Root extract of Hemsleya amabilis Diels suppresses renal cell carcinoma cell growth through inducing apoptosis and G2/M phase arrest via PI3K/AKT signaling pathway

Abstract

Ethnopharmacological relevance: Hemsleya amabilis Diels, belongs to cucurbitaceae, was traditional Chinese medicine (TCM). It is widely used to treat various diseases. However, these diseases may contribute to the development of RCC.

Aim of the study: investigated the anticancer activities of root extract of Hemsleya amabilis Diels (HRE), and elucidated the underlying molecular mechanism in vivo and in vitro.

Materials and methods: Dried Hemsleya amabilis Diels roots were extracted by ethyl acetate and used to treat RCC4, OS-RC-2 and ACHN cells. UHPLC-MS was used to analyze the chemical composition of the extract. CCK-8 and colony formation assay were used to investigate proliferation. PI staining was used to detect cell cycle. Annexin-V-FITC, AO/EB and TEM were used to evaluate apoptosis. Transwell and wound healing assays were used to evaluate migration and invasion. RNA-seq, Network pharmacology, autodocking for virtual screening and molecular dynamics simulation were used to analyze potential molecular mechanisms and active components of HRE inhibiting proliferation of RCC. LY294002 and UC2288 were used to inhibit PI3K and P21 expression, respectively. IGF-1 was used to activate PI3K. Xenograft tumor model was established to evaluate its anti-tumor potential in vivo. Immunohistochemistry and Western blot were used to test protein expression levels. H&E staining was used to explore the side effects of HRE in vivo. Applying bioinformatics to analyze the effect of P21 on RCC.

Results: HRE consists of 739 compounds. CCK-8 and colony formation assay showed that HRE significantly inhibited RCC cells proliferation. PI staining indicated that HRE caused G₂/M phase arrest. Annexin-V-FITC, AO/EB and TEM experiments revealed that HRE significantly promoted apoptosis of RCC cells. Transwell and wound healing assays showed that HRE can inhibit the migration and invasion of RCC cells. RNA-seq showed that HRE induced 230 gene changes. Network pharmacology analysis found the relationship between HRE-component-target-RCC. Auto-docking found that Epitulipinolide diepoxide in HRE can stably bind to PIK3CA (-7.22 kJ/mol), and molecular dynamics simulation verified the combination between Epitulipinolide diepoxide of PIK3CA. In RCC4 cells, pretreatment with IGF-1, attenuated HRE-induced apoptosis and G₂/M arrest. When pretreated with PIK3 inhibitor LY294002, the opposite result appears. Pretreatment with CDKN1A (P21) inhibitor UC2288 attenuated HRE-induced G₂/M arrest. Xenograft tumor model showed that HRE inhibited tumor growth. Western blot analysis indicated that HRE can regulating Bax, Bcl-2, PARP, cleared-PARP, Caspase-9, Caspase-8, Caspase-3, Survivin, Cyclin-B1, CDK1, N-cadherin, snail, slug, E-cadherin, MMP-9. Immunohistochemical staining showed that in the treated group, expression of E-cadherin, Bax, P21 was up-regulated, while N-cadherin, PI3K, AKT and Bcl-2 were down-regulated. H&E staining showed that compared to control groups, the main organs in the HRE-treated groups showed no histological abnormalities. The overall survival rate of RCC patients in the high-expression group of P21 was higher than in the low-expression group of P21 on bioinformatics analysis.

Conclusions: HRE inhibited RCC migration and invasion through EMT, and inhibited proliferation in vivo and in vitro. In addition, HRE inhibited proliferation through promoting apoptosis and P21-induced G₂/M phase arrest via PI3K/AKT signaling pathway. Overall, these results suggest that HRE may be a promising chemotherapy agent for RCC.

Keywords: CDKN1A (P21); HRE; PI3K/AKT signaling pathway; Proliferation; RCC; Renal cell carcinoma; Root extract of Hemsleya amabilis dielsd.