Nanoparticles for Augmenting Therapeutic Potential and Alleviating the Effect of Di(2-ethylhexyl) Phthalate on Gastric Cancer.

Changes in diet culture and modern lifestyle contributed to a higher incidence of gastrointestinal-related diseases, including gastritis, implicated in the pathogenesis of gastric cancer. This observation raised concerns regarding exposure to di(2-ethylhexyl) phthalate (DEHP), which is linked to adverse health effects, including reproductive and developmental problems, inflammatory response, and invasive adenocarcinoma. Research on the direct link between DEHP and gastric cancer is ongoing, and further studies are required to establish a conclusive association. In our study, extremely low concentrations of DEHP exerted significant effects on cell migration by promoting the epithelial-mesenchymal transition in gastric cancer cells. This effect was mediated by the modulation of the PI3K/AKT/mTOR and Smad2 signaling pathways. To address the DEHP challenges, our initial design of TPGS-conjugated fucoidan, delivered via pH-responsive nanoparticles, successfully demonstrated binding to the P-selectin protein. This achievement has not only enhanced the antigastric tumor efficacy but has also led to a significant reduction in the expression of malignant proteins associated with the condition. These findings underscore the promising clinical therapeutic potential of our approach.

Nanoparticle-enhanced postbiotics: Revolutionizing cancer therapy through effective delivery.

AIM: Gastric cancer contributes to cancer-related fatalities. Conventional chemotherapy faces challenges due to severe adverse effects, prompting recent research to focus on postbiotics, which are safer biomolecules derived from nonviable probiotics. Despite promising in vitro results, efficient in vivo delivery systems remain a challenge. This study aimed to design a potential nanoparticle (NP) formulation encapsulating the Lacticaseibacillus paracasei GMNL-133 (SGMNL-133) isolate to enhance its therapeutic efficacy in treating gastric cancer. MAIN METHODS: We successfully isolated GMNL-133 (SGMNL-133) by optimizing the lysate extraction and column elution processes for L. paracasei GMNL-133, resulting in substantial enhancement of its capacity to inhibit the proliferation of gastric cancer cells. Additionally, we developed a potential NP utilizing arginine-chitosan and fucoidan encapsulating SGMNL-133. KEY FINDINGS: This innovative approach protected the SGMNL-133 from degradation by gastric acid, facilitated its penetration through the mucus layer, and enabled interaction with gastric cancer cells. Furthermore, in vivo experiments demonstrated that the encapsulation of SGMNL-133 in NPs significantly enhanced its efficacy in the treatment of orthotopic gastric tumors while simultaneously reducing tissue inflammation levels. SIGNIFICANCE: Recent research highlights postbiotics as a safe alternative, but in vivo delivery remains a challenge. Our study optimized the extraction of the lysate and column elution of GMNL-133, yielding SGMNL-133. We also developed NPs to protect SGMNL-133 from gastric acid, enhance mucus penetration, and improve the interaction with gastric cancer cells. This combination significantly enhanced drug delivery and anti-gastric tumor activity.

Targeting nanoparticle-conjugated microbubbles combined with ultrasound-mediated microbubble destruction for enhanced tumor therapy.

The stress of the abnormal stromal matrix of solid tumors is a major limiting factor that prevents drug penetration. Controlled, accurate, and efficient delivery of theranostic agents into tumor cells is crucial. Combining ultrasound with nanocarrierbased drug delivery systems have become a promising approach for targeted drug delivery in preclinical cancer therapy. In this study, to ensure effective tumor barrier penetration, access to the tumor microenvironment, and local drug release, we designed targeted nanoparticle (NP)-conjugated microbubbles (MBs); ultrasound could then help deliver acoustic energy to release the NPs from the MBs. The ultrasound-targeted MB destruction (UTMD) system of negatively charged NPs was conjugated with positively charged MBs using an ionic gelation method. We demonstrated the transfer of targeted NPs and their entry into gastric cancer cells through ligand-specific recognition, followed by enhanced cell growth inhibition owing to drug delivery-induced apoptosis. Moreover, the UTMD system combining therapeutic and ultrasound image properties can effectively target gastric cancer, thus significantly enhancing antitumor activity, as evident by tumor localization in an orthotopic mouse model of gastric cancer. The combination of ultrasound and NP-based drug delivery systems has become a promising approach for targeted drug delivery in preclinical cancer therapy.

Active Targeting of P-Selectin by Fucoidan Modulates the Molecular Profiling of Metastasis in Docetaxel-Resistant Prostate Cancer.

The standard of care for prostate cancer (PCa) is androgen deprivation therapy (ADT). Although hormone-sensitive PCa is curable by ADT, most conditions progress to castration-resistant prostate cancer (CRPCa) and metastatic CRPCa (mCRPCa). Front-line docetaxel has been administered to patients with CRPCa and mCRPCa. Nevertheless, docetaxel resistance after half a year of therapy has emerged as an urgent clinical concern in patients with CRPCa and mCRPCa. We verified the mechanism by which docetaxel-resistant PCa cells (DU/DX50) exhibited significant cell migration and expression of malignant tumor-related proteins. Our study shows that the biological activity of fucoidan has an important application for docetaxel-resistant PCa cells, inhibiting IL-1R by binding to P-selectin and reducing the expression levels of NF-κB p50 and Cox2 in this metastasis-inhibiting signaling pathway. Furthermore, the combined treatment of fucoidan and docetaxel showed significant anticancer and synergistic effects on the viability of DU/DX50 cells, which is relevant for overcoming the current limitations and improving treatment outcomes. Overall, fucoidan-based combination chemotherapy may exert beneficial effects and facilitate the treatment of docetaxel-resistant PCa.

Silencing of argininosuccinate lyase inhibits colorectal cancer formation.

Arginine and nitric oxide (NO) are important mediators of tumorigenesis in various types of cancer. Dysregulation of NO content by argininosuccinate lyase (ASL) has been previously demonstrated to inhibit the proliferation of liver and breast cancer cells. However, the function of ASL in colon cancer is not well defined. The present study aimed to determine the effect of ASL on colon cancer. Western blot analysis indicated that ASL expression was induced by endoplasmic reticulum stress in HCT116 and SW480 colon cancer cells. Additionally, the expression of ASL in colon cancer tissues was enhanced compared with that in the adjacent normal tissues, and the patients with colon cancer with higher ASL expression exhibited poorer survival rates. Transfection of ASL-targeting short hairpin RNA (shRNA) into HCT116 cells inhibited cell proliferation and decreased anchorage-independent growth in a soft agar assay. In addition, when injected subcutaneously into NOD/SCID mice, stable transfectant ASL-downregulated HCT116 cells exhibited decreased in vivo tumorigenic ability. Flow cytometric analysis of cell cycle progression indicated that ASL-targeting shRNA induced G2/M arrest, and western blot analysis showed that the inhibition of ASL was accompanied by cyclin A2 degradation. Furthermore, ASL-targeting shRNA resulted in increased autophagosomes and decreased NO levels. Inhibition of NO by the NO synthase inhibitor L-NMMA significantly reduced cell proliferation and colony formation. In summary, the results of the present study indicated that ASL-targeting shRNA-induced growth inhibition is associated with decreased cyclin A2 expression and NO content in colon cancer.

Argininosuccinate lyase interacts with cyclin A2 in cytoplasm and modulates growth of liver tumor cells.

Arginine is a critical amino acid in specific cancer types including hepatocellular carcinoma (HCC) and melanoma. Novel molecular mechanisms and therapeutic targets in arginine metabolism-mediated cancer formation await further identification. Our laboratory has previously demonstrated that arginine metabolic enzyme argininosuccinate lyase (ASL) promoted HCC formation in part via maintenance of cyclin A2 protein expression and arginine production for channeling to nitric oxide synthase. In this study, we investigated the mechanism by which ASL regulates cyclin A2 expression. We found that ASL interacted with cyclin A2 in HCC cells and the localization of their interaction was in the cytoplasm. Mutation of essential residues for enzymatic activity of ASL did not affect the binding of ASL to cyclin A2. Moreover, the mutant ASL retained the ability to restore the decreased tumorigenicity caused by ASL shRNA. Furthermore, overexpression of ASL conferred resistance to arginine deprivation therapy. Finally, the important pathways and potential therapeutic targets in ASL-regulated HCC were identified by bioinformatics analyses with Metacore database and Connectivity Map database. Our analyses suggested that bisoprolol, celecoxib, and ipratropium bromide, are potential therapeutics for ASL-regulated HCC formation. Thus, ASL interacts with cyclin A2 in cytoplasm, and may promote HCC formation through this non-enzymatic function. Overexpression of ASL may be a contributing factor in drug resistance for arginine deprivation therapy.

MST3 promotes proliferation and tumorigenicity through the VAV2/Rac1 signal axis in breast cancer.

MST3 (mammalian STE20-like kinase 3) belongs to the Ste20 serine/threonine protein kinase family. The role of MST3 in tumor growth is less studied; therefore, we investigates the function of MST3 in breast cancer. Here, we demonstrate that MST3 is overexpressed in human breast tumors. Online Kaplan-Meier plotter analysis reveals that overexpression of MST3 predicts poor prognosis in breast cancer patients. Knockdown of MST3 with shRNA inhibits proliferation and anchorage-independent growth in vitro. Downregulation of MST3 in triple-negative MDA-MB-231 and MDA-MB-468 breast cancer cells decreases tumor formation in NOD/SCID mice. MST3 interacts with VAV2, but not VAV3, as demonstrated by co-immunoprecipitation and confocal microscopy. By domain mapping of MST3, we determine that the proline-rich region of MST3 (353KDIPKRP359) interacts with the SH3 domain of VAV2. Mutation of the two proline residues in this domain significantly attenuates the interaction between MST3 and VAV2. Overexpression of wild-type MST3 (WT-MST3), but not proline-rich-deleted MST3 (∆P-MST3), enhances the proliferation rate and anchorage-independent growth of MDA-MB-468 cells. Overexpression of MST3 increases VAV2 phosphorylation and GTP-Rac1, whereas downregulation of MST3 or delivery of ∆P-MST3 results in a reduction of VAV2 and Rac1 activation. Knockdown of MST3 inhibits cyclin D1 protein expression. The Rac1 inhibitor EHop-016 attenuates cell proliferation induced by WT-MST3. Finally, Knockdown of MST3 or Rac1 inhibitor decreases cyclin D protein expression, which is important for tumor growth. These results indicate that MST3 interacts with VAV2 to activate Rac1 and promote the tumorigenicity of breast cancer.

Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer.

CD90 is used as a marker for cancer stem cell in liver cancer. We aimed to study the mechanism by which CD90 promoted liver cancer progression and identify the new therapeutic targets on CD90 signal pathway. Ectopic expression of CD90 in liver cancer cell lines enhanced anchorage-independent growth and tumor progression. Furthermore, CD90 promoted sphere formation in vitro and upregulated the expression of the cancer stem cell marker CD133. The CD133 expression was higher in CD45-CD90+ cells in liver cancer specimen. The natural carcinogenic molecules TGF-ß-1, HGF, and hepatitis B surface antigen increased the expression of CD90 and CD133. Inhibition of CD90 by either shRNA or antibody attenuated the induction of CD133 and anchorage-independent growth. Lentiviral delivery of CD133 shRNA abolished the tumorigenicity induced by CD90. Ectopic expression of CD90 induced mTOR phosphorylation and AMPK dephosphorylation. Mutation of integrin binding-RLD domain in CD90 attenuated the induction of CD133 and anchorage-independent growth. Similar results were observed after silencing ß3 integrin. Signaling analyses revealed that AMPK/mTOR and ß3 integrin were required for the induction of CD133 and tumor formation by CD90. Importantly, the energy restriction mimetic agent OSU-CG5 reduced the CD90 population in fresh liver tumor sample and repressed the tumor growth. In contrast, sorafenib did not decrease the CD90+ population. In conclusion, the signal axis of CD90-integrin-mTOR/AMPK-CD133 is critical for promoting liver carcinogenesis. Molecules inhibiting the signal axis, including OSU-CG5 and other inhibitors, may serve as potential novel cancer therapeutic targets in liver cancer.

Argininosuccinate lyase is a potential therapeutic target in breast cancer.

Arginine is a non-essential amino acid that modulates nitric oxide production and cancer homeostasis. In our previous study, we observed that blocking argininosuccinate lyase (ASL) attenuates tumor progression in liver cancer. However, the role of ASL in human breast cancer has been studied to a lesser degree. In the present study, we investigated the effect of targeting ASL in breast cancer. We found that ASL was induced by ER stress and was significantly upregulated in breast cancer tissues compared to that in the corresponding normal tissues. Downregulation of ASL inhibited the growth of breast cancer in vitro and in vivo. The level of cell cycle-related gene, cyclin A2, was reduced and was accompanied by a delay in G2/M transition. ASL shRNA-induced cell inhibition was rescued by exogenous cyclin A2. Furthermore, autophagy was observed in the cells expressing ASL shRNA, and inhibition of autophagy reduced cell growth, indicating that autophagy played a cell survival role in the ASL knockdown cells. Moreover, inhibition of ASL reduced NO content. Introduction of the NO donor partially restored the growth inhibition by ASL shRNA. Thus, the mechanism induced by ASL shRNA which occurred in human breast cancer may be attributed to a decrease in cyclin A2 and NO.

Attenuation of argininosuccinate lyase inhibits cancer growth via cyclin A2 and nitric oxide.

Arginine biosynthesis and nitric oxide (NO) production are important for cancer homeostasis. Degradation of arginine may be used to inhibit liver tumors with low argininosuccinate synthetase (ASS) expression. In this report, we investigated an alternative therapeutic approach by targeting argininosuccinate lyase (ASL). ASL is transcriptionally induced by endoplasmic reticulum stress and is overexpressed in some human liver tumors. Knockdown of ASL expression by short hairpin RNA (shRNA) in three liver cancer cell lines, ML-1, HuH-7, and HepG2, decreased colony formation in vitro and tumor growth in vivo. Furthermore, lentiviral infection of ASL shRNA inhibited tumor growth in a therapeutic animal tumor model. Analysis of ASL shRNA on the cell-cycle progression revealed a G2-M delay. Among cell-cycle regulatory molecules, cyclin A2 expression was reduced. Reintroduction of exogenous cyclin A2 restored the cell growth in ASL-knockdown cells. Autophagy was observed in the cells treated with ASL shRNA, as shown by an increase in LC3-II levels and autophagosome formation. The total cellular arginine level was not altered significantly. Inhibition of autophagy further attenuated cell growth, suggesting that autophagy induced by ASL shRNA plays a feedback prosurvival function. Knockdown of ASL reduced NO content, and addition of NO donor partially recovered the growth inhibition by ASL shRNA. In summary, downregulation of ASL attenuated tumor growth and the inhibition was mainly mediated by a decrease of cyclin A2 and NO.

Thermal injury-induced priming effect of neutrophil is TNF-alpha and P38 dependent.

Priming response of neutrophil in clinical-related conditions and its mechanism has not been clarified. This study is to determine if thermal injury-induced priming effect of neutrophil is TNF-alpha and p38 dependent. In Experiment 1, bone marrow neutrophil of wild-type (WT) mice and TNF receptor superfamily, member 1A (Tnfrsf1a-/-) mice were harvested and treated with TNF-alpha, platelet activating factor (PAF) first, then with or without N-formyl-Met-Leu-Phe (fMLP). Reactive oxygen species (ROS) production and p38 phosphorylation were evaluated. In Experiment 2, ROS of neutrophil from WT and Tnfrsf1a-/- mice at 3 or 15 h after thermal injury with or without fMLP treatment were assayed. In Experiment 3, p38 and p44/42 phosphorylation, CXCR2 and macrophage inflammatory protein-2 expression, apoptotic ratio, and activating protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB) activation of neutrophil from WT and Tnfrsf1a-/- mice at 3 h after thermal injury were tested. FMLP treatment after TNF-alpha or PAF incubation of neutrophil increased ROS of PAF-treated but not TNF-alpha-treated neutrophil. PAF treatment increased ROS of neutrophil in WT and Tnfrsf1a-/- mice. FMLP increased ROS of neutrophil of WT mice at 3 h after thermal but not that of Tnfrsf1a-/- mice. TNF-alpha and PAF increased p38 phosphorylation of neutrophil in WT but not that in Tnfrsf1a-/- mice. Thermal injury increased p38 phosphorylation, NF-kappaB activation, and decreased apoptosis of neutrophil at 3 h after thermal injury in WT but not in Tnfrsf1a-/- mice. Thermal injury also induced AP-1 activation and ROS production on neutrophil at 3 and 15 h after thermal injury, respectively, in WT and Tnfrsf1a-/- mice. Collectively, fMLP stimulates ROS of neutrophil through TNF-alpha signaling; PAF stimulates that of neutrophil through both TNF-alpha-dependent and TNF-alpha-independent pathway. Thermal injury induces a TNF-alpha-dependent priming effect and a TNF-alpha-independent activation effect on neutrophil at 3 and 15 h after thermal injury, respectively. NF-kappaB signaling pathway plays an important role in neutrophil activation. Thermal injury also induces TNF-alpha-dependent delay apoptosis and TNF-alpha-independent AP-1 activation of neutrophil at 3 h after thermal injury. Taken together with the TNF-alpha-dependent p38 and NF-kappaB activation in primed neutrophil, we conclude that thermal injury-induced priming effect of polymorphonuclear neutrophil is TNF-alpha and p38 dependent.

Hypertonic saline enhances host defense to bacterial challenge by augmenting Toll-like receptors.

OBJECTIVE: To determine whether hypertonic saline infusion modulates thermal injury-induced bacterial translocation and host response to bacterial challenge through the augmentation of Toll-like receptors (TLRs). DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the mice. INTERVENTIONS: In experiment 1, mice underwent burn were given with 10 mL/kg hypertonic saline (7.5% NaCl), 10 mg/kg saline (N/S1), or 80 mL/kg saline (N/S2) at 4 or 8 hrs after burn. At 24 hrs after burn, mesenteric lymph nodes were harvested for bacterial translocation assay. In experiment 2, mice receiving hypertonic saline or saline after thermal injury received peritoneal challenge with Escherichia coli, and bacterial clearance was measured. In experiment 3, peritoneal cells from mice receiving hypertonic saline or saline after thermal injury were incubated with E. coli, and bacterial count, TLR2, TLR4, MIP2, CXCR2, pp38, and ERK expression were evaluated. In experiment 4, reactive oxygen species production, CXCR2, MIP2, TLR2, and TLR4 expression of bone marrow neutrophil from mice receiving hypertonic saline or saline treatment after thermal injury were evaluated. In experiment 5, neutrophil were cultured with hypertonic saline or N/S and incubated with E. coli. TLR2 and TLR4 expression and bacterial count were evaluated. In experiment 6, mice were fed with oral antibiotics with or without lipopolysaccharide, a TLR ligand, supplements. At 24 hrs after burn, mesenteric lymph nodes were harvested for bacterial translocation assay, and neutrophils were harvested for TLR2 and TLR4 protein assay. MEASUREMENTS AND MAIN RESULTS: Hypertonic saline decreased thermal injury-induced bacterial translocation. Hypertonic saline increased bacterial clearance, phagocytic activity, and TLR2, TLR4, CXCR2, pp38, and p44/42 expression of peritoneal cells. Hypertonic saline treatment at 4 or 8 hrs after thermal injury decreased reactive oxygen species production of neutrophil. Hypertonic saline injection increased TLR2, TLR4, and pp38 expression of neutrophil. In vitro treatment of neutrophil with hypertonic saline increased phagocytic activity and TLR2 and TLR4 expression. Commensal depletion with oral antibiotics decreased TLR2 and TLR4 expression of neutrophil; lipopolysaccharide increased TLR4 expression of neutrophil and decreased thermal injury-induced bacterial translocation. CONCLUSIONS: Restoration of extracellular fluid in burn shock with hypertonic saline decreased thermal injury-induced bacterial translocation. Hypertonic saline increased the phagocytic activity and TLR2, TLR4, CXCR2, pp38, and P44/42 expression of peritoneal cells. Hypertonic saline decreased reactive oxygen species but increased TLR2, TLR4, and pp38 expression and phagocytic activity of bone marrow neutrophil. Stimulation of the TLRs with lipopolysaccharide in commensal depleted mice increased TLRs expression of neutrophil and decreased thermal injury-induced bacterial translocation. Taken together with the fact that stimulation of TLRs with hypertonic saline increases phagocytic activity of systemic inflammatory cells, we conclude that TLRs play a critical role in the innate immunity by recognizing bacteria and that hypertonic saline enhances host response to bacterial challenge by increasing TLRs of inflammatory cells.