Anticancer Properties of Alpha Solanine against Breast Cancer Stem Cells: Bioinformatic and In Vitro Studies

Monica Hana Widyardhita, Dr. apt. Dyaningtyas Dewi Pamungkas Putri, M.Sc.

2025 | Skripsi | FARMASI

Kanker payudara menyumbang 12 persen dari seluruh kejadian kanker pada tahun 2020 dan menyebabkan sekitar 658.000 kematian di seluruh dunia yang diduga disebabkan oleh kekambuhan akibat keberadaan sel punca kanker payudara atau breast cancer stem cells (BCSC). Kehadiran BCSC dalam populasi tumor utuh telah lama ditandai oleh ekspresi biomarka stemness, seperti CD133 dan ALDH1, yang pada akhirnya memicu terjadinya resistensi dan metastasis. Berdasarkan permasalahan tersebut, pencarian terapi yang secara spesifik menargetkan BCSC mengarah pada suatu senyawa glikoalkaloid steroidal, Alfa Solanine, yang telah dilaporkan memiliki aktivitas antiproliferatif terhadap kanker payudara baik secara in vitro maupun in vivo. Alfa Solanine menunjukkan prospek terapeutik dalam mengatasi BCSC melalui modulasi ekspresi gen dan aktivitas seluler yang berperan dalam proses metastasis dan resistensi. Oleh karena itu, penelitian ini bertujuan untuk mengidentifikasi target hub-gene potensial dari Alfa Solanine terhadap BCSC dan menganalisis efek sitotoksiknya terhadap BCSC.

Penelitian ini dilakukan secara simultan melalui pendekatan bioinformatika dan uji in vitro. Pada tahap awal, penyaringan gen dilakukan menggunakan basis data GeneCards, CHEMBL, SwissTargetPrediction, SEA, TargetNet, PubChem, dan SuperPred, kemudian gen yang beririsan diidentifikasi menggunakan Interactivenn. Analisis functional enrichment dilakukan menggunakan Shiny GO 0.8 sedangkan analisis interaksi protein-protein (PPI) dan hub-gene network dilakukan dengan menggunakan STRING dan Cytoscape serta plug-in Cytohubba. Selain itu, genetic alterations dari 10 hub-gene teratas dianalisis lebih lanjut menggunakan cBioPortal. Setelah studi bioinformatika, aktivitas antikanker Alfa Solanine terhadap BCSC diuji melalui uji in vitro menggunakan model MCF7 mammosphere. Setelah pembentukan MCF7 mammosphere (kultur sel 3D), karakterisasi BCSC dilakukan menggunakan RT-qPCR untuk membandingkan ekspresi mRNA CD133 dan ALDH1 pada kultur sel 3D dibandingkan dengan 2D. Selanjutnya, uji sitotoksisitas MTT dilakukan untuk memperoleh nilai IC₅₀ dan persentase viabilitas sel setelah perlakuan Alfa Solanine pada kedua model sel. Analisis statistik dilakukan menggunakan unpaired t-test untuk menentukan signifikansi data yang diperoleh.

Berdasarkan rangkaian prosedur bioinformatika, sepuluh hub-gene teratas dari Alfa Solanine terhadap BCSC yang diidentifikasi adalah STAT3, HSP90AA1, ESR1, TNF, KRAS, HIF1A, NFKB1, RELA, PTK2, dan RAF1. Dari sepuluh gen tersebut, protein PTK2, KRAS, RAF1, dan HIF1A ditemukan memiliki interaksi erat dengan CD133 dan ALDH1. Selanjutnya, BCSC berhasil diperkaya secara in vitro melalui pertumbuhan MCF7 mammosphere (MS) atau sel 3D pada non-adherent plate. CD133, sebagai salah satu penanda BCSC, mengalami overekspresi pada MCF7 MS dibandingkan dengan MCF7 2D yang mengindikasikan kemampuan MCF7 MS dalam merepresentasikan BCSC. Meskipun nilai IC₅₀-nya lebih tinggi pada MCF7 MS, efek sitotoksik Alfa Solanine terbukti secara signifikan lebih rendah pada MCF7 MS dibandingkan MCF7 2D pada konsentrasi 10 µM dan 20 µM. Dengan demikian, dapat disimpulkan bahwa Alfa Solanine berpotensi menghambat BCSC pada model MCF7 MS.

Breast cancer (BC) accounts for 12% of cancer incidences in 2020 and a total of 658,000 deaths worldwide presumably due to recurrence caused by BC stem cells (BCSC). BCSC presence in bulk tumor population has long been characterised by the expression of stemness biomarkers, including CD133 and ALDH1, that eventually initiate resistance and metastasis. Based on the existing problems, a search for BCSC-targeted therapy falls to a steroidal glycoalkaloid compound called Alpha Solanine with reported BC antiproliferation activity in vitro and in vivo. Alpha Solanine presents therapeutic prospects to combat BCSC by modulating the metastasis and resistance gene expressions and cellular activities. Therefore, this research aims further to identify the potential target hub-genes of Alpha Solanine against BCSC and analyse the cytotoxic effect of Alpha Solanine towards BCSC.

This research was conducted simultaneously through bioinformatic and in vitro studies. Initially, gene screening was performed using GeneCards, CHEMBL, SwissTargetPrediction, SEA, TargetNet, PubChem, and SuperPred, followed by acquiring intersecting genes using Interactivenn. Functional enrichment was studied through Shiny GO 0.8 while protein-protein interaction (PPI) and hub-gene network analysis were conducted using STRING and Cytoscape with Cytohubba plug-in respectively. Moreover, genetic alterations of top 10 hub-genes were further analysed using cBioportal. Following the bioinformatic study, the anticancer activity of Alpha Solanine against BCSC was assessed through in vitro assay using MCF7 mammosphere. After generating the MCF7 mammospheres (3D cell culture), BCSC characterization was conducted using RT-qPCR to compare the mRNA expression of CD133 and ALDH1 in 3D cell cultures relative to the 2D. Afterwards, MTT cytotoxic assay was performed to obtain IC₅₀ values and percent cell viability after Alpha Solanine treatment in both cell models. Statistical analysis was carried out using unpaired t-test to conclude the data’s significances.

From the series of bioinformatic procedures, top 10 hub-genes of Alpha Solanine against BCSC were STAT3, HSP90AA1, ESR1, TNF, KRAS, HIF1A, NFKB1, RELA, PTK2, and RAF1. From the top 10 hub-genes, PTK2, KRAS, RAF1, and HIF1A proteins were found to be closely interacted with CD133 and ALDH1. Afterwards, BCSC were successfully enriched in vitro by growing MCF7 mammosphere (MS) or 3D cells in non-adherent plates. CD133, one of the BCSC markers, was overexpressed in MCF7 MS relative to the MCF7 2D, indicating MCF7 MS ability to represent BCSC. Despite having IC₅₀ higher in MCF7 MS, the cytotoxic effect of Alpha Solanine was significantly lower in MCF7 MS compared to MCF7 2D at concentrations of 10 µM and 20 µM. Therefore, it is concluded that Alpha Solanine potentially inhibited BCSC in MCF7 MS.

 

Kata Kunci : Anticancer, Alpha Solanine, Breast cancer stem cells (BCSC), Bioinformatic, Mammospheres