Sreeparna Banerjee

Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2–MSH6 complex

Cadmium (Cd2+) is a known carcinogen that inactivates the DNA mismatch repair (MMR) pathway. In this study, we have tested the effect of Cd2+ exposure on the enzymatic activity of the mismatch binding complex MSH2–MSH6. Our results indicate that Cd2+ is highly inhibitory to the ATP binding and hydrolysis activities of MSH2–MSH6, and less inhibitory to its DNA mismatch binding activity. The inhibition of the ATPase activity appears to be dose and exposure time dependent. However, the inhibition of the ATPase activity by Cd2+ is prevented by cysteine and histidine, suggesting that these residues are essential for the ATPase activity and are targeted by Cd2+. A comparison of the mechanism of inhibition with N-ethyl maleimide, a sulfhydryl group inhibitor, indicates that this inhibition does not occur through direct inactivation of sulfhydryl groups. Zinc (Zn2+) does not overcome the direct inhibitory effect of Cd2+ on the MSH2–MSH6 ATPase activity in vitro. However, the increase in the mutator phenotype of yeast cells exposed to Cd2+ was prevented by excess Zn2+, probably by blocking the entry of Cd2+ into the cell. We conclude that the inhibition of MMR by Cd2+ is through the inactivation of the ATPase activity of the MSH2–MSH6 heterodimer, resulting in a dominant negative effect and causing a mutator phenotype.

Celecoxib-loaded liposomes: effect of cholesterol on encapsulation and in vitro release characteristics

CLX (celecoxib) is a highly hydrophobic non-steroidal anti-inflammatory drug with high plasma protein binding. We describe here the encapsulation of CLX in MLVs (multilamellar vesicles) composed of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and variable amounts of cholesterol. The effects of cholesterol content on liposome size, percentage drug loading and in vitro drug release profiles were investigated. Differential scanning calorimetry and FTIR (Fourier-transform infrared) spectroscopy were used to determine molecular interactions between CLX, cholesterol and DSPC. The phase transition temperature (Tm) of vesicles was reduced in a synergistic manner in the presence of both CLX and cholesterol. Encapsulation efficiency, loading and release of CLX decreased with increasing cholesterol content. FTIR results indicated that this decrease was due to a competition between CLX and cholesterol for the co-operativity region of the phospholipids. In the presence of cholesterol, CLX was pushed further into the hydrophobic core of the bilayer. However, MLVs prepared with DSPC only (without cholesterol) exhibited the lowest ability for drug retention after 72 h. Our results indicated that CLX, without the requirement of modifications to enhance solubilization, can be encapsulated and released from liposomal formulations. This method of drug delivery may be used to circumvent the low bioavailability and systemic side effects of oral CLX formulations.

15-Lipoxygenase-1 expression suppresses the invasive properties of colorectal carcinoma cell lines HCT-116 and HT-29.

Colorectal carcinoma (CRC) is often lethal when invasion and/or metastasis occur. 15‐Lipoxygenase‐1 (15‐LO‐1), a member of the inflammatory eicosanoid pathway, oxidatively metabolizes linoleic acid and its expression is repressed in CRC. In this study, we investigated the hypothesis that the lack of 15‐LO‐1 expression in CRC cells might contribute to tumorigenesis. Therefore we introduced 15‐LO‐1 into HCT‐116 and HT‐29 cells that do not have detectable levels of 15‐LO‐1. Our data indicate that expression of 15‐LO‐1 significantly decreased cell proliferation and increased apoptosis. In addition, we observed a reduction in adhesion to fibronectin, anchorage‐independent growth on soft agar, cellular motility and ability to heal a scratch wound, and migratory and invasive capacity across Matrigel. 15‐LO‐1 expression also reduced the expression of metastasis associated protein‐1, a part of the nucleosome remodeling and histone deacetylase silencing complex. We propose that 15‐LO‐1 expression in CRC might contribute to the inhibition of metastatic capacity in vitro and can be exploited for therapeutic purposes. (Cancer Sci 2009;100: 2283–2291)

MTA-1 expression is associated with metastasis and epithelial to mesenchymal transition in colorectal cancer cells

Although metastasis associated protein 1 (MTA1) has been widely linked to tumor metastasis, the relevant mechanisms remain to be elucidated, especially in colorectal cancer (CRC). Here, we have investigated the link between MTA1, metastasis and epithelial–mesenchymal transition (EMT) in CRC. Eighteen normal colon tissues and 91 resected tumor samples were analyzed for MTA1 expression by immunohistochemistry (IHC). IHC indicated low or no nuclear MTA1 expression in the normal tissues and significantly higher expression in Grade II, Grade III and liver metastasis tumors. No statistically significant difference was observed in MTA1 expression between Grade III and liver metastatic tumors. To demonstrate the functional importance of MTA1 in vitro, the gene was silenced in HCT-116 cells and LoVo cells and overexpressed in HCT-116 cells. MTA1 overexpression in HCT-116 cells enhanced proliferation, adhesion to fibronectin, motility, migration, invasion through Matrigel, anchorage-independent growth, neoangiogenesis and induced a loss of apoptosis. Silencing of MTA1 resulted in a reversal of all of these features. Mechanistically, MTA1 silencing caused an increase in the epithelial markers E-cadherin and ZO-1 and a decrease in the mesenchymal marker vimentin while MTA1 overexpression caused an increase in vimentin expression. Moreover, MTA1 enhanced the expression of Snai1 and Slug; silencing of MTA1 reduced their recruitment to the promoter of E-cadherin, thereby leading to its expression. MTA1 is highly expressed in higher grade tumors and is important in the orchestration of various phenotypic changes in CRC, most likely by inducing EMT. This further corroborates its role as a master regulator in tumorigenesis.

15‐lipoxygenase‐1 exerts its tumor suppressive role by inhibiting nuclear factor‐kappa B via activation of PPAR gamma

15‐Lipoxygenase‐1 (15‐LOX‐1) is an enzyme of the inflammatory eicosanoid pathway whose expression is known to be lost in colorectal cancer (CRC). We have previously shown that reintroduction of the gene in CRC cell lines slows proliferation and induces apoptosis (Cimen et al. [2009] Cancer Sci 100: 2283–2291). We have hypothesized that 15‐LOX‐1 may be anti‐tumorigenic by the inhibition of the anti‐apoptotic inflammatory transcription factor nuclear factor kappa B. We show here that ectopic expression of 15‐LOX‐1 gene in HCT‐116 and HT‐29 CRC cell lines inhibited the degradation of inhibitor of kappa B (IκBα), decreased nuclear translocation of p65 and p50, decreased DNA binding in the nucleus and decreased transcriptional activity of Nuclear factor kappa B (NF‐κB). As the 15‐LOX‐1 enzymatic product 13(S)‐HODE is known to be a peroxisome proliferator‐activated receptor gamma (PPARγ) agonist, and NF‐κB can be inhibited by PPARγ, we examined whether activation of PPARγ was necessary for the abrogation of NF‐κB activity. Our data show that the inhibition of both early and late stages of NF‐κB activation could rescued by the PPARγ antagonist GW9662 indicating that the inhibition was most likely mediated via PPARγ. J. Cell. Biochem. 112: 2490–2501, 2011.

The NF‐κB target genes ICAM‐1 and VCAM‐1 are differentially regulated during spontaneous differentiation of Caco‐2 cells

Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cell–cell and cell–microenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco‐2 cell line, which undergoes spontaneous re‐differentiation when grown past confluency, we observed a loss of VCAM‐1 (vascular cell adhesion molecule 1) mRNA expression, while ICAM‐1 (intercellular cell adhesion molecule 1) mRNA expression was seen to increase over the course of differentiation. Protein kinase Cθ (PKCθ) acted downstream of protein kinase Cα (PKCα) to inactivate inhibitor of κB (IκB) and activate nuclear factor κB (NF‐κB) in undifferentiated cells, and this pathway was inhibited in the differentiated cells. The increase in ICAM‐1 mRNA expression in the differentiated cells was due to increased promoter recruitment of C/EBPβ, which transcriptionally up‐regulated ICAM‐1 mRNA. However, protein expression of ICAM‐1 was found to decrease over the course of differentiation due to degradation in the proteasome and lysosome. Immunohistochemistry using tumor samples from colon cancer patients indicated that non‐transformed matched normal cells (well‐differentiatied) showed no ICAM‐1 expression, but the poorly differentiated tumor cells showed higher expression. Functionally, a decrease in adhesion to human umbilical vein endothelial cells was observed in the differentiated Caco‐2 cells. Thus, regulation of ICAM‐1 and VCAM‐1, although both NF‐κB target genes, appears to be different over the course of epithelial differentiation in Caco‐2 cells.

Eicosanoid pathway in colorectal cancer: Recent updates

Enzymatic metabolism of the 20C polyunsaturated fatty acid (PUFA) arachidonic acid (AA) occurs via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways, and leads to the production of various bioactive lipids termed eicosanoids. These eicosanoids have a variety of functions, including stimulation of homeostatic responses in the cardiovascular system, induction and resolution of inflammation, and modulation of immune responses against diseases associated with chronic inflammation, such as cancer. Because chronic inflammation is essential for the development of colorectal cancer (CRC), it is not surprising that many eicosanoids are implicated in CRC. Oftentimes, these autacoids work in an antagonistic and highly temporal manner in inflammation; therefore, inhibition of the pro-inflammatory COX-2 or 5-LOX enzymes may subsequently inhibit the formation of their essential products, or shunt substrates from one pathway to another, leading to undesirable side-effects. A better understanding of these different enzymes and their products is essential not only for understanding the importance of eicosanoids, but also for designing more effective drugs that solely target the inflammatory molecules found in both chronic inflammation and cancer. In this review, we have evaluated the cancer promoting and anti-cancer roles of different eicosanoids in CRC, and highlighted the most recent literature which describes how those molecules affect not only tumor tissue, but also the tumor microenvironment. Additionally, we have attempted to delineate the roles that eicosanoids with opposing functions play in neoplastic transformation in CRC through their effects on proliferation, apoptosis, motility, metastasis, and angiogenesis.

Epidermal growth factor receptor-targeted immunoliposomes for delivery of celecoxib to cancer cells.

Cyclooxygenase-2 (COX-2) is highly expressed in many different cancers. Therefore, the inhibition of the COX-2 pathway by a selective COX-2 inhibitor, celecoxib (CLX), may be an alternative strategy for cancer prevention and therapy. Liposomal drug delivery systems can be used to increase the therapeutic efficacy of CLX while minimizing its side effects. Previous studies have reported the encapsulation of CLX within the non-targeted long circulating liposomes and functional effect of these formulations against colorectal cancer cell lines. However, the selectivity and internalization of CLX-loaded liposomes can further be improved by grafting targeting ligands on their surface. Cetuximab (anti-epidermal growth factor receptor - EGFR - monoclonal antibody) is a promising targeting ligand since EGFR is highly expressed in a wide range of solid tumors. The aim of this study was to develop EGFR-targeted immunoliposomes for enhancing the delivery of CLX to cancer cells and to evaluate the functional effects of these liposomes in cancer cell lines. EGFR-targeted ILs, having an average size of 120nm, could encapsulate 40% of the CLX, while providing a sustained drug release profile. Cell association studies have also shown that the immunoliposome uptake was higher in EGFR-overexpressing cells compared to the non-targeted liposomes. In addition, the CLX-loaded-anti-EGFR immunoliposomes were significantly more toxic compared to the non-targeted ones in cancer cells with EGFR-overexpression but not in the cells with low EGFR expression, regardless of their COX-2 expression status. Thus, selective targeting of CLX with anti-EGFR immunoliposomes appears to be a promising strategy for therapy of tumors that overexpress EGFR.

Matrix metalloprotease 16 expression is downregulated by microRNA-146a in spontaneously differentiating Caco-2 cells

Cellular differentiation in the gut is vital in maintaining the cellular and functional specialization of the epithelial layer. MicroRNAs (miRNAs) have recently emerged as one of the key players in orchestrating the differentiation process in the gut. Using the spontaneously differentiating Caco‐2 cell line, we observed an increased expression of miR‐146a but not miR‐146b in the course of differentiation. Bioinformatic analyses revealed that the membrane type matrix metalloprotease 16 (MMP16, MT3‐MMP) was a predicted target of miR‐146a and a decrease in the mRNA and protein expression of MMP16 was observed in the course of differentiation. Transfection of a luciferase reporter vector containing the 3′UTR of MMP16 showed decreased luciferase activity due to miR‐146a expression. With forced expression of miR‐146a in undifferentiated Caco‐2 cells, a decrease in the mRNA and protein levels of MMP16 and a lower gelatinase activity in a gelatin zymogram were observed. Additionally, forced expression of miR‐146a in HT‐29 colon cancer cells also resulted in decreased expression of MMP16, along with a decrease in the invasion through Matrigel. Taken together, we have shown here that MMP16 is regulated by miR‐146a in spontaneously differentiated Caco‐2 cells. As MMP16 activates the zymogen of MMP2, which is known to degrade extracellular matrix proteins, the regulation of MMP16 by miR‐146a may account, at least in part, for lower motility of well‐differentiated cells.

Monoclonal antibodies for targeted therapy in colorectal cancer.

Traditional therapeutic regimens of solid tumors such as chemotherapy and radiotherapy often do not distinguish between malignant and normal tissues, resulting in considerable side-effects. Monoclonal antibodies (mAbs), targeted against antigens dysregulated in cancers, have therefore generated great interest in both clinical and research settings. The antibodies are either chimeric or human(ized) and can bind to and inhibit target proteins overexpressed in both solid tumors and hematological malignancies. Some of these mAbs have shown efficacy in patients who are refractory to traditional chemotherapy. Examples of FDA approved antibodies against metastatic colorectal cancer include cetuximab, panitumumab and bevacizumab. This review summarizes the current knowledge of mAbs targeting growth factors in colorectal cancer and the importance of carefully screening patients to select candidates who will benefit most from these therapies.