The hippo kinases MST1/2 in cardiovascular and metabolic diseases: A promising therapeutic target option for pharmacotherapy

Cardiovascular diseases (CVDs) and metabolic disorders are major components of noncommunicable diseases, causing an enormous health and economic burden worldwide. There are common risk factors and developmental mechanisms among them, indicating the far-reaching significance in exploring the corresponding therapeutic targets. MST1/2 kinases are well-established proapoptotic effectors that also bidirectionally regulate autophagic activity. Recent studies have demonstrated that MST1/2 influence the outcome of cardiovascular and metabolic diseases by regulating immune inflammation. In addition, drug development against them is in full swing. In this review, we mainly describe the roles and mechanisms of MST1/2 in apoptosis and autophagy in cardiovascular and metabolic events as well as emphasis on the existing evidence for their involvement in immune inflammation. Moreover, we summarize the latest progress of pharmacotherapy targeting MST1/2 and propose a new mode of drug combination therapy, which may be beneficial to seek more effective strategies to prevent and treat CVDs and metabolic disorders.

Radix Scrophulariae Extracts Exert Effect on Hyperthyroidism via MST1/Hippo Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To explore the mechanism of Radix Scrophulariae (RS) extracts in the treatment of hyperthyroidism rats by regulating proliferation, apoptosis, and autophagy of thyroid cell through the mammalian sterile 20-like kinase 1 (MST1)/Hippo pathway.@*METHODS@#Twenty-four rats were randomly divided into 4 groups according to a random number table: control, model group, RS, and RS+Hippo inhibitor (XMU-MP-1) groups (n=6 per group). Rats were gavaged with levothyroxine sodium tablet suspension (LST, 8 μ g/kg) for 21 days except for the control group. Afterwards, rats in the RS group were gavaged with RS extracts at the dose of 1,350 mg/kg, and rats in the RS+XMU-MP-1 group were gavaged with 1,350 mg/kg RS extracts and 1 mg/kg XMU-MP-1. After 15 days of administration, thyroid gland was taken for gross observation, and histopathological changes were observed by hematoxylin-eosin staining. The structure of Golgi secretory vesicles in thyroid tissues was observed by transmission electron microscopy. The expression of thyrotropin receptor (TSH-R) was observed by immunohistochemistry. Terminal-deoxynucleoitidyl transferase mediated nick end labeling assay was used to detect cell apoptosis in thyroid tissues. Real-time quantity primer chain reaction and Western blot were used to detect the expressions of MST1, p-large tumor suppressor gene 1 (LATS1), p-Yes1 associated transcriptional regulator (YAP), proliferating cell nuclear antigen (PCNA), G1/S-specific cyclin-D1 (Cyclin D1), B-cell lymphoma-2 (Bcl-2), Caspase-3, microtubule-associated proeins light chain 3 II/I (LC3-II/I), and recombinant human autophagy related 5 (ATG5). Thyroxine (T4) level was detected by enzyme-linked immunosorbent assay.@*RESULTS@#The thyroid volume of rats in the model group was significantly increased compared to the normal control group (P<0.01), and pathological changes such as uneven size of follicular epithelial cells, disorderly arrangement, and irregular morphology occurred. The secretion of small vesicles by Golgi apparatus was reduced, and the expressions of receptor protein TSH-R and T4 were significantly increased (P<0.01), while the expressions of MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 were significantly decreased (P<0.01). The expressions of Bcl-2, PCNA, and cyclin D1 were significantly increased (P<0.01). Compared with the model group, RS extracts reduced the volume of thyroid gland, improved pathological condition of the thyroid gland, promoted secretion of the secretory vesicles with double-layer membrane structure in thyroid Golgi, significantly inhibited the expression of TSH-R and T4 levels (P<0.01), upregulated MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 expressions (P<0.01), and downregulated Bcl-2, PCNA, and Cyclin D1 expressions (P<0.01). XMU-MP-1 inhibited the intervention effects of RS extracts (P<0.01).@*CONCLUSION@#RS extracts could inhibit proliferation and promote apoptosis and autophagy in thyroid tissues through MST1/Hippo pathway for treating hyperthyroidism.

Expression and clinical significance of MST1 in cervical cancer / 天津医药

Objective To investigate the expression and clinical significance of mammalian sterile 20-like kinase 1 (MST1) in cervical cancer. Methods Immunohistochemical method was applied to detect the expression level of MST1 protein in specimens of cervical cancer tissues (n=139) and pericarcinomatous tissues (n=20, with≥4 cm distance from the primary tumor's edge). Western blot assay and qPCR were used to detect the protein and mRNA transcription expression levels of MST1 in 20 pairs of cervical cancer tissues and pericarcinomatous tissues, respectively. The correlation between MST1 expression, clinic pathological features and the prognosis were analyzed. Results MST1 was mainly expressed in cytoplasm. The positive expression rate of MST1 was significantly lower in cervical cancer tissues (27%, 38/139) than that in pericarcinomatous tissues (80%, 16/20,χ2=21.62, P<0.01). The expressions levels of MST1 protein and mRNA were both lower in the cervical cancer tissues (P<0.01). In cervical cancer, the positive expression rate of MST1 inⅠb+Ⅱa stage was higher than that ofⅡb+Ⅳstage (P<0.05), the positive expression rate of MST1 in lymph node metastasis was lower than that of without lymph node metastasis (P < 0.05). Values of age, tumor size, histological type and differentiation degree showed no significant difference to positive expression rate of MST1. Moreover, the negative expression of MST1 displayed a significantly poorer overall survival time than that of positive expression of MST1 (Log-rank χ2=28.35, P < 0.01). Conclusion MST1 shows a lower expression in cervical cancer, which may be a new target for clinical treatment and prognosis of cervical cancer.

Protective effect and mechanism of MST1 inhibition on kidney tissue in diabetic rats induced by streptozotocin / 中华肾脏病杂志

Objective To investigate the protective effect and mechanism of MST1 inhibition on kidney tissue in diabetic rats,and to find a new therapeutic target for diabetic nephropathy.Methods Total of 54 male SD rats enrolled in this study were divided into 3 groups including normal control (group A,n=18),MST1 inhibition group (Group B,n=18) and diabetes group (group C,n=18).Diabetes was induced by a single streptozotocin (STZ,50 mg/kg) injection in group B and group C.rats in group B received lentiviral vector contain Mst1 interference RNA (shRNA) and the rats in group C received empty vector.The end of 4th,8th and 12th week after modeling were considered as time points in this study.At each time point,the level of 24 hours urine protein (24-HUP),blood glucose and serum creatinine were examined.Pathological changes were observed with HE stain; Injury of podocyte and glomerular basement membrane (GBM) were examined with transmission electron microscope (TEM).The intensity and location of MST1 in kidney tissue were detected by immunohistochemistry.The level of MST1,Phosphorylated-MST1,nephrin,Caspase-3 and FasL were detected by western bloting.Results (1) At the starting point,there were no significant differences among groups in terms of weight,activity,eating and drinking.Since the end of 72nd hour after modeling,the levels of glucose in both group B and group C,compared to those in group A,significantly increased (P ＜ 0.05).There was no significant difference between group B and group C for glucose level at each time point (P ＞ 0.05); the level of 24-HUP increased significantly since the end of 4th week after modeling,and the level in group C was higher than its counterpart in group B at the same point (P ＜ 0.05); (2) There was no significant pathological lesion observed in group A.Without obvious K-W nodular changes,mesangial proliferation was observed in group B and group C.It was shown by TEM that podocyte fusion and thickening of the GBM could be found in group B and group C.The pathological change in group B was better than that in group C; (3) Compared to group A,it was shown by western blot that the levels of MST1,Phosphorylated-MST1,Caspase-3 and FasL in group B and group C were significantly higher (P ＜ 0.05),and the levels of nephrin in group B and group C were significantly lower (P ＜ 0.05) since the end of 4th week after modeling.Meanwhile,the levels of MST1,Phosphorylated-MST1,Caspase-3 and FasL in group B were significantly lower than that in group C at each time point (P ＜ 0.05),the level of nephrin in group B was significantly higher than the one in group C; (4) It was shown by immunohistochemistry that there was low MST1 expression in normal condition,especially in cytoplasm of tubular epithelial cells.The level of MST1 in group B and group C significantly increased after modeling,and the change could be the same as Western blot shown.Conclusions MST1 pathway could be involved in kidney injury induced by diabetes.MST1 inhibition could alleviate the kidney injury in STZ-induced diabetes animal model.

Construction of inducible lentiviral expression vector containing MST1 gene and its expression in Huh-7 cell line / 复旦学报（医学版）

Objective To construct inducible lentiviral vector containing MST1 gene, which provides an ideal model for further study of the relationship between liver cancer and MST1 gene. Methods We cloned MST1 into inducible lentiviral vector. Two lentiviral vectors (pLVPT-tTRKRAB-MST1 and pLV-tTRKRAB-Red) with package plasmids were contransfected into 293FT respectively, and the lentiviral viruses were harvested from 293FT. Viruses were used to infect liver cancer cell line (Huh-7) in tandem. We used doxcycline to induce the expression of target gene MST1 which was indentified by Western blot after 7-day cell culture. Results The recombinant inducable lentiviral vector containing MST1 gene was successfully constructed. The lentiviruses were also obtained and mediated by 293FT, which were highly efficient to infect liver cell lines Huh-7. The expression of MST1 was identified under Dox induction. Conclusions The recombinant inducable lentiviral vector containing MST1 gene has been successfully constructed. It is viable to obtain inducible cell lines Huh-7 with MST1 gene expression under Dox induction.

Effects of MST1 on cell proliferation and apoptosis of human breast carcinoma cell line MCF-7 / 肿瘤

Objective: To elucidate effects of mammalian sterile 20-like kinase 1 (MST1) gene on cell proliferation and apoptosis of human breast carcinoma cell line MCF-7. Methods: This study constructed plasmid pCMV-FLAG-MST1 and transfected it into human breast carcinoma cell line MCF-7 via mediation by Lipofectamine 2000. We detected the efficiency of transfection by Western blotting. The effect of MST1 on cell proliferation was measured by MTT assay at 12, 24, 36, and 48 h after transfection. The effect of MST1 on cell proliferation was also determined by BrdU incorporation assay at 36 h after transfection. Cisplatin was added into cultured cells at 36 h to induce apoptosis. Forteen hours later cell apoptosis was detected by Annexin V staining. Results: This study successfully constructed plasmid pCMV-FLAG-MST1. The MTT and BrdU incorporation assay showed that the cell proliferation was significantly inhibited after transfection of pCMV-FLAG-MST1. Annexin V staining demonstrated that cell apoptotic rate was relatively higher after overexpression of MST1. Conclusion: In summary, overexpression of MST1 decreases cell proliferation and induced apoptosis of human breast carcinoma cell line MCF-7.