Cell Cycle Analysis Research Articles

Enhancing Effects of Olaparib by Alpha- and Beta-Emitting Radionuclides, X-Rays, and Ultraviolet A Light in Combination with Ortho-IodoHoechst in a Prostate Cancer Cell Model

Background: New therapeutic strategies for metastatic castration-resistant prostate cancer (mCRPC) have been developed in the past to achieve the best response rates. Most recently, the use of combination therapies has been explored to optimize patient outcomes. Poly(ADP-ribose) polymerase inhibitors (PARPi) may help to treat mCRPC more effectively. Objectives: This study aimed to determine whether the combination of a PARPi with different radiation qualities results in different levels of radiosensitization of PC-3 cells. Methods: The radiosensitizing potential of Olaparib in combination with 177Lu, 223Ra, X-rays and photodynamic therapy (PDT) using the UVA light-activated photosensitizer ortho-iodoHoechst33258 (oIH) was evaluated by determining the clonogenic survival, DNA damage and cell cycle analysis. Results: Here, we show that this combination strategy differentially sensitized PC-3 cells to different radiation qualities. The combination of 177Lu with Olaparib increased the numbers of persistent double-strand breaks (DSBs) by a factor of 3.3 and cell death in PC-3 cells. Overall, the β-emitter 177Lu indicated a higher radiosensitization efficacy compared to 223Ra, with X-rays corresponding to dose modification factors (DMF) of 1.77, 1.17 and 1.16 respectively. Even in the case of the α-emitter 223Ra, the effects were much less pronounced than for 177Lu. PARPi also showed a slight potentiation of the cytotoxic effects both in co-treatment with X-rays and with PDT. Conclusions: The results of our study indicate a potential role for Olaparib in further optimizing the PSMA radioligand therapy (PRLT) outcomes. However, further evaluation of the combination of PARPi with PRLT is needed to gain more insights into improving the benefit to patients suffering from mCRPC.

Investigation of Apoptotic and Anticancer Effects of 2-substituted Benzothiazoles in Breast Cancer Cell Lines: EGFR Modulation and Mechanistic Insights.

Benzothiazole derivatives, a class of heterocyclic compounds, exhibited diverse biological activities influenced by substituents in the thiazole ring. This study aimed to synthesize these compounds with two functional groups to investigate their potential as anticancer agents, particularly against breast cancer. While previous research demonstrated the efficacy of 2-substituted benzothiazoles against glioma and cervical and pancreatic cancer cells, there is a gap in studies targeting breast cancer. The synthesized compounds were tested in vitro using MCF-7, MDA-MB-231, and MCF-10A cell lines, with Doxorubicin as the positive control. Various assays were conducted, including Annexin V/PI, cell cycle analysis, wound healing, and measurement of mitochondrial membrane potential. Protein expression of EGFR and transcription levels of apoptosis-related genes (Bax and Bcl-xL) and cancer progression-related genes (JAK, STAT3, ERK, AKT, mTOR) were analyzed. Additionally, the balance between antioxidants and oxidants was evaluated by measuring TAS and TOS levels. Our findings revealed that benzothiazole compounds significantly inhibited breast cancer cell growth by reducing cell motility, disrupting mitochondrial membrane potential, and inducing cell cycle arrest in the sub-G1 phase. These compounds increased reactive oxygen species accumulation, leading to cell death. Furthermore, they decreased EGFR protein levels, increased Bax gene transcription, and downregulated the expression of genes such as JAK, STAT3, ERK, AKT, and mTOR. In conclusion, benzothiazole derivatives exhibited potent inhibitory effects on breast cancer in vitro by promoting apoptosis, downregulating EGFR activity, and modulating key signaling pathways, including JAK/STAT, ERK/MAPK, and PI3K/Akt/mTOR. These results highlighted the potential of benzothiazole derivatives as novel therapeutic agents for breast cancer treatment.

The efficacy of infrared diode laser in enhancing the regenerative potential of human periodontal ligament stem cells (hPDLSCs)

BackgroundThe present study aimed to investigate the effects of infrared diode laser irradiation on the proliferation and differentiation capacity of periodontal ligament stem cells (hPDLSCs), which are optimal cell sources for periodontal regeneration.MethodshPDLSCs were isolated and characterized by flow cytometric analysis of mesenchymal stem cell markers, and their trilineage differentiation capacity was tested. hPDLSCs were then cultured and irradiated with infrared diode laser (970 nm) at a power of 200 mW and a fluence of 4 J/cm2 for 3 s. MTT assay was performed to assess cellular proliferation. Cell cycle analysis was performed, and the impact of infrared diode laser irradiation on the stemness and osteogenic differentiation potential of hPDLSCs was evaluated via RT‒PCR.ResultsInfrared diode laser application enhanced the stemness, viability, proliferation, and differentiation of PDLSCs. Stem cell markers (OCT4, SOX2, and NANOG) were significantly upregulated in hPDLSCs exposed to laser irradiation. There was significant overexpression of RUNX2, ALP, OPN, and OCN on day 14 after laser application.ConclusionsThese findings provide valuable insights into the specific applications of infrared diode lasers to effectively regenerate periodontal tissues. The results can aid in the development of precise clinical protocols aimed at enhancing osseointegration and promoting tissue regeneration. Ultimately, the combination of infrared diode laser with hPDLSCs is promising for stimulating periodontal regeneration.

Curcumin protects porcine granulosa cells and mouse ovary against reproductive toxicity of aflatoxin B1 via PI3K/AKT signaling pathway

Aflatoxin B1 (AFB1) is a widespread food contaminant with toxic effects on female reproductive system. This concerns the human public health and the development of the livestock industry. Effective and feasible measures against reproductive toxicity of AFB1 are also unknown. Curcumin has been shown to improve animal production performance and alleviate toxicity of AFB1 in other tissues. The study aimed to reveal the potential mechanism of curcumin in alleviating AFB1 exposure-triggered reproductive toxicity in porcine. Porcine GCs and matured oocytes in vitro and mouse ovary in vivo were cultured as model and then exposed to AFB1 and curcumin. The transcriptomics for porcine GCs was performed. The apoptosis, cell cycle, ROS content and mitochondrial membrane potential level analysis were determined using probe staining and flow cytometry. QRT-PCR and western blotting were analyzed for the genes expression. Oocytes maturation and parthenogenetic activation were performed to evaluate the quality of oocytes. The data showed that AFB1 exposure significantly inhibited porcine GCs viability and exhibited concentration-dependent relationship from 1 to 16 μM. Curcumin supplementation efficiently reversed the inhibition of cell viability. Further analysis indicated that curcumin application alleviated AFB1-exposed porcine GCs and mouse ovary mitochondria dysfunction, oxidative stress, cell cycle arrest, and apoptosis. We found that PI3K/Akt signaling pathway plays a vital role in AFB1-induced porcine GCs toxicity through bioinformatic analysis. The data not only confirmed the correlation of PI3K/Akt signaling pathway and oxidative stress, cell cycle arrest and apoptosis, but also consistent with the results that curcumin supplementation could markedly activate the PI3K/Akt signal inhibited by AFB1. Moreover, curcumin reversed AFB1-impaired cumulus-oocyte complex expansion, oocytes maturation rate, blastocyst rate and formation. Our study demonstrated that curcumin supplementation can protect porcine GCs and mouse ovary from toxicity of AFB1 by targeting the PI3K/AKT signaling pathway and provides a viable treatment approach for AFB1-induced female reproductive toxicity.

ZSTK474 targeting PIK3R3 inhibits the Wilms' tumor through G0 / G1 phase arrest.

Wilms' tumor (WT), also known as nephroblastoma, is the predominant form of primary malignant renal cancer. The unfavorable prognoses linked to anaplastic nephroblastoma and recurrent nephroblastoma emphasize the crucial requirement for the exploration of innovative treatment modalities for WT. Our study conducted one-way Cox regression and Kaplan-Meier analyses using TARGET-WT nephroblastoma data to identify differentially expressed genes in nephroblastoma and evaluate their prognostic relevance. Utilizing the Connectivity Map database, ZSTK474 emerged as a viable therapeutic option for WT. The effect of ZSTK474 on WT and related underlying mechanisms were further investigated through in vitro and in vivo investigations. The in vivo experiment results indicated that ZSTK474 effectively inhibited subcutaneous tumor growth in WT mice. CCK-8 assays revealed two nephroblastoma cell lines exhibited half-inhibitory concentrations of 2μM and 2.51μM for ZSTK474, respectively. ZSTK474 was shown to inhibit the migration and invasion capabilities of WT cells in both Transwell and wound healing assays. Flow cytometry apoptosis and TUNEL assays demonstrated that ZSTK474 induced apoptosis in WT cells. Cell cycle analysis revealed that ZSTK474 led to the induction of G0/G1 phase arrest. Sequencing of ZSTK474-treated WiT49 cells suggested that the impact of ZSTK474 on WT might be mediated by the PI3K/Akt pathway, specifically by inhibiting PIK3R3. Knock-down of PIK3R3 confirmed that ZSTK474 downregulated PIK3R3, reducing Akt phosphorylation, cyclin D and CDK4 levels and elevating P21 expression in nephroblastoma cells. However, current research has limitations, including a lack of understanding of the long-term effects and potential resistance mechanisms of new therapies. This research provides insight into the potential of ZSTK474 and other PI3K inhibitors for treating nephroblastoma.

The molecular anti-metastatic potential of CBD and THC from Lebanese Cannabis via apoptosis induction and alterations in autophagy

The medicinal plant Cannabis sativa L. (C. sativa) is currently being extensively studied to determine the full extent of its therapeutic pharmacological potential. Δ9-tetrahydocannabinol (THC) and cannabidiol (CBD) are the most thoroughly investigated compounds. We aimed to explore the anticancer activity of cannabinoids mixture isolated from the Lebanese C. sativa plant in ratios comparable to the local medicinal plant, to elucidate its mechanism of action in breast cancer cells in vitro. Cells were subjected to cytotoxicity assay, cell cycle analysis, Annexin V/PI dual staining, cell death ELISA, immunofluorescence, in addition to western blot analysis of apoptotic and autophagy markers. We further evaluated the anti-metastatic effect of cannabinoids on MDA-MB-231 using the scratch wound-healing, trans-well migration and invasion assays. Our results revealed the promising therapeutic benefits of CBD/THC on inhibiting the growth of breast cancer cells by promoting cellular fragmentation, phosphatidylserine translocation to the outer membrane leaflet and DNA fragmentation in both cell lines while inhibiting the motility of the triple negative breast cancer cells. In our study, CBD/THC mixture was found to exhibit a pro-apoptotic activity via the activation of the mitochondrial apoptotic pathway, independent from ROS production while also suggesting the activation of a caspase-dependent apoptotic pathway. Even though autophagy was altered upon exposure to the cannabinoid mixture, our data suggested that it is not the mechanism responsible of inducing cell death. In conclusion, our study demonstrates the promising therapeutic benefits of CBD and THC isolated from the Lebanese C. sativa plant on breast cancer cells in vitro.

Nimbolide Induces Cell Apoptosis via Mediating ER Stress-Regulated Apoptotic Signaling in Human Oral Squamous Cell Carcinoma.

Human oral squamous cell carcinoma (OSCC) poses a significant health challenge in Asia, with current therapeutic strategies failing to improve the survival rates for OSCC patients sufficiently. To elucidate the effects of Nimbolide on OSCC cell proliferation and apoptosis, we performed a series of experiments, including cell proliferation assays, annexin V/PI assays, and cell cycle analysis. We further investigated nimbolide's role in modulating endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) production, and mitochondrial dysfunction using flow cytometry. Additionally, Western blotting was used to detect apoptosis-related protein expression. Our findings reveal that nimbolide exerts its anti-proliferative effects on OSCC cells by inducing apoptosis. The nimbolide increased intracellular ROS levels and acceleration of cellular calcium accumulation, respectively promoting endoplasmic reticulum stress and cancer cell apoptosis. Furthermore, nimbolide activates the caspase cascade by altering the mitochondrial membrane potential and apoptotic protein expression, thereby inhibiting the viability of tumor cells. Our data show that Nimbolide suppresses tumor growth through the induction of ROS production, ER stress, and mitochondrial dysfunction, resulting in apoptosis in OSCC cells. Overall, our study highlights nimbolide as a potential natural compound for OSCC therapy.

The cyclin-G associated kinase (GAK) is a novel mitotic kinase and therapeutic target in diffuse large B-cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma and the most frequently diagnosed hematologic malignancy in the United States. DLBCL exhibits significant molecular and clinical heterogeneity, and at least a third of patients are left uncured with standard frontline chemoimmunotherapy. As such, there is a critical need to identify novel targeted therapies to improve outcomes. We conducted a phenotypic screen of kinase inhibitors against DLBCL cell lines and non-malignant controls. We identified the cyclin G-associated kinase (GAK) as a tumor-selective, readily druggable target whose inhibition killed DLBCL cell lines, while sparing non-malignant blood cells. Upon investigation of GAK's cellular function, we discovered that inhibition results in G2/M-phase cell cycle arrest. Immunofluorescent confocal microscopy revealed significant chromosome misalignment and spindle distortion in DLBCL cells following GAK-inhibition, disrupting progression through mitosis. Analysis of RNA-seq data from clinical samples showed increased GAK expression associates strongly with RB1 deficiency in DLBCL cases, suggesting dependency on GAK for proper mitotic progression linked to retinoblastoma associated protein (RB) loss of function, a common DLBCL driver. In cell-cycle analyses and under microscopy, RB-deficient DLBCL cells treated with an exquisitely selective GAK inhibitor showed complete arrest at G2/M, pronounced distortion of mitotic spindles, and widespread chromosomal damage. Finally, in vivo studies of DLBCL xenograft-bearing NSG mice achieved a dramatic tumor-burden reduction in response to targeted GAK inhibition. These results reveal a novel cell cycle kinase suitable for therapeutic exploitation in DLBCL patients and linked to the common, undruggable biomarker of RB loss of function.

IKKα affects the susceptibility of primary human osteoarthritis chondrocytes to oxidative stress-induced DNA damage by tuning autophagy

The functional derangement affecting human chondrocytes during osteoarthritis (OA) onset and progression is sustained by the failure of major homeostatic mechanisms. This makes them more susceptible to oxidative stress (OS), which can induce DNA damage responses and exacerbate stress-induced senescence. The knockdown (KD) of IκB kinase α (IKKα), a dispensable protein in healthy articular cartilage physiology, was shown to increase the survival and replication potential of human primary OA chondrocytes. Our recent findings showed that the DNA Mismatch Repair pathway only partially accounts for the reduced susceptibility to OS of IKKαKD cells. Here we therefore investigated other ROS-mediated DNA damage and repair mechanisms. We exposed IKKαWT and IKKαKD chondrocytes to sub-cytotoxic hydrogen peroxide and evaluated the occurrence of double-strand breaks (DSB), 8-oxo-2′-deoxyguanosine (8-oxo-dG) and telomere shortening. ROS exposure was able to significantly increase the number of γH2AX foci (directly related to the number of DSB) in both cell types, but IKKα deficient cells undergoing cell division were able to better recover compared to their IKKα proficient counterpart. 8-oxo-dG signal proved to be the highest DNA damage signal among those investigated, located in the mitochondria and with a slightly higher intensity in IKKα proficient cells immediately after OS exposure. Furthermore, ROS significantly reduced telomere length both in IKKαWT and IKKαKD, with the former showing more pervasive effects, especially in dividing cells. Assessment of the HIF-1α>Beclin-1>LC3B axis after recovery from OS showed that IKKα deficient cells exhibited a more efficient autophagic machinery that allowed them to better cope with oxidative stress, possibly through the turnover of damaged mitochondria. Higher Beclin-1 levels likely helped in rescuing dividing cells (identified by coupled cell cycle analysis) because of Beclin-1's involvement in both autophagy and mitotic spindle organization. Therefore, our data further confirm the higher capacity of IKKαKD chondrocytes to cope with oxidative stress-induced DNA damage.

Anticancer Effect of Cycas media: Molecular Basis Through Modulation of PI3K/AKT/mTOR Signaling Pathway

Many researchers are focusing on screening the biological activities of plants owing to their safety and possible pharmacological actions. Consequently, we aimed to explore the antiproliferative and cytotoxic properties of Cycas media methanolic extract on HepG2 cell lines. Moreover, we also explore the antitumor action against the experimentally induced solid Ehrlich carcinoma (SEC) model and investigate the possible involved molecular mechanisms. Also, the antibacterial action of the extract was elucidated. Different concentrations of the extract were incubated with HepG2 to determine cytotoxicity, followed by cell cycle analysis. The in vivo experiment was accomplished by grouping the animals into four different groups (n = 10); normal control, SEC, C. media 100, and C. media 200. The extract was administered at 100 and 200 mg/kg. Tumor volume, tumor inhibition rate, toxicity profile, and antioxidant biomarkers were determined. Moreover, the PI3K/AKT/mTOR signaling pathway was investigated as a possible underlying antitumor mechanism. The tumor control group showed a remarkable upregulation for PI3K, p-AKT, and p-mTOR, along with downregulation for the antioxidant SOD and GPX4, as well as decreased levels of GSH and MDA. C. media extract reversed these parameters to a significant level and the higher dose showed a superior antitumor effect. C. media extract showed antiproliferative effects against HepG2 cells, along with a suppressive action on the PI3K/AKT/mTOR pathway and an antioxidant effect. Additionally, C. media had antibacterial consequences against S. aureus isolates with minimum inhibitory concentrations from 32 to 128 µg/mL. It also caused a noteworthy growth delay as well as a notable reduction in the membrane integrity of S. aureus isolates. These beneficial outcomes suggest C. media to have potential antitumor and antibacterial activities.

Neurosurgical application of olaparib from a thermo-responsive paste potentiates DNA damage to prolong survival in malignant glioma.

There is increased pan-cancer specific interest in repurposing the poly adenosine diphosphate-ribose polymerase-1 (PARP-1) inhibitor, olaparib, for newly diagnosed or recurrent isocitrate dehydrogenase wild type glioblastoma. We explore whether intra-cavity delivery of olaparib confers a survival benefit in a pre-clinical high-grade glioma model. Primary tumor RNA sequencing data was used to determine PARP-1 as a target in the glioblastoma infiltrative margin. We assessed radiosensitization conferred by olaparib alone and concomitant to genotoxic insults in vitro using clonal growth assays, cell cycle analysis and immunocytochemistry, and in vivo upon post-surgical delivery from a temperature-sensitive polymeric paste. RNA-sequencing confirmed PARP-1 as a viable therapy target in glioblastoma infiltrative disease. Acute exposure of glioma cells to olaparib impaired proliferation and induced late-stage apoptosis associated with DNA damage in vitro, potentiated by radiation. Using high-grade glioma orthotopic allografts, a long-term overall survival benefit was observed upon interstitial olaparib delivery concomitant with radiotherapy, compared to systemic olaparib and standard glioblastoma treatment. Combined delivery of olaparib with either temozolomide or etoposide increased long-term survival, suggestive of olaparib functioning as DNA damage sensitizer. Collectively, our data support a rationale for localized olaparib delivery concomitant with the current clinical regimen for malignant glioma treatment.

Advantages of Cell Proliferation and Immune Regulation in CD146+NESTIN+ HUMSCs: Insights from Single-Cell RNA Sequencing.

The heterogeneity of stem cells is a significant factor inhibiting their clinical application, as different cell subpopulations may exhibit substantial differences in biological functions. We performed single-cell sequencing on HUMSCs from three donors of different gestational ages (22+5 weeks, 28 weeks, 39 weeks). We also compared the data with single-cell sequencing data from BMSCs from two public databases. The content of CD146+Nestin+ MSCs in preterm HUMSCs (22+5W: 30.2%, 28W: 25.8%) was higher than that in full-term HUMSCs (39W: 0.5%) and BMSCs (BMSC1: 0, BMSC2: 0.9%). Cell cycle analysis indicated a higher proportion of cells in the proliferative G2M phase in CD146+Nestin+ MSCs (40.8%) compared to CD146+Nestin- MSCs (20%) and CD146-Nestin- MSCs (12.5%). Degree of differentiation assessment suggested that CD146+Nestin+ MSCs exhibited lower differentiation than other cell subpopulations. Differential gene analysis revealed that CD146+Nestin+ MSCs overexpressed immune regulation-related factors. GO and KEGG enrichment analysis of modules identified by WGCNA suggested enrichment in pathways related to cellular immune regulation, antimicrobial activity, and proliferation. Immune-related gene analysis indicated that CD146+Nestin+ MSCs exhibited expression of multiple immune-related genes associated with "Antimicrobials," "Cytokines," and "Cytokine Receptors." Gene regulatory network analysis revealed high expression of immune-related regulators RELB, GAPB1, and EHF in CD146+Nestin+ MSCs.Our study provides a single-cell atlas of preterm HUMSCs, demonstrating the expression of CD146+Nestin+ MSCs across different tissues and confirming their advantages in cellular proliferation, antimicrobial activity, immune regulation, and low differentiation at the RNA level. This contributes valuable insights for the clinical application of HUMSCs.

Protective Effects of Nettle Tea on SKOV-3 Ovarian Cancer Cells Through ROS Production, Apoptosis Induction, and Motility Inhibition Without Altering Autophagy.

Urtica dioica L. (UD), also known as the stinging nettle, has long been used in traditional medicine for its wide range of health benefits. The current study focuses on the effect of nettle tea on the growth and proliferation of one of the most aggressive ovarian adenocarcinoma cell line, SKOV-3 cells. To examine this, cytotoxicity, cell cycle analysis, and ROS assays were performed, along with Annexin V/PI dual staining, cell death ELISA, Western blot analysis, and motility assays. The results showed that a UD aqueous extract (UDAE) can inhibit the growth and proliferation of SKOV-3 cells in a dose- and time-dependent manner by promoting cellular fragmentation. This was accompanied by an increase in two apoptotic hallmarks, the flipping of phosphatidylserine to the outer membrane leaflet and DNA fragmentation as revealed by cell death ELISA. This aqueous extract showed a pro-oxidant activity while also activating the extrinsic caspase-dependent apoptotic pathway with no alteration in autophagy markers. Furthermore, the extract showed promising inhibitory effect on the migratory capacities of aggressive ovarian cancer cells, in vitro.

The proliferation and viability of human periodontal ligament stem cells cultured on polymeric scaffolds can be improved by low-level laser irradiation.

This study assessed the impact of low-level laser irradiation on the viability and proliferation of human periodontal ligament stem cells (hPDLSCs) cultivated on polylactic acid (PLA) scaffolds. hPDLSCs were obtained, characterized, and grown on the surface of PLA films produced via the solvent casting technique. The study involved two groups: the control group, which was not exposed to radiation, and the laser group, which was irradiated with a diode laser (InGaAIP) with a power of 30 mW, a wavelength of 660 nm, and a single dose of 1 J/cm² emitted continuously. Cell viability was assessed 24 and 48 hours after irradiation using the Alamar blue and Live/Dead assays. Flow cytometry was used to assess cell cycle events, and scanning electron microscopy (SEM) was used to evaluate the interaction between cells and the biomaterial. The results revealed a statistically significant increase in cell metabolic activity in the laser group compared with the control group at 24 hours (p <0.05) and 48 hours (p <0.001), as indicated by the Alamar blue assay. The Live/Dead assay also revealed a greater density of viable cells in the laser group. The cell cycle analysis revealed a significant increase in the number of cells in the proliferative phase (G2/M) in the laser group compared with the control group (p <0.001). The SEM images demonstrated that the irradiated group had a greater concentration of cells while still maintaining their cell shape and projections. This study demonstrated that photobiomodulation can increase the viability and proliferation of periodontal stem cells cultured on PLA scaffolds, suggesting the potential of this protocol for future studies on periodontal tissue engineering.

Silencing of ERRα gene represses cell proliferation and induces apoptosis in human skin fibroblasts.

Estrogen‑related receptor (ERR) is an orphan nuclear receptor structurally akin to the estrogen receptor. ERR is expressed in tissues with active energy metabolism and regulates intracellular metabolic functions. Additionally, ERRs are known to be strongly expressed in the epidermis of skin tissue, but their functions are unknown. The present study investigated the function of ERRα in human skin fibroblasts. ERRα expressed in human dermal fibroblast TIG113 was knocked down using small interfering (si)RNA and gene expression was comprehensively analyzed using microarrays 48 h later. Pathway analysis was performed using Wikipathways on genes exhibiting expression changes of ≥1.5‑fold. Expression of cell cycle‑related and apoptosis‑related genes was compared using reverse transcription‑quantitative PCR. After treating TIG113 cells with siERRα for 72 h, cell proliferation was assessed using the Cell Counting Kit‑8 or a scratch wound healing assay and apoptotic cells were measured using the Poly Caspase Assay Kit. Cell cycle analysis was performed using flow cytometry. The expression of the ERRα gene was suppressed by siRNA. The expression of genes associated with cell cycle‑related pathways were decreased while that of those associated with apoptosis‑related pathways increased. Furthermore, the expression of cell cycle‑related genes such as cell division cycle 25C, cyclin E and cyclin B1 was decreased and the expression of apoptosis‑related genes such as caspase3 and Fas cell surface death receptor was increased. Cell proliferation was suppressed and the number of apoptotic cells increased ~2‑fold in ERRα‑knockdown TIG113 cells. Cell cycle analysis revealed that the number of cells in the Sub‑G1 phase increased and that in the S and G2/M phases decreased. The present study suggested that ERRα is an essential for the survival of human skin fibroblasts.

DNA hypermethylation of COL4A1 in ultraviolet-B-induced age-related cataract models in vitro and in vivo.

To explore the DNA methylation of COL4A1 in ultraviolet-B (UVB)-induced age-related cataract (ARC) models in vitro and in vivo. Human lens epithelium B3 (HLEB3) cells and Sprague Dawley rats were exposure to UVB respectively. The MTT assay was utilized to evaluate cell proliferation. Flow cytometry was employed for analysis of cell apoptosis and cell cycle. COL4A1 expression in HLEB3 cells and anterior lens capsules were assessed using Western blot and reverse transcription-polymerase chain reaction (RT-PCR). The localization of COL4A1 in HLEB3 cells was determined by immunofluorescence. The methylation status of CpG islands located in COL4A1 promoter was verified using bisulfite-sequencing PCR (BSP). DNMTs and TETs mRNA levels was examined by RT-PCR. UVB exposure decreased HLEB3 cells proliferation, while increased the apoptosis rate and cells were arrested in G0/G1 phase. COL4A1 expression was markedly inhibited in UVB treated cells compared to the controls. Hypermethylation status was detected in the CpG islands within COL4A1 promoter in HLEB3 cells subjected to UVB exposure. Expressions of DNMTs including DNMT1/2/3 were elevated in UVB treated HLEB3 cells compared to that in the controls, while expressions of TETs including TET1/2/3 showed the opposite trend. Results from the UVB treated rat model further confirmed the decreased expression of COL4A1, hypermethylation status of the CpG islands at promoter of COL4A1 and abnormal expression of DNMT1/2/3 and TET1/2/in UVB exposure group. DNA hypermethylation of COL4A1 promoter CpG islands is correlated with decreased COL4A1 expression in UVB induced HLEB3 cells and anterior lens capsules of rats.

Antagonist of Growth Hormone-Releasing Hormone Receptor MIA-690 Suppresses the Growth of Androgen-Independent Prostate Cancers

The development of resistance remains the primary challenge in treating castration-resistant prostate cancer (CRPC). GHRH receptors (GHRH-R), which are coupled to G-proteins (GPCRs), can mediate EGFR transactivation, offering an alternative pathway for tumour survival. This study aimed to evaluate the effects of the GHRH-R antagonist MIA-690, in combination with the EGFR inhibitor Gefitinib, on cell viability, adhesion, gelatinolytic activity, and the cell cycle in advanced prostate cancer PC-3 cells. The findings demonstrate a synergistic effect between MIA-690 and Gefitinib, leading to the inhibition of cell viability, adhesion, and metalloprotease activity. Cell cycle analysis suggests that both compounds induce cell cycle arrest, both individually and in combination. Furthermore, similar effects of the GHRH-R antagonist MIA-690 combined with Gefitinib were observed in PC-3 tumours developed by subcutaneous injection in athymic nude mice 36 days post-inoculation. These results indicate that combined therapy with a GHRH-R antagonist and an EGFR inhibitor exerts a stronger antitumor effect compared to monotherapy by preventing transactivation between EGFR and GHRH-R in CRPC.

New S-substituted-3-phenyltetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one scaffold with promising anticancer activity profile through the regulation and inhibition of mutated B-RAF signaling pathway.

Novel 3-phenyltetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives were synthesized and screened for their antiproliferative activity against a panel of 60 cancer cell lines. Derivatives 5b, 5f, and 9c showed significant antitumor activity at a single dose with mean growth inhibition of 55.62%, 55.79%, and 71.40%, respectively. These compounds were further investigated against HCT-116, colon cancer cell line, and FHC, normal colon cell line. Compound 9c showed the highest activity with IC50 = 0.904 ± 0.03 µM and SI = 20.42 excelling doxorubicin which scored IC50 = 2.556 ± 0.09 µM and SI = 6.19. Compound 9c was also the most potent against B-RAFWT and mutated B-RAFV600E with IC50 = 0.145 ± 0.005 and 0.042 ± 0.002 µM, respectively in comparison with vemurafenib with IC50 = 0.229 ± 0.008 and 0.038 ± 0.001 µM, respectively. The cell cycle analysis showed that 9c increased the cell population and induced an arrest in the cell cycle of HCT-116 cancer cells at the G0-G1 stage with 1.23-fold. Apoptosis evaluation showed that compound 9c displayed an 18.18-fold elevation in total apoptosis of HCT-116 cancer cells in comparison to the control. Compound 9c increased the content of caspase-3 by 3.52-fold versus the control. A molecular modeling study determined the binding profile and interaction of 9c with the B-RAF active site.

Cell-cycle phase progression analysis identifies three unique phenotypes in soft tissue sarcoma

PurposeLoddo et al. (Br J Cancer 100:959–70, 2009) established the prognostic significance of cell cycle markers and "Cell-Cycle Phenotypes" in breast carcinoma. This study aims to 1) identify prognostic cell-cycle markers in sarcoma, and 2) assess the prognostic potential of specific cell-cycle phenotypes in sarcoma.MethodsTissue samples from 128 soft tissue sarcomas were stained for four cell cycle-specific markers: Mcm2, Geminin, Plk1, and H3S10ph. Only primary soft tissue tumors (liposarcoma, leiomyosarcoma, synovial sarcoma, and undifferentiated pleomorphic sarcoma) were included in the analysis. Any tumor coming from a recurrent or metastatic lesion were excluded from the analysis. Three cell-cycle phenotypes (I, II, III) were derived from marker expression patterns. Prognostic significance was evaluated in a subset of primary soft tissue sarcomas using Cox regression for survival analysis.ResultsCompared to phenotype I, the phenotype III tumors had a decreased 5-year overall survival (HR 6.81 [2.36–19.61]; p = < 0.001), 5-year disease-free survival (HR 1.07 (1.02–1.18); p = 0.004), and 5-year metastasis-free survival (HR 4.34 [1.58–11.93]; p = 0.004).High expression of Plk1 was associated with decreased 5-year overall survival (HR: 4.04 CI [1.21–6.67; p = 0.02) and 5-year metastasis-free survival (HR: 2.91 CI [1.15–7.37]; p = 0.03). Geminin was also found to have a decreased 5-year overall survival (HR:2.84 CI [1.21–6.67]; p = 0.02). No statistical difference in prognostication were noted between phenotypes and the AJCC system.ConclusionsWe identified three unique sarcoma cell cycle phenotypes that have prognostic significance. This performs similarly to the AJCC staging system.

Enhancing radiosensitivity in osteosarcoma via CDKN2C overexpression: A mechanism involving G1 phase arrest mediated by inhibition of CDK4 expression and Thr172 phosphorylation

BackgroundThe limited radiosensitivity of osteosarcoma poses a challenge in applying radiotherapy, necessitating the search for effective radiosensitizing targets. MethodsThe lentiviral vectors were employed to establish CDKN2C-overexpressing (CDKN2C-OE) and CDKN2C-negative control (CDKN2C-NC) HOS and U2OS osteosarcoma cells. Cells were treated with or without irradiation (IR) to assess radiosensitization via viability, proliferation, apoptosis, and cell cycle analysis. A mouse model with subcutaneous tumors from CDKN2C-OE and CDKN2C-NC HOS cells evaluated tumor growth post-IR. Immunohistochemical staining and Western blot analysis were conducted to confirm model establishment and explore mechanisms. ResultsCDKN2C-OE combined with IR inhibited cell viability and proliferation, promoting apoptosis in vitro and inhibiting tumor growth in vivo. CDKN2C-OE inhibited G1 phase progression post-IR by suppressing Cyclin-dependent kinase 4 (CDK4) expression and Thr172 phosphorylation, reducing retinoblastoma protein (RB) phosphorylation at Ser807/811. CDKN2C-OE did not primarily impact the cell cycle by regulating the expression of CDK6 and Cyclin D1. Furthermore, when CDKN2C-OE was combined with IR, the expression of BAX, Caspase-3, and its active cleavage product, cleaved Caspase-3, was upregulated. ConclusionsOur research results indicate that overexpression of CDKN2C enhances radiosensitivity in osteosarcoma through the induction of G1 phase arrest and subsequent apoptosis. G1 phase arrest is mediated by the suppression of CDK4 expression and Thr172 phosphorylation, which consequently affects the expression of phosphorylated RB at the Ser807/811 sites.