• Journal of Oral Medicine and Pain
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• v.32 no.3
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• pp.251-261
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• 2007

Bile acids and synthetic its derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. Previous studies have been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis inducing activity on various cancer cells in vitro. It wasn't discovered those materials have apoptosis induced effects on YD9 human oral squamous carcinoma cells. The present study was done to examine the synthetic bile acid derivatives(HS-1199, HS-1200) induced apoptosis on YD9 cells and such these apoptosis events. We administered them in culture to YD9 cells. Tested YD9 cells showed several lines of apoptotic manifestation such as activation of caspase-3, degradation of DFF, production of poly (ADP-ribose) polymerase(PARP) cleavage(HS-1200 only), DNA degradation(HS-1200 only), nuclear condensation, inhibition of proteasome activity, reduction of mitochondrial membrane potential(HS-1200 only) and the release of cytochrome c and AIF to cytosol. Between two synthetic CDCA derivatives, HS-1200 showed stronger apoptosis-inducing effect than HS-1199. Therefore HS-1200 was demonstrated to have the most efficient antitumor effect. Taken collectively, we demonstrated that a synthetic CDCA derivative HS-1200 induced caspases-dependent apoptosis via mitochondrial pathway in human oral sqauamous carcinoma cells in vitro. Our data therefore provide the possibility that HS-1200 could be considered as a novel therapeutic strategy for human orall squamous carcinoma from its poweful apoptosis-inducing activity.

• Radiation Oncology Journal
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• v.22 no.2
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• pp.145-154
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• 2004

Purpose : To examine whether a synthetic bile acid derivatives (HS-1200) sensitizes the radiation-induced apoptosis in human breast cancer cells (MCF-7) and to investigate the underlying mechanism. Materials and Methods : Human breast cancer cells (MCF-7) in exponential growth phase were treated with HS-1200 for 24 hours at 37$^{\circ}C$ with 5$\%$ CO$_{2}$ in air atmosphere. After removal of HS-1200, cells were irradiated with 2$\~$8 Gy X-ray, and then cultured Ii drug-free media for 24-96 hours. The effect of radiation on the clonogenicity of MCF-7 cells was determined with clonogenic cell survival assay with 16$\mu$M of HS-1200. The induction of apoptosis was determined using agarose gel electrophoresis and Hoechst staining. The expression level of apoptosis-related molecules, such as PARP, Bax, Bcl-2, Bak and AIF, were assayed by Western blotting analysis with 40$\mu$M of HS-1200 combined with 8 Gy irradiation. To examine the cellular location of cytochrome c, bax and AIF immunofluorescent stainings were undertaken. Results : Treatment of MCF-7 cells with 40$\mu$M of HS-1200 combined with 8 Gy irradiation showed several changes associated with enhanced apoptosis by agarose gel electrophoresis and Hoechst staining. HS-1200 combined with 8 Gy irradiation treatment also enhanced production of PARP cleavage products and increased Bax/Bcl-2 ratio by Western blotting. Loss of mitochondrial membrane potential ($\Delta$$\psi$$_{m}$) and increased cytochrome c staining indicated that cytochrome c had been released from the mitochondria in HS-1200 treated cells. Conclusion : We demonstrated that combination treatment with a synthetic chenodeoxycholic acid derivative HS-1200 and irradiation enhanced radiation-induced apoptosis of human breast cancer cells (MCF-7). We suggest that the increased Bax/Bcl-2 ratio In HS-1200 co-treatment group underlies the increased radio sensitivity of MCF-7 cells. Further futures studies are remained elusive.

• Radiation Oncology Journal
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• v.20 no.4
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• pp.367-374
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• 2002

Purpose : To investigate the growth inhibitory effects, and the underlying mechanism of human colon cancer cell (HT-29) death, induced by a new synthetic bile acid derivative (HS-1200). Materials and Methods : Human colon cancer cells (HT-29), in exponential growth phase, were treated with various concentrations of a new synthetic bile acid derivative (HS-1200). The growth inhibitory effects on HT-29 cells were examined using a frypan blue exclusion assay. The extent of apoptosis was determined using agarose gel electrophoresis, TUNEL assays and Hoechst staining. The apoptotic cell death was also confirmed by Western blotting of PARP, caspase-3 and DNA fragmentation factor (DFF) analysis. To investigate the involvement of mitochondria, we employed immunofluorescent staining of cytochrome c and mitochondrial membrane potential analyses. Results : The dose required for the half maximal inhibition $(IC_{50})$ of the HT-29 cell growth was $100\~150\;{\mu}M$ of HS-1200. Several changes, associated with the apoptosis of the HT-29 cells, were reveal by the agarose gel eletrophoresis, TUNEL assays and Hoechst staining, following their treatment with $100\;{\mu}M$ of HS-1200. HS-1200 treatment also induced caspase-3, PARP and DFF degradations, and the western blotting showed the processed caspase-3 p20, PARP p85 and DFF p30 and p11 cleaved products. Mitochondrial events were also demonstrated. The cytochrome c staining indicated that cytochrome c had been released from the mitochondria in the HS-1200 treated cells. The mitochondrial membrane potential $(\Delta\Psi_m)$ was also prominently decreased in the HS-1200 treated cells. Conclusion : These findings suggest that the HS-1200 - induced apoptosis of human colon cancer cells (HT-29) is mediated via caspase and mitochondrial pathways.

• International Journal of Oral Biology
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• v.38 no.3
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• pp.93-100
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• 2013

Bile acids and synthetic bile acid derivatives induce apoptosis in various kinds of cancer cells and thus have anticancer properties. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, few data are available regarding the role of autophagy in oral cancers and there have been no reports of autophagic cell death in OSCCs (oral squamous cell carcinoma cells) induced by HS-1200, a synthetic bile acid derivative. We thus examine whether HS-1200 modulates autophagy in OSCCs. Our findings indicate that HS-1200 has anticancer effects in OSCCs, and we observed in these cells that autophagic vacuoles were visible by monodansylcadaverine (MDC)and acridine orange staining. When we analyzed HS-1200-treated OSCC cells for the presence of biochemical markers, we observed that this treatment directly affects the conversion of LC-3II, degradation of p62/SQSTM1 and full-length beclin-1, cleavage of ATG5-12 and the activation of caspase. An autophagy inhibitor suppressed HS-1200-induced cell death in OSCCs, confirming that autophagy acts as a pro-death signal in these cells. Furthermore, HS-1200 shows anticancer activity against OSCCs via both autophagy and apoptosis. Our current findings suggest that HS-1200 may potentially contribute to oral cancer treatment and thus provide useful information for the future development of a new therapeutic agent.

• International Journal of Oral Biology
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• v.37 no.3
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• pp.91-102
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• 2012

Bcl-2 protects tumor cells from the apoptotic effects of various anti-neoplastic agents. Increased expression of Bcl-2 has been associated with a poor response to chemotherapy in various malignancies, including leukemia. Hence, bypassing the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against cancer cells, including leukemic cells. This study was undertaken to examine whether the anticancer drug, cisplatin and the synthetic chenodeoxycholic acid (CDCA) derivative, HS-1200 show anti-tumor activity in U937 and U937/Bcl-2 cells. Viability assays revealed that HS-1200 overcomes the resistance conferred by Bcl-2 in human leukemic U937 cells. Various apoptosis assessment assays further demonstrated that HS-1200 overcomes the resistance conferred by Bcl-2 in human leukemic U937 cells by inducing apoptosis. In addition HS-1200, but not cisplatin, overcomes the anti-apoptotic effects of Bcl-2 in Bcl-2 over-expressing human leukemic cells (U937/Bcl-2 cells). Notably, we observed that the HS-1200-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with a suppression of the anti-apoptotic effects of Bcl-2 in human leukemic cells over-expressing this protein (U937/Bcl-2 cells). Furthermore, HS-1200 was found to induce the association between PML and SUMO-1, Daxx, Sp100, p53 or CBP in the aggregated PML-NBs of U937/Bcl-2 cells. Thus, PML protein and the formation of mature PML-NBs could be considered as therapeutic targets that may help to bypass the resistance to apoptosis conferred by Bcl-2. Elucidating the exact mechanism by which PML regulates Bcl-2 will require further work.

• Journal of Oral Medicine and Pain
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• v.38 no.3
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• pp.221-233
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• 2013

Bile acids are polar derivatives of cholesterol essential for the absorption of dietary lipids and regulate the transcription of genes that control cholesterol homeostasis. Recently it have been identified the synthetic chenodeoxycholic acid (CDCA) derivatives HS-1200 and cisplatin showed apoptisis-inducing activity on various cancer cells in vivo and in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with HS-1200 and cisplatin on human tongue squamous cell carcinoma cells (SCC25 cells). To investigate whether the co-treatment with HS-1200 and cisplatin compared to each single treatment efficiently reduces the viability of SCC25 cells, MTT assay was conducted. The induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and an analysis DNA hypoploidy. Westen blot analysis and immunofluorescent staining were also performed to evaluate the expression levels and the translocation of apoptosis-related proteins following this co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. In this study, co-treatment with HS-1200 and cisplatin on SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensations, DNA fragmentation, reduction of MMP and proteasome activity, the increase of Bax and the decrease of Bcl-2, decrease of DNA content, the release of cytochrome c into cytosol, translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-9, caspase-7, caspase-3, PARP and DFF45 (ICAD) whereas each single treated SCC25 cells did not show these patterns. Although the single treatment of $25{\mu}M$ HS-1200 and $4{\mu}g/ml$ cisplatin for 24 h did not induce apoptosis, the co-treatment of these reagents prominently induced apoptosis. Therefore our data provide the possibility that the combination therapy with HS-1200 and cisplatin could be considered as a novel therapeutic strategy for human squamous cell carcinoma.

• Journal of Oral Medicine and Pain
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• v.35 no.3
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• pp.165-175
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• 2010

Valproic acid (VPA) is a well-known anticonvulsive agent and has been used in the treatment of epilepsy for almost 30 years. VPA emerged in 1997 as an antineoplastic agent as well, when findings indicated the substance inhibited proliferation and induced differentiation of primitive neuroectocdermal tumor cells in vivo (Cinatl et al., 1997). Antitmor activity of VPA is associated with its targeting histone deacetylases. Bile acids and their synthetic derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with the histone deacetylases inhibitor, VPA and a CDCA derivative, HS-1200 on human osteosarcoma (HOS) cells. Cell viability was evaluated by trypan-blue exclusion. Induction and augmentation of apoptosis were confirmed by Hoechst staining, flow cytometry (DNA hypoploidy and MMP change), Westen blot analysis and immunofluorescent staining. In this study, HOS cells co-treated with VPA and HS-1200 showed several lines of apoptotic manifestation such as nuclear condensations, the reduction of MMP, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF onto nuclei, and activation of caspase-7, caspase-3 and PARP whereas each single treated HOS cells did not. Although the single treatment of 1 mM VPA or $25\;{\mu}M$ HS-1200 for 48 h did not induce apoptosis, the co-treatment of them induced prominently apoptosis. Therefore our data provide the possibility that combination therapy of VPA and HS-1200 could be considered as a novel therapeutic strategy for human osteosarcoma.

• Analytical Science and Technology
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• v.21 no.2
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• pp.93-101
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• 2008

The application of solid phase microextraction (SPME) is generally conducted by directly immersing the fiber into the liquid sample or by exposing the fiber in the head space (HS). The extraction temperature, the time of incubation, and application of stirring are often designated to be the most important parameters for achieving the best extraction efficiencies of HS-SPME analysis. In this study, relative importance of these three analytical parameters involved in the HS-SPME method is evaluated using a polydimethylsiloxane/carboxen (PDMS/CAR) fiber. To optimize its operation conditions the competing relationships between different parameters were investigated by comparing the extraction efficiency based on the combination of three parameters and two contracting conditions: (1) heating the sample at 30 vs. 50 C, (2) exposing samples at two durations of 10 vs. 30 min, and (3) application of stirring vs. no stirring. According to our analysis among 8 combination types of HS-SPME method, an extraction condition termed as S50-30 condition ((1) 1200 rpm stirring, (2) $50^{\circ}C$ exposure temp, and (3) 30 min exposure duration) showed maximum recovery rate of 45.5~68.5% relative to an arbitrary reference of direct GC injection. According to this study, the employment of stirring is the most crucial factor to improve extraction efficiency in the application of HS-SPME.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.229.1-229.1
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• 2003

The anti proliferative effects of bile acids and their derivatives on HT -29 human colon cancer cells were investigated. Ursodeoxycholic acid (UDCA) and its synthetic derivatives, HS-1030 and HS-1183, and chenodeoxycholic acid (CDCA) and its synthetic derivatives, HS-1199 and HS-1200 were employed for this study. General evaluations focusing on cell cycle were conducted in HT -29 human colon adenocarcinoma cell line (p53 mutant type). (omitted)

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.246.1-246.1
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• 2002

We studied the effects of ursodeoxycholic acid (UDCA) and its synthetic derivatives. HS-l030 and HS-1183. and chenodeoxycholic acid (CDCA) and its synthetic derivatives, HS-1199 and HS-1200. on the human colon adenocarcinoma cell line. HT -29 (p53 mutant type). The effects on cell viability and growth were assessed by MTT assay and cell growth study. While UDCA and CDCA exhibited no significant effect, their novel derivatives inhibited the proliferation of HT-29 cell line in a concentration- and time-dependent manners. (omitted)