Effect of various natural products on growth of bladder cancer cells: two promising mushroom extracts

Alternative Medicine Review, March, 2007 by Sensuke Konno

Abstract

Despite the availability of several therapeutic options, a safer and more effective modality is urgently needed for treatment of bladder cancer. Specific immunotherapy is effective, but severe side effects limit its clinical use and underscore the need for unconventional therapies using less toxic substances. Many natural substances are touted for their medicinal aspects and side effect profiles, and some of these have been well characterized for their biological and medicinal properties. Accordingly, the effects on bladder cancer cells in vitro were investigated. Eight commercially available natural products were tested for possible effects on the growth of human bladder cancer T24 cells. This study demonstrated that two mushroom extracts, GD- and PL-fractions, induced a significant (>90%) growth reduction in 72 hours, whereas the remaining six products had no effect. Interestingly, non-toxic concentrations of the GD- or PL-fractions, when combined with a non-toxic concentration of vitamin C, became highly cytotoxic, resulting in >90-percent cell death. Thus, vitamin C appears to act synergistically with these fractions to potentiate their bioactivity (cytotoxicity). No other products tested demonstrated such a synergistic potentiation with vitamin C. The present study indicates that GD- and PL-fractions appear to have the most potent cytotoxic effect on human bladder cancer T24 cells. It is thus plausible that these substances could be used, solely or combined with conventional modalities, for the treatment of superficial bladder cancer.

Introduction

Bladder cancer is the second-most common urological malignancy in the United States, next to prostate cancer. (1) Currently, transitional cell carcinoma (TCC) is the most prevalent type (~90%) of bladder cancer. Annually, 50,000 new cases are diagnosed and more than 10,000 people die of this disease. (2) Approximately 80 percent of TCC presents as superficial bladder tumors, while 15 percent are invasive and five percent present as metastatic disease. (1) Although endoscopic transurethral resection (TUR) is often performed as the primary therapy, 50-75 percent of patients have a recurrence within five years and approximately 10 percent progress to invasive disease. (3) TCC is characterized by a multifocal origin and an acquired resistance to drugs, factors that likely contribute to the high incidence of disease relapse. (4,5) Thus, the primary therapeutic objectives are to prevent multiple recurrences, as well as progression to a more advanced, invasive disease.

Several cytotoxic and immune-modifying agents are used intravesically to treat bladder cancer. Among currently available therapeutic options, intravesical administration of Bacillus Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis, is the most effective immunotherapy for superficial bladder cancer and carcinoma in situ. (6,7) BCG therapy has been shown to alter disease progression, reduce recurrence, and increase survival. (8) Adjuvant intravesical BCG therapy following TUR is now the established therapy for superficial bladder cancer, resulting in a 40-percent reduction in cancer recurrence, compared to other viable options (<20-percent reduction). (8,9) However, this benefit should be measured against potentially severe side effects, including cystitis, fever, allergic reactions, sepsis, and even death. (10,11) These drawbacks do limit BCG therapy in clinical practice and demonstrate the need for a non-toxic, safe, effective treatment modality with minimal side effects.

Various medicinal aspects of natural agents have been claimed or passed down through folklore. Such natural substances include herbs, mushrooms, flowers, fruits, plant seeds, seaweeds, algae, teas, bark, shark cartilage, etc. In some cases, sufficient scientific studies have not been performed to demonstrate the biological properties of these natural substances, although several studies have characterized the medicinal properties of some natural agents. For example, the maitake (Grifola frondosa) mushroom has been extensively studied. (12) In particular, its antitumor activity was well-documented in a study using tumor-bearing mice, (13) which demonstrated activation of various immune effectors including macrophages, cytotoxic T-lymphocytes, and natural killer cells. (14) Accordingly; this preliminary study investigates whether commercially available natural products have potential anticancer effects on human bladder cancer T24 cells in vitro.

Materials and Methods

Human bladder cancer T24 cells, derived from a patient with TCC, were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in McCoy's 5a medium containing 10-percent fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 [micro]g/mL). Culture medium was routinely changed every 3-4 days and the passage of cells was performed weekly. For experiments, cells were seeded in six-well culture plates at an initial cell density of 1x[10.sup.5] cells/mL. T24 cells were cultured for 72 hours with varying concentrations (0-1,000 [micro]g/mL) of eight natural, commercially available products (Table 1). Cell morphology/appearance was monitored daily and cell number and viability were assessed at 72 hours using the trypan blue exclusion test and Alamar Blue cell viability test (Biosource International, Camarillo, CA), respectively.

Results

This dose-dependent study reveals a 25-percent reduction in cell growth with 120 [micro]g/mL of GD-fraction and >90-percent growth reduction (due to cell death) with [greater than or equal to] 240 [micro]g/mL of GD (Figure 1). No effects were observed with GD concentrations up to 60 [micro]g/ mL. Similarly, PL-fraction induced 30-, 80-, and >90-percent growth reduction at 50, 100, and [greater than or equal to] 200 [micro]g/mL, respectively (Figure 2). PL at 20 [micro]g/mL had no effect.

[FIGURES 1-2 OMITTED]

In contrast, five other products (see Table 1 for product description)--ARBX, ABE, ASC, MSK, and AHC--were found to have little effect on T24 cell growth, even at the highest concentration of 1,000 lag/ mL (Figure 3). Interestingly, YBG at 20 [micro]g/mL showed a severe cytotoxic effect (>90% cell death). However, since YBG was originally prepared with vitamin C, this result should be interpreted with caution. Our pilot study confirmed that vitamin C by itself had a significant cytotoxic effect on T24 cells, especially when its concentration went beyond 500 [micro]M (Table 2). In fact, calculations revealed the above-lethal dosage of 2,400 [micro]M (2.4 mM) of vitamin C was included in 20 [micro]g/mL of YBG. Thus, a cytotoxic effect of YBG is more likely attributable to an extremely high concentration of vitamin C, not to its bioactive component ([beta]-glucan).