Drug Resistance in Hepatocellular Carcinoma: Theoretical Basis and Therapeutic Aspects.

Primary liver cancer is one of the most common malignant tumors with high mortality and increasing incidence worldwide. Currently, chemotherapy is an important comprehensive treatment for moderate or advanced liver cancer. Despite the effective therapeutic effects initially achieved by chemotherapy, the high phenotypic and molecular heterogeneity of liver cancer cells facilitates resistance to conventional chemotherapy or targeted therapy and even leads to multidrug resistance (MDR), which is one of the major obstacles for clinical chemotherapy. Drug resistance exhibits multiple and complex molecular mechanisms to antagonize therapy under pharmacological pressure, including overexpression of drug efflux transporters, downstream adaptive response (such as apoptosis, autophagy, and endoplasmic reticulum stress), dysfunction of DNA damage repair (DDR), epigenetic modification, tumor microenvironment (TME) as well as extracellular matrix (ECM). In this paper, we summarize the recent research progress and intervention strategies for drug resistance in hepatocellular carcinoma (HCC), which will provide a promising therapeutic strategy for overcoming MDR in liver cancer.

NAMPT promotes the malignant progression of HBV-associated hepatocellular carcinoma through activation of SREBP1-mediated lipogenesis.

Metabolic reprogramming is a hallmark of cancer. The nicotinamide phosphoribosyltransferase (NAMPT)-mediated salvage pathway maintains sufficient cellular NAD levels and is required for tumorigenesis and development. However, the molecular mechanism by which NAMPT contributes to HBV-associated hepatocellular carcinoma (HCC) remains not fully understood. In the present study, our results showed that NAMPT protein was obviously upregulated in HBV-positive HCC tissues compared with HBV-negative HCC tissues. NAMPT was positively associated with aggressive HCC phenotypes and poor prognosis in HBV-positive HCC patients. NAMPT overexpression strengthened the proliferative, migratory, and invasive capacities of HBV-associated HCC cells, while NAMPT-insufficient HCC cells exhibited decreased growth and mobility. Mechanistically, we demonstrated that NAMPT activated SREBP1 (sterol regulatory element-binding protein 1) by increasing the expression and nuclear translocation of SREBP1, leading to the transcription of SREBP1 downstream lipogenesis-related genes and the production of intracellular lipids and cholesterol. Altogether, our data uncovered an important molecular mechanism by which NAMPT promoted HBV-induced HCC progression through the activation of SREBP1-triggered lipid metabolism reprogramming and suggested NAMPT as a promising prognostic biomarker and therapeutic target for HBV-associated HCC patients.

NAMPT promotes hepatitis B virus replication and liver cancer cell proliferation through the regulation of aerobic glycolysis.

Nicotinamide phosphoribosyltransferase (NAMPT) is a critical rate-limiting enzyme involved in NAD synthesis that has been shown to contribute to the progression of liver cancer. However, the potential role and mechanism of NAMPT in hepatitis B virus (HBV)-associated liver cancer remain unclear. The present study assessed the expression of NAMPT in HBV-positive and -negative liver cancer cells, and investigated whether HBV-induced NAMPT expression is dependent on HBV X protein (HBx). In addition, the role of NAMPT in HBV replication and transcription, and in HBV-mediated liver cancer cell growth was explored. The effects of NAMPT on the glycolytic pathway were also evaluated. Reverse transcription-quantitative PCR and western blotting results revealed that NAMPT expression levels were significantly higher in HBV-positive liver cancer cells than in HBV-negative liver cancer cells, and this effect was HBx-dependent. Moreover, the activation of NAMPT was demonstrated to be required for HBV replication and transcription. The NAMPT inhibitor FK866 repressed cell survival and promoted cell death in HBV-expressing liver cancer cells, and these effects were attenuated by nicotinamide mononucleotide. Furthermore, the inhibition of NAMPT was associated with decreased glucose uptake, decreased lactate production and decreased ATP levels in HBV-expressing liver cancer cells, indicating that NAMPT may promote the aerobic glycolysis. Collectively, these findings reveal a positive feedback loop in which HBV enhances NAMPT expression and the activation of NAMPT promotes HBV replication and HBV-mediated malignant cell growth in liver cancer. The present study highlights the important role of NAMPT in the regulation of aerobic glycolysis in HBV-mediated liver cancer, and suggests that NAMPT may be a promising treatment target for patients with HBV-associated liver cancer.

IQGAP1 promotes anoikis resistance and metastasis through Rac1-dependent ROS accumulation and activation of Src/FAK signalling in hepatocellular carcinoma.

BACKGROUND: Hepatitis B virus (HBV) has a crucial role in the progression of hepatocellular carcinoma (HCC). Tumour cells must develop anoikis resistance in order to survive before metastasis. This study aimed to investigate the mechanism of IQGAP1 in HBV-mediated anoikis evasion and metastasis in HCC cells. METHODS: IQGAP1 expression was detected by immunohistochemistry, real-time PCR and immunoblot analysis. Lentiviral-mediated stable upregulation or knockdown of IGAQP1, immunoprecipitation, etc. were used in function and mechanism study. RESULTS: IQGAP1 was markedly upregulated in HBV-positive compared with HBV-negative HCC cells and tissues. IQGAP1 was positively correlated to poor prognosis of HBV-associated HCC patients. IQGAP1 overexpression significantly enhanced the anchorage-independent growth and metastasis, whereas IQGAP1-deficient HCC cells are more sensitive to anoikis. Mechanistically, we found that HBV-induced ROS enhanced the association of IQGAP1 and Rac1 that activated Rac1, leading to phosphorylation of Src/FAK pathway. Antioxidants efficiently inhibited IQGAP1-mediated anoikis resistance and metastasis. CONCLUSIONS: Our study indicated an important mechanism by which upregulated IQGAP1 by HBV promoted anoikis resistance, migration and invasion of HCC cells through Rac1-dependent ROS accumulation and activation of Src/FAK signalling, suggesting IQGAP1 as a prognostic indicator and a novel therapeutic target in HCC patients with HBV infection.

PO-322 exerts potent immunosuppressive effects in vitro and in vivo by selectively inhibiting SGK1 activity.

BACKGROUND AND PURPOSE: Immunosuppressive drugs have shown great promise in treating autoimmune diseases in recent years. A series of novel oxazole derivatives were screened for their immunosuppressive activity. PO-322 [1H-indole-2,3-dione 3-(1,3-benzoxazol-2-ylhydrazone)] was identified as the most effective of these compounds. Here, we have investigated the mechanism(s) underlying the inhibition of T-cell proliferation in vitro by PO-322, as well as its effects on the delayed-type hypersensitivity (DTH) response and imiquimod-induced dermatitis in vivo. EXPERIMENTAL APPROACH: T-cell proliferation and apoptosis were analysed with flow cytometry. Cell viability was assessed with a CCK-8 assay. Protein kinase activity was assessed by SelectScreen Kinase Profiling Services. The phosphorylation of signal-regulated molecules was measured by Western blot. Cytokine levels were determined by elisa. The effect of PO-322 on DTH and imiquimod-induced dermatitis was evaluated in BALB/c mice. KEY RESULTS: PO-322 inhibited human T-cell proliferation with anti-CD3/anti-CD28 mAbs or alloantigen without significant cytotoxicity. Importantly, PO-322 was a selective inhibitor of the serum- and glucocorticoid-regulated kinase 1 (SGK1) and decreased NDRG1 phosphorylation but not p70S6K, STAT5, Akt, or ERK1/2 phosphorylation. Furthermore, PO-322 inhibited IFN-γ, IL-6, and IL-17 expression but not IL-10 expression. Finally, treatment with PO-322 was safe and effective for ameliorating the DTH response and imiquimod-induced dermatitis in mice. CONCLUSIONS AND IMPLICATIONS: PO-322 exerted immunosuppressive activity in vitro and in vivo by selectively inhibiting SGK1 activity. PO-322 represents a potential lead compound for the design and development of new drugs for the treatment of autoimmune diseases.

Activation of Toll-Like Receptor 3 Induces Interleukin-1 Receptor Antagonist Expression by Activating the Interferon Regulatory Factor 3.

Toll-like receptor 3 (TLR3) is a sensor of endogenous cell necrosis during the process of acute inflammation. Interleukin (IL)-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine and can negatively regulate the pathogenesis of inflammation. However, whether and how activation of TLR3 can regulate IL-1Ra expression has not been clarified. Here, we show that poly(I:C) induces IL-1Ra expression in primarily cultured human fibroblast-like synoviocytes and other types of cells. Induction of IL-1Ra by poly(I:C) was dependent on TLR3, but was independent of melanoma differentiation--associated protein 5 or retinoic acid-inducible gene I. Interferon regulatory factor 3 (IRF3) directly binds to the IL-1Ra promoter and promotes IL-1Ra expression in response to poly(I:C) stimulation. Induction of IL-1Ra by poly(I:C) was abolished by the inhibition of the NF-κB signaling, attenuated by the inhibition of the PI3K-Akt signaling, enhanced by inhibition of the ERK1/2 or MSK1/2 activation, but was independent of the p38 MAPK signaling. Treatment with poly(I:C) or Sendai virus elevated the levels of serum IL-1Ra in wild-type, but not in TLR3-/- or IRF3-/- mice. Our findings may provide new insights into the intrinsic anti-inflammatory function of TLR3 and double-stranded RNA-induced IL-Ra expression by TLR3 and its regulation.

A benzoxazole derivative PO-296 inhibits T lymphocyte proliferation by the JAK3/STAT5 signal pathway.

Immunosuppressants have shown striking achievements in treating autoimmune diseases in recent years. It is urgent to develop more immunosuppressants to provide more options for patients. PO-296 [2-(6-chlorobenzo[d]oxazol-2-yl)-4,5,6,7-tetrahydro-2H-indazol-3-ol] was identified as a novel benzoxazole derivative. We observed that it exhibits an obvious immunosuppressive activity to T lymphocytes. PO-296 significantly inhibited the proliferation of activated human T lymphocyte without cytotoxicity. Moreover, PO-296 did not affect the expression of cluster of differentiation (CD)-25 or CD69 but induced T lymphocyte cycle arrest in the G0/G1 phase. Furthermore, PO-296 inhibited interleukin (IL)-6, IL-17, and interferon gamma expression but had no effect on IL-2, IL-4, or IL-10. Yet, importantly, PO-296 inhibited the phosphorylation of signal transducer and activator of transcription 5 (STAT5), increased the phosphorylation of p70S6K, but did not affect the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mitogen-activated protein kinase pathway. In conclusion, these findings indicate that PO-296 inhibits human activated T-lymphocyte proliferation by affecting the janus kinase 3 (JAK3)/STAT5 pathway. PO-296 possesses a potential lead compound for the design and development of new immunosuppressants for the treatment of autoimmune diseases.

K313, a novel benzoxazole derivative, exhibits anti-inflammatory properties via inhibiting GSK3ß activity in LPS-induced RAW264.7 macrophages.

Benzoxazole and its derivatives have been widely studied in recent years due to their various biological properties. A previous study has demonstrated that K313 (1H-indole-2,3-dione 3-(1,3-benzoxazol-2-ylhydrazone)), a novel benzoxazole derivative, inhibits T cell proliferation to yield immunosuppressive effects. However, there are no related reports about its anti-inflammatory effects. In the present study, we investigated the anti-inflammatory properties and the underlying molecular mechanism of K313 in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. K313 dose-dependently (5, 10, and 20 µM) inhibited LPS-stimulated nitric oxide (NO), interleukin (IL)-6, tumor necrosis factor (TNF)-α, and 3-nitrotyrosine (3-NT) production and significantly decreased the gene transcription levels of inducible nitric oxide (iNOS), IL-6, and TNF-α. In addition, the results showed that the inflammatory cytokines suppressed by K313 were not regulated by p65 NF-κB, ERK1/2, AKT, or p38 MAPK. Instead, K313 increased phosphorylation of glycogen synthase kinase-3 beta (GSK-3ß) (Ser9) resulting in GSK-3ß deactivation. Moreover, in LPS-stimulated RAW264.7 macrophages, K313 and lithium chloride (LiCl) had a synergistic effect on the anti-inflammatory response. These results indicated that K313 exhibited anti-inflammatory properties and revealed the potential mechanism. K313 can increase GSK-3ß (Ser9) phosphorylation to decrease GSK-3ß activation in LPS-induced RAW264.7 macrophages.