This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakashio, A. Articles by Tsuruo, T. Search for Related Content PubMed PubMed Citation Articles by Nakashio, A. Articles by Tsuruo, T.

Prevention of Phosphatidylinositol 3'-Kinase-Akt Survival Signaling Pathway during Topotecan-induced Apoptosis¹

Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032 [A. N., N. F., S. R., S. S., T. T.]; SmithKline Beecham Seiyaku K. K., Tokyo 102-0075 [A. N.]; and Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170-8455 [T. T.], Japan

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The serine/threonine kinase Akt (also known as protein kinase B) is a downstream effector of phosphatidylinositol-3'-kinase [PI(3)K] that is recognized as the major mediator of survival signals that protect cells from undergoing apoptosis. In the course of examining the target molecules of the topoisomerase I inhibitor topotecan, we found that topotecan treatment promoted Akt dephosphorylation that led to the inactivation of Akt in human lung cancer A549 cells. Transfection of the constitutively active akt cDNA into A549 cells resulted in the reduction of the cytotoxic effect of topotecan, indicating that inhibition of the Akt pathway played an important role in exhibition of topotecan-mediated cytotoxic effects. Further analysis of Akt dephosphorylation revealed that topotecan treatment suppressed upstream kinases of Akt, 3-phosphoinositide-dependent protein kinase 1, and PI(3)K. Overall, the results demonstrate that topotecan exhibited its cytotoxic effects by down-regulating the PI(3)K-Akt survival signaling pathway in addition to inhibiting topoisomerase I.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A number of growth factors, such as insulin, insulin-like growth factor I, platelet-derived growth factor, and interleukins, have been reported to promote cell survival. The characterization of survival signal transduction pathways stimulated by these factors has revealed that PI(3)K3 is involved in protecting cells from undergoing apoptotic cell death (reviewed in Refs. 1 and 2 ). Blockade of PI(3)K activity suppresses growth factor-mediated cell survival (3) . After growth factor stimulation, the activated PI(3)K phosphorylates inositol lipids at the D-3 position of the inositol ring. The generated phospholipid second messengers, PtdIns-3,4,5-P₃ and PtdIns-3,4-P₂, raise a diverse set of cellular responses (4) . The major targets of PtdIns-3,4,5-P₃ and PtdIns-3,4-P₂ are PH domain-containing proteins (4, 5, 6) .

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Topotecan treatment decreased Akt kinase activity. A, A549 cells were treated with the indicated concentrations of topotecan for 48 h. Akt was then immunoprecipitated with protein G-agarose conjugated with an anti-Akt antibody. Akt kinase activity was evaluated as described in "Materials and Methods." Data represent the mean of triplicate determinations. A549 (B) and A549/CPT (C) cells were treated with the indicated concentrations of topotecan for 48 h. Cell lysates were subjected to SDS-PAGE, followed by Western blot analysis with an anti-phospho-Akt (Ser473) antibody (top panels) or an anti-Akt antibody (bottom panels). D, A549 cells were treated with the indicated concentrations of topotecan for 48 h. Cell lysates were subjected to SDS-PAGE, followed by Western blot analysis with an anti-phospho-IB (Ser32) antibody (top panel) or an anti-IB antibody (bottom panel). Results shown (B—D) are representative of at least two independent experiments.

Topotecan [10-hydroxy-9-dimethylaminomethyl-(S)-camptothecin], a water-soluble camptothecin analogue, is a novel topoisomerase I inhibitor that has shown activity against numerous human tumor cell lines and xenografts (17, 18, 19, 20, 21) . Topotecan has also shown clinical activity in small cell and non-small cell bronchogenic carcinoma, ovarian carcinoma, and myeloid leukemia (reviewed in Ref. 22 ) and has been approved for the treatment of ovarian cancer and small cell lung cancer.

The susceptibility of cells to undergo chemotherapy-induced apoptosis appears to be dependent on the balance between proapoptotic and antiapoptotic signals. Therefore, it is possible that a chemotherapeutic agent may induce apoptosis not only by increasing the proapoptotic signal but also by decreasing the antiapoptotic signal, such as the PI(3)K-Akt survival pathway. Down-regulation of the PI(3)K-Akt pathway has been observed in apoptosis induced by hyperosmotic stress, -irradiation, UV radiation, and cell-permeable ceramide (23) . Therefore, we have investigated the effects of topotecan on the PI(3)K-Akt survival signaling pathway.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials.
Topotecan was obtained from SmithKline & Beecham (King of Prussia, PA) and dissolved in sterile water. Camptothecin was a generous gift from Yakult (Tokyo, Japan). Z-Asp and Z-VAD were purchased from Funakoshi (Tokyo, Japan).

Cell Culture Conditions.
Human lung cancer cell line A549 and its camptothecin-resistant subline A549/CPT (24 , 25) were cultured at 37°C in a humidified atmosphere of 5% CO₂ and 95% air in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Biocell, Carson, CA), 2 mM L-glutamine (Life Technologies, Inc., Grand Island, NY), and 100 µg/ml kanamycin.

Expression Vector Construction.
Human full-length akt1 cDNA was generated by reverse transcription-PCR, as described previously (26) . Active forms of akt cDNAs, E40K-akt or T308D/S473D-akt, were generated by converting the Glu⁴⁰ codon to Lys codon or by substituting both Thr³⁰⁸ and Ser⁴⁷³ codons with Asp codons in full-length akt cDNA, respectively. Converting the Lys¹⁷⁹ codon to Met codon produced the kinase-dead form of akt cDNA, KD-akt. These cDNAs were subcloned into a pFLAG-CMV-2 vector (Kodak, New Haven, CT).

Transient Transfection.
Five µg of pHook-1 (Invitrogen, Carlsbad, CA) and 5 µg of pFLAG-CMV-2 vector containing E40K-akt, T308D/S473D-akt, or KD-akt cDNA were transiently cotransfected into A549 cells using Superfect transfection reagent according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The cells transfected with pHook-1 displayed a single-chain antibody (sFv) against 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one on the cell surface. After transfection for 24 h, the transfected cells were isolated from culture by binding to magnetic beads coated with 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (Capture-Tec Beads; Invitrogen). The bound cells were further incubated with or without 1 µM topotecan for 48 h. The viable cell number was counted by using the trypan blue dye exclusion method.

Western Blot Analysis.
Cells were solubilized with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 3 mM EGTA, 12 mM ß-glycerophosphate, 150 mM sodium chloride, 50 mM sodium fluoride, 1 mM sodium vanadate, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, and 0.1% 2-mercaptoethanol. The cell lysates were then applied to a 10–20% gradient polyacrylamide gel. The electrophoresed proteins were transblotted onto a nitrocellulose membrane. After blocking, the membranes were incubated with an anti-Akt antibody (New England Biolabs, Beverly, MA), an anti-phospho-Akt (Ser⁴⁷³) antibody (New England Biolabs), an-anti-IB (MAD-3) antibody (Transduction Laboratories, Lexington, KY), an anti-phospho-IB (Ser³²) antibody (New England Biolabs), an anti-MAPK antibody (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-phospho-MAPK antibody (Promega, Madison, WI), an anti-PARP p85 fragment antibody (Promega), an anti-PDK1 antibody (Transduction Laboratories), or an anti-PI(3)K-p85 antibody (Transduction Laboratories). The membrane was then incubated with an appropriate peroxidase-conjugated secondary antibody and developed with the enhanced chemiluminescence mixture (Amersham, Buckinghamshire, United Kingdom).

FACScan Analysis of Cell Cycle Distribution.
Cells were harvested, washed with PBS, and fixed in 70% ethanol. The fixed cells were washed with PBS and resuspended in 1 mg/ml RNase A in PBS, followed by incubation at 37°C for 30 min. Cells were stained with propidium iodide solution (50 µg/ml propidium iodide, 0.1% sodium citrate, and 0.1% NP40) for 30 min at 23°C. The cells were then resuspended and analyzed using a Becton Dickinson FACScan flow cytometer with Cell Quest software (Braintree, MA).

MTT Assay.
The sensitivity of A549 and A549/CPT cell lines to topotecan was evaluated by inhibition of cell growth after incubation at 37°C for 72 h with various concentrations of topotecan. The cytotoxicity was estimated using the MTT colorimetric assay.

Measurement of Caspase Activity.
The caspase activity in the cell lysates was measured as described previously, with slight modifications (26) . In brief, cells were harvested and lysed with caspase lysis buffer [10 mM HEPES (pH 7.4), 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1 propanesulfonic acid, and 5 mM DTT]. The cell lysate was then incubated with 20 µM DEVD-AMC (Peptide Institute, Osaka, Japan) in caspase assay buffer [20 mM HEPES (pH 7.4), 10% glycerol, and 2 mM DTT] for 1 h at 37°C. The AMC released from the fluorogenic substrate was excited at 380 nm, and the emission was measured at 460 nm using a Hitachi fluorescence spectrophotometer (model F-2000; Hitachi, Tokyo, Japan).

Measurement of Akt Kinase Activity and PDK1 Kinase Activity.
A549 cells were treated with or without topotecan for 48 h. Then cells were solubilized with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM ß-glycerophosphate, 50 mM sodium fluoride, 0.5 mM sodium vanadate, 5 mM Na PP_i, 1 µM microcystin, 0.1% 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of aprotinin, pepstatin, and leupeptin. Cell lysates were reacted to protein G-Sepharose that had been conjugated with an anti-Akt antibody (New England Biolabs) or an anti-PDK1 antibody (Upstate Biotechnology, Lake Placid, NY) for 2 h at 4°C. The beads were washed three times with lysis buffer. The Akt kinase activity was estimated by measuring incorporation of [-³²P]ATP into the peptide of glycogen synthase kinase 3 (Upstate Biotechnology). We measured PDK1 kinase activity indirectly through activation of unactive serum- and glucocorticoid-inducible kinase using a PDK1 kinase assay kit according to the manufacturer’s instructions (Upstate Biotechnology).

PI(3)K Assay.
PI(3)K activity was determined as described previously (27) . Briefly, cells were harvested and solubilized with lysis buffer containing 20 mM Tris-HCl (pH 7.5), 145 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 0.5% NP40, 100 mM sodium fluoride, 0.5 mM sodium vanadate, and 10 µg/ml each of aprotinin and leupeptin. Cell lysates were incubated with agarose conjugated with an antiphosphotyrosine (PY20) antibody (Transduction Laboratories) for 2 h at 4°C. The beads were then washed sequentially three times with wash buffer A [Tris-HCl (pH 7.5), 150 mM NaCl, 0.01% NP40, and 100 µM sodium vanadate], wash buffer B [100 mM Tris-HCl (pH 7.5), 500 mM LiCl, and 100 µM sodium vanadate], and wash buffer C [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, and 100 µM sodium vanadate]. The beads were then resuspended in 50 µl of kinase assay buffer [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, and 20 mM MgCl₂], and the kinase reaction was initiated by the addition of 20 µg of phosphatidylinositol and 50 µM ATP containing 20 µCi of [-³²P]ATP. The samples were incubated for 20 min at 23°C, and the reactions were terminated by the addition of 20 µl of 8 N HCl. The samples were then extracted with 160 µl of chloroform-methanol (1:1), and the organic phase was concentrated by evaporation. The resultant lipid fractions were resolved by TLC in chloroform-methanol-water-ammonium hydroxide (60:47:11.3:2). The phosphorylated products were then visualized by autoradiography.

Statistical Analysis.
Statistical significance was calculated using Student’s t test. A probability value of <0.05 was considered to be significant.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Topotecan Treatment Induced Apoptosis in Human Lung Cancer A549 Cells.
Topotecan is a water-soluble camptothecin analogue. When human lung cancer A549 cells were treated with topotecan for 72 h, the viable cell number was decreased in a dose-dependent manner (Fig. 1 A, ). In contrast, camptothecin-resistant A549/CPT cells exhibited resistance to a topotecan-induced decrease in viable cell number (Fig. 1 A, •). It has been reported that A549/CPT cells expressed a similar amount of topoisomerase I as the parental A549 cells and are resistant to apoptosis induced by chemotherapeutic agents such as camptothecin, cisplatin, or Adriamycin (24 , 25) . When cells were treated with 10 µM topotecan, about 70% of A549/CPT cells remained viable, whereas the viable cell number of parental A549 cells decreased to 20%. To examine whether A549 cells underwent apoptosis when the cells were treated with topotecan, cells were stained with propidium iodide, followed by examination of the appearance of the sub-G₁ population using flow cytometry (Fig. 1B) . As shown in Fig. 1C (), topotecan treatment increased the number of apoptotic sub-G₁ fraction of A549 cells in a dose-dependent manner. Therefore, A549 cells underwent apoptosis after topotecan treatment. In contrast, A549/CPT cells exhibited resistance to topotecan-induced apoptosis because only 20% of A549/CPT cells underwent apoptosis after topotecan treatment for 48 h (Fig. 1 A, •). The number of sub-G₁ fraction in A549/CPT cells was smaller than that in A549 cells (Fig. 1 C, ).

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Cytotoxic effects of topotecan on A549 and its resistant subline, A549/CPT. A, A549 () and A549/CPT (•) cells were treated with the indicated concentrations of topotecan for 72 h. The viable cell number was determined by MTT assay as described in "Materials and Methods." Each point represents a mean ± SE of three independent experiments. B, A549 (top panels) and A549/CPT (bottom panels) cells were treated with the indicated concentrations of topotecan for 48 h. Cells were stained with propidium iodide, followed by analysis with FACScan flow cytometry as described in "Materials and Methods." C, A549 () and A549/CPT () cells were treated with the indicated concentrations of topotecan for 48 h. The percentage of the sub-G1 population of A549 cells was determined by propidium iodide staining and FACScan flow cytometry as shown in B.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Activation of caspase-3 by topotecan treatment. A, A549 () and A549/CPT () cells were treated with the indicated concentrations of topotecan for 48 h. Cell lysates were incubated with the fluorogenic-labeled tetrapeptide DEVD-AMC (20 µM) for 1 h at 37°C. The increase in DEVDase activity in the cell lysates was determined as described in "Materials and Methods." The vertical bars represent the SD value of triplicate determinations. B, A549 (Lanes 1–4) and A549/CPT (Lanes 5–8) cells were incubated with 0 (Lanes 1 and 5), 0.1 (Lanes 2 and 6), 1 (Lanes 3 and 7), or 10 µM (Lanes 4 and 8) topotecan for 48 h. Cell lysates were subjected to SDS-PAGE, followed by Western blot analysis with an anti-PARP p85 fragment-specific antibody. Results shown are representative of at least two independent experiments.

To estimate the role of Akt inactivation during topotecan-mediated cytotoxicity, we examined the effect of topotecan on A549 cells transfected with active form of akt cDNAs. Constitutively active akt cDNA, T308D/S473D-akt, was generated by replacing the both PDK phosphorylation sites Thr³⁰⁸ and Ser⁴⁷³ with Asp, leading to an elevated kinase level (29) . Another constitutively active akt cDNA, E40K-akt, was generated by point mutation at Glu⁴⁰ with Lys, which results in an increased affinity of the PH domain for phospholipids (29) . We also constructed the kinase-dead form of akt cDNA, KD-akt, by substituting Met for Lys¹⁷⁹ (29) . These cDNAs in pFLAG-CMV-2 vector were transfected into A549 cells together with pHook-1 plasmid. Due to the low transfection efficiency of A549 cells (1%), transfected cells were isolated using Capture-Tec Beads, followed by treatment with topotecan for 48 h. The expression of transfected FLAG-tagged Akt was confirmed by Western blot analysis (Fig. 4B , arrowhead). The expression level of KD-akt was somewhat lower than that of active-form of Akt (T308D/S473D and E40K). Because KD-akt behaves as a dominant negative, we could not overexpress KD-akt in A549 cells. As shown in Fig. 4A , expression of the active form of Akt (T308D/S473D and E40K) significantly suppressed topotecan-induced cell death and increased the viable cell number (P < 0.05). Transfection of KD-akt cDNA had no effect on the sensitivity to topotecan. The result indicates that Akt inactivation plays an important role in the exhibition of topotecan-mediated cytotoxic effects. The lower viability of mock-transfectant (Fig. 4A) compared with nontransfected cells (Fig. 1A) after topotecan treatment might be caused by the difference in cell density during topotecan treatment or by mechanical stress during Capture-Tec Bead selection.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Overexpression of constitutively active Akt decreased topotecan-induced cell death. A549 cells were transfected with empty pFLAG-CMV-2 vector (Mock), pFLAG-CMV-2 vector containing the constitutively active form of akt cDNA E40K-akt (E40K) or T308D/S473D-akt (T308D/S473D), or pFLAG-CMV-2 vector containing the kinase-dead form of akt cDNA, KD-akt (KD), together with pHook-1 plasmid. After transfection for 24 h, transfected cells were enriched using Capture-Tec Beads. The transfected cells were further cultured with or without 1 µM topotecan for 48 h. A, viable cell number was counted by using the trypan blue dye exclusion method. Viable cell numbers of untreated control cells were normalized as 100%. Mean viable cell numbers of untreated mock, E40K-akt, T308D/S473D-akt, and KD-akt transfectants were 3.2 x 104, 4.6 x 104, 4.1 x 104, and 2.3 x 104 cells, respectively. Data represent the mean ± SE for three independent experiments. *, P < 0.05 (Student’s t test; n = 3) compared with control mock transfectants. B, 24 h after transfection, the lysates of A549 cells were subjected to SDS-PAGE, followed by Western blot analysis with an anti-Akt antibody. The transfected FLAG-tagged Akt is indicated by the arrowhead.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Topotecan treatment decreased the amount of phospho-Akt before caspase-3 activation. A549 cells were treated with 1 µM topotecan. At the indicated time points, cells were harvested. A, the cell lysates were subjected to Western blot analysis with an anti-phospho-Akt (Ser473) antibody, an anti-Akt antibody, an anti-phospho-MAPK antibody, or an anti-MAPK antibody. B, cell lysates were incubated with the fluorogenic-labeled tetrapeptide DEVD-AMC (20 µM) for 1 h at 37°C. The increase in DEVDase activity in the cell lysates was determined as described in "Materials and Methods." The vertical bars represent the SD value of triplicate determinations.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Effects of caspase inhibitors on topotecan-induced Akt dephosphorylation. A549 cells were incubated with or without topotecan (1 µM) for 48 h. In some experiments, cells were treated with topotecan (1 µM) together with either Z-Asp (100 µg/ml) or Z-VAD (100 µg/ml). A, the cell lysates were subjected to SDS-PAGE, followed by Western blot analysis with an anti-phospho-Akt (Ser473) antibody (top panel) or an anti-Akt antibody (bottom panel). Results shown are representative of at least two independent experiments. B, cell lysates were incubated with the fluorogenic-labeled tetrapeptide DEVD-AMC (20 µM) for 1 h at 37°C. The increase in DEVDase activity in the cell lysates was determined as described in "Materials and Methods." The vertical bars represent the SD value of triplicate determinations. C, the percentage of the sub-G1 population of A549 cells was determined by propidium iodide staining and FACScan flow cytometry as described in "Materials and Methods."

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Topotecan suppresses PDK1 and PI(3)K kinase activity. A549 cells were incubated with the indicated concentration of topotecan for 48 h. A, PDK1 kinase activity was evaluated as described in "Materials and Methods." The value of PDK1 kinase activity in untreated A549 cell lysates was normalized as 100%. The vertical bars represent the SD value of triplicate determinations. Cell lysates were subjected to SDS-PAGE, followed by Western blot analysis with an anti-PDK1 antibody. B, PI(3)K kinase activity was assessed by incubating the phosphatidylinositol with the immunoprecipitated phosphotyrosine-containing proteins in the presence of 50 µM ATP containing 20 µCi of [-32P]ATP. The lipid fractions were resolved by TLC followed by autoradiography. Cell lysates were subjected to SDS-PAGE, followed by Western blot analysis with an anti-PI(3)K p85 antibody.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Topotecan, a water-soluble semisynthetic camptothecin analogue, is a potent inhibitor of topoisomerase I, an enzyme necessary for DNA replication. Topoisomerase I induces transient single-strand DNA breaks that lead to relaxation of supercoiled DNA, which is an essential step for DNA replication and RNA transcription. Topotecan interfere with DNA replication by stabilizing the covalent complex between topoisomerase I and DNA, preventing the religation of enzyme-linked single-strand DNA breaks (30) . Although topotecan is known to inhibit topoisomerase I, neither mRNA levels of the enzyme nor cleavable DNA complex formation predicts tumor cell responses to topotecan in vitro (31) . Moreover, despite the similar topoisomerase I expression in the resistant subline A549/CPT and in parental A549 cells (24 , 25) , A549/CPT showed decreased sensitivity to topotecan when compared with parental A549 cells (Fig. 1) . Because the topoisomerase I level is not altered in the A549/CPT resistant subline (24 , 25) , decreased sensitivity to topotecan may not be the consequence of decreased induction of DNA damage. Thus, topoisomerase I inhibition is not the sole mechanism by which topotecan exerts its cytotoxic effects on tumor cells.

Apoptosis is a major mode of cell death in response to drug treatment (32 , 33) , and resistance to apoptosis induction has been proposed as a critical mechanism of drug resistance (34, 35, 36) . The pattern of tumor response after in vivo topotecan treatment was reported to correlate with the ability of the drug to induce apoptosis but not with its in vitro antiproliferative activity (37) . As shown in Fig. 2 , topotecan induced apoptosis in A549 cells with caspase-3 activation. Thus, we examined the possibility that topotecan exerted its cytotoxic effects by interfering with some antiapoptotic machinery. To clarify the hypothesis, we examined Akt kinase activity. The serine/threonine kinase Akt has been shown to play a central role in promoting survival and blocking apoptosis induced by diverse apoptotic stimuli (7) . When we measured Akt kinase activity, we found that topotecan decreased Akt kinase activity in a dose-dependent manner (Fig. 3, A and D) . Because Akt is activated by phosphorylation of Thr³⁰⁸ and Ser⁴⁷³ residues, we performed Western blot analysis with an anti-phospho-Akt (Ser⁴⁷³) antibody and found the dephosphorylation of Akt after topotecan treatment in A549 cells (Fig. 3B) but not in A549/CPT cells (Fig. 3C) . These results suggest that the cytotoxic effects of topotecan might be mediated in part by suppression of Akt kinase activity.

To confirm the results, we generated constitutively active E40K-akt and T308D/S473D-akt cDNAs. T308D/S473D-akt cDNA was generated by replacing the both PDK phosphorylation sites Thr³⁰⁸ and Ser⁴⁷³ with negatively charged Asp by mimicking a phosphorylated state, leading to an elevated kinase level (29) . Another constitutively active akt cDNA, E40K-akt, was generated by point mutation at Glu⁴⁰ with Lys, which results in an increased affinity of the PH domain for phospholipids (29) . Transient transfection with constitutively active Akt resulted in a reduction of the cytotoxic effect of topotecan (Fig. 4) . Therefore, it was indicated that inhibition of Akt kinase activity might play a part in the underlying mechanisms responsible for the apoptotic effects of topotecan.

We therefore examined the possibility that topotecan down-regulated Akt kinase activity by inactivating upstream kinases that could phosphorylate Akt at the Thr³⁰⁸ and Ser⁴⁷³ residues. PDK1 was known to be a kinase that phosphorylates Akt at the Thr³⁰⁸ residue. Recently, Balendran et al. (9) reported that the interaction of the PRK2 fragment with PDK1 converted PDK1 from the kinase that could phosphorylate only the Thr³⁰⁸ residue of Akt to the kinase that could phosphorylate both the Thr³⁰⁸ and Ser⁴⁷³ residues of Akt. However, PDK1 kinase activity was constitutive, and its kinase activity was not affected by the product of PI(3)K, PtdIns-3,4,5-P₃, or PtdIns-3,4-P₂. The dependence of Akt kinase activity on PI(3)K was thought to reflect the ability of PtdIns-3,4,5-P₃ and PtdIns-3,4-P₂ to increase the colocalization of PDK1 and Akt to the membrane and the ability to displace the inhibitory Akt PH domain (1) . We therefore examined the change in PDK1 and PI(3)K kinase activities after topotecan treatment. Interestingly, topotecan (at concentrations up to 1 µM) suppressed the kinase activity of both PDK1 and PI(3)K without affecting their expression levels (Fig. 7, A and B) . Thus, topotecan might exert its cytotoxic effects in part by down-regulating PI(3)K-Akt survival pathways. Because PDK1 was known to be a constitutively active kinase, the mechanisms of PDK1 suppression by topotecan must be clarified in future. Recently, phosphorylation of PDK1 at the Ser²⁴¹ residue was reported to be essential for PDK1 activity (38) . It may be possible that topotecan affected some kinases or phosphatases of PDK1 to suppress the kinase activity of PDK1.

When A549 cells were treated with topotecan, a decrease in the level of phosphorylated active Akt was observed before caspase-3 activation (Fig. 5) and was not affected by caspase inhibitor (Fig. 6) , indicating that Akt inhibition is an upstream event of caspase-3 activation in topotecan-induced apoptosis. These observations were consistent with the finding that activated Akt could phosphorylate and inactivate caspase-9, which is an initiator caspase that is activated by the release of cytochrome c from mitochondria in response to various apoptotic stimuli (14) . Akt is also associated with cell survival by phosphorylating proapoptotic Bad, transcription factor FKHRL1, and IB kinase (13 , 15 , 16) . Therefore, inactivating Akt turned on diverse sets of apoptosis machinery. That might be the reason why topotecan showed a broad spectrum of antitumor activity against human tumor cell lines and xenografts.

In conclusion, we found Akt inactivation after topotecan treatment. Akt inactivation might be important for topotecan-induced apoptosis in tumor cells. Because increased Akt kinase activity was found in some tumor cell lines, topotecan might be effective in the clinical treatment of these tumor cells. Recently, Plo et al. (39) reported that daunorubicin treatment activates the Akt pathway in human acute myeloid leukemia cells. In the future, we need to examine whether or not down-regulation of Akt kinase activity is a topotecan-specific phenomena.

	ACKNOWLEDGMENTS

 
We thank Dr. Randall K. Johnson (SmithKline Beecham) for helpful discussions and critical review of the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by a special grant for advanced research on cancer and a grant-in-aid for cancer research from the Ministry of Education, Science, Sports and Culture of Japan.

² To whom requests for reprints should be addressed, at Laboratory of Biomedical Research, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Phone: 81-3-5841-7861; Fax: 81-3-5841-8487; E-mail: ttsuruo{at}iam.u-tokyo.ac.jp

³ The abbreviations used are: PI(3)K, phosphatidylinositol-3'-kinase; PDK, 3-phosphoinositide-dependent protein kinase; Z-Asp, benzyloxycarbonyl-Asp-CH₂OCO-2,6,-dichlorobenzene; Z-VAD, benzyloxycarbonyl-Val-Ala-Asp-CH₂OCO-2,6,-dichlorobenzene; DEVD-AMC, acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspart-7-amino-4-methylcoumarin; PtdIns-3,4,5-P₃, phosphatidylinositol-3,4,5-triphosphate; PtdIns-3,4-P₂, phosphatidylinositol-3,4-bisphosphate; PRK2, protein kinase C-related kinase 2; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly(ADP-ribose) polymerase; CMV, cytomegalovirus; DEVDase, caspase-3-like protease.

Received 12/13/99. Accepted 7/20/00.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Alessi D. R., Cohen P. Mechanism of activation and function of protein kinase B. Curr. Opin. Genet. Dev., 8: 55-62, 1998.[Medline]
2. Heldin C. H. Dimerization of cell surface receptors in signal transduction. Cell, 80: 213-223, 1995.[Medline]
3. Yao R., Cooper G. M. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science (Washington DC), 267: 2003-2006, 1995.[Abstract/Free Full Text]
4. Vanhaesebroeck B., Leevers S. J., Panayotou G., Waterfield M. D. Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem. Sci., 22: 267-272, 1997.[Medline]
5. Toker A., Cantley L. C. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature (Lond.), 387: 673-676, 1997.[Medline]
6. Rodriguez-Viciana P., Warne P. H., Khwaja A., Marte B. M., Pappin D., Das P., Waterfield M. D., Ridley A., Downward J. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell, 89: 457-467, 1997.[Medline]
7. Franke T. F., Kaplan D. R., Cantley L. C. PI3K: downstream AKTion blocks apoptosis. Cell, 88: 435-437, 1997.[Medline]
8. Bellacosa A., Testa J. R., Staal S. P., Tsichlis P. N. A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science (Washington DC), 254: 274-277, 1991.[Abstract/Free Full Text]
9. Balendran A., Casamayor A., Deak M., Paterson A., Gaffney P., Currie R., Downes C. P., Alessi D. R. PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr. Biol., 9: 393-404, 1999.[Medline]
10. Alessi D. R., James S. R., Downes C. P., Holmes A. B., Gaffney R. P. J., Reese C. B., Cohen P. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B. Curr. Biol., 7: 261-269, 1997.[Medline]
11. Stephens L., Anderson K., Stokoe D., Erdjument-Bromage H., Painter G. F., Holmes A. B., Gaffney P. R., Reese C. B., McCormick F., Tempst P., Coadwell J., Hawkins P. T. Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science (Washington DC), 279: 710-714, 1998.[Abstract/Free Full Text]
12. Le Good J. A., Ziegler W. H., Parekh D. B., Alessi D. R., Cohen P., Parker P. J. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science (Washington DC), 281: 2042-2045, 1998.[Abstract/Free Full Text]
13. Datta S. R., Dudek H., Tao X., Masters S., Fu H., Gotoh Y., Greenberg M. E. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell, 91: 231-241, 1997.[Medline]
14. Cardone M. H., Roy N., Stennicke H. R., Salvesen G. S., Franke T. F., Stanbridge E., Frisch S., Reed J. C. Regulation of cell death protease caspase-9 by phosphorylation. Science (Washington DC), 282: 1318-1321, 1998.[Abstract/Free Full Text]
15. Brunet A., Bonni A., Zigmond M. J., Lin M. Z., Juo P., Hu L. S., Anderson M. J., Arden K. C., Blenis J., Greenberg M. E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell, 96: 857-868, 1999.[Medline]
16. Romashkova J. A., Makarov S. S. NF-B is a target of AKT in anti-apoptotic PDGF signalling. Nature (Lond.), 401: 86-90, 1999.[Medline]
17. Houghton P. J., Cheshire P. J., Myers L., Stewart C. F., Synold T. W., Houghton J. A. Evaluation of 9-dimethylaminomethyl-10-hydroxycamptothecin against xenografts derived from adult and childhood solid tumors. Cancer Chemother. Pharmacol., 31: 229-239, 1992.[Medline]
18. Planchon S. M., Wuerzberger S., Frydman B., Witiak D. T., Hutson P., Church D. R., Wilding G., Boothman D. A. ß-Lapachone-mediated apoptosis in human promyelocytic leukemia (HL-60) and human prostate cancer cells: A p53-independent response. Cancer Res., 55: 3706-3711, 1995.[Abstract/Free Full Text]
19. Kaufmann S. H., Svingen P. A., Gore S. D., Armstrong D. K., Cheng Y. C., Rowinsky E. K. Altered formation of topotecan-stabilized topoisomerase I-DNA adducts in human leukemia cells. Blood, 89: 2098-2104, 1997.[Abstract/Free Full Text]
20. Kaufmann S. H., Gore S. D., Letendre L., Svingen P. A., Kottke T., Buckwalter C. A., Jones R. J., Grochow L. B., Burke P. J., Donehower R. C., Rowinsky E. K. Factors affecting topotecan sensitivity in human leukemia samples. Ann. N. Y. Acad. Sci., 803: 128-142, 1996.[Medline]
21. Traganos F., Seiter K., Feldman E., Halicka H. D., Darzynkiewicz Z. Induction of apoptosis by camptothecin and topotecan. Ann. N. Y. Acad. Sci., 803: 101-110, 1996.[Medline]
22. Kollmannsberger C., Mross K., Jakob A., Kanz L., Bokemeyer C. Topotecan, a novel topoisomerase I inhibitor: pharmacology and clinical experience. Oncology (Basel), 56: 1-12, 1999.[Medline]
23. Zundel W., Giaccia A. Inhibition of the anti-apoptotic PI(3)K/Akt/Bad pathway by stress. Genes Dev., 12: 1941-1946, 1998.[Abstract/Free Full Text]
24. Sugimoto Y., Tsukahara S., Oh-hara T., Isoe T., Tsuruo T. Decreased expression of DNA topoisomerase I in camptothecin-resistant tumor cell lines as determined by a monoclonal antibody. Cancer Res., 50: 6925-6930, 1990.[Abstract/Free Full Text]
25. Zhang Y., Fujita N., Tsuruo T. p21^Waf1/Cip1 acts in synergy with bcl-2 to confer multidrug resistant in a camptothecin-selected human lung cancer cell line. Int. J. Cancer, 83: 790-797, 1999.[Medline]
26. Rokudai S., Fujita N., Hashimoto Y., Tsuruo T. Cleavage and inactivation of antiapoptotic Akt/PKB by caspases during apoptosis. J. Cell. Physiol., 182: 290-296, 2000.[Medline]
27. Whitman M., Downes C. P., Keeler M., Keller T., Cantley L. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature (Lond.), 332: 644-646, 1988.[Medline]
28. Lazebnik Y. A., Kaufmann S. H., Desnoyers S., Poirier G. G., Earnshaw W. C. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature (Lond.), 371: 346-347, 1994.[Medline]
29. Aoki M., Batista O., Bellacosa A., Tsichlis P., Vogt P. K. The Akt kinase: molecular determinants of oncogenicity. Proc. Natl. Acad. Sci. USA, 95: 14950-14955, 1998.[Abstract/Free Full Text]
30. Hsiang Y. H., Liu L. F. Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res., 48: 1722-1726, 1988.[Abstract/Free Full Text]
31. Dubrez L., Goldwasser F., Genne P., Pommier Y., Solary E. The role of cell cycle regulation and apoptosis triggering in determining the sensitivity of leukemic cells to topoisomerase I and II inhibitors. Leukemia (Baltimore), 9: 1013-1024, 1995.[Medline]
32. Hickman J. A. Apoptosis induced by anticancer drugs. Cancer Metastasis Rev., 11: 121-139, 1992.[Medline]
33. Kerr J. F. R., Winterford C. M., Harmon B. V. Apoptosis: its significance in cancer and cancer therapy. Cancer (Phila.), 73: 2013-2026, 1994.[Medline]
34. Kataoka S., Naito M., Tomida A., Tsuruo T. Resistance to antitumor agent-induced apoptosis in a mutant of human myeloid leukemia U937 cells. Exp. Cell Res., 215: 199-205, 1994.[Medline]
35. Seimiya H., Mashima T., Toho M., Tsuruo T. c-Jun NH₂-terminal kinase-mediated activation of interleukin-1ß-converting enzyme/CED-3-like protease during anticancer drug-induced apoptosis. J. Biol. Chem., 272: 4631-4636, 1997.[Abstract/Free Full Text]
36. Chen Z., Naito M., Mashima T., Tsuruo T. Activation of actin-cleavable interleukin 1ß-converting enzyme (ICE) family protease CPP-32 during chemotherapeutic agent-induced apoptosis in ovarian carcinoma cells. Cancer Res., 56: 5224-5229, 1996.[Abstract/Free Full Text]
37. Caserini C., Pratesi G., Tortoreto M., Bedogne B., Carenini N., Supino R., Perego P., Righetti S. C., Zunino F. Apoptosis as a determinant of tumor sensitivity to topotecan in human ovarian tumors: preclinical in vitro/in vivo studies. Clin. Cancer Res., 3: 955-961, 1997.[Abstract]
38. Casamayor A., Morris N. A., Alessi D. R. Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo. Biochem. J., 342: 287-292, 1999.
39. Plo I., Bettaieb A., Payrastre B., Mansat-De Mas V., Bordier C., Rousse A., Kowalski-Chauvel A., Laurent G., Lautier D. The phosphoinositide 3-kinase/Akt pathway is activated by daunorubicin in human acute myeloid leukemia cell lines. FEBS Lett., 452: 150-154, 1999.[Medline]

This article has been cited by other articles:

				R. Saito, M. T. Krauze, C. O. Noble, D. C. Drummond, D. B. Kirpotin, M. S. Berger, J. W. Park, and K. S. Bankiewicz Convection-enhanced delivery of Ls-TPT enables an effective, continuous, low-dose chemotherapy against malignant glioma xenograft model Neuro-oncol, July 1, 2006; 8(3): 205 - 214. [Abstract] [Full Text] [PDF]

				D. Xiao and S. V. Singh Diallyl trisulfide, a constituent of processed garlic, inactivates Akt to trigger mitochondrial translocation of BAD and caspase-mediated apoptosis in human prostate cancer cells Carcinogenesis, March 1, 2006; 27(3): 533 - 540. [Abstract] [Full Text] [PDF]

				H. Asanuma, T. Torigoe, K. Kamiguchi, Y. Hirohashi, T. Ohmura, K. Hirata, M. Sato, and N. Sato Survivin Expression Is Regulated by Coexpression of Human Epidermal Growth Factor Receptor 2 and Epidermal Growth Factor Receptor via Phosphatidylinositol 3-Kinase/AKT Signaling Pathway in Breast Cancer Cells Cancer Res., December 1, 2005; 65(23): 11018 - 11025. [Abstract] [Full Text] [PDF]

				K. Katayama, N. Fujita, and T. Tsuruo Akt/Protein Kinase B-Dependent Phosphorylation and Inactivation of WEE1Hu Promote Cell Cycle Progression at G2/M Transition Mol. Cell. Biol., July 1, 2005; 25(13): 5725 - 5737. [Abstract] [Full Text] [PDF]

				Y. Luo, A. R. Shoemaker, X. Liu, K. W. Woods, S. A. Thomas, R. de Jong, E. K. Han, T. Li, V. S. Stoll, J. A. Powlas, et al. Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo Mol. Cancer Ther., June 1, 2005; 4(6): 977 - 986. [Abstract] [Full Text] [PDF]

				B. R. Balsara, J. Pei, Y. Mitsuuchi, R. Page, A. Klein-Szanto, H. Wang, M. Unger, and J. R. Testa Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions Carcinogenesis, November 1, 2004; 25(11): 2053 - 2059. [Abstract] [Full Text] [PDF]

				O. David, J. Jett, H. LeBeau, G. Dy, J. Hughes, M. Friedman, and A. R. Brody Phospho-Akt Overexpression in Non-Small Cell Lung Cancer Confers Significant Stage-Independent Survival Disadvantage Clin. Cancer Res., October 15, 2004; 10(20): 6865 - 6871. [Abstract] [Full Text] [PDF]

				M. Fu, M. Rao, K. Wu, C. Wang, X. Zhang, M. Hessien, Y.-G. Yeung, D. Gioeli, M. J. Weber, and R. G. Pestell The Androgen Receptor Acetylation Site Regulates cAMP and AKT but Not ERK-induced Activity J. Biol. Chem., July 9, 2004; 279(28): 29436 - 29449. [Abstract] [Full Text] [PDF]

				A. Rapisarda, B. Uranchimeg, O. Sordet, Y. Pommier, R. H. Shoemaker, and G. Melillo Topoisomerase I-Mediated Inhibition of Hypoxia-Inducible Factor 1: Mechanism and Therapeutic Implications Cancer Res., February 15, 2004; 64(4): 1475 - 1482. [Abstract] [Full Text] [PDF]

				T.-D. Way, M.-C. Kao, and J.-K. Lin Apigenin Induces Apoptosis through Proteasomal Degradation of HER2/neu in HER2/neu-overexpressing Breast Cancer Cells via the Phosphatidylinositol 3-Kinase/Akt-dependent Pathway J. Biol. Chem., February 6, 2004; 279(6): 4479 - 4489. [Abstract] [Full Text] [PDF]

				G. Petrangolini, G. Pratesi, M. De Cesare, R. Supino, C. Pisano, M. Marcellini, V. Giordano, D. Laccabue, C. Lanzi, and F. Zunino Antiangiogenic Effects of the Novel Camptothecin ST1481 (Gimatecan) in Human Tumor Xenografts Mol. Cancer Res., October 1, 2003; 1(12): 863 - 870. [Abstract] [Full Text] [PDF]

				J. Asakuma, M. Sumitomo, T. Asano, T. Asano, and M. Hayakawa Selective Akt Inactivation and Tumor Necrosis Factor-related Apoptosis-inducing Ligand Sensitization of Renal Cancer Cells by Low Concentrations of Paclitaxel Cancer Res., March 15, 2003; 63(6): 1365 - 1370. [Abstract] [Full Text] [PDF]

				S. Rokudai, N. Fujita, O. Kitahara, Y. Nakamura, and T. Tsuruo Involvement of FKHR-Dependent TRADD Expression in Chemotherapeutic Drug-Induced Apoptosis Mol. Cell. Biol., December 15, 2002; 22(24): 8695 - 8708. [Abstract] [Full Text] [PDF]

				G. W. Krystal, G. Sulanke, and J. Litz Inhibition of Phosphatidylinositol 3-Kinase-Akt Signaling Blocks Growth, Promotes Apoptosis, and Enhances Sensitivity of Small Cell Lung Cancer Cells to Chemotherapy Mol. Cancer Ther., September 1, 2002; 1(11): 913 - 922. [Abstract] [Full Text] [PDF]

<

A. S. Clark, K. West, S. Streicher, and P. A. Dennis Constitutive and Inducible Akt Activity Promotes Resistance to Chemotherapy, Trastuzumab, or Tamoxifen in Breast Cancer Cells Mol. Cancer Ther., July 1, 2002; 1(9): 707 - 717. [Abstract] [Full Text] [PDF]