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. 2019 Apr;110(4):1431-1441.
doi: 10.1111/cas.13960. Epub 2019 Mar 1.

Sulfasalazine could modulate the CD44v9-xCT system and enhance cisplatin-induced cytotoxic effects in metastatic bladder cancer

Koichiro Ogihara  1 , Eiji Kikuchi  1 , Shogo Okazaki  2 , Masayuki Hagiwara  1 , Toshikazu Takeda  1 , Kazuhiro Matsumoto  1 , Takeo Kosaka  1 , Shuji Mikami  3 , Hideyuki Saya  2 , Mototsugu Oya  1
Affiliations

Sulfasalazine could modulate the CD44v9-xCT system and enhance cisplatin-induced cytotoxic effects in metastatic bladder cancer

Koichiro Ogihara et al. Cancer Sci. 2019 Apr.

Abstract

The prognostic role of CD44v9, a variant isoform of CD44 and a new cell surface marker of cancer stem cells, remains unclear in bladder cancer (BC) patients. Furthermore, limited information is available on the functional role of sulfasalazine (SSZ), which could modulate the CD44v9-xCT system in order to enhance cisplatin (CDDP)-induced cytotoxicity and inhibit the metastatic potential of BC. CD44v9 protein expression was examined immunohistochemically in 63 muscle invasive BC (MIBC) patients who underwent radical cystectomy. CD44v9 expression was independently associated with disease recurrence and cancer-specific death in MIBC. Cytotoxic effects, glutathione levels, and reactive oxygen species production by SSZ and CD44v9 and phospho-p38MAPK protein expression by SSZ with or without CDDP were assessed in MBT-2V cells with highly metastatic potential. Sulfasalazine exerted cytotoxic effects against MBT-2V cells by inhibiting glutathione levels and inducing the production of reactive oxygen species. Sulfasalazine in combination with CDDP appeared to exert strong cytotoxic effects against MBT-2V cells by inhibiting CD44v9 expression and upregulating phospho-p38MAPK expression. The inhibitory effects of SSZ with or without CDDP were also investigated using an MBT-2V lung metastatic model. In the murine lung metastatic BC model, SSZ significantly prolonged animal survival. Furthermore, the combination of SSZ with CDDP exerted stronger inhibitory effects on the establishment of lung tumor nodules than SSZ or CDDP alone. CD44v9 expression could be a clinical biomarker for predicting poor outcomes in MIBC patients. Sulfasalazine in combination with CDDP has potential as a novel therapy against metastatic BC.

Keywords: CD44v9; bladder cancer; cisplatin; metastasis; sulfasalazine.

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Conflict of interest statement

Eiji Kikuchi received honoraria for lecture fees from MSD, Chugai Pharmaceutical, Ono Pharmaceutical, Taiho Pharmaceutical, Astra Zeneca, Astellas Pharma, Japan BCG Laboratory, and Nippon Kayaku. The other authors have no conflict of interest.

Figures

Figure 1
Figure 1
Representative immunostaining for CD44v9 in radical cystectomy specimens of muscle invasive bladder cancer (MIBC) patients and prognostic significance of CD44v9 expression on disease recurrence and cancer‐specific death. A, Low expression of CD44v9 in radical cystectomy specimens of MIBC. B,C, High expression of CD44v9 in MIBC. Scale bar = 100 μm. D,E, Kaplan‐Meier curves of recurrence‐free survival (D) and cancer‐specific survival (E) according to CD44v9 expression (low expression of CD44v9 vs high expression of CD44v9)
Figure 2
Figure 2
Sulfasalazine selectively inhibits cell proliferation, decreases glutathione (GSH) synthesis, increases reactive oxygen species (ROS) levels, and enhances cisplatin‐induced cytotoxic effects in MBT‐2V cells. A, Cytotoxic effects of sulfasalazine (SSZ) in MBT‐2V cells. Cells were exposed to various concentrations of SSZ for 48 h. B, Cytotoxic effects of SSZ in the presence or absence of N‐acetylcysteine (NAC, an antioxidant). Cells were exposed to various concentrations of SSZ with or without NAC (3 μmol/L) for 48 h. C, Intracellular GSH levels of MBT‐2V cells treated with the vehicle control, 300 or 400 μmol/L SSZ, and 100 μmol/L L‐buthionine‐sulfoximine (BSO) for 24 h. D, Quantitative analysis of ROS production by MBT‐2V cells treated with the vehicle control, 300 or 400 μmol/L SSZ, and 100 μmol/L BSO for 24 h. E, Cytotoxic effects of SSZ (300 μmol/L), cisplatin (CDDP) (10 μmol/L), and their combinations in MBT‐2V cells for 48 h. F, Expression of CD44v9, phospho‐p38MAPK, and total p38MAPK protein in MBT‐2V cells treated with the vehicle control, SSZ alone (300 μmol/L), CDDP alone (10 μmol/L), and their combinations detected by western blotting. G,H, Signal intensities of CD44v9 and phospho‐p38MAPK protein expression in each group was quantified. All data are shown as mean ± SE. *P < .01, **P < .001
Figure 3
Figure 3
Survival analysis of sulfasalazine (SSZ) treatment and antitumor effects for lung tumor nodules of SSZ alone, cisplatin alone, and their combinations in a murine lung metastasis model. A, Lung tumor nodules were generated by injecting 2 × 105 MBT‐2V cells into the tail veins of female C3H/HeN mice on day 0. The i.p. administration (2 days on/1 day off) of SSZ (500 mg/kg) or the vehicle control (PBS) was started on day 3 (n = 18 each group). Survival analysis was evaluated by Kaplan‐Meier curve between the SSZ treatment group and the control group. B, In the lung metastasis model, mice were classified into 4 groups: vehicle control, SSZ alone (500 mg/kg), cisplatin alone (2 mg/kg, every fifth day), and their combinations (n = 10 each group). Mice were killed on day 15, and the number of lung tumor nodules was counted macroscopically. *P < .01, **P < .001. C, Representative lungs extracted from mice treated with the vehicle control, SSZ alone, cisplatin alone, and their combination
Figure 4
Figure 4
Representative microscopic findings of lungs extracted from mice treated with the vehicle control, sulfasalazine alone, cisplatin alone, and their combinations in a murine lung metastasis model. A,B, H&E staining (A) and immunostaining (B) for CD44v9 in lung tissue of mice treated with the vehicle control, sulfasalazine alone, cisplatin alone, and their combinations. Scale bar = 100 μm. C, Density of CD44v9 in lung tumor nodules of the 4 treatment groups. All data are shown as mean ± SE. *P < .05, **P < .01, ***P < .001
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