| Objective:Mantle cell lymphoma (MCL), deriving from B cells, is a special subtypeof Non-Hogkin's lymphoma. It is characterized as high aggressive, poorprognosis and incurable. Although new therapies, such as bortezomib,rituximab, have been applied on clinic, the prognosis of patients have notremarkable improvement. Therefore, new therapies are urgently needed.Histone deacetylase inhibitors (HDACis) target as making histone acetylation,inducing apoptosis and cell cycle arrest, inhibiting the proliferation of tumorcells. Moreover, HDACis interact synergistically with some antineoplasticdrugs. The purpose of this study is to investigate the influence of SAHA withbortezomib on human MCL cell line Z-138, explore the mechanism, look fornew therapy for MCL and provide theory accordances and experiment datasfor clinic.Methods:1. Cell culture of Z-138The human MCL cell line Z-138cells were maintained in RPMI1640,containing10%fetal bovine serum,100units/ml penicillin and100μg/mlstreptomycin. Z-138cells were cultured at37℃in a humidified of5%CO2atmosphere. The cells were passaged every two or three days. All experimentswere using logarithmically growing cells, and cells' viability≥95%tested bytrypan blue staining.2. The effect of SAHA and/or bortezomib on Z-138cells tested by MTT2.1experiment of SAHA inhibited the growth of Z-138Z-138cells, which in exponential growth state, were seeded in96-wellplates,at a density of2×105cells/ml. The control groups were added100μl the cell nutrient fluid, and the experimental groups were added differentconcentrations of SAHA, made the final concentrations were0.10,0.25,0.50,0.75,1.00μM, respectively. Four hours before the ending point, added10μlMTT in each well, then centrifugate the plate. After this, discarding thesupernatants, adding150μl DMSO to each well, shaking the plate until theprecipitate dissolved. Lastly, using the elisa reader to measure each well'sabsorbance at570nm.2.2bortezomib inhibited the proliferation of Z-138cellsTaking Z-138cells in logarithm vegetal period, divided into the controlgroups and the experimental groups, randomly. The control groups were added100μl cell culture, while the experimental groups were exposed to1,2,4,6,8,10nM bortezomib, respectively, then using the above method to measure theabsorbance of each well.2.3SAHA and bortezomib synergistically inhibited the growth of Z-138cellsThe Z-138cells were seeded in96-well plates, then divided these cellsinto four groups: control group, SAHA group, bortezomib group, SAHA andbortezomib group, randomly. Using MTT test to measure the absorbance ofeach well.3.Assessment of apoptosis and cell cycleThe extent of apoptosis were evaluated by propidium iodide (PI) staining.Cells were cultured in the presence or absence of SAHA and/or bortezomib for36h. After washing the cells, staining cells with PI (50mg/ml). Both sampleswere analyzed by FCM to detect cell apoptosis ratio and cell cycle.4.Test the expression of NF-κB (p65) proteinTake Z-138cells, divided into the control groups and the experimentalgroups. The experimental groups were exposed to SAHA and/or bortezomibfor24h, while the control groups were added only cell culture. After washingthe cells with4℃PBS, added NF-κB (p65) antibody to each group. Half anhour later, added deuto-antibody into each group, then using FCM to test theexpression of NF-κB (p65) protein. 5. Test the expression of bcl-2proteinTake Z-138cells, divided into the control group, SAHA group,bortezomib group and SAHA+bortezomib group, randomly. Z-138cells wereexposed to SAHA and/or bortezomib for24h. After washing the cells with4℃PBS, added bcl-2antibody, lefting cells reaction with bcl-2antibody at37℃for half an hour, added deuto-antibody to each group, then using FCM to testthe expression of bcl-2protein.6. Test the expression of CD20Z-138cells, which in exponential growth state, were cultured in culturebottle, at a density of5×105cells/ml. Divided Z-138cells into control groupsand experimental groups. The control groups were added cell nutrient fluid,while the experimental groups were added different concentrations of SAHA,made the final concentrations are0.005,0.010,0.100μM, respectively.Twenty-four hours later, collecting and washing the Z-138cells, adding CD20antibody. One hour later, using FCM to test the expression of CD20.Results:1. Under a certain dose scope, SAHA inhibited the proliferation of Z-138cells, in a time and concentration-dependent manner. While the concentrationwas0.10,0.25,0.50,0.75,1.00μM, the correspondence inhibition ratio for24h was (3.50±0.51)%,(9.95±0.99)%,(25.33±1.44)%,(38.43±1.74)%,(50.16±1.80)%;48h cell inhibition ratio was (6.65±1.18)%,(16.65±1.45)%,(43.15±1.59)%,(62.97±2.38)%,(79.82±3.42)%;72h cell inhibition ratiowas(11.16±1.11)%,(28.64±2.74)%,(64.34±3.63)%,(83.24±4.30)%,(86.74±2.87)%, the difference was significant (P<0.05).2. Under a certain dose scope, bortezomib suppressed the growth of Z-138cells, in a time and concentration-dependent manner. While theconcentration of bortezomib was1,2,4,6,8,10nM, the correspondence cellinhibition ratio for24h was (3.52±0.65)%,(8.41±0.77)%,(18.61±1.44)%,(49.48±1.09)%,(66.33±2.00)%,(83.24±4.24)%;48h cell inhibition ratiowas (4.65±0.28)%,(11.24±0.39)%,(23.24±1.24)%,(58.71±3.36)%,(74.92±3.54)%,(87.73±1.65)%;72h cell inhibition ratio was (6.73±0.62)%, (16.84±1.16)%,(31.31±0.53)%,(68.13±1.48)%,(82.63±1.72)%,(90.45±2.44)%, the difference was significant (P<0.05).3.SAHA interacted synergistically with bortezomib on inhibiting theproliferation of Z-138cells. For example, the cell inhibition ratio was(6.65±1.18)%,(4.65±0.28)%and (30.55±0.58)%for0.10μM SAHA,1nMbortezomib and the combination of both drugs, respectively. While theinhibition ratio for0.25μM SAHA,2nM bortezomib and the combination ofboth drugs was (16.65±1.45)%,(11.24±0.39)%and (50.79±1.99)%.4.Both SAHA and bortezomib could induce the apoptosis of Z-138cellsand cell cycle arrest at G2/M stage, in a concentration-dependent manner.Along with the concentration adding, the ratio of apoptosis was raising, thecell number at G2/M stage was increasing. Combination SAHA andbortezomib, cell apoptosis ratio was markable increasing. Simultaneously,more cells were arrested at G2/M stage. The control group,0.25μM SAHAgroup,2nM bortezomib group and0.25μM SAHA+2nM bortezomib, thecorresponding apoptosis ratio was (0.48±0.06)%,(2.25±0.21)%,(1.90±0.11)%,(23.79±1.46)%for36h, the difference was significant (P<0.05). Thecontrol group,1.00μM SAHA group,10nM bortezomib group and1.00μMSAHA+10nM bortezomib, the ratio of cells at G2/M stage was (9.07±0.86)%,(10.93±0.38)%,(16.47±1.06)%,(32.33±0.55)%for36h,respectively, the difference was significant (P<0.05).5.Under a certain concentration scope, both SAHA and bortezomib coulddownregulate the expression of NF-κB (p65) and bcl-2, while combinationthem, the expression of NF-κB (p65) and bcl-2was markable downregulated.The control group,0.50μM SAHA group,6nM bortezomib group and0.50μM SAHA+6nM bortezomib group treat Z-138cells for24h, the expressionof NF-κB (p65) was474.08±10.11,401.22±11.24,388.34±11.28,242.78±17.12, respectively, the difference was significant (P<0.01). Thecontrol group,0.50μM SAHA group,6nM bortezomib group and0.50μMSAHA+6nM bortezomib group treat Z-138cells for24h, the expression ofbcl-2was444.57±9.81,413.14±9.61,417.52±10.47,372.77±8.98, respectively, the difference was significant (P<0.01).6.At low dose, SAHA could uprelugate the expression of CD20. Afterdifferent concentration SAHA treatment Z-138cells for24h, the expression ofCD20was increased. The CD20expression of the control group,0.005μMSAHA group and0.010μM SAHA group was498.14±6.32,517.70±5.98and559.49±5.21, respectively, the difference was significant (P<0.05).Conclusions:1.Under a certain concentration scope, SAHA could inhibit theproliferation of human MCL cell line Z-138and induce cell cycle arrest, in atime and concentration-dependent manner. The possible mechanism wasdownregulate the expression of NF-κB (p65) and bcl-2protein.2.Under a certain concentration scope, combination SAHA withbortezomib could synergistically inhibit the proliferation of Z-138cells, thepossible mechanism was synergistically downregulate the expression of NF-κB (p65) and bcl-2protein.3.At low dose, SAHA could obviously upregulate the expression ofCD20, increase the sensitivity of the cells to rituximab, may ameliorate cellresistance to rituximab and synergistic with rituximab, but need futher study toconfirmed. |
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