Tadashi Asakura

Chemotherapeutic Agents That Induce Mitochondrial Apoptosis

In cancer chemotherapy, it is necessary to design an agent that suppresses or inhibits the targets that influence cell growth and apoptosis. We focus on the apoptotic pathway via mitochondria in this article. In this pathway, c-Jun N-terminal kinase (JNK), one of the stress activated protein kinases, is predominantly activated by apoptotic stimuli. JNK activity is inhibited by the binding of glutathione S-transferase P1-1 (GST P1-1) through protein-protein interactions. It has been noted that GST P1-1 overexpression plays an important role in carcinogenesis and in part in the MDR phenotype. We show several useful modifications of an anticancer agent that suppress the enzyme activity and expression of GST P1-1. The release of cytochrome c from mitochondria to the cytosol during apoptosis is mediated by the mitochondrial permeability transition pore, which is a protein complex formed by the voltage-dependent anion channel, members of the pro- and anti- apoptotic Bax-Bcl-2 protein family, cyclophilin D, and adenine nucleotide (ADP/ATP) translocators. We propose some drugs, including a proteasome inhibitor that can triger the permeability transition.

Caspase-3 activation during apoptosis caused by glutathione-doxorubicin conjugate.

SummaryGlutathione–doxorubicin (GSH–DXR) effectively induced apoptosis in rat hepatoma cells (AH66) at a lower concentration than DXR. After 24 h of drug treatment, DNA fragmentation of the cells was observed at the concentration of 1.0 μM DXR or 0.01 μM GSH–DXR. Increase in caspase-3 activity and DNA fragmentation were observed within 12 h and 15 h after treatment with either drug. Intracellular caspase-3 activity was increased in a dose-dependent manner after treatment with DXR or GSH–DXR, and caspase-3 activity correlated well with the ability to induce DNA fragmentation. When the cells were treated with either DXR or GSH–DXR for only 6 h, apoptotic DNA degradation and caspase-3 activation occurred 24 h after treatment. DNA fragmentation caused by these drugs was prevented completely by simultaneous treatment with the caspase-3 inhibitor, acetyl–Asp–Glu–Val–Asp-aldehyde (DEVD-CHO), at 10 μM. By contrast, DNA fragmentation was not prevented by the caspase-1 inhibitor, acetyl–Tyr–Val–Ala–Asp–aldehyde (YVAD-CHO), at the same concentration as DEVD-CHO, and caspase-1 was not activated at all by the treatment of AH66 cells with both DXR and GSH–DXR. These results demonstrate that DXR and GSH–DXR induce apoptotic DNA fragmentation via caspase-3 activation, but not via caspase-1 activation, and that GSH–DXR enhances the activation of caspase-3 approximately 100-fold more than DXR. Moreover, the findings suggested that an upstream apoptotic signal that can activate caspase-3 is induced within 6 h by treating AH66 cells with the drug.

Conformational change in the active center region of GST P1-1, due to binding of a synthetic conjugate of DXR with GSH, enhanced JNK-mediated apoptosis

Treatment of cells with a synthetic conjugate of DXR with GSH via glutaraldehyde (GSH-DXR) caused cytochrome c to be released from the mitochondria to the cytosol following potent activation of caspase-3 and -9 by typical DNA fragmentation. This apoptosis was regulated by the JNK-signaling pathway. In the present experiment, binding of GSH-DXR to GST P1-1 allosterically led to the disappearance of its enzyme activity and activated the kinase activity of JNK without dissociation of the JNK-GST P1-1 complex. The recombinant GST P1-1 molecule with a mutation in the active center region (W38H and C47S) lost its GST activity when bound to JNK to the same degree as the wild-type, with the mutated GST P1-1 molecule failing to inhibit the activity of JNK. It has been reported that JNK-signaling is regulated by GST P1-1 via interaction with the C-terminus. We confirmed that GST P1-1 deletion mutant (Δ194–209) and a site-directed mutant (R201A) in the C-terminal region failed to bind and inhibit JNK. These results indicated that not only binding of the C-terminal region of GST P1-1 to the JNK molecule, but also the active center region of GST P1-1 play important roles in the regulation of JNK enzyme activity. The findings suggested that allosteric inhibition of GST P1-1 activity by the binding of GSH-DXR following conformational change may activate JNK and induce apoptosis via the mitochondrial pathway in the cells.

Efflux of γ-aminobutyric acid from and appearance of free arachidonic acid inside synaptosomes

When synaptosomes were depolarized in the presence of Ca2+, or when Ca2+ was added to synaptosomes pretreated with Ca2+ ionophore (A23187), free arachidonic acid was clearly increased within synaptosomes, and at the same time an efflux of γ-aminobutyric acid from synaptosomes was observed. Moreover, when synaptosomes labelled with [14C]arachidonic acid were depolarized in the presence of Ca2+, there was a significant decrease in the radioactivity of the fatty acid of phosphatidylinositol and phosphatidylcholine. Exogenously added arachidonic acid, but not other fatty acids, stimulated the efflux of γ-aminobutyric acid in the absence of Ca2+. These observations suggest that the release of arachidonic acid from phospholipids is an intrinsic part of the biochemical mechanism that modulates the γ-aminobutyric acid efflux.

Hypoxia promotes glycogen synthesis and accumulation in human ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) has several significant characteristics based on molecular features that are distinct from those of ovarian high-grade serous carcinoma. Cellular glycogen accumulation is the most conspicuous feature of OCCC and in the present study its metabolic mechanism was investigated. The amount of glycogen in cells cultured under hypoxia increased significantly and approximately doubled after 48 h (P<0.01) compared to that under normoxic conditions. Periodic acid-Schiff positive staining also demonstrated intracellular glycogen storage. Western blot analysis revealed that HIF1α, which was overexpressed and stabilized under hypoxic conditions, led to an increase in the levels of cellular glycogen synthase 1, muscle type (GYS1), and conversely to a decrease in inactive phosphorylated GYS1 at serine (Ser) 641. Additional increases were observed in both protein phosphatase 1, which dephosphorylates and thereby induces GYS1 enzyme activity, and glycogen synthase kinase 3 beta (GSK3β) phosphorylated at Ser9, which is inactive on phosphorylation of GYS1 and subsequently induces its enzyme activity. By contrast, the level of PYGM-b decreased. These results indicated that the glycogen accumulation under a hypoxic environment resulted in the promotion of glycogen synthesis, but did not lead to inhibition of glycogen degradation and/or consumption. Under hypoxic conditions, HAC2 cells showed activation of the PI3K/AKT pathway caused by a mutation in exon 20 of PIK3CA, encoding the catalytic subunit p110α of PI3K. The resulting activation of AKT (phosphoSer473) also plays a role as a central enhancer in glycogen synthesis through suppression of GSK3β via phosphorylation at Ser9. Hypoxia decreased the cytocidal activity of cisplatin and doxorubicin to various degrees. In conclusion, the hypoxic conditions together with HIF1 expression and stabilization increased the intracellular glycogen contents and resistance to the anticancer drugs.

DRUG CONJUGATE OF DOXORUBICIN WITH GLUTATHIONE IS A POTENT REVERSER OF MULTIDRUG RESISTANCE IN RAT HEPATOMA CELLS

A recent study has suggested that degraded adducts smaller than 2 kDa in molecular weight of bovine serum albumin (BSA)-conjugated doxorubicin (DXR) (BSA-DXR) might exhibit cytotoxicity against multidrug resistant (MDR) cells. To investigate this notion further, intracellular accumulation and cytotoxicity of DXR coupled to several small peptides, such as glycylglycine (diGly), glycylglycylglycine (triGly), reduced glutathione (GSH) and oxidized glutathione (GSSG), were investigated using DXR-sensitive (AH66P) and DXR-resistant (AH66DR) rat hepatoma cell lines. Against both AH66P and AH66DR cells, diGly-conjugated DXR (diGly-DXR) and triGly-conjugated DXR (triGly-DXR) demonstrated the same cytotoxic activity as DXR, and the accumulation of both conjugates in the two cell lines was almost similar to that of DXR. After treatment of AH66DR cells with 5 microM verapamil [an inhibitor of P-glycoprotein (Pgp)], the intracellular levels of diGly-DXR and triGly-DXR were markedly increased and consequent cytotoxicity was improved. On the other hand, GSH-conjugated DXR (GSH-DXR) showed 9- and 7.5-fold more cytotoxic activity than BSA-DXR against AH66P and AH66DR cells, respectively. GSH-DXR accumulated rapidly in AH66DR cells, probably by the same mechanism as in AH66P cells, because the treatment of AH66DR cells with verapamil did not cause a significant increase in the intracellular drug level as compared with that in cells treated without verapamil. The levels of cytotoxicity and accumulation of GSSG-DXR were the same as those of BSA-DXR for both cell lines. These results indicate that GSH-DXR exerts potent cytotoxicity against both cell lines among the peptide DXR conjugates examined because of the rapid uptake and high accumulation of GSH-DXR similar to that of DXR without efflux.

Glutathione-doxorubicin conjugate expresses potent cytotoxicity by suppression of glutathione S-transferase activity: comparison between doxorubicin-sensitive and -resistant rat hepatoma cells.

The cytotoxic mechanism of a conjugate of doxorubicin (DXR) and glutathione (GSH) via glutaraldehyde (GSH-DXR) was investigated using DXR-sensitive (AH66P) and -resistant (AH66DR) rat hepatoma cells. GSH-DXR accumulated in AH66DR cells as well as in AH66P cells without efflux by P-gp and exhibited the potent cytocidal activity against both cells compared with DXR. To examine whether thiol from GSH-DXR affected the expression of cytotoxicity, two conjugates of DXR, with modified peptides containing alanine or serine substituted for cysteine in GSH were prepared and their cytotoxicities determined. Substitution of these amino acids for cysteine resulted in an approximately two- to fourfold reduction in cytotoxic activity against both cell lines compared with the effect of GSH-DXR. Depletion of intracellular GSH by treatment of both cells with buthionine sulphoximine did not change the cytotoxic activity of DXR, BSA-DXR or GSH-DXR. By co-treating the cells with tributyltin acetate, an inhibitor of glutathione S-transferase (GST), and either DXR, BSA-DXR or GSH-DXR, the cytotoxicity was markedly increased. Interestingly, GSH-DXR showed non-competitive inhibition of GST activity and its IC50 value was 1.3 microM. These results suggested that the inhibition of GST activity by GSH-DXR must be an important contribution to the expression of potent cytotoxicity of the drug.

Suppression of GST-P by treatment with glutathione-doxorubicin conjugate induces potent apoptosis in rat hepatoma cells.

A conjugate of doxorubicin and glutathione via glutaraldehyde (GSH‐DXR) inhibited glutathione S‐transferase (GST) activity of rat hepatoma AH66 cells, and treatment of the cells with GSH‐DXR induced caspase‐3 activation and DNA fragmentation. After treatment of AH66 cells with 0.1 μM GSH‐DXR, GST‐P (placental type of rat GST isozymes) mRNA and its protein increased transiently and then decreased thereafter compared with the levels in nontreated cells. Caspase‐3 activation and DNA fragmentation were induced following the suppression of GST‐P expression by treatment with GSH‐DXR. When the cells were treated with 100 μM ethacrynic acid (ECA), an inhibitor of GST, DNA fragmentation and caspase‐3 activation were observed. In contrast, treatment of AH66 cells with a low concentration of ECA (1 μM) that showed little inhibition of GST activity induced slight, but significantly enhanced expression and activity of GST‐P, and consequent prevention of DXR‐ and GSH‐DXR‐induced DNA fragmentation. Overexpression of GST‐π (placental type of human GST isozymes) by transfection of GST‐π sense cDNA into AH66 cells decreased sensitivities to DXR and GSH‐DXR, and the suppression of GST‐P by transfection of the antisense cDNA into the cells increased drug sensitivity. On the other hand, there was little change in drug sensitivity caused by overexpression of site‐directedly mutated GST‐P in which the active‐site residue Tyr39 was replaced with His (W39H) or the substrate‐binding site residue Cys48 was replaced with Ser (C48S) by transfection of those cDNAs into AH66 cells. These results suggested that the suppression of GST‐P in AH66 cells treated with GSH‐DXR must play an important role in the induction of apoptosis.

Glutathione-S-transferase-pi expression regulates sensitivity to glutathione-doxorubicin conjugate.

We have reported that glutathione-doxorubicin conjugate (GSH-DXR) exhibited potent cytotoxicity against tumor cells and inhibited glutathione-S-transferase (GST) enzyme activity. In order to determine whether or not the expression of GST-π lowered the cytotoxicity of GSH-DXR, cytocidal activity of the conjugate was examined using tumor cells in which the level of GST-π expression was regulated by transfecting GST-π cDNA in the correct or reverse direction and comparing with that of DXR. Enhancement of GST-π expression by transfecting GST-π sense cDNA into human hepatoblastoma HepG2 cells in which GST-π expression was extremely low caused an increase in GST activity from 0.26 to 55.0 nmol/mg/min and a marked reduction in transfectant sensitivity to GSH-DXR to 1/120 (0.15-18 nM IC50) although the sensitivity to DXR was slightly decreased to 1/2.6 (380-990 nM IC50). By contrast, a high GST-π-expressing human colon cancer cell line, HT29, showed a decrease in GST enzyme activity from 72.0 to 45.9 nmol/mg/min after transfecting GST-π antisense cDNA and a marked improvement in transfectant sensitivity to GSH-DXR was observed (28-2.9 nM IC50) compared with the transfectant sensitivity to DXR (1020-320 nM IC50). Additionally, the expression of GST-π in HepG2 cells caused a decrease in GSH-DXR-induced activation of caspase-3, which was an apoptotic marker, whereas the suppression of GST-π in HT29 cells showed an increase in caspase-3 activation. These results suggested that the cytocidal efficacy of GSH-DXR, but not that of DXR, was controlled by the level of GST-π expression in the cells.

PHARMACOKINETIC ANALYSIS OF PROTEIN-CONJUGATED DOXORUBICIN (DXR) AND ITS DEGRADED ADDUCTS IN DXR-SENSITIVE AND -RESISTANT RAT HEPATOMA CELLS

After treatment of AH66DR cells with the multidrug resistance (MDR) phenotype with bovine serum albumin (BSA)- conjugated [14C]doxorubicin (DXR), accumulation of the drug in the secondary lysosomal fraction increased as a function of time up to 24 h without any significant increase of the drug in other organellae. By contrast, AH66P cells showed a marked increase in accumulation of the drug in the mitochondrial fraction, and a moderate increase in the lysosomal and nuclear fractions. The intracellular degradation of the internalized conjugate was assessed by HPLC gel filtration as molecular change of the drug. The initial molecular mass (Mr) of BSA-con|ugated [14C]DXR was estimated to be 70 kDa; however, the secondary lysosomal fraction contained mainly three peaks of [14C]compounds ranging from 3 to 70 kDa. The [14C]compound extracted from the nuclear and mitochondrial fractions showed only one peak, which was estimated to be smaller than 2 kDa. By contrast, the cytosolic fraction contained mainly two peaks of [14C]compounds, which were smaller than 2 kDa and larger than 500 kDa. These results indicated that the intracellular distribution of the administered drug, based probably on the drug-traffic mechanism in the cells, was quite different between the two cell lines, but some of the biochemical characteristics of the degraded compounds from each subcellular fraction were similar because the degradation processes in each fraction might be almost identical. The possibility of lysosomal degradation of the protein-conjugated DXR leading to expression of cytotoxicity was also confirmed by the fact that only lysosomal digestable poly-L-lysine-conjugated DXR exhibited dosedependent cytotoxicity against both cell lines in marked contrast to the cells treated with poly-D-lysine-conjugated DXR. It was concluded that lysosomal breakdown of protein-conjugated DXR, which had been taken up by endocytosis, and the liberation of the degraded active adducts of the conjugate without efflux by the MDR pump mechanism must be an essential stage in the development of the cytotoxicity against tumor cells with or without the MDR phenotype.