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thesis entitled

Immune system activation by
Cisplatin and “Poly-plat”
presented by

Kyunghec Burkitt

has been accepted towards fulﬁllment
of the requirements for

MS degree in 20010343)-

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aAYllb 132911}0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

moo WWW“

WUNE SYSTEM ACTIVATION BY CISPLATIN
AND “POLY-PLAT”

By

Kyunghee Burkitt

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Zoology

2000

ABSTRACT
IMMUNE SYSTEM ACTIVATION BY CISPLATIN AND “POLY-PLAT”
By

Kyunghee Burkitt

Murine peritoneal macrophages, human ﬁbroblasts and human ovarian
teratocarcinoma cells were treated with cisplatin (10 ug/ml) and “poly-plat”

(10 pig/ml) for 2h and cultured for 2-96h. Aﬁer “poly-plat” treatment, macrophages
developed cytoplasmic extensions much faster and also secrete higher levels of
interleukin-2 (IL-2), compared to cisplatin. “Poly-plat” treated human ﬁbroblasts also
showed signiﬁcant release of IL-2 (138 pg/ml), compared to cisplatin treated (64
pg/ml) or normal controls (38 pg/ml) after 8h of treatment. However, human ovarian
teratocarcinoma cells didn’t show signiﬁcant increase of IL-2 levels after “poly-plat”
treatment except only aﬁer 2h and 24h of treatment. Human ﬁbroblasts demonstrated
a 53% increase in cell numbers after “poly-plat” treatment, compared to 37% after
cisplatin. However, after “poly-plat” treatment, human ovarian carcinoma cells
showed an initial decrease (15%) in cell numbers and then an increase (23%) up to 24h
post-treatment. Based on our studies, we propose that “poly-plat” is more effective in

activation of the macrophages and human ﬁbroblasts in terms of various cytokines.

To my family

iii

ACKNOWLEDGEMENTS

The last four years at MSU have brought me many challenges and beautiful memories.
First of all, I would like to say a special thank to Dr. S.K. Aggarwal for his guidance and
support. I had so many difﬁculties the last couple of years. At times, I almost gave up.
But, I learned three valuable things, independence, conﬁdence and above all, modesty.
There were always people around me who supported me during difﬁcult situation.

Dr. Tanaka, Dr. Ross, and Judy Smith deserve my thanks for their special care and

direction. I also can’t forget about the loving and endless support from my family in Korea
I thank my father for his ﬁnancial support and critical guidance, and my mother for her warm
and cheerful mental support and my brother for his deep love, and ﬁnally my best friend
kyung-min for her support. There is also another best ﬁiend and my loving husband, Kevin
G. Burkitt, who is a my best critic and also counselor at all times as he will be in the future.

I really thank him for his devotion through my studies.

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

Page
LIST OF TABLES vi
LIST OF FIGURES vii
INTRODUCTION 1
MATERIALS AND METHODS 3
Cell culture 3

Drug treatment 4

Cell counting 4

Slide preparation 4

IL-2 analysis 5

RESULTS 6
DISCUSSION 24
CONCLUSION 28

 

 

REFERENCES 29

LIST OF TABLES
Table Page

1 Number of human ﬁbroblasts after drug treatment 14

 

2 Number of human ovarian teratocarcinoma cells after drug treatment ----------- 18

vi

Figure

10

ll

12

LIST OF FIGURES

Light micrographs of murine peritoneal macrophages at 2h after
treatment with cisplatin (10 ug/ml) and “poly-plat”(10 ug/ml)

Page

11

 

Light micrographs of murine peritoneal macrophages at 16h after
treatment with cisplatin (10 pg/ml) and “poly-plat” (10 pg/ml)

12

 

Light micro graphs of murine peritoneal macrophages at 24h after
treatment with cisplatin (10 ug/ml) and “poly-plat” (10 ug/rnl)

13

 

Light micrographs of human ﬁbroblasts immediately after treatment with
cisplatin (10 ug/ml) and “poly-plat” (10 ug/ml)

15

 

Light micrographs of human ﬁbroblasts at 8h after treatment with
cispaltin (10 ug/ml) and “poly-plat” (10 ug/ml)

16

 

Light micro graphs of human ﬁbroblasts at 16h aﬁer treatment with
cisplatin (10 ug/ml) and “poly-plat” (10 ug/rnl)

17

 

Light micrographs of normal human teratocarcinoma cells before
drug treatment and at 24h after no treatment

19

 

Light micrographs of human teratocarcinoma cells at 24h after treatment
with cisplatin (10 pg/ml) and “poly-plat” (10 ug/ml)

20

 

Light micrographs of human teratocarcinoma cells at 48h after treatment
with cisplatin (10 pg/ml) and “poly-plat” (10 ug/ml)

20

 

IL-2 analysis of murine peritoneal macrophages treated with
cisplatin (10 ug/ml) and “poly-plat” (10 ug/ml)

21

 

IL-2 analysis of human ﬁbroblasts treated with cisplatin(10 ug/rnl) and
“Poly-Plat” (10 lug/ml)

 

IL-2 analysis of human ovarian teratocarcinoma cells treated
with (10 ug/ml) and “poly-plat” (10 ug/ml)

22

23

 

vii

INTRODUCTION

Cisplatin [cis-dichlorodiammine-platinum(II)], a broad spectrum anticancer drug,
presently is being used for the treatment of testicular and ovarian cancers (1, 2). One of
the major proposed mechanisms of this anti-tumor activity is interaction with DNA that
results in the inhibition of DNA replication and transcription (3).

Recent studies have demonstrated that immune system activation might be another
mechanism of anti-tumor activity of this drug (4). There are many immune system cells
such as macrophages, lymphocytes and monocytes, of which macrophages are one of the
most important immune system cells because of their phagocytic activity and also the
release of various cytokines (5).

Cisplatin has been shown to activate murine peritoneal macrophages in vitro and in
vivo (4, 6). Activated macrophages mediate tumor lysis by lysosomal transfer to tumor
cells and through the release of various cytokines such as IL-la and TNF-a and
interleukin-2 (4, 5).

Interleukin-2 (IL-2) was originally described as “T cell growth factor” (7). IL-2
differentiates T cells as well as stimulates NK (natural killer) cells and LAK (lymphocyte
activated killer) cells (8-11). Therapeutic application of IL-2 injection has been involved
in the treatment of neoplastic conditions (12), infectious diseases such as hepatitis B
(13) and malignancies associated with AIDS (14). Recent studies have demonstrated
that transfected human ﬁbroblast with the IL-2 gene are stimulated to induce high levels
of IL-2 which increases the population of CD3+, CD56+ lymphocytes to ﬁght tumor cells

(15). IL-2 appears to have an important role not only in activating immune responses,

but also in gene therapy which has been used for the treatment of various cancers (16-
18).

Poly-[trans-l, 2-diaminocyclohexane platinum]-carboxyamylase (poly-plat), a
second generation analog of cisplatin, contains only one ﬁﬁh the amount of platinum
compared to cisplatin and has been shown to be less toxic and more effective as an
anticancer agent (19). Recent studies show that “poly-plat” is more eﬁective in
activating macrophages than cisplatin by inducing higher levels of cytokines such as IL-
la and TNF-a (20, 21).

In the present study we describe the results of treating various cell lines from
normal (Human ﬁbroblasts, Murine peritoneal macrophages) and tumor cells (Human
ovarian teratocarcinoma cells) after “poly-plat” treatment, and compare those after

cisplatin treatments.

MATERIALS AND METHODS

Cell culture

1. Macrophages: Swiss webster mice were anesthetized using C02 and killed by
cervical dislocation. Peritoneal macrophages were isolated by injecting 5ml of a chilled
Dulbecco’s modiﬁed eagle medium (DMEM;Gibco, NY) into the peritoneal cavity.
After gently massaging the abdomen wall, the peritoneal ﬂuid was aspirated. Almost
equal amounts of cells (2x106 cells/ml) were seeded onto glass coverslips and then were
placed in 35mm Petri dishes and incubated for 2h at 37°C. After 2h of incubation, the
coverslips were washed with phosphate buﬂ‘er solution (PBS; pH 7.2) to remove non-
adherent cells, leaving the attached macrophages. Cells were then incubated in ﬁesh

DMEM with 10% fetal bovine serum for further experiment.

2. Human ﬁbroblasts and human ovarian teratocarcinoma: Human ﬁbroblasts and
ovarian teratocarcinoma cells were obtained from Dr. C.C. Chang of Michigan State
University. Cell cultures were incubated in a DMEM with 10% fetal bovine serum at
37°C in a 5% C02 incubator. When cells were conﬂuent in culture ﬂasks, cells were
then trypsinized and subcultured into plastic culture dishes with an equal number of cells

in each culture dish .

Dgg treatment
Plated cells were treated with cisplatin (10 ug/ml) and “poly-plat” (10 ug/ml)
prepared in 0.85% NaCl for 2h at 37°C in a 5% C02 incubator. After drug treatment,

the drug containing medium was replaced by normal DMEM with 10% fetal bovine

8611111].

Cell counting

Cells in various cultures (human ﬁbroblasts and human ovarian teratocarcinoma
cells) were counted before and after drug treatment. Subsequently, counts were
maintained after 8, 16, 24, 36, 48, 72 and 96h of treatment. To count cells at each time
interval, cells were washed with PBS and trypsinized with 50% trypsin-EDTA diluted
with PBS. Cell suspension (200w) was mixed with 500p] of trypan blue (0.4%) and
300m of HBBS. Cells were counted in the 16 square leukocyte chamber to determine
the cells/ml. Cells/ml were calculated using the following formula: average counted cells
per square (n=9) x dilution factor x 104. Cells/ml were multiplied by the original volume

of ﬂuid (10ml) in the culture dish to get the number of total cells.

Slide preparation

In order to visualize changes in cell population (human ﬁbroblasts and human
ovarian teratocarcinoma cells) after any drug treatment, same number of cells were
plated onto 18mm glass coverslips. Aﬁer 2h incubation on the coverslips, cells were
washed with a phosphate buffer solution (PBS) to remove non-adherent cells.

Coverslips were then transferred into the plastic culture dish with normal medium and

drugs were added for 2h. Coverslips were then removed from the culture dish, washed,
and put on normal medium. Sample coverslips were removed from these cultures at
intervals of 8, 16, 24, and 48h. These were ﬁxed in 2% (0.02g/rnl) glutaraldehyde in
0.05M phosphate buffer (pH 7.4) for 2 minutes and then rinsed in distilled water.

Methylene blue was used to help visualize cellular details.

IL—2 Assay

The drug containing medium was replaced by normal medium and aliquots of the
supernatant were collected at 0.5, 1, 2, 4, 8, 16, 24, 72, 96h post-treatment from various
cell cultures. An enzyme-linked immunosorbent assay (ELISA) kit (Pharrnagen, San
Diego, CA) was used for IL-2 assay. Monoclonal anti-mouse IL-2 was added to each
well of microplate, and then the antibody was incubated overnight at 4°C. After
incubation, the antibody was aspirated from each well and washed with 0.05% PBT
(PBS with 0.05% Tween-20) to remove non-adherent materials. IL-2 standards
(200,100, 50, 25, 12.5, 6.3 and 3.1 pg/ml) and samples (cell culture supematants) were
added to each well and incubated for 2h at room temperature. All samples were then
washed with 0.05% PBT and working detector (Detection Ab + Avidin-horseradish
peroxidase conjugate) was added to bind the primary and secondary antibody complex.
A substrate solution (Tetramethylbenzidine and Hydrogen Peroxide) was added to give
color change. The reaction was stopped by adding stop solution, 2 N HzSO4.
Absorbance was read at 450nm Standard curves were generated with IL-2 (3.1-

200pg/ml) provided in the kit and linear regression analysis was performed twice.

RESULTS

Morphologcal chaaggs aﬁer drugtreatment on murine peritoneal macrophages

Morphological changes of cisplatin and “poly-plat” treated murine peritoneal
macrophages were observed at 2, 16, 24h post-treatment (Fig 1-3).

No extensions were observed in the normal (Fig 1A) or the cisplatin treated
cultures aﬂer 2h post-treatment (Fig 1B). However, short extension formations were
observed after “poly-plat” treatment (Fig 1C). Similarly, no extension formation was
observed even at 16h post-treatment in either the normal (Fig 2A) or the cisplatin (Fig
2B) treated macrophages. However, pseudopod formation was dramatically increased
after “poly-plat” treated macrophages (Fig 2C).

Both normal and cisplatin treated macrophages demonstrated pseudopod formation
only after 24h post-treatment. About 20—30% of the total population of non-treated
macrophages formed short extensions (Fig 3A) and cisplatin treated macrophages
showed between short and medium developed extensions on 50-60% of total cell
population (Fig 3B). “Poly-plat” treated mcrophages demonstrated cytoplasmic

extension in 70-80% of the cells. These extensions were long and slender (Fig 3C).

Changes in cell population i_n drug treated human ﬁbrobLasta

Numbers of normal and drug treated human ﬁbroblasts were counted before
treatment with cisplatin or “poly-plat” and immediately after treatments. Subsequently

cell counts were made at 8, 16, 24, 36 and 48h post-treatment (Tablel). “Poly-plat”

treated ﬁbroblasts showed a greater increase in cell numbers compared to cisplatin
treated ﬁbroblasts. An initial increase of about 15% was observed immediately after
“poly-plat” treatment, at the same time, normal cells and cisplatin treated ﬁbroblasts
showed only 1% and 11% increase respectively. There were continuous increases in cell
numbers of cisplatin and “poly-plat” treated ﬁbroblasts up to 24h post- treatment when
numbers started to decrease while the normal cells continued to multiply.

To visualize any changes in cell population, human ﬁbroblasts were observed using
light microscopy immediately after drug treatments and also after 8 and 16 of cisplatin or
“poly-plat” treatment (Fig 4-6). To show the increase in the number of cells observed at
each time interval, we used percentages (based on Table 1) to describe changes in cell
numbers. Immediately after “poly-plat” treatment, there was observed a 15% increase in
the ﬁbroblastic cells (Fig 4C), while cisplatin treated ﬁbroblasts demonstrated an 11%
increase (Fig 43). Normal ﬁbroblasts demonstrated a minimal increase of no more than
1% (Fig 4A).

A greater increase in cell numbers was observed after 8h of treatments (Fig 5).
“Poly-plat” treated cells appeared very compact (Fig 5C), about a 23% increase in cell
numbers ﬁom normal ﬁbroblasts (Fig 5A). However, cisplatin treated cells (Fig 5B)
showed only a 15% increase ﬁ'om normal cells. After 16h of treatments, cisplatin
(Fig 6B) and “poly-plat” (Fig 6C) treated cells showed an 8% and 15% increase,

respectively, compared to the normal cells (Fig 6A).

Changes in cell mpulation on drug treated human ovarian teratocarcinoma cells

Nomial cells and drug treated human ovarian teratocarcinoma cells were counted
before and after 2h of drug treatments. Subsequently, cell counts were made at 8, 16,
24, 36, 48, 72 and 96h post- treatment (Table 2). Normal cells were nmintained in
cultures without any treatments for comparison at each time interval. Cisplatin treated
ovarian carcinoma cells showed a continuous decrease in cell numbers at each of the
time intervals. However, there was an initial decrease of about 15% in cell numbers
after 2h of “poly-plat” treatment. “Poly-plat” treated cells then showed a gradual
increase to about 23% from the initial decrease up to 24h post-treatment. After 24h of
“poly-plat” treatment, however, there was a gradual decrease in cell numbers reaching
close to those after 96h of cisplatin treatment.

To visualize any changes in cell population, human ovarian teratocarcinoma cells
were observed using light microscopy before any treatments and also after 24 and 48h of
treatments with cisplatin or “poly-plat” (Fig 7-9). To show the increase in the number
of cells observed at each time interval, we used percentages (based on Table 2) to see
changes in cell numbers on the glass slide. After 24h of treatment, non-treated tumor
cells (Fig 73) showed twice the number of cells (141% increase) compared to those
before treatment (Fig 7A) while 59% of the cells were destroyed after cisplatin
treatment (Fig 8A). However, cell numbers increased to about 5% after 24h of “poly-
plat” treatment (Fig 8B) from those before treatment (Fig 7A). After 48h of treatment,
“poly-plat” showed about a 19% decrease in cell numbers (Fig 9B) from 24h post-

treatment whereas those of cisplatin treatment stayed almost the same (Fig 9A).

IL-2 release

1. Macrophages: There was an increase in the IL-2 level detected in the supematants of
the macrophages treated with either cisplatin (10 ug/ml) or “poly-plat” (10 ug/ml) for
the various times tested (Fig 10). Signiﬁcant differences in the IL-2 level between
cisplatin and “poly-plat” treatment were observed after 4h of treatment. The greatest
increase (1 66pg/ml) in the IL-2 level of “poly-plat” treatment was observed after 16h of
treatment. The IL-2 level of both cisplatin and “poly-plat” decreased to 81pg/ml and

125pg/ml after 24h of treatment.

2. Human ﬁbroblasts: No signiﬁcant differences in the IL-2 level of normal, cisplatin
and “poly-plat” treated ﬁbroblasts were observed until 2h post-treatment (Fig 11). After
4h of treatment, “poly-plat” treated cells released IL-2 (52pg/ml) at the rate of about 9
times that of untreated ﬁbroblasts. The IL-2 level reached a maximum (138 pg/ml) after
8h of “poly-plat” treatment. However, the IL-2 level of cisplatin treated cells showed
only half(64 pg/ml) of the IL-2 level compared to “poly-plat” treated cells. The IL-2
level of both cisplatin and ‘poly-plat’ treated cells decreased to 25 and 31 pg/ml,

respectively, after 16h of treatment.

3. Human ovarian teratocarcinoma: IL-2 levels of up to 82 pg/ml were observed after
0.5h of “poly-plat” treatment compared to normal (43 pg/ml) and after cisplatin
treatment (58 pg/rnl). This level increased up to 104 pg/ml after 2b of “poly-plat”
treatment. Interestingly, normal cells also demonstrated an increase in the IL-2 levels

(97 pg/ml) after 1h in normal medium without any treatment. After 2h of “poly-plat”

treatment, IL-2 levels decreased (37 pg/ml) until 16h post-treatment (Fig 12). There
was again an increase in the IL-2 levels after 24h of “poly-plat” (104 pg/ml) and after
cisplatin treatment (97 pg/ml). However, this increase did not remain constant. After

48h of treatment, it was lower than its peak at 24h post-treatment (see Fig 12).

10

 

F igl. Light micrographs showing murine peritoneal macrophages at 2h in normal
medium after (A) no treatment, (B) cisplatin (10 pig/ml) and (C) “poly-plat” (10 ug/ml)
treatment for 2h. Note the short cytoplasmic extension formation (arrows) after “poly-
plat” treatment, while normal and cisplatin treated macrophages show no extension
formation. Final magniﬁcation: x2000 (A, B, C), Bar = 7.5 urn

 

9&1? $1.4»!

 

Fig 2. Light micrographs showing murine peritoneal macrophages at 16 h in nonml
medium after (A) no treatment, (B) cisplatin (10 rig/ml) for 2h, (C) “poly-plat”

(10 rig/ml) for 2h. Note the cytoplasmic extensions (arrows) were observed after
“poly-plat” treatment as bidirectional in their formation, while no cytoplasmic extensions
were observed in normal or cisplatin treated macrophages. Final magniﬁcation: x2000
(A, B, C), Bar = 7.5 pm

x 7\ A_-\ Aﬂ.\

 

Fig 3. Light micrographs showing macrophages at 24h in normal medium after (A) no
treatment, (B) cisplatin (10 ug/rnl) and (C) “poly-plat” (10 ug/rnl) treatment for 2h
Note the cytoplasmic extensions (arrows) after normal, cisplatin and “poly-plat”
treatment. “Poly-plat” treated macrophages have a more developed extension formation
compared to normal and cisplatin treated macrophages. Final magniﬁcation: x2000 (A,
B, C), Bar = 7.5 pm

TABLE 1. Number of human ﬁbroblasts before and after drug treatments

 

 

Cell numbers
Treatment before after treatment (hr)
treatment 0‘ 8 16 24 36 48

 

Control 0.88x106 0.89x10"1.02x106 1.10x106 1.47x106 1.58x106 2.13x106
CDDP ------ 0.98x106 1.18x106 1.19x10" 1.22x106 1.19x1061.13x106

Poly-plat ------ 1.01x106 1.25x10° 1.27x106 1.35x10“ 1.25x1o6 1.16x106

 

* Speciﬁes the number just after 2h of drug treatment, when the cells were placed in a
normal medium without any drug. Results are averages of experiments done in
triplicate.

14

 

Fig 4. Light micrographs of human ﬁbroblasts showing changes in the number of cells
immediately after (A) no treatment, (B) cisplatin (10 ug/ml) and (C) “poly-plat” (10
uglml) treatment for 2h. Note the increase in cell numbers were observed immediately
after cisplatin (0.98x106 cells/ml) and “poly-plat” (1.01x106 cells/ml) treatment. The
cell numbers are much higher after “poly-plat”, compared to cisplatin.

Final magniﬁcation: x200 (A, B, C), Bar = 37.5 urn

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Fig 5. Light micrographs showing human ﬁbroblasts at 8b in normal medium after (A)
no treatment, (B) cisplatin (10 rig/ml) and (C) “poly-plat” (10 rig/ml) treatment for 2h.
Note the large increase (1.25x106 cells/ml) in cell numbers alter 8h of “poly-plat”
treated ﬁbroblasts. Final magniﬁcation: x200 (A, B, C), Bar = 37.5 um

 

Fig 6. Light micrographs showing human ﬁbroblasts at 16h in nomial medium after (A)
no treatment, (B) cisplatin (10 rig/ml) treatment for 2h, (C) “poly-plat” (10 rig/ml)
treatment for 2h. Note the clump formation in “poly-plat” treated ﬁbroblasts probably
due to a large increase in cell number (1.27x106 cells/ml). Final magniﬁcation: x200 (A,
B, C), Bar = 37.5 um

TABLE 2. Number of human ovarian teratocarcinoma cells before and after drug

 

 

treatments
Cell number
Treatment before after treatment (hr)
treatment 0’ 8 16 24 36 4s 72 96

 

Control 4.1x10° 4.2x106 7x106 8.9er6 9.9xro6 10.4x106 12x10616.5x10 22.1x10"
CDDP ------ 4x106 3.9x106 2.6x10° 1.7x106 0.5er6 0.41xro6 0.4x10‘5 0.4x106

Poly-plat ------ 3.5x106 3.8x106 3.9xro6 4.3xro" 3.5x106 2.9x106 2.3x106 0.5x106

 

* Speciﬁes the number just after 2h of drug treatment, when the cells were placed in a
normal medium without any drug. Results are average of experiments done in triplicate.

l8

 

Fig 7. Light micrographs showing human teratocarcinoma cells in normal medium
immediately before any drug treatment (Fig A: 4.1x106 cells/ml) and after 24h of
incubation in normal medium without any drug treatment (Fig B: 9.9x 10° cells/ml).
Note the increase in cell numbers alter 24h incubation. Final magniﬁcation: x200,
Bar = 37.5 um

 

Fig 8. Light micrographs showing human teratocarcinoma cells at 24h in normal
medium after (A) cisplatin (10 rig/ml) and (B) “poly-plat” (10 ug/ml) treatment for 2h.
Number of cells greatly decreased (1.7x106 cells/ml) after cisplatin treatment, however,
there was an increase in cell number (4.3x106 cells/ml) after “poly-plat” treatment.
Final magniﬁcation: x200 (A, B), Bar = 37.5 um

 

Fig 9. Light micrographs showing human teratocarcinoma cells at 48h in normal
medium after (A) cisplatin (10 rig/ml) and (B) “poly-plat” (10 ug/ml) treatment for 2h.
Note the continuous decrease (0.41 x106 cells/ml) in cell numbers with cisplatin
treatment while there was less of decrease in cell numbers (2.9x 106 cells/ml) with “poly-
plat” treatment. Final magniﬁcation: x200 (A, B), Bar = 37.5um

20

Macrophage IL-2 Assay

 

 

 

 

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8
Hours post-treatment

Fig 10. Bar graph* showing IL-2 release in the supernatants of macrophages treated
with either cisplatin (10 ug/ml) or “poly-plat” (10 ug/ml) at various time intervals. Note
the gradual increase in IL-2 for “poly-plat” treatment up to 16h post-treatment with the
highest level at 16h of “poly-plat” treatment. Cisplatin treatment induced a gradual
increase through 16h but is still less than that of “poly-plat” treatment.

* Each bar represents the mean ﬁ'om 2 sets of readings.

21

Human Fibroblasts IL-2 Assay

 

 

 

 

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Figure 11. Bar graph* showing IL-2 release in the supematants of human ﬁbroblasts
treated either with cisplatin (10 jig/ml) or “poly-plat” (10 rig/ml) at various time
intervals. Note the sudden increase (138 pg/rnl) after 8h of “poly-plat” treatment
compared to cisplatin. After 16h of “poly-plat” treatment, Il—2 levels (31 pg/ml)
dramatically decreased.

* Each bar represents the mean ﬁom 2 sets of readings.

22

Human Ovarian Terabcarcinoma lL-2 Auay

 

 

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1 2 4 8 16 24

Hours post-treatment

Figure 12. Bar graph* showing IL-2 release in the supernatants of human ovarian

teratocarcinoma cells treated with either cisplatin (10 rig/ml) or “poly-plat” (10ug/ml) at
various time intervals. Highest IL-2 level (104 pg/ml) was observed after 2h and 24h of
“poly-plat” treatment. Cisplatin also induced IL-2 releases reaching a peak after 24h of

treatment .

* Each bar represents the mean ﬁom 2 sets of readings.

23

DISCUSSION

Cisplatin, a broad spectrum anticancer drug, can activate murine peritoneal
macrophages in vitro and in vivo (4, 6). Activated macrophages seek out tumor cells
through cytoplasmic extensions and lysosomal transfer to the tumor cells causing tumor
cell death (4). In order to increase the efﬁcacy, second generation analogs of cisplatin
have been reported. “Poly-plat”, one such analog of cisplatin, has been shown to
activate macrophages better than cisplatin by releasing signiﬁcant levels of cytokines like
IL-lor and TNF-lor (20, 21). IL-1 and TNF-la have been known to be involved in
direct antitumor activity and increase the expression of MHC (major histocompatibility
complex) molecules on the cell surface (22, 23) .

In the study of macrophage activation, it has been demonstrated that macrophages
activated by “poly-plat” increase cytoplasmic extension formation and also increase the
release of cytokine, interleukin-1a, better than cisplatin (20). Other studies have shown
that “poly-plat” generates 10 times the number of lysosomes compared to cisplatin,
which are transferred to tumor cells through the cytocrine process (19).

In the present study, we have observed cytoplasmic extension formation within 2h
after “poly-plat” treatment while cisplatin treatment induces only after 24h. It was
observed that there was a greater release of IL-2 from macrophages after “poly-plat”
treatment compared to cisplatin. Studies have demonstrated that the ability of IL-2 to
destroy tumor cells is based on various effects on immune response to tumor cells
through activation of cytotoxic lymphocytes and increased surface antigen or receptors

on tumor cells involved in tumor cell recognition and lysis (24, 25). Since IL-2

24

stimulates the immune system (7, 26), investigators have used IL-2 gene expression
vectors which have been transfected into ﬁbroblasts in order to stimulate IL-2 release.
Released IL-2 from the ﬁbroblasts stimulate an increase in cell population of CD3+
CD56+ lymphocytes which are involved in tumor cell lysis(15).

“Poly-plat” can stimulate not only macrophages, but also non-immune system cells
like human ﬁbroblasts. Studies on cell population of ﬁbroblasts show that a 53%
increase in cell ntunber was observed after 24h of “poly-plat” treatment, while there was
only a 38% increase after cisplatin treatment. An increase in cell population of
ﬁbroblasts seems to be followed by maximum release of IL-2 (138 pg/ml). Recent
studies have demonstrated that T cells are stimulated to express high amnity IL-2
receptor and secrete IL-2 in the presence of an antigen (27). The binding of IL-2 to IL-
2 receptor signals the T cells to release IL-2 which in turn induces T cell proliferation
(27). Since IL-2 receptors have been detected on human ﬁbroblasts (28), it is possible
that our observation of cell proliferation on human ﬁbroblasts might be due to IL-2
interaction with IL-2 receptor.

Unlike murine macrophages or human ﬁbroblasts, “poly-plat” treated human
ovarian teratocarcinoma cells didn’t show signiﬁcant release of IL-2. “Poly-plat”
treatment increased the release of IL-2 to a maximum level (104 pg/ml) after 2h of
treatment, decreasing until 16h followed by another peak level after 24h.
Correspondingly, tumor cell population decreased by 15% after 2h of “poly-plat”
treatment with a slight increase (23%) subsequently through 24h. However, a
signiﬁcant decrease (88%) was seen until 96h post-treatment. It has been suggested that

IL-2 interaction with IL-2 receptor results in cell proliferation of human breast

25

carcinoma cell line through autocrine regulation on the cells (29). Similar situations
probably apply to tumor cells in our study.

IL-2 receptors consist of three structural domains (or, [3, 7). When IL-2 binds IL-2
receptors, serine-rich region in the B chain seems to be involved in two signal
transduction pathways: activating tyrosine kinase of src family and induction of c-myc
(30). C-myc is the early response gene which is induced within 15 minutes of growth
factor treatment. When growth factor is present, concentration of Myc protein is
elevated and then reaches to a new steady value which triggers cells to escape G0 phase
and to proliferate (31). Based on these ﬁndings, we suggest that proliferation of human
ﬁbroblasts and human ovarian tumor cells in our study might be due to the increased
expression of c-myc followed by binding of IL-2 to IL-2 receptors.

Cells that are treated with anticancer agents activate an injury response that
eventually induces cell death by apoptosis (3 2-36). After 24h of “poly-plat” treatment,
an almost 3 times faster rate of tumor cell death was observed than with cisplatin
treatment. One of the possible mechanisms in increasing cell death might be the
involvement of nitric oxide (37, 38). Nitric oxide is generated in mammalian cells by the
conversion of L-arginine to L-citrulline. Since nitric oxide released from activated
macrophages has been demonstrated to be involved in killing tumor cells (37, 38), it is
possible that nitric oxide might be released from “poly-plat” treated tumor cells (ovarian
teratocarcinoma cells). Higher production of nitric oxide might have occurred after
“poly-plat” treatment as compared to cisplatin treatment, thereby causing increased
tumor cell death Changes in the level of nitric oxide has not been studied after “poly-

plat” treatment on ovarian cancer cells. Studies on the mechanisms which control

26

positive and negative regulation on the tumor cells needs to be investigated. Further
studies of the level of interleukin-2 receptor and nitric oxide on the different tumor cells

may be required to answer this question.

27

CONCLUSION

This study focused on treating various cell cultures (macrophages, human
ﬁbroblasts and human ovarian carcinoma cells) with cisplatin and “poly-plat” to see the
difference in level of cytokine (IL-2) and also in cell numbers before and after
treatments. “Poly-plat” treated macrophages stimulated cytoplasmic extensions more
eﬁiciently than cisplatin and also released signiﬁcant levels of cytokine, IL-Z. We have
also found that “poly-plat” stimulates cell proliferation (human ﬁbroblasts) followed by
increased level of IL-2.

Based on these ﬁndings, we suggest that “poly-plat” is more effective in
macrophages activation and also inducing higher levels of IL-2 ﬁom different cell

cultures, compared to cisplatin.

28

10

ll

12

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