CANINE TRANSMISSIBLE VENEREAL TUMOR: MULTIDRUG RESISTANCE ASSOCIATED GENE AND PROTEIN EXPRESSIONS DURING VINCRISTINE SULFATE ADMINISTRATION

Abstract

The thesis was aimed to: 1) retrospectively find internal and external factors affecting treatment outcome after administrating vincristine sulfate which is the chemotherapeutic drug of choice for canine transmissible venereal tumor (cTVT), 2) study mechanism of drug resistance via ABC-transporter (p-glycoprotein (P-gp), multidrug resistance protein-1 (MRP1) and multidrug resistance protein-2 (MRP2), and 3) clinically evaluate the use of l-asparaginase as a combined drug with vincristine sulfate in animals tolerated to previous vincristine administration. Factors were retrieved from medical records of 157 dogs infected with cTVT and divided into three categories; host general background, tumor details, and treatment details. The male and female dogs (75 and 82 dogs, respectively) aged (6.4±3.61 years) affected with genital and extra-genital cTVT tumor masses of various diameters, and treated with vincristine (5.0±2.6 shots) during various seasons were analyzed for tumor mass complete remission (CR) or partial remission (PR). The age, gender and breed had no effect on treatment outcome or number of vincristine shots. The tumors at the genital area were prone to CR than the others (P = 0.08). The tumors of diameter >6 cm required higher number of vincristine shots than that of diameter <2 cm (P = 0.05). Moreover, there was a tendency that CR was observed with lower average daily ambient temperature at 60 days after enrolled (Temp60) and lower maximal average daily ambient temperature (MaxTemp60) than the PR cases (P = 0.08 and P = 0.07, respectively). The results contributed to the prognosis of the treatment with vincristine sulfate particularly when the treatment is influenced by tumor site, tumor size, and climate. The second study was performed in 12 cTVT affected dogs during vincristine treatments and 4 dogs resistant to vincristine therapy. Tumor samples were submitted to mRNA quantification via quantitative realtime polymerase chain reaction (qPCR), immunolocalization by immunohistochemistry (IHC) and protein expression level analysis by western blot analysis (WB). The MDR1, MRP1 and MRP2 mRNAs and proteins were detected in all samples. During vincristine treatments, there was no significant difference in all mRNA expressions. The lower expression of P-gp was observed at week3 after the start of treatment whereas MRP1 and MRP2 proteins were not different. In comparison with the untreated group, the resistance group demonstrated lower MDR1 mRNA expression leading to the lower confirmed by IHC and WB. MRP2 protein (IHC) was significant higher in the resistant group but MRP1 protein expression was no significant difference between the two groups. The results suggested that MRP2 likely involves multidrug resistance mechanism of cTVT. Drugs transported through MRP2-related mechanism should be avoided in the cTVT resistance cases. The third part of this study was aimed to combine l-asparaginase with the standard treatment in resistance cTVT. The resistance cases were composed of three cases that had been treated with vincristine and did not achieve a complete remission. L-asparaginase at 10,000IU/m2 BSA was combined with vincristine injections. All cases were achieved complete remission and the recurrence was not observed six months after complete treatment. This finding suggested that L-asparaginase in combination with vincristine at every 2 weeks provided a promising result in the resistance cTVT with minimal side effect when compared with the other anticancer drugs suggested previously. In conclusion, tumor diameter, tumor location and daily ambient temperature during treatment might influence the treatment outcome in cTVT. The MRP2 might be involved in the vincristine resistance mechanism in cTVT and the combination of L-asparaginase with vincristine is an alternative treatment for vincristine-resistance cTVT.