Proteasome Inhibitor PS-341 (Bortezomib) Induces Calpain-dependent IκBα Degradation*

The proteasome, a key component of the ubiquitin-proteasome pathway, has emerged as an important cancer therapeutic target. PS-341 (also called Bortezomib or Velcade) is the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma and is currently being tested in many clinical trials against other types of cancers. One proposed mechanism by which PS-341 exerts its anticancer effect is inactivation of nuclear factor-κB (NF-κB) through prevention of IκBα degradation. In this study, we show that PS-341 at concentrations that effectively inhibited the growth of human cancer cells, instead of increasing IκBα stability, paradoxically induced IκBα degradation. As a result, PS-341 facilitated p65 nuclear translocation and increased NF-κB activity. Moreover, IκBα degradation by PS-341 occurred early before induction of apoptosis and could not be inhibited by a pan-caspase inhibitor or caspase-8 silencing; however, it could be prevented with calpain inhibitors, calcium-chelating agents, calpain knockdown, or calpastatin overexpression. In agreement, PS-341 increased calpain activity. These data together indicate that PS-341 induces a calpain-mediated IκBα degradation independent of caspases. In the presence of a calpain inhibitor, the apoptosis-inducing activity of PS-341 was dramatically enhanced. Collectively, these unexpected findings suggest not only a novel paradigm regarding the relationship between proteasome inhibition and NF-κB activity but also a strategy to enhance the anticancer efficacy of PS-341.

cell lymphoma. Many preclinical studies have demonstrated that PS-341 alone or in combination with other cancer therapeutic agents induces apoptosis in a variety of human cancer cells in vitro, including both hematologic and solid tumor malignancies, and inhibits the growth of tumor xenografts in vivo. Currently, there are many ongoing clinical trials that test the anticancer efficacy of PS-341 or its combinations with other agents in several types of cancers, including lung cancer (2,6,7).
Nuclear factor-B (NF-B) 5 is a transcription factor that regulates the expression of a large number of genes involved in multiple biological processes, including apoptosis (8,9). Its activation is often associated with cell resistance to apoptosis via up-regulation of the expression of multiple anti-apoptotic genes such as Bcl-2 and c-FLIP (9,10). Therefore, NF-B has long been an attractive target for cancer therapy. It is well known that IB␣ ubiquitination and its subsequent degradation by the 26 S proteasome leading to nuclear translocation of p65 and an increase in NF-B DNA binding are the primary mechanisms of NF-B activation in cells upon stimulation (9,10). Thus, a putative mechanism by which PS-341 induces apoptosis and exerts its anticancer activity is to inhibit NF-B activation via prevention of IB␣ degradation (1,5).
PS-341 either as a single drug or in combination with other agents has been demonstrated to be active in inducing growth arrest and apoptosis in lung cancer cells in vitro and in inhibiting the growth of lung tumors in animal models (6,7). To date, PS-341 is the only proteasome inhibitor to be evaluated in clinical trials against lung cancer and to show activity when used both as a single agent and in combination regimens (7). However, the mechanisms by which PS-341 induces apoptosis or growth arrest in lung cancer, particularly the role of NF-B in these events, remain unclear. Therefore, we have been interested in studying how PS-341 induces apoptosis in human cancer cells, particularly lung cancer cells. This study particularly focused on the role of NF-B inhibition in PS-341-induced apoptosis in human lung cancer cells. Unexpectedly, we found that PS-341, instead of preventing IB␣ degradation, induces calpain-dependent IB␣ degradation in human lung cancer cells and other types of cancer cells. Moreover, we demonstrate that PS-341-induced IB␣ degradation results in NF-B activation and impacts cell responses to PS-341-induced apoptosis. Thus, this study sug-gests a novel mechanism underlying PS-341-mediated biological functions and a novel strategy to enhance the anticancer efficacy of PS-341.
Cell Lines and Cell Culture-All cell lines used in this study were originally purchased from the American Type Culture Collection (ATCC; Manassas, VA) and kept in our laboratory. They were grown in monolayer culture in RPMI 1640 medium supplemented with 5% fetal bovine serum at 37°C in a humidified atmosphere consisting of 5% CO 2 and 95% air.
Gene Silencing with Small Interfering RNA-The nonsilencing control and caspase-8 small interfering RNA duplexes were described previously (12). Calpain regulatory subunit (SC-29887) and calpastatin (SC-29889) siRNAs were purchased from Santa Cruz Biotechnology. Transfection of these siRNA duplexes was conducted in 6-mm dishes using the HiPerFect transfection reagent (Qiagen, Valencia, CA) following the manufacturer's fast forward protocol. Briefly, 1 ϫ 10 6 cells were seeded into a 60-mm culture dish 1 h prior to transfection. The given siRNA at 20 nM was mixed with 40 l of HiPerFect transfection reagent, diluted with Opti-MEM (Invitrogen) medium, and then added to the cells. After 38 h post-transfection, the cells were treated with 50 nM PS-341 for an additional 10 h before collecting the cells for Western blotting. Gene silencing effects and other protein expression were evaluated by Western blot analysis as described above.
Cell Survival Assay-Cells were seeded in 96-well cell culture plates and treated the next day with the agents indicated. The viable cell number was determined using the sulforhodamine B assay, as described previously (13).
Detection of Apoptosis-Apoptosis was evaluated by annexin V staining using annexin V-PE apoptosis detection kit purchased from BD Biosciences following the manufacturer's instructions.
Proteasome Activity Assay-Fluorometric measurement of 20 S proteasome activity was the same as we described previously (14).
Calpain Activity Assay-Intracellular calpain activity was measured as described previously (15,16). Cells in a 96-well plate or in chamber slides were exposed to PS-341 for a given time and then loaded with 10 M t-butoxycarbonyl-Leu-Metchloromethylcoumarin. After a 30 -60-min incubation, cellular fluorescence was then quantified with a SPECTRAmax dualscanning microplate spectrofluorometer (Molecular Devices Co., Sunnyvale, CA) with 351-nm excitation and 430-nm emission filters or recorded under a fluorescent microscope.
Transient Transfection and Reporter Activity Assay-The pNF-B-luc reporter plasmid, which contains the luciferase reporter gene driven by a basic promoter element (TATA box) joined to seven repeats of an NF-B-response element, was purchased from Stratagene (La Jolla, CA). A pCH110 plasmid encoding ␤-galactosidase was purchased from GE Healthcare and used for normalization. The procedures for transfection, luciferase activity assay, and ␤-galactosidase assay were described previously (18). Adenoviral Infection-Adenovirus harboring an empty vector (Ad-CMV) or a super repressor of IB␣ (Ad-SRIB␣) was generously provided by Dr. L. W. Chung (Emory University, Atlanta, GA). The amplification, purification, and infection of adenovirus were described previously (17).

PS-341 Reduces IB␣ Levels by Induction of Its Proteolysis
while Inhibiting Proteasome Activity-IB␣ is known to be degraded via a ubiquitin-proteasome mechanism (9). PS-341 as a proteasome inhibitor theoretically should stabilize IB␣ protein through inhibition of proteasome activity. When examining the effects of PS-341 on IB␣ degradation in two human lung cancer cell lines, we surprisingly found that PS-341 decreased IB␣ levels while increasing p-IB␣ levels in a timedependent fashion. In H157 cells, PS-341 substantially increased p-IB␣ levels at 1 h followed by a great decrease in IB␣ levels after 2 h. In H460 cells, a drastic p-IB␣ increase was accompanied by a substantial reduction of IB␣ levels after a 2-h treatment. In both cell lines, the IB␣ reduction was sustained up to 12 h (Fig. 1A). By measuring the 20 S proteasome activity, we found that PS-341, under the same experimental conditions, effectively inhibited the 20 S proteasome activity starting from 0.5 h post-treatment in both tested cell lines (Fig.  1B). Moreover, we examined the effects of different concentrations of PS-341 on modulation of IB␣ levels. As presented in In the other two lung cancer cell lines, PS-341 also effectively reduced the levels of IB␣ (Fig. 1D). Moreover, we examined the effects of PS-341 on IB␣ degradation in other types of solid tumor cell lines, including prostate (DU145), cervical (HeLa), breast (MCF-7), and head and neck cancer (SqCC/Y1), to determine whether IB␣ reduction by PS-341 is a common phenomenon. In agreement with the findings in lung cancer cell lines, PS-341 at both low (50 nM) and high (1000 nM) concentrations decreased IB␣ levels while increasing p-IB␣ levels in all of the tested cell lines ( Fig. 2A). Similar results were also obtained in several myeloma (U226, B226, and RPM/8226) cell lines (Fig. 2B).
Furthermore, we determined whether other proteasome inhibitors exerted similar effects as PS-341 in modulation of IB␣ levels. We used three proteasome inhibitors MG132, epoxomycin, and ALLN in this experiment and found that all these agents increased p-IB␣ levels and decreased IB␣ levels in both H157 and H460 cells (Fig. 2C). Thus, it appears that reduction of IB␣ levels is a common phenomenon for proteasome inhibitors.
We noted that a short form of a protein band could be detected in the Western blot analysis for IB␣ from cells exposed to either PS-341 ( Fig. 1C and Fig. 2, A and B) or other proteasome inhibitors (Fig. 2C). This short form of protein appeared or was increased as the IB␣ levels were decreased. Given that IB␣ can be cleaved by a calpain-dependent mechanism (19,20), it is very likely that this short form of protein is a cleaved form of IB␣. Thus, it appears that inhibition of proteasome with either PS-341 or other inhibitors reduced IB␣ through promotion of its cleavage.
We also examined the effects of cisplatin and docetaxel, two cancer chemotherapeutic agents, on IB␣ levels in our cell systems in comparison with PS-341. Under the tested conditions, we found that only PS-341, but not cisplatin and docetaxel, increased p-IB␣ levels and decreased IB␣ levels with increased levels of cleaved fragment (supplemental Fig. S1), suggesting that IB␣ degradation is unique to PS-341 treatment.
PS-341 Induces Calpain-dependent IB␣ Proteolysis Independent of Caspases-IB␣ has been shown to be degraded through proteasome-independent mechanisms (e.g. caspase or calpain protease) (19 -22). In our study, we noted that cleavage of PARP, a hallmark of apoptosis, was detected only at 8 h after PS-341 treatment, which was far later than IB␣ reduction (after 1 h) (Fig. 1A). Moreover, strong cleavage of IB␣ was detected in DU145 and MCF-7 cells, in which PARP cleavage was minimally detected or not detected, whereas relatively weak IB␣ cleavage was detected in 293FT, HeLa, and SqCC/Y1 cells in which PARP was strongly cleaved (Fig. 2A). These results together suggest that the observed IB␣ cleavage is unlikely due to caspase activation. Indeed, in the presence of the pan-caspase inhibitor, Z-VAD-FMK, which at the tested concentration abolished PS-341-induced caspase-3 activation (supplemental Fig. S2), PS-341 still effectively induced IB␣ reduction in both H157 and H460 cells (Fig. 3A). A previous study suggested that caspase-8 is involved in IB␣ cleavage (23). Thus, we also specifically addressed whether caspase-8 is involved in mediating PS-341-induced IB␣ degradation through inactivating caspase-8 by knocking down caspase-8 expression. As presented in Fig. 3A (lower panel), knockdown of caspase-8 failed to prevent PS-341-induced IB␣ reduction, indicating that caspase 8 is not involved in IB␣ degradation induced by PS-341. Collectively, these data strongly suggest that PS-341 induces a caspase-independent IB␣ proteolysis.
In addition, we determined whether the trypsin-like protease and cysteine protease inhibitor, leupeptin, could prevent PS-341-induced IB␣ degradation. As presented in Fig. 3A, PS-341-induced IB␣ reduction could not be prevented by leu- peptin in either H157 or H460 cells, indicating that PS-341 decreases IB␣ levels independent of leupeptin-sensitive proteases as well.
Finally, we investigated whether PS-341 induces IB␣ degradation through a calpain-mediated mechanism. To this end, we first examined whether the presence of calpain inhibitors abolished the ability of PS-341 to induce IB␣ degradation. As presented in Fig. 3B, the presence of either EST or PD150506 prevented IB␣ from reduction induced by PS-341 in both H157 and H460 cells. In agreement, another calpain inhibitor calpeptin inhibited PS-341-induced IB␣ reduction in H460 cells as well. Furthermore, we silenced the expression of the calpain regulatory subunit, which is a common regulatory subunit for both -calpain (calpain 1) and m-calpain (calpain 2) (24), and we examined its effect on PS-341-induced IB␣ degradation. We assumed that knockdown of the calpain regulatory subunit would inhibit both -calpain and m-calpain activity. In agreement with the effects of the calpain inhibitors, as discussed above, knockdown of calpain regulatory subunit abolished the ability of PS-341 to reduce IB␣ levels (Fig. 3C). These results clearly indicate that PS-341 induces calpain-dependent IB␣ proteolysis.
It is known that calpains are calcium-dependent cysteine proteases (24). Thus, we further looked at the impact of calcium depletion on PS-341-induced IB␣ degradation. The presence of the calcium-chelating agent BAPTA or EGTA alone in part prevented IB␣ reduction by PS-341; however, the co-presence of both EGTA and BAPTA abolished the ability of PS-341 to decrease IB␣ levels (Fig. 3D). These results again support the critical role of calpains in mediating PS-341-induced IB␣ degradation.
Moreover, we determined whether PS-341 indeed increases calpain activity. As presented in Fig. 4, A  and B, PS-341 significantly increased the proteolytic hydrolysis of the calpain-specific fluorogenic substrate, t-butoxycarbonyl-Leu-Met-chloromethylcoumarin, exhibiting increased fluorescence. These results indicate that PS-341 increases intracellular calpain activity.
In the presence of the calpain inhibitor PD150606, PS-341 did not increase calpain activity (i.e. fluorescence intensity) (Fig. 4C), indicating the specificity of the assay for calpain activity. This result provides another piece of evidence supporting involvement of calpain in PS-341-induced IB␣ degradation.
PS-341 Induces IB␣ Proteolysis Involving Down-regulation of Calpastatin-To gain insight into the mechanism underlying proteasome inhibition-induced calpain activation, we further examined whether PS-341 modulates the levels of calpastatin, a well known endogenous calpain inhibitor (25), because proteasome inhibition was reported to reduce calpastatin levels (26). In Western blotting, we detected multiple sizes (e.g. ϳ110, 70, and 40 kDa) of calpastatin, which are identical to published observations (27,28). siRNA-mediated silencing of calpastatin substantially reduced the levels of these proteins, indicating that they are all specific calpastatin proteins. Meanwhile, IB␣ levels were drastically reduced in calpastatin-silenced cells (Fig. 5A). PS-341 treatment decreased the levels of calpastatin accompanied by an increase in p-IB␣ levels and reduction of IB␣ in a time-dependent manner (Fig. 5B). Enforced expression of ectopic calpastatin increased basal levels of IB␣ and prevented IB␣ from degradation by PS-341 (Fig. 5C). Collectively, these results indicate that calpastatin inhibition is involved in mediating IB␣ proteolysis by PS-341.
IB␣ Phosphorylation Is Required for PS-341-induced IB␣ Degradation-Given that inhibition of the proteasome with different proteasome inhibitors increases p-IB␣ levels, we then asked whether IB␣ phosphorylation is required for proteasome inhibition-initiated IB␣ degradation. To address this issue, we took advantage of SRIB␣, which carries serine-toalanine mutations at residues 32 and 36 and thus cannot be phosphorylated at Ser-32 and Ser-36. We first infected two lung cancer cell lines H460 and H157 with Ad-SRIB␣ and then treated cells with either PS-341 or MG132 to determine  whether PS-341 or MG132 could still induce SRIB␣ degradation. For up to 12 h of treatment, neither PS-341 nor MG132 decreased the levels of SRIB␣; instead, both agents increased the levels of SRIB␣ (Fig. 6A). Accordingly, we noted that the endogenous IB␣ levels were not reduced or only slightly reduced (Fig. 6A). Thus, these results clearly indicate that IB␣ phosphorylation at Ser-32 and Ser-36 is required for proteasome inhibition-initiated IB␣ degradation.

. Induction of IB␣ degradation by PS-341 in other types of cancer cells lines (A and B) and by other proteasome inhibitors in human lung cancer cells (C).
PS-341 Activates NF-B-Since IB␣ degradation is an essential step for p65 nuclear translocation and subsequent NF-B activation (9, 21), we then questioned whether IB␣ reduction by PS-341 resulted in NF-B activation. To this end, we analyzed p65 localization in cells treated with PS-341 using immunofluorescent staining. As shown in Fig. 6B, we primarily detected p65 staining in the cytoplasm in DMSO-treated H460 or H157 cells. However, a strong nuclear staining of p65 was detected in PS-341-treated H460 or H157 cells, indicating that PS-341 induces p65 nuclear translocation in these cell lines. Moreover, we examined NF-B activity in both H460 and H157 cell lines treated with PS-341 and found that PS-341 increased NF-B luciferase activity in a dose-dependent manner (Fig. 6C).
c-FLIP, survivin, and COX-2 are NF-B-regulated genes (9,29,30). PS-341 increased the levels of these proteins; however, the effects of PS-341 on increasing the levels of these proteins were abolished or attenuated in cells infected with Ad-SRIB␣ (supplemental Fig. S3). These results clearly indicate that PS-341 modulates the expression of these proteins through an IB␣ degradation-or NF-B activationdependent mechanism. Taken together with the above findings, we conclude that PS-341 increases NF-B activity.
Moreover, we further analyzed the effects of PS-341 on p65 nuclear translocation in more cancer cell lines. Consistently, PS-341 induces p65 nuclear translocation in every tested cell line (supplemental Fig. S4). These data again support the notion that PS-341 activates NF-B.

Inhibition of Calpain Augments the Effect of PS-341 on Induction of
Apoptosis-Activation of NF-B is generally associated with apoptosis resistance, although it can be proapoptotic under certain conditions (8,9,22,31,32). Therefore, we investigated the impact of IB␣ degradation on PS-341-induced apoptosis. To do this, we compared the effects of PS-341 on cell survival and apoptosis in the absence and presence of a calpain inhibitor. As presented in Fig. 7A, the calpain inhibitor PD150606 itself had a very weak effect on decreasing cell numbers. The combination of PS-341 with PD150606 was much more potent than PS-341 alone in decreasing cell numbers in both tested cell lines. Consistently, the combination of PS-341 with PD150606 caused the most apoptotic cells compared with PS-341 alone in both cell lines (Fig. 7B). For example, in H460 cells, PS-341 alone induced 13% apoptosis; however, the combination of PS-341 and PD150606 caused more than 40% apoptosis (Fig. 7B). Thus, it is clear that the presence of PD150606 substantially enhances the effect of PS-341 on induction of apoptosis, implying that inhibition of calpain activation may augment the anticancer activity of PS-341.

DISCUSSION
Although inhibition of IB␣ degradation and NF-B activation is a putative mechanism for action of PS-341, no study has been done to demonstrate this mechanism in lung cancer cells. Our efforts in this regard have generated surprising results that instead of inhibiting IB␣ degradation PS-341 actually potently decreases IB␣ levels and results in NF-B activation. These findings are consistent with three previous studies demonstrating that PS-341 as well other proteasome inhibitors induce IB␣ degradation and activate NF-B in endometrial carcinoma cell lines (33), the HepG2 liver cancer cell line (23), and multiple myeloma cells (34). We also found that PS-341 decreased IB␣ levels in other types of cancer cell lines, including myeloma, prostate, cervical, breast, and head and neck cancer cells. One previous study shows that PS-341 indeed increases IB␣ levels in human glioblastoma multiforme cells (35). Nevertheless, it seems that induction of IB␣ degradation by PS-341 is likely to be a common event in many types of cancer cells.
It is known that activated IB kinase (IKK) complex phosphorylates IB␣ on serines 32 and 36, leading to subsequent ubiquitination and proteasome-mediated degradation of IB␣. Our results clearly show that PS-341 induces IB␣ phosphorylation on serines 32 and 36 accompanied with reduction of IB␣ levels. Moreover, we found that PS-341 failed to induce degradation of the IB␣ mutant, SR-IB␣, in which serines 32 and 36 are mutated to alanines; rather, it increased the levels of SR-IB␣ (Fig.  6A). Thus, we conclude that PS-341 induces phosphorylation-dependent IB␣ degradation. This finding is in agreement with previous work demonstrating that PS-341 and other proteasome inhibitors induce IKK-dependent IB␣ phosphorylation and degradation in other types of cancer cell lines through detection of phosphorylated IKKs and inhibition of IKKs with small molecules or siRNA (33,34,36). We assume that PS-341 may induce an IKK-dependent IB␣ phosphorylation and degradation in lung cancer cells as well.  Before this study, it was unclear why PS-341 as a proteasome inhibitor induces IB␣ degradation. It is worth mentioning that other proteasome inhibitors such as MG132 were also shown to induce IB␣ degradation in colon cancer (36) and endometrial carcinoma cells (33). Our own data demonstrate that the proteasome inhibitors MG132, epoxomycin, and ALLN increase IB␣ phosphorylation and decrease IB␣ levels in human lung cancer cells as well. Thus, it is likely that proteasome inhibition is associated with PS-341-induced IB␣ phosphorylation and degradation.
IB␣ can also be degraded by proteases (e.g. calpain) or caspases (21,22) in addition to proteasome. A recent study shows that PS-341 induces IB␣ degradation involving caspase-8 in HepG2 cells (23). Our study demonstrates that PS-341 induces IB␣ degradation or proteolysis independent of caspase activation, including caspase-8 activation based on the following facts. First, IB␣ phosphorylation and degradation apparently occurred much earlier (at 2 h) than PARP cleavage (at 8 h) (Fig. 1A). Second, strong IB␣ cleavage occurred in the cell lines where PARP cleavage was not detected or only minimally detected ( Fig. 2A). Third, the presence of the pan-caspase inhibitor Z-VAD-FMK failed to prevent PS-341-induced IB␣ degradation (Fig. 3A). Finally, inactivation of caspase-8 by siRNA-mediated knockdown of caspase-8 did not inhibit PS-341-induced IB␣ degradation (Fig. 3A, lower panel).
It has been documented that IB␣ can undergo calpain-mediated proteolysis, which is parallel to the ubiquitination-proteasome mechanism (19). Calpain-mediated IB␣ proteolysis generates an ϳ30-kDa cleaved IB␣ intermediate in a previous report (19). In our study, we also detected an ϳ30-kDa cleaved band of IB␣ in cells exposed to PS-341 or other proteasome inhibitors ( Figs. 1 and 2). The presence of the calpain inhibitor EST, calpeptin, or PD150606 abolished the ability of PS-341 to induce IB␣ degradation (Fig. 3B). In addition, knockdown of the calpain regulatory subunit with a calpain regulatory siRNA abolished the ability of PS-341 to decrease IB␣ levels (Fig. 3C). Consistently, the calcium chelators EGTA and BAPTA inhibited PS-341-induced IB␣ degradation as well (Fig. 3D) because calpains are calcium-dependent proteases. Moreover, we detected increased calpain activity in cells exposed to PS-341 (Fig. 4). Together, these results clearly indicate that PS-341 induces IB␣ proteolysis through a calpain-dependent mechanism. Thus, it is likely that inhibition of the proteasome may favor calpain activation, leading to IB␣ degradation.
It is well known that calpain activity is negatively regulated by its endogenous inhibitor calpastatin (25). It was previously shown that proteasome inhibition induced calpastatin degradation, leading to calpain activation and subsequent Tau protein degradation in neuroblastoma cells (26). Similarly, we found that knockdown of calpastatin substantially decreased IB␣ levels, further supporting the notion that calpain can cause IB␣ degradation. Importantly, PS-341 decreased the levels of calpastatin accompanied with IB␣ degradation (Fig. 5B). Consistently, overexpression of an exogenous calpastatin elevated basal IB␣ levels and prevented IB␣ from degradation by PS-341 (Fig. 5C). Thus, it appears that proteasome inhibition down-regulates or inhibits calpastatin, leading to calpain activation and subsequent IB␣ degradation. The cells were lysed for preparation of whole-cell protein lysates and subsequent Western blot analysis. The migration difference between the endogenous IB␣ and SR-IB␣ is likely due to mutation (i.e. amino acid change) and phosphorylation inability. B, given cell lines were treated with DMSO or 50 nM PS-341 for 4 h and then subjected to immunofluorescent staining of p65. C, indicated cell lines were transfected with pNF-B-luc plasmid together with ␤-galactosidase plasmid as an internal control using FuGENE 6 transfection reagent. On the 2nd day, the cells were treated with the indicated concentrations of PS-341 for 8 h and then subjected to cell lysis and luciferase activity assay. Columns, means of triplicate determinations; Bars, ϮS.D.
Because NF-B regulates the expression of multiple anti-apoptotic genes, its activation is often associated with apoptosis resistance (9,10,32). In agreement with the finding in endometrial carcinoma cells (33) and multiple myeloma cells (34), we have also shown that PS-341 induces p65 nuclear translocation and increases NF-B activity (Fig. 5) in our cell systems. Moreover, PS-341 increased the expression of survivin, c-FLIP, and COX-2, all of which are NF-B-regulated genes (9,29,30). Importantly, suppression of IB␣ degradation by expressing SR-IB␣ abrogated the increase of these proteins by PS-341 (supplemental Fig. S3), furthering the notion that PS-341 activates NF-B in our cell systems. In endometrial carcinoma cells, NF-B activation does not mediate cytotoxic effects of proteasome inhibitors because neither IKK␣ nor IKK␤ small hairpin RNA protected cells from death induced by these inhibitors (33). In multiple myeloma cells, inhibition of IKK␤ enhances PS-341-induced cytotoxicity (34). In human lung cancer cells, we found that the presence of a calpain inhibitor (e.g. PD150606), which inhibits PS-341-induced IB␣ degradation, substantially enhanced the effects of PS-341 on decreasing cell survival and inducing apoptosis (Fig. 7), implying that IB␣ degradation consequently confers cell resistance to undergo apoptosis, presumably through activation of NF-B. Thus, our findings in this regard may suggest a novel therapeutic strategy for enhancing the anticancer activity of PS-341 by preventing calpain activation.
In summary, we report a novel finding that PS-341 induces calpain-dependent IB␣ degradation in human lung cancer as well other types of cancer cells. As a result, PS-341 activates NF-B, which can confer apoptotic resistance and counteract the apoptosis inducing activity of PS-341. Thus, prevention of calpain-dependent IB␣ degradation will substantially augment the apoptosis inducing activity of PS-341.