Bing Xia

[Effect of PI3Kδ inhibitor CAL-101 on myeloma cell lines and preliminary study of synergistic effects with other new drugs].

OBJECTIVE To investigate the proliferation inhibitory role and mechanism of PI3Kδ inhibitor CAL-101 on multiple myeloma (MM) cells, and to provide new therapeutic options for MM treatment. METHODS MM cell lines U266 and RPMI8226 cells were treated with various concentrations of CAL-101. MTT assay and CalcuSyn software were performed to determine the inhibitory effect of CAL-101 and the synergistic effect with PCI- 32765, SAHA (suberoylanilide hydroxamic acid), BTZ (Bortezomib) on MM cells. The protein expression level of p-AKT, p-ERK, AKT, ERK and PI3Kδ processed by CAL-101 were analyzed by Western blot. RESULTS CAL-101 at concentration of 15, 20, 25, 30 and 40 μmol/L could induce significant dose-dependent proliferation inhibition on U266 cells after treatment for 48 hours. The cell proliferation inhibition rates were (33.54 ± 1.23)%, (41.72 ± 1.78)%, (53.67 ± 2.01)%, (68.97 ± 2.11)% and (79.25 ± 1.92)%, respectively. Similar results were found in RPMI8226 cell line. Western blots showed high expression level of p-AKT, p-ERK, AKT, ERK and PI3Kδ in cell lines and MM primary cells. p-AKT and p-ERK protein expression levels were down-regulated significantly by CAL-101 treatment. Synergistic effect has been verified between CAL-101 and PCI-32765, SAHA and Bortezomib in U266 cell line, and PCI-32765, Bortezomib in RPMI8226 cell line with CI values less than 1. CONCLUSION CAL-101 could inhibit proliferation of MM cell lines. High levels of p-AKT, p-ERK, AKT, ERK and PI3Kδ protein expression were observed in both cell lines and primary cells. Down-regulation of p-AKT and p-ERK probably related with the mechanism of CAL-101 in MM cell proliferation inhibition. CAL-101 has significant synergistic effect with PCI-32765, SAHA and BTZ.

Toll-like receptor 4-induced inflammatory responses contribute to the tumor-associated macrophages formation and infiltration in patients with diffuse large B-cell lymphoma

To evaluate the expression of tumor-associated macrophages (TAMs) and Toll-like receptor 4 (TLR4) in diffuse large B-cell lymphoma (DLBCL) and their correlation with patient clinical characteristics, we detected using immunohistochemistry in 81 specimens of patients with DLBCL. The correlation between protein expression levels and clinical parameters, as well as the association between CD68 and TLR4 were analyzed. The number of CD68 TAMs was closely related to β2-microglobulin (P = .028 and P < .05), whereas there was no significant correlation between the number of CD68 TAMs and other clinical factors. Toll-like receptor 4 was related to tumor size and peripheral blood lymphocyte to monocyte ratio. The Spearman correlation coefficient indicated a significant positive correlation between CD68 TAMs and TLR4 expression (r = 0.240; P = .038, P = .05). These results, on one hand, indicated that TLR4-induced inflammatory responses may affect TAM infiltration and accumulation, and that TAMs and TLR4 may interact to play important roles in DLBCL microenvironment regulating the tumor growth, but, on the other hand demonstrated that both of TAMs and TLR4 had not only one side on DLBCL growth.

Synergistic suppression of the PI3K inhibitor CAL-101 with bortezomib on mantle cell lymphoma growth

Objective To investigate the effects of CAL-101, particularly when combined with bortezomib (BTZ) on mantle cell lymphoma (MCL) cells, and to explore its relative mechanisms. Methods MTT assay was applied to detect the inhibitory effects of different concentrations of CAL-101. MCL cells were divided into four groups: control group, CAL-101 group, BTZ group, and CAL-101/BTZ group. The expression of PI3K-p110σ, AKT, ERK, p-AKT and p-ERK were detected by Western blot. The apoptosis rates of CAL-101 group, BTZ group, and combination group were detected by flow cytometry. The location changes of nuclear factor kappa-B (NF-κB) of 4 groups was investigated by NF-κB Kit exploring. Western blot was applied to detect the levels of caspase-3 and the phosphorylation of AKT in different groups. Results CAL-101 dose- and time-dependently induced reduction in MCL cell viability. CAL-101 combined with BTZ enhanced the reduction in cell viability and apoptosis. Western blot analysis showed that CAL-101 significantly blocked the PI3K/AKT and ERK signaling pathway in MCL cells. The combination therapy contributed to the inactivation of NF-κB and AKT in MCL cell lines. However, cleaved caspase-3 was up-regulated after combined treatment. Conclusion Our study showed that PI3K/p110σ is a novel therapeutic target in MCL, and the underlying mechanism could be the blocking of the PI3K/AKT and ERK signaling pathways. These findings provided a basis for clinical evaluation of CAL-101 and a rationale for its application in combination therapy, particularly with BTZ.

Approaches to optimize gene therapy for the treatment of hematologic malignancies: Overcoming the obstacles

Gene transfer and oncolytic viruses provide new therapeutic approaches for the treatment of hematologic malignancies. However, it is still too early to introduce gene delivery or oncolytic viruses into standard clinical protocol. It is very important to discuss the obstacles that gene transfer and oncolytic virotherapy face for the further clinical application for the treatment of hematologic malignancies, and updating the advances made to overcome them. The major concerns in this review include the approaches of the development of immuno-stimulatory gene transfer mediated-vaccination for leukemia therapy, RNAi-based therapy for leukemia and enhancement of sensitivity of target malignant cells to virotherapy and alteration of host immune response to favor oncolytic viruses. We conclude with a perspective on the future of the gene therapy and virotherapy for the treatment of hematologic malignancies, emphasizing the problems we should solve and the technological requirements for further clinical applications.

Analysis of Clinical Characteristics and Prognosis of 46 Elderly Patients with Peripheral T Cell Lymphoma (PTCL)

OBJECTIVE To investigate the clinical characteristics and prognostic factors of patients with peripheral T cell lymphoma (PTCL). METHODS The clinical data of 46 elderly PTCL patients admitted in Tianjin Medical University Cancer Hospital from April 2008 to August 2014 were collected, the clinical features, prognostic factors and treatments, as well as followed-up outcome were analyzed retrospectively. Survival analysis was performed by Kaplan-Meier method, and the COX proportional hazard model was used to perform multivariate analysis. RESULTS The median survival time was 11 months, and the expected 1-year, 2-year and 3-year overall survival rate (OS) was 50%, 36% and 33%, respectively. Univariate analysis showed that the age, ECOG score, Charlson Comorbidity Index Score, the efficacy and course of chemotherapy were all the prognostic indicators affecting the OS and progression free survival (PFS) in this cohort of elderly patients. Multivariate analysis indicated that ECOG score and course of chemotherapy were the independent prognostic indicators affecting the OS and PFS (P < 0.05). CONCLUSION ECOG score and course of chemotherapy are of great significance for predicting the prognosis in elderly PTCL patients. The elderly patientss general condition and completion of a certain intensity of chemotherapy are an important measure to prolong survival time in elderly PTCL patients.

[Role of CXCR4/STAT3 in mesenchymal stromal cell-mediated drug resistance of acute leukemia cells].

目的 探讨CXCR4/STAT3在骨髓基质细胞介导的急性髓系白血病(AML)细胞耐药中的作用。 方法 将AML细胞系U937、KG1a和原代AML细胞与健康供者来源的骨髓基质细胞共培养，单独培养的细胞作为对照组。通过膜联蛋白Ⅴ (Annexin Ⅴ)／碘化丙锭(PI)双染色法比较共培养前后AML细胞对米托蒽醌诱导的凋亡的差异，并通过流式细胞术、实时定量PCR及Western blot法检测共培养前后AML细胞CXCR4、磷酸化STAT3(p-STAT3)蛋白的表达；在培养体系中加入STAT3的特异性抑制剂Cucurbitacin Ⅰ或CXCR4拮抗剂AMD3100，检测AML细胞对米托蒽醌诱导的凋亡的变化，并观察AMD3100对p-STAT3表达的影响。 结果 U937、KG1a细胞与骨髓基质细胞共培养后，对米托蒽醌诱导的凋亡率均低于对照组[U937：(20.08±1.53)%对(45.33±1.03)%，P=0.004; KG1a：(25.60 ± 1.82)%对(40.33±3.29)%，P=0.003]。共培养后，Western blot方法检测发现AML细胞p-STAT3的蛋白表达明显上调，流式细胞术和Western blot检测结果显示CXCR4蛋白表达显著上调，实时定量PCR检测显示CXCR4 mRNA水平明显增高；STAT3的特异性抑制剂Cucurbitacin Ⅰ或CXCR4拮抗剂AMD3100加入共培养体系后，原代AML细胞对米托蒽醌诱导的凋亡率较对照组显著升高，差异均有统计学意义(P值均<0.05)，且加入AMD3100后AML细胞p-STAT3蛋白的表达显著下降。 结论 AML细胞与骨髓基质细胞共培养可导致p-STAT3、CXCR4蛋白表达的上调，从而导致AML细胞对化疗药物的耐药；针对STAT3、CXCR4的联合靶向治疗可能为AML的治疗提供新思路。OBJECTIVE To explore the role of CXCR4/STAT3 in mesenchymal stromal cell (MSC)-mediated drug resistance of AML cells. METHODS AML cell lines U937 and KG1a and primary AML cells were co-cultured with MSC from bone marrow of healthy donors. The AML cell lines cultured alone were used as control. Apoptosis induced by mitoxantrone was measured by flow cytometry. Expression of CXCR4 and STAT3 protein were detected by Western blot. After incubated with STAT3 inhibitor Cucurbitacin I or CXCR4 antagonist AMD3100, the apoptosis of AML cells induced by mitoxantrone was evaluated. RESULTS Apoptosis of AML cells (U937 and KG1a) and primary AML cells induced by mitoxantrone significantly decreased in cocultured group than that of control group [U937 cells: (20.08±1.53)% vs (45.33 ± 1.03)% , P=0.004; KG1a cells: (25.60 ± 1.82)% vs (40.33 ± 3.29)% , P=0.020]. Expression of phosphorylated STAT3 and CXCR4 protein in AML cells were upregulated in cocultured group. After addition of Cucurbitacin I into the co-culture system, the apoptosis rate of primary AML cells significantly increased. Similar results of the apoptosis rates were also detected when the inhibitor of CXCR4 AMD3100 was added to overcome the stromal cell-mediated drug resistance. Besides, the expression of p-STAT3 in AML cells after incubated with AMD3100 decreased significantly. CONCLUSIONS AML cells cocultured with MSC leads to the up-regulation of phosphorylated STAT3 and CXCR4 proteins, which resulted in AML cells resistance to chemotherapeutic drugs. Therefore targeting STAT3 or CXCR4 could be a new therapeutic strategy of AML.