Diterpenoid C of Radix Curcumae: An inhibitor of proliferation and inducer of apoptosis in human colon adenocarcinoma cells acting via inhibiting MAPK signaling pathway

Abstract

Context: Radix Curcumae is a traditional Chinese medicine that possesses antitumor properties, from which a new compound, diterpenoid C, was previously isolated and characterized.

Objective: In this study, using human colon adenocarcinoma SW620 cells, we further investigated the antitumor effects of diterpenoid C and the underlying mechanisms.

Materials and methods: Cell proliferation was assessed with the MTT assay. Cell apoptosis and cell-cycle progression were analyzed with flow cytometry. The expression of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), and their phosphorylated forms, as well as caspase-3 protein levels were examined with Western blots.

Results: Diterpenoid C could inhibit the proliferation of SW620 cells in a dose- and time-dependent manner. The median inhibitory concentration (IC₅₀) at 24, 48, and 72 h were 28.31, 15.58, and 6.14 μg/ml, respectively. The inhibition of proliferation was found to be statistically significant as compared with the well-established drugs 5-fluorouracil (5-Fu) and oxaliplatin (L-OHP) (p < 0.01). Diterpenoid C also induced apoptosis and arrested cell cycle. It showed the highest apoptosis rate (98.20 ± 0.91%) at 70 μg/ml, at 72 h. Meanwhile, diterpenoid C suppressed the phosphorylation of ERK, JNK, and p38 MAPK proteins, and markedly induced the cleavage of caspase 3.

Discussion and conclusion: Diterpenoid C inhibits proliferation and induces apoptosis of cancer cells by suppressing the MAPK signaling pathway and inducing apoptotic factor caspase-3. Our results suggest that this novel compound might become a potent chemotherapeutic agent for the treatment of colon cancer and further studies are warranted.

• Apoptosis
• caspase-3
• colon cancer
• diterpenoid C
• MAPK
• SW620 cell
• Zingiberaceae

Introduction

Human colon carcinoma is the third highest incidence and death rate among all types of cancers identified globally (Kohler et al., 2011; Siegel et al., 2013). According to the multi-step theory of tumourigenesis, it develops through a series of stages as follows: normal mucosa, proliferative mucosa, polyp, carcinoma in situ, and infiltrating carcinoma (Fearon, 2011). Many signal transduction pathways are involved in these processes; particularly, the MAPK pathway plays a very important role and its dysregulation contributes to the oncogenesis of colon carcinoma (Slattery et al., 2012; Zenonos & Kyprianou, 2013).

Currently, aggressive chemotherapy and radiotherapy are commonly used to treat late-stage colon carcinoma. However, the outcomes are still not satisfactory (Ngan et al., 2012; Saltz, 2008). For such patient, the 5-year survival rate is low, as compared with the high cost of treatment (Heitman et al., 2010; Ritchie et al., 2013). In addition, the reported side effects are usually severe, and the cancer cells are reported often to develop drug resistance (Crawford, 2013). Therefore, exploring anti-cancer remedies are considered mandatory, and currently medicinal plants used in traditional Chinese medicine are considered as a new strategy for cure.

Radix Curcumae is the dry root of Curcuma wenyujin Y. H. Chen et C. Ling (Zingiberaceae) which is commonly used in TCM. A wide range of pharmacological functions, including antiatheroscloresis, liver protection, radioresistance, and antiallergy, are attributed to C. wenyujin. Recently, its anti-tumor effects have attracted increasing scientific attention. Extracts of Radix Curcumae can inhibit the formation and development of gastric cancer in vivo and in vitro (Cao et al., 2013; Lu et al., 2008). For example, its ether- and ethanol-extracts, curcumin and elemene, respectively, are powerful anti-cancer ingredients (Bar-Sela et al., 2010; Cao et al., 2013; Dai et al., 2013; Li et al., 2010).

Previously, we isolated and purified a new compound, diterpenoid C, from Radix Curcumae, and characterized its chemical properties that are distinctly different from curcumin and elemene (Ma et al., 2009). In continuation of our work, now we explored the anti-tumor potential and mechanism of action of the newly isolated diperpenoid C. The study included the treatment effect on the proliferation, apoptosis, and cell cycle of human colon carcinoma SW620 cells. The effect of diperpenoid C. on MAPK pathways in these cells is also a part of the current investigations and the results are presented in the current communication.

Methods

Reagents, instruments, and equipments

L-15 medium, penicillin, streptomycin, l-glutamine, fetal bovine serum (FBS), and phosphate buffered saline (PBS) were purchased from Invitrogen (New York, NY). All the primary antibodies used in the study, including antibodies against phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, caspase-3, and β-actin, were purchased from Cell Signaling Technology (Danvers, MA). The secondary antibodies, horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology (Paso Robles, CA). 5-Fluorouracil (5-Fu) was purchased from Tianjin Kingyork Group (Tianjin, China), l-OHP was purchased from Jiangsu Hengrui Medicine Corporation (Jiangsu, China), and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit was purchased from Sigma (St Louis, MO). Diterpenoid C, molecular weight 380, formula C₂₂H₃₆O₅ (Ma et al., 2009), was isolated from a TCM C. Wenyujin, and its structure is as follows.

The major instruments and equipment used in this study included a light microscope (OLYMPUS, Center Valley, PA), a CO₂ incubator (Thermo, Waltham, MA), a biological safety operation counter (Thermo, Waltham, MA), a centrifuge (Thermo, Waltham, MA), a microplate reader (BioTek Instruments Inc., Winooski, VT), and a fluorescence-activated cell sorting (FACS) caliber (Becton Dickinson, Franklin Lakes, CA, USA).

Cell line and culture conditions

Human SW620 colon carcinoma cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shangai, China) and maintained in Leibovitz’s L-15 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin, and streptomycin (the complete medium). Cells were grown at 37 °C in a humidified cell culture incubator under 5% CO₂, and exponentially growing cells were used for the following experiments.

For cell treatment, diterpenoid C, in powder form, was reconstituted in dimethyl sulfoxide (DMSO) to produce a stock solution (10 mg/ml) and was then further diluted with culture medium to various concentrations as working solutions for each experiment. 5-Fu and L-OHP, two chemotherapeutic agents, were included in the experiment as the positive control, and their stock solution in DMSO and working solutions in the complete medium were made similarly as for diterpenoid C. To minimize the effect of DMSO on cell growth, the final concentration of DMSO in the culture media was 0.1%.

Cell proliferation assay

Cell proliferation was assessed by the MTT assay. SW620 cells were seeded at 1 × 10⁴/well and HUVEC cells were seeded at 5 × 10³/well into 96-well plates in the complete media. After 24 h, media were replaced with fresh medium containing 10–70 μg/ml diterpenoid C in a final volume of 200 μl. Cells were treated in triplicates, and medium without drugs was added into some cells as the negative control. The blank wells with medium alone (without cells) served as a blank control. Cells treated with the same concentration of 5-Fu and L-OHP were used as a positive control. At 24, 48, and 72 h, the culture media were removed, 20 μl MTT (5 mg/ml) in PBS was added to all the wells, and the incubation continued for another 4 h at 37 °C. The purple-blue MTT formazan precipitates in wells were dissolved in 150 μl DMSO after oscillating for 10 min, and then the absorbance (A) was recorded in a micro plate reader at 490 nm. Since the absorbance is proportional to the cell growth rate, the inhibition rate on cell proliferation can be calculated by the formula:

Assessment of apoptosis and cell-cycle distribution

Cells were stained by annexin V, using the annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions, and percentages of annexin V-fluorescein positive cells were estimated by flow cytometry analysis. Briefly, cells were seeded at 6 × 10⁵/well into 60 mm dishes in complete media. After 24 h, media were replaced with diterpenoid C working solution at 40, 55, and 70 μg/ml in a final volume of 3 ml. Cells were treated in triplicates, medium without drug was added into some cells as the negative control, and blank wells with medium alone without cells were used as the blank control. At 12, 24, 48, and 72 h, cells were digested by EDTA-free trypsin, rinsed two times in PBS, pelleted by centrifugation (2000 rpm for 5 min), and then resuspended in ice-cold binding buffer at a concentration of 10⁶ cells/ml. The cell suspension (500 μl) was mixed with 5 μl of Annexin V-FITC solution, incubated for 5–15 min at room temperature in the dark, and subjected to FACS analysis for apoptotic rate. Another 500 μl aliquot was first fixed with ice-cold 75% ethanol for at least 1 h, and then treated with 1 ml PI solution. After incubation for 20 min, the mixture was analyzed by FACS to determine cell-cycle distribution.

Western blot

Western blot analysis was performed according to the instructions provided by Cell Signaling Technology (Danvers, MA) with minor modifications. Briefly, cells (2 × 10⁵/well) were seeded in 50 ml flasks, incubated with 40, 55, and 70 μg/ml diterpenoid C for 48 h in a final volume of 5 ml. Cells were then lysed with protein lysis buffer RIPA. Equal amounts of total proteins in the cell lysates (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) for about 1.5 h at 80–100 V. The separated proteins were then transferred electrophoretically to PVDF membrane for 2.5 h at 250 mA. The membranes were blocked with TTBS containing 5% BSA overnight, and then incubated for 2 h with primary antibodies diluted in TTBS at 4 °C. After washing with TTBS buffer, cells were incubated with suitable secondary antibodies for 1 h. To verify equal loading of samples, the membranes were subsequently incubated with monoclonal antibody against β-actin followed by a horseradish peroxidase conjugated sheep anti-mouse IgG. Protein bands were visualized after the blots were reacted with the ECL western blotting detection reagents.

Statistical analysis

All data were subject to statistical analyses using SPSS 17.0 software package (SPSS Inc., Chicago, IL). Single comparison between two groups was made by independent sample t-test, and multiple comparisons between data sets were performed using a one-way analysis of variance (ANOVA) followed by least-significant difference (LSD) test. Differences were considered to be significant at the p < 0.05 level.

Results

Diterpenoid C inhibits proliferation of human SW620 colon carcinoma cells

As shown in Figure 1, diterpenoid C, 5-Fu, and L-OHP can all inhibit the proliferation of SW620 cells in dose- and time-dependent manners. The IC₅₀ for diterpenoid C were 28.31, 15.58, and 6.14 μg/ml, respectively, at 24, 48, and 72 h. The inhibitory effect of diterpenoid C was significantly stronger than that of 5-Fu and L-OHP used as the positive control at all the time points (p < 0.01). At 60 μg/ml, diterpenoid C showed the highest inhibition at 72 h, and reduced 95.30 ± 1.5% of cell growth. It is also interesting to note that diterpenoid C only exerted mild cytotoxic effects on non-tumorigenic HUVEC cells, with IC₅₀ value >200 μg/ml at 48 h, while 5-Fu and L-OHP had a more potent cytotoxic effect on HUVEC cells, with an IC₅₀ value of 127.837 and 100.953 at 48 h, respectively (Figure 2). It is distinct that diterpenoid C exhibited a desirable selective cytotoxic profile on human colon adenocarcinoma SW620 cells.

Figure 1. Effects of diterpenoid C, 5-Fu, and L-OHP on the proliferation of SW620 cells. N = 3,  ± s. (A) Three drugs at 24 h. (B) Three drugs at 48 h. (C) Three drugs at 72 h. *p < 0.01 versus the 5-Fu group, Δp < 0.01 versus the L-OHP group.

Figure 2. Effects of diterpenoid C, 5-Fu, and L-OHP on the proliferation of HUVEC cells at 48 h. n = 3,  ± s.

Diterpenoid C induces apoptosis of SW620 cells

After treatment with diterpenoid C at different concentrations for 48 h, under a light microscope, the effect of SW620 cells displayed was that dose- and time-dependent morphological changes which are consistent with cell death such as shrinking, dissociation from the culture dishes, and moreover, releasing cellular debris at the group exposed to the highest concentration (70 μg/ml). Apoptosis of the cells in response to diterpenoid C was quantified with annexin V-PI double staining followed by flow cytometry analysis. As shown in Figure 3(A), cells treated with diterpenoid C for 12 h resulted in a clear shift from live cell population to early and late apoptotic cell population, and this effect was augmented at 24, 48, and 72 h (Figure 3B–D). Diterpenoid C induced a significant increase in the early and late apoptotic cells dose and time dependently. In contrast, cells received no treatment showed no significant cell death.

Figure 3. Flow cytometry analysis for apoptosis of SW620 cells induced by diterpenoid C. n = 3,  ± s. (A(a–d)) Diterpenoid C at 12 h. (B(a–d)) Diterpenoid C at 24 h. (C(a–d)) Diterpenoid C at 48 h. (D(a–d)) Diterpenoid C at 72 h. *p < 0.01 versus the control group.

Diterpenoid C induces cell-cycle arrest at G₁ and S phases in SW620 cells

The effect of different concentrations of diterpenoid C on cell-cycle progression was studied after 24 and 48 h of drug exposure. Diterpenoid C treatment resulted in a dose- and time-dependent accumulation of cells in G₁ and S phases with concomitant loss from the G₂ phase. The changes in cell cycle were much more dramatic at 48 h (Figure 4). We can also notice the hypodiploid apoptosis peak at 55 and 70 μg/ml, at 48 h, demonstrating the markedly apoptotic induction effect of diterpenoid C. These results were well consistent with the anti-proliferation effect of diterpenoid C in SW620 cells.

Figure 4. Flow cytometry analysis of cell-cycle distribution effected by diterpenoid C. n = 3,  ± s. (A(a–d)) Diterpenoid C at 24 h. (B(a–d)) Diterpenoid C at 48 h. *p < 0.01 versus the control group, Δp < 0.01 versus the 24 h group.

Diterpenoid C inhibits MAPK pathway and activates caspase 3 in SW620 cells

As is shown in Figure 5(A–C), following lower concentration treatment of diterpenoid C (40 μg/ml), ERK, and JNK levels were almost similar to that of control, and p38 MAPK level even remained unchanged at any concentration group. At the same time, diterpenoid C could inhibit phosphorylation of ERK, JNK, and p38 MAPK after 48 h, with a more distinct response observed at high concentration (55 and 70 μg/ml). In contrast, we can clearly find the proteolytic activation of pro-caspases 3 in SW620 cells exposed to diterpenoid C, thus markedly induced the cleavage of caspase 3, and the activating effect was dose dependent (Figure 5D). These results were in agreement with the anti-proliferation and pro-apoptotic effect of diterpenoid C in SW620 cells.

Figure 5. Effects of diterpenoid C on MAPK signaling pathway and caspase-3 in SW620 cells treated for 48 h. Representative western-blot images showing protein levels for ERK and phosphor-ERK (A), JNK and phosphor-JNK (B), p38 and phosphor-p38 (C), pro-caspase 3 and cleaved caspase 3 (D), as indicated. Graphs under each Western-blot image shows protein levels for each protein normalized with that of β-actin. Data are presented as mean ± SD from three repeated measurements; *p < 0.05 versus the control group, **p < 0.01 versus the control group.

Discussion

Radix Curcumae, one of the commonly used traditional Chinese medicine in clinics, has attracted great scientific attention due to its anticancer activity. In our previous studies, Radix Curcumae extracts decocted by water, petroleum ether, or ethanol can inhibit the proliferation and growth of human gastric cancer SGC-7901 cells; while the ether and ethanol soluble extracts even induce apoptosis of these cells (Xu et al., 2004a). The apoptosis promoting effects may be mediated through down-regulating the expression of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) to reduce microvascular density in focal tumors, increasing the level of somatostatin in the circulation and tissues, and inhibiting the secretion of insulin-like growth factors I and II (He et al., 2004; Lu et al., 2010; Xu et al., 2004b).

A number of studies have explored the utility of active constituents in radix curcumae extracts. The main ingredient of ethanol extract curcumin exerts its anticancer function by targeting cancer cells themselves, or cancer-related proteins (Guo et al., 2013; Li et al., 2014), genes (Nasiri et al., 2013) or components in signal transduction pathways (Han et al., 2012). The major component of petroleum ether extract elemene also induces apoptosis of cancer cells via multiple mechanisms. It can block cell-cycle progression through inhibiting the expression of Bcl-2 and VEGF, and therefore activates cytochrome C and caspase signaling cascades (Chen et al., 2011; Li et al., 2010; Wang et al., 2005). In order to identify and develop other potentially novel anticancer components from Radix Curcumae, we further purified a new diterpenoid C from petroleum ether extract, and identified its chemical structure and properties that are different from those of curcumin and elemene (Ma et al., 2009). Our preliminary data showed that it promotes apoptosis of SGC7901 cancer cells by arresting cell cycle and blocking the activation of caspase cascade.

In the present study, diterpenoid C showed a strong inhibition of the proliferation of colon adenocarcinoma cells, which is more potent than that of 5-Fu and L-OHP, the efficient chemotherapy drugs for colorectal cancer widely used in the clinic, at all the time points. In addition, diterpenoid C only exhibited mild cytotoxic effects on non-tumorigenic HUVEC cells, which demonstrated its satisfactory selective cytotoxic profile on SW620 cells. Our present results also reveal that diterpenoid C arrests cell-cycle progression of the cancer cells at G₁ and S stages and promotes apoptosis. These results suggest that the newly isolated diterpenoid C should be the major active component in Radix Curcumae, and it might serve as a potent chemotherapeutic agent for colon cancer treatment.

Although carcinogenesis is a complex process involving many cellular components and undergoing multiple stages (Migheli & Migliore, 2012; Pritchard & Grady, 2011), the transformation from normal colon epithelial cells to cancerous cells is often associated with over-activation of MAK kinase pathways. MAP kinases are serine/threonine protein kinases that transduce a wide range of extracellular stimuli to diversify intracellular signaling events to regulate gene expressions and various cellular activities such as cell proliferation, growth, differentiation, and cell survival/apoptosis (Cobb & Goldsmith, 1995; Zenonos & Kyprianou, 2013). Three members in the MAPK superfamily have been identified in mammalian cells, namely ERK, SAPK/JNK, and p38MAPK.

Overexpression or over-activation of these MAPKs has been implicated in the carcinogenesis in many digestive system tumors (Hsieh et al., 2007; Mishra et al., 2010; Slattery et al., 2013; Wang et al., 2010). ERK responds to mitogenic stimuli and plays a particularly critical role in cell growth, survival and differentiation (Lai et al., 2010; Mao et al., 2008); while JNK/p38MAPK are activated mostly in response to a variety of stress signals and pathological damages as well as mitogenic stimuli. Their effects can be manifest by promoting or inhibiting apoptosis, which mainly depends on the signal patterns and/or cell subtypes (Chatzinikolaou et al., 2010; Zhang et al., 2010).

Caspase-3 is the downstream protein component of the classical apoptotic pathway. It exists as the inactive enzyme precursor in normal physiological conditions, and it becomes activated by hydrolyzation and forms a heterodimer upon stimulation of extracellular pro-apoptotic signals. Caspase-3 heterodimer is the most critical apoptosis mediator that initiates most apoptosis events (Alenzi et al., 2010). The activity of caspase-3 has been shown to be a marker for early diagnosis, histological grading and prognosis of various tumors.

Through western blot analysis, we could observe the attenuation of phosphorylated ERK, JNK, p38 MAPK and pro-caspase 3 proteins, as well as the augmentation of cleaved caspase 3 protein. We found that with the treatment of diterpenoid C, the phosphorylation of ERK, JNK, and p38 MAPK in SW620 cells was inhibited, while the proteolysis of pro-caspases 3 was dramatically activated. Therefore, it is possible that diterpenoid C exerts its antitumor function through down-regulating cell proliferation pathway mediated by the ERK signaling, inhibiting the protective effects of the JNK/p38 pathway, and activating the apoptosis triggered by caspase-3.

Conclusions

Our experiments have established that diterpenoid C is a potent growth inhibitor of colon adenocarcinoma SW620 cells. Its anticancer activity involves induction of apoptosis through inhibition of MAPK pathways and activation of caspase-3 apoptotic factor. Although more in-depth research is needed to explore its clinical application, this new Radix Curcumae-derived component, diterpenoid C, may be a new generation of anticancer therapy against colon cancer.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported by Traditional Chinese Medicine Science and Technology Plan Projects of Zhejiang Province (2009CB014).