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Supplementary Fig. 1. Genipin enhanced oxaliplatin sensitivity, but not geniposide in HCT116. Supplementary Fig. 2. Genipin increased oxaliplatin-induced apoptosis in DLD-1. Supplementary Fig. 3. Genipin induced the activation of the ROS-ER stress-JNK pathway. Supplemental Fig. 4 Combination of oxaliplatin and genipin decreased tumor growth in vivo.

Despite an increase in the survival rate of patients with cancer owing to the use of current chemotherapeutic agents, adverse effects of cancer therapies remain a concern. Combination therapies have been developed to increase efficacy, reduce adverse effects, and overcome drug resistance. Genipin is a natural product derived from Gardenia jasminoides, which has been associated with anti-inflammatory, anti-angiogenic, and anti-proliferative effects; hypertension; and anti-ischemic brain injuries. However, the enhancement of oxaliplatin sensitivity by genipin remains unexplored. Our study showed that a combination of genipin and oxaliplatin exerts synergistic antitumor effects in vitro and in vivo in colorectal cancer cell lines through the reactive oxygen species (ROS)/endoplasmic reticulum (ER) stress/BIM pathway. Importantly, the combination did not affect normal colon cells. BIM knockdown markedly inhibited apoptosis induced by the combination. In addition, genipin induced ROS by inhibiting superoxide dismutase 3 activity. These findings suggest that genipin may be a novel agent for increasing the sensitivity of oxaliplatin against colorectal cancer. The combination of oxaliplatin and genipin hold significant therapeutic potential with minimal adverse effects.

Purpose: The purpose of this study was to analyze retinal capillary parameters using optical coherence tomography angiography (OCTA) of the affected eye and the fellow eye of unilateral normal tension glaucoma (NTG) patients and compare the findings with eyes from a normal control group.Methods: A retrospective cross-sectional study was carried out on patients diagnosed with unilateral NTG (24 affected eyes and 24 fellow eyes each) and normal individuals (29 eyes, the control group). OCTA was used to measure the vascular density (VD) and perfusion density (PD) of the macular area and the peripapillary area.Results: In the superficial capillary plexus, the fellow eye group of unilateral NTG patients showed a decrease in VD of the inner-inferior and PD of the inner-inferior and outer-average peripapillary area, compared with the normal control group (p = 0.008, p < 0.001, and p = 0.001). In the affected NTG eye group, the VD (p = 0.014, p = 0.011, p < 0.001, p < 0.001, and p < 0.001) and PD (p = 0.017, p = 0.023, p < 0.001, p = 0.001, and p < 0.001) of the total, inner-average, inner-inferior, and outer-inferior peripapillary area, and the outer-inferior macular area decreased compared to the fellow eye and normal control group, as well as the VD of the outer-average peripapillary area (p = 0.010). The PD of the outer-average peripapillary area (p = 0.003); the VD (p = 0.041, p = 0.008, p = 0.006) and the PD (p = 0.013, p < 0.001, p = 0.001) of the total, inner-inferior, and outer-average macular area; and the PD of the outer-temporal macular area (p = 0.003) were lower than the normal control group. There was no difference in the VD or PD obtained from the deep capillary plexus of the macular area among the groups.Conclusions: It is useful to observe retinal capillary parameters using OCTA for patients with unilateral NTG.

Background Quantitative recognition of visual field loss is important for glaucoma patients and has implications for follow-up management. This study aimed to identify the characteristic patterns of 10 − 2 visual field (VF) test results and effectively express central VF loss using a machine learning approach. Methods We obtained 7,927 10 − 2 VF test data from 3,328 patients in five hospitals. We propose a hybrid approach that combines archetypal analysis (AA) and fuzzy c-means (FCM) to identify characteristic patterns and decompose VF without loss. To demonstrate the clinical usefulness of our approach, mean deviation (MD) change prediction was performed through supervised learning using decomposition coefficients change and a linear mixed model was performed to examine the relationship between the MD slope and baseline decomposition coefficients. Results We identified ten characteristic and representative archetypes (AT) for the central VF test results. FCM decomposition results outperformed the AA-only approach in MD change prediction based on mean squared error (MSE) and Pearson correlation coefficient (PCC) prediction evaluation metrics (all P ≤ 0.039). In the linear mixed model, the FCM is more suitable in the prediction of MD slope compared with the AA model for both Akaike (AIC) and Bayes information criteria (BIC) (AIC decrease:20.31, BIC decrease:13.33). The FCM baseline coefficients of AT 3, and AT 4 were significantly associated with a faster MD slope (both P ≤ 0.026). Conclusions In this study, we used a hybrid approach of unsupervised learning to identify hidden aspects of central VF loss via a characteristic archetype and lossless decomposition. We believe that our approach can help discover hidden clinical features of glaucoma.

Phloretin is one of the apple polyphenols with anticancer activities. Since tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves important roles in inducing apoptosis, the present study examined the effect of phloretin on TRAIL-induced apoptosis in colon cancer cells. Treatment with both phloretin and TRAIL markedly suppressed the survival of cancer cells from several colon cancer cell lines compared with that of cells treated with either TRAIL or phloretin. Additionally, decreased numbers of colonies were observed following addition of phloretin and TRAIL. Furthermore, TRAIL- and phloretin-treated HT-29-Luc cells exhibited decreased luciferase activity. Increased apoptosis was observed in phloretin- and TRAIL-treated HT-29-Luc colon cancer cells, accompanying elevated levels of cleaved poly(ADP-ribose) polymerase, and caspase-3, -8 and -9. The expression levels of MCL1 apoptosis regulator BCL2 family member (Mcl-1) were decreased following addition of phloretin in colon cancer cells. In addition, overexpression of Mcl-1 in phloretin- and TRAIL-treated HT-29-Luc cells resulted in increased cell survival. Treatment of HT-29-Luc cells with a combination of cycloheximide (CHX) and phloretin led to a more prominent decrease in Mcl-1 expression compared with that in cells treated with CHX alone, while Mcl-1 expression was recovered by treatment with MG132. Binding of ubiquitin with Mcl-1 was verified using immunoprecipitation. Intraperitoneal injection of both TRAIL and phloretin into tumor xenografts was associated with a decreased tumor volume compared with that following injection with either TRAIL or phloretin. Overall, the present results suggest a synergistic effect of phloretin on TRAIL-induced apoptosis in colon cancer cells.

Simple Summary Cannabidiol (CBD), one of the compounds present in the marijuana plant, has antitumor properties. However, the effect of CBD on breast cancer remains unclear. The aim of this study was to assess the effects of CBD for the angiogenesis and stemness of breast cancer cells by decreasing the expression of hypoxia-induced factor-1α (HIF-1α) through the Src/von Hippel–Lindau tumor suppressor protein (VHL) interaction. CBD can suppress angiogenesis and stem cell-like properties of breast cancer through Src/VHL/HIF-1α signaling. Abstract To assess the effect of Cannabidiol (CBD) on the angiogenesis and stemness of breast cancer cells as well as proliferation. Methods: mRNA level and the amount of protein of vascular endothelial growth factor (VEGF) were determined by qRT-PCR and ELISA. The angiogenic potential of breast cancer cells under hypoxic conditions was identified by the HUVEC tube formation assay. The degradation of HIF-1α by CBD and the Src/von Hippel–Lindau tumor suppressor protein (VHL) interaction were assessed by a co-immunoprecipitation assay and Western blotting. To identify the stemness of mamospheres, they were evaluated by the sphere-forming assay and flow cytometry. Results: CBD can suppress angiogenesis and stem cell-like properties of breast cancer through Src/VHL/HIF-1α signaling. CBD may potentially be utilized in the treatment of refractory or recurrent breast cancer.

Hypoxia, or the deficiency of oxygen, in solid tumors is majorly responsible for the progression of cancer and remains unaffected by chemotherapy, but still requires definitive definition of the hypoxia signaling. Hypoxia disrupts the complete folding of mitochondrial proteins, leading to several diseases. The present study confirms that hypoxia activates the Hedgehog pathway in colorectal cancer (CRC), considering its role in cancer epithelial to mesenchymal transition, migration, and invasion. The activity of hypoxia-mediated Gli-1, a Hedgehog signaling factor in hypoxia, was confirmed by in vitro western blotting, immunofluorescence staining, wound-healing assay, and matrigel invasion assay, as well as by in vivo xenograft models (n = 5 per group). The Gli-1 mechanism in hypoxia was analyzed via mass spectrometry. Hypoxia enhanced the interaction of Gli-1 and T-complex protein 1 subunit beta (CCT2), as observed in the mass spectrometric analysis. We observed that reduction in CCT2 inhibits tumor induction by Gli-1. Ubiquitination-mediated Gli-1 degradation by β-TrCP occurs during incomplete folding of Gli-1 in hypoxia. The human CRC tissues revealed greater CCT2 expression than did the normal colon tissues, indicating that higher CCT2 expression in tumor tissues from CRC patients reduced their survival rate. Moreover, we suggest that CCT2 correlates with Gli-1 expression and is an important determinant of survival in the CRC patients. The results reveal that CCT2 can regulate the folding of Gli-1 in relation to hypoxia in CRC.

A major problem of colorectal cancer (CRC) targeted therapies is relapse caused by drug resistance. In most cases of CRC, patients develop resistance to anticancer drugs. Cetuximab does not show many of the side effects of other anticancer drugs and improves the survival of patients with metastatic CRC. However, the molecular mechanism of cetuximab resistance is not fully understood. Methods: EPHB3-mediated cetuximab resistance was confirmed by in vitro western blotting, colony-forming assays, WST-1 colorimetric assay, and in vivo xenograft models (n = 7 per group). RNA-seq analysis and receptor tyrosine kinase assays were performed to identify the cetuximab resistance mechanism of EPHB3. All statistical tests were two-sided. Results: The expression of EFNB3, which upregulates the EPHB3 receptor, was shown to be increased via microarray analysis. When resistance to cetuximab was acquired, EPHB3 protein levels increased. Hedgehog signaling, cancer stemness, and epithelial-mesenchymal transition signaling proteins were also increased in the cetuximab-resistant human colon cancer cell line SW48R. Despite cells acquiring resistance to cetuximab, STAT3 was still responsive to EGF and cetuximab treatment. Moreover, inhibition of EPHB3 was associated with decreased STAT3 activity. Co-immunoprecipitation confirmed that EGFR and EPHB3 bind to each other and this binding increases upon resistance acquisition, suggesting that STAT3 is activated by the binding between EGFR and EPHB3. Protein levels of GLI-1, SOX2, and Vimentin, which are affected by STAT3, also increased. Similar results were obtained in samples from patients with CRC. Conclusion: EPHB3 expression is associated with anticancer drug resistance.

Our team has previously reported a series of quinazoline-based lapatinib hybrids as potent kinase-targeting anticancer agents. Among them, AF8c showed a relatively safe profile in colorectal cancer (CRC) cells. In this study, we delineate a novel anticancer activity of AF8c in CRC cells. AF8c mediated p53-dependent apoptosis of CRC cells via the generation of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS), as well as activation of nuclear respiratory factor 2 alpha subunit (Nrf2) and death receptor 5 (DR5), among others. The silencing of DR5 attenuated the expression levels of Nrf2 and partially inhibited AF8c-induced apoptosis. Additionally, upregulation of Nrf2 by AF8c evoked apoptosis through a decrease in antioxidant levels. Treatment of a CRC mice model with AF8c also resulted in the upregulation of DR5, Nrf2, and CHOP proteins, subsequently leading to a significant decrease in tumor burden. In comparison with lapatinib, AF8c showed higher cellular antiproliferative activity at the tested concentrations in CRC cells and synergized TRAIL effects in CRC cells. Overall, our results suggest that AF8c-induced apoptosis may be associated with DR5/Nrf2 activation through ER stress and ROS generation in CRC cells. These findings indicate that AF8c represents a promising polypharmacological molecule for the treatment of human CRC.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to behave as an attractive anti-cancer agent in various cancers. Despite its promise TRAIL has limitations such as short half-life and rapid development of resistance. In this regard, approaches to sensitizers of TRAIL that can overcome the limitations of TRAIL are necessary. However, the molecular targets and mechanisms underlying sensitization to TRAIL-induced apoptosis are not fully understood. Here, we propose that reactive oxygen species modulator-1 (Romo1) as an attractive sensitizer of TRAIL. Romo1 is a mitochondrial inner membrane channel protein that controls reactive oxygen species (ROS) production, and its expression is highly upregulated in various cancers, including colorectal cancer. In the present study, we demonstrated that Romo1 inhibition significantly increased TRAIL-induced apoptosis of colorectal cancer cells, but not of normal colon cells. The combined effect of TRAIL and Romo1 inhibition was correlated with the activation of mitochondrial apoptosis pathways. Romo1 silencing elevated the protein levels of BCL-2-associated X protein (Bax) by downregulating the ubiquitin proteasome system (UPS). Romo1 inhibition downregulated the interaction between Bax and Parkin. Furthermore, Romo1 knockdown triggered the mitochondrial dysfunction and ROS generation. We validated the effect of combination in tumor xenograft model in vivo. In conclusion, our study demonstrates that Romo1 inhibition induces TRAIL-mediated apoptosis by identifying the novel mechanism associated with the Bax/Parkin interaction. We suggest that targeting of Romo1 is essential for the treatment of colorectal cancer and may be a new therapeutic approach in the future and contribute to the drug discovery.

Genipin, a major component of Gardenia jasminoides Ellis fruit, has been shown to inhibit the growth of gastric, prostate, and breast cancers. However, the anti-proliferative activity of genipin in colorectal cancer (CRC) has not been characterized. Herein, we demonstrated that genipin inhibits the proliferation of CRC cells and that genipin suppressed the Hedgehog pathway. Further investigation showed that p53 and NOXA protein levels were increased during inhibition of Hedgehog pathway-mediated apoptosis in CRC cells. We also showed that p53 modulated the expression of NOXA during genipin-induced apoptosis, and suppression via SMO also played a role in this process. Subsequently, GLI1 was ubiquitinated by the E3 ligase PCAF. In a xenograft tumor model, genipin suppressed tumor growth, which was also associated with Hedgehog inactivation. Taken together, these results suggest that genipin induces apoptosis through the Hedgehog signaling pathway by suppressing p53. These findings reveal a novel regulatory mechanism involving Hedgehog/p53/NOXA signaling in the modulation of CRC cell apoptosis and tumor-forming defects.

According to recent studies, Cannabidiol (CBD), one of the main components of Cannabis sativa, has anticancer effects in several cancers. However, the exact mechanism of CBD action is not currently understood. Here, CBD promoted cell death in gastric cancer. We suggest that CBD induced apoptosis by suppressing X-linked inhibitor apoptosis (XIAP), a member of the IAP protein family. CBD reduced XIAP protein levels while increasing ubiquitination of XIAP. The expression of Smac, a known inhibitor of XIAP, was found to be elevated during CBD treatment. Moreover, CBD treatment increased the interaction between XIAP and Smac by increasing Smac release from mitochondria to the cytosol. CBD has also been shown to affect mitochondrial dysfunction. Taken together, these results suggest that CBD may have potential as a new therapeutic target in gastric cancer.

Despite the importance of radiation therapy, there are few radiation-related markers available for use in clinical practice. A larger catalog of such biomarkers is required to help clinicians decide when radiotherapy should be replaced with a patient-specific treatment. Arachidonate 15-lipoxygenase (15-LOX-1) enzyme is involved in polyunsaturated fatty acid metabolism. When colorectal cancer (CRC) cells were exposed to radiation, 15-LOX-1 was upregulated. To verify whether 15-LOX-1 protects against or induces DNA damage, we irradiated sh15-LOX-1 stable cells. We found that low 15-LOX-1 is correlated with radioresistance in CRC cells. These data suggest that the presence of 15-LOX-1 can be used as a marker for radiation-induced DNA damage. Consistent with this observation, gene-set-enrichment analysis based on microarray experiments showed that UV_RESPONSE was decreased in sh15-LOX-1 cells compared to shCon cells. Moreover, we discovered that the expression of the histone H2A variant macroH2A2 was sevenfold lower in sh15-LOX-1 cells. Overall, our findings present mechanistic evidence that macroH2A2 is transcriptionally regulated by 15-LOX-1 and suppresses the DNA damage response in irradiated cells by delaying H2AX activation.

Background: Although the anticancer activity of Korean Red Ginseng (KRG) has been known in various cancers, the mechanism of KRG-induced apoptosis is unknown in colorectal cancer (CRC). In our study, we examined whether KRG induces apoptosis in CRC cells. Methods: In the cell viability assay, the concentration of the appropriate KRG extracts was fixed at 2.5 mg/mL in numerous CRC cells. This fixed concentration was in other experiments, and it was confirmed that the KRG extracts induce apoptosis in CRC cells. Results: We found that KRG induced Noxa activation and apoptosis and increased endoplasmic reticulum stress via reactive oxygen species production. This indicated that KRG efficiently enhanced cell death in CRC cells. Conclusion: Our results show that KRG can be used as a possible anticancer drug for patients with CRC

Oxaliplatin is an anticancer drug administered to colorectal cancer (CRC) patients in combination with 5-fluorouracil and antibodies (bevacizumab and cetuximab), thereby significantly improving the survival rate of CRC. However, due to various side effects associated with the above treatment strategy, the need for combinatorial therapeutic strategies has emerged. Based on the demand for new combinatorial therapies and the known antitumor effects of the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), we investigated the Oxaliplatin and DHA combination for its effect. Our results indicated that DHA further enhanced Oxaliplatin-induced cell viability and autophagic cell death, in vitro and in vivo. Oxaliplatin and DHA also increased the expression of Sestrin 2 (SESN2) and endoplasmic reticulum (ER) stress related C/EBP homologous protein (CHOP). Additionally, treatment with Oxaliplatin and DHA enhanced the binding of CHOP to the promotor region of SESN2, increasing SESN2 expression. These results suggested that DHA enhanced Oxaliplatin-induced reduction in cell viability and increase in autophagy via activating SESN2 and increasing ER stress. Thus, SESN2 may be an effective preclinical target for CRC treatment.

Cannabidiol, a major non-psychotomimetic compound derived from Cannabis sativa, is a potential therapeutic agent for a variety of diseases such as inflammatory diseases, chronic neurodegenerative diseases, and cancers. Here, we found that the combination of cannabidiol and TNF-related apoptosis-inducing ligand (TRAIL) produces synergistic antitumor effects in vitro. However, this synergistic effect was not observed in normal colonic cells. The levels of ER stress-related proteins, including C/EBP homologous protein (CHOP) and phosphorylated protein kinase RNA-like ER kinase (PERK) were increased in treatment of cannabidiol. Cannabidiol enhanced significantly DR5 expression by ER stress. Knockdown of DR5 decreased the combined effect of cannabidiol and TRAIL. Additionally, the combination of TRAIL and cannabidiol decreased tumor growth in xenograft models. Our studies demonstrate that cannabidiol enhances TRAIL-induced apoptosis by upregulating DR5 and suggests that cannabidiol is a novel agent for increasing sensitivity to TRAIL.

Although oxaliplatin is an effective chemotherapeutic drug for colorectal cancer (CRC) treatment, patients often develop resistance to it. Therefore, a new strategy for CRC treatment is needed. The purpose of this study was to determine the effect of cannabidiol (CBD), one of the components of the cannabis plant, in overcoming oxaliplatin resistance in CRC cells. We established oxaliplatin-resistant cell lines, DLD-1 R and colo205 R, in CRC DLD-1 and colo205 cells. Autophagic cell death was induced when oxaliplatin-resistant cells were treated with both oxaliplatin and CBD. Additionally, phosphorylation of nitric oxide synthase 3 (NOS3) was increased in oxaliplatin-resistant cells compared to that in parent cells. Combined treatment with oxaliplatin and CBD reduced phospho-NOS3 levels and nitric oxide (NO) production and resulted in the production of reactive oxygen species (ROS) by reducing the levels of superoxide dismutase 2, an antioxidant present in the mitochondria, causing mitochondrial dysfunction. Taken together, these results suggest that elevated phosphorylation of NOS3 is essential for oxaliplatin resistance. The combination of oxaliplatin and CBD decreased NOS3 phosphorylation, which resulted in autophagy, by inducing the overproduction of ROS through mitochondrial dysfunction, thus overcoming oxaliplatin resistance.

Natural products have shown great promise in sensitizing cells to TNF-related apoptosis-inducing ligand (TRAIL) therapy. Sea cucumber (SC) extracts possess antitumor activity, and hence their potential to sensitize colorectal cancer (CRC) cells to TRAIL therapy was evaluated. This study used Western blotting to evaluate the combination effects of SC and TRAIL in CRC, and determined the molecular mechanism underlying these effects. SC fractions and TRAIL alone did not affect apoptosis, however, combined treatment dramatically induced the apoptosis of CRC cells, but not of normal colon cells. Combined treatment induced the expression of apoptotic proteins (poly (ADP-ribose) polymerase (PARP), caspase 3, and 8), and this effect was markedly inhibited by the ubiquitination of X-linked inhibitor of apoptosis protein (XIAP). SC did not affect the mRNA levels, but it increased proteasomal degradation and ubiquitination of the XIAP protein. Furthermore, SC induced reactive oxygen species (ROS) production, thereby activating c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER) stress-related apoptotic pathways in CRC. Altogether, our results demonstrate that the SC F2 fraction may sensitize CRC cells to TRAIL-induced apoptosis through XIAP ubiquitination and ER stress.

Oxaliplatin is used for treatment in combination with many drugs. However, the survival rate is still low due to side effects and drug resistance. Therefore, the combination with natural products was required for increasing efficacy and reducing side effects. Genipin, a natural product derived from the Gardenia jasminoides, associated with anti-angiogenic, anti-proliferative, hypertension, inflammatory, and the Hedgehog pathway. It is not known that genipin increases the therapeutic effect of oxaliplatin in gastric cancer. In this study, we found that genipin sensitizes oxaliplatin-induced apoptosis for the first time using colony forming assay, FACS analysis, and western blotting in gastric cancer. Additionally, genipin induced p53 expression in AGS, MKN45, and MKN28 cells. Also, genipin induced autophagy and LC3 expression. Knockdown of LC3 decreased cell death enhanced by the combination of oxaliplatin and genipin. In summary, we showed that genipin increases the oxaliplatin-induced cell death via p53-DRAM autophagy. Based on this, we suggest that genipin is a sensitizer of oxaliplatin.

Imatinib is a powerful tyrosine kinase inhibitor that specifically targets BCR‑ABL, c‑KIT, and PDGFR kinases, and is used in the treatment of chronic myelogenous leukemia, gastrointestinal stromal tumors, and other types of cancers. However, the possible anticancer effects of imatinib in gastric cancer have not yet been explored. The present study evaluated the in vitro effects of imatinib on gastric cancer cells and determined the molecular mechanism underlying these effects. We determined that imatinib induced mitochondria‑mediated apoptosis of gastric cancer cells by involving endoplasmic reticulum (ER) stress‑associated activation of c‑Jun NH2‑terminal kinase (JNK). We also found that imatinib suppressed cell proliferation in a time‑ and dose‑dependent manner. Cell cycle analysis revealed that imatinib‑treated AGS cells were arrested in the G2/M phase of the cell cycle. Moreover, imatinib‑treated cells exhibited increased levels of phosphorylated JNK, and of the transcription factor C/EBP homologous protein, an ER stress‑associated apoptotic molecule. Results of cell viability assays revealed that treatment with a combination of imatinib and chemotherapy agents irinotecan or 5‑Fu synergistically inhibited cell growth, compared with treatment with any of these drugs alone. These data indicated that imatinib exerted cytotoxic effects on gastric cancer cells by inducing apoptosis mediated by reactive oxygen species generation and ER stress‑associated JNK activation. Furthermore, we revealed that imatinib induced the apoptosis of gastric cancer cells by inhibiting platelet‑derived growth factor receptor signaling. Collectively, our results strongly support the use of imatinib in the treatment of treating gastric cancer.

// Seong Hye Park 1, * , Dae-Hee Lee 1, 2, * , Jung Lim Kim 2 , Bo Ram Kim 1 , Yoo Jin Na 1 , Min Jee Jo 1 , Yoon A. Jeong 2 , Suk-Young Lee 2 , Sun Il Lee 3 , Yong Yook Lee 4 , Sang Cheul Oh 1, 2 1 Brain Korea 21 Program for Biomedicine Science, Korea University College of Medicine, Korea University, Seoul, Republic of Korea 2 Division of Oncology/Hematology, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea 3 Department of Surgery, Korea University Guro Hospital, Korea University College of Medicine, Seoul, Republic of Korea 4 The Korean Ginseng Research Institute, Daejeon, Republic of Korea * These authors contributed equally to this work Correspondence to: Sang Cheul Oh, email: sachoh@korea.ac.kr Keywords: metformin, TRAIL, CRC, Mcl-1, Noxa Received: December 20, 2015 Accepted: July 06, 2016 Published: August 09, 2016 ABSTRACT Metformin is an anti-diabetic drug with a promising anti-cancer potential. In this study, we show that subtoxic doses of metformin effectively sensitize human colorectal cancer (CRC) cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), which induces apoptosis. Metformin alone did not induce apoptosis, but significantly potentiated TRAIL-induced apoptosis in CRC cells. CRC cells treated with metformin and TRAIL showed activation of the intrinsic and extrinsic pathways of caspase activation. We attempted to elucidate the underlying mechanism, and found that metformin significantly reduced the protein levels of myeloid cell leukemia 1 (Mcl-1) in CRC cells and, the overexpression of Mcl-1 inhibited cell death induced by metformin and/or TRAIL. Further experiments revealed that metformin did not affect mRNA levels, but increased proteasomal degradation and protein stability of Mcl-1. Knockdown of Mule triggered a significant decrease of Mcl-1 polyubiquitination. Metformin caused the dissociation of Noxa from Mcl-1, which allowed the binding of the BH3-containing ubiquitin ligase Mule followed by Mcl-1ubiquitination and degradation. The metformin-induced degradation of Mcl-1 required E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Our study is the first report indicating that metformin enhances TRAIL-induced apoptosis through Noxa and favors the interaction between Mcl-1 and Mule, which consequently affects Mcl-1 ubiquitination.

Background Genipin is a compound derived from gardenia fruit extract. Although Genipin has anti-tumor effects in various cancers, its effect and mechanism in gastric cancer remain unclear. Here, we investigated the relationship between the anticancer effect of Genipin and signal transducer and activator of transcription (Stat3)/myeloid cell leukemia-1 (Mcl-1) in human gastric cancers. Methods MTT assays were performed to determine the cell viability of gastric cancer and gastric epithelial cell lines (AGS, MKN45, SNU638, MKN74, HFE-145). A TUNEL assay and Western blotting were carried out to investigate apoptosis. Stat3 activity was measured by proteome profiler phospho kinase array, immunofluorescence and immunoblotting. Mitochondria function was monitored with an XF24 analyzer and by flow cytometry, confocal microscopy using fluorescent probes for general mitochondrial membrane potential (MMP). Results Genipin induced apoptosis in gastric cancer cells, including AGS and MKN45 cells. Genipin also reduced Mcl-1 mRNA and protein levels. Furthermore, we found that phosphorylation of Stat3 is regulated by Genipin. Additionally, the protein level of phospho Janus kinase 2 (JAK2) was decreased by Genipin treatment, indicating that the Stat3/JAK2/Mcl-1 pathway is suppressed by Genipin treatment in gastric cancer cells. Mcl-1 is closely related to mitochondrial function. These findings suggest that Genipin contributes to the collapse of mitochondrial functions like MMP. Conclusions Genipin induced apoptosis by suppressing the Stat3/Mcl-1 pathway and led to mitochondrial dysfunction. Our results reveal a novel mechanism for the anti-cancer effect of Genipin in gastric cancer. Electronic supplementary material The online version of this article (10.1186/s12885-019-5957-x) contains supplementary material, which is available to authorized users.

Disabled tumor suppressor genes and hyperactive oncogenes greatly contribute to cell fates during cancer development because of their genetic alterations such as somatic mutations. However, little is known about how tumor suppressor genes react to diverse oncogenes during tumor progression. Our previous study showed that RUNX3 inhibits invasiveness by preventing vascular endothelial growth factor secretion and suppressed endothelial cell growth and tube formation in colorectal cancer (CRC). Hedgehog signaling is crucial for the physiological maintenance and self-renewal of stem cells, and its deregulation is responsible for their tumor development. The mechanisms that inhibit this pathway during proliferation remain poorly understood. Here, we found that the tumor suppressor RUNX3 modulates tumorigenesis in response to cancer cells induced by inhibiting oncogene GLI1 ubiquitination. Moreover, we demonstrated that RUNX3 and GLI1 expression were inversely correlated in CRC cells and tissues. We observed a direct interaction between RUNX3 and GLI1, promoting ubiquitination of GLI1 at the intracellular level. Increased ubiquitination of GLI1 was induced by the E3 ligase β-TrCP. This novel RUNX3-dependent regulatory loop may limit the extent and duration of Hedgehog signaling during extension of the tumor initiation capacity. On the basis of our results, identification of agents that induce RUNX3 may be useful for developing new and effective therapies for CRC.

Genipin is known as a compound derived from gardenia fruit extract. Although Genipin is known to have an anti-tumor effects in various cancers, its effect and mechanism are unknown in gastric cancer. In our studies, Genipin induced apoptosis in gastric cancer cells including AGS and MKN45 cells. Genipin reduced Mcl-1 mRNA levels and confirmed with transfection of Mcl-1 siRNA. Furthermore, We found that phospho Stat3 is regulated by Genipin through proteome profiler phosphor-kinase array. In addition, protein level of phosphor JAK2 decreased in Genipin treatment. The results elucidate that Stat3/JAK2/Mcl-1 signaling pathway was suppressed with Genipin treatment in gastric cancer cells. Mcl-1 is known to be closely related to the function of mitochondria. Genipin has been shown to result in the collapse of mitochondrial function such as mitochondrial membrane potential. In conclusion, Genipin induced apoptosis by suppressing Stat3/JAK2/Mcl-1 pathway and led to mitochondrial dysfunction. Our finding implies that a novel mechanism of anti-cancer effect of Genipin in gastric cancer can be identified. Citation Format: Min Jee Jo, Soyeon Jeong, Hye Kyeong Yun, Dae Yeong Kim, Bo Ram Kim, Jung Lim Kim, Yoo Jin Na, Seong Hye Park, Yoon A Jeong, Bu Gyeom Kim, Hassan Ashktorab, Duane T Smoot, Jun Young Heo, Jeongsu Han, Dae-Hee Lee, Sang Cheul Oh. Genipin enhanced apoptosis of gastric cancer by mitochondrial dysfunction and downregulation of Stat3/JAK2/Mcl-1 signaling pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A021. doi:10.1158/1535-7163.TARG-19-A021

Although oxaliplatin is an effective chemotherapeutic drug for colorectal cancer (CRC) treatment, patients often develop resistance to it. Therefore, a new strategy for CRC treatment is needed. The purpose of this study was to determine the effect of cannabidiol (CBD), one of the components of the cannabis plant, in overcoming oxaliplatin resistance in CRC cells. We established oxaliplatin-resistant cell lines, DLD-1 R and colo205 R, in CRC DLD-1 and colo205 cells. Autophagic cell death was induced when oxaliplatin-resistant cells were treated with both oxaliplatin and CBD. Additionally, phosphorylation of nitric oxide synthase 3 (NOS3) was increased in oxaliplatin-resistant cells compared to that in parent cells. Combined treatment with oxaliplatin and CBD reduced phospho-NOS3 levels and NO production and resulted in the production of reactive oxygen species (ROS) by reducing the levels of superoxide dismutase 2, an antioxidant present in the mitochondria, causing mitochondrial dysfunction. Taken together, these results suggest that elevated phosphorylation of NOS3 is essential for oxaliplatin resistance. The combination of oxaliplatin and CBD decreased NOS3 phosphorylation, which resulted in autophagy, by inducing the overproduction of ROS through mitochondrial dysfunction, thus overcoming oxaliplatin resistance. Citation Format: Bu Gyeom Kim, Soyeon Jeong, Dae Yeong Kim, Bo Ram Kim, Jung Lim Kim, Seong Hye Park, Yoo Jin Na, Min Jee Jo, Hye Kyeong Yun, Yoon A Jeong, Sun Il Lee, Yoon A Jeong, Hong Jun Kim, Han Do Kim, Dae Hyun Kim, Dae-Hee Lee, Sang Cheul Oh. Cannabidiol overcomes oxaliplatin resistance by enhancing autophagy through reduction NOS3 and SOD2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A130. doi:10.1158/1535-7163.TARG-19-A130

Multifunctional, antiproliferative small molecules are deemed to offer pharmacodynamic and pharmacokinetic benefits over combination therapy, in addition to reducing toxicity, cancer resistance, and therapy costs. In this study, we conducted an in vitro cellular screen of our recently reported series of EGFR/HER2 inhibitors. Due to its preferentially apoptosis triggering in colorectal cancer (CRC) cells compared to normal colon cells, the compound AF8c was selected for further evaluation. Analysis of the AF8c mode of action in CRC cells revealed that it mediates apoptosis via the generation of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS), as well as selective activation of nuclear respiratory factor 2 alpha subunit (Nrf2) and death receptor 5 (DR5), but not DR4. Silencing of DR5 attenuated the expression levels of Nrf2 and partially inhibited AF8c-induced apoptosis. Additionally, upregulation of Nrf2 by AF8c evoked apoptosis through a decrease in antioxidant levels. Treatment of mice with AF8c also resulted in the upregulation of DR5, Nrf2, and CHOP proteins, subsequently leading to a significant decrease in tumor burden. Overall, our results suggest that AF8c-induced apoptosis may be associated with DR5/Nrf2 activation through ER stress and ROS generation in CRC. These findings indicate that AF8c represents a promising polypharmacological molecule for the treatment of human CRC. Moreover, it could also be a potential starting point for the understanding of the structural features of quinazoline-based ErbB family inhibitors targeting the TRAIL cascade. Citation Format: Soyeon Jeong, Ahmed Karam Farag, Hye Kyeong Yun, Yoon A Jeong, Dae Yeong Kim, Min Jee Jo, Seong Hye Park, Bo Ram Kim, Jung Lim Kim, Dae-Hee Lee, Eun Joo Roh, Sang Cheul Oh. AF8c, a pan-ErbB family inhibitor induces apoptotic cell death by stimulating DR5/Nrf2 via ROS in colorectal cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A111. doi:10.1158/1535-7163.TARG-19-A111

Hong Jun Kim,1 Min Jee Jo,2 Bo Ram Kim,2 Jung Lim Kim,2 Yoon A Jeong,2 Yoo Jin Na,2 Seong Hye Park,2 Suk-young Lee,1 Dae-Hee Lee,1,2 Baek-hui Kim,3 Young Do Yoo,4 Sang Cheul Oh1,2 1Division of Oncology and Hematology, Department of Internal Medicine, College of Medicine, Korea University, Seoul, Republic of Korea; 2Laboratory of Oncology, Graduate School of Medicine, Korea University, Seoul, Republic of Korea; 3Department of Pathology, College of Medicine, Korea University, Seoul, Republic of Korea; 4Laboratory of Molecular Cell Biology, Graduate School of Medicine, Korea University, Seoul, Republic of Korea Introduction: Reactive oxygen species modulator-1 (Romo1) is a protein that modulates levels of reactive oxygen species (ROS) and has been reported to affect cancer cell invasion and proliferation via persistent inflammation. Several studies have demonstrated the clinical application of Romo1 as a prognostic marker in non-small cell lung cancer (NSCLC); however, there have been no studies investigating the mechanism by which Romo1 adversely affects the prognosis of these patients. Methods: We examined Romo1, ROS, and vascular endothelial growth factor (VEGF) in tumor tissues immunohistochemically. We conducted survival analyses of patients who had curative resection (n=30) in accordance with clinical parameters including levels of Romo1 expression. Results: Romo1 levels were associated with serologic inflammatory markers and high lymphatic metastatic tendencies. Significantly longer disease-free survival (68.7 vs 24.2 months, P=0.031) and overall survival (92.7 vs 51.6 months) were observed in the group with low Romo1 compared with high Romo1. Survival outcomes were also significantly associated with serologic inflammatory markers. Spearman’s correlation analyses demonstrated significant positive correlations of Romo1 expression with VEGF-C (P=0.008, R=0.478) and ROS (P=0.016, R=0.436) in tumor samples. Conclusion: The current study demonstrates that Romo1 induces lymphatic metastasis of NSCLC by modulating persistent inflammation and oxidative stress (ROS)/VEGF signaling. Lymphatic metastasis associated with elevated Romo1 was shown to be a key reason for unfavorable survival rates. Keywords: non–small cell lung carcinoma, inflammation, reactive oxygen species modulator-1, vascular endothelial growth factor A, reactive oxygen species

Macroautophagy is induced under various stresses to remove cytotoxic materials, including misfolded proteins and their aggregates. These protein cargoes are collected by specific autophagic receptors such as SQSTM1/p62 (sequestosome 1) and delivered to phagophores for lysosomal degradation. To date, little is known about how cells sense and react to diverse stresses by inducing the activity of SQSTM1. Here, we show that the peroxiredoxin-like redox sensor PARK7/DJ-1 modulates the activity of SQSTM1 and the targeting of ubiquitin (Ub)-conjugated proteins to macroautophagy under oxidative stress caused by TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10). In this mechanism, TNFSF10 induces the N-terminal arginylation (Nt-arginylation) of the endoplasmic reticulum (ER)-residing molecular chaperone HSPA5/BiP/GRP78, leading to cytosolic accumulation of Nt-arginylated HSPA5 (R-HSPA5). In parallel, TNFSF10 induces the oxidation of PARK7. Oxidized PARK7 acts as a co-chaperone-like protein that binds the ER-derived chaperone R-HSPA5, a member of the HSPA/HSP70 family. By forming a complex with PARK7 (and possibly misfolded protein cargoes), R-HSPA5 binds SQSTM1 through its Nt-Arg, facilitating self-polymerization of SQSTM1 and the targeting of SQSTM1-cargo complexes to phagophores. The 3-way interaction among PARK7, R-HSPA5, and SQSTM1 is stabilized by the Nt-Arg residue of R-HSPA5. PARK7-deficient cells are impaired in the targeting of R-HSPA5 and SQSTM1 to phagophores and the removal of Ub-conjugated cargoes. Our results suggest that PARK7 functions as a co-chaperone for R-HSPA5 to modulate autophagic removal of misfolded protein cargoes generated by oxidative stress.

RUNX3 is frequently inactivated by DNA hypermethylation in numerous cancers. Here, we show that RUNX3 has an important role in modulating apoptosis in immediate response to tumor necrosis factor-related apoptosis-including ligand (TRAIL). Importantly, no combined effect of TRAIL and RUNX3 was observed in non-cancerous cells. We investigated the expression of the death receptors (DRs) DR4 and DR5, which are related to TRAIL resistance. Overexpression of RUNX3 increased DR5 expression via induction of the reactive oxygen species (ROS)-endoplasmic reticulum (ER) stress-effector CHOP. Reduction of DR5 markedly decreased apoptosis enhanced by the combined therapy of TRAIL and RUNX3. Interestingly, RUNX3 induced reactive oxygen species production by inhibiting SOD3 transcription via binding to the Superoxide dismutase 3 (SOD3) promoter. Additionally, the combined effect of TRAIL and RUNX3 decreased tumor growth in xenograft models. Our results demonstrate a direct role for RUNX3 in TRAIL-induced apoptosis via activation of DR5 and provide further support for RUNX3 as an anti-tumor.

Despite an increase in the survival rate of patients with cancer owing to the use of current chemotherapeutic agents, adverse effects of cancer therapies remain a concern. Combination therapies have been developed to increase efficacy, reduce adverse effects, and overcome drug resistance. Genipin is a natural product derived from Gardenia jasminoides, which has been associated with anti-inflammatory, anti-angiogenic, and anti-proliferative effects; hypertension; and anti-ischemic brain injuries. However, the enhancement of oxaliplatin sensitivity by genipin remains unexplored. Our study showed that a combination of genipin and oxaliplatin exerts synergistic antitumor effects in vitro and in vivo in colorectal cancer cell lines through the reactive oxygen species (ROS)/endoplasmic reticulum (ER) stress/BIM pathway. Importantly, the combination did not affect normal colon cells. BIM knockdown markedly inhibited apoptosis induced by the combination. In addition, genipin induced ROS by inhibiting superoxide dismutase 3 activity. These findings suggest that genipin may be a novel agent for increasing the sensitivity of oxaliplatin against colorectal cancer. The combination of oxaliplatin and genipin hold significant therapeutic potential with minimal adverse effects.

Bone morphogenetic proteins (BMPs) have been involved in metastatic progression and tumorigenesis of many cancer types. However, it remains unclear how BMP-2 contributes to the initiation and development of these cancers. Here, we investigated the role of BMP-2 in colon cancer stem cell (CSC) development from colon cancer cells. We also determined the effects of BMP-2 on CSC development and epithelial-mesenchymal transition (EMT) in human colon cancer cell lines HCT-116 and SW620. We found that BMP-2 enhanced sphere formation of colon cancer cells without serum. Also, BMP-2-induced spheres displayed up-regulation of stemness markers (CD133+ and EpCAM+) and increased drug resistance, hallmarks of CSCs. Importantly, expression of EMT activators p-Smad1/5 and Snail and N-cadherin was increased in the spheres' cells, indicating that BMP-2 signaling might result in CSC self-renewal and EMT. Furthermore, siRNA-mediated knockdown of signal transducer and activator of transcription 3 (STAT3) in HCT-116 cells reversed BMP-2-induced EMT and stem cell formation. Taken together, our results suggest that the BMP-2 induced STAT3-mediated induction of colon cancer cell metastasis requires an EMT and/or changes in CSC markers.

Background Reactive oxygen species modulator-1 (Romo1) is a novel protein that has been reported to be crucial for cancer cell proliferation and invasion. However, its clinical implications in colorectal cancer patients are not well-known. For the first time, we investigated the association between Romo1 and the clinical outcomes of colorectal cancer patients. Study We examined Romo1 expression in resected tumor tissues immunohistochemically and assessed it with histological scores. We conducted survival analyses for patients who had curative resection (n = 190) in accordance with clinical parameters including level of Romo1 expression, and we examined the association between Romo1 expression and cell invasion using Matrigel invasion assay in colorectal cancer cells. Results We observed significantly longer mean disease-free survival in the low Romo1 group compared with the high Romo1 group (161 vs 127.6 months, p = 0.035), and the median overall survival of the low Romo1 group was significantly longer than that of the high Romo1 group (196.9 vs 171.3 months, p = 0.036). Cell invasiveness decreased in the Romo1 knockdown colorectal cancer cells in contrast to the controlled cells. Romo1 overexpression in tumor tissue was associated with a high lymph node ratio between the metastatic and examined lymph nodes (p = 0.025). Conclusions Romo1 overexpression in tumor tissue was significantly associated with survival in curatively resected colorectal cancer patients, suggesting Romo1 expression as a potential adverse prognostic marker. Increased Romo1 expression was found to be associated with high lymph node ratio. Cancer invasiveness appeared to be a key reason for the poor survival related to highly expressed Romo1.

Background Multifunctional, antiproliferative small molecules are deemed to offer pharmacodynamic and pharmacokinetic benefits over combination therapy, in addition to reducing toxicity, cancer resistance, and therapy costs. In this study, we conducted an in vitro cellular screen of our recently reported series of EGFR/HER2 inhibitors. Methods Cytotoxicity induced by AF8c was confirmed using WST-1 assay. An Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit, TUNEL assay, and Western blotting were carried out to invastigate apoptosis. A human phosphorylated kinase array was used to identify proteins differentially expressed between control cells and AF8c-treated cell lines. ROS generation was monitored by flow cytometry, confocal microscopy using dihydroethidium (DHE) and MitoSOX. For in vivo tumor xenograft study, four-week-old female BALB/c nude mice were injected subcutaneously with either HT29 Luc+ or HCT116 Luc+ cells (1 × 107 cells in 100 μl PBS). When the tumor had reached approximately 100 mm3 in size, the mice were randomly divided into three groups (n = 8): DMSO-treated, treated with 10 mg/kg AF8c, and treated with 20 mg/kg AF8c. Results Analysis of the AF8c mode of action in CRC cells revealed that it mediates apoptosis via the generation of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS), as well as selective activation of nuclear respiratory factor 2 alpha subunit (Nrf2) and death receptor 5 (DR5), but not DR4. Silencing of DR5 attenuated the expression levels of Nrf2 and partially inhibited AF8c-induced apoptosis. Additionally, upregulation of Nrf2 by AF8c evoked apoptosis through a decrease in antioxidant levels. Treatment of mice with AF8c also resulted in the upregulation of DR5, Nrf2, and CHOP proteins, subsequently leading to a significant decrease in tumor burden. Conclusions AF8c-induced apoptosis may be associated with DR5/Nrf2 activation through ER stress and ROS generation in CRC. These findings indicate that AF8c represents a promising polypharmacological molecule for the treatment of human CRC. Moreover, it could also be a potential starting point for the understanding of the structural features of quinazoline-based ErbB family inhibitors targeting the TRAIL cascade. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Natural products have shown great promise in sensitizing cells to TNF-related apoptosis-inducing ligand (TRAIL) therapy. Sea cucumber (SC) extracts possess antitumor activity, and hence their potential to sensitize CRC cells to TRAIL therapy was evaluated. This study used Western blotting to evaluate the combination effects of SC and TRAIL in CRC and determined the molecular mechanism underlying these effects. SC fractions and TRAIL alone had no effect on apoptosis; however, combined treatment dramatically induced apoptosis of CRC cells, but not normal colon cells. Combined treatment induced expression of apoptotic proteins (poly (ADP-ribose) polymerase (PARP), caspase 3, and 8), and this effect was markedly inhibited by ubiquitination of X-linked inhibitor of apoptosis protein (XIAP). SC did not affect the mRNA levels, but increased proteasomal degradation and ubiquitination of XIAP protein. Furthermore, SC induced reactive oxygen species (ROS) production, thereby activating c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER) stress-related apoptotic pathways in CRC. Taken together, our results demonstrate that the SC fraction may sensitize CRC cells to TRAIL-induced apoptosis through XIAP ubiquitination-ER stress. Citation Format: Jung lim Kim, Seong Hye Park, Soyeon Jeong, Bo Ram Kim, Yoo Jin Na, Min Jee Jo, Yoon A Jeong, Hye Kyeong Yun, Dae Yeong Kim, Bu Gyeom Kim, Sang Cheul Oh, Dae-Hee Lee. Sea cucumber (Stichopus japonicas) F2 enhanced TRAIL-induced apoptosis via XIAP ubiquitination-ER stress in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1866.

Cannabidiol (CBD), one of the compounds present in the marijuana plant, has anti-tumor properties, but its mechanism is not well known. The authors aimed to evaluate the apoptotic action of CBD in colorectal cancer cells (CRC), and focused on its effects on the novel pro-apoptotic Noxa-reactive oxygen species (ROS) signaling pathway. CBD experiments were performed using the colorectal cancer cell lines HCT116 and DLD-1. CBD induced an apoptosis by regulating many pro- and anti-apoptotic proteins, of which Noxa showed a significantly higher expression. To understand the relationship between Noxa and CBD-induced apoptosis, authors downregulated Noxa levels using siRNA and confirmed that the expression of apoptosis markers was decreased. After ROS production was blocked, the level of Noxa also decreased, suggesting that ROS is involved in the regulation of Noxa. The Noxa and ROS are well-known pro-apoptotic signaling agents. As a result, CBD was found to induce apoptosis in a Noxa-and-ROS-dependent manner. The effects of CBD treatment were re-demonstrated in vivo, thus confirming its role as a novel, reliable anticancer drug Citation Format: Hye Kyeong Yun, Soyeon Jeong, Yoon A Jeong, Min Jee Jo, Jung lim Kim, Seong Hye Park, Bo Ram Kim, Yoo Jin Na, Dae Young Kim, Bu Gyeom Kim, Dae Hee Lee, Sang Cheul Oh. Cannabidiol-induced apoptosis is mediated by activation of Noxa in human colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 714.

Radiotherapy is the principal treatment method for inducing cell death in various cancer types such as lung cancer; however, this therapeutics is frequently associated with occurrence of resistance such as recurrences or metastasis due to recent studies that radiotherapy might promote cancer progression and metastasis. Recurrences or metastasis is major cause of treatment failure of cancer, but its intra-/inter-cellular molecular mechanisms was not identified clearly yet due to the fact that genetic characteristics of cancer are extremely diverse according to individuals. Therefore, the objective of this study is to investige the modifications of intracellular signaling/genetic profiling in ionizing radiation (IR)-irradiated microenvironment of non-small-cell lung cancer (NSCLC), and to provide new therapeutic target for treatment of metastatic cancer. Previously, we have studied that IR treatment increased migration and invasion via induction of epithelial-mesenchymal transition (EMT) in A549 cells. With the same system, we found the increase of expression level of receptor interacting protein 1 (RIP1) in IR-treated A549, and this increased expression of RIP1 is associated with IR-induced EMT induction in vitro. NCI-H460 and A549 overexpressing RIP1 was established, and these transfectants also showed increased of migration and invasion via EMT induction. Signaling analyses indicated activation of EGFR-Src-STAT3 pathway is required for the EMT induction, and RIP1 exerts effect on this pathway resulting in activation of EGFR- RIP1-Src-STAT3 pathway. The down-regulation of RIP1 expression by treatment of siRNA, pharmaceutical inhibitor for RIP1 kinase, or dominant negative mutant of the kinase suppressed not only the activation of EGFR-RIP1-Src-STAT3, but also the EMT induction; this suggests that RIP1 might be one of key molecules in IR-induced migration/invasion. Additionally, DNA profiling analyses of RIP1-overexpressed NCI-H460 and A549 indicated that IL-1β is the most prominent over-expressed gene in common. IR treatment also stimulated IL-1β expression and its upstream NF-κB activation via increase of RIP1 followed by induction of EMT and cancer invasion/migration. Supernatants of stable cells including secreted IL-1β treatment promoted invasion/migration of NCI-H460 and A549 cells via EMT induced by activation of EGFR- RIP1-Src-STAT3 pathway. Taken together, our present results imply that RIP1 signals through EGFR- RIP1-Src-STAT3 axis and also through RIP1-NF-κB-IL-1β pathway. This also suggest that these two pathways are connected with common molecule, RIP1, which is novel signaling pathway and activations of these two pathways contribute to new autocrine signaling involving IL-1β secretion; therefore, RIP1 has a role as a novel biomarker as well as a molecular target for suppressing lung cancer metastasis. Citation Format: Jeong Hyun Cho, Jung Lim Kim, Hong-Duck Um, Sang-Gu Hwang, EunAh Lee, Sang-Churl Oh, Jong Kuk Park. RIP1 and its signaling network regulate radiation-induced modification of cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1973.

Recent studies have determined that inactivation of runt‑related transcription factor 3 (RUNX3) expression is highly associated with lymph node metastasis and poor prognosis in various types of cancer. However, the mechanism of RUNX3-mediated suppression of tumor metastasis remains unclear. Herein, we aimed to clarify the effect of RUNX3 on metastasis and angiogenesis in colorectal cancer (CRC). Firstly, we found that the reduction in expression of RUNX3 in CRC tissues when compared with tumor adjacent normal colon tissues, as indicated by reduced RUNX3 staining, was significantly correlated with tumor-node-metastasis (TNM) stage. Secondly, we demonstrated that RUNX3 overexpression inhibited CRC cell migration and invasion resulting from the upregulation of matrix metalloproteinase-2 (MMP-2) and MMP-9 expression. In contrast, the knockdown of RUNX3 reduced the inhibition of migration and invasion of CRC cells. Finally, we found that restoration of RUNX3 decreased vascular endothelial growth factor (VEGF) secretion and suppressed endothelial cell growth and tube formation in CRC cells. All in all, our findings may provide insight into the development of RUNX3 for CRC metastasis diagnostics and therapeutics.

Disabled tumor suppressor genes and hyperactive oncogenes contribute considerably to cell fates during cancer development via genetic alterations such as somatic mutations. To date, little is known about how tumor suppressor genes react to diverse oncogenes during tumor progress. Our previous report showed that RUNX3 inhibits metastasis by preventing vascular endothelial growth factor (VEGF) secretion and suppressed endothelial cell growth and tube formation in colorectal cancer. Hedgehog signaling is crucial for the physiological maintenance and self-renewal of stem cells, and its deregulation is responsible for their tumor development. The mechanisms that inhibit this pathway during the proliferation are poorly understood. Here, we show that the tumor suppressor RUNX3 modulates tumorigenesis in response to cancer cells induced by inhibition by oncogene Gli-1 ubiquitination. Moreover, we demonstrated that RUNX3 and Gli-1 expression had inverse correlation in colorectal cancer cells and tissues. We observed a direct interaction between RUNX3 and Gli-1, promoting ubiquitination of Gli-1 at the intracellular level. The increased ubiquitination of Gli-1 was induced through the E3 ligase β-TrCP. This novel RUNX3-dependent regulatory loop may limit the extent and duration of Hedgehog signaling during extension of tumor initiation capacity. Base on this research, a search for agents that can induce RUNX3 might result in new and effective therapies for colorectal cancer. Citation Format: Bo Ram Kim, Yoo Jin Na, Jung Lim Kim, Soyeon Jeong, Seong Hae Park, Min Jee Jo, YoonA Jeong, Dae-Hee Lee, Sang Cheul Oh. RUNX3 inhibits invasiveness and stemness through downregulation of Gli-1 in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 102.