Topotecan (TpT) is a major inhibitory compound of topoisomerase (topo) I that plays important roles in gene transcription and cell division. We have previously reported that heparin and heparan sulfate (HS) might be transported to the cell nucleus and they can interact with topoisomerase I. We hypothesized that heparin and HS might interfere with the action of TpT. To test this hypothesis we isolated topoisomerase I containing cell nuclear protein fractions from normal liver, liver cancer tissues, and hepatoma cell lines. The enzymatic activity of these extracts was measured in the presence of heparin, liver HS, and liver cancer HS. In addition, topo I activity, cell viability, and apoptosis of HepG2 and Hep3B cells were investigated after heparin and TpT treatments. Liver cancer HS inhibited topo I activity in vitro. Heparin treatment abrogated topo I enzyme activity in Hep3B cells, but not in HepG2 cells, where the basal activity was higher. Heparin protected the two hepatoma cell lines from TpT actions and decreased the rate of TpT induced S phase block and cell death. These results suggest that heparin and HS might interfere with the function of TpT in liver and liver cancer.
Heparin and heparan sulfate (HS) are polysulfated sugars, members of glycosaminoglycans (GAGs), present in animal and human tissue in free or protein bound forms.
Heparan sulfate glycanated proteins are found in the extracellular matrix and on the cell surface [
The cellular role of HS has been studied for years without a major breakthrough achieved [
We reported that heparin and liver HS inhibit the plasmid relaxation activity of topoisomerase I enzyme in vitro [
Surgical specimens from cancer patients were sent to our department for histological diagnosis and were used with the permission of the regional ethical committee. The samples were frozen in liquid nitrogen and stored at −80°C until used.
American Tissue Type Culture Collection HepG2 and Hep3B cell lines were used after 12–15 passages. Cells were plated at a density of
Unless specified otherwise, the chemicals were purchased from Merck (Darmstadt, Germany). Hind III and Klenow DNA polymerase enzymes were obtained from Promega (Madison, USA). Topotecan was a gift of SmithKline Beecham (King of Prussia, USA). Heparin was purchased from Sigma (Steinheim, Germany).
Protein concentration was determined by using the Coomassie protein assay kit of Pierce (Rockford, USA). Recombinant topo I and polyclonal human anti-topo I IgG (scl-70) from Topogen (Columbus, USA) were used for western blot.
Mitochondrial succinate dehydrogenase activity [
Morphology of the two hepatoma cell lines was studied either by growing them onto coverslips or by preparing cytospin slides. Cells were visualized with hematoxyline-eosine staining.
HepG2 and Hep3B cells were washed twice with PBS then suspended in a buffer containing 0.1% sodium citrate, 0.1% Triton X-100, and 0.05 mg/mL ribonuclease, pH 7.7, at 106 cell/mL density. Before the analysis, the cells were stained with 50
Cell cycle parameters were measured on a FACScan flow cytometer (Becton Dickinson, San Jose, USA) scanning the propidium-iodide signals and the forward and side scatter parameters. The Multicycle software of Robinovitch (Phoenics Flow San Diego, USA) was used for analyzing the results.
Nuclei from liver specimens were isolated on saccharose gradient, according to Hogeboom [
ATP independent relaxation of supercoiled pBR322 plasmid Stratagene (La Jolla, USA) was done in standard 30
pBR322 plasmid was linearized with EcoRI enzyme and then end-labeled with 5
pBluescript plasmid was digested with Hpa II restriction enzyme. One of the restriction fragments with 516 base pair was separated and labeled with DIG-11 dUTP and terminal deoxynucleotide transferase (Roche, Mannheim, Germany), as suggested by the manufacturer. Based on its sequence analysis the labeled fragment contained 8 potential topoisomerase I binding sequences [
The assays have been run in triplicates and statistical significance has been calculated based on data distribution (normal or non-parametric) using a Student’s
To assess the interference of heparin with TpT, the two hepatoma cell lines were treated with 1
Effects of heparin and TpT on HepG2 (panel a) and Hep3B (panel b) cell numbers after 48–120 h incubation (results of 3 independent experiments). After 48 h of plating, serum has been withdrawn and the action of heparin and TpT was studied under serum-free conditions. Cells were grown for 72 h in the presence of 1
Although heparin inhibited the proliferation of both hepatoma lines, no changes in cell cycle parameters were discernable. TpT induced dramatic G1-S phase block and cell death in both cell lines. Both effects were reduced when TpT was administered together with heparin (Table
(a) Effect of heparin and TpT on cell cycle parameters of HepG2 and Hep3B cells (results of 3 independent experiments). Concentrations used: TpT 1
% of cells | HepG2 | Hep3B | ||||
---|---|---|---|---|---|---|
Treatment | G1 | S | G2 | G1 | S | G2 |
Control | 52,6 ± 4,08 | 35,7 ± 3,29 | 11,6 ± 2,19 | 78,7 ± 0,42 | 15,9 ± 0,14 | 5,5 ± 0,28 |
1 |
15,8 ± 1,31 | 80,8 ± 15,51 | 4,2 ± 0,071 | 2,06 ± 0,071 | 65,6 ± 0,991 | 13,7 ± 1,061 |
100 |
58,9 ± 1,39 | 28,3 ± 3,58 | 12,8 ± 0,59 | 81,1 ± 0,28 | 14 ± 0,28 | 5 ± 0,07 |
Hp + TpT | 14,5 ± 0,45 | 70,2 ± 3,973 | 15,2 ± 0,68 | 35,4 ± 0,1 | 56,4 ± 2,332 | 8,1 ± 2,4 |
1Student’s
2Student’s
3Student’s
%-of cells in apoptotic gate | HepG2 | Hep3B |
---|---|---|
Control | 5,65 ± 0,595 |
|
1 |
18,67 ± 3,531 |
|
100 |
9,06 ± 0,162 |
|
Hp + TpT |
|
|
1Student’s
2Student’s
3Student’s
The topo I enzymatic activities of surgically removed human liver and hepatocellular carcinomas as well as those of two hepatoma cell lines were studied. Nuclear extracts from peritumoral liver specimens showed low topo I activity. In contrast, 200 ng of nuclear protein from liver cancer resulted in total relaxation of the pBR322 plasmid. An identical amount of protein from peritumoral liver left more than half of the plasmid unattached. The activity in HepG2 cells was as high as in the primary liver cancer. Interestingly the less differentiated Hep3B hepatoma cell line retained only moderated topoisomerase I activity (Figures
Differences of topoisomerase I activity in liver samples (a) and in hepatoma cells (b) (representative image of three independent experiments). Topoisomerase I activities in liver samples (a) and hepatoma cell lines (b) of 250 and 500 ng nuclear extract from peritumoral liver (a 1, a 2), hepatocellular carcinoma (a 3, a 4), HepG2 (b 1, b 2), and Hep3B cells (b 3, b 4). The activities of the specimens are related to the amounts of topoisomerase I protein in the cell nuclear extracts, as it is demonstrated on a western blot (c). Lane 1: Hep3B, lanes 2 and 3: HepG2, lanes 4 and 6: hepatocellular carcinomas, and lanes 5 and 7: peritumoral liver tissues. In addition to the 120 kDa band of the whole protein, the antibody reacts with more degradation products of the enzyme, including the 67 kDa catalytic fragment. A: plasmid control without cell nuclear extract. R: relaxed, S: supercoiled plasmid.
Western blots loaded with 15
Differences of topoisomerase I plasmid cleavage activity trapped by TpT in liver samples and in hepatoma cells (representative image of three independent experiments). pBR322 plasmid was linearized and end-labeled with 5
Nuclear extracts with high topoisomerase I activity from HepG2 cells and a surgically removed hepatoma were used to assess the inhibitory potential of commercial heparin, normal liver HS, human hepatocellular carcinoma, and peritumoral liver tissue GAG specimens on topo I plasmid relaxation and TpT-trapped cleavage reaction. All GAG specimens but HS from liver carcinoma inhibited the plasmid relaxation assay in a dose-dependent manner. In this measure, commercial heparin was the most effective confirming an earlier report [
In cleavage reactions, the efficacy of heparin and normal liver HS was identical. Peritumoral liver GAG inhibited the cleavage better than the HCC GAG did (not shown). When this experiment was repeated by using isolated peritumoral and HCC HS, the result was similar (Figure
Inhibitory effect of heparin, normal and peritumoral liver heparane sulfate (HS), and hepatocellular carcinoma heparane sulfate on the TpT induced topo I cleavage reaction (representative image of three independent experiments). The origin of nuclear extracts: (a): hepatocellular carcinoma; (b): HepG2 cells; (c): Hep3B cells. The reaction mixture contained pBR322 plasmid DNA, nuclear extracts as described, 100
(HCC)
(HepG2)
(Hep3B)
We also studied if this phenomenon, observed in a cell-free system, can also be detected when cell cultures were treated with heparin. To this end, hepatoma cell lines were treated with 100
Effect of heparin treatment on topoisomerase I plasmid relaxation activity of HepG2 and Hep3B cells (representative image of three independent experiments). Cells were plated to 6 well plates at
As topoisomerase I is a heparin-binding protein, we tested if heparin competed with the DNA for the enzyme. Changes in the electrophoretic mobility shift indicated that 10
Competition of heparin and DNA for topoisomerase I (representative image of three independent experiments). Effect of heparin on the DNA gel retardation produced by 10 U purified topoisomerase I protein. DNA fragment of 516 base pair size with 8 potential topo I-binding sequences was end-labeled with digoxigenin-UTP. Thirty-five ng DNA was incubated with 10 U topoisomerase I alone or in the presence of 10 and 100 ng heparin. Samples were run or 1% agarose, blotted to nylon membrane, and developed with antidigoxigenin alkaline phosphatase. 1: control DNA, without protein; 2 and 3: DNA with topoisomerase I; 4 and 5: DNA and topoisomerase I, with 10 and 100 ng heparin, respectively.
Our previous studies on various human cancer specimens revealed that the increase in the amount of proteoglycans and their sugar components is one of the most striking features of these tumors [
Furthermore, not only GAGs but also proteoglycans have been detected in the nucleus [
The evidences for the regulatory significance of heparin and HS justify our efforts to look for physiological or pathological cell nuclear functions where heparan sulfates could be involved [
The HepG2 cell line with high and the Hep3B cell line with moderate enzymatic activity served as a model to test this hypothesis. In support of the results obtained in a cell-free system, heparin exposure abolished the moderate topoisomerase I activity of the Hep3B cell line, while HepG2 cells retained their enzymatic activities. As the efficacy of TpT depends on the actual activity of topoisomerase I, it was reasonable to expect that, if administered together, heparin will interfere with the action of the drug. Certainly, using the Hep3B cell line with low topoisomerase I activity, the growth inhibitory effect of TpT decreased in the presence of heparin, while only a modest, transient heparin protection has been achieved on the HepG2 cell line. However, the mechanism of action was still a question.
Heparin alone did not affect the cell cycle. More likely, its protective effects against TpT action were related to its topo I binding capacity. In an assay mixture containing heparin, recombinant topo I and labeled DNA heparin appeared to compete with DNA for the binding of topo I. Thus, in the presence of heparin or HS a lower proportion of topoisomerase I could be bound covalently to DNA by TpT, thus preventing DNA fragmentation. The binding capacity and the amount of heparin or heparan sulfate can determine the proportion of topoisomerase I that will not interact with DNA. The decrease in dead cell fraction after a combined TpT-heparin treatment provided further support to this hypothesis.
Our results are in a good agreement with those clinical observations that aimed to treat liver tumors with TpT. Similarly to the HepG2 cell line, hepatoblastomas respond well for TpT treatment [
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work has been supported by the Hungarian National Science Fund (OTKA) Project no. 100904. The authors acknowledge the proofreading of Professor László Ötvös.