Novel Drug Resistance-associated Protein and new plant derived modulators for overcoming drug resistance in human tumor cells

Cheppail Ramachandran Ph.D., Steven J. Melnick, MD, Ph.D., Thangaiyan Rabi, Ph.D., and Enrique Escalon, MD,

Research Institute, Miami Children's Hospital, Miami, FL 33155

Aim: Multidrug resistance (MDR) is one of the major limiting factors for the success of chemotherapy in human malignancies. Investigations were performed to identify novel drug resistance-associated genes and novel modulators for overcoming drug resistance in tumor cells. Method: We have used molecular subtractive hybridization technique for identifying and cloning novel cDNAs uniquely expressed in drug resistant cell line. One of the clones was characterized and its role in conferring drug resistance phenotype was analyzed by transfection studies. We have also identified two plant-derived drugs for reversing drug resistance in human tumor cells by flow cytometric and photolabeling techniques. Results: We have identified five novel cDNA sequences (pMEL487, pMEL884, pMEL1006, pMEL1082 and pMEL1094) uniquely expressed in drug resistant human melanoma cell line (FCCM-9) by subtractive hybridization against drug sensitive cell line (HM-1). The full-length (1.3 Kb) novel drug resistance-associated protein (DRP) was obtained by screening a 5’-stretch human breast carcinoma library. DRP gene codes for a 1.1 kb mRNA species, which is overexpressed in drug resistant cell lines such as FCCM-9, CEM/VLB, and MCF-7/TH without gene amplification. DRP has sites for N-linked glycosylation, N-myristoylation, protein kinase C and caesin kinase II phosphorylation, ATP-binding, plasma membrane attachment and distant similarity at the 3’-end with cyclophilin A-binding cyclophilin protein. DRP is overexpressed in kidney, liver and lung, but not in skin, breast, mucosa and artery tissues. Transfection of CEM and NIH3T3 cell lines with DRP gene conferred drug resistance (9-10 fold) phenotype. Polyclonal antibody (DRP81) raised against a 22 aminoacid oliogopeptide hybridized with a 50 kDa protein in DRP-positive cell lines. In our studies on MDR, we have also identified two novel drugs of plant origin, amooranin and thaliblastine, capable of modulating anthracyline resistance in tumor cells. While thaliblastine is an alkaloid isolated from Thalictrum species, amooranin is a triterpenoid from Amoora rohituka, a tropical medicinal tree gown wild in India. Even though both these compounds are structurally unrelated, they are cytotoxic in tumor cells by inducing a G2 arrest. Furthermore, both these drugs overcome doxorubicin resistance by increasing cellular drug accumulation. Amooranin also induced apoptosis by activation of general caspases, especially inducer caspase-8. Conclusions: The newly cloned DRP gene may be a good prognostic marker for the drug resistance phenotype of a large number of human tumors that can not be explained by the already known drug transporters and markers. The newly identified efflux blocking drugs, amooranin and thaliblastine might have great potential for pre-clinical development for overcoming multidrug resistance in human malignancies

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Novel Therapies, Part 1.

This presentation received an honorable mention in our poster contest and was recognized with the Symposium Presidents' Award for Scientific Excellence.