Streptokinase increases the sensitivity of colon cancer cells to chemotherapy by gemcitabine and cisplatine in vitro

Streptokinase increases the sensitivity of colon cancer cells

to chemotherapy by gemcitabine and cis-platine in vitro

Vladimir Bobeka,b,*, Daniela Pinterovaa, Katarina Kolostovaa, Michael Boubelika,c,

Joshua Douglasa, Petr Teysslerd, Jiri Pavlaseke, Josef Kovarika

aDepartment of Tumor Biology, Third Faculty of Medicine, Charles University, Ruska 87,

Prague 10, 100 34, Czech RepublicbDepartment of Thoracic Surgery, Medical Academy, Grabiszynska 105, Wroclaw, 53–439, Poland

cInstitute of Molecular Genetics, Academy of Sciences, Flemingovo nam.2, Prague 6, 166 37, Czech RepublicdClinic of Children and Adult Orthopaedic Surgery and Traumatology, Faculty Hospital in Motol, V Uvalu 84,

Prague 5, 150 06, Czech RepubliceDepartment of Gynaecology and Obstetrics, Third Faculty of Medicine, Charles University, Rusaka 87,

Prague 10, 100 34, Czech Republic

Received 10 April 2005; received in revised form 19 May 2005; accepted 24 May 2005

Abstract

The aim was to determine the effect of fybrinolytic therapy by streptokinase on chemotherapy and radiation response in human

colon cancer cells.The cellswere treatedwithdifferent concentrations of gemcitabine,cis-platine and streptokinase, at a singleuse or

in combinations. Radiation was tested at a dose 0.5, 5 and 15 Gy in three different schedules. The chemotherapy showed higher

cytotoxic effect in combination with streptokinase. On the other hand, the combination of chemotherapy with streptokinase and

radiotherapy provide no improvement in sensitivity of cancer cells to treatment. The data suggest that fybrinolytic therapy could

influence the effect of chemotherapy.

q 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Chemotherapy; Gemcitabine; Radiation; Streptokinase; Colon cancer

1. Introduction

Colon cancer is one of the most common human

cancers, with approximately 600,000 new cases

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights re

doi:10.1016/j.canlet.2005.05.030

* Corresponding author. Address: Department of Tumor Biology,

Third Faculty of Medicine, Charles University Prague, Ruska 87,

Prague 10034, Czech Republic. Tel.: C420 267 102 662;

fax: C420 267 102 650.

E-mail address: [email protected] (V. Bobek).

diagnosed worldwide each year, accounting for 9% of

all cancers. Surgery is the only universally accepted

treatment offering any hope of cure for colorectal

cancer.About 85%ofpatients diagnosedwithcolorectal

cancer can undergo surgical resection.Metastases to the

regional lymph nodes are found in 40–70% of cases at

the time of resection. About 85% of all recurrences that

are designed to occur in colorectal cancer are evident

within 3 years after surgical resection [1]. This study

Cancer Letters 237 (2006) 95–101

www.elsevier.com/locate/canlet

served.

V. Bobek et al. / Cancer Letters 237 (2006) 95–10196

was designed to test the hypothesis that fibrinolytic

drugs increase significantly the colon cancer cell

sensitivity to often used chemotherapeutics in in vitro

experiments.

Gemcitabine (2 0,2 0-difluorodeoxycytidine, dFdC)

is a synthetic pyrimidine nucleoside analogue that has

a structure very similar to that of deoxycytidine and

cytosine arabinoside [2]. In clinical use, gemcitabine

is active against a variety of solid tumors such as

cancer of the pancreas, lung head and neck, bladder,

breast and ovary. It is activated intracellularly by

deoxycytidine kinase, which adds multiple phosphate

groups to the 5 0 position of the ribose group. The

diphosphate and triphosphate forms of the drug

play an important role in the inhibition of DNA

synthesis [3,4]. Gemcitabine was widely tested as

radiosensitizer in various biological models [3,5,6].

cis-Platine (cis-diaminedichloroplatinum(II), CP),

a DNA-damaging agent, is widely used anticancer

drug. CP binds to DNA, primarily to guanines

nucleotides resulting in formation of intra- and inter-

strand adducts and in cross-linking DNA to proteins

[7,8]. Chemotherapeutic effect of CP relies primarily

on its ability to induce apoptosis in tumor cells [9].

The old fibrinolytic agent streptokinase is an

extracellular enzyme produced by various strains of

b-hemolytic streptococci. The enzyme is a single-chain

polypeptide that exerts its fibrinolytic action indirectly

by activating the circulatory plasminogen. Streptoki-

nase consists of multiple structural domains (i.e. a-, b-and g-domains) with different associated functional

properties. Streptokinase is known to activate plasmi-

nogen system both ways, by fibrin-dependent and -

independent mechanisms [10,11]. Streptokinase is

widely used therapeutically for the treatment of acute

myocardial infarction and pleural empyema.

Recent studies have shown the effect of the

plasminogen activation in cancer therapy [12]. A

significant antitumor effect using chemotherapy in

combination with fibrinolytic agents was observed in

many studies, case and case cohort studies [12–14].

Adjuvant single therapy, in the form of radiotherapy or

chemotherapy has not shown increased survival, but

combined chemotherapy with fibrinolytic therapy may

be effective in reducing tumor and metastasis growth in

patients with solid tumors [12,15].

The aims of this study were (i) to quantify the

effect of fybrinolytic agent on the cytotoxic action of

gemcitabine and cis-platine (ii) to examine the effect

of streptokinase on radiotherapy (iii) to determine the

optimal doses and sequence of drug, radiation and

timing on proliferation of in vitro growing colon

cancer cells.

2. Materials and methods

2.1. Cell culture and media

The human colon cancer cell line HT29 was

maintained in exponential growth as monolayer in

Dulbecco’s Modified Eagle Medium (Sigma, Czech

Republic) supplemented with 10% fetal bovine serum

as described elsewhere. The cells were grown at 37 8C,

in a humidified atmosphere containing 5% CO2.

2.2. Chemicals

Stock solutions of gemcitabine (Lilly, France), cis-

platine (Pharmachemie, the Netherlands) and strepto-

kinase (Hoechst Marion Roussel, Germany) were

prepared freshly in culture medium and diluted before

each experiment.

2.3. Growth inhibition

Cells were treated with different concentrations of

gemcitabine (range from 2.4 to 0.003 mg/ml), cis-

platine (0.25–0.063 mg/ml) and streptokinase (2000–

63 IU/ml), at a single concentration or in combi-

nations (Tables 1 and 2). Plates were incubated for 4,

24 and 72 h. The survival of drug-treated cells was

corrected for cell growing in culture medium without

any tested agents, drug or streptokinase. After these

periods the MTT assay was carried out to determine

the amount of cells, which survived.

2.4. Radiosensitization by gemcitabine and cis-platine

Radiation was given using a 60Co source at a dose

0.5, 5 and 15 Gy. We tested three concentrations of

each drug alone and four combinations of drug with

streptokinase together with radiation (Table 2).

Besides 24 h treatment before radiation, which is

the schedule commonly used for in vitro experiments

with gemcitabine and radiation treatment, alternative

Table 1

Used concentrations of tested agents

Gemcitabine

(mg/ml)

G1 G2 G3 G4 G5 G6

2.4 1.2 0.6 0.3 0.03 0.003

Streptokinase

(IU/ml)

S1 S2 S3 S4 S5 S6

2000 1000 500 250 125 63

V. Bobek et al. / Cancer Letters 237 (2006) 95–101 97

schedules were used (Fig. 1). Different gemcitabine

treatment times, 24 and 5 h before radiation and 5 h

after radiation, were compared. Cells were incubated

in 96 well plates 24 h before radiation (RT), while

period of drug action was different (Fig. 1). Four

identical plates were used for each drug and different

doses (0, 0.5, 5 and 15 Gy) of radiotherapy were

compared. The radiation was followed by the 24 h

incubation period and then the MTT assay was

performed. Radiation survival of drug-treated cells

was corrected to the number of cells killed by drug

itself (dose of radiation 0 Gy).

Table 2

Used concentrations of tested agents

cis-platine

(mg/ml)

Pt1 Pt2 Pt3

0.25 0.125 0.063

Gemcitabine

(mg/ml)

G2 G3 G5

1.2 0.6 0.03

Streptokinase

(IU/ml)

S1 S4 S6

2000 250 63

Fig. 1. Different schedules for treatment of HT29 cells. The full line

described the drug treatment, the dotted line described the

cultivation without drugs.

2.5. MTT proliferation assay

This method provides a quantitative measure-

ment of the number of cells with metabolically

active mitochondria and is based on the mitochon-

drial reduction of tetrazolium bromide salt (MTT)

to purple formazan crystals. The formazan crystals

are solubilized and the resulting colored solution is

quantified by measurement of absorbance at

570 nm. An increase in the number of living cells

results in an increase of purple formazan product

that is monitored by absorbance increase.

Cells were plated in 96-well plates at a final density

of 2!104 cells/well. Cells were treated with different

concentrations of drugs, streptokinase or combi-

nations of drug with streptokinase. Different incu-

bation times with drugs or combinations with

streptokinase, 24, 5 h before radiation and 5 h after

radiation, were tested. MTT was added (final

concentration 0.45 mg/ml) for 4 h. We did triplicates

for each concentration. After this incubation period,

the solubilization solution was added and the plates

were incubated overnight in humidified atmosphere

(37 8C, 5% CO2). An ELISA plate reader at 570 nm

with reference wavelength 690 nm measured the

absorbance of the formazan product.

2.6. Statistical analysis

The one-way ANOVA with Dunnett’s post-test

was performed using GraphPad Prism version 4.00 for

Windows (GraphPad Software, San Diego, CA, USA)

to analyze the difference of inhibition effect between

drugs and combinations of drugs with streptokinase.

The P value !0.05 was considered as significant.

3. Results

3.1. The effect of streptokinase on the cytotoxic

action of gemcitabine and cis-platine

For both drugs, gemcitabine and cis-platine, we

obtained clear concentration-dependent growth

Fig. 2. The percentage of alived colon cancer cells after treatment with gemcitabine, cis-platine or drug combination with streptokinase or

(effect of combined therapy with streptokinase).

Table 3

The P-value

Combinations P value after

4 h

P value after

24 h

P value after

72 h

G1CS1 ns ns ns

G1CS6 !0.05 ns ns

G2CS5 ns !0.01 ns

G3CS3 !0.05 !0.05 ns

G4CS6 !0.01 !0.01 ns

G5CS5 !0.01 !0.01 !0.01

G6CS1 !0.05 !0.01 !0.01

G6CS2 !0.01 !0.01 !0.01

V. Bobek et al. / Cancer Letters 237 (2006) 95–10198

inhibition of cancer cells. In all experiments the

inhibition of proliferation was proportional to the

exposure time. The streptokinase in independent

application had no or even small stimulating effect

in this cancer cell line (Fig. 2). Different effects of

combinations with fybrinolytic agents were observed

between gemcitabine and cis-platine. The best growth

inhibition effect was obtained using the combination

of gemcitabine and streptokinase. We obtained better

results in combination with streptokinase for all tested

concentrations of gemcitabine (Fig. 2). Different

effect was also observed for different incubation

time, we tested 4, 24 and 72 h incubation time.

According to our results, the inhibition of prolifer-

ation using chemotherapy in combination with

streptokinase was the most effective for the 24 h

treatment of the cells. For this incubation time, we

obtained statistically significant data, which confirm

the better effect of combinations with streptokinase

for all concentrations of gemcitabine except the

highest one and as well the better inhibition effect

for lower concentration of cis-platine (Table 3).

Pt2CS1 ns ns ns

Pt2CS6 ns ns ns

Pt3CS1 ns ! 0.05 ns

Pt3CS6 ns ! 0.05 ns

ns-not significant.

3.2. The effect of streptokinase on radiotherapy

We tested three different treatment schedules for

chemotherapy combined with radiotherapy (Fig. 1).

The best inhibition effect on proliferation for both

drugs was obtained when the incubation time of drug

with streptokinase was 24 h before radiation. We did

not observe any stronger inhibitive effect on prolifer-

ation for alternative schedules (data not shown).

The most effective combination was the gemcitabine

with streptokinase used in lower concentrations (e.g.

G2CS6) (Fig. 3). Generally, we determinated that the

positive effects of streptokinase on chemotherapy

were observed at lower concentrations of drugs and

Fig. 3. (A and B) The percentage of living colon cancer cells after applied chemotherapy and radiotherapy, #P-value !0.01; *P-value !0.05.

V. Bobek et al. / Cancer Letters 237 (2006) 95–101 99

without any radiation. The radiation 0.5 and 5 Gy had

small or no effect on combination of streptokinase

with gemcitabine and no effect on proliferation in

combination with cis-platine (Fig. 3). The radiation

15 Gy had inhibitive effect but in spite of this high

dose of radiation we found only mild effect in -

comparison to results without any radiation. Our

results suggest that the combination of fibrinolytic

agent with radiotherapy will probably not have any

effect on treatment and we were not able to confirm

gemcitabine like radiosensitiser. For better compari-

son of the inhibition effect for cis-platine, the drug

combination with streptokinase is shown against cis-

platine alone and not against control (Fig. 3).

4. Discussion

In the past, when the fibrinolytic and anticoagulant

therapy was used to treat tromboembolic disease in

patients with different kinds of cancer, it was

discovered that these agents could have anticancer

effect, e.g. reduction of the primary cancer or

influencing the metastatic process [16]. In the 19th

century, it was described that there are some changes

in the coagulation system and impaired fybrinolysis in

patients with malignancy [17].

In our study we proved inhibition effect of the

fybrinolytic therapy in combination with chemother-

apy by gemcitabine or cis-platine on proliferation rate

V. Bobek et al. / Cancer Letters 237 (2006) 95–101100

of colon cancer cells in vitro. We decided to choose

combination of gemcitabine and streptokinase accord-

ing to our previous results [15] and we tested cis-

platine commonly used as a chemotherapeutic drug

in colon cancer therapy. It has been shown that the

cancer cells can activate the clotting system by their

ability to produce procoagulant substances, inhibitors

and other molecules associated with this system [18].

The changes in expression of different genes caused

by fybrinolytic therapy could be one reason for the

higher cancer cell sensitivity against chemotherapy. It

has been established that elevated plasminogen

activator inhibitor-1 (PAI-1) levels in cancers are

associated with poor prognosis. As well the poly-

morphism of PAI-1 has been studied for association

with colorectal cancer risk and prognosis [19].

Surprisingly, it was published that plasmin induced

the formation of multicellular spheroids in breast

cancer cells, which were more resistant to the

chemotherapy, doxorubicin and paclitaxel, but the

proliferative rate did not change [20]. This implies

that active proteinase plasmin, which has the ability to

degrade most extracellular proteins, could affect the

sensitivity of cancer on cellular level.

Further we found that the combination of

chemotherapy with streptokinase and additional

radiotherapy provides no improvement in sensitivity

of cancer cells. It was shown in several studies that

some anticancer drugs could cause that the radio-

therapy is more effective. The radiosensitizing effect

could be related to enhanced apoptosis [21]. On the

other hand, it was suggested that gemcitabine might

cause the differences in cell cycle distribution.

Gemcitabine can block the cells in early S-phase

and this may play a role in enhancing radiosensitivity

[3]. In our experiments, we even detected small

stimulating effect of lower radiation doses for

combination of chemotherapy with streptokinase

(Fig. 3A and B). Some other authors published

unusual radiation response of cancer cells treated by

gemcitabine [6]. We suggest that it is really important

to establish the treatment schedule and the dose of

chemotherapy for each cancer cell line. And it can

happen that for some specific cases the combination of

chemotherapy with radiotherapy improves signifi-

cantly the treatment.

According to our results, the application of

fibrinolytic therapy positively influences the

anticancer treatment of the colon cancer cells in vitro.

However, the complex biochemical mechanisms of

the fibrinolysis in cancer patients are not completely

understood yet. Furthermore, the amount of the

available clinical data is still relatively small and

therefore the question whether or not routinely to

consider the coagulation and/or fibrinolysis system as

therapeutic targets in cancer patients is the matter to

be answered [22].

Acknowledgements

This research work was supported by League

Against Cancer Czech Republic 2004/2005.

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