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Renal fibrosis is the pathological hallmark of chronic kidney disease that is influenced by numerous factors. Arrest of renal tubular epithelial cells (RTECs) in G2/M phase is closely correlated with the progression of renal fibrosis; however, the mechanisms mediating these responses remain poorly defined. In this study, we observed that human leukocyte antigen-F adjacent transcript 10 (FAT10) deficiency abolished hypoxia-induced upregulation of checkpoint kinase 1 (CHK1) expression in RTECs derived from FAT10

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic–lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2–M arrest and DNA damage. The VRK1–VRK2 synthetic–lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line–derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. Significance: A paralog synthetic–lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Background: Coumarin is a functional compound with a pronounced wide range of biological activities and has recently been shown to have anticancer effects on various human cancer cells. Cisplatin is widely used in treating many cancers, but its effectiveness is limited due to acquired resistance and dose-related side effects. Objective: This study aimed to reveal the chemosensitizing ability of novel synthesized coumarin-triazole hybrid compounds (3a-f) compared to the cisplatin in A549, MCF-7, and HeLa cancer cells. Methods: Cytotoxicity was determined by MTT assay. Lactate dehydrogenase (LDH), antioxidant/oxidant status, and DNA fragmentation were determined spectrophotometrically using commercial kits. Muse™ Cell Analyzer was used to assess cell cycle progression. Pro/anti-apoptotic gene expressions were determined by Real-Time qPCR. The antiangiogenic activity was determined by VEGF expression and Hen's chorioallantoic membrane model. Results: Compounds 3c, -d, -e, and -f potentiated the cisplatin-induced cytotoxicity by increasing LDH release and DNA fragmentation, inducing G2/M cell cycle arrest, overproducing oxidative stress, and decreasing cellular antioxidant levels. These compounds combined with cisplatin caused upregulation in the pro-apoptotic Bax, Bıd, caspase-3, caspase-8, caspase-9, Fas, and p53 gene expressions while downregulating anti-apoptotic DFFA, NFkB1, and Bcl2 gene expressions. These combinations caused vascular loss and a reduction in VEGF expression. Conclusion: These results suggest that a combinational regimen of coumarin compounds with cisplatin could enhance the effect of cisplatin in A549 cells. Besides, these compounds exhibit relatively low toxicity in normal cells, thus decreasing the dose requirement of cisplatin in cancer treatments.

Small cell lung cancer (SCLC) is a highly aggressive disease with a poor prognosis. Although most patients initially respond to topoisomerase inhibitors, resistance rapidly emerges. The aim, therefore, is to overcome resistance to topoisomerase I (irinotecan) or II (etoposide) inhibitors in SCLCs.To identify key factors in the chemoresistance of SCLCs, we established four cell lines resistant to etoposide or an active metabolite of irinotecan, SN-38, from SCLC cell lines and evaluated RNA profiles using parental and newly established cell lines.We found that the drug efflux protein, ATP-binding cassette sub-family B member 1 (ABCB1), was associated with resistance to etoposide, and ATP-binding cassette sub-family G member 2 (ABCG2) was associated with resistance to SN-38 by RNA sequencing. The inhibition of ABCB1 or ABCG2 in each resistant cell line induced synergistic apoptotic activity and promoted drug sensitivity in resistant SCLC cells. The ABC transporter inhibitors, elacridar and tariquidar, restored sensitivity to etoposide or SN-38 in in vitro and in vivo studies, and promoted apoptotic activity and G2-M arrest in resistant SCLC cells.ABC transporter inhibitors may be a promising therapeutic strategy for the purpose of overcoming resistance to topoisomerase inhibitors in patients with SCLC.

Zearalenone is a mycotoxin produced by fungi of the genus Fusarium, which has severe toxicity on animal and human health including reproduction. Previous study showed that zearalenone exposure inhibited oocyte polar body extrusion, while in present study we found that high dose zearalenone disturbed oocyte meiosis resumption. Our results showed that a high concentration of 100 μM zearalenone reduced the rate of germinal vesicle (GV) breakdown in mouse oocytes. Further analysis indicated that zearalenone caused the decrease of Cyclin B1 and CDK1 expression, indicating MPF activity was affected, which further induced G2/M arrest, and this could be rescued by the inhibition of Wee1 activity. We found that the oocytes under high concentration of zearalenone showed lower γ-H2A.X expression, suggesting that DNA damage repair was disturbed, which further activated of DNA damage checkpoints. This could be confirmed by the altered expression of CHK1 and CHK2 after zearalenone treatment. Moreover, the organelles such as mitochondria, ribosome, endoplasmic reticulum and Golgi apparatus were diffused from germinal vesicle periphery after zearalenone exposure, indicating that zearalenone affected protein synthesis, modification and transport, which further induced the arrest of G2/M transition. Taken together, our results showed that high dose of zearalenone exposure induced G2/M transition defect by affecting organelle function-related CHK1/2-Wee1-MPF pathway.

The effects of ethephon on the reproductive systems of mammalian females are still ambiguous. This study was conducted to evaluate the toxic effects of ethephon on the female reproductive system.Forty female C57 mice were used as experimental subjects and evenly divided into 8 groups, which were fed with mixed ethephon (0, 107.3, 214.5, and 429 mg/kg bw/day) and pure water. After 20 and 40 days of gavage, the mice were weighed and individual organ coefficients of the ovaries were measured. Enzyme-linked immunoassay was used to detect the serum levels of serum sex hormones. The cell cycle distribution and rate of apoptosis of mouse ovarian tissues were examined using flow cytometry.Ethephon intoxication significantly decreased serum levels of progesterone (P) and oestradiol (E2) and increased the serum levels of luteinizing hormone (LH). The serum levels of follicle-stimulating hormone (FSH) decreased and then increased over time. In addition, ethephon significantly inhibited the apoptosis rate in the ovary and caused G0/G1 and G2/M arrest.These results indicate that prolonged exposure to ethephon may have negative effects on the female reproductive system.

Acidic nucleoplasmic DNA binding protein 1 (AND-1, also known as WD repeat and HMG-box DNA-binding protein 1, WDHD1) plays an important role in DNA replication and repair, but the relationship between AND-1 and radiosensitivity is not well understood. This research explored the impact of AND-1 on the radiosensitivity of non-small cell lung cancer (NSCLC) for the first time. NSCLC cells were treated with AND-1 siRNA or a new AND-1 inhibitor, CH-3, and clonogenic survival assay was used to characterize cell radiosensitivity. Cell cycle and apoptosis were examined by flow cytometry. DNA damage was detected by comet assay, immunofluorescence, and homologous recombination (HR) repair assay. Finally, the radiosensitization effect of CH-3 was investigated in vivo in a xenograft tumor model. The results showed that AND-1 inhibition significantly increased the radiosensitivity of NSCLC cells. Mechanistically, AND-1 inhibitor (CH-3) induced G2/M phase arrest by regulating the ATM signaling pathway and enhanced irradiation-induced DNA damage by inhibiting the DNA HR repair pathway. CH-3 enhanced the radiosensitivity of NSCLC cells in vivo. The development of radiosensitizers that target AND-1 may provide an alternative strategy to inhibit NSCLC.

Ribosomal S6 kinase has been shown to play a key role in cellular resistance to endocrine therapy in prostate cancer through its regulation of YB-1/androgen receptor (AR) signaling. PMD-026, an oral first-in-class small molecule kinase inhibitor, is the first identified ribosomal S6 kinase inhibitor. This study investigated the effect of PMD-026 on YB-1/AR signaling and its antitumor effect in prostate cancer in vitro and in vivo. Castration-resistant prostate cancer 22Rv1 cells that express high-level AR variants were used in this study. The effect of PMD-026 on YB-1/AR signaling was investigated by quantitative real-time PCR and western blot analysis. The effects of PMD-026 on prostate cancer cells were investigated by cytotoxicity analysis, apoptosis assay, and cell cycle assay in vitro and a mouse castration model in vivo. PMD-026 decreased YB-1 phosphorylation as well as AR V7 mRNA and AR variant expressions in 22Rv1 cells. PMD-026 suppressed cell proliferation alone and in combination with the second-generation antiandrogens enzalutamide and darolutamide by inducing cellular apoptosis and G2/M arrest. In a mouse xenograft model, PMD-026 suppressed tumor growth, and the combination of PMD-026 and enzalutamide inhibited tumor growth more prominently than single treatments. Our results demonstrate an excellent antitumor effect of the novel ribosomal S6 kinase inhibitor PMD-026 and the combination effect with the antiandrogen enzalutamide in castration-resistant prostate cancer. These findings warrant a clinical trial of PMD-026 in prostate cancer patients.

Hepatocellular carcinoma (HCC) is one of the largest causes of cancer-related deaths worldwide owing to the limitation of effective treatment options. The ubiquitin-proteasome system has been rapidly recognized as a frequent target of deregulation leading to cancers. Enhanced DNA damage response (DDR) promotes HCC growth and prevents chemosensitivity, and ubiquitin E3 ligases are key modulators in DDR. Therefore, a better understanding of how E3 ligases regulate cell growth and DNA damage may provide novel insights in understanding the oncogenic mechanism and improving the efficacy of DNA damage therapeutic agents. Here, we performed a high-content RNAi screening targeting 52 DDR-related E3 ligases in HCC and found that ring finger protein 4 (RNF4) was essential for HCC growth. RNF4 was highly expressed in HCC tissues, and the expression levels of RNF4 were associated with poor outcomes. RNF4 silencing significantly suppressed the cell growth, and subsequently induced G2/M arrest and apoptosis of HCC cells in vitro; RNF4 silencing also demonstrated the tumor-suppressive efficacy on HCC in vivo. Moreover, RNF4 silencing increased DNA damage, and rendered HCC cells more sensitive to DNA damage drugs and radiation. We found RNF4 functionally interacts with p62, and mechanistic analyses indicated that RNF4 silencing triggered the nuclear enrichment of p62. Moreover, the p62 nuclear targeting was required for increased DNA damage and growth suppression mediated by RNF4 silencing. Thus, our findings suggest RNF4 is essential for HCC proliferation via preventing nuclear translocation of p62. RNF4 silencing promotes DNA damage and may serve as a novel strategy to suppress cell growth and increase the sensitivity of DNA damage therapeutic agents in HCC.

Drug resistance to molecular targeted agents, such as lenvatinib, is an important issue. The aim of this study was to explore the mechanism of lenvatinib resistance and to investigate potential drugs that may improve the treatment of lenvatinib-resistant (LR) hepatocellular carcinoma (HCC).LR cells were developed by long-term culture under lenvatinib exposure. We analyzed the biological characteristics of LR cells in vitro, and investigated the antitumor effects and endogenous mechanisms of cisplatin in LR cells.The proliferative potential of LR cells was enhanced by activation of ERK signaling and changes in several miRNAs. Cisplatin inhibited cell proliferation of LR cells and induced G2/M cell cycle arrest. Furthermore, cisplatin triggered the DNA damage response, via the ATM/ATR-Chk1/Chk2 signaling pathway.Proliferation of LR cells was induced upon ERK signaling activation. Cisplatin exerted antitumor effects in LR cells and was involved in the regulation of miRNAs associated with drug resistance.

Obesity is associated with elevated levels of free fatty acids (FFAs). Excessive saturated fatty acids (SFAs) exhibit significant deleterious cytotoxic effects in many types of cells. However, the effects of palmitic acid (PA), the most common circulating SFA, on cell cycle progression in neuronal cells have not been well-examined. The aim of this study was to examine whether PA affects the proliferation and cell cycle progression in mouse neuroblastoma Neuro-2a (N2a) cells. Our studies found that 200 µM PA significantly decreased DNA synthesis and mitotic index in N2a cells as early as 4 h following treatment. 24 h treatment with 200 µM PA significantly decreased the percentage of diploid (2 N) cells while dramatically increasing the percentage of tetraploid (4 N) cells as compared to the BSA control. Moreover, our studies found that 24 h treatment with 200 µM PA increased the percentage of binucleate cells as compared to the BSA control. Our studies also found that unsaturated fatty acids (UFAs), including linoleic acid, oleic acid, α-linolenic acid, and docosahexaenoic acid, were able to abolish PA-induced decrease of 2 N cells, increase of 4 N cells, and accumulation of binucleate cells. Taken together, these results suggest that PA may affect multiple aspects of the cell cycle progression in N2a cells, including decreased DNA synthesis, G2/M arrest, and cytokinetic failure, which could be abolished by UFAs. Abbreviations: 4-PBA, 4-Phenylbutyric Acid; ALA, α-linolenic acid; BrdU, 5-bromo-2’-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; FFA, free fatty acids; FITC, fluorescein isothiocyanate; LA, linoleic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N2a, Neuro-2a; NAC, N-acetyl cysteine; OA, oleic acid; PA, palmitic acid; pHH3, Phosphorylation of histone H3; PI, propidium iodide; SFA, saturated fatty acids; PUFA, polyunsaturated fatty acids; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; UFA, unsaturated fatty acids

It is vital to remove residual tumor cells after resection to avoid the recurrence and metastasis of osteosarcoma. In this study, a mineral nanomedicine, europium-doped calcium fluoride (CaF

BackgroundThe use of fenbendazole (FBZ) in terminal cancer patients has recently increased, as anthelminthic drugs, such as FBZ and benzimidazole, exhibit anti-tubulin effects in tumour cells.ObjectivesThe present study evaluated the in vitro anti-cancer effects of FBZ in five canine melanoma cell lines originating from the oral cavity (UCDK9M3, UCDK9M4, UCDK9M5, KMeC and LMeC).MethodsFive canine melanoma cell lines were treated with FBZ and analysed with cell viability assay, cell cycle analysis, western blot assay and immunofluorescence staining to identify apoptotic effect, cell cycle arrest, microtubule disruption and mitotic slippage.ResultsCell viability was reduced in all melanoma cell lines in a dose-dependent manner after FBZ treatment. Through cell cycle analysis, G2/M arrest and mitotic slippage were identified, which showed a time-dependent change. All treatment concentrations induced increased cleaved PARP signals in western blot analysis compared to the control groups. Immunofluorescence of cells treated for 24h revealed defects in microtubule structure, multinucleation or macronucleation. With the exception of UCDK9M3, the melanoma cells showed mitotic slippage and post-slippage death, indicative of mitotic catastrophe.ConclusionsThese results indicate that FBZ exhibits anti-cancer effects in vitro against canine melanoma cells; however, further in vivo studies regarding the clinical applications of FBZ are required.

PARP1 and Chk1 inhibitors have been shown to be synergistic in different cancer models in relatively short time treatment modes. However, the consequences of long-term/repeated treatments with the combinations in cancer models remain unclear. In this study, the synergistic cytotoxicity of their combinations in 8 tumor cell lines was confirmed in a 7-day exposure mode. Then, pancreatic Capan-1 cells were repeatedly treated with the PARP1 inhibitor olaparib, the Chk1 inhibitor rabusertib or their combination for 211–214 days, during which the changes in drug sensitivity were monitored at a 35-day interval. Unexpectedly, among the 3 treatment modes, the combination treatments resulted in the highest-grade resistance to Chk1 (~14.6 fold) and PARP1 (~420.2 fold) inhibitors, respectively. Consistently, G2/M arrest and apoptosis decreased significantly in the resulting resistant variants exposed to olaparib. All 3 resistant variants also unexpectedly obtained enhanced migratory and invasive capabilities. Moreover, the combination treatments resulted in increased migration and invasion than olaparib alone. The expression of 124 genes changed significantly in all the resistant variants. We further demonstrate that activating CXCL3-ERK1/2 signaling might contribute to the enhanced migratory capabilities rather than the acquired drug resistance. Our findings indicate that repeated treatments with the rabusertib/olaparib combination result in increased drug resistance and a more aggressive cell phenotype than those with either single agent, providing new clues for future clinical anticancer tests of PARP1 and Chk1 inhibitor combinations.

Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC), and approximately 20% of patients experience treatment failure due to tumour radioresistance. However, the exact regulatory mechanism remains poorly understood. Here, we show that the deubiquitinase USP44 is hypermethylated in NPC, which results in its downregulation. USP44 enhances the sensitivity of NPC cells to radiotherapy in vitro and in vivo. USP44 recruits and stabilizes the E3 ubiquitin ligase TRIM25 by removing its K48-linked polyubiquitin chains at Lys439, which further facilitates the degradation of Ku80 and inhibits its recruitment to DNA double-strand breaks (DSBs), thus enhancing DNA damage and inhibiting DNA repair via non-homologous end joining (NHEJ). Knockout of TRIM25 reverses the radiotherapy sensitization effect of USP44. Clinically, low expression of USP44 indicates a poor prognosis and facilitates tumour relapse in NPC patients. This study suggests the USP44-TRIM25-Ku80 axis provides potential therapeutic targets for NPC patients., Radiotherapy is the mainstay treatment for nasopharyngeal carcinoma (NPC). Here the authors show that the deubiquitinase, USP44, increases radiosensitivity of NPC cells by promoting the degradation of Ku80, and thus enhancing the levels of DNA damage.

PRKCSH, also known as glucosidase II beta, functions as a contributor to lung tumorigenesis by regulating the cell cycle in a p53-dependent manner under severe environmental stress. However, the prognostic value and molecular mechanisms by which the level of PRKCSH is significantly increased in cancer cells are not clearly understood. Here, we first generated a biological profile of PRKCSH expression changes in cancers by analysing bioinformatic data from cancer databases. We found that higher PRKCSH expression was correlated with a poorer prognosis and greater infiltration of most immune cell types in patients with lung cancer. In particular, PRKCSH expression showed significant negative correlations with the level of STAT6 (

BACKGROUND Head and neck cancer (HNC) is common worldwide. Given poor outcomes for patients with HNC, research into targeted therapies is needed. Ataxia telangiectasia mutated (ATM) is a DNA damage kinase which is activated by double-strand DNA breaks. We tested the effects of a novel ATM inhibitor on HNC cell lines and xenografts. MATERIALS AND METHODS p53-Binding protein 1 and phosphorylated ATM were localized in cultured cells by immunofluorescence microscopy. Protein expression was determined by western blot. Tumor xenografts were established by injecting HNC lines into immunocompromised mice. Tumor sections were characterized by immunohistochemistry. Apoptotic cells were determined by terminal transferase-mediated dUTP nick-end labeling assay. RESULTS ATM inhibition increased double-strand DNA breaks at replication foci in HNC cell lines. ATM inhibition affected cell-cycle regulatory protein expression, blocked cell-cycle progression at the G2/M phase and resulted in apoptosis. CONCLUSION ATM inhibition may be therapeutically useful in treating HNC.

Dual inhibition of tubulin and poly(ADP-ribose) polymerase-1 (PARP-1) may become an attractive approach for cancer therapy. Here, we discover a dual tubulin/PARP-1 inhibitor (termed as TP-3) using structure-based virtual screening. TP-3 shows strong dual inhibitory effects on both tubulin and PARP-1. Cellular assays reveal that TP-3 shows superior antiproliferative activities against human cancer cells, including breast, liver, ovarian, and cervical cancers. Further studies indicate that TP-3 plays an antitumor role through multiple mechanisms, including the disturbance of the microtubule network and the PARP-1 DNA repairing function, accumulation of DNA double-strand breaks, inhibition of the tube formation, and induction of G2/M cell cycle arrest and apoptosis. In vivo assessment indicates that TP-3 inhibits the growth of MDA-MB-231 xenograft tumors in nude mouse with no notable side effects. These data demonstrate that TP-3 is a dual-targeting, high-efficacy, and low-toxic antitumor agent.

PURPOSE Doxorubicin (DOX) is a common chemotherapeutic agent, with toxic side effects, and chemoresistance. Combination chemotherapy is a successful approach to overcome these limitations. Here, we investigated the effects of pioglitazone (PGZ), a PPARγ agonist, and/or DOX on the viability, cell cycle, apoptosis on THP-1 cells and normal human monocytes (NHMs). METHODS MTT assay was used to evaluate the cytotoxicity of DOX and/or PGZ. Cell cycle progression and apoptosis induction were examined by PI or Annexin V-PI double staining, and analyzed by flow cytometry. Quantitative RT-PCR was used to evaluate the changes in the mRNA expression of cell cycle progression or apoptosis-associated genes including P27, P21, CDK2, P53, BCL2 and FasR. RESULTS DOX, PGZ and DOX + PGZ exerted their cytotoxic effects in a dose- and time-dependent manner with low toxicity on NHMs. The cell growth inhibitory effects of DOX were in association with G2/M arrest, while PGZ executed S phase arrest. PGZ treatment enhanced G2/M among DOX-treated combinations with moderate elevation in the S phase. DOX, PGZ and combined treatments induced apoptosis (mostly late phase) in a dose-dependent manner. All treatments resulted in the significant overexpression of p21, p27, p53 and FasR genes and downregulation of CDK2. DOX + PGZ combined treatments exhibited the most significant changes in mRNA expression. CONCLUSION We demonstrated that the antiproliferative, cell cycle regulation and apoptosis-inducing capacity of DOX was enhanced by PGZ in THP-1 leukemia cells in a dose-dependent manner. Therefore, the combination of DOX + PGZ could be used as a novel combination to target AML.

Background The human miR-17-92 polycistron is the first reported and most well-studied onco-miRNA with a cluster of seven miRNAs. miR-17-5p, a member of the miR-17-92 family, plays an important role in tumor cell proliferation, apoptosis, migration and invasion. However, few studies have shown the role of miR-17-5p in the cell cycle of head and neck squamous cell carcinoma (HNSCC). Methods RT-qPCR was used to detect miR-17-5p expression levels in 64 HNSCC tissues and 5 cell lines. The relationship between the expression of miR-17-5p in the tissues and the clinical characteristics of the patients was analyzed. HNSCC cells were transfected with an miR-17-5p mimic or inhibitor to evaluate cell cycle distribution by flow cytometry. Cell cycle distribution of cells transfected with target gene was evaluated using flow cytometry. Dual-luciferase reporter assay was used to detect the regulatory effect of miR-17-5p on target gene expression. Results In the present study, we found that miR-17-5p expression in HNSCC tissues and cell lines was remarkably increased, and miR-17-5p is related to recurrence in HNSCC patients. Silencing miR-17-5p blocked HNSCC cells in G2/M phase, whereas its overexpression propelled cell cycle progression. More importantly, we verified that miR-17-5p negatively regulated CCNG2 mRNA and protein expression by directly targeting its 3’UTR. Conclusion These findings suggest that miR-17-5p might act as a tumor promoter and prognostic factor for recurrence in HNSCC patients.

Background Various types of normal and cancer cells undergo senescence in response to carboplatin and paclitaxel, which are considered the gold standard treatments in ovarian cancer management. Surprisingly, the effect of these drugs on ovarian cancer cell senescence remained unknown. Methods The experiments were conducted on primary high-grade serous ovarian cancer cells. Molecular markers of senescence were evaluated using cytochemistry and immunofluorescence. Cell cycle distribution was analyzed using flow cytometry. Expression of cyclins and signaling pathways was tested using western blot. Telomere length and telomerase activity were measured using qPCR, and the colocalization of telomeres with DNA damage foci using immuno-FISH. Oxidative stress-related parameters were quantified using appropriate fluorescence probes. Production of cancerogenic agents was analyzed using qPCR and ELISA. Results Carboplatin applied with paclitaxel induces senescence of ovarian cancer cells in vitro. This activity was reflected by permanent G2/M growth arrest, a high fraction of cells expressing senescence biomarkers (SA-β-Gal and γ-H2A.X), upregulated expression of p16, p21, and p53 cell cycle inhibitors, and decreased expression of cyclin B1. Neither telomere length nor telomerase activity changed in the senescent cells, and the majority of DNA damage was localized outside telomeres. Moreover, drug-treated cancer cells exhibited increased production of STAT3 protein, overproduced superoxide and peroxides, and increased mitochondrial mass. They were also characterized by upregulated ANG1, CCL11, IL-6, PDGF-D, TIMP-3, TSP-1, and TGF-β1 at the mRNA and/or protein level. Conclusions Our findings imply that conventional chemotherapy may elicit senescence in ovarian cancer cells, which may translate to the development of a cancer-promoting phenotype, despite the inability of these cells to divide.

The current study was designed to unveil the anticancer effects of naringenin against breast cancer MDA-MB-231 cells. Cytotoxic effects were estimated via MTT viability assay. Clonogenic assay was performed to assess clonogenic potential of MDA-MB-231 cells. Apoptosis was examined via AO/EB staining, quantified via annexin V/PI staining and western blotting was performed to monitor apoptosis allied protein expressions. Cell cycle was analyzed through flow cytometric analysis. Transwell chambers assay was executed for determination of cell migration and cell invasion tendency of MDA-MB-231 breast cancer cells. Results indicated significant anticancer potential of naringenin drug against MDA-MB-231 cells. On evaluation of cell proliferation rate of breast cancer cells by MTT assay, it was observed that naringenin inhibited proliferation rate in dose as well as time dependent manner. AO/EB staining assay revealed potential morphological changes indicating apoptotic cell death. Annexin V/PI staining assay revealed increased apoptotic cell percentage with increased drug doses. The apoptosis inducing potential of naringenin drug was observed to be mediated via caspase activation. Flow cytometric analysis predicted cell cycle arrest at G2/M phase of cell cycle. Further cell migration as well as cell invasion tendency of MDA-MB-231 cells was reduced to minimum upon application of naringenin drug.

PURPOSE: Insulin like growth factor receptor 1 (IGF-1R) has been documented to play a key role in radiation response, thereby offering an attractive drug target to enhance tumor sensitivity to radiotherapy. Here, we investigated wether knockdown of IGF-1R can sensitize colorectal cancer (CRC) cell lines to radiation. MATERIAL AND METHODS: Human colon carcinoma SW480 and HT-29 cells were transfected with specific small interference RNA (siRNA) to mediate IGF-1R depletion. The expression of IGF-1R mRNA and protein among transfected and untransfected cells was detected by Western blot analysis. Changes in cell proliferation and radiosensitivity were evaluated by the clonogenic survival assay. NVP-ADW742, an IGF-1R inhibitor, in combination with radiation was studied. RAD51, a measure for homologous recombination repair, and 53BP1, a maker for non-homologous end-joining (NHEJ), were determined by immunofluorescence for double-strand breaks (DSB) repair pathways. Cell cycle was also examined in the IGF-1R knockdown and IGF-1R-inhibited cells. RESULTS: CRC cell lines were selectively sensitized to radiation after siRNA-mediated IGF-1R depletion. NVP-ADW742 efficiently increases cancer cell response to radiation. Furthermore, initial formation of RAD51 foci after IR, and 53BP1 foci were significantly reduced in IGF-1R-depleted or with IGF-1R Inhibitor CRC cell lines. Lastly, IGF-1R-depleted or with IGF-1R Inhibitor caused more G2 phase cell arrest. CONCLUSION: Our findings demonstrate that depletion of IGF-1R lead to an increase in radiosensitivity in CRC.

Anaplastic thyroid cancer (ATC) is an extremely aggressive tumor associated with poor prognosis due to a lack of efficient therapies. In Japan, lenvatinib is the only drug approved for patients with ATC; however, its efficacy is limited. Therefore, novel therapeutic strategies are urgently required for patients with ATC. The present study aimed to identify compounds that enhance the antiproliferative effects of lenvatinib in ATC cells using a compound library. IRAK1/4 Inhibitor I was identified as a candidate compound. Combined treatment with lenvatinib and IRAK1/4 Inhibitor I showed synergistic antiproliferative effects via the induction of cell cycle arrest at G2/M phase in the ATC cell lines 8305C, HTC/C3, ACT‐1, and 8505C. Furthermore, IRAK1/4 Inhibitor I enhanced the inhibition of ERK phosphorylation by lenvatinib in 8305C, HTC/C3, and 8505C cells. In an HTC/C3 xenograft mouse model, tumor volume was lower in the combined IRAK1/4 Inhibitor I and lenvatinib group compared with that in the vehicle control, IRAK1/4 Inhibitor I, and lenvatinib groups. IRAK1/4 Inhibitor I was identified as a promising compound that enhances the antiproliferative and antitumor effects of lenvatinib in ATC., We screened for novel compounds that could enhance the antiproliferative effects of lenvatinib in anaplastic thyroid cancer (ATC), which is an extremely aggressive tumor with poor prognosis. We identified IRAK1/4 Inhibitor I as a candidate compound and examined its combined use with lenvatinib. In our HTC/C3 xenograft mouse model, tumor volume was significantly lower in the combined IRAK1/4 Inhibitor I and lenvatinib group compared with that in the control, IRAK1/4 Inhibitor I alone, and lenvatinib alone groups.

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a bifunctional enzyme located in the mitochondria. MTHFD2 has been reported to be overexpressed in several malignant tumors and is implicated in cancer development. This study aimed to investigate the effect of MTHFD2 on ovarian cancer progression. The expression of MTHFD2 was detected by bioinformatic analysis, immunohistochemistry, RT‐qPCR (real‐time quantitative PCR analysis), and western blot analysis. The effects of MTHFD2 depletion on cell proliferation, migration, and invasion were determined through in vitro experiments. Cell cycle progression and apoptosis were accessed by flow cytometry. The related signaling pathway protein expression was determined by western blot analysis. We found that MTHFD2 is highly expressed in both ovarian cancer tissues and cell lines. MTHFD2 deletion suppressed cell proliferation and metastasis. Knockdown of MTHFD2 induces cell apoptosis and G2/M arrest, whereas the number of cells in S phase increased with MTHFD2 overexpression. Mechanically, our results indicate that an inhibitory effect of MTHFD2 knockdown may be mediated by the downregulation of cyclin B1/Cdc2 complex and the inhibitory effect on its activity. Additionally, MTHFD2 could regulate cell growth and aggressiveness via activation of STAT3 and the STAT3‐induced epithelial–mesenchymal transition signaling pathway. These findings indicate that MTHFD2 is overexpressed in ovarian cancer and regulates cell proliferation and metastasis, presenting an attractive therapeutic target., Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is overexpressed in ovarian cancer tissues and cell lines. MTHFD2 depletion inhibits cell growth and metastasis and induces G2/M arrest and apoptosis in ovarian cancer cell lines. MTHFD2 regulates cyclin B1/Cdc2 complex and activates the STAT3 and STAT3‐induced epithelial–mesenchymal transition signaling pathway. Taken together, our results suggest that targeting MTHFD2 maybe served as a novel approach for ovarian cancer treatment.

Cisplatin is a potent chemotherapeutic used for the treatment of many types of cancer, but it has nephrotoxic side effects leading to acute kidney injury and subsequently chronic kidney disease (CKD). Previous work has focused on acute kidney tubular injury induced by cisplatin, whereas the chronic sequelae post-injury has not been well-explored. In the present study, we established a kidney fibroblast model of CKD induced by repeated administration of cisplatin (RAC) as a clinically relevant model. In NRK-49F rat kidney fibroblasts, RAC upregulated α-smooth muscle actin (α-SMA) and fibronectin proteins, suggesting that RAC induces kidney fibroblast-to-myofibroblast transformation. RAC also enhanced cell size, including the cell attachment surface area, nuclear area, and cell volume. Furthermore, RAC induced p21 expression and senescence-associated β-galactosidase activity, suggesting that kidney fibroblasts exposed to RAC develop a senescent phenotype. Inhibition of p21 reduced cellular senescence, hypertrophy, and myofibroblast transformation induced by RAC. Intriguingly, after RAC, kidney fibroblasts were arrested at the G2/M phase. Repeated treatment with paclitaxel as an inducer of G2/M arrest upregulated p21, α-SMA, and fibronectin in the kidney fibroblasts. Taken together, these data suggest that RAC transforms kidney fibroblasts into myofibroblasts through G2/M arrest and cellular senescence.

The mechanisms underlying fibrogenic responses after injury are not well understood. Epithelial cell cycle arrest in G2/M after injury is a key checkpoint for determining wound-healing leading to either normal cell proliferation or fibrosis. Here, we identify a kidney- and liver-enriched circular RNA, circBNC2, which is abundantly expressed in normal renal tubular cells and hepatocytes but significantly downregulated after acute ischemic or toxic insult. Loss of circBNC2 is at least partially mediated by upregulation of DHX9. Gain- and loss-of-function studies, both in vitro and in vivo, demonstrate that circBNC2 acts as a negative regulator of cell G2/M arrest by encoding a protein that promotes formation of CDK1/cyclin B1 complexes. Restoring circBNC2 in experimentally-induced male mouse models of fibrotic kidney and liver, decreases G2/M arrested cell numbers with secretion of fibrotic factors, thereby mitigating extracellular matrix deposition and fibrosis. Decreased expression of circBNC2 and increased G2/M arrest of epithelial cells are recapitulated in human ischemic reperfusion injury (IRI)-induced chronic kidney disease and inflammation-induced liver fibrosis, highlighting the clinical relevance. These findings suggest that restoring circBNC2 might represent a potential strategy for therapeutic intervention in epithelial organ fibrosis.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death, which indicates that efficient intervention agents or strategies against HCC are urgently needed. In the present study, we firstly found that a combination of gefitinib (an ep i dermal growth factor receptor (EGFR) inhibitor) and B I 6727 (a pol o -like kinase 1 (PLK1) inhibitor) could significantly inhibit cell proliferation of HCC cells, which attenuated acquired resistance of gefitinib in HCC cells. Interestingly, our results showed that these anti-tumor effects of gefitinib in combination with BI6727 were associated with G2/M arrest. Moreover, further study revealed that BI6727 could downregulate the protein levels of cell division cycle 25C (Cdc25C) via ubiquitination-dependent pathway, which subsequently induced G2/M arrest. Furthermore, two critical checkpoints proteins ataxia telangiectasia-mutated (p-ATM)/ ATM and Rad-3 related(p-ATR) and another hallmark phosphorylated H2AX (γ-H2AX ) of DNA damage were positively regulated in HCC cells exposed to gefitinib in combination with BI6727. These results indicated that co-treatment induced G2/M arrest was closely related to DNA damage. In summary, the present study discovered that gefitinib synergizing with BI6727 could significantly facilitate DNA damage and overcome acquired resistance of HCC cells to gefitinib. Our study provides a promising approach for the combination of EGFR inhibitors and PLK1 inhibitors in the clinical treatment for HCC.

Background Seaweed polysaccharides have been recommended as anticancer supplements and for boosting human health; however, their benefits in the treatment of triple-negative breast cancers (TNBCs) and improving immune surveillance remain unclear. Olaparib is a first-in-class poly (ADP-ribose) polymerase inhibitor. Oligo-Fucoidan, a low-molecular-weight sulfated polysaccharide purified from brown seaweed (Laminaria japonica), exhibits significant bioactivities that may aid in disease management. Methods Macrophage polarity, clonogenic assays, cancer stemness properties, cancer cell trajectory, glucose metabolism, the TNBC 4T1 cells and a 4T1 syngeneic mouse model were used to inspect the therapeutic effects of olaparib and Oligo-Fucoidan supplementation on TNBC aggressiveness and microenvironment. Results Olaparib treatment increased sub-G1 cell death and G2/M arrest in TNBC cells, and these effects were enhanced when Oligo-Fucoidan was added to treat the TNBC cells. The levels of Rad51 and programmed death-ligand 1 (PD-L1) and the activation of epidermal growth factor receptor (EGFR) and adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) facilitate drug resistance and TNBC metastasis. However, the combination of olaparib and Oligo-Fucoidan synergistically reduced Rad51 and PD-L1 levels, as well as the activity of EGFR and AMPK; consistently, TNBC cytotoxicity and stemness were inhibited. Oligo-Fucoidan plus olaparib better inhibited the formation of TNBC stem cell mammospheroids with decreased subpopulations of CD44high/CD24low and EpCAMhigh cells than monotherapy. Importantly, Oligo-Fucoidan plus olaparib repressed the oncogenic interleukin-6 (IL-6)/p-EGFR/PD-L1 pathway, glucose uptake and lactate production. Oligo-Fucoidan induced immunoactive and antitumoral M1 macrophages and attenuated the side effects of olaparib, such as the promotion on immunosuppressive and protumoral M2 macrophages. Furthermore, olaparib plus Oligo-Fucoidan dramatically suppressed M2 macrophage invasiveness and repolarized M2 to the M0-like (F4/80high) and M1-like (CD80high and CD86high) phenotypes. In addition, olaparib- and Oligo-Fucoidan-pretreated TNBC cells resulted in the polarization of M0 macrophages into CD80(+) M1 but not CD163(+) M2 macrophages. Importantly, olaparib supplemented with oral administration of Oligo-Fucoidan in mice inhibited postsurgical TNBC recurrence and metastasis with increased cytotoxic T cells in the lymphatic system and decreased regulatory T cells and M2 macrophages in tumors. Conclusion Olaparib supplemented with natural compound Oligo-Fucoidan is a novel therapeutic strategy for reprogramming cancer stemness, metabolism and the microenvironment to prevent local postsurgical recurrence and distant metastasis. The combination therapy may advance therapeutic efficacy that prevent metastasis, chemoresistance and mortality in TNBC patients.

WEE1 plays an important role in the regulation of cell cycle G2/M checkpoints and DNA damage response (DDR). Inhibition of WEE1 can increase the instability of the genome and have anti–tumor effects in some solid tumors. However, it has certain limitations for multiple cancer cells from different lineages. Therefore, we consider the use of synthetic lethal interactions to enhance the therapeutic effect. Our experiments proved that WEE1 inhibitor (WEE1i) can activate the ataxia telangiectasia and RAD3‐related (ATR) pathway and that blockage of ATR dramatically sensitized the WEE1i‐induced cell death. The tumor‐selective synthetic lethality between bioavailable WEE1 and ATR inhibitors led to tumor remission in vivo. Mechanistically, the combination promoted the accumulation of cytosolic double‐strand DNA, which subsequently activated the stimulator of the interferon gene (STING) pathway and induced the production of type I interferon and CD8+ T cells, thereby inducing anti–tumor immunity. Furthermore, our study found that immune checkpoint programmed death‐ligand 1 is upregulated by the combination therapy, and blocking PD‐L1 further enhances the effect of the combination therapy. In summary, as an immunomodulator, the combination of WEE1i with ATR inhibitor (ATRi) and immune checkpoint blockers provides a potential new approach for cancer treatment., Combined application of WEE1i and ATRi in vivo and in vitro can inhibit tumor growth. Mechanistically, the combination promoted the accumulation of cytoplasmic dsDNA, which activated the STING‐TBK1‐IRF3 signal axis and induced the production of type I interferon, thereby inducing anti–tumor immunity. The effects of the combination were further enhanced by immune checkpoint PD‐L1 blockade.

Previous studies reported the critical role of the brefeldin A–inhibited guanine nucleotide exchange protein 3–prohibitin 2 (BIG3‐PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3‐PHB2 in OS malignancy. BIG3‐PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen‐dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3‐PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose‐dependent suppression of OS cell growth, migration, and invasion resulting from G2/M‐phase arrest and in PARP cleavage, ultimately leading to PARP‐1/apoptosis‐inducing factor (AIF) pathway activation–dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3‐PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3‐PHB2 complex might regulate PARP‐1/AIF pathway–dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS., In this study, we focused on understanding the critical role of the mitochondrial BIG3‐PHB2 complex in osteosarcoma (OS) cell proliferation and survival. Its complex disruption by the specific dominant‐peptide inhibitor causes mitochondrial dysfunction, resulting in apoptotic cell death and decreases in the migration and invasion abilities of OS cells. These findings suggest that inhibiting the BIG3‐PHB2 complex formation may be a new therapeutic strategy for the treatment of OS.

Multidrug resistance (MDR) is the main cause of chemotherapy failure in the treatment of colon cancer and the high expression of drug efflux protein P-gp is one of the main factors of MDR. P-gp expression is regulated by the signal transducer and activator of transcription 3 (STAT3) signaling pathway. In this study, human colon cancer oxaliplatin-resistant cells were treated with oxaliplatin combined with the natural product erianin. Then, we evaluated the impact of erianin on drug resistance, and explored the relationship between erianin-related oxaliplatin resistance and the Janus kinase 2/STAT3 signaling pathway in vitro. Our research showed that erianin could significantly inhibit the proliferation of human colon cancer oxaliplatin-resistant cells, and suppress the cell cycle of oxaliplatin-resistant cells in the G2/M phase, indicating that erianin could regulate the MDR phenotype of oxaliplatin-resistant cells, and its mechanism might be the inhibition of STAT3 signaling pathway and the significant reduction of P-gp expression. However, this study provides a theoretical basis for the clinical application of erianin in platinum-based chemotherapy for colon cancer.

We previously reported a potent tubulin inhibitor CH-2-77. In this study, we optimized the structure of CH-2-77 by blocking metabolically labile sites and synthesized a series of CH-2-77 analogues. Two compounds, 40a and 60c, preserved the potency while improving the metabolic stability over CH-2-77 by 3- to 4-fold (46.8 and 29.4 vs 10.8 min in human microsomes). We determined the high-resolution X-ray crystal structures of 40a (resolution 2.3 Å) and 60c (resolution 2.6 Å) in complex with tubulin and confirmed their direct binding at the colchicine-binding site. In vitro, 60c maintained its mode of action by inhibiting tubulin polymerization and was effective against P-glycoprotein-mediated multiple drug resistance and taxol resistance. In vivo, 60c exhibited a strong inhibitory effect on tumor growth and metastasis in a taxol-resistant A375/TxR xenograft model without obvious toxicity. Collectively, this work showed that 60c is a promising lead compound for further development as a potential anticancer agent.

PURPOSE: Recent preclinical studies suggest combining the HSP90 inhibitor AT13387 (Onalespib) with radiation (IR) against colon cancer and head and neck squamous cell carcinoma (HNSCC). These studies emphasized that AT13387 downregulates HSP90 client proteins involved in oncogenic signaling and DNA repair mechanisms as major drivers of enhanced radiosensitivity. Given the large array of client proteins HSP90 directs, we hypothesized that other key proteins or signaling pathways may be inhibited by AT13387 and contribute to enhanced radiosensitivity. Metabolomic analysis of HSP90 inhibition by AT13387 was conducted to identify metabolic biomarkers of radiosensitization and whether modulations of key proteins were involved in IR-induced tumor vasculogenesis, a process involved in tumor recurrence. METHODS AND MATERIALS: HNSCC and non-small cell lung cancer cell lines were used to evaluate the AT13387 radiosensitization effect in vitro and in vivo. Flow cytometry, immunofluorescence, and immunoblot analysis were used to evaluate cell cycle changes and HSP90 client protein’s role in DNA damage repair. Metabolic analysis was performed using liquid chromatography−Mass spectrometry. Immunohistochemical examination of resected tumors post-AT13387 and IR treatment were conducted to identify biomarkers of IR-induced tumor vasculogenesis. RESULTS: In agreement with recent studies, AT13387 treatment combined with IR resulted in a G2/M cell cycle arrest and inhibited DNA repair. Metabolomic profiling indicated a decrease in key metabolites in glycolysis and tricarboxylic acid cycle by AT13387, a reduction in Adenosine 5’-triphosphate levels, and rate-limiting metabolites in nucleotide metabolism, namely phosphoribosyl diphosphate and aspartate. HNSCC xenografts treated with the combination exhibited increased tumor regrowth delay, decreased tumor infiltration of CD45 and CD11b+ bone marrow−derived cells, and inhibition of HIF-1 and SDF-1 expression, thereby inhibiting IR-induced vasculogenesis. CONCLUSIONS: AT13387 treatment resulted in pharmacologic inhibition of cancer cell metabolism that was linked to DNA damage repair. AT13387 combined with IR inhibited IR-induced vasculogenesis, a process involved in tumor recurrence post-radiotherapy. Combining AT13387 with IR warrants consideration of clinical trial assessment. © 2021 Elsevier Inc. All rights reserved.

Using a panel of cancer cell lines, we characterized a novel degrader of AKT, MS21. In mutant PI3K–PTEN pathway cell lines, AKT degradation was superior to AKT kinase inhibition for reducing cell growth and sustaining lower signaling over many days. AKT degradation, but not kinase inhibition, profoundly lowered Aurora kinase B (AURKB) protein, which is known to be essential for cell division, and induced G2–M arrest and hyperploidy. PI3K activated AKT phosphorylation of AURKB on threonine 73, which protected it from proteasome degradation. A mutant of AURKB (T73E) that mimics phosphorylation and blocks degradation rescued cells from growth inhibition. Degrader-resistant lines were associated with low AKT phosphorylation, wild-type PI3K/PTEN status, and mutation of KRAS/BRAF. Pan-cancer analysis identified that 19% of cases have PI3K–PTEN pathway mutation without RAS pathway mutation, suggesting that these patients with cancer could benefit from AKT degrader therapy that leads to loss of AURKB. Significance: MS21 depletes cells of phosphorylated AKT (pAKT) and a newly identified AKT substrate, AURKB, to inhibit tumor growth in mice. MS21 is superior to prior agents that target PI3K and AKT due to its ability to selectively target active, pAKT and sustain repression of signaling to deplete AURKB. This article is highlighted in the In This Issue feature, p. 2945

LncRNA RHPN1-AS1 (RHPN1-AS1) has been confirmed to promote tumor progression in multiple cancers and is upregulated in prostate cancer (PCa), but whether it has an effect on PCa progression remains unclear. In this study, we found that PCa patients with high RHPN1-AS1 expression had a shorter survival time, and RHPN1-AS1 was significantly upregulated in PCa tissues and cells. Based on informatics analysis we predicted that miR-7-5p binds to 3'UTR of RHPN1-AS1 and epidermal growth factor receptor (EGFR) and verified it by luciferase reporter gene assay. Subsequently, we transfected PCa cells with RHPN1-AS1 overexpression vector (RHPN1-AS1), knockdown plasmids (sh-RHPN1-AS1) and/or miR-7-5p mimics or inhibitor and/or overexpression vector (EGFR) or small interfering RNA of EGFR (si-EGFR) or its control, and found that overexpression of RHPN1-AS1 inhibited miR-7-5p expression and promoted EGFR expression, silencing RHPN1-AS1 inhibited proliferation and invasion, and induced G2/M arrest, apoptosis and autophagy in PCa cells. 3MA (an inhibitor of autophagy)-mediated autophagy inhibition attenuated RHPN1-AS1 inhibition-induced apoptosis. Overexpression miR-7-5p or silencing EGFR promoted LC3-I to LC3-II conversion, enhanced autophagy activity, induced cleaved-caspase-3 expression and apoptosis in PCa cells. Furthermore, overexpression of RHPN1-AS1 promoted phosphorylation of phosphatidylinositol 3-kinase (PI3K), AKT and mTOR, inhibited LC3-I to LC3-II conversion and reduced apoptosis in PCa cells, while GSK2126458 (an inhibitor of PI3K) reversed the effect of RHPN1-AS1 on PCa cells. In summary, RHPN1-AS1 acted as a ceRNA of miR-7-5p to upregulate EGFR expression, silencing RHPN1-AS1 suppressed PCa tumor progression by inducing autophagy and apoptosis in PCa cells through the miR-7-5p/EGFR/PI3K/AKT/mTOR pathway.

Cancer and malaria are both global health threats. Due to the increase in the resistance to the known drugs, research on new active substances is a priority. Here, we present the design, synthesis, and evaluation of the biological activity of harmicens, hybrids composed of covalently bound harmine/β-carboline and ferrocene scaffolds. Structural diversity was achieved by varying the type and length of the linker between the β-carboline ring and ferrocene, as well as its position on the β-carboline ring. Triazole-type harmicens were prepared using Cu(I)-catalyzed azide-alkyne cycloaddition, while the synthesis of amide-type harmicens was carried out by applying a standard coupling reaction. The results of in vitro biological assays showed that the harmicens exerted moderate antiplasmodial activity against the erythrocytic stage of P. falciparum (IC50 in submicromolar and low micromolar range) and significant and selective antiproliferative activity against the MCF-7 and HCT116 cell lines (IC50 in the single-digit micromolar range, SI > 5.9). Cell localization experiments showed different localizations of nonselective harmicene 36 and HCT116-selective compound 28, which clearly entered the nucleus. A cell cycle analysis revealed that selective harmicene 28 had already induced G1 cell cycle arrest after 24 h, followed by G2/M arrest with a concomitant drastic reduction in the percentage of cells in the S phase, whereas the effect of nonselective compound 36 on the cell cycle was much less pronounced, which agreed with their different localizations within the cell.

The two estrogen receptors ERα and ERβ are nuclear receptors that bind estrogen (E2) and function as ligand-inducible transcription factors. They are homologues and can form dimers with each other and bind to the same estrogen-response element motifs in the DNA. ERα drives breast cancer growth whereas ERβ has been reported to be anti-proliferative. However, they are rarely expressed in the same cells, and it is not fully investigated to which extent their functions are different because of inherent differences or because of different cellular context. To dissect their similarities and differences, we here generated a novel estrogen-dependent cell model where ERα homodimers can be directly compared to ERβ homodimers within the identical cellular context. By using CRISPR-cas9 to delete ERα in breast cancer MCF7 cells with Tet-Off-inducible ERβ expression, we generated MCF7 cells that express ERβ but not ERα. MCF7 (ERβ only) cells exhibited regulation of estrogen-responsive targets in a ligand-dependent manner. We demonstrated that either ER was required for MCF7 proliferation, but while E2 increased proliferation via ERα, it reduced proliferation through a G2/M arrest via ERβ. The two ERs also impacted migration differently. In absence of ligand, ERβ increased migration, but upon E2 treatment, ERβ reduced migration. E2 via ERα, on the other hand, had no significant impact on migration. RNA sequencing revealed that E2 regulated a transcriptome of around 800 genes via each receptor, but over half were specific for either ERα or ERβ (417 and 503 genes, respectively). Functional gene ontology enrichment analysis reinforced that E2 regulated cell proliferation in opposite directions depending on the ER, and that ERβ specifically impacted extracellular matrix organization. We corroborated that ERβ bound to cis-regulatory chromatin of its unique proposed migration-related direct targets ANXA9 and TFAP2C. In conclusion, we demonstrate that within the same cellular context, the two ERs regulate cell proliferation in the opposite manner, impact migration differently, and each receptor also regulates a distinct set of target genes in response to E2. The developed cell model provides a novel and valuable resource to further complement the mechanistic understanding of the two different ER isoforms.

Renal fibrosis is a serious condition that results in the development of chronic kidney diseases. The MEN1 gene is an epigenetic regulator that encodes the menin protein and its role in kidney tissue remains unclear.Kidney histology was examined on paraffin sections stained with hematoxylin-eosin staining. Masson's trichrome staining and Sirius red staining were used to analyze renal fibrosis. Gene and protein expression were determined by quantitative real-time PCR (qPCR) and Western blot, respectively. Immunohistochemistry staining in the kidney tissues from mice or patients was used to evaluate protein levels. Flow cytometry was used to analyze the cell cycle distributions and apoptosis. RNA-sequencing was performed for differential expression genes in the kidney tissues of the Men1f/f and Men1∆/∆ mice. Chromatin immunoprecipitation sequencing (ChIP-seq) was carried out for identification of menin- and H3K4me3-enriched regions within the whole genome in the mouse kidney tissue. ChIP-qPCR assays were performed for occupancy of menin and H3K4me3 at the gene promoter regions. Luciferase reporter assay was used to detect the promoter activity. The exacerbated unilateral ureteral obstruction (UUO) models in the Men1f/f and Men1∆/∆ mice were used to assess the pharmacological effects of rh-HGF on renal fibrosis.The expression of MEN1 is reduce in kidney tissues of fibrotic mouse and human diabetic patients and treatment with fibrotic factor results in the downregulation of MEN1 expression in renal tubular epithelial cells (RTECs). Disruption of MEN1 in RTECs leads to high expression of α-SMA and Collagen 1, whereas MEN1 overexpression restrains epithelial-to-mesenchymal transition (EMT) induced by TGF-β treatment. Conditional knockout of MEN1 resulted in chronic renal fibrosis and UUO-induced tubulointerstitial fibrosis (TIF), which is associated with an increased induction of EMT, G2/M arrest and JNK signaling. Mechanistically, menin recruits and increases H3K4me3 at the promoter regions of hepatocyte growth factor (HGF) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts5) genes and enhances their transcriptional activation. In the UUO mice model, exogenous HGF restored the expression of Adamts5 and ameliorated renal fibrosis induced by Men1 deficiency.These findings demonstrate that MEN1 is an essential antifibrotic factor in renal fibrogenesis and could be a potential target for antifibrotic therapy.

Ajwa dates (Phoenix dactylifera L.) have been described in traditional and alternative medicine to provide several health benefits, but their mechanism of apoptosis induction against human triple-negative breast cancer MDA-MB-231 cells remains to be investigated. In this study, we analyzed the phytoconstituents in ethanolic Ajwa Dates Pulp Extract (ADPE) by liquid chromatography-mass spectrometry (LC–MS) and investigated anticancer effects against MDA-MB-231 cells. LC–MS analysis revealed that ADPE contained phytocomponents belonging to classes such as carbohydrates, phenolics, flavonoids and terpenoids. MTT assay demonstrated statistically significant dose- and time-dependent inhibition of MDA-MB-231 cells with IC50 values of 17.45 and 16.67 mg/mL at 24 and 48 h, respectively. Hoechst 33342 dye and DNA fragmentation data showed apoptotic cell death while AO/PI and Annexin V-FITC data revealed cells in late apoptosis at higher doses of ADPE. More importantly, ADPE prompted reactive oxygen species (ROS) induced alterations in mitochondrial membrane potential (MMP) in ADPE treated MDA-MB-231 cells. Cell cycle analysis demonstrated that ADPE induced cell arrest in S and G2/M checkpoints. ADPE upregulated the p53, Bax and cleaved caspase-3, thereby leading to the downregulation of Bcl-2 and AKT/mTOR pathway. ADPE did not show any significant toxicity on normal human peripheral blood mononuclear cells which suggests its safe application to biological systems under study. Thus, ADPE has the potential to be used as an adjunct to the mainline of treatment against breast cancer.

Hepatocellular carcinoma is one of the most common malignancies and leading cancer-associated deaths worldwide. Ozone has been proposed as a promising therapeutic agent in the treatment of various disorders. The purpose of this paper is to assess the potential anticancer effects of the ozone on liver cancer cells. The liver cancer cell line of bel7402 and SMMC7721 was used in this study. Proliferation was evaluated using the CCK-8 and the colony formation assay. Wond healing assay and transwell assay without Matrigel were used to evaluate their migration ability. Flow cytometry was used for cell cycle analysis and reactive oxygen species (ROS) determination. Glutathione detection kit was used for measurement of glutathione level. Protein expression was estimated by western blot analysis. Ozone treatment inhibited liver cancer cell proliferation, colony formation. Ozone induced G2/M phase cell cycle arrest, which could be elucidated by the change of protein levels of p53, p21, Cyclin D1, cyclin B1, cdc2, and CDK4. We also found that ozone treatment inhibited migration ability by inhibiting EMT-relating protein. Ozone also induced ROS accumulation and decreased glutathione level decreased, which contributed to the inactivation of the PI3K/AKT/NF-κB pathway. Finally, we found that pre-treatment of liver cancer cells with N-acetylcysteine resisted ozone-induced effects. Ozone restrains the proliferation and migration potential and EMT process of liver cancer cells via ROS accumulation and PI3K/AKT/NF-κB suppression.

Background To determine the effects of concurrent irradiation and T-DM1 on HER2-positive breast cancer cell lines. Methods Five human breast cancer cell lines (in vitro study) presenting various levels of HER2 expression were used to determine the potential therapeutic effect of T-DM1 combined with radiation. The toxicity of T-DM1 was assessed using viability assay and cell cycle analysis was performed by flow cytometry after BrdU incorporation. HER2 cells were irradiated at different dose levels after exposure to T-DM1. Survival curves were determined by cell survival assays (after 5 population doubling times). Results The results revealed that T-DM1 induced significant lethality due to the intracellular action of DM1 on the cell cycle with significant G2/M phase blocking. Even after a short time incubation, the potency of T-DM1 was maintained and even enhanced over time, with a higher rate of cell death. After irradiation alone, the D10 (dose required to achieve 10% cell survival) was significantly higher for high HER2-expressing cell lines than for low HER2-expressing cells, with a linearly increasing relationship. In combination with irradiation, using conditions that allow cell survival, T-DM1 does not induce a radiosensitivity. Conclusions Although there is a linear correlation between intrinsic HER2 expression and radioresistance, the results indicated that T-DM1 is not a radiation-sensitizer under the experimental conditions of this study that allowed cell survival. However, further investigations are needed, in particular in vivo studies before reaching a final conclusion.

CDK1 plays key roles in cell cycle progression through the G2/M phase transition and activation of homologous recombination (HR) DNA repair pathway. Accordingly, various CDK1 inhibitors have been developed for cancer therapy that induce prolonged G2 arrest and/or sensitize cells to DNA damaging agents in tumor cells, resulting in cell death. However, CDK1 inhibition can induce resistance to DNA damage in certain conditions. The mechanism of different DNA damage sensitivity is not completely understood. We performed immunofluorescence and flow cytometry analysis to investigate DNA damage responses in human tumor cells during low and high dose treatments with RO-3306, a selective CDK1 inhibitor. This comparative investigation demonstrated that RO-3306-induced G2 arrest prevented cells with DNA double-strand breaks from transitioning into the M-phase and that the cells maintained their DNA repair capacity in G2-phase, even under RO-3306 dose-dependent DNA repair inhibition. These findings reveal that CDK1 inhibitor-induced DNA repair inhibition and cell cycle control, which regulate each other during the G2/M phase transition determine the cellular sensitivity to DNA damage, providing insight useful for developing clinical strategies targeting CDK1 inhibition in tumor cells.

Background Radiotherapy is one of the main strategies for the treatment of esophageal squamous cell carcinoma (ESCC). However, treatment failure often occurs due to the emergence of radioresistance. In this study, we report a key regulator of radiation sensitivity, termed TAB182 that may become an ideal biomarker and therapeutic target to overcome radioresistance. Materials and Methods By applying qRT‐PCR and immunohistochemical staining, the expression of TAB182 was detected in patient tissues. We next assessed the influence of TAB182 downregulation to radiosensitivity using clonogenic survival assay and γ‐H2A.X foci analysis in TE‐1, TE‐10, and radioresistant TE‐1R cell lines after ionizing radiation. To unveil the mechanism underlying, TAB182 interacting proteins were identified by mass spectrometry following co‐immunoprecipitation. Furthermore, flow cytometry and western blot assay were applied to validate the identified proteins. Results Our results demonstrated that the expression of TAB182 is higher in cancer tissues than normal tissues and elevated expression of TAB182 correlates with poor outcomes of postoperative radiotherapy. Downregulation of TAB182 sensitized cancer cells to ionizing radiation, particularly in radioresistant TE‐1R cells that spontaneously overexpress TAB182. Mechanically, TAB182 interacts with FHL2 to induce G2‐M arrest through wiring the CHK2/CDC25C/CDC2 signaling pathway. Finally, overexpression of shRNA‐resistant TAB182 restored the checkpoint and radioresistance. Conclusion TAB182 potentiates the radioresistance of ESCC cells by modulating the G2‐M checkpoint through its interaction with FHL2. Thus, TAB182 may become an ideal biomarker and therapeutic target of ESCC radiotherapy., Our findings suggest a novel mechanism of radioresistance of ESCC in which TAB182 modulates G2‐M checkpoint to potentiate ESCC radioresistance. Therefore, TAB182 may serve as an ideal biomarker to predict prognosis of postoperative radiotherapy and may become a potent therapeutic target to sensitize ESCC to radiotherapy.

BackgroundForkhead transcription factors control cell growth in multiple cancer types.Foxd1is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported.MethodsTranscriptome data from the TCGA database was used to correlateFOXD1expression with patient survival.FOXD1was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of theFOXD1pathway with cell cycle progression was investigated through correlation analysis using the TCGA database.ResultsFOXD1expression level in ccRCC correlated inversely with patient survival. Knockout ofFOXD1in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-OFOXD1null. In vivo, xenografted 786-OFOXD1nullshowed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-OFOXD1nullhad an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-OFOXD1null, and increased DNA damage. Genes correlate withFOXD1in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation.ConclusionsWe show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression ofFOXD1and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.

Fuchs endothelial corneal dystrophy (FECD) is an age-related disease whereby progressive loss of corneal endothelial cells (CEnCs) leads to loss of vision. There is currently a lack of therapeutic interventions as the etiology of the disease is complex, with both genetic and environmental factors. In this study, we have provided further insights into the pathogenesis of the disease, showing a causal relationship between senescence and endothelial-mesenchymal transition (EMT) using in vitro and in vivo models. Ultraviolet A (UVA) light induced EMT and senescence in CEnCs. Senescent cells were arrested in G2/M phase of the cell cycle and responsible for the resulting profibrotic phenotype. Inhibiting ATR signaling and subsequently preventing G2/M arrest attenuated EMT. In vivo, UVA irradiation induced cell cycle re-entry in post mitotic CEnCs, resulting in senescence and fibrosis at 1- and 2-weeks post-UVA. Selectively eliminating senescent cells using the senolytic cocktail of dasatinib and quercetin attenuated UVA induced fibrosis, highlighting the potential for a new therapeutic intervention for FECD.

Melanoma is aggressive, highly metastatic, and potentially fatal. In the case of patients with advanced melanoma, it is difficult to expect a good prognosis, since this cancer has low sensitivity to chemotherapy and radiation therapy. The use of natural ingredients may enhance existing therapies. Centipedegrass extract (CGE) which contains phenolic structures and C-glycosyl flavones, has been shown to have anti-inflammatory effects and anti-cancer effects. The purpose of this study was to evaluate the radio sensitizing effects of CGE in combination with ionizing radiation (IR). Two melanoma cell lines were exposed to IR after treatment with CGE at concentrations that were not toxic alone. The effects of CGE + IR on cell survival, cell cycle, and apoptotic cell death were examined using MTT and Muse® Cell Analyzer, and fluorescence microscopy. Molecular signaling mechanisms were explored by western blots. Our findings showed that co-treatment of CGE + IR reduced the survival of melanoma cells more than IR alone. Also, cell cycle arrest in CGE-treated cells was enhanced and these cells became more radiosensitive. CGE + IR increased apoptotic cell death more than IR alone. Western blot results showed that the effect of CGE + IR involved MAPKs (ERK1/2, p38, and JNK) pathway. Our study suggests that CGE + IR treatment enhanced radio-sensitization and cell death of melanoma cells via cell cycle arrest and the MAPKs pathway.

A series of heteroleptic copper(II) complexes of the composition [Cu(L1–5)Cl]X, where X = ClO4 and/or PF6 and [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazol-1-yl))-(6-methyl-(2-pyridylmethyl))]amine (L1), [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazol-1-yl))-(3,4-dimethoxy-(2-pyridylmethyl))]amine (L2), [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazol-1-yl)-(2-quinolymethyl)]amine (L3), [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazolyl)-(di(3,5-dimethyl-1H-pyrazol-1-yl-methyl))]amine (L4) and [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazol-1-yl)-(5-methyl-3-phenyl-1H-pyrazol-1-yl-methyl)]amine (L5), were prepared and thoroughly characterized including single-crystal X-ray diffraction technique. The in vitro cytotoxicity of complexes against A2780, A2780R, HOS and MCF-7 human cancer cell lines was evaluated using the MTT test. The results revealed that complexes [Cu(L1)Cl]PF6 (1-PF6), [Cu(L2)Cl]ClO4 (2-ClO4) and [Cu(L3)Cl]PF6 (3-PF6) are the most effective, with IC50 values ranging from 1.4 to 6.3 μM, thus exceeding the cytotoxic potential of metallodrug cisplatin (IC50 values ranging from 29.9 to 82.0 μM). The complexes [Cu(L4)Cl]PF6 (4-PF6) and [Cu(L5)Cl]PF6 (5-PF6) showed only moderate cytotoxicity against A2780, with IC50 = 53.6 μM, and 33.8 μM, respectively. The cell cycle profile, time-resolved cellular uptake, interactions with small sulfur-containing biomolecules (cysteine and glutathione), intracellular ROS production, induction of apoptosis and activation of caspases 3/7 were also evaluated in the case of the selected complexes. It has been found that the best performing complexes 1 and 2 cause cell arrest in the G2/M phase and induce apoptosis via the increase in production of ROS, dominantly due to the overproduction of superoxide.

A functional p53 protein plays an important role in killing tumor cells. Previous studies showed that chemotherapeutic drug, paclitaxel (PTX), showed anti-tumor activity through inducing G2/M arrest and apoptosis by targeting microtubules in tumor cells. However, PTX was not sensitive to p53-inactivated papillary thyroid carcinoma (PTC) cells by inducing G2/M arrest only. Recombinant adenovirus-p53 (rAd-p53) was used to increase the level of p53, which significantly increased the sensitivity of PTC cells to PTX by inducing S arrest, G2/M arrest and apoptosis. To discuss the anti-tumor mechanism of rAd-p53 + PTX and found p53 activation was necessary for anti-tumor effect of PTX in PTC cells. There was high level of p53 in rAd-p53-treated PTC cells. rAd-p53 + PTX increased the level of p21, p-ATM and γ-H2AX and decreased the level of Cyclin D1/E1, suggesting p53 activated p21 which negatively regulated cyclins to induce S arrest response to DNA damage in PTC cells. rAd-p53 + PTX increased the levels of cleaved-PARP-1, cleaved -Caspase 3, and BAX and decreased the level of BCL-XL, suggesting p53 regulates the expression of BAX/BCL-XL to mediate DNA damage-induced apoptosis in PTC cells. Furthermore, rAd-p53 + PTX showed significant tumor inhibition in TPC-1 xenograft model, with an inhibitory rate of 79.39%. TUNEL assay showed rAd-p53 + PTX induced notable apoptosis in tumor tissues. rAd-p53 showed good sensitization of PTX in vitro and in vivo through inducing DNA damage induced-apoptosis indicated p53-dependent apoptosis was essential for the antitumor effect of PTX in PTC.

Endometrial cancer (EC) is the most common gynecological cancer, and one of the most important causes of cancer‑related deaths in women worldwide. The long‑term survival rate is lower in advanced‑stage and recurrent EC, therefore it is important to identify new anticancer drugs. Garcinol, a polyisoprenylated benzophenone, is a promising anticancer drug for various cancer types but its effects on EC remain unclear. To investigate the anticancer effects of garcinol on EC, cell proliferation and cell cycle were assessed by real‑time cell proliferation, cell counting, and colony formation assays, flow cytometric analysis, and 5‑ethynyl‑2'‑deoxyuridine (EdU) incorporation assay, in EC Ishikawa (ISH) and HEC‑1B cell lines. Western blotting was used to evaluate the expression of cell cycle‑related protein cyclins, cyclin‑dependent kinase and tumor suppression proteins. Garcinol inhibited ISH and HEC‑1B cell proliferation in a dose‑dependent manner, and induced ISH and HEC‑1B cell cycle arrest at the G1 phase and G2/M phase, respectively, and decreased the S phase and DNA synthesis in these two cell lines. Following garcinol treatment the expression levels of p53 and p21 were increased, while the expression levels of CDK2, CDK4, cyclin D1 and cyclin B1 were gradually decreased in a dose‑dependent manner in both ISH and HEC‑1B cells. In addition, the expression levels of phosphorylated c‑JUN N‑terminal kinase (JNK) and p‑c‑JUN were significantly increased in both types of cells. Collectively, garcinol can induce EC cell cycle arrest and may be a promising candidate for EC chemotherapy.