Li Wu1, Ke Xin Cao2, Zi Hui Ni3, Wei Dong Li4, Zhi Peng Chen5, Hai Bo Cheng6, Xiao Liu7. 1. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China; Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China. Electronic address: wuli@njucm.edu.cn. 2. Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China. Electronic address: ckx@njucm.edu.cn. 3. Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China. Electronic address: nizihuideyouxiang@163.com. 4. Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China; Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China. Electronic address: liweidong0801@163.com. 5. Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China; Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China. Electronic address: czpcpu2000@hotmail.com. 6. The First Clinical Medical College, Nanjing University of Chinese Medicine, China; Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing, China. Electronic address: hbcheng@njucm.edu.cn. 7. Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China; Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China. Electronic address: liuxiao04_0@163.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Dahuang zhechong pill (DHZCP) is a famous traditional Chinese medicinal prescription from the "Synopsis of Prescriptions of the Golden Chamber (Jin Kui Yao Lue)",Lue)", an ancient Chinese medical classic. DHZCP is commonly used for clinical treatment of liver cancer by promoting blood circulation to dissolve blood stasis and by removing pathogenic vegetations.vegetations. DHZCP-based treatment has been derived from Traditional Chinese Medicine (TCM) and is officially recorded in the Chinese Pharmacopoeia. AIM OF THE STUDY: The aim of this study was to investigate the ability of DHZCP to reverse doxorubicin (DOX) resistance of SMMC-7721 cells in a xenograft mouse model, and to explore the underlying mechanisms. MATERIALS AND METHODS: Liquid chromatography-mass spectrometry was used to verify the composition of DHZCP. H&E staining was used to observe the pathological changes in hepatocellular carcinoma samples. Intracellular DOX accumulation was observed as intrinsic fluorescence by microscopy. Cell apoptosis was detected by the TUNEL assay. Human antibody arrays were used to analyze the expression of apoptotic- and angiogenic-related proteins. ATP levels were assessed and western blots were used to detect the protein expression of key enzymes of energy metabolism. RESULTS: DHZCP significantly reduced the tumor volume and weight of subcutaneous xenografts of drug-resistant hepatoma cells, and combining DHZCP with lower doses of DOX significantly increased the content of DOX in tumor tissue, increased the apoptosis of hepatoma cells, and reversed Dox resistance. With respect to 43 apoptosis-associated proteins, DHZCP regulated the expression of 5 of them. When combined with low-dose DOX, the expression of 40 apoptosis-related proteins was significantly altered. With respect to 23 angiogenesis-associated proteins, DHZCP upregulated the expression of endostatin and inhibited the expression of matrix metallopeptidase 9. When combined with low-dose DOX, DHZCP significantly downregulated protein expression of urokinase receptor, as well as vascular endothelial growth factor receptors 2 and 3. Especially, DHZCP significantly inhibited the expression of key enzymes of the tricarboxylic acid cycle and of oxidative phosphorylation, reducing the level of ATP in tumor tissue. CONCLUSIONS: DHZCP inhibited the growth of DOX-resistant hepatocellular carcinoma subcutaneous xenografts in nude mice and promoted increased apoptosis caused by DOX, thus reversing DOX resistance. This was associated with a decline in energy metabolism and regulated expression of pro-apoptotic proteins.
ETHNOPHARMACOLOGICAL RELEVANCE: Dahuang zhechong pill (DHZCP) is a famous traditional Chinese medicinal prescription from the "Synopsis of Prescriptions of the Golden Chamber (Jin Kui Yao Lue)",Lue)", an ancient Chinese medical classic. DHZCP is commonly used for clinical treatment of liver cancer by promoting blood circulation to dissolve blood stasis and by removing pathogenic vegetations.vegetations. DHZCP-based treatment has been derived from Traditional Chinese Medicine (TCM) and is officially recorded in the Chinese Pharmacopoeia. AIM OF THE STUDY: The aim of this study was to investigate the ability of DHZCP to reverse doxorubicin (DOX) resistance of SMMC-7721 cells in a xenograft mouse model, and to explore the underlying mechanisms. MATERIALS AND METHODS: Liquid chromatography-mass spectrometry was used to verify the composition of DHZCP. H&E staining was used to observe the pathological changes in hepatocellular carcinoma samples. Intracellular DOX accumulation was observed as intrinsic fluorescence by microscopy. Cell apoptosis was detected by the TUNEL assay. Human antibody arrays were used to analyze the expression of apoptotic- and angiogenic-related proteins. ATP levels were assessed and western blots were used to detect the protein expression of key enzymes of energy metabolism. RESULTS: DHZCP significantly reduced the tumor volume and weight of subcutaneous xenografts of drug-resistant hepatoma cells, and combining DHZCP with lower doses of DOX significantly increased the content of DOX in tumor tissue, increased the apoptosis of hepatoma cells, and reversed Dox resistance. With respect to 43 apoptosis-associated proteins, DHZCP regulated the expression of 5 of them. When combined with low-dose DOX, the expression of 40 apoptosis-related proteins was significantly altered. With respect to 23 angiogenesis-associated proteins, DHZCP upregulated the expression of endostatin and inhibited the expression of matrix metallopeptidase 9. When combined with low-dose DOX, DHZCP significantly downregulated protein expression of urokinase receptor, as well as vascular endothelial growth factor receptors 2 and 3. Especially, DHZCP significantly inhibited the expression of key enzymes of the tricarboxylic acid cycle and of oxidative phosphorylation, reducing the level of ATP in tumor tissue. CONCLUSIONS: DHZCP inhibited the growth of DOX-resistant hepatocellular carcinoma subcutaneous xenografts in nude mice and promoted increased apoptosis caused by DOX, thus reversing DOX resistance. This was associated with a decline in energy metabolism and regulated expression of pro-apoptotic proteins.