17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry
Aim: Peritoneal or retro-peritoneal sarcomatosis related malignant ascites formation is a rare but serious consequence of the locoregional metastatic event. The present work aimed to study the effect of the Hsp90 inhibitor (17-AAG), an ansamycin analog, on cell cycle and DNA replication specific cha...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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irk-123456789-1386882018-06-20T03:05:58Z 17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry Chaklader, M. Das, P. Pereira, J.A. Law, A. Chattopadhyay, S. Chatterjee, R. Mondal, A. Law, S. Original contributions Aim: Peritoneal or retro-peritoneal sarcomatosis related malignant ascites formation is a rare but serious consequence of the locoregional metastatic event. The present work aimed to study the effect of the Hsp90 inhibitor (17-AAG), an ansamycin analog, on cell cycle and DNA replication specific chaperone-clients interaction in the event of peritoneal sarcoma related malignant ascites formation in mouse model at the late stage of malignant growth. Methods: We administered 17-AAG, an Hsp90 inhibitor, divided doses (330 μg/kg b.w./day for first five days then next ten days with166 μg/kg b.w./day) through intra-peritoneal route of inbred Swiss albino mice bearing full grown peritoneal malignant ascites of sarcoma-180. Our study was evaluated by peripheral blood hemogram analysis, malignant ascitic cytology, cell viability test, survival time and mitotic indexing. Furthermore, flowcytometric HSP90, TERT, CyclinD1, PCNA and GM-CSF expression analysis has been considered for special objective of the study. Results: Our experimental efforts reduced the aggressive proliferation of malignant ascites by drastic downregulation of TERT and cyclin D1 on the verge of cell cycle entry along with DNA replication processivity factor PCNA by directly modulating their folding machinery — heat shock protein 90. Consequently, we observed that malignant ascitic cells became error prone during the event of karyokinesis and produced micronucleus containing malignant cells with low viability. Peripheral neutrophilia due to over-expression of GM-CSF by the peritoneal malignant ascites were also controlled by the treatment with 17-AAG and overall, the treatment modality improved the median survival time. Conclusion: Finally we can conclude that 17AAG administration might serve as a prospective pharmacological agent for the management of peritoneal sarcoma related malignant ascites and throws light towards prolonged survival of the patients concerned. 2012 Article 17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry / M. Chaklader, P. Das, J.A. Pereira, A. Law, S. Chattopadhyay, R. Chatterjee, A. Mondal, S. Law // Experimental Oncology. — 2012. — Т. 34, № 2. — С. 90-96. — Бібліогр.: 24 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138688 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Chaklader, M. Das, P. Pereira, J.A. Law, A. Chattopadhyay, S. Chatterjee, R. Mondal, A. Law, S. 17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry Experimental Oncology |
| description |
Aim: Peritoneal or retro-peritoneal sarcomatosis related malignant ascites formation is a rare but serious consequence of the locoregional metastatic event. The present work aimed to study the effect of the Hsp90 inhibitor (17-AAG), an ansamycin analog, on cell cycle and DNA replication specific chaperone-clients interaction in the event of peritoneal sarcoma related malignant ascites formation in mouse model at the late stage of malignant growth. Methods: We administered 17-AAG, an Hsp90 inhibitor, divided doses (330 μg/kg b.w./day for first five days then next ten days with166 μg/kg b.w./day) through intra-peritoneal route of inbred Swiss albino mice bearing full grown peritoneal malignant ascites of sarcoma-180. Our study was evaluated by peripheral blood hemogram analysis, malignant ascitic cytology, cell viability test, survival time and mitotic indexing. Furthermore, flowcytometric HSP90, TERT, CyclinD1, PCNA and GM-CSF expression analysis has been considered for special objective of the study. Results: Our experimental efforts reduced the aggressive proliferation of malignant ascites by drastic downregulation of TERT and cyclin D1 on the verge of cell cycle entry along with DNA replication processivity factor PCNA by directly modulating their folding machinery — heat shock protein 90. Consequently, we observed that malignant ascitic cells became error prone during the event of karyokinesis and produced micronucleus containing malignant cells with low viability. Peripheral neutrophilia due to over-expression of GM-CSF by the peritoneal malignant ascites were also controlled by the treatment with 17-AAG and overall, the treatment modality improved the median survival time. Conclusion: Finally we can conclude that 17AAG administration might serve as a prospective pharmacological agent for the management of peritoneal sarcoma related malignant ascites and throws light towards prolonged survival of the patients concerned. |
| format |
Article |
| author |
Chaklader, M. Das, P. Pereira, J.A. Law, A. Chattopadhyay, S. Chatterjee, R. Mondal, A. Law, S. |
| author_facet |
Chaklader, M. Das, P. Pereira, J.A. Law, A. Chattopadhyay, S. Chatterjee, R. Mondal, A. Law, S. |
| author_sort |
Chaklader, M. |
| title |
17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry |
| title_short |
17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry |
| title_full |
17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry |
| title_fullStr |
17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry |
| title_full_unstemmed |
17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry |
| title_sort |
17-aag mediates targeting of hsp90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin d1 during cell cycle entry |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2012 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138688 |
| citation_txt |
17-AAG mediates targeting of HSP90 limits tert activity in peritoneal sarcoma related malignant ascites by downregulating cyclin D1 during cell cycle entry / M. Chaklader, P. Das, J.A. Pereira, A. Law, S. Chattopadhyay, R. Chatterjee, A. Mondal, S. Law // Experimental Oncology. — 2012. — Т. 34, № 2. — С. 90-96. — Бібліогр.: 24 назв. — англ. |
| series |
Experimental Oncology |
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| fulltext |
90 Experimental Oncology 34, 90–96, 2012 (June)
17-AAG MEDIATED TARGETING OF HSP90 LIMITS TERT ACTIVITY
IN PERITONEAL SARCOMA RELATED MALIGNANT ASCITES
BY DOWNREGULATING CYCLIN D1 DURING CELL CYCLE ENTRY
M. Chaklader, P. Das, J.A. Pereira, A. Law, S. Chattopadhyay, R. Chatterjee, A. Mondal, S. Law*
Stem Cell Research and Application Unit, Department of Biochemistry and Medical Biotechnology,
Calcutta School of Tropical Medicine, Kolkata-700073, West Bengal, India
Aim: Peritoneal or retro-peritoneal sarcomatosis related malignant ascites formation is a rare but serious consequence of the lo-
coregional metastatic event. The present work aimed to study the effect of the Hsp90 inhibitor (17-AAG), an ansamycin analog,
on cell cycle and DNA replication specific chaperone-clients interaction in the event of peritoneal sarcoma related malignant as-
cites formation in mouse model at the late stage of malignant growth. Methods: We administered 17-AAG, an Hsp90 inhibitor,
divided doses (330 μg/kg b.w./day for first five days then next ten days with166 μg/kg b.w./day) through intra-peritoneal route
of inbred Swiss albino mice bearing full grown peritoneal malignant ascites of sarcoma-180. Our study was evaluated by periph-
eral blood hemogram analysis, malignant ascitic cytology, cell viability test, survival time and mitotic indexing. Furthermore,
flowcytometric HSP90, TERT, CyclinD1, PCNA and GM-CSF expression analysis has been considered for special objective of the
study. Results: Our experimental efforts reduced the aggressive proliferation of malignant ascites by drastic downregulation of TERT
and cyclin D1 on the verge of cell cycle entry along with DNA replication processivity factor PCNA by directly modulating their
folding machinery — heat shock protein 90. Consequently, we observed that malignant ascitic cells became error prone during the
event of karyokinesis and produced micronucleus containing malignant cells with low viability. Peripheral neutrophilia due to over-
expression of GM-CSF by the peritoneal malignant ascites were also controlled by the treatment with 17-AAG and overall, the
treatment modality improved the median survival time. Conclusion: Finally we can conclude that 17AAG administration might serve
as a prospective pharmacological agent for the management of peritoneal sarcoma related malignant ascites and throws light towards
prolonged survival of the patients concerned.
Key Words: 17-AAG, HSP90, TERT, sarcoma-180, malignant ascites, neutrophilia.
Malignant peritoneal ascites formation from
the primary neoplastic growth is a very critical and
troublesome medical emergency faced by most of the
medical and surgical oncologists. In the context of car-
cinomatosis related malignant peritoneal ascites gen-
eration is a common feature [1]. In the modern clinical
practice, peritoneal or retro peritoneal sarcomatosis
related malignant ascites formation and its proper
management is actually less discussed issue. Perito-
neal leiomyosarcoma, liposarcoma, carcinosarcoma
gastrointestinal stromal tumor and primary perito-
neal spindle cell sarcoma have primitive mesodermal
origin and can promote peritoneal malignant ascites
growth [2–5]. One unusual example of the peritoneal
sarcomatosis is peritoneal Kaposi’s sarcoma in HIV
patient which can initiate peritoneal malignant ascites
as a result of metastasis [6]. However, irrespective
of the peritoneal sarcomatous origin of ascites, the
main objective of our present study was two folds,
1) to understand the pathobiology of the peritoneal
sarcomatosis related ascites formation and 2) to find
out the proper molecular target for pharmacological
management of the said pathophysiology.
In this context, very few pharmacological and
surgical options are available to combat the situation
properly. Management of peritoneal ascites by locore-
gional treatment is an attractive option as the malignant
ascites remains confined to the intra-peritoneal cavity
during most of their natural history. Most of the con-
ventional routes of chemotherapy administration are
either intravenous or oral which are very effective but
have high risk of side effects too where loco-regional
chemotherapeutic treatment can be a better option.
So to bring down the unwholesome effect on non target
organs, locoregional chemotherapeutic administration
should be considered wherever it is possible [7–9].
In our study, we also showed the use of intraperitoneal
route of chemotherapy administration to manage the
experimentally induced malignant ascites.
As the drug resistance is a matter of concern
along with route of drug administration, the choice
of pharmacological agents and pharmacological target
to be taken under consideration. The present study
considers chaperone-Hsp90 which is the main phar-
macological target. Additionally we also consider the
status of some important Hsp90 specific clients in the
event of malignant ascites formation and progression.
In general, chaperones are ubiquitous, highly con-
served proteins which play a major role in the evolution
of most of the modern proteins [10–12]. Chaperones
are vital for all kind of cells for their whole lifespan and
it saves the cells from proteotoxic damages coming
from the external environment. Besides the assistance
of folding and refolding of protein, chaperones are
Received: April 30, 2012.
*Correspondence: E-mail: msuj2002@yahoo.co.in
Abbreviations used: GM-CSF — granulocyte macrophage-colony
stimulating factor; Hsp90 — heat shock protein 90; ILS — in-
creased life span; MI — mitotic index; MRA — malignancy related
ascites; MST — median survival time; PCNA — proliferating cell
nuclear antigen; 17-AAG — 17-allylamino-17-demethoxygeldana-
mycin; TERT — telomerase reverse transcriptase.
Exp Oncol 2012
34, 2, 90–96
Experimental Oncology 34, 90–96, 2012 (June) 91
also helpful for the cellular remodeling by maintaining
the cytoplasmic meshwork i.e. microtubular lattice
[13–15]. Chaperones target newly synthesized pro-
teins, mutant proteins, newly damaged proteins and
competing cytoplasmic lattice during cellular remodel-
ing. These above mentioned protein machineries are
collectively referred as “Clients”, which include cell
surface receptor to various cytokines and huge array
of protein molecules. In this scenario, pharmaco-
logical targeting of overloaded Hsp90, the molecular
chaperone, causes simultaneous degradation of client
proteins via an apparently understood mechanism
and results in retardation of neoplastic progression
[16–18]. Here we tried to unearth the mechanistic
insight of Hsp90 inhibitor i.e. 17-AAG mediated neo-
plastic growth inhibition by modulating telomerase re-
verse transcriptase (TERT) and cyclin D1. Both, TERT
and Cyclin D1 control cell cycle and cell proliferation
of normal and neoplastic cells. Previously we reported
that intraperitoneal administration of vincristine sul-
fate indirectly reduced TERT expression in experi-
mentally induced sarcomatosis by arresting the cells
at G2/M phase stage of cell cycle [9]. Previously it was
also reported that 17-AAG inhibit telomerase assem-
bly and activity in vitro in human JR8 melanoma cell
line [19]. Our present study considered direct target-
ing of TERT in vivo by inhibiting the Hsp90 through
17-allylamino-17-demethoxygeldanamycin (17-AAG),
which is a derivative of ansamycin family of antitu-
mor antibiotic — geldanamycin. Currently 17-AAG
is in Phase II clinical trial. In Phase-I trial, pharmaco-
dynamic activity of the drug was evaluated by mea-
suring Hsp70 activation and/or degradation of CDK4,
a kinase, which phosphorylates Cyclin D1 [20–24].
In normal condition, CDK4 mediated phosphorylation
of Cyclin D1 promotes cells to complete G1 phase
of cell cycle and enter in S phase. We wanted to study
the Cyclin D1 status after 17-AAG administration in the
context of sarcomatosis related peritoneal ascites
formation. Our study also addressed the peritoneal
sarcomatosis related peripheral blood neutrophilia due
to over expression of GM-CSF by peritoneal malignant
ascitic sarcoma. We have followed up the possible
modulation of GM-CSF production after 17-AAG ad-
ministration in the sarcoma bearing mice as GM-CSF
is one of the clients of Hsp90.
Ultimately, our present study tried to reduce chap-
erone overload in neoplastic condition like experimen-
tally induced sarcomatosis related peritoneal ascites
in mouse model by geldanamycin derivatives 17-AAG
and hopefully it will bring some new light to those pa-
tients having aforementioned secondary neoplastic
complicacy.
MATERIAL AND METHODS
Animals. 10–12 weeks old Swiss albino mice
(Mus musculus) weighing 20 to 24 g were selected
from an inbred colony maintained under controlled
room temperature (22±2°C) in the animal house of our
institute. During the course of the study the animals
were fed on a diet consisting of 25.0% protein, 10.0%
fiber, 5.0% fat, 9.0% minerals and access to water
ad libitum, under standard conditions with a 12 h light
dark period. Maximum six animals were housed in cage
containing sterile paddy husk as bedding through
out the experiment. The procedures followed were
in agreement with the approved guide for the care
and use of laboratory animals and Institutional Animal
Ethical Committee (IAEC).
Sarcoma-180 transplantation. In this experiment,
Crocker’s sarcoma (ascitic sarcoma-180) was taken
as the source of malignant peritoneal ascites develop-
ment. The ascitic fluid was drawn using an 18-gauge
needle into sterile syringe. 100 μL of ascitic fluid was
tested for microbial contamination, then viability was
determined by erythrosin-B exclusion test and cells
were counted using hemocytometer. The ascitic fluid
was suitably diluted in sterile phosphate buffer saline
to get a concentration of 3 × 106 cells/ml of ascitic sus-
pension. This was injected intraperitoneally to obtain
malignant ascitic effusion within 12 to 14 days.
Chemotherapy administration in vivo. We have
used an Hsp90 inhibitor, 17-AAG in two divided doses
(330 μg/kg b.w./day for first five days then next ten
days with166 μg/kg b.w./day) through intra-peritoneal
route of inbred Swiss albino mice bearing full grown
peritoneal malignant ascites of sarcoma-180 after
twelve days of inoculation. The above drug administra-
tion schedule with fully developed tumor burden mice
was considered for mimicking the late stage condition
of malignant peritoneal ascites complicacy in human
being regarding any peritoneal or retroperitoneal
sarcomatosis.
Peripheral blood hemogram study. We have
randomly selected experimental and control animals
from respective cages for blood hemogram profiling.
Approximately 200 μL of blood was collected in hepa-
rinized vial by tail vein puncture from both the groups
of animals. The hemoglobin concentration, total white
blood cells (WBC), red blood cells, platelets, reticu-
locytes and differential WBC count were performed
as per standard laboratory techniques.
Malignant ascitic cytology study. Malignant
peritoneal ascitic fluid was drawn from both 1) control
untreated malignant ascites bearing animals and 2)
from 17-AAG treated malignant ascites bearing ani-
mals and washed by phosphate-buffered saline (PBS).
Slides were prepared to study the cytology without
damaging the cell membrane stained by Giemsa.
Erythrosin-B dye exclusion study. After 12–
14 days of S-180 inoculation, 1.0 to 2.0 ml of malignant
ascitic fluid was collected from control and 17-AAG
treated animals and diluted in PBS to maintain a cell
density of 1.0×106/ml. The survival rate of tumor cells
was evaluated by the Erythrosin-B (Sigma) dye ex-
clusion technique using a hemocytometer (Rohem
India) under an optical binocular light microscope
(×400 magnification, Olympus).
Survival study. Chemotherapeutic efficacy can
be accessed by median survival time (MST) and
92 Experimental Oncology 34, 90–96, 2012 (June)
increased life span (%ILs) of tumor bearing animals
by the following formula.
%ILS = MST of the treated group ─ MST of the
control group × 100 / MST of the control group
Enhancement of life span (25% and above)
of treated group over the control was considered
as effective antitumor response. Additionally, we also
studied growth pattern of the treated and untreated
animals at regular interval which helped us to correlate
the effect of 17-AAG on tumor burden of the animals.
Mitotic index study. Intraperitoneal treatment
of 17-AAG might caused metaphase arrest of ma-
lignant peritoneal ascites in the peritoneum within
90 min of drug administration. Peritoneal ascitic fluid
was collected by aspiration and washed with normal
saline (0.9% NaCl) followed by further processing
in hypotonic solution of 0.075M KCl at 37˚C for about
15–25 min. Thereafter, cells were treated with acetic-
methanol (1:3) fixative solution following vigorous
vortexing to avoid the cell coagulation. The process
was repeated for three times to increase the number
of fixed cells. Slides were prepared through fixation
by heat followed by Giemsa staining.
Mitotic indexing:
Mitotic Index (MI) = TDC × 100/TC
(TDC= Total dividing cells, TC=Total Cell)
Cell cycle analysis. Flowcytometric cell cycle
pattern was studied from 17-AAG treated and 17-
AAG untreated control cell population by propidium
iodide staining kit as per manufacturer instruction.
Here, 20 000 events were analyzed by BD-FACS Cal-
libur (Becton Dickenson, USA), using Cell Quest Pro
software.
Flowcytometric analysis of malignancy related
ascites for Hsp90, TERT, CyclinD1, PCNA and GM-
CSF expression. In both the groups Hsp90, TERT,
Cyclin D1, PCNA and GM-CSF expression pattern
in peritoneal malignant ascites were studied by 15 min-
utes 1% Para-formaldehyde fixation and permiabiliza-
tion by 90% ice-cold methanol followed by intracellular
staining as described later. Five samples were incu-
bated for 30 min with 2 μL rabbit anti Hsp90 antibody
(Cell Signaling Technology, Canada) , rabbit anti TERT
antibody (Santa Cruz Biotechnology, USA), Anti Cyclin
D1-FITC tagged antibody (Abcam, UK), PCNA (Santa
Cruz Biotechnology, USA), and Anti GM-CSF –FITC an-
tibody (Biolegend, USA) respectively followed by addi-
tion of 2 μL anti rabbit IgG-Alexafluor-488 (Invitrogen,
USA) in untagged primary antibody coupled cells and
incubated further in dark for 30–35 min. Excess fluo-
rescence was then washed off with PBS. Samples were
analyzed by BDFACS Callibur (Becton Dickenson,
USA) using Cell Quest Pro software.
Statistics. All the value of hemogram, cell viability,
mitotic index and FACS data were expressed as mean
± SD (standard deviation). Statistical analysis was
performed by Paired t Test (2α = 0.05) and each ex-
periment was performed three times.
RESULTS
Peripheral blood. Untreated sarcomatosis re-
lated ascites bearing animals showed leukemia like
condition during peritoneal sarcoma burden condi-
tion and mostly sudden overshoot of hemoglobin,
reticulocytes, RBC and WBC count. Mainly, in differ-
ential count, untreated animals showed an unusual
elevation of neutrophils in comparison to lymphocyte
population. In the normal Swiss albino strain of mouse,
peripheral blood comprised of a higher proportion
of lymphocyte (60–70%) over neutrophils (25–35%).
On the other hand, treatment with 17-AAG (after
12 days of experimentally induced peritoneal sarco-
matosis) showed a suppression of sudden neutrophilia
in the differential count along with normal standards
of total Hb, Reticulocyte, RBC and WBC count. All
the results of comparative hemogram were verified
by Student’s‘t’ test and were statistically significant
(Fig. 1 a, b, and Table 1).
a
b
Fig. 1. (a) represented peripheral blood neutrophilia in the un-
treated group of animals with sarcomatosis related malignant
ascites in peritoneum. Appearance of banded nucleated neu-
trophils in peripheral blood of untreated animals was the mani-
festation of malignant cell mediated GM-CSF over production.
In contrary, (b) showed almost reversed hematological picture
in the 17-AAG treated group of animals, which revealed a normal
neutrophil count along with standard nuclear segmentation
Malignant ascitic cytology. Light microscopy
of untreated and 17-AAG treated malignant peritoneal
sarcomatosis related ascites showed the presence
of usual large cells with high cytoplasmic to nuclear
ratio. 17-AAG treated malignant cells showed cytoplas-
Experimental Oncology 34, 90–96, 2012 (June) 93
mic-nuclear asynchrony by producing syncytial cells.
Additionally, a number of micronuclei were observed
inside the 17-AAG treated syncytial malignant cells.
Cytoplasmic basophilia was also evident in untreated
Table1. Peripheral blood hemogram of untreated and 17-AAG treated groups of animals
Parameters 9th days 15th days 18th days 24th days 30th days 45th days
P value (Each ex-
periment repeat-
ed for 3 times)
Hemoglobin (g/dl)
Untreated S-180 animal 16.93±0.34 10.44±0.40 15.64± 0.6 ** ** ** P<0.0001Treated S-180 animal **** 12.75±0.53 11.54±1.0 10.50±0.5 12.60±0.1 12.75±0.2
Reticulocyte (%)
Untreated S-180 animal 1.56±0.1 1.38±0.3 1.42± 0.34 ** ** ** P<0.0001Treated S-180 animal **** 1.26±0.06 0.77± 0.1 1.95±0.3 1.92 0.15 1.68±0.13
Total R Total RBC count (×106 cell/ μL)
Untreated S-180 animal 13.87±0.1 6.16±0.20 6.55±0.30 ** ** ** P<0.001Treated S-180 animal **** 6.54±0.12 3.19±0.11 2.26±0.21 2.88±0.1 4.59±0.05
Total WBC count (×104 cell/μL)
Untreated S-180 animal 7.8±0.5 6.7±0.13 6.7±0.20 ** ** ** P<0.0001Treated S-180 animal **** 5.3±0.21 5.6±0.14 1.94± 0.3 3.96±0.23 4.11±0.5
Differential count (%)
Untreated
P<0.0001
Lymphocyte
Neutrophils
Eosinophils
Basophiles
Monocyte
10±1.3
87±1.5
0
0
3±0.2
11±2.1
86±2.3
0
0
3±1.1
15±1.24
84±1.5
0
0
1± 0.01
** ** **
Treated
Lymphocyte
Neutrophils
Eosinophils
Basophiles
Monocyte
****
20±1.2
75±0.5
0
0
5±0.01
28±2.0
71±1.3
0
0
1±0.2
40±0.5
60±0.75
0
0
0
53±2.1
40±1.3
0
0
7±1.2
47±2.0
53±.0.5
0
0
0
Notes: ** Untreated malignant ascites bearing animals more or less survived up to 18 days from 1st day of passaging.**** We started the 17-AAG treatment
after 12 days of tumor passaging.
b
d
a
c
Fig. 2. (a) and (b) showed ascites cytology in the untreated group of animals. High cytoplasmic to nuclear ratio was evident from
(a). Cytoplasmic basophilia was also documented in (b) with a few contaminating lymphocytes. (c) and (d) revealed the ascites
cytology after 17-AAG treatment, which made malignant sarcoma cells error prone during the event of karyokinesis. Additionally,
cytoplasmic to nuclear ratio was altered and as a result heterogeneous population of malignant cells appeared in the peritoneum
of treated animals (c). Faulty karyokinesis was manifested by nuclear-cytoplasmic asynchrony and formation of micronucleus
containing syncytial cells (d)
94 Experimental Oncology 34, 90–96, 2012 (June)
malignant ascites in comparison to 17-AAG treated as-
cites. Furthermore, it was observed that geldanamycin
treatment also deformed the uniformity of the plasma
membrane of the experimental cells (Fig. 2 a–d).
Erythrosin-B dye exclusion study for cell viabil-
ity. In comparison to the untreated animals (99.53%±
0.6) the ascitic cell viability of the 17-AAG treated ani-
mals decreased to 43.71%±1.5 which was statistically
significant (P < 0.0001) (Fig. 3) and correlated well with
the efficacy of the 17-AAG treatment.
0
10
20
30
40
50
60
70
80
90
100
Untreated 17-AAG treated
%
o
f L
ive
tu
m
or
c
el
ls
Fig. 3. Erythrosin-B dye exclusion test for cell viability as-
sessment revealed that untreated malignant cells were more
or less 100% viable in comparison to the 17-AAG treated group.
On an average 44% cells were viable inside the peritoneum of the
treated group of animals
Survival study. MST and %ILS varied significantly
in both groups. MST value of untreated group was
10 days whereas the treated group MST showed
23 days. The survival range also improved from
3–11 days in untreated to 14–45 days in the treated
group. Increased life span (%ILs) was observed in the
17-AAG treated group (130%) in comparison to the
untreated group (Table 2).
Table 2. Effect of 17-AAG on MST and % increased life span (%ILS) of treated
peritoneal sarcomatosis related ascites bearing mice
Groups MST (Days) (Survival range) % ILS
Untreated 10 (3–10) _
17-AAG treated 23 (14–45) 130
Mitotic index of untreated and 17-AAG treated
peritoneal sarcoma. We designed the present experi-
ment to evaluate the effect of 17-AAG on the tumor cell
mitosis and chromosome equipartioning mechanism
during the event of metaphase. The untreated group
showed 60.57% ± 1.91 MI in comparison to 17-AAG
treated mice, which had 35.21 ± 1.30% MI (Fig. 4, a,
b). Most of the cells of the treated group were found
in the interphase stage of cell cycle in comparison
to the untreated animals (Fig. 4, b).
Cell cycle analysis. Unlike 0.24% Go/G1 cells
of untreated malignant ascites, 17AAG treated as-
cites showed 79.98% cells halted in G0/G1 phase.
On the other hand high percentage of S phase cells
(92.57%) was evidenced in untreated sample whereas
17AAG treatment reduced the number of S phase cells
(18.24%) in treated group of animals. G2/M phase
specific cells were 7.27% in case of untreated sam-
ple, whereas only 1.98% 17AAG treated cells were
in G2/M phase stage (Fig. 5).
Prophase
38%
Metaphase
10%
Anaphase
8%
Telophase
14%
Interphase
30%
Prophase
16%
Metaphase
7%
Anaphase
2%
Telophase
10%
Interphase
65%
a
b
Fig. 4. Mitotic index (MI) of untreated group (a) represented
60.57% cells in mitosis and very negligible amount of cells
were present in metaphase which demonstrated the rapid cell
cycling nature of untreated malignant cells. Nearly 38% cells
were in Interphase stage. (b) revealed the MI of 17-AAG treated
group where 17-AAG treatment blocked the cells at Interphase
(65%) and very low amount of cells were at metaphase (7%)
0 200
200
160
120
80
40
20
400 600
FL2-A
Co
un
ts
800 1000 0 200
200
160
120
80
40
20
400 600
FL2-A
Co
un
ts
800 1000
Fig. 5. Flowcytometric cell cycle study of untreated and 17AAG
treated malignant ascites. M1, M2 and M3 are representative
virtual marker of G0/G1, S and G2/M phases respectively
Flowcytometric analysis of Hsp 90 and its cli-
ent proteins. Flowcytometric study of Hsp90, TERT,
Cyclin D1, PCNA and GM-CSF expression pattern
in peritoneal malignant sarcoma related ascites in the
17-AAG treated group showed downregulation of TERT
(60.01 ± 1.22), cyclin D1(3.32 ± 0.24), PCNA(60.23 ±
0.02), GM-CSF (61.43 ± 0.4) and Hsp90 (209 ± 2.65)
expression in comparison to the untreated group
(TERT (172.90 ± 2.07), cyclin D1 (14.01± 0.2), PCNA
(73.80 ± 0.1), GM-CSF (70.15 ± 1.2) and Hsp90 ex-
pression (248 ± 2.65)] (Fig. 6 and Table 3 ).
Table 3. Flowcytometrically determined mean fluorescence intensity (MFI)
values of Hsp90 and its clients represented comparative expression pat-
tern in untreated and treated groups of animals
Target proteins MFI of untreat-
ed samples
MFI of treated
samples
P value (Each experiment
repeated for 3 times)
Hsp90 209 ± 2.65 46.32 ± 2.1 P<0.0001
TERT 172.90 ± 2.07 60.01 ± 1.22 P=0.0003
Cyclin D1 14.01± 0.2 3.32 ± 0.24 P=0.0001
PCNA 73.80 ± 0.1 60.23 ± 0.02 P<0.0001
GM-CSF 70.15 ± 1.2 61.43 ± 0.4 P<0.0001
DISCUSSION
In the present study we emphasized on the forma-
tion of peritoneal sarcomatosis (sarcoma-180) related
malignant ascites and its associated pathophysiologies
in syngenic adult Swiss albino mice. In our previous
study we already showed that intraperitoneal vincristine
administration reduced malignant ascite burden inside
Experimental Oncology 34, 90–96, 2012 (June) 95
the murine peritoneum by downregulating cellular
senescence switch — TERT, a main regulatory com-
ponent of the telomerase enzyme [9]. In corroboration
with the previous study, here we again targeted TERT
by a more efficient manner which was related to antago-
nizing Hsp90 by its newly developed pharmacological
inhibitor-17-AAG. The Hsp90 inhibitor 17-AAG is the
derivative of ansamycin and it competes with cellular
ATP to bind at NH2-terminal ATP/ADP binding domain
of Hsp90 to block its chaperone functions, leading
to destabilization and proteosomal degradation of the
client proteins (e.g. TERT, cyclin D1 etc). Deregulation
of the client’s folding procedure by Hsp90 brought
a number of direct and indirect consequences inside
the cells. Our present work also revealed the changes
step by step in the event like sarcoma related malignant
ascites development inside the murine peritoneum.
0 50
200
160
120
80
40
20
100 150
Cyclin D1
Co
un
ts
200 250 0 50
200
160
120
80
40
20
100 150
GM-CSF
Co
un
ts
200 250
0 50
200
160
120
80
40
20
100 150
HSP 90
Co
un
ts
200 250
0 50
200
160
120
80
40
20
100 150
TERT
Co
un
ts
200 250
0 50
200
160
120
80
40
20
100 150
PCNA
Co
un
ts
200 250
Isotype
Untreated
17-AAG treated
Fig. 6. Comparative flowcytometric analysis of Hsp90 and its client
proteins in the untreated and 17-AAG treated groups of animals
In the untreated group we observed abnormal periph-
eral blood hemogram with a huge overshoot of neutrophil
population in comparison to lymphocytes. Furthermore
this peripheral blood neutrophilia was steadily present
and often, in some experimental animals, lymphocytes
and other granulocytes were totally replaced by afore-
mentioned neutrophilia in peripheral blood during differ-
ential count. The most possible explanation of the per-
sisting peripheral neutrophilia in ascitic sarcoma bearing
mice was due to the constant over stimulation of resident
bone marrow neutrophil population by endogenous
GM-CSF which was not found in normal condition. The
overshoot of GM-CSF production as evidenced by FACS
analysis furthered validated the reason of neutrophilia
in untreated group. The immature marrow neutrophil
pool (mostly banded neutrophils) shifted from endosteal
niche to vascular niche to peripheral blood due to higher
expression of GM-CSF. The reason behind the over
expression of GM-CSF by sarcoma cells is still obscure.
Constitutive expression of any protein is actually the result
of mutation and these mutated proteins usually crowd the
cellular environment rapidly. Ramification of the unfolded
mutated protein is generally managed by over expressed
Hsp90 in response to oncogenic stress. So, in the present
study we targeted the Hsp90 by 17-AAG, resulted in de-
regulation in refolding process of GM-CSF by competitive
binding with Hsp90 and produced faulty unstable non-
functional proteins (GM-CSF) in cellular environment and
consequently stopped the procedure of over stimulation
of marrow neutrophil storage. Ultimately the beneficial
effect of 17-AAG was documented in the post treatment
hemogram of the treated experimental mice.
We observed micronucleus formation and nuclear-
cytoplasmic asynchrony in the treated group in com-
parison to the untreated group. Nuclear-cytoplasmic
asynchrony with more than two nucleoli was due to the
deregulation at cytokinesis and polyploidy induc-
tion in malignant cells. But, multinucleated cells with
presence of one or two or three micronuclei and one
large nucleus were the manifestation of improper as-
sortment of chromosome during metaphase stage.
It evidenced deregulation of Hsp90 and cytoskeletal
protein interaction during metaphase in presence
of Hsp90 inhibitor like 17-AAG. Interestingly no sig-
nificant amounts of malignant cells were found at the
metaphase stage during the mitosis study and mitotic
indexing. Our previous study related to vincristine re-
vealed that the vinca alkaloid reduced the mitotic index
of malignant cells by metaphase blocking mechanism
[9]. The present study established the decrease
of MI of the malignant cells without blocking at meta-
phase stage by 17-AAG treatment. However, in com-
parison to the untreated group, treated group showed
higher percentage of cells at interphase stage which
was furthered explained by flowcytometric investiga-
tion of cell cycle specific marker protein expression.
We have considered the status of a few important
cell cycle specific proteins by flow cytometry e.g.
cyclinD1, PCNA and TERT to clarify the increase of in-
terphase cells. Cyclin D1 was used as G0/G1 phase
marker. PCNA was used for S-phase specific marker
where DNA replication takes place and the TERT was
used as the late S phase to early G2 phase entry
marker which causes cellular immortality. The 17-AAG
treated group showed drastic downregulation of Cyclin
D1 in comparison to the untreated group which signi-
fied the direct competition of Cyclin D1 with 17-AAG
for the N-terminal of Hsp90 and the remnant unfolded
Cyclin D1 degraded soon. As a result of Cyclin D1 de-
generation, significant amount of cells were blocked
at G0/G1 stage which might be the possible reason for
the increased interphase cell count during MI.
96 Experimental Oncology 34, 90–96, 2012 (June)
A moderate depression of PCNA was observed
in the treated group in comparison to the untreated
group. 17-AAG treatment hampered PCNA expression
and stability during the DNA replication. Downregu-
lated PCNA in 17-AAG treated group also signified that
very few numbers of cells with active DNA replication
procedure were present in S phase. The TERT expres-
sion data also added an extra dimension in the context
of cell cycling status of the treated group. Unlike un-
treated group, treated group showed a sharp down-
regulation of TERT expression, which manifested two
things: 1) 17-AAG efficiently blocked Hsp90 guided
client processing mechanism for which cancer cell
proliferation and self renewed process was under
limitation as both were being maintained by TERT. 2)
Low viability of treated peritoneal sarcoma cells due
to improper DNA end replication.
In conclusion, we can say that 17-AAG treatment
mediated controlling of a number of oncoprotein
expression along with Hsp90 itself increase the life
expectancy of the treated animals group in compari-
son to untreated one. Targeting Hsp90 by 17-AAG also
caused overcrowding of misfolded protein in malignant
cells which ultimately reduced tumor burden in mice
by diminished tumor cell viability and flowcytometric
TERT expression study would be considered as a novel
diagnostic and prognostic tool for sarcoma progression.
CONFLICT OF INTEREST
All authors unanimously declared no potential
conflict of interest.
ACKNOWLEDGEMENT
We are thankful to the Director of the Calcutta
School of Tropical Medicine.
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