Scabraside D Extracted from Holothuria scabra Induces Apoptosis and Inhibits Growth of Human Cholangiocarcinoma Xenografts in Mice

Scabraside D, a sulfated triterpene glycoside extract from sea cucumber Holothulia scabra, shows various biological activities, but effects on human cholangiocarcinoma cells have not previously been reported. In the present study, we investigated the activity of scabraside D against human cholangiocarcinoma (HuCCA) both in vitro and for tumor growth inhibition in vivo using a xenograft model in nude mice. Scabraside D (12.5-100 μg/mL) significantly decreased the viability and the migration of the HuCCA cells in a dose-dependent manner, with 50% inhibitory concentration (IC50) of 12.8 ± 0.05 μg/mL at 24 h. It induced signs of apoptotic cells, including shrinkage, pyknosis and karyorrhetic nuclei and DNA fragmentation on agarose gel electrophoresis. Moreover, by quantitative real-time PCR, scabraside D effectively decreased Bcl 2 while increasing Bax and Caspase-3 gene expression levels suggesting that the scabraside D could induce apoptosis in HuCCA cells. In vivo study demonstrated that scabraside D (1 mg/kg/day, i.p. for 21 days) significantly reduced growth of the HuCCA xenografts without adverse effects on the nude mice. Conclusively, scabraside D induced apoptosis in HuCCA cells and reduced the growth of HuCCA xenographs model. Therefore, scabraside D may have potential as a new therapeutic agent for cholangiocarcinoma.     

Cytotoxicity,Cell Cycle Arrest,and Apoptosis Induction Activity of Ethyl-p-methoxycinnamate in Cholangiocarcinoma Cell

Objective: To investigate cytotoxic activity of ethyl-p-methoxycinnamate (EPMC) including its effect on p-glycoprotein (multidrug resistance-1: mdr-1 gene) in human cholangiocarcinoma cell. Methods: Cytotoxic activity of EPMC against human cholangiocarcinoma (CL-6), fibroblast (OUMS-36T-1F), and colon cancer (Caco-2) cell lines were assessed using MTT assay. Selectivity index (SI) was determined as the ratio of IC50 (concentration that inhibits cell growth by 50%) of EPMC in OUMS-36T-1F and that in CL-6 cell. Cell cycle arrest and apoptosis in CL-6 cells were investigated by flow cytometry and fluorescent microscopy. Effect of EPMC on mdr-1 gene expression in CL-6 and Caco-2 was determined by real-time PCR. Results: The median (95% CI) IC50 values of EPMC in CL-6, OUMS-36T-1F, and Caco-2 were 245.5 (243.1-266.7), 899.60 (855.8-966.3) and 347.0 (340.3-356.9) µg/ml, respectively. The SI value of the compound for the CL-6 cell was 3.70. EPMC at IC50 inhibited CL-6 cell division and induced apoptosis compared to untreated control. EPMC exposure did not induce mdr-1 gene expression in both CL-6 and Caco-2 cells. Conclusion: The results suggest the potential role of EPMC in cholangiocarcinoma with a low possibility of drug resistance induction.     

Scabraside D Derived from Sea Cucumber Induces Apoptosis and Inhibits Metastasis via iNOS and STAT-3 Expression in Human Cholangiocarcinoma Xenografts

Scabraside D, a sulfated triterpene glycoside, was extracted from the sea cucumber Holothuria scabra. It shows anti-proliferation in many of cancer cell lines, but the function and mechanisms of action of scabraside D in human cholangiocarcinoma (HuCCA) have not previously determined. In this study, we investigated the activity of scabraside D on HuCCA cell apoptosis, lymphangiogenesis and metastasis in a nude mouse model. Scabraside D induced signs of apoptosis, such as cell shrinkage, nuclear condensation, nuclear fragmentation and DNA fragmentation on TUNEL assays, while effectively decreasing expression of BCl-2 but increasing caspase-3 gene level expression. Immunohistochemistry revealed that scabraside D significantly reduced lymphatic vessel density (LVD). Moreover, scabraside D treatment significantly decreased VEGF-C, MMP-9 and uPA gene expression, which play important roles in the lymphangiogenesis and invasion of cancer cells in metastasis processes. Quantitative real-time PCR showed that scabraside D significantly decreased iNOS and STAT-3 gene expression. This study demonstrated that scabraside D plays a role in activation of HuCCA tumor apoptosis and inhibition of lymphangiogenesis, invasion and metastasis through decreasing BCl-2, MMP-9, uPA and VEGF-C and increasing caspase-3 expression by suppression of iNOS and STAT-3 expression. Therefore, scabraside D could be a promising candidate for cholangiocarcinoma treatment.     

Sida rhombifolia Exerts Anti-Proliferative and Pro-Apoptotic Effects in Human Liver Cancer HepG2 Cells in Vitro

Purpose: Modern research revealed that plants belonging to the Sida rhombifolia family (Malvaceae) contain biologically active compounds that make them prone to discovering and developing anticancer drugs. This study aimed to evaluate the apoptosis effects of S. rhombifolia extracts in HepG2 Cell Line was performed. Methods: The extractions were prepared, and an MTT assay was applied to evaluate its role in decreasing the viability of HepG2 and HFF cells. Phenolic compounds were analyzed using High-performance liquid chromatography (HPLC). FlowCytometry and RT-qPCR evaluated apoptosis was performed to measure the mRNA expression of pro-and anti-apoptotic mediators. Results: The results can be summarized as EtOAc extract was more cytotoxic against the HepG2 cells (IC50= 364.3 µg/mL) compared to MeOH and HEX extracts (720.2 µg/mL) (560.4 µg/mL) with less cytotoxicity in HFF cells (353.2 µg/mL). The HPLC analysis results revealed most phenolic compounds, such as Epicatechin(1.3 mg/g). The EtOAc extract (300 μg/mL) induced 34% apoptosis in HepG2 cells. RT-qPCR data showed upregulation of the proapoptotic gene (Bax) and increased Bax/BCL-2 ratio by S. rhombifolia EtOAc extract (300 μg/mL). Conclusion: In conclusion, the EtOAc extract of S. rhombifolia is capable of inducing apoptosis in HepG2 cells through modulation of the mitochondrial pathway, which explains their antitumor activity.     

Evaluation of In Vitro Cytotoxic Property Against Cholangiocarcinoma Cell Line and GC/MS Analysis from Leaf of Erythrophleum succirubrum Gagnep

Objective: Plants are valuable sources of new pharmaceuticals. Secondary metabolites of the genus Erythrophleum exhibit cytotoxicity and may have therapeutic value. The cytotoxic activity of ethanolic leaf extract of Erythrophleum succirubrum Gagnep. against a human cholangiocarcinoma cell line was assessed. Methods: Crude extract of E. succirubrum was prepared by ethanol extraction. The ethanolic leaf extract of E. succirubrum was evaluated for cytotoxicity against the human cholangiocarcinoma cell line KKU-M213 using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays. The chemical composition of E. succirubrum leaf extract was analyzed using GC/MS. Result: The ethanolic leaf extract of E. succirubrum reduced the viability of KKU-M213 cells in a dose- and time-dependent manner. It showed high cytotoxicity, with IC50 values of 65.22 ± 1.18 µg/mL and 1.19 ± 1.38 µg/mL at exposure times of 24 and 96 h, respectively. GC/MS analysis of the ethanolic leaf extract of E. succirubrum identified 22 components. The main constituents identified were Cyclohexanone, 2-[2-nitro-1-(2-naphthyl)ethyl]-(14.79%) followed by allomycin (14.65%), mome inositol (14.30%), campesterol (11.80%) and ethyl linolenate (10.83%), respectively. Conclusion: Five major groups of compounds were found, with lipids dominating, followed by carbohydrates, benzenoids, phenylpropanoids, polyketides and organoheterocyclic compounds. Many of the bioactive components discovered in the ethanolic leaf extract of E. succirubrum might be responsible for its cytotoxic properties.     

Brazilin Isolated from Caesalpina Sappan Wood Induces Intrinsic Apoptosis on A549 Cancer Cell Line by Increasing p53, caspase-9, and caspase-3

Objective: Lung cancer is the leading cause of death among cancer patients. The majority of lung cancer is the Non-Small Lung Carcinoma (NSLC). This study evaluated the potency of brazilin isolated from Caesalpinia sappan wood to induce apoptosis on non-small lung carcinoma cell line, A549, by examining the expression of p53, caspase-9, and caspase-3. Methods: Brazilin was isolated from Caesalpinia sappan wood following a guided assay and it was determined by using Brazilin®SIGMA as standard. The activity of brazilin on the growth of A549 cell line was analysed by MTT assay and the apoptosis was evaluated by flowcytometer following Annexin V (FITC) and PI staining. The expression of p53, caspase-9, and caspase-3 was examined by immunocytochemistry. Result: The IC50 of brazilin on A549 cell line was 43µg/mL. Cell treatment with 20 µg/mL and 40 µg/mL of brazilin significantly increased early apoptosis (p<0.001). Cell treatment with 40 µg/mL  of Brazilin significantly increased late apoptosis (p<0.001). Brazilin significantly increased the expression of p53, Caspase-9, and caspase-3 (p<0.001). Conclusion: This study showed evidence of the activity of brazilin to induce intrinsic apoptosis on a NSLC cell line A549.     

Addition of Gallic Acid Overcomes Resistance to Cisplatin in Ovarian Cancer Cell Lines

Objective: Ovarian cancer is one of the leading causes of cancer-related mortality in women, and is often associated with drug resistance. Therefore, finding effective drugs, including naturally derived compounds, is urgently needed. Herein, we aimed to test the anti-cancer potential of gallic acid monohydrate (GA) and its congeners on cisplatin-sensitive (A2780S), and resistant (A2780CP) ovarian cancer and normal ovarian (HOSE6-3) cell lines. Methods: Cytotoxicity was assessed by AlamarBlue and CCK08 assays by exposing cells to different concentrations of cisplatin (0-21µg/mL), GA and its congeners (0-100µg/mL), and a combination of GA and cisplatin. Apoptosis was estimated by Hoechst stain and monitoring the relative RNA expression of the apoptotic effector caspase-3 using qRT-PCR. Results: GA decreased cell viability in a concentration-dependent manner in all cell lines, with an IC50 of 19.39µg/mL (A2780S), 35.59 µg/mL (A2780CP), and 49.32µg/mL (HOSE6-3). GA displayed higher cytotoxicity than its congeners. An apoptotic rate estimation of approximately 20% and 30% was obtained in A2780S and A2780CP. While the cytotoxicity observed with cisplatin and GA was comparable, combining the two enhanced the cytotoxicity significantly, especially in the A2780CP cell line (p<0.05). Conclusion: These data suggest that GA may help overcome the resistance. Hence, the cytotoxic effects of GA, especially on chemo-resistant ovarian cancer cells merit further investigation.     

Evaluation of Apoptotic Gene Expression in Hepatoma Cell Line (HepG2) Following Nisin Treatment

Objective: The present study aims to examine the effects of nisin on the survival and apoptosis of the hepatoma cell line HepG2 and to investigate possible apoptosis pathways activated by nisin. Materials and Methods: For this purpose, viability and apoptosis of the cells were accomplished by the nisin treatment using the MTT assay and Annexin-V-fluorescein/propidium iodide (PI) double staining, respectively. Additionally, the human apoptosis PCR array was performed to determine pathways or genes activated by nisin during possible apoptosis. Results: The results of the present study showed that nisin was able to decrease cell viability (IC50 ~ 40 µg/ml) in a dose-dependent manner and could induce apoptosis in HepG2 cells. PCR data indicated a considerable increase in the expression of genes, such as caspase and BCL2 families, involved in the induction of apoptosis. Conclusions: The data from this study showed that overexpression of genes involved in the intrinsic pathway of apoptosis, especially caspase-9 and BID, increased apoptosis in HepG2 cells treated by nisin, compared to the control group.     

Reversal Effect of Dihydromyricetin on Multiple Drug Resistance in SGC7901/5-FU Cells

Background: One of the most common treatment for gastric cancer is chemotherapy, however, multiple drug resistance (MDR) induce the therapeutic effect which result in the failure of anticancer therapy. Dihydromyricetin (DMY) was reported to have antitumor activities on various human cancer cells in vitro, our previous studies demonstrated that DMY combined with mitomycin has inhibitory effect on proliferation of gastric carcinoma cells. However, the underlying role of DMY reversing the MDR of gastric carcinoma is poor understood. The aim of this study was to evaluate the reversal effect of DMY on MDR and investigate the molecular mechanisms in vitro. Methods: Using MTT assay, we identified the toxicity of DMY on SGC7901 and SGC7901/5-FU cells. The effect of DMY on 5-FU induced apoptosis was evaluated by flow cytometry analysis. Using RT-PCR and Western blot, we determined the MDR1 mRNA and protein expression. Results: DMY induced growth inhibition in both SGC7901 and SGC7901/5-FU cells, the IC50 value was 13.64±1.15 µg/mL, 20.69±1.82 µg/mL respectively. DMY treatment sensitized SGC7901/5-FU cells to cytotoxicity of 5-FU. The combination of DMY with 5-FU increased the apoptosis rate (9.91%, 16.67%) comparing with 5-FU alone (5.25%). Comparing with the control group, the MDR1 mRNA and protein expression in SGC7901/5-FU cells after treatment of DMY decreased significantly (P< 0.05). Conclusion: In brief, our study demonstrated that DMY effectively reversed multi-drug resistance occurring in SGC7901/5-FU cells cultured in vitro, and the potential mechanism was involved in the downregulation of the MDR1 expression.     

Cytotoxicity Studies of the Crude venom and Fractions of Persian Gulf Snail (Conus textile) on Chronic Lymphocytic Leukemia and Normal Lymphocytes

Background: Marine animals have been considered by many researchers due to their various pharmacological effects. One group of marine animals that have been studied is cone snails. The conotoxin obtained from these marine animals has various therapeutic effects. Methods: This study was designed to investigate the apoptotic effects of crude venom of Conus textile and its fractions (A and B) on chronic lymphocytic leukemia (CLL) cells. Accordingly, parameters such as cell viability, reactive oxygen species (ROS) level, collapse in mitochondrial membrane potential (MMP), lysosomal membrane damage and caspase-3 activation were evaluated. Results: The results showed that the crude venom (50, 100 and 200 µg/ml) from Conus textile and its fraction B (50, 100 and 200 µg/ml) significantly reduced viability in CLL B-lymphocyte. In addition, exposure of CLL B-lymphocyte to fraction B (50, 100 and 200 µg/ml) was associated with an increase in the level of ROS, the collapse of the MMP, damage to the lysosomal membrane, and activation of caspase-3. Conclusion: According to results, it was concluded that fraction B from crude venom of Conus textile causes selective toxicity on CLL B-lymphocyte with almost no effect on a normal lymphocyte. Furthermore, this venom fraction could be a promising candidate for induction of apoptosis in patients with CLL through the mitochondrial pathway.     

Anticancer Activity of Solvent Extracts of Hexogonia glabra against Cervical Cancer Cell Lines

Objective: In this study, we aimed to harness some solvent extracts of one wild mushroom Hexagonia glabra and test their anti-cancer activity against cervical human cell lines, namelyHeLa, SiHa, and CaSki. Methods: It includes cell morphological study by microscope, nuclear morphology by DAPI staining under fluorescence microscopy, apoptosis assay by fluorescence technique, anti-proliferation by MTT assay and expression of apoptotic and anti-apoptotic genes by Western blotting and cell cycle analysis was done. Results: The selected cervical cancer cells were treated separately with 150 µg/mL of three extracts, namely of ethanolic (EE), ethyl acetate (EAE), and water extract (WE) and exhibited features like round, shrink and dead. All extracts caused apoptosis in cell lines and EE had the highest effect in this regard. The percentages of apoptotic cells in HeLa, SiHa and CaSki, at the same concentration of EE were 79.23, 75.42, and 76.36% respectively. Cytotoxicity assay showed that all three extracts (50 – 250 μg/mL) were potent for inhibition of cell growth of three cell lines and again EE had the highest effect. The percentages of cell growth inhibition in HeLa, SiHa, and CaSki cells treated with EE at 24 h at 50 µg/mL were 45.79±4.11, 41.66±4.03, and 36.72±2.67, while they were 74.23±7.45, 62.31±5.97, and 54.23±5.04 at 150 µg/mL concentration. At 250 µg/mL concentration, the percentages of cell growth inhibition were 94.25 ±8.11, 90.02 ±8.67, and 85.43±6.21, respectively. The expression of apoptotic gene (Caspase 3, 9) and tumor guard gene (p53), as their proteins in Western blotting increased . However, anti-apoptotic BcL2 gene of all cell lines was decreased following treatment with extracts. In addition, the cell cycle analysis (CaSki cell) showed that treatment (EE) arrested at G2/M check point cell cycle. Conclusion: All extracts of this mushroom were active in arresting growth of three cell lines and EE had the highest effect, indicating that this mushroom can be a valuable source of anticancer agents.     

Atractylodin and β-eudesmol from Atractylodes lancea (Thunb.) DC. Inhibit Cholangiocarcinoma Cell Proliferation by Downregulating the Notch Signaling Pathway

Objective: Notch signaling pathway has been reported to be involved in the development and progression of various types of cancer, including cholangiocarcinoma (CCA).  Compounds that modulate this signaling pathway could be promising candidates for CCA treatment and control. The study investigated the antiproliferative activities and modulatory effects of atractylodin and β-eudesmol, the two bioactive compounds of Atractylodes lancea (Thunb.) DC. , on Notch signaling and upstream molecules (Notch1 and Notch2 receptors, JAG1, mTOR, PI3K, and YAP), and downstream molecules (Snail) in HuCCT-1 (CCA cell line) and OUMS-36T-1 (normal fibroblast cell line). Gemcitabine (standard drug for CCA), and Notch inhibitors (DAPT and zebularine) were included in the experiments as positive control compounds. Methods: The antiproliferative activity was evaluated using MTT assay.  mRNA and protein expression of Notch signaling molecules were evaluated using real-time PCR and Western blot analysis. Results: Atractylodin and β-eudesmol moderately inhibited HuCCT-1  cell growth with IC50 (concentration that inhibits cell growth by 50%) of 29.00 ± 6.44 and 16.80 ± 4.41 µg/ml (mean±SD), respectively. The direction and extent of the modulatory effects on mRNA and protein expression in the CCA cell line varied with the signaling molecules. Notch1 receptor was shown to be the most promising target of atractylodin and β-eudesmol in CCA. The level of gene expression was significantly downregulated (0.042 to 0.195 fold of control) after treating HuCC-T1 cells with both compounds at low and high concentrations. The extent and change in Notch1 gene expression correlated well with protein expression. Conclusion: The notch signaling pathway could be a promising target of atractylodin and β-eudesmol in CCA.     

Conjugation of Cetuximab – Puromycin and Its Target-Specific Effect on Triple Negative Breast Cancer Cell Lines

Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also toxic for normal cell, not specific to the target cells. Consequently, chemotherapy has various side effects. Monoclonal antibody (MAb) has been developed for specific therapy which only has killing effect in cancer cells, but the survival rate of most MAbs around 20%. Therefore, in clinical practice, MAbs administration should combine with chemotherapeutic agents. For effectiveness of therapy and to minimalize adverse effects, anticancer agent with selective cytotoxic effect on target cells is needed, the immunotoxin. Objective: This study introduces a novel approach to conjugate monoclonal antibody (Cetuximab) and toxin (Puromycin), in order to selectively inhibit proliferation of triple negative breast cancer (TNBC) and to enhance the efficacy of MAb in target cells killing. Methods: Cetuximab was conjugated with Puromycin using a linker, i.e SATP (Succinimidyl-acetylthiopropionate) and tested on triple negative breast cancer cell lines (MDA-MB-231) which expressed EGFR (epidermal growth factor receptor). Cetuximab is MAb which targets EGFR. MCF-7 was used as control cells since it has low or no EGFR expression. Cell counting were conducted as viability assay at 24 hours, 48 hours, and 72 hours after treatment. Results: The results showed significant reduction of live cells number in Conjugate 20 µg/mL cultured in MDA-MB-231 compared to MCF-7 after 24 hours, 48 hours, and 72 hours incubation. In all time period of incubation, significant reduction of MDA-MB-231 live cells number was also observed in Conjugate 20 µg/mL compared to Cetuximab 20 µg/mL. Conclusion: Synthesized conjugate showed its target-specific effect in TNBC and improved the efficacy of Cetuximab on TNBC. In the future, this conjugate can be a potential anticancer therapy in treating triple-negative breast cancer.     

A Phytochemicals Approach Towards the Treatment of Cervical Cancer Using Polyphenols and Flavonoids

Aim: Despite enormous progress in cancer biology, oncologists are still struggling to retrieve the methods and drugs to cure cancer which remains a global threat to humans. Plant-derived natural compounds, also known as phytochemicals, carry therapeutic potential and could be taken as dietary supplements, which are a radical way to resist as well as cure cancer. The present study reveals the anti-cancerous potential of a few phytochemical constituents under in vitro conditions and their mode of action on cervical cancer. SiHa was treated with phytochemicals viz. Quercetin dihydrate, Gallic Acid, and Naringin with varying concentrations to assess their cytotoxicity potential by various methods and also to elucidate their IC50 values. Methods: All three compounds reduced the cell number and viability, along with alterations in cancer cell morphology. The diagnosed IC50 values of the compounds were 160µM, 200µM, and 1500µM for Quercetin dihydrate, Gallic Acid, and Naringin, respectively. DNA fragmentation assay and gene expression analysis were also used to assess apoptosis and anti-proliferative activity of compounds. Results: We found fragmented DNA in treated cells as assessed by gel electrophoresis assay. These phytochemicals elicit an apoptotic response in SiHa cells by significantly up-regulating the gene expression level of p53 and p21 (p-value <0.005). Conclusion: Considering the anti-cancer and anti-proliferative potential of Quercetin dihydrate, Gallic Acid, and Naringin on the cervical cancer cell line, these phytochemicals could be used as an alternative or concurrent cancer therapeutic approach. However, further in-depth elucidation of their mode of action, safety, and efficacy should be explored.     

Pregnane Steroids from the Leaves of Melia Azedarach and Apoptotic Activity against T47D Cells

Objective: Nature has provided us with many pharmaceutical resources so far. Breast cancer shows an increasing trend in the world for the last decade and becomes one of five leading causes of death. Among the plants, Melia azedarach L. has been used widely in traditional medicine for many ailments including breast cancer. Following our previous findings that the ethyl acetate fraction was the most active cytotoxic fraction against T47D cells, we aimed to isolate the cytotoxic compounds and further elucidate their apoptotic mechanisms. Methods: The compounds were isolated through a series of chromatography with cytotoxicity evaluations. Identification of the isolated compounds was achieved by intensive spectroscopic analysis such as NMR, MS, and IR spectra. Cytotoxicity was evaluated by MTT method using doxorubicin as a reference compound. The expression of apoptosis-related factors was quantified by flow cytometry and immunocytochemistry. Results: Two isomers of pregnane steroids with molecular weight 330.2087 (C21H30O3) were isolated from the EtOAc extract. Spectroscopic analysis revealed the structures as 17-ethylene-3,4-dihydroxy-14-methyl-18-norandrostene-16-one (1) and 17-ethylene-3,4-dihydroxy-5-pregnene-16-one (2), respectively. These compounds showed moderate cytotoxicity (IC50 172.9 and 62.2 µg/mL, respectively) comparable to doxorubicin (IC50 3.08 µg/mL). The execution of apoptosis may be related to the increase of the ratio of BAX/bcl-2 of the cells.  Conclusion: The EtOAc fraction of Melia azedarach L. leaves and the isolated 5-pregnene-16-one steroids are promising reagents for breast cancer treatment by introducing apoptosis to tumor cells. However, further researches are required to highlight its safety and usage in vivo.     

Estrogen Receptor Beta (ERβ) May Act as Mediator in Apoptotic Induction of Grape Seed Extract (GSE)

Background: Grape seed extract is a complex mixture of polyphenols. Its anti-tumor effects have been reported by several studies. Estrogen receptors (ERs) are commonly considered as important markers for breast cancer. The present study aimed to evaluate the apoptotic effects of GSE on MCF7 breast cancer cells and assessed the expression of ERβ during treatment of cells with GSE. Material and Methods: The half maximal inhibitory concentration (IC50) of GSE in MCF7 breast cancer cells were calculated by treating cells with serial dilution of GSE for 48 hours and cell viability evaluated using MTT assay. Then cells assigned to three groups: control (no treatment), DMSO (cells treated with 0.05% of DMSO) and GSE group (cells treated with of GSE for 48 hours). The apoptosis assay was performed by detecting Annexin V protein by flow cytometry. The gene expression of ERβ and caspase-3 was evaluated by Real-Time PCR. Results: Cells in GSE group treated with GSE IC50 concentration for 48 hours. Annexin V staining assay, represented early apoptosis detected by flow cytometry analysis showed significantly higher expression (p<0.01) than control and DMSO groups. Moreover, results of Real-Time PCR showed a significant expression in ERβ and caspase-3 genes in GSE group compared to control and DMSO groups (Fold change = 2.3 and 3.5, respectively). Conclusion: GSE may induce apoptosis in MCF7 human breast cancer cells by activation of ERβ gene.     

Anticancer Activity of Combretum fragrans F. Hoffm on Glioblastoma and Prostate Cancer Cell Lines

Background: Cancer incidence has been growing in an alarming rate worldwide and new therapeutics are needed, particularly for intractable and chemoresistant cases. We evaluated the cytotoxic effects of Combretum fragrans F. Hoffm (Combretaceae) on glioblastoma (U87MG and C6) and prostate (PC-3) cancer cell lines. Methods: The cytotoxic effect of the methanolic extract of the stem bark of Combretum fragrans was assessed using XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) test. Expressions of Akt and ERK1/2 were determined using Western blot technique, while Caspase-3/7 kits were used to evaluate caspase-3/7 activity. Results: C. fragrans extract inhibited the proliferation of U87 (IC50 = 20.13 µg/mL), C6 (IC50 = 12.17 µg/mL), and PC-3 (IC50 = 11.50 µg/mL) cells. Treatment with the extract resulted in lower levels (p < 0.001) of phospho-ERK1/2 and phospho-Akt in U87 cells, and instead, higher levels of phospho-ERK1/2 (p < 0.001) in C6 and PC-3 cells. An increase in caspase-3/7 activity was observed, mainly after 24 hours of treatment, indicating the activation of apoptotic processes. Conclusion: Altogether, these results suggest that C. fragrans have potent anticancer properties. This plant should be further investigated for developing new anticancer drugs.     

Aqueous Extract of Origanum majorana at Low Temperature (0°C) Promotes Mitochondrial Fusion and Contributes to Induced Apoptosis in Human Breast Cancer Cells

Marjoram plants have varied pharmacological properties because they contain antioxidants. In the present study, to evaluate the effect of Origanum majorana, gathered from Abha, Saudi Arabia, on the growth of human breast cancer cells using MCF7. Fresh aerial parts from Origanum majorana were extracted at a low temperature (0 ℃/6 hours). Human MCF7 breast cancer cells were then treated with 4 separate fluctuated concentrations of 0, 50, 150, 200 and 350 µg/mL for 24 and 48 hours. The findings showed that Origanum majorana aqueous extract contained absolute phenolic content (TPC) was 58.24 mg equivalent/g DW, and the complete flavonoid content (TFC) 35.31 mg GAE equivalent/g DW in the Origanum majorana aqueous extract. The endurance of MCF7 cells after incubation with aqueous extract diminished, indicating that Origanum majorana is tumour cell selective. Origanum majorana extract increased the mRNA Expression of Apoptotic Genes in MCF7. Majorana aqueous extract expanded the activity of Caspase-7 action specifically at higher concentrations, 150, 200, and 350 µg/ml. Our findings indicate that Origanum majorana could induce apoptosis of human breast cancer cells. This is the first study that provides a basis for the use of aqueous Origanum majorana extracted at low temperature (0 °C/6 hours) as more effective anticancer treatment.     

Dryocrassin ABBA Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells Through a Caspase-Dependent Mitochondrial Pathway

Background: Biological and pharmacological activities of dryocrassin ABBA, a phloroglucinol derivative extracted from Dryopteris crassirhizoma, have attracted attention. In this study, the apoptotic effect of dryocrassin ABBA on human hepatocellular carcinoma HepG2 cells was investigated. Materials and Methods: We tested the effects of dryocrassin ABBA on HepG2 in vitro by MTT, flow cytometry, real-time PCR, and Western blotting. KM male mice were used to detect the effect of dryocrassin ABBA on H22 cells in vivo. Results: Dryocrassin ABBA inhibited the growth of HepG2 cells in a concentration-dependent manner. After treatment with 25, 50, and 75 g/mL dryocrassin ABBA, the cell viability was 68%, 60% and 49%, respectively. Dryocrassin ABBA was able to induce apoptosis, measured by propidium iodide (PI)/annexin V-FITC double staining. The results of real-time PCR and Western ting showed that dryocrassin ABBA up-regulated p53 and Bax expression and inhibited Bcl-2 expression which led to an activation of caspase-3 and caspase-7 in the cytosol, and then induction of cell apoptosis. In vivo experiments also showed that dryocrassin ABBA treatment significantly suppressed tumor growth, without major side effects. Conclusions: Overall, these findings provide evidence that dryocrassin ABBA may induce apoptosis in human hepatocellular carcinoma cells through a caspase-mediated mitochondrial pathway.