Intrinsic, Cancer Cell-Selective Toxicity of Organic Photothermal Nanoagent: A Simple Formulation for Combined Photothermal Chemotherapy of Cancer

Nano-agent-mediated photothermal therapy (PTT) combined with chemotherapy has been proposed as an effective strategy against cancer. However, chemotherapeutic agents often cause serious side effects. Herein, a novel PTT nanoagent (Cy5.5–MSA–G250) with unanticipated intrinsic tumor-selective cytotoxicity is developed. The Cy5.5–MSA–G250 nanoparticles (NPs) are created by mixing mouse serum albumin (MSA) and coomassie brilliant blue (G250) and then conjugated with cyanine 5.5 (Cy5.5). As expected, Cy5.5–MSA–G250 NPs can efficiently kill cancer cells in vitro and in vivo by PTT. Meanwhile, we accidentally discover that Cy5.5–MSA–G250 have intrinsic specific cytotoxicity against tumor cells but not against normal cells. Moreover, the tumor-specific cytotoxicity of Cy5.5–MSA–G250 is much stronger than that of cytarabine, an FDA-approved anticancer drug. In vivo experiments also prove that Cy5.5–MSA–G250 NPs can effectively eliminate residual tumor cells and prevent metastasis. Further study indicates that selective induction of G1 cell cycle arrest and inhibition of DNA duplication in tumor cells may be the possible mechanism of the tumor cell-selective cytotoxicity of Cy5.5–MSA–G250 NPs. In addition, direct visualization, low systematic toxicity, good biodegradation, and efficient body excretion further make Cy5.5–MSA–G250 NPs attractive for in vivo applications. Taken together, Cy5.5–MSA–G250 NPs are proven to be a promising platform for combined photothermal chemotherapy

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Phosphatases can dephosphorylate phosphorylated kinases, leading to their inactivation, and ferroptosis is a type of cell death. Therefore, our aim is to identify phosphatases associated with ferroptosis by analyzing the differentially expressed genes (DEGs) of the Luminal A Breast Cancer (LumABC) cohort from the Cancer Genome Atlas (TCGA). An analysis of 260 phosphatase genes from the GeneCard database revealed that out of the 28 DEGs with high expression, only the expression of pyruvate dehydrogenase phosphatase 2 (PDP2) had a significant correlation with patient survival. In addition, an analysis of DEGs using gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis revealed a significant variation in the expression of ferroptosis-related genes. To further investigate this, we analyzed 34 ferroptosis-related genes from the TCGA-LumABC cohort. The expression of long-chain acyl-CoA synthetase 4 (ACSL4) was found to have the highest correlation with the expression of PDP2, and its expression was also inversely proportional to the survival rate of patients. Western blot experiments using the MCF-7 cell line showed that the phosphorylation level of ACSL4 was significantly lower in cells transfected with the HA-PDP2 plasmid, and ferroptosis was correspondingly reduced (p 2+. Immunoprecipitation experiments further revealed that the phosphorylation level of ACSL4 was only significantly reduced in cells where PDP2 and ACSL4 co-precipitated. These findings suggest that PDP2 may act as a phosphatase to dephosphorylate and inhibit the activity of ACSL4, which had been phosphorylated and activated in LumABC cells. Further experiments are needed to confirm the molecular mechanism of PDP2 inhibiting ferroptosis.</div

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Phosphatases can dephosphorylate phosphorylated kinases, leading to their inactivation, and ferroptosis is a type of cell death. Therefore, our aim is to identify phosphatases associated with ferroptosis by analyzing the differentially expressed genes (DEGs) of the Luminal A Breast Cancer (LumABC) cohort from the Cancer Genome Atlas (TCGA). An analysis of 260 phosphatase genes from the GeneCard database revealed that out of the 28 DEGs with high expression, only the expression of pyruvate dehydrogenase phosphatase 2 (PDP2) had a significant correlation with patient survival. In addition, an analysis of DEGs using gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis revealed a significant variation in the expression of ferroptosis-related genes. To further investigate this, we analyzed 34 ferroptosis-related genes from the TCGA-LumABC cohort. The expression of long-chain acyl-CoA synthetase 4 (ACSL4) was found to have the highest correlation with the expression of PDP2, and its expression was also inversely proportional to the survival rate of patients. Western blot experiments using the MCF-7 cell line showed that the phosphorylation level of ACSL4 was significantly lower in cells transfected with the HA-PDP2 plasmid, and ferroptosis was correspondingly reduced (p 2+. Immunoprecipitation experiments further revealed that the phosphorylation level of ACSL4 was only significantly reduced in cells where PDP2 and ACSL4 co-precipitated. These findings suggest that PDP2 may act as a phosphatase to dephosphorylate and inhibit the activity of ACSL4, which had been phosphorylated and activated in LumABC cells. Further experiments are needed to confirm the molecular mechanism of PDP2 inhibiting ferroptosis.</div

ACSL4 expression is correlated with the prognosis of LumABC patients.

(A) Forest plot showing the univariate Cox proportional hazards regression analysis of 16 ferroptosis-related genes based on data from the TCGA-LumABC cohort. (B) Kaplan-Meier survival of the three ferroptosis-related genes based on data from the TCGA-LumABC cohort.</p

GO and KEGG analysis reveal abnormalities in multiple pathways associated with ferroptosis.

(A-C) Results of the GO analysis, showing the first fifteen cellular compartments (A), biological processes (B), and molecular functions (C), respectively. (D) Results of the KEGG pathways analysis, showing the first ten pathways. (E) Visualization of the ferroptosis pathway in the KEGG analysis, showing the connections and interactions among various molecules related to ferroptosis.</p

Correlation between ACSL4 and PDP2 expression.

(A) ferroptosis-related genes with expression data in the TCGA-LumABC cohort. (B and C) Volcano plots (B) and heatmaps (C) showing differentially expressed genes (DEGs) among the ferroptosis-related genes in the TCGA-LumABC cohort. (D) Correlation analysis of the five phosphatases and the fifteen ferroptosis-related genes.</p

PDP2 is highly expressed in LumABC tissues, and its expression is inversely correlated with patient prognosis.

(A) Volcano plot showing the differentially expressed genes (DEGs) between LumABC and adjacent tissues based on data from the TCGA-LumABC cohort. (B) Forest plot showing the LASSO and univariate Cox regression analyses of five phosphatase-related genes based on data from the TCGA-LumABC cohort. (C) Risk diagnostic plot showing the diagnostic values of the five phosphatase genes. (D) Kaplan-Meier survival of the five phosphatases based on gene expression and clinical survival data from the TCGA-LumABC cohort.</p

GSEA analysis indicates the inhibition of ferroptosis in the TCGA-LumABC cohort.

(A) Thw results of the GSEA analysis show the first twenty pathways. (B) Out of the twenty pathways, three pathways related to ferroptosis are blocked.</p

PDP2 inhibits ferroptosis through dephosphorylating phosphorylated ACSL4.

MCF-7 cells were treated with the indicated plasmids and erastin. (A) Immunoblotting detection of PDP2, ACSL4, and phosphorylated ACSL4 (pACSL4), showing typical images in three replicates. (B) Flow cytometry detection of lipid peroxides using C11-BODIPY (n = 5). (C) Flow cytometry analysis of the proportion of 7-AAD-positive dead cells (n = 5). (D) Transmission electron microscopy observation of cell morphology, with white arrows indicating ferroptotic mitochondria and yellow arrows indicating normal mitochondria. (E) Flow cytometry detection of the fluorescence intensity of Fe2+ in cells stained with FerroOrange (n = 5). (F) Co-immunoprecipitation with anti-HA (PDP2) and immunoblotting detection of PDP2, ACSL4, and pACSL4, respectively. The flow cytometry data represent the mean ± SD from three independent experiments and were analyzed using two-way ANOVA followed by Tukey’s post-hoc multiple comparison analysis. Statistical significance is denoted as *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001.</p