SSPH I, a Novel Anti-Cancer Saponin, Inhibits Autophagy and Induces Apoptosis via ROS Accumulation and ERK1/2 Signaling Pathway in Hepatocellular Carcinoma Cells.

INTRODUCTION: Saponin of Schizocapsa plantaginea Hance I (SSPH I), a novel bioactive phytochemical isolated from the rhizomes of Schizocapsa plantaginea, has been demonstrated to exhibit anti-cancer activity against various tumors in preclinical studies. However, the molecular mechanisms involved in the suppression of hepatocellular carcinoma (HCC) are poorly understood. The present study aimed at analyzing the effects of SSPH I on autophagy and apoptosis in vitro. METHODS: MTT and colony forming assays were used to detect cell viability and cell proliferation. Hoechst 33,258 staining and flow cytometry were used to determine apoptosis and ROS production. The apoptosis and autophagy-related protein expression levels were evaluated via Western blot assay. Characteristics of autophagy and apoptosis were observed by transmission electron microscopy. Lysosomal activity was stained with Lyso-Tracker Red and Magic Red Cathepsin B. RESULTS: The results showed that SSPH I exhibited potent anti-cancer activity and proliferation in HepG2 and BEL-7402 cells and inhibited HepG2 cells through inhibiting autophagy and promoting apoptosis. The mechanistic study indicated that the inhibition of autophagy of SSPH I was mediated by blocking autophagosome-lysosome fusion. Additionally, we found that SSPH I could mediate the activation of MAPK/ERK1/2 signaling pathway, and the use of NAC (ROS inhibitor) and U0126 (MEK1/2 inhibitor) converted the effect of SSPH I on apoptosis and autophagy in HepG2 cells. CONCLUSION: These data suggest that SSPH I induces tumor cells apoptosis and reduces autophagy in vitro by inducing ROS and activating MAPK/ERK1/2 signaling pathway, indicating that SSPH I might be a novel agent for the treatment of HCC.

The epigallocatechin gallate derivative Y6 reduces the cardiotoxicity and enhances the efficacy of daunorubicin against human hepatocellular carcinoma by inhibiting carbonyl reductase 1 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Green tea is the most ancient and popular beverage worldwide and its main constituent epigallocatechin-3-gallate (EGCG) has a potential role in the management of cancer through the modulation of cell signaling pathways. However, EGCG is frangible to oxidation and exhibits low lipid solubility and bioavailability, and we synthesized a derivative of EGCG in an attempt to overcome these limitations. AIM OF THE STUDY: The anthracycline antibiotic daunorubicin (DNR) is a potent anticancer agent. However, its severe cardiotoxic limits its clinical efficacy. Human carbonyl reductase 1 (CBR1) is one of the most effective human reductases for producing hydroxyl metabolites and thus may be involved in increasing the cardiotoxicity and decreasing the antineoplastic effect of anthracycline antibiotics. Accordingly, in this study, we investigated the co-therapeutic effect of Y6, a novel and potent adjuvant obtained by optimization of the structure of EGCG. MATERIAL AND METHODS: The cellular concentrations of DNR and its metabolite DNRol were measured by HPLC to determine the effects of EGCG and Y6 on the inhibition of DNRol formation. The cytotoxic effects of EGCG and Y6 were tested by MTT assay in order to identify non-toxic concentrations of them. To understand their antitumor and cardioprotective mechanisms, hypoxia-inducible factor-1α (HIF-1α) and CBR1 protein expression was measured via Western blotting and immunohistochemical staining while gene expression was analyzed using RT-PCR. Moreover, PI3K/AKT and MEK/ERK signaling pathways were analyzed via Western blotting. HepG2 xenograft model was used to detect the effects of EGCG and Y6 on the antitumor activity and cardiotoxicity of DNR in vivo. Finally, to obtain further insight into the interactions of Y6 and EGCG with HIF-1α and CBR1, we performed a molecular modeling. RESULTS: Y6(10 µg/ml or 55 mg/kg) decreased the expression of HIF-1α and CBR1 at both the mRNA and protein levels during combined drug therapy in vitro as well as in vivo, thereby inhibiting formation of the metabolite DNRol from DNR, with the mechanisms being related to PI3K/AKT and MEK/ERK signaling inhibition. In a human carcinoma xenograft model established with subcutaneous HepG2 cells, Y6(55 mg/kg) enhanced the antitumor effect and reduced the cardiotoxicity of DNR more effectively than EGCG(40 mg/kg). CONCLUSIONS: Y6 has the ability to inhibit CBR1 expression through the coordinate inhibition of PI3K/AKT and MEK/ERK signaling, then synergistically enhances the antitumor effect and reduces the cardiotoxicity of DNR.

The epigallocatechin gallate derivative Y6 inhibits human hepatocellular carcinoma by inhibiting angiogenesis in MAPK/ERK1/2 and PI3K/AKT/ HIF-1α/VEGF dependent pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypervascularity has been considered as one of the major features of many solid tumors. Green tea is one of the commonly drink resources in China, and its active component, Epigallocatechin gallate (EGCG), exhibits antiangiogenic activities in various experimental tumor models. However, EGCG has many shortages, e.g., relatively unstable, low lipid solubility, poor bioavailability, and short duration of action. AIM OF THE STUDY: To overcome the shortages of EGCG for antiangiogenic antitumor usage, our study developed a novel EGCG derivate, Y6(5,3',4',3″,4″,5″-6-0-ethyl-EGCG). The underlying mechanism was also elucidated. MATERIAL AND METHODS: we evaluated the effects of EGCG, Y6 on HCC and angiogenesis in vivo and in vitro. Moreover, to understand their antitumor mechanisms, key factors within angiogenesis-related signaling pathways (MAPK/ERK1/2, PI3K/AKT, HIF-1 VEGF) were analyzed by using western blot, immunohistochemistry (IHC), quantitative real-time quantitative PCR (RT-PCR). HepG2 xenograft model and the chorioallantoic membrane (CAM) were used to investigate the effects of Y6 and EGCG on tumors and anti-angiogenesis in vivo. Micro-vessel density (MVD) was analyzed by IHC of CD34 staining. IHC, qRT-PCR and Western blot were used to detect the expression of HIF-1α and VEGF protein in tumor tissues. The protein levels of MAPK/ERK1/2, PI3K/AKT, HIF-1α, and VEGF in tumor tissues were detected by western blot. RESULTS: Our results demonstrated that both EGCG and Y6 displayed antiangiogenetic and antitumor effects against HCC cells in vitro and in vivo. We found that rather than equal amount of EGCG, Y6 displayed better abilities in inhibiting the growth of HCC tumor cells, as well as inhibiting the growth of neovascularization in the chick embryos and HepG2 xenograft tumors bearing-mice, based on the data obtained from MTT assay, immunohistochemistry (IHC), chick chorioallantoic membrane (CAM) assays. In the comparison of equivalent dose of EGCG, qRT-PCR data showed that Y6 induced more significant decrease of the mRNA levels of HIF-1α and VEGF in supernatant-treated SMMC-7721 cells under hypoxic condition, as well as in the in xenograft tumor tissues; whereas Y6 also significantly reduced the protein levels of MAPK/ERK1/2, PI3K/AKT, HIF-1α, and VEGF to a greater extent than EGCG, determined by western blotting assay. CONCLUSIONS: our work suggests that the new EGCG derivate Y6 could significantly inhibit tumor growth and angiogenesis which is possibly involved with the signaling intervention of MAPK/ERK1/2 and PI3K/AKT/HIF-1α/VEGF pathways, and is supposed to be a potential therapeutic reagent for anti-angiogenesis treatment of solid tumors.

[Relationship between obstructive sleep apnea-hypopnea syndrome and high sensitivity C-reactive protein in non-obese subjects].

OBJECTIVE: To evaluate the relationship between high sensitivity C-reactive protein (hs-CRP) level and the severity of obstructive sleep apnea-hypopnea syndrome (OSAHS) in non-obese patients. METHODS: A total of 65 patients with suspected OSAHS were recruited. Those with a history of cardiovascular or cerebrovascular events, arterial hypertension, chronic obstructive pulmonary disease, diabetes mellitus and smoking were excluded. All subjects were examined by polysomnography. Subjects with an apnea-hypopnea index (AHI) ≥ 5 were considered to have OSAHS. They were divided into mild, moderate and severe OSAHS groups and those with an AHI < 5 were accepted into the control group. The serum level of hs-CRP, a biomarker for cardiovascular disease, was measured with peripheral venous blood samples. RESULTS: There were 53 males and 12 females with a mean age 44.3 ± 12.2 years and a body mass index (BMI) <25 kg/m(2). The serum levels of hs-CRP were significantly higher in OSAHS patients than those of the controls (geometric mean [95% confidence interval] 0.416 [0.288-0.600] vs 0.749 [0.559-1.003] mg/L, P < 0.05). Mean levels of hs-CRP did not differ between the group with mild-to-moderate OSAHS and the control group. However, the severe OSAHS group had a higher level of hs-CRP than the control group (P = 0.004). A significant positive relationship existed between hs-CRP and AHI (r = 0.407, P = 0.001) . According to stepwise multivariate analysis, only AHI was a reliable predictor for elevated serum level of hs-CRP among all these related factors (R(2) = 0.166, P = 0.001). CONCLUSION: The severity of OSAHS is associated with an elevated serum level of hs-CRP in non-obese patients.

Prevention of osteoporosis in mice after ovariectomy via allograft of microencapsulated ovarian cells.

It is believed that estrogen deficiency is one of the major risk factors associated with osteoporosis. To investigate the effects of the transplantation of microencapsulated ovarian cells in estrogen-deficient mice, ovarian cells from female Kunming (KM) mice (6-weeks old) were separated, cultured, and microencapsulated with alginic acid-polylysine-alginic acid. Female KM mice (8-weeks old) were randomly separated into three groups: intact (normal), ovariectomized (OVX), and treatment (OVX+ implantation). Microencapsulated ovarian cells were found to secrete estrogen at normal levels in vitro. Ninety days after transplantation, serum estradiol levels in the OVX group were significantly lower, and the trabecular bone amount and volume were decreased when compared with the normal group. The expression of alkaline phosphatase in chondrocytes appeared lower, while the expression of matrix metalloproteinase 9 (MMP-9) in the bone matrix was higher. The ratio of MMP-9-positive chondrocytes and osteoblasts to osteoclasts was significantly lower than that of the normal group. The concentrations of hydroxyproline (Hyp), Ca, and P in the left femurs of the OVX group were lower than those of the normal group. However, the aforementioned changes were not seen in the treatment group. In conclusion, microencapsulated ovarian cells survive well after transplantation and secrete estrogen in vivo, and they can prevent in some degree osteoporosis caused by ovariectomy.