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Peran TMEPAI (transmembrane prostate androgen-induced) yang diinduksi TGF-β pada resistensi sel kanker payudara triple negative terhadap doksorubisin = The role of TGF-β-induced TMEPAI (transmembrane prostate androgen-induced) in the resistance of triple negative breast cancer cell to doxorubicin.

Latar belakang: Terapi farmakologi kanker payudara triple negative (KPTN) terbatas pada obat sitostatika seperti doksorubisin. Namun, resistensi doksorubisin sulit dihindari. Salah satu jalur signaling yang berperan penting pada resistensi KPTN terhadap doksorubisn adalah TGF-β. TMEPAI (transmembran prostate androgen-induced protein), regulator negatif sekaligus gen target pada jalur TGFβ diduga merupakan salah satu kunci dalam resistensi KPTN terhadap doksorubisin. Metode: Teknik CRISPR-Cas9 digunakan untuk menghilangkan TMEPAI pada galur sel KPTN, BT549. Sel diberi perlakuan TGF-β 2 ng/mL dan doksorubisin selama 24 jam. Pengukuran konsentrasi sitotoksik doksorubisin pada 50% populasi sel (CC50) dilakukan terhadap sel KPTN wildtype (WT) dan knock out (KO). Setelah itu, sel dipanen dan dihitung. Real Time-Polymerase Chain Reaction (RT-PCR) dan western blot (WB) digunakan untuk mengukur tingkat ekspresi penande proliferasi, apoptosis, EMT, dan transporter. Selain itu, SMAD yang terfosforilasi dan aktivitas PI3K/Akt juga dipelajari. Hasil: Galur sel yang tidak memiliki TMEPAI (KO) berhasil diperoleh dari sel KPTN, BT549. Sel WT terbukti lebih resistan terhadap doksorubisin dibandingkan sel KO yang ditunjukkan dengan peningkatan CC50 dan Ki-67. TMEPAI menurunkan efek apoptosis doksorubisin dengan memodulasi ekspresi bcl-2 dan kaspase-3, namun tidak kaspase-9 dan bax. TMEPAI mengurangi efek doksorubisin dengan menekan fosforilasi SMAD. Namun TMEPAI meningkatkan penghambatan PI3K/Akt oleh doksorubisin. TMEPAI juga meningkatkan EMT dan transporter efluks yang diinduksi oleh doksorubisin. Kesimpulan: TMEPAI berperan dalam resistensi sel KPTN terhadap doksorubisin melalui aktivasi jalur sinyal TGF-β non-canonical beserta protein dan gen targetnya.
Kata Kunci: Doksorubisin, TMEPAI, Kanker payudara triple negative, TGF-β


Background: Pharmacological therapy for triple negative breast cancer (TNBC) is limited to cytostatic drugs such as doxorubicin. However, resistance to doxorubicin is unavoidable. In TNBC, TGF-ß signaling pathway plays important role in doxorubicin resistance. TMEPAI, as a negative regulator and target gene in TGF-ß pathways, are thought to have vital role in determining the response of TNBC to doxorubicin. Methods: CRISPR-Cas9 system was used to knock-out TMEPAI in BT549 triple negative breast cancer cells. The cells were treated with TGF-β 2 ng/mL and doxorubicin for 24 hours. Doxorubicin 50% cytotoxic concentration (CC50), was determined in wildtype and knock-out cells. Afterwards, the cells were harvested and counted. RT-PCR and western blots were used to analyze the expression levels of proliferation, apoptosis, EMT markers and transporters. Furthermore, Smad phosphorylation and PI3K/Akt activity were also determined. Results: Knock-out TMEPAI were successfully established in BT549 triple negative breast cancer cells. TMEPAI wild type TNBC cells were shown to be more resistant to doxorubicin as compared to KO-TMEPAI cells, as demonstrated by the increasing of CC50 and Ki-67 expression. TMEPAI reduce the apoptotic effect of doxorubicin by modulating bcl-2, caspase-3, but not caspase-9 and bax. TMEPAI attenuates the effect of doxorubicin by suppressing Smad phosphorylation. However, TMEPAI enhanced the inhibition of PI3K/Akt by doxorubicin. TMEPAI is also shown to magnify EMT markers and drug efflux transporters induced by doxorubicin. Conclusion: TMEPAI plays a role in the resistance of triple negative breast cancer cell to doxorubicin by activating TGF-ß non canonical signaling pathways and its downstream genes and proteins.
Keywords: Doxorubicin, TMEPAI, Triple-negative breast cancer, TGF-β