Correction: Systemic Delivery of MicroRNA-101 Potently Inhibits Hepatocellular Carcinoma In Vivo by Repressing Multiple Targets.

[This corrects the article DOI: 10.1371/journal.pgen.1004873.].

CD133+ liver cancer stem cells resist interferon-gamma-induced autophagy.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most fatal malignancies worldwide, and CD133 is a popular cancer stem cell (CSC) marker for HCC. CD133(+) CSCs have been reported to resist conventional chemo- and radiotherapy, but little is known about their response to immune surveillance. Interferon-gamma (IFN-γ) is one of key cytokines that the immune system produce to eradicate cancer cells, so we investigated the function of IFN-γ on CD133+ HCC CSCs in this study. METHODS: The response of CD133(+) cells to IFN-γ was performed with functional assays (cell proliferation assay and tumor formation in nude mice), flow cytometry, immunofluorescence staining and RNA interference. RESULTS: We found that IFN-γ inhibited the proliferation of cell lines with low percentage of CD133(+) cells (wild-type human cells, BEL7402, QGY7701) but it did not affect the proliferation of cell lines with high percentage of CD133(+) cells (wild-type human cells, Huh7, PLC8024) in vivo and in vitro (nude mice). Flow cytometry analysis demonstrated that the percentage of CD133+ cells increased after IFN-γ treatment of low CD133(+) cell lines. Furthermore, IFN-γ induced the autophagy of low CD133(+) cell lines to decrease proliferation. CONCLUSION: CD133(+) HCC CSCs resisted IFN-γ-induced autophagy, which might also be a mechanism through which CSCs resist immune eradication.

MiR-449a suppresses the epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma by multiple targets.

BACKGROUND: Increasing evidence indicates that Epithelial-mesenchymal transition (EMT) can be regulated by microRNAs (miRNAs). MiR-449a is a liver abundant miRNA. However, the role of miR-449a in the metastasis of hepatocellular carcinoma (HCC) remains largely unknown. METHODS: The expression levels of miR-449a were first examined in HCC cell lines and tumour tissues by real-time PCR. The in vitro and in vivo functional effect and underlying molecular mechanisms of miR-449a were examined further. RESULTS: In the present study, we found that miR-449a was significantly decreased in HCC cells and tissues, especially in those with the portal vein tumor thrombus. In HCC cell lines, stable overexpression of miR-449a was sufficient to inhibit cell motility in vitro, and pulmonary metastasis in vivo. In addition, ectopic overexpression of miR-449a in HCC cells promoted the expression of epithelial markers and reduced the levels of mesenchymal markers. Further studies revealed that the reintroduction of miR-449a attenuated the downstream signaling of Met, and consequently reduced the accumulation of Snail in cell nucleus by targeting the 3'-untranslated regions (3'-UTR) of FOS and Met. CONCLUSIONS: Our data highlight an important role of miR-449a in the molecular etiology of HCC, and implicate the potential application of miR-449a in cancer therapy.

The Effect of the Modified Eighth Section of Eight-Section Brocade on Osteoporosis in Postmenopausal Women: A Prospective Randomized Trial.

Osteoporosis and related fragility fractures represent a serious and global public health problem. To evaluate whether the modified eighth section of Eight-section Brocade (MESE) exercise could improve the symptom and indexes associated with osteoporosis in postmenopausal women. Guangzhou and Liuzhou hospital of traditional Chinese medicine in China. Women (n = 198) aged 50 to 75 years were randomized into Control, Ca, MESE, and MESE + Ca. Subjects in Ca and MESE groups were separately asked to consume thrice daily Calcium Carbonate Chewable D3 tablet and to perform thrice daily MESE exercise by 7 repetitions per time for 12 months. Subjects in MESE + Ca group performed such the combined treatment project for 12 months. Body height and Hospital for Special Surgery (HSS) scores of both knees, chronic back pain visual analogue scale scores (VAS), bone mineral density (BMD) at L2 to L4 and the left femoral neck, 3-feet Up and Go Test (3') and one-leg Stance (OLS). In our study, the improvement in chronic back pain of the patients in Ca, MESE, and MESE + Ca group was better than that in control group. There was 1.9% and 1.7%, 2.3%, and 2.1% net profit in left femoral neck and lumbar BMD after the treatment for 12 months in MESE and MESE + Ca groups. For the balance capacity, the subjects in MESE and MESE + Ca groups secured much better performance than those in Ca and control group after the treatment for 12 months (P < 0.001, P < 0.001). The treatment of MESE exercise is the most effective for the improvement of the symptom and indexes in postmenopausal women. Importantly, the low attrition and the high exercise compliance indicate that MESE exercise is safe, feasible, and well tolerated by postmenopausal women.

Systemic delivery of microRNA-101 potently inhibits hepatocellular carcinoma in vivo by repressing multiple targets.

Targeted therapy based on adjustment of microRNA (miRNA)s activity takes great promise due to the ability of these small RNAs to modulate cellular behavior. However, the efficacy of miR-101 replacement therapy to hepatocellular carcinoma (HCC) remains unclear. In the current study, we first observed that plasma levels of miR-101 were significantly lower in distant metastatic HCC patients than in HCCs without distant metastasis, and down-regulation of plasma miR-101 predicted a worse disease-free survival (DFS, P<0.05). In an animal model of HCC, we demonstrated that systemic delivery of lentivirus-mediated miR-101 abrogated HCC growth in the liver, intrahepatic metastasis and distant metastasis to the lung and to the mediastinum, resulting in a dramatic suppression of HCC development and metastasis in mice without toxicity and extending life expectancy. Furthermore, enforced overexpression of miR-101 in HCC cells not only decreased EZH2, COX2 and STMN1, but also directly down-regulated a novel target ROCK2, inhibited Rho/Rac GTPase activation, and blocked HCC cells epithelial-mesenchymal transition (EMT) and angiogenesis, inducing a strong abrogation of HCC tumorigenesis and aggressiveness both in vitro and in vivo. These results provide proof-of-concept support for systemic delivery of lentivirus-mediated miR-101 as a powerful anti-HCC therapeutic modality by repressing multiple molecular targets.

Prostate tumor overexpressed 1 is a novel prognostic marker for hepatocellular carcinoma progression and overall patient survival.

The gene prostate tumor overexpressed 1 (PTOV1) was first found to be upregulated in prostate cancer. This upregulation increased tumor cell proliferation, retinoic acid resistance, and migration. This study investigated the expression and prognostic significance of PTOV1 in hepatocellular carcinoma (HCC). Real-time Polymerase Chain Reaction and western blot analysis were performed to examine PTOV1 expression in 11 HCC cell lines and 2 normal hepatic cell lines. PTOV1 expression levels were also determined in 8 pairs of tissue samples taken from primary HCC tumors and the matched adjacent noncancerous liver tissue from the same patient. Immunohistochemistry assays assessed PTOV1 protein expression in paraffin-embedded clinical samples taken from 215 HCC patients. The correlation of PTOV1 expression with the clinicopathological parameters was evaluated along with the prognostic impact of PTOV1 expression in these HCC patients. PTOV1 mRNA and protein were overexpressed in HCC cell lines compared with normal liver cell lines and were overexpressed in primary HCC samples compared with the matched noncancerous liver tissue samples. In the paraffin-embedded tissue samples from 215 HCC patients, PTOV1 protein expression was significantly correlated with T classification, N classification, clinical stage, and serum α-fetoprotein. HCC patients with higher PTOV1 expression had shorter survival times than patients with lower PTOV1 expression. Our study demonstrated that PTOV1 overexpression is correlated with increased aggressiveness of HCC and could be a prognostic biomarker for patients with HCC.

Transducin ß-like 1 X-linked receptor 1 suppresses cisplatin sensitivity in nasopharyngeal carcinoma via activation of NF-κB pathway.

BACKGROUND: Transducin ß-like 1 X-linked receptor 1 (TBL1XR1) is an important transcriptional cofactor involved in the regulation of many signaling pathways, and is associated with carcinogenesis and tumor progression. However, the precise role of TBL1XR1 in these processes is not well understood. METHODS: We detected the expression of TBL1XR1 protein and mRNA in nasopharyngeal carcinoma (NPC) cell lines and biopsies by western blotting, real-time PCR and immunohistochemical staining (IHC). Overexpression of TBL1XR1 in NPC enhanced chemoresistance to cisplatin using two NPC cell lines in vitro and in vivo. RESULTS: TBL1XR1 was upregulated in NPC cell lines and clinical samples. The expression of TBL1XR1 was correlated with several clinicopathological factors including clinical stage, T classification, N classification and patient survival. Univariate and multivariate analysis revealed that TBL1XR1 was an independent prognostic factor for patient survival. In vitro and in vivo studies demonstrated that TBL1XR1 high expression induced resistance to cisplatin-induced apoptosis in NPC cells. Furthermore, we found that TBL1XR1 activated the NF-κB pathway and promoted transcription of genes downstream of NF-κB, especially anti-apoptotic genes. CONCLUSIONS: Upregulation of TBL1XR1 induces NPC cells resistance to cisplatin by activating the NF-κB pathway, and correlates with poor overall survival of NPC patients. TBL1XR1 has a pivotal role in NPC and could be a valuable prognostic factor as well as a novel biomarker for tailoring appropriate therapeutic regimes.

Anti-tumour activity of longikaurin A (LK-A), a novel natural diterpenoid, in nasopharyngeal carcinoma.

BACKGROUND: Longikaurin A is a natural ent-kaurene diterpenoid isolated from Isodon genus. The ent-kaurene diterpenoids isolated from medicinal plants have been shown to have anti-disease effects. The present study was designed to examine the anti-tumour effects of longikaurin A (LK-A) in nasopharyngeal carcinoma in vitro and in vivo. METHODS: Apoptosis and cell cycle arrest were determined by flow cytometry analysis of the cells treated with Longikaurin A. The proteins of apoptosis signaling pathway were detected by western blotting analysis. Finally, we examined whether LK-A exhibits anti-tumour activity in xenograft models. RESULTS: Longikaurin A inhibited the cell growth by inducing apoptosis and cell cycle arrest. At low concentrations, longikaurin A induced S phase arrest and at higher concentrations, longikaurin A induced caspase-dependent apoptosis by regulating apoptotic molecules. Finally, longikaurin A significantly inhibited the tumour growth of CNE2 xenografts in vivo and showed no obvious effect on the body weights of the mice. CONCLUSION: Our results suggest that Longikaurin A exhibited anti-tumour activity in nasopharyngeal carcinoma in vitro and in vivo.

The inhibition of autophagy sensitises colon cancer cells with wild-type p53 but not mutant p53 to topotecan treatment.

BACKGROUND: Topotecan produces DNA damage that induces autophagy in cancer cells. In this study, sensitising topotecan to colon cancer cells with different P53 status via modulation of autophagy was examined. METHODOLOGY/PRINCIPAL FINDINGS: The DNA damage induced by topotecan treatment resulted in cytoprotective autophagy in colon cancer cells with wild-type p53. However, in cells with mutant p53 or p53 knockout, treatment with topotecan induced autophagy-associated cell death. In wild-type p53 colon cancer cells, topotecan treatment activated p53, upregulated the expression of sestrin 2, induced the phosphorylation of the AMPKα subunit at Thr172, and inhibited the mTORC1 pathway. Furthermore, the inhibition of autophagy enhanced the anti-tumour effect of topotecan treatment in wild-type p53 colon cancer cells but alleviated the anti-tumour effect of topotecan treatment in p53 knockout cells in vivo. CONCLUSIONS/SIGNIFICANCE: These results imply that the wild-type p53-dependent induction of cytoprotective autophagy is one of the cellular responses that determines the cellular sensitivity to the DNA-damaging drug topotecan. Therefore, our study provides a potential therapeutic strategy that utilises a combination of DNA-damaging agents and autophagy inhibitors for the treatment of colon cancer with wild-type p53.

Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2.

Stemlike cells have been isolated by their ability to efflux Hoechst 33342 dye and are called the side population (SP). We evaluated the effect of axitinib on targeting cancer stemlike cells and enhancing the efficacy of chemotherapeutical agents. We found that axitinib enhanced the cytotoxicity of topotecan and mitoxantrone in SP cells sorted from human lung cancer A549 cells and increased cell apoptosis induced by chemotherapeutical agents. Moreover, axitinib particularly inhibited the function of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) and reversed ABCG2-mediated multidrug resistance (MDR) in vitro. However, no significant reversal effect was observed in ABCB1-, ABCC1- or lung resistance-related protein (LRP)-mediated MDR. Furthermore, in both sensitive and MDR cancer cells axitinib neither altered the expression of ABCG2 at the mRNA or protein levels nor blocked the phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2. In nude mice bearing ABCG2-overexpressing S1-M1-80 xenografts, axitinib significantly enhanced the antitumor activity of topotecan without causing additional toxicity. Taken together, these data suggest that axitinib particularly targets cancer stemlike cells and reverses ABCG2-mediated drug resistance by inhibiting the transporter activity of ABCG2.

The putative tumour suppressor microRNA-124 modulates hepatocellular carcinoma cell aggressiveness by repressing ROCK2 and EZH2.

BACKGROUND: Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-124) in hepatocellular carcinoma (HCC). OBJECTIVE: To determine the status of miR-124 expression and its underlying mechanisms in the pathogenesis of HCC. METHODS: The expression levels of miR-124 were first examined in HCC cell lines and tumour tissues by real-time PCR. The in vitro and in vivo functional effect of miR-124 was examined further. A luciferase reporter assay was conducted to confirm target associations. RESULTS: The expression levels of miR-124 were frequently reduced in HCC cells and tissues, and low-level expression of miR-124 was significantly associated with a more aggressive and/or poor prognostic phenotype of patients with HCC (p<0.05). In HCC cell lines, stable overexpression of miR-124 was sufficient to inhibit cell motility and invasion in vitro, and suppress intrahepatic and pulmonary metastasis in vivo. In addition, ectopic overexpression of miR-124 in HCC cells inhibited epithelial-mesenchymal cell transition, formation of stress fibres, filopodia and lamellipodia. Further studies showed that miR-124 could directly target the 3'-untranslated region (3'-UTR) of both ROCK2 and EZH2 mRNAs, and suppress their mRNA and protein expressions. These findings suggest that miR-124 plays a critical role in regulating cytoskeletal events and epithelial-mesenchymal cell transition and, ultimately, inhibits the invasive and/or metastatic potential of HCC, probably by its direct target on ROCK2 and EZH2 genes. These results provide functional and mechanistic links between the tumour suppressor miRNA-124 and the two oncogenes ROCK2 and EZH2 on the aggressive nature of HCC. CONCLUSION: These data highlight an important role for miR-124 in the regulation of invasion and metastasis in the molecular aetiology of HCC, and suggest a potential application of miR-124 in prognosis prediction and cancer treatment.

Distribution, characterization, and induction of CD8+ regulatory T cells and IL-17-producing CD8+ T cells in nasopharyngeal carcinoma.

BACKGROUND: CD8+ effector cells often have an antitumor function in patients with cancer. However, CD8+Foxp3+ regulatory T cells (Tcregs) and interleukin (IL)-17-producing CD8+ T cells (Tc17 cells) also derive from the CD8+ T cell lineage. Their role in the antitumor response remains largely unknown. In the present study, we aimed to investigate the distribution, characterization, and generation of CD8+ Tcregs and Tc17 cells in NPC patients. METHODS: Peripheral blood and tumor biopsy tissues from 21 newly diagnosed patients with nasopharyngeal carcinoma (NPC) were collected, along with peripheral blood from 21 healthy donors. The biological characteristics of Tcregs and Tc17 cells from blood and tumor tissues were examined by intracellular staining, tetramer staining and fluorescence-activated cell sorting (FACS) analysis. The suppressive function of Tcregs was investigated using a proliferation assay that involved co-culture of sorted CD8+CD25+ T cells with naïve CD4+ T cells in vitro. RESULTS: We observed an increased prevalence of Tcregs and Tc17 cells among tumor-infiltrating lymphocytes (TILs) and different distribution among peripheral blood mononuclear cells (PBMCs) in NPC patients. Cytokine profiles showed that the Tcregs expressed a high level of IL-10 and low level of transforming growth factor ß, whereas Tc17 cells expressed a high level of tumor necrosis factor α. Interestingly, both subsets expressed a high level of interferon γ in TILs, and the Tcregs suppressed naïve CD4+ T cell proliferation by a cell contact-dependent mechanism in vitro. Moreover, we demonstrated the existence of Epstein-Barr virus latent membrane protein (LMP) 1 and LMP2 antigen-specific Tcregs in NPC. CONCLUSIONS: Our data provide new insights into the composition and function of CD8+ T-cell subsets in NPC, which may have an important influence on NPC immunotherapy.

Combination of salinomycin and gemcitabine eliminates pancreatic cancer cells.

Previous research has documented that a subpopulation of pancreatic cancer cells, named cancer stem cells (CSCs), harbor stem cell-like properties. Here, we examined the efficacy of combined treatments of salinomycin and gemcitabine in human pancreatic cancer cells. Salinomycin inhibited the growth of CSCs, while gemcitabine suppressed the viability of non-CSCs. Consistently, in vivo studies showed that salinomycin combined with gemcitabine could eliminate the engraftment of human pancreatic cancer more effectively than the individual agents. These data indicated that administration of salinomycin, which targets CSCs, may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine to eradicate pancreatic cancer.

Euphorbia factor L1 reverses ABCB1-mediated multidrug resistance involving interaction with ABCB1 independent of ABCB1 downregualtion.

Euphorbia factor L1 (EFL1) belongs to diterpenoids of genus Euphorbia. In this article, its reversal activity against ABCB1-mediated MDR in KBv200 and MCF-7/adr cells was reported. However, EFL1 did not alter the sensitivity of KB and MCF-7 cells to chemotherapeutic agents. Meanwhile, EFL1 significantly increased accumulation of doxorubicin and rhodamine 123 in KBv200 and MCF-7/adr cells, showing no significant influence on that of KB and MCF-7 cells. Furthermore, EFL1 could enhance the ATP hydrolysis activity of ABCB1 stimulated by verapamil. At the same time, EFL1 inhibited the efflux of ABCB1 in KBv200 and MCF-7/adr cells. In addition, EFL1 did not downregulate expression of ABCB1 in KBv200 and MCF-7/adr cells either in mRNA or protein level.

Prognostic significance and therapeutic implications of centromere protein F expression in human nasopharyngeal carcinoma.

BACKGROUND: Our recent cDNA microarray data showed that centromere protein F (CENP-F) is significantly upregulated in primary cultured nasopharyngeal carcinoma (NPC) tumor cells compared with normal nasopharyngeal epithelial cells. The goal of this study was to further investigate the levels of CENP-F expression in NPC cell lines and tissues to clarify the clinical significance of CENP-F expression in NPC as well as the potential therapeutic implications of CENP-F expression. METHODS: Real-time RT-PCR and western blotting were used to examine CENP-F expression levels in normal primary nasopharyngeal epithelial cells (NPEC), immortalized nasopharyngeal epithelial cells and NPC cell lines. Levels of CENP-F mRNA were determined by real-time RT-PCR in 23 freshly frozen nasopharyngeal biopsy tissues, and CENP-F protein levels were detected by immunohistochemistry in paraffin sections of 202 archival NPC tissues. Statistical analyses were applied to test for prognostic associations. The cytotoxicities of CENP-F potential target chemicals, zoledronic acid (ZOL) and FTI-277 alone, or in combination with cisplatin, in NPC cells were determined by the MTT assay. RESULTS: The levels of CENP-F mRNA and protein were higher in NPC cell lines than in normal and immortalized NPECs. CENP-F mRNA level was upregulated in nasopharyngeal carcinoma biopsy tissues compared with noncancerous tissues. By immunohistochemical analysis, CENP-F was highly expressed in 98 (48.5%) of 202 NPC tissues. Statistical analysis showed that high expression of CENP-F was positively correlated with T classification (P < 0.001), clinical stage (P < 0.001), skull-base invasion (P < 0.001) and distant metastasis (P = 0.012) inversely correlated with the overall survival time in NPC patients. Multivariate analysis showed that CENP-F expression was an independent prognostic indicator for the survival of the patient. Moreover, we found that ZOL or FTI-277 could significantly enhance the chemotherapeutic sensitivity of NPC cell lines (HONE1 and 6-10B) with high CENP-F expression to cisplatin, although ZOL or FTI-277 alone only exhibited a minor inhibitory effect to NPC cells. CONCLUSION: Our data suggest that CENP-F protein is a valuable marker of NPC progression, and CENP-F expression is associated with poor overall survival of patients. In addition, our data indicate a potential benefit of combining ZOL or FTI-277 with cisplatin in NPC suggesting that CENP-F expression may have therapeutic implications.

Characterization of a novel mechanism of genomic instability involving the SEI1/SET/NM23H1 pathway in esophageal cancers.

Amplification of 19q is a frequent genetic alteration in many solid tumors, and SEI1 is a candidate oncogene within the amplified region. Our previous study found that the oncogenic function of SEI1 was associated with chromosome instability. In this study, we report a novel mechanism of genomic instability involving the SEI1-SET-NM23H1 pathway. Overexpression of SEI1 was observed in 57 of 100 of esophageal squamous cell carcinoma cases. Functional study showed that SEI1 had strong tumorigenic ability, and overexpression of SEI1 could induce the genomic instability by increasing micronuclei formation and reducing the number of chromosomes. Further study found that SEI1 was able to upregulate SET expression and subsequently promote the translocation of a small amount of NM23H1 from the cytoplasm to the nucleus. Nuclear NM23H1 can induce DNA damage through its DNA nick activity. Unlike CTL attack, only a small amount of NM23H1 translocated into the nucleus (<10%) induced by the overexpression of SEI1. Further study found that the small amount of NM23H1 only induced minor DNA damage and subsequently increased genomic instability, rather than inducing irreparable DNA damage and initiating apoptosis by CTL attack. Sister chromatid exchange experiment found that the translocation of small amount of NM23H1 into the nucleus induced by the overexpressions of SEI1/SET could increase the frequency of sister chromatid exchange. In addition, overexpression of SEI1 was associated with poor prognosis of esophageal squamous cell carcinoma. Taken together, these findings define a novel mechanism of genomic instability and malignant progression in esophageal cancers, a deadly disease of increasing incidence in developed countries.