Research ArticleExperimental Studies

siPGK1 Sensitizes Chemoresistant Human Ovarian Cancer Cell Lines to Cisplatin

CHARLOTTE LEPLEUX, EDWIGE ABEILARD-LEMOISSON, MARYLINE DUVAL, PHILIPPE ICARD and HUBERT LINCET
Anticancer Research October 2012, 32 (10) 4277-4286;

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Abstract

Enhanced glycolysis provides essential intermediates for cancer cell proliferation. Its inhibition could be a promising approach for destroying tumors, especially those developing in hypoxic conditions, which are presumably the most chemoresistant. In hypoxic cells, glycolysis provides the main part of ATP. Phosphoglycerate kinase-1 (PGK1) catalyzes a crucial reaction of glycolysis that reconstitutes the two molecules of ATP previously consumed. PGK1 inhibition could arrest growth or kill hypoxic and/or chemoresistant cells. We tested siPGK1 transfection in two human ovarian cancer cells lines of increasing chemoresistance, and showed that: Expression of PGK1 was significantly reduced and associated with blockade of cell growth in the G1 phase; siPGK1 associated with cisplatin was more effective than cisplatin-alone at inhibiting proliferation of chemoresistant cells; siPGK1 -alone and -associated with cisplatin strongly increased expression of the BH3-only pro-apoptotic protein BCL-2 Interacting Mediator of cell death (BIM). PGK1 might be a key target for sensitizing chemoresistant cells to cisplatin.

Glycolysis plays a main role in energy metabolism of cancer cells, which take-up glucose at higher rates than normal cells, and transform a significant part to lactate, even in the presence of oxygen. This phenomenon, referred to as aerobic glycolysis or the Warburg effect, is a common feature of tumor growth (1, 2). Enhanced glycolysis provides an increased supply of various precursors (such as ribose, glycerol and serine) required for nucleotide, protein and lipid synthesis (3-5). Cancer cells must produce great quantities of macromolecules and lipids to proliferate, and this metabolism must be sustained by a continuous production of ATP and co-factors (NAD+, NADPH, H+). Thus, glycolysis plays a critical role in tumor development, and several studies have shown that its inhibition could be a promising approach for cancer treatment (6-10).

Among the glycolytic enzymes, phosphoglycerate kinase-1 (PGK1) catalyzes a crucial step of glycolysis, transferring a phosphate group from 1,3-biphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate. At this step, glycolysis has reached a break-even point because the two molecules of ATP which have been previously consumed are now reformed. This reaction could be of essential importance for producing ATP in hypoxic cells, where oxidative phosphorylation is slowed-down or arrested. Interestingly, overexpression of PGK1 has been observed in cisplatin-resistant ovarian cancer cell lines, and has been associated with a poor outcome of patients with multiple drug-resistant lung adenocarcinomas (11, 12). Thus, PGK1 might be an interesting target, inhibition of which could be able to sensitize cancer cells to chemotherapy, especially hypoxic cells which are likely to be the most chemoresistant ones (13, 14).

Ovarian cancer is the fifth most frequent cause of cancer-related death in women, often diagnosed at an advanced stage (75%), and is associated with poor survival (15-17). Despite a good response to surgery and to first-line platinum-based chemotherapy, the 5-year survival rates for stage III and IV disease are 20%-30%, mainly due to intrinsic chemoresistance or to progressively-acquired resistance. In the present study, we investigated whether the small-inhibitory (si) RNA to PGK1 could sensitize human ovarian carcinoma cell lines to cisplatin.

Materials and Methods

Cell lines and culture conditions. The IGROV1 cell line was established from a human ovarian adenocarcinoma and was obtained from the European Collection of Cell Cultures (ECACC) (Sigma-Aldrich, Saint Quentin Fallavier, France). The variant, highly chemoresistant cell line, IGROV1-R10, was established through a protocol that mimics the clinical protocol of cisplatin treatment leading to a complete cisplatin refractory state of cells (18). Cells were grown in RPMI medium supplemented with 4500 mg/l glucose, 2 mM Glutamax™, 1 mM sodium pyruvate, 10% fetal calf serum, 33 mM sodium bicarbonate (Invitrogen, Cergy-Pontoise, France). Cells were maintained at 37°C in a 5% CO2 humidified atmosphere and split twice a week by trypsinization.

siRNA synthesis and transfection. All siRNAs used in these studies were chemically synthesized by Eurogentec (Liege, Belgium) and received as annealed oligonucleotides. The sequence of the double-stranded RNA used to inhibit PGK1 expression (noted siPGK1) was; sense: 5’ gagcuaaaguugcagacaa-3’ and anti-sense: 5’ uugucugcaac uuuagcuc 3’. The sequence of the control siRNA siGFP was; sense: 5’ gacguaaacggccacaagutt-3’ and anti-sense: 5’ acuuguggccgu uuacguctt-3’. The control siRNA does not bear any homology with any relevant human genes. According to the manufacturer's instruction, exponentially growing cells were seeded (2.5×105 cells per 25 cm2/flask) the day before reaching 30-50% confluency at the time of transfection. Briefly, the transfecting INTERFERin™ reagent (Polyplus Transfection, Strasbourg, France) was added to siRNA (20 nM) diluted in Opti-MEM® reduced serum medium (Invitrogen, Cergy-Pontoise, France) and complex formation was allowed to proceed for 15 min at room temperature before being applied to cells. The next day, cell media were changed to remove the transfecting reagent. At the indicated time, cells were trypsinized and washed with cold PBS. Cell pellets were used directly or stored at −80°C for later use. At least two independent experiments were carried out and typical results are shown.

Analysis of cellular DNA content by flow cytometry. Preparation of cells: After exposure, detached cells were collected separately. Adherent cells were then harvested by trypsin/EDTA dissociation. Adherent and detached cells were pooled and washed in phosphate buffered saline (PBS) before being fixed in 70% ethanol and stored at −20°C until analysis. Before flow cytometric analysis, cells were washed in PBS and incubated for 30 min at a temperature of 37°C in PBS in order to allow the release of low molecular weight (m.w.) DNA, characteristic of apoptotic cells. After centrifugation at 4000 ×g for 10 min, the cell pellets were resuspended and stained with propidium iodide (PI) using DNA Prep Coulter Reagent Kit (Beckman-Coulter, Villepinte, France), at a final density of 106 cells/ml. Instrument settings: The samples were analyzed using an EPICS XL flow cytometer (Beckman Coulter, Villepinte, France) equipped with an argon laser at 15 mW. PI-stained cells were analyzed using 488 nm excitation. A 620-nm band pass filter was placed on the red fluorescence of PI. Computerized gating was applied on the side and forward scatter to exclude very small debris and on pulse width and integral peak of red fluorescence to eliminate aggregates. All samples were analyzed at a flow rate lower than 100 events per second and with a sheath pressure of 30 psi.

Data analysis: the EXPO 32 Acquisition Software (Beckman Coulter, Villepinte, France) was run for data acquisition.

Nuclear morphology study. After treatment, detached cells were separately collected and adherent cells were dissociated by trypsin/EDTA. Cells were then pooled and collected on a polylysine-coated glass slide by cytocentrifugation, fixed in an ethanol/chloroform/acetic acid solution (6:3:1), and incubated for 15 min, at room temperature, with a 1 μg/ml aqueous solution of 4’,6-diamidino-2-phenyl-indole (DAPI). Slides were then extensively washed in distilled water, mounted in Mowiol and analyzed under a fluorescence microscope.

Western immunoblotting. Adherent cells were rinsed with deionized water and lysed with lysis buffer [pH 8.8, 30 mM Tris buffer containing 6 M urea, 2 M thiourea, 2% CHAPS and 1x protease inhibitor mixture]. Western blots were carried out as previously described in detail (5). The membrane was either incubated overnight at 4°C in Tween-TBS-milk 5% with primary antibodies PGK1 (1:2000; Abcam, Cambridge, UK), PARP (1:1000; Cell Signaling Technology, Beverly, MA, USA), BCL-xL (1:1000), BIM (1:1000; Cell Signaling Technology), tubulin (1:5000, Sigma-Aldrich). After three washes with Tween-TBS and one with TBS, immunoreactivity was detected by enhanced chemiluminescence (ECL Prime kit; GE Healthcare, Velizy-Villacoublay, France).

Results

siPGK1 transfection significantly reduces the expression of PGK1 on human ovarian IGROV1 carcinoma cells. To determine whether siPGK1 could be down-regulated in ovarian cancer cells, total cellular extracts of IGROV1 cells previously transfected with siPGK1 (20 nM) 48 h before were analyzed by western blot. Analysis revealed a significant reduction of PGK1 protein expression in these transfected cells. No modification of PGK1 expression was induced in control cells [non-transfected or transfected with small-inhibitory Green Fluorescent Protein (siGFP)] (Figure 1A). Densitometric analysis (Figure 1B) showed a more than 60% reduction in the expression level of PGK1 in cells 48 h after siPGK1 transfection.

The reduced expression of PGK1 induces a blockage of cell growth and cell cycle in IGROV1 and IGROV1-R10 cells. IGROV1 and IGROV1-R10 cells which were cultured in monolayers and transfected with siPGK1, at 48 h after transfection, revealed the presence of many rounded cells, suggesting detached cells (Figure 2). In both lines, especially 72 h after siPGK1 transfection, cell layers were less confluent as compared to control cell lines (Figure 2). In comparison, the morphological features of layers of cells transfected with siGFP showed transfection and did not affect IGROV1 cell growth at any time.

DNA histogram analysis of IGROV1 cells transfected with siPGK1 showed a decrease in the proportion of viable cells in the S phase at 48 h, with an increase in those at G1 phase (Figure 3). However, this increase of G1 phase cells was transient, followed by a decrease occurring at 72 h, whereas the number of apoptotic cells increased from 4.2% to 10.4% (Figure 3). In the IGROV1-R10 cell line, similar results were obtained (data not shown).

Figure 1.
Figure 1.

Western blot analysis was performed 48 h after 20 nM small-inhibitory phosphoglycerate kinase 1 (siPGK1) transfection. As compared to non-transfected or small-inhibitory Green Fluorescent Protein (siGFP)-transfected cells, this transfection induced a reduction in the expression of PGK1 in IGROV1 cells (A), which was quantified by densitometric analysis (B).

siPGK1 treatment associated with cisplatin was more effective than cisplatin-alone on IGROV1 and IGROV1-R10 cells. At 24 h after siPGK1 transfection, IGROV1 and IGROV1-R10 cells were exposed to 5 μg/ml of cisplatin (referred as C5) (Figure 4). In IGROV1 cells, 24 h after this combined treatment (i.e. 48 h after transfection), the percentage of viable cells slightly decreased as compared to control cells (non transfected or transfected with siGFP, or cells treated with C5) (Figure 5A). At 72 h, the cytotoxicity was higher, both in cells treated by C5 alone and by combined treatment (siPGK1 plus C5), with 52% and 30% of viable cells remaining, respectively.

In IGROV1-R10 cells, this combined treatment (siPGK1 plus C5) also demonstrated a cytotoxic effect which increased from 48 h to 72 h, with only 30% of viable cells remaining at 72 h (Figure 5B). In contrast, cells exposed to C5-alone, siGFP or siPGK1 alone, demonstrated a transient inhibitory effect at 48 h, with a restarting of proliferation at 72 h, the percentage of viable cells exceeding 50% at this time.

siPGK1 treatment associated with cisplatin was more efficient at arresting cells in the G1 phase. Cytometric analysis confirmed the inhibition of IGROV1 and IGROV1-R10 cell growth by combined treatment, with a strong cellular arrest in the G1 phase, which persisted at 72 h (Figures 6A and 7). As the described above, IGROV1-R10 cells treated with C5 only exhibited a loss of blockage in the G2-M phases, with a restarting of proliferation at 72 h (Figure 7). In both cell lines treated with the combination of siPGK1 plus C5, the blockage in the G1 phase was accompanied by the appearance of a sub G0-G1 cell population, due to apoptotic cells, which was at least two-fold that of the control groups (i.e. C5 alone or siGFP plus C5) (Figure 6A and 7). In IGROV1 cells, the rate of cellular death, taking into account not only the sub G0-G1 apoptotic cells population but also apoptotic cells in the G2-M phases, reached 24.3% after siPGK1 plus C5, as compared to control groups which exhibited only 12% of cell death (Figure 6B). Similar results were obtained with IGROV1-R10 cells (data not shown).

siPGK1 treatment associated with cisplatin increased the expression of the BH3-only protein BIM. In IGROV1-R10 cells, by western blot, we studied the expression of various proteins which confirm apoptosis induction (PARP) or play a role in this process, as anti-apoptotic factors [Myeloid Cell Leukemia 1 (MCL-1), BCL-xL] or pro-apoptotic agents (BIM). Analysis confirmed the efficiency of siPGK1 which clearly reduced PGK1 expression at 72 h after transfection, both alone and when associated with cisplatin, although to a lesser degree (Figure 8). As compared to control groups, (C5 or siGFP plus C5), the siPGK1 plus C5 combination showed neither modulation of the state of PARP, nor PARP cleavage fragments (Figure 8B). In response to siPGK1 transfection, BCL-xL expression slightly increased as compared to that of control groups (Figure 8A), whereas it was not modified after siPGK1 plus C5 treatment (Figure 8B). MCL-1 protein expression was up-regulated in response to siPGK1 transfection (Figure 8A), but was not modified in response to siPGK1 8A), and was not modified in response to siPGK1 plus C5 (Figure 8B). BIM protein expression strongly increased after siPGK1-alone, and after siPGK1 plus C5 treatment.

Discussion

Chemoresistance is of paramount importance in cancer, because in the absence of an effective chemotherapy, other treatments (surgery, radiotherapy) are most often doomed to failure. This is particularly true for ovarian cancer, where survival is generally less than 50% at five years, as for many other types of solid cancer. Cisplatin is a chemotherapeutic drug which forms intra- and inter-strand adducts with DNA, and is widely used in the treatment of solid tumors, such as ovarian cancer. The development of resistance to cisplatin remains a major hurdle to successful therapy. Although mechanisms of chemoresistance are multiple, it has been recognized for a long time that hypoxic cells, like those found in highly hypoxic core parts of tumors, are the most chemo- and radio-resistant cells (19, 20). To survive these hard conditions, lacking O2, cells necessarily adopt strategies of resistance, such as enhanced glycolysis or overexpression of antiapoptotic factors. It is likely that hypoxic cells are the most chemoresistant cells (13, 14). In cases of severe hypoxia, glycolysis becomes the main cause, if not the unique route, of ATP generation. Under such conditions, PGK1 regenerates the two molecules of ATP previously consumed, and thus, plays a crucial role in the functioning of glycolysis. PGK1 is a glycolytic enzyme that transforms 1,3 biphosphoglycerate to 3-phosphoglycerate (3-PG). Of importance, PGK1 has been found to be overexpressed in chemoresistant cells (5, 21). This enzyme would also appear to be a multifunctional molecule, implicated in tumor biology (22), angiogenesis (23), DNA replication and/or repair in mammalian nucleus (24, 25), and cell growth and/or metastasis (26). Moreover, PGK1 is also linked to reactive oxygen species (ROS) generation (27), which in turn improves, the stability of hypoxia-inducible factor-1 (HIF-1), resulting in accumulation of PGK1 (28).

Figure 2.
Figure 2.

Effects of small-inhibitory phosphoglycerate kinase-1 (siPGK1) and small-inhibitory Green Fluorescent Protein (siGFP) transfection in IGROV1 (A) and IGROV1-R10 (B). Cellular morphology 48 or 72 h after the beginning of transfection can be seen. Scale bars=20 μm.

Figure 3.
Figure 3.

Effects of small-inhibitory phosphoglycerate kinase-1 (siPGK1) and small-inhibitory Green Fluorescent Protein (siGFP) transfection in IGROV1 cell line on cell-cycle distribution, 48 or 72h h after the beginning of transfection.

In this study, we tested siPGK1 transfection in two human ovarian cancer cells lines, IGROV1 and IGROV1-R10, of increasing chemoresistance. Although siPGK1 reduced the expression of PGK1 to around 60%, this incomplete inhibition clearly induced the arrest of cell growth. This effect could be related to the provoked reduction of ATP and/or to the lack of 3-PG entering the serine pathway, which is involved in de novo protein and folate synthesis, two crucial pathways for cancer cell growth. Moreover, PGK1 under expression would promote the accumulation of DNA damage by unknown mechanisms (25), slowing down cellular proliferation.

As we previously reported, like others (6, 8, 9, 29),

glycolysis inhibition could be a promising approach for sensitizing chemoresistant cells to cisplatin. In the current study, we confirmed the benefit of associating cisplatin with a glycolytic inhibitor for overwhelming chemoresistance. Indeed, cisplatin associated with siPGK1, induced more cell death, which was of particular interest in our highly chemoresistant IGROV1-R10 cells. The decrease of ATP might induce cells to repair DNA damage induced by cisplatin or to expel chemotherapeutic drugs, a process requiring ATP (30, 31). Moreover, the lack of ATP favors the unfolding protein accumulation induced by cisplatin, leading to endoplasmic reticulum (ER) stress. ER stress is characterized by induction of the pro-apoptotic protein BIM (32), which plays a critical role in the initiation of apoptosis in response to multiple death stimuli, including growth factor deprivation (33). In our study, siPGK1-alone and -associated with cisplatin strongly increased BIM expression. This expression is known to trigger the translocation of BAX to the mitochondria and to facilitate cell killing through the activation of BAX (34). It is noteworthy that BIM is linked to MCL-1 and neutralizes the antiapoptotic effect of this protein. In our study, although we observed an increase of MCL-1 under treatment with siPGK1 alone and when associated with cisplatin, it is likely that the activation of BIM was the dominant effect. However, the mechanisms by which the combination of siPGK1 with cisplatin exerts antitumor activity remain to be elucidated.

Figure 4.
Figure 4.

Protocol of exposure regarding the treatment by cisplatin (5 μg/ml=C5), administered 24 h after transfection. The culture was arrested either 48 h or 72 h after the beginning of transfection.

Figure 5.
Figure 5.

The percentage of viable cells at 48 and 72 h in IGROV1 (A) and IGROV1-R10 (B) in control groups as compared to cisplatin treatment-alone or -associated with 20 nM small-inhibitory phosphoglycerate kinase 1 (siPGK1).

Figure 6.
Figure 6.

Effect on cell-cycle distribution of the combination of 20 nM small-inhibitory phosphoglycerate kinase-1 (siPGK1) and (C5) in IGROV1 cells at 48 and 72 h after transfection. DNA content histograms are expressed either by flow cytometry (A), or as forward scatter (FS), as a function of propidium iodide fluorescence (FL3) (B).

Figure 7.
Figure 7.

Effect on cell-cycle distribution of the combination of 20 nM small-inhibitory phosphoglycerate kinase-1 (siPGK1) and C5 treatment of IGROV1-R10 cells at 48 and 72 h after transfection. DNA content histograms were determined by flow cytometry.

Figure 8.
Figure 8.

Analysis of PARP, PGK1, MCL-1, BCL-xL and BIM protein expressions in IGROV1-R10 cells, at 72 h by western blotting, after transfection alone by siRNA (A), or after exposure to cisplatin-alone or combined with siRNA (B).

Finally, our preliminary results demonstrated that siPGK1 can be interestingly combined with cisplatin to overcome chemoresistance of ovarian cancer. These results reinforce the interest in using and developing glycolytic inhibitors (26), especially these targeting PGK1, as new strategies for combating cancer development and chemoresistance.

  • Received June 25, 2012.
  • Revision received August 21, 2012.
  • Accepted August 22, 2012.

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siPGK1 Sensitizes Chemoresistant Human Ovarian Cancer Cell Lines to Cisplatin
CHARLOTTE LEPLEUX, EDWIGE ABEILARD-LEMOISSON, MARYLINE DUVAL, PHILIPPE ICARD, HUBERT LINCET
Anticancer Research Oct 2012, 32 (10) 4277-4286;
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