Annals of Oncology 28: 2119–2127, 2017

doi:10.1093/annonc/mdx318
Published online 23 June 2017

ORIGINAL ARTICLE

Prevention of chemotherapy toxicity by agents that

neutralize or degrade cell-free chromatin

I. Mittra*, K. Pal, N. Pancholi, A. Shaikh, B. Rane, P. Tidke, S. Kirolikar, N. K. Khare, K. Agrawal, H. Nagare &
N. K. Nair
Translational Research Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Sector 22, Utsav Chowk – CISF Road,
Kharghar, Navi Mumbai, Raigad, Maharashtra 410210, India

*Correspondence to: Prof. Indraneel Mittra, Translational Research Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre,
Navi-Mumbai 410210, India. Tel: þ91-97-69-76-97-20; E-mail: indraneel.mittra@gmail.com

Background: Toxicity associated with chemotherapy is a major therapeutic challenge and is caused by chemotherapy-induced
DNA damage and inflammation. We have recently reported that cell-free chromatin (cfCh) fragments released from dying cells
can readily enter into healthy cells of the body to integrate into their genomes and induce DNA double-strand breaks,
apoptosis and inflammation in them. We hypothesized that much of the toxicity of chemotherapy might be due to release of
large quantities of cfCh from dying cells that could trigger an exaggerated DNA damage, apoptotic and inflammatory response
in healthy cells over and above that caused by the drugs themselves.
Methods: We tested this hypothesis by administering cfCh neutralizing/degrading agents namely, anti-histone antibody
complexed nanoparticles, DNase I and a novel DNA degrading agent—Resveratrol-Cu concurrently with five different
chemotherapeutic agents to examine if chemotherapy-induced toxicity could be minimized.
Results: We observed (i) significant reduction in chemotherapy-induced surge of cfCh in blood; (ii) significant reduction in
chemotherapy-induced surge of inflammatory cytokines CRP, IL-6, IFNc and TNFa in blood; (iii) abolition of chemotherapy-
induced tissue DNA damage (cH2AX), apoptosis (active caspase-3) and inflammation (NFjB and IL-6) in multiple organs and
peripheral blood mononuclear cells; (iv) prevention of prolonged neutropenia following a single injection of adriamycin and
(v) significant reduction in death following a lethal dose of adriamycin.
Conclusion: Our results suggest that toxicity of chemotherapy is caused to a large extent by cfCh released from dying cells and
can be prevented by concurrent treatment with cfCh neutralizing/degrading agents.
Key words: chemotherapy toxicity, prevention of chemotherapy toxicity, dying cells, cell-free chromatin, neutralizing cell-free
chromatin, degrading cell-free chromatin

Introduction
Fifteen million new cases of cancer are detected globally each year
[1], the majority of whom receive some form of chemotherapy
and suffer varying degrees of its toxic side-effects.
Chemotherapy-induced toxicity results from systemic DNA
damage and inflammation of healthy cells [2–4]. Symptoms of
toxicity include bone marrow suppression, sore mouth, nausea
and vomiting, diarrhoea, loss of appetite, fatigue, hair loss, steril-
ity and nerve damage [5]. Several antidotes are usually prescribed
to mitigate the toxic symptoms of chemotherapy with varying
degrees of success [5]. We have recently reported that cell-free
chromatin (cfCh) released from dying cells can readily enter into
healthy cells of the body to integrate into their genomes to induce
DNA double-strand breaks apoptosis and inflammation in them
[6, 7]. We show here that chemotherapy-induced toxicity can be
traced to these findings in that most of the toxic side-effects
of chemotherapy are caused by cfCh released from dying cells.
The initial round of chemotherapy-induced cell death triggers a
cascading effect whereby the dead cells release more cfCh causing
further rounds of DNA damage, apoptosis and inflammation
thereby exaggerating or amplifying the toxic effects of chemo-
therapy. We also show that chemo-toxicity can be largely pre-
vented by cfCh neutralizing/degrading agents administered
concurrently with chemotherapy.

VC The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

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 Original article
Methods
Institutional Animal Ethics Committee approval
The experimental protocol was approved by the Institutional Animal
Ethics Committee of Advanced Centre for Treatment, Research and
Education in Cancer, Tata Memorial Centre, Navi Mumbai, India and
experiments were carried out according to the Committee’s animal safety
guidelines.
Chemotherapy drugs and dosage
The chemotherapy drugs used in this study were: adriamycin (10 mg/kg),
cyclophoshamide (100 mg/kg), cisplatin (20 mg/kg), methotrexate
(25 mg/kg) and paclitaxel (10 mg/kg). All drugs were administered as a
single i.p. dose.
Preparation of cfCh neutralizing/degrading
agents
Pullulan-histone antibody nanoconjugates (CNPs) were synthesized as
described by us earlier except that we exclusively used H4 IgG for prepar-
ing CNPs [6, 8]. We have shown that CNPs specifically bind to chromatin
fragments and inactivate them both in vitro and in vivo [6, 8].
DNase I. Bovine pancreatic DNase I was obtained from Sigma-Aldrich
(Catalogue No. DN25-1G).
Resveratrol-copper (R-Cu). Resveratrol (R) is a plant polyphenol which
has been widely investigated for its antioxidant properties [9]. We and
Annals of Oncology
others have shown that R acts as a pro-oxidant in the presence of copper
(Cu) by its ability to reduce Cu (II) to Cu (I) thereby generating a free
radical [10, 11]. Because of this pro-oxidant property, R-Cu can degrade
genomic DNA [12], and this pro-oxidant property is retained even when
the molar concentration of Cu is reduced more than 10 000-fold with re-
spect to that of R [12]. The detailed methodology for preparing R-Cu for
in vitro and in vivo experiments is given in supplementary materials,
available at Annals of Oncology online.
Isolation of cfCh from mouse serum and
assessment of biological activity
The method for cfCh isolation from serum has been described by
us in detail earlier [6]. Biological activity of the isolated cfCh [6] was as-
certained by treating NIH3T3 mouse fibroblast cells with the cfCh iso-
lates (10 ng of DNA) and analysing the activation of H2AX, active
caspase-3, NFjB and IL-6 at 6 h by indirect immunofluorescence
as described earlier [6, 7]. Detailed methodology for isolation of cfCh
from mouse serum and assessment of biological activity is given in sup-
plementary materials, available at Annals of Oncology online. The list of
sources and catalogue numbers of the primary and secondary antibodies
used is given in supplementary Table S1, available at Annals of Oncology
online.
Estimation of serum cfCh and inflammatory
cytokines following adriamycin treatment
Serum cfCh was estimated using the Cell Death Detection ELISA
PLUS kit (Roche Diagnostics GmbH, Mannheim, Germany) [6] and

gH2AX Active caspase-3

80 80
NIH3T3 NIH3T3
NIH3T3 + cfCh NIH3T3 + cfCh
NIH3T3 + cfCh + CNPs NIH3T3 + cfCh + CNPs
60 NIH3T3 + cfCh + DNase I 60 NIH3T3 + cfCh + DNase I
NIH3T3 + cfCh + R-Cu NIH3T3 + cfCh + R-Cu
MFI

MFI

40 40 *
* * * * *
* * * * * * *
* * * * * * * *
* * * * * * *
* * *
20 20 *

0 0

80 NFkB IL-6

NIH3T3 NIH3T3
NIH3T3 + cfCh NIH3T3 + cfCh
60 NIH3T3 + cfCh + CNPs 60 NIH3T3 + cfCh + CNPs
NIH3T3 + cfCh + DNase I NIH3T3 + cfCh + DNase I
NIH3T3 + cfCh + R-Cu NIH3T3 + cfCh + R-Cu
MFI

40 40
*
* * * * *
MFI

* * * * * * * *
* * * * * * * *
* * * * * * *
20 * 20 * *

0 0

Figure 1. Activation of H2AX, active caspase-3, NFjB and IL-6 in NIH3T3 cells treated with cfCh (10 ng DNA) isolated from mouse serum at
6h with and without concurrent treatment with CNPs, DNase I and R-Cu. The bars represent mean 6 SEM values of duplicate experiments in
each group. Statistical comparison was carried out using unpaired Student’s t-test. ****P < 0.0001.

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 Annals of Oncology Original article
A 1.50

1.25 Control
Adriamycin
Absorbance at 405nm
1.00 Adriamycin + CNPs
Adriamycin + DNase I

0.75 Adriamycin + R-Cu

0.50
** **
* **
0.25 ** * *
*
0

B CRP IL-6
Control Control
60000 500
Adriamycin Adriamycin
Adriamycin + CNPs Adriamycin + CNPs
50000
Adriamycin + DNase I 400 Adriamycin + DNase I
* Adriamycin + R-Cu NS Adriamycin + R-Cu
40000 * NS
* * 300
* * *
ng/ml

* pg/ml
30000 * *
* * *
* * 200
20000 *
* *
100
*
10000

0 0

IFNY TNFa
300 Control
60 Control
Adriamycin
Adriamycin
250 Adriamycin + CNPs
50 Adriamycin + CNPs
Adriamycin + DNase I
Adriamycin + DNase I *
Adriamycin + R-Cu
40 Adriamycin + R-Cu 200
* *
* *
pg/ml

* *
pg/ml

30 * * * 150
* * *
*
20 100

10 50

0 0

Figure 2. Prevention of cfCh and inflammatory cytokine surge in blood at 18 h following a single i.p. injection of adriamycin by concurrent
treatment with CNPs, DNase I and R-Cu. (A) Prevention of cfCh. Results are expressed in arbitrary units in the form of absorbance value in
spectrophotometer. (B) Prevention of inflammatory cytokines. The bars represent mean values 6 SEM of five animals in each group.
Statistical comparison was carried out using unpaired Student’s t-test. * ¼ p < 0.05; ** ¼ p < 0.01; *** ¼ p < 0.001; **** ¼ p < 0.0001.

results expressed in arbitrary units in the form of absorbance value
in spectrophotometer. Inflammatory cytokines viz., CRP, IL-6, TNF-a
and IFN-c were estimated by enzyme-linked immunosorbent
assay (ELISA) according to vendor’s protocol (BD and R&D biosciences
ELISA kits). Mean values (6SEM) were determined and compared be-
tween groups using unpaired Student’s t-test. Details of estimation of
serum cfCh and inflammatory cytokines following adriamycin treatment
are given in supplementary materials, available at Annals of Oncology
online.
Detection of cellular DNA-damage, apoptosis and
inflammation in various organs, tissues and
peripheral blood mononuclear cells
Detailed methodology for detecting cellular DNA-damage, apoptosis and
inflammation in various organs, tissues and peripheral blood mononu-
clear cells (PBMCs) is given in supplementary materials, available at
Annals of Oncology online. Activation of the various parameters was
examined by indirect immuno-fluorescence [6, 7].

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 Original article Annals of Oncology
Assessment of neutropenia following a single injection of adriamycin. We first determined
Detailed methodology for assessing neutropenia is given in supplemen-
the time-point when the maximum surge of cfCh and inflamma-
tary materials, available at Annals of Oncology online. tory cytokines occurred following adriamycin challenge (10 mg/
kg) (supplementary Figure 2A and B, available at Annals of
Oncology online). In case of cfCh, we observed two peaks occur-
Survival analysis ring at 6 and 18 h, while in case of inflammatory cytokines (IL-6),
Forty animals were divided into 4 groups of 10 mice each and all groups we observed a single peak that occurred at 18 h. We chose the 18 h
received a lethal dose of adriamycin (20 mg/kg). CNPs, DNase I and time-point for our experiments using the cfCh inhibitors. We in-
R-Cu were administered in doses and frequencies as described above.
jected mice with adriamycin (10 mg/kg) with and without con-
Survival between groups was compared by Kaplan–Meier survival ana-
lysis using log-rank test. current treatment with CNPs, DNase I and R-Cu and collected
blood samples by orbital puncture at 18 h. The marked rise in
serum cfCh that occurred following adriamycin injection could
be completely neutralized by concurrent treatment with the three
Results cfCh neutralizing/degrading agents (Figure 2A) (P < 0.001). We
Since we had earlier demonstrated that cfCh isolated from serum also observed a marked rise in inflammatory cytokines CRP, IL-
of human subjects can phosphorylate H2AX and active caspase-3 6, IFN-c and TNF-a following adriamycin challenge. These could
[6], we first investigated whether mouse cfCh was also capable of be significantly inhibited by CNPs, DNase I and R-Cu (Figure
activating DNA damage and apoptosis when added to cells in cul- 2B) (P < 0.01–0.0001).
ture. We also examined whether mouse cfCh could, in addition, We next examined whether CNPs, DNase I and R-Cu could
activate inflammatory cytokines. Figure 1 and supplementary prevent tissue DNA damage and inflammation following admin-
Figure S1, available at Annals of Oncology online, show that add- istration of 5 different chemotherapy agents namely, adriamycin,
ition of 10 ng DNA equivalent of cfCh isolated from mouse serum cyclophosamide, cisplatin, methotrexate and paclitaxel. A single
when added to NIH3T3 cells caused an approximately threefold dose of adriamycin induced remarkable cellular activation of
increase in activities of cH2AX, active caspase-3, NFjB and IL-6 H2AX, active Capsase-3, NFjB and IL-6 in lung, liver, heart,
at 6 h. All these activities could be neutralized by simultaneous brain, ovary, skin and small intestine (Figure 3 and supplemen-
addition of cfCh neutralizing/degrading agents namely, CNPs, tary Figures S3A–D, available at Annals of Oncology online)
DNase I and R-Cu (P < 0.0001). (P < 0.01–0.0001). Concurrent administration of CNPs, DNase I
We next examined whether cfCh neutralizing/degrading agents and R-Cu was highly effective in preventing DNA damage, apop-
namely, CNPs, DNase I and R-Cu could prevent the systemic re- tosis and inflammation in all organs examined (P < 0.05 to
lease of cfCh and inflammatory cytokines into the circulation <0.0001). In most cases the three cfCh neutralizing/degrading

γH2AX 0.2 NFκB

Control Control
Adriamycin Adriamycin
Adriamycin + CNPs 0.16 Adriamycin + CNPs
0.24 Adriamycin + DNase I Adriamycin + DNase I
Adriamycin + R-Cu Adriamycin + R-Cu
0.2
0.12
0.16
MFI
MFI

0.12 0.08

0.08 * **
** ***
** ** *** ** ** ** 0.04 ** ** * * **
* *** *** * ** * * ** * ** * * ** **
*
* ** ** **
0.04 ** ** ** **
* * ** ** ** *
** ** *** **
** ** ** ** ** ** *** ** *
* **
** **
*
** ** * * ** ** * * ** ** *** ** *** ** ** * **
* * * ** ** * * * * * ** ** * * * * ** *
0 0
Lung Liver Heart Brain Ovary Skin Small Lung Liver Heart Brain Ovary Skin Small
intestine intestine

Active caspase-3 IL6

0.16 Control Control
Adriamycin 1.2 Adriamycin
Adriamycin + CNPs Adriamycin + CNPs
0.12 Adriamycin + DNase I Adriamycin + DNase I
1
Adriamycin + R-Cu Adriamycin + R-Cu
0.8
MFI

0.08 **
MFI

0.6 *
** **
* ** ** **
* ** ** 0.4 ** ** * **
0.04 * ** ** ** ** ** ** ** **
** ** * * ** * * ** * ** * ** ** **
** ** ** * * ** ** ** * ** ** ** *** ** ** ** ** ** ** ** ** * ** **
** * * ** * ** ** ** ** 0.2 ** ** ** ** *** * ** ** **
**
*
** ** **
***
* * * **
* *** * ** ** * *** ** ** ** * **
*
**
** *
0
Lung Liver Heart Brain Ovary Skin Small Lung Liver Heart Brain Ovary Skin Small
intestine intestine

Figure 3. Prevention of cellular DNA damage (cH2AX), apoptosis (active caspase 3) and inflammation (NFjB and IL-6) in various tissues fol-
lowing a single i.p. injection of adriamycin by concurrent treatment with CNPs, DNase I and R-Cu. Estimation of cH2AX and active caspase-3
were done at 24 h whereas NFjB and IL-6 were estimated at 72 h. The bars represent mean values 6 SEM of five animals in each group.
Statistical comparison was carried out using unpaired Student’s t-test. **P <0.01; ***P <0.001; ****P <0.0001.

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 Annals of Oncology
agents could reduce the activated levels of the tissue parameters
to those obtained in control animals (supplementary Table S2,
available at Annals of Oncology online). These data show that
cfCh neutralizing/degrading agents could inhibit adriamycin-
induced DNA damage, apoptosis and inflammation indicating
that cfCh were responsible for inducing these cellular
pathologies.
With respect to the other chemotherapy agents namely, cyclo-
phosamide, cisplatin, methotrexate and paclitaxel, we restricted
Original article
our investigation to the study of activation of H2AX and NFjB in
brain and PBMCs (Figure 4A–D and supplementary Figures
S4A–D, available at Annals of Oncology online). All four chemo-
therapeutic agents induced DNA damage and inflammation in
brain and PBMCs, albeit to varying degrees (P < 0.05 to
<0.0001). However, in each case, the administration of CNPs,
DNase I and R-Cu concurrently with the respective chemothera-
peutic agents caused significant reduction in the activation of
these parameters (P < 0.05 to <0.0001).

A γ H2AX
0.20 Control Control
0.20
Cyclophosphamide Cyclophosphamide
Cyclophosphamide + CNPs Cyclophosphamide + CNPs
0.15 0.15
Cyclophosphamide + DNase I Cyclophosphamide + DNase I
Cyclophosphamide + R-Cu Cyclophosphamide + R-Cu
MFI

MFI
0.10 0.10

* *
0.05 * * * 0.05 * * *
* * * * * * * *
* * * * * * * *
* * * * *
* *
0.00 0.00
Brain PBMCs
NFκB
0.15 Control 0.3 Control
Cyclophosphamide Cyclophosphamide
Cyclophosphamide + CNPs Cyclophosphamide + CNPs
0.10 Cyclophosphamide + DNase I 0.2 Cyclophosphamide + DNase I
Cyclophosphamide + R-Cu Cyclophosphamide + R-Cu
MFI

MFI

0.05 * * 0.1 *
* * * *
* * * * *
* * * * * *
* * * *
* *
0.00 0.0
Brain PBMCs
B 0.25 γ H2AX
0.20
Control Control
0.20 Cisplatin Cisplatin
0.15
Cisplatin + CNPs Cisplatin + CNPs
0.15 Cisplatin + DNase I Cisplatin + DNase I
MFI
MFI

Cisplatin + R-Cu 0.10 Cisplatin + R-Cu

0.10
0.05 * * * *
0.05 * * * * *
* * * * * * *
* * * * * * *
0.00
* 0.00
Brain PBMCs
NFκB
0.20 Control 0.3
Cisplatin Control
0.15 Cisplatin + CNPs Cisplatin
Cisplatin + DNase I 0.2 Cisplatin + CNPs
Cisplatin + R-Cu Cisplatin + DNase I
MFI

MFI

0.10 *
* * Cisplatin + R-Cu
* * *
* * * 0.1 *
0.05 * * *
* * * *
* * *
* *
* *
0.00 0.0
Brain PBMCs

Figure 4. Prevention of cellular DNA damage (cH2AX) and inflammation (NFjB) in brain and PBMCs of mice following a single i.p. injection
of cyclophosphamide (A), cisplatin (B), methotrexate (C) and paclitaxel (D) by concurrent treatment with CNPs, DNase I and R-Cu. All animals
were sacrificed at 72 h except in case of the cH2AX-cisplatin experiment wherein animals were sacrificed at 24 h. The bars represent mean
values 6 SEM of five animals in each group. Statistical comparison was carried out using unpaired Student’s t-test. *P <0.05; **P <0.01;
***P <0.001; ****P <0.0001.

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 Original article Annals of Oncology
C γ H2AX
0.20 0.20
Control Control
Methotrexate Methotrexate
0.15 Methotrexate + CNPs 0.15 Methotrexate + CNPs
Methotrexate + DNase I Methotrexate + DNase I

MFI
MFI Methotrexate + R-Cu Methotrexate + R-Cu
0.10 0.10

** ** ** ** ** **
0.05
** ** ** ** 0.05 *
** **
0.00 0.00
Brain PBMCs

NFκB
0.20 0.20
Control Control
Methotrexate Methotrexate
0.15 Methotrexate + CNPs 0.15 Methotrexate + CNPs
Methotrexate + DNase I Methotrexate + DNase I
MFI

Methotrexate + R-Cu

MFI
Methotrexate + R-Cu
0.10 0.10
* ** **
** ** ** **
0.05 0.05 * *
** ** **
* * *
0.00 0.00
Brain PBMCs

D γ H2AX
0.25 Control 0.20 Control
Paclitaxel Paclitaxel
0.20 Paclitaxel + CNPs Paclitaxel + CNPs
Paclitaxel + DNase I 0.15 Paclitaxel + DNase I
0.15 Paclitaxel + R-Cu Paclitaxel + R-Cu
MFI

MFI

** 0.10
0.10 ** ** **
** ** **
** 0.05 ** ** **
0.05 ** **
** ** **
0.00 0.00
Brain PBMCs

NFκB
0.20 0.15 Control
Paclitaxel
Control Paclitaxel + CNPs
0.15 Paclitaxel Paclitaxel + DNase I
Paclitaxel + CNPs 0.10 Paclitaxel + R-Cu
**
MFI

MFI

Paclitaxel + DNase I **
0.10 **
Paclitaxel + R-Cu ** ** ** **
0.05 **
0.05 ** ** ** **
* * * *
0.00 0.00
Brain PBMCs

Figure 4. Continued.

We next examined the effect of CNPs, DNase I and R-Cu
in preventing neutropenia caused by adriamycin. A single injec-
tion of the drug (10 mg/kg) induced a significant reduction in
total leukocyte count that reached a nadir at 24 h and remained
low for several days (Figure 5A–C). Concurrent administration
of CNPs, DNase I and R-Cu prevented neutropenia to a great ex-
tent throughout its course. Values at most time-points, with
a few exceptions, were statistically significant (P < 0.05 to
<0.0001).
Finally, we show that death from a lethal dose of adriamycin
(20 mg/kg) could be prevented or delayed by concurrent admin-
istration of cfCh neutralizing/degrading agents (Figure 6). All
animals in the control (adriamycin alone) group died by day 6,
whereas 50% and 30% of animals treated with R-Cu and DNase I,
respectively, survived (P < 0.0001 and P < 0.05, respectively).
With respect to the group of mice treated with CNPs, although all
animals ultimately died, their survival was significantly pro-
longed (P < 0.01).

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 Annals of Oncology Original article
A Adriamycin
14000 ** Adriamycin + CNPs
** **
** **
12000 * **
**
10000 ns
**
** * **

TLC/mm3
8000 ** * **
*
*
6000 ns **
** **
*
4000

2000

0 24 48 72 96 120 144 168 192 216 240

Time (h)
B
16000 Adriamycin
** Adriamycin + DNase I
**
14000
**
12000 ** ** **
* *
ns
10000 ns
TLC/mm3

8000 ** ** **
* ** **
* * *
ns
6000 ns

4000

2000

0 24 48 72 96 120 144 168 192 216 240

Time (h)
C 12000
* Adriamycin
Adriamycin + R-Cu
10000 ns
**
** **
* ** ** ** **
8000 ** * ns *
** *
TLC/mm3

** *
6000

4000

2000

0 24 48 72 96 120 144 168 192 216 240

Time (h)

Figure 5. Prevention of neutropenia over time following a single injection of adriamycin by concurrent treatment with CNPs, DNase I and
R-Cu. TLC, total leukocyte count. Statistical analysis was carried out using unpaired Student’s t-test at each time point (n ¼ 5 mice at each
time-point).

Discussion
It is the current belief that toxic side-effects of chemotherapy-
induced systemic cellular DNA damage and inflammation are en-
tirely caused by the drugs themselves. However, a puzzle relating
to chemotherapy toxicity has remained unaddressed; this is,
while the half-life of most chemotherapeutic agents is around
24 h [5], their toxic effects last for several days. For example, bone
marrow suppression following chemotherapy reaches a nadir
around 7–14 days and sore mouth is at its worst between 5 and 14
days [5]. For these reasons, chemotherapy cycles are usually
spaced at 3-weekly intervals to allow for tissue recovery to occur.
Our results suggest a different mechanism underlying chemo-
therapy toxicity. They suggest that the cellular damage and in-
flammation that are directly attributable to chemotherapy
drugs are marginal and that most of the toxic side-effects of
chemotherapy are induced by cfCh released from the initial

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 Original article
100
80
Percent survival
60
40
20
0
0 1 2 3
Figure 6. Survival of mice treated with a single lethal dose of adriamycin with and without concurrent treatment with CNPs, DNase I and
R-Cu. Comparison between groups was conducted by Kaplan–Meier survival analysis using log-rank test. Control versus CNPs (P < 0.01); con-
trol versus DNase I (P < 0.05); control versus R-Cu (P < 0.0001) (n ¼ 10 mice in each group).
round of drug-induced cell death triggering a cascading effect
whereby dead cells release more cfCh causing further rounds of
DNA damage, apoptosis and inflammation thereby exaggerating
or amplifying the toxic effects. This proposal helps to explain why
the duration of toxic effects of chemotherapy are disproportion-
ally prolonged compared with the half-life of the drugs
themselves.
In confirmation of our earlier report using cfCh isolated from
serum of human subjects [6], we demonstrate that cfCh isolated
from mouse serum can also activate cH2AX and active caspase 3
when added to cells in culture (Figure 1, upper two panels and
supplementary Figure S1, available at Annals of Oncology online,
upper two panels). Although inflammation is a major side-effect
of chemotherapy, the mechanism by which chemotherapy-
induced inflammation is triggered is poorly understood [2, 13].
We show that cfCh are directly responsible for inducing inflam-
mation and that the activation of pro-inflammatory cytokines
NFjB and IL-6 can be abrogated by concurrent treatment with
cfCh degrading/neutralizing agents (Figure 1, lower two panels
and supplementary Figure S1, available at Annals of Oncology on-
line lower two panels).
We demonstrate that CNPs, DNase I and R-Cu can virtually
abolish tissue DNA damage, apoptosis and inflammation result-
ing from chemotherapy. There was remarkable suppression of
these toxic pathologies in all tissues examined namely, lung, liver,
heart, brain, ovary, skin small intestine and PBMCs (Figures 3
and 4). cfCh release from dying cells following chemotherapy
treatment appears also to be responsible for bone marrow sup-
pression. The prolonged neutropenia following a single injection
of adriamycin which lasted for 8–10 days supports our hypothesis
that the initial round of cell death inflicted by adriamycin caused
a cascading effect whereby cfCh released from the initial round of
cell-death catalysed several further rounds of cfCh-induced cell
death resulting in a prolonged neutropenic effect. In any event,
we show that neutropenia could be prevented to a great extent by
concurrent administration of CNPs, DNase I and R-Cu. Finally,
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Annals of Oncology
Adriamycin
Adriamycin + CNPs
Adriamycin + DNase I
Adriamycin + R-Cu
4 5 6 7 8 9 10
Day
we show that the above agents can prevent adriamycin induced
lethality thereby implicating cfCh in causing death of mice fol-
lowing high-dose chemotherapy.
It is likely that suppression of DNA damage, apoptosis and in-
flammation by cfCh neutralizing/degrading agents is affected at
two levels. The first is mediated via a reduction in the blood levels
of cfCh (Figure 2A and B), while the second is mediated at a cellu-
lar level wherein cfCh released from dying cells are neutralized/
degraded and prevented from inducing damaging effects on by-
stander living cells. In support of the latter proposal, we have re-
cently shown that cfCh from dying cells can activate DNA
damage and inflammation in surrounding cells and that these
could be prevented by cfCh neutralizing/degrading agents both
in vitro and in vivo [7].
It could be argued that the agents that we have used to neutral-
ize/degrade cfCh may themselves be responsible for inactivating
the cytotoxic drugs thereby suppressing their toxic side-effects.
This is unlikely for two reasons: first, CNPs, DNase I and R-Cu
have different chemical compositions and modes of action with
respect to cfCh inactivation. It is unlikely that these three agents
will have same drug inactivating action. Second, we have used
five different cytotoxic drugs each one with a different mode of
action. It is again improbable that CNPs, DNase I and R-Cu,
which themselves have different modes of action, will interfere
with the diverse pathways involved in induction of cytotoxic ef-
fects of five disparate drugs.
Our results have significant clinical implications. They sug-
gest that cardiac, gastro-intestinal, neurological, cutaneous and
reproductive side-effects of chemotherapy which include car-
diac failure, nausea, vomiting, diarrhoea, neuro-toxicity, steril-
ity and hair-loss can be potentially prevented by administration
of cfCh neutralizing/degrading agents administered concur-
rently with chemotherapy. Similarly, bone marrow suppression
and the resultant neutropenia as well as the occasional fatality
resulting from chemotherapy may also appear to be preventable.
If our results are successfully translated in to the clinic, it might
Volume 28 | Issue 9 | 2017
 Annals of Oncology
help to bring relief to thousands of patients who are prescribed
chemotherapy for cancer worldwide every day.
Funding
This work was supported by the Department of Atomic Energy,
Government of India, through its grant CTCTMC to Tata
Memorial Centre awarded to IM (Project No. 900011).
Disclosure
The authors have declared no conflicts of interest.
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