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Experimental Therapeutics, Preclinical Pharmacology |
Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: The KM20 cell line, established from a metastatic colon cancer, was treated in vitro with NaBT, gemcitabine, or 5-fluorouracil either alone or in combination with the PI3k inhibitors wortmannin or LY294002; DNA fragmentation and cell viability were measured. As further indicators of apoptosis, protein was extracted to determine caspase-9 and caspase-3 activation and cleavage of poly(ADP-ribose) polymerase. In addition, the effect of NaBT and wortmannin on in vivo KM20 tumor growth was determined.
Results: We demonstrate that inhibition of PI3k enhanced NaBT-mediated apoptosis and decreased KM20 cell viability; the nonspecific caspase inhibitor zVAD-fmk blocked the induction of apoptosis by the combination treatment. Either wortmannin or LY294002, combined with NaBT, enhanced activation of caspase-9 and caspase-3 and the subsequent cleavage of poly(ADP-ribose) polymerase. Furthermore, inhibition of PI3k increased the sensitivity of KM20 cells to gemcitabine and 5-fluorouracil. Wortmannin alone inhibited KM20 xenograft growth in vivo; the combination of wortmannin and NaBT demonstrated an enhanced effect compared with either agent alone.
Conclusions: Our results are the first to show that inhibition of PI3k enhances NaBT-mediated colon cancer cell apoptosis through the activation of caspase-9 and caspase-3. Moreover, these findings suggest that agents that selectively target the PI3k pathway may enhance the effects of standard chemotherapeutic agents and provide novel adjuvant treatment for selected colon cancers.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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130,000 new cases diagnosed per year and 57,000 deaths estimated this year secondary to this disease (1)
. Although many patients presenting with colorectal cancer may undergo surgical resection for possible cure, and advances in multimodality therapy have improved survival for patients with stage II, III, and IV disease, 50% of patients with cancers of the colon and rectum die from their disease (2)
. A better understanding of the signaling pathways contributing to colorectal cancer proliferation will provide targets for potentially new therapeutic agents.
One pathway that has been shown to contribute to cancer proliferation and survival is the PI3k3
signaling pathway. PI3k, a ubiquitous lipid kinase involved in receptor signal transduction by tyrosine kinase receptors, is involved in a number of diverse cellular processes such as growth and transformation, membrane ruffling, vesicular trafficking, and cell survival (3, 4, 5, 6, 7)
. PI3k catalyzes the phosphorylation of the inositol ring at the D3 position in a variety of phosphoinositide substrates forming 3-phosphorylated phosphoinositides (8)
. Activation of PI3k, a down-regulator of the Ras signaling pathway, is necessary for actin cytoskeletal rearrangement, which is associated with the transformed phenotype, and is increased in 86% of human colorectal cancers (9)
. Furthermore, activation of PI3k induces colon cancer proliferation (10, 11, 12)
. Increased PI3k activity can convert differentiated human gastric and colon carcinoma cells to a less differentiated and more malignant phenotype (13)
. Downstream of PI3k, Akt2 (PKBß) is amplified and overexpressed in some ovarian cancers (14)
. Similarly, Akt3 (PKB
) is overexpressed in some steroid hormone-insensitive breast cancers (15)
. Current evidence suggests that activation of Akt/PKB in cancers appears to reflect increased PI3k activity rather than the consequence of primary overexpression.
In addition to playing a role in cancer cell proliferation, the PI3k pathway also contributes to the differentiation of certain cell types. Inhibition of PI3k induces B16 melanoma cell differentiation as well as endocrine differentiation of human fetal undifferentiated cells (16 , 17) . Recently, we have shown that inhibition of PI3k, using the chemical inhibitor wortmannin, enhances NaBT-mediated enterocyte-like differentiation of HT29 and Caco-2 human colon cancer cells (18) . NaBT, a short chain fatty acid, is produced in the colon by breakdown of dietary fiber (19) . Our laboratory, as well as others, has shown that NaBT treatment of certain colon cancers induces differentiation by mechanisms that include G1 cell cycle arrest and stimulation of cell cycle-related proteins (20 , 21) . In addition, NaBT, which also inhibits HDA activity, results in growth inhibition of certain colon cancers and has been proposed as a potential anticancer agent (22, 23, 24) . Compounds possessing HDA inhibiting activity are thought to represent a novel class of agents for treatment of human cancers (25) .
Because PI3k inhibition can augment the differentiation of certain colon cancer cells induced by NaBT, we postulate that this combination treatment may enhance apoptosis and the antitumor effects of NaBT on colon cancer cells in vitro and in vivo. In this study, we demonstrate that inhibition of PI3k, using the wortmannin or LY294002 chemical inhibitor, augments NaBT-induced apoptosis and cell proliferation in the KM20 human colon cancer cell line in vitro. The combination of NaBT with wortmannin or LY294002 enhanced activation of caspase-9 and caspase-3 and the subsequent cleavage of PARP. In addition, we show that inhibition of PI3k significantly increased KM20 cell apoptosis in combination with the gemcitabine or 5-FU chemotherapeutic agent. We have also used another human colon cancer, HCT116, to show that, similar to KM20 cells, the combination of the PI3k inhibitor (wortmannin or LY294002) with NaBT augmented NaBT-mediated apoptosis. Moreover, treatment with wortmannin or a combination of wortmannin and NaBT inhibited the growth of KM20 colon cancer xenografts. These findings suggest that agents targeting the PI3k pathway may represent novel therapies for the adjuvant treatment of colon cancer and enhance the effect of other chemotherapeutic agents.
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MATERIALS AND METHODS |
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Cell Culture.
The human colon cancer cell line KM20 (derived from a Dukes’ D colon cancer) was obtained from Dr. Isaiah Fidler (M. D. Anderson Cancer Center, Houston, TX; Ref. 26
), and human colon cancer cell line HCT116 was obtained from American Type Culture Collection (Manassas, VA). KM20 cells were grown in minimum Eagle’s medium supplemented with 1% sodium pyruvate and 1% nonessential amino acids, and HCT116 cells were maintained in McCoy’s 5a medium; both were supplemented with 10% fetal bovine serum and cultured at 37°C under an atmosphere containing 5% CO2.
Proliferation Assay.
Cells were plated at a density of 2 x 105 cells/well in 12-well plastic plates in a volume of 2 ml/well. After 12 h, cells were pretreated with wortmannin or LY294002 PI3k inhibitor for 4 h, followed by the inhibitor alone or combined with NaBT. After 24 h of incubation, adherent cells were detached by rapid trypsinization and counted in a Hausser chamber.
DNA Fragmentation Assay.
Cells were plated in 96-well plates 24 h before treatment. After treatment, DNA fragmentation was evaluated by examination of cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) using a Cell Death Detection ELISAPlus kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions.
Protein Preparation and Western Blot Analysis.
Western immunoblot analyses were performed as described previously (27)
. Cells were lysed with TNN buffer [50 mM Tris-HCI (pH 7.5), 150 mM NaCl, 0.5 mM NP40, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 25 µg/ml each of aprotinin, leupeptin, and pepstatin A] at 4°C for 30 min. Lysates were clarified by centrifugation (10,000 x g for 30 min at 4°C), and protein concentrations were determined using the Bradford method (28)
. Briefly, total protein (100 µg) was resolved on a 10% polyacrylamide gel and transferred to Immobilon-P nylon membranes as described previously (29)
. Filters were incubated overnight at 4°C in a blocking solution (Tris-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20), followed by a 1-h incubation with primary antibodies. Filters were washed three times in a blocking solution and incubated with horseradish peroxidase-conjugated second antibodies for 1 h. After three additional washes, the immune complexes were visualized by the enhanced chemiluminescence detection system.
In Vivo Experiments.
For in vivo studies, 7-week-old male BALB/c athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN) were used. The mice were housed in an environment with controlled temperature (22°C), humidity, and a 12-h light/dark cycle. The mice were fed standard chow (Formula Chow 5008; Purina Mills, St. Louis, MO) and tap water ad libitum. Mice received s.c. injection of 1 x107 KM20 cells in the right flanks. After tumors reached 150 mm3 (day 5), mice were randomized into four groups (n = 6–10 mice/group) to receive either vehicle (control), NaBT (2 g/kg), wortmannin (1.5 mg/kg), or NaBT plus wortmannin. NaBT was dissolved with PBS, and wortmannin was dissolved in 0.1% Tween 20 with 10% ethanol. NaBT was injected s.c., and wortmannin was administered p.o. once daily, 5 days/week, for 3 weeks. Mice were sacrificed on day 33 after tumor cell injections. Tumor growth was assessed biweekly by measuring the two greatest perpendicular tumor dimensions using vernier calipers (Mitutozo, Tokyo, Japan). Tumor volume was calculated as follows: tumor volume (mm3) = [tumor length (mm) x tumor width (mm)2]/2.
Statistical Analysis.
Results are expressed as the mean ± SE. The data in Figs. 1
, 2
, and 6
were analyzed using Kruskal-Wallis test with Bonferroni adjustment for the number of comparisons. The data in Figs. 4
and 5
were analyzed using unpaired Student’s t test. All tests were assessed at the 0.05 level of significance.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). First, we determined the effect of the differentiating agent NaBT on the induction of KM20 cell death as assessed by a sensitive DNA fragmentation assay 24 h after treatment (Fig. 1A)
. Treatment with NaBT produced a dose-dependent increase in apoptosis, demonstrating that NaBT can induce cell death in these colon cancer cells, as we (29)
and others (22
, 23)
have shown in other colon cancer cell lines.
Next, we determined whether inhibition of PI3k using wortmannin, a fungal metabolite that is a covalent inhibitor of the catalytic p110 subunit of PI3k (30)
, augments the effect of NaBT on KM20 cell death (Fig. 1B)
. Wortmannin (400 nM) alone had no effect on KM20 cell death; however, the combination of wortmannin with NaBT (5 mM) produced a dose-dependent augmentation of NaBT-mediated apoptosis. To further confirm this effect of wortmannin on KM20 cell death, we used another PI3k inhibitor, LY294002, which is structurally unrelated to wortmannin (7)
. Similar to wortmannin, the combination of LY294002 increased NaBT-mediated cell death (Fig. 1C)
. In fact, the effect of LY294002 on cell death was even more dramatic than that of wortmannin, particularly at concentrations of >5 µM.
Inhibition of PI3k enhanced NaBT-mediated DNA fragmentation in KM20 cells, suggesting an increased killing effect by the combination of these agents. To assess the effect of these treatments on KM20 cell viability, we next assayed the effect of the PI3k inhibitors on the proliferation of KM20 cells. Cells were pretreated with wortmannin or LY294002 PI3k inhibitor for 4 h, followed by the inhibitors alone or combined with NaBT for 24 h. Both PI3k inhibitors and NaBT (5 mM) individually decreased KM20 cell viability; however, the combination of wortmannin or LY294002 with NaBT significantly augmented the inhibitory effects of these agents (Fig. 1D)
. To assess whether increasing concentrations of wortmannin and LY294002 exhibit a dose-response effect in the inhibition of the downstream Akt/PKB protein, we next performed Western blots to determine expression of activated (i.e., phosphorylated) Akt. Increasing concentrations of either wortmannin or LY294002 inhibited phosphorylated Akt in a dose-dependent fashion, whereas steady-state Akt levels were not affected (Fig. 1E)
.
Taken together, these findings demonstrate that inhibition of PI3k can augment the apoptotic effect of NaBT on KM20 cell death, suggesting the PI3k pathway as a potential target for therapeutic intervention.
Involvement of Caspase Activation in KM20 Cell Death Induced by the Combination of NaBT and PI3k Inhibition.
Inhibition of PI3k augments the apoptotic effect of NaBT in the aggressive KM20 cell line. The mechanism of apoptosis is remarkably conserved across species and is mediated by sequential activation of initiator and effector caspases (31)
. To confirm that these agents are inducing apoptosis through caspase activation, we next treated KM20 cells with the pan-caspase inhibitor zVAD-fmk (32)
before treatment with the PI3k inhibitor and NaBT. As previously demonstrated, either wortmannin (400 nM) or LY294002 (20 µM) enhanced the apoptotic effect of NaBT (Fig. 2A)
. Treatment with zVAD-fmk effectively inhibited apoptosis induced by the combination of NaBT with the PI3k inhibitors, suggesting a role for caspase activation in the augmented induction of apoptosis by the PI3k inhibitors.
To confirm the inhibitory effect of wortmannin and LY294002 on the PI3k/Akt pathway, we assessed their effects on the activation of the downstream Akt/PKB protein. Western blots were performed to determine the levels of phosphorylated Akt/PKB in untreated KM20 cells or cells treated with wortmannin (400 nM) or LY294002 (20 µM) alone or combined with NaBT (Fig. 2B)
. As noted previously, levels of phosphorylated Akt are increased in untreated KM20 cells. Treatment with either wortmannin or LY294002 inhibited Akt phosphorylation. NaBT alone resulted in a slight increase in Akt phosphorylation, as has been described previously (18)
; however, both of the PI3k inhibitors inhibited Akt phosphorylation in NaBT-treated KM20 cells. The blot was stripped and reprobed for steady-state levels of Akt, demonstrating no significant change in nonphosphorylated Akt with the various treatments.
Activation of Caspase-9 and Caspase-3 and PARP Cleavage in KM20 Cells.
The initiator caspases (caspase-1, -2, -4, -5, -8, -9, and -10) have long prodomains and function in targeting and regulating apoptosis (33)
. The effector caspases (caspase-3, -6, and -7) have short prodomains and induce apoptosis by cleavage of the DNA repair enzyme PARP (33)
. To further determine the molecular mechanisms and caspases involved in the apoptotic effect of these agents on KM20 cells, we next determined activation of the initiator caspases (caspase-8 and -9) and the effector caspase (caspase-3; Fig. 3
). The combination of wortmannin (400 nM) or LY294002 (20 µM) with NaBT (5 mM) resulted in activation of caspase-9, as demonstrated by its autocleavage. In contrast to caspase-9, we detected no activation of caspase-8, as demonstrated by the absence of cleavage with any of the treatments. A similar analysis of caspase-3 demonstrated that the combination of wortmannin or LY294002 with NaBT resulted in enhanced cleavage of pro-caspase-3 as noted by the Mr 24,000 and Mr 17,000 cleavage products (i.e., active caspase-3). In addition, cleavage of PARP (Mr 81,000 cleavage product) was noted using a combination of wortmannin or LY294002 with NaBT. The blot was stripped and reprobed with human vinculin, demonstrating relatively equal protein loading (34)
. Taken together, these results demonstrate that the initiator caspase-9 and effector caspase-3 are involved in mediating the augmented induction of apoptosis by the combination of NaBT and the PI3k inhibitors.
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Next, KM20 cells were treated with either gemcitabine (80 µM) or 5-FU (32 µM) individually or in combination with wortmannin (400 nM) or LY294002 (20 µM; Fig. 4
). Gemcitabine had no effect on KM20 cell DNA fragmentation; in contrast, the combination of gemcitabine and either wortmannin or LY294002 significantly increased KM20 cell death (Fig. 4A)
. Treatment with the pan-caspase inhibitor zVAD-fmk effectively inhibited apoptosis induced by the combination of gemcitabine with the PI3k inhibitors. Similarly, the combination of either wortmannin or LY294002 with 5-FU significantly increased KM20 cell death compared with treatment with 5-FU alone (Fig. 4B)
. Treatment with zVAD-fmk effectively inhibited apoptosis. Similar to our results with NaBT, these findings demonstrate that inhibition of PI3k can potentiate the apoptotic effect of gemcitabine and 5-FU on KM20 cell death, further suggesting the PI3k pathway as a potential target for therapeutic intervention.
PI3k Inhibition Augments NaBT-induced Apoptosis in HCT116 Cells.
To determine whether the enhanced apoptotic effect noted in KM20 cells treated with the combination of PI3k inhibition and NaBT was limited to this cell line, we next treated human colon cancer HCT116 cells with either wortmannin (400 nM), LY294002 (20 µM), or NaBT (2.5 mM), individually or in combination, and DNA fragmentation was assessed (Fig. 5A)
. Similar to KM20 cells, neither wortmannin nor LY294002 alone induced apoptosis, whereas NaBT alone significantly increased HCT116 cell death; the combination of wortmannin or LY294002 with NaBT resulted in an enhanced apoptotic effect compared with treatment with NaBT alone. The inhibitory effect of wortmannin and LY294002 on the PI3k/Akt pathway was confirmed by Western blots demonstrating inhibition of phosphorylated Akt levels; in contrast, steady-state Akt levels were not affected (Fig. 5B)
. As noted in KM20 cells, NaBT treatment alone resulted in a slight increase in Akt phosphorylation (Fig. 5C)
. Therefore, these findings demonstrate that PI3k inhibition potentiates NaBT-mediated apoptosis in both KM20 and HCT116 colon cancer cells.
The Combination of Wortmannin and NaBT Effectively Inhibits the in Vivo Growth of KM20 Colon Cancer Xenografts.
To this point, our findings have shown augmentation of KM20 cell death in vitro using the combination of PI3k inhibitors with NaBT. We next determined the effect of wortmannin and NaBT individually or in combination on KM20 tumor growth in vivo. Athymic nude mice bearing KM20 xenografts were randomized into four groups: (a) control; (b) NaBT (2 g/kg); (c) wortmannin (1.5 mg/kg); and (d) NaBT plus wortmannin. Treatment was initiated when the tumors reached a mean volume of 150 mm3 (day 5); tumor volumes were determined twice a week for >4 weeks. Either NaBT or wortmannin alone produced a significant antitumor effect in KM20 cells within 8 days, and the combination of wortmannin plus NaBT showed an additional inhibitory effect compared with either agent alone after 21 days of treatment (Fig. 6A)
. In control (i.e., saline-injected) mice, the mean tumor volume increased >500% within 21 days after treatment (Fig. 6B)
. In mice treated with NaBT or wortmannin alone, mean tumor volumes increased 200 and 100%, respectively. In contrast, when NaBT and wortmannin were combined, the tumors actually regressed 20% during the treatment period. Our in vivo results demonstrate that, similar to our in vitro findings, inhibition of PI3k enhances the inhibitory effect of NaBT on the growth of the aggressive KM20 colon cancer cell line.
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DISCUSSION |
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The increased activity of PI3k and the downstream protein Akt/PKB suggests a role for this pathway in enhanced cancer cell survival (7
, 10
, 11
, 38)
. In fact, PI3k activity was increased in 86% of human colon cancers in one study (9)
. In this study, we did not identify an obvious inhibitory effect of either wortmannin or LY294002, when used as single agents, on the in vitro growth of KM20 or HCT116 human colon cancer cells. Consistent with our findings, Schultz et al. (39)
found relatively minimal effects of wortmannin alone with a panel of human colon cancer cell lines. In contrast, certain cancers appear to be sensitive to the effects of PI3k inhibitors when used as single agents. Inhibition of PI3k signaling by wortmannin decreased thyroid cancer cell viability (38)
as well as non-small cell lung cancer cell viability (40)
.
In contrast to the PI3k inhibitors alone, the combination of PI3k inhibitors with the short chain fatty acid NaBT enhanced apoptosis, as demonstrated by increased DNA fragmentation and reduced cell viability. In addition, the combination of wortmannin and NaBT increased caspase-9 and caspase-3 activities and PARP cleavage. These findings suggest that the combination treatment is more effective than treatment with either agent alone. NaBT treatment can induce differentiation and cell death through a number of mechanisms, including inhibition of HDAs (20 , 21 , 41 , 42) . As we (18) and others (43) have shown, NaBT also stimulates Akt activity; therefore, the enhanced effect of the PI3k inhibitors may be through the inhibition of NaBT-mediated Akt activity.
Similar to our findings, the inhibition of PI3k has been shown to sensitize certain cancers to the effects of other therapeutic agents. In some instances, the addition of PI3k inhibitors can significantly enhance the apoptotic effects produced by single agent treatment. For example, inhibition of PI3k enhances gemcitabine-induced apoptosis in human pancreatic cancer cells (43) . Furthermore, the PI3k inhibitors wortmannin and LY294002 sensitize resistant prostate cancers to the effect of tumor necrosis factor-related apoptosis-inducing ligand, a tumor necrosis factor family member that induces apoptosis in a number cancers, and enhance doxorubicin-induced apoptosis in certain types of sarcomas (44) . Wortmannin has been shown to be an effective radiosensitizer of some cancer cells (45, 46, 47) . In this regard, we also show that PI3k inhibition enhances the apoptotic effect of gemcitabine and 5-FU, two commonly used chemotherapeutic agents (35, 36, 37) , in the KM20 colon cancer cell line, which was resistant to the effect of these agents when used individually. Collectively, these findings suggest that the selective inhibition of PI3k may be useful in combination with chemotherapeutic agents that target other cellular pathways or radiation treatment.
Our results demonstrate inhibition of Akt/PKB phosphorylation in untreated and NaBT-treated KM20 cells using either wortmannin or LY294002. Although both agents have been shown to inhibit other enzymes in addition to PI3k, such inhibition usually occurs at much higher concentrations than those used in our experiments (48, 49, 50) . Wortmannin at concentrations ranging from 20 nM to 2 µM appears to be specific for PI3k and fails to inhibit PI4-kinase, protein kinase A, protein kinase C, and protein kinase G (48) . However, it is possible that wortmannin, at the concentrations used in this study, may inhibit DNA-dependent protein kinase, a member of the PI3k family (51) , and contribute to enhancement of NaBT-induced apoptosis. Future studies will better delineate the potential role of these kinases in the inhibitory effect exerted by PI3k inhibitors.
In contrast to its effects in vitro, wortmannin administered as a single agent in vivo significantly inhibited KM20 tumor growth. The combination of wortmannin and NaBT completely suppressed tumor growth during the treatment period. Consistent with our findings, Arbiser et al. (52) noted inhibitory effects of wortmannin in vivo. These antiproliferative effects appeared to be due, in part, to the inhibition of angiogenesis. Therefore, it is possible that PI3k inhibition has multiple cellular effects in vivo with potential multiple applications in the treatment of solid tumors. The findings in our study suggest that the inhibition of PI3k signaling may be beneficial in the treatment of colon cancer. Moreover, the apparent enhanced apoptotic effect of PI3k inhibitors in combination with NaBT may provide a novel therapeutic regimen.
In summary, we show that PI3k inhibition enhances apoptosis induced by NaBT in the colon cancer cell lines KM20 and HCT116. Furthermore, PI3k inhibition enhances the apoptotic effect of the chemotherapeutic agents gemcitabine and 5-FU in the aggressive KM20 cell line, which is resistant to either of these agents alone. Our findings suggest that targeting the PI3k/Akt pathway may have therapeutic potential for certain chemoresistant colon cancers by enhancing the apoptotic effect of the fatty acid NaBT or standard chemotherapeutic agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants RO1 DK48498, RO1 AG10885, and PO1 DK305608. M. M. K. was a recipient of a Stjepcevich Scholarship Award. 
2 To whom requests for reprints should be addressed, at Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0536. Phone: (409) 772-5612; Fax: (409) 747-4819; E-mail: mevers{at}utmb.edu
3 The abbreviations used are: PI3k, phosphatidylinositol 3'-kinase; NaBT, sodium butyrate; HDA, histone deacetylase; PARP, poly(ADP-ribose) polymerase; 5-FU, 5-fluorouracil; PKB, protein kinase B.
Received 9/19/01; revised 1/18/02; accepted 2/28/02.
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REFERENCES |
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is required for DNA synthesis induced by some, but not all, growth factors. Proc. Natl. Acad. Sci. USA, 91: 9185-9189, 1994. (p85-p110) in cell survival and for phosphatidylinositol 3-kinase ß (p85-p110ß) in de novo DNA synthesis of human colon carcinoma cells. Oncogene, 19: 5083-5090, 2000.[CrossRef][Medline]
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, P < 0.05 compared with NaBT alone. E, KM20 cells were treated with wortmannin or LY294002 as described in "Materials and Methods." Cells were lysed, and Western blots were performed using anti-phospho-Akt. The same membranes were stripped and reprobed with anti-Akt antibody.
