2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione isolated from Averrhoa carambola L. root inhibits high glucose-induced EMT in HK-2 cells through targeting the regulation of miR-21-5p/Smad7 signaling pathway.

OBJECTIVE: 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione (DMDD) isolated from Averrhoa carambola L. root, has been proven therapeutic effects on diabetic kidney disease (DKD). This research aims to assess DMDD's effects on DKD and to investigate its underlying mechanisms, to establish DMDD as a novel pharmaceutical agent for DKD treatment. METHODS: The human renal tubular epithelial (HK-2) cells were induced by high glucose (HG) to mimic DKD and followed by DMDD treatment. The cytotoxicity of DMDD was assessed using the Cell Counting Kit-8 (CCK-8) assay. The migratory capacity of HK-2 cells was evaluated through transwell and scratch-wound assays. To investigate the effect of Smad7 and miR-21-5p, lentiviral transfection was employed in HK-2 cells. Additionally, the expression of proteins related to epithelial-mesenchymal transition (EMT) and TGFß1/Smad2/3 pathway was checked by QRT-PCR, Western blot, and immunofluorescence techniques. RESULTS: This study has shown that DMDD significantly suppresses cell migration and the expression of Vimentin, α-SMA, TGFß1, and p-Smad2/3 in HK-2 cells under HG conditions. Concurrently, DMDD enhances the protein expression of E-cadherin and Smad7. Intriguingly, the therapeutic effect of DMDD was abrogated upon Smad7 silencing. Further investigations revealed that DMDD effectively inhibits miR-21-5p expression, which is upregulated by HG. Downregulation of miR-21-5p inhibits the activation of the TGFß1/Smad2/3 pathway and EMT induced by HG. In contrast, overexpression of miR-21-5p negates DMDD's therapeutic benefits. CONCLUSION: DMDD mitigates EMT in HG-induced HK-2 cells by modulating the miR-21-5p/Smad7 pathway, thereby inhibiting renal fibrosis in DKD. These findings suggest that DMDD holds promise as a potential therapeutic agent for DKD.

Insights into the eradication of drug resistant Staphylococcus aureus via compound 6-nitrobenzo[cd]indole-2(1H)-ketone.

6-Nitrobenzo[cd]indole-2(1H)-ketone (compound C2) exhibits an excellent germicidal effect against methicillin-resistant Staphylococcus aureus (MRSA). Mechanism studies show that C2 induces ROS over-production, cell membrane damage, and ATP and virulence factor down-regulation in bacteria. More importantly, C2 can inhibit biofilm formation and accelerate wound healing in a mouse infection model induced by MRSA.

Targeting Telomerase Enhances Cytotoxicity of Salinomycin in Cancer Cells.

Salinomycin exhibits significant systemic adverse reactions such as tachycardia and myoglobinuria in mammals, which hinders its application as a drug for human cancers. Although many strategies aimed at increasing salinomycin's toxicity to cancer cells have been identified to allow a lower dose of salinomycin to be used, they often cause normal cell damage by themselves. Thus, it is urgent to find more effective methods to increase salinomycin's toxicity to cancer cells with little influences on normal cells. Telomerase, which is expressed highly in most cancer cells rather than normal somatic cells, plays central roles in cancer cell fate regulation. Targeting telomerase represents a potential method for enhancing salinomycin's cytotoxicity to cancer cells with little effects on normal cells. Herein, we improve the toxicity of salinomycin against cancer cells by telomerase inhibition BIBR1532 (BIBR), which binds to the active site of telomerase reverse transcriptase. We find that a non-toxic dose of BIBR can enhance cytotoxicity of salinomycin in MCF-7 and MDA-MB-231 cells. Moreover, BIBR enhances mammosphere formation inhibition mediated by salinomycin in MCF-7 and MDA-MB-231 cells. Further studies show that BIBR enhances tumor growth inhibition induced by salinomycin in vivo. To our knowledge, this is the first example that targeting telomerase improves anti-cancer effects of salinomycin.

Clinical outcomes and immune phenotypes associated with STK11 co-occurring mutations in non-small cell lung cancer.

Background: STK11 mutation in non-small cell lung cancer (NSCLC) is associated with worse survival as well as primary resistance to PD-1/PD-L1 targeting immunotherapy. We hypothesize that co-occurring mutations and tumor mutation burden (TMB) may impact response to therapy and prognosis. Methods: Forty-one patients with STK11-mutated NSCLC seen in our Thoracic oncology clinic with available next-generation sequencing tumor data were included in the analysis. Data from the Cancer Genome Atlas (TCGA) was used for survival and immune gene expression analysis. Overall and progression-free survival (PFS) was estimated by the Kaplan-Meier method and compared using a log-rank test. Results: In the 41 patients included, common co-occurring alterations with STK11 were KRAS (54%), TP53 (44%), CDKN2A (37%) and KEAP1 (27%). Overall 17 patients received locoregional therapy with surgery or radiation with median OS of 8.6 years and there was no significant difference in clinical outcomes with KRAS and TP53 co-occurring mutations. Response to both chemotherapy and immunotherapy was poor across all co-occurring mutations. However, TP53 co-mutation was associated with improved clinical benefit with immunotherapy. Patients with higher TMB had longer PFS with immunotherapy. In TCGA survival analysis, tumors with STK11 mutation with or without KRAS co-mutation were associated with worse survival (P<0.05) but tumors with STK11/TP53 co-mutation did not have worst survival compared to STK11 wild type tumors. Moreover, co-occurring mutations had significant effect on intratumoral immune status with both STK11 alone and STK11/KRAS co-mutated tumors showing more enrichment for wound healing immune subtype while STK11/TP53 co-mutated tumors showed more enrichment for IFN-g immune subtype. Conclusions: Our retrospective analysis in patients with STK11-mutated NSCLC found that both TMB and co-occurring mutations may be predictors for response to immunotherapy with worse outcomes in patients with low TMB or KRAS co-mutation and improved outcomes with TP53 co-mutation. Patients with STK11-mutated NSCLC also demonstrate chemotherapy resistance but have similar outcomes with localized treatment compared to STK11 wild type tumors. Moreover, co-mutations with KRAS or TP53 significantly alter tumor immune landscape of STK11-mutated tumors and therefore response to immunotherapy.

Plasma miR-1273g-3p acts as a potential biomarker for early Breast Ductal Cancer diagnosis.

Circulating miRNAs presenting in plasma in a stable manner have been demonstrated their potential role as a promising biomarkers in many human diseases, such as Alzheimer's disease, melanoma and ovarian carcinoma. However, few circulating miRNAs could be used for breast ductal cancer diagnosis. Here, we identified miR-1273g-3p as a biomarker for detecting breast ductal cancer. We detected miR-1273g-3p levels in the plasma of 39 sporadic breast ductal cancer patients and 40 healthy donors by Stem-loop Quantitative Real-time PCR (qRT-PCR). The results showed the plasma miR-1273g-3p level were significantly up-regulated in breast ductal cancer patients compared with healthy donors (p=0.0139). Receiver operating characteristic (ROC) curve also revealed the significantly diagnostic ability of miR-1273g-3p in patients (p=0.0414). In addition, the plasma level of miR-1273g-3p was closely related to IIIB-IIIC TNM stage. We also confirmed the higher expression level of miR-1273g-3p in breast cancer cell lines MCF-7 (4.872±0.537) than normal breast cells (Hs 578Bst). Taken together, miR-1273g-3p could represent as a potential biomarker for early breast ductal cancer diagnosis.

Phase Ib/II study of hydroxychloroquine in combination with chemotherapy in patients with metastatic non-small cell lung cancer (NSCLC).

OBJECTIVES: Activation of cell survival pathways such as autophagy represents a potential resistance mechanism to chemotherapy in NSCLC. Preclinical studies report that autophagy inhibition suppresses lung tumor development and progression. We report the safety and efficacy for adding autophagy inhibitor, hydroxychloroquine, to chemotherapy in a phase Ib/II single-arm study in patients with metastatic NSCLC. PATIENTS AND METHODS: We treated patients with untreated metastatic NSCLC with carboplatin, paclitaxel (and bevacizumab if criteria met) and hydroxychloroquine 200 mg BID. Patients continued on hydroxychloroquine (+/- bevacizumab) maintenance after 4-6 cycles of therapy. RESULTS: We enrolled 40 patients, 8 on phase Ib and 32 on phase II. Forty-three percent were female; 50% with squamous histology. Median age was 62 years (range, 43-73). Thirteen patients developed ≥grade 3 treatment-related adverse event. Common adverse events (all grades) were neutropenia (35%), neuropathy (32.5%), and anemia (32.5%). The objective response rate (ORR) was 33% in the 30 patients (phase II) evaluable for response. Additionally, 20% of the patients demonstrated stable disease (clinical benefit rate of 53%). The median PFS was 3.3 months (95% CI 2.1-6.8 months). In 9 patients with KRAS positive tumors, ORR was 44% and median PFS was higher than expected at 6.4 months (95% CI 1.8-15.6). CONCLUSIONS: Addition of hydroxychloroquine is safe and tolerable with a modest improvement in clinical responses compared to prior studies. Autophagy inhibition may overcome chemotherapy resistance in advanced NSCLC and further study in a more molecularly selected population such as KRAS-positive tumors is warranted.

Role of autophagy in suppression of inflammation and cancer.

Autophagy is a crucial component of the cellular stress adaptation response that maintains mammalian homeostasis. Autophagy protects against neurodegenerative and inflammatory conditions, aging, and cancer. This is accomplished by the degradation and intracellular recycling of cellular components to maintain energy metabolism and by damage mitigation through the elimination of damaged proteins and organelles. How autophagy modulates oncogenesis is gradually emerging. Tumor cells induce autophagy in response to metabolic stress to promote survival, suggesting deployment of therapeutic strategies to block autophagy for cancer therapy. By contrast, defects in autophagy lead to cell death, chronic inflammation, and genetic instability. Thus, stimulating autophagy may be a powerful approach for chemoprevention. Analogous to infection or toxins that create persistent tissue damage and chronic inflammation that increases the incidence of cancer, defective autophagy represents a cell-intrinsic mechanism to create the damaging, inflammatory environment that predisposes to cancer. Thus, cellular damage mitigation through autophagy is a novel mechanism of tumor suppression.

Cdc2 and Mos regulate Emi2 stability to promote the meiosis I-meiosis II transition.

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.

Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A.

Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest.

BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.

Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis.

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. We show here that DNA-responsive checkpoints also activate PP2A/B56delta phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Our data suggest that creation of a 14-3-3 "sink," consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56delta as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis.

Cloning and Expression of Human CD20 Gene on NIH-3T3 Cell Membrane.

CD20 is a specific antigen expressed on normal and neoplastic B cells exclusively. Recent researches showed that in B cell leukemia, CD20 was over-expressed. Therefore monoclonal antibody (McAb) to CD20 may be of clinical value in diagnosis and treatment of some leukemias and lymphomas. In this study, the full length gene of CD20 cDNA were cloned from total RNA of Raji cells, inserted into an eukaryotic expression vector pcDNA3.1, forming a recombinant plasmid pcDNA3.1/CD20. NIH-3T3 cells were transfected with pcDNA3.1/CD20 and selected with G418 for the transfected cells. Alkaline phosphatase against alkaline phosphatase assay(APAAP) experiments showed that the selected cells could express the human CD20 onto its surface. Balb/c mice were immunized with CD20( ) NIH-3T3 cells once three weeks for 3 shuts. Indirect immunofluorescence experiments were done with the Raji cells and the serum of the immunized mice, and the results showed that the spleen of the immunized mice could be used to prepare the McAb to CD20.