Bioprospeksi Khamir Probiotik dangke sebagai Antikanker Payudara melalui Pendekatan Genomik, Metabolomik, dan Uji Sitotoksisitas terhadap Sel MCF-7

Abstract

Kanker payudara masih merupakan kanker ganas dengan prevalensi tertinggi pada perempuan. Terapi konvensional seperti kemoterapi dan terapi hormon sering menimbulkan efek samping berat, sehingga diperlukan eksplorasi agen terapeutik alternatif berbasis bahan alam yang lebih aman. Khamir probiotik dari produk fermentasi tradisional diketahui berpotensi menghasilkan senyawa bioaktif antikanker. Penelitian ini bertujuan mengungkap potensi dua isolat khamir probiotik dangke, yaitu SDR 2.37.2 (Pichia norvegensis) dan SDR 2.37.4 (Kluyveromyces marxianus), sebagai kandidat agen terapeutik antikanker payudara melalui pendekatan multi-omik terintegrasi. Metode meliputi karakterisasi morfologi (SEM), Whole Genome Sequencing (WGS) pada SDR 2.37.4, identifikasi metabolit ekstraseluler (GC-MS/LC-HRMS), farmakologi jaringan dan penambatan molekuler terhadap protein target (ESR1), serta uji MTT pada sel MCF-7. Hasil WGS menunjukkan genom SDR 2.37.4 memiliki total panjang 10,76 Mb dengan kandungan GC 40,18% dan tingkat kelengkapan 96,7% (BUSCO), serta teridentifikasi sebagai Kluyveromyces marxianus dengan nilai OrthoANI 98,89%. Genom ini tidak mengandung gen resistensi antimikroba maupun faktor virulensi, tetapi memiliki empat BGCs terpenoid atau poliketida yang homolog dengan clavaric acid (78%) dan squalestatin S1 (53%), serta gen fungsional pendukung sifat probiotik meliputi toleransi panas (HSP82, SSA4), stres oksidatif (SOD1, SOD2), toleransi asam dan garam empedu (HOG1, PBS2), serta adhesi (FLO5, ALA1). Profil metabolomik berhasil mengidentifikasi berbagai senyawa kandidat bioaktif, dengan farmakologi jaringan menentukan ESR1, MDM2, dan SIRT1 sebagai protein target utama. Penambatan molekuler terhadap ESR1 menunjukkan empat senyawa dengan afinitas tertinggi (energi ikatan = -6 kkal/mol), yaitu methylmalonic acid (-6,867 kkal/mol), 4-methyl-2H,3H,4H-pyrido[3,2-b][1,4]oxazine-7-carboxylic acid (-6,349 kkal/mol), succinic anhydride (-6,102 kkal/mol), dan pyruvic acid (-6,041 kkal/mol). Uji MTT menunjukkan ekstrak kedua isolat memiliki IC50 >1000 µg/mL (aktif lemah menurut kriteria NCI), namun pada konsentrasi 1000 µg/mL SDR 2.37.2 menekan viabilitas sel hingga 53,02%, lebih baik dibandingkan SDR 2.37.4 (58,96%). Simpulan penelitian ini yaitu SDR 2.37.4 memiliki profil genomik yang aman dan mendukung sifat probiotik serta produksi senyawa bioaktif, sedangkan SDR 2.37.2 menunjukkan aktivitas sitotoksik relatif lebih baik meskipun tergolong aktif lemah. Integrasi data genomik, metabolomik, farmakologi jaringan, dan validasi in vitro secara konsisten mendukung pengembangan kedua isolat sebagai kandidat agen antikanker payudara yang aman, dengan potensi lebih diarahkan pada mekanisme preventif yang memerlukan validasi lebih lanjut secara in vivo.

Breast cancer remains the most prevalent malignancy in women. Conventional therapies such as chemotherapy and hormonal therapy often cause severe side effects, necessitating the exploration of safer alternative therapeutic agents from natural sources. Probiotic yeasts from traditional fermented products are known to have the potential to produce anticancer bioactive compounds. This study aimed to uncover the potential of two dangke probiotic yeast isolates, SDR 2.37.2 (Pichia norvegensis) and SDR 2.37.4 (Kluyveromyces marxianus), as breast cancer therapeutic candidates through an integrated multi-omics approach. Methods included morphological characterization (SEM), Whole Genome Sequencing (WGS) on SDR 2.37.4, identification of extracellular metabolites (GC-MS and LC-HRMS), network pharmacology and molecular docking against target proteins (ESR1), and MTT assay on MCF-7 cells. WGS results revealed that the SDR 2.37.4 genome had a total length of 10.76 Mb with 40.18% GC content and 96.7% completeness (BUSCO), and was identified as K. marxianus with an OrthoANI value of 98.89%. The genome contained no antimicrobial resistance or virulence factor genes, but harbored four Biosynthetic Gene Clusters (BGCs) producing terpenes and polyketides with similarity to clavaric acid (78%) and squalestatin S1 (53%). Functional genes supporting probiotic properties were also detected, including heat tolerance (HSP82, SSA4), oxidative stress response (SOD1, SOD2), acid and bile salt tolerance (HOG1, PBS2), and adhesion (FLO5, ALA1). Metabolite profiling identified various bioactive compound candidates. Network pharmacology identified ESR1, MDM2, and SIRT1 as the main target proteins, with molecular docking against ESR1 revealing four compounds with the highest affinity (binding energy = -6 kcal/mol): methylmalonic acid (-6.867 kcal/mol), 4-methyl-2H,3H,4H-pyrido[3,2-b][1,4]oxazine-7-carboxylic acid (-6.349 kcal/mol), succinic anhydride (-6.102 kcal/mol), and pyruvic acid (-6.041 kcal/mol). The MTT assay revealed that both isolates exhibited IC50 > 1000 µg/mL (weakly active according to NCI criteria). However, at 1000 µg/mL, SDR 2.37.2 suppressed cell viability to 53.02%, slightly better than SDR 2.37.4 (58.96%). This study concludes that SDR 2.37.4 possesses a genomically safe profile supporting probiotic properties and bioactive compound production, while SDR 2.37.2 exhibits relatively better cytotoxic activity, although both are classified as weakly active. The integration of genomic, metabolomic, network pharmacology, and in vitro validation data consistently supports the development of both isolates as safe anti-breast cancer candidates, with their potential directed toward a preventive mechanism that requires further in vivo validation.