PARP Protein Research Articles

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.

Recurrent Loss of Macrodomain Activity in Host Immunity and Viral Proteins.

Protein post-translational modifications (PTMs) are an important battleground in the evolutionary arms races that are waged between the host innate immune system and viruses. One such PTM, ADP-ribosylation, has recently emerged as an important mediator of host antiviral immunity. Important for the host-virus conflict over this PTM is the addition of ADP-ribose by PARP proteins and removal of ADP-ribose by macrodomain-containing proteins. Interestingly, several host proteins, known as macroPARPs, contain macrodomains as well as a PARP domain, and these proteins are both important for the host antiviral immune response and evolving under very strong positive (diversifying) evolutionary selection. In addition, several viruses, including alphaviruses and coronaviruses, encode one or more macrodomains. Despite the presence of the conserved macrodomain fold, the enzymatic activity of many of these proteins has not been characterized. Here, we perform evolutionary and functional analyses to characterize the activity of macroPARP and viral macrodomains. We trace the evolutionary history of macroPARPs in metazoans and show that PARP9 and PARP14 contain a single active macrodomain, whereas PARP15 contains none. Interestingly, we also reveal several independent losses of macrodomain enzymatic activity within mammalian PARP14, including in the bat, ungulate, and carnivore lineages. Similar to macroPARPs, coronaviruses contain up to three macrodomains, with only the first displaying catalytic activity. Intriguingly, we also reveal the recurrent loss of macrodomain activity within the alphavirus group of viruses, including enzymatic loss in insect-specific alphaviruses as well as independent enzymatic losses in two human-infecting viruses. Together, our evolutionary and functional data reveal an unexpected turnover in macrodomain activity in both host antiviral proteins and viral proteins.

The Antioxidant Activity of Betanin protects MRC-5 cellsAgainst Cadmium Induced Toxicity.

Cadmium (Cd) can induce both acute and chronic effects in the lungs depending on the time and the exposure route. Betanin is a component derived from the roots of red beets and it is well-known for its antioxidant and anti-apoptosis effects. The current study aimed to survey the protective effects of betanin on cell toxicity induced by Cd. Different concentration of Cd alone and in combination with betanin was assessed in MRC-5 cells. The viability and oxidative stress were measured using resazurin and DCF-DA methods respectively. Apoptotic cells were assessed by PI staining of the fragmented DNA and western blot analysis detected the activation of caspase 3 and PARP proteins. Cd exposure for 24h declined viability and increased ROS production in MRC-5 cells compared to the control group (p < 0.001). Also, Cd (35μM) elevated DNA fragmentation (p < 0.05), and the level of caspase 3-cleaved and cleaved PARP proteins in MRC-5 cells (p < 0.001). Co-treatment of cells with betanin for 24h significantly enhanced viability in concentrations of 1.25 and 2.5μM (p < 0.001) and 5μM (p < 0.05) and declined ROS generation (1.25 and 5μM p < 0.001, and 2.5μM p < 0.01). As well as, betanin reduced DNA fragmentation (p < 0.01), and the markers of apoptosis (p < 0.001) compared to the Cd-treated group. In conclusion, betanin protects lung cells against Cd-induced toxicity through antioxidant activity and inhibition of apoptosis.

Secondary Metabolites from Dendrobium nobile and Their Activities Induce Metabolites Apoptosis in OSC-19 Cells.

To identify potential drug candidates, secondary metabolites of Dendrobium nobile were performed. As a result, two previously undescribed phenanthrene derivatives with a spirolactone ring (1 and 2), along with four known compounds, N-trans-cinnamoyltyramine (3), N-trans-p-coumaroyltyramine (4), N-trans-feruloyltyramine (5), and moscatilin (6), were isolated from Dendrobium nobile. The structures of the undescribed compounds were elucidated using NMR spectroscopy, electronic circular dichroism (ECD) calculations, and extensive spectroscopic data analysis. The cytotoxic effects of compounds on human tongue squamous cells OSC-19 were determined using MTT at concentrations of 2.5 μM, 5 μM, 10 μM, and 20 μM. Compound 6 exhibited potent inhibitory activity against OSC-19 cells with an IC50 of 1.32 μM. Migration assays and western blot assays demonstrated that compound 6 effectively inhibited migration by down-regulating MMP2 and MMP9 at concentrations of 0.5 μM and 1 μM. To investigate its effect on apoptosis, we performed AO/PI staining, flow cytometry, and WB experiments. The results showed that increasing concentrations led to increased red fluorescence, decreased green fluorescence, increased apoptosis rate, decreased expression of bcl-2, caspase 3, caspase 9, and parp proteins, and increased bax expression. Furthermore, the phosphorylation of JNK and P38 was activated, suggesting that compound 6 may induce apoptosis via the MAPK pathway.

Abstract 2184: Short Peptides derived from MIEN1 and their analogs exhibit anti-cancer activity in breast and prostate cancer cells

Abstract Migration and invasion enhancer 1 (MIEN1) is overexpressed in various cancers and plays an important role in cancer cell migration and invasion. Conserved regions of ITAM and prenylation motif in MIEN1 were used as a template to identify anti-cancer peptides. RNA-seq, qPCR analysis and western blots showed changes in the transcriptome and protein expression after peptide treatment. The two bioactive peptides inhibited genes and proteins responsible for cancer cell migration and invasion in both MDA-MB-231 breast cancer and DU-145 prostate cancer cells. The mechanism of the action of the peptides involves the inhibition of key pathways like Epithelial-Mesenchymal transition and Epidermal Growth Factor-mediated NF-κB pathway to exert their anti-cancer activity. Interestingly, the peptides targeted the same signal transduction pathways followed by parental MIEN1 to show its cancer properties. Thus, the two peptides acted as dominant negative effectors of MIEN1 activity. Additionally, Peptide-treatments induced apoptosis in mice groups bearing tumors derived from MDA-MB-231 cells as evidenced by increased levels of cleaved caspase-3 and PARP proteins in western blots. Interestingly, the MIEN1 mRNA and protein expression was lowered in the in vivo breast cancer tumor models which remained unchanged in MDA-MB-231 breast cancer cells indicating an improved therapeutic activity in living system. The peptides did not cause any toxicity in the mice group which received peptides only, at three times the dose used during in vivo assays. Citation Format: AMIT Kumar Tripathi, Jamboor K. Vishwanatha. Short Peptides derived from MIEN1 and their analogs exhibit anti-cancer activity in breast and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2184.

Abstract 6285: Preclinical efficacy of CDK7 inhibitor-based combinations in cellular models of advanced myeloproliferative neoplasms (MPN)

Abstract Advanced MPN are characterized by driver mutations in JAK2, c-MPL or calreticulin (CALR) gene, with hyperactive JAK-STAT5/3 and NFkB signaling. Co-occurrence of mutations in chromatin/transcriptional regulators, including DNMT3A, TET2, ASXL1, EZH2, SRSF2, RUNX1 and TP53, creates the dysregulated transcriptome, which is responsible for the increased growth, survival and therapy-refractoriness in MPN blastic phase (BP). Treatment with JAK inhibitors (JAKis), e.g., ruxolitinib, venetoclax and hypomethylating agents are ineffective in improving survival in MPN-BP. CDK7 is the crucial catalytic component of the general transcript initiation factors TFIIH, which is recruited to the transcription start-sites along with RNA-pol II (RNAP2) to initiate transcription. Complexed with cyclin H, CDK7 phosphorylates CDK1, 2, 4 and 6, as well as serine 5 and 7 in the C terminal domain (CTD) of RNAP2, thus facilitating transcript initiation. CDK7 also phosphorylates CDK9, which in turn phosphorylates serine 2 of the CTD of RNAP2, and phosphorylates and inactivates the negative elongation factors, thus enabling productive mRNA transcript elongation of growth and survival promoting oncogenes. In MPN-BP, these include RUNX1, Bcl-xL, MCL1, CDK4/6, PIM1 and MYC. CRISPR-gRNA dependency-screen (DepMap) demonstrated that CDK7 is a dependency in the Advanced-AML cell lines, SET2 and HEL cells. Present studies demonstrate that treatment with ATP-competitive, covalent CDK7 inhibitors (CDK7i) SY-1365, and its clinical grade counterpart SY-5609, dose-dependently inhibits cell cycle, growth and induces lethality in SET2 and HEL as well as patient-derived (PD), CD34+ MPN-sAML cells. ChIP-Seq analysis showed that SY-1365 treatment caused log2 fold-reduction in p-Ser2-RNAP2 occupancy on the DNA of MYC, PIM1 and MCL1 genes in HEL cells. RNA-Seq analysis showed that SY1365 treatment caused negative enrichment of gene-sets of MYC and E2F targets, as well as inflammatory response, IL6-JAK-STAT3 and mTORC1 signaling. SY-1365 treatment also dose-dependently depleted protein expressions of p-Ser5 RNAP2, p-Ser2 RNAP2, c-Myc, c-Myb, RUNX1, PU.1, MCL1 and Bcl-xL, while inducing cleavage of PARP protein. As compared to the parental JAKi-sensitive, in vitro generated JAKi-persister/resistant (P/R) SET2/RuxP cells were significantly more sensitive to SY-1365-induced apoptosis. Co-treatment with CDK7i and ruxolitinib or the BET inhibitor OTX015 was synergistically lethal in PD MPN-BP cells (n=7). In an aggressive xenograft model of HEL-GFP/Luc cells in NSG mice, compared to the vehicle control, monotherapy with SY5609 significantly reduced the MPN sAML burden and improved survival of the NSG mice without causing host toxicity. These findings demonstrate promising activity and support the rationale to further evaluate the in vivo efficacy of CDK7i-based combinations against advanced MPN. Citation Format: Christopher P. Mill, Warren C. Fiskus, Christine E. Birdwell, John A. Davis, Kaberi Das, Kapil N. Bhalla. Preclinical efficacy of CDK7 inhibitor-based combinations in cellular models of advanced myeloproliferative neoplasms (MPN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6285.

Abstract 419: Elevated Akt/mTOR signaling drives therapy resistance in ALM

Abstract Metastatic acral lentiginous melanoma (ALM) is the most lethal subtype of cutaneous melanoma and effective therapies for patients have not been established. Studies revealed a link between the dysregulation of CDK4/6 and ALM mortality, however CDK4/6 inhibitors (CDK4i/6i) have proved ineffective in patients with advanced ALM. The tumor suppressor Rb was recently shown to have a novel suppressive role towards mTORC2. Here, we elucidate the role of a non-canonical tumor suppressor function of Rb in negative regulation of mTOR in CDK4i/6i-resistance. We hypothesize that the loss of Rb induced by CDK4i/6i triggers phosphorylation of Akt and downstream mTORC1 signaling pathways. We characterized baseline activity of the PI3K/AKT/mTOR pathway in a panel of ALM cell lines, finding heterogeneity across signaling. We immunoblotted for key proteins involved in the signal reprogramming hypothesized: pRb, pAkt, pS6 to define activation status and found the loss of Rb phosphorylation preceded the enrichment of pAKT, supporting the non-canonical role of Rb tumor suppression and the Rb-mTORC2 signaling axis as intrinsic CDK4i/6i mechanism. In parallel, we assessed the essential protein-protein interactions required for mechanistic mTORC1 activation—mTOR and Rheb GTPase. The enrichment of these interactions shortly after introducing CDK4i/6i supported our hypothesized mechanism. Translational utility of CDK4i/6i and mTORi dual inhibition was determined via long-term Colony Formation Assays (CFAs) using Palbociclib, rapamycin and sapanisertib (TAK228), a catalytic mTOR inhibitor. We generated Palbociclib (acquired)-resistant ALM cell lines and investigated the pharmacological inhibition of PI3K signaling pathways through immunoblot and colony-formation assays. Concurrent mTORi and CDK4i/6i inhibition showed effective reduction in pRb, pS6, Cyclin D and increases in Cleaved PARP protein, suggesting apoptotic cell death was elevated with combined CDK4i/6i+mTORi treatment. Similarly, CFAs revealed dual inhibition elicited superior antiproliferative effect over the monotherapy treatments. CFAs with acquired-resistance cell lines also showed that combination inhibition decreased colony formation, showing the potential utility of concurrent CDK4i/6i and mTORi as an approach against acquired resistance. Our results show the efficacy of combined inhibition as an anti-tumor strategy. Citation Format: Jeremy A. Bravo Narula, Vito W. Rebecca. Elevated Akt/mTOR signaling drives therapy resistance in ALM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 419.

In- Silico Drug Design and Molecular Docking Studies of Poly ADP-Ribose Polymerase (PARP-1) Inhibitors as Anticancer Agents

Background of the Topic: Drug discovery employs bioinformatics and computational biology (CADD) approaches for the identification and optimization of lead compounds. The PARP-1 is the member of the PARP family. PARP-1 is the enzyme that repairs the DNA damage in cancer cell hence it is selected as the target for the study. PARP inhibitors were shown the prominent results in the treatment of different kinds of malignancies due to loss of function of BRCA1/2 genes.&#x0D; Methodology: Based on literature review, database search, ADME and MD simulation, top 10 selected ligands were screened and tested against PARP-1 (4R6E) protein using molecular docking (Glide and Gold software), protein ligand interaction by LIGPLOT analysis. Molecular simulation studies were performed using DESMOND software afterwards on the top compound (CHEMBL378794).&#x0D; Results: All top 10 compounds showed binding affinity based inhibitory potential against the PARP-1 protein. The highest binding affinities from top 10 compounds towards anticancer targets were exhibited by CHEMBL378794 and CHEMBL245559. &#x0D; Discussion: The comparative analysis of 4R6E and the identified hits using MD simulation exhibited better stability of the binding domain with these molecules suggesting their strong interaction and selectivity towards PARP-1. &#x0D; Conclusion: This study showed the significant results in cancer treatment and their strong interaction with PARP-1 binding BRCT domain. Hence, these molecules could further be carried forward to explore their potency against PARP-1 sensitive cancer.

Antimutagenic 1,4-Dihydropyridine AV-153 Normalizes Expression of GLUT1, GLUT4, INOS, PARP1, and Gamma H2AX Histone in Myocardium of Rats with Streptozotocin Model of Diabetes Mellitus

Abstract Diabetic cardiomyopathy increases the risk of heart failure and worsens prognosis for diabetes mellitus (DM) patients. Its development depends on many factors, including modification of nitric oxide production and impaired DNA repair. The goal of the present work was to study in vivo effects of a 1,4-dihydropyridine AV-153, known as antimutagen and DNA-binder, on DNA integrity, and on the expression of several proteins involved in glucose transport, nitric oxide metabolism, and DNA repair in myocardium in diabetic rats. DM was induced in rats by streptozotocin (STZ) injection. Expression of proteins was studied by means of immunohistochemistry. Development of the STZ-induced DM significantly induced PARP1 and gamma H2AX histone, markers of DNA breakage, protein expression in heart tissue, while AV-153 administration decreased PARP1 and H2AX protein expression. In this model of diabetes, myocardial expression of iNOS was also significantly increased, but administration of AV-153 reduced it to normal levels. AV-153 also upregulated the expression of insulin-dependent GLUT4 and insulin-independent GLUT1 glucose transporters up to normal level in diabetic rats. Thus, AV-153 appears to be prospective for creation of a remedy for prevention of diabetic cardiomyopathy.

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.

The Leaf Essential Oil of Myrtus communis subsp. tarentina (L.) Nyman: From Phytochemical Characterization to Cytotoxic and Antimigratory Activity in Human Prostate Cancer Cells.

The aim of this study was to investigate the chemical profile and the cytotoxic activity in two castration-resistant prostate cancer (CRPC) cell lines of the leaf essential oil in Myrtus communis subsp. tarentina (L.) Nyman (EO MT), which was cultivated at the Ghirardi Botanical Garden (Toscolano Maderno, Brescia, Italy). The leaves were air-dried and extracted by hydrodistillation with a Clevenger-type apparatus, and the EO profile was characterized by GC/MS. For the cytotoxic activity investigation, we analyzed the cell viability by MTT assay, and the apoptosis induction by Annexin V/propidium iodide assay/Western blot analysis of cleaved caspase 3 and cleaved PARP proteins. Moreover, the cellular migration was analyzed by Boyden's chamber assay and the distribution of actin cytoskeleton filaments by immunofluorescence. We identified 29 total compounds; the main compound classes were oxygenated monoterpenes, monoterpene hydrocarbons, and sesquiterpenes. The main constituents were α-pinene, α-humulene, α-terpineol, durohydroquinon, linalool, geranyl acetate, and β-caryophyllene. We found that EO MT was able to reduce cellular viability, activating an apoptotic process, and to decrease the migratory capacity of CRPC cells. These results suggest that it might be interesting to further investigate the effects of single compounds present in EO MT for their possible use in prostate cancer treatment.

In Silico Investigation of the Molecular Mechanism of PARP1 Inhibition for the Treatment of BRCA-Deficient Cancers.

The protein PARP1, which plays a crucial role in DNA repair processes, is an attractive target for cancer therapy, especially for BRCA-deficient cancers. To overcome the acquired drug resistance of PARP1, PARP1 G-quadruplex (G4) identified in the PARP1-promotor region is gaining increasing attention. Aiming to explore the molecular mechanism of PARP1 inhibition with PARP1 G4 and PARP1 as potential targets, a comparative investigation of the binding characteristics of the newly identified G4 stabilizer MTR-106, which showed modest activity against talazoparib-resistant xenograft models and the FDA-approved PARP1 inhibitor (PARPi) talazoparib, were performed through molecular simulations. Combined analyses revealed that, relative to the groove binding of talazoparib, MTR-106 induced the formation of a sandwich framework through stacking with dT1 and the capping G-pair (dG2 and dG14) of PARP1 G4 to present largely enhanced binding affinity. For the binding with PARP1, although both were located in the catalytic pocket of PARP1, MTR-106 formed more extensive interactions with the surrounding PARP1 residues compared to talazoparib, in line with its increased binding strength. Importantly, vdW interaction was recognized as a decisive factor in the bindings with PARP1 G4 and PARP1. Collectively, these findings demonstrated the ascendancy of MTR-106 over talazoparib at the atomic level and revealed that the dual targeting of PARP1 G4 and PARP1 might be pivotal for PARPi that is capable of overcoming acquired drug resistance, providing valuable information for the design and development of novel drugs.

PARP Inhibitors and Proteins Interacting with SLX4.

PARP inhibitors are small molecules currently used with success in the treatment of certain cancer patients. Their action was first shown to be specific to cells with DNA repair deficiencies, such as BRCA-mutant cancers. However, recent work has suggested clinical interest of these drugs beyond this group of patients. Preclinical data on relationships between the activity of PARP inhibitors and other proteins involved in DNA repair exist, and this review will only highlight findings on the SLX4 protein and its interacting protein partners. As suggested from these available data and depending on further validations, new treatment strategies could be developed in order to broaden the use for PARP inhibitors in cancer patients.

The Role of the WGR Domain in the Functions of PARP1 and PARP2

The PARP1 and PARP2 proteins are members of the poly(ADP-ribose) polymerase family involved in the regulation of DNA repair and replication, RNA processing, ribosome biogenesis, transcription, cell division, and cell death. PARP1 and PARP2 are promising targets for the development of anticancer drugs and can be used in the treatment of cardiovascular, neurodegenerative, and other disorders. The WGR domain has been shown to play a central role in the functioning of PARP1 and PARP2 proteins. This review considers the mechanisms of functioning of WGR domains in the PARP1 and PARP2 proteins, which have several similar and specialized properties. Understanding these processes is of great interest to fundamental science and can contribute to the development of more effective and selective inhibitors of PARP1 and PARP2.

PARP1 controls the transcription of CD24 by ADP-ribosylating the RNA helicase DDX5 in pancreatic cancer.

The PARP1 protein plays a key role in DNA damage repair and ADP-ribosylation to regulate gene expression. Strategies to target PARP1 have rapidly been developed for cancer treatment. However, the role of the innate immune response in targeted anti-PARP1 therapy remains poorly understood. In this work, we aimed to elucidate the regulatory mechanism underlying the immunogenicity of PARP1 and explore efficient therapeutic strategies to enhance the antitumor effect of PARP inhibitors. The relationships between PARP1 expression and immunosuppressive factors were examined by qRTPCR and immunoblot analysis. DNA pull-down, chromatin immunoprecipitation-quantitative PCR (ChIPqPCR) and luciferase reporter assays were employed to reveal the mechanism by which the expression of the immune checkpoint regulator CD24 is regulated by PARP1. Phagocytosis assays and pancreatic cancer animal models were applied to evaluate the therapeutic effect of simultaneous disruption of PARP1 and the antiphagocytic factor CD24. Upregulation of the innate immunosuppressive factor CD24 was observed in pancreatic cancer during PARP1 inhibition. The activating effect of targeting CD24 on macrophage phagocytosis was verified. Then, we showed that PARP1 attenuated the transcription of CD24 by ADP-ribosylating the transcription factor DDX5 in pancreatic cancer. Combined blockade of PARP1 and the antiphagocytic factor CD24 elicited a synergetic antitumor effect in pancreatic cancer. Our research provided evidence that combination treatment with PARP inhibitors and CD24 blocking monoclonal antibodies (mAbs) could be an effective strategy to improve the clinical therapeutic response in pancreatic cancer.

Long non-coding RNA AC018926.2 regulates palmitic acid exposure-compromised osteogenic potential of periodontal ligament stem cells via the ITGA2/FAK/AKT pathway.

Although obesity has been proposed as a risk factor for periodontitis, the influence of excessive fat accumulation on the development of periodontitis and periodontal recovery from disease remains largely unknown. This study investigated the cellular response of periodontal ligament stem cells (PDLSCs) to elevated levels of a specific fatty acid, namely, palmitic acid (PA). The mechanism by which PA exposure compromises the osteogenic potential of cells was also explored. It was found that exposure of PDLSCs to abundant PA led to decreased cell osteogenic differentiation. Given that long non-coding RNAs (lncRNAs) play a key role in the stem cell response to adverse environmental stimuli, we screened the lncRNAs that were differentially expressed in PDLSCs following PA exposure using lncRNA microarray analysis, and AC018926.2 was identified as the lncRNA that was most sensitive to PA. Next, gain/loss-of-function studies illustrated that AC018926.2 was an important regulator in PA-mediated osteogenic differentiation of PDLSCs. Mechanistically, AC018926.2 upregulated integrin α2 (ITGA2) expression and therefore activated ITGA2/FAK/AKT signalling. Further functional studies revealed that inactivation of ITGA2/FAK/AKT signalling by silencing ITGA2 counteracted the pro-osteogenic effect induced by AC018926.2 overexpression. Moreover, the results of bioinformatics analysis and RNA immunoprecipitation assay suggested that AC018926.2 might transcriptionally regulate ITGA2 expression by binding to PARP1 protein. Our data suggest that AC018926.2 may serve as a therapeutic target for the management of periodontitis in obese patients.

PARP1 inhibitors induce pyroptosis via caspase 3-mediated gasdermin E cleavage

The identification of PARP1 as a therapeutic target for BRCA1/2-deficient cells has led to a paradigm shift for the treatment of human malignancies with BRCA1/2 mutations. However, our understanding of the mechanism of action of PARP1 inhibitors (PARPi) is still evolving. It is being increasingly appreciated that the immunomodulatory function of PARPi is a critical contributor of the anti-tumor effects of these compounds. Here, we identify a novel cell death effector pathway for PARPi where PARPi induces inflammatory pyroptosis that is mediated by caspase 3-dependent cleavage of GSDME. Caspase 3 is activated upon PARPi treatment which directly cleaves GSDME and, subsequently induces pyroptosis. Genetic and pharmacological experiments show that the presence of the PARP1 protein with uncompromised DNA binding capability is required for PARPi-induced pyroptosis, suggesting that PARP1 trapping is a key driver of this phenomenon. Importantly, we show that PARPi-induced GSDME cleavage and pyroptosis occurred only in the BRCA1-deficient cells, but not in those reconstituted with BRCA1 wild-type (WT). These findings suggest that pyroptosis could be a novel aspect of the immunomodulatory function of PARPi. Our studies could also offer new insights to the potential biomarkers and therapeutic strategies to achieve better anti-tumor effects of PARPi for BRCA-deficient tumors with low GSDME expression.

Cell migration induces apoptosis in osteosarcoma cell via inhibition of Wnt-β-catenin signaling pathway

The current design scheme on anti-cancer materials is mainly through tuning the mechanical properties of the materials to induce apoptosis in cancer cells, with the involvement of Rho/ROCK signaling pathway. We hypothesize that tuning the motility is another potential important approach to modifying the tumor microenvironment and inducing tumor apoptosis. To this aim, we have prepared RGD-modified substrates to regulate cell motility through modification of RGD with different concentrations, and systematically examined the effect of motility on the apoptosis of tumor cells, and the potential involvement of Wnt signaling pathway. Our studies indicated that RGD modification could be readily used to tune the motility of cancer cells. High RGD concentration significantly suppressed the migration of cancer cells, leading to significantly increased apoptosis rate, about three times of that of the unmodified samples. Western-blot analysis also showed that cell with low motility expressed more caspase-3 and PARP proteins. Further RNA sequence study strongly suggested that low motility inhibited the canonical Wnt signaling pathway, which in turn led to the activation of the mitochondria-associated caspase signaling pathway, and ultimately to the apoptosis of osteosarcoma cells. Activation of the Wnt-β-catenin pathway through HLY78 significantly suppressed the apoptosis of MG-63 cells, further suggesting the critical role of Wnt pathway in motility-regulated-apoptosis of tumor cells. Our findings shed insights to understand the underlying mechanisms that induced the tumor cell apoptosis, and might provide new strategy for designing the novel anti-tumor materials.

Alkaloid from Alstonia yunnanensis diels root against gastrointestinal cancer: Acetoxytabernosine inhibits apoptosis in hepatocellular carcinoma cells.

Liver cancer belongs to Gastrointestinal (GI) malignancies which is a common clinical disease, a thorny public health problem, and one of the major diseases that endanger human health. Molecules from natural products (NPs) or their derivatives play an increasingly important role in various chronic diseases such as GI cancers. The chemical composition of the Alstonia yunnanensis Diels roots was studied using silica column chromatography, gel chromatography, recrystallization, and HPLC, and the compounds were structurally identified by modern spectral analysis using mass spectrometry (MS) and nuclear magnetic resonance (1H-, 13C-, HMQC-, HMBC-, and 1H-1HCOSY-NMR), ultraviolet and visible spectrum (UV), and electronic Circular Dichroism (ECD). Acetoxytabernosine (AC), an indole alkaloid with antitumor activity, was isolated from Alstonia yunnanensis Diels root. The current study aimed to investigate the influence of AC on the cell proliferation of BEL-7402 and SMMC7721 and to elucidate the underlying mechanism. The absolute configuration of AC was calculated by ECD (electronic circular dichroism). The effects of AC on the viability of different tumor cell lines were studied by the SRB method. The death mode of human hepatoma cells caused by AC was studied by TUNEL cell apoptosis detection and AnnexinV-FITC/PI double staining image. Mitochondrial membrane potential was detected by JC-1. The effects of AC on the expression of apoptosis-related proteins (Caspase9, Caspase3, and Parp-1) in SMMC7721 and BEL-7402 cells were detected by western blot. It was found that the absolute configuration of AC is 19(s), 20(s)-Acetoxytabernosine. AC could induce apoptosis of SMMC7721 and BEL-7402, and block the replication of DNA in the G1 phase. Under the treatment of AC, the total protein expression of apoptosis-related proteins (Caspase9, Caspase3, and Parp-1) significantly decreased in SMMC7721 and BEL-7402. The results suggested that AC induced apoptosis through a caspase-dependent intrinsic pathway in SMMC7721 and BEL-7402, and natural product-based drug development is an important direction in antitumor drug discovery and research.

Nicotinamide prevention in diabetes-induced alterations in the rat liver.

Objective. The study was performed to elucidate whether nicotinamide (NAm) can attenuate the diabetes-induced liver damage by correction of ammonia detoxifying function and disbalance of NAD-dependent processes in diabetic rats. Methods. After four weeks of streptozotocin-induced diabetes, Wistar male rats were treated for two weeks with or without NAm. Urea concentration, arginase, and glutamine synthetase activities, NAD+ levels, and NAD+/NADH ratio were measured in cytosolic liver extracts. Expression of parp-1 gene in the liver was estimated by quantitative polymerase chain reaction and PARP-1 cleavage evaluated by Western blotting. Results. Despite the blood plasma lipid peroxidation products in diabetic rats were increased by 60%, the activity of superoxide dismutase (SOD) was reduced. NAm attenuated the oxidative stress, but did not affect the enzyme activity in diabetic rats. In liver of the diabetic rats, urea concentration and arginase activity were significantly higher than in the controls. The glutamine synthetase activity was decreased. Decline in NAD+ level and cytosolic NAD+/NADH ratio in the liver of diabetic rats was observed. Western blot analysis demonstrated a significant up-regulation of PARP-1 expression accompanied by the enzyme cleavage in the diabetic rat liver. However, no correlation was seen between mRNA expression of parp-1 gene and PARP-1 protein in the liver of diabetic rats. NAm markedly attenuated PARP-1 cleavage induced by diabetes, but did not affect the parp-1 gene expression. Conclusions. NAm counteracts diabetes-induced impairments in the rat liver through improvement of its detoxifying function, partial restoration of oxidative stress, NAD+ level, normalization of redox state of free cytosolic NAD+/NADH-couples, and prevention of PARP-1 cleavage.