Sulforaphane has an additive anticancer effect to FOLFOX in highly metastatic human colon carcinoma cells.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Patients with CRC may need chemotherapy (CTx) in a neoadjuvant, adjuvant or palliative setting through the course of the disease. Unfortunately, its effect is limited by chemoresistance and chemotoxicity. Novel more effective and non­toxic CTx regimens are needed to further improve CRC treatment outcomes. Thus, the present study was designed to test the hypothesis that non­toxic sulforaphane (SF) is effective against CRC and has additive effects in combination with conventional 5­fluorouracil, oxaliplatin and folinic acid (FOLFOX) CTx in vitro. Highly metastatic human colon cancer cells, CX­1, and fibroblasts were treated with FOLFOX ± SF. Cell viability was assessed using an MTT assay. The level of apoptosis and the expression of apoptotic proteins were measured by TUNEL assay and quantitative PCR analysis. Aldehyde dehydrogenase isoform 1 (ALDH1) and multidrug resistance protein 2 (MRP2) levels were evaluated. The ability of cells to form spheroids was measured in three­dimensional cell culture. SF alone and in combination with FOLFOX effectively decreased the viability of the CX­1 cells, promoted apoptosis within the CX­1 cells, prevented cellular spheroid formation and decreased ALDH1 activity. However, SF promoted MRP2 expression and protein levels. In conclusion, SF together with conventional FOLFOX has additive anticancer effects against highly metastatic human CRC in vitro.

Nanosilver inhibits the progression of pancreatic cancer by inducing a paraptosis-like mixed type of cell death.

Silver has been in clinical use since ancient times and silver nanoparticles (AgNPs) have attracted attention in cancer therapy. We investigated the mechanisms by which AgNPs inhibit pancreatic ductal adenocarcinoma (PDAC). AgNPs were synthesized and 3 human PDAC and 2 nonmalignant primary cell lines were treated with AgNPs. MTT, MAPK, colony, spheroid and scratch assays, Western blotting, TEM, annexin V, 7-AAD, and H2DCFDA staining, FACS analysis, mRNA array and bioinformatics analyses, tumor xenograft transplantation, and immunohistochemistry of the treated cells were performed. We found that minimal AgNPs amounts selectively eradicated PDAC cells within a few hours. AgNPs inhibited cell migration and spheroid and colony formation, damaged mitochondria, and induced paraptosis-like cell death with the presence of cytoplasmic vacuoles, dilation of the ER and mitochondria, ROS formation, MAPK activity, and p62 and LC3b expression, whereas effects on the nucleus, DNA fragmentation, or caspases were not detectable. AgNPs strongly decreased tumor xenograft growth without side effects and reduced the expression of markers for proliferation and DNA repair, but upregulated paraptosis markers. The results highlight nanosilver as complementary agent to improve the therapeutic efficacy in pancreatic cancer.

Broccoli sprout supplementation in patients with advanced pancreatic cancer is difficult despite positive effects-results from the POUDER pilot study.

Pancreatic ductal adenocarcinoma is a highly aggressive malignancy with short survival and limited therapeutic options. Broccoli sulforaphane is a promising new treatment due to the results of recent epidemiological, experimental and patient studies. Upon approval from the ethics committee and registration at ClinicalTrials.gov, 40 patients with palliative chemotherapy were placed into a placebo and treatment group in an unblinded fashion. Fifteen capsules with pulverized broccoli sprouts containing 90 mg/508 µmol sulforaphane and 180 mg/411 µmol glucoraphanin or methylcellulose were administered daily for up to 1 year. Twenty-nine patients were included in the treatment group and 11 patients were in the placebo group; these patients were followed for up to 1 year. The patient characteristics, overall survival and feasibility were assessed. Compared to those of the placebo group, the mean death rate was lower in the treatment group during the first 6 months after intake (day 30: 0%/18%, day 90: 0%/25%, and day 180: 25%/43%), and Kaplan-Meier analysis revealed a higher survival rate. There was a high drop-out rate (72% in the treatment group and 55% in the placebo group) after 1 year. We concluded from the Karnofsky index that the broccoli sprouts did not impact patient's self-care and overall abilities severely. The intake of 15 capsules daily was difficult for some patients, and the broccoli sprouts sometimes increased digestive problems, nausea and emesis. We did not obtain statistically significant results (p = 0.291 for the endpoint at day 180), but the knowledge about the feasibility is the basis for the development of new sulforaphane drugs.

Melatonin promotes sorafenib-induced apoptosis through synergistic activation of JNK/c-jun pathway in human hepatocellular carcinoma.

Melatonin has been shown to exert anticancer activity on hepatocellular carcinoma (HCC) through its antiproliferative and pro-apoptotic effect in both experimental and clinical studies, and sorafenib is the only approved drug for the systemic treatment of HCC. Thus, this study was designed to investigate the combined effect of melatonin and sorafenib on proliferation, apoptosis, and its possible mechanism in human HCC. Here, we found that both melatonin and sorafenib resulted in a dose-dependent growth inhibition of HuH-7 cells after 48 hours treatment, and the combination of them enhanced the growth inhibition in a synergistic manner. Colony formation assay indicated that co-treatment of HuH-7 cells with melatonin and sorafenib significantly decreased the clonogenicity compared to the treatment with single agent. Furthermore, FACS and TUNEL assay confirmed that melatonin synergistically augmented the sorafenib-induced apoptosis after 48 hours incubation, which was in accordance with the activation of caspase-3 and the JNK/c-jun pathway. Inhibition of JNK/c-jun pathway with its inhibitor SP600125 reversed the phosphorylation of c-jun and the activation of caspase-3 induced by co-treatment of HuH-7 cells with melatonin and sorafenib in a dose-dependent manner. Furthermore, SP600125 exhibited protective effect against apoptosis induced by the combination of melatonin and sorafenib. This study demonstrates that melatonin in combination with sorafenib synergistically inhibits proliferation and induces apoptosis in human HCC cells; therefore, supplementation of sorafenib with melatonin may serve as a potential therapeutic choice for advanced HCC.

Delivery of improved oncolytic adenoviruses by mesenchymal stromal cells for elimination of tumorigenic pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies and has poor therapeutic options. We evaluated improved oncolytic adenoviruses (OAds), in which the adenoviral gene E1B19K was deleted or a TRAIL transgene was inserted. Bone marrow mesenchymal stromal cells (MSCs) served as carriers for protected and tumor-specific virus transfers. The infection competence, tumor migration, and oncolysis were measured in cancer stem cell (CSC) models of primary and established tumor cells and in tumor xenografts. All OAds infected and lysed CSCs and prevented colony formation. MSCs migrated into PDA spheroids without impaired homing capacity. Xenotransplantation of non-infected PDA cells mixed with infected tumor cells strongly reduced the tumor volume and the expression of the proliferation marker Ki67 along with a necrotic morphology. Adenoviral capsid protein was detected in tumor xenograft tissue after intravenous injection of infected MSCs, but not in normal tissue, implying tumor-specific migration. Likewise, direct in vivo treatment correlated with a strongly reduced tumor volume, lower expression of Ki67 and CD24, and enhanced activity of caspase 3. These data demonstrate that the improved OAds induced efficient oncolysis with the OAd-TRAIL as most promising candidate for future clinical application.

Sulforaphane, quercetin and catechins complement each other in elimination of advanced pancreatic cancer by miR-let-7 induction and K-ras inhibition.

Pancreatic ductal adenocarcinoma (PDA) has the worst prognosis of all malignancies, and current therapeutic options do not target cancer stem cells (CSCs), which may be the reason for the extreme aggressiveness. The dietary agents sulforaphane and quercetin enriched e.g., in broccoli, and the main and best studied green tea catechin EGCG hold promise as anti-CSC agents in PDA. We examined the efficacy of additional catechins and the combination of these bioactive agents to stem cell features and miRNA signaling. Two established and one primary PDA cell line and non-malignant pancreatic ductal cells were used. Whereas each agent strongly inhibited colony formation, the catechins ECG and CG were more effective than EGCG. A mixture of green tea catechins (GTCs) significantly inhibited viability, migration, expression of MMP-2 and -9, ALDH1 activity, colony and spheroid formation and induced apoptosis, but the combination of GTCs with sulforaphane or quercetin was superior. Following treatment with bioactive agents, the expression of miR-let7-a was specifically induced in cancer cells but not in normal cells and it was associated with K-ras inhibition. These data demonstrate that sulforaphane, quercetin and GTC complement each other in inhibition of PDA progression by induction of miR-let7-a and inhibition of K-ras.

Triptolide reverses hypoxia-induced epithelial-mesenchymal transition and stem-like features in pancreatic cancer by NF-κB downregulation.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies characterized by an intense tumor stroma with hypoperfused regions, a significant inflammatory response and pronounced therapy resistance. New therapeutic agents are urgently needed. The plant-derived agent triptolide also known as "thunder god vine" has a long history in traditional Chinese medicine for treatment of rheumatoid arthritis and cancer and is now in a clinical phase II trial for establishing the efficacy against a placebo. The authors mimicked the situation in patient tumors by induction of hypoxia in experimental models of pancreatic cancer stem cells (CSCs) and evaluated the therapeutic effect of triptolide. Hypoxia led to induction of colony and spheroid formation, aldehyde dehydrogenase 1 (ALDH1) and NF-κB activity, migratory potential and a switch in morphology to a fibroblastoid phenotype, as well as stem cell- and epithelial-mesenchymal transition-associated protein expression. Triptolide efficiently inhibited hypoxia-induced transcriptional signaling and downregulated epithelial-mesenchymal transition (EMT) and CSC features in established highly malignant cell lines, whereas sensitive cancer cells or nonmalignant cells were less affected. In vivo triptolide inhibited tumor take and tumor growth. In primary CSCs isolated from patient tumors, triptolide downregulated markers of CSCs, proliferation and mesenchymal cells along with upregulation of markers for apoptosis and epithelial cells. This study is the first to show that triptolide reverses EMT and CSC characteristics and therefore may be superior to current chemotherapeutics for treatment of PDA.

Sulforaphane and related mustard oils in focus of cancer prevention and therapy.

The plant family Brassicaceae, formerly Cruciferae, contains mustard oil glycosides, from which mustard oils are enzymatically hydrolyzed. Mustard oils offer protection from pests, microorganisms and fungi. More than 120 different mustard oils with various biological functions are known. Since ancient times, these substances are used as natural antibiotics, antiviral drugs and antimycotics. The antioxidative effect of mustard oils contributes to protection from DNA damage. Epidemiological and experimental studies have shown preventive and therapeutic effects of crucifers or isolated substances thereof. Particularly well studied is the mustard oil sulforaphane, which is contained in high concentrations in broccoli and its sprouts. As has been shown in mice recently, sulforaphane also targets the most malignant cancer stem cells, which are not affected by conventional cancer treatments. Based on these promising results, the first prospective clinical studies with cancer patients and sulforaphane-enriched broccoli sprouts have now been initiated in the United States.

Synergistic activity of sorafenib and sulforaphane abolishes pancreatic cancer stem cell characteristics.

Recent evidence suggests that pancreatic cancer and other solid tumors contain a subset of tumorigenic cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. Sorafenib (SO) is a promising new multikinase inhibitor for treatment of advanced kidney and liver cancers. We report here targeting of pancreatic cancer stem cells (CSC) by SO and the development of a strategy to enhance this effect. Although SO administration diminished clonogenicity, spheroid formation, aldehyde dehydrogenase 1 (ALDH1) activity, growth on immunodeficient mice, proliferation, and angiogenesis and induced apoptosis, we observed SO-induced activation of NF-kappaB associated with survival and regrowth of spheroids. For enhanced elimination of CSC characteristics by SO, we cotreated cells with sulforaphane (SF). This broccoli isothiocyanate was recently described to eliminate pancreatic CSCs by downregulation of NF-kappaB activity without inducing toxic side effects. On combination treatment, SF completely eradicated SO-induced NF-kappaB binding, which was associated with abrogated clonogenicity, spheroid formation, ALDH1 activity, migratory capacity, and induction of apoptosis. In vivo, combination therapy reduced the tumor size in a synergistic manner. This was due to induction of apoptosis, inhibition of proliferation and angiogenesis, and downregulation of SO-induced expression of proteins involved in epithelial-mesenchymal transition. Our data suggest that SF may be suited to increase targeting of CSCs by SO.

The alkaloid emetine as a promising agent for the induction and enhancement of drug-induced apoptosis in leukemia cells.

Emetine, a natural alkaloid from Psychotria ipecacuanha, has been used in phytomedicine to induce vomiting, and to treat cough and severe amoebiasis. Certain data suggest the induction of apoptosis by emetine in leukemia cells. Therefore, we examined the suitability of emetine for the sensitisation of leukemia cells to apoptosis induced by cisplatin. In response to emetine, we found a strong reduction in viability, an induction of apoptosis and caspase activity comparable to the cytotoxic effect of cisplatin. Moreover, emetine had an additive effect and increased cisplatin-induced apoptosis. Mechanistically, we demonstrate by DNA array analysis that emetine alone or together with cisplatin down-regulates several anti-survival genes and up-regulates several pro-apoptotic signalling molecules along with other effects on signalling. These data show that emetine is a strong inducer of apoptosis in leukemia cells and could be a suitable cytotoxic drug alone or in combination with other chemotherapeutics to sensitise leukemia cells to apoptosis.

Rocaglamide derivatives are potent inhibitors of NF-kappa B activation in T-cells.

Crude extracts from different Aglaia species are used as anti-inflammatory remedies in the traditional medicine of several countries from Southeast Asia. Because NF-kappaB transcription factors represent key regulators of genes involved in immune and inflammatory responses, we supposed that the anti-inflammatory effects of Aglaia extracts are mediated by the inhibition of NF-kappaB activity. Purified compounds of Aglaia species, namely 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one aglain congener were tested for their ability to inhibit NF-kappaB activity. We show that a group of rocaglamides represent highly potent and specific inhibitors of tumor necrosis factor-alpha (TNFalpha) and phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB-dependent reporter gene activity in Jurkat T cells with IC(50) values in the nanomolar range. Some derivatives are less effective, and others are completely inactive. Rocaglamides are able to suppress the PMA-induced expression of NF-kappaB target genes and sensitize leukemic T cells to apoptosis induced by TNFalpha, cisplatin, and gamma-irradiation. The suppression of NF-kappaB activation correlated with the inhibition of induced IkappaB(alpha) degradation and IkappaB(alpha) kinase activation. The level of interference was determined and found to be localized upstream of the IkappaB kinase complex but downstream of the TNF receptor-associated protein 2. Our data suggest that rocaglamide derivatives could serve as lead structures in the development of anti-inflammatory and tumoricidal drugs.