Teruyoshi Saito

The hyaluronan synthesis inhibitor 4-methylumbelliferone exhibits antitumor effects against mesenchymal-like canine mammary tumor cells

Hyaluronan (HA), a principal constituent of the extracellular matrix (ECM), mediates growth and metastasis of tumor cells. The role of HA in the epithelial-mesenchymal transition (EMT) is well known, and increased ECM remodeling is observed in mesenchymal-like cells. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) is anti-tumorigenic for various malignant tumors. However, the antitumor effect of 4-MU against canine mammary tumor cells that possess a mesenchymal-like phenotype is unclear. We examined the antitumor effect of 4-MU on CF41.Mg mesenchymal-like canine mammary tumor cells. We investigated the influence of 4-MU on the expression of HA synthase (HAS) 1-3 mRNA and observed dose-dependent downregulation of HAS2 mRNA at 24-72 h; in contrast, HAS3 expression was elevated at 24 h. Thus, 4-MU inhibited HA synthesis via HAS2 repression. 4-MU also inhibited cell proliferation and induced apoptosis in the CF41.Mg cells. Our experiments showed that 4-MU-induced apoptosis in CF41.Mg cells involved induction of the pro-apoptotic gene BAX. We also assessed motility and found that 4-MU reduced chemokinesis and chemotaxis in CF41.Mg cells. Our data suggest that 4-MU may serve as a candidate therapeutic agent for the treatment of canine mammary tumors. Since 4-MU exhibits antitumor activity in mesenchymal-like cells, it may also be a useful inhibitor of canine mammary tumor invasion and metastasis.

Fragmented hyaluronan is an autocrine chemokinetic motility factor supported by the HAS2-HYAL2/CD44 system on the plasma membrane.

Hyaluronan (HA) is synthesized by HA synthase (HAS) 1, HAS2 and HAS3, and degraded by hyaluronidase (HYAL) 1 and HYAL2 in a CD44-dependent manner. HA and HYALs are intricately involved in tumor growth and metastasis. Random cell movement is generally described as chemokinesis, and represents an important step at the beginning of tumor cell liberation from the primary site. To investigate the roles of HAS2 and HYAL2/CD44 in cell motility, we examined HeLa-S3 cells showing spontaneous chemokinesis. HeLa-S3 cells expressed HAS2 and HAS3. siRNA-mediated knockdown of HAS2 decreased spontaneous chemokinesis of HeLa-S3 cells. Although HeLa-S3 cells secreted 50 ng/ml of high molecular weight (HMW)-HA (peak: 990 kDa) into the culture supernatant after 6 h of culture, exogenously added HMW-HA did not enhance spontaneous chemokinesis of the cells. These observations suggested that HeLa-S3 cells may have a self-degrading system for HA to regulate their spontaneous chemokinesis. To examine this possibility, we investigated the effects of siRNA-mediated knockdown of HYAL2 or CD44 on the spontaneous chemokinesis of HeLa-S3 cells. Knockdown of either molecule decreased the spontaneous chemokinesis of the cells. Low molecular weight (LMW)-HA (23 kDa) reversed the HYAL2 siRNA-mediated reduction in spontaneous chemokinesis of HeLa-S3 cells to the level in control cells stimulated with the same HA. These findings indicate that the HAS2-HYAL2/CD44 system may support spontaneous chemokinesis of human cancer cells through self-degradation of HMW-HA to produce LMW-HA by an autocrine mechanism. Consequently, our study may further expand our understanding of HA functions in cancer.

Usefulness of selective COX-2 inhibitors as therapeutic agents against canine mammary tumors

Cyclooxygenase-2 (COX-2) is a key enzyme for converting arachidonic acids to prostanoids, which are known to be induced during inflammation and cancer initiation. Previously, it has been reported that COX inhibitors, such as aspirin, reduce the incidence of human colorectal cancer; therefore, it is widely believed that COX-2 is a potential therapeutic and chemoprevention target for several types of human cancer. However, whether selective COX-2 inhibitors have antitumor effects against canine mammary tumor cells remains unclear. In the present study, to elucidate the antitumor effect of selective COX-2 inhibitors against canine mammary tumors, we investigated the antitumor effects of meloxicam, etodolac and celecoxib using COX-2-expressing canine mammary tumor (CF33) cells. We analyzed the effects of selective COX-2 inhibitors on COX-2 protein expression levels in CF33 cells. Celecoxib (100 µM) was found to induce downregulation of COX-2 protein expression. We examined the effect of selective COX-2 inhibitors on CF33 cell proliferation. All the selective COX-2 inhibitors suppressed CF33 cell growth. Specifically, etodolac and celecoxib inhibited cell proliferation via a decrease in S-phase cells and an increase in G0/G1 arrest. We examined the apoptotic effect of selective COX-2 inhibitors on CF33 cells. Our data suggested that etodolac and celecoxib induced apoptosis in CF33 cells. In particular, celecoxib led to apoptosis mediated by the activation of the mitochondrial apoptosis pathway, including the upregulation of BAX expression, downregulation of Bcl-2 expression and activation of caspase-3/7. Furthermore, celecoxib increased the percentages of cells in both early apoptosis and late apoptosis. Our results revealed that celecoxib induced apoptosis and cell cycle arrest in CF33 cells. The data suggested that celecoxib is the most viable candidate as a therapeutic agent for the treatment of canine mammary tumors. Furthermore, our findings provide the first indication that COX-2 inhibition can provide a new therapeutic strategy for treating canine mammary tumors.

4-methylumbelliferone leads to growth arrest and apoptosis in canine mammary tumor cells.

Hyaluronan (HA), a major component of the extracellular matrix (ECM), is synthesized by HA synthase (HAS) 1, HAS2 and HAS3 and is intricately involved in cell growth and metastasis. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) has been reported to exhibit anticancer properties in various types of malignant tumors. However, the underlying mechanisms at the molecular and cellular levels remain unclear. In this study, to establish an animal model for studying the function of HA in human breast cancer, we investigated the antitumor effects of 4-MU using canine mammary tumor (CF33) cells. First, we investigated the effects of 4-MU on HA production in CF33 cells. Quantitative analysis of HA in culture media showed that 4-MU inhibited HA synthesis, accompanied by downregulation of HAS2 mRNA levels, in a dose-dependent manner at 24-72 h. Additionally, we observed a 4-MU-mediated decrease in the extent of the cell-associated HA matrix. We examined the effect of 4-MU on cell growth and apoptosis in CF33 cells. 4-MU markedly inhibited cell proliferation and induced apoptosis in CF33 cells. In particular, our experiments showed that the mechanism of 4-MU-induced apoptosis in CF33 cells involved increased levels of expression of pro-apoptotic BAX mRNA and protein molecules. These data suggest that 4-MU may be a candidate therapeutic agent for the treatment of canine mammary tumors. Furthermore, this study provides the first indication that the canine mammary tumor may be a suitable model for comparative study of the function of HA in human breast cancer.

Celecoxib exerts antitumor effects in canine mammary tumor cells via COX‑2‑independent mechanisms.

Celecoxib plays antitumor roles via multiple mechanisms in a variety of human cancers. The aim of this study was to clarify the mechanism of action of celecoxib in canine mammary tumors. We examined the antitumor effects of celecoxib in AZACB canine mammary tumor cells expressing low levels of cyclooxygenase‑2 (COX‑2) to minimize the effect of COX‑2 on its activity. Our data revealed that celecoxib inhibited cell proliferation mainly via COX‑2‑independent mechanisms. Specifically, celecoxib decreased the proportion of cells in S phase and increased G2/M arrest, which was associated with increased expression of the cyclin‑dependent kinase inhibitors (CDKIs) p21 and p27. In addition, treatment with celecoxib downregulated COX‑2 expression, and induced apoptosis via both the intrinsic and extrinsic pathways. These findings suggest that celecoxib might be a useful agent for the treatment of canine mammary tumors, regardless of COX‑2 expression. In the future, it might be possible to use a combination of celecoxib and other antitumor agents to treat canine mammary tumors.

Visualization of the activity of Rac1 small GTPase in a cell

Rho family G proteins including Rac regulate a variety of cellular functions, such as morphology, motility, and gene expression. Here we developed a fluorescence resonance energy transfer-based analysis in which we could monitor the activity of Rac1. To detect fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan fluorescent protein fused Cdc42-Rac1-interaction-binding domain of Pak1 protein were used as intermolecular probes of FRET. The fluorophores were separated with linear unmixing method. The fluorescence resonance energy transfer efficiency was measured by acceptor photobleaching assisted assay. With these methods, the Rac1 activity was visualized in a cell. The present findings indicate that this approach is sensitive enough to achieve results similar to those from ratiometric fluorescence resonance energy transfer analysis.

Connexin 43-dependent tumor-suppressing effect of the Bowman-Birk protease inhibitor on M5076 ovarian sarcoma-bearing mice.

The present study was designed to confirm whether the Bowman-Birk inhibitor (BBI) induces an increase in p27 accumulation without S phase kinase-associated protein 2 (skp2) degradation by means of the expression of connexin (Cx) 43 as a gap junctional intercellular communication (GJIC)-dependent pathway in mice with M5076 ovarian sarcoma. M5076 ovarian sarcomas (1x105 cells/animal) were subcutaneously transplanted onto the backs of BDF1 mice receiving 10, 20 or 40 mg/kg of purified BBI intraperitoneally. Relative tumor weight (p<0.01, r=0.503) was negatively correlated with the dose of BBI. In contrast, the relative density of Cx43 mRNA (p<0.01, r=0.570) and Cx43 (p<0.01, r=0.718) was positively correlated with the dose of BBI, as were p21 (p<0.01, r=0.633), p27 (p<0.01, r=0.561) and skp2 (p<0.01, r=0.733). We therefore suggest that the anti-carcinogenic effects of BBI induce negative growth control by means of an increase in p27 accumulation caused by the expression of Cx43 as a GJIC pathway.

Abstract 1393: CD44 has dual functions to enhance the hyaluronan-induced chemokinesis in cancer cells as an associated molecule of hyaluronidase2-mediated HA catabolism and a hyaluronan receptor for RhoGTPase activation

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Major oncogenic signaling pathways are involved in the alteration of hyaluronan (HA) metabolism of cancer cells, such as Erb B, Wnt, TGF-β and p53 signalings. HA is a widely distributed extracellular matrix glycosaminoglycan and changes biological activities depending on its molecular sizes. Our recent study has shown that fragmented low molecular weight (LMW) HA (approximately 20 kDa) is an autocrine chemokinetic motility factor supported by the HA synthethase2-hyaluronidase2 / CD44 system on the plasma membrane of the cancer cells. In HeLaS3 cells, LMW-HA stimulation effectively enhanced cell spreading and random cell movement (chemokinesis) among different molecular sizes of HA (3, 23, 230, 940-kDa). Biochemically, LMW HA effectively provoked a RhoA activation, recurrent Cdc42 and Rac1 activation, and sustained phosphorylation of ERK1/2. Constrastly, 220 kDa HA did not enhance chemokinesis of HeLaS3 cells with similar activation profiles of Rac1 and ERK 1/2 except for the progressive decrease of Cdc42 and RhoA activation. The transfection of CD44 si-RNA to HeLaS3 cells abolished the enhanced chemokinesis with the remarkable decrease of Rho GTPase activation even in LMW HA stimulation. Accordingly, our study showed that CD44 has dual functions to enhance the HA-induced chemokinesis in cancer cells as an associated molecule of hyaluronidase2-mediated HA catabolism and a HA receptor for RhoGTPase activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1393. doi:10.1158/1538-7445.AM2011-1393

Abstract C74: Activated RhoA, Rac1 and ERK1/2 participate in chemokinesis triggered by fragmented 23 kDa hyaluronan

Tumors generally possess high levels of hyaluronan (HA), but have a reduced size compared with normal tissue. Experimentally increased HA induces tumor growth and metastasis in xenograft models (review by Toole, BP, 2004). Our study sought to elucidate the possible molecular mechanism of HA‐mediated cell motility in a human epithelial cell line, HeLaS3, using a Boyden chamber system. We showed that among the different molecular masses of HA (3, 23, 230 and 940 kDa) (Seikagaku Kogyo Co.) 23 kDa HA was the most potent in chemokinesis, while 230 and 940 kDa HAs had no detectable effect on chemokinesis. With a drop of 23‐kDa HA, sparsely adhered HeLaS3 cells spread, exhibiting multiple surface extensions in various directions, and altered lamellipodia. HeLaS3 cells exhibited fine cortical F‐actin at the lamellipodia and formed many small focal complexes instead of large focal adhesions at the base of membrane protrusions at 1–20 minutes after 23 kDa HA stimulation. These morphological alterations are reminiscent of Rac1 activated fibroblasts (Rottner, 1999. To properly address the modulation of Rho GTPases in HA‐induced chemokinesis, we evaluated cells carrying dominant negative mutants of either RhoA cDNA (dnRhoA) or Rac1 cDNA (dnRac1). The transduction of either dnRhoA or dnRac1 into HeLaS3 cells caused a dramatic decrease in chemokinesis with or without HA stimulation, compared with mock‐transduced control. To biochemically confirm Rho GTPase activation, we measured GTP‐RhoA and GTP‐Rac1 levels after stimulation with 23 kDa HA using ELISA based Rho GTPases activation assay kits (Cytoskeleton, Inc.). GTP‐Rho A increased after 23kD HA ligation, had a small peak at 1 minute, decreased, and then returned to baseline by 60 min. After 23kD HA stimulation, Rac1 activation was observed in dual peaks of 1.8–2 fold increases at 3 and 15 minutes; thereafter, it became near to baseline by 60 minutes. It has been shown that HA can modulate cell migration involving hyaladherin‐mediated signaling through ERK1/2 (Vigetti, 2008, Tolg, 2006). Initially, we examined the effect of the ERK inhibitor PD98059 in our system. A treatment of 6.6 µg/ ml PD98059 suppressed 23 kDa HA‐induced chemokinesis compared with the control group. Western blotting of the cell lysates showed sustained ERK1/2 phosphorylation over 120 minutes. This study indicates that sustained ERK1/2 phosphorylation is required for 23 kDa HA‐induced chemokinesis. Here we show that fragmented 23 kDa HA elicits enhanced chemokinesis and changes the shape of epithelial cells by weak Rho A activation prior to the recurrent Rac1 activation and the sustained phosphorylation of ERK1/2. Our unpublished study also indicates that hyaluronidase‐2‐mediated catabolism could play a significant role in HA oligosaccharide generation with the association of CD44 in epithelial cells. This indicates the possibility that fragmented HA acts as a mediator in the autocrine/paracrine mechanism. Our study showed that signals initiated by fragmented HA‐hyaladherin interactions induce chemokinesis of epithelial cells, expanding our understanding of the functions of HA in cancer cell biology. Citation Information: Cancer Res 2009;69(23 Suppl):C74.