The ability of neoplastic cells to adapt and maintain viability in the presence of cytotoxic agents is a major characteristic of drug resistance.  Our research program has investigated the processes that initiate and sustain drug resistance in tumors of neuroectodermal and mesodermal origin.  A primary objective of these studies has been to define the molecular, enzymatic, genetic and epigenetic mechanisms that underlie development of drug resistance and to identify strategies that will prevent or reverse these adaptive responses.

 

Research projects currently in progress are summarized below:

 

          (Investigators:  B. L. Mirkin, A. Rebbaa, P. Chou and S. Clark)

 

An adaptive response observed in different types of cancer cells incubated with chemotherapeutic drugs is the secretion of molecules that exert a cytoprotective effect on drug-sensitive cells by inhibiting apoptosis and other cytotoxic responses.  Our findings have shown that the heparin-binding proteins, midkine and pleiotrophin, are selectively secreted by neuroblastoma and other neoplastic cells, depending on whether they are drug-sensitive or drug-resistant.  The drug-sensitive cells secrete primarily pleiotrophin and very small amounts of midkine, whereas the drug-resistant cells secrete extremely high concentrations of both neurotrophins.  A schematic diagram describing the signal transduction pathways by which secreted midkine inhibits apoptosis is shown below (Figure 1).  In vitro studies utilizing co-culture systems and small inhibitory RNA (siRNA) for midkine and pleiotrophin have confirmed that drug-resistant cells, but not drug-sensitive cells, are able to humorally mediate cytoprotection in drug-sensitive cells due to the secretion of midkine.  A major goal of this project will be to demonstrate that the humoral transfer of drug resistance occurs in vivo using human neuroblastoma cells explanted into SCID mice.

 

 

 

 

 

          (Investigators:  A. Rebbaa, F. Chu and X. Zheng)

 

This laboratory has demonstrated that cellular ability to proliferate in a toxic environment is a prerequisite for the formation of drug resistant tumors, leading to the hypothesis that targeting the genes that control irreversible proliferation arrest (also called senescence) would represent a rational approach to overcome resistance of cancer cells to chemotherapy.  Previous work from this laboratory indicated that inhibition of senescence (by over-expression of the longevity gene, Sirt1), rendered cancer cells resistant to drugs through induction of the multidrug resistance gene mdr1.  Inversely, forcing drug resistant cancer cells to undergo senescence through inhibition of the lysosomal cathepsin L induced senescence and suppressed drug resistance in vitro and in vivo. Based on these findings, the hypothesis was made that drug resistant cancer cells must have acquired an elaborate anti-senescence defense, and thus, would represent a compelling source for the search of novel anti-senescence genes. The discovery of such genes could have application not only for the treatment of cancer but also to prevent other aging associated diseases. The findings are also relevant to the understanding of the intricate relationship between cancer and aging in general. Genetic functional screenings are currently underway to identify and characterize such anti-senescence genes.

 

Investigations to understand the mechanisms of drug-induced cellular senescence, apoptosis and autophagy as well as the influence of cancer cell micro-environment on chromatin remodeling and its consequences on development of drug resistance are also carried out in this laboratory.

 

          (Investigators:  B. L. Mirkin, D. Garrett and S. Clark)

 

The efflux transporters P-glycoprotein (P-gp) and multi-drug resistance protein (MRP-1), which pump drugs out of cancer cells, are present in human neuroblastoma cells that have become resistant to specific chemotherapeutic agents.  We have shown that the uptake and concentration of anti-cancer drugs in drug-resistant cells was significantly decreased when compared to that of drug-sensitive cells.  This was reversed by treating drug-resistant tumor cells with verapamil, an agent which greatly increased the uptake of cytotoxic drugs and restored virtually all anti-cancer activity.  These studies have been expanded in an effort to identify novel pharmacologic compounds that effectively antagonize the activity of efflux transporters.  A model describing the cellular localization and actions of efflux transporters in neuroblastoma cells is presented in Figure 2.

 

 

 

 

          (Investigators:  M. B. Madonna, Y. Y. Qiu, R. Kabre and B. L. Mirkin)

 

Recent studies have demonstrated that the transcription of total caspase 3 exerts an important impact on apoptosis.  The putative role of caspase 3 transcription in apoptosis has never been linked to effects induced by EGF.  Incubating wild-type neuroblastoma cells with EGF for 24 hours increased total caspase 3 protein expression and a similar but attenuated response was seen in doxorubicin resistant cells.  This observation demonstrated that EGF can modulate the expression of total caspase 3 during apoptosis.  Further study of this phenomenon will clarify the link between proteins with seemingly opposed biological functions, i.e., EGF, a growth factor with mitogenic actions, and caspase 3, an effector in apoptosis.

 

 

 

            (Investigators:  B. L. Mirkin, S. Nguyen and D. D. Smith)

 

In conjunction with the Institute of Nanotechnology and Department of Chemistry of Northwestern University, collaborative studies have been carried out to determine the potential cytotoxic activity of drug conjugates consisting of doxorubicin coupled to nanoparticles with unique types of chemical linkages.  The theoretical basis for this study is to determine whether a specific type of bond between doxorubicin and a nanoparticle can affect cytotoxic potency of the conjugate by facilitating cleavage within the cell and/or release into specific subcellular organelles.  This concept is based on data showing that drug resistant cells have a lower intracellular pH, not only in the cytosol, but in their subcellular organelles.  Consequently, linkages that are disrupted at an acid pH would tend to release the free drug to a greater extent, making it available for increased entry into the nucleus.

 

Thus far, seven analogues are being synthesized and initial data has shown that the cytotoxic potency of the doxorubicin conjugate exceeded that of non-conjugated doxorubicin in drug resistant cells.

 

          (Investigators:  H. Li, S. Clark and B. L. Mirkin)

 

The establishment of stable tumor cell lines that can be identified by fluorescent proteins is in progress.  In contrast to conventional transfections, these cells are infected using viral constructs which undergo genomic integration.  A stable GFP (green) drug-sensitive human neuroblastoma cell (SKN-SH) line has been developed and is being used for in vitro and in vivo experimental studies.  In addition, an RFP (red) drug-resistant human neuroblastoma cell line (SKNSH-DOX6) is being generated and will be used for similar studies.  The goal of this project is to facilitate morphologic analyses in tumor cell models using transgenic mice as the recipients of GFP and RFP labeled human neuroblastoma cell heterografts.

 

3.         Antagonism of Drug Efflux Transporters by Cyclo-oxygenase Inhibitors.

            (Investigators:  B. L. Mirkin, A. Rasmuson, J. Johnson, P. Kogner and S. Clark)

 

The development of novel inhibitors of MRP-1 drug efflux transporters is in progress with members of the Childhood Cancer Research Unit of the Department of Women’s and Child’s Health in Stockholm, Sweden.  We are investigating the potential utility of using cyclo-oxygenase inhibitors to prevent the efflux transport of drugs in resistant cell lines.