Dietary flavonoids inhibit the anticancer effects of the proteasome inhibitor bortezomib

Abstract 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.

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Human B Cell Lines and Primary B Cells Actively Secrete Granzyme B in Response to IL-2 Family Cytokines.

Blood ◽

10.1182/blood.v110.11.1340.1340 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1340-1340

Author(s):

Bernd Jahrsdoerfer ◽

Sue E. Blackwell ◽

Thomas Simmet ◽

George J. Weiner

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Granzyme B ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Main Function ◽

Dependent Manner ◽

Unexpected Findings

Abstract It is widely believed that the main function of B cells is antibody secretion, but not cellular cytotoxicity. Recently we found that human B cells activated with interleukin 21 (IL-21) and antibodies to the B cell receptor (BCR) or immunostimulatory oligonucleotides (CpG ODN) develop a phenotype similar to that of cytotoxic T lymphocytes. B cells treated in such a way start to secrete large amounts of granzyme B (GrB) instead of antibodies and, as in the case of B-chronic lymphocytic leukemia (B-CLL), acquire the capability to induce apoptosis in bystander B-CLL cells in a GrB-dependent manner. Using FACS and ELISpot analyses we could now demonstrate that GrB is actively secreted by B cells in a time-dependent manner and that IL-21 is not the only cytokine that induces GrB in B cells. Also cytokine combinations such as IL-10 and IL-4 as well as IL-10 and IFN-alpha induce GrB in normal B cells and various B cell lines including MEC-1 (CLL), ARH-77 (plasma cell leukemia) and Namalwa (Burkitts lymphoma). We conclude that IL-21 and further cytokines can induce B cells to produce functional granzyme B. Further studies are required to elucidate the interactions with B lymphocytes of cells producing these cytokines such as CD4+ T cells, regulatory T cells, NKT cells and plasmacytoid dendritic cells. Our unexpected findings could have significant implications on our understanding of the role of B cells in immune regulation and for a variety of immune phenomena including auto-, cancer and infectious immunity.

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FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] Propane 1, 3-diol hydrochloride), Mediates Cytotoxicity through Caspase Independent and Protein Phosphatase 2A Dependent Mechanisms in Chronic Lymphocytic Leukemia and Lymphoblastic Leukemia/Lymphoma.

Blood ◽

10.1182/blood.v108.11.2095.2095 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2095-2095

Author(s):

Qing Liu ◽

Xiaobin Zhao ◽

Frank Frissora ◽

Yihui Ma ◽

Ramasamy Santhanam ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Lines ◽

Protein Phosphatase ◽

Lymphocytic Leukemia ◽

Pp2a Activity ◽

Phosphatase 2A ◽

Induced Apoptosis ◽

Pp2a Activation

Abstract FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] propane 1, 3-diol hydrochloride) is a synthetic compound produced by modification of a natural immunosuppressant, ISP-1. It is an immunosuppressive agent that is being developed to prevent organ transplant rejection. Recent studies indicate additional role for FTY720 in inducing cell apoptosis. We demonstrate here a novel mechanism by which FTY720 mediates cytotoxic effects in cell lines representing different B cell malignancies and primary B cells from chronic lymphocytic leukemia (CLL) patients. FTY720 induced apoptosis as detected by annexin V/ propidium iodide staining in representative B cell lines and CLL patient derived CD19+ B cells in time and dose dependent manner (p<0.0001, untreated vs 10mM-treated CLL cells, n=15). In contrast to previous reports in T cell lines, FTY720 induced cytotoxicity in Raji cell line and primary CLL cells is independent of activation of caspase 3, 8 and 9 or poly-ADP ribose polymerase cleavage. Further, pan-caspase inhibitor Z-VAD-fmk rescued these cells from fludarabine but not FTY720 induced apoptosis (p=0.001 fludarabine vs fludarabine+z-VAD-fmk; p=0.99 FTY720 vs FTY720+z-VAD-fmk, n=5). Over-expression of Bcl-2 failed to protect transformed B-cells from FTY720 induced apoptosis suggesting Bcl-2 independent cytotoxic effect. Interestingly, FTY720 induced consistent increase in protein phosphatase 2a (PP2a) activity and concentrations of okadaic acid that inhibited the FTY720-induced PP2A activity also resulted in inhibition of FTY720-mediated cytotoxicity in B cell lines and primary CLL cells, indicating a role for PP2A activation in FTY720 induced cytotoxicity. Consistent with its activation of PP2A, FTY720 induced dephosphorylation of of Erk1/2 in CLL B cells. Further, FTY720 treatment resulted in significant in-vivo therapeutic efficacy associated with prolonged survival in a xenograft SCID mouse model of disseminated B cell lymphoma/leukemia (median survival time for FTY720 treated mice was 47 days (95 %CI 39–53) compared to 18 days in placebo controls (95% CI 17–19) P<0.0001-FTY720 vs placebo). These results provide first evidence for a PP2A dependent and caspase independent cytotoxicity of FTY720 in B cells. The novel caspase and Bcl-2 independent mechanism of cytotoxicity concurrent with identification of PP2a activation as a surrogate marker of cell killing provide further justification for clinical development of this agent in lymphoid leukemia.

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Haloperidol Induced Cell Cycle Arrest and Apoptosis in Glioblastoma Cells

Biomedicines ◽

10.3390/biomedicines8120595 ◽

2020 ◽

Vol 8 (12) ◽

pp. 595

Author(s):

Fotios Papadopoulos ◽

Rafaela Isihou ◽

George A. Alexiou ◽

Thomas Tsalios ◽

Evrysthenis Vartholomatos ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Combined Treatment ◽

Caspase 8 ◽

Dependent Manner ◽

Anticancer Activities ◽

Tumor Cell Death ◽

Trypan Blue Exclusion ◽

Innovative Strategy ◽

Induced Apoptosis

Although several antipsychotic drugs have been shown to possess anticancer activities, haloperidol, a “first-generation” antipsychotic drug, has not been extensively evaluated for potential antineoplastic properties. The aim of this study was to investigate the antitumoral effects of haloperidol in glioblastoma (GBM) U87, U251 and T98 cell lines, and the effects of combined treatment with temozolomide (TMZ) and/or radiotherapy, using 4 Gy of irradiation. The viability and proliferation of the cells were evaluated with trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis, using the annexin-propidium iodide (PI), and cell cycle, cluster of differentiation (CD) expression and caspase-8 activation were measured using flow cytometry. Treatment with haloperidol significantly reduced cell viability in U87, U251 and T98 GBM cell lines. Haloperidol induced apoptosis in a dose-dependent manner, inhibited cell migration and produced an alteration in the expression of CD24/CD44. The additional effect of haloperidol, combined with temozolomide and radiation therapy, increased tumor cell death. Haloperidol was observed to induce apoptosis and to increase caspase-8 activation. In conclusion, haloperidol may represent an innovative strategy for the treatment of GBM and further studies are warranted in glioma xenograft models and other malignancies.

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Resistance to BTK inhibition by ibrutinib can be overcome by preventing FOXO3a nuclear export and PI3K/AKT activation in B-cell lymphoid malignancies

Cell Death and Disease ◽

10.1038/s41419-019-2158-0 ◽

2019 ◽

Vol 10 (12) ◽

Cited By ~ 10

Author(s):

Isha Kapoor ◽

Yue Li ◽

Arishya Sharma ◽

Huayuan Zhu ◽

Juraj Bodo ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Nuclear Export ◽

Acquired Resistance ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Nuclear Accumulation ◽

Lymphoid Malignancies ◽

Ibrutinib Resistance ◽

Induced Apoptosis

AbstractChronic activation of the Bruton’s tyrosine kinase (BTK)-mediated B-cell receptor (BCR) signaling is a hallmark of many B-cell lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL). Ibrutinib, an FDA approved, orally administered BTK inhibitor, has demonstrated high response rates, however, complete responses are infrequent and acquired resistance to BTK inhibition can emerge. In this study, we generated ibrutinib-resistant (IB-R) cell lines by chronic exposure of CLL and activated B-cell (ABC)-DLBCL cells to ibrutinib in order to investigate the mechanism of acquired resistance to ibrutinib. IB-R cell lines demonstrated downregulation of FOXO3a and PTEN levels and activation of AKT, with their levels being low in the nuclei of resistant cells in comparison to the sensitive counterparts. Inhibition of PI3K and AKT using idelalisib and MK2206, respectively increased ibrutinib-induced apoptosis in IB-R cells by downregulation of pAKT473 and restoring FOXO3a levels, demonstrating the importance of these cell survival factors for ibrutinib-resistance. Notably, the exportin 1 inhibitor, selinexor synergized with ibrutinib in IB-R cells and restored nuclear abundance of FOXO3a and PTEN, suggesting that nuclear accumulation of FOXO3a and PTEN facilitates increase in ibrutinib-induced apoptosis in IB-R cells. These data demonstrate that reactivation of FOXO3a nuclear function enhances the efficacy of ibrutinib and overcomes acquired resistance to ibrutinib. Together, these findings reveal a novel mechanism that confers ibrutinib resistance via aberrant nuclear/cytoplasmic subcellular localization of FOXO3a and could be exploited by rational therapeutic combination regimens for effectively treating lymphoid malignancies.

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Mediation of apoptosis by and antitumor activity of lumiliximab in chronic lymphocytic leukemia cells and CD23+ lymphoma cell lines

Blood ◽

10.1182/blood-2007-03-082024 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1594-1602 ◽

Cited By ~ 57

Author(s):

Nuzhat I. Pathan ◽

Peter Chu ◽

Kandasamy Hariharan ◽

Carolyn Cheney ◽

Arturo Molina ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Antitumor Activity ◽

B Cell ◽

Cell Lines ◽

Human Monoclonal Antibody ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Synergistic Cytotoxicity ◽

Induced Apoptosis

AbstractLumiliximab is a chimeric macaque-human monoclonal antibody to CD23, a protein expressed on virtually all chronic lymphocytic leukemia (CLL) cells. We examined the ability of lumiliximab to mediate apoptosis, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity against primary CLL cells and CD23-expressing B-cell lines. Our data suggest that lumiliximab kills CLL cells and CD23-expressing B cells predominantly by apoptosis, which occurs through the intrinsic pathway. Lumiliximab-induced apoptosis was accompanied by the down-regulation of antiapoptotic proteins Bcl-2, Bcl-XL, and XIAP, activation of Bax, and release of cytochrome c from the mitochondria. We also found that the addition of lumiliximab to rituximab or fludarabine results in synergistic cytotoxicity of primary CLL cells and CD23-expressing B-cell lines. We investigated the in vivo activity of lumiliximab in a human disseminated CD23+ B-cell lymphoma SCID mouse model and found greater antitumor activity with it than with control antibody. We also found that paralysis-free survival was greater with lumiliximab plus rituximab or fludarabine than with any of those agents alone. These results suggest that lumiliximab may be an effective treatment alone or in combination with rituximab or chemotherapy agents in CLL or other CD23-overexpressing B-cell malignancies.

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Pharmacologic Targeting MCL1 with AZD5991 Induces Apoptosis and Mitochondrial Dysfunction in Non-Hodgkin Lymphoma (NHL) Cells

Blood ◽

10.1182/blood-2020-134951 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 33-33

Author(s):

Tingting Liu ◽

Fei Xu ◽

Elana Thieme ◽

Vi Lam ◽

Guang Fan ◽

...

Keyword(s):

B Cells ◽

Mitochondrial Dysfunction ◽

B Cell ◽

Cell Lines ◽

Small Molecule ◽

Research Funding ◽

Synthetic Lethality ◽

Lymphocytic Leukemia ◽

Dependent Manner ◽

Oncology Research

BCL2 family proteins determine cell fate and comprise pro-apoptotic "initiators" (NOXA, BIM, PUMA), anti-apoptotic "guardians" (BCL2, MCL1, BCLX) and pro-apoptotic effectors (BAX/BAK). Venetoclax, a BCL2 inhibitor, received regulatory approval in therapy of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia. However, venetoclax is modestly effective in NHL. MCL1 is a short-lived pro-survival protein that is frequently overexpressed in NHL, leading to increased cell survival and drug resistance. MCL1 transgenic mice develop lymphoma, mostly of the diffuse large B-cell (DLBCL) type. Thus, selective targeting MCL1 represents a promising pharmacologic strategy in NHL. AZD5991 is a small molecule inhibitor highly selective towards MCL1 (Tron et al, 2018) in clinical trials. Here we explored pre-clinical activity of AZ5591 in NHL. Experiments were conducted in activated B-cell (ABC)-like (OCI-LY3, U-2932) and germinal center (GC)-like (OCI-LY18/19, SUDHL4/6/10, VAL) DLBCL cell lines; parental and ibrutinib-resistant Mino mantle cell lymphoma (MCL) cell lines. Peripheral blood mononuclear cells were isolated from patients with CLL and MCL and co-cultured with BAFF- or CD40L-expressing stroma. AZD5991 was obtained from Activ Biochem Ltd. MCL1 expression was detected in 10 tested DLBCL cell lines, highly expressed in 7/10. Meanwhile, BCLX was upregulated in eight, and BCL2 in 5/10 lines. Four cell lines expressed all three proteins. To confirm relevance of this data, we conducted analysis of primary DLBCL lymph nodes (n=30). MCL1 and BCLX were expressed in 50% and 80% of GC-like, and 10% and 25% of non-GC tumors, respectively. Treatment with AZD5991 restricted growth of DLBCL cells in a dose-dependent manner. GC-like cell lines VAL, SU-DHL4 and SU-DHL6 were highly susceptible with IC50~0.2 µM. Interestingly, they expressed relatively low levels of MCL1. Similarly, parental and ibrutinib-resistant Mino MCL cells were susceptible to MCL1 inhibition, with IC50 of 0.1 µM and 0.5 µM, respectively. Meanwhile, SU-DHL10, OCI-LY19 (GC-like), OCI-LY3 and U-2932 (ABC-like) cells were resistant to AZD5591. Consistent with its mechanism of action, immunoprecipitation assays showed that AZD5591 displaced BIM from MCL1 in NHL cells. We then evaluated MCL1 inhibition in primary neoplastic B-cells. CLL and MCL cells from patients were co-cultured with either CD40L- or BAFF-expressing stroma for 24 h. While CD40L predominantly induced BCLX, BAFF upregulated MCL1 in those cells. Consistent with this, BAFF-stimulated cells were highly sensitive to AZD5991, while CD40L-stimulated cells exhibited resistance. Since MCL1 acts in balance with pro-apoptotic effectors BAX and BAK at the mitochondrial membrane, we assessed the physiological effects of MCL1 inhibition on the mitochondrial function. Treatment with AZD5991 induced mitochondrial depolarization and led to a reduction in mitochondrial mass as well as increased generation of reactive oxygen species. This was accompanied by a decrease in cellular maximal respiratory capacity in both DLBCL and parental/ibrutinib-resistant MCL cells. Meanwhile, the rate of glycolysis was not significantly impacted. Interestingly, MCL1 inhibition induced mitophagy in sensitive (VAL) but not resistant cells (OCI-LY3). Next, we evaluated AZD5991 for synthetic lethality in a functional MTS-based screening assay using a panel of 189 small molecule inhibitors that target a variety of distinct signaling pathways activated in cancer (Tyner et al, 2018). AZD5991 demonstrated synergy with other BH3-mimetics. Co-treatment of DLBCL cells with BCL2/X inhibitors AZ4320, venetoclax and navitoclax overcame resistance to AZD5991. In summary, MCL1 inhibition using selective BH3-mimetic AZD5991 restricts cell proliferation and induces apoptosis in a subset of DLBCL, ibrutinib-resistant MCL cells and primary neoplastic B cells. MCL1 inhibition leads to mitochondrial dysfunction and mitophagy. Resistance to MCL1 inhibition may be overcome by concurrent targeting of alternative anti-apoptotic proteins (BCL2/X) in NHL. Disclosures Tyner: Syros: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Aptose: Research Funding; Constellation: Research Funding; AstraZeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Petra: Research Funding; Agios: Research Funding. Danilov:Abbvie: Consultancy; Celgene: Consultancy; Rigel Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Research Funding; Aptose Biosciences: Research Funding; Astra Zeneca: Consultancy, Research Funding; Verastem Oncology: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; TG Therapeutics: Consultancy; Nurix: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Karyopharm: Consultancy.

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Targeting the mTORC2 signaling complex in B cell malignancies

10.1101/564500 ◽

2019 ◽

Author(s):

Wei Liao ◽

Gwen Jordaan ◽

Angelica Benavides-Serrato ◽

Brent Holmes ◽

Joseph Gera ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

B Cell Malignancies ◽

Signaling Complex ◽

Mantle Cell ◽

Pkc Alpha ◽

Induced Apoptosis ◽

Mtorc2 Complex

AbstractHyperactive PI3 kinase-Akt (PI3K-Akt) signaling has an important role in cell growth and resistance to apoptosis in B cell malignancies. Inhibition of this pathway by blocking PI3K activity, and or inhibiting mTORC1/2 signaling complexes is an active area of research in B cell leukemia/lymphoma such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). With a tissue-scan array, the expression of Rictor is a component of the mTORC2 complex was determined by quantitative PCR in a number of B cell malignancies. Rictor was found to be over-expressed in CLL and MCL cells as compared to normal B cells with no over-expression in Hodgkins and non-Hodgkins lymphomas. Inactivation of Rictor was performed by shRNA in two Mantle cell lines and these stable Rictor knockdown cell lines demonstrated a slower growth of cells as compared to scrambled shRNA control. In addition, there was a decrease of mTORC2 signaling and B cell receptor (BCR) cross-linking mediated Akt (Ser473) and NDRG1 (Thr 346) phosphorylation. To specifically disrupt the mTORC2 signaling complex and target Rictor overexpression, previously identified inhibitors that block Rictor and MTOR interaction in a yeast two-hybrid system were analyzed. Treatment of primary CLL specimens with these inhibitors followed by immunoprecipitation experiments confirmed the disruption of the mTORC2 complex. These inhibitors also induced apoptosis in CLL specimens and were more effective than rapamycin, an MTOR inhibitor and pp242, an mTORC1 and 2 inhibitors, at equimolar concentrations. Treatment of CLL specimens with the lead inhibitor, compound#6, resulted in inhibition of p-Akt, p-GSK 3 beta, p-PKC alpha, p-Foxo1, and p-Foxo3, with minimal effect on the phosphorylation of an mTORC1 target gene, S6 kinase. In comparison with Idelalisib (CAL-101), a clinically approved PI3Kinase p110 delta inhibitor in CLL, comp#6 is more effective in inducing apoptosis in primary CLL specimens at equimolar concentrations (mean 51.2, SD 21.7 as compared to mean 26.9, SD 17.2). The data support the effectiveness of these novel inhibitors that specifically disrupt the mTORC2 complex in primary CLL specimens.

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Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL

Blood ◽

10.1182/blood-2007-08-110445 ◽

2008 ◽

Vol 111 (5) ◽

pp. 2797-2805 ◽

Cited By ~ 57

Author(s):

Feng-Ting Liu ◽

Samir G. Agrawal ◽

John G. Gribben ◽

Hongtao Ye ◽

Ming-Qing Du ◽

...

Keyword(s):

B Cells ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Resistant Cell ◽

Protein Levels ◽

Bax Protein ◽

Degradation Activity ◽

Potent Drug ◽

Induced Apoptosis

Proapoptotic Bcl-2 family member Bax is a crucial protein in the induction of apoptosis, and its activation is required for this process. Here we report that Bax is a short-lived protein in malignant B cells and Bax protein levels decreased rapidly when protein synthesis was blocked. Malignant B cells were relatively resistant to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)–induced apoptosis, and this correlated with low basal Bax protein levels. Furthermore, during treatment with TRAIL, the resistant cell lines showed prominent Bax degradation activity. This degradation activity was localized to mitochondrial Bax and could be prevented by truncated Bid, a BH3-only protein; in contrast, cytosolic Bax was relatively stable. The proteasome inhibitor bortezomib is a potent drug in inducing apoptosis in vitro in malignant B-cell lines and primary chronic lymphocytic leukemic (CLL) cells. In CLL cells, bortezomib induced Bax accumulation, translocation to mitochondria, conformational change, and oligomerization. Accumulation and stabilization of Bax protein by bortezomib-sensitized malignant B cells to TRAIL-induced apoptosis. This study reveals that Bax instability confers resistance to TRAIL, which can be reversed by Bax stabilization with a proteasome inhibitor.

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