Non-toxic Natural Agent Research Articles

Exopolysaccharide from Porphyridium cruentum (purpureum) is Not Toxic and Stimulates Immune Response against Vibriosis: The Assessment Using Zebrafish and White Shrimp Litopenaeus vannamei.

Exopolysaccharides, or extracellular polysaccharides (EPS, sPS), represent a valuable metabolite compound synthesized from red microalgae. It is a non-toxic natural agent and can be applied as an immunostimulant. The toxicity test of exopolysaccharides from Porphyridium has been done in vivo using zebrafish (Danio rerio) embryonic model, or the ZET (zebrafish embryotoxicity test). The administration of extracellular polysaccharides or exopolysaccharides (EPS) from microalgae Porphyridium cruentum (synonym: P. purpureum) to shrimps Litopenaeus vannamei was investigated to determine the effect of this immunostimulant on their non-specific immune response and to test if this compound can be used as a protective agent for shrimps in relation to Vibrio infection. For immune response, exopolysaccharides were given to shrimps via the immersion method on day 1 and booster on day 8. Shrimp hemocytes were taken on day 1 (EPS administration), day 7 (no treatment), day 8 (EPS booster) and day 9 (Vibrio infection) and tested for their immune response on each treatment. The result shows that the EPS is not toxic, as represented by the normal embryonic development and the mortality data. In the Pacific white shrimps, an increase in the values of all immune parameters was shown, in line with the increasing EPS concentration, except for the differential hemocyte count (DHC). In detail, an increase was noted in total hemocytes (THC) value, phagocytotic activity (PA) and respiratory burst (RB) in line with the EPS concentration increase. These results and other previous studies indicate that EPS from Porphyridium is safe, enhances immune parameters in shrimp rapidly, and has the ability to act as an immunostimulant or an immunomodulator. It is a good modulator for the non-specific immune cells of Pacific white shrimps, and it can be used as a preventive agent against vibriosis.

Diosmin Regulates Oxidative Stress and Inflammatory Marker Levels in N-Methyl-N-Nitrosourea-Induced Gastric Carcinogenesis in Rats.

Gastric cancer is the most typical oncological illness globally. Though the incidence of gastric cancer has dropped dramatically over the last few years, the survival rate is yet concerning due to poor diagnostic strategy. The etiology of gastric cancer is majorly associated with dietary factors. For this reason, application of nontoxic natural agents with anticancer effects for patients is needed. Diosmin has been commonly utilized to treat various diseases both traditionally and now clinically. However, its effect on gastric carcinogenesis remains unclear. The effects of diosmin were used to evaluate how N-methyl-N-nitrosourea (MNU) induces gastric carcinogenesis in rats. The general and gross assessment of MNU in experimental animals was tabulated. Biological tumor markers (gastrin, ALP, LDH, and γ-GT) were examined. Oxidative markers (LPO) and antioxidative markers (GSH, vitamin E, and vitamin C) were determined. In addition, inflammatory cytokines (thioredoxin, glutaredoxin, NF-κB, TNF-α, IL-6, PGE2) were explored and justified with histopathological studies. Overall, the results showed positive anticancer activity demonstrated by improved body weight, reduced tumor rate, decreased oxidative marker value by increased antioxidative rate, and suppressed tumor biomarkers and inflammatory cytokines. The histopathological analysis was congruent with the data observed. Our conclusion that diosmin exerts its anticancer activity by up-regulation of antioxidants which might be responsible for amelioration of oxidative stress and inflammatory cytokines in carcinogenesis to prevent gastric cancer.

Capsaicin Inhibits Multiple Bladder Cancer Cell Phenotypes by Inhibiting Tumor-Associated NADH Oxidase (tNOX) and Sirtuin1 (SIRT1).

Bladder cancer is one of the most frequent cancers among males, and its poor survival rate reflects problems with aggressiveness and chemo-resistance. Recent interest has focused on the use of chemopreventatives (nontoxic natural agents that may suppress cancer progression) to induce targeted apoptosis for cancer therapy. Capsaicin, which has anti-cancer properties, is one such agent. It is known to preferentially inhibit a tumor-associated NADH oxidase (tNOX) that is preferentially expressed in cancer/transformed cells. Here, we set out to elucidate the correlation between tNOX expression and the inhibitory effects of capsaicin in human bladder cancer cells. We showed that capsaicin downregulates tNOX expression and decreases bladder cancer cell growth by enhancing apoptosis. Moreover, capsaicin was found to reduce the expression levels of several proteins involved in cell cycle progression, in association with increases in the cell doubling time and enhanced cell cycle arrest. Capsaicin was also shown to inhibit the activation of ERK, thereby reducing the phosphorylation of paxillin and FAK, which leads to decreased cell migration. Finally, our results indicate that RNA interference-mediated tNOX depletion enhances spontaneous apoptosis, prolongs cell cycle progression, and reduces cell migration and the epithelial-mesenchymal transition. We also observed a downregulation of sirtuin 1 (SIRT1) in these tNOX-knockdown cells, a deacetylase that is important in multiple cellular functions. Taken together, our results indicate that capsaicin inhibits the growth of bladder cancer cells by inhibiting tNOX and SIRT1 and thereby reducing proliferation, attenuating migration, and prolonging cell cycle progression.

Abstract P5-14-02: Efficacy of tabebuia avellandae extract on a cell culture model for triple negative breast cancer

Abstract Background: The triple negative breast cancer (TNBC), a molecular subtype of clinical breast cancer consisting of epithelial cells that lack estrogen receptor-α (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) expression, is non-responsive to either endocrine therapy or HER-2 targeted therapy. Chemotherapy is frequently associated with long-term systemic toxicity, acquired tumor resistance and resultant compromised treatment efficacy. These aspects emphasize a need to identify efficacious non-toxic agents for secondary prevention/therapy of TNBC. Non-fractionated aqueous extract from the inner bark of the Tabebuia avellandae (TA) tree found in the Amazon rainforest, available as a dietary supplement under the name of Taheebo or Pau d’arco, has documented efficacy in a cell culture model for the Luminal A breast cancer subtype, as well as for several other cancers. Present study examines the inhibitory effects of the TA extract, and identifies possible mechanistic targets for its efficacy in a cell culture model for TNBC. Nutritional Supplement, Experimental Model and Biomarkers: Lyophilized powder of non-fractionated TA extract from Taheebo Japan, Osaka, Japan, provides the source material for the study. The ER-/PR-/HER-2- MDA-MB-231 cell line represents the cell culture model for TNBC. Anchorage dependent growth, cell cycle progression, status of cell cycle regulatory proteins and anchorage independent colony formation, represent the quantitative biomarkers for efficacy. Results: Relative to the non-tumorigenic 184-B5 human mammary epithelial cells, the tumor derived MDA-MB-231 cells exhibited decreased population doubling time, increased saturation density, decreased G1: S+G2/M ratio and increased S+G2/M: Sub G0 ratio, indicating loss of homeostatic growth control. Additionally, unlike 184-B5 cells, MDA-MB-231 cells exhibited increased anchorage independent growth in vitro and tumor development in vivo, indicating enhanced cancer risk. Treatment of MDA-MB-231 cells with TA resulted in a substantial dose dependent cytostatic growth arrest (IC50:1.0%; IC90: 2.5%). Cell cycle analysis of TA treated cells revealed G1 arrest, leading to a progressive dose dependent increase in the G1: S+G2/M ratio. Mechanistically, TA decreased Cyclin D1 expression and attenuated RB phosphorylation, predicting Cyclin D-CDK4-pRB pathway as a molecular target for efficacy. Furthermore, TA effectively inhibited anchorage independent growth in a dose dependent manner. Conclusions: Present data demonstrated pronounced efficacy of TA as a naturally occurring nutritional substance in a cell culture model for TNBC, and validated TA as a promising non-toxic natural agent for secondary prevention/therapy of clinical TNBC. Citation Format: Nitin T Telang, Hareesh B Nair, George YC Wong. Efficacy of tabebuia avellandae extract on a cell culture model for triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-14-02.

Targeting Epigenetically Deregulated miRNA by Nutraceuticals: Focusing on Cancer Prevention and Treatment

In recent years, the epigenetic alterations, especially the regulation of microRNA (miRNA), have received much attention in the context of tumor development and progression. High level of oncogenic miRNAs and low expression of tumor-suppressive miRNAs are commonly observed in cancer cells. These aberrant expressions of miRNAs are intimately associated with tumor development and progression. The expression level of miRNAs could be controlled by epigenetic regulations. The hypermethylation at the promoter region of a tumor-suppressive miRNA could cause decreased expression of miRNA via the blockade of transcriptional elements. The histone modification could also indirectly cause reduced expression of tumor-suppressive miRNAs, resulting in the high expression of oncogenes. Interestingly, emerging evidences have demonstrated that several non-toxic natural agents known as nutraceuticals including isoflavone, curcumin, resveratrol, and 3,3′-diindolylmethane could significantly decrease the level of DNA hypermethylation in the promoter of miRNAs or modulate histone, leading to the alteration of miRNA expression which is believed to be associated with inhibition of tumor development and progression. Therefore, administration of these nutraceuticals combined with conventional chemotherapeutics could be a promising therapeutic strategy for fighting the war against cancers.

Molecular Mechanisms of Silibinin-Mediated Cancer Chemoprevention with Major Emphasis on Prostate Cancer

Despite advances in early detection, prostate cancer remains the second highest cancer mortality in American men, and even successful interventions are associated with enormous health care costs as well as prolonged deleterious effects on quality of patient life. Prostate cancer chemoprevention is one potential avenue to alleviate these burdens. It is a regime whereby long-term treatments are intended to prevent or arrest cancer development, in contrast to more direct intervention upon disease diagnosis. Based on this intention, cancer chemoprevention generally focuses on the use of nontoxic chemical agents which are well-tolerated for prolonged usage that is necessary to address prostate cancer's multistage and lengthy period of progression. One such nontoxic natural agent is the flavonoid silibinin, derived from the milk thistle plant (Silybum marianum), which has ancient medicinal usage and potent antioxidant activity. Based on these properties, silibinin has been investigated in a host of cancer models where it exhibits broad-spectrum efficacy against cancer progression both in vitro and in vivo without noticeable toxicity. Specifically in prostate cancer models, silibinin has shown the ability to modulate cell signaling, proliferation, apoptosis, epithelial to mesenchymal transition, invasion, metastasis, and angiogenesis, which taken together provides strong support for silibinin as a candidate prostate cancer chemopreventive agent.

The Role of EMT in Pancreatic Cancer Progression

The Role of EMT in Pancreatic Cancer Progression

Green tea catechin, epigallocatechin-3-gallate (EGCG): Mechanisms, perspectives and clinical applications

An expanding body of preclinical evidence suggests EGCG, the major catechin found in green tea (Camellia sinensis), has the potential to impact a variety of human diseases. Apparently, EGCG functions as a powerful antioxidant, preventing oxidative damage in healthy cells, but also as an antiangiogenic and antitumor agent and as a modulator of tumor cell response to chemotherapy. Much of the cancer chemopreventive properties of green tea are mediated by EGCG that induces apoptosis and promotes cell growth arrest by altering the expression of cell cycle regulatory proteins, activating killer caspases, and suppressing oncogenic transcription factors and pluripotency maintain factors. In vitro studies have demonstrated that EGCG blocks carcinogenesis by affecting a wide array of signal transduction pathways including JAK/STAT, MAPK, PI3K/AKT, Wnt and Notch. EGCG stimulates telomere fragmentation through inhibiting telomerase activity. Various clinical studies have revealed that treatment by EGCG inhibits tumor incidence and multiplicity in different organ sites such as liver, stomach, skin, lung, mammary gland and colon. Recent work demonstrated that EGCG reduced DNMTs, proteases, and DHFR activities, which would affect transcription of TSGs and protein synthesis. EGCG has great potential in cancer prevention because of its safety, low cost and bioavailability. In this review, we discuss its cancer preventive properties and its mechanism of action at numerous points regulating cancer cell growth, survival, angiogenesis and metastasis. Therefore, non-toxic natural agent could be useful either alone or in combination with conventional therapeutics for the prevention of tumor progression and/or treatment of human malignancies.

Abstract 3690: Asiatic acid is an effective alternative agent targeting growth, angiogenesis and invasiveness of malignant gliomas

Abstract Malignant gliomas are one of the most devastating and incurable tumors with approximately 77% of patients dying within first year of diagnosis. This poor prognosis is due to characteristics of glioma cells to proliferate uncontrollably, sustain excessive angiogenesis and invade aggressively. There has been no improvement in survival of glioma patients in past few decades and existing therapies, with tremendous side-effects, have largely failed. This suggests that immediate additional and alternative strategies are warranted to control this deadly malignancy. In this regard, preventing or inhibiting growth and progression of gliomas through non-toxic natural agents is one such strategy. An agent suited ideally against advance glioma is Asiatic Acid (AsA), the active constituent in Centella asiatica; both Centella asiatica extract and pure AsA are extensively used in traditional medicine to treat neurological disorders. Here we analyzed the anti-cancer efficacy of AsA against glioma cells both in vitro and in vivo. AsA treatment (10-100 microM, 6-24 hrs) significantly decreased cell viability in U87, LN18 and U118 glioma cells, together with strong apoptotic cell death. AsA treatment (6 hrs) in glioma cells caused PARP, Bid and caspases (3, 9 and 8) cleavage, activated JNK1/2, and decreased phosphorylated-Akt and survivin. Use of pan-caspase inhibitor (z-VAD.fmk) confirmed that AsA-induced apoptosis is caspase-dependent. Next, we studied whether AsA could target the pro-angiogenic effects of glioma cells. We observed that AsA treatment at non-cytotoxic doses significantly impairs potency of glioma cells (U87 and LN18) to promote capillary formation by HUVEC (human umbilical vein endothelial cells) and HBMEC (human brain microvascular endothelial cells) on matrigel. Further, AsA (5-20 microM) significantly inhibited VEGF-induced growth of HUVEC and HMBEC. We also observed remarkable anti-migratory effects of AsA treatment (5-20 microM) in wound healing assay and anti-invasive potential in Boyden chambers against glioma cells. In a novel assay developed by us, we further established the inhibitory effects of AsA on fibronectin-induced motility in glioma cells. Additional mechanistic studies revealed that AsA strongly increases intracellular Ca++ levels; and antiproliferative and antimigratory effects of AsA were largely compromised in the presence of Ca++ chelating agent BAPTA. We also established for the first time in vivo anti-cancer efficacy of AsA using xenograft model. AsA feeding (30 mg/kg body weight dose in saline) for 5 weeks significantly decreased U87 xenografts volume by 54% (p value less than 0.05) and strongly inhibited tumor vasculature without causing any adverse effects to animals. Together, these results suggest that AsA could be an effective alternative agent against malignant glioma and recommend its clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3690. doi:10.1158/1538-7445.AM2011-3690

Abstract 3780: Plumbagin a non-toxic natural agent, induces apoptosis and inhibits the growth of prostate cancer LNCaP and C4-2 cells via blocking of both androgen and non-androgen activation of androgen receptor signaling

Abstract Prostate cancer (PCa) is one of the most commonly diagnosed cancers and leading cause of cancer related death in men in Western countries including the Unites States. Androgen receptor (AR)-mediated signaling plays a critical role in the development and progression of PCa. Most androgen independent (AI) prostate tumors continue to express AR as well as its target gene PSA, which indicates that these tumor cells maintain functional AR signaling. Evidence suggests that AR signaling is activated through androgen and androgen bypass mechanism. We believe that identification of non-toxic natural agents capable of blocking both androgen and non-androgen activation of AR signaling could be effective for the treatment of PCa. We have previously shown that a novel plant derived napthoquinone plumbagin (PL) induces apoptosis and inhibits growth of PCa cells in in vitro as well as in in vivo model systems (Cancer Research 2008, PMID: 18974148). We present here that PL inhibits both androgen and non-androgen activation of AR signaling in PCa cells. We observed that PL inhibits synthetic androgen methyltrienolone (R1881) induced growth of androgen-dependent (AD) LNCaP and AI C4-2 cells. PL treatment was found to inhibit protein levels of AR and AR-target gene PSA and accelerates AR degradation. To analyze the effect of PL on decreased AR transactivation, transient transfections using androgen responsive reporters PSA-luc were carried out. Treatment of LNCaP and C4-2 cells with R1881 led to induction of PSA reporter in these cells. Co-treatment of LNCaP and C4-2 cells with PL led to a dose-dependent decrease in AR transactivation as reflected by a decrease in the PSA reporter activity. These results suggest that PL interferes with androgen-induced transactivation function of AR. It has been reported that IL-6 signaling acts as a ligand for the activation of AR signaling. IL-6 induced phosphorylated Stat3 interacts with the N-terminal domain of AR and activates its target genes. We have previously shown that PL treatment inhibits Stat3 phosphorylation in PCa DU-145 cells xenograft tumor tissues (Cancer Research 2008, PMID: 18974148). Therefore, we hypothesized that PL treatment will inhibit Stat3 and AR N-terminal domain interaction in PCa cells. Reciprocal immunoprecipitation/blotting experiments revealed that PL treatment inhibited IL-6 induced interaction of Stat3 with AR N-terminal domain in both the PCa LNCaP and C4-2 cells. PL treatment also inhibited IL-6 induced Tyr 705 and Ser727 phosphorylation of Stat3. These results establish that PL has the potential to block the androgen and non-androgen activation of AR signaling in PCa and provide further evidence that PL could be used as a chemopreventive natural agent in ongoing anti-hormone therapy of PCa. (Support: NIH grants, CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3780.

Increased therapeutic potential of an experimental anti-mitotic inhibitor SB715992 by genistein in PC-3 human prostate cancer cell line.

BackgroundKinesin spindle proteins (KSP) are motor proteins that play an essential role in mitotic spindle formation. HsEg5, a KSP, is responsible for the formation of the bipolar spindle, which is critical for proper cell division during mitosis. The function of HsEg5 provides a novel target for the manipulation of the cell cycle and the induction of apoptosis. SB715992, an experimental KSP inhibitor, has been shown to perturb bipolar spindle formation, thus making it an excellent candidate for anti-cancer agent. Our major objective was a) to investigate the cell growth inhibitory effects of SB715992 on PC-3 human prostate cancer cell line, b) to investigate whether the growth inhibitory effects of SB715992 could be enhanced when combined with genistein, a naturally occurring isoflavone and, c) to determine gene expression profile to establish molecular mechanism of action of SB715992.MethodsPC-3 cells were treated with varying concentration of SB715992, 30 μM of genistein, and SB715992 plus 30 μM of genistein. After treatments, PC-3 cells were assayed for cell proliferation, induction of apoptosis, and alteration in gene and protein expression using cell inhibition assay, apoptosis assay, microarray analysis, real-time RT-PCR, and Western Blot analysis.ResultsSB715992 inhibited cell proliferation and induced apoptosis in PC-3 cells. SB715992 was found to regulate the expression of genes related to the control of cell proliferation, cell cycle, cell signaling pathways, and apoptosis. In addition, our results showed that combination treatment with SB715992 and genistein caused significantly greater cell growth inhibition and induction of apoptosis compared to the effects of either agent alone.ConclusionOur results clearly show that SB715992 is a potent anti-tumor agent whose therapeutic effects could be enhanced by genistein. Hence, we believe that SB715992 could be a novel agent for the treatment of prostate cancer with greater success when combined with a non-toxic natural agent like genistein.

Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells.

The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53- and p21-independent G(2)/M phase arrest and apoptosis in HCT-116(p53(+/+)), HCT-116(p53(-/-)) and HCT-116(p21(-/-)) cell lines. We further investigated the association of the beta-catenin-mediated c-Myc expression and the cell-cell adhesion pathways in curcumin-induced G(2)/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of beta-catenin, decreased transactivation of beta-catenin/Tcf-Lef, decreased promoter DNA binding activity of the beta-catenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G(2)/M phase arrest. The decreased transactivation of beta-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of beta-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell-cell adhesion proteins beta-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell-cell adhesion pathways, resulting in G(2)/M phase arrest and apoptosis in HCT-116 cells.