Artesunate inhibits intestinal tumorigenesis through inhibiting wnt signaling.

Artesunate (ART) is a clinically approved antimalarial drug and was revealed as a candidate of colorectal cancer chemopreventive agents in our drug screening system. Here, we aimed to understand the suppressive effects of ART on intestinal tumorigenesis. In vitro, ART reduced T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter transcriptional activity. In vivo, ART inhibited intestinal polyp development. We found that ART reduces TCF1/TCF7 nuclear translocation by binding the Ras-related nuclear protein (RAN), suggesting that ART inhibits TCF/LEF transcriptional factor nuclear translocation by binding to RAN, thereby inhibiting Wnt signaling. Our results provide a novel mechanism through which artesunate inhibits intestinal tumorigenesis.

Very Long-Term Treatment with a Lactobacillus Probiotic Preparation, Lactobacillus casei Strain Shirota, Suppresses Weight Loss in the Elderly.

Weight loss, often observed in the elderly, is associated with increased risks of various diseases. No large and long-term human study has been conducted to demonstrate the health maintenance-related effects of lactic acid bacteria preparations. To reveal the potential benefit of long-term lactic acid, the effects of bacteria-based probiotics for health maintenance were examined. This observational study included the participants from a previous clinical study designed to evaluate the effects of wheat bran biscuits or Lactobacillus preparation, 3 g/day biolactis powder (BLP), in preventing colorectal tumor. The participants were provided an option to continue treatment with BLP on an outpatient basis after completion of the study. The 380 patients who completed the study were contacted and asked to participate in the present study and those who consented were surveyed for cancer incidence, treatment compliance, lifestyle, weight, and other variables. Informed consent was obtained from 237 of the 380 (62.4%) patients. The mean follow-up period was 7913 days (21.7 years). Cancer developed in 24 of 128 (18.8%) patients in the BLP extension group and 24 of 109 (22.0%) patients in the non-BLP extension group (risk ratio 0.88 [95% confidence interval 0.53-1.47]). Although no significant difference was observed, the cumulative cancer incidence rose at a slightly lower rate in the BLP extension group. Both groups showed a significant weight decrease over the course of 20 years, although the decrease in the BLP extension group was only 1.4 kg, compared with 2.8 kg in the non-BLP extension group. Very long-term treatment with a Lactobacillus probiotic preparation suppressed weight loss in the elderly.

Coffee prevents proximal colorectal adenomas in Japanese men: a prospective cohort study.

This prospective cohort study aimed to show that coffee prevents the recurrence of colorectal tumors (adenomas, precursors of colorectal cancer, and early-stage colorectal cancers) as well as colorectal cancer. The present study included 307 patients who participated in a clinical study that required endoscopy to remove a colorectal tumor. The amount of coffee consumed by the patients at study inclusion and the frequency of colorectal tumors, as detected by colonoscopy over the subsequent 4 years, were assessed. Coffee consumption was determined using a diet survey that included 3-consecutive-day food records. The risk of colorectal tumor recurrence was significantly lower (odds ratio=0.21; 95% confidence interval, 0.06-0.74) in patients who consumed more than three cups of coffee per day compared with those who consumed no coffee. No correlation was observed between the examined factors, including green tea and black tea intake and the amount of caffeine consumed. In subanalysis divided by the tumor location within the colorectum, the odds ratio of colorectal tumor recurrence in the proximal colon showed a tendency toward reduction as coffee consumption increased; however, increased coffee consumption significantly increased colorectal tumor recurrence in the distal colon. We showed that high coffee consumption reduced the overall occurrence of colorectal tumors, affected by the reduction in the proximal colon.

Resibufogenin Induces G1-Phase Arrest through the Proteasomal Degradation of Cyclin D1 in Human Malignant Tumor Cells.

Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB) protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3ß (GSK-3ß) induces the proteasomal degradation of cyclin D1. The addition of GSK-3ß inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3ß.

A pilot, randomized, placebo-controlled, double-blind phase 0/biomarker study on effect of artepillin C-rich extract of Brazilian propolis in frequent colorectal adenoma polyp patients.

OBJECTIVE: Brazilian propolis, a folk medicine, is used worldwide as an alternative medicine to prevent colon cancer. The objective of the study was to test in a small pilot biomarker study in a high-risk group the safety and efficacy of propolis for colon cancer prevention, which has not been evaluated in humans. METHODS: Subjects with adenoma polyps recently removed from the colon were randomly assigned to a propolis group of 15 and a placebo group of 16. In a double-blind study, the propolis group received capsules containing 165 µmol artepillin C and 150 µmol other polyphenols per day for 3 months. Prior to and at the end of the experiments, their blood was analyzed using biochemical tests, and specimens from the normal-appearing sigmoid colon mucosa were biopsied endoscopically to examine the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and mRNA expressions of proliferating cell nuclear antigen, cyclin D1, and Bax. RESULTS: Propolis extract significantly increased the mRNA level of cyclin D1 in the sigmoid colon mucosa, and the other biomarkers remained unchanged. Blood biochemical tests showed significantly higher activity of creatine phosphokinase (CPK), 143 ± 52 units/ml in the propolis group and 104 ± 38 units/ml in the placebo group (p = 0.026), at the end of the study. The increase in CPK activity in the propolis group was due to the increase of the myocardial band form of CPK. On the other hand, laxative treatment prior to endoscopic biopsy significantly increased 8-OHdG levels. CONCLUSIONS: The results from our pilot study did not provide evidence that Brazilian propolis was effective in preventing changes occurring during early stages of colon cancer. In contrast, propolis may have detrimental side effects on muscle tissue, including myocardial cells.

A novel MEK1/2 inhibitor induces G1/S cell cycle arrest in human fibrosarcoma cells.

Blockade of the ERK pathway has antitumor effects against malignant tumor cells. In this study, we investigated the antitumor activity of JTP-70902, a novel specific MEK inhibitor, against human fibrosarcoma cells in which the ERK pathway is constitutively activated. JTP-70902 was synthesized at Japan Tabacco. Human fibrosarcoma HT1080 cells were cultured. JTP-70902 was added at various concentrations. The number of viable cells was counted employing a trypan blue dye exclusion test. Unsynchronized cells were exposed to JTP-70902 for 24 h. The nuclei were stained with propidium iodide. The DNA content was measured using a FACSCalibur flow cytometer. Protein extraction and Western blot analysis were performed. (1) A dose-dependent inhibition of cell growth was observed at concentrations of 10 nM or more. Forty-eight hours after the treatment, the growth of HT1080 cells was completely inhibited by 200 nM JTP-70902. (2) FACS analysis revealed that a 24-h exposure to JTP-70902 increased the population of G1/S phase cells in a dose-dependent manner. (3) The phosphorylation of ERK was inhibited by JTP-70902. Furthermore, after the treatment with JTP-70902, p21WAF1/CIP1 and p27KIP1 protein expression increased and the phosphorylation of RB was reduced. Our results showed that JTP-70902 inhibits cell growth and induces cell cycle arrest in human Ras mutant fibrosarcoma cells. These results indicate that JTP-70902 might be an attractive compound for molecular-targeting chemotherapy for malignant soft tissue tumors with the activation of the Ras-MEK-ERK pathway.

Activity of mangosteen xanthones and teleocidin a-2 in death receptor expression enhancement and tumor necrosis factor related apoptosis-inducing ligand assays.

A screening study using a luciferase assay to identify natural products that enhance death receptor 5 (DR5) expression was carried out, and bioassay-guided fractionation of two organisms, the pericarp of Garcinia mangostana (mangosteen) and actinomycete CKK609 strain, led to the isolation of eight xanthone derivatives (1-8) and teleocidin A-2 (9). Among them, compounds 1, 2, and 5, isolated from G. mangostana, and 9, from the actinomycete, showed potent DR5 promoter activity. Furthermore, we revealed that combined treatment with gartanin (5) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) showed a potentiation effect in sensitizing TRAIL-resistant human gastric adenocarcinoma (AGS) cells. Thus, the present results suggested that 5 has the ability to overcome TRAIL resistance via the up-regulation of DR5 and may be an effective sensitizer of TRAIL-resistant cells.

Death receptor 5 promoter-enhancing compounds isolated from Catimbium speciosum and their enhancement effect on TRAIL-induced apoptosis.

The TRAIL/death-receptor signaling pathway has been considered a promising target for selective cancer therapy, although some malignant tumors exhibit TRAIL resistance. We previously found that isoflavonoid enhanced TRAIL-induced apoptosis in TRAIL-resistant cells, which is achieved through up-regulation of death receptor 5 (DR5). In our screening program targeting DR5 promoter enhancement activity, activity-guided fractionations of the extract of Catimbium speciosum led to the isolation of six compounds. Of the isolates, cardamomin (6), the most potent compound, enhanced the expressions of DR5 and DR4 and decreased the Bcl-xL level in TRAIL-resistant DLD1 cells. The combination of 6 and TRAIL synergistically enhanced TRAIL-induced apoptosis against TRAIL-resistant cells upon the activation of caspase-8, 9, and 3. In addition, enhancement of apoptosis by 6 was inhibited by human recombinant DR5/Fc and DR4/Fc chimera proteins, TRAIL-neutralizing fusion proteins, indicating that 6 sensitize TRAIL-resistant cells to TRAIL through the induction of DR5 and DR4. Also, up-regulation of DR5 by 6 paralleled that of CCAAT/enhancer-binding protein-homologous protein (CHOP).

Nitric oxide sensitizes tumor cells to TRAIL-induced apoptosis via inhibition of the DR5 transcription repressor Yin Yang 1.

Treatment of TRAIL-resistant tumor cells with the nitric oxide donor DETANONOate sensitizes the tumor cells to TRAIL-induced apoptosis concomitantly with DR5 upregulation. The mechanism of sensitization was examined based on the hypothesis that DETANONOate inhibits a transcription repressor Yin Yang 1 (YY1) that negatively regulates DR5 transcription. Treatment of the prostate carcinoma cell lines with DETANONOate inhibited both NF-kappaB and YY1 DNA-binding activities concomitantly with upregulation of DR5 expression. The direct role of YY1 in the regulation of TRAIL resistance was demonstrated in cells treated with YY1 siRNA resulting in TRAIL-induced apoptosis. The role of YY1 in the transcriptional regulation of DR5 was examined in cells treated with a DR5 luciferase reporter system (pDR5) and two constructs, namely, the pDR5/-605 construct with a deletion of the putative YY1 DNA-binding region (-1224 to -605) and a construct pDR5-YY1 with a mutation of the YY1 DNA-binding site. A significant (3-fold) augmentation of luciferase activity over baseline transfection with pDR5 was observed in cells transfected with the modified constructs. ChIP analysis corroborated the YY1 binding to the DR5 promoter. In vivo, tissues from nude mice bearing the PC-3 xenograft and treated with DETANONOate showed inhibition of YY1 and upregulation of DR5. The present findings demonstrate that YY1 negatively regulates DR5 transcription and expression and these correlated with resistance to TRAIL-induced apoptosis. DETANONOate inhibits both NF-kappaB and YY1 and in combination with TRAIL reverses tumor cell resistance to TRAIL apoptosis.

The plant alkaloid cryptolepine induces p21WAF1/CIP1 and cell cycle arrest in a human osteosarcoma cell line.

We previously established a bioassay method to screen for compounds that activate the promoter activity of p21(WAF1/CIP1), a potent inhibitor of cyclin-dependent kinases, in a p53-independent manner. As an activator of p21(WAF1/CIP1) promoter activity, we isolated cryptolepine (CLP: 5-methyl indolo (2,3b)-quiniine), an indoloquinoline alkaloid, from the traditional Ayurvedic medicinal plant Sida cordifolia. We show here that CLP induces the expression of p21(WAF1/CIP1) with growth arrest in p53-mutated human osteosarcoma MG63 cells. Four micromolar of CLP completely inhibited the growth of MG63 cells and caused G2/M-phase arrest. CLP up-regulated the expression of p21(WAF1/CIP1) at both mRNA and protein levels in a dose-dependent manner. Using several mutant p21(WAF1/CIP1) promoter constructs, we found that the CLP-responsive element is an Sp1 site at -82 relative to the transcription start site of the p21(WAF1/CIP1) promoter. These findings suggest that CLP arrests the growth of MG63 cells by activating the p21(WAF1/CIP1) promoter through the specific Sp1 site in a p53-independent manner. In addition, CLP-mediated cell cycle arrest was reduced by the knockout of the p21(WAF1/CIP1) gene in human colon cancer HCT116 cells, suggesting that the cell cycle arrest by CLP was at least partially mediated through the induction of p21(WAF1/CIP1) expression. Although we need further study of chemotherapeutic effect in vivo, these results raise the possibility that CLP might be a suitable chemotherapeutic agent for treatment of osteosarcoma.

Sesamin, a lignan of sesame, down-regulates cyclin D1 protein expression in human tumor cells.

Sesamin is a major lignan constituent of sesame and possesses multiple functions such as antihypertensive, cholesterol-lowering, lipid-lowering and anticancer activities. Several groups have previously reported that sesamin induces growth inhibition in human cancer cells. However, the nature of this growth inhibitory mechanism remains unknown. The authors here report that sesamin induces growth arrest at the G1 phase in cell cycle progression in the human breast cancer cell line MCF-7. Furthermore, sesamin dephosphorylates tumor-suppressor retinoblastoma protein (RB). It is also shown that inhibition of MCF-7 cell proliferation by sesamin is correlated with down-regulated cyclin D1 protein expression, a proto-oncogene that is overexpressed in many human cancer cells. It was found that sesamin-induced down-regulation of cyclin D1 was inhibited by proteasome inhibitors, suggesting that sesamin suppresses cyclin D1 protein expression by promoting proteasome degradation of cyclin D1 protein. Sesamin down-regulates cyclin D1 protein expression in various kinds of human tumor cells, including lung cancer, transformed renal cells, immortalized keratinocyte, melanoma and osteosarcoma. Furthermore, depletion of cyclin D1 protein using small interfering RNA rendered MCF-7 cells insensitive to the growth inhibitory effects of sesamin, implicating that cyclin D1 is at least partially related to the antiproliferative effects of sesamin. Taken together, these results suggest that the ability of sesamin to down-regulate cyclin D1 protein expression through the activation of proteasome degradation could be one of the mechanisms of the antiproliferative activity of this agent.

Chemotherapeutic drugs sensitize cancer cells to TRAIL-mediated apoptosis: up-regulation of DR5 and inhibition of Yin Yang 1.

Several chemotherapeutic drugs in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) result in reversal of resistance to TRAIL-mediated apoptosis through up-regulation of DR5 expression. The promoter of DR5 has one putative binding site for the transcription repressor Yin Yang 1 (YY1), and thus, we hypothesized that the sensitizing drugs may inhibit YY1. We have found that treatment of tumor cells with various chemotherapeutic drugs inhibited nuclear factor-kappaB. We examined whether drugs also inhibit YY1 activity and whether YY1 inhibition correlates with up-regulation of DR5 expression and sensitization of cells to TRAIL-induced apoptosis. The TRAIL- and drug-resistant prostate carcinoma PC-3 cell line was treated with CDDP, VP-16, ADR, and vincristine. DR5 luciferase reporter constructs and small interfering RNA against YY1 were used to determine the role of YY1 in DR5 transcription. Pretreatment of PC-3 cells and other tumor cell lines with various chemotherapeutic drugs sensitized the cells to TRAIL-induced apoptosis concurrently with up-regulation of DR5 expression and inhibition of YY1 expression and its DNA-binding activity. The baseline luciferase activity in PC-3 cells transfected with the wild-type DR5 reporter was significantly augmented in cells transfected with DR5 constructs carrying deletions or mutation in the YY1-binding site. Treatment with drug enhanced DR5 wild-type luciferase activity, with no increase in cells transfected with the YY1-deleted or YY1-mutated constructs. Cells transfected with YY1 small interfering RNA showed up-regulation of DR5 expression and sensitization to TRAIL-mediated apoptosis. The findings provide evidence that drug-induced sensitization of tumor cells to TRAIL is mediated, in part, by inhibition of the transcription repressor YY1 and up-regulation of DR5 expression. Hence, YY1 may be a potential therapeutic target to reverse resistance to TRAIL-induced apoptosis.

The significance of the expression of dihydropyrimidine dehydrogenase in prostate cancer.

OBJECTIVE: To measure dihydropyrimidine dehydrogenase (DPD), an enzyme involved in the metabolism of 5-fluorouracil (5-FU), expression in prostate cancer and determine whether 5-chloro-2,4-dihydroxypyridine (CDHP), a potent inhibitor of DPD, enhances the antitumoral activity of 5-FU against prostate cancer. PATIENTS, MATERIALS AND METHODS: In all, 44 prostate tissue specimens were obtained from men who had a radical prostatectomy alone for prostate cancer, and 38 specimens from men who had had neoadjuvant hormonal therapy. We analysed the cancerous tissue and normal prostate tissue for DPD expression using immunohistochemistry, and determined its prognostic significance. In cultured human prostate cancer lines (DU145 and LNCaP), we compared the cytotoxicity of 5-FU/CDHP with that of 5-FU alone. Finally, in experiments on immunodeficient mice, we studied the effect of oral administration of tegafur, a pro-drug for 5-FU, with or without CDHP on the growth of tumours introduced by injection of DU145 cells. RESULTS: The expression of DPD was significantly higher in cancerous than normal prostate tissue; 36 of 44 (82%) specimens of prostate cancer expressed DPD, whereas only 25 of 44 (57%) specimens of normal prostate tissue expressed DPD. For men with prostate cancer who had radical prostatectomy alone, men with negative DPD expression tended to have a longer recurrence-free survival than those with positive expression; there were no recurrences in men with prostate cancer and negative DPD expression in the 5-year follow-up. DPD expression was significantly lower in men with prostate cancer who received neoadjuvant hormonal therapy. In vitro treatment of human prostate cancer cell lines with 5-FU/CDHP showed more cytotoxicity than with 5-FU treatment alone. Finally, DU145 tumours in mice treated with tegafur and CDHP were significantly smaller than in mice given tegafur alone. CONCLUSION: The present study showed that DPD expression is elevated in prostate cancer, and indicate that DPD inhibitors might enhance the antitumour activity of 5-FU against prostate cancer.

Dihydroflavonol BB-1, an extract of natural plant Blumea balsamifera, abrogates TRAIL resistance in leukemia cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many transformed cells but not in normal cells and, hence, has emerged as a novel anticancer agent. Previously, we showed that although most adult T-cell leukemia/lymphoma (ATLL) cells express the TRAIL death receptor DR4 (TRAIL-R1) or DR5 (TRAIL-R2), they are resistant to TRAIL. Thus, in this study, we tried to find natural products that can overcome TRAIL resistance. Among more than 150 materials screened, a dihydroflavonol that was extracted from Blumea balsamifera (BB-1) exhibited the most striking synergism with TRAIL. Treatment of the TRAIL-resistant ATLL cell line KOB, with a combination of BB-1 and TRAIL, resulted in apparent apoptosis that was not observed on treatment with either agent alone. Furthermore, pretreatment with BB-1 followed by TRAIL further augmented the synergism. BB-1 increased the level of TRAIL-R2 promoter activity and surface protein expression in a p53-independent manner. TRAIL-R2 siRNA inhibited the synergism, indicating that sensitization was caused by the increase of TRAIL-R2 expression. More interestingly, similar effects were observed in other leukemia cell lines by exactly the same mechanisms. These results suggest that combined treatment with BB-1 and TRAIL may be a new strategy for cancer therapy.

Activation of protein kinase C promotes human cancer cell growth through downregulation of p18(INK4c).

p18(INK4c), a member of INK4 family of cyclin-dependent kinase inhibitors, negatively regulates the cyclin D-cyclin-dependent kinase 4/6 complexes which promote G1/S transition by phosphorylating the retinoblastoma tumor-suppressor gene product. Several recent studies using p18(INK4c)-null mice revealed that the p18(INK4c) plays an important role in cell proliferation and tumor development. We report here that 12-O-tetradecanoylphorbol-13-acetate (TPA), widely used as a protein kinase C (PKC) activator, suppresses the expression of p18(INK4c) through its promoter, accompanied by the induction of human cancer cell growth. Reduction of p18(INK4c) using small interfering RNA (siRNA) also enhanced cell growth, suggesting that p18(INK4c) is a critical target of TPA. Ro 31-8425, a potent and highly specific PKC inhibitor abrogated the suppressive effect of TPA on p18(INK4c) gene expression. However, the expression of dominant-negative c-Jun (TAM-67) did not inhibit the action of TPA on p18(INK4c). These findings suggest that activation of PKC promotes human cancer cell growth through downregulation of p18(INK4c) in an AP-1 activation-independent manner. These results suggest that the accelerated cellular proliferation of some human tumors caused by enhanced PKC activity at least partially involves the suppression of p18(INK4c), which is a ubiquitously expressed cyclin-dependent kinase inhibitor.

Isoliquiritigenin inhibits the growth of prostate cancer.

OBJECTIVE: Isoliquiritigenin, one of the components in the root of Glycyrrhiza glabra L., is a member of the flavonoids, which are known to have an anti-tumor activity in vitro and in vivo. In this study, we investigated the anti-tumor effect of isoliquiritigenin on prostate cancer in vitro. METHODS: DU145 and LNCaP prostate cancer cell lines were used as targets. We examined the effects of isoliquiritigenin on cell proliferation, cell cycle regulation and cell cycle-regulating gene expression. Further, we investigated the effects of isoliquiritigenin on the GADD153 mRNA and protein expression, and promoter activity. RESULTS: Isoliquiritigenin significantly inhibited the proliferation of prostate cancer cell lines in a dose-dependent and time-dependent manner. Fluorescence-activated cell sorting (FACS) analysis indicated that isoliquiritigenin induced S and G2/M phase arrest. Isoliquiritigenin enhanced the expression of GADD153 mRNA and protein associated with cell cycle arrest. Further, isoliquiritigenin stimulated transcriptional activity of GADD153 promoter dose-dependently. CONCLUSION: These findings suggest that isoliquiritigenin is a candidate agent for the treatment of prostate cancer and GADD153 may play an important role in isoliquiritigenin-induced cell cycle arrest and cell growth inhibition.