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PG.2 MORINGA OLEIFERA LEAVES WITH A NEW ANTICANCER ACTIVITY

MORINGA OLEIFERA LEAVES WITH A NEW ANTICANCER ACTIVITY

 

cDNA Synthesis and PCR Amplification

Cells (1 × 105) were seeded in a 6-well culture plate for 1 d before treatment with the MOL extract. After an additional 2-d incubation, total RNA was isolated from the cells by using the High Pure RNA isolation kit (Roche, Basel, Switzerland). To generate first-strand cDNA from the total RNA (1 µg) by using oligo dT, a cDNA synthesis kit (Maxim RT Premix Kit-Oligo dT Primer, iNtRON Biotechnology, Korea) was employed. The resulting cDNAs were amplified with different primers (Table 1) by using Maxim PCR Premix Kit-iTaq (iNtRON Biotechnology, Korea). The amplified polymerase chain reaction (PCR) products were analyzed by 1.5% agarose gel electrophoresis and then photographed under UV light (Smart gel imaging analysis system; Beijing Sage Creation Science And Technology Co. Ltd).

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Table 1. Primers used in this study.

doi:10.1371/journal.pone.0095492.t001

Western Blot Analysis

Cells (1×105) were seeded in a 6-well culture plate for 1 d before treatment with the MOL extract. After an additional 2-d incubation, the cells were collected and lysed for western blot analysis. Antibodies for western blot analysis were purchased from Cell Signaling Technology (β-actin, cat. no. cs4967; Akt, cat. no. cs9272; p-JNK, cat. no. cs9251; p-Erk, cat. no. cs9101; p-IκBα: cat. no. cs2859; SOX2: cat. no. cs3579; and cleaved Notch1, cat. no. cs2421), from Santa Cruz Biotechnology (p53, cat. no. sc-126; cyclinD1, cat. no. sc-753; Notch1, cat. no. sc-6014; NF-κB, cat. no. sc-109; β-catenin, cat. no. sc-796; and c-Myc, cat. no. sc-761), and from Merck Millipore (Oct4, cat. no. mab4305). Protein concentration was determined with the Bradford method (Protein Assay Dye Reagent Concentrate, Bio-Rad Laboratories, cat. no. 500-0006). After cell lysis, equal amounts of proteins (20–80 µg) were separated on a 8–12% SDS polyacrylamide gel according to the size of the proteins and transferred to a nitrocellulose membrane (Whatman). The blots were blocked for 16 h at 4°C with blocking buffer (10% nonfat milk in Tris-buffered saline [TBS] buffer containing 0.1% Tween 20 [TBS-T]). After the membrane was washed with 3 times with TBS-T, it was incubated at room temperature for 2 h with a horseradish peroxidase-labeled secondary antibody and visualized using the ECL kit (GE Healthcare, cat. no. RPN1237). To confirm the transfer of proteins to the nitrocellulose membrane, the membrane was stained with Ponceau S solution (Sigma, cat. no. P7170-1L) for 5 min. The stained membrane was then washed with DW several times, and proper gel transfer was verified.

Identification of Proteins on SDS-PAGE Gels

Cells (1×105) were seeded in a 6-well culture plate for 1 d before treatment with the MOL extract. The cells were lysed after an additional 2-d incubation. After cell lysis, equal amounts of proteins (20 µg) were separated on a 10% SDS polyacrylamide gel and stained with Comassie blue. The stained gel was destained with a mixed solution of methanol and acetic acid. Bands at around 60–80 kDa on the gel were directly sliced with a knife and treated with trypsin. The tryptic peptides produced by the in-gel digestion were analyzed using an LTQ mass spectrometer (Thermo Finnigan, San Jose, CA) coupled with an Eksigent-Nano-Ultra-UPLC (Eksigent Technologies, CA). Proteins were identified using the UniPort Program (http://www.uniprot.org).

Microarray Preparation

All the oligonucleotides corresponding to 23,753 coding sequences were resuspended in printing buffer (Telechem International, Inc., USA) at a final concentration of 50 pmol/µL. Resuspended oligonucleotides were spotted onto silanized glass slides (UltraGAPS™, Corning Lifesciences, MA) by using a robotic microarrayer (OmniGrid II, GeneMachines, CA) at 20–25°C with 40% humidity.

Preparation of the cDNA Probe and Microarray Hybridization

Cells (1×105) were seeded in a 6-well culture plate for 1 d before treatment with the MOL extract. After an additional 2-d incubation, total RNA was extracted. The synthesis of target cDNA probes and hybridization were performed as previously described [26]. Each 20 µg aliquot of total RNA was mixed with 5 µg of pdN6 primer (Amersham Biosciences, UK) in 15.4 µL of RNase-free water and incubated at 65°C for 10 min. cDNA was synthesized in the presence of 3 µL of Cy3- or Cy5-dUTP (1 mM each; NEN Life Science Products, Boston, USA) at 42°C for 2 h. The fluorescent-labeled cDNA was purified using a PCR purification kit (Qiagen). Both Cy3 and Cy5-labeled cDNAs were concentrated into a final volume of 27 µL by using Microcon YM-30 (Millipore Corp. USA). The hybridization mixture (80 µL) contained 20 µL of 20× SSC, 8 µL of 1% SDS, 24 µL formamide (Sigma, USA), 10 µg of salmon sperm DNA (Invitrogen Corp.) and 27 µL of labeled cDNA solution. The hybridization mixture was heated at 100°C for 2–3 min and immediately applied onto microarrays. The arrays were hybridized at 42°C for 12–16 h in a humidified hybridization chamber (Array Chamber X, GenomicTree Inc., Korea). The hybridized microarrays were washed, and quantification was performed using an Axon 4000B scanner (Axon Instruments, CA).

Data Acquisition and Analysis

The hybridization images were used for quantification with GenePix Pro 4.0 (Axon Instruments, CA). The average fluorescence intensity for each spot was calculated, and the local background was subtracted. All data normalization and statistical analyses were performed using GeneSpring 6.1 (Silicon Genetics, USA). Genes were filtered according to their intensity in the control channel. Intensity-dependent normalization (LOWESS) was performed, where the ratio was reduced to the residual of the Lowess fit of the intensity vs. ratio curve. The fold-change values were calculated by dividing the median of the normalized signal channel intensity by the median of the normalized control channel intensity. The ratios of fold changes were calculated by dividing the value of fold change with the value of fold change between relevant conditions. A value above 2.0 was considered significantly different. The parametric ANOVA test was performed using the Benjamini and Hochberg false discovery rate corrections below p values of 0.05 or 0.01 to identify genes differentially expressed across the samples. Hierarchical clustering was performed by similarity measurements based on Pearson correlations around 0. The correlation analysis was performed using Pearson correlation (from −1 to 1). The microarray data from these experiments are available for download athttp://218.48.131.23/Servelet/GeNet with a guest login.

Results

Antiproliferative Effects of Soluble MOL Extract on Lung Cancer Cell A549

To determine the inhibitory effect on human lung cancer cell A549, various soluble extracts from MOL were prepared using different extraction times. I fixed the temperature at 4°C and investigated the effects of different extraction times (0–24 hours) by MTT analysis. As shown in Figure 1, cell growth inhibition was the highest with the MOL extract obtained after extraction for 5 min.

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Figure 1. Inhibitory effects of the MOL extract on the proliferation of A549 lung adenocarcinoma cells.

The MOL extract was obtained after extraction with cold DW for 5-d incubation. Data were averaged from 3 independent experiments, and the variations were below 10%.

doi:10.1371/journal.pone.0095492.g001

Next, I investigated the changes in the cell cycle and apoptosis in A549 cells. After 48 h-treatment, 200, 300, and 400 µg/mL MOL extracts increased the average sub-G1 population for 6 independent experiments by 21%, 65%, and 93%, respectively (Figure 2A). Western blot analysis showed that caspase-3 was downregulated and that cleaved caspase-3 was upregulated upon MOL treatment in a dose-dependent manner.

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Figure 2. Induction of apoptosis in A549 cells by MOL treatment.

(A) FAScan analysis. The numbers in the data boxes indicate the G0/G1 fraction. (B) Western blot analysis of caspase-3. Data were averaged from 3 independent experiments, and the variations were below 10%.

doi:10.1371/journal.pone.0095492.g002

The cytotoxic effect of MOL extract against A549 cells was also examined using microscopy and clonogenic assays. As shown in Figure 3A, cells treated with more than 200 µg/mL of the MOL extract could not adhere onto the culture dishes and showed a typical apoptotic form. The survival ability of A549 cells following treatment with the MOL extracts was assessed using the clonogenic survival assay, in which a total of 3,000 cells were allowed to adhere for 24 h and were subsequently treated with increasing concentrations of MOL extract. After 7 d, the colonies were fixed and stained with crystal violet (0.1% w/v) for 1 min, washed with DW, and photographed. As shown in Figure 3B, the fraction of A549 surviving colonies significantly decreased with increasing amounts of the MOL extract, compared to the results for nontreated cells. No colony was observed above 100 µg/mL concentration.

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Figure 3. Inhibitory effect of MOL treatment on cell proliferation.

(A) Microscopic view (×1,000) and number of cells calculated by the cell counter. (B) Colony-formation assay and number of colonies.

doi:10.1371/journal.pone.0095492.g003

Detection of Intracellular ROS

Next, since the antioxidant effects of MOL extracts have been proven for several cellular and molecular targets associated with cell death and cell survival [27][28][29][30], I measured the intracellular ROS level. A fluorometric assay was used to determine intracellular levels of ROS. The production of ROS following treatment with soluble MOL extracts for 48 h was measured using the cell-permeable oxidation-sensitive dye 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (Invitrogen). As shown in Figure 4, the MOL extract induced a significant decrease in ROS concentration as compared to the untreated control group (blue plot) in a dose-dependent manner, indicating that MOL has free radical-scavenging abilities.

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Figure 4. ROS determination by DCFH-DA treatment.

Trypsinized cells grown for 48

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Housekeeping Gene or Protein as a Control

I investigated the expression of apoptosis-related mRNAs and proteins induced by MOL treatment. First, I tested the expression of housekeeping genes or proteins (e.g., β-actin) as a control by reverse transcription (RT)-PCR and western blot analysis, respectively. However, as shown in the first and second gels of Figure 5A, the expression level of β-actin was notably lower in 300 µg/mL MOL-treated cells than in untreated cells, in spite of loading equal amounts of sample on the gels. These phenomena were repeatedly observed in many experiments. In order to confirm that I loaded the same concentration of protein for the western blot analysis, total protein bands were visualized by Ponceau S (Sigma) staining of the membrane, ruling out any problem in gel loading and protein transfer to the membrane (Figure 5B). The reason why abnormal band patterns relative to the 300 µg/mL MOL-treated cells were observed will be discussed below in the context of the next experiments. However, subsequently, I added more RNA and proteins to the 300 µg/mL MOS-treated sample in order to obtain comparable expression levels among all the samples (third and fourth gels in Figure 5A).

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Figure 5. Western blot analysis.

Cells were grown for 48(A) β-actin expression. (B) Ponceau S solution staining. (C)–(F) Expression levels of different proteins.

doi:10.1371/journal.pone.0095492.g005

Signaling Pathway

A549 were exposed to 0–300 µg/mL of the soluble MOL extract and mRNA and protein expression was analyzed by RT-PCR and western blot, respectively. Akt, p-IkB, NF-kB, p-Erk, β-catenin, and cyclin D1 were significantly downregulated in MOL-treated cells in a dose-dependent manner (Figure 5C and D). However, c-Jun N-terminal kinases (JNKs), which can be activated by inflammatory signals, changes in levels of ROS, ultraviolet radiation, protein-synthesis inhibitors, and a variety of stresses, were increased by MOL treatment.

Forced expression of Sox, Oct4, Klf4, or c-Myc generates induced pluripotent stem (iPS) cells[31], while silencing of the SOX2 gene reduces the tumorigenic property of A549 cells with attenuated expression of c-MYC, WNT1, WNT2, and NOTCH1 in xenografted NOD/SCID mice[32][33]. Therefore, I investigated the protein and gene expression of the abovementioned molecules, including that of Notch1 [33] and its active intracellular domain, cleaved Notch1[34][35][36][37]. Interestingly, as shown in Figure 5E and F, the levels of all the proteins tested showed a significant decrease. Additionally, the dose-dependent decrease in mRNA expression was also consistent with the MOL treatment.

Significant Decrease in Protein Expression because of MOL Treatment

As shown in Figure 5A, the expression of the housekeeping protein β-actin was considerably low even if equal amounts of proteins were loaded and normal transfer of proteins to the membrane occurred. For more in-depth analysis, I examined the SDS-PAGE gel patterns. As shown in Figure 6, overall band intensities were similar regardless of the MOL concentrations used, suggesting that relatively similar concentrations of proteins were loaded in each well. However, interestingly, the levels of most of the proteins decreased, with the exception of some proteins in the 61.5–90.5 kDa regions. The main proteins in this range were identified as heat-shock proteins: heat shock cognate 71 kDa protein (69 kDa), heat shock 70 kDa protein (68 kDa), heat shock protein HSP 90-beta (83 kDa) and heat shock protein, mitochondrial (61 kDa).

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Figure 6. SDS-PAGE analysis for total cellular proteins.

Total proteins in cells treated with or without the MOL extract were measured. Equal amounts of total proteins (20 µg) were separated on a 10% SDS polyacrylamide gel, stained with Comassie brilliant blue, and destained. The box highlights the molecular weight region of interest.

doi:10.1371/journal.pone.0095492.g006

Significant Downregulation of Gene Expression by MOL Treatment

I showed that MOL led to a significant decrease in the levels of most proteins. Therefore, I investigated total gene e

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