Epidermoid KB Research Articles

Enhanced intracellular drug delivery of pH-sensitive doxorubicin/poly(ethylene glycol)-block-poly(4-vinylbenzylphosphonate) nanoparticles in multi-drug resistant human epidermoid KB carcinoma cells.

A pH-sensitive nanoparticle was prepared using our original amphiphilic block copolymer, poly(ethylene glycol)-b-poly(4-vinylbenzylphosphonate) (PEG-b-PVBP), which possesses phosphate groups as a side chain of its hydrophobic segment (termed here, phosphate nanoparticle (PNP)). The cationic anticancer drug, doxorubicin (DOX) was incorporated into a PNP (DOX@PNP), and its loading capacity was 320 mg g-1-PNP. Electrostatic and hydrophobic interactions in the core of the PNP might act synergistically to significantly improve its loading capacity. The cytotoxicity of the DOX@PNP was examined using the drug-sensitive human epidermoid KB carcinoma cell line (KB-3-1) and two different multi-drug resistance (MDR) KB cell lines (P-glycoprotein (P-gp) overexpressed (KB-C-2) and multidrug resistance protein 1 (MRP1) overexpressed (KB/MRP) cell lines). The DOX@PNP displayed a lower cytotoxic activity than free DOX against KB-3-1 cells. In contrast, the DOX@PNP showed a higher cytotoxic activity than free DOX against MDR cells. Of particular note, the cytotoxicity of the DOX@PNP against KB-C-2 cells was much higher than that against KB/MRP cells, suggesting that different mechanisms of drug reflux via the ATP binding cassette (ABC) transporting system play an important role in nanoparticle-assisted chemotherapy. Observation with confocal laser scanning microscopy (CLSM) indicated that the DOX@PNP was taken up by cells via the endocytosis pathway. The DOX@PNP was initially localized in the late endosome and lysosome, with the subsequent release of DOX from the DOX@PNP in response to the acidic pH of the late endosome and lysosome. Quantitative analysis using flow cytometry confirmed that the uptake of the DOX@PNP into KB-C-2 cells was much higher than that into KB/MRP cells, which was one of the reasons for the enhanced toxicity of the DOX@PNP against KB-C-2 cells compared to that against KB/MRP cells. Reflux of the liberated free DOX in the cytosol, via an endosomal membrane transporter, is considered one of the reasons for the low efficiency of DOX@PNP chemotherapy against KB/MRP cells. However, compared to the free DOX dose, a high dose of the DOX@PNP was effectively delivered to the nuclei of the KB/MRP cells. On the basis of these results, the pH-sensitive DOX@PNP is anticipated as one of the effective chemotherapeutic drugs with enhanced cytotoxicity for multiple types of MDR cancer cells.

Platinum(II) complexes of 8-quinolylmethylphosphonates: Synthesis, characterization and antitumour activity

A series of novel platinum(II) complexes of diethyl (8-dqmp) and monoethyl (8-Hmqmp) ester of 8-quinolylmethylphosphonic acid has been prepared and studied. It was shown that molecular or ionic complexes could be isolated by reaction of these organophosphorus ligands with [PtX 4] 2− (X = Cl, Br), depending on the acidity of the reaction solution. In the neutral medium diester formed dihalide adducts, trans-[Pt(8-dqmp) 2X 2] ( 1 and 2), with N-bonded ligand through the quinoline nitrogen. Under acidic conditions (pH < 3) both ligands gave the quinolinium salt complexes [LH] 2[PtX 4] ( 3 and 4, L = 8-dqmp; 7 and 8, L = 8-Hmqmp), with protonated quinoline ligand as cation and tetrahalidoplatinate complex as anion. By heating in methanol complexes 3 and 4 were converted into the corresponding dimeric hexahalidodiplatinum complexes, [8-Hdqmp] 2[Pt 2X 6] ( 5 and 6). The chelate complex [Pt(8-mqmp) 2] ( 9), with monoester ligand bonded through the quinoline nitrogen and the deprotonated phosphonic acid oxygen and forming two seven-membered {N, O} chelate rings, was obtained in neutral and basic media by reaction of platinum(II) halides either with sodium or hydrochloride salt of this monoester. The complexes were identified and characterized by elemental and thermogravimetric analyses, conductometric measurements, and by spectroscopic studies. In vitro antitumour activity of complexes was evaluated against the human epidermoid KB and murine leukaemia L1210 cell lines. These results were compared with those obtained for the palladium(II) complexes of the same phosphonate ligands and with those of platinum(II) and palladium(II) complexes of diethyl and monoethyl 2-quinolylmethylphosphonate, in order to correlate the structural and biological properties of quinoline-based aminophosphonate compounds.

Synthesis and evaluation of a technetium-99m labeled cytotoxic bombesin peptide conjugate for targeting bombesin receptor-expressing tumors

Conjugation of the cytotoxic drugs to receptor-binding peptides is an attractive approach for the targeted delivery of cytotoxic peptide conjugates to tumor cells. In an attempt to develop an efficient peptide-based radiopharmaceutical for targeting bombesin (BN) receptor-expressing tumors (i.e., breast and prostate), we have prepared by solid-phase peptide synthesis, a novel BN analog derived from the universal sequence of BN and conjugated to a widely characterized antineoplastic agent, methotrexate (MTX). MTX-BN, after radiolabeling with (99m)Tc via stannous-tartrate exchange, showed a good stability against cysteine and histidine transchelation as well as a high in vitro metabolic stability in human plasma. In vitro cell-binding and internalization on MDA-MB-231, MCF-7, T47-D breast cancer and PC-3 prostate cancer cell lines demonstrated high affinity and specificity of (99m)Tc-MTX-BN towards both human breast and prostate cancer cells (binding affinities in nanomolar range). In addition, the radioconjugate displayed a significant internalization (values ranged between 19-35%) into the tumor cells. In vivo biodistribution and clearance kinetics in Balb/c mice are characterized by an efficient clearance from the blood and excretion mainly through the renal-urinary pathway with some elimination via the hepatobiliary system. In vivo tumor uptake in nude mice bearing MDA-MB-231 cells was 2.70+/-0.44% ID/g at 1 h, whereas in nude mice with human epidermoid KB cells the accumulation in the tumor was found to be 1.48+/-0.31% ID/g at 1 h post injection. The tumor uptake was always higher than in the blood and muscle, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. The accumulation/retention in the major organs (i.e., lungs, stomach, liver, intestines, etc.) was low to moderate (<6% ID/g) in both healthy and tumor-bearing mice. However, the uptake/retention in the kidneys was rather high (up to 11.05+/-1.80% ID/g), which is of a concern, particularly for radionuclide therapy. This initial study towards the development of a novel cytotoxic BN conjugate suggest that the combination of favorable in vitro and in vivo properties may render (99m)Tc-MTX-BN a potential candidate for the targeted imaging and eventually for radionuclide therapy (when labeled with an appropriate radionuclide) of BN receptor-positive tumors and deserves further evaluation.

Kaempferia pandurata Roxb. inhibits Porphyromonas gingivalis supernatant-induced matrix metalloproteinase-9 expression via signal transduction in human oral epidermoid cells

Ethnopharmacological relevance Kaempferia pandurata Roxb., an edible tropical medicinal plant (Zingiberaceae), has been traditionally used to treat dental caries. However, its efficacy on inhibition of matrix metalloproteinase (MMP)-9 for preventing periodontal inflammation has been rarely studied to date. Aim of the study This present study was focused to investigate the effects of the ethanolic extract of Kaempferia pandurata Roxb. on inhibition of Porphyromonas gingivalis supernatant-induced MMP-9 expression through activation of mitogen-activated protein kinase (MAPK) signaling pathways in human oral epidermoid KB cells. Materials and methods MMP-9 expression and its signaling pathways in KB cells induced by Porphyromonas gingivalis supernatant were determined by gelatin zymogram, Western blotting and reporter gene assays. Results Kaempferia pandurata significantly decreased MMP-9 expression at both protein and mRNA levels in a dose-dependent manner. Kaempferia pandurata interfered Porphyromonas gingivalis supernatant-induced MMP-9 expression in KB cells by downregulating MAPK phosphorylation (extracellular signal-related kinase 1/2, p38 kinase and c-Jun N-terminal kinase), inhibiting transcriptional expression (Elk1, c-Jun and c-Fos), and blocking AP-1 and NF-κB activities. Conclusions Kaempferia pandurata could be employed as a candidate for MMP-9 inhibitor with therapeutic potential for treatment of periodontal inflammation.

Inhibitory Effect of Panduratin A on c-Jun N-Terminal Kinase and Activator Protein-1 Signaling Involved in Porphyromonas gingivalis Supernatant-Stimulated Matrix Metalloproteinase-9 Expression in Human Oral Epidermoid Cells

Porphyromonas gingivalis, a type of Gram-negative periodontopathogen, causes periodontal disease by activating intracellular signaling pathways that produce excessive inflammatory responses such as matrix metalloproteinases (MMPs). Recently, we reported that panduratin A, a chalcone compound isolated from Kaempferia pandurata ROXB., caused the decreased levels of MMP-9 secretion, protein, and gene expression in human oral epidermoid KB cells exposed to P. gingivalis supernatant. In this study, we clarified if mitogen-activated protein kinase (MAPK) signaling mediated MMP-9 expression by examining the effect of specific MAPK inhibitors, i.e. U0126, SB203580, and SP600125, on P. gingivalis supernatant-stimulated MMP-9 expression in KB cells. We next elucidated the molecular mechanism by which panduratin A attenuated signaling pathways involved in MMP-9 expression by performing gelatin zymography, Western blotting, reverse transcription-polymerase chain reaction, and promoter assays. Exposure of KB cells to P. gingivalis supernatant up-regulated the expression of MMP-9 protein and gene, and activation of activator protein-1 (AP-1) element, MAPK phosphorylation (extracellular signal-related kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK)), and transcription factors (Elk1, c-Jun, and c-Fos). A JNK inhibitor (SP600125) significantly attenuated MMP-9 gene expression and AP-1 activity in KB cells in response to P. gingivalis supernatant. Similar to SP600125, panduratin A was found to strongly suppress the level of phosphorylated JNK and block AP-1 activity in P. gingivalis supernatant-stimulated KB cells. In summary, JNK and AP-1 are the major signaling for P. gingivalis supernatant-stimulated MMP-9 expression in KB cells, and panduratin A markedly down-regulates MMP-9 expression through inhibition of these signaling.

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-α effects in inducing apoptosis on human cancer cells through the generation of oxidative stress

It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNα) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNα had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O 2−, nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNα could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.

Imaging Pharmacodynamics of the α-Folate Receptor–Targeted Thymidylate Synthase Inhibitor BGC 945

The assessment of tissue-specific pharmacodynamics is desirable in the development of tumor-targeted therapies. Plasma deoxyuridine (dUrd) levels, a measure of systemic thymidylate synthase (TS) inhibition, has limited application for studying the pharmacodynamics of novel TS inhibitors targeted to the high affinity alpha-folate receptor (FR). Here, we have evaluated the utility of [(18)F]fluorothymidine positron emission tomography ([(18)F]FLT-PET) for imaging the tissue pharmacodynamics of BGC 945, an FR-targeted antifolate TS inhibitor; the nontargeted antifolate BGC 9331 was used for comparison. TS inhibition by both drugs induced a concentration-dependent increase in [(3)H]thymidine uptake in FR-positive human epidermoid KB cells. Membrane-associated equilibrative nucleoside transporter type 1 levels increased from 55,720 +/- 6,101 to 118,700 +/- 5,193 and 130,800 +/- 10,800 per cell at 100 mug/mL of BGC 9331 and BGC 945, respectively, suggesting this as a potential mechanism of increased nucleoside uptake. In keeping with these in vitro findings, tumor [(18)F]FLT accumulation in KB xenografts increased by >/=2-fold after drug treatment with maximal levels at 1 to 4 hours and 4 to 24 hours after BGC 9331 and BGC 945 treatment, respectively. Of interest to FR targeting, BGC 9331, but not BGC 945, induced accumulation of [(18)F]FLT uptake in intestine, a proliferative and TS-responsive tissue. For both drugs, quantitative changes in tumor [(18)F]FLT uptake were associated with increased tumor dUrd levels. In conclusion, we have validated the utility of [(18)F]FLT-PET to image TS inhibition induced by antifolates and shown the tumor-specific activity of BGC 945. This imaging biomarker readout will be useful in the early clinical development of BGC 945.

Structure and Total Synthesis of Aspernigerin: A Novel Cytotoxic Endophyte Metabolite

Aspernigerin (1), a novel cytotoxic alkaloid consisting of an unprecedented structural framework has been isolated from the extract of a culture of Aspergillus niger IFB-E003, an endophyte in Cyndon dactylon. Its structure was elucidated on the basis of comprehensive NMR spectral analysis and confirmed by single-crystal X-ray analysis. Aspernigerin (1) has been shown to be cytotoxic to the tumor cell lines nasopharynyeal epidermoid KB, cervical carcinoma Hela, and colorectal carcinoma SW1116 with corresponding IC(50) values of 22, 46, and 35 microM, respectively. A feasible total synthetic route for aspernigerin (1) has been established for further pharmacological research.

The apoptosis effect of hispolon from Phellinus linteus (Berkeley & Curtis) Teng on human epidermoid KB cells

Phellinus linteus (Berkeley & Curtis) Teng, a well-known fungus of the genus Phellinus in the family of Hymenochaetaceae, is being increasingly used to treat a wide variety of disease processes such as oral ulcer, gastroenteric disorder, inflammation, lymphatic disease, and various cancers. However, the mechanism underlying its anti-oral cancer effect is poorly understood. In the present study, we prepared the ethanol extract of Phellinus linteus as a crude drug, and then obtained the active component hispolon by bioassay-guided isolation. Hispolon showed a dose-dependent inhibition of human epidermoid KB cell proliferation with IC50 of 4.62 ± 0.16 μg/ml. Furthermore, it was revealed that hispolon could induce human epidermoid KB cell apoptosis with the characteristic of a DNA ladder, and with a significant increase of sub-G1. This process was accompanied by the collapse of mitochondrial membrane potential, the release of cytochrome c and the activation of Caspase-3. These results demonstrated that hispolon induced the death of KB cells through a mitochondria mediated apoptotic pathway. We propose that Phellinus linteus and its effective components could be used as an anti-oral cancer drug for future studies.

Antimalarial Compounds from Grewia bilamellata

Bioassay-directed fractionation led to the isolation of 12 compounds from a sample of the dried leaves, twigs, and stems of Grewia bilamellata. Five of the isolates, 3alpha,20-lupandiol (1), grewin (2), nitidanin (4), 2alpha,3beta-dihydroxy-olean-12-en-28-oic acid (5), and 2,6-dimethoxy-1-acetonylquinol (6), showed varying degrees of in vitro antimalarial activity against Plasmodium falciparum, but were devoid of significant cytotoxicity to the human oral epidermoid KB cancer cell line. Of the 12 isolates, compounds 1, 2, and 3 (bilagrewin) were determined to be a new triterpene, a new coumarinolignan, and a new neolignan, respectively. Other known compounds isolated in this study were 8-O-4' neolignan guaiacylglycerol-beta-coniferyl ether isomers (threo and erythro), cleomiscosin D, icariol A(2), ciwujiatone, and daucosterol. The structures of 1-3 were elucidated and identified on the basis of spectroscopic data including 1D and 2D NMR analysis.

Docetaxel induces p53-dependent apoptosis and synergizes with farnesyl transferase inhibitor r115777 in human epithelial cancer cells

Docetaxel (Taxotere, DTX) is a promoter of apoptosis in cancer cells. Since cytotoxic mechanisms of DTX are not yet fully understood, we have investigated the effects of DTX on apoptosis and ras-->Erk-mediated signal transduction in human epidermoid KB, colon HT-29 and breast HCC1937 cancer cells. We have found that the exposure to 0.78 or 1.56 or 2.5 ng/ml DTX for 48 h induced apoptosis and growth inhibition in about 50 % of KB, HCC1937 and HT-29 cell population, respectively. In these experimental conditions, PARP and caspase 3 cleavage was also showed in all cell lines. KB and HCC1937 cells express a wild type p53 while HT-29 display a mutated form. Interestingly, we have found that DTX reduces the expression of mutated p53 in HT-29 and increases the expression of wild type in KB and HCC1937 cells. Moreover, DTX reduces ubiquitination of the wild type p53 in KB and HCC1937 cells and increases the ubiquitin-conjugated form of mutated p53 in HT-29 cells. Furthermore, exposure of cancer cells to DTX for 48 h increases the expression and activity of Ras and up-regulates Raf-1 and the phosphorylated isoforms of Erk-1/2. On the bases of these data, we have hypothesized that the increased activity of the ras-->erk-dependent pathway induced by DTX could be a protective signalling from the apoptosis caused by the drug. Therefore, we have used R115777, a farnesyl transferase inhibitor that inactivates ras, in combination with DTX. The combined treatment with DTX and R115777 resulted in a strong synergism in growth inhibition in the three cell lines. These data suggest the use of the combination in these therapeutic settings even if further experiments are required for the clinical translation.

Post-translational modifications of eukaryotic initiation factor-5A (eIF-5A) as a new target for anti-cancer therapy.

Eukaryotic translation initiation factor 5A (eIF-5A) is the only cell protein that contains the unusual basic amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed by the transfer of the butylamine portion from spermidine to the epsilon-amino group of a specific lysine residue of eIF-5A precursor and the subsequent hydroxylation at carbon 2 of the incoming 4-aminobutyl moiety. Agents that reduce cell hypusine levels inhibit the growth of mammalian cells. These observations suggest that hypusine is crucial for proliferation and transformation of eukaryotic cells. Here we have studied whether the inhibition of hypusine synthesis can potentiate the anti-cancer activity of the anti-tumour agents interferon-alpha (IFN alpha) and cytosine arabinoside (ara-C). We have found that IFN alpha increased epidermal growth factor receptor (EGF-R) expression, but reduced S phase and proliferative marker expression in human epidermoid KB cells and that this effect was antagonised by epidermal growth factor (EGF). Growth inhibition induced by IFN alpha was paralleled by decreased hypusine synthesis and, when EGF counteracted anti-proliferative effects, a reconstitution of hypusine levels was recorded. We also studied the effects of IFN alpha on the cytotoxicity of the recombinant toxin TP40 which inhibits elongation factor 2, another step of protein synthesis, through EGF-R binding and internalisation; IFN alpha induced an about 27-fold increase of TP40 cytotoxicity in KB cells. Ara-C, another antineoplastic agent commonly used in haematologic malignancies, induced both apoptosis and iron depletion in human acute myeloid leukaemic cells. The combination of ara-C and of the iron chelator desferioxamine, a strong inhibitor of hypusine synthesis, had a synergistic activity on apoptosis in these cells. The data strongly suggest that the post-translational modifications of eIF-5A could be a suitable target for the potentiation of the activity of anti-cancer agents.

Protein kinase C-independent correlation between P-glycoprotein expression and volume sensitivity of Cl- channel.

The possible correlation between P-glycoprotein (PGP) and volume-sensitive Cl- channel was examined in a pair of cell lines: a subline of the human epidermoid KB cell (KB-3-1) and the corresponding MDR1-transfected cell line (KB-G2). Western blot analysis and indirect immunofluorescence studies indicated that KB-G2, but not KB-3-1, exhibits the PGP expression. Patch-clamp whole-cell recordings showed that osmotic swelling activates Cl- currents not only in PGP-expressing but also in PGP-lacking cells. The amplitude of the maximal current was indistinguishable between both cells. Activation of protein kinase C (PKC) or loading with a PKC inhibitor failed to affect the swelling-induced activation of the Cl- currents in both cells. The relation between whole-cell Cl- currents and cell size measured simultaneously showed that volume sensitivity of the Cl- channel was augmented by the PGP expression irrespective of the activity of PKC on the plasma membrane. A similar increase in volume sensitivity of the Cl- channel was also induced by the expression of the ATP hydrolysis-deficient PGP mutant, K433M. We conclude that P-glycoprotein does not represent the volume-sensitive Cl- channel but that its expression modulates volume sensitivity of the Cl- channel in a manner independent of its ATPase activity or of the protein kinase C activity.

Alpha-Interferon potentiates epidermal growth factor receptor-mediated effects on human epidermoid carcinoma KB cells.

The molecular mechanisms underlying the growth inhibition of human tumor cells induced by recombinant interferon-alpha (IFN alpha) are mostly unknown. It has been proposed that this effect could be related to down-regulation and/or impaired function of peptide growth factor receptors (PGF-Rs) in tumor cells exposed to IFN alpha. However, we have previously described that IFN alpha-induced growth inhibition of human epidermoid carcinoma cells is paralleled by up-regulation of epidermal growth factor receptor (EGF-R). Here we report that an increase in EGF-R synthesis is detectable after 3 hr of exposure to cytostatic concentration of IFN alpha in epidermoid KB tumor cells. In these experimental conditions IFN alpha does not depress and even potentiates EGF-R function. IFN alpha-treated KB cells retain sensitivity to the cytotoxic activity of the anti-EGF-R 225 monoclonal antibody (MAb), which acts through receptor blockade, and are sensitized to the growth-promoting effect of EGF. EGF-induced tyrosine (tyr) phosphorylation both of total cellular protein extracts and of the immunoprecipitated EGF-R is increased in IFN alpha-treated cells. We conclude that a cross-talk between IFN alpha and EGF occurs in KB cells since IFN alpha, at cytostatic concentration, potentiates the effects mediated by the EGF-R.

Non-P-glycoprotein-mediated multidrug-resistant human KB cells selected in medium containing adriamycin, cepharanthine, and mezerein.

Human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500, and C-A1000, were isolated in selection medium containing increasing concentrations of adriamycin, 1 microgram/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100 nM of mezerein, a protein kinase C activating agent. One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D, VP-16, and colchicine. The accumulation of adriamycin and vincristine was decreased in C-A500 cells and the efflux of adriamycin from C-A500 was enhanced compared with parental KB-3-1 cells. These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpress MDR1. Multidrug-resistance-associated protein (MRP) and MRP mRNA were expressed in the adriamycin-resistant KB cells, C-A120, C-A500, and C-A1000, but not in parental KB-3-1 and revertant C-AR cells. The MRP gene was amplified in all the MDR cells that overexpressed MRP mRNA. DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells. These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincristine and that both the increased expression of MRP and decreased levels of topoisomerase II underlie the drug resistance in C-A120, C-A500, and C-A1000 cell lines.

Active Efflux System for Cisplatin in Cisplatin‐resistant Human KB Cells

Mutants, KCP‐4 and PC‐5, resistant to an anticancer agent, cisplatin, were selected in multiple steps from human epidermoid KB carcinoma cells and human prostate PC‐3 carcinoma cells, respectively. KCP‐4 and PC‐5 were 63 and 10 fold more resistant to cisplatin than the parental cells, respectively. KCP‐4 cells exhibited increased resistance to cisplatin analogues and were also slightly cross‐resistant to melphalan, cyclophosphamide, mitomycin C and methotrexate. KCP‐4 cells were not cross‐resistant to doxorubicin, daunorubicin, vincristine or CdSO4. The accumulations of cisplatin in KCP‐4 cells and PC‐5 in medium containing 50 μM cisplatin were approximately 20% of those in the parental cells. Revertant analysis suggested that a defect in cisplatin accumulation may be related to cisplatin resistance in PC‐5 cells. The uncoupling agent of oxidative phosphorylation, 2,4‐dinitrophenol, increased the accumulation of cisplatin in KCP‐4 and cisplatin‐resistant human prostate carcinoma PC‐5 cells to nearly the same level as in their parental KB‐3‐1 and human prostate carcinoma PC‐3 cells without 2,4‐dinitrophenol, but did not increase accumulation in KB‐3‐1 and PC‐3 cells. Addition of glucose in the medium inhibited the enhancement of cisplatin accumulation in KCP‐4 cells by 2,4‐dinitrophenol. Enhanced active efflux of cisplatin from KCP‐4 cells was observed. A cell‐cell hybridization test showed that the cisplatin resistance and the accumulation defect behaved as codominant traits. These data suggest that an active efflux system for cisplatin exists in cisplatin‐resistant KCP‐4 cells.

In vitro susceptibility to anticancer agents of the human KB carcinoma cell line transfected with COX-2 cDNA

In order to investigate the malignant phenotype of cyclooxygenase (COX)-2 overexpressing cancer cells, a human epidermoid KB carcinoma cell line minimally expressing COX-2 protein was transfected with human COX-2 cDNA. In this study, we used a COX-2 transfected clone KB/COX-2 and a neomycin-transfected clone KB/neo as the control. When we examined the susceptibility to anticancer agents, there was no difference between these two clones in vincristine, bleomycin and 5-fluorouracil, although KB/COX-2 showed a 2.5-fold resistance to cisplatin (CDDP) as compared with KB/neo. The IC50 for CDDP was 4.3 microM in KB/COX-2 and 1.7 microM in KB/neo. Treatment with small interfering RNA (siRNA) mediated the inhibition of COX-2 significantly increasing the level of susceptibility to CDDP in COX-2 siRNA as compared to that of the control siRNA. The expression of MRP1 and MRP2 was stronger in KB/COX-2 than in KB/neo by Western blot analysis. In addition, apoptosis induction by CDDP was at a lower level in KB/COX-2 (31%) than in KB/neo (38%). These results suggested that the overexpression of COX-2 increases the intracellular production of MRP1 and MRP2 and causes drug resistance to CDDP.