The tricyclic antidepressant amitriptyline is cytotoxicto HTB114 human leiomyosarcoma and inducesp75NTR-dependent apoptosisGrazia Pula*, Alessandra Pistilli*, Claudia Montagnoli, Anna M. Stabile,Maria G. Rambotti and Mario Rende

Nerve growth factor (NGF) receptors, TrKA and p75NTR,

are being investigated in cancer therapy. Our previous data

show that, in HTB114 uterine leiomyosarcoma cells,

p75NTR-dependent apoptosis is inducible by cytotoxic

drugs and can suppress nerve growth factor-dependent

growth. Although amitriptyline can kill cancer cells and

bind TrKA/B, its effects on p75NTR-dependent apoptosis

are unknown. The aim of this paper was to evaluate the

antineoplastic potential of amitriptyline, and the role

of p75NTR-dependent apoptosis in the chemoresistant uterine

HTB114 leiomyosarcoma. Using proliferation assays and

fluorescence-activated cell sorting analysis, we found

that amitriptyline caused a marked reduction in HTB114

cell viability, associated with the parallel upregulation

of p75NTR expression. This converted the TrKA+-proliferating

cells into TrKA + /p75NTR + , leading to downregulation

of TrKA-prosurvival signaling (AKT) and activation of

p75NTR-dependent apoptosis (through caspase-3).

Overall, we provide novel evidence that HTB114 uterine

leiomyosarcoma cells are highly sensitive to amitriptyline,

supporting the role of p75NTR-dependent apoptosis as

a novel cytotoxic mechanism of this drug and of p75NTR as

an inducible stress receptor and a novel target in clinical

oncology. Anti-Cancer Drugs 00:000–000 �c 2013 Wolters

Kluwer Health | Lippincott Williams & Wilkins.

Anti-Cancer Drugs 2013, 00:000–000

Keywords: amitriptyline, apoptosis, cancer, cancer therapy, neurotrophins,nerve growth factor, p75NTR, TrKA, uterine leiomyosarcoma

Anatomy Section, Department of Medico-Surgical Specialties and Public Health,School of Medicine, University of Perugia, Perugia, Italy

Correspondence to Grazia Pula, MD, PhD, Anatomy Section, Departmentof Medico-Surgical Specialties and Public Health, School of Medicine,University of Perugia, Via del Giochetto, 06126 Perugia, ItalyTel: + 39 075 585 7449; fax: + 39 075 5857454; e-mail: pula@unipg.it

*Grazia Pula and Alessandra Pistilli contributed equally to this work.

Received 15 January 2013 Revised 6 June 2013Revised form accepted 12 June 2013

IntroductionUterine leiomyosarcoma is an aggressive tumor, which is

radioresistant and chemoresistant. Generally, resistance

to chemotherapy is linked to the ability of cancer cells to

repair DNA damage and develop adaptations, which

promote survival in toxic environments. Thus, finding

alternative strategies to ‘switch off ’ the survival pathways

of these cells may provide a clinical benefit. Furthermore,

investigation of existing drugs that are currently pre-

scribed for other indications can prove cost-effective.

Like other developmental factors, neurotrophins (NTs)

have been increasingly linked to cancer [1–6]. In fact,

accumulating evidences show that tumors can develop

autonomous secretion of NTs, especially nerve growth

factor (NGF) and brain derived neurotrophic factor

(BDNF). This triggers an autocrine loop, leading to

constitutive activation of their cognate receptors, unrest-

ricted growth, and resistance to apoptosis. Thus,

neurotrophin receptors (NTRs) have become promising

targets in cancer therapy [7,8]. There are two type of

NTRs, namely, the NT-selective tropomyosin receptor

kinases (TrKA, TrKB, and TrKC) and the nonselective

p75NTR, which binds all NTs [9]. The TrKs are tyrosine-

kinase receptors. They are encoded by proto-oncogenes

and activate the classic signaling pathways of growth

factor receptors, including (a) the mitogenic p38 mito-

gen-activated protein kinase pathway and (b) the

prosurvival phosphatidylinositol 3-kinase (PI3K)/AKT

pathway, which also inhibits apoptosis [10]. They can

also trigger Ca2 + release through the phospholipase C-g/

phosphatidylinositol bisphosphate/inositol trisphosphate/

diacylglycerol pathway [9]. In contrast, p75NTR is a death

receptor of the tumor necrosis factor (TNF) receptor

superfamily, with a functional death domain [5,11,12].

However, this receptor is also a stem cell marker

(CD271) [13], associated with cancer growth [13,14],

and can form complexes with different coreceptors

activating multiple pathways. Therefore, whereas the

TrKs are generally prosurvival, the role of p75NTR is more

complex and enigmatic [11–15] and it can induce either

(a) apoptosis, through c-jun N-terminal kinase (JNK)/

caspase-3, -6, and -9, or (b) survival, through NF-kB (NF-

kB), depending on crosstalk with the TrKs and other

coreceptors [13,15,16]. This ambiguity has been de-

scribed in other death receptors [17] and reflects the

different signaling molecules expressed by differentiated

Supplemental digital content is available for this article. Direct URL citationsappear in the printed text and are provided in the HTML and PDF versions of thisarticle on the journal’s website (www.anti-cancerdrugs.com).

Preclinical report 1

0959-4973 �c 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/CAD.0b013e328364312f

and cancer (or stem) cells. In fact, the final cellular

response results from the integration of multiple

variables, including the presence of mature/immature

NTs, TrK isoforms, transactivating receptors, coreceptors

and adapters, and the balance between TrKs/p75NTR, all

of which can be significantly dysregulated by the

oncogenic transformation [6].

Generally, because of the higher Kd of the TrKs versus

p75NTR (pmol/l vs. nmol/l, respectively), NTs bind the

TrKs preferentially. Activating mutations, upregulation or

ectopic expression of the TrKs, have been linked to

cancer [6,18]. TrK activation can induce growth, invasion,

metastasis, ankoisis resistance, and angiogenesis [6], all of

which promote survival under limiting conditions, and

multidrug resistance [6,18,19]. In addition, overexpres-

sion of the TrKs and constitutive activation of their

prosurvival pathways (especially AKT) can inhibit apop-

tosis [2–4,20], causing apoptosis resistance. TrK inhibitors

(i.e. CEP-701, AZ7451) [7] are therefore investigated in

clinical trials for cancer therapy (http://clinicaltrialsfeeds.org).

However, previous work from our group and others has

shown that upregulation of p75NTR can restore apoptosis

in susceptible cancers and could represent an alternative

antineoplastic strategy [2–4,20].

Sarcomas are among the tumors characterized by auto-

crine activation of the NGF/TrKA/AKT axis [2–4,21].

In the human HTB114 uterine leiomyosarcoma, this self-

sustaining loop leads to a constitutive receptor imbalance

– that is high-TrKA/low-p75NTR - promoting malignant

growth and survival [3,4]. Recently, the BDNF/TrKB axis

has also been linked to uterine leiomyosarcoma growth

[22]. In HTB114 cells, p75NTR-dependent apoptosis is

inducible by both TrK inhibitors and gene therapy.

Interestingly, we observed that the proapoptotic effect

of TrK blockade was associated with the upregulation of

p75NTR/caspase-3, rather than AKT inhibition [3,4],

confirming the key role of crosstalk in NGF receptor

signaling. Upregulation of p75NTR by injury and trau-

ma [23] is known to trigger apoptosis in the nervous

system (see the Discussion section). We therefore

postulated that the same mechanism could be used to

kill susceptible cancers. In fact, p75NTR may act as an

inducible stress receptor [24] in dysfunctional cells,

leading to p75NTR-dependent apoptosis as a final common

pathway for both cellular injuries and drug cytotoxicity.

We therefore sought to investigate whether other

cytotoxic drugs could induce p75NTR-dependent apopto-

sis in cancer cells. Tricyclic antidepressants (TCAs) are

currently prescribed to control depression and neuro-

pathic pain. However, although their potential relation

with cancer is controversial in epidemiological stu-

dies [25–30], they possess documented antineoplastic

effects. These drugs are cytotoxic to cancer cell lines

in vitro [31–34] and can reverse chemoresistance in

animal models [35–37]; furthermore, a preliminary

clinical trial has investigated the TCA chlorimipramine

as a potential therapy for glioma patients [38]. Although

the antidepressive effects of the TCAs have been

attributed to the inhibition of monoamine transporters,

their exact mechanism of action is unknown. On the basis

of their cationic amphiphilic properties, accumulation

into intracellular organelles is an off-target effect leading

to acute oxidant damage, through inhibition of the

mitochondrial complex III [33,39,40]. However, cytotoxi-

city has also been attributed to nonmitochondrial damage,

through cell cycle arrest [41], and autophagy [42]. These

diverse mechanisms may confer different sensitivities

to TCAs.

Although TCA cytotoxicity has been documented in

different cancers [31,39–46], these drugs have never

been tested in uterine leiomyosarcoma. Furthermore,

their potential modulation of p75NTR-dependent apop-

tosis has never been explored. Because of its known

antineoplastic activity [38–41] and as it has recently been

shown to bind TrK-A/B receptors [47], amitriptyline

appeared to be the ideal candidate.

The aim of this report was to investigate p75NTR-

dependent apoptosis and the NGF/TrKA/p75NTR axis in

HTB114 human uterine leiomyosarcoma cells exposed to

cytotoxic concentrations of amitriptyline. Our data show

for the first time the antineoplastic potential of

amitriptyline in human uterine leiomyosarcoma. Further-

more, we provide novel and interesting evidence that the

drug induces the membrane expression of p75NTR, which

has already been shown to trigger apoptosis in this cancer

cell model [2–4].

Materials and methodsReagents

Human recombinant b-NGF was obtained from Alomone

Labs (Zion, Israel). Amitriptyline was from Sigma-Aldrich

(Milan, Italy) and was solubilized in dimethylsulfoxide

(DMSO; Sigma) before adding to cells. Trypan blue and

the CellTrace CSFE Cell Proliferation kit (C34554) were

from Gibco BRL (Invitrogen, Monza, Italy). BSA was

from Santa Cruz Biotechnology (Santa Cruz, California,

USA). The fluorescein isothiocyanate (FITC) Annexin V

apoptosis detection kit and the cytofix/cytoperm kit were

from BD (Milan, Italy).

The following antibodies were used: phycoerythrin (PE)-

conjugated anti-human TrKA mouse monoclonal antibody

(R&D Systems, Abingdon, Virginia, USA); FITC-con-

jugated anti-human p75NTR mouse monoclonal antibody

(Alomone Labs); PE-conjugated anti-AKT mouse mono-

clonal antibody; and PE-conjugated anti-active caspase-3

rabbit monoclonal antibody (BD).

Cells were cultured in minimum essential medium

(MEM), containing 1 g/l glucose and supplemented with

antibiotic/antimycotic, L-glutamine (2 mmol/l), MEM

2 Anti-Cancer Drugs 2013, Vol 00 No 00

nonessential amino acid solutions (100�), sodium pyr-

uvate, and 10% fetal bovine serum. Cells were harvested

in Dulbecco’s phosphate-buffered saline (D-PBS) con-

taining 2 mmol/l EDTA. All culture media and supple-

ments were from Gibco BRL (Invitrogen).

Cancer cell lines

The HTB114 human leiomyosarcoma cell line, derived

from human uterine leiomyosarcoma, was from the

American Type Culture Collection (ATCC, Manassas,

Virginia, USA) and was cultured in MEM medium

according to ATCC instructions.

Amitriptyline treatment

For all experiments, cells were seeded in duplicate in six-

multiwell plates (Corning, Milan, Italy), at a density of

100 000 cells/well, and cultured for 24 h at 371C in a

humidified atmosphere of 5% CO2 to promote adhesion.

Amitriptyline, or its vehicle DMSO, was then added to

the culture medium at the indicated concentrations, and

cells were further incubated for the indicated times (see

below). Cells were then harvested, using 2 mmol/l EDTA

in D-PBS (D-PBS/EDTA), and analyzed, as described

below. All experiments included positive controls, treated

in parallel with b-NGF (10 ng/ml).

CFSE proliferation assay and vital counts

Cell cultures were set up in parallel for the carboxy-

fluorescein succinimidyl ester (CFSE) proliferation assay

and vital counts, as described, to have comparable

conditions. For the proliferation assay, cells were pre-

labeled with the CSFE probe, according to the manu-

facturer’s instructions, before plating, and then seeded

into multiwells. After 24 h culture, serial concentrations

of amitriptyline (0.5, 50, 100, 500, and 1000 mmol/l), or its

vehicle DMSO, were added to the culture medium and

cells were further incubated for 24, 48, 72 h, and up to 7

days. Cells were then harvested in D-PBS/EDTA and

analyzed by cytofluorimetric [fluorescence-activated cell

sorting (FACS)] scan. Trypan blue vital count was

performed as described in Rende et al. [2].

Determination of apoptosis by Annexin V labeling

Cells were seeded as described. After 24 h, serial

concentrations (0.5, 50, 100, 500, and 1000 mmol/l) of

amitriptyline, or its vehicle DMSO, were then added to

the culture medium and the cells were further incubated

for 24, 48, and 72 h. Cells were then harvested, labeled

using the FITC Annexin V apoptosis detection kit,

according to the manufacturer’s instructions, and ana-

lyzed by FACS scan.

Determination of NGF-receptor expression

Cells were seeded as described. After 24 h, 50 mmol/l

amitriptyline, or its vehicle DMSO, was added to the

culture medium, and cells were further incubated for 24,

48, and 72 h. Cells were then harvested, preincubated

with 0.5% BSA in D-PBS for 15 min at room temperature

to minimize unspecific staining, followed by centrifuga-

tion, and 1-h incubation at 41C with either the PE-

conjugated anti-human TrKA antibody (1:10) or the

FITC-conjugated anti-human p75NTR antibody (1:10),

in 0.5% BSA in D-PBS (D-PBS/BSA). Cells were then

washed in D-PBS/BSA and analyzed by FACS scan. All

experiments included negative controls, incubated with

human nonimmune immunoglobulins G (1:10).

Determination of caspase-3 and AKT activation

Cells were seeded as described. After 24 h, serial concentra-

tions (0.5, 50, 100, 500, and 1000mmol/l) of amitriptyline, or

its vehicle DMSO, were added to the culture medium, and

cells were further incubated for 24, 48, and 72 h. Cells were

then harvested in D-PBS/EDTA and fixed-permeabilized

using the cytofix/cytoperm kit according to the manufac-

turer’s instructions. Cells were then incubated for 1 h at 41C

with either the PE-conjugated anti-AKTantibody or the PE-

conjugated anti-active caspase-3 antibody in perm/wash

buffer (from the same kit). Samples were then rinsed in

the same buffer and analyzed by FACS scan.

Statistical analysis

Duplicate samples were used in each experiment. Data

are expressed as mean±SD of three separate experi-

ments. Student’s t-test was used to compare two groups.

A probability value of P less than 0.05 indicates statistical

significance.

ResultsAmitriptyline reduces cell viability and proliferation,

and induces apoptosis in HTB114 cells.

Cell viability and proliferation were analyzed by cell counts

and CSFE assay, respectively (Fig. 1). As shown in Fig. 1a,

a trend toward reduced cell viability was already apparent at

24 h in cells treated with 50mmol/l amitriptyline. The cell

viability decreased below 15% of controls (13%, P < 0.05)

at 48 h and approached 0% (2%, P < 0.05) at 72 h (Fig. 1b).

The IC50 was B4mmol/l at 48 h (Fig. 1b).

Cytotoxicity was confirmed by parallel CFSE proliferation

assays. The proliferative index is calculated by measuring

the reduction in the mean fluorescence intensity of cells

labeled with the fluorescent dye CSFE, which is

consequent to the dilution of the dye after cell division.

Measurement of the duplication rate of viable cells allows

a better estimate of the antiproliferative effect, without

interference of cell death. As shown in Fig. 1c, amitripty-

line significantly reduced proliferation at 48 h versus

controls and almost suppressed it (– 92.5%, P < 0.05) in

cells exposed to 500 mmol/l drug for 72 h. The dose–

response curves seem to show a more prominent effect on

cell viability as compared with cell proliferation (see the

Discussion section).

The analysis of apoptosis, using the Annexin V assay

(Fig. 2), confirmed the antineoplastic effect. Apoptosis was

Antineoplastic activity of amitriptyline Pula et al. 3

time and dose dependent, starting at 48 h and reaching

a plateau at 72 h, when greater than 80% of the cells exposed

to 100mmol/l drug were apoptotic. The dose–response

curves are in agreement with proliferation data.

In contrast, exogenous b-NGF (10 ng/ml) (Supplemental

Fig. 1, http://links.lww.com/ACD/A12) slightly increased cell

proliferation (+ 29% vs. controls at 48 h, P < 0.05%, data

not shown) and a trend toward increased cell counts

(+ 15% vs. controls at 48 h) was also observed, confirming

its mitogenic role in HTB114 cells. The limited effect

suggests that autocrine secretion is already sufficient to

sustain growth and survival in these cells. In agreement

with our previous data, no apoptosis was observed in cells

exposed to exogenous b-NGF for up to 7 days.

Amitriptyline upregulates p75NTR expression, converting

the TrKA + HTB114 cells into TrKA + /p75NTR + ,

and inducing cell death.

Our previous work has shown that, although expressing

both NGF receptors, HTB114 cells constitutively upre-

gulate TrKA while downregulating p75NTR [2–4]. FACS

analysis confirmed the high TrKA/p75NTR prosurvival ratio

(64/14% at 24 h) of proliferating control cells (Fig. 3a and

b), which was further increased as cells progressed along

the log-growth phase (Fig. 3c). In contrast, exposure to

50mmol/l amitriptyline upregulated p75NTR time depen-

dently (Fig. 3). The effect was already significant at 24 h,

versus controls (34 vs. 14%, P < 0.05, respectively), and

was further increased at 72 h, both versus 24 h (72 vs. 34%,

P < 0.05, respectively) and versus controls (72 vs. 9.3%,

P < 0.05, respectively). Thus, whereas proliferating cells

‘switched off ’ their p75NTR (9.9%, at 72 h), the cells

exposed to amitriptyline ‘switched it on’ to almost the

same levels of TrKA (72 vs. 82% at 72 h, respectively).

This ‘switch’ led to a marked reduction in the TrKA/

p75NTR prosurvival ratio at 72 h versus controls (1.1 vs.

7.8, P < 0.05, respectively, Fig. 3c).

Interestingly, FACS analysis also showed that p75NTR is

always coexpressed with TrKA (Fig. 4), in HTB114 cells,

and therefore the double positives (TrKA + /p75NTR + )

Fig. 1

∗

∗

∗∗

∗

∗

∗

0

50 000

100 000

150 000

200 000

0 h 24 h 48 h 72 h

cell

coun

ts (c

ells

/ml)

CONTRAMI 50NGF

∗∗

0.010.020.030.040.050.060.070.080.090.0

100.0

0 50 500 1000

prol

ifera

tive

inde

x (%

of c

ontr

ols)

Amitriptyline (μmol/l)

48 h72 h

0.010.020.030.040.050.060.070.080.090.0

100.0

1 10 100 1000

Via

bilit

y (%

of c

ontr

ols)

Amitriptyline (μmol/l)

24 h

48 h

72 h

∗

∗

IC50

0

(a)

(b)

(c)

Amitriptyline reduces HTB114 leiomyosarcoma cell viability and proliferation. Cell viability and proliferation were analyzed in parallel by vital counts(a, b) and the CFSE proliferation assay (c). Cells were seeded in six-multiwell plates, at a density of 100 000 cells/well. After 24 h culture, serialconcentrations of amitriptyline, or its control vehicle DMSO, were added to the culture medium and cells were further incubated for 24, 48, or 72 h.Cells were then harvested and analyzed as described in the Materials and methods section. Cells treated with b-NGF (10 ng/ml) were used aspositive controls. Data in (b) and (c) have been normalized to controls (n = 3). *Significantly different from controls (P < 0.05). **Significantly differentfrom NGF (P < 0.05). AMI 50, 50 mmol/l amitriptyline; CONTR, control cells; CFSE, carboxyfluorescein succinimidyl ester; DMSO, dimethylsulfoxide;IC50, drug concentration reducing cell viability by 50%; NGF, nerve growth factor.

4 Anti-Cancer Drugs 2013, Vol 00 No 00

represent a subpopulation of TrKA + cells. This led to the

identification of three different subpopulations (or

phenotypes), on the basis of the expression of different

combinations of NGF receptors (Fig. 5), namely: (a) the

double positives (TrKA + /p75NTR + ); (b) the TrKA + -

single positives; and (c) the double negatives (TrKA – /

p75NTR – ). The distribution of the three phenotypes in

the 24 h-control population was B14/50/35%, respec-

tively. However, these figures changed significantly in

cells exposed to amitriptyline and b-NGF, as shown

in Table 1 and Fig. 5.

The high-TrKA/p75NTR ratio of proliferating control cells,

reflecting the high proportion of TrKA + -single positives

(Fig. 5), suggests that unopposed TrKA signaling can

boost cell growth. In fact, proliferating control cells

progressively upregulated their overall TrKA (71%, at

72 h) while further suppressing their already low p75NTR

(9.3%, at 72 h). Therefore, the TrKA + -single positives

increased accordingly (61.7 vs. 50%, P < 0.05, at 72 vs.

24 h, respectively), at the expense of the other two

subpopulations, namely, the double positives (9 vs. 14%,

at 72 vs. 24 h, respectively) and the double negatives (29

vs. 36%, P < 0.05, at 72 vs. 24 h, respectively). In contrast,

amitriptyline reduced the TrKA/p75NTR ratio to B1 : 1

(1.1) at 72 h, meaning that almost all TrKA + cells

coexpressed p75NTR (Fig. 5). This phenotype switch

occurred at the expense of the TrKA + -single positives

(the proliferating cells), which decreased to 10% at 72 h,

from 61.7% of controls (P < 0.05). In other words,

amitriptyline converted the HTB114 cells from prolifer-

ating TrKA + -single positives into ‘agonizing’ double

positives (72 vs. 9.3%, P < 0.05, amitriptyline-treated

vs. control cells double positives at 72 h, respectively),

suggesting that coexpression of p75NTR interferes with

TrKA prosurvival signaling, inducing cell death.

As expected, exogenous b-NGF upregulated both the

overall TrKA expression (81.7%, P < 0.05, at 24 h,

Supplemental Fig. 1, http://links.lww.com/ACD/A12) and

the proportion of TrKA + -single positives, thereby con-

firming its mitogenic role. Because of the rapid extra-

cellular degradation of b-NGF, the effect was transient

and peaked at 24 h.

Coexpression of p75NTR in TrKA + -HTB114 cells, induced

by amitriptyline, downregulated AKT and activated

caspase-3

To elucidate the mechanism of action of amitriptyline, we

next analyzed the downstream effectors of TrKA and

p75NTR receptors by FACS analysis (Fig. 6). As already

mentioned, in HTB114 cells, TrKA-dependent survival

involves AKT, whereas p75NTR-dependent apoptosis is

associated with the activation of caspase-3 [3,4]. We

found that, in the proliferating control cell population,

composed of about 50% of TrKA + -single positives, AKT

was constitutively ‘on’ (38%), whereas caspase-3 was

almost suppressed (4%). As expected, the coexpression of

p75NTR, induced by amitriptyline, dose dependently

‘switched on’ caspase-3 (25% at 500 mmol/l) while

‘switching off ’ AKT (15% at 500 mmol/l). These effects

started at 24 h and reflected the upregulation of p75NTR,

supporting the hypothesis that coexpression of this

receptor negatively regulates TrKA signaling, switching

off its survival pathways. These data confirm our previous

observations of caspase-3-dependent apoptosis induced

by upregulation of p75NTR in HTB114 cells.

As expected, exogenous b-NGF (10 ng/ml) significantly

upregulated AKT at 1 h and up to 24 h (Supplemental

Figs 1 and 2, http://links.lww.com/ACD/A12, http://links.lww.com/ACD/A13), reflecting TrKA expression and further

confirming its prosurvival role in these cells. The effect

was transient because of the rapid degradation of NGF

in the culture medium (data not shown).

DiscussionOur study shows considerable in-vitro activity of the TCA

amitriptyline in the HTB114 uterine leiomyosarcoma cell

line, confirming the antineoplastic potential of this drug.

This is the first report of amitriptyline cytotoxicity to

human uterine leiomyosarcoma, which is an aggressive

cancer, and highly resistant to nonsurgical therapies.

In our in-vitro model, amitriptyline (50 mmol/l) markedly

suppressed cell viability and proliferation, and induced

apoptosis. Cytotoxicity was dose and time dependent.

The concentrations used in this report are in the range

Fig. 2

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

10 100 1000

Apo

ptos

is (%

)

Amitriptyline (μmol/l)

24 h48 h72 h96 h7 days

0

Amitriptyline induces time-dependent and dose-dependent apoptosisin HTB114 leiomyosarcoma cells. Apoptosis was determined by theAnnexin V assay, as described in the Materials and methods section.Cells were seeded in six-multiwell plates, at a density of 100 000 cells/well. After 24 h, serial concentrations of amitriptyline (50, 100, 500, and1000mmol/l), or its vehicle DMSO, were added to the culture mediumand cells were further incubated for 24, 48, 72, 96 h, and up to 7 days.Cells were then harvested, labeled with FITC-conjugated Annexin V,and analyzed by FACS scan (n = 3). DMSO, dimethylsulfoxide;FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate.

Antineoplastic activity of amitriptyline Pula et al. 5

reported in in-vitro studies of cancer cells [31,39–41,43–46].

The dose–response curves seem to indicate a more

prominent effect on cell viability (i.e. lower IC50 and a

shorter latency), versus cell proliferation and apoptosis.

These discrepancies could have two possible explanations.

First of all, they may reflect the mechanism of the CSFE

assay, which measures the proliferative index of viable cells,

whereas viability refers to the overall cell population. As only

a small fraction of cells survived amitriptyline cytotoxicity at

48 and 72 h, we would expect the proliferative rates to be

lower, and more similar to cell viability data, if measured in

the entire cell population. In addition, the dose–response

curves may reflect a dual antineoplastic mechanism – both

cytotoxic and antiproliferative – in agreement with the

multiple effects described for amitriptyline, including both

mitochondrial apoptosis [39] and nonmitochondrial cell

cycle arrest [41].

The second and more relevant novelty of this study is the

cytotoxic mechanism used by amitriptyline in these cells,

which appeared to be associated with the upregulation of

p75NTR, leading to p75NTR-dependent apoptosis. In fact, we

found that most proliferating HTB114 leiomyosarcoma cells

only express TrKA, which is also upregulated, suggesting that

unopposed TrKA signaling promotes cancer growth and

survival. However, following exposure to amitriptyline, all

TrKA+ cells coexpressed p75NTR and unopposed TrKA

signaling virtually disappeared. Coexpression of both NGF

Fig. 3

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

2 00 000

0AMI 50

1 50 000

1 00 000

50 000

024 48 72

24 h 48 h 72 h

24 h 48 h 72 h

TrK

A-p

ositi

ve c

ells

(%)

p75N

TR-p

ositi

ve c

ells

(%)

CONTR AMI 50

3438

24 h 48 h 72 h

∗

∗∗

∗

∗

Cel

l cou

nts

0

1

2

3

4

5

6

7

8

9

10

TrKA/p75NTR

Pro survival

TrK

A/p

75N

TR ra

tio

∗ ∗∗

∗∗

CONTR AMI 50

CONTR AMI 50

(a) (b)

(c)

Amitriptyline upregulates p75NTR in HTB114 leiomyosarcoma cells, shifting the TrKA/p75NTR receptor balance from prosurvival to proapoptotic.NGF-receptor expression was analyzed by FACS scan, as described in the Materials and methods section. (a) TrKA. (b) p75NTR, inset, cell counts.(c) TrKA/p75NTR receptor balance. Cells were seeded in six-multiwell plates, at a density of 100 000 cells/well. After 24 h, 50mmol/l amitriptyline,or its control vehicle DMSO, was added to the culture medium, and cells were further incubated for 24, 48, or 72 h. Cells were then harvested,labeled with either the PE-conjugated anti-human TrKA antibody (a) or the FITC-conjugated anti-human p75NTR antibody (b), and analyzed by FACSscan. The p75NTR/TrKA expression ratio, reflecting the prosurvival balance, was calculated using data from (a) and (b); (c) n = 3. *Significantlydifferent from controls (P < 0.05). **Significantly different from 24 h (P < 0.05). AMI 50, 50 mmol/l amitriptyline, CONTR, control cells (n = 3).DMSO, dimethylsulfoxide; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; NGF, nerve growth factor; NTR, neurotrophinreceptor; PE, phycoerythrin; TrKA, tropomyosin receptor kinase A.

6 Anti-Cancer Drugs 2013, Vol 00 No 00

receptors on the same cell was paralleled by downregulation

of AKT and activation of caspase-3, suggesting that p75NTR

suppressed TrKA prosurvival pathways (AKT), thereby

triggering caspase-3-dependent apoptosis. In other words,

in our model, coexpression of p75NTR appeared to convey

a death signal that antagonized and prevailed over the

Fig. 4

TrKA+

21%

55%

21%

55%

19%

21%

49% 25%

20%TrKA

FL1H FL1H

FL2H

FL2H

FL2H

DN

TrKA+/p75NTR+

p75NTR+

p75NTR

C AMI 50104

103

102

101

100

100100

100101 102 103 104

FL1H100

100

101

101

102

102

103

103

104

104

10%

55% 30%

FL2H

FL1H100

100

101

101

102

102

103

103

104

104

40%

51% 7%

FL2H

FL1H100

100

101

101

102

102

103

103

104

104

17%

30% 51% 72 h

48 h

24 h

FL2H

FL1H100

100

101

101

102

102

103

103

104

104

104

103

102

101

101 102 103 104

Amitriptyline alters the NGF-receptor phenotype of HTB114 cells. FACS analysis of TrKA and p75NTR receptor expression (from one of theexperiments described in Fig. 3), showing the differential distribution of the three subpopulations – that is, TrKA single positives, TrKA/p75NTR doublepositives, and double negatives – in amitriptyline-treated and control HTB114 cells. TrKA and p75NTR signals were recorded in the FL2-H and FL1-Hchannels, respectively. The horizontal cut-off line separates the TrKA-negative cells (lower quadrants) from the TrKA-positive cells (upper quadrants).The vertical cut-off line separates the p75NTR-negative cells (left quadrants) from the p75NTR-positive cells (right quadrants). The upper left quadrantrepresents the TrKA single positive cells, the upper right quadrant represents the TrKA/p75NTR double positive cells, the lower left quadrantrepresents the double negative cells, and the lower right quadrant represents the p75NTR-single positive cells. Data are representative of a singleexperiment. AMI 50, 50mmol/l amitriptyline; C, control cells; DN, double negatives; FACS, fluorescence-activated cell sorting; NGF, nerve growthfactor; NTR, neurotrophin receptor; p75NTR + , p75NTR-positive cells; TrKA, tropomyosin receptor kinase A; TrKA + , TrKA-single positive cells;TrKA + /p75NTR + , double positive cells.

Antineoplastic activity of amitriptyline Pula et al. 7

prosurvival pathways of NGF/TrKA, providing further insight

into NGF receptor crosstalk. Although we have already

described inducible p75NTR-dependent apoptosis in

HTB114 uterine leiomyosarcoma [2–4], this mechanism

has never been proposed for amitriptyline.

In support of our hypothesis, we also found that

coadministration of noncytotoxic concentrations of ami-

triptyline and exogenous b-NGF suppressed the mito-

genic effects of the latter in HTB114 cells. This

antiproliferative effect was again associated with down-

regulation of AKT and upregulation of both p75NTR

expression and apoptosis (Supplemental Fig. 2, http://links.lww.com/ACD/A13), suggesting functional antagonism.

Although this scenario is different from that described in

PC12 cells [47], distinct intracellular pathways may be

involved.

Overall, our data suggest that p75NTR-dependent apop-

tosis may represent a novel cytotoxic mechanism of

amitriptyline in HTB114 cells and possibly in human

uterine leiomyosarcomas in general (and other cancers

alike, Supplemental Fig. 3, http://links.lww.com/ACD/A14).

As p75NTR-dependent apoptosis is also activated by

Fig. 5

TrKA p75NTR

Survival and growth

p75NTR−

dependentapoptosis

24 h48 h

72 h

0

10

20

30

40

50

60

70

80

90

100

C AMI 50 C AMI 50 C AMI 50

TrKA+ TrKA+/p75NTR+ TrkA−/p75NTR−

Pos

itive

cel

ls (%

)

24 h

48 h

72 h

∗

∗

∗

∗ ∗

∗

∗

∗∗

∗∗ ∗∗

Amitriptyline converts HTB114 cells from proliferating TrKA + -singlepositives into apoptotic TrKA + /p75NTR + -double positives.NGF-receptor expression in amitriptyline-treated and control cells.Data from Table 1 have been plotted to show the effects of amitriptylineon TrKA + cells. *Significantly different from controls (P < 0.05).**Significantly different from 24 h (P < 0.05). AMI 50, 50mmol/lamitriptyline; C, control cells (n = 3); NGF, nerve growth factor;NTR, neurotrophin receptor; TrKA, tropomyosin receptor kinase A.

Table 1 Expression of NGF receptors in HTB114 cells exposed to 50 lmol/l amitriptyline

TrKA + TrKA + /p75NTR + TrKA-/p75NTR –

C AMI 50 C AMI 50 C AMI 50

24 h 50.0 (±2.9) 17.5 (±3.5)* 14.2 (±1.7) 34.0 (±1.4)* 35.7 (±2.9)** 48.5 (±2.1)*48 h 44.3 (±3.2) 33.0 (±2.8)* 17.3 (±0.6) 38.0 (±1.4)* 38.3 (±2.9)** 29.0 (±1.4)*72 h 61.7 (±1.5)** 10.0 (±7.1)*, ** 9.3 (±1.15)** 72.0 (±9.9)* 29.0 (±1.7)** 18.0 (±2.8)*, **

Expression of NGF receptors in control and amitriptyline-treated HTB114 human uterine leiomyosarcoma cells. Cells were seeded into six-multiwell plates, at a density of100 000 cells/well. After 24 h culture, 50 mmol/l amitriptyline, or its control vehicle DMSO, was added to the culture medium. Cells were further incubated for theindicated times and analyzed by FACS scan, as described in the Materials and methods section.Data are expressed as percentage of positive cells in the overall population±SD.AMI 50, amitriptyline 50 mmol/l; C, controls (n = 3); DMSO, dimethylsulfoxide; FACS, fluorescence-activated cell sorting; NGF, nerve growth factor; NTR, neurotrophinreceptor; TrKA, tropomyosin receptor kinase A.*P < 0.05 versus controls.**P < 0.05 versus 24 h.

Fig. 6

0

10

20

30

40

50

60

70

80

90

100

0 0.5 50 500 1000

Pos

itive

cel

ls (%

)

Amitriptyline (μmol/l)

24 h

AKTCASPASE-3

∗ ∗∗∗

∗ ∗

The coexpression of p75NTR and TrKA downregulates AKT andactivates caspase-3, inducing death in HTB114 cells. The expressionof AKT and active caspase-3 was analyzed by FACS scan, as describedin the Materials and methods section. Cells were seeded in six-multiwellplates, at a density of 100 000 cells/well. After 24 h, 50 mmol/lamitriptyline, or its control vehicle DMSO, was added to the culturemedium and cells were further cultured for 24 h. Cells were thenharvested, fixed-permeabilized, incubated with either anti-AKT oranti-active caspase-3 PE-conjugated antibodies, and analyzed byFACS scan (n = 3). *Significantly different from controls (P < 0.05).DMSO, dimethylsulfoxide; FACS, fluorescence-activated cell sorting;NTR, neurotrophin receptor; PE, phycoerythrin; TrKA, tropomyosinreceptor kinase A.

8 Anti-Cancer Drugs 2013, Vol 00 No 00

different cytotoxic agents (Trk inhibitors) [3,4],

we propose that the activation of this inducible death

receptor (activated by cellular stressors) may represent

a potential therapeutic strategy in susceptible cancers

(Fig. 7).

As already mentioned, the role of p75NTR is still

enigmatic, as it can modulate divergent signaling path-

ways by forming complexes with different coreceptors to

induce specific responses [11,12]. Depending on the cell

type, p75NTR can act synergistically, antagonistically, or

independently from TrKs. In the presence of mature

NTs, it can function as a coreceptor for the TrKs,

increasing their selectivity and prosurvival signaling. In

central neurons, it can form trimeric complexes with the

Nogo receptor-1 (NgR1) and the leucine-rich repeat and

Ig domain containing 1 (Lingo-1) receptor, which can

suppress neurite outgrowth through the Ras homolog

gene family member A (RhoA) [15]. Apoptosis can be

induced in different ways, including forming complexes

with sortilin, which are activated by pro-NTs [15], or

acting as a dependence receptor activated by the absence

of ligand (e.g. apoptosis by NTs withdrawal), or binding

non-NT ligands produced by pathogenic processes (e.g.

prion peptides, viruses, b-amyloid peptides). Thus,

p75NTR-dependent apoptosis can be triggered either by

its ligands (i.e. pro-NTs, mature NTs, or non-NT ligands)

or by their absence (e.g. NTs withdrawal) depending on

the extracellular signals and on the signaling apparatus

expressed by the cell.

In the nervous system, p75NTR-dependent apoptosis is

induced in response to damage. This receptor is ex-

pressed during neural development and ‘switched off ’ in

adult life, but it is re-expressed with neuronal injury and

death [23], suggesting the reactivation of a develop-

mental program by lesion-induced plasticity. Multiple

cellular stressors can induce re-expression of p75NTR in

neuronal and glial cells, which is sometimes associated

with the upregulation of pro-NTs [48]. In this setting,

the ‘toxic’ effects of pro-NTs seem to predominate over

the ‘trophic’ effect of mature NTs, despite the presence

of functional TrKs. These observations have led to the

hypothesis that p75NTR-dependent apoptosis could

represent a homeostatic mechanism to eliminate da-

maged cells – similar to Fas-dependent apoptosis

associated with inflammation – and possibly a ‘class

effect’ of death receptors [23].

Fig. 7

TrKA p75NTR

Autocrine NGF release

AmitriptylineTrK blockade

p75NTR gene transfer

TrKA

TRKA - dependentproliferation and survival

p75NTR - dependent apoptosisand cell death

AKT ‘on’caspase -3 ‘off’

Oncogenictransformation

Suppressionof cancer growth

Caspase-3 ‘on’

TrKA

NGF

HTB114 human uterine leiomyosarcoma

In HTB114 uterine leiomyosarcoma cells, p75NTR behaves as a stress/death receptor, which is inducible by cytotoxic drugs to trigger cell death.HTB114 cells are characterized by autocrine activation of the NGF/TrKA/AKT axis, leading to constitutive upregulation of TrKA and downregulationof p75NTR, which promotes unrestricted growth and survival. In these cells, the upregulation of p75NTR by different cytotoxic drugs, includingamitriptyline or TrK inhibitors [2,3], or by gene transfer [2], induces apoptosis and cell death. We therefore suggest that p75NTR acts as a stressreceptor, inducible by cellular injury, which can be pharmacologically upregulated to target the survival pathways of susceptible cancer cells.NGF, nerve growth factor; NTR, neurotrophin receptor; TrKA, tropomyosin receptor kinase A.

Antineoplastic activity of amitriptyline Pula et al. 9

We propose that this same ‘homeostatic’ mechanism of

p75NTR-dependent apoptosis could also be used to kill

cancer cells. In fact, re-expression of p75NTR can suppress

growth in tumors characterized by constitutive upregula-

tion of the TrKs and downregulation of p75NTR (e.g.

uterine leiomyosarcoma, prostatic adenocarcinoma, gas-

tric cancer, hepatocellular carcinoma, and bladder can-

cer [2–4,20,49–51]). Indeed, we found that PC3 prostatic

carcinoma was also sensitive to amitriptyline (Supple-

mental Fig. 3, http://links.lww.com/ACD/A14). As the same

mechanism of p75NTR-dependent apoptosis seems to be

inducible by both cellular injury and unrelated cytotoxic

agents, in neural and extraneural cells, it might represent

a conserved ‘final common pathway’ to trigger cell suicide

in dysfunctional cells.

Our data provide a rationale for investigating p75NTR-

dependent apoptosis in susceptible cancers. However, this

strategy represents both an opportunity and a challenge.

First of all, the molecular mechanisms leading to the

upregulation of p75NTR warrant further investigation. In

neurons, the hypo-osmotic stress can upregulate p75NTR

through Sp1 [52], a transcription factor expressed in

development and cancer, and regulating genes that control

differentiation, proliferation, and apoptosis [53]. Brain

injury has recently been shown to upregulate p75NTR

through the proinflammatory cytokines interleukin-1b and

tumor necrosis factor-alpha (TNF-alpha) [54]; the effect

is both cytokine and cell type specific. Cytokine secretion

has been documented in the activated myometrium,

where it seems to trigger delivery [55,56] (which is also

a cellular stress). In HTB114 cells, p75NTR can be

upregulated by both TrK blockade [3,4] and PI3K

inhibitors (our unpublished observations). Further, pro-

NGF can also upregulate p75NTR and induce apoptosis in

these cells (our unpublished observations). Secretion of

both pro-NGF and b-NGF has been documented in rat

myometrium and pro-NGF release increases at the end of

pregnancy (also a stressful condition), paralleled by the

decrease of b-NGF [57], thereby promoting delivery.

Thus, although the inhibition of TrK signaling and the

activation of caspase-3 can explain the p75NTR-dependent

apoptosis induced by amitriptyline in leiomyosarcoma

cells, we cannot rule out the possibility that other stress-

inducible factors, such as pro-NGF and/or cytokines,

might be released following exposure to the drug, further

contributing toward p75NTR-dependent apoptosis.

Second, as for any antineoplastic chemotherapy, concerns

might be raised about the potential side effects caused by

the pharmacological modulation of p75NTR in non-

neoplastic tissues. For example, the activation of the

NgR/Lingo-1/p75NTR receptor complex has been linked

to neurodegeneration. As already mentioned, this path-

way inhibits neurite outgrowth in the central nervous

system (CNS) and seems to be one of the reasons why

central neurons cannot regenerate following injury [58].

However, the role of p75NTR in neurodegenerative

diseases is still unclear. For example, in the C57BL/6

experimental autoimmune encephalomyelitis model – a

murine model for the human multiple sclerosis – p75NTR

knockout mice developed a more severe (or even lethal)

disease and concomitant CNS inflammation, compared

with wild-type mice [59]. Thus, despite the initial

enthusiasm for p75NTR, the studies targeting this path-

way in neurodegenerative diseases are currently focused

on Lingo and NgR [60–62]. In fact, while binding of

myelin inhibitory proteins to NgR1/Lingo-1 activates

RhoA, leading to inhibition of neurite outgrowth, binding

of NTs to p75NTR seems to suppress this effect [58,63],

suggesting that the functional role of this pathway may

depend on the extracellular ligands interacting with the

complex [64]. Moreover, functional redundancy has been

shown for p75NTR [65], which is generally absent in adult

neurons, where most NgR1/Lingo-1 complexes contain

its functional homologue TAJ/TROY – also a member of

the TNF receptor family [66]. Although we did not test

the effects of amitriptyline on TROY, this receptor does

not interact with the TrKs, whereas the upregulation of

p75NTR by cytotoxic drugs is apparently associated with

TrK inhibition ([4] and our unpublished observations).

Therefore, amitriptyline should not impact significantly

on this pathway in the adult CNS. In fact, neurodegen-

eration has never been reported in the postmarketing

experience with this drug.

Another potential safety concern, however, might be

associated with the risk of unwanted apoptosis in non-

neoplastic tissues. A potential pathway could be the

formation of p75NTR/sortilin complexes, which are

activated by pro-NTs. Sortilin-dependent apoptosis has

been studied in the nervous system. Our preliminary data

show that, despite a functional p75NTR/sortilin apparatus,

pro-NGF-dependent apoptosis is low in HTB114 cells,

especially compared with apoptosis associated with the

mere overexpression of p75NTR; furthermore, the two

pathways do not seem to be synergistic (our unpublished

observations). Although we did not test the effects of

amitriptyline on sortilin, we could find no evidence in the

literature of a potential modulation of sortilin by

antidepressants.

Although we have no direct evidence of amitriptyline (or

other antidepressants) cytotoxicity in non-neoplastic cells

through upregulation of p75NTR, unwanted apoptosis could

be a possibility, as for any systemic chemotherapy. The

animal studies investigating the antineoplastic effects of

antidepressants did not report specific organ toxicity;

however, in-vitro cytotoxicity to non-neoplastic fibroblasts

is described at the concentrations used in our report [67].

The interactions between antidepressants and NTs have

only been studied in the brain, where they have been

shown to affect NT levels. The only report investigating

the potential modulation of NT receptors [47] was limited

10 Anti-Cancer Drugs 2013, Vol 00 No 00

to mouse brain and found that amitriptyline binds neuronal

TrK-A/B, but does not bind p75NTR. The drugs were used

at noncytotoxic concentrations (i.e. 500mmol/l in vitro), and

apoptosis was not observed. Otherwise, there are no data on

a potential modulation of p75NTR by these drugs. However,

it is also possible that p75NTR-dependent apoptosis might

be a ‘homeostatic’ mechanism related to development or

cellular dysfunction, such as cancer. In fact, we have

preliminary evidence that, in certain human pathologies,

associated with cellular stress and severe inflammation,

p75NTR is upregulated but does not induce apoptosis (our

unpublished observations). This has also been shown in

oligodendrocytes, where p75NTR induces apoptosis under

specific conditions (i.e. after spinal cord injury), but does

not invariably lead to cell death, suggesting that the cellular

response to p75NTR may depend on the state of maturation

or activation of the cell [15]. In fact, although we did not

test non-neoplastic cells, we also found variable in-vitro

modulation of p75NTR expression and apoptosis by

amitriptyline in other cancer cell lines, suggesting that

the effects of the drug are dependent on the cell line (our

unpublished observations).

Conclusion

Our study shows considerable in-vitro antineoplastic

activity of the TCA amitriptyline in the HTB114 uterine

leiomyosarcoma cell line and provides novel evidence that

cytotoxicity is mediated by p75NTR-dependent apoptosis.

Our data suggest that p75NTR-dependent apoptosis may

be a novel strategy in cancer therapy in uterine

leiomyosarcoma, and possibly other cancers, characterized

by autocrine activation of TrKs. Further research in

xenograft models is warranted to confirm these findings

and to assess the safety profile of the drug for this

indication.

AcknowledgementsThis study was funded by Fondazione Cassa di Risparmio

di Terni (CARIT), Italy.

Conflicts of interest

There are no conflicts of interest.

References1 Nakagawara A. Trk receptor tyrosine kinases: a bridge between cancer and

neural development. Cancer Lett 2001; 169:107–114.2 Rende M, Brizi E, Conner J, Provenzano C, Sanna PP. NGF influences

differentiation and proliferation of myogenic cells in vitro via TrKA. Int J DevNeurosci 2000; 18:869–885.

3 Rende M, Pistilli A, Stabile AM, Terenzi A, Cattaneo A, Ugolini G, Sanna P.Role of NGF and its receptors in non-nervous cancer growth: efficacy of atyrosine kinase inhibitor (AG879) and neutralizing antibodies anti-TrKA andanti-NGF: an in vitro and in vivo study. Anticancer Drugs 2006; 17:929–941.

4 Stabile AM, Montagnoli C, Pistilli A, Rambotti MG, Pula G, Rende M.Antiproliferative and pro-apoptotic effects of the Trk-inhibitor GW441756 inhuman myosarcomas and prostatic carcinoma. Curr Signal Transduct Ther2013; 8:74–83.

5 Molloy NH, Read DE, Gorman AM. Nerve growth factor in cancer cell deathand survival. Cancers 2011; 3:510–530.

6 Thiele CJ, Li Z, McKee AE. On Trk – the TrkB signal transduction pathway isan increasingly important target in cancer biology. Clin Cancer Res 2009;15:5962–5967.

7 Gschwind A, Fischer OM, Ullrich A. The discovery of receptor tyrosinekinases: targets for cancer therapy. Nat Rev Cancer 2004; 4:361–370.

8 Wang T, Yu D, Lamb ML. Trk kinase inhibitors as new treatments for cancerand pain. Expert Opin Ther Pat 2009; 3:305–319.

9 Skaper SD. The biology of neurotrophins: signaling pathways, and functionalpeptide mimetics of neurotrophins and their receptors. CNS Neurol DisordDrug Targets 2008; 7:46–62.

10 Holgado-Madruga M, Moscatello DK, Emlet DR, Dieterich R, Wong AJ.Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activationand the promotion of cell survival by nerve growth factor. Proc Natl Acad SciUSA 1997; 94:12419–12424.

11 Barrett GL. The p75 neurotrophin receptor and neuronal apoptosis. ProgNeurobiol 2000; 61:205–229.

12 Arevalo JC, Wu SH. Neurotrophin signaling: many exciting surprises! CellMol Life Sci 2006; 63:1523–1537.

13 Kuci S, Kuci Z, Kreyenberg H, Deak E, Putsch K, Huenecke S, et al. CD271antigen defines a subset of multipotent stromal cells withimmunosuppressive and lympho-hematopoietic engraftment-promotingproperties. Haematologica 2010; 95:651–659.

14 Boiko AD, Razorenova OV, van de Rijn M, Swetter SM, Johnson DL, Ly DP,et al. Human melanoma-initiating cells express neural crest nerve growthfactor receptor CD271. Nature 2010; 466:133–137.

15 Cragnolini A, Friedman WJ. The function of p75NTR in glia. Trends Neurosci2008; 31:99–104.

16 Segal RA, Greenberg ME. Intracellular signaling pathways activated byneurotrophic factors. Annu Rev Neurosci 1996; 19:463–489.

17 Peter ME, Budd RC, Desbarats J, Hedrick SM, Hueber AO, Newell MK,et al. The CD95 receptor: apoptosis revisited. Cell 2007; 129:447–450.

18 Lagadec C, Meignan S, Adriaenssens E, Foveau B, Vanhecke E, Romon R,et al. TrkA overexpression enhances growth and metastasis of breastcancer cells. Oncogene 2009; 28:1960–1970.

19 Lee J, Jiffar T, Kupferman ME. A novel role for BDNF-TrkB in the regulation ofchemotherapy resistance in head and neck squamous cell carcinoma. PLoSOne 2012; 7:e30246.

20 Papatsoris AG, Liolitsa D, Deliveliotis C. Manipulation of the nerve growthfactor network in prostate cancer. Expert Opin Investig Drugs 2007;44:303–309.

21 Werrbach-Perez K, Perez-Polo JR. De novo synthesis of NGF subunits inS-180 mouse sarcoma cell line. Neurochem Res 1987; 12:875–883.

22 Makino K, Kawamura K, Sato W, Kawamura N, Fujimoto T, Terada Y.Inhibition of uterine sarcoma cell growth through suppression ofendogenous tyrosine kinase B signaling. PLoS One 2012; 7:e41049.

23 Ibanez C, Simi A. p75 neurotrophin receptor signaling in nervous systeminjury and degeneration: paradox and opportunity. Trends Neurosci 2012;35:431–440.

24 Bhakar AL, Roux PP, Lachance C, Kryl D, Zeindler C, Barkeri PA. The p75neurotrophin receptor (p75NTR) alters tumor necrosis factor-mediated NF-kB activity under physiological conditions, but direct p75NTR-mediated NF-kB activation requires cell stress. J Biol Chem 1999; 274:21443–21449.

25 Tamim HM, Mahmud S, Hanley JA, Boivin JF, Stang MR, Collet JP.Antidepressants and risk of prostate cancer: a nested case–control study.Prostate Cancer Prostatic Dis 2007; 11:53–60.

26 Toh S, Garcia Rodriguez L, Hernandez-Dıaz S. Use of antidepressants andrisk of lung cancer. CCC 2007; 18:1055–1064.

27 Xu W, Tamim H, Shapiro S, Stang MR, Collet JP. Use of antidepressants andrisk of colorectal cancer: a nested case–control study. Lancet Oncol 2006;7:301–308.

28 Fulton-Kehoe D, Rossing MA, Rutter C, Mandelson MT, Weiss NS. Use ofantidepressant medications in relation to the incidence of breast cancer.Br J Cancer 2006; 94:1071–1078.

29 Walker AJ, Card T, Bates TE, Muir K. Tricyclic antidepressants and theincidence of certain cancers: a study using the GPRD. Br J Cancer 2011;104:193–197.

30 Chubak J, Boudreau DM, Rulyak SJ, Mandelson MT. Colorectal cancer riskin relation to antidepressant medication use. Int J Cancer 2011; 128:227–232.

31 Arimochi H, Morita K. Characterization of cytotoxic actions of tricyclicantidepressants on human HT29 colon carcinoma cells. Eur J Pharmacol2006; 541:17–23.

32 Xia Z, Bergstrand A, DePierre JW, Nassberger L. The antidepressantsimipramine, clomipramine, and citalopram induce apoptosis in human acutemyeloid leukemia HL-60 cells via caspase-3 activation. J Biochem MolToxicol 1999; 13:338–347.

Antineoplastic activity of amitriptyline Pula et al. 11

33 Daley E, Wilkie D, Loesch A, Hargreaves IP, Kendall DA, Pilkington GJ, et al.Chlorimipramine: a novel anticancer agent with a mitochondrial target.Biochem Biophys Res Commun 2005; 328:623–632.

34 Levkovitz Y, Gil-Ad I, Zeldich E, Dayag M, Weizman A. Differential inductionof apoptosis by antidepressants in glioma and neuroblastoma cell lines:evidence for p-c-Jun, cytochrome c, and caspase-3 involvement. J MolNeurosci 2005; 27:29–42.

35 Tsuruo T, Iida H, Nojiri M, Tsukagoshi S, Sakurai Y. Potentiation ofchemotherapeutic effect of vincristine in vincristine resistant tumor bearing miceby calmodulin inhibitor clomipramine. J Pharmacobiodyn 1983; 6:145–147.

36 Merry S, Hamilton TG, Flanigan P, Freshney RI, Kaye SB. Circumventionof pleiotropic drug resistance in subcutaneous tumors in vivo with verapamiland clomipramine. Eur J Cancer 1991; 27:31–34.

37 Pommerenke EW, Volm M. Reversal of doxorubicin-resistance in solidtumors by clomipramine. In Vivo 1995; 9:99–101.

38 Beaney RP, Gullan RW, Pilkington GJ. Therapeutic potential ofantidepressants in malignant glioma: clinical experience with clomipramine[abstract]. J Clin Oncol 2005; 23:1535.

39 Cordero MD, Sanchez-Alcazar JA, Bautista-Ferrufino MR, Carmona-Lopez MI,Illane M, Rıos MJ, et al. Acute oxidant damage promoted on cancer cells byamitriptyline in comparison with some common chemotherapeutic drugs.Anticancer Drugs 2010; 21:932–944.

40 Higgins SC, Pilkington GJ. The in vitro effects of tricyclic drugs anddexamethasone on cellular respiration of malignant glioma. Anticancer Res2010; 30:391–397.

41 Mao X, Hou T, Cao B, Wang W, Li Z, Chen S, et al. The tricyclicantidepressant amitriptyline inhibits D-cyclin transactivation and inducesmyeloma cell apoptosis by inhibiting histone deacetylases: in vitro and insilico evidence. Mol Pharmacol 2011; 79:672–680.

42 Jeon SH, Kim SH, Kim Y, Kim YS, Lim Y, Lee YH, et al. The tricyclicantidepressant imipramine induces autophagic cell death in U-87MGglioma cells. Biochem Biophys Res Commun 2011; 413:311–317.

43 Varga A, Nugel H, Baehr R, Marx U, Hever A, Nacsa J, et al. Reversal of multidrugresistance by amitriptyline in vitro. Anticancer Res 1996; 16:209–211.

44 Pilkington GJ, Parker K, Murray SA. Approaches to mitochondrially mediatedcancer therapy. Semin Cancer Biol 2008; 18:226–235.

45 Pilkington GJ, Akinwunmi J, Amar S. The role of tricyclic drugs in selectivetriggering of mitochondrially-mediated apoptosis in neoplastic glia: atherapeutic option in malignant glioma? Radiol Oncol 2006; 40:73–85.

46 Parker KA, Glaysher S, Hurren J, Knight LA, McCormick D, Suovouri A, et al.The effect of tricyclic antidepressants on cutaneous melanoma cell lines andprimary cell cultures. Anticancer Drugs 2012; 23:65–69.

47 Jang SW, Liu X, Chan CB, Weinshenker D, Hall RA, Xiao G, et al.Amitriptyline is a TrkA and TrkB receptor agonist that promotes TrkA/TrkBheterodimerization and has potent neurotrophic activity. Chem Biol 2009;6:644–656.

48 Lebrun-Julien F, Bertrand MJ, De Backer O, Stellwagen D, Morales CR,Di Polo A, et al. ProNGF induces TNFa-dependent death of retinal ganglioncells through a p75NTR non-cell-autonomous signaling pathway. Proc NatlAcad Sci USA 2010; 107:3817–3822.

49 Jin H, Pan Y, Zhao L, Zhai H, Li X, Sun L, et al. P75 neurotrophin receptorsuppresses the proliferation of human gastric cancer cells. Neoplasia 2007;9:471–478.

50 Yuanlong H, Haifeng J, Xiaoyin Z, Jialin S, Jie L, Li Y, et al. The inhibitoryeffect of p75 neurotrophin receptor on growth of human hepatocellularcarcinoma cells. Cancer Lett 2008; 268:110–119.

51 Khwaja F, Djakiew D. Inhibition of cell-cycle effectors of proliferation inbladder tumor epithelial cells by the p75NTR tumor suppressor. MolCarcinog 2003; 36:153–160.

52 Ramos A, Ho WC, Forte S, Dickson K, Boutilier J, Favell K, et al. Hypo-osmolar stress induces p75NTR expression by activating Sp1-dependenttranscription. J Neurosci 2007; 27:1498–1506.

53 Deniaud E, Baguet J, Chalard R, Blanquier B, Brinza L, Meunier J, et al.Overexpression of transcription factor Sp1 leads to gene expressionperturbations and cell cycle inhibition. PLoS One 2009; 4:e7035.

54 Choi S, Friedman WJ. Inflammatory cytokines IL-1b and TNF-a regulatep75NTR expression in CNS neurons and astrocytes by distinct cell-type-specific signaling mechanisms. ASN Neuro 2009; 1:e00010.

55 Mittal P, Romero R, Tarca AL, Gonzalez J, Draghici S, Xu Y, et al.Characterization of the myometrial transcriptome and biological pathwaysof spontaneous human labor at term. J Perinat Med 2010; 38:617–643.

56 Shynlova O, Lee YH, Srikhajon K, Lye S. Physiologic uterine inflammationand labor onset: integration of endocrine and mechanical signals. ReprodSci 2013; 2:154–167.

57 Lobos E, Gebhardt C, Kluge A, Spanel-Borowski K. Expression of nervegrowth factor (NGF) isoforms in the rat uterus during pregnancy:accumulation of precursor proNGF. Endocrinology 2005; 146:1922–1929.

58 Mathew SJ, Haubert D, Kronke M, Leptin M. Looking beyond death: amorphogenetic role for the TNF signalling pathway. J Cell Sci 2009;122:1939–1946.

59 Kust B, Mantingh-Otter I, Boddeke E, Copray S. Deficient p75 low-affinityneurotrophin receptor expression does alter the composition of cellularinfiltrate in experimental autoimmune encephalomyelitis in C57BL/6 mice.J Neuroimmunol 2006; 174:92–100.

60 Jepson S, Vought B, Gross CH, Gan L, Austen D, Frantz JD, et al. LINGO-1,a transmembrane signaling protein, inhibits oligodendrocyte differentiationand myelination through intercellular self-interactions. J Biol Chem 2012;287:22184–22195.

61 Mi S, Sandrock A, Miller RH. LINGO-1 and its role in CNS repair. Int JBiochem Cell Biol 2008; 40:1971–1978.

62 McDonald CL, Bandtlow C, Reindl M. Targeting the Nogo receptor complexin diseases of the central nervous system. Curr Med Chem 2011; 18:234–244.

63 Barker PA. P75NTR is positively promiscuous: novel partners and newinsights. Neuron 2004; 42:529–533.

64 Blaise S, Kneib M, Rousseau A, Gambino F, Chenard MP, Messadeq N,et al. In vivo evidence that TRAF4 is required for central nervous systemmyelin homeostasis. PLoS One 2012; 7:e30917.

65 Mandemakers WJ, Barres BA. Axon regeneration: it’s getting crowded at thegates of TROY. Curr Biol 2005; 15:R302–R305.

66 Shao Z, Browning JL, Lee X, Scott ML, Shulga-Morskaya S, Allaire N, et al.TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor1 and regulates axonal regeneration. Neuron 2005; 45:353–359.

67 Moreno-Fernandez AM, Cordero MD, De Miguel M, Delgado-Rufino MD,Sanchez-Alcazar JA, Navas P. Cytotoxic effects of amitriptyline in humanfibroblasts. Toxicology 2008; 243:51–58.

12 Anti-Cancer Drugs 2013, Vol 00 No 00