Abstract
Hepatocellular carcinoma (HCC) is the most common malignancy of the liver. It is unfortunate that HCCs are highly refractory to conventional chemotherapy, radiation therapy, and even immunotherapy. Thus, novel therapeutic targets need to be sought for the successful treatment of HCCs. We now report that (±)-(3aRS,4SR)-2-(2-chloro-4-methylsulfonylphenyl)-4′-chloro-3α,4-diethoxy-flavane[4,3-d]-D1,9b-1,2,3-thiadiazoline (MSFTZ), a synthesized flavanone derivative, induced growth arrest and apoptosis of HCCs both in vitro and in vivo. MSFTZ induced a time- and dose-dependent increase in HCC apoptosis through caspase-3 activation and poly(ADP-ribose) polymerase-1 cleavage. Activation of caspase-9 induced by MSFTZ suggested that MSFTZ-induced signaling was mediated through a mitochondrial death pathway. In addition, we observed an elevation of reactive oxygen species (ROS) and a consequent loss of mitochondrial membrane potential, further suggesting that MSFTZ-induced death signaling was mediated through a mitochondrial oxygen stress pathway. These events were associated with a decrease and increase in Bcl-2 and Bax expression, respectively, as well as phosphorylation of mitogen-activated protein kinase (MAPK) and activation of p53-MDM2 pathway. However, the antioxidant N-acetylcysteine opposed MSFTZ-mediated mitochondrial dysfunction, caspase activation, Bcl-2/Bax modulation, and apoptosis, supporting the role of ROS in the apoptotic process. We were surprised that we failed to observe the protective effect of N-acetylcysteine against MSFTZ-induced MAPK activation. Furthermore, MSFTZ had an antitumor effect in vivo by 34.8 to 78.7% reduction of tumor size in SMMC-7721-xenografted nude mice. We conclude that MSFTZ induces HCC cell apoptosis both in vivo and in vitro via caspase- and ROS-dependent mitochondrial pathway. In addition, MSFTZ has potential as a novel therapeutic agent for the treatment of HCC.
Footnotes
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This work was supported by the National Natural Science Foundation of China Grant 30572211, the Technology and Science Bureau of Zhejiang Province Foundation Grant 2006C24006, the Health Bureau of Zhejiang Province Foundation Grant WKJ2006-2-015, and the Natural Science Foundation of Zhejiang Province R20512.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.135657.
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ABBREVIATIONS: HCC, hepatocellular carcinoma; MSFTZ, (±)-(3aRS,4SR)-2-(2-chloro-4-methylsulfonylphenyl)-4′-chloro-3α,4-diethoxy-flavane-[4,3-d]-D1,9b-1,2,3-thiadiazoline; ROS, reactive oxygen species; ΔΨm, mitochondrial membrane potential; MAPK, mitogen-activated protein kinase; DMSO, dimethyl sulfoxide; NAC, N-acetylcysteine; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide; carboxy-DCFDA, 5-(and-6)-carboxy-2′-7′-dichlorofluorescin diacetate; XIAP, X-linked inhibitor of apoptosis protein; PARP, poly(ADP-ribose) polymerase; ERK, extracellular signal-regulated kinase; p-, phosphorylated; T/C%, 100 × (mean RTV of treated group)/(mean RTV of control group); RTV, relative tumor volume; PI, propidium iodide; PBS, phosphate-buffered saline; AO, acridine orange; EB, ethidium bromide.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received December 19, 2007.
- Accepted March 4, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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