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UBE2L6/UBCH8 and ISG15 attenuate autophagy in esophageal cancer cells.

ABSTRACT: Esophageal cancer remains a poor prognosis cancer due to advanced stage of presentation and drug resistant disease. To understand the molecular mechanisms influencing response to chemotherapy, we examined genes that are differentially expressed between drug sensitive, apoptosis competent esophageal cancer cells (OE21, OE33, FLO-1) and those which are more resistant and do not exhibit apoptosis (KYSE450 and OE19). Members of the ISG15 (ubiquitin-like) protein modification pathway, including UBE2L6 and ISG15, were found to be more highly expressed in the drug sensitive cell lines. In this study, we evaluated the contribution of these proteins to the response of drug sensitive cells. Depletion of UBE2L6 or ISG15 with siRNA did not influence caspase-3 activation or nuclear fragmentation following treatment with 5-fluorouracil (5-FU). We assessed autophagy by analysis of LC3II expression and Cyto-ID staining. Depletion of either ISG15 or UBE2L6 resulted in enhanced endogenous autophagic flux. An increase in autophagic flux was also observed following treatment with cytotoxic drugs (5-FU, rapamycin). In ISG15 depleted cells, this increase in autophagy was associated with improved recovery of drug treated cells. In contrast, UBE2L6 depleted cells, did not show enhanced recovery. UBE2L6 may therefore influence additional targets that limit the pro-survival effect of ISG15 depletion. These data identify UBE2L6 and ISG15 as novel inhibitors of autophagy, with the potential to influence chemosensitivity in esophageal cancer cells.

SUBMITTER: Falvey CM

PROVIDER: S-EPMC5410320 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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UBE2L6/UBCH8 and ISG15 attenuate autophagy in esophageal cancer cells.

Falvey Chloe M CM O'Donovan Tracey R TR El-Mashed Shereen S Nyhan Michelle J MJ O'Reilly Seamus S McKenna Sharon L SL

Oncotarget 20170401 14

Esophageal cancer remains a poor prognosis cancer due to advanced stage of presentation and drug resistant disease. To understand the molecular mechanisms influencing response to chemotherapy, we examined genes that are differentially expressed between drug sensitive, apoptosis competent esophageal cancer cells (OE21, OE33, FLO-1) and those which are more resistant and do not exhibit apoptosis (KYSE450 and OE19). Members of the ISG15 (ubiquitin-like) protein modification pathway, including UBE2L ...[more]

PMID: 28186990

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Project description:Background:Dihydroartemisinin (DHA), a derivate of artemisinin, is an effective antimalarial agent. DHA has been shown to exert anticancer activities to numerous cancer cells in the past few years, while the exact molecular mechanisms remain to be elucidated, especially in esophageal cancer. Methods:Crystal violet assay was conducted to determine the cell viability of human esophageal cancer cell line Eca109 treated with DHA. Tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Soft agar and crystal violet assays were used to measure the tumorigenicity of Eca109 cells. Flow cytometry was performed to evaluate ROS or cell cycle distribution. GFP-LC3 plasmids were delivered into Eca109 cells to visualize autophagy induced by DHA under a fluorescence microscope. The mRNA and protein levels of each gene were tested by qRT-PCR and western blot, respectively. Results:Our results proved that DHA significantly reduced the viability of Eca109 cells in a dose- and time-dependent manner. Further investigation showed that DHA evidently induced cell cycle arrest at the G2/M phase in Eca109 cells. Mechanistically, DHA induced intracellular ROS generation and autophagy in Eca109 cells, while blocking ROS by an antioxidant NAC obviously inhibited autophagy. Furthermore, we found that telomere shelterin component TRF2 was down-regulated in Eca109 cells exposed to DHA through autophagy-dependent degradation, which could be rescued after autophagy was blocked by ROS inhibition. Moreover, the DNA damage response (DDR) was induced obviously in DHA treated cells. To further explore whether ROS or autophagy played a vital role in DHA induced cell cycle arrest, the cell cycle distribution of Eca109 cells was evaluated after ROS or autophagy blocking, and the results showed that autophagy, but not ROS, was essential for cell cycle arrest in DHA treated cells. Conclusion:Taken together, DHA showed anticancer effect on esophageal cancer cells through autophagy-dependent cell cycle arrest at the G2/M phase, which unveiled a novel mechanism of DHA as a chemotherapeutic agent, and the degradation of TRF2 followed by DDR might be responsible for this cell phenotype.

Project description:Salvia chinensia Benth (Shijianchuan in Chinese, SJC) has been used as a traditional anti-cancer herb. SJC showed good anti-esophageal cancer efficacy based on our clinical application. However, the current research on SJC is minimal, and its anti-cancer effect lacks scientific certification. This study aims to clarify the inhibitory effect of SJC on esophageal cancer and explore its underlying mechanism. Q-Orbitrap high-resolution LC/MS was used to identify the primary chemical constituents in SJC. Cell proliferation and colony formation assays showed that SJC could effectively inhibit the growth of esophageal tumor cells in vitro. To clarify its mechanism of action, proteomic and bioinformatic analyses were carried out by combining tandem mass labeling and two-dimensional liquid chromatography-mass spectrometry (LC-MS). Data are available via ProteomeXchange with identifier PXD035823. The results indicated that SJC could activate AMPK signaling pathway and effectively promote autophagy in esophageal cancer cells. Therefore, we further used western blotting to confirm that SJC activated autophagy in esophageal cancer cells through the AMPK/ULK1 signaling pathway. The results showed that P-AMPK and P-ULK1 were significantly up-regulated after the treatment with SJC. The ratio of autophagosomes marker proteins LC3II/I was significantly increased. In addition, the expression of the autophagy substrate protein P62 decreased with the degradation of autophagosomes. Using lentiviral transfection of fluorescent label SensGFP-StubRFP-LC3 protein and revalidation of LC3 expression before and after administration by laser confocal microscopy. Compared with the control group, the fluorescence expression of the SJC group was significantly enhanced, indicating that it promoted autophagy in esophageal cancer cells. Cell morphology and the formation of autophagosomes were observed by transmission electron microscopy. Our study shows that the tumor suppressor effect of SJC is related to promoting autophagy in esophageal tumor cells via the AMPK/ULK1 signaling pathway.

Project description:Salvia chinensia Benth. (Shijianchuan in Chinese, SJC) has been used as a traditional anti-cancer herb. SJC showed good anti-esophageal cancer efficacy based on our clinical application. However, the current research on SJC is minimal, and its anti-cancer effect lacks scientific certification. This study aims to clarify the inhibitory effect of SJC on esophageal cancer and explore its underlying mechanism. Q-Orbitrap high-resolution LC/MS was used to identify the primary chemical constituents in SJC. Cell proliferation and colony formation assays showed that SJC could effectively inhibit the growth of esophageal tumor cells in vitro. To clarify its mechanism of action, proteomic and bioinformatic analyses were carried out by combining tandem mass labeling and two-dimensional liquid chromatography-mass spectrometry (LC-MS). The results indicated that SJC could activate AMPK signaling pathway and effectively promote autophagy in esophageal cancer cells. Therefore, we further used WB to confirm that SJC activated autophagy in esophageal cancer cells through the AMPK / ULK1 signaling pathway. The results showed that P-AMPK and P-ULK1 were significantly up-regulated after the treatment with SJC. The ratio of autophagosomes marker proteins LC3II/I was significantly increased. In addition, the expression of the autophagy substrate protein P62 decreased with the degradation of autophagosomes. Using lentiviral transfection of fluorescent label SensGFP-StubRFP-LC3 protein and revalidation of LC3 expression before and after administration by laser confocal microscopy. Compared with the control group, the fluorescence expression of the SJC group was significantly enhanced, indicating that it promoted autophagy in esophageal cancer cells. Cell morphology and the formation of autophagosomes were observed by transmission electron microscopy. Our study shows that the tumor suppressor effect of SJC is related to promoting autophagy in esophageal tumor cells via the AMPK/ULK1 signaling pathway.

Dataset Information

UBE2L6/UBCH8 and ISG15 attenuate autophagy in esophageal cancer cells.

Publications

UBE2L6/UBCH8 and ISG15 attenuate autophagy in esophageal cancer cells.

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