The Novel Akt Inhibitor API-1 Induces c-FLIP Degradation and Synergizes with TRAIL to Augment Apoptosis Independent of Akt Inhibition

API-1 (pyrido[2,3-d]pyrimidines) is a recently identified small-molecule inhibitor of Akt, which acts through binding to Akt and blocking its membrane translocation (1). A previous study has shown that API-1 possesses promising anticancer activity, evidenced by its ability to suppress cell growth, induce apoptosis, and inhibit the growth of cancer xenografts, particularly those with activated Akt, in nude mice (1).

TRAIL (also called APO-2L) is a member of the TNF family and is currently being tested in phase I oncology trials due to its unique ability to trigger apoptosis in various types of cancer cells with limited toxicity toward normal cells (2, 3). However, many primary tumors are inherently resistant to TRAIL-mediated apoptosis and require additional sensitization (4, 5).

TRAIL initiates apoptosis by binding to cell surface death receptor 4 (DR4) or 5 (DR5); this induces oligomerization of the death receptors and formation of the death-inducing signaling complex (DISC), involving recruitment of the adaptor molecule FADD and subsequent caspase-8. DISC assembly promotes the autocleavage and activation of caspase-8, leading to further activation of the effector caspases (e.g., caspase-3) that eventually drive apoptotic death (6). Cellular FLICE-inhibitory protein (c-FLIP) is a truncated form of caspase-8 that lacks enzymatic activity. It can also be recruited to DISC but suppresses apoptosis by blocking the activation of caspase-8 through competing with caspase-8 for binding to FADD (7). It has been well documented that elevated c-FLIP expression protects cells from death receptor–mediated apoptosis, whereas downregulation of c-FLIP by chemicals or siRNA sensitizes cells to death receptor–mediated apoptosis (7, 8). Therefore, c-FLIP acts as a key inhibitor of TRAIL/death receptor–induced apoptosis. c-FLIP has multiple splice variants, however, only 2 of them have been well characterized at the protein levels: the 26 kDa short form (c-FLIP_S) containing 2 death effector domains and the 55 kDa long form (c-FLIP_L) containing an inactive caspase-like domain in addition to the 2 death effector domains (7, 9). The levels of c-FLIP, including both FLIP_L and FLIP_S are regulated by ubiquitin/proteasome-mediated degradation (10–12).

Although cancer cells possess intrinsic resistance to TRAIL, many anticancer agents can sensitize cancer cells to TRAIL-induced apoptosis through various mechanisms such as induction of DR5 and/or DR4 expression and/or downregulation of c-FLIP levels (13, 14). Akt has been suggested to positively regulate c-FLIP expression because activation or suppression of Akt accordingly increased or decreased the levels of c-FLIP (15). Recently, Akt1 was shown to directly interact with FLIP_L and to phosphorylate it at S273, leading to stabilization of FLIP_L (16). Thus, the current study primarily focused on determining whether API-1 negatively regulates c-FLIP levels and sensitizes cancer cells to TRAIL-induced apoptosis. Moreover, we have revealed the mechanisms by which API-1 reduces c-FLIP levels and enhances TRAIL-induced apoptosis.

API-1 (NSC177233) was obtained from the National Cancer Institute (Bethesda, MD). API-2 (17) was provided by Dr. J.Q. Cheng (H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL). MK2206 was purchased from Active Biochem. The soluble recombinant human TRAIL was purchased from PeproTech, Inc. The proteasome inhibitor MG132 and the protein synthesis inhibitor cycloheximide were purchased from Sigma Chemical Corporation. Monoclonal anti-FLIP antibody (NF6) was obtained from Alexis Biochemicals. Mouse monoclonal anti-caspase-8 and rabbit polyclonal anti-caspase-9, anti-PARP, and anti-Akt antibodies were purchased from Cell Signaling Technology, Inc. Mouse monoclonal anti-caspase-3 antibody was purchased from Imgenex. Rabbit polyclonal anti-DR5 antibody was obtained from ProSci Inc. Mouse monoclonal anti-DR4 antibody (B-N28) was purchased from Diaclone. Rabbit monoclonal anti-p-Akt (S473) antibody was purchased from Epitomics, Inc. Both polyclonal and monoclonal anti-actin antibodies were purchased from Sigma Chemical Corporation.

Human non–small cell lung carcinoma (NSCLC) cell lines (H157, Calu-1, and H1299) and head and neck squamous cell carcinoma (HNSCC) cell lines (22A, Tr146, and SqCC/Y1) were described in our previous work (18). H157 cells were recently authenticated by Genetica DNA Laboratories, Inc. by analyzing short tandem repeat DNA profile. The other cell lines have not been authenticated. The H157-LacZ-5, H157-FLIP_L-21, and H157-FLIP_S-1 stable transfectants were established as described previously (19, 20). The 22A cells (pool) stably expressing LacZ, FLIP_L, and FLIP_S were described previously (21). These cell lines were cultured in PMRI-1640 or Dulbecco's Modified Eagle's Medium (DMEM)/Ham's F-12 medium containing 5% FBS at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

Cells were seeded in 96-well cell culture plates and treated the next day with the given agents. The viable cell number was determined by sulforhodamine B (SRB) assay as described previously (22). Combination index (CI) for drug interaction (e.g., synergy) was calculated with the CompuSyn software (ComboSyn, Inc.). Apoptosis was evaluated with the Annexin V-PE Apoptosis Detection Kit purchased from BD Biosciences. We also detected caspase and PARP cleavage by Western blot analysis as described later, as additional indicators of apoptosis.

The procedures for preparation of whole-cell protein lysates and for Western blotting were the same as described before (23, 24). The quantification of Western blotting results was done with NIH ImageJ software.

H157-FLIP_L-21 cells were transfected with hemagglutinin (HA)-ubiquitin plasmid with Lipofectamine 2000 transfection reagent (Invitrogen) based on the manufacturer's instructions. After 24 hours, the cells were treated with API-1 or API-1 plus MG132 for 3 hours. Cells were collected and lysed for immunoprecipitation of Flag-FLIP_L using Flag M2 monoclonal antibody (Sigma) as previously described (19, 25), followed by detection of ubiquitinated FLIP_L with Western blot analysis using anti-HA antibody (Abgent).

We first evaluated the single-agent activity of API-1 on the growth of a panel of NSCLC and HNSCC cell lines. A 3-day exposure to API-1 effectively inhibited the growth of 5 (H1299, H157, SqCC/Y1, 22A, and Tr146) of 6 tested cancer cell lines (Fig. 1A). The effective concentrations that decreased cell numbers by 50% (IC₅₀s) ranged between 2 and 5 μmol/L for these sensitive cell lines (Fig. 1A). Calu-1 was relatively insensitive to API-1 with an IC₅₀ value greater than 10 μmol/L.

We then determined whether API-1 induces apoptosis in these cell lines. Treatment of the representative H1299, Calu-1, and SqCC/Y1 cell lines with different concentrations of API-1 for 24 hours dose dependently increased Annexin V–positive (or apoptotic) cells in H1299 and SqCC/Y1 cells (>40% at 10 μmol/L) but did so only minimally in Calu-1 cells (<15% at 10 μmol/L; Fig. 1B). In agreement, we detected dose-dependent increase in cleavage of caspase-8, caspase-9, caspase-3, and PARP in H1299 and SqCC/Y1 cells, but this was not apparent in Calu-1 cells (Fig. 1C). These results clearly indicate that API-1 effectively induces apoptosis in API-1–sensitive NSCLC and HNSCC cell lines.

Because API-1 effectively activates caspase-8, we then asked whether API-1 modulates the levels of key proteins (e.g., c-FLIP, DR4, and DR5) involved in the death receptor–mediated apoptotic pathway. As presented in Fig. 2A, API-1 at 5 μmol/L reduced the levels of c-FLIP in H157, H1299, SqCC/Y1, and Tr146 cells but not in Calu-1 cells. c-FLIP levels in 22A cells were too low to be detected. API-1 did not increase the expression of either DR5 or DR4 in any of the cell lines. Rather, API-1 reduced the levels of DR4 in some cell lines (H1299, SqCC/Y1, and Tr146). Moreover, we conducted detailed dose-course and time-course studies of the effects of API-1 on the levels of c-FLIP, DR4, and DR5 in H157 cells. We found that API-1 could reduce c-FLIP levels even at 1.25 μmol/L (Fig. 2B). The apparent reduction of c-FLIP in cells occurred after 4-hour exposure to API-1 (Fig. 2C). Under these conditions, we did not see that API-1 increased the expression of either DR4 or DR5 (Fig. 2B and C). Thus, API-1 downregulates c-FLIP levels without induction of DR4 and DR5 expression in NSCLC and HNSCC cells.

In addition, we compared the effects of API-1 on Akt phosphorylation among the 6 NSCLC and HNSCC cell lines. As presented in Fig. 2A, API-1 inhibited the phosphorylation of Akt in all of the cell lines, albeit with varied potencies. Thus, it is clear that API-1 inhibits Akt phosphorylation.

Given that API-1 reduces c-FLIP levels, we determined whether API-1 enhances TRAIL-induced apoptosis. As presented in Fig. 3A, the combination of API-1 at 5 or 2.5 μmol/L and TRAIL (25–100 ng/mL) was much more effective than either agent alone in decreasing the survival of the tested NSCLC and HNSCC cell lines, except for Calu-1 cells. The CIs for these combinations were less than 1 (Supplementary Fig. S1), indicating that the combination of API-1 and TRAIL synergistically decreased the survival of these cancer cells. In agreement, the combination of API-1 (e.g., 5 μmol/L) and TRAIL (e.g., 20 ng/mL) was also much more potent than either agent alone in increasing cleavage of caspase-8, caspase-9, caspase-3, and PARP in Western blot analysis (Fig. 3B) and in increasing the proportion of Annexin V–positive cells as detected by the Annexin V assay (Fig. 3C) in 2 representative cell lines, H1229 and 22A. For example, we detected approximately 5% and 12% of apoptotic cells in cells treated with TRAIL and API-1, respectively, but more than 50% of apoptotic cells in H1299 cells exposed to the combination of API-1 and TRAIL (Fig. 3C), which is clearly greater than the sum of apoptosis induced by both single agents, further indicating that the combination of API-1 and TRAIL exerts more than additive (i.e., synergistic) apoptosis-inducing activity. Taken together, it is clear that the API-1 synergizes with TRAIL to induce apoptosis in NSCLC and HNSCC cells.

To show whether c-FLIP downregulation contributes to enhancement of TRAIL-induced apoptosis by API-1, we compared the effect of API-1 plus TRAIL on cell survival and apoptosis induction among 22A stable transfectants that express LacZ (control), FLIP_L, and FLIP_S. As shown earlier, the combination of API-1 and TRAIL was more effective than either agent alone in decreasing the survival of 22A-LacZ cells but not of 22A-FLIP_L or 22A-FLIP_S cells (Fig. 4A). In agreement, the combination of API-1 and TRAIL was much more effective in increasing Annexin V–positive (apoptotic) cells and inducing the cleavage of caspase-8, caspase-9, caspase-3, and PARP in 22A-LacZ cells than in 22A-FLIP_S and 22A-FLIP_L cells (Fig. 4B and C). Similar results were also generated from H157 cells that stably express LacZ or FLIP_L (Supplementary Fig. S2). Enforced expression of FLIP_L attenuated the ability of API-1 to augment the effect of TRAIL on decreasing cell survival (Supplementary Fig. S2B), on increasing apoptotic populations (Supplementary Fig. S2C), and on inducing cleavage of caspase-8, caspase-9, caspase-3, and PARP (Supplementary Fig. S2D). These data taken together indicate that overexpression of c-FLIP protects cells from apoptosis induced by the API-1 and TRAIL combination, implying that c-FLIP downregulation contributes to enhancement of TRAIL-induced apoptosis by API-1.

We also determined whether overexpression of c-FLIP confers resistance to API-1 alone and found that enforced expression of FLIP_L or FLIP_S did not affect the ability of API-1 to decrease cell survival in both H157 and 22A cells (data not shown). This suggests that c-FLIP downregulation may not be sufficient for API-1 to trigger apoptosis.

To elucidate the mechanism by which API-1 reduces c-FLIP levels, we first tested whether proteasomal degradation is involved in this process, as c-FLIP is known to be regulated by an ubiquitin/proteasome-dependent mechanism. Thus, we treated H157 cells with API-1 in the absence and presence of the proteasome inhibitor MG132 and then detected c-FLIP with Western blot analysis. In the absence of MG132, API-1 decreased c-FLIP levels; however, the presence of MG132 increased basal levels of c-FLIP, particularly FLIP_S, and prevented c-FLIP from reduction by API-1 (Fig. 5A). These data suggest that API-1 induces c-FLIP reduction through a proteasome-dependent mechanism. We next determined whether API-1 increases c-FLIP degradation by measuring its stability. To this end, cycloheximide was added to H157 cells 5 hours after dimethyl sulfoxide (DMSO) or API-1 treatment. The cells were then harvested at the indicated times after cycloheximide for analysis of the c-FLIP degradation rate. These data shown in Fig. 5B and C revealed that the half-lives of FLIP_S and FLIP_L in DMSO-treated samples were about 34 and 120 minutes, respectively; on the contrary, in API-1–treated samples, their half-lives were reduced to approximately 24 and 69 minutes, respectively. Therefore, it is apparent that API-1 reduces c-FLIP protein stability. Furthermore, we determined whether API-1 increases c-FLIP ubiquitination. As presented in Fig. 5D, the highest level of ubiquitinated FLIP_L was detected in H157-FLIP_L-21 cells (which stably express ectopic flag-FLIP_L) treated with API-1 plus MG132 compared with API-1 alone or MG132 alone, indicating that API-1 increases c-FLIP ubiquitination. Collectively, we conclude that API-1 facilitates ubiquitin/proteasome-mediated c-FLIP degradation, leading to downregulation of c-FLIP.

Because API-1 is an Akt inhibitor, we wanted to know whether the effects of API-1 on reducing c-FLIP levels and enhancing TRAIL-induced apoptosis are consequences of Akt inhibition. To this end, we tested whether 2 other allosteric Akt inhibitors, MK2206 (26) and API-2 (17), can also reduce c-FLIP levels and enhance TRAIL-mediated cell killing. Both MK2206 and API-2 at concentrations ranging from 1 to 5 μmol/L (MK2206) or 2.5 to 10 μmol/L (API-2) effectively inhibited Akt phosphorylation in H1299 and H157 cells but did not reduce c-FLIP levels in these cell lines (Fig. 6A and B). MK2006 did not increase the expression of DR5 or DR4 either (Fig. 6A). API-2 did not increase DR5 expression but elevated DR4 levels (Fig. 6B). When combined with TRAIL, both MK2206 and API-2 exhibited only minimal increase in cell killing (i.e., decreasing cell survival) in comparison with cell-killing effects by either agent alone (Fig. 6C and D). Thus, it is clear that other Akt inhibitors do not function in the same manner as API-1 in downregulating c-FLIP and in enhancing TRAIL-inducing cell killing.

In this study, we have shown that API-1 effectively inhibits the growth of most NSCLC and HNSCC cell lines tested, with IC₅₀ values ranging from 1 to 5 μmol/L. Moreover, API-1 effectively induces apoptosis in some NSCLC and HNSCC cell lines (Fig. 1). Thus, API-1 possesses promising single-agent activity against NSCLC and HNSCC cells. When combined with TRAIL, synergistic induction of apoptosis, including decreased cell survival, induction of caspase cleavage, and increased Annexin V–positive cells, occurred in most of the tested cell lines (Fig. 3). To the best of knowledge, this is the first report of the synergistic induction of apoptosis by the combination of API-1 and TRAIL in cancer cells. Given that TRAIL is being tested as a cancer therapeutic agent in clinical trials (6, 27), the further study of the potential application of the API-1 and TRAIL combination in cancer therapy (e.g., NSCLC and HNSCC) is warranted. Recently, targeting the Akt protein kinase or the TRAIL-mediated apoptotic pathway has been emerged as attractive strategies for cancer chemoprevention (13, 28–30). Indeed, a phase 0 chemoprevention trial on an orally active Akt inhibitor has been successfully conducted recently (31). Thus, the potential of the API-1 alone or in combination with TRAIL in cancer chemoprevention needs investigation as well.

We noted that, among the tested cancer cell lines, Calu-1 was the only cell line that exhibited resistance to API-1 alone or the combination of API-1 and TRAIL (Figs. 1 and 3). Thus understanding of the mechanisms by which API-1 induces apoptosis, including modulation of TRAIL-induced apoptosis, will be very helpful for guiding effective application of API-1 in future treatment of cancer in the clinic.

It is well known that cells can die of apoptosis primarily through the extrinsic death receptor–induced pathway and/or the intrinsic mitochondria-mediated pathway. Cross-talk between these 2 pathways is mediated by the truncated proapoptotic protein Bid (32). The activation of caspase-8 is the key step in the death receptor–mediated apoptosis, whereas caspase-9 activation is the key even in the mitochondria-mediated apoptotic pathway. Activated caspase-8 can also induce caspase-9 activation through Bid-mediated activation of the mitochondria-mediated apoptotic pathway (32). In this study, we found that API-1 activated both caspase-8 and caspase-9 (Fig. 1C), suggesting that API-1 either activates the death receptor–mediated apoptotic pathway or both the death receptor- and mitochondria-mediated apoptotic pathways, leading to induction of apoptosis.

DR4, DR5, and c-FLIP are key components in the regulation of TRAIL-induced apoptosis (6, 33). Modulation of the levels of these proteins in general (e.g., upregulation of DR4 and/or DR5 and/or downregulation of c-FLIP) results in sensitization of cancer cells to TRAIL-induced apoptosis (13, 14). We found that API-1 reduced c-FLIP levels without increasing DR4 or DR5 expression in the sensitive cancer cell lines (Fig. 2). Interestingly, Calu-1 cells, which are relatively resistant to API-1 or API-1 plus TRAIL, expressed the highest basal levels of c-FLIP, which was not reduced by API-1 (Fig. 2A). These results suggest that c-FLIP downregulation may play a critical role in mediating apoptosis induced by API-1 or by the combination of API-1 and TRAIL. Enforced expression of ectopic FLIP_L or FLIP_S did not confer resistance to API-1 alone, but indeed attenuated or abolished the effect of API-1 on enhancing TRAIL-induced apoptosis in both 22A and H157 cells (Fig. 4 and Supplementary Fig. S2). Therefore, c-FLIP downregulation may not be sufficient for API-1 to initiate apoptosis, suggesting that other mechanisms are needed for API-1–induced apoptosis. However, it is clear that c-FLIP downregulation apparently plays a critical role in mediating synergistic induction of apoptosis by API-1 and TRAIL.

It is known that enhancement of TRAIL-induced apoptosis can be achieved through other mechanisms (e.g., inhibition of Bcl-2 family members) beyond downregulation of c-FLIP (34). Here, we claim a critical role of c-FLIP downregulation in mediating enhancement of TRAIL-induced apoptosis by API-1 but does not exclude other potential mechanisms. We noted that c-FLIP protein was not detected in 22A cells (Fig. 2) and API-1 clearly enhanced TRAIL-induced apoptosis in this cell line (Fig. 3). It is possible that the downregulation of c-FLIP by API-1 in this cell line was not detected because of the sensitivity limitation of the assay. Of course, whether other mechanisms play a more important role than downregulation of c-FLIP in mediating enhancement of TRAIL-induced apoptosis by API-1 in this cell line cannot be ruled out and needs further investigation.

It is known that c-FLIP, including FLIP_L and FLIP_S, are rapidly turnover proteins subjected to regulation through ubiquitin/proteasome-mediated protein degradation (10–12). Some small molecules negatively regulate c-FLIP levels through this mechanism (19, 35, 36). In this study, we found that API-1 failed to decrease c-FLIP levels in the presence of a proteasome inhibitor, increased c-FLIP ubiquitination, and reduced the stability of c-FLIP protein (Fig. 5). Thus, we conclude that API-1 reduces c-FLIP levels by facilitating its degradation through the ubiquitin/proteasome-dependent pathway. In the current study, we cannot rule out additional mechanisms accounting for c-FLIP downregulation induced by API-1 such as transcriptional regulation even though they are unlikely to be the primary mechanisms.

It has been suggested that Akt positively regulates c-FLIP expression (15). Recently, Akt1 was shown to directly interact with FLIP_L and to phosphorylate it at S273, leading to stabilization of FLIP_L (16). Given that API-1 is an Akt inhibitor, it is reasonable to speculate that API-1 may downregulate c-FLIP due to its Akt-inhibitory activity. To explore this, we tested the effects of 2 additional Akt inhibitors, MK2206 and API-2, on modulation of c-FLIP levels and TRAIL-induced apoptosis. Unfortunately, both MK2206 and API-2 failed to reduce c-FLIP levels or to detectably enhance TRAIL-induced cell killing although they effectively reduced p-Akt levels (Fig. 6), suggesting that inhibition of Akt does not necessarily result in c-FLIP downregulation and enhancement of TRAIL-induced apoptosis. Accordingly, we suggest that the effects of API-1 on downregulation of c-FLIP and enhancement of TRAIL-induced apoptosis are unlikely secondary to Akt inhibition. Furthermore, we noted that API-1 downregulation of c-FLIP is not associated with its activity against Akt. In Calu-1 cells, API-1 did not reduce c-FLIP levels but inhibited Akt phosphorylation (Fig. 2A). These data further support the notion that API-1 downregulates c-FLIP independent of Akt inhibition.

In summary, the current study has revealed a novel function of API-1 that induces c-FLIP degradation and synergizes with TRAIL to induce apoptosis of cancer cells. Moreover, our results warrant further evaluation of the potential of API-1 and TRAIL combination against cancer in the clinic.

No potential conflicts of interest were disclosed.

Conception and design: F.R. Khuri, S.-Y. Sun

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): B. Li, H. Ren

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B. Li, H. Ren, F.R. Khuri, S.-Y. Sun

Writing, review, and/or revision of the manuscript: F.R. Khuri, S.-Y. Sun

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P. Yue

Study supervision: F.R. Khuri, S.-Y. Sun

The authors thank Dr. J.Q. Cheng for generously providing some reagents and Dr. A. Hammond for editing the manuscript.

The study was supported by Georgia Cancer Coalition Distinguished Cancer Scholar award (to S.-Y. Sun) and National Cancer Institute NIH SPORE P50 grant CA128613 (project 2 to S.-Y. Sun and F.R. Khuri).

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.