An integrated analysis of dysregulated SCD1 in human cancers and functional verification of miR-181a-5p/SCD1 axis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC), one of the most lethal cancers, has become a global health issue. Stearoyl-coA desaturase 1 (SCD1) has been demonstrated to play a crucial role in human cancers. However, pan-cancer analysis has revealed little evidence to date. In the current study, we systematically inspected the expression patterns and potential clinical outcomes of SCD1 in multiple human cancers. SCD1 was dysregulated in several types of cancers, and its aberrant expression acted as a diagnostic biomarker, indicating that SCD1 may play a role in tumorigenesis. We used ESCC as an example to demonstrate that SCD1 was dramatically upregulated in tumor tissues of ESCC and was associated with clinicopathological characteristics in ESCC patients. Furthermore, Kaplan-Meier analysis showed that high SCD1 expression was correlated with poor progression-free survival (PFS) and disease-free survival (DFS) in ESCC patients. The protein-protein interaction (PPI) network and module analysis by PINA database and Gephi were performed to identify the hub targets. Meanwhile, the functional annotation analysis of these hubs was constructed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Functionally, the gain-of-function of SCD1 in ESCC cells promoted cell proliferation, migration, and invasion; in contrast, loss-of-function of SCD1 in ESCC cells had opposite effects. Bioinformatic, QPCR, Western blotting and luciferase assays indicated that SCD1 was a direct target of miR-181a-5p in ESCC cells. In addition, gain-of-function of miR-181a-5p in ESCC cells reduced the cell growth, migratory, and invasive abilities. Conversely, inhibition of miR-181a-5p expression by its inhibitor in ESCC cells had opposite biological effects. Importantly, reinforced SCD1 in miR-181a-5p mimic ESCC transfectants reversed miR-181a-5p mimic-prevented malignant phenotypes of ESCC cells. Taken together, these results indicate that SCD1 expression influences tumor progression in a variety of cancers, and the miR-181a-5p/SCD1 axis may be a potential therapeutic target for ESCC treatment.

A novel therapeutic approach for endometriosis using adipose-derived stem cell-derived conditioned medium- A new hope for endometriotic patients in improving fertility.

Introduction: Endometriosis is defined as the growth of endometrial glands and stromal cells in a heterotopic location with immune dysregulation. It usually leads to chronic pelvic pain and subfertility. Although various treatments are available, the recurrence rate remains high. Adipose tissue is an abundant source of multipotent mesenchymal adipose-derived stem cells (ADSCs). ADSCs display effects on not only tissue regeneration, but also immune regulation. Thus, the current study aims to test the effects of ADSCs on the growth of endometriosis. Methods: ADSCs isolated from lipoaspiration-generated adipose tissue and their conditioned medium (ADSC-CM) were subjected to quality validation, including karyotyping as well as growth promotion and sterility tests for microbial contamination under Good Tissue Practice and Good Manufacturing Practice regulations. An autologous endometriosis mouse model was established by suturing endometrial tissue to peritoneal wall followed by treating with DMEM/F12 medium, ADSC-CM, ADSCs or ADSC-CM+ADSCs for 28 days. The area of endometriotic cysts and the degree of pelvic adhesion were measured. ICAM-1, VEGF and caspase 3 expression was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Moreover, the mice were allowed to mate and deliver. The pregnancy outcomes were recorded. The ADSC-CM was subjected to proteomics analysis with further data mining with Ingenuity Pathway Analysis (IPA). Results: Both ADSC-CM and ADSCs passed quality validation. ADSC-CM reduced the area of endometriotic cysts. The inhibition by ADSC-CM was obliterated by adding ADSCs. The presence of ADSCs with or without ADSC-CM increased the peritoneal adhesion. ADSC-CM inhibited ICAM-1 and VEGF mRNA and protein expression, whereas the addition of ADSCs not only did not inhibit by itself, but also blocked the inhibition by ADSC-CM. The resorption rate was reduced by ADSC-CM. The number of live birth/dam and the survival rate of pup at 1 week-old were both increased by ADSC-CM in mice with endometriosis. IPA demonstrated that PTX3 was potentially critical for the inhibition of endometriosis by ADSC-CM due to its anti-inflammatory and antiangiogenic properties as well as its importance in implantation. Conclusion: ADSC-CM inhibited endometriosis development and improved pregnancy outcomes in mice. Potential translation to clinical treatment for human endometriosis is expected.

Combinatory effects of current regimens and Guizhi Fuling Wan on the development of endometriosis.

OBJECTIVE: Endometriosis, defined as the growth of endometrial glands and stromal cells in a heterotopic location under the cyclic influence of ovarian hormones, is a common gynecological disorder manifested by chronic pelvic pain and infertility. In traditional Chinese medicine, endometriosis is characterized by stagnation of vital energy (qi) and blood stasis. Guizhi Fuling Wan (GFW) was first described in Chinese canonical medicine to treat disorders associated with stagnation of qi and blood stasis, including endometriosis. Therefore, the current study aimed to test the effects of combining GFW with western medicine on the suppression of endometriosis. MATERIALS AND METHODS: Endometriosis was generated by suturing endometrial tissue on the peritoneal wall of C57BL/6JNarl mice. The mice were subsequently treated with either GFW or current hormonal therapies or in combination for 28 days. RESULTS: Endometriosis development was inhibited by GFW, Gestrinone, Visanne, GFW + Gestrinone or GFW + medroxyprogesterone acetate (MPA). The expression of intercellular adhesion molecule 1 (ICAM-1) was inhibited by GFW, Gestrinone, MPA, Visanne, GFW + Gestrinone, GFW + MPA and GFW + Visanne. Vascular endothelial growth factor (VEGF) expression was inhibited by GFW, Gestrinone, Visanne, GFW + Gestrinone and GFW + MPA. Both ICAM-1- and VEGF-reducing effects of GFW were attenuated by western medicines. Administration of GFW, MPA, Visanne, GFW + MPA and GFW + Visanne also correspondingly reduced macrophage population in peritoneal fluid. GFW, MPA, Visanne, GFW + MPA and GFW + Visanne enhanced B-cell population in peritoneal fluid. CONCLUSION: The current study reveals the therapeutic effects of GFW on endometriosis. However, the combination of GFW and current hormonal therapies potentially impedes the efficacy of each individual agent in treating endometriosis.

Transplantation of 3D adipose-derived stem cell/hepatocyte spheroids alleviates chronic hepatic damage in a rat model of thioacetamide-induced liver cirrhosis.

Cirrhosis refers to irreversible liver damage where healthy tissue is replaced by scar tissue, resulting in impaired liver function. There is no cure and current treatments only prevent further liver damage; thus, novel therapeutic options are urgently needed. Here, we report a new approach that enables the formation of self-assembled 3D spheroids of adipose-derived stem cells (ADSCs) and murine hepatocytes (AML12) via reconstituted collagen fibers. Compared with the spheroids formed in the commercially available EZSHERE dish, the collagen fiber-based ADSC/hepatocyte spheroids offer a notable benefit in structure formation and paracrine factor secretion. To test the regenerative capability of the collagen fiber-based 3D ADSC/hepatocyte spheroids, a rat model of thioacetamide (TAA)-induced liver cirrhosis was employed. The transplantation of the collagen fiber-based 3D ADSC/hepatocyte spheroids show an improvement in liver function and ameliorates pathological liver cirrhosis in TAA-treated rats. In summary, our data show collagen fiber-based self-assembled 3D ADSC/hepatocyte spheroids to possess the excellent regenerative capacity in response to TAA-induced liver injury, promising an alternative therapeutic strategy for liver cirrhosis.

TGF-ß1 Neutralization Improves Pregnancy Outcomes by Restoring Endometrial Receptivity in Mice with Adenomyosis.

The objective of this research is to study the effects of TGF-ß1 inhibition on endometrial receptivity and pregnancy outcomes in mice with adenomyosis. Experiments were done using a mouse model of adenomyosis which took place in a hospital-affiliated laboratory. The mouse model used for this research is ICR mouse. Adenomyosis was induced by oral gavage of tamoxifen (TAM) from postnatal days (PNDs) 1 to 4 in ICR mice. Bilateral intrauterine injection of anti-TGF-ß1-neutralizing antibody or isotype IgG or PBS was performed at PND42. The mice were then either sacrificed or mated at PND64 followed by sacrificing at gestational day (GD) 4 or proceeding to delivery. Implantation numbers, rate of dams with live birth, live birth numbers, survival at 1 week old, and pup mortality rate after weaning were recorded. Collagen was demonstrated by Masson's trichrome and Van Gieson's stains. Uterine expression of a receptivity marker, leukemia inhibitory factor (LIF), was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemistry (IHC). Anti-TGF-ß1 treatment increased the mean implantation numbers, fecundity rate, the rate of dams with live birth, pup survival rate at 1 week old, and pup mortality rate after weaning. Collagen expression in uteri with adenomyosis was attenuated by anti-TGF-ß1 treatment. Increased LIF expression by anti-TGF-ß1 treatment was detected by qRT-PCR, Western blot, and IHC. The results suggest that inhibition of TGF-ß1 improves pregnancy outcomes by restoring endometrial receptivity in mice with adenomyosis.

Reparative and toxicity-reducing effects of liposome-encapsulated saikosaponin in mice with liver fibrosis.

Saikosaponin d (SSd), a primary active component of the Chinese herb Bupleurum falcatum, has antitumor and antiliver fibrosis effects. However, the toxicity of SSd at high doses can induce conditions such as metabolic disorders and hemolysis in vivo, thus hampering its clinical use. The present study investigated the toxicity-reducing effects of liposome encapsulation of pure SSd and the therapeutic action of SSd-loaded liposomes (Lipo-SSd) in liver fibrosis in vitro and in vivo. Lipo-SSd (diameter, 31.7 ± 7.8 nm) was prepared at an entrapment efficiency of 94.1%. After 10-day incubation, a slow release profile of 56% SSd from Lipo-SSd was observed. The IC50 of SSd on hepatic stellate cells was approximately 2.9 µM. Lipo-SSd exhibited much lower cytotoxicity than did pure SSd. In the in vivo toxicity assay, Lipo-SSd significantly increased mice survival rate and duration compared with pure SSd at the same dose. These in vitro and in vivo data indicate that liposomal encapsulation can reduce the cytotoxicity of SSd. The histopathological analysis results demonstrated that in mice with thioacetamide-induced liver fibrosis, Lipo-SSd exerted more obvious fibrosis- and inflammation-alleviating and liver tissue-reparative effects than did pure SSd; these effects are potentially attributable to the sustained release of SSd. In conclusion, Lipo-SSd fabricated here have antiliver fibrosis effects and lower toxicity compared with that of pure SSd.

Effects of Hericium erinaceus Mycelium Extracts on the Functional Activity of Purinoceptors and Neuropathic Pain in Mice with L5 Spinal Nerve Ligation.

Neuropathic pain is a serious clinical problem that is difficult to treat. Purinoceptors (P2Rs) transduce pain perception from the peripheral to the central nervous system and play an important role in the transmission of neuropathic pain signals. We previously found that the crude extracts of Hericium erinaceus mycelium (HE-CE) inhibited P2R-mediated signaling in cells and reduced heat-induced pain in mice. The present study explored the effects of HE-CE on neuropathic pain. We used adenosine triphosphate (ATP) as a P2R agonist to generate Ca2+ signaling and neuronal damage in a cell line. We also established a neuropathic mouse model of L5 spinal nerve ligation (L5-SNL) to examine neuropathic pain and neuroinflammation. Neuropathic pain was recorded using the von Frey test. Neuroinflammation was evaluated based on immunohistofluorescence observation of glial fibrillary acidic protein (GFAP) levels in astrocytes, ionized calcium-binding adaptor molecule1 (iba1) levels in microglia, and IL-6 levels in plasma. The results show that HE-CE and erinacine-S, but not erinacine-A, totally counteracted Ca2+ signaling and cytotoxic effects upon P2R stimulation by ATP in human osteosarcoma HOS cells and human neuroblastoma SH-SY5Y cells, respectively. SNL induced a decrease in the withdrawal pressure of the ipsilateral hind paw, indicating neuropathic pain. It also raised the GFAP level in astrocytes, the iba1 level in microglia, and the IL-6 level in plasma, indicating neuroinflammation. HE-CE significantly counteracted the SNL-induced decrease in withdrawal pressure, illustrating that it could relieve neuropathic pain. It also reduced SNL-induced increases in astrocyte GFAP levels, microglial iba1 levels, and plasma IL-6 levels, suggesting that HE-CE reduces neuroinflammation. Erinacine-S relieved neuropathic pain better than HE-CE. The present study demonstrated that HE inhibits P2R and, thus, that it can relieve neuropathic pain and neuroinflammation.

The Effects of Anti-TGF-ß1 on Epithelial-Mesenchymal Transition in the Pathogenesis of Adenomyosis.

Adenomyosis is defined as the presence of endometrial glands and stroma in the myometrium. The mechanisms associated with the pathogenesis of adenomyosis remain unclear. Epithelial-mesenchymal transition (EMT) is characterized by losing cell polarity and cell-cell adhesion together with gaining migratory and invasive properties of stromal cells to become mesenchymal stem cells. Transforming growth factor-ß1 (TGF-ß1), an anti-inflammatory cytokine secreted by multiple cell types, plays a crucial role in embryogenesis and tissue homeostasis. The induction of EMT and ultimate fibrosis by TGF-ß1 is suggested to play a critical role in the pathogenesis of adenomyosis. Thus, this study aims to demonstrate the occurrence of EMT in and the effects of anti-TGF-ß1 on the pathogenesis of adenomyosis. ICR mice were fed with 1 µg/g body weight of tamoxifen (TAM) by in the first 4 postnatal days (PNDs). Subsequently, the right and left uterine horns were correspondingly injected with or without 10 µg of anti-TGF-ß1 neutralizing antibody on PND42 followed by sacrifice on PND64. E-cadherin, vimentin, and α-smooth muscle actin (α-SMA) expression in the uteri was evaluated by qRT-PCR, Western blot, and immunohistochemistry. Clusters of endometrial glands and increased numbers of vimentin-positive stromal cells in the disrupted α-SMA-positive myometrium were observed in the uteri from TAM-treated mice. Numbers of stromal cells in the myometrium and the disrupted myometrial continuity were reduced by anti-TGF-ß1. Moreover, uterine expression of E-cadherin and vimentin/α-SMA was increased and decreased by anti-TGF-ß1 treatment, respectively. Anti-TGF-ß1 successfully inhibits EMT and the development of adenomyosis in mouse uteri.

Carnosic acid impedes cell growth and enhances anticancer effects of carmustine and lomustine in melanoma.

Carnosic acid (CA), a major polyphenolic diterpene present in Rosmarinus officinalis, has been reported to have multiple functions, including antitumor activity. The MTT assay, BrdU incorporation, wound healing, and colony formation were used to detect melanoma B16F10 cell growth and proliferation. Flow cytometry was used for cell cycle detection. p21 and p27 expression was detected by Western blotting. B16F10 cell xenograft model was established, and treated with CA, carmustine (BCNU), or lomustine (CCNU). The present study found that CA exhibits significant growth inhibition and cell cycle arrest in melanoma B16F10 cells. We also found that CA triggers cell cycle arrest at G0/G1 phase, and enhances p21 expression. Additionally, CA can enhance BCNU- and CCNU-mediated cytotoxicity and cell cycle arrest in B16F10 cells. Finally, we found that CA inhibits tumor growth, and reduces the values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in vivo The present study study concluded that CA may be safe and useful as a novel chemotherapeutic agent.

Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes), Modulates Purinoceptor-Coupled Calcium Signaling and Murine Nociceptive Behavior.

Hericium erinaceus is well known for the neurotrophic effect it confers by promoting nerve growth factor biosynthesis. We discovered a novel bioactivity of H. erinaceus in its ability to suppress adenosine triphosphate (ATP)-induced calcium signaling in neuronal PC12 cells. ATP, known primarily as a neurotransmitter, also acts on purinoceptors (P2 purinergic receptor [P2R]) to generate the cellular calcium signaling and secretion that mediate P2R physiological manifestations, including pain. Chronic pain reduces quality of life. However, constant analgesic administration can cause liver and kidney injury, as well as loss of the analgesic effect because of desensitization. In this study we investigated the analgesic potential of H. erinaceus through measurements of ATP-induced Ca2+ signaling in cell lines and observation of pain behaviors in mice. In P2R-coupled Ca2+ signaling measurements, extracts of H. erinaceus mycelia (HEEs) blocked ATP-induced Ca2+ signaling in both rat PC12 cells and human HOS cells. HEEs completely blocked ATP-induced Ca2+ signaling in human HOS cells, suggesting that this effect of HEEs is exerted through the P2R subtypes present in HOS cells, which include the P2X4, P2X7, P2Y2, and P2Y4 subtypes. In observations of animal behavior during pain, HEEs significantly reduced heat-induced pain, including postponing both the tail-flick response to heat stimulation and the paw-lifting response to a hot plate. This study demonstrates novel characteristics of H. erinaceus in reducing nociceptive behavior and blocking the functional activity of P2R. Further studies are required to verify this linkage and its molecular mechanisms.

Metformin disrupts malignant behavior of oral squamous cell carcinoma via a novel signaling involving Late SV40 factor/Aurora-A.

Conventional therapeutic processes in patient with OSCC are associated with several unfavorable effects leading to patients with poor survival rate. Metformin has been shown to protect against a variety of specific diseases, including cancer. However, the precise roles and mechanisms underlying the therapeutic effects of metformin on OSCC remain elusive. In the current study, in vitro and xenograft model experiments revealed that metformin inhibited growth and metastasis of oral cancer cells. Importantly, metformin-restrained tumorigenesis of oral cancer was accompanied with strong decrease of both Aurora-A and Late SV40 Factor (LSF) expressions. Furthermore, LSF contributed to Aurora-A-elicited malignancy behaviors of oral cancer via binding to the promoter region of Aurora-A. A significant correlation was observed between LSF and Aurora-A levels in a cohort of specimens of oral cancer. These findings showed that a novel LSF/Aurora-A-signaling inhibition supports the rationale of using metformin as potential OSCC therapeutics.

Saikosaponin d induces cell death through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian hepatic stellate cells.

BACKGROUND: Saikosaponin d (SSd) is one of the main active triterpene saponins in Bupleurum falcatum. It has a steroid-like structure, and is reported to have pharmacological activities, including liver protection in rat, cell cycle arrest and apoptosis induction in several cancer cell lines. However, the biological functions and molecular mechanisms of mammalian cells under SSd treatment are still unclear. METHODS: The cytotoxicity and apoptosis of hepatic stellate cells (HSCs) upon SSd treatment were discovered by MTT assay, colony formation assay and flow cytometry. The collage I/III, caspase activity and apoptotic related genes were examined by quantitative PCR, Western blotting, immunofluorescence and ELISA. The mitochondrial functions were monitored by flow cytometry, MitoTracker staining, ATP production and XF24 bioenergetic assay. RESULTS: This study found that SSd triggers cell death via an apoptosis path. An example of this path might be typical apoptotic morphology, increased sub-G1 phase cell population, inhibition of cell proliferation and activation of caspase-3 and caspase-9. However, the apoptotic effects induced by SSd are partially blocked by the caspase-3 inhibitor, Z-DEVD-FMK, suggesting that SSd may trigger both HSC-T6 and LX-2 cell apoptosis through caspase-3-dependent and independent pathways. We also found that SSd can trigger BAX and BAK translocation from the cytosol to the mitochondria, resulting in mitochondrial function inhibition, membrane potential disruption. Finally, SSd also increases the release of apoptotic factors. CONCLUSIONS: The overall analytical data indicate that SSd-elicited cell death may occur through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian HSCs, and thus can delay the formation of liver fibrosis by reducing the level of HSCs.

Discordance of Mutation Statuses of Epidermal Growth Factor Receptor and K-ras between Primary Adenocarcinoma of Lung and Brain Metastasis.

Mutations on epidermal growth factor receptor (EGFR) of adenocarcinomas of lung have been found to be associated with increased sensitivity to EGFR tyrosine kinase inhibitors and K-ras mutations may correlate with primary resistance. We aimed to explore the discordant mutation statuses of EGFR and K-ras between primary tumors and matched brain metastases in adenocarcinomas of lung. We used a sensitive Scorpion ARMS method to analyze EGFR mutation, and Sanger sequencing followed by allele-specific real-time polymerase chain reaction to analyze K-ras mutation. Forty-nine paired tissues with both primary adenocarcinoma of lung and matched brain metastasis were collected. Thirteen patients (26.5%) were discordant for the status of EGFR between primary and metastatic sites. K-ras gene could be checked in paired specimens from 33 patients, thirteen patients (39.6%) were discordant for the status of K-ras. In primary lung adenocarcinoma, there were 14 patients of mutant EGFR had mutant K-ras synchronously. This study revealed that the status of EGFR mutation in lung adenocarcinomas is relatively consistent between primary and metastatic sites compared to K-ras mutation. However, there are still a few cases of adenocarcinoma of lung showing discordance for the status of EGFR mutation. Repeated analysis of EGFR mutation is highly recommended if tissue from metastatic or recurrent site is available for the evaluation of target therapy.

Suppression of Aurora-A-FLJ10540 signaling axis prohibits the malignant state of head and neck cancer.

BACKGROUND: Head and neck cancer (HNC) is a highly invasive cancer. Aurora-A has been reported for a number of malignancies. However, the identity of downstream effectors responsible for its aggressive phenotype in HNC remains underinvestigated. METHODS: The mRNA and protein expression levels of Aurora-A and FLJ10540 were assessed in HNC specimens and cell lines using RT-qPCR, western blot, Oncomine, and microarray database analysis. The downstream molecular mechanisms of Aurora-A were confirmed by RT-qPCR, western blot, luciferase reporter, confocal microscopy analyses, immunoprecipitation, colony formation, cell viability, and xenograft model. Cellular functions in response to Aurora-A-modulated downstream targets such as FLJ10540 and MMPs were examined in vitro and in vivo, including cell growth, motility and chemosensitivity. Aurora-A/FLJ10540/MMPs expression was determined in cancer and adjacent normal tissues from HNC patients by immunohistochemistry approach. RESULTS: In the current study, Aurora-A exhibited similar gene expression profiles with FLJ10540 by using accessibly public microarray and Oncomine database analysis, raising the possibility that these molecules might coordinately participate in cancer progression and metastasis of HNC. These two molecules connection were also examined in cell lines and tissues of HNC. Aurora-A overexpression could not only bind to the promoter of FLJ10540 to induce FLJ10540 expression, but also increase both mRNA and protein levels of MMP-7 and MMP-10 in HNC cells. Conversely, depletion of Aurora-A expression by using siRNA or Aurora-A kinase inhibitor, MLN8237, suppressed FLJ10540, MMP-7 and MMP-10 mRNA and protein expressions in vitro and in vivo. In addition, the FLJ10540-PI3K complex was destroyed by inhibition the Aurora-A kinase activity. Forced overexpression of FLJ10540 in Aurora-A-depleted or in MLN8237-treated HNC cells attenuated the effect on cytotoxicity to cisplatin. Elevated Aurora-A expression in HNC cells led to the characteristics of more aggressive malignancy, including enhanced chemoresistance and increased the abilities of proliferation, migration and invasion, which was required for FLJ10540/MMP-7 or FLJ10540/MMP-10 expressions. Finally, immunohistochemical analysis of human HNC specimens showed a significant positively correlation among Aurora-A, FLJ10540, MMP-7 and MMP-10 expressions. CONCLUSION: Together, our findings define a novel mechanism by which Aurora-A promotes cell malignancy, with potential implications for understanding the clinical action of Aurora-A.

Aurora-A signaling is activated in advanced stage of squamous cell carcinoma of head and neck cancer and requires osteopontin to stimulate invasive behavior.

The clinical significances, cellular effects, and molecular mechanisms by which Aurora-A mediate its invasive effects in HNSCC are still unclear. Here, we found that Aurora-A expression is significantly higher in tumor tissues on 14-microarray of HNSCC in Oncomine-databases. The activity of Aurora-A was not only found in HNSCC specimens, but also significantly correlated with advanced-T-classification, positive-N-classification, TNM-stage and the poor 5-year survival rate. HNSCC-microarray profile showed that osteopontin and Aurora-A exhibited positive correlation. Stimulation of HNC cells with osteopontin results in an increase in Aurora-A expression where localized at the centrosome. Functionally, Aurora-A had the abilities to stimulate cell motility in HNC cells through increase ERK1/2 activity under osteopontin stimulation. Conversely, depletion of Aurora-A expression by siRNAs suppressed ERK1/2 activity as well as inhibition of cell invasiveness. Treatment with anti-CD44 antibodies in HNC cells not only caused a decrease of mRNA/protein of Aurora-A and ERK1/2 activity upon osteopontin stimulation, but also affected the abilities of Aurora-A-elicited cell motility. Finally, immunohistochemical/Western-blotting analysis of human aggressive HNSCC specimens showed a significant positively correlation between osteopontin-Aurora-A and ERK1/2. These findings suggest that Aurora-A is not only an important prognostic factor but also a new therapeutic target in the osteopontin/CD44/ERK pathway for HNSCC treatment.

Saikosaponin a and saikosaponin d inhibit proliferation and migratory activity of rat HSC-T6 cells.

The proliferation and migration of hepatic stellate cells (HSCs) profoundly impact the pathogenesis of liver inflammation and fibrogenesis. As a perennial herb native to China, Bupleurum falcatum is administered for its anti-inflammatory, antipyretic, and antihepatotoxic effects. Saikosaponin a (SSa) and Saikosaponin d (SSd) are the major active components of triterpene saponins in Bupleurum falcatum. This study analyzes how SSa and SSd affect rat HSC-T6 cell line proliferation and migration. Experimental results indicate that, in addition to suppressing HSC-T6 proliferation, wound healing activity and cell migration in a time- and dose-dependent manner, SSa and SSd significantly induce apoptosis. Additionally, SSa and SSd decreased the expressions of extracellular matrix-regulated kinase 1/2 (ERK1/2), platelet-derived growth factor receptor 1 (PDGFR1), and subsequently transforming growth factor-ß1 receptor (TGF-ß1R), α-smooth muscle actin, TGF-ß1 and connective tissue growth factor. They also decreased phosphorylation of p38 (p-p38) and ERK1/2 (p-ERK1/2) of HSC-T6. Furthermore, both SSa and SSd can block PDGF-BB and TGF-ß1-induced cell proliferation and migration of HSC-T6. These results suggest that SSa and SSd may inhibit proliferation and activation of HSC-T6, and the modulated mechanisms warrant further study.

Oncogenic fibulin-5 promotes nasopharyngeal carcinoma cell metastasis through the FLJ10540/AKT pathway and correlates with poor prognosis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential and locoregional recurrence, although the molecular alterations that are driving NPC metastasis remain unclear at this time. This study aimed to examine the expression of fibulin-5 in NPC, correlate the results with clinicopathological variables and survival, and to investigate the role of fibulin-5 in human NPC cell lines. MATERIAL AND METHODS: Standard semi-quantitative-RT-PCR, quantitative-RT-PCR, immunoblotting, and immunohistochemistry were used to investigate the mRNA and protein expression profiles of fibulin-5 in normal and NPC tissues. Immunohistochemistry of fibulin-5 was correlated with clinicopathological characteristics by univariate analyses. NPC cells overexpressing fibulin-5 or fibulin-5-siRNA cells were generated by stable transfection to characterize the molecular mechanisms of fibulin-5-elicited cell growth and metastasis. RESULTS: Our results demonstrated that fibulin-5 overexpression in NPC specimens and significantly correlated with advanced tumor metastasis indicating a poor 5-year overall survival. Fibulin-5 was mainly expressed in the nucleus in human NPC specimens and cell lines. Functionally, fibulin-5 overexpression yielded fast growth in NPC cells. In addition, fibulin-5 promotes cell metastasis in NPC cells through increased FLJ10540 and phosphor-AKT activity. In contrast, siRNA depletion of fibulin-5 suppressed FLJ10540 expression and phosphor-AKT activity. Suppression of either fibulin-5 or FLJ10540 can cause significant inhibition with regards to cell motility in NPC cells. Finally, immunohistochemical analysis of human aggressive NPC specimens showed a significant and positive correlation between fibulin-5 and FLJ10540 expression. CONCLUSION: Higher fibulin-5 expression is not only an important indicator of poor survival, but also contributes to the development of new therapeutic strategies in the FLJ10540/AKT pathway for NPC treatment.

Overexpression of Rap-1A indicates a poor prognosis for oral cavity squamous cell carcinoma and promotes tumor cell invasion via Aurora-A modulation.

The functions of Rap-1A in oral carcinogenesis are largely unexplored. In this study, we examined the expression of Rap-1A at different malignant stages of oral cavity squamous cell carcinoma (OCSCC). Semiquantitative RT-PCR, quantitative RT-PCR, and Western blotting were used to evaluate Rap-1A mRNA and protein expressions, respectively, in paired OCSCC patient specimens. To determine the possible correlation between Rap-1A expression and various clinical characteristics, 256 samples from patients with OCSCC were evaluated by immunohistochemical staining. Strong Rap-1A expression was a significant prognostic marker and predictor of aggressive OCSCC. The overall and disease-specific 5-year survival rates were significantly correlated with strong expression of Rap-1A (P < 0.001). Functionally, overexpressed Rap-1A could promote oral cancer cell migration and invasion by Transwell chambers and wound healing assay. Conversely, the suppression of Rap-1A expression using Rap-1A-mediated siRNA was sufficient to decrease cell motility. Furthermore, our data also illustrated that Aurora-A could not only induce mRNA and protein expressions of Rap-1A for enhancing cancer cell motility but also co-localize and form a complex with Rap-1A in the oral cancer cell line. Finally, immunohistochemical staining, indirect immunofluorescence, and Western blotting analysis of human aggressive OCSCC specimens revealed a significantly positive correlation between Rap-1A and Aurora-A expression. Taken together, our results suggest that the Aurora-A/Rap-1A pathway is associated with survival, tumor progression, and metastasis of OCSCC patients.

FLJ10540 is associated with tumor progression in nasopharyngeal carcinomas and contributes to nasopharyngeal cell proliferation, and metastasis via osteopontin/CD44 pathway.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is well-known for its highly metastatic characteristics, but little is known of its molecular mechanisms. New biomarkers that predict clinical outcome, in particular the ability of the primary tumor to develop metastatic tumors are urgently needed. The aim of this study is to investigate the role of FLJ10540 in human NPC development. METHODS: A bioinformatics approach was used to explore the potentially important regulatory genes involved in the growth/metastasis control of NPC. FLJ10540 was chosen for this study. Two co-expression strategies from NPC microarray were employed to identify the relationship between FLJ10540 and osteopontin. Quantitative-RT-PCR, immunoblotting, and immunohistochemistry analysis were used to investigate the mRNA and protein expression profiles of FLJ10540 and osteopontin in the normal and NPC tissues to confirm microarray results. TW01 and Hone1 NPC cells with overexpression FLJ10540 or siRNA to repress endogenous FLJ10540 were generated by stable transfection to further elucidate the molecular mechanisms of FLJ10540-elicited cell growth and metastasis under osteopontin stimulation. RESULTS: We found that osteopontin expression exhibited a positive correlation with FLJ10540 in NPC microarray. We also demonstrated comprehensively that FLJ10540 and osteopontin were not only overexpressed in NPC specimens, but also significantly correlated with advanced tumor and lymph node-metastasis stages, and had a poor 5-year survival rate, respectively. Stimulation of NPC parental cells with osteopontin results in an increase in FLJ10540 mRNA and protein expressions. Functionally, FLJ10540 transfectant alone, or stimulated with osteopontin, exhibited fast growth and increased metastasis as compared to vehicle control with or without osteopontin stimulation. Conversely, knockdown of FLJ10540 by siRNA results in the suppression of NPC cell growth and motility. Treatment with anti-CD44 antibodies in NPC parental cells not only resulted in a decrease of FLJ10540 protein, but also affected the abilities of FLJ10540-elicited cell growth and motility in osteopontin stimulated-NPC cells. CONCLUSIONS: These findings suggest that FLJ10540 may be critical regulator of disease progression in NPC, and the underlying mechanism may involve in the osteopontin/CD44 pathway.

Solanum incanum extract (SR-T100) induces human cutaneous squamous cell carcinoma apoptosis through modulating tumor necrosis factor receptor signaling pathway.

BACKGROUND: The Solanum species herbs have been used to treat cancer for centuries; however, the underlying mechanisms and effectiveness in vivo remain unclear. OBJECTIVES: SR-T100, extracted from the Solanum incanum, contains solamargine alkaloid as the main active ingredient. Here, we investigated the apoptosis-inducing effects of SR-T100 for targeting squamous cell carcinoma (SCC) in vitro and in vivo. METHODS: We elucidated the mechanism by which SR-T100 induces apoptosis of human SCCs (A431, SCC4, SCC9, and SCC25) cells. The efficacy and safety issues were addressed regarding topical treatment of SR-T100 on UVB-induced cutaneous SCC of hairless mice and actinic keratoses (AKs) of human. RESULTS: SR-T100 induces apoptosis in human SCCs cell lines by up-regulating the expressions of tumor necrosis factor receptors (TNFRs) and Fas, and downstream adaptors FADD/TRADD of the TNF-α and Fas ligand signaling cascades. SR-T100 also triggered the mitochondrial apoptotic pathway, as up-regulated cytochrome c and Bax, down-regulated Bcl-X(L). Animal experiments showed that all papillomas (35/35) and 27 of 30 UVB-induced microinvasive SCCs in hairless mice disappeared within 10 weeks after once-daily application of topical SR-T100. Furthermore, 13 patients, who suffered with 14 AKs, were treated with once-daily topical SR-T100 gel and 10 AKs cured after 16 weeks, showing negligible discomforts. CONCLUSION: Our studies indicate that SR-T100 induces apoptosis of SCC cells via death receptors and the mitochondrial death pathway. The high efficacy of SR-T100 in our preclinical trial suggests that SR-T100 is a highly promising herb for AKs and related disorders.