Japanese Dental Science Review (2010) 46, 173187

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

journal homepage: www.elsevier.com/locate/jdsr

Review article

Apoptosis-inducing activity and tumor-specificity

of antitumor agents against oral squamous cell
carcinoma
Hiroshi Sakagami *
Division of Pharmacology, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry,
Sakado, Saitama 350-0283, Japan
Received 4 August 2009; received in revised form 2 November 2009; accepted 13 January 2010

KEYWORDS
Oral squamous cell
carcinoma;
Apoptosis;
Autophagy;
Structure

Summary This review searched previous works of apoptosis induction in human oral squamous
cell carcinoma (OSCC) cells. Many chemotherapeutic drugs, natural products, metabolic inhibitors,
hormone receptor ligands and gene manipulations induced the apoptosis directly or indirectly by
reverting the anti-apoptotic pathways. However, these antitumor agents, sometimes induced
incomplete apoptosis or other types of cell death characterized by lack of caspase activation
and internucleosomal DNA fragmentation, depending on the target cells or the chemical structure
of inducers. There were very few investigations that have compared the cytotoxicity of these
antitumor substances against OSCC cells with that of normal oral cells. Furthermore, their
apoptosis-inducing activity has not been correlated well with tumor-specificity (higher cytotoxicity
against tumor cells versus normal cells). These accumulated evidences provide the cautionary note
against the apoptosis-oriented research.
# 2010 Japanese Association for Dental Science. Published by Elsevier Ireland.
Open access under CC BY-NC-ND license.

Contents
1.
2.

3.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Classification of three major types of cell death
2.1. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Autophagy . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Necrosis . . . . . . . . . . . . . . . . . . . . . . . . . .
Apoptosis inducers . . . . . . . . . . . . . . . . . . . . . . .
3.1. Apoptosis inducers against OSCC (Table 1)
3.1.1. Chemotherapeutic drugs. . . . . . . .
3.1.2. Natural products . . . . . . . . . . . . .

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* Tel.: +81 49 279 2758; fax: +81 49 285 5171.

E-mail address: sakagami@dent.meikai.ac.jp.
1882-7616 # 2010 Japanese Association for Dental Science. Published by Elsevier Ireland. Open access under CC BY-NC-ND license.
doi:10.1016/j.jdsr.2010.01.004

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174

4.
5.

6.
7.

8.

H. Sakagami
3.1.3. Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.4. Hormone receptor ligands . . . . . . . . . . . . . . . . .
3.1.5. Gene manipulations . . . . . . . . . . . . . . . . . . . . . .
3.1.6. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Head and neck squamous cell carcinoma (HNSCC) (Table
3.3. Salivary gland carcinoma . . . . . . . . . . . . . . . . . . . . . . . .
Anti-apoptotic mechanism in OSCC (Table 3) . . . . . . . . . . . . . .
Potentiators of apoptosis (Table 4). . . . . . . . . . . . . . . . . . . . . .
5.1. Chemotherapeutic drugs. . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Induction of non-apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tumor-specificity (Table 5). . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1. Flavonoid-related polyphenols . . . . . . . . . . . . . . . . . . . .
7.2. Coumarins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3. Tannins-related compounds . . . . . . . . . . . . . . . . . . . . . .
7.4. Terpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5. Ketones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.6. Synthetic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.7. Antitumor antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.8. Plant extracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Squamous cell carcinoma (SCC) is a form of cancer of the
carcinoma type that may occur in many different organs,
including the skin, lips, mouth, esophagus, urinary bladder,
prostate, lungs, vagina, and cervix. It is a malignant tumor of
squamous epithelium, and invasive cancers are able to
spread to other organs and cause metastasis. Most cases of
head and neck cancer are due to SCC, and caused by tobacco
and alcohol, and human papilloma virus. Oral cancer is one of
the most disfiguring types of cancer, since the surgical
removal of the tumor may result in facial distortion.
Oral squamous cell carcinoma (OSCC) is the fifth most
common cancer worldwide, with the number of cases consistently increasing in developing countries. Despite focused
efforts to improve therapy, 5-year survival rates for persons
with advanced-stage OSCC remain discouragingly low. OSCC,
like other types of cancer, is a genetic disease, resulting in
the loss of differentiation, and possibly generated by the
decline of apoptotic potential and immunity [1]. Aggressive
OSCC with a high score of malignancy showed reduced
expression of the tumor suppressor gene phosphatase and
tensin (PTEN) homologue deleted on chromosome 10 [2], p53
positivity and low apoptotic index [3], and increased expression of anti-apoptotic proteins such as survivin [4] and Bcl-2
[5]. Down-regulation of heat shock protein 27 enhanced the
transformation of oral epithelial dysplasia into OSCC, possibly by impairing the protective mechanism against mutagenesis induced by environmental factors [6]. Carcinogenesis of
OSCC is related to the overexpression of prolyl isomerase Pin
1 [7], a stronghold for the therapy of Alzheimers disease.
These data suggest that OSCC may be produced by an imbalance of the regulation between cell survival and apoptosis.
Early detection combined with strategies for local intervention, such as chemoprevention prior to SCC development,

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could dramatically improve clinical outcomes. Microarray

analysis of small noncoding microRNAs (miRNAs) in the
patient can be a useful prognostic markers [8].
Recently, the targeted elimination of OSCC cells by inducing apoptosis has emerged as a valued strategy to combat
oral cancer. Studies utilizing a variety of chemical or biological interventions demonstrated promising results for induction of apoptosis in oral malignant cells [9]. There are at least
three types of cell death: apoptosis, autophagy and necrosis
[10,11]. The present review surveys recent studies of the
type of cell death (either apoptosis or non-apoptosis) induced
by various antitumor agents in OSCC and the relationship to
their tumor-specificity.

2. Classification of three major types of cell

death
2.1. Apoptosis
Apoptosis is characterized by morphological changes such as
cell shrinkage, pyknosis, densed cytoplasm, tightly packed
organelles, chromatin condensation, and loss of cell surface
microvilli [12]. Markers of apoptosis include DNA fragmentation (assessed by TUNEL method on fixed cell slide preparations, proportion of sub-G1 cells on FACS analysis, ladder
pattern on agarose gel electrophoresis), decline of mitochondrial membrane potential, cytochrome c release from mitochondria, activation of caspase-3 using specific synthetic
substrate or poly(ADP-ribose)polymerase (PARP).

2.2. Autophagy
Autophagic cell death is characterized by the sequestration
of cytoplasm and organelles in double or multimembrane
vesicles and delivery to the cells own lysosomes for subse-

Apoptosis-inducing activity and tumor-specificity of antitumor agents against oral squamous cell carcinoma
quent degradation [13]. The process of autophagy depends
on both continuous protein synthesis and the continuous
presence of ATP [14]. Markers of autophagy is formation
of phagophore and autophagosome detected by electron
microscopy, Atg8/LC3 western blotting and ubiquitin-like
protein conjugation systems (increase in the amount of
LC3-II, and Atg12Atg5 conjugation) or fluorescent microscopy (increase in punctuate LC3 (or Atg18), TOR and Atg1
kinase activity) [11].

2.3. Necrosis
Necrosis is an uncontrolled and passive process that usually
affects large fields of cells. Necrotic cell injury is mediated by
two main mechanisms, interference with the energy supply
of the cell and direct damage to cell membranes [10].
Necrosis is characterized by cell swelling, formation of cytoplasmic vacuoles, distended endoplasmic reticulum, formation of cytoplasmic blebs, condensed swollen or ruptured
mitochondria, disaggregation and detachment of ribosomes,
disrupted organelle membranes, swollen and ruptured lysosomes and eventually disruption of the cell membrane [15].

3. Apoptosis inducers
3.1. Apoptosis inducers against OSCC (Table 1)
3.1.1. Chemotherapeutic drugs
Chemotherapeutic drugs are the most frequently used apoptosis inducers of OSCC. 5-FU, peplomycin, cisplatin (CDDP) and
g-ray, NO generator (ONOO, SIN-1 and SNP) induced apoptosis
in OSCC cells (OSC-4) [16]. These treatments slightly increased
the intracellular concentration of NO and O2 levels, and
induced the nitration of cytochrome c and caspase-3. CDDP
induced the rapid decline of surviving gene expression [17] and
augmentation of apoptosome molecules (cytochrome c and
Apaf-1) and activation of caspase-9 and capase-3, but no
change in NF-kB nor the expression of anti-apoptotic proteins
(TRAF-1, TRAF-2, c-FLIP). This suggests that cisplatin exerts its
apoptotic action by the mitochondria-mediated activation of
caspases but not by the activation of caspases due to the
inhibition of NF-kB activity that follows the suppression of
anti-apoptotic proteins [18]. Low-dose of CDDP enhanced the
Fas/CD95) expression and increased susceptibility to apoptosis
in oral cancer cell line (UT-SCC-20A) [19].
Carboplatin (CBDCA) induced apoptosis in human welldifferentiated tongue squamous carcinoma cell lines (SCC-9
and SCC-25). The carboplatin-induced apoptosis was obliterated by neutralizing anti-Fas (APO-1/CD95) and anti-Fas
ligand (FasL). Anti-Fas antibody itself was inactive, but in the
presence of carboplatin, markedly enhanced the apoptosis.
There was large enhancement of FADD expression, but no
alterations in Fas or FasL expression upon carboplatin treatment. Suppression of FADD expression using the specific
antisense oligonucleotide resulted in a failure of carboplatin
induction of cell death. These data suggest that carboplatin
may switch nonfunctional Fas to functional Fas by up-regulation of FADD expression, resulting in activation of Fas-sensitive pathway leading to apoptosis [20]. Carboplatin induced
apoptosis in OSCC (MIT7) probably through the cleavage of
Bax-alpha and Bcl-x(L) [21].

175

Paclitaxel, an antimitotic chemotherapeutic agent,

induced apoptosis in human OSCC cell line (NB-1) by downregulation of Bcl-2 and up-regulation of Bax [22]. Docetaxel
induced Bax expression at both protein and mRNA levels in
not all OSCC cell lines and clinical cases, but the enhanced
expression of Bax has been suggested to be contributed to the
antitumor effects of chemotherapy [23].
Peplomycin and bleomycin induced apoptosis in OSCC
(SSKCN) [24]. Pingyangmycin (bleomycin A) induced growth
arrest at G2-M phase of the cell cycle and apoptosis in OSCC
(KB cells) [25].
137
Cs irradiation, 5-fluorouracil (5-FU) or CDDP enhanced
the apoptosis of OSCC induced by interleukin-2-activated
lymphocytes [26]. Low-dose (0.5-Gy) of radiation caused
enhanced apoptosis in human OSCC cells (T-167, T-409) without induction of transmembrane protein P-glycoprotein
(MDR1), while higher dose (2-Gy) of radiation increased
expression of MDR1 by NF-kB, NF-Y. Low-dose fractionated
radiation can be used as an adjuvant for chemotherapy [27].
Heat stress induced 100-fold increase in the production of
heat shock proteins, and prevented the NF-kB activation
(nuclear translocation), may ultimately promote apoptosis
in OSCC Ca9-22 cell line [28]. Hyperthermia induced apoptosis in OSCC cells (wild-type p53), accompanied by decrease
of the gene expression of IL-12. On the other hand, IL-12Rb1
increased in the mutated p53 cells. p53 status is a useful
candidate for a predictive indicator of the effectiveness in
hyperthermic therapy [29].
Molecular inhibition of epidermal growth factor receptor
(EGFR) signaling is a promising cancer treatment strategy.
Treatment of OSCC cells with an anti-EGFR monoclonal antibody, Cetuximab (C225, Erbitux) and an EGFR tyrosine kinase
inhibitor (TKI), AG1478, which target the extracellular and
intracellular domains of the receptor, respectively, inhibited
phosphorylation of EGFR and its downstream effector molecule Akt and amplified the induction of Fas-mediated apoptosis. The pro-apoptotic activity of EGFR inhibitors in OSCC
cells depends on the extrinsic pathway of the caspase cascade accompanied by the down-regulation of cellular FLICEinhibitory protein (c-FLIP) [30].
3.1.2. Natural products
Essential oil of a perennial small herbal plant induced apoptosis via MAPK activation, based on the inhibition of MAPK
kinase inhibitor [31]. Soft coral extract induced apoptosis in
OSCC (SCC4, SCC9, SCC25) [32]. Ethanol extract of freezedried black raspberries suppressed the proliferation of OSCC
cell lines without affecting viability, inhibited the production
of vascular endothelial growth factor (VEGF), and induced
both apoptosis and terminal differentiation. This extract may
be a promising candidate for use as a chemopreventive agent
in persons with oral epithelial dysplasia [33].
Theaflavin-3,30 -digallate, a polyphenol in black tea,
induced apoptosis in HSC-2 cell by its prooxidant action (elevation of reactive oxygen species (ROS) production) whereas
normal human gingival fibroblast (GN) was more resistant [34].
Green tea polyphenol induced apoptosis in OSCC cell line
accompanied by the gradual decline of mitochondrial function. The cells deleted of caspase-3 gene did not undergo
apoptosis. This suggests that green tea polyphenol-induced
apoptosis is a mitochondria-targeted, caspase-3-executed
mechanism [35]. Green tea polyphenols (catechins) induced

176
apoptosis in OSCC cells, and p58/KIP2 is a determinant prosurvival factor for cell protection from green tea polyphenolinduced apoptosis [36]. Curcumin alone or in combination with
tea polyphenols inhibited the oral carcinogenesis in hamsters,
possibly by suppressing the cell proliferation, induction of
apoptosis and angiogenesis [37].
Stilbenes (resveratrol, piceatannol, rhaponticin), stilbene
trimers (sophorastilbene A, ()-e-viniferin), a stilbene dimer
((+)-a-viniferin) and flavonoids (kaempferol, fisetin, quercetin, isoliquiritigenin, butein) induced activation of caspase-3, 8 and -9, and internucleosomal DNA fragmentation in human
promyelocytic leukemia HL-60 and OSCC (HSC-2) cells [38].
Ovatodiolide, a diterpenoid from a Chinese herb, induced
G2/M arrest and apoptosis in OSCC cell line Ca9-22 by Nacetyl-L-cysteine (NAC)-inhibitable ROS generation [39]. Shikonin, a naphthoquinone pigment isolated from the Chinese
herbal therapeutic, Zicao, has been shown to exhibit antioxidant and anticancer effects. Shikonin induced apoptosis in
human OSCC (Tca8113) cells via inactivation of NF-kB pathway [40]. Paradol, a pungent phenolic compound found in
ginger and other Zingiberaceae plants, induced apoptosis in
OSCC (KB) cells through a caspase-3-dependent mechanism
[41].
3.1.3. Inhibitors
Cyclooxygenase-2 (COX-2), an enzyme that catalyzes the
synthesis of prostaglandins (PGs), is made inducible by various stimuli such as inflammation. COX-2 is commonly overexpressed in a variety of premalignant and malignant
conditions including oral leukoplakia and SCC. Overexpression of COX-2 plays an important role in the development and
progression of different cancers. Immunohistochemical analysis demonstrated that expression of COX-2 and E2 promoterbinding factor-1 (E2F-1) proteins was increased in both oral
dysplasia and SCC compared to the normal epithelium. A
selective COX-2 inhibitor, NS398, suppressed PGE2 level, and
expressions of COX-2, pRb and E2F-1 proteins, and induced
G1 arrest and apoptosis [42]. Apoptosis-inducing activity of
four selective COX-2 inhibitors (celecoxib, NS-398, nimesulide, meloxicam) was compared using OSCC (KB) cells. Celecoxib and NS-398 strongly suppressed the proliferation of KB
cells at 10100 mM, whereas nimesulide and meloxicam are
less potent. All COX-2 inhibitors increased COX-2 protein
expression but suppressed PGE2 production. Only celecoxib
induced apoptosis. Celecoxib is a good therapeutic candidate
for treating OSCC through the suppression of cell proliferation and the induction of apoptosis in a COX-2 independent
manner [43]. Sulindac, a non-steroidal anti-inflammatory
drug (NSAID), induced growth inhibition and apoptosis in
OSCC (SCCa), and caused up-regulation of the protein and
mRNA expression levels of COX-2 and PPARg. Treatment of
antisense PPARg oligonuleotides abolished sulindacs growth
inhibitory effect, suggesting that up-regulation of PPARg
expression and activation may be, at least partially, responsible for sulindacs antiproliferative effect [44].
Protein kinase C (PKC) inhibitor safingol induced apoptosis
in OSCC cells, accompanied by release of endonuclease G
from mitochondria and translocated to nucleus [45]. Safingol
induced the apoptosis in OSCC by mechanism involved with
increase of Bmi and decrease of Bcl-xL and phosphorylated
focal adhesion kinase (FAK) [46]. UCN-01 (7-hydroxystaurosporine), a PKC inhibitor, also induced apoptosis and G1 arrest

H. Sakagami
in OSCC cell lines. HSC-3 (primary-type OSCC) were less
sensitive than LMF4 cells (metastatic-type OSCC)[47].
Protein phosphatase inhibitors, okadaic acid (OA) and
calyculin A (CA), induced apoptosis in three OSCC cell lines
(SCC-25, SCCKN, SCCTF) [48]. Okadaic acid induced apoptosis
in OSCC (SCC-25) cells by enhancing the Fas and FasL expression [49].
Flavopiridol, a synthetic flavone, inhibited the growth (by
reducing the expressions of cyclin A, cyclin B and cyclin D1,
and cyclin-dependent kinases (CDK) activation kinase and
CDC25C) and induced apoptosis (by activating the Bcl-x pathway) in OSCC cells [50].
Proteasome inhibitors, carbobenzoxy-L-leucyl-L-leucyl-Lnorvalinal, or lactacystin, induced apoptosis in OSCC cells,
but this was inhibited by antisense p27Kip1 oligonucleotide.
The accumulation of p27Kip1 may play an important role in
the apoptosis induced by proteasome inhibitor [51].
3.1.4. Hormone receptor ligands
15-Deoxy-D12, D14-prostaglandin (PG) J2, a proliferator-activated receptor g (PPARg) ligand, induced growth inhibition
and apoptosis in OSCC cells, whereas rosiglitazone and ciglitazone, thiazolidinedione family of PPARg activators, did not
exert a growth inhibitory effect. 15-PGJ2, but not other
PPARg activators, induced significant reduction of both phosphorylated and unphosphorylated Stat3 (signal transducer
and activator of transcription). This suggests that 15-PG
induces apoptosis in OSCC by PPARg-independent pathways
[52]. Cyclopentenone 15-Deoxy-D12, D14 PGJ2 induced apoptosis in OSCC (B88) cell lines by down-regulation of the
constitutive and IL-6-mediated JAK (Janus kinase) phosphorylation as well as Stat3 phosphorylation. The apoptosis-inducing activity of this compound required the a,b-unsaturated
ketone structure within the cyclopentenone ring [52].
All-trans retinoic acid (ATRA) and 1a,25(OH)2vitamin D3
(calcitriol) suppressed the cell proliferation and induced
apoptosis in OSCC (KB) cells, upregulated sensitivity of the
chemotherapeutics drugs and down-regulated several angiogenesis factors and survivin [53]. Retinoic acid induced
apoptosis in OSCC (Tca83) cells by enhancement of Fas gene
transcription and translation, without change in FasL expression [54].
3.1.5. Gene manipulations
RNAi targeting urokinase-type plasminogen activator receptor (u-PAR) induced apoptosis as well as oral cancer invasion
and metastasis [55]. Stable transfection of intracellular fragment of Notch induced G0G1 cell cycle arrest and apoptosis
in human tongue cancer cell line (Tca8113), accompanied by
down-regulation of Wnt-b-catenin, increase of p21 and p53
expression, and decrease in Skp2 (S-phase kinase-associated
protein 2) and Bcl-2 (B-cell lymphocytic-leukaemia protooncogene 2) expression [56].
3.1.6. Others
Nitric oxide (NO) donor, nitroprusside (SNP), induced keratinocyte differentiation markers at lower concentrations,
while it induced apoptosis at higher concentrations in human
immortalized keratinocytes (IHOK) and primary oral cancer
cells (HK4) [57]. NO generator (ONOO, SIN-1 and SNP)
induced apoptosis in OSCC (OSC-4) cells [16].

Apoptosis-inducing activity and tumor-specificity of antitumor agents against oral squamous cell carcinoma
Selenium (Se) compounds induced apoptosis in OSCC (HSC3) cells, accompanied by the loss of mitochondrial membrane
potential and glutathione (GSH), without a concurrent
increase in ROS. The apoptosis-inducing activity of Se was
diminished by GSH or compounds that elevated the intracellular GSH level, but augmented by buthionine sulfoximine
that reduced GSH level. These data suggest possible role of
GSH in the mitochondrial apoptosis of OSCC caused by Se
[58].
Arsenic trioxide induced toxic damage and apoptosis characterized by caspase-3 activation, mitochondrial transmembrane potential collapse and G2/M arrest, but no change in
p16, p53 and Bcl-2, in OSCC cell line. Tubulins and mitochondria may be the chief action position of arsenic trioxide [59].
Taurolidine, a derivative of the amino acid taurine,
induced apoptosis in two OSCC cell lines (SCC4, SCC15), in
contrast to the reference group treated with povidone iodine
or the untreated control group [60].

177

reduced. Intraperitoneal administration of vitamin E succinate slowed the growth of JHU-022 solid tumor xenograft in
immunodeficient mice [65].
An active component of OK-432, a streptococcal preparation, induced apoptosis in human HNSCC cell line by the
activation of caspases through p53-independent pathway
via TLR4 signaling [66].
The expression of E1A induced down-regulation of the
expression of EGFR that was overexpressed in four HNSCC cell
lines (Table 2). Overexpression of an exogenously introduced
EGFR blocked E1A induction of apoptosis in these cells. These
data suggest that E1A induces apoptosis by novel pathway
involving EGFR [67].
Oral administration of vesnarinone, a cardiotonic, to a
patient with histopathological recurrent oral cancer with
well-differentiated squamous cell carcinoma, resulted in
complete remission of the tumour [68].

3.3. Salivary gland carcinoma

3.2. Head and neck squamous cell carcinoma
(HNSCC) (Table 2)
Cetuximab is a chimeric mouse/human monoclonal antibody
used to treat metastatic colorectal cancer and head and neck
cancer. The antibody binds to the EGFR, a signalling protein
that normally controls cell division. In some cancers, this
receptor is altered to cause uncontrolled cell division. Cetuximab blocks EGFR and stops the uncontrolled cell division.
Cetuximab induced apoptosis in HNSSC by inhibiting the MAPK
and Janus kinase (JAK)/STAT-3 pathways [61].
Combination treatment with 13-retinoic acid, interferona2a and a-tocopherol apparently further inhibited the
growth of five squamous cell carcinoma of the head and neck
(SCCHN) in comparison to any single agent and two-drug
combinations. Three-drug combination induced significant
accumulation of the cells at S-phase and induced apoptosis.
These data supported the promising outcomes of the phase III
trial with a combination of these three drugs in patients with
advanced head and neck cancer in the adjuvant settings [62].
Galanin is a neuropeptide present in humans and other
mammals. It is a peptide consisting of a chain of 29 amino
acids (or 30 amino acids in humans).Galanin is formed by the
cleavage of a prepropeptide encoded by a gene known as
GAL. Galanin induced apoptosis in galanin receptor-transfected HNSCC cell line with mutant p53 [63].
Alterations in histone acetylation status have been implicated in carcinogenesis. Histone deacetylase inhibitors, such
as suberoylanilide hydroxamic acid, can potentially reactivate aberrantly silenced genes by restoring histone acetylation and allowing gene transcription. Suberoylanilide
hydroxamic acid induced both mitochondrial pathway of
apoptosis (cytochrome c release, caspase-3, caspase-9 activation) and extrinsic apoptosis pathway (increased Fas and
Fas ligand (FasL) expression, activation of caspase-8, cleavage of Bid), having comparatively little activity against
precancerous and normal oral cells [64].
Vitamin E succinate (a-TOS) induced apoptosis in five
HNSCC cell lines (JHU-011, JHU-013, JHU-019, JHU-022,
JHU-029) accompanied by elevation of ceramide, sphingomyelinase activity and p53. On the other hand, the amounts
of nuclear factor kB, Bcl-2, and Bcl-X(L) proteins were

Slindac, a non-steroidal anti-inflammatory drug (NSAID),

induced a significant decrease in cell proliferation and an
increase in apoptosis in two salivary gland adenocarcinoma
cell lines (HSY, HSG) [69].

4. Anti-apoptotic mechanism in OSCC

(Table 3)
OSCC (Ho-1-N-1 SP) cells survived even under treatment with
various agents (5-FU, carboplatin), possibly due to high level
expression of ABC transporters (ABCB1, ABCG2) and antiapoptotic protein (CFLAR, BCL2, BCL2A1) [70].
Hyperthermia-resistant human squamous cell carcinoma
(SAS) cell: higher expression of Bcl-2, Bcl-xL, NF-kB, COX-2,
STAT3, IL-6, IKKa/1) [71]. The imbalance between expression
of anti-apoptotic and pro-apoptotic Bcl-2 family genes may
promote survival in the oral cell lines [72]. The frequency of
bcl-2 expression was associated with tumor histologic grade
and decrease in apoptotic index in tongue SCC [73].
()-Epigallocatechin-3-gallate (EGCG), a major component of green tea, induced p57, a cyclin-dependent kinase
and apoptosis inhibitor, in a dose- and time-dependent manner in normal keratinocytes, but not in OSCC (SCC25, OSC2)
cells. The chemopreventive effect of green tea polyphenols
may involve p57-mediated cell cycle regulation in normal
epithelial cell [74].
Paired box gene 9 (Pax9) and c-myb are transcription factors
that regulate the expression of the genes involved in mediating
cell proliferation, resistance to apoptosis, and migration. Pax9
and c-myb expressions in KB cells seem to be essential for cell
growth, and survival was enhanced by c-myb. Disruption of the
function of c-myb and Pax9 induced G0 arrest [75].
The mouse double minute 2 (MDM2) plays a pivotal role in
radiotherapy by down regulating p53. A functional T-to-G
polymorphism at nucleotide 309 in MDM promoter intron 1
(SNP309) influences transcription activity. A G-to-C SNP at p53
codon 72 results in an Arg/Pro polymorphism, which is associated with apoptosis induction potential and p53 mutation
status. The MDM2 SNP309 G/G polymorphism was associated
with poor overall survival in advanced OSCC and the overall
survival and disease-free survival of irradiated patients. The

178
Table 1

H. Sakagami
Substances that induce apoptosis in OSCC cells.

Inducers
Antitumor agents
5-FU
Cisplatin (CDDP)
Carboplatin (CBDCA)
Paclitaxel
Docetaxel
Peplomycin, bleomycin
Radiation, g-ray, Cs
Heat shock, hyperthermia
Anti-EGFR mAb, EGFR TKI
Natural products
Essential oil
Soft coral extracts
Black raspberry extract
Theaflavin-3,30 -digallate
Green tea polyphenols
Curcumin
Stilbenes, flavonoids
Diterpenoid
Shikonin, a naphthoquinone pigment
Paradol
Inhibitors
Selective COX-2 inhibitor, NS398, celecoxib
PKC inhibitor safingol, 7-hydroxystaurosporine
Phosphatase inhibitors: Okadaic acid calyculin A
CDK inhibitor, flavopiridol
Proteasome inhibitors
Ligands to hormone receptors
Cyclopentenone 15-deoxy-D12, D14- PGJ2
All-trans retinoic acid
1a,25(OH)2vitamin D3

Possible mechanism

Survivin #, FADD ", no change in NF-kB CDKIs "

Cleavage of Bax-alpha and Bcl-x(L)
BAX "
G2-M block
IL-12 #
c-FLIP #

p57/KIP2 #

NF-kB #

Fas receptor ", FasL "

CDKs #
p21Kip1 accumulation
JAK signalling #, Stat3 #
Survivin #, VEGF #

Gene manipulation
siRNA targeting u-PAR
Notch overexpression
Others
NO generator, ONOO, SIN-1 and SNP
Selenium compounds
Arsenic trioxide
Taurolidine

Reference
[16,26,97]
[16,17,18,26,80]
[20,21]
[22]
[23]
[16,24,25]
[16,26,27]
[28,29]
[30]
[31]
[32]
[33]
[34]
[35,36,74]
[37]
[38]
[39]
[40]
[41]
[42,43,44]
[45,46,47]
[48,49]
[50]
[51]
[52]
[53]
[53]
[55]
[56]
[16,57]
[58]
[59]
[60]

GSH #

combination of MDM2 SNP309 G/G and p53 codon 72 Arg/Arg

polymorphism is associated with the worst overall survival and
disease-free survival. Both MDM2 SNP309 and p53 codon 72 SNP
could be useful factors for evaluating the outcome of advanced
OSCC treated with adjuvant radiation [76].
p58/KIP2 is a determinant pro-survival factor for cell protection from green tea polyphenol-induced apoptosis [36].
The nu/nu mice inoculated with Mn-SOD antisense-transfected SCC cells showed prolonged survival time, as compared with mice inoculated with control vector-transfected
SCC cells after treatment with antitumor agents such as 5-FU,
peplomycin, CDDP or g-ray. SCC cells transfected with MnSOD antisense group showed higher incidence of apoptosis
than that transfected with empty vector. Mn-SOD may act as
a negative regulator of apoptosis [77]. OSCC cell lines with
lower Mn-SOD activity were easily committed to apoptosis

upon treatment with 5-FU, peplomycin or g-rays, than those

with higher Mn-SOD. This suggests that Mn-SOD negatively
regulates the apoptosis by these inducers [78].

5. Potentiators of apoptosis (Table 4)

Apoptosis induction may be potentiated by reversing the
anti-apoptotic mechanism.

5.1. Chemotherapeutic drugs

CDDP administered for 24 h before 5-FU treatment for 48 h
induced apoptosis in human oral cancer cells (B88) more
efficiently than simultaneous administration of CDDP and 5FU or the sequential treatment of 5-FU followed by CDDP [79].

Apoptosis-inducing activity and tumor-specificity of antitumor agents against oral squamous cell carcinoma
Table 2

Apoptosis induction in HNSCC cells.

Inducers

Possible mechanism

Cetuximb
Retinoic acid + IFN-a + vitamin E
Gelanin
HDA inhibitor, suberoylanilide hydroxamic acid
Vitamin E succinate
OK-432, a streptococcal preparation
Expression of E1A
Vesnarinone, cardiotonic (clinical trial)

Table 3

Reference

Ceramide ", p53 ", NF-kB #, Bcl-2 #, Bcl-xl #

p53-independent pathway via TLR4 signaling
EGFR #

Anti-apoptotic mechanism in OSCC cells.

Anti-apoptotic mechanism

Reference

ABC transporters: ABCB1, ABCG2

Bim ", Bcl-xL #, phosphorylated FAK #
Bcl-2, Bcl-xL, NF-kB, COX-2, STAT3,
IL-6, IKKa/1
Pax9, c-myb
SNP 309 and p53 codon 72 polymorphisms
Cyclin-dependent kinase inhibitor p57/KIP2
Mn-SOD

[70]
[46]
[71,73]
[75]
[76]
[36,74]
[77,78]

Tamoxifen alone induced a transient G1 arrest by upregulation of cyclin-dependent kinase inhibitors (CDKIs) such
as p21/Waf-1, p27/Kip1 and p15/INK4a in HNSCC cell lines
(HN5, HN6), and sensitized the cells to apoptosis induced by
CDDP. Tamoxifen stimulated the secretion of TGF-b1, and
anti-TGF-b1 blocking antibody prevented both the blockade
of cellular proliferation and increased expression of CDKIs.
This suggests the role of TGF-b1 for the tamoxifen stimulation of cisplatin-induced apoptosis [80].
Hyperthermia is used as one of the treatment modalities
for various types of cancers, but the acquisition of thermotolerance in cancer, through the induction of heat shock
proteins (Hsps), renders hyperthermia less effective. PreTable 4

179

[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]

treatment of HSC-2 cells with IFN-g suppressed Hsp27 transcription and promoter activity, and enhanced the induction
of cell death by hyperthermia and cisplatin treatment [81].

5.2. Inhibitors
Etodolac, a selective COX-2 inhibitor, enhanced the carboplatin (CBDCA)-induced apoptosis of OSCC (SCC-25) cell line
through the suppression of FAP-1 (anti-apoptotic tyrosine
phosphatase) expression, although etodolac alone did not
induce the apoptosis [82].
Histone deacetylase inhibitor, suberoylanilide hydroxamic
acid, enhanced CDDP-induced apoptosis in the OSCC cell line
(HSC-3), by rapidly up-regulating the endoplasmic reticulum
(ER) stress-associated events such as the sustained phosphorylation of eukaryotic transition initiation factor-2 (eIF2), activation of protein phosphatase 1 and Akt dephosphorylation
[83]. Co-administration of low-dose cisplatin (4 mg/ml) and
suberoylanilide hydroxamic acid (2 mM) synergistically induced
cytotoxicity and apoptosis in OSCC (Tca8113, KB) cell lines [84].

5.3. Others
Resveratrol (trans-resveratrol) is a phytoalexin produced
naturally by several plants when under attack by pathogens

Potentiators of apoptosis induction.

Potentiator
Antitumor agents
Cisplatin
Cs, 5-FU, CDDP
Tamoxifen
IFN-g
Inhibitors
Selective COX-2 inhibitor, etodolac
HDA inhibitor, suberoylanilide
hydroxamic acid
Others
Resveratrol
Glycerol
Infection of wild-type p53-encoding
adenovirus

Inducer

Possible mechanism

Reference

Agonistic Fas antibody

(CH11), 5-FU
LAK
Cisplatin
Hyperthermia, cisplatin, ATLA

Soluble Fas", Bcl-2#

[19,79]

CDKIs", TGF-b1 secretion "

Hsp27 transcription and
promoter activity #

[26]
[80]
[81,88]

Carboplatin (CBDCA)
CDDP

FAP-1 #
Protein phosphatase ", Akt
dephosphorylation

[82]
[83,84]

Vincristin, adriamycin,
paclitaxel
X-irradiation
X-irradiation

Bcl-2 #, MDR1 #

[85]
[86]
[87]

180
such as bacteria or fungi, and found in the skin of red grapes
and is a constituent of red wine. Resveratrol down-regulated
the expression of Bcl-2 and MDR1, and enhanced the sensitivity of human epidermoid carcinoma KBv200 cells [85].
Pretreatment with glycerol enhanced the effectiveness of
X-irradiation in Ca9-22 cells bearing a mutant p53. Glycerol
restored the DNA-binding activity of mutant p53 for a p53consensus sequence to levels similar to that of wild-type p53
[86].
Infection with wild-type p53-encoding adenovirus alone, or
X-irradiation alone, significantly inhibited the growth of OSCC
(HSC-4 and SAS) cells, but combined treatment was most
effective, even in mutant p53-accumulated HSC-4 cells [87].
ATLA and interferon-g synergistically stimulated the apoptosis in OSCC cell lines [88].

6. Induction of non-apoptosis
As compared with the studies of apoptosis induction in OSCC,
those of non-apoptosis induction are limited. Morin
(3,5,7,20 ,40 -pentahydroxyflavone) induced G2/M arrest,
without induction of apoptosis in human OSCC cells, via
inhibition of AKT activation [89]. Intratumoral laser illumination of OSCC transplated nude mice induced necrosis, but not
apoptosis, in OSCC cells [90].
We have recently reported that a,b-unsaturated ketones
such as 1-trichloroacetyl-3-bromo-2-methoxyazulene and 1trichloroacetyl-3-chloro-2-ethoxyazulene induced autophagic cell death characterized by the vacuolization detected by
transmission electron microscopy, and the granular distribution of acridine orange and translocation of LC3-GFP into the
autophagosome of OSCC (HSC-4) cells. Although HL-60 cells
are easily committed to apoptosis by many inducers, this cell
line did not express apoptosis markers, such as internucleosomal DNA fragmentation and caspase activation, upon treatment with 4,4-dimethyl-2-cyclopenten-1-one, a-methyleneg-butyrolactone, 5,6-dihydro-2H-pyran-2-one, 3,3,3-trifluoro-2-hydroxy-1-phenyl-1-propanone, codeinone (an oxidative product of codeine) or morphinone (an oxidative
metabolite of morphine) [91]. We found that 3,3,3-trifluoro-2-hydroxy-1-phenyl-1-propanone induced autophagic
cell death (characterized by acridine and LC3-GFP accumulation in the autophagosome) in human OSCC cell lines (HSC-2,
HSC-4) [92]. Furthermore, morphinone induced the formation of autophagosome engulfing organelles in HL-60 cells.
The addition of N-acetyl-L-cysteine (NAC) reduced the cytotoxic activity of codeinone by 30-folds, whereas other antioxidants (cysteine, ascorbate, catalase) and metals (FeCl3,
CoCl2, CuCl2) were almost inactive. Similarly, the cytotoxic
activity of 4,4-dimethyl-2-cyclopenten-1-one, a-methyleneg-butyrolactone, 5,6-dihydro-2H-pyran-2-one was almost
completely eliminated by NAC. This suggests that the cytotoxicity of a,b-unsaturated ketones is generated by the
interaction between the b-position of the a,b-unsaturated
carbonyl moiety and SH group of any targeted molecules (the
so-called non-sterically hindered Michael acceptor). We
also found that morphinone induced a significant reduction of
mitochondrial size. It is not clear at present whether this
resulted from the perturbation of mitochondrial morphogenesis or the fragmentation of mitochondria as recently
reported in Bax/Bak double knockout mouse embryonic

H. Sakagami
fibroblast [93]. It has been recently reported that autophagy
selectively degrades mitochondria, blocking the vicious cycle
between defective mitochondria and ROS. These data suggest the mitochondrial target of autophagy.

7. Tumor-specificity (Table 5)
There are very few reports available that have dealt with the
tumor-specificity of natural and synthetic compounds. Theaflavin-3,30 -digallate showed a concentration and time-dependent inhibition, with the OSCC cells more sensitive than the
normal GN46 fibroblasts [34]. Morin showed higher cytotoxicity
against OSCC than normal oral mucosa cells (NOMC) (TS = 1.6)
whereas quercetin, kaempferol, datiscetin and galangin
showed little or no tumor-specificity (TS = 1.03, 1.06, 1.00
and 1.08, respectively) [89]. Chemopreventive effects of green
tea polyphenols may involve p57-mediated cell cycle regulation in normal epithelial cells [74]. Histone deacetylase inhibits
suberoylanilide hydroxamic acid induced growth inhibition and
apoptosis induction in HNSCC cell lines, but had limited effects
on premalignant and normal cells [64]. Based on these backgrounds, we have surveyed a total of 1000 compounds for their
tumor-specificity. The tumor-specific cytotoxicity index (TS)
was determined by the ratio of the mean 50% cytotoxic concentration (CC50) against normal human oral cells (gingival
fibroblast (HGF), pulp cell (HPC), periodontal ligament fibroblast (HPLF)) to that against human OSCC cell lines (HSC-2,
HSC-3, HSC-4, NA, Ca9-22), submandibular gland carcinoma
(HSG) [38,94,95] (Table 5). Preparations of normal cells from
the extracted teeth and periodontal tissues have been
approved by the intramural ethic committee after obtaining
the informed consent from the donors. The compounds analyzed were mostly isolated and identified by our groups.

7.1. Flavonoid-related polyphenols

Flavonoids, generally, showed weak tumor-specificity
(TS = 1.24.0). Licochalcone B, a chalcone derivative without an isoprenoid group, showed the highest TS value of 31.7.
Isoprenoid-substituted chalcone, prenylated isoflavone and
genistein had higher cytotoxicity, but without high tumorspecificity, suggesting that the prenylation itself is not necessary for the tumor-specificity. Among the benzophenones,
compounds with two isoprenoid groups had higher cytotoxicity than the monoprenylated compound, but with minor TS
values. Anthraquinones exhibited relatively higher TS values.
Among them, emodin and aloe-emodin, without glycosylation, were the most potent (TS = 8.5 and >18.6, respectively). Stilbenes (resveratrol, piceatannol, rhaponticin),
stilbene trimers (sophorastilbene A, ()-e-viniferin), a stilbene dimer ((+)-a-viniferin) (TS = 1.43.6) and flavonoids
(kaempferol, fisetin, quercetin, isoliquiritigenin, butein)
(TS = 1.44.7) gave lower TS values.

7.2. Coumarins
Coumarin itself and its 7-hydroxy-, 6-methoxy-7-hydroxy and
5,6-dimethoxy-derivatives were relatively non-toxic to all
cell lines. Its 6,7-dihydroxy derivatives (esculetin) revealed a
tumor cell line-specific cytotoxicity (TS > 5.1). Higher
tumor-specificity of 4-methyl (TS > 8.3), 3,4-dimethyl

Apoptosis-inducing activity and tumor-specificity of antitumor agents against oral squamous cell carcinoma
Table 5

181

Tumor-specificity of various groups of compounds against OSCC cells.

Compounds

TS (range) (no. of compounds)

Flavonoids
Flavones, flavonols, isoprenylated flavonoids
Flavonoids
Isoprenylated flavonoids
2-Arylbenzofurans
Benzophenones
Xanthones
Anthraquinones
Phenylbutanone glucoside
Stilbene glucoside
Naphthalene glucosides
Stilbenes

1.2  0.6 (0.33.2) (n = 36)

3.3  4.0 (0.831.7) (n = 27)
2.1  0.4 (1.63.0) (n = 22)
1.2  0.2 (1.01.5) (n = 6)
1.7  0.4 (1.22.3) (n = 5)
1.3  0.4 (n = 9)
3.8  4.9 (1.018.6) (n = 13)
2.4 (1.53.3) (n = 2)
1.8  0.8 (1.02.6) (n = 3)
1.3 (1.11.4) (n = 2)
3.0  1.2 (1.44.7) (n = 6)

Coumarins

2.4  3.0 (1.011.0) (n = 23)

Tannin-related compounds
Procyanidins
Flavonoids
Monohydrolyzable tannins
Oligomeric hydrolysable tannins
Macrocyclic ellagitannins

4.8  2.3
1.1  0.1
1.5  0.5
1.4  0.2
4.4  2.7

(1.07.4)
(1.01.2)
(1.02.5)
(1.21.5)
(2.38.2)

Terpens
Triterpenes
Triterpene glycosides
Triterpenes, triterpene glycosides, chromones
Cycloartane glycosides
Furostaol glycosides

1.5  0.7
1.4  0.5
1.0  0.1
1.1  0.2
2.5  4.1

(0.72.8) (n = 8)
(1.02.4) (n = 21)
(0.81.3) (n = 20)
(0.91.4) (n = 7)
(0.417.0) (n = 17)

Ketones
a,b-Unsaturated ketones
Cyclice a,b-unsaturated ketones
a-Hydroxyketones
b-Diketones
Trifluoromethylketones
Azulenequinones

1.2  0.3 (0.61.9) (n = 26)

>229.0 (n = 1)
5.7  6.0 (1.017.6) (n = 8)
1.8  1.4 (0.36.3) (n = 22)
2.6  1.6 (n = 6)
2.6  2.3 (1.010.2) (n = 27)

Bacterial products
Anthracyclines
Mitomycin C
Bleomycin, peplomycin
Nocobactines

>167  89 (n = 4)
>29 (n = 1)
>3.8  0.2 (n = 2)
62.0 (43.980.0) (n = 2)

Others
Vitamin K2 derivatives
Prenylalcohols
Azulenes
Trihaloacetylazulenes
Tropolones
Berberines
3,5-Dibenzoyl-1,4-dihydropyridines
Styrylchromones
Isoxazole derivatives

1.9  0.2 (1.7-2.0) (n = 3)

1.3  0.3 (1.0-1.8) (n = 5)
1.7  1.0 (0.85.7) (n = 27)
6.5  10.7 (n = 26)
2.6  1.8 (1.09.9) (n = 27)
3.8 (3.64.0) (n = 2)
>43.0 (>33 to >53) (n = 2)
7.3  6.1 (1.117.4) (n = 6)
1.2  0.2 (0.91.6) (n = 24)

Antioxidants
Epigallocatechin gallate (EGCG)
Catechin
Quercetin
Isoliquiritigenin
Ascorbic acid (vitamin C)
Gallic acid
Chlorogenic acid

4.1 (n = 1)
1.0 (n = 1)
3.3 (n = 1)
4 (n = 1)
2.5 (n = 1)
1.1 (n = 1)
1.7 (n = 1)

(n = 6)
(n = 4)
(n = 7)
(n = 3)
(n = 4)

182

H. Sakagami

Table 5 (Continued )

Compounds
Curcumin
Morin
Quercetin
Kaemferol
Datiscetin
Galangin

TS (range) (no. of compounds)

1.7 (n = 1)
1.64 (n = 1)
1.06 (n = 1)
1.06 (n = 1)
1.00 (n = 1)
1.08 (n = 1)

Data derived from [38,94,95].

(TS > 9.3) and 3,4-cycloalkyl derivatives (TS > 11.0) than
6,7-dihydroxycoumarin may be due to their higher lipophilicity.

7.3. Tannins-related compounds

Procyanidin B-2 (MW 578) (TS = 5.8), procyanidin C-1 (MW
866) (TS = >6.7) and procyanidin oligomers (MW 3170)
(TS > 7.4) had higher cytotoxicity and tumor-specific cytotoxicity against OSCC cells than did catechin (MW 290)
(TS = 1.0), ()-epicatechin (MW 290) (TS = 4.0) and ()-epigallocatechin gallate (EGCG) (MW 458) (TS = 4.1).
Among hydrolyzable tannins (which contain glucose in the
core of the molecule), three oligomeric hydrolyzable tannins
(MW 18541873) had an approximately two-fold higher cytotoxicity than seven monomeric hydrolyzable tannins. Macrocyclic hydrolyzable tannins (oenothein B, woodfordin C,
camelliin B, woodfordin D) (MW 15682506) exhibited
one order higher cytotoxicity than monomeric hydrolyzable
tannins. The tumor-specific cytotoxicity of macrocylic hydrolyzable tannins (TS = 2.78.2) was two- or three-fold higher
than monomeric (TS = 1.02.5) or oligomeric hydrolyzable
(TS = 1.21.5) tannins. On the other hand, gallic acid (MW
170) (TS = 1.1), methylgallate (MW 184) (TS = 1.3), ellagic acid
(MW 302) (TS = 1.0) and chlorogenic acid (MW 354) (TS = 1.7)
had much lower tumor-specific cytotoxicity. These data suggest
that a more condensed structure, with (hydrolyzable tannins)
or without glucose in the molecule (condensed tannins such as
procyanidin oligomers), increases the tumor-specificity.

7.4. Terpenoids
Generally, terpenoids had low tumor-specificity (TS = 0.5
2.4), except for oleanolic acid (TS > 2.8) and 22a-methoxyfurostanol monodesmosides (TS = 7.4, >17,4).

7.5. Ketones
The cytotoxic activity of a,b-unsaturated ketones declined
with the introduction of methyl group at C-3 (b) or the
addition of N-acetyl-L-cysteine (NAC), suggesting that their
cytotoxicity is generated by the interaction between the C-3
and SH group of targeted molecules. Codeinone and morphinone (oxidative metabolites of codeine or morphine, respectively) containing a a,b-unsaturated ketone backbone
showed lower tumor-specificity (TS = 3.8 and 3.1, respectively). Cyclic a,b-unsaturated ketones, such as 3-arylidene1-(4-nitrophenylmethylene)-3,4-dihydro-1H-naphthalen-2ones exhibited unusually high tumor-specificity (TS > 229).

Among 8 hydroxyketones, deferiprone (TS > 17.6), mimosine (TS > 7.9), tropolone (TS > 4.1) and hinokitiol
(TS = 10.7) had the highest tumor-specific cytotoxicity. On
the other hand, maltol (TS = 1.0), kojic acid (TS = 1.4), 3methyl-1,2-cyclopentanedione (TS = 1.3), 1,2-cyclohexanedione (TS = 1.2) had lower tumor-specificity.
Among 23 b-diketones, 3-formylchromone was the most
tumor-specific (TS = 6.3), followed by (+)- and ()-3-(trifluoroacetyl)camphor (TS = 4.4), 4,4,4-trifluoro-1-phenyl1,3-butanedione (TS = 3.4) and ()-3-(trifluoroacetyl)camphor (TS = 3.3); others including curcumin were much less
active (TS = 0.92.2).
Among 27 azulenequinone derivatives, 3-phenoxy-1,5azulenequinone (TS > 8.5) and 7-isopropyl-3-(4-methylanilino)-2-methyl-1,5-azulenequinone (TS = 10.2) had highest
tumor-specificity and apoptosis-inducing activity (caspase
activation). Among 27 tropolone derivatives, 5-aminotropolone was the most tumor-specific (TS = 9.9) and had the
highest apoptosis-inducing activity.

7.6. Synthetic compounds

Among 27 azulene derivatives, 2-acetylaminoazulene
(TS > 3.6),
diethyl
2-chloroazulene-1,3-dicarboxylate
(TS > 5.7) and methyl 7-isopropyl-2-methoxyazulene-1-carboxylate (TS = 2.4) had the highest tumor-specificity. Chlorination of azulene resulted in the elevation of both
cytotoxicity and tumor-specificity, while fluorination of the
same compound was not so effective.
There was no apparent difference between the cytotoxic
activity of 2-methoxyazulenes and 2-ethoxyazulenes. Trichloroacetylazulenes generally gave higher cytotoxicity
and tumor-specificity as compared with the corresponding
trifluoroacetylazulenes. Substitution of chlorine, bromine or
iodine at the C-3 position further enhanced their cytotoxicity
to four tumor cell lines. Among 20 trihaloacetylazulene
derivatives, 1-trichloroacetyl-3-bromo-2-methoxyazulene
and 1-trichloroacetyl-3-chloro-2-ethoxyazulene had the
highest tumor-specificity (TS = >3.5 and >2.5, respectively).
Berberines exhibited some tumor-specificity (TS = 3.6
4.0). Two 3,5-dibenzoyl-1,4-dihydropyridines had higher
tumor-specificity, but only weakly induced apoptosis markers
(DNA fragmentation, caspase activation). All six styrylchromones had higher cytotoxic activity against tumor cell lines
than against normal cells. Styrylchromones, with one to
three methoxy groups, had higher tumor-specificity and
water solubility (TS = 517) and induced DNA fragmentation
and caspase-3, -8 and -9 activation. Twenty-four 3-acetyland 3-benzoylisoxazole derivatives exhibited much lower
tumor-specificity (TS = 0.91.6).

Apoptosis-inducing activity and tumor-specificity of antitumor agents against oral squamous cell carcinoma
Flavopiridol, a synthetic flavone, induced apoptosis (subG1 DNA content, DNA fragmentation, PARP cleavage) via
activation of Bcl-x in OSCC cell lines [50].

7.7. Antitumor antibiotics

Doxorubicin, an anthracycline antibiotic isolated from Streptomyces peucetius var. caesius has been used for the treatment of cancer of the bladder, breast (in combination with
other anticancer agents) and prostate, but is, however, suspected to be a human carcinogen. Doxorubicin, used as a
positive control in our screening system, exhibited the highest
tumor-specific cytotoxic activity (TS = 255.0). It activated
caspase-3, -8 and -9 in both OSCC (HSC-2) and promyelocytic
leukemia HL-60 cells, but only induced internucleosomal DNA
fragmentation in HL-60 cells. Western blot analysis showed
that doxorubicin did not significantly change the intracellular
concentration of Bcl-2, Bax or Bad in HL-60 cells. Real time
PCR analysis showed that HPC cells (normal) expressed the
highest amount of mdr1 mRNA, followed by HSC-2
(tumor) > HGF (normal) > HSC-3 (tumor) > HPLF (normal) > HSG (tumor) > HL-60 (tumor). These data suggest that
mdr1 expression in the tumor cells seems to be unrelated
to the tumor-specificity of doxorubicin. Other anthracyclines
such as mitoxantrone (TS > 259) and daunorubicin (TS > 164)
produced comparable tumor-specificity with doxorubicin.
Idarubicin exhibited slightly lower cytotoxicity and tumorspecificity (TS = 47). On the other hand, the tumor-specificity
of other groups of antitumor antibiotics such as mitomycin C
(TS > 29), bleomycin (TS > 3.7) and peplomycin (TS > 4.0)
was much lower. This confirms the antitumor potential of
anthracylines for the treatment of OSCC.
Nocobactins NA-a (NBNAa) and NA-b (NBNAb) are mycobactin-like siderophores, which may play a role in the uptake
of iron from the proteins of the host by chelation of ferric ion
(Fe3+). These compounds exhibited high tumor-specificity
index (TS = 80.0 and 43.9, respectively).

7.8. Plant extracts

Poly-herbal extracts of Himalaya (HD-12, DLH-3073) exhibited highly tumor-specific cytotoxicity to tumor cell lines
(TS = >1070 and >106, respectively) [96]. These extracts
produced radicals under alkaline condition and scavenged
O2. The tumor-specificity and antioxidant properties suggest their medicinal efficacy. The identification of the active
principle is essential.

8. Conclusion
The present review article demonstrated that OSCC cells can
be committed to either apoptosis or non-apoptosis, depending on the type or sensitivity of target cells. OSCC showed
different susceptibility of apoptosis, upon treatment with 5FU [97]. There was a considerable variation (approximately
1020-fold) in drug-sensitivity of five OSCC cell lines. The
sensitivity to mitomycin C is in the following order (from
sensitive to resistant): HSC-2 (CC50 = 3.5 mM) > HSC-3
(9.7 mM) > Ca9-22
(16.4 mM) > HSC-4
(18.0 mM) > NA
(37.8 mM). The sensitivity to bleomycin is in the following
order: HSC-2 (CC50 = 4.6 mM) > HSC-3 (6.3 mM) > HSC-4

183

(77.4 mM) > NA (91.6 mM) > Ca9-22 (111.6 mM). The sensitivity to peplomycin is in the following order: HSC-2
(CC50 = 9.9 mM) > HSC-3 (25.2 mM) > NA (143.2 mM) > HSC4 (175.8 mM) > Ca9-22 (216.9 mM) [94]. In general, OSCC
cells are relatively resistant to Fas-mediated apoptosis; this
may be due to a lower expression of FAS [5] or the cellular
FLICE-inhibitory protein (c-FLIP) [98]. A cyclooxygenase
(COX)-2 inhibitor (NS398) induced G0/G1 arrest, but no
apoptosis in OSCC cells [99]. On the other hand, human
glioblastoma cell lines (M059J, M059K, U373-MG and T98G)
has been reported to more exclusively commit to autophagy
(characterized by autophagosome formation, the accumulation of Agp8p/Aut7p and LC3 (Atg8 homolog) in autophagosome, and the inhibition of cell death by 3-methyladenine, an
autophagic inhibitor), upon exposure to radiation
(137Cs)[100], arsenic trioxide [101], ceramide [102] or temozolomide (a new alkylating agent) [103] or doxorubicin [104].
Anthracyclines, with the highest tumor-specificity, also
induced non-apoptotic cell death in acute myeloblastic leukemia [105], cardiomyocytes [106] and breast cancer cells
[107].
Another factor that determines the type of cell death is
the category of compounds that is used. a,b-Unsaturated
ketones (2-cyclohexen-1-one, 2-cyclopenten-1-one, 4,4dimethyl-2-cyclopenten-1-one, 2-cyclohepten-1-one, amethylene-g-butyrolactone,
6-dihydro-2H-pyran-2-one,
methyl 2-oxo-2H-pyran-3-carboxylate, codeinone, morphinone) and 3,5-dibenzoyl-1,4-dihydropyridines activated caspase only marginally in HL-60 cells. Morphinone, an oxidative
metabolite of morphine, similarly induced non-apoptotic cell
death, accompanied by a significant reduction of mitochondrial size.
There is evidence that modulation of one form of cell
death may lead to another. Various stimuli (such as etoposide, TNF, hyperthermia, UV irradiation, ascorbic acid,
hydrogen peroxide) induced apoptosis (internucleosomal
DNA fragmentation) in various human leukemia cell lines
at relatively lower concentrations, whereas it induced necrotic cell death (smear pattern of DNA fragmentation) at higher
concentrations [108]. Recently, cross-talk between apoptotic
and autophagic pathways has been suggested [109,110].
This review reveals that tumor-specificity and apoptosis
induction do not always correlate with each other. This means
that having an apoptosis-inducing activity does not guarantee
its antitumor activity. Similar is true for autophagy-inducing
compounds, since a,b-unsaturated ketones which exclusively
induce non-apoptosis, have shown broad range of tumor-specificity (TS = 1200). The tumor-specific cytotoxicity may be
affected by various factors such as the co-existence of both
hydrophilic and hydrophobic groups in the same molecule, the
presence of an isoprenyl group, a halogen and/or a polycyclic
structure, a highly condensed structure, and lipophilicity. The
expression of multi-drug resistant proteins and drug metabolizing enzymes may also play an important role. External
factors that may affect the tumor-specificity include the type
of serum, oxygen concentration, metallic ion presence/concentration and external pressure. The cytotoxic activity of
curcumin and nobobactins was significantly inhibited by the
addition of FeCl3 due to the chelate formation. Systematization of the relationship between various factors mentioned
above and tumor-specificity may contribute to the quest for
more active compounds.

184
Both the resistance of tumor cells to anticancer drugs and
the dose-related toxicity remain the most important problems in the chemotherapy of clinical OSCC. Researchers
have been seeking a combinative treatment regimen to
improve the effect of chemotherapy. 13-Retinoic acid, interferon-a2a and a-tocopherol synergistically induced apoptosis
in squamous cell carcinoma of the head and neck (SCCHN)
may be a basis of the promising outcomes of the phase III trial
with a combination of these three drugs in patients with
advanced head and neck cancer in the adjuvant settings [62].

Conflict of interest
There is no financial and personal relationships with other
people or organizations that could inappropriately influence
this review article.

Acknowledgement
This article is supported in part by a Grant-in-Aid from the
Ministry of Education, Science, Sports and Culture of Japan
(Grant no: Sakagami, No. 19592156).

References
[1] Vogelstein B, Kinzler K. Cancer genes and the pathways they
control. Nat Med 2004;10:78999.
[2] Squarize CH, Castilho RM, Santos Jr PD. Immunohistochemical
evidence of PTEN in oral squamous cell carcinoma and its
correlation with the histological malignancy grading system.
J Oral Pathol Med 2002;31:37984.
[3] Tokman B, Gultekin SE, Sezer C, Alpar R. The expression of p53,
p16 proteins and prevalence of apoptosis in oral squamous cell
carcinoma. Correlation with mode of invasion grading system.
Saudi Med J 2004;25:192230.
[4] Jane C, Nerurkar AV, Shirsat NV, Deshpande RB, Amrapurkar
AD, Karjodkar FR. Increased survivin expression in high-grade
oral squamous cell carcinoma: a study in Indian tobacco
chewers. J Oral Pathol Med 2006;35:595601.
[5] Wang X, Sun SZ, Zhang XY, Ma BL. The expression of Fas and bcl2 in hamster buccal carcinogenesis. Shanghai Kou Qiang Yi Xue
2005;14:1558.
[6] Leonardi R, Pannone G, Margo G, Kudo Y, Takata T, Lo Muzio L.
Differential expression of heat shock protein 27 in normal oral
mucosa, oral epithelial dysplasia and squamous cell carcinoma. Oncol Rep 2002;9:2616.
[7] Miyashita H, Mori S, Motegi K, Fukumoto M, Uchida T. Pin 1 is
overexpressed in oral squamous cell carcinoma and its levels
correlate with cyclin D1 overexpression. Oncol Rep 2003;
10:45561.
[8] Childs G, Fazzari M, Kung G, Kawachi N, Brabdwein-Gensler M,
McLemore M, et al. Low-level expression of microRNAs let-7d
and miR-205 are prognostic markers of head and neck squamous cell carcinoma. Am J Pathol 2009;174:73645.
[9] Hsu S, Singh B, Schuster G. Induction of apoptosis in oral cancer
cells: agents and mechanisms for potential therapy and prevention. Oral Oncol 2004;40:46173.
[10] Elmore S, Apoptosis:. A review of programmed cell death.
Toxicol Pathol 2007;35:495516.
[11] Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G,
Askew DS, et al. Guidelines for the use and interpretation of
assays for monitoring autophagy in higher eukaryotes. Autophagy 2008;4:15175.
[12] Hacker G. The morphology of apoptosis. Cell Tissue Res 2000;
301:517.

H. Sakagami
[13] Noda T, Suzuki K, Ohsumi Y. Yeast autophagosomes: de novo
formation of a membrane structure. Trends Cell Biol 2002;
12:2315.
[14] Ohsumi Y. Molecular dissection of autophagy: two ubiquitinlike systems. Nat Rev Mol Cell Biol 2001;2:2116.
[15] Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview
of cell death. Am J Pathol 1995;146:315.
[16] Ueta E, Kamatani T, Yamamoto T, Osaki T. Tyrosine-nitration of
caspase 3 and cytochrome c does not suppress apoptosis
induction in squamous cell carcinoma cells. Int J Cancer
2003;103:71722.
[17] Xu JH, Huang HZ, Pan CB, Zhang B, Zhang LT. Role of surviving
gene on the apoptosis of Tca8113 cells induced by cisplatin.
Hua Xi Kou Qiang Yi Xue Za Zhi 2007;25:1736 [in Chinese].
[18] Azuma M, Tamatani T, Ashida Y, Takashima R, Harada K, Sato M.
Cisplatin induces apoptosis in oral squamous carcinoma cells by
the mitochondria-mediated but not the NF-kB-suppressed
pathway. Oral Oncol 2003;39:2829.
[19] Sundelin K, Roberg K, Gre
kansson L. Effects of
nman R, Ha
cisplatin, alpha-interferon, and 13-cis retinoic acid on the
expression of Fas (CD95), intercellular adhesion molecule-1
(ICAM-1), and epidermal growth factor receptor (EGFR) in oral
cancer cell lines. J Oral Pathol Med 2007;36:17783.
[20] Mishima K, Nariai Y, Yoshimura Y. Carboplatin induces Fas (APO1/CD95)-dependent apoptosis of human tongue carcinoma
cells: sensitization for apoptosis by upregulation of FADD
expression. Int J Cancer 2003;105:593600.
[21] Itoh M, Chiba H, Noutomi T, Takada E, Mizuguchi J. Cleavage of
Bax-alpha and Bcl-x(L) during carboplatin-mediated apoptosis
in squamous cell carcinoma cell lines. Oral Oncol 2000;
36:27785.
[22] Nonaka M, Ikeda H, Fujisawa A, Uehara M, Inokuchi T. Induction
of apoptosis by paclitaxel in human oral carcinoma cells. Int J
Oral Maxillofac Surg 2006;35:64952.
[23] Takemura K, Noguchi M, Ogi K, Tokino T, Kubota H, Miyazaki A,
et al. Enhanced Bax in oral SCC in relation to antitumor effects
of chemotherapy. J Oral Pathol Med 2005;34:939.
[24] Okamura H, Morimoto H, Haneji T. Peplomycin-induced apoptosis in oral squamous carcinoma cells depends on bleomycin
sensitivity. Oral Oncol 2001;37:37985.
[25] Tai KW, Chou MY, Hu CC, Yang JJ, Chang YC. Induction of
apoptosis in KB cells by pingyangmycin. Oral Oncol 2000;
36:2427.
[26] Yamamoto T, Yoneda K, Ueta E, Doi S, Osaki T. Enhanced
apoptosis of squamous cell carcinoma cells by interleukin-2activated cytotoxic lymphocytes combined with radiation and
anticancer drugs. Eur J Cancer 2000;36:200717.
[27] Shareef MM, Brown B, Shajahan S, Sathishkumar S, Arnold SM,
Mohiuddin M, et al. Lack of P-glycoprotein expression by lowdose fractionated radiation results from loss of nuclear factorkappaB and NF-Y activation in oral carcinoma cells. Mol Cancer
Res 2008;6:8998.
[28] King TA, Ghazaleh RA, Juhn SK, Adams GL, Ondrey FG. Induction of heat shock protein 70 inhibits NF-kappa-B in squamous
cell carcinoma. Otolaryngol Head Neck 2005;13:709.
[29] Yasumoto J, Kirita T, Takahashi A, Ohnishi K, Imai Y, Yuki K,
et al. Apoptosis-related gene expression after hyperthermia
in human tongue squamous cell carcinoma cells harboring
wild-type or mutated-type p53. Cancer Lett 2004;10:
4151.
[30] Iwase M, Takaoka S, Uchida M, Yoshiba S, Kondo G, Watanabe H,
et al. Epidernal growth factor receptor inhibitors enhance
susceptibility to Fas-mediated apoptosis in oral squamous cell
carcinoma cells. Oral Oncol 2008;44:3618.
[31] Cha JD, Jeong MR, Kim HY, Lee JC, Lee KY. MAPK activation is
necessary to the apoptotic death of KB cells induced by the
essential oil isolated from Artemisia iwayomogi. J Ethnopharmacol 2009;123:30814.

Apoptosis-inducing activity and tumor-specificity of antitumor agents against oral squamous cell carcinoma
[32] Liang CH, Wang GH, Liaw CC, Lee MF, Wang SH, Cheng DL, et al.
Extracts from Cladiella australis, Clavularia viridis and Klyxum
simplex (soft corals) are capable of inhibiting the growth of
human oral squamous cell carcinoma cells. Mar Drugs 2008;
6:595606.
[33] Rodrigo KA, Rawal Y, Renner RJ, Schwartz SJ, Tian Q, Larsen
PE, et al. Suppression of the tumorigenic phenotype in human
oral squamous cell carcinoma cells by an ethanol extract
derived from freeze-dried black raspberries. Nutr Cancer
2006;54:5868.
[34] Schuck AG, Ausubel MB, Zuckerbraun HL, Babich H. Theaflavin3,30 -digallate, a component of black tea: an inducer of oxidative stress and apoptosis. Toxicol In Vitro 2008;22:598609.
[35] Hsu S, Lewis J, Singh B, Schoenlein P, Osaki T, Athar M, et al.
Green tea polyphenol targets the mitochondrial in tumor cells
inducing caspase 3-dependent apoptosis. Anticancer Res
2003;23:15339.
[36] Hsu S, Yu FS, Lewis J, Singh B, Borke J, Osaki T, et al. Induction
of p57 is required for cell survival when exposed to green tea
polyphenols. Anticancer Res 2002;22:411520.
[37] Li N, Chen X, Han C, Chen J. Chemopreventive effect of tea and
curcumin on DMBA-induced oral carcinogenesis in hamsters.
Wei Sheng Yan Jiu 2002;31:3547 [in Chinese].
[38] Sakagami H, Kobayashi M, Chien C-H, Kanegae H, Kawase M.
Selective toxicity and type of cell death induced by various
natural and synthetic compounds in oral squamous cell carcinoma. In Vivo 2007;21:31120.
[39] Hou YY, Wu ML, Hwang YC, Chang FR, Wu YC, Wu CC. The
natural diterpenoid ovatodiolide induces cell cycle arrest and
apoptosis in human oral squamous cell carcinoma Ca9-22 cells.
Life Sci 2009;85:2632.
[40] Min R, Tong J, Wenjun Y, Wenhu D, Xiaojian Z, Jiacai H, et al.
Growth inhibition and induction of apoptosis in human oral
squamous cell carcinoma Tca-8113 cell lines by Shikonin was
partly through the inactivation of NF-kappa B pathway. Phytother Res 2008;22:40715.
[41] Keum YS, Kim J, Lee KH, Park KK, Surh YJ, Lee JM, et al.
Induction of apoptosis and capase-3 activity by chemopreventive [6]-paradol and structurally related compounds in KB
cells. Cancer Lett 2002;177:414.
[42] Du Y, Zhang S, Wang Z, Zhou W, Luan M, Yang X, et al. Induction
of apoptosis and cell cycle arrest by NS398 in oral squamous
cell carcinoma cells via downregulation of E2 promoter-binding factor-1. Oncol Rep 2008;20:60511.
[43] Ko SH, Choi GJ, Lee JH, Han YA, Lim SJ, Kim SH. Differential
effects of selective cyclooxygenase-2 inhibitors in inhibiting
proliferation and induction of apoptosis in oral squamous cell
carcinoma. Oncol Rep 2008;19:42533.
[44] Nikitakis NG, Hebert C, Lopes MA, Reynolds MA, Sauk JJ.
PPARgamma-mediated antineoplastic effect of NSAID sulindac
on human oral squamous carcinoma cells. Int J Cancer
2002;98:81723.
[45] Hamada M, Sumi T, Iwai S, Nakazawa M, Yura Y. Induction of
endonuclease G-mediated apoptosis in human oral squamous
cell carcinoma cells by protein kinase C inhibitor safingol.
Apoptosis 2006;11:4756.
[46] Noda T, Iwai S, Hamada M, Fujita Y, Yura Y. Induction of
apoptosis of detached oral squamous cell carcinoma cells by
safingol. Possible role of Bim, focal adhesion kinase and endonuclease G. Apoptosis 2009;14:28797.
[47] Otsubo A, Bhawal UK, Nomura Y, Mitani Y, Ozawa K, Kuniyasu H,
et al. UCN-01 (7-hydroxystaurosporine) induces apoptosis and
G1 arrest of both primary and metastatic oral cancer cell lines
in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2007;103:3917.
[48] Fujita M, Seta C, Fukuda J, Kobayashi S, Haneji T. Induction of
apoptosis in human oral squamous carcinoma cell lines by
protein phosphatase inhibitors. Oral Oncol 1999;35:4018.

185

[49] Goto K, Fukuda J, Haneji T. Okadaic acid stimulates apoptosis

through expression of Fas receptor and Fas ligand in human
oral squamous carcinoma cells. Oral Oncol 2002;38:1622.
[50] Mihara M, Shintani S, Nakashiro K, Hamakawa H. Flavopiridol, a
cyclin dependent kinase (CDK) inhibitor, induces apoptosis by
regulating Bcl-x in oral cancer cells. Oral Oncol 2003;39:4955.
[51] Kudo Y, Takata T, Ogawa I, Kaneda T, Sato S, Takekoshi T, et al.
p27Kip1 accumulation by inhibition of proteasome function
induces apoptosis in oral squamous cell carcinoma cells. Clin
Cancer Res 2000;6:91623.
[52] Siavash H, Nikitakis NG, Sauk JJ. Abrogation of IL-6-mediated
JAK signalling by the cyclopentenone prostaglandin 15dPGJ(2) in oral squamous carcinoma cells. Br J Cancer 2004;
91:107480.
[53] Sakate K, Takagi E, Ishii A, Kato Y, Imagawa Y, Kimura Y, et al.
Anti-tumor effect of vitamin A an D on head and neck squamous
cell carcinoma. Auris Nasus Larynx 2003;30:40312.
[54] Gaun W, Yu S, Gao Y. Alternation on Fas/FasL gene expression
in oral squamous cell carcinoma cell line Tca83 by retinoid
acid. Zhonghua Kou Qiang Yi Xue Za Zhi 2001;36:2158.
[55] Zhou H, Tang Y, Liang X, Yang X, Yang J, Zhu G, et al. RNAi
targeting urokinase-type plasminogen activator receptor inhibits metastasis and progression of oral squamous cell carcinoma in vivo. Int J Cancer 2009;125:45362.
[56] Duan L, Yao J, Wu X, Fan M. Growth suppression induced by
Notch 1 activation involves Wnt-beta-catenin down-regulation
in human tongue carcinoma cells. Biol Cell 2006;98:47990.
[57] Lee SK, Kim HS, Lee HJ, Lee J, Jeon BH, Jun CD, et al. Dual
effect of nitric oxide in immortalized and malignant human
oral keratinocytes: induction of apoptosis and differentiation.
J Oral Pathol Med 2006;35:35260.
[58] Takahashi M, Sato T, Shinohara F, Echigo S, Rikiishi H. Possible
role of glutathione in mitochondrial apoptosis human oral
squamous cell carcinoma caused by inorganic selenium compounds. Int J Oncol 2005;27:48995.
[59] Guo J, Zhang ZY. Investigation of apoptosis mechanism of
arsenic trioxide on oral squamous cell carcinoma. Zhoughua
Kou Qiang Yi Xue Za Zhi 2003;38:203 [in Chinese].
[60] Petrovic L, Schlegel KA, Ries J, Park J, Diebel E, Schultze-Mosgau
S, et al. In vitro effect of taurolidine on squamous cell carcinoma
in the oral cavity. Mund Kiefer Gesichtschir 2003;7:1027.
[61] Bonner JA, Raisch KP, Trummell HQ, Robert F, Meredith RF,
Spencer SA, et al. Enhanced apoptosis with combination C225/
radiation treatment serves as the impetus for clinical investigation in head and neck cancer. J Clin Oncol 2000;18(21
Suppl.):47S53S.
[62] Zhang X, Chen ZG, Khuri FR, Shin DM. Induction of cell cycle
arrest and apoptosis by a combined treatment with 13-cisretinoic acid, interferon-alpha2a, and alpha-tocopherol in
squanous cell carcinoma of the head and neck. Head Neck
2007;29:35161.
[63] Kanazawa T, Kommareddi PK, Iwashita T, Kumar B, Misawa K,
Misawa Y, et al. Galanin receptor subtype 2 suppresses cell
proliferation and induces apoptosis in p53 mutant head and
neck cancer cells. Clin Cancer Res 2009;15:222230.
[64] Gillenwater AM, Zhong M, Lotan R. Histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis through
both mitochondrial and Fas (Cd95) signaling in head and neck
squamous carcinoma cells. Mol Cancer Ther 2007;6:296775.
[65] Gu X, Song X, Dong Y, Cai H, Walters E, Zhang R, et al. Vitamin E
succinate induces ceramide-mediated apoptosis in head and
neck squamous cell carcinoma in vitro and in vivo. Clin Cancer
Res 2008;15:18408.
[66] Tano T, Okamoto M, Oshikawa T, Ahmed SU, Sasai A, Sato M.
Induction of apoptosis in human head and neck cancer cell lines
by an active component of OK-432 through p53-independent
pathway via toll-like receptor (TLR)4 signalling. Gan To Kagaku
Ryoho 2005;32:15624 [in Japanese].

186
[67] Flinterman M, Ga
ken J, Farzaneh F, Tavassoli M. E1A-mediated
suppression of EGFR expression and induction of apoptosis in
head and neck squamous carcinoma cell lines. Oncogene
2003;22:196577.
[68] Sato M, Harada K, Yura Y, Azuma M, Kawamata H, Iga H, et al.
The treatment with differentiation- and apoptosis-inducing
agent, vesnarinone, of a patient with oral squamous cell
carcinoma. Apoptosis 1997;2:3138.
[69] Nikitakis NG, Scheper MA, Papanikolaou VS, Sauk JJ. The
oncogenic effects of constitutive Stat3 signaling in salivary
gland cancer cells are mediated by survivin and modulated by
the NSAID sulindac. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 2009;107:82636.
[70] Yajima T, Ochiai H, Uchiyama T, Takano N, Shibahara T, Azuma
T. Resistance to cytotoxic chemotherapy-induced apoptosis in
side population cells of human oral squamous cell carcinoma
cell line Ho-1-N-1. Int J Oncol 2009;35:27380.
[71] Kajihara A, Takahashi A, Ohnishi K, Imai Y, Yamakawa N,
Yasumoto J, et al. Protein microarray analysis of apoptosisrelated protein expression following heat shock in human
tongue squamous cell carcinomas containing different p53
phenotypes. Int J Hyperthermia 2008;24:60512.
[72] Mallick S, Patil R, Gyanchandani R, Pawar S, Palve V, Kannan S,
et al. Human oral cancers have altered expression of Bcl-2
family members and increased expression of the anti-apoptotic splice variant of Mcl-1. J Pathol 2009;217:398407.
[73] Yao L, Iwai M, Furuta I. Correlations of bcl-2 and p53 expression
with the clinicopathological features in tongue squamous cell
carcinomas. Oral Oncol 1999;35:5662.
[74] Hus S, Lewis JB, Borke JL, Singh B, Dickinson DP, Caughman GB,
et al. Chemopreventive effects of green tea polyphenols
correlate with reversible induction of p57 expression. Anticancer Res 2001;37438.
[75] Lee JC, Sharma M, Lee YH, Lee NH, Kim SY, Yun JS, et al. Pax9
mediated cell survival in oral squamous carcinoma cell enhanced by c-myb. Cell Biochem Funct 2008;26:8929.
[76] Tu HF, Chen HW, Kao SY, Lin SC, Liu CJ, Chang KW. MDM2 SNP
309 and p53 codon 72 polymorphisms are associated with the
outcome of oral carcinoma patients receiving postoperative
irradiation. Radiother Oncol 2008;87:24352.
[77] Ueta E, Yoneda K, Kimura T, Tatemoto Y, Doi S, Yamamoto T,
et al. Mn-SOD antisense upregulates in vivo apoptosis of
squamous cell carcinoma cells by anticancer drugs and gamma-rays regulating expression of the BCL-2 family proteins,
COX-2 and p21. Int J Cancer 2001;94:54550.
[78] Ueta E, Yoneda K, Yamamoto T, Osaki T. Manganese superoxide
dismutase negatively regulates the induction of apoptosis by 5fluorouracil, peplomycin and gamma-rays in squamous cell
carcinoma cells. Jpn J Cancer Res 1999;90:55564.
[79] Azuma M, Harada K, Supriatno, Tamatani T, Motegi K, Ashida Y,
et al. Potentiation of induction of apoptosis by sequential
treatment with cisplatin followed by 5-fluorouracil in human
oral cancer cells. Int J Oncol 2004;24:144955.
[80] Tavassoli M, Soltaninia J, Rudnicka J, Mashanyare D, Johnson N,
Ga
ken J. Tamoxifen inhibits the growth of head and neck
cancer cells and sensitizes these cells to cisplatin-induced
apoptosis: role of TGF-beta 1. Carcinogenesis 2002;23:
156975.
[81] Oba M, Yano S, Shuto T, Suico MA, Eguma A, Kai H. IFN-gamma
down-regulates Hsp27 and enhances hyperthermia-induced
tumor cell death in vitro and tumor suppression in vivo. Int
J Oncol 2008;32:131724.
[82] Mishima K, Nariai Y, Yoshimura Y. Etodolac, a selective cyclooxygenase-2 inhibitor, enhances carboplatin-induced apoptosis of human tongue carcinoma cells by down-regulation of
FAP-1 expression. Oral Oncol 2005;41:7781.
[83] Suzuki M, Endo M, Shinohara F, Echigo S, Rikiishi H. Enhancement
of cisplatin cytotoxicity by SAHA involves endoplasmic reticulum

H. Sakagami

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

[97]
[98]

[99]

stress-mediated apoptosis in oral squamous cell carcinoma cells.

Cancer Chemother Pharmacol 2009;64:111522.
Shen J, Huang C, Jiang L, Gao F, Wang Z, Zhang Y, et al.
Enhancement of cisplatin induced apoptosis by suberoylanilide
hydroxamic acid in human oral squamous cell carcinoma cell
lines. Biochem Pharmacol 2007;73:19019.
Quan F, Pan CE, Zhang SQ, Yan LY, Yu L. Reversal effect of
resveratrol on chemotherapy resistance in KBv200 cell line and
underlying mechanisms. Zhonghua Er Bi Yan Hou Tou Jing Wai
Ke Za Zhi 2009;44:638 [in Chinese].
Imai Y, Ohnishi K, Yasumoto J, Kajiwara A, Yamakawa N,
Takahashi A, et al. Glycerol enhances radiosensitivity in a
human oral squamous cell carcinoma cell line (Ca9-22) bearing
a mutant p53 gene via Bax-mediated induction of apoptosis.
Oral Oncol 2005;41:6316.
Wakasa T, Inoue T, Kawai N, Murakami J, Kishi K, Fukui K. The
combination of ionizing radiation and expression of a wild type
p53 gene via recombinant adenovirus induced a prominent
tumour suppressing effect in human oral squamous cell carcinoma. Br J Radiol 2002;75:65762.
Chen W, He R, Zhang Z, Lin G, Zhou X. Enhanced efficacy of the
combination of all-trans-retinoic acid and gamma-interferon
in inhibiting proliferation of Tca8113 cells. Chin J Dent Res
1999;2:5460.
Brown J, OPrey J, Harrison PR. Enhanced sensitivity of human
oral tumours to the flavonol, morin, during cancer progression:
involvement of the Akt and stress kinase pathways. Carcinogenesis 2003;24:1717.
Schleier P, Berndt A, Berndt A, Dahse R, Zenk W, Hyckel P, et al.
Experimental 5-aminolevulinic acid-induced photodynamic
therapy (ALA-PDT) of oral carcinomas. Procedure in treatment
of solid tumors and elucidation of cell death. Mund Kiefer
Gesichtschir 2001;5:98104 [in German].
Sakagami H, Kawase M, Wakabayashi H, Kurihara T. Factors
that affect the type of cell death induced by chemicals.
Autophagy 2007;3:4935.
Ideo A, Hashimoto K, Shimada J, Kawase M, Sakagami H. Type
of cell death induced by a-trifluoromethyl acyloins in oral
squamous cell carcinoma. Anticancer Res 2009;29:17582.
Karbowski M, Norris KL, Cleland MM, Jeong S-Y, Youle RJ. Role
of Bax and Bak in mitochondrial morphogenesis. Nature
2006;443:65862.
Sakagami H, Chowdhury SA, Suzuki F, Hashimoto K, Hatano H,
Takekawa F, et al. Tumor-specific cytotoxic activity of polyphenols, terpenoids, ketones and other synthetic compounds. In:
Motohashi N, editor. Functional polyphenols and carotenes with
antioxidative action. Kerala, India: Research Signpost; 2005. p.
13376.
Sakagami H, Kobayashi M, Ishihara M, Kikukchi H, Nakamura Y,
Kawase M, et al. Tumor-specificity and type of cell death induced
by heterocycles. In: Motohashi N, editor. Topics in heterocyclic
chemistry, vol. 15. Berlin Heidelberg: Springer-Verlag; 2008. p.
17399.
Miyamoto M, Sakagami H, Minagawa K, Kikuchi H, Nishikawa H,
Satoh K, et al. Tumor-specificity and radical scavenging
activity of poly-herbal formula. Anticancer Res 2002;22:
121723.
Tong D, Poot M, Hu D, Oda D. 5-Fluorouracil-induced apoptosis
in cultured oral cancer cells. Oral Oncol 2000;36:23641.
Kondo G, Iwase M, Watanabe H, Uchida M, Takaoka S, Ohashi M,
et al. Enhancement of susceptibility to Fas-mediated apoptosis
in oral squamous cell carcinoma cells by phosphatidylinositol
3-kinase inhibitor. Oral Oncol 2006;42:74552.
Minter HA, Eveson JW, Huntley S, Elder DJ, Hague A. The
cyclooxygenase 2-selective inhibitor NS398 inhibits proliferation of oral carcinoma cell lines by mechanism dependent and
independent of reduced prostaglandin E2 synthesis. Clin Cancer Res 2003;9:188597.

Apoptosis-inducing activity and tumor-specificity of antitumor agents against oral squamous cell carcinoma
[100] Daido S, Yamamoto A, Fujiwara K, Sawaya R, Kondo S, Kondo Y.
Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy. Cancer Res 2005;65:436875.
[101] Kanzawa T, Zhang L, Xiao L, Germano IM, Kondo Y, Kondo S.
Arsenic trioxide induces autophagic cell death in malignant
glioma cells by upregulation of mitochondrial cell death protein BNIP3. Oncogene 2005;24:98091.
[102] Daido S, Kanzawa T, Yamamoto A, Takeuchi H, Kondo Y, Kondo
S. Pivotal role of the cell death factor BNIP3 in ceramideinduced autophagic cell death in malignant glioma cells.
Cancer Res 2004;64:428693.
[103] Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S.
Role of autophagy in temozolomide-induced cytotoxicity
for malignant glioma cells. Cell Death Differ 2004;11:
44857.
[104] Hopkins-Donaldson S, Yan P, Bourloud KB, Muhlethaler A, Bodmer
JL, Gross N. Doxorubicin-induced death in neuroblastoma does
not involve death receptors in S-type cells and is caspaseindependent in N-type cells. Oncogene 2002;21:61327.
[105] Carter BZ, Kornblau SM, Tsao T, Wang RY, Schober WD, Milella
M, et al. Caspase-independent cell death in AML: caspase

[106]

[107]

[108]

[109]

[110]

187

inhibition in vitro with pan-caspase inhibitors or in vivo by

XIAP or Survivin does not affect cell survival or prognosis. Blood
2003;102:417986.
Lim CC, Zuppinger C, Guo X, Kuster GM, Helmes M, Eppenberger HM, et al. Anthracyclines induce calpain-dependent titin
proteolysis and necrosis in cardiomyocytes. J Biol Chem
2004;279:82909.
Bilir A, Altinoz MA, Erkan M, Ozmen V, Aydiner A. Autophagy
and nuclear changes in FM3A breast tumor cells after epirubicin, medroxyprogesterone and tamoxifen treatment in vitro.
Pathobiology 2001;69:1206.
Yanagisawa-Shiota F, Sakagami H, Kuribayashi N, Iida M, Sakagami T, Takeda M. Endonuclease activity and induction of DNA
fragmentation in human myelogenous leukemic cell lines.
Anticancer Res 1995;15:25966.
Cui Q, Tashiro S, Onodera S, Ikejima T. Augmentation of
oridonin-induced apoptosis observed with reduced autophagy.
J Pharmacol Sci 2006;101:2309.
Li D, Cui Q, Chen SG, Wu LJ, Tashiro S, Onodera S, et al.
Inactivation of ras and changes of mitochondrial membrane
potential contribute to oridonine-induced autophagy in a 431
cells. J Pharmacol Sci 2007;105:223.