[Effects of the phosphoinostitide-3'-kinase delta inhibitor, CAL-101, in combination with Bortezomib on mantle lymophma cells and exploration of its related mechanism].

OBJECTIVE: To investigate the effect of CAL-101, a selective inhibitor of PI3Kδ, in combination with bortezomib on the proliferation and apoptosis in human mantle cell lymphoma cell lines Z138, HBL-2 and Jeko-1 in vitro, to explore its mechanisms and provide the foundation for effective treatment strategies against mantle cell lymphoma. METHODS: MTT assay was applied to detect the inhibitory effects of CAL-101 and bortezomib either alone or combined on Z138, HBL-2 and Jeko-1 cells. Calcusyn software was used to analyze the synergistic cytotoxicity. Western blot was used to detect the expression of PI3K-p110σ and p-Akt, Akt, p-ERK and ERK proteins after the cells were exposed to different concentrations of CAL-101. Flow cytometry was employed to assess the apoptosis rate. NF-κB kit was used to determine the changes of location of NF-κB P65, and Western blot was applied to detect the level of caswpase-3 and the phosphorylation of Akt in different groups. RESULTS: CAL-101 and BTZ inhibited the proliferation of Z138, HBL-2 and Jeko-1 cells in a dose- and time-dependent manner. CAL-101/BTZ combination induced significantly synergistic cytotoxicity in the MCL cells. The results of Western blot assay showed that CAL-101 significantly blocked the phosphorylation of Akt and ERK in the MCL cell lines. In addition, CAL-101 combined with BTZ induced pronounced apoptosis (P < 0.01). For example, after the Z138 cells exposed to the drugs for 48 h, the apoptosis rates of the control, CAL-101, BTZ and CAL-101 + BTZ groups were: (2.6 ± 1.8)%, (40.0 ± 3.0)%, (34.0 ± 1.0)%, and (67.4 ± 1.0)%, respectively; and when drug treatment was given to HBL-2 cells over 96 h, the apoptosis rates of these four cell groups were (7.4 ± 0.6)%, (30.7 ± 5.7)%, (12.0 ± 1.0)%, and (85.0 ± 4.0)%, respectively. The combination therapy contributed to the enhanced inactivity of nuclear factor-κB (NF-κB) and Akt inactivation in the MCL cell lines (P < 0.05), however, the casepase-3 activity was up-regulated. CONCLUSIONS: The combination of CAL-101 and bortezomib is muchmore effective in inhibiting proliferation and promoting apoptosis of mantle cell lymphoma cell lines (Z138, HBL-2 and Jeko-1), which may be mediated through inhibiting PI3K/Akt signaling pathway and the transcription of NF-κB.

[Inhibitory effects of Hes1 on acute myeloid leukemia cells].

OBJECTIVE: To elucidate the impact of Hes1 on the proliferation and apoptosis of acute myeloid leukemia (AML) cells. METHODS: The expression levels of Hes1 and p21 in AML patient samples and myeloid leukemia cell lines were analyzed by real-time PCR. Hes1 was up-regulated by retrovirus transfection in AML cell lines and the proliferation capacity were assayed by MTT, cell cycle by Hoechst/PY, apoptosis by AnnexinV. RESULTS: The expression of Hes1 in primary AML cells and HL-60, U937, KG1a cell lines were 0.67 ± 0.24, 0.59 ± 0.43, 0.42 ± 0.03, and 0.32 ± 0.26, respectively, and p21 were 0.54 ± 0.01, 0.44 ± 0.12, 0.36 ± 0.12, and 0.59 ± 0.43, respectively. Hes1 expression levels after transduction in HL-60, U937, KG1a were 4.9 ± 0.2, 5.2 ± 0.4, 5.8 ± 0.5, respectively. Induced activation of Hes1 led to AML cells growth arrest and apoptosis, which was associated with an enhanced p21 expression. Besides, activated Hes1 led to AML cells growth inhibition in vivo. CONCLUSION: Hes1 could mediate growth arrest and apoptosis in AML cells, which may be a novel target for AML.

Coexpression of MYC and BCL-2 predicts prognosis in primary gastrointestinal diffuse large B-cell lymphoma.

AIM: To investigate whether MYC and BCL-2 coexpression has prognostic significance in primary gastrointestinal diffuse large B-cell lymphoma (PGI-DLBCL) patients, and explore its associations with patients' clinical parameters. METHODS: Fresh and paraffin-embedded tumor tissue samples from 60 PGI-DLBCL patients who had undergone surgery at the Tianjin Medical University Cancer Institute and Hospital from January 2005 to May 2010 were obtained, and 30 lymphoid tissue samples from reactive lymph nodes of age- and sex-matched patients represented control samples. Staging and diagnostic procedures were conducted according to the Lugano staging system. All patients had been treated with three therapeutic modalities: surgery, chemotherapy, or radiotherapy. Expression of MYC and BCL-2 were detected at both protein and mRNA levels by immunohistochemistry and real-time RT-PCR. RESULTS: Positive expression levels of MYC and BCL-2 proteins were detected in 35% and 45% of patients, respectively. MYC+/BCL-2+ protein was present in 30% of patients. MYC and BCL-2 protein levels were correlated with high MYC and BCL-2 mRNA expression, respectively (both P<0.05). We found that advanced-stage disease (at IIE-IV) was associated with MYC and BCL-2 coexpression levels (P<0.05). In addition, MYC+/BCL-2+ patients had more difficulty in achieving complete remission than others (P<0.05). Presence of MYC protein expression only affected overall survival and progression-free survival (PFS) when BCL-2 protein was coexpressed. The adverse prognostic impact of MYC+/BCL-2+ protein on PFS remained significant (P<0.05) even after adjusting for age, Lugano stage, international prognostic index, and BCL-2 protein expression in a multivariable model. CONCLUSION: MYC+/BCL-2+ patients have worse chemotherapy response and poorer prognosis than patients who only express one of the two proteins, suggesting that assessment of MYC and BCL-2 expression by immunohistochemistry has clinical significance in predicting clinical outcomes of PGI-DLBCL patients.

c-Myc plays part in drug resistance mediated by bone marrow stromal cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant and aggressive disease not sensitive to chemotherapy. The dynamic interaction between AML cells and bone marrow (BM) microenvironment plays a critical role in response of this disease to chemotherapy. It is reported that mesenchymal stromal cells (MSC) are essential component of bone marrow microenvironment which affects the survival of AML cells. The aim of our research is to elucidate the mechanism of drug resistance of AML cells associated with MSC. We found that adhesion of AML cell lines U937, KG1a and primary AML cells to MSC inhibited cytotoxic drug-induced apoptosis. Western blot showed that c-Myc of AML cells cocultured with stroma was up-regulated. Treatment with 10058-F4, a small molecule inhibitor of MYC-MAX heterodimerization, or c-Myc siRNA significantly induced apoptosis. Western blot analysis further showed that inhibition of c-Myc induced expression of caspases-3, cleavage of PARP and reduced expression of Bcl-2, Bcl-xL and vascular endothelial growth factor (VEGF). Thus, we conclude that MSCs protected leukemia cells from apoptosis, at least in part, through c-Myc dependent mechanisms, and that c-Myc contributed to microenvironment-mediated drug resistance in AML. In summary, we declared that c-Myc is a potential therapeutic target for overcoming drug resistance in AML.

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.

Targeting Bruton's tyrosine kinase signaling as an emerging therapeutic agent of B-cell malignancies.

It is becoming increasingly evident that B-cell receptor (BCR) signaling is central to the development and function of B cells. BCR signaling has emerged as a pivotal pathway and a key driver of numerous B-cell lymphomas. Disruption of BCR signaling can be lethal to malignant B cells. Recently, kinase inhibitors that target BCR signaling have induced notable clinical responses. These inhibitors include spleen tyrosine kinase, mammalian target of rapamycin, phosphoinositide 3'-kinase and Bruton's tyrosine kinase (BTK). Ibrutinib, an oral irreversible BTK inhibitor, has emerged as a promising targeted therapy for patients with B-cell malignancies. The present review discusses the current understanding of BTK-mediated BCR signaling in the biology and pathobiology of normal and malignant B cells, and the cellular interaction with the tumor microenvironment. The data on ibrutinib in the preclinical and clinical settings is also discussed, and perspectives for the future use of ibrutinib are outlined.

[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.

Downregulated expression of Dicer1 predicts inferior survival in primary gastrointestinal diffuse large B-cell lymphoma treated with CHOP-like regimen and rituximab.

The aim of this study was to detect the expression levels of Dicer1, Drosha, DGCR8, and Ago2 messenger ribonucleic acids (mRNAs) in patients with primary gastrointestinal diffuse large B-cell lymphoma (PGI-DLBCL) and determine their associations with clinical parameters and prognostic significance. The mRNA level expressions of Dicer1, Drosha, DGCR8, and Ago2 were detected by real-time quantitative polymerase chain reaction. Immunohistochemical staining of CD10, BCL6, and MUM1 was performed using EnVision™ system. The clinicopathologic features and follow-up data were analyzed using Kaplan-Meier estimator. The results show that the expression of Dicer1 (P=0.001), Drosha (P=0.01), DGCR8 (P=0.02), and Ago2 (P=0.002) mRNAs in cancer tissues of patients with PGI-DLBCL was significantly lower than those in normal tissues of healthy controls. Among the expression of CD10, BCL6, and MUM1, 27.4% (17/62) of the patients belonged to the germinal center B-cell (GCB) subtype and 72.6% (45/62) belonged to the non-GCB subtype. Dicer1 expression was significantly decreased in the non-GCB subgroup (P=0.02) and in the high International Prognostic Index (3-5 score) subgroup (P=0.03). Kaplan-Meier analysis showed that the low-Dicer1 subgroup had a shorter overall survival (P=0.02) and shorter progression-free survival (P=0.015) than the high-Dicer1 subgroup. Multivariate analysis identified Dicer1 as an independent prognostic factor in PGI-DLBCL. In Conclusion, Dicer1, Drosha, DGCR8, and Ago2 play key roles in the pathogenesis of PGI-DLBCL. Dicer1, an independent prognostic factor for predicting shortened survival of patients with PGI-DLBCL, can be used as a biomarker to guide the prognosis.