Hyaluronan nanogel co-loaded with chloroquine to enhance intracellular cisplatin delivery through lysosomal permeabilization and lysophagy inhibition.

Hyaluronan (HA) has been widely used to construct nanocarriers for cancer-targeted drug delivery, due to its excellent biocompatibility and intrinsic affinity towards CD44 that is overexpressed in most cancer types. However, the HA-based nanocarriers are prone to trapping in lysosomes following the HA-mediated endocytosis, which limited the delivered drug to access its pharmacological action sites and subsequently compromised the therapeutic efficacy. To overcome this intracellular obstacle, here we demonstrated the co-loading of chloroquine (CQ) in HA nanogel could efficiently promote the intracellular delivery of cisplatin. The cisplatin coordination with HA generated the nanogel that could also co-encapsulate CQ (HA/Cis/CQ nanogel). Compared with cisplatin-loaded HA nanogel (HA/Cis), HA/Cis/CQ significantly promoted the lysosomal escape of cisplatin as well as enhanced tumor inhibition in the triple-negative breast cancer model. Mechanism studies suggested that co-delivery of CQ not only induced the lysosomal membrane permeabilization but also inhibited the lysophagy, which collectively contributed to the lysosomal instability and cisplatin escape. This HA/Cis/CQ nanogel elicited less toxicity compared with the combination of free Cis and CQ, thus suggesting a promising HA nanocarrier to boost the cisplatin delivery towards cancer-targeted therapy.

Magnetically-activated, nanostructured cellulose for efficient capture of circulating tumor cells from the blood sample of head and neck cancer patients.

In this report, the relative efficiency of cellulose nanocrystals (CNCs) and nanofibers (CNFs) to capture circulating tumor cells (CTCs) from the blood sample of head and neck cancer (HNC) patients was evaluated. Detection and enumeration of CTCs are critical for monitoring cancer progression. Both types of nanostructured cellulose were chemically modified with Epithelial Cell Adhesion Molecule (EpCAM) antibody and iron oxide nanoparticles. The EpCAM antibody facilitated the engagement of CTCs, promoting entrapment within the cellulose cage structure. Iron oxide nanoparticles, on the other hand, rendered the cages activatable via the use of a magnet for the capture and separation of entrapped CTCs. The efficiency of the network structures is shown in head and neck cancer (HNC) patients' blood samples. It was observed that the degree of chemical functionalization of hydroxyl groups located within the CNCs or CNFs with anti-EpCAM determined the efficiency of the system's interaction with CTCs. Further, our result indicated that inflexible scaffolds of nanocrystals interacted more efficiently with CTCs than that of the fibrous CNF scaffolds. Network structures derived from CNCs demonstrated comparable CTC capturing efficiency to commercial standard, OncoDiscover®. The output of the work will provide the chemical design principles of cellulosic materials intended for constructing affordable platforms for monitoring cancer progression in 'real time'.

Multifunctional nanogel based on carboxymethyl cellulose interfering with cellular redox homeostasis enhances phycocyanobilin photodynamic therapy.

The redox homeostasis defense mechanism of tumor cells is one of the prime reasons for the unsatisfactory effect of photodynamic therapy (PDT). So far, little attention has been paid to this obstacle. In this work, we reported a synthesizing simple yet versatile nanogel (BCPS), synthesized by cystamine dihydrochloride functionalized sodium carboxymethylcellulose (CMC-SS), bovine serum albumin, and Phycocyanobilin self-assembly. The BCPS reduced the levels of glutathione molecules by reacting with glutathione, thereby interfering with intracellular redox homeostasis and enhancing the sensitivity of tumor cells to PDT. The BCPS was shown to possess excellent serum stability, high blood compatibility, low toxic side effects, and higher reactive oxygen species (ROS) utilization. After irradiation, the BCPS could significantly increase intracellular ROS level by approximately 1.6-fold and decrease the IC50 to HeLa cells by approximately 1.5-fold, compared to the pre-functional drugs BCP. This proposed strategy, based on increasing the utilization rate of ROS in tumor cells is promising for application potentials in tumor therapy.

Transcriptomics and molecular docking reveal the potential mechanism of lycorine against pancreatic cancer.

BACKGROUND: Pancreatic cancer is an extremely malignant digestive tumor, however, owing to its high drug resistance of pancreatic cancer, the search for more effective anti-pancreatic cancer drugs is urgently needed. Lycorine, an alkaloid of natural plant origin, exerts antitumor effects on a variety of tumors. PURPOSE: This study aimed to investigate the therapeutic effect of lycorine on pancreatic cancer and elucidate its potential molecular mechanism. METHODS: Two pancreatic cancer cell lines, PANC-1 and BxPC-3, were used to investigate the therapeutic effects of lycorine on pancreatic cancer in vitro using the CCK8 assay, colony formation assay, 5-Ethynyl-2'- deoxyuridine (EdU) incorporation assay, flow cytometry, and western blotting. Transcriptome sequencing and gene set enrichment analysis (GSEA) were used to analyze the differentially expressed genes and pathways after lycorine treatment. Molecular docking, quantitative real-time PCR (qRT-PCR), oil red O staining, small interfering RNA (siRNA) transfection, and other experiments were performed to further validate the differentially expressed genes and pathways. In vivo experiments were conducted to investigate lycorine's inhibitory effects and toxicity on pancreatic cancer using a tumor-bearing mouse model. RESULTS: Lycorine inhibited the proliferation of pancreatic cancer cells, caused G2/M phase cycle arrest and induced apoptosis. Transcriptome sequencing and GSEA showed that lycorine inhibition of pancreatic cancer was associated with fatty acid metabolism, and aldehyde dehydrogenase 3A1 (ALDH3A1) was a significantly enriched target in the fatty acid metabolism process. ALDH3A1 expression was significantly upregulated in pancreatic cancer and was closely associated with prognosis. Molecular docking showed that lycorine binds strongly to ALDH3A1. Further studies revealed that lycorine inhibited the fatty acid oxidation (FAO) process in pancreatic cancer cells and induced cell growth inhibition and apoptosis through ALDH3A1. Lycorine also showed significant suppressive effects in tumor-bearing mice. Importantly, it did not result in significant toxicity to liver and kidney of mice, demonstrating its therapeutic potential as a safe antitumor agent. CONCLUSION: Lycorine inhibited pancreatic cancer cell proliferation, blocked the cell cycle, and induced apoptosis by targeting ALDH3A1. FAO inhibition was identified for the first time as a possible mechanism for the anticancer effects of lycorine. These findings enrich the theory of targeted therapy for pancreatic cancer, expand our understanding of the pharmacological targets of lycorine, and provide a reference for exploring its natural components.

Exosomal microRNA profiling revealed enhanced autophagy suppression and anti-tumor effects of a combination of compound Phyllanthus urinaria and lenvatinib in hepatocellular carcinoma.

BACKGROUND: Compound Phyllanthus urinaria (CP), a traditional Chinese herbal remedy, possesses strong anti-cancer effects and is extensively employed in the clinical management of hepatocellular carcinoma (HCC). While lenvatinib and other oral tyrosine kinase inhibitors have been authorized as initial treatments for advanced unresectable HCC, the survival of patients is ultimately restricted due to the gradual development of drug resistance. Fortunately, the co-administration of CP and lenvatinib holds promise for anti-cancer applications. PURPOSE: Our objective was to understand the molecular-level mechanisms of bioactive phytocompounds in CP, in order to explore the anti-HCC effects of combining CP and lenvatinib treatment and reveal the underlying mechanisms. Furthermore, we discovered new miRNAs associated with autophagy that are common to both HepG2-derived exosomes and HepG2 cells. These miRNAs play a role in the advancement of HCC and were identified through the utilization of CP and lenvatinib. METHODS: To assess the anti-HCC effects of CP in combination with lenvatinib, both an in vitro CCK-8 assay and an in vivo xenograft model assay were performed. TEM, NTA, and nano-flow cytometry were employed for the identification of isolated exosomes. To ascertain the miRNA expression patterns in HepG2 cells and HepG2-derived exosomes, miRNA-sequencing analysis was conducted. Further investigation involved the use of real-time PCR, examination of the fusion protein GFP-mRFP-LC3, TEM analysis, and western blotting. RESULTS: In vitro and in vivo, the combination of CP and lenvatinib showed a stronger and more powerful impact on HCC compared to either CP or lenvatinib alone. The combination of CP and lenvatinib had a significant impact on autophagy-related miRNAs in HepG2-derived exosomes and HepG2 cells, as demonstrated by cellular and exosomal miRNA sequencing. Additional tests indicated that the increased inhibition of autophagy in HepG2 cells subjected to CP treatment, as well as the combination of CP and lenvatinib, was accomplished through the regulation of Beclin-1, LC3-II, and P62 expression. CONCLUSION: In conclusion, our results indicate that the combination of CP and lenvatinib can effectively inhibit HCC by promoting the exosome-mediated suppression of autophagy. This novel therapeutic option is highly efficient and durable, making it a promising treatment for HCC. Moreover, the miRNAs that are differentially expressed and associated with exosome-mediated autophagy, which have been discovered in this study, could potentially be targeted for clinical treatment of HCC.

Matrine induces ferroptosis in cervical cancer through activation of piezo1 channel.

BACKGROUND: Cervical cancer, which is a significant public health concern in women, currently lacks effective therapeutic drugs. Matrine, a constituent of the traditional Chinese herb Sophora flavescentis Radix, is known for its anti-cervical cancer properties and ability to induce programmed cell death. The induction of cancer cell ferroptosis, which is a novel cell death pattern, can become an effective clinical therapy for tumor in the future. However, the effect of matrine on ferroptosis in cervical cancer remains to be elucidated. PURPOSE: In this study, we investigated whether matrine induces ferroptosis in cervical cancer and elucidated the underlying mechanisms. METHODS: We established an SiHa-derived tumor-bearing mouse model using CB17 severe combined immunodeficient (SCID) mice and administered a group of matrine (25, 50, and 75 mg/kg) and cisplatin (2 mg/kg). We meticulously tracked alterations in body weight and tumor size and evaluated liver and kidney health using haematoxylin and eosin (H&E) staining. Using Gene Expression Omnibus (GEO) Dataset (GSE201309), we evaluated the relationship between the effects of matrine on malignant tumor cells and ferroptosis. In vitro, tetrazolium-based colorimetric (MTT), lactate dehydrogenase (LDH) and colony formation assays were used to study the effects of matrine on SiHa cell activity and cytotoxicity. We assessed ferroptosis-related protein abundance using western blotting and ferroptosis-related indices in cells using confocal immunofluorescence microscopy. The interaction of matrine with a protein linked to ferroptosis was studied using cellular thermal shift assay (CETSA). The effects of matrine on Piezo1 expression were investigated using calcium imaging. We also used Piezo1-specific siRNA to explore the role of Piezo1 in ferroptosis. RESULTS: Matrine administration effectively inhibited tumor growth in a SiHa-derived tumor-bearing mouse model without inducing noticeable harm. The analysis results of GEO data set show matrine-induced effects in tumor cells were indeed involved in the process of ferroptosis. Treatment with matrine resulted in a significant reduction in GPX4 protein levels and a concurrent increase in lipid peroxide and Fe2+ content, suggesting matrine-induced modulation of ferroptosis. Matrine promoted SiHa cell death in vitro, as evidenced by the results of MTT and LDH assays. Cell death coincides with increases in intracellular Fe2+, reactive oxygen species (ROS), and lipid peroxides. Our study also revealed significant upregulation of Piezo1 expression through the action of matrine, whereas transferrin receptor (Tfr) and System Xc- (xCT) expression and interaction remained unaffected. We provided further evidence that matrine induces calcium influx through the Piezo1 channel, thereby potentially influencing ferroptosis. Transfection with Piezo1 siRNA reversed the effects of matrine in SiHa cell. CONCLUSIONS: Our findings indicate that matrine exerts a protective effect against cervical cancer by inducing ferroptosis through the activation of Piezo1, but not xCT or Tfr.

FTIR Microspectroscopy as a new probe to study human uterine lesions: Characterization of tumor cell lines from uterine smooth muscle cells and evaluation of EPA and DHA in vitro treatments.

During their life, women are likely to develop uterine diseases, which often compromise their fertile and perimenopausal age. Besides benign lesions like leiomyomas, several malignant neoplasms can occur, such as the uterine leiomyosarcoma, which represents the most frequent malignancy among the rarest uterine cancers. It presents several variants similar to both benign and malignant neoplasms, and sometimes it shares symptoms with the benign counterpart. In this scenario, for a correct diagnosis and a successful prognosis, it is mandatory to detect new reliable markers which strengthen histopathological outcomes and let define a more appropriate and less harmful therapy. Based on this concerning evidence, in the present study, Fourier Transform Infrared Microspectroscopy has been exploited at a cellular level on uterine leiomyoma and leiomyosarcoma cell lines to (1) identify specific spectral biomarkers able to distinguish between benign and malignant lesions, and (2) evaluate the efficacy of eicosapentaenoic and docosahexaenoic acids (respectively EPA and DHA), already successfully tested. Results evidenced reliable differences in the spectral signature of benign and malignant cells, mainly in terms of lipids and nucleic acids composition. Moreover, even if EPA and DHA seemed to exert different effects on the tested cell lines, no cytotoxic and/or anti-apoptotic actions were observed after omega-3 based treatments.

FZKA reverses gefitinib resistance by regulating EZH2/Snail/EGFR signaling pathway in lung adenocarcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzheng Kang-Ai (FZKA) decoction is mainly composed of 12 components with different types of herbs. In the last decade, FZKA has been used as an adjuvant treatment for lung cancer in clinical practice. Our previous studies have confirmed that FZKA shows a strong anti-cancer activity, significantly increases the clinical efficacy of gefitinib and reverses gefitinib resistance in non-small cell lung cancer (NSCLC). However, the molecular mechanism still needs to be further elucidated. AIM OF THE STUDY: The aim of this study was to investigate the role and mechanism by which FZKA inhibited the cell growth, proliferation and invasion of lung adenocarcinoma(LUAD) and reversed the acquired resistance of gefitinib for the therapy in LUAD. MATERIALS AND METHODS: Cell viability assay and EDU assay were used for detecting of cell viability and cell proliferation. Transwell assay was performed to measure cell invasion. Western Blot and qRT-PCR were used for protein and gene expression test. The gene promoter activity was determined by dul-luciferase reporter assay. The in situ expression of protein was measured by cell immunofluorescence. Stabilized cell lines were established for stable overexpression of EZH2. Transient transfection assay was used for gene silence and overexpression. Xenograft tumors and bioluminescent imaging were used for in vivo experiments. RESULTS: FZKA significantly inhibited the cell viability, proliferation and cell invasion of LUAD, the combination of FZKA and gefitinib had a great synergy on the above processes. Moreover, FZKA significantly decreased EZH2 mRNA and protein expression, FZKA reversed the resistance of gefitinib by down-regulation of EZH2 protein. ERK1/2 kinase mediated the down-regulation of EZH2 reduced by FZKA. In addition, FZKA decreased the expression of Snail and EGFR by decreasing EZH2. Overexpression of Snail and EGFR significantly reversed the effect of FZKA-inhibited cell invasion and cell proliferation. More important, the combination of FZKA and gefitinib enhanced the inhibitory effect on EZH2, Snail and EGFR proteins. Furthermore, the growth inhibition and reversal of gefitinib resistance induced by FZKA were further validated in vivo. Finally, the expression and clinical correlation of EZH2,EGFR and Snail in cancer patients were further validated using bioinformatics analysis. CONCLUSIONS: FZKA significantly suppressed tumor progression and reversed gefitinib resistance by regulating the p-ERK1/2-EZH2-Snail/EGFR signaling pathway in LUAD.

Celastrus orbiculatus Thunb. extract targeting DJ-1 inhibits non-small cell lung cancer invasion and metastasis through mitochondrial-induced ROS accumulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Celastrus orbiculatus Thunb. is an ancient traditional Chinese herb with a long history of medicinal use. The ethyl acetate extract of Celastrus orbiculatus Thunb. (COE) has been shown to have anti-tumor effects in various preclinical studies. However, the anti-invasive and metastatic efficacy of COE in non-small cell lung cancer (NSCLC) and the mechanism by which COE regulates cellular oxidation levels are yet to be elucidated. AIM: To study the anti-dissemination effect of COE on NSCLC and to elucidate the molecular mechanism of COE in regulating cellular oxidation levels and its effect on lung cancer invasion and metastasis. METHODS: CCK-8 assay was used to detect the toxic effects of COE on NSCLC. Transwell assay and high-content imaging was used to detect the Motility of NSCLC. Transmission electron microscopy and three-dimensional (3D) imaging of mitochondrial fluorescence were employed to detect the number and structure of mitochondria. JC-1 probe was used to detect the level of mitochondrial membrane potential. Firefly luciferase assay was used to detect the level of total intracellular ATP. MitoSox probe and DCFH-DA probe were applied to detect the level of reactive oxygen species (ROS) inside the mitochondria and the total intracellular ROS, respectively. Immunohistochemistry was used to detect protein expression in xenograft tumors. RESULTS: COE inhibited motility and induced DJ-1 downregulation in NSCLC at low toxic concentrations, and the antiseptic effect of COE was reduced significantly after the overexpression of DJ-1. COE induced structural disruption of mitochondria in NSCLC and accumulation of superoxide compounds, decreased the volume of membrane potential depolarization, and impaired energy production, ultimately leading to a large accumulation of ROS at the cellular level. The antioxidant acetylcysteine (NAC) significantly reversed the antiseptic capacity of COE. In a xenograft tumor model, protein expression of DJ-1, E-cadherin, N-cadherin, and MMP-2 in COE group was significantly changed compared to the model group. CONCLUSION: In the present study, COE inhibited NSCLC invasion and metastasis and was associated with the downregulation of DJ-1 and elevated ROS. COE-mediated downregulation of DJ-1 may be the primary cause of mitochondrial structural and functional dysfunction in NSCLC, eventually leading to ROS accumulation.

Inhibitory effect of Lonicera japonica-derived exosomal miR2911 on human papilloma virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Lonicera japonica Thunb. has been used as a traditional medicinal herb in China for thousands of years for its heat-clearing and detoxification effects. In recent years, experimental and clinical studies have shown that some Lonicera japonica-containing Chinese medicine prescriptions have been used to treat intraepithelia neoplasia caused by human papilloma virus (HPV) infection. However, its bioactive molecules and mechanism of action have not been fully explored. AIM OF THE STUDY: In this study, Lonicera japonica-derived exosomes was extracted and exosomal miR2911 was identified. Bioinformatic analysis indicated that miR2911 potentially binds to the sequence of HPV. The mechanism of miR2911 action on HPV and the effect of exosomal miR2911 on HPV-induced cervical cancer cells were investigated. METHODS: The potential targets of miR2911 on the HPV sequence were predicted and confirmed by using RNAhybrid and dual-luciferase reporter assays. Lonicera japonica exosomes were characterized by transmission electronic microscopy and zeta sizer analysis. RT-qPCR was used to measure miR2911 concentration in various tissues and exosomes. Synthetic miR2911 and GFP-E6/E7 plasmids were transfected into HEK293T cells to examine the effect of miR2911 on E6/E7 gene expression. The effects of miR2911 on endogenous E6/E7 mRNA and protein levels were detected in HPV16/18-positive cervical cancer cells by RT-qPCR and Western blotting. The proliferation and apoptosis of CaSki, SiHa and HeLa cells by the treatment of miR2911 or miR2911-containing exosomes were examined by CCK8, colony formation and flow cytometry assays. RESULTS: MiR2911 is found to be enriched in various Lonicera japonica tissues, and is stably present in Lonicera japonica-derived exosomes. It is observed that MiR2911 directly binds to E6 and E7 oncogenes of HPV16/18, leading to the suppression of their protein expression. In addition, the endogenous E6/E7 mRNA and protein levels were significantly decreased by using miR2911 treatment in HPV16/18-positive cervical cancer cells. Furthermore, both miR2911 and miR2911-containing exosomes inhibited cell proliferation of SiHa, CaSki and HeLa cells, meanwhile inducing the cell apoptosis through E6/E7-p53/Caspase3 axis. CONCLUSION: Our findings indicate that miR2911, an active component present in Lonicera japonica exosomes, inhibits proliferation and induces apoptosis of cervical cancer cells by targeting the E6/E7 genes of HPV16/18. Thus, Lonicera japonica-derived exosomal miR2911 has implications for the development of novel therapeutic strategies for the treatment of HPV-associated cervical cancers.

Identification of lncRNA PCAT19 as potential novel biomarker for colorectal cancer.

Long non-coding RNAs have been implicated in biological processes, and are dysregulated in types of cancer. Studies have shown that PCAT19 and CKMT2-AS1 lncRNAs promote tumor growth, invasion, and metastasis by regulating signaling pathways and modulating the gene expression. This study investigated the expression levels of lncRNAs PCAT19 and CKMT2-AS1 in colorectal tumors and normal tissues. First, Using GEPIA2 database, we compared the expression level of target lncRNAs between primary colon adenocarcinoma tumor and normal tissues. Then, the expression levels of lncRNAs PCAT19 and CKMT2-AS1 were detected in 35 colorectal tumors and paired adjacent tissues using qRT-PCR. A receiver operating characteristic (ROC) curve was used to evaluate the value of these lncRNAs as biomarkers. Statistical analysis based on GEPIA2 showed that both lncRNAs PCAT19 and CKMT2-AS1 were significantly decreased in colon adenocarcinoma compared to the normal group (P < 0.001). Experimental analysis showed that the expression level of lncRNA PCAT19 was decreased in colorectal tumors (p < 0.0001) compared to normal tissues. While the expression level of lncRNA CKMT2-AS1 did not change in tumor tissues, it decreased in non-metastatic tumors compared to normal tissues (p = 0.04). The significantly downregulation of lncRNA PCAT19 expression in both metastatic and non-metastatic colorectal tumors compared to normal tissue suggests that PCAT19 may play a role in the carcinogenesis and progression of colorectal cancer and may provide potential therapeutic targets for colorectal cancer. Based on the results of ROC curve analysis, lncRNA PCAT19 may also serves as a novel potential good biomarker in diagnosis colorectal cancer (AUC = 0.94, p < 0.0001) but no significant was found for lncRNA CKMT2-AS1 (p > 0.05).

Transcription Factor E2F1 Enhances Hepatocellular Carcinoma Cell Proliferation and Stemness by Activating GINS1.

Present studies report that high expression of GINS complex subunit 1 (GINS1) is notably pertinent to poor survival for hepatocellular carcinoma (HCC), but it remains unclear how GINS1 affects the progression of HCC. This study aims at investigating the mechanism by which GINS1 affects HCC cell proliferation and stemness. We performed bioinformatics analysis for determining GINS1 expression in HCC tissues, as well as the HCC patients' survival rate with different expression levels of GINS1. E2F transcription factor 1 (E2F1) was predicted as the upstream transcription factor of GINS1, and the binding relation between the two was verified by chromatin immunoprecipitation and dual-luciferase reporter assays. Quantitative real-time polymerase chain reaction was adopted to evaluate the expression of GINS1 and E2F1. The protein expression levels of GINS1, E2F1, and cell stemness-related genes (SOX-2, NANOG, OCT4, and CD133) were detected by Western blot. Afterward, the proliferative capacity and stemness of HCC tumor cells were determined through colony formation, cell counting kit-8, and sphere formation assays. Our study found the high expression of GINS1 and E2F1 in HCC, and overexpressed GINS1 markedly enhanced the sphere formation and proliferation of HCC cells, while silencing GINS1 led to the opposite results. Besides, E2F1 promoted the transcription of GINS1 by working as an upstream transcription factor. The results of the rescue experiment suggested that overexpressed E2F1 could offset the suppressive effect of GINS1 silencing on HCC cell stemness and proliferation. We demonstrated that the transcription factor E2F1 accelerated cell proliferation and stemness in HCC by activating GINS1 transcription. The results can provide new insight into the GINS1-related regulatory mechanism in HCC, which suggest that it may be an effective way for HCC treatment by targeting the E2F1/GINS1 axis.

Root extract of Hemsleya amabilis Diels suppresses renal cell carcinoma cell growth through inducing apoptosis and G2/M phase arrest via PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hemsleya amabilis Diels, belongs to cucurbitaceae, was traditional Chinese medicine (TCM). It is widely used to treat various diseases. However, these diseases may contribute to the development of RCC. AIM OF THE STUDY: investigated the anticancer activities of root extract of Hemsleya amabilis Diels (HRE), and elucidated the underlying molecular mechanism in vivo and in vitro. MATERIALS AND METHODS: Dried Hemsleya amabilis Diels roots were extracted by ethyl acetate and used to treat RCC4, OS-RC-2 and ACHN cells. UHPLC-MS was used to analyze the chemical composition of the extract. CCK-8 and colony formation assay were used to investigate proliferation. PI staining was used to detect cell cycle. Annexin-V-FITC, AO/EB and TEM were used to evaluate apoptosis. Transwell and wound healing assays were used to evaluate migration and invasion. RNA-seq, Network pharmacology, autodocking for virtual screening and molecular dynamics simulation were used to analyze potential molecular mechanisms and active components of HRE inhibiting proliferation of RCC. LY294002 and UC2288 were used to inhibit PI3K and P21 expression, respectively. IGF-1 was used to activate PI3K. Xenograft tumor model was established to evaluate its anti-tumor potential in vivo. Immunohistochemistry and Western blot were used to test protein expression levels. H&E staining was used to explore the side effects of HRE in vivo. Applying bioinformatics to analyze the effect of P21 on RCC. RESULTS: HRE consists of 739 compounds. CCK-8 and colony formation assay showed that HRE significantly inhibited RCC cells proliferation. PI staining indicated that HRE caused G2/M phase arrest. Annexin-V-FITC, AO/EB and TEM experiments revealed that HRE significantly promoted apoptosis of RCC cells. Transwell and wound healing assays showed that HRE can inhibit the migration and invasion of RCC cells. RNA-seq showed that HRE induced 230 gene changes. Network pharmacology analysis found the relationship between HRE-component-target-RCC. Auto-docking found that Epitulipinolide diepoxide in HRE can stably bind to PIK3CA (-7.22 kJ/mol), and molecular dynamics simulation verified the combination between Epitulipinolide diepoxide of PIK3CA. In RCC4 cells, pretreatment with IGF-1, attenuated HRE-induced apoptosis and G2/M arrest. When pretreated with PIK3 inhibitor LY294002, the opposite result appears. Pretreatment with CDKN1A (P21) inhibitor UC2288 attenuated HRE-induced G2/M arrest. Xenograft tumor model showed that HRE inhibited tumor growth. Western blot analysis indicated that HRE can regulating Bax, Bcl-2, PARP, cleared-PARP, Caspase-9, Caspase-8, Caspase-3, Survivin, Cyclin-B1, CDK1, N-cadherin, snail, slug, E-cadherin, MMP-9. Immunohistochemical staining showed that in the treated group, expression of E-cadherin, Bax, P21 was up-regulated, while N-cadherin, PI3K, AKT and Bcl-2 were down-regulated. H&E staining showed that compared to control groups, the main organs in the HRE-treated groups showed no histological abnormalities. The overall survival rate of RCC patients in the high-expression group of P21 was higher than in the low-expression group of P21 on bioinformatics analysis. CONCLUSIONS: HRE inhibited RCC migration and invasion through EMT, and inhibited proliferation in vivo and in vitro. In addition, HRE inhibited proliferation through promoting apoptosis and P21-induced G2/M phase arrest via PI3K/AKT signaling pathway. Overall, these results suggest that HRE may be a promising chemotherapy agent for RCC.

Antrodia salmonea suppresses epithelial-mesenchymal transition/metastasis and Warburg effects by inhibiting Twist and HIF-1α expression in Twist-overexpressing head and neck squamous cell carcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Antrodia salmonea (AS), linked to the genus Taiwanofungus, is a medicinal fungus, and exhibits anti-inflammatory, anti-oxidant, and tumor inhibiting properties. AIM OF THE STUDY: In this study, we investigated the metabolic reprogramming and anti-metastasis/epithelial-mesenchymal transition (EMT) effects of AS exposure in Twist-overexpressing head and neck squamous cell carcinoma (HNSCC, OECM-1 and FaDu-Twist) cells. MATERIALS AND METHODS: MTT assay, Western blot, migration/invasion assay, immunofluorescence, glucose uptake assay, lactate assay, oxygen consumption rate (OCR)/Extracellular acidification rate (ECAR) assay, Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS), and qRT-PCR experimental techniques were used to evaluate the therapeutic potential of AS treatment in HNSCC cells. RESULTS: This study showed that AS exhibits anti-EMT and anti-metastatic effects as well as metabolic reprogramming in Twist-overexpressing HNSCC cells. AS exposure inhibited Twist and hypoxia-inducible factor-1α (HIF-1α) protein and/or mRNA expression in Twist-overexpressing OECM-1 and FaDu-Twist cells. AS markedly suppressed EMT by enhancing the expression of E-cadherin; while the N-cadherin was suppressed. Furthermore, glucose uptake and lactate accumulation, together with HIF-1α-regulated glycolysis genes were diminished by AS in OECM-1 cells. AS decreased the ECAR, and enhanced the OCR together with basal respiration, ATP production, maximal respiration, and spare respiratory capacity under normoxia and hypoxia (CoCl2) in OECM-1 cells. There was a marked reduction in the level of glycolytic intermediate's; while TCA cycle metabolites were increased by AS treatment in OECM-1 cells. CONCLUSIONS: We concluded that AS treatment suppresses EMT/metastasis and Warburg effects through Twist and HIF-1α inhibition in Twist-overexpressing HNSCC cells.

Highly bright stable organic radicals encapsulated by amphiphilic polypeptide for efficient near-infrared phototheranostics.

Stable organic radical molecules have received extensive attention due to their unique electronic structure and photophysical properties, and the highly fluorescent quantum efficiency has great appeal to bioimaging. However, still scarce reports on the application of them on the therapy of tumors, especially theranostics. Here, 3,6-dibromocarbazole modified tris (2,4,6-trichlorophenyl) methane radical (TB) has been synthesized with high NIR fluorescence quantum efficiency, and free radical nanoparticles (NPs) have been prepared using the precursor of the radical doping strategy. The free radical molecule TB and its precursor molecule HTB were mixed in proportion and encapsulated with an amphiphilic polypeptide (PEG-PAsp) to obtain the NPs. The 4% NPs can achieve a high fluorescence quantum efficiency (18.68%) in the NIR region. In addition, the NPs also have a good ability to produce reactive oxygen species (ROS) under either normoxia or hypoxia conditions, which makes it possible for photodynamic therapy (PDT). Interestingly, the NPs also show preferable photothermal ability (PCE = 42.39%) for photothermal therapy (PTT). Both in vitro and in vivo studies reveal that the as-prepared radical NPS show a NIR fluorescence imaging-guided synergistic PTT and type I/II PDT to tumors. It provides new strategies and new clues for the application of free radical molecules in the theranostics of tumors.

Effect of alpha lipoic acid on epithelial mesenchymal transition in SKOV-3 cells.

BACKGROUND: Ovarian cancer is the fifth leading cause of cancer-related death in women. Patients are usually diagnosed with advanced tumor metastass. Epithelial over cancer cells spread from primary tumor by undergoing epithelial mesenchymal transition (EMT). It has been suggested that alpha lipoic acid (ALA), a natural antioxidant lipophilic compound, reduces the oxidative stress by causing apoptosis and inhibition of proliferation of cell in cancer cells. The aim of our study was to establish a transforming growth factor ß1 (TGF ß1) dependent epithelial mesenchymal transition model in the SKOV-3 ovarian adenocarcinoma cell line which is an epithelial subtype of ovarian cancer and to investigate the effects of alpha lipoic acid on EMT and ovarian cancer migration. METHODS: For establish an EMT model, SKOV-3 cells were treated with different dose of TGF ß1 and XTT cell viability kit was used to find IC 50 dose of ALA. Four different groups that are control, TGF ß1, ALA and ALA + TGF ß1 were created. Changes in the expression of genes related to EMT markers that are E-cadherin, vimentin, Snail, Slug, Twist and Zeb were analyzed with quantitative real-time PCR. These proteins were determined with the immunocytochemistry method. The migration capacity was analyzed with wound healing assay. Matrigel invasion capacity test was used to show invasion and colonization test to show colonization. RESULTS: The dose of TGF ß1 was determined 100 ng/ml at 72 h, the IC50 dose of ALA 219.033 µM at 48 h was determined. EMT markers in the TGF ß1 group were compatible with EMT and it was shown to inhibit EMT in the groups given ALA. According to wound healing, colonization and invasion experiments, proliferation and invasion increased in TGF ß1 group, but decreased in ALA and combined groups (p < 0.05). CONCLUSION: These results indicate that ALA suppresses the metastasis of ovarian cancer cells by regulating EMT, implying that ALA might be a potential therapeutic agent for the treatment of ovarian cancer.

Exosomes secreted by metastatic cancer cells promotes epithelial mesenchymal transition in small cell lung carcinoma: The key role of Src/TGF-ß1 axis.

Exosome-mediated epithelial mesenchymal transition (EMT) is key to cancer metastasis. c-Src is involved in the secretion of exosomes and initiation of EMT. Effects of exosomes from metastatic non-small cell lung carcinoma (NSCLC) cells on the EMT process in primary NSCLC cells were assessed. Levels of c-Src in NSCLC tissues were detected and the influence of exosomes from metastatic NSCLC cells on the exosome secretion and EMT process in primary NSCLC cells was assessed. The expression of c-Src was modulated, and the influence on the secretion of exosomes and EMT initiation was evaluated. The level of c-Src was higher in NSCLC specimen and NSCLC cells with promoted EMT process. The suppression of c-Src inhibited secretion of exosomes. Exosomes from metastatic NSCLC cells enhanced migration and invasion abilities of primary NSCLC cells, which had identical effects to c-Src overexpression. The suppression of c-Src inhibited growth and metastasis of solid tumors as well as secretion of exosomes, while the injection of exosomes with c-Src overexpression promoted lung metastasis. TGF-ß1 restored the invasion and migration abilities even with c-Src knockdown. The exosomes from metastatic NSCLC cells with high c-Src expression of can increase c-Src level in primary NSCLC cells, contributing to the promoted EMT process through TGF-ß1 pathway.

Expression of CD44 is regulated by ELF3 in 5-FU treated colorectal cancer cells.

The development of chemoresistance in colorectal cancer (CRC) cells was usually thought to be inevitable as a result of continuing exposure to chemotherapeutic drugs. The existence of cancer stem cells (CSCs) within CRC tissues was recently suggested to play importance roles for this process. In this study, in order to mimic a dose schedule used in clinic (continuous infusion), low dose of fluorouracil (IC10 of 5-FU) was used to treat CRC cells. Our results showed that the expression of CD44, including some other CSCs markers were all increased after 5-FU treatment. The stemness properties of survived CRC cells were also observed to be enhanced. RNA-seq analysis revealed that ELF3, one of the members of ETS (E26 transformation-specific) transcription activator family, was increased along with CD44 after 5-FU treatment of CRC cells. Results from dual-luciferase reporter assay revealed that the transcription of CD44 could be activated by ELF3 in CRC cells. The induced CD44 expression in 5-FU treated CRC cells could also be decreased after the expression of ELF3 was inhibited. Moreover, it could be observed that the expression of ELF3 is significantly higher in CD44+ CRC cells. Taken together, our results suggested that CD44 expression might be regulated by ELF3 and could be induced after 5-FU treatment of CRC cells. Inhibition of ELF3 might be a promising treatment method when it was used in combination with chemotherapeutics to overcome chemoresistance formation during CRC treatment in clinic.

DNMT1-mediated NR3C1 DNA methylation enables transcription activation of connexin40 and augments angiogenesis during colorectal cancer progression.

Colorectal cancer (CRC) continues to be a major contributor to cancer-related mortality. Connexin 40 (CX40) is one of the major gap junction proteins with the capacity in regulating cell-to-cell communication and angiogenesis. This study investigates its role in angiogenesis in CRC and explores the regulatory mechanism. Aberrant high CX40 expression was detected in tumor tissues, which was associated with a poor prognosis in CRC patients. Elevated CX40 expression was detected in CRC cell lines as well. Conditioned medium of SW620 and HT29 cell lines was used to induce angiogenesis of human umbilical vein endothelial cells (HUVECs). CX40 knockdown in CRC cells reduced angiogenesis and mobility of HUVECs and blocked CRC cell proliferation, mobility, and survival. Following bioinformatics predictions, we validated by chromatin immunoprecipitation and luciferase assays that nuclear receptor subfamily 3 group C member 1 (NR3C1), which was poorly expressed in CRC samples, suppressed CX40 transcription. The poor NR3C1 expression was attributive to DNA hypermethylation induced by DNA methyltransferase 1 (DNMT1). Restoration of NR3C1 suppressed the pro-angiogenic effect, proliferation and survival, and tumorigenic activity of CRC cells, which were, however, rescued by CX40 upregulation. Collectively, this study demonstrates that transcription activation of CX40 upon DNMT1-mediated NR3C1 DNA methylation potentiates angiogenesis in CRC.