Modulation of autophagy: a Phase II study of vorinostat plus hydroxychloroquine versus regorafenib in chemotherapy-refractory metastatic colorectal cancer (mCRC).

BACKGROUND: In metastatic colorectal cancer (mCRC), regorafenib (RGF), a multi-kinase inhibitor with angiogenic inhibition has modest effects on survival. We reported that autophagy modulation using hydroxychloroquine (HCQ), enhances the anticancer activity of the histone deacetylase inhibitor, vorinostat (VOR), in mCRC, is well tolerated, and has comparable activity to RGF. Thus, we conducted a prospective study of VOR/HCQ versus RGF in mCRC. METHODS: This is a randomised, controlled trial of VOR 400 mg and HCQ 600 mg orally daily versus RGF 160 mg orally daily (3 weeks on/1 week off), every 4 weeks, in patients with mCRC. PRIMARY ENDPOINT: median progression-free survival (mPFS). Secondary endpoints: median overall survival (mOS); adverse events; pharmacodynamic analyses. RESULTS: From 2/2015-10/2017, 42 patients were randomised to VOR/HCQ and RGF. Median age was 58.4 years. mPFS on VOR/HCQ was 1.9 months versus 4.35 months with RGF (P = 0.032). There was no difference in mOS (P = 0.9). Treatment was tolerated in both arms. In both arms, there was improved anti-tumour immunity. CONCLUSIONS: VOR/HCQ had an inferior PFS when compared to RGF, although there was an increase in anti-tumour immunity in mCRC. VOR/HCQ has a favourable safety profile, and immune or tumour biomarkers may be used to identify clinical benefit of autophagy modulation in mCRC. CLINICAL TRIAL REGISTRATION: NCT02316340.

The Highly Pure Neem Leaf Extract, SCNE, Inhibits Tumorigenesis in Oral Squamous Cell Carcinoma via Disruption of Pro-tumor Inflammatory Cytokines and Cell Signaling.

Oral squamous cell carcinoma (OSCC) is a deadly disease that comprises 60% of all head and neck squamous cell cancers. The leaves of the Neem tree (Azadirachta indica) have been used in traditional Ayurvedic medicine for centuries to treat numerous oral maladies and are known to have significant anti-inflammatory properties. We hypothesize that a highly pure super critical CO2 Neem leaf extract (SCNE) prevents initiation and progression of OSCC via downregulation of intra-tumor pro-inflammatory pathways, which promote tumorigenesis. Hence, we investigated the anticancer effects of SCNE using in vitro and in vivo platforms. OSCC cell lines (SCC4, Cal27, and HSC3) were treated with SCNE while inflammation, proliferation, and migration were analyzed over time. SCNE treatment significantly inhibited OSCC cell proliferation and migration and reduced MMP activity in vitro, suggesting its potential to inhibit tumor growth and metastasis. The preventive effects of SCNE in ectopic xenograft and 4NQO-1 (4-Nitroquinoline-1-oxide) carcinogen-induced mouse models of OSCC were also evaluated. Indeed, xenografted nude mice showed significant reduction of OSCC tumor volumes. Likewise, SCNE significantly reduced the incidence of tongue dysplasia in the 4NQO-1 OSCC initiation model. In both OSCC animal models, SCNE significantly depressed circulating pro-cancer inflammatory cytokines (host and tumor-secreted) including NFkB, COX2, IL-1, IL-6, TNFα, and IFNγ. In addition, we demonstrate that SCNE downregulates STAT3 and AKT expression and activity in vitro. We also demonstrate that the primary active component, nimbolide (NIM), has significant anticancer activity in established OSCC xenografts. Lastly, we show that SCNE induces an M1 phenotype in tumor associated macrophages (TAMS) in vivo. Taken together, these data strongly support SCNE as means of preventing OSCC via downregulation of pro-cancer inflammatory cascades and NIM as a potential new therapy for existing OSCC.

Disruption of Epigenetic Silencing in Human Colon Cancer Cells Lines Utilizing a Novel Supercritical CO2 Extract of Neem Leaf (Azadirachta indica).

BACKGROUND/AIM: Aerial parts and seeds of the neem tree (Azadirachta indica) have long been used in traditional medicine such as Ayurveda for health-related purposes. Our interest in neem bioactives lies in their potential use as standalone anticancer agents, or as adjuvants to standard therapy. The aim of the present study was to explore a supercritical CO2 extract (SCNE) of neem leaf and a prominent liminoid in neem leaf, nimbolide, for epigenetic activity. MATERIALS AND METHODS: Human colorectal cancer cell lines (HCT116 and HT29) were cultured for 48 h in the presence of neem extract or nimbolide and evaluated for growth inhibition and evidence of suppression of histone deacetylation and DNA methylation. RESULTS: Both SCNE and nimbolide suppressed the proliferation of colon cancer cells by inducing epigenetic modifications. CONCLUSION: Neem leaf contains bioactive constituents which modify epigenetic activity.

A supercritical CO2 extract of neem leaf (A. indica) and its bioactive liminoid, nimbolide, suppresses colon cancer in preclinical models by modulating pro-inflammatory pathways.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death in men and women in the United States. Anti-inflammatory blockade has been proven to be a promising avenue of colorectal cancer prevention. However, NSAIDs while effective in curbing CRC risk are too toxic for long-term use in cancer prevention. The Neem tree (Azadirachta indica) is rich in liminoid terpenoids, collectively known as azadiractoids and has been shown to have anti-inflammatory effects. To explore a role of neem in CRC, human colon cancer cell lines HCT116 and HT29 cells were treated with purified Super Critical Neem Extract (SCNE) or the neem liminoid, nimbolide. SCNE treatment resulted in a dose dependent inhibition of CRC cell proliferation and an increase in apoptosis. Treatment with SCNE and nimbolide decreased the expression of transcriptional factors, STAT3 and NF-κB which plays a major role in gene regulation of multiple cellular processes. Protein expression of COX1, IL-6, and TNF-α were decreased on treatment with SCNE in CRC cells. Western blots and Zymogram assays results revealed anti-invasive effect by decreased expression of MMP2 and MMP9 proteins in CRC cells. Overall, these data confirm a potential anti-cancer effect of SCNE, reducing cell proliferation, inflammation, migration, and invasion in human colon cancer cells. Confirming these indications, we found that treatment of mice bearing HT29 and HCT116 xenografted tumors exhibited striking inhibition of colon tumor growth. Clearly we must explore the effect of neem in preclinical animal models for anti-cancer therapy.

Therapeutic effects of the euglenoid ichthyotoxin, euglenophycin, in colon cancer.

Colorectal cancer (CRC) remains one of the most commonly diagnosed cancers and the 3rd leading cause of cancer-related mortality. The emergence of drug resistance poses a major challenge in CRC care or treatment. This can be addressed by determining cancer mechanisms, discovery of druggable targets, and development of new drugs. In search for novel agents, aquatic microorganisms offer a vastly untapped pharmacological source that can be developed for cancer therapeutics. In this study, we characterized the anti-colorectal cancer potential of euglenophycin, a microalgal toxin from Euglena sanguinea. The toxin (49.1-114.6 µM) demonstrated cytotoxic, anti-proliferative, anti-clonogenic, and anti-migration effects against HCT116, HT29, and SW620 CRC cells. We identified G1 cell cycle arrest and cell type - dependent modulation of autophagy as mechanisms of growth inhibition. We validated euglenophycin's anti-tumorigenic activity in vivo using CRL:Nu(NCr)Foxn1nu athymic nude mouse CRC xenograft models. Intraperitoneal toxin administration (100 mg/kg; 5 days) decreased HCT116 and HT29 xenograft tumor volumes (n=10 each). Tumor inhibition was associated with reduced expression of autophagy negative regulator mechanistic target of rapamycin (mTOR) and decreased trend of serum pro-inflammatory cytokines. Together, these results provide compelling evidence that euglenophycin can be a promising anti-colorectal cancer agent targeting multiple cancer-promoting processes. Furthermore, this study supports expanding natural products drug discovery to freshwater niches as prospective sources of anti-cancer compounds.

Natural Agents Used in Chemoprevention of Aerodigestive and GI Cancers.

Aerodigestive cancers are on an increasing level in both occurrence and mortality. A major cause in many of these cancers is disruption of the inflammatory pathway, leading to increased cell proliferation, and epigenetic silencing of normal regulatory genes. Here we review the research on several natural products: silibinin, silymarin, quercetin, neem & nimbolide, gingerol, epigallatecatechin-3- gallate, curcumin, genistein and resveratrol conducted on aerodigestive cancers. These types of cancers are primarily those from oral cavity, esophagus/windpipe, stomach, small and large intestine, colon/rectum and bile/pancreas tissues. We report on the utilization in vivo and in vitro systems to research these dose effects on the inflammatory and epigenetic pathway components within the aerodigestive cancer. To follow up on the basic research we will discuss remaining research questions and future directions involving these natural products as putative stand alone or in combination with clinical agents.

Reduction in promotor methylation utilizing EGCG (epigallocatechin-3-gallate) restores RXRα expression in human colon cancer cells.

Silencing of regulatory genes through hypermethylation of CpG islands is an important mechanism in tumorigenesis. In colon cancer, RXRα, an important dimerization partner with other nuclear transcription factors, is silenced through this mechanism. We previously found that colon tumors in ApcMin/+ mice had diminished levels of RXRα protein and expression levels of this gene were restored by treatment with a green tea intervention, due to reduced promoter methylation of RXRα. We hypothesized that CIMP+ cell lines, which epigenetically silence key regulatory genes would also evidence silencing of RXRα and EGCG treatment would restore its expression. We indeed found EGCG to restore RXRα activity levels in the human cell lines, in a dose dependent manner and reduced RXRα promoter methylation. EGCG induced methylation changes in several other colon cancer related genes but did not cause a decrease in global methylation. Numerous epidemiological reports have shown the benefits of green tea consumption in reducing colon cancer risk but to date no studies have shown that the risk reduction may be related to the epigenetic restoration by tea polyphenols. Our results show that EGCG modulates the reversal of gene silencing involved in colon carcinogenesis providing a possible avenue for colon cancer prevention and treatment.

Adaptations to chronic rapamycin in mice.

Rapamycin inhibits mechanistic (or mammalian) target of rapamycin (mTOR) that promotes protein production in cells by facilitating ribosome biogenesis (RiBi) and eIF4E-mediated 5'cap mRNA translation. Chronic treatment with encapsulated rapamycin (eRapa) extended health and life span for wild-type and cancer-prone mice. Yet, the long-term consequences of chronic eRapa treatment are not known at the organ level. Here, we report our observations of chronic eRapa treatment on mTORC1 signaling and RiBi in mouse colon and visceral adipose. As expected, chronic eRapa treatment decreased detection of phosphorylated mTORC1/S6K substrate, ribosomal protein (rpS6) in colon and fat. However, in colon, contrary to expectations, there was an upregulation of 18S rRNA and some ribosomal protein genes (RPGs) suggesting increased RiBi. Among RPGs, eRapa increases rpl22l1 mRNA but not its paralog rpl22. Furthermore, there was an increase in the cap-binding protein, eIF4E relative to its repressor 4E-BP1 suggesting increased translation. By comparison, in fat, there was a decrease in the level of 18S rRNA (opposite to colon), while overall mRNAs encoding ribosomal protein genes appeared to increase, including rpl22, but not rpl22l1 (opposite to colon). In fat, there was a decrease in eIF4E relative to actin (opposite to colon) but also an increase in the eIF4E/4E-BP1 ratio likely due to reductions in 4E-BP1 at our lower eRapa dose (similar to colon). Thus, in contrast to predictions of decreased protein production seen in cell-based studies, we provide evidence that colon from chronically treated mice exhibited an adaptive 'pseudo-anabolic' state, which is only partially present in fat, which might relate to differing tissue levels of rapamycin, cell-type-specific responses, and/or strain differences.

Chemoprevention in gastrointestinal physiology and disease. Targeting the progression of cancer with natural products: a focus on gastrointestinal cancer.

The last decade has witnessed remarkable progress in the utilization of natural products for the prevention and treatment of human cancer. Many agents now in the pipeline for clinical trial testing have evolved from our understanding of how human nutritional patterns account for widespread differences in cancer risk. In this review, we have focused on many of these promising agents arguing that they may provide a new strategy for cancer control: natural products once thought to be only preventive in their mode of action now are being explored for efficacy in tandem with cancer therapeutics. Natural products may reduce off-target toxicity of therapeutics while making cancers more amenable to therapy. On the horizon is the use of certain natural products, in their own right, as mitigants of late-stage cancer, a new frontier for small-molecule natural product drug discovery.

Green tea polyphenol epigallocatechin 3-gallate, contributes to the degradation of DNMT3A and HDAC3 in HCT 116 human colon cancer cells.

BACKGROUND: Colon cancer is still the second leading cause of cancer deaths in the United States. Epigenetic gene silencing involving DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) plays an important role in the progression of colon cancer. MATERIALS AND METHODS: In the present study we found that the sensitivity of colon cancer cells to methylation plays a role in its response to alternative therapy involving the green tea polyphenol, epigallocatechin 3-gallate. HDAC and DNMT protein expression were reduced when methylation-sensitive HCT 116 human colon cancer cells was treated with EGCG, but was relatively stable in the HT-29 cell line. This decrease in expression may be partially explained by our finding that DNMT3A and HDAC3 are degraded in the methylation-sensitive colon cancer cells in part by inhibiting their association with the E3 ubiquitin ligase, UHRF1. CONCLUSION: These findings provide a rationale for the development of a targeted therapy for methylation-sensitive colon cancer that can include EGCG in combination with other DNMT and HDAC inhibitors.

Nutraceutical use in late-stage cancer.

Access to a wealth of information on the internet has led many cancer patients to use complementary methods as an adjunct to traditional therapy for cancer, with, and more often, without informing their primary caregiver. Of the common complementary modalities, the use of dietary supplements appears to be highly prevalent in patients in active treatment for cancer, and later in cancer survivors. Emerging research suggests that some plant-based agents may, indeed, impact late-stage cancer, influencing molecular processes corrupted by tumor cells to evade detection, expand clonally, and invade surrounding tissues. The intent of this article is to review some of the current science underpinning the use of nutraceuticals in the latter stages of cancer.

Aberrant crypt foci: the case for inclusion as a biomarker for colon cancer.

Aberrant crypt foci (ACF) are one of the earliest histopathological manifestations of colon cancer. In this review, we critically present the molecular, cellular, histopathological, and chemopreventive evidence that ACF are relevant biomarkers for colon cancer. The laboratory and clinical evidence are highly suggestive that ACF are in the pathway leading to colon cancer, but not all ACF will do so. The possible fate and outcome of ACF in the progression toward colon cancer may be dependent on a number of features that define their predictive power for the prevention or progression of cancer.

AtCCX3 is an Arabidopsis endomembrane H+ -dependent K+ transporter.

The Arabidopsis (Arabidopsis thaliana) cation calcium exchangers (CCXs) were recently identified as a subfamily of cation transporters; however, no plant CCXs have been functionally characterized. Here, we show that Arabidopsis AtCCX3 (At3g14070) and AtCCX4 (At1g54115) can suppress yeast mutants defective in Na(+), K(+), and Mn(2+) transport. We also report high-capacity uptake of (86)Rb(+) in tonoplast-enriched vesicles from yeast expressing AtCCX3. Cation competition studies showed inhibition of (86)Rb(+) uptake in AtCCX3 cells by excess Na(+), K(+), and Mn(2+). Functional epitope-tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. In Arabidopsis, AtCCX3 is primarily expressed in flowers, while AtCCX4 is expressed throughout the plant. Quantitative polymerase chain reaction showed that expression of AtCCX3 increased in plants treated with NaCl, KCl, and MnCl(2). Insertional mutant lines of AtCCX3 and AtCCX4 displayed no apparent growth defects; however, overexpression of AtCCX3 caused increased Na(+) accumulation and increased (86)Rb(+) transport. Uptake of (86)Rb(+) increased in tonoplast-enriched membranes isolated from Arabidopsis lines expressing CCX3 driven by the cauliflower mosaic virus 35S promoter. Overexpression of AtCCX3 in tobacco (Nicotiana tabacum) produced lesions in the leaves, stunted growth, and resulted in the accumulation of higher levels of numerous cations. In summary, these findings suggest that AtCCX3 is an endomembrane-localized H(+)-dependent K(+) transporter with apparent Na(+) and Mn(2+) transport properties distinct from those of previously characterized plant transporters.

Nutritional impact of elevated calcium transport activity in carrots.

Nutrition recommendations worldwide emphasize ingestion of plant-based diets rather than diets that rely primarily on animal products. However, this plant-based diet could limit the intake of essential nutrients such as calcium. Osteoporosis is one of the world's most prevalent nutritional disorders, and inadequate dietary calcium is a known contributor to the pathophysiology of this condition. Previously, we have modified carrots to express increased levels of a plant calcium transporter (sCAX1), and these plants contain approximately 2-fold-higher calcium content in the edible portions of the carrots. However, it was unproven whether this change would increase the total amount of bioavailable calcium. In randomized trials, we labeled these modified carrots with isotopic calcium and fed them to mice and humans to assess calcium bioavailability. In mice feeding regimes (n = 120), we measured (45)Ca incorporation into bones and determined that mice required twice the serving size of control carrots to obtain the calcium found in sCAX1 carrots. We used a dual-stable isotope method with (42)Ca-labeled carrots and i.v. (46)Ca to determine the absorption of calcium from these carrots in humans. In a cross-over study of 15 male and 15 female adults, we found that when people were fed sCAX1 and control carrots, total calcium absorption per 100 g of carrots was 41% +/- 2% higher in sCAX1 carrots. Both the mice and human feeding studies demonstrate increased calcium absorption from sCAX1-expressing carrots compared with controls. These results demonstrate an alternative means of fortifying vegetables with bioavailable calcium.

Increased calcium bioavailability in mice fed genetically engineered plants lacking calcium oxalate.

Bioavailable calcium affects bone formation and calcification. Here we investigate how a single gene mutation altering calcium partitioning in the model forage crop Medicago truncatula affects calcium bioavailability. Previously, the cod5 M. truncatula mutant was identified which contains identical calcium concentrations to wild-type, but contains no oxalate crystals. In this study, equal number of male and female mice were randomly grouped and then fed one of four 45Ca-containing diets: M. truncatula extrinsically or intrinsically labeled, and cod5 extrinsically or intrinsically labeled. Absorption of the tracer was determined in the legs one day after consumption. The absorption was similar in the M. truncatula and cod5 extrinsically labeled diets; however, in the intrinsically labeled diets, calcium absorption was 22.87% (P < 0.001) higher in mice fed cod5. Our study presents the first genetic evidence demonstrating the nutritional impact of removing oxalate crystals from foods.

Up-regulation of a H+-pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistant crop plants.

Engineering drought -resistant crop plants is a critically important objective. Overexpression of the vacuolar H(+)-pyrophosphatase (H(+)-PPase) AVP1 in the model plant Arabidopsis thaliana results in enhanced performance under soil water deficits. Recent work demonstrates that AVP1 plays an important role in root development through the facilitation of auxin fluxes. With the objective of improving crop performance, we expressed AVP1 in a commercial cultivar of tomato. This approach resulted in (i) greater pyrophosphate-driven cation transport into root vacuolar fractions, (ii) increased root biomass, and (iii) enhanced recovery of plants from an episode of soil water deficit stress. More robust root systems allowed transgenic tomato plants to take up greater amounts of water during the imposed water deficit stress, resulting in a more favorable plant water status and less injury. This study documents a general strategy for improving drought resistance of crops.

Functional and regulatory analysis of the Arabidopsis thaliana CAX2 cation transporter.

The vacuolar sequestration of metals is an important metal tolerance mechanism in plants. The Arabidopsis thaliana vacuolar transporters CAX1 and CAX2 were originally identified in a Saccharomyces cerevisiae suppression screen as Ca2+/H+ antiporters. CAX2 has a low affinity for Ca2+ but can transport other metals including Mn2+ and Cd2+. Here we demonstrate that unlike cax1 mutants, CAX2 insertional mutants caused no discernable morphological phenotypes or alterations in Ca2+/H+ antiport activity. However, cax2 lines exhibited a reduction in vacuolar Mn2+/H+ antiport and, like cax1 mutants, reduced V-type H+ -ATPase (V-ATPase) activity. Analysis of a CAX2 promoter beta-glucoronidase (GUS) reporter gene fusion confirmed that CAX2 was expressed throughout the plant and strongly expressed in flower tissue, vascular tissue and in the apical meristem of young plants. Heterologous expression in yeast identified an N-terminal regulatory region in CAX2, suggesting that Arabidopsis contains multiple cation/H+ antiporters with shared regulatory features. Furthermore, despite significant variations in morphological and biochemical phenotypes, cax1 and cax2 lines both significantly alter V-ATPase activity, hinting at coordinate regulation among transporters driven by H+ gradients and the V-ATPase.

Efficient and genotype-independent Agrobacterium--mediated tomato transformation.

An efficient method to transform five cultivars of tomato (Lycopersicon esculentum), Micro-Tom, Red Cherry, Rubion, Piedmont, and E6203 is reported. A comparison was made of leaf, cotyledon, and hypocotyl explants on 7 different regeneration media without Agrobacterium tumefaciens cocultivation and on 11 different media with cocultivation. Although all cultivars and explants formed callus and regenerated on the initial 7 media, cocultivation with A. tumefaciens significantly reduced the callus induction and regeneration. From these experiments, a transformation methodology using either hypocotyls or cotyledons cultured for one day on BA 1 mgL-1, NAA 0.1 mgL-1 and 3 days cocultivation with the Agrobacterium on this same medium followed by a transfer to a medium with zeatin 2 mgL-1 and IAA 0.1 mgL-1 for 4-6 weeks resulted in a greater than 20% transformation frequency for all five cultivars tested. In this transformation method, no feeder layers of tobacco, petunia or tomato suspension cultures were used, and the subculture media was minimal. Stable integration and transmission of the transgene in T1 generation plants were confirmed by Southern blot analysis. This procedure represents a simple, efficient and general means of transforming tomato.