Effect of irinotecan on HMGB1,MMP9 expression,cell cycle,and cell growth in breast cancer (MCF-7) cells

Abstract

Irinotecan is a natural alkaloid agent widely used in cancer therapy. High-mobility group protein B1 as a non-histone chromosomal protein plays a fundamental role in gene expression and inflammation. In this study, the effect of irinotecan on high-mobility group protein B1 and MMP9 content, gene expression, cell cycle, and cell growth in human breast cancer cells (MCF-7) was investigated. The cells were exposed to various concentrations of irinotecan and the viability determined by trypan blue exclusion and 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazolium bromide assays. High-mobility group B proteins were extracted from the control and drug-treated cells and analyzed by immunoblot. High-mobility group protein B1 and MMP9 messenger RNA expression was studied by reverse transcription polymerase chain reaction. The results demonstrated reduction of cell viability upon increasing irinotecan concentration, up-regulated high-mobility group protein B1 gene expression, and down-regulated MMP9 mRNA. Although the content of high-mobility group protein B1 was decreased in chromatin extract upon drug action, no high-mobility group protein B1 release to extracellular space was detected by immunoblot analysis. Irinotecan decreased H3K9 acetylation and increased poly ADP-ribose polymerase fragmentation to 89 kDa and anion superoxide production suggesting induction of apoptosis in these cells. Propidium iodide staining of the cells 24 h after the drug treatment revealed arrest of the cells in S-phase. From the results, it is concluded that overexpression of high-mobility group protein B1 in the presence of irinotecan precedes breast cancer cells into apoptosis and in this response the binding of irinotecan to chromatin or high-mobility group protein B1 may condense/aggregate chromatin, preventing high-mobility group protein B1 release from chromatin.

Keywords

Breast cancer cells irinotecan gene expression high-mobility group B1 chromatin

Introduction

High-mobility group (HMG) proteins are the most abundant group of non-histone proteins in chromatin in which their name comes from their high electrophoretic mobility in polyacrylamide gel.¹ HMG proteins are divided into three main families according to their DNA-binding domain including HMGA, HMGB, and HMGN. Among them HGMB is the largest group with three families: high-mobility group B1 (HMGB1), HMGB2, and HMGB3. They have two DNA-binding motif, HMG-box A and B, and an acidic C-terminal domain.^2,3 HMGB proteins act as architectural elements in chromatin, bend DNA structure, and direct assembly of proteins on DNA as a chaperone, playing important role in replication, transcription, recombination, and DNA repair.³ In addition, HMGB1 represents different functions in the cell and apart from its binding to DNA and chromatin structure, translocates into extracellular space acting as a signaling molecule (cytokine) in inflammation, cell proliferation, invasion, angiogenesis, and late apoptosis in tumor cells.^4–7

Irinotecan (CPT-11) is a semisynthetic derivative of camptothecin (CPT), which is synthesized in 1983 with the addition of piperidino–carbonyloxy side chain at the position 10 of A ring and ethyl group at position 7 of the B ring of camptothecin (Figure 1). It is a potent chemotherapeutic agent, widely used in the treatment of solid tumors such as colorectal, lung, cervical, leukemia, and breast cancer.^8,9 It is an inhibitor of topoisomerase I (Topo-1) as it binds to DNA-Topo-I complex, generates a gap in DNA structure and therefore prevents DNA replication and ultimately leads to cell death.^10,11 Irinotecan alone or in combination with other drugs induces apoptosis through changes in expression of Bax, caspase-9, Bcl2, and surviving genes in cancer cells.^12–14

Figure 1.

(a) Chemical formula of irinotecan (C₃₃H₃₈N₄O₆) and its effect on the viability of MCF-7 cells obtained from 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. The cells at a density of 10⁶ cells/mL were cultured for 24 and 48 h in the absence and presence of varying concentrations of irinotecan. (b) Acridine orange/ethidium bromide staining of the cells treated with irinotecan and analyzed by fluorescence microscope. A–C are 0, 40, and 160 µg/mL irinotecan, respectively. Data are presented as means ± SD of three experiments.

In this study, we attempted to explore the biological mechanism of irinotecan action via HMGB1, MMP9, and its cytotoxicity in MCF-7 breast cancer cells. The results demonstrate evaluation of HMGB1 expression and reduction of its content in the cells extract. Also irinotecan represents toxic effect by inducing apoptosis and ultimately arrests the cells in S-phase.

Materials and methods

Irinotecan was purchased from Helale Ahmar, Tehran, Iran (manufactured by Vianex S.A., plant C. Greece). Before use, it was diluted at a concentration of 2 mg/mL and stored at −20°C in the dark. Trypan blue, 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazolium bromide (MTT), proteinase K, EcoR1-Hind III digested DNA marker, anti-rabbit IgG-HRP, cytochrome C, superoxide dismutase, ethidium bromide, trypsin, and Hoechst were purchased from Sigma Chemical Company (Becton Dickinson, San Diego, CA, USA). Poly ADP-ribose polymerase (PARP) antibody that only detects 89-kDa fragment and anti Histone H₃ lysine 9 acetylation (H3K9Ac) were purchased from Abcam (Cambridge, UK). HMGB1 antibody was prepared and purified in our Laboratory (Tehran, Iran). HMGB1 protein was prepared from calf thymus as described by Goodwin et al.¹ and used as a marker. Annexin V-propidium iodide (PI) apoptosis detection kit was obtained from Roche (Mannheim, Germany). Fetal calf serum (FCS) and RPMI-1640 medium were purchased from Gibco (Denmark). RPMI-1640 supplemented with 3.7 g/L NaHCO₃, 30 mg/L asparagine, 100 U/mL penicillin, and 10 mg/mL streptomycin (Gibco, Invitrogen, Carlsbad, CA, USA) pH 7.2 was prepared and after sterilization by 0.2-µm Millipore filter kept at 4°C before use. FCS was from Gibco (Denmark). Human breast cancer cell line MCF-7 was obtained from the Pasteur Institute of Iran. The cells were cultured in RPMI-1640 medium by incubation in fully humidified condition with 5% CO₂ at 37°C. The cells were sub-cultured in long phase of growth by trypsinization.

Cytotoxicity and nuclear morphology assay

MCF-7 cells (1 × 10⁶ cells/mL) were cultured in the absence and presence of various concentrations of irinotecan (0–160 µg/mL) for 24 and 48 h. The cells were mixed with trypan blue (0.4%, w/v) and viability determined using hemocytometer. Also MTT assay was performed following the method of Mosmann.¹⁵ The cells (10⁴ cells/well) were cultured in RPMI in 96-well cell culture plates and treated with various concentrations of the drug (0–160 µg/mL). Then, 10 µL of MTT (5 mg/mL in H₂O) was added to each well and the cells were incubated for 4 h in humidified condition and 5% CO₂ at 37°C. The medium was removed, 150 µL dimethyl sulfoxide (DMSO) was added to each well to solubilize the resulting formazan crystals, and the absorbance monitored at 570 nm by Bio Tek ELIZA microplate reader. Cell viability was estimated as the percentage of the control. All assays were performed in triplicate.

For morphological study, drug-treated cells and the controls were washed with cold phosphate-buffered saline (PBS), stained with fluorescence solution containing 100 µg ethidium bromide or 100 µg acridine orange in 1 mL PBS (pH 7.2), and the cells and nuclear morphology were examined under Zeiss fluorescence microscope.

RNA extraction and real-time PCR

Total RNA was extracted from the drug-treated cells and the controls using RNX-Plus solution (Sina clone, Iran) following the manufacturer’s instruction. The RNA (2 µg) was then reverse transcribed into complementary DNA (cDNA) using two-step reverse transcription polymerase chain reaction (RT-PCR) Kit (Vivantis, Malaysia) according to the manufacturer’s protocol. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in final volume of 20 µL using quantitative polymerase chain reaction (qPCR) Green-Master (Jena Bioscience, Germany). PCR profile was 95°C for 3 min as pre-denaturation step, 40 amplification cycles at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s. Final extension was carried out at 72°C for 10 min.

Gene-specific PCR primers were as follows: the HMGB1 forward: 5′-ACAGCCATTGCAGTACATTGA-3′, HMGB1 reverse: 5′-ATGCTCCTCCCGACAAGTTT-3′, MMP9 forward: 5′-TCTTCCCTGGAGACCTGAGAAC-3′, MMP9 reverse: 5′-TCTTCCCTGGAGACCTGAGAAC-3′, β-Actin forward: 5′-CAAGATCATTGCTCCTCCTG-3′, β-Actin reverse: 5′-ATCCACATCTGCTGGAAGG-3′ (Sina Clone). All gene sequences were acquired from the NCBI website and Primer3, and oligo analyzer and gene runner programs were used to design primers for the specific genes and their specificity analyzed by the NCBI Blast Program. β-Actin was used as an endogenous control and quantitation of gene expression determined using ΔΔC_t calculation, where C_t is the threshold cycle. HMGB1, MMP9, and β-actin mRNA expression levels were then analyzed as fold-change by the 2^−ΔΔCt method.¹⁶

Extraction of HMGB1 protein, PARP, and DNA from the cells

HMGB1

MCF-7 cells (1 × 10⁶) were incubated in the presence and absence of various concentrations of irinotecan and HMG proteins were extracted by 0.35 M NaCl in 10 mM Tris-HCl (pH 7.5) containing 1/40 ratio cocktail protease inhibitor (Sigma, St. Louis, MO, USA) according to the method of Goodwin et al.¹ To detect release of HMGB1, the culture media were centrifuged and released HMGB1 into the culture medium was extracted by 5% perchloric acid (PCA) which selectively solubilizes HMG proteins. Then, the proteins were precipitated from the extracts using 12% trichloroacetic acid (TCA), applied on 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and analyzed by western blot using HMGB1 antibody.

PARP

Cells treated with different concentrations of the drug and the control were detached and suspended for 1 h in extraction buffer containing 62 mM Tris-HCl, 2% SDS, 10% glycerol, 4 M urea, 0.3% bromophenol blue, and 5% β-mercaptoethanol at 4°C. The samples eppendorfed for 5 min at 4°C and the supernatants were loaded on 12% SDS-polyacrylamide gel. The unstained gel was immunoblotted against anti PARP antibody specific for 89-kDa fragment.

DNA extraction and agarose gel

Drug-treated cells and the controls were washed with PBS and suspended in 0.5 mL of lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM NaCl, 1% SDS, 0.2 mM proteinase k) for 3 h at 37°C. DNA was extracted once with phenol, once with chloroform, and precipitated with cold absolute ethanol (2.5 volume) in the presence of 0.1 volume of 3 M sodium acetate. The samples were incubated at −20°C for 2 h and centrifuged at 8000g for 5 min at 4°C. After washing with 70% ethanol, DNA samples were air dried, solubilized in TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), after adding DNA sample solvent, heated at 65°C and electrophoresed on 1.2% agarose gel running at 100 V for1 h. The gel was stained with ethidium bromide (0.5 µg/mL), visualized under UV light, and photographed.

SDS-PAGE and Western blot

The extracted proteins were analyzed on 15% polyacrylamide gel at 100 V according to Laemmli.¹⁷ The proteins were transferred to nitrocellulose membrane by electro transfer at 4°C for 4 h. The membranes were incubated in blocking buffer with 1% (w/v) gelatin in Tris-NaCl buffer for 1 h at 37°C and washed three times (each 5 min) with the same buffer. The membranes were incubated overnight with anti-rabbit-specific antibodies (HMGB1, PARP, H3K9Ac individually), washed three times with Tris-NaCl/Tween (0.05%), and incubated with peroxidase-conjugated goat anti-rabbit IgG for 2 h at room temperature. After three times washing, the membranes were incubated with the substrate solution of 3,3′-diaminobenzidine (DAB) in 0.1 M PBS, 10 µL H₂O₂ for 5 min at room temperature and the reaction stopped by adding distilled water. The membranes were dried, photographed, and intensity of the bands quantified using Image-J software.

Quantitative analysis of DNA fragmentation and anion superoxide

DNA fragmentation was assayed by diphenylamine (DPA) reaction and agarose gel. The cells were treated with various concentrations of the drug and lysed in lysis buffer containing 10 mM Tris-HCl, 1 mM EDTA, 0.2% triton X-100, and centrifuged at 8000g for 10 min at 4°C. The pellets (B) were suspended in 0.5 mL of lysis buffer, 0.5 mL of 25% TCA was added to the supernatants (A) and the pellets (B) and incubated for 24 h at 4°C. The samples were then centrifuged at 8000g for 10 min (4°C), and the pellets were suspended in 80 µL of 5% TCA and incubated at 83°C for 20 min. The reaction was followed by adding 160 µL DPA solution (150 mg DPA in 10 mL acetic acid, 150 µL sulfuric acid, and 50 µL acetaldehyde 1.6%) to each sample and incubated 24 h at room temperature. Absorbance was read at 600 nm using Bio Tek ELIZA microplate reader and the percentage of DNA fragmentation was estimated as: % fragmented DNA = abs A/abs A + abs B × 100, A and B are absorbance of supernatant and pellet, respectively.

The production of anion superoxide was estimated according to the method of Mayo and Curnutte.¹⁸ The cells were incubated in the absence and presence of irinotecan for 3 h, and after washing with PBS, 200 µL cytochrome C (160 µM) and 10 µL phorbolmyristate 13-acetate (10⁻⁶ M) were added to each sample and 17 µL of superoxide dismutase (1 µg/mL) to the control. The samples were then incubated at 37°C for 10 min, centrifuged at 8000g (4°C) for 2 min, and the absorbance monitored at 550 nm by ELIZA microplate reader.

Flow cytometry

The control and drug-treated cells (1 × 10⁶) were stained with annexin V/PI according to the manufacturer’s protocol for 20 min in the dark at room temperature. Flow cytometry analysis was performed using FAC Scan flow cytometer (Becton Dickinson), and the percentage of apoptotic cells was determined by Win MDI software.

For cell cycle analysis, the cells (1 × 10⁶) were cultured in the absence and presence of irinotecan, fixed with 70% ethanol at 4°C for 2 h, and stained in PBS containing 20 µg/mL PI and 20 µg/mL RNase at 37°C for 30 min in the dark. The cell cycle profile was performed by a FAC Scan flow cytometry, and data were analyzed using Win MDI software.

Results

Toxic effect of irinotecan on MCF-7 cells

Breast cancer MCF-7 cells were cultured in the absence and presence of various concentrations of irinotecan for 24 and 48 h and viability determined by trypan blue exclusion and MTT assays. As is shown in Figure 1(a) in the absence of the drug, the cells represent about 100% viability but in the presence of irinotecan, viability is decreased to 85.5% at 10 µg/mL and 58% at 160 µg/mL of irinotecan. When the cells were cultured for longer time (48 h), viability of viable cells is decreased to 33% when 160 µg/mL drug was used. MTT assay also confirmed the result, and as is seen in Figure 1(a), there is correlation between trypan blue and MTT assays.

For morphological analysis, the cells were labeled with acridine orange/ethidium bromide (AO/ET) 24 h after irinotecan was applied. Dual staining was examined under fluorescence microscope and the result is shown in Figure 1(b). Fluorescent acridine orange penetrates the live and dead cells and emits green fluorescence; however, ethidium bromide penetrates into dead cells and emits red fluorescence. As is seen in Figure 1(b), the viable cells in the absence of irinotecan are green and have intact nuclei, but upon addition of the drug, the cells loss their integrity and their color is changed into green and orange, which is an indication of apoptotic cells with condensed chromatin and fragmented nuclei but necrotic cells show red color with no condensed chromatin and are mostly disrupted.

The effect of irinotecan on HMGB1 expression and its content in the cell

High-mobility group box protein 1 (HMGB1) is described as a non-histone DNA-binding protein having different functions depending on its location and cell types. HMGB1 expression varies in different cancer cells and plays important role in chemotherapy.^4,6,19 To evaluate the expression of HMGB1 mRNA, total RNA was extracted from MCF-7 cells cultured in the absence and presence of various concentrations of irinotecan, converted to cDNA and analysis of gene expression performed by qPCR. β-actin was used to establish normalized expression (ΔΔCt). As is seen in Figure 2(a), HMGB1 expression in the presence of irinotecan is significantly increased as the drug concentration raised, thus at 160 µg/mL of the drug, about twofold increase in the expression is observed compared to the control (marked 0).

Figure 2.

The effect of irinotecan on (a) HMGB1 mRNA expression and (b) MMP9 expression in MCF-7 cells. The cells were treated with various concentrations of the drug, total RNA was isolated, expressed by qPCR. β-actin used as a reference gene. (c) Immunoblot of HMGB1 extracted from the cells in the absence and presence of irinotecan. The gel was 15% SDS-PAGE. Drug concentrations are indicated at the top of the gel. (d) Relative bands intensity analyzed by Image-G. The results are means ± SD of at least two experiments.

Since irinotecan is used in the treatment of metastatic breast cancer cells and MMP9 is one of the most important proteins in metastasis,²⁰ the expression of this protein was also estimated in the identical condition as used for HMGB1. The result in Figure 2(b) shows that, in contrast to HMGB1, MMP9 mRNA expression is down-regulated by increasing irinotecan concentration. At low concentrations of the drug (≤40 mg/mL), the level of MMP9 expression is similar to the control, but upon raising drug concentration, it is decreased thus at 160 µg/mL of irinotecan the mRNA expression is about 50% of the control.

Up-regulation of HMGB1 expression in the presence of irinotecan in MCF-7 cells encouraged us to investigate whether the drug has any effect on HMGB1 content in the cells and its possible release to extracellular space. For this purpose, HMGB proteins were extracted from the drug-treated cells and controls and immunoblotted against HMGB1 antibody. As is seen in Figure 2(c), the control (lane 0, in the absence of irinotecan), a band in HMGB1 position, is observed compared to thymus HMGB1 protein (lane M). Upon addition of the drug, the content of HMGB1 protein is gradually decreased thus at concentrations higher than 40 µg/mL, the band intensity is reduced and then completely disappears on the gel. Estimation of relative band intensities (Figure 2(d)) confirmed the result thus at 10, 20, and 40 µg/mL of the drug, 81%, 67%, and 52% intensity is observed and at higher concentrations it is reduced to zero.

As the result obtained from the HMGB1 content analysis is opposite to its expression outlined above, and that it has been shown that HMGB1 is passively released during late apoptosis and necrosis into extracellular space,^4,21 it was necessary to found out whether HMGB1 is secreted outside the cell under exposure to irinotecan. Therefore, the cell culture media after drug treatment were collected and HMGB proteins were extracted and immunoblotted against HMGB1 antibody. The blot pattern was negative and as no band was detected in HMGB1 position, data are not shown.

Overexpression of HMGB1 genome on one hand and reduction of HMGB1 content in the presence of irinotecan on the other hand raised a question whether high concentrations of the drug influences chromatin somehow that prevents extraction of HMGB proteins. Acetylation removes positive charges of histones, thereby transfer condensed chromatin to relaxed one. It is known that acetylation of histone H3 on K9 is an epigenetic marker of active genes.²² In order to analyze the effect of irinotecan on epigenetic changes of H3k9Ac, the cells were treated with various concentrations of irinotecan and modification of histone H3 analyzed by immunoblot against anti H3K9Ac. As is seen in Figure 3, the control (lane 0) represents a thick band of H3K9Ac against H3K9Ac antibody, but the content of H3K9Ac is decreased as irinotecan concentration increased until at high concentrations of the drug, only a faint band at H3K9Ac position is observed. Relative band intensity estimation (Figure 3 marked H3K9Ac) shows that at 10 µg/mL of irinotecan, the content of H3K9Ac is significantly diminished to 63% thus at 320 µg/mL only 19% of H3K9Ac content is detected.

Figure 3.

Immunoblot of HMGB1, H3K9Ac, and PARP proteins extracted from the MCF-7 cells (10⁶/mL) in the absence and presence of various concentrations of irinotecan. Analysis performed with HMGB1 antiserum, rabbit anti mouse H3K9Ac, and antibody against 89-kDa fragment of cleaved PARP. Irinotecan concentration in µg/mL is shown on the top of each gel. The protein level in the control and treated cells was quantified by Image-J software (n = 3).

PARP is a nuclear enzyme with a molecular weight of 116 kDa. During apoptosis, it is cleaved by caspases to 24 and 89 kDa fragments that are useful markers for apoptosis detection in cells under exposure to toxicants.²³ To investigate the effect of irinotecan on PARP cleavage, it was extracted from the control and drug-treated cells and immunoblotted against 89-kDa fragment antibody. As is seen in Figure 3 (marked PARP), in the presence of low concentrations of the drug, no band is observed but upon increasing irinotecan concentration (>40 µg/mL), 89-kDa fragment of PARP is appeared and its content is increased upon enhancement of the drug concentration. Its content at 80, 160, and 320 µg/mL is 65%, 80%, and 100%, respectively, as estimation of relative band intensity confirmed it.

Irinotecan induces apoptosis in MCF-7 cells

Cleavage of PARP demonstrates that irinotecan induces apoptosis in the MCF-7 cells. For this purpose, the control and drug-treated cells were stained with annexin V/PI and analyzed by flow cytometry (Figure 4(a)). In this procedure, viable cells are annexin V⁻ and PI⁻, early apoptotic cells are annexin V⁺ and PI⁻, and necrotic cells are annexin V⁺ and PI⁺. The percent of apoptotic and necrotic cells were estimated, and the result is shown in Figure 4(b). As is seen, in the absence of the drug, the cells are 98% viable and the amount of apoptotic and necrotic cells are nearly zero. Upon addition of the drug, a shift occurs from live cells (lower-left quadrant) to early apoptotic cells (lower-right quadrant) as irinotecan concentration is increased. Also percent of apoptotic cells is increased to 10%, 21%, and 35% after exposure to 40, 80, and 160 µg/mL of the drug, whereas necrosis is negligible and does not exceed 8%.

Figure 4.

(a) Flow cytometry profiles of the cells 24 h after irinotecan exposure, stained with Annexin V/PI. (A–D) are 0, 40, 80, and 160 µg/mL irinotecan, respectively. (b) Histogram showing percentages of apoptotic and necrotic cells in the control and drug-treated cells. (c) Data are means ± SD of three experiments. Cell cycle analysis of the cells fixed in 70% ethanol, stained with PI and analyzed by flow cytometry assay. The percent of the cells in Go/G1, S, and G2/M phases were analyzed by Win MDI software. Profiles (A–C) are 0, 40, and 160 µg/mL irinotecan, respectively. Data are means ± SD of three experiments.

To determine the mechanism of irinotecan-induced cell death, cell cycle analysis using PI staining and flow cytometry was used. Figure 4(c) demonstrates the diagrams. In the control, distribution is 60%, 22%, and 19% cells in G1, S, and G2 cycles, respectively, whereas exposure to irinotecan resulted in increase in the cell accumulation in S-phase, thus distribution for 160 µg/mL is 53%, 38%, and 8% for G1, S, and G2 phases, respectively.

To assess whether irinotecan induces DNA damage, quantitative DNA fragmentation was examined by DPA assay and agarose gel. As is shown in Figure 5(a), as the drug concentration is raised, the content of DNA fragmentation is gradually increased and at 80 µg/mL of the drug, 35% fragmentation is achieved. On agarose gel (Figure 5(b)), DNA of the cells in the absence of irinotecan represent a band at 4800 bp. The pattern of DNA fragmentation in the presence of the drug up to 80 µg/mL is not significant but at higher concentrations induces DNA fragmentation, as smears in the presence of the drug is more extensive.

Figure 5.

(a) DNA fragmentation analyzed by diphenylamine reaction (■) and superoxide anion production (Δ) of the cells in the absence and presence of various concentrations of irinotecan. Data are means ± SD of three experiments. (b) 1.5% agarose gel electrophoresis of DNA extracted from the drug-treated cells and control. The drug concentration is shown at the top of each lane and M is EcoR1 digested DNA marker.

Determination of anion superoxide production and cytochrome C reduction in the control and drug-treated cells revealed that the amount of anions production is remarkably enhanced upon increasing irinotecan concentration (Figure 5(a)). Thus, threefold increase is observed when 160 µg/mL irinotecan is used compared to untreated cells.

Discussion

High-mobility group protein 1(HMGB1) is a conserved non-histone protein of chromatin with different functions. It binds to DNA molecule in nuclei via its two HMG-box domains A and B and regulates chromatin activity such as replication, transcription, repair, and remodeling.³ In the extracellular space, it is as a cytokine that plays potential role in apoptosis, autophagy, and metastasis.^4,24,25 Irinotecan is a potent anticancer drug in the treatment of solid tumors, and its biological activity is related to its binding to DNA-Topo I complex.^10,11 But to date, no work has been published on the effect of this drug on chromatin proteins. In this study, we have explored the effect of irinotecan on HMGB1 protein expression and its possible toxicity toward apoptosis in MCF-7 cells. We have demonstrated for the first time that HMGB1 is essential activator of cellular response to genotoxicity caused by irinotecan. The finding makes HMGB1 as a potential target for modulating activity of chemotherapeutic drugs.

The results demonstrate that irinotecan has toxicity on MCF-7 cells at high concentrations. The IC₅₀ value obtained is 100 µg/mL after 48 h exposure. Various IC₅₀ values have been reported for irinotecan on different cancer cells depending on cell type, time of exposure, and the number of cells. For example, IC₅₀ value of irinotecan alone in colon cancer cells is about 62 µM after 72 h.²⁶ IC₅₀ value of irinotecan in MCF-7 breast cancer cells is higher (about 94 µM) suggesting that breast cancer cells are slightly resistance to this drug. Also analysis of fluorescent staining of the cells exposed to the drug clearly exhibit apoptotic events, condensation of chromatin, and nuclear fragmentation.

HMGB1 expression in the cells exposed to irinotecan is up-regulated compared to the control; however, the content of HMGB1 protein in the cells is reduced when analyzed by western blot. Overexpression of HMGB1 has been observed in some cancer cells. It has been reported that HMGB1 is overexpressed and released following chemotherapy in cell of hematological malignances.²⁷ Also overexpression of HMGB1 in K562 leukemia cells sustains Bcl-2 protein expression and inhibits Adriamycin-induced activation of caspase-3 and 9.²⁸ It has been shown that overexpression of HMGB1 affects cell cycle progression in MCF-7 cells and sensitizes breast cancer cells to cisplatin family treatment.^29,30 In contrast down regulation of HMGB1 expression results in apoptosis in prostate cancer cells via caspase-3-dependent pathway.³¹ Unlike the reports that overexpression of HMGB1 is always accompanied by its translocation to extracellular space, in our experimental condition, no HMGB1 release from MCF-7 cells exposed to irinotecan was observed. HMGB1 has dual function depending on its subcellular location. It is normally located in the nucleus, binds to DNA and acts as a chromatin gene regulator establishing nuclear function.³ In addition, it acts as an extracellular signaling molecule that is critical in inflammation and carcinogenesis.²⁴ The only explanation for overexpression of HMGB1 in irinotecan-treated cells and lack of its release to extracellular space is that irinotecan proceeds breast cancer cells into apoptosis in which PARP cleavage, reactive oxygen species (ROS) production, and DNA fragmentation as well as flow cytometry confirmed it. No release of HMGB1 in the presence of irinotecan is in agreement with the finding that HMGB1 release is a specific marker of necrosis with cell death, necrosis can lead to release of HMGB1 by a passive mechanism while with apoptosis, and HMGB1 is only released during secondary necrosis (late apoptosis) when cell barrier breaks down.³² Also flow cytometry indicates that the percent of necrosis in the cells exposed to even high concentrations of irinotecan is negligible, which confirms the occurrence of apoptosis in irinotecan-treated cells. MMP9 is a protease that degrades extracellular matrix in tumor cells in metastasis process.²⁰ Down-regulation of MMP9 mRNA expression in the presence of the drug suggests that irinotecan can reduce metastasis, consistent with the fact that irinotecan is useful in the treatment of metastatic breast cancer.

Reduction of HMGB1 content in irinotecan-treated cells is not related to HMGB1 release into extracellular space, but it is suggested that possibly binding of irinotecan to chromatin and HMGB proteins condenses chromatin structure somehow that HMGB1 protein cannot be extracted by manual procedures. H3K9Ac modification is reduced in a dose-dependent manner confirming chromatin condensation because H3K9Ac is one of the epigenetic markers of condense and relax chromatin.³³

From the results, it is concluded that irinotecan, as an anticancer drug, can be used in breast cancer chemotherapy in combination with potent drugs. Although it enhances HMGB1 gene expression, but at the present time, there is no evidence concerning that the produced mRNA is translated to HMGB1 protein. Hence, it is possible that by occurrence of apoptosis, HMGB1 mRNA may be degraded, what normally happens to DNA molecule. Irinotecan induces apoptosis in MCF-7 cells and HMGB1 may play a role in this process. The level of necrosis in the drug-treated cells is very low and no release of HMGB1 is observed and the cells are arrested in S-phase. Although the results are useful in understanding the mechanism of irinotecan action at genome level and its genotoxicity, more work is needed to elucidate the real mechanism of HMGB1 expression in breast cancer cells upon chemotherapy.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors would like to acknowledge the financial support of University of Tehran for this research under grant #27/6/6401017.

References

Goodwin

Sanders

Johns

. A new group of chromatin-associated proteins with a high content of acidic and basic amino acids. Eur J Biochem 1973; 38: 9–14.

Zhang

Wang

. HMG modifications and nuclear function. Biochim Biophys Acta 2010; 1799: 28–36.

Reeves

. Nuclear functions of the HMG proteins. Biochim Biophys Acta 2010; 1799: 3–14.

Tang

Kang

Zeh

. High-mobility group box 1 and cancer. Biochim Biophys Acta 2010; 1799: 131–140.

Andersson

Pisetsky

. HMGB1: a multifunctional alarmin driving autoimmune and inflammatory disease. Nat Rev Rheumatol 2012; 8: 195–202.

Kang

Zhang

Zeh

. HMGB1 in cancer: good, bad, or both? Clin Cancer Res 2013; 19: 4046–4057.

Ulloa

Batliwalla

Gregersen

. High mobility group box chromosomal protein 1 as a nuclear protein, cytokine, and potential therapeutic target in arthritis. Arthritis Rheum 2003; 48: 876–881.

Chabot

. Clinical pharmacokinetics of irinotecan. Clin Pharmacokinet 1997; 33: 245–259.

Ramirez

Koch

Edenfield

. Irinotecan-induced dysarthria: a case report and review of the literature. J Oncol Pharm Pract. Epub ahead of print 23 February 2016. DOI: 10.1177/1078155216634181.

10.

Mathijssen

Loos

Verweji

. Pharmacology of topoisomerase 1 inhibitors irinotecan (CPT-11) and topotecan. Curr Cancer Drug Targets 2002; 2: 103–123.

11.

Sasine

Savaraj

Feun

. Topoisomerase 1 inhibitors in the treatment of primary CNS malignancies: an update on recent trends. Anticancer Agents Med Chem 2010; 10: 683–696.

12.

Rudolf

John

Cervinka

. Irinotecan induces senescence and apoptosis in colonic cells in vitro. Toxicol Lett 2012; 214: 1–8.

13.

Wulaningsih

Wardhana

Watkins

. Irinotecan chemotherapy combined with fluoropyrimidines versus irinotecan alone for overall survival and progression-free survival in patients with advanced and/or metastatic colorectal cancer. Cochrane Database Syst Rev 2016; 2: CD008593.

14.

Takano

Yamamoto

Tabata

. Impact of UGT1A1 genotype upon toxicities of combination with low-dose irinotecan plus platinum. Asia Pac J Clin Oncol 2016; 12: 115–124.

15.

Mosmann

. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55–63.

16.

Livak

Schmittgen

. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods 2001; 25: 402–408.

17.

Laemmli

. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–685.

18.

Mayo

Curnutte

. Kinetic microplate assay for superoxide production by neutrophils and other phagocytic cells. Methods Enzymol 1990; 186: 567–575.

19.

Smolarczyk

Cichoń

Jarosz

. [HMGB1—its role in tumor progression and anticancer therapy]. Postepy Hig Med Dosw 2012; 66: 913–920.

20.

Song

Zhou

. Prognostic value of matrix metalloproteinase 9 expression in breast cancer patients: a meta-analysis. Asian Pac J Cancer Prev 2013; 14: 1615–1621.

21.

Gauley

Pisetsky

. The translocation of HMGB1 during activation and cell death. Autoimmunity 2009; 42: 299–301.

22.

Koch

Andrews

Flicek

. The landscape of histone modifications across 1% of the human genome in five human cell lines. Genome Res 2007; 17: 691–707.

23.

Isabelle

Moreel

Gagné

. Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry. Proteom Sci 2010; 8: 22–32.

24.

Ellerman

Brown

de Vera

. Masquerader: high mobility group box-1 and cancer. Clin Cancer Res 2007; 13: 2836–2848.

25.

Liu

Yang

Kang

. HMGB1-induced authophagy promotes chemotherapy resistance in leukemia cells. Leukemia 2011; 25: 23–31.

26.

Jang

Hong

Jang

. Synergistic effects of simvastatin and irinotecan against colon cancer cells with or without irinotecan resistance. Gastroenterol Res Pract 2016; 2016: 1–9.

27.

Yang

Zhang

. [HMGB1-a as potential target for therapy of hematological malignancies]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2014; 22: 560–564.

28.

Xie

. [Role of high mobility group box 1 in adriamycin-induced apoptosis in leukemia K562 cells]. Ai Zheng 2008; 27: 929–933.

29.

Yoon

Lee

Yoon

. Effects of HMGB1 overexpression on cell cycle progression in MCF-7 cells. J Korean Med Sci 2004; 19: 321–326.

30.

Liang

Lippard

. Steroid hormones induce HMG1 overexpression and sensitize breast cancer cells to cisplatin and carboplatin. Proc Natl Acad Sci USA 2000; 97: 5768–5772.

31.

Gnanasekar

Thirugnanam

Ramaswamy

. Short hairpin RNA constructs targeting high mobility group box-1 expression leads to inhibition of prostate cancer cell survival and apoptosis. Int J Oncol 2009; 34: 425–431.

32.

Bell

Jiang

Reich

. The extracellular release of HMGB1 during apoptotic cell death. Am J Physiol Cell Physiol 2006; 291: C1318–C1325.

33.

Zentner

Henikoff

. Regulation of nucleosome dynamics by histone modifications. Nat Struct Mol Biol 2013; 20: 259–266.