Single-Cell Profiling Reveals Immune-Based Mechanisms Underlying Tumor Radiosensitization by a Novel Mn Porphyrin Clinical Candidate, MnTnBuOE-2-PyP5+ (BMX-001).

Manganese porphyrins reportedly exhibit synergic effects when combined with irradiation. However, an in-depth understanding of intratumoral heterogeneity and immune pathways, as affected by Mn porphyrins, remains limited. Here, we explored the mechanisms underlying immunomodulation of a clinical candidate, MnTnBuOE-2-PyP5+ (BMX-001, MnBuOE), using single-cell analysis in a murine carcinoma model. Mice bearing 4T1 tumors were divided into four groups: control, MnBuOE, radiotherapy (RT), and combined MnBuOE and radiotherapy (MnBuOE/RT). In epithelial cells, the epithelial-mesenchymal transition, TNF-α signaling via NF-кB, angiogenesis, and hypoxia-related genes were significantly downregulated in the MnBuOE/RT group compared with the RT group. All subtypes of cancer-associated fibroblasts (CAFs) were clearly reduced in MnBuOE and MnBuOE/RT. Inhibitory receptor-ligand interactions, in which epithelial cells and CAFs interacted with CD8+ T cells, were significantly lower in the MnBuOE/RT group than in the RT group. Trajectory analysis showed that dendritic cells maturation-associated markers were increased in MnBuOE/RT. M1 macrophages were significantly increased in the MnBuOE/RT group compared with the RT group, whereas myeloid-derived suppressor cells were decreased. CellChat analysis showed that the number of cell-cell communications was the lowest in the MnBuOE/RT group. Our study is the first to provide evidence for the combined radiotherapy with a novel Mn porphyrin clinical candidate, BMX-001, from the perspective of each cell type within the tumor microenvironment.

Targeting Na-H exchanger 1 overcomes nuclear factor kappa B-mediated tumor resistance to radiotherapy.

Intrinsic or acquired radioresistance often limits the efficacy of radiation therapy (RT), thereby leading to local control failure. Cancerous cells have abnormal pH dynamics due to high metabolic demands, but it is unclear how pH dynamics contribute to radioresistance. In this study, we investigated the role of Na-H exchange 1 (NHE1), the major intracellular pH (pHi) regulator, in RT response. We observed that RT increased NHE1 expression and modulated pHi in MDA-MB-231 human breast cancer cells. When combined with RT, pharmacological NHE1 inhibition by 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) reduced pHi and clonogenic survival. EIPA attenuated radiation-damaged DNA repair, increasing G2/M cell cycle arrest. The combination of EIPA and RT increased apoptotic cell death while decreasing phosphorylation of NF-κB p65. Similarly, the knockdown of NHE1 increased radiosensitivity with lower pHi and increased apoptosis. Consistent with in vitro data, the EIPA plus RT inhibited the growth of MDA-MB-231 xenograft tumors in mice to a greater extent than either EIPA or RT alone. EIPA abrogated the RT-induced increase in NHE1 and phospho-NF-κB p65 expression in tumor tissues. Such coincidence of increased NHE1 level, pHi, and NF-κB activation was also found in radioresistant MDA-MB-231 cells, which were reversed by EIPA treatment. Bioinformatics analysis of RNA sequencing data revealed that inhibiting NHE1 reversed three core gene networks that were up-regulated in radioresistant cells and correlated with high NHE1 expression in patient samples: NF-κB, senescence, and extracellular matrix. Taken together, our findings suggest that NHE1 contributes to RT resistance via NF-κB-mediated signaling networks, and NHE1 may be a promising target for improving RT outcomes.

Immunomodulation of HDAC Inhibitor Entinostat Potentiates the Anticancer Effects of Radiation and PD-1 Blockade in the Murine Lewis Lung Carcinoma Model.

Although the combination of radiotherapy and immunotherapy has proven to be effective in lung cancer treatment, it may not be sufficient to fully activate the antitumor immune response. Here, we investigated whether entinostat, a histone deacetylase inhibitor, could improve the efficacy of radiotherapy and anti-PD-1 in a murine syngeneic LL/2 tumor model. A total of 12 Gy of X-rays administered in two fractions significantly delayed tumor growth in mice, which was further enhanced by oral entinostat administration. Flow cytometry-aided immune cell profiling revealed that entinostat increased radiation-induced infiltration of myeloid-derived suppressor cells and CD8+ T cells with decreased regulatory T-cells (Tregs). Transcriptomics-based immune phenotype prediction showed that entinostat potentiated radiation-activated pathways, such as JAK/STAT3/interferon-gamma (IFN-γ) and PD-1/PD-L1 signaling. Entinostat augmented the antitumor efficacy of radiation and anti-PD-1, which may be related to an increase in IFN-γ-producing CD8+ T-cells with a decrease in Treg cells. Comparative transcriptomic profiling predicted that entinostat increased the number of dendritic cells, B cells, and T cells in tumors treated with radiation and anti-PD-1 by inducing MHC-II genes. In conclusion, our findings provided insights into how entinostat improves the efficacy of ionizing radiation plus anti-PD-1 therapy and offered clues for developing new strategies for clinical trials.

Immunomodulatory effect of splenectomy in lung cancer mouse xenograft models receiving radiation therapy.

PURPOSE: This study aims to investigate the effect of splenectomy on radiation-mediated growth inhibition and immune modulation in lung cancer xenograft models. MATERIALS AND METHODS: Human non-small cell lung cancer H1299 cells and murine Lewis lung carcinoma LL/2-luc cells were injected into the right hind leg of BALB/c-nude mice and C57BL/6 mice, respectively. Splenectomy or sham operation was performed prior to tumor cell injection or before and after irradiation during tumor growth. Irradiation was delivered with 2-3 fractions of 6 Gy X-ray using a linear accelerator. Flow cytometry analysis was performed for immune cell profiling. RESULTS: Splenectomy prior to tumor injection or at early stage inhibited growth of LL/2-luc tumors but not that of H1299 tumors; however, it did not enhance the antitumor effect of radiation regardless of intervention timing. Flow cytometry analysis showed monocytic myeloid-derived suppressor cells (MDSCs) and activated CD8+ T cells increased after irradiation in the tumors of splenectomized mice, compared to those of sham-operated mice. Administration of anti-PD-1 (programmed death-1) antibodies improved the ability of splenectomy to attenuate the growth of irradiated tumors. CONCLUSION: Splenectomy has paradoxical effects on radiation-induced tumor growth inhibition, depending on tumor types and intervention timing, but it has an immune-modulating effect when combined with radiation.

Antigen-Capturing Mesoporous Silica Nanoparticles Enhance the Radiation-Induced Abscopal Effect in Murine Hepatocellular Carcinoma Hepa1-6 Models.

Immunomodulation by radiotherapy (RT) is an emerging strategy for improving cancer immunotherapy. Nanomaterials have been employed as innovative tools for cancer therapy. This study aimed to investigate whether mesoporous silica nanoparticles (MSNs) enhance RT-mediated local tumor control and the abscopal effect by stimulating anti-cancer immunity. Hepa1-6 murine hepatocellular carcinoma syngeneic models and immunophenotyping with flow cytometry were used to evaluate the immune responses. When mice harboring bilateral tumors received 8 Gy of X-rays on a single tumor, the direct injection of MSNs into irradiated tumors enhanced the growth inhibition of irradiated and unirradiated contralateral tumors. MSNs enhanced RT-induced tumor infiltration of cytotoxic T cells on both sides and suppressed RT-enhanced infiltration of regulatory T cells. The administration of MSNs pre-incubated with irradiated cell-conditioned medium enhanced the anti-tumor effect of anti-PD1 compared to the as-synthesized MSNs. Intracellular uptake of MSNs activated JAWS II dendritic cells (DCs), which were consistently observed in DCs in tumor-draining lymph nodes (TDLNs). Our findings suggest that MSNs may capture tumor antigens released after RT, which is followed by DC maturation in TDLNs and infiltration of cytotoxic T cells in tumors, thereby leading to systemic tumor regression. Our results suggest that MSNs can be applied as an adjuvant for in situ cancer vaccines with RT.

MnTnHex-2-PyP5+, Coupled to Radiation, Suppresses Metastasis of 4T1 and MDA-MB-231 Breast Cancer via AKT/Snail/EMT Pathways.

Tumor migration and invasion induced by the epithelial-to-mesenchymal transition (EMT) are prerequisites for metastasis. Here, we investigated the inhibitory effect of a mimic of superoxide dismutase (SOD), cationic Mn(III) ortho-substituted N-n-hexylpyridylporphyrin (MnTnHex-2-PyP5+, MnHex) on the metastasis of breast cancer in cellular and animal models, focusing on the migration of tumor cells and the factors that modulate this behavior. Wound healing and Transwell migration assays revealed that the migration of mouse mammary carcinoma 4T1 cells was markedly reduced during the concurrent treatment of MnHex and radiation therapy (RT) compared with that of the control and RT alone. Bioluminescence imaging showed that MnHex/RT co-treatment dramatically reduced lung metastasis of 4T1 cells in mice, compared with the sham control and both single treatments. Western blotting and immunofluorescence showed that MnHex treatment of 4T1 cells reversed the RT-induced EMT via inhibiting AKT/GSK-3ß/Snail pathway in vitro, thereby decreasing cell migration and invasion. Consistently, histopathological analyses of 4T1 tumors showed that MnHex/RT reduced Snail expression, blocked EMT, and in turn suppressed metastases. Again, in the human metastatic breast cancer MDA-MB-231 cell line, MnHex inhibited metastatic potential in vitro and in vivo and suppressed the RT-induced Snail expression. In addition to our previous studies showing tumor growth inhibition, this study demonstrated that MnHex carries the ability to minimize the metastatic potential of RT-treated cancers, thus overcoming their radioresistance.

Manganese Ferrite Nanoparticles Enhance the Sensitivity of Hepa1-6 Hepatocellular Carcinoma to Radiation by Remodeling Tumor Microenvironments.

We evaluated the effect of manganese ferrite nanoparticles (MFN) on radiosensitization and immunologic responses using the murine hepatoma cell line Hepa1-6 and the syngeneic mouse model. The clonogenic survival of Hepa1-6 cells was increased by hypoxia, while being restricted by ionizing radiation (IR) and/or MFN. Although MFN suppressed HIF-1α under hypoxia, the combination of IR and MFN enhanced apoptosis and DNA damage in Hepa1-6 cells. In the Hepa1-6 syngeneic mouse model, the combination of IR and MFN notably limited the tumor growth compared to the single treatment with IR or MFN, and also triggered more frequent apoptosis in tumor tissues than that observed under other conditions. Increased expression of PD-L1 after IR was not observed with MFN alone or the combination of IR and MFN in vitro and in vivo, and the percentage of tumor-infiltrating T cells and cytotoxic T cells increased with MFN, regardless of IR, in the Hepa1-6 syngeneic mouse model, while IR alone led to T cell depletion. MFN might have the potential to overcome radioresistance by alleviating hypoxia and strengthening antitumor immunity in the tumor microenvironment.

Therapeutic Potential of (-)-Agelamide D, a Diterpene Alkaloid from the Marine Sponge Agelas sp., as a Natural Radiosensitizer in Hepatocellular Carcinoma Models.

Radiation therapy (RT) is an effective local treatment for unresectable hepatocellular carcinoma (HCC), but there are currently no predictive biomarkers to guide treatment decision for RT or adjuvant systemic drugs to be combined with RT for HCC patients. Previously, we reported that extracts of the marine sponge Agelas sp. may contain a natural radiosensitizer for HCC treatment. In this study, we isolated (-)-agelamide D from Agelas extract and investigated the mechanism underlying its radiosensitization. (-)-Agelamide D enhanced radiation sensitivity of Hep3B cells with decreased clonogenic survival and increased apoptotic cell death. Furthermore, (-)-agelamide D increased the expression of protein kinase RNA-like endoplasmic reticulum kinase/inositol-requiring enzyme 1α/activating transcription factor 4 (PERK/eIF2α/ATF4), a key pathway of the unfolded protein response (UPR) in multiple HCC cell lines, and augmented radiation-induced UPR signaling. In vivo xenograft experiments confirmed that (-)-agelamide D enhanced tumor growth inhibition by radiation without systemic toxicity. Immunohistochemistry results showed that (-)-agelamide D further increased radiation-induced ATF4 expression and apoptotic cell death, which was consistent with our in vitro finding. Collectively, our results provide preclinical evidence that the use of UPR inducers such as (-)-agelamide D may enhance the efficacy of RT in HCC management.

Checkpoint Kinase 1 (CHK1) Inhibition Enhances the Sensitivity of Triple-Negative Breast Cancer Cells to Proton Irradiation via Rad51 Downregulation.

Due to a superior dose conformity to the target, proton beam therapy (PBT) continues to rise in popularity. Recently, considerable efforts have been directed toward discovering treatment options for use in combination with PBT. This study aimed to investigate the targeting of checkpoint kinase 1 (CHK1), a critical player regulating the G2/M checkpoint, as a promising strategy to potentiate PBT in human triple-negative breast cancer (TNBC) cells. Protons induced cell-cycle arrest at the G2/M checkpoint more readily in response to increased CHK1 activation than X-rays. A clonogenic survival assay revealed that CHK1 inhibition using PF-477736 or small interfering RNA (siRNA) enhanced the sensitivity toward protons to a greater extent than toward X-rays. Western blotting demonstrated that PF-477736 treatment in the background of proton irradiation increased the pro-apoptotic signaling, which was further supported by flow cytometry using annexin V. Immunofluorescence revealed that proton-induced DNA double-strand breaks (DSBs) were further enhanced by PF-477736, which was linked to the downregulation of Rad51, essential for the homologous recombination repair of DSBs. Direct inactivation of Rad51 resulted in enhanced proton sensitization. Collectively, these data suggest that targeting CHK1 may be a promising approach for improving PBT efficacy in the treatment of TNBC.

Anti-Tumor Effects of Wee1 Kinase Inhibitor with Radiotherapy in Human Cervical Cancer.

Although the concurrent use of a chemotherapeutic agent and radiotherapy improves survival in patients with locally advanced or recurrent cervical cancer, severe side effects related to chemotherapy are frequent and may result in a low quality of life for the patients. In this study, we investigated the effects of a combination of Wee1 inhibitor (AZD1775) and irradiation in cervical cancer. In vitro effects of AZD1775 with irradiation in human cervical cancer cells were assessed by clonogenic survival and apoptosis assays. The effects on DNA damage response signaling and the cell cycle were also explored. Tumor growth delay was evaluated to investigate the in vivo effects of AZD1775 with irradiation in cervical cancer mouse models, including xenografts and patient-derived xenografts (PDXs). The co-treatment of AZD1775 and irradiation significantly decreased clonogenic survival and increased apoptosis in cervical cancer cells. These effects were associated with G2 checkpoint abrogation which resulted in persistent DNA damage. Both in the xenografts and the PDXs, the co-treatment significantly decreased tumor growth compared tothe irradiation alone (p < 0.05). These results demonstrate that the Wee1 inhibitor (AZD1775) can be considered as a potential alternative as a radiosensitizer in cervical cancer instead of a chemotherapeutic agent such as cisplatin.

NOD2 Supports Crypt Survival and Epithelial Regeneration after Radiation-Induced Injury.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) affords stem cell protection and links microbes to intestinal epithelial regeneration. We investigated whether NOD2 status is associated with crypt survival and intestinal epithelial regeneration independent of microbiota-derived molecules. To assess crypt survival, a clonogenic microcolony assay was performed with 15 Gy of X-ray irradiation. The fractional crypt survival rate (46.0 ± 15.5% vs. 24.7 ± 9.2%, p < 0.01) and fractional EdU-positive crypt survival rate (29.8 ± 14.5% vs. 9.79 ± 4.37%, p = 0.015) were significantly decreased in the NOD2-/- mice compared with the wild-type (WT) mice at 3.5 days after irradiation. To evaluate intestinal epithelial regeneration capability, organoid reconstitution assays were performed. Small bowel crypts of the WT and NOD2-/- mice were isolated and seeded into Matrigel for 3D culture. In the organoid reconstitution assays, the number of organoids formed did not differ between the NOD2-/- and WT mice. Organoid formation ability was also assessed after exposure to 5 Gy irradiation. Organoid formation ability was significantly decreased in the NOD2-/- mice compared with the WT ones after exposure to 5 Gy irradiation (33.2 ± 5.9 vs. 19.7 ± 8.8/well, p < 0.01). NOD2 supports crypt survival after potentially lethal irradiation damage and is associated with intestinal epithelial regeneration.

Targeting DNA-dependent protein kinase sensitizes hepatocellular carcinoma cells to proton beam irradiation through apoptosis induction.

Recent studies have highlighted the implications of genetic variations in the relative biological effectiveness (RBE) of proton beam irradiation over conventional X-ray irradiation. Proton beam radiotherapy is a reasonable radiotherapy option for hepatocellular carcinoma (HCC), but the impact of genetic difference on the HCC RBE remains unknown. Here, we determined proton RBE in human HCC cells by exposing them to various doses of either 6-MV X-rays or 230-MeV proton beams. Clonogenic survival assay revealed variable radiosensitivity of human HCC cell lines with survival fraction at 2 Gy ranging from 0.38 to 0.83 and variable proton RBEs with 37% survival fraction ranging from 1.00 to 1.48. HCC cells appeared more sensitive to proton irradiation than X-rays, with more persistent activation of DNA damage repair proteins over time. Depletion of a DNA damage repair gene, DNA-PKcs, by siRNA dramatically increased the sensitivity of HCC cells to proton beams with a decrease in colony survival and an increase in apoptosis. Our findings suggest that there are large variations in proton RBE in HCC cells despite the use of a constant RBE of 1.1 in the clinic and targeting DNA-PKcs in combination with proton beam therapy may be a promising regimen for treating HCC.

Comparison of Proton and Photon Beam Irradiation in Radiation-Induced Intestinal Injury Using a Mouse Model.

When radiotherapy is applied to the abdomen or pelvis, normal tissue toxicity in the gastrointestinal (GI) tract is considered a major dose-limiting factor. Proton beam therapy has a specific advantage in terms of reduced doses to normal tissues. This study investigated the fundamental differences between proton- and X-ray-induced intestinal injuries in mouse models. C57BL/6J mice were irradiated with 6-MV X-rays or 230-MeV protons and were sacrificed after 84 h. The number of surviving crypts per circumference of the jejunum was identified using Hematoxylin and Eosin staining. Diverse intestinal stem cell (ISC) populations and apoptotic cells were analyzed using immunohistochemistry (IHC) and a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay, respectively. The crypt microcolony assay revealed a radiation-dose-dependent decrease in the number of regenerative crypts in the mouse jejunum; proton irradiation was more effective than X-ray irradiation with a relative biological effectiveness of 1.14. The jejunum is the most sensitive to radiations, followed by the ileum and the colon. Both types of radiation therapy decreased the number of radiosensitive, active cycling ISC populations. However, a higher number of radioresistant, reserve ISC populations and Paneth cells were eradicated by proton irradiation than X-ray irradiation, as shown in the IHC analyses. The TUNEL assay revealed that proton irradiation was more effective in enhancing apoptotic cell death than X-ray irradiation. This study conducted a detailed analysis on the effects of proton irradiation versus X-ray irradiation on intestinal crypt regeneration in mouse models. Our findings revealed that proton irradiation has a direct effect on ISC populations, which may result in an increase in the risk of GI toxicity during proton beam therapy.

Fucoidan-Manganese Dioxide Nanoparticles Potentiate Radiation Therapy by Co-Targeting Tumor Hypoxia and Angiogenesis.

Tumor hypoxia is a major mechanism of resistance to radiation therapy (RT), which is associated with poor prognosis in affected cancer patients. Various approaches to treat hypoxic and radioresistant cancers, including pancreatic cancer, have shown limited success. Fucoidan, a polysaccharide from brown seaweed, has antitumor and antiangiogenesis activities. Here, we discuss the development of fucoidan-coated manganese dioxide nanoparticles (Fuco-MnO2-NPs) and testing of the therapeutic potential with RT using pancreatic cancer models. In vitro data showed that Fuco-MnO2-NPs generated oxygen efficiently in the presence of H2O2 and substantially suppressed HIF-1 expression under a hypoxic condition in human pancreatic cancer cells. Fuco-MnO2-NPs reversed hypoxia-induced radioresistance by decreasing clonogenic survival and increasing DNA damage and apoptotic cell death in response to RT. In a BxPC3 xenograft mouse model, the combination treatment with Fuco-MnO2-NPs and RT resulted in a greater tumor growth delay than RT alone. Fucoidan-coated NPs, but not naked ones, further suppressed tumor angiogenesis, as judged by immunohistochemistry data with diminished expression of phosphorylated vascular endothelial growth factor receptor 2 (VEGFR2) and CD31. These data suggest that Fuco-MnO2-NPs may potentiate the effects of RT via dual targeting of tumor hypoxia and angiogenesis, and they are of great clinical potential in the treatment of hypoxic, radioresistant pancreatic cancer.

Feasibility study of Fe3O4/TaO x nanoparticles as a radiosensitizer for proton therapy.

We investigated the feasibility of using multifunctional Fe3O4/TaO x (core/shell) nanoparticles, developed for use in contrast agents for computed tomography (CT) and magnetic resonance imaging (MRI), as dose-enhancing radiosensitizers. First, to verify the detectability of Fe3O4/TaO x nanoparticles in imaging, in vivo tests were conducted. Approximately 600 mg kg-1 of 19 nm-diameter Fe3O4/TaO x nanoparticles dispersed in phosphate-buffered saline was injected into the tail vein of six Balb/c mice used as tumour (4T1 mammary carcinoma cell) models. Three mice underwent MRI (BioSpec 70/20 USR, Bruker, Billerica, MA, USA) and micro-CT (Inveon, Siemens Preclinical, Knoxville, TN, USA) before and after the injection. The difference between the pre- and post-injection images was quantified by finding the correlation coefficient. The aorta, blood vessel, and liver were clearly seen in the MRI and micro-CT images 60 min after intravenous injection of Fe3O4/TaO x nanoparticles, but the tumour region was not visible in the CT images until after 24 h. There were large differences between the pre- and post-injection images. Second, the therapeutic enhancement dose of nanomaterials was computed via Monte Carlo simulation. Monoenergetic 70- and 150 MeV proton beams irradiated x-ray contrast agent (iodine, BaSO4), MRI contrast agent (gadolinium, Fe3O4), Au, Fe3O4/TaO x (core/shell) nanoparticles and water located at the centre of a 4 × 4 × 4 µm3 water phantom, upon which the dose enhancement ratio (DER) (dose with/without nanoparticles) was computed. When 70 MeV protons irradiated the Au, gadolinium, Fe3O4/TaO x , Fe3O4, iodine, and BaSO4 nanoparticles, the DERs at 1 nm were 15.76, 7.68, 7.82, 6.17, 4.85, and 5.51, respectively. Fe3O4/TaO x nanoparticles have the potential to be used as a multifunctional agent that enhances tumour detection and increases the dose. Dose enhancement with Fe3O4/TaO x was half that with Au. However, Fe3O4/TaO x is much cheaper than Au, and it is expected that tumour targeting combined with magnetic field could overcome the low DER.

Publisher Correction: Valproic Acid Sensitizes Hepatocellular Carcinoma Cells to Proton Therapy by Suppressing NRF2 Activation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Valproic Acid Sensitizes Hepatocellular Carcinoma Cells to Proton Therapy by Suppressing NRF2 Activation.

Although efficacy of combined histone deacetylase (HDAC) inhibitors and conventional photon radiotherapy is being tested in clinical trials, their combined effect with proton beam radiotherapy has yet to be determined. Here, we compared combined effect of valproic acid (VPA), a class I and II HDAC inhibitor and antiepileptic drug with proton and photon irradiation in hepatocellular carcinoma (HCC) cells in vitro and in vivo. We found that VPA sensitized more Hep3B cells to proton than to photon irradiation. VPA prolonged proton-induced DNA damage and augmented proton-induced apoptosis. In addition, VPA further increased proton-induced production of intracellular reactive oxygen species and suppressed expression of nuclear factor erythroid-2-related factor 2 (NRF2), a key transcription factor regulating antioxidant response. Downregulation of NRF2 by siRNA transfection increased proton-induced apoptotic cell death, supporting NRF2 as a target of VPA in radiosensitization. In Hep3B tumor xenograft models, VPA significantly enhanced proton-induced tumor growth delay with increased apoptosis and decreased NRF2 expression in vivo. Collectively, our study highlights a proton radiosensitizing effect of VPA in HCC cells. As NRF2 is an emerging prognostic marker contributing to radioresistance in HCC, targeting NRF2 pathway may impact clinical outcome of proton beam radiotherapy.

Expression of fatty acid synthase is regulated by PGC­1α and contributes to increased cell proliferation.

We previously demonstrated that overexpression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) promotes increased cell proliferation and tumorigenic potential through upregulation of specificity protein 1 (Sp1) and acyl-CoA-binding protein (ACBP). Fatty acid synthase (FASN) is a key enzyme in fatty acid biosynthesis, and its expression in various cancers is associated with survival, poor prognosis and cancer recurrence. In the present study, we evaluated whether PGC-1α regulated FASN expression in human colorectal cancer (SNU-C4 and HT-29) cells. We also examined whether cell proliferation was inhibited by shRNA­induced FASN knockdown in SNU-C4 and HT-29 cells. In all tested cell lines, FASN-shRNA knockdown inhibited cell proliferation, decreased antioxidant enzyme expression, and increased apoptosis and production of H2O2­induced reactive oxygen species (ROS). These findings indicated that FASN expression may enhance cell proliferation by regulating antioxidant enzyme production and resistance to ROS-induced apoptosis. We further provided evidence that FASN expression was regulated indirectly through upregulation of Sp1 and SREBP-1c by PGC-1α. Overall, our results revealed that FASN expression, mediated by PGC-1α, may play a positive role in cancer cell proliferation.

Mechanism of the Antitumor and Radiosensitizing Effects of a Manganese Porphyrin, MnHex-2-PyP.

AIMS: Cationic manganese (Mn)-substituted N-pyridylporphyrin-based potent mimics of the family of superoxide dismutases (SODs) protect normal tissues from injury related to ionizing radiation (IR) by reducing levels of reactive oxygen and nitrogen species (ROS/RNS). Furthermore, Mn-porphyrins have demonstrated antitumor and radiosensitizing effects on cancer cells by promoting IR-induced tumor vasculature damage and apoptotic processes. In this study, we explored the underlying mechanisms of Mn-porphyrin-mediated tumor radiosensitization using murine mammary carcinoma 4T1 and melanoma B16 cells in vitro and in vivo. RESULTS: Combination treatment with MnTnHex-2-PyP and IR substantially reduced cell viability, clonogenic cell survival, and DNA damage repair and synergistically increased IR-induced apoptosis of 4T1 and B16 cells. MnTnHex-2-PyP in combination with IR caused a significant delay in growth of 4T1 and B16 xenograft tumors. MnTnHex-2-PyP dose-dependently enhanced IR-mediated production of H2O2-derived species, but not superoxide. Catalase overexpression reversed MnTnHex-2-PyP-enhanced ROS production and apoptosis. Demonstrated suppression of phosphorylation of several mitogen-activated protein (MAP) kinases and activation of NF-κB by MnTnHex-2-PyP/IR, which presumably inhibited activation of the antiapoptotic pathway, are in agreement with our other data on the apoptosis of cancer cells. Innovation and Conclusions: MnTnHex-2-PyP exerted a radiosensitizing effect on 4T1 and B16 tumor models in vitro and in vivo via pro-oxidative actions and therefore bears a large therapeutic potential. When combined with IR, it attenuated DNA damage repair and triggered a shift from prosurvival pathways to apoptotic cell death, likely due to increased ROS production and disturbed cellular redox balance, acting at the level of nuclear factor κB (NF-κB). Antioxid. Redox Signal. 27, 1067-1082.

Ceramide as a Target of Marine Triterpene Glycosides for Treatment of Human Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous myeloid clonal disorder exhibiting the accumulation of immature myeloid progenitors in the bone marrow and peripheral blood. Standard AML therapy requires intensive combination chemotherapy, which leads to significant treatment-related toxicity. The search for new, low toxic marine agents, inducing the generation of ceramide in leukemic cells is a new approach to improve the therapy of leukemia. This review focuses on the metabolism of sphingolipids, the role of ceramide in treating leukemia, and the antitumor activity, related to ceramide metabolism, of some marine metabolites, particularly stichoposides, triterpene glycosides extracted from sea cucumbers of the family Stichopodiidae.