High-risk HPVs, microbiota and epithelial carcinogenesis: state of the art and research contribution of in vitro 3D models

Persistent high-risk human papillomavirus (HR-HPV) infection is associated with anogenital and head & neck squamous epithelial (HNSCC) tumors, which altogether cause about 550,000 new cases every year. Several evidences suggest that the microbiota could have a role on the inflammatory, epithelial mesenchymal transition and tumorigenesis processes promoted by HR-HPV infection, yet the mechanisms involved remain to be clarified. In this review we report the state of the art on this topic and on the most promising in vitro developed models for studying the host-pathogen interactions. Using MEDLINE, several terms were searched and combined to select the most pertinent papers. The investigation was limited to the international indexed articles published in PubMed in the last 10 years. This review reports the latest knowledge in the field of the microbial-associated anogenital tumors and HNSCC. In addition, we also discuss the in vitro epithelial culture systems that reproduce the pathophysiological features of the tumoral microenvironment and the in vivo response to microbial agents, thus representing a useful tool for analyzing at cellular and molecular levels the role played by infective agents in tumorigenesis

Role of the Microbiota in Skin Neoplasms: New Therapeutic Horizons

The skin and the gut are regularly colonized by a variety of microorganisms capable of interacting with the immune system through their metabolites and influencing the balance between immune tolerance and inflammation. Alterations in the composition and diversity of the skin microbiota have been described in various cutaneous diseases, including skin cancer, and the actual function of the human microbiota in skin carcinogenesis, such as in progression and metastasis, is currently an active area of research. The role of Human Papilloma Virus (HPV) in the pathogenesis of squamous cell carcinoma is well consolidated, especially in chronically immunosuppressed patients. Furthermore, an imbalance between Staphylococcus spp., such as Staphylococcus epidermidis and aureus, has been found to be strongly related to the progression from actinic keratosis to squamous cell carcinoma and differently associated with various stages of the diseases in cutaneous T-cell lymphoma patients. Also, in melanoma patients, differences in microbiota have been related to dissimilar disease course and prognosis and may affect the effectiveness and tolerability of immune checkpoint inhibitors, which currently represent one of the best chances of a cure. From this point of view, acting on microbiota can be considered a possible therapeutic option for patients with advanced skin cancers, even if several issues are still open

EVALUATION OF THE INTERACTION AMONG HPV16 E6 AND E7 ONCOPROTEINS AND THE DNA DAMAGE SENSOR 53BP1 IN 2D AND 3D EPITHELIAL CULTURES BY THE PROXIMITY LIGATION ASSAY

INTRODUCTION. Human papillomaviruses (HPV) group several viruses able to infect squamous stratified epithelia and cause benign papillomas, warts and anogenital lesions. They also correlate to oropharyngeal and anogenital malignancies, mainly promoted by the high risk (HR) \u3b1-HPV16 E6E7 oncoproteins. Despite scientific progresses and the development of vaccines, these tumors are still common and represent one of the major causes of women\u2019s death. Host\u2019s cell replication fidelity depends by the DNA Damage Response (DDR). Unlike from other DNA viruses, HR-HPVs encourage cells proliferation without inactivating the DDR: the mechanisms at the basis haven\u2019t been clarified yet. During HPV16 infection, E6 binds and degrades p53 through the E6AP LXXLL domain. Similarly, E7 competes with E2F1-pRb interaction, thus inactivating pRb, and promoting the linking the pRb-like proteins CBP/p300 and p107, that also harbor a LXXLL sequence. Unfortunately, E6 E7 role in the DDR activation is not elucidated yet. EXPERIMENTAL MODEL. To gain new information, we reproduced an in vitro 3D HPV16-E6E7 infected epithelium, already characterized for HPVs studies, and checked for cellular and viral markers, among them HPV16E6E7 oncoproteins and the double strand breaks (DSB) sensor 53BP1; we then made a Co-IF for E6 and E7 with 53BP1. Since E6 and E7 both interact with LXXLL containing proteins, we analyzed 53BP1 BRCT2 domain and we explored the binding hypothesis via the in situ PLA technique in 2D in CaSki and E6E7HPV16 keratinocytes and in the 3D model. RESULTS. The in vitro infected epithelium resembles the in vivo tissue. E6E7HPV16, both in basal and differentiated strata, induce a 53BP1 increase in nuclear foci. After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the binding in all the employed cellular models. CONCLUSION. Our results add new information on HPV16 oncoproteins capability in overcome cellular defense strategies

In Vitro Reconstructed Human Epithelial Models for Microbial Infection Research: Why Do We Need them?

In the last 50 years, the Replacement, Reduction and Refinement principles have become a framework for conducting high quality academic, pre-clinical, clinical and industrial research experimentation studies, in order to respond to the European Union legislative demand of alternatives to animal-based experimentation, often difficult to translate to human applications, expensive and not ethically approved. Thanks to the improvement of cellular isolation protocols, culture and co-culture conditions, together with the increased clinical demand, several novel in vitro three-dimensional tissue engineered human epithelial models, able to create sophisticate pre-clinical tests and produce results more reliable than the traditional bi-dimensional flat cell culture systems, have been developing also for microbial infection research purposes

Growth Conditions Influence Lactobacillus Cell-Free Supernatant Impact on Viability, Biofilm Formation, and Co-Aggregation of the Oral Periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis

Fusobacterium nucleatum and Porphyromonas gingivalis human periodontopathogens play a leading part in oral squamous cell carcinoma through cell proliferation, invasion, and persistent inflammation promotion and maintenance. To explore how the activity of Lactobacillus-derived cell-free supernatants (CFSs) can be influenced by growth medium components, CFSs were produced both in the standard MRS and the novel animal-derivative-free "Terreno Industriale Lattobacilli" (TIL) media, and in vitro screened for the containment of F. nucleatum and P. gingivalis both single and co-cultured and also for the interference on their co-aggregation. The viability assay demonstrated that the Limosilactobacillus reuteri LRE11 and Ligilactobacillus salivarius LS03 MRS-produced CFSs were significantly more effective against single and co-cultured pathogens. All the other CFSs significantly improved their efficacy when produced in TIL. Both MRS- and TIL-produced CFSs significantly inhibited the single and co-cultured pathogen biofilm formation. Only Levilactobacillus brevis LBR01 CFS in MRS specifically reduced F. nucleatum and P. gingivalis co-aggregation, while viable LBR01, LS03, and LRE11 in MRS significantly co-aggregated with the pathogens, but only LS03 cultivated in TIL improved this effect. This work paves the way to better consider environmental growth conditions when screening for probiotic and postbiotic efficacy as crucial to pathogen aggregation, adhesion to the host's niches, and exclusion

Bioactive glasses functionalized with polyphenols: in vitro interactions with healthy and cancerous osteoblast cells

Bioactive glasses are widely studied as biomaterials for bone contact applications. In this research work, the opportunity to modify the surface of a bioactive glass with polyphenols (gallic acid, and natural polyphenols extracted from red grape skin and green tea leaves) has been investigated in order to induce a selective anti-tumor activity in vitro. The presence of surface grafted molecules has been optically proved by fluorescence microscopy exploiting their autofluorescence. Direct and indirect cytotoxicity assays have been performed with human bone osteosarcoma cells (U2OS) and human fetal pre-osteoblasts (hFOB), as well as the quantification of oxygen and nitrogen reactive species (RONS) engendered from cells in response to the materials. Finally, the DNA damage of U2OS cells upon contact with the bioactive glass has been evaluated in order to verify any selective cytotoxic activity of functionalized materials against cancer cells. Results showed a selective cytotoxic activity of functionalized bioactive glasses toward osteosarcoma cells that was particularly evident when cells were cultivated directly onto glasses surface. Moreover, the presence of grafted polyphenols increased the RONS production and induced a permanent DNA damage on the U2SOS cells while they promote a certain anti-inflammatory action toward hFOB. These preliminary results suggest polyphenols grafted bioactive glasses as promising material for bone substitution in cancer treatment

Microbiota, Oxidative Stress, and Skin Cancer: An Unexpected Triangle

: Mounting evidence indicates that the microbiota, the unique combination of micro-organisms residing in a specific environment, plays an essential role in the development of a wide range of human diseases, including skin cancer. Moreover, a persistent imbalance of microbial community, named dysbiosis, can also be associated with oxidative stress, a well-known emerging force involved in the pathogenesis of several human diseases, including cutaneous malignancies. Although their interplay has been somewhat suggested, the connection between microbiota, oxidative stress, and skin cancer is a largely unexplored field. In the present review, we discuss the current knowledge on these topics, suggesting potential therapeutic strategies

In Vitro Selection of Lactobacillus and Bifidobacterium Probiotic Strains for the Management of Oral Pathobiont Infections Associated to Systemic Diseases

: The human oral pathobionts Aggregatibacter actinomycetemcomitans, Streptococcus mitis and Streptococcus mutans, in dysbiosis-promoting conditions, lead to oral infections, which also represent a threat to human systemic health. This scenario may be worsened by antibiotic misuse, which favours multi-drug resistance, making the research on pathogen containment strategies more than crucial. Therefore, we aimed to in vitro select the most promising probiotic strains against oral pathogen growth, viability, biofilm formation, and co-aggregation capacity, employing both the viable probiotics and their cell-free supernatants (CFSs). Interestingly, we also assessed probiotic efficacy against the three-pathogen co-culture, mimicking an environment similar to that in vivo. Overall, the results showed that Lactobacillus CFSs performed better than the Bifidobacterium, highlighting Limosilactobacillus reuteri LRE11, Lacticaseibacillus rhamnosus LR04, Lacticaseibacillus casei LC04, and Limosilactobacillus fermentum LF26 as the most effective strains, opening the chance to deeper investigation of their action and CFS composition. Altogether, the methodologies presented in this study can be used for probiotic efficacy screenings, in order to better focus the research on a viable probiotic, or on its postbiotics, suitable in case of infections

Effects of Probiotics Administration on Human Metabolic Phenotype

The establishment of the beneficial interactions between the host and its microbiota is essential for the correct functioning of the organism, since microflora alterations can lead to many diseases. Probiotics improve balanced microbial communities, exerting substantial healthpromoting effects. Here we monitored the molecular outcomes, obtained by gut microflora modulation through probiotic treatment, on human urine and serum metabolic profiles, with a metabolomic approach. Twenty-two subjects were enrolled in the study and administered with two different probiotic types, both singularly and in combination, for 8 weeks. Urine and serum samples were collected before and during the supplementation and were analyzed by nuclear magnetic resonance (NMR) spectroscopy and statistical analyses. After eight weeks of treatment, probiotics deeply influence the urinary metabolic profiles of the volunteers, without significantly altering their single phenotypes. Anyway, bacteria supplementation tends to reduce the differences in metabolic phenotypes among individuals. Overall, the effects are recipient-dependent, and in some individuals, robust effects are already well visible after four weeks. Modifications in metabolite levels, attributable to each type of probiotic administration, were also monitored. Metabolomic analysis of biofluids turns out to be a powerful technique to monitor the dynamic interactions between the microflora and the host, and the individual response to probiotic assumption

Cytotoxic activity of a plant extract on cancer cells

Chemoprevention by natural products may be considered a promising approach to cancer control and management [1]. Many studies have demonstrated antiproliferative, cytostatic and cytotoxic activities of phytochemicals against cancer cells [2]. In this study, a plant extract from Arctium lappa, Berberis vulgaris and Eschscholtia californica was tested as potential anticancer agent. The antitumoral activity of this plant extract was tested on four human cancer cell lines: MCF-7 (breast carcinoma cells), Huh-7 (hepatic carcinoma cells), HTB-43 (oropharyngeal carcinoma cells) and ECV- 304 (urinary bladder carcinoma cells). The efficacy of the extract was compared to the common chemotherapeutic agent cyclophosphamide. Three plant extract concentrations were tested: 800, 650 and 450 ng/ml; for cyclophosphamide, three concentrations were assayed, according to literature data: 1300, 1000 and 850 ng/ml [3]. In addition, plant extract and cyclophosphamide were tested on two primary cell lines as controls, human gingival fibroblasts and human mammary fibroblasts. Cell viability was evaluated by the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma] colorimetric assay and the new xCELLigence system (Roche) for real-time monitoring of cell viability. All concentrations of plant extract exhibited a high level of cytotoxicity on MCF-7, Huh-7, HTB-43 and ECV-304 cancer cells, similar to cyclophosphamide, though they slightly reduced viability of human gingival and mammary fibroblasts. Conversely, the conventional chemotherapeutic drug showed a marked cytotoxicity on control cells. The potential of the plant extract has been demonstrated in vitro on various types of cancers, suggesting a possible use of this natural product as a promising anticancer agent. Further studies are needed to ascertain its efficacy in vivo and to elucidate its mechanism(s) of action at molecular and biochemical levels