[Corrigendum] PRIMA­1met induces autophagy in colorectal cancer cells through upregulation of the mTOR/AMPK­ULK1­Vps34 signaling cascade.

Following the publication of the above article, the authors contacted the Editorial Office to explain that the strips of ß­actin, LC3 and p62 proteins of the RKO cell line shown in Fig. 2A and B, and those of the SW620 cell line shown in Fig. 3A and B, were assembled in these figures incorrectly. To rectify the presentation of these two figures, the authors proposed that they replace the strips of ß­actin and p62 proteins in the original Figs. 2B and 3B with the ß­actin bands from one of the repeated western blotting experiments.  The revised and corrected versions of Figs. 2 and 3 are shown on the next page. The authors wish to emphasize that these corrections do not grossly affect either the results or the conclusions reported in this work. The authors all agree to the publication of this Corrigendum, and are grateful to the Editor of Oncology Reports for granting them the opportunity to correct the errors that were made during the assembly of these figures. Lastly, the authors apologize to the readership for any inconvenience these errors may have caused. [Oncology Reports 45: 86, 2021; DOI: 10.3892/or.2021.8037].

Repurposing the Pentameric B-Subunit of Shiga Toxin for Gb3-Targeted Immunotherapy of Colorectal Cancer by Rhamnose Conjugation.

Globotriaosylceramide (Gb3 or CD77) is a tumor-associated carbohydrate antigen implicated in several types of cancer that serves as a potential cancer marker for developing target-specific diagnosis and therapy. However, the development of Gb3-targeted therapeutics has been challenging due to its carbohydrate nature. In the present work, taking advantage of its natural pentamer architecture and Gb3-specific targeting of shiga toxin B subunit (StxB), we constructed a pentameric antibody recruiting chimera by site-specifically conjugating StxB with the rhamnose hapten for immunotherapy of colorectal cancer. The Sortase A-catalyzed enzymatic tethering of rhamnose moieties to the C terminus of Stx1B and Stx2B had very moderate effect on their pentamer architectures and thus the resultant conjugates maintained the potent ability to bind to Gb3 antigen both immobilized on an assay plate and expressed on colorectal cancer cells. All StxB-rhamnose constructs were capable of efficiently mediating the binding of rhamnose antibodies onto HT29 colorectal cancer cells, which was further shown to be able to induce cancer cell lysis by eliciting potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vitro. Finally, the best StxB-rhamnose conjugate, i.e. 1B-3R, was confirmed to be able to inhibit the colorectal tumor growth using a HT29-derived xenograft murine model. Taken together, our data demonstrated the potential of repurposing StxB as an excellent multivalent scaffold for developing Gb3-targeted biotherapeutics and StxB-rhamnose conjugates might be promising candidates for targeted immunotherapy of Gb3-related colorectal cancer.

Role of TRIM22 in ulcerative colitis and its underlying mechanisms.

Ulcerative colitis (UC) is a common chronic recurrent inflammatory disease, which seriously threatens human life and health. Therefore, the present study aimed to explore the role of tripartite motif­containing (TRIM)22 in UC and its potential mechanism. C57BL/6 mice and HT­29 cell models of UC were constructed using 2% dextran sulphate sodium (DSS). The protein and mRNA expression levels were detected by western blotting and reverse transcription­quantitative PCR, respectively. Cell transfection was performed to overexpress Kruppel­like factor 2 (KLF2), or knockdown KLF2, TRIM22 and TRIM30 expression. The levels of inflammatory factors were evaluated by enzyme­linked immunosorbent assays. Cell Counting Kit­8 and TUNEL staining assay were employed to assess cell viability and apoptosis. Dual­luciferase reporter assay and chromatin immunoprecipitation assay were performed to determine the binding ability of the TRIM22 promoter to KLF2. The results revealed that DSS increased the expression levels of TRIM22 in HT­29 cells and TRIM30 in mice. Short hairpin RNA (sh)­TRIM30 could inhibit the NF­κB pathway, and reduce the levels of TNF­α, IL­6 and IFN­Î³. Furthermore, KLF2 expression was downregulated in the cell model of UC, and the luciferase assay confirmed that the 3' untranslated region of TRIM22 was a direct target of KLF2. The ChIP assay also verified the binding of KLF2 with the TRIM22 promoter. Notably, knockdown of KLF2 reversed the enhancing effects of sh­TRIM22 on the viability of DSS­treated HT­29 cells. In addition, compared with in the DSS + sh­TRIM22 group, the protein expression levels of phosphorylated (p)­NF­κB and p­IκBα were increased in the DSS + sh­TRIM22 + sh­KLF2 group, as were the levels of TNF­α, IL­6 and IFN­Î³. In conclusion, TRIM22 was upregulated in DSS­induced HT­29 cells. TRIM22 knockdown increased DSS­induced HT­29 cell viability and decreased apoptosis and inflammation; this was reversed by knockdown of KLF2. These findings suggested that TRIM22 may promote disease development through the NF­κB signaling pathway in UC and could be inhibited by KLF2 transcription.

RIP3 knockdown inhibits necroptosis of human intestinal epithelial cells via TLR4/MyD88/NF-κB signaling and ameliorates murine colitis.

BACKGROUND: Ulcerative colitis (UC) is a common inflammatory bowel disease, during which cell necroptosis plays key roles in driving inflammation initiation and aggravation. Previous studies reported Receptor Interacting Protein Kinase 3 (RIP3)-mediated necroptosis in multiple diseases, and RIP3 protein in Paneth cells significantly enriched in the intestines of both humans and mice. Therefore, we hypothesized targeting RIP3 to inhibit necroptosis may depress UC. METHODS: We classified clinical UC samples according to the modified Truelove & Witts criterion. The expression of RIP3 was measured by western blot and immunohistochemistry. Cell proliferation and apoptosis were analyzed by MTT assay and flow cytometry. ROS production and the secretion of inflammatory cytokines were measured by DCFH-DA probe and ELISA assay. TLR4/MyD88/NF-κB signaling pathway was analyzed by western blot. We established experimental colitis model in RIP3 knockout and wild-type mice and disease activity index (DAI) score was calculated. The expression and distribution of tight junction protein were analyzed by immunofluorescence. The ratio of CD4+Foxp3+ T cells in the spleen was detected by flow cytometry. Oxidative damage of mouse colon was assessed by detecting the levels of SOD, MDA and MPO. Data were analyzed by one-way ANOVA or student's t test. RESULTS: The expression of RIP3 in human colon is positively associated with the severity of UC. RIP3 inhibitor GSK872 or RIP3 knockdown reverses the inhibitory effect of TNF-α on proliferation and the promoting effect of TNF-α on apoptosis and necrosis in human intestinal epithelial cells. In addition, RIP3 deficiency inhibits the secretion of inflammatory cytokines (IL-16, IL-17 and IFN-γ) and ROS production induced by TNF-α. In vivo, RIP3 inhibitor Nec-1 effectively improves DSS-induced colitis in mice. In mechanism, RIP3 depression could upregulate the proportion of CD4+Foxp3+ immunosuppressive Treg cells in the spleen while suppressed TLR4/MyD88/NF-κB signaling pathway and ROS generation, and all these anti-inflammation factors together suppress the secretion of inflammatory cytokines and necroptosis of intestinal epithelial cells. CONCLUSIONS: This study preliminarily explored the regulating mechanism of RIP3 on UC, and Nec-1 may be a promising drug to alleviate the inflammation and necroptosis of the colon in UC patients.

Nanobody-Based Bispecific Neutralizer for Shiga Toxin-Producing E. coli.

Currently, no specific therapeutics are available for foodborne Shiga toxin-producing Escherichia coli (STEC) infections that cause severe gastroenteritis and life-threatening complications of hemolytic uremic syndrome (HUS). As STEC attachment to intestinal epithelium might increase the host absorption of Shiga toxins and severity of the disease, we were inspired to develop a bispecific neutralizer capable of blocking its Shiga toxin and adhesin intimin simultaneously. Two nanobodies against the B subunit of Shiga toxin 2 (Stx2B) and the C terminus of Intimin (IntC280) were genetically fused together as the bispecific neutralizer, and it can be efficiently produced in a conventional E. coli expression system. We demonstrated that each of the nanobody modules in the bispecific format showed increased antigen binding capability and was able to functionally neutralize the binding of Stx2B or IntC280 to the respective host receptors even in the presence of the two virulence factors together. Moreover, the bispecific neutralizer was relatively stable to harsh storage conditions and gastrointestinal pH extremes. Taking into account its easy and economical production and superior pharmaceutical properties, we believe that a nanobody-based bispecific neutralizer would be more favorable and practical to be developed as a therapeutic to fight STEC in the developing world.

Dinitrophenol-mediated modulation of an anti-PD-L1 VHH for Fc-dependent effector functions and prolonged serum half-life.

Single-domain antibodies, VHHs or nanobodies, represent a promising set of alternatives to conventional therapeutic antibodies, gaining substantial attention in the field of cancer immunotherapy. However, inherent drawbacks of nanobodies such as fast clearance from blood circulation and lack of immune effector functions often led to unsatisfactory therapeutic efficacy. We previously reported that dinitrophenyl modification of an anti-EGFR VHH conferred Fc-dependent immune effector functions and elongated serum half-life on it through recruiting of hapten antibodies, resulting in improved immunotherapy efficacy in vivo. In the present work, we further tested the versatility of this approach in the case of an anti-PD-L1 blockade VHH (KN035). Site-specific dinitrophenyl conjugation did not impair the binding capacity of KN035 portion to PD-L1, but indirectly restored its immune effector functions, manifested by the observed antibody dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement-dependent cytotoxicity against PD-L1 positive tumor cells. Significant delay of blood clearance of dinitrophenylated KN035 was evidenced by the prolonged half-life of ca. 22 h. This approach, using small hapten molecule conjugation, loaded additional antibody-mediated tumor killing mechanisms to PD-L1 blockade VHH and therefore improved efficacy is anticipated in the future in vivo therapeutic studies. Thus, our results underscore the power of this versatile approach for achieving desirable properties of VHH-based or similar therapeutics.

PRIMA­1met induces autophagy in colorectal cancer cells through upregulation of the mTOR/AMPK­ULK1­Vps34 signaling cascade.

p53­reactivation and induction of massive apoptosis­1, APR­017 methylated (PRIMA­1met; APR246) targets mutant p53 to restore its wild­type structure and function. It was previously demonstrated that PRIMA­1met effectively inhibited the growth of colorectal cancer (CRC) cells in a p53­independent manner, and distinctly induced apoptosis by upregulating Noxa in p53­mutant cell lines. The present study including experiments of western blotting, acridine orange staining and transmission electron microscopy revealed that PRIMA­1met induced autophagy in CRC cells independently of p53 status. Importantly, PRIMA­1met not only promoted autophagic vesicle (AV) formation and AV­lysosome fusion, but also increased lysosomal degradation. Furthermore, Cell Counting Kit­8 assay, colony formation assay and small interfering RNA transfection were performed to investigate the underling mechanisms. The study indicated that activation of the mTOR/AMPK­ULK1­Vps34 autophagic signaling cascade was key for PRIMA­1met­induced autophagy. Additionally, autophagy served a crucial role in the inhibitory effect of PRIMA­1met in cells harboring wild­type p53, which was closely associated with the increased expression of Noxa. Taken together, the results determined the effect of PRIMA­1met on autophagy, and further revealed mechanistic insights into different CRC cell lines. It was concluded that PRIMA­1met­based therapy may be an effective strategy for CRC treatment.

Water oxidation catalyzed by a charge-neutral mononuclear ruthenium(iii) complex.

A new charge-neutral Ru(iii) complex RuL(pic)3 (1) (H3L = 3,6-di-tert-butyl-9H-carbazole-1,8-dicarboxylic acid, pic = 4-picoline) was synthesized and fully characterized. This complex promoted chemical and photochemical water oxidation efficiently with turnover frequencies of 0.28 s-1 and 5 min-1, respectively. In particular, for photochemical water oxidation, complex 1 showed excellent stability and good activity. The X-ray crystal structure, electrochemical results, and the detection of the RuIV-OH intermediate by high-resolution mass spectrometry revealed that complex 1 exchanged its 4-picoline ligand with water at the RuIII state to form the authentic water oxidation catalyst. The kinetics studies suggested a reaction mechanism involving nucleophilic attack by a water molecule.

Analysis of the association of single nucleotide polymorphisms of interleukin-23 receptor (IL-23R) and inflammatory bowel disease in a Chinese Han cohort.

Inflammatory bowel disease (IBD) is a chronic, complex genetic disease with rapidly increasing prevalence in China. The interactions of genetic, environmental, and microbial factors contribute to the development of IBD, however, the precise etiologies of IBD are not well understood yet. Interleukin-23 receptor (IL-23R) encodes a subunit of receptor for IL-23, which is an important proinflammatory cytokine. In this study, we investigated the relationship between the single nucleotide polymorphism (SNP) of IL-23R gene and IBD in Chinese Han population. We genotyped three nonsynonymous IL-23R SNPs with amino acid changes (rs11209026, p.Arg381Gln; rs41313262 p.Val362Ile and rs11465797 p.Thr175Asn) in 198 patients with IBD (124 UC and 74 CD) and 100 healthy controls. The prevalence of the A allele in IL-23R Arg381Gln of CD appeared less than controls, but it was not statistically significant (2.70% vs. 6.00%, p > 0.05). There was no statistical difference between UC and controls (5.65% vs. 6.00%, p = 0.91). The p.Val362Ile variant was present in 2.42% of UC patients, in 2.70% of CD patients, which was similar in the control (2.00%). There was no statistical difference among these three groups. We did not detect Thr175Asn (rs11465797 c.524 C>A) in all the three groups. In conclusion, our study demonstrated that the p.Val362Ile and Arg381Gln were not associated with susceptibility to IBD in Chinese Han population.

Efficient molecular ruthenium catalysts containing anionic ligands for water oxidation.

Two new mononuclear Ru complexes RuII(bipa)(pic)3 (1; H2bipa = 6-(1H-benzo[d]imidazol-2-yl)picolinic acid, pic = 4-picoline) and RuII(pbic)(pic)3 (2; H2pbic = 2-(pyridin-2-yl)-1H-benzo[d]imidazole-7-carboxylic acid, pic = 4-picoline) based on anionic ligands were successfully synthesized, and characterized using NMR spectroscopy, mass spectrometry, and X-ray crystallography. These catalysts showed high activities and stabilities in water oxidation in homogeneous systems with a high turnover number of 2100 and a turnover frequency of 0.21 s-1 for complex 1. The O-O band formation mechanism involved water nucleophilic attack. An active catalytic intermediate, i.e., RuIV-OH, was detected using high-resolution mass spectrometry.

Artemisinin derivative artesunate induces radiosensitivity in cervical cancer cells in vitro and in vivo.

OBJECTIVE: Cervical cancer is the third most common type of cancer in women worldwide and radiotherapy remains its predominant therapeutic treatment. Artesunate (ART), a derivative of artemisinin, has shown radiosensitization effect in previous studies. However, such effects of ART have not yet been revealed for cervical cancer cells. METHODS: The effect of ART on radiosensitivity of human cervical cancer cell lines HeLa and SiHa was assessed using the clonogenic assay. Cell cycle progression and apoptosis alterations were analyzed by flow cytometry. For in vivo study, HeLa or SiHa cells were inoculated into nude mice to establish tumors. Tissues from xenografts were obtained to detect the changes of microvessel density, apoptosis and cell cycle distribution. Microarray was used to analyze differentially expressed genes. RESULTS: ART increased the radiosensitivity of HeLa cells (SER=1.43, P<0.001) but not of SiHa cells. Apoptosis and the G2-M phase transition induced by X-ray irradiation (IR) were enhanced by ART via increased Cyclin B1 expression in HeLa cells. Tumor growth of xenografts from HeLa but not SiHa cells was significantly inhibited by irradiation combined with ART (tumor volume reduction of 72.34% in IR+ART group vs. 41.22% in IR group in HeLa cells and 48.79% in IR+ART group vs. 44.03% in IR alone group in SiHa cells). Compared with the irradiated group, cell apoptosis was increased and the G2/M cell cycle arrest was enhanced in the group receiving irradiation combined with ART. Furthermore, compared with radiation alone, X-ray irradiation plus ART affected the expression of 203 genes that function in multiple pathways including RNA transport, the spliceosome, RNA degradation and p53 signaling. CONCLUSION: ART potently abrogates the G2 checkpoint control in HeLa cells. ART can induce radiosensitivity of HeLa cells in vitro and in vivo.

Visible light driven water splitting in a molecular device with unprecedentedly high photocurrent density.

A molecular water oxidation catalyst (2) has been synthesized and immobilized together with a molecular photosensitizer (1) on nanostructured TiO2 particles on FTO conducting glass, forming a photoactive anode (TiO2(1+2)). By using the TiO2(1+2) as working electrode in a three-electrode photoelectrochemical cell (PEC), visible light driven water splitting has been successfully demonstrated in a phosphate buffer solution (pH 6.8), with oxygen and hydrogen bubbles evolved respectively from the working electrode and counter electrode. By applying 0.2 V external bias vs NHE, a high photocurrent density of more than 1.7 mA·cm(-2) has been achieved. This value is higher than any PEC devices with molecular components reported in literature.