Acacetin Inhibits Cell Proliferation and Induces Apoptosis in Human Hepatocellular Carcinoma Cell Lines

Human hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of death across the world. Recent evidence suggests that STAT3 regulates proliferative, survival, metastasis, and angiogenesis genes in HCC. Novel agents that suppress STAT3 activation can be used to prevent or treat HCC. We used a functional proteomics tumor pathway technology platform and multiple HCC cell lines to investigate the effects of acacetin (ACN) on STAT3 activation, protein kinases, phosphatases, products of STAT3-regulated genes, and apoptosis. ACN was found to inhibit STAT3 activation in a dose- and time-dependent manner in HCC cells. Upstream kinases c-Src, Janus-activated kinase 1, and Janus-activated kinase 2 were also inhibited. The ACN inhibition of STAT3 was abolished by vanadate treatment, suggesting the involvement of tyrosine phosphatase activity. ACN was found to suppress the protein expression of genes involved in proliferation, survival, and angiogenesis via STAT3 inhibition. ACN appears to be a novel STAT3 inhibitor and may be a promising therapeutic compound for application in the treatment of HCC and other cancers.     

UVA and UVB Radiation Differentially Regulate Vascular Endothelial Growth Factor Expression in Keratinocyte-derived Cell Lines and in Human Keratinocytes

Vascular endothelial growth factor (VEGF) is a central regulator of neoangiogenesis in inflammatory and neoplastic conditions. Ultraviolet irradiation is one of the mainstays of dermatological therapy for various inflammatory skin diseases. In the present study we have compared the effects of UV irradiation on the production of VEGF by keratinocytes (KC) and by the KC-derived cell lines A431 and HaCaT. Irradiation of A431 and HaCaT cells with both UVA (10 J/cm2 and 20 J/cm2) and UVB (8 mJ/cm2 and 16 mJ/cm2) led to strong upregulation of VEGF mRNA and protein. Induction of VEGF by UVA and UVB in these cells was mediated by different pathways, i.e. the generation of free radicals and the secretion of (a) soluble factor(s), respectively. Unlike KC-derived cell lines, no increase in VEGF production was observed in KC in primary culture after irradiation with the same UV doses. Increasing the irradiation dose in these cells of UVA to 40 J/cm2 led to a marked decrease in soluble VEGF, whereas doses as high as 32 mJ/cm2 UVB only minimally affected VEGF levels. Reduction of VEGF production by KC might contribute to the effect of UVA irradiation in inflammatory skin diseases. The differential response of primary KC and autonomously growing KC-derived cell lines to the induction of VEGF by UV light could favor neoangiogenesis in the vicinity of epidermal tumor cells in vivo, thereby endowing them with a growth advantage over normal cells.     

Naringenin Induces ROS-Mediated ER Stress,Autophagy, and Apoptosis in Human Osteosarcoma Cell Lines

Osteosarcoma, a primary bone tumor, responds poorly to chemotherapy and radiation therapy in children and young adults; hence, as the basis for an alternative treatment, this study investigated the cytotoxic and antiproliferative effects of naringenin on osteosarcoma cell lines, HOS and U2OS, by using cell counting kit-8 and colony formation assays. DNA fragmentation and the increase in the G2/M phase in HOS and U2OS cells upon treatment with various naringenin concentrations were determined by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and Annexin V/propidium iodide double staining, respectively. Flow cytometry was performed, and 2′,7′-dichlorodihydrofluorescein diacetate, JC-1, and Fluo-4 AM ester probes were examined for reactive oxygen species (ROS) generation, mitochondrial membrane potential, and intracellular calcium levels, respectively. Caspase activation, cell cycle, cytosolic and mitochondrial, and autophagy-related proteins were determined using western blotting. The results indicated that naringenin significantly inhibited viability and proliferation of osteosarcoma cells in a dose-dependent manner. In addition, naringenin induced cell cycle arrest in osteosarcoma cells by inhibiting cyclin B1 and cyclin-dependent kinase 1 expression and upregulating p21 expression. Furthermore, naringenin significantly inhibited the growth of osteosarcoma cells by increasing the intracellular ROS level. Naringenin induced endoplasmic reticulum (ER) stress-mediated apoptosis through the upregulation of ER stress markers, GRP78 and GRP94. Naringenin caused acidic vesicular organelle formation and increased autophagolysosomes, microtubule-associated protein-light chain 3-II protein levels, and autophagy. The findings suggest that the induction of cell apoptosis, cell cycle arrest, and autophagy by naringenin through mitochondrial dysfunction, ROS production, and ER stress signaling pathways contribute to the antiproliferative effect of naringenin on osteosarcoma cells.     

温度及药物诱导肿瘤细胞凋亡的实验观察

将微量热法和透射电镜技术相结合,研究了温度对人卵巢癌细胞代谢的影响,不同温度下癌细胞超微结构的变化和加抗肿瘤药物(嘧福绿)的协同作用.从宏观到微观,深入探讨了热疗与化疗协同作用对肿瘤细胞杀伤作用的机理.实验结果表明,在高温下细胞代谢降低,肿瘤细胞的形态发生一系列恶性变化,且高热可能诱导肿瘤细胞的凋亡;热疗加化疗的协同作用则能加速肿瘤细胞的凋亡,使细胞的能量代谢更低,随着作用时间的延长,最终导致细胞死亡.     

Apoptosis in leukocytes induced by UVA in the presence of 8-methoxypsoralen,chlorpromazine or 4,6,4'-trimethylangelicin

Although 8-methoxypsoralen (8-MOP) has been successfully used in extracorporeal photochemotherapy (ECP) of several T cell-mediated diseases, the exact mechanism of the drug therapeutic action has not been established. We have studied in vitro apoptotic activity of 8-MOP, and for comparison of 4,6,4'-trimethylangelicin (TMA) and chlorpromazine (CPZ) as alternative photosensitizers for potential use in photopheresis. However, while 8-MOP and CPZ are known for their immune suppressive activity, TMA does not exhibit such an activity in an animal model for ECP. Apoptosis and necrosis were measured in both Jurkat cells and primary rat leukocytes under conditions comparable to those used in the animal model to suppress contact hypersensitivity (CHS). Cells were irradiated with UVA (200 kJ/m(2)) after treatment with 8-MOP, CPZ or TMA (300 ng/ml). Flow cytometric analysis (annexin-V-FLUOS/propidium iodide) and fluorescence microscopy examinations, using acridine orange/propidium iodide, indicated that the number of cells undergoing apoptosis or necrosis increased significantly after 24 h following treatment. Similar results were observed irrespective of the cell type and photosensitizer used. The results of the present study, combined with previous observations with the animal model for ECP, suggest that apoptosis is not likely to be a critical step in the cascade of events leading to immunosuppression.     

Betulinic Acid Modulates the Expression of HSPA and Activates Apoptosis in Two Cell Lines of Human Colorectal Cancer

Betulinic acid (BA) is a pentacyclic triterpene usually isolated from botanical sources. Numerous studies have reported the inhibitory effect of BA against human colorectal cancer cells (CRC). However, its effect on the expression of the molecular chaperone HSPA is unclear. The aim of this research is to investigate the anti-cancer activities of BA purified from Piper retrofractum and study its effect on the expression of HSPA in colorectal cancer HCT116 and SW480 cells. The viability of both cancer cells was reduced after they were treated with an increasing dosage of BA. Flow cytometry assay revealed that levels of cell apoptosis significantly increased after incubation with BA in both cancer cells. Pro-apoptotic markers including Bax, cleaved-caspase-3 and cleaved-caspase-9 were increased while anti-apoptotic marker Bcl-2 was decreased after BA treatment. Western blot also showed that the expression of HSPA fluctuated upon BA treatment, whereby HSPA was increased at lower BA concentrations while at higher BA concentrations HSPA expression was decreased. Preliminary molecular docking assay showed that BA can bind to the nucleotide binding domain of the HSP70 at its ADP-bound state of the HSP70. Although further research is needed to comprehend the BA-HSPA interaction, our findings indicate that BA can be considered as potential candidate for the development of new treatment for colorectal cancer.     

Synergetic Effect of Tumor Treating Fields and Zinc Oxide Nanoparticles on Cell Apoptosis and Genotoxicity of Three Different Human Cancer Cell Lines

Cancer remains a leading cause of death worldwide, despite extraordinary progress. So, new cancer treatment modalities are needed. Tumor-treating fields (TTFs) use low-intensity, intermediate-frequency alternating electric fields with reported cancer anti-mitotic properties. Moreover, nanomedicine is a promising therapy option for cancer. Numerous cancer types have been treated with nanoparticles, but zinc oxide nanoparticles (ZnO NPs) exhibit biocompatibility. Here, we investigate the activity of TTFs, a sub-lethal dose of ZnO NPs, and their combination on hepatocellular carcinoma (HepG2), the colorectal cancer cell line (HT-29), and breast cancer cell lines (MCF-7). The lethal effect of different ZnO NPs concentrations was assessed by sulforhodamine B sodium salt assay (SRB). The cell death percent was determined by flow cytometer, the genotoxicity was evaluated by comet assay, and the total antioxidant capacity was chemically measured. Our results show that TTFs alone cause cell death of 14, 8, and 17% of HepG2, HT-29, and MCF-7, respectively; 10 µg/mL ZnO NPs was the sub-lethal dose according to SRB results. The combination between TTFs and sub-lethal ZnO NPs increased the cell death to 29, 20, and 33% for HepG2, HT-29, and MCF-7, respectively, without reactive oxygen species increase. Increasing NPs potency using TTFs can be a novel technique in many biomedical applications.     

The Induction and Analysis of Micronuclei and Cell Killing by Ultraviolet-B Radiation in Human Peripheral Blood Lymphocytes

The DNA-damaging potential of ultraviolet-B (UVB) radiation was investigated by analyzing the frequency and origin of micronuclei (MN) in cytokinesis-blocked, binucleated (BN) peripheral blood lymphocytes (PBL) and cloning efficiencies (CE) of PBL after exposure to different fluences of UVB. In total, PBL obtained from five normal donors were investigated. The PBL from all donors showed a dose-related, linear-quadratic increase in the frequency of MN per 1000 BN cells and in the frequency of micronucleated BN cells. In two experiments the origin of UVB-induced MN was studied by analyzing MN for the presence or absence of centromeres by applying the MN assay in combination with a centromeric probe and fluorescence in situ hybridization. This revealed, for the first time, that UVB-induced MN were centromere negative, indicating that UVB acted exclusively as a clastogenic agent in the tested dose range. The PBL from all donors showed a clear dose-dependent decrease in CE, after UVB exposure. The UVB-exposed PBL from all donors showed an inverse relationship between the induction of MN and the decrease in CE, but regression analysis revealed no correlation between the induction of MN and the decrease in cell survival. It is concluded that UVB has a clastogenic and cytotoxic effect on PBL.     

Application of Oil-in-Water Nanoemulsion Carrying Size-Defined Gold Nanoparticles Synthesized by Non-thermal Plasma for the Human Breast Cancer Cell Lines Migration and Apoptosis

Plasma Chemistry and Plasma Processing - In this work, the gold nanoparticles (AuNPs) were synthesized using pulse-modulated radio-frequency atmospheric pressure glow discharge (pm-rf-APGD). By...     

Coexpression of Vimentin and Cytokeratins in Human Melanoma Cell Lines

Moscow University Chemistry Bulletin - Abstract—Molecular heterogeneity in seven human metastatic melanoma cells lines is revealed using a double immunofluorescent assay by flow cytometry. It...     

Induction of Cell Killing, Mutation and umu Gene Expression by 6-Mercaptopurine or 2-Thiouracil with UVA Irradiation

Abstract— When Escherichia coli cells were irradiated by UVA in the presence of 6-mercaptopurine (6-MP) or 2-thiouracil (S²Ura), two kinds of repair-deficient strains of recA ⁻ and uvrA ⁻ were killed more efficiently than the parental wild-type strain having normal repair capacities. In addition, these agents with UVA exposure greatly induced the incidence of mutations in the uvrA ⁻ strain as compared with the wild-type strain but not the uvrA ⁻ strain. Furthermore, the induction of expression of umuDC genes was investigated in two Salmonella typhimurium strains, TA1S35 and TA1538, carrying a pSK1002 plasmid. In these systems, it is easy to measure β-galactosidase activities for the induced activities of SOS responses. These agents with UVA exposure also induced expression of the umuDC genes. These results suggest that 6-MP and S²Ura with UVA induce DNA damage which is repairable by the excision repair mechanism.     

DNA Photodamage, Repair, Gene Induction and Genotoxicity Following Exposures to 254 nm UV and 8-Methoxypsoralen Plus UVA in a Eukaryotic Cell System

The induction and repair of different types of photodamage and photogenotoxicity in eukaryotic cells have been the subject of many studies. Little is known about possible links between these phenomena and the induction of DNA damage-inducible genes. We explored this relationship using the yeast Saccharomyces cerevisiae, a pertinent eukaryotic model. Previous results showed that the photogenotoxic potential of 8-methoxypsoralen (8-MOP) plus UVA is higher than that of UV (254 nm). Moreover, the induction of the ribonucleotide reductase gene RNR2 by UV and 8-MOP plus UVA in an RNR2-LACZ fusion strain and the formation of DNA double-strand breaks (dsb) as repair intermediates after such treatments suggest that the latter process could involve a signal for gene induction. To further substantiate this, we measured the induction of the DNA repair gene RAD51 in RAD51-LACZ fusion strains using the dsb repair and recombination deficient mutant rad52 and the corresponding wild type, and we determined the formation of dsb by pulsed-field gel electrophoresis. After treatments, the resealing of dsb formed as repair intermediates was impaired in the rad52 mutant. At equal doses, i.e. the same number of lesions, the induction of the RAD51 gene by UV or 8-MOP plus UVA was significantly reduced in the rad52 mutant as compared with the wild type. The same was true when equitoxic doses were used. Thus, the RAD52 repair pathway appears to play an important role not only in dsb repair but also in gene induction. Furthermore, the signaling pathways initiated by DNA damage and its processing are somewhat linked to the photogenotoxic response.     

UVA II Exposure of Human Skin Results in Decreased Immunization Capacity, Increased Induction of Tolerance and a Unique Pattern of Epidermal Antigen-Presenting Cell Alteration

Abstract— The risks incurred from increased exposure to UVA II (320-340 nm) (i.e. during sunscreen use and extended outdoor exposure, tanning parlors) are not well understood. Therefore, we explored the effects of UVA II on skin immune responses in humans. After a single local exposure (4 minimum erythemal dose [MED]) using a xenon arc lamp filtered with a narrow bandpass filter (335 ± 5 nm full width at half maximum), individuals were contact-sensitized with dinitrochlorobenzene (DNCB) through a UVA II exposure site or through normal skin. UVA II induced a marked decrease in the magnitude of skin immune responses (P < 0.0001). The UVA II group had only 29% successful sensitizations, as compared to 83% in the control group. The percentage of individuals who remained tolerant to DNCB after two sensitizations was 23.6% for the UVA II-exposed group, as compared to 3.8% in the controls (P= 0.006). UVA II also uniquely altered the type of antigen-presenting cells present in the epidermis. Human leukocyte antigen (HLA)-DR+ cells in control epidermal cell suspensions (C-EC) comprised a single, homogeneous population of Langerhans cells (LC) with the phenotype: CD1a^hi DR^mid CD11b^? CD36^? (1.5 ± 0.3% of EC). UVA II irradiation reduced the number of such LC to 0.6 ± 0.2% of EC. Although cells expressing the macrophage phenotype: CD1a DR^hi CD11b+ CD36+ were increased in UVA II skin, relative to C-EC, these comprised only 10.1 ± 6.1% of the DR+ cells, which is less than that after UVB exposure. Also distinct from UVB, a third population was found in UVA II-EC, which exhibited a novel phenotype: CD1a+ DR+ CD36+ CDllb+; these comprised 11.1 ± 6.9% of the DR+ UVA II-EC. In conclusion, despite the above differences in infiltrating DR cells, both UVB and UVA II reduce the skin's ability to support contact sensitization, induce active suppression (tolerance) and induce a reduction in LC.     

Production of Terretonin N and Butyrolactone I by Thermophilic Aspergillus terreus TM8 Promoted Apoptosis and Cell Death in Human Prostate and Ovarian Cancer Cells

The anticancer activity of terretonin N (1) and butyrolactone I (2), obtained from the thermophilic fungus Aspergillus terreus TM8, was intensively studied against prostate adenocarcinoma (PC-3) and ovary adenocarcinoma (SKOV3) human cell lines. According to this study, both compounds showed potent cytotoxicity towards ovarian adenocarcinoma cells (SKOV3) with IC₅₀ 1.2 and 0.6 μg/mL, respectively. With respect to metastatic prostate cells (PC-3), the two compounds 1 and 2 showed a significantly promising cytotoxicity effect with IC₅₀ of 7.4 and 4.5 μg/mL, respectively. The tested fungal metabolites showed higher rates of early and late apoptosis with little or no necrotic apoptotic pathway in all treated prostate adenocarcinoma (PC-3) and ovary adenocarcinoma (SKOV3) human cell lines, respectively. The results reported in this study confirmed the promising biological properties of terretonin N (1) and butyrolactone I (2) as anticancer agents via the induction of cellular apoptosis. However, further studies are needed to elucidate the molecular mechanism by which cellular apoptosis is induced in cancer cells.     

Cytotoxic and Photocytotoxic Effects of Cercosporin on Human Tumor Cell Lines

Cercosporin is a naturally occurring perylenequinone. Although other perylenequinones have been extensively studied as photosensitizers in photodynamic therapy of cancer (PDT), cercosporin has been studied in this light only within the remits of phytopathology. Herein, we investigated the photocytotoxicity of cercosporin against two glioblastoma multiforme (T98G and U87) and one breast adenocarcinoma (MCF7) human cell lines. Cercosporin was found to be a potent singlet oxygen producer upon 532 nm excitation, while its cell loading was similar for MCF7 and U87, but approximately threefold higher for T98G cells. The subcellular localization of cercosporin was in all cases in both mitochondria and the endoplasmic reticulum. Light irradiation of cercosporin‐incubated cells around 450 nm showed that T98G cells were more susceptible to cercosporin PDT, mainly due to their higher cercosporin uptake. Metabolic studies before and 1 h following cercosporin PDT showed that cercosporin PDT instigated a bioenergetic collapse in both the respiratory and glycolytic activities of all cell lines. In the dark, cercosporin exhibited a synergistic cytotoxicity with copper only in the most respiratory cell lines (MCF7 and T98G). Cercosporin is a potent photosensitizer, but with a short activation wavelength, mostly suitable for superficial PDT treatments, especially when it is necessary to avoid perforations.     

Induction of Apoptosis by Ribosome Inactivating Proteins

Apoptotic cell death is a fundamental process in the development and physiological homeostasis of multicellular organisms. It is associated with control of cell numbers in tissues and organs during development, with cell turnover, and with response to infection. Molecules that trigger this process in continuously proliferating cancer cells can be used as chemotherapeutic agents. Ribosome inactivating proteins (RIPs) that inhibit translation in a cell by depurinating (N-glycosidase activity) the 28S rRNA are known to serve as apoptosis inducers. However, the role of depurination activity of the RIPs in apoptosis induction is still controversial. Presently, there are three different hypotheses which propose that depurination is: (1) essential, (2) essential but not the sole factor, or (3) not essential for apoptosis induction. This article reviews various experimental outcomes on the importance of N-glycosidase activity of RIPs in the induction of apoptosis.     

Yamogenin-Induced Cell Cycle Arrest,Oxidative Stress,and Apoptosis in Human Ovarian Cancer Cell Line

Steroidal saponins are a group of compounds with complex structures and biological activities. They have anti-inflammatory, antimicrobial, fungicidal, and antitumor properties. Yamogenin is one of the spirostane saponins and occurs in Trigonella foenum-graecum, Asparagus officinalis, and Dioscorea collettii. It is a stereoisomer of diosgenin—a well-known compound whose activity and mechanisms of action in cancer cells are determined. However, the antitumor effect of yamogenin is still little known, and the mechanism of action has not been determined. In this study, we evaluated the effect of yamogenin on human ovarian cancer SKOV-3 cells in vitro by determining the cellular factors that trigger cell death. The viability of the cells was assessed with a Real-Time xCELLigence system and the cell cycle arrest with flow cytometry. The activity of initiator and executioner caspases (-8, -9, and -3/7) was estimated with luminometry and flow cytometry, respectively. The mitochondrial membrane depolarization, the level of oxidative stress, and DNA damage in the yamogenin-treated cells were also evaluated by flow cytometry. Genes expression analysis at the mRNA level was conducted with Real-Time PCR. Bid activation and chromatin condensation were estimated with fluorescent microscopy. The obtained results indicate that yamogenin has cytotoxic activity in SKOV-3 cells with an IC₅₀ value of 23.90 ± 1.48 µg/mL and strongly inhibits the cell cycle in the sub-G1 phase. The compound also triggers cell death with a significant decrease in mitochondrial membrane potential, an increase in the level of oxidative stress (over two times higher in comparison to the control), and activation of caspase-8, -9, -3/7, as well as Bid. The results of genes expression indicate that the Tumor Necrosis Factor (TNF) Receptor Superfamily Members (TNF, TNFRSF10, TNFRSF10B, TNFRSF1B, and TNFRSF25), Fas Associated via Death Domain (FADD), and Death Effector Domain Containing 2 (DEDD2) were significantly upregulated and their relative expression was at least two times higher than in the control. Our work shows that yamogenin induces apoptosis in ovarian cancer cells, and both the extrinsic and mitochondrial—intrinsic pathways are involved in this process.