Won-Suk Jang

Coexistent mutations of KRAS and PIK3CA affect the efficacy of NVP-BEZ235, a dual PI3K/MTOR inhibitor, in regulating the PI3K/MTOR pathway in colorectal cancer

Colorectal cancer (CRC) with mutational activation of KRAS is observed frequently. In addition, PIK3CA mutations commonly coexist with KRAS mutations and lead to additive activation of the PI3K/MTOR signaling pathway. Here, we investigated how CRC cells that harbor KRAS and PIK3CA mutations affect sensitivity to inhibition of PI3K/MTOR with NVP‐BEZ235 (BEZ235). We selected CRC patient samples and assessed their mutational status. CRC patients with KRAS or PIK3CA mutations show activation of AKT and MTOR, particularly when KRAS and PIK3CA mutations coexist. Suppression of PI3K/MTOR by BEZ235 results in a growth inhibitory effect and enhanced apoptosis via BIM activation in KRAS mutant cells. Mutational activation of KRAS when accompanied by a PIK3CA mutation converges at PI3K/MTOR pathway activation, resulting in resistance to BEZ235. BIM knockdown blocked the apoptotic response to BEZ235 in KRAS mutant cells, suggesting that PI3K inhibition leads to BIM accumulation. Moreover, BEZ235 treatment resulted in induction of FOXO3A activity and its induced transcription of BIM activation, which sensitized cells to cytotoxic agents leading to apoptosis in double mutant cells in vitro and in vivo. Taken together, our data suggest that targeting PI3K/MTOR sensitizes cells to apoptosis, implying that activation of PI3K/MTOR signaling via KRAS or PIK3CA mutation is an important pathway in CRC cell growth. Based on these results, coexistent KRAS and PIK3CA mutations confer resistance to BEZ235 via suppression of BIM‐induced apoptosis, suggesting that combined treatment with conventional chemoagents is a potential strategy in the clinic.

The dual PI3K and mTOR inhibitor NVP-BEZ235 exhibits anti-proliferative activity and overcomes bortezomib resistance in mantle cell lymphoma cells

Mantle cell lymphoma (MCL) is one of the most difficult B-cell lymphomas to be treated. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is constitutively activated in MCL and plays a critical role in tumor growth and survival. However, single targeted agent mTOR has limited efficacy in treating MCL. Here, we investigate for the first time potential efficacy of NVP-BEZ235 (BEZ235) in treating MCL by simultaneously targeting Akt and mTOR. In this study, phosphorylated Akt and mTOR level were elevated in tissue samples from MCL patients and in MCL cell lines. We also generated bortezomib-resistant MCL cell lines and found increased phosphorylation of Akt and mTOR. Individual inhibition of PI3K or mTOR had limited anti-proliferative effects, whereas dual inhibition with BEZ235 effectively inhibited cell growth. The effect of BEZ235 was synergistic and sensitized the cells to the cytotoxic effects of conventional agents. Furthermore, BEZ235 could overcome acquired resistance to bortezomib in MCL cells and suppress the activated Akt/mTOR pathway. Therefore, these data suggest that the Akt/mTOR pathway plays a key role in the growth and survival of MCL cells and that these proteins may need to be simultaneously targeted for effective treatment of the disease. Our findings suggest that BEZ235 may be an effective agent for the treatment of MCL.

Development of a New Minipig Model to Study Radiation-Induced Gastrointestinal Syndrome and its Application in Clinical Research

Because of insufficient clinical data regarding acute radiation damage after single high-dose radiation exposure, acute radiation-induced gastrointestinal (GI) syndrome remains difficult to treat. The goal of this study was to establish an appropriate and efficient minipig model to study high-dose radiation-induced GI syndrome after radiation exposure. For endoscopic access to the ileum, ileocutaneous anastomosis was performed 3 weeks before irradiation in six male Göttingen minipigs. Minipigs were locally irradiated at the abdominal area using a gamma source as follows: 1,000 cGy (n = 3) and 1,500 cGy (n = 3). Endoscopic evaluation for the terminal ileum was periodically performed via the ileocutaneous anastomosis tract. Pieces of tissue were serially taken for histological examination. The irradiated intestine presented characteristic morphological changes over time. The most obvious changes in the ileum were mucosal atrophy and telangiectasia from day 1 to day 17 after abdominal irradiation. Microscopic findings were characterized as architectural disorganization, loss of villi and chronic active inflammation. Increase in cyclooxygenase-2 (COX-2) expression was closely correlated with severity of tissue damage and inflammation. Particularly, the plasma citrulline level (PCL), a potential marker for radiation-induced intestinal damage, was significantly decreased the day after irradiation and recovered when irradiated mucosa was normalized. Our results also showed that PCL changes were positively correlated with microscopic changes and the endoscopic score in radiation-induced mucosal damage. In conclusion, the ileocutaneous anastomosis model using the minipig mimics human GI syndrome and allows the study of sequential changes in the ileum, the main target tissue of abdominal irradiation. In addition, PCL could be a simple biomarker for radiation-induced intestinal damage.

Granulocyte-colony stimulating factor ameliorates irradiation-induced suppression of hippocampal neurogenesis in adult mice

Granulocyte-colony stimulating factor (G-csf) is a member of the hematopoietic growth factor family and demonstrates neuroprotective functions in neurodegenerative diseases. This study evaluated the radioprotective effects of G-csf in the suppression of hippocampal neurogenesis in adult mice undergoing irradiation. The radioprotective effects were assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and immunohistochemical markers of neurogenesis, including the proliferating cell marker Ki-67 and the immature progenitor neuron marker doublecortin (DCX). Acute exposure to cranial irradiation (5Gy γ-rays) induced neural apoptosis and inhibited neurogenesis in the dentate gyrus (DG) of the adult mouse hippocampus. Pretreatment with G-csf (100μg/kg every 12h subcutaneously on three consecutive days) attenuated neural apoptosis and decreased the number of Ki-67- and DCX-positive cells in the DG of the irradiated mouse hippocampus. Therefore, G-csf inhibited the detrimental effects of irradiation on hippocampal neurogenesis, suggesting that G-csf administration has potential therapeutic utility in brain irradiation.

Mitigating effects of hUCB-MSCs on the hematopoietic syndrome resulting from total body irradiation

This study evaluated the clinical and pathologic effects of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the recovery from total body irradiation by comparing it with the effects of granulocyte-colony stimulating factor (G-CSF), an efficacious drug in the treatment of acute bone marrow radiation syndrome. BALB/c mice were treated with G-CSF or hUCB-MSCs after they were irradiated with 7 Gy cobalt-60 γ-rays. Circulating blood counts, histopathologic changes in the bone marrow, and plasma level of Flt-3L and transforming growth factor (TGF-β1) were monitored in the postirradiation period. Hematologic analysis revealed that the peripheral leukocyte counts were markedly increased in the hUCB-MSCs-treated group, whereas G-CSF-treated mice did not recover significantly. Moreover, differential counts showed that hUCB-MSC treatment has regenerative effects on white blood cells, lymphocytes, and monocytes compared with the irradiated group. Treatment with hUCB-MSCs or G-CSF significantly increased immunoreactivity of Ki-67 until 3 weeks after total body irradiation. However, at 3 weeks, the number of Ki-67 immunoreactive cells significantly increased in the hUCB-MSCs-treated group compared with the G-CSF-treated group. Furthermore, hUCB-MSC treatment significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGF-β1, whereas G-CSF treatment failed to decrease the plasma Flt-3L levels at 2 weeks after irradiation. Based on the differences in circulating blood cell reconstitution and cell density of bone marrow, the authors suggest that MSC treatment is superior to G-CSF treatment for hematopoietic reconstitution following sublethal dose radiation exposure.

A combination of methotrexate and irradiation promotes cell death in NK/T-cell lymphoma cells via down-regulation of NF-κB signaling.

Nasal NK/T-cell lymphoma (NKTL) is a highly aggressive disease. Although radiotherapy is the first-line of treatment for NKTL, the clinical outcome is poor. Thus, there is a need for an effective radiosensitizer to improve the survival rate of patients. NF-κB activation contributes to cell survival as well as chemo- and radio-resistance in various cancer cells. In NKTL, the constitutive activation of NF-κB is also a critical factor. In the present study, we used two EBV-expressing NKTL cell lines (Hank-1 and NK-92) to evaluate the radiosensitizing effect of methotrexate (MTX), highlighting the role of NF-κB. Combined treatment of MTX and IR significantly induced apoptosis and growth inhibition in both NKTL cells. The synergistic cytotoxicity was correlated with blocking nuclear NF-κB and suppressing expression of NF-κB-mediated anti-apoptotic proteins. These data suggest that the combined treatment with MTX and IR can inhibit IR-induced NF-κB activation in NKTL cells. Taken together, co-treatment with MTX and IR may provide a therapeutic advantage for patients with NKTL.

In vivo characterization of early-stage radiation skin injury in a mouse model by two-photon microscopy.

Ionizing radiation (IR) injury is tissue damage caused by high energy electromagnetic waves such as X-ray and gamma ray. Diagnosis and treatment of IR injury are difficult due to its characteristics of clinically latent post-irradiation periods and the following successive and unpredictable inflammatory bursts. Skin is one of the many sensitive organs to IR and bears local injury upon exposure. Early-stage diagnosis of IR skin injury is essential in order to maximize treatment efficiency and to prevent the aggravation of IR injury. In this study, early-stage changes of the IR injured skin at the cellular level were characterized in an in vivo mouse model by two-photon microscopy (TPM). Various IR doses were applied to the mouse hind limbs and the injured skin regions were imaged daily for 6 days after IR irradiation. Changes in the morphology and distribution of the epidermal cells and damage of the sebaceous glands were observed before clinical symptoms. These results showed that TPM is sensitive to early-stage changes of IR skin injury and may be useful for its diagnosis.

Development of a minipig model for lung injury induced by a single high-dose radiation exposure and evaluation with thoracic computed tomography

Radiation-induced lung injury (RILI) due to nuclear or radiological exposure remains difficult to treat because of insufficient clinical data. The goal of this study was to establish an appropriate and efficient minipig model and introduce a thoracic computed tomography (CT)-based method to measure the progression of RILI. Göttingen minipigs were allocated to control and irradiation groups. The most obvious changes in the CT images after irradiation were peribronchial opacification, interlobular septal thickening, and lung volume loss. Hounsfield units (HU) in the irradiation group reached a maximum level at 6 weeks and decreased thereafter, but remained higher than those of the control group. Both lung area and cardiac right lateral shift showed significant changes at 22 weeks post irradiation. The white blood cell (WBC) count, a marker of pneumonitis, increased and reached a maximum at 6 weeks in both peripheral blood and bronchial alveolar lavage fluid. Microscopic findings at 22 weeks post irradiation were characterized by widening of the interlobular septum, with dense fibrosis and an increase in the radiation dose–dependent fibrotic score. Our results also showed that WBC counts and microscopic findings were positively correlated with the three CT parameters. In conclusion, the minipig model can provide useful clinical data regarding RILI caused by the adverse effects of high-dose radiotherapy. Peribronchial opacification, interlobular septal thickening, and lung volume loss are three quantifiable CT parameters that can be used as a simple method for monitoring the progression of RILI.

Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor

AIM To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells. METHODS Crypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1 (ENR) or ENR/CHIR99021/VPA (ENR-CV). For subculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging. RESULTS We established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging. CONCLUSION The maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.

Claudin-3 expression in radiation-exposed rat models: A potential marker for radiation-induced intestinal barrier failure

The molecular events leading to radiation-induced intestinal barrier failure are not well known. The influence of the expression of claudin proteins in the presence and absence of neurotensin was investigated in radiation-exposed rat intestinal epithelium. Wistar rats were randomly divided into control, irradiation, and irradiation+neurotensin groups, and bacterial translocation to the mesenteric lymph node and expression of claudins were determined. Irradiation led to intestinal barrier failure as demonstrated by significant bacterial translocation. In irradiated terminal ilea, expression of claudin-3 and claudin-4 was significantly decreased, and claudin-2 expression was increased. Administration of neurotensin significantly reduced bacterial translocation and restored the structure of the villi as seen by histologic examination. Among the three subtype of claudins, only claudin-3 expression was restored. These results suggest that the therapeutic effect of neurotensin on the disruption of the intestinal barrier is associated with claudin-3 alteration and that claudin-3 could be used as a marker in evaluating radiation-induced intestinal injury.