O6-Alkylguanine-DNA Alkyltransferase Maintains Genome Integrity by Forming DNA-Protein Cross-Links during Inflammation-Associated Peroxynitrite-Mediated DNA Damage.

Inflammation is an early immune response against invading pathogens and damaged tissue. Although beneficial, uncontrolled inflammation leads to various diseases including cancer in a chronic setting. Peroxynitrite (PN) is a major reactive nitrogen species generated during inflammation. It produces various DNA lesions including 8-nitro-guanine which spontaneously converts into abasic sites, resulting in DNA strand breakage, and is suspected to be mutagenic. Here, we report the discovery of a previously unrecognized function of the human repair protein O6-alkylguanine-DNA alkyltransferase (hAGT or MGMT). We showed that hAGT through its active site nucleophilic Cys145 thiolate spontaneously reacts with 8-nitro-guanine in DNA to form a stable DNA-protein cross-link (DPC). Interestingly, the process of DPC formation provided protection from PN-mediated genome instability, growth arrest, and apoptosis. The Cys145 mutant of hAGT failed to form a DPC and was unable to protect cells from inflammation-associated PN-mediated cytotoxicity. Gel-shift, dot blot, and UV-vis assays showed formation of a covalent linkage between PN-damaged DNA and hAGT through its active site Cys145. Finally, expression of hAGT was found to be significantly increased by induced macrophages and PN. The data presented here clearly demonstrated hAGT as a dual-function protein that along with DNA repair is capable of maintaining genomic integrity and providing protection from the toxicity caused by PN-mediated DNA damage. Although DPCs are known to be detrimental to the cell, recently, multiple pathways have been identified in normal cells for their repair.

Opposing Interplay between Nuclear Factor Erythroid 2-Related Factor 2 and Forkhead BoxO 1/3 is Responsible for Sepantronium Bromide's Poor Efficacy and Resistance in Cancer cells: Opportunity for Combination Therapy in Triple Negative Breast Cancer.

Survivin, a cancer-cell-specific multifunctional protein, is regulated by many oncogenic signaling pathways and an effective therapeutic target. Although, several types of survivin-targeting agents have been developed over the past few decades, none of them received clinical approval. This could be because survivin expression is tightly controlled by the feedback interaction between different signaling molecules. Of the several signaling pathways that are known to regulate survivin expression, the phosphatidylinositol 3-kinase/AKT serine-threonine kinase/forkhead boxO (PI3K/AKT/FoxO) pathway is well-known for feedback loops constructed by cross-talk among different molecules. Using sepantronium bromide (YM155), the first of its class of survivin-suppressant, we uncovered the existence of an interesting cross-talk between Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) and FoxO transcription factors that also contributes to YM155 resistance in triple negative breast cancer (TNBC) cells. Pharmacological manipulation to interrupt this interaction not only helped restore/enhance the drug-sensitivity but also prompted effective immune clearance of cancer cells. Because the YM155-induced reactive oxygen species (ROS) initiates this feedback, we believe that it will be occurring for many ROS-producing chemotherapeutic agents. Our work provides a rational explanation for the poor efficacy of YM155 compared to standard chemotherapy in clinical trials. Finally, the triple drug combination approach used herein might help reintroducing YM155 into the clinical pipeline, and given the high survivin expression in TNBC cells in general, it could be effective in treating this subtype of breast cancer.

Targeting prostate cancer via therapeutic targeting of PIM-1 kinase by Naringenin and Quercetin.

PIM-1 kinase belongs to the Ser/Thr kinases family, an attractive therapeutic target for prostate cancer. Here, we screened about 100 natural substances to find potential PIM-1 inhibitors. Two natural compounds, Naringenin and Quercetin, were finally selected based on their PIM-1 inhibitory potential and binding affinities. The docking score of Naringenin and Quercetin with PIM-1 is -8.4 and - 8.1 kcal/mol, respectively. Fluorescence binding studies revealed a strong affinity (Ka values, 3.1 × 104 M-1 and 4.6 × 107 M-1 for Naringenin and Quercetin, respectively) with excellent IC50 values for Naringenin and Quercetin (28.6 µM and 34.9 µM, respectively). Both compounds inhibited the growth of prostate cancer cells (LNCaP) in a dose-dependent manner, with the IC50 value of Naringenin at 17.5 µM and Quercetin at 8.88 µM. To obtain deeper insights into the PIM-1 inhibitory effect of Naringenin and Quercetin, we performed extensive molecular dynamics simulation studies, which provided insights into the binding mechanisms of PIM-1 inhibitors. Finally, Naringenin and Quercetin were suggested to serve as potent PIM-1 inhibitors, offering targeted treatments of prostate cancer. In addition, our findings may help to design novel Naringenin and Quercetin derivatives that could be effective in therapeutic targeting of prostate cancer.

Thymoquinone, artemisinin, and thymol attenuate proliferation of lung cancer cells as Sphingosine kinase 1 inhibitors.

Sphingosine-1-phosphate (S1P) formed via catalytic actions of sphingosine kinase 1 (SphK1) behaves as a pro-survival substance and activates downstream target molecules associated with various pathologies, including initiation, inflammation, and progression of cancer. Here, we aimed to investigate the SphK1 inhibitory potentials of thymoquinone (TQ), Artemisinin (AR), and Thymol (TM) for the therapeutic management of lung cancer. We implemented docking, molecular dynamics (MD) simulations, enzyme inhibition assay, and fluorescence measurement studies to estimate binding affinity and SphK1 inhibitory potential of TQ, AR, and TM. We further investigated the anti-cancer potential of these compounds on non-small cell lung cancer (NSCLC) cell lines (H1299 and A549), followed by estimation of mitochondrial ROS, mitochondrial membrane potential depolarization, and cleavage of DNA by comet assay. Enzyme activity and fluorescence binding studies suggest that TQ, AR, and TM significantly inhibit the activity of SphK1 with IC50 values of 35.52 µM, 42.81 µM, and 53.68 µM, respectively, and have an excellent binding affinity. TQ shows cytotoxic effect and anti-proliferative potentials on H1299 and A549 with an IC50 value of 27.96 µM and 54.43 µM, respectively. Detection of mitochondrial ROS and mitochondrial membrane potential depolarization shows promising TQ-induced oxidative stress on H1299 and A549 cell lines. Comet assay shows promising TQ-induced oxidative DNA damage. In conclusion, TQ, AR, and TM act as potential inhibitors for SphK1, with a strong binding affinity. In addition, the cytotoxicity of TQ is linked to oxidative stress due to mitochondrial ROS generation. Overall, our study suggests that TQ is a promising inhibitor of SphK1 targeting lung cancer therapy.

Design, synthesis, molecular docking and anti-proliferative activity of novel phenothiazine containing imidazo[1,2-a]pyridine derivatives against MARK4 protein.

A series of novel phenothiazine-containing imidazo[1,2-a]pyridine derivatives were designed and synthesized under metal-free conditions in excellent yield. These derivatives were effectively transformed further into N-alkyl, sulfoxide, and sulfone derivatives. Derivatives were deployed against human microtubule affinity regulating kinase (MARK4), some molecules play crucial roles in cell-cycle progression such as G1/S transition and regulator of microtubule dynamics. Hence, molecules have shown excellent MARK4 inhibitory potential. Molecules with excellent IC50 values were selected for further studies such as ligand interactions using fluorescence quenching experiments for the binding constant. The highest binding constant was calculated as K = 0.79 × 105 and K = 0.1 × 107 for compounds 6a and 6h, respectively. Molecular docking, cell cytotoxicity, mitochondrial reactive oxygen species measurement and oxidative DNA damage were also studied to understand the mechanism of action of the molecules on cancer cells. It was found that the designed and synthesized compounds played anti-cancer roles by binding and inhibiting MARK4 protein.

Pre-implementation perceptions of clinicians regarding a patient portal in inpatient addictions settings: A qualitative descriptive study.

BACKGROUND: Patient portals have been implemented in many organizations to support patient engagement and empowerment. However, their use in inpatient mental health and addiction settings is relatively new and has not been extensively studied. To address this gap, this study explored clinician perspectives of implementing a patient portal in inpatient addiction settings. METHODS: The study followed qualitative descriptive methodology and used the Consolidated Framework for Implementation Research (CFIR) to guide the research. Interviews were conducted with clinicians working in an inpatient addictions service at a large mental health and addictions teaching hospital in Toronto, Canada. Data analysis was performed using directed content analysis and the CFIR domains. RESULTS: Twelve clinicians participated in semi-structured interviews. Participants included prescribers (such as physicians and nurse practitioners), registered nurses, allied health clinicians, and leadership. Participants had positive attitudes toward the patient portal, believing it would benefit patients and support consistency in healthcare. However, they also expressed reservations about its relevance and value during short inpatient admissions. Clinicians perceived the patient portal as compatible with existing workflows, enhancing patient empowerment and facilitating access to medical documentation. Concerns were raised about potential negative impacts on therapeutic rapport, particularly if patients disagreed with or were upset by the contents of their notes. Adaptations to the portal, such as improving documentation templates and providing detailed medication information, were suggested. Participants also highlighted advantages of the portal, including secure communication and access to laboratory results. CONCLUSIONS: Clinicians generally had positive attitudes toward implementing a patient portal. However, concerns about maintaining therapeutic rapport and the relevance of information to patients were identified, and adaptations were suggested to improve the utility of a portal in the context of short inpatient stays. The findings provide insights into clinician perspectives and can inform the implementation of patient portals in inpatient addiction settings.