CDK4/6 inhibition sensitizes MEK inhibition by inhibiting cell cycle and proliferation in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is not sensitive to most chemotherapy drugs, leading to poor chemotherapy efficacy. Recently, Trametinib and Palbociclib have promising prospects in the treatment of pancreatic cancer. This article aims to explore the effects of Trametinib on pancreatic cancer and address the underlying mechanism of resistance as well as its reversal strategies. The GDSC (Genomics of Drug Sensitivity in Cancer) and CTD2 (Cancer Target Discovery and Development) were utilized to screen the potential drug candidate in PDAC cell lines. The dose-increase method combined with the high-dose shock method was applied to induce the Trametinib-resistant PANC-1 and MIA PaCa-2 cell lines. The CCK8 proliferation assay, colony formation assay, flow cytometry, and western blot were conducted to verify the inhibitory effect of Trametinib and Palbociclib. RNA-seq was performed in resistant PDAC cell lines to find the differential expression genes related to drug resistance and predict pathways leading to the reversal of Trametinib resistance. The GDSC and CTD2 database screening revealed that Trametinib demonstrates a significant inhibitory effect on PDAC. We found that Trametinib has a lower IC50 than Gemcitabine in PDAC cell lines. Both Trametinib and Gemcitabine can decrease the proliferation capacity of pancreatic cells, induce cell cycle arrest, and increase apoptosis. Simultaneously, the phosphorylation of the AKT and ERK pathways were inhibited by the treatment of Trametinib. In addition, the RNA-seq of Trametinib-induced resistance PDAC cell lines reveals that the cyclin-dependent kinase (CDK)-RB-E2F regulatory axis and G2/M DNA damage checkpoint might lead the drug resistance. Besides, the combination of Trametinib with Palbociclib could inhibit the proliferation and cell cycle of both resistant cells lines and also restore the sensitivity of drug-resistant cells to Trametinib. Last but not least, the interferon-α and interferon-γ expression were upregulated in resistance cell lines, which might lead to the reversal of drug resistance. The study shows Trametinib has a critical inhibitory effect on PDAC. Besides, the combination of Trametinib with Palbociclib can inhibit the proliferation of PDAC-resistant cells.

Comprehensive assessment of goat adipose tissue-derived mesenchymal stem cells cultured in different media.

Mesenchymal stem cells (MSCs) have demonstrated potential in treating livestock diseases that are unresponsive to conventional therapies. MSCs derived from goats, a valuable model for studying orthopaedic disorders in humans, offer insights into bone formation and regeneration. Adipose tissue-derived MSCs (ADSCs) are easily accessible and have a high capacity for expansion. Although the choice of culture media significantly influences the biological properties of MSCs, the optimal media for goat ADSCs (gADSCs) remains unclear. This study aimed to assess the effects of four commonly used culture media on gADSCs' culture characteristics, stem cell-specific immunophenotype, and differentiation. Results showed that MEM, DMEM/F12, and DMEM-LG were superior in maintaining cell morphology and culture parameters of gADSCs, such as cell adherence, metabolic activity, colony-forming potential, and population doubling. Conversely, DMEM-HG exhibited poor performance across all evaluated parameters. The gADSCs cultured in DMEM/F12 showed enhanced early proliferation and lower apoptosis. The cell surface marker distribution exhibited superior characteristics in gADSCs cultured in MEM and DMEM/F12. In contrast, the distribution was inferior in gADSCs cultured in DMEM-LG. DMEM/F12 and DMEM-LG culture media demonstrated a significantly higher potential for chondrogenic differentiation and DMEM-LG for osteogenic differentiation. In conclusion, DMEM/F12 is a suitable culture medium for propagating gADSCs as it effectively maintains cell morphology, growth parameters, proliferation and lower apoptosis while exhibiting desirable expression patterns of MSC-specific markers. These findings contribute to optimising culture conditions for gADSCs, enhancing their potential applications in disease treatment and regenerative medicine.

Methylated lncRNAs suppress apoptosis of gastric cancer stem cells via the lncRNA-miRNA/protein axis.

BACKGROUND: Long noncoding RNAs (lncRNAs) play essential roles in the tumorigenesis of gastric cancer. However, the influence of lncRNA methylation on gastric cancer stem cells (GCSCs) remains unclear. METHODS: The N6-methyladenosine (m6A) levels of lncRNAs in gastric cancer stem cells were detected by methylated RNA immunoprecipitation sequencing (MeRIP-seq), and the results were validated by MeRIP-quantitative polymerase chain reaction (qPCR). Specific sites of m6A modification on lncRNAs were detected by single-base elongation- and ligation-based qPCR amplification (SELECT). By constructing and transfecting the plasmid expressing methyltransferase-like 3 (METTL3) fused with catalytically inactivated Cas13 (dCas13b) and guide RNA targeting specific methylation sites of lncRNAs, we obtained gastric cancer stem cells with site-specific methylation of lncRNAs. Reverse transcription (RT)-qPCR and Western blot were used for detecting the stemness of treated gastric cancer stem cells. RESULTS: The site-specific methylation of PSMA3-AS1 and MIR22HG suppressed apoptosis and promoted stemness of GCSCs. LncRNA methylation enhanced the stability of PSMA3-AS1 and MIR22HG to suppress apoptosis of GCSCs via the PSMA3-AS1-miR-411-3p- or MIR22HG-miR-24-3p-SERTAD1 axis. Simultaneously, the methylated lncRNAs promoted the interaction between PSMA3-AS1 and the EEF1A1 protein or MIR22HG and the LRPPRC protein, stabilizing the proteins and leading to the suppression of apoptosis. The in vivo data revealed that the methylated PSMA3-AS1 and MIR22HG triggered tumorigenesis of GCSCs. CONCLUSIONS: Our study revealed the requirement for site-specific methylation of lncRNAs in the tumorigenesis of GCSCs, contributing novel insights into cancer development.

Beta hydroxybutyrate induces lung cancer cell death, mitochondrial impairment and oxidative stress in a long term glucose-restricted condition.

BACKGROUND: Metabolic plasticity gives cancer cells the ability to shift between signaling pathways to facilitate their growth and survival. This study investigates the role of glucose deprivation in the presence and absence of beta-hydroxybutyrate (BHB) in growth, death, oxidative stress and the stemness features of lung cancer cells. METHODS AND RESULTS: A549 cells were exposed to various glucose conditions, both with and without beta-hydroxybutyrate (BHB), to evaluate their effects on apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) levels using flow cytometry, and the expression of CD133, CD44, SOX-9, and ß-Catenin through Quantitative PCR. The activity of superoxide dismutase, glutathione peroxidase, and malondialdehyde was assessed using colorimetric assays. Treatment with therapeutic doses of BHB triggered apoptosis in A549 cells, particularly in cells adapted to glucose deprivation. The elevated ROS levels, combined with reduced levels of SOD and GPx, indicate that oxidative stress contributes to the cell arrest induced by BHB. Notably, BHB treatment under glucose-restricted conditions notably decreased CD133 expression, suggesting a potential inhibition of cell survival through the downregulation of CD133 levels. Additionally, the simultaneous decrease in mitochondrial membrane potential and increase in ROS levels indicate the potential for creating oxidative stress conditions to impede tumor cell growth in such environmental settings. CONCLUSION: The induced cell death, oxidative stress and mitochondria impairment beside attenuated levels of cancer stem cell markers following BHB administration emphasize on the distinctive role of metabolic plasticity of cancer cells and propose possible therapeutic approaches to control cancer cell growth through metabolic fuels.

Iron deficiency in astrocytes alters cellular status and impacts on oligodendrocyte differentiation.

Iron deficiency (ID) has been shown to affect central nervous system (CNS) development and induce hypomyelination. Previous work from our laboratory in a gestational ID model showed that both oligodendrocyte (OLG) and astrocyte (AST) maturation was impaired. To explore the contribution of AST iron to the myelination process, we generated an in vitro ID model by silencing divalent metal transporter 1 (DMT1) in AST (siDMT1 AST) or treating AST with Fe3+ chelator deferoxamine (DFX; DFX AST). siDMT1 AST showed no changes in proliferation but remained immature. Co-cultures of oligodendrocyte precursors cells (OPC) with siDMT1 AST and OPC cultures incubated with siDMT1 AST-conditioned media (ACM) rendered a reduction in OPC maturation. These findings correlated with a decrease in the expression of AST-secreted factors IGF-1, NRG-1, and LIF, known to promote OPC differentiation. siDMT1 AST also displayed increased mitochondrial number and reduced mitochondrial size as compared to control cells. DFX AST also remained immature and DFX AST-conditioned media also hampered OPC maturation in culture, in keeping with a decrease in the expression of AST-secreted growth factors IGF-1, NRG-1, LIF, and CNTF. DFX AST mitochondrial morphology and number showed results similar to those observed in siDMT1 AST. In sum, our results show that ID, induced through two different methods, impacts AST maturation and mitochondrial functioning, which in turn hampers OPC differentiation.

Combined OLA1 and CLEC3B Gene Is a Prognostic Signature for Hepatocellular Carcinoma and Impact Tumor Progression.

Hepatocellular carcinoma (HCC), partly because of its complexity and high heterogeneity, has a poor prognosis and an extremely high mortality rate. In this study, mRNA sequencing expression profiles and relevant clinical data of HCC patients were gathered from different public databases. Kaplan-Meier survival curves as well as ROC curves validated that OLA1|CLEC3B was an independent predictor with better predictive capability of HCC prognosis compared to OLA1 and CLEC3B separately. Further, the cell transfection experiment verified that knockdown of OLA1 inhibited cell proliferation, facilitated apoptosis, and improved sensitivity of HCC cells to gemcitabine. In this study, the prognostic model of HCC composed of OLA1/CLEC3B genes was constructed and verified, and the prediction ability was favorable. A higher level of OLA1 along with a lower level of CEC3B is a sign of poor prognosis in HCC. We revealed a novel gene pair OLA1|CLEC3B overexpressed in HCC patients, which may serve as a promising independent predictor of HCC survival and an approach for innovative diagnostic and therapeutic strategies.

Selective Targeting of Regulated Rhabdomyosarcoma Cells by Trinuclear Ruthenium(II)-Arene Complexes.

The use of benzimidazole-based trinuclear ruthenium(II)-arene complexes (1-3) to selectively target the rare cancer rhabdomyosarcoma is reported. Preliminary cytotoxic evaluations of the ruthenium complexes in an eight-cancer cell line panel revealed enhanced, selective cytotoxicity toward rhabdomyosarcoma cells (RMS). The trinuclear complex 1 was noted to show superior short- and long-term cytotoxicity in RMS cell lines and enhanced selectivity relative to cisplatin. Remarkably, 1 inhibits the migration of metastatic RMS cells and maintains superior activity in a 3D multicellular spheroid model in comparison to that of the clinically used cisplatin. Mechanistic insights reveal that 1 effectively induces genomic DNA damage, initiates autophagy, and prompts the intrinsic and extrinsic apoptotic pathways in RMS cells. To the best of our knowledge, 1 is the first trinuclear ruthenium(II) arene complex to selectively kill RMS cells in 2D and 3D cell cultures.

Peptide Inhibitor Targeting the Extraterminal Domain in BRD4 Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

BRD4 is associated with a variety of human diseases, including breast cancer. The crucial roles of amino-terminal bromodomains (BDs) of BRD4 in binding with acetylated histones to regulate oncogene expression make them promising drug targets. However, adverse events impede the development of the BD inhibitors. BRD4 adopts an extraterminal (ET) domain, which recruits proteins to drive oncogene expression. We discovered a peptide inhibitor PiET targeting the ET domain to disrupt BRD4/JMJD6 interaction, a protein complex critical in oncogene expression and breast cancer. The cell-permeable form of PiET, TAT-PiET, and PROTAC-modified TAT-PiET, TAT-PiET-PROTAC, potently inhibits the expression of BRD4/JMJD6 target genes and breast cancer cell growth. Combination therapy with TAT-PiET/TAT-PiET-PROTAC and JQ1, iJMJD6, or Fulvestrant exhibits synergistic effects. TAT-PiET or TAT-PiET-PROTAC treatment overcomes endocrine therapy resistance in ERα-positive breast cancer cells. Taken together, we demonstrated that targeting the ET domain is effective in suppressing breast cancer, providing a therapeutic avenue in the clinic.

Critical roles of long noncoding RNA H19 in cancer.

Long noncoding RNAs (lncRNAs) are a category of noncoding RNAs characterized by their length, often exceeding 200 nucleotides. There is a growing body of data that indicate the significant involvement of lncRNAs in a wide range of disorders, including cancer. lncRNA H19 was among the initial lncRNAs to be identified and is transcribed from the H19 gene. The H19 lncRNA exhibits significant upregulation in a diverse range of human malignancies, such as breast, colorectal, pancreatic, glioma, and gastric cancer. Moreover, the overexpression of H19 is frequently associated with a worse prognosis among individuals diagnosed with cancer. H19 has been shown to have a role in facilitating several cellular processes, including cell proliferation, invasion, migration, epithelial-mesenchymal transition, metastasis, and apoptosis. This article summarizes the aberrant upregulation of H19 in human malignancies, indicating promising avenues for future investigations on cancer diagnostics and therapeutic interventions.

Ocrelizumab Alters Cytotoxic Lymphocyte Function While Reducing EBV-Specific CD8+ T-Cell Proliferation in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The role of B cells in the pathogenic events leading to relapsing multiple sclerosis (R-MS) has only been recently elucidated. A pivotal step in defining this role has been provided by therapeutic efficacy of anti-CD20 monoclonal antibodies. Indeed, treatment with anti-CD20 can also alter number and function of other immune cells not directly expressing CD20 on their cell surface, whose activities can contribute to unknown aspects influencing therapeutic efficacy. We examined the phenotype and function of cytotoxic lymphocytes and Epstein-Barr virus (EBV)-specific immune responses in people with R-MS before and after ocrelizumab treatment. METHODS: In this prospective study, we collected blood samples from people with R-MS (n = 41) before and 6 and 12 months after initiating ocrelizumab to assess the immune phenotype and the indirect impact on cytotoxic functions of CD8+ T and NK cells. In addition, we evaluated the specific anti-EBV proliferative responses of both CD8+ T and NK lymphocytes as surrogate markers of anti-EBV activity. RESULTS: We observed that while ocrelizumab depleted circulating B cells, it also reduced the expression of activation and migratory markers on both CD8+ T and NK cells as well as their in vitro cytotoxic activity. A comparable pattern in the modulation of immune molecules by ocrelizumab was observed in cytotoxic cells even when patients with R-MS were divided into groups based on their prior disease-modifying treatment. These effects were accompanied by a significant and selective reduction of CD8+ T-cell proliferation in response to EBV antigenic peptides. DISCUSSION: Taken together, our findings suggest that ocrelizumab-while depleting B cells-affects the cytotoxic function of CD8+ and NK cells, whose reduced cross-activity against myelin antigens might also contribute to its therapeutic efficacy during MS.

[Effects of gelatin methacrylate anhydride hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells in the treatment of full-thickness skin defect wounds in mice].

Objective: To investigate the effects of gelatin methacrylate anhydride (GelMA) hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUCMSCs-sEVs) in the treatment of full-thickness skin defect wounds in mice. Methods: This study was an experimental study. hUCMSCs-sEVs were extracted by ultracentrifugation, their morphology was observed through transmission electron microscope, and the expression of CD9, CD63, tumor susceptibility gene 101 (TSG101), and calnexin was detected by Western blotting. The human umbilical vein endothelial cells (HUVECs), the 3rd and 4th passages of human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDFs) were all divided into blank control group (routinely cultured) and hUCMSC-sEV group (cultured with the cell supernatant containing hUCMSCs-sEVs). The cell scratch test was performed and the cell migration rates at 6, 12, and 24 h after scratching were calculated, the cell Transwell assay was performed and the number of migration cells at 12 h after culture was calculated, and the proportion of proliferating cells was detected by 5-acetylidene-2'-deoxyuridine and Hoechst staining at 24 h after culture, with sample numbers being all 3. The simple GelMA hydrogel and the GelMA hydrogel loaded with hUCMSCs-sEVs (hereinafter referred to as hUCMSC-sEV/GelMA hydrogel) were prepared. Then the micromorphology of 2 kinds of hydrogels was observed under scanning electron microscope, the distribution of hUCMSCs-sEVs was observed by laser scanning confocal microscope, and the cumulative release rates of hUCMSCs-sEVs at 0 (immediately), 2, 4, 6, 8, 10, and 12 d after soaking hUCMSC-sEV/GelMA hydrogel in phosphate buffer solution (PBS) were measured and calculated by protein colorimetric quantification (n=3). Twenty-four 6-week-old male C57BL/6J mice were divided into PBS group, hUCMSC-sEV alone group, GelMA hydrogel alone group, and hUCMSC-sEV/GelMA hydrogel group according to the random number table, with 6 mice in each group, and after the full-thickness skin defect wounds on the back of mice in each group were produced, the wounds were performed with PBS injection, hUCMSC-sEV suspenson injection, simple GelMA coverage, and hUCMSC-sEV/GelMA hydrogel coverage, respectively. Wound healing was observed on post injury day (PID) 0 (immediately), 4, 8, and 12, and the wound healing rates on PID 4, 8, and 12 were calculated, and the wound tissue was collected on PID 12 for hematoxylin-eosin staining to observe the structure of new tissue, with sample numbers being both 6. Results: The extracted hUCMSCs-sEVs showed a cup-shaped structure and expressed CD9, CD63, and TSG101, but barely expressed calnexin. At 6, 12, and 24 h after scratching, the migration rates of HEKs (with t values of 25.94, 20.98, and 20.04, respectively), HDFs (with t values of 3.18, 5.68, and 4.28, respectively), and HUVECs (with t values of 4.32, 19.33, and 4.00, respectively) in hUCMSC-sEV group were significantly higher than those in blank control group (P<0.05). At 12 h after culture, the numbers of migrated HEKs, HDFs, and HUVECs in hUCMSC-sEV group were 550±23, 235±9, and 856±35, respectively, which were significantly higher than 188±14, 97±6, and 370±32 in blank control group (with t values of 22.95, 23.13, and 17.84, respectively, P<0.05). At 24 h after culture, the proportions of proliferating cells of HEKs, HDFs, and HUVECs in hUCMSC-sEV group were significantly higher than those in blank control group (with t values of 22.00, 13.82, and 32.32, respectively, P<0.05). The inside of simple GelMA hydrogel showed a loose and porous sponge-like structure, and hUCMSCs-sEVs was not observed in it. The hUCMSC-sEV/GelMA hydrogel had the same sponge-like structure, and hUCMSCs-sEVs were uniformly distributed in clumps. The cumulative release rate curve of hUCMSCs-sEVs from hUCMSC-sEV/GelMA hydrogel tended to plateau at 2 d after soaking, and the cumulative release rate of hUCMSCs-sEVs was (59.2±1.8)% at 12 d after soaking. From PID 0 to 12, the wound areas of mice in the 4 groups gradually decreased. On PID 4, 8, and 12, the wound healing rates of mice in hUCMSC-sEV/GelMA hydrogel group were significantly higher than those in the other 3 groups (P<0.05); the wound healing rates of mice in GelMA hydrogel alone group and hUCMSC-sEV alone group were significantly higher than those in PBS group (P<0.05). On PID 8 and 12, the wound healing rates of mice in hUCMSC-sEV alone group were significantly higher than those in GelMA hydrogel alone group (P<0.05). On PID 12, the wounds of mice in hUCMSC-sEV/GelMA hydrogel group showed the best wound epithelization, loose and orderly arrangement of dermal collagen, and the least number of inflammatory cells, while the dense arrangement of dermal collagen and varying degrees of inflammatory cell infiltration were observed in the wounds of mice in the other 3 groups. Conclusions: hUCMSCs-sEVs can promote the migration and proliferation of HEKs, HDFs, and HUVECs which are related to skin wound healing, and slowly release in GelMA hydrogel. The hUCMSC-sEV/GelMA hydrogel as a wound dressing can significantly improve the healing speed of full-thickness skin defect wounds in mice.

A potential strategy for bladder cancer treatment: inhibiting autophagy to enhance antitumor effects of Nectin-4-MMAE.

Research and development on Nectin-4 antibody-drug conjugates (ADC) have been greatly accelerated since the approval of enfortumab vedotin to treat uroepithelial cancer. During the course of this study, we identified that autophagy serves as a cytoprotective mechanism during Nectin-4-MMAE treatment and proposed a strategy to enhance the antitumor effects of Nectin-4-MMAE in bladder cancer. Nectin-4-MMAE rapidly internalized into bladder cancer cells in 30 minutes and released MMAE, inducing the onset of caspase-mediated apoptosis and leading to the inhibition of tumor cell growth. Transcriptomics showed significant alterations in autophagy-associated genes in bladder cancer cells treated with Nectin-4-MMAE, which suggested autophagy was activated by Nectin-4-MMAE. Furthermore, autophagy activation was characterized by ultrastructural analysis of autophagosome accumulation, immunofluorescence of autophagic flux, and immunoblotting autophagy marker proteins SQSTM1 and LC3 I/II. Importantly, inhibiting autophagy by LY294002 and chloroquine significantly enhances the cytotoxicity effects of Nectin-4-MMAE in bladder cancer cells. Additionally, we detected the participation of the AKT/mTOR signaling cascade in the induction of autophagy by Nectin-4-MMAE. The combination of Nectin-4-MMAE and an autophagy inhibitor demonstrated enhanced antitumor effects in the HT1376 xenograft tumor model. After receiving a single dose of Nectin-4-MMAE, the group that received the combination treatment showed a significant decrease in tumor size compared to the group that received only one type of treatment. Notably, one mouse in the combination treatment group achieved complete remission of the tumor. The combination group exhibited a notable rise in apoptosis and necrosis, as indicated by H&E staining and immunohistochemistry (cleaved caspase-3, ki67). These findings demonstrated the cytoprotective role of autophagy during Nectin-4-MMAE treatment and highlighted the potential of combining Nectin-4-MMAE with autophagy inhibitors for bladder cancer treatment.

PICK1 inhibits the malignancy of nasopharyngeal carcinoma and serves as a novel prognostic marker.

Although many important advances have been made in the treatment of nasopharyngeal carcinoma (NPC) in recent years, local recurrence and distant metastasis remain the main factors affecting NPC prognosis. Biomarkers for predicting the prognosis of NPC need to be urgently identified. Here, we used whole-exon sequencing (WES) to determine whether PICK1 mutations are associated with the prognosis of NPC. Functionally, PICK1 inhibits the proliferation and metastasis of NPC cells both in vivo and in vitro. Mechanistically, PICK1 inhibited the expression of proteins related to the Wnt/ß-catenin signaling pathway. PICK1 restrained the nuclear accumulation of ß-catenin and accelerated the degradation of ß-catenin through the ubiquitin-proteasome pathway. The reduced PICK1 levels were significantly associated with poor patient prognosis. Hence, our study findings reveal the mechanism by which PICK1 inactivates the Wnt/ß-catenin signaling pathway, thereby inhibiting the progression of NPC. They support PICK1 as a potential tumor suppressor and prognostic marker for NPC.

TMEM9 promotes lung adenocarcinoma progression via activating the MEK/ERK/STAT3 pathway to induce VEGF expression.

Abnormal Transmembrane protein 9 (TMEM9) expression has been identified in various human tumors. However, the prognostic potential and mechanistic role of TMEM9 in lung adenocarcinoma (LUAD) remain unclear. Here, we first found a significant upregulation of TMEM9 in LUAD tissues, and TMEM9 expression was positively correlated with microvessel density (MVD), T stage, and clinical stage. Survival analysis demonstrated TMEM9 was an independent indicator of poor prognosis in LUAD patients. In addition, downregulation of TMEM9 suppressed tumor growth and metastasis in vitro and in vivo models, and reduced HUVEC proliferation, migration, and tube formation in a cancer cell/HUVEC coculture model. Furthermore, TMEM9 upregulated VEGF expression, and VEGF-neutralizing antibodies reversed HUVEC angiogenesis and cancer cell migration ability caused by overexpression of TMEM9. In contrast, recombinant VEGF (rVEGF) abolished the inhibitory effect of TMEM9-knockdown LUAD cells on HUVEC angiogenesis and tumor cell migration. Moreover, we showed that TMEM9 upregulated VEGF expression by activating the mitogen-activated protein kinase/extracellular signal-regulated kinase/STAT3 (MEK/ERK/STAT3) pathway. Together, our study provides mechanistic insights into the role of TMEM9 in LUAD and highlights the potential of targeting the TMEM9/MEK/ERK/STAT3/VEGF pathway as a novel therapy for preventing LUAD progression.

Apigenin and its combination with Vorinostat induces apoptotic-mediated cell death in TNBC by modulating the epigenetic and apoptotic regulators and related miRNAs.

Triple-negative breast cancer (TNBC) is a metastatic disease and a formidable treatment challenge as it does not respond to existing therapies. Epigenetic regulators play a crucial role in the progression and metastasis by modulating the expression of anti-apoptotic, pro-apoptotic markers and related miRNAs in TNBC cells. We have investigated the anti-TNBC potential of dietary flavonoid 'Apigenin' and its combination with Vorinostat on MDA-MB-231 cells. At Apigenin generated ROS, inhibited cell migration, arrested the cell cycle at subG0/G1 phases, and induced apoptotic-mediated cell death. Apigenin reduced the expression of the class-I HDACs at the transcriptomic and proteomic levels. In the immunoblotting study, Apigenin has upregulated pro-apoptotic markers and downregulated anti-apoptotic proteins. Apigenin inhibited the enzymatic activity of HDAC/DNMT and increased HAT activity. Apigenin has manifested its effect on miRNA expression by upregulating the tumor-suppressor miR-200b and downregulation oncomiR-21. Combination study reduced the growth of TNBC cells synergistically by modulating the expression of epigenetic and apoptotic regulators. Molecular docking and MD simulations explored the mechanism of catalytic inhibition of HDAC1 and HDAC3 and supported the in-vitro studies. The overall studies demonstrated an anti-TNBC potential of Apigenin and may help to design an effective strategy to treat metastatic phenotype of TNBC.

Comparative analysis of SEC61A1 mutant R236C in two patient-derived cellular platforms.

SEC61A1 encodes a central protein of the mammalian translocon and dysfunction results in severe disease. Recently, mutation R236C was identified in patients having autosomal dominant polycystic liver disease (ADPLD). The molecular phenotype of R236C was assessed in two cellular platforms. Cells were immortalized by retroviral transduction of an oncogene (UCi) or reprogrammed to induced pluripotent stem cells (iPSC) that were differentiated to cholangiocyte progenitor-like cells (CPLC). UCi and CPLC were subjected to analyses of molecular pathways that were associated with development of disease. UCi displayed markers of epithelial cells, while CPLCs expressed typical markers of both cholangiocytes and hepatocytes. Cells encoding R236C showed a stable, continuous proliferation in both platforms, however growth rates were reduced as compared to wildtype control. Autophagy, cAMP synthesis, and secretion of important marker proteins were reduced in R236C-expressing cells. In addition, R236C induced increased calcium leakiness from the ER to the cytoplasm. Upon oxidative stress, R236C led to a high induction of apoptosis and necrosis. Although the grade of aberrant cellular functions differed between the two platforms, the molecular phenotype of R236C was shared suggesting that the mutation, regardless of the cell type, has a dominant impact on disease-associated pathways.

CHOP regulated by METTL14-m6A affects cell cycle arrest and regorafenib sensitivity in HCC cells.

BACKGROUND: Regorafenib, a multi-targeted kinase inhibitor, has been used in the treatment of Hepatocellular carcinoma (HCC). The purpose of this study is to investigate the mechanism of Regorafenib in HCC. METHODS: Regorafenib's impact on the sensitivity of HCC cells was assessed using CCK8. Differential gene expression analysis was performed by conducting mRNA sequencing after treatment with Regorafenib. The m6A methylation status of CHOP and differential expression of m6A methylation-related proteins were assessed by RIP and Western Blot. To explore the molecular mechanisms involved in the therapeutic effects of Regorafenib in HCC and the impact of METTL14 and CHOP on Regorafenib treatment, we employed shRNA/overexpression approaches to transfect METTL14 and CHOP genes, as well as conducted in vivo experiments. RESULTS: Treatment with Regorafenib led to a notable decrease in viability and proliferation of SK-Hep-1 and HCC-LM3 cells. The expression level of CHOP was upregulated after Regorafenib intervention, and CHOP underwent m6A methylation. Among the m6A methylation-related proteins, METTL14 exhibited the most significant downregulation. Mechanistic studies revealed that Regorafenib regulated the cell cycle arrest in HCC through METTL14-mediated modulation of CHOP, and the METTL14/CHOP axis affected the sensitivity of HCC to Regorafenib. In vivo, CHOP enhanced the anticancer effect of Regorafenib. CONCLUSION: The inhibition of HCC development by Regorafenib is attributed to its modulation of m6A expression of CHOP, mediated by METTL14, and the METTL14/CHOP axis enhances the sensitivity of HCC to Regorafenib. These findings provide insights into the treatment of HCC and the issue of drug resistance to Regorafenib.

Synergistic effect of human uterine cervical mesenchymal stem cell secretome and paclitaxel on triple negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer and, despite its adverse effects, chemotherapy is the standard systemic treatment option for TNBC. Since, it is of utmost importance to consider the combination of different agents to achieve greater efficacy and curability potential, MSC secretome is a possible innovative alternative. METHODS: In the present study, we proposed to investigate the anti-tumor effect of the combination of a chemical agent (paclitaxel) with a complex biological product, secretome derived from human Uterine Cervical Stem cells (CM-hUCESC) in TNBC. RESULTS: The combination of paclitaxel and CM-hUCESC decreased cell proliferation and invasiveness of tumor cells and induced apoptosis in vitro (MDA-MB-231 and/or primary tumor cells). The anti-tumor effect was confirmed in a mouse tumor xenograft model showing that the combination of both products has a significant effect in reducing tumor growth. Also, pre-conditioning hUCESC with a sub-lethal dose of paclitaxel enhances the effect of its secretome and in combination with paclitaxel reduced significantly tumor growth and even allows to diminish the dose of paclitaxel in vivo. This effect is in part due to the action of extracellular vesicles (EVs) derived from CM-hUCESC and soluble factors, such as TIMP-1 and - 2. CONCLUSIONS: In conclusion, our data demonstrate the synergistic effect of the combination of CM-hUCESC with paclitaxel on TNBC and opens an opportunity to reduce the dose of the chemotherapeutic agents, which may decrease chemotherapy-related toxicity.

siRNA treatment targeting integrin α11 overexpressed via EZH2-driven axis inhibits drug-resistant breast cancer progression.

BACKGROUND: Breast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets. METHODS: Transcriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target. RESULTS: Integrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours. CONCLUSION: Our findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.

The Role of Copper-Induced M2 Macrophage Polarization in Protecting Cartilage Matrix in Osteoarthritis.

BACKGROUND The pathological mechanism of osteoarthritis is still unclear. The regulation of the immune microenvironment has been of growing interest in the progression and treatment of osteoarthritis. Macrophages with different phenotypes, producing different cytokines, have been linked to the mechanism of cartilage injury in osteoarthritis. Copper ions play a role in the immune response and are involved in the pathological mechanisms of osteoarthritis by affecting the metabolism of the cartilage matrix. Bioactive glass (BG) is an osteogenic material with superior biocompatibility. Here, we report on the regulatory behavior of macrophages using a copper-based composite BG material. MATERIAL AND METHODS Cu-BGC powder was prepared by sol-gel method, and scaffolds were fabricated and characterized using 3D printing. Macrophage cultures grown with Cu-BGC were examined for cell culture and proliferation. The effect of Cu-BGC on the degradation metabolism of chondrocytes, cultured in the environment of inflammatory cytokine IL-1ß, was determined. In addition, the morphology of macrophages, secretion of inflammatory cytokines, and expression of surface markers were examined. RESULTS The results show that Cu-BGC promotes macrophage proliferation at a range of concentrations and increases the secretion of anti-inflammatory cytokines while inhibiting proinflammatory cytokines. At the same time, M2-type cell surface markers are definitely expressed and the morphology of macrophages is altered. In addition, Cu-BGC inhibited the degradation metabolism of chondrocytes in the inflammatory environment induced by IL-1ß. CONCLUSIONS These results suggest that Cu-BGC induced macrophage polarization into an M2 type anti-inflammatory phenotype, and inhibition of immune injury response may play a role in delaying cartilage matrix damage in osteoarthritis.