Dual functions of NDRG1 in cell growth and invasion

Dual functions of NDRG1 in cell growth and invasion
Hsiao-Fang Li (李曉芳)1, Ann-Joy Cheng (鄭恩加)2
Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, 333,
School of Medical Technology, Chang Gung University, Taoyuan 333, Taiwan
Head neck cancer (HNC) is a top ten leading cancers in Taiwan. Since this
cancer usually occurs in the middle age male, at the high peak of life responsibility, it
has tremendous impact of family and society. Previously through differential display
analysis, we have identified several genes associated with HNC including NDRG1
(N-myc downstream-regulated gene-1), which is over-expression in HNC.
In the
present study, we further investigate cellular function of this gene leads to cancer
development. NDRG1 expression was modulated by shRNA and full-length plasmid
transfection and the potential alterations of cellular phenotypes were examined.
Results showed that NDRG1 suppressed cell growths by 14% to 55%.
phenomenon was confirmed by colony formation. However, by using wound healing
assay the cell migration increased (17% ~ 43%) and by using matrigel invasion assay
the invasion increased (7 ~ 12 folds). Both migration and invasion were significantly
enhanced. These results suggest that NDRG1 plays dual functions in tumorigenesis
with multiple aspects.
Background, Significance and Study Aims
Head-neck cancer
Epidemiology and etiology
Head-neck cancers (HNCs) include oral cavity cancer, laryngeal cancer and
nasopharyngeal cancer [1]. Most of the HNCs begin in squamous cells that line the
mucosal surfaces in the head and neck area. Some of the HNCs which begin in other
types of cells are called adenocarcinomas. HNCs are identified by the area where they
begin. As determined by the international classification of diseases, tenth revision, and
clinical modification (ICD-10-CM) HNCs are separated from C00 to C14. Depend on
the tumor location, HNCs can be classification as oral cavity includes lips (C00.x),
base of tongue (C01), unspecified parts of tongue (C02.x), gums (C03.x), floor of
mouth under the tongue (C04.x), hard palate (C05.x), buccal mucosa (C06.x), salivary
glands (C08.x), tonsils (C09.x), oropharynx (C10.x), nasopharynx (C11.x) and
hypopharynx (C13.x) [2].
The worldwide incidence exceeds half a million cases annually, the incidental
rates for HNCs show a wide variation range, with females generally showing much
lower rates (about one-third) than males. HNCs are highly prevalent in India,
comprising 35-40% of all malignancies, compared to 2-4% in Western countries. In
Taiwan, the incidence rate accounts for about 10% of all malignancies, and is
increasing annually [3]. Besides the incident rate, the tumor sites of HNCs are discrete
from various countries. Cancers of tongue and buccal mucosa constitute the majority
of HNCs in India. In contrast, the Western registries show cancers of the mouth floor
are the most frequent, with cancer of gum or tongue being rare. In Taiwan, the
majority of head-neck cancer occurs in buccal and tongue [4-5]. These differences
may be resulted from the distinct exposure of carcinogens and possibly the genetic
Association of carcinogen exposure with head- neck cancer
About 90 percent of head-neck cancers arise after prolonged exposure to specific
factors. These carcinogens include tobacco, alcohol, marijuana or cigarette smoking,
betel quid and human papillomavirus (HPV) [6-7]. Tobacco quid chewing leads to a
six-fold increase in risk for oral cavity cancer [8]. Alcohol abuse is known to be the
second largest risk factor for the development of oral cancer. A recent study classified
40% of head-neck cancer patients as alcoholic's [9]. Tobacco and alcohol use are the
most important risk factors for head-neck cancers, particularly those of the oral cavity,
oropharynx, hypopharynx, and larynx [10-13]. People who use both tobacco and
alcohol are at greater risk for developing these cancers than people who use either
tobacco or alcohol alone [12]. Smoking is postulated to have a greater carcinogenic
effect on the upper aerodigestive tract (oral cavity and larynx) than on the lower
airways. Betel quid represents a cheap pharmacologically addicting stimulant, widely
consumed in South East Asia. In Taiwan, approximately 85% of all head-neck cancer
patients are associated with this habit [14]. In India, head-neck cancer patients usually
combine with tabacco chewing, whereas in Taiwan, patients often together with
smoking. Epideminological studies have been reported the association of betel
chewing and head-neck cancer in many Southeast Asian, such as India, Pakistan,
Malaysia, and Taiwan [15]. It was further reported that inclusion of tobacco into the
quid increased the risk of carcinoma formation from 4 to 29 times. The risk of
head-neck cancer is increased by the use of alcohol, betel nut, or tobacco 10-, 18-, and
28-fold, respectively; combined use of these substances increases the risk up to
123-fold [16]. Recently, the statistical data evidence has been shown that Human
Papillomavirus (HPV) infection is playing an increasing important role in oral cancer
[17]. Besides, high-risk HPV types are a risk factor in about 26% of head-neck
squamous cell carcinomas independent of other known risk factors such as alcohol
and tobacco [18-20].
Therapy of head-neck cancer
The overall 5-year survival rate for patients with HNCs is among the lowest of
the major cancers, and has not changed during the past two decades. In Taiwan, the
incidence of HNCs has become the 6th leading cancer, the 4th leading cancer in male,
and is still increasing in the recent years [3]. Since HNCs usually occurs in the male
in the prime of life, at the high peak of society responsibility, it has tremendous
impact of family and society. The traditional therapies of HNCs include surgery,
radiotherapy and chemotherapy. Although the medical science has been progressed,
treatment of this disease has not significantly improved over the past decades. Until
the 1990s, the standard treatment for locally advanced disease was surgery [21]. Now
a day, more limited and less invasive operations are performed to remove tumors and
to try to preserve as much normal function as possible. In many cases, additional
therapies such as radiation or chemotherapy closely follow or occasionally come
before cancer surgery.
Chemotherapy is to interfering the growth and reproduction of cancer cells by
drugs. These chemical drugs by various pathways to effects the cancer cells and can
be given in a single drug or in combination with other drugs. Chemotherapy can also
accompany with radiation therapy. The primary purpose of radiation therapy is to
eliminate localized cancers pinpointed at a specific site with minimal damage of
healthy tissues. Radiation therapy can be used alone or used in conjunction with
surgery or chemotherapy with low mortality rate [22]. In general, the survival rate for
combination therapy is greater than any single type of therapy. Radiation therapy is
useful for organ and function preservation, especially for the critical and sophisticated
diseases such as head-neck cancers.
NDRG1 is a head-neck cancer associated gene
Previously, through differential display technique, we have globally surveyed
and identified several cancer-associated genes using matched normal and tumor
tissues from patients with head and neck cancer [23]. In this assay, 7 pairs of tumor
and normal tissues were used. We have identified 30 differential expressed genes from
the gel display results. After blasting their sequences through GenBank databases for
identification search, we have found eight genes that have not been reported in head
and neck cancer, as NPM, CDK1, NDRG1, HMGCR, EF1A, NAC and DSG3
(up-regulated), and CHES1 (down-regulated). The gene information is summarized in
the Table 1.
We further examine the definitive frequency information of each gene in head
and neck cancer patients, the mRNA expressions were measured in 52 normal and
cancer pair tissues by using comparative reverse transcription real-time quantitative
PCR method. Relative expression levels of these genes are shown in the Figure 1A,
and the frequency of the gene alterations in head and neck cancer samples are
summarized and shown in the Figure 1B.
In summary, we found that eight genes, NPM, CDK1, NDRG1, HMGCR,
EF1A, NAC, DSG3 and CHES1, were differentially expressed in head and neck
cancer. NPM, CDK1, NDRG1 and DSG3 are more significantly over-expressed and
with greater frequency than HMGCR, EF1A and NAC. CHES1 is significantly
under-expressed in cancer tissues. The mRNA expression level of NDRG1 in cancer
tissue was higher than normal tissue about 67.3%. This suggests that NDRG1 play
important roles in the development of head and neck cancer. In which, NDRG1 is our
focus in the present proposal study.
NDRG1 is a carcinogenic gene with multiple functions
Discovery of NDRG1
N-myc downstream-regulated gene-1 (NDRG1) is a novel protein named as RTP
(reducing agents and tunicamycin-responsive protein) that had been identified in 1996
by Kokame et al. [24]. In 1997, Drg1 (differentiation-related gene 1) has been
reported as a molecular markers for differentiation of normal and neoplastic colon
epithelium [25]. There were two scientific groups whom had reported respectively,
one was that Cap43 (calcium activated protein, 43kDa) gene can be induced by nickel
not only in vitro but also in several rat organs after oral exposure to NiCl 2 [26] the
other was that rit42 (reduced in tumor, 42 kDa) may play a growth inhibitory role, and
down-regulation may contribute to the tumor malignant phenotype [27]. Taken
together, these genes had been previously isolated as a homocysteine-inducible gene
in human endothelial cells (RTP). It had been reported that there were two
homological genes in mice named TDD5 [28] and Ndr1 [29] respectability. It also has
the same phenomenon in plants and roundworms [30]. The gene RTP, also called
Drg1/Cap43/rit42/TDD5/Ndr1 was evolutionarily conserved in humans, mice, rats,
roundworms, and plant. Furthermore, it has been found that RTP can expressed in
human’s brain, heart, prostate, kidney, lung, liver, skeletal muscle, placental and so on.
This finding indicated that RTP was not tissue specific [24, 31-32]. These two
characteristics of RTP suggest that this protein might serve an essential, housekeeping
function. At a later time, HUGO Nomenclature Committee had unified the official
name for this gene as NDRG1 [32], referred to as NDRG1 hereafter.
The biochemical features of NDRG1
NDRG1 which localized at human chromosome 8q24.3 [27] expressed a 3.0-kb
mRNA included a 1182bp open reading frame, translated 394 amino acids that encode
a protein with molecular mass of 43kDa. NDRG1 mainly distributed in cytoplasm [24]
but it also associated with the cellular membrane or nucleus [25, 31]. At present,
NDRG1 has seven or more phosphorylation sites and is phosphorylated by PKA
(protein kinase A) [30]. Because the high content of serines and threonines in NDRG1,
suggested that NDRG1 may have potential phosphorylation site for PKC (protein
kinase C) ,casein kinase II and tyrosine kinase [26]. The open reading frame of
NDRG1 encoded a three-tandem repeats of “GTRSRSHTSE” in its C-terminal region
[24], that’s pretein sequence was shown as Figure 2. The function of the three-tandem
repeats in NDRG1 hasn’t be reported however it can be phosphorylated by SGK1
(serum- and glucocorticoid-induced kinase 1) at Thr346, Thr356 and Thr366 or by GSK3
(glycogen synthase kinase 3) at Ser342, Ser352 and Ser362 [33]. The phosphorylation of
NDRG1 may associate with its function.
In mice, NDRG1 promoter activity is repressed by the combination of N-myc
and c-myc. However, the effect of N-myc and c-myc may not depend on the direct
binding to the NDRG1 promoter [29]. In human, Musuda et al. reported that Sp1 on
the NDRG1 promoter was involved in its basal gene expression activity [34]. Besides
the regulation of NDRG1 by its promoter, there still have other factors that would
affect the expression of NDRG1. The expression of NDRG1 was controlled by several
reagents which affecting DNA methylation and histone acetylation, such as ligands of
PPARγ (peroxisome proliferator-activated receptorγ) and the retinoid X receptor [35].
Furthermore, the expression level of NDRG1 has been associated with homocysteine,
cysteine, tunicamycin, β-mercaptoethanol [24], okadaic acid, Ni2+ [26],
lysophosphatidylcholine [36], Ca2+[37], DNA damage agents, p53 [27], androgen [38],
forskolin, phorbol myristate acetate [39], vitamin A, vitamin D3 [40]. When cell under
the condition of hypoxia the mRNA or protein level of NDRG1 would be induced
[41-42], so NDRG1 has been consider as a stress-responsive gene [30, 39, 43].
The association of NDRG1 with diseases
NDRG1 has been reported associated with many physiologipathological status,
including arteriosclerosis [24], cell differentiation, apoptosis, tumorigenesis,
metastasis and embryo development [29]. However the reports of NDRG1 functions
in regulation of the homeostatic conditions were not consistent. For example, NDRG1
were induced when cell were in the crisis state such as DNA damage [27] or in
chromosomal translocation in acute lymphoblastic leukemia [44]. On the other hand,
NDRG1 expression was decreased when colon cancer cells were treated with
flavopiridol which induced apoptosis [45].
As for the association of cancer, NDRG1 has been postulated as a tumor
suppressor. It has been proposed that NDRG1 was down regulated in several tumor
cell lines, including breast, prostate [27, 43, 46], colorectal [25, 48] and renal cancer
[34]. Furthermore, NDRG1 expression was associated with cellular differentiation
status [25, 27, 38, 39, 48], and overexpression of which suppress cell proliferation
[35]. Furthermore, NDRG1 was thought a metastasis suppressor gene for prostate
cancer and colon cancer [35, 46], since its expression was inverse correlated with the
metastatic progression [46].
Paradoxically, NDRG1 has been thought play oncogenic function. The
expression of NDRG1 was increased in mouse skin tumors, as well as in hyperplastic
mouse skin [49]. Moreover, NDRG1 protein is expressed at high levels in a variety of
cancers, including lung, brain, melanoma, liver, prostate, breast, and renal cancers
[50]. NDRG1 protein expression correlated the possibility of lymph node metastasis
in colorectal cancer [47].
Study Aims
As state above, the NDRG1 function was obscure in various reports. This may
be resulted from different study models and different assay conditions. In the present
study, we intend to investigate cellular functions of NDRG1 and to clarify its role in
oncogenesis of HNC. Two specific aims will be accomplished to unveil such
Cellular level of study. To examine the cellular effects of the NDRG1 in HNC
Biochemical level of study. To examine the biochemical pathways that leads to
the specific function of NDRG.
Aim #1: Functional study of NDRG1
Different endogenous levels of NDRG1 in various HNC cell lines
To choose proper cell lines as study material, the expression level of NDRG1 in
7 HNC cell lines were examine as Fadu, Detroit, NPC076, NPCBM1, NPCBM2, SAS
and OECM1. As shown in the Figure 4, after normalization with the β-actin. BM2
cells showed highest expression of NDRG1, while OECM1 showed the lowest, which
were suitable for siRNA or overexpression modulation. Therefore, these two cell lines
were chosen for study.
The efficiency of NDRG1-FL and NDRG1-siRNA
Two NDRG1 plasmids were constructed into a CMV promoter-driven vector, as
shown in the Figure 5A, cDNA full length (pNDRG1-FL) and the full length clone
with the deletion of 3R domain at the c-terminal end (pEGFP-△C). The expression
levels of these NDRG1 plasmids were examined by western blot analysis. As shown
in the Figure 5B, these two clones have highly expressions of NDRG1. On the other
hands, 3 NDRG1-siRNA plasmids with different target sequences on NDRG1 were
designed (Figure 3A). The knockdown efficiency of NDRG1-siRNA was determined
by western blot analysis. As shown in the Figure 6B, all the NDRG1-siRNA plasmids
could suppress the protein expressions.
NDRG1 promotes cell migration
The wound healing migration assay was applied to evaluate cell migration status.
As shown in Figure 8A, expression of NDRG1 by pEGFP-FL in OECM1 cells
increased migration ability by up to 20% at 16 hours. Conversely, expression of
pGSH1-siNDRG1 in BM2 cells decreased mobility rate (Figure 8B). These results
suggest that the NDRG1 promotes the migration ability in HNC cells.
NDRG1 promotes cell invasion
The Matrigel invasion assay was used to examine cell invasive capability. As
shown in the Figure 9A, transfection of NDRG1 cDNA (pEGFP-FL) in OECM1 cells
increased invasion ability by up to 10 folds at day 2. Conversely, although various
levels, three pGSH1-siNDRG1 clones (pGSH1-si225, pGSH1-si580 and
pGSH1-si980) also substantially decreased invasion ability (Figure 9B). Furthermore,
the cells permanently expressing NDRG1 dramatically increased invasion ability by 6
folds at day 2 (P<0.01)(Figure 9C). These data suggest that NDRG1 promotes
invasion ability in HNC cells.
Jemal, A., et al., Cancer Statistics, 2009. CA Cancer J Clin, 2009.
International Classification of Diseases, Tenth Revision, Clinical Modification.
2009: World Health Organization (WHO).
Cancer Causes of Death. 2009, Department of Health, E.Y., Taiwan, R.O.C
Sturgis, E.M., Q. Wei, and M.R. Spitz, Descriptive epidemiology and risk factors
for head and neck cancer. Semin Oncol, 2004. 31(6): p. 726-33.
Chang, J.T., et al., Enzyme immunoassay for serum autoantibody to survivin and
its findings in head-and-neck cancer patients. Clin Chem, 2004. 50(7): p. 1261-4.
Spitz, M.R., Epidemiology and risk factors for head and neck cancer. Semin Oncol,
1994. 21(3): p. 281-8.
Ragin, C.C., F. Modugno, and S.M. Gollin, The epidemiology and risk factors of
head and neck cancer: a focus on human papillomavirus. J Dent Res, 2007. 86(2):
p. 104-14.
Dikshit, R.P. and S. Kanhere, Tobacco habits and risk of lung, oropharyngeal and
oral cavity cancer: a population-based case-control study in Bhopal, India. Int J
Epidemiol, 2000. 29(4): p. 609-14.
Deleyiannis, F.W., et al., Alcoholism: independent predictor of survival in patients
with head and neck cancer. J Natl Cancer Inst, 1996. 88(8): p. 542-9.
Andre, K., et al., Role of alcohol and tobacco in the aetiology of head and neck
cancer: a case-control study in the Doubs region of France. Eur J Cancer B Oral
Oncol, 1995. 31B(5): p. 301-9.
Iribarren, C., et al., Effect of cigar smoking on the risk of cardiovascular disease,
chronic obstructive pulmonary disease, and cancer in men. N Engl J Med, 1999.
340(23): p. 1773-80.
Murata, M., et al., A nested case-control study on alcohol drinking, tobacco
smoking, and cancer. Cancer Detect Prev, 1996. 20(6): p. 557-65.
Winn, D.M., Smokeless tobacco and aerodigestive tract cancers: recent research
directions. Adv Exp Med Biol, 1992. 320: p. 39-46.
14. Liao, C.T., et al., Lack of correlation of betel nut chewing, tobacco smoking, and
alcohol consumption with telomerase activity and the severity of oral cancer.
Chang Gung Med J, 2003. 26(9): p. 637-45.
15. Jafarey, N.A., Z. Mahmood, and S.H. Zaidi, Habits and dietary pattern of cases of
carcinoma of the oral cavity and oropharynx. J Pak Med Assoc, 1977. 27(6): p.
16. Ko, Y.C., et al., Betel quid chewing, cigarette smoking and alcohol consumption
related to oral cancer in Taiwan. J Oral Pathol Med, 1995. 24(10): p. 450-3.
17. Campisi, G. and L. Giovannelli, Controversies surrounding human papilloma
virus infection, head & neck vs oral cancer, implications for prophylaxis and
treatment. Head Neck Oncol, 2009. 1(1): p. 8.
18. Applebaum, K.M., et al., Lack of association of alcohol and tobacco with
HPV16-associated head and neck cancer. J Natl Cancer Inst, 2007. 99(23): p.
19. Ritchie, J.M., et al., Human papillomavirus infection as a prognostic factor in
carcinomas of the oral cavity and oropharynx. Int J Cancer, 2003. 104(3): p.
20. Schwartz, S.R., et al., Human papillomavirus infection and survival in oral
squamous cell cancer: a population-based study. Otolaryngol Head Neck Surg,
2001. 125(1): p. 1-9.
21. Forastiere, A.A., et al., Concurrent chemotherapy and radiotherapy for organ
preservation in advanced laryngeal cancer. N Engl J Med, 2003. 349(22): p.
22. Teshima, T., et al., Incidence of other primary cancers in 1,569 patients with
pharyngolaryngeal cancer and treated with radiation therapy. Strahlenther Onkol,
1992. 168(4): p. 213-8.
23. Chang, J.T., et al., Identification of differentially expressed genes in oral squamous
cell carcinoma (OSCC): overexpression of NPM, CDK1 and NDRG1 and
underexpression of CHES1. Int J Cancer, 2005. 114(6): p. 942-9.
24. Kokame, K., H. Kato, and T. Miyata, Homocysteine-respondent genes in vascular
endothelial cells identified by differential display analysis. GRP78/BiP and novel
genes. J Biol Chem, 1996. 271(47): p. 29659-65.
25. van Belzen, N., et al., A novel gene which is up-regulated during colon epithelial
cell differentiation and down-regulated in colorectal neoplasms. Lab Invest, 1997.
77(1): p. 85-92.
26. Zhou, D., K. Salnikow, and M. Costa, Cap43, a novel gene specifically induced by
Ni2+ compounds. Cancer Res, 1998. 58(10): p. 2182-9.
27. Kurdistani, S.K., et al., Inhibition of tumor cell growth by RTP/rit42 and its
responsiveness to p53 and DNA damage. Cancer Res, 1998. 58(19): p. 4439-44.
Lin, C.T., et al., Characterization of seven newly established nasopharyngeal
carcinoma cell lines. Lab Invest, 1993. 68(6): p. 716-27.
Shimono, A., T. Okuda, and H. Kondoh, N-myc-dependent repression of ndr1, a
gene identified by direct subtraction of whole mouse embryo cDNAs between wild
type and N-myc mutant. Mech Dev, 1999. 83(1-2): p. 39-52.
Agarwala, K.L., et al., Phosphorylation of RTP, an ER stress-responsive
cytoplasmic protein. Biochem Biophys Res Commun, 2000. 272(3): p. 641-7.
Lachat, P., et al., Expression of NDRG1, a differentiation-related gene, in human
tissues. Histochem Cell Biol, 2002. 118(5): p. 399-408.
32. Zhou, R.H., et al., Characterization of the human NDRG gene family: a newly
identified member, NDRG4, is specifically expressed in brain and heart. Genomics,
2001. 73(1): p. 86-97.
33. Murray, J.T., et al., Exploitation of KESTREL to identify NDRG family members as
physiological substrates for SGK1 and GSK3. Biochem J, 2004. 384(Pt 3): p.
34. Masuda, K., et al., Downregulation of Cap43 gene by von Hippel-Lindau tumor
suppressor protein in human renal cancer cells. Int J Cancer, 2003. 105(6): p.
35. Guan, R.J., et al., Drg-1 as a differentiation-related, putative metastatic
suppressor gene in human colon cancer. Cancer Res, 2000. 60(3): p. 749-55.
36. Sato, N., et al., Changes of gene expression by lysophosphatidylcholine in vascular
endothelial cells: 12 up-regulated distinct genes including 5 cell growth-related, 3
thrombosis-related, and 4 others. J Biochem, 1998. 123(6): p. 1119-26.
37. Salnikow, K., T. Kluz, and M. Costa, Role of Ca(2+) in the regulation of
nickel-inducible Cap43 gene expression. Toxicol Appl Pharmacol, 1999. 160(2): p.
38. Ulrix, W., et al., The differentiation-related gene 1, Drg1, is markedly upregulated
by androgens in LNCaP prostatic adenocarcinoma cells. FEBS Lett, 1999.
455(1-2): p. 23-6.
39. Xu, B., L. Lin, and N.S. Rote, Identification of a stress-induced protein during
human trophoblast differentiation by differential display analysis. Biol Reprod,
1999. 61(3): p. 681-6.
40. Piquemal, D., et al., Differential expression of the RTP/Drg1/Ndr1 gene product in
proliferating and growth arrested cells. Biochim Biophys Acta, 1999. 1450(3): p.
41. Park, H., et al., Hypoxia induces the expression of a 43-kDa protein (PROXY-1) in
normal and malignant cells. Biochem Biophys Res Commun, 2000. 276(1): p.
42. Salnikow, K., et al., Carcinogenic metals induce hypoxia-inducible
factor-stimulated transcription by reactive oxygen species-independent
mechanism. Cancer Res, 2000. 60(13): p. 3375-8.
43. Segawa, T., et al., Androgen-induced expression of endoplasmic reticulum (ER)
stress response genes in prostate cancer cells. Oncogene, 2002. 21(57): p.
44. Rutherford, M.N., et al., The leukemogenic transcription factor E2a-Pbx1 induces
expression of the putative N-myc and p53 target gene NDRG1 in Ba/F3 cells.
Leukemia, 2001. 15(3): p. 362-70.
45. Motwani, M., et al., Drg1, a novel target for modulating sensitivity to CPT-11 in
colon cancer cells. Cancer Res, 2002. 62(14): p. 3950-5.
46. Bandyopadhyay, S., et al., The Drg-1 gene suppresses tumor metastasis in prostate
cancer. Cancer Res, 2003. 63(8): p. 1731-6.
47. Wang, Z., et al., Correlation of N-myc downstream-regulated gene 1
overexpression with progressive growth of colorectal neoplasm. World J
Gastroenterol, 2004. 10(4): p. 550-4.
48. van Belzen, N., et al., Expression of differentiation-related genes in colorectal
cancer: possible implications for prognosis. Histol Histopathol, 1998. 13(4): p.
Gomez-Casero, E., et al., Regulation of the differentiation-related gene Drg-1
during mouse skin carcinogenesis. Mol Carcinog, 2001. 32(2): p. 100-9.
Cangul, H., et al., Enhanced overexpression of an HIF-1/hypoxia-related protein
in cancer cells. Environ Health Perspect, 2002. 110 Suppl 5: p. 783-8.
Colwill, K., et al., The Clk/Sty protein kinase phosphorylates SR splicing factors
and regulates their intranuclear distribution. EMBO J, 1996. 15(2): p. 265-75.
Liao, S.K., et al., Chromosomal abnormalities of a new nasopharyngeal carcinoma
cell line (NPC-BM1) derived from a bone marrow metastatic lesion. Cancer Genet
Cytogenet, 1998. 103(1): p. 52-8.
Irizarry, R.A., et al., Exploration, normalization, and summaries of high density
oligonucleotide array probe level data. Biostatistics, 2003. 4(2): p. 249-64.
Ashburner, M., et al., Gene ontology: tool for the unification of biology. The Gene
Ontology Consortium. Nat Genet, 2000. 25(1): p. 25-9.
McManus, M.T., et al., Small interfering RNA-mediated gene silencing in T
lymphocytes. J Immunol, 2002. 169(10): p. 5754-60.
Hannon, G.J., RNA interference. Nature, 2002. 418(6894): p. 244-51.
Material and Methods
Cells and Cell culture
Six head-neck cancer cell lines either devived from oral cavity cancer OEC-M1,
KB, nasopharyngeal carcinomas NPC-BM2 [52], NPC076 [28] and
pharyngolaryngeal cancer cell lines FaDu and Detroit 562 were used for the study.
OEC-M1, KB and NPC076 were kindly provided by Dr. I. Chang in the Yang-Ming
University of Taiwan. NPC-BM2 was generously provided by Dr. S.K. Liao in Chang
Gung University of Taiwan. The cells OEC-M1 and MPC-BM2 were maintained in
RPMI Medium 1640 (Gibco BRL, Bethesda,ML, USA) supplemented with 10% fetal
bovine serum and 1% antibiotics (10,000 units/ml penicillin, 10,000 μg/ml
streptomycin, and 25 μg/ml amphotericin B) ; KB, Fadu and Dtroue562 cell were
incubate in MEM (Invitrogen Life technologies, Brazil) supplemented with 10% fetal
calf serum and 1% antibiotics; NPC076 cells were incubated in DMEM medium
contained10% fetal bovine serum and 1% antibiotics, and were cultured at 37℃ in a
humidified atmosphere containing 5% CO2.
RNA extraction
Total RNA was extracted with TRIzol reagent (Gibco BRL, Rockville, MD, USA)
following the manufacturer’s instructions. The concentration, purity, and amount of
total RNA were determined by ultraviolet spectrometry.
Total protein extraction
Cell protein extracted by subcellular fractionation. Cellular proteins were
extracted by incubation for 30 min at 4oC with iced-cold CHAPS lysis buffer (10 Mm
Tris, pH 7.4, 1 mM MgCl2, 1 mM EGTA, 150 mM NaCl, 0.5% CHAPS and 10%
glycerol) then homogenized and incubated on ice for 30 minutes. After centrifuging at
14,000 g for 30 min at 4℃, the supernatant was harvested for protein quantification
The protein concentration was determined using Protein Assay Dye Reagent (Bio-Rad,
Hercules, CA, USA).
Cloning of the NDRG1-RNAi plasmid
The existing approaches to suppress specific gene expression in mammalian cells,
mainly antisense and dominant-negative, have shown inefficient and inconsistent,
compared to currently developed technique: RNA interference (RNAi) [55-56]. RNAi,
with its characteristic efficacy at very low concentrations, in the low nanomolar range,
holds great promise for knock down genes in mammalian cell cultures. In this study,
we use pGSH1-GFP vector (Genlantis, Inc., San Diego, CA, USA) to generate the
siRNA plasmid. We will construct an RNAi expression vector in which 19-nt sense
and an antisense sequences against a target gene under the control of H1 promoters.
Because of secondary structure and other factors, some target sequences are more
potent than others so not all RNAi target sequence are equally potent. Therefore we
design 3 specific NDRG1 RNAi oligonucleotides will be annealed to pGSH1-GFP
vector sequence in Figure5A and Figure5B. The annealing generated sites
corresponding to the blunt end and the overhang will match to the BamHI- and
NotI-digested pGSH1-GFP vector. The ligation reaction between the annealing
oligonucleotide and pGSH-GFP at the BamHI and NotI cloning sites will generate
NDRG1-RNAi plasmid. Gene sequence information can be obtained from GeneBank
database and the BLAST sequence analysis programs will be used to survey candidate
RNAi sequence for gene specificity.
Western blotting
For western blot analysis, 30μg of cellular protein was prepared. All samples
were boiled at 95oC for 5 min, and subjected to 10% SDS-polyacrylamide gel for
electrophoresis. The protein from the electrophoretic gel were transferred to a
nitrocellulose membrane and blocked at 37 ℃, 1 hr with 5% non-fat milk in PBST
solution (phosphate buffer saline plus 0.1 % Triton X-100). After blocking, the
membrane would be washed twice with PBST, the membrane was then incubated
with 1:1000 dilution of first antibodies (rabbit anti-NDRG1, Invitrogen, Carlsbad, CA,
USA) at 4℃ for 2 hours. After incubated, the membrane was washed again and
incubated with 1:5000 dilution of second antibodies (anti-rabbit IgG, Santa Cruz,
Delaware Avenue, CA, USA) which conjugated with horseradish peroxidase, at 4℃
for 1 hr.
Membrane was treated with ECL developing solution (Amersham
Pharmacia Biotech, Piscataway, NJ, USA) and exposed to X-ray film. Actin
expression was used as an internal control.
Cellular transfection
Cells were seeded in 2×105 on 10-cm dish and incubated for 24 hours prior to
transfection. Cells were transfected with the mixture of 6 μg plasmid DNA and 6 μl
LipofectaminTM 2000 reagent (Invitrogen, Carlsbad, CA, USA) in 4 ml OPTI-MEM
medium (Gibco BRL, Bethesda,ML, USA) and incubate in 37oC, 5% CO2. After 6 hrs
the culture medium will be aspirated and wash the cells with phosphate buffer saline
(Gibco BRL, Bethesda,ML, USA), following by changing to complete culture
medium, cells were continuously incubated for 3 days. Cell numbers and viability
were determined.
Table 1. Cloned cDNA fragments and matched genes in the GenBank database
Figure 1. Relative expression of the seven genes in oral cancer patients. (A)
Relative expression levels of the seven genes. NPM, CDK1, NDRG1, HMGCR,
EF1A and NAC were over-expressed in tumor samples as compared to normal
counterparts, while CHES1 was under-expressed in tumors. (B) The percentage of the
differentially expressed genes in tumor samples as compared to normal counterparts.
Figure 2. The protein sequence of NDRG1. The open reading frame of NDRG1
encoded a three-tandem repeats of “GTRSRSHTSE” in its C-terminal region. The.
three-tandem repeats in NDRG1 can be phosphorylated by SGK1at Thr346, Thr356 and
Thr366 and by GSK3 at Ser342, Ser352 and Ser362 respectively.
Figure 3. Similarity of 3R domain in NDRG1 and RS domain in SR protein
family. The amino acid sequence of 3R domain and RS domain of different SR
protein (splicing factor1, 4, 5, 5, 7 and 10).
Figure 4. Endogenous NDRG1 in head-neck cancer cell lines. Western blotting
analysis for NDRG1 in variety head-neck cancer cell lines including, Detroit, Fadu,
BM2, BM1, 076, SAS and OECM1. The result was quantification and normalized
with β-actin.
Figure 6. Depletion of NDRG1 by RNAi strategy. (A) Specific NDRG1-RNAi
oligonucleotides will be annealed to pGSH1-GFP vector sequence.
(pGSH1-GFP-si225: 5’-gau ccg agu uug aug ucc agg agc gaagcuug gcu ccu gga cau
caa acu cgg auc uu-3’; pGSH1-GFP-si580: 5’-gau ccc tcg auu ugc ucu aaa caa
gaagcuug uug uuu aga gca aau cga agg gau cuu-3’, pGSH1-GFP-si980: 5’-gau ccg
aga cca cuc ucc uca aga gaagcuug ucu uga gga gag ugg ucu cgg auc uu-3’) (B)
Knockdown efficiency of NDRG1-siRNA. V: pGSH1-GFP, si225: pGSH1-si225,
pGSH1-si580, pGSH1-si980
Figure 8. NDRG1 promote HNC cell motility ability. The wound healing assay
shows the cell migration ability. (A) NDRG1 expression in OECM1 cell promotes the
cell motility. (B) Depletion NDRG1 in BM2 cell decrease the cell motility.
Figure 9. NDRG1 promote HNC cell invasion ability. (A) Expression NDRG1 in
OECM1 cell, dramatically increase the HNC cell invasion ability. (B) Depletion
NDRG1 in BM2 cell, decrease the cell invasion ability (p<0.05). (C) Expression
NDRG1 permanently in OECM1 cells, increase HNC cell invasion ability
significantly (p<0.01).