Cheliensisin A Inhibits EGF-Induced Cell Transformation with Stabilization of p53 Protein Via a Hydrogen Peroxide/Chk1-Dependent Axis

Abstract

Cheliensisin A (Chel A), a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been shown to induce of apoptosis in human promyelocytic leukemia HL-60 cells with Bcl-2 downregulation. Yet the potential chemopreventive effect of Chel A has not been explored. Here we demonstrated that Chel A treatment with various concentrations (0.5, 1.0, 2.0, and 4.0 μM) for 3 weeks could dramatically inhibit EGF-induced cell transformation in Cl41 cells (IC50 approximately 2.0 μM). Also, co-incubation of Cl41 cells with Chel A (2.0 and 4.0 μM) for 48 hours could induce cell apoptosis in a caspase-3-dependent manner. Mechanically, Chel A treatment could result in increased p53 phosphorylation at Ser15 and elevated p53 total protein expression. Moreover, we found that p53 induction by Chel A was regulated at the protein degradation level, but not at either the transcription or the mRNA level. Further studies showed that p53 stabilization by Chel A was mediated via induction of phosphorylation and activation of Chk1 protein at Ser345. This notion was substantiated by the results that transfection of dominant negative mutant of Chk1 (GFP-Chk1 D130A) significantly attenuated the p53 protein expression, cell apoptosis, and inhibition of cell transformation by Chel A. Finally, increased hydrogen peroxide was found to mediate Chk1 phosphorylation at Ser345, p53 protein induction, cell apoptotic induction, and transformation inhibition following Chel A treatment. Taken together, our studies identify Chel A as a chemopreventive agent with the understanding of the molecular mechanisms involved.

Loss of p27 upregulates MnSOD in a STAT3-dependent manner, disrupts intracellular redox activity and enhances cell migration

“…CRE luciferase reporter plasmids containing the CRE sequences from the intracisternal A-type particle element, as well as a mutant version, were described previously (Galien et al, 1994;Gupta et al, 2001). Expression plasmids for SOD, mitochondrially targeted catalase (Mito-Catalase) and catalase, as well as the parental control vector pSV-Zeo were kindly provided by Juan Andres Melendez (Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY) (Rodríguez et al, 2000;Ding et al, 2006;Zhang et al, 2013). The plasmid encoding dominantnegative ERK2, which contained a K52R mutation of rat ERK2, was as published previously (Frost et al, 1994).…”

Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA stability

“…Cells were maintained in a humidified incubator at 37°C with 5% CO 2 atmosphere[45]. The antibodies specific against phospho-c-Jun, c-Jun, phospho-JNKs, JNKs, phospho-ERKs, ERKs, phospho-p38, p38, phospho-MKK7, MKK7, phospho-MKK4, MKK4, PP2A A subunit, XIAP, SP1, β-Actin and GAPDH were purchased from Cell Signaling Technology Inc (Beverly, MA).…”

“…The plates were incubated at 37 °C in 5% CO2 for 3 weeks. The colonies with more than 32 cells were scored and the results were presented as colonies/10 4 cells[45, 47, 51]. …”

“…The fixed cells were washed twice with PBS, and then suspended in Propidium Iodide staining solution (Propidium Iodide 50 mg /ml, RNAse A 10 mg /ml, and 0.1% Triton X-100) (Sigma-Aldrich Chemical, St. Louis, MO) for at least 1 hour at 4°C. The DNA content was determined by means of Flow Cytometric Analysis with the Epics XL FACS (Beckman Coulter, Miami, FL) and EXPO 32 software as described previously [45, 51, 55]. …”

“…The primers for mouse β-actin are 5'-ATA TCG CTG CGC TGG TCG TC-3' and 5'-AGG ATG GCG TGA GGG AGA GC-3'. The PCR products were separated onto 3% agarose gels, stained with EB, and scanned the images from a UV light as described previously[45, 52, 60-61]. …”