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

Jae-Hyoun Park(박재현),Gyoo-Cheon Kim(김규천),Hyun-Ho Kwak(곽현호),In-Ryoung Kim(김인령),Seung-Eun Lee(이승은),Jin Chung(정진),Hae-Ryoun Park(박혜련),Sang-Hun Shin(신상훈),Soo-Hyun Choi(조수현),Chul-Hoon Kim(김철훈),Chang-Ok Nam( 대한체질인류학회 2008 대한체질인류학회지 Vol.21 No.1

식물인 Pistiacia lentiscus L. var. Chia.는 그리이스 키오스 섬의 남부지방에서만 서식하며, Chios gum mastic (CGM)으로 알려져 있는 수지를 만들어 낸다. Pistacia lentiscus 나무의 줄기와 잎에서 추출한 천연물질인 CGM은 과거 수세기 동안 지중해와 중동 지역 국가들에서 음식 첨가물과 치료약으로 광범히 하게 사용되어 왔었다. 본 연구는 사람구강편평상피암종세포(YD9 cells)에서 CGM의 세포독성과 성장억제 효과, 그리고 세포주기의 변형과 세포자멸사(apoptosis)에 대한 분자생물학적 기전을 알기 위해서 수행하였다. YD9 세포와 사람정상각화세포(HaCaT)의 생존률 측정은 MTT법을 시행하였고, YD9 세포의 성장억제를 확인하기 위해서는 clonogenic assay를 사용하였다. 세포자멸사가 유도되는 YD9 세포를 관찰하기 위해서 hoechst 염색법과 DNA 전기영동법을 사용하였다. 그리고 YD9 세포에 CGM을 적용한 후, Western blot 분석, 세포면역화학염색, 공점레이저주사현미경 검경, FACScan flow cytometry, 사립체막 전위변화, proteasome 활성도 측정 등을 시행하였다. CGM으로 처리된 YD9 세포는 용량 의존적인 세포 성장억제와 세포자멸사에 의한 세포죽음을 보였고, caspase-9, caspase-3, PARP 그리고 DFF45 (ICAD)의 파괴와 분절의 생성, DNA의 조각남, 핵 응축, 사립체막전위의 감소, Bax와 Bcl-2의 분율의 변화, cytochrome c의 사립체에서의 세포질로의 유리, AIF와 DFF40 (CAD)의 핵으로의 이동과 같은 세포자멸사 증거를 보였다. Flow cytometry 분석에서는 cyclin D1, cyclin D3, Cdk2 그리고 Cdk4의 발현의 감소와 p21<SUP>WAF1/CIP1</SUP>와 p53의 발현 증가와 관계있는 것으로 보여지는 G1 세포주기 정지를 보였다. 이러한 결과는 CGM이 세포주기 관련 단백질들의 변형을 유도한 G1 세포주기정지와 사립체와 caspase 경로를 통한 세포자멸사를 유도함을 명확하게 증명하고 있다. 이와 같은 강력한 세포주기 정지와 세포자멸사 유도능은 CGM이 사람구강편평세포암종의 새로운 치료전략으로서의 가능성을 높여 준다고 생각한다. 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<SUP>WAF1/CIP1</SUP> 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.

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

Ji-Hak Min(민지학),Min-Jeong Kim(김민정),In-Ryoung Kim(김인령),Seung-Eun Lee(이승은),Hyun-Ho Kwak(곽현호),Gyoo-Cheon Kim(김규천),Hae-Ryoun Park(박혜련),Sang-Hun Shin(신상훈),Chul-Hoon Kim(김철훈),Na-Young Jeong(정나영),Hongsuk Suh 대한체질인류학회 2008 대한체질인류학회지 Vol.21 No.2

Chios gum mastic(CGM)은 그리이스 지역에서만 서식하는 Pistacia lenticulus 나무의 줄기와 잎에서 추출한 수지상의 천연 추출물이다. 합성 chenodeoxycholic acid(CDCA) 유E씨가 여러 가지 암세포에 유도한 세포자멸사 연구들이 보고되어져 왔다. 본 연구는 천연물질인 CGM과 항성 CDCA 유도체인 HS-1200의 병용처리가 사람골육종세포에 효과적인 상승 세포자멸사 효과가 있는지를 알기 위해서 수행되었다. CDM과 HS-1200의 병용처리가 단독처리에 비해서 효과적인 세포생존율 감소가 있는지 확인하기 위해서 MIT법을 시행하였고, 세포자멸사의 유도와 증가를 확인하기 위해서 Hoechst 염색법과 DNA 전기영동법을 사용하였다. 병용처리 때, 세포자멸사에 관계하는 단백질의 발현 변화와 세포내에서의 이동을 밝혀내기 위해서 Western bot 분석과 면역형광염색법을 수행하였다. 더 나아가서 proteasome 활성도와 사립체막 전위 변화를 측정하였다. 병용처리 된 사람골육종세포는 단독처리 된 사람골육종세포에서 거의 관찰할 수 없었던 많은 핵 응축, DNA 조각남, 사립체막 전위와 proteasome 활성도의 감소, DNA 양의 감소, cytochrome c의 세포질로의 유리, AIF와 DFF40(CAD)의 핵으로의 이동, caspase-7, caspase-3 그리고 PARP의 활성화와 같은 세포자멸사 증거를 보였다. 24시간 동안 40㎍/mL CGM과 25μM HS-1200을 각기 단독처리 한 결과에서는 세포자멸사를 유도 못했으나, 병용처리한 결과에는 아주 탁월한 세포자멸사의 유도를 보였다. 이러한 병용처리 결과는 사람골육종의 새로운 치료적 전략으로 응용될 수 있다고 생각한다. 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 HDS cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and DNA hypoploidy, Westen blot analysis and immuno-fluorescent 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 ㎍/㎖, CGM or 25 μM HS-1200 for 24 h did not induce apoptosis, the co-treatment 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.

Mechanism underlying Chios gum mastic-induced cell cycle arrest and apoptosis of the G361 human melanoma cell line

김인령,강해미,유수빈,박봉수 대한구강해부학회 2017 대한구강해부학회지 Vol.38 No.1

Chios gum mastic (CGM) is a resin extracted from the stem and leaves of Pistacia lentiscus L. var. chia. It has been used as a traditional medicine in many Mediterranean countries. Recently, numerous researches have demonstrated that CGM induces cell cycle arrest and apoptosis in many cancer cells. In the present study, an alteration of the cell cycle and induction of apoptosis by CGM treatment on malignant melanoma was investigated. CGM treatment showed the inhibition of cell viability in a dose- and time-dependent manner on the G631 melanoma cell line. Apoptotic hallmarks, such as nuclear condensation and DNA fragmentation, were also identified in CGM-treated cells. Several lines of apoptotic manifestation were demonstrated. The proapoptotic factor Bax was increased in a time-dependent manner, leading to MMP loss, proteasome activity reduction, AIF translocation, and cytochrome c release. Activation of caspases, such as caspase-9, -7, and -3, led to the cleavage of PARP and DFF45 (ICAD). DFF40 (CAD) was translocated into the nucleus from the cytoplasm of the CGM-treated G361 cells. CGM halted the cell cycle progression by G1 arrest and the reduction in the level of cyclin-dependent kinases (CDKs). Accumulation of p53 protein was not observed in cells treated with CGM for 24–72 h. The level of p27KIP1 was increased by CGM treatment only for an initial 24-h period. Cyclin D1, D3, and E protein levels were diminished in CGM-treated cells. Therefore, it is possible that CGM treatment could serve as a novel therapeutic strategy against human melanoma.

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

Bo-Young Lee,Kee-Hyun Lee,In-Ryoung Kim,Yong-Ho Kim,Hae-Ryoun Park,Bong-Soo Park KOREAN ACADAMY OF ORAL BIOLOGY 2014 International Journal of Oral Biology Vol.39 No.2

Chios Gum Mastic (CGM) is a natural resin extracted fromthe leaves of Pistacia lentiscus, a plant endemic to the Greekisland of Chios. It has been used by traditional healers, and ithas antibacterial, antifungal properties, and therapeuticbenefits for the skin. The CGM reduces the formation ofdental plaque and bacterial growth in oral saliva, and recentstudies have demonstrated the role of antioxidant activity ofCGM. Although CGM has been widely investigated, itsprotective effect against oxidative-damage to keratinocytes,as well as the relationship between CGM and autophagy, hasnot been investigated. The aim of this study was to assess theprotective effect of CGM against H2O2-induced oxidativestress and to evaluate the autophagic features induced byCGM in human keratinocytes. The pretreatment with CGMsignificantly reduced apoptosis in H2O2-exposed HaCaTcells. It promoted the degradation of caspase-3, caspase-8,and caspase-9; and it induced the formation of the processedPARP. The treatment with CGM caused an increase invesicle formation compared to control group. The level ofp62 was reduced and the conversion of LC3-I to LC3-II wasincreased in CGM treated HaCaT cells. Also, the treatment with CGM increased cleavage of ATG5-ATG12 complex. Insummary, CGM helps the cells to survive under stressfulconditions by preventing apoptosis and enhancingautophagy. Besides, the present investigation providesevidence to support the antioxidant potential of CGM in vitroand opens up a new horizon for future experiments.

Mechanism underlying Chios gum mastic-induced apoptosis on SCC25 human tongue squamous cell carcinoma cell line

Lee, Seung-Eun,Hur, Young-Joo,Kim, In-Ryoung,Kwak, Hyun-Ho,Kim, Gyoo-Cheon,Shin, Sang-Hun,Kim, Chul-Hoon,Park, Bong-Soo KOREAN ACADAMY OF ORAL BIOLOGY 2009 International Journal of Oral Biology Vol.34 No.2

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 known also to induce cell cycle arrest and apoptosis in some cancer cells. In this study, we further investigated the induction and mechanisms underlying the apoptotic response to CGM treatment in the SCC25 human tongue squamous cell carcinoma cell line. The viability of SCC25 cells, human normal keratinocytes(HaCaT cells) and human gingival fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay, respectively. Staining with Hoechst and hemacolor dyes and TUNEL assays were employed to detect SCC25 cells undergoing apoptosis. SCC25 cells were treated with CGM, and this was followed by western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, MMP activity and proteasome activity analyses. CGM treatment of SCC25 cells was found to result in a time- and dose-dependent decrease in cell viability, a dose-dependent inhibition of cell growth, and apoptotic cell death. Interestingly, CGM showed a remarkable level of cytotoxicity in SCC25 cells but not in normal cells. Tested SCC25 cells also showed several lines of apoptotic manifestation. Taken together, our present findings demonstrate that CGM strongly inhibits cell proliferation by modulating the expression of G1 cell cycle-related proteins and induces apoptosis via the proteasome, mitochondria and caspase cascades in SCC25 cells.

Effects of Chios mastic gum and exercise on physical characteristics, blood lipid markers, insulin resistance, and hepatic function in healthy Japanese men

Tomoko Fukazawa,Ilias Smyrnioudis,Masayuki Konishi,Hyeon Ki Kim,Mio Nishimaki,Mi Xiang,Shizuo Sakamoto 한국식품과학회 2018 Food Science and Biotechnology Vol.27 No.3

The effects of Chios mastic gum (Pistacia lentiscus var. Chia) and exercise on physical characteristics, blood lipid markers, insulin resistance, and hepatic function were investigated in healthy, non-smoking Japanese men aged C 40 years. Participants were allocated to control (C, 5 g/day placebo powder, n = 7), mastic (M, 5 g/day mastic powder, n = 7), or mastic plus physical activity groups (M ? PA, 5 g/day mastic powder and 30-min exercise three times/week, n = 7), and measurements were taken at baseline, 3 and 6 months. Serum triglycerides were significantly reduced at 3 months in M and M ? PA compared with C (P\0.05). Serum insulin and homeostatic model assessment of insulin resistance values were significantly reduced at 3 and 6 months in M ? PA, and at 6 months in M, compared with C (P\0.05). These results indicate that Chios mastic gum intake for 6 months reduced serum triglyceride and insulin concentrations, and additional exercise enhanced the effect on insulin.

Antiviral effect of Chios mastic gum through inhibiting host cell entry of influenza A virus

Yong-Bin Cho,In-Gu Lee,Yong-Hyun Joo,Hoyeon Jeon,Young Hyun Lim,Dong-In Kim,Young-Jin Seo 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Chios mastic gum (CMG) is a natural resin made from the sap of the mastic tree (Pistacia lentiscus), a Mediterranean crop. Although CMG has been used to treat gastrointestinal malfunctions and bacterial infections for more than 2500 years, little is known about its antiviral activity. CMG is known to influence multiple cellular processes such as cell proliferation, differentiation, and apoptosis. Since virus replication is completely dependent on the host cellular metabolism, CMG possibly regulates virus infectivity. Influenza, commonly known as ‘flu’, is an infectious respiratory disease in humans caused by influenza virus infections. In this study, we evaluated CMG’s potential as an antiviral drug to treat influenza A virus (IAV) infection. CMG dramatically reduced the cytopathogenic effect and increased the cell viability upon IAV infection. Also, CMG suppressed the expression of viral RNAs and proteins. Interestingly, CMG interfered with the early stage of the virus life cycle after viral attachment. Importantly, the intranasal administration of CMG greatly reduced viral replication in the lungs of IAV-infected mice. Moreover, CMG treatment ameliorated morbidity and mortality of the IAV-infected mice. Collectively, these results suggest that CMG displays a potent anti-IAV activity by blocking the early stage of viral replication. Thus, mastic gum could be used as a novel therapeutic agent to treat IAV infection. Furthermore, we believe that this study could expand the application of CMG to other viruses.

Evaluation of Chios Mastic Gum on Lipid and Glucose Metabolism in Diabetic Mice

Ioannis Georgiadis,Theodore Karatzas,Laskarina-Maria Korou,George Agrogiannis,Ioannis S. Vlachos,Alkisti Pantopoulou,Irene P. Tzanetakou,Nikolaos Katsilambros,Despina N. Perrea 한국식품영양과학회 2014 Journal of medicinal food Vol.17 No.3

Chios mastic gum (MG), a resin produced from Pistacia lentiscus var. Chia, is reported to possess beneficial cardiovascular and hepatoprotective properties. This study investigated the effect of crude Chios MG on metabolic parameters in diabetic mice. Streptozotocin-induced diabetic 12-week-old male C57bl/6 mice were assigned to three groups: NC (n = 9) control; LdM (n = 9) animals receiving low dose mastic for 8 weeks (20 mg/kg body weight [BW]); and HdM (n = 9) animals receiving high dose mastic (500 mg/kg BW) for the same period. Serum lipid and glucose levels were determined at baseline, at 4 and 8 weeks. Serum total protein, adiponectin, and resistin levels were also measured at the end of the experiment. Histopathological examination for liver, kidney, aorta, and heart lesions was performed. After 4 weeks, MG administration resulted in decreased serum glucose and triglyceride levels in both LdM and HdM, whereas BW levels were reduced in LdM group compared with controls. At the end of the experiment, LdM presented significantly lower serum glucose, cholesterol, low-density lipoprotein cholesterol, and triglyceride levels and improved high-density lipoprotein cholesterol levels compared with control group. HdM group had ameliorated serum triglyceride levels. Hepatic steatosis observed in control group was partially reversed in LdM and HdM groups. MG administered in low dosages improves glucose and lipid disturbances in diabetic mice while alleviating hepatic damage.

Effects of Chios mastic gum and exercise on physical characteristics, blood lipid markers, insulin resistance, and hepatic function in healthy Japanese men

Fukazawa, Tomoko,Smyrnioudis, Ilias,Konishi, Masayuki,Takahashi, Masaki,Kim, Hyeon Ki,Nishimaki, Mio,Xiang, Mi,Sakamoto, Shizuo 한국식품과학회 2018 Food Science and Biotechnology Vol.27 No.3

The effects of Chios mastic gum (Pistacia lentiscus var. Chia) and exercise on physical characteristics, blood lipid markers, insulin resistance, and hepatic function were investigated in healthy, non-smoking Japanese men aged ${\geq}40$ years. Participants were allocated to control (C, 5 g/day placebo powder, n = 7), mastic (M, 5 g/day mastic powder, n = 7), or mastic plus physical activity groups (M + PA, 5 g/day mastic powder and 30-min exercise three times/week, n = 7), and measurements were taken at baseline, 3 and 6 months. Serum triglycerides were significantly reduced at 3 months in M and M + PA compared with C (P < 0.05). Serum insulin and homeostatic model assessment of insulin resistance values were significantly reduced at 3 and 6 months in M + PA, and at 6 months in M, compared with C (P < 0.05). These results indicate that Chios mastic gum intake for 6 months reduced serum triglyceride and insulin concentrations, and additional exercise enhanced the effect on insulin.

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

Byung-Chan Koo,Duck-Han Kim,In-Ryoung Kim,Gyoo-Cheon Kim,Hyun-Ho Kwak,Bong-Soo Park KOREAN ACADAMY OF ORAL BIOLOGY 2011 International Journal of Oral Biology Vol.36 No.1

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.