Many studies have shown that maternal embryonic leucine zipper kinase (MELK) plays a critical role in the proliferation and development of cancer cells. 4 As a result, MELK has become a potential molecular target for cancer therapy, including liver cancer and breast cancer. 5,6 A well-known MELK inhibitor, OTS167, has been reported to exhibit effective growth suppression in mice xenograft models for a number of cancer types, including lung cancer, kidney cancer, and acute myeloid leukemia. 7–11 Currently, some clinical studies, such as NCT01910545 and NCT02926690, are being performed to evaluate the therapeutic potential of OTS167. 12,13 For example, Chung and his colleagues have shown that an orally administrative MELK-targeting inhibitor could suppress the growth of tumor-initiating cells and has the potential to be widely used for the treatment of a variety of cancer types, including PC. 14 As the role
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Not only cancer stem cell markers, but other MELK-related proteins such as DEPDC1, and FOXM1 as well as major tumor-suppressive proteins, p21 and p53, also showed drastic alterations in their protein levels with treatment of OTS167 both in cell lines and in xenograft tumor tissues. It is reported that suppression of MELK by siRNA activated the p53 pathway and induced cell cycle arrest , and our results also indicated the strong activation of p53 and p21 in p53 wild-type cancer cells with treatment of MELK inhibitor. However, the activation of p21 was also observed in p53-mutant cells although it was relatively weak in p53-mutated cancer cells. These data indicated that p21 might be activated by MELK suppression regardless to the p53 status. As described above, cancer cells with p53 wild-type showed relatively higher sensitivity to OTS167 than p53-mutant cells. Although further validation is required, MELK inhibition may be enhanced in the presence of wild-type p53 since p53 can activate multiple downstream genes involved in growth arrest or apoptosis . Moreover, changes in the levels of proteins examined here were observed even at an early time-point of the treatment and could be used for monitoring the antitumor effect. Thus, the clinical efficacy in the patients who will be treated with OTS167 might be predictable by the analysis of these markers in biopsy samples at the relatively early stage of treatment.
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Maternal embryonic leucine zipper kinase (MELK), that plays a critical role in maintenance of cancer stem cells (CSCs), is predominantly expressed in various types of human cancer including small cell lung cancer (SCLC). SCLC usually acquires resistance to anti-cancer drugs and portends dismal prognosis. We have delineated roles of MELK in development/progression of SCLC and examined anti-tumor efficacy of OTS167, a highly potent MELK inhibitor, against SCLC. MELK expression was highly upregulated in both SCLC cell lines and primary tumors. siRNA-mediated MELK knockdown induced significant growth inhibition in SCLC cell lines. Concordantly, treatment with OTS167 exhibited strong cytotoxicity against eleven SCLC cell lines with IC 50 of < 10 nM. As similar to siRNA knockdown, OTS167 treatment induced cytokinetic defects with intercellular bridges, and in some cell lines we observed formation of neuronal protrusions accompanied with increase of a neuronal differentiation marker (CD56), indicating that the compound induced differentiation of cancer cells to neuron- like cells. Furthermore, the MELK inhibition decreased its downstream FOXM1 activity and Akt expression in SCLC cells, and led to apoptotic cell death. OTS167 appeared to be more effective to CSCs as measured by the sphere formation assay, thus MELK inhibition might become a promising treatment modality for SCLC.
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We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also reported to be highly upregulated in multiple types of human cancer. It was implied to play indispensable roles in cancer cell survival and indicated its involvement in the maintenance of tumor-initiating cells. We conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC 50 of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin- like), which we identified as novel MELK substrates and are important for stem-cell characteristics and invasiveness. The compound suppressed mammosphere formation of breast cancer cells and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. This MELK inhibitor should be a promising compound possibly to suppress the growth of tumor-initiating cells and be applied for treatment of a wide range of human cancer.
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The two molecules, TOPK and MELK, have shown similar expression patterns; they are up-regulated in various types of cancer including cancer stem cell- enriched tumors and more importantly their expressions are hardly detectable in normal organs except in the testis [11, 20]. Moreover, MELK expression levels were strongly correlated with those of forkhead box protein M1 (FOXM1) known as an important transcriptional factor and a master regulator of mitosis in cancer stem cells [21, 22]. These results suggest a possible close link among TOPK, MELK, and FOXM1 in a growth regulation pathway in cancer cells, which may provide a new strategy for successful treatment of cancer patients. Hence, we have developed TOPK inhibitors (OTS514 and OTS964) and a MELK inhibitor (OTS167) that showed therapeutic potentials in pre-clinical models of human cancer [23, 24].
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Given the preferential upregulation of MELK in cancers and recent experimental data suggesting the positive role of MELK on cancer cell growth, MELK has been re- cognized as a potential therapeutic target for brain, colo- rectal, lung, and ovarian cancers . In light of these preclinical studies, novel therapeutics have been devel- oped to selectively target MELK in cancers. As demon- strated by Compound 1 (C1) and OTSSP167, discoveries of novel Small Molecule Drugs (SMDs) that inhibit MELK can be potential therapies for cancers with high levels of MELK expression [36,39,40]. For example, OTSSP167 suppressed mammosphere formation of breast cancer cells, and suppressed tumor growth in xenograft studies of breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration . Currently, OTSSP167 is in a Phase I, single-center, cohort dose escalation trial for patients with any locally advanced or metastatic solid tumor malignancies re- fractory to current treatments (clinicaltrials.gov identi- fier: NCT01910545). This study, being conducted by OncoTherapy Science Inc. at the University of Chicago since August 2013, is the first human trial of a MELK inhibitor. The final data collection for primary outcomes is estimated to occur by December 2015, the results of which are highly anticipated.
For cell culture samples, total RNA was isolated using TRIZOL reagent (Invitrogen, Paisley, UK). For clinical samples, paired tumour and adjacent normal breast epithelial tissues were col- lected from a total of 21 female patients with ductal breast cancer, rapidly frozen and stored at -140°C. All samples were examined histologically, and samples grossly contaminated with adipocytes, or with noncancerous tissue in the case of tumour samples, were excluded from the study. Total RNA was isolated using 1.4 M guanidine thiocyanate/0.5% sodium dodecyl sulphate/25 mM ethylenediamine tetraacetic acid/50 mM Tris–Cl (pH 7.5) [24,25]. For all samples, isolated RNA was treated with RQ1 RNase-free DNase (Promega, South- ampton, Hampshire, UK), and was reverse transcribed using random hexamer priming and SuperScript II Reverse Tran- scriptase (Invitrogen), according to the supplied protocols. Real-time PCR was conducted using the Sensimix (dT) DNA kit (Quantace, Finchley, London, UK) and Taq Man Gene Expression Assays (assay codes Hs00609872_g1 for Fau, Hs00373302_m1 for Bcl-G, Hs00207681_m1 for MELK, Hs00167441_m1 for ALAS1, and Hs99999901_m1 for 18S; Applied Biosystems, Foster City, CA, USA), as recommended by the manufacturers, and was run on an ABI Prism Sequence
Tabel 1 laat het rekenschema zien, dat vastgesteld is om de praktische kostprijs voor biologische melk te berekenen (Evers et al., 2008). De opbouw van deze praktische kostprijs is in principe gebaseerd op de berekening van de kritieke melkprijs. De kritieke melkprijs is de prijs die een veehouder voor zijn melk moet ontvangen om juist aan al zijn betalingsverplichtingen te voldoen. Hierbij gaat het alleen om uitgaven en inkomsten en worden berekende kostenposten (kosten die geen uitgaven zijn) volledig buiten beschouwing gelaten.
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Results: Of the 84 patients, 55 (65.1%) had high-grade serous carcinomas. Seventy-three (86.7%) patients underwent NGS at the time of diagnosis, and 11 (13.3%) underwent NGS upon relapse. The most common genetic alterations were in TP53 (64%), PIK3CA (15%), and BRCA1/2 (13%), arising as single nucleotide variants and indels. MYC amplification (27%) was the most common copy num- ber variation and fusion. Fifty-seven (67.9%) patients had more than one actionable alteration other than TP53. Seven (8.3%) cases received matched-target therapy based on the following sequencing results: BRCA1 or 2 mutation, poly ADP ribose polymerase inhibitor (n=5); PIK3CA mutation, AKT inhibitor (n=1); and MLH1 mutation, PD-1 inhibitor (n=1). Fifty-three (63.0%) patients had a possibility of treatment change, and 8 (9.5%) patients received genetic counseling.
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MELK is also known as murine protein serine/threo- nine kinase 38 (MPK38)  and Eg3 protein (pEg3) . MELK is a cell cycle-dependent protein kinase that belongs to the KIN1/PAR-1/MARK family of serine- threonine kinases [15–17]. It is localised in the cyto- plasm and nucleus during interphase and at the cell cortex during anaphase and telophase . MELK, activated via autophosphorylation, phosphorylates sev- eral substrates which modulates intracellular signalling in several cellular and biological processes. Further- more, MELK interacts by binding to the following mol- ecules: a) cell cycle protein (CDC25B), inducing cell accumulation in G2 , b) zinc finger-like protein 9 (ZPR9), a physiological substrate of MELK kinase in vivo , c) mitosis-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), enhancing kinase activity , d) members of the Bcl-2 family of proapoptotic genes (Bcl-GL), conferring resistance to apoptosis [21, 22], e) inhibitor of protein Ser/Thr phosphatase-1 (NIPP1), transcription and splicing fac- tor, regulating cell cycle progression through pre-mRNA processing ,f ) PDK1, an enzyme responsible for Akt/ PKB loop activation, inhibiting its activity and function, g) Smad proteins, Smad2, Smad3, Smad4, and Smad7, intra- cellular signalling mediators of the TGF-β signalling pathway, regulating their activity , and h) p53, tumour suppressor, enhancing p53-dependent apoptosis and arresting cycle cell by modulating p53 stability .
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To find drug- likeness of inhibitors we used OSIRIS Property Explorer tool. It allows you draw chemical structure of compounds and calculates various drug relevant properties such as Molecular weight, toxicity risk assessment, clogp, solubility logS, drug likeness and drug likeness score. According to prediction, compounds with higher Molecular weights are less likely to be absorbed and therefore to ever reach the place of action. Drugs those have molecular weight below 450 are more promising. In the present study all the inhibitors which show minimum docking score possess low molecular weight (>450). Toxicity risk assessment is an indication about a compound whether it is mutagenic, tumourigenic, irritant effect or it posses any reproductive effect. In our study most of the compounds showed LR except PKR inhibitor, Akt Inhibitor V, Triciribine and STO-609. The logP value of a compound, which is the logarithm of its partition coefficient between n-octanol and water log(c octanol /c water ), is a well established measure of the
PDE-5 inhibitor, sildenafil having strong proconvulsant activity in MES and INH induced animal models of epilepsy and also PDE-3 & 4 inhibitors, such as cilostazol and rolipram possess less proconvulsant action. The probable mode of action may be the cGMP levels elevated by certain excitatory amino acids and may allow one to imply that an excitable state exists in the neuronal cells through the action of the G-protein as well as the ion channel. Although the findings here seem to indicate divergent features of the excitable neuronal cells, they may provide clues to observing a close association of receptors, enzymes and channels of the membranes in exploring the provocation of seizures.
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