Histidine Enhances the Anticancer Effect of Gemcitabine against Pancreatic Cancer via Disruption of Amino Acid Homeostasis and Oxidant-Antioxidant Balance.

Due to the severe toxicity posed by chemotherapeutic drugs, adjuvant nutritional intervention has gained increased attention in the treatment of pancreatic cancer (PC). Amino acid (AA) metabolism is aberrantly regulated in PC and circulating histidine (His) levels are low in PC patients. We hypothesized that His uptake and/or metabolism is dysregulated in PC and that combining His with gemcitabine (Gem), a drug used in the treatment of PC, will enhance the anti-cancer effects of Gem. We performed in vitro and in vivo studies to determine the anticancer effect of the combination of His and Gem against lethal PC. We demonstrate that circulating His levels are low in both human subjects and genetically engineered mice exhibiting pancreatic tumors. Interestingly, the expression of histidine ammonia lyase, an enzyme involved in His catabolism, is higher in PC compared to normal subjects. His + Gem exerts a more potent cytotoxic effect in PC cells compared to individual treatments. His treatment results in a profound increase in His accumulation, accompanied by a depletion of a number of AAs, promoting cancer cell survival and/or glutathione (GSH) synthesis. His but not Gem increases hydrogen peroxide and depletes cellular GSH. Supplementation with GSH protects cells against His + Gem-induced cytotoxicity. Further, our in vivo studies demonstrate that His + Gem potently reduced tumor mass and improved mouse survival. Taken together, our data suggest that PC cells exhibit an aberrant His uptake/accumulation which, in turn, leads to oxidative stress and depletion of AA pool, thereby enhancing the anticancer effect of Gem.

Erratum: Selective targeting of the TLR4 co-receptor, MD2, prevents colon cancer growth and lung metastasis: Erratum.

[This corrects the article DOI: 10.7150/ijbs.39098.].

Triptonide Modulates MAPK Signaling Pathways and Exerts Anticancer Effects via ER Stress-Mediated Apoptosis Induction in Human Osteosarcoma Cells.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignancy arise from bone and is one of the causes of cancer-related deaths. Triptonide (TN), a diterpenoid epoxide presented in Tripterygium wilfordii, is shown to possess a broad spectrum of biological properties. METHODS: In this study, we investigate the growth inhibitory effect of TN against human OS cells and its underlying molecular mechanism of action. RESULTS: Findings of our in vitro study revealed that TN exhibited a dose-dependent cytotoxic effect in MG63 and U-2OS cells. ROS-mediated cytotoxic effect was achieved in OS cells treated with TN which was reversed upon NAC treatment. Significantly, increased expression of PERK, p-EIF2, GRP78, ATF4 and CHOP in TN-treated OS cells unfolds the molecular mechanism of TN targets ER stress-mediated apoptosis. Modulation of ERK MAPK pathway was also observed as evidenced by the increased phosphorylation of ERK (p-ERK) and p-p38 in TN-treated OS cells. CONCLUSION: Altogether, the outcome of the study for the first time revealed that TN exhibited its potential chemotherapeutic effects through ROS-mediated ER stress-induced apoptosis via p38 and ERK MAPK signaling pathways.

Selective targeting of the TLR4 co-receptor, MD2, prevents colon cancer growth and lung metastasis.

Toll-like receptor (TLR) signaling is an emerging pathway in tumor cell invasion and metastasis. Myeloid differentiation protein-2 (MD2) contributes to ligand recognition and activation of TLRs in response to exogenous microbial insults or endogenous agents. We hypothesized that blocking MD2 using a specific inhibitor would prevent TLR4-mediated inflammatory responses and metastatic cancer growth. Here, we report that a MD2 inhibitor, L6H21, inhibited migration and invasion of LPS-activated colon cancer CT26.WT cells. These activities were accompanied by inhibition of nuclear factor-κB (NF-κB) activation, and thereby inhibition of the production of pro-inflammatory cytokines and adhesive molecules in colon cancer cells. Furthermore, L6H21 inhibited CT26.WT metastasis to the lung in BALB/c mice as well as suppressed colitis-induced colon cancer induced by azoxymethane/dextran sulfate sodium (AOM/DSS). Taken together, our results demonstrated that L6H21 suppressed tumor invasion and metastasis through blocking TLR4-MD2/NF-κB signaling axis. These findings reveal that inhibition of MD2 may be an important target for the development of colon cancer therapies.

Allylated Curcumin Analog CA6 Inhibits TrxR1 and Leads to ROS-Dependent Apoptotic Cell Death in Gastric Cancer Through Akt-FoxO3a.

BACKGROUND: Gastric cancer is one of the leading causes of cancer-related deaths. Allylated monocarbonyl analogs of curcumin (MACs) have been reported to selectively inhibit a broad range of human cancers including gastric cancer. However, the precise molecular mechanisms underlying the inhibitory activities of MACs are not fully known. METHODS: In this study, we examined the anti-tumor activities of an allylated MAC, CA6, on gastric cancer cells and gastric cancer xenograft mouse model. The potential molecular anti-tumor mechanisms of CA6 were also elucidated. RESULTS: Our data show that CA6 exhibited significant cytotoxicity in gastric cancer cells, which was seen as an induction of G2/M cell cycle arrest and apoptosis. These activities were mediated through an elaboration of ROS levels in gastric cancer cells and induction of endoplasmic reticulum stress. CA6 increased ROS levels through directly binding to and inhibiting thioredoxin reductase R1 (TrxR1). Also, CA6-generated ROS inhibited Akt and activated forkhead O3A (FoxO3a), causing cytotoxicity in gastric cancer cells. Finally, CA6 treatment dose-dependently reduced the growth of gastric cancer xenografts in tumor-bearing mice, which was associated with reduced TrxR1 activity and increased ROS in the tumor. CONCLUSION: In summary, our studies demonstrate that CA6 inhibited gastric cancer growth by inhibiting TrxR1 and increasing ROS, which in turn activated FoxO3a through suppressing Akt. CA6 is a potential candidate for the treatment of gastric cancer.

Indole-2-Carboxamide Derivative LG25 Inhibits Triple-Negative Breast Cancer Growth By Suppressing Akt/mTOR/NF-κB Signalling Pathway.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer which is associated with poor patient outcome and lack of targeted therapy. Our laboratory has synthesized a series of indole-2-carboxamide derivatives. Among this series, compound LG25 showed a favorable pharmacological profile against sepsis and inflammatory diseases. In the present study, we investigated the chemotherapeutic potential of LG25 against TNBC utilizing in vitro and in vivo models. METHODS: Changes in cell viability, cell cycle phases and apoptosis were analyzed using MTT, clonogenic assay, FACS and Western blotting assays. The anti-cancer effects of LG25 were further determined in a xenograft mouse model. RESULTS: Our findings reveal that LG25 reduced TNBC viability in a dose-dependent manner. Flow cytometric and Western blot analyses showed that LG25 enhances G2/M cell cycle arrest and induced cell apoptosis. In addition, LG25 treatment significantly inhibited Akt/mTOR phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB). These inhibitory activities of LG25 were confirmed in mice implanted MDA-MB-231 TNBC cells. CONCLUSION: Our studies provide experimental evidence that indole-2-carboxamide derivative LG25 effectively targeted TNBC in preclinical models by inducing cell cycle arrest and apoptosis, through suppressing Akt/mTOR/NF-κB signaling pathway.

Pharmacological Aspects and Potential Use of Phloretin: A Systemic Review.

Over the past two decades, many researchers have concluded that a diet rich in polyphenolic compounds plays an important therapeutic role in reducing the risk of cancer, cardiovascular disease, inflammation, diabetes, and other degenerative diseases. Polyphenolic compounds have been reported to be involved in neutralization of reactive oxygen species and charged radicals, and have anticarcinogenic effects, hepatoprotective effects, low-glycaemic response, and other benefits. The benefits of fruits and vegetables may be partly attributable to polyphenolic compounds, which have antioxidant and free radical scavenging properties. Fruits such as apples contain a variety of phytochemicals, including (+)-catechin and (-)-epicatechin, phlorizin, phloretin quercetin, cyanidin-3-Ogalactoside, chlorogenic acid, and p-coumaric acid, all of which are strong antioxidants. Phloretin, a natural phenolic compound, is a dihydrochalcone, which is present in the apple. It exhibits a wide variety of activities such as antioxidative, anti-inflammatory, anti-microbial, anti-allergic, anticarcinogenic, anti-thrombotic, and hepatoprotective, besides being involved in the activation of apoptotic associated gene expression and signal transduction in molecular pathways. Despite a multitude of clinical studies, new efforts are needed in clinical research to determine the complete therapeutic potential of phloretin.

Alantolactone promotes ER stress-mediated apoptosis by inhibition of TrxR1 in triple-negative breast cancer cell lines and in a mouse model.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor clinical outcome and currently no effective targeted therapies are available. Alantolactone (ATL), a sesquiterpene lactone, has been shown to have potential anti-tumour activity against various cancer cells. However, the underlying mechanism and therapeutic effect of ATL in the TNBC are largely unknown. In the present study, we found that ATL suppresses TNBC cell viability by reactive oxygen species (ROS) accumulation and subsequent ROS-dependent endoplasmic reticulum (ER) stress both in vitro and in vivo. Thioredoxin reductase 1 (TrxR1) expression and activity of were significantly up-regulated in the TNBC tissue specimens compare to the normal adjacent tissues. Further analyses showed that ATL inhibits the activity of TrxR1 both in vitro and in vivo in TNBC and knockdown of TrxR1 in TNBC cells sensitized ATL-induced cell apoptosis and ROS increase. These results will provide pre-clinical evidences that ATL could be a potential therapeutic agent against TNBC by promoting ROS-ER stress-mediated apoptosis through partly targeting TrxR1.

Induction of immune responses by a novel recombinant fusion protein of enterovirus A71 in BALB/c mice.

Fusion protein technology is used in biotechnology and medical developments. In this study, recombinant fusion proteins from enterovirus A71 (EV-A71) subgenotype B5, Thailand were designed based two surface proteins (VP1 and VP2) and an internal protein (VP4), and named "VP0" (consisting of VP4-VP2) and "EV71" (consisting of VP4-VP2-VP1), respectively. The recombinant fusion proteins VP0 and EV71 were expressed in insect cells and successfully produced and secreted into the media. Both recombinant fusion proteins were shown to have immunogenic properties in BALB/c mice when formulated with Freund's complete/incomplete adjuvant (FA). Interestingly, EV71 formulated with FA- induced a level of IgG antibodies level similar to that induced by the recombinant protein VP1 formulated with FA (the positive control). Our results showed that VP1 alone is better at eliciting a strong cell-mediated immune response. Nontheless, EV71 formulated with FA was capable of inducing lymphocyte proliferation and increasing the cytokine-related mRNA expression levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), and IL-10 in mice after immunization. Additionally, the number of CD4+ and CD8+ T lymphocyte cells after stimulation with purified EV71 in splenic cell culture showed highly specific CD4+ and CD8+ T-cell production. We suggest that EV71, which consists of VP4-VP2-VP1, could be used as the foundation for developing a novel recombinant fusion protein-based vaccine for EV-A71.

Curcuminoid B63 induces ROS-mediated paraptosis-like cell death by targeting TrxR1 in gastric cells.

Gastric cancer is one of the leading causes of cancer-related deaths. Chemotherapy has improved long-term survival of patients with gastric cancer. Unfortunately, cancer readily develops resistance to apoptosis-inducing agents. New mechanisms, inducing caspase-independent paraptosis-like cell death in cancer cells is presently emerging as a potential direction. We previously developed a curcumin analog B63 as an anti-cancer agent in pre-clinical evaluation. In the present study, we evaluated the effect and mechanism of B63 on gastric cancer cells. Our studies show that B63 targets TrxR1 protein and increases cellular reactive oxygen species (ROS) level, which results in halting gastric cancer cells and inducing caspase-independent paraptotic modes of death. The paraptosis induced by B63 was mediated by ROS-mediated ER stress and MAPK activation. Either overexpression of TrxR1 or suppression of ROS normalized B63-induced paraptosis in gastric cancer cells. Furthermore, B63 caused paraptosis in 5-fluorouracil-resistant gastric cancer cells, and B63 treatment reduced the growth of gastric cancer xenografts, which was associated with increased ROS and paraptosis. Collectively, our findings provide a novel strategy for the treatment of gastric cancer by utilizing TrxR1-mediated oxidative stress generation and subsequent cell paraptosis.

Supplementation of p-coumaric acid exhibits chemopreventive effect via induction of Nrf2 in a short-term preclinical model of colon cancer.

Suppression of colorectal cancer by means of chemoprevention is gaining great attention owing to promising outcomes with less adverse effects in preclinical and clinical trials. The present study aims to explore the mechanism of chemoprevention by p-coumaric acid (p-CA) in a short-term preclinical model of colon cancer. 1,2-dimethylhydrazine-administered rats supplemented with p-CA showed downregulation of the expression of colonic proteins, namely, cyclin B1, cdc2 and mdm2, which regulate cell cycle, and immediate early response genes, namely, c-fos, c-jun and c-myc, which regulate cell proliferation. Apoptosis induction was also observed in the colon of p-CA-supplemented rats as assessed by the Bax/Bcl-2 ratio. Immunohistochemistry, immunoblotting and real-time polymerase chain reaction analysis revealed that supplementation of p-CA improved the in-vivo detoxification potential by modulating the cytoplasmic-to-nuclear ratio of nuclear factor erythroid 2-related factor 2, favouring the induction of genes responsible for cytoprotection and detoxification. The outcome of these findings suggests that p-CA inhibited polyp formation by improving the process of detoxification and apoptosis in the colon of 1,2-dimethylhydrazine-administered rats.

Antiproliferative effect of p-Coumaric acid targets UPR activation by downregulating Grp78 in colon cancer.

p-CA is a naturally occurring phenolic acid present in most plants and in all commonly consumed vegetables and fruits. Here we demonstrated the anti-cancer effect of the food borne phytochemical p-CA both in vitro and in vivo models of colon cancer using growth rate and tumor incidence as endpoints. Glucose regulated protein (GRP78) induction and UPR activation plays a key role in oncogenic progression, therefore increased dependence of cancer cells on these UPR signaling pathways for survival can be exploited for anti-cancer research. Hence we investigated the effect of p-CA on Grp78 a molecular chaperone often upregulated in colon cancer and its impact on unfolded protein response (UPR). Administration of the procarcinogen 1,2- dimethylhydrazine (DMH) causes Grp78 upregulation and tumor adaptation via UPR activation. The adaptive activity of UPR activates antiapoptotic NF-κB that results in upregulation of the markers of inflammation and angiogenesis. Supplementation of p-CA downregulated Grp78 and activated UPR mediated apoptosis both in in vitro and in vivo models of colon cancer. Further we observed that p-CA significantly reduced inflammation by decreasing the expression of cytokines COX-2, IL-6, TNF-α and PGE2 as analyzed by q-PCR and also reduced the expression of p-p65 and p-IκBα as analyzed by western blot. Further mechanistic insights revealed that p-CA inhibits Grp78 upregulation in cancer cells through activation of PERK-eIF2α-ATF-4-CHOP pathway that culminates in apoptosis inducing effect of p-CA.

Novel allylated monocarbonyl analogs of curcumin induce mitotic arrest and apoptosis by reactive oxygen species-mediated endoplasmic reticulum stress and inhibition of STAT3.

Curcumin is a promising active compound from a natural source and is extensively being tested in clinical trials because of its bio-functional properties. However, poor bioavailability has hampered its clinical application. Numerous attempts have been made in our laboratory to discover analogs of curcumin with enhanced bioavailability and superior pharmacological activity. In this study, we have investigated a new series of allylated monocarbonyl analogs of curcumin (MAC) and tested their effect on gastric cancer cells. Our results show six MAC that selectively targeted cancer cell lines to inhibit growth and induce apoptosis. This activity was achieved by generation of reactive oxygen species (ROS) by MAC. We selected one effective MAC (CA10) for further investigation and show that CA10 inhibits cell growth by causing G2/M cell cycle arrest and induction of apoptotic death. CA10 induced ROS generation and subsequent activation of endoplasmic reticulum (ER) stress and inhibition of signal transducer and activator of transcription 3 (STAT3) phosphorylation, inhibits cancer cell proliferation. These anti-tumor activities of CA10 were confirmed in gastric cancer xenografts. CA10 induced ROS, activated the ER stress pathway and inhibited STAT3 phosphorylation and gastric xenografts tumor growth in mice. Our studies provide experimental evidence that MAC CA10 effectively targets gastric cancer in preclinical models by enhancing ROS and ROS-mediated signaling.

Curcuminoid EF24 enhances the anti-tumour activity of Akt inhibitor MK-2206 through ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction in gastric cancer.

BACKGROUND AND PURPOSE: Gastric cancer is one of the leading causes of morbidity and mortality worldwide. Akt is an anti-apoptotic kinase that plays a dynamic role in cell survival and is implicated in the pathogenesis of gastric cancer. MK-2206, the first allosteric inhibitor of Akt, is in clinical trials for a number of cancers. Although preclinical studies showed promise, clinical trials reported it had no effect when given alone at tolerated doses. The aim of our study was to delineate the effects of MK-2206 on gastric cancer cells and explore the ability of combination treatments to enhance the anti-tumour activity of MK-2206. EXPERIMENTAL APPROACH: SGC-7901, BGC-823 cells and immunodeficient mice were chosen as a model to study the treatment effects. Changes in cell viability, apoptosis and ROS, endoplasmic reticulum stress and mitochondrial dysfunction in the cells were analysed by MTT assays, ROS imaging and FACSCalibur, electron microscopy, JC-1 staining and western blotting. KEY RESULTS: MK-2206 induced apoptotic cell death through the generation of ROS. We utilized ROS production to target gastric cancer cells by combining MK-2206 and an ROS inducer EF24. Our in vitro and in vivo xenograft studies showed that combined treatment with MK-2206 and EF24 synergistically induced apoptosis in gastric cancer cells and caused cell cycle arrest. These activities were mediated through ROS generation and the induction of endoplasmic reticulum stress and mitochondrial dysfunction. CONCLUSION AND IMPLICATIONS: Targeting ROS generation by using a combination of an Akt inhibitor and EF24 could have potential as a therapy for gastric cancer.

Increased Intracellular Reactive Oxygen Species Mediates the Anti-Cancer Effects of WZ35 via Activating Mitochondrial Apoptosis Pathway in Prostate Cancer Cells.

BACKGROUND: The limited treatment option for recurrent prostate cancer and eventual resistant to conventional chemotherapy drugs has fueled continued interest in finding new anti-neoplastic agents. WZ35, a chemical analog of curcumin, had been demonstrated to have high chemical stability and potential anticancer effects in gastric cancer cells. The present study aimed to investigate the anti-prostate cancer effects of WZ35 in vitro and in vivo as well as the underlying mechanism. METHODS: Two prostate cancer cell lines RM-1 and DU145 were utilized to test the anti-cancer effects of WZ35 and the underlying mechanism. MTT assay was used to assess the cytotoxic effect of WZ35. Cell cycle distribution, apoptosis, alteration of ROS, and [Ca2+ ]i level were evaluated using flow cytometry. Western blotting assay was applied to measure the levels of proteins associated with apoptosis and cell cycle. Immunofluorescence staining and Electron micrographs were used to evaluate activation of mitochondrial apoptosis pathway. Tumor models in nude mice were induced by injection of RM-1 prostate cancer cells to test the in vivo anticancer action of WZ35. RESULTS: Our results showed that WZ35 treatment induced loss of cell viability, cell apoptosis, and G2/M cycle arrest in both RM-1 and DU145 cells, coupled with ROS overproduction, intracellular calcium surge, and activation of mitochondrial apoptosis pathway in RM-1 cells. Interestingly, all above changes induced by WZ35 were completely reversed by ROS blockage. In addition, prevention of [Ca2+ ]i elevation by BAPTA/AM also inhibited activation of mitochondrial apoptosis pathway induced by WZ35. In vivo studies, WZ35 treatment significantly inhibited RM-1 homograft tumor growth along with increased ROS accumulation, mitochondrial disruption, and cell apoptosis in tumor tissues. CONCLUSIONS: In conclusion, this work provides a novel anticancer candidate for the treatment of prostate cancer and demonstrated that increased ROS mediate the anti-cancer effects of WZ35 via activating mitochondrial apoptosis pathway. Importantly, this work also reveals that targeting ROS generation might be an effective strategy in human androgen-resistant prostate cancer treatment. Prostate 77:489-504, 2017. © 2016 Wiley Periodicals, Inc.

Piperlongumine as a direct TrxR1 inhibitor with suppressive activity against gastric cancer.

Piperlongumine (PL), a natural alkaloid isolated from the fruit of long pepper, is known to selectively kill tumor cells while sparing their normal counterparts. However, the cellular target and potent anticancer efficacy of PL in numerous types of human cancer cells have not been fully defined. We report here that PL may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, PL induces a lethal endoplasmic reticulum stress and mitochondrial dysfunction in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to PL treatment, and PL displays synergistic lethality with GSH inhibitors (BSO and Erastin) against gastric cancer cells. In vivo, PL treatment markedly reduces the TrxR1 activity and tumor cell burden. Remarkably, TrxR1 was significantly overexpressed in gastric cancer cell lines and human gastric cancer tissues. Targeting TrxR1 with PL thus discloses a previously unrecognized mechanism underlying the biological activity of PL and provides an in-depth insight into the action of PL in the treatment of gastric cancer.