Induced Apoptosis In HeLa Cells Research Articles

Exploring the therapeutic potential of edible vegetables, fruits, and spices against cancer in various cell lines.

Cancer is rapidly becoming the leading cause of death globally. This study aimed to identify edible foods with cytotoxic and/or antioxidant activities that can prevent cancer when consumed in a regular diet. Sixty-eight edible foods were purchased from the local market, and the materials were extracted with 80% methanol. The cytotoxic activity of the extracts was evaluated using MTT on HeLa, H2228, HEK293, and H3122 cell lines. To study apoptosis, triple fluorescence labeling with DAPI, Annexin V, and propidium iodide was used. The phenolic content, antioxidant capacity, and free radical scavenging capabilities were studied using conventional spectrophotometric techniques. Among the edible foods, carrot, pointed gourd, wax gourd, ficus, apple, lemon, cumin seed, and white peppercorn showed moderate cytotoxicity in HeLa cells. The growth of HeLa cells was significantly inhibited dose-dependently by tomato, banana, Indian spinach, guava, lemon peel, and coriander (IC50, 24.54, 17.89, 13.18, 9.33, 1.23, and 2.96 µg/mL, respectively). Tomato, Indian spinach, lemon peel, and coriander exerted significant dose-dependent inhibition of H2228, HEK293, and H3122 cell proliferation. The tomato, Indian spinach, lemon peel, and coriander extracts induced HeLa cell apoptosis. White peppercorn, amaranth, apple, wax gourd, cumin seed, taro, and lemon peel contained significant amounts of polyphenols and showed high antioxidant activity. White peppercorn, apple, coriander, lemon peel, and ficus significantly scavenged DPPH free radicals (IC50 values of 10.23, 12.02, 13.49, 13.8, and 14.0 µg/mL, respectively). The overall results suggest that the daily intake of these antioxidant-rich cytotoxic foods can prevent or reduce the risk of cancer.

Synthesis, characterization, and cytotoxicity of Pt (Ⅱ) and Pd (Ⅱ) complexes with 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline as ligand

Pt(II) and Pd(II) complexes have emerged as highly potent anticancer drugs, and their efficacy in treating of various types cancer has garnered increasing attention. Herein, we report two trapezoid planar structures of promising complexes with 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline as a ligand. The structures of complexes 1 and 2 were studied by a series of characterizations, including single-crystal X-ray diffraction, thermogravimetric analysis and infrared spectroscopy. Fluorescence and UV–vis spectra, revealed the hypochromic phenomenon, which demonstrates the capacity of the complexes 1 and 2 interacting with ct-DNA. Furthermore, the ability of the complexes 1 and 2 to cut DNA was validated by the agar gel electrophoresis experiment. The cytotoxicity of 1 was assessed using flow cytometry analysis and morphology analysis. It is worth noting that, complex 1, had obvious advantages in inducing HeLa cell apoptosis, and the number of apoptotic cells (49.5 %) is significantly higher than that in the control group (1.6 %), after 24 h. Moreover, 1 can generate singlet oxygen and hydroxyl radical to damage cells under light irradiation, thus achieving an excellent to induced HeLa cells apoptosis. Overall, this work illustrated that complexes 1 and 2 are promising candidates for inducing apoptosis in HeLa cells.

In-vitro, in-silico studies on the proposed penasulfate A and its derivatives as dual inhibitors of α-glucosidases and human cancer cells

α-Glucosidase inhibitors have been receiving significant attention from the scientific community in recent years due to their potential in treating a variety of diseases, including diabetes, cancer, and obesity. The synthetic penasulfate A, a novel α-glucosidase inhibitor, displayed outstanding inhibitory activity against α-glucosidases G0660 and G5003 with IC50 values of 11.00 and 9.62 µM, respectively. Two derivatives of the synthetic penasulfate A, namely compound 2 and 3, also inhibited α-glucosidase G0660 with IC50 values of 1101.00 and 111.00 µM separately. Enzyme kinetic experiments showed penasulfate A acted as a mixed inhibitor against α-glucosidase. In addition, MTT cytotoxicity assay revealed that all three synthetic compounds exhibited potent anti-proliferative activity against eight human cancer cell lines, hepatocellular carcinoma cell line (HepG2), breast cancer cell line (MCF-7), colon cancer cell line (HCT116), colorectal cancer cell line (HT29), prostate cancer cell line (DU145), lung cancer cell line (A549), ovarian cancer cell line (SKOV-3) and cervical cancer cell line (HeLa), with IC50 values ranging from 0.04 to 6.77 µM after treatment with compounds for 48 h. Cell morphology observations indicated that anti-proliferative effect of the compounds was induced by apoptosis, which was mostly associated with the late apoptosis state (Q2), confirmed by flow cytometry. Subsequent mechanistic investigation indicated that the synthetic compounds triggered Hela cell apoptosis by reducing its mitochondrial membrane potential, thus leading to an increase in intracellular levels of reactive oxygen species (ROS). Additionally, analysis of apoptosis-related factors verified that the synthetic compounds induced Hela cell apoptosis by upregulating the activity of the protein caspase-3. Furthermore, the binding affinity between compounds and relevant proteins was expressed in total scores and the pose of ligand from the docked complex was visualized using Surflex-Dock in SYBYL-X software. Molecular dynamic simulations were also applied to assess the stability of the targeting enzyme-compound 1 complex. These findings suggest that these compounds could offer promising new lead derivatives for further potential hypoglycemic and anticancer drug discovery.

Cyclometalated ruthenium (II) complexes induced HeLa cell apoptosis through intracellular reductive injury

The main challenge of cancer chemotherapy is the resistance of tumor cells to oxidative damage. Herein, we proposed a novel antitumor strategy: cyclic metal‑ruthenium (Ru) complexes mediate reductive damage to kill tumor cells. We designed and synthesized Ru(II) complexes with β-carboline as ligands: [Ru (phen)2(NO2-Ph-βC)](PF6) (RuβC-7) and [Ru(phen)2(1-Ph-βC)](PF6) (RuβC-8). In vitro experimental results showed that RuβC-7 and RuβC-8 can inhibit cell proliferation, promote mitochondrial abnormalities, and induce DNA damage. Interestingly, RuβC-7 with SOD activity could reduce intracellular reactive oxygen species (ROS) levels, while RuβC-8 has the opposite effect. Accordingly, this study identified the reductive damage mechanism of tumor apoptosis, and may provide a new ideas for the design of novel metal complexes.

Oridonin inhibits Hela cell proliferation via downregulation of glutathione metabolism: a new insight from metabolomics.

This study aims to elucidate Oridonin' s inhibitory mechanism to cervical cancer using metabolomics methods and pharmacological assays. Network pharmacology and KEGG pathway analysis are used to identify overlapped targets and involved metabolic pathways. UPLC-MS/MS metabolomics analysis is used to determine altered metabolites after Oridonin treatment. Other bioassays are also employed to uncover the changes in critical molecules that are highly related to altered metabolites. Seventy-five overlapped targets are identified between Oridonin and cervical cancer. Twenty-one metabolites involved in tricarboxylic acid cycle glutathione metabolism, branched-chain amino acid metabolism and so on changes significantly after Oridonin treatment. Oridonin treatment significantly reduces the content of cysteine and inhibit the catalytic activity of glutamine-cysteine ligase subunit, a rate-limiting enzyme for the synthesis of glutathione. As a result, the content of glutathione is also reduced. The antioxidant enzyme glutathione peroxidase 4 which uses glutathione as a cofactor, is inactivated, resulting in a burst release of reactive oxygen species. The ATP content is also significantly reduced in Hela cells after Oridonin treatment. This study finds that Oridonin treatment induces Hela cell apoptosis possibly via inhibition of the glutathione metabolism.

Novel Peptide from the Hydrolysate of Hybrid Sturgeon (Acipenseridae) Spinal Cord: Isolation, Identification, and Anti-proliferative Effects in Human Cervix Cancer Cells.

Anti-proliferative peptides have recently attracted attention for their excellent bioactivity and biocompatibility. In this paper, five novel anti-proliferative peptides were identified from the hydrolysate of hybrid sturgeon spinal cord (HSSC). In addition, the structure-activity relationship of the novel anti-proliferative peptides was explored. In vitro experiments indicated that the peptide "VDSVLDVVRK" presented the highest inhibition of HeLa cell growth in all samples (IC50 = 2.5 μM). VDSVLDVVRK showed a random coil secondary structure and nanomicelles in the tumor microenvironment. Transmission electron microscopy results confirmed that nanomicelles disassemble as the concentration of VDSVLDVVRK decreases. Furthermore, VDSVLDVVRK could induce HeLa cell apoptosis by increasing the expression of Cyt-c (98.65 ± 1.85%, p < 0.01) and caspase-9 (39.85 ± 1.81%, p < 0.01). In this study, the anti-proliferative mechanism of the HSSC peptide was discussed, which provided a theoretical basis for the research and development of anti-proliferative functional food.

Drimia calcarata Bulb Extracts Deactivate the PI3K Signalling Pathway in Cervical HPV-18 Positive HeLa Cells

• D. calcarata extracts promoted proliferation of HPV types 16 and 18 positive Ca-Ski cells through maintaining the expression of STAT3, CLB1 and CDC2 and maintaining the activated levels of PI3K. • Water extract induced apoptosis and G2/M cell cycle arrest of HeLa cells through increased expression of the tumour suppressor p53. • Both methanol and water extracts deactivated the PI3K signalling pathways in HeLa cells. • D. calcarata plant extracts could be useful for therapeutic purpose against human HPV-18 positive gynaecologic cancers. The efficacy of Drimia calcarata against the human cervical cancer remains unexplored and less understood. The present study focused on investigating the cytotoxic effect of D. calcarata bulb extracts on cervical cancer cells. The growth inhibitory effects of D. calcarata extracts were determined using the MTT, Ki67 and PI3K Activation assays. Apoptosis induction was assessed using fluorescence microscopy, the Muse Ⓡ Cell Analyser and gene expression analysis by RT-PCR. The cytotoxicity of the fractions was evaluated using MTT assay and the apoptosis induction using Muse Ⓡ Cell Analyser. Both methanol extract (ME) and water extract (WE) showed safety against noncancerous KMST-6 and HEK-293 cells. The extracts exhibited anticancer activity against HeLa, with no significant cytotoxic effect against the Ca-Ski cells. The WE increased the Ki67 positive Ca-Ski population, while both ME and WE arrested HeLa cells at G2/M phase, and Ca-Ski cells in G0/G1 phase. AO/EB staining, Annexin V and Caspase 3/7 Activation revealed that the extracts significantly induced apoptosis in HeLa cells. In HeLa cells, the ME downregulated TP53 variants, while WE upregulated both TP53 variants in HeLa cells. Both extracts decreased the STAT5A and STAT5B mRNA expression in HeLa cells; however, these extracts upregulated cancer-promoting STAT3 in Ca-Ski cells. Additionally, these extracts inactivated the PI3K signalling pathway in HeLa cells but not in Ca-Ski cells. The resistance of the Ca-Ski cells to the D. calcarata extracts may be due to the upregulation of STAT3 and activated PI3K signalling pathway. The cytotoxicity and apoptosis induction in HeLa cells by D. calcarata extracts may be attributed due to downregulation of STAT5A survival mechanisms. Water fractions 1, 2, 3 and 6 and methanol fractions 1 and 2 reduced cell viability of HeLa cells. Water fractions 2, 3 and 6 and methanol fractions 1 and 3 induced apoptosis, which was preceded by secondary necrosis. However, water fraction 1 and methanol fraction 2 led to most cells undergoing necrotic cell death. There are several compounds that can be credited with the anticancer activities of the ME and WE extracts since several fractions exhibited cytotoxicity against the HeLa cells. These findings suggest that the D. calcarata extracts have anticancer activities, and thus, could be useful for therapeutic purposes against human HPV-18 positive gynaecologic cancers.

α-Pinene inhibits the growth of cervical cancer cells through its proapoptotic activity by regulating the miR-34a-5p/Bcl-2 signaling axis.

Among gynecological tumors, cervical cancer (CC) has the second-highest prevalence and mortality rate. α-Pinene is a bicyclic monoterpenoid compound extracted from pine needles that carried promising anticancer properties. Nevertheless, its effect on CC and the underlying mechanism has not yet been elucidated. Therefore, we investigated the effect of α-Pinene on apoptosis in CC via in vitro assays of flow cytometry (FCW), terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL) assay, quantitative real-time polymerase chain reaction(qRT-PCR), and Western blot. Following that, we detected the proapoptotic function of α-Pinene on HeLa cells in vivo by TUNEL assay and immunofluorescence staining. Our results displayed that the α-Pinene inhibited the growth of HeLa cells and stalled the cells in the G0/G1 phase. Interestingly, we also detected that α-Pinene induced HeLa cells to apoptosis. The results investigated that α-Pinene induced HeLa cells apoptosis along with up-regulating the expression of Bax, Bid, caspase-9, caspase-3, miR-34a-5p, and down-regulating the expression of Bcl-2 in vitro. At the same time, the expression levels of target genes in vivo were consistent with those in vitro. Our experiment proved that α-Pinene promoted apoptosis, which will be used to hopefully maximize the therapeutic strategies in clinical studies in CC.

Gelsemium elegans cyclic peptide induces human cervical carcinoma cells apoptosis through intrinsic and extrinsic pathways.

Four novel Gelsemium elegans cyclic peptides (GEPs) were isolated in an antihuman cervical carcinoma activity tracking method, and their amino acid sequences were identified. The GEP-1 cyclic-(Trp-Leu-His-Val)-peptide inhibited HeLa cell proliferation in a dose- and time-dependent manner. GEP-1 induced intracellular reactive oxygen species (ROS) overproduction and induced HeLa cells apoptosis in a caspase-dependent manner. GEP-1 also induced collapse of the mitochondrial membrane potential and promoted the mitochondrial release of cytochrome c (cyt c), apoptosis-inducing factor (AIF), and endonuclease G (Endo G) in HeLa cells. Furthermore, GEP-1 triggered the extrinsic death receptor-dependent pathway, which was characterized by activating Fas and FADD. Notably, GEP-1 is a potential antihuman cervical carcinoma peptide.

Warangalone Induces Apoptosis in HeLa Cells via Mitochondria‐Mediated Endogenous Pathway

Cervical cancer as one of the major malignant tumors seriously threatens women's health. More than 270,000 women die of cervical cancer each year. Warangalone is an isoflavone compound isolated from Cudrania tricuspidata with excellent antitumor activity. In this research, we investigated the molecular mechanism of warangalone‐induced apoptosis in HeLa cells. The results show that warangalone can selectively and effectively inhibit HeLa cells proliferation. Warangalone can effectively inhibit the invasion and migration of HeLa cells. Furthermore, warangalone was confirmed to activate p53 and mitogen‐activated protein kinase (MAPK) family signaling pathways to cause apoptosis. In this case, the expression of the B‐cell lymphoma‐2 (Bcl‐2) family is regulated, and caspase‐3 is eventually cleaved, finally triggering the mitochondrial apoptosis. In conclusion, warangalone can induce HeLa cells apoptosis via a mitochondria‐mediated endogenous pathway, which represented the potential therapeutic effect of warangalone on cervical cancer.

Osthole Induces Apoptosis and Inhibits Proliferation, Invasion, and Migration of Human Cervical Carcinoma HeLa Cells.

Purpose To study the effect of osthole extract on proliferation, migration, invasion, and apoptosis of human cervical carcinoma HeLa cells and investigate its underlying mechanism. Methods HeLa cells were exposed to osthole at various concentrations. Cell viability, migration, and invasion were detected by MTT assay, scratch wound-healing assay, and invasion assay, respectively. The proportion of cells undergoing apoptosis was analyzed by flow cytometry. Western blot and RT-qPCR were performed to determine changes in the expression of key factors in the Wnt/β-catenin signaling pathway. Results The osthole extract effectively inhibited the proliferation, migration, and invasion potential of HeLa cells in a dose-dependent manner. The rate of apoptosis induction in HeLa cells treated with the osthole extract for 48 h was significantly higher than that of the untreated controls. Outcomes of the western blotting analysis and RT-qPCR showed that the expression of β-catenin, c-Myc, cyclin D1, survivin, and MMP-9 was significantly inhibited. Conclusion Osthole could significantly inhibit the malignant behavior of HeLa cells and induce cellular apoptosis. Inactivation of the Wnt/β-catenin signaling pathway by osthole may be a mechanism to control cancer metastasis.

Studies on the Mechanism of Alloimperatorin on the Proliferation and Apoptosis of HeLa Cells.

Alloimperatorin is a compound extracted from the traditional Chinese medicine (Angelica dahurica), which has exhibited anticancer activity. However, its precise molecular mechanism of anticancer remains unclear. Alloimperatorin-induced apoptosis of cervical cancer cells and its molecular mechanism were investigated in the present study. Cholecystokinin octapeptide (CCK-8) was employed to evaluate the cytotoxicity of alloimperatorin on HeLa, SiHa, and MS-751 cells. Flow cytometry was used to assess apoptosis induced by alloimperatorin. The mechanism of apoptosis was verified by mitochondrial membrane potential, Western blotting, and fluorescent PCR. The results of the study showed that alloimperatorin reduced the activity of HeLa cells. The calculated IC50 at 48 hours was 116.9 μM. Compared with the control group, alloimperatorin increased the apoptotic rate of HeLa cells and reduced the mitochondrial membrane potential of HeLa cells. The Western blot results showed that alloimperatorin promotes the expression of caspase3, 8, 9 and that Bax apoptotic proteins reduce PARP expression, procaspase3, 8, 9, and BCL-2 proteins and reduces the cyt-c in the mitochondria expression. The results demonstrated that alloimperatorin can induce HeLa cell apoptosis through mitochondria and extrinsic apoptotic pathways.

Prenylated flavonoids from Ficus hirta induces HeLa cells apoptosis via MAPK and AKT signaling pathways

A pair of undescribed enantiomers, (±) ficusflavonid A (1a/1b), along with five known analogues, were isolated from the roots of Ficus hirta. Their structures were determined by the analysis of extensive spectroscopic data (including UV, IR, HRESIMS and NMR). Two enantiomers (1a and 1b) were successfully separated by chiral chromatographic column and their absolute configurations were assigned by the comparison of experimental and calculated ECD data. The cytotoxicity of all the isolates against HeLa, MCF-7, HepG2 and H460 cell lines were evaluated by MTT assay. Among them, 4 suppressed the proliferation of HeLa cells with the IC50 value of 28.88 μM. Furthermore, the apoptotic effect of 4 on HeLa cells and the level of several crucial proteins in AKT/MAPKs signaling pathways were analyzed by flow cytometer and western blot assay. As a result, 4 induced HeLa cell apoptosis in a dose dependent manner and significantly increased the protein levels of p-JNK and p-p38, whereas distinctly reduced the expression of p-AKT, and p-ERK. Thus, compound 4 might induce HeLa cells apoptosis via MAPK and AKT signaling pathways, which could be considered as a potential leading compound for the development of anticancer drugs.

Studies on apoptosis induced by B-norcholesteryl benzimidazole compounds in HeLa cells

Certain B-norcholesteryl benzimidazole compounds were found to mediate marked anti-tumor proliferative effects in vitro in our earlier study. Here, the mechanism of action of these anti-tumor effects was evaluated using HeLa human cervical cancer cells. Methods for detecting cell invasion and migration, Annexin V-PI double staining, cell cycle status, and mitochondrial membrane potential Δψm were employed. These compounds were confirmed to significantly inhibit the proliferation of HeLa cells in vitro. Compound 1 induced apoptosis in S phase, compound 2induced apoptosis in the G0/G1 phase and compound 3 induced late apoptosis in the G2/M phase. These compounds induced HeLa cell apoptosis through depolarization of mitochondrial membrane potential Δψm in a dose-dependent manner. B-norcholesteryl benzimidazole compounds induced morphological changes in HeLa cells and inhibited proliferation, invasion and metastasis. Apoptosis was promoted by mechanisms involving p21 and p53 in this cervical cancer cell line.

Geographical variations of triterpenoid contents in Cyclocarya paliurus leaves and their inhibitory effects on HeLa cells

This research investigated the variations of triterpenoid contents in Cyclocarya paliurus leaves and their underlying anticancer activity and mechanism. Leaf samples were collected from twelve geographical locations in China for the experiments. Significant variation in the triterpenoid content and growth inhibition on HeLa cells were observed among the C. paliurus populations. Antiproliferation effects of the leaf samples were significantly affected by the pentacyclic triterpenoids contents except for oleanolic acid. Among the pentacyclic triterpenoids, hederagenin exhibited the best anticancer activity with IC50 of 17.42 μg/mL. Based on the results, pentacyclic triterpenoids may induce HeLa cell apoptosis via mitochondrial pathway. RNA sequencing indicated that the ACTB, ATF4, TUBA1B and TUBA1C genes might be the key ones involved in the apoptosis induction by cyclocaric acid B and hederagenin. These results provided useful information for application of C. paliurus in functional beverage and pharmaceutical industries.

Synthesis, characterization, crystal structure, DNA and human serum albumin interactions, as well as antiproliferative activity of a Cu(II) complex containing a Schiff base ligand formed in situ from the Cu(II)‐induced cyclization of 1,5‐bis(salicylidene)thiocarbohydrazide

The reaction of Cu(OAc)2·H2O with o‐HOC6H4‐C(H)N‐NH‐C(S)‐NH‐NC(H)‐C6H4OH‐o in methanol resulted in the formation of a dinuclear Cu(II) complex [(o‐phen)Cu(HL)Cu(HL)] (1) in the solution where HL2− is a new O−NN− ligand formed in situ from cyclization of the Schiff base and the isolated product was found to be a dimer of 1 forming a tetranuclear compound in solid due to axial coordination of bridging phenolate O− as revealed from X‐ray crystallography. Electronic spectroscopic study using calf thymus DNA as well as fluorescence quenching study of ethidium bromide bound DNA in presence of 1 showed intercalative DNA binding with Kb = 1.12 × 105 M−1 and Kapp = 8.80 × 105 M−1, respectively. This compound was found to be efficient in hydrolytic as well as oxidative DNA cleavage. The binding interaction between complex 1 and human serum albumin (HSA) was investigated using fluorescence emission, absorption, and molecular docking studies. 3D fluorescence studies revealed that HSA structure was altered at secondary and tertiary levels. The binding constant Ka value (~106 M−1) suggested a strong binding affinity of 1 to HSA, and the bimolecular quenching constant kq value (1013 M−1 s−1) suggested a static quenching mechanism operative in the quenching of the intrinsic fluorescence of the protein. Such high kq value may also be accounted by Förster resonance energy transfer (FRET) process. The distance r (3.22 nm) between donor HSA and acceptor complex 1 calculated from FRET theory suggested that they are close to each other. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay with human cervical cancer HeLa cells and human breast cancer MCF7 cells (IC50 = 2.2 and 1.2 μM, respectively) suggested remarkable in vitro anticancer activity of 1. Cell death in HeLa cells was induced via apoptosis as revealed from nuclear staining assays using Hoechst 33342 and acridine orange/propidium iodide dual staining, and apoptosis induction in HeLa cells happened through reactive oxygen species accumulation.

Development of poly ADP-ribose polymerase-1 inhibitor with anti-cervical carcinoma activity

This research aimed to discover and identify new poly ADP-ribose polymerase-1 (PARP) inhibitors with potent anti-cervical carcinoma activity, and then explore their potential biological roles on cervical carcinoma cell. For this purpose, we identified a new PARP inhibitor from a high-throughput virtual screening method and found that the compound strongly inhibited cervical carcinoma HeLa cell. Cell proliferation was evaluated by an MTT assay, and the cell apoptosis was assessed by flow cytometry. Results showed that PARP1 is a poly ADP-ribose catalyzing enzyme in eukaryotic cells, which is activated during DNA damage and repair, and plays an important role in DNA repair and cell apoptosis. Herein we report the first discovery of a new PARP inhibitor from a high-throughput virtual screening method, then the compound was measured its anti-cervical carcinoma activity by using an MTT assay, which suggested that the compound strongly inhibited HeLa cell proliferation, the IC50 value is 0.65 µM. In addition, the compound induced HeLa cell apoptosis in a dose-response manner. All these data suggested that the compound is a promising lead compound, which deserves further investigation. It is concluded that the compound discover herein is a promising PARP-1 inhibitor with potent anti-cervical carcinoma activity, which deserves further investigation.

Inducing Tumor Cell Apoptosis: Mediated Survivin miRNA by Ultrasound and Cationic Lipid Contrast Agent.

Non-viral delivery systems is a promising method for gene or drug delivery. Polyethyleneimine (PEI) is a double-edged sword. It internalizes itself into the cell through endocytosis and promotes gene transfer efficiency. However, the strong positive charge also makes PEI highly toxic to cells. Ultrasound-targeted microbubble destruction (UTMD) is a promising non-viral method for gene and drug delivery, but its efficiency still needs to be improved. The aim of this study was to explore a system that combines ultrasound with non-viral gene delivery for the treatment of cervical cancer HeLa cells. In this study, we synthesized a kind of cationic ultrasound contrast agent(CUCA) that the physical and chemical properties, gene carrying capacity and cytotoxicity were verified. On the basis of previous studies, we further optimized the following transfusion parameters including ultrasound parameters, microbubble concentration, plasmid concentration, cell density and other parameters. The experiment was designed to compare the following six groups: (1) Plasmid group (P group), plasmid 15 μg; (2) PEI + plasmid group (PEI + P group),1 μl of PEI containing 10 nmol nitrogen and 1 μg of DNA containing 3 nmol phosphate for a PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection; (3) Ultrasound + plasmid group (US + P), plasmid 15 μg; (4) Ultrasound + cationic liposomal ultrasound contrast agent + plasmid group (UTMD + P group), plasmid 15 μg and cationic liposomal ultrasound contrast agent 5%; (5) Ultrasound + cationic liposomal ultrasound contrast agent + PEI + plasmid group (UTMD + PEI + P group), PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection and cationic liposomal ultrasound contrast agent 5%; and (6) Blank group, no treatment), The influence on Hela cells was observed under microscope, the efficiency of apoptosis was measured by flow cytometry, and cell viability was tested in CCK 8. The optimized transfection parameters can improve the transfection efficiency of ultrasound combined with C-UCA to a certain extent, but its transfection efficiency is still lower than that of branched polyethyleneimine (bPEI) 25 kDa. By investigating the effect of HeLa cells apoptosis induced by UTMD in combination with PEI mediated survivin miRNA, we found that both PEI alone and ultrasound in combination with CUCA were able to transfect cells with survivin miRNA to effectively induce HeLa cell apoptosis. However, the synergistic effect between the two methods was not significant. In contrast, the combined use of ultrasound, C-UCA and PEI may significantly reduce the transfection efficiency of UTMD and PEI, and the specific mechanism remains to be further studied.

Molecular Detection of Cordycepin-Induced HeLa Cell Apoptosis with Surface-Enhanced Raman Spectroscopy

Cordycepin, extracted from the medicinal mushroom Cordyceps militaris, was shown to induce cancer cell apoptosis. Yet its anticancer function was limitedly evaluated, and the mechanism was not entirely elucidated. In this study, we demonstrated the effectiveness of cordycepin in inducing apoptosis in HeLa cells and investigated its apoptosis-inducing mechanism through label-free surface-enhanced Raman spectroscopy (SERS). SERS spectral changes revealed detailed molecular changes in both early and late stages of apoptosis. Importantly, SERS characteristic peaks at 805 and 1438 cm−1, which were assigned to RNA, continued to decrease significantly from early to late apoptosis stages. It indicated that cordycepin induced HeLa cell apoptosis mainly through interfering with RNA production, potentially by restraining the translation of RNA encoding ribosomal proteins. Meanwhile, apoptotic cells and their apoptosis stage could be easily differentiated by SERS-based principal component analysis (PCA). Furthermore, the morphological changes of early and late-stage apoptotic cells were illustrated by differential interference contrast and fluorescence microscopic imaging. Therefore, the natural ingredient, cordycepin, could serve as a promising anticancer candidate. Our biological model of cell apoptosis in vitro, the non-invasive and rapid SERS technique, combined with PCA analysis, could be a powerful tool in the investigation of cell apoptosis mechanisms and anticancer drug screening.

Methyl protodioscin from Polygonatum sibiricum inhibits cervical cancer through cell cycle arrest and apoptosis induction

Methyl protodioscin (MPD) is a steroid saponin which has been well known for its pharmacological properties. Herein, we evaluated the anti-cancer activity of MPD for proliferation inhibition and apoptosis induction in Hela cells. MPD was purified from the rhizoma of Polygonatum sibiricum primarily and identified by HPLC, UPLC-TOF-MS/MS and NMR analysis, respectively. Results showed that MPD repressed cell proliferation at IC50 of 18.49 μM, altered cell morphology, arrested the cell cycle in G2/M phase, facilitated the generation of intracellular ROS and led to cell apoptosis in a concentration-dependent manner. Furthermore, MPD treatment promoted death receptor pathway and mitochondrial pathway efficiently. The inhibition of Caspase-8 and Caspase-9 proteins in these pathways abolished the apoptosis significantly, further demonstrated the mechanism of MPD-induced apoptosis. These findings offer novel information that MPD may be considered as a possible natural anti-cancerous agent in the form of functional foods or medicinal products.