The Synergistic Effects of Corbicula fluminea and Sarcodia montagneana on Alleviating Systemic Inflammation and Osteoarthritis Progression.

Osteoarthritis (OA) is a progressive disease that causes pain, stiffness, and inflammation in the affected joints. Currently, there are no effective treatments for preventing the worst outcomes, such as synovitis or cartilage degradation. Sarcodia montagneana and Corbicula fluminea are common species found in the ocean or in freshwater areas. Their extracts are demonstrated to possess both antioxidative and anti-inflammatory functions. This study aimed to investigate the synergistic effects of the extracts of Sarcodia montagneana (SME) and Corbicula fluminea (FCE) on reducing local and systemic inflammation, as well as their efficacy in OA symptom relief. An in vitro monocytic LPS-treated THP-1 cell model and in vivo MIA-induced mouse OA model were applied, and the results showed that the combinatory usage of SME and FCE effectively suppressed IFN-γ and TNF-α production when THP-1 cells were treated with LPS. SME and FCE also significantly decreased the systemic TNF-α level and joint swelling and prevented the loss of proteoglycan in the cartilage within the joints of OA mice. The data shown here provide a potential solution for the treatment of osteoarthritis.

A flexible liposomal polymer complex as a platform of specific and regulable immune regulation for individual cancer immunotherapy.

BACKGROUND: The applicability and therapeutic efficacy of specific personalized immunotherapy for cancer patients is limited by the genetic diversity of the host or the tumor. Side-effects such as immune-related adverse events (IRAEs) derived from the administration of immunotherapy have also been observed. Therefore, regulatory immunotherapy is required for cancer patients and should be developed. METHODS: The cationic lipo-PEG-PEI complex (LPPC) can stably and irreplaceably adsorb various proteins on its surface without covalent linkage, and the bound proteins maintain their original functions. In this study, LPPC was developed as an immunoregulatory platform for personalized immunotherapy for tumors to address the barriers related to the heterogenetic characteristics of MHC molecules or tumor associated antigens (TAAs) in the patient population. Here, the immune-suppressive and highly metastatic melanoma, B16F10 cells were used to examine the effects of this platform. Adsorption of anti-CD3 antibodies, HLA-A2/peptide, or dendritic cells' membrane proteins (MP) could flexibly provide pan-T-cell responses, specific Th1 responses, or specific Th1 and Th2 responses, depending on the host needs. Furthermore, with regulatory antibodies, the immuno-LPPC complex properly mediated immune responses by adsorbing positive or negative antibodies, such as anti-CD28 or anti-CTLA4 antibodies. RESULTS: The results clearly showed that treatment with LPPC/MP/CD28 complexes activated specific Th1 and Th2 responses, including cytokine release, CTL and prevented T-cell apoptosis. Moreover, LPPC/MP/CD28 complexes could eliminate metastatic B16F10 melanoma cells in the lung more efficiently than LPPC/MP. Interestingly, the melanoma resistance of mice treated with LPPC/MP/CD28 complexes would be reversed to susceptible after administration with LPPC/MP/CTLA4 complexes. NGS data revealed that LPPC/MP/CD28 complexes could enhance the gene expression of cytokine and chemokine pathways to strengthen immune activation than LPPC/MP, and that LPPC/MP/CTLA4 could abolish the LPPC/MP complex-mediated gene expression back to un-treatment. CONCLUSIONS: Overall, we proved a convenient and flexible immunotherapy platform for developing personalized cancer therapy.

Transdermal nanolipoplex simultaneously inhibits subcutaneous melanoma growth and suppresses systemically metastatic melanoma by activating host immunity.

Benefit for clinical melanoma treatments, the transdermal neoadjuvant therapy could reduce surgery region and increase immunotherapy efficacy. Using lipoplex (Lipo-PEG-PEI-complex, LPPC) encapsulated doxorubicin (DOX) and carrying CpG oligodeoxynucleotide; the transdermally administered nano-liposomal drug complex (LPPC-DOX-CpG) would have high cytotoxicity and immunostimulatory activity to suppress systemic metastasis of melanoma. LPPC-DOX-CpG dramatically suppressed subcutaneous melanoma growth by inducing tumor cell apoptosis and recruiting immune cells into the tumor area. Animal studies further showed that the colonization and growth of spontaneously metastatic melanoma cells in the liver and lung were suppressed by transdermal LPPC-DOX-CpG. Furthermore, NGS analysis revealed IFN-γ and NF-κB pathways were triggered to recruit and activate the antigen-presenting-cells and effecter cells, which could activate the anti-tumor responses as the major mechanism responsible for the therapeutic effect of LPPC-DOX-CpG. Finally, we have successfully proved transdermal LPPC-DOX-CpG as a promising penetrative carrier to activate systemic anti-tumor immunity against subcutaneous and metastatic tumor.

Anticancer peptide Q7 suppresses the growth and migration of human endometrial cancer by inhibiting DHCR24 expression and modulating the AKT-mediated pathway.

Endometrial cancer is one of the most common malignancy affecting women in developed countries. Resection uterus or lesion area is usually the first option for a simple and efficient therapy. Therefore, it is necessary to find a new therapeutic drug to reduce surgery areas to preserve fertility. Anticancer peptides (ACP) are bioactive amino acids with lower toxicity and higher specificity than chemical drugs. This study is to address an ACP, herein named Q7, which could downregulate 24-Dehydrocholesterol Reductase (DHCR24) to disrupt lipid rafts formation, and sequentially affect the AKT signal pathway of HEC-1-A cells to suppress their tumorigenicity such as proliferation and migration. Moreover, lipo-PEI-PEG-complex (LPPC) was used to enhance Q7 anticancer activity in vitro and efficiently show its effects on HEC-1-A cells. Furthermore, LPPC-Q7 exhibited a synergistic effect in combination with doxorubicin or paclitaxel. To summarize, Q7 was firstly proved to exhibit an anticancer effect on endometrial cancer cells and combined with LPPC efficiently improved the cytotoxicity of Q7.

The Lysosome in Malignant Melanoma: Biology, Function and Therapeutic Applications.

Lysosomes are membrane-bound vesicles that play roles in the degradation and recycling of cellular waste and homeostasis maintenance within cells. False alterations of lysosomal functions can lead to broad detrimental effects and cause various diseases, including cancers. Cancer cells that are rapidly proliferative and invasive are highly dependent on effective lysosomal function. Malignant melanoma is the most lethal form of skin cancer, with high metastasis characteristics, drug resistance, and aggressiveness. It is critical to understand the role of lysosomes in melanoma pathogenesis in order to improve the outcomes of melanoma patients. In this mini-review, we compile our current knowledge of lysosomes' role in tumorigenesis, progression, therapy resistance, and the current treatment strategies related to lysosomes in melanoma.

The Effects of Freshwater Clam (Corbicula fluminea) Extract on Serum Tumor Necrosis Factor-Alpha (TNF-α) in Prediabetic Patients in Taiwan.

Freshwater clam extract (FCE) is a functional food that regulates the immune system and has been demonstrated in numerous studies to display desirable anti-tumor necrosis factor-alpha (TNF-α) responses. In addition, excess TNF-α production is positively associated with type 2 diabetes. However, few longitudinal clinical studies evaluating the efficiency and toxicity of FCE are available. This article reports that patients with prediabetes who received FCE had a desirable outcome of a reduction in serum TNF-α for a long period. This was a double-blind, randomized, parallel clinical trial conducted using FCE intervention and placebo groups, and 36 patients with prediabetes were enrolled. Two grams of FCE or placebo was consumed daily for 180 consecutive days. The serum of the participants was collected at four time points (0M: before the intervention; 3M: after 3 months of intervention; 6M: after 6 months of intervention; 12M: 6 months after cessation of intervention at 6M). A serum TNF-α concentration higher than 4.05 pg/mL was defined as a cut-off value. FCE reduced serum TNF-α in all participants at 6M and 12M. Moreover, FCE significantly suppressed serum TNF-α concentrations at 6M and 12M and inhibited TNF-α release with time series in subjects with elevated TNF-α values. FCE intervention effectively reduced serum TNF-α and persistently sustained the effects for half a year in patients with prediabetes. Gas chromatography-mass spectrometry (GS-MS) analysis revealed that the major components of FCE were phytosterols and fatty acids, which exerted anti-inflammatory and anti-TNF-α abilities. Hence, FCE has the potential to be developed as a natural treatment for prediabetic patients in Taiwan.

Surfactin induces ER stress-mediated apoptosis via IRE1-ASK1-JNK signaling in human osteosarcoma.

Osteosarcoma, one of primary bone tumor in children and young adults, has poor prognosis and drug resistances to chemotherapy. In order to reinforce the conventional therapies and antagonize the osteosarcoma in patients, a novel strategy is required for developing a new treatment. In this study, surfactin, a natural product from Bacillus subtilis, showed the efficiency of cell death in osteosarcoma, but not in normal cells. Surfactin triggers ER stress mechanism by promoting the aberrant Ca2+ release from ER lumen and ER-signaling to mitochondrial dysfunction following caspases activation mediating cell apoptosis. Surfactin-induced ER stress not only upregulated of glucose-regulated protein 78/94 and IRE1-ASK1-JNK pathway but also leading to calpains and Bcl-2 proteins family involving the release of cytochrome c. The releases into cytosol trigger the cleavage of caspase-9 and caspase-3 to induce cell apoptosis. In this study, surfactin demonstrated the potential functions to trigger the ER stress, ER stress-associated IRE1-ASK1-JNK signaling pathway, mitochondrial dysfunction, and caspase activations leading to programmed cell apoptosis. Importantly, implicating the signaling pathway that regulates the connection between ER stress and mitochondrial dysfunction causing apoptosis associated with surfactin. These results indicated a potential application of surfactin strengthen current conventional therapies.

Enhancement of cytotoxicity and induction of apoptosis by cationic nano-liposome formulation of n-butylidenephthalide in breast cancer cells.

Breast cancer is the second most common malignancy in women. Current clinical therapy for breast cancer has many disadvantages, including metastasis, recurrence, and poor quality of life. Furthermore, it is necessary to find a new therapeutic drug for breast cancer patients to meet clinical demand. n-Butylidenephthalide (BP) is a natural and hydrophobic compound that can inhibit several tumors. However, BP is unstable in aqueous or protein-rich environments, which reduces the activity of BP. Therefore, we used an LPPC (Lipo-PEG-PEI complex) that can encapsulate both hydrophobic and hydrophilic compounds to improve the limitation of BP. The purpose of this study is to investigate the anti-tumor mechanisms of BP and BP/LPPC and further test the efficacy of BP encapsulated by LPPC on SK-BR-3 cells. BP inhibited breast cancer cell growth, and LPPC encapsulation (BP/LPPC complex) enhanced the cytotoxicity on breast cancer by stabilizing the BP activity and offering endocytic pathways. Additionally, BP and LPPC-encapsulated BP induced cell cycle arrest at the G0/G1 phase and might trigger both extrinsic as well as intrinsic cell apoptosis pathway, resulting in cell death. Moreover, the BP/LPPC complex had a synergistic effect with doxorubicin of enhancing the inhibitory effect on breast cancer cells. Consequently, LPPC-encapsulated BP could improve the anti-cancer effects on breast cancer in vitro. In conclusion, BP exhibited an anti-cancer effect on breast cancer cells, and LPPC encapsulation efficiently improved the cytotoxicity of BP via an acceleration of entrapment efficiency to induce cell cycle block and apoptosis. Furthermore, BP/LPPC exhibited a synergistic effect in combination with doxorubicin.

Positively Charged Nanoparticle Delivery of n-Butylidenephthalide Enhances Antitumor Effect in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second and sixth leading cause of cancer death in men and woman in 185 countries statistics, respectively. n-Butylidenephthalide (BP) has shown anti-HCC activity, but it also has an unstable structure that decreases its potential antitumor activity. The aim of this study was to investigate the cell uptake, activity protection, and antitumor mechanism of BP encapsulated in the novel liposome LPPC in HCC cells. BP/LPPC exhibited higher cell uptake and cytotoxicity than BP alone, and combined with clinical drug etoposide (VP-16), BP/LPPC showed a synergistic effect against HCC cells. Additionally, BP/LPPC increased cell cycle regulators (p53, p-p53, and p21) and decreased cell cycle-related proteins (Rb, p-Rb, CDK4, and cyclin D1), leading to cell cycle arrest at the G0/G1 phase in HCC cells. BP/LPPC induced cell apoptosis through activation of both the extrinsic (Fas-L and Caspase-8) and intrinsic (Bax and Caspase-9) apoptosis pathways and activated the caspase cascade to trigger HCC cell death. In conclusion, the LPPC complex improved the antitumor activity of BP in terms of cytotoxicity, cell cycle regulation and cell apoptosis, and BP/LPPC synergistically inhibited cell growth during combination treatment with VP-16 in HCC cells. Therefore, BP/LPPC is potentially a good candidate for clinical drug development or for use as an adjuvant for clinical drugs as a combination therapy for hepatocellular carcinoma.

Genome-wide DNA methylation analysis using MethylCap-seq in canine high-grade B-cell lymphoma.

DNA methylation is a comprehensively studied epigenetic modification and plays crucial roles in cancer development. In the present study, MethylCap-seq was used to characterize the genome-wide DNA methylation patterns in canine high-grade B-cell lymphoma (cHGBL). Canine methylated DNA fragments were captured and the MEDIUM-HIGH and LOW fraction of methylated DNA was obtained based on variation in CpG methylation density. In the MEDIUM-HIGH and LOW fraction, 2144 and 1987 cHGBL-specific hypermethylated genes, respectively, were identified. Functional analysis highlighted pathways strongly related to oncogenesis. The relevant signaling pathways associated with neuronal system were also revealed, echoing recent novel findings that neurogenesis plays key roles in tumor establishment. In addition, 14 genes were hypermethylated in all the cHGBL cases but not in the healthy dogs. These genes might be potential signatures for tracing cHGBL, and some of them have been reported to play roles in various types of cancers. Further, the distinct methylation pattern of cHGBL showed a concordance with the clinical outcome, suggesting that aberrant epigenetic changes may influence tumor behavior. In summary, our study characterized genome-wide DNA methylation patterns using MethylCap-seq in cHGBL; the findings suggest that specific DNA hypermethylation holds promise for dissecting tumorigenesis and uncovering biomarkers for monitoring the progression of cHGBL.

Secoiridoid Glucosides and Anti-Inflammatory Constituents from the Stem Bark of Fraxinus chinensis.

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), and 3'',4''-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4-26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), 3'',4''-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 µg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

Dynamic Hip Screw with Trochanter-Stabilizing Plate Compared with Proximal Femoral Nail Antirotation as a Treatment for Unstable AO/OTA 31-A2 and 31-A3 Intertrochanteric Fractures.

BACKGROUND: The dynamic hip screw (DHS) with the addition of an angular stable trochanter-stabilizing plate (TSP) has been considered the ideal treatment for the unstable intertrochanteric fracture type. However, there have been few comparisons between DHS+TSP augmentation with intramedullary (IM) nailing. The aim of this retrospectively registered study was to compare the clinical outcomes of patients with the unstable type of intertrochanteric fractures treated with DHS+TSP or IM nailing (proximal femoral nail antirotation (PFNA)). METHODS: From June 2013 to April 2018, 358 patients with proximal femur fracture AO/OTA type 31A2 and 31A3 treated with PFNA or DHS+TSP and followed for ≥10 months postoperatively were included. The surgical-dependent outcome evaluation included the operation time, intraoperative blood loss, postoperative decrease in hemoglobin, and blood transfusion amount. Functional status was also measured. Radiographic findings and postoperative complications were recorded and analyzed. RESULT: The operation time was significantly shorter in the DHS+TSP group than that in the PFNA group for both A2 and A3 fractures (A2 type: 84.0 vs.96.4 min; p < 0.05; A3 type: 102.4 vs.116.1 min; p < 0.05). Postoperative decrease in hemoglobin was more significant in the PFNA group than that in the DHS+TSP group for both fracture types (A2 type: -1.88 vs. -1.29 (mg/dL); p < 0.05; A3 type: -1.63 vs. -1.04 (mg/dL); p < 0.05). However, the patients treated with DHS+TSP had significantly more residual pain than those treated with PFNA during the final follow-up (Visual Analog Scale score, A2 type: 28.4 vs.23.2; p < 0.05; A3 type: 27.5 vs.23.6; p < 0.05) and complained of greater implant irritation. CONCLUSION: We found that DHS+TSP was associated with less operation time and less postoperative decrease in hemoglobin but more residual pain and implant irritation than those of PFNA. As a treatment for unstable intertrochanteric fracture, DHS+TSP provided ideal surgical outcomes which were not inferior to the PFNA.

A panel of eight microRNAs is a good predictive parameter for triple-negative breast cancer relapse.

Rationale: Triple-negative breast cancer (TNBC), which has the highest recurrence rate and shortest survival time of all breast cancers, is in urgent need of a risk assessment method to determine an accurate treatment course. Recently, miRNA expression patterns have been identified as potential biomarkers for diagnosis, prognosis, and personalized therapy. Here, we investigate a combination of candidate miRNAs as a clinically applicable signature that can precisely predict relapse in TNBC patients after surgery. Methods: Four total cohorts of training (TCGA_TNBC and GEOD-40525) and validation (GSE40049 and GSE19783) datasets were analyzed with logistic regression and Gaussian mixture analyses. We established a miRNA signature risk model and identified an 8-miRNA signature for the prediction of TNBC relapse. Results: The miRNA signature risk model identified ten candidate miRNAs in the training set. By combining 8 of the 10 miRNAs (miR-139-5p, miR-10b-5p, miR-486-5p, miR-455-3p, miR-107, miR-146b-5p, miR-324-5p and miR-20a-5p), an accurate predictive model of relapse in TNBC patients was established and was highly correlated with prognosis (AUC of 0.80). Subsequently, this 8-miRNA signature prognosticated relapse in the two validation sets with AUCs of 0.89 and 0.90. Conclusion: The 8-miRNA signature predictive model may help clinicians provide a prognosis for TNBC patients with a high risk of recurrence after surgery and provide further personalized treatment to decrease the chance of relapse.

Amphiregulin promotes cisplatin chemoresistance by upregulating ABCB1 expression in human chondrosarcoma.

Chondrosarcomas are well known for their resistance to chemotherapeutic agents, including cisplatin, which is commonly used in chondrosarcomas. Amphiregulin (AR), a ligand of epidermal growth factor receptor (EGFR), plays an important role in drug resistance. We therefore sought to determine the role of AR in cisplatin chemoresistance. We found that AR inhibits cisplatin-induced cell apoptosis and promotes ATP-binding cassette subfamily B member 1 (ABCB1) expression, while knockdown of ABCB1 by small interfering RNA (siRNA) reverses these effects. High phosphoinositide 3-kinase (PI3K), Akt and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation levels were observed in cisplatin-resistant cells. Pretreating chondrosarcoma cells with PI3K, Akt and NF-κB inhibitors or transfecting the cells with p85, Akt and p65 siRNAs potentiated cisplatin-induced cytotoxicity. In a mouse xenograft model, knockdown of AR expression in chondrosarcoma cells increased the cytotoxic effects of cisplatin and also decreased tumor volume and weight. These results indicate that AR upregulates ABCB1 expression through the PI3K/Akt/NF-κB signaling pathway and thus contributes to cisplatin resistance in chondrosarcoma.

Antitumor Effects of N-Butylidenephthalide Encapsulated in Lipopolyplexs in Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third most common type of cancer and the second most common cause of cancer-related death in the world. N-Butylidenephthalide (BP), a natural compound, inhibits several cancers, such as hepatoma, brain tumor and colon cancer. However, due to the unstable structure, the activity of BP is quickly lost after dissolution in an aqueous solution. A polycationic liposomal polyethylenimine and polyethylene glycol complex (LPPC), a new drug carrier, encapsulates both hydrophobic and hydrophilic compounds, maintains the activity of the compound, and increases uptake of cancer cells. The purpose of this study is to investigate the antitumor effects and protection of BP encapsulated in LPPC in CRC cells. The LPPC encapsulation protected BP activity, increased the cytotoxicity of BP and enhanced cell uptake through clathrin-mediated endocytosis. Moreover, the BP/LPPC-regulated the expression of the p21 protein and cell cycle-related proteins (CDK4, Cyclin B1 and Cyclin D1), resulting in an increase in the population of cells in the G0/G1 and subG1 phases. BP/LPPC induced cell apoptosis by activating the extrinsic (Fas, Fas-L and Caspase-8) and intrinsic (Bax and Caspase-9) apoptosis pathways. Additionally, BP/LPPC combined with 5-FU synergistically inhibited the growth of HT-29 cells. In conclusion, LPPC enhanced the antitumor activity and cellular uptake of BP, and the BP/LPPC complex induced cell cycle arrest and apoptosis, thereby causing death. These findings suggest the putative use of BP/LPPC as an adjuvant cytotoxic agent for colorectal cancer.

Lipoplex-based targeted gene therapy for the suppression of tumours with VEGFR expression by producing anti-angiogenic molecules.

BACKGROUND: The anti-angiogenic fusion protein RBDV-IgG1 Fc (RBDV), which comprises the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A), has shown antitumour effects by reducing angiogenesis in vivo. This study used the cationic lipoplex lipo-PEG-PEI-complex (LPPC) to simultaneously encapsulate both the RBDV targeting protein and the RBDV plasmid (pRBDV) without covalent bonds to assess VEGFR targeting gene therapy in mice with melanoma in vivo. RESULTS: LPPC protected the therapeutic transgene from degradation by DNase, and the LPPC/RBDV complexes could specifically target VEGFR-positive B16-F10 cells both in vitro and in vivo. With or without RBDV protein-targeting direction, the pRBDV-expressing RBDV proteins were expressed and reached a maximal concentration on the 7th day in the sera after transfection in vivo and significantly elicited growth suppression against B16-F10 melanoma but not IgG1 control proteins. In particular, LPPC/pRBDV/RBDV treatment with the targeting molecules dramatically inhibited B16-F10 tumour growth in vivo to provide better therapeutic efficacy than the treatments with gene therapy with IgG1 protein targeting or administration of a protein drug with RBDV. CONCLUSIONS: The simultaneous combination of the LPPC complex with pRBDV gene therapy and RBDV protein targeting might be a potential tool to conveniently administer targeted gene therapy for cancer therapy.

Putative Abscopal Effect in Three Patients Treated by Combined Radiotherapy and Modulated Electrohyperthermia.

Purpose: True abscopal responses from radiation therapy are extremely rare; the combination of immune checkpoint inhibitors with radiation therapy has led to more reports of the abscopal effect, but even in this setting, the genuine magnitude remains unknown and is still considered generally uncommon. We report the occurrence of what appears to be putative, durable abscopal tumor responses with associated auto-immune systemic reactions resulting from the combination of local radiotherapy (RT) and modulated electrohyperthermia (mEHT). Materials and Methods: Data from advanced cancer patients treated palliatively with RT and mEHT between January and December 2017 were collected as part of a post-marketing safety monitoring program of mEHT therapy. We specified a minimum RT dose of 30 Gy and at least four mEHT treatments for reporting toxicities, which was the primary aim of the larger study. Results: Thirty-three patients treated with RT and mEHT, both applied to the same lesion, were included. The median RT dose was 45.5 Gy in 20 fractions (fxs) and the median number of mEHT treatments was 12 (range, 4-20). Most patients had subsequent systemic therapy after one course of RT and mEHT. Three patients (9.1%) developed autoimmune toxicities. Case number 1 received RT and mEHT only; case number 2 had two cycles of concurrent low dose chemotherapy during RT; and case number 3 received concurrent immune checkpoint inhibitors. None of the three patients received any further systemic treatment due to obvious treatment-related autoimmune reactions which occurred rapidly after RT; one had autoimmune hepatitis, one had dermatitis herpetiformis and the third developed severe myasthenia gravis. Interestingly, what we surmise to be long-lasting abscopal responses outside the irradiated area, were noted in all three patients. Conclusion: RT combined with mEHT could putatively result in enhancing immune responsiveness. These preliminary observational findings lead to the generation of a hypothesis that this combination induces both an in-situ, tumor-specific immune reaction and an anti-self-autoimmune reaction, in at least a small proportion of patients, and of those who experience the auto-immune response, tumor response is a concomitant finding. Mechanisms underlying this phenomenon need to be investigated further.

ZnO Nanoparticles Induced Caspase-Dependent Apoptosis in Gingival Squamous Cell Carcinoma through Mitochondrial Dysfunction and p70S6K Signaling Pathway.

Zinc oxide nanoparticles (ZnO-NPs) are increasingly used in sunscreens, food additives, pigments, rubber manufacture, and electronic materials. Several studies have shown that ZnO-NPs inhibit cell growth and induce apoptosis by the production of oxidative stress in a variety of human cancer cells. However, the anti-cancer property and molecular mechanism of ZnO-NPs in human gingival squamous cell carcinoma (GSCC) are not fully understood. In this study, we found that ZnO-NPs induced growth inhibition of GSCC (Ca9-22 and OECM-1 cells), but no damage in human normal keratinocytes (HaCaT cells) and gingival fibroblasts (HGF-1 cells). ZnO-NPs caused apoptotic cell death of GSCC in a concentration-dependent manner by the quantitative assessment of oligonucleosomal DNA fragmentation. Flow cytometric analysis of cell cycle progression revealed that sub-G1 phase accumulation was dramatically induced by ZnO-NPs. In addition, ZnO-NPs increased the intracellular reactive oxygen species and specifically superoxide levels, and also decreased the mitochondrial membrane potential. ZnO-NPs further activated apoptotic cell death via the caspase cascades. Importantly, anti-oxidant and caspase inhibitor clearly prevented ZnO-NP-induced cell death, indicating the fact that superoxide-induced mitochondrial dysfunction is associated with the ZnO-NP-mediated caspase-dependent apoptosis in human GSCC. Moreover, ZnO-NPs significantly inhibited the phosphorylation of ribosomal protein S6 kinase (p70S6K kinase). In a corollary in vivo study, our results demonstrated that ZnO-NPs possessed an anti-cancer effect in a zebrafish xenograft model. Collectively, these results suggest that ZnO-NPs induce apoptosis through the mitochondrial oxidative damage and p70S6K signaling pathway in human GSCC. The present study may provide an experimental basis for ZnO-NPs to be considered as a promising novel anti­tumor agent for the treatment of gingival cancer.

Encapsulated n-Butylidenephthalide Efficiently Crosses the Blood-Brain Barrier and Suppresses Growth of Glioblastoma.

BACKGROUND: n-Butylidenephthalide (BP) has anti-tumor effects on glioblastoma. However, the limitation of BP for clinical application is its unstable structure. A polycationic liposomal polyethylenimine (PEI) and polyethylene glycol (PEG) complex (LPPC) has been developed to encapsulate BP for drug structure protection. The purpose of this study was to investigate the anti-cancer effects of the BP/LPPC complex on glioblastoma in vitro and in vivo. METHODS: DBTRG-05MG tumor bearing xenograft mice were treated with BP and BP/LPPC and then their tumor sizes, survival, drug biodistribution were measured. RG2 tumor bearing F344 rats also treated with BP and BP/LPPC and then their tumor sizes by magnetic resonance imaging for evaluation blood-brain barrier (BBB) across and drug therapeutic effects. After treated with BP/LPPC in vitro, cell uptake, cell cycle and apoptotic regulators were analyzed for evaluation the therapeutic mechanism. RESULTS: In athymic mice, BP/LPPC could efficiently suppress tumor growth and prolong survival. In F334 rats, BP/LPPC crossed the BBB and led to tumor shrinkage. BP/LPPC promoted cell cycle arrest at the G0/G1 phase and triggered the extrinsic and intrinsic cell apoptosis pathways resulting cell death. BP/LPPC also efficiently suppressed VEGF, VEGFR1, VEGFR2, MMP2 and MMP9 expression. CONCLUSION: BP/LPPC was rapidly and efficiently transported to the tumor area across the BBB and induced cell apoptosis, anti-angiogenetic and anti-metastatic effects in vitro and in vivo.