Genipin exhibits anti-invasive effect in orthotopically HCC-implanted mice. A

<p>shows Genipin has no toxic effect on the orthotopically HCC implanted model. MHCC97L cells were subcutaneously injected into the right flank of the nude mice to allow xenografted tumor growth. Once the solid tumor reaches about 1 cm in diameter, it was dissected out and cut into 1–2 mm small cubes, which was then implanted to the left liver lobe of the nude mice to allow orthotopical growth of HCC. Seven days later, the nude mice was randomized into two groups (n = 5). Treatment was conducted seven days after implantation (genipin 30 mg/kg/2 days, i.p.) and lasted for 3 weeks, while control group received same volume of PBS. Body weight of the mice was examined once per two days. No significant loss in body weight during treatment could be observed, indicating genipin may not exhibit potent toxicity to the mice. <b>B</b> shows that genipin treatment could reduce the tumor size in HCC-implanted mice. <b>C</b> shows that genipin suppresses tumor cell invasion to the normal tissue in orthotopically HCC implanted model. The liver of mice was dissected out and histological analysis was conducted with H & E staining. Significantly reduction of intrahepatic invasion of MHCC97L could be observed in genipin-treated mice.</p

Activation of p38 MAPK signaling by genipin confers the overexpression of TIMP-1 in HCC cells. A

<p>shows that genipin activates p38 MAPK signaling in dose- and time- dependent manner. Cells were treated with genipin for 12 for 24H then protein was collected. The activation of p38 MAPK was analyzed by immunoblotting. <b>B</b> shows that inhibition of p38 MAPK repressed mRNA transcripts up-regulated by genipin treatment. Cells were treated with genipin for 12H in the presence or absence with SB202190 (20 µM) then total RNA was collected. The TIMP-1 mRNA transcripts were analyzed by qRT-PCR. *p<0.05, **p<0.01 when compared with control; <b>C</b> shows that inhibition of p38 MAPK suppressed the up-regulation of TIMP-1 protein expression induced by genipin. Cells were treated with genipin for 24H in the presence or absence with SB202190 (20 µM) then protein was collected for immunoblotting analysis. <b>D</b> shows that inhibition of p38 MAPK reduced TIMP-1 secretion. Cells were treated with genipin for 12, 24 or 48H in the presence or absence with SB202190 (20 µM) then medium was collected and TIMP-1 secretion was analyzed by ELISA assay. *p<0.05, **p<0.01 when compared with control.</p

Primers used for real-time quantitative PCR.

<p>Primers used for real-time quantitative PCR.</p

Upregulation of TIMP-1 expression is responsible for the MMP-2 inhibition by genipin in HCC cells.

<p><b>A</b> shows that inhibition of TIMP-1 expression by RNA interference abolishes inhibition of MMP-2 activity by genipin. Cells were transfected with either scr siRNA or siRNA against TIMP-1 followed by genipin treated for 24H. The suppression of TIMP-1 by siRNA was verified as Fig. 3C shows. Medium was then collected for gelatin zymography analysis. Intensity of the band was quantified. *p<0.05, **p<0.01 when compared with control; <b>B</b> shows that inhibition of TIMP-1 attenuates genipin-suppressed HCC cell motility. Distance between the gap was quantified. *p<0.05, **p<0.01 when compared with control; <b>C</b> shows that inhibition of TIMP-1 expression attenuates HCC cell invasiveness suppressed by genipin. The invaded cell number was quantified. *p<0.05, **p<0.01 when compared with control.</p

Genipin suppresses hepatocellular carcinoma cell migration and invasion at non-toxic doses.

<p><b>A</b> shows that genipin exhibits no potent cytotoxicity to human heaptocellular carcinoma cells HepG2 and MHCC97L. Cells were seeded in 96-well culture plate and treated with genipin for 24 h or 48 h. Cell viability was determined by MTT assay. <b>B</b> shows that genipin remarkably reduce HepG2 and MHCC97L cell motility in dose-dependent manner. Cells were seeded in 12-well culture plate with 100% confluence. Wound healing assay was introduced to determine the cell motility in the presence of genipin. The distance between the gap was quantified and the results were shown. *p<0.05, **p<0.01 when compared with control; <b>C</b> shows that genipin could inhibit cell invasion through extracellular matrix. Cells were seeded in the chamber of transwells coated with Matrigel matrix. The invasiveness of cells was determined by counted cells passing through Matrigel matrix to the basal side of transwells. *p<0.05, **p<0.01 when compare with vehicle group.</p

Overall scheme of the regulatory network involved in the inhibitory effect of genipin on HCC invasion.

<p>Overall scheme of the regulatory network involved in the inhibitory effect of genipin on HCC invasion.</p

Genipin treatment up-regulates the expression of TIMP-1 in HCC cells.

<p><b>A</b> shows the quantitative real-time PCR screening of endogenous MMP-2/9 inhibitors. Cells were treated with genipin for 24H and total RNA was collected for anlaysis. The result shows that genipin particularly up-regulate TIMP-1 mRNA transcription but has no effect on TIMP-2, -3, -4 and RECK mRNA transcript in HepG2 and MHCC97L cells. <b>B</b> shows that genipin exposure is responsible for the increased secretion of TIMP-1 in HepG2 and MHCC97L cells. The cells were transfected with scr negative control siRNA or siRNA against TIMP-1. Then cells were treated with genipin for 24H then medium was collected for ELISA determination of TIMP-1. *p<0.05,p<0.01 when compared with control; <b>C</b> shows that genipin up-regulates expression of TIMP-1. The cells were transfected with scr negative control siRNA or siRNA against TIMP-1. Then cells were treated with genipin for 24H and cell lysates were analyzed with immunoblotting.</p

CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

<div><p>Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by <em>in vitro</em> and <em>in vivo</em> assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. <em>FOXN4</em>, <em>GLI1</em>), immune response (<em>CCR7</em>, <em>IL8</em>) and transmembrane transport (e.g. <em>ABCC3</em>, <em>ABCC11</em>) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 <em>in vitro</em>. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients.</p> </div

Immunohistochemical analysis of CCR7 and CD44 expression in primary NPC.

<p>Representative primary NPC cases with high (A), medium (B), low (C) expression of CCR7. (D) Primary NPC with absence of CCR7 expression was shown. CCR7 staining were detected in few infiltrating lymphocytes, but not in the tumor cells. Primary tumors with high (E) and medium (F) CD44 expression were shown. In (G) and (H), weak CD44 expression was detected in the tumor cells while strong CD44 staining in infiltrating lymphocytes was commonly found.</p

In vivo tumorigenic capacity of sphere-forming cells and unselected parental cells of C666-1 in nude mice.

<p>N/A – data not available.</p