Systematic optimization and evaluation of culture conditions for the construction of circulating tumor cell clusters using breast cancer cell lines.

BACKGROUND: Circulating tumor cell (CTC) clusters play a critical role in carcinoma metastasis. However, the rarity of CTC clusters and the limitations of capture techniques have retarded the research progress. In vitro CTC clusters model can help to further understand the biological properties of CTC clusters and their clinical significance. Therefore, it is necessary to establish reliable in vitro methodological models to form CTC clusters whose biological characteristics are very similar to clinical CTC clusters. METHODS: The assays of immunofluorescence, transmission electron microscopy, EdU incorporation, cell adhension and microfluidic chips were used. The experimental metastasis model in mice was used. RESULTS: We systematically optimized the culture methods to form in vitro CTC clusters model, and more importantly, evaluated it with reference to the biological capabilities of reported clinical CTC clusters. In vitro CTC clusters exhibited a high degree of similarity to the reported pathological characteristics of CTC clusters isolated from patients at different stages of tumor metastasis, including the appearance morphology, size, adhesive and tight junctions-associated proteins, and other indicators of CTC clusters. Furthermore, in vivo experiments also demonstrated that the CTC clusters had an enhanced ability to grow and metastasize compared to single CTC. CONCLUSIONS: The study provides a reliable model to help to obtain comparatively stable and qualified CTC clusters in vitro, propelling the studies on tumor metastasis.

Rosmarinic acid in combination with ginsenoside Rg1 suppresses colon cancer metastasis via co-inhition of COX-2 and PD1/PD-L1 signaling axis.

Metastasis of colorectal cancer (CRC) is a leading cause of mortality among CRC patients. Elevated COX-2 and PD-L1 expression in colon cancer tissue has been linked to distant metastasis of tumor cells. Although COX-2 inhibitors and immune checkpoint inhibitors demonstrate improved anti-tumor efficacy, their toxicity and variable therapeutic effects in individual patients raise concerns. To address this challenge, it is vital to identify traditional Chinese medicine components that modulate COX-2 and PD-1/PD-L1: rosmarinic acid (RA) exerts striking inhibitory effect on COX-2, while ginsenoside Rg1 (GR) possesses the potential to suppress the binding of PD-1/PD-L1. In this study we investigated whether the combination of RA and GR could exert anti-metastatic effects against CRC. MC38 tumor xenograft mouse model with lung metastasis was established. The mice were administered RA (100 mg·kg-1·d-1, i.g.) alone or in combination with GR (100 mg·kg-1·d-1, i.p.). We showed that RA (50, 100, 150 µM) or a COX-2 inhibitor Celecoxib (1, 3, 9 µM) concentration-dependently inhibited the migration and invasion of MC38 cells in vitro. We further demonstrated that RA and Celecoxib inhibited the metastasis of MC38 tumors in vitro and in vivo via interfering with the COX-2-MYO10 signaling axis and inhibiting the generation of filopodia. In the MC38 tumor xenograft mice, RA administration significantly decreased the number of metastatic foci in the lungs detected by Micro CT scanning; RA in combination with GR that had inhibitory effect on the binding of PD-1 and PD-L1 further suppressed the lung metastasis of colon cancer. Compared to COX-2 inhibitors and immune checkpoint inhibitors, RA and GR displayed better safety profiles without disrupting the tissue structures of the liver, stomach and colon, offering insights into the lower toxic effects of clinical traditional Chinese medicine against tumors while retaining its efficacy.

Capsaicin orchestrates metastasis in gastric cancer via modulating expression of TRPV1 channels and driving gut microbiota disorder.

The association between capsaicin, the major natural pungent compound of chili peppers, and gastric cancer progression has engendered conflicting findings. In this work, we sought to explore the character of a high capsaicin diet in gastric cancer metastasis and its possible mechanism. The impact of high capsaicin consumption on gastric cancer metastasis was investigated in vivo (xenograft mouse and zebrafish models) and in vitro (biochemical and molecular assays). It was demonstrated that high diet of capsaicin gave rise to accelerate tumor metastasis, which was partially mediated by elevating the expression of transient receptor potential vanilloid 1 (TRPV1) in gastric cancer cells. Importantly, we found that genetic depletion of TRPV1 could reduce gastric cancer metastasis by diminishing the motility of tumor cells in vitro, but acted poorly in xenograft mouse model. Considering the distribution of capsaicin in vivo, 16S rRNA sequencing and fecal microbiota transplantation (FMT) were used to appraise whether the gut microbiota involved in the high capsaicin diet induced metastasis. It was demonstrated that the level of Firmicutes and Clostridiales was expressively boosted following the high consumption of capsaicin. This microbial shift contributed to the increased peripheral 5-hydroxytryptamine (5-HT) levels, yielding the aggravated metastatic burden. Collectively, our findings highlighted the potential risk of high capsaicin diet in promoting gastric cancer metastasis by virtue of modulating TRPV1 expression and gut microbiota composition, indicating the importance of controlled consumption of chili peppers for patients with gastric cancer. Video Abstract.

Capsaicin shapes gut microbiota and pre-metastatic niche to facilitate cancer metastasis to liver.

Dietary factors are fundamental in tumorigenesis throughout our lifetime. A spicy diet has been ambiguous on the development of cancers, especially in the study of colon cancer metastasis. Here, we utilized a mouse metastasis model to test the potential role of capsaicin in influencing metastasis. Long-term continuous administration of capsaicin diet (300 mg/kg) to mice promotes the formation of liver pre-metastatic niche to facilitate the metastasis of colon cancer cells. Bacteria translocation to liver is clearly observed. Capsaicin increases intestinal barrier permeability and disrupts gut vascular barrier by altering the composition of gut microbiota. Capsaicin not only changes the abundance of mucin-related bacteria like Akkermanisa and Muribaculaceae, but also bacteria involved in bile acids metabolism. Dysregulated bile acids profile is related to the recruitment of natural killer T (NKT) cells in pre-metastatic niche, primary bile acid α-Muricholic acid can enhance the recruitment of NKT cells, while secondary bile acids Glycoursodeoxycholic acid and Taurohyodeoxycholic acid impair the recruitment of NKT cells. These findings reveal long term consumption of capsaicin increases the risk of cancer metastasis through modulating the gut microbiota. Capsaicin (300 mg/kg) disrupts gut barrier and promotes the translocation of bacteria to liver, while altered bile acids metabolism affects the recruitment of NKT cells in liver, forming a pre-metastatic niche and promoting cancer metastasis.

Pharmacological manipulation of Ezh2 with salvianolic acid B results in tumor vascular normalization and synergizes with cisplatin and T cell-mediated immunotherapy.

Tumor vasculature is characterized by aberrant structure and function, resulting in immune suppressive profiles of tumor microenvironment (TME) through limiting immune cell infiltration into tumors. The defective vascular perfusion in tumors also impairs the delivery and efficacy of chemotherapeutic agents. Targeting abnormal tumor blood vessels has emerged as an effective therapeutic strategy to improve the outcome of chemotherapy and immunotherapy. In this study, we demonstrated that Salvianolic acid B (SalB), one of the major ingredients of Salvia miltiorriza elicited vascular normalization in the mouse models of breast cancer, contributing to improved delivery and response of chemotherapeutic agent cisplatin as well as attenuated metastasis. Moreover, SalB in combination with anti-PD-L1 blockade retarded tumor growth, which was mainly due to elevated infiltration of immune effector cells and boosted delivery of anti-PD-L1 into tumors. Mechanistically, tumor cell enhancer of zeste homolog 2 (Ezh2)-driven cytokines disrupted the endothelial junctions with diminished VE-cadherin expression, which could be rescued in the presence of SalB. The restored vascular integrity by SalB via modulating the interactions between tumor cells and endothelial cells (ECs) offered a principal route for achieving vascular normalization. Taken together, our data elucidated that SalB enhanced sensitivity of tumor cells to chemotherapy and immunotherapy through triggering tumor vascular normalization, providing a potential therapeutic strategy of combining SalB and chemotherapy or immunotherapy for patients with breast cancer.

Crosstalk between traditional Chinese medicine-derived polysaccharides and the gut microbiota: A new perspective to understand traditional Chinese medicine.

Polysaccharide is a kind of macromolecule polymer composed of monosaccharides connected by glycosidic bonds. Traditional Chinese medicine (TCM), composed of various bioactive ingredients, is usually rich in polysaccharides. In recent years, extensive research on TCM polysaccharides has demonstrated their pharmacological effects. Polysaccharides can hardly be catabolized by enzymes encoded by the human genome but can be degraded to absorbable metabolites by bacteria inhabiting the colon. Hence, the gut microbiota plays a vital role in degrading TCM polysaccharides into short-chain fatty acids (SCFAs) which exert physiological functions locally and systemically. Besides, TCM polysaccharides can also modulate the composition and activities of the gut microbiota by promoting the growth of beneficial bacteria and inhibiting the colonization of pathogenic bacteria, ultimately restoring gut homeostasis and improving human health. In this review, we discuss the extraction and pharmacological effects of TCM polysaccharides, various functions of the gut microbiota, and the interactions between TCM polysaccharides and the gut microbiota, illuminating the mechanisms of TCM polysaccharides modulating host physiology via the gut microbiota. To firmly establish the clinical efficacy of TCM polysaccharides, further high-quality studies especially clinical trials are needed. Generally, discussion on the interplay between TCM polysaccharides and the gut microbiota is expected to elucidate their application prospects and inspire new thoughts in the development of TCM.

TRPV4 Promotes Metastasis in Melanoma by Regulating Cell Motility through Cytoskeletal Rearrangement.

The abnormal expression of Transient Receptor Potential cation channel subfamily V member 4 (TRPV4) is closely related to the progression of multiple tumors. In addition, TRPV4 is increasingly being considered a potential target for cancer therapy, especially in tumor metastasis prevention. However, the biological correlation between TRPV4 and tumor metastasis, as well as the specific role of TRPV4 in malignant melanoma metastasis, is poorly understood. In this study, we aimed to examine the role of TRPV4 in melanoma metastasis through experiments and clinical data analysis, and the underlying anticancer mechanism of Baicalin, a natural compound, and its inhibitory effect on TRPV4 with in vivo and in vitro experiments. Our findings suggested that TRPV4 promotes metastasis in melanoma by regulating cell motility via rearranging the cytoskeletal, and Baicalin can inhibit cancer metastasis, whose mechanisms reverse the recruitment of activated cofilin to leading-edge protrusion and the increasing phosphorylation level of cortactin, which is provoked by TRPV4 activation.

Thermo-Transient Receptor Potential Channels: Therapeutic Potential in Gastric Cancer.

Over the last decade, researchers have found abnormal expression of transient receptor potential (TRP) channels. In particular, members of the thermally sensitive subclass (thermo-TRPs) are involved in many disease processes. Moreover, they have a vital role in the occurrence and development of gastric cancer (GC). Accordingly, thermo-TRPs constitute a major pharmacological target, and the elucidation of the mechanisms underlying their response to physiological stimuli or drugs is key for notable advances in GC treatment. Therefore, this paper summarizes the existing literature about thermo-TRP protein expression changes that are linked to the incidence and progression of GC. The review also discusses the implication of such association to pathology and cell physiology and identifies potential thermo-TRP protein targets for diagnosis and treatment of GC.

Peptide Derived from AHNAK Inhibits Cell Migration and Proliferation in Hirschsprung's Disease by Targeting the ERK1/2 Pathway.

Hirschsprung's disease (HSCR) is characterized by the lack of ganglion cells in the distal part of the digestive tract. It occurs due to migration disorders of enteric neural crest cells (ENCCs) from 5 to 12 weeks of embryonic development. More and more studies show that HSCR is a result of the interaction of multiple genes and the microenvironments, but its specific pathogenesis has not been fully elucidated. Studies have confirmed that many substances in the intestinal microenvironment, such as laminin and ß1-integrin, play a vital regulatory role in cell growth and disease progression. In addition to these high-molecular-weight proteins, research on endogenous polypeptides derived from these proteins has been increasing in recent years. However, it is unclear whether these endogenous peptides have effects on the migration of ENCCs and thus participate in the occurrence of HSCR. Previously, our research group found that compared with the normal intestinal tissue, the expression of AHNAK protein in the stenosed intestinal tissue of HSCR patients was significantly upregulated, and overexpression of AHNAK could inhibit cell migration and proliferation. In this study, endogenous peptides were extracted from the normal control intestinal tissue and the stenosed HSCR intestinal tissue. The endogenous polypeptide expression profile was analyzed by liquid chromatography-mass spectrometry, and multiple peptides derived from AHNAK protein were found. We selected one of them, "EGPEVDVNLPK", for research. Because there is no uniform naming system, this peptide is temporarily named PDAHNAK (peptide derived from AHNAK). This project aims to clarify the potential role of PDAHNAK in the development of HSCR and to further understand its relationship with its precursor protein AHNAK and how they contribute to the development of HSCR.

Relieving Sore Throat Formula Exerts a Therapeutic Effect on Pharyngitis through Immunoregulation and NF-κB Pathway.

Relieving Sore Throat Formula (RSTF) is a formula approved by the China Food and Drug Administration and has been used for the treatment of pharyngitis in clinic for many years. However, the potential pharmacological mechanism still remains unknown. We combined multiple methods including bioinformatics data digging, network pharmacology analysis, and pathway analysis to predict the potential target of RSTF. We verified our in silico prediction results with an in vivo/vitro antibacterial effect test, mouse phagocytic index test, proliferation, transformation, and migration of mouse spleen lymphocytes. Alteration of NF-κB pathway was determined by Western blotting, immunofluorescence, and PCR. The in vivo experiments demonstrated that the RSTF could significantly relieve the symptoms of pharyngitis. A rat saliva secretion test showed that RSTF can effectively relieve the xerostomia symptom. A phenol red excretion test showed that RSTF has an eliminating phlegm effect. A hot plate method and granuloma experiment proved that RSTF also have analgesic and anti-inflammatory effects. In silico prediction demonstrates that 70 active compounds of RSTF were filtered out through ADME screening and 84 putative targets correlated with different diseases. Pathway enrichment analysis showed that the candidate targets were mostly related to the response to bacteria and immunity signalling pathways, which are known contributors to pharyngitis. Experimental results confirmed that RSTF exerted therapeutic effects on pharyngitis mainly by antibacterial effect and downregulation of NF-κB activities. It is demonstrated both in silico and in vivo/vitro that RSTF exerted therapeutic effects on pharyngitis mainly through an antibiotic effect and downregulation of NF-κB signalling pathway.

Beta-elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation.

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta-elemene (ß-elemene), an approved drug for complementary cancer therapy, exerted distinct anti-metastatic activity dependent on PKM2. The results indicated that ß-elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. ß-Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that ß-elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of ß-elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose-1,6-bisphosphate (FBP) and L-cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and ß-elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti-metastatic agent from natural compounds.

Zhile Capsule Exerts Antidepressant-Like Effects through Upregulation of the BDNF Signaling Pathway and Neuroprotection.

Major depressive disorder is now becoming a common disease in daily life, and most patients do not have satisfactory treatment outcomes. We herein evaluated the therapeutic effects of Zhile capsule and clarified the molecular mechanism. A rat model of chronic unpredictable mild stress-induced depression was established to assess the antidepressant-like effects of Zhile by using the sucrose preference test, open field test, forced swim test, tail suspension test and HPLC. Systems pharmacology was then performed to unravel the underlying mechanism which was confirmed by western blot, enzyme-linked immunosorbent assay, and qPCR. Zhile alleviated depression-like behaviors by upregulating the cAMP-CREB-BDNF (brain-derived neurotrophic factor) axis to exert neuroprotective effects. It may be beneficial to depressive patients in clinical practice.

Targeting Thioredoxin System with an Organosulfur Compound, Diallyl Trisulfide (DATS), Attenuates Progression and Metastasis of Triple-Negative Breast Cancer (TNBC).

BACKGROUND/AIMS: Metastasis is the leading cause resulting in high mortality in triple negative breast cancer (TNBC) patients. Cancer cells are skilled at utilizing thioredoxin (Trx) system as an efficient antioxidant system to counteract oxidative damage, facilitating the occurrence of metastasis. Here, we identified an organosulfur compound named DATS isolated from garlic, that inhibits the expression of Trx-1 and the enzyme activity of Trx reductase in breast cancer cells. METHODS: Tissue microarray of breast cancer patients and immunohistochemical method were used to analyze the role of Trx-1 in breast cancer metastasis. Spotaneous metastasis model and experimental metastasis model combined with HE staining, immunohistochemistry were used to verify in vivo anti-metastatic effect of DATS as well as its regulation on thioredoxin. Western blot, immunofluorescence, redox state assessment and detection of enzyme activity were employed to determine the effect of DATS on thioredoxin system. Trx-1 siRNA interference was used to investigate the conclusive evidence that Trx-1 was the target of DATS. RESULTS: In agreement with reduced Trx-1 nuclear translocation from cytoplasm by DATS, the production of reduced form of Trx-1 was dramatically decreased. Furthermore, in vivo, DATS administration was observed to significantly suppress spontaneous and experimental metastasis in nude mice. Delivery of DATS also resulted in decreased expression of Trx-1 as the direct target, as well as expression of NF-κB and MMP2/9 in primary tumor and lung tissue. Notably, the effects of DATS on the expression of downstream metastasis-associated genes were mediated by Trx-1, as demonstrated by the combination use of DATS and Trx-1 siRNA. CONCLUSION: Collectively, this present study indicates that targeting Trx system with DATS may provide a promising strategy for treating metastasis of TNBC.

Lactate dehydrogenase is associated with 28-day mortality in patients with sepsis: a retrospective observational study.

BACKGROUND: Sepsis is a major health care problem, which affects millions of people around the world. Glucose metabolic reprogramming of immune cells plays a crucial role during advancement of sepsis. However, the association between glucose metabolic reprogramming and mortality in patients with sepsis is unclear. Lactate dehydrogenase (LDH) catalyzes the last step of glycolysis. Investigating the relationship between LDH and mortality is important to understand the effect of metabolic reprogramming on prognosis of patients with sepsis. METHODS: A total of 192 patients with sepsis were included in our study. Data on characteristics of patients, biochemical variables, and inflammatory mediator were collected. Association between the level of serum LDH and 28-day mortality was also analyzed. The correlations between serum LDH, interleukin-1ß, creatinine, PaO2/FiO2, and lactate were also observed. The association between LDH and the risk of death was further analyzed. Moreover, receiver operating characteristic curve was depicted to compare the accuracy in prediction of LDH and other variables. RESULTS: There were statistic difference in 28-day mortality between elevated LDH group and normal LDH group (P = 0.021). Level of serum LDH was an independent risk factor for death of patients with sepsis (hazard ratio 1.005, 95% confidence interval 1.002-1.007, P = 0.001). There were significant correlations between LDH, interleukin-1ß (r = 0.514, P = 0.000), creatinine (r = 0.368, P = 0.000), PaO2/FiO2 (r = -0.304, P = 0.000), and lactate (r = 0.560, P = 0.000). The receiver operating characteristic curves showed that the area under the LDH curve for prediction for mortality was 0.783. CONCLUSIONS: Serum LDH is probably associated with 28-day mortality in patients with sepsis.

Cryptotanshinone inhibition of mammalian target of rapamycin pathway is dependent on oestrogen receptor alpha in breast cancer.

Cryptotanshinone (CPT) has been demonstrated to inhibit proliferation and mammalian target of rapamycin (mTOR) pathway in MCF-7 breast cancer cells. However, the same results are unable to be repeated in MDA-MB-231 cells. Given the main difference of oestrogen receptor α (ERα) between two types of breast cancer cells, It is possibly suggested that CPT inhibits mTOR pathway dependent on ERα in breast cancer. CPT could significantly inhibit cell proliferation of ERα-positive cancer cells, whereas ERα-negative cancer cells are insensitive to CPT. The molecular docking results indicated that CPT has a high affinity with ERα, and the oestrogen receptor element luciferase reporter verified CPT distinct anti-oestrogen effect. Furthermore, CPT inhibits mTOR signalling in MCF-7 cells, but not in MDA-MB-231 cells, which is independent on binding to the FKBP12 and disrupting the mTOR complex. Meanwhile, increased expression of phosphorylation AKT and insulin receptor substrate (IRS1) induced by insulin-like growth factor 1 (IGF-1) was antagonized by CPT, but other molecules of IGF-1/AKT/mTOR signalling pathway such as phosphatase and tensin homolog (PTEN) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) were negatively affected. Finally, the MCF-7 cells transfected with shERα for silencing ERα show resistant to CPT, and p-AKT, phosphorylation of p70 S6 kinase 1 (p-S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) were partially recovered, suggesting ERα is required for CPT inhibition of mTOR signalling. Overall, CPT inhibition of mTOR is dependent on ERα in breast cancer and should be a potential anti-oestrogen agent and a natural adjuvant for application in endocrine resistance therapy.

Diallyl trisulfides, a natural histone deacetylase inhibitor, attenuate HIF-1α synthesis, and decreases breast cancer metastasis.

Intratumoral hypoxia promotes the distant metastasis of cancer subclones. The clinical expression level of hypoxia-inducible factor-1α (HIF-1α) reflects the prognosis of a variety of cancers, especially breast cancer. Histone deacetylase (HDAC) inhibitors can target HIF-1α protein due to von Hippel-Lindau (VHL) protein-dependent degradation. Dietary organosulfur compounds, such as those in garlic, have been reported as HDAC inhibitors. The effects of diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS) on the ratio of firefly/Renilla luciferase activity in hypoxic MDA-MB-231 cells were determined. The mRNA expressions of HIF-1α target genes ANGPTL4, LOXL4, and LOX in hypoxic MDA-MB-231 cells were significantly down-regulated by DATS. DATS attenuated the metastatic potential of MDA-MB-231 cells in hypoxia-induced embryonic zebrafish, xenograft, and orthotopic tumors. Endothelial cell-cancer cell adhesion, wound healing, transwell, and tube formation assays showed that DATS dose-dependently inhibited the migration and angiogenesis of MDA-MB-231 cells in vitro. The expressions of L1CAM, VEGF-A, and EMT-related proteins (Slug, Snail, MMP-2) were inhibited by DATS. DATS dose-dependently inhibited HIF-1α transcriptional activity and hypoxia-induced hematogenous metastasis of MDA-MB-231 cells. It reduced the protein expression of HIF-1α, which did not involve inhibition of HIF-1α mRNA expression or ubiquitin proteasome degradation. Efficient inhibition of HIF-1α expression was required for DATS to resist breast cancer.

Xanthatin triggers Chk1-mediated DNA damage response and destabilizes Cdc25C via lysosomal degradation in lung cancer cells.

Previous studies had shown that xanthatin, a natural xanthanolide sesquiterpene lactone, could induce mitotic arrest and apoptosis in non-small cell lung cancer (NSCLC) cells. Here, we examined whether the DNA damage response (DDR) could be a primary cytotoxic event underlying xanthatin-mediated anti-tumor activity. Using EdU incorporation assay in combination with novel imaging flow cytometry, our data indicated that xanthatin suppressed DNA replication, prevented cells from G2/M entry and increased the spot count of γH2AX nuclear foci. Given that checkpoint kinase 1 (Chk1) represents a core component in DDR-mediated cell cycle transition and the phosphorylation on Ser-345 is essential for kinase activation and function, we surprisingly found xanthatin distinctly modulated Ser-345 phosphorylation of Chk1 in A549 and H1299 cells. Further investigation on Cdc25C/CDK1/CyclinB1 signaling cascade in the absence or presence of pharmacological DDR inhibitors showed that xanthatin directly destabilized the protein levels of Cdc25C, and recovery of p53 expression in p53-deficient H1299 cells further intensified xanthatin-mediated inhibition of Cdc25C, suggesting p53-dependent regulation of Cdc25C in a DDR machinery. Moreover, exogenous expression of Cdc25C was also substantially repressed by xanthatin and partially impaired xanthatin-induced G2 arrest. In addition, xanthatin could induce accumulation of ubiquitinated Cdc25C without undergoing further proteasomal degradation. However, an alternative lysosomal proteolysis of Cdc25C was observed. Interestingly, lysosome-like vesicles were produced upon xanthatin treatment, accompanied by rapid accumulation of lysosomal associated membrane protein LAPM-1. Furthermore, vacuolar proton (V)-ATPases inhibitor bafilomycin A1 and lysosomal proteases inhibitor leupeptin could remarkably overturn the levels of Cdc25C in xanthatin-treated H1299 cells. Altogether, these data provide insight into how xanthatin can be effectively targeted DDR molecules towards certain tumors.

Cryptotanshinone, a novel tumor angiogenesis inhibitor, destabilizes tumor necrosis factor-α mRNA via decreasing nuclear-cytoplasmic translocation of RNA-binding protein HuR.

Cryptotanshinone (CT), one major lipophilic component isolated from Salvia miltiorrhiza Bunge, has shown to possess chemopreventive properties against various types of cancer cells. In this study, CT was shown to be a potent anti-angiogenic agent in zebrafish, and mouse models and could limit tumor growth by inhibiting tumor angiogenesis. We further found that CT could inhibit the proliferation, migration, angiogenic sprouting, and tube formation of HUVECs. In addition, we demonstrated that CT could lower the level of TNF-α due to the destabilization of TNF-α mRNA, which associated with regulating 3'-untranslated region (3'-UTR) of TNF-α and preventing the translocation of RNA binding protein, HuR, from the nucleus to the cytoplasm. Moreover, the underlying mechanism responsible for the regulation in angiogenesis by CT was partially related to the suppression of NF-κB, and STAT3 activity. Based on the abilities of CT in targeting tumor cells, inhibiting angiogenesis, and destroying tumor vasculature, CT is worthy of further investigation for preventive, and therapeutic purposes in cancer. © 2015 Wiley Periodicals, Inc.

The angiogenic responses induced by release of angiogenic proteins from tumor cell-activated platelets are regulated by distinct molecular pathways.

There is mounting evidence that tumor angiogenesis can be regulated by platelets (Plts), which serve as major sources and delivery vehicles of many proangiogenic cytokines including transforming growth factor-ß and vascular endothelial growth factor. Although considerable progress has been made in understanding the role for Plt secretion in tumor angiogenesis, very little is known about the precise mechanisms underlying cancer cell induction of Plt granule release. Here, we demonstrated that nonsmall cell lung cancer (NSCLC) cells directly induced Plt secretion of several angiogenic regulatory cytokines that promoted angiogenesis in concert. Moreover, we discovered that these Plt-derived angiogenesis modulators were regulated by different molecular pathways and could be largely inhibited by combination of multiple signaling inhibitors. Our present studies indicated that manipulation of Plt secretion of angiogenic cytokines without compromising hemostatic functions could provide a novel option for management of tumor angiogenesis and metastasis in NSCLC patients with thrombocytosis.

Synergistic potentiation of the anti-metastatic effect of a Ginseng-Salvia miltiorrhiza herbal pair and its biological ingredients via the suppression of CD62E-dependent neutrophil infiltration and NET formation.

INTRODUCTION: The combination of the roots of ginseng and Salvia miltiorrhiza is an effective approach for treating metastatic cancer in patients with Qi stagnation and blood stasis patterns. However, the molecular mechanism underlying the combined use of ginseng and Salvia miltiorrhiza is unknown. OBJECTIVES: This study unveils the pharmacological foundation of ginseng and Salvia miltiorrhiza by examining the involvement of neutrophils in the critical process of tumor hematogenous metastasis. Additionally, by employing a reverse pharmacology research model (effect-target-constituent), potential potent components were screened, and the dominant component formulations were determined. METHODS: An experimental lung metastatic model was constructed to compare the antitumor effects of ginseng and Salvia miltiorrhiza. RNA sequencing was employed to identify pivotal biological events and key targets, while the detection of CD62E expression and neutrophil extracellular traps (NETs) release was used to screen for effective substances in ginseng and Salvia miltiorrhiza. In addition, a comprehensive array of in vitro and in vivo experiments was conducted to explore the underlying mechanisms and therapeutic significance. RESULTS: Compared with single-herb use, the use of ginseng or Salvia miltiorrhiza significantly reduced tumor metastasis, which was accompanied by reduced neutrophil infiltration into the lungs. Cryptotanshinone (CPT), an active constituent of Salvia miltiorrhiza, can inhibit neutrophil adhesion and recruitment to lung tissue by downregulating the expression of E-selectin (CD62E) in endothelial cells. Moreover, the ginseng -derived ginsenoside Rg1 mitigated the formation of NETs in lung tissues and reversed the protumor effects of NETs. We further explored the efficacy of combination therapy with Rg1 and CPT, which also reduced tumor metastasis in vivo. CONCLUSION: Ginseng and Salvia miltiorrhiza exhibited a mutual potentiation of the anti-metastatic effect by suppressing both early and late stages of neutrophil-initiated metastasis cascade. Rg1 and CPT represent the synergistic ingredients from ginseng and Salvia miltiorrhiza, respectively.