Diffuse large B-cell lymphoma (DLBCL) is the most common type of adult lymphoma. It is a group of malignant tumors with a large number of clinical manifestations and prognoses. Therefore, it is necessary to explore its unknown potential therapeutic targets. Histone deacetylase inhibitor (HDACi) is a novel drug for the treatment of DLBCL, however pan-HDACis cannot be ignored because of their clinical efficacy. By contrast, specific HDACi is well-tolerated, and LMK-235 is a novel HDACi that is a specific inhibitor of HDAC4 and HDAC5. In this study, we investigated the up-regulation of BCLAF1 through NF-κB signaling pathways in LMK-235, mediating the apoptosis of two diffuse large B-cell lymphoma cell lines, OCI-LY10 and OCI-LY3. Further studies showed that BCLAF1 expression was increased in DLBCL cells after treatment with the NF-κB inhibitor Bay11-7082. The combination of Bay11-7082 and siRNA si-HDAC4 significantly increased BCLAF1 expression and further increased apoptosis. These results indicate that BCLAF1 plays an important role in LMK-235-mediated apoptosis and may be a potential target for the treatment of diffuse large B-cell lymphoma.
Imatinib (IM) resistance has become a critical problem for the treatment of patients with relapsed chronic myeloid leukaemia (CML), so novel therapies are in need. Various isotypes of protein kinases C (PKCs) are up-regulated in CML and related with BCR-ABL regulating several signalling pathways that are crucial to malignant cellular transformation. However, it is still unknown whether PKC isotypes play crucial roles in IM resistance. Therefore, we herein used a PKC pan-inhibitor staurosporine (St). To protect normal cells from damage, a proper dose of St was used, at which IM-resistant CML cells were selectively killed in combination with IM but normal cells survived. The IM resistance of CML cells was best reversed by 4 nM St alone, mainly depending on the G2/M phase arrest. Cell cycle-related proteins p21, CDK2, cyclin A and cyclin B were down-regulated. Meanwhile, PKC-a was more significantly decreased than other PKC isotypes at this concentration. The PKC-a-dependent G2/M phase arrest was induced by down-regulation of CDC23, an important regulator of mitotic progression. Low-dose St also reversed IM resistance in vivo. In conclusion, low-dose St selectively increased the sensitivity of IM-resistant CML to IM by arresting cell cycle in the G2/M phase through PKC-a-dependent CDC23 inhibition.
<b><i>Purpose:</i></b> HDAC3, which is associated with smurf2, has been shown to be associated with poor prognosis in B-ALL. This study examined the efficacy of targeting HDAC3 combined with MG-132 as a possible therapeutic strategy for B-ALL patients. <b><i>Methods:</i></b> Real-time PCR and western blot were used to measure the expression of smurf2 and HDAC3 from B-ALL patients bone marrow samples. Sup-B15 and CCRF-SB cells were treated with MG-132, small interfering RNA of smurf2 or HDAC3. A plasmid designed to up-regulate smurf2 expression was transfected into B-ALL cells. Flow cytometry and western blot were used to measure variation due to these treatments in terms of apoptosis and cell cycle arrest. <b><i>Results:</i></b> Expression of Smurf2 and HDAC3 mRNA were inversely related in B-ALL patients. Up-regulation of smurf2 or MG-132 influenced HDAC3, further inhibiting the JAK/signal transducer and activator of transcription 3 (STAT3) signal pathway and inducing apoptosis in B-ALL cells. When we treated Sup-B15 and CCRF-SB cells with siHDAC3 and MG-132 for 24 h, silencing HDAC3 enhanced the apoptosis rate induced by MG-132 in B-ALL cells and further inhibited the JAK/STAT3 pathway. Furthermore, MG-132 was observed to cause G2/M phase arrest in B-ALL cells and inhibited the JAK/STAT3 pathway, leading to apoptosis. <b><i>Conclusions:</i></b> Silencing of HDAC3 enhanced the sensitivity of B-ALL cells to MG-132. The combination of targeting HDAC3 and MG-132 may provide a new avenue for clinical treatment of acute B lymphocytic leukaemia and improve the poor survival of leukaemia patients.
Purpose Smad ubiquitin regulatory factor 2(Smurf2) as a member of ubiquitinated E3 ligase was certificated to regulate the ubiquitination level of target gene. In this study, smurf2 as tumor suppressor was studied in acute B lymphoblastic leukemia, Histone deacetylases 3(HDAC3) which was connected with smurf2 was related with poor prognosis. It is further proved that smurf2 and HDAC3 were expected to be therapeutic targets for B-ALL cells and improve the prognosis of patients with B-ALL. Methods Realtime-PCR and westernblot were used to detect the expression of smurf2 and HDAC3 in healthy donors and B-ALL patients by us. Sup-B15 and CCRF-SB were treated with MG-132, small interfering RNA of smurf2 or HDAC3. Up-regulating smurf2 plasmid was also transfected to B-ALL cells by lipo3000. Flow cytometry and westernblot were used to validate the difference of apoptosis and cell cycle. Results Smurf2 mRNA in B-ALL patients was significantly lower than that in healthy donors. HDAC3 was higher in B-ALL patients than that in healthy donors. Protein level of smurf2 and HDAC3 was consistent with mRNA level. Up-regulating smurf2 by plasmid or MG-132 and silencing smurf2 could influence HDAC3, which further depressed JAK/STAT3 signal pathway inducing apoptosis of B-ALL cells. Sup-B15 and CCRF-SB were treated with siHDAC3 and MG-132 for 24h, we discovered that silencing HDAC3 enhanced the apoptosis level of B-ALL cells MG-132 inducing by depressing JAK/STAT3 pathway. It was expected to become a therapeutic target for improving the clinical prognosis of B-ALL patients. On the other hand, we found that MG-132 caused G2/M phase arrest in B-ALL cells and successfully inhibited the JAK/STAT3 pathway leading to apoptosis. Conclusions Silencing HDAC3 inhibited the JAK/STAT3 pathway and further enhanced apoptosis in B-ALL cells MG-132 inducing. HDAC3 and smurf2 were expected to become therapeutic targets for clinical treatment of acute B lymphocytic leukemia and improve survival rate of leukemia patients. Key words Smurf2; HDAC3; Apoptosis; Prognosis; Disclosures No relevant conflicts of interest to declare.
Myelodysplastic syndrome (MDS) patients would have a chance to become acute myeloid leukemia (AML) when undergoing chemotherapy or waiting for the hematopoietic stem cell transplantation. These patients were not sensitive to demethylation therapy and we should explore deeper mechanisms. According to the WPSS scoring system, we divided 58 MDS patients into four different groups. Real-time PCR results showed the expression of EZH2 or HO-1 in MDS patients were higher than that in normal donors (P<0.05). Even HO-1 and EZH2 were simultaneously increased in some patients, especially in high-risk and extremely very high-risk groups. The linear correlation analysis result of them was 0.42 (P<0.05). In addition, Laser scanning confocal microscopy results also indicated that they were both present in the nucleus of tumor cells. Therefore, we speculated that there was a correlation between EZH2 and HO-1 in MDS patients. Using the High-throughput sequencing to analyze MDS cells, we found that the conversion of MDS to AML may be related to EZH2. The EZH2 in converted MDS patients were significantly higher than that of other MDS patients (P<0.05). Among these patients, we also found that HO-1 and EZH2 were also positively correlated. We found the new EZH2 inhibitor JQEZ5 could significantly promote tumor cells apoptosis in this part of patients. When the concentration of JQEZ5 was 10 umol/mL, the apoptosis rate of tumor cells reached 46.7% after 24 hours (P<0.05). Apoptosis rate was positively correlated with the concentration of JQEZ5 (P<0.05). And tumor cells were significantly inhibited in the G0/G1 cell phase. Cell cycle regulatory genes (CDK4 and CDK6) and apoptosis regulatory genes (Caspase-3 and Caspase-9) would changed. The expression of P15 and P53 would also be affected. In order to verify the malignant degree of MDS cells whether be related to the expression of EZH2. We injected MDS cells into 10 mice. Compared to the control group, MDS cells that highly express EZH2 infiltrated the spleen of experimental mice. Interestingly, the spleens of the experimental group were significantly reduced (0.2CM-0.4CM) and the spleens weight of the experimental group was reduced by 0.028g-0.12g compared to the control group spleens weight. These cells also significantly shortened the survival days of mice and reduced their body weight. Although control mice could survived for 30 days without disease, the time of the experimental mice was significantly shortened (18-25 days). Even One of them survived only 15 days. The results of immunohistochemistry indicated that EZH2 was related to the pRB-E2F pathway. Using the E2F inhibitor HLM006474, we proved HO-1 could regulated EZH2 through the pRB-E2F pathway. Our previous experiments indicated that HO-1 could help leukemia cells resistance and proliferation. The effect of JQEZ5 would be affected when we used hemin and zinc protoporphyrin to regulate HO-1 in MDS cells. The EZH2 was significantly inhibited by JQEZ5 after HO-1 was silenced by siRNAs. Also, the apoptosis rate of MDS cells increased and the cell cycle was arrested in the G0/G1 phase. However, when HO-1 expression was up-regulated by lentivirus, the effects of JQEZ5 were attenuated. MDS patients are frequently in a state of HO-1 enrichment during chemotherapy. HO-1 stimulates MDS patients to transcribe and activate excess EZH2 through pRB-E2F pathway, which increases the chances of becoming AML. Therefore, the conversion of MDS may be attributed to EZH2. In addition, considering HO-1 could promote the expression of EZH2, HO-1 may be a target for enhancing the effects of EZH2 inhibitors on MDS and the influence of HO-1 should be considered in the treatment of patients with high-risk and extremely very high-risk MDS. Disclosures No relevant conflicts of interest to declare.
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