Xingming Deng

Phosphorylation of Bcl2 and regulation of apoptosis

Members of the Bcl2 family of proteins are important regulators of programmed cell death pathways with individual members that can suppress (eg Bcl2, Bcl-XL) or promote (eg Bax, Bad) apoptosis. While the mechanism(s) of Bcl2’s anti-apoptotic function is not yet clear, introduction of Bcl2 into most eukaryotic cell types will protect the recipient cell from a wide variety of stress applications that lead to cell death. There are, however, physiologic situations in which Bcl2 expression apparently fails to protect cells from apoptosis (eg negative selection of thymocytes). Further, Bcl2 expression in patient tumor samples does not consistently correlate with a worse outcome or resistance to anticancer therapies. For example, patient response and survival following chemotherapy is independent of Bcl2 expression at least for pediatric patients with ALL. These findings indicate that simple expression of Bcl2 may not be enough to functionally protect cells from apoptosis. The finding that Bcl2 is post-translationally modified by phosphorylation suggests another level of regulation of function. Recent studies have shown that agonist-activated phosphorylation of Bcl2 at serine 70 (single site phosphorylation), a site within the flexible loop domain (FLD), is required for Bcl2’s full and potent anti-apoptotic function, at least in murine IL-3-dependent myeloid cell lines. Several protein kinases have now been demonstrated to be physiologic Bcl2 kinases indicating the importance of this post-translational modification. Since Bcl2 phosphorylation has been found to be a dynamic process involving both a Bcl2 kinase(s) and phosphatase(s), a mechanism exists to rapidly and reversibly regulate Bcl2’s activity and affect cell viability. In addition, multisite Bcl2 phosphorylation induced by anti-mitotic drugs like paclitaxel may inhibit Bcl2 indicating the potential wide range of functional consequences that this post-translational modification may have on function. While post-translational mechanisms other than phosphorylation may also regulate Bcl2’s function (eg ubiquitination), this review will focus on the regulatory role for phosphorylation and discuss its potential clinical ramifications.

Novel Role for JNK as a Stress-activated Bcl2 Kinase

Interleukin (IL)-3-induced Bcl2 phosphorylation at Ser70 may be required for its full and potent antiapoptotic activity. However, in the absence of IL-3, increased expression of Bcl2 can also prolong cell survival. To determine how Bcl2 may be functionally phosphorylated following IL-3 withdrawal, astress-activated Bcl2 kinase (SAK) was sought. Results indicate that anisomycin, a potent activator of the stress kinase JNK/SAPK, can induce Bcl2 phosphorylation at Ser70 and that JNK1 can be latently activated following IL-3 withdrawal to mediate Bcl2 phosphorylation. JNK1 directly phosphorylates Bcl2 in vitro, co-localizes with Bcl2, and collaborates with Bcl-2 to mediate prolonged cell survival in the absence of IL-3 or following various stress applications. Dominant-negative (DN)-JNK1 can block both anisomycin and latent IL-3 withdrawal-induced Bcl2 phosphorylation (>90%) and potently enhances cell death. Furthermore, low dose okadaic acid (OA), a potent protein phosphatase 1 and 2A inhibitor, can activate the mitogen-activated protein kinases JNK1 and ERK1/2, but not p38 kinase, to induce Bcl2 phosphorylation and prolong cell survival in factor-deprived cells. Since PD98059, a specific MEK inhibitor, can only partially inhibit OA-induced Bcl2 phosphorylation but completely blocks OA-induced Bcl2 phosphorylation in cells expressing DN-JNK1, this supports the conclusion that OA may stimulate Bcl2 phosphorylation via a mechanism involving both JNK1 and ERK1/2. Collectively, these findings indicate a novel role for JNK1 as a SAK and may explain, at least in part, how functional phosphorylation of Bc12 can occur in the absence of growth factor.

Mono- and multisite phosphorylation enhances Bcl2's antiapoptotic function and inhibition of cell cycle entry functions

Bcl2 functions to suppress apoptosis and retard cell cycle entry. Single-site phosphorylation at serine 70 (S70) is required for Bcl2s antiapoptotic function, and multisite phosphorylation at threonine 69 (T69), S70, and S87 has been reported to inactivate Bcl2. To address this apparent conflict and identify the regulatory role for Bcl2 phosphorylation in cell death and cell cycle control, a series of serine/threonine (S/T) → glutamate/alanine (E/A) mutants including T69E/A, S70E/A, S87E/A, T69E/S70A/S87A (EAA), T69A/S70E/S87A (AEA), T69A/S70A/S87E (AAE), T69E/S70E/S87E (EEE), and T69A/S70A/S87A (AAA) was created to mimic or abrogate, respectively, either single-site or multisite phosphorylation. The survival and cell cycle status of cells expressing the phosphomimetic or nonphosphorylatable Bcl2 mutants were compared. Surprisingly, all of the E but not the A Bcl2 mutants potently enhance cell survival after stress and retard G1/S cell cycle transition. The EEE Bcl2 mutant is the most potent, indicating a possible cumulative advantage for multisite phosphorylation of Bcl2 in survival and retardation of G1/S transition functions. Because the E-containing Bcl2 mutants, but not the A-containing mutants, can more potently block cytochrome c release from mitochondria during apoptotic stress, even at times when steady-state expression levels are similar for all mutants, we conclude that phosphorylation at one or multiple sites within the flexible loop domain of Bcl2 not only stimulates antiapoptotic activity but also can regulate cell cycle entry.

A Functional Role for Nicotine in Bcl2 Phosphorylation and Suppression of Apoptosis

Nicotine is not only a major component in tobacco but is also a survival agonist that inhibits apoptosis induced by diverse stimuli including chemotherapeutic drugs. However, the intracellular mechanism(s) involved in nicotine suppression of apoptosis is unclear. Bcl2 is a potent antiapoptotic protein and tumor promotor that is expressed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells. It is possible that nicotine may regulate Bcl2 to stimulate cell survival. Here we report that nicotine can induce Bcl2 phosphorylation exclusively at the serine 70 site in association with prolonged survival of SCLC H82 cells expressing wild-type but not the phosphorylation-deficient S70A mutant Bcl2 after treatment with chemotherapeutic agents (i.e. cisplatin or VP-16). Nicotine induces activation of PKCα and the MAPKs ERK1 and ERK2, which are physiological Bcl2 kinases. Furthermore, ET-18-OCH3, a specific phospholipase C (PLC) inhibitor, blocks nicotine-stimulated Bcl2 phosphorylation and promotes apoptosis, suggesting that PLC may be involved in nicotine activation of Bcl2 kinases. Using a genetic approach, the gain-of-function S70E mutant, which mimics Ser70 site phosphorylation in the flexible loop domain, potently enhances chemoresistance in SCLC cells. Thus, nicotine-induced cell survival results, at least in part, from a mechanism that involves Bcl2 phosphorylation. Therefore, novel therapeutic strategies for lung cancer in which Bcl2 is expressed may be used to abrogate the anti-apoptotic activity of Bcl2 by inhibiting multiple upstream nicotine-activated pathways.

Reversion of multidrug resistance by co-encapsulation of doxorubicin and curcumin in chitosan/poly(butyl cyanoacrylate) nanoparticles

Co-encapsulated doxorubicin (DOX) and curcumin (CUR) in poly(butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared with emulsion polymerization and interfacial polymerization. The mean particle size and mean zeta potential of CUR-DOX-PBCA-NPs were 133 ± 5.34 nm in diameter and +32.23 ± 4.56 mV, respectively. The entrapment efficiencies of doxorubicin and curcumin were 49.98 ± 3.32% and 94.52 ± 3.14%, respectively. Anticancer activities and reversal efficacy of the formulations and various combination approaches were assessed using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide assay and western blotting. The results showed that the dual-agent loaded PBCA-NPs system had the similar cytotoxicity to co-administration of two single-agent loaded PBCA-NPs (DOX-PBCA-NPs+CUR-PBCA-NPs), which was slightly higher than that of the free drug combination (DOX+CUR) and one free drug/another agent loaded PBCA-NPs combination (DOX+CUR-PBCA-NPs or CUR+DOX-PBCA-NPs). The simultaneous administration of doxorubicin and curcumin achieved the highest reversal efficacy and down-regulation of P-glycoprotein in MCF-7/ADR cell lines, an MCF-7 breast carcer cell line resistant to adriamycin. Multidrug resistance can be enhanced by combination delivery of encapsulated cytotoxic drugs and reversal agents.

Bcl2's Flexible Loop Domain Regulates p53 Binding and Survival

ABSTRACT p53 not only functions as a transcription factor but also has a direct, apoptogenic role at the mitochondria. We have discovered that DNA damage-induced p53-Bcl2 binding is associated with decreased Bcl2-Bax interaction and increased apoptotic cell death in a mechanism regulated by Bcl2s flexible loop regulatory domain (FLD), since purified p53 protein can disrupt the Bcl2/Bax complex by directly binding to a negative regulatory region of the FLD (amino acids [aa] 32 to 68). Deletion of the negative regulatory region (Δ32-68) abolishes Bcl2-p53 binding and enhances Bcl2s antiapoptotic function. Conversely, removal of a positive regulatory region (aa 69 to 87) of the FLD, which contains the Bcl2 phosphorylation site(s) T69, S70, and S87, enhances Bcl2-p53 binding and significantly abrogates Bcl2s survival activity. The phospho-mimetic T69E/S70E/S87E (EEE) but not the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutant displays a reduced capacity to bind p53 and potently inhibits p53-induced cytochrome c release from isolated mitochondria. Furthermore, the FLD-only aa32-87 and aa32-68 peptides but not the aa69-87 peptide can directly bind p53 in vitro. p53-induced cytochrome c release occurs through a mechanism involving Baxs integral insertion into the outer mitochondrial membrane. Either DNA damage to cells or expression of p53 selectively targeted to the mitochondria results in Bcl2-p53 binding followed by exposure of Bcl2s BH3 domain in association with inactivation of Bcl2s antiapoptotic function, indicating a conformational change in Bcl2 can occur upon direct ligation of p53. Thus, Bcl2s FLD contains both positive and negative regulatory regions which functionally regulate Bcl2s antiapoptotic activity by affecting Bax or p53 binding.

Bcl2 negatively regulates DNA double-strand-break repair through a nonhomologous end-joining pathway.

Bcl2 can enhance susceptibility to carcinogenesis, but the mechanism(s) remains fragmentary. Here we discovered that Bcl2 suppresses DNA double-strand-break (DSB) repair and V(D)J recombination by downregulating Ku DNA binding activity, which is associated with increased genetic instability. Exposure of cells to ionizing radiation enhances Bcl2 expression in the nucleus, which interacts with both Ku70 and Ku86 via its BH1 and BH4 domains. Removal of the BH1 or BH4 domain abrogates the inhibitory effect of Bcl2 on Ku DNA binding, DNA-PK, and DNA end-joining activities, which results in the failure of Bcl2 to block DSB repair as well as V(D)J recombination. Intriguingly, Bcl2 directly disrupts the Ku/DNA-PKcs complex in vivo and in vitro. Thus, Bcl2 suppression of the general DSB repair and V(D)J recombination may occur in a mechanism by inhibiting the nonhomologous end-joining pathway, which may lead to an accumulation of DNA damage and genetic instability.

Ceramide Regulates Protein Synthesis by a Novel Mechanism Involving the Cellular PKR Activator RAX

The sphingolipid ceramide is an important second signal molecule and potent apoptotic agent. The production of ceramide is associated with virtually every known stress stimulus, and thus, generation of this sphingolipid has been suggested as a universal feature of apoptosis. Recent studies suggest that an important component of cell death following diverse stress stimuli (e.g. interleukin-3 withdrawal, sodium arsenite treatment, and peroxide treatment) is the activation of the double-stranded RNA-activable protein kinase, PKR, resulting in the inhibition of protein synthesis (Ito, T., Jagus, R., and May, W. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7455–7459). The recently discovered cellular PKR activator, RAX, is phosphorylated in association with PKR activation (Ito, T., Yang, M., and May, W. S. (1999) J. Biol. Chem. 274, 15427–15432). Since RAX is phosphorylated by an as yet undetermined SAPK and ceramide is a potent activator of SAPKs such as JNK, a role for ceramide in the activation of RAX might be possible. Results indicate that overexpression of exogenous RAX potentiates ceramide-induced killing. Furthermore, ceramide can potently inhibit protein synthesis. Since ceramide potently promotes RAX and eukaryotic initiation factor-2α phosphorylation, a possible role for ceramide in this process may involve the activation of PKR by RAX. Since 2-aminopurine, a serine/threonine kinase inhibitor that has previously been shown to inhibit PKR, blocks both the potentiation of ceramide killing by RAX and ceramide-induced inhibition of protein synthesis, ceramide appears to promote PKR activation, at least indirectly. Collectively, these findings suggest a novel role for ceramide in the regulation of protein synthesis and apoptosis.

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.

Disruption of STAT3 by Niclosamide Reverses Radioresistance of Human Lung Cancer

A major challenge affecting the outcomes of patients with lung cancer is the development of acquired radioresistance. However, the mechanisms underlying the development of resistance to therapy are not fully understood. Here, we discovered that ionizing radiation induces phosphorylation of Janus-associated kinase (JAK)-2 and STAT3 in association with increased levels of Bcl2/Bcl-XL in various human lung cancer cells. To uncover new mechanism(s) of radioresistance of lung cancer, we established lung cancer cell model systems with acquired radioresistance. As compared with radiosensitive parental lung cancer cells (i.e., A549, H358, and H157), the JAK2/STAT3/Bcl2/Bcl-XL survival pathway is significantly more activated in acquired radioresistant lung cancer cells (i.e., A549-IRR, H358-IRR, and H157-IRR). Higher levels of STAT3 were found to be accumulated in the nucleus of radioresistant lung cancer cells. Niclosamide, a potent STAT3 inhibitor, can reduce STAT3 nuclear localization in radioresistant lung cancer cells. Intriguingly, either inhibition of STAT3 activity by niclosamide or depletion of STAT3 by RNA interference reverses radioresistance in vitro. Niclosamide alone or in combination with radiation overcame radioresistance in lung cancer xenografts. These findings uncover a novel mechanism of radioresistance and provide a more effective approach to overcome radioresistance by blocking the STAT3/Bcl2/Bcl-XL survival signaling pathway, which may potentially improve lung cancer outcome, especially for those patients who have resistance to radiotherapy. Mol Cancer Ther; 13(3); 606–16. ©2013 AACR.