Study of antimicrobial and anticancer activity of new synthetic and natural tools

Dottorato di Ricerca in Medicina Traslazionale. Ciclo XXIXInfectious diseases and cancer are the two disease groups that representing the major cause of death worldwide. Unfortunately, antibiotic resistance is the biggest threat in the first case; in fact, new resistance mechanisms continuously are emerging and spreading globally, threatening the ability to treat common infectious diseases. A growing list of infections caused by bacteria, viruses, parasites etc. are becoming harder and harder to treat, and sometimes impossible, as antibiotics become less effective. Without urgent action, we are heading for a post-antibiotic era, in which common infections and minor injuries can once again kill the human population. Concerning cancer, Resistance to chemotherapy and molecularly targeted therapies is a major problem in current research. Drugs side effects and toxicity to normal body cells is also an important threat in cancer treatments. In this regard, these problems are at the forefront of scientific research and technological innovation and are leading to the development of new therapeutic approaches against cancer and infectious disease with fewer side effects and lesser resistance problems. The aim of the present study was to investigate on the new compounds in order to find new possible therapeutic agents against bacteria, parasites and cancer. Infectious diseases are caused by microorganisms such as bacteria, parasites, viruses etc.; in particular, bacterial infectious diseases are caused by either Gram +ve or Gram -ve bacteria. Certainly, antibiotics are the main weapon against infectious bacterial diseases; however, the uncontrolled use of antibiotics to control infections in humans, animals and in agriculture caused the development of drug resistance by bacterial populations. Besides this, infections caused by Gram -ve bacteria are difficult to treat due to the presence of a protective outer membrane consisting of lipopolysaccharides. Therefore, it is clear that there is a need to develop novel classes of antibacterial agents capable of killing bacteria through mechanisms unlike those of the known classes of antibiotics. Then, scientists are currently searching for new approaches to treat infectious diseases, particularly those caused by Gram -ve bacteria, focusing on exactly how the pathogens change and how drug resistance evolves. Since ancient times, metal complexes have been used as antibacterial compounds, metallic silver and silver salts are good examples of this. Silver compounds are particularly interesting since their antibacterial activity can be altered by changing the ligand associated with the silver complex. To date, among silver derivatives, silver sulfadiazine remains one of the most commonly-used antibacterial drugs. Therefore, metal N-heterocyclic carbene (MNHC) complexes appeared as an emerging field of research in medicinal chemistry where NHC complexes of coinage metals (Cu, Au, and Ag) proved to be better antimicrobial agents. Herein, it was investigated the, in vitro, antibacterial activity of the newly synthesized silver (Ag) complexes, Iodide[N-methyl-N-(2-hydoxy-cyclopentyl-imidazole-2ylidine]silver(I), Iodide[N-methyl-N-(2-hydoxy-cyclohexyl)-imidazole-2-ylidine]silver(I) and Iodide[N-methyl- N-(2-hydoxy-2-phenyl)ethyl-imidazole-2-ylidine]silver(I), namely AgL6, AgL18 and AgL20, against two Gram +ve (Staphylococcus aureus, Streptococcus pyogenes) and three Gram -ve (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) bacteria. Among these, AgL6 showed good antibacterial activity against both Gram +ve and Gram -ve bacteria. However, the minimum inhibitory concentration (MIC) value was 32 μg/mL for Gram +ve and 16 μg/mL for Gram -ve bacteria, which was higher than that displayed by commercial drug, used as control (Silver Sulfadiazine, AgSD). We therefore hypothesized that the poor activity is due to the poor intake of the compound. In order to enhance its antibacterial activity, we have developed “a pharmaceutically-oriented device”, a nanocarrier as a tool for targeted drug delivery. Here it was described, for the first time, the production of a polymer nanostructure in which dextran, a biopolymer, and oleate residues represent the hydrophilic and hydrophobic parts, respectively. This nanoparticle was loaded with AgL6 and the antibacterial activity has been investigated. The results were very interesting, with MIC values being reduced four-fold for both Gram +ve and Gram -ve bacteria. Surprisingly, these values were two-fold lower than for silver sulfadiazine. Briefly, our results showed that K. pneumoniae and E. coli are the most susceptible bacteria to AgL6, followed by P. aeruginosa. In conclusion, the investigated compound AgL6 showed excellent potentiality against bacterial infections. According to the World Health Organization (WHO), 17 diseases caused by bacteria and parasites have been classified as neglected tropical diseases (NTDs). NTDs are endemic in 149 tropical and subtropical countries and affect more than 1 billion people, including 875 million children. These diseases are responsible for over 500,000 deaths per year and are characterized by severe pain and long term disability. Human African Trypanosomiasis (sleeping sickness) is an important disease among them and is caused by two parasites of the genus Trypanosome: Trypanosome brucei rhodesiense and Trypanosome brucei gambiense. Trypanosomiasis is a disease with a devastating socio-economic impact in sub-Saharan Africa through direct infection of humans and livestock. This disease is fatal if left untreated. Current therapy relies on five drugs that have many limitations among which acute toxicity, problems with oral absorption and emergence of trypanosomal resistance, this latter is a major concern owing to the absence of vaccines and therapeutic alternatives. Therefore pharmaceutical research is aimed at the discovery of new drugs, although the investment in this therapeutic area is not attractive owing to the prospect of poor financial returns. Many pharmaceutical industries have already utilized an opportunistic approach by utilizing drugs long since used for other diseases, a process known as “repurposing” of the drug. It is estimated that over half of the drugs used today are derived from natural sources. In the present study, in a search for molecules with trypanocida activity, it was screened 2000 natural extracts from Fungi and Actinomycetes. The extracts showing activity were selected, and the active compound was identified by liquid chromatography and mass spectroscopy. Chaetocin is one of the molecules identified which showed good trypanocidal activity when tested in vitro. Chaetocin is already used as an antibacterial and anticancer drug, here it was repurposed as drug against trypanosomiasis. The results were very surprising because the trypanocidal activity was in the nanomolar range; the IC50 value was found to be 8.3 nM. Next, it was investigated on its mechanism of action. In chaetocin treated cells, morphological changes and chromatin degradation were identified, by fluorescence microscopy and cell-cycle arrest during the G2 phase was proven by cytometry analysis. Finally, it was hypothesized that the enzyme histone methyl transferase, an important enzyme acting in the G2 phase, could be the target for this drug.This study displayed that chaetocin could have great potentiality in the fight against the deadly trypanosomiasis. However, further studies will be needed to reveal whether this compound can cross the blood-brain barrier. In the third part of this thesis it was evaluated the synthesis and anticancer activity of some phosphonium salts. Phosphonium salts are a class of lipophilic cationic molecules that accumulate preferentially in mitochondria and inhibit the growth of human cancer cell lines The aim of the present study was to investigate the effects of a lipophilic phosphonium salt, (11-methoxy, 11-oxoundecyl)triphenylphosphonium bromide (MUTP) along with two other newly synthesized phosphine oxide salts, 3,3’-(methylphosphoryl)dibenzenaminium chloride and 3,3’-(phenylphosphoryl)dibenzenaminium chloride (SBAMPO and SBAPPO) on proliferation, in two human cancer cell lines: human breast cancer cells (MCF-7) and human uterine cervix adenocarcinoma cells (HeLa) and to elucidate their mechanism. The cancer cell mitochondrial membrane potential is relatively high when compared to normal cells, this force the phosphonium salts to accumulate, preferencially, in the mitochondria and inhibit their function. The results showed that only MUTP exhibits anti-proliferative effects on both cell lines, without affecting normal breast epithelial cell proliferation. More specifically, it was demonstrated that MUTP treatment of breast cancer cells is associated with impaired cell cycle progression, as determined by cytometry analysis. The G1/S cell cycle arrest was confirmed by an increased expression level of two proteins involved in cell cycle regulation, p21 and p53. Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers. Therefore, in order to characterize the mitochondrial function in MCF7 cells, after MUTP treatment, the cells were stained with specific metabolic probes and analyzed by FACS. The outcomes displayed that MUTP treatment decreased mitochondrial mass and mitochondrial membrane potential and increased the ROS production. In agreement with these findings, the reduction in the expression of the mitochondrial oxidative pathway (OXPHOS) enzymes revealed a bioenergetics failure, induced by MUTP, in treated cells. TUNEL assay, DNA Laddering and Western blot analysis of caspase-3, caspase-9 and Bax confirmed the apoptotic effect of MUTP treatment. Taken together, all these data suggest that MUTP may be capable of selectively targeting neoplastic cell growth and therefore has potential applications as an anticancer agent.Università della Calabri

Diets Differently Regulate Tumorigenesis in Young E0771 Syngeneic Breast Cancer Mouse Model

Breast cancer (BC) is the most diagnosed cancer type, accounting for one in eight cancer diagnoses worldwide. Epidemiological studies have shown that obesity is associated with increased risk of BC in post-menopausal women, whereas adiposity reduces the risk of BC in premenopausal women. The mechanistic link between obesity and BC has been examined by combining murine BC models with high-fat diet (HFD) induced obesity. However, the effect of adiposity (not obesity) induced by a short period of HFD consumption on BC pathogenesis is not well understood. In the current study, we examined the effects of different diet compositions on BC pathogenesis using a young E0771 syngeneic BC mouse model fed on either an HFD or regular diet (RD: a low-fat high-carbohydrate diet) for a short period (4 weeks) before implanting mammary tumors in mice. We analyzed the effect of diet composition on the onset of tumor growth, metastasis, and metabolic and immune status in the tumor microenvironment (TME) using various methods including in vivo bioluminescence imaging and immunoblotting analyses. We showed for the first time that a short-term HFD delays the onset of tumorigenesis by altering the immune and metabolic signaling and energy mechanism in the TME. However, RD may increase the risk of tumorigenesis and metastasis by increasing pro-inflammatory factors in the TME in young mice. Our data suggest that diet composition, adipogenesis, and loss of body fat likely regulate the pathogenesis of BC in a manner that differs between young and post-menopausal subjects

Diets Differently Regulate Pulmonary Pathogenesis and Immune Signaling in Mice during Acute and Chronic <i>Mycobacterium tuberculosis</i> Infection

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection persists as a leading cause of mortality and morbidity globally, especially in developing and underdeveloped countries. The prevalence of TB-DM (diabetes mellitus) is higher in low- and middle-income countries where TB and DM are most prevalent. Epidemiological data suggest that slight obesity reduces the risk of TB, whereas DM increases the risk of pulmonary TB. Diets can alter the levels of body fat mass and body mass index by regulating systemic adiposity. Earlier, using a transgenic Mtb-infected murine model, we demonstrated that loss of body fat increased the risk of pulmonary bacterial load and pathology. In the present study, we investigated whether increased adiposity alters pulmonary pathology and bacterial load using C57BL/6 mice infected with HN878 Mtb strain and fed a medium-fat diet (MFD). We analyzed the effects of MFD on the lung during acute and chronic infections by comparing the results to those obtained with infected mice fed a regular diet (RD). Histological and biochemical analyses demonstrated that MFD reduces bacterial burden by increasing the activation of immune cells in the lungs during acute infection and reduces necrosis in the lungs during chronic infection by decreasing lipid accumulation. Our data suggest that slight adiposity likely protects the host during active TB infection by regulating immune and metabolic conditions in the lungs

Susceptibility of Fat Tissue to SARS-CoV-2 Infection in Female hACE2 Mouse Model

The coronavirus disease (COVID-19) is a highly contagious viral illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 has had a catastrophic effect globally causing millions of deaths worldwide and causing long-lasting health complications in COVID-19 survivors. Recent studies including ours have highlighted that adipose tissue can act as a reservoir where SARS-CoV-2 can persist and cause long-term health problems. Here, we evaluated the effect of SARS-CoV-2 infection on adipose tissue physiology and the pathogenesis of fat loss in a murine COVID-19 model using humanized angiotensin-converting enzyme 2 (hACE2) mice. Since epidemiological studies reported a higher mortality rate of COVID-19 in males than in females, we examined hACE2 mice of both sexes and performed a comparative analysis. Our study revealed for the first time that: (a) viral loads in adipose tissue and the lungs differ between males and females in hACE2 mice; (b) an inverse relationship exists between the viral loads in the lungs and adipose tissue, and it differs between males and females; and (c) CoV-2 infection alters immune signaling and cell death signaling differently in SARS-CoV-2 infected male and female mice. Overall, our data suggest that adipose tissue and loss of fat cells could play important roles in determining susceptibility to CoV-2 infection in a sex-dependent manner

Biopolymeric self-assembled nanoparticles for enhanced antibacterial activity of Ag-based compounds

Microbial infections are still one of the major causes of death among world population. For this reason, there is an urgent need of new antimicrobial agents that express antibacterial activity. Silver-based compounds are known to be toxic against bacteria. One of the most representative examples is silver sulfadiazine. In our article we report the antibacterial activity of silver Nheterocyclic carbene complexes, tested againt a wide spectrum of microorganisms. MIC and MBC were calculated for each compound. We have also produced a dextran oleate conjugate, that was employed for the preparation of self-assembled biopolymeric nanoparticles

Phosphonium Salt Displays Cytotoxic Effects Against Human Cancer Cell Lines

Aims/Objective: Phosphonium salts are compounds whose structural characteristics enable them to cross the plasma and mitochondrial membrane with ease. Cancer cells have higher plasma membrane potentials than normal cells; phosphonium salts selectively accumulate in the mitochondria of neoplastic cells and inhibit mitochondrial function. Method: In the present work, we investigated the cytotoxic activity of lipophilic phosphonium salt (11- methoxy11-oxo-undecyl) triphenylphosphonium bromide (MUTP) as well as of the two new phosphine oxide salts, 3,3'-(methylphosphoryl) dibenzenaminium chloride (SBAMPO) and 3,3' (phenylphosphoryl) dibenzenaminium chloride (SBAPPO) on the proliferation of breast cancer cell line (MCF-7) and human uterin cervix adenocarcinoma cells (HeLa). Result: We showed that only MUTP exhibits antiproliferative effects on both cell lines, without affecting the normal breast epithelial cell proliferation. More specifically, we demonstrated that MUTP treatment of breast cancer cells is associated with impaired cell-cycle progression and metabolically induces mitochondrial damage and triggers apoptotic cell death in MCF-7 and HeLa cells. Taken together, these findings suggest that MUTP may be capable of selectively targeting neoplastic cell growth and therefore has potential applications as anticancer agent

Biopolymeric self-assembled nanoparticles for enhanced antibacterial activity of Ag-based compounds

Microbial infections still remain one of the main issues for human health. The rapid development of resistance towards the most common antimicrobial drugs in bacteria represents today a challenge in the infections management. In the present work we have investigated the antibacterial activity of a group of compounds, namely silver N-heterocyclic carbene complexes, against a broad spectrum of bacteria. For the most promising compound, a biopolymeric nanocarrier has been developed, in order to potentiate the metal complex activity against both Gram +ve and Gram −ve. The polymeric nanovehicle is based on dextran, modified with oleic acid residues, that confer amphiphilic properties to the polysaccharide. We have characterized the obtained biomaterial and studied its ability to self-assemble into nanoparticles in aqueous environment. Next, the transdermal diffusion analyses have been carried out to evaluate the ability of the polymeric particles to penetrate tissues. Thanks to the strategy adopted, we have fabricated an antibacterial system to which K. pneumoniae and E. coli are the most sensitive