Structure-activity relationship of Triterpenes and derived Glycosides against cancer cells and mechanism of apoptosis induction

Abstract Triterpenoids possess a wide range of biological effects. Here, the cytotoxic activities of 55 triterpenes and derived glycosides against BEL-7404 and SGC-7901 cells were assessed, and structure-activity relationships were analysed accordingly. Nine of them effectively inhibited the two cell lines. In particular, compounds 49 and 52 inhibited BEL-7404 cells as efficiently as 5’-fluorouracil (IC50 values 0.46 and 1.48, respectively). Moreover, we found that compounds 49 and 52 induced apoptosis in BEL-7404 cells. Indeed, DNA fragmentation assay showed a time-dependent degradation of DNA after treatment of cells with compounds 49 and 52. In addition, Bax gene expression levels were increased after treatment with these compounds, in a concentration-dependent manner. Taken together, our findings suggested that compounds 49 and 52 induce apoptosis in BEL-7404 cells by upregulating the Bax gene without affecting Bcl-2 gene expression.


Introduction
Natural products have historically served as a major source of new leads for pharmaceutical development, especially in cancer therapy (Yang et al. 2007;Costa et al. 2008). Triterpenoids possess a wide range of biological effects, including anti-inflammatory, hepatoprotective, analgesic, antimicrobial, antimycotic, virostatic, immunomodulatory and tonic properties (Dzubak et al. 2006). However, systematic studies assessing the cytotoxic effects of these compounds are scarce.
Apoptosis is a selective process of physiological cell deletion that regulates the balance between cell proliferation and cell death. Out-of-control cell growth has been reported in many cancer cells (Ming et al. 2007). Therefore, developing new therapeutic and preventive strategies targeted at apoptosis could be effective in controlling the proliferation and invasiveness of cancer cells (Oh & Lee 2004;Akter et al. 2016;Zhang et al. 2016).
Previous research has demonstrated that the BCL-2 family of proteins play important roles in apoptosis through the balance between the anti-apoptotic (e.g. Bcl-2, Bcl-xL and Mcl-1) and pro-apoptotic (e.g. Bak, Bax, Bad and Bid) members (Vargas-Roig et al. 2008). Strategies for countering Bcl-2 family in cancer include upregulating the opposing proapoptotic family members such as Bax with p53-, Mda7 (IL-24)-or Bax-adenovirus gene therapy for loco-regional cancer control (Reed 2003). Therefore, it might be possible to induce Bax or Bak gene expression using drugs (Letai et al. 2002).
In this work, we screened 55 triterpenoids for cytotoxicity against SGC-7901 and BEL-7404 cells. Some of them showed significant inhibitory effects. In order to identify their mechanisms of action, the effects of these compounds on DNA degradation as well as Bax and BCL-2 protein expression were evaluated. Interestingly, DNA degradation occurred after cell treatment with both 20 α-Hydroxydammar-24-en-3one (49) and 3-oxo-urs-12-ene (52) from 12 h incubation, and extensively at 48 h. In addition, the two active compounds upregulated Bax protein levels without altering Bcl-2 expression. These results indicated that compounds 49 and 52 induce apoptosis via Bax upregulation, independently from Bcl-2. Our findings provide a basis for further research on the mitochondrial pathway in BEL-7404 cells after apoptosis induction by active compounds.
Multiple studies have suggested that triterpenes are cytotoxic to several cancer cell lines in vitro and in vivo (Sakurai et al. 2003;Liu et al. 2004;Chiang et al. 2005;Tian et al. 2005;Jayaprakasha et al. 2008;Yamaguchi et al. 2008). For example, increasing evidence indicates that ginsenoside is responsible for many biological activities, including antitumour activities, observed in cell cultures and in vivo following intraperitoneal or intravenous injection of experimental animals (Al-Fatimi et al. 2007). Here, we assessed 55 triterpenes and related glycosides, whose antitumour activity (Table S1) was significantly correlated with their structures.
The 55 compounds included 52 and 2 pentacyclic and tetracyclic triterpenes, respectively, and an 18, 19-seco-ursane. Among them, 47 were saponins containing one to six sugar molecules. Interestingly, highly polar compounds, linked to two to four sugar molecules at the C-3 position, presented less cytotoxicity than those linked to a hydroxyl group or one sugar molecule (Table S1 and Figure S1). The cytotoxic activities of ursane type triterpenoids, as a whole, were higher than those obtained for oleanane type compounds. These findings corroborate the results reported for triterpenes and related glycosides against a plant virus (Tobacco mosaic virus). Cytotoxic activity against BEL-7404 cells was significantly affected by the C-3, C-23 and C-19 positions of triterpenes. At the same concentration (200 μg/mL), compounds 10, 12, 32 and 45 showed higher cytotoxicity compared with other active molecules such as 6, 42, 43 and 49-53. With hydroxyl or monosaccharide at the C-3 position, hydroxyl at C-19, or conjugation with double bond of C-12, 13, the compounds displayed the best cytotoxic activity against the two cell lines. Although compounds 49 and 52 at 200 μg/mL showed medium cytotoxic activities, they had similar IC 50 values to those of 5-FU or CDPP towards BEL-7404 cells. These pronounced activities should be much suitable to normal cells because the compounds act by chemical cytotoxicity in a non-selective manner.
Regarding the antitumour activities of compounds 18-25 ( Figure S7), oblonganoside M (18) showed an overtly higher activity (inhibition rates of 74.91% and 75.09% towards BEL-7404 and SGC-7901, respectively) compared with the other saponins, especially compound 20, which was completely inactive. The difference between them lies mainly in the monosaccharide residue: others had two or three monosaccharide residues. What's more, cytotoxic activity appeared to decrease with increasing number of sugar molecules. Our current hypothesis is that there is a negative relationship between cytotoxicity and the number of sugar molecules in saponins, which was also demonstrated by the activities of compounds 1-5, 6-9 and 10-17 ( Figure S4-6). Compounds 1-5 ( Figure S4) with 3-5 sugars were weakly active or completely inactive. As shown in Figure S5, except kudinoside (6) that showed an inhibitory activity higher than 60%, the remaining compounds with three or more sugars showed only a slight activity or were completely inactive. However, ziyuglucoside (12) with two sugars had high inhibitory activity at 86.95% and 83.51% against BEL-7404 and SGC-7901, respectively, while pomolic acid-28-O-β-D-glucopyranosyl ester (10) with only one sugar showed even higher activity (81.77% and 86.27%). As for compounds 26-34 ( Figure  S8), oblonganoside J (32) showed high inhibitory rates of 78.22% and 87.91% against BEL-7404 and SGC-7901, respectively. The others showed inhibition rates < 44.98%. The main difference between them is that oblonganoside J (32) has a special substituent at C-3. Compounds 35-41 ( Figure S9) were only weakly active or completely inactive at the tested concentrations. Compounds 42-47 were all triterpenoid saponins ( Figure S10).

Apoptotic levels and Bax gene expression-induced apoptotic effects
Apoptosis is characterised by the degradation of chromosomal DNA at internucleosomal linkages. Typical DNA laddering was observed in cells treated with 20α-Hydroxydammar-24-en-3-one (49) and 3-oxo-urs-12-ene (52) ( Figure S2). DNA degradation exhibited a time-dependent manner, after treatment with 50 μg/mL 3-oxo-urs-12-ene (52): chromosomal DNA began to degrade at 12 h, and continued to 48 h; afterwards, DNA degradation was almost complete. Compound 49 had a similar effect. These data indicated that compounds 49 and 52 inhibited cell proliferation in BEL-7404 cells. No obvious DNA degradation occurred in BEL-7404 cells induced by compounds 50, 51 and 53.
We selected the two most potent compounds (49 and 52) to assess their effects on the apoptosis-related Bcl-2 family of proteins. Bax mRNA levels (Reed 2003;Motomura et al. 2008) were increased after treatment with 20α-Hydroxydammar-24-en-3one (49) and 3-oxours-12-ene (52). In addition, extensive DNA degradation was observed after treating cells with both compounds at 48 h ( Figure S2B). These findings suggested that both compounds induce apoptosis in BEL-7404 cells via upregulation of the Bax gene, with the triggered apoptotic signalling pathway mainly related to mitochondrial pathway (Vargas-Roig et al. 2008).
We did not detect Bcl-2 mRNA, using three different primer pairs, which is consistent with previous reports. Indeed, Bcl-2 gene expression levels vary with hepatoma cell type: some hepatoma cells highly express Bcl-2, whereas this gene is scarcely detected in others (Li et al. 2003). Also, during the apoptosis of some hepatoma cells, it was found that the expression of bcl-2 and bcl-xs was lack, whereas the expression of bcl-xl, bax and bad was variable. That means the gene expression of bcl-2 family had specificity in human hepatoma cells (Luo et al. 2000).
In this work, we only determined the cytotoxic activities of the compounds in vitro, which may not be able to reach target cells in vivo. Ongoing research in our laboratory aims at examining other members in the Bcl-2 family of proteins (e.g. Bak and Bcl-xL) in order to fully characterise the associations of Bcl-2 family members with various signalling pathways in this setting. Such studies would allow further understanding of the molecular mechanisms of apoptosis induced by 20α-Hydroxydammar-24-en-3one (49) and 3-oxo-urs-12-ene (52), and provide the basis for drug molecular therapy in hepatocellular carcinoma.
DAB substrate Kit was purchased from Wuhan Boster Biological Technology, Ltd (Wuhan, Hubei Province, China). Apoptotic DNA Fragment Extraction Kit, RNA isoReagent Kit, One- Step RT-PCR Kit and SYBR Premix Ex Taq Kit were obtained from TaKaRa (Dalian, Liaoning Province, China).

Cell lines and culture
The human gastric cancer cell line SGC-7901 and human hepatoma cell line BEL-7404 were provided by Fujian Medical University, and maintained in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin solution, in a humid environment containing 5% CO 2 at 37 °C.
Compounds were dissolved in DMSO and diluted to the required concentrations before use. Cells grown in media containing equivalent amounts of DMSO with no compound served as negative controls.

MTT assay
Cancer cells were plated onto 96-well culture dishes at a density of 1 × 10 3 /well in 180 μL medium. After plating, the cells were allowed to attach for 24 h, before treatment with compounds at concentrations ranging from 12.5 to 200 μg/mL (using DMSO as vehicle at maximum concentration of 0.1%). After 72 h incubation, 20 μL of 2 mg/mL MTT was added to each well, followed by additional four hours of incubation. Finally, the medium was carefully aspirated, 200 μL DMSO was added to each well, and absorbance read at 570 nm on a microtiter plate reader (Twentyman et al. 1989). 5'-fluorouracial (5-FU) at 50 μg/mL and cisplatin (CDDP) at 20 μg/mL were used as positive controls. Experiments were performed in triplicate. Cytotoxicity was expressed as IC 50 (concentration achieving 50% cytotoxicity, extrapolated from the linear regression analysis of experimental data).

DNA fragmentation and electrophoresis
The apoptotic response was evaluated by DNA fragmentation measurements. Cells were treated with compounds for the indicated times. Then, DNA was extracted using the Apoptotic DNA Fragment Extraction kit, and analysed electrophoretically on 1.5% agarose gel containing ethidium bromide. DNA Molecular Weight Marker was loaded in parallel in each experiment.

Real-time PCR
Real-time PCR was performed with SYBR Premix Ex Taq Kit as described previously (Jiang et al. 2007), with β-actin used as an internal control. The relative abundance of mRNA expression of a control sample was set to 1, and values for the other samples were calculated accordingly. The fluorescence signals were read at the end of each extension step. The threshold cycle was determined for each sample using the exponential growth phase, and the baseline signal from the fluorescence versus the cycle number plots. To ensure that a single product was amplified, melt curve analysis was performed on the PCR products whenever each PCR run was done.

Statistical analysis
Data are mean ± standard deviation (SD) of triplicate samples. Statistical analysis was performed using SPSS 13.0. One-way ANOVA was used to compare groups. p < 0.05 was considered statistically significant.

Conclusion
Nine compounds effectively inhibited the two cell lines. In particular, compounds 49 and 52 inhibited BEL-7404 cells as efficiently as 5′-fluorouracil (IC 50 values 0.46 and 1.48, respectively). Moreover, compounds 49 and 52 induced apoptosis in BEL-7404 cells. Indeed, DNA fragmentation assay showed a time-dependent degradation of DNA after treatment of cells with 49 and 52. In addition, Bax gene expression levels were increased after treatment with these compounds, in a concentration-dependent manner. Taken together, our findings suggest that 49 and 52 induce apoptosis in BEL-7404 cells by upregulating the Bax gene without affecting Bcl-2 gene expression.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Foundation of Key Laboratory of the Tibet Plateau Biotechnology, Ministry of Education (No. 2011), andFujian Natural Science Foundation (No. 2012J01088).