sub-G1 Cell Population Research Articles

Analysis of Selenium Levels in Osteosarcoma Patients and the Effects of Se-Methylselenocysteine on Osteosarcoma Cells In Vitro

The form of selenium appears to be important for preventing cancer in humans. Here, we evaluated selenium levels in the serum and bone tissue samples from osteosarcoma patients using atomic absorption spectrometry. The in vitro effects of Se-methylselenocysteine (Se-MSC) on growth, cell cycle status, and apoptosis of osteosarcoma cells were assessed using the WST-1 assay, Hoechst 33342/propidium iodide staining, and flow cytometry, respectively. In osteosarcoma cases, the mean serum selenium levels in osteosarcoma tissue and normal bone were 0.08 mg/kg and 0.03 mg/kg, respectively (P < 0.05). Serum selenium levels in osteosarcoma and non-osteosarcoma cases were 0.09 mg/L and 0.08 mg/L, respectively (P > 0.05). Se-MSC-treated MG63 cells showed altered cellular morphology, decreased viability in a time- and dose-dependent manner, and an increase in the sub-G1 cell population. Se-MSC also downregulated Bcl-2 expression and upregulated Bax. Se-MSC inhibited the proliferation of the drug-resistant osteosarcoma cell line Saos-2/MTX300 and enhanced the inhibitory effect of pirarubicin on MG63 cells. Our data demonstrate that selenium levels are significantly higher in osteosarcoma tissue than normal bone tissue in osteosarcoma patients. The results also support the anticancer effects of Se-MSC in osteosarcoma. Further development of Se-MSC as an ancillary chemotherapy agent in osteosarcoma is warranted.

Tumstatin induces apoptosis mediated by Fas signaling pathway in oral squamous cell carcinoma SCC-VII cells.

Oral squamous cell carcinoma is a cancer originating in the tissues lining the mouth and lips. The present study investigated the effects of recombinant tumstatin, an anti-angiogenic agent with distinct antitumor activity, on oral squamous cell carcinoma SCC-VII cells. Apoptosis was characterized by YO-PRO-1 staining, sub-G1 population, and DNA fragmentation analysis. Apoptotic mechanism of tumstatin was also investigated. The antitumor activity of tumstatin was further evaluated using an SCC-VII animal model. Recombinant tumstatin was found to decrease the viability of SCC-VII cells in a dose-dependent manner. The number of cells stained with the apoptotic marker YO-PRO-1, the sub-G1 cell population and the level of apoptotic DNA fragmentation increased in the SCC-VII cells following treatment with recombinant tumstatin. In addition, recombinant tumstatin treatment increased the expression of the Fas gene at the transcript and protein levels, and the inhibition of cell viability by recombinant tumstatin was suppressed by a neutralizing anti-Fas antibody. Furthermore, treatment with recombinant tumstatin decreased the volume and weight of tumors in C3H/HeJ mice implanted with SCC-VII cells. In conclusion, the results indicated that tumstatin induced apoptosis that is mediated by the Fas signaling pathway in SCC-VII cells and inhibited tumor growth in an SCC-VII animal model.

Ursolic acid benzaldehyde chalcone leads to inhibition of cell proliferation and arrests cycle in G1/G0 phase in ovarian cancer

&lt;p&gt;In the present study, the effect of ursolic acid benzaldehyde chalcone (UABC) on ovarian carcinoma cells was studied. The results revealed that ovarian carcinoma cells on UABC treatment increased Sub-G1 cell population, increased rate of cell apoptosis and morphological changes in mitochondrial membrane. In OVCAR 432 cells treatment with UABC increased the Sub-G1 cell population to 72.3% and growth inhibition rate of &amp;gt;72%. Treatment with 20 µM of UABC for 48 hours, led to an induction of apoptosis in 67.2% and induced morphological changes in OVCAR 432 cells. The Western blot results showed high concentration of cytochrome c in the cell cytosol after 48 hours of UABC treatment. Treatment of RMS-13 cells with UABC resulted in inhibition of GLI1, GLI2, PTCH1, and IGF2 genes. In addition, we found a significant reduction in hedgehog activity of RMS-13 cells after UABC treatment by means of a hedgehog-responsive reporter assay. Therefore, UABC can be a promising agent for the treatment of ovarian carcinoma.&lt;/p&gt;

Indole-3-carbinol as inhibitors of glucocorticoid-induced apoptosis in osteoblastic cells through blocking ROS-mediated Nrf2 pathway

Apoptosis of osteoblasts induced by glucocorticoid (GC) has been identified as a main cause of osteoporosis, bone loss and fractures, and the oxidative stress was found as an important contributor. Therefore, natural or synthetic agents with antioxidant activities can antagonize GCs-induced apoptosis in osteoblasts, and thus demonstrate the potential application to reverse osteoporosis. In this study, we showed that, indole-3-carbinol (I3C), a natural product found in broadly consumed plants of the Brassica genus, could block the cytotoxic effects of dexamethasone (Dex), and elucidated the underlying molecular mechanisms. Firstly, we showed that, I3C could effectively suppress Dex-induced cytotoxicity and apoptotic cell death in osteoblastic cells, as evidenced by the decrease in Sub-G1 cell population. Treatment of the cells with Dex resulted in activation of caspase-3/-8/-9 and subsequent cleavage of PARP, which was also effectively blocked by co-incubation of I3C. Moreover, exposure to Dex triggered a rapid onset and time-dependent superoxide overproduction in osteoblastic cells, which was effectively suppressed by addition of I3C. Excess intracellular ROS induced by Dex significantly suppressed the expression levels of Nrf2 and the downstream effectors, HO1 and NQO1, but these changes could be reversed by I3C. Knockdown of Nrf2 using siRNA silencing technique significantly reversed the protective effects of I3C against Dex-induced apoptosis and ROS generation. Taken together, I3C can reverse cytotoxicity of Dex through blocking ROS overproduction and enhancement of Nrf2 expression. This study may provide a safe and good strategy for molecular intervention of GCs-induced osteoporosis by using natural products.

Liposomal co-delivery of curcumin and albumin/paclitaxel nanoparticle for enhanced synergistic antitumor efficacy

Paclitaxel (PTX) and curcumin (CUR) are potent chemotherapeutic agents used in the treatment of cancer. In the present study, hybrid polymer–lipid nanoparticles co-loaded with PTX and CUR were developed to investigate the therapeutic potential of a combination drug regimen. For this purpose, PTX-loaded albumin nanoparticles (APN) were prepared and encapsulated in PEGylated hybrid liposomes containing CUR (CL-APN) via a thin-film hydration technique. CL-APN was nanosized with a uniform spherical morphology. PTX and CUR release was sustained and occurred in a sequential manner, wherein CUR was expected to downregulate the nuclear factor NF-κB and Akt pathways and increase the therapeutic efficacy of PTX. The ratiometric combination of PTX and CUR was significantly more cytotoxic than the individual drugs. Importantly, dual-drug-loaded nanocarriers exhibited a superior cytotoxic effect than a cocktail combination at a lower dose. CL-APN induced significantly higher early and late apoptosis, induced a stronger G2/M arrest, and significantly increased the subG1 cell population. By combining CUR, an effective NF-κB inhibitor, with PTX, a powerful anticancer drug, in a polymer–lipid hybrid nanoparticle system, we could improve the therapeutic efficacy in cancer treatments. Our results showed that such co-loaded delivery systems could serve as a promising therapeutic approach to improve clinical outcomes against various malignancies.

Epigenetic Regulation of KLHL34 Predictive of Pathologic Response to Preoperative Chemoradiation Therapy in Rectal Cancer Patients

Prediction of individual responsiveness to preoperative chemoradiation therapy (CRT) is urgently needed in patients with poorly responsive locally advanced rectal cancer (LARC). Candidate methylation genes associated with radiosensitivity were identified using a 3-step process. In the first step, genome-wide screening of methylation genes was performed in correlation with histopathologic tumor regression grade in 45 patients with LARC. In the second step, the methylation status of selected sites was analyzed by pyrosequencing in 67 LARC patients, including 24 patients analyzed in the first step. Finally, colorectal cancer cell clones with stable KLHL34 knockdown were generated and tested for cellular sensitivity to radiation. Genome-wide screening identified 7 hypermethylated CpG sites (DZIP1 cg24107021, DZIP1 cg26886381, ZEB1 cg04430381, DKK3 cg041006961, STL cg00991794, KLHL34 cg01828474, and ARHGAP6 cg07828380) associated with preoperative CRT responses. Radiosensitivity in patients with hypermethylated KLHL34 cg14232291 was confirmed by pyrosequencing in additional cohorts. Knockdown of KLHL34 significantly reduced colony formation (KLHL34 sh#1: 20.1%, P=.0001 and KLHL34 sh#2: 15.8%, P=.0002), increased the cytotoxicity (KLHL34 sh#1: 14.8%, P=.019 and KLHL34 sh#2: 17.9%, P=.007) in LoVo cells, and increased radiation-induced caspase-3 activity and the sub-G1 population of cells. The methylation status of KLHL34 cg14232291 may be a predictive candidate of sensitivity to preoperative CRT, although further validation is needed in large cohorts using various cell types.

Effects of thonningianin A in natural foods on apoptosis and cell cycle arrest of HepG-2 human hepatocellular carcinoma cells.

The anti-cancer activities of Thonningianin A on the HepG-2 human hepatocellular carcinoma cell line were evaluated by MTT assay, flow cytometry, quantitative real-time PCR and western blotting. Results showed that Thonningianin A effectively inhibited the proliferation of HepG-2 cells by inducing apoptosis, as evidenced by increase in the sub-G1 cell population, DNA fragmentation, and increase in the content of reactive oxygen species. Activation of caspase-9 and the subsequent activation of caspase-3 indicated that Thonningianin A-induced apoptosis is caspase-dependent. Thonningianin A also disrupted the mitochondrial membrane potential (Δψm) and down-regulated the Bcl-xL mRNA expression in HepG-2 cells. Thonningianin A induced cell cycle arrest by changing the cyclin D1 and CDK4 mRNA expression levels. Moreover, western blotting showed that Thonningianin A significantly down-regulated the NF-kappa-B cell survival pathway, along with up-regulation of the expression level of phosphorylated P38 and down-regulation of the expression level of phosphorylated ERK. The anti-cancer activity of Thonningianin A was confirmed by the characteristic patterns of DNA fragmentation and cell cycle arrest, suggesting that Th A is an effective antitumor ingredient in natural plant foods, and is worthy of further study.

Pfaffosidic Fraction from Hebanthe paniculata Induces Cell Cycle Arrest and Caspase-3-Induced Apoptosis in HepG2 Cells.

Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng) show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A–F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27KIP1 overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27KIP1 overexpression, besides induction of apoptosis through caspase-3 activation.

Inhibitory activities of Perilla frutescens britton leaf extract against the growth, migration, and adhesion of human cancer cells.

BACKGROUND/OBJECTIVESPerilla frutescens Britton leaves are a commonly consumed vegetable in different Asian countries including Korea. Cancer is a major cause of human death worldwide. The aim of the current study was to investigate the inhibitory effects of ethanol extract of perilla leaf (PLE) against important characteristics of cancer cells, including unrestricted growth, resisted apoptosis, and activated metastasis, using human cancer cells.MATERIALS/METHODSTwo human cancer cell lines were used in this study, HCT116 colorectal carcinoma cells and H1299 non-small cell lung carcinoma cells. Assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were performed for measurement of cell growth. Soft agar and wound healing assays were performed to determine colony formation and cell migration, respectively. Nuclear staining and cell cycle analysis were performed for assessment of apoptosis. Fibronectin-coated plates were used to determine cell adhesion.RESULTSTreatment of HCT116 and H1299 cells with PLE resulted in dose-dependent inhibition of growth by 52-92% (at the concentrations of 87.5, 175, and 350 µg/ml) and completely abolished the colony formation in soft agar (at the concentration of 350 µg/ml). Treatment with PLE at the 350 µg/ml concentration resulted in change of the nucleus morphology and significantly increased sub-G1 cell population in both cells, indicating its apoptosis-inducing activity. PLE at the concentration range of 87.5 to 350 µg/ml was also effective in inhibiting the migration of H1299 cells (by 52-58%) and adhesion of both HCT116 and H1299 cells (by 25-46%).CONCLUSIONSThese results indicate that PLE exerts anti-cancer activities against colon and lung cancers in vitro. Further studies are needed in order to determine whether similar effects are reproduced in vivo.

Doxorubicin synergizes with 34.5ENVE to enhance antitumor efficacy against metastatic ovarian cancer.

Novel therapeutic regimens are needed to improve dismal outcomes associated with late-stage ovarian cancer. Oncolytic viruses are currently being tested in patients with ovarian cancer. Here, we tested the therapeutic efficacy of combining doxorubicin with 34.5ENVE, an oncolytic herpes simplex virus transcriptionally driven by a modified stem cell-specific nestin promoter, and encoding for antiangiogenic Vasculostatin-120 (VStat120) for use against progressive ovarian cancer. Antitumor efficacy of 34.5ENVE was assessed in ovarian cancer cell lines, mouse ascites-derived tumor cells, and primary patient ascites-derived tumor cells by standard MTT assay. The ability of conditioned medium derived from 34.5ENVE-infected ovarian cancer cells to inhibit endothelial cell migration was measured by a Transwell chamber assay. Scope of cytotoxic interactions between 34.5ENVE and doxorubicin were evaluated using Chou-Talalay synergy analysis. Viral replication, herpes simplex virus receptor expression, and apoptosis were evaluated. Efficacy of oncolytic viral therapy in combination with doxorubicin was evaluated in vivo in the murine xenograft model of human ovarian cancer. Treatment with 34.5ENVE reduced cell viability of ovarian cancer cell lines, and mouse ascites-derived and patient ascites-derived ovarian tumor cells. Conditioned media from tumor cells infected with 34.5ENVE reduced endothelial cell migration. When combined with doxorubicin, 34.5ENVE killed synergistically with a significant increase in caspase-3/7 activation, and an increase in sub-G1 population of cells. The combination of doxorubicin and 34.5ENVE significantly prolonged survival in nude mice bearing intraperitoneal ovarian cancer tumors. This study indicates significant antitumor efficacy of 34.5ENVE alone, and in combination with doxorubicin against disseminated peritoneal ovarian cancer.

Combination Treatment of Renal Cell Carcinoma with Belinostat and 5-Fluorouracil: A Role for Oxidative Stress Induced DNA Damage and HSP90 Regulated Thymidine Synthase

Despite several therapeutic options renal cell carcinoma is associated with a poor clinical outcome. Therefore, we investigated whether combining 5-fluorouracil with the histone deacetylase inhibitor belinostat would exert a synergistic effect on renal cell carcinoma cells invitro and invivo. We used SN12C cells treated with 5-fluorouracil and/or belinostat invitro and in xenograft experiments invivo. Cell viability and death mechanisms were assessed by MTS assay and Western blot. To investigate the role of reactive oxygen species we used H2DCF-DA, reactive oxygen species scavengers and the roGFP2 construct. Belinostat potentiated the anticancer effect of 5-fluorouracil. It synergistically induced apoptosis by activating caspases and increasing the subG1 cell population. Effects on reactive oxygen species mediated DNA damage included decreased thioredoxin expression and increased levels of TBP-2, γ-H2AX and Ac-H3. Furthermore, belinostat attenuated the 5-fluorouracil mediated induction of thymidylate synthase via HSP90 hyperacetylation. Co-administration of 5-fluorouracil with belinostat similarly reduced tumor volume and weight, and increased γ-H2AX and Ac-H3 levels in the SN12C xenograft model. In combination with 5-fluorouracil the targeted inhibitor of histone deacetylase synergistically inhibited renal cancer cell growth by the blockade of thymidylate synthase induction and the induction of reactive oxygen species mediated DNA damage invitro and invivo. Our results suggest that combined treatment with belinostat and 5-fluorouracil may represent a promising new approach to renal cancer.

Withaferin A induces apoptosis through the generation of thiol oxidation in human head and neck cancer cells.

Withaferin A is a steroidal lactone purified from the Indian medicinal plant, Withania somnifera. Withaferin A has been shown to inhibit the proliferation, metastasis, invasion and angiogenesis of cancer cells. In the present study, we investigated whether withaferin A induces apoptosis in the human head and neck cancer cells, AMC-HN4. Withaferin A markedly increased the sub-G1 cell population and the cleavage of poly(ADP-ribose) polymerase (PARP), which are markers of apoptosis. Pan-caspase inhibitor, z-VAD-fmk (z-VAD), markedly inhibited the withaferin A-induced apoptosis. However, the withaferin A-induced increase in the expression of COX-2 was not affected by treatment with z-VAD. Furthermore, withaferin A upregulated cyclooxygenase-2 (COX-2) expression. The COX-2 inhibitor, NS-398, reduced the withaferin A-induced production of prostaglandin E2. However, treatment with NS-398 did not affect the sub-G1 population and the cleavage of PARP. In addition, the withaferin A-induced apoptosis was independent of reactive oxygen species production. Thiol donors [N-acetylcysteine (NAC) and dithiothreitol (DTT)] reversed withaferin A-induced apoptosis. Therefore, our data suggest that withaferin A induces apoptosis through the mechanism of thiol oxidation in head and neck carcinoma cells.

Sensitization of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant primary prostate cancer cells by isoegomaketone from Perilla frutescens.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently in clinical trials as a cancer treatment due to its ability to induce apoptosis selectively in cancer cells. Nevertheless, the risk of developing resistance warrants the development of sensitizers that can overcome resistance to TRAIL. In this study, isoegomaketone (1) acted as a synergistic TRAIL sensitizer by mediating up-regulation of DR5 expression in primary prostate cancer RC-58T/h/SA#4 cells. Combined with 1, TRAIL exhibited enhanced apoptotic activity in a human prostate cancer cell line designated RC-58T/h/SA#4, as indicated by increases in annexin V-positive and sub-G1 cell populations as well as condensation of chromatin or apoptotic bodies. Combined treatment also activated caspases-8, -9, and -3; increased the protein levels of Bax, AIF, and cytosolic cytochrome c; and induced PARP cleavage while reducing Bcl-2 protein expression. Human recombinant DR5 Fc chimera efficiently attenuated 1-induced apoptosis, thereby demonstrating the critical role of DR5 in 1-mediated apoptotic cell death. Furthermore, DR5 expression induced by 1 was mediated via a ROS-independent pathway that required CHOP and p53. Overall, these findings provide evidence that 1 potentiates TRAIL-mediated apoptosis through up-regulation of DR5 via a ROS-independent pathway. This suggests that 1 has potential for increasing the effectiveness of prostate cancer therapy with TRAIL.

인간 대장암 HT-29 세포에서 제주조릿대의 세포사멸 효과

Sasa quelpaertensis Nakai (Korean name, Jeju-Joritdae) is one of the most abundant plants on Mt. Halla, Jeju Island, and it has long been used in traditional medicines. Recent studies have reported it as possessing various beneficial functions, including anti-inflammatory, anti-diabetic, anti-hypertension, anti-gastritis, anti-oxidant, and anti-cancer effects. However, the molecular mechanisms of its anti-cancer activity have not been clearly elucidated. In this study, we investigated the anti-cancer effects and mechanism of S. quelpaertensis on human colon cancer HT-29 cells. Cell growth inhibition by S. quelpaertensis was determined by MTT assay. Apoptosis was performed by DNA fragmentation, flow cytometry with propidium iodide staining (PI), and reverse transcription-polymerase chain reaction (RT-PCR) to confirm the anti-apoptotic factors, such as inhibitor of apoptosis (IAP) family members. NO • production was determined by Griess assay. S. quelpaertensis treatment resulted in the timeand dose-dependent inhibition of the cell viability of HT-29 cells by inducing apoptosis, as evidenced by the accumulation of the sub-G1 cell population stained by PI, as well as the ladder-like DNA fragmentation in a dose-dependent manner. S. quelpaertensis-inducing apoptosis was accompanied by the induction of S cell cycle arrests, increasing NO concentrations, and the down-regulation of IAPs, including X-chromosome-linked IAP (XIAP), cellular IAP-1 (cIAP-1), cIAP-2, and survivin. Taken together, these findings have important implications for future clinical developments of S. quelpaertensis in colon cancer treatment.

Bergamottin, a natural furanocoumarin obtained from grapefruit juice induces chemosensitization and apoptosis through the inhibition of STAT3 signaling pathway in tumor cells

Persistent activation of signal transducers and activator of transcription 3 (STAT3) has been closely related to growth, survival, proliferation, metastasis, and angiogenesis of various cancer cells, and thus its inhibition can be considered a potential therapeutic strategy. In this study, we investigated the role of bergamottin (BGM) obtained from grapefruit juice in abrogating the constitutive STAT3 activation in multiple myeloma (MM) cells. This suppression was mediated through the inhibition of phosphorylation of Janus-activated kinase (JAK) 1/2 and c-Src. Pervanadate reversed the BGM induced down-regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, BGM induced the expression of the tyrosine phosphatase SHP-1, and gene silencing of the SHP-1 by small interfering RNA abolished the ability of BGM to inhibit STAT3 activation, suggesting a critical role for SHP-1 in the action of BGM. BGM also downregulated the expression of STAT3-regulated gene products such as COX-2, VEGF, cyclin D1, survivin, IAP-1, Bcl-2, and Bcl-xl in MM cells. This correlated with induction of substantial apoptosis as indicated by an increase in the sub-G1 cell population and caspase-3 induced PARP cleavage. Also, this agent significantly potentiated the apoptotic effects of bortezomib and thalidomide in MM cells. Overall, these results suggest that BGM is a novel blocker of STAT3 activation pathway thus may have a potential in therapy of MM and other cancers.

Involvement of mitogen-activated protein kinase pathway in T-2 toxin-induced cell cycle alteration and apoptosis in human neuroblastoma cells.

T-2 toxin is the most toxic trichothecene and a frequent contaminant in many agriculture products. Dietary ingestion represents the most common route of T-2 toxin exposure in humans. T-2 toxin exposure leads to many pathological conditions like nervous disorders, cardiovascular alterations, immune depression and dermal inflammation. However, the neuronal toxicity of T-2 toxin in vitro remains unclear. In the present study, we investigated the mechanism of T-2 toxin-induced apoptosis in human neuroblastoma cells (IMR-32). T-2 toxin was cytotoxic at a low concentration of 10 ng/ml. The 50% inhibitory concentration (IC50) of T-2 toxin was found to be 40 ng/ml as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, crystal violet dye exclusion test and lactate dehydrogenase (LDH) leakage. T-2 toxin increased intracellular reactive oxygen species generation as early as 15 min and peaked at 60 min as analyzed by flow cytometry. Annexin V + propidium iodide staining showed time-dependent increase in percent apoptotic cells. DNA gel electrophoresis showed oligonucleosomal DNA fragmentation typical of apoptotic cells. Additionally, casapse-3 activation and PARP cleavage indicated involvement of mitochondrial mediated caspase-dependent pathway of apoptosis. Cell cycle analysis revealed time-dependent increase in sub-G1 population of cells and significant up-regulation of CDK2, CDK6, cyclin A and p21 messenger RNA (mRNA) levels. Exposure to T-2 toxin induced the phosphorylation of extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase and c-jun N-terminal kinases (JNK). Analysis of human phospho-mitogen-activated protein kinase (MAPK) antibody array revealed time-dependent increase in phosphorylation. Upstream of ERK pathway Grb2, Ras and Raf and downstream transcription factors c-fos and c-jun were significantly up-regulated. Z-VAD-FMK and MAPK inhibitors (PD 98059, SB 203580 and ZM 336372) exposure prior to T-2 toxin treatment significantly decreased percent of apoptotic cells compared to only T-2 toxin-exposed cells. Results of the present study show that T-2 toxin at nanogram concentrations can induce apoptosis in human neuronal cells through multiple signal transduction pathways. The study provides possible leads for developing therapeutic approaches to prevent T-2 toxin-induced neurotoxicity.

Synergistic Apoptosis-Inducing Effects on A375 Human Melanoma Cells of Natural Borneol and Curcumin

This study was to investigate the synergistic effect of NB/Cur on growth and apoptosis in A375 human melanoma cell line by MTT assay, flow cytometry and Western blotting. Our results demonstrated that NB effectively synergized with Cur to enhance its antiproliferative activity on A375 human melanoma cells by induction of apoptosis, as evidenced by an increase in sub-G1 cell population, DNA fragmentation, PARP cleavage and caspase activation. Further mechanistic studies by Western blotting showed that after treatment of the cells with NB/Cur, up-regulation of the expression level of phosphorylated JNK and down-regulation of the expression level of phosphorylated ERK and Akt contributed to A375 cells apoptosis. Moreover, NB also potentiated Cur to trigger intracellular ROS overproduction and the DNA damage with up-regulation of the expression level of phosphorylated ATM, phosphorylated Brca1 and phosphorylated p53. The results indicate the combinational application potential of NB and Cur in treatments of cancers.

Antitumor activity and induction of TP53-dependent apoptosis toward ovarian clear cell adenocarcinoma by the dual PI3K/mTOR inhibitor DS-7423.

DS-7423, a novel, small-molecule dual inhibitor of phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR), is currently in phase I clinical trials for solid tumors. Although DS-7423 potently inhibits PI3Kα (IC50 = 15.6 nM) and mTOR (IC50 = 34.9 nM), it also inhibits other isoforms of class I PI3K (IC50 values: PI3Kβ = 1,143 nM; PI3Kγ = 249 nM; PI3Kδ = 262 nM). The PI3K/mTOR pathway is frequently activated in ovarian clear cell adenocarcinomas (OCCA) through various mutations that activate PI3K-AKT signaling. Here, we describe the anti-tumor effect of DS-7423 on a panel of nine OCCA cell lines. IC50 values for DS-7423 were <75 nM in all the lines, regardless of the mutational status of PIK3CA. In mouse xenograft models, DS-7423 suppressed the tumor growth of OCCA in a dose-dependent manner. Flow cytometry analysis revealed a decrease in S-phase cell populations in all the cell lines and an increase in sub-G1 cell populations following treatment with DS-7423 in six of the nine OCCA cell lines tested. DS-7423-mediated apoptosis was induced more effectively in the six cell lines without TP53 mutations than in the three cell lines with TP53 mutations. Concomitantly with the decreased phosphorylation level of MDM2 (mouse double minute 2 homolog), the level of phosphorylation of TP53 at Ser46 was increased by DS-7423 in the six cell lines with wild-type TP53, with induction of genes that mediate TP53-dependent apoptosis, including p53AIP1 and PUMA at 39 nM or higher doses. Our data suggest that the dual PI3K/mTOR inhibitor DS-7423 may constitute a promising molecular targeted therapy for OCCA, and that its antitumor effect might be partly obtained by induction of TP53-dependent apoptosis in TP53 wild-type OCCAs.

A novel selenadiazole derivative induces apoptosis in human glioma cells by dephosphorylation of AKT.

Selenadiazole derivatives are synthetic organoselenium compounds with improved anticancer activity and greater selectivity than inorganic selenium. In this study, 4-(benzo[c][1,2,5]selenadiazol-6-yl)-benzene-1,2-diamine (BSBD) was shown to induce time- and dose-dependent apoptosis in SWO-38 human glioma cells by accumulation of a sub-G1 cell population, DNA fragmentation, nuclear condensation, caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage. Further mechanistic investigation showed that BSBD treatment induced dephosphorylation of AKT and DNA damage-mediated activation of p53, leading to extensive apoptosis through the mitochondrial pathway. Our findings suggest that BSBD represents a potential human glioma therapeutic.

Antiproliferative effects of n-butyl-β-D-fructofuranoside from Kangaisan on Bel-7402 cells

Aims:Kangaisan is a powdered compound prescription of Traditional Chinese Medicine which has been used in cancers for many years in Hubei province, China. The purpose of this study was to investigate the antitumor effects of Kangaisan and screen bioactive components.Materials and Methods:3-(4,5-Dimethythiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, DNA (Deoxyribonucleic acid) fragmentation assay, Western blot, and real time-polymerase chain reaction were used to investigate the antiproliferation effect of n-butyl-β-D-fructofuranoside on Bel-7402 cells.Statistical Analysis:All experiments were performed in triplicate and the results were expressed as mean ± standard deviation. Statistical analysis was performed with analysis of variance using Origin 8.0 software.Results:It was illustrated that treatment of Bel-7402 cells with various concentrations of n-butyl-β-D-fructofuranoside resulted in growth inhibition in both a dose-dependent and time-dependent manner. The arrest of G0/G1 phase was also induced (P < 0.05). The increasing of sub-G1 cell population indicated the apoplectic characteristic (P < 0.05). Furthermore, the emerging of DNA fragmentation and the increase of Bax/Bcl-2 ratio and p53 expression suggested the possible mitochondrial apoptotic pathway (P < 0.05).Conclusions:The results illustrate that Kangaisan showed anticancer effects and n-butyl-β-D-fructofuranoside extracted from Kangaisan can suppress Bel-7402 cells via interfering cell cycle and by inducing apoptosis.