1 (pWO:TTBK1) and TTBK2 (TTBK2 GFP pFLAP dest) sequences RNA was purified from flash-frozen cell pellets using TRIzol Reagent (Life Technologies) according to the manufacturer's protocol. cDNA was made using iScript Reverse Transcription Supermix (Bio-Rad). qPCR was performed on an 7900HT Real Time PCR System (Applied Biosystems) using iTaq Universal SYBR Green Supermix (Bio-Rad).De-identified post-mortem brain tissue used in this study was determined to be an exempt from IRB review by the VA Puget Sound Health Care System Human Research Protection Program Director on December 29, 2011. Tissue used for these studies was obtained from the University of Washington Alzheimer's Disease Research Center brain bank , and the Indiana Alzheimer Disease Center brain bank, where consent for autopsy and permission for use of tissue in scientific experiments was obtained. FTLD and ALS cases were selected on the basis of having an autopsy-confirmed diagnosis of FTLD and FTLD-related disorders or ALS. Control samples were from de-identified neurologically healthy control participants, who were of a similar age.Primary antibodies used for immunohistochemistry were anti-TTBK1 , anti-TTBK2 , and anti-phospho TDP-43 409/410 ). In order to minimize variability, sections from all cases  were stained simultaneously for each antibody. Immunostained sections were analyzed using the computerized image analysis system, MicroComputer Imaging Device . Blinded assessment of optical density measurements were obtained relative to the proportional area for TTBK1 and TTBK2 immunostaining in frontal cortex grey matter (three separate readings per case). Data were averaged and are represented as mean +/− SEM. A two tailed Student's t-test was used to assess differences in TTBK1 and TTBK2 expression between cases and controls. For double label immunohistochemistry experiments, sections were first immunostained with anti-phospho TDP-43 and reaction product was visualized with nickel enhanced DAB (black). Sections were then immunostained with anti-TTBK1 or TTBK2 and visualized with DAB alone (brown). For double label immunofluorescence experiments, AlexaFluor 488 goat anti-rabbit and AlexaFluor 594 goat anti-mouse secondary antibodies (Molecular Probes) were used and autofluoresence was quenched with 0.1% Sudan Black S1 FigureC. elegans were harvested into 96-well plates prior to immunoblot analysis. Gene names and locations of kinases tested are presented in Table S1. Two rows from each plate were tested in alternating wells for each immunoblot. Labels above individual wells describe Row and Column information for each sample. Plate numbers are indicated at the left of the immunoblot. α-tubulin antibody is used as a load control. Candidates confirmed on repeat testing are boxed in blue.Immunoblot results from primary kinase RNAi screen. Populations of RNAi treated (PDF)Click here for additional data file.S2 FigureFull immunoblots of total TDP-43 levels. Full immunoblots from (PDF)Click here for additional data file.S3 Figuredkf-2 mammalian homologs identified by BLAST.  Cladogram of vertebrate homologs of the C. elegans proteins H05L14.1 and dkf-2. The entire C. elegans amino acid sequences of H05L14.1 or dkf-2 were compared against non-redundant reference sequences from the RefSeq protein database . (A) 2878 active hits were identified by BLAST with similarity to H05L14.1, and subsequently filtered to include only sequences from C. elegans or phylum chordata. The top 50 hits underwent multiple sequence alignment, alignment refinement, phylogenetic reconstruction, and are displayed in a cladogram, with branch support values in red dkf-2, filtered, and graphed as above. Homo sapiens GI# 5031689 is PRKD3. (C) H05L14.1 kinase domain has 40% identity to human TTBK1 and TTBK2. (D) dkf-2 has more than 70% identity to human PRKD2 and PRKD3. Sequence identity was calculated using Clustal W method for multiple sequence alignment. (E) Alignment report for H05L14.1, TTBK1, and TTBK2 kinase domain, including boxes around sequence that matches the consensus. (F) Alignment report for dkf-2, PRKD2, and PRKD3. Reports were generated using Lasergene MegAlign software for protein sequence analysis and alignment.H05L14.1 and (PDF)Click here for additional data file.S4 FigureIn vitro kinase assay controls. (A) Tau is a known substrate of TTBK2 E. coli were incubated with equivalent amounts of TTBK2 enzyme purified from cultured cells by two commercial suppliers . Phosphorylation was assessed by reactivity with AT270, a phospho-tau antibody recognizing tau phosphorylated at Thr181. (B) Purified TTBK1, TTBK2, and CDC7 can also phosphorylate TDP-43 at serines 403 and 404  in an in vitro kinase assay. (C) Histone H1 is a known substrate of PRKD2 (PDF)Click here for additional data file.S5 Figure(A) Phosphorylated TDP-43 is localized in a large discrete cytoplasmic aggregate following TTBK2 overexpression in HEK293 cells. (B) TTBK2 and phosphorylated TDP-43 co-localize in cells overexpressing TTBK2.(PDF)Click here for additional data file.S6 Figure(A) Treatment of HEK293 cells with a variety of cellular stressors failed to produce phosphorylated TDP-43. Bafilomycin and wortmannin are inhibitors of autophagy, PSI is a general proteasome inhibitor, cadmium chloride is a heavy metal, taxol is an inhibitor of microtubule dynamics, rotenone blocks the mitochondrial electron transport chain (creating reactive oxygen species (ROS)), pepstatin inhibits aspartic proteases, and paraquat catalyzes formation of ROS. (B) TTBK1 is reduced by nearly 80% following siRNA treatment in NSC-34 cells. Quantitative PCR measurements (qPCR) for TTBK1 mRNA levels are displayed in arbitrary units for an untreated control and cells treated with TTBK1 siRNA. (C) siRNA targeting TTBK1 reduce levels of TTBK1 protein, as detected by immunoblot. (D) TTBK1 protein levels are reduced by an average of 46%, following siRNA treatment in NSC-34 cells. Quantitation of TTBK1 protein levels from three independent experiments is graphed in arbitrary units of band intensity.(PDF)Click here for additional data file.S7 FigureAbcam, ab103944). (B) Antibodies tested for TTBK2 were TTBK2 Antibody N-term , Anti-Tau tubulin kinase 1 antibody , and TTBK2 Polyclonal Antibody . Antibodies underlined/bold above were used in further experiments and are boxed in red in the figure. (C–J) Peptide blocking experiments with the cognate immunizing peptide further demonstrates specificity of the selected TTBK1 and TTBK2 antibodies. Anti-TTBK1  (C–F) and anti-TTBK2  (G–J) were pre-incubated with a 50 fold excess of the blocking peptide  before proceeding with the immunostaining protocol and compared with immunostaining using antibody alone . ALS spinal cord  and FTLD frontal cortex . Scale bar = 100 um.Antibody Validation. (A) Studies examining expression of TTBK1 used commercially sourced antibodies including Anti-TTBK1 , Anti TTBK#1 , Anti-TTBK1 ((PDF)Click here for additional data file.S8 FigTTBK1/2 co-localize with phosphorylated TDP-43 in aggregates in ALS spinal cord. Double-label immunofluorescence of ALS spinal cord demonstrates additional neurons that significantly co-localize TTBK1 (upper panel) or TTBK2 (lower panel) with phospho-TDP-43 within neuronal cytoplasmic inclusions. Significance was determined using Pearson coefficient of colocalization.(PDF)Click here for additional data file.S1 TableC. elegans prior to testing by immunoblot . Control RNAi for each plate are highlighted in purple. L4440: empty vector RNAi control. unc-22: positive control for effective RNAi treatments, causing a strong paralyzed phenotype in treated TDP-43 worms. TDP-43: RNAi targeting the TDP-43 transgene is a positive control for suppression of TDP-43 phenotypes. Kinase RNAi treatments that caused C. elegans sterility or growth arrest, causing insufficient sample for protein detection by immunoblot, are highlighted in green. Kinase RNAi treatments that decreased TDP-43 phosphorylation in initial testing are highlighted in blue. Kinase RNAi treatments that reproducibly decreased TDP-43 phosphorylation in multiple independent experiments have names that are bolded and underlined.Kinase genes tested by immunoblot. 96-well plate locations of each RNAi treated population of (XLSX)Click here for additional data file.


Delineate the feasibility of simultaneous, dual selective “targeted” chemotherapeutic delivery and determine if this molecular strategy can promote higher levels anti-neoplastic cytotoxicity than if only one covalent immunochemotherapeutic is selectively “targeted” for delivery at a single membrane associated receptor over-expressed by chemotherapeutic-resistant mammary adenocarcinoma.neu utilizing a rapid multi-phase synthetic organic chemistry reaction scheme. Determination that 96% or greater gemcitabine or epirubicin content was covalently bond to immunoglobulin fractions following size separation by micro-scale column chromatography was established by methanol precipitation analysis. Residual binding-avidity of gemcitabine-(C4-amide)-[anti-EG-FR] applied in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] was determined by cell-ELIZA utilizing chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) populations. Lack of fragmentation or polymerization was validated by SDS-PAGE/immunodetection/chemiluminescent autoradiography. Anti-neoplastic cytotoxic potency was determined by vitality stain analysis of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) monolayers known to uniquely over-express EGFR (2 × 105/cell) and HER2/neu (1 × 106/cell) receptor complexes. The covalent immunochemotherapeutics gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] were applied simultaneously in dual-combination to determine their capacity to collectively evoke elevated levels of anti-neoplastic cytotoxicity. Lastly, the tubulin/microtubule inhibitor mebendazole evaluated to determine if it’s potential to complemented the anti-neoplastic cytotoxic properties of gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu].Gemcitabine and epirubicin were covalently bond to anti-EGFR and anti-HER2/4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] produced greater levels of anti-neoplastic cytotoxicity than either of the covalent immunochemotherapeutics alone. The benzimidazole microtubule/tubulin inhibitor, mebendazole complemented the anti-neoplastic cytotoxicity of gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu].Dual-combination of gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] produced higher levels of selectively “targeted” anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) than either covalent immunochemotherapeutic alone. The benzimidazole tubulin/microtubule inhibitor, mebendazole also possessed anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) and complemented the potency and efficacy of gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu].The dual-combination of gemcitabine-(C The anthracycline class chemotherapeutics intercalate between DNA strands to exert their mechanism-of-action that in turn inhibits DNA and RNA synthesis in addition to triggers topoisomerasae II mediated DNA cleavage resulting in the promotion of cell death. Binding to cell membranes and plasma proteins may also be involved in the cytotoxic properties of the anthracyclines which is complemented by outright injury to neoplastic cells secondary to the generation of highly reactive free radicals species. Each of these mechanisms-of-action collectively promotes apoptosis. In their clinical usage, the anthracycline are among the most potent and clinically effective class of chemotherapeutics for the treatment of mammary carcinoma, ovarian carcinoma, colon carcinoma, and acute myeloid leukemia.Gemcitabine is a deoxycytadine nucleotide analog that intra-cellularly has a mechanism-of-action that involves it being triphosphoralated in a manner that allows it to substitute for cytadine during DNA transcription resulting in incorporation into DNA strands and inhibit the biochemical activity of DNA polymerase. A second mechanism-of-action for gemcitabine involves inhibition and inactivation of ribonucleotide reductase and ultimately the suppression of deoxyribonucleotide synthesis in concert with diminished DNA repair and reduced DNA transcription. Each of these mechanisms-of-action collectively promotes cellular apoptosis. Features of the pharmacokinetic profile for gemcitabine include a brief plasma half-life because it is rapidly deaminated to an inactive metabolite that is rapidly eliminated through renal excretion into the urine–3.In clinical oncology, the anthracycline chemotherapeutics are commonly administered to treat breast cancer and many other neoplastic conditions due to their superior level of potency. Gemcitabine is administered for the treatment certain leukemias and potentially lymphoma conditions in addition to a spectrum of adenocarcinomas and carcinomas affecting the lung , pancreas, urinary bladder and esophagus. Gemcitabine has a brief plasma half-life because it is rapidly deaminated to an inactive metabolite that is rapidly eliminated through renal excretion into the urine–3. Despiin-vivo in “free form” they still pose a high risk frequency for toxic sequelae that can ultimately limit the extent and duration of therapeutic intervention and epirubicin-(C3-amide)-[anti-HER2/neu] were applied simultaneously in a dual-combination to detect their potential to evoke additive or synergistic levels of anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Complementary investigations delineated the potential for benzimidazole tubulin/microtubule inhibitors to complement the anti-neoplastic cytotoxicity of gemcitabine-(C4-amide)-[anti-EGFR] applied in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu]. Investigations ultimately demonstrated how gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] can selectively “target” the delivery of two different chemotherapeutic agents at two different unique or over-expressed receptors over-expressed by neoplastic cell types. The anti-neoplastic cytotoxicity of gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] is complemented by the biological activity of benzimidazole tubulin/microtubule inhibitors.Covalent immunochemotherapeutics that possess properties of selective “targeted” delivery have traditionally been synthesized utilizing the anthracyclines–85. wherrapeutics. Even lerapeutics. Given t4-amine of gemcitabine  or the C3 α-monoamine on the carbohydrate-type moiety of epirubicin was reacted at a 2.5:1 molar-ratio with the amine-reactive N-hydroxysuccinimide ester “leaving” complex of succinimidyl 4,4-azipentanoate  in the presence of triethylamine  utilizing dimethylsulfoxide as an anhydrous organic solvent system  in buffer  were combined at a 1:10 molar-ratio with either the UV-photoactivated gemcitabine-(C4-amide) or epirubicin-(C3-amide) intermediate (Phase-1 end product) and were initially allowed to gently mix by constant stirring for 5 minutes at 25° C in the dark. The photoactivated group of the gemcitabine-(C4-amide) or epirubicin-(C3-amide) reactive intermediates was covalently bonded to chemical groups associated with sides chains of amino acid residues in the sequence of anti-EGFR or anti-HER2/neu monoclonal immunoglobulin fractions during a 15 minute exposure to UV light at 354 nm (reagent activation range 320–370 nm) in combination with constant gentle stirring -[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] preparations respectively entailed initial protein precipitation of the covalent immunochemotherapeutics with methanol:acetonitrile (1:9 v/v) and subsequent measurement of gemcitabine (absorbance: 265–268nm),[3-amide)-[anti-HER2/neu][4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu].Quantification of the amount of covalently bound gemcitabine or epirubicin was performed at previously described for gemcitabine--[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] immunochemotherapeutics was determined by measuring absorbance at 280nm in combinations with utilizing a 235nm -vs- 280nm standardized reference curve in order to accommodate for any potential absorption profile over-lap at 280nm between immunoglobulin and the chemotherapeutic moieties of gemcitabine and epirubicin.Determination of the immunoglobulin concentration for the covalent gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] immunochemotherapeutics in addition to reference control anti-EGFR and anti-HER2/neu immunoglobulin fractions were adjusted to a standardized protein concentration of 60µg/ml and then combined 50/50 v/v with conventional SDS-PAGE sample preparation buffer (Tris/glycerol/bromphenyl blue/SDS) formulated without 2-mercaptoethanol or boiling. Each covalent immunochemotherapeutic, the reference control immunoglobulin fraction (0.9µg/well) and a mixture of pre-stained reference control molecular weight markers were then developed by non-reducing SDS-PAGE (11% acrylamide) performed at 100 V constant voltage at 3°C for 2.5 hours.Covalent gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] immunochemotherapeutics following mass/size-dependent separation by non-reducing SDS-PAGE were equilibrated in tank buffer devoid of methanol. Mass/size-separated gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] contained in acrylamide SDS-PAGE gels were then transferred laterally onto sheets of nitrocellulose membrane at 20 volts (constant voltage) for 16 hours at 2° to 3°C with the transfer manifold packed in crushed ice.Covalent gemcitabine- at 4°C for 15 minutes followed by incubation in TBS blocking buffer solution  containing bovine serum albumin (5%) for 16 hours at 2 for 16 hours at 2° to 3°C applied in combination with gentle horizontal agitation. Prior to further processing, nitrocellulose membranes were vigorously rinsed in Tris buffered saline .Nitrocellulose membranes following BSA-block and serial rinsing were then incubated with biotinylated goat anti-murine IgG  at 4°C for 18 hours applied in combination with gentle horizontal agitation. Nitrocellulose membranes were then vigorously rinsed in TBS  followed by incubation in blocking buffer . Blocking buffer was decanted from nitrocellulose membrane blots which were then rinsed in TBS  before incubation with strepavidin-HRPO  at 4°C for 2 hours applied in combination with gentle horizontal agitation. Prior to chemiluminescent development nitrocellulose membranes were vigorously rinsed in Tris buffered saline . Following development with conjugated HR-PO-strepavidin each nitrocellulose membrane was then incubated with HRPO chemiluminescent substrate . Chemiluminescent autoradiography images were acquired by exposing radiographic film (Kodak BioMax XAR) to nitrocellulose membranes sealed within transparent ultra-clear re-sealable plastic bags.ex-vivo model for neoplastic disease. Populations of the mammary adenocarcinoma (SKBr-3) were propagated at >85% level of confluency in 150-cc2 tissue culture flasks containing McCoy's 5a Modified Medium supplemented with fetal bovine serum (10% v/v) and penicillin-streptomycin at a temperature of 37° C under a gas atmosphere of air (95%) and carbon dioxide (5% CO2). Trypsin or any other biochemically active enzyme fraction were not used to facilitate harvest of mammary adenocarcinoma SKBr-3 cell suspensions for seeding of tissue culture flasks or multi-well tissue culture plates. Growth media was not supplemented with growth factors, growth hormones or any other type of growth stimulant. Characteristic features and biological properties of the mammary adenocarcinoma (SKBr-3) cell line includes chemotherapeutic-resistance, overexpression of epidermal growth factor receptor 1 , and high over-expression of epidermal growth factor receptor 2 .The human mammary adenocarcinoma (SKBr-3) was utilized as an 5 cells/well and allowed to form a confluent adherent monolayer over a period of 48 hours. The growth media content of each individual well was removed manually by pipette and cellular monolayers were then serially rinsed (n = 3) with PBS followed by their stabilization onto the plastic surface of 96-well plates with paraformaldehyde . Stabilized cellular monolayers were then incubated with covalent gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] immunochemotherapeutics formulated at gradient concentrations of 0.1, 0.25, 0.5, 1.0, 5.0 and 10µg/ml in tissue culture growth media (200µl/well). Direct contact incubation between (SKBr-3) cellular monolayers and gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] was performed at 37° C over an incubation period of 3-hours using a gas atmosphere of air (95%) and carbon dioxide (5% CO2). Following serial rinsing with PBS (n = 3), development of stabilized mammary adenocarcinoma (SKBr-3) monolayers entailed incubation with β-galactosidase conjugated goat anti-mouse IgG (1:500 dilution) for 2 hours at 25° C with residual unbound immunoglobulin removed by serial rinsing with PBS (n = 3). Final cell ELISA development required serial rinsing (n = 3) of stabilized (SKBr-3) monolayers with PBS followed by incubation with nitrophenyl-β-D-galactopyranoside substrate . Absorbance within each individual well was measured at 410nm (630nm reference wavelength) after incubation at 37° C for a period of 15 minutes.Cell suspensions of mammary adenocarcinoma (SKBr-3) were seeded into 96-well microtiter plates in aliquots of 2 × 104-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] were formulated in growth media at standardized chemotherapeutic-equivalent concentrations of 10−10, 10−9, 10−8, 10−7, and 10−6 M . Each chemotherapeutic-equivalent concentration of covalent immunochemotherapeutic was then transferred in triplicate into 96-well microtiter plates containing mammary adenocarcinoma (SKBr-3) monolayers and growth media (200µl/well). Covalent immunochemotherapeutics where then incubated in direct contact with monolayer populations of mammary adenocarcinoma (SKBr-3) for a period of 182-hours (37° C under a gas atmosphere of air (95%) and carbon dioxide/CO2 (5%). Following the initial 96-hour incubation period, mammary adenocarcinoma (SKBr-3) populations were replenished with fresh tissue culture media with or without covalent immunochemotherapeutics or benzimidazole tubulin/microtubule inhibitors.Individual preparations of gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] were measured by removing all contents within the 96-well microtiter plates manually by pipette followed by serial rinsing of monolayers (n = 3) with PBS and incubation with 3--2,5-diphenyl tetrazolium bromide vitality stain reagent formulated in RPMI-1640 growth media devoid of pH indicator or bovine fetal calf serum (MTT: 5mg/ml). During an incubation period of 3–4 hours at 37° C under a gas atmosphere of air (95%) and carbon dioxide (5% CO2) the enzyme mitochondrial succinate dehydrogenase was allowed to convert the MTT vitality stain reagent to navy-blue formazone crystals within the cytosol of mammary adenocarcinoma (SKBr-3) populations . Contents of the 96-well microtiter plate was then removed, followed by serial rinsing with PBS (n = 3). The resulting blue intracellular formazone crystals were dissolved with DMSO (300µl/well) and then the spectrophotometric absorbance of the blue-colored supernatant measured at 570nm using a computer-integrated microtiter plate reader.Cytotoxic potencies for gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] by micro-scale desalting/buffer exchange column chromatography consistently yields covalent immunochemotherapeutic preparations that contained <4.0% of residual chemotherapeutic that was not covalently bound to immunoglobulin[4-amide)-[anti-HER2/neu] immunochemotherapeutic was 2.78 utilizing the organic chemistry reaction scheme to form an amide bond at the cytosine-like mono-amine of gemcitabine and synthesis of the UV-photoactivated gemcitabine-(C4-amide) intermediate (4-amide)-[anti-HER2/neu] was relatively larger than the 1.1-to-1 gemcitabine molar-incorporation-index attained during the synthesis of gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu][Size-separation of gemcitabine--[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] immunochemotherapeutics by SDS-PAGE in combination with immunodetection analysis (Western blot) and chemiluminescent autoradiography recognized a single primary condensed band of 150-kDa between a molecular weight range of 5.0-kDa to 450-kDa (4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] directly corresponds with the known molecular weight/mass of reference control anti-HER2/neu monoclonal immunoglobulin fractions -[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] on the external surface membrane of adherent mammary adenocarcinoma (SKBr-3) populations was measured by cell-ELISA (4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] was detected with progressive increases in standardized total immunoglobulin-equivalent concentrations formulated at 0.010, 0.025, 0.050, 0.250, and 0.500µg/ml (amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] for over-expressed EGFR (2.2 × 105 per cell) and highly over-expressed HER2/neu (1 × 106 per cell) trophic receptor sites on the exterior surface membrane of mammary adenocarcinoma (SKBr-3) populations 4-amide)-[anti-EGFR] after 182-hours was consistently greater than epirubicin-(C3-amide)-[anti-HER2/neu] following a 96-hour incubation period against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) at and between chemotherapeutic-equivalent concentrations of 10−13 M and 10−7 M (3-amide)-[anti-HER2/neu] is relatively more potent than gemcitabine-(C4-amide)-[anti-EGFR] over a direct contact incubation period of 96-hours (4-amide)-[anti-EGFR] produced progressively higher levels of anti-neoplastic cytotoxicity of 0.0% at 10−14 M  to 41.4% at 10−8 M  followed by a relatively more rapid increase from 41.4% to 90.1% at and between 10−8 M and 10−6 M  respectively (3-amide)-[anti-R2/neu] at and between chemotherapeutic-equivalent concentrations of 10−9 M and 10−6 M  respectively (3-amide)-[anti-HER2/neu] following a 182-hour incubation period produced essentially identical levels of anti-neoplastic cytotoxicity that varied between 7.7% and 9.3% residual survival for chemotherapeutic-equivalent concentrations at and between 10−14 M and 10−6 M (Anti-neoplastic cytotoxicity of gemcitabine-(Cd 10−7 M . Based o96-hours . Gemcitaectively . Alternaectively . Epirubid 10−6 M .4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] compared to gemcitabine-(C4-amide)-[anti-EGFR] alone were somewhat similar. Rapid progressive increases in anti-neoplastic cytotoxicity for gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] from 0.0% to 91.9% were detected at and between the chemotherapeutic-equivalent concentrations of 10−11 M and 10−6 M  respectively (4-amide)-[anti-EGFR] created progressive and substantial increases in anti-neoplastic cytotoxicity from 0.0% to 90.1% at and between the chemotherapeutic-equivalent concentrations of 10−14 M and 10−6 M  respectively (4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] were very similar to gemcitabine-(C4-amide)-[anti-EG-FR] at and between the chemotherapeutic concentrations of 10−10 M  and the maximum concentration of 10−6 M  respectively (The anti-neoplastic cytotoxicity profiles for gemcitabine-(Cectively  and 5. Aectively  and 5. Lectively  and 5.4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] formulated as a chemotherapeutic-standardized 50/50 dual-combination following a 182-hour incubation period appeared distinctly different than those detected for only epirubicin-(C3-amide)-[anti-HER2/neu] after a 96-hour incubation period especially at the chemotherapeutic-equivalent concentrations of 10−10 M, 10−9 M and 10−8 M (4-amide)- [anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] at 182-hours and epirubicin-(C3-amide)-[anti-HER2/neu] after a 96-hour at and between the chemotherapeutic-equivalent concentrations of 10−14 M and 10−11 M were essentially identical based on values of 5.6% and 0.4%  respectively (4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] produced greater levels of anti-neoplastic cytotoxicity compared to epirubicin-(C3-amide)-[anti-HER2/neu] alone based on the measured values of 20.5% -vs-0.4% at 10−10 M ; 49.5% -vs- 0.0% at 10−9 M ; 51.3% -vs- 9.8% at 10−8 M ; and 89.1% -vs-66.9% at 10−7  respectively (4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu], and epirubicin-(C3-amide)-[anti-HER2/neu] alone had nearly identical maximum values of 88.5% -vs- 91.9% at 10−6 M  respectively (3-amide)-[anti-HER2/neu] following a 182-hour incubation period produced nearly identical levels of anti-neoplastic cytotoxicity that varied between 7.7% and 9.3% residual survival for chemotherapeutic-equivalent concentrations at and between 10−14 M and 10−6 M (The anti-neoplastic cytotoxicity profiles for gemcitabine-(Cd 10−8 M . Anti-neectively . Gemcitaectively . Both geectively . Epirubid 10−6 M .4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] consistently displayed a trend for exerting greater selectively “targeted” anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma than did gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] at and between the chemotherapeutic-equivalent concentrations of 10−9 M and 10−6 M (4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] compared to gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] was 49.5% -vs-24.5%  at 10−9 M; 51.3% -vs- 66.7%  at 10−8 M; 11.6% -vs- 69.9%  at 10−7 M; and 91.9% -vs- 83.7.5%  at 10−6 M respectively  for the dual-combinations of gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] and gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] respectively -[anti-EGFR] and epirubicin-(C4-amide)-[anti-HER2/neu] each possessed potent levels of anti-neoplastic potency as a function of both chemotherapeutic-equivalent concentration and the duration of the contact incubation period (4-amide)-[anti-EGFR] was relatively more potent than many if not most analogous epirubicin with epirubicin-(C3-amide)-[anti-HER2/neu] formulated as a 50/50 dual-combination following a 182-hour incubation period was increased when evaluated in combination with mebendazole  following a 96-hour incubation period (4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] was detectably more potent at the chemotherapeutic-equivalent concentrations of 10−10 M , 10−9 M , and 10−8 M  respectively (4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] with and without mebendazole at the chemotherapeutic-equivalent concentrations of 10−7 M  and 10−6 M  respectively  at final concentrations formulated at and between the range of 0.05µM to 2.5µM . Mean an“free” non-covalently bound form of the chemotherapeutic moeity. -benzaldehyde derivative with epirubicin-(C3-amide)-[anti-HER2/neu] to exert slightly greater levels of selectively “targeted” anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) at the chemotherapeutic-equivalent concentrations of 10−9 M, 10−8 M and 10−7 M compared to gemcitabine-(C4-amide)-[EGFR] alone (4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] also produced greater levels of selectively “targeted” anti-neoplastic cytotoxicity compared to the dual-combination of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] at the chemotherapeutic-equivalent concentrations 10−9 M, 10−8 M, 10−7 M and 10−6 M although the results at 10−6 M were not significantly different  internalization by mechanisms of receptor-mediated-endocytosis; and the [vi] degree that neoplastic cell vitality and proliferation characteristics are dependent upon over-expression of specific membrane trophic receptor complexes.A variety of molecular mechanisms and cellular processes likely account for the levels of anti-neoplastic cytotoxicity produced by dual-combinations of covalent immunochemotherapeutics. Most notable in this regard is the interdependent interactions between aspects related to cancer cell biology, and properties of the corresponding immunoglobulin component of covalent immunochemotherapeutics. Cancer cell biology variables that are influential in this regard include; [4-amide)-[anti-EGFR], epirubicin-(C3-amide)-[anti-HER2/neu] and similar covalent immunochemotherapeutics, their corresponding immunoglobulin component can innately exert an array of properties that contribute significantly to their capacity to achieve maximum anti-neoplastic cytotoxic potency. Covalent immunochemotherapeutics like gemcitabine-(C4-amide)-[anti-EGFR][3-amide)-[anti-HER2/neu] and epirHER2/neu] that posdocytosis.4-amide)-[anti-EGFR], epirubicin-(C3-amide)-[anti-HER2/neu], and analogous covalent immunochemotherapeutics effectively facilitates selective “targeted” chemotherapeutic delivery and continual deposition of the chemotherapeutic moiety onto the exterior surface membrane of neoplastic cell populations. The decision regarding which site or sites on the external surface membrane of neoplastic cells is to be selected to facilitate selective “targeted” chemotherapeutic delivery is important because it determines several critical attributes. In general theory and practice the immunoglobulin component of covalent immunochemotherapeutics can promote selective “targeted” chemotherapeutic delivery only if it possesses binding-avidity specifically for an antigenic “site” that is either uniquely expressed or relatively over-expressed on the external surface membrane of cancer cells compared to normal healthy tissues and organ systems. In mammary adenocarcinoma (SKBr-3) EGFR (2.2 × 105/cell) is over-expressed and HER2/neu (1 × 106/cell) is highly over-expressed compared to normal/healthy tissues and organ systems. Similar membrane-associated antigenic “sites” that are over-expressed by neoplastic cell types include CD19 (B-cell lymphoma), CD20 (chronic lymphocytic leukemia), CD22 , CD33 (acute myelogenous leukemia), CD52 (chronic lymphocytic leukemia), CD74 , carcinoembryonic antigen (CEA: LoVo colon carcinoma), cervical carcinoma cell-surface antigen , chondroitin sulfate proteoglycan (metastatic melanoma), epidermal growth factor receptor .In parallel with suppression of trophic membrane receptor function the IgG immunoglobulin component of gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] are essentially incapable of passively diffusing across the intact structure of the lipid bilayer membrane in neoplastic cell populations. The immunoglobulin component of gemcitabine-(C4-amide)-[anti-EGFR], epirubicin-(C3-amide)-[anti-HER2/neu] and other covalent immunochemotherapeutics can facilitate not only the selective “targeted” delivery and deposition of chemotherapeutics on the external surface membrane of neoplastic cell types, but it can also initiate active trans-membrane intracellular transport of chemotherapeutic moieties. Trans-membrane intracellular transport of covalent immunochemotherapeutics is possible if their immunoglobulin component physically binds to (antigenic) “sites” on the exterior surface membrane of neoplastic cells that are known to be internalized by mechanisms similar or identical to those observed following selective binding of endogenous receptor ligands or immunoglobulin fraction (e.g. EGF or anti-EGFR → EGFR). Such qualities are often a characteristic of trophic membrane receptor complexes like EGFR, HER2/neu, IGF-1R, and VEGFR that are each over-expressed by several neoplastic cell types including adenocarcinomas and carcinomas that affect the breast, ovary, prostate, lung and intestine. Selective binding of endogenous receptor ligands and immunoglobulin fractions at their corresponding receptor “sites” on the external surface membrane of neoplastic cells initiates internalization by receptor-mediated-endocytosis phenomenon. [4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] affords several important outcomes and attributes. First, receptor-mediated-endocytosis stimulated by the binding of the immunoglobulin component reduces the risk of covalent immunochemotherapeutics like gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] from simply coating the exterior surface membrane of neoplastic cell populations. Such a prerequisite is only necessary if the chemotherapeutic moiety exerts a mechanism-of-action that is dependent upon entry into the cytosol or nuclear environments. Second, internalization of covalent immunochemotherapeutics by receptor-mediated-endocytosis at endogenous trophic receptor “sites” aids in facilitating progressive and continual active transmembrane transport and subsequent intra-cellular accumulation of the chemotherapeutic moiety.Due to their relatively massive size (molecular weight), gemcitabine--[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] are actively transported across intact external membrane structures of neoplastic cell types. Given a single trophic membrane receptor type in a single neoplastic cell type this assumption is largely considered to be relatively accurate. However, EGFR, HER2/neu, IGF-1R, VEGFR and other trophic membrane receptor sites in different neoplastic cell types are likely internalized by mechanisms of receptor-mediated-endocytosis at relatively unequal rates and are also subsequently re-expressed and replenished at different rates following internalization. Specific data remains rather limited about the receptor-mediated-endocytosis of covalent immunochemotherapeutics like epirubicin-[anti-HER2/neu][neu],,85,102 eHER2/neu] gemcitabeu][neu],,98 or gey per day. Acknowly per day fold gre4-amide)-[anti-EGFR] or epirubicin-(C3-amide)-[anti-HER2/neu] that are directed at more than one receptor (antigenic) “site” expressed on the exterior surface membrane of neoplastic cell populations represents one potential approach to achieving this objective -[anti-EGFR] or epirubicin-(C3-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) and potentially other chemotherapeutic-resistant cancer populations can be attributed to covalent bonding of the chemotherapeutic moieties to a delivery platform that has a much larger molecular weight . Covalent bonding of chemotherapeutics to large molecular weight platforms of this size imparts physical alterations that through mechanisms of steric hinderance inhibit the biological function of entities that can utilize the “free” form of chemotherapeutics as a substrate. In this manner, the biochemical activity of degradative enzymes is suppressed (e.g. gemcitabine inactivation by cytosine deaminase) as is the capacity of P-glycoprotein (MDR-1: multi-drug resistance protein) or epirubicin-(C3-amide)-[anti-HER2/neu] to the influence of P-glycorpotein, this same attributes imposes steric hinderance phenomenon that suppresses chemotherapeutic metabolism. As a consequence, chemotherapeutic moieties like gemcitabine that are covalently bound to immunoglobulin are less vulnerable to biochemical degradation by enzyme fractions like cytadine deaminase, and deoxycytidylate deaminase (following gemcitabine phosphorylation) which both impose rapid deamination reactions. Lastly, the large mass size of immunoglobulin fractions is greater than the glomerular filtration molecular weight cut of (MWCO = 50-kDa) which in turn effectively decreases the renal clearance (rate and extent) of chemotherapeutic moieties associated with covalent immunochemotherapeutics in a manner that substantially prolongs their plasma pharmacokinetic profile.Similar in concept to the large molecular weight immunoglobulin fractions decreasing the vulnerability of gemcitabine and epirubicin moieties within gemcitabine-(C4-amide)-[anti-HER2/neu],[3-amide)-[anti-HER2/neu] with epirubicin-(C3-amide)-[anti-HER2/neu] based on the levels of anti-neoplastic cytotoxicity detected at and between the chemotherapeutic equivalent concentrations of 10−9 M and 10−7 M  or highly over-expressed  because of their wider margin-of-safety. Given this perspective covalently bonding chemotherapeutics to large molecular weight delivery platforms provides a potential opportunity to also administer total chemotherapeutic dosage levels for the resolution of resistant forms of neoplastic disease that could not be safely resolved with the same total dosage of the “free” non-protein bound form of the chemotherapeutic agent.neu (highly over-expressed) function on the external surface membrane of mammary adenocarcinoma (SKBr-3) utilizing multiple covalent immunochemotherapeutics represents an approach for attaining additive and synergistic levels of anti-neoplastic efficacy. Selectively “targeted” inhibition of different membrane trophic receptors with multiple covalent immunochemotherapeutics like the dual-combination of gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-C3-amide)-[anti-HER2/neu] is important because EGFR, HER2/neu, IGFR, and VEGFR are over-expressed by many neoplastic cell types where they directly or indirectly regulate proliferation rate, metastatic characteristics, and chemotherapeutic resistance. Inhibiting the function of more than one trophic receptor over-expressed on the exterior surface membrane of neoplastic cell populations is also important because a single immunoglobulin fraction like anti-EGFR, anti-HER2/neu, anti-IGFR, and anti-VEGFR usually are only capable of reducing cancer cell vitality and decreasing rates of proliferation but are generally incapable of evoking cytotoxic resolution of neoplastic disease with epirubicin-(C3-amide)-[anti-HER2/neu] the possible levels of additive or synergistic interactions between chemotherapeutic moieties and immunoglobulin fractions would include; [i] gemcitabine and anti-EGFR, [ii] gemcitabine and anti-HER2/neu; [iii] gemcitabine, anti-EGFR, and anti-HER2/neu; [iv] epirubicin and anti-EGFR, [v] epirubicin and anti-HER2/neu; [vi] epirubicin, anti-EGFR, and anti-HER2/neu; [vii] gemcitabine, epirubicin and anti-EGFR; [viii] gemcitabine, epirubicin and anti-HER2/neu; and/or [ix] gemcitabine, epirubicin, anti-EGFR and anti-HER2/neu.Additive and synergistic levels of anti-neoplastic cytotoxicity can be attained when immunoglobulin fractions like anti-EGFR, anti-HER2/otoxicity,161,162.sphamide,,48 docetsphamide,;48 etoposphamide,;48 or viide, and epirubicin-(C3-amide)-[anti-HER2/neu] are selectively “targeted” in-vivo at EGFR and HER2/neu that are highly over-expressed by chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) it results in initiation of several innate immune responses that produce a variable degree of anti-neoplastic cytotoxicity. Most prominent in this regard is an additive or synergistic levels of anti-neoplastic cytotoxicity potentially attained through the combined interdependent effects of several immune-dependent processes that can include; [i] complement-mediated cytolysis; [ii] opsonization subsequent to the formation of IgG/receptor/complement complexes on the exterior surface of neoplastic cell membranes (e.g. macrophage mediated phagocytosis); and [iii] antibody dependent cell-mediated cytotoxicity (ADCC) which is classically mediated through natural killer lymphocytes (NK cells) but participation in this response can also include macrophages, neutrophils and eosinophils. In ADCC responses the immune cells involved in this phenomenon release cytotoxic components that are known to additively and synergistically enhance the cytotoxic anti-neoplastic activity of conventional chemotherapeutic agents. and epirubicin-(C3-amide)-[anti-HER2/neu], it is technically very difficult to simultaneously and accurately simulate these three immune-dependent responses utilizing ex-vivo models for neoplastic disease. In clinical environments immunoglobulin fractions when utilized for selective “targeted” delivery of therapeutic pharmaceuticals or diagnostic imaging agents in nuclear medicine frequently are biochemically modified with enzymes like papain in order to cleave (remove) the Fc segment of the IgG molecule. In effect, such biochemical modifications minimize non-selective binding at Fc receptors expressed by cells within the RE system (mononuclear phagocytic system) physically residing within the spleen and liver. Unfortunately, such biochemical modifications create a covalent immunochemotherapeutic composed of just F(ab’)2 or Fab’ fragment which would have less of a capacity to induce activation of the complement cascade , neoplastic cell opsonization (e.g. macrophage Fc receptor dependent binding), or ADCC phenomenon (e.g. NK lymphocyte Fc receptor dependent binding).Due to potential for complement-mediated lysis, ADCC and opsonization to all contribute to enhance the levels of anti-neoplastic cytotoxicity of covalent immunochemotherapeutics like gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] in-vivo presents an opportunity to potentially attain still another plane of additive and synergistic anti-neoplastic cytotoxicity that involves; [i] gemcitabine, trophic membrane receptor inhibition, and innate immune response activation; and/or [ii] epirubicin, trophic membrane receptor inhibition, and innate immune response activation. In support of this concept, immune cell populations that are involved in ADCC phenomenon release cytotoxic components that are known to additively and synergistically enhance the cytotoxic anti-neoplastic activity of conventional chemotherapeutic agents[in-vivo compared to their evaluation in ex-vivo tissue culture based models for neoplastic disease even when the same identical cancer cell type (xenographs) are utilized[4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] collectively serve to explain how the dual-combination of these two covalent immunochemotherapeutics produced additive levels of anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary-adenocarcinoma (SKBr-3) when utilized as an ex-vivo model for neoplastic disease (4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] were formulated as a 50:50 chemotherapeutic-equivalent combination the anti-neoplastic cytotoxicity levels were intermediate between levels detected for each of the two individual covalent immunochemotherapeutics -[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] collectively serve to explain how the dual-combination of these two covalent immunochemotherapeutics have the potential to induced levels of anti-neoplastic cytotoxicity that were greater than for either of the covalent immunochemotherapeutics alone (4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] over a prolonged period of time and especially following in-vivo administration (e.g. IV injection).Each of the qualities and properties of selective “targeted” chemotherapeutic delivery and complementary interactions afforded by gemcitabine--[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu]. ex-vivo incubation period during which time neoplastic cells are in direct and simultaneous contact with each individual covalent immunochemotherapeutic.Almost invariably, levels of anti-neoplastic cytotoxicity can be increased by prolonging the 4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu]. In contrast to chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) anti-neoplastic cytotoxicity of gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] would likely have been higher if it had been measured utilizing an entirely different neoplastic cell type such as pancreatic carcinoma,[4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] compared to gemcitabine in chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) populations -[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] that was not chemotherapeutic-resistant. Majority of published descriptions to date that report in current literature the efficacy of covalent immunochemotherapeutics or analogous biopharmaceutical agents utilize human neoplastic cell populations that are chemotherapeutic-resistant. Rare exceptions have been the application of chemotherapeutic-resistant metastatic melanoma M21 ; in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] would likely have been substantially greater if either cellular proliferation had been assessed with [3H]-thymidine, or an ATP-based assay method was alternatively applied as an analysis modality because of their reportedly >10-fold greater sensitivity in detecting early cell injury compared to MTT vitality stain based assay methods.[Anti-neoplastic cytotoxicity of gemcitabine--[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] would likely of been greater if their efficacy had been delineated in-vivo against human neoplastic xenographs in animal hosts as a model for human cancer. In such a scenario, added levels of selective “targeted” anti-neoplastic cytotoxicity is attained through antibody-dependent cell cytotoxicity (ADCC), complement mediated cytolysis, and immunoglobulin initiated opsonization similar to what has been observed with anti-CD20 and anti-CD52 administered for the treatment of certain forms of leukemia.Lastly, as previously eluded to, a high degree of probability suggests that the anti-neoplastic cytotoxicity of gemcitabine--[anti-EG-FR] and epirubicin-(C3-amide)-[anti-HER2/neu] produced higher levels of anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma than did the dual covalent immunochemotherapeutic combination alone (3-amide)-[anti-HER2/neu][4-amide)-[anti-HER2/neu] myeloma,HER2/neu],189–191 HER2/neu],189 or mHER2/neu].4-amide)-[anti-EG-FR] with epirubicin-(C3-amide)-[anti-HER2/neu] presents several opportunities for inducing lower frequencies of severe in-vivo sequelae. The covalent bonding of chemotherapeutics to a high-molecular weight delivery platform to facilitate selective “targeted” delivery effectively avoids innocent high-level exposure of normal tissues and organ systems to the chemotherapeutic moiety thereby reducing the risk and frequency of severe dose-dependent sequelae (e.g. anthracycline cardiotoxicity). Utilization of “carrier” platforms like immunoglobulin fractions that have a significantly larger mass/size (IgG MW ≅ 150-kDa) than gemcitabin, epirubicin and other conventional small molecular weight chemotherapeutics employed in the synthesis of covalent immunochemotherapeutics also decreases the risk of chemotherapy-associated toxicity by several mechanisms. The much large molecular weight of gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] drastically reduces the rate and extent that gemcitabine and epirubicin are removed from the plasma compartment and excreted into the urine by renal glomerular filtration (MWCO ≅ 50-kDa). In this manner highly renal-toxic chemotherapeutics can potentially be covalently bound to large molecular weight “targeting” platforms  to increase their utility as a form of systemic anti-cancer therapy. The substantially lower rate and extent that large molecular weight covalent immunochemotherapeutics like gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] are eliminated from the intra-vascular compartment by renal excretion in turn significantly prolongs and extends the chemotherapeutic moiety pharmacokinetic profile (plasma T1/2) and therefore contact with neoplastic lesions for longer periods of time per systemic dose. The potential for covalent immunochemotherapeutics to promote greater cytosol concentrations of the chemotherapeutic moiety than can be achieved by simple passive diffusion of the “free” chemotherapeutic from the extracellular fluid compartment can provide a level of safety because they provide an opportunity for more rapid resolution of neoplastic conditions at lower total dose requirements. Lastly, selectively “targeted” chemotherapeutic delivery allows the use of additive or synergistic chemotherapeutic combinations that otherwise could not previously be utilized in combination with one another due to dose-limiting sequelae.The dual-combination of gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] at least conceptually can therefore afford the attributes of greater anti-neoplastic cytotoxicity and prolongation of chemotherapeutic moiety pharmacokinetic profiles that can subsequently serve collectively as the basis for reducing individual and total dosage requirements. Improved margins-of-safety are therefore possible with gemcitabine-(C4-amide)-[anti-EGFR], epirubicin-(C3-amide)-[anti-HER2/neu] and analogous covalent immunochemotherapeutics through a combination of selective “targeted” anti-neoplastic cytotoxicity that avoids high-level innocent exposure of normal tissues and organ system, and reductions in total dosage requirements necessary for disease resolution when implemented for intervention in clinical oncology. The benzimidazole tubulin/microtubule inhibitor, mebendazole can potentially complement and enhance these properties of dual gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] combinations in part because of it’s anti-neoplastic cytotoxicity and because it may have a wider margin-of-safety than many if not most conventional chemotherapeutic agents.Covalent immunochemotherapeutics like the dual-combination of gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] both exerted selectively “targeted” anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) populations (4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] exerted at least additive levels of anti-neoplastic cytotoxicity which during the relatively brief incubation periods applied is primarily a result of the mechanisms-of-action for the two chemotherapeutic moieties, and also the anti-trophic properties of both anti-EGFR and anti-HER2/neu -[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] not only serves as a molecular strategy for increasing cytosol concentrations of small molecular weight chemotherapeutics beyond levels attainable by simple passive diffusion following intravenous infusion at clinically relevant dosages, it also represents an approach for reducing exposure of innocent tissues and organ systems. Mebendazole further complements these therapeutic attributes by evoking a higher level of anti-neoplastic cytotoxic against chemotherapeutic resistant mammary adenocarcinoma (SKBr-3) when applied in combination with both gemcitabine-(C4-amide)-[anti-EGFR] with epirubicin-(C3-amide)-[anti-HER2/neu] compared to the dual-combination of only the two covalent immunochemotherapeutics. Collectively, research investigations with gemcitabine-(C4-amide)-[anti-EGFR], epirubicin-(C3-amide)-[anti-HER2/neu] and mebendazole demonstrate therapeutic options for more effectively resolving chemotherapeutic-resistant neoplastic conditions within a more expedient time frame at lower total dosage levels.Simultaneous dual-combination of gemcitabine-(C


Percutaneous edge-to-edge repair with MitraClip has been recommended as an alternative to conventional mitral valve repair in symptomatic severe mitral regurgitation at very high surgical risk. Data on the impact of MitraClip on left ventricular volumes are scarce. Therefore we aimed to assess left ventricular remodeling following MitraClip.Consecutive patients scheduled for MitraClip were prospectively studied. Patients with pacemaker or ICD were excluded. Transthoracic echocardiography and CMR at 1.5 Tesla were performed before and 6 months after MitraClip procedure. Left ventricular volumes were measured using transthoracic echocardiography (biplane Simpson's method) and short-axis SSFP CMR images covering the left ventricle. Mitral insufficiency was graded using color Doppler flow mapping (grade 1-4).CMR was performed in 16 patients with symptomatic severe (grade 3 or 4) functional mitral regurgitation (median 73 years (range 45-87 years), M/F: 7/9, left ventricular ejection fraction (EF), 31% (20-73%), logistic EuroSCORE 23% (10-35%)). MitraClip reduced mitral regurgitation , but failed in 2 patients . MitraClip caused intracavitary metallic artefacts at the base of the heart, but did not impede endocardial border definition. No change in left ventricular volumes was measured following successful MitraClip implantation (enddiastolic volume before MitraClip: 221 ml (102-334 ml) versus following MitraClip: 215 ml  NS; endsystolic volume: 154 ml (31-248 ml) versus 145 ml (40-240 ml) NS and ejection fraction: 31% (20-73%) versus 32% (16-61%) NS). Negative remodeling occurred in both patients with MitraClip failure . There was a high correlation between biplane Simpson's method and CMR assessed left ventricular enddiastolic  and endsystolic  volumes.CMR allowed precise assessment of left ventricular volumes. Despite reduction of mitral regurgitation, no change of left ventricular volumes was measured at 6 months follow-up.N/A.


Severe trauma induces a blunted immune response associated with an enhanced susceptibility to nosocomial infections . Within Following ethical approval and consent, 30 ICU patients admitted following trauma and 16 healthy age and sex-matched controls were recruited. Blood samples were obtained within 2 hours of injury and at 24 hours. miRs were isolated using PAXGene (Qiagen). miRs were selected on the basis of their miRBase target prediction scores for the promoters of IL-10, IL-12 and TNFα. Six miRs selected for analysis were miR374b, -202 and 125a3p (IL-10), -410 and -21 (IL-12) and -454 (TNFα). qPCR was used to quantify candidate miRs and the results were normalised relative to small nucleolar RNAs U6/RNU44. Infections were assessed using predefined criteria [Twenty three patients (77%) developed an infection, 15 (50%) were shocked (base deficit ≥ 6 mEq/L) on admission and 6 (20%) died. Within 2 hours, expressions of miR-202, -125a3p, -21 and -454 were reduced  in patients compared to healthy controls. This reduction was maintained  24 hours after injury. At 24 hours, miR-202 was down-regulated  in shocked compared to non-shocked patients. Decreased miR-374b expression on admission was associated with subsequent development of pneumonia (p=0.009).Expression of miRs complementary to cytokine promoters varies significantly following severe traumatic injury and is associated with clinical outcomes. Reduction in inhibitory miRs could partly explain increased IL-10 expression and provide a mechanistic link between severe trauma, the observed immunosuppressive phenotype and an increased incidence of nosocomial infections. In vitro studies are now needed to invoke causation.


PRL-3, a metastasis-associated phosphatase, is known to exert its oncogenic functions through activation of PI3K/Akt, which is a key regulator of the rapamycin-sensitive mTOR complex 1 (mTORC1), but a coherent link between PRL-3 and activation of mTOR has not yet been formally demonstrated. We report a positive correlation between PRL-3 expression and mTOR phospho-activation in clinical tumour samples and mouse models of cancer and demonstrate that PRL-3 increased downstream signalling to the mTOR substrates, p70S6K and 4E-BP1, by increasing PI3K/Akt-mediated activation of Rheb-GTP via TSC2 suppression. We also show that PRL-3 increases mTOR translocation to lysosomes via increased mTOR binding affinity to Rag GTPases in an Akt-independent manner, demonstrating a previously undescribed mechanism of action for PRL-3. PRL-3 also enhanced matrix metalloproteinase-2 secretion and cellular invasiveness via activation of mTOR, attributes which were sensitive to rapamycin treatment. The downstream effects of PRL-3 were maintained even under conditions of environmental stress, suggesting that PRL-3 provides a strategic survival advantage to tumour cells via its effects on mTOR. PRL-3 was shown previously to promote autophagy in ovarian cancersTo investigate the relationship of PRL-3 with mTOR activity, we analysed 12 sets of PRL-3-positive tumours and matched normal tissue samples from gastric cancer patients for protein expression levels of PRL-3 and phosphorylation status of Thr37/46 of 4E-BP1, a direct substrate/effector of mTOR and indicator of mTOR oncogenic activityin vivo observations suggested the existence of a correlation between PRL-3 expression levels, mTOR activity, and mTOR activation-associated phosphorylation.We further investigated the above finding in the spontaneous mouse mammary tumour virus (MMTV) transgenic model, which harbours the polyomavirus middle T oncoprotein (PyMT) under transcriptional control of the MMTV promoter-enhancer, resulting in the formation of palpable mammary tumours in mice as early as 6 weeks of age27PRL-3 was shown previously to promote autophagy in ovarian cancersThe mTOR pathway integrates multiple environmental cues to regulate translation in response to stress. Deprivation of oxygen or nutrients, particularly amino acids, results in reduced mTOR phosphorylation and inhibition of downstream effectors of protein translation, including 4E-BP1 and p70S6K29319To validate whether Rheb was involved in PRL-3-mediated TSC2 suppression and activation of mTOR, we used a Rheb activation assay to directly study the levels of active Rheb (Rheb-GTP) in PRL-3-overexpressing cells. Relative to HCT116-Vec or HCT116-C104S cells, Rheb-GTP levels were higher in HCT116-PRL-3 cells under basal, hypoxic, or serum starved conditions , lane 5.AKT transcripts, or inhibition of Akt kinase using a small-molecule antagonist. siRNA-mediated AKT depletion reduced PRL-3-driven phosphorylation of TSC2, mTOR, and its downstream effectors, 4E-BP1 and p70S6K -mediated depletion of d p70S6K , lane 4.d p70S6K , lane 4.d p70S6K . CollectDespite a reduction in overall mTOR phosphorylation in cells grown under hypoxia, serum deprivation, or amino acid starvation, the PRL-3-driven increase in phosphorylation of the mTOR substrates 4E-BP1 and p70S6K under each of these stressors consistently appeared greater compared to the increase in mTOR S2448 phosphorylation itself . This inThe Rag small GTPases have been reported to be critical regulators of mTOR lysosomal relocalisation and activation. Upon amino acid stimulation, Rag GTPases associate with mTORC1 as heterodimers, recruiting it to late endosomal and lysosomal compartments for subsequent Rheb-mediated activationT54L-RagDQ121L heterodimersDN) or empty vector (Ctrl) and checked phosphorylation levels of various components of the Akt-mTOR signalling pathway. Overexpression of RagDN did not rescue mTOR phosphorylation levels . Importantly, rapamycin treatment suppressed invasiveness of PRL-3-overexpressing cells to similar levels as the control cells  are a group of enzymes which can degrade ECM and activate a number of growth factors, thus playing important roles in tumour invasion and metastasis393941n = 566). Supporting our in vitro data, PRL-3 expression positively correlated with expression of MMP-2 (p = 1.287E-4) Thus, PRL-3 expression appears to correlate with an mTOR-dependent invasive phenotype characterized by the increased expression of MMP-2. Furthermore, in colon cancer patients, MMP-2 expression had prognostic value for patient survival when PRL-3 was highly expressed  or mutant  PIK3CAIn matched tumour-normal patient cancer samples, we observed a striking correlation between PRL-3 expression and the ratio of phosphorylated/total 4E-BP1, a PRL-3 activates PI3K/Akt signalling via various pathways, including downregulation of phosphatase and tensin homologue (PTEN)13PRL-3-driven mTOR activation, in turn, could promote the metastatic development of cancers in at least two ways: increased motility and increased invasiveness. Both these pro-metastatic characteristics could be effectively inhibited by treatment with rapamycin, which selectivity inhibits mTORC1 but not mTORC2. Because of the importance of mTORC1 in protein translation and cell growth, PRL-3 likely exploits mTOR to upregulate the production of proteins involved in motile and invasive behaviour, especially under stress conditions. Indeed, PRL-3 overexpression resulted in a mTORC1-dependent increase in production of oncogenic MMP-2, a collagenase involved in the invasion and metastasis cascade and a marker of poor prognosis in multiple cancers51in vitro and in vivo, especially under oxygen- and nutrient-deprived conditions. Moreover, PRL-3 achieves this unconventionally via regulation of the two independent arms of mTORC1 activation, PI3K/Akt-driven Rheb-GTP accumulation, as well as enhanced Rag GTPase-mediated mTORC1 recruitment to endomembranes for Rheb-mediated activation. Such pleiotropic regulation by PRL-3 in driving cancer progression cements the urgent need to discover novel agentsFor decades, mTOR has emerged as an attractive target of anticancer therapy. Our data demonstrate an oncogenic role of PRL-3 on mTOR activation 2. For serum depletion  experiments, cells were washed twice in PBS and changed to RPMI-1640 medium without FBS, supplemented with 1% (v/v) penicillin/streptomycin. For hypoxia experiments, cells were placed in a GasPak EZ Gas Pouch  containing ≤1% O2. For amino acid starvation (AA-) experiments, cells were washed twice in PBS, once with Earle’s Balanced Salt Solution (EBSS), and finally incubated with EBSS medium for 1 h at 37 °C with 5% CO2. It should be noted that compared to RPMI-1640, EBSS not only lacks amino acids, but also lacks vitamins and has a different salt composition. For conditioned medium analysis, cells were cultured in RPMI-1640 medium without FBS for 24 h and medium was collected and condensed using centrifugal concentrators. Where indicated, cells were treated with DMSO-solubilised rapamycin , DMSO-solubilized Akt Inhibitor VIII , or DMSO alone .Human colon cancer cell lines , human cervical cancer cells (HeLa), human melanoma cells (G361), and human breast cancer cells (MCF7) were purchased from ATCC . Human ovarian cancer cells (A2780) were purchased from ECACC . Cell lines expressing EGFP (Vec), EGFP-PRL-3 (PRL-3), or EGFP-PRL-3 C104S (PRL-3 C104S) were generated by transfection of cells with pEGFP-C1 vector , pEGFP-C1-PRL-3 or pEGFP-C1-PRL-3 (C104S) constructs, respectively, using Lipofectamine 2000 , followed by G-418 selection (1 mg/mL) for 2 weeks. HCT116 cells stably expressing scrambled or PRL-3-targeting shNA constructs  were generated by puromycin selection (1 μg/mL) for 2 weeks. Cells were grown in RPMI-1640 medium supplemented with 10% FBS  and 1% (v/v) penicillin/streptomycin in a humidified incubator at 37 °C with 5% COAKT-targeting  and control siRNA were from Cell Signaling Technologies. siRNA  was transiently transfected using jetPRIME  following the manufacturer’s recommended protocol.Antibodies against mTOR (#2983), p-mTOR S2448 (#2971), Akt (#4691), p-Akt S473 (#4060), raptor (#2280), 4E-BP1 (#9644), p-4E-BP1 T37/46 #2855), p-p70S6K T389 (#9234), TSC2 (#4308), and p-TSC2 S939 (#3615) were purchased from Cell Signaling Technologies . Anti-p70S6K (#611260) antibody was purchased from BD Biosciences . Anti-LAMP2 (#ab25631) antibody was purchased from Abcam . Antibodies against GST (#sc-138) and GFP (sc-9996) were purchased from Santa Cruz Biotechnology . HRP-conjugated sheep anti-mouse (#515-035-062) and goat anti-rabbit (#111-035-045) antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. , whilst AlexaFluor568-conjugated goat anti-mouse (#A-11004) and AlexaFluor633-conjugated goat anti-rabbit (#A-21071) antibodies were from Life Technologies . Generation of anti-PRL3 antibody, p-p70S6Western blots were performed as describedImmunofluorescence analysis was carried out as previously describedg, 15 min), and incubated with glutathione beads overnight at 4 °C with rotation. The beads were washed three times with lysis buffer and finally eluted with reduced glutathione (25 mM). Elutes were subjected to western blotting with the indicated antibodies.GST-RagB and GST-RagC vectors were co-transfected into HCT116-EGFP or HCT116-EGFP-PRL-3 stable cells. The next day, culture media was replaced to incubate cells under normoxia (24 h), hypoxia (24 h), serum-free (24 h), or amino acid-free (1 h) conditions. Lysates were harvested, clarified by high speed centrifugation . Lysates were clarified by centrifugation  before equivalent amounts of lysates (2 mg) were subjected to immunoprecipitation with a configuration-specific monoclonal antibody that specifically recognizes RheB-GTP, but not RheB-GDP. Western blotting and densitometry analysis was subsequently used to characterize the proportion of active Rheb (as reflected by the Rheb-GTP/Rheb ratio) present in cellular extracts.Analysis of intracellular Rheb-GTP levels was done using a Rheb activation assay kit (Abcam), according to the manufacturer’s instructions. Briefly, 2.5 × 104 HCT116-EGFP or HCT116-EGFP-PRL-3 cells were seeded into μ-dishes  and cultured under normal conditions until they reached confluence. Subsequently, inserts were removed to yield standardized 500 μm-wide gaps. ‘Wounded’ cell monolayers were washed in PBS and subsequently cultured in low-serum medium (0.5% FBS) with 100 nM rapamycin or 0.1% DMSO for 48 h. Images were acquired sequentially every 24 h using an Axiovert 200M inverted microscope (Carl Zeiss AG).3.5 × 104 HCT116-EGFP or HCT116-EGFP-PRL-3 cells were suspended in complete medium containing 100 nM rapamycin or 0.1% DMSO and seeded into BioCoat Matrigel invasion chambers with 8.0 μm PET membranes . After incubation for 24 h at 37 °C, non-invading cells were removed from the upper surface of the membrane by a cotton swab, and membrane inserts were washed three times with PBS and fixed in 4% paraformaldehyde for 15 min. Subsequently, membranes were cut, mounted inverted on glass slides, and observed for EGFP fluorescence under an LSM700 confocal microscope (Carl Zeiss AG). The numbers of invaded cells in four ramdomly-chosen fields were counted. Data were presented as mean ± SD of cell number/field and statistically analyzed using the Student’s t-test.1 × 10Human gastric tissue samples were obtained with patient consent from the National University Hospital-National University of Singapore (NUH-NUS) Tissue Repository. Experimental procedures were approved by the Institutional Review Board (IRB) of NUH-NUS for research use and conducted in accordance with approved guidelines and regulations.For the isolation of mammary tissue lysates, the uppermost pair of mammary glands from wild-type FVB/N mice or MMTV-PyMT mice  were surgically removed, rinsed in PBS, and homogenised in RIPA lysis buffer using a Polytron homogenizer  prior to western blot analysis. All animal studies were approved by an Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with approved guidelines and regulations of the Biological Resource Centre, A*STAR, Singapore.n = 566) were performed using SPSS 19.0 software . Correlation between PRL-3 and MMP-2, MMP-7, or MMP-9 gene expression was analysed by Spearman Correlation. The association between PRL-3 expression and relapse-free survival (RFS) was analysed by Kaplan-Meier analysis.Statistical analyses of the colon cancer patient dataset GSE40967 .