Synergistic Effects of Chios Gum Mastic Extract and Low Level Laser Therapy on Osteoblast Differentiation

In the present study, we evaluated the effect of CGM on osteogenic differentiation of cultured osteoblasts, and determined whether combination treatment with LLLT had synergistic effects on osteogenic differentiation. The results indicated that CGM promoted proliferation, differentiation, and mineralization of osteoblasts at the threshold concentration of 10 µg/ml; whereas, CGM showed cytotoxic properties at concentrations above 100 µg/ml. ALP activity and mineralization were increased at concentrations above 10 µg/ml. CGM in concentrations up to 10 µg/ml also increased the expression of osteoblast-activated factors including type I collagen, BMP-2, RUNX2, and Osterix. The CGM (50 µg/ml) and LLLT (80 mW for 15 sec) combination treatment group showed the highest proliferation levels, ALP activity, and mineralization ratios. The combination treatment also increased the levels of phosphorylated forms of p38, ATF2, PKD, ERK, and JNK. In addition, the osteoblast differentiation factors including type I collagen, BMP-2, RUNX2, and Osterix protein levels were clearly increased in the combination treatment group. These results suggested that the combination treatment of CGM and LLLT has synergistic effects on the differentiation and mineralization of osteoblastic cells.

The Inhibition of Oxidative Stress by Chios Gum Mastic is Associated with Autophagy

Chios Gum Mastic (CGM) is a natural resin extracted from the leaves of Pistacia lentiscus, a plant endemic to the Greek island of Chios. It has been used by traditional healers, and it has antibacterial, antifungal properties, and therapeutic benefits for the skin. The CGM reduces the formation of dental plaque and bacterial growth in oral saliva, and recent studies have demonstrated the role of antioxidant activity of CGM. Although CGM has been widely investigated, its protective effect against oxidative-damage to keratinocytes, as well as the relationship between CGM and autophagy, has not been investigated. The aim of this study was to assess the protective effect of CGM against H2O2-induced oxidative stress and to evaluate the autophagic features induced by CGM in human keratinocytes. The pretreatment with CGM significantly reduced apoptosis in H2O2-exposed HaCaT cells. It promoted the degradation of caspase-3, caspase-8, and caspase-9; and it induced the formation of the processed PARP. The treatment with CGM caused an increase in vesicle formation compared to control group. The level of p62 was reduced and the conversion of LC3-I to LC3-II was increased in CGM treated HaCaT cells. Also, the treatment with CGM increased cleavage of ATG5-ATG12 complex. In summary, CGM helps the cells to survive under stressful conditions by preventing apoptosis and enhancing autophagy. Besides, the present investigation provides evidence to support the antioxidant potential of CGM in vitro and opens up a new horizon for future experiments.

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.

A Natural Product, Chios Gum Mastic, Induces the Death of HL-60 Cells via Apoptosis and Cell Cycle Arrest

Chios gum mastic (CGM) is produced from Pistiacia lentiscus L var chia, which grows only on Chios Island in Greece. CGM is a kind of resin extracted from the stem and leaves, has been used for many centuries in many Mediterranean countries as a dietary supplement and folk medicine for stomach and duodenal ulcers. CGM is known to induce cell cycle arrest and apoptosis in some cancer cells. This study was undertaken to investigate the alteration of the cell cycle and induction of apoptosis following CGM treatment of HL-60 cells. The viability of the HL-60 cells was assessed using the MTT assay. Hoechst staining and DNA electrophoresis were employed to detect HL-60 cells undergoing apoptosis. Western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, MMP activity and proteasome activity analyses were also employed. CGM treatment of HL-60 cells was found to result in a dose- and time-dependent decrease in cell viability and apoptotic cell death. Tested HL-60 cells showed a variety of apoptotic manifestations and induced the downregulation of G1 cell cycle-related proteins. Taken collectively, our present findings demonstrate that CGM strongly induces G1 cell cycle arrest via the modulation of cell cycle-related proteins, and also apoptosis via proteasome, mitochondrial and caspase cascades in HL-60 cells. Hence, we provide evidence that a natural product, CGM could be considered as a novel therapeutic for human leukemia.

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.

Chios Gum Mastic Induces Cell Cycle Arrest and Apoptosis in Human Osteosarcoma Cells / 대한해부학회지

Chios gum mastic (CGM) is a resin produced from the stem and leaves of Pistiacia lentiscus L var chia, a plant which grows only on Chios Island in Greece. CGM has been used for many centuries as a dietary supplement and folk medicine for stomach and duodenal ulcers in many Mediterranean countries and is also known to induce cell cycle arrest and apoptosis in some cancer cells. This study was undertaken to investigate the alteration of the cell cycle and induction of apoptosis by CGM treatment on human osteosarcoma (HOS) cells. The viability and the growth inhibition of HOS cells were assessed by the MTT assay and clonogenic assay respectively. The hoechst staining, TUNEL assay and DNA electrophoresis were conducted to observe the HOS cells undergoing apoptosis. HOS cells were treated with CGM, and Western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, mitochondrial membrane potential change and proteasome activity were conducted. CGM treatment of HOS cells was found to result in a dose- and time-dependent decrease in cell viability, a dose-dependent inhibition of cell growth, and apoptotic cell death. Tested HOS cells also showed several lines of apoptotic manifestation and G1 arrest in cell cycle progression. In summary, this study clearly demonstrated that CGM induces G1 cell cycle arrest via the modulation of cell cycle-related proteins, and apoptosis via proteasome, mitochondrial and caspase cascades in HOS cells. Therefore, our data provide the possibility that a natural product, CGM could be considered as a novel therapeutic strategy for human osteosarcoma.

Apoptotic Effect of Co-Treatment with a Natural Product, Chios Gum Mastic, and a Synthetic Chenodeoxycholic Acid Derivative, HS-1200, on Human Osteosarcoma Cells / 체질인류학회지

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 induced apoptosis in a few cancer cells in vitro. 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 co-treatment with a natural product, CGM and a CDCA derivative, HS-1200 on human osteosarcoma (HOS) cells. To investigate whether the co-treatment of CGM and HS-1200 compared with each single treatment efficiently reduced the viability of HOS cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and DNA hypoploidy, Westen blot analysis and immunofluorescent staining were performed to study the alterations of the expression level and translocation of apoptosis-related proteins in co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. In this study, HOS cells co-treated with CGM and HS-1200 showed several lines of apoptotic manifestation whereas each single treated HOS cells did not. Although the single treatment of 40 microgram/mL CGM or 25 micrometer HS-1200 for 24 h did not induce apoptosis, the cotreatment of them induced prominently apoptosis. Therefore our data provide the possibility that combination therapy of CGM and HS-1200 could be considered as a novel therapeutic strategy for human osteosarcoma.

Apoptotic Effect of Co-Treatment with a Natural Product, Chios Gum Mastic, and a Proteasome Inhibitor, Lactacystin, on Human Osteosarcoma Cells / 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 was reported that CGM induced apoptosis in a few cancer cells in vitro. Since recent studies indicated the synergistic interactions between the apoptotic stimulus and a proteasome inhibitor, the ubiquintin-proteasome pathway has become an attractive target in cancer therapy. And to date, there has been no report of the synergistic apoptotic effect between CGM and a proteasome inhibitor to become an attractive target in cancer therapy. Therefore, this study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM, and a proteasome inhibitor, lactacystin, on human osteosarcoma (HOS) cells. To investigate whether the co-treatment of CGM and lactacystin compared with each single treatment efficiently induced apoptosis on HOS cells, MTT assay, DNA electrophoresis, Hoechst staining, DNA hypoploidy assay, Westen blot analysis, immunofluorescent staining, proteasome activity and mitochondrial membrane potential (MMP) change were performed. In this study, HOS cells co-treated with CGM and lactacystin showed several lines of apoptotic manifestation such as nuclear condensation, 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, and activation of caspase-7, caspase-3, PARP and DFF45 (ICAD) whereas each single treated HOS cells hardly showed. We presented data indicating that the co-treatment of CGM and lactacystin induced potentially apoptosis whereas each single treatment did slightly. Moreover, the co-treatment of CGM and lactacystin potentiated the inhibition of proteasome activity. Therefore, our data provide the possibility that combination therapy of CGM and lactacystin could be considered as a novel therapeutic strategy for human osteosarcoma.

Chios Gum Mastic Induces Cell Cycle Arrest and Apoptosis in YD9 Human Oral Squamous Carcinoma Cells / 체질인류학회지

Chios gum mastic (CGM) is obtained from the stem and leaves of Pistacia lentiscus trees and has been extensively used for centuries in Mediterranean and Middle Eastern countries, both as a dietary supplement and herbal remedy. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying modulation of cell cycle and induction of apoptosis in YD9 human oral squamous carcinoma cell line treated with CGM. The viability of YD9 cells and human normal keratinocyes (HaCaT cells), and the growth inhibition of YD9 cells were assessed by the MTT assay and clonogenic assay respectively. The hoechst staining and DNA electrophoresis were conducted to observe the YD9 cells undergoing apoptosis. YD9 cells were treated with CGM, and Western blotting, immunocytochemistry, confocal microscopy and FACScan flow cytometry were conducted. Mitochondrial membrane potential change and proteasome activity were measured. CGM treatment on YD9 cells resulted in a does-dependent inhibition of cell growth and induced apoptotic cell death. And tested YD9 cells showed several lines of apoptotic manifestation. Flow cytometric analysis revealed that CGM resulted in G1 arrest in cell cycle progression which was associated with decrease in the protein expression of cyclin D1, cyclin D3, Cdk2 and Cdk4, and increase in the protein expression of p21(WAF1/CIP1) and p53. These results demonstrate that CGM induces G1 the cell cycle arrest via the modulation of cell cycle-related proteins, and apoptosis via mitochondria and caspase pathway in YD9 cells, suggesting that CGM can be considered as a novel therapeutic strategy for human oral squamous cell carcinoma from its strong cell cycle arrest and apoptosis-inducing activity.