The effects of recombinant human interferon-gamma on a panel of human bladder cancer cell lines

We have examined the Major Histocompatibility Complex class II antigen inducing capabilities of recombinant human interferon-gamma, on a panel of human transitional cell carcinoma lines which have been raised from original tumours of varying histological grades: RT4 (grade 1), RT112 (grade 2) and MGH-U1 (grade 3). Cells were examined for class II antigens using an indirect immunofluorescent staining method and analysed on a fluorescence activated cell sorter. Twenty percent of RT4 cells constitutively expressed class II antigen. Both RT112 and MGH-U1 were repeatedly found to be negative for this antigen prior to treatment with interferon-gamma. Following treatment with interferon-gamma all three lines showed an increase in class II antigen expression, which was consistently dependent on both the length of incubation and concentration of interferon-gamma. A differential susceptibility was found amongst the three cell lines which may relate to the histological grade of the parent tumor.     

Lactobacillus species is more cytotoxic to human bladder cancer cells than Mycobacterium Bovis (bacillus Calmette-Guerin)

PURPOSE: We determined if Lactobacillus species has growth inhibitory effects in human bladder cancer cell lines and how this effect compares with the known effects of Mycobacterium bovis, that is bacillus Calmette-Guerin (BCG). MATERIALS AND METHODS: The growth of MGH and RT112 cells were determined by cell counts after 24, 48 and 72 hours of exposure to L. casei strain Shirota (Yakult, Singapore) or L. rhamnosus strain GG (National Collection of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland) (1 x 10 and 1 x 10 cfu) or BCG (1 x 10 cfu) in the presence and absence of streptomycin. Annexin-V was used to monitor the presence of pre-apoptotic cells. RESULTS: L. rhamnosus GG inhibited MGH proliferation and it was cytotoxic to RT112 cells (p <0.05). L. casei Shirota was cytotoxic to the 2 cell lines (p <0.05). BCG had a similar cytotoxic effect in MGH cells as Lactobacillus species but was not as effective in RT112 cells. Streptomycin abrogated the cytotoxic effect of Lactobacillus species but not that of BCG. Cytotoxic activity was not found in Lactobacilli culture supernates but it was induced in the presence of mammalian cells. L. rhamnosus GG induced apoptosis in RT112 but not in MGH cells. No apoptotic cells were detected after treatment with L. casei Shirota. CONCLUSIONS: Lactobacillus species induced cytotoxic effects in bladder cancer cells. Unlike BCG, it requires bacterial protein synthesis. Like BCG, L. casei Shirota induces cell death primarily via necrosis. The cytoxicity of these lactobacilli in bladder cancer cells raises the possibility of using this species of bacteria as intravesical agents for treating bladder cancer.     

An investigation of factors influencing the in vitro induction of LAK activity against a variety of human bladder cancer cell lines.

We investigated the sensitivity of transitional cell carcinoma cells, derived from the human bladder, to lymphokine activated killer cells. Recombinant interleukin-2 activated peripheral blood mononuclear cells were studied for their ability to mediate the cytolysis of a panel of four established human bladder transitional cell carcinoma cell lines. Lymphokine activated killer activity was assessed using a standard four hour chromium release assay. All four bladder cancer cell lines proved to be susceptible to lymphokine activated killer mediated cytolysis. This was found to be dependent upon the dose of cytokine and upon the duration of the activation period. The four cell lines were differentially susceptible to lysis (specific cytotoxicity at effector to target ratio of 40:1; RT112 = 22.9%, RT4 = 49.2%, MGH-U1 = 49.1%, EJ18 = 62.3%). The varying susceptibility of lymphokine activated killer mediated cytotoxicity was found to be independent of the histological grade of the parent tumour or the donor of effector cells. Both interferon-alpha and tumour necrosis factor-alpha also elicited lymphokine activated killer cell activity, although the maximum specific cytotoxicity achieved was considerably lower than that obtained with interleukin-2 alone. Interleukin-2, at optimal concentration, and tumour necrosis factor-alpha were found to behave synergistically in the generation of lymphokine activated killer effectors. However, concentrations of tumour necrosis factor-alpha higher than 100 Uml.-1 resulted in a decrease in specific cytotoxicity. These findings suggest a possible use of adoptive immunotherapy in human bladder cancer and indicate the optimum conditions for the generation of such effector cells.     

Progesterone: a novel adjunct to intravesical chemotherapy

OBJECTIVE: To investigate the effect of progesterone on multidrug-resistant urothelial cell lines, as the failure of intravesical chemotherapeutic drugs is often caused by multidrug resistance (MDR), mediated by the drug efflux pump P-glycoprotein (PGP), the function of which can be down-regulated by various compounds including steroid hormones. MATERIALS AND METHODS: Two urothelial cell lines (RT112S and MGH-U1S) and their MDR sublines (RT112R, to cisplatin; and MGH-U1R, a cell line expressing PGP) were used to assess the cytotoxic effects of progesterone, epirubicin and their combination. Cytotoxicity was assessed using a tetrazolium-based assay and in situ confocal microscopy. RESULTS: Cell lines sensitive to epirubicin (MGH-U1S, RT112S and RT112R) required a much lower dose of epirubicin to kill half the cells than did the MDR cell line. Progesterone was intrinsically cytotoxic to all cell lines with little difference among them. Combined therapy had no cumulative effect on epirubicin-sensitive cell lines, but reversed MDR in the MGHU1R cell line, both assessed by confocal microscopy and by the tetrazolium assay. CONCLUSIONS: Progesterone can reverse MDR in urothelial cells in vitro. This, combined with its effects on cell differentiation and apoptosis, together with its safety and tolerability compared to other MDR agents, suggests it may be a valuable adjunct to intravesical chemotherapy.     

The effect of hyperthermia on mitomycin-C induced cytotoxicity in four human bladder cancer cell lines

INTRODUCTION: Hyperthermia and mitomycin-C (MMC) have given very encouraging results in several clinical studies for the treatment of superficial transitional cell carcinoma of the bladder. However, a synergistic effect of hyperthermia and MMC on the decrease of cell proliferation has never been demonstrated accurately in vitro. We investigated the effect of MMC versus MMC combined with hyperthermia on the cytotoxicity in four human bladder cancer cell lines. MATERIAL AND METHODS: The RT112, RT4, 253J and T24 human bladder cancer cell lines were seeded in 96-well microtiter plates at 2.0 x 10(4) cells per well and were left to attach for 24 hours. The cells were then treated for 60 minutes with MMC concentrations ranging from 0 to 400 microg/ml at a temperature of 37 degrees C or 43 degrees C. After treatment cells were rinsed three times with culture medium and left for 24 hours in the incubator. Dimethyl thiazolyl tetrazolium (MTT) solution was added and after 4 hours of incubation the MTT containing media was aspired from all wells and 100 microl of dimethyl sulfoxide was added to each well. A spectrum analyses was performed at 595 nm light wavelength. RESULTS: A decrease of cell proliferation after treatment with increasing concentrations MMC was demonstrated. Hyperthermia has a synergistic effect on the decrease of cell proliferation by different concentrations MMC. In the cells treated without MMC no significant difference in the extent of cell killing at 37 degrees C and 43 degrees C was observed. Furthermore, no difference was observed between cells with a p53 protein mutation (RT112 and T24) or without a p53 protein mutation (253J and RT4). Conclusion: A clear synergistic effect of MMC and hyperthermia has been demonstrated in four human bladder cancer cell lines.     

Effects of bacillus Calmette-Guèrin and interferon alpha-2B on cytokine production in human bladder cancer cell lines

PURPOSE: To determine the effects of live BCG, autoclaved BCG and interferon alpha-2b on cytokine production in human bladder cancer cell lines. MATERIALS AND METHODS: The release of nine cytokines from the human bladder cancer cell lines, RT4, RT112, SD, MGH and J82, was measured by ELISA assay. The mRNA level of IL-6 and GM-CSF was determined by RT-PCR. RESULTS: BCG and/or interferon alpha-2b differentially increased IL-1beta, IL-6, IL-8, GM-CSF and TNF-alpha production in the bladder cancer cells. High grade cell lines were more responsive to BCG whereas low grade lines were more sensitive to interferon alpha-2b. This correlated with cytotoxicity and growth inhibition induced by these agents. BCG could also induce low levels of IFN-alpha production in all the cell lines. Compared with live BCG, autoclaved BCG had no antiproliferative effect on MGH cells and was less effective in stimulating the production of IL-6, IL-8 and GM-CSF. However, autoclaved BCG was as effective as live BCG in inhibiting growth and stimulating IL-6 and TNF-alpha production of J82 cells. The combination of BCG and interferon alpha-2b also completely suppressed TGF-beta1 production in the MGH and RT112 cell lines. CONCLUSIONS: The combination of BCG and interferon alpha-2b has additive effects in cytokine production from bladder cancer cells. This correlates with cytotoxicity and growth inhibition induced by these agents.     

Soluble Fas and Fas-ligand in bladder cancer in vitro and in vivo

Circulating soluble Fas (sFas) and expression of Fas-ligand on cancer cells are mechanisms of immune escape. The aim of the present study was to investigate expression and production of Fas and Fas-ligand on bladder cancer cell lines of different grade as a basic mechanism of their secretion in vivo. sFas and sFas-ligand serum levels of patients with different stage of bladder cancer were examined to determine the possible clinical use of these molecules as tumor markers. Bladder cancer cell lines RT4 (G1), RT112 (G1), T24 (G3) and SUP (G4) were analyzed by flowcytometry for Fas and Fas-ligand expression. To determine if the Fas-ligand gene is transcribed in these bladder cancer cell lines, RT-PCR was performed on mRNA extracted from these cell lines. Production of sFas and sFas-ligand was examined in cell culture supernatants of the cancer cells as well as in the serum of 62 patients with bladder cancer by a specific ELISA test. We demonstrate that Fas is expressed in similar levels on all human bladder carcinoma cell lines. In T24 (G3) and SUP (G4) cell lines we were able to detect the Fas-ligand protein, whereas no Fas-ligand protein could be found in RT4 and RT112 (G1) cells. Fas-ligand mRNA was expressed in all bladder cancer cell lines. Furthermore, all bladder cancer cell lines produce sFas but no sFas-ligand in spite of mRNA expression. The range of sFas levels in the serum of all patients with bladder cancer was large and did not show a correlation to the histopathological stage of bladder cancer. Although there is in vitro evidence that sFas and Fas-ligand play a role in bladder cancer, no correlation between the sFas and s Fas-ligand serum levels and the histopathological stage of bladder cancer could be found. Therefore, serum sFas and sFas-ligand have to date limited clinical relevance.     

In vitro study of the effect of hyperthermia on normal bladder cell line and on five different transitional cell carcinoma cell lines.

Intraluminal hyperthermia is potentially useful in the management of superficial bladder cancer. The potential inhibitory effect of hyperthermia on various human bladder cancer cell lines, normal human bladder cells and the murine MBT-2 bladder cancer cell line has been studied in vitro. These cell lines were exposed for one hour to 43 +/- 0.5C and compared to controls. Cell survival was assessed comparing the cell growth curve and colony formation. The human transitional cell carcinoma (TCC) cell lines vary in their sensitivity to heat. MGH-U1 was the most heat sensitive cell line. The human A-1698, CUB-2, UM-UC-3 and the murine MBT-2 lines were heat insensitive. We conclude that the cytocidal effect of hyperthermia in bladder transitional cell carcinoma is variable. Further experiments using the combination of hyperthermia and intravesical anticancer agents are in progress.     

The cytostatic effect of 9-cis-retinoic acid, tretinoin, and isotretinoin on three different human bladder cancer cell lines in vitro

Retinoids have been shown to have activity in both preclinical and clinical bladder cancer studies but their exact role in its treatment and prevention remains obscure. In this study cytostatic activity of a novel 9-cis-retinoic acid (9-cis-RA) was compared with two other retinoids: tretinoin and isotretinoin, in three different bladder cancer cell lines: RT4 (well differentiated), 5637 (moderately differentiated) and T24 (poorly differentiated). The three retinoids were incubated at concentrations of 0.3, 3 and 30 μg/ml with bladder cancer cells in microtitre plates for 3 and 6 days. The cytostatic effect was estimated by using luminometric measuring of ATP activity of viable cells in suspension. Compared with the older retinoids, tretinoin and isotretinoin, the highest concentration of 9-cis-RA had a cytostatic efficacy in all three bladder cancer cell lines tested. A clear dose–response relationship was observed in isotretinoin-treated cultures after 6 days and in all 9-cis-RA-treated cultures. Tretinoin was either ineffective or had a stimulating effect on poorly differentiated tumour cells. To conclude, isotretinoin and 9-cis-RA had a cytostatic effect on human bladder cancer cells in vitro. However, the possibility of stimulating cancer growth at small doses, at least with tretinoin, and toxicity at high doses must be considered when planning clinical trials. Received: 17 July 1997 / Accepted: 26 June 1998     

The nuclear membrane in multidrug resistance: microinjection of epirubicin into bladder cancer cell lines

OBJECTIVE: To assess whether microinjecting epirubicin into cells showing multidrug resistance (MDR, common to many cancers, including bladder cancer, with resistance to, e.g. anthracyclines and mitomycin C) spares the nucleus, as when these drugs accumulate, distribution in MDR cells characteristically spares the nucleus, suggesting that the nuclear membrane is responsible for excluding cytotoxic drugs from MDR nuclei. MATERIALS AND METHODS: Nuclear exclusion of drugs is an important feature of resistance in MDR cells, as many MDR-susceptible drugs have cytotoxic actions within the nucleus. Drug accumulation in 'classical' P-glycoprotein-mediated MDR cells is greatly reduced by efflux. Microinjection of epirubicin into the cytoplasm of MDR cells bypasses the P-glycoprotein efflux pump on the plasma membrane. Nuclear sparing would directly implicate the nuclear membrane in this phenomenon. Because of their fluorescence properties, which allow study by confocal microscopy and flow cytometry, anthracyclines have also been used extensively to investigate MDR. Thus sensitive (MGH-U1 and RT112) and MDR (MGH-U1R and MGH-U1-MMC) bladder cancer cell lines were used. Adherent cells from each cell line were individually microinjected with epirubicin (0.5 mg/mL) and a 77 kDa fluorescein isothiocyanate (FITC)-dextran (0.5 mg/mL). The pattern of nuclear epirubicin uptake in injected cells was then evaluated by confocal microscopy. The 77 kDa FITC-dextran allowed easier identification of injected cells and was also excluded from their nuclei. RESULTS: Sensitive bladder cancer cell lines all showed a nuclear accumulation pattern of epirubicin, consistent with their normal uptake after exposure to epirubicin. The MDR cell lines showed the characteristic nuclear-sparing pattern of epirubicin uptake, similar to the normal uptake pattern after epirubicin exposure. The 77 kDa FITC-dextran showed clearly which cells had been microinjected, and was excluded from the nuclei of all injected cells. Cell viability was confirmed by acridine-orange staining after initial visualization of injected cells. CONCLUSION: The nuclear membrane is responsible for the nuclear exclusion of epirubicin in MDR cells. Further work is necessary to determine the mechanisms involved.     

Evaluation of CA19-9 as a tumor marker in urothelial malignancy

OBJECTIVE: Carbohydrate antigen 19-9 (CA19-9) is known to be a marker with a high positive rate in pancreatic cancer. There are limited data on the use of CA19-9 as a tumor marker in bladder carcinoma. We tested the expression of CA19-9 in transitional cell carcinoma (TCC) cell lines and bladder cancer patients to determine its usefulness in clinical applications. MATERIAL AND METHODS: The expression of CA19-9 was determined in six TCC cell lines and 42 bladder carcinoma tissues using two approaches: immunohistochemistry and enzyme immunoassay (EIA) analysis. EIA was used for testing CA19-9 levels in spent medium of cultured TCC cells and the urine of bladder tumor patients. RESULTS: The CA19-9 value was low in spent media of the MGH-U1, MGH-U1R and MGH-U3 cell lines, but high in that of reactivity in MGH-U4 cells, while negative reactivity was found in high-grade MGH-U1 and MGH-U1R cells, both of which were derived from a stage B, grade 3 TCC. High incidences of negative CA19-9 staining were found in high-grade and invasive tumor tissues: 69.6% (16/23) and 70.8% (17/24), respectively. The sensitivity and specificity of urinary CA19-9 for detecting tumor recurrence were 83.3% and 50.8%, respectively. However, urinary tract infection also resulted in a high false-positive rate. CONCLUSION: CA19-9 is promising for use as a biomarker for the detection and monitoring of low-grade and low-stage bladder cancer, with the proviso that patients to be tested should be free of infection.     

Chemosensitivity testing on human bladder cancer cell lines, using MTT-assay

Sensitivity of human transitional cancer cells to anticancer agents was evaluated utilizing cultured cell lines. T-24, MGH-U1 and KU-1. Simultaneously, chemosensitivity tests combined with 42 degrees C hyperthermia were performed. Cells inoculated in 96-well multiplates for 48 hours, were exposed to graded concentrations of doxorubicin (DOX), mitomycin C (MMC), bleomycin (BLM), peplomycin (PEP), cis-diamminedichloroplatinum (II) (CDDP) for 2 to 48 hours. After additional culture for 48 hours, viable cell numbers were estimated by the dye exclusion assay (DEA) and tetrazolium-based colorimetric assay (MTT-assay). In 2-hour exposure, most of anti-cancer agents did not significantly suppress the growth of the cell lines. Only DOX suppressed the cell growth. In 6-hour and 48-hour exposure, DOX, MMC and CDDP showed significant growth inhibitory effect on the transitional cancer cell lines. The effect of BLM and PEP was insufficient. The hyperthermia of 42 degrees C enhanced the growth inhibitory effect of MMC and CDDP, but did not influence the effect of DOX. In comparison of DEA and MTT-assay, viable cell numbers measured by DEA well correlated with the optical density in MTT-assay. Since MTT-assay is a semiautomated, rapid and inexpensive assay with good reproducibility, it can be a useful substitute for DEA in chemosensitivity testing of cancer cells.     

Antiproliferative activities of interferons against human bladder carcinoma cell lines in vitro

The antiproliferative effect of interferons against 5 human bladder carcinoma cell lines, RT112, T24, RT4, 647V and HT1197, was determined in vitro. Each of these human bladder carcinoma cell lines except 647V was sensitive to human interferons in liquid media. The antiproliferative effect of interferons was observed only upon continuous exposure, not after 1 hour. Partially purified, naturally produced interferon beta was more inhibitory of cell growth than naturally produced interferon alpha. Interferon alpha 54, 76, 61, 6L and 1 purified to homogeneity were as effective as naturally produced, partially pure interferon alpha. Although interferon beta, produced by recombinant DNA technology and purified to homogeneity, was not equivalent in effectiveness to naturally produced interferon beta, its antiproliferative activity was greater than interferon alpha 54 for 3 of 4 cell lines tested. Antimitotic effects may underlie, at least in part, the potential therapeutic activity of interferons for bladder carcinoma.     

The liposome-incorporating cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guéin can directly enhance the susceptibility of cancer cells to lymphokine-activated killer cells through up-regulation of natural-killer group 2, member D ligands

OBJECTIVE

? To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural‐killer group 2, member D (NKG2D) ligands by R8‐liposome‐bacillus Calmette‐Guéin (BCG)‐cell wall skeleton (CWS) treatment.

MATERIALS AND METHODS

? The T24 cells and RT‐112 cells were co‐cultured with R8‐liposome‐BCG‐CWS and BCG for 2, 4, or 6 h, and then the surface expression of NKG2D ligands was analyzed using TaqMan real‐time quantitative RT‐PCR. ? Peripheral blood mononuclear cells were obtained with a conventional preparation kit, and then lymphokine‐activated killer (LAK) cells were generated from these purified peripheral blood mononuclear cells via interleukin‐2 stimulation. ? The anti‐tumour effect of LAK cells against untreated and R8‐liposome‐BCG‐CWS co‐cultured with cells of the human bladder cancer cell lines T24 and RT‐112 was analyzed using the cytotoxic WST‐8 assay method at 4 h of culture at various effector/target (E : T) ratios.

RESULTS

? Major histocompatibility complex class I‐related chain B (MICB) expression was increased ≈1.5‐fold on T24 cells and RT‐112 cells with BCG. ? UL‐16‐binding protein (ULBP) 1 expression was also increased ≈1.5‐fold on T24 cells and RT‐112 cells with BCG. R8‐liposome‐BCG‐CWS increased the surface expression of MICB 2.2‐fold on T24 cells but did not increase it significantly on RT‐112 cells. ? ULBP1 expression was increased ≈2.2‐fold on RT‐112 cells, although no differences were observed between the expression of ULBP2 and 3 with R8‐liposome‐BCG‐CWS. ? T24 cells that were co‐cultured with R8‐liposome‐BCG‐CWS showed an ≈1.3‐fold increase in sensitivity to cytolysis by LAK cells at an E : T ratio of 4 and RT‐112 cells showed an ≈1.4‐fold increase at an E : T ratio of 2.

CONCLUSIONS

? In the present study, the induction of surface NKG2D ligands by R8‐liposome‐BCG‐CWS rendered cancer cells more susceptible to cytolysis by LAK cells. ? T24 cells and RT‐112 cells, even when cultured singly in the absence of immune cells, can directly respond to R8‐liposome‐BCG‐CWS. ? The results obtained in the present study may therefore indicate a novel adoptive immunotherapy against bladder cancers.     

Expression of adhesion molecules by bladder cancer cells: modulation by interferon-gamma and tumour necrosis factor-alpha.

The constitutive expression by eight human bladder cancer cell lines of the cell adhesion molecules intercellular adhesion molecule-1 and intercellular adhesion molecule-2 was studied using monoclonal antibody probes in conjunction with flow-cytometry. Tumour lines of low grade (G1) did not constitutively express intercellular adhesion molecule-1, rather they were found to express intercellular adhesion molecule-2. The G2 cells expressed no intercellular adhesion molecule-2, however, a low percentage did express intercellular adhesion molecule-1. High grade cells (G3) only expressed intercellular adhesion molecule-1 on their cell surface but at higher levels than the G2 cell line. Exposure of the bladder cancer cell lines to interferon-gamma induced and augmented the expression of intercellular adhesion molecule-1 by all except one of the cell lines (UMUC3). Intercellular adhesion molecule-2 expression remained unaltered. The modulation of intercellular adhesion molecule-1 expression was dependent on the concentration of interferon-gamma and the duration of stimulus. De novo intercellular adhesion molecule-1 expression, induced by interferon-gamma, was rapid (< 4 hours) with only a short period of stimulation being required (< 10 seconds). The rapid increase in expression of intercellular adhesion molecule-1 required de novo protein synthesis and was not the result of release of intercellular adhesion molecule-1 from an intracellular pool. Interferon-gamma and tumour necrosis factor-alpha were found to act synergistically in the induction and augmentation of intercellular adhesion molecule-1 expression. Optimal induction occurred with 10 Uml-1 of both molecules. These results suggest a correlation between constitutive adhesion molecule expression and the histopathological grade of the tumour. The implications of these findings for Bacillus Calmette Guerin and interferon-gamma immunotherapy of bladder cancer is discussed.     

Antiproliferative effects of interferons against human bladder carcinoma cell lines in vitro

In order to evaluate the antiproliferative effects of recombinant human interferon-gamma 2c (rHu IFN-gamma 2c), recombinant human interferon-gamma (rHu IFN-gamma), natural interferon-beta (IFN-beta), and their combination with cytotoxic agents, 17 different human bladder carcinoma cell lines were tested in vitro. The antiproliferative effects were compared in evaluating the tumor cell inhibiting potency of the different interferon (IFN) classes. It could be demonstrated that interferons have inhibiting effects on the bladder cancer cell multiplication rate, yet there are significant differences in the susceptibility of different IFN preparations on different cell lines. The cell lines BT1, RT4, EJ, 468P, 253J, SD, TCCSUP, and SW1738 can be defined as sensitive, T24, 647V, VM-CUB2, and J82 as semisensitive, HT1376, 5637, VM-CUB1, 639V and SW1710 as resistant upon treatment with IFN. The combination of rHu IFN-alpha 2c, IFN-beta, and rHu IFN-gamma seems to be more effective than treatment with rHu IFN-alpha 2c alone. The cytotoxic effect of doxorubicin on bladder cancer cells can be intensified by combining it with IFN.     

Expression of interferon-γ receptors on bladder cancer cells: does it correlate with biological response?

Summary Previously we have shown a differential biological response of three human bladder cancer cell lines (RT4, RT112 and MGH-U1) to gamma interferon (IFN-). The present study examines the relationship between the biological response and the expression of the interferon- receptor on the tumour cell surface. Using a competitive radioligand binding assay and Scatchard analysis, we measured the number and affinity of the IFN- receptors on each of the above cell lines. Individual cells from each line expressed large numbers (29,100–41,800) of high-affinity receptors (k _d =2.4–3.9×10¹⁰M). There was no statistically significant difference in either of these parameters between the three lines. We therfore conclude that the biological response of these bladder lines to IFN- does not relate to the number or affinity of its receptor on the plasma membrane of these tumour cells.     

Antineoplastic activity of honey in an experimental bladder cancer implantation model: In vivo and in vitro studies

OBJECTIVES: The antitumor effect of bee honey against bladder cancer was examined in vitro and in vivo. Methods: Three human bladder cancer cell lines (T24, 253J and RT4) and one murine bladder cancer cell line (MBT-2) were used in these experiments. In an in vitro study, the antitumor activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay, 5-Bromodeoxyuridine (BrdU) labeling index and flowcytometry (FCM). In the in vivo study, cancer cells were implanted subcutaneously in the abdomens of mice, and the effects were assessed by the tumor growth. RESULTS: In vitro studies revealed significant inhibition of the proliferation of T24 and MBT-2 cell lines by 1-25% honey and of RT4 and 253J cell lines by 6-25% honey. BrdU labeling index was significantly lower. FCM showed lower S-phase fraction, as well as absence of aneuploidy compared with control cells. In the in vivo studies, intralesional injection of 6 and 12% honey as well as oral ingestion of honey significantly inhibited tumor growth. CONCLUSION: Bee honey is an effective agent for inhibiting the growth of T24, RT4, 253J and MBT-2 bladder cancer cell lines in vitro. It is also effective when administered intralesionally or orally in the MBT-2 bladder cancer implantation models. Our results are promising, and further research is needed to clarify the mechanisms of the antitumor activity of honey.     

Is gamma-linolenic acid an effective intravesical agent for superficial bladder cancer? In vitro cytotoxicity and in vivo tolerance studies

The essential fatty acid gamma-linolenic acid (GLA) is an effective cytotoxic agent when applied topically and for prolonged periods to tumour cells. Topical application, by intravesical therapy, is firmly established in the treatment of superficial bladder cancer. However, this form of therapy is limited to a maximum duration of 2?h. At such a short drug exposure time, does GLA retain its cytotoxicity? We have examined this question by exposing the superficial bladder cancer cell lines MGH-U1 and RT112 to meglumine-GLA (MeGLA) for time intervals ranging from 30?min to 2?h, at drug concentrations ranging from 1000 to 1.95?μg/ml. The MTT viable biomass assay was used to assess cell kill. Greater than 90% inhibition was observed at a concentration of 125?μg/ml (IC?>?90), at 2?h drug exposure. At shorter drug exposure times, higher drug concentrations were needed to induce the same effect. At 1?h drug exposure, the IC?>?90 was recorded at 500?μg/ml. In vivo intravesical tolerance studies were conducted in rats. Rats exposed to 2.5?mg/ml MeGLA intravesically for 2?h or less remained well and bladder histology showed minimal changes. This study confirms that GLA retains its cytotoxicity at short drug exposure times and is well tolerated by normal bladder mucosa in vivo. Bladder mucosa tolerated >10× the concentration required for the IC?>?90 in vitro. MeGLA is therefore a feasible intravesical agent for superficial bladder cancer.