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Bantari WK Wardhani,1,2 Melva Louisa,3 Yukihide Watanabe,4 Rianto Setiabudy,3 Mitsuyasu Kato4 1Biomedical Sciences, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia; 2Department of Pharmacology, Faculty of Military Pharmacy, Indonesia Defense University, West Java, Indonesia; 3Department of Pharmacology and Therapeutics, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia; 4Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, Faculty of Medicine, University of Tsukuba, Tsukuba, JapanCorrespondence: Melva LouisaDepartment of Pharmacology and Therapeutics, Faculty of Medicine Universitas Indonesia, Jl. Salemba Raya No. 6, Jakarta Pusat, Jakarta, 10430, IndonesiaTel +62 21 3193-481Email melva.louisa@gmail.comIntroduction: Epithelial–mesenchymal transition (EMT) and overexpression of drug efflux transporters have been reported to cause doxorubicin resistance. Our previous study indicated that TMEPAI (transmembrane prostate androgen-induced protein) attenuated doxorubicin sensitivity in triple-negative breast cancer cells. However, how TMEPAI contributes to doxorubicin resistance in TNBC remains unclear. Thus, the present study aimed to elucidate the mechanism of TMEPAI in doxorubicin resistance in triple-negative breast cancer cells.Methods: We used BT549, triple-negative cells wild type (WT), and BT549 TMEPAI knock-out. Both cells were treated with TGF-β 2 ng/mL for 24 hours, followed by TGF-β 2 ng/mL and doxorubicin 12.9 nM for another 24 hours. Afterward, the cells were harvested and counted. Cells were further lysed and used for RT-PCR and Western blot analysis. We determined the expression levels of proliferation, apoptosis, EMT markers, and drug efflux transporters. Additionally, we investigated the expressions of PI3K as well as SMAD3 and AKT phosphorylation.Results: TNBC cells were shown to be less sensitive to doxorubicin in the presence of TMEPAI. TMEPAI was shown to alleviate the mRNA expressions of apoptosis markers: Bax, Bcl2, Caspase-3, and Caspase-9. Our results indicated that the presence of TMEPAI greatly amplifies EMT and increases drug efflux transporter expressions after doxorubicin treatment. Furthermore, our findings demonstrated that TMEPAI reduced the action of doxorubicin in inhibiting SMAD3 phosphorylation. TMEPAI was also shown to modify the effect of doxorubicin by reducing PI3K expressions and Akt phosphorylation in triple-negative breast cancer cells.Conclusion: Our findings indicate that TMEPAI promotes EMT and drug efflux transporters at least in part by shifting doxorubicin action from SMAD3 phosphorylation reduction to PI3K/AKT inhibition in triple-negative breast cancer cells.Keywords: PMEPAI, TGF-β, SMAD3, vimentin, E-cadherin, drug efflux transporters

Dewi Selvina Rosdiana,1 Rianto Setiabudy,1 Rizka Andalusia,2,3 Djajadiman Gatot,4 Melva Louisa,1 Saptawati Bardosono,5 Instiaty Instiaty1 1Department of Pharmacology and Therapeutics, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; 2Dharmais National Cancer Hospital, Jakarta, Indonesia; 3Drug Registration Directorate, National Agency for Drug and Food Control, Jakarta, Indonesia; 4Division of Hematology-Oncology, Department of Pediatrics, Faculty of Medicine, Universitas Indonesia/Cipto Mangunkusumo Hospital, Jakarta, Indonesia; 5Department of Nutrition, Faculty of Medicine, University of Indonesia, Jakarta, IndonesiaCorrespondence: Dewi Selvina RosdianaDepartment of Pharmacology and Therapeutics, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Jakarta, 10430, IndonesiaTel +62 21 31930481Fax +62 21 3920947Email dewiselvina79@gmail.comPurpose: Hematotoxicity monitoring in children with acute lymphoblastic leukemia (ALL) is critical to preventing life-threatening infections and drug discontinuation. The primary drug that causes hematotoxicity in ALL children is 6-mercaptopurine (6-MP). Genetic variability of the drug-metabolizing enzymes of 6-MP, thiopurine S-methyltransferase (TPMT), is one factor that might increase the susceptibility of children to hematotoxicity. The present study aimed to determine the variability in TPMT genotypes and phenotypes and its association with the occurrence of hematotoxicity in ALL children in maintenance therapy.Patients and Methods: A cross-sectional study was conducted at Cipto Mangunkusumo and Dharmais National Cancer Hospital, Jakarta, Indonesia, from June 2017 to October 2018. We included ALL patients, 1– 18 years, who were receiving at least one month of 6-MP during maintenance therapy according to the Indonesian protocol for ALL 2013. Direct sequencing was used to determine TPMT*3A, *3B, and *3C genotypes, and LC-MS/MS analysis was performed to measure the plasma concentrations of 6-MP and its metabolites. Association analysis between the TPMT genotype and hematotoxicity was evaluated using the unpaired t-test or Mann–Whitney’s test.Results: The prevalence of neutropenia, anemia, and thrombocytopenia in ALL children during maintenance therapy was 51.9%, 44.3%, and 6.6%, respectively. We found a low frequency of TPMT*3C, which is 0.95%. No association was found between hematotoxicity and TPMT genotypes or age, nutritional status, serum albumin levels, risk stratification, the daily dose of 6-MP, and cotrimoxazole co-administration. However, hematotoxicity was associated with 6-methylmercaptopurine (6-MeMP) plasma concentrations and the ratio 6-MeMP/6-thioguanine (6-TGN). We also found no association between TPMT genotypes and TPMT phenotypes.Conclusion: The 6-MeMP/6-TGN ratio is associated with hematotoxicity in ALL children during maintenance therapy but is not strong enough to predict hematotoxicity.Keywords: thiopurine methyltransferase, mercaptopurine, methylmercaptopurine, thioguanine, neutropenia

Bantari WK Wardhani, 1 Meidi Utami Puteri, 2 Yukihide Watanabe, 2 Melva Louisa, 3 Rianto Setiabudy, 3 Mitsuyasu Kato 2 1Doctoral Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; 2Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences and Faculty of Medicine, University of Tsukuba, Tsukuba, Japan; 3Department of Pharmacology and Therapeutics, Faculty of Medicine, Universitas Indonesia, Jakarta, IndonesiaCorrespondence: Melva LouisaDepartment of Pharmacology and Therapeutics, Faculty of Medicine Universitas Indonesia, Jl Salemba Raya No. 6, Jakarta Pusat, Jakarta 10430, IndonesiaTel +62 21 31930481Email melva.louisa@gmail.comPurpose: Triple-negative breast cancer (TNBC) is a refractory type of breast cancer with poor prognosis and limited choice for treatment. Previous studies had shown that TNBC has high expressions of transmembrane prostate androgen-induced protein (TMEPAI). TMEPAI was known to be induced by TGF-β/Smad signaling and have tumorigenic functions that converting TGF-β from tumor suppressor to tumor promoter and inducing epithelial–mesenchymal transition (EMT). Therefore, we aimed to define the role of TMEPAI in triple-negative breast cancer cells treatment using several anti-cancers in the presence of TGF-β.Methods: TMEPAI-knock out (KO) was carried out in a triple-negative breast cancer cell, BT549. TMEPAI editing was developed using the CRISPR-Cas9 system using two combinations of sgRNA to remove exon 4 of the TMEPAI gene entirely. Genotyping and proteomic analysis were performed to check the establishment of the TMEPAI-KO cells. Wild type (WT) and KO cells were used to determine inhibitory concentration 50% (IC 50) of several anti-cancers: doxorubicin, cisplatin, paclitaxel, and bicalutamide in the presence of TGF-β treatment.Results: KO cells were successfully established by completely removing the TMEPAI gene, which was proven in genomic and proteomic analysis. Further, in TMEPAI-KO cells, we found a significant reduction of IC 50 for doxorubicin and paclitaxel, and minimal effects were seen for cisplatin and bicalutamide. Our findings suggest that TGF-β-induced TMEPAI attenuates the response of TNBC to doxorubicin and paclitaxel, but not to cisplatin and bicalutamide.Conclusion: TGF-β induced TMEPAI contributes to the reduced response of TNBC treatment to doxorubicin and paclitaxel, but minimal on cisplatin and bicalutamide. Further study is needed to confirm our findings in other growth factor-induced cells, as well as in in vivo model.Keywords: TMEPAI, TGF-β, doxorubicin, paclitaxel