Wanxin Peng

Autophagy contributes to ING4-induced glioma cell death

Previous studies suggest that ING4, a novel member of ING (inhibitor of growth) family, can inhibit brain tumor growth. However, whether autophagy is involved in ING4-induced cell death still remains unknown. In this study, we found that in addition to apoptosis, autophagy also contributed to cell death induced by ING4. Autophagy levels were elevated following the exposure to Ad-ING4, including enhanced fluorescence intensity of monodansylcadervarine (MDC), a specific in vivo marker for autophagic vacuoles, and increased expression levels of the LC3-II and Beclin-1, wheras the autophagic levels were attenuated following the pretreatment of 3-MA, the inhibitor of autophagy, which significantly decreased the Ad-ING4-induced cell death compared with caspase inhibitor zVAD. Furthermore, ING4 also induced mitochondrial dysfunction, such as mitophagy, collapse of mitochondrial membrane potential and the intracellular ROS, which indicated that mitochondria might be associated with the process of autophagic cell death of glioma cells. Finally, the relationship among Bax, Bcl-2, Beclin-1 and caspase family proteins levels were analyzed in glioma cells U251MG and LN229 infected with Ad-ING4 or Ad-lacZ. It is suggested that both autophagy and apoptosis could contribute to ING4-induced glioma cell death, and mitochondria might play an important role in this process. Our findings reveal novel aspects of the autophagy in glioma cells that underlie the cytotoxic action of ING4, possibly providing new insights in the development of combinatorial therapies for gliomas.

Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells.

Previous studies suggest that early growth response gene-1 (Egr-1) plays an important role in hypoxia-induced drug-resistance. However, the mechanism still remains to be clarified. Herein, we investigated the role of Egr-1 in hypoxia-induced autophagy and its resulted hypoxia-driven chemo-resistance in Hepatocellular Carcinoma (HCC) cells. Our data demonstrated that Egr-1 was overexpressed in HCC tissues and cells and conferred them drug resistance under hypoxia. Mechanistically, Egr-1 transcriptionally regulated hypoxia-induced autophagy by binding to LC3 promoter in HCC cells, which resulted in resistance of HCC cells to chemotherapeutic agents; while dominant negative Egr-1 could inhibit autophagy level, and thus enhanced the sensitivity of HCC cells to chemotherapeutic agents, indicating that hypoxia-induced Egr-1 expression enhanced drug resistance of HCC cells likely through autophagy. Accordingly, it is suggested that a mechanism of hypoxia/Egr-1/autophagy axis might be involved in drug resistance in HCC.

FoxM1-mediated RFC5 expression promotes temozolomide resistance

Although methylguanine-DNA-methyltransferase (MGMT) plays an important role in resistance to temozolomide (TMZ) in glioma, 40% of gliomas with MGMT inactivation are still resistant to TMZ. The underlying mechanism is not clear. Here, we report that forkhead box M1 (FoxM1) transcriptionally activates the expression of DNA repair gene replication factor C5 (RFC5) to promote TMZ resistance in glioma cells independent of MGMT activation. We showed that RFC5 expression is positively correlated with FoxM1 expression in human glioma cells and FoxM1 is able to transcriptionally activate RFC expression by interaction with the RFC5 promoter. Furthermore, knockdown of FoxM1 or RFC5 partially re-sensitizes glioma cells to TMZ. Consistently, thiostrepton, a FoxM1 inhibitor, in combination with TMZ significantly inhibits proliferation and promotes apoptosis in glioma cells. Taken together, these findings suggest that the FoxM1-RFC5 axis may mediate TMZ resistance and thiostrepton may serve as a potential therapeutic agent against TMZ resistance in glioma cells.

MeCP2 suppresses LIN28A expression via binding to its methylated-CpG islands in pancreatic cancer cells

LIN28A aberrant expression contributes to the development of human malignancies. However, the LIN28A expression profile remains to be clarified. Herein, we report that LIN28A expression is directly associated with the methylation status of its two CpG island sites in pancreatic cancer cells. First, Bisulfite sequencing reveals that PANC1 cells possess the higher methylation rate at LIN28A CpG islands compared with SW1990 and PaTu8988 cells. Subsequently, LIN28A expression is increased at both mRNA and protein levels in pancreatic cancer cells treated with 5-Aza-2′-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor. Further Chromatin immunoprecipitation (ChIP) assays indicate that methyl-CpG-binding protein 2 (MeCP2) binds preferentially to the two hypermethylated CpG islands sites at LIN28A promoter compare to MBD3. Expectedly, MeCP2 knockdown transcriptionally activates LIN28A expression in above cells, rather than MBD3 knockdown. Moreover, LIN28A overexpression remarkably improves OCT4, NANOG and SOX2 expression, and the ability of sphere and colony formation, and enhances the capacities of invasion in PaTu8988 and SW1990 cells, whereas LIN28A knockdown significantly inhibits the above malignant behaviors in PANC1 cells. These findings suggest that LIN28A is epigenetically regulated via MeCP2 binding to methylated-CpG islands, and may play a crucial role in pancreatic cancer progression.

Knockdown of autophagy-related gene LC3 enhances the sensitivity of HepG2 cells to epirubicin

Hepatocellular carcinoma (HCC) is a major public health problem. Despite new chemotherapeutic treatments, drug resistance remains a major clinical obstacle to successful treatment in HCC patients. Therefore, novel therapeutic targets and new modalities of treatment are urgently required. In this study, tetracycline-inducible lentivirus-mediated RNA interference (RNAi) was employed to knock down microtubule-associated protein 1 light chain 3 (LC3) gene, which encodes a key protein in the induction of autophagy, to study the protective function of autophagy in liver cancer tolerant to epirubicin. The effect of combined treatment with lentiviral shLC3 and epirubicin on cell growth and chemosensitivity to epirubicin in the HCC cell line HepG2 were also investigated. The results demonstrated that lentivirus-mediated LC3 silencing significantly inhibited cell proliferation. In addition, combined treatment with lentiviral shLC3 and epirubicin significantly decreased the survival rate of HepG2 cells, compared with that following treatment with either agent alone. Overall, the results from this study suggest for the first time, to the best of our knowledge, that LC3 plays a key role in HCC tumorigenesis, and is a novel therapeutic target for HCC.

LncRNA UCA1 promotes migration and invasion in pancreatic cancer cells via the Hippo pathway

Although overexpression of the long non-coding RNA (lncRNA) UCA1 has been implicated in several human cancers, its biological function in pancreatic cancer remains to be clarified. In this study, we reported that UCA1 expression was significantly increased in pancreatic cancer tissues and correlated with clinicopathological features, tumor stage, and poorer patient outcome. We further showed that UCA1 promoted cell migration and invasion of pancreatic cancer cells. Importantly, we found that UCA1 overexpression inhibited YAP phosphorylation, and increased YAP expression. Mechanistically, UCA1 interacted with MOB1, Lats1, and YAP, forming shielding composites. Moreover, we demonstrated that UCA1 increased YAP nuclear localization and stabilization, and improved TEAD luciferase activity. In turn, YAP promotes UCA1 expression. Collectively, the present study provides insights into the mechanistic regulation of UCA1 promoting pancreatic cancer progression through the Hippo signaling pathway. UCA1 may serve as a candidate biomarker for poor prognosis and a target for new pancreatic cancer therapies.

Methyl-CpG-binding domain 3 inhibits epithelial-mesenchymal transition in pancreatic cancer cells via TGF-β/Smad signalling.

Background:Methyl-CpG-binding domain 3 (MBD3) is an aberrant expression in human malignancies. However, the role of MBD3 in pancreatic cancer progression remains to be clarified. In this study, we investigated the effects of MBD3 on the epithelial–mesenchymal transition (EMT), and the underlying mechanism in pancreatic cancer cells.Methods:The abilities of migration and invasion were examined by transwell and BD Matrigel invasion assays. EMT and TGF-β/Smad signalling were evaluated.Results:First, we find that MBD3 expression is lower in pancreatic cancer tissues than that in non-tumour tissues, and patients with lower MBD3 levels survive significantly less than those with higher levels. Subsequently, we find that MBD3 knockdown promotes the abilities of migration and invasion, while MBD3 overexpression inhibits the above abilities. Also, MBD3 knockdown remarkably increases mesenchymal markers expression of Vimentin, α-SMA, Snail, N-cadherin, β-catenin, and downregulates epithelial markers expression of E-cadherin. On the contrary, MBD3 overexpression results in the opposite effects. Further evidence reveals that MBD3 knockdown up-regulates expression of TGF-β, and then activates p-Smad2 and p-Smad3, while MBD3 overexpression results in downregulation of TGF-β, p-Smad2, and p-Smad3.Conclusions:MBD3 inhibits EMT in pancreatic cancer cells probably via TGF-β/Smad signalling, and may be a new candidate target for diagnostics and prognosis of pancreatic cancer.

Scoparone Protects Against Pancreatic Fibrosis via TGF-β/Smad Signaling in Rats.

Background/Aims: This study was to investigate the influence of scoparone on pancreatic fibrosis in vitro and in vivo. Methods: Pancreatic stellate cells (PSCs) were isolated from pancreas tissue blocks, and cultured for 3-5 generations for the experiment. PSCs were treated with scoparone in different doses as experimental groups, salvianolic acid B as a positive control and PBS as a blank group. We measured the effects of scoparone on cellular proliferation, oxidative stress, epithelial-mesenchymal transition (EMT), and pancreatic fibrosis. Cellular oxidative stress was detected by using commercially available kits. The impact of scoparone on EMT and fibrosis was detected through immunofluorescence or western blotting. Results: Compared with the control group, scoparone significantly inhibited stellate cell proliferation, and reduced MDA, the expression of mesenchymal makers, and increased the levels of SOD and the expression of E-cadherin (P < 0.05). Western blot analysis showed that scoparone downregulated the expression of TGF-β and p-smad2/3, and upregultated the expression of smad7 (P < 0.05). Conclusion: Scoparone can reduce the levels of oxidative stress, repress pancreatic stellate cells activation, and alleviate fibrosis by regulating TGF-β/Smad pathway.

Furin promotes epithelial-mesenchymal transition in pancreatic cancer cells via Hippo-YAP pathway

Furin, a well-characterized proprotein convertase, plays an important role in many diseases and links to tumor metastasis. However, the role of furin in pancreatic cancer progression remains to be elucidated. In the present study, we found that furin promotes the growth and the epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. First, we found that furin knockdown significantly inhibited proliferation, invasion and migration in BxPC3 and SW1990 cells, while furin overexpression promoted the above behavior in PANC1 and PaTu8988 cells. Further evidence revealed that furin knockdown resulted in the upregulation of E-cadherin (epithelial marker), and the downregulation of N-cadherin and Vimentin (mesenchymal markers) in BxPC3 and SW1990 cells, whereas furin overexpression remarkably led to the opposite effects in PANC1 and PaTu8988 cells. Furthermore, our data showed that Furin knockdown, furin inhibitor D6R or overexpression significantly affected YAP phosphoration level and total YAP protein level, indicating that furin was involved in Hippo-YAP pathway. It is suggested that furin promotes epithelial-mesenchymal transition in pancreatic cancer cells probably via Hippo-YAP pathway and may be a potential target for anti-pancreatic cancer.

ADAM17 promotes epithelial-mesenchymal transition via TGF-β/Smad pathway in gastric carcinoma cells

Although a disintegrin and metalloproteinase-17 (ADAM17) overexpression has been demonstrated in numerous human tumors including gastric cancer, its role in gastric cancer development remains to be clarified. In the present study, we identify that ADAM17 activates TGF-β/Smad signaling to promote epithelial-mesenchymal transition (EMT) in gastric cancer cells. We found that ADAM17 promotes proliferation, migration and invasion in gastric carcinoma cells. Subsequently, we revealed that silencing ADAM17 induces the expression of epithelial marker of E-cadherin and downregulates expression of mesenchymal markers including N-cadherin, vimentin and Snail in MGC803 and MKN45 cells, whereas ADAM17 overexpression reverses these changes in BGC823 and HGC27 cells. Furthermore, ADAM17 knockdown significantly inhibits the expression of TGF-β and its downstream signaling molecules p-Smad2 and p-Smad3 in MGC803 and MKN45 cells. Consistently, ADAM17 overexpression reversed these changes in BGC823 and HGC27 cells. These results suggest that ADAM17 promotes epithelial-mesenchymal transition via the TGF-β/Smad pathway. Collectively, the present study demonstrates that ADAM17 plays a critical role in the development of gastric cancer and provides a potential therapeutic target for gastric cancer.