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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.

Cannabidiol (CBD), one of the compounds present in the marijuana plant, has anti-tumor properties, but its mechanism is not well known. This study aimed to evaluate the apoptotic action of CBD in colorectal cancer (CRC) cells, and focused on its effects on the novel pro-apoptotic Noxa-reactive oxygen species (ROS) signaling pathway. CBD experiments were performed using the CRC cell lines HCT116 and DLD-1. CBD induced apoptosis by regulating many pro- and anti-apoptotic proteins, of which Noxa showed significantly higher expression. To understand the relationship between Noxa and CBD-induced apoptosis, Noxa levels were downregulated using siRNA, and the expression of apoptosis markers decreased. After ROS production was blocked, the level of Noxa also decreased, suggesting that ROS is involved in the regulation of Noxa, which along with ROS is a well-known pro-apoptotic signaling agents. As a result, CBD induced apoptosis in a Noxa-and-ROS-dependent manner. Taken together, the results obtained in this study re-demonstrated the effects of CBD treatment in vivo, thus confirming its role as a novel, reliable anticancer drug.

Objectives Rapid detection of carbapenemase-producing Enterobacterales (CPE) is important to control spread of the resistance. We previously reported that imipenem disks prepared from injectable imipenem-cilastatin could rapidly detect KPC- and NDM-type carbapenemases. In the present study, we evaluated performance of disks of IPM and combined disks of imipenem-tazobactam and imipenem-EDTA, which were prepared from powders of imipenem and inhibitors. Methods Isolates of Enterobacterales were recovered from specimens of patients at a tertiary care hospital in Korea during January 2017 and March 2018. Routine CPE detection was performed by the CPE surveillance personnel whereas evaluation of the Disk carbapenemase test (DCT) was performed by the other personnel without knowing the results of surveillance. The DCT was carried out by pressing disks on to colonies and rehydrating in Petri plates and observing color change. Results The DCT differentiated 688 of 694 (sensitivity 99.1%) carbapenemase-producing isolates in 2.5–20 min: 630 with KPC, 51 with NDM, three with IMP, one with VIM, two with KPC and IMP, and one with NDM and OXA-181. The DCT failed to detect six OXA- 48-like enzyme-producing isolates, but the modified method using 96-well flat-bottom microplates with mineral oil cover detected all 29 OXA-48-like enzyme-producing isolates in 20–120 min. The DCT was negative for all 440 ertapenem-nonsusceptible, carbapenemase gene-negative isolates (specificity 100%). Conclusion The procedure of DCT is simple and can differentiate isolates of Enterobacterales with KPC-, NDM-, IMP- and VIM-type carbapenemases rapidly, and the modified DCT can detect isolates with OXA-48-like enzymes rapidly.

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.

Amplification of fibroblast growth factor receptor2 (FGFR2) has been regarded as a druggable target in gastric cancer (GC). Despite known potential of AZD4547, a selective inhibitor of FGFR 1-3, to suppress tumorigenic effects of activated FGFR2, resistance to the targeted agent has been an unresolved issue. This study was performed to elucidate the mechanism of AZD4547 resistance in GC cells. SNU-16 cells were used to establish an AZD4547-resistant GC cell line, SNU-16R. Elevated phosphorylation of EphB3 was confirmed using the Human Phospho-Receptor Tyrosine Kinase Array kit. A tyrosine kinase inhibitor (TKI) of EphB3 was used to investigate the effects of suppressed EphB3 activity in the SNU-16R cell line. SNU-16R cells exhibited upregulated phosphorylation of EphB3. Treatment of SNU-16R cells with the EphB3 TKI resulted in induction of apoptosis, decreased cellular viability, and cell cycle arrest at sub-G1 phase. SNU-16R cells expressed upregulated levels of N-cadherin, vimentin, Snail, matrix metalloproteinase 2 (MMP-2), and MMP-9, and reduced levels of E-cadherin, characteristic of epithelial to mesenchymal transition (EMT). Matrigel invasion assay also demonstrated the increased invasiveness of SNU-16R cells. EphB3 TKI treatment inhibited EMT of SNU-16R cells. Activation of mammalian target of rapamycin (mTOR) through the Ras-ERK1/2 pathway was suggested as the signal transduction mechanism downstream EphB3 by showing enhanced phosphorylation of Raf-1, MEK1/2, ERK1/2, mTOR and its downstream substrates in SNU-16R cells. As expected, EphB3 TKI decreased phosphorylation of these proteins. Our data suggest phosphorylation of mTOR through signaling by EphB3 is a potential mechanism of AZD4547 resistance in GC cells.

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.

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.

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.

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.

This study demonstrates that combined treatment with subtoxic doses of Codium extracts (CE), a flavonoid found in many fruits and vegetables, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induces apoptosis in TRAIL-resistant colorectal cancer (CRC) cells. Effective induction of apoptosis by combined treatment with CE and TRAIL was not blocked by Bcl-xL overexpression, which is known to confer resistance to various chemotherapeutic agents. While TRAIL-mediated proteolytic processing of procaspase-3 was partially blocked in various CRC cells treated with TRAIL alone, co-treatment with CE efficiently recovered TRAIL-induced caspase activation. We observed that CE treatment of CRC cells did not change the expression of anti-apoptotic proteins and pro-apoptotic proteins, including death receptors (DR4 and DR5). However, CE treatment markedly reduced the protein level of the short form of the cellular FLICE-inhibitory protein (c-FLIPS), an inhibitor of caspase-8, via proteasome-mediated degradation. Collectively, these observations show that CE recovers TRAIL sensitivity in various CRC cells via down-regulation of c-FLIPS.

// 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.

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

The molecular epidemiology and antimicrobial resistance of Clostridioides difficile were studied in South Korea in 2017 as part of a National Surveillance System. From February to May 2017, all non-duplicate isolates of C. difficile were recovered from patients who were suspected to have C. difficile infection and collected from 6 referral hospitals representing the 6 regions in South Korea. We performed PCRs for the toxin gene, PCR ribotyping, multilocus sequence typing (MLST), antimicrobial susceptibility testing by agar dilution according to the recommendations of the CLSI and detection of antimicrobial resistance genes such as ermB, catD, tetM, vanZ and nimR by PCR. Of 331 C. difficile isolates, 257 (77.6%) were toxigenic and the prevalence of strains producing binary toxin (CDT) was 5.1% (13/257). A total of 52 different ribotype (RT) patterns were found. RT018 was the most common (25.1% of all isolates), and RT014/020, RT002 and RT012 were also common. RT010 was most common non-toxigenic strain. MLST analysis of randomly selected 72 C. difficile isolates identified 46 sequence types (STs), of which three were new and not in the PubMLST library. There was a good correlation between MLST and RT as following: ST1 (RT027), ST8 (RT002), ST11 (RT078), ST17 (RT018), ST35 (RT046), ST37 (RT017), ST42 (RT106), ST53 (RT103), ST81 (RT369), and ST99 (RT070). All toxigenic isolates were susceptible to metronidazole and vancomycin (MIC ≤ 2 mg/L). For rifaximin, 24% of toxigenic isolates were resistant. Of randomly selected 106 toxigenic isolates, resistance rates for ampicillin, cefotetan, clindamycin, imipenem, chloramphenicol, tetracycline, and moxifloxacin were 48%, 46%, 64%, 54%, 0%, 6% and 52% respectively and frequencies of various resistance genes were 62.3% for ermB, 0.9% catD and 10.4% tetM. RTs018, 002, 017 and 369 showed high MICs to various antimicrobial agents and multi-drug resistance was common also.

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.

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.