Effect of Smilax china L.-containing serum on the expression of POLD1 mRNA in human hepatocarcinoma SMMC-7721 cells

Abstract

Bock greenbrier rhizome, also known as Smilax china L. rhizome, induces heat clearing and detoxification and dispels wind dampness. Additionally, this Chinese medicine has been shown to function as an anticancer compound in various types of cancer. The aim of the present study was to investigate the mechanism by which Smilax china L.-containing serum suppresses SMMC-7721 human hepatocellular carcinoma (HCC) cell growth as well as to determine its effect on the expression of DNA polymerase δ catalytic subunit gene 1 (POLD1). SMMC-7721 human HCC cells were cultured with serum containing various amounts of Smilax china L. for 24 h. The cells were also cultured in blank serum or serum containing a drug used in Western medicine (cyclophosphamide; CTX) as a positive control. HCC cell growth and proliferation were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution and apoptosis were analyzed by flow cytometry, and the expression of POLD1 mRNA was detected by quantitative polymerase chain reaction (PCR). The number of cells following culture with Smilax china L.-containing serum was observed to be decreased. There was significant growth inhibition in the Smilax china L.-treated cells (shown in the high concentration serum group, volume fraction 30%), which was significantly different from the inhibition observed in the control group (P<0.05). Among the various cell cycle phases following culture, the percentage of cells in the S phase was significantly increased, and the percentage of cells in the G0/G1 phase was decreased; these percentages were significantly different from the percentages of the control cells (P<0.05). The results obtained following quantitative PCR showed a significant reduction in POLD1 expression. Smilax china L.-containing serum directly suppressed cell growth and induced the apoptosis of human HCC cells. However, the number of cells in the S phase was reduced. This mechanism is suggested to be associated with the suppression of POLD1 expression.

Keywords: POLD1; SMMC-7721 human hepatocarcinoma cell; Smilax china L.; drug-containing serum.

Figure 1.

Percentage of apoptotic SMMC-7721 cells following treatment with serum from various groups. (A) Blank low-concentration group; (B) blank high-concentration group; (C) CTX low-concentration group; (D) CTX high-concentration group; (E) Smilax china L. low-concentration group; (F) Smilax china L. high-concentration group. CTX, cyclophosphamide.

Figure 2.

Cell cycle distribution of SMMC-7721 cells following treatment with serum from various groups. (A) Blank low concentration group; (B) blank high concentration group; (C) CTX low concentration group; (D) CTX high concentration group; (E) Smilax china L. low concentration group; (F) Smilax china L. high concentration group. CTX, cyclophosphamide.

Figure 3.

Diagrams of the reference genes used and the target genes investigated in quantitative polymerase chain reaction (PCR) analyses. (A) Internal control standard curve; (B) POLD standard curve; (C) internal control solubility curve; (D) POLD solubility curve; (E) internal control amplification curves; (F) POLD amplification curves compositions and multi-interference factors. POLD1, DNA polymerase δ catalytic subunit gene 1.