Anticancer Properties and Mechanisms of Singly-Protonated Dehydronorcantharidin Silver Coordination Polymer in a Bladder Cancer Model

Abstract

Bladder cancer is the most common malignant urinary system tumor. Chemotherapy is frequently used as a treatment regimen for patients with bladder cancer, however, new and effective drugs for bladder cancer need to be developed. The present study examined the effects and mechanisms of Ag-SP-DNC, a silver and singly-protonated dehydronorcantharidin complex, on bladder cancer in vitro and in vivo. It was identified that Ag-SP-DNC suppressed cell proliferation and induced apoptosis in bladder cancer cells in vitro, a suppression associated with G0/G1 phase arrest and elevated intracellular reactive oxygen species (ROS) levels. Furthermore, Ag-SP-DNC enhanced the cleaved caspase-3 levels, disrupted the mitochondrial transmembrane potential balance, and induced intracellular calcium overload. The Ag-SP-DNC-induced bladder cancer cell apoptosis was significantly decreased following treatment with a broad caspase inhibitor, zVAD-fmk. In addition, treatment of MB49 tumor-bearing mice with Ag-SP-DNC significantly inhibited tumor growth and decreased the anti-apoptosis and cell cycle promotion protein levels in the tumor. The results of the present study suggested that Ag-SP-DNC elicits a strong anticancer effect against bladder cancer, and can therefore be used as a promising treatment for bladder cancer.

Keywords: Ag-SP-DNC; apoptosis; bladder cancer; caspase-3; cell cycle.

FIGURE 1

Ag-SP-DNC shows potent growth inhibition in bladder cancer cells in vitro. (A) Chemical structure of Ag-SP-DNC. (B) Inhibitory rate histogram of MB49 treated with Ag-SP-DNC for 48 h. Data are mean ± SEM; n = 3. ***p < 0.001. (C) Inhibitory rate histogram of T24 treated with Ag-SP-DNC for 48 h. Data are mean ± SEM; n = 3. ***p < 0.001.

FIGURE 2

Ag-SP-DNC shows potent growth inhibition in bladder cancer cells in vitro. (A) Representative images of MB49 treated with Ag-SP-DNC or carboplatin for 48 h. The representative fields were photographed at 100x magnification.(B) Representative images of T24 treated with Ag-SP-DNC or carboplatin for 48 h. The fields were photographed at 100x magnification. (C,D) Ki67 expression of MB49 cells was measured by flow cytometry. (C) Representative histograms were shown. (D) Cell associated mean relative fluorescence intensities. Data are mean ± SEM; n = 3. *p < 0.05, **p < 0.01. (E,F) Ki67 expression of T24 cells was measured by flow cytometry. (E) Representative histograms were shown. (F) Cell associated mean relative fluorescence intensities. Data are mean ± SEM; n = 3. **p < 0.01, ***p < 0.001.

FIGURE 3

Ag-SP-DNC induces cell cycle arrest at G0/G1 stage in bladder cancer cells. Bladder cancer cells were treated with Ag-SP-DNC for 24 h, then stained with PI and analyzed using flow cytometry. (A) A fluorescence pattern of PI stained MB49 cells with or without Ag-SP-DNC treatment. (B) The statistical data of cell cycle distribution. Data are mean ± SEM; n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. (C) A fluorescence pattern of PI stained T24 cells with or without Ag-SP-DNC treatment. (D) The statistical data of cell cycle distribution. Data are mean ± SEM; n = 3. *p < 0.05, **p < 0.01.

FIGURE 4

Ag-SP-DNC induces bladder cancer cells apoptosis. The bladder cancer cells were treated with different dose of Ag-SP-DNC for 48 h and then stained with annexin V-FITC/PI followed by flow cytometry analysis. (A) The fluorescence pattern of annexin V-FITC and PI stained MB49 cells. (B) Percentages of annexin V positive cells for different treatments. Data are mean ± SEM; n = 3. ***p < 0.001. (C) The fluorescence pattern of annexin V-FITC and PI stained T24 cells. (D) Percentages of annexin V positive cells for different treatments. Data are mean ± SEM; n = 3. **p < 0.01, ***p < 0.001.

FIGURE 5

In vivo therapy to orthotopic bladder tumors. (A) In vivo bioluminescence images of MB49 tumor-bearing mice with different treatments. (B) Photograph of all collected bladders collected on day 14 post implantation. (C) Weight of all collected bladders collected on day 14 post implantation. Data are expressed as the mean ± SEM; n = 5. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Western blot of MCL-1, BCL-XL, cleaved caspase-3, Cyclin D1, ERK1/2 phosphorylation and total ERK1/2 levels of MB49 tumors. Tumors were excised and homogenized in lysis buffer on day 14 postimplantation.

FIGURE 6

Ag-SP-DNC induces the mitochondrial disruption, ROS production and DNA fragmentation of bladder cancer cells. Bladder cancer cells were treated with Ag-SP-DNC for 24 h. The levels of ROS were measured by DCFH-DA staining and flow cytometric analyses. (A,B) ROS levels of MB49 cells were measured by flow cytometry. (A) Representative histograms were shown. (B) Cell associated mean relative fluorescence intensities. Data are expressed as the mean ± SEM; n = 3. **p < 0.01, ***p < 0.001. (C,D) ROS levels of T24 cells were measured by flow cytometry. (C) Representative histograms were shown. (D) Cell associated mean relative fluorescence intensities. Data are expressed as the mean ± SEM; n = 3. *p < 0.05, ***p < 0.001. Bladder cancer cells were treated with Ag-SP-DNC for 24 h, then harvested and stained with JC-1 followed by flow cytometry analysis. (E,F) JC-1 staining of MB49 cells were measured by flow cytometry. (E) The fluorescence pattern of JC-1 stained MB49 cells. (F) Green/Red fluorescence intensities. Data are expressed as the mean ± SEM; n = 3. *p < 0.05, ***p < 0.001. (G,H) JC-1 staining of T24 cells were measured by flow cytometry. (G) The fluorescence pattern of JC-1 stained T24 cells. (H) Green/Red fluorescence intensities. Data are mean ± SEM; n = 3. ***p < 0.001. Bladder cancer cells were treated with Ag-SP-DNC for 24 h, and then harvested and processed by TUNEL staining using flow cytometry analysis. (I,J) TUNEL levels of MB49 cells were measured by flow cytometry. (I) Representative histograms were shown. (J) Cell associated mean relative fluorescence intensities. Data are expressed as the mean ± SEM; n = 3. ***p < 0.001. (K,L) TUNEL levels of T24 cells were measured by flow cytometry. (K) Representative histograms were shown. (L) Cell associated mean relative fluorescence intensities. Data are mean ± SEM; n = 3. *p < 0.05, ***p < 0.001.

FIGURE 7

Ag-SP-DNC induces bladder cancer cells apoptosis through cleaved caspase-3. Bladder cancer cells were treated with Ag-SP-DNC for 24 h. The levels of cleaved caspase-3 were measured by flow cytometric analyses. (A,B) Cleaved caspase-3 levels of MB49 cells were measured by flow cytometry. (A) Representative histograms were shown. (B) Cell associated mean relative fluorescence intensities. Data are expressed as the mean ± SEM; n = 3. ***p < 0.001. (C,D) Cleaved Caspase-3 levels of T24 cells were measured by flow cytometry. (C) Representative histograms were shown. (D) Cell associated mean relative fluorescence intensities. Data are expressed as the mean ± SEM; n = 3. ***p < 0.001. (E–H) The bladder cancer cells were treated with Ag-SP-DNC and caspases inhibitor zVAD-fmk for 48 h, then harvested and stained with Annexin V-FITC and PI followed by flow cytometry analysis. (E) The fluorescence pattern of Annexin V-FITC and PI stained MB49 cells. (F) Percentages of Annexin V positive cells for different treatments. Data are mean ± SEM; n = 3. ***p < 0.001. (G) The fluorescence pattern of Annexin V-FITC and PI stained T24 cells. (H) Percentages of Annexin V positive cells for different treatments. Data are mean ± SEM; n = 3. ***p < 0.001.

FIGURE 8

Ag-SP-DNC induces bladder cancer cells intracellular free Ca²⁺ release. Bladder cancer cells were treated with Ag-SP-DNC for 24 h. The levels of intracellular free Ca²⁺ were measured by Fluo-4/AM staining and flow cytometric analyses. (A,B) Intracellular free Ca²⁺ of MB49 cells were measured by flow cytometry. (A) Representative histograms were shown. (B) Cell associated mean relative fluorescence intensities. Data are mean ± SEM; n = 3. ***p < 0.001. (C,D) Intracellular free Ca²⁺ of T24 cells were measured by flow cytometry. (C) Representative histograms were shown. (D) Cell associated mean relative fluorescence intensities. Data are mean ± SEM; n = 3. **p < 0.01, ***p < 0.001.