Susan McDonnell

Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion.

Endotoxin/lipopolysaccharide (LPS), a cell wall component of Gram‐negative bacteria, is a potent inflammatory stimulus. We previously reported that LPS increased the growth of experimental metastases in a murine tumor model. Here, we examined the effect of LPS exposure on key determinants of metastasis—angiogenesis, tumor cell invasion, vascular permeability, nitric oxide synthase (NOS) and matrix metalloproteinase 2 (MMP2) expression. BALB/c mice bearing 4T1 lung metastases were given an intraperitoneal (i.p.) injection of 10 μg LPS or saline. LPS exposure resulted in increased lung weight and incidence of pleural lesions. LPS increased angiogenesis both in vivo and in vitro. Vascular permeability in lung tissue was increased 18 hr after LPS injection. LPS increased inducible nitric oxide synthase (iNOS) and MMP2 expression in lung tumor nodules. 4T1 cells transfected with green fluorescent protein (4T1‐GFP) were injected via lateral tail vein. LPS exposure resulted in increased numbers of 4T1‐GFP cells in mouse lung tissue compared to saline controls, an effect blocked by the competitive NOS inhibitor, NG methyl‐L‐arginine (NMA). LPS‐induced growth and metastasis of 4T1 experimental lung metastases is associated with increased angiogenesis, vascular permeability and tumor cell invasion/migration with iNOS expression implicated in LPS‐induced metastasis.

Selection with melphalan or paclitaxel (Taxol) yields variants with different patterns of multidrug resistance, integrin expression and in vitro invasiveness

A melphalan-resistant variant (Roswell Park Memorial Institute (RPMI)-2650Ml) and a paclitaxel-resistant variant (RPMI-2650Tx) of the drug-sensitive human nasal carcinoma cell line, RPMI-2650, were established. The multidrug resistance (MDR) phenotype in the RPMI-2650Tx appeared to be P-glycoprotein (PgP)-mediated. Overexpression of multidrug resistant protein (MRP) family members was observed in the RPMI-2650Ml cells, which were also much more invasive in vitro than the parental cell line or the paclitaxel-resistant variant. Increased expression of alpha(2), alpha(5), alpha(6), beta(1) and beta(4) integrin subunits, decreased expression of alpha(4) integrin subunit, stronger adhesion to collagen type IV, laminin, fibronectin and matrigel, increased expression of MMP-2 and MMP-9 and significant motility compared with the parental cells were observed, along with a high invasiveness in the RPMI-2650Ml cells. Decreased expression of the alpha(2) integrin subunit, decreased attachment to collagen type IV, absence of cytokeratin 18 expression, no detectable expression of gelatin-degrading proteases and poor motility may be associated with the non-invasiveness of the RPMI-2650Tx variant. These results suggest that melphalan exposure can result in not only a MDR phenotype, but could also make cancer cells more invasive, whereas paclitaxel exposure resulted in MDR without increasing the in vitro invasiveness in the RPMI-2650 cells.

The Role of the Matrix Metalloproteinase Stromelysin in the Progression of Squamous Cell Carcinomas

The expression of the metalloproteinase stromelysin correlates with the progression of chemically induced squamous cell carcinomas. We demonstrate that the expression of activated stromelysin in papilloma-derived cells enhances in vitro cell invasion. We also demonstrate that the Ha-ras oncogene induces the transcription of the stromelysin gene through an AP-1 dependent pathway. The hypothesis is that alterations in oncogenes and suppressor genes influence stromelysin expression and thus influence subsequent steps of tumor invasion and metastasis.

Examining the relationship between cancer invasion/metastasis and drug resistance.

Studies of cancer invasion/metastasis and drug resistance have in the past generally proceeded along the separate pathways of research. Recently, however, interest has been focused on the possible relationship between drug resistance and cancer invasion and metastasis. A relationship between these two phenotypes has been demonstrated by two types of observation: firstly, some tumor cells selected for resistance to drugs are more invasive/metastatic relative to non-resistant parental cells; secondly, in some cases, secondary (more metastatic) tumors are more resistant to chemotherapeutic drugs than their primary counterparts. In other instances reported in the literature, no correlation is seen between drug exposure/resistance and cancer invasion/metastasis. The possibility that treatment with some chemotherapeutic drugs may be able to promote cancer invasion and metastasis needs further investigation because of its potential clinical relevance. A better understanding of any relationship between drug resistance and cancer invasion could lead to more effective cancer treatment.

Enhanced in vitro invasiveness and drug resistance with altered gene expression patterns in a human lung carcinoma cell line after pulse selection with anticancer drugs

The human lung carcinoma cell line DLKP was exposed to sequential pulses of 10 commonly used chemotherapeutic drugs (VP‐16, vincristine, taxotere, mitoxantrone, 5‐fluorouracil, methotrexate, CCNU, BCNU, cisplatin and chlorambucil); resulting cell lines exhibited resistance to the selecting agents (ranging approx. 1.5‐ to 36‐fold) and, in some cases, cross‐resistance to methotrexate (approx. 1.4‐ to 22‐fold), vincristine (1.6‐ to 262‐fold), doxorubicin (Adriamycin, approx. 1.1‐ to 33‐fold) and taxotere (approx. 1.1‐ to 36‐fold). Several of the variants displayed collateral sensitivity to cisplatin. A marked increase in in vitro invasiveness and motility was observed with variants pulsed with mitoxantrone, 5‐fluorouracil, methotrexate, BCNU, cisplatin and chlorambucil. There was no significant change in invasiveness of cells pulsed with VP‐16, vincristine, taxotere or CCNU. All of the pulse‐selected variants showed elevated levels of MDR‐1/P‐gp protein by Western blot analysis, although mdr‐1 mRNA levels were not increased (except for DLKP‐taxotere). In DLKP‐taxotere, MRP1 protein levels were also greatly elevated, but mrp1 mRNA levels remained unchanged. BCRP was upregulated in DLKP‐mitoxantrone at both the mRNA and protein levels. Gelatin zymography, Western blot and RT‐PCR showed that DLKP and its variants secreted MMPs 2, 9 and 13. MMP inhibition assays suggested that MMP‐2 plays a more important role than MMPs 9 and 13 in cell invasion of these DLKP drug‐resistant variants in vitro. These results indicate that drug exposure may induce not only resistance but also invasiveness in cancer cells.

Lensless multispectral digital in-line holographic microscope

An compact multispectral digital in-line holographic microscope (DIHM) is developed that emulates Gabors original holographic principle. Using sources of varying spatial coherence (laser, LED), holographic images of objects, including optical fiber, latex microspheres, and cancer cells, are successfully captured and numerically processed. Quantitative measurement of cell locations and percentage confluence are estimated, and pseudocolor images are also presented. Phase profiles of weakly scattering cells are obtained from the DIHM and are compared to those produced by a commercially available off-axis digital holographic microscope.

Role of matrix metalloproteinases in invasion and metastasis: biology, diagnosis and inhibitors

The processes of tumour invasion and subsequent metastasis are the most lethal aspects of cancer. Whilst many factors are involved, the matrix metalloproteinases (MMPs) have been implicated as key-rate limiting enzymes in the invasive process. This family consisting of eight members of similar structure, can be roughly divided into three groups based on substrate specificity. All are secreted in a latent form and require proteolytic cleavage for activation. The expression of these enzymes is regulated at the transcriptional level by a variety of growth factors and oncogenes. They are also regulated at the protein level by a family of specific inhibitors called the tissue inhibitors of metalloproteinases (TIMPs). Studies in human tumour samples have shown a positive correlation between metalloproteinase expression and metastatic potential. The levels of metalloproteinase expression have been manipulated using molecular biology techniques in several cell lines and shown a similar correlation. These results suggest that an understanding of metalloproteinase expression and proteolytic activity may lead to the development of effective therapeutic agents with the potential to reduce the incidence of metastatic cancer.

MAGE‐D4B is a novel marker of poor prognosis and potential therapeutic target involved in breast cancer tumorigenesis

Melanoma‐associated antigen (MAGE) family members are generally described as tumor‐specific antigens. An association between MAGE‐D4B and breast cancer has yet to be reported and the functional role of the encoded protein has never been established. We performed microarray analysis of 104 invasive breast tumors and matched non‐cancerous breast biopsies. qPCR was used for validation in an independent biobank. To investigate the biological relevance of MAGE‐D4B in breast tumorigenesis, its phenotypic effects were assessed in vitro. Overall, MAGE‐D4B was detected in 43% of tumors while undetected in normal breast tissue. MAGE‐D4B was found to correlate with tumor progression and to be an independent prognostic marker for poor outcome in term of relapse‐free and overall survival, with potential predictive relevance in relation to response to chemotherapy. RNA interference‐mediated knockdown of MAGE‐D4B significantly hampered the invasive properties of Hs578T cells by affecting anchorage‐independent growth, adhesion, migration and invasion affecting anchorage‐independent growth, adhesion, migration and invasion and by modulating expression of invasion‐suppressor gene E‐cadherin.

The design and synthesis of guanosine compounds with in vitro activity against the colon cancer cell line SW480: non-taxane derived mimics of taxol?

In the course of our investigation into the use of taxol as a lead compound to design new molecules with anti-cancer activity, we have synthesized four compounds based on protected guanosine coupled to taxol isoserine side-chain analogues. These analogues show in vitro anti-cancer activity against the colon cancer cell line SW480 that their constituent parts do not.

Digital inline holography of biological specimens

Digital Holography is the technique of numerically reconstructing a three-dimensional (3D) image containing both amplitude and phase information from a two dimensional (2D) interference pattern recorded by the CCD. In this paper, we study the effects of varying the coherence length by using light from two types of sources (1) coherent laser light and (2) spatially filtered incoherent light from a Light Emitting Diode (LED). We present results using both calibrated test objects and biological samples with view to developing a 3D object recognition and classification system.