Chondroitin sulfate-functionalized selenium nanoparticle-induced S-phase cell cycle arrest and apoptosis in HeLa Cells.

This study aimed to evaluate the anti-cervical cancer activity of chondroitin sulfate-functionalized selenium nanoparticles (SeCS) and to elucidate their action mechanism. Cytotoxic effect of SeCS on HeLa cells was assessed by MTT assay. Further molecular mechanism of SeCS was analyzed by flow cytometric assay and western blotting. The results showed that treatment with SeCS resulted in a dose- and time-dependent inhibition in the proliferation of HeLa cells. The data obtained from flow cytometry demonstrated that SeCS inhibited HeLa cell growth via the induction of S-phase arrest and cell apoptosis. Further mechanism analysis found that SeCS down-regulated expression levels of cyclin A and CDK2 and up-regulated p21 expression, which contributed to S arrest. Moreover, SeCS increased the level of Bax and decreased the expression of Bcl-2, resulting in the release of cytochrome C from mitochondria and activating caspase-3/8/9 for caspase-dependent apoptosis. Meanwhile, intracellular reactive oxygen species (ROS) levels were elevated after SeCS treatment, suggesting that ROS might be upstream of SeCS-induced S-phase arrest and cell apoptosis. These data show that SeCS has anti-tumor effects and possesses the potential to become a new therapeutic agent or adjuvant therapy for cancer patients. PRACTICAL APPLICATION: In our previous study, we used chondroitin sulfate to stabilize nano-selenium to obtain SeCS to improve the bioactivity and stability of nano-selenium. We found that it possessed an inhibitory effect on HeLa cells. However, the molecular mechanism remains unclear. This study elucidated the mechanism of SeCS damage to HeLa cells. SeCS has the potential to become a new therapeutic agent or adjuvant therapy for cancer patients.

Mechanisms used by cancer cells to tolerate drug-induced replication stress.

Activation of oncogenes in cancer cells forces cell proliferation, leading to DNA replication stress (RS). As a consequence, cancer cells heavily rely on the intra S-phase checkpoint for survival. This fundamental principle formed the basis for the development of inhibitors against key players of the intra S-phase checkpoint, ATR and CHK1. These drugs are often combined with chemotherapeutic drugs that interfere with DNA replication to exacerbate RS and exhaust the intra S-phase checkpoint in cancer cells. However, drug resistance impedes efficient clinical use, suggesting that some cancer cells tolerate severe RS. In this review, we describe how an increased nucleotide pool, boosted stabilization and repair of stalled forks and firing of dormant origins fortify the RS response in cancer cells. Notably, the vast majority of the genes that confer RS tolerance are regulated by the E2F and NRF2 transcription factors. These transcriptional programs are frequently activated in cancer cells, allowing simultaneous activation of multiple tolerance avenues. We propose that the E2F and NRF2 transcriptional programs can be used as biomarker to select patients for treatment with RS-inducing drugs and as novel targets to kill RS-tolerant cancer cells. Together, this review aims to provide a framework to maximally exploit RS as an Achilles' heel of cancer cells.

Different Cell Responses to Hinokitiol Treatment Result in Senescence or Apoptosis in Human Osteosarcoma Cell Lines.

Hinokitiol is a tropolone-related compound isolated from the heartwood of cupressaceous plants. It is known to exhibit various biological functions including antibacterial, antifungal, and antioxidant activities. In the study, we investigated the antitumor activities of hinokitiol against human osteosarcoma cells. The results revealed that hinokitiol treatment inhibited cell viability of human osteosarcoma U-2 OS and MG-63 cells in the MTT assay. Further study revealed that hinokitiol exposure caused cell cycle arrest at the S phase and a DNA damage response with the induction of γ-H2AX foci in both osteosarcoma cell lines. In U-2 OS cells with wild-type tumor suppressor p53, we found that hinokitiol exposure induced p53 expression and cellular senescence, and knockdown of p53 suppressed the senescence. However, in MG-63 cells with mutated p53, a high percentage of cells underwent apoptosis with cleaved-PARP expression and Annexin V staining after hinokitiol treatment. In addition, up-regulated autophagy was observed both in hinokitiol-exposed U-2 OS and MG-63 cells. As the autophagy was suppressed through the autophagy inhibitor chloroquine, hinokitiol-induced senescence in U-2 OS cells was significantly enhanced accompanying more abundant p53 expression. In MG-63 cells, co-treatment of chloroquine increased hinokitiol-induced apoptosis and decreased cell viability of the treated cells. Our data revealed that hinokitiol treatment could result in different cell responses, senescence or apoptosis in osteosarcoma cell lines, and suppression of autophagy could promote these effects. We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors might provide more precise and efficacious therapies in hinokitiol-related trials for treating osteosarcoma.

LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint.

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Dichloromethane fractions of Calystegia soldanella induce S­phase arrest and apoptosis in HT­29 human colorectal cancer cells.

Calystegia soldanella is a halophyte and a perennial herb that grows on coastal sand dunes worldwide. Extracts from this plant have been previously revealed to have a variety of bioactive properties in humans. However, their effects on colorectal cancer cells remain poorly understood. In the present study, the potential biological activity of C. soldanella extracts in the colorectal cancer cell line HT­29 was examined. First, five solvent fractions [n­hexane, dichloromethane (DCM), ethyl acetate, n­butanol and water] were obtained from the crude extracts of C. soldanella through an organic solvent extraction method. In particular, the DCM fraction was demonstrated to exert marked dose­ and time­dependent inhibitory effects according to results from the cell viability assay. Data obtained from the apoptosis assay suggested that the inhibition of HT­29 cell viability induced by DCM treatment was attributed to increased apoptosis. The apoptotic rate was markedly increased in a dose­dependent manner, which was associated with the protein expression levels of apoptosis­related proteins, including increased Fas, Bad and Bax, and decreased pro­caspase­8, Bcl­2, Bcl­xL, pro­caspase­9, pro­caspase­7 and pro­caspase­3. A mitochondrial membrane potential assay demonstrated that more cells became depolarized and the extent of cytochrome c release was markedly increased in a dose­dependent manner in HT­29 cells treated with DCM. In addition, cell cycle analysis confirmed S­phase arrest following DCM fraction treatment, which was associated with decreased protein expression levels of cell cycle­related proteins, such as cyclin A, CDK2, cell division cycle 25 A and cyclin dependent kinase inhibitor 1. Based on these results, the present study suggested that the DCM fraction of the C. soldanella extract can inhibit HT­29 cell viability whilst inducing apoptosis through mitochondrial membrane potential regulation and S­phase arrest. These results also suggested that the DCM fraction has potential anticancer activity in HT­29 colorectal cells. Further research on the composition of the DCM fraction is warranted.

Intrinsic S phase checkpoint enforced by an antiproliferative oncosuppressor cytokine.

The cell cycle is strictly programmed with control mechanisms that dictate order in cell cycle progression to ensure faithful DNA replication, whose deviance may lead to cancer. Checkpoint control at the G1/S, S/G2 and G2/M portals have been defined but no statutory time-programmed control for securing orderly transition through S phase has so far been identified. Here we report that in normal cells DNA synthesis is controlled by a checkpoint sited within the early part of S phase, enforced by the ßGBP cytokine an antiproliferative molecule otherwise known for its oncosuppressor properties that normal cells constitutively produce for self-regulation. Suppression of active Ras and active MAPK, block of cyclin A gene expression and suppression of CDK2-cyclin A activity are events which while specific to the control of a cell cycle phase in normal cells are part of the apoptotic network in cancer cells.

Activation of ATM/Chk2 by Zanthoxylum armatum DC extract induces DNA damage and G1/S phase arrest in BRL 3A cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum armatum DC is a traditional medicinal plant. It is widely used in clinical treatment and disease prevention in China, India and other regions. Modern studies have reported the phytotoxicity, cytotoxicity and the animal toxicity of Zanthoxylum armatum DC, and the damage of genetic material has been observed in plants, but the detailed mechanism has not been explored. Besides, the toxicity of normal mammalian cells has not been evaluated. AIM OF THE STUDY: To evaluate the effects and underlying mechanism of genetic material damage in BRL 3A cells induced by Zanthoxylum armatum DC. MATERIALS AND METHODS: Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry was used for identification of compounds in methanol extract of Zanthoxylum armatum DC. BRL 3A cells were incubated with different concentrations of methanol extract of Zanthoxylum armatum DC (24 h). The cytotoxicity of extract was assessed with cell viability, LDH release rate, and ROS production. The damage of genetic material was assessed with OTM value of comet cells, cell cycle and the expression levels of p-ATM, p- Chk2, Cdc25A, and CDK2. RESULTS: Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry investigation revealed the presence of compounds belonging to flavonoid, fatty acid and alkaloid groups. The viability of BRL 3A cells was reduced in a time-dose dependent manner treated by methanol extract of Zanthoxylum armatum DC. It increased LDH release rate and ROS production, activated the DNA double strand damage marker of γH2AX and produced comet cells. In addition, methanol extract of Zanthoxylum armatum DC caused ATM-mediated DNA damage, further phosphorylated Chk2, inhibited cell cycle related proteins, and arrested the G1/S cycle. CONCLUSIONS: Methanol extract of Zanthoxylum armatum DC induces DNA damage and further leads G1/S cell cycle arrest by triggering oxidative stress in the BRL 3A cells. This study provides some useful evidences for its development as an antitumor drug via activation of ATM/Chk2.

Pillar-Based Mechanical Induction of an Aggressive Tumorigenic Lung Cancer Cell Model.

Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 µm diameter and 16 µm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 µm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.

A novel oral inhibitor for one-carbon metabolism and checkpoint kinase 1 inhibitor as a rational combination treatment for breast cancer.

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.

X-ray repair cross-complementing protein 1 (XRCC1) loss promotes ß-lapachone -induced apoptosis in pancreatic cancer cells.

BACKGROUND: ß-lapachone (ß-lap), the NQO1 bioactivatable drug, is thought to be a promising anticancer agent. However, the toxic side effects of ß-lap limit the drug use, highlighting the need for a thorough understanding of ß-lap's mechanism of action. ß-lap undergoes NQO1-dependent futile redox cycling, generating massive ROS and oxidative DNA lesions, leading to cell death. Thus, base excision repair (BER) pathway is an important resistance factor. XRCC1, a scaffolding component, plays a critical role in BER. METHODS: We knocked down XRCC1 expression by using pLVX-shXRCC1 in the MiaPaCa2 cells and BxPC3 cells and evaluated ß-lap-induced DNA lesions by γH2AX foci formation and alkaline comet assay. The cell death induced by XRCC1 knockdown + ß-lap treatment was analysed by relative survival, flow cytometry and Western blotting analysis. RESULTS: We found that knockdown of XRCC1 significantly increased ß-lap-induced DNA double-strand breaks, comet tail lengths and cell death in PDA cells. Furthermore, we observed combining XRCC1 knockdown with ß-lap treatment switched programmed necrosis with ß-lap monotherapy to caspase-dependent apoptosis. CONCLUSIONS: These results indicate that XRCC1 is involved in the repair of ß-lap-induced DNA damage, and XRCC1 loss amplifies sensitivity to ß-lap, suggesting targeting key components in BER pathways may have the potential to expand use and efficacy of ß-lap for gene-based therapy.

Novel chemotherapeutic agent FX-9 activates NF-κB signaling and induces G1 phase arrest by activating CDKN1A in a human prostate cancer cell line.

BACKGROUND: The aminoisoquinoline FX-9 shows pro-apoptotic and antimitotic effects against lymphoblastic leukemia cells and prostate adenocarcinoma cells. In contrast, decreased cytotoxic effects against non-neoplastic blood cells, chondrocytes, and fibroblasts were observed. However, the actual FX-9 molecular mode of action is currently not fully understood. METHODS: In this study, microarray gene expression analysis comparing FX-9 exposed and unexposed prostate cancer cells (PC-3 representing castration-resistant prostate cancer), followed by pathway analysis and gene annotation to functional processes were performed. Immunocytochemistry staining was performed with selected targets. RESULTS: Expression analysis revealed 0.83% of 21,448 differential expressed genes (DEGs) after 6-h exposure of FX-9 and 0.68% DEGs after 12-h exposure thereof. Functional annotation showed that FX-9 primarily caused an activation of inflammatory response by non-canonical nuclear factor-kappa B (NF-κB) signaling. The 6-h samples showed activation of the cell cycle inhibitor CDKN1A which might be involved in the secondary response in 12-h samples. This secondary response predominantly consisted of cell cycle-related changes, with further activation of CDKN1A and inhibition of the transcription factor E2F1, including downstream target genes, resulting in G1-phase arrest. Matching our previous observations on cellular level senescence signaling pathways were also found enriched. To verify these results immunocytochemical staining of p21 Waf1/Cip1 (CDKN1A), E2F1 (E2F1), PAI-1 (SERPNE1), and NFkB2/NFkB p 100 (NFKB2) was performed. Increased expression of p21 Waf1/Cip1 and NFkB2/NFkB p 100 after 24-h exposure to FX-9 was shown. E2F1 and PAI-1 showed no increased expression. CONCLUSIONS: FX-9 induced G1-phase arrest of PC-3 cells through activation of the cell cycle inhibitor CDKN1A, which was initiated by an inflammatory response of noncanonical NF-κB signaling.

Increasing anticancer effect in vitro and vivo of liposome-encapsulated iridium(III) complexes on BEL-7402 cells.

The studies of iridium (III) complexes as potent anticancer reagents have attracted great attention. Here, a new iridium (III) complex [Ir(bzq)2(PYIP)](PF6) (Ir1, bzq = benzo[h]quinoline, PYIP = 2-(pyren-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) was synthesized and its liposomes (Ir1Lipo) was prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the cytotoxic activity of Ir1 and Ir1Lipo on HepG2, SGC-7901, BEL-7402, HeLa, B16, A549 and normal NIH3T3 cells. The complex Ir1 displays no obvious inhibitory effect on the growth of BEL-7402 cells, while the Ir1Lipo shows significant cytotoxic activity on BEL-7402 cells (IC50 = 2.6 ± 0.03 µM). In further studies, Ir1Lipo induced apoptosis by the mitochondrial pathways, such as increasing intracellular reactive oxygen species (ROS) content and intracellular Ca2+ level, decreasing the mitochondrial membrane potential (MMP). In addition, after incubation with Ir1Lipo, the colony formation of BEL-7402 cells was significantly inhibited. Moreover, flow cytometry was used to detect the impact of Ir1Lipo on cell cycle distribution, and western blot was used to detect the expression of caspases and Bcl-2 (B-cell lymphoma-2) family proteins. Furthermore, Ir1Lipo exhibited significant antitumor activity in vivo with an inhibitory rate of 65.8%. These results indicated that Ir1Lipo induces apoptosis in BEL-7402 cells through intrinsic mitochondrial pathway.

ATP1A1 Mutant in Aldosterone-Producing Adenoma Leads to Cell Proliferation.

The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA.

Systematic evaluation of the antitumor activity of three ruthenium polypyridyl complexes.

Ruthenium-containing complexes have emerged as good alternative to the currently used platinum-containing drugs for malignant tumor therapy. In this work, cytotoxic effects of recently synthesized ruthenium polypyridyl complexes [Ru(bpy)2(CFPIP)](ClO4)2 (bpy = 2,2'-bipyridine, CFPIP = (E)-2-(4-fluorostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline, Ru(II)-1), [Ru(phen)2(CFPIP)](ClO4)2 (phen = 1,10-phenanthroline, Ru(II)-2) and [Ru(dmb)2(CFPIP)](ClO4)2 (dmb = 4,4'-dimethyl-2,2'-bipyridine, Ru(II)-3) toward different tumor cells were investigated in vitro and compared with cisplatin, the most widely used chemotherapeutic drug against hepatocellular carcinoma (HepG-2). The results demonstrate that target complexes show excellent cytotoxicity against HepG-2 cells with low IC50 value of 21.4 ± 1.5, 18.0 ± 2.1 and 22.3 ± 1.7 µM, respectively. It was important noting that target Ru(II) complexes exhibited better antitumor activity than cisplatin (IC50 = 28.5 ± 2.4 µM) against HepG-2 cells, and has no obvious toxicity to normal cell LO2. DNA binding results suggest that Ru(II)-1, Ru(II)-2 and Ru(II)-3 interact with CT DNA (calf thymus DNA) through intercalative mode. Complexes exerted its antitumor activity through increasing anti-migration and inducing cell cycle arrest at the S phase. In addition, the apoptosis was tested by AO (acridine orange)/EB (ethidium bromide) staining and flow cytometry. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and colocalization tests were also evaluated by ImageXpress Micro XLS system. Overall, the results show that the ruthenium polypyridyl complexes induce apoptosis in HepG-2 cells through ROS-mediated mitochondria dysfunction pathway.

CXCL15/Lungkine has suppressive activity on proliferation and expansion of multi-potential, erythroid, granulocyte and macrophage progenitors in S-phase specific manner.

Cytokines/chemokines regulate hematopoiesis, most having multiple cell actions. Numerous but not all chemokine family members act as negative regulators of hematopoietic progenitor cell (HPC) proliferation, but very little is known about such effects of the chemokine, CXCL15/Lungkine. We found that CXCL15/Lungkine-/- mice have greatly increased cycling of multi cytokine-stimulated bone marrow and spleen hematopoietic progenitor cells (HPCs: CFU-GM, BFU-E, and CFU-GEMM) and CXCL15 is expressed in many bone marrow progenitor and other cell types. This suggests that CXCL15/Lungkine acts as a negative regulator of the cell cycling of these HPCs in vivo. Recombinant murine CXCL15/Lungkine, decreased numbers of functional HPCs during cytokine-enhanced ex-vivo culture of lineage negative mouse bone marrow cells. Moreover, CXCL15/Lungkine, through S-Phase specific actions, was able to suppress in vitro colony formation of normal wildtype mouse bone marrow CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-GEMM. This clearly identifies the negative regulatory activity of CXCL15/Lungkine on proliferation of multiple types of mouse HPCs.

Alkylaminophenol and GPR17 Agonist for Glioblastoma Therapy: A Combinational Approach for Enhanced Cell Death Activity.

Drug resistance and tumor heterogeneity limits the therapeutic efficacy in treating glioblastoma, an aggressive infiltrative type of brain tumor. GBM cells develops resistance against chemotherapeutic agent, temozolomide (TMZ), which leads to the failure in treatment strategies. This enduring challenge of GBM drug resistance could be rational by combinatorial targeted therapy. Here, we evaluated the combinatorial effect of phenolic compound (2-(3,4-dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), GPR17 agonist 2-({5-[3-(Morpholine-4-sulfonyl)phenyl]-4-[4-(trifluoromethoxy)phenyl]-4H-1,2,4-triazol-3-yl}sulfanyl)-N-[4-(propan-2-yl)phenyl]acetamide (T0510.3657 or T0) with the frontline drug, TMZ, on the inhibition of GBM cells. Mesenchymal cell lines derived from patients' tumors, MMK1 and JK2 were treated with the combination of THTMP + T0, THTMP + TMZ and T0 + TMZ. Cellular migration, invasion and clonogenicity assays were performed to check the migratory behavior and the ability to form colony of GBM cells. Mitochondrial membrane permeability (MMP) assay and intracellular calcium, [Ca2+]i, assay was done to comprehend the mechanism of apoptosis. Role of apoptosis-related signaling molecules was analyzed in the induction of programmed cell death. In vivo validation in the xenograft models further validates the preclinical efficacy of the combinatorial drug. GBM cells exert better synergistic effect when exposed to the cytotoxic concentration of THTMP + T0, than other combinations. It also inhibited tumor cell proliferation, migration, invasion, colony-forming ability and cell cycle progression in S phase, better than the other combinations. Moreover, the combination of THTMP + T0 profoundly increased the [Ca2+]i, reactive oxygen species in a time-dependent manner, thus affecting MMP and leading to apoptosis. The activation of intrinsic apoptotic pathway was regulated by the expression of Bcl-2, cleaved caspases-3, cytochrome c, HSP27, cIAP-1, cIAP-2, p53, and XIAP. The combinatorial drug showed promising anti-tumor efficacy in GBM xenograft model by reducing the tumor volume, suggesting it as an alternative drug to TMZ. Our findings indicate the coordinated administration of THTMP + T0 as an efficient therapy for inhibiting GBM cell proliferation.

Engineering Exosomes Endowed with Targeted Delivery of Triptolide for Malignant Melanoma Therapy.

Malignant melanoma is considered the most aggressive skin carcinoma with invasive growth patterns. Triptolide (TPL) possesses various biological and pharmacological activities involved in cancer treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce cancer cell apoptosis by binding to DR5 highly expressed on cancer cells. Exosomes are natural nanomaterials with low immunogenicity, nontoxicity, and excellent biocompatibility and have been extensively used as emerging delivery vectors for diverse therapeutic cargos. Herein, a delivery system based on TRAIL-engineered exosomes (TRAIL-Exo) for loading TPL for targeted therapy against malignant melanoma is proposed and systematically investigated. Our results showed that TRAIL-Exo/TPL could improve tumor targetability, enhance cellular uptake, inhibit proliferation, invasion, and migration, and induce apoptosis of A375 cells through activating the extrinsic TRAIL pathway and the intrinsic mitochondrial pathway in vitro. Moreover, intravenous injection of TRAIL-Exo/TPL significantly suppressed tumor progression and reduced the toxicity of TPL in the melanoma nude mouse model. Together, our research presents a novel strategy for high-efficiency exosome-based drug-delivery nanocarriers and provides an alternative dimension for developing a promising approach with synergistic therapeutic efficacy and targeting capacity for melanoma treatment.

Antitumor Effect of Organometallic Half-Sandwich Ru(II)-Arene Complexes Bearing a Glutathione S-Transferase Inhibitor.

The facile modification of the ligands in organometallic Ru(II)-arene complexes offers more opportunities to optimize their pharmacological profiles. Herein, three Ru(II)-arene complexes containing a glutathione S-transferase (GST) inhibitor (NBDHEX) in chelate ligand have been designed and synthesized in this study. In vitro results indicated that the ligation with NBDHEX significantly increased the activities and selectivities of the organometallic Ru(II)-arene complexes against tumor cells, especially complex 3, which was the most active compound among the tested compounds. DFT calculations and hydrolysis results demonstrated that complex 3 with more alkyl groups in the arene ligand has increased electron density at the Ru(II) center as compared with complexes 1 and 2, thus resulting in the improved hydrolysis rate, which may be responsible for its higher anticancer activity. Further studies showed that complexes 1-3 can cause the loss of the mitochondrial membrane potential and upregulate the expression of Bcl-2 and Bax in A549 cells, suggesting that complexes 1-3-induced cell death may be mediated via the mitochondrial apoptotic pathway. Thus, these findings suggested that simultaneous modification of the chelate ligands and arene rings in the organometallic Ru(II)-arene complexes is an effective way to improve their pharmacological properties.

Low-Molecular-Weight Fucoidan as Complementary Therapy of Fluoropyrimidine-Based Chemotherapy in Colorectal Cancer.

This study investigated the roles of low-molecular-weight fucoidan (LMWF) in enhancing the anti-cancer effects of fluoropyrimidine-based chemotherapy. HCT116 and Caco-2 cells were treated with LMWF and 5-FU. Cell viability, cell cycle, apoptosis, and migration were analyzed in both cell types. Potential mechanisms underlying how LMWF enhances the anti-cancer effects of fluoropyrimidine-based chemotherapy were also explored. The cell viability of HCT116 and Caco-2 cells was significantly reduced after treatment with a LMWF--5FU combination. In HCT116 cells, LMWF enhanced the suppressive effects of 5-FU on cell viability through the (1) induction of cell cycle arrest in the S phase and (2) late apoptosis mediated by the Jun-N-terminal kinase (JNK) signaling pathway. In Caco-2 cells, LMWF enhanced the suppressive effects of 5-FU on cell viability through both the c-mesenchymal-epithelial transition (MET)/Kirsten rat sarcoma virus (KRAS)/extracellular signal-regulated kinase (ERK) and the c-MET/phosphatidyl-inositol 3-kinases (PI3K)/protein kinase B (AKT) signaling pathways. Moreover, LMWF enhanced the suppressive effects of 5-FU on tumor cell migration through the c-MET/matrix metalloproteinase (MMP)-2 signaling pathway in both HCT116 and Caco-2 cells. Our results demonstrated that LMWF is a potential complementary therapy for enhancing the efficacies of fluoropyrimidine-based chemotherapy in colorectal cancers (CRCs) with the wild-type or mutated KRAS gene through different mechanisms. However, in vivo studies and in clinical trials are required in order to validate the results of the present study.

A subset of CB002 xanthine analogs bypass p53-signaling to restore a p53 transcriptome and target an S-phase cell cycle checkpoint in tumors with mutated-p53.

Mutations in TP53 occur commonly in the majority of human tumors and confer aggressive tumor phenotypes, including metastasis and therapy resistance. CB002 and structural-analogs restore p53 signaling in tumors with mutant-p53 but we find that unlike other xanthines such as caffeine, pentoxifylline, and theophylline, they do not deregulate the G2 checkpoint. Novel CB002-analogs induce pro-apoptotic Noxa protein in an ATF3/4-dependent manner, whereas caffeine, pentoxifylline, and theophylline do not. By contrast to caffeine, CB002-analogs target an S-phase checkpoint associated with increased p-RPA/RPA2, p-ATR, decreased Cyclin A, p-histone H3 expression, and downregulation of essential proteins in DNA-synthesis and DNA-repair. CB002-analog #4 enhances cell death, and decreases Ki-67 in patient-derived tumor-organoids without toxicity to normal human cells. Preliminary in vivo studies demonstrate anti-tumor efficacy in mice. Thus, a novel class of anti-cancer drugs shows the activation of p53 pathway signaling in tumors with mutated p53, and targets an S-phase checkpoint.