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Trifolin induces apoptosis via extrinsic and intrinsic pathways in the NCI-H460 human non-small cell lung-cancer cell line.


Background: Trifolin (kaempferol-3-O-galactoside), which is a galactose-conjugated flavonol, exhibits antifungal and anticancer effects. However, the mechanisms underlying its anticancer activities have not yet been examined.

Purpose: In this study, the anticancer effects of trifolin were examined in human lung cancer cells. Methods: Cytotoxicity was determined by evaluating cell viability. Apoptosis was analyzed through flow cytometry and western blotting analysis. Death receptors and inhibitors of apoptosis were evaluated through RT-PCR.

Results: Trifolin induced apoptosis in NC1-H460 human non-small cell lung cancer (NSCLC) cells by inhibiting the survival pathway and inducing the intrinsic and extrinsic apoptosis pathways. Trifolin decreased levels of Akt/p-Akt, whereas levels of expression of phosphatidylinositide 3-kinase (PI3K), cyclin Dl, cyclin E, and cyclin A were not altered. Trifolin initiated cytochrome c release by inducing mitochondrial outer membrane permeabilization (MOMP). Trifolin increased Bcl-2-associated X protein (Bax) levels and decreased b-cell lymphoma 2 (Bcl-2) levels, while the levels of Bcl-xL were not altered. In addition, trifolin increased the levels of the death receptor involving the Fas/Fas ligand (FasL) and Fas-associated protein with the death domain (FADD), which consequently activated caspase-8, caspase-9, caspase-3, and the proteolytic cleavage of poly (ADP-ribose) polymerase (PARP).

Conclusion: These results suggested that trifolin induced apoptosis via death receptor-dependent and mitochondria-dependent pathways and that trifolin can be used as a therapeutic agent in human lung cancer.








Lung cancer is the leading cause of cancer deaths worldwide. Because little is known about how to treat and prevent lung cancer, many studies of lung cancer are still required. Commonly, lung cancer cells have chromosomal abnormalities, such as defects of the p53 and pRb cancer suppressor genes, which result in the failure of the regulation of apoptotic signals and cell proliferation (Carbone and Robinson, 2003; Thafeni et al., 2012). Lung cancer is classified as a non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) (Carbone and Robinson, 2003). SCLC commonly occurs in bronchial regions and results in malignant cancer. The 5-year survival rate of a patient with SCLC is under 21%, which is very low. NSCLC, which accounts for approximately 85% of all lung cancer cases (Herbst et al., 2008), includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Adenocarcinoma commonly occurs in pulmonary alveoli, while squamous cell carcinoma, which is caused by smoking, is mainly found in the large airway and bronchial regions of male patients (Cross, 2012). Large cell carcinoma occurs in any part of the lung, and its growth and metastasis is aggressive (Schuller, 1989; Yamamoto et al., 1987). In this study, large cell carcinoma NCI-H460 cells were used.

The development of alternative lung cancer agents is required. Plant extracts have begun to receive attention as therapeutic agents in cancer treatment due to their capacity to inhibit tumor growth, angiogenesis, and metastasis with minimal side effects. Plants contain several alkaloids, glycosides, and polyphenols, which are secondary metabolites that often found in fruits, vegetables, and even phytogenic beverages (Galluzzo and Marino, 2006). Trifolin, which is also known as kaempferol-3-O-galactoside (Fig. 1A), is a flavonol, which is a type of flavonoid (Vierstra et al., 1982). Flavonoids are relatively abundant in the human diet and in promising anticancer agents (Havsteen, 2002; Middleton et al., 2000; Ren et al., 2003). Trifolin is a galactoside-conjugated kaempferol that results from conjugation by kaempferol 3-O-galactosyltransferase according to the following formula: UDP-galactose + kaempferol [right arrow] UDP + kaempferol 3-O-beta-D-galactoside (Miller et al., 1999). Trifolin is found in many plants, such as Diospyros blancoi (Candolle, 1844), Camptotheca acuminata (Perdue, 1966), Euphorbia condylocarpa (Roshchin, 1977), and Consolida oliveriana (Arboretum, 1967). The bark and leaves of Diospyros blancoi are used for the treatment of itchy skin, while the bark is used for the treatment of coughs, fevers, dysentery and diarrhea (Ragasa et al., 2009). It has been reported the antifungal activity of Camptotheca acuminata (Li et al., 2005). In addition, the anticancer property of Consolida oliveriana against some human cell lines HL-60, U-937 and SK-MEL-l also has been reported (Diaz et al., 2008). However, there is no report regarding on medicinal property of Euphorbia condylocarpa. Kaempferol, which is the mother compound, has been studied in various fields (Devi et al., 2015), and many studies have suggested that kaempferol reduces the risk of various cancers and that it has antibacterial, antifungal, and antioxidant abilities (Ackland et al., 2005; Calderon-Montano et al., 2011; Devi et al., 2015; Kataoka et al., 2001). In lung cancer cells, kaempferol upregulates the levels of the proapoptotic proteins b-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and Bcl-2-associated death protein (Bad), while it down-regulates the levels of the antiapoptotic proteins Bcl-2 and b-cell lymphoma-extra large (Bd-xL). These changes result in an increase in apoptosis in the cancer cells (Kim and Choi, 2013). However, the effects of trifolin on cancer cells have not yet been studied. Only a few studies have reported that trifolin exhibits antifungal and antioxidant activities and that trifolin acetate can induce cell death in human leukemia cells (Torres et al., 2008). Therefore, in this study, we investigated the anticancer effects of trifolin in the NC1-H460 human NSCLC cell line.

Results and discussion

Growth inhibitory effects of trifolin in human NSCLC NCI-H460 cells

Plant extracts have the potential to become anticancer therapeutic agents because of their abilities to inhibit tumor growth, angiogenesis, and metastasis with few side effects (Cragg et al., 1997). Importantly, these effects are demonstrated by flavonoids, which are components of plant extracts. This study examined the effects of trifolin, which is a type of flavonoid that is conjugated to galactoside (Fig. 1A).

First, the viability of the trifolin-treated NCI-H460 cells was evaluated. NCI-H460 cells were treated with various concentrations of trifolin up to 50 [micro]M to investigate the time- and dose-dependent effects of trifolin. The cell viability of the trifolin-treated NCI-H460 cells was analyzed with a MTS assay and compared to untreated control cells. The results revealed that trifolin decreased cell viability to less than 50% when the cells were exposed to the highest concentration for 48 h, whereas no significant cytotoxic effects were observed after the 24 h treatment, except for treatment with 50 [micro]M of trifolin (Fig. IB). The micrographic analysis revealed that the cell density was also decreased after 48 h (Fig. 1C). Because the treatment with trifolin for 48 h was more effective than treatment for 24 h, this condition was used for the remaining experiments in this study.

Sub-G1 accumulation and down-regulation of the survival pathway by trifolin

Precise evaluations of cellular DNA content by flow cytometry can allow identification of the cell cycle phases. In addition, PI staining assays, which are based on the principle that apoptotic cells, among their other typical features, are characterized by DNA fragmentation, are widely used in evaluations of apoptosis (Riccardi and Nicoletti, 2006). In order to investigate whether the cell growth inhibitory effects induced by trifolin would be due to cell cycle arrest or apoptosis, the cell cycle was analyzed with PI staining and flow cytometry, showing that the cell populations in the Sub-G1 phase were increased after trifolin treatment, whereas the cell populations in G1 phase were decreased. However, there was no significant alteration in G2/M phase. The population in the Sub-G1 phase demonstrated ceils with hypo-diploid ([less than or equal to] 2 N) fragmented DNA, which is a marker of apoptosis (Figs. 2A, B). However, the other cell cycle phases were not altered. In addition, modulators of the cell cycle and factors involved in the survival pathway were analyzed. In addition, western blotting was performed to determine the effects on the levels of cell cycle-related proteins. The protein levels of all of the cell cycle modulators, including cyclin Dl, cyclin E, and cyclin A, were not altered (Fig. 2C). Furthermore, the protein levels of the survival pathway factors were confirmed with western blotting. The protein levels of Akt/p-Akt were decreased, and PI3K was not altered (Fig. 2D).

Trifolin induces chromatin condensation, DNA fragmentation, and cytoplasmic shrinkage

In order to investigate if cell apoptosis was induced by trifolin, DAPI staining and annexin V/PI double staining were performed. The morphology of the cells' nuclei was observed with DAPI staining, which showed that treatment with trifolin induced chromatin condensation in NCI-H460 cells (Fig. 3A). In order to investigate the shrinkage of the cytoplasm and DNA fragmentation, which is evidence of apoptosis, annexin V/PI staining was performed. It showed that nuclear fragmentation and cytoplasmic shrinkage were increased with trifolin treatment (Fig. 3B). These results suggested that trifolin induced apoptosis. Thus, the apoptosis signaling pathway was then analyzed, and the apoptotic pathway was activated. PARP, which is a key factor in DNA repair and programmed cell death, the initiator caspase-9, and caspase-3 which has a central role in cell apoptosis, were found to be cleaved with trifolin treatment (Fig. 3C).

Trifolin induced apoptosis via mitochondrial outer membrane permeabilization (MOMP) and death receptor mediated apoptosis pathway

To elucidate whether apoptosis was mediated by the mitochondrial apoptotic pathway, the levels of anti-/pro-apoptotic proteins were evaluated with western blotting. And in order to detect MOMP alterations, JC-1 staining was performed with flow cytometry. The results showed that the regulators of MOMP, including p-p53, Bax, and Bcl-2, were modulated (Figs. 4A, B). Consequently, MOMP was triggered, and cytochrome c was released into the cytosol (Fig. 4C, D). RT-PCR, which was performed to elucidate the effects of trifolin on the death receptor-mediated extrinsic apoptosis pathway, showed that concurrently, the mRNA levels of death receptors, including Fas and FADD, were increased (Fig. 4E). Additionally, caspase-8, which plays a major role in the extrinsic pathway, was activated, and inhibitors of apoptosis, including cFLIP and cIAP-1, were down-regulated (Figs. 4F, G).

We performed knock-out experiments by using siRNAs of p53, Fas, and clAP-1 in order to elucidate detailed mechanism of the anti-cancer effect of trifolin. In these experiments, NC1-H460 cells were tranfected with one of the siRNAs, than treated with trifolin. In case of cIAP-1, which play an inhibitory role in caspase activation. siRNA treatment induced decrease of cell viability more than negative control group and SC (scrambled) siRNA groups (Fig. 5A). We also treated caspase inhibitors. As shown in Fig. 5B, pro-forms of caspase-8, 9 were recovered. Consequently, pro-form of PARP was recovered in caspase-8, 9 inhibitors treated groups (Fig 5B). Taken all these results, it was revealed that p53, Fas and clAP-1 were involved in apoptosis signaling pathway induced by trifolin treatment. In addition, caspase inhibitors treatment showed that caspase-8, 9 had a crucial role in trifolin induced apoptosis.


Taken together, these results showed that trifolin induced apoptosis via the intrinsic and extrinsic signaling pathways. In addition, trifolin suppressed the P13K/Akt survival pathway. These findings suggested that trifolin has potential as a therapeutic agent for human lung cancer.

Materials and methods


Trifolin, propidium iodide (PI), and the 4',6-diamidino-2-phenylindole (DAPI) stain were obtained from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). The CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTS)] was purchased from Promega Corporation (Madison, WI, USA). NEPER Nuclear and Cytoplasmic Extraction Reagents were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Antibodies specific to PARP, caspase-3, caspase-9, p53, Bcl-2, Bcl-xL, Bax, proliferating cell nuclear antigen, and cytochrome c were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody and anti-mouse IgG HRP-conjugated secondary antibody were purchased from EMD Millipore Corporation (Billerica, MA, USA). Antibodies against PI3K, Akt, p27, p21, cyclin Dl, cyclin A, cydin E, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Finally, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolycarbocyanine chloride (JC-1) was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Caspase-8 inhibitor Z-IETD-FMK and Caspase-9 inhibitor Z-LEHD-FMK were purchased from R&D System (Minneapolis, MN, USA).

Cell culture

The human NSCLC cell line NCI-H460 was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI medium (Welgene, Inc., Gyeongsan-si, Korea) that was supplemented with heat-inactivated 10% (v/v) fetal bovine serum (GE Healthcare Life Sciences, Logan, UT, USA) and incubated in humidified conditions of 5% C02 and 37 [degrees]C. The cells used in these studies were subjected to fewer than 20 cell passages.

MTS assay

Cell viability was quantified with a MTS assay. Approximately 0.7 x [10.sup.4] cells were seeded in each well of a 96-well plate containing 100 [micro]l of RPMI that was supplemented with 10% fetal bovine serum, penicillin (100 [micro]g/ml), and streptomycin (0.25 [micro]g/ml) for overnight growth. After 20 h, various concentrations of kaempferol3-O-galactoside were added and incubated for an additional 48 h. Medium (100 [micro]l) was removed, and 20 [micro]l of MTS (2 mg/) added to the cells for 30 min to 1 h at 37[degrees]C. Optical density was measured at 492 nm with an ELISA reader (Apollo LB 9110, Berthold Technologies GmbH, Zug, Switzerland).

Cell cycle analysis

The cell-cycle distribution was analyzed with PI staining and flow cytometry. NCI-H460 cells (7.5 x [10.sup.4] cells/ml) were seeded in each well of a 60[phi] plate and exposed to various concentrations of trifolin for 48 h. The cells were harvested, fixed with ice-cold 70% ethanol, and stored at -20[degrees]C until analysis. After fixation, the cells were washed twice with cold phosphate-buffered solution (PBS) and centrifuged, and the supernatant was discarded. The pellet was re-suspended and stained in PBS containing 50 [micro]g/ml PI and 100 [micro]g/ml RNase A for 20 min in the dark. The DNA content was analyzed with flow cytometry on a FACSCalibur with the CellQuest software (BD Biosciences, San Jose, CA, USA). The sub-Gl population of hypo-diploid ([less than or equal to] 2 N) fragmented DNA is an important indicator of apoptosis.

Analysis of mitochondrial membrane potential

The MOMP was measured with JC-1 staining and flow cytometry. The NCI-H460 cells were seeded in 60[phi] plates (7.5 x [10.sup.4] cells/well) and treated with various concentrations of trifolin. After 48 h, the supernatants were transferred to 1.5 ml tubes, washed with warm PBS, and trypsinized. The cells were collected with the tube supernatants. JC-1 (5 [micro]g/ml) was mixed until the precipitate disappeared. The cells were incubated in the dark for 10 min at 37[degrees]C. After 10 min, the cells were centrifuged at 300 x g for 5 min at 4[degrees]C, washed twice with cold PBS, and re-suspended with 200 [micro]l of PBS. The solutions were sorted with the FACSCalibur instrument and analyzed with the CellQuest software (BD Biosciences). All steps were performed under reduced lighting.

Western blotting analysis

NCI-H460 cells were lysed in 1 ml of lysis buffer containing 50 mM Tris (pH 7.4), 1 mM of ethylenediaminetetraacetic acid, 1% NP-40, 1500 mM of sodium chloride, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, and protease inhibitor cocktail. The lysates were clarified by centrifugation at 17.010 x g for 30 min at 4[degrees]C. The protein concentrations were estimated with a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with a Bio-wave ultraviolet/visible Spectrophotometer (Biochrom Ltd., Cambridge, UK). Equal amounts of the lysates were loaded onto 10%-15% gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein bands were transferred to polyvinylidene difluoride membranes (EMD Millipore Corporation). The membranes were blocked with Tris-buffered saline containing Tween-20 (TBST) (2.7 M NaCI, 1 M Tris-HCl, 53.65 mM KCl, and 0.1% Tween-20, pH 7.4) and 5% skim milk for 1 h at room temperature. The membranes were incubated overnight at 4[degrees]C with primary antibodies specific to each target protein. After washing with TBST three times, HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were incubated with the membranes for 1 h at room temperature. After washing three times with TBST, the blots were exposed with a WEST-ZOL (plus) Western Blot Detection System (iNtRON Biotechnology, Seongnam-si, Korea).

Reverse transcription-polymerase chain reaction (RT-PCR)

The cells were lysed in 1 ml of easy-BLUE Total RNA Extraction Kit (iNtRON Biotechnology). The RNA was isolated according to the manufacturer's instructions. Oligo(dT)-primed RNA (5 [micro]g) was reverse transcribed with M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). The RT-PCR analysis was performed with a PCR thermal cycler Dice instrument (Takara Bio Inc., Kusatsu, Shiga, Japan) with the following primer sets: Fas: 5'-AGG GAT TGG AAT TGA GCA AG-3' (forward), 5'-ATG GGC TIT GTC TGT GTA CT-3' (reverse); FasL: 5'-AGT CCA CCC CCT GAA AAA AA-3' (forward), 5'-ATT CCA TAG GTG TCT TCC CA-3' (reverse); GAPDH: 5'-GGC TGC TIT TAA CTC TGG TA-3' (forward), 5'-TGG AAG ATG GTG ATG GGA TT-3' (reverse); tumor necrosis factor-related apoptosis-inducing ligand (TRAIL): 5IL necrosis factor-related apoptosis-inducing ligand (GG GATTC CTC TGG CT-3' (reverse); death receptor 5 (DR5): 5th receptor 5 GTC GGG GT-3' (forward), 5'-TGG TGC AGG GAC TTC TCT CT-3' (reverse); cellular inhibitor if apoptosis-1 (cIAP-1): 5'-CAG GTC CCT CGT ATC AAA ACS' (forward), 5'-TAA AAA CCA GCA CGA GCA AG-3' (reverse); cellular fas-associated protein with death domain (FADD)-like interleukin (IL)-1[beta]-converting enzyme-inhibitory protein ([cFLIP.sub.L]): 5'-CGA GCA CCG AGA CTA CGA CA-3' (forward), 5 CCG AGA CTA CGA CACAC ATA GT-3' (reverse); and FADD; 5ADD: rd), 5 CCGAA GAC CTG TG-3' forward), 5'-GGG GTA TCT GTC CTC GAT GC -3' (reverse).

DAPI staining

NCI-H460 cells (0.5 x [10.sup.4] cells/ml) were seeded in each well of slide plate and incubated overnight. And cells were treated with various concentrations of trifolin. After 48 h, plates were washed with PBS and cells were fixed with 4% para-formaldehyde and stained with DAPI staining solution for 10 s. The plates were washed with PBS and completely dried. Plates were mounted with cover slip. The stained cells were observed using fluorescence microscopy (Olymous, Tokyo, Japan).

Small interfering RNA (siRNA) transfection

We performed knock-out experiments by using siRNA of p53, Fas and cIAP-1 in order to elucidate detailed mechanism of the anti-cancer effect of trifolin. NCI-H460 cells (5 x [10.sup.4] cells/ml) were seeded in each well of a 600 plates and were transfected with one of the siRNAs than, cells were exposed to trifolin 50 [micro]M for 48 h.

Caspase inhibitor assay

The apoptosis mechanism was analyzed with caspase inhibitors. NCI-H460 cells (5 x [10.sup.4] cells/ml) were seeded in each well of a 600 plates and were treated with each 100 nM caspase-8 and caspase-9 inhibitors. After 2h, 50 [micro]M trifolin was added to each plate. After 48 h, cells were harvested and were used in western blot analyses.



Article history:

Received 13 January 2016

Revised 4 May 2016

Accepted 27 May 2016

Conflict of interest

The authors state that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.


This research was supported by the basic program (2015R1A2A2A09001137) of the National Research Foundation of Korea (NRF) and KKRIBB GM1221251. D.Y. Yoon was supported by the Priority Research Centres Program (2012-0006686) from the NRF.


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Min-Je Kim (a), Sae-Bom Kwon (a), Man-Sub Kim (a), Seung Won Jin (a), Hyung Won Ryu (b), Sei-Ryang Oh (b), Do-Young Yoon (a), *

(a) Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Gwangjin-gu, Seoul 05029, Republic of Korea

(b) Natural Medicine Research Center, KRIBB, Cheongwon-gu, Cheongju-si, Republic of Korea

Abbreviations: NSCLC, non-small cell lung cancer; MOMP, mitochondrial outer membrane permeabilization; PARP, poly (ADP-ribose) polymerase; Bax, Bcl-2-associated X protein; FasL, Fas ligand; FADD, Fas-associated protein with the death domain.

* Corresponding author: Tel.: +82 2 450 4119, fax: +82 2 444 4218.

E-mail address: (D.-Y. Yoon).


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Author:Kim, Min-Je; Kwon, Sae-Bom; Kim, Man-Sub; Jin, Seung Won; Ryu, Hyung Won; Oh, Sei-Ryang; Yoon, Do-Yo
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9SOUT
Date:Sep 15, 2016
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