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. 2018 Sep;109(9):2801-2810.
doi: 10.1111/cas.13728. Epub 2018 Aug 16.

High expression of CD44v9 and xCT in chemoresistant hepatocellular carcinoma: Potential targets by sulfasalazine

Fumitaka Wada  1   2 , Hironori Koga  1   2 , Jun Akiba  3 , Takashi Niizeki  1 , Hideki Iwamoto  1   2 , Yu Ikezono  1   2 , Toru Nakamura  1   2 , Mitsuhiko Abe  1   2 , Atsutaka Masuda  1   2 , Takahiko Sakaue  1   2 , Toshimitsu Tanaka  1   2 , Tatsuyuki Kakuma  4 , Hirohisa Yano  3 , Takuji Torimura  1   2
Affiliations

"V体育ios版" High expression of CD44v9 and xCT in chemoresistant hepatocellular carcinoma: Potential targets by sulfasalazine

Fumitaka Wada et al. Cancer Sci. 2018 Sep.

Abstract

CD44v9 is expressed in cancer stem cells (CSC) and stabilizes the glutamate-cystine transporter xCT on the cytoplasmic membrane, thereby decreasing intracellular levels of reactive oxygen species (ROS). This mechanism confers ROS resistance to CSC and CD44v9-expressing cancer cells VSports手机版. The aims of the present study were to assess: (i) expression status of CD44v9 and xCT in hepatocellular carcinoma (HCC) tissues, including those derived from patients treated with hepatic arterial infusion chemoembolization (HAIC) therapy with cisplatin (CDDP); and (ii) whether combination of CDDP with sulfasalazine (SASP), an inhibitor of xCT, was more effective on tumor cells than CDDP alone by inducing ROS-mediated apoptosis. Twenty non-pretreated HCC tissues and 7 HCC tissues administered HAIC therapy with CDDP before surgical resection were subjected to immunohistochemistry analysis of CD44v9 and xCT expression. Human HCC cell lines HAK-1A and HAK-1B were used in this study; the latter was also used for xenograft experiments in nude mice to assess in vivo efficacy of combination treatment. CD44v9 positivity was significantly higher in HAIC-treated tissues (5/7) than in non-pretreated tissues (2/30), suggesting the involvement of CD44v9 in the resistance to HAIC. xCT was significantly expressed in poorly differentiated HCC tissues. Combination treatment effectively killed the CD44v9-harboring HAK-1B cells through ROS-mediated apoptosis and significantly decreased xenografted tumor growth. In conclusion, the xCT inhibitor SASP augmented ROS-mediated apoptosis in CDDP-treated HCC cells, in which the CD44v9-xCT system functioned. As CD44v9 is typically expressed in HAIC-resistant HCC cells, combination treatment with SASP with CDDP may overcome such drug resistance. .

Keywords: anticancer drug resistance; cancer stem cell; combinational therapy; oxidative stress; sphere formation V体育安卓版. .

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V体育官网入口 - Figures

Figure 1
Figure 1
Expression of xCT, CD44s, and CD44v9 in human hepatocellular carcinoma (HCC) tissues. A, xCT is highly expressed in poorly differentiated HCC tissue (Poor), followed by moderately differentiated (Mod) and well‐differentiated tissues (Well). NT, non‐tumorous tissue. Scale bars, 100 μm. B, Immunostaining score shows significantly highest expression of xCT in Poor. formula image P < 0.01; C, Limited number of CD44v9‐positive HCC cells was observed among several CD44s‐positive cells in Poor. Scale bars, 100 μm. D, CD44v9 was clearly localized to the cytoplasmic membrane of HCC cells (magnified view of the squared area in C)). Scale bar, 20 μm. E, Immunostaining scores for 30 HCC tissues studied. CD44v9 was positive in only 2 HCC: untreated with neoadjuvant chemotherapy and presurgical radiation therapy formula image P < 0.01; n.s., not significant. F, (left upper panel) H&E staining of HCC tissue after hepatic arterial infusion chemoembolization (HAIC) treatment shows a viable tumor cell cluster surrounded by necrotic tissue area. Scale bar, 500 μm. (right upper panel) Magnified view of the squared area in the left upper panel. Scale bar, 50 μm. (left lower panel) CD44v9‐positive HCC cells were observed at the periphery of the tumor pretreated with neoadjuvant HAIC therapy. Scale bar, 50 μm. (right lower panel) xCT was strongly positive in the same tumor area in the serial section. Scale bar, 50 μm. Graph shows that CD44v9 positivity was significantly higher in HAIC‐pretreated HCC tissues (71.4%) than in those without presurgical treatments (10.0%). formula image P < 0.01
Figure 2
Figure 2
Western blotting for CD44v9 and xCT in human hepatocellular carcinoma (HCC) cell lines. A, HAK‐1B strongly and weakly expresses xCT and CD44v9, respectively, compared to HAK‐1A, a well‐differentiated HCC cell line. The colon cancer cell line HCT‐116 served as a positive control for both molecules. B, Cancer cells forming cancer spheres (S) strongly express both CD44v9 and xCT compared to those under attached conditions (A). C, Condensation of both molecules was universally observed in other liver cancer cell lines, such as Huh7 and HepG2. Phase contrast images of attached and sphere‐forming cells are shown at the bottom
Figure 3
Figure 3
Inhibition of cell proliferation by cisplatin (CDDP) plus sulfasalazine (SASP) in hepatocellular carcinoma (HCC) cells. A, Synergistic inhibition of cell proliferation is significant at ≥40 μM CDDP in combination with 100 μM SASP in HAK‐1B cells (formula image P < 0.01). B, A synergistic inhibitory effect was seen even in sphere‐forming HAK‐1B cells. As described in Materials and Methods, imaging analysis of the number of cell aggregates/spheres per well was conducted and the cell aggregate area (%) is shown. formula image P < 0.01
Figure 4
Figure 4
Reactive oxygen species (ROS)‐dependent apoptosis enhanced by sulfasalazine (SASP). A, SASP significantly increases intracellular ROS levels in HAK‐1B cells cotreated with cisplatin (CDDP) in a dose‐dependent way. N‐Acetylcysteine (NAC) completely inhibited ROS production. formula image P < 0.001. B, Decrease in intracellular glutathione (GSH) content was slow but significant in cells exposed to CDDP and 100 μM SASP. formula image P < 0.01. C, In western blotting, the apoptosis markers cleaved (Cl) poly (ADP‐ribose) polymerase (PARP) and cleaved caspase (Cas)‐3 were clearly observed in cells treated with both CDDP and SASP. When these cotreated cells were exposed to NAC, apoptosis was remarkably suppressed (lane 5)
Figure 5
Figure 5
Xenograft experiments in nude mice. A, Representative xenografted tumors (arrows) in nude mice are shown. Black arrows, control (DMSO); blue, cisplatin (CDDP)‐treated; and red, CDDP/sulfasalazine (SASP)‐treated. B, Size of xenografted tumors was significantly decreased from weeks 1‐5 by treatment of CDDP alone, compared with that of controls (DMSO) (P < 0.01 at weeks 1, 2, and 3; P < 0.05 at weeks 4 and 5). At week 2 after beginning combinatorial treatment, tumor growth began decreasing compared to that observed in mice treated with CDDP alone. After 4 weeks of dual treatments, tumor growth was significantly suppressed compared to that in mice treated with CDDP alone (formula image P < 0.001). C, Consistent with the tumor growth inhibition, immunohistochemistry analysis of cleaved caspase‐3 showed significantly highest positivity in tumor tissues of mice treated with both CDDP and SASP, suggesting that the drug combination also induces robust apoptosis in vivo (formula image means P < 0.001 or P < 0.01). D, CD44v9‐positive tumor cells were eliminated from xenografted tumors exposed to both CDDP and SASP (left bottom). Scale bar, 50 μm. The staining score was calculated as described in Materials and Methods (formula image P < 0.05; formula image P < 0.001)
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