Treatment Of Cells Research Articles

Two mTOR inhibitors, rapamycin and Torin 1, differentially regulate iron-induced generation of mitochondrial ROS.

It is generally believed that gene-environment interaction may contribute to neurodegeneration. Of particular note is that iron overload may be one of the risk factors for neurodegeneration. However, the mechanisms underlying iron-associated neurotoxicity are not fully understood. Here we explored the effects of mechanistic target of rapamycin (mTOR) inhibition in iron-stressed human neuroblastoma cells. Two mTOR inhibitors, rapamycin and Torin 1, had similar effects in cells exposed to a relatively low concentration of iron. At a higher concentration of iron, Torin 1, instead of rapamycin, could further aggravate iron-induced cytotoxicity, and mitochondrial ROS levels were significantly higher in Torin 1-treated cells. These results suggest that mTOR inhibition may not be able to alleviate iron-induced neurotoxicity.

Effect of melatonin on bovine theca cells in vitro

Melatonin affects granulosa cell function in several species but its function in theca cells is less clear, particularly in monotocous animals. Thus, the objectives of this study were to determine the effects of melatonin on theca cell steroidogenesis, gene expression and cell proliferation in a monotocous species, namely cattle. Ovaries were collected from a local bovine abattoir, from which theca cells were isolated from large (8-22mm) follicles and treated with various hormones in serum-free medium for 24h or 48h. Melatonin caused a dose-dependent inhibition (P<0.05) of LH+insulin-like growth factor 1 (IGF1)-induced androstenedione and progesterone production. Also, melatonin inhibited (P<0.05) LH+IGF1-induced expression of steroidogenic acute regulatory protein (StAR) mRNA (via real-time polymerase chain reaction) in theca cells, but it had no effect (P>0.10) on cytochrome P450 11A1 (CYP11A1) and cytochrome P450 17A1 (CYP17A1) mRNA abundance. In LH+IGF1-treated theca cells, melatonin decreased caspase 3 (CASP3) mRNA to levels similar to those observed in LH-treated theca cells. In contrast, melatonin increased (P<0.05) the number of bovine theca cells in both LH- and LH+IGF1-treated cultures. In conclusion, melatonin may act as an endocrine regulator of ovarian function in cattle by stimulating theca cell proliferation and inhibiting differentiation via inhibition of hormone-induced steroidogenesis.

Cell-Cycle Position of Single MYC-Driven Cancer Cells Dictates Their Susceptibility to a Chemotherapeutic Drug.

While many tumors initially respond to chemotherapy, regrowth of surviving cells compromises treatment efficacy in the long term. The cell-biological basis of this regrowth is not understood. Here, we characterize the response of individual, patient-derived neuroblastoma cells driven by the prominent oncogene MYC to the first-line chemotherapy, doxorubicin. Combining live-cell imaging, cell-cycle-resolved transcriptomics, and mathematical modeling, we demonstrate that a cell's treatment response is dictated by its expression level of MYC and its cell-cycle position prior to treatment. All low-MYC cells enter therapy-induced senescence. High-MYC cells, by contrast, disable their cell-cycle checkpoints, forcing renewed proliferation despite treatment-induced DNA damage. After treatment, the viability of high-MYC cells depends on their cell-cycle position during treatment: newborn cells promptly halt in G1 phase, repair DNA damage, and form re-growing clones; all other cells show protracted DNA repair and ultimately die. These findings demonstrate that fast-proliferating tumor cells may resist cytotoxic treatment non-genetically, by arresting within a favorable window of the cell cycle.

Epigenetic changes in the early stage of silica-induced cell transformation

The increasing use of nanomaterials in numerous domains has led to growing concern about their potential toxicological properties, and the potential risk to human health posed by silica nanoparticles remains under debate. Recent studies proposed that these particles could alter gene expression through the modulation of epigenetic marks, and the possible relationship between particle exposure and these mechanisms could represent a critical factor in carcinogenicity. In this study, using the Bhas 42 cell model, we compare the effects of exposure to two transforming particles, a pyrogenic amorphous silica nanoparticle NM-203 to those of the crystalline silica particle Min-U-Sil® 5. Short-term treatment by Min-U-Sil® 5 decreased global DNA methylation and increased the expression of the two de novo DNMTs, DNMT3a and DNMT3b. NM-203 treatment affected neither the expression of these enzymes nor DNA methylation. Moreover, modified global histone H4 acetylation status and HDAC protein levels were observed only in the Min-U-Sil® 5-treated cells. Finally, both types of particle treatment induced strong c-Myc expression in the early stage of cell transformation and this correlated with enrichment in RNA polymerase II as well as histone active marks on its promoter. Lastly, almost all parameters that were modulated in the early stage were restored in transformed cells suggesting their involvement mainly in the first steps of cell transformation.

Chemical Constituents of the Roots and Rhizomes of Saposhnikovia divaricata and their Cytotoxic Activity

Phytochemical investigation of the MeOH extract of the roots and rhizomes of Saposhnikovia divaricata (Umbelliferae) resulted in the isolation of six chromons (1-6)-and five polyacetylene derivatives (7-11). Compounds 9 and 11 were isolated from S. divaricate for the first time. The chromon derivatives -(1-6) were evaluated for their cytotoxic activity against HL-60 human promyclocytic leukemia cells. Compound 1 (3'-O-angeloylhamaudol) showed the most potent cytotoxic activity with an IC₅₀ value of 4.41 μM and was found to induce apoptotic cell death in HL-60 cells. The loss of mitochondrial membrane potential, release of cytochrome c into the cytoplasm, and activation of caspase-9 in the 1-treated HL-60 cells suggests that I induces apoptosis through the mitochondial-dependent apoptotic pathway.

Cinnamaldehyde Derivatives Inhibit Coxsackievirus B3-Induced Viral Myocarditis.

The chemical property of cinnamaldehyde is unstable in vivo, although early experiments have shown its obvious therapeutic effects on viral myocarditis (VMC). To overcome this problem, we used cinnamaldehyde as a leading compound to synthesize derivatives. Five derivatives of cinnamaldehyde were synthesized: 4-methylcinnamaldehyde (1), 4-chlorocinnamaldehyde (2), 4-methoxycinnamaldehyde (3), α-bromo-4-methylcinnamaldehyde (4), and α-bromo-4-chlorocinnamaldehyde (5). Neonatal rat cardiomyocytes and HeLa cells infected by coxsackievirus B3 (CVB3) were used to evaluate their antiviral and cytotoxic effects. In vivo BALB/c mice were infected with CVB3 for establishing VMC models. Among the derivatives, compound 4 and 5 inhibited the CVB3 in HeLa cells with the half-maximal inhibitory concentrations values of 11.38 ± 2.22 μM and 2.12 ± 0.37 μM, respectively. The 50% toxic concentrations of compound 4 and 5-treated cells were 39-fold and 87-fold higher than in the cinnamaldehyde group. Compound 4 and 5 effectively reduced the viral titers and cardiac pathological changes in a dose-dependent manner. In addition, compound 4 and 5 significantly inhibited the secretion, mRNA and protein expressions of inflammatory cytokines TNF-α, IL-1β and IL-6 in CVB3-infected cardiomyocytes, indicating that brominated cinnamaldehyde not only improved the anti-vital activities for VMC, but also had potent anti-inflammatory effects in cardiomyocytes induced by CVB3.

Insights into the cytotoxic activity of the phosphane copper(I) complex [Cu(thp)4][PF6

The phosphane Cu(I) complex [Cu(thp)4][PF6], 1 (thp=tris(hydroxymethyl)phosphane) shows notable in vitro antitumour activity against a wide range of solid tumours. Uptake experiments performed in 1-treated colon cancer cells by atomic absorption spectrometry, reveal that the antiproliferative activity is consistent with the intracellular copper content. The solution chemistry of this agent, investigated by means of X-ray Absorption Spectroscopy and spectrophotometric titrations in aqueous media, indicates that 1 is labile giving coordinative unsaturated [Cu(thp)n]+ species (n=3 and 2) at micromolar concentrations. [Cu(thp)n]+ are reactive species that yield the mixed-ligand complex [Cu(thp)2(BCS)]− (BCS: bathocuproinedisulphonate(2-)) upon interaction with N,N-diimine. Analogously, [Cu(thp)n]+ interact with the methionine-rich peptide sequence (Ac-MMMMPMTFK-NH2; Pep1), relevant in the recruiting of physiological copper, giving [Cu(thp)(Pep1)]+ and [Cu(Pep1)]+ species. The formation of these adducts was assessed by electrospray mass spectrometry in the positive ion mode and validated by density functional theory investigations. The possibility to trans-chelate Cu(I) from pure inorganic [Cu(thp)n]+ assemblies into more physiological adducts represents a pathway that complex 1 might follow during the internalization process into cancer cells.

Synthesis, characterisation and cytotoxicity studies of Manganese(I) and Rhenium(I) based metallacrown ethers

Manganese(I) and Rhenium(I) based metallacrown ethers [M(CO)3Br(μ-pcatgd)] (1, M=Mn; 2, M=Re) were synthesised by the reaction of M(CO)5Br (M=Mn, Re) and pyridine appended bidentate ligand crafted with flexible polyether spacer. The self-assembled mononuclear compounds 1 and 2 were characterised by IR, NMR, UV–Visible absorption and emission spectroscopic techniques. Molecular structure of 2 was determined by single-crystal X-ray diffraction methods and the molecular masses of 1 and 2 were confirmed by ESI–mass spectrometry. Anticancer activities of compounds 1 and 2 were investigated by in vitro cytotoxicity studies against different cancer cell lines as well as on normal cells. Compounds 1 and 2 showed a broad-spectrum inhibition on few cancer cells that were comparable to cisplatin. Compound 1 exhibited selective inhibition of cancer cells upon irradiation at λmax 365nm in dose-dependent manner. Morphological studies showed the induction of apoptosis in compound 1-treated cancer cells upon irradiation. The UV–Visible absorption spectroscopy-based myoglobin assay further supported the nature of compound 1 as photoactivatable CO releasing molecule (PhotoCORM).

Synthesis of apoptotic chalcone analogues in HepG2 human hepatocellular carcinoma cells.

Eight chalcone analogues were prepared and evaluated for their cytotoxic effects in human hepatoma HepG2 cells. Compound 5 had a potent cytotoxic effect. The percentage of apoptotic cells was significantly higher in compound 5-treated cells than in control cells. Exposure to compound 5 for 24h induced cleavage of caspase-8 and -3, and poly (ADP-ribose) polymerase (PARP). Our findings suggest that compound 5 is the active chalcone analogue that contributes to cell death in HepG2 cells via the extrinsic apoptotic pathway.

Hypoxia-preconditioned mesenchymal stem cells attenuate bleomycin-induced pulmonary fibrosis.

IntroductionIdiopathic pulmonary fibrosis is a progressive diffuse parenchymal lung disorder of unknown etiology. Mesenchymal stem cell (MSC)-based therapy is a novel approach with great therapeutic potential for the treatment of lung diseases. Despite demonstration of MSC grafting, the populations of engrafted MSCs have been shown to decrease dramatically 24 hours post-transplantation due to exposure to harsh microenvironments. Hypoxia is known to induce expression of cytoprotective genes and also secretion of anti-inflammatory, anti-apoptotic and anti-fibrotic factors. Hypoxic preconditioning is thought to enhance the therapeutic potency and duration of survival of engrafted MSCs. In this work, we aimed to prolong the duration of survival of engrafted MSCs and to enhance the effectiveness of idiopathic pulmonary fibrosis transplantation therapy by the use of hypoxia-preconditioned MSCs.MethodsHypoxic preconditioning was achieved in MSCs under an optimal hypoxic environment. The expression levels of cytoprotective factors and their biological effects on damaged alveolar epithelial cells or transforming growth factor-beta 1-treated fibroblast cells were studied in co-culture experiments in vitro. Furthermore, hypoxia-preconditioned MSCs (HP-MSCs) were intratracheally instilled into bleomycin-induced pulmonary fibrosis mice at day 3, and lung functions, cellular, molecular and pathological changes were assessed at 7 and 21 days after bleomycin administration.ResultsThe expression of genes for pro-survival, anti-apoptotic, anti-oxidant and growth factors was upregulated in MSCs under hypoxic conditions. In transforming growth factor-beta 1-treated MRC-5 fibroblast cells, hypoxia-preconditioned MSCs attenuated extracellular matrix production through paracrine effects. The pulmonary respiratory functions significantly improved for up to 18 days of hypoxia-preconditioned MSC treatment. Expression of inflammatory factors and fibrotic factor were all downregulated in the lung tissues of the hypoxia-preconditioned MSC-treated mice. Histopathologic examination observed a significant amelioration of the lung fibrosis. Several LacZ-labeled MSCs were observed within the lungs in the hypoxia-preconditioned MSC treatment groups at day 21, but no signals were detected in the normoxic MSC group. Our data further demonstrated that upregulation of hepatocyte growth factor possibly played an important role in mediating the therapeutic effects of transplanted hypoxia-preconditioned MSCs.ConclusionTransplantation of hypoxia-preconditioned MSCs exerted better therapeutic effects in bleomycin-induced pulmonary fibrotic mice and enhanced the survival rate of engrafted MSCs, partially due to the upregulation of hepatocyte growth factor.

Discriminative fluorescence sensing of biothiols in vitro and in living cells.

Simultaneous discriminative sensing of biothiols in vitro and in living cells has remained challenging. Herein, we report a new sulfonamide-based self-quenched fluorescent probe 1 for this purpose with high sensitivity and good selectivity. Treatment of 1 with cysteine (Cys), homocysteine (Hcy), or glutathione (GSH) yields aminoluciferin, 2-cyano-6-aminobenzothiazole homocysteine (CBTHcy), or 2-cyano-6-aminobenzothiazole (CBT), turning "on" the fluorescence at wavelengths of 522, 517, or 490 nm, respectively. Kinetic study indicated that 1 reacts with Cys faster than with Hcy or GSH. With these unique properties of 1, we applied 1 for highly sensitive sensing of Cys, Hcy, and GSH among other 19 natural amino acids (AAs) with good selectivity. Confocal fluorescence microscopic imaging of 1-treated HepG2 cells at two channels (522 ± 8 and 490 ± 8 nm), together with quantitative analysis, indicated that the "turn-on" fluorescence was induced by intracellular Cys-dominating condensation and reduction of 1 but not by intracellular GSH-dominating reduction of 1. This suggests that 1 could be applied for discriminative sensing of intracellular Cys from the abundant GSH. Further development of 1 might bring about an efficient tool for probing cellular functions that relate to biothiols.

6-Acetonyldihydrochelerythrine Is a Potent Inducer of Apoptosis in HCT116 and SW620 Colon Cancer Cells.

6-Acetonyldihydrochelerythrine (1), a benzophenanthridine alkaloid, isolated from the methanol extract of Zanthoxylum capense, displayed potent cytotoxic activity in human HCT116 and SW620 colon carcinoma cells, to a higher extent than 5-fluorouracil (5-FU), the cornerstone chemotherapeutic agent in colon cancer. Cytotoxicity of 1 was evaluated by MTS, lactate dehydrogenase (LDH), and Guava ViaCount assays. Interestingly, 1 significantly induced cytotoxicity in both cell lines, leading to a significant increase in LDH release, as compared to 5-FU. Further, Guava ViaCount flow cytometry assays demonstrated that 1 significantly increased cell death, as shown by the presence of a significantly higher population of apoptotic cells in both cell lines, as compared to cells exposed to 5-FU. Furthermore, evaluation of nuclear morphology by Hoechst staining of 1-treated HCT116 and SW620 cells confirmed flow cytometry results, demonstrating a marked induction of apoptotic cell death by 1, again to a further extent than that elicited by 5-FU. In addition, immunoblot analysis to ascertain the molecular events triggered by 1 exposure was performed. The results show that 1 exposure reduced the steady-state expression and activation of the pro-survival proteins ERK5 and Akt and increased the steady-state expression of p53 in both HCT116 and SW620 cells. Changes in ERK5 or Akt activation can be ascertained by evaluating the ratio of p-ERK5/ERK5 or p-Akt/Akt. In addition, exposure to 1 reduced expression of XIAP, Bcl-XL, and Bcl-2, while increasing the cleavage of poly(ADP-ribose) polymerase in both cell lines. Collectively, the data indicate that 6-acetonyldihydrochelerythrine (1) is a potent inducer of apoptosis in HCT116 and SW620 cell lines, highlighting its potential relevance in colon cancer.

Elimination of undifferentiated cancer cells by pluripotent stem cell inhibitors

Elimination of undifferentiated cancer cells by pluripotent stem cell inhibitors

Reply to “Heparan Sulfate in Baculovirus Binding and Entry of Mammalian Cells”

(1), we investigated the interaction ofbaculovirus and mammalian cell surface heparan sulfate pro-teoglycans (HSPG). The data show that baculovirus requiresHSPG sulfation, particularly N- and 6-O-sulfation, to bind andtransduce mammalian cells. We also show that baculovirus asso-ciates specifically with syndecan-1 (SDC-1) but not with othersyndecans or glypicans.As discussed in the article, HS has previously been shown to beinvolved in glycoprotein 64 (gp64)-mediated baculovirus bindingonto mammalian cells. Heparin and heparinase I and II treatmentof cells have also been shown to prevent the virus binding (2, 3).The role of HS in baculovirus entry was further studied in ourarticle (1). Binding and baculovirus-mediated gene delivery wereseen to decrease after NaClO

Withaferin A, a Steroidal Lactone from Withania somnifera, Induces Mitotic Catastrophe and Growth Arrest in Prostate Cancer Cells

Cell cycle deregulation is strongly associated with the pathogenesis of prostate cancer. Clinical trials of cell cycle regulators that target either the G0/G1 or G2/M phase to inhibit the growth of cancers including prostate cancer are increasing. The present study focused on the cell cycle regulatory potential of the withanolide withaferin A (1) on prostate cancer cells. Compound 1 induced G2/M arrest in both prostate cancer cell lines (PC-3 and DU-145) when treated for 48 h. The G2/M arrest was accompanied by upregulation of phosphorylated Wee-1, phosphorylated histone H3, p21, and Aurora B. On the other hand, downregulation of cyclins (A2, B1, and E2) and a reduction in phosphorylated Cdc2 (Tyr15) were observed in 1-treated prostate cancer cells. In addition, decreased levels of phosphorylated Chk1 (Ser345) and Chk2 (Thr68) were evident in prostate cancer cells on treatment with 1. These results suggest that activation of Cdc2 leads to arrest in the M phase, with abnormal duplication, and initiation of mitotic catastrophe that results in cell death. In conclusion, these results show clearly the potential of 1 as a regulator of the G2/M phase of the cell cycle and as a therapeutic agent for prostate cancer.

Bryostatin 5 induces apoptosis in acute monocytic leukemia cells by activating PUMA and caspases

Acute leukemia is a malignant clonal hematopoietic stem cell disease. In the current study, we examined the effects of bryostatin 5 on acute monocytic leukemia cells in vitro and in vivo. We also explored the mechanisms and pathways underlying the increase in apoptosis induced by bryostatin 5. Bryostatin 5 inhibited the growth of primary acute monocytic leukemia cells and U937 cells in a dose- and time-dependent manners. Bryostatin 5 also induced an increase in apoptosis and a decrease in the mitochondrial membrane potential (MMP) in U937 cells. Transmission electron microscopy (TEM) revealed that bryostatin 5-treated cells displayed typical apoptotic characteristics (chromatin condensation, karyopyknosis and formation of crescents and apoptotic bodies). In addition, bryostatin 5 increased the expression of P53 upregulated modulator of apoptosis (PUMA) and slightly increased P53 expression. Bryostatin 5 also significantly decreased Bcl-XL expression and significantly increased the expression levels of Bak, Bax, cleaved caspase 9 and cleaved caspase 3. The pro-apoptotic activity of bryostatin 5 in U937 cells was inhibited by PUMA siRNA and z-LEHD-fmk (a specific caspase 9 inhibitor). In addition, the PUMA siRNA significantly affected the expression of cleaved caspase 9, whereas z-LEHD-fmk had little effect on the expression of PUMA. The results suggest that PUMA is located upstream of caspase 9 in this apoptotic signaling pathway. These novel findings provide mechanistic insight into the induction of apoptosis by bryostatin 5 and might facilitate the development of clinical strategies to enhance the therapeutic efficacy of treatments for acute monocytic leukemia.

MR-Guided HIFU Treatment of Symptomatic Uterine Fibroids Using Novel Feedback-Regulated Volumetric Ablation: Effectiveness and Clinical Practice

To evaluate a novel feedback-regulated volumetric sonication method in MR-guided HIFU treatment of symptomatic uterine fibroids. 27 fibroids with an average volume of 124.9 ± 139.8 cc in 18 women with symptomatic uterine fibroids were ablated using the new HIFU system Sonalleve (1.5 T MR system Achieva, Philips). 21 myomas in 13 women were reevaluated 6 months later. Standard (treatment) cells (TC) and feedback-regulated (feedback) cells (FC) with a diameter of 4, 8, 12, and 16 mm were used and compared concerning sonication success, diameter of induced necrosis, and maximum achieved temperature. The non-perfused volume ratio (NPV related to myoma volume) was quantified. The fibroid volume was measured before, 1 month, and 6 months after therapy. Symptoms were quantified using a specific questionnaire (UFS-QoL). In total, 205 TC and 227 FC were applied. The NPV ratio was 23 ± 15 % (2 - 55). The TC were slightly smaller than intended (-3.9 ± 52 %; range, -100 - 81), while the FC were 20.1 ± 25.3 % bigger (p = 0.02). Feedback mechanism is less diversifying in diameter (p < 0.001). Overall, the FC correlate well with the planned treatment diameter (r = 0.79), other than the TC (r = 0.38). Six months after therapy, the fibroid volume was reduced by 45 ± 21 % (5 - 100) (p = 0.001). The symptoms decreased significantly (p = 0.001). No serious adverse events were recorded. Use of volumetric sonication leads to homogenous heating and sufficient necrosis. It is a safe and effective therapy for treating symptomatic uterine fibroids. Successful sonication of feedback cells leads to more contiguous necrosis in diameter and a less diversifying temperature. ▶ MR-guided HIFU ablation of symptomatic uterine fibroids is a valuable treatment option. ▶ By non-invasive HIFU fibroid volumes can be reduced and symptoms improved. ▶ The novel feedback-regulated treatment cells offer advantages over standard treatment cells.

2-Methoxyjuglone Induces Apoptosis in HepG2 Human Hepatocellular Carcinoma Cells and Exhibits in Vivo Antitumor Activity in a H22 Mouse Hepatocellular Carcinoma Model

In order to discover anticancer agents from natural sources, an ethanol-soluble extract of the root bark of Juglans cathayensis was investigated and showed cytotoxic effects against various human cancer cell lines. A subsequent phytochemical study on the EtOAc-soluble fraction determined 2-methoxyjuglone (1) as one of the main active constituents. Compound 1 was shown to be cytotoxic against HepG2 cells. Morphological features of apoptosis were observed in 1-treated HepG2 cells, including cell shrinkage, membrane blebbing, nuclear condensation, and apoptotic body formation. Cell cycle analysis with propidium iodide staining showed that 1 induced cell cycle arrest at the S phase in HepG2 cells. Flow cytometric analysis with annexin V and propidium iodide staining demonstrated that 1 induced HepG2 cell apoptotic events in a dose-dependent manner (0-8 μg/mL). Western blot analysis of apoptosis-related proteins revealed that 1 induces HepG2 cell apoptosis through mitochondrial cytochrome c-dependent activation of the caspase-9 and caspase-3 cascade pathway (intrinsic pathway). An in vivo experiment using tumor-bearing mice showed that treatment with 1 at 0.5 and 1.0 mg/kg per day decreased the tumor mass by 56% and 67%, respectively.

In vitro and in vivo anticancer properties of cucurbitacin isolated from Cayaponia racemosa

Context: Cucurbitacins are a group of triterpenoids that have a cucurbitane skeleton with a wide range of biological activities.Objectives: This study evaluated the anticancer properties of one cucurbitacin isolated from Cayaponia racemosa Cong. (Cucurbitaceae), 2β,3β,16α,20(R),25-pentahydroxy-22-oxocucurbita-5-en (1), with in vitro and in vivo models.Materials and methods: In vitro cytotoxic activity was determined with human leukemia (HL60) and normal blood cells (PBMC). Sarcoma 180 was used as in vivo model.Results: The cucurbitacin (1) reduced the number of viable cells; however, there was no changed in the number of non-viable cells at 5 µg/mL. Selectivity towards cancer cells was suggested by the absence of activity on normal proliferating lymphocytes at the concentrations tested (IC50 >25 µg/ml). Morphological analysis of compound 1-treated cells showed typical apoptotic features, such as intense deposition of granules in the cytoplasm (eosinophilia), DNA fragmentation and irregularities in the plasma membrane. In addition, the cells treated with compound 1 presented intense vacuolization and disruption of the plasma membrane. Acridine orange/Ethidium bromide staining confirmed these findings, revealing an increased number of apoptotic cells. In the Sarcoma 180 tumor model, compound 1 showed 52 and 62% of antitumor activity, either alone (25 mg/kg/day) or in association with the chemotherapeutic agent 5-FU (10 + 10 mg/kg/day), respectively. Moreover, either alone or associated with 5-FU, treatment with compound 1 caused an increase in spleen weight and morphological alterations related to immunostimulatory properties.Conclusion: These data indicate that these naturally occurring compounds have anticancer potential.

Stellettin A Induces Endoplasmic Reticulum Stress in Murine B16 Melanoma Cells

Isomalabaricanes are a small class of rearranged triterpenoids obtained from marine sponges. Most of these are cytotoxic to tumor cells, but the underlying mechanism is not clear. In this study, it was demonstrated that stellettin A (1), obtained from Geodia japonica, inhibited the growth of B16F10 murine melanoma cells by the induction of endoplasmic reticulum stress and accumulation of unfolded proteins. Immunoblotting analysis revealed abnormal glycosylation patterns of two melanoma marker proteins, tyrosinase and tyrosinase-related protein 1, and the retention of these proteins in the endoplasmic reticulum. Compound 1 induced the upregulation of the unfolded protein chaperone, glucose-regulated protein 78, in a dose-dependent manner. Increase of autophagosome-associated protein light chain 3 (LC3) in a membrane-bound form (LC3II) and its immunofluorescence co-localization with tyrosinase suggest the possible removal of deglycosylated and unfolded proteins by autophagy of the cells. There was no change in either the expression of the apoptosis marker protein Bcl-2 or the appearance of apoptotic nuclei in 1-treated cells. Taken together, 1 is an endoplasmic reticulum stressor that inhibits the growth of B16 melanoma cells by induction of abnormal protein glycosylation and autophagy.