Delta-tocotrienol suppresses Notch-1 pathway by upregulating miR-34a in nonsmall cell lung cancer cells

Authors


Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating various cellular functions by transcriptional silencing. miRNAs can function as either oncogenes or tumor suppressors (oncomirs), depending on cancer types. In our study, using miRNA microarray, we observed that downregulation of the Notch-1 pathway, by delta-tocotrienol, correlated with upregulation of miR-34a, in nonsmall cell lung cancer cells (NSCLC). Moreover, re-expression of miR-34a by transfection in NSCLC cells resulted in inhibition of cell growth and invasiveness, induction of apoptosis and enhanced p53 activity. Furthermore, cellular mechanism studies revealed that induction of miR-34a decreased the expression of Notch-1 and its downstream targets including Hes-1, Cyclin D1, Survivin and Bcl-2. Our findings suggest that delta-tocotrienol is a nontoxic activator of mir-34a which can inhibit NSCLC cell proliferation, induce apoptosis and inhibit invasion, and thus offering a potential starting point for the design of novel anticancer agents.

Lung cancer is the leading cause of death among all malignant diseases, with nonsmall cell lung carcinoma (NSCLC) reported to have a 5-year survival rate of only 16%, accounting for 80% of all lung cancer cases.1 Clinical data have demonstrated that 30% of NSCLC cases have increased Notch activity, whereas 10% have gain-of-function mutation of the Notch-1 gene.2 After a series of proteolytic cleavages, the active form of Notch translocates from the cell membrane into the nucleus.3 Subsequently, Notch combines with other transcription factors to regulate the expression of its target genes, such as cyclin D1, Bcl-2 and Survivin.4, 5 As Notch signaling regulates critical cell fate decisions, alterations in Notch signaling are associated with tumorigenesis. Indeed, Notch expression has been reported to be upregulated in different types of cancers including colon, lung, head and neck and pancreatic cancers.2, 6–8 Overexpression of Notch-1 has been shown to inhibit apoptosis in many human cancers,9, 10 suggesting its potential as a therapeutic target. Recently, Notch-1 has been reported to stimulate survival of NSCLC cells during hypoxia by activating the IGF pathway.11 As a Notch downstream target, cyclin D1 expression is another indicator of poor prognosis in resectable NSCLC.12 Cyclin D1 is cell-cycle regulator protein expressed during the G1 phase and drives the G1/S phase transition. Similarly, overexpressions of cyclin D1 have been found in other types of cancers such as breast, bladder and colorectal cancers.13–15

MicroRNAs (miRNAs) are small noncoding RNAs that are involved in posttranscriptional gene regulation.16 These molecules silence expression of their target genes by directly interacting with the 3′-untranslated region of mRNA and promoting RNA degradation as well as inhibiting transcription. Accumulating data demonstrate that miRNAs play important roles in cancers by regulating the expression of various oncogenes and tumor suppressor genes.17, 18 For example, reduced expression of let-7 has been shown to be associated with shortened postoperative survival in lung cancer patients.19 Therefore, it is important to investigate the relationship between miRNA and the Notch signaling pathway. It is also important to find novel agents that could regulate the miRNA and Notch-1 pathway which could be useful for the treatment of NSCLC in the future.

It has been demonstrated that tocotrienols can induce apoptosis by inhibiting multiple signaling pathways such as the EGFR, NF-κB, MAPK and PI3K/AKT pathways.20 Previously, we provided experimental evidence showing that delta-tocotrienol inhibited Notch-1 signaling, cell proliferation, invasion and induced apoptosis in NSCLC cells.21 In our study, we report that inhibition of NSCLC cell growth and induction of apoptosis by delta-tocotrienol owing to modulation of Notch-1 pathway occurs via alteration of specific miRNA expression.

Material and Methods

Cell culture, reagents and antibodies

Human NSCLC cell lines (A549 and H1650) obtained from ATCC were grown in RPMI1640 medium (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in 5% CO2. Pure delta-tocotrienol was a kind gift from American River Nutrition (American River Nutrition, Hadley, MA). Protease inhibitor cocktail was obtained from Sigma (St. Louis, MO). Primary antibodies for cyclin D1, β-actin and cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin) were purchased from Cell Signaling Technology (Danvers, MA). Primary antibodies against Notch-1, Hes-1, Survivin, Bcl-2 and p53 were bought from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibodies were purchased from Bio-Rad Laboratories (Hercules, CA).

miRNA microarray analysis

miRNA expression of 84 miRNA was measured using the RT2 miRNA PCR array system (SABiosciences, MD) according to the manufacturer's instructions. The Eppendorf realplex 4 system (Hauppauge, NY) was used for all PCR reactions. Data analysis was performed using the RT2 Profiler PCR Array Data Analysis (SABiosciences, MD). The expressions of all the miRNAs were normalized to hsa-SNORD-44. Further, DIANA-microT was used to predict the target genes.

miRNA real-time reverse transcriptase-PCR

To validate the altered expression of the miRNA (miR-34a) that was found by miRNA array analysis, we first converted the miRNA to cDNA using RT2 First-Stand cDNA Synthesis Kit (SABiosciences, MD). This was followed by real-time miRNA reverse transcriptase-PCR (RT-PCR) analysis using miR-34a and snord-44 primers from SABiosciences (SABiosciences, MD) to validate data from the microarrays.

miRNA-34a transfection

A549 and H1650 cells were seeded in six-well plates at a density of two million per well for 24 hr and then transfected with pre-miRNA-34a (miR-34a), miRNA-negative control (negative control) or miRNA-34 inhibitor (AS-miR-34a) at a final concentration of 10 nmol/L using DharmaFect Transfection Reagent (Dharmacon, CO). For the combination treatment of miRNA-34 inhibitor and delta-tocotrienol, A549 and H1650 cells were transfected with AS-miR-34a for 6 hr, and then delta-tocotrienol stock solution was added to each well for a final concentration of 20 μM. After 72 hr of incubation, the cells were subjected to different experiments as outlined below.

Cell viability studies by MTS assay

The A549 and H1650 cells (5 × 103) were seeded in a 96-well culture plate. After overnight incubation, the medium was removed and replaced with transient transfection medium containing either negative control, miR-34a, delta-tocotrienol or the combination of delta-tocotrienol and Antisense (AS)-miR-34a. After 72 hr of incubation, 20 μL of CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI) was added to each well. After 2-hr incubation at 37°C in a humidified, 5% CO2 atmosphere, the absorbance at 490 nm was recorded on ELx800 plate reader (Bio-Tek, Winooski, VT). Each variant of the experiment was performed in triplicate.

Histone/DNA ELISA for the detection of apoptosis

The Cell Death Detection ELISA Kit (Roche, Palo Alto, CA) was used to quantify apoptosis in NSCLC cells. Briefly, 2 × 105 cells were seeded in six-well plates. After 24-hr incubation, cells were treated with transient transfection medium containing either negative control, miR-34a, delta-tocotrienol or the combination of delta-tocotrienol and AS-miR-34a for 72 hr. The cells were then lysed, and cytoplasmic histone/DNA fragments were extracted and incubated in microtiter plate modules coated with antihistone antibody. To detect the immobilized histone/DNA fragment, peroxidase-conjugated anti-DNA antibody was used before color development with ABTS substrate for peroxidase. The spectrophotometric absorbance of the samples at 405 nm was determined by using ELx800 plate reader (Bio-Tek, Winooski, VT).

Clonogenic assay

Cells (2 × 105) were seeded in six-well plates for 24 hr. Subsequently, the cells were cultured with transfection medium containing either negative control or miR-34a for 72 hr. This was followed by counting of the viable cells which were then plated in 100-mm dishes at 1,000 cells per plate. The cells were then incubated for 21 days at 37°C in a 5% CO2 incubator. All the colonies were fixed in 4% paraformaldehyde and stained with 2% crystal violet.

Annexin V-FITC method for apoptosis analysis

Annexin V-FITC apoptosis detection kit (BD, San Jose, CA) was used to measure the apoptotic cells. Briefly, A549 and H1650 cells were incubated in the presence of either negative control, miR-34a, delta-tocotrienol or the combination of delta-tocotrienol and AS-miR-34a for 72 hr. Cells were trypsinized, washed twice with ice-cold PBS and resuspended in 1× binding buffer at a concentration of 105/mL cells in a total volume of 100 μL. After that, 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added. All the samples were kept in the dark for 20 min at room temperature. Finally, 400 μL of 1× binding buffer was added to each tube and the number of apoptotic cells was analyzed by flow cytometry (BD, San Jose, CA).

Flow cytometry and cell-cycle analysis

Four million cells were seeded in 100-mm dish incubated overnight. Subsequently, all the cells were starved for another 24 hr. After that, the cells were released to transient transfection medium containing either negative control, miR-34a, delta-tocotrienol or the combination of delta-tocotrienol and AS-miR-34a for 72 hr, followed by collection and fixing with ice-cold 70% (v/v) ethanol for 24 hr. After centrifugation at 3,000g for 5 min, the cell pellet was washed with PBS (pH = 7.4) and resuspended in PBS containing PI (50 μg/mL) and DNase-free RNase (1 μg/mL). Samples were then incubated at room temperature for 2 hr, and the DNA content was determined by flow cytometry using a FAC Scan flow cytometer (BD, San Jose, CA).

Cell invasive assay

BD Biocoat invasion kit (BD, San Jose, CA) was used to evaluate the tumor invasive ability. Two million cells were seeded in six-well plates. Cells were then cultured with transient transfection medium containing negative control or miR-34a for 72 hr. Subsequently, 0.5 × 105 cells of A549 and H1650 with basal media were transferred into the upper chamber of each six-well plate. In the meantime, 3 mL of culture medium with 10% FBS was added into each lower chamber of the six-well plate. After 20-hr incubation, the cells in the upper chamber were removed using a cotton swab. Each experimental condition was performed in triplicate. The cells were fixed in 4% paraformaldehyde and stained with 2% crystal violet for 10 min. The stain in the cells was then dissolved in 20% acetic acid and the absorbance measured using ELx800 plate reader (Bio-Tek, Winooski, VT) at 570 nm.

Protein extraction and Western blotting

A549 and H1650 cells were treated with negative control, miR-34a, delta-tocotrienol, AS-miR-34a or the combination of delta-tocotrienol and AS-miR-34a for 72 hr to evaluate the effects of treatment on Notch-1, Hes-1, Bcl-2, cyclin D1 and β-actin expressions. Cells were lysed in the cold lysis buffer for 30 min on ice. Protein concentrations were determined using the Bradford protein assay kit (Bio-Rad Laboratories, CA). Each sample contained 50 μg of total cell lysates. The samples were subjected to 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (Whatman, Clifton, NJ) using transfer buffer (25 mM Tris, 190 mM glycine and 20% methanol) in a Hoefer TE70XP transfer apparatus (Holliston, MA). The membranes were incubated for 1 hr at room temperature with 5% nonfat dried milk in 1× TBS buffer containing 0.1% Tween (TBS-T). Subsequently, the membranes were incubated over night at 4°C with primary antibodies (1:1,000). The membranes were washed three times with TBS-T, and subsequently incubated with the secondary antibodies (1:5,000) containing 2% bovine serum albumin (BSA) for 2 hr at room temperature. The signal intensity was measured by chemiluminescent imaging using a chemiDoc XRS imager (Bio-Rad Laboratories, CA).

Immunostaining assay and confocal microscopy

Single-cell suspensions of A549 and H1650 cells were prepared and plated in Millicell® EZ slide (Millipore, MA). After transfection with negative control or miR-34a as described above, the cells were washed with 1× PBS, and fixed with 4% paraformaldehyde for immunofluorescence staining. After washing three times with 1× PBS, cells were blocked in PBS containing 1% BSA for 2 hr at room temperature and incubated with a mouse anti-p53 in blocking buffer for 2 hr at room temperature. Cells were then incubated with Alexa Fluor 488-conjugated antimouse IgG (1:50 dilution) for 1 hr at room temperature and mounted with 30 μL of the ProLong Gold antifade reagents (Invitrogen, CA). The p53-labeled cells were photographed under Nikon Eclipse 80i confocal microscope (Nikon, CA) using software Nikon Elements built in the microscope.

Data analysis

Results were analyzed using GraphPad Prism 4.0 (Graph Pad Software, La Jolla, CA) and are expressed as means ± SEM. Statistical comparisons between groups were conducted using one-way ANOVA. Values of p < 0.05 were considered to be statistically significant and individual p-values are shown in the figures.

Results

Treatment of NSCLC cells with delta-tocotrienol showed increased expression of miR-34a

To investigate the differences in miRNA expression in NSCLC cell line (H1650) upon treatment by delta-tocotrienol, we conducted a miRNA array analysis using the RT2 miRNA PCR array system (SABiosciences, MD). We found that miR-34a expression was fourfold higher in the H1650 cells treated with delta-tocotrienol compared to untreated H1650 cell (Fig. 1a). The results from the miRNA array were validated by miRNA RT-PCR analysis upon treatment of delta-tocotrienol (Figs. 1b and 1c). As shown in Figure 1b, there was a significant (p < 0.05) increase in miR-34a expression in the delta-tocotrienol-treated A549 cells compared to controls in dose-dependent and time-dependent manner. Similarly, a significant increase in miR-34a expression was also observed in H1650 cells in a dose- and time-dependent manner (Fig. 1c). As delta-tocotrienol has been shown to have anticancer effects in different cancer cell lines,21, 22 these results suggest that miR-34a could be an inhibitory molecule for cancer development and progression and that delta-tocotrienol could inhibit the progression of NSCLC through induction of miR-34a in NSCLC cells. Based on the results of these PCR data, a 20-μM concentration of delta-tocotrienol was selected for the evaluations of its effects in further experiments.

Figure 1.

Delta-tocotrienol induces the overexpression of miR-34a on NSCLC cells. (a) MicroRNA microarray data (a) of H1650 cell treated with or without of delta-tocotrienol. The cut-off lines represent fourfold change between the control and the delta-tocotrienol-treated H1650 cell. The plot was automatically generated by uploading the CT value to the Qiagen website. Differences in the relative expression of microRNA were analyzed between control and delta-tocotrienol-treated H1650 cell. (b,c) The microarray data were validated in NSCLC cells, A549 (b) and H1650 (c) using RT-PCR. The left panel in (b) and (c) show a time-dependent comparative expression of miR-34a with 15-μM delta-tocotrienol treatment. The right panels in both figures represent a dose-dependent response of comparative expression of miR-34a at 72-hr time point. *p < 0.05, **p < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

The efficiency of re-expression of miR-34a in NSCLC cells

To elucidate the role of miR-34a in the proliferation of NSCLC, we tested the transfection efficiency of miR-34a in NSCLC cells. As shown in Figures 2a and 2b, the relative expression of miR-34a was induced after 4-hr transfection in both A549 and H1650 cell lines. The relative expressions of miR-34 after 72-hr transfection were about 118- and 120-fold higher than the controls in the A549 and H1650 cell lines, respectively.

Figure 2.

Antiproliferative effects by miR-34a re-expression in the NSCLC cells. (a,b) Time response of transfection efficiencies of miRNA-34a at a final concentration of 10 nmol/L, normalized to Snord-44 in A549 (a) and H1650 (b) cell lines. (c,d) Cell viability of human NSCLC cell lines A549 (c) and H1650 (d) cells using the MTS colorimetric assay. NC: negative control; 34a: pre-miR-34a; AS-34a: Antisense miR-34a; DT3: delta-tocotrienol. Vertical bars indicate the mean cell count ± SEM (n = 3). *p < 0.05 is considered significant as compared to negative controls. (e,f) Photomicrographic differences in colony formation by clonogenic assay depicting cell survival of human NSCLC cell lines A549 and H1650 cells. NC: negative control; 34a: pre-miR-34a; AS-34a: Antisense miR-34a; DT3: delta-tocotrienol. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Re-expression of miR-34a inhibited proliferation of NSCLC cells

To investigate the role of miR-34a in the regulation of cell proliferation, we transfected A549 and H1650 cells with negative control, miR-34a, delta-tocotrienol or the combination of delta-tocotrienol and AS-miR-34a for 72 hr followed by MTS assay. We found that re-expression of miR-34a significantly inhibits cell proliferation in A549 and H1650 cells. The re-expression of miR-34a for 72 hr resulted in approximately 60% of cell growth inhibition relative to negative control in both A549 and H1650 cell lines. As shown in Figures 2c and 2d, there was no significant difference in cell viability of cells transfected with the negative control and the control cells. The negative control was, therefore, used as the control for further experiments. Conversely, transfection of both A549 and H1650 cells with AS-miR-34a, knockdown of miR-34a, resulted in a loss of sensitivity to delta-tocotrienol treatment. In A549 cells, cell proliferation was inhibited by 74% with delta-tocotrienol alone, whereas the combination treatment of AS-miR-34a and delta-tocotrienol reduced it by only 57%. Similarly, for the H1650 cells, inhibition of proliferation decreased from 80% induced by delta-tocotrienol alone to 69% induced by combination treatment of AS-miR-34a and delta-tocotrienol. Taken together, these results indicate that re-expression of miR-34a in NSCLC cells can inhibit cell proliferation as compared to the controls.

To confirm the effects of miR-34 re-expression on cells growth, clonogenic assays on A549 and H1650 were performed. Figures 2e and 2f show significant inhibition of colony formation by miR-34a re-expression compared to the negative control. Overall, the results from the clonogenic assay were consistent with the MTS data shown in Figures 2c and 2d, confirming that miR-34a significantly inhibits the proliferation of NSCLC cells

Induction of apoptosis by re-expression of miR-34a

As inhibition of cell growth could also result from apoptosis induced by re-expression of miR-34a, we further investigated whether re-expression of miR-34a could induce apoptosis by two different approaches. As shown in Figure 3a, our histone/DNA ELISA data demonstrate that apoptosis induced by re-expression of miR-34 in A549 cells is about 1.8-folds greater than that induced in the control. Similarly, in the H1650 cell line (Fig. 3b), the re-expression of miR-34a induced approximately three times the amount of apoptosis as compared to the control.

Figure 3.

Induction of apoptotic effects by re-expression of miR-34a in the NSCLC cells. (a,b) A549 and H1650 cells were transfected with containing negative control, miR-34a, delta-tocotrienol or the combination of delta-tocotrienol and AS-miR-34a for 72 hr. The apoptosis of both cell lines was determined by histone/DNA ELISA. (c) H1650 cell was transfected with negative control, miR-34a, delta-tocotrienol or the combination of delta-tocotrienol and AS-miR-34a for 72 hr. Apoptosis was determined by Annexin V-FITC analysis. The percentage of dead cells (upper left quadrant), live cells (lower left quadrant), cells in late apoptosis (PI+/Annexin V+; upper right quadrant) and cells in early apoptosis (PI-/Annexin V+; lower right quadrant) is indicated. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

The ELISA data were further confirmed by Annexin V/PI staining analysis in H1650 cells (Fig. 3c). Consistent with our ELISA data, the re-expression of miR-34a initiated about 18% apoptosis as compared to 10% in the control cells. Conversely, a decrease in apoptosis from 61% in control cells treated with delta-tocotrienol to 55% in the miR-34a knockdown cells, under treatment with delta-tocotrienol was observed. Collectively, our results suggest that miR-34 re-expression caused a statistically significant (p < 0.05) increase in the percentage of apoptotic cells in NSCLC cell lines.

Analysis of cell-cycle distribution after re-expression of miR-34a

To further investigate cell growth inhibition by re-expression of miR-34a, cell-cycle distributions were examined using PI staining followed by flow cytometry. As shown in Figures 4a and 4b, both A549 and H1650 cell lines showed increased G0–G1 arrest patterns after re-expression of miR-34a. For A549 cells (Fig. 4a), there were about 63% cells in the G0–G1 phase in the miR-34 overexpression group compared to 55% in control cells. A similar response was observed in the H1650 cells (Fig. 4b) with about 75% of cells in the G0–G1 phase in the miR-34a overexpression group as compared to 57% in control cells. In an effort to confirm our results, we treated both the A549 and the H1650 cells with delta-tocotrienol alone or with the combination of delta-tocotrienol and AS-miR-34a. As shown in Figure 4a, we found that knockdown of miR-34a deceased the proportion of cells in the G0–G1 phase from 77 to 65% upon treatment with delta-tocotrienol in A549 cells. Similarly, knockdown of miR-34a caused the decease in the fraction of cells in G0–G1 arrest from 62 to 50% upon treatment with delta-tocotrienol in the H1650 cell line (Fig. 4b).

Figure 4.

Re-expression of miR-34a induces cell-cycle arrest at G0–G1 phase. (a,b) Cell-cycle distributions analyzed by using flow cytometry in A549 (a) and H1650 (b) cells after 72-hr incubation. NC: negative control; 34a: pre-miR-34a; AS-34a: Antisense miR-34a; DT3: delta-tocotrienol. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

miR-34a transfection or delta-tocotrienol treatment suppressed Notch-1 and its downstream gene expression

As the previous data from our group demonstrated that delta-tocotrienol inhibited cell growth by downregulation of the Notch-1 pathway,21 we used DIANA-microT to predict whether Notch-1 pathway is the target genes of miR-34a. To investigate the role of miR-34a in the regulation of cellular signaling, A549 and H1650 cells were transfected with miR-34a for 72 hr. As shown in Figures 5a and 5b, we found that re-expression of miR-34a in A549 and H1650 cells resulted in the downregulation of Notch-1 and its downstream molecules such as Hes-1, Bcl-2 and Survivin. We also found that the expression of cyclin D1, a key regulator of G1–S cell-cycle transition, was reduced.

Figure 5.

Downregulation of Notch-1 and its target genes by re-expression of miR-34a and cell invasion. (a,b) The expressions of Notch-1, Hes-1, Cyclin D1, Survivin, Bcl-2 and β-actin protein were analyzed by Western blotting analysis followed by chemiluminescence detection in A549 (a) and H1650 (b) cells after 72-hr treatment. NC: negative control; 34a: pre-miR-34a; AS-34a: Antisense miR-34a; DT3: delta-tocotrienol. (c,d) Inhibition of NSCLC cells invasion ability by miR-34 re-expression using Matrigel-coated inserts in A549 (c) and H1650 (d) cells. NC: negative control; 34a: pre-miR-34a; Cells that invaded to the lower surface of the insert over a period of 20 hr were stained with crystal violet dye. Five random fields were counted for the number of invaded NSCLC cells. Cell invasion is presented (lower panels) as means ± S.E.M of three independent experiments.*p < 0.05, **p < 0.01.

To gain further molecular insight, we assessed whether inactivation of miR-34a by its specific inhibitor could lessen the effects of delta-tocotrienol. We found that downregulation of miR-34a opposes the effects induced by delta-tocotrienol. As shown in Figures 5a and 5b, addition of delta-tocotrienol to miR-34a knockdown cells only partially restored delta-tocotrienols ability to decrease Notch-1 and its downstream signaling molecules, such as Hes-1, Surivivin and Bcl-2. Taken together, our findings suggest that delta-tocotrienol inhibited cell proliferation and induced apoptosis by downregulating the Notch-1 pathway through miR-34a overexpression in NSCLC cells.

Inhibition of cell invasion by overexpression of miR-34a

Although the effect of miR-34 re-expression on antiproliferation and induction of apoptosis have been shown,23 its effects on tumor cell invasion have not been evaluated so far. Using invasion assay, we found that the invasive capacity of A549 (p < 0.01) and H1650 (p < 0.05) cells was significantly decreased by the re-expression of miR-34a compared to the controls. As shown in Figure 5c, re-expression of miR-34a in A549 inhibits invasive capability by 28%. Similarly, re-expression of miR-34a in H1650 cells (Fig. 5d) decreases its invasive ability by 20%.

Re-expression of miR-34a promotes p53 activity

P53, a tumor suppressor gene, has been shown to play important role in tumor progression24 and drug responses.25 To determine the effects of miR-34 re-expression on the transcriptional activities of the p53, A549 and H1650 cells were transiently transfected with miR-34a or negative control (Figs. 6a and 6b). We determined the subcellular colocalizations of p53 by immunofluorescence and confocal microscopy. Consistent with our apoptosis analysis, DAPI staining demonstrated that re-expression of miR-34 induced greater apoptosis in both NSCLC cell lines. In addition, confocal microscopy data showed that p53 activity was increased and colocalized in the nucleus by re-expression of miR-34a as compared to the control in both cell lines. Taken together, these results suggest that miR-34 induced apoptosis via the activation of the p53 pathway.

Figure 6.

Immunoreactivity with p53 by fluorescent immunocytochemistry. MiR-34a re-expression induces the p53 expression and apoptosis in NSCLC cells. Fluorescence microscopy analysis showing p53 expressions in A549 (a) and H1650 (b) cells upon transfection with miR-34a or negative control. The cells were mounted with antifade mounting medium and analyzed by confocal microscopy (resolution, 40×).

Discussion

Cancer is a genetic disease resulting from the failure in the regulation of cell growth. For diseases to occur, the genes which regulate cell growth and differentiation must be altered so as to transform a normal cell to a cancer cell.26 Accumulating evidence reveals that pathogenesis of cancer is a multistep process of sequential alterations in several, often many, oncogenes, tumor-suppressor genes, or miRNA in human cancers including lung cancer.27, 28 MiR-34a has been shown to be associated with cancer cell proliferation and drug resistance through E2F in colon cancer cells.29 In addition, miR-34a was reported to be downregulated in different cancer cell lines including neuro, melanoma, kidney, breast and pancreatic cancer cells.30–32 Recently, a study showed that NSCLC patients with upregulated miR-34a had better prognosis for survival.33 In our study, we found that miR-34 can be induced by the treatment of delta-tocotrienol in NSCLC cells. We also found that re-expression of miR-34a in A549 and H1650 NSCLC cells inhibits proliferation, induces apoptosis and initiates G0/G1 cell-cycle arrest. Following from our previous data that demonstrated the anticancer potential of delta-tocotrienol in NSCLC cell lines,21 the current results confirm the activity of miR-34a as a tumor suppressor and its potential role as one of the key players in the inhibition of NSCLC cells by delta-tocotrienol. Moreover, the anticancer effects were associated with depressed Notch-1 signaling. We, therefore, propose that delta-tocotrienol suppresses the Notch-1 pathway by upregulating miR-34a in NSCLC cells.

The molecular mechanisms involved in the miR-34-mediated inhibition of cell proliferation and invasion remain unclear. From our results, we believe that miR-34a inhibits cell proliferation and invasion partly through the regulation of Notch-1 signaling pathway. From the DIANA-microT database, we found that Notch-1 is the predicted target of miR-34a. Although miR-34 has been reported to suppress the glioma cell proliferation,23 regulation of cancer proliferation and invasion in NSCLC by Notch-1 is unknown. In our study, we found that the re-expression of miR-34a by transfection suppressed the expression of Notch-1 and its target genes including Hes-1, Survivin and Bcl-2 in NSCLC cells.

As a key G1–S cell-cycle transition regulator, overexpression of cyclin D1 has been shown to promote cell growth and is associated with chemotherapeutic drug resistance.34, 35 In our study, we found that treatment with delta-tocotrienol can reduce the expression of cyclin D1 (Figs. 5a and 5b) in NSCLC cells. Moreover, transfection with miR-34a also inhibited the cyclin D1 expression in both NSCLC cell lines (Figs. 5a and 5b). As cyclin D1 is required for G1–S transition, our cell-cycle data consistently showed that re-expression of miR-34a in NSCLC cells induces G1–S arrest. Specifically, in the lower cyclin D1 expression, the higher percentage of cells was arrested in G0–G1 phase compared to control.

Loss of function of p53, a well-recognized tumor suppressor gene, is associated with the pathogenesis of different types of human malignancies.24, 25 Previous studies have shown that p53 controls cell-cycle progression, apoptosis, DNA repair and angiogenesis through upregulating miR-34a.30 Other studies have demonstrated that suppression of p53 pathway is associated with the activated Notch-1.33, 36 In combination, these studies suggest that there might be cross-talk between the p53 and the Notch-1 pathways which need further investigations. In our study, we found that upregulation of miR-34a by transfection could induce the activation of p53, which translocates into the nucleus and promotes apoptosis in NSCLC (Fig. 6). In addition, this activation of p53 is associated with downregulation of Notch-1 expression. We found that delta-tocotrienol could downregulate Notch-1 expression and upregulate the miR-34a expression. Thus, we propose that delta-tocotrienol suppresses the Notch-1 pathway by upregulating miR-34a in NSCLC cells.

In conclusion, our results demonstrate that delta-tocotrienol can upregulate miR-34a expression, inhibit cancer cell proliferation, induce apoptosis and reduce cancer cell invasion, at least in part due to downregulation of Notch-1, the molecular target that is predominately activated in NSCLC. Very recently, miR-34a was found to be downregulated in glioblastoma multiforme cells and was shown to inhibit cell growth by targeting the Notch-1 pathway. Moreover, knockdown of notch-1 showed similar cellular functions as overexpression of miR-34a both in vitro and in vivo.36 These data corroborate some of the work presented here. However, further in vivo studies in appropriate animal models for NSCLC are needed to establish whether delta-tocotrienol could be useful in combination with conventional chemotherapeutics or conventional targeted agents such as cisplatin and erolitinib for the treatment of NSCLC.

Acknowledgements

Delta-tocotrienol was provided by American River Nutrition, Inc (Hadley, MA).

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