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Cytotoxic and pro-oxidative effects of Imperata cylindrica aerial part ethyl acetate extract in colorectal cancer in vitro.

ABSTRACT

Background: Colorectal cancer (CRC) is the third most common cancer. Its global incidence and mortality have been on the rise. Recent strategy of therapies has involved the use of non-steroid anti-inflammatory drugs and cyclooxygenase-selective inhibitors. Aerial parts of Imperata cylindrical L. Raeusch (IMP) have been used as an anti-inflammatory agent in traditional Chinese medicine.

Hypothesis: Asarachidonate acid cascadeis often involved in inflammation-related malignancy and IMP is an anti-inflammatory agent, hence it is hypothesized that IMP aerial part ethyl acetate extract exerts cytotoxic effects on colorectal cancer cells in vitro.

Study design: The HT-29 adenocarcinoma cell line was used to elucidate its pro-apoptotic activities. Flow cytometry and fluorescent microscopy were performed to assess cell cycle arrest and the accumulation of reactive oxygen species (ROS). The mRNA and hormone levels of arachidonate acid pathways were studied via quantitative reverse transcription PCR (qRT-PCR) and ELISA.

Results: The 50% growth inhibitory effect ([GI.sub.50]) of the IMP extract on HT-29 was measured with a value of 14.5 [micro]g/ml. Immuno-blot and caspase-3/7 activity assay showed the pro-apoptotic effect of IMP on the activation of caspase cascade. G2/M arrest was observed via flow cytometry. The ROS activity was modulated by the IMP extraction a concentration-dependent manner in HT-29 cells. The IMP extract increased [PGE.sub.2] and [PGF.sub.2[alpha]] levels qRT-PCR revealed that transcripts of rate-limiting [PGE.sub.2[alpha]] and [PGF.sub.2[alpha]]-biosynthetic enzymes--COX-1, mPGES1 and AKR1C3 were notably up-regulated. Among the prostanoid receptors, EP] and FP transcripts were up-regulated while EP4 transcripts decreased. The findings suggest that the proliferative effect of PGE2, which is generally believed to associate with heightened DNA synthesis and cross-talk with MAPK pathways, is likely triggered by the pro-apoptotic or -oxidative effects exerted by IMP extract in HT-29 cells. Concurring with this notion, indomethacin (COX-l/2-inhibitor) was demonstrated to potentiate the cytotoxic effect of IMP extract ([GI.sub.50] [??] 10.8 [micro]g/ml). The results show that the cytotoxic effect of IMP extract predominates over the influence of proliferative prostanoids released by challenged colorectal cancer cells, and may present a potential source for development of novel anti-cancer drugs.

Keywords:

Imperata cylindrica

Colorectal cancer

Prostanoids

Cytotoxicity

Oxidative stress

Introduction

Colorectal cancer is ranked the third and fourth amongst neoplasm in term of prevalence and death rate (Ferlay et al., 2013). Its tumorigenesis is generally believed to be associated with or, in most cases, preceded by chronic inflammation and adenoma polyp formation along the distal colon (Philip et al., 2004), hence endoscopic colon examination is the common procedure for detection of these early signs (Rutter et al., 2004), as it helps with both prevention via surgical removal or diagnosis via collection for further histological examination of abnormal growth or tissues.

Arachidonate acid (ARA) metabolites are important lipid mediators regulating inflammation, analgesia, angiogenesis and cell proliferation [Bogatcheva et al., 2005], The common precursor ARA released by phospholipases (PLA2) then initiates either cyclooxygenase (COX) or lipoxygenase (LOX) pathways to yield the 2 classes of eicosanoids--prostanoids (PGs) or leukotrienes (LTs), respectively. COX and LOX catalyze the conversion of ARA into unstable intermediates [PGH.sub.2] and [LTA.sub.4], which then underwent rapid conversion into PGs (includes prostaglandin D2, E2, F2[alpha], 12 ([PGD.sub.2]; [PGE.sub.2]; [PGF.sub.2[alpha]]; [PGI.sub.2]), and thromboxanes ([TXA.sub.2])) and LTs (e.g. leukotriene C4 ([LTC.sub.4])) via their specific terminal synthases, respectively. Amongst these eicosanoids, [PGE.sub.2] has been most frequently implicated in cancer tumorigenesis, proliferation, metastasis and immune-surveillance in previous studies (Wang and DuBois, 2006). Significantly up-regulated level of [PGE.sub.2] is often observed in neoplastic tissues, including breast, colon and ovarian cancer cells (Wang and DuBois, 2006). And non-steroidal anti-inflammatory drugs (NSAIDs) targeting COXs have been widely used in cancer prevention and treatment (Thun et al., 2002), and in recent decades, COX-2-specific inhibitors and antagonists for [PGE.sub.2] receptors (EPs) have been studied as potential targets in chemotherapies or prevention.

Imperata cylindrica L. Rausch (IMP), commonly known as cogon grass or spear grass, is a perennial rhizomatous plant that can grow on soils with a vast range of nutrients, moisture and pH (Santoso et al., 1996). Despite ecological concerns over its extensive vegetation (Gordon, 1998), its rhizome has been widely used in traditional Chinese medicine as a diuretic and anti-inflammatory agent (Pharmacopeia Committee of P. R. C., 2010), of which responses are closely linked to eicosanoids. There is little information on the use of its aerial part. Previous study showed that its aerial part which was extracted with ethyl acetate demonstrated the potential in growth inhibition of cancer in vitro (Colotta et al 2009). Hence, the growth-inhibiting, pro-apoptotic and pro-oxidative effects of IMP extract in colorectal cancer cell line especially the eicosanoid biosynthesis and signaling were investigated.

Materials and methods

Cells, chemicals & reagents

HT-29 and WRL68 cell line were obtained from ATCC (Manassas, VA). Methanol (RCI, Labscan, Thailand), trifluoroacetic acid (Sigma Aldrich, St. Louis, MO) were of HPLC grade. All other reagents were of analytical reagent grade purchased from Sigma Aldrich. Apoptosis antibodies sampler kit and antibody against caspase-8 were purchased from Cell signaling (Danvers, MA) and Santa Cruz (Dallas, TX), respectively.

IMP extract preparation

Imperata cylindrical L Raeusch(lMP) was obtained from a local vendor. 200 g of its aerial parts was boiled with 61 of 70% ethanol for 1.5 h twice. The 70% ethanol extract was then condensed to 100 ml in a rotary evaporator (R-210, BUCH1, Switzerland) with 120 mbar at 60[degrees]C. It was partitioned with ethyl acetate (4 x 100 ml) to yield the final ethyl acetate extract. The extract was dried in the rotary evaporator and frozen at -80[degrees]C deep freezer overnight before lyophilization. IMP extract powder was kept at -20[degrees]C for a long-term storage and reconstituted with DMSO freshly before any experiment.

HPLC analysis

Individual components in the IMP extract were determined using high-performance liquid chromatography (HPLC). 30 mg of sample was dissolved in 1 ml solution [acetonitrile (CAN): DMSO = l: 1, v/v] by sonication for 5 min. It was then filtered through a 0.22 [micro]M polypropylene filter and 20 [micro]l of the filtrate was injected into the analytical HPLC column (ALLTIMA C18.5 [micro]M, 250x10 mm i.d.) and quantified on a HP1100 series HPLC system equipped with a UV detector at 323 nm. The elution profile (Fig. 1) was programmed with the gradient mobile phase composed of solvent A (0.1% trifluoroacetic acid) and solvent B (methanol). The gradient for separation was programmed as follows: Omin, 65% B, at a flow rate of 1.5 ml/min;5-10min, 70% B, at a flow rate of 1.5 ml/min; 15 min, 80% B, at a flow rate of 1.0 ml/min; 25-35 min, 85% B, at a flow rate of 0.8 ml/min; 40-50 min, 100% B, at a flow rate of 2.0 ml/min, and then was held for additional 5 min.

Viability assay

HT-29 or WRL68 cells were seeded at 8 x [10.sup.3] cells per 96-well (~20% confluence) and incubated overnight. The medium was then replaced by either 0.05% DMSO [as solvent control (SC)] or various dosages of IMP extract, alone or in combination with various concentrations of indomethacin (Indo) or N-acetyl cysteine (NAC), and incubated for 24 h. The medium was removed at the end of incubation, and the cells were washed in phosphate-buffered saline (PBS) once before the addition of MTT (5 [micro]g/ml)-supplemented incomplete RPMI1640 medium (1:100, v/v) (Gibco, Carlsbad, CA). The cells were incubated for 4 h, and then the medium was replaced by DMSO. MTT crystals were solubilized with gentle shaking and subjected to absorbance measurement at 540 nm (TECAN infinite M200, Switzerland). Each treatment or control was performed in 4 replicates. Mean absorbance readings from solvent control groups were defined as 100% viability and data from treatment groups were shown as % viability relative to the controls.

Cell cycle analysis

Cells were seeded at 1 x [10.sup.6] cells per well on a 6-well plate. After overnight incubation, cells were treated with IMP extract for 48 h. The cells were washed with PBS once and trypsinized into a single cell suspension. The harvested cells were washed with PBS twice before fixation with 70% ethanol for 1 h. The cells were centrifuged at 500 x g for 5 min and washed twice with PBS before addition of propidium iodide (final cone: 40 [micro]g/ml) with RNase A (8 [micro]g/ml; Life Technologies). The cells were incubated in dark at 37[degrees]C for 15 min. Flow cytometry was performed on FACSVerse (BD Biosciences) and the cell cycle distribution was then analyzed by ModFit (Allen, 1990).

Reactive oxidative species (ROS) assay

HT-29 or WRL68 cells were seeded on a coverslip in a 6-well plate at 3 x [10.sup.5] cells/well. After overnight incubation, various concentrations of IMP, or in combination with various concentrations of Indo or 4 mM NAC, were administered and incubated for 24 h. ROS assay was performed according to manufacturer's protocols. Cells were washed with warm HBSS once before 25-min incubation with 25 [micro]M carboxy-[H.sub.2]DCFDA and additional 5-min incubation with 5 [micro]M Hoechst 33342. The coverslips were washed thrice with warm HBSS and mounted for fluorescent imaging at 485/530 nm (ex/em) by Nikon E80i (Nikon, Tokyo, Japan).

Caspase-3/7 activity assay

Cells were cultured and incubated with drugs by the same procedures conducted for viability assays, except caspase 3 reagent was added and incubated for 3 h at room temperature before fluorescence reading at 485/530 nm (ex/em) by TECAN infinite M200.

Real-time RT-PCR

HT-29 cells were seeded in 6-well plates at a cell density of 1 x [10.sup.6] cells/well (70% confluence). After overnight incubation, the medium was removed and the cells washed twice with PBS. Solvent control (0.005% DMSO), or IMP at low dose (50 [micro]g/ml), intermediate dose (75 [micro]g/ml) and high dose (100 [micro]g/ml) was added to the assay wells and incubated for 24, 48 or 72 h. The medium was collected after incubation for subsequent ELISA assays, while the cells were rinsed with PBS before lysis. Total RNA was extracted using Qiagen RNeasy plus mini kit following manufacturer's protocols (Qiagen, Valencia, CA). Reverse transcription-real time PCR was performed using Takara One-step SYBR Primescript RT-PCR Kit II. 15 ng total RNA, 5 [micro]l 2X Buffer, 0.4 [micro]l enzyme mix, 0.4 [micro]l each of forward and reverse primers (10 ([micro]M), 0.2 [micro]l ROX II were added in a 10[micro]l reaction mix. The PCR was conducted with an initial cycle of reverse transcription at 42[degrees]C 5 min and denaturation at 95[degrees]C 10 s, followed by 40 cycles of 2-step amplification at 95[degrees]C 5 s and 60[degrees]C 34 s. Melt curve was generated after each PCR and only one peak was observed for each product (data not shown). [2.sup.-[DELTA][DELTA]CT] method was used to calculate the relative mRNA expression level as described previously (Livak and Schmittgen, 2001). GAPDH transcript levels were used to normalize the target transcript abundance. The results were expressed as the mean fold mRNA expression relative to the mRNA expression level of SC after 24 h incubation.

Immuno-blot and ELISA

Immuno-blot was carried out following the recommended protocol from Abeam. In brief, cells cultured in 100 mm dishes at 8 x [10.sup.6] cells/dish (70% confluence) were exposed to various concentrations of IMP extract for 48 h. Harvested cells were lysed by nonidet-P40 lysis buffer (150 mM sodium chloride, 1% Triton X-100, 50 mM Tris, pH 8) supplemented with protease inhibitor cocktails (Roche). The total proteins were quantified according to DC protein assay according to manufacturer's protocols (Biorad). Normalized protein samples were denatured in boiling water for 10 min and fractionated by 10-12% SDS-PAGE. Proteins were transferred from the gel to a 0.25 [micro]M PVDF membrane at 12 V for 30 min by Novex Semi-dry blotter (Invitrogen). Incubation with antibodies was performed (Cell signaling). Amersham ECL prime western blotting detection reagent (GE, Fairfield, CT) was used to develop the signals and the protein chemi-luminescent bands were visualized after exposure to Fuji Super RX film. Supernatants collected from treated cells were used for ELISA of [PGE.sub.2], [PGF.sub.2[alpha]], and CYLST, according to the manufacturer's protocols (Abeam).

Statistical analysis

Data are presented as means [+ or -] SD. Groups were compared by one-way ANOVA, followed by Dunnett's test. Differences between treatment groups were considered significant at p < 0.05.Nonlinear regression test was applied to the viability assays to obtain a fit curve with [R.sup.2] >0.98.

Results and discussion

IMP extract

Imperata cylindrica (IMP) has long been described in traditional Chinese medicine as an anti-inflammatory and analgesic agent (Pharmacopeia Committee of P. R. C., 2010). The HPLC profile of the IMP extract was shown in Fig. 1. As eicosanoids are important local lipid mediators regulating inflammatory stimuli and other closely-associated physiological responses, such as growth proliferation, angiogenesis and immune cell infiltration, this study focused on the scarcely documented effects of IMP aerial part extract on CRC which tumorigenesis, proliferation, metastasis and recurrence are strongly related to eicosanoid biosynthesis and signaling (Qiao et al., 1995).

IMP extract inhibits cell proliferation and promotes G2/M cell cycle arrest in vitro

In accordance with our preliminary screening results, IMP extract demonstrated a significant growth inhibitory effect on HT-29 cells, a colorectal adenocarcinoma cell line. MTT viability assay showed that the [GI.sub.50] of IMP on HT-29 is 14.45 [+ or -] 4.20 [micro]g/ml, while its [GI.sub.50] on the normal cell line, WRL68, is >100 [micro]g/ml (Fig. 2). Cell cycle analysis supported that IMP extract causes cell cycle arrest at G2/M phase dose-dependently, with its effect most prominent after 48 h incubation (Fig. 3A, B and C). Cell population in S phase also demonstrated a considerable rise at the highest dose tested (Fig. 3B). This may be attributed to a surge in endogenous [PGE.sub.2] level after 24 h incubation (Fig. 4A, B and C). [PGE.sub.2] is suggested to exert its mitogenic effect on colorectal cancer via enhancing its DNA synthesis in the nano-molar range (Loffler et al., 2008), which agrees with the current ELISA results (3000[rho]g/ml; ~8.51 nm). However, the time lag in response was comparatively shorter than that described in the previous study (72 h) (Loffler et al., 2008), apparent within 24-48 h of detectable [PGE.sub.2] increase.

IMP extract triggers ROS accumulation and cell apoptosis

To elucidate other aspects of the growth inhibitory effects of IMP extract observed in vitro, the pro-oxidative and apoptotic responses were investigated. IMP extract-treated cells showed dose-dependent morphological changes such as cell shrinkage and formation of apoptotic bodies, along with ROS accumulation (Fig. 5A-H), in support of its cytotoxic effects on HT-29 cells. There was no obvious increase of ROS level observed in the normal cell line, WRL68 (Fig. 5M-P). NAC, a ROS scavenger, reduced the ROS accumulation in IMP-treated HT-29 cells (Fig. 5I-L) and was shown to alleviate the cytotoxic effect of IMP on the cancer cell line via MTT viability assay (Fig. 2). The results show that IMP extract elicited a bell-shaped dose-dependent activation of caspase-3/7 via caspase3/7 activity assay (Fig. 6), in which the effect declines at higher concentrations ([greater than or equal to] 50-100 [micro]g/ml), possibly due to cell death triggered by caspase-independent pathways with necrotic characteristics (Essmann et al., 2003) when the concentration is much higher than the [GI.sub.50] (14.45 [micro]g/ml) was administered. Caspase-3/7 assays were conducted when cells were grown at 20% confluence, same as in the viability assays, for convenience of comparison between assays, while qRT-PCR, immuno-blot, ELISA and cell cycle analyses were performed when cells were grown at 80% confluence to maximize the cells harvested, with a concentration range proportional to cell confluence to compensate against PG-induced protective effect (discussed in the next section). Immuno-blot findings showed that dose-dependent up-regulations of cleaved caspase-3, 9 and PARP (Fig. 7), while no changes were recorded in cleaved caspase-7 protein level. It suggests IMP would trigger the intrinsic apoptotic pathway mediated via initiator caspase, caspase-9. In addition, pro-caspase-3 and 8 protein expression were up-regulated despite normalized GAPDH expression between samples, which may be related to the transcriptional up-regulation (Fig. 7). In addition, TNFR transcripts were shown to be dose-dependently up-regulated upon IMP treatment. The results imply that TNFaR-caspase-8 mediated extrinsic apoptotic signaling (Vandenabeele and Melino, 2012).

Effect of IMP extract on endogenous eicosanoid biosynthesis and signaling

Owing to the correlation between eicosanoids and endothelial cancer development (Bogatcheva et al., 2005), the two eicosanoid biosynthetic pathways and signaling receptors were studied, including the key rate-limiting enzymes COXs (COX-1 and COX-2) and LOX (ALOX5), the class-specific terminal synthases--mPGES1, mPGES-2, cPGES, PGFS (alias: AKR1C), PTGDS, HPGDS, PTGIS, TXBA2S and LTC4S, and their respective receptors--[EP.sub.1], [EP.sub.2], [EP.sub.3], [EP.sub.4], FP, DP, IP, CYSLTR1 and CYSLTR2 (see Suppl. info 1 for primers used). Among them, significant IMP-induced dose-dependent up-regulation of COX-1, mPGES-1 and PGFS transcripts were observed at all time-points studied (Fig. 8A & B, Suppl. info 2), in accordance to a consistent surge in endogenous [PGE.sub.2] and [PGF.sub.2[alpha]] level detected via ELISA (Fig. 4). On the other hand, down-regulation of COX-2 represents an interesting finding (Suppl. info 2). Although it is long-presumed that COX-1 is constitutive and COX-2 is inducible by inflammatory stimuli such as cytokines (Morita, 2002), the over-expression of COX-1 protein was described to occur more often than that of COX-2 protein in breast tumors, another epithelial cancer model, possibly induced by estrogen (Gibson et al., 2005; Hwang et al., 1998). Similarly, COX-1 transcript up-regulation was found in cervical carcinoma (Sales et al., 2002). In addition, its up-regulation was believed to promote crypt stem cell survival and proliferation, and epithelial integrity, particularly during wound repair (Cohn et al., 1997). When it is over-expressed in vitro, COX-1 induced an autocrine/paracrine up-regulation of COX-2 and mPGES-1 transcript and concomitant enhanced [PGE.sub.2] biosynthesis via cAMP-linked receptors, [EP.sub.2] and [EP.sub.4] (Sales et al., 2002). Hence, IMP-induced [PGE.sub.2] and [PGF.sub.2[alpha]] biosynthesis is unlikely to involve any pro-inflammatory cytokines which induce COX-2 upregulation, concurring with the anti-inflammatory actions of the herbal extract. As PGs are released upon biosynthesis, the accumulating [PGE.sub.2] and [PGF.sub.2[alpha]] most likely require a sustained transcriptional up-regulation of its rate-limiting enzyme, COX-1, via an autocrine/paracrine loop. The findings imply that COX-2 is either not involved in this signaling loop, or its transcription is selectively inhibited by constituents in IMP extract. Given the undetectable level of [EP.sub.2] and down-regulation of [EP.sub.4] in IMP-treated cells, the only two up-regulated [PGE.sub.2] and [PGF.sub.2[alpha]] receptors, the [Ca.sup.2+]--linked [EP.sub.1] and FP would transduce the signal from PGs, presenting a mechanism different from that previously observed in cervical cancer (Fig. 8A, B, C and D). Previous studies have shown that cAMP-linked [EP.sub.4] increase resistance to apoptosis and anchorage but have no effect on cell proliferation (Hawcroft et al., 2006), hence IMP-induced [EP.sub.4] down-regulation may contribute, at least in part, to its pro-apoptotic effect on HT-29 cells. While the effect of [PGF.sub.2[alpha]] on CRC growth remains debatable (Colotta et al., 2009; Hawcroft et al., 2006), cross-talk of the [Ca.sup.2+]-linked [EP.sub.1] with other signaling pathways, especially the EGFR-MAPK pathway, is often implicated in [PGE.sub.2]-associated mitogenic activity in gastrointestinal cancer (Pai et al., 2002), which may represent the retaliating proliferative response from IMP-challenged cells. To reduce the protective effect of PGs, pre-treatment with indomethacin (Indo), a NSAID and COX-inhibitor, significantly potentiates the cytotoxic effect of IMP on HT-29 cells by over 25% reduction in term of [GI.sub.50] (Fig. 1).

In conclusion, IMP aerial part extract represents a potential source for anti-proliferative, pro-oxidative and apoptotic agents in combating CRC and other endothelial cancers with aberrant [PGE.sub.2] biosynthesis and/or signaling.

http://dx.doi.org/10.1016/j.phymed.2016.02.015

ARTICLE INFO

Article history:

Received 18 June 2015

Revised 26 January 2016

Accepted 14 February 2016

Abbreviations: CRC, colorectal cancer; DOX, doxorubicin; IMP, Imperata cylindrica; Indo, indomethacin; MTT, 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; LTs, leukotrienes; PGs, prostanoids; ROS, reactive oxygen species.

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgments

This work is supported in part by a research grant no. 6903088 from Keenway Industries Ltd.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.02.015.

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Amy Ho Yan Kwok, Yan Wang Wing Shing Ho *

School of Life Sciences, The Chinese University of Hong Kong, Hong Kong

* Corresponding author. Tel.: +852 3943 6114; fax: +852 2603 7732.

E-mail address: ws203ho@cuhk.edu.hk (W.S. Ho).

Fig. 2. Viability assay of HT-29 and WRL68 cells under IMP
and-or indomethacin or NAC treatment. Indomethacin (Indo), a
NSAID and COX-inhibitor, which is used in combination with
IMP on HT-29 cells to test if it potentiates the cytotoxic
effect of IMP by abolishing the putative protective effect
of PCs. The effect of NAC, a ROS scavenger, is tested on
IMP-treated HT-29 cells to elucidate the correlation between
ROS accumulation and cell viability, IMP extract
demonstrated has a preferential cytotoxic effect on the
colorectal cancer cells, HT-29.

Mean absorbance readings from solvent control groups were
defined as 100% viability and data from treatment groups
were shown as % viability relative to the controls. Shaded
region represents the reduction in [GI.sub.50] between
treatment with IMP alone and that with pre-treatment of
indomethacin at the highest dose (100 [micro]M).

Cell line                   HT29                   WRL68

Indo (mM)    --      --     10      50      100    --
NAC(mM)      --      4      --      --      --     --

[GI.sub.50] o14.45   >100   10.76   10.06   6.87   >100
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Author:Kwok, Amy Ho Yan; Wang, Yan; Ho, Wing Shing
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9HONG
Date:May 15, 2016
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