Hypericin-mediated photodynamic therapy inhibits metastasis and EMT of colorectal cancer cells by regulating RhoA-ROCK1 signaling pathway.

Colorectal cancer (CRC) is significantly contributed to global cancer mortality rates. Treating CRC is particularly challenging due to metastasis and drug resistance. There is a pressing need for new treatment strategies against metastatic CRC. Photodynamic therapy (PDT) offers a well-established, minimally invasive treatment option for cancer with limited side effects. Hypericin (HYP), a potent photosensitizer for PDT, has been documented to induce cytotoxicity and apoptosis in various types of cancers. However, there are few reports on the inhibitory effects of HYP-mediated PDT on the metastatic ability of CRC cells. Here, we evaluate the inhibitory effects of HYP-mediated PDT against metastatic CRC cells and define its underlying mechanisms. Wound-healing and Transwell assays show that HYP-mediated PDT suppresses migration and invasion of CRC cells. F-actin visualization assays indicate HYP-mediated PDT decreases F-actin formation in CRC cells. TEM assays reveal HYP-mediated PDT disrupts pseudopodia formation of CRC cells. Mechanistically, immunofluorescence and western blotting results show that HYP-mediated PDT upregulates E-cadherin and downregulates N-cadherin and Vimentin. HYP-mediated PDT also suppresses key EMT regulators, including Snail, MMP9, ZEB1 and α-SMA. Additionally, the expressions of RhoA and ROCK1 are downregulated by HYP-mediated PDT. Together, these findings suggest that HYP-mediated PDT inhibits the migration and invasion of HCT116 and SW620 cells by modulating EMT and RhoA-ROCK1 signaling pathway. Thus, HYP-mediated PDT presents a potential therapeutic option for CRC.

Molecular mechanism of honeysuckle + forsythia in treatment of acute lung injury based on network pharmacology.

The pathogenesis of acute lung injury (ALI) is complex and it is a common critical illness in clinical practice, seriously threatening the lives of critically ill patients, for which no specific molecular marker exists and there is a lack of effective methods for the treatment of ALI. The present study aimed to investigate the mechanism of action of honeysuckle and forsythia in treatment of acute lung injury (ALI) based on network pharmacology and in vitro modeling. The active ingredients and targets of honeysuckle and forsythia were predicted using traditional Chinese medicine systems pharmacology, PubChem and Swiss Target Prediction databases, and the Cytoscape 3.7.2 software was used to construct a drug-component-potential target network. The potential targets were imported into the Search tool for recurring instances of neighboring genes) database to obtain protein-protein interactions and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Targets analysis using Database for Annotation, Visualization, and Integrated Discovery. AutoDock Vina 1.1.2 software was used for docking between key active ingredients and the target proteins to analyze the binding ability of the active ingredients to the primary targets in honeysuckle and forsythia. A total of 64 male BALB/c mice were randomly divided into control, model, positive drug (Lianhua-Qingwen capsule), honeysuckle, forsythia, honeysuckle + forsythia high-, medium- and low-dose groups. Lipopolysaccharide (LPS) was used to induced an ALI model. The lung tissues of the mice were stained with hematoxylin-eosin and the serum levels of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured 4 h after the LPS administration. Reverse transcription-quantitative PCR and western blotting were used to detect NF-κB mRNA and protein expression, respectively. Active ingredients of honeysuckle and forsythia acted on 265 common targets in ALI, which regulated NF-κB, tumor necrosis factor-α (TNF-α) and PI3K-AKT signaling pathway, HIF-1 signalling pathway to slow the inflammatory response in treatment of ALI. In the positive drug group, honeysuckle, forsythia group, honeysuckle + forsythia high-, medium- and low-dose groups, lung tissue damage were significantly decrease compared with the model group, and inflammatory cell infiltration was reduced. Compared with the model group, honeysuckle + forsythia groups experienced decreased damage caused by the LPS and inflammation in the lung tissues and significantly decreased TNF-α and NF-κB and MDA concentration and significantly increased the SOD and GSH-Px activities. The mechanism of the effect of honeysuckle and forsythia on ALI may be mediated by inhibition of TNF-α and NF-κB expression and the activation of antioxidant mechanisms to decrease production of pro-inflammatory cytokines in lung tissue, thus treating ALI.

Total saponins from Rhizoma Panacis Majoris inhibit proliferation, induce cell cycle arrest and apoptosis and influence MAPK signalling pathways on the colorectal cancer cell.

Colorectal cancer (CRC) ranks third in incidence and second in mortality among all types of cancer, and due to its insidious onset and lack of early symptoms, it is usually diagnosed at a later stage. Saponins, a class of compounds abundant in plants, have been reported to possess prominent anti­tumour properties. The use of ginsenoside Rg3 in the clinical setting was authorized by the National Medicinal Products Administration of China. In the present study, total saponins from Rhizoma Panacis Majoris (RPMTG) were prepared, and the pharmacological mechanisms underlying the anti­CRC effects of RPMTG were investigated. The effect of RPMTG on the proliferation, cell cycle progression and apoptosis of HCT116 and SW620 cells were detected by MTT, flow cytometry and western blotting assays, and it was demonstrated that RPMTG could inhibit the proliferation of HCT116 and SW620 cells with IC50 values of 315.8 and 355.1 µg/ml, respectively, induce cell cycle arrest in the S and G0/G1 phase, and trigger apoptosis by downregulating the expression of the anti­apoptotic proteins Bcl­2, Bcl­xL and induced myeloid leukaemia cell differentiation protein Mcl­1, and increasing the expression of the pro­apoptotic proteins Bax and Bad, cleaved caspased­3 and poly(ADP)­ribose polymerase. These findings suggested that RPMTG induced apoptosis through mitochondrial­related pathways. In addition, RPMTG also decreased the expression of phosphorylated (p)­extracellular signal­regulated kinase and increased p­c­Jun N­terminal kinase (p­JNK) and p­p38. Moreover, the effects of RPMTG on cell proliferation and apoptosis were partially reversed when the JNK and p38 mitogen­activated protein kinase (MAPK) pathways were inhibited, indicating that RPMTG triggered apoptosis mainly via regulating JNK and p38 MAPK signalling. Therefore, RPMTG may have potential as an anti­CRC agent, and further evaluations are needed.

Anti-Rheumatoid Arthritic Effects of Paris Saponin VII in Human Rheumatoid Arthritis Fibroblast-Like Synoviocytes and Adjuvant-Induced Arthritis in Rats.

In the pathogenesis of rheumatoid arthritis (RA), rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) have tumor-like characteristics, mainly manifested by hyperproliferation and resistance to apoptosis and then it will erode the bone and cartilage, eventually leading to joint destruction. Paris saponin VII (PS VII) is an active compound derived from a traditional herbal medicine named Trillium tschonoskii Maxim, which has anti-tumor, analgesic, and immunomodulatory effects. However, its anti-RA effect has not yet been reported. This study was to investigate the effect of PS VII on two rheumatoid arthritis fibroblast-like synoviocytes lines (RA-FLS and MH7A) and adjuvant-induced arthritis (AIA) in rats. In vitro, the effects of PS VII on the proliferation, cell cycle, and apoptosis of RA-FLS and MH7A cells were detected by MTT, flow cytometry, and western blot analysis. In vivo, the effect of PS VII on the weight of the rat, paw swelling, ankle joint diameter, arthritis index, serum inflammatory cytokines (TNF-α, IL-6, and IL-1ß), histopathological assessment and apoptosis proteins in the synovial tissues were evaluated in AIA rats. The in vitro studies showed that PS VII inhibited the proliferation of RA-FLS and MH7A cells, induced S phase arrest and triggered cell apoptosis mainly through the mitochondrial apoptotic pathway and the regulation of JNK and p38 MAPK pathways. The in vivo studies revealed that PS VII could improve ameliorate body weight, paw swelling, ankle joint diameter, reduce the spleen and thymus index, suppress the production of TNF-α, IL-6 and IL-1ß, improve histopathological changes and regulate the expressions of apoptosis proteins in AIA Rats. In conclusion, PS VII could inhibit the proliferation and trigger apoptosis of RA-FLS and MH7A cells by regulating the mitochondrial apoptosis pathway and the JNK and p38 MAPK pathways, and alleviate the symptoms of RA, signifying it to be one of the potential anti-RA therapeutics.

Hypericum perforatum L. Regulates Glutathione Redox Stress and Normalizes Ggt1/Anpep Signaling to Alleviate OVX-Induced Kidney Dysfunction.

Menopause and associated renal complications are linked to systemic redox stress, and the causal factors remain unclear. As the role of Hypericum perforatum L. (HPL) in menopause-induced kidney disease therapy is still ambiguous, we aim to explore the effects of HPL on systemic redox stress under ovariectomy (OVX)-induced kidney dysfunction conditions. Here, using combined proteomic and metabolomic approaches, we constructed a multi-scaled "HPL-disease-gene-metabolite" network to generate a therapeutic "big picture" that indicated an important link between glutathione redox stress and kidney impairment. HPL exhibited the potential to maintain cellular redox homeostasis by inhibiting gamma-glutamyltransferase 1 (Ggt1) overexpression, along with promoting the efflux of accumulated toxic amino acids and their metabolites. Moreover, HPL restored alanyl-aminopeptidase (Anpep) expression and metabolite shifts, promoting antioxidative metabolite processing, and recovery. These findings provide a comprehensive description of OVX-induced glutathione redox stress at multiple levels and support HPL therapy as an effective modulator in renal tissues to locally influence the glutathione metabolism pathway and subsequent redox homeostasis.

Hypericin-mediated photodynamic therapy inhibits growth of colorectal cancer cells via inducing S phase cell cycle arrest and apoptosis.

Colorectal cancer (CRC) is one type of cancer with high morbidity and mortality worldwide. Photodynamic therapy (PDT), a promising new therapeutic approach for cancer, induces tumor damage through photosensitizer-mediated oxidative cytotoxicity. Hypericin is a powerful photosensitizer with pronounced tumor-localizing properties. In this study, we investigated the phototoxic effects of hypericin-mediated PDT (HYP-PDT) in HCT116 and SW620 cells. We validated that HYP-PDT inhibited cell proliferation, triggered intracellular reactive oxygen species generation, induced S phase cell cycle arrest and apoptosis of HCT116 and SW620 cells. Mechanistically, the results of western blot showed that HYP-PDT downregulated CDK2 expression through decreasing the CDC25A protein, which resulted in the decrease of CDK2/Cyclin A complex. Additionally, HYP-PDT induced DNA damage as evidenced by ATM activation and upregulation of p-H2AX. Further investigation showed that HYP-PDT significantly increased Bax expression and decreased Bcl-2 expression, and then, upregulated the expression of cleaved caspase-9, cleaved caspase-3 and cleaved PARP, thereby inducing apoptosis in HCT116 and SW620 cells. In conclusion, our results indicated that the CDC25A/CDK2/Cyclin A pathway and the mitochondrial apoptosis pathway were involved in HYP-PDT induced S phase cell cycle arrest and apoptosis in colorectal cancer cells, which shows HYP could be a probable candidate used for treating colorectal cancer.

[Quantitative determination of nine furanocoumarins for quality evaluation of Angelica dahurica from different habitats].

A UPLC method has been developed for simultaneous determination of nine furanocoumarins of Angelica dahurics,and was used for quality evaluation of A. dahurica from different habitats. ACQUITY UPLC BEH C18 chromatographic column was employed,the separation was performed with the mobile phase consisting of acetonitrile and water,and the detection wavelength was set at254 nm. This method was used to simultaneously determine the content of xanthotoxol,oxypeucedaninhydrate,byak-angelicin,psoralen,xanthotoxin,bergapten,oxypeucedanin,imperatorin and isoimperatorin in A. dahurica from different habitats. Then,the further quality assessment of the drug was carried out by similarity evaluation,cluster analysis( CA),principal component analysis( PCA),and orthogonal partial least squares discriminant analysis( OPLS-DA). The content order of measured furanocoumarins from high to low was: oxypeucedanin>imperatorin>isoimperatorin>oxypeucedaninhydrate>bergapten>byak-angelicin>xanthotoxin>xanthotoxol>psoralen,with the mean content 2. 844,1. 277,0. 649 2,0. 216 2,0. 129 8,0. 062 68,0. 052 68,0. 019 30,0. 018 19 mg·g-1,respectively. There were difference between the batches of the drug,and the quality was influenced by smouldering sulphur based on the results of chemical pattern recognition and content determination. Finally,six active ingredients were recognized as the quality makers using OPLS-DA method. The validated UPLC fingerprint combined with chemical pattern recognition method can be used in the quality control and evaluation of A. dahurica.

Polyelectrolyte-Mediated Nontoxic AgGa xIn1- xS2 QDs/Low-Density Lipoprotein Nanoprobe for Selective 3D Fluorescence Imaging of Cancer Stem Cells.

Cancer stem cells, which are a population of cancer cells sharing common properties with normal stem cells, have strong self-renewal ability and multi-lineage differentiation potential to trigger tumor proliferation, metastases, and recurrence. From this, targeted therapy for cancer stem cells may be one of the most promising strategies for comprehensive treatment of tumors in the future. We design a facile approach to establish the colon cancer stem cells-selective fluorescent probe based on the low-density lipoprotein (LDL) and the novel AgGa xIn(1- x)S2 quantum dots (AGIS QDs). The AGIS QDs with a high crystallinity are obtained for the first time via cation-exchange protocol of Ga3+ to In3+ starting from parent AgInS2 QDs. Photoluminescence peak of AGIS QDs can be turned from 502 to 719 nm by regulating the reaction conditions, with the highest quantum yield up to 37%. Subsequently, AGIS QDs-conjugated LDL nanocomposites (NCs) are fabricated, in which a cationic polyelectrolyte was used as a coupling reagent to guarantee the electrostatic self-assembly. The structural integrity and physicochemical properties of the LDL-QDs NCs are found to be maintained in vitro, and the NCs exhibit remarkable biocompatibility. The LDL-QDs can be selectively delivered into cancer stem cells that overexpress LDL receptor, and three-dimensional imaging of cancer stem cells is realized. The results of this study not only demonstrate the versatility of nature-derived lipoprotein nanoparticles, but also confirm the feasibility of electrostatic conjugation using cationic polyelectrolyte, allowing reseachers to design nanoarchitectures for targeted diagnosis and treatment of cancer.

Combined SEP and anti-PD-L1 antibody produces a synergistic antitumor effect in B16-F10 melanoma-bearing mice.

The increased PD-L1 induces poorer prognosis in melanoma. The treatment with PD-1/PD-L1 antibodies have a low response rate. The combination immunotherapies are the encouraging drug development strategy to receive maximal therapeutic benefit. In this study, we investigated the enhanced antitumor and immunomodulatory activity of combined SEP and αPD-L1 in B16-F10 melanoma-bearing mice. The results shown that combined SEP and αPD-L1 presented significant synergistic antitumor effects, increased the frequency of CD8+ and CD4+ T cells in spleen and tumor, cytotoxic activity of CTL in spleen, and IL-2 and IFN-γ levels in splenocytes and tumor. The combination treatment also produced synergistic increase in P-ERK1/2 level in spleen. Immunohistochemistry shown that SEP induced the PD-L1 expression in melanoma tissue possibly by promoting IFN-γ excretion, which led to the synergistic anti-tumor effects of aPD-L1 and SEP. Furthermore, in the purified T lymphocyte from the naive mice, the combination of SEP and αPD-L1 had more potent than SEP or αPD-L1 in promoting T lymphocyte proliferation and cytokines secretion including IL-2 and IFN-γ, at least partially by activating MEK/ERK pathway. Our study provides the scientific basis for a clinical trial that would involve combination of anti-PD-L1 mAb and SEP for sustained melanoma control.

The CCR2 3'UTR functions as a competing endogenous RNA to inhibit breast cancer metastasis.

Diverse RNA transcripts acting as competing endogenous RNAs (ceRNAs) can co-regulate each other's expression by competing for shared microRNAs. CCR2 protein, the receptor for CCL2, is implicated in cancer progression. However, we found that a higher CCR2 mRNA level is remarkably associated with prolonged survival of breast cancer patients. These conflicting results prompted us to study the non-coding function of CCR2 mRNA. We found that the CCR2 3' untranslated region (UTR) inhibited MDA-MB-231 and MCF-7 cell metastasis by repressing epithelial-mesenchymal transition (EMT) in vitro, and suppressed breast cancer metastasis in vivo Mechanistically, the CCR2 3'UTR modulated the expression of the RhoGAP protein STARD13 via acting as a STARD13 ceRNA in a microRNA-dependent and protein coding-independent manner. The CCR2 3'UTR blocked the activation of RhoA-ROCK1 pathway, which is the downstream effector of STARD13, and thus decreased the phosphorylation level of myosin light chain 2 (MLC2) and formation of F-actin. Additionally, the function of the CCR2 3'UTR was dependent on STARD13 expression. In conclusion, our results confirmed that the CCR2 3'UTR acts as a metastasis suppressor by acting as a ceRNA for STARD13 and thus inhibiting RhoA-ROCK1-MLC-F-actin pathway in breast cancer cells.This article has an associated First Person interview with the first author of the paper.

MicroRNA-125b inhibits AML cells differentiation by directly targeting Fes.

MicroRNA-125b (miR-125b) has been reported to be upregulated in several kinds of leukemia, suggesting that miR-125b plays a role in Leukemia development. In this study, it was shown that miR-125b expression level decreased in response to 1α, 25-dihydroxy-vitamin D3 (1,25D3) in a dose- and time-dependent manner and miR-125b blocked 1,25D3-induced monocytic differentiation of U937 cells. In addition, miR-125b decreased mRNA expression of myelomonocytic differentiation markers, including CD11c, CD18 and CD64 and arrested the cell cycle at the S phase in U937 and HL60 cells. Fes was identified as a novel direct target of miR-125b and miR-125b could also reduce the expression levels of PU.1 and macrophage colony-stimulating factor receptor (MCSFR). Furthermore, Fes was found to be involved in monocytic differentiation via upregulation of PU.1 and MCSFR and Fes siRNA could also inhibit 1,25D3-induced monocytic differentiation of U937 and HL60 cells and decrease mRNA expression of CD11c, CD18 and CD64. Importantly, the inhibition of Fes siRNA on 1,25D3-induced monocytic differentiation could be rescued by transfection with miR-125b inhibitor. Our data highlights an important role of miR-125b in AML progression, implying the potential application of miR-125b in AML therapy.

STARD13-correlated ceRNA network inhibits EMT and metastasis of breast cancer.

Competing endogenous RNAs (ceRNAs) network has been correlated with the initiation and development of cancer. Here, we identify CDH5, HOXD1, and HOXD10 as putative STARD13 ceRNAs and they display concordant patterns with STARD13 in different metastatic potential breast cancer cell lines and tissues. Notably, 3'UTRs of these genes suppress breast cancer metastasis via inhibiting epithelial-mesenchymal transition (EMT) in vitro and in vivo, which are activated through the crosstalk between STARD13 and its ceRNAs in 3'UTR- and miRNA-dependent manners. In addition, Kaplan-Meier survival analysis reveals that mRNA level of STARD13 and its ceRNAs is remarkably associated with survival of breast cancer patients. These results suggest that 3'UTRs of CDH5, HOXD1, and HOXD10 inhibit breast cancer metastasis via serving as STARD13 ceRNAs.

Competing endogenous RNA networks of CYP4Z1 and pseudogene CYP4Z2P confer tamoxifen resistance in breast cancer.

Patients with estrogen receptor α (ERα)-positive breast cancer can be treated with endocrine therapy using anti-estrogens such as tamoxifen; nonetheless, patients often develop resistance limiting the success of breast cancer treatment. The potential mechanisms remain elusive. In detail, many miRNAs have been associated with breast cancer tamoxifen resistance, but no studies have addressed the role of miRNA-mediated competitive endogenous RNAs network (ceRNET) in tamoxifen resistance. The ceRNET between CYP4Z1 and pseudogene CYP4Z2P has been revealed to promote breast cancer angiogenesis. However, its function in tamoxifen resistance remains unclear. Here we report CYP4Z1 and CYP4Z2P were downregulated in MCF-7 cells compared with tamoxifen-resistant MCF-7-TamR cells. Enforced upregulation of CYP4Z1- or CYP4Z2P-3'UTR level renders MCF-7 Cells resistant to tamoxifen. We find that overexpression of CYP4Z1- or CYP4Z2P-3'UTR enhances the transcriptional activity of ERα through the activation of ERα phosphorylation. Furthermore, we find that CYP4Z1- and CYP4Z2P-3'UTRs increase ERα activity dependent on cyclin-dependent kinase 3 (CDK3). Reporter gene and western blot assays revealed that CYP4Z1- and CYP4Z2P-3'UTRs act as CDK3 ceRNAs. More importantly, the blocking of CYP4Z1- and CYP4Z2P-3'UTRs reversed tamoxifen resistance in MCF-7-TamR cells. Our data demonstrates that the ceRNET between CYP4Z1 and pseudogene CYP4Z2P acts as a sub-ceRNET to promote CDK3 expression in ER-positive breast cancer and is a potential therapeutic target for treatment of tamoxifen-resistant breast cancer.

MALAT1 induced migration and invasion of human breast cancer cells by competitively binding miR-1 with cdc42.

Competitive endogenous messenger RNAs (ceRNAs) affect other RNAs transcription through competitively binding common microRNAs (miRNAs). In this study we identified long non-coding RNA (lncRNA) MALAT1 can function as a ceRNA of cell division cycle 42 (cdc42) 3'UTR in inducing migration and invasion of breast cancer cells via miR-1. We found that miR-1 bound both MALAT1 and cdc42 3'UTR directly. Further study showed that MALAT1 induced migration and invasion of breast cancer cells while reduced the level of cdc42. Our results suggest that MALAT1 regulated migration and invasion of breast cancer cells via affecting cdc42 through binding miR-1 competitively.

MiR-424 regulates monocytic differentiation of human leukemia U937 cells by directly targeting CDX2.

MiR-424 plays an important role via promoting the monocytic differentiation in many human leukemia cell lines. Here, we report that miR-424 decreased miR-125b expression to 36 % by directly targeting caudal type homeobox 2. However, miR-424 also decreased expression of Fes, PU.1 and colony-stimulating factor receptor (MCSFR). As Fes, PU.1 and MCSFR were down-regulated by over-expression of miR-125b (unpublished work), a similar effect of miR-424 and Fes siRNA on CD64, Egr-1, Egr-2 and CEBPA indicates that Fes may be an important downstream target of miR-424. We hypothesize that miR-424 promotes monocytic differentiation by regulating other critical factors and miR-424 has high affinity for these factors. For the first time, the molecular mechanism of miR-424 during monocytic differentiation of U937 cells has been elucidated in this study.

[Effect of magnesium isoglycyrrhizinate on allergy contact dermatitis (ACD) in mice].

OBJECTIVE: To investigate the effect of magnesium isoglycyrrhizinate (MgIG) on allergy contact dermatitis (ACD) in mice. METHOD: The model of ACD was sensitized and challenged by 1% dinitrofluorobenzene(DNFB).48 SPF grade mice were divided into 6 groups randomly: a control group, a model group, three dosage groups and a positive group. The drug was injected through vena caudalis. The change of ear's swelling and the scores of ear's thickness and erythema of each mouse was observed. The level of INF-gamma, IgE, IL-4 in serum was detected by ELISA method. Then the pathologic change of mice ears was using HE staining examined under light microscope. RESULT: MgIG could decrease (P < 0.05) the ear's swelling, the scores of ear's thickness and erythema, and INF-gamma and IgE level in mice serum. It was observed that MgIG could significantly alleviate the infiltrate of inflam cell and the hemangiectasis in ear tissue. CONCLUSION: Certain concentration of MgIG has significant therapeutic effect on ACD in mice. Therapeutic mechanism of MgIG may be relevant with the suppression of INF-gamma and IgE.