Polyphyllin H Reverses Paclitaxel Resistance in Breast Cancer by Binding Membrane Cholesterol to Inhibit Both ABCB1 and ABCC3

<b>Background/Objectives</b>: Breast cancer is the most prevalent malignancy among women, and paclitaxel (PTX) is a first-line chemotherapeutic, but chemoresistance driven by ATP-binding cassette (ABC) transporters limits its efficacy. Single-target ABC inhibitors fail due to toxicity and cooperative transporter activity, creating an urgent need for safe multi-target strategies. Membrane cholesterol-rich lipid rafts support ABC transporter function, making cholesterol a key chemoresistance target. This study explored a cholesterol-targeted approach for overcoming PTX resistance. <b>Methods</b>: A PTX-resistant breast cancer line (MCF-7/PTX) showing ABCB1/ABCC3 co-upregulation and enriched cholesterol rafts was established. The effects of Polyphyllin H (PPH), a steroidal saponin from <i>Paris polyphylla</i>, were compared with lovastatin, a biosynthetic cholesterol inhibitor. In vitro and in vivo assays investigated Polyphyllin H's cholesterol binding and effects on transporters, PTX accumulation, and tumor growth. <b>Results</b>: PPH directly binds membrane cholesterol, disrupting lipid rafts, downregulating ABCB1/ABCC3, reducing drug efflux, and increasing intracellular PTX to restore sensitivity. PPH showed superior cholesterol-binding and resistance-reversal efficacy than lovastatin, with faster, stronger PTX-enhanced cytotoxicity and tumor suppression. <b>Conclusions</b>: PPH reverses PTX resistance by targeting cholesterol-lipid rafts to inhibit multiple ABC transporters. This offers a safer adjuvant for PTX-based breast cancer therapy and a translational framework for other drug-resistant malignancies.