Feng-Ting Liu

Dietary flavonoids inhibit the anticancer effects of the proteasome inhibitor bortezomib

Dietary flavonoids have many health-promoting actions, including anticancer activity via proteasome inhibition. Bor-tezomib 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. Dietary flavonoids, quercetin and myricetin, which are abundant in plasma, inhibited bortezomib-induced apoptosis of primary CLL and malignant B-cell lines in a dose-dependent manner. This inhibitory effect was associated with chemical reactions between quercetin and the boronic acid group, -RB(OH)2, in bortezomib. The addition of boric acid diminished the inhibitory effect of both quercetin and plasma on bortezomib-induced apoptosis. The protective effect was also reduced when myeloma cell lines, but not B-cell lines, were preincubated with quercetin, indicating a direct effect of quercetin on myeloma cells. At high doses, quercetin itself induced tumor cell death. These data indicate that dietary flavonoids limit the efficacy of bortezomib, whereas supplemental inorganic boric acid is able to reverse this. The complex interactions between quercetin, tumor cells, and bortezomib mean caution is required when giving dietary advice to patients.

Extracellular HMGB1 promotes differentiation of nurse-like cells in chronic lymphocytic leukemia.

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.

CD160 signaling mediates PI3K-dependent survival and growth signals in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (CLL) expresses CD160, a glycosylphosphatidylinositol-linked receptor found on normal natural killer (NK) and T cells, but not B cells. CD160 is a multifunctional molecule in normal lymphocytes, but its role in CLL biology is unknown. In vitro, CLL cells undergo rapid spontaneous apoptosis, which CD160 activation protected against-mean cell viability increased from 67% to 79% (P < .001). This was associated with up-regulation of Bcl-2, Bcl-xL, and Mcl-1, but not Bax. As expected from these changes in Bcl-2/Bax and Bcl-xL/Bax ratios, CD160 triggering reduced mitochondrial membrane potential collapse and cytochrome c release. CD160 stimulation also induced DNA synthesis, cell cycle progression, and proliferation. B-cell antigen receptor (BCR)-induced CLL proliferation was generally greater than with CD160, but marked variation was seen. Both BCR and CD160 signaling led to CLL secretion of interleukin-6 (IL-6) and IL-8, although CD160 induced greater increases of IL-6 (51-fold) and IL-8 (15-fold). Survival and activation signals mediated by CD160 showed dose-dependent suppression by phosphoinositide-3 kinase (PI3K) inhibitors. Thus, in vitro, CLL cells can use the CD160 pathway for survival and activation, mimicking CD160 signaling in normal NK and CD8(+) T cells. Establishing the pathophysiologic relevance of these findings may reveal new therapeutic targets.

Potential mechanisms of leukemia cell resistance to TRAIL-induced apopotosis

There are many factors contributing to the resistance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. However, it is not clear whether the mechanism of resistance to TRAIL is constitutive or inductive. Therefore, the purpose of this study was to investigate the resistant mechanisms to TRAIL at different levels in the apoptotic pathway. The human T-lymphoblastic leukemic CEM cell line showed more resistant to TRAIL-induced apoptosis compared with the human chronic myeloid leukemic K562 cell line. Lower level of constitutive caspase-8 expression in the CEM cell line led to a poor response to both TRAIL-induced activation of caspase-3 and reduction in the mitochondrial membrane potential (ΔΨm). There was no significant difference in the constitutive levels of NF-κB in CEM and K562 cell lines. However, CEM cells showed a faster response to TRAIL-induced NF-κB activation than K562 cells. TRAIL-induced regulation of Bcl-2 family of proteins included an up-regulation in Bcl-2/Bcl-XL and a down-regulation in Bax. IAPs, such as XIAP, cIAP-1, cIAP-2 and Survivin were all up-regulated during the treatment with TRAIL. In summary, our data suggest that the leukemic cells resistance to TRAIL-induced apoptosis might be due to the deficiency in the constitutive caspase-8 expression. Development of potential resistance to apoptosis by TRAIL can occur in both TRAIL-resistant and TRAIL-sensitive leukemic cells.

Liposomal encapsulation diminishes daunorubicin-induced generation of reactive oxygen species, depletion of ATP and necrotic cell death in human leukaemic cells

Summary. In this study, we tested the mechanisms of daunorubicin (DNR)‐ and the liposomal encapsulated daunorubicin (DaunoXome or DNX)‐induced killing in three human leukaemic cell lines, K562, K/Bax and CEM. DNX showed less cytotoxicity in leukaemic cells than conventional DNR. The intracellular accumulation of DNX was 10 times less than conventional DNR during exposure to drugs for up to 5 h. Cell cycle analysis indicated that DNR induced concentration‐dependent G2/M arrest, apoptosis and necrosis. However, DNX induced G2/M arrest and apoptosis but not necrotic cell death, even at a higher concentration. DNR‐ or DNX‐induced activation of caspase‐9 and ‐3 was detected at concentrations that induced apoptosis and necrosis. The sensitivity of leukaemic cells to DNR‐ and DNX‐induced apoptosis correlated with the activation of caspases and the reduction of mitochondrial membrane potential (ΔΨm), but not the depletion of ATP and the generation of reactive oxidative species (ROS). DNX did not provoke ROS generation and ATP depletion in leukaemic cells. We conclude that the liposomal encapsulation of DNR restricts the intracellular accumulation speed and therefore diminishes ROS generation, ATP depletion and necrotic cell death. This may have implications for the cause of cardiotoxicity seen with DNR, its main dose‐limiting step.

Increase in the ratio of mitochondrial Bax/Bcl-XL induces Bax activation in human leukemic K562 cell line

The p53- and Bcl-2-negative leukemic K562 cell line showed resistant to DNA damage-induced Bax activation and apoptosis. The constitutive balanced ratio of Bax/Bcl-XL in K562 mitochondria allowed the formation of active Bax and cytochrome c release from mitochondria in the presence of a BH3-only protein, tBid, in a cell-free system. Bax transfection led to Bax undergoing a conformational change, translocation to mitochondria and homo-oligomerization but not apoptosis in the K562 cell line. After treatment with UV light, while Bcl-XL but not Bax translocated to mitochondria in K562, both Bax and Bcl-XL translocated to mitochondria in the Bax stable transfectant K/Bax cells. The increased ratio of Bax/Bcl-XL in K/Bax mitochondria led to an increased conformationally changed Bax, formation of the homo-multimer of Bax-Bax, and a reduced hetero-dimerization of Bax-Bcl-XL. Increased proportion of active Bax was accompanied with increased percentage of apoptosis. We therefore demonstrate that direct increase in the ratio of mitochondrial Bax/Bcl-XL can induce Bax activation in the p53- and Bcl-2-negative leukemic cells. Increased Bcl-XL translocation and failure in Bax translocation from cytosol to mitochondria play important roles in preventing Bax activation.

BH3-domain mimetic compound BH3I-2¢ induces rapid damage to the inner mitochondrial membrane prior to the cytochrome c release from mitochondria

Summary. The Bcl‐2 family proteins are major regulators of cell survival and death in human leukaemia. BH3‐containing peptides induce apoptosis by binding to the hydrophobic pocket of the anti‐apoptotic proteins, such as Bcl‐2 or Bcl‐XL. A small cell‐permeable compound, BH3I‐2′ (3‐iodo‐5‐chloro‐N‐[2‐chloro‐5‐((4‐chlorophenyl)sulphonyl)phenyl]‐2‐hydroxybenzamide), has been recently reported to have a function similar to Bak BH3 peptide. BH3I‐2′ induces apoptosis by disrupting interactions mediated by the BH3 domain, between pro‐apoptotic and anti‐apoptotic members of the Bcl‐2 family. This study found that BH3I‐2′ induced cytochrome c release from the mitochondrial outer membrane in a Bax‐dependent manner and that this correlated with the sensitivity of leukaemic cells to apoptosis. Moreover, it also induced rapid damage to the inner mitochondrial membrane, represented by a rapid collapse of mitochondrial membrane potential (ΔΨm), prior to the cytochrome c release. This occurred both in whole cells and isolated mitochondria, and was not associated with the sensitivity of cells to BH3I‐2′‐induced apoptosis. Exogenous Bcl‐2 or Bcl‐XL neutralized BH3I‐2′in vitro and diminished its effect on the inner mitochondrial membrane. Our results indicate that BH3I‐2′ not only induces cytochrome c release from the outer mitochondrial membrane but also damages the inner mitochondrial membrane, probably by interacting with Bcl‐2 family proteins.

Differential and tumor-specific expression of CD160 in B-cell malignancies

CD160 is a human natural killer (NK)-cell-activating receptor that is also expressed on T-cell subsets. In the present study, we examined 811 consecutive cases of B-cell lymphoproliferative disorders (B-LPDs), and demonstrated CD160 expression in 98% (590 of 600) of chronic lymphocytic leukemia (CLL) cases, 100% (32 of 32) of hairy cell leukemia (HCL) cases, 15% (5 of 34) of mantle cell lymphoma (MCL) in the leukemic phase, and 16% (23 of 145) of other B-LPD cases. CD160 transcript and protein were absent in the normal B-cell hierarchy, from stem cells, B-cell precursors, maturing B cells in the germinal center, and circulating B cells, including CD5(+)CD19(+) B1 cells in umbilical cord. CD160 positivity was significantly higher in CLL and HCL in terms of percentage (65.9% and 67.8%, respectively, P < .0001) and median fluorescence intensity (552 and 857, respectively, P < .0001) compared with all other B-LPD cases. Lymph node CLL samples were also CD160(+). Using the disease-specific expression of CD5, CD23, and CD160, a score of 3 characterized CLL (diagnostic odds ratio, 1430); a score of 0 excluded CLL, MCL, and HCL; and the CD23/CD5 ratio differentiated CLL from leukemic CD23(+) MCL. In the B-cell lineage, CD160 is a tumor-specific antigen known to mediate cellular activation signals in CLL, and is a novel target for therapeutic manipulation and monitoring of minimal residual disease.

Why bortezomib cannot go with ‘green’?

Eat more ‘green’ or eat ‘five a day’ is one of the most important healthy lifestyle behaviours in the 21 century. Aiming to fight cancer effectively, more than half patients use vitamins or herbs concurrently with conventional anticancer treatment. Flavonoids or polyphenols existing in vegetables, fruits and green tea are common plant pigments with antioxidant properties and considered acting as cancer preventing or anti-cancer agents. Recently it was found that some flavonoids and vitamin C in diet or supplements have antagonistic effect with the anti-cancer drug bortezomib. Bortezomib is a specific inhibitor for proteasome and is currently used for treatment of relapsed and refractory multiple myeloma. Despite its successful rates in treating multiple myeloma and other solid tumors, it is unable to kill leukemic cells in the blood. It was recently revealed that some flavonoids and vitamin C present in green leaves and green teas in the blood can neutralize bortezomib by directly interaction between two chemicals. Here we summarize why dietary flavonoids should be avoided in patients who take bortezomib as chemotherapeutic drug.

Generation of reactive oxygen species is not involved in idarubicin-induced apoptosis in human leukaemic cells

The anthracycline antibiotic idarubicin (IDA) induces double‐stranded DNA breaks, the generation of reactive oxygen species (ROS) and apoptosis in human leukaemic cells. It is unclear whether the generation of ROS is associated with the apoptotic process. Using the T‐lymphoblastic leukaemic CEM cell line, we found that IDA‐induced DNA breaks were correlated with final cell death. The reduction in mitochondrial membrane potential (ΔΨm) and the generation of ROS occurred simultaneously with IDA‐induced activation of caspase‐9 and caspase‐3. Inhibition of caspases by a pan‐caspase inhibitor, benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone (Z‐VAD‐fmk) completely blocked IDA‐induced reduction of ΔΨm, apoptosis and final cell death. Interestingly, ROS generation was significantly enhanced by Z‐VAD‐fmk. ROS generation was neither caspase dependent nor part of the apoptotic process. IDA‐mediated reduction in ΔΨm is caspase dependent and is not a consequence of the generation of ROS. These results indicate that IDA‐induced generation of ROS and apoptosis are separate events. Inhibition of caspases facilitates IDA‐mediated generation of ROS.