Berberine ameliorates lipopolysaccharide-induced acute lung injury via the PERK-mediated Nrf2/HO-1 signaling axis.

A fundamental element of acute lung injury (ALI) is the inflammatory response, which can affect the entire respiratory system, including the respiratory tract and alveoli. Berberine has gained attention because of its anti-inflammatory effects. Nuclear factor-erythroid 2-related factor 2 (Nrf2) and endoplasmic reticulum (ER) stress are involved in lung injury. Nrf2 also acts as a protein kinase-like ER kinase (PERK) substrate in heart disease. Therefore, this study investigated the effect of berberine against lipopolysaccharide (LPS)-induced ALI and the role of the PERK-mediated Nrf2/HO-1 signaling axis. Berberine promoted Nrf2 nuclear translocation and phosphorylation in vitro. After LPS stimulation, this effect was further enhanced, whereas inflammatory factor (IL-6 and IL-8) release and reactive oxygen species generation were significantly decreased. Berberine effectively alleviated lung injury by reducing lung edema and neutrophil infiltration. Berberine also significantly reduced histopathological inflammatory changes via inhibition of ER stress and activation of Nrf2 signaling. Thapsigargin-induced ER stress and small interference RNA (siRNA)-mediated Nrf2 inhibition abrogated the protective effects of berberine in vitro, whereas siRNA-mediated suppression of ER stress and sulforaphane-induced Nrf2 activation further improved those effects. Importantly, ER stress induction led to Nrf2 activation, whereas PERK depletion partly reduced the level of Nrf2 phosphorylation and translocation in LPS-induced cells. Therefore, berberine inhibits LPS-induced ALI through the PERK-mediated Nrf2/HO-1 signaling axis.

Icariin protects cardiomyocytes against ischaemia/reperfusion injury by attenuating sirtuin 1-dependent mitochondrial oxidative damage.

BACKGROUND AND PURPOSE: Icariin, a major active ingredient in traditional Chinese medicines, is attracting increasing attention because of its unique pharmacological effects against ischaemic heart disease. The histone deacetylase, sirtuin-1, plays a protective role in ischaemia/reperfusion (I/R) injury, and this study was designed to investigate the protective role of icariin in models of cardiac I/R injury and to elucidate the potential involvement of sirtuin-1. EXPERIMENTAL APPROACH: I/R injury was simulated in vivo (mouse hearts), ex vivo (isolated rat hearts) and in vitro (neonatal rat cardiomyocytes and H9c2 cells). Prior to I/R injury, animals or cells were exposed to icariin, with or without inhibitors of sirtuin-1 (sirtinol and SIRT1 siRNA). KEY RESULTS: In vivo and in vitro, icariin given before I/R significantly improved post-I/R heart contraction and limited the infarct size and leakage of creatine kinase-MB and LDH from the damaged myocardium. Icariin also attenuated I/R-induced mitochondrial oxidative damage, decreasing malondialdehyde content and increasing superoxide dismutase activity and expression of Mn-superoxide dismutase. Icariin significantly improved mitochondrial membrane homeostasis by increasing mitochondrial membrane potential and cytochrome C stabilization, which further inhibited cell apoptosis. Sirtuin-1 was significantly up-regulated in hearts treated with icariin, whereas Ac-FOXO1 was simultaneously down-regulated. Importantly, sirtinol and SIRT1 siRNA either blocked icariin-induced cardioprotection or disrupted icariin-mediated mitochondrial homeostasis. CONCLUSIONS AND IMPLICATIONS: Pretreatment with icariin protected cardiomyocytes from I/R-induced oxidative stress through activation of sirtuin-1 /FOXO1 signalling.

Scutellaria barbata D. Don inhibits migration and invasion of colorectal cancer cells via suppression of PI3K/AKT and TGF-ß/Smad signaling pathways.

Metastasis is one of the most aberrant behaviors of cancer cells. Patients with cancers, including colorectal cancer (CRC), have a higher risk of tumor recurrence and cancer-related mortality once metastasis is diagnosed. Existing treatment strategies fail to cure cancer mostly due to the onset of metastasis. Therefore, metastasis remains a challenge in cancer treatment. Some complementary and alternative medical therapies using traditional Chinese medicine have been demonstrated to be clinically effective in cancer treatment. Scutellaria barbata D. Don (SB) is a promising medicinal herb. It was previously reported that the ethanol extract of SB (EESB) is able to promote apoptosis, and inhibit cell proliferation and angiogenesis in human colon cancer cells. However, the anticancer effect of SB and the underlying mechanism require further investigation, particularly its role against metastasis. To further elucidate the antimetastatic effect of SB, MTT and Transwell assays were used in the present study to evaluate the effect of EESB on the proliferation, migration and invasion of the CRC cell line HCT-8. In addition, western blot analysis was performed to detect the expression of matrix metalloproteinases (MMPs), cadherins and other metastasis-associated proteins. EESB significantly reduced HCT-8 cell viability and attenuated the migration and invasion ability of HCT-8 cells in a dose-dependent manner. In addition, EESB decreased the expression of MMP-1, MMP-2, MMP-3/10, MMP-9 and MMP-13, and proteins in the phosphoinositide 3-kinase (PI3K)/AKT and transforming growth factor (TGF)-ß/Smad pathways, but not the epithelial-mesenchymal transition (EMT)-related factors E-cadherin and N-cadherin. In conclusion, the results suggested that SB inhibits CRC cell metastasis via the suppression of PI3K/AKT and TGF-ß/Smad signaling pathways, which may represent a mechanism by which SB exerts an anticancer effect.

Hedyotis diffusa Willd suppresses metastasis in 5­fluorouracil­resistant colorectal cancer cells by regulating the TGF­ß signaling pathway.

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract, and threatens the survival and health of patients with CRC. Chemotherapy remains one of the main therapeutic approaches for patients with CRC; however, drug resistance limits the long­term use. CRC cells with multi­drug resistance (MDR) exhibit increased survival times and metastatic potential, which may lead to the recurrence and metastasis of CRC. In addition, MDR is one of the major causes of chemotherapy failure in clinical treatment. Hedyotis diffusa Willd (HDW) has been used in the treatment of inflammation­associated diseases and malignant tumors, including CRC. The authors previously demonstrated that HDW could reverse MDR in CRC cells; however, its underlying mechanism, particularly in MDR­associated metastasis, remains to be elucidated. In the present study, the drug­resistant CRC cell line HCT­8/5­fluorouracil (5­FU) was used to investigate the effect of HDW on the growth and metastasis of cancer cells. Cell viability was assessed using the MTT assay. Cell adhesion potential was evaluated using adhesion experiments. Cell migration was assessed using wound healing and Transwell assays. The mRNA and protein expression levels of crucial factors in the transforming growth factor­ß (TGF­ß) signaling pathway, including TGF­ß, Mothers against decapentaplegic homolog 4 (SMAD4), neural (N)­cadherin, and epithelial (E)­cadherin, were analyzed using the reverse transcription­semi­quantitative polymerase chain reaction and western blotting, respectively. The results demonstrated that the HCT­8/5­FU cell line was more resistant to 5­FU and thus can be used as the resistant cell model. HDW was able to inhibit the viability, and adhesive, migratory and invasion potential of the HCT­8/5­FU cells. In addition, HDW was able to downregulate the expression of TGF­ß, SMAD4 and N­cadherin, and upregulate E­cadherin, at the gene and protein level. In conclusion, the results demonstrated that HDW may suppress the metastasis of 5­FU­resistant CRC cells via regulation of the TGF­ß signaling pathway, which was also considered to be one of the underlying mechanisms of its anti­CRC effect.

Scutellaria barbata D. Don inhibits 5-fluorouracil resistance in colorectal cancer by regulating PI3K/AKT pathway.

5-Fluorouracil (5-FU) resistance or multidrug resistance (MDR) has become a major obstacle in clinical treatment of cancers including colorectal cancer (CRC). Aberrant activation of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway may lead to unlimited growth and chemoresistance in CRC cells, which thus could be a promising therapeutic target. As a long-term used traditional Chinese folk-medicine, Scutellaria barbata D. Don (SB) processes specific anticancer activity, but its activity against cancer chemoresistance is less known. Therefore, using a 5-FU-resistant CRC cell line HCT-8/5-FU, in this study we evaluated the therapeutic efficacy of the ethanol extracts of SB (EESB) against 5-FU resistance and explored the possible molecular mechanisms. We found that EESB significantly suppressed proliferation and promoted apoptosis in HCT-8/5-FU cells. Additionally, EESB displayed remarkable effect enhancing the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine­123 (Rh­123) in HCT-8/5-FU cells. Furthermore, EESB obviously downregulated the expression of cyclin D1, Bcl-2 and ABCG2, while upregulated p21 and Bax expression. Moreover, EESB showed a prominent suppressive effect on the activation of PI3K/AKT pathway. The findings suggested that Scutellaria barbata D. Don was able to inhibit chemoresistance in colorectal cancer by suppression of the PI3K/AKT pathway.

Spica Prunellae extract suppresses the growth of human colon carcinoma cells by targeting multiple oncogenes via activating miR-34a.

Spica Prunellae is the spike of the herb Prunella vulgaris L. in traditional Chinese medicine which is often used for the treatment of various cancers including colorectal cancer. In the present study, we found that a key tumor suppressor, microRNA-34a (miR-34a) is involved in the antitumor activity for Spica Prunellae. Human colon carcinoma HCT-8 cells treated with an ethanol extract of Spica Prunellae (EESP) had significantly decreased cell proliferation and viability, in a dose-dependent manner. Flow cytometry analysis with Annexin V/PI staining analysis revealed that EESP treatment could induce apoptosis of HCT-8 cells. The level of miR-34a was upregulated in HCT-8 cells following EESP treatment, whereas expression levels of its target genes Notch1, Notch2 and Bcl-2 were downregulated. Inhibition of miR-34a rescued the expression of these target genes. These results revealed that Spica Prunellae can suppress the growth of HCT-8 cells by targeting Notch1, Notch2 and Bcl-2 via activation of miR-34a.

Chloroform fraction of Scutellaria barbata D. Don inhibits the growth of colorectal cancer cells by activating miR­34a.

Scutellaria barbata D. Don (SB) is a well known formula in traditional Chinese medicine, which exhibits potent anticancer effects on various cancers. Many miRNAs play crucial roles in the regulation of cancer, for instance, miR­34a functions as a tumor suppressor, and is often downregulated during cancer. In this study, we investigated the role of ECSB in suppressing the growth of human colon cancer HCT­8 cells, and whether this is mediated by regulation of miR­34a and its downstream target genes, using real-time PCR and western blot analysis. ECSB treatment significantly inhibited the proliferation of HCT­8 cells and promoted apoptosis in a dose-dependent manner. In addition, ECSB treatment significantly increased the level of miR­34a expression and decreased the levels of Bcl-2, Notch1/2 and Jagged1 expression. Furthermore, knockdown of miR­34a expression through transfection of anti-miR­34a oligonucleotide was significantly reversed by ECSB treatment. Likewise, knockdown of miR­34a resulted in significant upregulation of Bcl-2, Notch1/2 and Jagged1 expression, which was reversed following ECSB treatment. Therefore, this study reveals that ECSB inhibited cancer cell growth via promoting apoptosis and inhibiting proliferation, through regulation of miR­34a. These findings further support the use of ECSB as an effective therapeutic agent against colon cancer.

Chloroform extract of Hedyotis diffusa Willd inhibits viability of human colorectal cancer cells via suppression of AKT and ERK signaling pathways.

Hedyotis diffusa Willd (HDW) is a widely used traditional Chinese medicine in clinical therapy to treat various types of cancer, including colorectal cancer (CRC), but its effective polar fractions and functional mechanisms remain unclear. The aim of the present study was to determine the most effective extract of HDW and to investigate its effects on the regulation of CRC cell proliferation and apoptosis, as well as to investigate the underlying molecular mechanisms. The results demonstrated that the chloroform extract of HDW (CEHDW) exhibited the most anticancer ability. Furthermore, results of the MTT assay, colony formation, carboxyfluorescein diacetate succinimidyl ester assay and annexin V/propidium iodide staining suggested that CEHDW significantly inhibits proliferation and promotes apoptosis in the SW620 CRC cell line. Additionally, reverse transcription-polymerase chain reaction and western blot analysis demonstrated that CEHDW treatment downregulated the expression of Survivin, proliferating cell nuclear antigen, Cyclin D1, cyclin-dependent kinase 4 and B-cell lymphoma 2 (Bcl-2), and upregulated the expression of Bcl-2-associated X protein at the mRNA and protein levels. CEHDW also decreased the phosphorylation of protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK), which indicated that the suppression of the AKT and ERK signaling pathways may be one of the underlying molecular mechanisms by which CEHDW exhibited its anticancer effect. Thus, CEHDW may be a promising agent for anticancer therapy.

Hedyotis diffusa willd extract suppresses colorectal cancer growth through multiple cellular pathways.

The development of colorectal cancer (CRC) is strongly associated with the imbalance of various intracellular signal transduction cascades, including protein kinase B (AKT), mitogen-activated protein kinase 1 (MAPK), signal transducer and activator of transcription 3 (STAT3), as well as crosstalk between these signaling networks. At present, anti-tumor agents are often single-targeted and therefore are not always therapeutically effective. Moreover, long-term use of these anti-tumor agents often generates drug resistance and potential side effects. These problems highlight the urgent need for the development of novel and more effective anti-cancer drugs. Hedyotis diffusa Willd (HDW) has been used as a major component in traditional Chinese medicine for the clinical treatment of colorectal cancer, with a limited number of adverse effects. However, the molecular mechanisms, which underlie its anti-cancer activity, still require further elucidation. In the present study, using xenograft models and various different human CRC cell lines, the efficacy of the ethanol extract of HDW (EEHDW) against tumor growth was evaluated, and its underlying molecular mechanisms of action were investigated. It was demonstrated that EEHDW was able to inhibit cancer growth in vivo and in vitro. Furthermore, EEHDW was able to suppress the activation of several CRC-associated signaling pathways and was able to regulate the expression of various inflammatory and angiogenic factors. This resulted in the induction of apoptosis and inhibition of cellular proliferation, as well as tumor angiogenesis. The present study demonstrated that EEHDW is able to exhibit anti-cancer activity due to its ability to affect multiple intracellular targets, which suggests that it may be a novel multi-potent therapeutic agent for the treatment of colorectal cancer.

Pien Tze Huang suppresses VEGF-C-mediated lymphangiogenesis in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. The majority of patients are not suitable for surgery due to the presence of metastatic disease at the time of diagnosis, which has led to a high mortality rate for patients with CRC. Lymphangiogenesis, formation of new lymphatic vessels, plays an critical role in cancer progression particularly in cancer metastasis. Vascular endothelial growth factor-C (VEGF-C) has been previously demonstrated to play a pivotal role in cancer metastasis and therefore has become an attractive target for anticancer treatments. Pien Tze Huang (PZH) is a well-known traditional Chinese formula, which has exhibited significant therapeutic effects against CRC. However, the molecular mechanisms underlying its anticancer effects, particularly in regards to antimetastasis activity, still require further elucidation. In the present study, we evaluated the effects of PZH on cell migration and VEGF-C expression using various human CRC cell lines. Moreover, using a VEGF­C-stimulated human lymphatic endothelial cell (HLEC) model, we demonstrated that PZH suppresses lymphangiogenesis by attenuating cell migration and tube formation. This indicates that PZH possesses significant antimetastatic activity. Moreover, suppression of lymphangiogenesis by PZH via the downregulation of VEGF-C may be a potential molecular mechanism by which PZH inhibits metastasis in CRC.

Inhibitory effects of Hedyotis diffusa Willd. on colorectal cancer stem cells.

Cancer stem cells (CSCs) are proposed to be closely correlated with the development and progression of tumors, as well as with chemo- and radioresistance. Targeting CSCs may therefore be a promising potential strategy for the treatment of cancer. Currently, natural products have received great interest due to their therapeutic efficacy and reduced adverse effects compared with modern chemotherapeutics. As a significant component of a number of traditional Chinese medicine formulas, the medicinal herb Hedyotis diffusa Willd. (HDW) has long been utilized in China to clinically treat a variety of malignancies, including colorectal cancer (CRC). Previously, the authors of the present study reported that HDW suppressed CRC growth through multiple mechanisms, including promoting apoptosis, and inhibiting cell proliferation and tumor angiogenesis. To additionally investigate its mode of action, the present study isolated a stem-like side population (SP) from colorectal cancer HT-29 cells to investigate the effect of ethanol extract of HDW on CSCs. It was observed that HDW was able to markedly downregulate the expression of CSC marker leucine-rich repeat-containing G-protein coupled receptor 5 and also significantly decrease the proportion of SP in HT-29 cells, in a dose-dependent manner. Furthermore, HDW treatment significantly and dose-dependently inhibited the viability and sphere formation, and induced cell morphological changes of isolated HT-29 SP cells. In addition, HDW greatly suppressed the messenger RNA expression of several critical genes that mediate CSC features, including ATP-binding cassette, sub-family B, member 1, ß-catenin, c-Myc, proliferating cell nuclear antigen and survivin. In conclusion, the present study indicates that HDW may exert inhibitory effects on cancer stem cells.

Pien Tze Huang Inhibits Hypoxia-Induced Angiogenesis via HIF-1 α /VEGF-A Pathway in Colorectal Cancer.

Hypoxia-induced angiogenesis plays an important role in the development and metastasis of solid tumors and is highly regulated by HIF-1α/VEGF-A pathway. Therefore, inhibiting tumor angiogenesis via suppression of HIF-1α/VEGF-A signaling represents a promising strategy for anticancer treatment. As a traditional Chinese medicine formula, Pien Tze Huang (PZH) has long been used as a folk remedy for cancer in China and Southeast Asia. Previously, we reported that PZH inhibits colorectal cancer (CRC) growth both in vivo and in vitro. To elucidate the antitumor mechanisms of PZH, in the present study we used human umbilical vein endothelial cells (HUVEC) and colorectal carcinoma HCT-8 cells to evaluate the effects of PZH on hypoxia-induced angiogenesis and investigated the underlying molecular mechanisms. We found that PZH could inhibit hypoxia-induced migration and tube formation of HUVEC cells in a dose-dependent manner, although the low concentrations of PZH had no effect on HUVEC viability. Moreover, PZH inhibited hypoxia-induced activation of HIF-1α signaling and the expression of VEGF-A and/or VEGFR2 in both HCT-8 and HUVEC cells. Collectively, our findings suggest that PZH can inhibit hypoxia-induced tumor angiogenesis via suppression of HIF-1α/VEGF-A pathway.

Pien Tze Huang inhibits liver metastasis by targeting TGF-ß signaling in an orthotopic model of colorectal cancer.

Metastasis is the leading cause of cancer-related mortality in almost all types of cancers, including colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) is a critical process during the metastatic cascade. This process may be a potential target for the diagnosis and treatment of CRC. Pien Tze Huang (PZH), a well-known traditional Chinese formula, has been demonstrated to be clinically effective in treating various types of human malignancies, including CRC. Our published data suggest that PZH can induce apoptosis, as well as inhibit cell proliferation and tumor angiogenesis, thus suppressing CRC growth in vitro and in vivo. We evaluated the therapeutic efficacy of PZH against CRC metastasis using a CRC liver metastasis mouse model to further explore the mechanisms underlying the antitumor action of PZH. MTT, migration, and Matrigel invasion assays were used to assess the effect of PZH on cell viability, migration and invasion. We then established an orthotopic liver metastasis model of colon cancer using microsurgical techniques. Mice were intragastrically administered 234 mg/kg/day dose of either PZH or saline for 14 days. The body and tumor weights of the mice were measured after they were sacrificed. Moreover, we examined the effect of PZH inhibition on liver metastasis. Finally, EMT-related proteins and the TGF-ß signaling pathway were assessed using immunohistochemical staining (IHS). The present data revealed that PZH significantly inhibited the migration and invasion of CT-26 cells in a dose-dependent manner, which affirmed the inhibitory effect of PZH on CRC cell metastasis. No significant change was observed between the in vivo primary tumor growth and body weight. However, the control group had five cases of liver metastasis (5/6), whereas one case was found in the PZH group (1/6). Thus, PZH exhibited therapeutic efficacy against CRC metastasis without apparent toxicity. The inhibitory effect of PZH on EMT resulted in an increase in E-cadherin expression, as well as a decrease in N-cadherin expression. In addition, PZH significantly inhibited TGF-ß, as well as the phosphorylation of Smad2/3 and Smad4 in the tumor tissues, indicating its suppressive action on TGF-ß signaling. These molecular effects ultimately resulted in the inhibition of cancer cell EMT and tumor metastasis.