Isosteviol derivatives induced apoptosis in Human lung cancer via targeting MEK/MAPK pathway:
An in vitro and in vivo study
of Health Sciences, Biomedical Program, Faculty of Science, college of Arts and Sciences, Qatar University, Doha, Qatar.
2 Department of Chemistry & Biochemistry, Ohio University, Athens, OH 45701,USA.
Results
Background and objectives
Diterpene derivatives 9 and 15 mediate apoptosis via Akt,
caspase-9 and caspase -3 signaling
Effect of Diterpene analogs on proliferation of lung
cancer cells
Background. Cancer metastasis is the major cause of cancer death. We
previously reported novel diterpenes derivatives which induced cytotoxicity in
lung cancer cells. The understanding of mechanisms which regulate lung cancer
sensitivity to our novel diterpene derivatives is necessary for development of
novel set of anticancer derivatives.
Aims. Investigate the molecular mechanisms of the optimized ring novel
diterpene derivatives on inhibition of proliferation, migration and tumor growth
in lung cancer in vitro and in vivo.
Results. Our data showed that novel MOM-ether analogs of isosteviol 8c and
9d decreased cell proliferation and induced apoptosis in H1299 lung cancer cells
more than p53 stably transfected H1299 cells. Flow cytometric analysis showed
that both diterpene derivatives 8c and 9d arrested the H1299 cells in G1 phase
which is further confirmed by increased expression level of p21. Moreover, both
diterpene derivatives 8c and 9d increased caspase-9 activity in H1299 cells and
the induction of apoptosis was significantly reduced after treating cells with
caspase-9 inhibitor LEHD-CHO. Both Diterpene derivatives 8c and 9d increased
Caspase 3 activities and induced Parp-1 cleavage in H1299 cells. Both derivative
8c and 9d reduced expression levels of AKT and Bcl-2 and increased expression
levels of Bax and Bad in H1299cells. Induction of apoptosis was significantly
reduced after treating H1299 with AKT inhibitor LY294002. In mice, oral
administration of diterpene derivative 9d inhibited the growth of xenograft
tumors, invasion, migration, and anchorage-independent growth in tumor
tissues without affecting body weight and it decreased the expression levels of
VEGF, MMP-9, MEK and MAPK in tumor tissues.
Conclusion. Based on previous results, our data support the development of
diterpenes derivatives as potential agent for lung cancer treatment via targeting
MEK/MAPK pathways
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Viable cells(%)
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10 11 12 13 14 15 16 17 18 19 20 29
0.25
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Viable cells (%)
1.25
1.5
1.75
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*
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9+LY29004 15+LY29004
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4.5
H1200 cells
NL-20 cells
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3.5
Log conc.
Diterpene derivatives
60
0.75
4
Induction of apoptosis (Abs at 405 nm)
c
Caspse 9 activties (% of control)
Viable cells(%)
9
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Induction of apoptosis (Abs at 405 nm)
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Induction of apoptosis(Abs at 405 nm)
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Caspase-3 activity(IU/ml)
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LEHD-CHO
LEHDCHO+ 9
LEHDCHO+ 15
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C
Figure 1 Diterpene derivatives reduced proliferation of H1299 cancer cells. Cell proliferation assay
was performed to detect living cells. H1299 cells were treated with 100 µM of the 16 derivatives for
24 h. Cells without derivative treatment were used as control. Each data point was an average of
results from thee independent experiments performed in triplicate and presented as M±SD(A). Dose
response curve was constructed and the % of viable cells was calculated at each concentration and
H1299 ells were treated with various concentrations of the potent derivatives 9 and 15 (B). MTT
assay with used to compare the cytotoxicity of Diterpene derivatives 9 and 15 between H1299 cells
and NL-20 cells (C). Induction of apoptosis in H1299 cancer cells by the two potent diterpene
derivatives according to the chosen concentration for 24 h duration interval. Induction of apoptosis
represents absorbance at 405 nm (D). H1299 cells were treated with the same concentrations of
derivatives for 24 h and induction of apoptosis was confirmed by appearance of
TUNEL positive cells in H1299 cells (E).
Experimental Design
As shown in Scheme 2, compound 7 was treated with HCl to remove the
MOM group. This provided a small set of analogs containing a free
hydroxyl group. The free hydroxyl group was then acylated to provide
12 additional analogs which are screened for anticancer activity.
Novel Diterpene derivatives 9 and 15 arrested cells in
G1 phase and increased p21 expression
9
15
Figure 3 Diterpene derivatives 9 and 15 induced apoptosis via Akt signaling and activation of caspases 9 and 3.
H1299 cells treated with Diterpene derivatives 9 and 15 for 24 hr and cells were harvested to investigate
expression of Bax, Bid, Akt and pAkt by Western blot analysis (A). Induction of apoptosis was determined after
pretreatment of 10 μM LY290042, a representative PI3K/Akt inhibitor for 1 h (B). Induction of apoptosis represents
absorbance at 405 nm. Each data point is the mean of three independent experiments and expressed as M±SD.
H1299 cells were treated with Diterpene derivatives 9 and 15 and Caspase-9 activity was determined with and
without derivatives (C) and H1299 cells were treated with and without Caspase -9 inhibitor and induction of
apoptosis was determined (D).Caspase-3 activity (As indicated by (DEVD-pNA) cleavage (E) and Parp-1 cleavage (F)
in H1299 cells were determined u[ on treating cells with Diterpene derivatives 9 and 15.
9d inhibited tumergencity in mice
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Percentage of growth
1Department
Ahmed M Malki1,,PhD Stephen C. Bergmeir2 PhD
Untreated
Treated
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G1
S
G2
Apoptosis
Cell cycle phases
The current study was come out to investigate the effect of novel
designed diterpene derivatives on cytotoxicity towards human
H1299 lung cancer cells, normal lung epithelial cells (NL-20) and an
animal model of lung cancer.
In vivo studies were performed to assess the anticancer effect of
Diterpene induced lung cancer bearing rats and we evaluated its
toxicity in different physiological processes.
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days
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Figure 4 Effect of Diterpene derivative treatment on growth of tumor volume in control untreated and
Diterpene derivative treated group in vivo. The data plotted as mean ±SEM ( n= 10).the tumor volumes
of the treated groups were significantly ( P < 0.05 ) lower than the negative control untreated group. Each
group of ten rats was used. Cells were treated with derivatives, total protein was extracted and used for
Western blotting analysis to determine the differences in VEGF,MEK,ERK, MMP-9 and MAPK expression
levels between treated and non-treated cells at the same
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30
The effects of the analogs were investigated by MTT , ELISA-based
apoptotic assay, terminal deoxynucleotidyl transferase dUTP nick end
labeling assay, immunofluoresence staining, flow cytometry, realtime reverse transcription polymerase chain reaction and Western
blot analysis
5
Figure 2 Diterpene derivatives 9 and 15 arrest H1299 cells at G1. The % of cell cycle phases
was determined in H1299 treatment with 20 µM and 30 µM of derivatives 9 and 15 respectively for
24 h (A). Each data point was an average of results from thee independent experiments performed in
triplicate and presented as M±SD. Cells were treated with derivatives, total protein was extracted
and used for Western blotting analysis to determine the differences in p21 expression levels between
treated and non-treated cells at the same condition(B), β-actin was used as loading control protein.
p21 in H1299 cells was immuno-stained using p21 monoclonal antibody and Texas Red conjugated
IgG as the secondary antibody (C).
Conclusion
 Diterpene derivatives 9 and 15 induced apoptosis in H1299 cancer cells
more than the normal lung epithelial NL-20 cells. Flow cytometric analysis
showed that both Diterpene derivatives 9 and 15 arrested the H1299 cells
in G1 phase.
 Moreover, both diterpene derivatives increased caspase-9 activity and the
induction of apoptosis was significantly reduced after treating cells with
caspase-9 inhibitor LEHD-CHO. Both Diterpene derivatives increased
Caspase 3 activities and induced Parp-1 cleavage in H1299 cells.
 To confirm the involvement of the PI3K/Akt pathway in Diterpene
derivatives-induced apoptosis, we investigated whether Derivatives 9 and
15 significantly induces apoptosis in the presence of LY290042, a
representative PI3K/Akt inhibitor, co-treatment with LY294002 and
Diterpene derivatives resulted in a marked decrease in apoptosis, indicating
that the PI3K/Akt pathway plays a role in regulating Diterpene derivativesinduced apoptosis of H1299 cells.
 In mice, oral administration of diterpene derivative 9d inhibited the
growth of xenograft tumors, invasion and it decreased the expression levels
of VEGF, MMP-9, MEK and MAPK in tumor tissues.
 Based on previous results, our data support the development of
diterpenes derivatives as potential agent for lung cancer treatment via
targeting MEK/MAPK pathways
Future directions
 The effects of the interesting novel analogs will be screened against various cancer cell
lines to investigate their selectivity.
 Investigational studies on the mechanism of action of these interesting analogs with
more cancer cell lines and in vivo studies are in progress.
Acknowledgements
I would like to thank Edison Institute of Biotechnology in Ohio University and Health
Sciences Department in Qatar University for supporting this work.