Qiaoyan Cai

Hedyotis diffusa Willd Inhibits Colorectal Cancer Growth in Vivo via Inhibition of STAT3 Signaling Pathway

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.

Scutellaria barbata D. Don Inhibits Tumor Angiogenesis via Suppression of Hedgehog Pathway in a Mouse Model of Colorectal Cancer

Angiogenesis, which plays a critical role during tumor development, is tightly regulated by the Sonic Hedgehog (SHH) pathway, which has been known to malfunction in many types of cancer. Therefore, inhibition of angiogenesis via modulation of the SHH signaling pathway has become very attractive for cancer chemotherapy. Scutellaria barbata D. Don (SB) has long been used in China to treat various cancers including colorectal cancer (CRC). Our published data suggested that the ethanol extract of SB (EESB) is able to induce apoptosis of colon cancer cells and inhibit angiogenesis in a chick embryo chorioallantoic membrane model. To further elucidate the precise mechanisms of its anti-tumor activity, in the present study we used a CRC mouse xenograft model to evaluate the effect of EESB on tumor growth and angiogenesis in vivo. Our current data indicated that EESB reduces tumor size without affecting on the body weight gain in CRC mice. In addition, EESB treatment suppresses the expression of key mediators of the SHH pathway in tumor tissues, which in turn resulted in the inhibition of tumor angiogenesis. Furthermore, EESB treatment inhibits the expression of vascular endothelial growth factor A (VEGF-A), an important target gene of SHH signaling and functioning as one of the strongest stimulators of angiogenesis. Our findings suggest that inhibition of tumor angiogenesis via suppression of the SHH pathway might be one of the mechanisms by which Scutellaria barbata D. Don can be effective in the treatment of cancers.

Hedyotis diffusa Willd extract suppresses Sonic hedgehog signaling leading to the inhibition of colorectal cancer 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.

Nitidine chloride inhibits hepatocellular carcinoma cell growth in vivo through the suppression of the JAK1/STAT3 signaling pathway.

Signal transducer and activator of transcription 3 (STAT3) is persistently activated in cancer cells and contributes to malignant progression in various types of cancer. The Janus-activated kinase (JAK) family phosphorylates STAT3 in response to stimulation by cytokines or growth factors. The JAK1-STAT3 signaling pathway plays an important role in cell proliferation and apoptosis. Nitidine chloride (NC) is a benzophenanthridine alkaloid that has been reported as an antitumor agent due to its its inhibitory effects on topoisomerase I. Using a mouse xenograft model of hepatocellular carcinoma (HCC), this study aimed to evaluate the effects of NC on tumor growth in vivo and to elucidate the underlying mechanisms. The analysis of the effects of NC on apoptosis in HCC tumor xenografts in mice was carried out by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay; the expression of Bcl-2, Bax, cyclin-dependent kinase (CDK)4, cyclin D1, p21 and proliferating cell nuclear antigen (PCNA) was analyzed by immunohistochemistry; and the protein expression of JAK1 and STAT3 was examined by western blot analysis. Our results revealed that treatment with NC decreased the tumor volume and tumor weight, suggesting that NC inhibits HCC cell growth in vivo. In addition, NC blocked the activation of JAK1-STAT3 in the tumor tissues, which in turn resulted in the induction of cancer cell apoptosis and the inhibition of proliferation. Consequently, treatment with NC downregulated the expression of cyclin D1, CDK4 and Bcl-2 and increased the level of p21 and Bax. Our data provide a molecular basis for the antitumor activity of NC.

Scutellaria Barbata D Don Inhibits Colorectal Cancer Growth via Suppression of Multiple Signaling Pathways

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.

Chloroform fraction of Scutellaria barbata D. Don promotes apoptosis and suppresses proliferation in human colon cancer cells

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.

Experimental study of low-frequency electroacupuncture-induced differentiation of bone marrow mesenchymal stem cells into chondrocytes.

In the present study, we investigated the effect of low-frequency electroacupuncture (EA) on the differentiation of bone mesenchymal stem cells (BMSCs) into chondrocytes and the molecular mechanism involved. We isolated BMSCs from Sprague-Dawley (SD) rat bone marrow. Third-generation SD rat BMSCs (P3 BMSCs) were harvested and characterized by flow cytometry with FITC staining. Data indicated that the positive rates of CD90 and CD45 were 98.22 and 1.91%, respectively, indicating the high purity of the BMSCs. The P3 BMSCs were treated with EA for 15 or 30 min daily for 7 or 14 days. Using optical microscopy and transmission electron microscopy, we found that EA induced morphological changes in the BMSCs, displaying typical morphology of early chondrocytes. In addition, we found that the cytoplasm and extracellular matrices were metachromatically stained by toluidine blue in the treated cells in a dose-dependent manner, indicating that EA treatment resulted in the expression of glycosaminoglycan. Furthermore, upon immunohistocytochemical staining and Western blotting, we found that EA treatment significantly and dose-dependently induced expression of chondrocyte-specific matrix protein type II collagen, which may have been mediated by the transcription factor Sox9, as the mRNA expression of Sox9 was found to be significantly increased after EA treatment. Taken together, these results suggest that EA can be employed as a novel non-drug-inducing method for the differentiation of BMSCs into chondrocytes.

Pien Tze Huang inhibits the proliferation, and induces the apoptosis and differentiation of colorectal cancer stem cells via suppression of the Notch1 pathway.

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.

Livistona chinensis seed suppresses hepatocellular carcinoma growth through promotion of mitochondrial-dependent apoptosis.

The Livistona chinensis seed has been used for centuries to clinically treat various types of cancer. However, the precise mechanism of its anticancer activity remains to be elucidated. In the present study, we evaluated the efficacy of the ethanol extract of Livistona chinensis seed (EELC) against tumor growth using a hepatocellular carcinoma (HCC) mouse xenograft model and an HCC cell line, HepG2, and investigated the molecular mechanisms mediating its biological activities. We found that EELC inhibited HCC growth both in vivo and in vitro, without apparent signs of toxicity. In addition, EELC treatment resulted in the induction of HCC cell apoptosis. Moreover, EELC-induced apoptosis was accompanied by the loss of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase in the pro-apoptotic Bax/Bcl-2 ratio. Our findings suggest that promotion of mitochondrial-dependent apoptosis in cancer cells may be one of the mechanisms by which the Livistona chinensis seed is effective in cancer treatment.

Pien Tze Huang inhibits the proliferation of colorectal cancer cells by increasing the expression of miR‑34c‑5p

MicroRNAs (miRNAs) are small, short endogenous non-coding RNA that act as oncogenes or tumor suppressors, and serve an important role in various human malignant cancers, including colorectal cancer (CRC). Evidence has indicated that miRNAs regulate the expression of various genes associated with human cancer, in particular the miR-34 family. A well-known traditional Chinese formula, Pien Tze Huang (PZH), has a significant clinical effect on CRC. Previous studies have demonstrated that PZH inhibits CRC growth in vitro and in vivo via multiple mechanisms, including the induction of apoptosis, inhibition of cell proliferation and tumor angiogenesis. To further elucidate the molecular mechanisms underlying the antitumor activity of PZH, in the present study its effects on cell proliferation and miRNA expression in human colon carcinoma (HCT)-8 cell lines was examined. It was observed that treatment with PZH inhibited cell viability and upregulated the expression of miR-34c-5p in HCT-8 cells. In addition, transfection with an miR-34c-5p mimic and treatment with PZH inhibited cell survival and arrested the cell cycle between the G0/G1 and S phase in HCT-8 cells. Furthermore, PZH treatment and transfection with miR-34c-5p downregulated the expression of cyclin-dependent kinase 4 and cMyc (a promoter of cell proliferation), and increased the expression of p53, which is a promoter of apoptosis. These results suggest that PZH may suppress proliferation in CRC cells by upregulating the expression of miR-34c-5p, which provides a novel perspective for understanding the mode of action of PZH.