Anti-cancer activity of DHA on gastric cancer—an in vitro and in vivo study

Treatment of gastric cancer remains a major challenge, and new anticancer drugs are urgently required. This study investigated whether dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, could inhibit the growth of gastric cancer both in vitro and in vivo. A series of in vitro experiments including MTT, colony-forming, wound healing, invasion, cell cycle, cellular senescence, and apoptosis assays were performed to examine the antiproliferative and antimetastatic effects of DHA on three gastric cancer cell lines, SGC-7901, BGC823, and MGC803. The result showed that the proliferation rate and colony-forming abilities of gastric cancer cells were significantly suppressed by DHA together with significant suppression of the expressions of proliferation markers (PCNA, cyclin E, and cyclin D1), and upregulation of p21 and p27. Moreover, DHA induced cellular senescence, G1 phase cell cycle arrest and hindered the migration and invasion of gastric cancer cells corresponding with downregulation of MMP-9 and MMP-2. Furthermore, DHA significantly induced apoptosis through suppressing Bcl-2 as well as activating caspase-9 and PARP. Treatment of gastric cancer cells with DHA increased miR-15b and miR-16 expression, caused a downregulation of Bcl-2, resulting in apoptosis of gastric cancer cells. In vivo, our data showed that DHA significantly inhibited the growth of SGC7901 cell-transplanted tumors. In summary, we have shown that DHA is able to inhibit the growth and metastasis of human gastric cancer. The modulation of miR-15b and miR-16 mediated the apoptosis effects of DHA in gastric cancer cells. Our work suggested that DHA has significant anticancer effects against gastric cancer both in vivo and in vitro, indicating that it is a promising therapy for human gastric cancer.     

Bullatacin — in vivo and in vitro experience in an ovarian cancer model

The cytotoxicity and antitumor effects of the acetogenin Bullatacin were evaluated in vitro in multiple ovarian cancer cell lines and in vivo in a murine ovarian teratocarcinoma (MOT) model in C3HeB/FeJ mice. The in vitro cytotoxicity of Bullatacin against four human ovarian epithelial tumor cell lines (OC-194, OC-222, OVCAR-3, and A-2780) was assessed in 48- and 72-h tetrazolium-dye (MTT) cytotoxicity assays. The percentage of cytotoxicity was determined on the basis of the mean optical density of the respective untreated cells and the dose effective against 50% of the cells (ED₅₀) was calculated for each cell line. In vivo experiments were performed on adult female C3HeB/FeJ mice, which were injected i.p. with 10⁵ MOT cells and varying amounts of Bullatacin given either in a single dose or in 5 subsequent doses over 72 h. All mice were observed for survival relative to that of the control groups, which were injected either with 10⁵ MOT cells with or without serial injections of vehicle or with vehicle only. All four epithelial ovarian cancer cell lines displayed sensitivity to Bullatacin. The relative cytotoxic effects were very heterogeneous, with the ED₅₀ value ranging between 10^–7 g/ml for OC-194 and 4 g/ml for the cisplatin-resistant cell line OVCAR-3 in a 72-h MTT cytotoxicity assay. All mice that had been injected i.p. with 10⁵ MOT cells and 1.4 mg/kg or more of Bullatacin died within the first 24 h after injection, whereas all mice that had received 600 g/kg of Bullatacin or less survived equally as long as the controls that had been injected with MOT only (21.1±0.9 days). Mice that had received Bullatacin at a dose ranging from 600 g/kg to 1.4 mg/kg either died during the 1st day postinjection or survived, but not longer than the MOT control group. Serial i.p. injections of Bullatacin again either led to death of the mice within 24–48 h of the last dose of Bullatacin or did not have any effect on the survival of the mice as compared with the respective control groups, which had been injected with the tumor and serial injections of vehicle (22.5±2.2 days). In summary, Bullatacin showed no effect on MOT-caused animal death in C3HeB/FeJ mice at nonlethal dose ranges, whether it was given as a single i.p. dose or serially over 72 h. In vitro, however, it proved to be a very potent cytotoxic agent in a variety of ovarian cancer cell lines. As compared with other chemotherapeutic agents, which we accept as having clinically important antitumor efficacy against ovarian cancer, such as cisplatin or carboplatin, Bullatacin demonstrated a very favorable ED_{50 in vitro}/LD_{50 in vivo} (the dose lethal to 50% of the mice) ratio.This work was supported by the Bertha Warshaver Rubin Research Fund and was presented at the 21st annual meeting of the Western Association of Gynecologic Oncologists, May 19–23, 1993, Santa Monica, California     

Host defense peptides for treatment of colorectal carcinoma – a comparative in vitro and in vivo analysis

Host defense peptides (HDP) constitute effector molecules of the innate immune system. Besides acting against microbia and fungi, they exhibit broad and selective oncolytic activity. The underlying mechanism is at least partially attributable to elevated surface-exposed levels of phosphatidylserine (PS) on tumor targets. In this study, comprehensive analysis of NK-2-based derivatives (C7A, C7A-D21K, and C7A-Δ) was done on patient-derived ultra-low passage colorectal carcinoma (CRC) cell lines. Peptides were designed to improve antitumoral potential. Mellitin was used as positive control and a non-toxic peptide (NK11) served as negative control. Subsequently, effectiveness of local HDP application was determined in xenopatients.Generally, CRC lines displayed a heterogeneous pattern of surface-exposed PS, which was usually below standard CRC cells. Of note, five out of seven cell lines were susceptible towards HDP-mediated lysis (lytic activity of peptides: C7A-D21K > C7A-Δ= C7A). Oncolytic activity correlated mostly with surface-exposed PS levels. Apoptosis as well as necrosis were involved in killing. In an in vivo experiment, substantial growth inhibition of HROC24 xenografts was observed after HDP therapy and, surprisingly, also after NK11 treatment.These promising data underline the high potential of HDPs for oncolytic therapies and may provide a rationale for optimizing preclinical treatment schedules based on NK-2.     

Does Lactoferrin Behave as an Immunohistochemical Oncofetal Marker in Bone and Cartilage Human Neoplasms?

By immunohistochemistry, lactoferrin (LF) has been extensively investigated in human neoplastic tissues; moreover, LF is able to promote bone growth in a murine model. Until now, no systematic studies on human osteocartilagineous fetal samples have been performed in comparison to corresponding neoplastic specimens to verify if LF may represent an oncofetal marker in this field of pathology. By a monoclonal antibody (clone 1A1; Biodesign International; w.d. 1:75) the distribution pattern of LF in bones of 25 human fetal tissues (8–34 gestation weeks), 10 adults (47–82 years) and 30 cartilage as well as 27 bone tumours (9–76 years) was analyzed. LF was encountered in 23/57 cases of osteocartilagineous tumors and namely in 10/10 giant cell tumours, 5/7 osteoid osteomas, 3/3 chondroblastomas, 3/3 chondromyxoid fibromas, 1/1 myeloma, 1/1 adamantinoma. No LF immunoexpression was detected in osteosarcomas, chondrosarcomas, ossifying fibromas, osteochondroma and enchondromas. In embryo-fetal tissues, LF immunoreactivity was localized in mesenchymal cells as well as in chondroblasts at the 8th gestational week and in immature osteocytes and osteoblasts up to the 18th gestation week, with a considerable decrease by the 24th week. No LF expression was found in any bone district since the 30th and up to the 34th week of gestation as well as in corresponding adult samples. Our findings indicate a role for LF as a bone growth regulator in the early phases of the human endochondral ossification, although the hypothesis of LF as oncofetal marker appears questionable in bone tumours.     

Differential effects of tumor–platelet interaction in vitro and in vivo in glioblastoma

An elevated platelet count is considered an independent predictor of short survival in glioblastoma and various other tumor entities. Prothrombotic activity of the tumor microcirculation resulting in platelet activation and release of cytokines from activated platelets has been suggested to play a role. This study was designed to analyze the effects of platelet-released cytokines on glioblastoma and endothelial cell proliferation and migration in vitro, and the influence of platelet count on glioblastoma growth and angiogenesis in vivo. In cultured human glioblastoma, umbilical cord and cerebral microvascular endothelial cells platelet-released cytokines significantly stimulated proliferation and migration as well as sprouting and formation of capillary-like structures. In vivo, glioblastoma cells were implanted in mice followed by platelet depletion starting 1 or 8 days later. Tumor volume, proliferative index, and vessel density analyzed 14 days after engraftment did not differ between animals with a normal and a low platelet count. Likewise, no effect of platelet depletion over 20 days upon the volume of intracerebrally growing tumors was observed in mice. Additionally, proliferative activity and vessel density determined in tumor samples from patients operated upon glioblastoma did not show any correlation with the patients’ preoperative platelet count. Thus, we conclude that distinct proliferation- and chemotaxis-stimulating effects of platelet-derived cytokines can be achieved in vitro, while the platelet count does not exert a major influence on tumor growth and tumor angiogenesis in GBM in vivo.     

Vasculogenic mimicry–potential target for glioblastoma therapy: an in vitro and in vivo study

Glioblastoma is one of the most angiogenic human tumors and characterized by microvascular proliferations. A better understanding of glioblastoma vasculature is needed to optimize anti-angiogenic therapy that has shown a promising but incomplete efficacy. The present study examined 48 glioblastomas by CD34 endothelial marker periodic acid–Schiff (PAS) dual staining and found non-endothelial cell-lined blood vessels that were formed by tumor cells (vasculogenic mimicry, VM) existing in a fraction of these tumors. We hypothesized that CD133-positive glioblastoma stem-like cells (GSCs) may play a pivotal role in glioblastoma VM formation and then demonstrated in vitro and in vivo that a subset of GSCs were capable of vasculogenesis. Moreover, we found that several growth factors involved in normal angiogenesis were expressed in GSCs. We describe here a new mechanism of alternative glioblastoma vascularization and open a new perspective for the anti-vascular treatment strategy.     

Retention of paclitaxel in cancer cells for 1 week in vivo and in vitro

Purpose: Clinically, the administration of paclitaxel for ovarian cancer on a dose-dense weekly schedule, rather than the conventional every-3-week schedule, might demonstrate greater tumor-cell death. Here, we investigate the pharmacokinetics and the pharmacodynamics of weekly paclitaxel in cancer cells in vivo and in vitro. Experimental design: Paclitaxel concentrations were measured by HPLC, and apoptotic cells were detected by TUNEL assay in paclitaxel-pretreated cervical cancer cells treated with paclitaxel (10 ng/ml) and in the tissues of cervical cancer patients treated with weekly paclitaxel (60 mg/m²/week). Polymerized tubulin was detected with a tubulin polymerization assay, and the BrdU cell proliferation assay was used to assess the effect of paclitaxel. Results: Paclitaxel remained in the cancer tissues of six patients for 6 days after the last medication. In vitro, paclitaxel was retained in all cell lines for 24 h after its removal from the medium, and paclitaxel was still detectable in CaSki cells on day 7. Simultaneous treatment with depolymerizing drugs inhibited the retention of paclitaxel in cells and paclitaxel-induced polymerization of tubulin. After paclitaxel treatment, apoptotic cells were detected in cancer tissues and CaSki cells for 1 week. Under high magnification, apoptotic cells on day 7 after paclitaxel treatment showed multinucleation. Conclusions: Paclitaxel is unusual in that it accumulates especially in cancer cells and induces apoptosis for 1 week in vivo and in vitro. On the other hand, paclitaxel could not be detected in cancer tissues after 2 weeks. The administration of paclitaxel on a weekly schedule, rather than the standard every-3-week schedule, might produce greater tumor-cell death.     

Influence of stromal–epithelial interactions on breast cancer in vitro and in vivo

Stromal cell-secreted chemokines including CCL2 have been implicated in the primary tumor microenvironment, as mediators of tumor cell migration, proliferation, and angiogenesis. Expression of CCL2 and its principal receptor CCR2 was analyzed by RQ-PCR in primary tumor cells and breast cancer cell lines. Breast cancer cell lines (MDA-MB-231, T47D) were co-cultured directly on a monolayer of primary breast tumor and normal stromal cells, retrieved using EpCAM+ magnetic beads, and changes in expression of CCL2, CCR2, MMP11, ELK1, VIL2, and Ki67 detected by RQ-PCR. Epithelial cell migration and proliferation in response to stromal cell-secreted factors was also analyzed. In vivo, tumor xenografts were formed by co-injecting T47D cells with primary tumor stromal cells. Following establishment, tumors were harvested and digested, epithelial cells retrieved and analyzed by RQ-PCR. Whole tumor tissue was also analyzed by immunohistochemistry for CD31 and the VIL2 encoded protein Ezrin. Tumor stromal cells expressed significantly higher levels of CCL2 than normal cells, with no CCR2 expression detected. Primary epithelial cells and breast cancer cell lines expressed elevated CCL2, with relative expression of CCR2 found to be higher than the ligand. Interaction of breast cancer epithelial cells with primary tumor, but not normal stromal cells, stimulated increased expression of CCL2 (8-fold), ELK1 (6-fold), VIL2 (6-fold), and MMP11 (17-fold). Factors secreted by stromal cells, including CCL2, stimulated a significant increase in epithelial cell migration, with no effect on cell proliferation in vitro observed. In vivo, the presence of stromal cells resulted in tumors of increased volume, mediated at least in part through neoangiogenesis demonstrated by immunohistochemistry (CD31). Admixed tumor xenografts exhibited increased expression of Ki67, MMP11, VIL2, and ELK1. Elevated Ezrin protein was also detected, with increased cytoplasmic localization. The results presented highlight mechanisms through which breast cancer epithelial cells can harness stromal cell biology to support tumor progression.     

Human umbilical cord blood stem cells show PDGF-D–dependent glioma cell tropism in vitro and in vivo

Despite advances in clinical therapies and technologies, the prognosis for patients with malignant glioma is poor. Neural stem cells (NSCs) have a chemotactic tropism toward glioma cells. The use of NSCs as carriers of therapeutic agents for gliomas is currently being explored. Here, we demonstrate that cells isolated from the umbilical cord blood show mesenchymal characteristics and can differentiate to adipocytes, osteocytes, and neural cells and show tropism toward cancer cells. We also show that these stem cells derived from the human umbilical cord blood (hUCB) induce apoptosis-like cell death in the glioma cell line SNB19 via Fas-mediated caspase-8 activation. From our glioma tropism studies, we have observed that hUCB cells show tropism toward glioma cells in vitro, in vivo, and ex vivo. We determined that this migration is partially dependent on the expression levels of platelet-derived growth factor (PDGF)-D from glioma cells and have observed that local concentration gradient of PDGF-D is sufficient to cause migration of hUCB cells toward the gradient as seen from our brain slice cultures. In our animal experiment studies, we observed that intracranially implanted SNB19 green fluorescent protein cells induced tropism of the hUCB cells toward themselves. In addition, the ability of these hUCBs to inhibit established intracranial tumors was also observed. We also determined that the migration of stem cells toward glioma cells was partially dependent on PDGF secreted by glioma cells and that the presence of PDGF-receptor (PDGFR) on hUCB is required for migration. Our results demonstrate that hUCB are capable of inducing apoptosis in human glioma cells and also show that glioma tropism and hUCB tropism toward glioma cells are partially dependent on the PDGF/PGGFR system.     

Silencing of IKKε using siRNA inhibits proliferation and invasion of glioma cells in vitro and in vivo

Recent studies implicated IKKε in the pathogenesis of many human cancers by promoting cell proliferation, increasing tumor angiogenesis and metastasis, and generating resistance to cell apoptosis. However, whether IKKε can influence the invasive ability and proliferation of glioma cells remains largely unknown. In this study, we showed that overexpression of IKKε is positively correlated to glioma pathological grade, suggesting that IKKε plays a role in tumor progression, rather than tumor initiation. Targeted knockdown of IKKε in human glioma cells using siRNA, was associated with inhibition of cell growth, cell cycle arrest and decreased cell invasion; however, notable apoptosis was not observed. Furthermore, we demonstrated that transposition of NF-κB p65 resulted in the alteration of these phenotypes. Tumor growth was attenuated in established subcutaneous gliomas in nude mice treated with IKKε siRNA in vivo. Collectively, our results suggest that deregulation of IKKε plays a pivotal role in the uncontrolled proliferation and malignant invasion of glioma cells in vitro and in vivo by targeting NF-κB. Silencing of IKKε using synthetic siRNAs may offer a novel therapeutic strategy for the treatment of glioma.     

Control of Atm−/− thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone

Aim Ataxia telangiectasia (A-T) is an autosomal recessive disease in humans caused by mutations in the Atm (A-T mutated) gene. The disease involves multiple organ systems, and is associated with a high incidence of leukemias and lymphomas that develop in childhood. We have reported previously that thymic lymphoma development in Atm knockout (Atm–/–) mice is associated with elevated spontaneous DNA synthesis in thymocytes, and that dexamethasone (Dex) attenuates the elevated DNA synthesis and prevents thymic lymphoma development. The primary objectives of the present study were (1) to investigate possible mechanisms underlying the tumor-suppressing effect of Dex on Atm–/– thymic lymphoma cells, and (2) to determine whether Dex is an effective tumor-suppressing treatment in mice bearing transplanted Atm–/– thymic tumors.Methods Establishment of a number of Atm–/– thymic lymphoma (ATL) cell lines from Atm–/– mice, cell proliferation assays, cell cycle analyses, Western blotting and Hoechst nuclear staining were used to analyze the effects of Dex on Atm–/– thymic lymphoma cells. Atm–/– tumor cells were transplanted into the right flanks of Atm+/+ mice prior to the initiation of Dex treatment.Results Atm–/– tumor cells were highly sensitive to Dex, both in culture and in vivo as ectopic tumors in mice. In cultured ATL-1 cells, Dex induced apoptosis, arrested the cell cycle at the G₁ phase and downregulated NF-B and multiple cell cycle regulators, while upregulating the NF-B inhibitor IB. In Atm+/+ mice transplanted subcutaneously with ATL-1 cells, tumor growth was either prevented completely or significantly suppressed by Dex treatment.Conclusions Our findings identify potential mechanisms by which Dex affects the proliferation and survival of ATL-1 cells in culture, and provide evidence that Dex can suppress the proliferation of Atm–/– thymic lymphoma cells growing in the body. Together these results add to our earlier published data suggesting that the cellular pathways regulated by Dex may be promising therapeutic targets for prevention and treatment of thymic lymphomas in A-T individuals.     

Effects of polyamine depletion by α-difluoromethylornithine on in vitro and in vivo biological properties of 4T1 murine mammary cancer cells

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anti-cancer therapy. Polyamine depletion by DFMO has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of STAT3, JNK, and ERK, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of anti-proliferative effect at the metastatic sites.     

Concomitant secretion of calcitonin, β-endorphin and ACTH from medullary thyroid carcinoma in vivo and in vitro

The present study was performed to investigate the possible synthesis of a common precursor molecule for calcitonin (CT), adrenocorticotropin (ACTH) and β-lipotropin (β-LPH)/β-endorphin (β-EP) by the human medullary thyroid carcinoma (MTC). In a patient with MTC but without Cushing's syndrome, the response of plasma CT, ACTH and cortisol levels to a calcium infusion, lysine vasopressin (LVP) and dexamethasone were measured. A parallel increase of these hormones in response to calcium and LVP was seen, while there was a paradoxical increase of CT during dexamethasone infusion. Incubation of MTC fragments obtained at surgery showed a significant correlation of the secretion of CT, ACTH and β-LPH/β-EP in response to calcium, LVP and dexamethasone. The concomitant release of these hormones in vivo and in vitro could be compatible with the synthesis of a common precursor molecule for CT, ACTH and β-LPH/β-EP in MTC, although this was not substantiated by gel-chromatography of the tumor extract. Corticotropin releasing factor, a regulator of the normal processing of pro-opiococorticoid precursor molecule in the anterior pituitary gland, is also able to activate ACTH, β-LPH/β-EP and calcitonin secretion from the malignant C-cell of the thyroid.     

Antitumor effects of minodronate,a third-generation nitrogen-containing bisphosphonate,in synergy with γδT cells in human glioblastoma in vitro and in vivo

Nitrogen-containing bisphosphonates (N-BPs), which prevent bone resorption, exert direct and γδT cell (GDT)-mediated antitumor effects against several tumor cell types, including glioblastoma (GBM). However, limited information is available regarding the antitumor effects of N-BPs in GBM. Specifically, the antitumor effects of minodronate (MDA), a third-generation N-BP, in GBM are yet unclear. This study aimed to investigate the antitumor effects of MDA in GBM in vitro and in vivo. We performed growth inhibition and apoptosis detection assays using the GBM cell lines U87MG and U138MG. Apoptosis inhibition assays were also conducted. In vivo xenograft assays were performed in highly immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Sug/Jic mice subcutaneously implanted with U87MG and U138MG cells. Growth inhibition and apoptosis detection assays demonstrated that MDA inhibited GBM cell growth via apoptosis, which was markedly enhanced by ex vivo expanded GDT. A pan-caspase inhibitor, z-VAD-fmk, inhibited MDA-induced U138MG apoptosis and MDA/GDT-induced U87MG and U138MG apoptosis. But z-VAD-fmk increased MDA-induced U87MG apoptosis. MDA/GDT-mediated apoptosis was blocked by the anti-T cell receptor (TCR) Vγ9, mevalonate pathway inhibitor, granzyme B inhibitor, and antitumor necrosis factor (TNF)-α. In vivo xenograft assays showed that combined intraperitoneal administration of MDA/GDT induced antitumor effects on unestablished U87MG-derived subcutaneous tumors. MDA exerted direct and GDT-mediated anti-GBM apoptotic effects in a caspase-dependent manner. GDT recognized MDA-exposed GBM cells via TCRVγ9 and induced apoptosis via granzyme B and TNF-α release. Because MDA elicited anti-GBM effects in synergy with GDT in vivo, a combination of MDA and ex vivo-generated GDT could be an effective treatment in patients with GBM.     

Berberine enhances radiosensitivity of esophageal squamous cancer by targeting HIF-1α in vitro and in vivo

Radiation therapy is an important treatment approach for esophageal squamous cell carcinoma (ESCC). However, how to promote radiation sensitivity in ESCC remains a challenge. This study aimed to evaluate the effects of berberine, a common used Chinese medicine, on the radiosensitivity of ESCC. ECSS cell line ECA109 and TE13 were subjected to hypoxia and/or ionizing radiation (IR), in the presence or absence of berberine treatment. Cell growth and survival, and apoptosis were evaluated. In addition, ECA109 cells were xenografted into nude mice and subjected to IR and/or berberine treatment. The expression of HIF-1α and VEGF was detected by western blot and immunohistochemical analysis. Our results showed that berberine increased radiosensitivity of ESCC cells and xenografts, and this was associated with the inhibition of HIF-1α and VEGF expression. These data suggest that berberine may be a potential radiotherapy sensitization drugs due to its significant anti-hypoxia activity.     

Fucoidan inhibition of lung cancer in vivo and in vitro: role of the Smurf2-dependent ubiquitin proteasome pathway in TGFβ receptor degradation

Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. In this study, we demonstrate that fucoidan reduces tumor size in LLC1-xenograft male C57BL/6 mice. Moreover, we found that LLC1-bearing mice continuously fed fucoidan showed greater antitumor activity than mice with discontinuous feeding. Fucoidan inhibited the in vitro growth of lung cancer cells. Transforming growth factor β (TGFβ) receptors (TGFRs) play important roles in the regulation of proliferation and progression, and high TGFRI expression in lung cancer specimens is associated with a worse prognosis. Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro. Moreover, fucoidan reduces TGFR downstream signaling events, including those in Smad2/3 and non-Smad pathways: Akt, Erk1/2, and FAK phosphorylation. Furthermore, fucoidan suppresses lung cancer cell mobility upon TGFβ stimulation. To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells. Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan, namely decreasing tumor growth by modulating the TGFR/Smad7/Smurf2-dependent axis, leading to TGFR protein degradation and inhibition of lung cancer cell progression in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent or dietary supplementation for lung cancer, acting via the Smurf2-dependent ubiquitin degradation of TGFβ receptors.     

β-elemene Induces Caspase-dependent Apoptosis in Human Glioma Cells in vitro through the Upregulation of Bax and Fas/ FasL and Downregulation of Bcl-2

Background: β-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects invarious cancer cell lines. However, the activity of β-elemene against glioma cells remains unclear. In the presentstudy, we assessed effects of β-elemene on human glioma cells and explored the underlying mechanism. Materialsand Methods: Human glioma U87 cells were used. Cell proliferation was determined with MTT assay andcolony formation assay to detect the effect of β-elemene at different doses and times. Fluorescence microscopywas used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cyclingwere analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed toinvestigated the influence of β-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experimentwas divided into two groups: the blank control group and β-elemne treatment group. Results: With increase inthe concentration of β-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentrationof inhibited cell viability (IC50) was 48.5 μg/mL for 24h. β-elemene could induce cell cycle arrest in the G0/G1phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation ofcaspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, whilethe anti-apoptotic Bcl-2 was downregulated after treatment with β-elemene at both mRNA and protein levels.Furthermore, proliferation and colony formation by U87 cells were inhibited by β-elemene in a time and doesdependentmanner. Conclusions: Our results indicate that β-elemene inhibits growth and induces apoptosis ofhuman glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasLand Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptoticcascades.