Human Epidermoid Carcinoma KB Cells Research Articles

Synthesis of Amphidinolide H

Amphidinolide H is a dinoflagellate metabolite with an exceptional cytotoxicity towards KB human epidermoid carcinoma cells. The closing stages of the Fürstner synthesis depicted required careful orchestration of the fragment linkage strategy and scrupulous orchestration of the protecting group regime.

Studies on the Total Synthesis of Amphidinolide O (III): A Stereoselective Synthesis of C1-C11 Fragment

The amphidinolides were isolated from the marine dinoflagellate Amphidinium sp., which produces a host of secondary metabolites endowed with potent cytotoxicity against various cancer cell lines. Amphidinolide O (1) displayed potent in vitro cytotoxicity against L1210 marine leukemia cells and human epidermoid carcinoma KB cells with 1.7 and 3.6 μg/mL of IC50s, respectively. In addition to our recent reports regarding to the synthesis of C12-C17 and C3-C11 fragments of amphidinolide O (1), we describe herein a new route to diastereoselective synthesis of C1-C11 fragment of 1. The retrosynthetic analysis of 1 led to the C1-C11 fragment 2 and C12-C17 fragment 3 (Scheme 1). The hemiketal 2 was expected from acyclic precursor 4 which, in turn, would be derived by diastereoselective aldol reaction between ketone 5 and aldehyde 6. The C1-C6 fragment 8 (equivalent to 5 in scheme 1) was prepared as summarized below (Scheme 2). Enolization of carboximide 7 with Bu2BOTf and EtN(i-Pr)2 was followed by reaction with aldehyde to provide the syn-aldol product in 86% yield (ds = > 97 : 3 by H NMR analysis) (Scheme 2). The aldol product was treated with N,O-dimethylhydroxylamine hydrochloride and Al(Me)3 to provide the Weinreb amide in 91% yield. The free hydroxyl group was protected as PMB ether, and finally Weinreb amide was converted to ketone 8 in 85% yield by reaction with MeMgCl. Synthesis of C7-C11 fragment 10 (equivalent to 6 in scheme 1) was completed via 5-step sequences (Scheme 3). Monoprotection of propane-1,3-diol (9), Swern oxidation of the remaining alcohol to aldehyde, and Wittig-olefination was undertaken to give α,β-unsaturated ester, which was subsequently reduced by DIBAL and oxidized to provide the C7-C11 fragment 10, another key intermediate in the next aldol reaction. Aldol-reactions of fragments 8 and 10 were investigated with four chiral boron reagents in ether at −78 C (Scheme 4), and the use of chlorodicyclohexylborane provided the desired product with the best diastereoselectivity (11a : 11b = 67 : 33) in 61% (11a) and 30% (11b) yield, respectively. Hydroxyl group-directed 1,3-anti reduction of either 11a or 11b with NaBH(OAc)3 provided the 5,7-anti diol 12a or 12b in 91% yield. The (5R,7S)-isomer 12a was treated with 2,2-dimethoxypropane in the presence of PPTS (Scheme 5). The PMB-

Isolation and characterization of arsenite-resistant human epidermoid carcinoma KB cells

Arsenic trioxide (As2O3) has been used with success in the treatment of acute promyelocytic leukemia. However, resistance to arsenite agents reduces their efficacy. We have isolated arsenite-resistant human epidermoid carcinoma KB cells, termed KAS. KAS cells were resistant to sodium arsenite (22-fold) and showed a reduced accumulation of arsenite as a result of an active efflux mechanism. Further analysis indicated that resistance of KAS cells extended to other drugs including cisplatin (17-fold), antimony potassium tartrate (11-fold) and doxorubicin (27-fold). Although increased expression of multidrug resistance protein 1 (MRP1) in KAS cells was confirmed by quantitative RT-PCR and immunoblot analysis, specific inhibitors of MRP1 did not completely eliminate arsenite resistance. The level of glutathione (GSH) in KAS cells was 3-fold higher than that in KB-3-1 cells, and the inhibition of GSH synthesis by buthionine sulfoximine (BSO) considerably increased the cytotoxic effect of arsenite on KAS cells. A pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridine-carboxylate P oxide (PAK-104P), partially reversed the arsenite resistance and increased the arsenite accumulation in KAS cells. We suggest that the increased level of GSH is involved in arsenite resistance and an as yet unidentified arsenite transporter is expressed in the arsenite-resistant KAS cells.

Prenylterphenyllin and Its Dehydroxyl Analogs, New Cytotoxic Substances from a Marine-derived Fungus Aspergillus candidus IF10

Three novel cytotoxic substances named prenylterphenyllin (1), 4''-deoxyprenylterphenyllin (2), and 4''-deoxyisoterprenin (3) were isolated from a cultured marine-derived fungus of Aspergillus candidus IF10 together with 4''-deoxyterprenin (4). Their chemical structures were elucidated on the basis of 2D NMR analysis. These compounds 1 approximately 4 showed cytotoxic activity against human epidermoid carcinoma KB cells (KB3-1) with IC(50) of 8.5, 3.0, 2.5, and 4.5 microg/ml, respectively.

Synthesis, antitumor activity and structure–activity relationships of a series of Ru(II) complexes

A series of octahedral Ru(II) polypyridyl complexes, [Ru(phen) 2L] 2+ (L = R-PIP and PIP = 2-phenylimidazo[4,5- f][1,10]phenanthroline) were synthesized and characterized by elementary analysis, 1H NMR and ES-MS, as well as UV–visible spectra and emission spectra. The antitumor activities of these complexes and their corresponding ligands were investigated against mouse leukemia L1210 cells, human oral epidermoid carcinoma KB cells, human promyelocytic leukemia cells (HL-60) and Bel-7402 liver cancer cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. It was found that the complexes [Ru(phen) 2L] 2+ (L = R-PIP) exert rather potent activities against all of these cell lines, especially for the KB cells (IC 50 = 4.7 ± 1.3 μM). The binding affinities of these Ru(II) complexes to CT-DNA (calf thymus DNA), as well as the DNA-unwinding properties on supercoiled pBR322 DNA were also investigated. The results showed that these Ru(II) polypyridyl complexes not only had an excellent DNA-binding property but also possessed a highly effective DNA-photocleavage ability. The structure–activity relationships and antitumor mechanism were also carefully discussed.

C35 Terpenoids from the Bark of Calocedrus macrolepis var. formosana with Activity against Human Cancer Cell Lines

Ferrugicadinol (1) and ferrugieudesmol (2), two new compounds with a unique C35 terpene skeleton, were isolated from the bark of Calocedrus macrolepis var. formosana. Their structures were elucidated principally from spectroscopic evidence. The two new C35 terpenes showed cytotoxicity against human oral epidermoid carcinoma KB cells with IC50 values ranging from 11 to 14 microM.

Involvement of PPARγ in Oxidative Stress-Mediated Prostaglandin E2 Production in SZ95 Human Sebaceous Gland Cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) is thought to play a role in sebaceous gland cell function. We previously demonstrated in human epidermoid carcinoma KB cells that UVB irradiation activates PPARgamma via the generation of multiple oxidized glycerophosphocholine species with PPARgamma ligand activity. UVB-induced cyclooxygenase 2 (COX-2) expression was also shown to be PPARgamma-dependent. We therefore reasoned that PPARgamma activation and PPARgamma-dependent COX-2 expression may occur as a general consequence of oxidative stress. The present studies were designed to examine the effects of the oxidant tert-butylhydroperoxide (TBH) on PPARgamma activation and COX-2 expression in SZ95 sebocytes. We first verified that functional PPARgamma is expressed and activated by UVB irradiation in these cells. We next demonstrated that TBH increased PPARgamma reporter activity in SZ95 sebocytes. Increased COX-2 protein, mRNA expression, and prostaglandin E(2) (PGE(2)) production was observed after TBH or PPARgamma agonist treatment. The ability of PPARgamma agonists and TBH to induce COX-2 expression and PGE(2) production was blocked by pretreatment with the specific PPARgamma antagonist GW9662. Finally, TBH and PPARgamma agonists failed to elicit a PGE(2) response in SZ95 sebocytes stably expressing a dominant-negative PPARgamma. This study illustrates the importance of the PPARgamma system in regulating cellular responses to oxidative stress.

Synthesis of Amphidinolide A

Amphidinolides are marine natural products displaying high cytotoxicity towards murine lymphoma L1210 and human epidermoid carcinoma KB cells.

Studies on the Total Synthesis of Amphidinolide O (II): A Stereoselective Synthesis of C1-C11 Fragment

The amphidinolides were isolated from the marine dinoflagellate Amphidinium sp., which produces a host of secondary metabolites endowed with potent cytotoxicity against various cancer cell lines. Amphidinolide O (1) displayed potent in vitro cytotoxicity against L1210 marine leukemia cells and human epidermoid carcinoma KB cells with 1.7 and 3.6 μg/mL of IC50s, respectively. In addition to our recent reports regarding to the synthesis of C12-C17 and C3-C11 fragments of amphidinolide O (1), we describe herein a new route to diastereoselective synthesis of C1-C11 fragment of 1. The retrosynthetic analysis of 1 led to the C1-C11 fragment 2 and C12-C17 fragment 3 (Scheme 1). The hemiketal 2 was expected from acyclic precursor 4 which, in turn, would be derived by diastereoselective aldol reaction between ketone 5 and aldehyde 6. The C1-C6 fragment 8 (equivalent to 5 in scheme 1) was prepared as summarized below (Scheme 2). Enolization of carboximide 7 with Bu2BOTf and EtN(i-Pr)2 was followed by reaction with aldehyde to provide the syn-aldol product in 86% yield (ds = > 97 : 3 by H NMR analysis) (Scheme 2). The aldol product was treated with N,O-dimethylhydroxylamine hydrochloride and Al(Me)3 to provide the Weinreb amide in 91% yield. The free hydroxyl group was protected as PMB ether, and finally Weinreb amide was converted to ketone 8 in 85% yield by reaction with MeMgCl. Synthesis of C7-C11 fragment 10 (equivalent to 6 in scheme 1) was completed via 5-step sequences (Scheme 3). Monoprotection of propane-1,3-diol (9), Swern oxidation of the remaining alcohol to aldehyde, and Wittig-olefination was undertaken to give α,β-unsaturated ester, which was subsequently reduced by DIBAL and oxidized to provide the C7-C11 fragment 10, another key intermediate in the next aldol reaction. Aldol-reactions of fragments 8 and 10 were investigated with four chiral boron reagents in ether at −78 C (Scheme 4), and the use of chlorodicyclohexylborane provided the desired product with the best diastereoselectivity (11a : 11b = 67 : 33) in 61% (11a) and 30% (11b) yield, respectively. Hydroxyl group-directed 1,3-anti reduction of either 11a or 11b with NaBH(OAc)3 provided the 5,7-anti diol 12a or 12b in 91% yield. The (5R,7S)-isomer 12a was treated with 2,2-dimethoxypropane in the presence of PPTS (Scheme 5). The PMB-

A novel bis-benzylidenecyclopentanone derivative, BPR0Y007, inducing a rapid caspase activation involving upregulation of Fas (CD95/APO-1) and wild-type p53 in human oral epidermoid carcinoma cells

BPR0Y007, a bis-benzylidenecyclopentanone derivative (2,5-bis- (4-hydroxy-3-methoxybenzylidene) cyclopentanone), was identified in our laboratory as a novel antineoplastic agent with a broad spectrum of antitumor activity against many human cancer cells. A previous study showed that BPR0Y007 inhibited DNA topoisomerase I (Top 1) activity and prevented tubulin polymerization. Notably, no cross-resistance with BPR0Y007 was observed in camptothecin-, VP-16- or vincristine-resistant cell lines. In this study, we further investigated the cellular and molecular events underlying the antitumoral function of this compound in human oral epidermoid carcinoma KB cells, focusing on the early cytotoxic effect. Treatment of KB cells with BPR0Y007-induced G 2/M phase arrest followed by sub-G 1 phase accumulation. Annexin-V–propidium iodide (PI) binding assay and DNA fragmentation assay further indicated that BPR0Y007-induced cell death proceeded through an apoptotic pathway as opposed to via necrosis. This compound produced a time-dependent activation of caspases-3 and -8, however, another caspase-3 initiator, caspase-9, was only marginally activated at later time point. We further demonstrated that the activation of the caspases cascade and nuclear fragmentation was not associated with inactivated Bcl-2 and perturbed mitochondrial membrane potential by BPR0Y007. The finding that BPR0Y007-induced apoptosis through a membrane-mediated mechanism was supported by up-regulated expression of Fas (CD95/APO-1), but not Fas-L. Furthermore, up-regulation of p53 and its affected gene, MDM2, in KB cells was found after BPR0Y007 exposure. Overall, our results demonstrated that the BPR0Y007 could induce an early cytotoxic apoptosis through a caspase-8-dependent but mitochondrial-caspase-9 independent pathway, and involving upregulation of p53.

A novel bis-benzylidenecyclopentanone derivative, BPR0Y007, inducing a rapid caspase activation involving upregulation of Fas (CD95/APO-1) and wild-type p53 in human oral epidermoid carcinoma cells

BPR0Y007, a bis-benzylidenecyclopentanone derivative (2,5-bis- (4-hydroxy-3-methoxybenzylidene) cyclopentanone), was identified in our laboratory as a novel antineoplastic agent with a broad spectrum of antitumor activity against many human cancer cells. A previous study showed that BPR0Y007 inhibited DNA topoisomerase I (Top 1) activity and prevented tubulin polymerization. Notably, no cross-resistance with BPR0Y007 was observed in camptothecin-, VP-16- or vincristine-resistant cell lines. In this study, we further investigated the cellular and molecular events underlying the antitumoral function of this compound in human oral epidermoid carcinoma KB cells, focusing on the early cytotoxic effect. Treatment of KB cells with BPR0Y007-induced G 2 /M phase arrest followed by sub-G 1 phase accumulation. Annexin-V–propidium iodide (PI) binding assay and DNA fragmentation assay further indicated that BPR0Y007-induced cell death proceeded through an apoptotic pathway as opposed to via necrosis. This compound produced a time-dependent activation of caspases-3 and -8, however, another caspase-3 initiator, caspase-9, was only marginally activated at later time point. We further demonstrated that the activation of the caspases cascade and nuclear fragmentation was not associated with inactivated Bcl-2 and perturbed mitochondrial membrane potential by BPR0Y007. The finding that BPR0Y007-induced apoptosis through a membrane-mediated mechanism was supported by up-regulated expression of Fas (CD95/APO-1), but not Fas-L. Furthermore, up-regulation of p53 and its affected gene, MDM2, in KB cells was found after BPR0Y007 exposure. Overall, our results demonstrated that the BPR0Y007 could induce an early cytotoxic apoptosis through a caspase-8-dependent but mitochondrial-caspase-9 independent pathway, and involving upregulation of p53.

Polymeric micelles of poly(2-ethyl-2-oxazoline)-block-poly(ε-caprolactone) copolymer as a carrier for paclitaxel

Polymeric micelles based on amphiphilic block copolymers of poly(2-ethyl-2-oxazoline) (PEtOz) and poly(ε-caprolactone) (PCL) were prepared in an aqueous phase. The loading of paclitaxel into PEtOz–PCL micelles was confirmed by 1H-NMR spectra. Paclitaxel was efficiently loaded into PEtOz–PCL micelles using dialysis method, and the loading content of paclitaxel in micelles was in the range 0.5–7.6 wt.% depending on the block composition of block copolymers, organic solvent used in the dialysis, and feed weight ratio of paclitaxel to block copolymer. The higher the content of hydrophobic block in the block copolymers, the higher the loading efficiency of micelles for paclitaxel. When acetonitrile was used as solvent, a higher drug loading efficiency was obtained than with THF. The loading efficiency decreased with increasing feed weight ratio of paclitaxel to block copolymer from 0.1:1 to 0.2:1. The hydrodynamic diameters of paclitaxel-loaded micelles were in the range 18.3–23.4 nm with narrow size distribution. The hemolysis test of PEtOz–PCL performed in vitro indicated that the toxicity of PEtOz–PCLs to lipid membrane was not significant compared with Tween 80, and was comparable to that observed with Cremophore EL. The proliferation inhibition activity of paclitaxel-loaded micelles for KB human epidermoid carcinoma cells was also evaluated in vitro. Paclitaxel-entrapped polymeric micelles exhibited comparable activity to that observed with Cremophore EL-based paclitaxel formulations in inhibiting the growth of KB cells.

Suppression of cofilin phosphorylation in insulin-stimulated ruffling membrane formation in KB cells.

Various cellular events such as cell motility and division are directed by the actin cytoskeleton under the control of its regulatory system. Cofilin is a low molecular weight actin-modulating protein that severs and depolymerizes F-actin and is shown to enhance actin filament dynamics. The activity of cofilin is negatively regulated by phosphorylation at Ser-3. In human epidermoid carcinoma KB cells, insulin treatment induces characteristic ruffling membranes, and it was reported that LIMK1, a cofilin kinase, was activated in these cells treated with insulin. Since cofilin is a key protein responsible for establishing the rapid turnover of actin filaments, it appears to be contradictory that cofilin is phosphorylated (inactivated) by a stimulus that is known to induce the highly dynamic actin structure, ruffling membranes. Therefore, we examined the phosphorylation state of endogenous cofilin in KB cells treated with insulin. The dephosphorylated form of cofilin increased with insulin treatment, as analyzed by nonequilibrium pH gradient gel electrophoresis (NEpHGE)-immunoblotting. Cell labeling with (32)P orthophosphate indicated that cofilin was being continuously phosphorylated and dephosphorylated, and that the apparent insulin-induced dephosphorylation was due to suppression of continuous phosphorylation and not to enhanced dephosphorylation. Further, we examined the localization of the phosphorylated form of cofilin using phospho-specific antibody raised against phosphorylated cofilin. Surprisingly, phosphorylated cofilin was concentrated in the ruffling membranes induced by insulin. These results suggest that the examination of the kinetics and spatial regulation of phosphorylation is critical for the elucidation of the role of cofilin and upstream kinases in actin reorganization.

Cinnamamide, an antitumor agent with low cytotoxicity acting on matrix metalloproteinase.

The antitumor activity of cinnamamide (CNM), an agent acting on matrix metalloproteinase (MMP), was investigated in the present study. CNM displayed low cytotoxicity. By the MTT assay the IC50 (50% inhibitory concentration) values of CNM on cell proliferation ranged from 1.29 to 1.94 mM in human oral epidermoid carcinoma KB cells, human hepatoma BEL-7402 cells and human fibrosarcoma HT-1080 cells. Moreover, the IC50 for human fetal lung 2BS cells reached 4.33 mM. The administration of CNM in the range of 50-150 mg/kg (i.p. or p.o.) showed moderate antitumor effects in mice. When administered i.p. or p.o., CNM (150 mg/kg) inhibited the growth of transplanted hepatoma 22 by 48.8 or 40.5%, respectively. At the dose of 100 mg/kg, CNM inhibited the growth of colon 26 carcinoma by 39.0% and that of Lewis lung carcinoma by 53.9%. In the Lewis lung carcinoma model, CNM at the dose of 100 mg/kg (i.p.) also reduced the lung metastasis by 59.1%. Gelatine zymography revealed that CNM was able to decrease the level of MMP-2 in conditioned medium of HT-1080 tumor cells in a concentration-dependent manner. These results indicate that CNM is an antitumor agent with low cytotoxicity acting on MMP and may serve as a lead compound in the development of antitumor drugs.

Interferon-alpha induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor.

We have demonstrated that interferon-alpha2-recombinant (IFNalpha) at growth inhibitory concentrations enhances the expression and signalling activity of the epidermal growth factor receptor (EGF-R) in human epidermoid carcinoma KB cells. Here we report that KB cells exposed to IFNalpha underwent apoptotic cell death and this effect was antagonized by EGF. We have also found that IFNalpha enhanced the expression of heat shock proteins (HSP) HSP-70, HSP-90 and HSP-27 and activated the NH2-terminal Jun kinase-1 (JNK-1) and p38 mitogen activated protein kinase, the target enzymes of a stress-dependent intracellular transduction pathway. Moreover, the overexpression of the wild-type JNK-1, obtained through plasmid transfection of KB cells, induced apoptosis which was potentiated by the exposure of wild-type JNK-1 (JNK-1wt)-transfected cells to IFNalpha. All these effects were neutralized by the addition of EGF to parental and JNK-1wt-transfected KB cells exposed to IFNalpha. In conclusion, EGF has a protective effect on KB cells from apoptosis while antagonizing a stress response elicited by IFNalpha and targeted on the stress pathway terminal kinases.

Synthesis of 2,8-disubstituted imidazo[1,5-a]pyrimidines with potent antitumor activity.

Seventeen 1,2,3,4-tetrahydroimidazo[1,5-a]pyrimidine derivatives bearing electron-withdrawing substituents were designed and synthesized by novel ring closure as potential antitumor agents. They were screened for their activities against mouse leukemia L1210 and human oral epidermoid carcinoma KB cell lines, and relationships of structure and antitumor activity in vitro are discussed. It was found that 8-thiocarbamoyl-1,2,3,4-tetrahydroimidazo[1, 5-a]pyrimidin-2(1H)-thione (8c) exhibited activity comparable to that of 5-fluorouracil against both L1210 and KB cells. The existence of both 2-thioxo and 8-substituent with a thioxo group in the molecule is crucial for the cytotoxicity against L1210 and KB cells. A novel procedure for introduction of a double bond between C-3 and C-4 in 8c was developed. Introduction of the 3,4-double bond increased the activity against L1210, but against KB cells the activity decreased by 4-fold. Cytotoxicity of compounds 8c and 8-thiocarbamoyl-1,2-dihydroimidazo[1,5-a]pyrimidin-2(1H)-thione (11c) against human solid tumor and leukemia cell lines was further evaluated. The saturation of the 3,4-double bond led to a significant increase in cytotoxicity against tumor cell lines tested.

Accumulation of cyclin B1, activation of cyclin B1-dependent kinase and induction of programmed cell death in human epidermoid carcinoma KB cells treated with taxol

Cyclin B1 plays a critical role in regulating cell-cycle progression from G2 through M phase (including exit from M phase). In this study, we investigated the relationship between taxol-induced M-phase arrest, disruption of the cyclin B1-regulation pathway and apoptosis in KB cells. Continuous exposure of KB cells to 0.5 microg/ml taxol caused mitotic arrest and >90% cell death at 48 hr. Mitotic blockade peaked at 24 hr, with 68% of cells in mitosis at that time compared with 3% at baseline, and decreased thereafter. Apoptosis assessed by morphological changes and DNA ladder fragmentation was a later event, peaking at 48 hr (later time points were not studied). Taxol also caused an increase in cyclin B1 accumulation, as assessed by Western blot analysis, and stimulated cyclin B1-dependent kinase. Cyclin B1 accumulation and kinase stimulation peaked at 12 and 24 hr, respectively, at which times they were 5-fold and 90-fold higher than in control untreated cells. These effects decreased thereafter. All taxol-induced cellular effects were abrogated by the protein and RNA synthesis inhibitors cycloheximide and actinomycin D. In contrast, the endonuclease inhibitors aurintricarboxilic acid and zinc markedly inhibited taxol-induced DNA ladder fragmentation without altering taxol-induced cell-cycle arrest, cyclin B1 accumulation, activation of cyclin B1 kinase activity and cytotoxicity. We conclude that taxol-induced stimulation of cyclin B1-dependent kinase activity parallels mitotic arrest, is more pronounced than mitotic arrest and precedes the induction of programmed cell death.

New polyketide peroxides from Okinawan marine sponge Plakortis sp.

New polyketide peroxides 2–4, isolated from an Okinawan sponge Plakortis sp., have been fully characterized by spectroscopic and chemical methods. The absolute stereochemistries have been determined by analysis of MTPA esters of the acyclic derivatives. Compound 2 was cytotoxic against human epidermoid carcinoma KB and murine lymphoma L1210 cells.

Combinations of paclitaxel and vinblastine and their effects on tubulin polymerization and cellular cytotoxicity: characterization of a synergistic schedule.

Paclitaxel (PTX) and vinblastine (VBL) represent 2 classes of drugs that target tubulin but have separate binding properties and opposing mechanisms of action. To evaluate the potential use of these agents together in a chemotherapeutic regimen, we investigated their effects on the dynamics of tubulin polymerization and cellular cytotoxicity, when administered singly or in combination. In human epidermoid carcinoma KB cells and MCF-7 breast carcinoma cells, we observed a time- and dose-dependent effect on cytoskeletal dynamics for both PTX and VBL. Tubulin polymerization induced by PTX was stable for more than 24 hr. When PTX treatment was followed by VBL, a time- and dose-dependent reversal of tubulin polymerization was observed. In contrast, rapid tubulin polymerization occurred when VBL was followed by PTX. When both agents were added simultaneously, a diminution of PTX-induced tubulin polymerization was observed with increasing doses of VBL; a maximum reduction was achieved when equal concentrations were used. Examination of the tubulin pattern by immunofluorescence in MCF-7 breast cancer cells confirmed and extended our findings. Bundle formation followed treatment with PTX. Addition of increasing concentrations of VBL prevented bundling; however, the normal cytoskeletal architecture was not restored. Cytotoxicity studies carried out using the median dose effect principles and the combination index analysis showed synergism when VBL and PTX were administered sequentially and antagonism for simultaneous administration. Our results demonstrate changes in tubulin dynamics following drug treatment and provide a rationale for combined PTX/VBL therapy after careful evaluation of the schedule of administration.

Synthesis of green fluorescent protein-ricin and monitoring of its intracellular trafficking.

We performed genetic engineering to fuse enhanced green fluorescent protein (EGFP) to the N terminus of RTA, expressed the fusion protein in Escherichia coli, purified and reassociated EGFP-RTA with plant RTB, and purified EGFP-ricin by size exclusion HPLC. The fusion heterodimer was able to bind galactosides, intoxicate cells, and show strong fluorescence. Mammalian cells incubated with EGFP-ricin showed strong cell surface fluorescence at 4 degrees C and, on incubation at 37 degrees C, distributed initially to endosomes and then to Golgi vesicles. Variable sensitivity of mammalian cells to ricin and ricin fusion proteins may be due in part to different patterns of intracellular routing. Cells were incubated with ricin or EGFP-ricin, and inhibition of protein synthesis was measured. Human hepatocellular carcinoma Hep3B cells were 10-fold more sensitive to ricin and 85-fold more sensitive to EGFP-ricin than human epidermoid carcinoma KB cells. Epifluorescence microscopy of cells incubated with EGFP-ricin showed greater localization of the fluorescence signal in the Golgi compartments in Hep3B cells than in KB cells. These data support a model requiring a Golgi-dependent step in cell intoxication by ricin. The work further identifies the usefulness of green fluorescent protein fusions in the study of retrograde transport of internalized peptides.