GL15 cells can be driven to metabolic decline by different strategies. An apoptotic pathway was induced following an imbalanced control of cell cycle progression or the alteration of cardiolipin CL synthesis in palmitate-treated cells Considering the emerging relevance of 3BP as an antitumor drug, we thought it would be of interest to unravel the biochemical mechanisms underlying 3BP effects in GL15 glioblastoma cells compared to antiglycolytic LND.
We found that 3BP exerts very early effects, compared to LND, both drugs leading cells to death, although by different routes. In GL15 cells, 3BP targets p53 to dephosphorylation and degradation, thus favoring an irreversible autophagic process. Adversely, LND orients GL15 cells towards autophagy that is eventually overcome by apoptosis, favored by p53 translocation to mitochondria. This process was paralleled by the decrease of p62 Fig. It is worth noting that the mitochondrial inhibitors did not influence significantly beclin 1 and cyt c expression.
B Expression of cyt c and the autophagic proteins p62, LC3, and beclin 1. Expression levels were analyzed by densitometry. In B , a representative blot is shown. After the treatments, cells were recovered and ATP was determined. The autophagic flux is confirmed by a significant decrease of p62 Fig. A parallel decrease was observed for p62 and beclin 1 expression, whereas cyt c and HK-II decreased after 2 hours, and VDAC1, an integral protein of the inner mitochondrial membrane, did not change Fig.
Protein expression was evaluated by Western blotting and analyzed by densitometry. C p53 protein expression and phosphorylation status at Ser and Ser F Akt phosphorylation status.
Representative blots are shown. Therefore, we analyzed 3BP effects on the p53 status in GL15 cells and, for comparison, in U87 and U glioblastoma cell lines. No variations of p53 total protein were observed within 2 hour 3BP treatment in any of the tested cell lines Fig. The p53 phosphorylation at Ser decreased in GL15 cells after 2 hours, but remained unchanged in U87 and U cells.
Adversely, phosphorylation at Ser decreased in each of the cell lines Fig. Dephosphorylation of p53 Ser is predictive of degradation, via interaction with MDM2 According to p-p53 Ser status, no p53 decrease was found in U87 and U cells Fig. As previously observed in GL15 cells 22 and confirmed in this study, 3BP produced Akt dephosphorylation also in U87 and U glioblastoma cell lines Fig.
Contrarily to GL15, where Akt inactivation could be predictive of the observed autophagic route, no autophagic response was observed in U87 and U cells Fig. An autophagic scenario was evidenced by acridine orange staining that showed acidic vesicular organelles in LND-treated cells, whereas control cells had the classical punctuate orange staining of acidic compartments. Moreover, after LND treatment a diffuse acidification was evident in cells presenting autophagolysosomes Fig.
A further evidence that LND induces autophagy in GL15 cells was obtained by immunofluorescence analysis of LC3 that, when localized in autophagosome membranes, appears as bright puncta. We found that LND induces a cellular phenotype in GL15 cells characterized by cytoplasm transparency, although with apparent integrity of the nuclear membrane not shown. The arrow indicates acidic vesicular organelles. Original magnification X. The arrows indicate bright puncta. LND caused cytoskeleton disorganization.
Full size image Since cytoskeleton alterations, as well as an increase of p62 expression in autophagic cells could predict the induction of an apoptotic route 25 , 26 we investigated a possible evolution of GL15 cells towards apoptosis. This trend was confirmed in U cells Fig. No caspase-3 activity was detected in GL15 cells after 48 and 72 hours of LND treatment not shown , thus excluding the involvement of the cyt c cascade in the onset of the apoptotic process.
The expression of p53 and of the caspase-independent pro-apoptotic mitochondrial protein AIF were not affected by LND. However, for prolonged incubation times control cells showed a significant p53 decrease Fig. Figure 4: LND induces apoptotic cell death in glioblastoma cells. The results shown are representative of three independent experiments. A p53 and AIF proteins expression was evaluated by Western blot analysis of total cell lysates.
For each protein, the ratio relative to actin was calculated by densitometric analysis. B Immunofluorescence analysis shows that LND modifies the cellular localization of p The arrows indicate co-localization of p53 with mitochondria aggregated in the perinuclear zone in LND-treated cells.
Images are representative of three experiments. C LND favors Akt dephosphorylation. Total Akt and pAkt expression was evaluated by Western blot analysis. A representative blot is shown. Full size image LND modifies the cellular localization of p53 in GL15 cells The binding of the tumor suppressor p53 to mitochondria promotes apoptosis Diffuse nuclear localization of p53, with the exclusion of nucleoli, and uniform cytoplasmic distribution of mitochondria were observed in control cells.
In LND-treated cells, p53 co-localized with mitochondria, which appeared aggregated in the perinuclear zone. Analysis of GL15 cell lipids Phospholipids constitute the primary components in membrane biogenesis during the autophagic process.
Our previous work showed that 3BP treatment of GL15 cells did not affect significantly phospholipids, with the exception of a marked decrease of CL The most relevant result was that phosphatidylinositol PI depletion occurred already at 4 hours and was complete at 45 ours of treatment. Moreover, at 45 hours the overall phospholipid content decreased but, with the exception of PI, the composition of phospholipid classes was not significantly affected Fig.
The phospholipid profile at 45 hours was confirmed by two-dimensional TLC analysis Fig. In A,B , the fatty acid composition of major phospholipids is indicated. In B , the insert is a zoom on cardiolipins. Full size image Discussion 3BP was first introduced as a specific alkylating agent for 2-ketodeoxyphosphogluconic aldolase and as a reactive probe in kinetic and stereochemical studies More recently 3BP has emerged as an energy blocker, thus representing a potential chemotherapeutic drug Indeed, 3BP contrasts hepatocellular carcinoma by acting preferentially against HK-II, which is highly expressed in liver carcinomas In addition, peculiar mitochondrial enzymes are also targeted by this drug 9.
New developments have extended the anticancer power of 3BP to brain tumors 22 , Shoshan recently published a state of the art review on this compound and its molecular targets Nevertheless, the biochemical mechanisms underlying the action of the drug are not yet clear. Both drugs induce autophagy, which constitutes the death modality in 3BP-treated cells, while in LND-treated cells autophagy eventually evolves into apoptosis, as a molecular mechanism for death.
Nevertheless, the lack of its contribution channels cells to an autophagic defense Fig. Anderson Cancer Center, Houston. For experiments, cells were collected from subconfluent monolayers in a solution of trypsin 0.
Cell Proliferation Assay Growth inhibition of the five cell lines by lonidamine and diazepam, alone or in combination, was studied by using the microculture 3- 4,5- dimethylthiazolyl -2,5-diphenyltetrazolium bromide MTT; Sigma Chemical Co. The amount of tetrazolium dye reduced to formazan is proportional to the number of viable cells. Briefly, 3 x cells were plated per well in well polystyrene plates ATGC, Noisy-le-Grand, France in culture medium and allowed to adhere overnight.
The medium was then replaced with fresh medium containing increasing concentrations of lonidamine Doridamine; supplied by F. Angelini S. After a hour incubation, the medium was removed, and the plates were washed with PBS.
Growth inhibition was quantified as the decrease in the amount of the tetrazolium dye reduced to formazan by the treated cells versus control cells; formazan was measured as absorbance at nm in an LP spectrophotometer J Bio, Les Ulis, France. Each assay was repeated twice. Culture and treatment conditions were as described for the MTT assay. Six identical samples were used for treatment, and 12 identical samples were used for control points. Cell Cycle Determination Cells were plated in cm2 plastic culture flasks and allowed to adhere for 24 hours.
The cell cycle was assessed according to Demarcq et al. The cells were then washed three times with PBS and collected after trypsinization. After washing, the cells were suspended in 0. Percentages of cells present in areas corresponding to each phase of the cell cycle were calculated.
The excitation wavelength was nm, and emission was read at nm Corrections for light scattering probefree cell suspensions were performed routinely. Membrane fluidity is expressed as , which is inversely related to fluidity. The data were analyzed by a discrete exponential components model. A nonlinear least-square routine was used, and the reduced c2 was taken as the goodness criterion of the fit.
At the end of the incubation period, all floating and attached cells were collected and washed three times in serum-free medium. DNA was extracted and analyzed by agarose gel electrophoresis as described Search for a sub-G1 population characteristic of cells undergoing apoptosis was done.
Briefly, 2 x glioma cells were used to prepare membranes for receptor-binding studies. The pellets were resuspended in 20 volumes of buffer, and the washing was repeated once more. Each sample contained 0. N-methyl D-glucamine did not exceed 0. SNARFAM, a rhodamine derivative that generates two pH-sensitive bands in its fluorescence emission spectrum, enables pHi determination by microspectrofluorometry under defined staining conditions The calibration curve was performed as described The pH equilibration technique of Thomas et al.
The care, housing, and handling of the mice were performed in accordance with the recommendations of the French Ethics Committee and under the supervision of authorized investigators. The TGOZ glioma was established by subcutaneous implantation of human tumor fragments into the scapular area of nude mice under ether anesthesia administered for a short period 5.
The karyotype was monitored to confirm the human origin of the tumor For therapeutic assays, the tumor-bearing mice were randomly divided into groups of ten.
The control group received injections of intralipid alone. All mice were weighed twice a week. Tumor growth was monitered by measuring two perpendicular diameters with a caliper.
The response of xenografted TGOZ tumors was also assessed by growth delay, expressed as T - C, which is defined as the difference in days between the median time for the tumors of treated T and control C animals to reach a volume five times greater than the volume recorded at the time of treatmentOn the other hand, it is known that in mitochondria of cancer cells HKII associates with the voltage-dependent anion channel VDAC and this association appears to protect tumor cells from mitochondrial outer membrane permeabilization. Abstract Abnormal metabolism is another cancer hallmark. Considering the emerging relevance of 3BP as an antitumor drug, we thought it would be of interest to unravel the biochemical mechanisms underlying 3BP effects in GL15 glioblastoma cells compared to antiglycolytic LND. Percentages of cells present in areas corresponding to each phase of the cell cycle were calculated. The cells were then washed three times with PBS and collected after trypsinization. It is worth noting that in 3BP-treated GL15 cells, although no PI disappearance occurred 22 , Akt was also dephosphorylated, which could correlate with the dramatic decrease of ATP levels.
The TGOZ glioma was established by subcutaneous implantation of human tumor fragments into the scapular area of nude mice under ether anesthesia administered for a short period 5. Similarly, 6-diazooxo-L-norleucine DON is a well-known inhibitor of the enzyme glutaminase GLS that was also clinically tested and deemed to be ineffective The slides were mounted with a coverslip.
This difference between normal and cancer cells provides a biochemical basis for new therapeutic strategies aimed to block the energy power plants of cells. At week 4, the mice were sacrificed in a CO2 chamber. For experiments, cells were collected from subconfluent monolayers in a solution of trypsin 0.
Lonidamine causes cell death by apoptosis triggering dissipation of the mitochondrial transmembrane potential, increases reactive oxygen species levels, increases DNA fragmentation, and leads to loss of cell viability. Expression levels were analyzed by densitometry.
Exp Mol Pathol. Apart from the oldest analogs of glutamine such as acivicin, DON, azaserine, and azotomycin which were clinically evaluated several decades ago and their development abandoned, only CB among the newest analogs has recently reached to phase I clinical trials. This finding substantiates the idea that neoplastic cells, including those growing in ascitic form, utilize mitochondrial oxidative phosphorylation as the main source of ATP for their biosynthetic processes.