p-Anilinoaniline enhancement of dioxin-induced CYP1A1 transcription and aryl hydrocarbon receptor occupancy of CYP1A1 promoter: role of the cell cycle.

The aryl hydrocarbon receptor (AhR) is targeted by ubiquitination for degradation by the proteasome shortly after its activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In silico screening identified p-anilinoaniline (pAA) as a putative inhibitor of an E2 ligase that partners with an E3 ligase implicated in AhR ubiquitination. We investigated whether pAA could modify AhR-dependent activation of its target gene CYP1A1. pAA (1-200 µM) alone did not affect AhR content, or stimulate CYP1A1 mRNA accumulation in human mammary epithelial MCF10A cultures. However, pretreatment with ≥100 µM pAA suppressed TCDD-induced CYP1A1 activation and AhR degradation via its functioning as an AhR antagonist. At a lower concentration (25 µM), pAA cotreatment increased TCDD-induced CYP1A1 mRNA accumulation, without inhibiting AhR turnover or altering CYP1A1 mRNA half-life. Whereas TCDD alone did not affect MCF10A proliferation, 25 µM pAA was cytostatic and induced a G(1) arrest that lasted ∼7 h and induced an S phase arrest that peaked 5 to 8 h later. TCDD neither affected MCF10A cell cycle progression nor did it alter pAA effects on the cell cycle. The magnitude of CYP1A1 activation depended upon the time elapsed between pAA pretreatment and TCDD addition. Maximal AhR occupancy of the CYP1A1 promoter and accumulation of CYP1A1 heterogeneous nuclear RNA and mRNA occurred when pAA-pretreated cultures were exposed to TCDD in late G(1) and early/mid S phase. TCDD-mediated induction of CYP2S1 was also cell cycle-dependent in MCF10A cultures. Similar studies with HepG2 cultures indicated that the cell cycle dependence of CYP1A1 induction is cell context-dependent.

Curcumin targets FOLFOX-surviving colon cancer cells via inhibition of EGFRs and IGF-1R.

Curcumin (diferuloylmethane), which has no discernible toxicity, inhibits initiation, promotion and progression of carcinogenesis. 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics, but produces an incomplete response resulting in survival of cells (chemo-surviving cells) that may lead to cancer recurrence. The present investigation was, therefore, undertaken to examine whether addition of curcumin to FOLFOX is a superior therapeutic strategy for chemo-surviving cells. Forty-eight-hour treatment of colon cancer HCT-116 and HT-29 cells with FOLFOX resulted in 60-70% survival, accompanied by a marked activation of insulin like growth factor-1 receptor (IGF-1R) and minor to moderate increase in epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER-2) as well as v-akt murine thymoma viral oncogene homolog 1 (AKT), cyclooxygenase-2 (COX-2) and cyclin-D1. However, inclusion of curcumin to continued FOLFOX treatment for another 48 h greatly reduced the survival of these cells, accompanied by a concomitant reduction in activation of EGFR, HER-2, IGF-1R and AKT, as well as expression of COX-2 and cyclin-D1. More importantly, EGFR tyrosine kinase inhibitor gefitinib or attenuation of IGF-1R expression by the corresponding si-RNA caused a 30-60% growth inhibition of chemo-surviving HCT-116 cells. However, curcumin alone was found to be more effective than both gefitinib and IGF-1R si-RNA mediated growth inhibition of chemo-surviving HCT-116 cells and addition of FOLFOX to curcumin did not increase the growth inhibitory effect of curcumin. Our data suggest that inclusion of curcumin in conventional chemotherapeutic regimens could be an effective strategy to prevent the emergence of chemoresistant colon cancer cells.

Curcumin synergizes with resveratrol to inhibit colon cancer.

Development and progression of many malignancies, including colorectal cancer, are associated with activation of multiple signaling pathways. Therefore, inhibition of these signaling pathways with noncytotoxic natural products represents a logical preventive and/or therapeutic approach for colon cancer. Curcumin and resveratrol, both of which inhibit the growth of transformed cells and colon carcinogenesis, were selected to examine whether combining them would be an effective preventive and/or therapeutic strategy for colon cancer. Indeed, the combination of curcumin and resveratrol was found to be more effective in inhibiting growth of p53-positive (wt) and p53-negative colon cancer HCT-116 cells in vitro and in vivo in SCID xenografts of colon cancer HCT-116 (wt) cells than either agent alone. Analysis by Calcusyn software showed synergism between curcumin and resveratrol. The inhibition of tumors in response to curcumin and/or resveratrol was associated with the reduction in proliferation and stimulation of apoptosis accompanied by attenuation of NF-kappaB activity. In vitro studies have further demonstrated that the combinatorial treatment caused a greater inhibition of constitutive activation of EGFR and its family members as well as IGF-1R. Our current data suggest that the combination of curcumin and resveratrol could be an effective preventive/therapeutic strategy for colon cancer.

Suppression of autophagy enhances the cytotoxicity of the DNA-damaging aromatic amine p-anilinoaniline.

p-Anilinoaniline (pAA) is an aromatic amine that is widely used in hair dying applications. It is also a metabolite of metanil yellow, an azo dye that is commonly used as a food coloring agent. Concentrations of pAA between 10 and 25 microM were cytostatic to cultures of the normal human mammary epithelia cell line MCF10A. Concentrations >or=50 microM were cytotoxic. Cytostatic concentrations induced transient G(1) and S cell cycle phase arrests; whereas cytotoxic concentrations induced protracted arrests. Cytotoxic concentrations of pAA caused DNA damage, as monitored by the alkaline single-cell gel electrophoresis (Comet) assay, and morphological changes consistent with cells undergoing apoptosis and/or autophagy. Enzymatic and western blot analyses, and binding analyses of fluorescent labeled VAD-FMK, suggested that caspase family members were activated by pAA. Western blot analyses documented the conversion of LC3-I to LC3-II, a post-translational modification involved in the development of the autophagosome. Suppression of autophagosome formation, via knockdown of ATG7 with shRNA, prevented pAA-induced vacuolization, enhanced the activation of pro-caspase-3, and increased susceptibility of ATG7-deficient cells to the cytostatic and cytotoxic activities of markedly lower concentrations of pAA. Cells stably transfected with a nonsense shRNA behaved like parental MCF10A cells. Collectively, these data suggest that MCF10A cultures undergo autophagy as a pro-survival response to concentrations of pAA sufficient to induce DNA damage.

Superinduction of CYP1A1 in MCF10A cultures by cycloheximide, anisomycin, and puromycin: a process independent of effects on protein translation and unrelated to suppression of aryl hydrocarbon receptor proteolysis by the proteasome.

Exposure of the human breast epithelial cell line MCF10A to > or = 1 microg/ml cycloheximide (CHX)-induced accumulations of CYP1A1 mRNA 6-fold greater than that achieved with only 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cotreatment with CHX and TCDD caused superinduction of CYP1A1 with accumulations of CYP1A1 mRNA 30-fold greater than that achieved with only TCDD. Similar results were obtained with the protein translation inhibitors anisomycin (ANS) and puromycin (PUR). Intra- and interinhibitor comparisons of dose/concentration response curves demonstrated the absence of a quantitative relationship between [3H]leucine incorporation and CYP1A1 induction/superinduction. The inducing/superinducing activities of CHX were suppressed by coincubation with the aryl hydrocarbon receptor (AhR) antagonists alpha-naphthoflavone and 3'-methoxy-4'-nitroflavone (PD168641). Electrophoretic mobility shift assays demonstrated that nuclear extracts from CHX-treated and CHX + TCDD cotreated cultures formed approximately 58 and approximately 340% of the AhR/DNA complexes obtained with TCDD-treated cultures, respectively. In contrast, rat liver extracts did not form AhR/DNA complexes after in vitro transformation with CHX. AhR turnover in TCDD-treated hepatoma 1c1c7 cultures was suppressed by cotreatment with CHX. In contrast, CHX or ANS treatment of MCF10A cultures induced AhR loss and enhanced AhR loss in cultures cotreated with TCDD. Cotreatment with N-benzoyloxycarbonyl-(Z)-Leu-Leu-leucinal (MG132) but not leptomycin B suppressed AhR loss. Hence, in MCF10A cells, CHX is not an AhR agonist but can superinduce CYP1A1 via an AhR-dependent mechanism; CYP1A1 superinduction by translation inhibitors is neither quantitatively related to effects on protein synthesis nor due to a generalized prevention of AhR proteolysis, and proteasome-mediated degradation of the activated AhR can occur in the nucleus.