In this communication we used multiple cultured cell models including genetically engineered, established cDDPR
, and patient-derived cell lines to demonstrate that reduced hCtr1-associated cDDP resistance can be overcome by Cu-lowering agents. We also present supportive results from animal work and bioinformatics from independent ovarian cancer patient dataset demonstrating the role of hCtr1 expression and cDDP sensitivity. The mechanism underlying differential upregulation of hCtr1 in cDDPR
variants over cDDPS
cells by Cu-lowering agents can be explained by the transcriptional regulation of hCtr1 expression within the context of the overall Cu homeostasis regulation network which consists of Cu-Sp1-hCtr1 loop (9
). Here, we demonstrated that Cu homeostatic regulation is confined within upper and lower ranges that constrain the magnitudes of hCtr1 regulation in response to Cu stressed conditions. cDDPR
cells with reduced hCtr1 levels possess high capacity of hCtr1 upregulation (~20-fold) by Cu chelation; whereas in cDDPS
cells which already express high hCtr1 levels, only limited capacity bywhich hCtr1 can be further upregulated (general <2-fold). These findings provide the mechanistic basis for the use of Cu chelation in overcoming cDDP resistance.
Our current findings may explain some seemingly contradictory published results. It has been reported that no induction of rCtr1 expression in the livers and intestines (26
) in the rats fed Cu-deficient diets, despite these organs showed >69% reduced Cu contents as correspondingly compared with those in animals fed Cu adequate diet. In another study, levels of mCtr1 expression were elevated in cervical tumors developed in the HPV16/E2 transgenic mice as compared with those in the cervix of wild-type animals. TM treatment did not further induce mCtr1 expression in these tumors (8
). These results can be explained because these tissues already produce elevated Ctr1 levels. Alternatively, it remains possible that different Ctr1 regulation mechanisms may exist between in vivo
and in vitro
systems, and between tumor tissues and normal counterparts. Further studies are needed to address these important issues.
is located on human chromosome 9q32. Another transcription unit with opposite direction encoding an FK506-binding protein-like transcript is located −201 bp upstream of the transcription start site of hCtr1
). This intergenic sequence controls the expression of both genes in response of Cu bioavailability (our unpublished data). While we found no single nucleotide polymorphism associated with the Sp1 binding sites within this region from the NCBI database; however, whether other genetic polymorphisms in the promoter region of hCtr1
that may contribute to differential regulation of hCtr1 by Cu chelation, particularly in the patient-derived ovarian cancer cells used in this study, remains to be critically evaluated.
Posttranslational regulation of hCtr1 expression by Cu stresses has also been described (18
). Nonetheless, this study demonstrated that hCtr1 mRNA levels are mostly correlated with hCtr1 protein levels which in turn correlated with cDDP sensitivity, although we also observed no strict correlations in some cases. These results suggest that transcriptional regulation may remain the major mechanism of hCtr1 regulation by Cu chelation.
While Cu chelation strategy targets cDDPR
tumor cells with reduced hCtr1 expression. It is important to note that mechanisms of cDDP resistance are multifactoral (28
). Notably, it has been reported that hCtr2 (for Cu storage) and ATP7A/ATP7B (for Cu efflux) can also transport cDDP (5
) and their overexpression is associated with cDDP resistance (5
). The complex cDDP resistance mechanisms suggest the need of using hCtr1 expression level as a guidance for the Cu chelation strategy in Pt drug chemotherapy.
Cu-lowering agents have been used for treating Wilson’s disease resulting from Cu accumulation because of genetic defect in ATP7B
. These chelating agents have also been used in clinical studies by targeting tumor angiogenesis (32
) because many angiogenic modulators require Cu as cofactor (34
). Combination therapy using Cu-lowering agents and Pt drugs may have additive antitumor effects but may also produces additional adverse cytotoxicities (18
). Carboplatin which is the second generation antitumor Pt drug has reduced cytotoxicities in many organs as compared with cDDP. The major adverse event in the trientine/carboplatin trial is myelosuppression but is medically manageable (11
). Several approaches have been proposed for improving the therapeutic index of Cu chelation therapy by enhancing the treatment efficacies of Pt drug through upregulating hCtr1 expression and reducing the unwanted adverse events (18
). Further research in this area may eventually lead to the development of effective strategies for using Cu chelation to combate Pt drug resistance in cancer chemotherapy.