Research ArticleExperimental Studies

Phosphatidylinositol Derivatives Induce Gastric Cancer Cell Apoptosis by Accumulating AIF and AMID in the Nucleus

MASATO OHYAMA, AYAKO TSUCHIYA, YOSHIKO KAKU, TAKESHI KANNO, TADASHI SHIMIZU, AKITO TANAKA and TOMOYUKI NISHIZAKI
Anticancer Research December 2015, 35 (12) 6563-6571;

Abstract

Background: The present study investigated the mechanism underlying the apoptosis of MKN28 human gastric cancer cells induced by the phosphatidylinositol (PI) derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidyl-D-1-inositol (diDCP-LA-D-PI) and its enantiomer diDCP-LA-L-PI. Materials and Methods: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, enzymatic caspase assay, real-time reverse transcription-polymerase chain reaction (RT-PCR), and western blotting were carried-out. Results: Both diDCP-LA-D-PI and diDCP-LA-L-PI induced caspase-independent apoptosis of MKN28 cells, with the potential for diDCP-LA-L-PI being much greater than that of diDCP-LA-D-PI. diDCP-LA-D-PI and diDCP-LA-L-PI accumulated apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) in the nucleus. Conclusion: diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis of MKN28 cells by accumulating AIF and AMID in the nucleus, with different potentials.

Phosphatidylinositol (PI), a component of the plasma membrane, participates in the regulation of a variety of cellular functions including cell proliferation, differentiation, migration, chemotaxis, phagocytosis, and survival. Little, however, is known about PI-induced apoptosis. To examine the anti-tumor effect of PI, we synthesized PI derivatives 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidyl-D-1-inositol (diDCP-LA-D-PI) with 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) at the α and β position, which exhibit stable bioactivities in vivo. In our earlier study, diDCP-LA-D-PI induced caspase-independent apoptosis of MKN28 human gastric cancer cells and Lu65 human lung cancer cells, with higher activities of mitogen-activated protein (MAP) kinase kinase (MEK) and ERK1/2 (1). In contrast, diDCP-LA-D-PI does not induce apoptosis of 786-O renal cancer cells and HuH-7 hepatoma cells, with lower activities of MEK and ERK1/2 (1). diDCP-LA-D-PI has the potential to enhance activity of protein phosphatase 2A (PP2A) (2). Therefore, diDCP-LA-D-PI appears to induce caspase-independent apoptosis at least in part by enhancing PP2A activity, to suppress MEK activity (1).

We also synthesized the diDCP-LA-D-PI enantiomer 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidyl-L-1-inositol (diDCP-LA-L-PI). Amazingly, diDCP-LA-L-PI has no effect on PP2A activity (2). The mechanism of different actions on PP2A activity between diDCP-LA-D-PI and diDCP-LA-L-PI is presently far from understandable. A plausible explanation for this is that PP2A contains the binding site for diDCP-LA-D-PI, but not for diDCP-LA-L-PI; in other words, PP2A contains a receptor to recognized diDCP-LA-D-PI, but not diDCP-LA-L-PI. An enhancement of PP2A activity is not obtained with the natural types of PIs 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol)(DOPI) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DPPI) (2).

Previously, we obtained data showing that DOPI and DPPI induce apoptosis of malignant pleural mesothelioma cells by accumulating apoptosis-inducing factor (AIF) in the nucleus (3). This suggests that diDCP-LA-D-PI-induced apoptosis of MKN 28 cells is not due only to suppression of MEK activity in association with enhanced PP2A activity. Then, we were prompted to see whether diDCP-LA-L-PI exerts its anticancer action and to understand the underlying mechanism. The present study was conducted to gain further insight into the antitumor action of diDCP-LA-D-PI and diDCP-LA-L-PI.

Figure 1.
Figure 1.

Chemical structure of diDCP-LA-D-PI and diDCP-LA-L-PI.

To address this issue, we carried-out 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, enzymatic caspase assays, real-time reverse transcription-polymerase chain reaction (RT-PCR), and western blotting. We herein showed that both diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis of MKN28 human gastric cancer cells by accumulating AIF and AIF-homologous mitochondrion-associated inducer of death (AMID) in the nucleus, with more beneficial effect of diDCP-LA-L-PI than that of diDCP-LA-D-PI.

Materials and Methods

Synthesis of diDCP-LA-D-PI and diDCP-LA-L-PI. We synthesized diDCP-LA-D-PI and diDCP-LA-L-PI by the methods previously described (Figure 1) (2).

Cell culture. MKN28 human gastric cancer cells, which were a gift from Dr. Tatematsu (Nagoya University, Japan), and Lu65 human lung cancer cells, which were purchased from Health Science Research Resources Bank (Osaka, Japan), were grown in RPMI-1640 medium supplemented with 10% (v/v) FBS. Penicillin (final concentration, 100 U/ml) and streptomycin (final concentration, 0.1 mg/ml) were added to the all the cultured media, and cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C.

Cell viability. To assess cell viability the MTT assay was performed by the method previously described (4).

TUNEL staining. TUNEL staining was performed by the method previously described (4). Briefly, fixed and permeabilized cells were reacted with terminal deoxynucleotidyl transferase and fluorescein isothiocyanate (FITC)-deoxyuridine triphosphate for 90 min at 37°C using an In Situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan). FITC signals were visualized with a confocal scanning laser microscope (LSM 510, Carl Zeiss Co., Ltd., Oberkochen, Germany).

Enzymatic assay of caspase activities. Caspase activity was enzymatically measured by the method as previously described (1). Briefly, cells were harvested before and after treatment with diDCP-LA-D-PI or diDCP-LA-L-PI, and then centrifuged at 800 × g for 5 min at 4°C. The pellet was centrifuged at 10,000 × g for 1 min at 4°C, and the supernatant was incubated in a caspase fluorometric assay kit (Ac-Asp-Glu-Val-Asp-MCA for a caspase-3 substrate peptide, Ac-Ile-Glu-Thr-Asp-MCA for a caspase-8 substrate peptide, and Ac-Leu-Glu-His-Asp-MCA for a caspase-9 substrate peptide) for 2 h at 37°C. Fluorescence was measured at an excitation wavelength of 380 nm and an emission wavelength of 460 nm with a fluorescence microplate reader (TECAN Infinite, Männedorf, Switzerland).

Real-time RT-PCR. Real-time RT-PCR was carried-out by the method previously described (4). Briefly, total RNAs from MKN28 cells were purified using a Sepasol-RNA I Super kit (Nacalai, Kyoto, Japan) and treated with RNase-free DNase I (2 units) for 30 min at 37°C to remove genomic DNAs. RNAs purified was incubated in a RT buffer containing random primers, dNTP, and Multiscribe Reverse Transcriptase for 10 min at 25°C and in turn, for 120 min at 37°C to synthesize the first-strand cDNA. Real-time RT-PCR was performed using a SYBR Green Realtime PCR Master Mix (Takara Bio, Otsu, Japan) and the Applied Biosystems 7900 real-time PCR detection system (ABI, Foster City, CA). Thermal cycling conditions were as follows: first step, 94°C for 4 min; the ensuing 40 cycles, 94°C for 1 s, 65°C for 15 s, and 72°C for 30 s. The expression level of each mRNA was normalized by that of GAPDH mRNA. Primers used for real-time RT-PCR are shown in Table I.

Separation into nuclear and cytosolic components. MKN28 cells were suspended in buffer A [25 mM MgCl2, 0.1% (v/v) Triton X-100, 1 mM dithiothreitol, and 10 mM HEPES, pH 7.6] and centrifuged at 1,100 ×g for 5 min at 4°C. The pellet and supernatant were used as nuclei- and cytosol-enriched components, respectively. Whether the nuclear and cytosolic components were successfully separated was confirmed by western blotting using an anti-Lamin A/C antibody, a nuclear marker.

Western blotting. Western blotting was carried out using antibodies against AIF (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), AMID (GeneTex, Inc., Irvine, CA, USA), or β-actin (SIGMA, Missouri, SL, USA) by a method as previously described (Immunoreactivity was detected with an ECL kit (Invitrogen, Carlsbad, CA, USA) and visualized using a chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). Protein concentrations for each sample were determined with a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA).

Statistical analysis. Statistical analysis was carried out using unpaired t-test and analysis of variance (ANOVA) followed by a Bonferroni correction.

Results

diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis of MKN28 human gastric cancer cells. diDCP-LA-D-PI and diDCP-LA-L-PI reduced MKN28 cell viability in a concentration (0.01-100 μM)-dependent manner, reaching nearly 50% and 0% of basal levels at 100 μM, respectively (Figure 2A). This indicates that diDCP-LA-L-PI induces MKN28 cell death, with the potential being greater than that for diDCP-LA-D-PI.

To confirm that the effect of the compounds is not limited to MKN28 cells, MTT assay was carried-out using a different cell line, the Lu65 human lung cancer cell. Expectedly, diDCP-LA-D-PI and diDCP-LA-L-PI reduced Lu65 cell viability in a concentration (0.01-100 μM)-dependent manner, reaching approximately 40% and 0% of basal levels at 100 μM, respectively (Figure 2B). This suggests that the compounds exhibit a broad anticancer effect.

To investigate the mechanism underlying the anticancer effect of diDCP-LA-D-PI and diDCP-LA-L-PI, we used a high concentration (100 μM) of the compounds for the following experiments. In the TUNEL staining, diDCP-LA-D-PI and diDCP-LA-L-PI significantly increased the number of TUNEL-positive cells in MKN28 and Lu65 cells as compared to that for untreated control cells (Figure 3A and B). This confirms that diDCP-LA-D-PI and diDCP-LA-L-PI induce apoptosis of MKN28 and Lu65 cells.

In the enzymatic assay of caspase activity, diDCP-LA-D-PI did not significantly activate caspase-3, -8, and -9 both in MKN28 and Lu65 cells (Figure 4A and C). Likewise, no significant activation of caspase-3, -8, and -9 in MKN28 and Lu65 cells was induced by diDCP-LA-L-PI (Figure 4B and D). Taken together, these results indicate that diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis of MKN28 and Lu65 cells.

View this table:
Table I.

Primers used for real-time RT-PCR.

diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis of MKN28 by accumulating AIF and AMID in the nucleus. In the real-time RT-PCR analysis, diDCP-LA-D-PI up-regulated expression of mRNAs for Fas, death receptor 4 (DR4), DR5, DR6, Puma, Mcl-1, and AMID in MKN28 cells (Figure 5). diDCP-LA-L-PI, on the other hand, up-regulated expression of mRNAs for Fas, tumor necrosis factor α (TNFα), TNF receptor 1 (TNFR1), TNFR2, DR4, DR5, Puma (Figure 5). In contrast, expression of mRNAs for FasL, Fas-associated death domain protein (FADD), TNFR1-associated death domain protein (TRADD), TNF-related apoptosis-inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), DR3, Bad, Bax, Bid, Hrk, Bcl-2, Bcl-XL, and AIF was not affected by these compounds (Figure 5). AIF and AMID induces caspase-independent apoptosis by accumulating in the nucleus (5-14). To address this point, we examined the effect of diDCP-LA-D-PI and diDCP-LA-L-PI on intracellular distribution of AIF and AMID in MKN28 cells. diDCP-LA-D-PI and diDCP-LA-L-PI did not affect expression of proteins for AIF and AMID (Figure 6A and C). Both diDCP-LA-D-PI and diDCP-LA-L-PI significantly increased nuclear localization of AIF in a treatment duration (6-24 h)-dependent manner (Figure 6B). A similar increase in the nuclear localization of AMID was obtained with diDCP-LA-D-PI and diDCP-LA-L-PI (Figure 6D). This indicates that diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis of MKN28 cells by accumulating AIF and AMID in the nucleus.

Figure 2.
Figure 2.

Effects of diDCP-LA-D-PI and diDCP-LA-L-PI on MKN28 and Lu65 cell viability. MKN28 (A) and Lu65 (B) cells were treated with diDCP-LA-D-PI or diDCP-LA-L-PI at the concentrations as indicated for 48 h, followed by MTT assay. In the graphs, each point represents the mean±SEM percentage of control cell viability (MTT intensities of cells untreated with diDCP-LA-D-PI or diDCP-LA-L-PI) (n=4 independent experiments).

Figure 3.
Figure 3.

TUNEL staining. TUNEL staining was carried-out on MKN28 (A) and Lu65 (B) cells treated with diDCP-LA-D-PI or diDCP-LA-L-PI at a concentration of 100 μM for 48 h. DIC, Differential interference contrast. Bars=100 μm. TUNEL-positive cells were counted in an area (0.4 mm x 0.4 mm) selected at random. In the graphs, each column represents the mean±SEM percentage of TUNEL-positive cells relative to total number of cells (n=4 independent experiments). p-Values are from unpaired t-test.

Figure 4.
Figure 4.

Effects of diDCP-LA-D-PI and diDCP-LA-L-PI on caspase activity. MKN28 (A)(B) and Lu65 (C)(D) cells were treated with diDCP-LA-D-PI or diDCP-LA-L-PI at a concentration of 100 μM for the indicated periods of time, and then the activities of caspase-3, -8, and -9 were enzymatically assayed. In the graphs, each point represents the mean±SEM ratio to basal caspase activities (before treatment with diDCP-LA-D-PI or diDCP-LA-L-PI) (n=4 independent experiments).

Discussion

In the present study, diDCP-LA-D-PI and diDCP-LA-L-PI reduced cell viability in a concentration (0.01-100 μM)-dependent manner for both MKN28 and Lu65 cells, with the potential for diDCP-LA-L-PI being greater than that for diDCP-LA-D-PI. diDCP-LA-D-PI and diDCP-LA-L-PI markedly increased TUNEL-positive cells in MKN28 and Lu65 cells, but no significant activation of caspase-3, -8, and -9 was obtained with these compounds. This indicates that diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis of MKN28 and Lu65 cells. It is presently unknown why there is difference in the anticancer potential between diDCP-LA-D-PI and diDCP-LA-L-PI. A plausible explanation for this is that this difference might be attributed to difference in the binding affinity of each compound to the responsible apoptosis-related receptor.

Figure 5.
Figure 5.

Real-time RT-PCR analysis. MKN28 cells were treated with diDCP-LA-D-PI or diDCP-LA-L-PI at a concentration of 100 μM for the indicated periods of time, and then real-time RT-PCR was carried-out. The mRNA quantity for each gene was calculated from the standard curve made by amplifying different amounts of GAPDH mRNA, and normalized regarding the average of independent basal mRNA quantity at 0 h as 1. In the graphs, each point represents the mean±SEM ratio relative to basal mRNA levels (n=4 independent experiments).

Apoptosis is initiated through the major extrinsic and intrinsic pathways, the former being related to death receptors on the plasma membrane and the latter to the mitochondria. Death receptors include Fas/Apo1/CD95, TNFR1, DR3/Apo3/WSL-1/LARD/TRAMP, DR4/TRAIL-R1, DR5/TRAIL-R2/TRICK2/KILLER, and DR6. The Bcl-2 family, on the other hand, is related to intrinsic mitochondrial apoptosis. The BH3-only Bcl-2 family members Bad, Bid, Puma, Hrk, and Noxa and the Bax subfamily member Bax serve as a pro-apoptotic executioner, but otherwise the Bcl-2 subfamily members Bcl-2, Bcl-XL, and Mcl-1 exert their anti-apoptotic action.

Figure 6.
Figure 6.

Effects of diDCP-LA-D-PI and diDCP-LA-L-PI on intracellular distribution of AIF and AMID. MKN28 cells were treated with diDCP-LA-D-PI or diDCP-LA-L-PI at a concentration of 100 μM for the indicated periods of time, and western blotting was carried-out using antibodies against AIF (A) and AMID (C). Signal intensities for total expression of AIF and AMID proteins were normalized to β-actin signal intensities. In the graphs, each column represents the mean±SEM normalized signal intensity for AIF and AMID (n=4 independent experiments). After treatment with diDCP-LA-D-PI or diDCP-LA-L-PI at a concentration of 100 μM for the indicated periods of time, cells were separated into their cytosolic and nuclear components and western blotting followed using antibodies against AIF (B) or AMID (D). In the graphs, each column represents the mean±SEM signal intensity for nuclear localization of AIF and AMID relative to that for total expression of each protein (n=4 independent experiments). p-Values, ANOVA followed by a Bonferroni correction.

In the real-time RT-PCR analysis, diDCP-LA-D-PI up-regulated expression of mRNAs for Fas, DR4, DR5, DR6, and Puma in MKN28 cells. Fas, activated by FasL, trigger apoptosis by activating caspase-8 through FADD-mediated recruitment of procaspase-8 and the effector caspase-3 (15). DR4 and DR5 are activated by TRAIL and in turn, recruit FADD, to activate caspase-8 and caspase-3 (16). DR6 is shown to engage apoptosis, but little is known about the relevant signaling. Puma disrupts mitochondrial membrane potentials, allowing cytochrome c release from the mitochondria to form an apoptotic protease activating factor 1 (Apaf-1) with dATP, causing activation of caspase-9 followed by the effector caspase-3. In the present study, diDCP-LA-D-PI did not activate caspase-3, -8, and -9. This suggests less possibility for the participation of Fas, DR4, DR5, DR6, and Puma in diDCP-LA-D-PI-induced apoptosis of MKN28 cells. diDCP-LA-L-PI, on the other hand, up-regulated expression of mRNAs for Fas, TNFα, TNFR1, DR4, DR5, and Puma in MKN28 cells. TNFR1, which is activated by TNFα, associates with FADD and activates caspase-8 and caspase-3, to induce apoptosis (17). In light of the fact that diDCP-LA-L-PI did not activate caspase-3, -8, and -9, however, Fas, TNFα, TNFR1, DR4, DR5, and Puma might have less contribution to diDCP-LA-L-PI-induced apoptosis of MKN28 cells.

Then, we focused on AIF and AMID as a target of diDCP-LA-D-PI and diDCP-LA-L-PI relevant to caspase-independent apoptosis (5-14). AIF is imbedded into the inner mitochondrial membrane, under normal conditions. When the proapoptotic Bcl-2 family molecules Bax and Bid are activated and make pores in the mitochondrial membranes or AIF undergoes proteolytic cleavage by cysteine proteases such as calpains and cathepsins, AIF is released from mitochondria and translocated into the nucleus. AIF arrived at the nucleus interacts with DNA and/or RNA, to cause chromatinolysis through recruitment of nucleases, or organizes a DNA-degrading complex with histone H2AX and CypA, in a caspase-independent manner (7, 8). Likewise, AMID is accumulated in the nucleus, to induce caspase-independent apoptosis (5, 9-14). diDCP-LA-D-PI and diDCP-LA-L-PI had no effect on expression of AIF and AMID mRNA and proteins in MKN28 cells. Both diDCP-LA-D-PI and diDCP-LA-L-PI significantly increased nuclear localization of AIF and AMID. This indicates that diDCP-LA-D-PI and diDCP-LA-L-PI could induce caspase-independent apoptosis of MKN28 cells by accumulation of AIF and AMID in the nucleus. The question that should be addressed is how diDCP-LA-D-PI and diDCP-LA-L-PI stimulate the release of AIF and AMID from the mitochondria and promote accumulation in the nucleus. To address this question, we are currently carrying out further experiments.

Conclusion

PI derivatives diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis in MKN28 human gastric cancer cells by accumulating AIF and AMID in the nucleus, with the potential of diDCP-LA-L-PI being greater than that of diDCP-LA-D-PI.

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Conflicts of Interest

    None of the Authors have any potential conflict of interest.

  • Received August 20, 2015.
  • Revision received September 15, 2015.
  • Accepted September 16, 2015.

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Phosphatidylinositol Derivatives Induce Gastric Cancer Cell Apoptosis by Accumulating AIF and AMID in the Nucleus
MASATO OHYAMA, AYAKO TSUCHIYA, YOSHIKO KAKU, TAKESHI KANNO, TADASHI SHIMIZU, AKITO TANAKA, TOMOYUKI NISHIZAKI
Anticancer Research Dec 2015, 35 (12) 6563-6571;
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