Gao Gx

Targeting Autophagy Augments In Vitro and In Vivo Antimyeloma Activity of DNA-Damaging Chemotherapy

Purpose: Although autophagy occurs in most tumor cells following DNA damage, it is still a mystery how this DNA-damaging event turns on the autophagy machinery in multiple myeloma (MM) and how the functional status of autophagy impacts on its susceptibility to death in response to DNA-damaging chemotherapy. Experimental Design: We investigate the effects of DNA damage on autophagy in MM cells and elucidate its underlying molecular mechanism. Then, we examined the impacts of pharmacologic or genetic inhibition of autophagy on DNA damage–induced apoptosis. Furthermore, the antimyeloma activity of autophagy inhibitor in combination with DNA-damaging agents was evaluated in MM xenograft models. Results: We showed that DNA-damaging drugs, doxorubicin and melphalan, induce caspase-dependent apoptosis and concurrently trigger Beclin 1–regulated autophagy in human MM cell lines H929 and RPMI 8226. Mechanistically, association of autophagy execution proteins Beclin 1 with class III phosphoinositide 3-kinase, which is inhibited by Bcl-2 recruitment, contributes directly to the autophagic process. Importantly, targeting suppression of autophagy by minimally toxic concentrations of pharmacologic inhibitors (hydroxychloroquine and 3-methyladenine) or short hairpin RNAs against autophagy genes, Beclin 1 and Atg5, dramatically augments proapoptotic activity of DNA-damaging chemotherapy both in vitro using MM cell lines or purified patient MM cells and in vivo in a human plasmacytoma xenograft mouse model. Conclusion: These data can help unravel the underlying molecular mechanism of autophagy in DNA-damaged MM cells and also provide a rationale for clinical evaluation of autophagy inhibitors in combination with DNA-damaging chemotherapy in MM. Clin Cancer Res; 17(10); 3248–58. ©2011 AACR.

Dysregulation of unfolded protein response partially underlies proapoptotic activity of bortezomib in multiple myeloma cells.

The 26S proteasome inhibitor, bortezomib, has shown remarkable therapeutic efficacy in multiple myeloma (MM), however, the mechanism by which this compound acts remains unknown. Here, we have demonstrated that bortezomib targets the prototypical expression of unfolded protein response (UPR) genes BiP, CHOP and XBP-1 at the mRNA and protein levels, resulting in induction of proapoptotic UPR outputs and suppression of cytoprotective UPR components, leading to caspase-dependent apoptosis in human MM H929 and 8226/S cell lines. Moreover, knockdown of XPB-1s, via lentivirus-mediated RNA interference approach, sensitises MM cells to apoptosis induction by bortezomib. Together, these data strongly suggest that dysregulated or disruptive UPR may, at least partly, underlie the antimyeloma activity of bortezomib.

Risk factors for multiple myeloma: A hospital-based case–control study in Northwest China

BACKGROUND The distinctive racial/ethnic and geographic distribution of multiple myeloma (MM) suggests that both family history and environmental factors may contribute to its development. METHODS A hospital-based case-control study consisting of 220 confirmed MM cases and 220 individually matched patient controls, by sex, age and hospital was carried out at 5 major hospitals in Northwest China. A questionnaire was used to obtain information on demographics, family history, and the frequency of food items consumed. RESULTS Based on multivariate analysis, a significant association between the risk of MM and family history of cancers in first degree relatives was observed (OR=4.03, 95% CI: 2.50-6.52). Fried food, cured/smoked food, black tea, and fish were not significantly associated with the risk of MM. Intake of shallot and garlic (OR=0.60, 95% CI: 0.43-0.85), soy food (OR=0.52, 95% CI: 0.36-0.75) and green tea (OR=0.38, 95% CI: 0.27-0.53) was significantly associated with a reduced risk of MM. In contrast, intake of brined vegetables and pickle was significantly associated with an increased risk (OR=2.03, 95% CI: 1.41-2.93). A more than multiplicative interaction on the decreased risk of MM was found between shallot/garlic and soy food. CONCLUSION Our study in Northwest China found an increased risk of MM with a family history of cancer, a diet characterized by low consumption of garlic, green tea and soy foods, and high consumption of pickled vegetables. The effect of green tea in reducing the risk of MM is an interesting new finding which should be further confirmed.

Increased expression of TIGIT on CD4+ T cells ameliorates immune-mediated bone marrow failure of aplastic anemia.

Aplastic anemia (AA) is an autoimmune disease in which T cell activation is suspected to play an important role. T cell immunoglobulin and ITIM (immunoreceptor tyrosine‐based inhibition motif) domain (TIGIT) is an inhibitory receptor, which exhibits inhibitory functions on the immune response. However, its role in AA has not been clearly determined. In the current study, we showed that the frequency of TIGIT‐positive CD4+ T cells was reduced in the vast majority of AA patients (85%, 17/20). In TIGIT‐silenced human CD4+ T cells, stimulation of agonistic anti‐TIGIT monoclonal antibody significantly facilitated cell proliferation, increased production of IL‐2 and IFN‐γ, and inhibited production of IL‐10. However, in TIGIT‐overexpressed human CD4+ T cells, cell proliferation and the production of IL‐2, IFN‐γ, and TNF‐α were significantly hindered; in contrast, the secretion of IL‐10 was improved. RT‐PCR and Western blotting showed that T‐bet expression in human CD4+ T cells was significantly decreased by TIGIT overexpression, but only slightly altered by TIGIT knockdown. In mouse models, lentivirus‐mediated TIGIT‐overexpressed CD4+ T cell transfer significantly rescued the decreased red blood cell count, attenuated the increase in serum INF‐γ and TNF‐α levels, and lengthened the median survival time. The mRNA levels of CD34, stem cell factor (SCF), and granulocyte/macrophage‐colony‐stimulating factor (GM‐CSF) in bone marrow mononuclear cells were also up‐regulated. In conclusion, increased expression of TIGIT could inhibit the function of CD4+ T cells in vitro and ameliorate immune‐mediated bone marrow failure of AA in vivo providing a new potential strategy for the treatment of AA. J. Cell. Biochem. 115: 1918–1927, 2014.

RhoA effector mDia1 is required for PI 3-kinase-dependent actin remodeling and spreading by thrombin in platelets

The RhoA effector mDia1 is involved in controlling the balance between filamentous and monomeric actin, but its role in modulating thrombin-induced actin remodeling and platelet spreading on fibrinogen matrices remains unclear. In this study, mDia1 was shown to translocate to the platelet cytoskeleton following thrombin stimulation, in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner. Anti-mDia1 loading or pretreatment with PI 3-kinase inhibitors essentially abrogated thrombin-elicited actin stress fiber formation, with a corresponding decrease in the proportion of platelets exhibiting a fully spread morphology. We also investigated the mechanisms underlying the effects of mDia1 on thrombin-induced actin remodeling and platelet spreading, and found that these involved PI 3-kinase-mediated induction of mDia1 interaction with RhoA. Collectively, these results suggest that the PI 3-kinase/RhoA/mDia1 axis is a critical pathway for coupling thrombin signaling to actin cytoskeletal remodeling during platelet spreading.

Bryostatin 5 induces apoptosis in acute monocytic leukemia cells by activating PUMA and caspases

Acute leukemia is a malignant clonal hematopoietic stem cell disease. In the current study, we examined the effects of bryostatin 5 on acute monocytic leukemia cells in vitro and in vivo. We also explored the mechanisms and pathways underlying the increase in apoptosis induced by bryostatin 5. Bryostatin 5 inhibited the growth of primary acute monocytic leukemia cells and U937 cells in a dose- and time-dependent manners. Bryostatin 5 also induced an increase in apoptosis and a decrease in the mitochondrial membrane potential (MMP) in U937 cells. Transmission electron microscopy (TEM) revealed that bryostatin 5-treated cells displayed typical apoptotic characteristics (chromatin condensation, karyopyknosis and formation of crescents and apoptotic bodies). In addition, bryostatin 5 increased the expression of P53 upregulated modulator of apoptosis (PUMA) and slightly increased P53 expression. Bryostatin 5 also significantly decreased Bcl-XL expression and significantly increased the expression levels of Bak, Bax, cleaved caspase 9 and cleaved caspase 3. The pro-apoptotic activity of bryostatin 5 in U937 cells was inhibited by PUMA siRNA and z-LEHD-fmk (a specific caspase 9 inhibitor). In addition, the PUMA siRNA significantly affected the expression of cleaved caspase 9, whereas z-LEHD-fmk had little effect on the expression of PUMA. The results suggest that PUMA is located upstream of caspase 9 in this apoptotic signaling pathway. These novel findings provide mechanistic insight into the induction of apoptosis by bryostatin 5 and might facilitate the development of clinical strategies to enhance the therapeutic efficacy of treatments for acute monocytic leukemia.

Increased 14-3-3ζ Expression in the Multidrug-Resistant Leukemia Cell Line HL-60/VCR as Compared to the Parental Line Mediates Cell Growth and Apoptosis in Part through Modification of Gene Expression

Background: Acute myeloid leukemia (AML) recurrence is largely a result of multidrug resistance (MDR). We aimed to examine the role of 14-3-3ζ in AML chemosensitivity using HL-60 and vincristine-resistant HL-60/VCR cells. Methods: The effects of 14-3-3ζ siRNA on the growth and cell cycle progression of HL-60 and HL-60/VCR cells were determined. The effect of 14-3-3ζ siRNA on topotecan (TPT)-induced apoptosis was evaluated by several assays. Results: Compared to HL-60 cells, HL-60/VCR cells had increased 14-3-3ζ mRNA and protein expression. Increased mdr-1 mRNA as well as mdr-1, Bcl-2 and Mcl-1 protein expression were observed in HL-60/VCR cells. In both HL-60 and HL-60/VCR cells, 14-3-3ζ was observed in the cytoplasm and nuclear compartments. 14-3-3ζ siRNA significantly reduced HL-60 and HL-60/VCR cell growth after 48 h and increased the proportion of cells in the G0/G1 phase. Moreover, 14-3-3ζ siRNA significantly increased the sensitivity of both HL-60 and HL-60/VCR cells to TPT, possibly through the inhibition of Bcl-2, Mcl-1 and mdr-1 protein expression. Conclusions: Silencing of 14-3-3ζ increased the sensitivity of both sensitive and resistant HL-60 cells to TPT-induced apoptosis, possibly through altering the expression of apoptosis-associated proteins, suggesting that it may be a potential target for MDR AML.

Distribution of lymphoid neoplasms in Northwest China: Analysis of 3244 cases according to WHO classification in a single institution

To explore the distribution of lymphoid neoplasms in Northwest China, the clinical and pathological data of lymphoma patients from 2006 to 2014 were analyzed according to the WHO classification in Xijing Hospital. Of the 3244 cases, mature B-cell neoplasms occupied 60.7%, while mature T/NK-cell neoplasms and Hodgkins lymphomas (HL) occupied 26.2% and 8.1%, respectively. The most common subtype of lymphoma was diffuse large B-cell lymphoma (35.0%), followed by extranodal NK/T-cell lymphoma, nasal type (ENKTCL) (12.9%) and marginal zone B-cell lymphoma (7.8%). Mixed cellularity (34.0%) was the most common subtype of HL. The especially high proportion of ENKTCL was the most outstanding feature of our study in comparison to previous reports. The mean age of all lymphoid neoplasms cases was 51years and most subtypes showed male predominance, with an average male-female ratio of 1.6. Extranodal lymphomas took up about 60% of all cases and gastrointestinal tract was the most frequently involved site. In conclusion, the distribution of lymphoid neoplasms of Northwest China showed some features similar to previous reports of China and other countries, but some subtypes presented distinct features.