Effect Of Curcumin On The Regulation Of EGFR, UBE1L And RARβ In Lung Cancer

Objective To explore the precise mechanism of downregulating epidermal growth factor receptor (EGFR) by curcumin-induced ubiquitin activating enzyme E1like (UBE1L) in Beas-2B cells.Method Western blotting assay was used to detect protein expression in cells and lung cancer tissues. UBE1L and EGFR mRNA expression were assessed using reverse transcription-polymerase chain reaction. Immunoprecipitation was used to identify the conjugation between EGFR and ISG15. Immunofluorescence staming and fluorescence-activated cell sorter (FACS) analysis were used to quantify EGFR surface labeling.Results The present study demonstrated that curcumin decreased EGFR expression in human bronchial epithelial (HBE) Beas-2B cells and lung cancer A549cells. For the first time, UBEIL was shown to be induced by curcumin in HBE cells. Interestingly, overexpression of UBEIL reduced EGFR at post-translational level in HBE cells. UBEIL triggered EGFR membrane internalization and promoted complex formation between ISG15and EGFR. Curcumin decreased EGFR downstream signaling pAKT and NF-κB. Overexpression or knockdown of UBEIL also resulted in downregulation or upregulation of PI3K/AKT/NF-κB correspondently. In human samples there was an inverse relationship between UBE1L and EGFR-AKT-NF-κB in non-small cell lung cancer tissues and adjacent tissues.Conclusion These results uncover a novel chemopreventive mechanism of curcumin in inducing UBE1L and downregulating EGFR signaling in HBE cells. Objective To explore the precise mechanism of curcumin, EGCG and resveratrol regulating ubiquitin activating enzyme E1-like (UBE1L) in Beas-2B cells.Method Western blotting assay was used to detect UBE1L, p-JNK, JNK and Nrf2protein expression in human bronchial epithelial cell Beas-2B cells. UBEIL mRNA expression was assessed using reverse transcription-polymerase chain reaction. Fluorescence-activated cell sorter (FACS) analysis was used to detect cellular ROS level.Results In the present study, we found that lower concentrations of curcumin, EGCG and resveratrol induced UBEIL protein expression in human bronchial epithelial cell Beas-2B cells. However, higher concentrations of curcumin, EGCG and resveratrol decreased UBEIL protein expression. Studies demonstrate that polyphenols exhibits antioxidative property at lower concentration while shows prooxidative character at higher concentration. Our results showed that curcumin, EGCG and resveratrol at the lower concentrations diminished celluar ROS, while at the higher concentrations generated ROS. To test whether UBEIL induction is mediate by redox status, an antioxidant N-acetylcysteine (NAC) and prooxidants as hydrogen peroxidant and tert-butyl hydroperoxide (tBHP) were treated to Beas-2B cells and UBEIL protein levels were detected. NAC was found to increase UBE1L protein level, while hydrogen peroxide and tBHP decline it, indicating the involvement of reactive oxygen species (ROS) in curucmin-mediated regulation of UBE1L expression. In addition, co-treatment of Beas-2B cells with curcumin and NAC led to enhanced increase protein expression of UBE1L compared to ever single treatment. Pretreatment of cells with curcumin could partially prevented the tBHP-induced decrease of UBE1L. A panel of kinase inhibitors were used to examine the contribution of MAP kinase activity to the induction of UBE1L by curcumin. Only inhibition of JNK significantly reduced UBE1L induction. Low concentrations of curcumin, EGCG and resveratrol induced p-JNK protein expression, while high concentrations of curcumin, EGCG and resveratrol decreased p-JNK protein expression. When knockdown of nuclear factor erythroid-2related factor-2(Nrf2),the protein level of UBE1L was down. Conclusion All these suggest that polyphenols-mediated regulation of UBE1L is by intracellular ROS status, which may through JNK/Nrf2signal pathway. Objective To explore the precise mechanism of curcumin regulating RARβ.Method Western blotting assay was used to detect RARβ protein expression. RARβ mRNA expression was assessed using reverse transcription-polymerase chain reaction. Methylation specific-polymerase chain reaction was used to detect methylation level of RARβ promoter region.Results Reduced expression of RARβ has been found in lung cancer cells and in lung cancer tissues. Curcumin and5-Aza-dC (DNA methyltransferase inhibitor) significantly elevate RARβ expression at both the mRNA and protein levels in two human lung cancer cell lines A549, H460and other tested cancer cells. Additionally, curcumin decrease RARβ promoter methylation in lung cancer A549and H460cells. Mechanistic study demonstrated that curcumin can downregulate the mRNA levels of DNMT3b in A549and H460cells.Conclusion This study reveals a novel molecular mechanism of curcumin as a chemo-preventive agent for lung cancer through hypomethylation reactivation of RARP by downregulate the mRNA levels of DNMT3b.