Pathogenesis of Hand-Foot Syndrome induced by PEG-modified liposomal Doxorubicin

Abstract

PEGL-DOX is an excellent treatment for recurrent ovarian cancer that rarely causes side-effects like cardiotoxicity or hair loss, but frequently results in Hand-Foot Syndrome (HFS). In severe cases, it can become necessary to reduce the PEGL-DOX concentration or the duration of the drug therapy, sometimes making it difficult to continue treatment. In this study, we prepared an animal model to compare the effects of DOX versus PEGL-DOX, and we noticed that only treatment with PEGL-DOX resulted in HFS, which led us to conclude that extravasation due to long-term circulation was one of the causes of HFS. In addition, we were able to show that the primary factor leading to the skin-specific outbreaks in the extremities was the appearance of reactive oxygen species (ROS) due to interactions between DOX and the metallic Cu(II) ions abundant in skin tissue. ROS directly disturb the surrounding tissue and simultaneously induce keratinocyte-specific apoptosis. Keratinocytes express the thermoreceptor TRPM2, which is thought to be able to detect ROS and stimulate the release of chemokines (IL-8, GRO, Fractalkine), which induce directed chemotaxis in neutrophils and other blood cells. Those cells and the keratinocytes then undergo apoptosis and simultaneously release IL-1β, IL-1α, and IL-6, which brings about an inflammatory state. In the future, we plan to develop preventative as well as therapeutic treatments by trapping the ROS.

Full text

RESEARCH ARTICLE
Pathogenesis of Hand-Foot Syndrome induced by PEG-modified
liposomal Doxorubicin
Noriyuki Yokomichi •Teruaki Nagasawa •Ariella Coler-Reilly •
Hiroyuki Suzuki •Yoshiki Kubota •Ryosuke Yoshioka •
Akiko Tozawa •Nao Suzuki •Yoko Yamaguchi
Received: 5 November 2012 / Accepted: 11 December 2012 / Published online: 6 February 2013
ÓThe Author(s) 2013. This article is published with open access at Springerlink.com
Abstract PEGL-DOX is an excellent treatment for
recurrent ovarian cancer that rarely causes side-effects like
cardiotoxicity or hair loss, but frequently results in Hand-
Foot Syndrome (HFS). In severe cases, it can become
necessary to reduce the PEGL-DOX concentration or the
duration of the drug therapy, sometimes making it difficult
to continue treatment. In this study, we prepared an animal
model to compare the effects of DOX versus PEGL-DOX,
and we noticed that only treatment with PEGL-DOX
resulted in HFS, which led us to conclude that extravasa-
tion due to long-term circulation was one of the causes of
HFS. In addition, we were able to show that the primary
factor leading to the skin-specific outbreaks in the
extremities was the appearance of reactive oxygen species
(ROS) due to interactions between DOX and the metallic
Cu(II) ions abundant in skin tissue. ROS directly disturb
the surrounding tissue and simultaneously induce kerati-
nocyte-specific apoptosis. Keratinocytes express the ther-
moreceptor TRPM2, which is thought to be able to detect
ROS and stimulate the release of chemokines (IL-8, GRO,
Fractalkine), which induce directed chemotaxis in neutro-
phils and other blood cells. Those cells and the keratino-
cytes then undergo apoptosis and simultaneously release
IL-1b, IL-1a, and IL-6, which brings about an inflamma-
tory state. In the future, we plan to develop preventative as
well as therapeutic treatments by trapping the ROS.
Keywords Hand-Foot Syndrome (HFS) Palmar-plantar
erythrodysesthesia (PPE) Pegylated liposomal
doxorubicin Drug delivery system (DDS) Reactive
oxygen species (ROS)
Introduction
Standard cancer treatments currently include surgery,
radiation therapy, and chemotherapies such as anticancer
drugs; however, there are advantages and disadvantages to
each treatment. The specific course of treatment is chosen
based on the cancer’s rate of progression, but in most cases,
the standard of care is to choose a chemotherapeutic anti-
cancer drug. Hair loss, pancytopenia, and nausea/vomiting
are the side-effects most typically seen with anticancer
drug treatment, but there have also been serious adverse
reactions in the skin. These reactions cause intense pain in
the hands and feet, and the phenomenon is known as Hand-
Foot Syndrome (HFS) or Palmar-Plantar Erythrodysesthe-
sia (PPE) [1]. In cases where HFS is severe, patients
experience difficulty walking and lose the ability to hold
objects in the hands, everyday life becomes significantly
impaired, and it may become difficult to continue the
cancer treatment.
Anticancer drugs that have been found to frequently
cause outbreaks of HFS include: fluoropyrimidines like
capecitabine, which is used to treat colorectal cancer and
N. Yokomichi (&)A. Tozawa N. Suzuki
Department of Obstetrics and Gynecology, St. Marianna
University School of Medicine, 2-16-1 Sugao, Miyamae,
Kawasaki, Kanagawa 216-8511, Japan
e-mail: n.yokomichi@gmail.com
n.yokomichi@marianna-u.ac.jp
T. Nagasawa H. Suzuki Y. Kubota R. Yoshioka 
Y. Yamaguchi
NANOEGGÒResearch Laboratories Inc, 2-16-1 Sugao,
Miyamae, Kawasaki, Kanagawa 216-8512, Japan
A. Coler-Reilly Y. Yamaguchi
Institute of Medical Science, St. Marianna University
School of Medicine, 2-16-1 Sugao, Miyamae,
Kawasaki, Kanagawa 216-8512, Japan
123
Human Cell (2013) 26:8–18
DOI 10.1007/s13577-012-0057-0

breast cancer; anthracycline, which is used against malig-
nant solid tumors; a PEG-modified liposomal doxorubicin
formulation (PEGL-DOX), which is used against recurrent
ovarian cancer [2]; docetaxel, which is used against a wide
variety of cancers; and sorafenib or sunitinib, which are
molecular target drugs used against kidney cancer. The
frequencies with which certain drugs can induce HFS are
ranked as follows: PEGL-DOX as the highest at approxi-
mately 78 %, next is capecitabine at 51–78 %, and finally
sorafenib at about 55 % [3].
Anticancer drugs are made to cause cellular malfunctions,
but the particular reasons for the occurrence of side effects
like pain in the hands and feet, reddening and cracking of the
skin, numbness, and erythema (red spots) are not understood
in much detail. For that reason, there are currently no
effective treatments, and the standard practice is to recom-
mend moisturizer, or topical steroids in severe cases.
PEGL-DOX is exceedingly effective in treating recurrent
ovarian cancer without causing side effects like cardiotox-
icity, neutropenia, anemia, alopecia, or nausea/vomiting.
However, because of the frequency with which HFS occurs,
there is a high risk that the patient’s quality of life will
severely decline. With regards to insuring the completion of
medical treatment, it is extremely important to establish
precautionary measures. Therefore, in order to elucidate the
mechanism of HFS outbreaks, we carried out in vivo
experiments to analyze skin histology in the extremities,
cytokine analyses, and in vitro experiments in skin cells.
Materials and methods
Chemicals
Doxorubicin (DOX, generic for Adriamycin) was obtained
from Nippon Kayaku (Tokyo, Japan). In order to make
PEGL-DOX (trade name Doxil
Ò
), a PEG-modified lipo-
somal formulation of DOX, lecithin and polyoxyethyle-
nated lecithin were obtained from NOF (Tokyo, Japan).
Methanol, chloroform, copper chloride, and Mayer’s
Hematoxylin and Eosin were purchased from Wako Pure
Chemical Industries (Osaka, Japan). Fluorescein sodium
salt was purchased for the fluorescent dye experiments
from Sigma-Aldrich (St. Louis, MO, USA). DeadEnd
TM
Fluorometric TUNEL System was purchased for TUNEL
staining from Promega (Madison, WI, USA).
Animals
Six-week-old female SD rats and hairless rats were pur-
chased from Japan SLC (Shizuoka, Japan), and were used
for experiments after 1 week of rest. The rats were raised
in independent cages in 50–60 % humidity, 23 ±1°C
environment with light from 0630 to 1830 hours (12-h
light–dark cycle) and were given ad libitum access to food
and water. All animal experiments were carried out in
accordance with the Guidelines for Animal Experimenta-
tion of St. Marianna University School of Medicine.
Cell culture
From in vitro experiment, pathogenesis of HFS was
expected to be a leakage of doxorubicin from peripheral
vascular in the dermal layer in skin. As it was assumed that
the cell in skin should be directly affected by doxorubicin
alone, all in vitro experiments have been performed with
doxorubicin alone.
HaCaT cell lines derived from human keratinocytes
were received from Dr. Masamitsu Ichihashi of the Kobe
University Graduate School of Medicine, Department of
Dermatology. Normal Human Dermal Fibroblasts (NHDF
cells) were purchased from KURABO Industries. HaCaT
were cultured using Dulbecco’s Modified Eagle Medium
(DMEM; Life Technologies, Carlsbad, CA, USA), and
NHDF were cultured using DMEM supplemented with
GlutaMAX
TM
Supplement I (Life Technologies, Carlsbad,
CA, USA). The media were supplemented with 10 % fetal
bovine serum, 100 U of penicillin, and 100 U of strepto-
mycin. The cells were cultured at 5 % CO
2
and 37 °C.
Preparation of the PEGL-DOX
PEGL-DOX was produced from raw materials in the lab-
oratory according to the Sadzuka method [4], but the steps
involving filtration to equalize the particle sizes and the
subsequent confirmation of the average particle size were
omitted. L-a-distearoylphosphatidyl-DL-glycerol (DSPG, a
PEG-modified lecithin) and lecithin were dissolved in a 1:4
methanol:chloroform solution and formed a thin film on the
surface of the flask after the solvent was removed using a
rotary evaporator in a 35 °C hot water bath. Aqueous DOX
solution and sorbitol/lactic-acid solution was then added to
the flask, and it was stirred for 15 min in a 60 °C hot water
bath. After 3 min of sonication, the final product was a
0.2 % weight/volume PEGL-DOX solution. Physico-
chemical properties of PEGL-DOX were confirmed the
following methods: (1) observation of a flow birefringence
under a polarizing plate, and (2) semi-transparent without
turbidity because of emulsified turbid appearance of lipo-
some without modified PEG.
Preparation of the HFS animal models
In order to determine whether the liposome formulation of
doxorubicin developed from HFS onset, we have carried
out comparative experiments with doxorubicin alone. To
Pathogenesis of Hand-Foot Syndrome 9
123

prepare the HFS animal models, PEGL-DOX and/or DOX
(at 10 and 5 mg/kg, respectively) was administered to SD
rats via the tail veins once every 3 days for 10 days. The
limbs were visually inspected and photographed 10 days
later. Afterwards, skin tissue samples from the hind limbs
were collected, fixed in formalin, and embedded in paraf-
fin. In non-clinical studies reported by dealers in DOXIL
Ò
,
they did not find onset HFS for each additive of the PEGL-
DOX preparation. Thus, in the present study, administra-
tion of the experiment in vivo was not performed for each
additive to confirm the onset of HFS.
Tissue Staining
The formalin-fixed paraffin-embedded skin tissue was
sliced into 4 lm sections, deparaffinized, rehydrated, H&E
stained, and then observed under an optical microscope.
Picrosirius Red staining was applied in order to observe the
state of the dermal collagen fibers and visualized under a
polarized light microscope. TUNEL staining with the
DeadEnd
TM
Fluorometric TUNEL System was used to
detect apoptosis and visualized under a fluorescence
microscope (BIOZERO; KEYENCE, Osaka, Japan).
Measurement of cytokine expression in vivo
and in vitro
Inflammatory cytokines and chemokines were measured in
order to investigate the origins of HFS. After the application
of the PEGL-DOX treatment, the hind-leg skin tissue was
collected and used to measure in vivo expression levels. First
the tissue was homogenized, then the samples were centri-
fuged, and finally the supernatant was analyzed using the Rat
Cytokine Antibody Array (RayBiotech,North Metro-Atlanta,
GA, USA). In addition, in vitro experiments were conducted
in order to clarify the influence of DOX on skin cells, spe-
cifically epithelial cells. HaCaT cells were cultured in media
supplemented with DOX and CuCl
2
, and the inflammatory
cytokines were measured using the Human Cytokine Anti-
body Array (RayBiotech). IL-8, GRO, and Fractalkine che-
mokines of the CXC family, which corresponds to the CINC3
rat chemokine family were quantified using the Luminex200
system (Millipore, Billerica, MA, USA). Similar experiments
were also carried out using NHDF.
Creation of a visualizable model of a PEG-modified
liposomal drug using fluorescein
In order to investigate the phenomenon by which HFS
develops selectively in the limbs rather than throughout the
whole body, fluorescein (FS) was used to create an easily
visualizable model of a PEG-modified liposomal drug. The
PEG-modified liposomal fluorescein (PEGL-FS) drug was
prepared exactly as the PEGL-DOX drug was prepared. FS
or PEGL-FS was administered to hairless rats via the tail
vein, and whole-body FS was visualized under a long-
wavelength ultraviolet lamp (UVGL-58 Handheld UV
Lamp; UVP, Upland, CA, USA). Observations commenced
immediately after drug administration, and photographs
were taken periodically. Skin samples were collected from
the soles of the hind-paws at 1, 7, and 24 h after drug
administration. The OCT compound-embedded tissue was
cut into 10-lm frozen sections and observed under a fluo-
rescence microscope.
Measurement of DOX toxicity in vitro
HaCaT and NHDF cell cultures were used to evaluate the
toxicity of DOX in vivo. DOX was added at various con-
centrations (0.1–10 lM) to the media, and the percentage
of viable cells was measured 24 h later using Cell Counting
Kit-8 (CCK8; Dojindo Laboratories, Kumamoto, Japan).
Results showed that 1.5 lM exhibited a moderate level of
toxicity that was appropriate for the toxicity tests to follow
(Fig. 1). To test the toxicity in the presence of copper ions,
various concentrations of copper chloride (50 or 375 lM)
were added to 1.5 lM DOX media. After 24 h of culturing,
the survival rate was again measured using CCK8. Finally,
to test the degree of inhibition of ROS by SODs, 100 lg/ml
SOD (Sigma-Aldrich) was added to the medium, and 12 h
later the survival rate was again measured.
Statistical analysis
Dunnett’s and Tukey’s multiple comparison tests were
used to analyze the results of in vitro experiments. The
software used was R v.2.15.1 [5].
Fig. 1 Survival rate of human skin cells following DOX treatments.
HaCaT and NHDF cells were cultured with DOX for 24 h before
counting. The values are presented as mean ±SD (n=3). Signif-
icantly different from control: *p\0.05, **p\0.01
10 N. Yokomichi et al.
123

Results
Injections of PEGL-DOX yielded an HFS-like disease
state
Single or multiple doses of high-dose (10 mg/kg) or low-
dose (5 mg/kg) DOX or PEGL-DOX were administered
intravenously to SD female rats, whose limbs were then
observed for signs of inflammation or redness. Changes in
appearance were compared within single-dose or multiple-
dose groups (Fig. 2a, b).
Within the single-dose group, immediately following
PEGL-DOX administration, reddening was observed in the
forepaws, hind-paws, ears, and at the tip of the nose;
however, no such change was observed after DOX
administration even after high-dose treatment (Fig. 2a).
The redness that appeared was transient and disappeared
after 5–10 min. Since the thickness of the rat limb skin is
very thin, we can normally see the blood vessel through the
skin. Because PEGL-DOX has a red color, observed tran-
sient redness after injection would correspond to the nat-
ural color of PEGL-DOX. Within the multiple-dose group,
inflammation was observed after multiple low-dose PEGL-
DOX treatments, and the change was even more striking
after high-dose treatments (Fig. 2b). This observed state of
inflammation, swelling, and dryness was judged to be
similar enough to human HFS to conclude that HFS had
indeed broken out in these rat limbs [6].
Skin tissue staining revealed multiple adverse affects
of high-dose PEGL-DOX
H&E staining clearly revealed the following effects of
multiple doses of high-dose PEGL-DOX as compared to an
untreated control group: a thinned or even absent granular
layer, a decrease in the number of cells between the basal
layer and the stratum spinosum, a rougher arrangement of
cells, and a thinning of the epithelial layer (Fig. 3a). On the
other hand, the dermal fibroblasts appeared relatively
unaffected. Picrosirius red staining, which stains collagen
fibers, revealed disarranged and broken collagen fibers in
the multiple-dose PEGL-DOX group (Fig. 3b). TUNEL
staining, which is a marker for apoptosis, showed that
apoptosis was induced in basal epidermal cells in the
PEGL-DOX group (Fig. 3c). In other words, the results of
the TUNEL staining imply that HFS is related to apoptosis
induced in epidermal cells.
Antibody array showed increased expression
of chemokines and inflammatory cytokines
The proteins expressed in the regions of the rat skin tissue
affected by the PEGL-DOX treatment were measured using
an antibody array. Markedly increased expression of mul-
tiple proteins was confirmed: the chemokines CINC3 and
Fractalkine, the IL-family-inhibitory IL-10, and inflam-
matory cytokines such as IL-1band IL-6 (Fig. 4).
PEGL-FS yielded more persistent fluorescence
than unaltered FS in rat paws
Immediately following administration of PEGL-FS or FS
(unaltered fluorescein), very strong fluorescence was
observed in the extremities. This fluorescence weakened
Fig. 2 Rat paws (SD, female, 7 weeks) after intravenous injection of
DOX or PEGL-DOX. aAppearance immediately after 10 mg/kg
injection. bAppearance after multiple doses. Doses were adminis-
tered once every 3 days, and photos were taken on the 10th day. Blue
circles indicate particularly inflamed areas
Pathogenesis of Hand-Foot Syndrome 11
123

over time but remained strong in the paws even after 3 h,
and a small amount of fluorescence remained after 7 h in
the PEGL-FS group (Fig. 5a).
Tissue sections at 1 h after treatment with either PEGL-
FS or FS exhibited fluorescence over the entire dermal
layer, indicating a high level of FS retention. It is assumed
that the FS leaked out of the capillaries in the dermal layer
(Fig. 5b). At this point, the PEGL-FS had already started to
spread to the epidermis and exhibit fluorescence there. In
sections at 7 h after treatment, the fluorescence had
become concentrated at the upper stratum corneum, which
suggested that the fluorescent dye had diffused from the
dermis through the epithelium and arrived at the stratum
corneum. In the PEGL-FS group, in contrast to the FS
Fig. 3 Tissue staining in rat (SD, female, 7 weeks) paw skin after
10 mg/kg PEGL-DOX injection. aH&E staining. Epidermal layer
was thinned with respect to control (epidermal layer is shown in blue
between the pink stratum corneum and lighter blue dermal layer).
bPicrosirius red staining. Color of stain in order of decreasing
strength and thickness of fibers: red,yellow,green. PEGL-DOX
group displayed disarranged and broken collagen fibers. cTUNEL
staining. Red marks nuclei, green marks apoptosis. Only basal cells
show signs of apoptosis. (a–c)scale bar 50 lm
12 N. Yokomichi et al.
123

group, a small but noticeable amount of fluorescence
remained in the dermis. In addition, the concentration of
fluorescence in the stratum corneum appeared slightly
higher in the PEGL-FS group than the FS group.
DOX and Cu(II) ions increased production
of chemokines and cytokines and lowered cell survival
rates, which were rescued by SOD
In the regions of the rat skin tissue affected by the PEGL-
DOX treatment were measured using an antibody-array,
markedly increased expression of multiple cytokines was
confirmed: the chemokines CINC3 and Fractalkine, the IL-
family-inhibitory IL-10, and inflammatory cytokines such
as IL-1band IL-6 (Fig. 4). Therefore, we studied this
mechanism in detail in vitro. HaCaT and NHDF cells were
treated with various concentrations of DOX (0.1–10 lM)
and survival rates were determined after 24 h. Results
showed that 1.5 lM exhibited a moderate level of toxicity
that was appropriate for toxicity tests to follow (Fig. 1).
The presence of the 1.5 lM DOX did not detectably
increase the production of chemokines in the HaCaT cells,
but the addition of Cu(II) ions caused increased production
of the aforementioned CXC chemokines GRO and IL-8,
with the production volume dependent on the Cu(II) ion
concentration (Fig. 6a). However, in the NHDF cells,
neither DOX alone nor the combination of DOX and Cu(II)
ions yielded increased production of chemokines; in fact,
DOX appeared to inhibit chemokine production.
The effects of DOX and Cu(II) ions on inflammatory
cytokine production varied across different cytokines and
different cell types (Fig. 6b). HaCaT cells produced IL-1a
and IL-6 in response to the presence of DOX, and pro-
duction was further amplified by the addition of Cu(II)
ions. The production of IL-1brose in the presence of DOX
combined with a very high concentration of Cu(II) ions. By
contrast, NHDF cells exhibited no noticeable response to
DOX alone, and the addition of Cu(II) ions stimulated an
increase in only IL-bproduction.
Without the addition of DOX, the survival rate of Ha-
CaT cells remained relatively constant across varying
concentrations of Cu(II) ions (Fig. 7). However, in the
presence of DOX, the survival rate of the cells rapidly
decreased with increasing Cu(II) ion concentration. By
contrast, the NDHF cells were relatively unaffected by the
combination of DOX and Cu(II) ions. The addition of
superoxide dismutase (SOD) improved this HaCaT cell
survival (Fig. 8).
Discussion
A variety of research has been conducted on the relation-
ship between Doxil
Ò
(trade name for PEGL-DOX) and
HFS, and many important discoveries have already been
made. Charrois et al. [7] have analyzed the pharmacoki-
netics of DOX in rat skin tissue and tumors after multiple
doses of Doxil
Ò
, and results have shown that the half-life
of Doxil
Ò
is particularly long in the paws. It has become
known that multiple doses of anticancer drugs, or possibly
a single large dose, can cause an accumulation of cell
damage and a speeding up of the cell cycle in keratino-
cytes, which can ultimately lead to an outbreak of HFS [8].
By using the skin of humans to whom fluorescence-tagged
Doxil
Ò
had been administered, Martschick et al. [9] have
discovered that Doxil
Ò
leaks out from the body in the
sweat. Both in vivo experiments in mice and rats and
in vitro experiments in HaCaT cells led to the conclusion
that DOX toxicity in the skin causes hair-loss via dena-
turation of the sebaceous line [10]. In addition, the idea that
Fig. 4 Rat Cytokine Antibody
Array. Skin tissue from HFS-
affected areas after multiple
10 mg/kg PEGL-DOX
injections. Results were
normalized to controls. Graph
shows increased production of
chemokines with respect to
control. The values are
presented as mean ±S.D
Pathogenesis of Hand-Foot Syndrome 13
123

Manganese SOD (MnSOD) can suppress apoptosis was
introduced in an experiment investigating DOX-induced
apoptosis in HaCaT cells [11]. Finally, it has been reported
that the coexistence of DOX and Cu(II) generates ROS,
which inflict oxidative damage on DNA [12].
As described above, while research on DOX, Doxil
Ò
,
and the skin is plentiful, little is known about why Doxil
Ò
/
PEGL-DOX frequently causes HFS or why the outbreaks
occur in the skin tissue. Moreover, there have been no
reports of effective treatments for this condition.
Fig. 5 Comparison of
photographs after intravenous
injection of 36 mg/kg 1.5 ml
PEGL-FS or FS in hairless rats
(female, 7 weeks). aPhotos
cropped from whole-body
visualization under long-
wavelength UV lamp
immediately after, 3 and 7 h
after injection. PEGL-FS
remained fluorescing in the
paws 7 h post-injection.
bTissue sections cut 1 h and
7 h after injection. PEGL-FS
diffused faster from the dermis
through the epidermis to the
stratum corneum, lingered in the
dermis longer, and showed
slightly higher fluorescence than
FS. Scale bar (b) 100 lm
14 N. Yokomichi et al.
123

The tendency of PEGL-DOX to induce HFS outbreaks in
rats where DOX did not led us to the theory that the dif-
ference in metabolic stability between the two drugs was the
cause of the difference in frequency of HFS outbreaks in
humans. In other words, the key difference is that the higher
metabolic stability of PEGL-DOX allows it to remain active
in the blood for a longer period of time, as illustrated by the
presence of PEGL-FS remaining in the rat extremities after
7 h (Fig. 4). This also means an increase in the frequency
with which the drug reaches the hands and feet.
Fig. 6 Changes in chemokine and cytokine production in HaCaT and
NHDF cells following addition of DOX or DOX ?Cu(II) ions to
culture medium. aProduction of chemokines IL-8, GRO, and
Fractalkine (ng/ml). HaCaT cells exhibited DOX- and Cu(II)-
dependent production of IL-8 and GRO. The values are presented
as mean ±SD (n=3). Significantly different from control:
*p\0.05, p\0.01. bProduction of cytokines IL-1a, IL-1b, and
IL-6 (ng/ml). HaCaT cells exhibited DOX/Cu(II)-dependent increased
production of all cytokines shown, and NHDF cells exhibited
substantially increased production of IL-1bonly
Fig. 7 Survival rate of human cells in the presence or absence of
1.5 lM DOX and varying concentrations of Cu(II) ions. aHaCaT
cells. Survival rate declined with increasing Cu(II) ion concentration
in the presence of DOX. bNHDF cells showed relatively little
response to DOX and Cu(II) ions. The values are presented as
mean ±SD (n=3). Significantly different from control: *p\0.05,
**p\0.01
Pathogenesis of Hand-Foot Syndrome 15
123

In fact, the capillaries are concentrated at the fingertips
and the soles of the feet, where the blood flow is high.
Unlike three-layer artery or arteriole walls, capillary walls
are composed of only a single layer of endothelial cells,
which makes capillary walls easy to penetrate with only
slight provocation. The capillaries are especially concen-
trated in the dermis, which suggests that anticancer drugs
might easily leak into the dermis and linger there at high
concentrations, as indicated by the results of the experi-
ment using the fluorescein drug model PEGL-FS (Fig. 4).
Doxorubicin is known as the most dangerous of all ves-
icant drugs, which are high-risk drugs capable of causing
tissue necrosis upon extravasation [13]. Therefore, the
accumulation of DOX in this tissue is extremely cytotoxic.
Many anticancer drugs cause DNA damage in order to
induce apoptosis in cancer cells. There have been many
experiments that show that apoptosis can be induced by
ROS generated directly or indirectly by anticancer drugs
[14–18]. It is said that DOX damages DNA by generating
ROS and inhibiting Topoisomerase II [12]. Furthermore, it
has been reported that the oxidative damage due to ROS was
magnified in the presence of Cu(II) ions in experiments
using the human promyelocytic leukemia cells [19]. Our
own results corroborated the pre-existing evidence that DOX
induces apoptosis in keratinocytes via ROS in the presence
of Cu(II) ions and that SOD rescues the cells from apoptosis
by capturing the ROS in the culture medium [11,20].
The effects of DOX and Cu(II) ions on cell viability and
chemokine production appear to be tightly correlated.
The results of our rat tissue staining experiments (Fig. 2)
indicate that DOX induces apoptosis in keratinocytes, but
not in dermal fibroblasts. Similarly, the results of our
cytotoxicity tests (Fig. 7) indicate that, while DOX does
not affect fibroblasts, it appears to kill keratinocytes, and it
appears to kill at a greater rate in the presence of Cu(II)
ions. As for chemokines, the pattern is similar. While
fibroblasts do not appear to produce chemokines even in
the presence of both DOX and Cu(II) ions, keratinocytes
produce the chemokines IL-8, GRO, and Fractalkine in
response to DOX, and IL-8 and GRO are produced in a
Cu(II) concentration-dependent manner (Fig. 6). The fact
that chemokine production spiked in areas of rat skin tissue
afflicted with HFS (Fig. 3) suggests that these chemokines
represent an important part of the mechanism by which
injection of PEGL-DOX leads to HFS.
Yamamoto et al. [20] have reported that the thermore-
ceptor TRPM2 is expressed on the surface of keratinocytes
and plays the role of sensing ROS in the surrounding
environment. In response to ROS, these receptors create
holes in the cell surface through which Ca
2?
ions flow into
the keratinocytes. The rise of the intracellular Ca
2?
ion
concentration due to this influx induces chemokine pro-
duction. This mechanism is thought to be responsible for
the increase in chemokine production in HFS-affected
tissues.
Chemokine production alone is not sufficient to produce
the typical HFS state of inflammation. Moreover, it is
thought that chemokines do not directly induce keratino-
cyte apoptosis, but, rather, death factor cytokines are a
necessary intermediary. Death factors known to induce
apoptosis include TNF-a, Fas ligand, lymphotoxin a, TNF-
related apoptosis-inducing ligand (TRAIL)/Apo2 ligand,
and Apo3 ligand [21]. Chemokines induce positive che-
motaxis in blood cells, which express these death factors.
For example, neutrophils expressing Fas ligand migrate to
the dermis in response to chemokines produced by kerati-
nocytes. These neutrophils undergo apoptosis in response
to ROS, and at the same time caspase-1 is activated inside
the neutrophils, and IL-1bis released from the cells [22].
While our fluorescent staining did not show this migration
of blood cells (Fig. 2), despite the presumably high level of
chemokine production, it is thought that these cells may
have undergone apoptosis and been taken up by macro-
phages, which would explain their absence in the tissue
staining photographs.
The prevailing view up until now was that cells under-
going apoptosis do not induce inflammation because they
are absorbed by phagocytes or surrounding cells; however,
the apoptosis of cells expressing death factors is a different
matter. It is known that keratinocytes also express death
factors [22], and it is thought that blood cells and kerati-
nocytes undergoing apoptosis due to the presence of ROS
cause inflammation by releasing IL-1b. Our results indicate
that fibroblasts also produce IL-1bin response to ROS
stimulation, and keratinocytes produce IL-1aand ILbin a
Fig. 8 Rescue of DOX/Cu(II)-induced decline in HaCaT cell
survival rate by SOD. The values are presented as mean ±SD
(n=3). Significantly different from DOX, DOX ?CuCl
2
, and
DOX ?CuCl
2
?SOD, respectively: *p\0.05, *p\0.01
16 N. Yokomichi et al.
123

manner dependent on Cu(II) ion concentration (Fig. 6). In
short, due to the presence of ROS, keratinocytes, blood
cells, and fibroblasts produce inflammatory cytokines,
which leads to vasodilation, rubefaction, fever, augmented
vascular permeability, and swelling; i.e., the HFS disease
state.
To summarize, the mechanism of HFS development that
we propose based on our research is as follows (Fig. 9): (1)
DOX penetrates the capillary walls and interacts with
Cu(II) ions in the skin to produce ROS; (2) these ROS
attack keratinocytes, which release chemokines and the
inflammatory cytokines IL-1b, IL-6, and IL-1a; the cyto-
kines induce apoptosis in the keratinocytes, and the che-
mokines induce positive chemotaxis in blood cells, which
in turn release IL-1band apoptose; (3) fibroblasts release
IL-1bin response to the presence of ROS; (4) ROS destroy
collagen fibers in addition to causing cell death; and (5) the
combination of inflammation and keratinocyte apoptosis
due to the accumulated cytokines and collagen destruction
in close proximity results in complete skin tissue
devastation.
In conclusion, we propose that HFS develops due to the
combination three primary factors: (1) the inherently strong
cytotoxicity of DOX, (2) the ability of PEGL-DOX to
remain in circulation for extended periods of time as a
PEG-modified liposome, and (3) the abundance of metal
ions in the skin tissue.
However, several factors remain to be investigated,
including the influences of different metal ions and the
effects of DDSs capable of remaining in circulation for
differing periods of time. Having elucidated a detailed
mechanism for HFS development, we are now ready to
begin the development of drugs to treat or prevent the
condition all together. A likely starting point is SOD,
which was able to rescue cell viability in the presence of
ROS, and therefore implies the potential for a treatment
that captures and removes ROS to prevent damage. In the
future, we plan to produce even more detailed research on
this subject.
Acknowledgments We appreciate the technical assistance of Mrs.
Mina Musashi and Ms. Satomi Kato.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
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