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Reserpine, a novel N6-methyladenosine regulator, reverses Lenvatinib resistance in hepatocellular carcinoma
Lei Zhao1, Heyao Ma2, Yuhui Jiang3
1Department of Hepatobiliary and Pancreatic Surgery, First Hospital of China Medical University, Shenyang, Liaoning 110001, China.
Background:
Hepatocellular carcinoma (HCC) is an aggressive malignancy and a growing global health problem. Reserpine (Res), a plant-derived hypertension drug, has been reported to possess anti-tumor efficacy. However, the role and function of Res in N6-methyladenosine (m6A) regulation and Lenvatinib (Len) resistance in HCC have not been clarified.
Purpose:
To verify whether Res can be used as a natural small-molecule regulator of m6A to reverse Len resistance in HCC.
Methods:
Dot blotting, Western blotting and m6A quantification were used to compare and analyze the differential expression of m6A and its methyltransferase METTL3. Western blotting, Real-Time PCR (RT-PCR), cellular thermal shift assay (CETSA) and molecular docking were used to explore the mechanism of interaction between Res and m6A. The effects of Res on the biological characteristics of Lenvatinib-resistant HCC cells were investigated through CCK-8, clone formation, and Transwell assays. Cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models were used to assess the ability of Res to reverse Len resistance in vivo. MeRIP m6A sequencing, PATHWAY analysis and Western blotting were used to analyze the downstream signaling pathways and genes involved in Res-mediated reversal of Len resistance.
Results:
Len resistance in HCC is related to the increased m6A level and the high expression of METTL3. Res affects the activity of METTL3 protein by binding to it, thereby downregulating the level of m6A. In vitro study showed that Res can sensitize HCC cells to the anti-tumor effects of Len treatment, including blocking proliferation, inhibiting migration, and inducing apoptosis. Len-resistant CDX and PDX models revealed that Res can reverse the resistant phenotype, with the tumor inhibition rates of 77.46 % and 62.1 %, respectively, when combined with Len treatment. Analysis of xenograft tissues showed that the combination of Res and Len down-regulates the m6A level, reduces proliferation biomarkers, and induces apoptosis, which is consistent with the in vitro data. Mechanistically, our preliminary results indicate that Res can up-regulate the SMAD3 level by down-regulating m6A in Len-resistant cells.
Conclusions:
Reserpine, a small-molecule regulator of m6A, reverses Lenvatinib-resistant phenotypes, including proliferation, migration and anti-apoptosis, in vitro and in vivo by targeting SMAD3 and down-regulating the m6A level in HCC.
Insights
Reserpine (Res) reverses Lenvatinib (Len) resistance in hepatocellular carcinoma (HCC) by regulating N6-methyladenosine (m6A) levels. This natural compound targets SMAD3, inhibiting proliferation and migration while promoting apoptosis in resistant HCC cells.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Hepatocellular carcinoma (HCC) presents a significant global health challenge due to its aggressive nature.
- Reserpine (Res), a known antihypertensive drug derived from plants, has demonstrated potential anti-tumor properties.
- The specific role of Res in modulating N6-methyladenosine (m6A) and overcoming Lenvatinib (Len) resistance in HCC remains largely unexplored.
Purpose of the Study:
- To investigate Reserpine's capacity as a natural small-molecule regulator of m6A to overcome Lenvatinib resistance in Hepatocellular Carcinoma.
- To elucidate the molecular mechanisms underlying Reserpine's action in Lenvatinib-resistant HCC.
Main Methods:
- Utilized dot blotting, Western blotting, and m6A quantification to assess m6A levels and METTL3 expression.
- Employed Western blotting, RT-PCR, CETSA, and molecular docking to explore Res-m6A interactions.
- Evaluated Res's impact on proliferation, migration, and apoptosis in resistant HCC cells using CCK-8, clone formation, and Transwell assays.
- Assessed Res's efficacy in reversing Len resistance in vivo using cell line-derived and patient-derived xenograft mouse models.
- Analyzed downstream signaling pathways via MeRIP m6A sequencing and pathway analysis.
Main Results:
- Lenvatinib resistance in HCC correlates with elevated m6A levels and high METTL3 expression.
- Reserpine binds to METTL3, reducing its activity and subsequently lowering m6A levels.
- In vitro, Reserpine sensitized HCC cells to Lenvatinib, inhibiting proliferation, migration, and inducing apoptosis.
- In vivo, Reserpine combined with Lenvatinib demonstrated significant tumor inhibition (77.46% in CDX, 62.1% in PDX models) by reducing proliferation and inducing apoptosis.
- Reserpine was found to up-regulate SMAD3 expression by decreasing m6A levels in Lenvatinib-resistant cells.
Conclusions:
- Reserpine acts as an m6A regulator, effectively reversing Lenvatinib-resistant phenotypes in Hepatocellular Carcinoma.
- Reserpine targets SMAD3 and reduces m6A levels, impacting proliferation, migration, and apoptosis both in vitro and in vivo.
- Reserpine holds promise as a therapeutic agent to overcome Lenvatinib resistance in HCC treatment.
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