Microbial metabolite sodium butyrate enhances the anti-tumor efficacy of 5-fluorouracil against colorectal cancer by modulating PINK1/Parkin signaling and intestinal flora.

Colorectal cancer (CRC) is a prevalent global health issue, with 5-fluorouracil (5-FU) being a commonly used chemotherapeutic agent for its treatment. However, the efficacy of 5-FU is often hindered by drug tolerance. Sodium butyrate (NaB), a derivative of intestinal flora, has demonstrated anti-cancer properties both in vitro and in vivo through pro-apoptotic effects and has shown promise in improving outcomes when used in conjunction with traditional chemotherapy agents. This study seeks to evaluate the impact and potential mechanisms of NaB in combination with 5-FU on CRC. We employed a comprehensive set of assays, including CCK-8, EdU staining, Hoechst 33258 staining, flow cytometry, ROS assay, MMP assay, immunofluorescence, and mitophagy assay, to detect the effect of NaB on the biological function of CRC cells in vitro. Western blotting and immunohistochemistry were used to verify the above experimental results. The xenograft tumor model was established to evaluate the in vivo anti-CRC activity of NaB. Subsequently, 16S rRNA gene sequencing was used to analyze the intestinal flora. The findings of our study demonstrate that sodium butyrate (NaB) exerts inhibitory effects on tumor cell proliferation and promotes tumor cell apoptosis in vitro, while also impeding tumor progression in vivo through the enhancement of the mitophagy pathway. Furthermore, the combined treatment of NaB and 5-fluorouracil (5-FU) yielded superior therapeutic outcomes compared to monotherapy with either agent. Moreover, this combination therapy resulted in the specific enrichment of Bacteroides, LigiLactobacillus, butyric acid-producing bacteria, and acetic acid-producing bacteria in the intestinal microbiota. The improvement in the intestinal microbiota contributed to enhanced therapeutic outcomes and reduced the adverse effects of 5-FU. Taken together, these findings indicate that NaB, a histone acetylation inhibitor synthesized through intestinal flora fermentation, has the potential to significantly enhance the therapeutic efficacy of 5-FU in CRC treatment and improve the prognosis of CRC patients.

5-Fluorouracil dose escalation generated desensitized colorectal cancer cells with reduced expression of protein methyltransferases and no epithelial-to-mesenchymal transition potential.

Background: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. In many cases, the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil (5-FU). The epithelial-to-mesenchymal transition (EMT) and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers. This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC. Materials and Methods: HCT-116, Caco-2, and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU. The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays. This was followed by a Western blot which analyzed the protein expressions of the epithelial marker E-cadherin, mesenchymal marker vimentin, and the EMT transcription factor (EMT-TF), the snail family transcriptional repressor 1 (Snail) in the parental and desensitized cells. Western blotting was also conducted to study the protein expressions of the protein methyltransferases (PMTs), Euchromatic histone lysine methyltransferase 2 (EHMT2/G9A), protein arginine methyltransferase (PRMT5), and SET domain containing 7/9 (SETD7/9) along with the global lysine and arginine methylation profiles. Results: The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU. The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells. This was reflected in the observed reduction in E-cadherin, vimentin, and Snail in the desensitized cell lines. Additionally, the protein expressions of EHMT2/G9A, PRMT5, and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment. Conclusion: This study showed that continuous, dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.

Selective but not pan-CDK inhibition abrogates 5-FU-driven tissue factor upregulation in colon cancer.

Thromboembolic events are complications in cancer patients and hypercoagulability has been linked to the tissue factor (TF) pathway, making this an attractive target. Here, we investigated the effects of chemotherapeutics and CDK inhibitors (CDKI) abemaciclib/palbociclib (CDK4/6), THZ-1 (CDK7/12/13), and dinaciclib (CDK1/2/5/9) alone and in combination regimens on TF abundance and coagulation. The human colorectal cancer (CRC) cell line HROC173 was treated with 5-FU or gemcitabine to stimulate TF expression. TF+ cells were sorted, recultured, and re-analyzed. The effect of treatment alone or in combination was assessed by functional assays. Low-dose chemotherapy induced a hypercoagulable state and significantly upregulated TF, even after reculture without treatment. Cells exhibited characteristics of epithelial-mesenchymal transition, including high expression of vimentin and mucin. Dinaciclib and THZ-1 also upregulated TF, while abemaciclib and palbociclib downregulated it. Similar results were observed in coagulation assays. The same anticoagulant activity of abemaciclib was seen after incubation with peripheral immune cells from healthy donors and CRC patients. Abemaciclib reversed 5-FU-induced TF upregulation and prolonged clotting times in second-line treatment. Effects were independent of cytotoxicity, senescence, and p27kip1 induction. TF-antibody blocking experiments confirmed the importance of TF in plasma coagulation, with Factor XII playing a minor role. Short-term abemaciclib counteracts 5-FU-induced hypercoagulation and eventually even prevents thromboembolic events.

Cetuximab plus FOLFOXIRI versus cetuximab plus FOLFOX as conversion regimen in RAS/BRAF wild-type patients with initially unresectable colorectal liver metastases (TRICE trial): A randomized controlled trial.

BACKGROUND: It remains unclear whether intensification of the chemotherapy backbone in tandem with an anti-EGFR can confer superior clinical outcomes in a cohort of RAS/BRAF wild-type colorectal cancer (CRC) patients with initially unresectable colorectal liver metastases (CRLM). To that end, we sought to comparatively evaluate the efficacy and safety of cetuximab plus FOLFOXIRI (triplet arm) versus cetuximab plus FOLFOX (doublet arm) as a conversion regimen (i.e., unresectable to resectable) in CRC patients with unresectable CRLM. METHODS AND FINDINGS: This open-label, randomized clinical trial was conducted from April 2018 to December 2022 in 7 medical centers across China, enrolling 146 RAS/BRAF wild-type CRC patients with initially unresectable CRLM. A stratified blocked randomization method was utilized to assign patients (1:1) to either the cetuximab plus FOLFOXIRI (n = 72) or cetuximab plus FOLFOX (n = 74) treatment arms. Stratification factors were tumor location (left versus right) and resectability (technically unresectable versus ≥5 metastases). The primary outcome was the objective response rate (ORR). Secondary outcomes included the median depth of tumor response (DpR), early tumor shrinkage (ETS), R0 resection rate, progression-free survival (PFS), overall survival (not mature at the time of analysis), and safety profile. Radiological tumor evaluations were conducted by radiologists blinded to the group allocation. Primary efficacy analyses were conducted based on the intention-to-treat population, while safety analyses were performed on patients who received at least 1 line of chemotherapy. A total of 14 patients (9.6%) were lost to follow-up (9 in the doublet arm and 5 in the triplet arm). The ORR was comparable following adjustment for stratification factors, with 84.7% versus 79.7% in the triplet and doublet arms, respectively (odds ratio [OR] 0.70; 95% confidence intervals [CI] [0.30, 1.67], Chi-square p = 0.42). Moreover, the ETS rate showed no significant difference between the triplet and doublet arms (80.6% (58/72) versus 77.0% (57/74), OR 0.82, 95% CI [0.37, 1.83], Chi-square p = 0.63). Although median DpR was higher in the triplet therapy group (59.6%, interquartile range [IQR], [50.0, 69.7] versus 55.0%, IQR [42.8, 63.8], Mann-Whitney p = 0.039), the R0/R1 resection rate with or without radiofrequency ablation/stereotactic body radiation therapy was comparable with 54.2% (39/72) of patients in the triplet arm versus 52.7% (39/74) in the doublet arm. At a median follow-up of 26.2 months (IQR [12.8, 40.5]), the median PFS was 11.8 months in the triplet arm versus 13.4 months in the doublet arm (hazard ratio [HR] 0.74, 95% CI [0.50, 1.11], Log-rank p = 0.14). Grade ≥ 3 events were reported in 47.2% (35/74) of patients in the doublet arm and 55.9% (38/68) of patients in the triplet arm. The triplet arm was associated with a higher incidence of grade ≥ 3 neutropenia (44.1% versus 27.0%, p = 0.03) and diarrhea (5.9% versus 0%, p = 0.03). The primary limitations of the study encompass the inherent bias in subjective surgical decisions regarding resection feasibility, as well as the lack of a centralized assessment for ORR and resection. CONCLUSIONS: The combination of cetuximab with FOLFOXIRI did not significantly improve ORR compared to cetuximab plus FOLFOX. Despite achieving an enhanced DpR, this improvement did not translate into improved R0 resection rates or PFS. Moreover, the triplet arm was associated with an increase in treatment-related toxicity. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03493048.

Apoptotic effect of 5-fluorouracil-doxorubicin combination on colorectal cancer cell monolayers and spheroids.

BACKGROUND: Drug combination studies help to improve new treatment approaches for colon cancer. Tumor spheroids (3D) are better models than traditional 2-dimensional cultures (2D) to evaluate cellular responses to chemotherapy drugs. The cultivation of cancer cells in 2D and 3D cultures affects the apoptotic process, which is a major factor influencing the response of cancer cells to chemotherapeutic drugs. In this study, the antiproliferative effects of 5-fluorouracil (5-FU) and doxorubicin (DOX) were investigated separately and in combination using 2D and 3D cell culture models on two different colon cancer cell lines, HT-29 (apoptosis-resistant cells) and Caco-2 2 (apoptosis-susceptible cells). METHODS: The effect of the drugs on the proliferation of both colon cancer cells was determined by performing an MTT assay in 2D culture. The apoptotic effect of 5-FU and DOX, both as single agents and in combination, was assessed in 2D and 3D cultures through quantitative real-time polymerase chain reaction analysis. The expression of apoptotic genes, such as caspases, p53, Bax, and Bcl-2, was quantified. RESULTS: It was found that the mRNA expression of proapoptotic genes was significantly upregulated, whereas the mRNA expression of the antiapoptotic Bcl-2 gene was significantly downregulated in both colon cancer models treated with 5-FU, DOX, and 5-FU + DOX. CONCLUSION: The results indicated that the 5-FU + DOX combination therapy induces apoptosis and renders 5-FU and DOX more effective at lower concentrations compared to their alone use. This study reveals promising results in reducing the potential side effects of treatment by enabling the use of lower drug doses.

Impact of the thyroid hormone T3 and its nuclear receptor TRα1 on colon cancer stem cell phenotypes and response to chemotherapies.

Colorectal cancers (CRCs) are highly heterogeneous and show a hierarchical organization, with cancer stem cells (CSCs) responsible for tumor development, maintenance, and drug resistance. Our previous studies showed the importance of thyroid hormone-dependent signaling on intestinal tumor development and progression through action on stem cells. These results have a translational value, given that the thyroid hormone nuclear receptor TRα1 is upregulated in human CRCs, including in the molecular subtypes associated with CSC features. We used an established spheroid model generated from the human colon adenocarcinoma cell line Caco2 to study the effects of T3 and TRα1 on spheroid formation, growth, and response to conventional chemotherapies. Our results show that T3 treatment and/or increased TRα1 expression in spheroids impaired the response to FOLFIRI and conferred a survival advantage. This was achieved by stimulating drug detoxification pathways and increasing ALDH1A1-expressing cells, including CSCs, within spheroids. These results suggest that clinical evaluation of the thyroid axis and assessing TRα1 levels in CRCs could help to select optimal therapeutic regimens for patients with CRC. Proposed mechanism of action of T3/TRα1 in colon cancer spheroids. In the control condition, TRα1 participates in maintaining homeostatic cell conditions. The presence of T3 in the culture medium activates TRα1 action on target genes, including the drug efflux pumps ABCG2 and ABCB1. In the case of chemotherapy FOLFIRI, the increased expression of ABC transcripts and proteins induced by T3 treatment is responsible for the augmented efflux of 5-FU and Irinotecan from the cancer cells. Taken together, these mechanisms contribute to the decreased efficacy of the chemotherapy and allow cells to escape the treatment. Created with BioRender.com .

Implementation of perioperative FLOT compared to ECX/EOX chemotherapy regimens in resectable esophagogastric adenocarcinomas: an analysis of real-world data.

BACKGROUND AND PURPOSE: Perioperative 5-FU, leucovorin, oxaliplatin, and docetaxel (FLOT) is recommended in resectable esophagogastric adenocarcinoma based on randomised trials. However, the effectiveness of FLOT in routine clinical practice remains unknown as randomised trials are subject to selection bias limiting their generalisability. The aim of this study was to evaluate the implementation of FLOT in real-world patients. METHODS: Retrospectively collected data were analysed in consecutive patients treated before or after the implementation of FLOT. The primary endpoint was complete pathological response (pCR) and secondary endpoints were margin-free resection (R0), overall survival (OS), relapse-free survival (RFS) tolerability of chemotherapy and surgical complications. RESULTS: Mean follow-up time for patients treated with FLOT (n = 205) was 37.7 versus 47.0 months for epirubicin, cis- or oxaliplatin, and capecitabine (ECX/EOX, n = 186). Surgical resection was performed in 88.0% versus 92.0%; pCR were observed in 3.8% versus 2.4%; and R0 resections were achieved in 78.0% versus 86.0% (p = 0.03) in the ECX/EOX and FLOT cohorts, respectively. Survival analysis indicated no significant difference in RFS (p = 0.17) or OS (p = 0.37) between the cohorts with a trend towards increased OS in performance status 0 (hazard ratio [HR] = 0.73, 95% confidence interval [CI]: 0.50-1.04). More patients treated with ECX/EOX completed chemotherapy (39% vs. 28%, p = 0.02). Febrile neutropenia was more common in the FLOT cohort (3.8% vs. 11%, p = 0.0086). 90-days mortality (1.2% vs. 0%) and frequency of anastomotic leakage (8% vs. 6%) were equal and low. INTERPRETATION: Patients receiving FLOT did not demonstrate improved pCR, RFS or OS. However, R0 rate was improved and patients in good PS trended towards improved OS.

Synthesis of Flavonols and Assessment of Their Biological Activity as Anticancer Agents.

A series of flavanols were synthesized to assess their biological activity against human non-small cell lung cancer cells (A549). Among the sixteen synthesized compounds, it was observed that compounds 6k (3.14 ± 0.29 µM) and 6l (0.46 ± 0.02 µM) exhibited higher potency compared to 5-fluorouracil (5-Fu, 4.98 ± 0.41 µM), a clinical anticancer drug which was used as a positive control. Moreover, compound 6l (4'-bromoflavonol) markedly induced apoptosis of A549 cells through the mitochondrial- and caspase-3-dependent pathways. Consequently, compound 6l might be developed as a candidate for treating or preventing lung cancer.

Modulators of the Nrf2 Signaling Pathway Enhance the Cytotoxic Effect of Standard Chemotherapeutic Drugs on Organoids of Metastatic Colorectal Cancer.

The activity of known modulators of the Nrf2 signaling pathway (bardoxolone and brusatol) was studied on cultures of tumor organoids of metastatic colorectal cancer previously obtained from three patients. The effect of modulators was studied both as monotherapy and in combination with standard chemotherapy drugs used to treat colorectal cancer. The Nrf2 inhibitor brusatol and the Nrf2 activator bardoxolone have antitumor activity. Moreover, bardoxolone and brusatol also significantly enhance the effect of the chemotherapy drugs 5-fluorouracil, oxaliplatin, and irinotecan metabolite SN-38. Thus, bardoxolone and brusatol can be considered promising candidates for further preclinical and clinical studies in the treatment of colorectal cancer.

Unveiling Discrepant and Rare Dihydropyrimidine Dehydrogenase (DPYD) Results Using an In-House Genotyping Test: A Case Series.

Fluoropyrimidine chemotherapy is a primary component of many solid tumor treatment regimens, particularly those for gastrointestinal malignancies. Approximately one-third of patients receiving fluoropyrimidine-based chemotherapies experience serious adverse effects. This risk is substantially higher in patients carrying DPYD genetic variants, which cause reduced fluoropyrimidine metabolism and inactivation (ie, dihydropyridine dehydrogenase [DPD] deficiency). Despite the known relationship between DPD deficiency and severe toxicity risk, including drug-related fatalities, pretreatment DPYD testing is not standard of care in the United States. We developed an in-house DPYD genotyping test that detects 5 clinically actionable variants associated with DPD deficiency, and genotyped 827 patients receiving fluoropyrimidines, of which 49 (6%) were identified as heterozygous carriers. We highlight 3 unique cases: (1) a patient with a false-negative result from a commercial laboratory that only tested for the c.1905 + 1G>A (*2A) variant, (2) a White patient in whom the c.557A>G variant (typically observed in people of African ancestry) was detected, and (3) a patient with the rare c.1679T>G (*13) variant. Lastly, we evaluated which DPYD variants are detected by commercial laboratories offering DPYD genotyping in the United States and found 6 of 13 (46%) did not test for all 5 variants included on our panel. We estimated that 20.4% to 81.6% of DPYD heterozygous carriers identified on our panel would have had a false-negative result if tested by 1 of these 6 laboratories. The sensitivity and negative predictive value of the diagnostic tests from these laboratories ranged from 18.4% to 79.6% and 95.1% to 98.7%, respectively. These cases underscore the importance of comprehensive DPYD genotyping to accurately identify patients with DPD deficiency who may require lower fluoropyrimidine doses to mitigate severe toxicities and hospitalizations. Clinicians should be aware of test limitations and variability in variant detection by commercial laboratories, and seek assistance by pharmacogenetic experts or available resources for test selection and result interpretation.

Prognostic and chemotherapeutic implications of a novel four-gene pyroptosis model in head and neck squamous cell carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers. Chemotherapy remains one dominant therapeutic strategy, while a substantial proportion of patients may develop chemotherapeutic resistance; therefore, it is particularly significant to identify the patients who could achieve maximum benefits from chemotherapy. Presently, four pyroptosis genes are reported to correlate with the chemotherapeutic response or prognosis of HNSCC, while no study has assessed the combinatorial predicting efficacy of these four genes. Hence, this study aims to evaluate the predictive value of a multi-gene pyroptosis model regarding the prognosis and chemotherapeutic responsiveness in HNSCC. Methods: By utilizing RNA-sequencing data from The Cancer Genome Atlas database and the Gene Expression Omnibus database, the pyroptosis-related gene score (PRGscore) was computed for each HNSCC sample by performing a Gene Set Variation Analysis (GSVA) based on four genes (Caspase-1, Caspase-3, Gasdermin D, Gasdermin E). The prognostic significance of the PRGscore was assessed through Cox regression and Kaplan-Meier survival analyses. Additionally, chemotherapy sensitivity stratified by high and low PRGscore was examined to determine the potential association between pyroptosis activity and chemosensitivity. Furthermore, chemotherapy sensitivity assays were conducted in HNSCC cell lines in vitro. Results: As a result, our study successfully formulated a PRGscore reflective of pyroptotic activity in HNSCC. Higher PRGscore correlates with worse prognosis. However, patients with higher PRGscore were remarkably more responsive to chemotherapy. In agreement, chemotherapy sensitivity tests on HNSCC cell lines indicated a positive association between overall pyroptosis levels and chemosensitivity to cisplatin and 5-fluorouracil; in addition, patients with higher PRGscore may benefit from the immunotherapy. Overall, our study suggests that HNSCC patients with higher PRGscore, though may have a less favorable prognosis, chemotherapy and immunotherapy may exhibit better benefits in this population.

Comparison of efficacy of neoadjuvant chemotherapy with gemcitabine plus cisplatin versus docetaxil, cisplatin, plus fluorouracil in locally advanced nasopharyngeal carcinoma.

Induction followed by concurrent chemoradiation (CCRT) is the standard of care for locally advanced nasopharyngeal carcinoma (LANPC). This study evaluated and compared the efficacy of two regimens of neoadjuvant chemotherapy along with CCRT in LANPC. Patients with LANPC were randomly divided in Group I (receiving neoadjuvant gemcitabine and cisplatin) and Group II (receiving neoadjuvant docetaxil, cisplatin and fluorouracil). Both groups also received concurrent single agent (i.e., cisplatin) chemotherapy and radiotherapy (70Gy). Treatment response was assessed at 8 weeks after the completion of CCRT using RECIST criteria. A total of 68 LANPC patients were enrolled. Group I comprised of 32 patients, with male to female ratio of 2.2, a mean (range, median) age of 38.6±11.3 (19-58, 36) years. Group II comprised of 36 patients, with male to female ratio of 3.5, mean (range, median) age of 40.9 ±11.6 (17-63, 40) years. The complete response was higher whereas the partial response was lower in Group I as compared to Group II (23/32 versus 16/36 and 06/32 versus 18/36, respectively). LANPC patients receiving gemcitabine plus cisplatin based neoadjuvant chemotherapy showed higher response, as compared with docetaxil, cisplatin and fluorouracil based neoadjuvant chemotherapy.

Intelligent nanovesicle for remodeling tumor microenvironment and circulating tumor chemoimmunotherapy amplification.

Imperceptible examination and unideal treatment effect are still intractable difficulties for the clinical treatment of pancreatic ductal adenocarcinoma (PDAC). At present, despite 5-fluorouracil (5-FU), as a clinical first-line FOLFIRINOX chemo-drug, has achieved significant therapeutic effects. Nevertheless, these unavoidable factors such as low solubility, lack of biological specificity and easy to induce immunosuppressive surroundings formation, severely limit their treatment in PDAC. As an important source of energy for many tumor cells, tryptophan (Trp), is easily degraded to kynurenine (Kyn) by indolamine 2,3- dioxygenase 1 (IDO1), which activates the axis of Kyn-AHR to form special suppressive immune microenvironment that promotes tumor growth and metastasis. However, our research findings that 5-FU can induce effectively immunogenic cell death (ICD) to further treat tumor by activating immune systems, while the secretion of interferon-γ (IFN-γ) re-induce the Kyn-AHR axis activation, leading to poor treatment efficiency. Therefore, a metal matrix protease-2 (MMP-2) and endogenous GSH dual-responsive liposomal-based nanovesicle, co-loading with 5-FU (anti-cancer drug) and NLG919 (IDO1 inhibitor), was constructed (named as ENP919@5-FU). The multifunctional ENP919@5-FU can effectively reshape the tumor immunosuppression microenvironment to enhance the effect of chemoimmunotherapy, thereby effectively inhibiting cancer growth. Mechanistically, PDAC with high expression of MMP-2 will propel the as-prepared nanovesicle to dwell in tumor region via shedding PEG on the nanovesicle surface, effectively enhancing tumor uptake. Subsequently, the S-S bond containing nanovesicle was cut via high endogenous GSH, leading to the continued release of 5-FU and NLG919, thereby enabling circulating chemoimmunotherapy to effectively cause tumor ablation. Moreover, the combination of ENP919@5-FU and PD-L1 antibody (αPD-L1) showed a synergistic anti-tumor effect on the PDAC model with abdominal cavity metastasis. Collectively, ENP919@5-FU nanovesicle, as a PDAC treatment strategy, showed excellent antitumor efficacy by remodeling tumor microenvironment to circulate tumor chemoimmunotherapy amplification, which has promising potential in a precision medicine approach.

The male reproductive toxicity after 5-Fluorouracil exposure: DNA damage, oxidative stress, and mitochondrial dysfunction in vitro and in vivo.

5-Fluorouracil (5-FU), a chemotherapeutic drug used to treat a variety of cancers, can enter the environment through different routes, causing serious public health and environmental concerns. It has been reported that 5-FU exposure adversely affects male reproductive function, and its effects on this system cannot be avoided. In this study, using western blotting and quantitative polymerase chain reaction studies, we found that 5-FU promoted testicular injury by inducing oxidative stress, which was accompanied by the inhibition of nuclear factor erythroid 2-related factor 2/antioxidant response element signaling. Accumulation of reactive oxygen species (ROS) aggravated 5-FU-mediated mitochondrial dysfunction and apoptosis in murine cell lines and testes, indicating oxidative stress and mitochondrial-dependent apoptotic signaling play crucial roles in the damage of spermatogenic cells caused. N-Acetyl-L-cysteine, an antioxidant that scavenges intracellular ROS, protected spermatogenic cells from 5-FU-induced oxidative damage and mitochondrial dysfunction, revealing the important role of ROS in testicular dysfunction caused by 5-FU. We found that 5-FU exposure induces testicular cell apoptosis through ROS-mediated mitochondria pathway in mice. In summary, our findings revealed the reproductive toxicological effect of 5-FU on mice and its mechanism, provided basic data reference for adverse ecological and human health outcomes associated with 5-FU contamination or poisoning.

VEGF-C and 5-Fluorouracil Improve Bleb Survival in a Rabbit Glaucoma Surgery Trabeculectomy Model.

Purpose: To evaluate VEGF-C-induced lymphoproliferation in conjunction with 5-fluorouracil (5-FU) antimetabolite treatment in a rabbit glaucoma filtration surgery (GFS) model. Methods: Thirty-two rabbits underwent GFS and were assigned to four groups (n = 8 each) defined by subconjunctival drug treatment: (a) VEGF-C combined with 5-FU, (b) 5-FU, (c) VEGF-C, (d) and control. Bleb survival, bleb measurements, and IOP were evaluated over 30 days. At the end, histology and anterior segment OCT were performed on some eyes. mRNA was isolated from the remaining eyes for RT-PCR evaluation of vessel-specific markers (lymphatics, podoplanin and LYVE-1; and blood vessels, CD31). Results: Qualitatively and quantitatively, VEGF-C combined with 5-FU resulted in blebs which were posteriorly longer and wider than the other conditions: vs. 5-FU (P = 0.043 for longer, P = 0.046 for wider), vs. VEGF-C (P < 0.001, P < 0.001) and vs. control (P < 0.001, P < 0.001). After 30 days, the VEGF-C combined with 5-FU condition resulted in longer bleb survival compared with 5-FU (P = 0.025), VEGF-C (P < 0.001), and control (P < 0.001). Only the VEGF-C combined with 5-FU condition showed a negative correlation between IOP and time that was statistically significant (r = -0.533; P = 0.034). Anterior segment OCT and histology demonstrated larger blebs for the VEGF-C combined with 5-FU condition. Only conditions including VEGF-C led to increased expression of lymphatic markers (LYVE-1, P < 0.001-0.008 and podoplanin, P = 0.002-0.011). Expression of CD31 was not different between the groups (P = 0.978). Conclusions: Adding VEGF-C lymphoproliferation to standard antimetabolite treatment improved rabbit GFS success and may suggest a future strategy to improve human GFSs.

Assessing outcomes of topical 5-fluorouracil as primary and adjuvant therapy for squamous cell carcinoma in-situ.

Cutaneous squamous cell carcinoma in-situ (SCCis) is an intraepithelial tumor with a good prognosis. Standard treatment includes both surgical and non-surgical interventions. We determined the clearance rate for SCCis and residual SCCis identified on frozen section during Mohs micrographic surgery (MMS) after treatment with topical fluorouracil 5% cream (5-FU). All MMS cases were initiated for biopsy-proven invasive squamous cell carcinoma (SCC). A retrospective chart review was conducted from January 2017-February 2024 at Columbia University Irving Medical Center (CUIMC) to identify patients with SCCis who were treated with topical 5-FU as primary therapy or adjuvant therapy (AT) for residual SCCis post-MMS for invasive SCC. 41 patients were included (80% males, 70.1 ± 11.8 years). The average follow-up time for the primary therapy group was 25.4 ± 12.8 months, and for the post-MMS AT group 22.5 ± 11.1 months. In the group treated with topical 5-FU as primary therapy (n = 28), 27 patients (96.43%, 95% confidence interval: 81.65-99.91%) achieved complete clearance. One patient had recurrence at 8 months post-treatment. Of the patients in the post-MMS adjuvant treatment group (n = 13), 12 (92.3% clearance, 95% confidence interval 63.97-99.81%) achieved complete clearance. One patient had recurrence at 8 months post-treatment. This study found that topical 5-FU cream is effective as both primary therapy for SCCis and as adjuvant therapy for residual SCCis following MMS of invasive SCC.

Correlation of gasdermin B staining patterns with prognosis, progression, and immune response in colorectal cancer.

BACKGROUND: Pyroptosis is a type of programmed cell death mediated by the gasdermin family. Gasdermin B (GSDMB), as a member of gasdermin family, can promote the occurrence of cell pyroptosis. However, the correlations of the GSDMB expression in colorectal cancer with clinicopathological predictors, immune microenvironment, and prognosis are unclear. METHODS: Specimens from 267 colorectal cancer cases were analyzed by immunohistochemistry to determine GSDMB expression, CD3+, CD4+, and CD8+ T lymphocytes, CD20+ B lymphocytes, CD68+ macrophages, and S100A8+ immune cells. GSDMB expression in cancer cells was scored in the membrane, cytoplasm, and nucleus respectively. GSDMB+ immune cell density was calculated. Univariate and multivariate survival analyses were performed. The association of GSDMB expression with other clinicopathological variables and immune cells were also analyzed. Double immunofluorescence was used to identify the nature of GSDMB+ immune cells. Cytotoxicity assays and sensitivity assays were performed to detect the sensitivity of cells to 5-fluorouracil. RESULTS: Multivariate survival analysis showed that cytoplasmic GSDMB expression was an independent favorable prognostic indicator. Patients with positive cytoplasmic or nuclear GSDMB expression would benefit from 5-fluorouracil based chemotherapy. The assays in vitro showed that high GSDMB expression enhanced the sensitivity of colorectal cancer cells to 5-fluorouracil. Patients with positive membranous or nuclear GSDMB expression had more abundant S100A8+ immune cells in the tumor invasive front. Positive nuclear GSDMB expression indicated more CD68+ macrophages in the tumor microenvironment. Moreover, GSDMB+ immune cell density in the stroma was associated with a higher neutrophil percentage but a lower lymphocyte counts and monocyte percentage in peripheral blood. Furthermore, the results of double immunofluorescence showed that GSDMB co-expressed with CD68 or S100A8 in stroma cells. CONCLUSION: The GSDMB staining patterns are linked to its role in cancer progression, the immune microenvironment, systemic inflammatory response, chemotherapeutic efficacy, and prognosis. Colorectal cancer cells with high GSDMB expression are more sensitive to 5-fluorouracil. However, GSDMB expression in immune cells has different effects on cancer progression from that in cancer cells.

WNT3 promotes chemoresistance to 5-Fluorouracil in oral squamous cell carcinoma via activating the canonical ß-catenin pathway.

BACKGROUND: 5-Fluorouracil (5FU) is a primary chemotherapeutic agent used to treat oral squamous cell carcinoma (OSCC). However, the development of drug resistance has significantly limited its clinical application. Therefore, there is an urgent need to determine the mechanisms underlying drug resistance and identify effective targets. In recent years, the Wingless and Int-1 (WNT) signaling pathway has been increasingly studied in cancer drug resistance; however, the role of WNT3, a ligand of the canonical WNT signaling pathway, in OSCC 5FU-resistance is not clear. This study delved into this potential connection. METHODS: 5FU-resistant cell lines were established by gradually elevating the drug concentration in the culture medium. Differential gene expressions between parental and resistant cells underwent RNA sequencing analysis, which was then substantiated via Real-time quantitative PCR (RT-qPCR) and western blot tests. The influence of the WNT signaling on OSCC chemoresistance was ascertained through WNT3 knockdown or overexpression. The WNT inhibitor methyl 3-benzoate (MSAB) was probed for its capacity to boost 5FU efficacy. RESULTS: In this study, the WNT/ß-catenin signaling pathway was notably activated in 5FU-resistant OSCC cell lines, which was confirmed through transcriptome sequencing analysis, RT-qPCR, and western blot verification. Additionally, the key ligand responsible for pathway activation, WNT3, was identified. By knocking down WNT3 in resistant cells or overexpressing WNT3 in parental cells, we found that WNT3 promoted 5FU-resistance in OSCC. In addition, the WNT inhibitor MSAB reversed 5FU-resistance in OSCC cells. CONCLUSIONS: These data underscored the activation of the WNT/ß-catenin signaling pathway in resistant cells and identified the promoting effect of WNT3 upregulation on 5FU-resistance in oral squamous carcinoma. This may provide a new therapeutic strategy for reversing 5FU-resistance in OSCC cells.

Efficacy and safety of hepatic arterial infusion chemotherapy combined with lenvatinib and PD-1 inhibitors for advanced hepatocellular carcinoma with macrovascular invasion.

BACKGROUND AND AIMS: The prognosis of hepatocellular carcinoma (HCC) with macrovascular invasion(MaVI)is poor, and the treatment is limited. This study aims to explore the efficacy and safety of hepatic arterial infusion chemotherapy (HAIC), combined with lenvatinib and programmed cell death-1(PD-1) inhibitor in the first-line treatment of HCC with MaVI. METHODS: From July 2020 to February 2022, we retrospectively analyzed consecutive patients with HCC with MaVI who received hepatic arterial infusion FOLFOX(oxaliplatin, 5-fluorouracil, and leucovorin)combined with lenvatinib and PD-1 inhibitor. The efficacy was evaluated by RECIST 1.1. Kaplan-Meier was used to explore the overall survival and progression-free survival (PFS), and the COX regression model was used to analyze the risk factors of PFS. Adverse events (AEs) were evaluated according to CTCAE5.0. RESULTS: Thirty-two patients with HCC complicated with MaVI were recruited from the Second Affiliated Hospital of Nanchang University. Among the patients treated with HAIC combined with lenvatinib and PD-1 inhibitor, ten patients (31.25%) got partial response, eighteen patients (56.25%) maintained stable disease and four patients (12.50%) suffered progressive disease during follow-up; and objective response rate was 31.25%, and disease control rate was 87.5%. The median PFS was 179 days. Univariate and multivariate Cox analysis showed that the extrahepatic metastases and Child-Pugh score were independent prognostic factors of PFS. Twenty-two (68.75%) patients suffered adverse reactions. The main AEs were elevated transaminase (46.87%), thrombocytopenia (40.63%), hypoalbuminemia (28.13%), nausea and vomiting (21.88%), leukopenia (18.76%), abdominal pain (15.63%), hypertension (15.63%) and fever (15.63%). There were seven cases (21.88%) that had grade 3 or above AEs; Among them, two cases with elevated transaminase (6.25%), leukopenia, thrombocytopenia, nausea and vomiting, abdominal pain, and diarrhea occurred in one case respectively. Moreover, no treatment-related death was observed. CONCLUSIONS: Hepatic arterial infusion of FOLFOX combined with lenvatinib and PD-1 inhibitor as the first-line treatment for HCC complicated with MaVI is effective, and adverse reactions are tolerable.

Squalene epoxidase promotes the chemoresistance of colorectal cancer via (S)-2,3-epoxysqualene-activated NF-κB.

BACKGROUND: While de novo cholesterol biosynthesis plays a crucial role in chemotherapy resistance of colorectal cancer (CRC), the underlying molecular mechanism remains poorly understood. METHODS: We conducted cell proliferation assays on CRC cells with or without depletion of squalene epoxidase (SQLE), with or without 5-fluorouracil (5-FU) treatment. Additionally, a xenograft mouse model was utilized to explore the impact of SQLE on the chemosensitivity of CRC to 5-FU. RNA-sequencing analysis and immunoblotting analysis were performed to clarify the mechanism. We further explore the effect of SQLE depletion on the ubiquitin of NF-κB inhibitor alpha (IκBα) and (S)-2,3-epoxysqualene on the binding of IκBα to beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) by using immunoprecipitation assay. In addition, a cohort of 272 CRC patients were selected for our clinical analyses. RESULTS: Mechanistically, (S)-2,3-epoxysqualene promotes IκBα degradation and subsequent NF-κB activation by enhancing the interaction between BTRC and IκBα. Activated NF-κB upregulates the expression of baculoviral IAP repeat containing 3 (BIRC3), sustains tumor cell survival after 5-FU treatment and promotes 5-FU resistance of CRC in vivo. Notably, the treatment of terbinafine, an inhibitor of SQLE commonly used as antifungal drug in clinic, enhances the sensitivity of CRC to 5-FU in vivo. Additionally, the expression of SQLE is associated with the prognosis of human CRC patients with 5-FU-based chemotherapy. CONCLUSIONS: Thus, our finding not only demonstrates a new role of SQLE in chemoresistance of CRC, but also reveals a novel mechanism of (S)-2,3-epoxysqualene-dependent NF-κB activation, implicating the combined potential of terbinafine for 5-FU-based CRC treatment.