Ursolic acid (UA) has been reported to possess anticancer activities. Although some of the anticancer activities of UA have been explained by its apoptosis‐inducing properties, the mechanisms ...underlying its anticancer actions are largely unknown. We have found that UA‐activated autophagy induced cytotoxicity and reduced tumor growth of cervical cancer cells TC‐1 in a concentration‐dependent manner. UA did not induce apoptosis of TC‐1 cells in vitro as determined by annexin V/propidium iodide staining, DNA fragmentation, and Western blot analysis of the apoptosis‐related proteins. We found that UA increased punctate staining of light chain 3 (LC3), which is an autophagy marker. LC3II, the processed form of LC3I which is formed during the formation of double membranes, was induced by UA treatment. These results were further confirmed by transmission electron microscopy. Wortmannin, an inhibitor of autophagy, and a small interfering RNA (siRNA) for autophagy‐related genes (Atg5) reduced LC3II and simultaneously increased the survival of TC‐1 cells treated with UA. We also found that LC3II was significantly reduced and that survival was increased in Atg5−/− mouse embryonic fibroblast (MEF) cells compared to Atg5+/+ MEF cells under UA treatment. However, silencing BECN1 by siRNA affected neither the expression of LC3II nor the survival of TC‐1 cells under UA treatment. These results suggest that autophagy is a major mechanism by which UA kills TC‐1 cells. It is Atg5 rather than BECN1 that plays a crucial role in UA‐induced autophagic cell death in TC‐1 cells. The activation of autophagy by UA may become a potential cancer therapeutic strategy complementing the apoptosis‐based therapies. Furthermore, regulation of Atg5 may improve the efficacy of UA in cancer treatment.
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Ursolic acid, a naturally occurring compound found in certain fruits and vegetables, possesses potent anticancer activity. But while capable of undermining cancer cells by inducing their death through apoptosis, other aspects of ursolic acid's anticancer mechanism are not fully understood. This study shows that the compound promotes cancer cell death by inducing Atg5‐dependent autophagy, a process that increasingly has been found to play a role in tumorigenesis. In TC‐1 cervical cancer cells, ursolic acid activated autophagy through PI3‐K signaling. The findings suggest that the compound could be a potent complementary therapy for cancers resistant to caspase‐dependent apoptosis.
MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that play important roles in various cellular functions and tumor development. Recent studies have indicated that miR-21 is one of the ...important miRNAs associated with tumor growth and metastasis, but the role and molecular mechanism of miR-21 in regulating tumor angiogenesis remain to be elucidated. In this study, miR-21 was overexpressed by transfecting pre-miR-21 into human prostate cancer cells and tumor angiogenesis was assayed using chicken chorioallantoic membrane (CAM). We found that overexpression of miR-21 in DU145 cells increased the expression of HIF-1α and VEGF, and induced tumor angiogenesis. AKT and extracellular regulated kinases (ERK) 1/2 are activated by miR-21. Inhibition of miR-21 by the antigomir blocked this process. Overexpression of the miR-21 target, PTEN, also inhibited tumor angiogenesis by partially inactivating AKT and ERK and decreasing the expression of HIF-1 and VEGF. The AKT and ERK inhibitors, LY294002 and U0126, suppressed HIF-1α and VEGF expression and angiogenesis. Moreover, inhibition of HIF-1α expression alone abolished miR-21-inducing tumor angiogenesis, indicating that HIF-1α is required for miR-21-upregulated angiogenesis. Therefore, we demonstrate that miR-21 induces tumor angiogenesis through targeting PTEN, leading to activate AKT and ERK1/2 signaling pathways, and thereby enhancing HIF-1α and VEGF expression; HIF-1α is a key downstream target of miR-21 in regulating tumor angiogenesis.
MiR‐637 (microRNA‐637) is a primate‐specific miRNA belonging to the small noncoding RNA family, which represses gene regulation at the post‐transcriptional expression level. Although it was ...discovered approximately 5 years ago, its biomedical significance and regulatory mechanism remain obscure. Our preliminary data showed that miR‐637 was significantly suppressed in four HCC cell lines and, also, in most of the hepatocellular carcinoma (HCC) specimens, thereby suggesting that miR‐637 would be a tumor suppressor in HCC. Simultaneously, the enforced overexpression of miR‐637 dramatically inhibited cell growth and induced the apoptosis of HCC cells. The transcription factor, signal transducer and activator of transcription 3 (Stat3), is constitutively activated in multiple tumors, and aberrant Stat3 activation is linked to the promotion of growth and desensitization of apoptosis. Our study showed that Stat3 tyrosine 705 phosphorylation and several Stat3‐regulated antiapoptotic genes were down‐regulated in miR‐637 mimics‐transfected and Lv‐miR637‐infected HCC cells. In addition, miR‐637 overexpression negatively regulated Stat3 phosphorylation by suppressing autocrine leukemia inhibitory factor (LIF) expression and exogenous LIF‐triggered Stat3 activation and rescued cell growth in these cells. A nude mice model also demonstrated the above‐described results, which were obtained from the cell model. Furthermore, we found that LIF was highly expressed in a large proportion of HCC specimens, and its expression was inversely associated with miR‐637 expression. Conclusion: Our data indicate that miR‐637 acted as a tumor suppressor in HCC, and the suppressive effect was mediated, at least in part, by the disruption of Stat3 activation. (HEPATOLOGY 2011)
Several microRNAs (miRNA) have been implicated in nasopharyngeal carcinoma (NPC), a highly invasive and metastatic cancer that is widely prevalent in southern China. In this study, we report that ...microRNA miR-26a is commonly downregulated in NPC specimens and NPC cell lines with important functional consequences. Ectopic expression of miR-26a dramatically suppressed cell proliferation and colony formation by inducing G(1)-phase cell-cycle arrest. We found that miR-26a strongly reduced the expression of EZH2 oncogene in NPC cells. Similar to the restoring miR-26 expression, EZH2 downregulation inhibited cell growth and cell-cycle progression, whereas EZH2 overexpression rescued the suppressive effect of miR-26a. Mechanistic investigations revealed that miR-26a suppressed the expression of c-myc, the cyclin D3 and E2, and the cyclin-dependent kinase CDK4 and CDK6 while enhancing the expression of CDK inhibitors p14(ARF) and p21(CIP1) in an EZH2-dependent manner. Interestingly, cyclin D2 was regulated by miR-26a but not by EZH2, revealing cyclin D2 as another direct yet mechanistically distinct target of miR-26a. In clinical specimens, EZH2 was widely overexpressed and its mRNA levels were inversely correlated with miR-26a expression. Taken together, our results indicate that miR-26a functions as a growth-suppressive miRNA in NPC, and that its suppressive effects are mediated chiefly by repressing EZH2 expression.
Glioblastoma (GBM) is the most lethal primary brain tumor and is highly resistant to current treatments. GBM harbors glioma stem cells (GSCs) that not only initiate and maintain malignant growth but ...also promote therapeutic resistance including radioresistance. Thus, targeting GSCs is critical for overcoming the resistance to improve GBM treatment. Because the bone marrow and X-linked (BMX) nonreceptor tyrosine kinase is preferentially up-regulated in GSCs relative to nonstem tumor cells and the BMX-mediated activation of the signal transducer and activator of transcription 3 (STAT3) is required for maintaining GSC self-renewal and tumorigenic potential, pharmacological inhibition of BMX may suppress GBM growth and reduce therapeutic resistance. We demonstrate that BMX inhibition by ibrutinib potently disrupts GSCs, suppresses GBM malignant growth, and effectively combines with radiotherapy. Ibrutinib markedly disrupts the BMX-mediated STAT3 activation in GSCs but shows minimal effect on neural progenitor cells (NPCs) lacking BMX expression. Mechanistically, BMX bypasses the suppressor of cytokine signaling 3 (SOCS3)-mediated inhibition of Janus kinase 2 (JAK2), whereas NPCs dampen the JAK2-mediated STAT3 activation via the negative regulation by SOCS3, providing a molecular basis for targeting BMX by ibrutinib to specifically eliminate GSCs while preserving NPCs. Our preclinical data suggest that repurposing ibrutinib for targeting GSCs could effectively control GBM tumor growth both as monotherapy and as adjuvant with conventional therapies.
Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to indentify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to ...inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.
•Under-expressed miR-138 is associated with poor clinical outcome of GBM patients.•MiR-138 inhibits tumor growth of GBM cells in vitro and in vivo.•EZH2, CDK6, E2F2 and E2F3 are direct targets of miR-138 in GBM cells.•An EZH2-CDK4/6-pRb-E2F1 signal loop is involved in GBM tumorigenicity.
To investigate expression, regulation, potential role and targets of miR-195 and miR-497 in breast cancer.
The expression patterns of miR-195 and miR-497 were initially examined in breast cancer ...tissues and cell lines by Northern blotting and quantitative real-time PCR. Combined bisulfite restriction analysis and bisulfite sequencing were carried out to study the DNA methylation status of miR-195 and miR-497 genes. Breast cancer cells stably expressing miR-195 and miR-497 were established to study their role and targets. Finally, normal, fibroadenoma and breast cancer tissues were employed to analyze the correlation between miR-195/497 levels and malignant stages of breast tumor tissues.
MiR-195 and miR-497 were significantly downregulated in breast cancer. The methylation state of CpG islands upstream of the miR-195/497 gene was found to be responsible for the downregulation of both miRNAs. Forced expression of miR-195 or miR-497 suppressed breast cancer cell proliferation and invasion. Raf-1 and Ccnd1 were identified as novel direct targets of miR-195 and miR-497. miR-195/497 expression levels in clinical specimens were found to be correlated inversely with malignancy of breast cancer.
Our data imply that both miR-195 and miR-497 play important inhibitory roles in breast cancer malignancy and may be the potential therapeutic and diagnostic targets.
Hpn is a small histidine‐rich cytoplasmic protein from Helicobacter pylori and has been recognized as a high‐risk factor for several cancers including gastric cancer, colorectal cancer, and MALT ...lymphoma. However, the relationship between Hpn and cancers remains elusive. In this study, we discovered that Hpn protein effectively suppressed cell growth and induced apoptosis in hepatocellular carcinoma (HCC). A two‐dimensional gel electrophoresis and mass spectrometry‐based comparative proteomics was performed to find the molecular targets of Hpn in HCC cells. It was identified that twelve proteins were differentially expressed, with USP5 being one of the most significantly downregulated protein. The P14ARF‐P53 signaling was activated by USP5 knockdown in HCC cells. Furthermore, USP5 overexpression significantly rescued the suppressive effect of Hpn on the viability of HCC cells. In conclusion, our study suggests that Hpn plays apoptosis‐inducing roles through suppressing USP5 expression and activating the P14ARF‐P53 signaling. Therefore, Hpn may be a potential candidate for developing novel anti‐HCC drugs.
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