CRISPR-Cas9-based genome-wide screening identified novel targets for treating sorafenib-resistant hepatocellular carcinoma: a cross-talk between FGF21 and the NRF2 pathway.

The treatment of hepatocellular carcinoma (HCC) has been dominated by multikinase inhibitors for more than a decade. However, drug resistance can severely restrict the efficacy of these drugs. Using CRISPR/CAS9 genome library screening, we evaluated Kelch-like ECH-associated protein 1 (KEAP1) as a key regulator of sorafenib's susceptibility in HCC. We also investigated whether KEAP1's knockdown can stabilize nuclear factor (erythroid-derived 2)-like 2 (NRF2) protein levels that led to sorafenib's resistance, including an NRF2 inhibitor that can synergize with sorafenib to abolish HCC's growth in vitro and in vivo. Furthermore, we clarified that fibroblast growth factor 21 (FGF21) is an important downstream regulator of NRF2 in HCC. Intriguingly, we observed that FGF21 bound to NRF2 through the C-terminus of FGF21, thereby stabilizing NRF2 by reducing its ubiquitination and generating a positive feedback loop in sorafenib-resistant HCC. These findings, therefore, propose that targeting FGF21 is a promising strategy to combat HCC sorafenib's resistance.

Photothermal therapy with AuNRs and EGFRmAb-AuNRs inhibits subcutaneous transplantable hypopharyngeal tumors in nude mice.

The present study aimed to investigate the effects of photothermal therapy with gold nanorods (AuNRs) or epidermal growth factor receptor monoclonal antibody­conjugated AuNRs (EGFRmAb­AuNRs) on hypopharyngeal carcinoma (HC) in nude mice. In addition, the associated signaling pathways were explored. Briefly, a subcutaneous transplantable hypopharyngeal tumor model was established in nude mice injected with FaDu human HC cells. A total of 70 nude mice were randomly divided into seven groups, each of which received a different treatment. Mice were treated with AuNRs, locally or through intravenous injection, whereas EGFRmAb or EGFRmAb­AuNRs were only administered locally. Near infrared spectroscopy (NIR) was also applied for plasmonic photothermal therapy (PPTT). The growth curve and the inhibitory rate for tumor growth were used to evaluate the effects of each treatment. Flow cytometry and the terminal­deoxynucleotidyl transferase dUTP nick end labeling assay were adopted to detect apoptosis of cells in the transplanted tumors. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to determine the mRNA and protein expression levels of target genes, respectively. Local treatment with AuNRs + NIR or EGFRmAb significantly inhibited tumor growth, and EGFRmAb conjugation further increased the inhibitory effects. Furthermore, there was a significant increase in apoptosis of tumor cells in the AuNRs + NIR, EGFRmAb and EGFRmAb­AuNRs + NIR groups; treatment with EGFRmAb­AuNRs + NIR induced the highest apoptotic effect. Mechanistic studies indicated that EGFRmAb­AuNRs + NIR may inhibit tumors through the AKT serine/threonine kinase (Akt) and DNA damage signaling pathways. In the AKT pathway, the mRNA and protein expression levels of phosphatase and tensin homolog were increased, whereas the expression levels of Akt and glycogen synthase kinase 3ß were decreased. In the DNA damage signaling pathway, the mRNA and protein expression levels of ATR serine/threonine kinase, checkpoint kinase 1 and p53 were enhanced, whereas phosphorylated­p53 protein expression was reduced. The present findings indicated that AuNRs + NIR inhibited HC tumor growth, and conjugating EGFRmAb to AuNRs further enhanced the inhibitory effects. EGFRmAb conjugation may increase the antitumor effects of AuNRs­induced PPTT by downregulating the phosphatidylinositol­3­kinase/Akt pathway and upregulating the DNA damage pathway. These findings may provide novel insights into tumor­targeting PPTT in vivo.

Photothermal treatment with EGFRmAb-AuNPs induces apoptosis in hypopharyngeal carcinoma cells via PI3K/AKT/mTOR and DNA damage response pathways.

Hypopharyngeal carcinoma (HC) is one of the most malignant tumors in the upper aerodigestive tract. Currently, there are no effective treatments for HC. Gold nanoparticles (AuNPs) are a promising tool that can be used for plasmonic photothermal therapy (PPTT), which refers to the use of electromagnetic radiation, most often in near infrared (NIR) region, for the treatment of various medical conditions including cancer. AuNPs have been proved to be a promising tool for NIR spectroscopy-mediated photothermal therapies. In this study, we chemically conjugated AuNPs with a monoclonal antibody (mAb) targeting the epidermal growth factor receptor (EGFR), a cell-surface receptor that is overexpressed in many cancers. We then assessed the effect of NIR photothermal treatment with the EGFRmAb-AuNPs in FaDu HC cells. Our data showed that nanoparticle conjugation with the EGFRmAb improved the specific targeting towards FaDu cells and reduced cytotoxicity towards normal (293 T) cells which do not overexpress the EGFR. A significant amount of our EGFRmAb-conjugated AuNPs could enter the nucleus. Moreover, the expression levels of double strand DNA break repair proteins, including p-ATR, p-CHK1, and p-CHK2 were increased following AuNPs treatment, indicating the presence of DNA damage. These findings suggest that the AuNPs can potentially disrupt genome integrity and induce apoptosis. In addition, EGFRmAb-AuNPs+NIR could induce FaDu cell apoptosis, accompanied by the inhibition of the PI3K/AKT/mTOR pathway and stimulation of DNA damage response. Based on these data, PPTT using the EGFRmAb-AuNPs could be a new promising treatment for HC.

[Determination of polysaccharides content of Gentiana farreri from different producing areas based on anthrone-sulfuric acid method].

Study a method for the detemination of the content of polysaccharides in Gentiana farreri, and analysis of the content of polysaccharides from different producing areas. The results showed that using the anthrone-sulfuric acid method, simple operation, accurate result. Sample was measured at 620 nm absorbance after anthrone-sulfuric acid color, at this wavelength, solution absorption and glucose showed a good linear relationship; The linearity was in the range of 0.01-0.07 g x L(-1) (r = 0.996 7). The recovery rate was 99.41%, with RSD of 2.0%. Considering the experimental conditions, to determine the solid-liquid ratio 1:60, extracting time 50 min, concentration of ethanol 80%. The mass fraction of polysaccharides was the highest to reached 0.743% in G. farreri from Gansu Xiahe. This experiment has laid a good foundation for further study on G. farreri.

Short-term microbiological effects of scaling and root planing and essential-oils mouthwash in Chinese adults.

OBJECTIVE: To assess the short-term effect of scaling and root planing (SRP) and essential-oils mouthwash on the levels of specific bacteria in Chinese adults. METHODS: Fifty Chinese adults with chronic periodontitis were randomly assigned to full-mouth SRP or a 7-d essential-oils mouthwash regimen. In addition, 22 periodontally healthy adults used essential-oils mouthwash for 7 d. Clinical examination and plaque/saliva sampling were performed at baseline and on Day 7. Quantitative real-time polymerase chain reaction (PCR) was used to measure Aggregatibacter actinomycetemcomitans (Aa), Fusobacterium nucleatum (Fn), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and total bacterial loads in saliva, supra- and sub-gingival plaque samples. RESULTS: The detection frequencies of four tested species remained unchanged after either treatment. However, the bacterial loads of Fn, Pg, and Pi were significantly reduced by SRP; the mean reduction of bacterial counts in saliva ranged from 52.2% to 62.5% (p<0.01), in supragingival plaque from 68.2% to 81.0% (p<0.05), and in subgingival plaque from 67.9% to 93.0% (p<0.01). Total bacterial loads were reduced after SRP in supra- and sub-gingival plaque (p<0.05). Essential-oils mouthwash reduced Fn levels in supragingival plaque by a mean of 53.2%, and reduced total bacterial loads in supra- and sub-gingival plaque (p<0.01). In subgingival plaque from periodontal patients, Pg and Pi reductions were high after SRP compared to essential-oils mouthwash (93.0% vs. 37.7% and 87.0% vs. 21.0%, p<0.05). No significant bacterial reduction was observed in periodontally healthy subjects using essential-oils mouthwash. CONCLUSIONS: SRP and essential-oils mouthwash both have an impact on saliva and gingival plaque flora in Chinese periodontitis patients in 7 d, with greater microbiological improvement by SRP.