Cyclovirobuxine D inhibits growth and progression of non­small cell lung cancer cells by suppressing the KIF11­CDC25C­CDK1­CyclinB1 G2/M phase transition regulatory network and the NFκB/JNK signaling pathway.

Lung cancer is the leading cause of cancer­related mortality worldwide. Non­small cell lung cancer (NSCLC) is the most common pathological subtype of lung cancer and is associated with low 5­year overall survival rates. Therefore, novel and effective chemotherapeutic drugs are urgently required for improving the survival outcomes of patients with lung cancer. Cyclovirobuxine D (CVB­D) is a natural steroidal alkaloid, used for the treatment of cardiovascular diseases in Traditional Chinese Medicine. Several studies have also demonstrated the antitumor effects of CVB­D. Therefore, in the present study, the therapeutic effects of CVB­D in lung cancer and the underlying mechanisms were investigated using the in vivo xenograft model of NSCLC in nude mice and in vitro experiments with the NSCLC cell lines. Bioinformatics analyses of RNA­sequencing data, and cell­based functional assays demonstrated that CVB­D treatment significantly inhibited in vitro and in vivo NSCLC cell proliferation, survival, invasion, migration, angiogenesis, epithelial­to­mesenchymal transition and G2/M phase cell cycle. CVB­D exerted its antitumor effects by inhibiting the KIF11­CDK1­CDC25C­cyclinB1 G2/M phase transition regulatory oncogenic network and the NF­κB/JNK signaling pathway. CVB­D treatment significantly reduced the sizes and weights and malignancy of xenograft NSCLC tumors in the nude mice. In conclusion, the present study demonstrated that CVB­D inhibited the growth and progression of NSCLC cells by inhibiting the KIF11­CDK1­CDC25C­CyclinB1 G2/M phase transition regulatory network and the NF­κB/JNK signaling pathway. Therefore, CVB­D is a promising drug for the treatment of NSCLC patients.

LINC00205, a YY1-modulated lncRNA, serves as a sponge for miR-26a-5p facilitating the proliferation of hepatocellular carcinoma cells by elevating CDK6.

OBJECTIVE: LINC00205, a bidirectional lncRNA, located at human chromosome 21q22.3, was recently characterized as an oncogenic molecule contributing to cell proliferation in several cancers, including hepatocellular carcinoma (HCC). In the present study, we aim to probe the new molecular mechanism for LINC00205 controlling the proliferation of HCC cells. PATIENTS AND METHODS: The expression status of LINC00205, miR-26a-5p, as well as CDK6 in HCC tissues/cell lines was determined by quantitative real-time PCR (qPCR). The cell proliferative activity was measured by using the Cell Counting Kit (CCK)-8 assay. Flow cytometry was performed to analyze cell cycle progression and apoptosis induction. The interaction among LINC00205, miR-26a-5p and CDK6, as well as transcription efficiency of LINC00205 promoter were examined by Dual-Luciferase reporter assay. Western blot was conducted to evaluate the protein levels of CDK6 in SNU-449 cells. The direct interplay between YY1 and LINC00205 promoter was detected by ChIP-qPCR. RESULTS: LINC00205 was strongly expressed in HCC tissues and cell lines. Elevated LINC00205 expression was positively associated with worse prognosis as well as pathological grade in HCC. Suppression of LINC00205 could impede the proliferation of HCC cells by triggering the G0/G1-phase cell cycle arrest and apoptosis in vitro. Mechanistically, we illustrated that LINC00205 could accelerate the proliferation of HCC cells by boosting CDK6 expression via sponging miR-26a-5p. Moreover, we unveiled that LINC00205 could be activated by transcription factor Yin Yang-1 (YY1) as its direct downstream target. CONCLUSIONS: LINC00205, a novel YY1-modulated lncRNA, can facilitate the proliferation of HCC cells through YY1/miR-26a-5p/CDK6 pathway, and may serve as a promising diagnostic biomarker and therapeutic target for HCC.

Induction of G2/M Cell Cycle Arrest via p38/p21Waf1/Cip1-Dependent Signaling Pathway Activation by Bavachinin in Non-Small-Cell Lung Cancer Cells.

Lung cancer is the most commonly diagnosed malignant cancer in the world. Non-small-cell lung cancer (NSCLC) is the major category of lung cancer. Although effective therapies have been administered, for improving the NSCLC patient's survival, the incident rate is still high. Therefore, searching for a good strategy for preventing NSCLC is urgent. Traditional Chinese medicine (TCM) are brilliant materials for cancer chemoprevention, because of their high biological safety and low cost. Bavachinin, which is an active flavanone of Proralea corylifolia L., possesses anti-inflammation, anti-angiogenesis, and anti-cancer activities. The present study's aim was to evaluate the anti-cancer activity of bavachinin on NSCLC, and its regulating molecular mechanisms. The results exhibited that a dose-dependent decrease in the cell viability and colony formation capacity of three NSCLC cell lines, by bavachinin, were through G2/M cell cycle arrest induction. Meanwhile, the expression of the G2/M cell cycle regulators, such as cyclin B, p-cdc2Y15, p-cdc2T161, and p-wee1, was suppressed. With the dramatic up-regulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, the expression and association of p21Waf1/Cip1 with the cyclin B/cdc2 complex was observed. Silencing the p21Waf1/Cip1 expression significantly rescued bavachinin-induced G2/M cell accumulation. Furthermore, the expression of p21Waf1/Cip1 mRNA was up-regulated in bavachinin-treated NSCLC cells. In addition, MAPK and AKT signaling were activated in bavachinin-added NSCLC cells. Interestingly, bavachinin-induced p21Waf1/Cip1 expression was repressed after restraint p38 MAPK activation. The inhibition of p38 MAPK activation reversed bavachinin-induced p21Waf1/Cip1 mRNA expression and G2/M cell cycle arrest. Collectively, bavachinin-induced G2/M cell cycle arrest was through the p38 MAPK-mediated p21Waf1/Cip1-dependent signaling pathway in the NSCLC cells.

Acetylshikonin Induces Apoptosis in Human Colorectal Cancer HCT-15 and LoVo Cells via Nuclear Translocation of FOXO3 and ROS Level Elevation.

Acetylshikonin, a naphthoquinone, is a pigment compound derived from Arnebia sp., which is known for its anti-inflammatory potential. However, its anticarcinogenic effect has not been well investigated. Thus, in this study, we focused on investigating its apoptotic effects against HCT-15 and LoVo cells, which are human colorectal cancer cells. MTT assay, cell counting assay, and colony formation assay have shown acetylshikonin treatment induced cytotoxic and antiproliferative effects against colorectal cancer cells in a dose- and time-dependent manner. DNA fragmentation was observed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Also, the increase of subG1 phase in cell cycle arrest assay and early/late apoptotic rates in annexin V/propidium iodide (PI) double staining assay was observed, which indicates an apoptotic potential of acetylshikonin against colorectal cancer cells. 2',7'-Dichlorofluorescin diacetate (DCF-DA) staining was used to evaluate reactive oxygen species (ROS) generation in acetylshikonin-treated colorectal cancer cells. Fluorescence-activated cell sorting (FACS) analysis showed that acetylshikonin induced an increase in reactive oxygen species (ROS) levels and apoptotic rate in a dose- and time-dependent manner in HCT-15 and LoVo cells. In contrast, cotreatment with N-acetyl cysteine (NAC) has reduced ROS generation and antiproliferative effects in colorectal cancer cells. Western blotting analysis showed that acetylshikonin treatment induced increase of cleaved PARP, γH2AX, FOXO3, Bax, Bim, Bad, p21, p27, and active forms of caspase-3, caspase-7, caspase-9, caspase-6, and caspase-8 protein levels, while those of inactive forms were decreased. Also, the expressions of pAkt, Bcl-2, Bcl-xL, peroxiredoxin, and thioredoxin 1 were decreased. Furthermore, western blotting analysis of cytoplasmic and nuclear fractionated proteins showed that acetylshikonin treatment induced the nuclear translocation of FOXO3, which might result from DNA damage by the increased intracellular ROS level. This study represents apoptotic potential of acetylshikonin against colorectal cancer cells via translocation of FOXO3 to the nucleus and upregulation of ROS generation.

Sesquiterpene Lactone Deoxyelephantopin Isolated from Elephantopus scaber and Its Derivative DETD-35 Suppress BRAFV600E Mutant Melanoma Lung Metastasis in Mice.

Melanoma is a highly metastatic disease with an increasing rate of incidence worldwide. It is treatment refractory and has poor clinical prognosis; therefore, the development of new therapeutic agents for metastatic melanoma are urgently required. In this study, we created a lung-seeking A375LM5IF4g/Luc BRAFV600E mutant melanoma cell clone and investigated the bioefficacy of a plant sesquiterpene lactone deoxyelephantopin (DET) and its novel semi-synthetic derivative, DETD-35, in suppressing metastatic A375LM5IF4g/Luc melanoma growth in vitro and in a xenograft mouse model. DET and DETD-35 treatment inhibited A375LM5IF4g/Luc cell proliferation, and induced G2/M cell-cycle arrest and apoptosis. Furthermore, A375LM5IF4g/Luc exhibited clonogenic, metastatic and invasive abilities, and several A375LM5IF4g/Luc metastasis markers, N-cadherin, MMP2, vimentin and integrin α4 were significantly suppressed by treatment with either compound. Interestingly, DET- and DETD-35-induced Reactive Oxygen Species (ROS) generation and glutathione (GSH) depletion were found to be upstream events important for the in vitro activities, because exogenous GSH supplementation blunted DET and DETD-35 effects on A375LM5IF4g/Luc cells. DET and DETD-35 also induced mitochondrial DNA mutation, superoxide production, mitochondrial bioenergetics dysfunction, and mitochondrial protein deregulation. Most importantly, DET and DETD-35 inhibited lung metastasis of A375LM5IF4g/Luc in NOD/SCID mice through inhibiting pulmonary vascular permeability and melanoma cell (Mel-A+) proliferation, angiogenesis (VEGF+, CD31+) and EMT (N-cadherin) in the tumor microenvironment in the lungs. These findings indicate that DET and DETD-35 may be useful in the intervention of lung metastatic BRAFV600E mutant melanoma.

Inhibition of Growth of Colon Tumors and Proliferation of HT-29 Cells by Warburgia ugandensis Extract through Mediating G0/G1 Cell Cycle Arrest, Cell Apoptosis, and Intracellular ROS Generation.

Warburgia ugandensis Sprague (W. ugandensis), widely distributed in Africa, is a traditional medicinal plant used for the treatment of various diseases including cancer. We intended to evaluate the anticolorectal cancer (CRC) activities of the crude extract from W. ugandensis (WUD) and reveal the underlying molecular mechanisms of its action. We found that WUD inhibited the proliferation of HT-29 and HCT116 cells in a time- and dose-dependent manner and induced intracellular ROS generation. The inhibitory effect of WUD on the proliferation of HT-29 and HCT116 cells could be attenuated by NAC (a ROS scavenger) in a dose-dependent manner. WUD induced G0/G1 phase arrest, down-regulated the protein expression of Cyclin D1 via ROS accumulation in HT-29 cells. In search of the molecular mechanism involved in WUD-induced Cyclin D1 down-regulation, it was found that WUD can suppress PI3K/Akt/GSK3ß signaling pathway in HT-29 cells. Next, it was found that WUD also activated apoptosis, poly-ADP ribose polymerase 1 (PARP1) cleavage and down-regulated pro-caspase 3 in HT-29 and HCT116 cells. Besides, WUD decreased the growth of colon tumors in vivo in the xenograft mouse model. We demonstrated for the first time that ROS and their modulation in the corresponding intracellular signaling could play a significant role in the potential activity of WUD against CRC cells.

Deoxypodophyllotoxin, a Lignan from Anthriscus sylvestris, Induces Apoptosis and Cell Cycle Arrest by Inhibiting the EGFR Signaling Pathways in Esophageal Squamous Cell Carcinoma Cells.

Deoxypodophyllotoxin (DPT) derived from Anthriscus sylvestris (L.) Hoffm has attracted considerable interest in recent years because of its anti-inflammatory, antitumor, and antiviral activity. However, the mechanisms underlying DPT mediated antitumor activity have yet to be fully elucidated in esophageal squamous cell carcinoma (ESCC). We show here that DPT inhibited the kinase activity of epidermal growth factor receptor (EGFR) directly, as well as phosphorylation of its downstream signaling kinases, AKT, GSK-3ß, and ERK. We confirmed a direct interaction between DPT and EGFR by pull-down assay using DPT-beads. DPT treatment suppressed ESCC cell viability and colony formation in a time- and dose-dependent manner, as shown by MTT analysis and soft agar assay. DPT also down-regulated cyclin B1 and cdc2 expression to induce G2/M phase arrest of the cell cycle and upregulated p21 and p27 expression. DPT treatment of ESCC cells triggered the release of cytochrome c via loss of mitochondrial membrane potential, thereby inducing apoptosis by upregulation of related proteins. In addition, treatment of KYSE 30 and KYSE 450 cells with DPT increased endoplasmic reticulum stress, reactive oxygen species generation, and multi-caspase activation. Consequently, our results suggest that DPT has the potential to become a new anticancer therapeutic by inhibiting EGFR mediated AKT/ERK signaling pathway in ESCC.

Arnicolide D Inhibits Triple Negative Breast Cancer Cell Proliferation by Suppression of Akt/mTOR and STAT3 Signaling Pathways.

Triple-Negative Breast Cancer (TNBC) is a most dangerous breast cancer subtype. The naturally occurring sesquiterpene lactone, arnicolide D (AD), has proven effective against a variety of tumors, however, the inhibitory effects of AD against TNBC and the underlying mechanisms remain unclear. In the present study, two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and an MDA-MB-231 xenograft mouse model were employed to investigate the anti-TNBC effects of AD in vitro and in vivo. Cell viability was assessed by MTT assay. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Protein levels were determined by immunoblotting. In vitro studies demonstrated that AD significantly decreased cell viability, and induced G2/M cell cycle arrest and apoptosis. In vivo assays showed that oral administration of 25 or 50 mg/kg AD for 22 days led to a reduction of tumor weights by 24.7% or 41.0%, without appreciable side effects. Mechanistically, AD inhibited the activation of Akt/mTOR and STAT3 signaling pathways. Based on our findings, AD is a promising candidate for development as an adjunctive therapeutic drug for TNBC.

Sanggenol L Induces Apoptosis and Cell Cycle Arrest via Activation of p53 and Suppression of PI3K/Akt/mTOR Signaling in Human Prostate Cancer Cells.

Prostate cancer is the most common cancer in Western countries. Recently, Asian countries are being affected by Western habits, which have had an important role in the rapid increase in cancer incidence. Sanggenol L (San L) is a natural flavonoid present in the root barks of Morus alba, which induces anti-cancer activities in ovarian cancer cells. However, the molecular and cellular mechanisms of the effects of sanggenol L on human prostate cancer cells have not been elucidated. In this study, we investigated whether sanggenol L exerts anti-cancer activity in human prostate cancer cells via apoptosis and cell cycle arrest. Sanggenol L induced caspase-dependent apoptosis (up-regulation of PARP and Bax or down-regulation of procaspase-3, -8, -9, Bid, and Bcl-2), induction of caspase-independent apoptosis (up-regulation of AIF and Endo G on cytosol), suppression of cell cycle (down-regulation of CDK1/2, CDK4, CDK6, cyclin D1, cyclin E, cyclin A, and cyclin B1 or up-regulation of p53 and p21), and inhibition of PI3K/Akt/mTOR signaling (down-regulation of PI3K, p-Akt, and p-mTOR) in prostate cancer cells. These results suggest the induction of apoptosis via suppression of PI3K/Akt/mTOR signaling and cell cycle arrest via activation of p53 in response to sanggenol L in prostate cancer cells.

Omega-3 polyunsaturated fatty acids suppress metastatic features of human cholangiocarcinoma cells by suppressing twist.

Cholangiocarcinoma (CCA) is a highly malignant cancer of the bile duct, which has a five-year survival rate less than 5% due to a high metastasis rate and lack of therapeutic options. Although omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to inhibit the proliferation of CCA cells, the effects on CCA metastasis have not been previously reported. In this study, we first assessed the proliferation, migration and invasion effects of n-3 PUFA-based fish oil on human CCA cells. Then, we investigated PUFA effects on metastasis in vivo by xenografting CCA cells into zebrafish larvae that overexpress a critical n-3 PUFA synthesis gene, Δ6 fatty acid desaturase. The results indicated that n-3 PUFA-based fish oil suppresses CCA cell growth, potentially by blocking the cell cycle at G2/M phase, and it inhibits migration and invasion potential with coincident downregulation of migration-related genes. Furthermore, zebrafish endogenous n-3 PUFAs appear to suppress CCA metastasis by inhibiting the expression of twist, a key regulator of tumor metastasis. Interestingly, only long chain n-3 PUFAs could inhibit the expression of twist in CCA cells. Together, our results suggest that n-3 PUFAs, especially DHA, may inhibit proliferation and metastasis of CCA cells by inhibiting the expression of twist.

Chinese Herbal Medicine Ganoderma tsugae Displays Potential Anti-Cancer Efficacy on Metastatic Prostate Cancer Cells.

Resistance to the current therapies is the main clinical challenge in the treatment of lethal metastatic prostate cancer (mPCa). Developing novel therapeutic approaches with effective regimes and minimal side effects for this fatal disease remain a priority in prostate cancer study. In the present study, we demonstrated that a traditional Chinese medicine, quality-assured Ganoderma tsugae ethanol extract (GTEE), significantly suppressed cell growth and metastatic capability and caused cell cycle arrest through decreasing expression of cyclins in mPCa cells, PC-3 and DU145 cells. GTEE also induced caspase-dependent apoptosis in mPCa cells. We further showed the potent therapeutic efficacy of GTEE by inhibiting subcutaneous PC-3 tumor growth in a xenograft model. The in vitro and in vivo efficacies on mPCa cells were due to blockade of the PI3K/Akt and MAPK/ERK signaling pathways associated with cancer cell growth, survival and apoptosis. These preclinical data provide the molecular basis for a new potential therapeutic approach toward the treatment of lethal prostate cancer progression.

Dihydrosanguinarine suppresses pancreatic cancer cells via regulation of mut-p53/WT-p53 and the Ras/Raf/Mek/Erk pathway.

BACKGROUND: There have been some reports implicating the pharmacologic action of Dihydrosanguinarine (DHSA), but little research including the effects of it on cancer cells. PANC-1 cells have mutations in K-Ras and TP53, which respectively express mutant K-Ras and p53 protein, and the mutations in Ras/p53 have been believed with closely relationship to the occurrence of various tumors. PURPOSE: To reveal the inhibition of Dihydrosanguinarine on pancreatic cancer cells (PANC-1 and SW1990) proliferation by inducing G0/G1 and G2/M phase arrest via the downregulation of mut-p53 protein, inducing apoptosis and inhibiting invasiveness through the Ras/Mek/Erk signaling pathway. METHODS: Human pancreatic cancer cell lines were cultured with cisplatin and DHSA. Then, cell proliferation, the cell cycle and apoptosis were measured by CCK-8 and flow cytometry. The migratory and invasive abilities of pancreatic cancer cells were evaluated by transwell assay. The expression levels of mRNA and protein were measured by RT-PCR and western blotting. RESULTS: The results showed that DHSA treatment inhibited cell proliferation, migration and invasion in a time- and dose-dependent manner and led to induction of cell cycle arrest and apoptosis. G0/G1 and G2/M phase arrest inhibited the viability of PANC-1 cells by downregulating the expression of mut-p53 protein. Decreased levels of C-Raf and Erk phosphorylation in DHSA-treated PANC-1 and SW1990 cells were observed in a time- and dose-dependent manner. However, the total expression of p53 and Ras proteins had a different change in PANC-1 and SW1990 cells. CONCLUSIONS: Our findings offer the novel perspective that DHSA inhibits pancreatic cancer cells through a bidirectional regulation between mut-p53/-Ras and WT-p53/-Ras to restore the dynamic balance by Ras and p53 proteins.

Antrodia cinnamomea, a Treasured Medicinal Mushroom, Induces Growth Arrest in Breast Cancer Cells, T47D Cells: New Mechanisms Emerge.

Reported cases of breast cancer have skyrocketed in the last decades with recent advances in examination techniques. Brest cancer has become the second leading cause of mortality among women worldwide, urging the scientific community to develop or find new drugs from natural sources with potent activity and a reasonable safety profile to tackle this ailment. Antrodia cinnamomea (AC) is a treasured medicinal fungus which has attracted attention due to its potent hepatoprotective and cytotoxic activities. We evaluated the antiproliferative activity of the ethanol extract of artificially cultured AC (EEAC) on breast cancer cells (T47D cells) in vivo and in vitro. Ethanol extract of artificially cultured AC inhibited T47D cells' proliferation mediated by cell cycle arrest at G1 phase as well induced autophagy. Immunoblotting assay confirmed that EEAC not only decreased the expression of the cell-cycle-related proteins but also increased the expression of transcription factor FOXO1, autophagic marker LC3 II, and p62. Ethanol extract of artificially cultured AC mediated endoplasmic reticulum stress by promoting the expression of IRE1 (inositol-requiring enzyme 1α), GRP78/Bip (glucose regulating protein 78), and CHOP (C/EBP homologous protein). Apart from previous studies, HDACs (histone deacetylases) activity was inhibited as demonstrated by a cell-free system, immunoblotting, and immunofluorescence assays following EEAC treatment. The in vivo studies demonstrated that EEAC decreased tumor volume and inhibited tumor growth without any significant side effects. High performance liquid chromatography profile demonstrated similar triterpenoids compared to the profile of wild AC ethanol extract. The multiple targets of EEAC on breast cancer cells suggested that this extract may be developed as a potential dietary supplement targeting this debilitating disease.

Nanoscale bubble ultrasound contrast agents-mediated suicide gene therapy system, Nanoscale bubble-LV5-YCD-TK/GCV/5-FC, effectively inhibits bladder cancer cell growth.

OBJECTIVE: Bladder cancer is the 2nd most common reason for human genitourinary cancer-associated mortality. This study aimed to investigate the effects of Nanoscale bubble ultrasound contrast agents-mediated yeast-cytosine-deaminase-thymidine kinase/ganciclovir (YCD-TK/GCV) or YCD-TK/5-fluorocytosine (5-FC) suicide gene therapy system on BIU-87 cell growth. MATERIALS AND METHODS: Targeted nanoscale bubble ultrasound contrast agents were prepared by utilizing thin-film hydration-sonication approach. Nanoscale bubble-LV5-YCD-TK/GCV(5-FC) was constructed and transfected to BIU-87 cells. Hematoxylin and eosin (HE) staining was used to evaluate inflammation. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to examine cell viability. Cell-cycle distribution was analyzed with cell cycle assay. Flow cytometry assay was utilized to test apoptosis of BIU-87 cells. YCD-TK expression was examined using Western blot and quantitative Real Time-PCR (qRT-PCR), respectively. RESULTS: YCD-TK highly expressed in Nanoscale bubble mediated suicide gene therapy system. Nanoscale bubble-mediated suicide gene therapy system significantly induced inflammatory response and apoptosis compared to that of Nanoscale bubble group (p<0.05). Nanoscale bubble mediated suicide gene therapy system significantly reduced cell viability compared to that of the Nanoscale bubble group (p<0.05). Nanoscale bubble mediated suicide gene therapy system significantly inhibited cell cycle arrest compared to that of the Nanoscale bubble group (p<0.05). Nanoscale bubble-LV5-YCD-TK/GCV/5-FC therapy system significantly reduced BIU-87 cell viability compared to that of the Nanoscale bubble-associated groups (p<0.05). CONCLUSIONS: Nanoscale bubble-mediated suicide gene therapy system, bubble-LV5-YCD-TK/GCV/5-FC, acts as a novel therapeutic strategy for bladder cancer treatment.

Phyla nodiflora L. Extracts Induce Apoptosis and Cell Cycle Arrest in Human Breast Cancer Cell Line, MCF-7.

Phyla nodiflora L. has been used as medicinal remedies for various ailments due to its antioxidant, anti-inflammatory, anti-bacterial, anti-tumor activity. Previously, we found that the plant extracts induced DNA fragmentation in MCF-7. This study was to investigate the modes of action of P. nodiflora in inhibiting breast cancer cells using leaf ethyl acetate (EA leaf), stem ethyl acetate (EA stem) and stem methanol (Met stem) extracts. The MTT assay showed that the anti-proliferative effects of P. nodiflora extracts were selective towards MCF-7 with a minimal effect on MCF10A. Morphological changes such as cell shrinkage and nuclear condensation were observed in treated cells. We found that induction of apoptosis by EA leaf and EA stem was mitochondrial-dependent while loss of mitochondrial membrane potential was not found in Met stem-treated cells. In addition, the expression levels of AIFM1, CASP9, CFLAR, and IGF1R were altered after treatment. Decreased BCL-2 expression was found in treated cells while BAX and caspases' expression was upregulated or maintained. All extracts caused perturbation of cell cycle at S phase by dysregulating the expression of cell cycle regulators such as CDKs and cyclins. Our findings indicate that P. nodiflora inhibits MCF-7 cells by inducing apoptosis and perturbing cell cycle.

Chidamide-induced ROS accumulation and miR-129-3p-dependent cell cycle arrest in non-small lung cancer cells.

BACKGROUND: Epigenetic therapy is a promising popular treatment modality for various cancers. Histone modification and miRNA should not be underestimated in lung cancer. This study aimed to investigate whether chidamide, a histone deacetylase inhibitor (HDACi), which inhibits telomerase activity and induces cell cycle arrest, influences ROS and miRNA production in non-small cell lung cancer (NSCLC) cells. METHODS: H1355 and A549 were treated with chidamide. The analysis of DNA content was measured by FACSCalibur equipped with a 488 nm laser. H1355 cells were transfected with miR-129-3p mimic by Lipofectamine2000. Telomerase activity was performed on the telomeric repeat amplification protocol (TRAP) assay. Detection of thymidylate synthase (TS), p21, p53, pRB, and ß-actin, were performed by western blot analysis. RESULTS: Our data showed that expression of TS, p21, and pRB were altered in the presence of chidamide by PCR and western blot. Using BrdU-incorporation analysis, we found that chidamide induced G1 arrest through the regulation of the TS gene by miR-129-3p. Chidamide was shown to suppress telomerase activity in the TRAP assay and reduced the expression of human telomerase reverse transcriptase (hTERT) by PCR and q-PCR in H1355 and A549 cells. Chidamide increased the generation of reactive oxygen species (ROS) by flow cytometry. N-acetyl cysteine (NAC), a ROS scavenger, attenuated chidamide-induced telomerase activity inhibition. CONCLUSION: Chidamide repressed telomerase activity through ROS accumulation and cell cycle arrest by miR-129-3p upregulation in both H1355 and A549 cells. This is the first study to demonstrate that chidamide induces miR-129-3p upregulation and ROS accumulation, leading to cell cycle arrest.

Astragaloside suppresses tumor necrosis factor receptor-associated factor 5 signaling pathway and alleviates neurodegenerative changes in retinal pigment epithelial cells induced by isoflurane.

Epidemiological studies showed that isoflurane, a general anesthetic widely used in surgery including those for the children, is associated with impairment of neurodevelopment and neurodegenerative diseases, such as Alzheimer's disease (AD) and age-related macular degeneration (AMD), which are related to the accumulation of reactive oxygen species (ROS). Astragaloside (AS) is an antioxidant derivative from a traditional Chinese herbal medicine Astragalus membraneaceus Bunge. In this study, we used retinal pigment epithelial cells, which share plenty of features with neurodegenerative diseases such as AD and AMD to investigate the effect of AS. Cell cycle re-entry and proapoptosis were seen in retinal pigment epithelium (RPE) cells treated with isoflurane, which was alleviated by pretreatment of AS. Further, tumor necrosis factor receptor-associated factor 5 (TRAF5) and downstream nuclear factor-κB (NF-κB) were investigated to elucidate the molecular mechanism underlying protective effect of AS. RPE cells exposed to isoflurane expressed higher TRAF5 and NF-κB than those pretreated with AS, suggesting a critical role of TRAF5 therein. In Morris water maze (MWM) assay, Sprague-Dawley rats pretreated with AS and then exposed to isoflurane spent less time in swimming to the platform, and their TRAF5 expression was significantly lower than those received anesthesia alone. Further studies on the consequence of forced downregulation or upregulation are warranted that may employ cutting-edge technologies such as optogenetics to overcome the difficulties in manipulating expression of TRAF5. Although the link between TRAF5 and neurodegeneration requires more in-depth investigations, our study provide a novel hint on the pathological mechanism of isoflurane and suggest a potential target for eliminating persistent side effect of anesthesia.

Carnosic acid regulates cell proliferation and invasion in chronic myeloid leukemia cancer cells via suppressing microRNA-708.

PURPOSE: Carnosic acid (CA) is an important polyphenol mainly isolated from the famous spice and the medicinal plant Rosmarinus officinalis. CA has been shown to exhibit tremendous pharmacological properties which include, but are not limited to, anticancer, antioxidant and anti-inflammatory activities. The current study was designed to evaluate the anticancer effects of CA against chronic myeloid leukemia (CML) which is one of the rare but deadly malignancies both in men and women. METHODS: CML KBM-7 cell line was used in this study. Cell viability was assessed by MTT assay. Apoptosis was detected by DAPI and annexin V/PI staining, cell cycle analysis by flow cytometry and cell invasion by Boyden chamber assay. The microRNA-780 expression was determined by quantitative RT-PCR. RESULTS: Our results indicated that CA exhibits significant anticancer activity on CML KBM-7 cells with an IC50 of 25 µM. The anticancer activity was due to induction of apoptosis and cell cycle arrest. Moreover, it was observed that CA inhibits the proliferation and invasion of CML KBM-7 cells which could mainly be due to downregulation of microRNA- 780 expression as indicated by the quantitative RT-PCR analysis. CONCLUSION: Taken together, we propose that carnosic acid could prove a potential lead compound in the treatment of CML and deserves further in vitro as well as in vivo study.

Formononetin inhibits colon carcinoma cell growth and invasion by microRNA­149­mediated EphB3 downregulation and inhibition of PI3K/AKT and STAT3 signaling pathways.

Formononetin (Form), a phytoestrogen extracted from the roots of Astragalus membranaceus, is one of the fundamental herbs used in traditional Chinese medicine because of its protective effects against certain malignant tumors. However, its role in colon carcinoma cells and the underlying molecular mechanisms have not been completely elucidated. The present study aimed to demonstrate that Form significantly inhibited the proliferation and invasion of the colon carcinoma cell lines SW1116 and HCT116. Mechanistic studies have suggested that Form suppresses colon carcinoma cell growth by downregulating cell cycle­associated protein (cyclin D1) expression and arresting the cell cycle at the G0­G1 checkpoint. Further studies revealed that treatment with Form inhibits matrix metalloproteinase (MMP)2 and MMP9 expression. Aditionally, the results demonstrated that Form significantly increased microRNA (miR)­149 expression. Following miR­149 overexpression in SW1116 and HCT116 cells using an miR­149 mimic, cell viability and Ephrin type­B receptor 3 (EphB3) levels decreased. Furthermore, the inhibitory effects of Form were associated with phosphatidylinositol 3­kinase (PI3K)/protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) signaling pathways. These results indicated the suppressive effect of Form on colon carcinoma cell proliferation and invasion, possibly via miR­149­induced EphB3 downregulation and the inhibition of the PI3K/AKT and STAT3 signaling pathways. Overall, Form may be used as a novel candidate for the clinical treatment of colorectal cancer in the future.

The root of Actinidia chinensis inhibits hepatocellular carcinomas cells through LAMB3.

The root of Actinidia chinensis, as traditional Chinese medicine, has been shown to inhibit cell proliferation in numerous cancer cells. However, the mechanisms underlying its inhibitory activity remain unclear. Death rates of hepatocellular carcinoma (HCC) are increasing, but therapies for advanced HCC are not well developed. We choose the extract from root of Actinidia chinensis (ERAC) to treat the HCC cell lines in vitro, displaying distinct effects on cell proliferation, S-phase cell cycle arrest, and apoptosis. LAMB3, the gene encoding laminin subunit beta-3, plays a key role in the proliferation suppression and S-phase cell cycle arrest of HepG2 cells treated with ERAC. The downstream genes ITGA3, CCND2, and TP53 in LAMB3 pathway show the same response to ERAC as LAMB3. Thus, LAMB3 pathways, along with extracellular matrix-receptor interaction, pathways in cancer, and focal adhesion, are involved in the ERAC-induced suppressive response in HepG2.