Jianping Jiang1, Bo Wang2, Jing Li2, Baodong Ye2, Shengyun Lin3, Wenbin Qian4, Letian Shan5, Thomas Efferth6. 1. The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, PR China; Zhejiang Chinese Medical University, Hangzhou 310053, PR China. 2. The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, PR China. 3. The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, PR China. Electronic address: lsyww2012@163.com. 4. The First Affiliated Hospital of Zhejiang University, Hangzhou 310003, PR China. 5. Zhejiang Chinese Medical University, Hangzhou 310053, PR China. Electronic address: letian.shan@hotmail.com. 6. University of Mainz, Mainz 55128, Germany.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa is an ethno-medicine used for anti-cancer treatment in the clinic of traditional Chinese medicine (TCM). The total coumarins of Hedyotis diffusa (TCHD) was a selected extract with observed antiproliferative activity, which has not been tested in treatment of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). AIM OF THE STUDY: This study aimed to evaluate the apoptosis-inducing effect of TCHD on human MDS cell line (SKM-1) and explore its action mechanism in association with caspase family and PI3K/Akt signaling pathway. MATERIALS AND METHODS: The chemical constituents and total coumarins content of TCHD were determined by High Performance Liquid Chromatography-tandem mass spectrometry (HPLC-MS/MS) and UV-vis spectrophotometry, respectively. MTT assay, Hoechst 33258 staining, and Annexin V-FITC/PI double labeling were applied to evaluate TCHD's efficacy on SKM-1 cells. Western blot analysis was also used to clarify the action mechanism of TCHD on protein expression level. RESULTS: Two compounds, p-coumaric acid and E-6-O-p-coumaroyl scandoside methyl ester, were identified in TCHD, and its total coumarins content reached 87.4%. By MTT assay, apoptosis-inducing effect of TCHD on SKM-1 cells was found in a dose-dependent manner after 24-48h treatment, with IC50 values of 104.48μg/ml and 100.66μg/ml, respectively. Morphological and flow cytometry observation also confirmed such effect of TCHD. Western blot analysis clarified its action mechanism associating with the activation of caspases and inhibition of PI3K/Akt pathway proteins. CONCLUSIONS: This is the first report regarding the apoptosis-inducing efficacy and mechanism of TCHD on SKM-1 cells, providing a promising candidate of TCM for MDS and AML therapy with fewer side effects.
ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa is an ethno-medicine used for anti-cancer treatment in the clinic of traditional Chinese medicine (TCM). The total coumarins of Hedyotis diffusa (TCHD) was a selected extract with observed antiproliferative activity, which has not been tested in treatment of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). AIM OF THE STUDY: This study aimed to evaluate the apoptosis-inducing effect of TCHD on humanMDS cell line (SKM-1) and explore its action mechanism in association with caspase family and PI3K/Akt signaling pathway. MATERIALS AND METHODS: The chemical constituents and total coumarins content of TCHD were determined by High Performance Liquid Chromatography-tandem mass spectrometry (HPLC-MS/MS) and UV-vis spectrophotometry, respectively. MTT assay, Hoechst 33258 staining, and Annexin V-FITC/PI double labeling were applied to evaluate TCHD's efficacy on SKM-1 cells. Western blot analysis was also used to clarify the action mechanism of TCHD on protein expression level. RESULTS: Two compounds, p-coumaric acid and E-6-O-p-coumaroyl scandoside methyl ester, were identified in TCHD, and its total coumarins content reached 87.4%. By MTT assay, apoptosis-inducing effect of TCHD on SKM-1 cells was found in a dose-dependent manner after 24-48h treatment, with IC50 values of 104.48μg/ml and 100.66μg/ml, respectively. Morphological and flow cytometry observation also confirmed such effect of TCHD. Western blot analysis clarified its action mechanism associating with the activation of caspases and inhibition of PI3K/Akt pathway proteins. CONCLUSIONS: This is the first report regarding the apoptosis-inducing efficacy and mechanism of TCHD on SKM-1 cells, providing a promising candidate of TCM for MDS and AML therapy with fewer side effects.