The differentiation inducers phenylacetate and phenylbutyrate modulate camptothecin sensitivity in colon carcinoma cells in vitro by intracellular acidification.

Aromatic fatty acids such as phenylbutyrate (PB) and its metabolite phenylacetate (PA) induce growth arrest, differentiation and apoptosis in solid tumor cells. Despite their antiproliferative action they were reported to exhibit a synergistic effect in combination with cytotoxic drugs like topotecan, and others. Since the activity of the camptothecines (CPTs) depends on local pH conditions, we investigated, whether PB/PA modulate CPT effects indirectly by affecting intracellular pH in SW620 and SW480 colon cancer cells. The results for the colon carcinoma cells show an antagonistic interaction for the combination of CPT and 0.25-5 mM PA in viability assays, resulting in an approximately 3-fold increase in IC50 (control: 20+/-7 nM). A synergistic effect with significantly increased numbers of late apoptotic/necrotic cancer cells (difference +21+/-4%) and 1.4-fold sensitization were detected upon inclusion of 2.5 mM PA during a 4-h CPT (10 micro;M) loading phase. In response to 0.25-1 mM PA/PB the cells exhibit a reversible decrease of pHi (0.1-0.31 pH units) in HEPES- or bicarbonate-buffered media. Dose-dependent acidification and pHi-recovery occurred following addition of PA and PB after an acid load and inhibition of the Na+/H+-antiporter and bicarbonate exchangers, pointing to a possible intracellular mechanism of cytoplasmic acidification. It is concluded that the synergistic modulation of CPT toxicity by short-term PA/PB treatment in colon carcinoma cells is caused by changes in intracellular pH, possibly affecting quantity and localization of the active closed lactone form of this drug.

Serum levels of vasoactive intestinal peptide (VIP) in patients with adenocarcinomas of the gastrointestinal tract.

BACKGROUND: VIP acts as a neuroendocrine mediator under physiological conditions, with an important role in water and electrolyte secretion in the gut. Recent findings suggest that VIP also promotes growth and proliferation of normal as well as malignant cells. We have investigated the VIP-serum levels in patients with pancreatic cancer and colonic adenocarcinoma as compared to healthy controls. This was accompanied by immunohistochemical investigations and in vitro experiments to further define the role of the peptide in pancreatic and colorectal cancer. MATERIALS AND METHODS: Serum levels of VIP were evaluated under standardized conditions in a total of 135 patients; 45 patients had metastatic colorectal cancer, 45 suffered from metastatic pancreatic cancer, and 45 healthy volunteers served as controls. Human pancreatic and colorectal carcinoma cell lines were incubated over 5 days with VIP in increasing concentrations. RESULTS: In healthy controls, a median VIP-serum level of 42.44 +/- 2.540 pg/ml (range, 12.9-98.5 pg/ml) was found, while patients with pancreatic cancer had a median level of 40.58 +/- 3.013 pg/ml (range, 6.9-102.4 pg/ml). In patients with cancer originating in the colon, however, a median serum level of 116 +/- 10.14 pg/ml (range, 51.6-487 pg/ml) was found. While no difference between healthy controls and patients with pancreatic cancer could be detected (p = 0.6381), a significant difference between patients with colorectal cancer and healthy controls (p < 0.0001) and patients with pancreatic cancer (p < 0.0001) was demonstrated. The median VIP-concentrations found in the patients sera for pancreatic and colonic tumor patient groups, 40 pg/ml and 115 pg/ml respectively, had no significant effect on the proliferation of PANC-1 and HT29, inhibited ASPC-1, BxPC3, COLO201 and HCT-15 cells, and stimulated the growth of one pancreatic (CAPAN-1) and one colonic (COLO320DM) cell line under these conditions. CONCLUSIONS: As opposed to pancreatic cancer and healthy controls, patients in our series had elevated serum VIP-levels. Further studies are warranted to evaluate whether VIP can be used as a tumor marker in this disease.

Measurements of free and total PSA, tissue polypeptide-specific antigen (TPS), and CYFRA 21-1 in prostate cancer patients under intermittent androgen suppression therapy.

BACKGROUND: The present study evaluated monthly measurements of free and total prostate-specific antigen (PSA), and the tumor proliferation markers tissue polypeptide-specific antigen (TPS) and cytokeratin fragment 21-1 (CYFRA 21-1) in patients with advanced prostate cancer receiving intermittent androgen suppression therapy (IAS). METHODS: Thirty-four men received alternating cycles of 8 month androgen suppression and treatment cessation (mean duration, 10.3 months) until PSA increased to >20 microg/l. Measurements of testosterone, percentage of free PSA, TPS, and CYFRA 21-1 were performed using ELISA and RIA assays. RESULTS: Periods of androgen suppression resulted in reversible reductions of testosterone (from 6 +/- 0.8 to <0.58 ng/ml), PSA (from 31.2 +/- 4.5 to <1.7 microg/l), and prostatic volume (mean reduction, 22.2 +/- 4.6%), indicating apoptotic regression of the tumors. Upon treatment cessation, testosterone increased to 6.1 +/- 0.56 ng/ml within 2 months, followed by an increase of PSA to 5.8 +/- 0.8 microg/l. The mean percentage of free PSA (15.1 +/- 2.6%) exhibited no significant change during the whole IAS cycle. TPS showed a decrease of 50% after 3 months, and CYFRA 21-1 a 25% decrease after 7 months of androgen suppression treatment. During treatment cessation, TPS exceeded the normal cutoff value of 90 U/l late in tumor regrowth (9-11 months), whereas CYFRA 21-1 remained below the normal cutoff value of 3.3 ng/ml. CONCLUSIONS: PSA is the best and most sensitive marker of prostate cancer regression and regrowth during IAS cycles of the markers tested in this study. Free PSA constitutes approximately 15% of total PSA (range, 5-32%), and its percentage showed no significant change during IAS cycles. The TPS and CYFRA 21-1 proliferation marker changes in IAS seem to be related mainly to effects on normal androgen-dependent tissues.

Nonsurgical management of malignant thymoma.

BACKGROUND: Thymoma is a rare tumor entity. Surgery remains the mainstay of treatment, but radiation and chemotherapy also have been applied widely in both the adjuvant and the palliative setting. The objective of this study was to review briefly the clinical trials available in the current literature utilizing nonsurgical oncologic treatment (radiotherapy and chemotherapy) either in patients with advanced (i.e., locally inoperable) or metastatic thymoma or as an adjunct to surgery. METHODS: A computerized (MEDLINE) and a manual search was performed to identify articles published on this topic between 1965-1998. Only articles with an English abstract were reviewed for inclusion; information abstracted included histologic proof of diagnosis, number of patients, dose and modality of treatment, assessment of response, response rate, survival duration, and side effects. RESULTS: Seventy-one trials were identified subsequently. These included 51 chemotherapy studies in a total of 410 patients (including 19 single agent trials and 32 combination chemotherapy trials) and 20 radiotherapy studies. In the adjuvant setting, radiation appeared to result in a higher survival rate compared with historic controls as well as excellent local control in patients with advanced stage of disease, whereas no apparent benefit was observed in patients with Masaoka et al. classified Stage I disease. The large majority of chemotherapeutic studies were case reports or Phase II trials of advanced disease, whereas no prospectively randomized trials were performed. Response rates were relatively heterogeneous and ranged between 24% and 100%, not including the results published in single case reports, and response rates >50% have been found consistently with the application of polychemotherapy. In the absence of randomized trials, multimodality approaches using induction chemotherapy followed by resection and consecutive radiation have produced highly promising results in terms of resectability and long term survival, even in patients with advanced disease. CONCLUSIONS: To the authors' knowledge, there is no standard approach to advanced thymoma apart from surgery (i.e., total resection whenever possible). Despite reports of long term disease control, symptomatic palliation, and encouraging survival data, the majority of studies involved only a small number of patients and were performed in a Phase II approach. Large scale, randomized trials to elucidate the potential of multimodality approaches clearly are needed, and patients with thymoma should be included in such studies.

Treatment of advanced pancreatic cancer with the long-acting somatostatin analogue lanreotide: in vitro and in vivo results.

Fourteen patients with metastatic pancreatic adenocarcinoma were treated with the long-acting somatostatin (SST) analogue lanreotide. No objective response was obtained, and the median survival was 4 months (range 1.8-7 months). Pancreatic cancer could not be visualized by means of SST-receptor (R) scintigraphy in our patients. In vitro data also demonstrated absence of SSTR2 expression, suggesting pancreatic cancer not to be a potential target for treatment with SST analogues.

Formation of a novel topotecan metabolite in the hormone-independent human prostate carcinoma cell lines DU-145 and PC-3.

To investigate the implications of drug metabolism on topotecan (TPT) resistance in prostate cancer cells, we measured the time-dependent uptake, metabolismrand efflux of TPT in the prostate cancer-derived cell lines DU-145 and PC-3 by HPLC. Exposure of DU-145 to 10 microM TPT resulted in a maximal intracellular concentration of TPT of 12.6 +/- 0.53 pmol/10(6) cells (t = 10 min) with a decrease to 4.4 +/- 0.25 pmol/10(6) cells after 2 hours. Incubation of PC-3 cells, however, revealed a more than 2-fold higher level of cytoplasmatic TPT (25.3 +/- 4.8 pmol/10(6) cells). In both cell lines, an intracellular metabolite was detectable after 30 minutes. Its concentration continuously increased reaching saturation after 6 hours (0.015 +/- 0.003 pmol/10(6) cells in DU-145 and 0.0059 +/- 0.0020 pmol/10(6) cells in PC-3 cells). Analysis of the culture supernatant of DU-145 and PC-3 cells revealed that this metabolite is secreted into the medium at increasing concentrations (0.220 +/- 0.025 and 0.079 +/- 0.008 pmol/10(6) cells, respectively). In accordance with the elevated formation of the TPT-metabolite in DU-145 cells, the expression of cytochrome P450 (CYP) isoenzymes CYP3A, CYP2B, CYP2D and CYP2E as measured by Western blot analysis was also higher in this cancer cell line. In conclusion, we found that TPT is rapidly taken up by the two prostate cancer cell lines and metabolized to a minor biotransformation product dependent on their content of cytochrome P450 isoenzymes. The structural identification of this TPT metabolite and the CYP isoenzyme(s) responsible for its formation remain to be elucidated.

P-glycoprotein-mediated multidrug resistance is modulated by pretreatment with chemosensitizers in HCT-8 carcinoma cells in vitro.

The effects of pretreatment with the multidrug resistance (MDR) modulators verapamil (VPM), tamoxifen (TMX), cyclosporin A (CsA), and SDZ PSC833 (PSC) on drug sensitivity of the P-glycoprotein (Pgp) expressing human ileocecal carcinoma cell line HCT-8 is described. Following pretreatment of 2, 16 and 48 h with the individual modulators, rhodamine 123 efflux (RHO), transepithelial vinblastine transport (VIN) across treated HCT-8 monolayers, and chemosensitivity to doxorubicin (DOX) were determined and compared to Pgp protein expression and phosphorylation. After 2 h, VPM, TMX, CsA and PSC inhibited RHO efflux and VIN transport and increased the chemosensitivity of HCT-8 to DOX significantly. Prolonged exposure failed to further increase inhibition of Pgp-mediated transport, but in contrast maximized phosphorylation of Pgp (16 h) and Pgp protein expression (48 h), respectively.

Hot spices influence permeability of human intestinal epithelial monolayers.

Indirect evidence suggests that hot spices may interact with epithelial cells of the gastrointestinal tract to modulate their transport properties. Using HCT-8 cells, a cell line from a human ileocoecal carcinoma, we studied the effects of spices on transepithelial electrical resistance (TER), permeability for fluorescein isothiocyanate (FITC)-labeled dextrans with graded molecular weight, and morphological alterations of tight junctions by immunofluorescence using an anti-ZO-1 antibody, a marker for tight junction integrity. Two different reactivity patterns were observed: paprika and cayenne pepper significantly decreased the TER and increased permeability for 10-, 20- and 40-kDa dextrans but not for -70 kDa dextrans. Simultaneously, tight junctions exhibited a discontinuous pattern. Applying extracts from black or green pepper, bay leaf or nutmeg increased the TER and macromolecular permeability remained low. Immunofluorescence ZO-1 staining was preserved. In accordance with the above findings, capsaicin transiently reduced resistance and piperine increased resistance, making them candidates for causing the effects seen with crude spice extracts. The observation that Solanaceae spices (paprika, cayenne pepper) increase permeability for ions and macromolecules might be of pathophysiological importance, particularly with respect to food allergy and intolerance.

Measurements of tissue polypeptide-specific antigen and prostate-specific antigen in prostate cancer patients under intermittent androgen suppression therapy.

The present study evaluated serial serum measurements of tissue polypeptide-specific antigen (TPS) in comparison with prostate-specific antigen (PSA) for assessment of tumour progression in patients with advanced prostate cancer receiving intermittent androgen suppression therapy (IAS). Twenty-three men were recruited into an IAS trial consisting of an initial 8 months of androgen suppression, followed by cycles of treatment cessation and resumption of therapy upon increases of PSA > 20 ng ml(-1) to prolong the hormone responsiveness of the tumour cells. Periods of androgen suppression resulted in reversible reduction in serum testosterone (< 1.8 nmol I(-1)) and PSA (< 4 ng ml(-1)) and decreases in tumour volume (mean reduction for first cycle 24 +/- 10%), indicating partial growth arrest and apoptotic regression of the tumours. In contrast to PSA values, non-specifically elevated TPS values were found in 8 of 23 patients. In 15 of 23 patients, TPS fell during periods of apoptotic tumour regression and increased simultaneously with testosterone and preceded the increases in PSA by 2 months during the period of treatment cessation. Although TPS represents a highly sensitive marker of tumour proliferation in this IAS clinical model of controlled tumour regression and regrowth, its low specificity compared with PSA limits its usefulness to monitoring of prostate cancer patients with proven absence of non-specific elevations of this marker.

Involvement of P-glycoprotein in the transmembrane transport of interleukin-2 (IL-2), IL-4, and interferon-gamma in normal human T lymphocytes.

The physiological role of the multidrug resistance P-glycoprotein (P-gp), which is expressed by normal human T lymphocytes, is still largely unknown. To investigate whether or not P-gp is involved in the transport of cytokines, peripheral blood lymphocytes were stimulated with phytohemagglutinin (PHA) in the absence or presence of P-gp inhibitors, and concentrations of cytokines (interleukin-2 [IL-2], IL-4, IL-6, interferon-gamma [IFN-gamma]) in the supernatants of these cultures were quantitated by enzyme-linked immunosorbent assay. P-gp inhibitors included verapamil (Ver), tamoxifen (Tmx), and the P-gp specific monoclonal antibody UIC2. Release of IL-2 was significantly suppressed by these inhibitors at concentrations that were also effective in blocking efflux of Rhodamine-123 from normal T lymphocytes. IL-2 mRNA expression in lymphocytes was not different between PHA control and the cultures with P-gp inhibitors. Ver and Tmx did not interfere with T-cell activation as determined by CD25 and CD69 expression. In a nonhematological model, the P-gp expressing HCT-8 adenocarcinoma cell line, exogenously added IL-2 was shown to exert an inhibitory effect on P-gp mediated Rhodamine-123 efflux. In addition, transepithelial transport of IL-2 by electrophysiologically tight and polarized HCT-8 monolayers was examined. A time-dependent flux of IL-2 across dense monolayers, which was partially inhibited by Ver, was observed. We also investigated whether or not P-gp inhibitors suppressed release of other cytokines produced by activated T cells (IL-4, IL-6, IFN-gamma). Release of IL-4 and IFN-gamma was significantly inhibited by Ver, Tmx, and UIC2; however, release of IL-6 remained unaffected. These data show P-gp mediated transmembrane flux of IL-2 in T lymphocytes and HCT-8 cells. We conclude that P-gp participates in the transport of cytokines (IL-2, IL-4, and IFN-gamma) in normal peripheral T lymphocytes.

In vitro antitumor activity of MIC2 protein-doxorubicin conjugates.

The pseudoautosomal encoded MIC2 glycoprotein is a tumor-associated antigen of Ewing's sarcoma (ES) and closely related tumors of unknown function. To investigate the use of this protein as selective drug carrier recombinant MIC2 was coupled to doxorubicin by a two step glutaraldehyde method (molar ratio DOX/MIC2 of 32 and 16). The conjugates showed dose-dependent cytostatic activity against the ES cell line SK-ES1, the peripheral neuroectodermal line KAL and the prostate cancer cell line PC-3 concurrent with reduced toxicity against normal lymphoblasts. In comparison to free doxorubicin the MIC2-doxorubicin conjugates exhibited highest activity against the PC-3 cell line. Confocal microscopy showed intracellular accumulation of MIC2 conjugates.

In vitro antiproliferative effects of albumin-doxorubicin conjugates against Ewing's sarcoma and peripheral neuroectodermal tumor cells.

The 3-5 year survival rates of patients with disseminated Ewing's sarcoma (ES) or the closely related peripheral primitive neuroectodermal tumors (PNET) remain low, even under aggressive treatment involving highly toxic multidrug chemotherapeutic regimens. ES and PNET are sensitive to doxorubicin, but may escape treatment by expression of the multidrug-resistant phenotype and/or other mechanisms. In this study, we have identified albumin as growth supporting factor for ES and PNET cells in IGF-I-supplemented serum-free tissue culture medium. To investigate the specificity and toxicity of albumin-based drug conjugates, doxorubicin was coupled to bovine serum albumin (BSA) by either a two step glutaraldehyde or carbodiimide-C4-spacer technique, yielding monomeric DOX-albumin conjugates with conjugation numbers ranging from 3-20 moles DOX/mole BSA. Cellular uptake of fluorescein-isothiocyanate-(FITC)-labeled albumin and DOX-albumin conjugates could be demonstrated by flow cytometric measurements of cell-associated fluorescence and confocal microscopy. The cytostatic activity of these conjugates against ES/PNET cell lines, a neuroblastoma (LAN-1) and prostate cancer carcinoma cell line (PC-3) and normal lymphoblasts was tested in short-term proliferation assays (48 h). The results show a high selectivity of the DOX-albumin conjugates for ES/PNET cell lines, with highest growth inhibition by conjugates with low DOX conjugation numbers (n = 3) in serum-supplemented medium (17-32 fold loss of activity compared to free DOX), followed by 20-DOX-C4-albumin in serum-free medium and low activity of the other conjugates. In conclusion, DOX-albumin conjugates inhibit the growth of ES/PNET cell lines selectively, showing low activity against the unrelated carcinoma line PC-3 and sparing normal lymphoblasts. The inverse correlation of activity and conjugation number demonstrates a low cytotoxic activity of DOX in acid-stable binding to monomeric albumin, pointing to a selective cytostatic activity of the modified albumin against ES and PNET cells, even in the presence of a 100 fold excess of unmodified serum albumin.