Regulasi Apoptosis oleh Kayu Bajakah dan Daun Cakar Ayam: Studi Molekuler Model Khamir dan Prediksi Aktivitas Antikanker

Abstract

Secara terpisah, kayu bajakah (Uncaria sp.) dan daun cakar ayam (Selaginella doederleinii Hieron) telah digunakan sebagai obat kanker tradisional. Riset sebelumnya menunjukkan aktivitas antiproliferasi dari kombinasi fraksi etil asetat kayu bajakah dan daun cakar ayam (FEA-BC), tetapi belum diketahui mekanisme molekuler yang mendasari kejadian ini. Riset ini bertujuan menentukan potensi antikanker FEA-BC melalui regulasi apoptosis pada model sel Saccharomyces cerevisiae dengan mengevaluasi hallmark apoptosis. Selain itu, riset ini juga memprediksi potensi FEA-BC dalam menghambat protein antiapoptosis manusia, yakni epidermal growth factor receptor (EGFR) dan cellular FLICE-inhibitory protein (cFLIPs).

Riset diawali dengan partisi bertingkat untuk memperoleh fraksi etil asetat, kemudian FEA-BC diformulasikan dengan rasio 1:1. Induksi apoptosis dilakukan dengan menginkubasi sel khamir dalam FEA-BC pada konsentrasi 5, 10, dan 100 ppm. Setelah 24 jam, kultur ditebar pada media nonfermentatif dengan glukosa minimal lalu jumlah koloni petite dihitung setelah 48 jam. Parameter yang dianalisis meliputi aktivitas mitokondria (rodamin B), perubahan permukaan sel (scanning electron microscope), eksternalisasi fosfatidilserin (Annexin V/FITC-PI), fragmentasi DNA (TUNEL), serta regulasi gen apoptosis FSH3, AIF1, dan YCA1. Di sisi lain, prediksi inhibisi protein antiapoptosis manusia EGFR dan cFLIPs dilakukan melalui penambatan dan dinamika molekuler.

Konsentrasi tertinggi FEA-BC, yakni 100 ppm menyebabkan kerusakan mitokondria ditandai dengan pembentukan koloni petite tertinggi (85%) dan penurunan intensitas pendaran mitokondria. Konsentrasi tersebut mengubah permukaan sel, menstimulasi eksternalisasi fosfatidilserin, serta menyebabkan kerusakan DNA yang masif. Konsentrasi 100 ppm meningkatkan ekspresi gen FSH3 (6 kali), AIF1 (2 kali), dan YCA1 (3 kali) yang meregulasi persinyalan apoptosis pada sel khamir. Meskipun demikian, konsentrasi 10 dan 5 ppm tetap mampu menginduksi apoptosis meskipun dengan aktivitas yang lebih rendah.

Selain itu, ditemukan peningkatan Analisis in silico mengidentifikasi 10-hidroksifeoforbid dan asam selaginelat sebagai inhibitor terbaik EGFR, tetapi kurang stabil secara dinamik (RMSD >3 Å), sedangkan asperglausida dan sotetsuflavon yang relatif menginhibisi cFLIPs memiliki kestabilan yang baik (RMSD <3 Å). Dari riset ini dapat disimpulkan bahwa FEA-BC berpotensi sebagai antikanker melalui peningkatan apoptosis jalur perusakan mitokondria pada sel model khamir dengan kemampuan yang dose dependent dibuktikan oleh terpenuhinya hallmark apoptosis. Selain itu, FEA-BC diprediksi mampu memodulasi jalur ekstrinsik apoptosis manusia dengan menghambat protein antiapoptosis yang terlibat.

Separately, bajakah wood (Uncaria sp.) and cakar ayam leaves (Selaginella doederleinii Hieron) have been traditionally used as anticancer herbal medicines. Previous studies reported the antiproliferative activity of the combination of ethyl acetate fractions of bajakah wood and cakar ayam leaves (FEA-BC), but the underlying molecular mechanism remains unclear. This study aimed to determine the anticancer potential of FEA-BC through apoptosis regulation in the Saccharomyces cerevisiae cell model by evaluating apoptosis hallmarks. In addition, this study predicted the potential of FEA-BC to inhibit human antiapoptotic proteins, namely epidermal growth factor receptor (EGFR) and cellular FLICE-inhibitory protein (cFLIPs).

The study began with sequential partitioning to obtain the ethyl acetate fraction, followed by FEA-BC formulation at a 1:1 ratio. Apoptosis induction was performed by incubating yeast cells with FEA-BC at concentrations of 5, 10, and 100 ppm. After 24 hours, cultures were plated on nonfermentative medium with minimal glucose, and petite colonies were counted after 48 hours. The analyzed parameters included mitochondrial activity (rhodamine B), cell surface changes (scanning electron microscope), phosphatidylserine externalization (Annexin V/FITC-PI), DNA fragmentation (TUNEL), and apoptosis gene regulation (FSH3, AIF1, and YCA1). Meanwhile, inhibition of human antiapoptotic proteins EGFR and cFLIPs was predicted through molecular docking and dynamics.

The highest FEA-BC concentration (100 ppm) caused mitochondrial damage, indicated by the highest petite colony formation (85%) and decreased mitochondrial fluorescence intensity. This concentration also altered cell surface morphology, stimulated phosphatidylserine externalization, and caused massive DNA damage. The 100 ppm concentration increased the expression of apoptosis-related genes FSH3 (6-fold), AIF1 (2-fold), and YCA1 (3-fold), which regulate apoptosis signaling in yeast cells. However, lower concentrations (10 and 5 ppm) were still able to induce apoptosis though with lower activity.

In addition, in silico analysis identified 10-hydroxypheophorbide and selaginellate acid as the best EGFR inhibitors, although they showed lower dynamic stability (RMSD >3 Å), while asperglauside and sotetsuflavone, which inhibited cFLIPs, exhibited good stability (RMSD <3 Å). This study concludes that FEA-BC has potential as an anticancer agent by enhancing mitochondrial pathway-mediated apoptosis in yeast model cells in a dose-dependent manner, as evidenced by the fulfillment of apoptosis hallmarks. Furthermore, FEA-BC is predicted to modulate the human extrinsic apoptosis pathway by inhibiting antiapoptotic proteins involved in cancer progression.