Effect And Mechanism Of Interferon A2b Combined With 5-Fluorouracil In The Treatment Of Ocular Surface Squamous Neoplasia

Part Ⅰ Clinical effects of interferon α-2b combined with 5-fluorouracil in ocular surface squamous neoplasiaObjective: This study evaluated the efficacy of combining interferon α-2b(IFNα2b)with 5-fluorouracil(5-FU)in squamous ocular surface neoplasia(OSSN).Methods: The data of 26(27 eyes)OSSN patients in the ophthalmology department of Wuhan Union Hospital were analyzed retrospectively from July 2016 to July 2021 receiving topical 1 million IU/m L IFNα2b plus 1% 5-FU.The main outcome measures were tumor response to medication,duration of treatment,recurrence rate,visual acuity changes and adverse reactions.Results: Twenty-six patients(17 male,9 female)had a mean age of 63.9(median,67;range 22–83)years.Complete tumor response was observed in 24 eyes(88.9%).Three eyes(11.1%)showed partial response to the chemotherapy agents and later underwent surgical tumor removal.Compared with previous data from large studies with monotherapy in the literature,the combination significantly shortened the time to OSSN tumor regression(mean 6.1 weeks,median 6 weeks,range 3 to 11 weeks)and reduced the number of 5-FU cycle(mean 1.8,median 2,range 1 to 3).No tumor recurrence was observed during follow-up at a mean time of 16.1 months(median 12 months,range 6 to 38 months).Conclusion: IFNα2b combined with 5-FU is a safe and effective treatment for OSSN.This method shorten the time of tumor regression,reduce the frequency of 5-FU cycle,providing a new way for clinical treatment of OSSN,which is worthy of popularization and application.Part Ⅱ pathological features of OSSN and expression of interferon receptors and key enzymes of 5-FU anabolismObjective: To explore the pathological characteristics of OSSN tumors and detect the expression of interferon receptor and key enzymes during 5-FU anabolism in OSSN tumor tissues.Methods: Fresh tumor tissue samples(6 cases)diagnosed as OSSN from June 2021 to January 2022 were collected and conjunctival tissue from body donors was taken as control group(3 cases).Pathological tissue specimens were prepared into frozen sections by Hematoxylin-eosin staining and immunofluorescence staining.According to the pathological manifestations of tissue,the specimens were divided into normal conjunctival group,papilloma group and squamous cell carcinoma group.The positive rates of type I interferon receptor,thymidylate synthase(TS),thymidine phosphorylase(TP)and dihydropyrimidine dehydrogenase(DPD)in 5-FU anabolism in each group were counted and analyzed.Results: In the six OSSN tumor specimens,four were diagnosed with papilloma by the pathology department of our hospital,one was papilloma with dysplasia and another one was diagnosed as SCC.IFNAR2 showed bright fluorescence and high expression in the cell membrane and interstitium of normal conjunctiva and OSSN tumor cells.Only two cases of OSSN showed low expression of IFNAR1.TP was mainly expressed in the stromal infiltrating margin of tumor cells with weak positive intensity.No TP expression was observed in the normal conjunctival tissue,and the TP expression difference between the two groups was statistically significant(P <0.05).TS and DPD are mainly localized in the cytoplasm of tumor cells and normal conjunctival epithelial cells,and occasionally in the nucleus.The positive rates of TS in donor conjunctival tissue and tumor tissue were 33.3% and 50%,respectively,and the positive rates of DPD were 33.3% and 83.3%.No significant differences of IFNAR1,IFNAR2,TS and DPD in OSSN and donor conjunctival tissue(P > 0.05).Conclusion: High expression of interferon receptor in tumor tissue may be part of the sensitivity of OSSN to IFNα2b treatment.TP was more highly expressed in OSSN tumor tissue compared to normal conjunctival tissue,consistent with an active proliferative state of tumor cells.Part Ⅲ Effects and mechanism of IFNα2b and 5-FU combination on proliferation,migration,apoptosis and cell cycle of Cal27 cell lineObjective: To explore the effects and related mechanisms of IFNα2b and 5-FU on proliferation,migration,apoptosis,and cycle of cells in human oral squamous cell carcinoma Cal27 cell line.Methods: In vitro experiments,Cal27 cell lines were treated with different concentrations of IFNα2b and 5-FU alone or in combination for 48 hours,cell activity was measured by CCK-8 method and effect-dose curve was drawn.Tumor cell migration was detected by cell scratch experiments and relative mobility was calculated.Cal27 cells were treated with nonfixed proportional combination,namely different concentrations of 5-FU and 100 IU/ml IFNα2b for 48 h,and the combination index(CI value)of both drugs was calculated by Compu Syn software.Western blot assay was used to detect the expression of the key enzymes TP,TS and DPD of 5-FU after treatment with IFNα2b at different doses.Cal27 tumor cells were treated with low,medium and high concentrations of IFNα2b(102,104,106 IU/ml),5-FU(0.2,2,20 μg/ml)and 100 IU/ml IFNα2b + 0.2 μg/ml 5-FU.Cell cycle and apoptosis were measured with flow cytometry by Annexin-V/PI double staining.TUNEL assay showed the apoptotic morphology and the proportion of apoptotic cells.Results: Both IFNα2b and 5-FU inhibited cell proliferation,of which the IC50 was 8*104 IU/ml and 7 μg/ml.Except for the lowest dose of 5-FU(0.032μg/ml)and the highest dose of 5-FU(2500 μg/ml),CI values of the other groups were all less than 1,showing synergistic effect.Low doses of IFNα2b(100 IU/ml)and 5-FU(0.2 μg/ml)both decreased the relative mobility of Cal27 cells and the inhibitory effect was more obvious when the two drugs were used in combination.The protein expression levels of TP and DPD in Cal27 cells were upregulated at a low concentration of IFN-α2b(100 IU/ml).With the increase of IFNα2b concentration,the expression levels of TP and DPD were dose-dependent.The proportion of G0/G1 phase cells in the medium and high dose IFN-α2b and 5-FU treatment groups was higher than that in the blank control group,while the proportion of S phase cells was reduced.Low-dose IFN-α2b(100 IU/ml)combined with 5-FU(0.2 μg/ml)inhibited cell proliferation in G0/G1 phase compared with 5-FU(0.2 μg/ml)alone.Medium and high doses of IFNα2b and each concentration of 5-FU could induce apoptosis,and the apoptosis rate increased with the increase of drug concentration.Low dose IFNα2b does not induce apoptosis of Cal27 tumor cells,but increase the sensitivity of tumor cells to 5-FU and increase the apoptosis rate of tumor cells.Conclusion: Both IFNα2b and 5-FU alone and in combination can inhibit the proliferation and migration of Cal27 tumor cells,causing cell apoptosis,inducing cell cycle arrest.Low doses of IFNα2b increase the antitumor effect of 5-FU on Cal27,showing a synergistic effect,the mechanism of which may be related to the up-regulation of the expression of TP,a key enzyme in 5-FU metabolism.