Tumor-intrinsic IFNα and CXCL10 are critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment.

Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only 10-30% of patients with various cancers. Literature has demonstrated that a "hot tumor" which contains high T lymphocytes in the tumor microenvironment exhibits a better response to immunotherapies than a "cold tumor." This study aimed to investigate whether tumor-intrinsic IFNα and CXCL10 determine the recruitment and activation of CD8+ T cells to become "hot tumor." In this study, we found that CXCL10 overexpressed in a variety of tumors including lung, colon, and liver tumors with a correlation with PD-L1. High PD-L1 and CXCL10 are associated with better survival rates in tumor patients receiving immunotherapies. IFNs-downstream transcriptional factor IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression. We demonstrated that IRF-1 and STAT1 were both bound with the promoters of PD-L1 and CXCL10, sharing the same signaling pathway and determining IFNs-mediated PD-L1 and CXCL10 expression. In addition, IFNα significantly increased activation marker IFNγ in PBMCs, promoting M1 type monocyte differentiation, CD4+ T, and CD8+ T cell activation. Particularly, we found that CD8+ T lymphocytes abundantly expressed CXCR3, a receptor of CXCL10, by flow cytometry, indicating that tumor-intrinsic CXCL10 potentially recruited CD8+ T in tumor microenvironment. To demonstrate the hypothesis, immunotherapy-sensitive CT26 and immunotherapy-resistant LL/2 were used and we found that CT26 cells exhibited higher IFNα, IFNγ, CXCL10, and PD-L1 levels compared to LL/2, leading to higher IFNγ expression in mouse splenocytes. Moreover, we found that CD8+ T cells were recruited by CXCL10 in vitro, whereas SCH546738, an inhibitor of CXCR3, inhibited T cell migration and splenocytes-mediated anti-tumor effect. We then confirmed that CT26-derived tumor was sensitive to αPD-L1 immunotherapy and LL/2-tumor was resistant, whereas αPD-L1 significantly increased T lymphocyte activation marker CD107a in CT26-derived BALB/c mice. In conclusion, this study revealed that CXCL10 expression is correlated with PD-L1 in tumors, sharing the same signaling pathway and associating with better immunotherapeutic efficacy. Further evidence in the syngeneic tumor models demonstrated that immunotherapy-sensitive CT26 intrinsically exhibited higher IFNα and CXCL10 compared to immunotherapy-resistant LL/2 to recruit and activate CD8+ T cells in the tumor microenvironment, exhibiting "hot tumor" characteristic of sensitizing αPD-L1 immunotherapies.

Radiotherapy enhances CXCR3highCD8+ T cell activation through inducing IFNγ-mediated CXCL10 and ICAM-1 expression in lung cancer cells.

Radiotherapy (RT) not only damages tumors but also induces interferon (IFN) expression in tumors. IFNs mediate PD-L1 to exhaust CD8+ T cells, but which also directly impact tumor cells and potentially activate anti-tumor immune surveillance. Little is known about the contradictory mechanism of IFNs in regulating CD8+ T-mediated anti-tumor activity in lung cancer. This study found that RT induced IFNs and CXCL9/10 expression in the RT-treated lung cancer cells. Specifically, RT- and IFNγ-pretreated A549 significantly activated CD8+ T cells, resulting in significant inhibition of A549 colony formation. RNAseq and consequent qPCR results revealed that IFNγ induced PD-L1, CXCL10, and ICAM-1, whereas PD-L1 knockdown activated CD8+ T cells, but ICAM-1 knockdown diminished CD8+ T cell activation. We further demonstrated that CXCR3 and CXCL10 decreased in the CD8+ T cells and nonCD8+ PBMCs, respectively, in the patients with lung cancer that expressed lower reactivation as co-cultured with A549 cells. In addition, inhibitors targeting CXCR3 and LFA-1 in CD8+ T cells significantly diminished CD8+ T cell activation and splenocytes-mediated anti-LL/2shPdl1. In conclusion, we validated that RT suppressed lung cancer and overexpress PD-L1, CXCL10, and ICAM-1, which exhibited different roles in regulating CD8+ T cell activity. We propose that CXCR3highCD8+ T cells stimulated by CXCL10 exhibit anti-tumor immunity, possibly by enhancing T cells-tumor cells adhesion through CXCL10/CXCR3-activated LFA-1-ICAM-1 interaction, but CXCR3lowCD8+ T cells with low CXCL10 in patients with lung cancer were exhausted by PD-L1 dominantly. Therefore, RT potentially activates CD8+ T cells by inducing IFNs-mediated CXCL10 and ICAM-1 expression in tumors to enhance CD8+ T-tumor adhesion and recognition. This study clarified the possible mechanisms of RT and IFNs in regulating CD8+ T cell activation in lung cancer.

Nicotine exhausts CD8+ T cells against tumor cells through increasing miR-629-5p to repress IL2RB-mediated granzyme B expression.

The mechanism exhausting CD8+ T cells is not completely clear against tumors. Literature has demonstrated that cigarette smoking disables the immunological activity, so we propose nicotine is able to exhaust CD8+ T cells. The CD8+ T cells from healthy volunteers with and without cigarette smoking and the capacity of CD8+ T cells against tumor cells were investigated. RNAseq was used to investigate the gene profiling expression in CD8+ T cells. Meanwhile, small RNAseq was also used to search novel microRNAs involved in the exhaustion of CD8+ T cells. The effect of nicotine exhausting CD8+ T cells was investigated in vitro and in the humanized tumor xenografts in vivo. We found that CD8+ T cells were able to reduce cell viability in lung cancer HCC827 and A549 cells, that secreted granzyme B, but CD8+ T cells from the healthy cigarette smokers lost anti-HCC827 effect. Moreover, nicotine suppressed the anti-HCC827 effect of CD8+ T cells. RNAseq revealed lower levels of IL2RB and GZMB in the exhausted CD8+ T cells. We identified that miR-629-5p was increased by nicotine, that targeted IL2RB. Transfection of miR-629-5p mimic reduced IL2RB and GZMB levels. We further validated that nicotine reduced granzyme B levels using a nuclear imaging technique, and demonstrated that nicotine exhausted peripheral blood mononuclear cells against HCC827 growth in the humanized tumor xenografts. This study demonstrated that nicotine exhausted CD8+ T cells against HCC827 cells through increasing miR-629-5p to suppress IL2RB.

Pro-apoptotic effect of haem oxygenase-1 in human colorectal carcinoma cells via endoplasmic reticular stress.

Several biological effects of haem oxygenase (HO)-1, including anti-inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO-1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO-1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)-3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT-15, LOVO and HT-29 cells in serum-free (SF) conditions with increased HO-1, but not heat shock protein 70 (HSP70) or HSP90. The addition of 10% foetal bovine serum (FBS) or 1% bovine serum albumin accordingly inhibited CoPP-induced apoptosis and HO-1 protein expression in human colon cancer cells. CoPP-induced apoptosis of colon cancer cells was prevented by the addition of the pan-caspase inhibitor, Z-VAD-FMK (VAD), and the Casp-3 inhibitor, Z-DEVD-FMK (DEVD). N-Acetyl cysteine inhibited reactive oxygen species-generated H2 O2 -induced cell death with reduced intracellular peroxide production, but did not affect CoPP-induced apoptosis in human colorectal carcinoma (CRC) cells. Two CoPP analogs, ferric protoporphyrin and tin protoporphyrin, did not affect the viability of human CRC cells or HO-1 expression by those cells, and knockdown of HO-1 protein expression by HO-1 small interfering (si)RNA reversed the cytotoxic effect elicited by CoPP. Furthermore, the carbon monoxide (CO) donor, CORM, but not FeSO4 or biliverdin, induced DNA ladders, and cleavage of Casp-3 and PARP proteins in human CRC cells. Increased phosphorylated levels of the endoplasmic reticular (ER) stress proteins, protein kinase R-like ER kinase (PERK), and eukaryotic initiation factor 2α (eIF2α) by CORM and CoPP were identified, and the addition of the PERK inhibitor, GSK2606414, inhibited CORM- and CoPP-induced apoptosis. Increased GRP78 level and formation of the HO-1/GRP78 complex were detected in CORM- and CoPP-treated human CRC cells. A pro-apoptotic role of HO-1 against the viability of human CRC cells via induction of CO and ER stress was firstly demonstrated herein.

STAT3 induces G9a to exacerbate HER3 expression for the survival of epidermal growth factor receptor-tyrosine kinase inhibitors in lung cancers.

BACKGROUND: HER3 mediates drug resistance against epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), resulting in tumor relapse in lung cancers. Previously, we demonstrated that EGFR induces HER3 overexpression, which facilitates the formation of cancer stem-like tumorspheres. However, the cellular mechanism through which EGFR regulates HER3 expression remains unclear. We hypothesized that EGFR downstream of STAT3 participates in HER3 expression because STAT3 contributes to cancer stemness and survival of EGFR-TKI resistant cancers. METHODS: First, RNAseq was used to uncover potential genes involved in the formation of lung cancer HCC827-derived stem-like tumorspheres. EGFR-positive lung cancer cell lines, including HCC827, A549, and H1975, were individually treated with a panel containing 172 therapeutic agents targeting stem cell-associated genes to search for potential agents that could be applied against EGFR-positive lung cancers. In addition, gene knockdown and RNAseq were used to investigate molecular mechanisms through which STAT3 regulates tumor progression and the survival in lung cancer. RESULTS: BBI608, a STAT3 inhibitor, was a potential therapeutic agent that reduced the cell viability of EGFR-positive lung cancer cell lines. Notably, the inhibitory effects of BBI608 were similar with those associated with YM155, an ILF3 inhibitor. Both compounds reduced G9a-mediated HER3 expression. We also demonstrated that STAT3 upregulated G9a to silence miR-145-5p, which exacerbated HER3 expression in this study. CONCLUSIONS: The present study revealed that BBI608 could eradicate EGFR-positive lung cancers and demonstrated that STAT3 enhanced the expression of HER3 through miR-145-5p repression by G9a, indicating that STAT3 is a reliable therapeutic target against EGFR-TKI-resistant lung cancers.

Epidermal growth factor induces STAT1 expression to exacerbate the IFNr-mediated PD-L1 axis in epidermal growth factor receptor-positive cancers.

The epidermal growth factor (EGF) receptor (EGFR) overexpressed in many cancers, including lung and head and neck cancers, and is involved in cancer cell progression and survival. PD-L1, increases in tumor cells to evade and inhibit CD8+ T cells, is a clinical immunotherapeutic target. This study investigated the molecular mechanism of EGF on regulating PD-L1 in EGFR-positive cancers and determined potential agents to reduce PD-L1 expression. RNA sequencing (RNAseq) and bioinformatics analysis were performed to determine potential driver genes that regulate PD-L1 in tumor cells-derived tumorspheres which mimicking cancer stem cells. Then, the specific inhibitors targeting EGFR were applied to reduce the expression of PD-L1 in vitro and in vivo. We validated that EGF could induce PD-L1 expression in the selected EGFR-positive cancers. RNAseq results revealed that STAT1 increased as a driver gene in KOSC-3-derived tumorspheres; these data were analyzed using PANTHER followed by NetworkAnalyst. The blockade of EGFR by afatinib resulted in decreased STAT1 and IRF-1 levels, both are transcriptional factors of PD-L1, and disabled the IFNr-STAT1-mediated PD-L1 axis in vitro and in vivo. Moreover, STAT1 knockdown significantly reduced EGF-mediated PD-L1 expression, and ruxolitinib, a JAK1/JAK2 inhibitor, significantly inhibited STAT1 phosphorylation to reduce the IFNr-mediated PD-L1 axis. These results indicate that EGF exacerbates PD-L1 by increasing the protein levels of STAT1 to enforce the IFNr-JAK1/2-mediated signaling axis in selected EGFR-positive cancers. The inhibition of EGFR by afatinib significantly reduced PD-L1 and may be a potential strategy for enhancing immunotherapeutic efficacy.

STAT3 exacerbates survival of cancer stem-like tumorspheres in EGFR-positive colorectal cancers: RNAseq analysis and therapeutic screening.

BACKGROUND: Cancer stem cells are capable of undergoing cell division after surviving cancer therapies, leading to tumor progression and recurrence. Inhibitory agents against cancer stem cells may be therapeutically used for efficiently eradicating tumors. Therefore, the aim of this study was to identify the relevant driver genes that maintain cancer stemness in epidermal growth factor receptor (EGFR)-positive colorectal cancer (CRC) cells and to discover effective therapeutic agents against these genes. METHODS: In this study, EGFR-positive cancer stem-like cells (CSLCs) derived from HCT116 and HT29 cells were used as study models for in vitro inductions. To identify the differential genes that maintain CSLCs, RNAseq analysis was conducted followed by bioinformatics analysis. Moreover, a panel containing 172 therapeutic agents targeting the various pathways of stem cells was used to identify effective therapeutics against CSLCs. RESULTS: RNAseq analysis revealed that 654 and 840 genes were significantly upregulated and downregulated, respectively, in the HCT116 CSLCs. Among these genes, notably, platelet-derived growth factor A (PDGFA) and signal transducer and activator of transcription 3 (STAT3) were relevant according to the cancer pathway analyzed using NetworkAnalyst. Furthermore, therapeutic screening revealed that the agents targeting STAT3 and Wnt signaling pathways were efficient in reducing the cell viabilities of both HCT116 and HT29 cells. Consequently, we discovered that STAT3 inhibition using homoharringtonine and STAT3 knockdown significantly reduced the formation and survival of HT29-derived tumorspheres. We also observed that STAT3 phosphorylation was regulated by epidermal growth factor (EGF) to induce PDGFA and Wnt signaling cascades. CONCLUSIONS: We identified the potential genes involved in tumorsphere formation and survival in selective EGFR-positive CRCs. The results reveal that the EGF-STAT3 signaling pathway promotes and maintains CRC stemness. In addition, a crosstalk between STAT3 and Wnt activates the Wnt/ß-catenin signaling pathway, which is also responsible for cancer stemness. Thus, STAT3 is a putative therapeutic target for CRC treatment.

Blocking heme oxygenase-1 by zinc protoporphyrin reduces tumor hypoxia-mediated VEGF release and inhibits tumor angiogenesis as a potential therapeutic agent against colorectal cancer.

BACKGROUND: Hypoxia in tumor niche is one of important factors to start regeneration of blood vessels, leading to increase survival, proliferation, and invasion in cancer cells. Under hypoxia microenvironment, furthermore, steadily increased hypoxia-inducible factor -1α (HIF-1α) is observed, and can increase vascular endothelial growth factor (VEGF) expression and promote angiogenesis. Zinc protoporphyrin (ZnPP), a heme oxygenase-1 (HO-1) inhibitor, is potential to inhibit tumor proliferation and progression. However, the mechanism of ZnPP in inhibition of tumor is not completely clear. We hypothesize that ZnPP may modulate HIF-1α through inhibiting HO-1, and then inhibit angiogenesis and tumor progression. This study aimed to dissect the mechanism of ZnPP in tumor suppression. RESULTS: We observed the amount of VEGF was increased in the sera of the colorectal cancer (CRC) patients (n = 34, p < 0.05). Furthermore, increased VEGF expression was also measured in colorectal cancer cells, HCT-15, culturing under mimicking hypoxic condition. It suggested that hypoxia induced VEGF production from cancer cells. VEGF production was significantly reduced from HCT-15 cells after exposure to HIF-1α inhibitor KC7F2, suggesting that HIF-1α regulated VEGF production. Moreover, we observed that the HO-1 inhibitor ZnPP inhibited the expressions of HIF-1α and VEGF coupled with cell proliferations of HCT-15 cells, suggesting that ZnPP blocked HIF-1α expression, and then inhibited the consequent VEGF production. In the xenograft model, we also observed that the animals exposed to ZnPP displayed much smaller tumor nodules and less degree of angiogenesis with decreased expression of the angiogenesis marker, αvß3 integrin, compared to that in normal control. CONCLUSIONS: This study demonstrated that VEGF level in serum was elevated in the patients with CRC. The HO-1 inhibitor, ZnPP, possessed the properties of anti-tumor agent by decreasing HIF-1α levels, blocking VEGF production, impairing tumor angiogenesis, and inhibiting tumor growth.

Engineering H2O2 Self-Supplying Platform for Xdynamic Therapies via Ru-Cu Peroxide Nanocarrier: Tumor Microenvironment-Mediated Synergistic Therapy.

Of the most common, hypoxia, overexpressed glutathione (GSH), and insufficient H2O2 concentration in the tumor microenvironment (TME) are the main barriers to the advancment of reactive oxygen species (ROS) mediated Xdynamic therapies (X = photo, chemodynamic, chemo). Maximizing Fenton catalytic efficiency is crucial in chemodynamic therapy (CDT), yet endogenous H2O2 levels are not sufficient to attain better anticancer efficacy. Specifically, there is a need to amplify Fenton reactivity within tumors, leveraging the unique attributes of the TME. Herein, for the first time, we design RuxCu1-xO2-Ce6/CPT (RCpCCPT) anticancer nanoagent for TME-mediated synergistic therapy based on heterogeneous Ru-Cu peroxide nanodots (RuxCu1-xO2 NDs) and chlorine e6 (Ce6), loaded with ROS-responsive thioketal (TK) linked-camptothecin (CPT). The Ru-Cu peroxide NDs (RCp NDs, x = 0.50) possess the highest oxygen vacancy (OV) density, which grants them the potential to form massive Lewis's acid sites for peroxide adsorption, while the dispersibility and targetability of the NDs were improved via surface modification using hyaluronic acid (HA). In TME, RCpCCPT degrades, releasing H2O2, Ru2+/3+, and Cu+/2+ ions, which cooperatively facilitate hydroxyl radical (•OH) formation and deactivate antioxidant GSH enzymes through a cocatalytic loop, resulting in excellent tumor therapeutic efficacy. Furthermore, when combined with laser treatment, RCpCCPT produces singlet oxygen (1O2) for PDT, which induces cell apoptosis at tumor sites. Following ROS generation, the TK linkage is disrupted, releasing up to 92% of the CPT within 48 h. In vitro investigations showed that laser-treated RCpCCPT caused 81.5% cell death from PDT/CDT and chemotherapy (CT). RCpCCPT in cancer cells produces red-blue emission in images of cells taking them in, which allows for fluorescence image-guided Xdynamic treatment. The overall results show that RCp NDs and RCpCCPT are more biocompatible and have excellent Xdynamic therapeutic effectiveness in vitro and in vivo.

Characterization of chicken-derived antibody against Alpha-Enolase of Streptococcus pneumoniae.

Streptococcus pneumoniae is a clinically relevant pathogen notorious for causing pneumonia, meningitis, and otitis media in immunocompromised patients. Currently, antibiotic therapy is the most efficient treatment for fighting pneumococcal infections. However, an arise in antimicrobial resistance in S. pneumoniae has become a serious health issue globally. To resolve the problem, alternative and cost-effective strategies, such as monoclonal antibody-based targeted therapy, are needed for combating bacterial infection. S. pneumoniae alpha-enolase (spEno1), which is thought to be a great target, is a surface protein that binds and converts human plasminogen to plasmin, leading to accelerated bacterial infections. We first purified recombinant spEno1 protein for chicken immunization to generate specific IgY antibodies. We next constructed two single-chain variable fragments (scFv) antibody libraries by phage display technology, containing 7.2 × 107 and 4.8 × 107 transformants. After bio-panning, ten scFv antibodies were obtained, and their binding activities to spEno1 were evaluated on ELISA, Western blot and IFA. The epitopes of spEno1 were identified by these scFv antibodies, which binding affinities were determined by competitive ELISA. Moreover, inhibition assay displayed that the scFv antibodies effectively inhibit the binding between spEno1 and human plasminogen. Overall, the results suggested that these scFv antibodies have the potential to serve as an immunotherapeutic drug against S. pneumoniae infections.

Preclinical Assessment of Enhanced Chemodynamic Therapy by an FeMnOx-Based Nanocarrier: Tumor-Microenvironment-Mediated Fenton Reaction and ROS-Induced Chemotherapeutic for Boosted Antitumor Activity.

In recent studies, iron-containing Fenton nanocatalysts have demonstrated significant promise for clinical use due to their effective antitumor activity and low cytotoxicity. A new approach was reported in this work utilizing cation exchange synthesis to fabricate FeMnOx nanoparticles (NPs) that boost Fenton reactions and responses to the tumor microenvironment (TME) for chemodynamic therapy (CDT) and chemotherapy (CT). Within the TME, the redox metal pair of Fe2+/Mn2+ helps break down endogenous hydrogen peroxide (H2O2) into very harmful hydroxyl radicals (•OH) while simultaneously deactivating glutathione (GSH) to boost CDT performance. To further enhance the therapeutic potential, FeMnOx NPs were encapsulated with thioketal-linked camptothecin (CPT-TK-COOH), a reactive oxygen species (ROS)-responsive prodrug, achieving a high CPT-loading capacity of up to 51.1%. Upon ROS generation through the Fenton reaction, the prodrug TK linkage was disrupted, releasing 80% of the CPT payload within 48 h. Notably, FeMnOx@CPT exhibited excellent dual-modal imaging capabilities, enabling magnetic resonance and fluorescence imaging for image-guided therapy. In vitro studies showed the cytocompatibility of FeMnOx NPs using MDA-Mb-231 and 4T1 cells, but in the presence of H2O2, they induced significant cytotoxicity, resulting in 80% cell death through CDT and CT effects. Upon intravenous administration, FeMnOx@CPT displayed remarkable tumor accumulation, which enhanced tumor suppression in xenografts through improved CDT and CT effects. Moreover, no significant adverse effects were observed in the FeMnOx NP-treated animals. In the current study, the FeMnOx@CPT anticancer platform, with its boosted •OH-producing capability and ROS-cleavable drug release, has been validated utilizing in vitro and animal studies, suggesting its capacity as a viable strategy for clinical trials.

Tumor targeting and therapeutic assessments of RNA nanoparticles carrying α9-nAChR aptamer and anti-miR-21 in triple-negative breast cancers.

Triple-negative breast cancer (TNBC) is highly aggressive with a poor prognosis because of a lack of cell markers as drug targets. α9-Nicotinic acetylcholine receptor (nAChR) is expressed abundantly in TNBC; thus, it is a valuable biomarker for TNBC detection and treatment. In this study, we utilized thermodynamically stable three-way junction (3WJ) packaging RNA (pRNA) as the core to construct RNA nanoparticles with an α9-nAChR RNA aptamer as a targeting ligand and an anti-microRNA-21 (miR-21) as a therapeutic module. We compared the configuration of the two RNA nanoparticles and found that 3WJ-B-α9-nAChR-aptamer fluorescent RNA nanoparticles (3WJ-B-α9-apt-Alexa) exhibited better specificity for α9-nAChR in TNBC cells compared with 3WJ-C-α9-nAChR. Furthermore, 3WJ-B-α9-apt-Alexa bound more efficiently to TNBC patient-derived xenograft (PDX) tumors than 3WJ fluorescent RNA nanoparticles (3WJ-Alexa) with little or no accumulation in healthy organs after systemic injection in mice. Moreover, 3WJ-B-α9-nAChR-aptamer RNA nanoparticles carrying anti-miR-21 (3WJ-B-α9-apt-anti-miR-21) significantly suppressed TNBC-PDX tumor growth and induced cell apoptosis because of reduced miR-21 gene expression and upregulated the phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4) proteins. In addition, no pathological changes were detected upon toxicity examination of treated mice. In conclusion, the 3WJ-B-α9-nAChR-aptamer RNA nanoparticles established in this study efficiently deliver therapeutic anti-miR-21, indicating their potential as a novel TNBC therapy.

Targeting to overexpressed glucose-regulated protein 78 in gastric cancer discovered by 2D DIGE improves the diagnostic and therapeutic efficacy of micelles-mediated system.

The survivals of gastric cancer (GC) patients are associated with early diagnosis and effective treatments. Therefore, it is urgent for the discovery of early GC biomarkers and tumor-targeting therapeutics. The aim of this study was to uncover putative tissue biomarkers of GC using 2D DIGE and then apply one of these specific markers in GC treatment. We found three putative biomarkers of GC with significant differences in expression level compared to adjacent normal tissue, including glucose-regulated protein 78 (GRP78) and glutathione s-transferase pi (GSTpi) with increased expression level, and alpha-1 antitrypsin (A1AT) with reduced expression level. The overexpressed GRP78 was used as a targeted protein for guiding the drugs to tumor cells, leading to more effective treatment for GC xenografts. Our results demonstrated that the designated GRP78-binding peptide based on the sequence, WIFPWIQL, was selectively prone to recognize and bind to GC MKN45 cells in vitro, and also improve the delivery efficiency of polymeric micelles-encapsulated drugs into tumor cells and displayed better therapeutic outcome in experimental animals. This strategy of GRP78-mediated drug targeting system may bring chemotherapeutic drugs with more precise targeting to tumor cells, leading to minimize side effects on patients after chemotherapy.

GMI-1070, a novel pan-selectin antagonist, reverses acute vascular occlusions in sickle cell mice.

Leukocyte adhesion in the microvasculature influences blood rheology and plays a key role in vaso-occlusive manifestations of sickle cell disease. Notably, polymorphonuclear neutrophils (PMNs) can capture circulating sickle red blood cells (sRBCs) in inflamed venules, leading to critical reduction in blood flow and vaso-occlusion. Recent studies have suggested that E-selectin expression by endothelial cells plays a key role by sending activating signals that lead to the activation of Mac-1 at the leading edge of PMNs, thereby allowing RBC capture. Thus, the inhibition of E-selectin may represent a valuable target in this disease. Here, we have tested the biologic properties of a novel synthetic pan-selectin inhibitor, GMI-1070, with in vitro assays and in a humanized model of sickle cell vaso-occlusion analyzed by intravital microscopy. We have found that GMI-1070 predominantly inhibited E-selectin-mediated adhesion and dramatically inhibited sRBC-leukocyte interactions, leading to improved microcirculatory blood flow and improved survival. These results suggest that GMI-1070 may represent a valuable novel therapeutic intervention for acute sickle cell crises that should be further evaluated in a clinical trial.

Preclinical Therapeutic Assessment of a New Chemotherapeutics [Dichloro(4,4'-Bis(2,2,3,3-Tetrafluoropropoxy) Methyl)-2,2'-Bipryridine) Platinum] in an Orthotopic Patient-Derived Xenograft Model of Triple-Negative Breast Cancers.

Cisplatin is one of the most common therapeutics used in treatments of several types of cancers. To enhance cisplatin lipophilicity and reduce resistance and side effects, a polyfluorinated bipyridine-modified cisplatin analogue, dichloro[4,4'-bis(2,2,3,3-tetrafluoropropoxy)methyl)-2,2'-bipryridine] platinum (TFBPC), was synthesized and therapeutic assessments were performed. TFBPC displayed superior effects in inhibiting the proliferation of several cisplatin-resistant human cancer cell lines, including MDA-MB-231 breast cancers, COLO205 colon cancers and SK-OV-3 ovarian cancers. TFBPC bound to DNA and formed DNA crosslinks that resulted in DNA degradation, triggering the cell death program through the PARP/Bax/Bcl-2 apoptosis and LC3-related autophagy pathway. Moreover, TFBPC significantly inhibited tumor growth in both animal models which include a cell line-derived xenograft model (CDX) of cisplatin-resistant MDA-MB-231, and a patient-derived xenograft (PDX) model of triple-negative breast cancers (TNBCs). Furthermore, the biopsy specimen from TFBPC-treated xenografts revealed decreased expressions of P53, Ki-67 and PD-L1 coupled with higher expression of cleaved caspase 3, suggesting TFBPC treatment was effective and resulted in good prognostic indications. No significant pathological changes were observed in hematological and biochemistry tests in blood and histological examinations from the specimen of major organs. Therefore, TFBPC is a potential candidate for treatments of patients suffering from TNBCs as well as other cisplatin-resistant cancers.

Improving Tirapazamine (TPZ) to Target and Eradicate Hypoxia Tumors by Gold Nanoparticle Carriers.

Tumor hypoxia is a hallmark of solid tumors and emerged as the therapeutic target for cancer treatments, such as a prodrug Tirapazamine (TPZ) activated in hypoxia. To increase tumor accumulation, gold nanoparticles (GNPs) were selected to conjugate with TPZ. In this study, we successfully formulated and assessed the biochemical and therapeutic roles of the conjugated gold nanoparticles-Tirapazamine (GNPs-TPZ) on therapeutic assessments of MKN45-induced xenograft animal model. The results indicated that GNPs-TPZ was a potential nanomedicine for selectively targeting hypoxia tumors coupled with decreased side effects on healthy tissue or organs. TPZ significantly reduced cell viability of hypoxic gastric cancer MKN45 cells, but not in cells incubated in normoxia condition. For improving tumor targeting efficiency, furthermore, the GNPs drug carrier was conjugated to TPZ via biding mediator bovine serum albumin (BSA), and we demonstrated that this conjugated GNPs-TPZ retained the unique characteristics of hypoxic toxin and possessed the adequate feature of systemic bio-distributions in animals. GNPs-TPZ nanoparticles revealed their superior affinity to hypoxia tumors in the MKN45 xenograft. Moreover, GNPs-TPZ treatments did not significantly alter the biochemical parameters of blood samples acquired from animals. Taken together, TPZ, a prodrug activated by hypoxia, was conjugated with GNPs, whereas BSA severed as an excellent binding agent for preparing the conjugated GNPs-TPZ nanomedicines. We demonstrated that GNPs-TPZ enhanced tumor targeting, resulting in higher therapeutic efficacy compared to TPZ. We suggest that it may sever as an adjuvant treatment or combined therapy with other chemotherapeutics for the treatment of cancer patients in the future.

Sorafenib suppresses radioresistance and synergizes radiotherapy-mediated CD8+ T cell activation to eradicate hepatocellular carcinoma.

Radiotherapy (RT) is applied to eradicate tumors in the clinic. However, hepatocellular carcinoma (HCC) exhibits resistance against RT. It is demonstrated that RT directly inhibits tumor growth but which induces type I interferons (IFNs) expression to phosphorylate STATs and increase STATs-downstream PD-L1 levels in the survival tumor cells. Since sorafenib is capable of suppressing STATs, we, therefore, hypothesize that sorafenib suppresses IFNs-mediated radioresistance and PD-L1 in the residual tumor cells and may synergistically enhance RT-mediated reactivation of CD8+ T immunological activity to eradicate HCC cells. We found that combined RT, sorafenib, and PBMCs significantly suppress the colony formation in the HCC cells, whereas CD8+ T cells expressed high granzyme B (GZMB) and perforin (PRF1) in co-cultured with RT-treated HCC cells. We demonstrated RT significantly inhibited HCC cell viability but induced IFNα and IL-6 expression in the RT-treated HCC cells, resulting in immune checkpoint PD-L1 and anti-apoptosis MCL1 and BCL2 overexpression in the non-RT HCC cells. We found that sorafenib decreased RT-PLC5 medium (RT-PLC5-m)-mediated cell growth by suppressing IFNα- and IL-6-mediated STAT1 and STAT3 phosphorylation. Sorafenib also reduced IFNα-mediated PD-L1 levels in HCC cells. Meanwhile, RT-PLC5-m reactivated CD8+ T cells and non-CD8+ PBMCs, resulting in high IFNγ and IL-2 levels in CD8+ T cells, and cytokines IFNα, IFNγ, IL-2, and IL-6 in non-CD8+ PBMCs. Particularly, CD8+ T cells expressed higher GZMB and PRF1 and non-CD8+ PBMCs expressed higher IFNα, IFNγ, IL-2, IL-6, CXCL9, and CXCL10 in co-cultured with RT-treated HCC cells compared to parental cells. Although we demonstrated that sorafenib slightly inhibited RT-mediated GZMB and PRF1 expression in CD8+ T cells, and cytokines levels in non-CD8+ PBMCs. Based on sorafenib significantly suppressed IFNα- and IL-6-mediated radioresistance and PD-L1 expression, we demonstrated that sorafenib synergized RT and immune surveillance for suppressing PLC5 cell viability in vitro. In conclusion, this study revealed that RT induced IFNα and IL-6 expression to phosphorylate STAT1 and STAT3 by autocrine and paracrine effect, leading to radioresistance and PD-L1 overexpression in HCC cells. Sorafenib not only suppressed IFNα- and IL-6-mediated PLC5 cell growth but also inhibited IFNα-mediated PD-L1 expression, synergistically enhancing RT-mediated CD8+ T cell reactivation against HCC cells.

Caffeic Acid Phenethyl Ester Inhibits Basal Lipolysis by Activating PPAR-Gamma and Increasing Lipid Droplet-Associated Perilipin in Mature Rat Adipocytes.

Abnormal lipolysis is correlated with metabolic syndrome. Caffeic acid phenethyl ester (CAPE), a natural product from honeybee hives, has been reported to improve metabolic syndrome. However, the effects of CAPE on lipolysis and perilipin-1 (the major lipid droplet-associated protein) in mature adipocytes were not clarified. In this study, mature adipocytes were isolated from the epididymal fat pads of male rats and incubated with CAPE to estimate lipolysis by measuring glycerol release. It was found that the basal lipolysis was inhibited by CAPE in a dose- and time-dependent manner. The lipid droplet-associated perilipin-1 and phosphorylated peroxisome proliferator-activated receptor (PPAR) gamma levels increased following CAPE treatment by Western blot analysis. Moreover, a specific PPAR-gamma inhibitor (T0070907) could partly reverse the effect of CAPE on basal lipolysis. Furthermore, treatment of adipocytes with dibutyryl-cAMP (db-cAMP) or isoproterenol (ISO) increased lipolysis, but the drug-induced lipolysis was abrogated by combination treatment with CAPE. The lipid droplet-associated perilipin-1 level was also decreased in the drug-induced groups but increased when combined treatment with CAPE. In conclusion, our results revealed that a decrease in basal lipolysis and an increase in lipid droplet-associated perilipin-1 levels induced by CAPE may be involved in the regulation of lipid metabolism through activation of PPAR-gamma in mature adipocytes.

Airway Exposure to 1,3-Beta-d-Glucan Induces Airway Hyperresponsiveness in Guinea Pigs.

1,3-Beta-d-glucan (ß-glucan) is a component of mold cell walls and is frequently found in fungi and house dust mites. The studies of ß-glucan are inconsistent, although it has been implicated in airway adverse responses. This study was carried out to determine whether airway hyperresponsiveness was seen 24 h after airway exposure to ß-glucan in guinea pigs. Two matching guinea pigs were exposed intratracheally to either ß-glucan or its vehicle. Twenty-four hours after intratracheal instillation, there was no difference between these two groups in the baseline of the total pulmonary resistance (R L), dynamic lung compliance (C dyn), arterial blood pressure, and heart rate. In contrast, the responses of R L to capsaicin injection were significantly increased in ß-glucan animals; capsaicin at the same dose of 3.2 µg/kg increased R L by 184% in vehicle animals and by 400% in ß-glucan animals. The effective dose 200% to capsaicin injection was lower in the ß-glucan animals. Furthermore, the increases in R L were partially reduced after transient lung hyperinflation to recruit the occluding airways; however, the R L induced by capsaicin injection after lung hyperinflation was significantly larger than the baseline in ß-glucan animals; also, the lung wet-to-dry ratio in capsaicin-injected animals was augmented in the ß-glucan group. Moreover, the airway hyperresponsiveness was accompanied by increases in neutrophils in the bronchoalveolar lavage fluid in the ß-glucan animals. Furthermore, the levels of substance P and the calcitonin gene-related peptide in the bronchoalveolar lavage fluid collected after capsaicin injection were increased in ß-glucan animals. We provide definitive evidence that ß-glucan can induce airway hyperresponsiveness in guinea pigs, and the neuropeptide releases play an important role in this airway hyperresponsiveness.

Combined impacts of histamine receptor H1 gene polymorphisms and an environmental carcinogen on the susceptibility to and progression of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most frequently encountered type of oral cancer. Histamine receptor H1 (HRH1) was reported to play a crucial role in OSCC carcinogenesis, but impacts of genetic variants of HRH1 on OSCC remain unclear. Herein, we investigated the association between functional single-nucleotide polymorphisms (SNPs) of HRH1 and OSCC susceptibility or clinicopathologic variables by logistic regression models. HRH1 genotypes at four loci (rs346074, rs346076, rs901865, and rs2606731) were analyzed by a TaqMan allelic discrimination assay, and we found that patients harboring HRH1 rs901865 T and rs346074 T alleles had a significantly lower risk of developing larger tumor sizes (>T2) under a dominant model. Based on the environmental carcinogen exposure status, we observed that HRH1 rs901865 polymorphic variants were also associated with a lower risk of developing more-advanced clinical stages (III or IV) in patients with a betel-quid-chewing habit. Moreover, genotype screening of rs901865 and rs346074 in OSCC cell lines showed that cells respectively carrying the CT and TT genotypes expressed lower HRH1 levels compared to cells carrying the CC genotype of rs901865 and rs346074. Furthermore, analyses of TCGA and GEO databases revealed that HRH1 expression levels were upregulated in head and neck squamous cell carcinoma (HNSCC) and OSCC tissues compared to normal tissues and were correlated with larger tumor sizes and poorer prognoses. These results indicated the involvement of HRH1 SNPs rs901865 and rs346074 in OSCC development and support the interaction between HRH1 gene polymorphisms and an environmental carcinogen as a predisposing factor for OSCC progression.