Objective To explore the role of ring finger protein 41(RNF41)in the initiation and progression of cholangiocarcinoma.Methods The expression levels of RNF41 mRNA and protein in tumor tissues and adjacent normal tissues from 84 CHOL patients who underwent total surgical resection at the Second Affiliated Hospital of Kunming Medical University and Kunming Ganmei Hospital between January 2010 and December 2016 were analyzed using bioinformatics,Western blot,and immunohistochemistry.The TIMER 2.0 database was used to analyze the impact of RNF41 on the prognosis and survival of CHOL patients and the relationship between RNF41 and tumor clinical characteristics.RNF41 siRNA was transfected into HCC9810,RBE,and HUCCT1 cells.CCK-8,Edu,colony formation,and Western blot assays were conducted to evaluate the changes in proliferation of cholangiocarcinoma cells between the RNF41 knockdown group and the control group.Transwell assays and detection of EMT and migration markers were performed to assess changes in the invasion ability of cholangiocarcinoma cells between the RNF41 knockdown and control groups.Western blot was used to examine the effect of RNF41 knockdown on epithelial-mesenchymal transition in cholangiocarcinoma cells.Twelve BALB/c mice were randomly divided into two groups:a control group and an RNF41 knockdown group,with six mice in each group.Tumor formation assays,Western blot assays,and immunohistochemistry staining were carried out to investigate the effect of RNF41 knockdown on tumor growth in nude mice.Results Real-time quantitative fluorescence PCR analysis revealed that the expression level of RNF41 mRNA in cholangiocarcinoma tissues was significantly higher than that in the corresponding adjacent non-tumor tissues(P<0.01),and this trend was corroborated at the protein level by immunohistochemical staining.Using the TIMER 2.0 database,we further analyzed the correlation between RNF41 expression and clinicopathological features,including histological grade,tumor stage,lymph node metastasis,and patient survival.The results indicated that elevated RNF41 expression was significantly associated with advanced histological grade and lymph node metastasis in cholangiocarcinoma(P<0.01).Survival analysis demonstrated that high RNF41 expression was closely linked to poor prognosis in patients with cholangiocarcinoma(CHOL).Functional assays,including CCK-8,EdU incorporation,and colony formation,showed that RNF41 knockdown significantly inhibited the proliferation of cholangiocarcinoma cells compared with the control group.Western blot analysis revealed that,following RNF41 silencing,the expression of the epithelial-mesenchymal transition(EMT)marker E-cadherin was markedly upregulated,whereas the levels of mesenchymal markers N-cadherin and MMP9 were significantly reduced(P<0.05).These findings were consistent with the results obtained from in vitro experiments(P<0.01).Moreover,in vivo studies showed that RNF41 knockdown suppressed subcutaneous tumor formation in nude mice(P<0.05).Conclusion RNF41 plays a critical role in promoting the occurrence and progression of cholangiocarcinoma and is closely associated with adverse clinicopathological features and poor prognosis in patients.The knockdown of RNF41 effectively suppresses the proliferation,invasion,epithelial-mesenchymal transition(EMT),and tumorigenicity of cholangiocarcinoma cells.

Since the approval of gemtuzumab ozogamicin,an antibody-drug conjugate(ADC)targeting CD33 in 2000,13 ADC drugs have been approved by the FDA.Although these drugs have clearly improved the survival of patients with various types of advanced cancers,their significant toxicity has compromised their therapeutic benefits.The adverse reactions of ADC drugs are complex and include on-target and off-target toxicities,where the payload drug is a determining factor.Antibody and linker may also affect the degree of toxicity.Combination therapy becomes an important strategy in anticancer treatment because of its increased efficiency,but treatment-related adverse reactions also increase accordingly.This review comprehensively analyzes the toxicity mechanisms of current ADC drugs and proposes various optimization strategies,including but not limited to optimizing linker molecules,upgrading antibody design,and changing drug administration strategies,to improve the overall safety profile of ADC drugs.

ObjectiveTo explore the effect of Tongxie Yaofang on the immune microenvironment of colorectal cancer in mice under chronic stress and the underlying mechanism. MethodA total of 40 male SPF BABL/C mice were randomized into normal group， stress group， Tongxie Yaofang group （13.65 g·kg^-1）， and Tongxie Yaofang-stress group （13.65 g·kg^-1）， with 10 in each group. Chronic restraint stress was induced in mice and administration （ig） of Tongxie Yaofang began after 7 days of stress. On the 14^th day， forced swim and tail suspension tests were used to examine the behavioral changes of mice after stress and the subcutaneous colorectal tumor was implanted in each group of mice. The effect of this prescription on the body mass and tumor volume of mice was observed. After the last administration， mouse serum and tumor samples were collected. The content of T lymphocytes （CD3⁺， CD4⁺， CD8⁺， and CD4⁺/CD8⁺） in tumor was detected by immunohistochemistry and flow cytometry and levels of corticosterone （CORT） in peripheral blood， and interleukin （IL）-2， interferon-γ （IFN-γ）， IL-6， and IL-10 in the serum were determined by enzyme-linked immunosorbent assay （ELISA）. The protein expression of inhibitor of nuclear factor-κB（IκB） kinase α/β （IKKα/β）， nuclear factor-κB （NF-κB）α （IκBα）， NF-κB p65， and phosphorylated （p）-NF-κB p65 was measured by Western blot. ResultCompared with the normal group， the stress group had large tumor volume （P<0.05）， low content of CD3⁺， CD4⁺， and CD4⁺/CD8⁺ （P<0.05， P<0.01）， high content of CD8⁺， low content of T helper 1 （Th1）-secreted IFN-γ （P<0.05）， high content of T helper 2 （Th2）-secreted IL-10 （P<0.05） and CORT （P<0.05）， high protein expression of p-NF-κB p65， NF-κB p65， and IKKα/β （P<0.05）， and low protein expression of IκBα （P<0.05）. Compared with the normal group， the Tongxie Yaofang group showed slow tumor growth， high content of CD3⁺， CD4⁺， and CD4⁺/CD8⁺ （P<0.01）， low content of CD8⁺ （P<0.05）， high content of Th1-secreted IL-2 and IFN-γ （P<0.05）， low content of Th2-secreted IL-6 and IL-10 （P<0.05）， low content of CORT， low protein expression of p-NF-κB p65， NF-κB p65， and IKKα/β （P<0.05）， and high protein expression of IκBα （P<0.01）. Tongxie Yaofang-stress group demonstrated slower tumor growth， higher content of CD3⁺， CD4⁺， and CD4⁺/CD8⁺ （P<0.01）， smaller content of CD8⁺ （P<0.05）， higher content of IL-2 and IFN-γ （P<0.05）， lower content of IL-6， IL-10 （P<0.05）， and CORT （P<0.05）， lower protein expression of p-NF-κB p65， NF-κB p65， and IKKα/β （P<0.05，P<0.01）， and higher protein expression of IκBα （P<0.01） than the stress group. ConclusionTongxie Yaofang can delay the growth of colorectal cancer under chronic stress and alleviate the deterioration of the immune microenvironment， possibly by inhibiting NF-κB signaling pathway， regulating the function of T lymphocyte subsets， and thus suppressing the secretion of pro-inflammatory factors.

ObjectiveTo observe the effect of berberine combined with evodiamine on the migration and invasion of colorectal cancer HCT116 and RKO cells and to explore the underlying mechanism. Methodcell counting kit-8 （CCK-8） assay was used to examine the proliferation of HCT116 and RKO cells treated by berberine （30 μmol·L^-1）， evodiamine （0.8 μmol·L^-1）， and combination of two （30 μmol·L^-1+0.8 μmol·L^-1）， respectively. Scratch assay and Transwell assay were employed to detect the migration and invasion of HCT116 and RKO cells treated with berberine， evodiamine， and the combination， separately. In addition， the protein expression of epithelial cadherin （E-cadherin）， neural cadherin （N-cadherin）， phosphatidylinositol 3-kinase （PI3K）， and protein kinase B （Akt） in HCT116 and RKO cells treated with the berberine， evodiamine， and the combination was respectively measured by Western blot. ResultCompared with the blank group， berberine alone and evodiamine alone had no significant inhibitory effect on the proliferation， migration， and invasion of HCT116 and RKO cells， while the combination showed significant inhibition （P<0.01）. Berberine alone and evodiamine alone had no remarkable influence on the expression of PI3K， N-cadherin， and E-cadherin in HCT116 and RKO cells， but the combination significantly reduced the expression of PI3K and N-cadherin （P<0.01） and increased the expression of E-cadherin （P<0.01） in HCT116 and RKO cells. Evodiamine alone also significantly suppressed the expression of Akt protein in HCT116 and RKO cells （P<0.05）， but the suppression was weaker than that of the combination. ConclusionThe combination of berberine and evodiamine can significantly inhibit the migration and invasion of colorectal cancer HCT116 and RKO cells and the two show synergy. The mechanism is the likelihood that the combination down-regulates the expression of PI3K and Akt.