Alpha-tubulin acetyltransferase/MEC-17 regulates cancer cell migration and invasion through epithelial-mesenchymal transition suppression and cell polarity disruption.

MEC-17, a newly identified alpha-tubulin-N-acetyltransferase 1, serves as the major α-tubulin acetyltransferase to promote α-tubulin acetylation in vitro and in vivo. Alteration of α-tubulin acetylation may be involved in morphology regulation, cell migration, and tumour metastasis. However, MEC-17's role in cell physiology and its effect on epithelial-mesenchymal transition (EMT) and cell polarity remain elusive. In the present study, we characterized the overexpressed or downregulated cell models through gene targeting as MEC-17 gain- or loss-of-function. Overexpression of MEC-17 enhanced the cell spreading area, suppressed pseudopods formation in a three-dimensional (3D) culture system, and inhibited cancer cell migratory and invasive ability and tumour metastasis by orthotopic lung cancer animal model. Furthermore, morphological change and migration inhibition of cancer cells were accompanied by EMT repression, Golgi reorientation, and polarity disruption caused by alteration of cdc42 activity via a decrease in Rho-GAP, ARHGAP21. By contrast, a reduction in endogenous MEC-17 accelerated the pseudopods formation and EMT, and facilitated cell migration and invasion. These results demonstrated the crucial role of MEC-17 in the modulation of intrinsic cell morphogenesis, migration, and invasive function through regulation of EMT and cell polarity.

MPT0B098, a Microtubule Inhibitor, Suppresses JAK2/STAT3 Signaling Pathway through Modulation of SOCS3 Stability in Oral Squamous Cell Carcinoma.

Microtubule inhibitors have been shown to inhibit Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in various cancer cells. However, little is known of the mechanism by which the microtubule inhibitors inhibit STAT3 activity. In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC). Treatment of various OSCC cells with MPT0B098 induced growth inhibition, cell cycle arrest and apoptosis, as well as increased the protein level of SOCS3. The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity. The inhibition of STAT3 activity led to sensitization of OSCC cells to MPT0B098 cytotoxicity, indicating that STAT3 is a key mediator of drug resistance in oral carcinogenesis. Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells. Taken together, our results provide a novel mechanism for the action of MPT0B098 in which the JAK2/STAT3 signaling pathway is suppressed through the modulation of SOCS3 protein level. The findings also provide a promising combinational therapy of MPT0B098 for OSCC.

Azaindolylsulfonamides, with a more selective inhibitory effect on histone deacetylase 6 activity, exhibit antitumor activity in colorectal cancer HCT116 cells.

A series of indolylsulfonylcinnamic hydroxamates has been synthesized. Compound 12, (E)-3-(3-((1H-pyrrolo[2,3-b]pyridin-1-yl)sulfonyl)phenyl)-N-hydroxyacrylamide, which has a 7-azaindole core cap, was shown to have antiproliferative activity against KB, H460, PC3, HSC-3, HONE-1, A549, MCF-7, TSGH, MKN45, HT29, and HCT116 human cancer cell lines. Pharmacological studies indicated that 12 functions as a potent HDAC inhibitor with an IC50 value of 0.1 µM. It is highly selective for histone deacetylase 6 (HDAC6) and is 60-fold more active than against HDAC1 and 223-fold more active than against HDAC2. It has a good pharmacokinetic profile with oral bioavailability of 33%. In in vivo efficacy evaluations in colorectal HCT116 xenografts, compound 12 suppresses tumor growth more effectively than SAHA (1, N-hydroxy-N'-phenyloctanediamide) and is therefore seen as a suitable candidate for further investigation.

MPT0B098, a novel microtubule inhibitor that destabilizes the hypoxia-inducible factor-1α mRNA through decreasing nuclear-cytoplasmic translocation of RNA-binding protein HuR.

Microtubule inhibitors have been shown to inhibit hypoxia-inducible factor-1α (HIF-1α) expression through inhibition translation or enhancing protein degradation. Little is known of the effect of microtubule inhibitors on the stability of HIF-1α mRNA. We recently discovered a novel indoline-sulfonamide compound, 7-aryl-indoline-1-benzene-sulfonamide (MPT0B098), as a potent microtubule inhibitor through binding to the colchicine-binding site of tubulin. MPT0B098 is active against the growth of various human cancer cells, including chemoresistant cells with IC50 values ranging from 70 to 150 nmol/L. However, normal cells, such as human umbilical vein endothelial cells (HUVEC), exhibit less susceptibility to the inhibitory effect of MPT0B098 with IC50 of 510 nmol/L. Similar to typical microtubule inhibitors, MPT0B098 arrests cells in the G2-M phase and subsequently induces cell apoptosis. In addition, MPT0B098 effectively suppresses VEGF-induced cell migration and capillary-like tube formation of HUVECs. Distinguished from other microtubule inhibitors, MPT0B098 not only inhibited the expression levels of HIF-1α protein but also destabilized HIF-1α mRNA. The mechanism of causing unstable of HIF-1α mRNA by MPT0B098 is through decreasing RNA-binding protein, HuR, translocation from the nucleus to the cytoplasm. Notably, MPT0B098 effectively suppresses tumor growth and microvessel density of tumor specimens in vivo. Taken together, our results provide a novel mechanism of inhibiting HIF-1α of a microtubule inhibitor MPT0B098. MPT0B098 is a promising anticancer drug candidate with potential for the treatment of human malignancies.

ROS-mediated p38alpha MAPK activation and ERK inactivation responsible for upregulation of Fas and FasL and autocrine Fas-mediated cell death in Taiwan cobra phospholipase A(2)-treated U937 cells.

The aim of the present study is to explore the signaling pathway associated with Naja naja atra phospholipase A(2) (PLA(2))-induced apoptotic death of human leukemia U937 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, and cytochrome c release were observed in PLA(2)-treated cells. PLA(2) treatment increased Fas and FasL protein expression, and upregulated transcription of Fas and FasL mRNA. Upon exposure to PLA(2), ROS generation, p38 MAPK activation, and ERK inactivation were found in U937 cells. Abolition of PLA(2)-induced ROS generation abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored ERK activation and viability of PLA(2)-treated cells. Block of p38 MAPK by SB202190 abolished PLA(2)-induced Fas/FasL upregulation and ERK inactivation, but not ROS generation. Activated ERK suppressed p38 MAPK activation and Fas/FasL protein expression. Selective inactivation or overexpression of p38alpha MAPK proved that upregulation of Fas/FasL and ERK inactivation were related to p38alpha MAPK activation. Deprivation of catalytic activity with PLA(2) blocked completely PLA(2)-induced Fas/FasL upregulation. Downregulation of FADD abolished PLA(2)-induced procaspase-8 degradation and rescued viability of PLA(2)-treated cells. Taken together, our results indicate that Fas/FasL upregulation in PLA(2)-treated U937 cells is elicited by ROS-mediated p38alpha MAPK activation and ERK inactivation, and suggest that autocrine Fas/FasL apoptotic mechanism is involved in PLA(2)-induced cell death. J. Cell. Physiol. 219: 642-651, 2009. (c) 2009 Wiley-Liss, Inc.

Genetic organization of Bungarus multicinctus protease inhibitor-like proteins.

The structural organization of the genes encoding Bungarus multicinctus protease inhibitor-like proteins (PILPs), PILP-1, PILP-2 and PILP-3, are reported in this study. Unlike PILP-2 and PILP-3, recombinant PILP-1 exhibited inhibitory activity on trypsin. PILP genes and B chain genes shared identical organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 chain and B2 chain genes were absent in that of PILP genes. Noticeably, intronic insertion in the second intron of B chain genes appeared in the promoter region of PILP-1 gene, but not in that of PILP-2 and PILP-3 genes. Comparative analyses of PILP genes and B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that PILP genes and B chain genes originate from a common ancestor, and that accelerated evolution may diversify PILP and B chain genes as that proposed for snake venom phospholipase A(2), neurotoxin and cardiotoxin genes.

B chain is a functional subunit of beta-bungarotoxin for inducing apoptotic death of human neuroblastoma SK-N-SH cells.

beta-Bungarotoxin (beta-Bgt), a presynaptic phospholipase A(2) (PLA(2)) neurotoxin isolated from the venom of Bungarus multicinctus, consists of A chain and B chain. The goal of the present study is to explore the functional contribution of the two subunits to the toxicity of beta-Bgt. beta-Bgt was found to induce apoptotic death of SK-N-SH cells via elevating intracellular Ca(2+) and intracellular ROS production. Moreover, an activation of p38 MAPK was associated with the cytotoxicity of beta-Bgt. SB202190 (p38 MAPK inhibitor), N-acetylcysteine (antioxidant reagent), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) (Ca(2+) chelator) and the inhibitors of Ca(2+) release from intracellular depots (ruthenium red and 2-aminoethoxydiphenyl borate) effectively attenuated the cytotoxicity of beta-Bgt. In sharp contrast to the inability of A chain, B chain was able to induce cytotoxic effects on SK-N-SH cells as beta-Bgt did. Abolishment of PLA(2) activity did not significantly alter the cytotoxic activity of beta-Bgt. MK801 (an NMDA receptor antagonist), antibodies against NMDA receptor and 4-aminopyridine (a potassium channel blocker) markedly reduced the cytotoxic effects of beta-Bgt, B chain and catalytically inactivated beta-Bgt. Moreover, antibodies against NMDA receptor blocked the binding of rhodamine-labeled beta-Bgt to SK-N-SH cells. Taken together, our data indicate that B chain is a functional subunit responsible for the cytotoxicity of beta-Bgt, and suggest that the cytotoxicity of beta-Bgt is mediated by NMDA receptor and potassium conductance.

Mutations on the N-terminal region abolish differentially the enzymatic activity, membrane-damaging activity and cytotoxicity of Taiwan cobra phospholipase A2.

To examine the functional contribution of the N-terminal region to the activities of Naja naja atra phospholipase A(2) (PLA(2)), studies on three N-terminally mutated PLA(2) were carried out in the present work. Removal of N-terminal heptapeptide caused a complete loss of membrane-damaging activity, whilst the mutants with an extra Met before Asn-1 or substituting Asn-1 with Met still retained approximately 40.9% and 82.9% membrane-damaging activity of the native enzyme, respectively. Mutations on the N-terminal region did not greatly affect the Ca(2+)-binding ability but caused a precipitous drop in PLA(2) activity. Moreover, the gross conformation of the mutants was different from that of the native enzyme as revealed by CD spectra. Nevertheless, the mutants as well as native PLA(2) induced apoptotic death of U937 cells, and the cytotoxicity of mutants was similar to or even greater than that of the native PLA(2). These results indicate that mutations on the N-terminus abolish the enzymatic activity, membrane-damaging activity and cytotoxicity of N. naja atra PLA(2) in different ways, and suggest a feasible approach to selective elimination of the multiple activities of PLA(2) enzymes.

Modification of Lys-6 and Lys-65 affects the structural stability of Taiwan cobra phospholipase A2.

To assess whether chemical modification of phospholipase A(2) (PLA(2)) enzymes may affect their fine structure and consequently alter their enzymatic activity, the present study was carried out. Both Lys-6 and Lys-65 in the Taiwan cobra (Naja naja atra) PLA(2) were selectively modified with trinitrobenzene sulfonate and pyridoxal-5'-phosphate (PLP), respectively. Incorporation of either trinitrophenylated (TNP) or PLP groups on Lys-6 and Lys-65 caused a drop in PLA(2) activity, but the Ca(2+)-binding ability and global conformation of modified derivatives were not significantly different from that of native enzyme. A distinct enhancement of stability was observed with native PLA(2) when thermal unfolding was conducted in the presence of 20 mM Ca(2+). Conformational transition induced by guanidine hydrochloride was also attenuated by the addition of Ca(2+). Conversely, a marked decrease in the structural stability was noted with modified derivatives, and the enhancing effect of Ca(2+) pronouncedly decreased. Together with the finding that the incorporated TNP and PLP groups did not equally affect enzymatic activity and structural stability of PLA(2), our data suggest that an alteration in the fine structure owing to the incorporated groups should contribute to the observed decrease in PLA(2) activity.

Mutagenesis studies on the N-terminus and Thr54 of Naja naja atra (Taiwan cobra) chymotrypsin inhibitor.

Ala-screening mutagenesis studies on Arg1, Pro2, Arg3, Phe4 and Thr54 of Naja naja atra (Taiwan cobra) chymotrypsin inhibitor showed that inhibitory potency and gross conformation of the mutants were not significantly different from those of wild-type inhibitor. Nevertheless, the R1A mutant had an appreciable decrease in the structural stability underlying thermal unfolding and urea-induced denaturation. Alternatively, deleting the first three residues at the N-terminus caused a reduction in structural stability as well as inhibitory potency. In sharp contrast to wild-type and other mutated inhibitors, R1A mutant and truncated mutant completely lost their inhibitory activity when the inhibitors were incubated with chymotrypsin for periods of up to 3 h. The loss of activity correlated with chymotryptic cleavage of inhibitors as evidenced by SDA-PAGE. Taken together, these results reflect that the globally structural rigidity of N. naja atra chymotrypsin inhibitor functionally affects the sustainable period in inhibiting chymotrypsin activity, and that the intact N-terminus might contribute to this event.

Divergence of genes encoding B chains of beta-bungarotoxins.

The structural organization of the genes encoding B2, B4, B5 and B6 chains of beta-bungarotoxins are reported in this study. These genes shared virtually identical overall organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 and B2 chains were absent in that of B4, B5 and B6 chains. RT-PCR analyses indicated that Bungarus multicinctus venom gland, heart, liver and muscle expressed the RNA transcripts showing sequence similarity with the intronic segment being deleted in B4, B5 and B6 chain genes. This reflects that the ancestral gene of the intronic segment might insert in multiple loci of B. multicinctus genome. Comparative analyses of B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that intron insertions or deletions occur with the evolution of B chains, and that accelerated evolution may diversify the protein-coding sequence of B chain genes same as snake phospholipase A2, neurotoxin and cardiotoxin genes.

Taiwan cobra chymotrypsin inhibitor: cloning, functional expression and gene organization.

A cDNA encoding chymotrypsin inhibitor was constructed from the cellular RNA isolated from the venom glands of Naja atra (Taiwan cobra). The resultant amino acid sequence showed that the mature protein is comprised of 57 amino acid residues with six cysteine residues. Cloned protein was expressed and isolated from the inclusion bodies of E. coli and refolded into a functional protein in vitro. Deleting the first three residues at its N-terminus caused a moderate increase in the inhibitory constant (K(i)) against chymotrypsin. The genomic DNA encoding the chymotrypsin inhibitor was amplified by PCR. The gene shares virtually an identical structural organization with the beta-bungarotoxin B1 chain (a snake Kunitz/BPTI neurotoxic homolog) gene. Moreover, the overall sequence identity of the N. atra chymotrypsin inhibitor and beta-bungarotoxin B1 chain genes was up to 83%. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.

The structural events associated with the binding of divalent cations to beta-bungarotoxin.

In order to address the mechanism why the Ca2+ was crucial for the manifestation of the phospholipase A2 (PLA2) activity of beta-bungarotoxin (beta-BuTx), four divalent cations were used to assess their influences on the catalytic activity and the fine structures of beta-BuTx. Substitution Mg2+ or Sr2+ for Ca2+ in the substrate solution was found to cause a decrease in the PLA2 activity to approximately 15 or 6% of that in the presence of Ca2+. However, only marginally detectable PLA2 activity was observed with the addition of Ba2+. The nonpolarity of 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of beta-BuTx markedly increased with the binding of cations to beta-BuTx. The negative ellipticity noted with the CD spectra of beta-BuTx increased upon the binding of cations too. With the exception of Ba2+, the order of the ability of cations to enhance the intensity of ANS fluorescence or increase the increment of negative ellipticity was Sr2+ > Ca2+ > Mg2+, which was the same order as the increase in their atomic radii. However, the energy transfer from Trp fluorescence emission to ANS was most effective upon the addition of Ca2+. Moreover, the extent of glutaraldehyde crosslinking between A chain and B chain decreased in the presence of cations. Nevertheless, the binding affinities of beta-BuTx for the four cations were similar. These results, together with the findings that the ANS molecule binds at the active site of the A chain in beta-BuTx, suggest that the binding of Ca2+ to beta-BuTx induces subtly conformational changes occurred at the active site for exerting the activity of beta-BuTx. Moreover, the change in the gross conformation induced by the binding of Ca2+ may affect the interaction between A chain and B chain, and consequently the activity of beta-BuTx as well.

Lys-64 of the A chain is involved in the enzymatic activity and neurotoxic effect of beta-bungarotoxin.

Two beta-bungarotoxin isotoxins BM12 and BM13 were isolated from Bungarus multicinctus (Taiwan banded krait) venom by sequential chromatography on ion-exchange and reverse phase columns. The two toxins have the same A chain, but different B chains. Different phospholipase A2 activity and different potencies in inhibiting the spontaneous enhancement of spontaneous synaptic current frequency and muscle contraction were observed for BM12 and BM13. Nevertheless, modification of Lys-64 in the A chain of BM12 and BM13 similarly reduced in their phospholipase A2 activity and toxicity. The modified derivatives retained their affinity with Ca2+ and their conformation as deduced by CD. These results suggest that Lys-64 of the A chain is involved in the phospholipase A2 activity and in the neurotoxic effect of beta-bungarotoxin.

Purification and characterization of Taiwan cobra venom proteins with weak toxicity.

Two proteins G2a and G2b with molecular masses of approximately 24 kDa were isolated from Naja naja atra (Taiwan cobra) venom using sequential chromatography on gel filtration, ion-exchanger and reverse phase columns. The results of Edman degradation and mass analysis revealed that G2a is a cysteine-rich protein reported previously, and G2b is a novel polypeptide. CD spectra showed that the gross conformation of G2a and G2b notably differed. G2a exhibited an activity higher than that noted with G2b on inhibiting carbachol-induced muscle contraction. However, the two proteins weakly blocked muscle contraction evoked by K+. The observations that the two proteins exhibit the toxic activity in the concentration of micromolar range suggest that they are inherently weak toxins as other snake venom cysteine-rich proteins.

Both A chain and B chain of beta-bungarotoxin are functionally involved in the facilitation of spontaneous transmitter release in Xenopus nerve-muscle cultures.

In the present study, Xenopus nerve-muscle cultures were used to explore the functional roles of A chain (a phospholipase A(2) subunit) and B chain (a non-phospholipase A(2) subunit) of Bungarus multicinctus beta-bungarotoxin. It was found that beta-bungarotoxin induced an increment of the frequency of spontaneous synaptic currents (SSCs) in the nerve-muscle cultures. Modification of beta-bungarotoxin with pyridoxal-5'-phosphate or substitution of Ca(2+) with Ba(2+) in buffer abolished the phospholipase A(2) activity of beta-bungarotoxin and the facilitatory phase of SSC as well. Antibodies that were directed specifically against A chain or B chain effectively inhibited phospholipase A(2) activity, and as a consequence the SSC frequency was not greatly different from the control rate. These results suggest that both A and B chains are indispensable parts of beta-bungarotoxin for inducing the facilitation of SSC frequency with Xenopus nerve-muscle cultures.