Effect Of Homoharringtonine On Induction Of Apoptosis And Differentiation In Diffuse Large B-cell Lymphoma Cell Lines

Background and ObjectiveDiffuse large B-cell lymphoma (DLBCL) is the most common subtype ofnon-Hodgkin’s lymphoma. DLBCL accounts for30%–40%of adult non-Hodgkin’slymphoma, which is a highly invasive and heterogeneous group of cancers.According to immune phenotypes, DCBCL is divided into three subtypes, namely,germinal centre B cell-like subtype (GCB), activated B cell-like subtype (ABC) andIII type. The prognosis of GCB-like subtype is significantly better than that of theother two subtypes. Immunological therapy of molecular targeted drugs CD20rituxancombined with classic chemotherapy regimens have significantly improved the rate ofcomplete remission and disease-free survival of DLBCL patients, but some patientsremain insensitive to immunological therapy or suffer early relapse.Homoharringtonine (HHT) is a plant alkaloid extracted from the total alkaloidsof Cephalotaxaceae. HHT is a highly efficient antitumor drug manufactured in ourcountry. HHT is mainly used in the treatment of acute myeloid leukaemia, chronicmyeloid leukaemia and myelodysplastic syndrome in clinical practice. Many studies have evaluated the effect of HHT on apoptosis of many types of tumour cells in vitro,but the function of HHT in DLBCL has not been investigated. In this study, weobserved the morphology and function of cells treated with HHT to discuss the effectsof HHT on the proliferation, differentiation and telomerase activity of human DLBCLcells in vitro. Moreover, the different effects of HHT between GCB and ABCimmunophenotypes were compared. The results provided anti-DLBCL effects ofHHT to enrich the spectrum of anti-tumour effects of HHT. The study will provide atheoretical basis and a laboratory data for clinical development of HHT in DLBCL asa single agent or in combination.Methods1. In this study, two immunophenotype cell lines SU-4and LY-3were used. Bothcell lines were treated with HHT in different concentrations and different time points.The cell apoptosis rate was detected by flow cytometry using Annexin V/FITCdouble-labelling technique. Cell morphology was observed after Wright stain. Fourdifferent tumour cell lines, namely, MCF-7, SGC-7901, KASUMI and K562, weretreated with HHT in the same concentration to measure the apoptosis rate forcomparison.2. Two immunophenotype cell lines SU-4and LY-3were used in this study. Bothcell lines were treated with20ng/mL HHT at different times, and the cell cyclechanges was detected by flow cytometry.3. Two immunophenotype cell lines SU-4and LY-3were treated with20ng/mLHHT at different times to detect antigens CD19, CD138, IgD and IgM changes at thecell surface by flow cytometry.4. Two immunophenotype cell lines SU-4and LY-3were treated with20ng/mLHHT at different times to detect telomerase activity changes by TRAP-silver staining.Results1. The apoptosis rates were significantly dose-dependent and time-dependentwhen cells were treated with HHT, and the induction of apoptosis in the ABC subtype was significant compared with that in the GCB subtype (P <0.01). The apoptosisrates showed no significant difference when the same HHT concentration was appliedon the MCF-7and SGC-7901cell lines. The48h apoptosis rate of K562cells was46.0767±3.7792. The24and48h apoptosis rates of Kasumi-1cells were59.4867±1.4009and74.0567±3.88472, respectively.2. When two immunophenotype cell lines were treated with40ng/mL HHT for24h, cell morphology changed significantly. Cells treated with HHT showedshrinkage, karyopyknosis, cytoplasmic vesicles and more apoptotic bodies.3. When two immunophenotype cell lines were treated with20ng/mL HHT atdifferent times, a hypodiploid apoptotic peak was observed before the G0/G1phasepeak of both cells. When cells were treated for24h or48h, the G0/G1phase of twoimmunophenotype cell lines was arrested significantly.4. As the treatment time of HHT increased, the positive expression of CD138,IgM and IgD in DLBCL cells gradually increased. The two types of DLBCL cellstreated with HHT for12h,24h or48h showing a significant difference comparedwith control (P <0.01).5. As the treatment time of HHT increased, the telomerase activity graduallyweakened. The telomerase activity of the ABC subtype disappeared after48h oftreatment with HHT, and was inhibited significantly compared with that of the GCBsubtype.Conclusions1. HHT had an obvious pro-apoptotic effect on DLBCL cells, and the effectswere dose-and time-dependent. The sensitivity of the ABC subtype was stronger thanthat of the GCB subtype. At the same concentration, HHT exhibited a strongpro-apoptotic effect on DLBCL compared with other tumours.2. HHT mainly acted on the S and G0/G1phases of DLBCL cells, blocking thecell cycle in the G0/G1phase.3. HHT stimulated the positive expression of CD19, CD138, IgM and IgD inDLBCL cells, and induced differentiation from DLBCL cells towards terminal pro-B cells.4. HHT demonstrated its anti-tumour effects by reducing telomerase activity.