British Journal of Pharmacology

Hydnocarpin D‐induced microRNA‐145‐5p down‐regulates SERPINE1 and suppresses metastasis in ovarian cancer

Background and Purpose Hydnocarpin D (HD) is a flavonolignan compound isolated from Hydnocarpus wightiana . This study was to investigate the effect of HD on ovarian cancer mediated by the regulation of SERPINE1 . Experimental Approach Cytotoxicity was assessed by the MTT assay. Cell migration and invasion were examined using wound healing and Boyden chamber assays. An orthotopic xenograft model was constructed to determine the inhibitory effect of HD on ovarian cancer in vivo. Differential gene expression was screened by RNA sequencing. A 3′‐UTR luciferase assay was performed to confirm the regulatory effect of microRNA‐145‐5p on SERPINE1 . Small interfering RNA and microRNA mimics/inhibitors were introduced to elucidate the mechanism. Key Results HD inhibited the migration and invasion of SKOV3 and OVCAR4 cells without showing cytotoxic effects. HD inhibited the growth and metastasis of ovarian cancer in vivo. RNA‐sequencing analysis suggested that HD suppressed metastasis by inhibiting SERPINE1 expression. Free uPA, which is not bound to PAI‐1 ( SERPINE1 ), was up‐regulated, whereas vitronectin, integrin α V and phosphorylated FAK, the downstream metastasis‐related signalling factors for PAI‐1, were down‐regulated after treatment with HD. SERPINE1 inhibition attenuated the effect of HD on reducing cell migration and rhPAI‐1 enhanced the effect of HD. We identified microRNA‐145‐5p as the post‐transcriptional repressor of SERPINE1 . MicroRNA‐145‐5p was up‐regulated by HD, and its overexpression enhanced the inhibition of PAI‐1 expression and migration by HD, whereas its inhibition had the opposite effect. Conclusion and Implications We showed that HD inhibits metastasis in ovarian cancer by up‐regulating microRNA‐145‐5p, which targets SERPINE1 , inhibiting vitronectin/integrin/FAK signalling.

PAR2 induces ovarian cancer cell motility by merging three signalling pathways to transactivate EGFR

Background and PurposeSpecific cellular functions mediated by GPCRs are often associated with signalling through a particular G protein or β‐arrestin. Here, we examine signalling through a GPCR, protease‐activated receptor 2 (PAR2), in a high‐grade serous ovarian cancer cell line (OV90).Experimental ApproachHuman ovarian cancer tissues (n = 1,200) and nine human ovarian cancer cell lines were assessed for PAR2 expression. PAR2 signalling mechanisms leading to cell migration and invasion were dissected using cellular assays, western blots, CRISPR‐Cas9 gene knockouts, pharmacological inhibitors of PAR2 and downstream signalling proteins in OV90 cancer cells.Key ResultsPAR2 was significantly overexpressed in clinical ovarian cancer tissues and in OV90 ovarian cancer cells. PAR2 agonists, an endogenous protease (trypsin) and a synthetic peptide (2f‐LIGRL‐NH2), induced migration and invasion of OV90 ovarian cancer cells through activating a combination of Gαq/11, Gα12/13 and β‐arrestin1/2, but not Gαs or Gαi. This novel cooperative rather than parallel signalling resulted in downstream serial activation of Src kinases, then transactivation of epidermal growth factor receptor (EGFR), followed by downstream MEK–ERK1/2–FOS/MYC/STAT3–COX2 signalling. Either a PAR2 antagonist (I‐191), CRISPR‐Cas9 gene knockouts (PAR2 or Gα proteins or β‐arrestin1/2), or inhibitors of each downstream protein attenuated human ovarian cancer cell motility.Conclusion and ImplicationsThis study highlights a novel shared signalling cascade, requiring each of Gαq/11, Gα12/13 and β‐arrestin1/2 for PAR2‐induced ovarian cancer cell migration and invasion. This mechanism controlling a cellular function is unusual in not being linked to a specific individual G protein or β‐arrestin‐mediated signalling pathway.

Resveratrol, a novel inhibitor of fatty acid binding protein 5, inhibits cervical cancer metastasis by suppressing fatty acid transport into nucleus and downstream pathways

Background and PurposeBecause of cervical cancer (CC) metastasis, the prognosis of diagnosed patients is poor. However, the molecular mechanisms and therapeutic approach for metastatic CC remain elusive.Experimental ApproachIn this study, we first evaluated the effect of resveratrol (RSV) on CC cell migration and metastasis. Via an activity‐based protein profiling (ABPP) approach, a photoaffinity probe of RSV (RSV‐P) was synthesized, and the protein targets of RSV in HeLa cells were identified. Based on target information and subsequent in vivo and in vitro validation experiments, we finally elucidated the mechanism of RSV corresponding to its antimetastatic activity.Key ResultsThe results showed that RSV concentration‐dependently suppressed CC cell migration and metastasis. A list of proteins was identified as the targets of RSV, through the ABPP approach with RSV‐P, among which fatty acid binding protein 5 (FABP5) attracted our attention based on The Cancer Genome Atlas (TCGA) database analysis. Subsequent knockout and overexpression experiments confirmed that RSV directly interacted with FABP5 to inhibit fatty acid transport into the nucleus, thereby suppressing downstream matrix metalloproteinase‐2 (MMP2) and matrix metalloproteinase‐9 (MMP9) expression, thus inhibiting CC metastasis.Conclusions and ImplicationsOur study confirmed the key role of FABP5 in CC metastasis and provided important target information for the design of therapeutic lead compounds for metastatic CC.

BBIT20 inhibits homologous DNA repair with disruption of the BRCA1–BARD1 interaction in breast and ovarian cancer

Background and PurposeAdvances in the treatment of triple‐negative breast and ovarian cancer remain challenging. In particular, resistance to the available therapy, by restoring or overexpressing the DNA repair machinery, has often been reported. New strategies to improve the therapeutic outcomes of these cancers are needed. Herein, we disclose the dregamine 5‐bromo‐pyridin‐2‐ylhydrazone (BBIT20), a natural monoterpene indole alkaloid derivative, as an inhibitor of homologous DNA repair.Experimental ApproachTo unveil BBIT20 antitumour activity and underlying molecular mechanism of action, two‐dimensional (2D) and three‐dimensional (3D) cell cultures, patient‐derived cell lines and xenograft mouse models were used.Key ResultsBBIT20 disrupted the BRCA1‐BARD1 interaction, triggering nuclear‐to‐cytoplasmic BRCA1 translocation, cell cycle arrest and downregulation of homologous DNA repair‐related genes and proteins, with subsequent enhancement of DNA damage, reactive oxygen species generation and apoptosis, in triple‐negative breast and ovarian cancer cells. BBIT20 also displayed pronounced antitumour activity in patient‐derived cells and xenograft mouse models of ovarian cancer, with low toxicity in non‐malignant cells and undetectable side effects in mice. Additionally, it did not induce resistance in triple‐negative breast and ovarian cancer and displayed marked synergistic effects with cisplatin and olaparib (a poly [ADP‐ribose] polymerase inhibitor), on 2D and 3D models of these cancer cells.Conclusion and ImplicationsThese findings add an inhibitor of the BRCA1‐BARD1 interaction to the list of DNA‐damaging agents. Importantly, either as a single agent or in combination therapy, BBIT20 reveals great potential in the personalized treatment of aggressive and resistant cancers, particularly triple‐negative breast and advanced ovarian cancer.

Enterolactone promotes efficacy of gemcitabine on epithelial ovarian cancer and ameliorates gut dysbacteriosis

Abstract Background and Purpose The effectiveness of conventional treatments for epithelial ovarian cancer (EOC) is very limited and their side effects are serious. Previous research has demonstrated the inhibitory effects of enterolactone (ENL) on EOC by inhibiting malignant angiogenesis. Gemcitabine (Gem) is a chemotherapeutic agent commonly used for the treatment of EOC with limited efficacy. In this study, we aimed to explore the combined inhibitory effects of ENL and Gem on EOC. Experimental approach We detected the proliferation ability of EOC cells after ENL/Gem/ENL + Gem by CCK8, crystal violet assays, migration and invasion ability by wound healing and transwell assays, in vivo evaluation of the anti‐neovascularisation efficacy of zebrafish and in vitro tube formation assays to detect angiogenesis, network pharmacology, Western‐blot and immunohistochemistry to analyse molecular pathways, and in vivo animal experiments on tumour progression. Key Results Our results demonstrated that the ENL and Gem combination synergistically inhibited the proliferation, migration and invasion of EOC. Tube formation and zebrafish neovascularization assays showed potent anti‐angiogenic activity of the ENL + Gem combination. In animal experiments, the combined use of ENL and Gem also synergistically inhibited tumour growth and in the meantime markedly reduced the side effects of Gem. ENL ameliorated gut dysbacteriosis of ovarian cancer animals, which significantly enhanced the synergistic anti‐cancer effect of ENL and Gem. Conclusions and Implications ENL and Gem synergistically inhibit the proliferation, migration, invasion, and angiogenesis of EOC by modulating the Akt‐Bax and Akt‐MMP9‐VEGFR‐2 pathways and ameliorating gut dysbacteriosis.

Inhibition of DNA polymerase eta‐mediated translesion DNA synthesis with small molecule sensitises ovarian cancer stem‐like cells to chemotherapy

AbstractBackground and PurposeChemoresistance and tumour relapse pose significant challenges in achieving successful chemotherapy outcomes. Targeting DNA polymerase eta (Pol ƞ)‐mediated mutagenic translesion DNA synthesis (TLS) has emerged as a promising strategy for improving chemotherapy. However, the identification of small molecule inhibitors targeting Pol ƞ ‐mediated TLS with high in vivo efficacy remains a challenge.Experimental ApproachThe small molecule was identified through in silico screening. Pol η inhibitory potential of the identified small molecule was validated by a fluorescent‐based reporter strand displacement assay. Flow cytometry was conducted to analyse the CD44 + CD117 + cancer stem‐like cell (CSC) population and live‐dead cell population. Xenograft mouse models were used to test the CSC sensitising potential.Key ResultsWe screened and identified chrysin as a small‐molecule inhibitor that sensitises ovarian cancer stem‐like cells to cisplatin treatment by inhibiting Pol ƞ ‐mediated TLS. Chrysin effectively inhibits Pol ƞ expression, mitigates cancer stem‐like cell enrichment and enhances cisplatin‐induced cell death both in vitro and in vivo. Furthermore, chrysin treatment reduces spontaneous and cisplatin‐induced mutagenesis. Pre‐treatment with chrysin attenuates cisplatin‐induced haematological toxicity and suppresses tumour growth in human ovarian cancer xenografts.Conclusions and ImplicationsThese results establish chrysin as a novel class of TLS inhibitors and highlight its potential as a chemotherapy adjuvant for overcoming chemoresistance and improving treatment outcomes in ovarian cancer.

IA‐0130, a novel 3‐(1,3‐diarylallylidene)oxindole derivative, alleviates ovarian cancer via inhibiting IL‐6/gp130/STAT3 signalling

Background and Purpose Dysregulation of the IL‐6/glycoprotein 130(gp130)/STAT3 signalling axis is implicated in several human diseases, particularly cancer. Notably, gp130, a single transducer of this signalling axis, is a target for ovarian cancer treatment. However, data regarding small‐molecule inhibitors of gp130 are lacking. Therefore, we aimed to identify a 3‐(1,3‐diarylallylidene)oxindole derivative that binds gp130 and reveal the anticancer mechanism acting on the IL‐6/gp130/STAT3 pathway in ovarian cancer. Experimental Approach We synthesised 24 derivatives based on the scaffold of 3‐(1,3‐diarylallylidene)oxindole, and derivatives that inhibit IL‐6 signalling were selected using HEK‐Blue™ IL‐6 cells. The binding of derivatives to gp130 was assessed using surface plasmon resonance. IA‐0130, with a strong gp130‐binding ability, was selected to observe its effect on the migration, invasion, cell cycle arrest and apoptosis of ovarian cancer cells in comparison to bazedoxifene, a known gp130‐binding derivative. Additionally, we examined the mechanism underlying the tumour suppressive effect of IA‐0130 in vivo. Key Results We found that IA‐0130 inhibited gp130/STAT3 phosphorylation in a concentration‐dependent manner in ovarian cancer cell line and also in ovarian cancer‐resistant cell line. By suppressing the expression of downstream target genes, IA‐0130 inhibited cancer cell growth, metastasis, and invasion and induced apoptosis, exhibiting anticancer effects. In a mouse xenograft model of human ovarian cancer, oral administration of IA‐0130 significantly delayed tumour growth. Conclusions and Implications IA‐0130 inhibits tumour growth, migration and metastasis by inhibiting IL‐6/gp130/STAT3 signalling in ovarian cancer by binding gp130. IA‐0130 holds therapeutic potential for treating ovarian cancer as well as anticancer drug‐resistant ovarian cancer.