Protein Levels Of PARP Research Articles

ARGININE DEPRIVATION SENSITISES GBM CELLS TO RADIATION THROUGH DOWNREGULATION OF DNA REPAIR PATHWAYS

Abstract AIMS Glioblastoma (GBM) is the most aggressive form of brain cancer, and therapy hasn’t changed for almost 20 years. We have previously shown that GBM tumours lack expression of argininosuccinate synthase 1 (ASS1), a key enzyme involved in arginine biosynthesis, due to promoter methylation. This renders them sensitive to systemic arginine depletion using agents such as pegylated arginine deiminase (ADI), allowing us to circumvent the issue of blood-brain-barrier (BBB) penetration. Furthermore, ADI appears to synergise with the DNA damaging agent temozolomide (TMZ), which is routinely used to treat patients with GBM. Here, we aimed to explore whether ADI can synergise with radiotherapy, and determine the mechanisms by which ADI is able to sensitise cells to DNA damaging agents. METHOD Primary GBM cell lines were treated with ADI in combination with radiation and underwent analysis using an RNA microarray, Qiagen DNA repair RT2 array, qPCR, western blotting, and proliferation assays in both 2D (SRB) and 3D (spheroid, CCK8) format. We performed orthotopic injection of primary GBM cells into the striatum of athymic nude mice, and randomised them to ADI, radiation, or the combination, plus controls. Tissue was analysed using IHC staining. RESULTS In ASS1 methylated primary GBM, treatment with ADI led to a significant change in the expression of ~500 genes with a fold change >2. Gene set enrichment analysis indicated that these genes were predominantly involved in cell cycle regulation, which is closely linked to the DNA damage and repair machinery. In another ASS1 methylated primary GBM cell line, an RT2 array revealed specific downregulation of genes involved in DNA repair. qPCR and western blot validation of these changes showed a significant decrease in the expression and protein levels of PARP1 and LIG1 with ADI treatment. CONCLUSION Arginine deprivation using ADI leads to a downregulation of PARP1 in GBM cells, sensitising them to radiotherapy.

Eurycomanone inhibits osteosarcoma growth and metastasis by suppressing GRP78 expression

Ethnopharmacological relevanceOsteosarcoma (OS) is characterized by rapid growth and frequent pulmonary metastasis. Eurycoma longifolia Jack, a flowering plant primarily found in Southeast Asian countries, is commonly used in traditional herbal medicine. Its root extract is mainly used for against cancer, malaria, parasites and other conditions. The active compound in its root extract, eurycomanone (EUR), has been proven to inhibit lung and liver cancer proliferation. Aim of the studyOur research aimed to investigate the inhibitory effect and underlying molecular mechanism of EUR on OS growth and metastasis. Materials and methodsIn vitro experiments: western blotting (WB) screened 41 compounds that inhibited GRP78 expression and evaluated the protein levels of GRP78, PARP, cleaved-PARP, MMP2, and MMP9. Cell proliferation was evaluated using CCK-8, EdU, colony formation assay, and cell apoptosis was assessed by flow cytometry. Transwell, wound healing, and tube formation assays were performed to determine the effect of EUR on tumor invasion, migration, and angiogenesis, respectively. Quantitative real-time polymerase chain (qRT-PCR) and dual-luciferase activity assays detected GRP78 mRNA stability and transcription levels post-EUR and thapsigargin treatment. RNA-Seq identified signaling pathways inhibited by EUR. In vivo experiments: effects of EUR in mice were evaluated by H&E staining to detect lung metastasis and potential toxic effects in tissues. Immunohistochemical (IHC) staining detected the expression of Ki-67, CD31, and cleaved caspase-3 in tumors. ResultsGRP78 is highly expressed in OS and correlated with poor prognosis. In vitro, eurycomanone (EUR) significantly downregulated GRP78 expression, inhibited cell proliferation, migration, invasion, tube formation, and induced apoptosis. Moreover, it enhanced trichostatin A (TSA) sensitivity and exhibited inhibitory effects on other cancer types. Mechanistically, EUR decreased GRP78 mRNA stability and transcription. In vivo, EUR inhibited proliferation and invasion in tibial and PDX models. ConclusionsOur study demonstrated that EUR inhibits the growth and metastasis of OS by reducing GRP78 mRNA stability and inhibiting its transcription, which offers a novel approach for clinical treatment of OS.

The effect mechanism of ergosterol from the nutritional mushroom Leucocalocybe mongolica in breast cancer cells: Protein expression modulation and metabolomic profiling using UHPLC-ESI-Q

The ergosterol from mushrooms has gained significant ethnopharmacological importance in various cultures, including China, Japan, and Europe. This compound has been found to possess immune-boosting and anti-inflammatory properties, making it useful in the treatment of immune disorders. In this study, we focused on investigating the potential anticancer properties of ergosterol isolated from the edible mushroom Leucocalocybe mongolica in breast cancer cell lines. The ergosterol was purified and identified using advanced analytical techniques such as ESI-MS and NMR. We conducted cell proliferation assays on 4 T1 breast cancer cells to assess the cytotoxic effects of ergosterol. Furthermore, we analyzed the transcription levels of BAX, caspase-7, BCL-2, STAT-3, and PARP proteins using real-time PCR and Western blot analysis. Additionally, we employed non-targeted ultra-high-performance liquid chromatography and high-resolution mass spectrometry (UPLC-MS/MS) to study the potential mechanisms underlying the anticancer effects of ergosterol at the metabolomics level. The results demonstrated a significant reduction in cell viability and the induction of apoptosis upon treatment with ergosterol, especially at higher concentrations (P < 0.05). Moreover, ergosterol affected the expression of cancer-related genes, upregulating pro-apoptotic proteins such as BAX, caspase-7, and PARP, while downregulating the anti-apoptotic proteins BCL-2 and STAT-3 (P < 0.05). Western blot analysis confirmed these findings and provided further evidence of ergosterol's role in inducing apoptosis. Metabolomics analysis revealed substantial changes in pathways related to amino acid, antioxidant, and carbohydrate metabolism. In conclusion, our study demonstrates that ergosterol exhibits anticancer effects by inducing apoptosis and modulating metabolic pathways in breast cancer cells.

Formononetin alleviates cerebral ischemia-reperfusion injury in rats by targeting the PARP-1/PARG/Iduna signaling pathway

Formononetin has been demonstrated to protect against cerebral ischemia–reperfusion injury, however its mechanism has to be further researched. This study examined the effect of formononetin on cerebral ischemia–reperfusion injury in rats using the PARP-1/PARG/Iduna signaling pathway. In male SD rats, a model of cerebral ischemia–reperfusion injury was developed. Animals were randomly assigned to one of eight groups: Sham operation, Sham operation + formononetin, MCAO, MCAO + formononetin, PARP inhibitor (PJ34) + MCAO, formononetin + PJ34 + MCAO, PARG inhibitor (Ethacridine lactate) + MCAO, and ethacridine lactate + formononetin. The neurological deficit test, TTC staining, HE staining, Nissl staining, TUNEL staining, and western blotting were utilized to assess formononetin's protective effects in MCAO rats. The data show that formononetin can effectively alleviate neurological dysfunction and pathological changes in brain tissue in rats with cerebral ischemia–reperfusion injury, reduce the area of cerebral infarction and neuronal apoptosis, decrease the protein levels of PARP-1, PARG, Caspase-3, P53, and AIF in brain tissue, and increase the protein levels of Iduna and p-AKT. As a result, we concluded that formononetin improves brain ischemia–reperfusion injury in rats by modulating the PARP-1/PARG/Iduna signaling pathway.

Abstract B030: PARP-1 inhibition results in ovarian and endometrial cancer cell death by disrupting the nucleolar localization of the RNA helicase DDX21 which is associated with decreased survival outcomes

Abstract Cancer cells with DNA repair defects (e.g., BRCA1/2 mutant cells) are vulnerable to PARP inhibitors (PARPi) due to induction of synthetic lethality. However, recent clinical evidence has shown that PARPi can prevent the growth of some cancers irrespective of their BRCA1/2 status, suggesting alternative mechanisms of action. One such mechanism involves DDX21, an RNA helicase that localizes to the nucleoli of cells. When ADP-ribosylated by PARP-1, DDX21 promotes rDNA transcription leading to ribosome biogenesis, protein translation, and ultimately the growth of breast cancer cells. Validating this pathway in other tumor types may lead to the expanded use of PARPi as a cancer therapeutic and the potential use of DDX21 nucleolar localization as a biomarker of clinical response to PARPi. For our first aim, we hypothesized that PARPi also blocks the DDX21/PARP-1/ribosome biogenesis pathway in ovarian and endometrial cancers and can thus be used to inhibit their growth. To test this, we are determining the effect of Niraparib, an inhibitor targeting PARP-1 activity, on DDX21 subcellular localization, ribosome biogenesis, and cell growth in different ovarian (OVCAR3, OVCAR4, and HCC5012) and endometrial (KLE, Ishikawa, and HEC-1-A) cancer cell lines. We are using Western blotting to assess the levels of DDX21 and ADP-ribosylation, and immunofluorescent staining to assess the localization of DDX21 to nucleoli before and after treatment with Niraparib. We are also using qPCR to determine the levels of rRNAs and Puromycin incorporation assays to monitor ribosome translation activity before and after treatment with Niraparib. Moreover, we are using cell growth assays to determine the effect of Niraparib on the proliferation of the ovarian and endometrial cancer cell lines. Our cell-based results indicate that the nucleolar localization of DDX21 is disrupted by Niraparib treatment, leading to a pan-nuclear distribution. Furthermore, Niraparib treatment in ovarian cancer cell lines causes decreased rRNA transcription, decreased protein translation, and decreased cell proliferation. For our second aim, we are assessing possible associations among DDX21 nucleolar localization, PARP-1 protein levels, and clinical outcomes in cancer samples from patients using immunohistochemical (IHC) staining for DDX21 and PARP-1. Analysis of endometrial cancer patient samples suggests a significant association among high DDX21 nucleolar localization, high PARP-1 expression, and decreased survival. Overall, our results suggest that Niraparib works, in part, by blocking the DDX21/PARP-1/ribosome biogenesis pathway leading to endometrial and ovarian cancer cell death. The potential use of DDX21 localization as a biomarker of response to PARPi and as a prognostic factor will be further explored by conducting additional laboratory and clinical studies. Citation Format: Marwa W. Aljardali, Kevin M. Kremer, Jessica Parker, Jayanthi S. Lea, W. Lee Kraus, Cristel V. Camacho. PARP-1 inhibition results in ovarian and endometrial cancer cell death by disrupting the nucleolar localization of the RNA helicase DDX21 which is associated with decreased survival outcomes [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B030.

Cinnamaldehyde protects cardiomyocytes from oxygen-glucose deprivation/reoxygenation-induced lipid peroxidation and DNA damage via activating the Nrf2 pathway.

Rapid restoration of perfusion in ischemic myocardium is the most direct and effective treatment for coronary heart disease but may cause myocardial ischemia/reperfusion injury (MIRI). Cinnamaldehyde (CA, C9H8O), a key component in the well-known Chinese medicine cinnamomum cassia, has cardioprotective effects against MIRI. This study aimed to observe the therapeutic effect of CA on MIRI and to elucidate its potential mechanism. H9C2 rat cardiomyocytes were pretreated with CA solution at 0, 10, and 100 μM, respectively and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Then the cell viability, the NF-κB and caspase3 gene levels, the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, superoxide dismutase (SOD) level, reactive oxygen species (ROS) generation, 4-hydroxynonenal (4-HNE), and malondialdehyde (MDA) were detected. The severity of DNA damage was assessed by tail moment (TM) values using alkaline comet assay. Besides, the DNA damage-related proteins and the key proteins of the Nrf2 pathway were detected by western blot. CA treatment increased the cell viability, GHS/GSSG ratio, SOD level, PARP1, Nrf2, PPAR-γ, and HO-1 protein levels of H9C2 cardiomyocytes, while reducing NF-κB, caspase3, ROS level, 4-HNE and MDA content, γ-H2AX protein level, and TM values. Inhibition of the Nrf2 pathway reversed the effect of CA on cell viability and apoptosis of OGD/R induced H9C2 cardiomyocytes. Besides, 100 μM CA was more effective than 10 μM CA. In the OGD/R-induced H9C2 cardiomyocyte model, CA can protect cardiomyocytes from MIRI by attenuating lipid peroxidation and repairing DNA damage. The mechanism may be related to the activation of the Nrf2 pathway.

Upregulated PARP1 confers breast cancer resistance to CDK4/6 inhibitors via YB-1 phosphorylation

BackgroundCyclic-dependent kinase (CDK) 4/6 kinases, as the critical drivers of the cell cycle, are involved in the tumor progression of various malignancies. Pharmacologic inhibitors of CDK4/6 have shown significant clinical prospects in treating hormone receptor-positive and human epidermal growth factor receptor-negative (HR + /HER2-) breast cancer (BC) patients. However, acquired resistance to CDK4/6 inhibitors (CDK4/6i), as a common issue, has developed rapidly. It is of great significance that the identification of novel therapeutic targets facilitates overcoming the CDK4/6i resistance. PARP1, an amplified gene for CDK4/6i-resistant patients, was found to be significantly upregulated during the construction of CDK4/6i-resistant strains. Whether PARP1 drives CDK4/6i resistance in breast cancer is worth further study.MethodPARP1 and p-YB-1 protein levels in breast cancer cells and tissues were quantified using Western blot (WB) analysis, immunohistochemical staining (IHC) and immunofluorescence (IF) assays. Bioinformatics analyses of Gene Expression Profiling Interactive Analysis (GEPIA), Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE) datasets were applied to explore the relationship between YB-1/PARP1 protein levels and CDK4/6i IC50. Cell Counting Kit-8 (CCK-8) and crystal violet staining assays were performed to evaluate cell proliferation rates and drug killing effects. Flow cytometry assays were conducted to assess apoptosis rates and the G1/S ratio in the cell cycle. An EdU proliferation assay was used to detect the DNA replication ratio after treatment with PARP1 and YB-1 inhibitors. A ChIP assay was performed to assess the interaction of the transcription factor YB-1 and associated DNA regions. A double fluorescein reporter gene assay was designed to assess the influence of WT/S102A/S102E YB-1 on the promoter region of PARP1. Subcutaneous implantation models were applied for in vivo tumor growth evaluations.ResultsHere, we reported that PARP1 was amplified in breast cancer cells and CDK4/6i-resistant patients, and knockdown or inhibition of PARP1 reversed drug resistance in cell experiments and animal models. In addition, upregulation of transcription factor YB-1 also occurred in CDK4/6i-resistant breast cancer, and YB-1 inhibition can regulate PARP1 expression. p-YB-1 and PARP1 were upregulated when treated with CDK4/6i based on the WB and IF results, and elevated PARP1 and p-YB-1 were almost simultaneously observed during the construction of MCF7AR-resistant strains. Inhibition of YB-1 or PAPR1 can cause decreased DNA replication, G1/S cycle arrest, and increased apoptosis. We initially confirmed that YB-1 can bind to the promoter region of PARP1 through a ChIP assay. Furthermore, we found that YB-1 phosphorylated at S102 was crucial for PARP1 transcription according to the double fluorescein reporter gene assay. The combination therapy of YB-1 inhibitors and CDK4/6i exerted a synergistic antitumor effect in vitro and in vivo. The clinical data suggested that HR + /HER2- patients with low expression of p-YB-1/PARP1 may be sensitive to CDK4/6i in breast cancer.ConclusionThese findings indicated that a ‘‘YB-1/PARP1’’ loop conferred resistance to CDK4/6 inhibitors. Furthermore, interrupting the loop can enhance tumor killing in the xenograft tumor model, which provides a promising strategy against drug resistance in breast cancer.

Effect of IFN‑γ encapsulated liposomes on major signal transduction pathways in the lymphocytes of patients with lung cancer.

Globally, lung cancer affected 2.2 million individuals and caused 1.8 million deaths in 2021. Lung cancer is caused by smoking, genetics and other factors. IFN-γ has anticancer activity. However, the mechanism by which IFN-γ has an effect on lung cancer is not fully understood. The present study aimed to assess the effect of IFN-γ on the peripheral lymphocytes of patients with lung cancer compared with healthy controls. The efficacy of IFN-γ against oxidative stress was assessed using a comet repair assay and the effects of IFN-γ on p53, PARP1 and OGG1 genes and protein levels in lymphocytes was evaluated by RT-qPCR and western blotting. DNA damage was significantly reduced in the lymphocytes of patients treated with IFN-γ. However, there was no effect in the cells of healthy individuals after treatment with naked IFN-γ [IFN-γ (N)] and liposomal IFN-γ [IFN-γ (L)]. Following treatment with IFN-γ (N) and IFN-γ (L), the p53, PARP1 and OGG1 protein and gene expression levels were significantly increased (P<0.001). It has been suggested that IFN-γ may induce p53-mediated cell cycle arrest and DNA repair in patients. These findings supported the idea that IFN-γ (N) and IFN-γ (L) may serve a significant role in the treatment of lung cancer, via cell cycle arrest of cancer cells and repair mechanisms.

Gamma-mangostin Protects S16Y Schwann Cells Against tert-Butyl Hydroperoxide-induced Apoptotic Cell Death.

Peripheral neuropathy is a common complication that affects individuals with diabetes. Its development involves an excessive presence of oxidative stress, which leads to cellular damage in various tissues. Schwann cells, which are vital for peripheral nerve conduction, are particularly susceptible to oxidative damage, resulting in cell death. Gamma-mangostin (γ-mangostin), a xanthone derived from Garcinia mangostana, possesses cytoprotective properties in various pathological conditions. In this study, we employed S16Y cells as a representative Schwann cell model to investigate the protective effects of γ-mangostin against the toxicity induced by tert-Butyl hydroperoxide (tBHP). Different concentrations of γ-mangostin and tBHP were used to determine non-toxic doses of γ-mangostin and toxic doses of tBHP for subsequent experiments. MTT cell viability assays, cell flow cytometry, and western blot analysis were used for evaluating the protective effects of γ-mangostin. The results indicated that tBHP (50 μM) significantly reduced S16Y cell viability and induced apoptotic cell death by upregulating cleaved caspase-3 and cleaved PARP protein levels and reducing the Bcl- XL/Bax ratio. Notably, pretreatment with γ-mangostin (2.5 μM) significantly mitigated the decrease in cell viability caused by tBHP treatment. Furthermore, γ-mangostin effectively reduced cellular apoptosis induced by tBHP. Lastly, γ-mangostin significantly reverted tBHP-mediated caspase-3 and PARP cleavage and increased the Bcl-XL/Bax ratio. Collectively, these findings highlight the ability of γ-mangostin to protect Schwann cells from apoptotic cell death induced by oxidative stress.

Mechanistic Studies of Arene-Ruthenium(II) Complexes with Carbothioamidopyrazoles as Alternative Cancer Drugs.

Arene-ruthenium(II) complexes with carbothioamidopyrazoles at the C-2 and C-5 positions have been recognized as chemotherapeutic agent alternatives to cisplatin and its oxaliplatin analogs. The aim of this study was to continue research on the biological aspect of arene-ruthenium(II) complexes and their anticancer activity. The present paper includes an additional 12 new tumor cells, analyzed by MTT, and employs a series of extended bioassays to better understand their potential mechanism of antitumor activity. The following tests were conducted: membrane permeability studies, intramolecular reactive oxygen and nitrogen species (ROS/RNS) assays, mitochondrial potential changes, DNA analysis by comet assay using the electrophoresis method, measurement of cleaved PARP protein levels, and determination of apoptotic and necrotic cell fractions by fluorescence microscopy. Additionally, the article presents lipophilicity studies based on RP-TLC and molecular docking studies. We hope that the presented data will prove useful in practical treatment, especially for patients with cancer.

Analysis of the PARP1, ADP-Ribosylation, and TRIP12 Triad With Markers of Patient Outcome in Human Breast Cancer

PARP inhibitors (PARPi) are increasingly used in breast cancer therapy, including high-grade triple-negative breast cancer (TNBC) treatment. Varying treatment responses and PARPi resistance with relapse currently pose limitations to the efficacy of PARPi therapy. The pathobiological reasons why individual patients respond differently to PARPi are poorly understood. In this study, we analyzed expression of PARP1, the main target of PARPi, in normal breast tissue, breast cancer, and its precursor lesions using human breast cancer tissue microarrays covering a total of 824 patients, including more than 100 TNBC cases. In parallel, we analyzed nuclear adenosine diphosphate (ADP)-ribosylation as a marker of PARP1 activity and TRIP12, an antagonist of PARPi-induced PARP1 trapping. Although we found PARP1 expression to be generally increased in invasive breast cancer, PARP1 protein levels and nuclear ADP-ribosylation were lower in higher tumor grade and TNBC samples than non-TNBCs. Cancers with low levels of PARP1 and low levels of nuclear ADP-ribosylation were associated with significantly reduced overall survival. This effect was even more pronounced in cases with high levels of TRIP12. These results indicate that PARP1-dependent DNA repair capacity may be compromised in aggressive breast cancers, potentially fueling enhanced accumulation of mutations. Moreover, the results revealed a subset of breast cancers with low PARP1, low nuclear ADP-ribosylation, and high TRIP12 levels, which may compromise their response to PARPi, suggesting a combination of markers for PARP1 abundance, enzymatic activity, and trapping capabilities might aid patient stratification for PARPi therapy.

Upregulation of Glutaminyl Cyclase Contributes to ERS-Induced Apoptosis in PC12 Cells.

Glutaminyl cyclase (QC) is responsible for converting the N-terminal glutaminyl and glutamyl of the proteins into pyroglutamate (pE) through cyclization. It has been confirmed that QC catalyzes the formation of neurotoxic pE-modified Aβ in the brain of AD patients. But the effects of upregulated QC in diverse diseases have not been much clear until recently. Here, RNA sequencing was applied to identify differentially expressed genes (DEGs) in PC12 cells with QC overexpressing or knockdown. A total of 697 DEGs were identified in QC overexpressing cells while only 77 in QC knockdown cells. Multiple bioinformatic approaches revealed that the DEGs in QC overexpressing group were enriched in endoplasmic reticulum stress (ERS) related signaling pathways. The gene expression patterns of 23 DEGs were confirmed by RT-qPCR, in which the genes related to ERS showed the highest consistency. We also revealed the protein levels of GRP78, PERK, CHOP, and PARP-1, and caspase family was significantly upregulated by overexpressing QC. Moreover, overexpressing QC significantly increased apoptosis of PC12 cells in a time dependent manner. However, no significant alteration was observed in QC knockdown cells. Therefore, our study indicated that upregulated QC could induce ERS and apoptosis, which consequently trigger diseases by catalyzing the generation of pE-modified mediators.

The protective effect of 17 β-estradiol on human uterosacral ligament fibroblasts from postmenopausal women with pelvic organ prolapse.

This study aims to explore the protective effects of 17 β-estradiol on the human uterosacral ligament fibroblasts (hUSLFs) under static or stretched conditions. The experiments were performed on hUSLFs derived from pelvic organ prolapse (POP) and non-POP patients. Fibroblasts were cultured after collagenase digestion and identified by morphological observation and immunocytochemical methods. 17 β-estradiol (10−10 M and 10−9 M) and mechanical stress induced by the FX-5000 T-cell stress loading system under a loading strain of 1/2 sin waveform uniaxial cyclic stress with a tensile strain of 20% and a frequency of 0.5 Hz were either or both applied on hUSLFs. Cell proliferation was measured by CCK8, and cell apoptosis and death were detected using Annexin V/7-AAD staining and flow cytometric analysis. We found that the fibroblasts growth rate of POP patients was significantly lower than controls. The cell apoptosis and death rate increased as the mechanical load intensifying. After 20% mechanical stretching for 24 h, the dead cell rate was higher in POP than control. Notably, 17 β-estradiol treatment reversed mechanical stress induced hUSLFs apoptosis and death in both POP and Control cells. The protein and mRNA levels of anti-apoptotic PARP1 (poly-ADP-ribose polymerase) and Bcl-2 were increased by estrogen treatment. Meanwhile, expression of estrogen receptor α, a target of Poly-ADP-Ribosylation of PARP1, was also enhanced by 17 β-estradiol under the mechanical load. In conclusion, estrogen application ameliorates the mechanical strain induced cell apoptosis and death in hUSLFs from POP patients. PARP1 might be involved in this protective process, providing novel insights into the mechanical biology of and possible therapies for POP.

Garmultin-A Incites Apoptosis in CB3 Cells Through miR-17-5p by Attenuating Poly (ADP-Ribose) Polymerase-1.

BackgroundLeukemia accounts for a large number of deaths, worldwide, every year. Treating this ailment is always a challenging job. Recently, oncogenic miRNA leading to apoptosis are highly promising targets of many natural products. In this study, Garmultin-A (GA), isolated from the bark of Garcinia multiflora, was elucidated for its anti-leukemic effect in CB3 cells.MethodsThe effect of the compound on CB3 cell viability was detected by MTT assay and apoptosis by FITC Annexin V/PI and Hochest 33258 staining. The western blot analysis assessed the BAX, BCL2, cMYC, pERK, and PARP-1 protein levels. Autodock analysis predicted the ligand–protein interactions. q-RT-PCR quantified the miR-17-5p expression. Luciferase assay confirmed the interaction between PARP-1 and miR-17-5p.ResultsWe uncover that GA leads to apoptosis by inducing overexpression of miR-17-5p and significantly downregulate PARP-1 protein levels in CB3 cells. The overexpression of miR-17-5p promotes apoptosis, and the miR-17-5p antagomirs restore GA-triggered apoptosis. Notably, we disclose that PARP-1 is a direct target of miR-17-5p. Increased pro-apoptotic and reduced anti-apoptosis protein levels were also observed in GA-treated CB3 cells.ConclusionThese results provide critical insights that GA could induce apoptosis in CB3 cells through targeting miR-17-5p by attenuating PARP-1. Thus, GA could act as a novel therapeutic agent for erythroleukemia.

Genomic and biological aspects of resistance to selective poly(ADP-ribose) glycohydrolase inhibitor PDD00017273 in human colorectal cancer cells.

Poly(ADP-ribose) glycohydrolase (PARG) is a key enzyme in poly(ADP-ribose) (PAR) metabolism and a potential anticancer target. Many drug candidates have been developed to inhibit its enzymatic activity. Additionally, PDD00017273 is an effective and selective inhibitor of PARG at the first cellular level. Using human colorectal cancer (CRC) HCT116 cells, we investigated the molecular mechanisms and tumor biological aspects of the resistance to PDD00017273. HCT116RPDD , a variant of the human CRC cell line HCT116, exhibits resistance to the PARG inhibitor PDD00017273. HCT116RPDD cells contained specific mutations of PARG and PARP1, namely, PARG mutation Glu352Gln and PARP1 mutation Lys134Asn, as revealed by exome sequencing. Notably, the levels of PARG protein were comparable between HCT116RPDD and HCT116. In contrast, the PARP1 protein levels in HCT116RPDD were significantly lower than those in HCT116. Consequently, the levels of intracellular poly(ADP-ribosyl)ation were elevated in HCT116RPDD compared to HCT116. Interestingly, HCT116RPDD cells did not exhibit cross-resistance to COH34, an additional PARG inhibitor. Our findings suggest that the mutated PARG acquires PDD00017273 resistance due to structural modifications. In addition, our findings indicate that PDD00017273 resistance induces mutation and PARP downregulation. These discoveries collectively provide a better understanding of the anticancer candidate PARG inhibitors in terms of resistance mechanisms and anticancer strategies.

Epithelial tumor compartment- and adjacent stromal compartment-specific expression of PARP1 in different anatomical sites in patients with HGSOC

High-grade serous ovarian carcinoma (HGSOC) is responsible for the majority of ovarian cancer-associated deaths. PARP (poly-(ADP-ribose) polymerase) inhibitors (PARPi) (olaparib, rucaparib and niraparib) have been approved in recent years to be used in both the frontline setting as maintenance therapy and in the recurrent setting for patients with ovarian cancer (OC). Previous studies have reported an increased expression of PARP1, a sensor for DNA damage, in OC; however, its compartment (epithelial tumor vs stroma)-, anatomical site (fallopian tube, omentum, ovary and serous tubal in situ carcinoma)- and histotype-specific expression have not been studied. Here, we showed that PARP1 protein levels are higher in epithelial tumors compared to adjacent non-malignant stroma at four different anatomical locations in HGSOC, with the most significant difference in PARP1 levels in omental metastases. Furthermore, we found that PARP1 protein levels are elevated in tumors relative to normal tissue in the fallopian tube, omentum and ovary in patients with HGSOC, again with the most significant difference in the omentum. We also showed that PARP1 expression is increased in a malignant and invasive stage in mouse ovarian surface epithelium cells, and that PARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial OC. These findings indicate that a better understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in OC is needed since PARP inhibitors are currently being used in the treatment of patients with OC.

High Expression of PARP1 in Tumor and Stroma Cells Predicts Different Prognosis and Platinum Resistance in Patients With Advanced Epithelial Ovarian Cancer.

ObjectiveThis study aimed to explore the roles of PARP1 mRNA and protein expression in platinum resistance and prognosis of EOC patients, and reveal the different roles of PARP1 protein in epithelial tumor and stroma cells.MethodsThe PARP1 mRNA expression of the EOC tissues was examined by RT-qPCR. The impacts of PARP1 expression on prognosis were measured by Kaplan-Meier and Cox regression. Receiver operating characteristic (ROC) curve analysis was employed for calculating the diagnostic value of PARP1 on platinum resistance. The microarray of formalin-fixed, paraffin-embedded (FFPE) tissues was processed for multiplex immunofluorescence to detect the protein levels of PARP1 and cytokeratin (CK).ResultsThe PARP1mRNA expression of EOC patients was higher in the platinum-resistant group compared with the sensitive group (P<0.01). Kaplan-Meier analysis demonstrated that high PARP1 mRNA expression was associated with poor survival of EOC patients. In Cox regression analyses, high PARP1 mRNA expression independently predicted poor prognosis (P=0.001, HR=2.076, 95%CI=1.373-3.140). The area under the ROC curve of PARP1 mRNA for predicting the platinum resistance in EOC patients was 0.649, with a sensitivity of 0.607 and specificity of 0.668. Furthermore, the protein expression of PARP1 was higher in the platinum-resistant group than in the sensitive group (P<0.01) and associated with a worse prognosis. Additionally, according to CK labeling, we observed that enhanced expression of PARP1 in the CK+ region was associated with platinum resistance and lower survival, but in CK- region, it predicted a good prognosis and platinum sensitivity.Conclusion PARP1 may be a potential biomarker to predict platinum resistance and prognosis for EOC patients, exerting different roles on epithelial tumor and stromal cells.

Diprenylated flavonoids from licorice induce death of SW480 colorectal cancer cells by promoting autophagy: Activities of lupalbigenin and 6,8-diprenylgenistein

Ethnopharmacological relevanceLicorice is a well-known herbal medicine, and we previously found that several licorice prenylated flavonoids could cause death of SW480 colorectal cancer cells by promoting autophagy. Given many kinds of prenylated flavonoids in licorice, the activities of other compounds deserve further investigation. In addition, the contribution of isoprenyl groups on the autophagy promotion activities has not been clarified. Aim of the studyThis study aimed to investigate whether lupalbigenin (LPB) and 6,8-diprenylgenistein (DPG), two licorice diprenylated flavonoids, could induce autophagic cell death of SW480 cells, and clarify the contribution of isoprenyl groups. Materials and methodsCytotoxic activities of LPB and DPG were tested by using an MTT method, and apoptosis induction effects were evaluated by PI-Annexin V staining-based flow cytometry and protein levels of caspase-3 and PARP-1. Autophagy promotion effects of LPB and DPG were assessed by protein levels of LC3, p62, Akt and mTOR as well as number of autophagosomes in cells, and autophagy inhibitor chloroquine (CQ) was involved to identify the role of autophagy on LPB or DPG-caused death of SW480 cells. In addition, two groups of structurally similar diprenylated, mono-prenylated and free flavonoids were obtained from licorice, which were used to investigate the contribution of isoprenyl groups on their autophagy promotion activities. ResultsBoth LPB and DPG significantly induced apoptosis of SW480 cells with strong cytotoxic activities, and meanwhile, they also promoted autophagy probably through the Akt/mTOR signaling pathway. Further studies indicated that LPB and DPG could induce autophagic cell death of SW480 cells. Moreover, isoprenyl groups contributed mainly to the cytotoxic and autophagy promotion activities of licorice prenylated flavonoids. ConclusionThis study reported for the first time that licorice diprenylated flavonoids LPB and DPG induced death of SW480 cells by promoting autophagy, which was mainly attributed to the isoprenyl groups. The results provided theoretical basis for researches on anti-colorectal cancer drugs and their structural modification.

Abstract 3244: Exploring the mechanisms of olaparib resistance in resistant-BRCA1 deficient murine ovarian cells

Abstract Background: High-grade serous ovarian carcinoma (HGSOC) is the most frequent histotype of ovarian cancer. More than half of HGSOC is characterized by homologous recombination deficiency (HRD) due to mutations in genes involved in this pathway, including BRCA1/2. Olaparib is a poly(ADP-ribose) polymerase inhibitor (PARPi) recently approved in front line and maintenance therapy in platinum-sensitive BRCA mutated ovarian cancer patients. Despite olaparib clinical benefits, this treatment is associated with the development of resistance. A better understanding of the mechanisms at its basis could help in finding strategies to delay or overcome/counteract it, possibly translatable in the clinical setting. Methods: We generated an olaparib resistant cell line (BRCA1-/- OlaR), starting from the BRCA1 and TP53 deleted murine ID8 cells (BRCA1-/-) by step wise increasing drug concentrations. Sensitive and resistant cell lines were biologically and pharmacologically characterized and the molecular mechanisms at the basis of olaparib resistance investigated. Cellular viability was assessed by MTS assay, for molecular studies real-time PCR, western blot and RAD51-foci immunofluorescence were performed. Results: BRCA1-/- OlaR cells were 24 fold time more resistant than BRCA1-/- cells and displayed a similar in vitro cell growth compared to the parental cells. Olaparib treatment at IC50 dose caused a longer and stronger G2-M block of the cell cycle in sensitive as compared to resistant cells. BRCA1-/- OlaR cells were cross-resistant to the other PARPis tested, suggesting a common mechanism of resistance. Platinum compounds showed similar cytotoxic activity in sensitive and resistant cells, while a partial cross-resistance was observed with doxorubicin. Interestingly, resistant cells were less responsive to a panel of DNA damage response (DDR) inhibitors (i.e. ATR, WEE1 and CHK1 inhibitors) compared to parental ones; however their combinations with olaparib were synergic in both the sensitive and resistant lines. A partial restoration of the HR pathway (increase in RAD51 foci formation after olaparib and IR treatment), a downregulation of PARP1 protein levels without mutation in PARP1 gene and an upregulation of the MDR transcript could be found in BRCA1-/- resistant cells, strongly supporting multiple heterogeneous mechanisms of resistance. Conclusions: The HR deficient cell line resistant to olaparib we generated showed multiple mechanisms of resistance. The combinations of olaparib with different DDR inhibitors were equally active in sensitive and resistant cells. Citation Format: Michela Chiappa, Martina Anselmi, Luca Russo, Monica Lupi, Nicolò Panini, Federica Guffanti, Giovanna Damia. Exploring the mechanisms of olaparib resistance in resistant-BRCA1 deficient murine ovarian cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3244.

Elucidating the Anticancer Mechanism of Arachidin‐1 in Triple‐Negative Breast Cancer Cells

Triple‐negative breast cancer (TNBC) is one of the deadliest forms of breast cancer that affects women worldwide. TNBC is unresponsive to standard hormonal cancer treatments, leading to high mortality rates. Treatment resistance and low survival rates necessitate investigating alternative treatment options for this type of cancer. Plant natural products, such as the prenylated stilbenoid arachidin‐1 from peanut, have shown cytotoxicity to certain cancer cells. To this end, the goal of this study was to examine the apoptotic effects of arachidin‐1 in TNBC cell lines MDA‐MB‐231 and MDA‐MB‐436. To produce arachidin‐1, peanut hairy root cultures were co‐treated with elicitors, and arachidin‐1 was purified from extracts of the culture medium by semi‐preparative high performance liquid chromatography. The apoptotic effects of arachidin‐1 were studied via western blotting by checking the protein levels of PARP, caspase‐8, caspase‐9, and survivin. Vinculin and GAPDH were used as protein loading controls. Results showed that as arachidin‐1 concentration increased, the amount of cleaved PARP also increased. Similarly, the levels of cleaved caspase‐9 increased as concentrations of arachidin‐1 increased. Whereas the level of survivin, an apoptosis inhibitor protein, decreased at increasing concentrations of arachidin‐1. The increased expression of cleaved PARP, cleaved caspase‐9, and reduced surviving expression in TNBC cells treated with arachidin‐1 represents the activation of intrinsic apoptosis in the cells. This accentuates the importance of expanding our understanding of the connection between stilbenoids and triple‐negative breast cancer cells. In future research, we will study the effect of arachidin‐1 on signaling pathways in TNBC cell lines.