Synthesis and biological activities of chlorin e6-based conjugate of fluorouracil as dual-mode antitumor photosensitizer / 药学实践杂志

Objective To design and synthesize the conjugate (compound 1) of chlorin e6 (compound 3) with fluorouracil (5-Fu) as novel pH-responsive dual-mode antitumor photosensitizer by acyl hydrazone bond coupling, based on literature reports that combination of 5-Fu and photosensitizer possess synergistic anti-tumor effect, and investigate its photodynamic antitumor activity and mechanism. Methods Lead compound 3 was obtained by alkali degradation with 25% KOH-CH3OH on pheophorbide a (compound 4) which was prepared through acid hydrolysis of chlorophyll a in crude chlorophyll extracts from silkworm excrement. Reflux reaction of 5-Fu with P2S5 in pyridine formed crude 4-thio-5-fluorouracil which was followed to react with hydrazine hydrate (N2H4·H2O) in CH3OH to give 5-fluorouracil-4-hydrazone (compound 2). Then, treatment of compound 3 i.e. acid alkali degradation product of chlorophyll a in silkworm excrement with EDC·HCl generated its 171- and 152 cyclic anhydride which was followed to directly react with intermediate compound 2 to successfully get title compound 1. In addition, its pH-responsive 5-Fu release and photodynamic antitumor activity and their mechanisms in vitro were investigated. Results Compound 1 could responsively release 5-Fu at pH 5.0, with a cumulative release rate of 60.3% within 24 h. It exhibited much higher phototoxicity against melanoma B16-F10 and liver cancer HepG2 cells than talaporfin and its precursor compound 3, with IC50 value being 0.73 μmol/L for B16-F10 cells and 0.90 μmol/L for HepG2 cells, respectively. Upon light irradiation, it also could significantly induce cell apoptosis and intracellular ROS level and block cell cycle in S phase. Its structure was confirmed by UV, 1H-NMR, ESI-MS and elemental analysis data. Conclusion The conjugate compound 1 of compound 3 and 5-Fu has the advantages of strong PDT anticancer activity, high therapeutic index (i.e. dark toxicity/phototoxicity ratio) and responsively release 5-Fu at pH 5.0 etc. which shows “unimolecular” dual antitumor effects of PDT and chemotherapy and is worthy of further research and development.

Application of 5-fluorouracil and bandage contact lens in pterygium excision combined with autogenous limbal stem cell transplantation / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To assess the clinical efficacy of 5-fluorouracil(5-FU)and bandage contact lens in the pterygium excision combined with autogenous limbal stem cell transplantation(ALSCT)in treating patients with pterygium.METHODS:Random controlled clinical trial. A total of 71 patients(71 eyes)of pterygium who treated at the department of ophthalmology in Qinhuangdao Haigang Hospital between May 2021 and November 2022 were included. They were divide into three groups, including 23 eyes received pterygium excision combined with ALSCT in group A, 24 eyes that were administered with 5-FU intraoperatively and postoperatively in group B, and 24 eyes that received both bandage contact lens and 5-FU in group C. Furthermore, comfort levels at 1, 3, 7, 14d postoperatively, corneal epithelial healing at 1, 3, 7, 14d and 1mo postoperatively, treatment outcomes and complications at 3～6mo postoperatively were compared among the three groups of patients.RESULTS:The comfort levels at 1, 3 and 7d postoperatively and corneal healing at 1 and 3d postoperatively of the group C were better than those of the groups A and B. There were no statistical significant differences in the comfort levels at 14d after surgery and corneal healing at 14d and 1mo after surgery among the three groups of patients. Over a 3～6mo follow-up period, group A experienced recurrence in 3 eyes, group B had 1 recurrence, while group C had no recurrence. There were no statistically significant differences in complication rates among the three groups of patients.CONCLUSIONS: The application of 5-FU combined with bandage contact lens can enhance postoperative comfort levels, promote corneal epithelial healing, and improve the success rate in pterygium excision combined with ALSCT.

Effect of baicalein combined with 5-FU on proliferation, apoptosis and migration of H446 cells by regulating ROS/MAPK signaling pathway / 中华内分泌外科杂志

Objective:To investigate the effects of baicalein combined with 5-FU on the proliferation, apoptosis and migration of small cell lung cancer H466 cells, and further to explore its sensitization mechanism.Methods:H466 cells were cultured in vitro and divided into blank control group, baicalein single treatment group, 5-FU single treatment group and combined treatment group. CCK-8 experiment was used to detect cell survival rate in each group; Flow cytometry was used to detect cell apoptosis and changes in ROS levels; Western blot was used to detect changes in the protein expression of the MAPK pathway.Results:CCK-8 results showed that the survival rates of the four groups of cells after 24 h treatment were 114.3% ± 2.8%, 79.8% ± 3.4%, 57.6% ± 1.8%, and 40.3% ± 2.7%. Compared with the other three groups, the combined treatment group had stronger inhibitory effect on the proliferation of H446 cells, and the difference was statistically significant ( P<0.001) . The proportion of apoptotic H446 cells in the combined treatment group was 49.2%±3.8%, which was significantly different from 33.9%±4.3% in the 5-FU single treatment group, and the difference was statistically significant ( P<0.001) . The inhibition rate of H446 cell migration in the combined treatment group was higher than that in the 5-FU single treatment group, and the difference was statistically significant ( P <0.001) . Combined treatment of baicalein and 5-FU can up-regulate ROS levels in H446 cells, thereby regulating the MAPK signaling pathway. Conclusion:The combined use of baicalein and 5-FU can increase the level of ROS in lung cancer H446 cells, activate the JNK and p38 signaling pathways, inhibit the ERK signaling pathway, and enhance 5-FU's proliferation inhibition, apoptosis induction and migration inhibition effects on H446 cells, which provides a certain experimental basis for the application of baicalein in small cell lung cancer.

Bioresponsive immune-booster-based prodrug nanogel for cancer immunotherapy

The combination of chemotherapy and immunotherapy motivates a potent immune system by triggering immunogenic cell death (ICD), showing great potential in inhibiting tumor growth and improving the immunosuppressive tumor microenvironment (ITM). However, the therapeutic effectiveness has been restricted by inferior drug bioavailability. Herein, we reported a universal bioresponsive doxorubicin (DOX)-based nanogel to achieve tumor-specific co-delivery of drugs. DOX-based mannose nanogels (DM NGs) was designed and choosed as an example to elucidate the mechanism of combined chemo-immunotherapy. As expected, the DM NGs exhibited prominent micellar stability, selective drug release and prolonged survival time, benefited from the enhanced tumor permeability and prolonged blood circulation. We discovered that the DOX delivered by DM NGs could induce powerful anti-tumor immune response facilitated by promoting ICD. Meanwhile, the released mannose from DM NGs was proved as a powerful and synergetic treatment for breast cancer in vitro and in vivo, via damaging the glucose metabolism in glycolysis and the tricarboxylic acid cycle. Overall, the regulation of tumor microenvironment with DOX-based nanogel is expected to be an effectual candidate strategy to overcome the current limitations of ICD-based immunotherapy, offering a paradigm for the exploitation of immunomodulatory nanomedicines.

Effect of Fuzheng Jiedu Prescription Combined with 5-Fu Against Postoperative Recurrence and Metastasis of Gastric Cancer in Mice： An Exploration Based on Endoplasmic Reticulum Stress / 中国实验方剂学杂志

Objective:To investigate the inhibitory effect of Fuzheng Jiedu prescription（FZJDP） combined with 5-fluorouracil （5-Fu） against postoperative recurrence and metastasis in gastric cancer-bearing mice and explore the possible mechanism based on changes in CD4⁺ T cells， CD8⁺ T cells， and regulatory T （Treg） cells of tumor microenvironment， endoplasmic reticulum stress （ERS） and phosphatidylinositol-3-kinase （PI3K）/protein kinase B（Akt）/mammalian target of rapamycin（mTOR） pathway. Method:Forty 615 mice were randomly divided into the model group，FZJDP （25 g·kg^-1） group，5-Fu（25 mg·kg^-1）group，and combined （25 g·kg^-1 FZJDP + 25 mg·kg^-1 5-Fu）group， with 10 mice in each group. Mouse forestomach carcinoma （MFC） cells were implanted into the inner side of footpad of the left hind paw and the transplanted tumor was then surgically excised to establish a postoperative recurrence model. Hematoxylin-eosin（HE） staining was conducted to observe the pathological changes in mice with gastric cancer recurrence and lung metastasis. The CD4⁺/CD8⁺ T cell ratio in recurrent tumor and the percentages of Treg cells ［CD4⁺，CD25⁺， and forkhead box protein P3 （FOXP3）⁺ cells］ in spleen were detected by flow cytometry. The contents of ERS-related proteins ［78-kDa glucose-regulated protein（GRP78），inositol-requiring enzyme 1 alpha （IRE1<italic>α</italic>），activating transcription factor 6（ATF6），and protein kinase R-like endoplasmic reticulum kinase（PERK）］ and the expression of related proteins in the PI3K/Akt/mTOR signaling pathway were determined by Western blot and immunohistochemistry （IHC）. Result:Compared with the model group， the combined group significantly increased the recurrent inhibition rate （<italic>P</italic><0.05）. The recurrent tumor weight was significantly decreased in each treatment group （<italic>P</italic><0.05）. The number of lung metastases and metastasis rate declined in each treatment group， and the lowest values were observed in the combined group， without any statistical significance. The CD4⁺/CD8⁺ T cell ratios in the FZJDP group and combined group were significantly elevated （<italic>P</italic><0.05）， while the percentages of Treg cells were reduced （<italic>P</italic><0.05）. However， 5-Fu resulted in a significant increase in Treg cell percentage （<italic>P</italic><0.05）. IHC results showed that the protein expression levels of ATF6 （<italic>P</italic><0.05，<italic>P</italic><0.01）， IRE1<italic>α</italic>（<italic>P</italic><0.05），and Akt （<italic>P</italic><0.01） in each treatment group were significantly down-regulated as compared with those in the model group. As revealed by Western blot， the GRP78 expression level in the 5-Fu group was lower than that in the model group （<italic>P</italic><0.05）， and the expression levels of PI3K， phosphorylated Akt （p-Akt）， and mTOR were significantly decreased in the 5-Fu group and the combined group （<italic>P</italic><0.05）. Conclusion:FZJDP combined with 5-Fu reduces postoperative recurrence and metastasis in tumor-bearing mice possibly by inhibiting PI3K/Akt/mTOR signaling pathway， diminishing ERS，and improving tumor immune microenvironment.

Identification of ferroptosis as a novel mechanism for antitumor activity of natural product derivative a2 in gastric cancer

Ferroptosis is a type of cell death accompanied by iron-dependent lipid peroxidation, thus stimulating ferroptosis may be a potential strategy for treating gastric cancer, therapeutic agents against which are urgently required. Jiyuan oridonin A (JDA) is a natural compound isolated from Jiyuan

The role of caveolin-1 in the biofate and efficacy of anti-tumor drugs and their nano-drug delivery systems

As one of the most important components of caveolae, caveolin-1 is involved in caveolae-mediated endocytosis and transcytosis pathways, and also plays a role in regulating the cell membrane cholesterol homeostasis and mediating signal transduction. In recent years, the relationship between the expression level of caveolin-1 in the tumor microenvironment and the prognostic effect of tumor treatment and drug treatment resistance has also been widely explored. In addition, the interplay between caveolin-1 and nano-drugs is bidirectional. Caveolin-1 could determine the intracellular biofate of specific nano-drugs, preventing from lysosomal degradation, and facilitate them penetrate into deeper site of tumors by transcytosis; while some nanocarriers could also affect caveolin-1 levels in tumor cells, thereby changing certain biophysical function of cells. This article reviews the role of caveolin-1 in tumor prognosis, chemotherapeutic drug resistance, antibody drug sensitivity, and nano-drug delivery, providing a reference for the further application of caveolin-1 in nano-drug delivery systems.

Hedgehog signaling in gastrointestinal carcinogenesis and the gastrointestinal tumor microenvironment

The Hedgehog (HH) signaling pathway plays important roles in gastrointestinal carcinogenesis and the gastrointestinal tumor microenvironment (TME). Aberrant HH signaling activation may accelerate the growth of gastrointestinal tumors and lead to tumor immune tolerance and drug resistance. The interaction between HH signaling and the TME is intimately involved in these processes, for example, tumor growth, tumor immune tolerance, inflammation, and drug resistance. Evidence indicates that inflammatory factors in the TME, such as interleukin 6 (IL-6) and interferon-

Curcumin mediates IL-6/STAT3 signaling pathway to repair intestinal mucosal injury induced by 5-FU chemotherapy for colon cancer / 中国中药杂志

This study aims to investigate the potential mechanism of curcumin in mediating interleukin-6(IL-6)/signal transducer and activator of transcription 3(STAT3) signaling pathway to repair intestinal mucosal injury induced by 5-fluorouracil(5-FU) chemotherapy for colon cancer. SD rats were intraperitoneally injected with 60 mg·kg~(-1)·d~(-1) 5-FU for 4 days to establish a model of intestinal mucosal injury. Then the rats were randomly divided into model group(equal volume of normal saline), curcumin low, medium and high dose groups(50, 100, 200 mg·kg~(-1)), and normal SD rats were used as control group(equal volume of normal saline). Each group received gavage administration for 4 consecutive days, and the changes of body weight and feces were recorded every day. After administration, blood was collected from the heart, and jejunum tissues were collected. The levels of serum interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by ELISA, and at the same time, the concentration of Evans blue(EB) in jejunum was measured. Hematoxylin-eosin(HE) staining was used to observe the pathological state of jejunum, and the length of jejunum villi and the depth of crypt were measured. The positive expression levels of claudin, occludin and ZO-1 were detected by immunohistochemistry. Western blot was used to detect the protein expression of IL-6, p-STAT3, E-cadherin, vimentin and N-cadherin in jejunum tissues. The results showed that, curcumin significantly increased body weight and fecal weight(P<0.05 or P<0.01), decreased fecal score, EB concentration, IL-1β and TNF-α levels(P<0.05 or P<0.01) in rats. In addition, curcumin maintained the integrity of mucosal surface and villi structure of jejunum to a large extent, and reduced pathological changes in a dose-dependent manner. Meanwhile, curcumin could increase the positive expression of occludin, claudin and ZO-1(P<0.05 or P<0.01), repair intestinal barrier function, downregulate the protein expression of IL-6, p-STAT3, vimentin and N-cadherin in jejunum tissues(P<0.05 or P<0.01), and upregulate the protein expression of E-cadherin(P<0.05). Therefore, curcumin could repair the intestinal mucosal injury induced by 5-FU chemotherapy for colon cancer, and the mechanism may be related to the inhibition of IL-6/STAT3 signal and the inhibition of epithelial-mesenchymal transition(EMT) process.

Simultaneous Determination of 5-FU and Its Metabolites 5-FUH2 in Human Plasma by UPLC-MS/MS and Appli- cation / 中国药房

OBJECTIVE：To establish the method for simultaneous determination of 5-fluorouracil（5-FU）and its metabolites 5-fluoro-5，6-dihydrouracil （5-FUH2） in human plasma ，and apply it in the clinic. METHODS ：After plasma samples were processed twice by ethyl acetate ，UPLC-MS/MS method was adopted using 5-bromouracil （5-Bru） as internal standard. The determination was performed on Acquity UPLC HSS T 3 column with mobile phase consisted of methanol-water （30 ∶ 70，V/V）at the flow rate of 0.3 mL/min. The column temperature was 20 ℃，and sample size was 5 μL. An electrospray ion source was used to carry out negative ion scanning with multiple reaction monitoring. The capillary voltage was 1.5 kV；the taper hole voltage was 20 V；the desolvent temperature was 450 ℃；the desolvent air flow was 850 L/h；the cone hole gas velocity was 50 L/h. The ion transitions for quantitative analysis were m/z 129.00→41.90（5-FU），m/z 130.87→82.92（5-FUH2），m/z 189.00→42.10（5-Bru）， respectively. From Aug. to Oct. 2020，10 patients with advanced colorectal cancer were treated with continuous intravenous drip of 5-FU for 46 hours were collected from Harbin Medicinal University Cancer Hospital. Steady-state plasma concentration of 5-FU and plasma concentration of 5-FUH2 were determined at 18-30 h of continuous intravenous drip. The area under the curve （AUC）for 5-FU and concentration ratio of 5-FUH2/5-FU were calculated. RESULTS ：The linear range of 5-FU and 5-FUH2 were 20 to 1 000 μg/mL（R 2＞0.990）. The quantification limits were 20 ng/mL. RSDs of precision test were all lower than 20％，and relative error ranged ±10％. The extraction recovery and matrix effects didn ’t affect the determination of substance to be measured. Among 10 patients with advanced colorectal cancer ，the steady-state concentration of 5-FU were 180.04-622.83 ng/mL，and AUC of 5-FU ranged from 8.28 to 28.65 mg·h/L. The concentration of 5-FUH2 ranged 336.48-948.43 ng/mL，and concentration ratio of 5-FUH2/ 5-FU ranged 0.93-4.21. AUC of 5-FU in 10 patients had about 3-4 fold individual differences. CONCLUSIONS ：The established method has good precision and accuracy ，high sensitivity ，and simple operation. It can be used for plasma monitoring of 5-FU in patients with advanced colorectal cancer.

Resectable locally advanced gastric adenocarcinoma: interim analysis

Background: The MAGIC and ACCORD 07 trials have established the role of perioperative chemotherapy in locally advanced gastric adenocarcinoma. A more recent study has demonstrated the superiority of the FLOT perioperative regimen. The best strategy to improve outcomes has yet to be determined. Aims of the study were to evaluate perioperative chemotherapy in terms of morbidity and tolerance of FLOT regimen with modification and histopathological responseMethods: This prospective study was started after ethical committee approval in February 2019 at a tertiary cancer center in South India for a period of 1 year up till February 2020. Patients fulfilling inclusion criteria were enrolled. Perioperative chemotherapy was given as scheduled regimen and adverse effects and response to preoperative chemotherapy were recorded. Radical D2 gastrectomy and histopathology assessed analysed by using IBM SPSS statistics ver. 21 and descriptive statistics used.Results: From February 2019 till February 2020, a total of 24 patients of newly diagnosed adenocarcinoma of the stomach of which 18 patients were nonmetastatic on workup. Moderately different (38.8%), well-differentiated in 11.2%, poorly differentiated in 50%. Total 66.7% were diagnosed as metastatic on staging laparoscopy, peritoneal wash cytology in 50% was negative. The cardiopulmonary resuscitation was seen in two patients.Conclusions: Even though it is an interim analysis with less number of patients enrolled, so far it can be concluded that all patients where surgery is planned should undergo peritoneal lavage cytology and FLOT regimen can be practised with acceptable morbidity. Long term results after completion of study will definitely throw more light.

siRNA knocking down the expression of NEK2 gene enhances the chemosensitivity of colorectal carcinoma cells to 5-FU / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨敲减中心体相关激酶2（never in mitosis A-related kinase 2，NEK2）对结直肠癌细胞5-FU化疗敏感性的影响及其可能的机制。方法：采用qPCR和Western blotting检测结直肠癌细胞中NEK2 mRNA及蛋白的表达水平。构建针对NEK2基因的小干扰RNA（siRNA）并转染至结直肠癌细胞HCT116及SW620，实验分为阳性干扰组1（转染NEK2 siRNA1）、阳性干扰组2（转染NEK2 siRNA2）和阴性对照组（转染si-NC），均用5-FU处理。采用CCK-8实验、V-FICT/PI Annexin双染色流式细胞术实验观察敲减NEK2基因对5-FU作用下结肠癌细胞的增殖、周期分布及凋亡的影响，采用Western blotting检测敲减NEK2基因对5-FU作用下结直肠癌细胞内Wnt/β-catenin信号通路相关蛋白表达的影响。结果：NEK2蛋白及mRNA在结直肠癌细胞HCT116、SW620中均呈高表达（P<0.05或P<0.01），转染NEK2 siRNA可高效抑制HCT116、SW620细胞中NEK2蛋白及mRNA表达（均P<0.01）。经不同浓度5-FU作用后，阳性干扰组1和阳性干扰组2的细胞存活率和IC50均显著低于阴性对照组（均P<0.01），细胞发生G0/G1期阻滞且凋亡率显著升高（均P<0.01），胞核β-catenin、c-myc和cyclin D1表达水平显著下降而胞质β-catenin表达水平升高（均P<0.01）。结论：敲减NEK2基因可有效提高人结直肠癌细胞对5-FU的化疗敏感性，该作用可能是通过调控Wnt/β-catenin信号通路相关蛋白表达来实现的。

Aspirin promotes apoptosis of esophageal cancer cells induced by 5-FU through inhibiting β-catenin/EMT signaling pathway / 中国药理学通报

Aim To investigate the synergistic effect of aspirin and 5-fluorouracil (5-FU) on proliferation and apoptosis of esophageal carcinoma ECA109 cells and further to explore the underlying mechanisms. Methods The ECA109 cells were cultured under different concentrations of aspirin (0. 062 5, 0. 125, 0. 25, 0.5, 1, 2 g · L-1)and 5-FU (0.01, 0.02, 0.1, 0.2, 0.5, 1 mg · L-1) respectively, then the cell proliferation was detected by CCK-8 assay. After that ECA109 cells were cultured with non-toxic low-concentration of aspirin (0. 125 g · L-1) and 5-FU (0. 1 mg · L-1). Flow cytometry analysis was used to detect the apoptotic rate. Western blot was used to evaluate the expression of apoptosis-related proteins and epithelial-mesenchymal transition (EMT)-related markers. Results Aspirin or 5-FU inhibited cell proliferation in a concentration-dependent manner. 5-FU enhanced apoptosis rate in ECA109 cells, with the down-regulation of Bcl-2 protein and up-regulation of cleaved caspase-3 protein, which was strengthened by aspirin. Furthermore, the synergistic effect of aspirin obviously downregulated the expression of N-cadherin and β-catenin protein while up-regulated the expression of E-cadherin. Conclusions Low-concentrations of aspirin and 5-FU have a synergistic effect on the proliferation and apoptosis of ECA109 cells through inhibiting β-catenin/ EMT signaling pathway, which might provide reference for esophagus cancer adjuvant chemotherapy.

Bioequivalence of capecitabine tablets in cancer patients / 中国临床药理学与治疗学

AIM: To evaluate the pharmacokinetics, bioequivalence, and safety of capecitabine tablet in cancer patients following single oral administration. METHODS: Based on an open-randomized two-period crossover designation, subjects were orally given capecitabine tablet (test or reference products, 0.6 g single dosage). Blood samples were then collected and the plasma concentrations of capecitabine and its active metabolite, 5-fluorouracil (5-FU) were examined by HPLC-MS/MS. The bioequivalence between the test and reference formulations were evaluated with the pharmacokinetic parameters determined by the Phoenix WinNonlin 7.0 software. RESULTS: The numbers of the major pharmacokinetic parameters in patients treated with test and reference products were similar. To analyze the numbers of Cmax, tmax, AUC0-t, AUC0-∞, the 90% confidence interval (CI) for Cmax, AUC0-t and AUC0-∞ were 84.48-106.70, 93.03-96.54 and 96.34-102.84, respectively. For the 5-FU, the 90%CI of the for Cmax, AUC0-t and AUC0-∞ were 84.32-99.67, 90.55-98.76 and 96.99-103.48, respectively. Both sets of numbers fell within the bioequivalent limit ranges of 80.00%-125.00%. No serious adverse event was observed. CONCLUSION: The current data indicate that the test and reference formulations of capecitabine tablets were bioequivalent in cancer patients.

Inhibitory effects of iridoid glycosides from Boschniakia rossica combined with 5-fluorouracil on human hepatoma HepG2 and SK-Hep1 cells epithelial-mesenchymal transition / 中草药

Objective: To study the inhibitory effect of iridoid glycosides from Boschniakia rossica (IGBR) combined with 5-Fu on epithelial-mesenchymal transition induced by TGF-β1 in human hepatoma SK-Hep1 and HepG2 cells, and compare the efficacy of drugs. Methods: The survival ability of HepG2 and SK-Hep1 cells was detected by MTT and the combination index (Q value) was calculated to judge the interaction of combined drugs. The EMT model of HepG2 and SK-Hep1 cells was established. The cell adhesion rate was detected by MTT. The expression of matrix metalloproteinase (MMP) MMP2, MMP7, MMP9, Snail, and Slug was detected by Western blotting. The localization and expression intensity of E-cadherin and Vimentin was detected by immunofluorescence. Results: MTT showed that compared with the control group, the 5-FU group, IGBR group and combination group cell survival ability were decreased (P < 0.05) at 48 h after administration; IGBR and 5-Fu had an additive or synergistic effect. Compared with the model group, the adhesion rate of 5-FU group, IGBR group and combination group was reduced (P < 0.05). Western blotting results showed that compared with the control group, the expression of MMP2, MMP7, MMP9, Snail, Slug were up-regulated (P < 0.05) in the model group. Compared with the model group, the expression of MMP2, MMP7, MMP9, Snail and Slug were down-regulated (P < 0.05) in 5-FU group, IGBR group and combination group. Compared with the control group, immunofluorescence showed that the E-cadherin fluorescence intensity was decreased in the model group, but the Vimentin fluorescence intensity was increased. Compared with the model group, the E-cadherin fluorescence intensity was increased in 5-FU group, IGBR group and combined group, but the Vimentin fluorescence intensity was decreased. Conclusion: IGBR and 5-Fu can inhibit human hepatoma EMT. The combined drugs have the combined effect on HepG2 cells and synergistic effect on SK-Hep1 cells. The therapeutic effect on SK-Hep1 cells is better than HepG2 cells.

Effect of DUOX2 on sensitivity of colorectal cancer cells to 5-fluorouracil / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of double oxidase 2 (DUOX2) on the sensitivity of colorectal cancer (CRC) cells to 5-fluorouracil (5-FU). Methods: CRC cell lines DLD-1, SW480, HCT116, SW620 and normal intestinal epithelial cell line NCM460 were selected, and the expression of DUOX2 in these cell lines were detected by qPCR. DUOX2 expression in HT-29 and HCT116 cells was stably knocked down by lentivirus infection technique. The knockdown efficiency was detected by qPCR and WB. Cells in sh-Control and sh-DUOX2 groups were treated with 5-FU at different concentrations (0, 5, 10, 20, 40, 80, 120 μg/ml). The effects of 5-FU on cell proliferation, apoptosis and cell cycle were detected by CCK-8 method and flow cytometry. HT29 cell transplanted xenograft model in nude mice was constructed to observe the effect of DUOX2 gene on the treatment efficacy of 5-FU. Results: the expression level of DUOX2 mRNA in CRC cells was significantly higher than that in NCM460 cells (P<0.05 or P<0.01). Compared with sh-Control group, the mRNA and protein expressions of DUOX2 in sh-DUOX2 group were significantly decreased (all P<0.01); the sensitivity of cells to 5-FU was enhanced, the apoptosis rate and the ratio of cells at G0/G1 phase were significantly increased (all P<0.01), and the ratio of cells at G2 and S phase was significantly decreased (all P<0.01). There was no significant difference in tumor volume and mass between sh-Control group and sh-DUOX2 group without 5-FU treatment (all P>0.05), but the volume and mass of transplanted tumor in sh-DUOX2+5-FU group after 5-FU treatment was significantly lower than that in sh-Control+5-FU group (all P<0.01). Conclusion: The sensitivity of CRC cells to 5-FU can be significantly enhanced by knocking down DUOX2 gene.

Study on synergistic anti-tumor effect of Shuangdan Capsules combined with 5-FU on hepatocellular carcinoma cells Huh-7 and xenograft mice / 中国中药杂志

This paper discussed the synergistic anti-tumor effect of Shuangdan Capsules combined with 5-fluorouracil(5-FU) on human liver cancer cell line Huh-7 and tumor bearing mice. The effects of Shuangdan Capsules combined with 5-FU on the activity and vascular endothelial growth factor(VEGF) receptor protein expression of Huh-7 cells were investigated, and the effects of drug combination on tube formation of HUVEC cell were also verified. In addition, the mice model of Huh-7 was established to observe the anti-tumor effect of drug combination and the distribution of tumor blood flow in tumor bearing mice by using molecular imaging. HPLC analysis showed that Shuangdan Capsules mainly consisted of danshensusodium, protocatechuic aldehyde, paeoniflorin, rosmarinic acid, alkannic acid, salvianolic acid B, and paeonol. In MTT experiment, the inhibition rate of Shuangdan Capsules(20 mg·L~(-1)) and 5-FU(1 μmol·L~(-1)) on Huh-7 cells was 60%, and the CI value was 0.59, suggesting that these two drugs had synergistic anti-hepatoma cells effect. The expression of VEGF receptor in Huh-7 cells was inhibited by the combination of these two drugs. In addition, the process of HUVEC was slow, and the number, length and area of the lumen branches decreased significantly. In vivo, Shuangdan Capsules combined with 5-FU inhibited the growth and prolongation of survival of Huh-7 cells in subcutaneous transplanted tumor nude mice; serum expression of CD31 and VEGF in nude mice were decreased, while caspase-3 was increased. Meanwhile, the drug combination significantly inhibited the expressions of MMP2 and VEGF in tumor tissues. Ultrasound showed that Shuangdan Capsules combined with 5-FU also inhibited tumor angiogenesis and reduced blood flow of tumor tissue. The results showed that Shuangdan Capsules combined with 5-FU may inhibit tumor angiogenesis by inhibiting VEGF and MMP2 expressions, thereby blocking tumor growth.

Effect and mechanism of allicin combined with 5-fluorouracil on proliferation and apoptosis of the MEC-1 cell line in mucoepidermoid carcinoma / 口腔疾病防治

Objective @#To investigate the effect and mechanism of allicin combined with 5-FU on proliferation inhibition and apoptosis of the mucoepidermoid carcinoma MEC-1 cell line in mucoepidermoid carcinoma in order to provide the corresponding basis for subsequent clinical drug application.@*Methods @# MEC-1 cells in the logarithmic growth phase were randomly divided into control groups and experimental groups. The control groups were PBS groups containing 0.1% DMSO, while the experimental groups were the allicin group, 5-FU group and combined drug group (the allicin combined with the 5-FU group). The proliferation inhibition rates of allicin, 5-FU and allicin combined with 5-FU in MEC-1 cells were detected by the CCK8 method at different concentrations (0, 25, 50, and 75 mg/L) for 24 h, and the IC50 value of allicin and 5-FU after 24 hours was calculated. The apoptotic rate of MEC-1 cells treated with allicin, 5-FU and allicin combined with 5-FU at different concentrations (0, 25, 50, and 75 mg/L) for 24 hours was measured by flow cytometry. The expression of Bax and Bcl-2 protein was determined by Western blot analysis of the IC50 concentration of allicin and 5-FU alone and in combination with MEC-1 cells for 24 hours. @*Results@#The growth inhibition rate and apoptosis rate of MEC-1 cells in the combined drug group were higher than those in the allicin group and the 5-FU alone group (P < 0.01). Allicin and 5-FU alone and in combination downregulated Bcl-2 protein and upregulated Bax protein expression, and the combined drug group had the largest ratio of Bax/Bcl-2 (P < 0.05). @*Conclusion @#Allicin and 5-FU both alone and in combination can inhibit the proliferation of and induce apoptosis in MEC-1 cells, and allicin can enhance the apoptosis of 5-FU in MEC-1 cells, which may be related to the apoptosis of the mitochondrial pathway.

Effect and molecular mechanism of Lin28 on 5-Fu sensitivity of hepatocellular carcinoma HepG2 cells / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect and mechanism of RNA binding protein Lin28 on the 5-fluorouracil (5-Fu) sensitivity of HepG2 cells. Methods: HepG2 cells were transfected with plasmid pcDNA3.1-Lin28 or si-Lin28 (small interfering RNA of Lin28). qPCR and Western blotting were used to detect the expression of Lin28 in HepG2 cells after transfection. Changes of cell proliferation in transfected cells after 5-Fu treatment was detected by CCK8 assay and the 50% inhibitory concentration (IC50) was calculated. Flow cytometry was used to detect apoptotic rate after 5-Fu treatment and the expression of apoptosis-related protein was assayed by Western blotting. The mRNA expressions of drug-resistant miRNAs (let-7a and let-7b), as well as cancer stem cell markers (Oct4, Nanog and Sox2) after transfection were detected by qPCR. Results: As compared to the HepG2/Vector cells, the mRNA and protein expressions of Lin28 were significantly up-regulated in HepG2/Lin28 cells (P<0.05 or P<0.01). Over-expression of Lin28 significantly suppressed the sensitivity of HepG2 cells to 5-Fu (IC50elevated obviously, P<0.05) and significantly increased cell proliferation while decreased apoptotic rate and expression of apoptotic-related protein caspase-3 (all P<0.01). As compared to si-control group, expression of Lin28 in HepG2/si-Lin28 cells was significantly down-regulated (P<0.01). Lin28 knockdown significantly reduced cell proliferation and IC50 of 5-Fu (all P<0.01) but increased apoptotic rate and expression of apoptosis-related protein (P<0.01). Compared with HepG2/Vector group, expressions of let-7a and let-7b, as well as cancer stem cell markers (Oct4, Nanog and Sox2) were significantly increased in HepG2/Lin28 cells (all P<0.01); while these molecules were significantly decreased in HepG2/si-Lin28 cells as comparing to si-control group (all P<0.01). Conclusion: Lin28 can modulate the chemosensitivity of HepG2 cells by regulating the expression of miRNAs and the formation of cancer stem cells. Targeting Lin28 might be a promising approach to improve the chemotherapy efficacy in HCC.

The gut microbiome: an orchestrator of xenobiotic metabolism

Microbes inhabiting the intestinal tract of humans represent a site for xenobiotic metabolism. The gut microbiome, the collection of microorganisms in the gastrointestinal tract, can alter the metabolic outcome of pharmaceuticals, environmental toxicants, and heavy metals, thereby changing their pharmacokinetics. Direct chemical modification of xenobiotics by the gut microbiome, either through the intestinal tract or re-entering the gut enterohepatic circulation, can lead to increased metabolism or bioactivation, depending on the enzymatic activity within the microbial niche. Unique enzymes encoded within the microbiome include those that reverse the modifications imparted by host detoxification pathways. Additionally, the microbiome can limit xenobiotic absorption in the small intestine by increasing the expression of cell-cell adhesion proteins, supporting the protective mucosal layer, and/or directly sequestering chemicals. Lastly, host gene expression is regulated by the microbiome, including CYP450s, multi-drug resistance proteins, and the transcription factors that regulate them. While the microbiome affects the host and pharmacokinetics of the xenobiotic, xenobiotics can also influence the viability and metabolism of the microbiome. Our understanding of the complex interconnectedness between host, microbiome, and metabolism will advance with new modeling systems, technology development and refinement, and mechanistic studies focused on the contribution of human and microbial metabolism.