Eriodictyol inhibits breast carcinogenesis by targeting circ_0007503 and repressing PI3K/Akt pathway.

BACKGROUND: Eriodictyol in citrus fruits, Eriodictyon californicum and several Chinese herbal medicines shows great promise for chronic disease prevention, including cancers. However, its role in chemopreventive activities against breast carcinogenesis is unknown. PURPOSE: In the present study, we investigated the chemopreventive effect and the underlying mechanism of eriodictyol on carcinogens-induced breast carcinogenesis in vivo and in vitro. METHODS: The carcinogenic transformation in MCF10A cells was induced by the environmental carcinogens in vitro. The chemopreventive effect in vivo was evaluated by using the experimental model of 1-methyl-1-nitrosourea (MNU)-induced mammary tumorigenesis in rats. The activation of the PI3K/Akt pathway was detected by western blot assay; the levels of circular RNAs (circRNAs) were measured by qRT-PCR. RESULTS: First, eriodictyol significantly reduces cells viability and induces apoptosis in breast cancer cells in a dose-dependent manner in vitro (P < 0.05). Next, eriodictyol could effectively suppress environmental carcinogens-induced acquisition of carcinogenic properties in human breast epithelial cell MCF10A (P < 0.05). In vivo, eriodictyol administration reduces the incidence of mammary tumor by 50% in carcinogen-treated female rats (P < 0.05). Further study revealed that eriodictyol represses the PI3K/Akt signaling pathway and down-regulates the level of circ_0007503 in breast cancer cells and in breast carcinogenesis (P < 0.01). When the effect of eriodictyol on circ_0007503 was blocked by transfection of a circ_0007503 over-expression plasmid, the cytotoxic effects and the suppression of the PI3K/Akt pathway of eriodictyol in breast cancer cells were significantly reduced (P < 0.05). CONCLUSION: Our data indicated that eriodictyol could effectively suppress breast carcinogenesis in vitro and in vivoThe mechanism may be attributed to targeting circ_0007503 and inhibiting PI3K/Akt pathway.

Kangxianruangan granule­containing serum mediated inhibition of hepatic oval cell differentiation into hepatocellular carcinoma cells via the Wnt­1/ß­catenin signaling pathway.

Hepatocellular carcinoma is a malignancy with poor clinical prognosis. Hepatic oval cells (HOCs) tend to differentiate into cancerous hepatocellular carcinoma cells (HCCs) in the tumor microenvironment. The purpose of the present study was to explore the role of kangxianruangan granule (KXRG)­containing serum in inhibiting the differentiation of HOCs into HCCs via the Wnt­1/ß­catenin signaling pathway. N­methyl­N'­nitro­N­nitrosoguanidine (MNNG) was applied to induce the transformation of the rat HOC cell line WB­F344 into HCCs. The overexpression plasmid, Wnt­1­up, was utilized to increase Wnt­1 expression. Subsequently, high, medium and low concentrations of KXRG were applied to MNNG­treated WB­F344 cells to assess the inhibitory effect of KXRG on cell differentiation. Flow cytometry was conducted to detect the cell cycle distribution, apoptotic rate and expression of cytokeratin­19 (CK­19) protein in cells. An immunofluorescence double staining protocol was used to detect the expression of Wnt­1 and ß­catenin. ELISAs were performed to detect α fetoprotein in the cell supernatants. Reverse transcription­quantitative PCR and western blotting were conducted to detect the mRNA and protein expression levels of Wnt­1, ß­catenin, Cyclin D1, C­myc, matrix metalloproteinase­7 (MMP­7), Axin2 and epithelial cell adhesion molecule (EpCAM) in cells. Compared with the normal group, the apoptotic rate, proportion of S phase cells, concentration of AFP in the cell supernatant, level of CK­19 protein, and mRNA and protein expression levels of Wnt­1, ß­catenin, Cyclin D1, C­myc, MMP­7, Axin2 and EpCAM were all significantly increased in the model group. Addition of KXRG significantly reduced the aforementioned indicators compared with the model group. Moreover, Wnt­1 overexpression further increased the aforementioned indicators compared with the model group, whereas KXRG significantly inhibited these effects. The results indicated that KXRG inhibited the differentiation of HOCs into HCCs via the Wnt­1/ß­catenin signaling pathway, which suggested the potential clinical application of KXRG for the prevention of hepatocellular carcinoma.

Entelon® (Vitis vinifera Seed Extract) Prevents Cancer Metastasis via the Downregulation of Interleukin-1 Alpha in Triple-Negative Breast Cancer Cells.

Interleukin-1 (IL1) is a proinflammatory cytokine and promotes cancer cell proliferation and invasiveness in a diversity of cancers, such as breast and colon cancer. Here, we focused on the pharmacological effect of Entelon® (ETL) on the tumorigenesis of triple-negative breast cancer (TNBC) cells by IL1-alpha (IL1A). IL1A enhanced the cell growth and invasiveness of TNBC cells. We observed that abnormal IL1A induction is related with the poor prognosis of TNBC patients. IL1A also increased a variety of chemokines such as CCL2 and IL8. Interestingly, IL1A expression was reduced by the ETL treatment. Here, we found that ETL significantly decreased the MEK/ERK signaling pathway in TNBC cells. IL1A expression was reduced by UO126. Lastly, we studied the effect of ETL on the metastatic potential of TNBC cells. Our results showed that ETL significantly reduced the lung metastasis of TNBC cells. Our results showed that IL1A expression was regulated by the MEK/ERK- and PI3K/AKT-dependent pathway. Taken together, ETL inhibited the MEK/ERK and PI3K/AKT signaling pathway and suppressing the lung metastasis of TNBC cells through downregulation of IL1A. Therefore, we propose the possibility of ETL as an effective adjuvant for treating TNBC.

Natural products in the reprogramming of cancer epigenetics.

Owing to the technological advancements, including next generation sequencing, the significance of deregulated epigenetic mechanisms in cancer initiation, progression and treatment has become evident. The accumulating knowledge relating to the epigenetic markers viz. DNA methylation, Histone modifications and non-coding RNAs make them one of the most interesting candidates for developing anti-cancer therapies. The reversibility of deregulated epigenetic mechanisms through environmental and dietary factors opens numerous avenues in the field of chemoprevention and drug development. Recent studies have proven that plant-derived natural products encompass a great potential in targeting epigenetic signatures in cancer and numerous natural products are being explored for their possibility to be considered as "epi-drug". This review intends to highlight the major aberrant epigenetic mechanisms and summarizes the essential functions of natural products like Resveratrol, Quercetin, Genistein, EGCG, Curcumin, Sulforaphane, Apigenin, Parthenolide and Berberine in modulating these aberrations. This knowledge along with the challenges and limitations in this field has potential and wider implications in developing novel and successful therapeutic strategies. The increased focus in the area will possibly provide a better understanding for the development of dietary supplements and/or drugs either alone or in combination. The interaction of epigenetics with different hallmarks of cancer and how natural products can be utilized to target them will also be interesting in the future therapeutic approaches.

Implications of Withaferin-A for triple-negative breast cancer chemoprevention.

Triple-negative breast cancer (TNBC) accounts for about 15 % of all breast cancer cases, and unlike other malignancies, it lacks definite prognostic markers. While improved survival responses have been documented with the ongoing therapeutic approaches, the development of tumor resistance mechanisms to these treatment options pose major challenges in the treatment of TNBC. Notably, naturally occurring medicinal compounds have been studied extensively for their anti-neoplastic activities in cancer models including breast cancer due to their safe and non-deleterious effects. Among various dietary compounds, Withaferin-A (WA), a phytochemical derived from an ayurvedic medicinal plant, Withania somnifera has been characterized to possess anti-inflammatory and anti-cancer properties. Importantly, multiple studies have shown that WA exhibits promising anti-tumoral activities against in-vitro and in-vivo experimental models of TNBC and that its combination has been documented to enhance chemotherapy efficacy. The current review highlights the mechanistic insights with recent updates including the pharmacokinetics parameters and implications of WA against breast cancer with major emphasis on TNBC.

Low-polar extract from seed of Cleistocalyx nervosum var. paniala modulates initiation and promotion stages of chemically-induced carcinogenesis in rats.

BACKGROUND: Cleistocalyx nervosum var. paniala is a local fruit mainly cultivated in the north of Thailand. Our previous study has reported that the methanol extract of C. nervosum seed presented antimutagenicity in a Salmonella mutation assay. The present study focused on the effect of a low-polar extract of C. nervosum seed on the early stages of diethylnitrosamine (DEN)- and dimethylhydrazine (DMH)-induced carcinogenesis in rats. METHODS: Dried C. nervosum seed powder was extracted using dichloromethane. To study its effect on the initiation stage of carcinogenesis of rats, they were fed with various doses of C. nervosum seed extract (CSE) for 21 days. DEN injection was used to initiate hepatocarcinogenesis and partial hepatectomy was performed to amplify mutated hepatocytes resulting in micronucleated hepatocyte formation. To study the role of CSE on the promotion stage, rats were injected with DEN and DMH to induce preneoplastic lesions and the numbers of glutathione S-transferase placental form (GST-P) positive foci in the liver and aberrant crypt foci (ACF) in the colon were measured. This was followed by CSE administration for 10 weeks. The inhibitory mechanisms of CSE on initiation and promotion stages, including xenobiotic metabolism, cell proliferation and apoptosis, were investigated. RESULTS: The total phenolic content in CSE was 80.34 ± 2.29 mg gallic acid equivalents (GAE) per g of extract and 2,4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone was found to be a major flavonoid. The main terpenoids in CSE were ß-selinene, α-selinene, γ-selinene and o-cymene while 24(Z)-methyl-25-homocholesterol was a major phytosterol. CSE significantly decreased the number of micronucleated hepatocytes in DEN-initiated rats and enhanced the activities of hepatic glutathione S-transferase and UDP-glucuronyltransferase. Furthermore, the formation of preneoplastic lesions in the liver and colon was statistically reduced by CSE. CSE also diminished cell proliferation in the liver and colon indicated by the number of PCNA positive cells. However, CSE did not alter the numbers of apoptotic hepatocytes and colonocytes in DEN- and DMH-initiated rats. CONCLUSIONS: The dichloromethane extract of C. nervosum seed demonstrated chemopreventive effects on chemically-induced carcinogenesis in both initiation and promotion stages in rats. The inhibitory mechanism might be involved in the modulation of hepatic detoxifying enzymes and suppression of hepatocyte and colonocyte proliferation.

The oxytocin receptor signalling system and breast cancer: a critical review.

Breast cancer is making up one-quarter of all new female cancer cases diagnosed worldwide. Breast cancer surgeries, radiation therapies, cytotoxic chemotherapies and targeted therapies have made significant progress and play a dominant role in breast cancer patient management. However, many challenges remain, including resistance to systemic therapies, tumour recurrence and metastasis. The cyclic neuropeptide oxytocin (OT) elicits a plethora of biological responses via the oxytocin receptor (OTR) in both the central and peripheral nervous system, including social bonding, stress, maternal behaviour, sexual activity, uterus contraction, milk ejection and cancer. As a typical member of the G protein-coupled receptor family, OTR represents also an intriguing target for cancer therapy. There is emerging evidence that OTR plays a role in breast cancer development and progression, and several breast cancer cell lines express OTR. However, despite supporting evidence that OT lowers breast cancer risks, its mechanistic role in breast cancer development and the related signalling pathways are not fully understood. Here, we review the current knowledge of the OT/OTR signalling system in healthy breast tissue as well as in breast cancer, and discuss OTR as a potential therapeutic target for breast cancer management.

The active fraction of Garcinia yunnanensis suppresses the progression of colorectal carcinoma by interfering with tumorassociated macrophage-associated M2 macrophage polarization in vivo and in vitro.

Colorectal cancer (CRC) is the third most common solid tumor worldwide and has shown resistance to several immunotherapies, particularly immune checkpoint blockade therapy, which is effective in many other types of cancer. Our previous studies indicated that the active fraction of Garcinia yunnanensis (YTE-17), had potent anticancer activities by regulating multiple signaling pathways. However, knowledge regarding the mechanism and effect of YTE-17 in the prevention of CRC is limited. This study tested the effects of YTE-17 on colon cancer development in vivo by using two murine models: the carcigenic azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CRC model and a genetically induced model using ApcMin/+ mice. Here, the tumor load, tumor number, histology, and even some oncogenes were used to evaluate the effect of YTE-17. The intragastric administration of YTE-17 for 12 weeks significantly decreased CRC incidence, tumor number and size, immunity, and some tumor-associated macrophage (TAM) markers, including CD206, Arg-1, IL-10, and TGF-ß. Importantly, the macrophages depletion by clodronate (CEL) also played a role in reducing the tumor burden and inhibiting tumor development, which were not affected by YTE-17 in the ApcMin/+ mice. Moreover, the YTE-17 treatment attenuated CRC cell growth in a co-culture system in the presence of macrophages. Consistently, YTE-17 effectively reduced the tumor burden and macrophage infiltration and enhanced immunity in the AOM/DSS and ApcMin/+ colon tumor models. Altogether, we demonstrate that macrophages in the microenvironment may contribute to the development and progression of CRC cells and propose YTE-17 as a new potential drug option for the treatment of CRC.

Dietary Ziziphus jujuba Fruit Attenuates Colitis-Associated Tumorigenesis: A Pivotal Role of the NF-κB/IL-6/JAK1/STAT3 Pathway.

Inflammatory bowel disease (IBD) including ulcerative colitis (UC) is one of the risk factors for the development of colitis-associated colon cancer (CAC). CAC is a type of colorectal cancer (CRC), the third leading cause of cancer death. Ziziphus jujuba (ZJ) fruit contains bioactive components such as polysaccharides, triterpenoid acid, and flavonoids, and it has shown anti-inflammatory property. The aim of the study was to investigate the protective effect of dietary ZJ on colitis-associated colorectal tumorigenesis in mice. Mice (n = 42, two sets) were injected with azoxymethane (AOM) followed by three cycles of 2% (w/v) dextran sulfate sodium (DSS) in drinking water to induce CAC. Simultaneously, those mice were fed with ZJ diet for 70 days (5% or 10% w/w). Data were analyzed by ANOVA followed by LSD Bonferroni test. Dietary ZJ decreased fecal blood, diarrhea, disease activity index (DAI), spleen weight (P < 0.001), and the number of tumors (P < 0.001). In addition, dietary ZJ increased colon length (P < 0.001) and suppressed the activation of NF-кB/IL-6/JAK1/STAT3 signaling pathway. In conclusion, we suggest that dietary ZJ attenuates inflammation by interfering NF-κB/IL-6/JAK1/STAT3 signaling pathway, thereby inhibits AOM/DSS-induced colon tumorigenesis in mice.

Lemongrass Extract Possesses Potent Anticancer Activity Against Human Colon Cancers, Inhibits Tumorigenesis, Enhances Efficacy of FOLFOX, and Reduces Its Adverse Effects.

Current chemotherapeutics for metastatic colorectal cancers have limited success and are extremely toxic due to nonselective targeting. Some natural extracts have been traditionally taken and have shown anticancer activity. These extracts have multiple phytochemicals that can target different pathways selectively in cancer cells. We have shown previously that lemongrass (Cymbopogon citratus) extract is effective at inducing cell death in human lymphomas. However, the efficacy of lemongrass extract on human colorectal cancer has not been investigated. Furthermore, its interactions with current chemotherapies for colon cancer is unknown. In this article, we report the anticancer effects of ethanolic lemongrass extract in colorectal cancer models, and importantly, its interactions with FOLFOX and Taxol. Lemongrass extract induced apoptosis in colon cancer cells in a time and dose-dependent manner without harming healthy cells in vitro. Oral administration of lemongrass extract was well tolerated and effective at inhibiting colon cancer xenograft growth in mice. It enhanced the anticancer efficacy of FOLFOX and, interestingly, inhibited FOLFOX-related weight loss in animals given the combination treatment. Furthermore, feeding lemongrass extract to APCmin/+ transgenic mice led to the reduction of intestinal tumors, indicating its preventative potential. Therefore, this natural extract has potential to be developed as a supplemental treatment for colorectal cancer.

Bufalin, a Traditional Chinese Medicine Compound, Prevents Tumor Formation in Two Murine Models of Colorectal Cancer.

Chemoprevention is cost-effective for colorectal cancer when targeted at intermediate- or high-risk populations. Bufalin is a cardiac glycoside extracted from the traditional Chinese medicine (TCM) "Chan Su," which has been used as an anticancer agent. On the basis of the relative safety of bufalin, we investigated whether bufalin could act as a chemoprophylactic agent to prevent colon tumorigenesis in two murine models, namely colitis-associated colorectal cancer and Apc germline mutation-developed colorectal cancer. Our results revealed that long-term (12-16 weeks) administration of low-dose bufalin (0.5 mg/kg) effectively suppressed tumorigenesis in both colorectal cancer models, accompanied by attenuated epithelial cell proliferation (reduced bromodeoxyuridine incorporation, lower levels of cyclin A, cyclin D1, cyclin E, and cyclin-dependent kinases-2/4, and higher levels of p21 and p27) and promoted apoptosis (increased TUNEL positivity and caspase-3/9 cleavages, reduced levels of Bcl-2, Bcl-xL and survivin, and increased levels of Bax and Bak). Bufalin also suppressed the expression of proinflammatory mediators [reduced levels of cyclooxygenase-2, tumor TNFα, IL1ß, IL6, C-X-C motif chemokine ligand (CXCL)-1, CXCL-2, and CXCL-5] in the colitis-associated colorectal cancer model. These effects were associated with the inhibition of oncogenic NF-κB and PI3K/Akt pathways. Our findings unveil a novel chemoprophylactic action of bufalin in colorectal cancer in vivo and provided efficacy data and mechanistic evidence for further clinical evaluation of this TCM compound for colorectal cancer chemoprevention in individuals at risk of colorectal cancer.

Fetal liver Mll-AF4+ hematopoietic stem and progenitor cells respond directly to poly(I:C), but not to a single maternal immune activation.

T(4;11) MLL-AF4 acute leukemia is one of the most aggressive malignancies in infant and pediatric populations. Epidemiological and functional studies have highlighted the influence of an overstimulation of the immune system on leukemia development. This study aimed at assessing if the cell-of-origin of t(4;11) MLL-AF4 acute leukemia is sensitive to a viral or bacterial mimic and if maternal immune activation can lead to a full-blown leukemia. To answer this, we used the Mll-AF4 pre-leukemia mouse model that initiates the expression of Mll-AF4 in the first definitive hematopoietic cells formed during embryonic development. We observed an increase in proliferation upon hematopoietic differentiation of fetal liver Mll-AF4+ Lineage-Sca1+ckit+ (LSK) cells exposed to the immune stimulants, poly(I:C) or LPS/lipopolysaccharide. This was accompanied by increased expression of a subset of MLL-AF4 signature genes and members of the Toll-like receptor signaling pathways in fetal liver Mll-AF4+ LSK exposed to poly(I:C), suggesting that the cell-of-origin responds to inflammatory stimuli. Maternal immune activation using a single dose of poly(I:C) did not lead to the development of leukemia in Mll-AF4+ and control offspring. Instead, aging MLL-AF4+ mice showed an increased proportion of T-lymphoid cells in the spleen, lost their B-lymphoid bias, and had decreased frequencies of hematopoietic stem and multipotent progenitor cells. Overall, this study suggests that the fetal liver Mll-AF4+ LSK cells are sensitive to direct exposure to inflammatory stimuli, especially poly(I:C); however, maternal immune activation induced by a single exposure to poly(I:C) is not sufficient to initiate MLL-AF4 leukemogenesis.

Sensitivity and Resistance of MET Exon 14 Mutations in Lung Cancer to Eight MET Tyrosine Kinase Inhibitors In Vitro.

BACKGROUND: MNNG HOS transforming gene (MET) exon 14 mutations in lung cancer, including exon 14 skipping and point mutations, have been attracting the attention of thoracic oncologists as new therapeutic targets. Tumors with these mutations almost always acquire resistance, which also occurs in other oncogene-addicted lung cancers. However, the resistance mechanisms and treatment strategies are not fully understood. METHODS: We generated Ba/F3 cells expressing MET exon 14 mutations by retroviral gene transfer. The sensitivities of these cells to eight MET-tyrosine kinase inhibitors (TKIs) were determined using a colorimetric assay. In addition, using N-ethyl-N-nitrosourea mutagenesis, we generated resistant clones, searched for secondary MET mutations, and then examined the sensitivities of these resistant cells to different TKIs. RESULTS: Ba/F3 cells transfected with MET mutations grew in the absence of interleukin-3, indicating their oncogenic activity. These cells were sensitive to all MET-TKIs except tivantinib. We identified a variety of secondary mutations. D1228 and Y1230 were common sites for resistance mutations for type I TKIs, which bind the active form of MET, whereas L1195 and F1200 were common sites for type II TKIs, which bind the inactive form. In general, resistance mutations against type I were sensitive to type II, and vice versa. CONCLUSIONS: MET-TKIs inhibited the growth of cells with MET exon 14 mutations. We also identified mutation sites specific for TKI types as resistance mechanisms and complementary activities between type I and type II inhibitors against those mutations. These finding should provide relevant clinical implication for treating patients with lung cancer harboring MET exon 14 mutations.

Houttuynia cordata Thunb. and its bioactive compound 2-undecanone significantly suppress benzo(a)pyrene-induced lung tumorigenesis by activating the Nrf2-HO-1/NQO-1 signaling pathway.

BACKGROUND: Lung cancer remains the most common cause of cancer-related deaths, with a high incidence and mortality in both sexes worldwide. Chemoprevention has been the most effective strategy for lung cancer prevention. Thus, exploring novel and effective candidate agents with low toxicity for chemoprevention is essential and urgent. Houttuynia cordata Thunb. (Saururaceae) (H. cordata), which is a widely used herbal medicine and is also popularly consumed as a healthy vegetable, exhibits anti-inflammatory, antioxidant and antitumor activity. However, the chemopreventive effect of H. cordata against benzo(a)pyrene (B[a]P)-initiated lung tumorigenesis and the underlying mechanism remain unclear. METHODS: A B[a]P-stimulated lung adenocarcinoma animal model in A/J mice in vivo and a normal lung cell model (BEAS.2B) in vitro were established to investigate the chemopreventive effects of H. cordata and its bioactive compound 2-undecanone against lung tumorigenesis and to clarify the underlying mechanisms. RESULTS: H. cordata and 2-undecanone significantly suppressed B[a]P-induced lung tumorigenesis without causing obvious systemic toxicity in mice in vivo. Moreover, H. cordata and 2-undecanone effectively decreased B[a]P-induced intracellular reactive oxygen species (ROS) overproduction and further notably protected BEAS.2B cells from B[a]P-induced DNA damage and inflammation by significantly inhibiting phosphorylated H2A.X overexpression and interleukin-1ß secretion. In addition, H. cordata and 2-undecanone markedly activated the Nrf2 pathway to induce the expression of the antioxidative enzymes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO-1). Nrf2 silencing by transfection with Nrf2 siRNA markedly decreased the expression of HO-1 and NQO-1 to diminish the reductions in B[a]P-induced ROS overproduction, DNA damage and inflammation mediated by H. cordata and 2-undecanone. CONCLUSIONS: H. cordata and 2-undecanone could effectively activate the Nrf2-HO-1/NQO-1 signaling pathway to counteract intracellular ROS generation, thereby attenuating DNA damage and inflammation induced by B[a]P stimulation and playing a role in the chemoprevention of B[a]P-induced lung tumorigenesis. These findings provide new insight into the pharmacological action of H. cordata and indicate that H. cordata is a novel candidate agent for the chemoprevention of lung cancer.

Preclinical Prevention Trial of Calcitriol: Impact of Stage of Intervention and Duration of Treatment on Oral Carcinogenesis.

The anticancer activity of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3 or calcitriol) has been widely reported in preclinical models. However, systematic investigation into the chemopreventive potential of calcitriol against the spectrum of oral carcinogenesis has not been performed. To address this gap in knowledge, we conducted a preclinical prevention trial of calcitriol in the 4-nitroquinoline-1-oxide (4NQO) oral carcinogenesis model. C57BL/6 mice were exposed to the carcinogen 4NQO in drinking water for 16 weeks and randomized to control (4NQO only) or calcitriol arms. Calcitriol (0.1 µg i.p, Monday, Wednesday, and Friday) was administered for (i) 16 weeks concurrently with 4NQO exposure, (ii) 10 weeks post completion of 4NQO exposure, and, (iii) a period of 26 weeks concurrent with and following 4NQO exposure. Longitudinal magnetic resonance imaging (MRI) was performed to monitor disease progression until end point (week 26). Correlative histopathology of tongue sections was performed to determine incidence and multiplicity of oral dysplastic lesions and squamous cell carcinomas (SCC). Vitamin D metabolites and calcium were measured in the serum using liquid chromatography-mass spectrometry (LC-MS/MS) and colorimetric assay, respectively. Renal CYP24A1 (24-hydroxylase) and CYP27B1 (1α-hydroxylase) expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Immunostaining of tongue sections for vitamin D receptor (VDR), CYP24A1, and Ki67 was also performed. Non-invasive MRI enabled longitudinal assessment of lesions in the oral cavity. Calcitriol administered concurrently with 4NQO for 16 weeks significantly (P < .001) decreased the number of premalignant lesions by 57% compared to 4NQO only controls. Mice treated with calcitriol for 26 weeks showed highest renal CYP24A1, lowest serum 1,25(OH)2D3 levels and highest incidence of invasive SCC. Immunohistochemistry revealed increased VDR, CYP24A1 and Ki67 staining in dysplastic epithelia compared to normal epithelium, in all four groups. Collectively, our results show that the effects of calcitriol on oral carcinogenesis are critically influenced by the stage of intervention and duration of exposure and provide the basis for exploring the potential of calcitriol for prevention of OSCC in the clinical setting.

Epimagnolin targeting on an active pocket of mammalian target of rapamycin suppressed cell transformation and colony growth of lung cancer cells.

Mammalian target of rapamycin (mTOR) has a pivotal role in carcinogenesis and cancer cell proliferation in diverse human cancers. In this study, we observed that epimagnolin, a natural compound abundantly found in Shin-Yi, suppressed cell proliferation by inhibition of epidermal growth factor (EGF)-induced G1/S cell-cycle phase transition in JB6 Cl41 cells. Interestingly, epimagnolin suppressed EGF-induced Akt phosphorylation strongly at Ser473 and weakly at Thr308 without alteration of phosphorylation of MAPK/ERK kinases (MEKs), extracellular signal-regulated kinase (ERKs), and RSK1, resulting in abrogation of the phosphorylation of GSK3ß at Ser9 and p70S6K at Thr389. Moreover, we found that epimagnolin suppressed c-Jun phosphorylation at Ser63/73, resulting in the inhibition of activator protein 1 (AP-1) transactivation activity. Computational docking indicated that epimagnolin targeted an active pocket of the mTOR kinase domain by forming three hydrogen bonds and three hydrophobic interactions. The prediction was confirmed by using in vitro kinase and adenosine triphosphate-bead competition assays. The inhibition of mTOR kinase activity resulted in the suppression of anchorage-independent cell transformation. Importantly, epimagnolin efficiently suppressed cell proliferation and anchorage-independent colony growth of H1650 rather than H460 lung cancer cells with dependency of total and phosphorylated protein levels of mTOR and Akt. Inhibitory signaling of epimagnolin on cell proliferation of lung cancer cells was observed mainly in mTOR-Akt-p70S6K and mTOR-Akt-GSK3ß-AP-1, which was similar to that shown in JB6 Cl41 cells. Taken together, our results indicate that epimagnolin potentiates as chemopreventive or therapeutic agents by direct active pocket targeting of mTOR kinase, resulting in sensitizing cancer cells harboring enhanced phosphorylation of the mTORC2-Akt-p70S6k signaling pathway.

Establishment of a multicomponent dietary bioactive human equivalent dose to delete damaged Lgr5+ stem cells using a mouse colon tumor initiation model.

Multicomponent therapy has gained interest for its potential to synergize and subsequently lower the effective dose of each constituent required to reduce colon cancer risk. We have previously showed that rapidly cycling Lgr5 stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk. In the present study, we quantified the dose-dependent synergistic properties of dietary n-3 polyunsaturated fatty acids (PUFA) and curcumin (Cur) to promote targeted apoptotic deletion of damaged colonic Lgr5 stem cells. For this purpose, both heterogeneous bulk colonocytes and Lgr5 stem cells were isolated from Lgr5-EGFP-IRES-CreER knock-in mice injected with azoxymethane (AOM). Isolated cells were analyzed for DNA damage (γH2AX), apoptosis (cleaved caspase-3), and targeted apoptosis (both γH2AX and cleaved caspase-3) at 12 h post-AOM injection. Comparison of the percentage of targeted apoptosis in Lgr5 stem cells (GFP) across a broad bioactive dose-range revealed an ED50 of 16.0 mg/day n-3 PUFA + 15.9 mg/day Cur. This corresponded to a human equivalent dose of 3.0 g n-3 PUFA + 3.0 g Cur. In summary, our results provide evidence that a low dose (n-3 PUFA + Cur) combination diet reduces AOM-induced DNA damage in Lgr5 stem cells and enhances targeted apoptosis of DNA-damaged cells, implying that a lower human equivalent dose can be utilized in future human clinical trials.

Xiao Tan He Wei Decoction reverses MNNG-induced precancerous lesions of gastric carcinoma in vivo and vitro: Regulation of apoptosis through NF-κB pathway.

In recent years, Chinese medicine has played an important role in the prognosis of gastric cancer. Precancerous lesions of gastric carcinoma (PLGC) is a class of gastric cancer which is closely related to the gastric mucosal pathology changes in the role of carcinogenic incentives, and plays key role in the progression of normal gastric mucosal cells into gastric cancerous cells. In current experiment, we explore the relationship between Chinese traditional medicine (Xiao Tan He Wei Decoction) and gastric cancer in the PLGC rat animal models and epithelial-mesenchymal transitioned GES-1 cells which were induced useing 1- Methyl-3-nitro-1-nitrosoguanidine (MNNG). PLGC rat model showed significant deterioration in the gastric mucosa with terrible growth rate in body weight and more atypical hyperplasia in gastric mucosa. MC cells, MNNG induced GES-1 cells which epithelial- mesenchymal-transition (EMT)-related proteins have a great change compare with normal GES-1 cells. The cells had characteristics of malignant cells including proliferation, invasion and metastasis ability. Our research founds that Xiao Tan He Wei Decoction could inhibit cell proliferation and increased apoptosis by increase the level of pro-apoptotic proteins like Bax and caspase-3 and decreased the level of anti-apoptotic protein Bcl-2, block the cells in G0/G1 phase simultaneously. Furthermore, Xiao Tan He Wei Decoction could inhibit nuclear factor kappa-light-chain-enhancer (NF-kB) activity and inhibit its transfer from the cytoplasm to the nucleus. However, when we incubated with NF-κB activator PMA, the effect of Xiao Tan He Wei Decoction was reversed. These results suggested that Xiao Tan He Wei Decoction could be used as a method for the treatment of gastric precancerous lesions, and possibly provide a theoretical basis for the clinical treatment of gastric cancer and gastric precancerous lesions.

Inhibition effect of glycyrrhiza polysaccharide (GCP) on tumor growth through regulation of the gut microbiota composition.

Glycyrrhiza Uralensis Polysaccharide (GCP), as a macromolecular polysaccharide extracted from the Traditional Chinese Medicine (TCM) - Licorice has been proved to inhibit tumor growth in vitro and in vivo; however, the specific anti-tumor mechanism of GCP needs to be further investigated. In this study, we explore the anti-tumor mechanism of GCP from the angle of gut microbiota. Colon carcinoma cells (CT-26) were used to set up a tumor-bearing mouse model. After 14 days of GCP treatment, the weights of tumors were significantly reduced. In addition, HE staining of tissue sections reflected that GCP could effectively inhibit tumor metastasis. 16SrRNA high-throughput sequencing of fecal samples showed a significant change between the model group and GCP group in the composition of gut microbiota. Subsequently, gut microbiota depletion and fecal transplantation experiments further confirmed the relationship between the anti-tumor effects of GCP and gut microbiota. Following depletion of gut microbiota, GCP cannot inhibit tumor growth. Fecal transplantation experiments found that transplanting the feces of GCP-treated mice, to a certain extent, could inhibit tumor growth and metastasis. These results indicate that Glycyrrhiza Polysaccharides exert anti-tumor effects by affecting gut microbiota composition.

Natural dietary compound naringin prevents azoxymethane/dextran sodium sulfate-induced chronic colorectal inflammation and carcinogenesis in mice.

Naringin, a natural occurring flavonoid compound, enriches in citrus fruits. We aimed to evaluate the inhibitory effect of naringin on colitis and chronic inflammation-driven carcinogenesis. Male C57BL/6 mice were exposed to AOM/DSS to induce colorectal inflammation and carcinogenesis. Naringin by oral administration prevented AOM/DSS-induced ulcerative colitis and carcinogenesis without significant side effects. Naringin attenuated the severity of colitis and colorectal adenomas through inhibiting myeloid-derived suppressor cells (MDSCs), pro-inflammatory mediators GM-CSF/M-CSF, IL-6 and TNF-α and the NF-κB/IL-6/STAT3 cascades in colorectal tissues. Naringin-treated mice exhibited normalized structures of colorectal tissues. Electron microscopy analysis showed the suppression of robust endoplasmic reticulum (ER) stress-induced autophagy. Naringin inhibited the secretion of the ER-spanning transmembrane proteins, such as GRP78 ATF6, IRE1α and activated PERK phosphorylated eIF-2α and complex of autophagosomes ATG3, ATG5, ATG7, ATG12, ATG16 and ATG16L1 in the colorectal mucosal cells. CONCLUSION: Naringin prevented colitis and colorectal carcinogenesis through suppressing robust ER stress-induced autophagy in colorectal mucosal cells. Naringin could develop a promising therapeutic agent for the prevention of ulcerative colitis and colorectal tumor.