Up-regulation Of Bax Expression Research Articles

Taccaoside induces apoptosis in hepatocellular carcinoma cell lines.

ObjectiveTaccaoside, a steroidal saponin, has been shown to be cytotoxic, although the mechanism of cytotoxicity remains unclear. This study examined the effect of taccaoside on the human hepatocellular carcinoma (HCC) cell lines SMMC-7721 and Bel-7404.MethodsThe antiproliferative effect of taccaoside were measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Cells were stained with Hoechst 33258 to observe morphology. Cell cycle and apoptosis were analysed by flow cytometry. Caspase activation was detected using specific assays, and PARP, Bax and Bcl-2 expression were analysed using western blotting.ResultsTaccaoside showed antiproliferative effect on HCC cell lines growth in a concentration- and time-dependent manner. Taccaoside arrested cell cycle in the G2/M phase and induced caspase-dependent apoptosis. Western blotting indicated that taccaoside upregulated Bax expression and downregulated Bcl-2 expression. PARP cleavage was observed following taccaoside treatment.ConclusionsThis study showed that taccaoside may inhibit HCC cell proliferation by inducing apoptosis.

Anti-tumor effects of (1 → 3)-β-d-glucan from Saccharomyces cerevisiae in S180 tumor-bearing mice

(1→3)-β-d-Glucan from Saccharomyces cerevisiae is a typical polysaccharide with various biological effects and is considered a candidate for the prevention and treatment of cancer in vitro. Research into the function of (1→3)-β-d-glucan in tumor-bearing animals in vivo, however, is limited. Here, we investigated the effects of (1→3)-β-d-glucan from S. cerevisiae on S180 tumor-bearing mice and on the immunity of the tumor-bearing host. The molecular mechanisms underlying the observed effects were investigated. (1→3)-β-d-Glucan was shown to exert anti-tumor effects without toxicity in normal mouse cells. The volume and weight of S180 tumors decreased dramatically following treatment with (1→3)-β-d-glucan, and treatment with the polysaccharide was furthermore shown to increase the tumor inhibition rate in a dose-dependent manner. Spleen index, T lymphocyte subsets (CD4 and CD8), as well as interleukins (IL)-2, (IL-2, IL-6), and tumor necrosis factor-α were assayed to detect the immunoregulatory and anti-tumor effects after (1→3)-β-d-glucan intragastrical administration. (1→3)-β-d-Glucan was shown to significantly potentiate the mouse immune responses by, among other effects, decreasing the ratio of CD4 to CD8. The expression levels of IL-2, IL-6, and TNF-α were also significantly increased by (1→3)-β-d-glucan. These results suggest that (1→3)-β-d-glucan enhances the host’s immune function during the tumor inhibition process. S180 tumor cells treated with (1→3)-β-d-glucan also exhibited significant apoptotic characteristics. (1→3)-β-d-glucan increased the ratio of Bax to Bcl-2 at the translation level by up-regulating Bax expression and down-regulating Bcl-2 expression, resulting in the initiation of cell apoptosis in S180 tumor-bearing mice. Taken together, these results indicate that the anti-tumor effects exerted by (1→3)-β-d-glucan may be attributed to the polysaccharide’s immunostimulating properties and apoptosis-inducing features. Further investigation into these properties and their associated mechanisms will contribute to the development of potent polysaccharide-based anti-tumor agents.

Abstract A28: Mechanistic study involving the combined antiproliferative effect of Etoricoxib- cyclooxygenase-2 inhibitor and cholecystokinin-2 receptor antagonist in human pancreatic cancer cells

Abstract Introduction: Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer death in United States. Due to poor prognosis and increasing incidences, over 80% of the patient are given palliative chemotherapy with combination drugs. Despite decades of efforts, the survival rate still remains 3-6%. The most common cause of pancreatic cancer are genetic alterations, cigarette smoking, obesity and lack of exercise. Patients with Type-2 Diabetes Mellitus are also on higher risk of pancreatic cancer. Although numerous chemotherapeutic drugs have been tested in this malignancy, survival of advanced pancreatic cancer has not improved over the past several decades. Because of the lack of effective treatment options available for this disease, identification of novel targets and approaches has been made. Recent studies have shown an association between the gastrointestinal hormone gastrin, the expression of Cholecystokinin -B/ gastrin receptor and an increased generation of COX-2 in colon-carcinoma cells and progression of gastric cancer, suggesting that gastrin has direct stimulating potencies. However, it remains unknown whether the combination of Cholecystokinin-2 (CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic antitumor effects on human gastric cancer. Therefore, for a better treatment and understanding the underlying mechanism that leads to pancreatic adenocarcinoma initiation, chemoresistance and progression, we further investigated the combined effect of Cyclooxygenase (COX-2) inhibitor and Cholecystokinin receptor-2 (CCK-2) antagonist on pancreatic cancer cells. Thus it is essential to recognize more effective targets/ receptors for treatment of pancreatic cancer. Materials & Methods: Antiproliferative effects were carried out using Cell Proliferation Kinetics, Trypan blue staining technique, Morphological analysis and MTT Assay. Apoptotic activity was confirmed by DNA Fragmentation, Western Blot analysis and Cell cycle analysis Result and Discussion: The present study demonstrate that COX-2 enzyme is a modulator of carcinogenesis, apoptosis and angiogenesis in pancreatic cancer and is such rational target for drug development. Similarly Cholecystokinin antagonists is also a potential target for developing novel strategies for pancreatic cancer and further defines the pathway affected by CCK-BR. To study the combinatorial effect of COX-2 inhibitor and Cholecystokinin antagonist we successfully purified Etoricoxib using HPLC and further characterized by spectroscopic techniques using FT-IR, NMR and Mass spectroscopy. The anti-proliferative activity of Etoricoxib was checked on various cell lines and it induced proliferation arrest in Miapaca-2, Panc-I, BXPC-3 and ASPC-1 both in time and concentration dependent manner. The IC50 values were found to be 280 μM (Miapaca-2), 400μM (Panc-1), 300μM (BXPC-3) and 450 μM (ASPC-1) cells. The IC50 of procured Cholecystokinin antagonists YM-022, LY 288513 and L-365-260 was found out to be 0.002μM, 0.001μM and 0.01μM on Panc-1 respectively. Similarly, combinatorial studies was carried out on Panc-1, BXPC-3, ASPC-1 and Miapaca-2 respectively using cytotoxicity and apoptotic techniques. It is thus, a probable insight in stating that it has lesser side effects and thus have selectivity for cancer cell toxicity. Conclusion: The combined effect of Cyclooxygenase-2 inhibitor and Cholecystokinin-2 Receptor antagonists was calculated using Chaou Tatlay method. A combination index was calculated. As a class NSAIDs possess analgesic, antipyretic, anti-inflammatory, and there is persuasive evidence that COX-2 inhibitor suppress cancer cell proliferation owing to their role in apoptosis, compelling evidence suggest that COX-2 over-expression promotes whereas COX-2 inhibition prevents tumor initiation and promotions. NSAIDs and COX-2 selective inhibitors may have different effects on cancer may be stage dependent therefore a better understanding of the critical COX related mechanisms of carcinogenesis, proliferation, apoptosis, inflammation, angiogenesis and tumor invasion will help to define the potential of COX-2 blockage in cancer therapy. Previous studies have shown that NSAIDs and specific COX-2 inhibitors are able to enhance the effect of certain cystostaic agents, a specific COX-2 inhibitor Celecoxib enhances the cystostaic effect of doxorubicin on tumor cell lines (Wijnggaarden J.V. et.al. 2007). Results in our lab showed that combination of doxorubicin and etoricoxib synergistically inhibited the proliferation of Hela and Miapaca cell lines. These results has indicated that the combination therapy of COX-2 selective inhibitors may be the potential chemotherapeutic strategy for cancer prevention. In the present study, human pancreatic cancer cell lines, in which the CCK-2 receptor and COX-2 were expressed, was applied to examine whether blockade of the CCK-2 receptor and COX-2 exerts synergistic anti-tumor effects on human pancreatic cancer in vitro. The detailed studies are under process. In conclusion, both Etoricoxib and YM-022 had growth inhibitory and apoptosis inductive effects on the pancreatic cancer cells through down regulation of Bcl-2 with simultaneously up-regulation of BAX expression which suggest that COX-2 inhibition is new molecular targets for effective therapy against pancreatic cancer. Citation Format: Manisha Sikka. Mechanistic study involving the combined antiproliferative effect of Etoricoxib- cyclooxygenase-2 inhibitor and cholecystokinin-2 receptor antagonist in human pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A28.

Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells

The food contaminant acrylamide (AA) is usually recognized as a probable human carcinogen. In addition, AA has also been found able to induce male infertility in animals. Interestingly, resent research work revealed that the toxic effect of AA on the ability of male reproduction in vivo may due to glycidamide (GA) which is the metabolite of AA. In this study, R2C Leydig cells was used to investigate the toxic effects of GA on progesterone production. GA caused dose-dependent inhibition on the cell growth, with IC25, IC50, and IC75 values found at 0.635, 0.872, and 1.198 mM, respectively. The results of single cell gel/Comet assay showed that GA significantly induced early-phase cell apoptosis, reduced progesterone production, as well as decreasing the protein expression of steroidogenic acute regulatory (StAR) in R2C cells. Furthermore, GA induced overproduction of intracellular reactive oxygen species (ROS), upregulated Bax expression, decreased mitochondrial membrane potential, and triggered mitochondria-mediated cell apoptosis. Consequently, the downstream effector caspase-3 was activated, resulting in Leydig cells apoptosis. Overall, our results showed that GA could damage R2C Leydig cells by the lesion of the ability of progesterone genesis and inducing cells apoptosis.

Iptakalim induces mitochondria-dependent apoptosis in hypoxic rat pulmonary arterial smooth muscle cells.

Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (HPH) is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Iptakalim (Ipt), an ATP sensitive potassium channel opener, ameliorates HPH in animal models. Here we investigated the effects of Ipt on proliferation and apoptosis of hypoxic rat PASMCs, and to determine the possible underlying mechanisms. Primary rat PASMCs were isolated and cultured. PASMCs were cultured for 24h in normoxia or hypoxia (5% O2) conditions with and without Ipt. Cell proliferation and cycle were determined by MTT assay and flow cytometry, respectively. Mitochondrial membrane potential (Δym) was detected by fluorescence microscope Western blot assays were used to examine the expression of cyclin D, CDK4, endothelin-1 (ET-1), hypoxia-inducible factor-1 (HIF-1), platelet-derived growth factor-BB (PDGF-BB), Bax, Bcl-2, cytochrome c (Cyt c), caspase-9, and caspase-3 in PASMCs. We found that hypoxia significantly stimulated proliferation and rendered resistance to apoptosis in PASMCs. Ipt suppressed proliferation and induced cell cycle arrest in hypoxia PASMCs. Ipt decreased the expression of cyclin D, CDK4, HIF-1, ET-1, and PDGF-BB in hypoxia PASMCs. It reversed the depolarization of Δψm in hypoxia PASMCs too. Ipt significantly upregulated Bax expression and downregulated Bcl-2 expression, and promoted the release of Cyt c from mitochondria to cytoplasm in hypoxia PASMCs. Furthermore, Ipt significantly activated the caspase cascades evidenced by increased expression of caspase-9 and caspase-3 in hypoxia PASMCs. Ipt could inhibit cell proliferation and induce apoptosis associated with cell cycle arrest, decreased ET-1, HIF-1, cyclin D, CDK4, PDGF-BB and Δψm, increased Bax/Bcl-2 ratio, enhanced Cyt c release, and activation of caspases in PASMCs under hypoxia status. Our data indicated that Ipt could be a therapeutic candidate for treatment of HPH.

EPA or DHA enhanced oxidative stress and aging protein expression in brain of d-galactose treated mice

Background:Effects of eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) upon fatty acid composition, oxidative and inflammatory factors and aging proteins in brain of d-galactose (DG) treated aging mice were examined.Methods:Each fatty acid at 7 mg/kg BW/week was supplied for 8 weeks. Brain aging was induced by DG treatment (100 mg/kg body weight) via daily subcutaneous injection for 8 weeks.Results:DG, EPA and DHA treatments changed brain fatty acid composition. DG down-regulated brain Bcl-2 expression and up-regulated Bax expression. Compared with DG groups, EPA and DHA further enhanced Bax expression. DG decreased glutathione content, increased reactive oxygen species (ROS) and oxidized glutathione (GSSG) production, the intake of EPA or DHA caused greater ROS and GSSG formation. DG treatments up-regulated the protein expression of p47phox and gp91phox, and the intake of EPA or DHA led to greater p47phox and gp91phox expression. DG increased brain prostaglandin E2 (PGE2) levels, and cyclooxygenase (COX)-2 expression and activity, the intake of EPA or DHA reduced brain COX-2 activity and PGE2 formation. DG enhanced brain p53, p16 and p21 expression. EPA and DHA intake led to greater p21 expression, and EPA only caused greater p53 and p16 expression. Conclusion: These findings suggest that these two PUFAs have toxic effects toward aging brain.

Pharbilignan C induces apoptosis through a mitochondria-mediated intrinsic pathway in human breast cancer cells

Pharbitidis Semen, the seed of Morning glory (Pharbitis nil), is a medicinal agent that has traditionally been used as a purgative in Korea. Pharbilignan C (PLC) is a dihydro[b]-benzofuran-type neolignan from Pharbitidis Semen, which reportedly exhibited the most potent cytotoxicity against human tumor cells. To further study the antiproliferative activity of PLC, its molecular mechanisms of action in two breast adenocarcinoma cells, MCF-7 and MDA-MB 231 cells were investigated. PLC inhibited the proliferation of MDA-MB 231 and MCF-7 cells, in order of sensitivity (IC50 of MDA-MB 231 cells: 7.0±2.0μM). PLC induced apoptosis in MDA-MB 231 cells with down regulation of Bcl-2 and up-regulation of Bax expression. It also decreased mitochondrial membrane potential accompanied with increasing initiator caspase, caspase-9 activation and executioner caspase, caspase-3 activation. This study demonstrates that PLC inhibited proliferation of MDA-MB 231 cells by inducing apoptosis via the mitochondria-mediated intrinsic pathway.

C-myc overexpression drives melanoma metastasis by promoting vasculogenic mimicry via c-myc/snail/Bax signaling.

High c-Myc expression is associated with tumor metastasis and poor prognosis in human melanoma. c-Myc upregulates Snail expression to promote EMT via the TGF-β/Snail/Ecadherin signal pathway. c-Myc leads to cell death by upregulating Bax expression causing a lower Bcl2/Bax ratio under severe hypoxic conditions. c-Myc promotes vasculogenic mimicry and linearly patterned programmed cell necrosis.

Co-expression of ING4 and P53 enhances hypopharyngeal cancer chemosensitivity to cisplatin invivo.

Hypopharyngeal cancer is a distinct type of malignant head and neck tumor, which exhibits low sensitivity to anti-cancer drugs. The importance of developing methods for reducing chemotherapy resistance, and improving and enhancing prognosis has previously been emphasized and is considered a challenge for effective clinical treatment of hypopharyngeal cancer. The current study investigated the effects of co-expression of inhibitor of growth protein 4 (ING4) and P53, a tumor suppressor gene, on chemosensitivity to cisplatin in human hypopharyngeal cancer xenografts in vivo, and the potential molecular mechanisms involved. A tumor model was established by injecting athymic nude mice with FADU human hypopharyngeal cancer cells. Five days after intratumoral and peritumoral injections of an empty adenoviral vector (Ad), Ad-ING4-P53, cisplatin, or a combination of Ad-ING4-P53 and cisplatin (Ad-ING4-P53 + cisplatin) every other day for 5 days, the mice were euthanized and their tumors, livers, and kidneys were removed. The tumor weights were used to calculate the inhibition rate, and the expression levels of ING4 and P53 were detected by reverse transcription-polymerase chain reaction. Additionally, apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry determined the levels ING4, P53, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) protein expression. The results demonstrated increased expression of ING4 and P53 in the Ad-ING4-P53 groups compared with PBS and Ad groups, indicating successful introduction of the genes into the tumor cells. Notably, the Ad-ING4-P53 + cisplatin group exhibited a higher inhibition rate compared with the four other groups. The results of immunohistochemistry analysis demonstrated that Bax expression was increased and Bcl-2 was decreased in the Ad-ING4-P53 + cisplatin group. This suggested that the enhanced cisplatin chemosensitivity with Ad-ING4-P53 gene therapy in hypopharyngeal cancer xenografts may be associated with apoptosis induction through upregulation of Bax expression and downregulation of Bcl-2. The results of the present study indicated that gene therapy combined with cisplatin treatment may be a promising treatment for human hypopharyngeal cancer.

Harmine combined with paclitaxel inhibits tumor proliferation and induces apoptosis through down-regulation of cyclooxygenase-2 expression in gastric cancer.

Cyclooxygenase-2 (COX-2) serves an important role in the carcinogenesis and progression of gastric cancer. Harmine (HM) and paclitaxel (PTX) are reported as promising drug candidates for cancer therapy, but whether a synergistic anti-tumor effect of HM combined with PTX exists in human gastric cancer remains unknown. The present study evaluated the effects of HM and/or PTX on cell proliferation and apoptosis in a gastric cancer cell line, SGC-7901. HM and PTX inhibited cell proliferation in a dose-dependent manner. Both HM and PTX alone induced apoptosis in gastric cancer cells. The combination of HM and PTX exerted synergistic effects on proliferation inhibition and apoptosis induction in SGC-7901 cells, with down-regulation of COX-2, PCNA and Bcl-2 and up-regulation of Bax expression. The results indicated that combination chemotherapy using HM with PTX exerts an anti-tumor effect for treating gastric cancer. The combination of the two drugs inhibits gastric cancer development more effectively than each drug alone through down-regulation of COX-2 expression.

Microcystin-LR induces mitochondria-mediated apoptosis in human bronchial epithelial cells.

The present study aimed to investigate the toxicity of microcystin-LR (MC-LR) and to explore the mechanism of MC-LR-induced apoptosis in human bronchial epithelial (HBE) cells. HBE cells were treated with MC-LR (1, 10, 20, 30 and 40 µg/ml) alone or with MC-LR (0, 2.5, 5 and 10 µg/ml) and Z-VAD-FMK (0, 10, 20, 40, 60, 80, 100, 120 and 140 µM), which is a caspase inhibitor, for 24 and 48 h. Cell viability was assessed via an MTT assay and the half maximal effective concentration of MC-LR was determined. The optimal concentration of Z-VAD-FMK was established as 50 µm, which was then used in the subsequent experiments. MC-LR significantly inhibited cell viability and induced apoptosis of HBE cells in a dose-dependent manner, as detected by an Annexin V/propidium iodide assay. MC-LR induced cell apoptosis, excess reactive oxygen species production and mitochondrial membrane potential collapse, upregulated Bax expression and downregulated B-cell lymphoma-2 expression in HBE cells. Moreover, western blot analysis demonstrated that MC-LR increased the activity levels of caspase-3 and caspase-9 and induced cytochrome c release into the cytoplasm, suggesting that MC-LR-induced apoptosis is associated with the mitochondrial pathway. Furthermore, pretreatment with Z-VAD-FMK reduced MC-LR-induced apoptosis by blocking caspase activation in HBE cells. Therefore, the results of the present study suggested that MC-LR is capable of significantly inhibiting the viability of HBE cells by inducing apoptosis in a mitochondria-dependent manner. The present study provides a foundation for further understanding the mechanism underlying the toxicity of MC-LR in the respiratory system.

Valproic acid enhances the antileukemic effect of cytarabine by triggering cell apoptosis.

Acute myeloid leukemia(AML) is an aggressive clonal malignancy of hematopoietic progenitor cells with a poor clinical outcome. The resistance of leukemia cells to contemporary chemotherapy is one of the most formidable obstacles to treating AML. Combining valproic acid(VPA) with other anti-leukemic agents has previously been noted as a useful and necessary strategy which can be used to specifically induce anticancer gene expression. In the present study, we demonstrated the synergistic antileukemic activities between VPA and cytarabine(Ara‑C) in a retrovirus-mediated murine model with MLL-AF9 leukemia, three leukemia cell lines (THP-1, K562 and HL-60) and seven primary human AML samples. Using RT-qPCR, we noted that the combination of VPA and Ara‑C significantly upregulated Bax expression and led to the arrest of leukemia cell proliferation, sub-G1 DNA accumulation and cell apoptosis, as demonstrated by flow cytometric analysis. Significantly, further experiments revealed that knockdown of Bax expression prevented VPA and Ara‑C‑induced cell apoptosis in THP-1 cells. The results of our present study demonstrated the synergistic antileukemic effect of combined VPA and Ara‑C treatment in AML, and thus we suggest that VPA be used an alternative treatment for AML.

MiR-27b attenuates apoptosis induced by transmissible gastroenteritis virus (TGEV) infection via targeting runt-related transcription factor 1 (RUNX1)

Transmissible gastroenteritis virus (TGEV), belonging to the coronaviridae family, is the key cause of the fatal diarrhea of piglets and results in many pathological processes. microRNAs (miRNAs) play a key role in the regulation of virus-induced apoptosis. During the process of apoptosis induced by TGEV infection in PK-15 cells, the miR-27b is notably down-regulated. Thus, we speculate that miR-27b is involved in regulating the process of apoptosis in PK-15 cells. In this study we demonstrated that the over-expression of miR-27b led to the inhibition of TGEV-induced apoptosis, reduction of Bax protein level, and decrease of caspase-3 and −9 activities. Conversely, silencing of miR-27b by miR-27b inhibitors enhanced apoptosis via up-regulating Bax expression and promoting the activities of caspase-3 and −9 in TGEV-infected cells. Subsequently, the runt-related transcription factor 1 (RUNX1) is a candidate target of miR-27b predicted by bioinformatics search. We further identified that the miR-27b directly bound to the 3′ UTR of RUNX1 mRNA and suppressed RUNX1 expression, which indicates RUNX1 is the direct target gene of miR-27b. The over-expression of RUNX1 increased apoptosis and knockdown RUNX1blocked apoptosis of viral-infected cells via regulating Bax expression and the activities of caspase-3 and −9. Our data reveal that miR-27b may repress the mitochondrial pathway of apoptosis by targeting RUNX1, indicating that TGEV may induce apoptosis via down-regulating miR-27b and that miR-27b may act as a target for therapeutic intervention.

Studies on chemoprotective effect of ginger extract in diethylnitrosamine induced hepatocarcinogenesis in rats

The present work was designed to evaluate anticarcinogenic effect of ginger extract (GE) in diethylnitrosamine (DEN) induced hepatocarcinogenesis in rats. Fifty one male Wistar albino rats were randomly allotted to four groups. DEN (0.01%) was given in drinking water ad libitum and GE (50 mg/kg BW) was administered in olive oil per os either alone or in combination for 90 days. Histopathology of liver sections revealed complete development of HCC in all animals of DEN treated group, while varying stages of progression of tumour in DEN+GE treated rats. Expression pattern of biomolecular markers such as PCNA, Bax and Bcl-2 were studied in liver sections to evaluate induction of HCC by DEN and chemoprotective activity of GE. Immunohistochemistry revealed overexpression of PCNA and Bcl-2 and reduced expression of Bax in DEN treated group. Concurrent treatment with GE significantly downregulated expression of PCNA and Bcl-2 and upregulated Bax expression. To conclude, present study demonstrated the anticancer properties of ginger extract in the form of partial chemoprotection with reduced tumor incidences, halted growth of altered hepatocellular foci, reduced cellular proliferation and induced apoptosis in HCC induced rats.

Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway.

Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3β activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy.

Isobavachalcone induces the apoptosis of gastric cancer cells via inhibition of the Akt and Erk pathways.

In the present study, the MGC803 gastric cancer cell line was used as an experimental model to evaluate the potential role of isobavachalcone (IBC) in cell apoptosis, migration and invasion. The inhibitory effects of IBC on cell proliferation were determined using a methylthiazolyltetrazolium assay. Cellular morphological changes were assessed using Wright-Giemsa staining, and cell apoptosis was evaluated by flow cytometric analysis. The results of the present study demonstrated that IBC inhibited the proliferation of MGC803 cells in a concentration- and time-dependent manner. Furthermore, the wound healing and Matrigel™ Transwell® invasion assays demonstrated that IBC decreased cell migration and invasion in a concentration-dependent manner. Western blotting was used to determine the protein expression levels of caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax), as well as the key protein kinases in the Akt and extracellular signal-regulated kinase (Erk) signaling pathways. During the IBC-induced apoptosis of MGC803 cells, transient activation of phosphorylated (p)-Akt and p-Erk inhibited the activation of Akt and Erk, upregulated Bax expression, downregulated Bcl-2 expression and activated caspase-3. These results suggest that IBC inhibited the growth of MGC803 gastric cancer cells by regulating the protein expression of caspase-3, Bcl-2 and Bax. In addition, inhibition of the Akt and Erk signaling pathways may be important mechanisms underlying the IBC-induced apoptosis of gastric cancer cells.

Abstract C7: Mechanistic studying involving the combined effect of Cyclooxygenase-2 inhibitor and Cholecystokinin -2 Receptor antagonist on Pancreatic cancer cell

Abstract Introduction: Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer death in United States. Due to poor prognosis and increasing incidences, over 80% of the patient are given palliative chemotherapy with combination drugs. Despite decades of efforts, the survival rate still remains 3-6%. Recent studies have shown an association between the gastrointestinal hormone gastrin, the expression of Cholecystokinin -B/ gastrin receptor and an increased generation of COX-2 in colon-carcinoma cells and progression of gastric cancer, suggesting that gastrin has direct stimulating potencies. However, it remains unknown whether the combination of Cholecystokinin -2 (CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic antitumor effects on human gastric cancer. Therefore, for a better treatment and understanding the underlying mechanism that leads to pancreatic adenocarcinoma initiation,we further investigated the combined effect of Cyclooxygenase (COX-2) inhibitor and Cholecystokinin receptor -2 (CCK-2) antagonist on pancreatic cancer cells. Thus it is essential to recognize more effective targets/ receptors for treatment of pancreatic cancer. Materials &Methods: Antiproliferative effects were carried out using Cell Proliferation Kinetics,Morphological analysis and MTT Assay. Apoptotic activity was confirmed by DNA Fragmentation, Western Blot analysis and Cell cycle analysis Result and Discussion: To study the combinatorial effect of COX-2 inhibitor and Cholecystokinin antagonist we successfully purified Etoricoxib using HPLC and further characterized by spectroscopic techniques.The anti-proliferative activity of Etoricoxib was checked on various cell lines and it induced proliferation arrest in Miapaca-2, Panc-I, BXPC-3 and ASPC-1 both in time and concentration dependent manner. combinatorial studies was carried out on Pancreatic cancer cells using cytotoxicity and apoptotic techniques. It is thus, a probable insight in stating that it has lesser side effects and thus have selectivity for cancer cell toxicity. Conclusion: The combined effect of Cyclooxygenase-2 inhibitor and Cholecystokinin-2 Receptor antagonists was calculated.The results has indicated that the combination therapy of COX-2 selective inhibitors may be the potential chemotherapeutic strategy for cancer prevention. In the present study, human pancreatic cancer cell lines, in which the CCK-2 receptor and COX-2 were expressed, was applied to examine whether blockade of the CCK-2 receptor and COX-2 exerts synergistic anti-tumor effects on human pancreatic cancer in vitro.In conclusion, both Etoricoxib and YM-022 had growth inhibitory and apoptosis inductive effects on the pancreatic cancer cells through down regulation of Bcl-2 with simultaneously up-regulation of BAX expression which suggest that COX-2 inhibition is new molecular targets for effective therapy against pancreatic cancer. Citation Format: Manisha Sikka, Madhu Chopra. Mechanistic studying involving the combined effect of Cyclooxygenase-2 inhibitor and Cholecystokinin -2 Receptor antagonist on Pancreatic cancer cell. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C7.

Inhibition of Glioblastoma Cell Growth In Vitro and In Vivo by Brucine, a Component of Chinese Medicine

Glioblastoma multiforme (GBM) is one of the most common glial cell tumors and has drawn more and more attention in the clinic in recent years. Brucine has been reported to significantly suppress gastric cancer, lung cancer, and prostate cancer growth in vivo by inducing cell apoptosis. Here, the effects of brucine on U251 human glioma cell growth were investigated in vitro by cell proliferation assay, FACs, and qPCR in a xenograft tumor model. Treatment with brucine reduced the expression of BCL-2 and cyclooxygenase-2 (COX-2), while upregulated BAX expression in U251 human glioma cells resulted in reduced glioma cell survival rate and inhibited the growth of xenograft tumors. We concluded that brucine has a suppressive effect on U251 human glioma cells in vitro and in vivo, which could help in understanding the role of brucine in glioma cells and guiding drug use in the clinic.

Evaluation of Effects of Metformin in Primary Ovarian Cancer Cells.

Ovarian cancer is the third most common cause of cancer in Indian women. Despite an initial 70-80% response rate, most patients relapse within 1-2 years and develop chemoresistance. Hence, identification or repositioning of drugs to resensitise ovarian cancer cells to existing chemotherapy is needed. Traditionally immortalized cell lines have been used in research, but these may contain genetic aberrations and chromosomal abnormalities serving as poor indicators of normal cell phenotype and progression of early-stage disease. The use of primary cells, maintained for only short periods of time in vitro, may serve as the best representative for studying in vivo conditions of the tissues from which they are derived. In this study we have attempted to evaluate the effect of metformin (an antidiabetic drug) in primary ovarian cancer cells because of its promising effect in other solid tumours. Primary cultures of epithelial ovarian cancer cells established from ascitic fluid of untreated ovarian cancer patients were used. The cells were treated with metformin at doses standardized by MTT assay and its ability to induce apoptosis was studied. The cells were analysed for apoptosis and apoptosis related proteins by flow cytometry and western blotting respectively. Metformin induced apoptosis in ovarian cancer cells, provoking cell cycle arrest in the G0/G1 and S phase. It induced apoptosis in ovarian cancer cells by, down-regulating Bcl-2 and up-regulating Bax expression. Metformin was able to induce apoptosis in primary ovarian cancer cells by modulating the expression of Bcl-2 family proteins. These data are relevant to ongoing translational research efforts exploring the chemotherapeutic potential of metformin.

β-Elemonic acid inhibits the cell proliferation of human lung adenocarcinoma A549 cells: The role of MAPK, ROS activation and glutathione depletion.

β-elemonic acid, a known triterpene, exhibits anti-inflammatory effects, yet research on the pharmacological effects of β-elemonic acid is rare. We investigated the anticancer effects and the related molecular mechanisms of β-elemonic acid on human non-small cell lung cancer (NSCLC) A549 cells. The effects of β-elemonic acid on the growth of A549 cells were studied using a 3-(4,5)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected using Annexin V staining. The effect of β-elemonic acid on the cell cycle of A549 cells was assessed using the propidium iodide method. The change in reactive oxygen species (ROS) was detected using a dichlorodihydrofluorescein diacetate (DCFH-DA) assay with microscopic examination. The expression levels of Bcl-2 family proteins, mitogen-activated protein kinase (MAPK) family proteins and cyclooxygenase 2 (COX-2) were detected using western blot analysis. Our data revealed that β-elemonic acid strongly induced human A549 lung cancer cell death in a dose- and time-dependent manner as determined by the MTT assay. β-elemonic acid-induced cell death was considered to be apoptotic when the phosphatidylserine exposure was observed using Annexin V staining. The death of human A549 lung cancer cells was caused by apoptosis induced by activation of ROS activity, increase in the sub-G1 proportion, downregulation of Bcl-2 expression, upregulation of Bax expression and inhibition of the MAPK signaling pathways. These results clearly demonstrated that β-elemonic acid inhibits proliferation by inducing hypoploid cells and cell apoptosis. Moreover, the anticancer effects of β-elemonic acid were related to the MAPK signaling pathway, ROS activation and glutathione depletion in human A549 lung cancer cells.