Coralyne Radiosensitizes A549 Cells by Upregulation of CDKN1A Expression to Attenuate Radiation Induced G2/M Block of the Cell Cycle

Coralyne is a synthetic analog of berberine related to protoberberine-isoquinoline alkaloids. Isoquinoline derivatives and analogs are renowned as potent radiosensitizers with potential medical application. In the present study, we investigated the effect of coralyne on the cell death, cytoskeletal changes and cell cycle progression of irradiated A549 cells. A clonogenic assay revealed that coralyne pretreatment decreased the viability of A549 cells in a time- and dose-dependent manner. Moreover, exposure to coralyne and ionizing radiation (IR) markedly altered the filamentous actin cytoskeletal architecture and integrin-β binding sites of A549 cells. Treatment with 1–25 µM coralyne in combination with 2 Gy of IR significantly reduced the percentage of cells in G2/M phase compared with 2 Gy IR alone. These results indicate that coralyne is a potent radiosensitizing agent that may find an application in medicine.     

Synthesis of New Biscoumarin Derivatives,In Vitro Cholinesterase Inhibition,Molecular Modelling and Antiproliferative Effect in A549 Human Lung Carcinoma Cells

A series of novel C4-C7-tethered biscoumarin derivatives (12a–e) linked through piperazine moiety was designed, synthesized, and evaluated biological/therapeutic potential. Biscoumarin 12d was found to be the most effective inhibitor of both acetylcholinesterase (AChE, IC₅₀ = 6.30 µM) and butyrylcholinesterase (BChE, IC₅₀ = 49 µM). Detailed molecular modelling studies compared the accommodation of ensaculin (well-established coumarin derivative tested in phase I of clinical trials) and 12d in the human recombinant AChE (hAChE) active site. The ability of novel compounds to cross the blood–brain barrier (BBB) was predicted with a positive outcome for compound 12e. The antiproliferative effects of newly synthesized biscoumarin derivatives were tested in vitro on human lung carcinoma cell line (A549) and normal colon fibroblast cell line (CCD-18Co). The effect of derivatives on cell proliferation was evaluated by MTT assay, quantification of cell numbers and viability, colony-forming assay, analysis of cell cycle distribution and mitotic activity. Intracellular localization of used derivatives in A549 cells was confirmed by confocal microscopy. Derivatives 12d and 12e showed significant antiproliferative activity in A549 cancer cells without a significant effect on normal CCD-18Co cells. The inhibition of hAChE/human recombinant BChE (hBChE), the antiproliferative activity on cancer cells, and the ability to cross the BBB suggest the high potential of biscoumarin derivatives. Beside the treatment of cancer, 12e might be applicable against disorders such as schizophrenia, and 12d could serve future development as therapeutic agents in the prevention and/or treatment of Alzheimer’s disease.     

Resistance of Lung Cancer Cells Grown as Multicellular Tumour Spheroids to Zinc Sulfophthalocyanine Photosensitization

Photodynamic therapy (PDT) is phototherapeutic modality used in the treatment of neoplastic and non-neoplastic diseases. The photochemical interaction of light, photosensitizer (PS) and molecular oxygen produces singlet oxygen which induces cell death. Zinc sulfophthalocyanine (ZnPcS_mix) has been shown to be effective in A549 monolayers, multicellular tumor spheroids (MCTSs) (250 µm) and not on MCTSs with a size of 500 µm. A549 cells used in this study were grown as MCTSs to a size of 500 µm in order to determine their susceptibility to PDT. ZnPcS_mix distribution in MCTSs and nuclear morphology was determined using a fluorescent microscope. Changes in cellular responses were evaluated using cell morphology, viability, proliferation, cytotoxicity, cell death analysis and mitochondrial membrane potential. Untreated MCTSs, showed no changes in cellular morphology, proliferation, cytotoxicity and nuclear morphology. Photoactivated ZnPcS_mix also showed no changes in cellular morphology and nuclear morphology. However, photoactivated ZnPcS_mix resulted in a significant dose dependant decrease in viability and proliferation as well as an increase in cell membrane damage in MCTSs over time. ZnPcS_mix photosensitization induces apoptotic cell death in MCTSs with a size of 500 µm and more resistantance when compared to monolayer cells and MCTSs with a size of 250 µm.     

Dibenzofuran, 4-Chromanone,Acetophenone, and Dithiecine Derivatives: Cytotoxic Constituents from Eupatorium fortunei

Five new compounds, eupatodibenzofuran A (1), eupatodibenzofuran B (2), 6-acetyl-8-methoxy-2,2-dimethylchroman-4-one (3), eupatofortunone (4), and eupatodithiecine (5), have been isolated from the aerial part of Eupatorium fortunei, together with 11 known compounds (6‒16). Compounds 1 and 2 featured a new carbon skeleton with an unprecedented 1-(9-(4-methylphenyl)-6-methyldibe nzo[b,d]furan-2-yl)ethenone. Among the isolates, compound 1 exhibited potent inhibitory activity with IC₅₀ values of 5.95 ± 0.89 and 5.55 ± 0.23 μM, respectively, against A549 and MCF-7 cells. The colony-formation assay demonstrated that compound 1 (5 μM) obviously decreased A549 and MCF-7 cell proliferation, and Western blot test confirmed that compound 1 markedly induced apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways.     

Intracellular Trafficking of Cationic Carbon Dots in Cancer Cell Lines MCF-7 and HeLa—Time Lapse Microscopy,Concentration-Dependent Uptake,Viability, DNA Damage,and Cell Cycle Profile

Fluorescent carbon dots (CDs) are potential tools for the labeling of cells with many advantages such as photostability, multicolor emission, small size, rapid uptake, biocompatibility, and easy preparation. Affinity towards organelles can be influenced by the surface properties of CDs which affect the interaction with the cell and cytoplasmic distribution. Organelle targeting by carbon dots is promising for anticancer treatment; thus, intracellular trafficking and cytotoxicity of cationic CDs was investigated. Based on our previous study, we used quaternized carbon dots (QCDs) for treatment and monitoring the behavior of two human cancer cell MCF-7 and HeLa lines. We found similarities between human cancer cells and mouse fibroblasts in the case of QCDs uptake. Time lapse microscopy of QCDs-labeled MCF-7 cells showed that cells are dying during the first two hours, faster at lower doses than at higher ones. QCDs at a concentration of 100 µg/mL entered into the nucleus before cellular death; however, at a dose of 200 µg/mL, blebbing of the cellular membrane occurred, with a subsequent penetration of QCDs into the nuclear area. In the case of HeLa cells, the dose-depended effect did not happen; however, the labeled cells were also dying in mitosis and genotoxicity occurred nearly at all doses. Moreover, contrasted intracellular compartments, probably mitochondria, were obvious after 24 h incubation with 100 µg/mL of QCDs. The levels of reactive oxygen species (ROS) slightly increased after 24 h, depending on the concentration, thus the genotoxicity was likely evoked by the nanomaterial. A decrease in viability did not reach IC 50 as the DNA damage was probably partly repaired in the prolonged G0/G1 phase of the cell cycle. Thus, the defects in the G2/M phase may have allowed a damaged cell to enter mitosis and undergo apoptosis. The anticancer effect in both cell lines was manifested mainly through genotoxicity.     

Development and Evaluation of Repurposed Etoricoxib Loaded Nanoemulsion for Improving Anticancer Activities against Lung Cancer Cells

In the present work, novel modality for lung cancer intervention has been explored. Primary literature has established the potential role of cyclooxygenase-2 (COX-2) inhibitor in regression of multiple forms of carcinomas. To overcome its poor water solubility and boost anticancer activity, etoricoxib (ETO) was chosen as a therapeutic candidate for repurposing and formulated into a nanoemulsion (NE). The prepared ETO loaded NE was characterized for the surface charge, droplet size, surface morphology, and in vitro release. The optimized ETO loaded NE was then investigated for its anticancer potential employing A549 lung cancer cell line via cytotoxicity, apoptotic activity, mitochondrial membrane potential activity, cell migration assay, cell cycle analysis, Caspase-3, 9, and p53 activity by ELISA and molecular biomarker analysis through RT-PCR test. The developed ETO-NE formulation showed adequate homogeneity in the droplet size distribution with polydispersity index (PDI) of (0.2 ± 0.03) and had the lowest possible droplet size (124 ± 2.91 nm) and optimal negative surface charge (−8.19 ± 1.51 mV) indicative of colloidal stability. The MTT assay results demonstrated that ETO-NE exhibited substantial anticancer activity compared to the free drug. The ETO-NE showed a substantially potent cytotoxic effect against lung cancer cells, as was evident from the commencement of apoptosis/necrotic cell death and S-phase cell cycle arrests in A549 cells. The study on these molecules through RT-PCR confirmed that ETO-NE is significantly efficacious in mitigating the abundance of IL-B, IL-6, TNF, COX-2, and NF-kB as compared to the free ETO and control group. The current study demonstrates that ETO-NE represents a feasible approach that could provide clinical benefits for lung cancer patients in the future.     

Albumin Suppresses Human Hepatocellular Carcinoma Proliferation and the Cell Cycle

Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC) is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP), p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma) were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD) with appropriate software (ModFit LT; BD). The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore). The mRNA levels of AFP relative to Alb(−): Alb(−), Alb(+), and Prionex, were 1, 0.7 ± 0.2 (p < 0.001 for Alb(−)), and 1 ± 0.3, respectively. The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(−) and p = 0.004 for Prionex), and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(−) and Prionex), and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+). More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+) than in Alb(−) (G0/G1: 60.9%, 67.7%, 61.5%; G2/M: 16.5%, 13.1%, 15.6%; S: 22.6%, 19.2%, 23.0%, Alb(−), Alb(+), Prionex, respectively). The same results were obtained in HepG2. Cell proliferation was inhibited in 5 g/dL albumin medium in both HepG2 cells and Hep3B cells in 24 h culture by counting cell numbers. The presence of albumin in serum reduces the phosphorylation of Rb proteins and enhances the expression of p21 and p57, following an increase in the G0/G1 cell population, and suppresses cell proliferation. These results suggest that albumin itself suppresses the proliferation of hepatocellular carcinoma.     

Tropinone-Derived Alkaloids as Potent Anticancer Agents: Synthesis,Tyrosinase Inhibition,Mechanism of Action,DFT Calculation,and Molecular Docking Studies

A new series of hybrid compounds with tropinone and thiazole rings in the structure was designed and synthesized as potential anticancer agents. They were tested against human multiple myeloma (RPMI 8226), lung carcinoma (A549), breast adenocarcinoma (MDA-MB-231), and mouse skin melanoma (B16-F10) cell lines. Toxicity was tested on human normal skin fibroblasts (HSF) and normal colon fibroblasts (CCD-18Co). The growth inhibition mechanism of the most active derivative was analyzed through investigation of its effect on the distribution of cell cycle phases and ability to induce apoptosis and necrosis in RPMI 8226 and A549 cancer cells. The tyrosinase inhibitory potential was assessed, followed by molecular docking studies. Compounds 3a–3h show high anticancer activity against MDA-MB-231 and B16-F10 cell lines with IC₅₀ values of 1.51–3.03 µM. Moreover, the cytotoxic activity of the investigated compounds against HSF and CCD-18Co cells was 8–70 times lower than against the cancer cells or no toxicity was shown in our tests, with derivative 3a being particularly successful. The mechanism of action of compound 3a in RPMI 8226 cell was shown to be through induction of cell death through apoptosis. The derivatives show ability to inhibit the tyrosinase activity with a mixed mechanism of inhibition. The final molecular docking results showed for IC₅₀ distinct correlation with experiment.     

Radiosensitization to γ-Ray by Functional Inhibition of APOBEC3G

The radiosensitization of tumor cells is one of the promising approaches for enhancing radiation damage to cancer cells and limiting radiation effects on normal tissue. In this study, we performed a comprehensive screening of radiosensitization targets in human lung cancer cell line A549 using an shRNA library and identified apolipoprotein B mRNA editing enzyme catalytic subunit 3G (APOBEC3G: A3G) as a candidate target. APOBEC3G is an innate restriction factor that inhibits HIV-1 infection as a cytidine deaminase. APOBEC3G knockdown with siRNA showed an increased radiosensitivity in several cancer cell lines, including pancreatic cancer MIAPaCa2 cells and lung cancer A549 cells. Cell cycle analysis revealed that APOBEC3G knockdown increased S-phase arrest in MIAPaCa2 and G2/M arrest in A549 cells after γ-irradiation. DNA double-strand break marker γH2AX level was increased in APOBEC3G-knocked-down MIAPaCa2 cells after γ-irradiation. Using a xenograft model of A549 in mice, enhanced radiosensitivity by a combination of X-ray irradiation and APOBEC3G knockdown was observed. These results suggest that the functional inhibition of APOBEC3G sensitizes cancer cells to radiation by attenuating the activation of the DNA repair pathway, suggesting that APOBEC3G could be useful as a target for the radiosensitization of cancer therapy.     

Combined Therapy with Dacarbazine and Hyperthermia Induces Cytotoxicity in A375 and MNT-1 Melanoma Cells

Melanoma is a drug-resistant cancer, representing a serious challenge in cancer treatment. Dacarbazine (DTIC) is the standard drug in metastatic melanoma treatment, despite the poor results. Hyperthermia has been proven to potentiate chemotherapy. Hence, this work analyzed the combined action of hyperthermia and DTIC on A375 and MNT-1 cell lines. First, temperatures between 40 °C and 45 °C were tested. The effect of DTIC on cell viability was also investigated after exposures of 24, 48, and 72 h. Then, cells were exposed to 43 °C and to the respective DTIC IC10 or IC20 of each time exposure. Overall, hyperthermia reduced cell viability, however, 45 °C caused an excessive cell death (>90%). Combinational treatment revealed that hyperthermia potentiates DTIC’s effect, but it is dependent on the concentration and temperature used. Also, it has different mechanisms from the treatments alone, delaying A375 cells at the G2/M phase and MNT-1 cells at the S and G2/M phases. Intracellular reactive oxygen species (ROS) levels increased after treatment with hyperthermia, but the combined treatment showed no additional differences. Also, hyperthermia highly increased the number of A375 early apoptotic cells. These results suggest that combining hyperthermia and DTIC should be more explored to improve melanoma treatment.     

Simulated Microgravity Inhibits the Proliferation of Chang Liver Cells by Attenuation of the Major Cell Cycle Regulators and Cytoskeletal Proteins

Simulated microgravity (SMG) induced the changes in cell proliferation and cytoskeleton organization, which plays an important factor in various cellular processes. The inhibition in cell cycle progression has been considered to be one of the main causes of proliferation inhibition in cells under SMG, but their mechanisms are still not fully understood. This study aimed to evaluate the effects of SMG on the proliferative ability and cytoskeleton changes of Chang Liver Cells (CCL-13). CCL-13 cells were induced SMG by 3D clinostat for 72 h, while the control group were treated in normal gravity at the same time. The results showed that SMG reduced CCL-13 cell proliferation by an increase in the number of CCL-13 cells in G0/G1 phase. This cell cycle phase arrest of CCL-13 cells was due to a downregulation of cell cycle-related proteins, such as cyclin A1 and A2, cyclin D1, and cyclin-dependent kinase 6 (Cdk6). SMG-exposed CCL-13 cells also exhibited a downregulation of α-tubulin 3 and β-actin which induced the cytoskeleton reorganization. These results suggested that the inhibited proliferation of SMG-exposed CCL-13 cells could be associate with the attenuation of major cell cycle regulators and main cytoskeletal proteins.     

A Genotoxic Stress-Responsive miRNA,miR-574-3p,Delays Cell Growth by Suppressing the Enhancer of Rudimentary Homolog Gene in Vitro

MicroRNA (miRNA) is a type of non-coding RNA that regulates the expression of its target genes by interacting with the complementary sequence of the target mRNA molecules. Recent evidence has shown that genotoxic stress induces miRNA expression, but the target genes involved and role in cellular responses remain unclear. We examined the role of miRNA in the cellular response to X-ray irradiation by studying the expression profiles of radio-responsive miRNAs and their target genes in cultured human cell lines. We found that expression of miR-574-3p was induced in the lung cancer cell line A549 by X-ray irradiation. Overexpression of miR-574-3p caused delayed growth in A549 cells. A predicted target site was detected in the 3′-untranslated region of the enhancer of the rudimentary homolog (ERH) gene, and transfected cells showed an interaction between the luciferase reporter containing the target sequences and miR-574-3p. Overexpression of miR-574-3p suppressed ERH protein production and delayed cell growth. This delay was confirmed by knockdown of ERH expression. Our study suggests that miR-574-3p may contribute to the regulation of the cell cycle in response to X-ray irradiation via suppression of ERH protein production.     

In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

Papaverine (PPV) is a benzylisoquinoline alkaloid isolated from Papaver somniferum that exerts antiproliferative activity. However, several questions remain regarding the biochemical pathways affected by PPV in tumourigenic cells. In this study, the influence of PPV on cell migration (light microscopy), expression of vascular endothelial growth factor (VEGF) B, VEGF R1, VEGF R2, and phosphorylated focal adhesion kinase (pFAK) were investigated using spectrophotometry in MDA-MB-231-, A549- and DU145 cell lines. The migration assay revealed that, after 48 h, PPV (100 µM) reduced cell migration to 81%, 91%, and 71% in MDA-MB-231-, A549-, and DU145 cells, respectively. VEGF B expression was reduced to 0.79-, 0.71-, and 0.73-fold after 48 h of exposure to PPV in MDA-MB-231-, A549- and DU145 cells, while PPV exposure of 48 h increased VEGF R1 expression in MDA-MB-231- and DU145 cells to 1.38 and 1.46. A fold decrease in VEGF R1 expression was observed in A549 cells to 0.90 after exposure to 150 µM. No statistically significant effects were observed on VEGF R2- and FAK expression after exposure to PPV. This study contributes to the understanding of the effects of a phytomedicinal alkaloid compound in cancer cells and may provide novel approaches to the application of non-addictive alkaloids.     

Repurposing Bedaquiline for Effective Non-Small Cell Lung Cancer (NSCLC) Therapy as Inhalable Cyclodextrin-Based Molecular Inclusion Complexes

There is growing evidence that repurposed drugs demonstrate excellent efficacy against many cancers, while facilitating accelerated drug development process. In this study, bedaquiline (BDQ), an FDA approved anti-mycobacterial agent, was repurposed and an inhalable cyclodextrin complex formulation was developed to explore its anti-cancer activity in non-small cell lung cancer (NSCLC). A sulfobutyl ether derivative of β-cyclodextrin (SBE-β-CD) was selected based on phase solubility studies and molecular modeling to prepare an inclusion complex of BDQ and cyclodextrin. Aqueous solubility of BDQ was increased by 2.8 × 10³-fold after complexation with SBE-β-CD, as compared to its intrinsic solubility. Solid-state characterization studies confirmed the successful incorporation of BDQ in the SBE-β-CD cavity. In vitro lung deposition study results demonstrated excellent inhalable properties (mass median aerodynamic diameter: 2.9 ± 0.6 µm (<5 µm) and fine particle fraction: 83.3 ± 3.8%) of BDQ-CD complex. Accelerated stability studies showed BDQ-CD complex to be stable up to 3 weeks. From cytotoxicity studies, a slight enhancement in the anti-cancer efficacy was observed with BDQ-cyclodextrin complex, compared to BDQ alone in H1299 cell line. The IC₅₀ values for BDQ and BDQ-CD complex were found to be ~40 µM in case of H1299 cell line at 72 h, whereas BDQ/BDQ-CD were not found to be cytotoxic up to concentrations of 50 µM in A549 cell line. Taken together, BDQ-CD complex offers a promising inhalation strategy with efficient lung deposition and cytotoxicity for NSCLC treatment.     

Modified Gold Nanoparticles for Efficient Delivery of Betulinic Acid to Cancer Cell Mitochondria

Advances in nanomedicine have seen the adaptation of nanoparticles (NPs) for subcellular delivery for enhanced therapeutic impact and reduced side effects. The pivotal role of the mitochondria in apoptosis and their potential as a target in cancers enables selective induction of cancer cell death. In this study, we examined the mitochondrial targeted delivery of betulinic acid (BA) by the mitochondriotropic TPP⁺-functionalized epigallocatechin gallate (EGCG)-capped gold NPs (AuNPs), comparing the impact of polyethylene glycol (PEG) and poly-L-lysine-graft-polyethylene glycol (PLL-g-PEG) copolymer on delivery efficacy. This included the assessment of their cellular uptake, mitochondrial localization and efficacy as therapeutic delivery platforms for BA in the human Caco-2, HeLa and MCF-7 cancer cell lines. These mitochondrial-targeted nanocomplexes demonstrated significant inhibition of cancer cell growth, with targeted nanocomplexes recording IC₅₀ values in the range of 3.12–13.2 µM compared to that of the free BA (9.74–36.31 µM) in vitro, demonstrating the merit of mitochondrial targeting. Their mechanisms of action implicated high amplitude mitochondrial depolarization, caspases 3/7 activation, with an associated arrest at the G0/G1 phase of the cell cycle. This nano-delivery system is a potentially viable platform for mitochondrial-targeted delivery of BA and highlights mitochondrial targeting as an option in cancer therapy.     

The Fight against Cancer by Microgravity: The Multicellular Spheroid as a Metastasis Model

Cancer is a disease exhibiting uncontrollable cell growth and spreading to other parts of the organism. It is a heavy, worldwide burden for mankind with high morbidity and mortality. Therefore, groundbreaking research and innovations are necessary. Research in space under microgravity (µg) conditions is a novel approach with the potential to fight cancer and develop future cancer therapies. Space travel is accompanied by adverse effects on our health, and there is a need to counteract these health problems. On the cellular level, studies have shown that real (r-) and simulated (s-) µg impact survival, apoptosis, proliferation, migration, and adhesion as well as the cytoskeleton, the extracellular matrix, focal adhesion, and growth factors in cancer cells. Moreover, the µg-environment induces in vitro 3D tumor models (multicellular spheroids and organoids) with a high potential for preclinical drug targeting, cancer drug development, and studying the processes of cancer progression and metastasis on a molecular level. This review focuses on the effects of r- and s-µg on different types of cells deriving from thyroid, breast, lung, skin, and prostate cancer, as well as tumors of the gastrointestinal tract. In addition, we summarize the current knowledge of the impact of µg on cancerous stem cells. The information demonstrates that µg has become an important new technology for increasing current knowledge of cancer biology.     

Distinct Immune Signatures Indicative of Treatment Response and Immune-Related Adverse Events in Melanoma Patients under Immune Checkpoint Inhibitor Therapy

To identify potential early biomarkers of treatment response and immune-related adverse events (irAE), a pilot immune monitoring study was performed in stage IV melanoma patients by flow cytometric analysis of peripheral blood mononuclear cells (PBMC). Overall, 17 patients were treated with either nivolumab or pembrolizumab alone, or with a combination of nivolumab and ipilimumab every three weeks. Of 15 patients for which complete response assessment was available, treatment responders (n = 10) as compared to non-responders (n = 5) were characterized by enhanced PD-1 expression on CD8⁺ T cells immediately before treatment (median ± median absolute deviation/MAD 26.7 ± 10.4% vs. 17.2 ± 5.3%). Responders showed a higher T cell responsiveness after T cell receptor ex vivo stimulation as determined by measurement of programmed cell death 1 (PD-1) expression on CD3⁺ T cells before the second cycle of treatment. The percentage of CD8⁺ effector memory (CD8⁺CD45RA⁻CD45RO⁺CCR7⁻) T cells was higher in responders compared to non-responders before and immediately after the first cycle of treatment (median ± MAD 39.2 ± 7.3% vs. 30.5 ± 4.1% and 37.7 ± 4.6 vs. 24.0 ± 6.4). Immune-related adverse events (irAE) were accompanied by a higher percentage of activated CD4⁺ (CD4⁺CD38⁺HLADR⁺) T cells before the second treatment cycle (median ± MAD 14.9 ± 3.9% vs. 5.3 ± 0.4%). In summary, PBMC immune monitoring of immune-checkpoint inhibition (ICI) treatment in melanoma appears to be a promising approach to identify early markers of treatment response and irAEs.     

Salvianolic Acid A,as a Novel ETA Receptor Antagonist,Shows Inhibitory Effects on Tumor in Vitro

Endothelin-1 (ET-1) autocrine and paracrine signaling modulate cell proliferation of tumor cells by activating its receptors, endothelin A receptor (ET_AR) and endothelin B receptor (ET_BR). Dysregulation of ET_AR activation promotes tumor development and progression. The potential of ET_AR antagonists and the dual-ET_AR and ET_BR antagonists as therapeutic approaches are under preclinical and clinical studies. Salvianolic acid A (Sal A) is a hydrophilic polyphenolic derivative isolated from Salvia miltiorrhiza Bunge (Danshen), which has been reported as an anti-cancer and cardio-protective herbal medicine. In this study, we demonstrate that Sal A inhibits ET_AR activation induced by ET-1 in both recombinant and endogenous ET_AR expression cell lines. The IC₅₀ values were determined as 5.7 µM in the HEK293/ET_AR cell line and 3.14 µM in HeLa cells, respectively. Furthermore, our results showed that Sal A suppressed cell proliferation and extended the doubling times of multiple cancer cells, including HeLa, DU145, H1975, and A549 cell lines. In addition, Sal A inhibited proliferation of DU145 cell lines stimulated by exogenous ET-1 treatment. Moreover, the cytotoxicity and cardio-toxicity of Sal A were assessed in human umbilical vein endothelial cells (HUVEC) and Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which proved that Sal A demonstrates no cytotoxicity or cardiotoxicity. Collectively, our findings indicate that Sal A is a novel anti-cancer candidate through targeting ET_AR.