MALDI-MSI of lipids in a model of breast cancer brain metastasis provides a surrogate measure of ischemia/hypoxia.

Breast cancer brain metastasis (BCBM) has an incidence of 10-30%. It is incurable and the biological mechanisms that promote its progression remain largely undefined. Consequently, to gain insights into BCBM processes, we have developed a spontaneous mouse model of BCBM and in this study found a 20% penetrance of macro-metastatic brain lesion formation. Considering that lipid metabolism is indispensable to metastatic progression, our goal was the mapping of lipid distributions throughout the metastatic regions of the brain. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of lipids revealed that, relative to surrounding brain tissue, seven long-chain (13-21 carbons long) fatty acylcarnitines, as well as two phosphatidylcholines, two phosphatidylinositols two diacylglycerols, a long-chain phosphatidylethanolamine, and a long-chain sphingomyelin were highly concentrated in the metastatic brain lesion In broad terms, lipids known to be enriched in brain tissues, such as very long-chain (≥ 22 carbons in length) polyunsaturated fatty acid of phosphatidylcholines, phosphatidylethanolamine, sphingomyelins, sulfatides, phosphatidylinositol phosphates, and galactosylceramides, were not found or only found in trace amounts in the metastatic lesion and instead consistently detected in surrounding brain tissues. The data, from this mouse model, highlights an accumulation of fatty acylcarnitines as possible biological makers of a chaotic inefficient vasculature within the metastasis, resulting in relatively inadequate blood flow and disruption of fatty acid ß-oxidation due to ischemia/hypoxia.

Direct facile screening of recombinant DNA vector constructs.

Direct efficient facile screening of bacterial transformants with the goal of selecting, retrieving, and using recombinant DNA is exemplified by simple visual-based colorimetric inspections or fluorescent protein-based assays. We describe pRedScript, which introduces the constitutive expression of a very bright red fluorescent protein into transformants. On agar plates, red colonies are simply visualized in ambient white light in stark contrast to recombinant transformants that are white. In addition, the bright red fluorescence of the reporter protein can also be harnessed as a sensitive signal for screening bacterial promoters during the development of optimized fermentation conditions.

Combined MR, fluorescence and histology imaging strategy in a human breast tumor xenograft model.

Applications of molecular imaging in cancer and other diseases frequently require the combination of in vivo imaging modalities, such as MR and optical imaging, with ex vivo optical, fluorescence, histology and immunohistochemical imaging to investigate and relate molecular and biological processes to imaging parameters within the same region of interest. We have developed a multimodal image reconstruction and fusion framework that accurately combines in vivo MRI and MRSI, ex vivo brightfield and fluorescence microscopic imaging and ex vivo histology imaging. Ex vivo brightfield microscopic imaging was used as an intermediate modality to facilitate the ultimate link between ex vivo histology and in vivo MRI/MRSI. Tissue sectioning necessary for optical and histology imaging required the generation of a three-dimensional reconstruction module for two-dimensional ex vivo optical and histology imaging data. We developed an external fiducial marker-based three-dimensional reconstruction method, which was able to fuse optical brightfield and fluorescence with histology imaging data. The registration of the three-dimensional tumor shape was pursued to combine in vivo MRI/MRSI and ex vivo optical brightfield and fluorescence imaging data. This registration strategy was applied to in vivo MRI/MRSI, ex vivo optical brightfield/fluorescence and histology imaging datasets obtained from human breast tumor models. Three-dimensional human breast tumor datasets were successfully reconstructed and fused with this platform.

Isogenic Cell Lines Derived from Specific Organ Metastases Exhibit Divergent Cytogenomic Aberrations.

Aneuploidy, a deviation in chromosome numbers from the normal diploid set, is now recognized as a fundamental characteristic of all cancer types and is found in 70-90% of all solid tumors. The majority of aneuploidies are generated by chromosomal instability (CIN). CIN/aneuploidy is an independent prognostic marker of cancer survival and is a cause of drug resistance. Hence, ongoing research has been directed towards the development of therapeutics aimed at targeting CIN/aneuploidy. However, there are relatively limited reports on the evolution of CIN/aneuploidies within or across metastatic lesions. In this work, we built on our previous studies using a human xenograft model system of metastatic disease in mice that is based on isogenic cell lines derived from the primary tumor and specific metastatic organs (brain, liver, lung, and spine). As such, these studies were aimed at exploring distinctions and commonalities between the karyotypes; biological processes that have been implicated in CIN; single-nucleotide polymorphisms (SNPs); losses, gains, and amplifications of chromosomal regions; and gene mutation variants across these cell lines. Substantial amounts of inter- and intra-heterogeneity were found across karyotypes, along with distinctions between SNP frequencies across each chromosome of each metastatic cell line relative the primary tumor cell line. There were disconnects between chromosomal gains or amplifications and protein levels of the genes in those regions. However, commonalities across all cell lines provide opportunities to select biological processes as druggable targets that could have efficacy against the primary tumor, as well as metastases.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy toward them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3X (DDX3), a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented support the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy towards them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3, a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented supports the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

Targeting host DEAD-box RNA helicase DDX3X for treating viral infections.

DDX3X or DDX3, a member of the DEAD (asp, glu, ala, asp) box RNA helicase family of proteins, is a multifunctional protein, which is usurped by several viruses and is vital to their production. To date, 18 species of virus from 12 genera have been demonstrated to be dependent on DDX3 for virulence. In addition, DDX3 has been shown to function within 7 of 10 subcellular regions that are involved in the metabolism of viruses. As such, due to its direct interaction with viral components across most or all stages of viral life cycles, DDX3 can be considered an excellent host target for pan-antiviral drug therapy and has been reported to be a possible broad-spectrum antiviral target. Along these lines, it has been demonstrated that treatment of virally infected cells with small molecule inhibitors of DDX3 blunts virion productions. On the other hand, DDX3 bolsters an innate immune response and viruses have evolved capacities to sequester or block DDX3, which dampens an innate immune response. Thus, enhancing DDX3 production or co-targeting direct viral products that interfere with DDX3's modulation of innate immunity would also diminish virion production. Here we review the evidence that supports the hypothesis that modulating DDX3's agonistic and antagonistic functions during viral infections could have an important impact on safely and efficiently subduing a broad-spectrum of viral infections.

Nrf2-deficiency creates a responsive microenvironment for metastasis to the lung.

The Nrf2 transcription factor is crucial for regulating the cellular defense against various carcinogens. However, relationship between host Nrf2 and cancer metastasis remains unexplored. To address this issue, we examined susceptibility of Nrf2-deficient mice to pulmonary cancer metastasis following implantation of the mouse Lewis lung carcinoma (3LL) cell line. Nrf2-deficient mice reproducibly exhibited a higher number of pulmonary metastatic nodules than wild-type mice did. The lung and bone marrow (BM) of cancer-bearing Nrf2-deficient mice contained increased numbers of inflammatory cells, including myeloid-derived suppressor cells (MDSCs), a potent population of immunosuppressive cells. MDSCs can attenuate CD8(+) T-cell immunity through modification of the T-cell receptor complex exploiting reactive oxygen species (ROS). MDSCs of Nrf2-deficient mice retained elevated levels of ROS relative to wild-type mice. BM transplantation experiments revealed functional disturbance in the hematopoietic and immune systems of Nrf2-deficient mice. Wild-type recipient mice with Nrf2-deficient BM cells showed increased levels of lung metastasis after cancer cell inoculation. These mice exhibited high-level accumulation of ROS in MDSCs, which showed very good coincidence to the decrease of splenic CD8(+) T-cells. In contrast, Keap1-knockdown mutant mice harboring high-level Nrf2 expression displayed increased resistance against the cancer cell metastasis to the lung, accompanied by a decrease in ROS in the MDSCs fraction. Our results thus reveal a novel function for Nrf2 in the prevention of cancer metastasis, presumably by its ability to preserve the redox balance in the hematopoietic and immune systems.

Choline kinase overexpression increases invasiveness and drug resistance of human breast cancer cells.

A direct correlation exists between increased choline kinase (Chk) expression, and the resulting increase of phosphocholine levels, and histological tumor grade. To better understand the function of Chk and choline phospholipid metabolism in breast cancer we have stably overexpressed one of the two isoforms of Chk-alpha known to be upregulated in malignant cells, in non-invasive MCF-7 human breast cancer cells. Dynamic tracking of cell invasion and cell metabolism were studied with a magnetic resonance (MR) compatible cell perfusion assay. The MR based invasion assay demonstrated that MCF-7 cells overexpressing Chk-alpha (MCF-7-Chk) exhibited an increase of invasion relative to control MCF-7 cells (0.84 vs 0.3). Proton MR spectroscopy studies showed significantly higher phosphocholine and elevated triglyceride signals in Chk overexpressing clones compared to control cells. A test of drug resistance in MCF-7-Chk cells revealed that these cells had an increased resistance to 5-fluorouracil and higher expression of thymidylate synthase compared to control MCF-7 cells. To further characterize increased drug resistance in these cells, we performed rhodamine-123 efflux studies to evaluate drug efflux pumps. MCF-7-Chk cells effluxed twice as much rhodamine-123 compared to MCF-7 cells. Chk-alpha overexpression resulted in MCF-7 human breast cancer cells acquiring an increasingly aggressive phenotype, supporting the role of Chk-alpha in mediating invasion and drug resistance, and the use of phosphocholine as a biomarker of aggressive breast cancers.

Neoplastic transformation of breast epithelial cells by genotoxic stress.

BACKGROUND: Exposure to genotoxic stresses such as radiation and tobacco smoke can cause increased cancer incidence rate as reflected in an in depth meta-analysis of data for women and breast cancer incidence. Published reports have indicated that exposures to low dose radiation and tobacco smoke are factors that contribute to the development of breast cancer. However, there is a scarcity of information on the combinatorial effects of low dose radiation and tobacco smoke on formation and progression of breast cancer. The combination of these two genotoxic insults can induce significant damage to the genetic material of the cells resulting in neoplastic transformation. METHODS: To study the effects of low dose ionizing radiation and tobacco smoke on breast cells, MCF 10A cells were treated either with radiation (Rad - 0.1 Gray) or cigarette smoke condensate (Csc - 10 microgram/ml of medium) or a combination of Rad + Csc. Following treatments, cells were analyzed for cell cycle distribution patterns and the ability to extrude the Hoechst 33342 dye. In addition, in vitro invasion and migration as well as mammosphere formation assays were performed. Finally, differential gene expression profiles were generated from the individual and combination treatment. RESULTS: Exposure of MCF 10A cells to the combination of radiation plus cigarette smoke condensate generated a neoplastic phenotype. The transformed phenotype promoted increased mammosphere numbers, altered cell cycle phases with a doubling of the population in S phase, and increased invasion and motility. Also, exclusion of Hoechst 33342 dye, a surrogate marker for increased ABC transporters, was observed, which indicates a possible increase in drug resistance. In addition, changes in gene expression include the up regulation of genes encoding proteins involved in metabolic pathways and inflammation. CONCLUSIONS: The results indicate that when normal breast cells are exposed to low dose radiation in combination with cigarette smoke condensate a phenotype is generated that exhibits traits indicative of neoplastic transformation. More importantly, this is the first study to provide a new insight into a possible etiology for breast cancer formation in individuals exposed to low dose radiation and tobacco smoke.

Artificial reporter gene providing MRI contrast based on proton exchange.

Existing magnetic resonance reporter genes all rely on the presence of (super)paramagnetic substances and employ water relaxation to gain contrast. We designed a nonmetallic, biodegradable, lysine rich-protein (LRP) reporter, the prototype of a potential family of genetically engineered reporters expressing artificial proteins with frequency-selective contrast. This endogenous contrast, based on transfer of radiofrequency labeling from the reporter's amide protons to water protons, can be switched on and off.

Divergent organ-specific isogenic metastatic cell lines identified using multi-omics exhibit differential drug sensitivity.

BACKGROUND: Monitoring and treating metastatic progression remains a formidable task due, in part, to an inability to monitor specific differential molecular adaptations that allow the cancer to thrive within different tissue types. Hence, to develop optimal treatment strategies for metastatic disease, an important consideration is the divergence of the metastatic cancer growing in visceral organs from the primary tumor. We had previously reported the establishment of isogenic human metastatic breast cancer cell lines that are representative of the common metastatic sites observed in breast cancer patients. METHODS: Here we have used proteomic, RNAseq, and metabolomic analyses of these isogenic cell lines to systematically identify differences and commonalities in pathway networks and examine the effect on the sensitivity to breast cancer therapeutic agents. RESULTS: Proteomic analyses indicated that dissemination of cells from the primary tumor sites to visceral organs resulted in cell lines that adapted to growth at each new site by, in part, acquiring protein pathways characteristic of the organ of growth. RNAseq and metabolomics analyses further confirmed the divergences, which resulted in differential efficacies to commonly used FDA approved chemotherapeutic drugs. This model system has provided data that indicates that organ-specific growth of malignant lesions is a selective adaptation and growth process. CONCLUSIONS: The insights provided by these analyses indicate that the rationale of targeted treatment of metastatic disease may benefit from a consideration that the biology of metastases has diverged from the primary tumor biology and using primary tumor traits as the basis for treatment may not be ideal to design treatment strategies.

Brain metabolites in cholinergic and glutamatergic pathways are altered by pancreatic cancer cachexia.

BACKGROUND: Cachexia is a major cause of morbidity in pancreatic ductal adenocarcinoma (PDAC) patients. Our purpose was to understand the impact of PDAC-induced cachexia on brain metabolism in PDAC xenograft studies, to gain new insights into the causes of cachexia-induced morbidity. Changes in mouse and human plasma metabolites were characterized to identify underlying causes of brain metabolic changes. METHODS: We quantified metabolites, detected with high-resolution 1 H magnetic resonance spectroscopy, in the brain and plasma of normal mice (n = 10) and mice bearing cachexia (n = 10) or non-cachexia (n = 9) inducing PDAC xenografts as well as in human plasma obtained from normal individuals (n = 24) and from individuals with benign pancreatic disease (n = 20) and PDAC (n = 20). Statistical significance was defined as a P value ≤0.05. RESULTS: The brain metabolic signature of cachexia-inducing PDAC was characterized by a significant depletion of choline of -27% and -21% as well as increases of glutamine of 13% and 9% and formate of 21% and 14%, relative to normal controls and non-cachectic tumour-bearing mice, respectively. Good to moderate correlations with percent weight change were found for choline (r = 0.70), glutamine (r = -0.58), and formate (r = -0.43). Significant choline depletion of -38% and -30%, relative to normal controls and non-cachectic tumour-bearing mice, respectively, detected in the plasma of cachectic mice likely contributed to decreased brain choline in cachectic mice. Similarly, relative to normal controls and patients with benign disease, choline levels in human plasma samples of PDAC patients were significantly lower by -12% and -20% respectively. A comparison of plasma metabolites from PDAC patients with and without weight loss identified significant changes in glutamine metabolism. CONCLUSIONS: Disturbances in metabolites of the choline/cholinergic and glutamine/glutamate/glutamatergic neurotransmitter pathways may contribute to morbidity. Metabolic normalization may provide strategies to reduce morbidity. The human plasma metabolite changes observed may lead to the development of companion diagnostic markers to detect PDAC and PDAC-induced cachexia.

Targeting DDX3 in Medulloblastoma Using the Small Molecule Inhibitor RK-33.

Medulloblastoma is the most common malignant tumor that arises from the cerebellum of the central nervous system. Clinically, medulloblastomas are treated by surgery, radiation, and chemotherapy, all of which result in toxicity and morbidity. Recent reports have identified that DDX3, a member of the RNA helicase family, is mutated in medulloblastoma. In this study, we demonstrate the role of DDX3 in driving medulloblastoma. With the use of a small molecule inhibitor of DDX3, RK-33, we could inhibit growth and promote cell death in two medulloblastoma cell lines, DAOY and UW228, with IC50 values of 2.5 µM and 3.5 µM, respectively. Treatment of DAOY and UW228 cells with RK-33 caused a G1 arrest, resulted in reduced TCF reporter activity, and reduced mRNA expression levels of downstream target genes of the WNT pathway, such as Axin2, CCND1, MYC, and Survivin. In addition, treatment of DAOY and UW228 cells with a combination of RK-33 and radiation exhibited a synergistic effect. Importantly, the combination of RK-33 and 5 Gy radiation caused tumor regression in a mouse xenograft model of medulloblastoma. Using immunohistochemistry, we observed DDX3 expression in both pediatric (55%) and adult (66%) medulloblastoma patients. Based on these results, we conclude that RK-33 is a promising radiosensitizing agent that inhibits DDX3 activity and down-regulates WNT/ß-catenin signaling and could be used as a frontline therapeutic strategy for DDX3-expressing medulloblastomas in combination with radiation.

Molecular imaging of metastatic potential.

If molecular imaging is to prove clinically useful it will have to surpass current, primarily anatomic techniques in terms of sensitivity and the ability to detect minimal changes in tissue. One of the most important tests for molecular imaging is to determine whether it can image the metastatic potential of tumors. Like all predictive endeavors, the imaging of such "potential" is a daunting task, but one that only molecular imaging--rather than standard, anatomic techniques--is likely to solve. Although difficult, imaging of metastatic potential is also arguably the most important task for molecular imaging of cancer because it is generally the dissemination of malignant tissue, not its prolonged residence in an inopportune site, which kills the patient. Below are examples of uses of molecular imaging of metastases as well as of metastatic potential, the former being a far more developed area of clinical inquiry.

Characterizing vascular parameters in hypoxic regions: a combined magnetic resonance and optical imaging study of a human prostate cancer model.

The integration of imaging technologies with the capabilities of genetic engineering has created novel opportunities for understanding and imaging cancer. Here, we have combined vascular magnetic resonance imaging (MRI) and optical imaging to understand the relationship between hypoxia and vascularization in a human prostate cancer model engineered to express enhanced green fluorescent protein (EGFP) under hypoxia. Characterization and validation of EGFP expression under hypoxic conditions was done in culture and in solid tumors in vivo. MRI measurements showed that vascular volume was significantly lower in fluorescing regions. These regions also frequently exhibited high permeability. These data were further supported by the detection of low vessel density in EGFP-positive regions, as determined by the distribution of intravascularly administered, fluorescence-labeled Lycopersicon esculentum lectin in frozen tumor sections. These observations are consistent with the possibility that regions of low vascular volumes are hypoxic, which induces increased expression of functionally active vascular endothelial growth factor, a potent vascular permeability factor.

Label-free Raman spectroscopy provides early determination and precise localization of breast cancer-colonized bone alterations.

Breast neoplasms frequently colonize bone and induce development of osteolytic bone lesions by disrupting the homeostasis of the bone microenvironment. This degenerative process can lead to bone pain and pathological bone fracture, a major cause of cancer morbidity and diminished quality of life, which is exacerbated by our limited ability to monitor early metastatic disease in bone and assess fracture risk. Spurred by its label-free, real-time nature and its exquisite molecular specificity, we employed spontaneous Raman spectroscopy to assess and quantify early metastasis driven biochemical alterations to bone composition. As early as two weeks after intracardiac inoculations of MDA-MB-435 breast cancer cells in NOD-SCID mice, Raman spectroscopic measurements in the femur and spine revealed consistent changes in carbonate substitution, overall mineralization as well as crystallinity increase in tumor-bearing bones when compared with their normal counterparts. Our observations reveal the possibility of early stage detection of biochemical changes in the tumor-bearing bones - significantly before morphological variations are captured through radiographic diagnosis. This study paves the way for a better molecular understanding of altered bone remodeling in such metastatic niches, and for further clinical studies with the goal of establishing a non-invasive tool for early metastasis detection and prediction of pathological fracture risk in breast cancer.

Twist overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer.

Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.

Delayed Progression of Lung Metastases Following Delivery of a Prodrug-activating Enzyme.

BACKGROUND: Chemotherapy is an effective option to treat recurrent or metastatic cancer but its debilitating side-effects limit the dose and time of exposure. Prodrugs that can be activated locally by an activating enzyme can minimize collateral damage from chemotherapy. We previously demonstrated the efficacy of a poly-L-lysine-based theranostic nanoplex containing bacterial cytosine deaminase (bCD) that locally converted 5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil in MDA-MB-231 primary tumor xenografts. MATERIALS AND METHODS: Here we used a more effective variant of bCD to target metastatic red fluorescence protein expressing MDA-MB-435 cells in the lungs. We used an intravenous injection of tumor cells and monitored tumor growth in the lungs for 5 weeks by which time metastatic nodules were detected with optical imaging. The animals were then treated with the bCD-nanoplex and 5-FC. RESULTS: We observed a significant decrease in metastatic burden with a single dose of the enzyme-nanoplex and two consecutive prodrug injections. CONCLUSION: These results are a first step towards the longitudinal evaluation of such a strategy with multiple doses. Additionally, the enzyme can be directly coupled to imaging reporters to time prodrug administration for the detection and treatment of aggressive metastatic cancer.

Role of DDX3 in the pathogenesis of inflammatory bowel disease.

When crypt stem cells of the gastrointestinal tract become injured, the result is increased synthesis of pro-inflammatory cytokines and matrix metalloproteinases by their progeny - the colonic epithelium. Chronic inflammation of the gastrointestinal tract is a characteristic of inflammatory bowel disease, which includes Crohn's Disease and Ulcerative Colitis. In our ongoing investigation to decipher the characteristic functions of a RNA helicase gene, DDX3, we identified high DDX3 expression by immunohistochemistry of colon biopsy samples, which included chronic/mild Morbus Crohn, active Morbus Crohn, Chronic/mild Colitis Ulcerosa and active Colitis Ulcerosa in epithelium and stromal compartments. We used a small molecule inhibitor of DDX3, RK-33, on two human colonic epithelial cell lines, HCEC1CT and HCEC2CT and found that RK-33 was able to decrease expression of MMP-1, MMP-2, MMP-3, and MMP-10. Moreover, forced differentiation of a human colonic cancer cell line, HT29, resulted in decreased DDX3 levels, indicating that DDX3 contributes to the modulation of colonic epithelium differentiation. In conclusion, our results revealed novel functions of DDX3 in inflammatory bowel disease and indicate a potential for using RK-33 as a systemic therapy to promote not only differentiation of transformed colonic epithelium but also to reduce MMP expression and thus elicit a decreased inflammatory response.