The human leukemia cell lines K562, CEM, CEM/VLB 100 , human leukemic blasts, and the bladder cancer J82 cell line have different sensitivities to UV light-induced apoptosis. It is reported that resistance to UV light-induced apoptosis occurs at a point in the apoptotic pathway upstream of caspase-3 but downstream of mitochondrial cytochrome c release. It is demonstrated that the block is due to deficiency of Apaf-1, a critical member of the apoptosome. Sensitivity to apoptosis was independent of caspase-9b or XIAP (inhibitors of apoptosis proteins) expression or levels of procaspase-9. IntroductionThe apoptosome, which includes the protein Apaf-1, cytochrome c/dATP, and procaspase-9, plays a central role in the apoptotic process. [1][2][3][4][5][6][7][8] Cytochrome c is released from mitochondria into the cytosol after the induction of apoptosis by many different stimuli, including CD95, tumor necrosis factor-␣, UV irradiation, and chemotherapeutic and DNA-damaging agents. 1,9,10 Released cytochrome c associates with Apaf-1 in the presence of dATP or ATP and induces the oligomerization of Apaf-1. 4,7,11,12 This recognizes inactive procaspase-9 and forms the apoptosome, triggering autocatalytic processing of procaspase-9. 4,6,13,14 Activated caspase-9 then processes effector caspases (caspase-3, -6, and -7), which in turn cause cell collapse by cleaving a specific set of substrates. By contrast, granzyme B-induced processing of procaspase-3 does not rely on the apoptosome. 15 Cells from Apaf-1 or procaspase-9 knock-out mice are resistant to several apoptotic stimuli. 8,[16][17][18][19] Deletions of Apaf-1 or caspase-9 in ras-and myc-transformed murine cells can replicate the tumorigenic effects of p53 deletion. 8 Oncogene (such as E1A)-transformed cells have increased levels of Apaf-1 and procaspase-9 protein expression and are sensitized to etoposide-induced apoptosis. 5 Inactivation of Apaf-1 and caspase-9 substantially reduces the number of cells required to form tumors. 8 Transfection of the Apaf-1 gene into leukemic cells increases the sensitivity of cells to etoposide-induced apoptosis. 20 An endogenous, alternatively spliced isoform of caspase-9 (named caspase-9b), which lacks the central large subunit caspase domain, can interact with the caspase recruitment domain of Apaf-1 and inhibit the apoptotic process. 21 Even in the presence of functional Apaf-1 and procaspase-9, inhibitors of apoptosis, such as XIAP (inhibitors of apoptosis proteins), can prevent the proteolytic processing of procaspase-3 by blocking the cytochrome c-induced activation of procaspase-9. 22 IAPs, in turn, can be regulated by the mitochondrial protein, Smac. 23,24 These data provide strong evidence that appropriate and functional levels of Apaf-1 or procaspase-9 proteins are crucial for the inhibition of tumor progression and for maintaining the sensitivity of tumor cells to apoptosis. However, it is unknown whether variations in constitutive levels of Apaf-1 have implications for human leukemia.Human leukemic cells differ widely ...

Key Points• HMGB1 and DNA released from CLL cells induce nurse-like cell differentiation.• This differentiation appears TLR9/RAGE dependent.Chronic lymphocytic leukemia (CLL) is a disease of an accumulation of mature B cells that are highly dependent on the microenvironment for maintenance and expansion. However, little is known regarding the mechanisms whereby CLL cells create their favorable microenvironment for survival. High-mobility group protein B-1 (HMGB1) is a highly conserved nuclear protein that can be actively secreted by innate immune cells and passively released by injured or dying cells. We found significantly increased HMGB1 levels in the plasma of CLL patients compared with healthy controls, and HMGB1 concentration is associated with absolute lymphocyte count. We therefore sought to determine potential roles of HMGB1 in modulating the CLL microenvironment. CLL cells passively released HMGB1, and the timing and concentrations of HMGB1 in the medium were associated with differentiation of nurse-like cells (NLCs). Higher CD68 expression in CLL lymph nodes, one of the markers for NLCs, was associated with shorter overall survival of CLL patients. HMGB1-mediated NLC differentiation involved internalization of both receptor for advanced glycation end products (RAGE) and Toll-like receptor-9 (TLR9). Differentiation of NLCs can be prevented by blocking the HMGB1-RAGE-TLR9 pathway. In conclusion, this study demonstrates for the first time that CLL cells might modulate their microenvironment by releasing HMGB1. (Blood. 2014;123(11):1709-1719 IntroductionMany cancers arise from sites of infection, chronic irritation, and inflammation. An inflammatory microenvironment is an important participant in the neoplastic process, fostering proliferation, survival, and migration for cancers, including chronic lymphocytic leukemia (CLL).1-4 Stressed, injured, or dying cells release damage-associated molecular patterns (DAMPs), which initiate noninfectious inflammatory responses. [5][6][7] The DAMP high-mobility group protein B1 (HMGB1) is a major player associating inflammation and cancer. 8,9 HMGB1 is a nuclear protein that can be released passively by damaged or dead cells or actively by immune cells and stressed cancer cells. [10][11][12][13][14] HMGB1 regulates transcription factors but also behaves as a proinflammatory cytokine mediating inflammation. 13,[15][16][17][18] Nonprotein DAMPs, including DNA, RNA, and ATP, are also released by damaged or dying cells. 6,7,19 DAMPs are associated with acute inflammatory responses, chronic inflammation, and wound healing, but are also important components of the disordered tumor microenvironment. 8,20 HMGB1 is a DNA-binding protein, and increased serum concentrations of the HMGB1-DNA complex can activate the immune system and cause systemic autoimmune disease via the receptor for advanced glycation end products (RAGE) and toll-like receptor-9 (TLR9). 21 Interactions of HMGB1-RAGE-TLR9 constitute a tripod that triggers nuclear factor kB (NF-kB) activation 22 and promotes ...

IntroductionBax is a critical element in the induction of apoptosis, and having adequate levels of intracellular Bax protein is crucial for cells to die by apoptosis in response to death signals. During the apoptotic process, Bax translocates from the cytosol to the mitochondria and undergoes conformational changes. Insertion into the mitochondrial membrane is essential for the proapoptotic activity of Bax. However, Bax protein has a shortened half-life in cancer cells because of greatly increased proteasomedependent degradation activity. 1 Low levels of, or absent, Bax protein in malignant cells are associated with significant resistance to cancer therapy. Instability of Bax has been found in several types of malignant cells, including Jurkat T-cell and pre-B acute lymphoblastic leukemia 697 cell lines, 2 primary chronic lymphocytic leukemic cells (CLL), 3 cervical cancer Hela cell line, 4 and advanced human prostate cancer. 1 Decrease of Bax protein was significantly correlated with a poor prognosis in prostate cancer 1 and primary superficialspreading melanoma. 5 Recently, studies have revealed that many proapoptotic molecules are substrates and targets for ubiquitin/proteasome degradation, including p53, 6 truncated Bid (tBid), 7 Bax, 1,4 ARTS [apoptosisrelated protein in the TGF-beta signaling pathway], 8 NOXA, 9 and Bim. 10 Alteration of the stability of these proteins through the ubiquitin/ proteasome-regulated pathway generally contributes to apoptosis resistance and poor prognosis in cancer cells. However, it is unclear whether the status of protein degradation by the ubiquitin/proteasome system could be altered by the treatment of cancer.The levels of all intracellular proteins are determined by the duality of regulation:protein synthesis versus degradation. Basal Bax protein levels or the ratios of Bax/Bcl-2 have been considered to be critical factors for the sensitivity of malignant B cells to anticancer drugs. 11,12 Killing of malignant cells by induction of Bax expression commonly occurs with DNA damaging agents via p53-dependent and p53-independent pathways. [13][14][15][16] Proteasome inhibitors also have the ability to increase or maintain Bax protein levels, which are critical for Bax activation, when used alone or in combination with other agents. [17][18][19][20] Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising anticancer agent. However, malignant B cells are resistant to TRAIL-induced apoptosis. 21,22 We previously found that Bax protein levels decline in leukemic cell lines during treatment with TRAIL. 23 It is unclear whether this could be one of the mechanisms of the resistance of malignant B cells to TRAIL.Bortezomib (PS-341, Velcade) is a novel, first-in-class proteasome inhibitor with antitumor activity against several hematologic and nonhematologic malignancies. 24 In vitro studies showed that Bortezomib selectively inhibits proteasome-dependent degradation of p53, 25 IB, 26 p21, 27 Noxa, 9,28 and TRAIL receptors DR4 and DR5. 29 Proteasome inhibitors h...

Dietary flavonoids have many healthpromoting actions, including anticancer activity via proteasome inhibition. Bortezomib is a dipeptide boronate proteasome inhibitor that has activity in the treatment of multiple myeloma but is not effective in chronic lymphocytic leukemia (CLL). Although CLL cells are sensitive in vitro to bortezomib-induced apoptosis when cultured in medium, the killing activity was blocked when cultured in 50% fresh autologous plasma. IntroductionBortezomib (Velcade) is a first-in-class proteasome inhibitor developed specifically for use as an antineoplastic agent. It is the most potent antineoplastic agent for the treatment of relapsed, refractory multiple myeloma. 1 A total of 73% of patients with myeloma responded to treatment with bortezomib combined with pegylated liposomal doxorubicin. 2 However, only 4 of 15 patients with acute leukemia showed a decrease in blast count. 3 The efficacy of bortezomib in chronic lymphocytic leukemia (CLL) appears to be related to IgV(H) and BCL-6 mutational status, 4 down-regulation of CD23, and inactivation of Notch 2. 5 However, we have previously shown that all primary CLL cells were sensitive to bortezomib in vitro. Proteasome inhibition increased Bax protein accumulation and Bax activation in CLL cells. Bortezomib also increased the sensitivity of CLL cells to TNF-related apoptosis-inducing ligand-induced apoptosis. 6 Although the effectiveness of bortezomib killing of CLL cells in vitro has also been reported by other groups, 4,5 it did not display substantial antitumor activity in patients with CLL. 7,8 The basis of this differential activity of bortezomib on CLL cells in vivo and in vitro is unknown. Autologous plasma is capable of maintaining survival of CLL cells in vitro and conferring resistance to chemo-radiotherapy, 9 and albumin in plasma plays an important role in this survival mechanism. 10 We sought to identify factors in the blood, which could prevent bortezomib-mediated killing of leukemic cells in the circulation.Quercetin is one of the most abundant flavonoids in the human diet and is a potent antioxidant. 11 Quercetin, abundant in human plasma, noncovalently binds to serum albumin. 12,13 It contributes to the prevention of human diseases by promoting relaxation of cardiovascular smooth muscle and protects low-density lipoprotein from oxidation. 14 Recent studies found that quercetin can induce apoptosis by inhibiting proteasome activation, 15 causing G 2 /M-phase arrest 16 and increasing p21 expression. 17 Other dietary flavonoids, such as myricetin, kaempferol, and apigenin, also have similar functions to quercetin with respect to proteasome inhibition and apoptosis induction. 15 In this study, we demonstrate that human plasma affects killing by bortezomib and that the dietary flavonoids, especially quercetin, inhibit bortezomib-induced apoptosis in malignant B-cell lines and primary CLL cells. This inhibitory activity of quercetin was associated with complex formation with bortezomib. However, in myeloma cell lines, quercetin al...

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