Expression of caspase-3 and c-myc in non-small cell lung cancer.
ABSTRACT Caspase-3 is a cysteine protease that plays an important role in the process of apoptotic cell death, but little has been studied clinically on caspase-3 in lung cancer. Increased c-myc expression can result in mitosis or apoptosis, and its contribution to the pathogenesis and prognosis of lung cancer has gained interest. In the present study, the expressions of caspase-3 and c-myc, along with their possible correlations with prognostic variables, were analyzed in resected non-small cell lung carcinomas (NSCLC).
Archival tumor tissues from 147 previously untreated NSCLC patients were examined by immunohistochemistry for the expressions of caspase-3 and c-myc proteins. Clinical information was obtained through the computerized retrospective database from the tumor registry.
The expressions of caspase-3 and c-myc were detected in 60 (88/147) and 16% (24/147) of tumors, respectively. No association was found between caspase-3 and c-myc expressions. A multivariate analysis demonstrated the N status and pathologic stage to be significantly correlated with poor survival (p-value=.018 and .002, respectively), but positive expression of caspase-3 was associated with a good prognosis (p=.03).
Our data suggest the involvement of caspase-3 in the tumorigenesis of NSCLC. It is also noteworthy that caspase-3 expression might be a favorable prognostic indicator in these tumors.
- SourceAvailable from: Geoffrey S Baird[Show abstract] [Hide abstract]
ABSTRACT: Lung cancer remains the most common cause of cancer-related mortality. We applied a highly multiplexed proteomic technology (SOMAscan) to compare protein expression signatures of non small-cell lung cancer (NSCLC) tissues with healthy adjacent and distant tissues from surgical resections. In this first report of SOMAscan applied to tissues, we highlight 36 proteins that exhibit the largest expression differences between matched tumor and non-tumor tissues. The concentrations of twenty proteins increased and sixteen decreased in tumor tissue, thirteen of which are novel for NSCLC. NSCLC tissue biomarkers identified here overlap with a core set identified in a large serum-based NSCLC study with SOMAscan. We show that large-scale comparative analysis of protein expression can be used to develop novel histochemical probes. As expected, relative differences in protein expression are greater in tissues than in serum. The combined results from tissue and serum present the most extensive view to date of the complex changes in NSCLC protein expression and provide important implications for diagnosis and treatment.PLoS ONE 01/2012; 7(4):e35157. · 3.53 Impact Factor
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ABSTRACT: Accumulating evidence is revealing an important role of microRNA (miRNA) in tumor progression and chemotherapeutic resistance. Dicer is a cytoplasmic endoribonuclease type III crucial for production of mature miRNAs. The aberrant expression of Dicer has also been reportedly associated with clinical aggressiveness, prognosis, and patient survival in various cancer types. However, the molecular mechanisms of Dicer in acquired gefitinib resistance are still not clear. In this study, we analyzed the protein level of Dicer between gefitinib-sensitive (PC9) and gefitinib-resistant (PC9/GR) non-small-cell lung cancer (NSCLC) cell lines by Western blot analysis. Silence and overexpression of the Dicer were performed to investigate the effects on gefitinib sensitivity, as assessed by (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and sub-G1 assay of flow cytometry. To further explore the mechanism of chemoresistance, we examined whether Dicer knockdown led to modulating specific miRNAs and its miRNA target genes. Dicer expression was significantly increased in PC9/GR compared with PC9 cells. Knockdown of Dicer restores gefitinib sensitivity in resistant cells, and overexpression of Dicer enhances resistance to gefitinib in sensitive cells. Silencing of Dicer induces sensitivity to gefitinib in NSCLC cells through the downregulation of miR-30b/c and miR-221/222 to increase the protein level of caspase-3, resulting in an increase in gefitinib-induced apoptosis. Dicer contributes to the resistance to gefitinib in lung cancer. These results indicate that Dicer may be a target for diagnosis and therapy of patients with resistance to gefitinib.Annals of Surgical Oncology 04/2014; · 4.12 Impact Factor
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ABSTRACT: MicroRNAs (miRNA) play an important role in tumorigenesis, proliferation, and differentiation. Altered miRNA expression in cancer indicates that miRNAs can function as tumor suppressors or oncogenes. MiR-449c downregulation in non-small cell lung cancer (NSCLC) compared with normal lung tissues was investigated in this study. NSCLC cell proliferation and invasion assays indicate that transfection of miR-449c expression plasmid inhibits the proliferation and invasion ability of NCI-H23 and NCI-H838 cells. In addition, miR-449c overexpression could suppress tumor growth in vivo. Morever, c-Myc was identified as a direct target gene of miR-449c. These findings clearly suggest that miR-449c downregulation and c-Myc amplification may be involved in the development of NSCLC.FEBS letters 03/2013; · 3.54 Impact Factor
Cancer Research and Treatment 2004;36(5):303-307
Expression of Caspase-3 and c-myc in Non-Small Cell Lung
Jin young Yoo, M.D.1, Chi Hong Kim, M.D.2, So Hyang Song, M.D.2, Byoung Yong Shim, M.D.2, Youn
Ju Jeong, M.D.3, Meyung Im Ahn, M.D.3, Suji Kim, M.D.4, Deog Gon Cho, M.D.5, Min Seop Jo, M.D.5,
Kyu Do Cho, M.D.5, Hong Joo Cho, R.N.2, Seok Jin Kang, M.D.1 and Hoon Kyo Kim, M.D.2
Vincent's Hospital, The Catholic University of Korea, Suwon, Korea
4Radiation Oncology and
5Thoracic Surgery, St.
Purpose: Caspase-3 is a cysteine protease that plays
an important role in the process of apoptotic cell death,
but little has been studied clinically on caspase-3 in lung
cancer. Increased c-myc expression can result in mitosis
or apoptosis, and its contribution to the pathogenesis and
prognosis of lung cancer has gained interest. In the
present study, the expressions of caspase-3 and c-myc,
along with their possible correlations with prognostic
variables, were analyzed in resected non-small cell lung
Materials and Methods: Archival tumor tissues from 147
previously untreated NSCLC patients were examined by
immunohistochemistry for the expressions of caspase-3
and c-myc proteins. Clinical information was obtained
through the computerized retrospective database from
the tumor registry.
Results: The expressions of caspase-3 and c-myc were
detected in 60 (88/147) and 16% (24/147) of tumors,
respectively. No association was found between
caspase-3 and c-myc expressions. A multivariate analysis
demonstrated the N status and pathologic stage to be
significantly correlated with poor survival (p-value=.018
and .002, respectively), but positive expression of cas-
pase-3 was associated with a good prognosis (p=.03).
Conclusion: Our data suggest the involvement of
caspase-3 in the tumorigenesis of NSCLC. It is also
noteworthy that caspase-3 expression might be a
favorable prognostic indicator in these tumors. (Cancer
Research and Treatment 2004;36:303-307)
Key Words: Caspase-3, c-myc, Lung cancer
Correspondence: Seok Jin Kang, M.D., Ph.D., Department of Patholo-
gy, St. Vincent's Hospital Catholic University, Suwon 442-723,
Korea. (Tel) +82-31-249-7591, (Fax) +82-31-244-6786, (E-mail)
Received August 19, 2004, Accepted September 15, 2004.
This work was supported in part by the 2004 Research Fund from the
St. Vincent’s Hospital and from the Lung Cancer Study Group, St. Vin-
In cancer, two major factors determine the kinetics of cell
turnover: cellular proliferation and apoptosis (programmed cell
death). Dysregulation of their cellular signaling pathways can
result in cellular transformation and cancer. Dysregulation of
apoptotic cell death has evolved as a critical key player, not
only for tumorigenesis, but also in the biological behavior of
cancer (1). The present study focuses on two molecules playing
important roles in the cellular pathways affecting apoptosis.
Recent studies have implicated a family of cysteine proteases,
called caspases, as being associated with the induction of
apoptosis; the activated caspases cleave key substrates to induce
apoptotic cell death. The activation of at least one caspase
appears to be an essential step in cellular apoptosis. Caspase-3
(also known as CPP32, YAMA and apopain) is synthesized as
an inactive proenzyme, which upon activation, is cleaved at the
ASP28-Ser29 and ASP175-Ser176, generating two subunits of
17kDa and 12kDa, respectively. The activated caspase-3 abro-
gates the effect of substrates that protect cellular integrity, such
as the DNA-repair enzyme poly (ADP-ribose) polymerase
(PARP), thereby inducing apoptotic cell death (2). Caspase-3
has been reported to be expressed in some malignant tumors,
including leukemia, malignant lymphoma, neuroblastoma and
Increased c-myc expression can result in mitosis or apoptosis,
depending on the availability of other critical growth stimuli.
In the presence of such stimuli, c-myc acts as a classic proto-
oncogene, stimulating mitosis; in their absence, it initiates ap-
optosis (7). Because the dysregulated expression of c-myc
contributes to the tumorigenesis of cervix, breast and colon
cancers, there has been growing interest in analyzing its
contribution to the pathogenesis and prognosis of lung cancer
Lung cancer is a major cause of cancer death worldwide, and
has become the leading cause of cancer death in Korea.
Although recent molecular studies have provided increased
understanding of the biology of lung cancer, the essential
genetic features, along with factors for prognosis, remain to be
304 Cancer Research and Treatment 2004;36(5)
Table 1. Clinicopathologic features and immunohistochemical studies in 147 patients with NSCLC
Age NS NS
≥66 (n=76) 47
Gender NS NS
T status NS NS
T1, T2 (n=61) 44
T3, T4 (n=86) 49
N status NS NS
N0 (n=22) 13
N1, N2, N3 (n=125)80
Pathologic stage NS NS
I, II (n=20) 14
III (n=127) 79
Histology NS NS
Squamous cell carcinoma (n=63)43
Large cell carcinoma (n=23)10
Negative p-value Positive Negativep-value
105 48 22
determined. To date, the literature contains limited data regard-
ing caspase-3 and c-myc in lung cancer. In this study, the
expressions of those proteins in formalin-fixed, paraffin-em-
bedded, lung tumor tissues from patients with non-small cell
lung carcinoma (NSCLC) were analyzed by immunohisto-
chemistry. The resulting data were evaluated for possible
correlations between their expressions and clinicopathological
MATERIALS AND METHODS
The tumor tissue samples were obtained from 147 NSCLC
patients who underwent surgical resection at the Catholic
University St Vincent's Hospital between January 2001 and
December 2002. No pre-operative radio- or chemotherapy had
been performed. Clinical information was obtained through a
computerized retrospective database of the tumor registry. All
patients had been followed up for overall survival. Follow-up
data were available for a time period ranging from 4 to 169
weeks, with a mean of 92 weeks. Thirty-six patients died
during follow-up, but 111 were alive at the time of the study.
Tissue samples were fixed in 10 % buffered formalin. After
routine embedding, light microscopy led to the final diagnosis.
One of the authors (JY) reviewed the histopathological diagno-
sis, according to the relevant WHO classifications, tumor grade
and quality of the tissue sections. For immunohistochemical
staining, a sensitive peroxidase- streptavidin method, as describ-
ed previously (11), was performed using the representative
sections of archival, formalin-fixed, paraffin-embedded tumor
specimens. Briefly, each block was cut into 4μm thick
sections, which were deparaffinized in xylene and rehydrated
in graded alcohols and water. Endogenous peroxidase was
blocked by soaking in 3% H2O2 at 45oC for 4 min. The slides
were heated to 120oC in a citrate buffer (2.1g/L, pH 6.0) for
15 min to unmask the antigen, and then treated with a protein
blocking reagent before overnight incubation, with primary
antibodies at a 1：50 dilution, at 4oC, as recommended by the
supplier. A polyclonal anti-caspase-3 antibody was purchased
for the expression of caspase-3 (Pharmingen, San Diego, CA).
This antibody recognizes both the inactive 32 kDa caspase-3
and the active 17 kDa fragment. Another primary antibody used
in the present study was that of anti-c-myc (NeoMarkers,
Fremont, CA). After extensive washing, the sections were
incubated at room temperature for 10 min, with biotinylated
anti-mouse immunoglobulin antibodies (Zymed, San Francisco,
CA) at a 1：20 dilution, and subsequently with streptavidin-
biotin peroxidase complexes at a 1：25 dilution. The reaction
products were visualized by immersing slides in 3, 3'-dia-
minobenzidine tetrahydrochloride and finally counterstained
All series included positive and negative controls. The tonsil
and tissue from breast carcinomas were used as the positive
controls for caspase-3 and c-myc, respectively. Negative con-
trols of the immunohistochemistry technique were performed
by replacing the primary antibodies with dilution buffer. The
immunoreactivity for caspase-3 was considered positive if
predominantly cytoplasmic, with some nuclear staining. Tumor
cells showing a nuclear staining pattern were interpreted as
positive for c-myc. Three observers initially reached agreement
in 89% of cases. For the discordant samples, slides were re-
viewed jointly, and a consensus reached.
Jin young Yoo, et al：Caspase-3 and c-myc in Lung Cancer 305
Fig. 2. Survival of patients with NSCLC according to the expressions of caspase-3 and c-myc (Kaplan-Meier method).
Fig. 1. Immunohistochemical staining of caspase-3 (A) and c-myc (B) in primary resected NSCLC. The majority of tumor cells show immunoreacti-
vity in their cytoplasm with some nuclear staining (A), and intense nuclear immunostaining (B) (magnification × 100).
Statistical analysis was carried out using the SSPS 11.5
software package (Seoul, Korea). Survival was measured in
weeks from the date of surgery. The influence of various
clinicopathological factors, including caspase-3 and c-myc
expression indices, on the survival was assessed with a Cox
proportional hazards model. Two-sided P values were deter-
mined by means of the log-rank test. The level of significance
was set at 0.05.
The clinical characteristics, along with the results of immu-
nohistochemical staining, are listed in Table 1. There were 106
men and 41 women, with a mean age of 66 years, ranging from
33 to 87. The T status distribution was as follows: 8% T1, 33%
T2, 8% T3 and 51% T4. Twenty-two patients had no lymph
node involvement. All the patients were staged at the time of
their surgery, according to the guidelines of the American Joint
Committee on Cancer. There were 15 patients in stage I, 5 in
stage II and 127 in stage III. According to the histologic type,
61 were adenocarcinomas, 63 squamous cell carcinomas and 23
large cell carcinomas.
Negative and positive controls for immunohistochemistry
showed the expected results. Caspase-3 expression was iden-
tified in 88 patients (60%) (Fig. 1A), whereas c-myc was
observed in 24 (16%) (Fig. 1B). No correlation was found
between caspase-3 expression and c-myc activation.
The median survival time was significantly longer in patients
with positive caspase-3 expression than in the negative patients
(66 vs. 27 weeks). In contrast, the patients with c-myc positive
tumors had a median survival time of 28 weeks; while this was
34 weeks in c-myc negative patients (data not shown), but the
difference was not statistically significant. A multivariate
analysis of the clinical and immunohistochemical data is sum-
marized in Table 2. Positive expression of caspase-3 was signi-
ficantly correlated with a good prognosis (p=.03), whereas
c-myc showed no association with survival (Fig. 2). It was
indicated that the N status, pathologic stage and caspase-3
expression were independent prognostic factors.
To determine whether the combination of caspase-3 and
c-myc expressions had any additional prognostic value, patients
306 Cancer Research and Treatment 2004;36(5)
Table 2. Cox regression analysis of survival in 147 patients with
(negative vs positive)
(negative vs positive)
95% confidence L o g - r a n k
were grouped with respect to their expression stati of both
variables. The median survival time for patients with caspase-3
positive/c-myc-positive, caspase-3-positive/c-myc-negative, cas-
pase-3-negative/c-myc-positive and caspase-3-negative/c-myc
-negative tumors were 47, 59, 28 and 38 weeks (data not sho-
wn), respectively. This difference, however, was not statistically
Multiple factors are responsible for the modulation of tumor
growth and the prognosis of patients with malignant tumors.
Cellular proliferation and apoptosis are two major factors
determining the kinetics of cell turnover in cancer. An imba-
lance between these factors is believed to underlie tumor de-
velopment and prognosis. Caspase-3 is one of the most impor-
tant molecules in the apoptosis cascade (12), but the association
between caspase-3 expression and prognosis of various
malignancy types is controversial. In esophageal squamous cell
carcinomas, caspase-3 expression was present in 60% of
tumors, and correlated with a favorable prognosis (13). High
levels of caspase-3 mRNA expression in neuroblastomas were
also associated with a favorable prognosis (5). In contrast, a
significant correlation with a higher risk of recurrence, as well
as no association with prognosis, was reported in colorectal and
liver cell carcinomas, respectively (14,15).
In the present study, caspase-3 was frequently expressed in
NSCLC, suggesting its possible involvement in tumor deve-
lopment. In addition, caspase-3 positive staining was a sig-
nificant prognostic factor in predicting survival in these tumors;
the median survival was longer for caspase-3 positive than for
caspase-3 negative patients (66 vs. 27 weeks, p=.019). This was
in keeping with the results of Koomagi et al. (16,17), who
found caspase-3 expression in 72% of NSCLC, with a favorable
prognosis. Takata et al.(18) demonstrated 58% (69/118)
caspase-3 immunoreactivity in lung cancer, which was con-
sistent with the results of our study (60%). However, in their
study, the 5-year survival rate for caspase-3 positive patients
was significantly lower than that for caspase-3 negative patients
(66.6 vs. 82.1%, p=.049). The reason for a poor prognosis in
patients with caspase-3 expression was explained as a result of
decreased caspase-3 mediated apoptotic cancer cell death; since
the antibodies they used recognized the “uncleaved” inactive
form only, with most of the caspase-3 expression considered
as the inactive form. Caspases are synthesized as “uncleaved”
proenzymes. Cleavage at specific aspartate residues converts
the proenzymes into biologically active cysteine proteases.
Therefore, apoptotic cell death mediated by caspase-3 is
induced only after the inactive 32 kDa caspase-3 is cleaved into
two active fragments of 17 and 12 kDa, respectively. However,
the antibodies used in the study of caspase-3 expression with
a favorable prognosis also react, not with the “cleaved”
caspase-3, but with the “uncleaved” caspase-3 (16). Further-
more, the polyclonal anti-caspase-3 antibodies used in our study
recognized both the pro- and activated caspase-3. The discre-
pancies in the prognostic significance of caspase-3 activity,
thus, may not be due to the use of different primary antibodies.
Further studies on the expression of the “uncleaved” and
“cleaved” caspase-3 in a larger series, with a long-term follow-
up, will clarify the role of the caspase-3 protein in the prog-
nostic implications of lung cancer.
The c-myc oncogene is known to regulate neoplastic
development and apoptotic cell death (7). Deregulation of
c-myc occurs in a broad range of human tumors, including lung
cancer. C-myc not only promotes G1 to S cell cycle progression
in a mechanism involving activation of cyclin-E/cdk2, but also
sensitizes cells to apoptosis. Its expression in the determination
of prognostic significance has infrequently been investigated in
NSCLC. Amplification of c-myc was reported to be associated
with lymph node metastasis, but not with the clinical outcome
(19,20). It was, however, recently shown that c-myc amplifi-
cation was related to a shortening of survival (21). Our study
demonstrated no difference in the median survival time of
patients with and without c-myc expression (28 weeks vs. 34
weeks), suggesting that c-myc alone may not contribute
critically to the progression of NSCLC. Other gene products
and/or additional factors dictating the outcome of c-myc
expression on survival may be required.
Interestingly, our patients with caspase-3-positive and c-myc-
negative tumors had a tendency for the most favorable prog-
nosis, while patients with caspase-3-negative and c-myc-
positive tumors tended to have the most unfavorable prognosis.
Although the combination of both gene expression stati pro-
vided no statistically significant prognostic information, extend-
ed analyses, including these proteins and other associated gene
products, may aid in the understanding of the novel mechanism
for complex molecular control in lung cancer, which may yield
useful prognostic information.
Caspase-3 is expressed relatively frequently in NSCLC,
suggesting a possible role in tumorigenesis, which may con-
stitute a prognostic factor in these tumors. However, c-myc
alone may not contribute critically to tumor development and/or
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