Bacteriophage-based particles carrying the TNF-related apoptosis-inducing ligand (TRAIL) gene for targeted delivery in hepatocellular carcinoma.

The TRAIL (Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand) is a promising candidate for cancer treatment due to its unique ability to selectively induce programmed cell death, or apoptosis, in cancer cells while sparing healthy ones. This selectivity arises from the preferential binding of the TRAIL to death receptors on cancer cells, triggering a cascade of events that lead to their demise. However, significant limitations in using the TRAIL for cancer treatment are the administration of the TRAIL protein that can potentially lead to tissue toxicity (off-target) and the short half-life of the TRAIL in the body which may necessitate frequent and sustained administration; these can pose logistical challenges for long-term treatment regimens. We have devised a novel approach for surmounting these limitations by introducing the TRAIL gene directly into cancer cells, enabling them to produce the TRAIL locally and subsequently trigger apoptosis. A novel gene delivery system such as a bacteriophage-based particle TPA (transmorphic phage/AAV) was utilized to address these limitations. TPA is a hybrid M13 filamentous bacteriophage particle encapsulating a therapeutic gene cassette with inverted terminal repeats (ITRs) from adeno-associated viruses (AAVs). The particle also showed a tumour targeting ligand, CDCRGDCFC (RGD4C), on its capsid (RGD4C.TPA) to target the particle to cancer cells. RGD4C selectively binds to αvß3 and αvß5 integrins overexpressed on the surface of most of the cancer cells but is barely present on normal cells. Hepatocellular carcinoma (HCC) was chosen as a model because it has one of the lowest survival rates among cancers. We demonstrated that human HCC cell lines (Huh-7 and HepG2) express αvß5 integrin receptors on their surface. These HCC cells also express death receptors and TRAIL-binding receptors. We showed that the targeted TPA particle carrying the transmembrane TRAIL gene (RGD4C.TPA-tmTRAIL) selectively and efficiently delivered the tmTRAIL gene to HCC cells resulting in the production of tmTRAIL from transduced cells and subsequently induced apoptotic death of HCC cells. This tumour-targeted particle can be an excellent candidate for the targeted gene therapy of HCC.

Brazilein inhibits epithelial-mesenchymal transition (EMT) and programmed death ligand 1 (PD-L1) expression in breast cancer cells.

Triple-negative breast cancer (TNBC) exhibits high levels of Epithelial-mesenchymal transition (EMT) and Programmed death ligand 1 (PD-L1) expression, which promotes immune escape and metastasis. Brazilein is a natural compound extracted from Caesalpinia sappan L., and has been demonstrated to be an anti-inflammatory anti- proliferative and apoptosis-inducer in various cancer cells. Here, we investigated the effect of brazilein on EMT and PD-L1 expression in breast cancer cells and its related molecular mechanisms using MCF-7 and MDA-MB-231 cells as a model. Since the AKT, NF-κB, and GSK3ß/ß-catenin signaling were reported to be important mechanisms in immune escape and metastasis, the effect of brazilein on these signaling pathways were also found out in our study. Firstly, brazilein was treated on breast cancer cells at various concentrations to study cell viability, apoptosis, and apoptosis proteins. Then, breast cancer cells were treated with non-toxic concentrations of brazilein to study its influence on EMT and expression of PD-L1 protein using MTT, flow cytometry, western blot, and wound healing analysis, respectively. We found that brazilein exerts an anti-cancer effect by reducing cell viability via induction of apoptosis, while it also downregulated EMT and PD-L1 through suppression of phosphorylation of AKT, NF-κB, and GSK3ß/ß-catenin. Moreover, the migration ability was diminished by inhibiting the activation of MMP-9 and MMP-2. Taken together, brazilein might delay cancer progression through inhibition of EMT, PD-L1, and metastasis suggesting it might be a potential therapeutic option in breast cancer patients having a high level of EMT and PD-L1.

Anticancer Activities of Hesperidin via Suppression of Up-Regulated Programmed Death-Ligand 1 Expression in Oral Cancer Cells.

Up-regulated expression of programmed death-ligand 1 (PD-L1) by interferon-gamma (IFN-γ) has been associated with promotion of cancer cell survival and tumor cell escape from anti-tumor immunity. Therefore, a blockade of PD-L1 expression can potentially be used as a molecular target for cancer therapy. The aim of this study was to investigate whether suppression of IFN-γ induced PD-L1 expression in two oral cancer cell lines, HN6 and HN15, by hesperidin effectively decreased cell proliferation and migration. Further, our objective was to elucidate the involvement of the signal transducer and activator of transcription 1 (STAT1) and STAT3 in the inhibition of induced PD-L1 expression by hesperidin. Our findings indicate that IFN-γ induced expression of PD-L1 protein in HN6 and HN15 via phosphorylation of STAT1 and STAT3 and that hesperidin significantly reduced that induction through suppression of phosphorylated STAT1 and STAT3 in both cell lines. Moreover, hesperidin also significantly decreased the viability, proliferation, migration, and invasion of both cell lines. In conclusion, hesperidin exerted anticancer effects against oral cancer cells through the suppression of PD-L1 expression via inactivation of the STAT1 and STAT3 signaling molecules. The findings of this study support the use of hesperidin as a potential adjunctive treatment for oral cancer.

Atorvastatin Attenuates Programmed Death Ligand-1 (PD-L1) Induction in Human Hepatocellular Carcinoma Cells.

Liver cancer is the sixth most common cancer worldwide with high morbidity and mortality. Programmed death ligand 1 (PD-L1) is a major ligand of programmed death 1 receptor (PD1), and PD1/PD-L1 checkpoint acts as a negative regulator of the immune system. Cancers evade the host's immune defense via PD-L1 expression. This study aimed to investigate the effects of tumor-related cytokines, interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) on PD-L1 expression in human hepatocellular carcinoma cells, HepG2. Furthermore, as atorvastatin, a cholesterol-lowering agent, is documented for its immunomodulatory properties, its effect on PD-L1 expression was investigated. In this study, through real-time RT-PCR, Western blot, and immunocytochemistry methods, PD-L1 expression in both mRNA and protein levels was found to be synergistically upregulated in HepG2 by a combination of IFNγ and TNFα, and STAT1 activation was mainly responsible for that synergistic effect. Next, atorvastatin can inhibit the induction of PD-L1 by either IFNγ alone or IFNγ/TNFα combination treatment in HepG2 cells. In conclusion, in HepG2 cells, expression of PD-L1 was augmented by cytokines in the tumor microenvironment, and the effect of atorvastatin on tumor immune response through inhibition of PD-L1 induction should be taken into consideration in cancer patients who have been prescribed atorvastatin.

Inhibition of programmed death ligand 1 (PD-L1) expression in breast cancer cells by sesamin.

Programmed death ligand 1 (PD-L1) is overexpressed in some metastatic breast cancer subtypes, specifically triple-negative breast cancer (TNBC). This feature can assist in the eradication of anti-tumor immunity, thereby enhancing the survival of the tumor. This study aims to explore how sesamin affects PD-L1 expression in breast cancer cells and its related molecular mechanisms. We found high levels of expression of PD-L1 in both mRNA and protein levels in the TNBC cell line, MDA-MB231, but not in the luminal type-breast cancer cell line, MCF-7. We then demonstrated the tumor suppressive effect of sesamin, which induced the inhibition of cell proliferation in MDA-MB231 cells. Additionally, sesamin triggered PD-L1 downregulation (both mRNA and protein) through the inhibition of AKT, NF-κB and JAK/Stat signaling in MDA-MB231 cells. Moreover, the migration ability of MDA-MB231 cells was effectively diminished by sesamin via inhibition of the activation of MMP-9 and MMP-2. In summary, this study demonstrated that sesamin suppresses MDA-MB231 breast cancer cells' proliferation and migration; and decreases the expression of PD-L1 via the downregulation of AKT, NF-κB, and JAK/Stat signaling. Therefore, sesamin may be an effective alternative and novel therapeutic option for immunotherapy in breast cancer cells with high PD-L1 expression.

Inhibitory Effect of Hesperidin on the Expression of Programmed Death Ligand (PD-L1) in Breast Cancer.

Programmed death ligand 1 (PD-L1) is overexpressed in the most aggressive breast cancer subtype, triple-negative breast cancer (TNBC), assisting the eradication of antitumor immunity, and thereby enhancing the survival of the tumor. This study explored how hesperidin affects PD-L1 expression, and thereby cancer progression in breast cancer cells. We found that MDA-MB231, the triple-negative breast adenocarcinoma cancer cell line, (high aggressiveness) has higher expression, in both mRNA and protein, of PD-L1 than that of the other breast cancer cell line, MCF-7 (low aggressiveness). Hesperidin inhibited cell proliferation in MDA-MB231 cells. Additionally, high expression of PD-L1 (both mRNA and protein) in aggressive cancer cells was strongly inhibited by hesperidin through inhibition of Akt and NF-κB signaling. Moreover, hesperidin treatment, by inhibiting activation of matrix metalloproteinases such as MMP-9 and MMP-2, suppressed the metastatic phenotype and cell migration in the PD-L1 high-expressing MDA-MB231 cells. In summary, hesperidin inhibits breast cancer cell growth through the inhibition of the expression of PD-L1 via downregulation of Akt and NF-κB signaling in TNBC. Moreover, hesperidin significantly suppresses cell migration of MDA-MB231 cells. Our findings reveal fresh insights into the anticancer effects of hesperidin which might have potential clinical implications.

Alpha-mangostin, an active compound in Garcinia mangostana, abrogates anoikis-resistance in human hepatocellular carcinoma cells.

Anoikis-resistance is a critical step in cancer progression, especially during the process of metastasis. During this phase, the cancer phenotype that causes cell survival in detachment conditions, drug resistance, and epithelial-to-mesenchymal transition (EMT) is altered. Inhibition of anoikis-resistance can potentially be the molecular target in cancer therapy. Alpha-mangostin, an active compound in Garcinia mangostana, has been reported for its cell-death induction and its chemosensitizing and anti-metastatic properties in many cancer cell types, such as ovarian cancer, lung cancer, and hepatocellular carcinoma. We, therefore, have investigated whether alpha-mangostin could sensitize anoikis in human hepatocellular carcinoma (HepG2). The established anoikis-resistant HepG2 displayed more aggressive malignant behaviors, including rapid proliferation, doxorubicin resistance, up-regulated anti-apoptotic protein levels, and EMT phenotype. Alpha-mangostin significantly sensitized anoikis in HepG2 through the inhibition of cell survival by induced caspase-9, caspase-8 and caspase-3 activities, increased pro-apoptotic protein (Bax, Bim, t-Bid) levels, and decreased anti-apoptotic protein (c-FLIP, Mcl-1) levels. Besides, alpha-mangostin significantly reduced cell re-adhesion and migration, matrix metalloproteinases-2 (MMP-2) and MMP-9 secretions, and EMT-involved protein (N-cadherin, αV, ß1 integrin, and vimentin) expressions. AKT and ERK signaling pathways were dramatically suppressed, which indicated that alpha-mangostin inhibited anoikis-resistance via the inhibition of these pathways in HepG2. These findings support the development of alpha-mangostin to be used in the treatment of anoikis-resistant liver cancer.

Induction of Human Hepatocellular Carcinoma HepG2 Cell Apoptosis by Naringin.

Naringin, a bioflavonoid found in Citrus seeds, inhibits proliferation of cancer cells. The objectives of this study were to investigate the mode and mechanism(s) of hepatocellular carcinoma HepG2 cell death induced by naringin. The cytotoxicity of naringin towards HepG2 cells proved dosedependent, measured by MTT assay. Naringintreated HepG2 cells underwent apoptosis also in a concentration related manner, determined by annexin Vfluorescein isothiocyanate (FITC) and propidium iodide (PI) employing flow cytometry. Mitochondrial transmembrane potential (MTP) measured using 3,3'dihexyloxacarbocyanine iodide (DiOC6) and flow cytometer was reduced concentrationdependently, which indicated influence on the mitochondrial signaling pathway. Caspase3, 8 and 9 activities were enhanced as evidenced by colorimetric detection of paranitroaniline tagged with a substrate for each caspase. Thus, the extrinsic and intrinsic pathways were linked in human naringintreated HepG2 cell apoptosis. The expression levels of proapoptotic Bax and Bak proteins were increased whereas that of the antiapoptotic BclxL protein was decreased, confirming the involvement of the mitochondrial pathway by immunoblotting. There was an increased expression of truncated Bid (tBid), which indicated caspase8 proteolysis activity in Bid cleavage as its substrate in the extrinsic pathway. In conclusion, naringin induces human hepatocellular carcinoma HepG2 cell apoptosis via mitochondriamediated activation of caspase9 and caspase8mediated proteolysis of Bid. Naringin anticancer activity warrants further investigation for application in medical treatment.

Hesperidin from Citrus seed induces human hepatocellular carcinoma HepG2 cell apoptosis via both mitochondrial and death receptor pathways.

Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was in a dose-dependent manner, and IC50 levels were determined. The sensitivity of human HepG2 cells was as follows: hesperidin > naringin > neohesperidin > naringenin. Hesperidin induced HepG2 cells to undergo apoptosis in a dose-dependent manner as evidenced by the externalization of phosphatidylserine and determined by annexin V-fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Hesperidin did not induce the generation of reactive oxygen species, which was determined by using 2',7'-dichlorohydrofluorescein diacetate and flow cytometry method. The number of hesperidin-treated HepG2 cells with the loss of mitochondrial transmembrane potential increased concentration dependently, using 3,3'-dihexyloxacarbocyanine iodide employing flow cytometry. Caspase-9, -8, and -3 activities were activated and increased in hesperidin-treated HepG2 cells. Bcl-xL protein was downregulated whereas Bax, Bak, and tBid protein levels were upregulated after treatment with hesperidin in a dose-dependent manner. In conclusion, the bioflavanone from Citrus seeds, hesperidin, induced human HepG2 cell apoptosis via mitochondrial pathway and death receptor pathway. Citrus seed flavonoids are beneficial and can be developed as anticancer drug or food supplement, which still needs further in vivo investigation in animals and human beings.

Enterocarpam-III induces human liver and breast cancer cell apoptosis via mitochondrial and caspase-9 activation.

An aristolactam-type alkaloid, isolated from Orophea enterocarpa, is enterocarpam-III (10-amino-2,3,4,6- tetramethoxyphenanthrene-1-carboxylic acid lactam). It is cytotoxic to various human and murine cancer cell lines; however, the molecular mechanisms remain unclear. The aims of this study were to investigate cytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells compared to normal murine fibroblast NIH3T3 cells. Cell viability was determined by MTT assay to determine IC10, IC20 and IC50 levels, reactive oxygen species (ROS) production with 2',7'-dichlorohydrofluorescein diacetate and the caspase-3, -8 and -9 activities using specific chromogenic (p-nitroaniline) tetrapeptide substrates, viz., DEVD-NA, IETD-NA and LEHD-NA and employing a microplate reader. Mitochondrial transmembrane potential (MTP) was measured by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC6) and using flow cytometry. The compound was cytotoxic to HepG2 and MDA-MB-231 cells with the IC50 levels of 26.0±4.45 and 51.3±2.05 µM, respectively. For murine normal fibroblast NIH3T3 cells, the IC50 concentration was 81.3±10.1 µM. ROS production was reduced in a dose-response manner in HepG2 cells. The caspase-9 and -3 activities increased in a concentration-dependent manner, whereas caspase-8 activity did not alter, indicating the intrinsic pathway activation. Enterocarpam-III decreased the mitochondrial transmembrane potential (MTP) dose-dependently in HepG2 cells, suggesting that the compound induced HepG2 cell apoptosis via the mitochondrial pathway. In conclusion, enterocarpam-III inhibited HepG2 and MDA-MB-231 cell proliferation and induced human HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway and induction of caspase-9 activity.

Cytotoxic aporphine alkaloids from leaves and twigs of Pseuduvaria trimera (Craib).

From ethyl acetate-methanol extracts of leaves and twigs of Pseuduvaria trimera a new aporphine alkaloid; 8-hydroxy-1,4,5-trimethoxy-7-oxoaporphine or 8-hydroxyartabonatine C (1) was isolated, together with the known 1,2,3-trimethoxy-4,5-dioxo-6a,7-dehydroaporphine (ouregidione, 2). Their structures were elucidated by a combination of spectral methods; mainly 2D NMR; IR and MS. Compounds 1 and 2 exhibited cytotoxic activity with IC50 values of 26.36±5.18 µM and 12.88±2.49 µM, respectively, for human hepatocellular carcinoma HepG2 cells, and 64.75±4.45 and 67.06±3.5 µM, respectively, for human breast cancer MDA-MB231 cells. Both compounds displayed anti-cancer activity but less than that of doxorubicin; a conventional chemotherapeutic drug, the IC50 levels of which were 2.21±1.72 and 1.83±0.09 µM for HepG2 and MDA-MB231 cells, respectively.

Cytotoxic and apoptotic-inducing effects of purple rice extracts and chemotherapeutic drugs on human cancer cell lines.

Pigmented rice is mainly black, red, and dark purple, and contains a variety of flavones, tannin, polyphenols, sterols, tocopherols, γ-oryzanols, amino acids, and essential oils. The present study evaluated the cytotoxic effects of purple rice extracts (PREs) combined with chemotherapeutic drugs on human cancer cells and mechanisms of cell death. Methanolic (MeOH) and dichloromethane (DCM) extracts of three cultivars of purple rice in Thailand: Doisaket (DSK), Nan and Payao (PYO), were tested and compared with white rice (KK6). Cytotoxicity was determined by 3-(4, 5-dimethyl)-2, 5-diphenyltetrazolium bromide (MTT) assay in human hepatocellular carcinoma HepG2, prostate cancer LNCaP and murine normal fibroblast NIH3T3 cells. MeOH-PYO-PRE was the most cytotoxic and inhibited HepG2 cell growth more than that of LNCaP cells but was not toxic to NIH3T3 cells. When PREs were combined with paclitaxel or vinblastine, they showed additive cytotoxic effects on HepG2 and LNCaP cells, except for MeOH-PYO-PRE which showed synergistic effects on HepG2 cells when combined with vinblastine. MeOH-PYO-PRE plus vinblastine induced HepG2 cell apoptosis with loss of mitochondrial transmembrane potential (MTP) but no ROS production. MeOH-PYO-PRE-treated HepG2 cells underwent apoptosis via caspase-9 and-3 activation. The level of γ-oryzanol was highest in DCM-PYO-PRE (44.17 mg/g) whereas anthocyanin content was high in MeOH-PYO-PRE (5.80 mg/g). In conclusion, methanolic Payao purple rice extract was mostly toxic to human HepG2 cells and synergistically enhanced the cytotoxicity of vinblastine. Human HepG2 cell apoptosis induced by MeOH-PYO-PRE and vinblastine was mediated through a mitochondrial pathway.

Stigmalactam from Orophea enterocarpa induces human cancer cell apoptosis via a mitochondrial pathway.

Stigmalactam, an aristolactam-type alkaloid extracted from Orophea enterocarpa, exerts cytotoxicity against several human and murine cancer cell lines, but the molecular mechanisms remain elusive. The aims of this study were to identify the mode and mechanisms of human cancer cell death induced by stigmalactam employing human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells as models, compared to normal murine fibroblasts. It was found that stigmalactam was toxic to HepG2 and MDA-MB-231 cells with IC50 levels of 23.0±2.67 µM and 33.2±4.54 µM, respectively, using MTT assays. At the same time the IC50 level towards murine normal fibroblast NIH3T3 cells was 24.4±6.75 µM. Reactive oxygen species (ROS) production was reduced in stigmalactam-treated cells dose dependently after 4 h of incubation, indicating antioxidant activity, measured by using 2',7',-dichlorohydrofluorescein diacetate and flow cytometry. Caspase-3 and caspase-9 activities were increased in a dose response manner, while stigmalactam decreased the mitochondrial transmembrane potential dose-dependently in HepG2 cells, using 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, indicating mitochondrial pathway-mediated apoptosis. In conclusion, stigmalactam from O. enterocarpa was toxic to both HepG2 and MDA-MB-231 cells and induced human cancer HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway.

Sawtehtetronenin from Goniothalamus sawtehii and its cytotoxicity.

A new acetogenin has been isolated from the ethyl acetate extract of leaves and twigs of G. sawtehii (Annonaceae). The structure of compound 1 was identified as sawtehtetronenin on the basis of spectral evidence (UV, IR, MS and 1H, and 13C NMR) and by comparison with related compounds. Sawtehtetronenin was found to be cytotoxic to human hepatocellular carcinoma HepG2 and breast cancer MDA-MB231 cells with IC50 values of 79.3 ± 11.9 µM and 108.1 ± 1.5 µM, respectively. Compound 1 was less toxic to both cell lines when compared with camptothecin, a chemotherapeutic drug.

Methoxyflavone derivatives modulate the effect of TRAIL-induced apoptosis in human leukemic cell lines.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various tumor cells, but does not affect normal cells or human leukemic cells, such as MOLT-4 and U937 cells, which are relatively resistant to TRAIL. Three flavonoids extracted from the rhizome of K. parviflora were 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF) and 3,5,7,3',4'-pentamethoxyflavone (PMF), and synthetic flavonoids including 5-methoxyflavone (5-MF) and 2'-methoxyflavone (2"-MF) were chosen for testing in this study. The aims of this study were to examine whether the treatment of TRAIL-resistant leukemia MOLT-4 and U937 cells, with methoxyflavone derivatives could enhance the apoptotic response and to identify the mechanism involved. METHODS: The cytotoxic effect of methoxyflavone (MF) derivatives in MOLT-4, U937 and peripheral blood mononuclear cells (PBMCs) was analyzed by the MTT assay. The induction of apoptosis and the reduction of mitochondrial transmembrane potential (ΔΨm) after staining with annexin V FITC and propidium iodide (PI), and 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)), respectively, were performed using flow cytometry. ROS production was determined by staining with 2',7'-dichlorofluorescin diacetate and processed with a flow cytometer. DR4, DR5, cFLIP, Mcl-1, BAX and Bid expression were demonstrated by immunoblotting. Caspase-8 and -3 activities were determined by using IETD-AFC and DEVD-AFC substrates and the fluorescence intensity was measured. RESULTS: All methoxyflavone derivatives were cytotoxic to MOLT-4, U937 cells and PBMCs, except DMF, TMF and PMF were not toxic to PBMCs. All MF derivatives induced human leukemic MOLT-4 cell apoptosis, but not in U937 cells. Percentage of MOLT-4 cells with (ΔΨm) was increased when treated with DMF, TMF, PMF, 5-MF and 2'-MF in the presence of TRAIL. 5-MF and 2'-MF enhanced TRAIL-induced apoptosis through the up-regulation of both DRs and the down-regulation of cFLIP and Mcl-1. Bid was cleaved and BAX was up-regulated, followed by the activation of caspase-8 and -3. Oxidative stress was also increased. 2'-MF gave the same result compared with 5-MF but with a less effect. CONCLUSION: Methoxyflavone derivatives enhanced TRAIL-induced apoptosis in human leukemic MOLT-4 cells through the death receptors and mitochondrial pathways.