Cancer Chemopreventive Effect of 2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone on Diethylnitrosamine-Induced Early Stages of Hepatocarcinogenesis in Rats

Abstract

2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) is a major compound in Cleistocalyx nervosum seed extract (CSE), which has been reported to have various biological activities, including anti-cancer activity. Therefore, this study attempted to evaluate whether DMC is a chemopreventive compound in CSE. Moreover, the preventive mechanisms of CSE and DMC in the DEN-induced early stages of hepatocarcinogenesis in rats were investigated. Male Wistar rats were intraperitoneally injected with DEN 50 mg/kg bw once a week for 8 weeks. Rats received CSE and DMC orally throughout the experiment. The number of glutathione S-transferase placental form (GST-P)-positive foci in the liver was measured. Furthermore, the preventive mechanisms of CSE and DMC on DEN-induced HCC, including cell proliferation and apoptosis, were investigated. Administering CSE at a dosage of 400 mg/kg bw and DMC at a dosage of 10 mg/kg bw significantly decreased the number and size of GST-P-positive foci and GST-P expression. In addition, DMC inhibited the development of preneoplastic lesions by decreasing cell proliferation and causing cell apoptosis; however, CSE inhibited the development of preneoplastic lesions by inducing cell apoptosis. In conclusion, DMC exhibited a cancer chemopreventive effect on the early stages of hepatocarcinogenesis by increasing cell apoptosis and reducing cell proliferation.

Keywords: 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone; Cleistocalyx nervosum var. paniala seed; cancer chemoprevention; hepatocarcinogenesis.

Figure 1

Effects of CSE and DMC on levels of (A) AST and (B) ALT in rats. The results are presented as mean ± SD. Diethylnitrosamine (DEN); normal saline solution (NSS); C. nervosum seed extract (CSE); 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC). * Significantly different compared to the NSS-treated group (p < 0.05). ** Significantly different compared to the DEN-treated group (p < 0.05).

Figure 2

Effects of CSE and DMC on glutathione S-transferase placental form (GST-P)-positive foci expression in rat liver. Diethylnitrosamine (DEN); normal saline solution (NSS); C. nervosum seed extract (CSE); 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC). (A) Immunohistochemistry; (B) Western blot analysis. Values are expressed as the mean ± SD. G1 through G8 are fully described in Section 4.3. * Significantly different compared to the NSS-treated group (p < 0.05). ** Significantly different compared to the DEN-treated group (p < 0.05).

Figure 3

Effects of CSE and DMC on cell proliferation and apoptosis in rat liver. The number of (A) PCNA-positive and (B) TUNEL-positive cells in rat liver. Values are expressed as the mean ± SD. Diethylnitrosamine (DEN); normal saline solution (NSS); C. nervosum seed extract (CSE); 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC). * Significantly different compared to the NSS-treated group (p < 0.05). ** Significantly different compared to the DEN-treated group (p < 0.05).

Figure 4

Effects of CSE and DMC on the expression of proteins associated with proliferation and apoptosis in rat liver. (A) PCNA and (B) cleaved caspase-3. Values are expressed as the mean ± SD. Diethylnitrosamine (DEN); normal saline solution (NSS); C. nervosum seed extract (CSE); 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC). * Significantly different compared to the NSS-treated group (p < 0.05). ** Significantly different compared to the DEN-treated group (p < 0.05).

Figure 5

A schematic overview of the experimental design for studying the cancer chemopreventive effects of CSE and DMC on the DEN-induced early stages of hepatocarcinogenesis in rats. Diethylnitrosamine (DEN); normal saline solution (NSS); C. nervosum seed extract (CSE); 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC).