Biochemical profile, selective cytotoxicity and molecular effects of Calendula officinalis extracts on breast cancer cell lines

Calendula officinalis extracts have been known to possess anti-tumor properties, but questions regarding their mechanisms of action still need to be answered. Therefore, the present study aims to investigate the selective cytotoxicity, the biochemical profile and the corresponding molecular effects of two extracts of C. officinalis: flowers and leaves, against several breast cancer cell lines in vitro. Dry flowers and leaves were subjected to ultrasonication assisted extraction in methanol 70%. The phenolic and volatile profiles of the extracts, determined by HPLC-MS and nontargeted GC-MS, revealed high levels of specific phenolic acids, flavonols and coumarin and several volatile compounds, including mono- and sesquiterpenes, ketones, aldehydes and esters. Both extracts proved to possess selective cytotoxic activities against tumor cells in comparison to healthy endothelial cells, according to the MTT assay. The flower extract was superior in terms of both cytotoxicity and selectivity when compared to the leaf extract, in accordance to their biochemical profiles. The gene expression pattern for 10 genes of interest was evaluated by RT-qPCR. The expression level of several genes involved in apoptosis (BCL2, BAX, BBC3, ZMAT3), and cell cycle progression (NFkB, CCND1, STAT3) was modulated by the treatment with both extracts. Therefore, C. officinalis extracts proved to be rich in compounds characterized by cancer-related cytotoxicity and are capable of inducing selective cytotoxicity on breast cancer cell lines

In Vitro Culture as a Stressful Factor Triggers Changes in Polyphenols, Flavonoids and Antioxidant Activity in Somatic Hybrids between Solanum tuberosum and S. bulbocastanum and their Respective Parents

Phenols, important secondary metabolites in plants, are responsible for specific defence mechanisms against abiotic stress, due to their strong antioxidant activity. Flavonoids, as part of phenolic group, are also involved in plant stress responses, being primarily responsible for photo-protection against UV solar radiation. Based on these premises, the plant response to optimized in vitro culture was evaluated, by quantifying the total polyphenolic content, the total flavonoid content and the antioxidant activity, both under in vitro and ex vitro conditions. Four closely related potato genotypes were analysed: the wild species Solanum bulbocatanum, S. tuberosum cv. ‘Rasant’ and two somatic hybrids between them. For all genotypes, both total polyphenolic content and antioxidant activity were increasing under optimized in vitro culture. The shoot responses were genotype dependent and the two somatic hybrids were intermediate between the parents both as morphology and reaction to in vitro stress. The somatic hybrid 1508/5, having morphology similar to potato, was reacting as the wild species, while the somatic hybrid 1508/2, with a similar morphology to the wild species was reacting as potato to in vitro stress. The somatic hybrid 1508/5 being also resistant to late blight is of interest for further use in pre-breeding. Total flavonoid content is decreasing under in vitro as compared to ex vitro conditions, UV-B radiation, the major trigger of flavonoid biosynthesis being absent in the fluorescent light. This study reveals the effect of in vitro culture on flavonoid content and details aspects of the biochemical parameters involved in plant in vitro stress

In Vitro Culture as a Stressful Factor Triggers Changes in Polyphenols, Flavonoids and Antioxidant Activity in Somatic Hybrids between Solanum tuberosum and S. bulbocastanum and their Respective Parents

Phenols, important secondary metabolites in plants, are responsible for specific defence mechanisms against abiotic stress, due to their strong antioxidant activity. Flavonoids, as part of phenolic group, are also involved in plant stress responses, being primarily responsible for photo-protection against UV solar radiation. Based on these premises, the plant response to optimized in vitro culture was evaluated, by quantifying the total polyphenolic content, the total flavonoid content and the antioxidant activity, both under in vitro and ex vitro conditions. Four closely related potato genotypes were analysed: the wild species Solanum bulbocatanum, S. tuberosum cv. ‘Rasant’ and two somatic hybrids between them. For all genotypes, both total polyphenolic content and antioxidant activity were increasing under optimized in vitro culture. The shoot responses were genotype dependent and the two somatic hybrids were intermediate between the parents both as morphology and reaction to in vitro stress. The somatic hybrid 1508/5, having morphology similar to potato, was reacting as the wild species, while the somatic hybrid 1508/2, with a similar morphology to the wild species was reacting as potato to in vitro stress. The somatic hybrid 1508/5 being also resistant to late blight is of interest for further use in pre-breeding. Total flavonoid content is decreasing under in vitro as compared to ex vitro conditions, UV-B radiation, the major trigger of flavonoid biosynthesis being absent in the fluorescent light. This study reveals the effect of in vitro culture on flavonoid content and details aspects of the biochemical parameters involved in plant in vitro stress

Phytochemical Profile and Selective Cytotoxic Activity of a <i>Solanum bulbocastanum</i> Dun. Methanolic Extract on Breast Cancer Cells

Solanum bulbocastanum is a wild potato species, intensively used in potato breeding programs due to its resistance to environmental factors. Thus, its biochemical profile and putative human health-related traits might be transferred into potato cultivars aimed for consumption. This study aims to assess the phytochemical profile and the selective cytotoxicity of an S. bulbocastanum extract against breast cancer cells. Dry leaves were subjected to ultrasonication-assisted extraction in methanol [70%]. The phenolic and glycoalkaloid profiles were determined by HPLC-PDA/-ESI+-MS. The volatile profile was investigated by nontargeted ITEX/GC-MS. The extract was tested against three breast cancer cell lines (MCF7, MDA-MB-231, HS578T) and a healthy cell line (HUVEC) by the MTT assay, to assess its selective cytotoxicity. The phenolic profile of the extract revealed high levels of phenolic acids (5959.615 µg/mL extract), and the presence of flavanols (818.919 µg/mL extract). The diversity of the volatile compounds was rather low (nine compounds), whereas no glycoalkaloids were identified, only two alkaloid precursors (813.524 µg/mL extract). The extract proved to be cytotoxic towards all breast cancer cell lines (IC50 values between 139.1 and 356,1 µg/mL), with selectivity coefficients between 1.96 and 4.96 when compared with its toxicity on HUVECs. Based on these results we conclude that the exerted cytotoxic activity of the extract is due to its high polyphenolic content, whereas the lack of Solanaceae-specific glycoalkaloids might be responsible for its high selectivity against breast cancer cells in comparison with other extract obtained from wild Solanum species. However, further research is needed in order to assess the cytotoxicity of the individual compounds found in the extract, as well as the anti-tumor potential of the S. bulbocastanum tubers

Breast Cancer-Delivered Exosomal miRNA as Liquid Biopsy Biomarkers for Metastasis Prediction: A Focus on Translational Research with Clinical Applicability

Metastasis represents the most important cause of breast cancer-associated mortality. Even for early diagnosed stages, the risk of metastasis is significantly high and predicts a grim outcome for the patient. Nowadays, efforts are made for identifying blood-based biomarkers that could reliably distinguish patients with highly metastatic cancers in order to ensure a closer follow-up and a more personalized therapeutic method. Exosomes are nano vesicles secreted by cancer cells that can transport miRNAs, proteins, and other molecules and deliver them to recipient cells all over the body. Through this transfer, cancer cells modulate their microenvironment and facilitate the formation of the pre-metastatic niche, leading to sustained progression. Exosomal miRNAs have been extensively studied due to their promising potential as prognosis biomarkers for metastatic breast cancer. In this review, we tried to depict an overview of the existing literature regarding exosomal miRNAs that are already validated as potential biomarkers, and which could be immediately available for the clinic. Moreover, in the last section, we highlighted several miRNAs that have proven their function in preclinical studies and could be considered for clinical validation. Considering the lack of standard methods for evaluating exosomal miRNA, we also discussed the challenges and the technical aspects underlying this issue

The Impact of miRNA in Colorectal Cancer Progression and Its Liver Metastases

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies with a high incidence and mortality rate. An essential challenge in colorectal cancer management is to identify new prognostic factors that could better estimate the evolution and treatment responses of this disease. Considering their role in cancer development, progression and metastasis, miRNAs have become an important class of molecules suitable for cancer biomarkers discovery. We performed a systematic search of studies investigating the role of miRNAs in colorectal progression and liver metastasis published until October 2018. In this review, we present up-to-date information regarding the specific microRNAs involved in CRC development, considering their roles in alteration of Wnt/&#946;catenin, EGFR, TGF&#946; and TP53 signaling pathways. We also emphasize the role of miRNAs in controlling the epithelial&#8315;mesenchymal transition of CRC cells, a process responsible for liver metastasis in a circulating tumor cell-dependent manner. Furthermore, we discuss the role of miRNAs transported by CRC-derived exosomes in mediating liver metastases, by preparing the secondary pre-metastatic niche and in inducing liver carcinogenesis in a Dicer-dependent manner

Chemical Structure, Sources and Role of Bioactive Flavonoids in Cancer Prevention: A Review

There has been a major shift in the collective mindset around the world in recent decades, both in terms of food and in terms of the treatment of chronic diseases. Increasing numbers of people are choosing to prevent rather than treat, which is why many consumers are choosing plant-based diets, mainly due to their bioactive compounds. A significant case of bioactive compound is flavonoids—a wide subclass of an even wider class of phytochemicals: polyphenols. Flavonoids are a broad topic of study for researchers due to their potential in the prevention and treatment of a broad range of cancers. The aim of this review is to inform/update the reader on the diversity, accessibility and importance of flavonoids as biomolecules that are essential for optimal health, focusing on the potential of these compounds in the prevention of various types of cancer. Along with conventional sources, this review presents some of the possible methods for obtaining significant amounts of flavonoids based on a slightly different approach, genetic manipulation

Critical Aspects Concerning the Development of a Pooling Approach for SARS-CoV-2 Diagnosis Using Large-Scale PCR Testing

The primary approach to controlling the spread of the pandemic SARS-CoV-2 is to diagnose and isolate the infected people quickly. Our paper aimed to investigate the efficiency and the reliability of a hierarchical pooling approach for large-scale PCR testing for SARS-CoV-2 diagnosis. To identify the best conditions for the pooling approach for SARS-CoV-2 diagnosis by RT-qPCR, we investigated four manual methods for both RNA extraction and PCR assessment targeting one or more of the RdRp, N, S, and ORF1a genes, by using two PCR devices and an automated flux for SARS-CoV-2 detection. We determined the most efficient and accurate diagnostic assay, taking into account multiple parameters. The optimal pool size calculation included the prevalence of SARS-CoV-2, the assay sensitivity of 95%, an assay specificity of 100%, and a range of pool sizes of 5 to 15 samples. Our investigation revealed that the most efficient and accurate procedure for detecting the SARS-CoV-2 has a detection limit of 2.5 copies/PCR reaction. This pooling approach proved to be efficient and accurate in detecting SARS-CoV-2 for all samples with individual quantification cycle (Cq) values lower than 35, accounting for more than 94% of all positive specimens. Our data could serve as a comprehensive practical guide for SARS-CoV-2 diagnostic centers planning to address such a pooling strategy

Circulating Small EVs miRNAs as Predictors of Pathological Response to Neo-Adjuvant Therapy in Breast Cancer Patients

Neo-adjuvant therapy (NAT) is increasingly used in the clinic for the treatment of breast cancer (BC). Pathological response to NAT has been associated with improved patients&rsquo; survival; however, the current techniques employed for assessing the tumor response have significant limitations. Small EVs (sEVs)-encapsulated miRNAs have emerged as promising new biomarkers for diagnosis and prediction. Therefore, our study aims to explore the predictive value of these miRNAs for the pathological response to NAT in BC. By employing bioinformatic tools, we selected a set of miRNAs and evaluated their expression in plasma sEVs and BC biopsies. Twelve miRNAs were identified in sEVs, of which, miR-21-5p, 221-3p, 146a-5p and 26a-5p were significantly associated with the Miller&ndash;Payne (MP) pathological response to NAT. Moreover, miR-21-5p, 146a-5p, 26a-5p and miR-24-3p were independent as predictors of MP response to NAT. However, the expression of these miRNAs showed no correlation between sEVs and tissue samples, indicating that the mechanisms of miRNA sorting into sEVs still needs to be elucidated. Functional analysis of miRNA target genes and drug interactions revealed that candidate miRNAs and their targets, can be regulated by different NAT regimens. This evidence supports their role in governing the patients&rsquo; therapy response and highlights their potential use as prediction biomarkers