Synergistic Cytotoxic Effects of Ganoderma lucidum and Bacillus Calmette Guérin on Premalignant Urothelial HUC-PC Cells and Its Regulation on Proinflammatory Cytokine Secretion

Abstract

Bacillus Calmette-Guérin (BCG) is conventionally used as an adjuvant immunotherapy to reduce the recurrence of bladder cancer. To address the issues of efficacy and safety, an ethanol extract of Ganoderma lucidum (GLe) was evaluated for its interaction with BCG. In a model of premalignant human uroepithelial cells (HUC-PC), GLe exerted immediate cytotoxic effects while BCG showed a delayed response, given that both were immunological active in inducing the secretion of interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1 (MCP-1). Synergistic cytotoxic effects were observed when cells were either coincubated with both drugs or firstly preincubated with GLe. Synergism between GLe and BCG was demonstrated to achieve a complete cytostasis in 24 hours, and such effects were progressed in the subsequent 5 days. However, the pretreatment of GLe resulted in suppression of IL-6, IL-8, and MCP-1 secretions without affecting the cytotoxicity. Given that numerous proinflammatory cytokines are associated with the high side effects toll of BCG, results herein suggested the potential implications of GL to supplement the BCG immunotherapy in bladder cancer, for better efficacy and reducing side effects.

Figure 1

The treatment schedules for experiments, illustrating 4 treatments (T1–T4) were tested. A 24-hour incubation was allowed for stabilizing the culture environment after seeding the cells. The initial cell seeding numbers of T1–T3 were halves of T4, because cell cultures for T1–T3 were maintained for an additional 24 hours in complete media, in order to align initial cell numbers for all treatment groups before the first day sampling (Day 0 as baseline), that is, approximately 2.5 × 10⁵ cells since doubling time for the HUC-PC cells was 24 hours. For T4, a range of GLe concentrations was administrated before the baseline, and this complete medium in the absence of GLe (0 μg/mL) was used as the baseline for this treatment group. Subsequent samples were collected on Day 1 (Day 3 after initial cell seeding) and Day 6 (Day 8 after initial cell seeding) following different treatments.

Figure 2

Showing the immediate and progressive cytotoxic effects exhibited by (a) BCG on day 1 (F = 4.908; statistically nonsignificant) and day 6 (F = 51.30; ***P < 0.001), and (b) GLe on day 1 (F = 18.69; ***P < 0.001) and day 6 (F = 118.5; ***P < 0.001). Results of BCG and GLe were statistically compared with the solvent media control according to the corresponding schedule (i.e., day 1 versus Day 1 and Day 6 versus Day 6), at 0 CFU containing BCG diluents (33% v/v) and at 0 μg/mL containing ethanol (0.1%; v/v), respectively. All experiments were performed in triplicate for reproducibility. F value of each treatment group was determined by one-way ANOVA, and Dunnett's posttest was followed to determine P values.

Figure 3

Showing the immediate and progressive cytotoxic effects exhibited by (a) coincubation of GLe with BCG on day 1 (F = 14.14; ***P < 0.01) and day 6 (F = 217.6; ***P < 0.001), and (b) pretreatment of GLe followed by BCG on day 1 (F = 169.8; ***P < 0.001) and day 6 (F = 30.52; ***P < 0.01). For all test conditions, BCG concentration was fixed at 1.2 × 10⁷ CFU for testing different concentrations of GLe. Results were statistically compared with control at 0 GLe concentration but treated with BCG at 1.2 × 10⁷ CFU, according to the corresponding schedule (i.e., day 1 versus day 1 and day 6 versus day 6). All experiments were performed in triplicate for reproducibility. F value of each treatment group was determined by one-way ANOVA, and Dunnett's posttest was followed to determine P values.

Figure 4

Dose-response curves of (a) BCG and (b) GLe generated by nonlinear regression, in order to define the sinGLe-agent effects as IC₇₀. On day 6 following drug cessation, and since only selected BCG concentrations were tested, the BCG cytotoxicity showed ranged from 60–73% when compared with the solvent control (i.e., BCG 0 CFU on day 6). In order to minimize the error, IC₇₀ was used for isobologram analysis. Isobolograms (c) were plotted for treatments 3 and 4 with the IC₇₀ determined for BCG and GLe. Concentrations of GLe and BCG were reflected on x- and y-axes, respectively. Area below the additive isobole (the line joining IC_70,BCG and IC_70,GLe) indicates synergistic interaction. Combination indices (CI) were calculated as CI = C_BCG,70/IC_70,BCG + C_GLe,70/IC_70,GLe according to Zhao et al. [14]. Specifically, C_BCG,70 and C_GLe,70 are the concentrations of BCG and GLe used in treatments 3 and 4 to achieve 70% drug effect. IC_70,BCG (fixed as 1.2 × 10⁷ CFU for all experiments) and IC_70,GLe (19.12 μg/mL in treatment 3; 12.37 μg/mL in treatment 4) are the concentrations for sinGLe agents to achieve the same effect.