Antioxidant properties and cytotoxic effects of Elephantorrhiza elephantina on cervical cancer cells

Chloroquine synergizes doxorubicin efficacy in cervical cancer cells through flux impairment and down regulation of proteins involved in the fusion of autophagosomes to lysosomes

BACKGROUND Cancer is one of the causes of high mortality. Breast and cervical cancers are two of the most frequent cancers affecting women around the world, including Indonesia. Natural materials such as rodent tuber (Typhonium flagelliforme) have anticancer potentials. The rodent tuber extract contains ribosome inactivating proteins (RIPs) capable of cutting the DNA or RNA of cancer cells and blocking the growth of cancer cells. The purpose of this study was to evaluate the cytotoxic effects of Typhonium flagelliforme Lodd extract on HeLa cervical cancer and Michigan Cancer Foundation-7 (MCF-7) breast cancer cells. METHODS Subjects were cultured cell lines of HeLa cells in Rosswell Park Memorial Institute (RPMI) and of MCF-7 cells in Dulbecco’s Minimum Essential Medium (DMEM). Rodent tuber ethanolic extract was diluted in dimethyl sulfoxide (DMSO). The cytotoxicity assay used the 3-(4,5-dimethyl thiazol-2-yl,5-diphenyl) tetrazolium bromide (MTT) method. Absorbance was read in an ELISA reader at a wavelength of 595 nm. RESULTS Rat tuber extract at all dilutions (500; 250; 125; 62.5; 31.25; 15.625;7.81; 3.9 i g/ mL) showed cytotoxic effects against HeLa and MCF-7 cells. Higher concentrations of the extract gave a higher proliferation inhibition effect. Calculated IC50 values of the extract by probit analysis were 30.19 ig/mL against HeLa cells and 5.586 i g/mL against MCF-7 cells. CONCLUSIONS Ethanolic extract of Typhonium flagelliforme Lodd has cytotoxic effects against HeLa cells and MCF-7 cells. The cytotoxic effects against MCF-7 cells are greater than the cytotoxic effects against HeLa cells.

The present PhD work aimed to contribute to the knowledge available on the antitumor properties of flavonoids through the in vitro evaluation of the bioactivity of some uncommon semi-synthetic derivatives against a panel of breast and cervical cancer cell lines that are well-established models for certain types of gynecological cancer. The work included the semi-synthetic preparation of seven naringenin oxime and oxime ether derivatives prior to their bioactivity testing, and the in vitro evaluation of nine protoflavone-chalcone hybrid compounds and their reference fragments against breast (hormone-dependent MCF-7 and triple-negative MDA-MB-231), and cervical (SiHa & HeLa) cancer cell lines in addition to human HL-60 leukemia cells and non-cancerous mouse embryonic fibroblasts (NIH/3T3). The work included cytotoxicity, cell cycle distribution, caspase-3 activity, and antioxidant activity evaluations. Further, the cytotoxic activity of the hybrid compounds was evaluated in comparison with that of their corresponding fragments in a virtual and experimental combination study. In summary, our work led to the following results.  Two naringenin oxime isomers and five oxime ether derivatives were synthesized, purified and characterized. Four of these compounds, such as the minor product naringenin Z-oxime, and the ethyl, allyl, and tert-butyl ethers of naringenin E-oxime were prepared as new compounds.  When evaluating the in vitro cytotoxicity of the prepared derivatives of naringenin, tert-butyl oxime ether (6) showed the most potent effects on different gynecological cancer cells, with significant activity against MCF-7 and HeLa cells.  The flow cytometric analysis of compound (6) on gynecological cancer cells revealed significant accumulation of cells in the hypodiploid (subG1) phase in HeLa & SiHa cell lines, indicating the apoptosis induction effect, and induced cycle suppression at G2/M stage in MCF-7 cancer cells. Further, the proapoptotic activity of this compound was confirmed in HeLa cells by detecting the increased activity of caspase-3.  To our surprise, naringenin methyl oxime ether was more potent in the ORAC assay than its parent compound, while all other analogs were up to an order of magnitude less active. This suggests good peroxyl radical scavenging capacity for this compound. There was no apparent correlation between the in vitro cytotoxic and antioxidant activities of the tested compounds, suggesting that their anticancer effects are likely not related to their antioxidant properties.  Four protoflavone hybrid compounds were identified as promising antitumor lead compounds based on their prominent in vitro cytotoxic effects and their selectivity on different breast and cervical cancer cells with antiproliferative effects better than cisplatin.  The most potent compounds have an intense proapoptotic effect on TNBC, as evidenced by flow cytometric investigation and caspase-3 activity. It was shown that compound 10c induces a considerable expansion in caspase-3 activity in a concentration-time dependent manner with a significant increase in the sub G1 phase.  A novel approach was used to evaluate the bioactivity of the hybrid compounds in comparison with that of their corresponding fragments. A virtual combination study was performed by using the Chou-Talalay method as a mathematical tool, and results were compared to the corresponding experimental combinations of the cells with non-coupled fragments. This gave valuable extra information as virtual combination index values and confirmed that the studied hybrid compounds are much more potent than what would be expected by a mathematical sum of the bioactivity of their fragments.  Based on the above, we demonstrated the use of a novel approach to evaluate the bioactivity of hybrid compounds in general, and suggested an extension of the applicability of the Chou-Talalay method, one of the currently available most popular platforms for drug-drug combination studies. Altogether, our present study provided valuable information about the antitumor potential of two series of unusual semi-synthetic flavonoid derivatives, and made a significant contribution to identifying a set of highly potent hybrid lead compounds obtained through fragment-based drug design. Further, by providing a novel use for an existing convenient and very widely used mathematical platform, we believe our study may have contributed to the research of hybrid compounds also in a more general manner.

NSAIDs have been reported to have anticancer effects against certain types of cancer, however, the mechanism behind NSAIDs action on cancer cells is unclear in many cases. The aim of this study was to investigate the cytotoxic effects of ibuprofen on cervical cancer (Hela) cells and evaluation of nitric oxide synthase2 (iNOS) gene expression level. In this in vitro study, the cells were divided into control (untreated) group and groups treated with 0.01, 0.1, 1, 2.5, 5, and 10 mg/mL of ibuprofen for 24 and 48 hours. MTT assay method was used to determine the cytotoxic effects of ibuprofen on Hela cells. The Griess method was used to quantify NOS activity, and iNOS gene expression level was evaluated using quantitative RT-PCR. The data were analyzed using ANOVA and Student's t-test. Ibuprofen had cytotoxic effects on Hela cells in a dose dependent manner. The cytotoxic dose of ibuprofen significantly increased the NOS activity and iNOS gene expression level. Our findings indicated that the cytotoxic effects of ibuprofen on cervical cancer cells is partly mediated by increased activation of NOS.

BackgroundAbrus precatorius possesses various therapeutic properties including anticancer potentials. This study evaluated the anti-proliferative activities of fractions of methanol root extract of A. precatorius on breast and cervical cancer cells and their immunomodulatory effect. Phytochemical screening was done by FTIR and GCMS. In vitro anti-proliferative effect was evaluated on human breast cancer (AU565) and cervical cancer (HeLa) cells and on murine fibroblast (NIH 3 T3) cells. Antioxidant activity was performed via DPPH radical scavenging assay. The immunomodulatory potential of fractions was evaluated by inhibition of phagocytes oxidative burst (ROS), Nitric oxide (NO) and proinflammatory cytokine TNF-α.ResultsA. precatorius fractions showed different chemical groups and were somewhat selective in antiproliferative activity against studied cancer cells. Ethyl acetate fraction showed the most significant antiproliferative activity with IC50 values of 18.10 μg/mL and 11.89 μg/mL against AU565 and HeLa cells respectively. Hexane fraction significantly (p < 0.05) inhibited HeLa cells (IC50 18.24 ± 0.16 μg/mL), whereas aqueous fraction showed mild inhibition (IC50 46.46 ± 0.14 μg/mL) on AU565 cell proliferation. All fractions showed no cytotoxicity against NIH-3 T3 murine fibroblast normal cells. All fractions showed potent and significant (p < 0.001) DPPH radical scavenging activity as well as suppressed phagocytic oxidative burst. Hexane (< 1 μg/mL), ethyl acetate (< 1 μg/mL), and butanol (5.74 μg/mL) fractions potently inhibited the cytokine TNF- α, hexane (< 1 μg/mL) and ethyl acetate (< 1 μg/mL) fractions also potently inhibited NO.ConclusionsThe antiproliferative activities and suppressive effect on the phagocytic oxidative burst, NO and proinflammatory cytokine might be due to the synergistic actions of bioactive compounds especially flavonoids present in the assayed fractions and therefore, suggest chemotherapeutic use of A. precatorius in cancer treatment.

Several studies have reported the anticancer effects of ibuprofen on cervical cancer cells, but the molecular pathway is still unclear in many aspects. This study aimed to investigate the effects of cytotoxic dose of ibuprofen on BAX, BCL-2, caspase-3, MMP-9, KAI-1 and NM23 gene expression levels and evaluation of caspase-3, -8 and -9 activity level in cervical cancer (HeLa) cells. Cervical cancer cells were divided into untreated (control) groups and ibuprofen treated groups. Expression levels of BAX, BCL-2, caspase-3, MMP-9, KAI-1 and NM23 genes were evaluated by Real-time PCR and caspase-3, -8 and -9 activity levels were determined using colorimetric method. Hoechst staining was used to confirm apoptosis. The data were statistically analyzed between groups using ANOVA and independent t-test. Significant increase in expression levels of caspases-3, and BAX/BCL-2 ratio, caspase-3, -8 and -9 activity level and a significant decrease in NM23, KAI-1 genes expression level were observed in HeLa cells treated with ibuprofen cytotoxic concentration. The apoptosis observed in HeLa cells was confirmed by Hoechst 333285 staining and flow cytometry analysis. Ibuprofen increased nuclear condensation and induced apoptosis in HeLa cells by both intrinsic and extrinsic apoptosis pathways.

Our research aims to assess the influence of erastin, a ferroptosis-inducing agent, on cervical cancer cells. Cervical cancer is a prevalent malignancy in females. Dysregulation of ferroptosis, a form of cell demise reliant on iron, is implicated in several cancers. The effect of erastin on HeLa and SiHa was detected by transwell assay, scratch test, and colony formation assay, while cell apoptosis was detected using flow cytometry. Cellular reactive oxygen species (ROS) generation was detected using the dichloro-dihydro-fluorescein diacetate assay. Sequencing analysis identified differentially expressed genes (DEGs), and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment analyses were employed to identify the target gene. Subsequently, the utilization of small interfering RNA (siRNA) was employed to suppress the targeted gene expression in HeLa cells, thereby effectively mitigating the impact of erastin on various cellular processes including invasion, colony formation, migration, and ROS generation. The findings indicate that erastin attenuates the viability of both HeLa cells (IC50 = 30.88µM) and SiHa cells (IC50 = 29.40µM). Treatment with erastin at 10µM inhibits the invasion, colony formation, and migration of both HeLa and SiHa cells within 24h. Ferrostatin-1 (1µM) notably alleviates the inhibitory effects of erastin of HeLa and SiHa cells. Upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target, heme oxygenase-1 (HO-1), was found in erastin-treated cells compared to the control group. When knocked down HO-1 in HeLa cells, effectively counteracting the effects of erastin on the invasion, colony formation, migration, and ROS production in HeLa cells. Our research demonstrates that erastin induces ferroptosis and the accumulation of ROS in cervical cancer cells by activating the Nrf2/HO-1 pathway, significantly reducing cell proliferation and motility. These findings propose a potential molecular mechanism of erastin-mediated cervical cancer development.

Sesamin induces ER stress-mediated apoptosis and activates autophagy in cervical cancer cells

Coconut water is considered to be nature’s elixir. It is a refreshing beverage that can quench thirst, and it is consumed as a health tonic, especially by people living in the tropics. As cancer prevalence increases, novel anticancer therapies are urgently needed, and the chemoprevention approach using natural products is gaining research attention. The goal of this study was to evaluate the potential chemopreventive effects of two varieties of freeze dried coconut water (FDCW) against cervical cancer (HeLa) cells. Both the MATAG FDCW and the Aromatic Dwarf (AD) FDCW varieties exerted anti-proliferative activity against HeLa cells with a inhibitory concentration of 100 µg/ml. After 72 h of treatment, observation under an inverted microscope showed typical apoptotic morphological alteration in HeLa cells exposed to MATAG FDCW, and features of both apoptosis and autophagy were observed in HeLa cells treated with AD FDCW. Fluorescence microscopy revealed the presence of condensed chromatin and apoptotic bodies in HeLa cells from both treatments. To evaluate the anti-proliferative activity over a prolonged treatment period, HeLa cells treated with each type of FDCW were incubated for 8 d. MATAG FDCW was able to continuously suppress HeLa cell proliferation for the entire experiment, whereas the effect of AD FDCW was not stable and the suppression effect decreased over time. These results suggest that MATAG FDCW has a better anti-proliferative effect than AD FDCW. However, both FDCW varieties demonstrated positive chemopreventive activity and should be considered as potential novel anticancer therapies.

Hypoxia regulates fibroblast function by changing intracellular signaling and secretion factors, that influence the states of nearby cells. In this work, we investigated how medium (CM) from human adult dermal fibroblasts (HDFs) cultured in normoxic and hypoxic conditions affected cervical cancer (HeLa) cells. The HeLa cells showed decreased cell viability, increased apoptosis, and cell cycle arrest in response to CM from hypoxic-cultured HDFs (H-CM) compared with CM from normoxic-cultured HDFs (N-CM). Among the proteins up-regulated (>2-fold) in H-CM compared with N-CM, lymphotoxin-beta receptor (LTBR) decreased the viability of HeLa cells. Among the intracellular proteins down-regulated (>2-fold) in HeLa cells treated with H-CM compared with N-CM, the most enriched biological process GO term and KEGG pathway were protein deubiquitination and hsa05166:HTLV-I infection, respectively. In the protein–protein interaction network of intracellular proteins with altered expression (>2-fold), 1 up-regulated (TNF) and 8 down-regulated (ESR1, MCL1, TBP, CD19, LCK, PCNA, CHEK1, and POLA1) hub proteins were defined. Among the down-regulated hub proteins, the most enriched biological process GO term and KEGG pathway were leading strand elongation and hsa05166:HTLV-I infection, respectively. This study reveals that H-CM had stronger anti-cancer effects on cervical cancer cells than N-CM and induced intracellular signaling patterns related to those enhanced anti-cancer effects.

BackgroundThe aim of this study is to investigate the effects of polyphenol extract from Phyllanthus emblica (PEEP) on cervical cancer cells and to explore the underlying mechanism.MethodsMTT assay was used to measure inhibition of proliferation of cervical cancer (HeLa) cells after treatment with PEEP at concentrations of 0, 50, 100, 150, and 200 mg/ml for 48 hours. HeLa cells were treated with PEEP (150 mg/ml) for 48 hours in the following analysis. Karyomorphism was assessed by immunofluorescence using DAPI staining, and cell apoptosis and cell cycle were assessed using flow cytometry. Three apoptotic marker proteins, namely, Fas, FasL, and cleaved caspase-8, were assessed by western blotting.ResultsPEEP inhibited the growth of HeLa cells, and the optimum concentration of PEEP was 150 mg/ml. In addition, the karyomorphism of HeLa cells after treatment with PEEP was abnormal. Furthermore, PEEP induced arrest of the HeLa cell cycle at G2/M phase, and triggered apoptosis. PEEP also induced significant Fas and FasL activation, and cleavage of caspase-8.ConclusionsOur study indicates that PEEP is effective in inhibiting HeLa cell proliferation by inducing cell cycle arrest at G2/M phase and inducing apoptosis.

The purpose of the present study is to figure out the role of miRNA-148a (miR-148a) in growth, apoptosis, invasion, and migration of cervical cancer cells by binding to regulator of ribosome synthesis 1 (RRS1). Cervical cancer and adjacent normal tissues, as well as cervical cancer cell line Caski, HeLa, C-33A, and normal cervical epithelial cell line H8 were obtained to detect the expression of miR-148a and RRS1. Relationship between miR-148a and RRS1 expression with clinicopathological characteristics was assessed. The selected Caski and HeLa cells were then transfected with miR-148a mimics, miR-148a inhibitors or RRS1 siRNA to investigate the role of miR-148a and RRS1 on proliferation, apoptosis, colony formation, invasion, and migration abilities of cervical cancer cells. Bioinformatics information and dual luciferase reporter gene assay was for used to detect the targetting relationship between miR-148a and RRS1. Down-regulated miR-148a and up-regulated RRS1 were found in cervical cancer tissues and cells. Down-regulated miR-148a and up-regulated RRS1 are closely related with prognostic factors of cervical cancer. RRS1 was determined as a target gene of miR-148a and miR-148a inhibited RRS1 expression in cervical cancer cells. Up-regulation of miR-148a inhibited cell proliferation, migration, and invasion while promoting apoptosis in Caski and HeLa cells. Our study suggests that miR-148a down-regulates RRS1 expression, thereby inhibiting the proliferation, migration, and invasion while promoting cell apoptosis of cervical cancer cells.

The aim of this study was to explore the function of long noncoding RNA (lncRNA) FAM13A-AS1 and its associated mechanism in cervical cancer. A total of 30 cervical cancer tissues and adjacent tissues were collected. Cervical cancer cell lines, including SiHa and HeLa, were transfected with constructs expressing LV-FAM13A-AS1, silencing RNA LV-siFAM13A-AS1, miRNA mimics, and miRNA inhibitors. RT-qPCR was used to detect the expression of FAM13A-AS1 in cervical cancer tissues, including SiHa, HeLa, and HUCEC cells. MTT, flow cytometry, and transwell assays were performed to explore the influence of FAM13A-AS1 on cervical cancer cell proliferation, apoptosis, invasion, and migration. A bioinformatics analysis and a dual-luciferase assay were carried to confirm the target relationship between FAM13A-AS1 or DDI2 and miRNA-205-3p. Finally, in vivo tumorigenesis experiments were performed in nude mice to explore the effect of FAM13A-AS1 expression on cervical cancer. Low FAM13A-AS1 expression and high miRNA-205-3p expression were observed in cervical cancer tissues and cell lines (SiHa and HeLa). Upregulating the expression of FAM13A-AS1 inhibited proliferation, migration, and invasion of SiHa and HeLa cells, while the apoptosis of SiHa and HeLa cells was increased. More importantly, LV-FAM13A-AS1 could improve tumor development in vivo. In addition, FAM13A-AS1 negatively regulated the expression of miRNA-205-3p, while miRNA-205-3p reduced DDI2 expression, and miRNA-205-3p mimic reversed the effects of FAM13A-AS1 overexpression in vitro. In conclusion, FAM13A-AS1 inhibits the progression of cervical cancer by targeting the miRNA-205-3p/DDI2 axis, suggesting that FAM13A-AS1 might be a potential target for cancer cell treatment.

The aim of this study was to investigate the antioxidant, cytotoxic, and antibacterial activities of ethanol extract (SRE) and its hexane (SRH), chloroform (SRC), ethyl acetate (SREA), and aqueous ethanol (SRAE) sub-extracts obtained from the fruits of Scabiosa rotata M. Bieb. Molecular docking studies were also performed to evaluate the interactions of the most active sub-extract with the human mitochondrial ABC transporter (ABCB10) protein, plays an important role in cellular oxidative stress regulation and mitochondrial function. Total phenolic (TPC) and flavonoid (TFC) contents and antioxidant radical scavenging activities (1, 1-diphenyl-2-picryl hydrazyl (DPPH) and 3-ethylbenzothiazoline-6 sulfonic acid (ABTS)) of all extracts were determined. Cytotoxic activity was evaluated on different cell lines like; HepG2 (liver cancer), MCF-7 (breast cancer), and HeLa (cervical cancer) cells. Antibacterial activity was tested against E. coli, P. aeruginosa, Enterococcus faecalis, and S. aureus. SREA showed the best antioxidant activity with IC50 values of 54.20 μg/mL and 48.56 μg/mL for DPPH and ABTS, respectively, and also had high TPC (499.06 mg GA/g) and TFC (327.45 mg QE/g). Furthermore, SREA showed significant cytotoxicity comparable to cisplatin with IC50 values of 51.29, 51.41, and 52.89 μg/mL for HepG2, MCF-7, and HeLa cells, respectively. However, none of the extracts showed antibacterial activity. The analysis using liquid chromatography-mass spectrometry (LC/MS–MS) on SREA found that the main phenolic compounds are chlorogenic acid, hesperidin, quinic acid, and isoorientin. Molecular docking of the major compounds showed that chlorogenic acid (2.21 mM) exhibited the strongest binding affinity, followed by hesperidin (5.83 mM), quinic acid (8.86 mM), and isoorientin (13 mM). These findings indicate the potential of SREA to be used as a natural antioxidant and anticancer agent.

BackgroundMangifera pajang Kosterm is a plant species from the mango family (Anacardiaceae). The fruits are edible and have been reported to have high antioxidant content. However, the detailed phytochemical studies of the plant have not been reported previously. This study investigates the phytochemicals and biological activities of different parts of Mangifera pajang.MethodsThe plant samples were extracted with solvents of different polarity to obtain the crude extracts. The isolated compounds were characterized using spectroscopic methods. The extracts and isolated compounds were subjected to cytotoxicity tests using human breast cancer (MCF-7), human cervical cancer (HeLa) and human colon cancer (HT-29) cells. The free radical scavenging activity test was conducted using the DPPH assay. Antimicrobial activity tests were carried out by using the disc diffusion method.ResultsPhytochemical investigation on the kernel, stem bark and leaves of Mangifera pajang led to the isolation of methyl gallate (1), mixture of benzaldehyde (2) and benzyl alcohol (3), mangiferonic acid (4), 3β-hydroxy-cycloart-24-ene-26-oic acid (5), 3β,23-dihydroxy-cycloart-24-ene-26-oic acid (6), lupeol(7) lupenone(8), β-sitosterol(9), stigmasterol(10), trans-sobrerol(11) and quercitrin (12). Crude ethyl acetate and methanol extracts from the kernel indicated strong cytotoxic activity towards MCF-7 and HeLa cells with IC50 values of less than 10 μg/mL, while petroleum ether, chloroform and ethyl acetate extracts of the stem bark showed strong to moderate activity against MCF-7, HeLa and HT-29 cancer cell lines with IC50 values ranging from 5 to 30 μg/mL. As for the antimicrobial assays, only the ethyl acetate and methanol extracts from the kernel displayed some inhibition against the microbes in the antibacterial assays. The kernel extracts showed highest free radical scavenging activity with IC50 values of less than 10 μg/mL, while the ethyl acetate and methanol extracts of leaves displayed only weak activity in the DPPH assays.ConclusionsPhytochemical investigations on various parts of Mangifera pajang have identified terpenoids and a flavonol derivative as major constituents. Bioassay studies have indicated that the crude extracts and isolated compounds have potential as naturally-derived anticancer and antimicrobial agents, besides possess high free radical scavenging activity.