Cell Proliferation In Response Research Articles

Identification and characterization of diacylglycerol kinase ζ as a novel enzyme producing ceramide-1-phosphate

Ceramide-1-phosphate (C1P) is a sphingolipid formed by the phosphorylation of ceramide; it regulates various physiological functions, including cell survival, proliferation, and inflammatory responses. In mammals, ceramide kinase (CerK) is the only C1P-producing enzyme currently known. However, it has been suggested that C1P is also produced by a CerK-independent pathway, although the identity of this CerK-independent C1P was unknown. Here, we identified human diacylglycerol kinase (DGK) ζ as a novel C1P-producing enzyme and demonstrated that DGKζ catalyzes the phosphorylation of ceramide to produce C1P. Analysis using fluorescently labeled ceramide (NBD-ceramide) demonstrated that only DGKζ among ten kinds of DGK isoforms increased C1P production by transient overexpression of the DGK isoforms. Furthermore, an enzyme activity assay using purified DGKζ revealed that DGKζ could directly phosphorylate ceramide to produce C1P. Furthermore, genetic deletion of DGKζ decreased the formation of NBD-C1P and the levels of endogenous C18:1/24:1- and C18:1/26:0-C1P. Interestingly, the levels of endogenous C18:1/26:0-C1P were not decreased by the knockout of CerK in the cells. These results suggest that DGKζ is also involved in the formation of C1P under physiological conditions.

Peripheral blood mononuclear cells reactivity in recent-onset type I diabetes patients is directed against the leader peptide of preproinsulin, GAD65271-285 and GAD65431-450.

T cell reactivity against pancreatic autoantigens is considered one of the main contributors to the destruction of insulin-producing cells in type 1 diabetes (T1D). Over the years, peptide epitopes derived from these autoantigens have been described in NOD mice and in both HLA class II transgenic mice and humans. However, which ones are involved in the early onset or in the progressive phases of the disease is still unclear. In this work we have investigated, in early-onset T1D pediatric patients and HLA-matched controls from Sardinia, the potential of preproinsulin (PPI) and glutamate decarboxylase 65 (GAD65)-derived peptides to induce spontaneous T cell proliferation responses of peripheral blood mononuclear cells (PBMCs). Significant T cell responses against PPI1-18, PPI7-19 and PPI31-49, the first two belonging to the leader sequence of PPI, and GAD65271-285 and GAD65431-450, were found in HLA-DR4, -DQ8 and -DR3, -DQ2 T1D children. These data show that cryptic epitopes from the leader sequence of the PPI and GAD65271-285 and GAD65431-450 peptides might be among the critical antigenic epitopes eliciting the primary autoreactive responses in the early phases of the disease. These results may have implications in the design of immunogenic PPI and GAD65 peptides for peptide-based immunotherapy.

The impact of biological age of red blood cell on in vitro endothelial activation markers.

Introduction: Blood donor characteristics influence red blood cell transfusion outcomes. As donor sex affects the distribution of young to old RBCs in the circulation, we hypothesized that the amount of circulating young RBCs in the blood product are associated with immune suppression. Materials and Methods: Blood samples were collected from healthy volunteers and density fractionated into young and old subpopulations. In an activated endothelial cell model, RBC adhesion to endothelium and secretion of endothelial activation markers were assessed. The impact of RBC biological age was also assessed in a T cell proliferation assay and in a whole blood stimulation assay. Results: After Percoll fractionation, young RBCs contained more reticulocytes compared to old RBCs. Young RBCs associated with lower levels of E-selectin, ICAM-1, and vWF from activated endothelial cells compared to old RBCs. RBC subpopulations did not affect T cell proliferation or cytokine responses following whole blood stimulation. Conclusion: Young RBCs contain more reticulocytes which are associated with lower levels of endothelial activation markers compared to old RBCs.

Ribonucleotide reductase subunit switching in hepatoblastoma drug response and relapse

Prognosis of children with high-risk hepatoblastoma (HB), the most common pediatric liver cancer, remains poor. In this study, we found ribonucleotide reductase (RNR) subunit M2 (RRM2) was one of the key genes supporting cell proliferation in high-risk HB. While standard chemotherapies could effectively suppress RRM2 in HB cells, they induced a significant upregulation of the other RNR M2 subunit, RRM2B. Computational analysis revealed distinct signaling networks RRM2 and RRM2B were involved in HB patient tumors, with RRM2 supporting cell proliferation and RRM2B participating heavily in stress response pathways. Indeed, RRM2B upregulation in chemotherapy-treated HB cells promoted cell survival and subsequent relapse, during which RRM2B was gradually replaced back by RRM2. Combining an RRM2 inhibitor with chemotherapy showed an effective delaying of HB tumor relapse in vivo. Overall, our study revealed the distinct roles of the two RNR M2 subunits and their dynamic switching during HB cell proliferation and stress response.

USP35 promotes cell proliferation and chemotherapeutic resistance through stabilizing FUCA1 in colorectal cancer

Ubiquitin-specific-processing proteases 35 (USP35) is an under-characterized deubiquitinase and its role in colorectal cancer (CRC) remains unclear. Here, we focus on delineating the impact of USP35 on CRC cell proliferation and chemo-resistance, as well as the possible regulatory mechanism. By examining the genomic database and clinical samples, we found that USP35 was overexpressed in CRC. Further functional studies showed that enhanced USP35 expression promoted CRC cell proliferation and resistance to oxaliplatin (OXA) and 5-fluorouracil (5-FU), whereas USP35 depletion impeded cell proliferation and sensitized cells to OXA and 5-FU treatments. Then, to explore the possible mechanism underlying USP35-triggered cellular responses, we performed co-immunoprecipitation (co-IP) followed by mass spectrometry (MS) analysis and identified α-L-fucosidase 1 (FUCA1) as a direct deubiquitiation target of USP35. Importantly, we demonstrated that FUCA1 was an essential mediator for USP35-induced cell proliferation and chemo-resistance in vitro and in vivo. Finally, we observed that nucleotide excision repair (NER) components (e.g., XPC, XPA, ERCC1) were up-regulated by USP35-FUCA1 axis, indicating a potential mechanism for USP35-FUCA1-mediated platinum resistance in CRC. Together, our results for the first time explored the role and important mechanism of USP35 in CRC cell proliferation and chemotherapeutic response, providing a rationale for USP35-FUCA1-targeted therapy in CRC.

Enhanced external counterpulsation improves dysfunction of forearm muscle caused by radial artery occlusion.

This study aimed to investigate the therapeutic effect of enhanced external counterpulsation (EECP) on radial artery occlusion (RAO) through the oscillatory shear (OS) and pulsatile shear (PS) models of human umbilical vein endothelial cells (HUVECs) and RAO dog models. We used high-throughput sequencing data GSE92506 in GEO database to conduct time-series analysis of functional molecules on OS intervened HUVECs, and then compared the different molecules and their functions between PS and OS. Additionally, we studied the effect of EECP on the radial artery hemodynamics in Labrador dogs through multi-channel physiological monitor. Finally, we studied the therapeutic effect of EECP on RAO at the histological level through Hematoxylin-Eosin staining, Masson staining, ATPase staining and immunofluorescence in nine Labrador dogs. With the extension of OS intervention, the cell cycle decreased, blood vessel endothelial cell proliferation and angiogenesis responses of HUVECs were down-regulated. By contrast, the inflammation and oxidative stress responses and the related pathways of anaerobic metabolism of HUVECs were up-regulated. Additionally, we found that compared with OS, PS can significantly up-regulate muscle synthesis, angiogenesis, and NO production related molecules. Meanwhile, PS can significantly down-regulate inflammation and oxidative stress related molecules. The invasive arterial pressure monitoring showed that 30Kpa EECP treatment could significantly increase the radial artery peak pressure (p = 0.030, 95%CI, 7.236-82.524). Masson staining showed that RAO significantly increased muscle interstitial fibrosis (p = 0.002, 95%CI, 0.748-2.128), and EECP treatment can reduce this change (p = 0.011, 95%CI, -1.676 to -0.296). ATPase staining showed that RAO significantly increased the area of type II muscle fibers (p = 0.004, 95%CI, 7.181-25.326), and EECP treatment could reduce this change (p = 0.001, 95%CI, -29.213 to -11.069). In addition, immunofluorescence showed that EECP increased angiogenesis in muscle tissue (p = 0.035, 95%CI, 0.024-0.528). EECP improves interstitial fibrosis and hypoxia, and increases angiogenesis of muscle tissue around radial artery induced by RAO.

Abstract P2-21-11: A high content imaging and analysis approach to screen compounds for novel drug targets in 3D breast cancer co-culture model

Abstract The desired effect of cancer therapeutics can only be conceived when applied to complex multicellular structures with different cell types and extracellular matrix (ECM) in three-dimensional (3D) space. Therefore, 3D cell culture systems have become very popular in drug screening and discovery. There is an immense demand for highly efficient and easy methods to produce 3D spheroids in any cell format. However, heterogeneity in size, extended cultivation times, and reproducibility for high throughput assays are limiting factors in the generation of spheroids. Recently we have established a naturally transformed breast cancer cell line, KAIMRC1 (1), from ductal breast carcinoma. We have developed a novel and easy method to produce spheroids (2) from KAIMRC1 cells in vitro, which can be used as a 3D model to study proliferation, differentiation, metabolism, cell death, and drug response of cells in the tumor microenvironment. We developed an in-vitro high content imaging (HCI) based cellular assay using commercially available compound panels to perform initial drug screening in the KAIMRC1 spheroids model. Our approach allows rapid screening of a panel of drugs to assess inhibitory effects on the growth of tumor cells in 3D cultures. To mimic the actual in-vivo tumor microenvironment, we are now developing an HCI-based assay utilizing 3D KAIMRC1 spheroids co-cultured with patient-derived fibroblast cells. 1. Ali, Rizwan, et al. “Isolation and Characterization of a New Naturally Immortalized Human Breast Carcinoma Cell Line, KAIMRC1.” BMC Cancer 17 (2017) 2. Rizwan Ali et al., (2021) New Born Calf Serum (NBCS) can induce spheroid formation in breast cancer KAIMRC1 cell line. Front. Mol. Biosci. DOI: 10.3389/fmolb.2021.769030 Citation Format: Rizwan Ali, Sarah Huwaizi, Arwa Alsubait, Mohamed Boudjelal. A high content imaging and analysis approach to screen compounds for novel drug targets in 3D breast cancer co-culture model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-11.

Abstract P3-06-05: Effects of isoflavone genistein, combined with anti-estrogen fulvestrant, on anti-PD1 therapy response against E0771 mammary tumors in mice

Abstract Although immune checkpoint blockers (ICBs) have revolutionized the treatment of many cancers, only less than 30% of patients respond to any ICB as a monotherapy. Estrogens, produced by the adipose tissue in the mammary glands, are immunomodulators in the tumor microenvironment (TME). Estrogens suppress anti-tumor immunity and activate immunosuppression through the estrogen receptor α (ERα) in the immune cells but activate anti-tumor immunity through ERβ. Genistein (GEN) is a biologically active isoflavone and is structurally similar to estradiol (E2). However, in contrast to E2, GEN binds preferentially to ERβ. Here we studied if GEN combined with ERα inhibitor fulvestrant (Fulv) alters response to anti-PD1 therapy in ERα negative, ERβ positive E0771 mouse mammary tumors. Syngeneic C57BL/6 mice were fed either a control (CON) diet or GEN supplemented diet for 5 weeks and then allografted E0771 mammary tumor cell into the 4th mammary fat pad. When tumors reached an average size of 16mm2, mice in both diet groups were further divided into 4 sub-groups treated with: 1) anti-PD1 (150µg or 100µg every 3 days for a total of 3 doses), 2) Fulv (1mg) once weekly, 3) anti-PD1+Fulv or 4) IgG control group. We found that three doses of 150µg of anti-PD1 alone effectively reduced tumor growth in the CON (p=0.0005) group and its efficacy was notably weakened in the GEN group (p=0.04).. Combination with Fulv did not improve PD1 response. These findings suggest that when using a highly effective anti-PD1 dose, GEN may reduce rather than improve the response, and anti-ERα does not add to anti-PD1’s effect in control of GEN group. A suboptimal dose (100 µg) of anti-PD1 alone did not inhibit E0771 tumor growth in mice fed CON diet, but in the GEN group the tumor inhibition by anti-PD1 alone almost reached statistical significance (p=0.057). The combination of suboptimal PD1+Fulv significantly reduced tumor growth compared with IgG (p=0.04) or Fulv (p=0.02). During GEN feeding, anti-PD1+Fulv was even more potent in inhibiting tumor growth when compared with IgG group (p=0.02) and Fulv group (p=0.007). Thus, when anti-PD1 alone is not effective, either Fulv or GEN will improve responsiveness to anti-PD1. In the suboptimal anti-PD1 dose experiment, GEN feeding increased CD8+ T cells (P=0.04) and decreased CD4+Foxp3+ cells (p=0.01) in the TME. Further, GEN reduced the proportion of immunosuppressive MDSC cells (CD3-CD11B+Ly6Ghigh6Clow) in the PD1+ Fulv group (p=0.01) in the Peyer’s patches. In vitro experiments showed that both GEN and Fulv, and especially their combination, inhibited the E0771 cells proliferation, indicating that these two treatments can also directly suppress the growth of ERα negative but ERβ positive breast cancer cells. Taken together, the data show that anti-estrogens and GEN in the diet may improve response to anti-PD1 therapy, possibly both through activation of tumor immune response and inhibition of tumor cell proliferation and induction of tumor cell death. Citation Format: Fabia de Oliveira Andrade, Vivek Verma, Ester Molina Hoyo, Karla Andrade de Oliveira, Pal Koak, Lu Jin, Leena Hilakivi-Clarke. Effects of isoflavone genistein, combined with anti-estrogen fulvestrant, on anti-PD1 therapy response against E0771 mammary tumors in mice [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-05.

Tectorigenin inhibits inflammation in keratinocytes by inhibition of NLRP3 inflammasome regulated by the TLR4/NF-κB pathway.

Psoriasis is a prevalent inflammatory skin disease characterized by excessive proliferation and abnormal differentiation of keratinocytes, and infiltration of inflammatory cells into the epidermis. However, the underlying mechanisms remain unclear. Tectorigenin is an active ingredient in traditional medicines and has anti-inflammatory activity. This research explored the effects of tectorigenin on the anti-inflammatory property, autophagy, and the underlying mechanisms in M5 ([IL-22, IL-17A, oncostatin M, IL-1α, and TNF-α])-stimulated HaCaT cells. The in vitro model of mixed M5 cytokines-stimulated HaCaT keratinocytes was established to investigate the phenotypic features in psoriasis. Cell viability was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, cell proliferative rate by EdU (5-ethynyl-2'-deoxyuridine) assay, and autophagy was detected by immunofluorescence staining. After M5 exposure, the proliferative rate, protein expression of autophagy, and signaling activities of NLR family pyrin domain containing 3 (NLRP3) inflammasome and toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) were measured. The latter were quantitated using quantitative PCR and western blot, respectively. The inflammatory response was detected by enzyme-linked immunosorbent assay (ELISA). Tectorigenin exerted a protective effect in ameliorating the hyperproliferation and inflammation of HaCaT keratinocytes induced by M5 cytokines. Furthermore, tectorigenin on keratinocytes seemed to inactivate NLRP3 inflammasome and inhibit cell proliferation and inflammation response via suppression of TLR4/NF-κB pathway. This study proves that tectorigenin may be a potential therapeutic candidate for psoriasis treatment in future.

CIRCULAR RNA CIRC_0099188 CONTRIBUTES TO LPS-INDUCED HPAEpiC CELL INJURY BY TARGETING THE MIR-1236-3P/HMGB3 AXIS.

Purpose: This study is designed to explore the role and mechanism of circ_0099188 in LPS-engendered HPAEpiC cells. Methods: Circ_0099188, microRNA-1236-3p (miR-1236-3p), and high mobility group box 3 (HMGB3) levels were measured using real-time quantitative polymerase chain reaction. Cell viability and apoptosis were assessed using cell counting kit-8 (CCK-8) and flow cytometry assays. Protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), cleaved-caspase 3, cleaved-caspase 9, and HMGB3 were determined using Western blot assay. IL-6, IL-8, IL-1β, and TNF-α levels were analyzed using enzyme-linked immunosorbent assays. After predicting using Circinteractome and Targetscan, the binding between miR-1236-3p and circ_0099188 or HMGB3 was verified using a dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. Results: Circ_0099188 and HMGB3 were highly expressed, and miR-1236-3p was decreased in LPS-stimulated HPAEpiC cells. Also, the downregulation of circ_0099188 might overturn LPS-triggered HPAEpiC cell proliferation, apoptosis, and inflammatory response. Mechanically, circ_0099188 is able to affect HMGB3 expression by sponging miR-1236-3p. Conclusion: Circ_0099188 knockdown might mitigate LPS-induced HPAEpiC cell injury by targeting the miR-1236-3p/HMGB3 axis, providing an underlying therapeutic strategy for pneumonia treatment.

Prevention of Cell Proliferation Signaling Via RNA Interference of c-myc Gene Expression in Colon Cancer In-vitro

Colon cancer is a cancer begins in the large intestine (colon). Its aggression is due to late diagnosis, so poor prognosis and higher mortality rates are reported. Colon cancer has become a thorny research area that requires more examination of cellular pathways involved in its emergency. Based on this, we sought to check the biological role of cMyc in colon cancer cell proliferation through targeting its expression by a respective siRNA using CaCo-2 cell lines. Notably, the cytotoxic potential of cMyc knockdown was monitored by cell imaging using an inverted microscope, the production of lactate dehydrogenase (LDH) and viability of the transfected cells using MTT assay. The relative gene expression of c-myc and other related factors such as Raf-1 was investigated using quantitative real-time PCR (qRT-PCR). ELISA assay has been used to achieve the production of pro and anti-inflammatory cytokines released from transfected cells. Interestingly, the transfection with a new designed siRNA antagonist cMyc markedly decreased CaCo-2 cell viability rate in a dose-dependent manner compared with the transfection with specific siRNA antagonist Luciferase gene, which served as control. Likewise, number of survived cells significantly reduced following transfection with siRNA against c-myc, while the relative production of LDH significantly increased. Importantly, the cytotoxic effect of the transfection protocol showed neglected influence in normal colon epithelial cells (NCM-460 cell lines) indicating the selective regulation of colon cancer cell proliferation by transfection with siRNA targeting c-myc. Noteworthy, the knockdown efficiency of c-myc showed more than 80% inhibition of its expression in CaCo-2 cells transfected with the siRNA antagonist c-myc indicated by qRT-PCR. The relative gene expression of Raf-1 significantly decreased in c-myc knockdown cells, while the relative expression of both P53 and Caspase 3, as cell death indicators, significantly upregulated compared with control-transfected cells. Furthermore, the transfection with siRNA targeting c-myc markedly increased the production of interleukin 1 alpha (IL-1α ) and IL-1β, as pro-inflammatory cytokines, while decreased the production of IL- 4 and IL-10, as anti-inflammatory cytokines. These data provide evidence for the involvement of c-myc gene expression in colon cancer cell proliferation and immune response during cancer development.

Combination of EphA2- and Wee1-Targeted Therapies in Endometrial Cancer.

EphA2 tyrosine kinase is upregulated in many cancers and correlated with poor survival of patients, including those with endometrial cancer. EphA2-targeted drugs have shown modest clinical benefit. To improve the therapeutic response to such drugs, we performed a high-throughput chemical screen to discover novel synergistic partners for EphA2-targeted therapeutics. Our screen identified the Wee1 kinase inhibitor, MK1775, as a synergistic partner to EphA2, and this finding was confirmed using both in vitro and in vivo experiments. We hypothesized that Wee1 inhibition would sensitize cells to EphA2-targeted therapy. Combination treatment decreased cell viability, induced apoptosis, and reduced clonogenic potential in endometrial cancer cell lines. In vivo Hec1A and Ishikawa-Luc orthotopic mouse models of endometrial cancer showed greater anti-tumor responses to combination treatment than to either monotherapy. RNASeq analysis highlighted reduced cell proliferation and defective DNA damage response pathways as potential mediators of the combination's effects. In conclusion, our preclinical findings indicate that Wee1 inhibition can enhance the response to EphA2-targeted therapeutics in endometrial cancer; this strategy thus warrants further development.

Interleukin-6 Gene Polymorphisms Influencing in hematological indices from sickle cell Anemia Patients

The homozygous hemoglobin SS is characterized Sickle Cell Anemia (SCA), altering the original structure of erythrocytes to a sickle shape. The hemoglobinopathies encompass all genetic diseases of hemoglobin and the SCA is the one that presents the greatest clinical manifestations variability and also the most severe ones, causing chronic hemolysis, vaso-occlusive crises and severe anemia in patients. The present study aimed to investigate the role of rs2069832, rs2069835, rs2069840, rs2069845 and rs2069849 polymorphisms in the Interleukin-6 gene in the hematological values of SCA patients treated at Fundação HEMOAM, Manaus, AM. The inclusion of patients was carried out through outpatient care at HEMOAM. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen) and molecular analyzes by TaqMan® probes on Applied Biosystems QuantStudio 6 Flex Real-Time PCR System. A total of 277 SCA patients were included in this study, having the female gender having a minimally higher frequency (55.3%). The mean age at diagnosis was approximately three years old, with brown race being the most predominant (77.6%). The rs2069832_AA and rs2069845_AA genotypes showed high values for red blood cell, hemoglobin and hematocrit indices, by having an important role as a protective factor for hemolysis in these patients. While the rs2069835_CC genotype showed decreased values for the same hematimetric indices, demonstrating to be a potential risk factor for increased hemolysis. No significant correlation in hematimetric indices was observed for the rs2069840 and rs2069849 genotypes. There are few studies correlating the genetic variants of the IL-6 gene in SCA in the state of Amazonas, however, it is known that IL-6 is involved in cell proliferation and greater response to inflammatory cytokines, and may modulate the clinical response in these patients, such as chronic hemolysis, vaso-occlusion and infections. Our work demonstrated associations of risk and protective IL-6 genotypes for possible hemolysis in patients with sickle cell anemia. We understand that an investigation with a larger number of patients would be recommended to elucidate the roles of the studied polymorphisms in sickle cell anemia. In addition, elucidating the role of Il-6 in sickle cell anemia may lead to the development of new strategies and therapies to prevent the systemic effects of excessive cytokine production and, consequently, reduce the severity of crises in these patients, providing better prognosis, clinical follow-up and welfare.

Long non-coding RNA LTCONS8875 regulates innate immunity by up-regulating IRAK4 in Miichthys miiuy (miiuy croaker)

In recent years, many studies have shown that long non-coding RNAs (lncRNAs) can regulate many biochemical processes, such as cell growth, proliferation, and immune response, which have attracted great attention. There are relatively many studies on lncRNA in mammals, while the research on lncRNA in lower vertebrates has just begun. In this study, we found a lncRNA, lncRNA LTCONS8875, related to innate immune response in Miichthys miiuy (miiuy croaker). Our results showed that lncRNA LTCONS8875 can up-regulate the expression of IRAK4 at the mRNA and protein levels, and significantly increase the production of inflammatory factors under LPS stimulation. Our research also confirmed that lncRNA LTCONS8875 plays an active role in regulating inflammation, cell proliferation, and cell viability. In summary, this research results showed that lncRNA LTCONS8875 can as an active regulatory role of innate immunity in miiuy croaker by up-regulating the expression of IRAK4, providing some insights for understanding the network mechanism of non-coding regulation of fish immunity.

66P PD-L1 expression following neoadjuvant chemotherapy is upregulated and serves as a prognostic factor in patients with advanced high-grade serous ovarian carcinoma

Patients with high-grade serous ovarian carcinoma (HGSOC) gradually acquire resistance to standard chemotherapy after recurrence. In our previous study of HGSOC, histone deacetylase (HDAC)6 upregulation led to poor prognosis, and programmed cell death ligand-1(PD-L1) expressions positively correlated with HDAC6 expression. We analyzed the expression of HDAC6 and PD-L1 pre- and post-chemotherapy to investigate their association with chemotherapy resistance. PD-L1 and HDAC6 expressions were immunohistochemically analyzed using clinical samples obtained before and after standard chemotherapy (combination of platinum and taxane agents) from 54 patients with HGSOC. The clinicopathological characteristics, including surgical status, RECIST status, and chemotherapy response score, were reviewed. High expression (≥ 5%) of PD-L1 was detected in 5 and 8 cases pre- and post-chemotherapy, respectively. The mean PD-L1 positive rate post-chemotherapy was 3.88%, which was significantly higher than 0.68% pre- chemotherapy (p=0.045). The high expression frequency of HDAC6 significantly increased from 4 patients pre-chemotherapy to 13 patients post-chemotherapy (p=0.019). High PD-L1 expression post-chemotherapy was significantly correlated with chemotherapy response score 3, signifying chemosensitivity. High post-chemotherapy PD-L1 expression led to poor progression-free (p=0.037) and overall survival (p=0.049), in the group with complete surgical resection. In HGSOC, residual tumors post-chemotherapy had enhanced expression of HDAC6 and PD-L1, which are associated with tumor immunity, cell proliferation, and hypoxic responses. PD-L1 was also correlated with patient prognosis. These results suggest that HDAC6 and PD-L1 may serve as therapeutic targets and prognostic factors for residual tumors after standard chemotherapy in HGSOC.

Enhancing plant-derived smart nanoinhibitor in targeting mammalian target of rapamycin (mTOR) in breast cancer using Curcuma longa-derived compound curcumin.

Breast cancer is a diverse female malignancy; its classification is based on clinical evidence and pathologicalelucidation. Large public drug screening data databases combined with transcriptome measures have helped developpredictive computational models. Breast cancer is frequent among women worldwide. Several genes increase breast cancerrisk. The Mammalian Target of Rapamycin (popularly known as mTOR) is a risk factor mutated in numerous breastcarcinoma types. This has caught the scientific community's focus, which is attempting to generate creative, potent, andbio-available ligands for future anti-cancer treatments to establish a practical therapeutic approach. mTOR is a proteinkinase involved in cell proliferation, survival, metabolism, and immune response. Activating mTOR promotes cancer growthand spread. To generate a bioavailable and effective mTOR inhibitor, we used computer-aided drug design to studychromones and flavonoids, two naturally occurring chemicals with many biological activities. We used Curcuma longaderivedtiny nano-molecules, which can be coated using liposomes to target mTOR to prevent breast cancer growth.The significant interactions of Curcumin were anticipated using molecular docking. It had the highest binding affinityat -12.26 kcal/mol. 100 nanoseconds of molecular dynamic modelling confirmed Curcumin and mTOR receptor interaction.Liposomes are a form of medicine carrier. To improve healthcare, more liposome-like nanostructures are being made.Nanostructures' interactions with living creatures are being studied. Half-life, tissue accumulation, and toxicity have beenstudied. Future medication distribution may use nanocarriers having a liposome-like form, enabling targeted nano-delivery.Curcumin's interaction with the active site increased the complex's structural stability during its expansion. Ourresults may help future investigations of Curcumin's efficacy as a possible lead treatment targeting mTOR receptors inbreast cancer. Using Curcumin as a potential anti-cancer drug with lipid-coated nano-particles allows for tailoredadministration.

P095 Newly discovered gut matrikines from Crohn’s disease intestinal tissue induce cell proliferation, activation and inflammatory response in intestinal myofibroblasts in vitro

Abstract Background The interaction between intestinal myofibroblasts (iMFBs) and extracellular matrix (ECM) changes contributes to Crohn’s disease (CD) fibrosis, but the exact mechanisms are not yet clear. Our group recently discovered a selection of matrix-derived peptides (matrikines) that specifically appear in the intestinal tissue of patients with CD fibrostenosis.1 Here, we evaluate the in vitro effect of 19 matrikines (with various bioactivity likelihood) on primary human intestinal myofibroblasts (IMFBs), key cells in CD fibrosis. Methods Primary human iMFBS (500K/mL, Passage 4, Novabiosis) were cultured in serum-free medium (SFM) (DMEM high glucose, 1% sodium pyruvate, 1% non-essential aminoacids, 1% Antibiotics-Antimycotics and 3mg Gentamicin), complete medium (SFM with 10% fetal bovine serum), PDGF-bb 10ng/mL or treated with each of the matrikines at 10, 50, 100 μg/mL for 24hours. The effect of each matrikine on cell proliferation, gene expression and migration was assessed by BrdU, qPCR and Transwell assays, respectively. RNA sequencing data was analysed using the GSEA software. Results The effect on iMFBs proliferation and viability was tested with each of the 19 matrikines at 3 different doses (Table 1). Among 9 most likely-bioactive matrikines (M1-M9) (Figure 1), M1, M4, M7, M8 and M9 induced the highest effect in cell proliferation and were selected for further experiments. Gene expression assays showed upregulation of ACTA2 with M1 or M7 and upregulation of COL1A1 with M1, M7 or M8, indicating activation of the iMFBs (Figure 2). Transwell assays also showed that M1, M4 and M8 induce iMFB cell migration. A total of 1948, 1024, 460, 570, and 2749 differentially expressed genes (DEGs) were identified in iMBFs treated with M1, M4, M7, M8 and M9 vs no treatment, showing enrichment in pathways related to “smooth muscle contraction”, “fibroblast growth factor”, “cell migration”, “collagen formation”, “elastin formation” and “ECM organization”; most of which were also enriched by complete medium and/or PDGFbb that served as positive controls. However, pathways related to “proinflammatory and profibrotic mediators” or “vascular permeability” were only enriched in iMFBs treated with M1, M4, M8 and M9 or M1 and M9, respectively (Figure 3). Conclusion A selection of newly discovered CD-specific matrikines induce activation of iMFBs as well as inflammatory response and endothelial changes, suggesting an important role and therefore their potential value as biomarkers in the progression of intestinal fibrostenosis.

Structural Insights into the Interactions of Belumosudil with Rho-Associated Coiled-Coil Containing Protein Kinases 1 and 2 Based on Molecular Docking, Molecular Dynamics Simulations, and Free Energy Calculations

Rho-associated coiled-coil containing protein kinases (ROCKs) are members of the cyclic adenosine monophosphate-dependent protein kinase/protein kinase G/protein kinase C family that participates in a variety of important physiological functions, including smooth muscle contraction, cell proliferation, cell adhesion, migration, and inflammatory responses. In this study, we focused on ROCK1 and ROCK2, which are targets of the Food and Drug Administration-approved inhibitor 2-(3-(4-((1[Formula: see text]-Indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-[Formula: see text]-isopropylacetamide (belumosudil). We constructed four representative belumosudil/ROCK complex structures by molecular docking. The interactions between belumosudil and ROCK were then investigated via molecular dynamics simulations and binding free energy calculations. It was found that belumosudil showed a stronger binding affinity toward ROCK2 than toward ROCK1. Binding free energy calculations and free energy decompositions suggested that the modification of various regions of the belumosudil structure may enhance its binding affinity with ROCK, in addition to improving the selectivity between ROCK1 and ROCK2. This work therefore provides useful information to aid future drug design.

Identification and analysis of key microRNAs derived from osteoarthritis synovial fluid exosomes.

Identification and analysis of key microRNAs derived from osteoarthritis synovial fluid exosomes.

Purinergic signaling: Diverse effects and therapeutic potential in cancer.

Regardless of improved biological insights and therapeutic advances, cancer is consuming multiple lives worldwide. Cancer is a complex disease with diverse cellular, metabolic, and physiological parameters as its hallmarks. This instigates a need to uncover the latest therapeutic targets to advance the treatment of cancer patients. Purines are building blocks of nucleic acids but also function as metabolic intermediates and messengers, as part of a signaling pathway known as purinergic signaling. Purinergic signaling comprises primarily adenosine triphosphate (ATP) and adenosine (ADO), their analogous membrane receptors, and a set of ectonucleotidases, and has both short- and long-term (trophic) effects. Cells release ATP and ADO to modulate cellular function in an autocrine or paracrine manner by activating membrane-localized purinergic receptors (purinoceptors, P1 and P2). P1 receptors are selective for ADO and have four recognized subtypes-A1, A2A, A2B, and A3. Purines and pyrimidines activate P2 receptors, and the P2X subtype is ligand-gated ion channel receptors. P2X has seven subtypes (P2X1-7) and forms homo- and heterotrimers. The P2Y subtype is a G protein-coupled receptor with eight subtypes (P2Y1/2/4/6/11/12/13/14). ATP, its derivatives, and purinoceptors are widely distributed in all cell types for cellular communication, and any imbalance compromises the homeostasis of the cell. Neurotransmission, neuromodulation, and secretion employ fast purinergic signaling, while trophic purinergic signaling regulates cell metabolism, proliferation, differentiation, survival, migration, invasion, and immune response during tumor progression. Thus, purinergic signaling is a prospective therapeutic target in cancer and therapy resistance.