Enzyme-treated red rice (Oryza sativa L.) bran extracts mitigate inflammatory markers in RAW 264.7 macrophage cells and exhibit anti-inflammatory efficacy greater/comparable to ferulic acid, catechin, γ-tocopherol, and γ-oryzanol.

ETHNOPHARMACOLOGICAL RELEVANCE: Rice (Oryza sativa L.), a staple food for a significant portion of the global population, has been recognized for its traditional medicinal properties for centuries. Rice bran, a by-product of rice milling, contains many bioactive compounds with potential pharmaceutical and therapeutic benefits. In recent years, research has highlighted the anti-inflammatory potential of rice bran, contributed by the bioactive components concentrated in their bran but, unfortunately, entrapped in the bran matrix, with limited bioavailability. Previous studies have reported that the enzymatic treatment of rice bran improves the bran's bioactive compound profile but did not investigate its impact on chronic conditions such as inflammation. AIM OF THE STUDY: This study investigates the anti-inflammatory effects of endo-1,4-ß-xylanase (ERB) and Viscozyme (VRB) treated red rice bran extracts against lipopolysaccharide-induced inflammation in RAW264.7 macrophages in comparison with non-enzyme-treated bran (CRB). Further established their efficacy with known anti-inflammatory compounds-ferulic acid (FA), catechin (CAT), γ-tocopherol (GTP), and γ-oryzanol (ORZ). MATERIALS AND METHODS: The RAW 264.7 macrophage cells were pre-treated with non-toxic concentrations (10-200 µg/mL) of FA, CAT, GTP, ORZ, CRB, ERB, and VRB, followed by inflammatory stimulation with LPS for 24 h. Further, the cell supernatant and pellets were harvested to study the anti-inflammatory effects by evaluating and measuring their efficacy in inhibiting pro-inflammatory cytokines (TNF-α, IL-6, IL-10, IL-1ß) and mediators (ROS, NO, PGE2, COX2, iNOS) through biochemical, ELISA, and mRNA expression studies. RESULTS: The findings showed that both ERB and VRB effectively inhibited the production of pro-inflammatory markers (TNF-α, IL-6) and mediators (ROS, NO, PGE2) by downregulating mRNA expressions of inflammatory genes (TNF-α, IL-1ß, IL-6, IL-10, COX2, iNOS) and demonstrated anti-inflammatory efficacy higher than CRB. On comparison, ERB demonstrated exceptional efficacy by causing a reduction of 48% in ROS, 20% in TNF-α, and 23% in PGE2 at 10 µg/mL, surpassing the anti-inflammatory capabilities of all the bioactive compounds, FA and ORZ, respectively. At the same time, VRB exhibited remarkable efficacy by reducing NO production by 52% at 200 µg/mL and IL-6 by 66% at 10 µg/mL, surpassing FA, CAT, ORZ, and GTP. Further, ERB downregulated the mRNA expression of IL-10 and iNOS, while VRB downregulated TNF-α, IL-1ß, and COX2 expression. Both extracts equally downregulated IL-6 expression at 10 µg/mL, demonstrating the efficacy more remarkable/on par with established anti-inflammatory compounds. CONCLUSIONS: Overall, enzyme-treated rice bran/extract, particularly ERB, possesses excellent anti-inflammatory properties, making them promising agents for alternatives to contemporary nutraceuticals/functional food against inflammatory diseases.

Repositioning of Montelukast to inhibit proliferation of mutated KRAS pancreatic cancer through a novel mechanism that interfere the binding between KRAS and GTP/GDP.

Pancreatic cancer is one of the most lethal cancer types with 5-year survival rate of ∼10.8%. Various KRAS mutations exist in ∼85% pancreatic cancer cell lines. Mutated KRAS is a major cause that leads cancer cell proliferation. Chemotherapy is still the major treatment for pancreatic cancer. Alternatively, repositioning old drug to inhibit mutated KRAS may be a cost-effective way for pancreatic cancer treatment. In this study, we choose mutated KRAS (G12D) as a target. Based on mutated KRAS GTP binding domain (hydrolyze GTP to GDP), we perform virtual screening on FDA-approved drugs. Montelukast shows strong binding affinity to mutated KRAS as well as interfering both GTP and GDP binding to mutated KRAS. Furthermore, Montelukast shows very strong anti-proliferation effect on mutated KRAS pancreatic cancer cells both in vitro and in vivo. Our results support repositioning of Montelukast as single agent for pancreatic cancer treatment.

SLC25A21 downregulation promotes KRAS-mutant colorectal cancer progression by increasing glutamine anaplerosis.

Emerging evidence shows that KRAS-mutant colorectal cancer (CRC) depends on glutamine (Gln) for survival and progression, indicating that targeting Gln metabolism may be a promising therapeutic strategy for KRAS-mutant CRC. However, the precise mechanism by which Gln metabolism reprogramming promotes and coordinates KRAS-mutant CRC progression remains to be fully investigated. Here, we discovered that solute carrier 25 member 21 (SLC25A21) expression was downregulated in KRAS-mutant CRC, and that SLC25A21 downregulation was correlated with poor survival of KRAS-mutant CRC patients. SLC25A21 depletion selectively accelerated the growth, invasion, migration, and metastasis of KRAS-mutant CRC cells in vitro and in vivo, and inhibited Gln-derived α-ketoglutarate (α-KG) efflux from mitochondria, thereby potentiating Gln replenishment, accompanied by increased GTP availability for persistent KRAS activation in KRAS-mutant CRC. The restoration of SLC25A21 expression impaired the KRAS-mutation-mediated resistance to cetuximab in KRAS-mutant CRC. Moreover, the arrested α-KG efflux that occurred in response to SLC25A21 depletion inhibited the activity of α-KG-dependent DNA demethylases, resulting in a further decrease in SLC25A21 expression. Our studies demonstrate that SLC25A21 plays a significant role as a tumor suppressor in KRAS-mutant CRC by antagonizing Gln-dependent anaplerosis to limit GTP availability for KRAS activation, which suggests potential alternative therapeutic strategies for KRAS-mutant CRC.

Plerixafor and resatorvid inhibit hepatitis B virus in vitro by upregulating elongation factor Tu GTP-binding domain containing 2.

Background: An increase in the demand for a functional cure has accelerated research on new methods of therapy for chronic hepatitis B, which is mainly focused on restoring antiviral immunity for controlling viral infections. Previously, we had described elongation factor Tu GTP-binding domain containing 2 (EFTUD2) as an innate immune regulator and suggested that it might be an antiviral target. Methods: In this study, we generated the Epro-LUC-HepG2 cell model for screening compounds that target EFTUD2. Plerixafor and resatorvid were screened from 261 immunity and inflammation-related compounds due to their ability to highly upregulate EFTUD2. The effects of plerixafor and resatorvid on hepatitis B virus (HBV) were examined in HepAD38 cells and HBV-infected HepG2-NTCP cells. Results: The dual-luciferase reporter assays showed that the EFTUD2 promoter hEFTUD2pro-0.5 kb had the strongest activity. In Epro-LUC-HepG2 cells, plerixafor and resatorvid significantly upregulated the activity of the EFTUD2 promoter and the expression of the gene and protein. In HepAD38 cells and HBV-infected HepG2-NTCP cells, treatment with plerixafor and resatorvid strongly inhibited HBsAg, HBV DNA, HBV RNAs, and cccDNA in a dose-dependent manner. Furthermore, the anti-HBV effect was enhanced when entecavir was administered along with either of the previous two compounds, and the effect could be blocked by knocking down EFTUD2. Conclusion: We established a convenient model for screening compounds that target EFTUD2 and further identified plerixafor and resatorvid as novel HBV inhibitors in vitro. Our findings provided information on the development of a new class of anti-HBV agents that act on host factors rather than viral enzymes.

[Current status and outlook of medical treatment for KRAS-mutated non-small cell lung cancer].

Lung cancer remains the leading cause of cancer-related deaths in men and women worldwide, and 85% of these patients have non-small cell lung cancer. In recent years, the clinical use of targeted drug therapy and immune checkpoint inhibitors has dramatically changed the treatment landscape for advanced NSCLC. The mechanism and the value of targeted therapies have been a hot topic of research, as KRAS is one of the earliest discovered and most frequently mutated oncogenes, which is activated by binding to GTP and triggers a series of cascade reactions in cell proliferation and mitosis. The KRAS protein acts as a molecular switch and is activated by binding to GTP, triggering a series of cascade responses in cell proliferation and mitosis. Clinically, patients with KRAS mutated NSCLC have poor response to systemic medical therapy and poor prognosis. Since the first report of KRAS gene in 1982, research on KRAS targeted therapeutics has been slow, and previous studies such as farnesyltransferase inhibitors and downstream protein inhibitors of KRAS signaling pathway have not achieved the expected results, making KRAS long defined as a "non-druggable target". The deeper understanding of the crystal structure of KRAS has led to the discovery of potential therapeutic sites for KRAS and the development of several drugs directly targeting KRAS, especially KRAS G12C inhibitors such as AMG510 (sotorasib) and MRTX849 (adagrasib), which have shown encouraging results in clinical trials. In recent years, studies on the therapeutic efficacy of immune checkpoint inhibitors for KRAS-mutated NSCLC have made some progress. In this review, we systematically introduce the basic understanding of RAS gene and clinical characteristics of KRAS mutated NSCLC patients, summarize the medical treatments for KRAS mutated NSCLC, including chemotherapy, anti-vascular drug therapy and tumor immunotherapy, and focus on the review and outlook of the research progress of KRAS targeted therapy.

Current status and outlook of medical treatment for KRAS-mutated non-small cell lung cancer / 中华肿瘤杂志

Lung cancer remains the leading cause of cancer-related deaths in men and women worldwide, and 85% of these patients have non-small cell lung cancer. In recent years, the clinical use of targeted drug therapy and immune checkpoint inhibitors has dramatically changed the treatment landscape for advanced NSCLC. The mechanism and the value of targeted therapies have been a hot topic of research, as KRAS is one of the earliest discovered and most frequently mutated oncogenes, which is activated by binding to GTP and triggers a series of cascade reactions in cell proliferation and mitosis. The KRAS protein acts as a molecular switch and is activated by binding to GTP, triggering a series of cascade responses in cell proliferation and mitosis. Clinically, patients with KRAS mutated NSCLC have poor response to systemic medical therapy and poor prognosis. Since the first report of KRAS gene in 1982, research on KRAS targeted therapeutics has been slow, and previous studies such as farnesyltransferase inhibitors and downstream protein inhibitors of KRAS signaling pathway have not achieved the expected results, making KRAS long defined as a "non-druggable target". The deeper understanding of the crystal structure of KRAS has led to the discovery of potential therapeutic sites for KRAS and the development of several drugs directly targeting KRAS, especially KRAS G12C inhibitors such as AMG510 (sotorasib) and MRTX849 (adagrasib), which have shown encouraging results in clinical trials. In recent years, studies on the therapeutic efficacy of immune checkpoint inhibitors for KRAS-mutated NSCLC have made some progress. In this review, we systematically introduce the basic understanding of RAS gene and clinical characteristics of KRAS mutated NSCLC patients, summarize the medical treatments for KRAS mutated NSCLC, including chemotherapy, anti-vascular drug therapy and tumor immunotherapy, and focus on the review and outlook of the research progress of KRAS targeted therapy.

Immunological influence of serum-free manufactured umbilical cord-derived mesenchymal stromal cells for steroid-resistant acute graft-versus-host disease.

This study investigated the safety, efficacy, and immunological influence of allogeneic umbilical cord-derived mesenchymal stromal cells (IMSUT-CORD) processed in serum-free medium and cryoprotectant, for treating steroid-resistant acute graft-versus-host disease (aGVHD). In a phase I dose-escalation trial, IMSUT-CORD were infused intravenously twice weekly over two cycles with up to two additional cycles. Four patients received a dose of 1 × 106 cells/kg, while three received 2 × 106/kg. Of 76 total adverse events, fourteen associated or possibly associated adverse events included 2 cases of a hot flash, headache, and peripheral neuropathy, 1 each of upper abdominal pain, hypoxia, increased γ-GTP, somnolence, peripheral vascular pain at the injection site, thrombocytopenia, hypertension, and decreased fibrinogen. At 16 weeks after the initial IMSUT-CORD infusion, three patients showed complete response (CR), two partial response (PR), one mixed response, and one no response. The overall response rate was 71.4%, and the continuous CR/PR rate was 100% for over 28 days after CR/PR. NK cell count significantly increased and correlated with treatment response, whereas IL-12, IL-17, and IL-33 levels decreased, but did not correlate with treatment response. CCL2 and CCL11 levels increased during IMSUT-CORD therapy. IMSUT-CORD are usable in patients with steroid-resistant aGVHD (UMIN000032819: https://www.umin.ac.jp/ctr ).

Green Tea Extract Containing Piper retrofractum Fruit Ameliorates DSS-Induced Colitis via Modulating MicroRNA-21 Expression and NF-κB Activity.

The aim of the present study was to examine the effect of green tea extract containing Piper retrofractum fruit (GTP) on dextran-sulfate-sodium (DSS)-induced colitis, the regulatory mechanisms of microRNA (miR)-21, and the nuclear factor-κB (NF-κB) pathway. Different doses of GTP (50, 100, and 200 mg/kg) were administered orally once daily for 14 days, followed by GTP with 3% DSS for 7 days. Compared with the DSS-treated control, GTP administration alleviated clinical symptoms, including the disease activity index (DAI), colon shortening, and the degree of histological damage. Moreover, GTP suppressed miR-21 expression and NF-κB activity in colon tissue of DSS-induced colitis mice. The mRNA levels of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were downregulated by GTP. Colonic nitric oxide (NO) and prostaglandin E2 (PGE2) production, and myeloperoxidase (MPO) activity were also lowered by GTP. Taken together, our results revealed that GTP inhibits DSS-induced colonic inflammation by suppressing miR-21 expression and NF-κB activity, suggesting that it may be used as a potential functional material for improving colitis.

Green Tea Polyphenols Cause Apoptosis and Autophagy in HPV-16 Subgene-Immortalized Human Cervical Epithelial Cells via the Activation of the Nrf2 Pathway.

Infection with human papillomavirus (HPV) is relatively common and certain high-risk HPV strains can induce epithelial dysplasia, increasing the risk of cervical cancer. Green tea polyphenol (GTP) preparations exhibit diverse anti-inflammatory, antioxidative, and antitumor properties In Vitro and In Vivo. Topical GTP application has been recommended as a treatment for genital warts, but the effect of GTP treatment on HPV infection and HPV-associated cancer remains to be established. The present study aimed to explore the mechanism by which GTP affected HPV type 16 (HPV-16)-positive immortalized human cervical epithelial cells. Survival, apoptosis, and autophagocytosis of these cells following GTP treatment was assessed using CCK-8 assay, flow cytometry, and monodansylcadaverine (MDC) staining. These cells were further transfected with an shRNA specific for Nrf2 to generate stable Nrf2-knockdown cells. The levels of Caspase-3, Bcl-2, Bax, P53, Rb, HPV-16 E6, HPV-16 E7, P62, Beclin1 and LC3B were determined via Western blotting. These analyses revealed that GTP treatment induced autophagy and apoptosis in HPV-16-positive cells, while Nrf2 gene knockdown reversed GTP-induced autophagic and apoptotic effects. Together, these results suggested that GTP could alleviate HPV infection and HPV-associated precancerous lesions In Vitro by regulating the Nrf2 pathway, highlighting the therapeutic potential of GTP in treating HPV infection.

Impact of klotho on the expression of SRGAP2a in podocytes in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is the major cause of kidney failure, and glomerular podocytes play critical roles in the pathogenesis of DN by maintaining the glomerular structure and filtration barrier. Klotho and Slit-Robo GTP activating protein 2a (SRGAP2a) have been indicated to play protective roles in reducing kidney injury, but whether there is an internal relationship between these two factors is unclear. METHODS: In this study, we cultured differentiated rat podocytes in vitro and measured the SRGAP2a expressions by immunofluorescence staining, quantitative real-time PCR (qRT-PCR) and western blotting, after siRNA-mediated transforming growth factor ß1 (TGF-ß1) silencing, TGF-ß1 overexpression and in the presence of a reactive oxygen species (ROS) inhibitor. And we detected the expressions of SRGAP2a, small mother against decapentaplegic (Smad)2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, and NAD(P)H oxidase 4 (NOX4), ROS levels and podocyte cytoskeletal remodelling under high glucose (HG) and exogenous klotho conditions. In addition, we performed haematoxylin-eosin (HE) staining and immunohistochemistry with diabetic rat models to confirm the in vitro results. RESULTS: The results indicated that SRGAP2a expression was significantly upregulated under siRNA-mediated TGF-ß1 silencing conditions or after adding a ROS inhibitor, but significantly downregulated with TGF-ß1 overexpression, in the presence of HG. The supplementation of exogenous klotho under HG conditions significantly increased the SRGAP2a expression, remodelled the actin cytoskeleton and altered the expressions of Smad2/3, p-Smad2/3, Smad7 and NOX4 and reduced the ROS generation in podocytes. Moreover, klotho administration protected kidney injury in DN rats. CONCLUSIONS: This study indicated that klotho may modulate the expression of SRGAP2a by regulating the ROS and TGF-ß1 signalling pathways and provided theoretical support for klotho protein as a novel therapeutic strategy for treating DN patients.

Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) inhibits the replication of human herpes viruses.

The creatine kinase/creatine phosphate (CK/CrP) system plays an important role in cellular energy homeostasis. CK isoenzymes, which reversibly generate ATP from CrP, are compartmentalized at cellular sites where energy is produced or utilized. It has been noted that the expression of CK is induced in cells infected by several DNA viruses, implicating a role for cellular energy modulation as an important step for efficient viral replication. A CK substrate analog, 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine; CCr), was tested in vitro for antiviral activity against a variety of herpes viruses and RNA viruses. Several members of the human herpes virus family were found to be sensitive to CCr, including herpes simplex types 1 and 2 (HSV-1 and HSV-2). varicella-zoster virus, and cytomegalovirus. When administered to mice infected vaginally with HSV-2, CCr significantly reduced mortality, reduced vaginal lesion scores, and lowered the titers of recoverable virus. This treatment combined with acyclovir appeared to enhance the antiviral effects of acyclovir. In a second model, mice infected intraperitoneally with HSV-2 and treated with CCr showed a significant increase in survival compared to placebo. We conclude that CCr is the first example of a new class of antiviral compounds that target the CK/CrP system.

Cytomegalovirus cultures during maintenance DHPG therapy for cytomegalovirus (CMV) retinitis in acquired immunodeficiency syndrome (AIDS).

Nine patients with acquired immunodeficiency syndrome (AIDS) and cytomegalovirus (CMV) retinitis on maintenance therapy with ganciclovir: 9(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG) at high dose (30 mg/kg/week) or low dose (20 mg/kg/week) were tested every 1-2 weeks for CMV isolation from blood, saliva, and urine. Duration of therapy ranged from 1.5 to 12 months (average 5.3 months). During pretreatment and low-dose and high-dose maintenance therapy, CMV was isolated from 48/59 (81%), 90/211 (43%), and 40/290 (14%) of specimens, respectively. Three patients with progressive retinitis had viraemia more frequently than did six patients with stable retinitis, CMV being isolated from 29/47 (62%) and 17/121 (14%) of blood samples, respectively.

Novel pharmacological strategies in the treatment of life-threatening cytomegalovirus infections. Clinical experience with continuous infusion 9-(1,3-dihydroxy-2-propoxymethyl) guanine.

Two novel antiviral pharmacologic strategies were used for therapy of life- and sight-threatening cytomegalovirus (CMV) infection; these were continuous drug infusion by portable pump and individualized patient regimen. 9-(1,3-Dihydroxy-2-propoxymethyl)-guanine (DHPG), an active and recently licensed antiviral drug against cytomegalovirus infection, was administered to five immunocompromised patients with chorioretinitis (all patients), colitis (two), and pneumonitis (three). Through dosage escalation, correlations between plasma levels, toxicity (i.e., myelosuppression), and clinical benefit were ascertained for therapy of acute disease (pneumonitis) as well as long-term therapy (chorioretinitis). Resolution of viremia, pneumonitis, colitis, and chorioretinitis was accomplished with steady-state plasma levels of DHPG approximating the mean ID50 of CMV isolates. The most notable clinical benefit was survival from CMV pneumonia and stabilization of vision. Although no adverse toxicity occurred during the DHPG continuous long-term therapy, survival was limited by the underlying disease.

Antiviral chemotherapy and chemoprophylaxis.

Antiviral compounds have been developed for use in chemoprophylaxis and chemotherapy of a variety of infections in humans, including those caused by influenza viruses, respiratory syncytial virus, and herpesviruses. The efficacy of several of these compounds has been demonstrated in rigorously controlled trials. Advances in molecular virology have led to the identification of biochemically defined, virus-specific functions that serve as appropriate targets for the future development of antiviral compounds. Clinical investigators and practicing physicians are now confronting questions previously raised with the use of antibacterial antibiotics. These questions concern appropriate routes of administration for antiviral compounds, optimal dosage regimens, risks of long-term prophylaxis, and the emergence of resistant organisms.

Absence of significant antiviral effects of beta-gamma-methylene GTP on encephalomyocarditis virus infection of L cells and mice.

The protein synthesis inhibitor beta-gamma-methylene guanosine triphosphate (Gpp-CH2p) is shown here to be ineffective as a 'leaky membrane' antiviral agent against encephalomyocarditis virus infection of L cells and mice. Studies with GppCH2p in encephalomyocarditis virus-infected L cells indicate that the cells only become permeable to the inhibitor late in infection because the compound significantly inhibits protein synthesis only when added at 4 h p.i. At this time 50 to 70% of the new infectious virus particles have already been synthesized, and this is reflected in maximum inhibition of virus yields of only about 40%. Moreover, comparison of inhibition of protein synthesis by GppCH2p in vitro and in cell cultures indicates that the intracellular concentration attained is only 0.25% of that in the medium. The lack of antiviral activity of GppCH2p in encephalomyocarditis virus-infected mice is probably due to leakiness of infected cells occurring too late for sufficient inhibition of virus synthesis to be obtained.