Exploration of the regulatory mechanism of PD-L1 expression in pMMR AGS gastric cancer cell by cytotoxic drugs and anti-angiogenic drugs

PLIN3 knockdown enhances T cell-mediated cytotoxicity in non-small cell lung cancer via autophagy-dependent PD-L1 degradation.

BACKGROUND Recently, immune checkpoint inhibitors (ICIs), especially those that targets PD-1/PD-L1, have significantly altered the treatment approach for NSCLC. Nevertheless, many patients experience different levels of resistance after receiving treatment with ICIs, which restrict their broader use in clinical practice. Therefore, to enhance the overall efficacy of ICIs, there is an immediate necessity to further explain the processes of immune evasion in NSCLC, especially the modulatory mechanism of PD-L1. METHODS IHC was used to evaluate the protein expression level of PLIN3 and PD-L1 in NSCLC tissues. The impact of PLIN3 on PD-L1 was analyzed in NCI-H1975 and NCI-H157 cells using western blot, flow cytometry and quantitative PCR (qPCR). Immunofluorescence was performed to examine the effect of PLIN3 on the autophagy level. The levels of Granzyme B (GZMB) and interferon-gamma (IFN-γ) secreted by T cell were assessed. Bioinformatic analyses, including immune infiltration estimation and TIDE score, were performed using TCGA data. RESULTS In this research, we discovered that elevated PLIN3 level was linked to decreased infiltration of CD4+ and CD8+ T cell, a higher TIDE score, and poorer immunotherapy response. In NSCLC tissues, there was a positive correlation between PLIN3 and PD-L1. Besides, the level of PLIN3 protein is significantly reduced in patients who achieve pathological complete response. In addition, PLIN3 knockdown markedly reduced the level of PD-L1 protein. Mechanistically, PLIN3 knockdown activated autophagy, and promoted PD-L1 degradation via the autophagic-lysosomal pathway, which consequently shortened its protein half-life. Furthermore, PLIN3 knockdown enhanced T-cell-mediated tumor killing and resulted in an increased secretion of the effector molecules, including granzyme B and IFN-γ. CONCLUSIONS In summary, our study has shown that targeting PLIN3 can induce autophagy, which promotes the degradation of PD-L1, ultimately leading to enhanced activation of T cells. This research is the first to investigate the function of PLIN3 in the immune microenvironment, revealing its critical function in immune evasion and highlighting its promise as a treatment target. This offers an innovative approach to enhance the efficacy of immunotherapy in lung cancer.

Targeting the MDM2/Caspase-3/GSDME axis: apatinib and cinobufagin synergistically induce pyroptosis to suppress gastric cancer progression.

Dual MAO A and HSP90 inhibitors enhance anti-PD-1 therapy and suppress colorectal cancer

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, yet their efficacy remains limited by tumor resistance, immune-related adverse events, and poor response in microsatellite stable colorectal cancer (CRC). To address these challenges, dual monoamine oxidase A (MAO-A) and heat shock protein 90 (HSP90) inhibitors, MPT1B098 and MPT1B099, were developed and evaluated for their therapeutic potential and immune-modulatory efficacy in CRC. Human and murine CRC cell lines were treated with MPT1B098 and MPT1B099 to assess their effects in cytotoxicity, cell migration, apoptosis, cell cycle arrest, and cell-surface PD-L1 expression. Western blotting was performed to evaluate key apoptotic and EMT-related markers. In vivo therapeutic efficacy and safety were evaluated using CT26 and MC38 subcutaneous syngeneic tumor models in BALB/c and C57BL/6 mice treated with MPT1B098, MPT1B099, and/or anti-PD1 antibodies. Tumor infiltration of CD8 + T cells and tissue histopathology were evaluated by immunohistochemistry and H&E staining. In vitro, MPT1B098 and MPT1B099 exhibited potent cytotoxicity against human and murine CRC cell lines with sub-micromolar IC 50 values, effectively inhibiting cell growth and migration while inducing apoptosis. Both compounds decreased cell-surface PD-L1 expression and modulated key apoptotic and EMT markers on Western blot. In vivo, both compounds were well tolerated at 10 mg/kg and significantly suppressed tumor growth in microsatellite instable and stable models. Notably, anti-PD1 monotherapy was effective only in MSI models, whereas MPT1B098 and MPT1B099 showed efficacy in both MSI and MSS tumors, with combination therapy producing a modest synergistic effect. Immunohistochemistry revealed increased CD8 + T cell infiltration following treatment, particularly with MPT1B099 in combination with anti-PD1 antibodies. These findings highlight the dual anti-tumor and immune-modulatory properties of MPT1B098 and MPT1B099, mediated in part through PD-L1 downregulation and enhanced intratumoral T cell infiltration. Dual targeting of MAO-A and HSP90 represents a promising novel strategy to overcome immunotherapy resistance in MSS CRC and enhance the therapeutic efficacy of immune checkpoint blockade.

Targeting the PLEC/TPM4 cytoskeletal axis: DT-13 inhibits colorectal cancer metastasis through direct binding to PLEC and inactivation of Akt/ERK signaling.

BACKGROUND Metastasis is the leading cause of colorectal cancer (CRC) mortality. Standard 5-fluorouracil (5-FU) has limited anti-metastatic efficacy and dose-dependent toxicities, underscoring the need for therapeutic alternatives with improved safety profiles. METHODS This study investigated the anti‑metastatic mechanism of Liriope muscari baily saponins C (DT‑13) in CRC. Cell viability, migration, and invasion were assessed in HCT116 cells. An orthotopic CRC model was established in BALB/c nude mice to compare the therapeutic efficacy and hematological safety of DT-13 against 5-FU. Direct targets of DT-13 were identified using chemical proteomics (small‑molecule pull‑down, cellular thermal shift assay, drug affinity responsive target stability assay) and molecular dynamics simulations. RESULTS DT-13 inhibited HCT116 proliferation (IC50 ∼19 μM) and reduced migration and invasion by 78 % and 90 % in vitro. In vivo, DT-13 achieved a comparable reduction in tumor volume to 5-FU (21 % of control volume), while exhibiting a favorable safety profile. Chemical proteomics identified plectin (PLEC) as a primary direct target of DT-13. Molecular docking revealed a binding affinity of -7.654 kcal/mol. Mechanistically, DT-13 occupied the calponin homology domain of PLEC, disrupting its interaction with tropomyosin-4 (TPM4). This led to TPM4 downregulation and subsequently inhibition of Akt and ERK signaling pathways, thereby suppressing the epithelial-mesenchymal transition process. CONCLUSION DT‑13 exerts anti-metastatic effects in CRC by disrupting the PLEC/TPM4 cytoskeletal scaffold. DT-13 achieved potent anti-metastatic efficacy with a significantly improved systemic safety profile compared to 5-FU. These findings highlight the clinical potential of DT-13 as a safer therapeutic alternative for patients with metastatic CRC.

Exploring New Therapeutic Targets in NSCLC: Unraveling the Interplay Between NTRK and PD-L1 Expression

Background: Precision medicine has introduced immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) as key therapies for lung cancer. However, the interaction between PD-L1 expression and NTRK rearrangements in non-small cell lung cancer (NSCLC) remains unclear. Methods: A cohort of 482 resected NSCLC cases was analyzed using tissue microarrays. Immunohistochemistry (IHC) assessed PD-L1 expression with four antibody clones and pan-TRK with one clone. Whole-exome sequencing (WES) identified NTRK mutations, copy number variations, and gene fusions. Associations with clinicopathological features and prognosis were evaluated using Cox regression and survival analyses. Potential molecular connections between NTRK and PD-L1 were explored through a multi-database interactome analysis using Cytoscape. Results: PD-L1 positivity (TPS ≥ 1%) varied by clone: 28-8 (41.5%), 22C3 (34.2%), SP263 (42.7%), and SP142 (10.4%). High PD-L1 expression (TPS ≥ 50%) correlated with poorer overall survival in SP142-assessed cases (p = 0.045). TRK expression was observed in 4.6% (22/482) of cases, predominantly in squamous cell carcinoma (72.7%). Co-expression of pan-TRK and PD-L1 was significant (p < 0.001), with 4.6% of tumors showing dual positivity. WES detected three NTRK rearrangements, with no direct association with survival. Conclusions: The co-expression of PD-L1 and pan-TRK observed in this cohort suggests a biologically relevant interaction that warrants further investigation into its potential role in treatment response and resistance mechanisms to ICIs and TKIs.

Thymoquinone Potentiates Docetaxel-Induced Antitumor Activity with the Involvement of ROS and PI3K/AKT Pathway Modulation in Triple-Negative Breast Cancer Cells

Background: Drug resistance and treatment-associated toxicity remain major limitations of conventional chemotherapy for triple-negative breast cancer (TNBC). Thymoquinone (TQ), a bioactive phytochemical derived from Nigella sativa, has demonstrated anticancer properties and may enhance the therapeutic efficacy of docetaxel (DTX) through complementary molecular mechanisms. Objective: To investigate whether TQ potentiates the antitumor activity of DTX in MDA-MB-231 TNBC cells by affecting apoptosis, oxidative stress, wound closure, and PI3K/AKT pathway-related gene expression. Methods: MDA-MB-231 TNBC cells and HaCaT keratinocytes were treated with TQ, DTX, or their combination. Cell viability was determined using the MTT assay, and drug interactions were evaluated by the Chou–Talalay combination index (CI) method. Apoptosis, intracellular reactive oxygen species (ROS) production, ROS rescue experiments using N-acetyl-L-cysteine (NAC), caspase-9 expression, wound closure, and gene-expression changes were assessed using Annexin V/PI flow cytometry, DCFH-DA-based flow cytometric and fluorescence analyses, immunocytochemistry, wound-healing assay, and quantitative real-time PCR (qRT-PCR), respectively. Bioinformatic analyses were performed to identify signaling pathways associated with the observed molecular alterations. Results: The TQ + DTX combination demonstrated synergistic cytotoxicity and significantly increased apoptotic cell death compared with either monotherapy. Combination treatment markedly enhanced intracellular ROS accumulation, whereas NAC pretreatment significantly attenuated ROS generation and partially reversed the cytotoxic and pro-apoptotic effects, suggesting the involvement of ROS in the observed antitumor effects. Caspase-9 immunoreactivity was markedly increased following combination treatment, suggesting the involvement of the intrinsic apoptotic pathway. Furthermore, the combination significantly suppressed wound closure and downregulated BCL2, PIK3CA, and AKT1 while upregulating BAX, CASP9, and PTEN. Bioinformatic analyses identified apoptosis, p53, PI3K/AKT, mTOR, and MAPK signaling as the principal pathways potentially associated with the observed gene expression changes. Conclusions: TQ potentiates the antitumor activity of DTX, with the involvement of oxidative stress, apoptotic signaling, suppression of wound closure, and regulation of PI3K/AKT pathway-related gene expression in TNBC cells. These findings provide evidence supporting further preclinical investigation of the TQ + DTX combination as a promising therapeutic strategy for triple-negative breast cancer.