Vascular Normalization Induced By Sinomenine Hydrochloride Results In Suppressed Mammary Tumor Growth And Metastasis

Background:Pathological angiogenesis is one of the hallmarks of cancers,including breast cancer.Unlike blood vessels in the normal tissues,blood vessel growth in tumor is not only stimulated,but these vessels are also structurally and functionally abnormal.The pathological angiogenesis results in a hostile tumor microenvironment that mediates many deleterious aspects of tumor behavior.Traditional antiangiogenic“vessel pruning”agents can aggravate intratumoral hypoxia and worsen malignancy.While normalized vasculature could not only alleviate intratumoral hypoxia,but also improve chemotherapy,radiotherapy and immunotherapy.Therefore,antiangiogenic“vessel normalizing”strategies are gaining attention.Vascular normalization is achieved with judicious doses of anti-angiogenic treatment,targeting mainly the VEGF signaling pathway.However,the clinical efficacies of these drugs are still not satisfactory.It is urgent to identify more-effective strategies to normalize the tumor vasculature for enhancing anti-cancer therapies.Sinomenine is an alkaloid extracted from the Chinese medicinal plant Sinomenium acutum.Sinomenine hydrochloride?SH?,the hydrochloride chemical form of sinomenine,has been utilized to treat rheumatism and rheumatoid diseases in clinic.Sinomenine has been demonstrated to mediate a wide range of pharmacological actions including anti-inflammatory,immunoregulatory,anti-angiogenic,anti-arrhythmic,as well as mild sedative and analgesic effects.Recent studies reported the anti-proliferative potential of sinomenine against a variety of cancer cells including hepatocellular carcinoma,lung cancer and synovial sarcoma and so on.Studies in rheumatoid arthritis demonstrated the anti-angiogenic effect of sinomenine,but the effect of sinomenine on solid tumor vasculature is still undetermined.Preclinical evidence showed that anti-angiogenic drugs could induce vascular normalization within a proper therapeutic window.Hereby,we want to investigate the anti-angiogenic effect of sinomenine on breast cancer,the normalization window of sinomenine and the underlying mechanisms.Methods:1.In vitro study:trypan blue exclusion assay and MTT assay were used to study the effect of SH on HUVECs viability;flow cytometry was utilized to determine the effect of SH on HUVECs cell cycle;wound healing assay and tube formation assay were carried out to test the effect of SH on HUVECs function.2.In vivo study:orthotopic breast cancer model was utilized to investigate the anti-tumoral and anti-metastatic effect of SH;immunofluorescence?IF?staining visualized under con-focal microscopy,vessel perfusion assay and immunohistochemistry?IHC?staining were performed to show the effect of SH on the morphology and function of tumor vascular;antibody array and enzyme-linked immuno sorbent assay?ELISA?were used to investigate the underlying mechanisms.Results:1.Trypan blue exclusion assay and MTT assay showed that SH has little effect on HUVECs viability;only with 1 mM SH for 48 h,HUVECs cell cycle was arrested in G₁/S phase;SH could inhibit HUVECs migration even at low concentration;SH reduced tube formation of HUVECs in a dose-dependent manner.2.50 and 100 mg/kg SH could reduce spontaneous metastasis,but only tumors in 100 mg/kg SH-treated group showed significant decreased,so 100 mg/kg is the best therapeutic dosage of SH in this model.3.IF staining showed that 100 mg/kg SH could reduce vessel area,increase vessel diameter and pericyte coverage—structure normalization;vessel perfusion assay and IHC staining indicated that 100 mg/kg SH could improve vessel perfusion and alleviate intratumoral hypoxia—function normalization;100 mg/kg SH could also enhance chemotherapy sensitivity;antibody array and ELISA showed that 100 mg/kg SH could inhibit bFGF while promote PF-4 expression and secretion;IF staining also found an increase of M1 TAMs,a decrease of M2 TAMs and MDSCs within 100 mg/kg SH-treated tumors.4.200 mg/kg SH didn't exhibit inhibitory effect on tumor progression;IF staining showed that vessel area decreased further but intratumoral hypoxia become aggravated—excessive pruning;ELISA indicated that 200 mg/kg SH up-regulated G-CSF,down-regulated GM-CSF;IF staining also found an increase of M2 TAMs and MDSCs within 200 mg/kg SH-treated tumors.Conclusion:1.SH exhibited anti-angiogenic effect on breast cancer.2.Vascular normalization induced by SH is dose dependent:?1?The normalization window of SH is 100 mg/kg,and this dosage could induce vascular normalization,thus alleviate intratumoral hypoxia,inhibit tumor growth and dissemination,while improving chemotherapy sensitivity and anti-tumor immunity.Critically underlying these activities is the ability of SH to restore the balance between angiogenesis stimulators and inhibitors.?2?200 mg/kg SH didn't exhibit inhibitory effect on tumor progression due to the immunosuppressive microenvironment caused by excessive vessel pruning,G-CSF upregulation and GM-CSF downregulation.