Autocrine phosphatase PDP2 inhibits ferroptosis by dephosphorylating ACSL4 in the Luminal A Breast Cancer.

Phosphatases can dephosphorylate phosphorylated kinases, leading to their inactivation, and ferroptosis is a type of cell death. Therefore, our aim is to identify phosphatases associated with ferroptosis by analyzing the differentially expressed genes (DEGs) of the Luminal A Breast Cancer (LumABC) cohort from the Cancer Genome Atlas (TCGA). An analysis of 260 phosphatase genes from the GeneCard database revealed that out of the 28 DEGs with high expression, only the expression of pyruvate dehydrogenase phosphatase 2 (PDP2) had a significant correlation with patient survival. In addition, an analysis of DEGs using gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis revealed a significant variation in the expression of ferroptosis-related genes. To further investigate this, we analyzed 34 ferroptosis-related genes from the TCGA-LumABC cohort. The expression of long-chain acyl-CoA synthetase 4 (ACSL4) was found to have the highest correlation with the expression of PDP2, and its expression was also inversely proportional to the survival rate of patients. Western blot experiments using the MCF-7 cell line showed that the phosphorylation level of ACSL4 was significantly lower in cells transfected with the HA-PDP2 plasmid, and ferroptosis was correspondingly reduced (p < 0.001), as indicated by data from flow cytometry detection of membrane-permeability cell death stained with 7-aminoactinomycin, lipid peroxidation, and Fe2+. Immunoprecipitation experiments further revealed that the phosphorylation level of ACSL4 was only significantly reduced in cells where PDP2 and ACSL4 co-precipitated. These findings suggest that PDP2 may act as a phosphatase to dephosphorylate and inhibit the activity of ACSL4, which had been phosphorylated and activated in LumABC cells. Further experiments are needed to confirm the molecular mechanism of PDP2 inhibiting ferroptosis.

Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells.

Gankyrin is found in high levels in triple-negative breast cancer (TNBC) and has been established to form a complex with the E3 ubiquitin ligase MDM2 and p53, resulting in the degradation of p53 in hepatocarcinoma cells. Therefore, this study sought to determine whether gankyrin could inhibit ferroptosis through this mechanism in TNBC cells. The expression of gankyrin was investigated in relation to the prognosis of TNBC using bioinformatics. Co-immunoprecipitation and GST pull-down assays were then conducted to determine the presence of a gankyrin and MDM2 complex. RT-qPCR and immunoblotting were used to examine molecules related to ferroptosis, such as gankyrin, p53, MDM2, SLC7A11, and GPX4. Additionally, cell death was evaluated using flow cytometry detection of 7-AAD and a lactate dehydrogenase release assay, as well as lipid peroxide C11-BODIPY. Results showed that the expression of gankyrin is significantly higher in TNBC tissues and cell lines, and is associated with a poor prognosis for patients. Subsequent studies revealed that inhibiting gankyrin activity triggered ferroptosis in TNBC cells. Additionally, silencing gankyrin caused an increase in the expression of the p53 protein, without altering its mRNA expression. Co-immunoprecipitation and GST pull-down experiments indicated that gankyrin and MDM2 form a complex. In mouse embryonic fibroblasts lacking both MDM2 and p53, this gankyrin/MDM2 complex was observed to ubiquitinate p53, thus raising the expression of molecules inhibited by ferroptosis, such as SLC7A11 and GPX4. Furthermore, silencing gankyrin in TNBC cells disrupted the formation of the gankyrin/MDM2 complex, hindered the degradation of p53, increased SLC7A11 expression, impeded cysteine uptake, and decreased GPX4 production. Our findings suggest that TNBC cells are able to prevent cell ferroptosis through the gankyrin/p53/SLC7A11/GPX4 signaling pathway, indicating that gankyrin may be a useful biomarker for predicting TNBC prognosis or a potential therapeutic target.

Interoceptive awareness: MBSR training alters information processing of salience network.

Mindfulness refers to a mental state of awareness of internal experience without judgment. Studies have suggested that each mindfulness practice may involve a unique mental state, but the underlying neurophysiological mechanisms remain unknown. Here we examined how distinct mindfulness practices after mindfulness-based intervention alter brain functionality. Specifically, we investigated the functional alterations of the salience network (SN) using functional magnetic resonance imaging (fMRI) among the two interoceptive mindfulness practices-breathing and body scan-associated with interoceptive awareness in fixed attention and shifted attention, respectively. Long-distance functional connectivity (FC) and regional homogeneity (ReHo) approaches were applied to measure distant and local neural information processing across various mental states. We hypothesized that mindful breathing and body scan would yield a unique information processing pattern in terms of long-range and local functional connectivity (FC). A total of 18 meditation-naïve participants were enrolled in an 8-week mindfulness-based stress reduction (MBSR) program alongside a waitlist control group (n = 14), with both groups undergoing multiple fMRI sessions during breathing, body scan and resting state for comparison. We demonstrated that two mindfulness practices affect both the long-distance FC SN and the local ReHo, only apparent after the MBSR program. Three functional distinctions between the mindfulness practices and the resting state are noted: (1) distant SN connectivity to occipital regions increased during the breathing practice (fixed attention), whereas the SN increased connection with the frontal/central gyri during the body scan (shifting attention); (2) local ReHo increased only in the parietal lobe during the body scan (shifting attention); (3) distant and local connections turned into a positive correlation only during the mindfulness practices after the MBSR training, indicating a global enhancement of the SN information processing during mindfulness practices. Though with limited sample size, the functional specificity of mindfulness practices offers a potential research direction on neuroimaging of mindfulness, awaiting further studies for verification.

Enhanced electroencephalography effective connectivity in frontal low-gamma band correlates of emotional regulation after mindfulness training.

Practicing mindfulness, focusing attention on the internal and external experiences occurring in the present moment with open and nonjudgement stance, can lead to the development of emotional regulation skills. Yet, the effective connectivity of brain regions during mindfulness has been largely unexplored. Studies have shown that mindfulness practice promotes functional connectivity in practitioners, potentially due to improved emotional regulation abilities and increased connectivity in the lateral prefrontal areas. To examine the changes in effective connectivity due to mindfulness training, we analyzed electroencephalogram (EEG) signals taken before and after mindfulness training, focusing on training-related effective connectivity changes in the frontal area. The mindfulness training group participated in an 8-week mindfulness-based stress reduction (MBSR) program. The control group did not take part. Regardless of the specific mindfulness practice used, low-gamma band effective connectivity increased globally after the mindfulness training. High-beta band effective connectivity increased globally only during Breathing. Moreover, relatively higher outgoing effective connectivity strength was seen during Resting and Breathing and Body-scan. By analyzing the changes in outgoing and incoming connectivity edges, both F7 and F8 exhibited strong parietal connectivity during Resting and Breathing. Multiple regression analysis revealed that the changes in effective connectivity of the right lateral prefrontal area predicted mindfulness and emotional regulation abilities. These results partially support the theory that the lateral prefrontal areas have top-down modulatory control, as these areas have high outflow effective connectivity, implying that mindfulness training cultivates better emotional regulation.

3'-epi-12ß-hydroxyfroside-mediated autophagy degradation of RIPK1/RIPK3 necrosomes leads to anergy of immunogenic cell death in triple-negative breast cancer cells.

Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various tumors. We previously found that 3'-epi-12ß-hydroxyfroside (HyFS), a novel cardenolide compound isolated by our group, could induce cytoprotective autophagy through inactivation of the Akt/mTOR pathway. However, whether HyFS can induce ICD remains unknown. In this study, we extend our work to further investigate whether HyFS could induce both autophagy and ICD, and we investigated the relationship between autophagy and ICD in three TNBC cell lines. Unexpectedly, compared to DIG, we found that HyFS could induce complete autophagy flux but not ICD in three human triple-negative breast cancer (TNBC) cell lines and one murine TNBC model. Inhibition of HyFS-induced autophagy resulted in the production of ICD in TNBC MDA-MB-231, MDA-MB-436, and HCC38 cells. A further mechanism study showed that formation of RIPK1/RIPK3 necrosomes was necessary for ICD induction in DIG-treated TNBC cells, while HyFS treatment led to receptor-interacting serine-threonine kinase (RIPK)1/3 necrosome degradation via an autophagy process. Additionally, inhibition of HyFS-induced autophagy by the autophagy inhibitor chloroquine resulted in the reoccurrence of ICD and reversion of the tumor microenvironment, leading to more significant antitumor effects in immunocompetent mice than in immunodeficient mice. These findings indicate that HyFS-mediated autophagic degradation of RIPK1/RIPK3 necrosomes leads to inactivation of ICD in TNBC cells. Moreover, combined treatment with HyFS and an autophagy inhibitor may enhance the antitumor activities, suggesting an alternative therapeutic for TNBC treatment.

Immunotherapy combining tumor and endothelium cell lysis with immune enforcement by recombinant MIP-3α Newcastle disease virus in a vessel-targeting liposome enhances antitumor immunity.

BACKGROUND: Several agents for oncolytic immunotherapy have been approved for clinical use, but monotherapy is modest for most oncolytic agents. The combination of several therapeutic strategies through recombinant and nanotechnology to engineer multifunctional oncolytic viruses for oncolytic immunotherapy is a promising strategy. METHODS: An endothelium-targeting iRGD-liposome encapsulating a recombinant Newcastle disease virus (NDV), which expresses the dendritic cell (DC) chemokine MIP-3α (iNDV3α-LP), and three control liposomes were constructed. MIP-3α, HMGB1, IgG, and ATP were detected by western blotting or ELISA. The chemotaxis of DCs was examined by Transwell chambers. The phenotypes of the immune cells were analyzed by flow cytometry. The antitumor efficiency was investigated in B16 and 4T1 tumor-bearing mice. Immunofluorescence and immunohistochemistry were used to observe the localization of liposomes, molecular expression and angiogenesis. Synergistic index was calculated using the data of tumor volume, tumor angiogenesis and tumor-infiltrating lymphocytes. RESULTS: Compared with NDV-LP, treatment with iNDV3α-LP and NDV3α-LP induced stronger virus replication and cell lysis in B16 and 4T1 tumor cells and human umbilical vein endothelial cells (HUVECs) with the best response observed following iNDV3α-LP treatment. B16 and 4T1 cells treated with iNDV3α-LP produced more damage-associated molecular pattern molecules, including secreted HMGB1, ATP, and calreticulin. Moreover, iNDV3α-LP specifically bound to αvß3-expressing 4T1 cells and HUVECs and to tumor neovasculature. Tumor growth was significantly suppressed, and survival was longer in iNDV3α-LP-treated B16-bearing and 4T1-bearing mice. A mechanism study showed that iNDV3α-LP treatment initiated the strongest tumor-specific cellular and humoral immune response. Moreover, iNDV3α-LP treatment could significantly suppress tumor angiogenesis and reverse the tumor immune suppressive microenvironment in both B16-bearing and 4T1-bearing mice. CONCLUSIONS: In this study, iNDV3α-LP had several functions, such as tumor and vessel lysis, MIP-3α immunotherapy, and binding to αvß3-expressing tumor and its neovasculature. iNDV3α-LP treatment significantly suppressed tumor angiogenesis and reversed the tumor immunosuppressive microenvironment. These findings offer a strong rationale for further clinical investigation into a combination strategy for oncolytic immunotherapy, such as the formulation iNDV3α-LP in this study.

Mindfulness Training Associated With Resting-State Electroencephalograms Dynamics in Novice Practitioners via Mindful Breathing and Body-Scan.

Objectives: Mindfulness-based stress reduction has been proven to improve mental health and quality of life. This study examined how mindfulness training and various types of mindfulness practices altered brain activity. Methods: Specifically, the spectral powers of scalp electroencephalography of the mindfulness-based stress reduction (MBSR) group (n=17) who underwent an 8-week MBSR training-including mindful breathing and body-scan-were evaluated and compared with those of the waitlist controls (n=14). Results: Empirical results indicated that the post-intervention effect of MBSR significantly elevated the resting-state beta powers and reduced resting-state delta powers in both practices; such changes were not observed in the waitlist control. Compared with mindful breathing, body-scanning resulted in an overall decline in electroencephalograms (EEG) spectral powers at both delta and low-gamma bands among trained participants. Conclusion: Together with our preliminary data of expert mediators, the aforementioned spectral changes were salient after intervention, but mitigated along with expertise. Additionally, after receiving training, the MBSR group's mindfulness and emotion regulation levels improved significantly, which were correlated with the EEG spectral changes in the theta, alpha, and low-beta bands. The results supported that MBSR might function as a unique internal processing tool that involves increased vigilant capability and induces alterations similar to other cognitive training.

Engineered porous/hollow Burkholderia pseudomallei loading tumor lysate as a vaccine.

Due to its size, shape, and inherent expression of pathogen-associated molecular patterns and invasion-assistant adhesion proteins, Burkholderia pseudomallei can easily attach to, and then be internalized by, dendritic cells (DCs), leading to more efficient antigen cross-presentation if modified as carrier. Herein, we engineered Burkholderia pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and adjuvant CpG (C) to be used as a tumor vaccine (SB-LC). We found that the adhesion proteins of Burkholderia pseudomallei promote internalization of the SB-LC vaccine by DCs, and result in enhanced DC maturation and antigen cross-presentation. SB-LC induces robust cellular and humoral antitumor responses that synergistically inhibit tumor growth with minimal adverse side effects in several tumor models. Moreover, SB-LC vaccination reverses the immunosuppressive tumor microenvironment, apparently as a result of CD8+-induced tumor ferroptosis. Thus, SB-LC is a potential model tumor vaccine for translating into a clinically viable treatment option.

A biotin-avidin-system-based virus-mimicking nanovaccine for tumor immunotherapy.

Virus is a nanosized pathogen and mainly composed of viral protein and nucleic acids. Under the pressure of long-term selection, mammals have gradually evolved effective immune mechanisms to defend themselves against viruses. In addition to recognizing viral proteins, immune system can also respond to viral sequence-specific nucleic acids, including CpG ODN, single- and double- strand RNA, and thereby enhancing the ability to remove infected viruses. Inspired by these immune mechanisms, we have attempted to develop a tracing virus-mimicking nanovaccine for tumor immunotherapy. This nanovaccine mainly consists of nucleic acids (CpG ODN), proteins (including tumor-associated antigen, and neutravidin (nAvidin) as skeleton materials for constructing nanovaccine and carriers for loading tumor-associated antigen and CpG ODN), and the dye molecules for assembling nAvidin to form nanoparticles comparable in size to viruses and tracing the vaccine in vitro and in vivo. The as-prepared nanovaccine efficiently induces the maturation of dendritic cell, the enhancement of antigen cross-presentation ability, and amplification of cytokine production in vitro. Furthermore, in vivo analysis clearly shows that it targets lymph nodes, successfully presents antigens to generate tumor-antigen-specific CD8+ T cells and induces a Th1-biased immune response. Most notably, this virus-mimicking nanovaccine significantly inhibits the growth of antigen-expressed tumor and prolongs the survival time of the antigen-expressed tumor bearing mice.

Mindfulness-based cognitive therapy on bereavement grief: Alterations of resting-state network connectivity associate with changes of anxiety and mindfulness.

Bereavement, the experience of losing a loved one, is one of the most catastrophic but inevitable events in life. It causes grief and intense depression-like sadness. Recent studies have revealed the effectiveness and proficiency of mindfulness-based cognitive therapy (MBCT) in emotional regulation among bereavement populations. MBCT improves the well-being of the bereaved by enhancing cognitive performances. Regarding the neural correlates of bereavement grief, previous studies focused on the alleviation of emotion-cognition interferences at specific brain regions. Here, we hypothesized that the bereavement grief fundamentally triggers global alterations in the resting-state brain networks and part of the internetwork connectivity could be reformed after MBCT intervention. We recruited 19 bereaved individuals who participated the 8-week MBCT program. We evaluated (a) the large-scale changes in brain connectivity affected by the MBCT program; as well as (b) the association between connectivity changes and self-rated questionnaire. First, after MBCT, the bereaved individuals showed the reduction of the internetwork connectivity in the salience, default-mode and fronto-parietal networks in the resting state but not under emotional arousal, implying the alleviated attention to spontaneous mind wandering after MBCT. Second, the alterations of functional connectivity between subcortical (e.g., caudate) and cortical networks (e.g., cingulo-opercular/sensorimotor) were associated with the changes of the mindfulness scale, the anxiety and the emotion regulation ability. In summary, MBCT could enhance spontaneous emotion regulation among the bereaved individuals through the internetwork reorganizations in the resting state.

Trait mindfulness and depressive symptoms in non-small cell lung cancer patients: the mediating roles of quality of life and meaning in life.

OBJECTIVE: The present study examined the potential mediating influences of meaning in life and quality of life in the relationship of trait mindfulness and depressive symptoms in lung cancer patients. Design: We adopted a cross-sectional design studying a sample of patients with non-small cell lung cancer, aged 20-65 years, and receiving cancer treatments or follow-up care. Main Outcome Measures: The outcome measures included Beck Depression Inventory-II, European Organisation for Research and Treatment of Cancer Core Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and lung cancer specific complementary measure (EORTC QLQ-LC13), Five Facet Mindfulness Questionnaire, and the meaning in life questionnaire. Results: Among 116 lung cancer patients, 26.72% of them had clinically significant depressive symptoms. The presence of meaning, quality of life (QOL) functioning and symptom distress mediated the relationship of trait mindfulness and depressive symptoms. Multiple mediation analyses found that the presence of meaning in life was the main mediator. Conclusion: The reductions of depressive symptoms might be related to trait mindfulness enhancing lung cancer patients' perceptions of meaning in life. A mindfulness program has the potential to improve depressive symptoms in people with lung cancer.

A recombinant oncolytic Newcastle virus expressing MIP-3α promotes systemic antitumor immunity.

BACKGROUND: The oncolytic Newcastle disease virus (NDV) is inherently able to trigger the lysis of tumor cells and induce the immunogenic cell death (ICD) of tumor cells and is also an excellent gene-engineering vector. The macrophage inflammatory protein-3α (MIP-3α) is a specific chemokine for dendritic cells (DCs). Thus, we constructed a recombinant NDV expressing MIP-3α (NDV-MIP3α) as an in vivo DC vaccine for amplifying antitumor immunities. METHODS: The recombinant NDV-MIP3α was constructed by the insertion of MIP-3α cDNA between the P and M genes. Western blotting assay and ELISA were used to detect MIP-3α, HMGB1, IgG, and ATP in the supernatant and sera. The chemotaxis of DCs was examined by Transwell chambers. The phenotypes of the immune cells (eg, DCs) were analyzed by flow cytometry. The antitumor efficiency of NDV-MIP3α was observed in B16 and CT26 tumor-bearing mice. Immunofluorescence and immunohistochemistry were applied to observe the ecto-calreticulin (CRT) and intratumoral attraction of DCs. Adoptive transfer of splenocytes and antibodies and depletion of T-cell subsets were used to evaluate the relationship between antitumor immunities and the role of the T-cell subtype. RESULTS: The findings show that NDV-MIP3α has almost the same capabilities of tumor lysis and induction of ICD as the wild-type NDV (NDV-WT). MIP-3α secreted by NDV-MIP3α could successfully attract DCs in vitro and in vivo. Both B16 and CT26 cells infected with NDV-MIP3α could strongly promote DC maturation and activation. Compared with NDV-WT, intratumoral injection of NDV-MIP3α and the adoptive transfer of T lymphocytes from mice injected with NDV-MIP3α resulted in a significant suppression of B16 and CT26 tumor growth. The NDV-MIP3α-induced production of tumor-specific cellular and humoral immune responses was dependent on CD8+ T cells and partially on CD4+ T cells. A significant reversion of tumor microenvironments was found in the mice injected with NDV-MIP3α. CONCLUSIONS: Compared with NDV-WT, the recombinant NDV-MIP3α as an in vivo DC vaccine demonstrates enhanced antitumor activities through the induction of stronger system immunities and modulation of the tumor microenvironment. This strategy may be a potential approach for the generation of an in vivo DC vaccine.

Toxicarioside O Inhibits Cell Proliferation and Epithelial-Mesenchymal Transition by Downregulation of Trop2 in Lung Cancer Cells.

Toxicarioside O (TCO), a natural product derived from Antiaris toxicaria, has been identified to be a promising anticancer agent. In this study, we aimed to investigate the effect of TCO on the proliferation and epithelial-mesenchymal transition (EMT) of lung cancer cells and its molecular mechanisms. Here, we indicated that TCO inhibits the proliferation of lung cancer cells both in vitro and in vivo. Our results demonstrated that TCO induces apoptosis in lung cancer cells. Moreover, we found that TCO suppresses EMT program and inhibits cell migration in vitro. Mechanistically, TCO decreases the expression of trophoblast cell surface antigen 2 (Trop2), resulting in inhibition of the PI3K/Akt pathway and EMT program. Overexpression of Trop2 rescues TCO-induced inhibition of cell proliferation and EMT. Our findings demonstrate that TCO markedly inhibits cell proliferation and EMT in lung cancer cells and provides guidance for its drug development.

Exopolysaccharide from Paecilomyces lilacinus modulates macrophage activities through the TLR4/NF­κB/MAPK pathway.

Multiple exopolysaccharides (EPSs) have been isolated from various organisms in extreme environments and have yielded a variety of activities. The present study evaluated the immunomodulatory capabilities of an EPS (termed PH­EPS) derived from the fungus Paecilomyces lilacinus PH0016, which was isolated from a tropical and hyperhaline environment in southern China. The macrophage RAW 264.7 cell line was used to investigate the mechanism of PH­EPS­induced macrophage activation. The results indicated that RAW 264.7 macrophages were activated by PH­EPS, in an effect slightly inferior to lipopolysaccharide (LPS), as evidenced by secretion of interleukin (IL)­1ß, tumor necrosis factor (TNF)­α and nitric oxide (NO), and by significantly increased phagocytosis in the cells treated with PH­EPS. Nuclear factor (NF)­κB p65 was significantly translocated into the nucleus in the PH­EPS­treated cells. In addition, expression of inducible NO synthase (iNOS) and IκB­α degradation were enhanced in PH­EPS­treated cells. The phosphorylation levels of p38, JNK and ERK were also significantly increased in the PH­EPS­treated cells. Furthermore, IL­1ß and TNF­α production was markedly decreased in PH­EPS­treated cells when the mitogen­activated protein kinase (MAPK) pathways were blocked by the inhibitor Dectin­1 and by antibodies against Toll­like receptor 4 (TLR4). The present results indicated that PH­EPS from Paecilomyces lilacinus possessed the capability of activating RAW 264.7 cells via the TLR4/NF­κB/MAPKs signaling pathway.

Impaired Autophagic Degradation of Transforming Growth Factor-ß-Induced Protein by Macrophages in Lattice Corneal Dystrophy.

Purpose: Lattice corneal dystrophy (LCD) is related to the denaturation of transforming growth factor-ß-induced protein (TGFBIp). Autophagic degradation of the denatured proteins by macrophages is one pathway to remove the denatured proteins. Thus, we investigated the role of autophagy in the degradation of mutant (MU) TGFBIp in macrophages. Methods: Corneas from participants were observed by slit-lamp photography and subjected to histopathologic and genetic analysis. Wild-type (WT) and MU TGFBIp were recombined and expressed. Macrophages from MU participants were isolated and cocultured with the recombinant TGFBIp. Colocalization of the two molecules was observed by immunofluorescent microscopy. Enzyme-linked immunosorbent assay, Western blotting, and flow cytometry were used to detect changes in molecule expression related to the phenotype and autophagy process. Results: Fourteen members from a family of 25 were identified as LCD sufferers. Significant TGFBIp aggregates and macrophage infiltration were found only in the corneas of LCD sufferers. Marker accumulation of TGFBIp was found in macrophages exposed to MU TGFBIp even at 5 hours after MU TGFBIp was withdrawn. High expressions of CD68 and CD36 were found in macrophages exposed to WT TGFBIp, but not to MU TGFBIp. Impaired autophagic flux due to defective autophagosome fusion to lysosomes was found in macrophages exposed to MU TGFBIp. Blockage of the autophagic process suppressed the expression of CD68 and CD36 in macrophages exposed to WT TGFBIp to levels similar to those found in macrophages exposed to MU TGFBIp. Conclusions: Our results suggested that reversion of the defective autophagic process in macrophages may be a therapeutic strategy for patients with LCD.

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

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

Autophagy plays a protective role against apoptosis induced by toxicarioside N via the Akt/mTOR pathway in human gastric cancer SGC-7901 cells.

Toxicarioside N (Tox N), a natural product extract from Antiaris toxicaria, has been reported to induce apoptosis in human gastric cancer cells. However, the mechanism and actual role of autophagy in Tox N-induced apoptosis of human gastric cancer cells remains poorly understood. In the current study, we demonstrated that Tox N could induce autophagy by inhibiting the Akt/mTOR signaling pathway in SGC-7901 cells. Moreover, we found that the inhibition of autophagy by 3-methyladenine, an autophagy inhibitor, enhanced Tox N-induced apoptotic cell death. However, the stimulation of autophagy by rapamycin, an autophagy activator, remarkably suppressed Tox N-induced apoptosis, suggesting that autophagy plays a protective role in Tox N-induced apoptosis. Thus, the results from this study suggested that Tox N combination with an autophagy inhibitor might be a promising strategy to enhance the anticancer activity of Tox N for the treatment of human gastric cancer.

Induction of enhanced immunogenic cell death through ultrasound-controlled release of doxorubicin by liposome-microbubble complexes.

Immunogenic cell death (ICD) is a specific kind of cell death that stimulates the immune system to combat cancer cells. Ultrasound (US)-controlled targeted release of drugs by liposome-microbubble complexes is a promising approach due to its non-invasive nature and visibility through ultrasound imaging. However, it is not known whether this approach can enhance ICD induced by drugs, such as doxorubicin. Herein, we prepared a doxorubicin-liposome-microbubble complex (MbDox), and the resultant MbDox was then characterized and tested for US-controlled release of Dox (MbDox+US treatment) to enhance the induction of ICD in LL/2 and CT26 cancer cells and in syngeneic murine models. We found that MbDox+US treatment caused more cellular uptake and nuclear accumulation of Dox in tumor cells, and more accumulation of Dox in tumor tissues. Enhanced induction of ICD occurred both in vitro and in vivo. MbDox+US treatment induced more apoptosis, stronger membrane exposure and the release of ER stress proteins and DAMPs in tumor cells, and increased DC maturation in vitro. In addition, MbDox+US treatment also resulted in stronger therapeutic effects in immunocompetent mice than in immunodeficient mice. Moreover, MbDox+US enhancement of ICD was also evidenced by a higher proportion of activated CD8+ T-lymphocytes but lower Treg in tumor tissues. Taken together, our results demonstrate that US-controlled release of ICD inducers into nuclei using liposome-microbubble complexes may be an effective approach to enhance the induction of ICD for tumor treatment.

3'-epi-12ß-hydroxyfroside, a new cardenolide, induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells.

Rationale: Cardenolides have potential as anticancer drugs. 3'-epi-12ß-hydroxyfroside (HyFS) is a new cardenolide structure isolated by our research group, but its molecular mechanisms remain poorly understood. This study investigates the relationship between its antitumor activities and autophagy in lung cancer cells. Methods: Cell growth and proliferation were detected by MTT, lactate dehydrogenase (LDH) release, 5-ethynyl-20-deoxyuridine (EDU) and colony formation assays. Cell apoptosis was detected by flow cytometry. Autophagic and signal proteins were detected by Western blotting. Markers of autophagy and autophagy flux were also detected by immunofluorescence, transmission electron microscopy and acridine orange staining. Real time RT-PCR was used to analyze the gene expression of Hsp90. Hsp90 ubiquitination was detected by coimmunoprecipitation. The antitumore activities of HyFS were observed in nude mice. Results: HyFS treatment inhibited cell proliferation and induced autophagy in A549 and H460 lung cancer cells, but stronger inhibition of cell proliferation and induction of cell apoptosis were shown when HyFS-mediated autophagy was blocked. The Hsp90/Akt/mTOR axis was found to be involved in the activation of HyFS-mediated autophagy. Evidence of direct interaction between Hsp90 and Akt was observed. HyFS treatment resulted in decreased levels of heat shock protein 90 (Hsp90) and phosphorylated Akt, overexpression of Hsp90 increased activation of autophagy, and inhibition of Hsp90 expression decreased autophagy. In addition, ubiquitin-mediated degradation of Hsp90 and subsequent dephosphorylation of its client protein Akt were also found in HyFS-treated lung cancer cells. Moreover, combination treatment with HyFS and chloroquine showed remarkably increased tumor inhibition in both A549- and H460-bearing mice. Conclusion: Our results demonstrate that HyFS induced cytoprotective autophagy through ubiquitin-mediated degradation of Hsp90, which further blocked the Akt/mTOR pathway in lung cancer cells. Thus, a combination of a HyFS-like cardenolide and an autophagic inhibitor is a potential alternative approach for the treatment of lung cancer.

Coroglaucigenin induces senescence and autophagy in colorectal cancer cells.

OBJECTIVES: Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti-cancer agent. However, the molecular mechanisms involved remain poorly understood. MATERIALS AND METHODS: Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA-ß-gal assay, western blotting and immunofluorescence were performed to determine CGN-induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN-induced senescence and autophagy. The anti-tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models. RESULTS: We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell-cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN-mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti-tumour effects in vivo. CONCLUSIONS: Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN-mediated anti-cancer therapy.