Mitomycin C Treatment Research Articles

3′,4′-Dihydroxyflavonol Inhibits Fibrotic Response in a Rabbit Model of Glaucoma Filtration Surgery

Post-operative fibrosis of the filtering bleb limits the success of glaucoma filtration surgery (GFS). To minimise subconjunctival scarring following GFS, treatment with antimetabolites such as Mitomycin C (MMC) has become standard practice; however, their use is associated with considerable side effects. This study aimed to investigate the anti-scarring properties of 3′,4′-dihydroxyflavonol (DiOHF). GFS was performed in New Zealand white rabbits who received eye drops of DiOHF three times daily and vehicle eye drops after surgery (n = 5) or a single intraoperative treatment of MMC (n = 5). Blebs were imaged immediately following surgery and on days 7, 15, 21, and 28 for clinical examination. On day 28, eyes were harvested to assess collagen deposition, expression of α-SMA, oxidative stress, angiogenesis, fibroblast activity, and inflammation in the conjunctiva/Tenon’s layer. At 7 and 28 days post-GFS, MMC-treated blebs were more ischaemic than DiOHF- or vehicle-treated blebs. On day 28, DiOHF treatment significantly suppressed collagen accumulation, CD31 expression, Vimentin expression, and CD45 expression compared to the vehicle control. No difference was observed in 3-Nitrotyrosine or αSMA expression between treatment groups. Treatment with DiOHF reduced conjunctival scarring and angiogenesis in rabbits with GFS, which was comparable to MMC. DiOHF may be a safer and more effective wound-modulating agent than conventional antifibrotic therapy in GFS.

Mechanisms underlying age-associated exacerbation of pulmonary veno-occlusive disease.

Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2-3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over 1 year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR/ISR axis could be a potential therapeutic strategy for PVOD.

The Influence of Dietary Isothiocyanates on the Effectiveness of Mitomycin C and Bacillus Calmette-Guérin in Treating Nonmuscle-Invasive Bladder Cancer.

Nonmuscle-invasive bladder cancer (NMIBC) has high recurrence rates and is often treated with mitomycin C (MMC) and bacillus Calmette-Guérin (BCG). Their efficacy relies on phase 2 enzyme metabolism and immune response activation, respectively. Dietary isothiocyanates, phytochemicals in cruciferous vegetables, are phase 2 enzyme inducers and immunomodulators, and may impact treatment outcomes. We investigated the modifying effects of cruciferous vegetable and isothiocyanate intake on recurrence risk following MMC or BCG treatment. Self-reported cruciferous vegetable intake, estimated isothiocyanate intake, and urinary isothiocyanate metabolites were collected from 1158 patients with incident NMIBC in the prospective Be-Well Study. Hazard ratios (HRs) and 95% CIs were calculated from Cox proportional hazards regression models for risk of first recurrences, and random effects Cox shared frailty models for multiple recurrences. Over median follow-up of 23 months, 343 (30%) recurrences occurred. Receipt of MMC and BCG was associated with decreased risks of first recurrence (MMC: HR = 0.58; 95% CI: 0.46-0.73; BCG: HR = 0.66; 95% CI: 0.49-0.88) and multiple recurrences (MMC: HR = 0.55; 95% CI: 0.44-0.68; BCG: HR = 0.72; 95% CI: 0.55-0.95). Patients receiving BCG and having high intake (>2.4 servings/mo), but not low intake, of raw cruciferous vegetables had reduced risk of recurrence (HR: 0.56; 95% CI: 0.36-0.86; P for interaction = .02) and multiple recurrences (HR: 0.51; 95% CI: 0.34-0.77; P for interaction < .001). The inverse association between MMC receipt and recurrence risk was not modified. For NMIBC patients who receive induction BCG, increasing consumption of raw cruciferous vegetables could be a promising strategy to attenuate recurrence risk.

Late Onset Corneal Haze Post Photorefractive Keratectomy

Introduction: Photorefractive keratectomy (PRK) is a surface ablation procedure to correct refractive errors. Regardless of the safety and efficacy, corneal haze may occur after PRK, and it remains one of the most feared complications because it can impair good visual outcomes. Case Presentation: A 20-year-old woman complained of blurred vision in her right eye (RE) after undergoing PRK elsewhere six months before admission with a history of refraction of S-12.75 C-1.75 x 10° preoperatively and S-0.75 postoperatively. Six-month postoperative uncorrected visual acuity (UCVA) was 0.01, best corrected visual acuity (BCVA) was 0.5 with S-7.00 C-0.75 x 50°. A slit lamp examination revealed grade three corneal haze. Anterior optical coherence tomography (OCT) showed the hyperreflective area with 132 μm deep into the stroma. The patient underwent phototherapeutic keratectomy (PTK) and mitomycin-C (MMC) treatment to a depth of 50 μm Ø6.5mm transition zone 0.5 mm. Two months later, UCVA was 0.2, BCVA was 0.63 with S-2.50 C-0.50 x 90°, and slit lamp examination revealed no haze remaining. Conclusions: This case illustrates the potential risk for corneal haze development, mainly when PRK is performed at greater treatment depths. However, with phototherapeutic keratectomy and mitomycin-C treatment, an excellent visual outcome and vision restoration can be obtained.

Mitomycin C vs. Bacillus Calmette–Guerin for treatment of intermediate-risk nonmuscle invasive bladder cancer patients—A comparative analysis from a single center

BackgroundInduction followed by 1 year maintenance instillation of intravesical Bacillus Calmette–Guerin (BCG) is the standard treatment for intermediate-risk (IR) nonmuscle invasive bladder cancer (NMIBC) patients. Few data exist on the efficacy of Mitomycin C (MMC) instillation in this setting. MethodsWe retrospectively analyzed 226 IR-NMIBC patients classified by the International Bladder Cancer Group (IBCG) and 250 IR-NMIBC intravescical treatment-naïve patients classified by the European Association of Urology (EAU). All patients received either a full induction course of BCG or 40 mg/40 ml MMC from 2012 to 2022. Optimal treatment was defined as 1-year maintenance for BCG and 11 monthly maintenance instillations for MMC. Kaplan–Meier analysis estimated recurrence-free survival (RFS) before and after inverse probability of treatment-weighting (IPTW) and progression-free survival (PFS). Multivariable Cox regression was used to evaluate difference in recurrence after adjustment for clinically relevant variables before and after IPTW. ResultsOptimal BCG and MMC courses were administered to 21% of IR-IBCG and 23% of IR-EAU patients. At 4-years, patients treated with optimal MMC and BCG treatment had similar RFS and PFS in both EAU and IBCG groups. Patients receiving nonoptimal BCG compared to optimal MMC exhibited lower 4-year RFS after IPTW (82% vs. 68% in EAU and 82% vs. 65% in IBCG). At 4-year optimal MMC had greater PFS non optimal BCG. Optimal MMC treatment predicted recurrence in EAU (adjusted and weighted HR 0.33, 95% CI, 0.11-0.98) and IBCG (adjusted and weighted HR 0.29, 95% CI, 0.08-0.97) groups compared to nonoptimal BCG. ConclusionsOptimal 40 mg/40 ml MMC treatment was as effective as optimal BCG in IR-IBCG and IR-EAU NMIBC patients, reducing both recurrence and progression compared to nonoptimal BCG. MMC could be a valid first line alternative to BCG for both IR-EAU and IR-IBCG intravescical treatment-naïve patients, during BCG shortages.

Modeling of FAN1-Deficient Kidney Disease Using a Human Induced Pluripotent Stem Cell-Derived Kidney Organoid System.

Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this study was to explore the possibility of using a human induced pluripotent stem cell (hiPSC)-derived kidney organoid system for modeling FAN1-deficient kidney disease, also known as KIN. We generated kidney organoids using WTC-11 (wild-type) hiPSCs and FAN1-mutant hiPSCs which include KIN patient-derived hiPSCs and FAN1-edited hiPSCs (WTC-11 FAN1+/-), created using the CRISPR/Cas9 system in WTC-11-hiPSCs. Kidney organoids from each group were treated with 20 nM of mitomycin C (MMC) for 24 or 48 h, and the expression levels of Ki67 and H2A histone family member X (H2A.X) were analyzed to detect DNA damage and assess the viability of cells within the kidney organoids. Both WTC-11-hiPSCs and FAN1-mutant hiPSCs were successfully differentiated into kidney organoids without structural deformities. MMC treatment for 48 h significantly increased the expression of DNA damage markers, while cell viability in both FAN1-mutant kidney organoids was decreased. However, these findings were observed in WTC-11-kidney organoids. These results suggest that FAN1-mutant kidney organoids can recapitulate the phenotype of FAN1-deficient kidney disease.

Effects of cyclophosphamide and mitomycin C on radiation-induced transcriptional biomarkers in human lymphoblastoid cells

Purpose Ionizing radiation (IR)-induced transcriptional changes are considered a potential biodosimetry for dose evaluation and health risk monitoring of acute or chronic radiation exposure. It is crucial to understand the impact of confounding factors on the radiation-responsive gene expressions for accurate and reproducible dose assessment. This study aims to explore the potential influence of exposures to chemotherapeutic agents such as cyclophosphamide (CP) and mitomycin C (MMC) on IR-induced transcriptional biomarkers. Methods The human B lymphoblastoid cells (AHH-1) were exposed to 0, 20, 50, 100, 200 and 500 μg/ml CP or 0, 0.025, 0.05, 0.1 and 1 μg/ml MMC, respectively. The appropriate concentrations of CP and MMC were added for 1 h before irradiation with 0, 2, 4 and 6 Gy of 60Co γ-rays at a dose rate of 1 Gy/min. Cell viability was evaluated by CCK-8 assay. The gene expression responses of 18 radiation-induced transcriptional biomarkers were examined at 24 h after exposures to CP and MMC, respectively. The expression levels of five crucial DNA interstrand crosslinks (ICLs) repair genes were also evaluated. The biodosimetry models were established based on the specific radiation-responsive gene combinations. Results The baseline transcriptional levels of the 18 selected genes were slightly affected by CP treatment in the absence of IR, while the transcript responses to IR could be inhibited as the concentration of CP up to 50 μg/ml. MMC treatment up-regulated the background levels in most radiation-responsive gene expressions. Of 18 genes, only the relative mRNA expression levels of CDKN1A and BBC3 were repressed after treatment with IR and MMC in combination. The relative mRNA level of RAD51 was significantly up-regulated after exposure to CP, while the expression of FANCD2, RAD51 and BLM showed an overall increase in response to MMC treatment. After irradiation, the relative mRNA expression levels of FANCD2, BRCA2 and RAD51 exhibited dose-dependent increases in IR alone and MMC treatment groups. In addition, the biodosimetry models were established using 2-4 radiation-responsive genes based on different radiation exposure scenarios. Conclusion Our findings suggested that IR-induced gene expression changes were slightly affected after exposure to a relatively low concentration of CP and MMC. Gene expression combinations might improve the broad applicability of transcriptional biodosimetry across diverse radiation exposures.

Antigenotoxic effect of hyperoside against Mitomycin C and hydrogen peroxide-induced genotoxic damage on human lymphocytes

Hyperoside is a flavonol glycoside isolated from various plant genera such as Hypericum and Crataegus. It has an important place in the human diet and is used medically to relieve pain and ameliorate cardiovascular functions. However, a comprehensive profile of the genotoxic and antigenotoxic effects of hyperoside is not known. The current study aimed to investigate the genotoxic and antigenotoxic effects of hyperoside against genetic damages induced by two genotoxins (MMC and H2O2) using chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) assays in human peripheral blood lymphocytes in vitro. Blood lymphocytes were incubated with 7.8–62.5 μg/mL concentrations of hyperoside alone and simultaneously with 0.20 μg/mL Mitomycin C (MMC) or 100 μM Hydrogen peroxide (H2O2). Hyperoside did not exhibit genotoxic potential in the CA, SCE, and MN assays. Moreover, it did not cause a decrease in mitotic index (MI) which is an indicator of cytotoxicity. On the other hand, hyperoside significantly decreased CA, SCE, and MN (except for MMC treatment) frequencies induced by MMC and H2O2. Hyperoside, increased mitotic index against both mutagenic agents at 24-h treatment when compared to positive control. Our results demonstrate that hyperoside exhibited antigenotoxic effects rather than genotoxic in vitro human lymphocytes. Therefore, hyperoside may be a potential preventive agent in inhibiting chromosomal and oxidative damage induced by genotoxic chemicals.

Evaluation of the effects of ethanol and mitomycin on survival of rat limbal stem cells: an in vitro study.

Ethanol and mitomycin C (MMC) are clinically used to treat corneal diseases such as LASEK and LASIK surgery. In this study, we investigated the effects of time-dependent alcohol and MMC in cultured rat limbal stem cells (LSCs) to determine the appropriate time for the use of this compound in the clinical setting. LSCs (N = 10 eyes) isolated from male Wistar rats were cultured and characterized; then, isolates were divided into three groups. One group was exposed to a 20% concentration of ethanol for 5, 10, 15, 20, 25, and 30s, and cell viability was assessed one, three, and five days following ethanol exposure using an MTT assay. To investigate the effect of MMC, cells in the second group were treated with 0.02% MMC in various periods (i.e., 15s, 30s, 60s, 90s, and 120s) and time-dependent responses of cultured LSCs were recorded. Cells in the third group were co-treated with ethanol and MMC; then, dose and time dependency was evaluated. In comparison with the viable cells in the control group, ethanol markedly decreased the viability of cells in a time-dependent manner in days one and three. On day five, the viability of LSCs was improved significantly (p < 0.05) in comparison with day one. The number of viable progenitor cells was significantly decreased after MMC treatment in a time-dependent manner, as determined by the MTT assay (p < 0.001). The use of mitomycin, along with alcohol, decreased cell viability in all groups treated with ethanol + MMC compared to the control on days one, three, and five (p < 0.0001). Our findings suggest that ethanol and MMC reduced cell viability in cultured LSCs in a time-dependent manner. In addition, when LSCs were exposed to alcohol alone, they had a better recovery process within 5days in comparison to when exposed to mitomycin alone or mitomycin + alcohol.

Mitomycin C enhanced the antitumor efficacy of Rocaglamide in colorectal cancer.

Rocaglamide (ROC), a natural phytochemical isolated from Aglaia species, is a translational inhibitor of de novo c-FLIP synthesis, which relieves the inhibition of c-FLIP dimerization with procasoase-8 and downstream activation. Unfortunately, a lot of cancer cells, especially colorectal cancer cells (CRC), exhibit marked resistance to Rocaglamide-induced cell death. Research has demonstrated that mitomycin C (MMC) has broad-spectrum anti-tumor activity that it can synergize with a wide range of clinical drugs to inhibit tumor growth. The current study investigated whether MMC combined with ROC could sensitize CRC cells with different ROC resistance to apoptosis. HCT116 and HT29, two different CRC cells, were treated with ROC and/or MMC, and the induction of apoptosis, inhibition of cell migration and invasion, arrest of cell cycle, induction of reactive oxygen species, and effects on Bcl-2 family signaling pathway were investigated. The results showed that low concentration of MMC combined with ROC significantly promoted HCT116 and HT29 cell apoptosis and inhibited cell proliferation by downregulating the expression of Bcl-2 and c-FLIP, upregulating the expression of Bax, activating the caspase cascade (involving the mitochondrial apoptosis pathway), arresting cell cycle in G1 phase, and increasing the level of reactive oxygen species (ROS). In addition, the viability and morphology of MRC-5 cells were not significantly affected by the combined treatment with ROC and MMC, indicating its safety. Therefore, it is concluded that the combination treatment of ROC and MMC is a highly effective tumor therapy and may offer a promising therapeutic strategy for the treatment of CRC.

Two routes of administration of mitomycin C in a trabeculectomy.

The introduction of mitomycin C (MMC) as an adjunct to trabeculectomy was a major advance in the ability to improve the Intra ocular pressure lowering efficiency of the procedure. The time tested traditional way of administration of MMC is via a sponges soaked in it, duration and concentration varies depending on risk of failure. A subconjunctival injection of MMC instead of these sponges is recently being used as a promising alternative. Here is the video demonstrating the 2 possible ways to prepare and apply MMC during a trabeculectomy surgery. Intraoperative injection of MMC in trabeculectomy has several advantages over conventional method ofsponge application. A large MMC treatment area produces more diffuse and elevated blebs. Large-area MMC application also seems to increase long-term success without increasing the complication rates in trabeculectomies. Direct and diffuse application of MMC by injection may promote less scarring and vascularization of the bleb. To achieve the same surface area of exposure with sponges, i.e.achieved with injection, the surgeon must use multiple sponges, all of which must be carefully collected thereafter. The injection method,therefore,eliminates the risk of retained sponges. injection of MMC may be as safe and as effective as conventional sponge application of MMC with comparable estimated complete treatment success with relatively lower complication rates. https://youtu.be/RLEbK4IeRtU.

Mitomycin C in Homologous Recombination Deficient Metastatic Pancreatic Cancer after Disease Progression on Platinum-Based Chemotherapy and Olaparib.

Recent efforts to personalize treatment with platinum-based chemotherapy and PARP inhibitors have produced promising results in homologous recombinant deficient (HRD) metastatic pancreatic cancer (MPC). However, new strategies are necessary to overcome resistance. The below case series documents patients treated at the HonorHealth Research Institute with a diagnosis of HRD MPC who received Mitomycin C (MMC) treatment from January 2013 until July 2018. Five HRD MPC patients treated with MMC were evaluated. All patients received at least one course of treatment. Mean age at MMC treatment initiation was 58 years. There were 3 females and 2 males. All patients had tumors that progressed on platinum-based chemotherapy, four patients had previous exposure to Olaparib. The median PFS was 10.1 months, and the median OS was 12.3 months. Responses were observed only in patients harboring BRCA2 mutations, no response was observed in the PALB2 mutation carrier. MMC in this heavily previously treated PC was safe, with overall manageable grade 2 gastrointestinal toxicities including nausea and vomiting, and G3 hematological toxicities including anemia and thrombocytopenia. Pancreatic cancer patients with HRD may benefit from MMC treatment. Further clinical investigation of MMC in pancreatic cancer is warranted.

Recurrent Germline Variant in RAD21 Predisposes Children to Lymphoblastic Leukemia or Lymphoma.

Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.

Effects of Valproic Acid and Mitomycin C Combination Therapy in a Rabbit Model of Minimally Invasive Glaucoma Surgery.

PurposeThis study aimed to compare the effectiveness of combination therapy consisting of low-dose mitomycin C (MMC) and valproic acid (VPA) against high-dose MMC for improving the scar phenotype in minimally invasive glaucoma surgery (MIGS).MethodsA rabbit model of MIGS incorporating the PreserFlo MicroShunt was treated with high (0.4 mg/mL) or low (0.1 mg/mL) doses of MMC or with combination therapy consisting of low-dose (0.1 mg/mL) MMC and VPA. Operated eyes were examined by live ocular imaging, histochemical evaluation, multiphoton quantitation of collagen characteristics, and molecular analyses.ResultsAlthough high-dose MMC obliterated the vasculature, combination therapy vastly improved the postoperative tissue morphology by maintaining the vasculature without increased vascularization. Combination therapy also altered collagen morphology and reduced encapsulation of the MicroShunt distal end, which remained at risk with MMC treatment alone. Multiphoton quantitation indicated that the combination therapy significantly reduced collagen density and fiber dimensions compared with monotherapy. At the molecular level, combination therapy significantly reduced Vegfa, Vegfc, and Vegfd expression and inhibited Col1a1 upregulation from baseline levels, all of which low-dose MMC alone was unable to achieve. Notably, COL1A1 protein levels appeared more consistently suppressed by combination therapy compared with high-dose MMC alone.ConclusionsCompared with high-dose MMC, combination therapy was less toxic by sparing the vasculature and potentially more effective in reducing scarring via the regulation of collagen content and organization.Translational RelevanceVPA may be combined with low-dose MMC to replace high-dose MMC to deliver safe and effective anti-scarring outcomes.

The hokW-sokW Locus Encodes a Type I Toxin-Antitoxin System That Facilitates the Release of Lysogenic Sp5 Phage in Enterohemorrhagic Escherichia coli O157.

The toxin-antitoxin (TA) genetic modules control various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. They also exist in mobile genetic elements, including prophages; however, their physiological roles remain poorly understood. Here, we demonstrate that hokW-sokW, a putative TA locus encoded in Sakai prophage 5 (Sp5) in enterohemorrhagic Escherichia coli O157: H7 Sakai strain, functions as a type I TA system. Bacterial growth assays showed that the antitoxic activity of sokW RNA against HokW toxin partially requires an endoribonuclease, RNase III, and an RNA chaperone, Hfq. We also demonstrated that hokW-sokW assists Sp5-mediated lysis of E. coli cells when prophage induction is promoted by the DNA-damaging agent mitomycin C (MMC). We found that MMC treatment diminished sokW RNA and increased both the expression level and inner membrane localization of HokW in a RecA-dependent manner. Remarkably, the number of released Sp5 phages decreased by half in the absence of hokW-sokW. These results suggest that hokW-sokW plays a novel role as a TA system that facilitates the release of Sp5 phage progeny through E. coli lysis.

Electrochemotherapy with Mitomycin C Potentiates Apoptosis Death by Inhibiting Autophagy in Squamous Carcinoma Cells.

Simple SummaryInternationally, electrochemotherapy is considered an innovative therapeutic strategy capable of increasing the efficacy of lyophobic drugs in many solid tumors. Our study aimed to verify the potentiating effect of electroporation (EP), in combination with the drug mitomycin C (MMC), in oral and laryngeal cancer cells that exhibited intrinsic drug resistance. Our study showed that EP increased MMC cytotoxicity on both lines compared to single treatment; the mechanism of action was due to EP inhibition of the autophagic process induced by MMC, thus favoring apoptosis. EP plus MMC treatment was also performed on two veterinary patients demonstrating its efficacy, tolerability, and clinical feasibility.We investigated the chemosensitizing effect of electroporation (EP), which, using electrical pulses, permeabilizes cancer cells to drugs. The study involved two human hypopharyngeal and tongue carcinoma cell lines. The surface and intracytoplasmic expression of P-gp were evaluated by flow cytometry, demonstrating that both lines were intrinsically resistant. After establishing the optimal dose of mitomycin C (MMC) to be used, in combination with EP, we showed, by both MTT assay and optical and electron scanning microscopy, the potentiating cytotoxic effect of EP with MMC compared to single treatments. Flow cytometry showed that the cytotoxicity of EP + MMC was due to the induction of apoptosis. In addition to verifying the release of cytochrome C in EP + MMC samples, we performed an expression analysis of caspase-3, caspase-9, Akt, pAkt, HMGB1, LC3I, LC3II, p62, Beclin1, and associated proteins with both apoptotic and autophagic phenomena. Our results were confirmed by two veterinary patients in whom the EP + MMC combination was used to control margins after the resection of corneal squamous carcinoma. In conclusion, we affirmed that the effect for which EP enhances MMC treatment is due to the inhibition of the autophagic process induced by the drug in favor of apoptosis.

Spontaneous and induced chromosomal aberrations in peripheral blood of women with endometriosis: evidence of genomic instability.

Endometriosis is a prevalent gynecological disease which can lead to certain types of cancers. We investigated the spontaneous and induced chromosomal aberrations in peripheral blood lymphocytes (PBL) of endometriosis patients. We performed a pilot study utilizing mitomycin C (MMC) to assess chromosomal instability in the peripheral blood of participants. The patient group consisted of 20 infertile endometriosis patients and the controls of 20 healthy fertile women. Blood samples were collected, and two distinct lymphocyte cultures were prepared to evaluate the baseline and the MMC induced chromosomal aberrations. The results showed a significant difference before and after MMC treatment in both groups (P<0.001) and also revealed that endometriosis patients are far more sensitive to MMC than controls (P<0.001). The significantly higher frequency of induced and spontaneous chromosomal aberrations in patients can be consider as a sign of genomic instability and the defect in DNA repair mechanisms, which can be both assumed as a driver of cancer development in endometriosis patients.

Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia.

Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.

Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples.

Phosphorylation of H2AX histone (γH2AX) represents an early event in the DNA damage response against double-strand breaks (DSB); hence, its measurement provides a surrogate biomarker of DSB. Recently, we reported initial steps in the standardization of γH2AX assay in peripheral blood leukocytes (PBL), addressing the possibility of using cryopreserved samples, and the need of phytohaemagglutinin (PHA) stimulation prior analysis (Toxicol Sci2015, 144:406-13). Validating the use of whole blood samples as cell specimen for this assay would be particularly useful for human population studies. Hence, in the current study we determined for the first time the feasibility of whole blood samples, both fresh and frozen, to be used in the γH2AX assay, evaluated by flow cytometry, and the convenience of PHA stimulation. Freshly collected and cryopreserved whole blood samples were treated with bleomycin (BLM), actinomycin-D (Act-D) and mitomycin C (MMC); half of the samples were previously incubated with PHA. Results were compared with those from PBL. Negative responses in MMC treatments were probably due to the quiescence of unstimulated cells, or to the short treatment time in PHA stimulated cells. Fresh whole blood samples exhibited a more intense response to BLM and Act-D treatments in stimulated cells, probably due to DSB indirectly produced from other less relevant types of DNA damage. Results obtained in frozen whole blood samples indicate that PHA stimulation is not advisable. In conclusion, this study demonstrates that whole blood samples can be used to assess DSB-related genotoxicity by the flow cytometry γH2AX assay.

Safety and efficacy of combination of suberoylamilide hydroxyamic acid and mitomycin C in reducing pro-fibrotic changes in human corneal epithelial cells

Corneal haze post refractive surgery is prevented by mitomycin c (MMC) treatment though it can lead to corneal endothelial damage, persistent epithelial defects and necrosis of cells. Suberanilohydroxamic acid (SAHA) however has been proposed to prevent corneal haze without any adverse effects. For clinical application we have investigated the short and long term outcome of cells exposed to SAHA. Human donor cornea, cultured limbal epithelial cells, corneal rims and lenticules were incubated with SAHA and MMC. The cells/tissue was then analyzed by RT-qPCR, immunofluorescence and western blot for markers of apoptosis and fibrosis. The results reveal that short term exposure of SAHA and SAHA + MMC reduced apoptosis levels and increased αSMA expression compared to those treated with MMC. Epithelial cells derived from cultured corneal rim that were incubated with the MMC, SAHA or MMC + SAHA revealed enhanced apoptosis, reduced levels of CK3/CK12, ∆NP63 and COL4A compared to other treatments. In SAHA treated lenticules TGFβ induced fibrosis was reduced. The results imply that MMC treatment for corneal haze has both short term and long term adverse effects on cells and the cellular properties. However, a combinatorial treatment of SAHA + MMC prevents expression of corneal fibrotic markers without causing any adverse effect on cellular properties.