Synergistic Antitumor Interaction Of Chidamide With Doxorubicin In Peripheral T-cell Lymphoma

Objiective:In this study, we first detected the frequency of HDAC2 expression in peripheral T-cell lymphoma-unspecified(PTCL-U) tissue and analyzed the relationship between HDAC2 and variable clinical parameters as well as prognosis of patients, and then sought to determine whether HDAC inhibitor CS055 inhibits proliferation、induces cell cycle arrest and apoptosis of Jurkat cells. Further, to explore the synergies and the related molecular mechanisms of CS055 in combination with doxorubicin on Jurkat cells, and provide new ideas for the treatment of PTCL, for improving the prognosis of the patients with PTCL.Methods:1. The expression of HDAC2 protein in PTCL-U tissue was detected by immunohistochemistry, and analysis of the relationship between HDAC2 expression and clinical features and prognosis of patients according to statistical software SPSS 17.0.2. MTS/PMS assay and Compu Syn software were used to investigate the effect of CS055 alone and combined with doxorubicin on the growth of Jurkat cells.3. PI staining and flow cytometry were used to investigate the effect of CS055 on the cell cycle of Jurkat cells.4. Annexin V/PI staining and flow cytometry were used to detect the effect of CS055 and combination of CS055 with doxorubicin on the apoptosis of Jurkat cells.5. Rhomdamine 123 staining was used to detect the effect of CS055 and combination of CS055 with doxorubicin on the mitochondrial membrane potential of Jurkat cells.6. The synergistic mechanism of CS055 to doxorubicin in Jurkat cells was evaluated by Western blotting.Results:1. Immunohistochemistry showed that the high expression rate of HDAC2 in PTCL-U tissue could reach to 70.3%(26/37).2. High HDAC2 expression had a statistically significant association with elevated LDH(P=0.025)and stages III-IV(P=0.001). No correlation with the sex, age of patients, B symptoms, bone marrow involvement, IPI score,β2-microglobulin(β2-MG), or Ki-67 was found(P>0.05).3. According to the Kaplan-Meier, high HDAC2 expression was associsted with significantly decreased survival time(32.1 vs 16.0, P=0.032).4. Univariate analysis identified that HDAC2 is an unfavorable prognostic factor for PTCL-U patients(P=0.032). However, multivariate Cox model analysis revealed that HDAC2 cannot be an independent prognostic factor(P=0.722).5. CS055 treatments caused growth arrest of Jurkat cells in a time- and dose-dependent manner, and CS055 synergistically enhanced doxorucibin-induced growth inhibition of Jurkat cells.6. PI staining and flow cytometry detection showed that CS055 treatments induced G0/G1 cell cycle arrest in a dose-dependent fashion in Jurkat cells. However, the ratio of Jurkat cells in G2/M phase was significantly increased by doxorucibin alone. Thus, regarding inducing cell cycle arrest, combination of CS055 with doxorubicin has not a synergistic effect.7. CS055 treatments of Jurkat cells resulted in dose-dependent induction of modest apoptosis, and CS055 significantly enhanced doxorucibin-induced apoptosis determined by flow cytometry analysis with Annexin V/PI staining. The difference was statistically significant(P<0.01).8. After stained by Rhodamine 123, CS055 treatments of Jurkat cells could result in decrease of mitochondria membrane potential, and combined treatments of Jurkat cells with CS055 and doxorucibin resulted in further mitochondria membrane potential loss compared to treatments with CS055 alone.9. Western blot analysis demonstrated the dose-dependent accumulation of acetylated histone H3 in Jurkat cells after treatment with CS055. However,CS055 treatment had no effects on protein levels for HDAC2. Treatments of Jurkat cells with combined CS055 and doxorubicin increased expression ofγH2AX, decreased expression of Bcl-2, and activated caspase-3, caspase-9 and PARP.Conclusion:1. HDAC2 is overexpression in PTCL-U tissue [high expression rate:70.3%(26/37)]. In addition, high HDAC2 expression has a statistically significant association with elevated LDH(P=0.025)and stages III-IV(P=0.001). Further,high HDAC2 expression is associsted with significantly decreased survival time(32.1 vs 16.0, P=0.032). Univariate analysis identified that HDAC2 is an unfavorable prognostic factor for PTCL-U patients(P=0.032). However,multivariate Cox model analysis revealed that HDAC2 cannot be an independent prognostic factor(P=0.722).2. CS055 induces growth inhibition, G0/G1 arrest, and apoptosis of Jurkat cells.3. CS055 synergistically augments doxorucibin-induced growth and apoptosis.4. The mechanisms of synergistic effect may involve the mitochondrial apoptotic pathway and DNA damage.