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Rubus imperialis (Rosaceae) is a Brazilian medicinal plant that already exhibited therapeutical perspectives. However, previous studies revealed cellular and/or genetic toxicity of extracts from aerial parts of this plant, as well as other species of the Rubus genus. Being 2ß,3ß-19α-trihydroxyursolic acid (2B) one of the major compounds of this plant, with proven pharmacological effect, it is important to investigate the biosafety of this isolated compound. Therefore, in the present study, (2B) was tested by several cytogenotoxic endpoints up to 20 µg/ml in human hepatoma HepG2/C3A cells. The test compound did not produce any decreased cell viability, DNA damage, chromosomal mutations, cell cycle changes, or apoptotic effects in the tested cells. Additionally, RT-qPCR analysis revealed the downregulation of CYP3A4 (metabolism), M-TOR (cell death), and CDKN1A (cell cycle) genes. Under the experimental conditions used, the 2B compound did not show cytogenotoxic activity after a single exposure to HepG2/C3A human cells.

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Rubus imperialis Chum. Schl. (Rosaceae) have demonstrated some pharmacological activities, including gastroprotective action. However, genotoxic effects of R. imperialis extract was also reported. Since niga-ichigoside F1 (NIF1) is a major compound of this plant species, and which has proven pharmacological properties, it is essential to investigate whether this compound is responsible for the observed toxicity. Therefore, the objective of this study was to analyze the effects of NIF1 on HepG2/C3A cells for possible cytogenotoxicity, cell cycle and apoptosis influence, and expression of genes linked to the DNA damage, cell cycle, cell death, and xenobiotic metabolism. The results showed no cytogenotoxic effects of NIF1 at concentrations between 0.1 and 20 µg/ml. Flow cytometry also showed no cell cycle or apoptosis disturbance. In the gene expression analysis, none of the seven genes investigated showed altered expression. The data indicate that NIF1 has no cytogenotoxic effects, and no interruption of the cell cycle, or induction of apoptosis, apparently not being responsible for the cytotoxic effects observed in the crude extract of R. imperialis.

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Piperlongumine (PLN) is a biologically active alkaloid/amide derived from Piper longum, with known promising anticancer activity. The aim of this study was to compare the antiproliferative activity of PLN in human breast MCF-7 adenocarcinoma cell line with effects in HB4a normal mammary epithelial non-tumor cell line. The parameters examined were cell growth, viability, reactive oxygen species (ROS) levels and DNA damage, as well as the effects on the modulating targets responsible through regulation of these pathways. PLN increased ROS levels and expression of the SOD1 antioxidant enzyme. PLN inhibited the expression of the antioxidant enzymes catalase, TRx1, and PRx2. The ability of PLN to inhibit antioxidant enzyme expression was associated with the oxidative stress response. PLN induced genotoxicity in both cell lines and upregulated the levels of GADD45A mRNA and p21 protein. The DNA damage response ATR protein was downregulated in both cell lines and contributed to an enhanced PLN genotoxicity. In HB4a cells, Chk1 protein, and mRNA levels were also decreased. In response to elevated ROS levels and DNA damage induction, the cells were arrested at the G2/M phase, probably in an attempt to promote cell survival. Although cell viability was reduced in both cell lines, only HB4a cells underwent apoptotic cell death, whereas other types of cellular death may be involved in MCF-7 cells. Taken together, these data provide insight into the anticancer mechanisms attributed to PLN effects, which acts as an inhibitor of DNA damage response (DDR) proteins and antioxidant enzymes.

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Zerumbone (ZER) is a phytochemical isolated from plants of the Zingiberaceae family. Numerous studies have demonstrated its diverse pharmacological properties, particularly its potent antitumorigenic activity. This study aimed to assess the antiproliferative effects of ZER on HT-29 cells cultivated in both two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture systems. The evaluation of growth (size), cell death, and cell cycle arrest in 3D spheroid HT-29 cells was correlated with mRNA expression data. Treatment of 2D cells revealed that ZER exhibited cytotoxicity at concentrations above 30 µM, and an IC50 of 83.54 µM (24-h post-ZER treatment) effectively suppressed cell migration. In the 3D model, ZER induced an increase in spheroid volume over a 72-h period attributed to disaggregation and reconfiguration of characteristic zones. Analysis of cell death demonstrated a significant rise in apoptotic cells after 24 h of ZER treatment, along with cell cycle arrest in the G1 phase. Furthermore, ZER treatment resulted in alterations in mRNA expression, affecting key signaling pathways involved in cell death (BCL2 and BBC3), endoplasmic reticulum stress (ERN1), DNA damage (GADD45A), cell cycle regulation (CDKN1A, NFKB1, MYC, and TP53), and autophagy (BECN1 and SQSTM1). These findings suggested that ZER holds promise as a potential candidate for the development of novel anticancer agents that can modulate crucial cell signaling pathways. Additionally, the use of the 3D culture system proved to be a valuable tool in our investigation.

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Curcumin is an antiproliferative phytochemical extracted from Curcuma longa L and which has been studied in preclinical drug screening using cell monolayers and animal models. However, several limitations of these culture systems may be overcome by performing screening with three-dimensional (3-D) cell culture. The aim of this study was to investigate the effects of curcumin on cytotoxicity and genotoxicity as well as spheroid growth using cervical adenocarcinoma HeLa cell spheroids by performing RT-PCR mRNA expression of genes involved in cell death (CASP3, CASP8, CASP9, PARP1, BBC3, BIRC5, BCL2, TNF), autophagy (BECN1, SQSTM1), cell cycle regulation (TP53, C-MYC, NF-kB, CDKN1A, m-TOR, TRAF-2), DNA damage repair (H2AFX, GADD45A, GADD45G), oxidative stress (GPX1), reticulum stress (EIF2AK3, ERN1), and invasion (MMP1, MMP9) was investigated. Curcumin was cytotoxic in a concentration-dependent manner. Curcumin-treated spheroids exhibited lower proliferative recovery and cell proliferation attenuation, as observed in the clonogenic assay. Further, no marked genotoxicity was detected. Curcumin-treated spheroids displayed reduced expression of BECN1 (2.9×), CASP9 (2.1×), and PARP1 (2.1×) mRNA. PARP1 inhibition suggested disruption of essential pathways of proliferation maintenance. Downregulated expression of CASP9 mRNA and unchanged expression of CASP3/8 mRNA suggested caspase-independent cell death, whereas downregulated expression of BECN1 mRNA indicated autophagic disruption. Therefore, curcumin exhibits the potential for drug development with antiproliferative activity to be considered for use in cancers.

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Plants with medicinal potential may also produce adverse effects in humans. This seems to be the case for the species Rubus rosifolius, where preliminary studies demonstrated genotoxic effects attributed to extracts obtained from leaves and stems of this plant using on HepG2/C3A human hepatoma cells as a model. Considering the beneficial properties of this plant as an antidiarrheal, analgesic, antimicrobial, and antihypertensive and its effects in the treatment of gastrointestinal diseases, the present study was developed with the aim of determining the cytotoxic and genotoxic potential of extracts of leaves and stems of R. rosifolius in primary without metabolic competence in human peripheral blood mononuclear cells (PBMC). Cell viability analyses at concentrations of between 0.01 and 100 µg/ml of both extracts did not markedly affect cell viability. In contrast, assessment of the genotoxic potential using the comet assay demonstrated significant damage to DNA within PBMC from a concentration of 10 µg/ml in the stem extract, and a clastogenic/aneugenic response without cytokinesis-block proliferation index (CBPI) alterations at concentrations of 10, 20, or 100 µg/ml for both extracts. Under our experimental conditions, the data obtained demonstrated genotoxic and mutagenic effects attributed to extracts from leaves and stems of R. rosifolius in cells in the absence of hepatic metabolism.

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AIMS: Hepatocellular Carcinoma (HCC) is a primary neoplasm derived from hepatocytes with low responsiveness and recurrent chemoresistance. Melatonin is an alternative agent that may be helpful in treating HCC. We aimed to study in HuH 7.5 cells whether melatonin treatment exerts antitumor effects and, if so, what cellular responses are induced and involved. MAIN METHODS: We evaluated the effects of melatonin on cell cytotoxicity and proliferation, colony formation, morphological and immunohistochemical aspects, and on glucose consumption and lactate release. KEY FINDINGS: Melatonin reduced cell motility and caused lamellar breakdown, membrane damage, and reduction in microvillus. Immunofluorescence analysis revealed that melatonin reduced TGF and N-cadherin expression, which was further associated with inhibition of epithelial-mesenchymal transition process. In relation to the Warburg-type metabolism, melatonin reduced glucose uptake and lactate production by modulating intracellular lactate dehydrogenase activity. SIGNIFICANCE: Our results indicate that melatonin can act upon pyruvate/lactate metabolism, preventing the Warburg effect, which may reflect in the cell architecture. We demonstrated the direct cytotoxic and antiproliferative effect of melatonin on the HuH 7.5 cell line, and suggest that melatonin is a promising candidate to be further tested as an adjuvant to antitumor drugs for HCC treatment.

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Rubus rosifolius, popularly known as "red mulberry", is a common medicinal plant in southern Brazil and is used as an antidiarrheal, analgesic, antimicrobial and antihypertensive, and to treat stomach diseases. The aim of this study was to analyze the R. rosifolius stem extract (RrSE) for possible in vitro cytotoxic and genotoxic effects, using the comet assay and the micronucleus test to assess genotoxicity, and flow cytometry to assess the impact on the cell cycle and apoptosis in HepG2/C3A cells, in addition to evaluating the expression of genes linked to the induction of DNA damage, cell cycle, apoptosis and metabolism of xenobiotics. The MTT assay observed no cytotoxic effects at concentrations between 0.01 and 100 µg/mL of the extract. However, genotoxic effects occurred in treatments with the extract from a 1 µg/mL concentration. Flow cytometry analysis revealed a significant increase in cells in the G2/M phase after treatment with 10 µg/mL, a decrease in cells in the G0/G1 phase in the treatment with 100 µg/mL, and a significant increase in total apoptotic cells. In the gene expression analysis, an increase in the CYP1A2 xenobiotics metabolizing gene expression was observed. Despite the promising pharmacological effects of R. rosifolius, the results revealed that the RrSE has genotoxic effect and induces apoptosis in HepG2/C3A cells, indicating danger in using this plant extract by humans.

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BACKGROUND: Phenotypic switching generates fungal colonies with altered morphology and allows pathogens to adapt to changing environments. OBJECTIVE: This study investigated the structure and genetic factors of switched morphotypes colonies in Candida tropicalis. METHODS: Morphotypes of C. tropicalis comprised the clinical strain 49.07 that exhibited smooth colony phenotype and switched (crepe and rough) morphotypes that showed colonies with marked structural variations, including wrinkled surface, depressions areas, and irregular edges (structured morphology). The morphotypes were analyzed for the presence and distribution of the extracellular matrix (ECM) at the ultrastructural level-SEM. The composition of the ECM and the percentage of hyphae in colonies were evaluated. The expression of EFG1 (Enhanced filamentous growth protein 1), WOR1 (White-opaque regulator 1), and BCR1 (Biofilm and cell wall regulator 1) in the morphotypes was measured by RT-qPCR. RESULTS: Colonies of the switched variants exhibited distinct arrangements of ECM compared to the smooth phenotype (clinical strain). In addition, rough variant colonies showed higher amounts of total carbohydrates and proteins in ECM (p < 0.05). Switched (crepe and rough) colonies exhibited a higher percentage of hyphae throughout their development (p < 0.05). The mRNA expression levels of EFG1, WOR1, and BCR1 in the rough morphotype were significantly higher than they were in the smooth morphotype. In addition, there was a positive correlation between the expression of these genes and filamentation (hyphae formation) of the rough morphotype (r2 > 0.9472, p < 0.05). CONCLUSION: Structural variations in switched morphotypes colonies of C. tropicalis seem to be associated with increased hyphae growth and the amount and distribution of ECM. Switched colonies have distinct expressions of the EFG1, WOR1, and BCR1 master regulators genes.

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Zerumbone (ZER) is a phytochemical with antioxidant and antiproliferative properties. This study evaluated the cytoxicity of ZER combined with chemotherapeutic agents and the expression of mRNA genes related to cell cycle, cell death, xenobiotic metabolism, DNA damage, and endoplasmic reticulum (ER) stress in HepG2/C3A cells. ZER was cytotoxic (IC50, 44.31 µM). ZER-induced apoptosis was related to BBC3 and ERN1 upregulation (ER stress), and its antiproliferative effects were attributable to MYC, IGF1, and NF-kB mRNA inhibition. ZER-induced G2/M arrest and DNA damage was associated with mRNA expression of cell cycle (CDKN1A) and DNA damage (GADD45A) genes. Increased CYP1A2 and CYP2C19 mRNA expression suggested ZER metabolization, and reduced CYP1A1 and CYP2D6 expression indicated a longer time of action of ZER in the cell, enhancing its pharmacological effect. ZER downregulated TP53, PARP1, BIRC5 (apoptosis), and MAP1LC3A (autophagy). In apoptosis assay, the data of the association treatments with ZER suggested antagonism. In cytotoxicity assay, the data of the association treatments with ZER suggested synergism action to cisplatin and antagonism action to doxorubicin and 5-fluorouracil. Thus, ZER has potential for application in chemotherapy as it modulates mRNA targets; however, it may not have the desired efficiency when combined with other chemotherapeutic agents.

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