Antiproliferative evaluation of tall-oil docosanol and tetracosanol over CHO-K1 and human melanoma cells

Background Polycosanols derived from plant species have traditionally been used in medicine as antiproliferative agents for treating various viruses (primarily the herpes simplex virus). However, few studies have studied their effects on hyperproliferative cell lines. In this work, the antiproliferative capacity of polycosanols from tall-oil pitch, obtained from black liquor soaps in the kraft pulping process of cellulose (specifically from Pinus radiata, Pinus taede, and Eucalyptus globulus), was evaluated on CHO-K1 and CRL-1974 human melanoma cell lines. Results The proliferative capacities and cell viabilities were measured for 72 and 140 h, respectively. Treatment with docosanol produced differential effects on the CHO-K1 and human melanoma cells and significantly affected their proliferation rates, but not their cell viabilities. Tetracosanol produced a significant negative effect on the proliferation of human melanoma cells, and this effect was less than that caused by docosanol. However, it had no effect on the proliferation of CHO-K1 cells and did not induce any significant effect on the viability of the studied cell lines. Conclusion Docosanol and tetracosanol induced antiproliferative effects on the studied cell lines and exhibited significantly greater effects on the oncogenic cell lines. Prior to this study, the capacity of these polycosanols has never been investigated. Future studies will be necessary to determine their mechanisms of action on these cell systems.

Isoliquiritigenin induced apoptosis in human melanoma A375 cells / 中国药理学通报

Aim To evaluate the mechanism of apopto-sis induced by the isoliquiritigenin in A375 human ma-lignant melanoma cells. Methods Sulforhodamine B ( SRB) method was used to determine the A375 cell viability;acridine orange/ethidium bromide ( AO/EB) and Hoechst 33258 staining were used to observe the morphological changes of apoptotic cells; flow cytome-try was used to detect A375 cell apoptotic rate;DCFH-DA was applied to determine the changes of total intra-cellular ROS in A375 cells;JC-1 method was used to measure the changes of mitochondrial membrane poten-tial;the kits methods were used to determine the con-tent of ATP, lactic acid and glucose in A375 cell which was treated with different concentrations of isoliquiritigenin. Results Isoliquiritigenin could in-hibit A375 cell proliferation in a concentration-depend-ent manner; A375 cells showed obvious apoptosis charateristics after treatment by isoliquiritigenin, and the apoptosis rate increased with increasing concentra-tion of isoliquiritigenin. The level of total intracellular ROS in A375 cells increased obviously after dealing with different concentrations of isoliquiritigenin;in ad-dition, the mitochondrial membrane potential, the lev-els of intracellular ATP,lactic acid and the level of glu-cose uptake all declined. Conclusions These find-ings demonstrate that isoliquiritigenin can induce apop-tosis of A375 cells. The mechanism may be related to elevation of ROS level and reduction of aerobic glycoly-sis level.

Purification of butin and butein from the Vernonia anthelmintica Willd and their effects on proliferation and melanogenesis of A375 human melanoma cells / 中国药学杂志

OBJECTIVE: To purify butin and butein from the Vernonia anthelmintica Willd, and preliminary study their effects on proliferation and melanogenesis of A375 human melanoma cells. METHODS: Selica gel and Sephadex LH-20 column chromatography methods were used to separate butin and butein; their effects on proliferation and melanogenesis of A375 human melanoma cells were studied by MTT method, enzyme method and NaOH method. RESULTS: Butin and butein were isolated and identified. In the concentration range of 0.50-10.00 μg · mL-1, butin and butein enhanced the proliferation of A375 human melanoma cells, and the effect of butein was stronger than butin(P < 0.05) ; in the concentration range of 0.10-1.00 μg · mL-1, both butin and butein increased the activity of tyrosinase; in the concentration range of 0.50-5.00 μg · mL-1, butein stimulated melanogenesis, but butin inhibited melanogenesis. CONCLUSION: Butin and butein may be the active components of Vernonia anthelmintica Willd; butein promotes the proliferation and stimulates the melanogenesis of A375 human melanoma cells, however, butin mildly inhibites the melanogenesis.

Apoptotic Effects of Co-Treatment with a Chios Gum Mastic and Eugenol on G361 Human Melanoma Cells

We investigated the synergistic apoptotic effects of co-treatments with Chios gum mastic (CGM) and eugenol on G361 human melanoma cells. An MTT assay was conducted to investigate whether this co-treatment efficiently reduces the viability of G361 cells compared with each single treatment. The induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining, and analyses of DNA hypoploidy. Western blot analysis and immunofluorescent staining were also performed to evaluate expression and translocation of apoptosis-related proteins following CGM and eugenol co-treatment. Proteasome activity and mitochondrial membrane potential (MMP) changes were also assayed.The results indicated that the co-treatment of CGM and eugenol induces multiple pathways and processes associated with an apoptotic response in G361 cells. These include nuclear condensation, DNA fragmentation, a reduction in MMP and proteasome activity, an increase of Bax and decrease of Bcl-2, a decreased DNA content, cytochrome c release into the cytosol, the translocation of AIF and DFF40 (CAD) into the nucleus, and the activation of caspase-9, caspase-7, caspase-3, PARP and DFF45 (ICAD). In contrast, separate treatments of 40 microg/ml CGM or 300 microM eugenol for 24 hours did not induce apoptosis. Our present data thus suggest that a combination therapy of CGM and eugenol is a potential treatment strategy for human melanoma.

ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells

Objective:To estimate electroporation (EP) influence on malignant and normal cells.Methods:Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following:250,1000,1750,2500 V/cm;50 μs by5 impulses for every case. The viability of cells after EP was estimated byMTT assay. The ultrastructural analysis was observed by transmission electron microscope (ZeissEM900). Results:In the current study we observed the intracellular effect followingEP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated byEP. Conversely, we showed thatEP in some conditions can stimulate cells to proliferation. Some changes induced byEP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters ofEP (250 and1000 V/cm). After applying higher electric field intensities (2500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications afterEP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters ofEP.Conclusions:We can claim thatEP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude thatEP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.

ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells

<p><b>OBJECTIVE</b>To estimate electroporation (EP) influence on malignant and normal cells.</p><p><b>METHODS</b>Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following: 250, 1 000, 1 750, 2 500 V/cm; 50 µs by 5 impulses for every case. The viability of cells after EP was estimated by MTT assay. The ultrastructural analysis was observed by transmission electron microscope (Zeiss EM 900).</p><p><b>RESULTS</b>In the current study we observed the intracellular effect following EP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP. Conversely, we showed that EP in some conditions can stimulate cells to proliferation. Some changes induced by EP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters of EP (250 and 1 000 V/cm). After applying higher electric field intensities (2 500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications after EP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP.</p><p><b>CONCLUSIONS</b>We can claim that EP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.</p>

Mechanism Underlying NaF-induced Apoptosis and Cell Cycle Arrest on G361 Human Melanoma Cell Line / 체질인류학회지

Fluoride is widely used in dentistry to prevent dental caries, even though the safety of fluoride is a controversial issue. There are no known adverse effects of long-term fluoride ingestion for caries prevention, but an overdose can cause serious acute toxicity. Nevertheless it is accepted that fluoride is an important material for oral health. This study was undertaken to investigate the modulation of cell cycle-related proteins and apoptosis induction underlying mechanism by NaF treatment on G361 human melanoma cell line. The viability of G361 cells and the growth inhibition of G361 cells were assessed by MTT assay and clonogenic assay respectively. Hoechst staining, DNA electrophoresis and TUNEL staining were conducted to observe G361 cells undergoing apoptosis. G361 cells were treated with NaF, and Western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, MMP activity and proteasome activity were performed. NaF treatment in G361 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. And tested G361 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, a significant shift of Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-7, caspase-6, caspase-3, PARP, Lamin A/C and DFF45 (ICAD). Furthermore, NaF resulted in down-regulation of the G1 cell cycle-related proteins, and up-regulation of p53. Taken collectively, our present findings demonstrate that NaF strongly inhibits cell proliferation by modulating the expression of the G1 cell cycle-related proteins and induces apoptosis via proteasome, mitochondria and caspase cascades in G361 cells.

Apoptotic Effect of Co-treatment with Chios Gum Mastic and HS-1200 on G361 Human Melanoma Cell Line / 대한해부학회지

Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. Recently it reported that CGM induce apoptosis in a few cancer cells in vitro. Bile acids and their synthetic derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. This study was undertaken to investigate the synergistic apoptotic effect of cotreatment with a natural product, CGM and a CDCA derivative, HS-1200 on G361 human melanoma cells. To investigate whether the co-treatment of CGM and HS-1200 compared with each single treatment efficiently reduced the viability of G361 cells, MTT assay was conducted. To investigate augmentation of apoptosis in G631 cells co-treated with CGM and HS-1200, DNA electrophoresis, Hoechst staining, proteasome activity assay, flow cytometry, Westen blot analyses, immunofluorescent staining and confocal microscopy were performed. In this study, G361 cells co-treated with CGM and HS-1200 showed several lines of apoptotic manifestation such as nuclear condensations, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, activation of caspase-9, caspase-3, PARP and DFF45 (ICAD), and up-regulation of Bax whereas each single treated G361 cells did not. Although the single treatment of 40 micro/mL CGM or 25 micro HS-1200 for 24 hrs did not induce apoptosis, the co-treatment of them induced prominently apoptosis. Therefore, combination therapy of CGM and HS-1200 could be considered, in the future, as an alternative therapeutic strategy for human melanoma.

Induction of Apoptosis by Genistein in Human Melanoma Cells / 대한해부학회지

Genistein is a naturally occurring isoflavone that has been identified predominantly in soybean. It has been found that genistein can inhibit the growth of various cancer cell lines. Melanoma continues to increase in incidence in many parts of the world and remains among the top six cancers as a cause of death and morbidity. Understanding and overcoming resistance mechanism(s) of melanoma to apoptosis would therefore facilitate identification of new therapeutic targets and development of new treatments. This study was undertaken to investigate whether genistein induced apoptosis on human melanoma cells (G361). Genistein had a significant dose- and time-dependent inhibitory effect on the viability of G361 cells. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by hoechst staining, and DNA electrophoresis. p53 levels were not altered by genistein treatment. Genistein treatment induced caspase-3 cleavage and activation. Poly (ADP-ribose)-polymerase (PARP) and DNA fragmentation factor 45 (DFF45), which are caspase-3 substrates, were cleaved during genistein-induced apoptosis. It was found that the caspase-6 substrate lamin A was cleaved, whose cleavage has been reported to be necessary for complete condensation of DNA during apoptosis. The expression level and phosphorylation of focal adhesion kinase (FAK) were reduced by genistein treatment. These results suggest that genistein may constitute a potential antitumor compound against melanoma occurring at oral mucosa and skin.

Synthetic Chenodeoxycholic Acid Derivative HS-1200-induced Apoptosis of Human Melanoma Cells / 체질인류학회지

Bile acids and their synthetic derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. It wasn't discovered those materials have apoptosis-inducing effects on G361 human melanoma cells. The present study was done to examine the synthetic bile acid derivatives, HS-1199 and HS-1200, induced apoptosis on G361 cells and such these apoptosis events. The viability of G361 cells was assessed by the MTT assay. Induction of apoptosis was confirmed by DNA electrophoresis and Hoechst staining. Westen blot analysis and immunofluorescent staining were performed to study the alterations in expression level and translocation of apoptosis-related proteins. Proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. Tested G361 cells showed several lines of apoptotic manifestation such as activation of caspase-3, DFF and PARP, DNA degradation (HS-1200 only), nuclear condensation, inhibition of proteasome activity, reduction of mitochondrial membrane potential, and the release of cytochrome c and AIF to cytosol. Between two synthetic derivatives, HS-1200 showed stronger apoptosis-inducing effect than HS-1199 did. Taken collectively, we here demonstrated for the first time that synthetic CDCA dedrivatives induce apoptosis of human melanoma cells through the proteasome, mitochondria and caspase pathway. Therefore our data provide the possibility that HS-1200 could be considered as a novel therapeutic strategy for human melanoma cells from its powerful apoptosis-inducing activity.

Caspases-dependent Apoptosis in Human Melanoma Cell by Eugenol / 대한해부학회지

Malignant melanoma is a highly metastatic tumor, resistant to chemotherapy and radiotherapy. Recent studies have suggested that many therapeutic agents used against cancer mediate their effects by induction of apoptosis of the cancer cells. Eugenol enhances the generation of tissue-damaging free radicals and inflammation or allergic reactions. In particular, it is more cytotoxic against cancer cells compared with normal fibroblasts. This study was performed to investigate whether the cytotoxic effect of eugenol is associated with the induction of apoptosis and involves activation of caspase in the human melanoma G361 cells. Eugenol-induced apoptosis was confirmed by MTT assay, Hemacolor stain, Hoechst stain, DNA electrophoresis, and Western blot analysis. Eugenol had a significant dose- and time-dependent inhibitory effect on the viability of G361 cells. Eugenol treatment induced caspase-3 and -6 cleavage, and activation. The caspase-3 substrates PARP and DFF45 are cleaved during eugenol-induced apoptosis. It was found that the casapase-6 substrate lamin A was cleaved, whose cleavage has been reported to be necessary for complete condesation of DNA during apoptosis. These results suggest that eugenol may constitute a potential antitumor compound against melanoma occurring in the skin and oral mucosa.

IL-1beta Acts in Synergy with Endogenous IL-1beta in A375-S2 Human Melanoma Cell Apoptosis Through Mitochondrial Pathway

Interleukin-1beta (IL-1beta) is a pivotal proinflammatory cytokine. To investigate the mechanism of IL-1beta-induced cell death in human malignant melanoma A375-S2 cells, MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, radioimmunoassay and Western blot analysis were carried out. IL-1beta did not only induce nuclear condensation and DNA fragmentation, but also increased degradation of two substrates of caspase-3, poly ADP-ribose polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Simultaneously, release of precursor of IL-1beta (pro-IL-1beta) and endogenous IL-1beta production were involved in the apoptotic process. IL-1beta enhanced the ratio of Bax/Bcl-2 and Bax/Bcl-xL expression and up-regulated apoptosis inducing factor (AIF) expression, which required the activation of downstream caspases. These results suggest that IL-1beta induces endogenous IL-1beta production, enhances cleavage of caspase downstream substrates and promotes mitochondria mediated apoptosis in A375-S2 cells.