Original Article

Oncogene (2007) 26, 6184–6193. doi:10.1038/sj.onc.1210448; published online 9 April 2007

There is a Retraction (27 June 2016) associated with this article.

Rituximab inhibits the constitutively activated PI3K-Akt pathway in B-NHL cell lines: involvement in chemosensitization to drug-induced apoptosis

E Suzuki1,2, K Umezawa2 and B Bonavida1

  1. 1Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA, USA
  2. 2Department of Applied Chemistry, Keio University, Hiyoshi, Kohoku-ku, Yokohama, Japan

Correspondence: Professor B Bonavida, Department of Microbiology, Immunology and Molecular Genetics, University of California at Los Angeles,10833 Le Conte Avenue, A2-060 CHS, Los Angeles, CA 90095-1747, USA. E-mail: bbonavida@mednet.ucla.edu

Received 29 August 2006; Revised 25 January 2007; Accepted 11 February 2007; Published online 9 April 2007.

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Abstract

Rituximab (chimeric anti-CD20 monoclonal antibody) is currently being used, alone or in combination with chemotherapy, in the treatment of B-non-Hodgkin's lymphoma (B-NHL). We have reported that rituximab treatment of B-NHL cell lines sensitizes the drug-resistant tumor cells to apoptosis by various chemotherapeutic drugs and chemosensitization was, in large part, owing to the selective inhibition of the anti-apoptotic Bcl-XL gene product. The constitutive activation of the Akt pathway in B-NHL results in overexpression and functional activation of Bcl-xL. Hence, we hypothesized that rituximab-induced inhibition of Bcl-xL expression and chemosensitization may result, in part, from its inhibitory activity of the Akt pathway. This hypothesis was tested using the drug-resistant Ramos and Daudi B-NHL cell lines. Time kinetic analysis revealed that treatment with rituximab inhibited phosphorylation of Akt, but not unphosphorylated Akt, and the inhibition was first detected at 6 h post-rituximab treatment. Similar time kinetics revealed rituximab-induced inhibition of p-PDK1, p-Bad, p-IKKalpha/beta and p-Ikappabetaalpha and no inhibition of unphosphorylated proteins. In addition, rituximab treatment resulted in significant increase of Bcl-xL–Bad heterodimeric complexes as compared to untreated cells. The role of the Akt pathway in the regulation of resistance was corroborated by the use of the Akt inhibitor, LY294002, and by transfection with siRNA Akt. Treatment of tumor cells with LY294002 or with Akt siRNA, but not control siRNA, resulted in inhibition of Bcl-xL expression and sensitization to drug-induced apoptosis. Although rituximab did not inhibit the Akt pathway nor sensitized the rituximab-resistant Ramos RR1 clone, treatment with LY294002 or Akt siRNA sensitized the clone to drug-induced apoptosis. The present findings demonstrate for the first time that rituximab inhibits the constitutively activated Akt pathway in B-NHL cell lines, and this inhibition contributes to sensitization of drug-resistant cells to apoptosis by chemotherapeutic drugs. The findings also identify the Akt pathway as target for therapeutic intervention in the reversal of rituximab and drug-resistant B-NHL.

Keywords:

rituximab, Akt pathway, chemosensitization, apoptosis

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Introduction

Non-Hodgkin's lymphoma (NHL) is a heterogeneous group of disorders that represents about 4% of all malignancies and ranks fifth in cancer incidence and mortality. Although the initial response rates to chemotherapy are high, relapses eventually occur and subsequent chemotherapy regimens are incapable of yielding long-term remission (Rogers, 2006). One of the candidate antigens that has been targeted for immunotherapy is CD20, a 297-amino-acid (32–37 kDa) unglycosylated phosphoprotein that spans the membrane four times (Ernst et al., 2005). CD20 is expressed specifically within the B-cell lineage from pre-B cells to mature B cells. Rituximab (chimeric antihuman CD20 antibody) mediates its antitumor activity by multiple mechanisms that include complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity and induction of apoptosis following CD20 cross-linking (Shan et al., 1998; Jazirehi and Bonavida, 2005). We have recently reported that rituximab sensitizes drug-resistant B-NHL cell lines to the apoptotic effects of various chemotherapeutic drugs via the selective downregulation of Bcl-xL expression. Downregulation of Bcl-xL expression was the result of inhibition of both the Raf/MEK/ERK1/2 and NF-kappaB survival pathways (Jazirehi et al., 2004, 2005).

Akt is a serine/threonine protein kinase that mediates various downstream effects of PI3-K. It plays a central role in signaling by the PI3-K pathway by regulating many biological processes, such as proliferation, apoptosis and cell growth (Cantley et al., 2002). In addition, the activated PI3K-Akt pathway provides major survival signals to lymphoma cells and many other cancer cells (Goswami et al., 2006; Toker and Yoeli-Lerner, 2006). Akt controls a variety of mechanisms that inhibit apoptosis and prolong cell survival, exerting a positive effect on NF-kappaB functions (Ozes et al., 1999; Osaki et al., 2004).

The Akt pathway is constitutively activated in most tumor cells and in B-NHL cell lines and in B-NHL derived from patients (Arranz et al., 1996). Bcl-xL expression and/or activity can be regulated by the Akt pathway (Uddin et al., 2004). The regulation by the PI3k pathway of Bcl-xL activity results from Akt-mediated phosphorylation (p) of Bad, and thus promoting p-Bad to interact with Bcl-xL and forming heterodimeric complexes. The regulation of Bcl-xL translation and transcription by the Akt pathway is indirect and under the control of NF-kappaB, and NF-kappaB is regulated by the Akt pathway (Cuni et al., 2004). Because our prior findings demonstrated that rituximab inhibits Bcl-xL expression via inhibition of both the ERK1/2 and NF-kappaB pathways (Jazirehi et al., 2004, 2005), we hypothesized that downregulation of Bcl-xL expression by rituximab may also involve the Akt pathway. Thus, as Bcl-xL regulates chemoresistance, we hypothesized that inhibition of the Akt pathway and Bcl-xL by rituximab could contribute to the sensitization of tumor cells to chemotherapy-induced apoptosis. This hypothesis was tested with B-NHL cell lines and the followings were investigated: (1) Does rituximab inhibit the Akt signaling pathway? (2) Does rituximab inhibit the phosphorylation of Bad and augment the complex formation between Bcl-xL and Bad? (3) Does inhibition of the Akt pathway inhibit Bcl-xL activity and expression? (4) Does the direct inhibition of the Akt pathway by siRNA Akt or a pharmacologic Akt inhibitor, Ly294002, mimic rituximab and sensitizes B-NHL cells to apoptosis by chemotherapeutic drugs? and (5) Does rituximab failure to sensitize rituximab-resistant clones can be reversed by Ly294002? The findings obtained were concordant with the above hypotheses.

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Results

Rituximab diminishes the constitutive activity of the PI3k-Akt signaling pathway in B-NHL cells

We have recently reported that rituximab treatment of B-NHL cell lines resulted in inhibition of the survival Raf-1/MEK/ERK1/2 and the NF-kappaB signaling pathways both of which led to the selective inhibition of the expression of the anti-apoptotic gene product Bcl-xL (Jazirehi et al., 2004, 2005). Hence, we explored if other signaling pathways that also regulate Bcl-xL expression and/or activity may also be inhibited by rituximab. The Akt pathway has been shown to regulate Bcl-xL transcriptional and post-transcriptional expression and activity (Hayakawa et al., 2000; Weintraub et al., 2004). Hence, we examined whether rituximab treatment inhibits the activity of the Akt pathway. Time kinetic analyses revealed that rituximab treatment of Ramos inhibited phospho(p)-Lyn (Tyr507), phosphorylation of Akt (p-Akt) (Ser473 and Thre308), p-PDK1 and p-Bad, although there was no detectable inhibition of non-phosphorylated proteins. The inhibition was first observed at 6 h post-rituximab treatment and was maintained up to 24 h (Figure 1a). The rituximab-mediated inhibition of p-Akt was direct as treatment of Ramos with Fc-devoid rituximab (Fab)'2 also inhibited p-Akt like rituximab (Figure 1b). The PI3K-Akt signaling pathway regulates the transcriptional expression of Bcl-xL via IkappaB kinase (IKK), IkappaB and NF-kappaB activation (Sugimori et al., 2005) and, thus, we expected that rituximab treatment of Ramos would also inhibit the phosphorylation of these gene products. Treatment of Ramos cells with rituximab diminished the levels of p-IKKalpha/beta and p-Ikappabetaalpha, but not the levels of non-phosphorylated proteins, and the inhibition was first detected at 6 h post-rituximab treatment and significant inhibition was observed after 24 h (Figure 1c). The level of IkappaBalpha was augmented 3 h following rituximab treatment as less degradation takes place. The findings above demonstrate that rituximab treatment of Ramos cells inhibits the constitutively activated Akt and NF-kappaB pathways in a time-dependent fashion as early as 6 h post-treatment.

Figure 1.
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Inhibition of the PI3K-Akt signaling pathway in Ramos cells by rituximab. (a) Ramos cells (1 times 107/ml) were treated with rituximab (20 mug/ml) for different times (0–24 h) and incubated at 37°C, and total cell lysates were prepared as described in Materials and methods. The cell lysates were examined by Western blotting for various unphosphorylated and phosphorylated (p) proteins of the Akt pathway. beta-actin was used as control for loading. (b) Ramos cells were treated with rituximab (20 mug/ml) or with equal amount of rituximab (Fab)'2 (20 mug/ml) for 20 h at 37° and cell lysates were prepared and examined for Akt and p-Akt (Ser473) by Western blotting. beta-actin was used as control. (c) Ramos cell lysates, prepared as described above in (a) were examined for levels for unphosphorylated and phosphorylated proteins of the NF-kappaB pathway by Western blotting. beta-actin was used as control. (d) Ramos cells (2 times 106/ml) were left untreated or were treated with rituximab (20 mug/ml) for 20 h and cell lysates were prepared. The cell lysates were immunoprecipitated with rabbit anti-Bcl-xL antibody and the precipitate was examined by Western blotting for Bad using rabbit anti-Bad antibody as described in Materials and methods. The IgG bands in this figure correspond to the immunoprecipitated rabbit anti-Bcl-xL IgG and its development by the secondary goat anti-rabbit IgG and then developed with HRP-goat anti-rabbit IgG. The Bcl-xL bands correspond to the level of Bcl-xL protein in the precipitate 20 h after treatment with rituximab. The above findings are representative of three independent experiments yielding similar results.

Full figure and legend (103K)

The Akt signaling pathway regulates Bcl-xL activity via phosphorylation of Bad, which normally dissociates from Bcl-xL, and dissociated Bcl-xL can thus exert its anti-apoptotic activity. Likewise, phosphorylation of Bad also promotes cell survival (Datta et al., 1999; Hayakawa et al., 2000). Therefore, we expected that treatment of Ramos with rituximab, which inhibited pBad (Figure 1a), would significantly enhance the physical association of Bad and Bcl-xL. This was determined by treating Ramos cell lysates with anti-Bcl-xL antibody and the immunoprecipitated complexes (Bcl-xL–Bad) were analysed. Clearly, treatment of lysates from rituximab-treated Ramos cells showed significant augmentation of Bad in the immunoprecipitate as compared to the Bad level in untreated cell lysates (Figure 1d). The lysates of rituximab-treated cells also contain less Bcl-xL as compared to untreated cells. These findings demonstrate that rituximab-induced inhibition of the Akt signaling pathway in Ramos cells significantly enhanced the complex formation of nonphosphorylated Bad with Bcl-xL concomitant with inhibition of Bcl-xL.

Treatment of Ramos cells with the Akt inhibitor LY294002 mimics rituximab-mediated inhibition of the Akt pathway

We examined the direct role of the Akt pathway in the regulation of Bcl-xL expression in Ramos cells using the Akt specific inhibitor, LY294002. Treatment of Ramos cells with LY294002 inhibited the Akt pathway beginning at 6 h post-treatment. There was inhibition of p-Akt (Ser473), p-Akt (Thr308), p-PDK1 and p-Bad in the absence of any inhibition of non-phosphorylated proteins (Figure 2a). In addition, treatment of Ramos cells with LY294002 inhibited Bcl-xL expression like rituximab (Figure 2b). Altogether, these findings suggest that rituximab-induced inhibition of the Akt pathway participates in the inhibition of Bcl-xL expression.

Figure 2.
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Inhibition of the Akt pathway in Ramos cells by LY294002. Similar studies were performed as above with rituximab (Figure 1a) except that LY294002 (25 muM) was used. Cell lysates were examined for unphosphorylated and phosphorylated proteins (a) and for Bcl-xL expression (b) by Western blotting. beta-actin was used as control for loading.

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Participation of rituximab-mediated inhibition of the Akt pathway in the chemosensitization of B-NHL cells to drug-induced apoptosis

Two approaches were examined to determine the role of the AKT pathway in chemosensitization, namely, the use of a pharmacologic-specific Akt inhibitor, Ly294002 and inhibition of Akt by siRNA.

(1) Inhibition of the Akt pathway by LY294002 mimics rituximab-mediated sensitization of Ramos and Daudi cells to drug-induced apoptosis.

Rituximab treatment of Ramos cells for 24 h significantly sensitized the cells to cis-platinum (CDDP)-induced apoptosis, and the sensitization was a function of the antibody concentration used. Treatment with single agent had no cytotoxic activity (Figure 3a). Similar findings were obtained with rituximab (Fab)'2 (Figure 3b) and thus, ruling out the role of Fc in chemosensitization. The findings with Ramos were not unique to this cell line as rituximab inhibited p-Akt (Ser473) in Daudi cells and sensitized the tumor cells to CDDP-induced apoptosis (Figure 3c). By comparison with rituximab, treatment of Ramos cells with various concentrations of LY294002 (10–30 muM) also sensitized Ramos cells to CDDP-induced apoptosis, and the sensitization was a function of the LY294002 concentration used (Figure 3d). Similar findings were also observed with cells treated with LY294002 for adriamycin (ADR)-induced apoptosis (Figure 3e). These findings demonstrate that the inhibition of the Akt pathway by rituximab participates in the sensitization of B-NHL cells to drug-induced apoptosis.

Figure 3.
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Chemosensitization of Ramos and Ramos RR1 by rituximab, rituximab (Fab)'2 and LY294002. (a) Ramos cells were treated with different concentrations of rituximab (0–20 mug/ml) for 20 h and then treated with CDDP (15 mug/ml) for an additional 24 h. The cells were then evaluated for apoptosis by the PI method as described in Materials and methods. P<0.001 represents the combination treatment of rituximab (20 mug/ml) and CDDP as compared to treatment with CDDP alone. P<0.005 represents the combination treatment of rituximab (15 mug/ml)+CDDP as compared to treatment with CDDP alone. (b) Similar experiments to those described in (a) above were performed except that rituximab (Fab)'2 (0–15 mug/ml) was used instead of rituximab. P<0.001 represents the combination of rituximab (Fab)'2+CDDP as compared to treatment with CDDP alone. (c) Daudi B-NHL cells were treated with rituximab (20 mug/ml) and incubated for 0–24 h. Total cell lysates were prepared and examined for phosphor-Akt (ser 473), Akt and beta-actin. Apoptosis was determined by the PI/Annexin V method as described in Materials and methods. (d) Similar experiments as those described above in (a) were performed except that different concentrations of Ly294002 (0–30 muM) were used instead of rituximab. P<0.005 represents the combination of LY294002 (20 and 30 muM) and CDDP as compared to treatment with CDDP alone. (e) Similar experiments as those described above in (c) were performed except that ADR (5 mug/ml) was used instead of CDDP. P<0.001 represents the combination of LY294002 (10 and 25 muM)+ADR (5 mug/ml) as compared to treatment with ADR alone. The above experiments are representative of three different experiments with similar findings.

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We have recently reported that rituximab resistant Ramos (RR) and Daudi (RR) clones, generated in our laboratory, did not respond to rituximab-induced signaling and, further, rituximab did not chemosensitize the cells to drug-induced apoptosis (Jazirehi et al., 2007). Thus, we examined whether rituximab treatment affects the Akt pathway in Ramos RR1 cells. There was no inhibition of p-Akt (Ser473) by rituximab in Ramos RR1 (Figure 4a, top) or chemosensitization to CDDP (Figure 4a, Bottom). However, treatment of Ramos RR1 with LY294002 significantly sensitized the Ramos RR1 cells to CDDP-induced apoptosis (Figure 4b). These findings demonstrate that rituximab resistance can be overcome by an inhibitor of the Akt pathway.

Figure 4.
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Ly294002, but not rituximab, sensitizes Ramos RR1 cells to CDDP-induced apoptosis. (a) Similar experiments as those described above in Figure 3c were performed except that the rituximab-resistant Ramos RR1 clone was used. Shown in the top, Western blot analyses for unphosphorylated and phosphorylated Akt and beta-actin was used a control for loading. The bottom figure shows the data on the failure of rituximab to sensitize Ramos RR1 cells to CDDP-induced apoptosis. (b) Ramos RR1 cells were treated with various concentrations of LY294002 (0–30 muM), and then treated with CDDP (15 mug/ml) and apoptosis was determined. P<0.005 represents the significance between the combination treatment and treatment with single agent. The above findings are representative of three independent experiments yielding similar results.

Full figure and legend (72K)

(2) Inhibition of the Akt pathway by Akt siRNA sensitized Ramos and Ramos RR1 cells to CDDP-induced apoptosis.

The direct role of the Akt pathway in the regulation of drug resistance was tested by transfecting Ramos cells with Akt siRNA as described in methods. Treatment of Ramos cells with Akt siRNA resulted in significant inhibition of p-Akt and Akt beginning at 48 h posttransfection. There was also concomitant inhibition of Bcl-xL expression. However, transfection with control siRNA had no detectable effect (Figure 5a, top). Treatment with Akt siRNA, but not with siRNA control, significantly sensitized the cells to CDDP-induced apoptosis and 5 mug Akt SiRNA was optimal (Figure 5a, bottom). The findings with Akt siRNA corroborated with the findings above with LY294002. Like with wild-type Ramos, treatment of Ramos RR1 with Akt siRNA inhibited p-Akt (Ser473) and Akt (Figure 5b, top) and sensitized the cells to CDDP-induced apoptosis (Figure 5b, bottom).

Figure 5.
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Direct role of the Akt pathway in chemosensitization of Ramos and Ramos RR1 cells to CDDP-induced apoptosis. (a, top). Ramos cells were transfected with control siRNA or Akt siRNA for different periods of time (0–72 h) as described in Materials and methods. Cell lysates were prepared and examined by Western blotting for p-Akt, Akt and Bcl-xL expression. beta-actin was used as control. (a, bottom). Ramos cells were transfected with control siRNA or different concentrations of Akt siRNA or control siRNA (0–20 nM) for 48 h. The cells were then left untreated or treated with CDDP (15 mug/ml) for an additional 24 h and the cells were examined for apoptosis as described. P<0.001 represents the combinations of Ramos cells transfected with Akt siRNA (5,10 and 20 muM) and CDDP as compared to treatment with CDDP alone. (b) Similar experiments as those describe in (a) above were performed with Ramos RR1 cells. P<0.01 represents the significance between transfected cells with Akt siRNA and CDDP and treatment with control siRNA. P<0.005 represents the significance between cells transfected with Akt siRNA and CDDP versus siRNA alone. The above findings are representative of three independent experiments, yielding similar results.

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Altogether, the above findings demonstrate that rituximab-induced inhibition of the Akt pathway contributes to rituximab-mediated chemosensitization in both wild-type and resistant clones.

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Discussion

The present findings demonstrate for the first time that treatment of B-NHL cell lines with rituximab significantly inhibited the constitutively activated PI3k-Akt signaling pathway. Inhibition of this pathway resulted in inhibition of both Bcl-xL activity and Bcl-xL expression. The role of the Akt pathway in rituximab-induced sensitization of drug-resistant B-NHL cells to apoptosis by chemotherapeutic drugs was corroborated by the use of both the PI3K inhibitor Ly294002 and by the use of silencer RNA for Akt. Treatment of Ramos RR1 with LY294002 or Akt SiRNA inhibited the Akt pathway and Bcl-xL expression, and sensitized the cells to drug-induced apoptosis. These findings established the involvement of the activated Akt pathway in the regulation of chemoresistance in B-NHL. Further, inhibitors of the Akt pathway sensitized both rituximab-sensitive and rituximab-resistant tumor cells. Further, the findings identify the Akt pathway as a potential novel target for therapeutic intervention in the treatment of rituximab and drug-resistant B-NHL when used alone or in combination with cytotoxic drugs.

Aberrant activation of the PI3K-Akt pathway has been widely implicated in many cancers. The PI3K-Akt signaling pathway regulates many normal cellular processes (Vivanco and Sawyers, 2002). The PI3K-Akt pathway is a key regulator of cell survival through multiple downstream targets. Ramos, Daudi and Ramos RR1 cells exhibit constitutively activated signaling of the Akt pathway. Rituximab treatment inhibited both pAkt (ser473) and p-Akt (Thr308) as early as 6 h. The inhibition of pPDK1 and p-Bad by rituximab followed the same time kinetics as those for Akt. Akt can phosphorylate the Bcl-2 family member Bad, causing its sequestration from the mitochondrial membrane by 14-3-3 protein (Datta et al., 1999). In its unphosphorylated form, Bad binds to and inactivates anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, leading to its proapoptotic function. Inhibition of pBad was initially observed at 6 h post-rituximab treatment. The inhibition of pBad in Ramos cells resulted in the augmented association of Bad with Bcl-xL to form more complexes. Complexes of Bad and Bcl-2 could not be detected in Ramos as these cells are deficient in Bcl-2 expression, and we have previously confirmed this finding (Jazirehi et al., 2004).

A cross talk between the PI3K and the NFkappaB pathway has been reported previously in a number of systems (Yin et al., 2006). Treatment with rituximab inhibited the phosphorylation of IKK and IkappaBalpha, indicating that rituximab inhibits the anti-apoptotic effect regulated by NFkappaB. Considerable controversy remains regarding the involvement of Akt in signal-induced IKK activation. Thus, it is possible that Akt phosphorylates IKK in a cell context and stimulation-dependent manner.

Previous findings demonstrated the roles of the Raf/ERK1/2 and NF-kappaB pathways in rituximab-induced sensitization to apoptosis by chemotherapeutic drugs Jazirehi et al., 2004, 2005). The direct role of the activated Akt pathway in the regulation of resistance was independently demonstrated by the use of LY294002 and siRNA. Both inhibited Bcl-xL expression and sensitized the tumor cells to drug-induced apoptosis. Ly294002 has long been used as a selective inhibitor of PI3K-mediated p-Akt (Poh and Pervaiz, 2005). The chemosensitization induced by LY294002 resembles that induced by rituximab with similar time kinetics as those observed with rituximab. These findings support the contribution of rituximab-induced inhibition of the Akt pathway in chemosensitization. Our present findings are consistent with other studies that have been reported with the use of LY294002 (Wetzker and Rommel, 2004).

We also demonstrate that rituximab-resistant Ramos RR1 cells were sensitized by LY294002 to drug-induced apoptosis. LY294002 inhibited the constitutive activity of p-Akt and Bcl-xL expression (Figure 4b). This finding is of potential therapeutic application in the treatment of drug and rituximab-resistant tumor cells by combination of Akt inhibitors and cytotoxic drugs. In addition to the role of LY294002 as an inhibitor, we showed that knockdown of Akt by siRNA significantly inhibited both Akt, p-Akt and reduced Bcl-xL expression, and the cells were sensitized to CDDP-induced apoptosis. Likewise, treatment of rituximab-resistant RR1 cells with Akt siRNA also sensitizes cells to CDDP-induced apoptosis (Figure 5b). Consistent with our findings, clinically, knockdown of Akt by antisense or siRNA significantly reduced tumor cell growth and invasiveness, and induced cell growth arrest and apoptosis in tumor cells overexpressing Akt (Cheng et al., 1996).

There was a strong correlation between Bcl-xL inhibition by rituximab treatment or by Akt inhibitors (LY294002 and Akt siRNA) and chemosensitization. Thus, Bcl-xL inhibition in tumor cells is of clinical significance. Zhao et al. (2004) investigated the clinical significance of Bcl-xL expression in patients with follicular lymphoma using real-time quantitative polymerase chain reaction. A high Bcl-xL level was significantly associated with progression and with the international prognostic index indicating high risk. Moreover, Bcl-xL gene overexpression was linked to short overall survival (Zhao et al., 2004). Bcl-xL is abundantly expressed in lymphoma (Xerri et al., 1996) and protects the cells from apoptosis induced by DNA-damaging agents.

Various mechanisms contribute to the activation of the Akt pathway in tumors, including perturbation of upstream tensin homologue deleted on chromosome 10 (PTEN) and phosphatidylinositol (3,4,5) triphosphate (PIP3) (Vivanco and Sawyers, 2002). Others include autocrine or paracrine stimulation of receptor tyrosine kinases and overexpression of growth factor receptors and/or Ras activation. It has been shown that Akt is activated constitutively by active Ras and Src (Datta et al., 1999). Rituximab inhibits the Src kinase pLyn (Jazirehi et al., 2004) and was confirmed here (Figure 1a), and it is possible that this inhibition is involved in the downstream inhibition of the Akt pathway by rituximab. The PTEN tumor suppressor is a negative regulator of the Akt pathway (Di Cristofano and Pandolfi, 2000). Targeting of Akt, directly or indirectly, inhibits cell proliferation, promotes apoptosis and/or increases sensitivity to chemotherapy (Tachiiri et al., 2000). The loss of PTEN expression has been reported in a variety of cancers (Rennie and Nelson, 1998; Marsit et al., 2005; Haiman et al., 2006). There are relatively a few studies exploring PTEN abnormalities in lymphoma. Preliminary findings indicated that rituximab treatment of Ramos resulted in PTEN induction. The roles of PTEN induction in Ramos by rituximab, in both the regulation of the Akt pathway and chemosensitization, are currently being investigated in our laboratory.

In summary, the present findings demonstrate that rituximab inhibits the constitutively activated Akt pathway in the Ramos B-NHL cells and results in inhibition of both Bcl-xL activity and expression, leading to reversal of drug resistance. A schematic diagram summarizing the findings of this study is illustrated in Figure 6. Our findings indicate that modulation of the PI3K-Akt pathway and components of the Akt signal transduction pathway by rituximab are responsible, in part, for chemosensitization. Blocking this pathway could impede the proliferation of tumor cells by increasing sensitivity of tumor cells to undergo apoptosis in response to other cytotoxic agents. Several inhibitors have been the subject of several investigations to intervene in the Akt pathway for tumor cell sensitization (Cheng et al., 2005). LY294002 or Akt siRNA inhibited the Akt pathway, and like rituximab, led to chemosensitization. Thus, this study identifies the components of the Akt pathway as targets for therapeutic intervention, when used alone or in combination with therapeutics, in the treatment of drug/rituximab resistance NHL. It is speculated that hyperactivation of the Akt pathway may also be involved in the pathogenesis of NHL and the development of drug/rituximab resistance. Thus, hyperactivation of this pathway may serve as a prognostic indicator in patients who are refractory to conventional therapies.

Figure 6.
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Schematic diagram of rituximab-mediated inhibition of the Akt pathway and chemosensitization. This diagram shows that B-NHL cells exhibit constitutively activated Akt and NF-kappaB pathways, and these are represented in dotted lines. Activation of these pathways results downstream in cytosolic Bcl-xL that is not complexed with p-Bad, and overexpression of functional Bcl-xL leads to chemoresistance. In contrast, treatment with rituximab inhibits these pathways resulting in augmentation of the association of Bcl-xL and Bad, as well as downregulation of Bcl-xL expression leading to chemosensitization.

Full figure and legend (49K)

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Materials and methods

Reagents

RPMI-1640, opti-MEM and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Rituximab (stock, 10 mg/ml) was obtained commercially. Rituximab (Fab)'2 was a generous gift from Dr. Chin (Idec Biogen, San Diego, CA, USA). LY294002, anti-Lyn, anti-phospho Lyn (Tyr507), anti-IKKalpha/beta, anti-Ikappabalpha/beta, anti-IKKbeta, anti-Bcl-xL, anti-Bad, anti-phospho-Bad (Ser136), anti-phospho PDK1, antihuman phospho-Akt (Ser473, Thr308), anti-Akt and anti-beta-actin antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Akt siRNA and control scramble siRNA were purchased from Sigma (MO, USA). Protein A-agarose was purchased from Pierce (Rockford, IL, USA). The Annexin V-FITC kit was purchased from Beckman Coulter (Marseille, France). The CD20+ human Burkitt's lymphoma B-cell lines Ramos and Daudi were obtained from ATCC (Manassas, VA, USA). The Ramos RR1 clone was generated in our laboratory following culture of Ramos in the presence of increasing concentrations of rituximab for 10 weeks and clones were isolated by limiting dilution (Jazirehi et al., 2007).

Drug pretreatment

This was done as discussed previously (Jazirehi et al., 2004).

Analysis of apoptosis

DNA fragmentation was detected by flow cytometric analysis after propidium iodide staining as described (Alas and Bonavida, 2001) or by the PI/Annexin V staining method. The PI/Annexin V double staining method was performed for analyses of data in Figure 3a–c, f and g. The PI method was performed for analyses in Figure 3d and e.

Western blotting

Analysis by Western blotting was done according to our previous studies (Jazirehi et al., 2004).

Immunoprecipitation

Ramos cells (2 times 106 cells) were collected and resuspended with lysis buffer (20 mM Tris–HCl (pH 8.0), 2 mM ethylenediaminetetraacetic acid, 3% NP-40, 100 mM NaCl, 50 mM NaF, 1 muM phenylmethylsulfonyl fluoride, 1 muM VO4, 5 mug/ml aprotinin, 5 mug/ml leupeptin). After 30 min of incubation on ice, samples were centrifuged at 10 000 g, for 10 min at 4°C to remove cellular debris. The concentration of the protein in the resulting supernatant was measured using the Bio-Rad Laboratories (Hercules, CA, USA) protein assay reagent and equalized with radioimmunoprecipitation (RIPA) buffer. One milligram of total lysate was incubated with 5 mul protein A agarose beads (Pierce, Rockford, IL, USA), centrifuged and the supernatant was collected. The supernatant was mixed with 15 mul of protein A agarose and 2 mug Bcl-xL mouse monoclonal antibody or normal mouse immunoglobulin G (IgG)-1 at 4 °C for 4 h. Following centrifugation, the beads were washed four times with RIPA buffer, and the beads were boiled in 50 mul Laemmli buffer and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted with rabbit anti-Bad antibody. After overnight incubation with the primary antibody, the membranes were washed and incubated with horseradish peroxidase -coupled secondary goat anti-rabbit antibody (Cell Signaling, MA, USA). Immunoreactive bands were detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, UK).

Transfection of Ramos with Akt siRNA

Ramos B-NHL cells and Ramos RR1 cells were cultured in 1 ml of Opti-MEM medium without antibiotics and FBS. Transfections were performed using Xtreme GENE reagent (Roche Diagnostics Corporation, Indianapolis, IN, USA), Akt siRNA (Sigma, MO, USA) and control scramble siRNA (Sigma, MO, USA) according to the manufacturer's instructions. To determine Akt siRNA-induced sensitization to CDDP-induced apoptosis following transfection, the cells were treated with CDDP for 24 h, then were processed for PI/Annexin V staining and analysed by flow cytometry as described above.

Statistical analysis

Assays were set up in triplicates and the results were expressed as the meanplusminuss.d. Statistical analysis and P-value determinations were done by two-tailed paired t-test with a confidence interval of 95% for determination of the significance of differences between the treatment groups. P<0.05 was considered to be significant.

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References

  1. Alas S, Bonavida B. (2001). Rituximab inactivates signal transducer and activation of transcription 3 (STAT3) activity in B-non-Hodgkin's lymphoma through inhibition of the interleukin 10 autocrine/paracrine loop and results in down-regulation of Bcl-2 and sensitization to cytotoxic drugs. Cancer Res 61: 5137–5144. | PubMed | ISI | ChemPort |
  2. Arranz E, Robledo M, Martfnez B, Gallego J, Remain A, Rivas C et al. (1996). Incidence of homogeneously staining regions in non-Hodgkin lymphomas. Cancer Genet Cytogenet 87: 1–3. | Article | PubMed | ISI | ChemPort |
  3. Cantley LC. (2002). The phosphoinositide 3-kinase pathway. Science 296: 1655–1657. | Article | PubMed | ISI | ChemPort |
  4. Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomare DA, Watson DK et al. (1996). Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Sci USA 93: 3636–3641. | Article | ChemPort |
  5. Cheng JQ, Lindsley CW, Cheng GZ, Yang H, Nicosia SV. (2005). The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 24: 7482–7492. | Article | PubMed | ISI | ChemPort |
  6. Cuni S, Perez-Aciego P, Perez-Chacon G, Vargas JA, Sanchez A, Martin-Saavedra FM et al. (2004). A sustained activation of PI3K/NF-kappaB pathway is critical for the survival of chronic lymphocytic leukemia B cells. Leukemia 18: 1391–1400. | Article | PubMed | ISI | ChemPort |
  7. Datta SR, Brunet A, Greenberg ME. (1999). Cellular survival: a play in three Akts. Genes Dev 13: 2905–2927. | Article | PubMed | ISI | ChemPort |
  8. Di Cristofano A, Pandolfi PP. (2000). The multiple roles of PTEN in tumor suppression. Cell 100: 387–390. | Article | PubMed | ISI | ChemPort |
  9. Ernst JA, Li H, Kim HS, Nakamura GR, Yansura DG, Vandlen RL. (2005). Isolation and Characterization of the B-Cell Marker CD20. Biochemistry 44: 15150–15158. | Article | PubMed | ISI | ChemPort |
  10. Goswami A, Ranganathan P, Rangnekar VM. (2006). The phosphoinositide 3-kinase/Akt1/Par-4 axis: a cancer-selective therapeutic target. Cancer Res 66: 2889–2892. | Article | PubMed | ISI | ChemPort |
  11. Haiman CA, Stram DO, Cheng I, Giorgi EE, Pooler L, Penney K et al. (2006). Common genetic variation at PTEN and risk of sporadic breast and prostate cancer. Cancer Epidemiol Biomarkers Prev 15: 1021–1025. | Article | PubMed | ISI | ChemPort |
  12. Hayakawa J, Ohmichi M, Kurachi H, Kanda Y, Hisamoto K, Nishio Y et al. (2000). Inhibition of BAD phosphorylation either at serine 112 via extracellular signal-regulated protein kinase cascade or at serine 136 via Akt cascade sensitizes human ovarian cancer cells to cisplatin. Cancer Res 60: 5988–5994. | PubMed | ISI | ChemPort |
  13. Jazirehi AR, Bonavida B. (2005). Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-CD20 mAb) in non-Hodgkin's lymphoma: implications in chemosensitization and therapeutic intervention. Oncogene 24: 2121–2143. | Article | PubMed | ISI | ChemPort |
  14. Jazirehi AR, Huerta-Yepez S, Cheng G, Bonavida B. (2005). Rituximab (chimeric anti-CD20 monoclonal antibody) inhibits the constitutive nuclear factor-{kappa}B signaling pathway in non-Hodgkin's lymphoma B-cell lines: role in sensitization to chemo. Cancer Res 65: 264–276. | PubMed | ISI | ChemPort |
  15. Jazirehi AR, Vega MI, Bonavida B. (2007). Development of rituximab-resistant lymphoma clones with altered cell signaling and cross-resistance to chemotherapy. Cancer Res 67: 1270–1281. | Article | PubMed | ISI | ChemPort |
  16. Jazirehi AR, Vega MI, Chatterjee D, Goodglick L, Bonavida B. (2004). Inhibition of the Raf-MEK1/2-ERK1/2 signaling pathway, Bcl-xL down-regulation, and chemosensitization of non-Hodgkin's lymphoma B cells by Rituximab. Cancer Res 64: 7117–7126. | Article | PubMed | ISI | ChemPort |
  17. Marsit CJ, Zheng S, Aldape K, Hinds PW, Nelson HH, Wiencke JK et al. (2005). PTEN expression in non-small-cell lung cancer: evaluating its relation to tumor characteristics, allelic loss, and epigenetic alteration. Hum Pathol 36: 768–776. | Article | PubMed | ISI | ChemPort |
  18. Osaki M, Oshimura M, Ito H. (2004). PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 9: 667–676. | Article | PubMed | ISI | ChemPort |
  19. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB. (1999). NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401: 82–85. | Article | PubMed | ISI | ChemPort |
  20. Poh TW, Pervaiz S. (2005). LY294002 and LY303511 sensitize tumor cells to drug-induced apoptosis via intracellular hydrogen peroxide production independent of the phosphoinositide 3-kinase-Akt pathway. Cancer Res 65: 6264–6274. | Article | PubMed | ISI | ChemPort |
  21. Rennie PS, Nelson CC. (1998). Epigenetic Mechanisms for Progression of Prostate Cancer. Cancer Metastasis Rev 17: 401–409 Review. | Article | PubMed | ISI | ChemPort |
  22. Rogers BB. (2006). Overview of non-Hodgkin's lymphoma. Semin Oncol Nurs 22: 67–72. | Article | PubMed |
  23. Shan D, Ledbetter JA, Press OW. (1998). Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 91: 1644–1652. | PubMed | ISI | ChemPort |
  24. Sugimori K, Matsui K, Motomura H, Tokoro T, Wang J, Higa S et al. (2005). BMP-2 prevents apoptosis of the N1511 chondrocytic cell line through PI3K/Akt-mediated NF-kappaB activation. J Bone Miner Metab 23: 411–419. | Article | PubMed | ISI | ChemPort |
  25. Tachiiri S, Sasai K, Oya N, Hiraoka M. (2000). Enhanced cell killing by overexpression of dominant-negative phosphatidylinositol 3-kinase subunit, Deltap85, following genotoxic stresses. Jpn J Cancer Res 91: 1314–1318. | PubMed | ChemPort |
  26. Toker A, Yoeli-Lerner M. (2006). Akt signaling and cancer: surviving but not moving on. Cancer Res 66: 3963–3966. | Article | PubMed | ISI | ChemPort |
  27. Uddin S, Hussain A, Al-Hussein K, Platanias LC, Bhatiaa KG. (2004). Inhibition of phosphatidylinositol 3'-kinase induces preferentially killing of PTEN-null T leukemias through AKT pathway. Biochem Biophys Res Commun 320: 932–938. | Article | PubMed | ISI | ChemPort |
  28. Vivanco I, Sawyers CL. (2002). The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2: 489–501. | Article | PubMed | ISI | ChemPort |
  29. Weintraub SJ, Manson SR, Deverman BE. (2004). Resistance to antineoplastic therapy. The oncogenic tyrosine kinase-Bcl-x(L) axis. Cancer Cell 5: 3–4. | Article | PubMed | ISI | ChemPort |
  30. Wetzker R, Rommel C. (2004). Phosphoinositide 3-kinases as targets for therapeutic intervention. Curr Pharm Des 10: 1915–1922. | Article | PubMed | ISI | ChemPort |
  31. Xerri L, Parc P, Brousset P, Schlaifer D, Hassoun J, Reed JC et al. (1996). Predominant expression of the long isoform of Bcl-x (Bcl-xL) in human lymphomas. Br J Haematol 92: 900–906. | Article | PubMed | ISI | ChemPort |
  32. Yin D, Woodruff M, Zhang Y, Whaley S, Miao J, Ferslew K et al. (2006). Morphine promotes Jurkat cell apoptosis through pro-apoptotic FADD/P53 and anti-apoptotic PI3K/Akt/NF-kappaB pathways. J Neuroimmunol 174: 101–107. | Article | PubMed | ISI | ChemPort |
  33. Zhao WL, Daneshpouy ME, Mounier N, Briere J, Leboeuf C, Plassa LF et al. (2004). Prognostic significance of bcl-xL gene expression and apoptotic cell counts in follicular lymphoma. Blood 103: 695–697. | Article | PubMed | ISI | ChemPort |
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Acknowledgements

This work was supported in part by a gift from the JCCC Rosenfield Fund under the directorship of David Leveton. We also thank Maggie Yang and Alina Katsman in the preparation of the manuscript.