Discovery of a nitroaromatic nannocystin with potent in vivo anticancer activity against colorectal cancer by targeting AKT1.

The development of targeted chemotherapeutic agents against colorectal cancer (CRC), one of the most common cancers with a high mortality rate, is in a constant need. Nannocystins are a family of myxobacterial secondary metabolites featuring a 21-membered depsipeptide ring. The in vitro anti-CRC activity of natural and synthetic nannocystins was well documented, but little is known about their in vivo efficacy and if positive, the underlying mechanism of action. In this study we synthesized a nitroaromatic nannocystin through improved preparation of a key fragment, and characterized its in vitro activity and in vivo efficacy against CRC. We first described the total synthesis of compounds 2-4 featuring Heck macrocyclization to forge their 21-membered macrocycle. In a panel of 7 cancer cell lines from different tissues, compound 4 inhibited the cell viability with IC values of 1-6 nM. In particular, compound 4 (1, 2, 4 nM) inhibited the proliferation of CRC cell lines (HCT8, HCT116 and LoVo) in both concentration and time dependent manners. Furthermore, compound 4 concentration-dependently inhibited the colony formation and migration of CRC cell lines. Moreover, compound 4 induced cell cycle arrest at sub-G1 phase, apoptosis and cellular senescence in CRC cell lines. In three patient-derived CRC organoids, compound 4 inhibited the PDO with IC values of 3.68, 28.93 and 11.81 nM, respectively. In a patient-derived xenograft mouse model, injection of compound 4 (4, 8 mg/kg, i.p.) every other day for 12 times dose-dependently inhibited the tumor growth without significant change in body weight. We conducted RNA-sequencing, molecular docking and cellular thermal shift assay to elucidate the anti-CRC mechanisms of compound 4, and revealed that it exerted its anti-CRC effect at least in part by targeting AKT1.

Tyrosine metabolic reprogramming coordinated with the tricarboxylic acid cycle to drive glioma immune evasion by regulating PD-L1 expression.

Due to the existence of the blood-brain barrier in glioma, traditional drug therapy has a poor therapeutic outcome. Emerging immunotherapy has been shown to have satisfactory therapeutic effects in solid tumors, and it is clinically instructive to explore the possibility of immunotherapy in glioma. We performed a retrospective analysis of RNA-seq data and clinical information in 1027 glioma patients, utilizing machine learning to explore the relationship between tyrosine metabolizing enzymes and clinical characteristics. In addition, we also assessed the role of tyrosine metabolizing enzymes in the immune microenvironment including immune infiltration and immune evasion. Highly expressed tyrosine metabolizing enzymes 4-hydroxyphenylpyruvate dioxygenase, homogentisate 1,2-dioxygenase, and fumarylacetoacetate hydrolase not only promote the malignant phenotype of glioma but are also closely related to poor prognosis. The expression of tyrosine metabolizing enzymes could distinguish the malignancy degree of glioma. More importantly, tyrosine metabolizing enzymes regulate the adaptive immune process in glioma. Mechanistically, multiple metabolic enzymes remodel fumarate metabolism, promote α-ketoglutarate production, induce programmed death-ligand 1 expression, and help glioma evade immune surveillance. Our data suggest that the metabolic subclass driven by tyrosine metabolism provides promising targets for the immunotherapy of glioma.

Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2.

AIM: To investigate the mechanism underlying the increase of hepatoma cell proliferation by hepatitis B virus X protein (HBx). METHODS: HepG2, H7402 and HepG2.2.15 cells, which constitutively replicated hepatitis B virus were used. The effects of HBx on hepatoma cell proliferation were examined using 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and MTT assay. The expression level of MEKK2 was measured using RT-PCR, Western blot and luciferase reporter gene assay. The activity of activator protein 1 (AP-1) was detected using luciferase reporter gene assay. The phosphorylation levels of JNK and c-Jun were measured using Western blot. The expression levels of HBx and MEKK2 in 11 clinical hepatocellular carcinoma (HCC) tissues were measured using real time PCR and Western blot. In addition, the expression of MEKK2 in 95 clinical HCC tissues was examined using immunohistochemistry. RESULTS: HBx significantly enhanced HepG2-X cell proliferation. In HepG2-X, H7402-X and HepG2.2.15 cells, the expression level of MEKK2 was remarkably increased. In HepG2.2.15 cells, HBx was found to activate JNK and AP-1, which were the downstream effectors of MEKK2 in HepG2-X and HepG2.2.15 cells. In 11 clinical HCC tissues, both HBx and MEKK2 expression levels were remarkably increased, as compared to those in the corresponding peritumor tissues. In 95 clinical HCC tissues, the rate of detection of MEKK2 was 85.3%. CONCLUSION: HBx promotes hepatoma cell proliferation via upregulating MEKK2, which may be involved in hepatocarcinogenesis.

Anti-hepatitis B virus X protein in sera is one of the markers of development of liver cirrhosis and liver cancer mediated by HBV.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC). However, the significance of circulating antibody to hepatitis B virus X antigen (anti-HBx) in sera remains unclear. In the present study, we examined the titers of anti-HBx (IgG) in the sera from 173 patients with chronic hepatitis B (CHB), 106 liver cirrhosis (LC), and 61 HCC by enzyme-linked immunosorbent assay (ELISA), respectively. Our data showed that the positive rates of anti-HBx were higher in sera of LC (40.6%) and HCC (34.4%) than those of CHB (10.4%), P < .05. In all 40 patients with anti-HBx+ out of 340 patients, 39 (97.5%) were HBsAg/HBeAg/anti-HBc+ and 1 (2.5%) was anti-HBs+ (P < .01), suggesting that anti-HBx in sera is a marker of HBV replication rather than a protective antibody. Thus, our findings reveal that circulating anti-HBx in sera is one of the markers of development of LC and HCC mediated by HBV.

Gene expression profiles of human liver cells mediated by hepatitis B virus X protein.

AIM: To demonstrate the gene expression profiles mediated by hepatitis B virus X protein (HBx), we characterized the molecular features of pathogenesis associated with HBx in a human liver cell model. METHODS: We examined gene expression profiles in L-O2-X cells, an engineered L-O2 cell line that constitutively expresses HBx, relative to L-O2 cells using an Agilent 22 K human 70-mer oligonucleotide microarray representing more than 21,329 unique, well-characterized Homo sapiens genes. Western blot analysis and RNA interference (RNAi) targeting HBx mRNA validated the overexpression of proliferating cell nuclear antigen (PCNA) and Bcl-2 in L-O2-X cells. Meanwhile, the BrdU incorporation assay was used to test cell proliferation mediated by upregulated cyclooxygenase-2 (COX-2). RESULTS: The microarray showed that the expression levels of 152 genes were remarkably altered; 82 of the genes were upregulated and 70 genes were downregulated in L-O2-X cells. The altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes. PCNA and Bcl-2 were upregulated in L-O2-X cells. Furthermore, we found that COX-2 upregulation in L-O2-X cells enhanced proliferation using the BrdU incorporation assay, whereas indomethacin (an inhibitor of COX-2) abolished the promotion. CONCLUSION: Our findings provide new evidence that HBx is able to regulate many genes that may be involved in the carcinogenesis. These regulated genes mediated by HBx may serve as molecular targets for the prevention and treatment of hepatocellular carcinoma.

Identification of a natural mutant of HBV X protein truncated 27 amino acids at the COOH terminal and its effect on liver cell proliferation.

AIM: To identify mutants of the hepatitis B virus (HBV) X (HBx) gene and investigate the effect of the natural mutant on liver cell proliferation. METHODS: We identified natural mutants of the HBx gene from 188 sera and 48 tissues of Chinese patients infected with HBV by PCR, respectively. Based on the identification of the mutants of HBx gene, we cloned the fragments of the mutants into the pcDNA3 vector. The biological activities of the mutants were investigated. RESULTS: We identified a natural mutant of the HBx gene with deletion from 382 to 401 base pairs from 3 sera out of 188 patients, which resulted in the expression deletion of the HBx protein from the 128th amino acid at the COOH terminal. The similar mutant with deletion from 382 base pair at the COOH terminal was identified from 5 cases of genomes out of 48 hepatocellular carcinoma tissues. Regarding the biological activities of the mutant, we found that the mutant of the HBx protein failed to induce apoptosis by transient transfection, but promoted proliferation of human liver immortalized L-O2 cells by stable transfection, compared with the wild-type HBx protein. The data showed that the proliferation of the mutant stably-transfected L-O2-X-Sera cells and fragment stably-transfected L-O2-XDelta127 cells was enhanced by the BrdU incorporation assay and flow cytometry analysis. Luciferase reporter gene assay showed that the transcriptional activities of NF-kappaB, survivin, and human telomerase reverse transcriptase were upregulated, and Western blot analysis revealed that the expression levels of c-Myc and proliferating cell nuclear antigen (PCNA) were upregulated in the cells. CONCLUSION: Our findings suggest that the natural HBx mutant truncated 27 amino acids at the COOH terminal promotes cell proliferation.

NF-kappaB downregulation may be involved the depression of tumor cell proliferation mediated by human mesenchymal stem cells.

AIM: It has been reported that stem cells are able to home to tumorigenesis and inhibit the proliferation of tumor cells. The purpose of our study was to demonstrate the molecular mechanism of the inhibitory proliferation of hepatoma cells and breast cancer cells mediated by human mesenchymal stem cells (hMSCs). METHODS: The proliferation of H7402 human hepatoma cells and MCF-7 human breast cancer cells was measured by the 5-bromodeoxyuridine (BrdU) incorporation assay and flow cytometry assay after the treatment with conditioned media from hMSCs culture, such as Z3 cells or BMMS-03 cells. The role of NF-kappaB or the phosphorylation of inhibitor kappaBalpha (p-IkappaBalpha) in the depression of hepatoma or breast cancer cells treated with conditioned media from Z3 cells or BMMS-03 cells was examined by reporter gene assay, quantitative real-time PCR, and Western blot analysis, respectively. RESULTS: The proliferation of H7402 cells and MCF-7 cells was decreased significantly by the BrdU incorporation assay and flow cytometry assay after treatment. The transcriptional activity and mRNA level of NF-kappaB were downregulated in the treated cells by reporter gene assay and quantitative real-time PCR in a dose-dependent manner. At the protein level, NF-kappaB and p-IkappaBalpha decreased in the treated cells by Western blot analysis. CONCLUSION: Conditioned media from hMSCs are able to inhibit the proliferation of tumor cells. NF-kappaB downregulation is one of reasons for the depression of tumor cell proliferation mediated by hMSCs.