Synthetic Tryptanthrin Derivatives Induce Cell Cycle Arrest and Apoptosis via Akt and MAPKs in Human Hepatocellular Carcinoma Cells

Abstract

Trytanthrin, found in Ban-Lan-Gen, is a natural product containing an indoloquinazoline moiety and has been shown to possess anti-inflammatory and anti-viral activities. Chronic inflammation and hepatitis B are known to be associated with the progression of hepatocellular carcinoma (HCC). In this study, a series of tryptanthrin derivatives were synthesized to generate potent anti-tumor agents against HCC. This effort yielded two compounds, A1 and A6, that exhibited multi-fold higher cytotoxicity in HCC cells than the parent compound. Flow cytometric analysis demonstrated that A1 and A6 caused S-phase arrest and downregulated the expression of cyclin A1, B1, CDK2, and p-CDC2. In addition to inducing caspase-dependent apoptosis, A1 and A6 exhibited similar regulation of the phosphorylation or expression of multiple signaling targets, including Akt, NF-κB, and mitogen-activated protein kinases. The anti-tumor activities of A1 and A6 were also attributable to the generation of reactive oxygen species, accompanied by an increase in p-p53 levels. Therefore, A1 and A6 have potential clinical applications since they target diverse aspects of cancer cell growth in HCC.

Keywords: ROS; apoptosis; cell cycle arrest; hepatocellular carcinoma; tryptanthrin.

Figure 1

Tryptanthrin as the lead compound to synthesize anti-tumor agents. (A) Structure of tryptanthrin. (B) Structures and potencies of the tryptanthrin derivatives (A1–A12) in Hep3B cells. Cell viability was examined via MTT assay and the IC₅₀ values were measured 50% relative to DMSO.

Figure 2

(A) Anti-proliferative effects of tryptanthrin, (B) A1, (C) A6, and (D) sorafenib in Hep3B cells. Cells were treated with the above compounds for 24 h or 48 h, and cell viability was detected via MTT assay. Points, means; bars, S.D. (n = 3). * p < 0.05, ** p < 0.01.

Figure 3

A1 and A6 induce cell cycle arrest. (A) Cell cycle analysis of A1- and A6-treated Hep3B cells for 48 h. Cell cycle population in each histogram was indicated. The first peak is the G1 phase and the second peak is the G2/M phase. Etoposide (5 μmol/L) was used as a positive control. (B) Effects of A1 and A6 on the phosphorylation/expression of cyclin A1, cyclin B1, CDC-2, and CDK2 in Hep3B cells.

Figure 4

A1 and A6 induce apoptosis in Hep3B cells. (A) Upper panel, flow cytometric analysis of apoptotic cells after treatment with A1 or A6 for 48 h in Hep3B cells. Lower panel, statistical analysis of apoptotic cells after treatment with A1 or A6 in Hep3B cells. Points, means; bars, S.D. (n = 3). * p < 0.05, ** p < 0.01. (B) Caspase-3 activation by A1 and A6 in Hep3B cells using flow cytometry. Saturosporine (Stauro., 25 nmol/L) was used as a positive control. Points, means; bars, S.D. (n = 3). * p < 0.05, ** p < 0.01. (C) Effects on caspase-8 after treatment with A1 and A6.

Figure 5

Effects of A1 and A6 on Akt/NF-κB and MAPKs in Hep3B cells. (A) Phosphorylation/expression of Akt, NF-κB, IκBα, ERK, JNK, and p38 after treatment of Hep3B cells with A1 or A6. (B) Western blot analysis of the nuclear expression of NF-κB. Proteins from nuclear cellular fractions were isolated from Hep3B cells treated with A1 or A6 for 48 h. Fibrillarin was used as a nucleus-specific loading control. (C) Phosphorylation and expression of p38 in cells treated with A6 (0.5 μmol/L) alone or pre-treated with SB203580 (20 μmol/L) for 15 min in Hep3B cells.

Figure 6

Analysis of reactive oxygen species (ROS) in A1- and A6-treated HCC cells. (A) Cells were treated with A1, A6, or DMSO for 3 h and stained with carboxy-DCFDA. H₂O₂ (300 μmol/L) was used as a positive control. (B) Left, cells were treated with A1 or in combination with N-acetylcysteine (NAC) or glutathione (GSH) for 3 h. Right, cells were treated with A6 or in combination with NAC or GSH for 3 h. Data are presented as the mean ± S.D. (n = 3). ** p < 0.01. (C) Expression of p-p53 and p53 after treatment with A1 and A6 in Hep3B cells and (D) SK-Hep1 cells. Phosphorylation and expression of p53 in Hep3B cells treated with (E) A1 (5 μmol/L) and (F) A6 (2.5 μmol/L) alone or co-treated with pifithrin-α (10 μmol/L) for 48 h.