Runzhi Zhu

Background: Liver cancer is a leading cause of cancer death worldwide, and novel prognostic factor is needed for early detection and therapeutic responsiveness monitoring. The orphan nuclear receptor NR0B2 was reported to suppress liver cancer development in a mouse model, and its expression levels were reduced in liver cancer tissues and cell lines due to hypermethylation within its promoter region. However, it is not clear if NR0B2 expression is associated with cancer survival or disease progression and how NR0B2 gene expression is regulated at the molecular level. Methods: Multiple cancer databases were utilized to explore NR0B2 gene expression profiles crossing a variety of human cancers, including liver cancers, on several publicly assessable bioinformatics platforms. NR0B2 gene expression with or without kinase inhibitor treatment was analyzed using the qPCR technique, and NR0B2 protein expression was assessed in western blot assays. Two human hepatocellular carcinoma cell lines HepG2 and Huh7, were used in these experiments. NR0B2 gene activation was evaluated using NR0B2 promoter-driven luciferase reporter assays. Results: NR0B2 gene is predominantly expressed in liver tissue crossing human major organs or tissues, but it is significantly downregulated in liver cancers. NR0B2 expression is mostly downregulated in most common cancers but also upregulated in a few intestinal cancers. NR0B2 gene expression significantly correlated with patient overall survival status in multiple human malignancies, including lung, kidney, breast, urinary bladder, thyroid, colon, and head-neck cancers, as well as liposarcoma and B-cell lymphoma. In liver cancer patients, higher NR0B2 expression is associated with favorite relapse-free and progression-free survival, especially in Asian male patients with viral infection history. In addition, NR0B2 expression negatively correlated with immune infiltration and PIK3CA and PIK3CG gene expression in liver cancer tissues. In HepG2 and Huh7 cells, NR0B2 expression at the transcription level was drastically reduced after MAPK inhibition but was significantly enhanced after PI3K inhibition. Conclusion: NR0B2 gene expression is altered mainly in most human malignancies and significantly reduced in liver cancers. NR0B2 is a prognosis factor for patient survival in liver cancers. MAPK and PI3K oppositely modulate NR0B2 expression, and NR0B2 gene upregulation might serve as a therapeutic responsiveness factor in anti-PI3K therapy for liver cancer.

Ovarian cancer (OC) is the second most frequent gynecological cancer and is associated with a poor prognosis because OC progression is often asymptoma-tic and is detected at a late stage. There remains an urgent need for novel targeted therapies to improve clinical outcomes in ovarian cancer. As a nitric oxide prodrug, JS-K is reported highly cytotoxic to human cancer cells such as acute myeloid leukemia, multiple myeloma and breast cancer. This study is aim to investigate the influence of JS-K on proliferation and apoptosis in ovarian cancer cells and explored possible autophagy-related mechanisms, which will contribute to future ovarian cancer therapy and supply theory support that JS-K holds great promise as a novel therapeutic agent against ovarian cancer. The cytotoxicity, extracellular ROS/RNS activity and apoptotic effect of JS-K and indicated inhibitors on ovarian cancer cells in vitro were evaluated by MTT assay, extracellular ROS/RNS assay, caspases activities assay and western blot. Further autophagy effect of JS-K and indicated inhibitors were examined by MTT assay, cell transfection, immunofluorescence analysis, transmission electron microscopy (TEM) analysis and western blot on ovarian cancer cells in vitro. In vivo, the BALB/c-nude female mice with SKOV3 ovarian cancer cells xenograft were used to examine the efficacy of JS-K treatment on tumor growth. PCNA and p62 proteins were analyzed by immunohistochemistry. In vitro, JS-K inhibited the proliferation of ovarian cancer cells, induced apoptosis and cell nucleus shrinkage, enhanced the enzymatic activity of caspase-3/7/8/9, and significantly increased the production of ROS/RNS in ovarian cancer A2780 and SKOV3 cells, these effects were attenuated by inhibition of NAC. In addition, JS-K induced autophagy-related proteins and autophagosomes changes in ovarian cancer A2780 and SKOV3 cells. In vivo, JS-K inhibited tumor growth, decreased p62 protein expression and increased the expression levels of PCNA in xenograft models which were established using SKOV3 ovarian cancer cells. Taken together, we demonstrated that ROS/RNS stress-mediated apoptosis and autophagy are mechanisms by which SKOV3 cells undergo cell death after treatment with JS-K in vitro. Moreover, JS-K inhibited SKOV3 tumor growth in vivo. An alternative therapeutic approach for triggering cell death in cancer cells could constitute a useful multimodal therapies for treating ovarian cancer, which is known for its resistance to apoptosis-inducing drugs.

JIB‑04 is a structurally unique small molecule, known to exhibit anticancer activity and to inhibit the growth of human lung cancer and prostate cancer cell lines. However, the anticancer effect of JIB‑04 against human hepatic carcinoma, and its underlying mechanisms, are still unclear. In the present study, MHCC97H and HepG2 cells were employed to investigate the anticancer effects of JIB‑04 on cell viability and apoptosis. Annexin V/PI staining, a CCK‑8 assay and western blot analysis demonstrated that JIB‑04 induced apoptosis in MHCC97H and HepG2 cells, which was evidenced by the expression of proapoptotic and apoptotic proteins including p53, Bak, Bax, caspase‑3 and caspase‑9. Subsequently, the expression trends of Bcl‑2 and p53 were reversed after co‑treatment with pifithrin‑α (PFT‑α, a p53 inhibitor). The results revealed that JIB‑04 suppressed the cell viability of MHCC97H and HepG2 cells in a concentration‑dependent manner. Meanwhile, it was also demonstrated that JIB‑04 effectively triggered MHCC97H and HepG2 cell apoptosis by downregulating Bcl‑2/Bax expression, and upregulating proapoptotic and apoptotic protein expression via the p53/Bcl2/caspase signaling pathway. JIB‑04 had effects on the inhibition of cell viability and the induction of apoptosis in MHCC97H and HepG2 cells. The underlying mechanism of action of JIB‑04 was associated with the p53/Bcl‑2/caspase signaling pathway. Our findings provide a foundation for understanding the anticancer effect of JIB‑04 on MHCC97H and HepG2 cells, and suggested that JIB‑04 may be a promising therapeutic agent in human liver cancer.

Tetramethylpyrazine (TMP) has been widely used in the treatment of ischemic cerebrovascular disease. However, the mechanism of TMP and how to increase its bioavailability need to be further explored. In our study, an in vivo microdialysis sampling technique coupled with ultra‐performance liquid chromatography–mass spectrometry method was developed to investigate the pharmacokinetic properties of TMP and its interaction with different doses of borneol (BO) in rats. Linearity of TMP in brain and blood dialysates exhibited good linear relationships over the concentration range of 0.991–555.14 ng/mL. The specificity, linearity, accuracy, precision, matrix effect and stability were within acceptable ranges. The results demonstrated that BO had a marked impact on the pharmacokinetic properties of TMP. After co‐administration, the areas under the concentration–time curve (AUC) of TMP in brain and blood were significantly increased. Meanwhile, the peak concentration of TMP in brain was also enhanced. The AUC_Brain/AUC_Blood of TMP, increased from 44% to 56 and 60.8% after co‐administration with BO (15 and 30 mg/kg). The pharmacodynamic results showed that TMP co‐administration with BO enhanced the cerebral blood flow during the period of ischemia and reduced the infarct volume. Overall, it might be an effective way to treat stroke to use TMP co‐administered with BO.

Diosmetin (DIOS), a flavonoid compound, is abundant in Citrus limon. Emerging studies have shown that DIOS is an effective compound implicated in multiple types of cancer. However, whether DIOS serves a role in hepatocellular carcinoma (HCC) is still obscure. HepG2 cells were used in the present study, and it was observed that DIOS exhibited antitumor activity against liver cancer cells. Western blotting was performed to evaluate cell apoptosis and survival-associated proteins, and the results demonstrated that DIOS treatment resulted in the activation of the p53‑dependent apoptosis pathway. Our results revealed that DIOS caused inhibition of the nuclear factor (NF)‑κB signaling pathway and downregulation of Notch3 receptor. Furthermore, by using small hairpin RNA‑Notch3, it was confirmed that DIOS inhibited the NF‑κB signaling pathway by inactivation of Notch3. In conclusion, the present results demonstrated that DIOS triggered cell apoptosis by activation of the p53 signaling pathway and inhibited the NF‑κB cell survival pathway by downregulation of Notch3 receptor expression. DIOS is a potential agent for prevention of HCC.

Hepatocellular carcinoma (HCC), which is a type of malignant tumor, is the fifth most common cancer in men and ninth in women worldwide. The aim of the present study was to investigate the antitumor effect of diosmetin (DIOS) in hepatocellular carcinoma HepG2 cells. The proliferation, apoptosis and autophagy rates of HepG2 cells were measured following treatment with DIOS. The effects of DIOS treatment on HepG2 cell proliferation and apoptosis rates were analyzed using MTT assays and Annexin V staining, respectively. The effect of DIOS treatment on autophagy levels was assessed using transmission electron microscopy, green fluorescent protein (GFP)‑microtubule‑associated protein 1 light chain (LC3) transfection and LysoTracker Red staining. Furthermore, bafilomycin A1 (BA1), an autophagy inhibitor, was used to assess the association between DIOS and cell autophagy, proliferation and apoptosis. In addition, the expression of autophagy‑related proteins [mammalian target of rapamycin (mTOR), phosphatidylinositol 3‑kinase, P70S6K, phosphoinositide‑dependent kinase‑1, extracellular signal‑regulated kinase, 5'‑AMP‑activated protein kinase and Akt] and apoptosis‑related proteins [B‑cell lymphoma (Bcl)‑2‑associated X protein, Bak, p53, Bcl‑2 and caspase‑3] were analyzed by western blotting. The results revealed that DIOS significantly inhibited proliferation (P<0.01) and induced apoptosis (P<0.001) in HepG2 cells. It was also demonstrated that DIOS triggered autophagy by regulating the mTOR pathway in HepG2 cells. Notably, following treatment of HepG2 cells with the autophagy inhibitor, BA1, the expression of apoptosis‑related proteins, including Bax, Bak and p53, were significantly decreased (P<0.05), and cell viability was recovered to a certain extent. In conclusion, DIOS inhibits cell proliferation and induces apoptosis in HepG2 cells via regulation of the mTOR pathway. Thus, the results of the current study indicate that DIOS may present a potential therapeutic agent for HCC treatment.

Chrysin is a major active ingredient of flavonoids, known to exhibit protective effects against various types of cancer. However, the anticancer role of chrysin against hepatocellular carcinoma (HCC) and the underlying molecular mechanisms remain unclear. In order to evaluate the effects of chrysin on cell viability and apoptosis in human HCC, HepG2 and QGY7701 cells were used in the present study. Cell viability was monitored using an MTT assay. In addition, an Annexin V‑fluorescein isothiocyanate/propidium iodide kit was used for the labeling of the apoptotic cells, which were then measured using flow cytometry. Western blotting was used to examine the protein expression of p53, B‑cell lymphoma‑2 (Bcl‑2), Bcl‑2‑associated X (Bax), Bcl‑2‑associated death promoter (Bad), Bcl‑2 homologous antagonist/killer (Bak), caspases‑3 and ‑9, and cleaved‑caspases‑3 and ‑9. The results of the present study revealed that chrysin suppressed the cell viability of HepG2 and QGY7701 cells in a concentration‑dependent manner. In addition, chrysin induced significant apoptosis in HepG2 and QGY7701 cells. Furthermore, it was demonstrated that chrysin treatment increased the expression of proapoptotic proteins, including p53, Bax, Bad and Bak, while it decreased the protein level of antiapoptotic protein Bcl‑2. It was also demonstrated that chrysin induced apoptosis in the HCC cells by regulating the p53/Bcl‑2/caspase‑9 signaling pathway. In conclusion, the results of the present study suggested that chrysin may be a potential candidate agent for the induction of cell apoptosis in human HCC.

Chemotherapy is an effective weapon in the battle against cancer. Nedaplatin (NDP) is an improved platinum-containing drug with lower cytotoxicity than other similar drugs. However, the repeated use of NDP results in substantial hepatocyte damage as well as drug resistance in hepatocellular carcinoma (HCC) cases. Therefore, the development of effective chemotherapy strategies that enhance tumor sensitivity to chemotherapeutics and reduce the secondary damage to liver cells is urgently needed. Dihydromyricetin (DHM), a natural flavonoid compound, has been shown to have antitumor activity with no obvious toxicity to normal cells in vitro and in vivo. In this study, DHM and NDP were combined to treat liver cancer cells; we found that DHM functions as a protector of normal cells compared with the use of NDP alone. In addition, the synergy of DHM with NDP enhanced the effect of NDP on the induction of HCC cell apoptosis. We found that the combination caused clear changes in the level of reactive oxygen species (ROS). Furthermore, we demonstrated that the combination of DHM and NDP activated the p53/Bcl-2 signaling pathway, which resulted in mitochondrial dysfunction and induced cell death and growth inhibition in HCC cells.

Notch3 functions as a regulator of cell self-renewal by interacting with the β-catenin pathway in hepatocellular carcinoma Notch3 functions as a regulator of cell self-renewal by interacting with the β-catenin pathway in hepatocellular carcinoma

To investigate the hepatoprotective effects of phycocyanobilin (PCB) in reducing hepatic injury and accelerating hepatocyte proliferation following carbon tetrachloride (CCl4) treatment. C57BL/6 mice were orally administered PCB 100 mg/kg for 4 d after CCl4 injection, and then the serum and liver tissue of the mice were collected at days 1, 2, 3, 5 and 7 after CCl4 treatment. A series of evaluations were performed to identify the curative effects on liver injury and recovery. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin and superoxide dismutase (SOD) were detected to indirectly assess the anti-inflammatory effects of PCB. Meanwhile, we detected the expressions of hepatocyte growth factor, transforming growth factor alpha (TGF-α), TGF-β, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), the factors which are associated with inflammation and liver regeneration. The protein expressions of proliferating cell nuclear antigen (PCNA), TNF-α and cytochrome C were detected by western blot. Furthermore, the survival rates were analyzed of mice which were administered a lethal dose of CCl4 (2.6 mg/kg) with or without PCB. In our research, PCB showed a strongly anti-inflammatory effect on CCl4-induced liver injury in mice. The ALT was significantly decreased after CCl4 treatment from day 1 (P < 0.01) and the AST was significantly decreased from day 2 (P < 0.001). Both albumin and liver SOD were increased from day 2 (P < 0.001 and P < 0.01), but serum SOD levels did not show a significant increase (P > 0.05). PCB protected the structure of liver from the injury by CCl4. TUNEL assay showed that PCB dramatically reduced the number of apoptotic cells after CCl4 treatment compared to the control (101.0 ± 25.4 vs 25.7 ± 6.4, P < 0.01). The result of western blotting showed that PCB could increase PCNA expression, decrease TNF-α and cytochrome C expression. Furthermore, data shows that PCB could improve the survival rate of acute liver failure (ALF) mice which were injected with a lethal dose of CCl4 (60.0% vs 20.0%). Our study indicated that PCB could be an ideal candidate for reversing acute liver injury or ALF.