The effect of short-chain fatty acids butyrate, propionate, and acetate on urothelial cell kinetics in vitro: potential therapy in augmentation cystoplasty.

The intestinal element of enterocystoplasty is affected by chronic inflammatory changes, which lead to excess mucus production, urinary tract infections, and stone formation. There is also an increased risk of malignancy. These inflammatory changes may be due to diversion colitis, which affects colonic segments excluded from the faecal stream and likewise may respond to intraluminal short-chain fatty acid (SCFA) therapy. The SCFAs have interesting antiproliferative, differentiating, and pro-apoptotic effects, which are protective against colorectal cancer and may influence the risk of malignancy in enterocystoplasty. Before intravesical therapy can be considered, the effect on normal urothelium must be investigated. Primary urothelial cells cultured from biopsy specimens and transformed urothelial (RT112 and MGH-U1) and intestinal cell lines (HT29 and CaCo-2) were incubated with SCFAs. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the residual viable biomass to assess cell proliferation. Proliferation of primary and transformed urothelial cells in culture was inhibited by all SCFAs in a similar time- and dose-dependent manner. The concentration of SCFA required to inhibit growth of primary cells by 50% (IC50) was 20 mM of butyrate, 120 mM of propionate, and 240 mM of acetate after incubation for 1 h. After 72 h the IC50 was 2 mM of butyrate, 4 mM of propionate, and 20 mM of acetate. Transformed urothelial and colon cancer cell lines demonstrated similar growth inhibition. Butyrate was the most potent inhibitor of cell proliferation, followed by propionate and then acetate. Growth inhibition is not an immediate cytotoxic effect, and urothelial cells show a degree of adaptation to butyrate and growth recovery after incubation with butyrate. In conclusion, butyrate- and propionate-induced growth inhibition is potentially clinically significant and may have therapeutically beneficial implications in vivo.

Intravesical pH: a potentially important variable affecting efficacy and the further development of anthracycline chemotherapy for superficial bladder cancer.

OBJECTIVE: To assess, using epirubicin-sensitive and multidrug resistant (MDR) derivatives of human bladder cancer cell lines in vitro, the probable effect of intravesical pH changes, with and without the MDR antagonist verapamil, on the uptake, intracellular distribution and cytotoxicity of epirubicin during intravesical chemotherapy. MATERIALS AND METHODS: Incubations for cytotoxicity testing were carried out in buffered medium containing epirubicin, at pH values of 6.0-8.5, with verapamil where appropriate. The cytotoxicity of epirubicin, with and without verapamil, was determined using the tetrazolium cytotoxicity assay. Intracellular epirubicin fluorescence was assessed using flow cytometry and confocal microscopy. Flow cytometric total intracellular epirubicin fluorescence was measured at pH 6.0, 6.4, 6.8, 7.2, and 7.6, and confocal microscopy was carried out at pH 6.0 and 8.0. The MDR-reversing agent verapamil was added at 100 micro g/mL to some incubations. RESULTS: Epirubicin cytotoxicity in resistant cell lines appears considerably enhanced by adding verapamil and further improved, especially in MDR cells, by alkalinization of the drug solution to pH 8.0. Flow cytometry results showed striking and consistent differences in epirubicin handling with pH. Sensitive cells can be induced to absorb considerably more drug at alkaline pH, whilst resistant cells show no such behaviour. Nuclear drug fluorescence was greater in sensitive cells at alkaline pH, but cytoplasmic drug fluorescence in the resistant cells was little changed by pH. Adding verapamil to resistant cells restored the sensitive phenotype of drug handling. CONCLUSION: Buffering epirubicin to an alkaline pH before intravesical application should increase its intrinsic cytotoxicity. The potential for synergy at certain drug combinations will be enhanced by applying these findings. MDR reversal and fatty acid augmentation of drug uptake are discussed as examples.

Intravesical meglumine gamma-linolenic acid in superficial bladder cancer: an efficacy study.

OBJECTIVES: Gamma-linolenic acid (GLA) is known to be cytotoxic to malignant cells. We assessed the efficacy of the novel intravesical formulation, meglumine gamma-linolenic acid (MeGLA), in a phase II trial, in patients with recurrent, superficial bladder cancer. PATIENTS AND METHODS: Thirty patients with recurrent, superficial transitional cell carcinoma (TCC) were recruited. The tumour pattern was recorded at flexible cystoscopy. Patients received a single intravesical instillation of 50ml of either 50mg (1mg/ml) (15 patients), or 125mg (2.5mg/ml) (15 patients) of MeGLA in water, retained for one hour. At subsequent cystoscopy, the tumour patterns were recorded, prior to undertaking routine cystodiathermy. Biopsies were obtained for histological assessment. Responses were divided into complete, partial or none. RESULTS: All 30 patients retained the drug for 1 hour without significant local or systemic side effects. There were 4 (13%) complete responses, 9 (30%) partial responses, and 17 (57%) non-responders. Histology showed no evidence of damage to surrounding urothelium. CONCLUSIONS: Our data confirms the safety and tolerability of MeGLA, which is consistent with findings from a previous phase I trial. A response rate of 43% also indicates that MeGLA has a significant cytotoxic effect against TCC and the results are similar to those obtained using standard, single-dose, intravesical regimens.

A model of superficial bladder cancer using fluorescent tumour cells in an organ-culture system.

OBJECTIVE: To develop a reproducible in vitro simulation of superficial bladder cancer for testing cytotoxic agents at clinically relevant concentrations. MATERIALS AND METHODS: Square explants (5 mm) of rat bladder were cultured in Petri dishes in minimal volumes of Waymouth's MB 752/1 medium supplemented with 10% fetal calf serum, antibiotics and glutamine. Parental and resistant MGH-U1 urothelial cancer cells were transfected with a green fluorescent protein (GFP) vector. Transfectants were purified by flow cytometry. Cells were seeded onto the prepared organ cultures and images obtained using confocal microscopy. The tumour colonies were confirmed using scanning electron microscopy. Conventional intravesical cytotoxic agents including epirubicin, mitomycin-C and estramustine were tested in the system. RESULTS: Colonies of GFP-MGH-U1 cells became established on the explants and were identified by confocal microscopy; the development of the colonies was then followed over several days. Staining the explant for viability allowed imaging of normal urothelium on the explant surface or surrounding skirt of urothelial cells. The conventional cytotoxic agents epirubicin, mitomycin C and estramustine showed the expected differential responses to parental and resistant cell types. The colonies were able to survive high concentrations of the drug, equivalent to those in clinical use. The colonies were imaged serially over a period of several days. CONCLUSION: This system provides a more realistic model for testing cytotoxic agents for use in intravesical therapy, by allowing clinically relevant concentrations of drugs to be tested. The differential properties of the parental and resistant cells are maintained. The model also enables the same tumour colony to be imaged over several days in culture. The model may also be adapted for use in testing the effects of drugs on normal urothelium and the study of the effects of growth factors.

Nonhomologous bioinjectable materials in urology: 'size matters'?

OBJECTIVE: To assess the factors that influence how particles might become fixed in tissues or migrate from them, by measuring the size of the injectable particles, their susceptibility to phagocytosis and their affinity for fibroblast attachment in culture. MATERIALS AND METHODS: The particle size of three types of particulate unphysiological bioinjectable material, i.e. Urocol (Genesis Medical, Ltd., London), Macroplastiquetrade mark (Uroplasty Ltd., Reading, UK) and Urethrin (Mentor Medical Systems, Wantage, UK) was analysed using phase-contrast light microscopy and confocal microscopy. Human monocytes from peripheral blood were incubated with the three materials in phagocytic studies, where ingestion was determined by confocal microscopy. A fibroblast cell line was used to ascertain the ability of the particles to act as a substrate for cell attachment in culture. RESULTS: The mean (SEM) maximum particle diameters of Macroplastique, Urethrin and Urocol were 209 (5.10) microm, 49 (1.52) microm and 14 (0.39) microm, respectively. Rat peritoneal macrophages and human peripheral blood monocytes commonly ingested Urocol particles; the phagocytosis of Urethrin was rare and that of Macroplastique was not detected. Fibroblasts adhered to Urocol paste and Urethrin particles, but not to Macroplastique. CONCLUSION: Published reports of particle size and phagocytosis are confusing, but a relationship clearly exists. Macroplastique is the largest particle and is least likely to be phagocytosed by human mononuclear phagocytes. Urocol paste is the slowest to dissipate in culture conditions; the flat surfaces of Urethrin, but not Macroplastique, can serve as a substrate for fibroblast anchorage.

Estramustine reversal of resistance to intravesical epirubicin chemotherapy.

OBJECTIVE: Failure of epirubicin treatment of superficial bladder cancer implies multidrug resistance (MDR) which is common. MDR is characterised by decreased cellular levels of drug. TCC cell lines sensitive to epirubicin and resistant to both epirubicin and mitomycin C were used to investigate augmented therapy by adding the MDR reversing agent estramustine to an in vitro model. METHODS: Cells were cultured as monolayers. Fluorescence analysis was performed by flow cytometry and confocal microscopy. Cells were exposed to epirubicin 20 microg/ml for 2 h and increasing amounts of estramustine. Cytotoxicity was determined under similar exposure conditions and MTT culture (dye reduction by live cells) allowed viable biomass to be read optically. RESULTS: Resistant cells accumulated eight times less epirubicin than sensitive cells. Confocal microscopy confirmed this for nuclear uptake. Accumulation in resistant cells can be increased to near-sensitive cell levels using 40 microg/ml estramustine. Image analysis of confocal fluorescence showed a shift from cytoplasm to nucleus. This correlated with increased cytotoxicity. CONCLUSION: Estramustine plus epirubicin chemotherapy can overcome MDR and may achieve more successful tumour killing in vivo. It may also prevent emergence of resistance. Primary TCC culture examination permits detection of sensitive and resistant cells and may predict outcome allowing a more logical treatment selection.

Haematuria and clot retention after transurethral resection of the prostate: a pilot study.

OBJECTIVE: To determine, prospectively, the effect of clinical factors on the duration of frank haematuria and the incidence of clot retention after transurethral resection of the prostate (TURP). PATIENTS AND METHODS: Seventy-nine men who underwent TURP in a 3-month period were entered into this study, during which the time to cessation of bleeding and the occurrence of clot retention(s) were recorded over a 4-week period. The effect of other clinical factors (histology, weight of tissue resected. operative duration, grade of surgeon and resection rate) was also assessed. RESULTS: Gross haematuria ceased in 47%, 73%, 96%, and 97% of patients at the end of the first, second, third and fourth weeks, respectively. The duration of postoperative bleeding was significantly associated with the weight of tissue resected and the operation time (P<0.001 and <0.05, respectively). Furthermore, five patients were re-admitted with clot retention, but there was no significant correlation between the occurrence of this morbidity and any of the other indices. CONCLUSION: Postoperative bleeding usually stops within 3 weeks of TURP. This period, which is about half the time hitherto assumed, is directly related to the size of the gland resected and the duration of the procedure. However, the occurrence of clot retention is not significantly associated with the duration of haematuria or any of the other clinical factors evaluated. Thus, a high fluid intake is mandatory for 3 weeks after TURP, but men who continue to bleed should be advised to continue with a high-fluid regimen until their urine is clear.

Water as a tumoricidal agent in bladder cancer. In vitro studies in parental and resistant cell lines.

OBJECTIVE: Water is widely used as a tumoricidal agent during transurethral resection of bladder tumour (TURBT). Despite this, recurrences are common. One possible explanation may be that while the parental bladder cancer cells are sensitive to water, their multidrug resistant (MDR) clones which occur commonly, may be resistant to it. If so, is this a preserve of the MDR phenotype or do other mechanisms confer resistance to water? METHODS: The parental human urothelial cancer cell lines (MGHU-1 and RT112) and their drug-resistant variants (MDR and non-MDR) were exposed to short pulses of water. The MDR status of these cells was verified using confocal microscopy to detect intracellular accumulation of fluorescent drug (epirubicin). A clonogenic assay was used to establish tumoricidal efficacy on cells in suspension. A thiozolyl blue (MTT) assay was used on adherent cells to assess the residual viable biomass. RESULTS: In the MDR clone of cells, colony counts were reduced by 20% after treatment with water as compared to a 60% reduction in the parental cell line. There was a similar reduction in colony counts between the parental RT112 and its non-MDR cisplatin resistant variant. CONCLUSION: Water is less effective on MDR cells. Resistance to water may be a function of the MDR phenotype.

Intravesical chemotherapy with gamma linolenic acid becomes a realistic prospect in serum-free applications: in vitro cytotoxicity and systemic absorption studies.

PURPOSE: To assess the cytotoxicity of Meglumine gamma linolenic acid (MeGLA) in serum-free application on 2 urothelial cancer cell lines, to examine whether the instant kill action of MeGLA is retained in a serum free environment, and to study the pharmacokinetics of intravesical instillation of gamma linolenic acid (GLA). MATERIALS AND METHODS: The 2 human urothelial cancer cell lines (MGH-U1 & RT112) were utilized in classical cytotoxicity assays in which drug exposure lasted 2 hours in serum or in serum-free application. The thiozolyl blue (MTT) assay was used to quantify the residual viable biomass 5 days later. Immediate cytotoxicity was also compared in serum and serum-free application. Four Wistar rats were used to study the intravesical absorption profile of tritiated GLA (3H-GLA). RESULTS: There was a 10-fold enhancement of the lytic efficacy of MeGLA in serum-free application and this enhancement was also observed in experiments assessing instant kill. There was a similar enhancement of efficacy seen in the multi-drug resistant (MDR) clone of cells. The absorption profile showed < 2% of instilled counts were absorbed and the commonest destination for the absorbed GLA was the liver. CONCLUSIONS: The cytotoxic action of MeGLA was enhanced in serum free application. This enhancement was maintained when cells expressed the MDR phenotype. There was limited absorption from the bladder. MeGLA is a feasible intravesical agent for use in superficial bladder cancer.

Effects of short-chain fatty acids on primary urothelial cells in culture: implications for intravesical use in enterocystoplasties.

The presence of inflammatory changes and mucopus production in an enterocystoplasty may be similar to the condition of diversion colitis and starvation diarrhea caused by a lack of luminal short-chain fatty acids (SCFAs). We postulate a therapeutic role for intravesical SCFA. Because this treatment will also contact the urothelium, we have assessed the effect on cellular proliferation by utilizing primary urothelial cells in culture. Primary urothelial cells were grown from biopsy samples of normal urothelium obtained intraoperatively. A cocktail of SCFA used in the treatment of diversion colitis was incubated with these cells for time intervals ranging from 30 minutes to 72 hours at drug concentrations ranging from 0.04 to 20 mmol/L butyrate equivalent (BE). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the residual viable biomass to assess growth inhibition. These experiments were repeated on cells grown on matrigel substrate. The human urothelial cancer line RT112 was likewise exposed to SCFAs to assess selectivity between primary and transformed cells. Primary urothelial cells in culture undergo growth inhibition when exposed to SCFAs. The concentration of SCFAs required to reduce the general biomass by 50% or more (IC> or =50) was 20 mmol/L BE when exposure was for 2 hours or less. When drug exposure was prolonged for 72 hours, the IC> or =50 was 2.5 mmol/L BE. Cells grown on matrigel had their growth similarly inhibited. The IC > or = 50 for the RT112 cell line was 2.5 mmol/L BE after 72 hours of drug incubation. Primary urothelial cells in culture undergo a time- and dose-dependent growth inhibition when exposed to SCFAs. This inhibition is particularly apparent at the higher doses similar to those in use in clinical practice. Cells grown on a matrigel substrate suffer growth attenuation similar to that affecting cells grown on polystyrene plates. In vivo assessment in a rodent intravesical model is advisable before considering instillations in patients.

Bladder cancer recurrence by implantation of exfoliated cells: is gamma-linolenic acid an effective tumoricidal agent?

OBJECTIVE: To compare the tumoricidal efficacy of meglumine gamma-linolenic acid (MeGLA), mitomycin C, epirubicin and water on two urothelial cell lines, and to establish the effect of serum protein levels derived from bladder cancer resection craters on the action of these agents. MATERIALS AND METHODS: The human urothelial cell lines MGHU-1 and RT112 and their drug-resistant variants were exposed to short pulses of aqueous MeGLA, mitomycin, epirubicin and water. Both adherent and suspended cells were exposed to these agents. The MTT viable biomass assay and a clonogenic assay were used to establish tumoricidal efficacy. These experiments were then repeated to assess the effect of added serum proteins on the test results. Estimates of protein in the waste irrigation fluid from 10 patients undergoing transurethral resection of bladder tumour (TURBT) were used to select the quantity of protein used in the study, to establish the clinical relevance. RESULTS: MeGLA caused > 95% reduction in the residual viable biomass of adherent cells, compared with < 50% reduction with any other agent. Both epirubicin and mitomycin were as effective as MeGLA in preventing colony formation from suspended drug-sensitive (parental) cells. However, using multidrug-resistant (MDR) cell lines, only MeGLA prevented any colony formation, although counts were greatly reduced by mitomycin and epirubicin. Water was least effective as a tumoricidal agent on both adherent and suspended cells. On the latter, water was markedly inactivated by adding 5% serum. TURBT waste irrigation fluid was found frequently to contain such quantities of serous fluid contamination, as shown by albumin estimates in waste fluid from 10 consecutive patients undergoing this procedure. CONCLUSION: MeGLA is an effective tumoricidal agent against both parental and MDR cell lines. Its efficacy is maintained in the presence of clinically relevant serum contamination.

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 microg/ml. The MTT viable biomass assay was used to assess cell kill. Greater than 90% inhibition was observed at a concentration of 125 microg/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 microg/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 > 10x the concentration required for the IC > 90 in vitro. MeGLA is therefore a feasible intravesical agent for superficial bladder cancer.