miR156-PvSPL2 controls culm development by transcriptional repression of switchgrass CYTOKININ OXIDASE/DEHYDROGENASE4.

Culm development in grasses can be controlled by both miR156 and cytokinin. However, the crosstalk between the miR156-SPL module and the cytokinin metabolic pathway remains largely unknown. Here, we found CYTOKININ OXIDASE/DEHYDROGENASE4 (PvCKX4) plays a negative regulatory role in culm development of the bioenergy grass Panicum virgatum (switchgrass). Overexpression of PvCKX4 in switchgrass reduced the internode diameter and length without affecting tiller number. Interestingly, we also found that PvCKX4 was always upregulated in miR156 overexpressing (miR156OE) transgenic switchgrass lines. Additionally, upregulation of either miR156 or PvCKX4 in switchgrass reduced the content of isopentenyl adenine (iP) without affecting trans-zeatin (tZ) accumulation. It is consistent with the evidence that the recombinant PvCKX4 protein exhibited much higher catalytic activity against iP than tZ in vitro. Furthermore, our results showed that miR156-targeted SPL2 bound directly to the promoter of PvCKX4 to repress its expression. Thus, alleviating the SPL2-mediated transcriptional repression of PvCKX4 through miR156 overexpression resulted in a significant increase in cytokinin degradation and impaired culm development in switchgrass. On the contrary, suppressing PvCKX4 in miR156OE transgenic plants restored iP content, internode diameter, and length to wild-type levels. Most strikingly, the double transgenic lines retained the same increased tiller numbers as the miR156OE transgenic line, which yielded more biomass than the wild type. These findings indicate that the miR156-SPL module can control culm development through transcriptional repression of PvCKX4 in switchgrass, which provides a promising target for precise design of shoot architecture to yield more biomass from grasses.

UDP-glycosyltransferase UGT96C10 functions as a novel detoxification factor for conjugating the activated dinitrotoluene sulfonate in switchgrass.

Dinitrotoluene sulfonates (DNTSes) are highly toxic hazards regulated by the Resource Conservation and Recovery Act (RCRA) in the United States. The trinitrotoluene (TNT) red water formed during the TNT purification process consists mainly of DNTSes. Certain plants, including switchgrass, reed and alfalfa, can detoxify low concentrations of DNTS in TNT red water-contaminated soils. However, the precise mechanism by which these plants detoxify DNTS remains unknown. In order to aid in the development of phytoremediation resources with high DNTS removal rates, we identified and characterized 1-hydroxymethyl-2,4-dinitrobenzene sulfonic acid (HMDNBS) and its glycosylated product HMDNBS O-glucoside as the degradation products of 2,4-DNT-3-SO3Na, the major isoform of DNTS in TNT red water-contaminated soils, in switchgrass via LC-MS/MS- and NMR-based metabolite analyses. Transcriptomic analysis revealed that 15 UDP-glycosyltransferase genes were dramatically upregulated in switchgrass plants following 2,4-DNT-3-SO3Na treatment. We expressed, purified and assayed the activity of recombinant UGT proteins in vitro and identified PvUGT96C10 as the enzyme responsible for the glycosylation of HMDNBS in switchgrass. Overexpression of PvUGT96C10 in switchgrass significantly alleviated 2,4-DNT-3-SO3Na-induced plant growth inhibition. Notably, PvUGT96C10-overexpressing transgenic switchgrass plants removed 83.1% of 2,4-DNT-3-SO3Na in liquid medium after 28 days, representing a 3.2-fold higher removal rate than that of control plants. This work clarifies the DNTS detoxification mechanism in plants for the first time, suggesting that PvUGT96C10 is crucial for DNTS degradation. Our results indicate that PvUGT96C10-overexpressing plants may hold great potential for the phytoremediation of TNT red water-contaminated soils.

High-resolution genome mapping and functional dissection of chlorogenic acid production in Lonicera maackii.

Amur honeysuckle (Lonicera maackii) is a widely used medicinal plant of the Caprifoliaceae family that produces chlorogenic acid. Research on this plant mainly focuses on its ornamental value and medicinal compounds, but a reference genome sequence and molecular resources for accelerated breeding are currently lacking. Herein, nanopore sequencing and high-throughput chromosome conformation capture (Hi-C) allowed a chromosome-level genome assembly of L. maackii (2n = 18). A global view of the gene regulatory network involved in the biosynthesis of chlorogenic acid and the dynamics of fruit coloration in L. maackii was established through metabolite profiling and transcriptome analyses. Moreover, we identified the genes encoding hydroxycinnamoyl-CoA quinate transferase (LmHQT) and hydroxycinnamoyl-CoA shikimic/quinate transferase (LmHCT), which localized to the cytosol and nucleus. Heterologous overexpression of these genes in Nicotiana benthamiana leaves resulted in elevated chlorogenic acid contents. Importantly, HPLC analyses revealed that LmHCT and LmHQTs recombinant proteins modulate the accumulation of chlorogenic acid (CGA) using quinic acid and caffeoyl CoA as substrates, highlighting the importance of LmHQT and LmHCT in CGA biosynthesis. These results confirmed that LmHQTs and LmHCT catalyze the biosynthesis of CGA in vitro. The genomic data presented in this study will offer a valuable resource for the elucidation of CGA biosynthesis and facilitating selective molecular breeding.

Overexpressing CYP81D11 enhances 2,4,6-trinitrotoluene tolerance and removal efficiency in Arabidopsis.

Phytoremediation is a promising technology for removing the high-toxic explosive 2,4,6-trinitrotoluene (TNT) pollutant from the environment. Mining dominant genes is the key research direction of this technology. Most previous studies have focused on the detoxification of TNT rather than plants' TNT tolerance. Here, we conducted a transcriptomic analysis of wild type Arabidopsis plants under TNT stress and found that the Arabidopsis cytochrome P450 gene CYP81D11 was significantly induced in TNT-treated plants. Under TNT stress, the root length was approximately 1.4 times longer in CYP81D11-overexpressing transgenic plants than in wild type plants. The half-removal time for TNT was much shorter in CYP81D11-overexpressing transgenic plants (1.1 days) than in wild type plants (t1/2 = 2.2 day). In addition, metabolic analysis showed no difference in metabolites in transgenic plants compared to wild type plants. These results suggest that the high TNT uptake rates of CYP81D11-overexpressing transgenic plants were most likely due to increased tolerance and biomass rather than TNT degradation. However, CYP81D11-overexpressing plants were not more tolerant to osmotic stresses, such as salt or drought. Taken together, our results indicate that CYP81D11 is a promising target for producing bioengineered plants with high TNT removing capability.

Construction and validation of a nomogram for HBV-related hepatocellular carcinoma: A large, multicenter study.

INTRODUCTION AND OBJECTIVES: We initiated this multicenter study to integrate important risk factors to create a nomogram for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) for clinician decision-making. PATIENTS AND METHODS: Between April 2011 and March 2022, 2281 HCC patients with an HBV-related diagnosis were included. All patients were randomly divided into two groups in a ratio of 7:3 (training cohort, n = 1597; validation cohort, n = 684). The nomogram was built in the training cohort via Cox regression model and validated in the validation cohort. RESULTS: Multivariate Cox analyses revealed that the portal vein tumor thrombus, Child-Pugh class, tumor diameter, alanine aminotransferase level, tumor number, extrahepatic metastases, and therapy were independent predictive variables impacting overall survival. We constructed a new nomogram to predict 1-, 2-, and 3-year survival rates based on these factors. The nomogram-related receiver operating characteristics (ROC) curves indicated that the area under the curve (AUC) values were 0.809, 0.806, and 0.764 in predicting 1-, 2-, and 3-year survival rates, respectively. Furthermore, the calibration curves revealed good agreement between real measurements and nomogram predictions. The decision curve analyses (DCA) curves demonstrated excellent therapeutic application potential. In addition, stratified by risk scores, low-risk groups had longer median OS than medium-high-risk groups (p < 0.001). CONCLUSIONS: The nomogram we constructed showed good performance in predicting the 1-year survival rate for HBV- related HCC.

Discovery of a Unique Flavonoid Biosynthesis Mechanism in Fungi by Genome Mining.

Flavonoids are important plant natural products with variable structures and bioactivities. All known plant flavonoids are generated under the catalysis of a type III polyketide synthase (PKS) followed by a chalcone isomerase (CHI) and a flavone synthase (FNS). In this study, the biosynthetic gene cluster of chlorflavonin, a fungal flavonoid with acetolactate synthase inhibitory activity, was discovered using a self-resistance-gene-directed strategy. A novel flavonoid biosynthetic pathway in fungi was revealed. A core nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) is responsible for the generation of the key precursor chalcone. Then, a new type of CHI catalyzes the conversion of a chalcone into a flavanone by a histidine-mediated oxa-Michael addition mechanism. Finally, the desaturation of flavanone to flavone is catalyzed by a new type of FNS, a flavin mononucleotide (FMN)-dependent oxidoreductase.

LATERAL BRANCHING OXIDOREDUCTASE, one novel target gene of Squamosa Promoter Binding Protein-like 2, regulates tillering in switchgrass.

Strigolactones (SLs) play a critical role in regulating plant tiller number. LATERAL BRANCHING OXIDOREDUCTASE (LBO) encodes an important late-acting enzyme for SL biosynthesis and regulates shoot branching in Arabidopsis. However, little is known about the function of LBO in monocots including switchgrass (Panicum virgatum L.), a dual-purpose fodder and biofuel crop. We studied the function of PvLBO via the genetic manipulation of its expression levels in both the wild-type and miR156 overexpressing (miR156OE ) switchgrass. Co-expression analysis, quantitative real-time polymerase chain reaction (qRT-PCR), transient dual luciferase assay, and chromatin immunoprecipitation-qPCR were all used to determine the activation of PvLBO by miR156-targeted Squamosa Promoter Binding Protein-like 2 (PvSPL2) in regulating tillering of switchgrass. PvLBOtranscripts dramatically declined in miR156OE transgenic switchgrass, and the overexpression of PvLBO in the miR156OE transgenic line produce fewer tillers than the control. Furthermore, we found that PvSPL2 can directly bind to the promoter of PvLBO and activate its transcription, suggesting that PvLBO is a novel downstream gene of PvSPL2. We propose that PvLBO functions as an SL biosynthetic gene to mediate tillering and acts as an important downstream factor in the crosstalk between the SL biosynthetic pathway and the miR156-SPL module in switchgrass.

Down-regulation of PvSAMS impairs S-adenosyl-L-methionine and lignin biosynthesis, and improves cell wall digestibility in switchgrass.

S-adenosyl- l-methionine (SAM) is the methyl donor involved in the biosynthesis of guaiacyl (G) and syringyl (S) lignins in vascular plants. SAM is synthesized from methionine through the catalysis of the enzyme S-adenosylmethionine synthase (SAMS). However, the detailed function of SAMS in lignin biosynthesis has not been widely investigated in plants, particularly in monocot species. In this study, we identified PvSAMS genes from switchgrass (Panicum virgatum L.), an important dual-purpose fodder and biofuel crop, and generated numerous transgenic switchgrass lines through PvSAMS RNA interference technology. Down-regulation of PvSAMS reduced the contents of SAM, G-lignins, and S-lignins in the transgenic switchgrass. The methionine and glucoside derivatives of caffeoyl alcohol were found to accumulate in the transgenic plants. Moreover, down-regulation of PvSAMS in switchgrass resulted in brownish stems associated with reduced lignin content and improved cell wall digestibility. Furthermore, transcriptomic analysis revealed that most sulfur deficiency-responsive genes were differentially expressed in the transgenic switchgrass, leading to a significant increase in total sulfur content; thus implying an important role of SAMS in the methionine cycle, lignin biosynthesis, and sulfur assimilation. Taken together, our results suggest that SAMS is a valuable target in lignin manipulation, and that manipulation of PvSAMS can simultaneously regulate the biosynthesis of SAM and methylated monolignols in switchgrass.

Clinical efficacy and safety of external radiotherapy combined with sorafenib in the treatment of hepatocellular carcinoma: a systematic review and meta-analysis.

INTRODUCTION AND OBJECTIVES: Both external radiotherapy and sorafenib are promising treatments for hepatocellular carcinoma (HCC). Nevertheless, the combined treatment of external radiotherapy and sorafenib has not been widely applied clinically due to potentially adverse effects. This meta-analysis aimed to evaluate the clinical efficacy and safety of external radiotherapy combined with sorafenib in the treatment of HCC. METHODS: Pubmed, MEDLINE, EMBASE, Cochrane Library, and Web of Science databases were searched. The primary and secondary observation endpoints were the end of survival and incidence of adverse events, respectively. 11 studies involving 664 patients were included in this meta-analysis. RESULTS: The results demonstrated that median overall survival (mOS) and median progression-free survival (mPFS) of the external radiotherapy combined with sorafenib (RS) group were 19.45 months and 8.20 months. The one- and two-year survival rates were 0.65 (95%CI: 0.55-0.76) and 0.40 (95%CI: 0.24-0.56). The incidence of adverse events was 0.34 (95%CI: 0.25-0.44). CONCLUSIONS: The findings demonstrated that the survival of the RS group was significantly improved and few severe adverse events were observed. Hence, it can be concluded that external radiotherapy combined with sorafenib is a safe, effective, and promising therapeutic option for HCC.

Genome-Wide Identification of Switchgrass Laccases Involved in Lignin Biosynthesis and Heavy-Metal Responses.

Plant laccase genes belong to a multigene family, play key roles in lignin polymerization, and participate in the resistance of plants to biotic and abiotic stresses. Switchgrass is an important resource for forage and bioenergy production, yet information about the switchgrass laccase gene family is scarce. Using bioinformatic approaches, a genome-wide analysis of the laccase multigene family in switchgrass was carried out in this study. In total, 49 laccase genes (PvLac1 to PvLac49) were identified; these can be divided into five subclades, and 20 of them were identified as targets of miR397. The tandem and segmental duplication of laccase genes on Chr05 and Chr08 contributed to the expansion of the laccase family. The laccase proteins shared conserved signature sequences but displayed relatively low sequence similarity, indicating the potential functional diversity of switchgrass laccases. Switchgrass laccases exhibited distinct tissue/organ expression patterns, revealing that some laccases might be involved in the lignification process during stem development. All five of the laccase isoforms selected from different subclades responded to heavy metal. The immediate response of lignin-related laccases, as well as the delayed response of low-abundance laccases, to heavy-metal treatment shed light on the multiple roles of laccase isoforms in response to heavy-metal stress.

Highly efficient detoxification of dinitrotoluene by transgenic switchgrass overexpressing bacterial nitroreductase.

Dinitrotoluene (DNT) has been extensively used in manufacturing munitions, polyurethane foams and other important chemical products. However, it is highly toxic and mutagenic to most organisms. Here, we synthesized a codon-optimized bacterial nitroreductase gene, NfsI, for plant expression. The kinetic analysis indicates that the recombinant NfsI can detoxify both 2,4-DNT and its sulfonate (DNTS), while it has a 97.6-fold higher catalytic efficiency for 2,4-DNT than DNTS. Furthermore, we overexpressed NfsI in switchgrass (Panicum virgatum L.), which is a multiple-purpose crop used for fodder and biofuel production as well as phytoremediation. The 2,4-DNT treatment inhibited root elongation of wild-type switchgrass plants and promoted reactive oxygen species (ROS) accumulation in roots. In contrast, overexpression of NfsI in switchgrass significantly alleviated 2,4-DNT-induced root growth inhibition and ROS overproduction. Thus, the NfsI overexpressing transgenic switchgrass plant removed 94.1% 2,4-DNT after 6 days, whose efficiency was 1.7-fold higher than control plants. Moreover, the comparative transcriptome analysis suggests that 22.9% of differentially expressed genes induced by 2,4-DNT may participate in NfsI-mediated 2,4-DNT detoxification in switchgrass. Our work sheds light on the function of NfsI during DNT phytoremediation for the first time, revealing the application potential of switchgrass plants engineered with NfsI.

Quantitative assessment of Bell's palsy-related facial thermal asymmetry using infrared thermography: A preliminary study.

The temperature distribution of normal human skin is symmetrical. Facial paralysis generally changes this thermal symmetry. The aim of this study is to analyze facial thermal asymmetry during the early onset of Bell's palsy, and to assess the feasibility of the diagnosis of early-onset Bell's palsy using infrared thermography (IRT). Fifteen subjects with Bell's palsy and 15 healthy volunteers were considered in this study. The infrared thermal images of the front, left, and right sides of all the subjects were collected and analyzed. Each group of facial thermograms was divided into 16 symmetrical regions of interest (ROIs) with respect to the left and right sides. Three different temperature difference calculation methods were used to express the degree of thermal symmetry between the left- and right-side ROIs, namely, the mean temperature difference (ΔTroi), maximum temperature difference (ΔTmax), and minimum temperature difference (ΔTmin). Among the facial ROIs, there were significant differences in the thermal symmetries of the frontal region, medial canthus region, and infraorbital region between subjects with and without Bell's palsy (p < 0.05). Based on the results, ΔTroi was more effective than the other two methods for the diagnosis of early-onset Bell's palsy. The area under the ROC curve (AUC) of ΔTroi in the infraorbital region was 0.818; and the sensitivity and specificity were 0.867 and 0.800, respectively. Subjects with early-onset Bell's palsy exhibited thermal asymmetry on the left and right sides of their faces. The diagnosis of early-onset Bell's palsy using IRT is therefore necessary. Nevertheless, more effective thermal symmetry analysis methods will be investigated further in future research.

Simultaneous regulation of F5H in COMT-RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis.

Ferulate 5-hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O-methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT-down-regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down-regulation of F5H in COMT-RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5-OH G units. Conversely, overexpression of F5H in COMT-RNAi transgenic plants reduced G units and increased 5-OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down-regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up-regulation and down-regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.

Alteration of S-adenosylhomocysteine levels affects lignin biosynthesis in switchgrass.

Methionine (Met) synthesized from aspartate is a fundamental amino acid needed to produce S-adenosylmethionine (SAM) that is an important cofactor for the methylation of monolignols. As a competitive inhibitor of SAM-dependent methylation, the effect of S-adenosylhomocysteine (SAH) on lignin biosynthesis, however, is still largely unknown in plants. Expression levels of Cystathionine γ-synthase (PvCGS) and S-adenosylhomocysteine hydrolase 1 (PvSAHH1) were down-regulated by RNAi technology, respectively, in switchgrass, a dual-purpose forage and biofuel crop. The transgenic switchgrass lines were subjected to studying the impact of SAH on lignin biosynthesis. Our results showed that down-regulation of PvCGS in switchgrass altered the accumulation of aspartate-derived and aromatic amino acids, reduced the content of SAH, enhanced lignin biosynthesis and stunted plant growth. In contrast, down-regulation of PvSAHH1 raised SAH levels in switchgrass, impaired the biosynthesis of both guaiacyl and syringyl lignins and therefore significantly increased saccharification efficiency of cell walls. This work indicates that SAH plays a crucial role in monolignol methylation in switchgrass. Genetic regulation of either PvCGS or PvSAHH1 expression in switchgrass can change intracellular SAH contents and SAM to SAH ratios and therefore affect lignin biosynthesis. Thus, our study suggests that genes involved in Met metabolism are of interest as new valuable targets for cell wall bioengineering in future.

Methylenetetrahydrofolate reductase modulates methyl metabolism and lignin monomer methylation in maize.

The brown midrib2 (bm2) mutant of maize, which has a modified lignin composition, contains a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. Here, we show that a MITE transposon insertion caused down-regulation of MTHFR, with an accompanying decrease in 5-methyl-tetrahydrofolate and an increase in 5, 10-methylene-tetrahydrofolate and tetrahydrofolate in the bm2 mutant. Furthermore, MTHFR mutation did not change the content of S-adenosyl methionine (SAM), the methyl group donor involved in the biosynthesis of guaiacyl and syringyl lignins, but increased the level of S-adenosyl homocysteine (SAH), the demethylation product of SAM. Moreover, competitive inhibition of the maize caffeoyl CoA O-methyltransferase (CCoAOMT) and caffeic acid O-methyltransferase (COMT) enzyme activities by SAH was found, suggesting that the SAH/SAM ratio, rather than the concentration of SAM, regulates the transmethylation reactions of lignin intermediates. Phenolic profiling revealed that caffeoyl alcohol glucose derivatives accumulated in the bm2 mutant, indicating impaired 3-O-methylation of monolignols. A remarkable increase in the unusual catechyl lignin in the mutant demonstrates that MTHFR down-regulation mainly affects guaiacyl lignin biosynthesis, consistent with the observation that CCoAOMT is more sensitive to SAH inhibition than COMT. This study uncovered a novel regulatory mechanism in lignin biosynthesis, which may offer an effective approach to utilizing lignocellulosic feedstocks in the future.

Integrated genome sequence and linkage map of physic nut (Jatropha curcas L.), a biodiesel plant.

The family Euphorbiaceae includes some of the most efficient biomass accumulators. Whole genome sequencing and the development of genetic maps of these species are important components in molecular breeding and genetic improvement. Here we report the draft genome of physic nut (Jatropha curcas L.), a biodiesel plant. The assembled genome has a total length of 320.5 Mbp and contains 27,172 putative protein-coding genes. We established a linkage map containing 1208 markers and anchored the genome assembly (81.7%) to this map to produce 11 pseudochromosomes. After gene family clustering, 15,268 families were identified, of which 13,887 existed in the castor bean genome. Analysis of the genome highlighted specific expansion and contraction of a number of gene families during the evolution of this species, including the ribosome-inactivating proteins and oil biosynthesis pathway enzymes. The genomic sequence and linkage map provide a valuable resource not only for fundamental and applied research on physic nut but also for evolutionary and comparative genomics analysis, particularly in the Euphorbiaceae.

Prognostic value of circulating tumor cells combined with neutrophil-lymphocyte ratio in patients with hepatocellular carcinoma.

BACKGROUND: Circulating tumor cell (CTC) count and neutrophil-to-lymphocyte ratio (NLR) are both closely associated with the prognosis of hepatocellular carcinoma (HCC). AIM: To investigate the prognostic value of combining these two indicators in HCC. METHODS: Clinical data were collected from patients with advanced HCC who received immune therapy combined with targeted therapy at the Department of Oncology, the Affiliated Hospital of Southwest Medical University, Sichuan, China, from 2021 to 2023. The optimal cutoff values for CTC programmed death-ligand 1 (PD-L1) (+) > 1 or CTC PD-L1 (+) ≤ 1 and NLR > 3.89 or NLR ≤ 3.89 were evaluated using X-Tile software. Patients were categorized into three groups based on CTC PD-L1 (+) counts and NLR: CTC-NLR (0), CTC-NLR (1), and CTC-NLR (2). The relationship between CTC-NLR and clinical variables as well as survival rates was assessed. RESULTS: Patients with high CTC PD-L1 (+) expression or NLR at baseline had shorter median progression-free survival (mPFS) and median overall survival (mOS) than those with low levels of CTC PD-L1 (+) or NLR (P < 0.001). Meanwhile, patients in the CTC-NLR (2) group showed a significant decrease in mPFS and mOS. Cox regression analysis revealed that alpha-fetoprotein (AFP), CTC PD-L1 (+), and CTC-NLR were independent predictors of OS. The time-dependent receiver operating characteristic curve showed that the area under the curve of CTC-NLR at 12 months (0.821) and 18 months (0.821) was superior to that of AFP and CTC PD-L1 (+). CONCLUSION: HCC patients with high CTC PD-L1 (+) or NLR expression tend to exhibit poor prognosis, and a high baseline CTC-NLR score may indicate low survival. CTC-NLR may serve as an effective prognostic indicator for patients with advanced HCC receiving immunotherapy combined with targeted therapy.

Anti-angiogenic and antitumor effects of anlotinib combined with bevacizumab for colorectal cancer.

BACKGROUND: The progression and metastasis of tumors are typically accompanied by angiogenesis. Crucially, vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) play a significant role in tumor-associated angiogenesis. In this study, the aim was to investigate the antitumor effect of combining bevacizumab (Bev) with anlotinib (An) on colorectal cancer (CRC). METHODS: The CCK-8 assay, EdU assay, and Annexin V staining were conducted to evaluate the proliferation and apoptosis of CRC cells in vitro. The migration capability of CRC cells and HUVECs was assessed using the Transwell assay. Additionally, the tube formation capability of HUVECs was investigated. Furthermore, the antitumor and antiangiogenic effects were evaluated in the BALB/c mice model using immunohistochemistry, TUNEL staining, and 18F-FDG PET/CT imaging. Finally, we analyzed the inhibitory effect of Bev and/or An on related signaling effectors through western blotting. RESULTS: The in vivo CRC mice model revealed that the combination of Bev + An significantly suppressed tumor formation and angiogenesis. Bev + An inhibited tumor glucose metabolism and increased the median survival period in tumor-bearing mice. Mechanistically, the expressions of VEGF, VEGFR2, PDGFR, and FGFR, as well as the phosphorylation levels of AKT, were inhibited after Bev+An treatment. In conclusion, the dual vertical targeting of VEGF and VEGFR in the CRC mice model strongly inhibited tumor growth and angiogenesis, with the suppression of the AKT signaling pathway playing a partial role.