Proliferative effect of Hachimijiogan, a Japanese herbal medicine, in C2C12 skeletal muscle cells.

BACKGROUND: Hachimijiogan (HJG), Ba-Wei-Di-Huang-Wan in Chinese, is one of the most popular herbal medicines in Japanese Kampo. HJG is often prescribed for the prevention and treatment of age-related diseases. Muscle atrophy plays an important role in aging-related disabilities such as sarcopenia. The purpose of this study was to investigate the possible beneficial effect of HJG on skeletal muscle. METHODS: Cells of murine skeletal muscle myoblast cell line C2C12 were used as an in vitro model of muscle cell proliferation and differentiation. The effect of HJG on C2C12 cell proliferation and differentiation was assessed. We counted the number of myotubes morphologically to assess the degree of differentiation. RESULTS: HJG treatment (200 µg/mL) for 3 days significantly increased C2C12 cell number by 1.23-fold compared with that of the control. HJG promoted the proliferation of C2C12 cells through activation of the ERK1/2 signaling pathway without affecting the Akt signaling pathway. HJG did not affect the differentiation of C2C12 cells. CONCLUSION: HJG had beneficial effects on skeletal muscle myoblast proliferation. These findings may provide a useful intervention for the prevention and treatment of sarcopenia.

The anti-diabetic drug metformin inhibits vascular endothelial growth factor expression via the mammalian target of rapamycin complex 1/hypoxia-inducible factor-1α signaling pathway in ELT-3 cells.

The aim of this study was to elucidate whether metformin can regulate the expression of vascular endothelial growth factor (VEGF) in rat-derived uterine leiomyoma cells (ELT-3 cells). In vitro studies were conducted using ELT-3 cells. Under normoxic conditions, metformin suppressed VEGF protein levels in the supernatant and cells in a dose-dependent manner. In hypoxia-mimicking conditions, VEGF and hypoxia-inducible factor-1α (HIF-1α) proteins were both highly expressed and were suppressed by the metformin treatment. Metformin did not affect HIF-1α mRNA levels, which indicated that its effects occurred at the post-translational level. Metformin inhibited mammalian target of rapamycin complex 1 (mTORC1) activity by phosphorylating the mTORC1 component raptor. This study revealed the anti-angiogenic activity of metformin in ELT-3 cells by suppressing the expression of VEGF via the mTORC1/HIF-1α pathway. These results indicate that metformin may represent an effective alternative in the future treatment of uterine leiomyomas.

Curcumin targets the AKT-mTOR pathway for uterine leiomyosarcoma tumor growth suppression.

BACKGROUND: Uterine leiomyosarcomas generally do not respond well to standard chemotherapy. We previously demonstrated that curcumin, the active ingredient derived from the herb Curcuma longa, inhibits uterine leiomyosarcoma cells in vitro via the inhibition of the AKT-mammalian target of rapamycin (mTOR) pathway. As a preclinical investigation, we performed an in vivo study using female nude mice to confirm the therapeutic potential of curcumin against uterine leiomyosarcoma. METHODS: Human leiomyosarcoma cells, SK-UT-1, were inoculated in female nude mice to establish subcutaneous tumors. Either vehicle control or 250 mg/kg curcumin was administered intraperitoneally every day for 14 consecutive days, and the mice were then killed. The tumors were measured every 2-3 days. The tumors were processed for immunohistochemical analyses to detect total AKT, phosphorylated AKT, total mTOR, phosphorylated mTOR, and phosphorylated S6. To detect apoptosis, the tumors were stained for cleaved PARP and TUNEL. Ki-67 immunohistochemistry was performed to determine cell viability of the tumors. RESULTS: Compared with the control, curcumin reduced uterine leiomyosarcoma tumor volume and mass significantly with a concordant decrease in mTOR and S6 phosphorylation. However, AKT phosphorylation was not significantly altered. Cleaved PARP and TUNEL staining increased significantly with curcumin administration, indicating the induction of apoptosis. There was no difference in Ki-67 staining between the two groups. CONCLUSION: Curcumin inhibited uterine leiomyosarcoma tumor growth in vivo by targeting the AKT-mTOR pathway for inhibition.

Curcumin induces cross-regulation between autophagy and apoptosis in uterine leiomyosarcoma cells.

OBJECTIVE: Uterine leiomyosarcoma (LMS) has an unfavorable response to standard chemotherapy. A natural occurring compound, curcumin, has been shown to have inhibitory effects on cancers. We previously demonstrated that curcumin reduced uterine LMS cell proliferation by targeting the AKT-mTOR pathway and activating apoptosis. To further explore the anticancer effect of curcumin, we investigated the efficacy of curcumin on autophagy in LMS cells. METHODS: Cell proliferation in human uterine LMS cell lines, SKN and SK-UT-1, was assessed after exposure to rapamycin or curcumin. Autophagy was detected by Western blotting for light chain 3 and sequestosome 1 (SQSTM1/p62) expression. Apoptosis was confirmed by Western blotting for cleaved poly (ADP-ribose) polymerase (PARP). RESULTS: Both rapamycin and curcumin potently inhibited SKN and SK-UT-1 cell proliferation in a dose-dependent manner. Curcumin induced autophagy and apoptosis in SKN and SK-UT-1 cells, whereas rapamycin, a specific mTOR inhibitor, did not. Curcumin increased extracellular signal-regulated kinase 1/2 activity in both SKN and SK-UT-1 cells, whereas PD98059, an MEK1 inhibitor, inhibited both the extracellular signal-regulated kinase 1/2 pathway and curcumin-induced autophagy. CONCLUSIONS: These experimental findings suggest that curcumin is a potent inhibitor of cell proliferation in uterine LMS and provide new insights about ongoing signaling events leading to the possible development of a new therapeutic agent.

Epigallocatechin-3-gallate potentiates curcumin's ability to suppress uterine leiomyosarcoma cell growth and induce apoptosis.

BACKGROUND: Uterine leiomyosarcoma (LMS) has an unfavorable response to standard chemotherapeutic regimens. Two natural occurring compounds, curcumin and epigallocatechin gallate (EGCG), are reported to have anti-cancer activity. We previously reported that curcumin reduced uterine LMS cell proliferation by targeting the AKT-mTOR pathway. However, challenges remain in overcoming curcumin's low bioavailability. METHODS: The human LMS cell line SKN was used. The effect of EGCG, curcumin or their combination on cell growth was detected by MTS assay. Their effect on AKT, mTOR, and S6 was detected by Western blotting. The induction of apoptosis was determined by Western blotting using cleaved-PARP specific antibody, caspase-3 activity and TUNEL assay. Intracellular curcumin level was determined by a spectrophotometric method. Antibody against EGCG cell surface receptor, 67-kDa laminin receptor (67LR), was used to investigate the role of the receptor in curcumin's increased potency by EGCG. RESULTS: In this study, we showed that the combination of EGCG and curcumin significantly reduced SKN cell proliferation more than either drug alone. The combination inhibited AKT, mTOR, and S6 phosphorylation, and induced apoptosis at a much lower curcumin concentration than previously reported. EGCG enhanced the incorporation of curcumin. 67LR antibody partially rescued cell proliferation suppression by the combination treatment, but was not involved in the EGCG-enhanced intracellular incorporation of curcumin. CONCLUSIONS: EGCG significantly lowered the concentration of curcumin required to inhibit the AKT-mTOR pathway, reduce cell proliferation and induce apoptosis in uterine LMS cells by enhancing intracellular incorporation of curcumin, but the process was independent of 67LR.

The antidiabetic drug metformin inhibits uterine leiomyoma cell proliferation via an AMP-activated protein kinase signaling pathway.

Uterine leiomyomas are the most common gynecological benign tumors and greatly affect reproductive health and wellbeing. Metformin is the most widely used antidiabetic drug in the world, and there is increasing evidence of a potential efficacy of this agent as an anticancer drug. In order to understand metformin's anti-tumorigenic potential better, in this study, we investigated the inhibitory effect of metformin and expression of key targets of metformin cell signaling in leiomyoma cells. Cell proliferation was assessed after exposure to metformin. Apoptosis was assessed by western blotting for cleaved-PARP and TUNEL staining. The expressions of phosphorylated AMPK and phosphorylated S6 were determined by western blotting. Metformin potently inhibited ELT-3 cell proliferation in a dose-dependent manner. Western blotting analysis demonstrated that metformin induced phosphorylation of AMPK and the inhibitory effect was attenuated with AMPK inhibitor, compound C. In parallel, treatment with metformin decreased phosphorylation of S6 protein. These experimental findings show that metformin is a potent inhibitor of cell proliferation in leiomyoma cells. This effect is mediated by AMPK activation and subsequent inhibition of the mTOR pathway. Thus, this study provides a possible mechanism of the action of metformin in the inhibition of leiomyoma cell growth.

Curcumin disrupts uterine leiomyosarcoma cells through AKT-mTOR pathway inhibition.

OBJECTIVE: Uterine leiomyosarcoma generally has an unfavorable response to standard chemotherapy. The loss of PTEN which results in constitutive AKT-mTOR activation causes an increase in leiomyosarcoma formation in mice. The active ingredient derived from the herb Curcuma longa, curcumin, shows antitumor properties in a variety of cancer cell lines by altering a number of oncogenic pathways. To explore the possibility of curcumin as an alternative to standard chemotherapy, we decided to investigate curcumin's antitumor effect on uterine leiomyosarcoma cells. METHODS: Human leiomyosarcoma cell lines, SKN and SK-UT-1, were cultured for in vitro experiments. Rapamycin or curcumin was added in different doses and their effect on cell growth was detected by MTS assay. The influence of rapamycin or curcumin on AKT, mTOR, p70S6 and S6 phosphorylation and protein expression was detected by Western Blotting. The ability of rapamycin or curcumin to induce apoptosis was determined by Western blotting using cleaved-PARP specific antibody, Caspase-3 activity assay and TUNEL assay. RESULTS: Both rapamycin and curcumin significantly reduced SKN cell proliferation. Curcumin inhibited mTOR, p70S6 and S6 phosphorylation similar with rapamycin. Cleaved PARP, caspase-3 activity and DNA fragmentation increased proportional with curcumin concentration. At a high concentration, curcumin significantly induced apoptosis in SKN cells, but not rapamycin. CONCLUSIONS: Curcumin inhibited uterine leiomyosarcoma cells' growth by targeting the AKT-mTOR pathway for inhibition. However, rapamycin, a specific mTOR inhibitor, did not induce apoptosis in SKN cells unlike curcumin that also has a pro-apoptotic potential in SKN cells.

Antiproliferative effect of adiponectin on rat uterine leiomyoma ELT-3 cells.

OBJECTIVE: Although fibroids greatly affect reproductive health, the pathophysiology and epidemiology are not well known. Recently, we have reported the relationship between uterine leiomyoma and metabolic syndrome. Many studies have indicated that reductions in plasma adiponectin levels play major roles in the development of metabolic syndrome. In this study, we investigated the significant repressive effect of adiponectin on rat uterine leiomyoma ELT-3 cells proliferation. STUDY DESIGN: Expression of adiponectin receptor 1 and receptor 2 was evaluated by RT-PCR and Western blot analysis. Cell proliferation was assessed by the MTS assay and cell counting. Apoptosis was evaluated by Hoechst staining and cleaved poly (ADP-ribose) polymerase (PARP). RESULTS: Adiponectin receptor 1 and receptor 2 were expressed in ELT-3 cells. Adiponectin repressed rat uterine leiomyoma ELT-3 cells cell proliferation without inducing apoptosis. CONCLUSION: The repression of adiponectin on leiomyoma cell proliferation in the rat may explain a crucial role of adiponectin in the association of metabolic syndrome with uterine leiomyoma.

Inhibitory effect of curcumin on uterine leiomyoma cell proliferation.

OBJECTIVE: Uterine leiomyomas are the most common gynaecological benign tumour and greatly affect reproductive health and wellbeing. They are the predominant indication for hysterectomy in premenopausal women. Curcumin, a well-known component of turmeric, has been reported to prevent various diseases such as cancer, diabetes and obesity. Previous study reported that curcumin represses the proliferation of several tumour cells. However, there has not been a precise characterisation of the curcumin-induced inhibition of uterine leiomyoma cells. In this study, we investigated the inhibitory effect of curcumin on leiomyoma cells proliferation. STUDY DESIGN: Eker rat-derived uterine leiomyoma cell lines (ELT-3 cells) were used. Cell proliferation was assessed by counting the number of cells and MTS assay. The activation of peroxisome proliferator-activated receptor-gamma (PPARγ) was evaluated by luciferase assay. RESULTS: We found that curcumin significantly inhibited ELT-3 cell proliferation. PPARγ was expressed in ELT-3 cells and curcumin acted as a PPARγ ligand. This inhibitory effect of curcumin was attenuated by the treatment of cells with PPARγ antagonist. CONCLUSION: These experimental findings in vitro show that the inhibitory effect of curcumin on ELT-3 cell proliferation occurs through the activation of PPARγ. Curcumin may be useful as an alternative therapy for uterine leiomyoma.

Establishment of a novel xenograft model for human uterine leiomyoma in immunodeficient mice.

Uterine leiomyomas are the most common gynecological benign tumor and greatly affect reproductive health and wellbeing, but the pathophysiology and epidemiology of uterine leiomyoma are poorly understood. One of the major reasons for the slow progress in leiomyoma research is the lack of a good in vivo model system. We therefore aimed to develop a novel model by transplanting human uterine leiomyoma xenografts in an immunodeficient mouse strain (NOD/SCID/gammac-null: NOG). Human uterine leiomyoma tissues were cut into small pieces and inserted subcutaneously into the right and left flanks of NOG mice. Estrogen supplementation was needed to maintain the features of uterine leiomyoma in xenografted tissues. After 4 weeks or 8 weeks of transplantation, xenografted tissues were harvested and analyzed regarding tissue morphology, collagen content, and proliferation and apoptosis of uterine leiomyoma smooth muscle cells. The xenografts that were harvested after 4 weeks and 8 weeks retained the histological architecture of original uterine leiomyoma tissue both in cellular and collagen components. The expression profiles of key markers of uterine leiomyoma were also maintained, including estrogen receptor, progesterone receptor, and alpha-smooth muscle actin, as judged by immunohistochemical staining. The proportion of proliferating cells was significantly increased (1.5-fold) in the xenografts after 8 weeks of transplantation, whereas that of the apoptotic cells remained unchanged. Importantly, the reproducible results were obtained with the tumor tissues derived from six patients. The present in vivo model may provide a useful tool for development of novel therapeutic strategies for uterine leiomyoma.

Aldosterone stimulates the proliferation of uterine leiomyoma cells.

OBJECTIVE: Although uterine leiomyomas are the most common gynaecological benign tumour and greatly affect reproductive health and well being, the pathophysiology and epidemiology of uterine leiomyomas are not well known. Elevated blood pressure has an independent, positive association with risk for clinically detected uterine leiomyomas. Aldosterone is a key biological peptide in the renin-angiotensin-aldosterone system that regulates blood pressure. In this study, we investigated the siginificant stimulatory effect of aldosterone on leiomyoma cells proliferation. STUDY DESIGN: This study investigated the potential role of aldosterone in the proliferation of ELT-3 leiomyoma cells. RESULTS: Aldosterone-induced ELT-3 leiomyoma cell proliferation and the expression of mineralocorticoid receptor (MR) were confirmed. Pre-incubating the cells with the MR blockers spironolactone or eplerenone effectively repressed aldosterone-induced and angiotensin II (Ang II)-induced cell proliferation. Treatment of aldosterone increased the levels of Ang II type-1 receptor mRNA. CONCLUSION: These experimental findings in vitro show the presence of complex regulation of Ang II and aldosterone induced leiomyoma cell proliferation.

Identification of activated transcription factors from microarray gene expression data of Kampo medicine-treated mice.

We propose an approach to identify activated transcription factors from gene expression data using a statistical test. Applying the method, we can obtain a synoptic map of transcription factor activities which helps us to easily grasp the system's behavior. As a real data analysis, we use a case-control experiment data of mice treated by a drug of Kampo medicine remedying degraded myelin sheath of nerves in central nervous system. Kampo medicine is Japanese traditional herbal medicine. Since the drug is not a single chemical compound but extracts of multiple medicinal herb, the effector sites are possibly multiple. Thus it is hard to understand the action mechanism and the system's behavior by investigating only few highly expressed individual genes. Our method gives summary for the system's behavior with various functional annotations, e.g. TFAs and gene ontology, and thus offer clues to understand it in more holistic manner.