|Protocatechualdehyde; Apoptosis; Cell-cycle; Lung
cancer; Cancer prevention
|Growth arrest and DNA damage-inducible (GADD) 45 and
GADD153 proteins have been implicated in DNA repair, cell cycle
regulation and growth arrest along with numerous other cellular
mechanisms. In recent years, evidence has emerged that proteins
encoded by these genes play pivotal roles in tumor suppression and
apoptotic cell death. The GADD45 expression is rapidly regulated at
both transcriptional and post-transcriptional levels in response to
genotoxic stress via p53 tumor suppressor pathway or by mRNA stability
as well as other growth-arrest signals . Several studies have correlated
GADD153 expression with cell death by treating the cells to different
stress conditions especially with DNA damaging agents. Because of the
involvement of GADD45 protein with the cell cycle, apoptosis, DNA
repair and stability makes it an obvious target for cancer therapy .
Previous studies show that GADD45 and GADD153 proteins play key
roles in the induction of programmed cell death and thus we sought to
investigate their role in human lung cancer. In this study two GADD
genes (GADD45 and GADD153) were studied representing a p53-
dependent and a p53-independent pathway respectively.
|Lung cancers are carcinomas that derive from epithelial cells with
small-cell lung carcinoma (SCLC), and non-small-cell lung carcinoma
(NSCLC) being the main types. The estimated deaths from lung cancer
is 159,480 (87,260 in men and 72,220 among women), accounting
for about 27% of all cancer deaths . Worldwide, there are 2.28
million new cases of lung cancer annually, with 1.59 million deaths,
making lung cancer a leading cause of cancer-related mortality and
account for an estimate of about 14% of all new cancers . Tobacco
use, particularly of smoking, is the main contributor to lung cancer;
however, a significant number of non-smoking individuals are reported
with lung cancer . Males show higher mortality rate because of increased risk of lung cancer associated with cigarette smoking .
approximately reporting 63,000 new lung cancer cases each year in
India . Impediment in early detection and diagnosis of advanced
stage is the main reason for high mortality . The best strategy is
to minimize the development of lung cancer using cancer preventive
agents. There is a strong interest in cancer prevention with widely
investigated natural products, such as capsaicin, green tea, turmeric,
cardamom and resveratrol.
|Protocatechualdehyde which is isolated from Phellinus gilvus, has
been investigated as a promising cancer preventive agent because of
its medicinal properties. This mushroom belongs to Hymenochaetaceae
Basidiomycetes, and has advantages over many Phellinus species
due to short growth period (3months), making production costeffective
[9-11]. Previous studies have established various biological
activities of protocatechualdehyde. Among the reported benefits of
protocatechualdehyde are inhibition of pulmonary inflammation,
prevention of intraperitoneal adhesion under infectious circumstances,
promotion of dermal wound healing, anti-platelet aggregation and
antitumor activities [9,10,12,13]. Protocatechualdehyde has been a subject of continuous interest because of its effective scavenging
property for oxygen free radicals [14,15]. DPPH (2,2-diphenyl-1-
picrylhydrazyl) free radical scavenging and xanthine-oxidase (XO)-
inhibitory activities, antioxidant/anti-inflammatory activities of
protocatechualdehyde have been evaluated. Apoptotic cell death
induced by protocatechualdehyde is demonstrated in different cells.
The exact molecular mechanisms of protocatechualdehyde are not
clearly understood [16-19]. Since protocatechualdehyde is well studied
to have therapeutic effects on various types of diseases, the cancer
preventive activity of protocatechualdehyde is of very much interest,
and experiments with protocatechualdehyde in animal models
indicate the protective nature against several diseases. Specifically,
protocatechualdehyde inhibited cell growth of various cancer cell
lines, and induced apoptosis of cancer cells [16,20-22]. Moreover,
protocatechualdehyde was shown effective on cell-cycle regulation of the
mammalian cell line . Based on these findings, we investigated the
molecular mechanisms of cancer preventive and anti-cancer activities
of protocatechualdehyde for lung cancer prevention in humans, and
this study provides the first report on role of protocatechualdehyde in
the prevention of lung cancer in human.
|Numerous reports have focused on evidence that the cancer
preventive activity is stimulated by altering the phases of cell cycle,
associated with the modulation of their regulatory components [23,24].
This suggests that these genes are induced by stimulation of GADD45
and GADD153 in response to DNA damage and growth arrest, which is
shown in many cell types. In this study we report the anti-proliferative
effects of protocatechualdehyde on human lung cancer cell lines PC-9
and A549 by inducing GADD45 and GADD153 by p53 independent
manner. These results add to our knowledge on importance of
protocatechualdehyde on lung cancer prevention.
|Materials and Methods
|Chemicals and reagents
|Protocatechualdehyde, 4’, 6-diamidino-2-phenylindole (DAPI),
and propidium iodide (PI) were purchased from Sigma-Aldrich Pvt.
Ltd. (India). Antibodies specific for GADD45, GADD153 and GAPDH
were obtained (Santa Cruz Biotechnology, Santa Cruz, CA).
|Cell culture and transfection
|The human non-small cell lung cancer cell lines A549 and PC-9
were cultured in DMEM-F12 supplemented with 10% HyClone fetal
bovine serum (FBS) (ThermoFisher Scientific, Fremont, CA, USA) in
an atmosphere of 5% CO2 at 37°C. Cells were grown in 75 cm2 culture
flasks and harvested in a solution of trypsin-EDTA at the logarithmic
|PC-9 cells were transfected with human GADD45 and
GADD153 specific siRNAs prepared by Invitrogen. Stealth
RNAi negative control duplex (Invitrogen) was used as a
control. The siRNA targeting GADD45 corresponds to the
sequence GAGCAGAAGACCGAAAGGAUGGAUA, and
the siRNA targeting GADD153 corresponds to the sequence
GAGAAUGAACGGCUCAAGCAGGAAA. PC-9 cells were
transfected using Lipofectamine RNAi max (Invitrogen), according
to manufacturer’s instruction. In brief, 1.4 × 106 PC-9 cells were
transfected with final concentration of 50 nM siRNA in six-well culture
plate in triplicates. Cells were treated with protocatechualdehyde
or vehicle 24 h after transfection. DAPI staining was performed for
apoptosis assay. Cells were lysed in lysis buffer containing 0.2% NP-
40. Total RNA was extracted from the cells lysate using TRIzol reagent
following the manufacturer’s instructions (Invitrogen).
|Cells lysate was estimated for total protein content by Bradford
assay. Total 10 μg of protein sample was run on 10% polyacrylamide
gel. Subsequently transferred to a polyvinylidene difluoride (PVDF)
(Millipore) membrane by semi-dry electroblotting. Blocked in 5%
nonfat dry milk in TBS with Tween 20 (0.05%). Blots were incubated at
RT for 1h in primary antibody at 1:1250 dilutions in blocking solution.
Following washing with TBST, membranes were incubated respective
horseradish peroxidase (HRP)-conjugated secondary antibody, and
visualized with ECL plus western blot detection kit (GE Healthcare).
|Cell viability and cell growth assay
|Trypan blue dye exclusion method: PC-9 cells (1 × 104/ml)
were cultured in 6-well plates for 24 h followed by treatment with
different concentrations of protocatechualdehyde or vehicle as control
supplemented with 10% FBS for 24-72 h with changing culture
medium every 24 h. Protocatechualdehyde was dissolved in DMSO
as stock solution and stored at -20°C. Trypan blue dye exclusion test
 was used to determine the inhibition of cell growth. The result was
expressed as a percentage, relative to treatment with vehicle control,
and the IC50 values were calculated using non-linear regression analysis
(percent survival versus concentration). Results shown are from at least
three independent experiments performed in replicates.
|MTT assay: Cell viability was evaluated by measuring the amount
of insoluble formazan formed in live cells based on the reduction of
MTT salt. Briefly, 100 μl PC-9 cell suspensions at 2 × 104 cell/ml were
seeded in 96 well micro titer plate (BD, USA). Protocatechualdehyde
in concentration range of 0-100 μg/ml were added. MTT reagent was
added after 72 h exposure followed by dissolution of formed formazan
crystal using DMSO (Sigma, USA). Optical density was read with
ELISA reader (LX-800) at 550 nm. The dose-response curve is plotted
and concentration that exhibited 50% inhibition of cell growth (IC50) is
calculated. Concentration that inhibits 50% of cell viability was used as
a parameter for cytotoxicity.
|To compare the effects of different concentration of
protocatechualdehyde on the cell cycle, the Cycle TEST™ PLUS DNA
Reagent Kit (Becton Dickinson, USA) was used. The cells (1 × 106 cells/
ml) were seeded in a 6-well plate and treated with protocatechualdehyde
for 24h. After harvesting, made to suspension, permeabilized with
trypsin buffer. Incubated with propidium iodide (1mg/ml) for 10 min
in dark. Cells were analyzed by flow cytometry (BD FACS Calibur,
USA) using BD Cell Quest acquisition and analysis software. Cell cycle
phase distribution of nuclear DNA was determined by analyzing at
least 10,000 cells per sample and the percentage of cells in G1, S and
|Identification and quantitation of apoptotic cells were done by
DAPI staining. PC-9 cells were cultured overnight at the density of 5
× 105 cells/ml in a 6-well culture plate and then treated with different
concentrations (0-100 μg/ml) of protocatechualdehyde for 24 h. The
cells were then fixed with 500 μL fixing solution (acetic acid: methanol;
1:3) for 5 min, dried, and stained with the DNA-specific fluorochrome
DAPI (2 μg/ml). Following 10 min of incubation, the cells were washed
with phosphate buffered saline (PBS), air-dried, mounted with 90%
(v/v) glycerol. The percentage of apoptotic cells was calculated using
fluorescence microscopy (Olympus, Japan) with at least 200 randomly
selected cells. Results are obtained from at least three independent
|Quantitation of cell cycle and apoptosis related gene
expression by Quantitative Real-Time Polymerase Chain
|Total RNA was extracted from cultured PC-9 cells treated with
various concentrations of protocatechualdehyde for 24 h using
the TRIzol reagent and then one μg of RNA subjected to reversetranscription
using SuperScript® III First-Strand Synthesis System
(Life Technologies, Inc.). To allow a quantitative determination of
relative gene expression levels for the apoptosis related genes . The
cDNA content of the samples was normalized, and the linear range
of amplification was determined using SYBR Green reagent for each
primer set PCR on 7300 Real Time PCR System (Applied Biosystems).
GAPDH expression was used as normalization control. At least two
independent experiments were performed to confirm the results. The
sequences of the primers used are provided in Table 1.
|Statistical analyses were performed with Student’s t-test. The results
were considered to be significant when p value was less than 0.05,
|Effects of protocatechualdehyde exposure on cell viability
and cell growth
|Protocatechualdehyde inhibited cell growth in PC-9 cells, when
exposed with various concentrations and the inhibition was dosedependent
manner shown in Figure 1. After 24 h treatment with 100
μM protocatechualdehyde, the percentage of viable cells was 47.3%
of the vehicle control and after 48h treatment with 75 and 100 μM
protocatechualdehyde viable cells reduced to 48.6 and 31.0% of control.
The lowest and highest dose with significant changes were observed at
50 μM and 100 μM respectively, above which no considerable changes
were observed and therefore these two doses were used in the study
for analysis of role of protocatechualdehyde on expression of gene of
|Protocatechualdehyde induce apoptosis of PC-9 Cells
|We determined the mechanisms of growth inhibition activity
of PC-9 cell lines and further investigated apoptosis of PC-9 cells by
morphological changes with DAPI staining. Cells were treated with
various concentrations of protocatechualdehyde and percentage
of apoptotic cells increased dose-dependent manner and cells
with condensed and fragmented nuclei were observed. After 48 h
treatment of cells with protocatechualdehyde at 25, 50, 75 and 100 μM
concentrations, apoptosis was induced as 11.4, 32.5, 41.5 and 62.3% of
cells respectively (Figure 2). From the above results it was confirmed
that protocatechualdehyde treated cells shows alterations in the
expression of GADD genes, growth inhibition, and stimulation of cell
cycle arrest and apoptosis.
|Effect of protocatechualdehyde on cell cycle regulation in
|Regulatory effects of protocatechualdehyde on the cell cycle were
evaluated in PC-9 cells. Protocatechualdehyde at a concentration
of 100 μΜ in PC-9 cells significantly increased the cells of G1 phase
from 46.6 to 63.2% (Figure 3, Table 2). PC-9 cells when treated with
protocatechualdehyde at concentrations of 25 and 50 μΜ for 24 h, an
accumulation of S phase respectively was observed (Figure 3, Table
2).The growth inhibition was linked to protocatechualdehyde induced
G1 and S phase arrests in the cell cycle in PC-9 cells.
|Protocatechualdehyde induced expression of genes associated
with cell cycle and apoptosis
|The eukaryotic cell cycle is regulated by the periodic synthesis
and destruction of cyclins and their cyclin-dependent kinases and negatively controlled by cyclin-dependent kinases inhibitors (called
CDIs) . Expressions of these cell-cycle regulatory genes like Cyclin
D1, CDK2, CDK4 and CDK6 were determined in protocatechualdehyde
treated PC-9 cells for understanding the mechanisms of apoptosis.
Expression of genes and fold changes were evaluated by quantitative
real-time PCR after 24 h of treatment with protocatechualdehyde in
PC-9 cells. Transcript levels of cyclin D1, CDK2, CDK4 and CDK6
genes found to be decreased, as shown in Figure 4. In particular, after
24 h protocatechualdehyde at 100 μΜ concentration, the mRNA levels
of cyclin D1, CDK2, CDK4 and CDK6 were down-regulated 0.28, 0.31,
0.42 and 0.61 fold, compared with that of vehicle control. However,
after 24 h treatment of protocatechualdehyde at a concentration of
100 μΜ significantly increased the mRNA levels of the CDK inhibitors
p21 and p27 to 17.46 and 5.96 fold (Figure 4). Protocatechualdehyde in PC-9 cells decreased the expression of Bcl-2 (Figure 4) and there was no
change in expression of Bax gene.
|Protocatechualdehyde induces GADD45 and GADD153 gene
|GADD45 and GADD153 genes were up regulated with
protocatechualdehyde treatment in PC-9 cell line. Though the
increased expression was time and dose dependent still 100 μΜ
protocatechualdehyde dosages increased 29.2 and 21.6 fold expression
after 24 h incubation (Figure 5). Protocatechualdehyde treated human
lung cancer cells A549 cells with wild type p53 also showed similar
increased expression of GADD45 and GADD153. These results
suggest p53-independent induction of GADD45 and GADD153 by
protocatechualdehyde. Above observation was confirmed at protein level by western blotting for GADD45 and GADD153 proteins and
shown increase in protein level as shown in Figure 5.
|Protocatechualdehyde on GADD45 and GADD153
|From the above observations it was clear that protocatechualdehyde
induces the GADD45 and GADD153 gene expression with
consequential in apoptotic induction. Functions and involvement of
GADD45 and GADD153 genes were explored to study the mechanisms of apoptotic induction using siRNA-mediated knockdown of GADD45
and GADD153. PC-9 cells were treated with GADD45 and GADD153
siRNAs for 24 h, and expressions of the GADD45 and GADD153
genes that were induced by 100 μΜ protocatechualdehyde was
inhibited (Figure 6A). Furthermore, protein production of GADD45
and GADD153 in PC-9 cells induced by protocatechualdehyde (was
also inhibited by the siRNAs (Figure 6B). It was apparent to study the
relationship between the reduction of GADD45 and GADD153 proteins
and its effect on the apoptotic induction by protocatechualdehyde in
|In comparison with the control experiments where the
percentage of apoptotic cells were 20.0 and 33.0%, siRNA induced
apoptosis in 24.0 and 35.6% of PC-9 cells treated with 50 and 100μM
protocatechualdehyde, respectively, at 24 h (Figure 6C). However,
PC-9 cells treated with GADD45 and GADD153 siRNA, showed
significantly lower sensitivity towards the apoptotic induction by
protocatechualdehyde: 11.0 and 11.8% at 50 μM, and 15.6 and 16.2% at
100 μM, respectively (Figure 6C). From the above observations it was
evident that, both the GADD45 and GADD153 genes play significant
role in protocatechualdehyde-induced apoptosis and shall be targeted
for controlling the apoptosis process in the PC-9 cells.
|In the present study, we determined the growth inhibitory activities
and IC50 values of protocatechualdehyde and induction of G1/S phase
cell cycle arrest and apoptosis in colon cancer PC-9 cells. Earlier studies
revealed that protocatechualdehyde was able to exert consistent antiproliferative
activity indicating arrest from G1 to S in various cancer
cells, such as human colorectal cancer cells and smooth muscle cells [16,28]. The G1/S checkpoint is primarily controlled by collective
kinase activities of the complex of cyclin D with CDK4, CDK6 and
cyclin E and these G1 kinases can in turn be regulated by cell cycle
inhibitors, which may cause the cells to arrest at the G1 phase .
To evaluate the cell cycle inhibitor effect of protocatechualdehyde, we
investigated changes in regulation of putative G1 cyclin such as cyclin
D1 and the cyclin dependent kinase CDK2, CDK4, CDK6 and observed
that cell-cycle arrest is associated with downregulation of these cell
cycle regulators . Nuclear protein Cyclin D1, an essential regulator
of G1 to S phase progression, is tightly associated, and aberrantly
expressed in numerous human cancers  including human lung
tumors, and several other types of human cancer . Cyclin D1 is
frequently overexpressed in various forms of cancer including nonsmall
cell lung carcinoma (NSCLC)  and decrease in the level of
expression of Cyclin D1 inhibits growth and reverses the transformed
phenotype of human esophageal cancer cells . Phosphorylation of
the retinoblastoma (Rb) protein during the G1/S transition of the cell
cycle appears to be initiated by Cyclin D/CDK4 and from this study
we believe that protocatechualdehyde exerts its effect by cell cycle arrest in G1 phase in PC-9 cells by reducing the expression of Cyclin
D1 and CDK4/6 genes. Also the expression of CDK2 is decreased,
demonstrating the importance of CDK2 in the regulation of the cell
cycle G1 checkpoint . CDK inhibitor such as p21 is found associated
with multiple Cyclin-CDK complexes, phosphorylated p21 binds
simultaneously to both cyclin and CDKs and cyclin/CDK complexes are
regulated by p21 . Indeed, p21 is a potent CDK inhibitor and displays
selectivity for G1, and over-expression of p21 arrest cells in the G1 phase
of the cell cycle [36,37]. Protocatechualdehyde reportedly affects cell
proliferation and apoptosis in human colorectal cancer cells and define
potential mechanisms by which protocatechualdehyde mediate growth
arrest and apoptosis of cancer cells. Protocatechualdehyde decreases
Cyclin D1 expression in protein and mRNA level and suppress
luciferase activity of Cyclin D1 promoter, indicating transcriptional
downregulation of cyclin D1 gene in human colorectal cancer cells.
It was recently reported that suppressed cell proliferation by G1
arrest and induced apoptosis in human colorectal cancer cells 
and the effect may mediated by apoptosis through upregulation of
p21, in view of the fact that upregulation p21 may promote apoptosis.
We hypothesized that protocatechualdehyde inhibited the growth of
PC-9 cells and induced G1/S arrest of the cell cycle by upregulation
of p21 expression. Another member of the CDK inhibitor family p27/
Kip1 binding to Cyclin E-CDK2 complexes and the binding dependent
phosphorylation of p27 is involved in the transition of G1 to S phase
resulting in tumor suppression [38,39]. Earlier studies present strong
evidence that the lack of functional p27 has a profound effect on
tumorigenesis and is a major factor in the development of human
tumors [40-43]. Up-regulated p27 protein activity in PC-9 cells treated
with protocatechualdehyde signifies that the p27 gene upregulation is
associated with cancer preventive activity .
|To explore the possible association between GADD45 and GADD153
genes with apoptosis in PC-9 cells treated with protocatechualdehyde,
we examined the knockdown of GADD45 and GADD153 gene
expressions by siRNA in PC-9 cells. However, no significant change
in the expression of cell-cycle regulating gene including p21, p27, cyclin
D1, CDK2, CDK4, and CDK6 were observed. From our result, the
observed G1/S cell-cycle arrest by protocatechualdehyde in PC-9
cells may be associated with the variation of cell-cycle regulators by
protocatechualdehyde. A significant observation in this study is that,
expression of GADD45 and GADD153 genes in both p53 mutated PC-9
cells and p53 wild type A549 cells was up-regulated by treatment with
protocatechualdehyde, indicating that p53 protein does not have direct
stimulatory effect on expression of GADD45 and GADD153. The present
study was designed to determine whether protocatechualdehyde
increased expression of the gene GADD 45 and GADD153, which are
induced by genotoxic stress through p53-dependent and -independent
pathways. GADD45 and GADD153 gene expressions are induced by
deoxycholate in both HCT-116 colonocytes with wild type p53 and p53
mutant HCT-15 colonocytes . GADD proteins have been shown to
inhibit proliferation and stimulate DNA repair and/or apoptosis .
The induced effect of protocatechualdehyde on these gene expressions
in PC-9 and A549 cells would be independent of p53 as it was reported
with other genotoxic agents. One observation reported by several
researchers involves correlation between upregulation of GADD45
and GADD153 and apoptotic induction in actively dividing cells [46-48]. Earlier study indicates that the induction of apoptosis in mice
exposed to hyperoxia was associated with the upregulation of GADD45
and GADD153 gene expressions . Natural products like curcumin
is reported to induce DNA damage, apoptosis by GADD153 gene in
human colon cancer cell lines .
|In this study, we show that treatment with siRNAs inhibits the
expression of GADD45 and GADD153 in PC-9 cells and induce
apoptosis, suggesting significant role of GADD gene family in the
mechanisms of cancer prevention. Protocatechualdehyde is also one of
the active components of cardamom, popular dietary spice . Data
from earlier studies indicate that regular consumption of medicinal
herbs like cardamom enhances activities like fibrinolysis, antioxidant
 and Natural killer (NK) cell  in humans. Therefore, the daily
consumption of cardamom will contribute to protocatechualdehyde
and protect against the risk of cancer. GADD45 and GADD153 are new
targets for protocatechualdehyde in lung cancer, and may be used in
lung cancer prevention and treatment in humans.
|This work was supported by Manipal University, Manipal India.
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