HDAC Inhibition Overcomes Acute Resistance to MEK Inhibition in BRAF-Mutant Colorectal Cancer by Downregulation of c-FLIPL

Purpose: Activating mutations in the BRAF oncogene are found in 8% to 15% of colorectal cancer patients and have been associated with poor survival. In contrast with BRAF-mutant (MT) melanoma, inhibition of the MAPK pathway is ineffective in the majority of BRAFMT colorectal cancer patients. Therefore, identification of novel therapies for BRAFMT colorectal cancer is urgently needed.

Experimental Design: BRAFMT and wild-type (WT) colorectal cancer models were assessed in vitro and in vivo. Small-molecule inhibitors of MEK1/2, MET, and HDAC were used, overexpression and siRNA approaches were applied, and cell death was assessed by flow cytometry, Western blotting, cell viability, and caspase activity assays.

Results: Increased c-MET-STAT3 signaling was identified as a novel adaptive resistance mechanism to MEK inhibitors (MEKi) in BRAFMT colorectal cancer models in vitro and in vivo. Moreover, MEKi treatment resulted in acute increases in transcription of the endogenous caspase-8 inhibitor c-FLIPL in BRAFMT cells, but not in BRAFWT cells, and inhibition of STAT3 activity abrogated MEKi-induced c-FLIPL expression. In addition, treatment with c-FLIP–specific siRNA or HDAC inhibitors abrogated MEKi-induced upregulation of c-FLIPL expression and resulted in significant increases in MEKi-induced cell death in BRAFMT colorectal cancer cells. Notably, combined HDAC inhibitor/MEKi treatment resulted in dramatically attenuated tumor growth in BRAFMT xenografts.

Conclusions: Our findings indicate that c-MET/STAT3-dependent upregulation of c-FLIPL expression is an important escape mechanism following MEKi treatment in BRAFMT colorectal cancer. Thus, combinations of MEKi with inhibitors of c-MET or c-FLIP (e.g., HDAC inhibitors) could be potential novel treatment strategies for BRAFMT colorectal cancer.

The photocatalytic decomposition of microcystin-LR using selected titanium dioxide materials

<p>Microcystins (cyclic heptapeptides) produced by a number of freshwater cyanobacteria are a potential cause for concern in potable water supplies due to their acute and chronic toxicity. TiO₂ photocatalysis is a promising technology for removal of these toxins from drinking water. It is, however, necessary to have a sufficient knowledge of how the catalyst materials cause the degradation of the toxins through the photocatalytic process. The present study reports microcystin degradation products of the photocatalytic oxidation by using a number of commercial TiO₂ powder (P25, PC50, PC500 and UV100) and granular (KO1, KO3, TiCat-C, TiCat-S) materials, so aiding the mechanistic understanding of this process. Liquid chromatography-mass spectrometry analysis demonstrated that the major destruction pathway of microcystin for all the catalysts tested followed almost the same pathway, indicating the physical properties of the catalysts had little effects on the degradation pathway of microcystin-LR. p>

Processes influencing surface interaction and photocatalytic destruction of microcystins on titanium dioxide photocatalysts

<p>Microcystins are a family of hepatotoxic peptides produced by freshwater cyanobacteria. Their occurrence in drinking water is of concern since chronic exposure to these toxins causes tumor promotion. It is therefore essential to establish a reliable treatment strategy that will ensure their removal from potable water. We have previously described the rapid destruction of microcystin-LR using TiO₂ photocatalysis, however, since there are at least 70 microcystin variants it is essential that the destruction of a number of microcystins be evaluated. In this study the dark adsorption and destruction of four microcystins was followed over a range of pH. All four microcystins were destroyed although the efficiency of their removal varied. The two more hydrophobic microcystins (-LW and -LF) were found to have high dark adsorption (98 and 91% at pH 4) in contrast to microcystin-RR, which was found to have almost no (only 2-3%) dark adsorption across all pH. p>

Mechanistic and toxicity studies of the photocatalytic oxidation of microcystin-LR

<p>Cyanobacterial toxins present in drinking water sources pose a considerable threat to human health. Conventional water treatment systems have proven unreliable for the removal of these toxins and hence new techniques have been investigated. Previous work has shown that TiO₂ photocatalysis effectively destroys microcystin-LR in aqueous solutions, however, a variety of by-products were generated. In this paper, we report a mechanistic study of the photocatalytic destruction of microcystin-LR. In particular, the toxicity by-products of the process have been studied using both brine shrimp and protein phosphatase bioassays. p>

Hydrogen peroxide enhanced photocatalytic oxidation of microcystin-lR using titanium dioxide

<p>Cyanobacterial toxins present in drinking water sources pose a considerable threat to human health. Conventional water treatment systems have proven unreliable for the removal of these toxins and hence new techniques have been investigated. Previous work has shown that TiO₂ photocatalysis effectively destroys microcystin-LR in aqueous solutions, however non-toxic by-products were detected. It has been shown that photocatalytic reactions are enhanced by utilisation of alternative electron acceptors. We report here enhanced photocatalytic degradation of microcystin-LR following the addition of hydrogen peroxide to the system. It was also found that hydrogen peroxide with UV illumination alone was capable of decomposing microcystin-LR although at a much slower rate than found for TiO₂. No HPLC detectable by-products were found when the TiO₂/UV/H₂O₂ system was used indicating that this method is more effective than TiO₂/UV alone. Results however indicated that only 18% mineralisation occurred with the TiO₂/UV/H₂O₂ system and hence undetectable by-products must still be present. At higher concentrations hydrogen peroxide was found to compete with microcystin-LR for surface sites on the catalyst but at lower peroxide concentrations this competitive adsorption was not observed. Toxicity studies showed that both in the presence and absence of H₂O₂ the microcystin solutions were detoxified. These findings suggest that hydrogen peroxide greatly enhances the photocatalytic oxidation of microcystin-LR.p>

The Involvement of Phycocyanin Pigment in the Photodecomposition of the Cyanobacterial Toxin, Microcystin-LR

<p>While investigating the destruction of the cyanobacterial hepatotoxin microcystin-LR in the presence of phycocyanin pigment via semiconductor photocatalysis, it became apparent that the pigment was catalysing the toxin decomposition. The mechanism of this process in terms of phycocyanin acting as a photo-oxygenation sensitizer via singlet oxygen and superoxide attack is explored. The absorption and fluorescence spectra of phycocyanin have been obtained and data on the properties of the excited state calculated. The established photo-oxygenation sensitizer rose bengal was also used as a catalyst for the photolytic decomposition of microcystin-LR to help elucidate the decomposition mechanism. p>

Detoxification of microcystins (cyanobacterial hepatotoxins) using TiO2 photocatalytic oxidation

<p>TiO₂ photocatalysis has been used to destroy microcystin-LR in aqueous solution. The destruction of this toxin was monitored by HPLC, and the disappearance was accompanied by the appearance of seven UV detectable compounds. Spectral analysis revealed that some of these compounds retained spectra similar to the parent compound suggesting that the Adda moiety, thought to be responsible for the characteristic spectrum, remained intact whereas the spectra of some of the other products was more radically altered. Six of the seven observed reaction products did not appear to undergo further degradation during prolonged photocatalysis (100 min). The degree to which microcystin-LR was mineralized by photocatalytic oxidation was determined. Results indicated that less than 10% mineralization occurred. Mass spectral analysis of the photocatalyzed microcystin-LR allowed tentative characterization of the reaction process and products. Reduction in toxicity due to the photocatalytic oxidation was evaluated using an invertebrate bioassay, which demonstrated that the disappearance of microcystin-LR was paralleled by a reduction in toxicity. These findings suggest that photocatalytic destruction of microcystins may be a suitable method for the removal of these potentially hazardous compounds from drinking water.p>

Processes influencing the destruction of microcystin-LR by TiO2 photocatalysis

<p>We have previously reported the effectiveness of TiO₂ photocatalysis in the destruction of the cyanotoxin microcystin-LR [P.K.J. Robertson, L.A. Lawton, B. Münch, J. Rouzade, J. Chem. Soc., Chem. Commun., 4 (1997) 393; P.K.J. Robertson, L.A. Lawton, B. Münch, B.J.P.A. Cornish, J. Adv. Oxid. Technol., in press]. In this paper we report an investigation of factors which influence the rate of the toxin destruction at the catalyst surface. A primary kinetic isotope effect of approximately 3 was observed when the destruction was performed in a heavy water solvent. Hydroxylated compounds were observed as products of the destruction process. No destruction was observed when the process was investigated under a nitrogen atmosphere.p>