Chelerythrine Chloride Downregulates beta-Catenin and Inhibits Stem Cell Properties of Non-Small Cell Lung Carcinoma

Plant secondary metabolites have been seen as alternatives to seeking new medicines for treating various diseases. Phytochemical scientists remain hopeful that compounds isolated from natural sources could help alleviate the leading problem in oncology-the lung malignancy that kills an estimated two million people annually. In the present study, we characterized a medicinal compound benzophenanthridine alkaloid, called chelerythrine chloride for its anti-tumorigenic activities. Cell viability assays confirmed its cytotoxicity and anti-proliferative activity in non-small cell lung carcinoma (NSCLC) cell lines. Immunofluorescence staining of β-catenin revealed that there was a reduction of nuclear content as well as overall cellular content of β-catenin after treating NCI-H1703 with chelerythrine chloride. In functional characterizations, we observed favorable inhibitory activities of chelerythrine chloride in cancer stem cell (CSC) properties, which include soft agar colony-forming, migration, invasion, and spheroid forming abilities. Interesting observations in chelerythrine chloride treatment noted that its action abides to certain concentration-specific-targeting behavior in modulating β-catenin expression and apoptotic cell death. The downregulation of β-catenin implicates the downregulation of CSC transcription factors like SOX2 and MYC. In conclusion, chelerythrine chloride has the potential to mitigate cancer growth due to inhibitory actions toward the tumorigenic activity of CSC in lung cancer and it can be flexibly adjusted according to concentration to modulate specific targeting in different cell lines

Identification of Phytochemical-Based beta-Catenin Nuclear Localization Inhibitor in NSCLC:Differential Targeting Population from Member of Isothiocyanates

Decades of research has convinced us that phytochemical compounds contained within the plant products are the real deal, and they provide benefits such as health maintenance an d cure to illnesses. One of the deadliest noncommunicable diseases today is lung cancer, hence its disease management still deserves attention. Wnt/beta-catenin pathway activation conferring cancer stem cell (CSC) activities to non-small cell lung carcinomas (NSCLCs) may explain why the disease is still difficult to cure. In the present study, we assessed several representatives of phytochemical categories consisting of alkaloids, chalcones and isothiocyanates for their inhibitory activity to nuclear localization of beta-catenin-an important event for Wnt/beta-catenin pathway activation, in lung cancer cell lines. Real-time cell analyzer confirmed that evodiamine (EVO), chelidonine (CHE), isoliquiritigenin (ISO), licochalcone-A (LICO), benzyl isothiocyanate (BI) and phenethylisothiocyanate (PI) exhibited anti-proliferative activities and cytotoxicities to adenocarcinoma cell line SK-LU-1 and human lung CSC primary cell line (HLCSC). Immunofluorescence assay identified that CHE, ISO, LICO, BI and PI were capable of reducing the number of cells harboring beta-catenin within the nuclei of these cells. We extended the characterizations of BI and PI in Wnt-dependent squamous cell carcinoma cell line NCI-H1703 on several CSC functions and found that BI was better at inhibiting soft agar colony formation as an output of self-renewal ability, whereas PI was more effective in inhibiting the growth of multicellular tumor spheroid model mimicking micrometastases. Both however were not able to inhibit migration and invasion of NCI-H1703. In conclusion, BI could potentially be used as a safer alternative to target undifferentiated CSCs as adjuvant therapy, whereas PI could be used as chemotherapy to remove bulk tumor

Apoptosis effect of girinimbine isolated from Murraya koenigii on lung cancer cells in vitro

Murraya koenigii Spreng has been traditionally claimed as a remedy for cancer. The current study investigated the anticancer effects of girinimbine, a carbazole alkaloid isolated from Murraya koenigii Spreng, on A549 lung cancer cells in relation to apoptotic mechanistic pathway. Girinimbine was isolated from Murraya koenigii Spreng. The antiproliferative activity was assayed using MTT and the apoptosis detection was done by annexin V and lysosomal stability assays. Multiparameter cytotoxicity assays were performed to investigate the change in mitochondrial membrane potential and cytochrome c translocation. ROS, caspase, and human apoptosis proteome profiler assays were done to investigate the apoptotic mechanism of cell death. The MTT assay revealed that the girinimbine induces cell death with an IC50 of 19.01 μM. A significant induction of early phase of apoptosis was shown by annexin V and lysosomal stability assays. After 24 h treatment with 19.01 μM of girinimbine, decrease in the nuclear area and increase in mitochondrial membrane potential and plasma membrane permeability were readily visible. Moreover the translocation of cytochrome c also was observed. Girinimbine mediates its antiproliferative and apoptotic effects through up- and downregulation of apoptotic and antiapoptotic proteins. There was a significant involvement of both intrinsic and extrinsic pathways. Moreover, the upregulation of p53 as well as the cell proliferation repressor proteins, p27 and p21, and the significant role of insulin/IGF-1 signaling were also identified. Moreover the caspases 3 and 8 were found to be significantly activated. Our results taken together indicated that girinimbine may be a potential agent for anticancer drug development

Chloroform Fraction of Centratherum anthelminticum (L.) Seed Inhibits Tumor Necrosis Factor Alpha and Exhibits Pleotropic Bioactivities: Inhibitory Role in Human Tumor Cells

We investigated the antioxidant potential, cytotoxic effect, and TNF-α inhibition activity with NF-κB activation response in a chloroform fraction of Centratherum anthelminticum seeds (CACF). The antioxidant property of CACF was evaluated with DPPH, ORAC, and FRAP assays, which demonstrated significant antioxidant activity. The cytotoxicity of CACF was tested using the MTT assay; CACF effective inhibitory concentrations (IC50) for A549, PC-3, MCF-7, and WRL-68 cells were 31.42 ± 5.4, 22.61 ± 1.7, 8.1 ± 0.9, and 54.93 ± 8.3 μg/mL, respectively. CACF effectively and dose-dependently inhibited TNF-α release, in vitro and in vivo. CACF inhibited TNF-α secretion in stimulated RAW264.7 macrophage supernatants with an IC50 of 0.012 μg/mL, without affecting their viability; the highest dose tested reduced serum TNF-α by 61%. Acute toxicity testing in rats revealed that CACF was non-toxic at all doses tested. Matching the cytotoxic activity towards a mechanistic approach, CACF dose-dependently exhibited in vitro inhibitory effects against the activation of NF-κB translocation in MCF-7 cells. Preliminary phytochemical screening with GC/MS analysis detected 22 compounds in CACF, of which morpholinoethyl isothiocyanate was the most abundant (29.04%). The study reveals the potential of CACF in the treatment of breast cancer and in oxidative stress conditions with associated inflammatory responses

In vitro anti-inflammatory, cytotoxic and antioxidant activities of boesenbergin A, a chalcone isolated from Boesenbergia rotunda (L.) (fingerroot)

The current in vitro study was designed to investigate the anti-inflammatory, cytotoxic and antioxidant activities of boesenbergin A (BA), a chalcone derivative of known structure isolated from Boesenbergia rotunda. Human hepatocellular carcinoma (HepG2), colon adenocarcinoma (HT-29), non-small cell lung cancer (A549), prostate adenocarcinoma (PC3), and normal hepatic cells (WRL-68) were used to evaluate the cytotoxicity of BA using the MTT assay. The antioxidant activity of BA was assessed by the ORAC assay and compared to quercetin as a standard reference antioxidant. ORAC results are reported as the equivalent concentration of Trolox that produces the same level of antioxidant activity as the sample tested at 20 µg/mL. The toxic effect of BA on different cell types, reported as IC50, yielded 20.22 ± 3.15, 10.69 ± 2.64, 20.31 ± 1.34, 94.10 ± 1.19, and 9.324 ± 0.24 µg/mL for A549, PC3, HepG2, HT-29, and WRL-68, respectively. BA displayed considerable antioxidant activity, when the results of ORAC assay were reported as Trolox equivalents. BA (20 µg/mL) and quercetin (5 µg/mL) were equivalent to a Trolox concentration of 11.91 ± 0.23 and 160.32 ± 2.75 µM, respectively. Moreover, the anti-inflammatory activity of BA was significant at 12.5 to 50 µg/mL and without any significant cytotoxicity for the murine macrophage cell line RAW 264.7 at 50 µg/mL. The significant biological activities observed in this study indicated that BA may be one of the agents responsible for the reported biological activities of B. rotunda crude extract

In Vitro and In Vivo Anti-Angiogenic Activities of Panduratin A

Targeting angiogenesis has emerged as an attractive and promising strategy in anti-cancer therapeutic development. The present study investigates the anti-angiogenic potential of Panduratin A (PA), a natural chalcone isolated from Boesenbergia rotunda by using both in vitro and in vivo assays.PA exerted selective cytotoxicity on human umbilical vein endothelial cells (HUVECs) with IC(50) value of 6.91 ± 0.85 µM when compared to human normal fibroblast and normal liver epithelial cells. Assessment of the growth kinetics by cell impedance-based Real-Time Cell Analyzer showed that PA induced both cytotoxic and cytostatic effects on HUVECs, depending on the concentration used. Results also showed that PA suppressed VEGF-induced survival and proliferation of HUVECs. Furthermore, endothelial cell migration, invasion, and morphogenesis or tube formation demonstrated significant time- and dose-dependent inhibition by PA. PA also suppressed matrix metalloproteinase-2 (MMP-2) secretion and attenuated its activation to intermediate and active MMP-2. In addition, PA suppressed F-actin stress fiber formation to prevent migration of the endothelial cells. More importantly, anti-angiogenic potential of PA was also evidenced in two in vivo models. PA inhibited neo-vessels formation in murine Matrigel plugs, and angiogenesis in zebrafish embryos.Taken together, our study demonstrated the distinctive anti-angiogenic properties of PA, both in vitro and in vivo. This report thus reveals another biological activity of PA in addition to its reported anti-inflammatory and anti-cancer activities, suggestive of PA's potential for development as an anti-angiogenic agent for cancer therapy

(−)-Kunstleramide, a New Antioxidant and Cytotoxic Dienamide from the Bark of Beilschmiedia kunstleri Gamble

A new dienamide, (2E,4E)-7-(3',4'-dimethoxyphenyl)-N-ethyl-6-(R)-hydroxyhepta- 2,4-dienamide, named (-)-kunstleramide (1), were isolated from the bark of Beilschmiedia kunstleri Gamble together with one neolignan: (+)-kunstlerone (2) and seven known alkaloids: (+)-nornuciferine (3), (-)-isocaryachine (4), (+)-cassythicine (5), (+)-laurotetanine (6), (+)-boldine (7), noratherosperminine (8), (+)-N-demethylphyllocaryptine (9). Their structures were established from spectroscopic techniques, most notably 1D- and 2D-NMR, UV, IR, OR, circular dichroism (CD) spectra and LCMS-IT-TOF. (-)-Kunstleramide (1) exhibited very poor dose-dependent inhibition of DPPH activity, with an IC50 value of 179.5 ± 4.4 μg/mL, but showed a moderate cytotoxic effect on MTT assays of A375, A549, HT-29, PC-3 and WRL-68 with EC50 values of 64.65, 44.74, 55.94, 73.87 and 70.95 µg/mL, respectively

Panduratin A, a Possible Inhibitor in Metastasized A549 Cells through Inhibition of NF-Kappa B Translocation and Chemoinvasion

In the present study, we investigated the effects of panduratin A (PA), isolated from Boesenbergia rotunda, on apoptosis and chemoinvasion in A549 human non-small cell lung cancer cells. Activation of the executioner procaspase-3 by PA was found to be dose-dependent. Caspase-3 activity was significantly elevated at the 5 µg/mL level of PA treatment and progressed to a maximal level. However, no significant elevated level was detected on procaspase-8. These findings suggest that PA activated caspase-3 but not caspase-8. Numerous nuclei of PA treated A549 cells stained brightly by anti-cleaved PARP antibody through High Content Screening. This result further confirmed that PA induced apoptotic cell death was mediated through activation of caspase-3 and eventually led to PARP cleavage. Treatment of A549 cells with PA resulted in a strong inhibition of NF-κB activation, which was consistent with a decrease in nuclear levels of NF-κB/p65 and NF-κB/p50 and the elevation of p53 and p21. Besides that, we also showed that PA significantly inhibited the invasion of A549 cells in a dose-dependent manner through reducing the secretion of MMP-2 of A549 cells gelatin zymography assay. Our findings not only provide the effects of PA, but may also be important in the design of therapeutic protocols that involve targeting of either p53 or NF-κB