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We assessed the role of the protein-coding gene chaperonin-containing TCP1 subunit 6A (CCT6A) in osteosarcoma, as this is currently unknown. Using data from the R2 online genomic analysis and visualization application, we found that CCT6A messenger ribonucleic acid (RNA) expression is increased in osteosarcoma tissue and cells. Transfection of CCT6A small interfering RNA into cultured osteosarcoma cells revealed that CCT6A knockdown attenuates cell growth, cell viability, cell survival, and induced apoptosis and cell cycle progression at the G0/G1 phases. Moreover, CCT6A knockdown downregulated phospho-protein kinase B (p-Akt), cyclinD1 and B-cell lymphoma-2, whereas upregulated Bcl-2-associated X-protein expression. Thus, CCT6A knockdown inhibits cell proliferation, induces cell apoptosis, and suppresses the Akt pathway.

Background: NUFIP1 is an RNA-binding protein that interacts with fragile X mental retardation protein (FMRP) in the messenger ribonucleoprotein particle (mRNP). We previously showed that NUFIP1 was upregulated in colorectal cancer (CRC), but how the protein may contribute to the disease and patient prognosis is unknown. Methods: Here we combine database analysis, microarray, quantitative PCR, and immunohistochemistry of patients' samples to confirm our previous findings on NUFIP1 overexpression in CRC, and to reveal that increased expression of NUFIP1 in CRC tissues correlated with worse overall, recurrence-free, event-free and disease-free survival in patients, as well as with more advanced CRC clinicopathological stage. Results: Loss of function analysis demonstrated that NUFIP1 knockdown suppressed cell growth in vitro and in vivo, inhibited cell viability and survival, and induced cell cycle arrest and apoptosis in vitro. In addition, as a natural anticancer triterpene from various fruits and vegetables, ursolic acid (UA) treatment suppressed cell proliferation, down-regulated NUFIP1 protein expression, and further enhanced the effects of NUFIP1 knockdown in CRC cells in vitro. NUFIP1 knockdown up-regulated the expression of 136 proteins, down-regulated the expression of 41 proteins, and enriched multiple signaling pathways including the senescence-associated heterochromatin foci (SAHF) pathway. Furthermore, NUFIP1 knockdown enhanced the expression of senescence-associated-β-galactosidase (SA-β-gal), the SAHF markers HP1γ and trimethylation (H3k9me3), and the senescence-related protein HMGA2. Conclusion: Our findings suggest that NUFIP1 is overexpressed in CRC and correlates with disease progression and poor patient survival. NUFIP1 may exert oncogenic effects partly by altering senescence. UA may show potential to treat CRC by down-regulating NUFIP1.

Cell division cycle associated 5 (CDCA5) is implicated in the development and progression of a variety of human cancers. Functional significance of CDCA5 in colorectal cancer (CRC), however, has not been investigated. Using a combination of on-line data mining, biochemistry, and molecular biology, we examined the potential oncogenic activity of CDCA5 and the underlying mechanisms. Experiments with human tissue sample showed increased CDCA5 expression in CRC vs. in noncancerous adjacent tissue, and association of CDCA5 upregulation in CRC tissues with shorter patient survival. Also, representative CRC cell-lines had higher CDCA5 expression vs. fetal colonic mucosal cells. CDCA5 knockdown using lentivirus-mediated shRNA inhibited the proliferation and induced apoptosis in cultured HCT116 and HT-29 cells, and suppressed the growth of xenograft in nude mice. CDCA5 knockdown decreased the expression of CDK1 and CyclinB1, increased caspase-3 activity, cleaved PARP and the Bax/Bcl-2 ratio. CDCA5 knockdown also significantly decreased phosphorylation of ERK1/2 and expression of c-jun. Taken together, these findings suggest a significant role in CRC progression of CRC, likely by activating the ERK signaling pathway.

Disruption of the intestinal mucosal barrier integrity is a pathogenic process in inflammatory bowel disease (IBD) development, and is therefore considered a drug discovery target for IBD. The well-known traditional Chinese formulation Qing Hua Chang Yin (QHCY) has been suggested as a potential therapeutic agent for the treatment of ulcerative colitis. However, the possible underlying molecular mechanisms regarding its therapeutic effect remain unclear. Consequently, the present study investigated the effects of QHCY on lipopolysaccharide (LPS)-induced loss of intestinal epithelial barrier integrity in vitro using the Caco-2 cell model of intestinal epithelium. QHCY reversed the LPS-induced decrease in transepithelial electrical resistance and significantly alleviated the increased fluorescently-labeled dextran 4 flux caused by LPS. Moreover, QHCY upregulated the mRNA and protein expression levels of occludin, zona occludens-1 and claudin-1 in LPS-exposed Caco-2 cells. In conclusion, QHCY was able to protect intestinal epithelial barrier integrity following an inflammatory insult; the protective effects of QHCY may be mediated by modulation of the expression of tight junction proteins.

Huoxin Pill (HXP), a Traditional Chinese Medicine, is used widely to treat patients with coronary heart disease and angina pectoris in China. However, the underlying protective mechanism of HXP on cardiac apoptosis and fibrosis has never been evaluated. Therefore, the aim of this study was to investigate the role of HXP in a myocardial infarction (MI) mouse model. The mice were randomly divided into 3 groups and subjected to surgical ligation of the left anterior descending (LAD) coronary artery or sham surgery (n = 6 for each group) and treated with HXP (50 mg/kg/day) or saline by gavage for 2 weeks. At 2 weeks post MI, we found that HXP significantly enhanced myocardial function and attenuated the increase of heart weight index (HWI) and pathological changes in MI mice. RNA-sequencing and KEGG pathway analyses identified 660 differentially expressed genes and multiple enriched signaling pathways including p53 and TGF-β. In support of these findings, HXP attenuated cardiac apoptosis and decreased p53 and Bax protein expression, while increasing Bcl-2 protein expression in cardiac tissues of MI mice. Furthermore, HXP treatment inhibited cardiac fibrosis and significantly down-regulated TGF-β1 protein expression and Smad2/3 phosphorylation in cardiac tissues. In summary, HXP can improve cardiac function in mice after MI by attenuating cardiac apoptosis and fibrosis partly via supression of the p53/Bax/Bcl-2 and TGF-β1/Smad2/3 pathways.

Qingda granule (QDG), simplified from Qingxuan Jiangya Decoction, is a well-known traditional Chinese medicine formula that has been used for decades to treat hypertension. However, the cardioprotective effects of QDG on Ang II-induced hypertension remain unknown. This study aimed to investigate the effects of QDG on hypertension-induced cardiac hypertrophy and apoptosis, as well as explore its underlying mechanisms. Mice were infused with Ang II (500 ng/kg/min) or saline solution as control, then administered oral QDG (1.145 g/kg/day) or saline for two weeks. QDG treatment attenuated the elevation in blood pressure caused by Ang II, as well as the decreased left ventricle ejection fractions and fractional shortening. Moreover, QDG treatment significantly alleviated the Ang II-induced elevation of the ratio of heart weight to tibia length, as well as cardiac injury, hypertrophy, and apoptosis. In cultured H9C2 cells stimulated with Ang II, QDG partially reversed the increase in cell surface area and number of apoptotic cells, up-regulation of hypertrophy markers ANP and BNP, and activation of caspases-9 and -3. QDG also partially reversed Ang II-induced accumulation of reactive oxygen species (ROS), depolarization of the mitochondrial membrane, release of cytochrome C, up-regulation of Bax, and decrease in levels of p-PI3K, p-AKT, and Bcl-2. These results suggest that QDG can significantly attenuate Ang II-induced hypertension, cardiac hypertrophy and apoptosis, and it may exert these effects in part by suppressing ROS production and activating the PI3K/AKT signaling pathway.

Early B cell factor 1 (EBF1) has been identified as an upstream transcription factor of the potential oncogene PNO1 and is involved in the growth of colorectal cancer (CRC) cells. However, its expression, biological function, and underlying mechanism of action in most solid tumors remain largely unknown. We postulated that EBF1 has a role in the pathophysiology of CRC. Analysis of EBF1 mRNA expression in CRC tumor samples from several public databases and directly from banked tissues revealed that EBF1 mRNA expression is lower in CRC tissue compared to non-cancerous colorectal tissue. Survival analysis of multiple datasets revealed that low EBF1 expression was correlated with shorter overall survival, relapse-free survival, and event-free survival in CRC patients. Transduction of lentivirus encoding full length EBF1 followed by in vitro and in vivo assays demonstrated that EBF1 over-expression in CRC cell lines suppresses cell growth by inhibiting cell viability, cell survival, and induces cell cycle arrest and apoptosis. Mechanistic investigation indicated that EBF1 over-expression down-regulates PNO1 mRNA and protein expression, as well as transcriptional activity while up-regulating the expression of p53 and p21 proteins. These findings suggest that EBF1 is a novel potential tumor suppressor in CRC with prognostic value for the identification of patients at high-risk of relapse.

Ethnopharmacological relevance Kuanxiong aerosol (KXA) is a common clinical drug based on Fangxiang Wentong (FXWT) therapy in the treatment of angina pectoris. However, the pharmacological mechanism of KXA in the prevention and treatment of myocardial injury (MI) is not clear. Aim of the study The purpose of this study was to explore the protective effect of KXA on isoproterenol (ISO)-induced MI in rats. Materials and methods The study included male Wistar Kyoto rats (age: 6 weeks). The rats were randomly divided into the following 5 groups (n = 6 per group): control group, ISO group, isosorbide mononitrate (ISMN) group (5 mg/kg), KXA-L group (0.1 mL/kg), and KXA-H group (0.3 mL/kg). The rats in the last three groups were given intragastric administration for 14 days, and rats in control group and ISO group were given the same amount of normal saline daily. ISO (120 mg/kg) was used to induce MI on the 13th and 14th days. We assessed electrocardiograms (ECGs), myocardial specific enzymes, histopathological changes, and apoptosis. Results We found that KXA reduced the increase in the ST-segment amplitude (elevation or depression) and the levels of myocardial marker enzymes induced by ISO in MI rats, improved the pathological changes in myocardial tissue, and reduced cardiomyocyte apoptosis. At the same time, KXA significantly inhibited the up-regulation of caspase-3 and Bax expression and down-regulation of Bcl-2 expression induced by ISO. RNA sequencing showed that 90 up-regulated genes induced by ISO were down-regulated after KXA treatment, whereas 27 down-regulated genes induced by ISO were up-regulated after KXA treatment. In addition, KEGG pathway enrichment analysis showed that the mitogen-activated protein kinase (MAPK) signaling pathway may be an important target of KXA in the treatment of ISO-induced MI in rats. The results of RNA sequencing verified by Western blot analysis showed that KXA significantly inhibited the activation of the ISO-induced MAPK pathway. Conclusions KXA improves cardiac function in MI rats by inhibiting apoptosis mediated by the MAPK signaling pathway.

The well-known traditional Chinese medicine formula Pien Tze Huang (PZH) has long been used to treat various malignancies, including colorectal cancer (CRC). It was recently reported that PZH possesses the ability to overcome multidrug resistance in CRC cells. In the present study, a 5-fluorouracil (5-FU) resistant human CRC cell line (HCT-8/5-FU) was used to further evaluate the effect of PZH on chemotherapy (chemo)-resistance and investigate the mechanisms through which this occurs. The results identified that PZH significantly reduced the viability and cell density of HCT-8/5-FU cells in a dose- and time-dependent manner (P

Chemotherapeutic agents, including 5-fluorouracil (5-FU), frequently cause intestinal mucositis as a side effect, leading to life quality reduction in colorectal cancer (CRC) patients and interruption of CRC treatment. Traditional Chinese medicines (TCMs) have recently received attention due to their relatively few adverse effects. Pien Tze Huang (PZH), a well-known TCM formulation first documented to have been prescribed >450 years ago, has been demonstrated to be clinically effective in treating various types of cancer including CRC. Although previous studies by our group reported that PZH possesses a variety of anti-cancer activities via multiple mechanisms, it has remained elusive whether it is able to reduce intestinal mucositis induced by 5-FU. The present study evaluated the effect of PZH on 5-FU-induced intestinal mucositis in CT-26 tumor-bearing xenograft mice and investigated the possible molecular mechanism. The results indicated that administration of PZH effectively alleviated the severity of 5-FU-induced diarrhea and morphological intestinal damages, but had no significant effect on body weight loss. In addition, a terminal deoxynucleotidyl transferase dUTP nick end labeling assay revealed that PZH treatment significantly inhibited cell apoptosis in the intestinal crypt. Furthermore, immunohistochemical staining showed that PZH treatment reduced the protein expression of pro-apoptotic B-cell lymphoma 2 (Bcl-2)-associated X protein but enhanced that of anti-apoptotic Bcl-2 in the intestinal crypt. Taken together, the results of the present study suggested that PZH effectively attenuates 5-FU-induced intestinal mucositis, which is in part associated with its inhibitory effect on cell apoptosis in the intestinal crypt.

Cancer stem cells (CSCs) possess properties of continuous self-renewal, multi-directional differentiation and natural chemoresistance, leading to the initiation, progression and relapse of cancer. The characteristics of CSCs are strongly associated with multiple cellular pathways such as Notch1 signaling. Therefore, targeting CSCs via suppressing the Notch1 pathway might represent a promising strategy for cancer treatment. The well-known traditional Chinese medicine (TCM) formula Pien Tze Huang (PZH) has long been used as an alternative remedy for various cancers including colorectal cancer (CRC). We previously reported that PZH contains a broad range of anticancer activities including an inhibitory effect on CSCs. To further elucidate the mode of action of PZH, in this study we isolated the stem-like side population (SP) from the human CRC SW480 cell line to investigate its effect on CSCs as well as the possible molecular mechanisms. As compared with non-SP cells, the isolated SW480 SP cells displayed stronger capacities of spheroid formation in vitro and tumorigenicity in vivo, demonstrating the stem cell-like features of SP cells. However, PZH treatment significantly decreased the percentage of SP cells in a dose-dependent manner. In addition, PZH significantly and does-dependently inhibited the viability and promoted the apoptosis and differentiation of the isolated SW480 SP cells. Moreover, PZH treatment profoundly reduced the mRNA and protein expression of Notch1 and Hes1 in the SP cells. Our findings suggest that PZH negatively modulates the characteristics of CSCs through suppression of the Notch1 signaling pathway.

Interleukin‑6 (IL‑6)/signal transducer and activator of transcription 3 (STAT3) pathway plays essential roles in the development of inflammatory diseases including ulcerative colitis (UC). Therefore, suppression of IL‑6/STAT3 signaling provides a promising therapeutic strategy in UC. Pien Tze Huang (PZH), a well‑known traditional Chinese formula, has been used in China and Southeast Asia for centuries as a folk remedy for various inflammatory diseases. However, the molecular mechanisms of its anti‑inflammatory effects remain to be elucidated. In the present study, we generated a mouse colitis model by using dextran sulfate sodium (DSS) and evaluated the therapeutic efficacy of PZH against UC by observing the clinical manifestations. We found that PZH obviously alleviated DSS‑induced colitis symptoms, including body weight loss, rectal bleeding and stool consistency. In addition, administration of PZH profoundly prevented DSS‑induced colon shortening, and ameliorated colonic histopathological changes such as mucosal ulceration, infiltration of inflammatory cells, crypt distortion and hyperplastic epithelium. Moreover, PZH markedly inhibited the serum level of the inflammatory biomarker serum amylase A (SAA) in UC mice. Furthermore, PZH treatment significantly inhibited DSS‑induced expression of IL‑6 in colon tissues. Finally, the increased phosphorylation level of STAT3, induced either by DSS in experimental mice or by IL‑6 in the differentiated human colorectal carcinoma cells, was significantly suppressed by PZH. These results suggest that the inhibition of IL‑6/STAT3 signaling is a potential mechanism by which PZH is used in the treatment of UC.

Hepatocellular carcinoma (HCC) is characterized by uncontrolled proliferation and the deregulation of apoptotic signaling, although its molecular pathogenesis is not fully characterized. The ability to inhibit excessive proliferation and induce the apoptosis of cancer cells are crucial characteristics of anticancer drugs. Pien Tze Huang (PZH) is a widely used traditional Chinese medicine for the treatment of various types of cancer, and has exhibited promising therapeutic effects in clinical trials of HCC. However, the underlying mechanisms for its action are unclear. In the present study, the aim was to explore the effect of PZH on the proliferation and apoptosis of the BEL-7402 HCC cell line, and the associated mechanisms. PZH treatment significantly inhibited BEL-7402 cell viability, confluence and clonogenicity, inducing cell cycle arrest and promoting apoptosis. In addition, PZH treatment suppressed the expression of the pro-proliferative genes cyclin D1 and cyclin-dependent kinase 4, and decreased the expression of the anti-apoptotic gene Bcl-2. PZH treatment also upregulated the expression of a key microRNA (miR), miR-16. The study demonstrated that PZH can effectively inhibit cancer cell proliferation and induce apoptosis in BEL-7402 HCC cells via the upregulation of the tumor suppressor miR-16.

Patrinia scabiosaefolia (PS) has long been used as an important component in traditional Chinese medicine formulas to treat gastrointestinal malignancies including colorectal cancer (CRC). We recently reported that PS can inhibit CRC growth through induction of apoptosis and inhibition of tumor angiogenesis. To further elucidate the mode of action of PS, in the present study, we used a CRC mouse xenograft model and a human CRC cell line HT-29 to evaluate the effect of the ethanol extract of PS (EEPS) on cancer cell proliferation and investigated the underlying molecular mechanisms. We found that EEPS inhibited CRC growth both in vivo and in vitro, which was associated with the inhibitory effects of EEPS on cancer cell proliferation. In addition, EEPS treatment significantly blocked G1 to S phase cell cycle progression in HT-29 cells. Moreover, EEPS treatment decreased the expression of pro-proliferative CyclinD1 and CDK4, at both the mRNA and protein levels. Thus, inhibition of cell proliferation via G1/S cell cycle arrest might be a potential mechanism whereby PS effectively treats cancers.

To evaluate the inhibitory effect of bear bile powder (BBP) on hepatocellular carcinoma (HCC) growth in vivo and investigate the underlying mechanisms. A HCC xenograft mouse model was developed by producing with huh7 cells. After 5 days following xenograft implantation, ten HCC xenograft mice were given intra-gastric administration with 10 mg/(kg•d) dose of BBP or saline for 3 weeks. Tumor growth in HCC xenograft mice was evaluated by measuring the tumor weight and volume. Cell apoptosis, proliferation or tumor angiogenesis were examined via immunohistochemical (IHC) staining for transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), proliferating cell nuclear antigen (PCNA) or cluster of differentiation 31 (CD31), respectively. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) were determined by Western blot. The mRNA and protein expressions of Bcl-2, Bax, Cyclin D1 and Cyclin-dependent kinase 4 (CDK4) in HCC tumor tissues were respectively determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The protein expression of vascular endothelial growth factor A (VEGF-A) in tumor tissues was examined by IHC staining. BBP treatment led to a significant decrease on tumor volume and tumor weight in HCC mice (P

Spica Prunellae has long been used as a significant component in numerous traditional Chinese medicine (TCM) formulas to clinically treat cancers. Previously, Spica Prunellae was shown to promote cancer cell apoptosis and inhibit angiogenesis in vivo and in vitro. To further elucidate the precise mechanism of its tumoricidal activity, the effect of the ethanol extract of Spica Prunellae (EESP) on the proliferation of human colon carcinoma HT-29 cells was elucidated and the underlying molecular mechanisms were investigated. The proliferation of HT-29 cells was evaluated using 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation analyses. The cell cycle was determined using fluorescence-activated cell sorting (FACS) with propidium iodide (PI) staining. The mRNA and protein expression of cyclin-dependent kinase 4 (CDK4) and cyclin D1 was examined using RT-PCR and western blotting, respectively. EESP was observed to inhibit HT-29 viability and survival in a dose- and time-dependent manner. Furthermore, EESP treatment blocked G1/S cell cycle progression and reduced the expression of pro-proliferative cyclin D1 and CDK4 at the transcriptional and translational levels. Altogether, these data suggest that the inhibition of cell proliferation via G1/S cell cycle arrest may be one of the mechanisms through which Spica Prunellae treats cancer.

Disrupted apoptosis not only confers a survival advantage to cancer cells but also causes resistance to chemotherapies. Therefore, inducing cell apoptosis has become a promising strategy for anticancer treatment. Patrinia scabiosaefolia (PS) has long been used to clinically treat various types of malignancies including colorectal cancer (CRC). However, the precise mechanism of its tumoricidal activity remains largely unclear. Using a CRC mouse xenograft model and a human colon carcinoma cell line, HT-29, in the present study, we evaluated the antitumor activities of an ethanol extract of Patrinia scabiosaefolia (EEPS), and investigated the underlying molecular mechanisms. We found that EEPS inhibited CRC growth both in vivo and in vitro, without apparent adverse side-effects. Moreover, EEPS treatment promoted apoptosis in CRC tumor tissues and in HT-29 cells, suggesting that the inhibitory effect of EEPS on tumor growth was due to its pro-apoptotic activity. Furthermore, EEPS treatment inhibited the expression of anti-apoptotic Bcl-2 but enhanced pro-apoptotic Bax expression at both transcriptional and translational levels. Finally, EEPS induced the loss of mitochondrial membrane potential and activation of caspase-9 and -3 in HT-29 cells. Taken together, data in this study suggest that induction of cancer cell apoptosis via the mitochondrial-dependent pathway may be one of the mechanisms whereby PS exerts anticancer activity.

Pien Tze Huang (PZH), a well-known traditional Chinese formula prescribed 450 years ago in the Ming Dynasty, has been used in China and Southeast Asia for centuries as a folk remedy for various types of cancer, including colorectal cancer (CRC). Recently, we reported that PZH is capable of inhibiting colon cancer growth both in vivo and in vitro via the promotion of apoptosis and inhibition of tumor angiogenesis. To elucidate the mechanism of the tumoricidal activity of PZH, its effect on the proliferation of human colon carcinoma Caco-2 cells was evaluated and the underlying molecular mechanism was investigated. Results showed that PZH inhibited Caco-2 cell viability and survival in a dose- and/or time-dependent manner. In addition, PZH treatment was found to block the G1/S cell cycle progression. Moreover, PZH suppressed the mRNA and protein expression of pro-proliferative Cyclin D1 and CDK4. Findings of the present study suggest that inhibition of cell proliferation via the G1/S cell cycle arrest is a potential mechanism by which PZH can be effective in the treatment of cancer.

Signal Transducer and Activator of Transcription 3 (STAT3), a common oncogenic mediator, is constitutively activated in many types of human cancers; therefore it is a major focus in the development of novel anti-cancer agents. Hedyotis diffusa Willd has been used as a major component in several Chinese medicine formulas for the clinical treatment of colorectal cancer (CRC). However, the precise mechanism of its anti-tumor activity remains largely unclear. Using a CRC mouse xenograft model, in the present study we evaluated the effect of the ethanol extract of Hedyotis diffusa Willd (EEHDW) on tumor growth in vivo and investigated the underlying molecular mechanisms. We found that EEHDW reduced tumor volume and tumor weight, but had no effect on body weight gain in CRC mice, demonstrating that EEHDW can inhibit CRC growth in vivo without apparent adverse effect. In addition, EEHDW treatment suppressed STAT3 phosphorylation in tumor tissues, which in turn resulted in the promotion of cancer cell apoptosis and inhibition of proliferation. Moreover, EEHDW treatment altered the expression pattern of several important target genes of the STAT3 signaling pathway, i.e., decreased expression of Cyclin D1, CDK4 and Bcl-2 as well as up-regulated p21 and Bax. These results suggest that suppression of the STAT3 pathway might be one of the mechanisms by which EEHDW treats colorectal cancer.

One of the most critical cellular signal transduction pathways known to malfunction in colorectal cancer is the interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) pathway. Scutellaria barbata D. Don (SB) is well-known traditional medicine in China that targets STAT3 signaling, and it has long been used to treat various types of cancer; however, the precise mechanism of its antitumor activity remains largely unclear. In order to further elucidate this underlying mechanism, an ethanol extract of SB (EESB) in cancer treatment. The aim of the present study was to evaluate the effects of EESB on the IL-6-inducible STAT3 pathway. We tested the dose-response association between EESB, IL-6-induced proliferaion and apoptosis using an MTT assay, colony formation and flow cytometry analysis in vitro. In addition, caspase-9 and caspase-3 activation was determined using a colorimetric assay, the activity of IL-6-induced STAT3 pathway was evaluated using western blot analysis, and the expression levels of cyclin D1, cyclin-dependent kinase 4, Bcl2 and Bcl2-associated X were determined using reverse transcription-polymerase chain reaction and western blot analysis. In the present study it was found that EESB could significantly inhibit the IL-6-mediated increase in STAT3 phosphorylation levels and transcriptional activity in HT-29 human colon carcinoma cells, resulting in the suppression of cell proliferation and the induction of apoptosis. In addition, treatment with EESB markedly inhibited the IL-6-induced upregulation of cyclin D1 and B-cell lymphoma-2, two key target genes of the STAT3 pathway. These results suggest that treatment with EESB could effectively inhibit the proliferation and promote the apoptosis of human colon carcinoma cells via modulation of the IL-6/STAT3 signaling pathway and its target genes.

Non‑alcoholic fatty liver disease (NAFLD) is a widespread health concern, and there is currently insufficient understanding regarding its pathogenesis and treatment. The present study aimed to explore the effects of Artemisia capillaris formula (ACF) on high‑fat diet‑induced hepatic steatosis and fatty acid‑induced intracellular lipid accumulation, by micro (mi)RNA regulation. A total of 72 Sprague‑Dawley rats were divided into six groups (n=12/group). One group was designated as the control group and fed a normal diet, and the remaining five groups were allowed ad libitum access to a high‑fat diet for eight weeks, in order to establish an NAFLD rat model. The rats were subsequently administered polyene phosphatidylcholine (PP; 0.076 g/kg body weight/day), low dose of ACF (0.462 g/kg body weight/day), middle dose of ACF (0.924 g/kg body weight/day) or high dose of ACF (1.848 g/kg body weight/day) intragastrically for four weeks. HepG2 human hepatocellular carcinoma cells were treated with oleic acid and palm acid, followed by treatment with various concentrations of ACF. Serum alanine transaminase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), high‑density lipoprotein cholesterol (HDL‑C), low‑density lipoprotein cholesterol (LDL‑C), and steatotic HepG2 human liver carcinoma cell TC and TG levels were measured. ACF and PP treatments attenuated high‑fat diet‑induced hepatic steatosis and fatty acid‑induced intracellular lipid accumulation. A modified high‑fat diet significantly increased ALT, AST, TG, TC, LDL‑C levels and decreased HDL‑C levels. Treatment with ACF and PP abrogated the increase in liver enzymes and TG, TC and LDL‑C levels, but did not influence HDL‑C levels in a high‑fat diet induced rat model of steotosis. Steatotic HepG2 cells exhibited significantly increased levels of both TG and TC. Treatment with ACF significantly decreased TC and TG levels in vivo, and ACF and PP treatment decreased the expression levels of fatty acid synthase (FASN) and increased miR‑122 in vivo and in vitro. In conclusion, these results suggested that ACF may inhibit hepatic steatosis via miR‑122‑induced downregulation of FASN in vivo and in vitro.

Tumor metastasis, a complex process involving the spread of malignant tumor cells from a primary tumor site to a distant organ, is a major cause of failure of cancer chemotherapy. Epithelial-mesenchymal transition (EMT) is a critical step for the initiation of cancer metastasis. The processes of EMT and metastasis are highly regulated by a double-negative feedback loop consisting of TGF-β1/ZEB pathway and miR-200 family, which therefore has become a promising target for cancer chemotherapy. Pien Tze Huang (PZH), a well-known traditional Chinese formula first prescribed in the Ming Dynasty, has been demonstrated to be clinically effective in the treatment of various types of human malignancy including colorectal cancer (CRC). Our published data proposed that PZH was able to induce apoptosis, inhibit cell proliferation and tumor angiogenesis, leading to the suppression of CRC growth in vitro and in vivo. To further elucidate the mode of action of PZH, in the present study we evaluated its effects on the metastatic capacities of human colorectal carcinoma HCT-8 cells and investigated the underlying molecular mechanisms. We found that PZH significantly inhibited the migration and invasion of HCT-8 cells in a dose-dependent manner. In addition, PZH treatment inhibited the expression of key mediators of TGF-β1 signaling, such as TGF-β1, Smad2/3 and Smad4. Moreover, PZH treatment suppressed the expression of ZEB1 and ZEB2, two critical target genes of TGF-β1 pathway, leading to a decrease in the expression of mesenchymal marker N-cadherin and an increased expression of epithelial marker E-cadherin. Furthermore, PZH treatment upregulated the expression of miR-200a, miR-200b and miR-200c. Collectively, our findings in this study suggest that PZH can inhibit metastasis of colorectal cancer cells via modulating TGF-β1/ZEB/miR-200 signaling network, which might be one of the mechanisms whereby PZH exerts its anticancer function.

Background The development of hepatic cancer is tightly regulated by multiple intracellular signaling pathways. Therefore, most currently-used anti-tumor agents, which typically target single intracellular pathway, might not always be therapeutically effective. Additionally, long-term use of these agents probably generates drug resistance and unacceptable adverse effects. These problems increase the necessity for the development of new chemotherapeutic approaches. Nitidine chloride (NC), a natural benzophenanthridine alkaloid, has been shown to inhibit cancer growth via induction of cell apoptosis and suppression of cancer angiogenesis. But the precise mechanisms of its tumorcidal activity are not well understood. Methods To further elucidate the precise mechanisms of its anti-tumor activity, using a hepatic cancer mouse xenograft model, the human hepatic cancer cell lines (HepG2, HCCLM3, Huh7), and umbilical vein endothelial cells (HUVEC), here we evaluate the effect of NC on tumor growth in vivo and in vitro and investigated the underlying molecular mechanisms. Results We found that NC treatment resulted in significant decrease in tumor volume and tumor weight respectively, but didn’t affect body weight changes. Additionally, NC treatment dose- and time-dependently reduced the cell viability of all three hepatic cell lines. Moreover, NC suppressed the activation of STAT3, ERK and SHH pathways; and altered the expression of critical target genes including Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR2. These molecular effects resulted in the promotion of apoptosis, inhibition of cell proliferation and tumor angiogenesis. Conclusions Our findings suggest that NC possesses a broad range of anti-cancer activities due to its ability to affect multiple intracellular targets, suggesting that NC could be a novel multi-potent therapeutic agent for the treatment of hepatic cancer and other cancers.

The pathogenic mechanisms underlying cancer development are complex and heterogeneous, involving multiple cellular signaling transduction pathways that usually function redundantly. In addition, crosstalk between these pathways generates a complicated and robust signaling network that is regulated by compensatory mechanisms. Given the complexity of cancer pathogenesis and progression, many of the currently used antitumor agents, which typically target a single intracellular pathway, might not always be effective on complex tumor systems. Moreover, long-term use of these agents often generates drug resistance and toxicity against normal cells. Therefore, the development of novel anticancer chemotherapies is urgently needed. Scutellaria barbata D Don (SB) is a medicinal herb that has long been used in China to treat various types of cancer. We previously reported that the ethanol extract of SB (EESB) is able to induce colon cancer cell apoptosis, inhibit cell proliferation and tumor angiogenesis via modulation of several pathways, including Hedgehog, Akt, and p53. To further elucidate the precise mechanisms of SB’s antitumor activity, using a colorectal cancer (CRC) mouse xenograft model in the present study, we evaluated the therapeutic efficacy and molecular mechanisms of EESB against tumor growth. We found that EESB reduced tumor volume and tumor weight but had no effect on body weight gain in CRC mice, demonstrating that EESB could inhibit colon cancer growth in vivo without apparent adverse effect. In addition, EESB treatment could significantly suppress the activation of several CRC-related pathways, including STAT3, Erk, and p38 signalings in tumor tissues, and alter the expression of multiple critical target genes such as Bcl-2, Bax, Cyclin D1, CDK4, and p21. These molecular effects lead to the induction of cancer cell apoptosis and inhibition of cell proliferation. Our findings demonstrate that SB possesses a broad range of antitumor activities because of its ability to affect multiple intracellular targets.

Accumulating evidence suggests that a small population of cells termed cancer stem cells (CSCs) are crucial in tumor development and drug resistance, leading to cancer relapse and metastasis and eventually the failure of clinical cancer treatment. Therefore, targeting CSCs is a promising approach for anticancer therapies. Due to the drug resistance and adverse effects of currently used chemo- therapies, traditional Chinese medicines (TCM) have recently received attention due to the relatively few side-effects. Thus, they have been used as important alternative remedies for various diseases, including cancer. Pien Tze Huang (PZH), a well-known TCM formula that was first prescribed more than 450 years ago in the Ming Dynasty, has been used in China and Southeast Asia for centuries as a folk remedy for various types of cancer. Previously, it was reported that PZH inhibits colon cancer growth via the promotion of cancer cell apoptosis and inhibition of cell proliferation and tumor angiogenesis, which is probably mediated by its regula- tory effect on multiple intracellular signaling pathways. To elucidate the mechanism of the tumoricidal activity of PZH, the aim of the present study was to investigate the effect of PZH on CSCs that were isolated as the side population (SP) from the HT-29 colorectal cancer cell line. The results demonstrated that PZH significantly and dose-dependently reduced the percentage of the colorectal cancer stem-like SP cells, decreased the viability and sphere-forming capacity of HT-29 SP cells, indicating that PZH is potent in suppressing the growth of colorectal cancer stem cells. Moreover, PZH treatment in HT-29 SP cells markedly inhibited the mRNA levels of ABCB1 and ABCG2, which are members of the ABC transporter superfamily, thereby contributing to the SP phenotype and multi-drug resistance. Findings of the present study suggest that inhibiting the growth of CSCs is a potential mechanism by which PZH can be used in cancer treatment.

Scutellaria barbata D. Don (SB) has long been used as a major component in numerous Chinese medical formulas to clinically treat various types of cancer. Previously, we reported that the extracts of SB were able to suppress colon cancer growth in vivo and in vitro, possibly by inducing cancer cell apoptosis and inhibiting cell proliferation and tumor angiogenesis. However, the anticancer mechanisms of its bioactive ingredients remain largely unclear. In the present study, using three human colon cancer cell lines SW620, HT-29 and HCT-8, the antitumor effect of different solvent fractions of SB were evaluated and the potential underlying molecular mechanisms were investigated. Using an 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, it was revealed that the chloroform fraction of SB (ECSB) exhibited the most potent inhibitory effect on the growth of all three colon cancer cell lines and SW620 cells exhibited the most sensitive response to ECSB treatment (IC50=65 µg/ml). In addition, by performing fluorescence-activated cell sorting, transmission electron microscopy and colony formation assays, it was observed that ECSB significantly induced apoptosis and inhibited proliferation in SW620 cells in a dose-dependent manner. Furthermore, ECSB treatment resulted in the upregulation of the pro-apoptotic Bax/Bcl-2 ratio and a decrease in the expression of the pro-proliferative cyclin D1 and cyclin-dependent kinase 4. The results from the present study may provide a scientific foundation for the development of novel anticancer agents from the bioactive ingredients in the ECSB.

Angiogenesis is an essential process for tumor development and metastasis, therefore inhibition of tumor angiogenesis has become a promising strategy for anticancer treatments. Patrinia scabiosaefolia, a well-known Oriental folk medicine, has been shown to be effective in the clinical treatment of gastrointestinal cancers. However, the precise mechanism of its tumoricidal activity remains largely unknown. Using a colorectal cancer (CRC) mouse xenograft model, the human colon carcinoma cell line HT-29 and human umbilical vein endothelial cells (HUVECs), in the present study we evaluated the effects of an ethanol extract of Patrinia scabiosaefolia (EEPS) on tumor angiogenesis in vivo and in vitro, and investigated the underlying molecular mechanisms. We found that EEPS treatment significantly reduced the tumor volume in CRC mice and decreased the intratumoral microvessel density in tumor tissues. In addition, EEPS inhibited several key processes of angiogenesis, including the proliferation, migration and tube formation of HUVECs. Moreover, EEPS treatment suppressed the expression of VEGF-A in CRC tumors and HT-29 cells. Collectively, our data suggest that Patrinia scabiosaefolia inhibits CRC growth likely via suppression of tumor angiogenesis.

Sonic hedgehog (SHH) signaling pathway promotes the process of angiogenesis, contributing to the growth and progression of many human malignancies including colorectal cancer (CRC), which therefore has become a promising target for cancer chemotherapy. Hedyotis diffusa Willd (HDW), as a well-known traditional Chinese herbal medicine, has long been used in China for the clinic treatment of various cancers. Recently, we reported that HDW can inhibit colorectal cancer growth in vivo and in vitro via suppression of the STAT3 pathway. In addition, we demonstrated the anti-angiogenic activity of HDW in vitro. To further elucidate the mechanism of the tumoricidal activity of HDW, by using a CRC mouse xenograft model we evaluated the in vivo effect of the ethanol extract of HDW (EEHDW) on tumor angiogenesis, and investigated the underlying molecular mechanisms. We found that EEHDW could significantly reduce intratumoral microvessel density (MVD), indicating its activity of antitumor angiogenesis in vivo. EEHDW suppressed the activation of SHH signaling in CRC xenograft tumors since it significantly decreased the expression of key mediators of SHH pathway. EEHDW treatment inhibited the expression of the critical SHH signaling target gene VEGF-A as well as its specific receptor VEGFR2. Taken together, we propose for the first time that Hedyotis diffusa Willd inhibits colorectal cancer growth in vivo via inhibition of SHH-mediated tumor angiogenesis.

Qingxuan Jiangya Decoction (QXJYD), a traditional Chinese medicine formula prescribed by academician Ke-ji Chen, has been used in China to clinically treat hypertension for decades of years. However, the molecular mechanisms of its action remain largely unknown. In this study, we examined the therapeutic efficacy of QXJYD against elevated systolic blood pressure in the spontaneously hypertensive rat (SHR) model, and investigated the underlying molecular mechanisms. We found that oral administration of QXJYD significantly reduced the elevation of systolic blood pressure in SHR but had no effect on body weight change. Additionally, QXJYD treatment significantly decreased the media thickness and ratio of media thickness/lumen diameter in the carotid arteries of SHR. Moreover, QXJYD remarkably promoted apoptosis of vascular smooth muscle cells and reduced the expression of anti-apoptotic B-cell leukemia/lymphoma 2. Furthermore, QXJYD significantly decreased the plasma Angiotensin II level in SHR. Collectively, our findings suggest that reversing vascular remodeling via inducing VSMC apoptosis could be one of the mechanisms whereby QXJYD treats hypertension.

IL-6/STAT3 is one of the most critical cellular signal transduction pathways known to malfunction in colorectal cancer (CRC). As a target gene of signal transducer and activator of transcription 3 (STAT3) signaling, suppressor of cytokine signaling 3 (SOCS3) can be quickly induced by interleukin-6 (IL-6) stimulation but it then strongly inhibits IL-6-mediated STAT3 activation, functioning as a negative feedback regulator of the IL-6/STAT3 pathway. Aberrant activation of STAT3 and/or reduced expression of SOCS are strongly correlated with carcinogenesis, which therefore becomes a promising target for the development of novel anticancer chemotherapies. Pien Tze Huang (PZH) is a well-known traditional Chinese formula that was first prescribed by a royal physician 450 years ago in the Ming Dynasty. It has been used in China and Southeast Asia for centuries as a folk remedy for various types of cancer including CRC. However, the precise mechanism of its antitumor activity remains largely unclear. In the present study, we found that PZH could significantly and dose-dependently inhibit IL-6-mediated increase of STAT3 phosphorylation levels and transcriptional activity in the human colon carcinoma HT-29 cells, resulting in the suppression of cell proliferation and the induction of apoptosis. In addition, PZH treatment profoundly inhibited IL-6-induced upregulation of cyclin D1 and Bcl-2, two key target genes of the STAT3 pathway. Moreover, PZH treatment increased the expression of SOCS3. These results suggest that PZH could effectively inhibit proliferation and promote apoptosis of human colon carcinoma cells via modulation of the IL-6/STAT3 signaling pathway and its target genes.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in cell survival and proliferation. Constitutive activation of STAT3 is strongly correlated with pathogenesis of various types of malignant tumors including colorectal cancer (CRC), and therefore is a major focus in the development of anti-cancer agents. Pien Tze Huang (PZH), a well-known traditional Chinese formula prescribed already in the Ming Dynasty, has been demonstrated to be clinically effective in the treatment of CRC. However, the precise mechanism of its anti-cancer activity remains largely unknown. In the present study we evaluated the efficacy of PZH against tumor growth in vivo in the CRC mouse xenograft model, and investigated the underlying molecular mechanisms. We found that administration of PZH reduced tumor volume and tumor weight but had no effect on body weight gain in CRC mice, demonstrating that PZH can inhibit colon cancer growth in vivo without apparent adverse effect. We also observed that PZH treatment inhibited the phosphorylation level of STAT3 in tumor tissues. Consequently, the inhibitory effect of PZH on STAT3 activation resulted in the up-regulation of Bax/Bcl-2 ratio as well as down-regulation of Cyclin D1 and CDK4 expression, leading to the induction of apoptosis as well as the inhibition of cell proliferation. These results suggest that promotion of cancer cell apoptosis and inhibition of proliferation via suppression of STAT3 pathway might be one of the mechanisms by which PZH treats colorectal cancer.