COX-2 inhibition demonstrates potent anti-proliferative effects on bladder cancer in vitro.

PURPOSE: The purpose of this work was to determine the in vitro effect of Rofecoxib and specific COX 1 and COX 2 inhibitors in regards to cell growth and apoptotic and necrotic activity. INTRODUCTION AND OBJECTIVE: Rofecoxib (Vioxx) is a nonsteroidal anti-inflammatory agent (NSAID) that selectively inhibits cyclooxygenase-2 (COX-2). The inducible isoform of COX-2 is overexpressed in many gastrointestinal and genitourinary tract tumors. We hypothesized that in vitro treatment with both COX-1 and COX-2 inhibitors would significantly reduce cellular proliferation of bladder cancer cells by apoptotic pathways. MATERIALS AND METHODS: Two human bladder cancer cell lines were grown in culture using standard techniques and treated with Rofecoxib at doses ranging from 125 microg/well serially diluted down to 8.0 microg/well. Catechin (COX 1 inhibitor) and NS398 (COX 2 inhibitor) were used at doses of 50 and 100 microM. Cell viability was measured by MTT at 24 and 72 h. Apoptosis was evaluated by the Annexin V FITC Assay. Statistical analysis was performed by ANOVA. RESULTS: Rofecoxib, Catechin, and NS398 all exhibited significant inhibition of cell growth when compared to the nontreated controls. Significant changes in apoptotic activity were observed in all agents tested in both the T24 and the TCCSUP cells. CONCLUSIONS: Selective COX-2 inhibition, using the well-tolerated and commercially available Rofecoxib (VIOXX) and specific COX 1 and 2 inhibitors, reduced the growth of human bladder cancer in vitro by apoptotic mechanisms. Further in vivo and human studies are warranted to evaluate the safety and clinical utility of this agent in patients with bladder cancer.

Enhanced immunocompetence by garlic: role in bladder cancer and other malignancies.

Of the many beneficial actions of garlic, inhibition of the growth of cancer is perhaps the most remarkable. Our previous animal studies demonstrated that aged garlic extract was highly effective, and unlike the approved immunotherapy for human bladder cancer, bacillus Calmette--Guérin (BCG), garlic was effective when added to the diet. To elucidate the mechanism of this antitumor effect, the literature describing antitumor and immune-enhancing effects of garlic is reviewed. Garlic can detoxify carcinogens by stimulation of cytochrome P(450) enzymes, antioxidant activity or sulfur compound binding. Studies demonstrate a direct toxic effect of garlic to sarcoma and gastric, colon, bladder and prostate cancer cells in tissue culture, but these effects cannot explain the inhibition of growth of transplanted cancer in animal models. The most likely explanation of this effect is immune stimulation. Comparison of the effects of garlic to BCG immunotherapy reveals many similarities. Both stimulate proliferation of lymphocytes and macrophage phagocytosis, induce the infiltration of macrophages and lymphocytes in transplanted tumors, induce splenic hypertrophy, stimulate release of interleukin-2, tumor necrosis factor-alpha and interferon-gamma, enhance natural killer cell, killer cell and lymphokine-activated killer cell activity. These activities represent effective stimulation of the immune response. Studies suggest that garlic may be useful in preventing the suppression of immune response that is associated with increased risk of malignancy. Data suggest that maintenance of immune stimulation can significantly reduce the risk of cancer. Clinical trials should be initiated to test the hypothesis that the immune stimulation and other beneficial effects of garlic are able to reduce the incidence of cancer.

Keyhole limpet hemocyanin immunotherapy of bladder cancer: laboratory and clinical studies.

BACKGROUND: Since the serendipitous observation by Olsson in 1974 that patients immunized with 5 mg of keyhole limpet hemocyanin (KLH) had a marked reduction in recurrence of superficial bladder cancer, multiple laboratory and clinical studies have confirmed the efficacy of KLH immunotherapy. RESULTS: In 1981, we reported that KLH immunotherapy reduced tumor growth and prolonged survival in the MBT-2 murine model of transitional cell carcinoma (TCC), and in 1988, Jurincic and co-workers demonstrated that KLH was superior to mitomycin C chemotherapy in preventing bladder tumor recurrence. Subsequent studies using Immucothel (Biosyn), crude KLH, and endotoxin-free KLH confirmed the efficacy of KLH immunotherapy in the MBT-2 murine bladder cancer model (p < 0.05), and resulted in up to 100% survival. CONCLUSIONS: To evaluate the efficacy of KLH immunotherapy in patients, a multicenter clinical trial was performed. Sixty-four patients with CIS or residual stage T(a), T(1) TCC, or both were enrolled in a phase I-II trial of escalating doses of weekly KLH given intravesically for 6 weeks. Patients were followed with cystoscopic examination, urine cytology, and bladder biopsy. Complete response was seen in 50% of patients with CIS, 20% of patients with residual T(a), T(1) TCC, and 33% of patients with both CIS and residual T(a), T(1) TCC. Responses occurred at all doses tested: 0.4, 2, 10 and 50 mg. No significant difference in response according to dose was noted, but optimal overall complete response was seen with a dose of only 2 mg. The toxicity of KLH is minimal. KLH appears to be a safe and highly effective immunotherapy for superficial bladder cancer.

The potential application of Allium sativum (garlic) for the treatment of bladder cancer.

Additional studies are needed to identify the active ingredients in Allium Sativum (garlic) that are responsible for the observed antitumor activity and immune stimulation. Garlic seems to detoxify chemical carcinogens and prevent carcinogenesis and can also directly inhibit the growth of cancer cells. Current data suggest that low molecular weight sulfur compounds and protein F4 have immune-stimulation properties. Garlic is reported to stimulate immunity, including macrophage activity, natural killer and killer cells, and LAK cells, and to increase the production of IL-2, TNF, and interferon-gamma. These cytokines are associated with the beneficial Th1 antitumor response, which is characteristic of effective cancer immunotherapies. As is true of BCG, garlic stimulates the proliferation of macrophages and lymphocytes and protects against the suppression of immunity by chemotherapy and ultraviolet radiation. Garlic is clearly not a panacea for cancer, but its broad range of beneficial effects are worthy of serious consideration in clinical trials for the prevention and treatment of cancer.

Allium sativum (garlic) treatment for murine transitional cell carcinoma.

BACKGROUND: Currently, immunotherapy with Bacillus Calmette-Guerin (BCG) is the most effective treatment for superficial bladder carcinoma, but treatment-related toxicity may limit its use in some patients. Alternative treatments are needed for patients who fail to respond to BCG immunotherapy. Allium sativum (AS), or garlic, is known to have a broad range of biologic activities, including immune stimulation and reported antitumor activity. For these reasons, the authors conducted a series of experiments designed to explore the possible therapeutic effects of AS in the MBT2 murine bladder carcinoma model. METHODS: C3H/HeN mice were randomized prior to initiation of each experimental protocol. Mice received 1 x 10(3) MBT2 cells in 0.1 mL RPMI-1640, administered subcutaneously into the right thigh, on Day 0 of the experiment. AS was injected at the site of tumor transplantation on Day 1 and at 2- to 7-day intervals up to Day 28. To evaluate the effects of oral AS in this model, treatment was initiated 30 days prior to tumor inoculation and continued for 30 days after tumor inoculation. Animals in all experiments were followed for tumor incidence, tumor growth, and survival. RESULTS: In the initial experiments, subcutaneous AS significantly reduced tumor volume compared with the saline control (P < 0.05). Unfortunately, treatment-related death was also observed, requiring reduction in the total dose of AS. Animals that received 5 weekly immunizations of AS (5 mg, 5 mg, 1 mg, 1 mg, and 1 mg; cumulative dose = 13 mg) had significantly reduced tumor incidence, tumor growth, and increased survival when compared with animals that received the saline control. No treatment-related deaths were observed with this treatment schedule. To determine whether systemic AS administration might be effective, orally administered AS was tested at doses of 5 mg, 50 mg, and 500 mg per 100 mL of drinking water. Mice that received 50 mg oral AS had significant reductions in tumor volume (P < 0.05) when compared with animals that received the saline control, and mice that received 500 mg oral AS had significant reductions in both tumor volume and mortality (P < 0.05). CONCLUSIONS: The significant antitumor efficacy of subcutaneous and oral AS warrants further investigation and suggests that AS may provide a new and effective form of therapy for transitional cell carcinoma of the bladder.

Apparent failure of current intravesical chemotherapy prophylaxis to influence the long-term course of superficial transitional cell carcinoma of the bladder.

During the 4 decades since the first introduction of intravesical chemotherapy, 3,899 patients were enrolled in 22 randomized prospective controlled studies. Of these 22 studies 13 reported a statistically significant benefit of intravesical chemotherapy. With varying followup, the reported decrease in the incidence of patients with tumor recurrence averaged only 14% (range -3 to +43%). Unfortunately, long-term (5-year) studies show no decrease in the incidence of recurrent tumor. Maintenance chemotherapy has failed to improve these results and data suggest that a single early postoperative instillation may, in fact, be most effective. Among 10 studies that include progression data none showed decreased tumor progression, and overall among 2,011 randomized patients progression occurred in 7.5% of those receiving intravesical chemotherapy and 6.9% of those treated by surgery alone. Since intravesical chemotherapy has been demonstrated in animal models to be carcinogenic, these data raise the concern that intravesical chemotherapy might possibly be carcinogenic in humans. In the absence of demonstrated long-term benefit we question the advisability of routine prophylactic intravesical chemotherapy.

Drug therapy of bacillus Calmette-Guérin sepsis.

Intravesical bacillus Calmette-Guérin (BCG) is widely used for the treatment of transitional cell carcinoma of the bladder. Although it is usually well tolerated, sepsis can occur, which has resulted in at least eight deaths [3]. The survival of Connaught BCG-infected mice treated with single and combination antibiotic and steroid therapy was evaluated. Triple-drug therapy with isoniazid, rifampin, and prednisolone resulted in 53% survival compared with 25% survival in the control group (P = 0.0209). A survival of only 10.5% was observed with treatment using prednisolone alone. This survival was worse than that of the control group (25%), and approached statistical significance (P = 0.0669). Our data suggest that BCG sepsis probably has components of both a hypersensitivity reaction and bacterial sepsis; they support the current use of combination antibiotic and steroid therapy for treatment of BCG sepsis in humans, but argue against treatment with steroids alone.

Megadose vitamins in bladder cancer: a double-blind clinical trial.

Epidemiological and laboratory studies suggest that vitamin supplements may be helpful in the prevention of some cancers but clinical trials to date have failed to demonstrate protection with naturally occurring vitamins. Without substantiation of the highly touted benefits of vitamins, few physicians who care for cancer patients have recommended their use. A total of 65 patients with biopsy confirmed transitional cell carcinoma of the bladder enrolled in a randomized comparison of intravesical bacillus Calmette-Guerin (BCG) with or without percutaneous administration was also randomized by closed envelope to therapy with multiple vitamins in the recommended daily allowance (RDA) versus RDA multivitamins plus 40,000 units vitamin A, 100 mg. vitamin B6, 2,000 mg. vitamin C, 400 units vitamin E and 90 mg. zinc. The addition of percutaneous BCG did not significantly lessen tumor recurrence but recurrence after 10 months was markedly reduced in patients receiving megadose vitamins. The 5-year estimates of tumor recurrence are 91% in the RDA arm and 41% in the megadose arm (p = 0.0014, Mantel-Cox). Overall recurrence was 24 of 30 patients (80%) in the RDA arm and 14 of 35 (40%) in the high dose arm (p = 0.0011, 2-tailed Fisher's exact test). Megadose vitamins A, B6, C and E plus zinc decrease bladder tumor recurrence in patients receiving BCG immunotherapy. Further research will be required to identify which ingredient(s) provide this protection.

Immunotherapy of murine bladder cancer with keyhole limpet hemocyanin (KLH).

Keyhole limpet hemocyanin (KLH) is a potent immunogen that is being evaluated as an immunotherapeutic alternative to BCG in the treatment of bladder cancer. In the mouse bladder tumor model (MBT2) intralesional KLH significantly reduced tumor incidence, growth rate, and mortality and exhibited antitumor activity similar to that achievable with BCG. Endotoxin contamination of KLH was not responsible for the antitumor activity, although endotoxin alone was shown to have anti-tumor activity in this animal model. Keyhole limpet hemocyanin is both safe and effective in the MBT2 model, and is an immunomodulator to consider for clinical trials.

Keyhole limpet hemocyanin immunotherapy of murine bladder cancer.

The current treatment of choice for superficial bladder cancer, bacillus Calmette-Guérin, has significant adverse side effects. We have compared two alternative immunotherapies--crude keyhole limpet hemocyanin (KLH) and Immucothel, a KLH modified for clinical use (Biosyn)--in an intralesional mouse model of bladder cancer (MBT2). Crude KLH required either immunization before tumor transplant or frequent intralesional therapy after transplantation to be effective. In addition, Immucothel required pre-immunization to be effective, and increasing the frequency and dosage of post-transplant immunization was not effective without pre-immunization. Preliminary investigations into the KLH-induced anti-tumor mechanism(s) suggest that natural killer cell activity may be involved. Both crude KLH and Immucothel appear to be effective immunotherapies of use in the treatment of transitional cell carcinoma.

Sequential immunocytological evaluation of murine transitional cell carcinoma during intralesional bacillus Calmette-Guerin and interleukin-2 immunotherapy.

The antitumor effect of intralesionally administered recombinant interleukin-2 was highly effective (90% complete response) in murine bladder cancer. We postulated that interleukin-2 may be integral to the Bacillus Calmette-Guerin-induced antitumor response in human bladder cancer. Flow cytometric evaluation of the tumor infiltrates was compared before and after intralesional treatment of an established, untreated murine bladder tumor model with recombinant interleukin-2, Bacillus Calmette-Guerin or saline. Large increases in the number of tumor infiltrating immune cells occurred between the day of randomization and the second day (one day after the first treatment) in all three groups. However, since tumor volume was reduced by treatment, the ratios of the immune cells to tumor volume was increased. The ratios of T(helper), T(cytotoxic)/suppressor cells, macrophages, and natural killer cells to tumor volume were 1.5 to 3.4 times higher in the interleukin-2 and Bacillus Calmette-Guerin groups in comparison to the saline group. The ratio of T(helper)/T(cytotoxic)/suppressor cells however, remained approximately the same despite treatment. Over the next 22 days all subpopulations of tumor infiltrating immune cells decreased in number and frequency to less than measurable levels. The similar modulation of infiltrating immune cell subpopulations by Bacillus Calmette-Guerin and interleukin-2 may indicate that the production of interleukin-2 is part of the tumor modulating mechanism of Bacillus Calmette-Guerin.

Antibiotic and steroid therapy of massive systemic bacillus Calmette-Guerin toxicity.

Intravesical bacillus Calmette-Guerin (BCG) is the most effective treatment of carcinoma in situ available today and is superior to chemotherapy in the prevention of bladder tumor recurrence. While therapy is generally well tolerated, serious and even life threatening toxicity can occur. Treatment options for serious infection include isoniazid, rifampin, ethambutol, and cycloserine, but shock may also be secondary to hypersensitivity and require the addition of corticosteroids. The morbidity and mortality of systemically BCG-infected mice treated with single and combined antimicrobial and/or corticosteroid therapies was evaluated. BCG immunized mice were unable to survive doses of BCG which were uniformly tolerated in naive mice. The addition of cycloserine increased survival in mice treated with isoniazid and rifampin, but optimal survival was achieved with isoniazid, rifampin, and prednisolone. These experimental results support the previously reported clinical success of isoniazid, rifampin and prednisolone in patients with septic BCG reactions.

Immunotherapy of murine transitional cell carcinoma of the bladder using alpha and gamma interferon in combination with other forms of immunotherapy.

BCG immunotherapy is very effective in the treatment of superficial transitional cell carcinoma of the bladder, but its significant toxicity may limit its use in some patients. In an effort to find less toxic and potentially more effective treatments we investigated the possible immunotherapeutic potential of combinations of Alpha Interferon (1000 IU) and Gamma Interferon (500 IU) with bacillus Calmette Guerin (BCG) (10(7) cfu), Interleukin-2 (4000 IU), and Keyhole Limpet Hemocyanin (50 micrograms.) in the MBT2 murine bladder cancer model. Significant reductions (p less than 0.05) in tumor incidence relative to the saline control, 83%, Day 35) was observed in groups receiving alpha interferon (42%), Keyhole limpet hemocyanin (42%), interleukin-2 (25%), alpha interferon + Keyhole limpet hemocyanin (17%), alpha interferon + interleukin-2 (33%), gamma interferon + BCG (42%), and gamma interferon + interleukin-2 (17%). All treatment groups with the exception of the group receiving gamma interferon had significantly reduced tumor volume (p less than 0.05) relative to the saline control. Combination treatment groups were significantly more effective than single agent treatments (p = 0.0057). The exhibited anti-tumor effect of these immunotherapeutic agents alone and in combination suggest that they may prove to be effective forms of immunotherapy for transitional cell carcinoma of the bladder.

Treatment of murine transitional cell carcinoma with intralesional interleukin 2 and murine interferon gamma.

The antitumor effect of intralesionally administered recombinant interleukin-2, alone or in combination with recombinant interferon gamma was studied in murine transitional cell carcinoma, MBT2. In the initial prophylactic model treatment was started at day one at the site of tumor inoculation. Maximal and significant reduction in tumor volume occurred in groups receiving 4,000 units of recombinant interleukin 2 and 10(7) colony forming units Bacillus Calmette Guerin (p less than 0.00001 vs saline control). In the same experiment, a reduction in tumor incidence and increase in survival occurred in groups receiving 4,000 units of recombinant interleukin 2, 1,000 units of recombinant interleukin 2 plus 2,000 units of recombinant interferon gamma, as well as 10(7) colony forming units Bacillus Calmette Guerin relative to saline control (p less than 0.005). The dose-response effect of recombinant interleukin 2 alone was also tested in a model of an established transitional cell carcinoma. Intralesional injection treatments were initiated after tumors were palpable. Reduction in tumor volume was observed in the group receiving 8,000 units of recombinant interleukin 2 (p = 0.01 vs saline control), but no significant advantage in survival was noted.

Immunotherapy of murine bladder cancer by irradiated tumor vaccine.

This investigation explored the efficacy of irradiated autologous mouse bladder tumor (Ir-MBT2) as an active specific immunotherapeutic agent and as adjuvant therapy with Bacillus Calmette-Guerin (BCG) against a subcutaneously transplanted murine bladder tumor. Tumor incidence was significantly reduced in groups receiving BCG (27%, p less than 0.005) or Ir-MBT2 with BCG (53%, p less than 0.025), compared to control (93%). Survival was significantly improved in groups treated with BCG (100%, p less than 0.005), 10(5) Ir-MBT2 with BCG (53%, p less than 0.01), or 10(7) Ir-MBT2 with BCG (47%, p less than 0.025) compared with control (13%). Surprisingly, Ir-MBT2 consistently reduced the efficacy of BCG alone. Ir-MBT2 alone (10(7)) appeared to enhance tumor growth. Autologous irradiated bladder tumor vaccine, alone or in combination with BCG, displayed no immunotherapeutic advantage. The use of irradiated tumor cell vaccine for bladder cancer therapy may reduce the results achievable with BCG alone.