Dichloromethane fraction from Gardenia jasminoides: DNA topoisomerase 1 inhibition and oral cancer cell death induction

Context: A growing body of evidence shows that compounds of plant origin have the ability to prevent cancer. The fruit of gardenia, Gardenia jasminoides Ellis (Rubiaceae), has long been used as a food additive and herbal medicine, and its pharmacological actions, such as protective activity against oxidative damage, cytotoxic effect, and anti-inflammatory and anti-tumor activity, have already been reported.

Objective: The purpose of the present study was to investigate the presence of DNA topoisomerase 1 inhibitor in various solvent fractions of Gardenia extract and examine the induction of oral cancer cell death upon treatment with Gardenia extract.

Materials and methods: The methanol extract of Gardenia was partitioned with n-hexane, dichloromethane, ethyl acetate, n-butanol, and water.

Results: In the DNA topoisomerase 1 assay, n-hexane and dichloromethane fractions inhibited topoisomerase 1 and led to a decrease in the cell viability of KB cells. The dichloromethane fraction (0.1?mg/mL) also showed 77% inhibition of cell viability in KB cells compared with HaCaT cells. Treatment with dichloromethane fraction led to apoptotic cell death as evidenced by flow cytometric analysis and morphological changes. In addition, treatment with Gardenia extract dichloromethane fraction led to the partial increase of caspase-3, caspase-8 and caspase-9 activities and the cleavage of poly (ADP-ribose) polymerase.

Conclusion: Taken together, these results suggest that the dichloromethane fraction from Gardenia extract induces apoptotic cell death by DNA topoisomerase 1 inhibition in KB cells. These findings suggest the possibility that Gardenia extract could be developed as an anticancer modality.     

Ethyl acetate extract from Glycosmis parva leaf induces apoptosis and cell-cycle arrest by decreasing expression of COX-2 and altering BCL-2 family gene expression in human colorectal cancer HT-29 cells

Abstract

Context: Glycosmis parva Craib (Rutaceae) is reported to have cytotoxic and anti-inflammatory activities by decreasing COX-2 expression.

Objective: To investigate the effect of G. parva on human colorectal cancer cells expressing COX-2, HT-29 cells.

Materials and methods: HT-29 cells were treated with ethyl acetate extract from the leaves of G. parva (GPE 6.25–100?µg/ml) for 24–72?h. Cell viability was evaluated by the resuzurin reduction assay. An apoptotic study was performed using annexinV/FITC-PI staining. The cell-cycle pattern was investigated by PI staining. The expression of BCL-2 family genes was analyzed by quantitative RT-PCR and expression of cyclins and COX-2 were done by RT-PCR.

Results: GPE at 6.25–100?µg/ml reduced HT-29 cell viability with IC₅₀ values of 69.49, 55.89, and 48.94?µg/ml at 24, 48, and 72?h, respectively. HT-29 apoptosis was induced by 18.23% at 100?µg/ml. Cells in S phase decreased by 5.22% and 13.28% at 50 and 100?µg/ml, respectively, causing G0/G1 (10.6% at 50?µg/ml) and G2/M (15.67% at 100?µg/ml) accumulation. GPE at 50?µg/ml downregulated cyclin A (11.46%), cyclin E (17.98%), BCL-2 (0.32-fold), and COX-2 (29.06%) expression with an increased BAK expression (1.79-fold).

Discussion and conclusion: GPE reduced HT-29 cell viability, inhibited cell proliferation, induced apoptosis, and arrested the cell cycle. Underlying mechanisms may involve decreases in COX-2, cyclin A, and cyclin E expression in addition to changes in BCL-2 family gene expression. Fundamental knowledge of GPE anticancer effects found in this study could lead to future use of this compound for colorectal cancer treatment.     

Potent anti-proliferative effects against oral and cervical cancers of Thai medicinal plants selected from the Thai/Lanna medicinal plant recipe database “MANOSROI III”

Abstract

Context: Thai/Lanna medicinal plant recipes have been used for the treatment of several diseases including oral and cervical cancers.

Objective: To investigate anti-proliferative activity on human cervical (HeLa) and oral (KB) cancer cell lines of medicinal plants selected from Thai/Lanna medicinal plant recipe database “MANOSROI III”.

Materials and methods: Twenty-three methanolic plant crude extracts were tested for phytochemicals and anti-proliferative activity on HeLa and KB cell lines for 24?h by the sulforhodamine B (SRB) assay at the doses of 1?×?10¹–1?×?10^?6?mg/ml. The nine extracts with the concentrations giving 50% growth inhibition (GI₅₀) lower than 100?µg/ml were further semi-purified by liquid/liquid partition in order to evaluate and enhance the anti-proliferative potency.

Results: All extracts contained steroids/triterpenoids, but not xanthones. The methanolic extracts of Gloriosa superba L. (Colchinaceae) root and Albizia chinensis (Osbeck) Merr. (Leguminosae–Mimosoideae) wood gave the highest anti-proliferative activity on HeLa and KB cell lines with the GI₅₀ values of 0.91 (6.0- and 0.31-fold of cisplatin and doxorubicin) and 0.16?µg/ml (28.78- and 82.29-fold of cisplatin and doxorubicin), respectively. Hexane and methanol–water fractions of G. superba exhibited the highest anti-proliferative activity on HeLa and KB cell lines with the GI₅₀ values of 0.15 (37- and 1.9-fold of cisplatin and doxorubicin) and 0.058?µg/ml (77.45- and 221.46-fold of cisplatin and doxorubicin), respectively.

Discussion and conclusion: This study has demonstrated the potential of plants selected from MANOSROI III database especially G. superba and A. chinensis for further development as anti-oral and cervical cancer agents.     

Protective effect of Salacia oblonga against tobacco smoke-induced DNA damage and cellular changes in pancreatic β-cells

Context: Tobacco smoking generates a tremendous amount of free radicals that induce oxidative stress (OS) in diabetics (pancreatic islet cells are defective). Salacia oblonga Wall. (Celastraceae) is a proven antioxidant and antidiabetic plant whose mechanism of action is yet to be explored.

Objective: The present study focuses on the protective ability of S. oblonga in tobacco smoke-induced oxidatively stressed pancreatic β-cell line.

Materials and methods: The RINm5f cell line was exposed to tobacco smoke concentrate (TSC) (0.5–10%, 24?h), plant extract (1–75?µg/ml, 3?h), and their combinations. Cell viability was determined through MTT assay. Microscopic analysis was carried out in unstained and nonyl acridine orange-stained cells. The effect of toxic doses of TSC on DNA integrity was analyzed through DNA fragmentation assay. The TSC-induced nitric oxide generation was determined spectrophototmetrically. The expression of anti-apoptotic protein Bcl-X under the above treatment conditions was carried out through RT-PCR.

Results: The LD₅₀ dose for TSC was found to be 1% TSC. Salacia oblonga extracts (10 and 15?µg/ml) were found to be optimum safe doses that significantly increased cell viability and decreased the nitric oxide production in TSC-treated cells. Pre-treatment with plant extract suppressed apoptosis through probable increase in the expression of anti-apoptotic protein Bcl-X in TSC-treated cells. Thus, the overall efficiency of plant extract in recovering cellular damage was proven.

Discussion and conclusion: The results suggest that TSC-induced cellular alterations are related to rise in nitric oxide and Bcl-X mRNA expression and propose that S. oblonga may confer significant cytoprotection against OS-mediated injury in β-cells.     

Antioxidant activity and cytotoxicity of Cyrtosperma johnstonii extracts on drug sensitive and resistant leukemia and small cell lung carcinoma cells

Context: The number of patients with cancer is increasing. New therapeutic agents to overcome drug-resistant tumors are urgently needed. Cyrtosperma johnstonii N.E. Br. (Araceae) is used for treatment of cancer in Thai traditional medicine. This study aimed to evaluate antioxidant activity and cytotoxicity of C. johnstonii extracts on human cancer cells.

Materials and methods: Dried powder of C. johnstonii rhizomes was extracted with several solvents. The 0.1?mg/ml extract solution was tested for antioxidant activity by 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. Color formation from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to determine cell viability. Standardization of the extract was performed by high-performance liquid chromatography (HPLC) with photodiode array detector at 254 and 360?nm. Cell cycle arrest was evaluated by flow cytometry after 5?min, 12?h and 24?h treated with 20 µg/ml of the acetone extract.

Results: The acetone extract exhibited the highest phenolic content and antioxidant activity (TEAC and EC values = 19.2?±?0.14 and 19.2?±?0.31?mM/mg, respectively). The IC₅₀ values for leukemia ranged from 11?±?1 to 29?±?3 µg/ml and from 5?±?2 to 6?±?0 µg/ml for small cell lung carcinoma cells. Cell cycle arrest occurred at the G2/M phase followed by apoptosis. HPLC analysis revealed that rutin is the major constituents of the extract.

Discussion and conclusion: The acetone extract of C. johnstoni is a promising source of natural antioxidants and anticancer. The extract inhibits cancer cells effectively with less effect on normal cells.     

Cytotoxic effects of four Caryophyllaceae species extracts on macrophage cell lines

Context: Saponins have been reported to possess antitumor properties, to inhibit angiogenesis and to induce tumor apoptosis.

Objective: To test the possible cytotoxic effect of crude extracts from four Caryophyllaceae species including Gypsophila paniculata L., Gypsophila trichotoma Wend., Saponaria officinalis L., and Dianthus sylvestris Wulffen on cultured monocyte/macrophage cell lines.

Materials and methods: After acid hydrolysis of the methanol-aqueous extracts, two representative prosaponins of the Caryophyllaceae, gypsogenin 3-O-glucuronide and quillaic acid 3-O-glucuronide were purified using solid-phase extraction (SPE), then identified by ultra-performance liquid chromatography–electrospray/mass spectrometry (UPLC-ESI/MS). Cytotoxic activity of the crude extracts at concentrations ranging from 0.1 to 200?µg/ml was evaluated on rat alveolar macrophage NR8383 and human monocytic THP-1 cell lines. Apoptosis was determined by measuring caspase-3 activity.

Results: Quantitative analysis by reversed-phase high-performance liquid chromatography (RP-HPLC) revealed a high content of gypsogenin 3-O-glucuronide in Gypsophila species roots (0.52–1.13% dry weight). At a concentration ≥10?µg/ml of crude extracts, a significant reduction of NR8383 and THP-1 cell lines viability was evidenced using the Trypan blue exclusion test. D. sylvestris extract exhibited the highest toxicity against THP-1 cells. Caspase-3 activation was evidenced after 4 and 24?h incubation of macrophages with 100?µg/ml of S. officinalis and G. trichotoma extracts, indicating apoptosis induction.

Discussion and conclusion: Crude extracts from the assayed species revealed cytotoxic effects toward macrophage cell lines. In Gypsophila species, gypsogenin 3-O-glucuronide derivatives could be responsible for the observed cytotoxicity. Therefore, crude extract of Caryophyllaceae is worth investigating for the potential development of agents against cancer cells.     

Schizandra chinensis extracts induce apoptosis in human gastric cancer cells via JNK/p38 MAPK activation and the ROS-mediated/mitochondria-dependent pathway

Abstract

Context: Schizandra chinensis Baill (Magnoliaceae) fruit extract (SCE) is considered a traditional herbal medicine for the treatment and alleviation of various diseases. Gastric cancer is the second most common cause of cancer-related death worldwide, and the first most common in Korea.

Objectives: This study investigates the mechanism of SCE-induced apoptosis in AGS human gastric cancer cells.

Materials and methods: SCE concentrations from 100 to 400?µg/ml were used. Cell viabilities were determined using MTT assay. Members of the Bcl-2 family and Bax were detected by Western blotting. RT-PCR was performed to measure the expression level of the Fas/FasL pro-apoptotic genes.

Results: SCE inhibited the proliferation AGS cells for 24 or 72?h (inhibition by 3.1%?±?5.2% at 100?µg/ml and 87.3%?±?7.6% at 400?µg/ml at 24?h and by 40.2%?±?5.3% 100?µg/ml and 95.3%?±?1.3% 400?µg/ml at 72?h) and increased the sub-G1 phase (25.3%?±?5.2% at 100?µg/ml and 370.2%?±?7.2% at 400?µg/ml) and the mitochondrial membrane depolarization (11.2%?±?2.1% at 100?µg/ml and 311.5%?±?6.1% at 400?µg/ml). The SCE-induced apoptotic cell death showed the down-regulation of Bcl-2, but up-regulation of Bax. Subsequently, SCE increased the expression level of Fas/FasL, activated caspase-9 and -3, and increased reactive oxygen species generation. Also, JNK II inhibitor or a p38 MAPK inhibitor inhibited SCE-induced cell death.

Discussion and conclusion: These results indicate that SCE might be an effective chemotherapeutic for the treatment of human gastric cancer.     

Evaluation of the cytotoxicity,cell-cycle arrest,and apoptotic induction by Euphorbia hirta in MCF-7 breast cancer cells

Context: Euphorbia hirta L. (Euphorbiaceae) has been used as a folk remedy in Southeast Asia for the treatment of various ailments.

Objective: The current study evaluates the cytotoxicity, cell-cycle arrest, and apoptotic induction by E. hirta in MCF-7 breast cancer cells.

Materials and methods: Cytotoxic activity of methanol extract of whole part of E. hirta was determined by the MTT assay at various concentrations ranging from 1.96 to 250.00?µg/mL in MCF-7 cells. Cell morphology was assessed by light and fluorescence microscopy. Apoptosis and cell-cycle distribution were determined by annexin V staining and flow cytometry. DNA fragmentation, caspase activity, and reactive oxygen species (ROS) assays were performed using the commercially available kits. To identify the cytotoxic fraction, E. hirta extract was subjected to bioassay-guided fractionation.

Results: Euphorbia hirta exhibited significant inhibition of the survival of MCF-7 cells and the half inhibitory concentration (IC₅₀) values was 25.26?µg/mL at 24?h. Microscopic studies showed that E. hirta-treated cells exhibited marked morphological features characteristic of apoptosis. Euphorbia hirta extract also had an ignorable influence on the LDH leakage and generating intracellular ROS. The flow cytometry study confirmed that E. hirta extract induced apoptosis in MCF-7 cells. Euphorbia hirta also resulted in DNA fragmentation in MCF-7 cells. Moreover, E. hirta treatment resulted in the accumulation of cells at the S and G₂/M phases as well as apoptosis. The caspase activity study revealed that E. hirta extract induced apoptosis through the caspase-3-independent pathway by the activation of caspase-2, 6, 8, and 9. Euphorbia hirta hexane fraction, namely HFsub4 fraction, demonstrated highest activity among all the fractions tested with an IC₅₀ value of 10.01?µg/mL at 24?h.

Discussion and conclusion: This study revealed that E. hirta induced apoptotic cell death and suggests that E. hirta could be used as an apoptosis-inducing anticancer agent for breast cancer treatment with further detailed studies.     

The effects of arctigenin on human rheumatoid arthritis fibroblast-like synoviocytes

Abstract

Context: Rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) play an important role in the initiation and progression of RA, which are resistant to apoptosis and proliferate in an anchorage-independent manner.

Objective: The effects of arctigenin on the proliferation and apoptosis of RAFLSs were explored.

Materials and methods: Arctigenin (0–160?µM) was used to treat RAFLSs for 48?h. Cell viability and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and annexin V/propidium iodide staining. Western blot analysis was performed to detect the changes in apoptosis-related genes.

Results and discussion: Arctigenin decreased cell viability by 23, 30, and 38% at the dose of 10, 20, and 30?µM, respectively. The half maximal inhibitory concentration (IC₅₀) of arctignein on RAFLSs was about 38?µM. Moreover, 9, 15, and 21% of RAFLSs are induced apoptosis by 10, 20, and 30?µM of arctigenin. The apoptotic response was due to the loss of mitochondrial membrane potential, coupled with the release of cytochrome C into cytoplasm, the up-regulation of pro-apoptotic protein, Bax, and down-regulation of antiapoptotic protein, B cell lymphoma 2 (Bcl-2). The activation of mitochondrial pathway in arctigenin-treated RAFLSs induced the cleavage of caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP). Additionally, arctigenin inhibited the nuclear translocation of p65, decreased the degradation of inhibitor of kappa B alpha (IκBα), and attenuated the phosphorylation of Akt.

Conclusion: Our results reveal that arctigenin inhibits cell proliferation and induces mitochondrial apoptosis of RAFLSs, which is associated with the modulation of NF-κB and Akt signaling pathways.     

Cytotoxicity of alkaloids isolated from Argemone mexicana on SW480 human colon cancer cell line

Context: Argemone mexicana Linn. (Papaveraceae) has been used as traditional medicine in India and Taiwan for the treatment of skin diseases, inflammations, bilious, fever, etc. Some alkaloids of A. mexicana have been screened for their cytotoxicity on different cancer cell lines.

Objective: The study investigates potential cytotoxic effects of alkaloids isolated from aerial part of A. mexicana on SW480 human colon cancer cell line.

Materials and methods: Six alkaloids, 13-oxoprotopine, protomexicine, 8-methoxydihydrosanguinarine, dehydrocorydalmine, jatrorrhizine, and 8-oxyberberine were isolated from the methanol extract of A. mexicana. Cytotoxicity of these alkaloids was studied on SW480 human colon cancer cell line at 1, 25, 50, 75, 100, 125, 150, and 200?µg/mL for 24 and 48?h. Cells were seeded in a 96-well micro-plate at a concentration of 2?×?10⁴ cells per well and MTS assay was performed to assess cytotoxicity in terms of cell viability.

Results: At 200?µg/mL, protomexicine and 13-oxoprotopine showed mild cytotoxicity (～24–28%) whereas dehydrocorydalmine exhibited moderate cytotoxicity (～48%). 8-Oxyberberine was mildly cytotoxic (～27%) at 24?h but was more potent (～76%) at 48?h. Jatrorrhizine and 8-methoxydihydrosanguinarine were most potent (～95–100%) in inhibiting the human colon cancer cell proliferation showing complete reduction in cell viability.

Discussion and conclusion: This is the first study on the effect of these alkaloids on SW480 human colon cancer cell line. This study indicates that some alkaloids of A. mexicana strongly inhibit the cell proliferation in human colon cancer cells, and it might be a basis for future development of a potent chemotherapeutic drug.     

Antiproliferative and apoptotic activities of extracts of Asclepias subulata

Abstract

Context: Asclepias subulata Decne. (Apocynaceae) is a shrub used in the Mexican traditional medicine for the treatment of cancer.

Objective: The objective of this study was to evaluate the antiproliferative activity of methanol extract of aerial parts of A. subulata and its fractions against different cancer cell lines. Additionally, we analyzed the mechanism of action of the active fractions.

Materials and methods: Methanol extract fractions were prepared by serial extraction with n-hexane, ethyl acetate, and ethanol. The antiproliferative activity of methanol extract and its fractions was evaluated, against several murine (M12.C3.F6, RAW 264.7, and L929) and human (HeLa, A549, PC-3, LS 180, and ARPE-19) cell lines by the MTT assay, using concentrations of 0.4–400?µg/mL for 48?h. Ethanol and residual fractions were separated using silica gel column. Apoptosis induction of cancer cells was evaluated by Annexin and JC-1 staining using flow cytometry.

Results: Methanol extract and its fractions showed antiproliferative activity against all human cancer cell lines tested. Methanol extract had the highest antiproliferative activity on A549 and HeLa cells (IC₅₀ values?<?0.4 and 8.7?µg/mL, respectively). Ethanol and residual fractions exerted significant antiproliferative effect on A549 (IC₅₀?<?0.4?µg/mL) and PC3 cells (IC₅₀ 1.4 and 5.1?µg/mL). Apoptotic assays showed that CEF7, CEF9, CRF6, and CRF5 fractions induced mitochondrial depolarization in A549 cells, 70, 73, 77, and 80%, respectively. Those fractions triggered the apoptosis mitochondrial pathway.

Conclusion: Our data show that A. subulata extracts have potent antiproliferative properties on human cancer cell lines. This plant should be considered an important source of potent anticancer compounds.     

Bioactivites of two common polyphenolic compounds: Verbascoside and catechin

Context: Natural products can present remarkable biological and pharmacological activities. In traditional medicine, plants have been used historically in treating cancer, infections, and other inflammatory conditions.

Objective: Verbascoside and catechin are widespread polyphenolic plant compounds that could play a role in the anti-inflammatory and health-promoting effects of plants and plant extracts.

Materials and methods: This study compares the potential cytotoxic effects of polyphenols verbascoside and catechin (6.25–200?µM) on human peripheral blood mononuclear cells (PBMC) for 48?h and myelomonocytic THP-1 and THP-1 Blue cells for 24?h. The effects of the compounds on immune activation markers such as indoleamine 2,3-dioxygenase (IDO) activity as well as on neopterin formation and nuclear factor-κB (NF-κB) activation were investigated. Cytotoxicity of the compounds was tested using Cell-Titer Blue assay.

Results: Verbascoside exhibited significant suppressive effects in mitogen-stimulated PBMC on tryptophan breakdown (>50?µM; IC₅₀ value: 58.6?µM) and the production of neopterin (>6.25?µM; IC₅₀ value: 217?µM). These effects correlated with a decline in cell viability, while THP-1 Blue cells were less sensitive. NF-κB activity was slightly enhanced at lower concentrations (<50?µM verbascoside) in stimulated cells and at the highest concentration used in unstimulated cells. Catechin had no relevant effects on cell viability and on the tested inflammation markers, except NF-κB activation in THP-1 Blue cells.

Discussion and conclusion: The results obtained show that verbascoside and catechin represent effective compounds which interfere with immunobiochemical pathways that are highly relevant for immunosurveillance and competing virus infections.     

In vitro evaluation of Spirulina platensis extract incorporated skin cream with its wound healing and antioxidant activities

Context: Algae have gained importance in cosmeceutical product development due to their beneficial effects on skin health and therapeutical value with bioactive compounds. Spirulina platensis Parachas (Phormidiaceae) is renowned as a potential source of high-value chemicals and recently used in skincare products.

Objective: This study develops and evaluates skin creams incorporated with bioactive S. platensis extract.

Materials and methods: Spirulina platensis was cultivated, the aqueous crude extract was prepared and in vitro cytotoxicity of S. platensis extract in the range of 0.001–1% concentrations for 1, 3 and 7?d on HS2 keratinocyte cells was determined. Crude extracts were incorporated in skin cream formulation at 0.01% (w/w) concentration and in vitro wound healing and genotoxicity studies were performed. Immunohistochemical staining was performed to determine the collagen activity.

Results: 0.1% S. platensis extract exhibited higher proliferation activity compared with the control group with 198% of cell viability after 3?d. Skin cream including 1.125% S. platensis crude extract showed enhanced wound healing effect on HS2 keratinocyte cell line and the highest HS2 cell viability % was obtained with this concentration. The micronucleus (MN) assay results indicated that S. platensis extract incorporated creams had no genotoxic effect on human peripheral blood cells. Immunohistochemical analysis showed that collagen 1 immunoreactivity was improved by increased extract concentration and it was strongly positive in cells treated with 1.125% extract incorporated skin cream.

Conclusions: The cell viability, wound healing activity and genotoxicity results showed that S. platensis incorporated skin cream could be of potential value in cosmeceutical and biomedical applications.     

Effect of shikonin on multidrug resistance in HepG2: The role of SIRT1

Abstract

Context: Overexpression of SIRT1 is considered to enhance the resistance of HepG2 cells to irradiation. Shikonin, a naturally occurring naphthoquinone compound, displays anticancer effects and circumvents cancer drug resistance.

Objectives: This study investigated the MDR reversal effect of shikonin induced by the overexpression of SIRT1.

Materials and methods: The overexpression of SIRT1 in HepG2 cells was established by lentivirus infection. Five days after transduction, real-time quantitative polymerase chain reaction and western blotting were used to detect the expression of SIRT1 and MDR1/P-gp. Drug resistance was also evaluated by flow cytometry after rhodamine-123 staining. On day 5, the multidrug resistance cells were treated by shikonin (10^?7, 10^?6, and 10^?5 µmol/L) one time. The cell viability was detected by the MTT assay, and apoptosis was evaluated by Hoechst 33342 staining and caspase-3 activity 24?h after shikonin treatment.

Results: Overexpression of SIRT1 decreased rhodamine-123 staining and successfully produced the R-HepG2 cell line. Compared with HepG2, the expression of MDR1/P-gp mRNA (3.45?±?0.35) and protein (1.40?±?0.05) were both upregulated in R-HepG2. Shikonin inhibited cell viability (from 93.9?±?2.1 to 66.7?±?1.5%), induced apoptosis of R-HepG2 (apoptotic ratio from 3.5?±?0.8 to 47.5?±?2.7%, caspase-3 activity from 103.5?±?1.9 to 329.2?±?14.9%, respectively), downregulated the mRNA and protein expression of SIRT1 and MDR1/P-gp, and decreased rhodamin 123 efflux.

Discussion and conclusion: In the present study, we demonstrated that shikonin is able to overcome drug resistance in hepatocellular carcinoma cells, and the mechanism is related to the SIRT1-MDR1/P-gp signaling pathway.     

Chan-Yu-Bao-Yuan-Tang induces apoptosis in NSCLC and SCLC cell lines via a mitochondria-mediated pathway

1. Previous clinical studies have shown efficacy and safety of the traditional Chinese medicinal herb extract Chan-Yu-Bao-Yuan-Tang (CYBYT) in lung cancer patients.

2. The effects of CYBYT on proliferation and apoptosis in the non-small cell lung cancer cell line NCI-H460 and the small cell lung cancer cell line NCI-H446 were investigated in vitro.

3. CYBYT significantly induced antiproliferative effects of NCI-H460 and NCI-H446 cells in a concentration- and time-dependent manner. The IC₅₀ values in NCI-H460 cells were 94.37 µg/mL (24?h) and 20.89 µg/mL (48?h), whereas in NCI-H446 cells IC₅₀ values were 214.72 µg/mL (24?h) and 114.58 µg/mL (48?h). Annexin V–FITC/PI staining showed CYBYT could significantly induce apoptosis in NCI-H460 and NCI-H446 cells, and the total apoptosis rates were positively correlated with the concentration and time of CYBYT treatment. Furthermore, treatment with the broad-spectrum caspase inhibitor (z-VAD-fmk) effectively protected NCI-H460 and NCI-H446 cells against CYBYT-triggered apoptosis. The apoptotic processes involved were a marked decrease in antiapoptotic protein Bcl-2 and an increase in proapoptotic protein Bax. The release of mitochondrial cytochrome c into the cytosol was also observed, which, in turn, resulted in the activation of caspase-9 and caspase-3.

4. CYBYT exerts antiproliferative and growth inhibition effects on NCI-H460 and NCI-H446 cells through the mitochondrial caspase-dependent cell death pathway.

     

Anticancer effect of 2,7-dihydroxy-3-methylanthraquinone on human gastric cancer SGC-7901 cells in vitro and in vivo

Context: 2,7-Dihydroxy-3-methylanthraquinone (DDMN) is reported to have a remarkable anticancer activity against gastric cancer SGC-7901 cells.

Objective: The objective of this study is to study the anticancer effect and mechanism of DDMN on SGC-7901 cells.

Materials and methods: The MTT assay was used to determine the effect of DDMN on cell viability of SGC-7901 cells, and the cytotoxic effect was evaluated by the IC₅₀ value. After treatment with different doses of DDMN (10, 20, and 40?μM) for 48?h, flow cytometry was used to investigate the apoptosis of SGC-7901 cells induced by DDMN. Further, western blotting was performed to study anticancer mechanism by assaying apoptosis-related proteins containing Mcl-1, Bcl-xl, Bcl-2, Bax, Bak, Bad, cytochrome c, caspase-3, and caspase-9. Finally, xenograft assay was used to further evaluate the effect of DDMN on SGC-7901 cells by determining body weight of nude mice, tumor volumes, and apoptosis-related proteins.

Results: These results suggest that DDMN can significantly inhibit (IC₅₀ value?=?20.92?μM) the proliferation of SGC-7901 cells and induce apoptosis of SGC-7901 cells demonstrated by flow cytometry analysis. Additionally, the results of western blotting indicated that DDMN can suppress the expression of anti-apoptotic proteins Bcl-xl and Bcl-2, increase the expression of pro-apoptotic proteins Bax, Bad (40?μM), caspase-3 and caspase-9, and evidently promote the release of cytochrome c from the mitochondria to the cytoplasm. The xenograft assay further confirmed that DDMN had significant anticancer effects on SGC-7901 cells.

Conclusion: DDMN had significant anticancer effect on SGC-7901 cells in vitro and in vivo related to mitochondria-mediated apoptosis.     

Wound healing activity of Pluchea indica leaf extract in oral mucosal cell line and oral spray formulation containing nanoparticles of the extract

Context: Pluchea indica (L.) Less (Asteraceae) is an herb used as a traditional medicine for wound healing. The chemical compounds found in Pluchea indica leaves are phenolic acids, flavonoids, anthocyanins and carotenoids.

Objective: This study investigates the effect of Pluchea indica leaf ethanol extract and its nanoparticles (NPs) on cytotoxicity, cell survival and migration of human oral squamous carcinoma cell line.

Materials and methods: Cell viability was measured using MTT assay to assess the effect of Pluchea indica leaf extract and NPs (1–500?μg/mL) on cytotoxicity and cell survival. The effect of Pluchea indica leaf extract and NPs on cell migration was determined by scratch assay. The % relative migration was calculated after 24, 48 and 72?h of treatment.

Results: The sizes of Pluchea indica leaf extract NPs were in a range of nanometers. NPs possessed negative charge with the polydispersity index (PDI) smaller than 0.3. After the treatment for 24, 48 and 72?h, Pluchea indica leaf extract had IC₅₀ value of 443.2, 350.9 and 580.5?μg/mL, respectively, whereas the IC₅₀ value of NPs after the treatment for 24, 48 and 72?h were 177.4, 149.2 and 185.1?μg/mL, respectively. The % relative migration of cells was significantly increased when the cells were treated with 62.5 and 125?μg/mL of the extract and 62.5?μg/mL of NPs.

Discussion and Conclusions: NPs increased cytotoxicity of the Pluchea indica leaf extract, increased the migration of cells at low concentration and increased colloidal stability of the extract in an oral spray formulation.     

Cytotoxic,antioxidant and antimicrobial effects of nine species of woundwort (Stachys) plants

Context: Woundwort (Stachys) plants from the Lamiaceae family have been used in folk medicine for various purposes.

Objective: This study was designed to analyze cytotoxic, antioxidant and antimicrobial activities of Stachys plants, because these fields have extensively benefited of drug discovery from natural sources.

Materials and methods: Nine Stachys plants were collected from different regions of Iran. Cytotoxic activities of methanol, 80% methanol and dichloromethane (DCM) extracts of these plants were assessed on three human cancer cell lines (HL-60, K562 and MCF-7 cells) with the MTT assay, while antioxidant and antimicrobial activities were determined on methanol extracts by DPPH and nutrient broth micro-dilution assays, respectively.

Results: DCM extract of St. pilifera Benth. had the lowest IC₅₀ in three cancer cell lines ranging from 33.1 to 48.2?µg/ml, followed by the 80% methanol extract of St. persica S.G.Gmel. ex C.A.Mey. (IC₅₀ range: 62.1–104.1?µg/ml) and DCM extract of St. byzantina C. Koch (IC₅₀ range: 62.7–131.0?µg/ml). St. byzantina. St. lavandulifolia Vahl., St. acerosa Boiss., St. obtusicrena Boiss. and St. persica showed lowest IC₅₀ values in the DPPH scavenging assay (135.1, 162.6, 164.7, 169.4 and 172.4?µg/ml, respectively), while their total phenolic contents were 23.9, 18.2, 18.6, 20.4, 27.8?mg equivalent of gallic acid in 1?g dry plant, respectively. The methanol extracts of St. byzantina and St. persica inhibited all six tested Gram-negative and Gram-positive bacterial strains.

Conclusion: Various Stachys species (especially St. byzantina and St. persica) are valuable sources of natural compounds with important biological properties.     

Potential nephroprotective effects of the Chinese herb Angelica sinensis against cisplatin tubulotoxicity

Abstract

Context: Acute kidney injury (AKI) is often encountered in patients receiving cisplatin (CisPt), a chemotherapeutic drug that induces numerous toxic side effects. Techniques used to limit nephrotoxicity during CisPt treatment are not fully effective; about a third of patients experience AKI. New nephroprotective strategies, including pharmacological approaches, must be developed.

Objective: The present study investigated the nephroprotective potential of Angelica sinensis (Oliv.) Diels (Apiaceae) root towards CisPt tubulotoxicity.

Materials and methods: HK-2 cells were incubated with CisPt (10?µM) and/or with a methanolic extract of A. sinensis (AS). Nephroprotective capacity was evaluated by means of cellular viability (resazurin assay) and apoptosis (annexin-V/PI staining), oxidative stress generation (H₂DCF-DA oxidation), Ki-67 index (immunofluorescence), cell cycle analysis (DNA staining), cell migration rate (scratch assay), extracellular matrix deposition (collagen determination), and β-catenin relocalization.

Results: CisPt decreased cell viability [76% versus Ctrl], which was associated with an increased apoptosis. Simultaneous treatment with 50?µg/ml AS enhanced cell survival [84% versus Ctrl] and decreased the apoptosis rate. AS could not alleviate CisPt-induced oxidative stress; but doses of 5 and 50?µg/ml raised the Ki-67 index [135 and 244% versus Ctrl] and cell migration rates [1.2 and 1.3-fold versus Ctrl]. Finally, both doses of AS limited the amount of collagen deposition [121.6 and 119.6% for 5 and 50?µg/ml, respectively, versus 131.0% for CisPt-treated cells] and prevented the relocalization of β-catenin from the membrane to the nucleus.

Conclusion: These results confirm the nephroprotective potential of A. sinensis and require further investigations aiming at identifying its active compounds.