Growth inhibitory, apoptotic and anti-inflammatory activities displayed by a novel modified triterpenoid, cyano enone of methyl boswellates
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- Ravanan, P., Singh, S.K., Rao, G.S.R.S. et al. J Biosci (2011) 36: 297. doi:10.1007/s12038-011-9056-7
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Triterpenoids are pentacyclic secondary metabolites present in many terrestrial plants. Natural triterpenoids have been reported to exhibit anti-inflammatory and anti-carcinogenic activities. Here, we show that modifications of ring A of boswellic acid (2 cyano, 3 enone) resulted in a highly active growth inhibitory, anti-inflammatory, pro-differentiative and anti-tumour triterpenoid compound called cyano enone of methyl boswellates (CEMB). This compound showed cytotoxic activity on a number of cancer cell lines with IC50 ranging from 0.2 to 0.6 μM. CEMB inhibits DNA synthesis and induces apoptosis in A549 cell line at 0.25 μM and 1 μM concentrations, respectively. CEMB induces adipogenic differentiation in 3T3-L1 cells at a concentration of 0.1 μM. Finally, administration of CEMB intra-tumourally significantly inhibited the growth of C6 glioma tumour xenograft in immuno-compromised mice. Collectively, these results suggest that CEMB is a very potent anti-tumour compound.
KeywordsCancer therapeuticscytotoxicitydifferentiationnatural products
cyano enone of methyl boswellates
Dulbucco’s modified eagle’s medium
Dulbucco’s phosphate buffer saline
fetal bovine serum
50% inhibitory concentration
- IFN IFN-γ
induced nitric oxide synthase
transforming growth factor factor-β
The discovery that 2-cyano-3,12-dioxoolean-1,9(11)-diene-28-oic acid (CDDO) (Honda et al.1998), synthesized from naturally abundant oleanolic acid, displayed diverse biological activities ranging from suppression of iNOS and COX2, cellular proliferation of malignant and premalignant cells, to induce differentiation of malignant and nonmalignant cells (Suh et al.1999), has renewed interest in the pentacyclic triterpenoids and natural products. CDDO and its derivatives are highly active in suppressing cellular proliferation of human leukaemia (Suh et al.1999; Konopleva et al.2002; Place et al.2003; Konopleva et al.2004), breast cancer (Honda et al.1999; Lapillonne et al.2003; Konopleva et al.2006) and several other cancer cells. In addition, 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) exhibits strong inhibitory activity against production of NO, induced by IFN-γ in mouse macrophages (IC50 = 0.1 nM) (Honda et al.1998 ; Suh et al.1999). CDDO was also found to induce monocytic differentiation of human myeloid leukaemia cells and adipogenic differentiation of mouse 3T3-L1 fibroblasts (Suh et al.1999; Wang et al.2000). It has been shown that CDDO and CDDO-Im inhibit inflammatory response and tumour growth in vivo (Place et al.2003). Notably, CDDO-Im is approximately 10-fold more potent than CDDO in inhibiting cancer cell proliferation and in inducing differentiation in leukaemia cells (Kim et al.2002). Various derivatives of CDDO also showed potential apoptotic activity on many types of cancer cell lines. For instance, CDDO induces apoptosis of acute myelogenous leukaemia (Konopleva et al.2004), osteosarcoma (Ito et al.2001) and skin cancer (Hail et al.2004). The methyl ester of CDDO, CDDO-Me, triggers apoptosis in lung carcinoma (Kim et al.2002) and prostate cancer cells (Hyer et al.2008) whereas its imidazole ester, CDDO-Im, induces apoptosis in pancreatic (Samudio et al.2005) and ovarian cancer (Petronelli et al.2009). Despite the multifunctional activities of synthetic triterpenoids, the molecular mechanisms that mediate the effects are not fully understood except that CDDO and its derivatives have been shown to induce apoptosis either by activating intrinsic mitochondria-dependent or extrinsic-death-receptor-mediated pathways, depending on the cell types (Ito et al.2000, 2001; Samudio et al.2006; Brookes et al.2007). In addition, it has recently been shown that CDDO-Me activates endoplasmic reticulum stress (ER stress) and thereby triggers the DR5-mediated apoptotic pathway (Zou et al.2008). CDDO and derivative compounds are being used in phase I/II clinical trials as novel cancer therapeutic agents (Liby et al.2007b; Hyer et al.2008). All these studies suggest the potential of modified triterpenoids in anti-inflammatory and anti-carcinogenic applications.
Although, CDDO and its derivatives show great promise as therapeutic molecules, it is important to explore other triterpenoids for their efficacy in these actions, to provide alternatives to CDDO. A recent publication suggests that modified betulinic acid also shows activities similar to those reported for CDDO (Liby et al.2007a). However, it is necessary to explore other triterpenoids because of uncertainties in their metabolic turnover and other potential side effects that may be dependent on the molecular species. To this end, we obtained several triterpenoids available from Indian plants and made chemical modifications that are similar to CDDO. The details of their synthesis, characterization and preliminary activities of these triterpenoids such as inhibition of IFN-γ-induced NO production and cytotoxicity have been reported earlier (Subba Rao et al.2008).
2 Materials and methods
2.1 Cell cultures and reagents
Dimethyl sulphoxide (DMSO), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), phosphoric acid, N-(1-naphthyl) ethyl-enediamine (NED) and sulfanilamide were purchased from Sigma. Dulbucco’s modified eagle’s medium (DMEM), DMEM-F12, RPMI, Dulbucco’s phosphate buffer saline (DPBS), certified fetal bovine Serum, penicillin-streptomycin, amphotericin B and trypsin were purchased from GIBCO. Caspase 8 and Caspase 3 Activity Assay Kits were from Sigma (Product code: CASP-8-F and CASP-3-F). 5-Bromo-2′-deoxyuridine (BrdU), anti-BrdU antibody (ab-3) and Texas red-conjugated goat antimouse antibody were purchased from Oncogene. Human cancer cell lines A549, A431, HT1080, HepG2, HeLa, HCT116, MCF-7, T47D, T84, CaCO2, JEG, PC3, mouse 3T3-L1, Rat C6 glioma cells, and human keratinocyte cell line HaCaT were routinely grown in DMEM with 10% FBS. Jurkat, HL-60, THP-1 and K562 cell lines were grown in RPMI with 10% FBS. Penicillin-streptomycin and amphotericin B were added in the culture to prevent microbial growth. CEMB was synthesized from boswellic acid as described previously. CEMB was dissolved in DMSO at 10 mM concentration and stored at −20°C. The dilutions were made in culture medium just before the treatments.
2.2 Nitric oxide assay
Two-month-old C57black6 mice were injected intraperitoneally with 4% thioglycolate route. On the third day, the mice were sacrificed by cervical dislocation, and 6 ml of 0.32 M sucrose solution was injected in to the peritoneal region, and the macrophages were collected. Cells were centrifuged, washed with DPBS and plated (1.5 × 105 cells/well) in a 96-well plate. The cells were treated with IFN-γ (10 U/ml) in the presence or absence of CEMB and incubated for 3 days. NO was rapidly oxidized to nitrite in the culture medium (RPMI + 10% FBS), and determination of nitrite concentration was used as a measure of NO production. Equal volumes of cell supernatants and Griess reagent (Ding et al.1990) were added to individual wells of a 96-well plate and the absorbance was measured at 590 nm in an ELISA reader. Nitrite concentration was estimated using a sodium nitrite standard curve. Nitrite concentration was normalized with total protein determined by the Bradford method. The experiments were performed multiple times and the result presented here are from three independent experiments. Graph Pad Prism software was used to assess the inhibitory concentration (IC50) of the compound.
2.3 Cytotoxicity assay using MTT reagent
The cells were plated in 96-well tissue culture clusters (seeded at densities ranging from 5 × 103 to 8 × 103 cells/well) depending on the cell line and incubated at 37°C in 5% CO2 atmosphere. After attachment of the cells (usually taking 3–4 h), different concentrations of the compound were added and incubated for 72 h. MTT solution (20 μl of 5 mg/ml) was added to each well and the incubation continued for an additional 3 h. The dark blue formazan crystals formed within the healthy cells were solubilized with DMSO, the plates read in ELISA plate reader (7520 Microplate Reader, Cambridge Technologies Inc.) at 550 nm and the absorbance was correlated with the cell number. Experiments were performed in triplicates and the values are the average of three (n = 3) independent experiments. The inhibitory concentration (IC50) of the compound was assessed by Graph Pad Prism software.
2.4 Cell proliferation assay using BrdU incorporation
BrdU incorporation assay was carried out as described earlier (Wajapeyee and Somasundaram 2003). In brief, cells were plated at a density of 1.2 × 104 cells/well in a 96-well plate. After 24 h, cells were treated with the compound for another 24 h and BrdU (20 μM) was added to the culture medium 4 h prior to the termination. The cells were washed with phosphate buffered saline (DPBS) and fixed with 70% ethanol for 5 min. The cells were washed again with DPBS and incubated with 2 N HCl + Triton × 100 to denature the chromosomal DNA. The cells were washed thrice to completely remove HCl, followed by incubation again with 1% BSA for blocking nonspecific binding. BrdU incorporation was determined by treating with anti-BrdU antibody and Texas red conjugated goat-antimouse IgG.
2.5 DNA fragmentation assay
Low-molecular-weight genomic DNA was extracted as described previously (Yawata et al.1998). In brief, approximately 1 × 106 cells were plated and treated with 1 μM (A549 cells) and 0.5 μM (HL60 cells) of CEMB for various treatment hours. All the cells (including floating cells) were harvested by trypsinization and washed with DPBS. Cells were lysed with the lysis buffer, andtreated 40 mg/ml RNase A and proteinase K at 37°C for 1 h. DNA was precipitated using isopropanol, and subjected on to 1.0% agarose gels. The gels were stained with 1 μg/ml ethidium bromide.
2.6 Caspase activity assay
Caspase assays were performed using fluorometric kits (Sigma-Aldrich, St. Louis, USA). The cells were treated with CEMB (1 μM) for 3, 6, 12 and 24 h and lysed with the lysis buffer (50 mM HEPES, pH 7.4, with 5 mM CHAPS and 5 mM DTT). Later, 100 μl of the lysate was incubated with the caspase 3 or caspase 8 peptide substrates (Ac-DEVD-AMC and Ac-IETD-AMC, respectively) for 1 h as described in the manufacturer’s protocol (Caspase 8 and Caspase 3 Assay Kits, Fluorometric,SIGMA). Caspase inhibitors were also used to confirm the result. The caspase activity was represented as quantity of fluorescent 7-amino-4-methylcoumerin (AMC) released/mg protein/h.
2.7 Oil red O staining
Induction of differentiation in 3T3-L1 cells was performed as described earlier (Wang et al.2000). In brief, 3T3-L1 fibroblasts cells were seeded in 6-well plate at a cell density of 0.4 million cells/well and cultured for around 4 days until it reached confluence. The culture medium was replaced with fresh medium once in 2 days. Cells were treated with CEMB at various concentrations starting from 0.05 μM to 0.25 μM. The standard method to induce adipogenesis (0.5 mM IBMX + 0.5 μM dexamethasone + 1.7 μM insulin) was also included in the experiment to compare the efficiency of CEMB-induced differentiation. To stain the triglycerides, cells were washed twice with PBS and fixed in 3.7% formaldehyde for 1 h followed by oil red O staining as described previously (Hansen et al.1999).
2.8 Tumour xenograft study
Female nude mice at approximately 2 months age and 20–25 g weight were used for tumour xenograft studies. C6 rat glioma cells were injected subcutaneously (3 × 106 cells/mouse) over the left flank of mice (day 0). After 4 days, treatment with CEMB was initiated with CEMB in DMSO, castor oil and PBS mixed in the ratio of 1:1:8 in 0.1 ml solution and injected at the site of the tumours. An equivalent number of mice were injected with vehicle control (DMSO, castor oil and PBS in the same proportions). The treatment was carried out twice daily (every 12 h) for the first 2 days and then daily (every 24 h) for 3 more days. On day 14, the mice were sacrificed and tumours was excised and weighed. A portion of the tumour was fixed in 10% buffered formalin for histological analysis.
3.1 CEMB inhibits NO production in primary mouse macrophages
3.2 Cytotoxic effect of CEMB in tumour cell lines
Cytotoxic effect of CEMB on cancer cell lines
% of cytotoxicity at 1 μM conc.
77.4 ± 11
71.7 ± 7
58.6 ± 7
83.7 ± 1
72.5 ± 6
78.5 ± 3
71.5 ± 9
65.5 ± 0.5
61.4 ± 11
86.6 + 3
79.1 ± 5
56.8 ± 16
68.4 ± 1
60.8 ± 5
82.8 ± 1
79.1 ± 5
Raji (Bcl-2 over expressed)
96.4 ± 0.4
95.0 ± 0.6
3.3 CEMB inhibits DNA synthesis preferentially in carcinoma cells
3.4 CEMB induces DNA fragmentation
3.5 CEMB activates caspase 8 and caspase 3
3.6 Induction of differentiation in 3T3-L1 cells by CEMB
3.7 CEMB suppresses tumour growth in xenografts mouse model
The results presented here, reveal that modification to the A ring of pentacylcic triterpenoid α and β boswellic acids (extracted from Boswellia serrata) potentiates the anti-inflammatory and anti-carcinogenic properties of the compound. As the separation of the β and α (60:40) isomers of boswellic acid is a tedious process, we performed the synthetic scheme on the mixture to generate the CEMB (Subba Rao et al.2008). In our study, we screened the compound CEMB on a number of cancer cell lines from different tissue origins. We found that CEMB is a potent cytotoxic agent (IC50 ~ 0.15–0.7 μM) by the MTT assay on several cell lines. We have also shown that CEMB inhibits DNA synthesis in lung carcinoma cell line A549 and this effect is comparatively less in an untransformed cell line such as HaCaT, particularly at 0.25 and 0.1 μM concentrations. This compound is also shown to possess anti-inflammatory property as it inhibits NO production (IC50 ~ 0.05 μM) induced by IFN-γ. A strong link between cancer and inflammation is well known in lung, colon, bladder, cervical, pancreas and stomach cancers (Aggarwal et al.2006; Lu et al.2006). NO is a multifactorial molecule in tumourogenesis, known to participate in carcinogenesis by inducing DNA damage, supporting tumour progression and suppression of immune response (Lala and Orucevic 1998). NO also plays an important role in angiogenesis, invasion and metastatic processes (Palmer et al.1988; Radomski et al.1990). Therefore, inhibiting NO production could facilitate cancer therapy. 3T3-L1 mouse fibroblasts cells show induction of adipogenic differentiation in the presence of differentiation inducers (IBMX, dexamethasone and insulin). In our study, using CEMB we have shown that in absence of these inducers, CEMB (0.05–0.1 μM) alone could induce differentiation.
In the present work, we have shown that CEMB induces cell death via apoptosis. Specifically, we have demonstrated that CEMB activates the death-receptor-mediated caspase 8 apoptotic pathway, leading to caspase 3 activation followed by DNA breakage. We have tested CEMB on caspase 8 deficient Jurkat cells and Bcl-2-overexpressed Raji cells. In MTT assay, CEMB was surprisingly sensitive even to the Jurkat cells that are caspase 8 deficient, indicating that CEMB can also trigger caspases-independent cell death mechanisms. Bcl-2 has been extensively studied for chemoresistance as it is able to suppress chemotherapy-induced apoptosis (Korsmeyer 1992; Yang and Korsmeyer 1996; Reed 1997). Interestingly, we observed that CEMB was effective even in Bcl-2-overexpressed cells, suggesting that CEMB is capable of inducing cell death in cells resistant to apoptosis. The actual mechanism of cell death in caspase-8-deficient and Bcl-2-overexpressed cells remains to be established. This is an important observation and extensive testing is warranted to confirm the effectiveness of this compound on drug-resistant tumour cells. Furthermore, we have tested the in vivo effect of CEMB on a xenograft tumour model in immuno-compromised mice. Our data show that CEMB significantly reduced the tumour size in a dose-dependent manner, suggesting the potential of this as an anti-cancer compound.
In summary, we conclude that CEMB is a novel synthetic triterpenoid compound showing potent cytotoxic, growth inhibitory, anti-inflammatory and pro-differentiative activities. CEMB induced cell death is via apoptosis in A549 cells, mediated by caspase 8. CEMB could effectively reduce the tumour size in xenograft mouse model. Thus we advocate further studies to explore the development of CEMB as an anti-tumour therapeutic agent.
We thank Dr Vani Santosh for her help with the histology of tumours. This project was funded by a grant support received from National Institute of Health (NIH)/FIRCA, USA. Infrastructure support received from DST-FIST, UGC-SAP and DBT Program Support, India, are acknowledged. Central Animal Facility at Indian Institute of Science is greatfully acknowledged for the supply of nude mice. Also, we thank the Indian National Science Academy, New Delhi, for the award of honarary scientist position to GSRSR.