Sea Cucumber Derived Triterpenoid Glycoside Frondoside A: A Potential Anti-Bladder Cancer Drug

Abstract

Bladder cancer is a highly recurrent disease and a common cause of cancer-related deaths worldwide. Despite recent developments in diagnosis and therapy, the clinical outcome of bladder cancer remains poor; therefore, novel anti-bladder cancer drugs are urgently needed. Natural bioactive substances extracted from marine organisms such as sea cucumbers, scallops, and sea urchins are believed to have anti-cancer activity with high effectiveness and less toxicity. Frondoside A is a triterpenoid glycoside isolated from sea cucumber, Cucumaria frondosa. It has been demonstrated that Frondoside A exhibits anti-proliferative, anti-invasive, anti-angiogenic, anti-cancer, and potent immunomodulatory effects. In addition, CpG oligodeoxynucleotide (CpG-ODN) has also been shown to have potent anti-cancer effects in various tumors models, such as liver cancer, breast cancer, and bladder cancer. However, very few studies have investigated the effectiveness of Frondoside A against bladder cancer alone or in combination with CpG-ODN. In this study, we first investigated the individual effects of both Frondoside A and CpG-ODN and subsequently studied their combined effects on human bladder cancer cell viability, migration, apoptosis, and cell cycle in vitro, and on tumor growth in nude mice using human bladder cancer cell line UM-UC-3. To interrogate possible synergistic effects, combinations of different concentrations of the two drugs were used. Our data showed that Frondoside A decreased the viability of bladder cancer cells UM-UC-3 in a concentration-dependent manner, and its inhibitory effect on cell viability (2.5 μM) was superior to EPI (10 μM). We also showed that Frondoside A inhibited UM-UC-3 cell migration, affected the distribution of cell cycle and induced cell apoptosis in concentration-dependent manners, which effectively increased the sub-G1 (apoptotic) cell fraction. In addition, we also demonstrated that immunomodulator CpG-ODN could synergistically potentiate the inhibitory effects of Frondoside A on the proliferation and migration of human bladder cancer cell line UM-UC-3. In in vivo experiments, Frondoside A (800 μg/kg/day i.p. for 14 days) alone and in combination with CpG-ODN (1 mg/kg/dose i.p.) significantly decreased the growth of UM-UC-3 tumor xenografts, without any significant toxic side-effects; however, the chemotherapeutic agent EPI caused weight loss in nude mice. Taken together, these findings indicated that Frondoside A in combination with CpG-ODN is a promising therapeutic strategy for bladder cancer.

Keywords: CpG oligodexynucleotides; Frondoside A; apoptosis; bladder cancer; cell migration; cell viability.

Figure 1

Inhibitions of cell viability and migration by Frondoside A (Fr A). Exponentially growing UM-UC-3 cells were treated with the indicated concentrations of Frondoside A for 24, 48, and 72 h (A). Cell viability was assayed as described in Materials and Methods. Frondoside A significantly suppressed the migration of UM-UC-3 cells, and cell mobility was measured at 0, 12, and 24 h (objective × 4) (B). Data are expressed as means ± S.E.M. * Significantly different at p < 0.05. ** Significantly different at p < 0.01. *** Significantly different at p < 0.001. n.s (not significant). “a” stands for versus blank control; “b” stands for versus EPI (10 μM).

Figure 2

Effects of Frondoside A (Fr A) on cell cycle in bladder cancer cells. Cells were treated with increasing concentrations of Frondoside A for 48 h, with EPI (10 μM) as positive control. The distribution of cell cycle (A–C). Data are expressed as mean ± S.E.M. * Significantly different at p < 0.05. ** Significantly different at p < 0.01. *** Significantly different at p < 0.001. n.s (not significant). “a” stands for versus blank control; “b” stands for versus EPI (10 μM).

Figure 3

Effects of Frondoside A (Fr A) treatment on apoptosis and cell morphology in UM-UC-3 cells. Cells were treated with increasing concentrations of Frondoside A for 48 h, and EPI (10 μM) as positive control. The induction of cell apoptosis (A) and the morphological changes (the pictures were taken at ×200 magnification) (B). Data are expressed as mean ± S.E.M. ** Significantly different at p < 0.01. *** Significantly different at p < 0.001.

Figure 4

CpG-ODN enhances the inhibition of Frondoside A (Fr A) on UM-UC-3 cell viability and migration. Cells were treated with Frondoside A, CpG-ODN, and Frondoside A combined with CpG-ODN for 24, 48, 72 h. The effects of combinations of Frondoside A and CpG-ODN were tested in parallel with the drugs used alone (A). Effects of drugs combination with Frondoside A, and the combinational index (CI) was calculated with the CompuSyn v.1.0. software (B). Frondoside A significantly suppressed the migration of UM-UC-3 cells, cell mobility was measured at 0, 12 and 24 h (objective × 4) (C), and columns are means; bars are S.E.M. * Significantly different at p < 0.05. *** Significantly different at p < 0.001. n.s (not significant). “a” stands for versus blank control; “b” stands for versus EPI (10 μM); “c” stands for versus Frondoside A (0.5 μM) alone.

Figure 4

CpG-ODN enhances the inhibition of Frondoside A (Fr A) on UM-UC-3 cell viability and migration. Cells were treated with Frondoside A, CpG-ODN, and Frondoside A combined with CpG-ODN for 24, 48, 72 h. The effects of combinations of Frondoside A and CpG-ODN were tested in parallel with the drugs used alone (A). Effects of drugs combination with Frondoside A, and the combinational index (CI) was calculated with the CompuSyn v.1.0. software (B). Frondoside A significantly suppressed the migration of UM-UC-3 cells, cell mobility was measured at 0, 12 and 24 h (objective × 4) (C), and columns are means; bars are S.E.M. * Significantly different at p < 0.05. *** Significantly different at p < 0.001. n.s (not significant). “a” stands for versus blank control; “b” stands for versus EPI (10 μM); “c” stands for versus Frondoside A (0.5 μM) alone.

Figure 5

Effects of Frondoside A (Fr A) and CpG-ODN on cell cycle, apoptosis and nuclei morphology in bladder cancer cells. Cells were treated with Frondoside A (0.5 μM) and different concentrations of CpG-ODN (0.001, 0.1, and 1 μM) individually and in combination for 48 h. The distribution of cell cycle (A). The induction of cell apoptosis (B) and the morphological changes (The pictures were made at ×200) (C). Data represent the mean ± S.E.M. * Significantly different at p < 0.05. ** Significantly different at p < 0.01. *** Significantly different at p < 0.001.

Figure 5

Effects of Frondoside A (Fr A) and CpG-ODN on cell cycle, apoptosis and nuclei morphology in bladder cancer cells. Cells were treated with Frondoside A (0.5 μM) and different concentrations of CpG-ODN (0.001, 0.1, and 1 μM) individually and in combination for 48 h. The distribution of cell cycle (A). The induction of cell apoptosis (B) and the morphological changes (The pictures were made at ×200) (C). Data represent the mean ± S.E.M. * Significantly different at p < 0.05. ** Significantly different at p < 0.01. *** Significantly different at p < 0.001.

Figure 5

Effects of Frondoside A (Fr A) and CpG-ODN on cell cycle, apoptosis and nuclei morphology in bladder cancer cells. Cells were treated with Frondoside A (0.5 μM) and different concentrations of CpG-ODN (0.001, 0.1, and 1 μM) individually and in combination for 48 h. The distribution of cell cycle (A). The induction of cell apoptosis (B) and the morphological changes (The pictures were made at ×200) (C). Data represent the mean ± S.E.M. * Significantly different at p < 0.05. ** Significantly different at p < 0.01. *** Significantly different at p < 0.001.

Figure 6

Effects of Frondoside A (Fr A), CpG-ODN individually and in combination in bladder cancer cells of TP53 signaling and Intrinsic pathways. Cells were treated with the indicated concentrations of Frondoside A and CpG-ODN for 48 h, EPI (10 μM) as positive control. Equal amounts of cell extracts were analyzed by Quantitative Real-Time Polymerase Chain Reaction assay with TP53, Bax, Bcl-2, Caspase 3, and CDK1A. Data represent the mean ± S.E.M. * Significantly different at p < 0.05. ** Significantly different at p < 0.01. *** Significantly different at p < 0.001.

Figure 7

The suppression on the tumor growth of human bladder cancer xenografts. Bladder cancer cells UM-UC-3 (3 million per mouse) were subcutaneously transplanted into immune-deficient nude mice to establish a xenograft tumor model. Mice were treated with different drugs in 14 days. Tumor volumes were measured every 2 days to assess the effect of treatments on tumor growth (A). Xenografts were resected, and the weights of the tumors were measured at the end of the observation period (B). The body weight of the mice was monitored every 2 days to observe the drugs toxicity, and the body weight was weighed prior to the death (C,D). Data represent the mean ± S.E.M. * Significantly different at p < 0.05.

Figure 8

Histopathologic sections of tumor tissues (H&E, ×200). Nude mice were treated with different drugs in 14 days. All of the animals were sacrificed after the treatments and the tumors were collected for histological analysis.