Cell Transformation Research Articles

Potential effects of a human milk oligosaccharide 6'-sialyllactose on angiotensin II-induced aortic aneurysm via p90RSK/TGF-β/SMAD2 signaling pathway.

The aberrant phenotypic transformation of vascular smooth muscle cells (VSMCs) is a key factor in the formation of aortic aneurysm (AA). This study aimed to explore theeffects of 6'-sialyllactose (6'-SL), a human milk oligosaccharide, on angiotensin II (Ang II)-induced VSMC dysfunction and AA formation both in vitro and in vivo. An AA model was established in male C57BL/6 mice challenged with Ang II via osmotic pumps and a lysyl oxidase inhibitor, β-aminopropionitrile (BAPN), in drinking water. The mice were administered with 6'-SL, FMK (a p90RSK inhibitor), or losartan (as a positive control). In vitro, VSMCs were pretreated with 6'-SL before Ang II stimulation. We found that p90RSK inhibition abolished Ang II/BAPN-induced thoracic AA and abdominal AA formation. Treatment with 100mg/kg 6'-SL significantly attenuated Ang II/BAPN-induced aortic dilatation. 6'-SL attenuated Ang II-induced collagen deposition, calcification, and immune cell accumulation. Consistently, 6'-SL downregulated p-p90RSK, p90RSK, and p-SMAD2, and mitigated VSMC contractility loss, as indicated by α-SMA expression in vivo. Interestingly, Ang II-induced transforming growth factor-beta (TGF-β) signaling pathway was suppressed by p90RSK inhibition in VSMCs. 6'-SL treatment significantly reduced TGF-β/SMAD2 targets, including dedifferentiation markers such asosteopontin and vimentin, and elastin degradation factors MMP2 and MMP9. Overexpression of p90RSK in VSMCs enhanced TGF-β and abrogated the effects of 6'-SL. Furthermore, 6'-SL co-treatment abolished high phosphate-induced calcification in vitro via p90RSK/TGF-β signaling pathway. Altogether, our findings suggest that 6'-SL could be a potential therapeutic candidate for protecting against Ang II-induced AA formation by inhibiting the p90RSK/TGF-β/SMAD2 signaling pathway.

Protease activated receptor 2 deficiency retards progression of abdominal aortic aneurysms by modulating phenotypic transformation of vascular smooth muscle cells via ERK signaling

Abdominal aortic aneurysm (AAA) is characterized by localized structural deterioration of the aortic wall, leading to progressive dilatation and rupture. Protease activated receptor 2 (PAR2) dependent signaling has been implicated in the pathophysiology of atherosclerosis through the regulation of smooth muscle cell function. However, its role in AAA remains unclear. This study investigates the function and potential mechanism of PAR2 in AAA progression. Angiotensin II (Ang II) and β-aminopropionitrile (BAPN) were administered to wild type (WT) mice to induce AAA. Increased PAR2 expression was observed in the aneurysmal tissues of these mice and in Ang II-treated vascular smooth muscle cells (VSMCs). We demonstrated that PAR2 deficiency markedly inhibited aorta dilatation and vascular remodeling in the AAA model relative to WT mice. Immunohistochemical staining showed significant upregulation of contractile markers and a reduction in synthetic markers in PAR2 knockout mice. Consistent with in vivo results, PAR2 knockdown diminished the effects of Ang II on VSMCs phenotypic switching, resulting in reduced proliferation and migration. Conversely, a PAR2 agonist (SLIGRL) induced the opposite effect, which was partially mitigated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor (PD98059). This study suggests that PAR2 deficiency restrains aortic expansion and mitigates adverse vascular remodeling in AAA models, mediated in part by the ERK signaling pathway, indicating that PAR2 could be a potential therapeutic target for mitigating AAA development or progression.

Contribution of ECT2 to Tubulointerstitial Fibrosis in the Progression of Chronic Kidney Disease.

Chronic kidney disease (CKD) is a complex disorder resulting from a combination of various environmental and genetic factors. Considerable efforts have been dedicated to elucidating its etiological mechanisms. Nevertheless, the pathogenic mechanism of CKD remains poorly understood, which hinders the development of effective therapeutic strategies. In this study, we aimed to identify novel mediators that could contribute to the development of CKD. The ClinVar, STRING, MEME Suite, TRRUST, bedtools, GEO, and R Studio databases and software were used to analyze their common features and investigate potential CKD disease genes. Transcriptomic analysis, immunohistochemistry, qRT-PCR, and Western blotting were utilized to further validate the role of ECT2 in kidney fibrosis. In total, 26 CKD disease genes were obtained from the ClinVar database, and the STRING, MEME Suite, TRRUST, bedtools, and GEO databases and software were used to analyze their common properties and explore potential CKD disease genes. Epithelial cell transforming sequence 2 (ECT2), cyclin B 1, caspase 7 and collagen alpha-1 (IV) were identified as potential candidates for CKD progression. Weighted correlation network analysis (WGCNA) subsequently revealed the relationships between potential genes and CKD. The results of the transcriptomic analysis further confirmed that ECT2 expression was greater in the kidney tissue of CKD patients than in that of healthy controls. Next, immunohistochemistry and Western blotting demonstrated that ECT2 was predominantly expressed in the renal tubules of a unilateral ureteral obstruction (UUO) mouse model. Consistently, in vitro experiments revealed that ECT2 was upregulated in TGF-β1-treated HK-2 cells. Moreover, ECT2 overexpression or knockdown in HK-2 cells altered the intensity of fibrosis markers. ECT2 significantly affects the development and progression of CKD, particularly in association with tubulointerstitial fibrosis.

Alleviating vascular calcification with Bushen Huoxue formula in rats with chronic kidney disease by inhibiting the PTEN/PI3K/AKT signaling pathway through exosomal microRNA-32.

Vascular calcification (VC) significantly raises cardiovascular mortality in chronic kidney disease (CKD) patients. VC is characterized by the phenotypic transformation of vascular smooth muscle cells (VSMCs) to osteoblast-like cells, mediated by exosomes derived from calcified VSMCs and the exosomal microRNAs (miRNA) which may trigger some signals to recipient VSMCs. Bushen Huoxue (BSHX) formula has demonstrated its clinical efficacy in CKD and its protective role in CKD-VC rats has also been observed. However, little is known about its underlying mechanism. To establish a VC model, aortic VSMCs from rats were induced to osteogenic differentiation by high-level phosphate (HP) in vitro. The expression of exosome and calcification makers were analyzed by western blot, including CD9, CD63, α-SMA, BMP-2, and Runx2, respectively. Differential expression of exosomal miRNAs in normal and HP-induced VSMCs were identified by using whole miRNA microarray technology. GO and KEGG analyses were performed to determine the significant enrichment of functions and signaling pathways in the target genes. In vivo, the CKD-VC rat model was established by administering adenine gavage combined with a high phosphorus diet. The rats were divided into normal control, model, low-dose BSHX, medium-dose BSHX, high-dose BSHX groups, and sevelamer groups. The blood biochemical parameters were measured. Renal histopathology and aortic calcification were observed. Western blot detected the levels of the calcification markers. Quantitative real-time PCR (qPCR) assay detected exosomal microRNA-32 (miR-32) mRNA expression in the aorta, the most differentially expressed exosomal miRNA previously identified. Phosphatase and tensin homolog located on chromosome ten (PTEN)/phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) signaling pathway components were also tested by western blot. Exosomal miRNA-32 and PI3K/AKT signaling pathways were highly differentially expressed between normal and HP-induced VSMCs. In vivo, BSHX improved blood biochemical parameters, renal histopathology, and aortic calcification in CKD-VC rats. BSHX increased the expression level of α-SMA and decreased the level of BMP-2 and Runx2. BSHX also lowered the expression level of exosomal miR-32 mRNA, enhanced PTEN expression, therefore, reduced p-PI3K and p-AKT levels in the aorta. BSHX alleviated VC in CKD rats by downregulating exosomal miR-32 expression in the aorta, thereby promoting PTEN expression and inhibiting the PI3K/AKT signaling pathway.

Revisiting Virchow’s triad: exploring the cellular and molecular alterations in cerebral venous congestion

BackgroundCerebral venous thrombosis (CVT) is a rare but serious condition that can lead to significant morbidity and mortality. Virchow’s triad elucidates the role of blood hypercoagulability, blood flow dynamics, and endothelial damage in the pathogenesis of CVT. Cerebral venous congestion (CVC) increases the risk of cerebral venous sinus thrombosis and can lead to recurrent episodes and residual symptoms. However, the precise mechanism by which blood congestion leads to thrombosis remains unclear. Our objective was to investigate the cellular and molecular alterations linked to CVC through analysis of the pathological morphology of venous sinus endothelial cells and transcriptomic profiling.ResultsThis study demonstrated a remarkable correlation between CVC and the phenotypic transformation of endothelial cells from an anticoagulant to a procoagulant state. The findings revealed that cerebral venous stasis results in tortuous dilatation of the venous sinuses, with slow blood flow and elevated pressure in the sinuses and damaged endothelial cells of the retroglenoid and internal jugular vein ligation (JVL) rat model. Mechanistically, analysis of transcriptomic results of cerebral venous sinus endothelial cells showed significant activation of platelet activation, complement and coagulation cascades pathway in the JVL rats. Furthermore, the expression of von Willebrand factor (vWF) and coagulation factor VIII (F8) in the complement and coagulation cascades and Fgg and F2 in the platelet activation was increased in the cerebral venous sinuses of JVL rats than in sham rats, suggesting that endothelial cell injury in the venous sinus induced by CVC has a prothrombotic effect. In addition, endothelial cell damage accelerates coagulation and promotes platelet activation. Significantly, the concentrations of vWF, F2 and F8 in venous sinus blood of patients with internal jugular vein stenosis were higher than in their peripheral blood.ConclusionCollectively, our data suggest that CVC can induce endothelial cell damage, which then exhibits a procoagulant phenotype and ultimately increases the risk of CVT. This research contributes to our understanding of the pathophysiology of CVC associated with procoagulant factors and reexamines the components of Virchow’s triad in the context of CVC.

Cyclin-D1 rearrangement as a secondary event in the large cell transformation of splenic marginal zone lymphoma with a TP53 deletion.

IGH::CCND1 translocation has been implicated as a preliminary finding in mantle cell lymphoma (MCL) and plasma cell myeloma, with cyclin D1 over-expression by immunohistochemistry stain, without gene rearrangement, reported in other hematologic neoplasms. CCND1 rearrangement has been reported infrequently as a secondary event in high-grade B-cell lymphomas. We present the case of an elderly man who was found to have a splenic marginal zone lymphoma with a heterozygous TP53 deletion. Years later at the time of relapse, the patient acquired a CCND1 rearrangement, in addition to apersistent TP53 deletion. Sequencing of the two lymphomas demonstrated clonal relatedness of the two processes. To our knowledge, this is the first report of a splenic marginal zone lymphoma witha TP53 deletion at diagnosis, evolving into a large B-cell lymphoma with a CCND1 rearrangement.

RSK2-mediated cGAS phosphorylation induces cGAS chromatin-incorporation-mediated cell transformation and cancer cell colony growth

Cyclic guanosine-adenosine monophosphate synthase (cGAS) is a key cytosolic DNA sensor that plays a pivotal role in the innate immune response. Although a decade of research on the cGAS has advanced our understanding of inflammasome formation, cytokine production, and signaling pathways, the role of cGAS in the nucleus remains unclear. In this study, we found that the nuclear localization of endogenous and stably expressed cGAS differed from transiently expressed cGAS, which mainly localized in the cytosol. In the nucleus, cGAS is tightly bound to chromatin DNA. The chromatin DNA binding of cGAS was dependent on RSK2. Our molecular mechanism study indicated that the N-lobe of RSK2 harboring 1–323 interacted with the NTase domain of cGAS harboring residues 213–330. This interaction increased RSK2-induced cGAS phosphorylation at Ser120 and Thr130, resulting in the tightly binding of cGAS to chromatin. Importantly, epidermal growth factor (EGF)-induced cell transformation and anchorage-independent colony growth showed an increase in growth factors, such as EGF or bFGF, in cGAS stable expression compared to mock expression. Notably, the cGAS-S120A/T130A mutant abolished the increasing effect of cell transformation of JB6 Cl41 cells and colony growth of SK-MEL-2 malignant melanoma cells. The results suggested that cGAS’s chromatin DNA binding, which is indispensable to RSK2-dependent phosphorylation of cGAS at Ser120/Thr130, provides the first clue to how cGAS may participate in chromatin remodeling in the nucleus.

Semaphorin 3D promotes pancreatic ductal adenocarcinoma progression and metastasis through macrophage reprogramming.

Axon guidance molecules are frequently altered in pancreatic ductal adenocarcinoma (PDA) and influence PDA progression. However, the molecular mechanism remained unclear. Using genetically engineered mouse models to examine semaphorin 3D (SEMA3D), we identified a dual role for tumor- and nerve-derived SEMA3D in the malignant transformation of pancreatic epithelial cells and invasive PDA development. Pancreatic-specific knockout of the SEMA3D gene from the KRASG12D and TP53R172H mutation knock-in, PDX1-Cre(KPC) mouse model demonstrated delayed tumor initiation, prolonged survival, absence of metastasis, and reduced M2 macrophage expression. Mechanistically, tumor- and nerve-derived SEMA3D indirectly reprograms macrophages through KRASMUT-dependent ARF6 signaling in PDA cells, resulting in increased lactate production, which is sensed by GPCR132 on macrophages to stimulate protumorigenic M2 polarization. Multiplex immunohistochemistry demonstrated increased M2-polarized macrophages proximal to nerves in SEMA3D-expressing human PDA tissue. This study suggests that altered SEMA3D expression leads to an acquisition of cancer-promoting functions, and nerve-derived SEMA3D is "hijacked" by PDA cells to support growth and metastasis in a KRASMUT-dependent manner.

The Level of Serum Selenium in Patients with Cervical Intraepithelial Neoplasia in Medical City/ Baghdad Teaching Hospital

Introduction: Selenium is an essential trace element involved in different physiological functions of the human body. An inverse relationship between serum selenium levels and cervical intraepithelial neoplasia has been reported. cervical intraepithelial neoplasia is regarded as a potentially premalignant transformation of squamous cells of the cervix. Objectives: To evaluate the relationship between the serum level of selenium and cervical intraepithelial neoplasia. Methods: A case-control study was conducted at Baghdad Teaching Hospital and Iraqi National Cancer Research Center in the University of Baghdad during the period from July 2021 to July 2022. A convenient sample of 100 women was enrolled in the current study and included case group which consisted of 50 women who were diagnosed with cervical intraepithelial neoplasia and control group which consisted of 50 women who did not have cervical intraepithelial neoplasia as confirmed through histological examination after punch biopsy by colposcopy. Results: The serum level of the selenium was significantly lower in the case groups compared to the control groups (P-value=0.001). In addition, the proportion of the participant with abnormal levels of selenium was significantly higher in the case group compared to the control group (P-value=0.001). In the case group, there was a significant difference between the cervical intraepithelial neoplasia grade regarding the serum level of selenium, the level of selenium significantly decreased with the progression of the disease (P-value=0.001). Conclusions: Selenium deficiency may significantly increase the incidence of cervical intraepithelial neoplasia. In addition, it significantly impacts the transformation to a higher degree as there was a significant difference between cervical intraepithelial neoplasia I, II, and III regarding the level of serum selenium.

New horizons in our understanding of precursor multiple myeloma and early interception.

Multiple myeloma is an incurable plasma cell malignancy that evolves over decades through the selection and malignant transformation of monoclonal plasma cells. The evolution from precursor states to symptomatic disease is characterized by an increasing complexity of genomic alterations within the plasma cells and a remodelling of the microenvironment towards an immunosuppressive state. Notably, in patients with advanced disease, similar mechanisms of tumour escape and immune dysfunction mediate resistance to modern T cell-based therapies, such as T cell-engaging bispecific antibodies and chimeric antigen receptor (CAR)-T cells. Thus, an increasing number of clinical trials are assessing the efficiency and safety of these therapies in individuals with newly diagnosed multiple myeloma and high-risk smoldering multiple myeloma. In this Review, we summarize the current knowledge about tumour intrinsic and extrinsic processes underlying progression from precursor states to symptomatic myeloma and discuss the rationale for early interception including the use of T cell-redirecting therapies.

Lipoleiomyoma of the uterus in a woman of reproductive age (clinical case)

Uterine lipoleiomyoma is one of the variants of uterine leiomyoma, which is histologically represented by the presence of mature fat and smooth muscle cells. The relevance of the coverage of this clinical case is determined by the extreme rarity of the development of uterine lipoleiomyoma in women of reproductive age. The purpose of this work was to record this clinical case in the world statistics of lipoleiomyoma, to evaluate methods of diagnosis and differential diagnosis, to determine the role of early diagnosis of uterine lipoleioma in the subsequent tactics and scope of surgical treatment. Data from the medical records of a 41-year-old inpatient were analyzed. In addition to the generally accepted clinical and biochemical methods of blood and urine examination, electrocardiogram, ultrasound examination of the pelvic organs, the level of ovarian tumor markers in the blood (CA 125, HE4 and the ROMA index) was determined. The diagnosis of the disease was based on the data of pathohistological and immunohistochemical studies. Based on the results of a review of the medical literature, analysis of articles obtained as a result of a search of PubMed, SCOPUS, Web of Science, MedScape databases, the current state of the problem is highlighted, literary data related to the incidence, features of the clinical course, diagnosis and treatment of uterine lipoleiomyoma are summarized. The clinical case presented in the article demonstrates an incidental finding of a uterine lipoleiomyoma in a woman of reproductive age, measuring 30x25x20 cm, originated subserously from the body and cervix of the uterus, in the area of its isthmus. Under this condition, it occupied the entire Douglas space, the area of the sacro-uterine ligaments and the parietal peritoneum, intimately adjacent to the sigmoid and rectum, to the ureters and iliac vessels. The peculiarity of this clinical case is that sonographically uterine lipoleiomyoma was hidden under the "mask" of a dermoid cyst of the right ovary. Macroscopically, it differed from a typical lipoleiomyoma by the purple-bluish color of its outer surface and soot-colored, fine-lobed spongy structure on the section. The diagnosis of uterine lipoleiomyoma was verified only on the basis of pathohistological and immuno­histochemical research. Microscopically, the lipoleiomyoma had a mesenchymal structure with a pronounced vascular component and consisted of mitotically inactive bundles of smooth muscle cells and mature adipocytes. Im­munohistochemically, a positive reaction for caldesmon, desmin, smooth muscle actin alpha of tumor cells and for S.100 (DAKO, polyclonal) fatty cells was detected, which confirmed the hypothesis of direct transformation of smooth muscle cells existing in the leiomyoma of the uterus into fatty cells. This clinical case should complement the global statistical indicators of diagnosis of uterine lipoleiomyoma in women of reproductive age. Lipoleiomyoma should be considered as the primary diagnosis in case of detection of a large uterine tumor in women with excess body weight and be removed immediately after diagnosis, otherwise it is impossible to exclude its malignancy. For the planned diagnosis of neoplasms of the female genital organs, preference should be given to non-invasive research methods: magnetic resonance or computer tomography with contrast enhancement. The problem of these tumors lies in their unpredictable histogenesis, the unexpected presence of fat in the microscopic structure, and in the visual similarity to sarcomas. Verification of the diagnosis is carried out on the basis of pathohistological and immunohistochemical studies of the tumor preparation. Uterine lipoleioma can have a purplie-bluish color and develop by a broad base from the body and cervix of the uterus, as a result of "lipomatous" metaplasia of the uterine leiomyoma existing in a woman. Regular preventive examinations of women of all ages are crucial for timely detection of this rare neoplasm.

Hepatitis B Virus X Protein Contributes to Hepatocellular Carcinoma via Upregulation of KIAA1429 Methyltransferase and mRNA m6A Hypermethylation of HSPG2/Perlecan.

Chronic hepatitis B virus (HBV) remains to be the most common risk factor of hepatocellular carcinoma (HCC). While previous work has primarily focussed on understanding the direct and indirect mechanisms of Hepatitis B virus X protein (HBx)-mediated hepatocarcinogenesis, from genetic and epigenetic perspectives, its influence on RNA modification mediated onset of liver malignancies is less well understood. This study explored the role of HBV-encoded HBx in altering the m6A methylome profile and its implications on the pathogenesis of HCC. We established HBx-expressing stable HCC cell lines, Huh7-HBx and HepG2-HBx, and explored the transcriptomic and epitranscriptomic profiles by RNA-seq and MeRIP-seq, respectively. Preliminary results suggest that HBx promotes liver cell proliferation, migration, survival and overall m6A methylation in HCC cells and is involved in modulating the extracellular matrix. We show that HBx mediates liver cell transformation by upregulating KIAA1429 methyltransferase. HBx also drives the expression and hypermethylation of the extracellular matrix protein HSPG2/Perlecan and promotes tumourigenesis. Furthermore, we observed a potential interaction between KIAA1429 and HSPG2 in HCC liver cancer cells and demands further investigation.

Effect of C-to-T transition at CpG sites on tumor suppressor genes in tumor development in cattle evaluated by somatic mutation analysis in enzootic bovine leukosis.

Oncogenic transformation of normal cells is caused by mutations and chromosomal abnormalities in cancer-related genes. Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma caused by bovine leukemia virus (BLV) infection in cattle. Although a small fraction of BLV-infected cattle develops EBL after a long latent period, the mechanisms for oncogenesis in EBL cattle remain largely unknown. In this study, we analyzed the types and patterns of somatic mutations in cancer cells from 36 EBL cases, targeting 21 cancer-related genes. Various somatic mutations were identified in eight genes, TP53, KMT2D, CREBBP, KRAS, PTEN, NOTCH1, MYD88, and CARD11. In addition, TP53 gene was found to be mutated in 69.4% of EBL cases, with most being biallelic mutations. In some cases, associations were observed between the ages at which cattle had developed EBL and somatic mutation patterns; young onset of EBL possibly occurs due to high impact mutations affecting protein translation and biallelic mutations. Furthermore, nucleotide substitution patterns indicated that cytosine at CpG sites tended to be converted to thymine in many EBL cases, which was considered to be the result of spontaneous deamination of 5-methylcytosine. These results demonstrate how somatic mutations have occurred in cancer cells leading to EBL development, thereby explaining its pathogenic mechanism. These findings will contribute to a better understanding and future elucidation of disease progression in BLV infection.IMPORTANCEEnzootic bovine leukosis (EBL) is a malignant and lethal disease in cattle. Currently, there are no effective vaccines or therapeutic methods against bovine leukemia virus (BLV) infection, resulting in severe economic losses in livestock industry. This study provides a renewed hypothesis to explain the general mechanisms of EBL onset by combining the previous finding that several integration sites of BLV provirus can affect the increase in survival and proliferation of infected cells. We demonstrate that two additional random events are necessary for oncogenic transformation in infected cell clones, elucidating the reason why only few infected cattle develop EBL. Further exploration of somatic mutation and BLV integration sites could support this hypothesis more firmly, potentially contributing to the development of novel control methods for EBL onset.

Assessment of the carcinogenic potential of particulate matter generated from 3D printing devices in Balb/c 3T3-1-1 cells

Recently, there have been reports of sarcoma occurring in a Korean science teachers who used a 3D printer with acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) filaments for educational purposes. However, limited toxicological research data on 3D printing make it challenging to confirm a causal relationship between 3D printing and cancer. Therefore, occupational accidents involving teachers who have developed sarcoma have not been officially recognized. To address this gap, we aimed to evaluate the carcinogenic potential of particulate matter produced from ABS and PLA filaments commonly used in 3D printing. We created a generator mimicking 3D printing to generate particulate matter, which was used as an experimental material. The collected particulate matter was exposed to an in vitro system to investigate genetic damage, effects on cell transformation, and changes in carcinogenesis-related genes. Various assays, such as the comet assay, cell transformation assays, microarray analysis, and glucose consumption measurement, were employed. Cytotoxicity tests performed to determine the exposure concentration for the comet assay showed that cell viability was 83.6, 62.6, 42.0, and 10.2% for ABS at exposure concentrations of 50, 100, 200, and 400 µg/mL, respectively. PLA showed 91.7, 80.3, 65.1, and 60.8% viability at exposure concentrations of 50, 100, 200, and 400 µg/mL, respectively. Therefore, 50 µg/mL was set as the highest concentration for both ABS and PLA, and 25 and 12.5 µg/mL were set as the medium and low concentrations, respectively. The comet assay showed no changes in genetic damage caused by the particulate matter. Cytotoxicity results performed to establish exposure concentrations in the transformation assay showed that ABS showed cell viability of 88.0, 77.4, 84.7, and 85.5% at concentrations of 1.25, 2.5, 5, and 10 µg/mL, respectively, but few cells survived at concentrations above 20 µg/mL. PLA showed minimal cytotoxicity up to a concentration of 20 µg/ml. Therefore, in the cell transformation assay, a concentration of 10 µg/mL for ABS and 20 µg/mL for PLA was set as the highest exposure concentration, followed by medium and low exposure concentrations with a common ratio of 2. In cell transformation assays, only one transformed focus each was observed for both ABS and PLA particulate matter-exposed cells. The microarray assay revealed changes in gene expression, with a 41.7% change at 10 µg/mL for ABS and an 18.6% change at 20 µg/mL for PLA compared to the positive control group. Analysis of carcinogenesis-related gene expression changes on days 1, 7, and 25 of the promotion phase revealed that in cells exposed to 5 µg/mL of ABS, RBM3 gene expression increased by 3.66, 3.26, and 3.74 times, respectively, while MPP6 gene expression decreased by 0.33, 0.28, and 0.38 times, respectively, compared to the negative control group. Additionally, the measurement of glucose consumption showed that it increased in cells exposed to ABS and PLA particulate matter. Our findings suggest that the carcinogenic potential of ABS- and PLA-derived particulate matter in 3D printing cannot be completely ruled out. Therefore, further research in other test systems and analysis of additional parameters related to carcinogenesis, are deemed necessary to evaluate the carcinogenic risk of 3D printers using these materials.

Fisetin reduces ovalbumin-triggered airway remodeling by preventing phenotypic switching of airway smooth muscle cells

BackgroundThe transformation of airway smooth muscle cells (ASMCs) from a quiescent phenotype to a hypersecretory and hypercontractile phenotype is a defining feature of asthmatic airway remodeling. Fisetin, a flavonoid compound, possesses anti-inflammatory characteristics in asthma; yet, its impact on airway remodeling and ASMCs phenotype transition has not been investigated.ObjectivesThis research seeked to assess the impact of fisetin on ovalbumin (OVA) induced asthmatic airway remodeling and ASMCs phenotype transition, and clarify the mechanisms through network pharmacology predictions as well as in vivo and in vitro validation.MethodsFirst, a fisetin-asthma-ASMCs network was constructed to identify potential targets. Subsequently, cellular and animal studies were carried out to examine the inhibitory effects of fisetin on airway remodeling in asthmatic mice, and to detemine how fisetin impacts the phenotypic transition of ASMCs.ResultsNetwork analysis indicated that fisetin might affect asthma via mediating the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) pathway. Intraperitoneal administration of fisetin in vivo reduced airway inflammation and remodeling, as shown by reduced inflammatory cells, decreased T helper type 2 (Th2) cytokine release, diminished collagen accumulation, mitigated airway smooth muscle thickening, and decreased expression of osteopontin (OPN), collagen-I and α-smooth muscle actin (α-SMA). Moreover, fisetin suppressed the PI3K/AKT pathway in asthmatic lung tissue. According to the in vitro data, fisetin downregulated the expression of the synthetic phenotypic proteins OPN and collagen-I, contractile protein α-SMA, and inhibited cellular migration, potentially through the PI3K/AKT pathway.ConclusionThese results suggest that fisetin inhibits airway remodeling in asthma by regulating ASMCs phenotypic shift, emphasizing that fisetin is a promising candidate for the treatment of airway smooth muscle remodeling.

DHA plays a protective role in cerebral ischemia–reperfusion injury by affecting macrophage/microglia type polarization

A close correlation exists between the macrophage/microglia(MΦ/MG) polarization states and the development of cerebral ischemia and reperfusion (I/R). Therefore it is of great significance to research on how to modulate the MΦ/MG states for improved patient outcomes. In particular, regulatory mechanisms involved in this process remain to be identified. Hereby, we aim to shed light on how docosahexaenoic acid (DHA) actively modulates the switch between M1 and M2 macrophage states by restraining the NACHT-LRR-PYD-containing protein three inflammasome (NALP3). We found that NALP3-positive cells were detected in clinical human cerebral infarction tissue samples and the mouse tMCAO model. In mice after DHA treatment, the number of NALP3-positive cells was significantly reduced, significantly decreasing infarct volume and improving the postoperative physical status of mice. NALP3-positive cells were found to be MΦ/MG after co-staining with CD11b. By extracting peritoneal macrophages, it was verified that DHA inhibited the activation of NALP3 and regulated the transformation of M1 and M2 cells, thereby reducing I/R injury.

Synthetic and Natural Inhibitors of Mortalin for Cancer Therapy.

Upregulation of stress chaperone Mortalin has been closely linked to the malignant transformation of cells, tumorigenesis, the progression of tumors to highly aggressive stages, metastasis, drug resistance, and relapse. Various in vitro and in vivo assays have provided evidence of the critical role of Mortalin upregulation in promoting cancer cell characteristics, including proliferation, migration, invasion, and the inhibition of apoptosis, a consistent feature of most cancers. Given its critical role in several steps in oncogenesis and multi-modes of action, Mortalin presents a promising target for cancer therapy. Consequently, Mortalin inhibitors are emerging as potential anti-cancer drugs. In this review, we discuss various inhibitors of Mortalin (peptides, small RNAs, natural and synthetic compounds, and antibodies), elucidating their anti-cancer potentials.

Small cell transformation in EGFR-mutated non-small cell lung cancer: DLL3 expression and efficacy of immune checkpoint inhibitors or tyrosine kinase inhibitors combined with chemotherapy

IntroductionSmall cell transformation (SCT) is a typical mechanism of adaptive resistance to third generation epidermal growth factor receptor inhibitors (EGFRi) which have become the standard of care for EGFR-driven non-small cell lung cancer (EGFR+ NSCLC). Little is known about the optimal management of SCT patients. This study aimed to compare outcomes under platinum/etoposide chemotherapy alone (chemo) or in combination with EGFR inhibitors (EGFRi+chemo) or immune checkpoint inhibitors (ICI+chemo). In addition, DLL3 expression was explored as potential novel therapeutic target. MethodsWe conducted a retrospective study on patients with EGFR+ NSCLC and SCT treated at 19 centers in Europe and the United States. A total of 47 patients were included of whom 17 received chemo, 20 ICI+chemo, and 10 EGFRi+chemo. We analyzed DLL3 expression by immunohistochemistry. ResultsIn the entire cohort, median overall survival (OS) from start of first SCT therapy was 11 months (95 % confidence interval [95 %CI] 9.1–12.9) and median progression-free survival (PFS) was 5 months (95 %CI 4.2–5.8). Median PFS was similar in all three groups (chemo and ICI+chemo 4 months, EGFRi+chemo 6 months), and 12-months PFS was 12 % (95 %CI 2 %−31 %), 13 % (95 %CI 0 %−43 %), and 0 % for ICI+chemo, EGFRi+chemo, and chemo, respectively. Median OS in the ICI+chemo group was 13 months (95 %CI 5.5–20.5) compared to 10 months (95 %CI 7.6–12.4) with chemo and EGFRi+chemo (95 %CI 8.1–11.9), respectively. Before and after SCT, 0 % and 93 % of tumors were DLL3-positive. ConclusionsOur results suggest that ICI+chemo and DLL3-targeting agents are worth further exploration in EGFR+ NSCLC undergoing SCT. Presented elsewherePart of this work has been presented at ESMO annual meeting in Madrid, Spain in October 2023 (Poster 1336 P).

Spatial multiomics atlas reveals smooth muscle phenotypic transformation and metabolic reprogramming in diabetic macroangiopathy

BackgroundDiabetic macroangiopathy has been the main cause of death and disability in diabetic patients. The mechanisms underlying smooth muscle cell transformation and metabolic reprogramming other than abnormal glucose and lipid metabolism remain to be further explored.MethodSingle-cell transcriptome, spatial transcriptome and spatial metabolome sequencing were performed on anterior tibial artery from 11 diabetic patients with amputation. Multi-omics integration, cell communication analysis, time series analysis, network analysis, enrichment analysis, and gene expression analysis were performed to elucidate the potential molecular features.ResultWe constructed a spatial multiomics map of diabetic blood vessels based on multiomics integration, indicating single-cell and spatial landscape of transcriptome and spatial landscape of metabolome. At the same time, the characteristics of cell composition and biological function of calcified regions were obtained by integrating spatial omics and single cell omics. On this basis, our study provides favorable evidence for the cellular fate of smooth muscle cells, which can be transformed into pro-inflammatory chemotactic smooth muscle cells, macrophage-like smooth muscle cells/foam-like smooth muscle cells, and fibroblast/chondroblast smooth muscle cells in the anterior tibial artery of diabetic patients. The smooth muscle cell phenotypic transformation is driven by transcription factors net including KDM5B, DDIT3, etc. In addition, in order to focus on metabolic reprogramming apart from abnormal glucose and lipid metabolism, we constructed a metabolic network of diabetic vascular activation, and found that HNMT and CYP27A1 participate in diabetic vascular metabolic reprogramming by combining public data.ConclusionThis study constructs the spatial gene-metabolism map of the whole anterior tibial artery for the first time and reveals the characteristics of vascular calcification, the phenotypic transformation trend of SMCs, and the transcriptional driving network of SMCs phenotypic transformation of diabetic macrovascular disease. In the perspective of combining the transcriptome and metabolome, the study demonstrates the activated metabolic pathways in diabetic blood vessels and the key genes involved in diabetic metabolic reprogramming.Graphical abstract

Diagnostic potential of miRNA-135A1 in human papillomavirus associated cervical lesions

Introduction. Human papillomavirus (HPV) infection with high-risk HPVs is an etiological factor in the development of cervical cancer, with HPV type 16 (HPV16) being the most common. The mechanisms leading to disruption of viral oncogene expression and initiation of epithelial cell transformation are poorly understood. Epigenetic regulatory factors, including cellular miRNAs, may play an important role in HPV-induced carcinogenesis, and aberrantly expressed miRNAs may be promising markers for the diagnosis of HPV-associated lesions.Aim. To search for miRNAs involved in the pathogenesis of HPV16-associated cervical cancer and to evaluate their diagnostic potential for the detection of cervical cancer and precancerous lesions.Materials and methods. MiRNA expression in clinical samples was assessed by both next generation sequencing and quantitative stem-loop polymerase chain reaction (sl-qPCR). Plasma miRNAs from patients with precancerous and cancerous lesions and healthy donors were analyzed using sl-qPCR. Loss of heterozygosity in cervical cancer samples was assessed by copy number ratio of MIR135A1 and ACTB genes. A total of 67 patients with cervical cancer, 21 with precancerous cervical lesions and 24 healthy donors were included in the study. The effect of DNA methylation on miRNA-135A1 expression was evaluated after treatment with a demethylating agent of the cervical HPV16-positive SiHa cell line. Changes in the expression of the HPV16 E6 oncogene were analyzed after transfection with synthetic analogues of the mature forms of miRNA-135А1 (miRNA-135a-3p and miRNA-135a-5p).Results. A significant decrease in the expression of miRNA-135A1 and miRNA-135A2 was detected in tumor tissue samples from HPV16-positive cervical cancer, which was confirmed by sl-qPCR in an independent panel of tumor samples. A decrease in miRNA-135A1 expression was shown to result from both loss of heterozygosity of the gene and aberrant DNA methylation. Transfection of mature forms of miRNA-135A1 into SiHa cells resulted in decreased expression of the E6 oncogene of HPV16. Blood plasma samples from patients with cervical cancer and precancerous lesions showed lower levels of miRNA-135a-3p than healthy donors, and ROC analysis indicated its high diagnostic potential.Conclusion. Levels of miRNA-135A1 are significantly reduced in cervical lesions, both in tumor tissue and plasma, and the ability of this miRNA to suppress the expression of the HPV16 E6 oncogene suggests its oncosuppressive properties. Thus, miRNA-135A1 can be used as a promising new marker for the diagnosis of HPV-associated lesions.