A novel prophylactic regimen is demanded for preventing bladder cancer recurrence, because of the high side-effect tolls of conventional adjuvant Bacillus Calmette-Guérin (BCG) immunotherapy, in addition to its only moderate efficacy. In vitro and animal studies have demonstrated the anti-cancer properties of a medicinal mushroom called Ganoderma lucidum (GL). In this study, a pre-malignant human uroepithelial cells (HUC-PC) model was utilized to compare the effectiveness between ethanol extract of GL (GLe) and BCG on interleukin-6 (IL-6) secretion and lactate dehydrogenase (LDH) cytotoxicity. Additionally, parameters relevant to the BCG efficacy and safety, including free soluble fibronectin (FN) and cell-surface glycosaminoglycans (GAGs) levels were tested, following the exposure of GLe to the cells. GLe at 100 μg/ml and BCG at 4.8 × 107 CFU were shown to induce equivalent levels of IL-6, suggesting the potential synergism, while the tested concentrations of GLe were non-cytotoxic. During the initial four hours of GLe exposure, the free FN concentrations in harvested media were significantly reduced that might facilitate the binding of BCG for uroepithelial internalization to enhance BCG efficacy. Furthermore, the cell membrane-bound GAGs levels of HUC-PC cells were significant increased in response to GLe to suggest cellular protection from BCG infection. In summary, current findings suggest the potential additive synergism of GLe with the BCG efficacy, as well as its protective effects, and thus reducing the BCG toxicity.
Ganoderma lucidum (GL), a popular ancient medicinal mushroom ranked as a superior tonic in traditional Chinese medicine, is commonly used for health promotion and longevity. Nowadays, the mushroom is being used by many cancer patients because of its perceived health benefits including immunomodulating properties and antitumorigenicity. In the recent years, our research team and collaborators have focused on bladder cancer, and reported a range of in vitro chemopreventive activities for GL. It was demonstrated that remarkable growth inhibitory effects via G2/M phase cell cycle arrest [
The recurrence rate of superficial transitional cell carcinoma (TCC) of bladder remains exceptionally high even with the effective transurethral resection (TUR) technique [
In the present study, the HUC-PC cell model is continuously being utilized, firstly to compare between the effectiveness of GLe and BCG on the cytotoxicity and IL-6 secretion. IL-6 has been suggested to be responsible for the cytotoxicity of BCG on several TCC cell lines [26,27]. Whether BCG and GLe would both be cytotoxic to HUC-PC was determined using the LDH cytotoxicity assay, if they are capable of inducing IL-6 secretion. Secondly, the effects of GLe on extracellular FN and cell surface GAGs are explored. These findings will aid in elucidating whether GLe is a possible candidate to supplement or even replace the BCG immunotherapy.
The active ingredients of RishiMax GLPTM G. lucidum (Pharmanex, Hong Kong) were commercially standardized to 13.5% polysaccharides and 6% triterpenes. Powdered G. lucidum from capsules was re-extracted as previously described [3,4]. GLe was dissolved freshly in absolute ethanol (0.1% v/v) and diluted with culture medium to make a 1000 μg/ml stock solution. The whole vial (dry weight 81 mg) of live attenuated BCG (IMMUCYST®, Aventi, Toronto, Ontario, Canada) was reconstituted with 3 ml of the accompanying diluents to make a suspension containing a minimal dosage of 6.6 × 108 colony forming units (CFU). A 4.8 × 107 CFU BCG stock solution was prepared with culture medium. For assays, working assay media were prepared by further diluting the stock solutions of GLe and BCG into the concentrations of test ranges. Solvent media containing the maximal amount of corresponding solvent, i.e. 0.1% v/v ethanol for GLe and 33% v/v diluents for BCG, were used as controls. Furthermore, GLe and BCG were checked using Limulus Amebocyte Lysate (LAL) endpoint chromogenic kit assay (CAPE CO, E. Falmouth, MA, USA) for lipopolysaccharides (LPS) contamination. GlucashieldTM buffer (CAPE COD) was used to reconstitute pyrochrome to inhibit possible (1,3)-β-D-glucan presented in samples, and thus avoiding potential interference in the assay. Aseptic techniques were strictly applied throughout the procedures.
The HUC-PC cell line was derived in the Department of Human Oncology, University of Wisconsin Medical School, and gifted by Dr. Rao from the University of California, Los Angeles. The cell line was cultured in F12 Ham enriched Dulbecco’s Modified Eagle’s Meium (F12/DMEM purchased from Sigma, St. Louis, MO) with 1% penicillin (10,000 μg/ml) and streptomycin (10,000 mg/ml) and 10% Fetal Bovine Serum (GIBCO BRL Isaland, New York, USA). Logarithmically growing HUC-PC cells were harvested and seeded in 96-well flat-bottle tissue culture plate (Greiner bio-one, Germany) at a concentration of 5 × 104 cells per microtitre well for cytotoxicity, FN and GAGs measurement. In parallel experiments, 1 × 106 cells were also seeded in 100-mm tissue culture dishes (Greiner bio-one, Germany) for IL-6 assay.
Cytotoxicity of GLe and BCG was assayed by measuring LDH released from cells with LDH Cytotoxicity Detection kit (TaKaRa Bio Inc., Shiga, Japan). Following the manufacturer’s instructions, the cells were incubated with assay media containing GLe or BCG for 24 hours in microtitre plate wells (Thermo Labsystems, Franklin, MA). No significant cytotoxicity was observed with the solvent controls. The release of LDH from cells was measured at 490 nm with reference wavelength at 690 nm, using TECAN SPECTRA Fluor Plus microplate reader (TECAN Austria GmbH, Grodig, Austria). Untreated cells were used as low controls to measure the spontaneous LDH release, and Triton X-100 treated cells were used as high controls to measure the maximum releasable LDH activity. No interference was observed from any test substances used in the assay. Cytotoxicity was calculated as a percentage of LDH release with the following formula:
Cultured supernatants were collected to measure the IL-6 secretion with the Endogen® Human IL-6 ELISA kit (Pierce Biotechnology Inc, Rockford, USA). The manufacturer’s instructions were followed. Culture medium was used to prepare the standard curve by serial dilutions (ranging from 0 pg/ml to 400 pg/ml). Absorbance of the reaction microplate wells was measured at 450 nm on microplate reader (TECAN, Austria).
Conditioned media were harvested from cell-seeded microtitre plate after four hours (for avoiding cytotoxic effects based on previous findings of apoptosis) of treatment with GLe. The TaKaRa Fibronectin EIA kit (TAKARA Bio Inc., Japan) was used for assay. Following the kit instructions, a 100 μl of sample/standard was added into an ELISA well coated with human anti-fibronectin and incubated for one hour at 37˚C. The microtitre wells were washed four times and then 100 μl of substrate solution was added and incubated for 15 minutes at room temperature. 1 N Sulphuric acid (H2SO4) was added to stop the reaction. Finally, absorbance was read against diluent blank at 450 nm, using TECAN SPECTRA Fluor Plus microplate reader (TECAN, Austria).
After four hours of incubation, same as for FN assay, membrane-bound GAGs from HUC-PC cells were extracted by a 0.1 M sodium acetate buffer at pH 5.8 in a microtitre plate overnight, in accordance with previous publication [
Each study group was run in triplicate and duplicated samples from each group were measured for each variable. Differences between means were determined using Student’s t-test (GraphPad Prism version 3.0 for Windows, San Diego California, USA). Statistical significance was sought at two tailed P-value of 0.05.
Results indicated that both BCG and GLe were clearly capable of inducing dose-dependent IL-6 secretion in the HUC-PC culture (
μg/ml or lower GLe concentration (
About 15% of the free FN in the cultured media was significantly (P < 0.01) reduced by GLe at concentrations of 40 - 100 μg/ml (
the other hand, the cell-membrane bound GAGs levels on HUC-PC cells were significantly (P < 0.01) increased (
In accordance with one of our recent publications [
The α5β1 integrin is a classic cellular receptor presented on the malignant urothelium for fibronectin (FN) [26,33]. Expression of α5 and β1 mRNA could be promoted by exogenous and autocrine IL-6, while competive inhibitors of FN inhibit BCG-induced NF-κB signaling pathways [33,40]. Furthermore, autocrine IL-6 enhanced BCG adherence to the 253J TCC cell line through the up-regulation of α5β1 integrin receptor for FN [
Furthermore, the expression of GAGs on the HUC-PC cell surface was also increased by GLe. Formerly known as mucopolysaccharides, GAGs are long unbranched polysaccharides, are highly anionic and are often bound to core proteins to become proteoglycans with varying properties of extracellular matrices of tissues [52,53]. GAGs are extremely hydrophilic and trap water at the outer layer of the umbrella urothelium, and this trapped water forms a gel as part of the mucosal barrier that interfaces urine and the bladder wall [54,55]. This provides a protective barrier that becomes highly impermable to any solutes, crystals and even bacteria in urine [54,56]. The disruption of this mucosal permeability is pathologically significant such that interstitial cystitis (IC) occurs [54, 57]. GAGs are also able to repair damaged bladder mucosa [
In summary, ethanol extract of GL exerted as a similar activator for IL-6 production as BCG in the HUC-PC cells. IL-6 was the only cytokine selected for measurement because it is the earliest cytokine that can be detected after BCG exposure to urothelial cells and it is also an indicator for successful BCG internalization by these cells. Current results suggested that combinational use of GLe and BCG may exert synergistic effects in several ways (
urothelial surface for subsequent internalization and IL-6 secretion, particularly for cells at normal or low grades. Nonetheless, the reduction of free FN, by itself may also suggest being tumor suppressive to inhibit the growth and progression by reducing unnecessary cell-substrate interactions; and thirdly, the increased cell surface GAGs expression provide additional protection from chemical and bacterial attacks, and thus is potential in reducing side-effects caused by BCG. Further experiments are underway to define the synergism of GLe and BCG as well as to investigate the underlying mechanisms. No doubt, the anticancer activities of GLe were demonstrated in the HUC-PC cell model to suggest the associations between IL-6 induction, FN reduction, and BCGGLe synergism were suggested. However, the cause-andeffect mechanism needs to be confirmed by further careful scientific investigation.
This project was supported by the Research Committee of the Hong Kong Polytechnic University for the postgraduate scholarship (RGH8) and Sir Edward Youde Memorial Fellowship awarded to Dr. John Yuen. The authors are grateful to Dr. J. Y. Rao (UCLA Medical Center, USA) for providing the HUC-PC cell line and professional advice.