Combined therapeutic effects of vinblastine and Astragalus saponins in human colon cancer cells and tumor xenograft via inhibition of tumor growth and proangiogenic factors.

Our previous study had demonstrated that Astragalus saponins (AST) could reduce the side effects of orthodox chemotherapeutic drugs, while concurrently promote antitumor activity. In the present study, we attempted to investigate the potential synergistic anticarcinogenic effects of AST and a vinca alkaloid vinblastine (VBL). Reduced expression of key proangiogenic and metastatic factors including VEGF, bFGF, metalloproteinase (MMP)-2, and MMP-9 was detected in VBL-treated colon cancer cells, with further downregulation by combined VBL/AST treatment. Subsequently, VBL or AST decreased LoVo cell invasiveness, with further reduction when the drugs were cotreated. Significant growth inhibition and cell cycle arrest at G2/M phase were achieved by either drug treatment with apparent synergistic effects. VBL-induced apoptosis was confirmed but found to be unrelated to induction of the novel apoptotic protein NSAID-activated gene 1. In vivo study in tumor xenograft indicates that combined VBL/AST treatment resulted in sustained regression of tumor growth, with attenuation of the neutropenic and anemic effects of VBL. In addition, downregulation of proangiogenic and proliferative factors was also visualized, with boosting effect by combined drug treatment. These findings have provided evidence that AST combined with adjuvant chemotherapeutics like VBL could alleviate cancer development through diversified modes of action, including the regulation of angiogenesis.

Astragalus saponins modulates colon cancer development by regulating calpain-mediated glucose-regulated protein expression.

BACKGROUND: Glucose-regulated proteins (GRP) are induced in the cancer microenvironment to promote tumor survival, metastasis and drug resistance. AST was obtained from the medicinal plant Astragalus membranaceus, which possesses anti-tumor and pro-apoptotic properties in colon cancer cells and tumor xenograft. The present study aimed to investigate the involvement of GRP in endoplasmic reticulum (ER) stress-mediated apoptosis during colon cancer development, with focus on the correlation between AST-evoked regulation of GRP and calpain activation. METHODS: The effects of AST on GRP and apoptotic activity were assessed in HCT 116 human colon adenocarcinoma cells. Calpain activity was examined by using a fluorescence assay kit. Immunofluorescence staining and immunoprecipitation were employed to determine the localization and association between calpains and GRP. GRP78 gene silencing was performed to confirm the importance of GRP in anticancer drug activities. The modulation of GRP and calpains was also studied in nude mice xenograft. RESULTS: ER stress-mediated apoptosis was induced by AST, as shown by elevation in both spliced XBP-1 and CHOP levels, with parallel up-regulation of GRP. The expression of XBP-1 and CHOP continued to increase after the peak level of GRP was attained at 24 h. Nevertheless, the initial increase in calpain activity as well as calpain I and II protein level was gradually declined at later stage of drug treatment. Besides, the induction of GRP was partly reversed by calpain inhibitors, with concurrent promotion of AST-mediated apoptosis. The knockdown of GRP78 by gene silencing resulted in higher sensitivity of colon cancer cells to AST-induced apoptosis and reduction of colony formation. The association between calpains and GRP78 had been confirmed by immunofluorescence staining and immunoprecipitation. Modulation of GRP and calpains by AST was similarly demonstrated in nude mice xenograft, leading to significant inhibition of tumor growth. CONCLUSIONS: Our findings exemplify that calpains, in particular calpain II, play a permissive role in the modulation of GRP78 and consequent regulation of ER stress-induced apoptosis. Combination of calpain inhibitors and AST could exhibit a more pronounced pro-apoptotic effect. These results help to envisage a new therapeutic approach in colon cancer by targeting calpain and GRP.

Astragalus saponins downregulate vascular endothelial growth factor under cobalt chloride-stimulated hypoxia in colon cancer cells.

BACKGROUND: Our ongoing research has revealed that total saponins extracted from the medicinal herb Radix Astragali (AST) exhibits significant growth-inhibitory and proapoptotic effects in human cancer cells. In the present study, the potential of AST in controlling angiogenesis was further investigated with elaboration of the underlying molecular mechanism in human colon cancer cell and tumor xenograft. RESULTS: AST decreased the protein level of VEGF and bFGF in HCT 116 colon cancer cells in a time- and dose-dependent manner. Among the Akt/mTOR signal transduction molecules being examined, AST caused PTEN upregulation, reduction in Akt phosphorylation and subsequent activation of mTOR. AST also suppressed the induction of HIF-1α and VEGF under CoCl2-mimicked hypoxia. These effects were intensified by combined treatment of AST with the mTOR inhibitor rapamycin. Despite this, our data also indicate that AST could attenuate cobalt chloride-evoked COX-2 activation, while such effect on COX-2 and its downstream target VEGF was intensified when indomethacin was concurrently treated. The anti-carcinogenic action of AST was further illustrated in HCT 116 xenografted athymic nude mice. AST significantly suppressed tumor growth and reduced serum VEGF level in vivo. In the tumor tissues excised from AST-treated animals, protein level of p-Akt, p-mTOR, VEGF, VEGFR1 and VEGFR2 was down-regulated. Immunohistochemistry has also revealed that AST effectively reduced the level of COX-2 in tumor sections when compared with that in untreated control. CONCLUSION: Taken together, these findings suggest that AST exerts anti-carcinogenic activity in colon cancer cells through modulation of mTOR signaling and downregulation of COX-2, which together reduce VEGF level in tumor cells that could potentially suppress angiogenesis.

Ginger extract and polaprezinc exert gastroprotective actions by anti-oxidant and growth factor modulating effects in rats.

BACKGROUND AND AIM: Contemporary medications used in the treatment of gastric ulcers involve the use of novel mucosal protective drugs. The present study aimed to investigate the gastroprotective effect of ginger extract and polaprezinc in a rat model of acetic acid-induced gastric ulcer. METHODS: 'Kissing' ulcers were induced in male Sprague-Dawley rats by using 60% acetic acid. Rhizoma Zingiber officinale (ginger) extract (1.5-5 g/kg) or polaprezinc (30 and 60 mg/kg) was orally given to the animals once daily for three consecutive days after ulcer induction. All animals were killed on day 5 by an overdose of ketamine. RESULTS: Both ginger extract and polaprezinc significantly reduce the gastric ulcer area in a dose-dependent manner, with concomitant attenuation of the elevated activities of xanthine oxidase and myeloperoxidase, as well as malondialdehyde level in the ulcerated mucosa. Nevertheless, only polaprezinc could restore the mucosal glutathione level. Polaprezinc also causes the overexpression of basic fibroblast growth factor, vascular endothelial growth factor and ornithine decarboxylase, whereas ginger extract only increases the expression of the two growth factors in the gastric mucosa. Furthermore, polaprezinc could consistently downregulate the protein expression of tumor necrosis factor (TNF)-α, interleukin-1ß, macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant-2α that have been activated in the ulcerated tissues, whereas ginger extract mainly inhibits the expression of the chemokines and to some extent TNF-α. CONCLUSION: Ginger extract and polaprezinc both show anti-oxidation that consequently alleviates gastric mucosal damage and promotes ulcer healing, which together serve as effective mucosal protective agents.

A novel anticancer effect of Astragalus saponins: Transcriptional activation of NSAID-activated gene.

Astragalus membranaceus has been used to ameliorate the side effects of antineoplastic drugs because of its immunomodulating nature. We had recently demonstrated that total Astragalus saponins (AST) possess anticarcinogenic and proapoptotic properties in human colon cancer cells and tumor xenograft. In this study, we identified NSAID-activated gene (NAG-1) as a potential molecular target of AST. The growth-inhibitory and proapoptotic effects of AST were assessed in a panel of human cancer cell lines. Hoechst 33342 nuclear staining, Annexin V-FITC/propidium iodide staining, Western immunoblotting, real-time PCR, luciferase reporter assay and electrophoretic mobility shift assay were conducted to determine the association of NAG-1 and related transcription factors with AST during its regulation of apoptotic activities. Moreover, the combined proapoptotic and NAG-1 promoting activities of AST and/or inhibitors of the PI3K-Akt pathway were also examined. AST caused overexpression of NAG-1, leading to PARP cleavage and apoptosis. The induction of NAG-1 promoter activity by the drug was associated with increased gene expression, in addition to prior increase in Egr-1 expression and DNA binding activity. AST-induced NAG-1 activation was intensified when PI3K inhibitor LY294002 or Akt inhibitor was co-treated and reversed by NAG-1 siRNA transfection. Nevertheless, the extent of NAG-1 induction could not be altered by the ERK inhibitor PD98059. Our results indicate that NAG-1 is a potential molecular target of AST in its antitumorigenic and proapoptotic actions, which would have additive effects when used along with PI3K-Akt inhibitors. The information obtained could facilitate future development of a novel target-specific chemotherapeutic agent with known molecular pathway.

Herbal diterpenoids induce growth arrest and apoptosis in colon cancer cells with increased expression of the nonsteroidal anti-inflammatory drug-activated gene.

Novel chemotherapeutic agents derived from active phytochemicals could be used as adjuvants and improve the anti-carcinogenicity of standard drug treatments. However, their precise mechanisms of action are sometimes unclear. In this study, the anti-carcinogenic effect of the herbal diterpenoid pseudolaric acid B (PAB) on the growth and apoptosis of colon cancer cells was investigated, and to compare that with the more toxic compound triptolide. PAB induced growth inhibition and apoptosis in HT-29 cells, which were associated with cell cycle arrest at the G(2)/M phase, modulation of cyclin expression and downregulation of the protooncogene c-myc. In addition, PAB also inhibited bcl-x(L) expression, induced cleavage of procaspase-3 and its substrate poly(ADP-ribose) polymerase (PARP), which together caused DNA fragmentation and nuclear chromatin condensation. Concomitantly, the modulation of the growth-related and apoptotic factors by PAB was accompanied by the increased protein and gene expression of the nonsteroidal anti-inflammatory drug-activated gene (NAG-1), which occurred along with cyclooxygenase-2 inhibition. The effects of PAB on PARP cleavage and NAG-1 overexpression were not reversible upon removal of the drug from the culture medium. Similar cytotoxic and pro-apoptotic effects were also attained by treating the HT-29 cells with another diterpenoid triptolide, but its actions on cell cycle progression and on the upstream transcriptional regulation of NAG-1 both took place in a less coherent manner. These findings exemplify the potential of herbal terpenoids, particularly PAB, in modulating colon cancer carcinogenesis through known molecular targets and precise mechanism of action.

Angiogenesis and Oxidative Stress in Metastatic Tumor Progression: Pathogenesis and Novel Therapeutic Approach of Colon Cancer.

BACKGROUND: When tumor cells are under hypoxic condition or other forms of oxidative stress, one of the survival mechanisms is to undergo angiogenesis, involving dissemination of existing vessels or neovascularization to antagonize the apoptotic drive and to facilitate migration to secondary sites. METHODS: This paper reveals the pathogenesis of tumor angiogenesis, particularly during hypoxia and other forms of oxidative stress in cancer cells. RESULTS: Following successive invasion of the extracellular matrix (ECM), new blood vessels penetrate and supply nutrients to tumor tissues for growth and metastasis. The metastatic power of cancers is determined by a series of angiogenic and metastatic factors. These factors could allow neoplastic tissues to survive and withstand the stress induced by hypoxia and/or disruption of the ECM, including vascular endothelial growth factor and matrix metalloproteinases that were found to be highly elevated in tumor tissues of colon cancer patients. These aggressive factors could be regulated by cancer signaling pathways such as the phosphatidylinositol 3-kinase/Akt/ mTOR cascade. In fact, mTOR (the mammalian target of rapamycin) acts as a central regulator of many cellular activities involving growth and differentiation through regulation of cell cycle progression, cell size, cell migration and survival, including those in tumor cells. Several novel therapeutic approaches that target the angiogenic drive of cancers have been introduced, including compounds derived from natural products and synthetic chemicals. CONCLUSION: This article highlights the importance of angiogenesis and oxidative stress on the development of advanced and metastatic colon cancers, and provides new insights on alternative and effective treatment options.

Astragalus membranaceus: A Review of its Protection Against Inflammation and Gastrointestinal Cancers.

Astragalus membranaceus is a major medicinal herb commonly used in many herbal formulations in the practice of traditional Chinese medicine (TCM) to treat a wide variety of diseases and body disorders. Among its diversified clinical applications, the potential use of this herb and its chemical constituents in treatments of inflammatory diseases and cancers has been actively investigated in recent years. Astragalus-based treatments have demonstrated significant amelioration of the toxicity induced by other concurrently administered orthodox drugs (e.g., immunosuppressants and cancer chemotherapeutics). The major components of Astragalus membranaceus are polysaccharides, flavonoids, and saponins. Contemporary use of Astragalus membranaceus mainly focuses on its immunomodulating, anti-oxidant, and anti-inflammatory, as well as anticancer effects. In this paper, we summarize the properties of Astragalus membranaceus and its major constituents in the biological system based on experimental and clinical studies. The antitumorigenic mechanisms of a novel Astragalus saponins extract called AST in treating various gastrointestinal cancers are highlighted. We discuss in detail how the Astragalus herb and AST influence the immune system, modulate various cancer signaling pathways, and interact with specific transcription molecules during protection against gastrointestinal inflammation and cancers. This information could help clinicians and scientists develop novel target-specific and effective therapeutic agents that are deprived of major systemic side effects, so as to establish a better treatment regimen in the battle against inflammatory diseases and cancers of the gut.

The diverse actions of nicotine and different extracted fractions from tobacco smoke against hapten-induced colitis in rats.

The etiology of ulcerative colitis (UC) remains unknown, although the risk of developing UC is apparently higher in non-smokers and ex-smokers. We have demonstrated in a colitis animal model that exposure to tobacco smoke could attenuate UC pathogenesis. The present study aimed to investigate and compare between the modes of action of nicotine and different fractions of tobacco smoke extract in the development of experimental colitis. The hapten 2,4-dinitrobenzene sulfonic acid (DNBS) was used to induce colitis in Sprague-Dawley rats. Results indicated that both tobacco smoke exposure and subcutaneous nicotine differentially reduced colonic lesion size, myeloperoxidase (MPO) activity, luminol-amplified free radical generation, and leukotriene B4 formation in the inflamed colon of colitis animals. These phenomena were accompanied by the downregulation of colonic interleukin (IL)-1beta and monocyte chemoattractant protein (MCP)-1 protein expression. By treating the colitis animals with various tobacco extracts, we further discovered that ethanol extract from filtered tobacco smoke could attenuate DNBS-evoked colonic damage and the elevated MPO activity, while at the same time it downregulated colonic IL-1beta and MCP-1 protein expression. In contrast, the highest dose of the chloroform extract from the cigarette filter caused aggravating effects and overexpression of the pro-inflammatory cytokines and chemokines. These data suggest that effective attenuation of DNBS-induced colitis by tobacco smoke could be due to its nicotine content and possibly other flavonoid components found in the ethanol smoke extract.

Delineation of the protective action of zinc sulfate on ulcerative colitis in rats.

The protective action of zinc compounds in Crohn's disease-like inflammatory bowel disease in animals has been shown. A similar action of zinc sulfate on ulcerative colitis has not been defined. The present study aimed to delineate the protective action of zinc sulfate and the pathogenic mechanisms of 2,4-dinitrobenzene sulfonic acid (DNBS)-induced ulcerative colitis in rats. Zinc sulfate at different concentrations was given either orally (p.o.) or rectally (p.r.) to rats at 42, 48, 66 and 72 h following the induction of colonic inflammation by DNBS. Rats were killed 96 h after instillation of DNBS rectally to assess the severity of colonic damage, myeloperoxidase and xanthine oxidase activities. The involvement of mast cell degranulation and histamine release in the pathogenesis of DNBS-induced colitis was determined by using a mast cell stabilizer (ketotifen) and histamine receptor blockers (terfenadine and ranitidine). DNBS given rectally produced inflammation and ulceration in rats with a pathology resembling ulcerative colitis. Myeloperoxidase activity but not xanthine oxidase activity was sharply increased by this agent. Intrarectal administration of zinc solution and parenteral injection of histamine blockers significantly reduced tissue damage and myeloperoxidase but not xanthine oxidase activity. Ketotifen, a mast cell stabilizer, also significantly decreased mucosal injury and myeloperoxidase activity in the colon. In conclusion, mast cell degranulation followed by histamine release plays an important role in the pathogenesis of DNBS-induced ulcerative colitis. Zinc given rectally has a therapeutic effect against this colitis model, perhaps through the reduction of inflammation and inhibition of the above pathogenic mechanisms.

Inflammatory bowel disease: etiology, pathogenesis and current therapy.

Ulcerative colitis (UC) and Crohn's disease (CD) constitute the two major groups of idiopathic disorders in inflammatory bowel disease (IBD). Environmental factors, genetic factors and immune responses have been considered as the major etiology of IBD. Despite the diversified pathogenesis of the disease, no guaranteed curative therapeutic regimen has been developed so far. This review summarizes the knowledge on the pathophysiology and current treatment approaches of IBD. Since IBD is caused by excessive and tissue- disruptive inflammatory reactions of the gut wall, down-regulation of the immune responses may allow the damaged mucosa to heal and reset the physiological functions of the gut back to normal. Current pharmacotherapy through modulation of neutrophil-derived factors, cytokines, adhesion molecules and reactive oxygen/nitrogen metabolites has been utterly described. Categories of treatment modalities include corticosteroids, aminosalicylates, immunomodulators, antibiotics, probiotics, and a series of unique novel agents. The use of anti-tumor necrosis factor monoclonal antibody (Infliximab), recombinant anti-inflammatory cytokines and related gene therapy has been covered. In addition, discussions on dietary supplementation and heparin treatment are also included. The anti-inflammatory and immunoregulatory potential of investigational agents such as nicotine and the filtered protective compounds from tobacco smoke, as well as active herbal medicinal compounds were tested in our previous experimental works, whereas promising findings have been presented here. With the discovery of novel target-oriented agents, more effective and relatively harmless approaches of IBD therapy could be established to achieve a curative outcome. Indeed, more experimental and clinical studies are needed to confirm the relevance of these therapies.

Identification of functional peptides from natural and synthetic products on their anticancer activities by tumor targeting.

Cancer cells can express specific membrane proteins, which act as biomarkers for chemotherapeutic targeting. Functional peptides possess unique properties that will ensure efficacy, selectivity, specificity and low toxicity when used as therapeutic agents. Therapeutic peptides have been derived in treatment of cancers through improvement of cellular uptake, drug targeting and vaccine development. Peptides from natural source have been used for chemoprevention and therapy of various cancers. These include peptides derived from food, marine products, venom components and other animal constituents. Besides, chemically- and recombinantly-synthesized peptides have also been produced and extensively studied in contemporary applications. Improvement of tumor targeting is essential for chemotherapeutic development. This can be achieved through enhancement of intracellular delivery and/or increased specific binding affinity to cancer cells by pore-forming and cytotoxic peptides. Cytotoxic peptides such as the Bcl-2 family members can induce receptor-specific binding to tumor cells and promote apoptosis by targeting lipid membranes. This approach has some limitations in targeting, penetration and localization within tumors. Cell-penetrating peptides (CPPs) belong to a new class of tumor-targeting peptides that can facilitate internalization of tumor markers and/or chemotherapeutic drugs. In order to overcome the problem of serum instability in classical CPPs (e.g. Tat), newer classes of CPPs has been recently introduced. Nevertheless, some cyclized CPPs can further enhance cellular uptake and binding selectivity when compared to activities of their linear counterpart, especially when treating chemoresistant tumors. This review compiles the use of effective tumor-targeting peptides including novel CPPs that represents new therapeutic strategies for the treatment of cancers.

Target-oriented mechanisms of novel herbal therapeutics in the chemotherapy of gastrointestinal cancer and inflammation.

A prominent group of effective cancer chemopreventive drugs has been derived from natural products having low toxicity while possessing apparent benefit in the disease process. It is plausible that there are multiple target molecules critical to cancer cell survival. Herbal terpenoids have demonstrated excellent target-specific anti-neoplastic functions by suppression of cell proliferation and induction of apoptosis. Transcriptional molecules in the NF-κB, MEK/ERK and PI3K/Akt/mTOR pathways are important molecular targets of chemotherapy that play distinctive roles in modulating the apoptosis cascades. It is recently suggested that NSAID-activated gene (NAG-1), a novel proapoptotic protein, is the upstream anti-carcinogenic target of NSAIDs, PPAR ligands and herbal chemotherapeutic agents that triggers some of the events mentioned above. Besides, angiogenesis, oxidative stress as well as inflammation are important factors that contribute to the development and metastasis of cancer, which could be actively modulated by novel agents of plant origin. The aim of the present review is to discuss and summarize the contemporary use of herbal therapeutics and phytochemicals in the treatment of human cancers, in particular that of the colon. The major events and signaling pathways in the carcinogenesis process being potentially modulated by natural products and novel herbal compounds will be evaluated, with emphasis on some terpenoids. Advances in eliciting the precise cellular and molecular mechanisms during the anti-tumorigenic process of novel herbal therapeutics will be of imperative clinical significance to increase the efficacy and reduce prominent adverse drug effects in cancer patients through target-specific therapy.

Effects of adrenaline in human colon adenocarcinoma HT-29 cells.

AIMS: Stress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved. MAIN METHODS: The effect of adrenaline on HT-29 cell proliferation was determined by [(3)H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E(2) (PGE(2)) release were determined by zymography and enzyme immunoassay, respectively. KEY FINDINGS: Adrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE(2) release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of ß-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a ß(1)- and ß(2)-selective antagonist, respectively. This signified the role of ß-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE(2) release in HT-29 cells. SIGNIFICANCE: These results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both ß(1)- and ß(2)-adrenoceptors by a COX-2 dependent pathway.

Astragalus saponins induce growth inhibition and apoptosis in human colon cancer cells and tumor xenograft.

Astragalus memebranaceus is used as immunomodulating agent in treating immunodeficiency diseases and to alleviate the adverse effects of chemotherapeutic drugs. In recent years, it has been proposed that Astragalus may possess anti-tumorigenic potential in certain cancer cell types. In this study, the anti-carcinogenic effects of Astragalus saponin extract were investigated in HT-29 human colon cancer cells and tumor xenograft. Our findings have shown that Astragalus saponins (AST) inhibit cell proliferation through accumulation in S phase and G2/M arrest, with concomitant suppression of p21 expression and inhibition of cyclin-dependent kinase activity. Besides, AST promotes apoptosis in HT-29 cells through caspase 3 activation and poly(ADP-ribose) polymerase cleavage, which is indicated by DNA fragmentation and nuclear chromatin condensation. Nevertheless, we also demonstrate the anti-tumorigenic effects of AST in vivo, of which the reduction of tumor volume as well as pro-apoptotic and anti-proliferative effects in HT-29 nude mice xenograft are comparable with that produced by the conventional chemotherapeutic drug 5-fluorouracil (5-FU). In addition, the side effects (body weight drop and mortality) associated with the drug combo 5-FU and oxaliplatin are not induced by AST. These results indicate that AST could be an effective chemotherapeutic agent in colon cancer treatment, which might also be used as an adjuvant in combination with other orthodox chemotherapeutic drugs to reduce the side effects of the latter compounds.