Microenvironment involved in FPR1 expression by human glioblastomas.

Formyl peptide receptor 1 (FPR1) activity in U87 glioblastoma (GBM) cells contributes to tumor cell motility. The present study aimed to evaluate the FPR1 expression in human GBM, the possibility to elicit agonist induced FPR1 activation of GBM cells and inhibit this activation with chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Immunohistochemistry was used to assess FPR1 expression in GBM patient samples, which was present in all 178 samples. Also FPR1 mRNA levels measured with quantitative PCR, could be detected in all 25 GBM patient samples tested. Activation of FPR1 in U87 cells, as measured by human mitochondrial-derived agonists, increased calcium mobilization, AKT and ERK1/2 phosphorylation, and ligand-induced migration. Inhibition of all responses could be achieved with CHIPS. Eight early passage human Groningen Glioma (GG) cell lines, isolated from primary GBM tissue were screened for the presence of FPR1. FPR1 mRNA and protein expression as well as receptor activation could not be detected in any of these early passage GG cell lines. However FPR1 was present in ex vivo tumors formed by the same GG cell lines after being implanted in mouse brains. FPR1 is highly expressed in human GBM specimens, it can be activated by human mitochondrial-derived agonists in U87 and inhibited with CHIPS. FPR1 cannot be detected in early passage GG cell lines in vitro, however when engrafted in the mouse brain these cells show FPR1 expression. These results suggest a role of the brain microenvironment in FPR1 expression in GBM.

Inhibition of formyl peptide receptor in high-grade astrocytoma by CHemotaxis Inhibitory Protein of S. aureus.

BACKGROUND: High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. METHODS AND RESULTS: FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I-IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). CONCLUSION: Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients.

Unravelling mechanisms of cisplatin sensitivity and resistance in testicular cancer.

Testicular cancer is the most frequent solid malignant tumour type in men 20-40 years of age. At the time of diagnosis up to 50% of the patients suffer from metastatic disease. In contrast to most other metastatic solid tumours, the majority of metastatic testicular cancer patients can be cured with highly effective cisplatin-based chemotherapy. This review aims to summarise the current knowledge on response to chemotherapy and the biological basis of cisplatin-induced apoptosis in testicular cancer. The frequent presence of wild-type TP53 and the low levels of p53 in complex with the p53 negative feed-back regulator MDM2 contribute to cisplatin sensitivity. Moreover, the high levels of the pluripotency regulator Oct4 and as a consequence of Oct4 expression high levels of miR-17/106b seed family and pro-apoptotic Noxa and the low levels of cytoplasmic p21 (WAF1/Cip1) appear to be causative for the exquisite sensitivity to cisplatin-based therapy of testicular cancer. However, resistance of testicular cancer to cisplatin-based therapy does occur and can be mediated through aberrant levels of the above mentioned key players. Drugs targeting these key players showed, at least pre-clinically, a sensitising effect to cisplatin treatment. Further clinical development of such treatment strategies will lead to new treatment options for platinum-resistant testicular cancers.

Influence of docosahexaenoic acid on cisplatin resistance in a human small cell lung carcinoma cell line.

In a sensitive, human, small cell lung carcinoma cell line (GLC4) and a cisplatin (CP)-resistant subline (GLC4-CP), the effect of co-culturing with docosahexaenoic acid (DCHA) on CP cytotoxicity was studied. Cells were cultured for 4 days, with 32 microM of DCHA added on days 1 and 3. Incorporation of DCHA into the cellular phospholipids was demonstrated by fatty acid analysis. Supplementation with DCHA led to almost a threefold decrease of resistance in GLC4-CP and had no influence on CP cytotoxicity in GLC4. After culturing with DCHA, cellular platinum (Pt); total Pt bound to DNA; and Pt-GG, Pt-AG, G-Pt-G, and Pt-GMP adduct contents increased in both lines, whereas interstrand cross-link formation was elevated only in GLC4-CP. These experiments demonstrate that DCHA reduces CP resistance. Although an effect on cellular membranes resulting in an increased CP uptake apparently was present, this mechanism does not seem to be responsible for resistance modulation. Rather, an effect on nuclear, probably DNA-related, structures is likely and leads to an increased formation of interstrand cross-links in GLC4-CP.

Role of free radicals in an adriamycin-resistant human small cell lung cancer cell line.

In two Adriamycin (Adr) resistant sublines (GLC4-Adr1 and GLC4-Adr2) of a human small cell lung carcinoma cell line, GLC4, cross-resistance for radiation was found. GLC4-Adr1 has an acquired Adr resistance factor of 44 after culturing without Adr for 20 days and GLC4-Adr2, the same subline cultured without Adr for 3 months, has a decreased but stable resistance factor of 8. One of the assumed mechanisms of Adr is that the effect is mediated through the formation of free radicals. Therefore free radical scavenging might play a role in these Adr resistant cell lines. Adr, H2O2, and X-ray induced cytotoxicity were evaluated. Glutathione (GSH) levels and activities of associated enzymes were determined as well as Adr, H2O2, and X-ray induced DNA breaks and repair. GSH level was decreased in GLC4-Adr1, but restored to the normal level in GLC4-Adr2. Superoxide dismutase, catalase, glutathione-peroxidase, and glutathione S-transferase were not elevated in the resistant sublines. Adr induced a decreased amount of DNA breaks in GLC4-Adr1 compared to GLC4. For X-ray and H2O2 a comparable amount of DNA damage was found. GLC4-Adr1 was able to repair DNA breaks induced by Adr, X-ray, and H2O2 better than GLC4. In conclusion, no increased enzyme capacity for detoxification of free radicals could be detected in the cytosol of the resistant cells. The resistance against free radicals in the GLC4-Adr1 line may at least in part be a result of increased DNA repair.

cis-diamminedichloroplatinum(ii) resistance in vitro and in vivo in human embryonal carcinoma cells.

In the embryonal carcinoma cell line Tera and its 3.7-fold cis-diamminedichloroplatinum(II) (CDDP)-resistant subline, Tera-CP, parameters were studied that might have changed in relation to induction of CDDP resistance. Phenotypes of both lines were embryonal carcinoma. Karyotypes were related with a decreased mean number of chromosomes and fewer copies of the short arm of chromosome 12 in Tera-CP. Tera-CP showed cross-resistance for melphalan and 4-hydroperoxycyclophosphamide and had an 1.4-fold increased glutathione (GSH) level, a 1.5-fold increased glutathione S-transferase (GST) activity, and a 1.4-fold increased GST pi expression compared to Tera. Tera-CP was cross-resistant to 5-fluorouracil, but thymidylate synthase activity was not increased. Topoisomerase I and II activities and c-myc RNA and protein expression were the same in both lines. Platinum accumulation was equal in both lines, and platinum-DNA binding was lower in Tera-CP than in Tera. Both cell lines were xenografted into nude mice and tumors showed marked differentiation. Tera-CP tumors were 2.8-fold resistant to CDDP compared to Tera tumors. In new cell lines derived from xenografts of Tera and Tera-CP CDDP sensitivity, GST activity and GSH level corresponded with their sensitivity and resistant origin. Tera-CP is a model of in vitro and in vivo CDDP resistance with the GSH/GST detoxifying system as an important mechanism. CDDP resistance could be induced without a concomitant increase in differentiation.

Effects of amiodarone, cyclosporin A, and PSC 833 on the cytotoxicity of mitoxantrone, doxorubicin, and vincristine in non-P-glycoprotein human small cell lung cancer cell lines.

The multidrug resistance (MDR) modulators amiodarone (AM), cyclosporin A (CsA), and PSC 833 were tested for their potential to modulate cytotoxicity of doxorubicin (DOX), vincristine (VCR), and mitoxantrone (MX) in a sensitive human small cell lung carcinoma cell line GLC4, in its DOX-resistant non-P-glycoprotein subline GLC4-Adr, and in its cisplatin-resistant subline GLC4-CDDP. GLC4-Adr, in which overexpression of the so-called multidrug resistance-associated protein has been demonstrated, is 91-fold resistant for DOX, 22-fold for VCR, and 7.5-fold for MX, compared with its sensitive cell line. AM previously modulated DOX and VCR resistance in the P-glycoprotein-positive human colon cancer cell line COLO 320. Cytotoxicity was studied in the microtiter well tetrazolium assay. In the small cell lung carcinoma cell lines described above, AM did not increase cytotoxicity of DOX, but increased VCR cytotoxicity; moreover, AM was shown to be a potent modulator of MX cytotoxicity. CsA did not potentiate DOX cytotoxicity, but, at a concentration of 4 microM, it modestly increased VCR cytotoxicity in GLC4. However, 0.8 and 4.0 microM CsA protected against MX cytotoxicity in GLC4 and GLC4-CDDP, but no effect was observed in GLC4-Adr. At the much higher ID10 concentration CsA modulated MX cytotoxicity 1.6-fold in GLC4-Adr and slightly in GLC4 and GLC4-CDDP. PSC 833, a nonimmunosuppressive CsA analogue, did not alter the cytotoxicity of DOX or MX in these cell lines, but potentiated VCR cytotoxicity in GLC4-Adr at a concentration of 0.4 microM. The modulation of MX cytotoxicity by AM and the protection by CsA was confirmed in a clonogenic assay. In the colony-forming unit granulocyte-monocyte assay, no additional MX toxicity on normal bone marrow by AM was observed. Flow cytometry of cellular MX fluorescence was performed in order to elucidate the mechanism behind the AM-induced increased MX cytotoxicity. This revealed an increase in cellular MX after 1-h incubation of MX combined with AM and an inhibition of efflux from GLC4 and GLC4-Adr; CsA and PSC 833 had no effect on MX efflux. An increase in MX-induced cleavable complexes by AM in GLC4 was observed using the K+/sodium dodecyl sulfate coprecipitation assay, but no effect of CsA was found. In conclusion, AM enhances MX and VCR cytotoxicity in these sensitive, non-P-glycoprotein DOX and cisplatin-resistant small cell lung carcinoma cell lines. It also inhibits efflux of MX and causes more MX-induced cleavable complexes.

Relationship of cellular glutathione to the cytotoxicity and resistance of seven platinum compounds.

The role of glutathione (GSH) in the effectiveness of and resistance to 7 platinum compounds [5 Pt(II) and 2 Pt(IV) drugs] was evaluated in a 8.6-fold cisplatin (CDDP)-resistant human small cell lung cancer cell line (GLC4/CDDP), the parent GLC4 line, a 3.7-fold CDDP-resistant human embryonal carcinoma cell line (Tera-CP), and the parent Tera line (NTera2/D1). Resistance factors for both CDDP-resistant cell lines were determined after continuous incubation (4 days) with CDDP. Continuous incubation with the other studied platinum drugs revealed complete cross-resistance for carboplatin (CBDCA) and zeniplatin but less for enloplatin (ENLO) and iproplatin in both models. Tetraplatin and lobaplatin showed, respectively, partial and complete cross-resistance in GLC4/CDDP but no cross-resistance in Tera-CP. GSH level, but not glutathione S-transferase activity, of the 4 cell lines correlated with platinum drug concentrations inhibiting cell survival by 50% after continuous incubation (r = 0.86, P < 0.05). GSH depletion by DL-buthionine-S,R-sulfoximine (BSO) increased sensitivity, as measured after a 4-h exposure to the drugs, of GLC4/CDDP for CDDP 2.0-fold, for CBDCA 1.7-fold, for zeniplatin 1.7-fold, and almost to the level of the sensitive GLC4 for ENLO, whereas no effect was observed for lobaplatin and the Pt(IV) compounds iproplatin and tetraplatin. BSO-modulating effect was higher in the sensitive GLC4 line for most compounds; therefore reduction of resistance could be achieved only for CDDP and ENLO. In contrast to GLC4, no modulation occurred in Tera. In Tera-CP BSO increased sensitivity for CDDP 1.5-fold, for CBDCA 1.9-fold, and for zeniplatin 1.2-fold; no effect was observed for ENLO, lobaplatin, and the Pt(IV) compounds. Reduction of CDDP resistance by BSO was known to occur with identical cellular platinum levels and higher Pt-DNA binding in GLC4/CDDP. However, pretreatment with BSO followed by 4 h ENLO incubation increased cellular platinum levels in both GLC4 and GLC4/CDDP while Pt-DNA binding remained unchanged. In conclusion, GSH reflected sensitivity to platinum-containing drugs. However, since the involvement of GSH differed between the models and the various platinum drugs, the effect of modulation with BSO was unpredictable.

Resistance mechanisms in three human small cell lung cancer cell lines established from one patient during clinical follow-up.

Mechanisms for resistance were studied in three classic type, human small cell lung cancer cell lines, GLC14, GLC16, and GLC19, that were established from one patient during clinical follow-up. Clinically the tumor changed from sensitive (GLC14) to completely resistant to (chemo)therapy (GLC19) during this period. The stain with JSB-1 antibody, detecting the Mr 170,000 multidrug resistance associated glycoprotein, was most pronounced in GLC16 and absent in GLC19. Intracellular Adriamycin (Adr) concentrations were decreased in GLC16 and GLC19 versus GLC14. Glutathione levels were 12.9, 15.5, and 16.6 micrograms/mg protein; total sulfhydryl groups were 36.5, 45.7, and 48.8 micrograms/mg protein; and glutathione S-transferase activity was 13, 29, and 43 nmol I-chloro-2,4-dinitrobenzene/min/mg protein for GLC14, GLC16, and GLC19, respectively. Incubation with DL-buthionine-S,R-sulfoximine increased Adr and cisplatin induced cytotoxicity, whereas X-ray induced cytotoxicity remained the same. Catalase activity increased from 0.88 to 1.73 to 3.83 mumol H2O2/min/mg protein in, respectively, GLC14, GLC16, and GLC19. Compared to GLC14 and GLC16, Adr induced a higher amount of DNA strand breaks in GLC19. In none of the three cell lines could Adr induced DNA strand breaks be repaired. X-ray induced a comparable amount of DNA strand breaks in all three cell lines but all cell lines were capable of repairing the X-ray induced DNA strand breaks within 90 min. It is concluded that a number of different mechanisms are operative and that some but not all of the observed changes in mechanisms for drug resistance in these lines correlate with the clinical data.

Phase I study of transforming growth factor-beta3 mouthwashes for prevention of chemotherapy-induced mucositis.

The purpose of this study was to establish the safety and tolerability of recombinant transforming growth factor-beta3 (TGF-beta3; CGP 46614) mouthwashes intended for prevention of chemotherapy-induced mucositis. Local effects were especially analyzed by objective and subjective measurements of mucositis. Secondary aims were analysis of potential systemic exposure and development of anti-TGF-beta3-antibodies. Eleven breast cancer patients received chemotherapy with 1.5 g/m2 cyclophosphamide i.v., 80 mg/m2 epirubicin i.v., and 1.0 g/m2 5-fluorouracil i.v. (n = 8) or 1.6 g/m2 carboplatin i.v., 480 mg/m2 thiotepa i.v., and 6 g/m2 cyclophosphamide i.v. divided over 4 days (n = 3). TGF-beta3 mouthwashes (10 ml; provided by Novartis, Basel, Switzerland) were administered for 4 days, four times a day, starting 1 day before chemotherapy. The dose was escalated in following patients from 25 microg/ml (n = 3) to 50 microg/ml (n = 3) and 100 microg/ml (n = 5). Clinically, the mucosa was scored objectively and according to WHO criteria. The percentage of viable oral epithelial cells was determined by trypan blue dye exclusion. Morphology of cells was assessed in buccal smears. Plasma samples were collected for determination of TGF-beta3 levels and anti-TGF-beta3-antibodies. Adverse events were recorded by the patient in a diary. Mouthwashes with TGF-beta3 were well tolerated. Three patients scored for mucositis > grade 0 (WHO grading criteria). The percentage of viable oral epithelial cells in patients treated with 1.5 g/m2 cyclophosphamide i.v., 80 mg/m2 epirubicin i.v., and 1.0 g/m2 5-fluorouracil i.v. was stable, whereas in patients treated with 1.6 g/m2 carboplatin i.v., 480 mg/m2 thiotepa i.v., and 6 g/m2 cyclophosphamide i.v. divided over 4 days, an increase was observed. The morphology of buccal cells showed a transient shift from mature to immature cells in the first week. Neither systemic absorption of TGF-beta3 nor development of TGF-beta3-antibodies was observed. TGF-beta3 mouthwashes were well tolerated and deserve further study in preventing chemotherapy-induced mucositis.

Targeting breast cancer through its microenvironment: current status of preclinical and clinical research in finding relevant targets.

It is increasingly evident that not only breast cancer cells, but also the tissue embedding these cells: the tumor microenvironment, plays an important role in tumor progression, metastasis formation and treatment sensitivity. This review focuses on the current knowledge of processes by which the microenvironment affects breast cancer, including formation of the metastatic niche, metabolic stimulation, stimulation of tumor cell migration, immune modulation, angiogenesis and matrix remodeling. The number of drugs targeting key factors in these processes is expanding, and the available clinical data is increasing. Therefore current strategies for intervention and prediction of treatment response are outlined. At present, targeting the formation of the metastatic niche and metabolic stimulation by the breast cancer microenvironment, are already showing clinical efficacy. Intervening in the stimulation of tumor cell migration and immune modulation by the microenvironment upcoming fields of great research interest. In contrast, targeting microenvironmental angiogenesis or matrix remodeling appears to be of limited clinical relevance in breast cancer treatment so far. Further research is warranted to optimize intervention strategies and develop predictive tests for the relevance of targeting involved factors within the microenvironment in order to optimally personalize breast cancer treatment.

Differential effects of all-trans-retinoic acid, docosahexaenoic acid, and hexadecylphosphocholine on cisplatin-induced cytotoxicity and apoptosis in a cisplantin-sensitive and resistant human embryonal carcinoma cell line.

Apart from modulation of tumor-cell drug sensitivity, induction of differentiation might be an alternative in the treatment of tumors resistant to cytotoxic drugs. In this report the capacity to induce differentiation and to modulate the cis-diamminedichloroplatinum(II) (CDDP) sensitivity of all-trans-retinoic acid (RA), docosahexaenoic acid (DCHA), and hexadecylphosphocholine (HePC) is examined in human germ-cell tumor cell lines. In the embryonal carcinoma cell line Tera-2 and its 3.7-fold CDDP-resistant subline Tera2-CP, we evaluated the effects of 96 h of pretreatment with RA (0.1 microM), DCHA (23 microM), and HePC (25 microM) on differentiation induction and on CDDP-induced cytotoxicity, DNA platination (4-h incubation), and apoptosis (continuous incubation). Without drug treatment, Tera2-CP showed less apoptosis than Tera-2. Pretreatment with RA decreased the cytotoxicity and apoptosis induced by CDDP without resulting in decreased DNA platination and increased differentiation in both cell lines. DCHA enhanced CDDP-induced cytotoxicity and apoptosis and did not affect the embryonal character of either cell line. HePC did not affect CDDP cytotoxicity or differentiation in either cell lines. Effects of the modulators on differentiation and on CDDP-induced cytotoxicity, DNA platination, and apoptosis did not differ between Tera-2 and Tera2-CP. RA can be applied for differentiation induction in CDDP-resistant germ-cell tumor models. However, in this model, RA reduced the apoptotic susceptibility. DCHA potentiated CDDP cytotoxicity in vitro; its in vivo modulatory capacity in germ-cell tumor cells deserves further study.

Combined modulation by leucovorin and alpha-2a interferon of fluoropyrimidine mediated growth inhibition.

UNLABELLED: One way to improve fluoropyrimidine activity is the use of leucovorin (LV). Another way is the use of alpha-2a interferon (alpha-IF). The mechanism of the alpha-IF effect on fluoropyrimidines has not yet been elucidated. Besides, only limited data area available on double modulation (LV and alpha-IF) of fluoropyrimidines. Therefore, the modulating capacity of both drugs was tested in a fluoropyrimidine resistant (COLO 320) and a sensitive (SW 948) cell line. Also, the binding capacity of thymidylate synthase (TS) to 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) and TS catalytic activity were studied in both cell lines as well as the effects of 5-fluorouracil (5-FU) and alpha IF on enzyme activity. COLO 320 had, compared to SW 948, a 7.5 fold higher FdUMP binding capacity to TS. TS activity was 4.4 and 11.3 fold higher at 10 and 1 mM substrate, respectively. In COLO 320 enhancement of 5-FU, either by LV or by alpha-IF, was not possible. Since LV did enhance 5-fluoro-2' deoxyuridine (FUdR) activity, it is conceivable that 5-FU mediated growth inhibition in COLO 320 is not TS mediated. SW 948 was sensitive to both modulating agents with a 2.4 fold lower IC50 for 5-FU/LV, 6.8 fold lower IC50 for 5-FU/alpha-IF and a 11.2 fold lower IC50 for 5-FU/LV/alpha-IF. Effects of LV and alpha-IF on FUdR were comparable but less pronounced, with a 3.4 fold lower IC50 for FUdR/LV/alpha-IF compared with FUdR alone. Thymidine, which circumvents TS inhibition, neutralized the synergistic effects of alpha-IF, indicating that alpha-IF enhancement is mediated via inhibition of DNA synthesis. However, no direct effects of alpha-IF on FdUMP binding or catalytic activity could be demonstrated. IN CONCLUSION: alpha-IF can increase 5-FU/LV mediated growth inhibition in fluoropyrimidine sensitive colorectal cancer cells. FdUMP binding capacity and catalytic activity of TS may predict sensitivity to (modulation of) fluoropyrimidines.

Thymidylate synthase levels in tumor biopsies from patients with colorectal cancer.

Catalytic activity and FdUMP binding characteristics of thymidylate synthase (TS) were determined in 22 tumor biopsies of patients to be treated (15) or just treated (7) for colorectal cancer with 5-fluorouracil and leucovorin. In 19 samples both parameters could be determined and were found to represent a wide range (15-20 fold). High values of activity correlated with no response, but low values of binding or activity were found in responders as well as in non-responders. It is concluded that the determination of parameters related to TS function is feasible in tumor biopsies and its relevance for clinical management deserves further study.

Effect of specific or random c-DNA priming on sensitivity of tyrosinase nested RT-PCR: potential clinical relevance.

The reverse transcriptase polymerase chain reaction (RT-PCR) can be of clinical relevance in identifying malignant melanoma cells in blood or tissues of patients at risk for disseminated melanoma. The diagnostic value of this marker however, is still controversial. The objective of this study was to compare and quantify the difference in sensitivity of the nested RT-PCR for tyrosinase, with respect to the method utilized to produce the template c-DNA. We found a difference of a factor 10 in favor of a specific priming versus a random one. We concluded that this difference can be exploited in the analysis of blood samples. However, in the analysis of lymph node specimens, where the chance of positivity due to tyrosinase positive non-melanoma cells is much higher, the choice of a highly sensitive assay should be made with caution.

Uptake mechanisms of L-3-[125I]iodo-alpha-methyl-tyrosine in a human small-cell lung cancer cell line: comparison with L-1.

The radiolabelled amino acid analogue L-3-[125I]iodo-alpha-methyl-tyrosine (IMT) is under evaluation in brain tumours, where it reflects amino acid transport activity, but is also taken up in many other tumour types. This study investigated the uptake mechanism of IMT in tumour cells not derived from brain tumours, in comparison with the native amino acid 14C-tyrosine (Tyr) from which IMT is derived. Human GLC4 small-cell lung cancer cells in log-phase were incubated with IMT and Tyr. Tracer uptake was determined in various buffers, incubation periods, concentrations of specific amino acid transport blockers, pH and temperature. IMT uptake was very fast, reaching a plateau within 5 min, while Tyr kept on accumulating for > 60 min. Based on steady-state experiments, > 90% of IMT uptake could be attributed to amino acid transport activity. The L transport system was the most important, both for IMT and Tyr. IMT uptake into GLC4 tumour cells is almost completely the result of amino acid transport activity (especially the L system) and is very similar to Tyr uptake. Therefore, also outside the brain, IMT is a metabolic tracer that may reflect the increased amino acid transport that is characteristic for malignant tumours.

Disruption of the MDM2-p53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway.

Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2-p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (∼threefold) and enhanced Fas/FasL interactions at the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.

The chemokine network, a newly discovered target in high grade gliomas.

Chemokines are small cytokines, characterised by their ability to induce directional migration of cells by binding to chemokine receptors. They are known to play a role in tumour development, angiogenesis and metastasis. Interestingly, the chemokine network also contributes to the progression of gliomas, mainly by intensifying their characteristic invasive character. The main hurdle in treatment of these tumours is their infiltration of surrounding tissues, hampering complete surgical tumour removal. Standard postsurgical treatment with radio- and chemotherapy is of limited effect. Therefore drugs that target the chemokine system in high grade gliomas might fill the gap existing in the current approach. This review presents the current knowledge of the role of chemokine network in the development of the central nervous system, in brain physiology and the involvement in brain tumour progression. Finally, current studies exploring new compounds targeting the chemokine network in cancer patient are discussed.

Effect of novobiocin on cisplatin cytotoxicity and DNA interstrand cross-link formation in a cisplatin-resistant, small-cell lung carcinoma cell line.

Studies were performed to determine whether novobiocin can be used to enhance cisplatin (CDDP) cytotoxicity in a human small-cell lung carcinoma cell line, GLC4/CDDP, resistant to CDDP. Continuous incubation with novobiocin enhanced the cytotoxicity of CDDP treatment 1.9-fold in the parental cell line GLC4, but had no effect on its cytotoxicity in the resistant cell line GLC4/CDDP. Short incubation with novobiocin enhanced the cytotoxicity of CDDP treatment in GLC4 and GLC4/CDDP by a factor of 4.1 and 2.8, respectively. Using the latter schedule, the amount of CDDP-induced DNA interstrand cross-links (DNA ISC) at 4 hr as well as at 24 hr after novobiocin and CDDP treatment was higher in GLC4 than in GLC4/CDDP. In this case, the amount of DNA ISC had increased 1.6-fold in GLC4 and 1.3-fold in GLC4/CDDP at 4 hr, and 2.7-fold and 1.4-fold, respectively, in these cell lines at 24 hr after treatment compared to CDDP treatment alone. Our results suggest an effect of novobiocin on the formation of DNA ISC. The decreased efficacy of novobiocin, an inhibitor of DNA topoisomerase (Topo) II catalytic activity, in GLC4/CDDP may be due to the increased Topo II activity previously found in the resistant cells. In the present study, we showed that increased Topo II activity was not due to changes in amounts of Topo II in nuclei or nuclear extracts of GLC4/CDDP. Further analysis of the chromatin, that includes Topo II, showed that the chromatin in nuclei of GLC4/CDDP was more sensitive to micrococcal nuclease digestion than GLC4. In addition, the amount of a 56-kDa protein was increased 2-fold in nuclei and nuclear matrices from GLC4/CDDP. The reduced efficacy of novobiocin to increase the CDDP cytotoxicity as well as the formation of DNA ISC in GLC4/CDDP compared to GLC4 may be due to changes in the chromatin structure of the resistant cells.

Modulation of cis-diamminedichloroplatinum(II) resistance: a review.

In this review an inventory is made of agents used to circumvent cis-diamminedichloroplatinum(II) (CDDP) resistance in vitro and in vivo. Agents that affect CDDP accumulation and membrane related systems, cytoplasmic defense mechanisms, as well as DNA accessibility and repair are reviewed. In resistant cell lines that have decreased accumulation, this can be restored by hyperthermic treatment. With or without effects on accumulation compounds that affect cell signal transduction often increase CDDP cytotoxicity. Calcium channel blockers and calmodulin inhibitors do not seem to be uniformly good modulators of CDDP resistance. For transduction modulators as well as cellular calcium affecting agents mechanisms are mainly unclear or controversial. Glutathione appears, with the now available agents, to be the most promising target for modulation of cytoplasmic defense mechanisms. At the nuclear level the inhibition of DNA repair related enzymes as well as the use of modified nucleosides to interfere with repair is studied in various cell lines. Results with these agents suggest opportunities for clinically feasible cytotoxicity modulation. DNA accessibility could in vitro be affected, but seems to be an unreliable target for modulation. Whenever possible the resistance mechanism affected and the mode of action of the modulator are discussed. As an alternative for modulation another method of overcoming CDDP resistance namely the application of CDDP analogues is considered.