Ying Tong

Despite advances in targeted therapies, treatment resistance and disease recurrence remain major obstacles in leukemia management. Structural modification of clinically successful kinase inhibitors represents a promising approach for discovering novel anti-leukemic agents with improved biological activities. In the present study, we investigated the anti-leukemic activity and mechanism of action of MO2455, a newly synthesized imatinib-derived compound, in monocytic leukemia U937, THP−1, and T-cell leukemia CCRF-CEM cells. MO2455 showed substantial cytotoxic activity against all three cell lines, with IC₅₀ values of 0.83 μM, 0.43 μM, and 0.57 μM in U937, THP−1, and CCRF-CEM cells, respectively. Treatment with MO2455 induced early mitochondrial membrane depolarization, internucleosomal DNA laddering, a significant accumulation of sub−G1 population and increased Annexin V positivity, suggesting typical apoptosis without cell cycle arrest in U937 cells. MO2455 induced γ-H2AX induction and upregulated the pro-apoptotic NOXA and BBC3 genes. THP−1 and CCRF-CEM cells displayed distinct large DNA fragmentation with smearing. In PBMCs from two healthy donors, IC₅₀ values were 0.99 and 0.60 μM, respectively, although the cells remained morphologically healthy without obvious extensive cell death. Overall, MO2455 exhibits substantial anti-leukemic activity and induces cell death through apoptosis and other mechanisms, supporting further investigation as an anti-leukemic agent.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) with radiation therapy can enhance the sensitivity of cancer cells by inhibiting DNA repair pathways. To determine the most suitable PARP inhibitor for radiosensitization in cancer cells, we compared various types of clinically used PARPis in lung cancer A549 cells. We found that most PARP inhibitors showed radiosensitization effects on A549 cells. ER10 values for talazoparib, olaparib rucaparib, ABT888 and niraparib were 1.5, 1.8, 2.8, 1.4, and 1.4, respectively. Talazoparib showed a radiosensitization effect at its lowest concentration. Talazoparib is a potent PARP inhibitor and has been used in clinical settings for several types of cancer as an anti-cancer agent. We thus focused on how talazoparib causes radiosensitization in lung cancer A549 cells. As a result of the combination of talazoparib and γ-irradiation, we observed an increased level of cellular senescence accompanied by a decrease in mitochondrial membrane potential. When the p21 gene was knocked down, both the decrease in mitochondrial membrane potential and senescence level were attenuated, suggesting that p21 is involved in senescence induction after γ-irradiation combined with talazoparib treatment. Taken together, we showed that PARP inhibitor talazoparib treatment in combination with γ-irradiation causes cellular senescence in lung cancer cells, involving p21 function.

Developing strategies for the radiosensitization of cancer cells by the inhibition of genes, which harbor low toxicity to normal cells, will be useful for improving cancer radiotherapy. Here, we focused on a β-site of amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1; β-secretase, memapsin-2). By functional inhibition of this peptidase by siRNA, it has also recently been shown that the DNA strand break marker, γH2AX foci, increased, suggesting its involvement in DNA damage response. To investigate this possibility, we knocked down BACE1 with siRNA in cancer cell lines, and sensitization to γ-irradiation was examined by a colony formation assay, γH2AX foci and level analysis, and flow cytometry. BACE1 knockdown resulted in the sensitization of HeLa, MDA-MB-231, U2OS, and SAOS cells to γ-irradiation in a diverse range. BACE1 knockdown showed a weak radiosensitization effect in osteosarcoma U2OS cells, which has a normal p53 function. HeLa and SAOS cells, which harbor p53 dysfunction, exhibited a greater level of radiosensitization. These results suggest that BACE1 may be a potential target for the radiosensitization in particular cancer cells.

Boron neutron capture therapy (BNCT) is a selective radiotherapy based on nuclear reaction that occurs when ¹⁰B atoms accumulated in cancer cells are irradiated by thermal neutrons, triggering a nuclear fission response leading to cell death. Despite its growing importance in cancer treatment, molecular characterization of its effects is still lacking. In this context, proteomics investigation can be useful to study BNCT effect and identify potential biomarkers. Hence, we performed proteomic analysis with nanoLC-MS/MS (liquid chromatography coupled to tandem mass spectrometry) on extracellular vesicles (EVs) isolated from SAS cultures treated or not with ¹⁰B-boronophenylalanine (BPA) and different doses of neutron irradiation, to study the cellular response related to both boron administration and neutrons action. Despite the interference of fetal bovine serum in the medium, we were able to stratify BPA− and BPA+ conditions and to identify EVs-derived proteins characterizing pathways potentially related to a BNCT effect such as apoptosis, DNA repair and inflammatory response. In particular, KLF11, SERPINA1 and SERPINF2 were up-regulated in BPA+, while POLE and SERPINC1 were up-regulated in BPA−. These results provide the first proteomic investigation of EVs treated with BNCT in different conditions and highlight the potentiality of proteomics for improving biomarkers identification and mechanisms understanding of BNCT.

Poly(ADP-ribose) polymerase (PARP) is involved in DNA repair and chromatin regulation. 5-Aza-2′-deoxycytidine (5-aza-dC) inhibits DNA methyltransferases, induces hypomethylation, blocks DNA replication, and causes DNA single strand breaks (SSBs). As the PARP inhibitor is expected to affect both DNA repair and transcriptional regulations, we investigated the effect of combinational use of PARP inhibitors on cytotoxicity of 5-aza-dC in human cancer cell lines. The combinational treatment of 5-aza-dC and PARP inhibitor PJ-34 exhibited a stronger cytotoxicity compared with their treatment alone in blood cancer HL-60, U937, and colon cancer HCT116 and RKO cells. Treatment with 5-aza-dC but not PJ-34 caused SSBs in HCT116 cell lines. Global genome DNA demethylation was observed after treatment with 5-aza-dC but not with PJ-34. Notably, in microarray analysis, combinational treatment with PJ-34 and 5-aza-dC caused dissimilar broad changes in gene expression profiles compared with their single treatments in both HCT116 and RKO cells. The profiles of reactivation of silenced genes were also different in combination of PJ-34 and 5-aza-dC and their single treatments. The results suggest that the combinational use of 5-aza-dC and PARP inhibitor may be useful by causing distinct transcriptional profile changes.

The radiosensitization of tumor cells is one of the promising approaches for enhancing radiation damage to cancer cells and limiting radiation effects on normal tissue. In this study, we performed a comprehensive screening of radiosensitization targets in human lung cancer cell line A549 using an shRNA library and identified apolipoprotein B mRNA editing enzyme catalytic subunit 3G (APOBEC3G: A3G) as a candidate target. APOBEC3G is an innate restriction factor that inhibits HIV-1 infection as a cytidine deaminase. APOBEC3G knockdown with siRNA showed an increased radiosensitivity in several cancer cell lines, including pancreatic cancer MIAPaCa2 cells and lung cancer A549 cells. Cell cycle analysis revealed that APOBEC3G knockdown increased S-phase arrest in MIAPaCa2 and G2/M arrest in A549 cells after γ-irradiation. DNA double-strand break marker γH2AX level was increased in APOBEC3G-knocked-down MIAPaCa2 cells after γ-irradiation. Using a xenograft model of A549 in mice, enhanced radiosensitivity by a combination of X-ray irradiation and APOBEC3G knockdown was observed. These results suggest that the functional inhibition of APOBEC3G sensitizes cancer cells to radiation by attenuating the activation of the DNA repair pathway, suggesting that APOBEC3G could be useful as a target for the radiosensitization of cancer therapy.

Boron neutron capture therapy (BNCT) is a non-invasive therapeutic technique for treating malignant tumors, however, methods to evaluate its therapeutic efficacy and adverse reactions are lacking. High mobility group box 1 (HMGB1) is an inflammatory molecule released during cell death. Therefore, we aimed to investigate HMGB1 as a biomarker for BNCT response, by examining the early responses of tumor cells to ¹⁰B-boronophenylalanine (BPA)-based BNCT in the Kyoto University Nuclear Reactor. Extracellular HMGB1 release was significantly increased in human squamous carcinoma SAS and melanoma A375 cells 24 h after neutron irradiation but not after γ-irradiation. At 3 days post-BPA-based BNCT irradiation in a SAS xenograft mouse model, plasma HMGB1 levels were higher than those in the non-irradiation control, and HMGB1 was detected in both nuclei and cytoplasm in tumor cells. Additionally, increased plasma HMGB1 levels post-BNCT irradiation were detected even when tumors decreased in size. Collectively, these results indicate that the extracellular HMGB1 release occurs at an early stage and is persistent when tumors are reduced in size; therefore, it is a potential biomarker for evaluating the therapeutic response during BNCT.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes dendritic cell differentiation from precursors, and consequently, enhances the antigen presentation process and adaptive immune responses. With such functions, GM-CSF has been used as immunotherapy in combination with radiotherapy for cancer treatment to augment the survival and activity of immune cells. However, an immune-suppressive tumor microenvironment may cause anergy of T cells. It has also been reported that GM-CSF contributes to the development of myeloid-derived suppressor cells from the precursors. In this study, to analyze the combined effect of GM-CSF and released factors from cancer cells after gamma-ray irradiation on bone marrow cell differentiation and dynamics, we established an in vitro culture system using mouse bone marrow cells, GM-CSF, and conditioned medium from gamma ray irradiated mouse melanoma B16 cells at 24 Gy. We analyzed the gene expression changes of the bone marrow-derived cells on day 6. The results showed that GM-CSF dose-dependently enhanced the differentiation of macrophages from bone marrow cells, their antigen-presenting function and polarization to type I. The results implied the induced macrophages from the bone marrow may potentially contribute to tumor immune responses in a systemic manner when GM-CSF is boosted during photon-beam radiation therapy.