KB Cell Culture Research Articles

Biodegradable nanoparticles combining cancer cell targeting and anti-angiogenic activity for synergistic chemotherapy in epithelial cancer

A biodegradable engineered nanoplatform combining anti-angiogenic activity and targeting of cancer cells to improve the anticancer activity of docetaxel (DTX) is here proposed. Indeed, we have developed biodegradable nanoparticles (NPs) of poly(ethylene glycol)-poly(ε-caprolactone), exposing on the surface both folate motifs (Fol) for recognition in cells overexpressing Folate receptor-α (FRα) and the anti-angiogenic hexapeptide aFLT1. NPs showed a size around 100 nm, the exposure of 60% of Fol moieties on the surface, and the ability to entrap DTX and sustain its release with time. NPs were stable in simulated biological fluids and slightly interacted with Fetal Bovine serum, especially in the formulation decorated with Fol and aFLT1. The presence of Fol on NPs did not impair the anti-angiogenic activity of aFLT1, as assessed by in vitro tube formation assay in HUVEC endothelial cells. In both 2D and 3D KB cell cultures in vitro, the cytotoxicity of DTX loaded in NPs was not significantly affected by Fol/aFLT1 double decoration compared to free DTX. Remarkably, NPs distributed differently in 3D multicellular spheroids of FRα-positive KB cancer cells depending on the type of ligand displayed on the surface. In particular, NPs unmodified on the surface were randomly distributed in the spheroid, whereas the presence of Fol promoted the accumulation in the outer rims of the spheroid. Finally, NPs with Fol and aFLT1 gave a uniform distribution throughout the spheroid structure. When tested in zebrafish embryos xenografted with KB cells, NPs displaying Fol/aFLT1 reduced DTX systemic toxicity and inhibited the growth of the tumor mass and associated vasculature synergistically. Overall, nanotechnology offers excellent ground for combining therapeutic concepts in cancer, paving the way to novel multifunctional nanopharmaceuticals decorated with bioactive elements that can significantly improve therapeutic outcomes.Graphical abstract

Effect of antiepileptic drugs and reactive oxygen species on folate receptor 1 (FOLR1)-dependent 5-methyltetrahydrofolate transport

Metabolic breakdown of valproate (VPA), carbamazepine (CBZ) and phenytoin (PHT) by the cytochrome P450 pathway generates toxic drug intermediates and reactive oxygen species (ROS). This mechanism has been suspected to play a role in the pathogenesis of secondary cerebral folate deficiency (CFD). Using KB-cell cultures, highly expressing the folate receptor 1 (FOLR1), the effect of antiepileptic drugs (AEDs) and reactive oxygen species (ROS) on the FOLR1 dependent 5-methyltetrahydrofolate (MTHF) uptake was studied. MTHF uptake is time and concentration dependent and shows saturation kinetics. At physiological MTHF concentrations the high-affinity FOLR1 represents the predominant mechanism for cellular incorporation, while at high MTHF concentrations other transport mechanisms participate in folate uptake. Exposure to PHT for more than 8 h led to a higher MTHF uptake and decreased cell count, whereas MTHF uptake remained unaltered by VPA and CBZ. However, exposure to superoxide and hydrogen peroxide radicals significantly decreased cellular MTHF uptake. By specific elimination and downregulation of FOLR1 using phosphatidyl-inositol-specific phospholipase C (PIPLC) and siRNA silencing, it was shown that ROS not only inhibited FOLR1 mediated MTHF uptake but also affected all other mechanisms of membrane-mediated MTHF uptake. Generation of ROS with the use of AED might therefore provide an additional explanation for the disturbed folate transfer across the blood–CSF barrier in patients with CFD.

Folate and Folate−PEG−PAMAM Dendrimers: Synthesis, Characterization, and Targeted Anticancer Drug Delivery Potential in Tumor Bearing Mice

Ligand-mediated targeting of drugs especially in anticancer drug delivery is an effective approach. Dendrimers, due to unique surface topologies, can be a choice in this context. In the present study, PAMAM (polyamidoamine) dendrimers up to fourth generation were synthesized and characterized through infrared (IR), nuclear magnetic resonance (NMR), electrospray ionization (ESI) mass spectrometric, and transmission electron microscopic (TEM) techniques. Primary amines present on the dendritic surface were conjugated through folic acid and folic acid-PEG (poly(ethylene glycol))-NHS (N-hydroxysuccinimide) conjugates. Tumor in mice was induced through the use of KB cell culture. Prepared dendritic conjugates were evaluated for the anticancer drug delivery potential using 5-FU (5-fluorouracil) in tumor-bearing mice. Approximately 31% of 5-FU was loaded in folate-PEG-dendritic conjugates. Results indicated that folate-PEG-dendrimer conjugate was significantly safe and effective in tumor targeting compared to a non-PEGylated formulation. Tailoring of dendrimers via PEG-folic acid reduced hemolytic toxicity, which led to a sustained drug release pattern as well as highest accumulation in the tumor area.

Cytotoxic Activity toward KB Cells of 2-Substituted Naphtho[2,3-b]furan-4,9-diones and Their Related Compounds

We investigated the cytotoxic activity of 2-substituted naphtho[2,3-b]furan-4,9-diones. We have previously synthesized 33 types of 2-substituted and related compounds, and the cytotoxic activity of these compounds was then examined by a KB cell culture assay. 2-(3-Furanoyl)benzoic acids and 1,4-naphthoquinones had no activity. 2-Acetyl-4,9-dimethoxynaphtho[2,3-b]furan 4 showed low activity. However, parent naphtho[2,3-b]furan-4,9-dione 2 and most 2-substituted derivatives exhibited cytotoxic activity. The parent structure was therefore for cytotoxicity. 2-Formylnaphtho[2,3-b]furan-4,9-dione 11 had particularly potent activity (ED50=0.09 microg/ml).

Two cyclopeptides from the seeds of Annona cherimola

Bioassay-guided fractionation of cytotoxic of methanol extract of the seeds of Annona cherimola provided two novel cyclic peptides, cherimolacyclopeptide E ( 1) and cherimolacyclopeptide F ( 2), which exhibited significant cytotoxic activity against the KB (human nasopharyngeal carcinoma) cell culture system. The peptide 1 and 2 were elucidated by MS/MS fragmentation experiments using a Q-TOF mass spectrometer equipped with an ESI source, extensive 2D NMR analyses and chemical degradation.

Cytotoxic Activities of O‐Cholesteryl‐O‐Phenyl‐N‐Phenylphosphoramidate and ItsOrganometallic Tin(lV) Derivatives

O-Cholesteryl-O-phenyl-N-phenylphosphoramidate (1) and four organotin (lV) derivatives of the ambidentate O-cholesteryl-O -phenyl phosphorothioate ligand formulated as Me3 SnOSPR’R”(2), Ph3SnOSPR’R”(3), O(CH2CH2S)2Sn(n-Bu)OSPR’R”(4), S(CH2CH2S)2Sn(n-Bu)OSPR’R”(5), (R’ = O-phenyl; R”= O-cholesteryl) were subjected to cytotoxicity screening against KB (nasopharingel carcinoma), OVCAR-5 (ovarium carcinoma) and SQC-1 UlSO (squamous cell cervix carcinoma) cell cultures. The results of the bioassay showed that these compounds possess potent antitumor activities against the studied human carcinoma cell lines.

An unusual nitrogenous terphenyl derivative from fruiting bodies of the basidiomycete Sarcodon leucopus.

A new nitrogenous metabolite with a p-terphenyl core, sarcodonin (3), has been isolated from fruiting bodies of the basidiomycete Sarcodon leucopus. The structure of 3 was determined through a careful study of its peracetate 4, by use of an array of spectroscopic techniques (including (15)N NMR, gradient-enhanced, and reverse-detected 2D NMR experiments) and chemical degradation. ROESY data and molecular mechanics (MM+) calculations led to assignment of the absolute configuration of 4. Compound 3 exhibited moderate cytotoxicity against KB and P-388 tumor cell cultures.

Short-time cytotoxicity of mussel extracts: A new bioassay for okadaic acid detection

Okadaic acid (OA), the main toxin responsible for diarrhoeic shellfish poisoning (DSP) has high cytotoxicity for KB cell cultures (apparent after 3 hr of contact), facilitating rapid detection in contaminated mussels. We developed a method to determine the mimimal active concentration (MAC) based on direct microscopic study of toxin-induced changes in cell morphology. A high correlation was found between the MAC of tested extracts and corresponding OA concentrations in mussel hepatopancreas as measured by high performance liquid chromatography. This technique is rapid and reproducible and does not require the use of living animals.

In vitro effects of β-carotene on human oral keratinocytes from precancerous lesions and squamous carcinoma

Human keratinocytes, obtained from bioptic specimens of healthy and preneoplastic oral mucosa, and from human cell lines from oral cavity tumors (KB and SCC-25) were treated with beta-carotene (10 microM). The colony forming efficiency (CFE), the proliferation rate and the frequency of micronucleated cells were measured in these cultures. CFE was significantly reduced (p less than 0.05) by beta-carotene treatment in cells from healthy mucosa and in KB cells. Decreases (p greater than 0.05; NS) were also observed in cells from pathological mucosa and in SCC-25 cells. Cell proliferation rate was not substantially affected by beta-carotene in all cultures. Finally, a decreased frequency of micronucleated cells was found in treated cultures, but significant reductions (p less than 0.05) were only observed in cultures from oral mucosa (healthy and pathological) as well as in KB cell cultures. Our results indicate that beta-carotene is able to reduce the clonogenic activity (CFE), even if it does not seem to influence cell proliferation, and that it has a protective effect against genotoxic damage.

ChemInform Abstract: An Easy Approach to Prepare Naphtho(2,3‐b)furan‐4,9‐diones.

AbstractCoupling of the hydroxynaphthoquinone (I) with the butanal (II) gives the butenylated derivative (III).

Effects of cultural conditions on hexavalent chromium uptake and the cytotoxicity thereof in KB cell culture.

To reveal the effects of cultural conditions on the cytotoxicity of hexavalent chromium, the uptake of sodium chromate (Na2CrO4) by KB cells and the colony-forming efficiency of the cells were examined under various cultural conditions. The results were summarized as follows: 1) The chromium uptake by the cells after a certain period of incubation with hexavalent chromium was inhibited with the decrease of the temperature (3 degrees, 20 degrees, 37 degrees C), increase of the serum concentration (0, 10, 20, 30%) and increase of pH (6.8-8.2) of the medium. In particular, low temperatures inhibited the chromium uptake by the cells remarkably. However, in relation to the serum addition, no marked effect was found. 2) The chromium uptake by the cells increased with the volume of the medium containing an identical concentration of chromium (2 ppm) and then reached saturation when it was about 0.23 microgram per 10(6) cells. On the other hand, the chromium uptake positively correlated with the concentration of chromium and the total chromium in the medium. 3) The difference of chromium uptake by the cells in different culture media was more marked at acidic pH than that at alkaline pH. However, there was no effect of calcium chloride and glucose concentrations on the uptake of chromium. The chromium uptake by the cells in Ca-Mg-free phosphate-buffered solution (PBS(-] was higher than that in other culture media. Consequently, the above results suggested that the chromium uptake by the KB cells might be affected by the various cultural conditions, especially by temperature, pH and medium volume.

Sesquiterpene lactones and other constituents from a cytotoxic extract of Michelia floribunda.

The pentane and CHCl3 fractions of a crude extract of Michelia floribunda exhibited cytotoxic activity when tested in KB and P388 tumor cell cultures. Repeated chromatography led to the isolation of three cytotoxic sesquiterpene lactones (costunolide, parthenolide, and santamarine) and a cytotoxic isoquinoline alkaloid (liriodenine). Inactive sesquiterpene lactones obtained during the course of this study included dihydroparthenolide and two new glucosides of dihydrotamaulipin A and dihydroreynosin (1 and 2). The structures of these new compounds were determined through interpretation of their spectroscopic data including 2D-NMR spectroscopy. Syringin was also isolated from the extract.

ChemInform Abstract: New Cytotoxic Seleno Derivatives of Guaianolides.

AbstractThe cytotoxicity of the title compounds (I) and (II) (prepared by standard procedures) is evaluated in vitro against KB cell cultures, showing that introduction of the 13‐Se‐Ph group generally leads to increased bioactivity as compared with the exo‐methylene analogues.

New cytotoxic selenoderivatives of guaianolides

A series of 13-phenylselenoderivatives of natural and semisynthetic guaianolides were prepared and their cytotoxicity tested in vitro against KB cell cultures. Generally the presence of the 13-Se-phenyl group led to an increased bioactivity ( ID 50) supporting the hypothesis that it may act as a “masked” α-methylene-γ-lactone group.

An Easy Approach to Prepare Naphtho [−2, 3-b] Furan-4, 9-diones

Abstract From the ethanol extract of the stem bark of Tabebuia cassinoide (Lam.) (Bignoniaceae), Rao and Kingston isolated two new quinones, 5 and 6, that showed activity in KB cell culture assay. The synthesis of these quinones were realized by two routes that would be amenable to relatively large-scale work.

Human tumor clonogenic assayを用いた制癌剤感受性試験とnatural human Tumor Necrosis Factorの抗腫瘍効果に関する研究

The human tumor clonogenic assay is a test of susceptibility to anticancer drugs which is known to correlate well with clinical effects. In this report, this assay was used to study the antitumor effects of several anticancer drugs and human tumor necrosis factor-α (nHuTNF-α), and the combination effect of nHuTNF-α and natural human interferon-α (nHuIFN-α). Susceptibility to exisiting anticancer drugs was studied in 94 cases involving 92 patients. Colony formation was obtained 50 cases (53.2%). Of these, susceptibility to ADM was found in 15 cases, to MMC in 8 cases and to 5-Fu in 11 cases. When the antitumor effect of nHuTNF-α was studied in 10 cases obtained from patients with malignant tumor, colony inhibition was shown in 9 cases of more than 30% with 100 units and more than 50% with 300 units. A synergistic effect was assessed with a combination of nHuTNF-α and nHuIFN-α against 4 cell lines (KB, Hep-2, KATO-III, PC-9). The results showed a synergistic effect in KB and Hep-2 cell culture system. From these results, nHuTNF-α seemed to be effective anticancer drugs. The synergistic antitumor effect may be expected if the combination of nHuTNF-α and nHuIFN-α was used clinically.

Constituents of Michelia Rajaniana. Two new germacranolide amides.

Six components have been isolated from the bark of Michelia rajaniana, and their structures have been determined by spectroscopic analysis. Four of the components have been reported previously: The germacranolide (-)-parthenolide and the oxoaporphinoid alkaloid liriodenine have been observed as constituents of several species, and (-)-bisparthenolidine and (+)-paramicholide have been reported recently by us as constituents of Paramichelia baillonii. The two new components 3 and 4, which are novel derivatives of parthenolide and contain an unusual N-acetyl substituent at C-13, have been given the names (+)-N-acetylparthenolidine and (+)-N-acetyl-8 alpha-hydroxyparthenolidine, respectively. The crude CHCl3 extract of the bark of M. rajaniana exhibited strong cytotoxicity in the KB cell culture assay.

Synthesis of Dimethoxyfuranonaftoquinones

Abstract Recently some furanonaphthoquinones were isolated from Tabebuia species2,3,4. The structures la, lb2, and li4 were assigned to three of these compounds (those of la and lb being later confirmed by synthesis3,5,6). However, for the three other isolated compounds the spectroscopic data did not permit a decision to be made between the 2,3,4 - - - 4 isomeric pairs of structure lc and Id, le and lf 3, and lg and lh 4. Compounds la, lb, and le (or If), were tested in the KB cell culture assay and shown to be more active cytotoxic agents than lapachol2,3, the probable biogenetic precursor of all of them.

Combined use of alpha-difluoromethylornithine and an inhibitor of S-adenosylmethionine decarboxylase in mice bearing P388 leukemia or Lewis lung carcinoma.

The antitumor and antimetastatic effects of α‐difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, combined with an inhibitor of S‐adenosylmethionine decarboxylase, either methylglyoxal bis(guanylhydrazone) (MGBG) or ethylglyoxal bis(guanylhydrazone) (EGBG), were studied in mice bearing P388 leukemia or Lewis lung carcinoma. Although EGBG is a more specific inhibitor of polyamine biosynthesis than the widely used MGBG, the antitumor effect of the DFMO‐EGBG combination on P388 leukemia‐bearing mice was less than that of the DFMO‐MGBG combination. The prolongation of survival time by the DFMOC1000 mg/kg)‐MGBG(25 mg/kg) combination was 2.65‐fold, while that of the DFMO(1000 mg/kg)‐EGBG(50 mg/kg) combination was 1.34‐fold. When mice were fed a polyamine‐deficient diet, stronger antitumor effects were exerted; the prolongation of survival time by the DFMO‐MGBG and the DFMO‐EGBG combinations was 2.89‐fold and 2.03‐fold, respectively. The antitumor effect of combined use of the two polyamine antimetabolites with mice on normal and polyamine‐deficient diets correlated with a decrease of polyamine charge contents in the tumor cells. The above in vivo results were confirmed clearly in the KB cell culture system. The antimetastatic activity of DFMO on Lewis lung carcinoma‐bearing mice was strengthened by the addition of MGBG or EGBG. The antimetastatic activity of the DFMO‐MGBG or DFMO‐EGBG combination did not parallel the polyamine charge contents in the primary tumor and blood.

Modified taxols, 4. Synthesis and biological activity of taxols modified in the side chain.

A number of taxol derivatives substituted at the 2' position of the side chain have been prepared and their biological activities determined in KB cell culture and/or the P-388 in vivo assay. The 2'-(t-butyldimethylsilyl)taxol 5 is essentially inactive, indicating the need for a free hydroxyl group at the 2' position for activity. Epimerization of the 2' position occurred on treatment of 2'-acetyltaxol derivatives with 1,5-diazabicyclo[5.4.0]undec-7-ene, but treatment of 2'-(2,2,2-trichloroethyloxycarbonyl) taxol derivatives with DBU yielded the novel cyclization products 11 and 12 and, after deprotection at the 7 position, 13. The derivative 13 is also essentially inactive in the KB test system. Two taxols with increased H2O solubility were prepared, the 2'-(beta-alanyl) derivative 15 and the 2'-succinyl derivative 16. Although both these derivatives were active in vivo and in vitro, the former was too unstable and the latter not active enough to make suitable H2O-soluble derivatives of taxol.