Reversal of resistance to cytotoxic cancer therapies: DHMEQ as a chemo-sensitizing and immuno-sensitizing agent

Abstract

The development of tumor cell resistance to conventional therapeutics is a major clinical problem. There is an urgent need to develop novel therapeutics to overcome resistance and save patients from tumor recurrences. Novel therapeutics are currently being developed based on better understanding of the underlying molecular mechanisms that govern resistance and the identification of targets that control resistance. One of the major factors that controls resistance is the transcription factor nuclear factor kappaB (NF-κB) that has been shown to be constitutively activated in the majority of cancers and is responsible, in large part, for tumor cell survival, growth and direct activation of anti-apoptotic gene products. The development of non-toxic inhibitors of NF-κB activity may result in diminishing the anti-apoptotic threshold of resistant tumor cells and leading to inhibition of tumor cell growth and cell death or sensitization to the apoptotic effects of cytotoxic therapeutics. The novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), selectively prevents the translocation of NF-κB into the nucleus and, hence, prevents its various transcriptional functions. Thus, DHMEQ is unlike many other NF-κB inhibitors that target gene products of the NF-κB pathway and it is also unlike proteasome inhibitors that prevent the degradation of pIκB. DHMEQ is a small molecule shown to be non-toxic in mice and rodents and exerts direct anti-tumor effects in vitro and in vivo as well as significant chemo- and immuno-sensitizing activities in resistant tumor cells. The present review summarizes studies that have used DHMEQ as a novel anti-cancer agent.

Introduction

The emergence of resistant tumor cells is partly due to the expansion of preexisting resistant cells or acquired resistance following conventional cancer treatments such as radiation, chemotherapy, immunotherapy and hormonal therapy. While these anti-cancer therapies may initially be effective against tumors, advanced and metastasized tumors poorly respond to subsequent conventional therapies. The challenges in treating cancer with conventional therapeutics have led to the development of novel molecular approaches aimed at addressing tumor cell resistance. Tumor cells have developed several mechanisms to confer resistance including mutation, gene amplification of a particular gene, DNA repair, upregulation of gene products that inactivate the drug, or upregulation of a multi-drug resistance pump that removes the drugs from the cell. Some drugs, such as the widely used cisplatin [cis-diamminedichloroplatinum (II), CDDP], elicit resistance by overexpressing or inactivating certain oncogenes that enable the cell to withstand drug-induced DNA damage. Because chemotherapeutic drugs and radiation have been shown to exert their cytotoxic effects by apoptosis, the key to chemoresistance lies in the dysregulation of apoptotic pathways.

In order to circumvent some of the problems associated with chemotherapy resistance, several alternative approaches have been proposed, such as cancer vaccines, monoclonal antibodies, recombinant cytokines, adoptive cellular infusions and gene therapy (Hodi and Dranoff, 2006, Tamm, 2006).

Most chemotherapeutic drugs were initially designed to stop DNA replication, but their method of killing is by apoptosis, or programmed cell death, via the regulation of proteins involved in the apoptotic signaling pathways. Immunotherapy, which also kills cells by apoptosis, has been the preferred alternative method because it uses the host's own immune system and is thus less toxic to the organ and is more tumor-selective (Ng and Bonavida, 2002). Current examples of immunotherapy include the targeting of the extrinsic apoptotic pathway using TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) to the cell surface death receptors DR4 and DR5 or by using monoclonal antibodies against DR4 and DR5 (Cretney et al., 2007). However, some tumor cells develop resistance to immunotherapy as well and drug-resistant tumors develop cross-resistance to immunotherapy (Ng and Bonavida, 2002). The resistance to immunotherapy is largely based on the ability of tumor cells to become resistant to apoptosis and on the constitutive upregulation of survival pathways.

Cancer cells are generally in a hyperactivated state for survival and proliferation through the activation of many genes. Some of the survival pathways also regulate apoptotic pathways. Survival hyperactivated pathways include the PI3K/Akt, the p38 MAPK, the Raf/MEK/ERK, and the IKK-α/NF-κB pathways (Faivre et al., 2006, Shukla et al., 2005). The activation of these pathways leads to cell survival and resistance to apoptosis. For example, phosphatidylinositol 3-kinase (PI3K) plays a role in signaling pathways important to cell survival, motility, proliferation, and tissue neovascularization and is upregulated in many cancers (Ghobrial et al., 2005). PI3K activates Akt, which in turn causes phosphorylation of certain proteins that lead to cell survival. For example, phosphorylation of IκB by Akt leads to activation of nuclear factor kappaB (NF-κB) to promote survival. Phosphorylation of Bad by Akt leads to its inactivation and blocking of the apoptotic signal (del Peso et al., 1997).

Nuclear factor kappaB is a transcription factor responsible for regulating many genes associated with apoptosis and inflammation (Karin, 2006). Without stimulation, NF-κB is usually inactive in the cytoplasm. It is activated by extracellular signals such as IL-1, TNF-α, lipopolysaccharide (LPS), lipopeptides, receptor tyrosine kinases and tumor promoters such as phorbol esters (Luo et al., 2005). For example, the signal transduction pathway from the TNF-α receptor to NF-κB activation starts with TNF-α binding to TNF receptor 1 (TNFR1). The cytoplasmic region of TNFR1 contains the death domain that binds to TNFR-associated death domain protein (TRADD). For induction of apoptosis, TRADD then activates the Fas-associated death domain-containing molecule (FADD), which in turn activates caspase 8. For activation of NF-κB, TRADD recruits receptor-interacting protein (RIP) and TNFR-associated factor-2 (TRAF2) (Devin et al., 2000). TRAF2 activates IKK, which induces the phosphorylation of IκB. IκB is an inhibitory protein that is bound to NF-κB. The phosphorylation of IκB induces its release from its complex with NF-κB after ubiquitination and degradation by proteasomes. Liberated NF-κB molecules having NLS (nuclear localization signal) regions then translocate to the nucleus, where they bind to the κB site of DNA. This leads to transcription of survival and anti-apoptotic genes (see Fig. 1, Fig. 2).

The focus of this review will be the importance of the constitutively activated NF-κB pathway in cancer and its role in cell survival, its activation of anti-apoptotic gene products and its use as a therapeutic target to reverse resistance. Finding a non-toxic strategy to block or modulate this survival pathway may result in modulation of the anti-apoptotic pathways and may sensitize the drug/immune-resistant tumor cells to apoptosis by chemotherapeutic/immunotherapeutic drugs (see Fig. 3, Fig. 4).