REVIEW ARTICLE

Oncoapoptotic Markers in Oral Cancer:Prognostics and Therapeutic Perspective

Anubhav Jain • Saurabh Bundela •

Ram P. Tiwari • Prakash S. Bisen

� Springer International Publishing Switzerland 2014

Abstract Oral cancer is one of the most commonly found

cancers in many South-Asian underdeveloped countries,

especially among men in comparison to women. When

considering the mortality rate among all types of existing

cancers, in India oral cancer is the primary reason for death

in men. Some of the major reasons contributing to the high

mortality rate are late diagnosis, lack of treatment options

and high prevalence of tobacco consumption. Oral cancer

is often diagnosed at a stage when cancer cells have already

become aggressive and become resistant to standard ther-

apeutic options. Progression, apoptosis, angiogenesis,

metastasis and invasion behold great capability to treat and

detect cancer at the molecular level. Dysregulation of

apoptosis is one of the most common molecular events

known to be associated with the development of oral

cancer. In this review, we discuss key apoptotic markers

which can be used as prognostic and/or therapeutic targets

in oral cancer.

Key Points

Apoptosis, tightly regulated by various molecular

factors, is an evolutionary conserved process which

maintains subtle balance between progression and

cell death.

The regulation of apoptosis involves tumor

suppressor genes, oncogenes, the Bcl-2 gene family,

receptor and mitochondrial apoptotic factors and

caspases.

Dysregulation of apoptosis is one of the most

common molecular events known to be associated

with the development of oral cancer and can be used

as a prognostic marker for oral cancer.

Therapeutic strategies, by leveraging apoptotic

machinery, are focused around promoting apoptosis

through activation of the death receptors, pro-

apoptotic factors and inhibition of anti-apoptotic

factors such as inhibitors of apoptosis (IAPs) and

Bcl-2.

1 Introduction

Oral cancer is an umbrella term which includes cancer of

the lip, tongue, mouth, oropharynx, piriform sinus, hypo-

pharynx and other ill-defined sites of the lip, oral cavity

and pharynx. Oral cancer has emerged as the top killer

among men in India; more than 20 % of cancer-related

A. Jain

Diagnostic R&D Division, RFCL Limited, New Delhi, India

S. Bundela

Department of Postgraduate Studies and Research in Biological

Sciences, Rani Durgawati Vishwavidyalaya, Jabalpur,

India

R. P. Tiwari

Rapid Diagnostic Group of Companies, New Delhi, India

P. S. Bisen (&)

School of Studies in Biotechnology, Jiwaji University,

Gwalior 474 011, India

e-mail: psbisen@gmail.com

P. S. Bisen

Defense Research & Development Establishment, DRDO,

Ministry of Defense, Government of India, Jhansi Road,

Gwalior 474 002, India

Mol Diagn Ther

DOI 10.1007/s40291-014-0104-5

deaths in India were due to oral cancer [1]. Oral cancer is

also the seventh most common form of cancer worldwide

and is estimated to increase by over 60 % in next two

decades [2]. Technological constraints do not allow cancers

of the oral cavity to be detected at an early and clinically

manageable stage. The understanding of risk factor asso-

ciated with oral cancer and unambiguous diagnosis of oral

cancer at an early stage is highly desirable to improve

survival rate. Several key genes have been implicated in

oral carcinogenesis. These implicated genes are involved in

processes such as cell proliferation, differentiation, and cell

survival. The study of signal transduction pathways for

mechanisms of apoptosis has significantly advanced our

understanding of human cancer, subsequently leading to

more effective treatments. Apoptosis is a critical process

responsible for maintaining the subtle balance between

progression and cell death. It is an evolutionary conserved

process which is tightly regulated by various molecular

factors in normal cells, and its dysregulation is associated

with diseases such as cancer, heart attack, liver failure,

stroke, etc. [3]. Cancer cells adapt various strategies to

survive and grow in the cellular microenvironment; eva-

sion of apoptosis is one such strategies. Chemotherapeutic

drugs act by killing cancerous cells which invariably

involves the apoptotic pathway. Dysregulation of factors

involved in the apoptotic pathway leads to the development

of chemoresistance [3, 4].

Factors involved in the initiation of apoptosis mainly act

through two pathways—extrinsic and intrinsic apoptotic

pathways [5] (Fig. 1). The extrinsic apoptotic pathway is

initiated by interaction between death ligands with extra-

cellular death receptors. Activated death receptors, along

with an adaptor protein such as FADD, and procaspase 8

form the death-inducible signaling complex (DISC), which

in turn leads to the activation of apoptosis effector protein

caspase 3 through caspase 8. The intrinsic apoptotic path-

way, which is also known as the mitochondrial pathway, is

initiated in response to DNA damage or cytotoxic stress.

The Bcl-2 protein family is the main regulator of the

intrinsic apoptotic pathway; it consists of both anti-apop-

totic and pro-apoptotic factors. The pro-apoptotic factors

(e.g. Bcl-2-associated protein X [Bax], Bak, Noxa, Puma)

are responsible for increasing mitochondrial membrane

permeability, whereas anti-apoptotic factors (e.g. Bcl-xL,

Bcl-2 and Mcl-1) are responsible for maintaining mito-

chondrial membrane integrity. The induction of the

intrinsic apoptotic pathway requires the presence of Bak or

Bax [6]. Activation of Bak and Bax is achieved either by

direct interaction with t-Bid or through neutralization of

Bcl-2 family proteins which keep a check on Bax and Bak.

BH3-only proteins such as Puma, Noxa, Bid and Bad, are

some of the important factors that are involved in the

neutralization of anti-apoptotic Bcl-2 proteins [7].

Mitochondria releases the inhibitor of inhibitors of apop-

tosis (IAPs), such as DIABLO/Smac and Omi/HtrA2,

apoptosis-inducing factor (AIF) and endonuclease G.

These proteins can contribute to apoptosis via caspase-

independent pathways to cell death. p53 is another

important regulator of apoptosis which mainly acts by

transcriptional regulation of apoptosis effector genes.

Among the IAP gene family protein, survivin is distinctly

identified as a critical protein with a dual role in the inhi-

bition of apoptosis and regulation of mitosis. These key

players of apoptosis can be exploited for therapeutic and/or

prognostic application in oral cancer. Activation of the

death receptors, pro-apoptotic factors (Caspases, p53) and

inhibition of anti-apoptotic factors such as IAPs and Bcl-2,

are some therapeutic strategies for controlling cancer.

Dysregulation of factors of apoptosis pathways can be used

as prognostic and/or prognostic markers for oral cancer

(Table 1).

2 Apoptotic Markers of Relevance to Prognosis

and Prediction of Outcome in Oral Cancer

2.1 Bcl-2 and Bcl-2-associated protein X (Bax)

Bcl-2 is a human proto-oncogene located on chromosome

18q21; it codes for Bcl-2 protein which is an integral

membrane protein located in the membranes of the endo-

plasmic reticulum (ER), nuclear envelope, and in the outer

membranes of mitochondria [8]. Bax is the most charac-

teristic death-promoting member of the Bcl-2 family. The

activation of the caspase cascade is triggered by the

translocation of Bax protein from the cytosol to the mito-

chondria, and the activated caspase cascade leads to cell

death [9]. The Bcl-2 family comprises cell death antago-

nists, such as Bcl-2 and Bcl-XL, and death agonists, such

as Bax and Bad. The Bcl-2 family proteins decide whether

a cell continues to survive or instead commits to death

through the mitochondrial apoptotic pathway, and carries

high prognostic significance in oral cancer. Bcl-2, Bax and

Bcl-2/Bax ratio can be used as effective biomarkers to

predict the prognosis of oral cancer. The 5-year survival

rate was significantly higher in patients with a ratio of Bcl-

2/Bax B1 than in those with Bcl-2/Bax [1

(76.79 ± 6.69 % vs. 59.26 ± 6.69 %; p = 0.0489) [10,

11]. The expression of Bcl-2 and Bax is reported to be a

significant predictor of invasiveness in oral cancer [12].

Bcl-2 and Bax expression were also found to be indepen-

dent prognostic factors for overall survival and cancer-

specific 5-year survival (CSS). Bcl-2 expression emerged

as an independent marker of favorable CSS, and expression

of Bcl-2 family members was found to be an important

marker of a favorable prognosis in oral squamous cell

A. Jain et al.

carcinoma (OSCC) [13]. In oral cancer, Bcl-2 is observed

from the initial stages of carcinogenesis up to the appear-

ance of metastasis [14–16]. The association between Bcl-2

expression and oral cancer has also been refuted in some

other studies. Yuen et al. [17] observed no association

between Bcl-2 expression and survival in OSCC. Another

study reported associations between increased Bcl-2

expression, neck metastasis and poor survival, but did not

detect any prognostic significance for Bax expression [18].

The lack of consensus in these studies may be attributed to

the lack of robust and reliable techniques for measuring

biomarker expression. Recently, automated quantitative

immunohistochemistry-based techniques, such as AQUA,

were used to eliminate observer bias in the measurement of

protein expression, and found Bax expression as an inde-

pendent prognostic marker in OSCC [19].

2.2 Survivin

Survivin is an important member of the IAP protein family

and has become the focus of various cancer research studies,

mainly due to its highly specific expression in most solid and

hematological malignancies [20]. Survivin negatively reg-

ulates apoptosis by inhibiting caspase activation. The over-

expression of survivin imparts survival benefit to cancer cells

and is therefore consistently associated with tumor aggres-

siveness and poor prognosis. For example, normal oral

mucosa and skin, including adnexal structures, are negative

for survivin. In contrast, one study found that 56 % and 64%

of oral and cutaneous squamous cell carcinomas,

respectively, were strongly positive for survivin [21]. The

expression of survivin is associated with larger, more poorly

differentiated tumors and lymph node metastasis.

The overexpression of survivin has been reported in

various human cancers, including oral cancer [22–24]. In a

study on OSCC, high survivin messenger RNA (mRNA)

expression was found to be correlated with poorer tumor

differentiation, higher clinical stage, and the presence of

lymph node metastasis (p\ 0.05). Multivariate analysis

showed that the status of survivin mRNA could be an

independent prognostic factor for OSCC patients (hazard

ratio [HR] 2.71; 95 % confidence interval 1.46–5.10;

p = 0.012), and upregulation of survivin is associated with

a poor prognosis and chemoresistance [25]. Clinicopatho-

logical analysis revealed a significant correlation between

survivin expression and lymph node metastasis (p = 0.006)

and proliferation (p\ 0.001) [26]. Survivin is found to be

prominently expressed in more than 80 % of OSCC, and its

expression often increased in poorly differentiated tumors

and it’s high expression correlated with poor survival rates

[27]. The difference between cytoplasmic and nuclear sur-

vivin is an indicator for survivin activity in tumor cells in

oral cancer patients and this difference may serve as a

predictive marker of outcome in oral cancer patients

undergoing multi-modality therapy [28]. The high expres-

sion of survivin in the oral cavity is an early predictor of

malignant transformation in pre-cancerous and cancerous

lesions [29]. In their study of OSCC in Taiwan, Lin et al.

found no significant correlation between survivin expres-

sion and patient age, sex, oral habits, cancer location, or

Fig. 1 Extrinsic and intrinsic

apoptotic pathway. Apart from

the intrinsic apoptotic pathway,

the extrinsic pathway is another

way through which cells

undergo apoptotic death. The

extrinsic pathway is triggered

by factors (death ligands)

located outside the cell, which

activates specific death

receptors on the cell surface.

The pro-apoptotic ligands

include Apo2L/TRAIL and

CD95L/FasL, which bind to

their cognate receptors, DR4/

DR5 and CD95/Fas,

respectively. The intrinsic

pathway is triggered in response

to stimuli generated due to DNA

damage or cytotoxic stress. Bcl-

2 family proteins and factors

such cytochrome-c, SMAC,

OMI, etc. located inside the

mitochondrion play an

important role in the intrinsic

pathway

Prognostic and Therapeutic Potential of Apoptotic Markers in Oral Cancer

TNM status, but patients with high survivin expression, an

advanced stage, a larger tumour size, or positive lymph

node metastases had a significantly shorter overall survival

than the others [30]. The expression of survivin is a strong

predictor of aggressive and invasive phenotype in oral

cancer patients, and therefore could be used for tailoring

therapeutic procedure [31].

2.3 p53

p53 holds a crucial position in the network of signaling

pathways that are essential for the regulation of cell

growth, and extrinsic and intrinsic apoptotic pathways [32,

33]. Transcriptional regulation of apoptosis effector genes

is the main route by which p53 regulates apoptosis. p53

activates transcription of various pro-apoptotic genes such

as Noxa, PUMA, Bax, Bad, and Bim. It represses tran-

scription of various anti-apoptotic factors such as survivin,

Bcl-2, and Bcl-xL. p53 activates apoptotic protease acti-

vating factor 1 (APAF1) and thereby contributes toward the

formation of apoptosome. p53 promotes the extrinsic

apoptotic pathway by activation of death receptors such as

tumor necrosis factor (TNF)-a, Fas, and DR5.

Deregulation of the p53 tumor suppression network is

observed in many tumor types, including oral cancer, and is

associated with poor prognosis [22, 34]. Mutation in p53 is

Table 1 Role of apoptotic markers in cancer diagnosis and therapy

Apoptotic markers Cancer prognosis Cancer treatment

Survivin Prognosis Transcription factors blockers: YM155, STA3 and SOX2 inhibitors

Lymph node metastasis Antisense oligonucleotides: LY2181308, EZN-3042

Invasiveness siRNA

miRNA (miRNA-34)

Tumor grading Oligopeptides (Shepherdin)

Targeting HSP90 to interfere with protein stabilityChemoresistance

Immunotherapy

p53 Prognosis Gene addition therapy (Ad-p53)

Oncolytic viruses (ONYX-015)

Diagnosis p53 activators: RITA, Nutin-3, MI-219, BDA

Mutant-type p53 reactivator: PRIMA-1, WR-1065, MIRA-1Treatment response

MDM2/p53 interaction blockers: benzodiazepinedione antagonistics

Mcl-1 Treatment response CDK inhibitors: flavopiridol, SNS-032, Sorafenib

Deubiquitinase inhibitors: WP1130

Radioresistance BH3 mimetics: obatoclax, gossypol, sabutoclax, BH3-M6

siRNA

Bcl-2 Prognosis Ribozyme

Interference with protein stabilitySurvival

Invasiveness

Cancer-specific 5-year survival

Bax Prognosis –

Survival

Invasiveness

Cancer-specific 5-year

survival

Fas/FasL Prognosis –

Tumor differentiation

Invasiveness

Tumor grading

Apoptotic marker

TRAIL – Esculetin

Monoclonal antibody

Bcl-2/Bax Prognosis –

Survival

A. Jain et al.

frequently reported in various cancers, including oral

cancer. The loss of function of p53 mutant proteins may

predict a significantly low pathological complete response

rate and suboptimal response to cisplatin-based neoadju-

vant chemotherapy in patients with oral cavity SCC [35].

In a study, 100 % of the oral cancers and 66 % (19/29) of

the pre-malignant lesions examined from Yemen and India

overexpressed p53 [36]. Also, co-expression and correla-

tion between p53 and Ki-67 have been demonstrated in oral

cancer, suggesting that alterations in the p53 protein might

lead to increased cell proliferation. Furthermore, overex-

pression of p53 has been suggested to be a reliable indi-

cator for OC development [37]. The credibility of p53 as an

independent prognostic factor is still not fully established

[38]; however, in several studies it has been reported to be

a significant clinical factor when used in combination with

other markers, such as cyclin D1 and epidermal growth

factor receptor (EGFR) [39]. Mutation in p53 has been

reported to be the most frequent genetic alterations in

human cancer [40]. p53 gene mutations may be better

predictors of recurrence than the expression of the protein,

and serum p53 levels may be more efficient prognosticators

than its tissue immunodetection [41]. The expression of

p53 in the dysplastic epithelium, in association with p16

and Ki-67, can be used as a marker to identify the pre-

cancerous stage of oral cancer [42]. Cox proportional

hazards model showed the presence of p53 antibodies to be

an independent prognostic factor (p = 0.020; HR 3.509)

[43, 44]. The mutations in p53 have been used in prediction

and/or screening of neoplastic samples in OSCC [40, 45].

There are many point mutations reported in p53 by various

research groups and it is difficult to identify a particular

point mutation that is specific for oral cancer, therefore

limiting its application as a cost-effective marker in the

early detection of oral cancer.

2.4 Fas/Fas Ligand

Fas ligand (FasL) is a death ligand that belongs to the TNF

family. It is predominantly expressed in activated T cells.

Activation of Fas with FasL induces the extrinsic apoptotic

pathway in normal and tumor cells. FasL is overexpressed,

and Fas receptor is under expressed, in OSCC, and this

expression pattern correlates with poorly differentiated

tumors [46–48]. High expression of FasL was found to be

associated with greater tumor size and advanced stages of

oral cancer [47]. The downregulation of Fas expression in

keratinocytes and lymphocytes of oral lichen planus (OLP)

specimens, together with upregulation of FasL, may serve

as an initial prognostic biomarker in oral cancer develop-

ment [49]. A significant positive correlation was found

between the argyrophilic nucleolar organizer region

(AgNOR) parameters and Fas/FasL expression as apoptotic

markers in the tumoral cells of oral and oropharyngeal SCC

(p\ 0.05) [50]. AgNOR count was found to be a strong

proliferation marker in patients with OSCC, and Fas and

FasL staining was useful in tumor grading [50].

2.5 Myeloid Cell Leukemia-1

Myeloid cell leukemia-1 (Mcl-1) is a protein encoded by

the MCL1 gene, which is an anti-apoptotic member of the

Bcl-2 family protein. It contains three BH domains and

differs from other anti-apoptotic members of the Bcl-2

family which contain four BH domains. Mcl-1 was found

to mediate resistance to apoptosis in gastric cancer cells by

blocking the mitochondrial pathway of cell death [51].

Mcl-1 was demonstrated to predict outcome in oral cancer

patients treated with definitive radiotherapy [52]. Expres-

sion of the Mcl-1L isoform was found to be significantly

associated with the radioresistance of OSCC [53]. Treat-

ment with Mcl-1L small interfering RNAs (siRNAs) alone

or in combination with ionizing radiation significantly

increased apoptosis in radioresistant oral squamous carci-

noma cells. The Bak/Mcl-1 ratio was reported to be a

useful biomarker to predict clinical outcomes of oral ver-

rucous hyperplasia (OVH) and oral leukoplakia (OL)

lesions treated with photodynamic therapy [54].

2.6 Other Apoptotic Markers

There are other factors from the apoptotic pathway which

can be used as diagnostic and/or prognostic markers for

oral cancer. Growth differentiation factor 15 (GDF15) is a

protein from the transforming growth factor (TGF)-bsuperfamily that has a regulatory role in inflammatory and

apoptotic pathways. A significant high serum level of

GDF15 was reported to be a potential diagnostic and

prognostic marker for oral cancer [55]. Caspases such as

caspase 8 and 9 are significantly expressed in OSCC.

Moreover the expression of caspase 7 was reported to be a

predictor of locoregional recurrence of OSCC [56].

X-linked IAP (XIAP) is reported to be prognostic, and a

treatment response marker [47, 57], and was also found to

be related to histological differentiation [58]. Loss of p21

expression was found to be associated with clinicopatho-

logical factors related to tumor progression, invasiveness,

aggressiveness, and malignancy [59, 60].

3 Apoptotic Markers in Oral Cancer Therapeutics

3.1 Gene Therapy

Gene therapy involves the manipulation of the existing

genetic material by transfer of genetic material into

Prognostic and Therapeutic Potential of Apoptotic Markers in Oral Cancer

targeted cells. Gene therapy is based on the premise that

‘the normal physiological state can be restored when the

gene exerts its normal function’. The comprehensive

understanding of molecular events involved in the devel-

opment of disease, along with the accessibility of target

organ/tissue for delivering therapeutic material, and in vivo

stability of therapeutic material to produce the desired

effect, are some of the major factors responsible for the

success of gene therapy. The easy accessibility of the oral

cavity makes oral cancer an ideal candidate for gene

therapy [61]. The gene therapy procedure typically

involves the following steps: (a) identification of the target

gene, which is involved in disease and qualifies to be a

candidate for gene therapy; (b) isolation and amplification

of the candidate gene; (c) cloning of the candidate gene on

a specific vector; and (d) delivery of candidate genes

through ex vivo or in vivo [4].

3.1.1 Gene Therapy Strategies to Induce Apoptosis in Oral

Cancer Cells

3.1.1.1 Gene Addition Therapy Cancer formation is

typically facilitated by inactivation of tumor suppressor

genes such as p53; gene addition therapy intents to coun-

teract this phenomenon by introduction of the tumor sup-

pressor gene to cancer cells with the help of the appropriate

delivery system. Alteration of p53 is known to be one of

the earliest events in the progressive development of oral

cancer from pre-cancerous lesions [62]. The feasibility and

efficacy of adenoviral-mediated p53 (Ad-p53) gene transfer

were found to be promising in the treatment of advanced

head and neck cancer [63, 64]. These results formed the

basis of human trials for designing clinical therapeutic

regimen of Ad-p53 gene therapy (ClincalTrials.gov num-

ber: NCT00064103).

3.1.1.2 Oncolytic Virus-Mediated Gene Therapy The

application of viruses in cancer therapy is based on the

observation that a cancer regression is seen in cancer

patients when infected with viruses. Advancements in

genetic manipulation techniques have made it possible to

design viruses that can selectively target and kill tumor

cells. The replication of the virus is critically dependent on

the E1B viral protein, and in the absence of a functional

p53 protein. Viruses without the E1B protein are unable to

replicate in the presence of the functional p53 pathway in

the host cell. The p53 pathway becomes dysfunctional in

oral cancer cells; this fact is leveraged in the treatment

strategy of utilizing the oncolytic virus ONYX-015, which

lacks the viral E1B protein [65]. ONYX-015 replicates in

tumor cells and thereby selectively lyses tumor cells

because of the absence of the functional p53 pathway in

these tumor cells. ONYX-015 is currently being evaluated

as a preventive treatment for pre-cancerous oral tissue,

since even in pre-cancerous cells p53 pathway-inactivating

mutations will allow ONYX-015 to destroy and eliminate

the pre-cancerous cells before a tumor develops. The effi-

cacy of ONYX-015 in cancer treatment can be further

enhanced in combination with chemotherapy [66, 67].

3.1.1.3 Antisense RNA This technology is based on post-

transcriptional silencing of the target mRNA with the help

of the introduction of sequence-specific antisense RNA,

also known as siRNA. Apoptosis is evaded in cancer cells

by involvement of survival factors such as Bcl-2, XIAP,

and survivin. These genes involved in the survival pathway

can be selectively inhibited by sequence-specific antisense

RNA. The effective inhibition of oral cancer growth

mediated via induction of apoptosis was observed after the

introduction of siRNA targeting survivin [68, 69]. The

selective silencing of Mcl-1 by siRNA resulted in inhibi-

tion of growth of OSCC accompanied with apoptosis [70].

The silencing of other survival genes such as Bcl-x or Bcl-

xL by siRNA has been reported to induce apoptosis in

various tumor cells and sensitize tumor cells to chemo-

therapy [71, 72]. Induction of apoptosis by an anti-Bcl-2

ribozyme, delivered by an adenovirus vector, and HSP70

oligonucleotide treatment to decrease expression of Bcl-2

has been reported in oral cancer cells [73, 74].

3.2 Targeted Therapy

Chemotherapy is a standard treatment option for various

cancers, including those of the oral cavity. The main

drawback of chemotherapy is indiscriminately killing

normal proliferating cells along with intended cancer cells,

which causes unavoidable toxicities. Owing to scientific

advancements made in the last couple of decades, we are

now in a better position as far as molecular understanding

of oral carcinogenesis is concerned. The detailed study and

understanding of molecular events of oral carcinogenesis

has gifted us with molecular targets which could form the

base for therapeutic intervention. However, this informa-

tion is not as comprehensive as in the case of other well-

studied cancers of breast, lung and colorectum.

Cetuximab is the only approved targeted therapy avail-

able for oral cancer. It targets EGFR, which is involved in

cell progression [75]. Although KRAS, NRAS and BRAF

mutations have been established as potential predictive

biomarkers for cetuximabin in colorectal cancer, little is

known about predictive markers for cetuximabin in oral

cancer. Other targeted therapy for oral cancer in clinical

trial phase targets molecules such as CD44 [GSK1120212]

[76], PPARgamma [Pioglitazone] (ClinicalTrials.gov

number NCT00951379), and EpCAM [Proxinium] (Clini-

calTrials.gov number NCT00272181). There are a host of

A. Jain et al.

potential targets from the apoptotic pathway which can be

exploited for therapeutic intervention in oral cancer; some

of the important targets are discussed here.

3.2.1 Targets of the Extrinsic Pathway

Death ligands (TNF-a, CD95L, TRAIL) and their receptors(DR-4 or TRAIL-R1 and DR-5 or TRAIL-R2) are important

targets from the extrinsic apoptotic pathway. Esculetin was

reported to enhance TRAIL-mediated apoptosis, in the oral

cancer cell line [77]. Monoclonal antibodies (HGS-ETR1,

HGS-ETR2, HGS-TR2J, TRA-8) with agonist function at

death receptors have also been used to induce apoptosis;

however, they have not been tested for oral cancer treat-

ment. Results from preclinical and clinical studies have

shown the potential utility of TRAIL-targeted therapies in

advanced cancers, including oral cancer [78–81].

3.2.2 Targets of Intrinsic Pathway

The intrinsic apoptotic pathway or mitochondrial pathway

is mainly regulated by Bcl-2 family proteins (Fig. 1). The

Bcl-2 protein family consists of anti-apoptotic (e.g. Bcl-xL,

Bcl-2 and Mcl-1) and pro-apoptotic (e.g. Bax, Bak, Noxa,

Puma) factors. The strategies used for inducing apoptosis

through the Bcl-2 family proteins are: (a) silencing or

downregulation of anti-apoptotic proteins by antisense

techniques; (b) release of pro-apoptotic protein from the

Bcl-2 complex, through the application of the BH3 domain

peptide; and (c) release of pro-apoptotic protein from the

Bcl-2 complex, through the application of synthetic small-

molecule drugs such as tetrocarcin A (TC-A) [82, 83],

antimycin A3 [84], and chelerythrine [85]. Small molecules

such as Gossypol, ABT-737, ABT-263, GX15-070 and

HA14-1 have been developed that directly interact with

anti-apoptotic Bcl-2 proteins. These agents mimic the

action of BH3 proteins and interact with anti-apoptotic Bcl-

2 proteins at their BH3-binding groove [86].

The execution of apoptosis involves initiator caspases

(caspase 8, 9, 10) and executioner caspases as caspase 3, 4

and 7. Activation of these caspases by external compound is

an alternative pathway for killing cancer cells through

apoptosis. Many research groups have attempted to find the

caspase activator based on analysis of compound screening

data from high-throughput screening (HTS) studies. In one

such screening study, dichlorobenzyl carbamates and in-

dolones were detected as strong caspase activators [87].

Maxim Pharmaceuticals’ MX-2060 series of caspase-acti-

vating compounds is reported to have caspase-activating

attributes [88]. Caspase activity is inhibited by a class of

regulatory proteins known as IAPs. Survivin and XIAP are

prominent members of the IAP family of proteins which are

currently been pursued by various research groups as

potential cancer therapeutic targets. Cisplatin induces

apoptosis in oral cancer cells through inhibition of XIAP

[89]. Caspase 9 contains the IAP-binding motif (IBM)

through which it binds with the BIR-3 domain of XIAP. A

domain homologous to IBM is found in mitochondrial

proteins SMAC and Omi/HtrA2. SMAC interacts with

XIAP and releases caspase 9 for inducing apoptosis. SMAC

mimetic drugs hold potential in cancer treatment [4].

3.2.3 Targeting p53

p53 is the key regulator of various critical pathways, including

apoptosis; it becomes dysfunctional in the cancerous condi-

tion, thereby imparting survival benefit to the cancer cell.

Restoration of wild-type p53 (WT p53) is a potential strategy

in anticancer therapy.MDM2acts as an inhibitor of p53 and is

found to be overexpressed in various cancers. Blocking of

p53/MDM2 interaction by a variety of agents, such as small-

molecule inhibitors, synthetic peptides and benzodiazepin-

edione antagonists [90–92], has led to p53-dependent apop-

tosis in cancer cells. Activation of mutant-type p53 (MT p53)

by small-molecule activators offers an opportunity to inhibit

tumor growth through p53-mediated pathways. Some of the

WTp53 activators are RITA [93], Nutlin-3 [94],MI-219 [95],

BDA [92], HLI98C [96], Tenovin-1 [97] and JJ78:12 [98].

Mutant-type p53 is unable to perform its normal function

because of the defect in its folding caused by various point

mutations associatedwith cancer. The function ofMTp53 can

be restored with the help of peptides and small-molecule

compounds which act as a stabilizer. They include synthetic

peptides derived from the C-terminus of p53 [99], as well as

peptides such as CDB3 [100, 101], and compounds isolated

fromchemical library screenings suchasPRIMA-1 [102] (p53

reactivation and induction of massive apoptosis) and CP-

31398 [103–106]. Some of the other small-molecule reacti-

vators ofMTp53 areMIRA-1 [107], ellipiticine [108], P53R3

[109] and WR-1065 [110].

3.2.4 Targeting Survivin

Survivin is an attractive target for oral cancer because of its

highly specific expression in cancer tissues [4]. Survivin

can be inhibited at multiple levels, such as (a) interference

with survivin gene (BIRC5) expression by inhibiting its

transcription factors; (b) post-transcriptional regulation by

antisense technologies; (c) regulating upstream regulators

of survivin; (d) disturbing protein stability by modulating

interacting partners; (e) modulating protein stability; and

(f) activation of the immune response. Sepantronium bro-

mide (YM155) can inhibit tumor growth by inhibiting

survivin promoter-reporter gene construct; it was found to

reverse cisplatin resistance in head and neck cancer cells

[111, 112]. The expression of survivin is also controlled by

Prognostic and Therapeutic Potential of Apoptotic Markers in Oral Cancer

transcriptional factors of research interest such as STAT3

and SOX2, and, therefore, targeting these transcription

factors offers an attractive strategy to inhibit survivin [113–

115]. Caspase 2 is shown to repress survivin by interfering

with its transactivation by nuclear factor kappa B (NF-jB)[116]. The post-transcriptional regulation of survivin can

be achieved with the help of antisense oligonucleotides

(ASOs), siRNAs, and micro RNAs (miRNAs). LY2181308

is survivin-directed ASO which was found to effectively

inhibit survivin and enhanced effect of gemcitabine, pac-

litaxel, and docetaxel [117]. EZN-3042 is an ASO that was

to effectively inhibit the expression of survivin [118].

siRNAs directed against survivin have also shown to

effectively inhibit survivin expression [119–121]. miRNA-

34a was found to negatively regulate survivin in gastric

cancer cells, and its modulation in cancer can be consid-

ered as an alternative strategy to inhibit survivin [122]. The

stability of survivin protein can be compromised by tar-

geting its chaperon proteins such as HSP90. Shepherdin is

an oligopeptide that inhibits the formation of survivin

HSP90 complex because of the presence of survivin

sequence from lysine 79 to glycine 83 [123]. Survivin is a

tumor-associated antigen (TAA) which is corroborated by

the presence of survivin-specific CD8? T cells and IgG

antibodies in cancer patients [124]. Survivin-derived epi-

topes have been used in vaccination, and have been shown

to elicit immune response against tumors [125, 126].

Application of peptide ligands is yet another exploratory

strategy to inhibit survivin [127].

3.2.5 Targeting Myeloid Cell Leukemia-1 (Mcl-1)

Although there is no drug that has been designed to directly

target Mcl-1, some drugs such as cyclin-dependent kinase

(CDK) inhibitors (flavopiridol [128], SNS-032 [129], so-

rafenib [130–132]) and deubiquitinase inhibitors (WP1130

[133, 134]) have been reported to elicit anti-cancer activity

by targeting Mcl-1 along with its intended targets. The

CDK inhibitor roscovitine significantly increased ABT-737

(BH3 mimetic drug) lethality in human leukemia cells

through a mechanism involving downregulation of Mcl-1

[135]. BH3 mimetic drugs targeting Mcl-1 are obatoclax

(GX15) [136], gossypol [137], sabutoclax (BI-97C1) [138]

and BH3-M6 [139]. The inhibition of survivin expression

by sequence-specific siRNA was found to induce apoptosis

and growth inhibition in OSCC [70].

4 Conclusions

The molecular events associated with apoptosis in cancer

cells are strategically tuned to support survival and pro-

liferation necessary for tumorous growth. These molecular

events are significantly different in cancer cells when

compared with normal cells, and therefore offer great

potential to be utilized as diagnostic/prognostic/therapeutic

markers. Barring a few gaps, our understanding of the role

of the apoptotic pathway in oral carcinogenesis is almost

complete, which has given us key apoptotic factors such as

p53, survivin, Bcl-2/Bax, Mcl-1 and Fas. It would be

interesting to see how many of these factors will be able to

cross the translational gap between bench and bedside.

Until now, there have been very few markers that have

been able to successfully cross this translational gap

because of inherent challenges in the path of biomolecules

to qualify as definitive biomarkers. Some of the challenges

are related to its specificity and ability to detect oral cancer

much before the clinical manifestation of cancer. More-

over, biomarkers should be available in body fluids such as

blood and saliva for easy detection through non-invasive

tests. Apoptotic markers such as p53, survivin, Bcl-2/Bax

and Mcl-1 have emerged as potential biomarkers for the

diagnosis and prognosis of oral cancer. These biomarkers

should also be studied in various combinations to explore

any hidden pattern that can form the basis of designing the

diagnostic application. With the accumulation of knowl-

edge about the role of apoptosis in oral carcinogenesis, we

should be able to, unambiguously, assign the role of the

biomarker as a single entity and its role in the group

towards contributing towards cancer development; such

knowledge would help the researcher to design precise

analytical tests with high specificity and sensitivity.

Oral cancer is currently managed by the integration of

traditional methods such as surgery, radiation therapy and

chemotherapy. The current treatment options have failed to

improve survival rate, which is why target-based therapies

need to be brought into clinical practice instead of tradi-

tional therapies. The targeted elimination of oral squamous

cell carcinoma cells by inducing apoptosis has emerged as

a valued strategy to combat oral cancer. Targeted therapies

require a detailed understanding of the underlying disease

process. Gene therapies such as ONYX-015, Ad-p53 and

antisense techniques should be treated as a turning point for

oral cancer treatment. The BH3 mimetic class of drugs

have the potential to inhibit multiple anti-apoptotic Bcl-2

family proteins, and could therefore prove to be attractive

treatment options, especially in cases when cancer cells

develop resistance to the drug targeting a single apoptotic

target. With a better understanding of the role of the

apoptotic pathway in oral cancer, we should expect tar-

geted therapies with better efficacy and minimal toxicity.

The success of therapies focused on apoptotic markers

would depend on the skillful combination of diverse drug

classes (such as gene therapy, BH3 mimetic, small-mole-

cule inhibitors, etc.) to effectively kill cancer cells without

developing resistance to therapy.

A. Jain et al.

Acknowledgements and Disclosures The authors are thankful to

the Council of Scientific and Industrial Research (CSIR), New Delhi,

for the award of Emeritus Scientist to Professor P.S. Bisen. No

sources of funding were used to prepare this article. The authors have

no conflicts of interest that are directly relevant to the content of this

article.

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