Search Results (131)

Black rockfish (Sebastes schlegelii) is a marine ovoviviparous teleost that exhibits significant sexual dimorphism, with females growing faster and reaching larger sizes than males. Establishing stable oogonial stem cells (OSCs) is critical for understanding germline stem cell dynamics and facilitating all-female breeding. In this study, we successfully isolated and cultured OSCs from S. schlegelii for 12 passages. These cells exhibited alkaline phosphatase activity, expressed germline marker genes (ddx4, cdh1, klf4), and maintained a diploid karyotype (2n = 48). Transcriptomic comparisons between early (P3) and late (P12) passages revealed significant metabolic dysfunction and cell cycle arrest in the late-passage cells. Specifically, the down-regulation of glutathione-related and glycolysis-related genes (gstm3, gstt1, mgst3, gsta1, gsta4, gsto1, gapdh) and key mitotic regulators (cdk1, chk1, cdk4, e2f3, ccne2, ccnb1) suggested that metabolic imbalance contributes to oxidative stress, resulting in cell cycle inhibition and eventual senescence. This study provides a marine fish model for investigating metabolism-cell cycle interactions in germline stem cells and lays the foundation for future applications in germ cell transplantation and all-female breeding. Full article

Caffeine, one of the most widely consumed bioactive compounds worldwide, is gaining recognition for its potential anticancer properties beyond its well-known neurological and metabolic effects. Mechanistically, caffeine exerts anti-tumor activity by modulating key cellular pathways involved in carcinogenesis, including the inhibition of phosphodiesterases, antagonism of adenosine A2A receptors, and disruption of the DNA damage response through ATR-Chk1 pathway inhibition. These actions collectively promote apoptosis, suppress tumor cell proliferation, and impair metastatic spread. In vitro and in vivo studies have demonstrated that caffeine can enhance the cytotoxic effects of chemotherapeutic agents and radiation therapy, suggesting a synergistic role in conventional cancer treatments. Epidemiological data further supports an inverse association between habitual caffeine consumption and the incidence of several cancers, notably liver, colorectal, breast, and prostate cancers. Among these, the most consistent experimental and clinical evidence exists for liver and colorectal cancer, where caffeine’s modulatory effects on inflammation and cell proliferation have been repeatedly observed. Additionally, caffeine’s anti-oxidant and anti-inflammatory properties may contribute to a microenvironment less conducive to tumor initiation and progression. While promising, the anticancer effects of caffeine are influenced by factors such as dosage, individual genetic variability, and cancer type, underscoring the need for further clinical investigation. This review explores the emerging role of caffeine as a potential chemopreventive and adjuvant therapeutic agent in oncology. Full article

Background: Hepatocellular carcinoma (HCC) remains a significant therapeutic challenge due to its complex molecular mechanisms and limited effective treatments. Although the Tibetan medicine Liuwei Muxiang Pill (LWMX) has shown efficacy in gastric and colorectal cancer, no study has yet demonstrated its potential anti-HCC activity. Objective: To evaluate the therapeutic effects of LWMX on HCC. Methods: Integrating network pharmacology, high-resolution mass spectrometry, machine learning, and molecular dynamics simulations, combined with in vitro experiments for mechanism validation. Results: The study identified (Checkpoint kinase 1) CHK1 as a key target of LWMX in HCC regulation and confirmed that quercetin can form a stable complex with CHK1. In vitro experiments demonstrated that LWMX and its active component quercetin significantly inhibit the proliferation and migration of HCC cells. Conclusions: The study establishes CHK1 as a key therapeutic target, confirming the potential of LWMX and its core component quercetin in the treatment of HCC. Full article

Background/Objectives: Melanoma has a rising incidence worldwide. Current treatments are effective, although the development of resistance is common. A novel anti-cancer treatment using checkpoint kinase 1 inhibitor (CHK1i), SRA737, in combination with low-dose hydroxyurea (LDHU), has been demonstrated to effectively kill tumour cells and promote an anti-tumour immune response through the treatment-induced release of pro-inflammatory chemokines and cytokines. These chemokines/cytokines modify the tumour microenvironment from an immunosuppressive to an inflamed state to recruit anti-tumour immune cells. Methods: A panel of human melanoma cell lines was assessed using a panel of chemokines and cytokine expression, and the mechanism of their regulation was investigated. Results: We demonstrate that SRA737 + LDHU upregulates pro-inflammatory chemokines in human melanoma cells in response to SRA737 + LDHU through the ATM-NF-κB signalling pathway. The increased chemokine expression corresponded to the increase in secretion of pro-inflammatory chemokines from tumour cells following SRA737 + LDHU treatment. However, inhibiting NF-κB and ATM did not affect SRA737 + LDHU-induced cell killing. Increased expression of non-NF-κB target genes with SRA737 + LDHU suggests that other transcriptional pathways are also activated and may contribute to the increasing cytokine/chemokine gene expression in response to treatment. Conclusions: SRA737 + LDHU upregulates pro-inflammatory chemokine expression through an ATM-NF-κB-dependent mechanism. Full article

Eliciting DNA damage in tumor cells continues to be one of the most successful strategies against cancer. This is the case for classical chemotherapy drugs and radiotherapy. In the modern era of personalized medicine, this strategy tries to identify specific vulnerabilities found in each patient’s tumor, to inflict DNA damage in certain cell contexts that end up in massive cancer cell death. Cells rely on multiple DNA repair pathways to fix DNA damage, but cancer cells frequently exhibit defects in these pathways, many times being tolerant to the damage. Key vulnerabilities, such as BRCA1/BRCA2 mutations, have been exploited with PARP inhibitors, leveraging synthetic lethality to selectively kill tumor cells and improving patients’ survival. In the DNA damage response (DDR) network, kinases ATM, ATR, Chk1, and Chk2 coordinate DNA repair, cell cycle arrest, and apoptosis. Inhibiting these proteins enhances tumor sensitivity to DNA-damaging therapies, especially in DDR-deficient cancers. Several small-molecule inhibitors targeting ATM/Chk2 or ATR/Chk1 are currently being tested in preclinical and/or clinical settings, showing promise in cancer models and patients. Additionally, pharmacological blockade of ATM/Chk2 and ATR/Chk1 axes enhances the effects of immunotherapy by increasing tumor immunogenicity, promoting T-cell infiltration and activating immune responses. Combining ATM/Chk2- or ATR/Chk1-targeting drugs with conventional chemotherapy, radiotherapy or immune checkpoint inhibitors offers a compelling strategy to improve treatment efficacy, overcome resistance, and enhance patients’ survival in modern oncology. Full article

Background: Esophageal cancer (EC) is the eighth most common cancer and the sixth most common cause of death worldwide. Esophageal squamous cell carcinoma (ESCC) comprises the majority of esophageal cancers globally, and 5-Fluorouraci (5-FU) is one of the commonly used chemotherapeutics for this type of cancer. Chemoresistance to drugs is a main obstacle in the successful treatment of this malignancy. Methods: In this study, we used the CRISPR/Cas9 screening method to determine the target gene related to 5-FU drug resistance in esophageal cancer. Results: Our research findings indicate that the loss of PFKFB3 can increase the resistance of different human esophageal squamous cell carcinoma cell lines to 5-FU through various pathways. Specifically, in KYSE-70 cells, loss of PFKFB3 can induce epithelial–mesenchymal transition (EMT) and prolong the S phase of the cell cycle, allowing cancer cells to evade the effects of 5-FU and develop resistance. In the KYSE-270 and KYSE-150 cell lines, loss of PFKFB3 can upregulate the expression of Slug and Mcl-1, indirectly regulate Chk1 and promote its autophosphorylation, which in turn inhibits apoptosis, thus counteracting the effects of 5-FU. Conclusions: Our research not only enriches our understanding of the biological characteristics of different ESCC cell lines but also provides new clinical insights for future personalized treatments. Assessing the status of PFKFB3 can help predict resistance to 5-FU in ESCC patients with different genetic backgrounds, allowing for more precise treatment planning. This personalized approach has the potential to improve treatment efficacy, reduce unnecessary drug use and side effects, and ultimately improve patient survival rates and quality of life. Full article

Formononetin (FM), an isoflavone with a range of anti-cancer activities, has not been fully elucidated regarding its anti-hepatocellular carcinoma (HCC) mechanisms. Therefore, this study aims to explore the underlying mechanisms of FM using a comprehensive pharmacology model based on computational technologies and omics technology. A network pharmacology approach was applied to detect the components and targets. A mathematical formula was used to evaluate the network contribution index (CI). Bioinformatics analysis was used to analyze clinical data related to HCC targets corresponding to the core component, and molecular docking simulations were conducted to assess binding activity. The results showed that FM induces oxidative DNA damage through ROS generation and triggers G2/M phase cell cycle arrest via the Chk1/Cdc25C/CDK1/CCNB1 signaling pathway. Subsequently, UPLC-MS/MS was applied for the analysis of differential metabolites and the exploration of distinct metabolic pathways. FM limited the synthesis of glutathione, promoted lipid peroxidation, and facilitated the generation of divalent iron. Finally, a colony formation assay, Western blot, and molecular dynamics simulation methods were executed to further validate the metabolomic results. FM exhibited a strong binding affinity for glutathione peroxidase 4 (GPX4). In addition, FM induces ferroptosis by inhibiting the p53/xCT/GPX4 signaling pathway. In vivo, FM could inhibit tumor growth. Conclusions: FM could induce DNA damage leading to cell cycle arrest and may also induce ferroptosis by regulating glutathione metabolism, thereby intervening in the occurrence and development of HCC, making it a promising candidate for HCC treatment. Full article

Medulloblastoma (MB) is one of the most common malignant pediatric brain tumors. Current therapy results in a poor prognosis for high-risk SHH/p53-mutated MB, emphasizing the importance of more effective therapeutic strategies. Here, we investigated the potential radiosensitizing effects of the checkpoint kinase inhibitors (Chk-is) prexasertib (Chk1/2) and SAR-020106 (Chk1) in human SHH/p53-mutated MB in vitro and in vivo. UW228 and DAOY cells were treated with Chk-is and irradiation (RT). Metabolic activity, proliferation, and apoptosis were determined at d3, and long-term clonogenicity was determined at d14. DNA damage was assessed after 1, 24, and 72 h. Patient-derived SHH/p53-mutated, luciferase-transfected MB cells were implanted orthotopically into NSG mice (d0). Fractionated therapy (daily, d7–11) was applied. Body weight (BW) was documented daily, tumor growth weekly, and proliferation at d42. In vitro, Chk-is exhibited a dose-dependent reduction in metabolic activity, proliferation, and clonogenicity and increased apoptosis. A combination of Chk-is with RT enhanced these antitumor effects, including proliferation, apoptosis, and clonogenicity, and increased residual DNA damage compared to RT alone. In vivo, tumor growth was delayed by Chk-is alone. Low-dose prexasertib enhanced RT-induced tumor growth inhibition. High-dose prexasertib and SAR-020106 showed opposite effects, at least at later time points (n = 3). BW assessments revealed that the treatment was well tolerated. Our data indicate a potential benefit of Chk-is in combination with RT in SHH/p53-mutated MB. However, high-dose Chk-is may compromise the RT effect, possibly through anti-proliferative activity. Furthermore, we demonstrate, for the first time, the intracranial antitumor activity of the Chk1-specific inhibitor SAR-020106. Full article

Hepatocellular carcinoma (HCC) cells critically depend on PARP1 and CHK1 activation for survival. Combining the PARP inhibitor (PARPi) olaparib with a CHK1 inhibitor (MK-8776, CHK1i) produced a synergistic effect, reducing cell viability and inducing marked oxidative stress and DNA damage, particularly in the HepG2 cells. This dual treatment significantly increased apoptosis markers, including γH2AX and caspase-3/7 activity. Both HCC cell lines exhibited heightened sensitivity to the combined treatment. The effect of drugs on the expression of proliferation markers in an olaparib-resistant patient-derived xenograft (PDX) model of ovarian cancer was also investigated. Ovarian tumors displayed reduced tissue growth, as reflected by a drop in proliferation marker Ki-67 levels in response to PARPi combined with CHK1i. No changes were observed in corresponding liver tissues using Ki-67 and pCHK staining, which indicates the absence of metastases and a hepatotoxic effect. Thus, our results indicate that the dual inhibition of PARP and CHK1 may prove to be a promising therapeutic approach in the treatment of primary HCC as well as OC tumors without the risk of liver metastases, especially in patients with olaparib-resistant tumor profiles. Full article

Epithelial ovarian cancer (EOC) remains a leading cause of gynecologic cancer mortality. Despite advances in treatment, metastatic progression and resistance to standard therapies significantly worsen patient outcomes. Epithelial–mesenchymal transition (EMT) is a critical process in metastasis, enabling cancer cells to gain invasive and migratory capabilities, often driven by changing miRNA expression involved in the regulation of pathological processes like drug resistance. Targeted therapies like PARP inhibitors (PARPi) have improved outcomes, particularly in BRCA-mutated and DNA repair-deficient tumors; however, resistance and limited efficacy in advanced stages remain challenges. Recent studies highlight the potential synergy of PARPi with DNA damage response (DDR) inhibitors, such as ATR and CHK1 inhibitors, which disrupt cancer cell survival pathways under stress. This study investigated the combined effects of olaparib with ATR and CHK1 inhibitors (ATRi and CHK1i) on migration, invasion, and EMT-related protein expression and miRNA expression in ovarian cancer cell lines OV-90 and SKOV-3. The results demonstrated enhanced cytotoxicity, inhibition of migration and invasion, and modulation of miRNAs linked to metastasis. These findings suggest that combination therapies targeting DNA repair and cell cycle pathways may offer a novel, more effective approach to managing advanced EOC and reducing metastatic spread. Full article

Lung cancer is a leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) is the most prevalent lung cancer subtype. Ciclopirox olamine (CPX), an off-patent fungicide, has been identified as a new anticancer agent. Prexasertib (PRE), a Chk1 inhibitor, is in phase 1/2 clinical trials in various tumors. The anticancer effect of the combination of CPX with PRE on NSCLC cells is unknown. Here, we show that CPX is synergistic with PRE in inhibiting cell proliferation and inducing apoptosis of NSCLC (A549 and A427) cells. Combined treatment with CPX and PRE significantly increased the cell population in the G1/G0 and sub-G1 phases, compared to the single treatment with CPX or PRE. Concurrently, the combined treatment downregulated the protein levels of cyclins (A, B1), cyclin-dependent kinases 4, 6, 2 (CDK4, CDK6, CDK2), cell division cycle 25 B, C (Cdc25B, Cdc25C), and upregulated the protein levels of the CDK inhibitors p21 and p27, leading to decreased phosphorylation of Rb. In addition, the combined treatment increased DNA damage, evidenced by increased expression of γH2AX. In line with this, the combined treatment induced more apoptosis than either single treatment. This was associated with increased expression of DR4, DR5, Fas, and FADD and decreased expression of survivin, resulting in activation of caspase 8 and caspase 3 as well as cleavage of poly (ADP ribose) polymerase (PARP). Taken together, the results suggest that inhibition of Chk1 with PRE can enhance the anticancer activity of CPX at least partly by decreasing cell proliferation and increasing apoptosis in NSCLC cells. Full article

Background: The survival rate of patients with Ewing sarcoma (EWS) has seen very little improvement over the past several decades and remains dismal for those with recurrent or metastatic disease. HDAC2, ALK, JAK1, and CDK4 were identified as potential targets using RNA sequencing performed on EWS patient tumors with the bioinformatic analysis of gene expression. Methods/Results: The pan-HDAC inhibitor Panobinostat was cytotoxic to all the Ewing sarcoma cell lines tested. Mechanistically, Panobinostat decreases the expression of proteins involved in the cell cycle, including Cyclin D1 and phospho-Rb, and DNA damage repair, including CHK1. Further, Panobinostat induces a G1 cell cycle arrest. The combination of Panobinostat with Doxorubicin or Etoposide, both of which are used as standard of care in upfront treatment, leads to a synergistic effect in EWS cells. The combination of Panobinostat and Doxorubicin induces an accumulation of DNA damage, a decrease in the expression of DNA damage repair proteins CHK1 and CHK2, and an increase in caspase 3 cleavage. The addition of Panobinostat to standard-of-care chemotherapy combinations significantly reduces cell viability compared to that of chemotherapy alone. Conclusions: Overall, our data indicate that HDAC2 is overexpressed in many EWS tumor samples and HDAC inhibition is effective in targeting EWS cells, alone and in combination with standard-of-care chemotherapy agents. This work suggests that the addition of an HDAC inhibitor to upfront treatment may improve response. Full article

Radiation therapy continues to be the cornerstone treatment for malignant brain tumors, the majority of which express wild-type p53. Therefore, the identification of drugs that promote the ionizing radiation (IR)-induced activation of p53 is expected to increase the efficacy of radiation therapy for these tumors. The growth inhibitory effects of CEP-1347, a known inhibitor of MDM4 expression, on malignant brain tumor cell lines expressing wild-type p53 were examined, alone or in combination with IR, by dye exclusion and/or colony formation assays. The effects of CEP-1347 on the p53 pathway, alone or in combination with IR, were examined by RT-PCR and Western blot analyses. The combination of CEP-1347 and IR activated p53 in malignant brain tumor cells and inhibited their growth more effectively than either alone. Mechanistically, CEP-1347 and IR each reduced MDM4 expression, while their combination did not result in further decreases. CEP-1347 promoted IR-induced Chk2 phosphorylation and increased p53 expression in concert with IR in a Chk2-dependent manner. The present results show, for the first time, that CEP-1347 is capable of promoting Chk2-mediated p53 activation by IR in addition to inhibiting the expression of MDM4 and, thus, CEP-1347 has potential as a radiosensitizer for malignant brain tumors expressing wild-type p53. Full article

Checkpoint kinases 1 and 2 (CHK1 and CHK2) are enzymes that are involved in the control of DNA damage. At the present time, these enzymes are some of the most important targets in the fight against cancer since their inhibition produces cytotoxic effects in carcinogenic cells. This paper proposes the use of spirostans (Sp), natural compounds, as possible inhibitors of the enzymes CHK1 and CHK2 from an in silico analysis of a database of 155 molecules (S5). Bioinformatics studies of molecular docking were able to discriminate between 13 possible CHK1 inhibitors, 13 CHK2 inhibitors and 1 dual inhibitor for both enzymes. The administration, distribution, metabolism, excretion and toxicity (ADMETx) studies allowed a prediction of the distribution and metabolism of the potential inhibitors in the body, as well as determining the excretion routes and the appropriate administration route. The best inhibition candidates were discriminated by comparing the enzyme-substrate interactions from 2D diagrams and molecular docking. Specific inhibition candidates were obtained, in addition to studying the dual inhibitor candidate and observing their stability in dynamic molecular studies. In addition, Highest Occupied Molecular Orbital—Lowest Unoccupied Molecular Orbital (HOMO-LUMO) interactions were analyzed to study the stability of interactions between the selected enzymes and spirostans resulting in the predominant gaps from HOMOCHKs to LUMOSp (Highest Occupied Molecular Orbital of CHKs—Lowest Unoccupied Molecular Orbital of spirostan). In brief, this study presents the selection inhibitors of CHK1 and CHK2 as a potential treatment for cancer using a combination of molecular docking and dynamics, ADMETx predictons, and HOMO-LUMO calculation for selection. Full article

Ovarian cancer is the most fatal of all the reproductive cancers within the female population, mainly due to its late diagnosis that limits surgery and medical treatment. Classically, ovarian cancer therapy has included conventional chemotherapy, and other therapeutic approaches are now being used to treat these patients, but the outcomes of the disease are still poor. Therefore, new strategies are needed to improve life expectancy and life quality of ovarian cancer patients. Considering that, we investigated the effect of the nutritional supplement Ocoxin Oral Solution (OOS) in ovarian cancer models. OOS contains several nutritional supplements, some of them with demonstrated antitumoral action. In vitro studies showed that OOS inhibited the proliferation of several ovarian cancer cell lines, especially of those representative of the endometrioid subtype, in a time- and dose-dependent manner. A fast cell death induction after OOS treatment was observed, and when the molecular mechanisms leading to this effect were investigated, an activation of the DNA damage checkpoint was detected, as shown by activation (phosphorylation) of CHK1 and CHK2 kinases that was followed by the phosphorylation of the target protein histone H2AX. When tested in animal models of ovarian cancer, OOS reduced tumor growth without any observed secondary effects. Moreover, such reduction in tumor proliferation was caused by the induction of DNA damage as corroborated by the in vivo phosphorylation of CHK2 and Histone H2AX. Finally, OOS potentiated the action of carboplatin or olaparib, the standard of care treatments used in ovarian clinics, opening the possibility of including OOS in combination with those standard of care agents in patients with ovarian cancer. Full article