BACKGROUND: Normal pubertal ovary displays all stages of follicular development and a biased BAX/BCL2 protein ratio in favor of pro-apoptotic BAX protein comparable to the adult ovary. However, adolescents suffering malignant extra-gonadal disease show a limited follicle development after cytotoxic drug treatment and a reduced capacity of in vitro follicle growth. We evaluated the expression of pro- and anti-apoptotic members of the BCL2 gene family, the FAS/FAS-L proteins from the extrinsic apoptosis pathway, the germ-cell-specific marker VASA, the pluripotency marker OCT3/4, and markers of early and late apoptosis in the ovary of pubertal patients with malignant extra-gonadal disease, which received or not pre-surgery chemotherapy, entering a cryopreservation program.
RESULTS: Ovarian biopsies from 12 adolescent girls were screened for follicle count and expression of VASA, OCT3/4, BAX, BCL2, MCL1L and S, cleaved-BID, FAS/FAS-L and CASPASE 3 through immunohistochemistry, western blot and RT-PCR. All stages of folliculogenesis, from primordial to antral follicle, were present in all 12 patients analyzed. VASA and most of the screened apoptosis-related genes showed a pattern of immune-expression comparable to that previously reported. OCT3/4 showed a cytoplasmic localization in the great majority of the primordial follicles; however, in some cases the localization was nuclear. In addition, OCT3/4B showed a significant reduction compared to OCT3/4A. Unexpectedly, BCL2 was detected at all stages of folliculogenesis, associated to the Balbiani's body in the primordial follicles, regardless of whether patients had or had not received chemotherapy, ruling out the possibility that its expression is a protective response to chemotherapy.
CONCLUSIONS: These findings reveal new information on the morphological status of the follicular reserve and the expression of apoptosis-related genes in histologically normal adolescent ovary from patients undergoing extragonadal cancer. The unexpected expression of apoptosis-inhibiting BCL2 protein, both in patients that had or had not received chemotherapy, opens a new avenue for thorough investigations. Moreover, the nuclear localization of OCT3/4 protein in primordial follicle-enclosed oocytes suggests a possible increased activity of ovarian stem cells in response to chemotherapy and/or extragonadal cancer. This new information can be essential for a better managing of in vitro culture of follicles that can be removed by filtration from preserved ovarian tissue, especially in girls that entered a cryopreservation program.

The present research seeks to investigate the process of mixing targeted gene delivery and transcriptional targeting. We have conjugated Polyethylenimine polymers (PEI) and molecules of poly (ethylene glycol). The next step was covalent attachment of anti-HER2 variables domains of camelid heavy chains antibodies (VHHs) or nanobodies (Nbs) to the distal terminals of NHS-PEG3500 in PEI-PEG nanoparticles. The whole procedure yielded PEI-PEG-Nb immunoconjugates. Having determined the properties of polyplexes, steps were taken to investigate the most efficient ratio of PEI polymers to pDNA molecules (N/P) so that the greatest rate of transfection may be obtained. This immune targeted nano biopolymer could condense the gene constructs that coded a transcriptionally targeted truncated -Bid (tBid) killer gene which was controlled by the breast cancer-specific MUC1 promoter. The favourable physicochemical properties matching both the size and zeta potential were observed in engineered polyplexes. Elevated transfection efficiency in HER2 positive cell lines using Nb-modified polyplexes were shown by the results of flow cytometry as compared against non-modified particles. 1.6 and 4.8 fold higher transfection efficiencies were observed in in vitro gene expression researches which used PEI-PEG-Nb/pGL4.50 compared to the situation when native PEI polymers were utilized in both BT-474 and SK-BR-3, respectively. A 2.22 and 3.62 fold rise in the level of tBid gene expression in BT-474 and SK-BR-3 cell lines relative to unmodified PEI treated cells was the result of transfection with PEI-PEG-Nb/pMUC1-tBid, respectively. In those HER2-positive cells which were transfected by targeted polyplexes, higher levels of cell death were observed. This fact points not only to the effective targeted delivery, but it is also indicative of transcriptional targeting efficiency of tBid killer gene when its expression is controlled by MUC1 promoter.

BACKGROUND: Locally advanced pancreatic cancer (LAPC) has a dismal prognosis with current treatment modalities and one-third of patients die from local progression of disease. Preclinical studies with orthotopic PC demonstrated dramatic synergy between radiotherapy (RT) and the poly(ADP-ribose) polymerase-1/2 inhibitor (PARPi), veliparib. We conducted a phase I trial of gemcitabine, radiotherapy and dose-escalated veliparib in LAPC.
METHODS: This was a single institution investigator-initiated open-label, single-arm phase 1 clinical trial (NCT01908478). Weekly gemcitabine with daily IMRT and veliparib dose escalated using a Bayesian adaptive design were administered in treatment naïve LA or borderline resectable PC. The primary end point was identification of the MTD. Secondary endpoints included efficacy, characterization of PAR levels using ELISA, DDR alterations with targeted next generation sequencing and transcriptome analysis, tumor mutation burden (TMB) and microsatellite instability (MSI) status.
FINDINGS: Thirty patients were enrolled. The MTD of veliparib was 40 mg BID with gemcitabine 400 mg/m
INTERPRETATION: This is the first report of a PARPi-chemoradiotherapy combination in PC. The regimen was safe, tolerable at the RP2D, and clinically active as an upfront treatment strategy in patients biologically unselected by upfront chemotherapy. Expression of the DDR transcripts, PARP3 and RBX1, were associated with OS suggesting validation in a follow up phase 2 study. FUND: Phase One Foundation; National Institutes of Health [1R01CA188480-01A1, P01 CA098912]. Veliparib was provided by Abbvie.

BACKGROUND: Licochalconce (LC) H is an artificial compound in the course of synthesizing LCC in 2013. So far, few studies on the effects of LCH have been found in the literature. Despite progress in treatment modalities for oral cancer, the cure from cancer has still limitations.
PURPOSE: The effects of LCH were investigated on human oral squamous cell carcinoma (OSCC) cells to elucidate its mechanisms.
STUDY DESIGN: We explored the mechanism of action of LCH by which it could have effects on JAK2/STAT3 signaling pathway.
METHODS: To confirm LCH anti-cancer effect, analyzed were MTT assay, DAPI staining, soft agar, kinase assay, molecular docking simulation, flow cytometry and Western blotting analysis.
RESULTS: According to docking and molecular dynamics simulations, the predicted pose of the complex LCH and JAK2 seems reasonable and LCH is strongly bound to active JAK2 with opened activation loop. The LCH inhibitor is surrounded by specific ATP-binding pocket in which it is stabilized by forming hydrogen bonds and hydrophobic interactions. It is shown that LCH plays as a competitive inhibitor in an active state of JAK2. LCH caused a dose-dependent decrease in phosphorylation of JAK2 and STAT3. More interestingly, LCH suppressed JAK2 kinase activity in vitro by its direct binding to the JAK2. LCH significantly inhibited the JAK2/STAT3 signaling pathway, causing the down-regulation of target genes such as Bcl-2, survivin, cyclin D1, p21 and p27. In addition, LCH inhibited cell proliferation and colony formation of OSCC cells in a dose- and time-dependent manner, as well as induction of cell apoptosis through extrinsic and intrinsic pathway. The induction of apoptosis in OSCC cells by LCH was evident in the increased production of ROS, loss of mitochondrial membrane potential, release of cyto c, variation of apoptotic proteins and activation of caspase cascade.
CONCLUSION: LCH not only induces apoptosis in OSCC cells through the JAK/STAT3 signaling pathway but also inhibits cell growth. It is proposed that LCH has a promising use for the chemotherapeutic agent of oral cancer.

Primary gastric lymphoma (PGL) represents a rare pathology, which can be easily misdiagnosed because of unspecific symptoms of the digestive tract. Histologically, PGL can vary from indolent marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) to aggressive diffuse large B-cell lymphoma (DLBCL). During the years, clinical trials revealed the important role of Helicobacter pylori (H. pylori) in the pathogenesis of gastric MALT lymphoma. Infection with Helicobacter pylori is an influential promoter of gastric lymphomagenesis initiation. Long-term studies revealed that eradication therapy could regress gastric lymphomas.

PURPOSE: We have shown that GPC3 overexpression in breast cancer cells inhibits in vivo tumor progression, by acting as a metastatic suppressor. GPC3-overexpressing cells are less clonogenic, viable and motile, while their homotypic adhesion is increased. We have presented evidences indicating that GPC3 inhibits canonical Wnt and Akt pathways, while non-canonical Wnt and p38MAPK cascades are activated. In this study, we aimed to investigate whether GPC3-induced Wnt signaling inhibition modulates breast cancer cell properties as well as to describe the interactions among pathways modulated by GPC3.
METHODS: Fluorescence microscopy, qRT-PCR microarray, gene reporter assay and Western blotting were performed to determine gene expression levels, signaling pathway activities and molecule localization. Lithium was employed to activate canonical Wnt pathway and treated LM3-GPC3 cell viability, migration, cytoskeleton organization and homotypic adhesion were assessed using MTS, wound healing, phalloidin staining and suspension growth assays, respectively.
RESULTS: We provide new data demonstrating that GPC3 blocks-also at a transcriptional level-both autocrine and paracrine canonical Wnt activities, and that this inhibition is required for GPC3 to modulate migration and homotypic adhesion. Our results indicate that GPC3 is secreted into the extracellular media, suggesting that secreted GPC3 competes with Wnt factors or interacts with them and thus prevents Wnt binding to Fz receptors. We also describe the complex network of interactions among GPC3-modulated signaling pathways.
CONCLUSION: GPC3 is operating through an intricate molecular signaling network. From the balance of these interactions, the inhibition of breast metastatic spread induced by GPC3 emerges.

Quercetin is one of the natural components from natural plant and it induces cell apoptosis in many human cancer cell lines. However, no available reports show that quercetin induces apoptosis and altered associated gene expressions in human gastric cancer cells, thus, we investigated the effect of quercetin on the apoptotic cell death and associated gene expression in human gastric cancer AGS cells. Results indicated that quercetin induced cell morphological changes and reduced total viability via apoptotic cell death in AGS cells. Furthermore, results from flow cytometric assay indicated that quercetin increased reactive oxygen species (ROS) production, decreased the levels of mitochondrial membrane potential (ΔΨ

AIMS: Luteolin, a naturally occurring flavonoid, possesses anti-cancer effects including induction of apoptosis. This study investigated the involvement of osteopontin (OPN) in luteolin-induced apoptosis in human hepatocellular carcinoma (HCC) SK-Hep-1 cells with high OPN expression.
MAIN METHODS: MTT assay was used to determine the cell viability. Cell cycle analysis was performed to identify apoptosis. Apoptosis was confirmed by detecting cytoplasmic histone-associated-DNA-fragments using a cell death detection ELISA
KEY FINDINGS: Cytotoxic effect of luteolin was higher in cancer cell line SK-Hep-1 cells than in normal cell line AML12 cells. Luteolin led a significantly increase in apoptosis accompanied by activation of caspase 8, -9 and -3 and cleavage of poly (ADP-ribose) polymerase (PARP), which was completely blocked by Z-VAD-FMK, a pan caspase inhibitor. Luteolin significantly downregulated the expression of X-linked inhibitor of apoptosis (XIAP), Mcl-1 and Bid. Furthermore, luteolin effectively decreased OPN expression at both mRNA and protein level. Exogenous OPN markedly blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of XIAP and Mcl-1 in luteolin-treated cells. Luteolin impaired the AKT pathway by inhibiting the phosphorylation of AKT. SC79, an AKT activator, blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of OPN, XIAP, Mcl-1 and Bid in luteolin-treated cells.
SIGNIFICANCE: These results demonstrated that luteolin inhibits the AKT/OPN pathway, thereby inducing caspase-dependent apoptosis in human HCC SK-Hep-1 cells with little toxicity.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the most common human malignant diseases in the world, and the mechanisms underlying HCC carcinogenesis and progression need further investigation. MicroRNAs play important roles in the development of cancer, and miR-500a is suggested to be deregulated in some types of cancer. However, the underlying molecular mechanisms of miR-500a in HCC remain unknown.
METHODS: The expression of miR-500a in HCC was analyzed in The Cancer Genome Atlas (TCGA) database and examined in 33 pairs of HCC tissues and matched nontumor tissues. The correlation between miR-500a expression and prognosis of HCC patients was analyzed from the survival data in TCGA. The mechanism of miR-500a upregulation in HCC was detected using chromatin immunoprecipitation-quantitative real-time PCR. The roles of miR-500a in HCC development were examined using a cell counting kit-8 assay in vitro and growth of transplanted tumors in nude mice in vivo. Apoptosis of HCC was detected using Annexin V/propidium iodide staining. The expression of BH3-interacting death agonist (BID) protein was examined using western blot analysis.
RESULTS: miR-500a expression was upregulated in HCC tissues, and high miR-500a expression was significantly correlated with the poor prognosis of HCC patients. Histone modifications in the promoter region of miR-500a may be responsible for its increased expression. Inhibition of miR-500a in HCC cell lines significantly promoted apoptosis, as well as inhibiting the proliferation of HCC cells and growth of transplanted tumors in nude mice. miR-500a directly targeted the 3' untranslated region of BID mRNA, and inhibition of miR-500a-promoted apoptosis was almost completely abolished by the administration of ABT-199 via the BID-mitochondria pathway.
CONCLUSION: Our results suggest that histone modifications in the promoter region of miR-500a may be responsible for the increased expression of miR-500a in HCC, which promotes cancer progression by targeting BID, indicating that miR-500a may be a potential prognostic predictor and therapeutic target for HCC patients.

l-asparagine essentially regulates growth and proliferation of cancer cells. l-asparaginase is an anti-cancer enzyme that deprives the cancer cells of l-asparagine. The purpose of this study was to explore the mechanism of a novel l-asparaginase from Pseudomonas fluorescens on l-asparagine deprivation mediated anti-proliferation, apoptosis in human gastric adenocarcinoma cells and to evaluate inhibition of angiogenesis. We observed that, the presence of extracellular l-asparagine was essential for the growth of AGS cells. l-asparagine deprivation by l-asparaginase induced metabolic stress, cytotoxicity and apoptosis by G0 phase cell-cycle arrest, modulated the mitochondrial membrane integrity, accelerated caspase-3 activation and instigated DNA damage. The RT-PCR analysis of pro-apoptosis genes: bak1, bax, bbc3, bik, pmaip1, bnip3l, apaf1, casp3, casp7 and casp9 were significantly higher (P < 0.05), while anti-apoptotic markers xiap, bid, mcl1, and death receptor genes tnf and tradd were significantly down-regulated (P < 0.05). Additionally, higher protein expressions of p53, caspase-3 and TEM analysis showing modulations in mitochondria confirmed intrinsic apoptosis pathway. The enzyme impeded tumor progression through inhibition of cell migration and vascular remodelling of endothelial cells. Our findings suggests that the action of l-asparaginase alters mitochondrial membrane permeability and auxiliary activates intrinsic apoptosis. Therefore, this mechanistic approach might be considered as a targeted enzymotherapy against gastric adenocarcinoma.

MYD88 is an adaptor protein known to involve in activation of NF-κB through IL-1 receptor and TLR stimulation. It consists of N-terminal death domain and C-terminal Toll/IL-R homology domain that mediates its interaction with IL-1R associated kinase and IL-1R/TLR, respectively. MYD88 contributes to various types of carcinogenesis due to its involvement in oncogene induced inflammation. In the present study, we have recognized two new alternatively spliced variants of MyD88 gene in mouse using bioinformatics tools and molecular biology techniques in combination. The newly identified non-coding exon (NE-1) from 5' upstream region alternatively splices with either exon E-2 or exon E-5 to produce two novel transcript variants MyD88N1 and MyD88N2 respectively. The transcript variant MyD88N1 was expressed in several tissues studied while the variant MyD88N2 was found to be expressed only in the brain. The analysis of the upstream region of novel exon by in silico approach revealed new promoter region PN, which possess potential signature sequences for diverse transcription factors, suggesting complex gene regulation. Studies of post translational modifications of conceptualized amino acid sequences of these isoforms revealed diversity in properties. Western blot analysis further confirmed the expression of protein isoform MYD88N1.

In this study, we investigated the role of microRNA-644a (miR-644a) in the growth and survival of hepatocellular carcinoma (HCC) cells. MiR-644a levels were lower in HCC tissues than in adjacent peri-cancerous tissues (n = 135). MiR-644a expression was inversely correlated with heat shock factor 1 (HSF1) expression, tumour diameter and TNM stage. Moreover, HepG2 and SMMC-7721 cell lines showed lower miR-644a expression than normal L-O2 hepatocytes. MiR-644a overexpression in HepG2 and SMMC-7721 cells increased apoptosis by downregulating HSF1. Dual luciferase reporter assays confirmed the presence of a miR-644a binding site in the 3'-untranslated region (3'-UTR) of HSF1. Xenograft tumours derived from SMMC-7721 cells transfected with a miR-664a mimic showed less growth than tumours derived from untransfected controls. Protein chip analysis revealed that miR-644a-overexpressing SMMC-7721 and HepG2 cells strongly expressed pro-apoptotic BH3-only proteins, such as BID, BAD, BIM, SMAC, Apaf-1 and cleaved caspases-3 and -9. These findings suggest miR-644a promotes apoptosis in HCC cells by inhibiting HSF1.

Thyroid cancer (TC) is the most common endocrine malignancy, accounting for approximately 90% of all malignancies of the endocrine system. Despite the fact that patients with TC tend to have good prognoses, the high incidence rate and lymph node metastases remain unresolved issues. Autophagy is an indispensable process that maintains intracellular homeostasis; however, the role of autophagy in several steps of the initiation and progression of TC has not yet been elucidated. In this study, we first identified several autophagy-related genes (ARGs) that were provoked in the onset of TC. Subsequently, a bioinformatics analysis hinted that these genes were markedly disturbed in several proliferative signaling pathways. Moreover, we demonstrated that the differentially expressed ARGs were closely related to several aggressive clinical manifestations, including an advanced tumor stage and lymph node metastasis. Our study further selected prognostic ARGs and developed a prognostic signature based on three key genes (ATG9B, BID and B1DNAJB1), which displayed a moderate ability to predict the prognosis of TC. On the whole, the findings of this study demonstrate that ARGs disrupt proliferation-related pathways and consequently lead to aggressive clinical manifestations. These findings provide insight into the potential molecular mechanisms of action of ARGs and their clinical significance, and also provide classification information of potential therapeutic significance.

PURPOSE: Alectinib is a selective and potent anaplastic lymphoma kinase (ALK) inhibitor that is active in the central nervous system (CNS). Alectinib demonstrated robust efficacy in a pooled analysis of two single-arm, open-label phase II studies (NP28673, NCT01801111; NP28761, NCT01871805) in crizotinib-resistant ALK-positive non-small-cell lung cancer (NSCLC): median overall survival (OS) 29.1 months (95% confidence interval [CI]: 21.3-39.0) for alectinib 600 mg twice daily (BID). We investigated exposure-response relationships from final pooled phase II OS and safety data to assess alectinib dose selection.
METHODS: A semi-parametric Cox proportional hazards model analyzed relationships between individual median observed steady-state trough concentrations (C
RESULTS: Overall, 92% of patients (n = 207/225) had C
CONCLUSION: Alectinib 600 mg BID provides systemic exposures at plateau of response for OS while maintaining a well-tolerated safety profile. This analysis confirms alectinib 600 mg BID as the recommended global dose for patients with crizotinib-resistant ALK-positive NSCLC.

SPAG9 is a novel tumor associated antigen, expressed in variety of malignancies. However, its role in ovarian cancer remains unexplored. SPAG9 expression was validated in ovarian cancer cells by real time PCR and Western blot. SPAG9 involvement in cell cycle, DNA damage, apoptosis, paclitaxel sensitivity and epithelial- mesenchymal transition (EMT) was investigated employing RNA interference approach. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro. Quantitative PCR and Western blot analysis revealed SPAG9 expression in A10, SKOV-3 and Caov3 compared to normal ovarian epithelial cells. SPAG9 ablation resulted in reduced cellular proliferation, colony forming ability and enhanced cytotoxicity of chemotherapeutic agent paclitaxel. Effect of ablation of SPAG9 on cell cycle revealed S phase arrest and showed decreased expression of CDK1, CDK2, CDK4, CDK6, cyclin B1, cyclin D1, cyclin E and increased expression of tumor suppressor p21. Ablation of SPAG9 also resulted in increased apoptosis with increased expression of various pro- apoptotic molecules including BAD, BID, PUMA, caspase 3, caspase 7, caspase 8 and cytochrome C. Decreased expression of mesenchymal markers and increased expression of epithelial markers was found in SPAG9 ablated cells. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro assays which showed that ablation of SPAG9 resulted in increased paclitaxel sensitivity and caused enhanced cell death. In vivo ovarian cancer xenograft studies showed that ablation of SPAG9 resulted in significant reduction in tumor growth. Present study revealed therapeutic potential of SPAG9 in ovarian cancer.

The compound (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI) is known as an inhibitor of dual specific phosphatase 1/6 and mitogen-activated protein kinase. However, its precise anti-lung cancer mechanism remains unknown. In this study, the effects of BCI on the viability of non-small cell lung cancer cell lines NCI-H1299, A549, and NCI-H460 were evaluated. We confirmed that BCI significantly inhibited the viability of p53(-) NCI-H1299 cells as compared to NCI-H460 and A549 cells, which express wild-type p53. Furthermore, BCI treatment increased the level of cellular reactive oxygen species and pre-treatment of cells with N-acetylcysteine markedly attenuated BCI-mediated apoptosis of NCI-H1299 cells. BCI induced cellular morphological changes, inhibited viability, and produced reactive oxygen species in NCI-H1299 cells in a dose-dependent manner. BCI induced processing of caspase-9, caspase-3, and poly ADP-ribose polymerase as well as the release of cytochrome c from the mitochondria into the cytosol. In addition, BCI downregulated Bcl-2 expression and enhanced Bax expression in a dose-dependent manner in NCI-H1299 cells. However, BCI failed to modulate the expression of the death receptor and extrinsic factor caspase-8 and Bid, a linker between the intrinsic and extrinsic apoptotic pathways in NCI-H1299 cells. Thus, BCI induces apoptosis via generation of reactive oxygen species and activation of the intrinsic pathway in NCI-H1299 cells.

NutramilTM Complex is a multicomponent food product that meets the requirements of a food for special medical purpose. As a complete, high-energy diet it consists of properly balanced nutrients, vitamins and minerals. The aim of this study was to assess the effect of NutramilTM Complex on breast and prostate carcinoma cells. Our results showed that NutramilTM Complex reduced the viability and proliferation of breast and prostate cancer cells and that this process was associated with the induction of apoptosis via activation of caspase signalling. Data showed elevated levels of p53 tumour suppressor, up-regulation of p38 MAPK and SAPK / JNK proteins and downregulation of anti-apoptotic ERK1/2, AKT1 and HSP27. Treatment with NutramilTM Complex also affected the expression of the BCL2 family genes. Results also showed down-regulation of anti-apoptotic BCL-2 and up-regulation of pro-apoptotic members such as BAX, BAD, BID. In addition, we also observed regulation of many other genes, including Iκβα, Chk1 and Chk2, associated with apoptotic events. Taken together, our results suggest activation of the mitochondrial apoptotic pathway as most likely mechanism of anti-carcinogenic activity of NutramilTM Complex.

We have previously shown that the human follicular lymphoma cell line, HF28GFP, is sensitive to TRAIL-mediated apoptosis. Nevertheless, when the same cells overexpress anti-apoptotic Bcl-2 family protein, Bcl-xL (HF28Bcl-xL), they become resistant to TRAIL. Thus, these cell lines help us to investigate the action of novel apoptosis inducing candidate drugs. In the present study, we examined the effects of MG-132 (a proteasome inhibitor), LiCl (a glycogen synthase kinase-3 inhibitor) and/or TRAIL on pro-apoptotic Bcl-2 family proteins such as Bim and Bid. Here we demonstrate that the combination of MG-132 and TRAIL induced significant apoptotic cell death in both cell lines, HF28GFP and HF28BclxL. Apoptosis correlated with a decrease of phospho-ERK1/2, the accumulation of Bim and translocation of truncated Bid (tBid) and jBid. In addition, the combination of MG-132 and TRAIL seemed to target other apoptotic factors, which led to the accumulation of active capsase-3. Furthermore, co-stimulation of LiCl and TRAIL induced apoptosis in HF28GFP cells. However, HF28Bcl-xL cells were far less sensitive to the combinatorial effects of LiCl and TRAIL. Interestingly, we observed that LiCl did not target Bim and Bid proteins. In conclusion, these data show that targeting of pro-apoptotic Bcl-2 family proteins simultaneously through a selective proteasome inhibition might help to overcome TRAIL resistance caused by overexpression of anti-apoptotic Bcl-2 family proteins. Moreover, the data may provide new strategies to develop targeted therapies against lymphomas.

BACKGROUND: The anticancer effects of histone deacetylase inhibitors (HDACi) vary between patients, and their molecular mechanisms remain poorly understood. Previously, we have identified heat shock 70 kDa protein 1A (HSPA1A, also known as HSP72) as the most overexpressed protein in valproic acid (VPA)-resistant cell lines. KNK437, an inhibitor of heat shock proteins, enhanced the cytotoxic effects of not only VPA but also vorinostat, another HDACi. However, the mechanisms underlying the role of HSP72 in resistance against HDACi remain largely unknown.
OBJECTIVE: The purpose of this study was to identify the mechanisms underlying the role of HSP72 in HDACi resistance.
METHODS: We established an HSP72-overexpressing Jurkat cell line and used it to assess the functional role of HSP72 following treatment with the HDACi vorinostat and VPA.
RESULTS: HDACi-induced apoptosis, assessed using annexin V assays, sub-G1 fraction analysis, and PARP cleavage, was significantly lower in HSP72-overexpressing cells than in control cells. The HDACi-induced upregulation in caspase-3, -8, and -9 activity, as well as the HDACi-induced reduction in mitochondrial membrane potential, were also suppressed following HSP72 overexpression. The basal expression levels of Bcl-2, phosphorylated Bad, and XIAP increased in HSP72-overexpressing cells, whereas HDACi-induced Bid truncation and the suppression of Bad expression. Furthermore, vorinostat-induced histone hyperacetylation was also diminished in HSP72-overexpressing cells.
CONCLUSION: These findings clearly demonstrate that HSP72 inhibits HDACi-induced apoptosis.

OBJECTIVE: Determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of veliparib combined with carboplatin and gemcitabine in patients with advanced ovarian cancer and other nonhematologic malignancies.
METHODS: In this phase I study, patients with metastatic or unresectable solid tumors and ≤2 prior chemotherapy regimens received veliparib combined with carboplatin area under the curve (AUC) 4 on day 1 and gemcitabine 800mg/m
RESULTS: Seventy-five patients were enrolled (ovarian cancer, n=54; breast cancer, n=12). Thirty-six patients with ovarian cancer (67%) had known germline BRCA mutations. Most common treatment-related adverse events (TRAEs; ≥60%) were thrombocytopenia, neutropenia, nausea, and anemia. Most common grade 3/4 TRAEs (≥40%) were neutropenia and thrombocytopenia. Dose-limiting toxicities were thrombocytopenia and neutropenia. The MTD/RP2D was established at veliparib 250mg with carboplatin AUC 4 plus gemcitabine 800mg/m
CONCLUSIONS: Veliparib plus carboplatin/gemcitabine is tolerated, with a safety profile similar to carboplatin and gemcitabine alone. Combination therapy demonstrated promising preliminary antitumor activity in platinum-sensitive ovarian cancer patients with germline BRCA mutations. Trial registration ID: NCT01063816.

BACKGROUND: Cutaneous T cell lymphoma (CTCL) comprises a heterogeneous group of skin-homing T cell tumors. The small guanosine triphosphate effector p21-activated kinase 1 (PAK1) plays an important role in many fundamental cellular functions, including cell motility, proliferation, and apoptosis. The expression of PAK1 is up-regulated in several types of human cancers. However, little is known about the role of PAK1 in the pathogenesis of CTCL.
OBJECTIVE: The aim of this study was to evaluate the expression pattern and underlying mechanism of PAK1 in CTCL.
METHODS: Quantitative real-time polymerase chain reaction(qRT-PCR) was used to detect PAK1 mRNA expression in the peripheral blood mononuclear cells (PBMCs) of patients with CTCL. The expression of PAK1 protein in CTCL tumor tissues was determined by immunohistochemistry. CTCL cell lines were treated with a small molecule inhibitor of PAK1, p21-activated kinase inhibitor III (IPA3), at concentrations of 2, 3.5 and 5 μM for 24 h. Hut 78 and HH CTCL cells were transfected with lentiviral-based PAK1 gene knockdown vectors. We determined the effects of PAK1 knockdown on cell proliferation and apoptosis in CTCL cells by MTS assay and flow cytometry. Animal experiments were performed to investigate the effects of PAK1 knockdown on the growth of tumors in vivo. Transcriptomic sequencing was performed to detect the direct downstream targets of PAK1 silencing. Reverse transcription polymerase chain reaction and western blot analysis were applied to verify the results of the transcriptomic analysis.
RESULTS: We detected PAK1 overexpression in PBMCs and skin lesions from patients with CTCL compared with benign inflammatory dermatoses (BID). Knockdown of PAK1 inhibited cell proliferation and promoted spontaneous apoptosis. In addition, the inhibitory effect of IPA3 was validated in the CTCL cell lines. Additionally, mice injected with PAK1-silenced cells presented with a decreased rate of tumor growth compared with the control groups. Moreover, the mRNA and protein expression of PUMA (BBC3) and p21 (CDKN1A) were increased in PAK1-silenced Hut 78 and HH cells.
CONCLUSIONS: Our data indicated that PAK1 is upregulated in CTCL. PAK1 silencing induced apoptosis and inhibited cell growth by stimulating the expression of PUMA and p21. Thus, PAK1 may be a potential tumor marker and therapeutic target of CTCL.

Lung cancer has high incidence and mortality rates worldwide. In the present study, the mechanisms by which hesperidin decreases the viability and induces the apoptosis of human non-small cell lung cancer (NSCLC) A549 cells were investigated. Initially, MTT and flow cytometric assays were performed to evaluate the effects of hesperidin on the viability and apoptosis of A549 cells and human normal lung epithelial BEAS-2B cells. The results revealed that hesperidin has no negative effects on the human normal lung epithelial BEAS-2B cells and the viability of cells treated with various concentrations of hesperidin was inhibited in a time- and dose-dependent manner compared with the control groups. Subsequently, the expression levels of proteins involved in the mitochondria-associated apoptotic pathway were studied by western blot analysis. Hesperidin was identified to induce A549 cell apoptosis by downregulating the levels of B-cell lymphoma-2 (Bcl-2) and Bcl extra large protein and simultaneously upregulating the levels of Bcl-2-associated X protein, BH3 interacting-domain death agonist (Bid), tBid, cleaved caspase-9, cleaved caspase-3 and cleaved poly(adenosine diphosphate ribose)polymerase. The effect of hesperidin on the cell cycle was assessed by flow cytometry. Hesperidin was observed to cause G0/G1 arrest of A549 cells by decreasing the expression of cyclin D1 and increasing the expression of p21 and p53. In summary, it was demonstrated that hesperidin induced apoptosis through the mitochondrial apoptotic pathway and induced G0/G1 arrest in human NSCLC A549 cells. Therefore, hesperidin may be developed as a potential therapeutic drug for the treatment of NSCLC.

Photodynamic therapy (PDT) is considered to be an advancing antitumor technology. PDT using hydrophilic/lipophilic tetra‑α-(4-carboxyphenoxy) phthalocyanine zinc (TαPcZn-PDT) has exhibited antitumor activity in Bel-7402 hepatocellular cancer cells. However, the manner in which p38 MAPK and caspase-9 are involved in the regulation of mitochondria-mediated apoptosis in the TαPcZn-PDT-treated LoVo human colon carcinoma cells remains unclear. Therefore, in the present study, a siRNA targeting p38 MAPK (siRNA-p38 MAPK) and the caspase‑9 specific inhibitor z-LEHD-fmk were used to examine the crosstalk between p38 MAPK and caspase-9 during mitochondria-mediated apoptosis in the TαPcZn-PDT‑treated LoVo cells. The findings revealed that the TαPcZn-PDT treatment of LoVo cells resulted in the induction of apoptosis, the formation of p38 MAPK/caspase-9 complexes, the activation of p38 MAPK, caspase-9, caspase-3 and Bid, the downregulation of Bcl-2, the reduction of mitochondrial membrane potential (ΔΨm), the upregulation of Bax and the release of apoptosis-inducing factor (AIF) and cytochrome c (Cyto c). By contrast, siRNA‑p38 MAPK or z-LEHD-fmk both attenuated the effects of TαPcZn-PDT in the LoVo cells. Furthermore, the results revealed that siRNA-p38 MAPK had more significant inhibitory effects on apoptosis and mitochondria compared with the effects of z-LEHD-fmk in TαPcZn-PDT-treated LoVo cells. These findings indicated that p38 MAPK plays the major regulatory role in the crosstalk between p38 MAPK and caspase-9 and that direct interaction between p38 MAPK and caspase-9 may regulate mitochondria-mediated apoptosis in the TαPcZn-PDT-treated LoVo cells.

Cardamonin has been demonstrated to have an inhibitory effect in many cancers, but its underlying mechanism remains elusive. Here, we studied, for the first time, the mechanism of cardamonin-induced nasopharyngeal carcinoma cell death both in vitro and in vivo. In our study, we showed that cardamonin inhibited cancer cell growth by inducing G2/M phase cell cycle arrest and apoptosis via accumulation of ROS. NF-κB activation was involved in breaking cellular redox homeostasis. Therefore, our results provided new insight into the mechanism of the antitumor effect of cardamonin, supporting cardamonin as a prospective therapeutic drug in nasopharyngeal carcinoma by modulating intracellular redox balance.

Natural flavonoids are proven to be powerful against various cancers, but few studies have investigated the potential effects of two flavonoids galangin and quercetin on a human gastric cancer cell line (SGC-7901). In vitro growth inhibition and apoptosis of the two flavonoids on the SGC-7901 cells as well as potential mechanism about apoptosis induction are reported in the present study. The assaying results showed that the two flavonoids at 40-200 μmmol/L for 24-72 hours conferred lower cell viability of 14.1-90.3% in dose- and time-dependent manner, and at 160 μmmol/L for 24-48 hours enhanced the proportion of apoptotic cells into 13.3-27.4% and 40.6-65.6%, respectively. Galangin was more powerful than quercetin to inhibit cell growth, induce apoptosis and decrease mitochondrial membrane potential (MMP). Oligonucleotide micro array, real-time RT-PCR and Western-blot analyses revealed expression changes of the genes and proteins in the treated cells, clarifying a mechanism related to apoptosis induction. The two flavonoids activated caspase-8, which cleaved Bid into tBid; simultaneously, Bax transferred from cytosol into mitochondria to decrease MMP; consequentially, cytochrome c released from mitochondria activated caspase-9, and then caspase-9 activated caspase-3, which executed the apoptosis. That is, the apoptosis occurred via a mitochondrial pathway involving caspase-8/Bid/Bax activation.

OBJECTIVE: An integrated analysis was undertaken to characterize the antitumor activity and safety profile of the oral poly(ADP-ribose) polymerase inhibitor rucaparib in patients with relapsed high-grade ovarian carcinoma (HGOC).
METHODS: Eligible patients from Study 10 (NCT01482715) and ARIEL2 (NCT01891344) who received a starting dose of oral rucaparib 600mg twice daily (BID) with or without food were included in these analyses. The integrated efficacy population included patients with HGOC and a deleterious germline or somatic BRCA1 or BRCA2 (BRCA1/2) mutation who received at least two prior chemotherapies and were sensitive, resistant, or refractory to platinum-based chemotherapy. The primary endpoint was investigator-assessed confirmed objective response rate (ORR). Secondary endpoints included duration of response (DOR) and progression-free survival (PFS). The integrated safety population included patients with HGOC who received at least one dose of rucaparib 600mg BID, irrespective of BRCA1/2 mutation status and prior treatments.
RESULTS: In the efficacy population (n=106), ORR was 53.8% (95% confidence interval [CI], 43.8-63.5); 8.5% and 45.3% of patients achieved complete and partial responses, respectively. Median DOR was 9.2months (95% CI, 6.6-11.6). In the safety population (n=377), the most frequent treatment-emergent adverse events (AEs) were nausea, asthenia/fatigue, vomiting, and anemia/hemoglobin decreased. The most common grade ≥3 treatment-emergent AE was anemia/hemoglobin decreased. Treatment-emergent AEs led to treatment interruption, dose reduction, and treatment discontinuation in 58.6%, 45.9%, and 9.8% of patients, respectively. No treatment-related deaths occurred.
CONCLUSIONS: Rucaparib has antitumor activity in advanced BRCA1/2-mutated HGOC and a manageable safety profile.

Several studies have demonstrated that cancer radiosensitivity is associated with the deregulation of c‑Myc, but the relationship between c‑Myc and Fas in radioresistance of lung adenocarcinoma remains unclear. In this study, we established radiation-resistant A549 cell model (A549/R), and investigated the roles of c‑Myc and Fas in radiation-induced cytotoxicity of A549 cells. Apoptosis detection showed that there were fewer apoptotic cells in A549/R cells treated with radiation than in A549 cells. Western blotting results demonstrated the inverse expression pattern of c‑Myc and Fas in A549 and A549/R cells. Suppression of c‑Myc expression by small interfering RNA (siRNA) displayed enhancement of Fas-mediated apoptosis in A549/R cells, accompanying a significant decrease of Bid, Bcl‑2, pro‑caspase‑8, -9 and -3 and increase of Bax. In contrast, Fas-mediated apoptosis was attenuated while Fas expression was suppressed by ectopic expression of c‑Myc in A549 cells. Moreover, decreased cell viability and increased induction of apoptosis were observed in A549/R cells followed by combinational treatment of c‑Myc siRNA and irradiation, whereas, upregulation of c‑Myc reduced the sensitivity of A549 cells to irradiation. These results indicated that c‑Myc and Fas regulated the sensitivity of A549 cells to irradiation by regulating caspase‑8-mediated Bid activation and the subsequent association with the mitochondrial pathway of apoptosis.

Constitutive activation of the pro-survival transcription factor NF-κB has been associated with resistance to both chemotherapy and radiation therapy in many human cancers, including prostate cancer. Our lab and others have demonstrated that the natural product parthenolide can inhibit NF-κB activity and sensitize PC-3 prostate cancers cells to X-rays in vitro; however, parthenolide has poor bioavailability in vivo and therefore has little clinical utility in this regard. We show here that treatment of PC-3 and DU145 human prostate cancer cells with dimethylaminoparthenolide (DMAPT), a parthenolide derivative with increased bioavailability, inhibits constitutive and radiation-induced NF-κB binding activity and slows prostate cancer cell growth. We also show that DMAPT increases single and fractionated X-ray-induced killing of prostate cancer cells through inhibition of DNA double strand break repair and also that DMAPT-induced radiosensitization is, at least partially, dependent upon the alteration of intracellular thiol reduction-oxidation chemistry. Finally, we demonstrate that the treatment of PC-3 prostate tumor xenografts with oral DMAPT in addition to radiation therapy significantly decreases tumor growth and results in significantly smaller tumor volumes compared to xenografts treated with either DMAPT or radiation therapy alone, suggesting that DMAPT might have a potential clinical role as a radiosensitizing agent in the treatment of prostate cancer.

Proteins of the B-cell lymphoma-2 (BCL-2) family control the intrinsic apoptosis pathway. The pro-apoptotic BCL-2 proteins BAX and BAK can commit a cell to its programmed death by permeabilizing the outer mitochondrial membrane (OMM) and subsequent initiation of the caspase cascade. Therefore, the activities of BAX and BAK are precisely controlled by a complex network of proteins inside and outside the BCL-2 family. Cells survive by constant control of dynamic translocation and retrotranslocation of BAX and BAK to the mitochondria and back into the cytosol. Recent insights into BAX/BAK shuttling, BCL-2 protein interactions, the role of BH3-only proteins in apoptosis signaling and the active BAX complex set the stage for the development of novel strategies in cancer therapy and the analysis of cellular predisposition to apoptosis.