Diaminobutoxy-substituted Isoflavonoid (DBI-1) Enhances the Therapeutic Efficacy of GLUT1 Inhibitor BAY-876 by Modulating Metabolic Pathways in Colon Cancer Cells.

Cancer cells undergo significant "metabolic remodeling" to provide sufficient ATP to maintain cell survival and to promote rapid growth. In colorectal cancer cells, ATP is produced by mitochondrial oxidative phosphorylation and by substantially elevated cytoplasmic glucose fermentation (i.e., the Warburg effect). Glucose transporter 1 (GLUT1) expression is significantly increased in colorectal cancer cells, and GLUT1 inhibitors block glucose uptake and hence glycolysis crucial for cancer cell growth. In addition to ATP, these metabolic pathways also provide macromolecule building blocks and signaling molecules required for tumor growth. In this study, we identify a diaminobutoxy-substituted isoflavonoid (DBI-1) that inhibits mitochondrial complex I and deprives rapidly growing cancer cells of energy needed for growth. DBI-1 and the GLUT1 inhibitor, BAY-876, synergistically inhibit colorectal cancer cell growth in vitro and in vivo. This study suggests that an electron transport chain inhibitor (i.e., DBI-1) and a glucose transport inhibitor, (i.e., BAY-876) are potentially effective combination for colorectal cancer treatment.

Nonadherence to Standard of Care for Locally Advanced Colon Cancer as a Contributory Factor for High Mortality Rates in Kentucky.

BACKGROUND: Kentucky has one of the highest mortality rates for colon cancer, despite dramatic improvements in screening. The National Comprehensive Cancer Network (NCCN) guidelines recommend operation and adjuvant chemotherapy for locally advanced (stage IIb/c and stage III) colon cancer (LACC). The purpose of this study was to determine the rate of nonadherence with current standard of care (SOC) and associated factors as possible contributors to mortality. METHODS: The Kentucky Cancer Registry database linked with administrative health claims was queried for individuals (20 years and older) diagnosed with LACC from 2007 to 2012. Bivariate and logistic regression of nonadherence was performed. Survival analysis was performed with Cox regression and Kaplan-Meier plots. RESULTS: A total of 1,404 patients with LACC were included. Approximately 42% of patients with LACC were noted to be nonadherent to SOC, with nearly all (95.7%) failing to receive adjuvant chemotherapy. After adjusting for all significant factors, we found the factors associated with nonadherence included the following: age older than 75 years, stage III colon cancer, high Charlson Comorbidity Index (3+), low poverty level, Medicaid coverage, and disability. Adherence to SOC is associated with a significant improvement in the 5-year survival rate compared with nonadherence (63.0% and 27.4%, respectively; p < 0.0001). CONCLUSIONS: Our study identified multiple factors associated with the failure of patients with LACC to receive SOC, particularly adjuvant chemotherapy, suggesting the need to focus on improving adjuvant chemotherapy compliance in specific populations. Nonadherence to LACC SOC is likely a major contributor to the persistently high mortality rates in Kentucky.

Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer.

Sterol regulatory element-binding proteins (SREBPs) belong to a family of transcription factors that regulate the expression of genes required for the synthesis of fatty acids and cholesterol. Three SREBP isoforms, SREBP1a, SREBP1c, and SREBP2, have been identified in mammalian cells. SREBP1a and SREBP1c are derived from a single gene through the use of alternative transcription start sites. Here we investigated the role of SREBP-mediated lipogenesis in regulating tumor growth and initiation in colon cancer. Knockdown of either SREBP1 or SREBP2 decreased levels of fatty acids as a result of decreased expression of SREBP target genes required for lipid biosynthesis in colon cancer cells. Bioenergetic analysis revealed that silencing SREBP1 or SREBP2 expression reduced the mitochondrial respiration, glycolysis, as well as fatty acid oxidation indicating an alteration in cellular metabolism. Consequently, the rate of cell proliferation and the ability of cancer cells to form tumor spheroids in suspension culture were significantly decreased. Similar results were obtained in colon cancer cells in which the proteolytic activation of SREBP was blocked. Importantly, knockdown of either SREBP1 or SREBP2 inhibited xenograft tumor growth in vivo and decreased the expression of genes associated with cancer stem cells. Taken together, our findings establish the molecular basis of SREBP-dependent metabolic regulation and provide a rationale for targeting lipid biosynthesis as a promising approach in colon cancer treatment.

Temperature induces significant changes in both glycolytic reserve and mitochondrial spare respiratory capacity in colorectal cancer cell lines.

Thermotherapy, as a method of treating cancer, has recently attracted considerable attention from basic and clinical investigators. A number of studies and clinical trials have shown that thermotherapy can be successfully used as a therapeutic approach for various cancers. However, the effects of temperature on cancer bioenergetics have not been studied in detail with a real time, microplate based, label-free detection approach. This study investigates how changes in temperature affect the bioenergetics characteristics (mitochondrial function and glycolysis) of three colorectal cancer (CRC) cell lines utilizing the Seahorse XF96 technology. Experiments were performed at 32°C, 37°C and 42°C using assay medium conditions and equipment settings adjusted to produce equal oxygen and pH levels ubiquitously at the beginning of all experiments. The results suggest that temperature significantly changes multiple components of glycolytic and mitochondrial function of all cell lines tested. Under hypothermia conditions (32°C), the extracellular acidification rates (ECAR) of CRC cells were significantly lower compared to the same basal ECAR levels measured at 37°C. Mitochondrial stress test for SW480 cells at 37°C vs 42°C demonstrated increased proton leak while all other OCR components remained unchanged (similar results were detected also for the patient-derived xenograft cells Pt.93). Interestingly, the FCCP dose response at 37°C vs 42°C show significant shifts in profiles, suggesting that single dose FCCP experiments might not be sufficient to characterize the mitochondrial metabolic potential when comparing groups, conditions or treatments. These findings provide valuable insights for the metabolic and bioenergetic changes of CRC cells under hypo- and hyperthermia conditions that could potentially lead to development of better targeted and personalized strategies for patients undergoing combined thermotherapy with chemotherapy.

The role of PI3K/mTOR inhibition in combination with sorafenib in hepatocellular carcinoma treatment.

BACKGROUND: Deregulated RAS/RAF/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PI-103 (a small molecule inhibitor of PI3K and mTOR) and sorafenib as single agents and in combination on HCC tumorigenesis in an in vivo xenograft model. MATERIALS AND METHODS: In vitro study: Huh7 proliferation was assayed by 3H-thymidine incorporation and by thiazolyl blue tetrazolium bromide (MTT) assay. Western blots were used to detect phosphorylation of the key enzymes in the two pathways. In vivo study: Human HCC cell line Huh7 was inoculated into nude mice s.c. and the mice were treated with sorafenib (20 mg/kg/day) and PI-103 (5 mg/kg, every 4 days). Tumor size was measured every other day. Tumors were isolated for western blot and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detection of apoptosis and signaling pathway enzymes. RESULTS: Our in vitro study found that combination of sorafenib and PI-103 additively inhibited Huh7 proliferation as compared to single-agent treatment. Sorafenib and PI-103 as single agents differentially inhibited or activated key enzymes (MEK, ERK, AKT, mTOR, and S6K) in PI3K/AKT/mTOR and RAS/RAF/MAPK signaling pathways. Combination of sorafenib and PI-103 inhibited all the key enzymes in the two pathways. Our in vivo study demonstrated significant differences between control group, mono-drug groups and drug-combination group (p<0.05). Combination of Sorafenib and PI-103 more efficiently inhibited tumorigenesis as compared to mono-drug treatments (p<0.032). CONCLUSION: The combination of PI-103 and sorafenib has the advantage over mono-drug therapy on inhibition of HCC cell proliferation and tumorigenesis by inhibiting both PI3K/AKT/mTOR and RAS/RAF/MAPK signaling pathways.

Sorafenib enhances the therapeutic efficacy of rapamycin in colorectal cancers harboring oncogenic KRAS and PIK3CA.

Activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling is associated with tumorigenesis and metastasis of colorectal cancer (CRC). The mammalian target of rapamycin (mTOR) kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis and metastasis of CRCs, indicating that mTOR inhibition may have therapeutic potential. Notwithstanding, many cancers, including CRC, demonstrate resistance to the antitumorigenic effects of rapamycin. In this study, we show that inhibition of mTORC1 with rapamycin leads to feedback activation of PI3K/Akt and Ras-MAPK signaling, resulting in cell survival and possible contribution to rapamycin resistance. Combination with the multikinase inhibitor, sorafenib, abrogates rapamycin-induced activation of PI3K/Akt and Ras-MAPK signaling pathways. Combination of rapamycin with sorafenib synergistically inhibits proliferation of CRC cells. CRCs harboring coexistent KRAS and PIK3CA mutations are partially sensitive to either rapamycin or sorafenib monotherapy, but highly sensitive to combination treatment with rapamycin and sorafenib. Combination with sorafenib enhances therapeutic efficacy of rapamycin on induction of apoptosis and inhibition of cell-cycle progression, migration and invasion of CRCs. We demonstrate efficacy and safety of concomitant treatment with rapamycin and sorafenib at inhibiting growth of xenografts from CRC cells with coexistent mutations in KRAS and PIK3CA. The efficacy and tolerability of combined treatment with rapamycin and sorafenib provides rationale for use in treating CRC patients, particularly those with tumors harboring coexistent KRAS and PIK3CA mutations.

Triptolide inhibits proliferation and migration of colon cancer cells by inhibition of cell cycle regulators and cytokine receptors.

BACKGROUND: Phytochemicals are an important source of emerging preventive and therapeutic agents for cancer. Triptolide/PG490, an extract of the Chinese herb Tripterygium wilfordii Hook F, is a potent anti-inflammatory agent that also possesses anticancer activity. While its antiproliferative effects are well-established, the potential antimigratory effects of triptolide have not been characterized. MATERIAL AND METHODS: Effects of triptolide on the proliferation and invasion of colon cancer cells and expression of cancer-related genes and proteins were assessed. RESULTS: Triptolide potently inhibited HT29 and HCT116 colon cancer cell growth and reduced basal and stimulated HCT116 migration through collagen by 65% to 80%. Triptolide inhibited mRNA expression of the positive cell cycle regulatory genes c-myc, and A, B, C, and D-type cyclins in multiple colon cancer cell lines. Additionally, we show that triptolide treatment decreased expression of VEGF and COX-2, which promote cancer progression and invasion, and inhibited the expression of multiple cytokine receptors potentially involved in cell migration and cancer metastasis, including the thrombin receptor, CXCR4, TNF receptors, and TGF-ß receptors. CONCLUSIONS: Triptolide is a potent inhibitor of colon cancer proliferation and migration in vitro. The down-regulation of multiple cytokine receptors, in combination with inhibition of COX-2 and VEGF and positive cell cycle regulators, may contribute to the antimetastatic action of this herbal extract.

Alternative medicine products as a novel treatment strategy for inflammatory bowel disease.

Inflammatory bowel disease (IBD) affects the mucosal lining of the gastrointestinal tract; the etiology is unknown and treatment is directed at systemic immunosuppression. Natural products, including medicinal herbs, have provided approximately half of the drugs developed for clinical use over the past 20 years. The purpose of our current study was to determine the effects of a novel combination of herbal extracts on intestinal inflammation using a murine model of IBD. Female Swiss-Webster mice were randomized to receive normal water or 5% dextran sulfate sodium (DSS) drinking water to induce colitis. Mice were treated with either a novel combination of herbal aqueous extracts or vehicle control per os (po) or per rectum (pr) every 24 hours for 7-8 days. Disease activity index score (DAI) was determined daily; mice were sacrificed and colons were analyzed by H & E staining, MPO assay, and cytokine (TNF-alpha, IL-6) ELISAs. Mice treated with the combination of herbal extracts, either po or pr, had significantly less rectal bleeding and lower DAI scores compared to the vehicle-treated group. Moreover, colonic ulceration, leukocytic infiltration, and cytokine levels (TNF-alpha and IL-6) were also decreased in the colons of herbal-treated mice, reflected by H & E staining, MPO assay, and cytokine ELISA. Treatment with the combination of medicinal herbs decreases leukocyte infiltration and mucosal ulceration, ameliorating the course of acute colonic inflammation. This herbal remedy may prove to be a novel and safe therapeutic alternative in the treatment of IBD.

Role of cyclooxygenase 2 in protein kinase C beta II-mediated colon carcinogenesis.

Elevated expression of protein kinase C beta II (PKC beta II) is an early promotive event in colon carcinogenesis (Gokmen-Polar, Y., Murray, N. R., Velasco, M. A., Gatalica, Z., and Fields, A. P. (2001) Cancer Res. 61, 1375-1381). Expression of PKC beta II in the colon of transgenic mice leads to hyperproliferation and increased susceptibility to colon carcinogenesis due, at least in part, to repression of transforming growth factor beta type II receptor (TGF-beta RII) expression (Murray, N. R., Davidson, L. A., Chapkin, R. S., Gustafson, W. C., Schattenberg, D. G., and Fields, A. P. (1999) J. Cell Biol., 145, 699-711). Here we report that PKC beta II induces the expression of cyclooxygenase type 2 (Cox-2) in rat intestinal epithelial (RIE) cells in vitro and in transgenic PKC beta II mice in vivo. Cox-2 mRNA increases more than 10-fold with corresponding increases in Cox-2 protein and PGE2 production in RIE/PKC beta II cells. PKC beta II activates the Cox-2 promoter by 2- to 3-fold and stabilizes Cox-2 mRNA by at least 4-fold. The selective Cox-2 inhibitor Celecoxib restores expression of TGF-beta RII both in vitro and in vivo and restores TGF beta-mediated transcription in RIE/PKC beta II cells. Likewise, the omega-3 fatty acid eicosapentaenoic acid (EPA), which inhibits PKC beta II activity and colon carcinogenesis, causes inhibition of Cox-2 protein expression, re-expression of TGF-beta RII, and restoration of TGF-beta1-mediated transcription in RIE/PKC beta II cells. Our data demonstrate that PKC beta II promotes colon cancer, at least in part, through induction of Cox-2, suppression of TGF-beta signaling, and establishment of a TGF-beta-resistant, hyperproliferative state in the colonic epithelium. Our data define a procarcinogenic PKC beta II --> Cox-2 --> TGF-beta signaling axis within the colonic epithelium, and provide a molecular mechanism by which dietary omega-3 fatty acids and nonsteroidal antiinflammatory agents such as Celecoxib suppress colon carcinogenesis.

Gastrin-releasing peptide is a growth factor for human neuroblastomas.

OBJECTIVE: To evaluate whether gastrin-releasing peptide (GRP) and GRP receptor (GRP-R) expression correlate with tumor behavior and to examine the mitogenic actions of GRP on neuroblastomas. SUMMARY BACKGROUND DATA: Neuroblastoma is the most common solid tumor of infants and children. Despite recent advances in multimodality treatment regimens, the survival for advanced-stage tumors remains dismal. Neuroblastomas are known to produce GRP; however, the proliferative effects of GRP on neuroblastomas have not been elucidated. METHODS: Sections of paraffin-embedded neuroblastomas from 33 patients were analyzed for GRP and GRP-R protein expression by immunohistochemistry. Functional binding of GRP-R to the Ca2+ signaling pathway was examined. In addition, the proliferative effect of GRP on neuroblastoma cells (SK-N-SH, IMR-32, SH-SY5Y, LAN-1) was determined. RESULTS: Immunohistochemical analysis showed GRP and GRP-R protein expression in neuroblastomas; an increased expression of GRP-R was noted in a higher percentage of undifferentiated tumors compared with tumors that were benign. GRP-R mRNA was confirmed in neuroblastoma cell lines. GRP treatment resulted in intracellular calcium [Ca2+]i mobilization in two cell lines (SK-N-SH, LAN-1). GRP treatment stimulated growth of all four neuroblastoma cell lines; this effect was inhibited in SK-N-SH cells by pretreatment with GRP antibody. CONCLUSIONS: These findings show increased GRP-R expression in the more aggressive and undifferentiated neuroblastomas. The synchronous expression of GRP and its receptor, GRP-R, suggests a role for these proteins in tumor growth. Moreover, these findings show enhanced proliferation of neuroblastoma cells in vitro after GRP treatment, suggesting that GRP may act as an autocrine and/or paracrine growth factor for neuroblastomas. Treatment with specific GRP-R antagonists may provide novel adjuvant therapy for neuroblastomas in children.

Activation of conventional PKC isoforms increases expression of the pro-apoptotic protein Bad and TRAIL receptors.

BACKGROUND: Pancreatic cancer is a leading cause of cancer death worldwide; current treatment options have been ineffective in prolonging survival. Agents that target specific signaling pathways (e.g., protein kinase C [PKC]) may regulate apoptotic gene expression rendering resistant cancers sensitive to the effects of other chemotherapeutic drugs. The purpose of our study was to assess the effect of PKC stimulation on apoptotic gene expression in pancreatic cancer cells. METHODS: The human pancreatic cancer cell line, PANC-1, was treated with PKC-stimulating agents, phorbol 12-myristate 13-acetate (PMA) or bryostatin-1, and analyzed for expression of apoptosis-related genes. RESULTS: Both PMA and bryostatin-1 induced expression of the pro-apoptotic gene Bad in a dose dependent fashion. The expression of Bad was blocked by the PKC inhibitors GF109203x, Gö6983, and Ro-31-8220, suggesting a role for the conventional isoforms of PKC. In addition, treatment with the MEK inhibitors PD98059 or UO126 reduced PMA-mediated induction of Bad gene expression. PMA also increased the expression of TRAIL receptors DR4 and DR5; this expression was inhibited by the PKC inhibitors GF109203x, Gö6983, and Ro-31-8220 and the MEK inhibitor UO126, suggesting a role for conventional PKC isoforms and MEK in the regulation of TRAIL receptor expression. CONCLUSIONS: PKC stimulation in PANC-1 cells increases expression of the pro-apoptotic gene Bad and the TRAIL receptors, DR4 and DR5, through both conventional PKC- and MEK-dependent pathways. Agents that stimulate PKC may sensitize pancreatic cancer cells to apoptosis and provide a potential adjuvant therapy for the treatment of chemoresistant pancreatic cancers.

Peroxisome proliferator-activated receptor gamma ligand inhibits cell growth and invasion of human pancreatic cancer cells.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in certain human cancers; ligand-induced PPARgamma activation can result in growth inhibition and differentiation in these cells. However, the precise mechanism for the antiproliferative effect of PPARgamma ligands is not entirely known. AIM OF STUDY: The purpose of this study was to examine the effect of PPARgamma ligands on pancreatic cancer cell growth and invasiveness. METHODS: The effect of two PPARgamma ligands, 15 deoxy-delta12,14 prostaglandin J2 (15d-PGJ2) and ciglitazone, on the growth of four human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Panc-1, and L3.6) was assessed. Expression of cell-cycle and apoptotic-related proteins was measured. Finally, the effect of 15d-PGJ2 on pancreatic cancer cell invasiveness and matrix metalloproteinase expression was determined. RESULTS: Both 15d-PGJ2 and ciglitazone inhibited the growth of all four pancreatic cancer cell lines in a dose- and time-dependent fashion. Treatment of BxPC-3 cells with 15d-PGJ2 resulted in a time-dependent decrease in cyclin D1 expression associated with a concomitant induction of p21waf1 and p27kip1. In addition, 15d-PGJ2 treatment induced apoptosis through activation of caspase-8, -9, and -3. Moreover, pancreatic cancer cell invasiveness was significantly suppressed after treatment with a nontoxic dose of 15d-PGJ2, which was associated with a reduction of MMP-2 and MMP-9 protein levels and activity. CONCLUSIONS: These results demonstrate that PPARgamma ligands have the dual advantage of inhibiting pancreatic cancer cell growth while reducing the invasiveness of the tumor cells, suggesting a potential role for these agents in the adjuvant treatment of pancreatic cancer.