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TMT-based proteomics analysis identifies RPLP1 as a key protein target in ursolic acid inhibition of colorectal cancer

ABSTRACT: Purpose: Ursolic Acid (UA), a triterpenoid extracted from Hedyotis Diffusa Willd. (HDW), is known for its anti-inflammatory, antioxidant, and antitumor effects. Nevertheless, the mechanisms underlying UA's anti-colorectal cancer (CRC) effects remain insufficiently understood. This study is aim to identify the key target proteins of UA and investigated their functions in CRC development. Methods: The cytotoxicity of three active components of HDW (UA, oleanolic acid (OA), and quercetin) on CRC cells was evaluated using the CCK-8 assay. Tandem mass tag (TMT)-based proteomics was employed to detect differentially expressed proteins (DEPs) in CRC cells after UA treatment. Bioinformatics analysis and high-content screening were used to identify UA’s key protein targets. The expression and role of RPLP1 in CRC cells were investigated, including the effects of RPLP1 knockdown and its combination with UA treatment on cell proliferation, migration, invasion, and apoptosis. Results: UA demonstrated superior inhibitory effects on CRC cells compared to OA and quercetin, highlighting it as a principal active ingredient of HDW. TMT-based proteomic analysis identified revealed 438 upregulated and 366 downregulated proteins after UA intervention. Among these, RPLP1 was identified as a critical target. UA inhibited RPLP1 expression in CRC cells, resulting in decreased cell proliferation, migration, and invasion. Furthermore, combination of UA treatment and RPLP1 knockdown exhibited synergistic effects in inhibiting CRC cell growth and migration, as well as promoting apoptosis. Conclusions: UA, a bioactive triterpenoid of HDW, inhibits CRC development by targeting and suppressing RPLP1 expression. These findings provide novel insights into the therapeutic of UA for CRC and highlight RPLP1 as a promising target for intervention.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colonocyte, Cell Culture

DISEASE(S): Colorectal Cancer

SUBMITTER: Limin Zhu

LAB HEAD: Li-Min Zhu

PROVIDER: PXD063684 | Pride | 2026-02-09

REPOSITORIES: Pride

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TMT-based proteomics analysis identifies RPLP1 as a key protein target in ursolic acid Inhibition of colorectal cancer.

Zhu Li-Min LM Shi Hai-Xia HX Xu Zhen-Ye ZY Deng Hai-Bin HB

Discover oncology 20250901 1

<h4>Purpose</h4>Ursolic Acid (UA), a triterpenoid extracted from Hedyotis Diffusa Willd. (HDW), is known for its anti-inflammatory, antioxidant, and antitumor effects. Nevertheless, the mechanisms underlying UA's anti-colorectal cancer (CRC) effects remain insufficiently understood. This study aimed to identify the key target proteins of UA and investigate their functions in CRC development.<h4>Methods</h4>The cytotoxicity of three active components of HDW (UA, oleanolic acid (OA), and quercetin ...[more]

PMID: 40889039

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Project description:Mass spectrometry-based omics technologies are of increasing relevance for drug discovery. In order to map drug treatments to biological pathways for mode of action (MoA) deconvolution, they rely on the determination of multiple testing corrected significant molecular events. The main contributors to statistical significance are the effect size and the variation of a given molecule during the drug treatment. While the former is largely determined by the biological system and the impact of the drug, the latter reflects the robustness of the experimental workflow. The impact of sample preparation and instrumentation on variation are well characterized, but the impact of cell culture conditions on variation of omics readouts is less explored, specifically related to subculture conditions during cell expansion. Here, we provide evidence that memory effects from subculture directly influence the variation of multilevel omics readouts, including oxylipins and fatty acids, proteins and phosphorylated sites, and phenotypic parameters, e.g. cell motility. When comparing drug treatments after homogeneous and heterogeneous subculture, the coefficients of variation (CVs) of controls increased from 1% to 3% for oxylipins and fatty acids, 11% to 23% for proteomics, 24% to 53% for phosphoproteomics and 6% to 14% for cell motility, respectively. The number of identifications remained comparable and the fold-change distributions were also largely similar after both subculture conditions. Therefore, the decrease in CVs seems to be the main contributor to the increased number of significant molecular events in drug treatments after homogeneous subculture. This in turn increased information content in the obtained perturbation network, based on proteomics and phosphoproteomics data, from 58 to 321 causal conjectures. Perturbation networks after homogeneous and heterogeneous subculture allowed for delineating the established MoA of arsenic trioxide in detail, including enhanced kinase signaling and reduced MYC-related gene expression, induction of heat shock proteins and cell cycle disruption. However, only the perturbation network after homogeneous subculture enabled the discovery of unprecedented drug effects, including down-regulation and quadruple dephosphorylation of the transcription factor RB1, among others. In summary, memory effects from subculture have broad relevance for MoA testing in drug discovery since this impact clearly manifests in supernatants, whole cell lysates and phenotypic properties and its control enables a considerably enhanced MoA deconvolution, facilitating discovery efforts.

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Dataset Information

TMT-based proteomics analysis identifies RPLP1 as a key protein target in ursolic acid inhibition of colorectal cancer

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TMT-based proteomics analysis identifies RPLP1 as a key protein target in ursolic acid Inhibition of colorectal cancer.

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