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Lee, Min Sang,Kim, Nak Won,Lee, Jung Eun,Kim, Myung Goo,Yin, Yue,Kim, Sun Young,Ko, Bo Sung,Kim, Aeseon,Lee, Jong Han,Lim, Su Yeon,Lim, Dong Woo,Kim, Sun Hwa,Park, Ji Won,Lim, Yong Taik,Jeong, Ji Hoon Elsevier 2018 Acta Biomaterialia: structure-property-function re Vol.81 No.-
<P><B>Abstract</B></P> <P>Direct delivery of proteins into cells has been considered an effective approach for treating the protein-related diseases. However, clinical use of proteins has still been limited due to their instability in the blood and poor membrane permeability. To achieve an efficient cellular delivery of the protein to target cells via a systemic administration, a multifunctional carrier system having desirable stability both in the blood stream and the cells, specific cell-targeting property and endosomal escape functions may be required. In this study, we prepared a catalytic nanoparticle containing an active enzyme by cross-tethering multiple superoxide dismutase (SOD) molecules with catechol-derivatized hyaluronic acid (HA). The permeable shell of hydrophilic HA chains effectively protects the enzyme from degradation in the blood after intravenous administration and provides an additional function for targeting hepatocytes expressing HA receptor (CD44). The structure and catalytic activity of the enzyme molecules in the nanoparticle were not significantly compromised in the nanoparticle. In addition, ultra-small calcium phosphate nanoparticles (USCaP, 2–5 nm) were crystalized and decorated on the surface of the nanoparticle for the efficient endosomal escape after cellular uptake. The SOD-containing nanoparticle fortified with USCaP was used for the treatment of acetaminophen (APAP)-induced fulminant hepatotoxicity and liver injury. The nanoparticle achieved the efficient hepatic cellular delivery of SOD via a systemic administration and resulted in efficient removal of reactive oxygen species (ROS) in the liver and remarkable improvement of APAP-induced hepatotoxicity and liver injury in animals.</P> <P><B>Statement of Significance</B></P> <P>Despite the enormous therapeutic potential, the intracellular delivery of proteins has been limited due to their poor membrane permeability and stability. In this study, we demonstrated an active enzyme-containing nanoparticle functionalized by hyaluronic acid and ultra-small size calcium phosphate nanoparticles (2–5 nm) for targeted cellular delivery of superoxide dismutase (SOD). The nanoparticle was designed to integrate all the essential functions, including serum stability, target specificity, and endosomal escape capability, for a systemic delivery of a therapeutic protein to the cells of the liver tissue. The intravenous administration of the nanoparticle efficiently removes reactive oxygen species (ROS) in the liver and remarkably improves the drug-induced hepatotoxicity and the progress of fulminant liver injury in an acetaminophen-overdose animal model.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
난소암 세포주에서 Mullerian Inhibiting Substance의 증식 억제 효과
류기성 ( Ki Sung Ryu ),서미영 ( Mi Young Seo ),조윤성 ( Yun Sung Jo ),김미란 ( Mee Ran Kim ),김진우 ( Jin Woo Kim ),한구택 ( Goo Taik Han ),이준모 ( Joon Mo Lee ),김장흡 ( Jang Heub Kim ) 대한산부인과학회 2006 Obstetrics & Gynecology Science Vol.49 No.11
목적: 임신 7주경 태아 고환의 미숙 Sertoli 세포에서 생산되어 Muller관을 퇴행시키는 Mullerian inhibiting substance (MIS)는 성 발달과 가임기 여성의 생식생리 기전을 조절하는 이외에 Muller관으로부터 발생하는 일부 종양과 세포주의 성장을 in vivo와 in vitro에서 억제한다는 사실이 밝혀졌다. 이에 본 연구자들은 난소상피암 세포주들에서 MIS type II 수용체 (MISR II)의 발현을 확인하고, MIS를 투여하여 MIS의 세포 증식 억제효과와 그 기전을 밝힘으로서 종양치료제로서 MIS의 임상적 사용에 대한 가능성을 알아보고자 하였다. 연구 방법: 난소암 세포주인 SKOV-3, OVCAR-3 및 OVCAR-8에서 면역조직화학염색법으로 MISR II의 발현여부를 확인하였다. 난소암 세포주의 생존도는 고순도 MIS를 투여하여 24시간과 48시간동안 배양한 후 MTT assay로 측정하였다. MIS에 의한 세포주기 변화는 DNA 염색 후 flow cytometer로 분석하였으며 세포자멸사는 annexin-V-FITC 염색방법을 이용하여 flow cytometer로 평가하였다. MIS에 의한 난소암 세포주의 성장억제 감수성 차이와 세포자멸사 기전을 알아보기 위해 western blot 분석법을 이용하였다. 결과: 모든 난소암 세포주에서 MISR II 발현이 관찰되었지만, 발현정도는 OVCAR-8에서 제일 강했다. 세포 생존도는 OVCAR-8만이 MIS의 처치 용량과 시간에 따라 감소하였고, OVCAR-3은 고농도 MIS로 48시간 투여한 경우에만 의의 있게 감소하였으나, SKOV-3은 반응하지 않았다. Flow cytometer로 조사한 세포주기 변화는 OVCAR-8에서 10 μg/mL MIS로 24시간 처치한 경우에 G1세포주기 정지 소견을 보였으며, 48시간 후에는 세포자멸사를 의미하는 sub G0G1분기 분획이 7.02%로 나타나, MIS는 세포주기의 변화를 먼저 일으킨 후 세포자멸사를 유발하였다. OVCAR-3에서는 48시간 후 sub G0G1분기가 3.32%였으나 SKOV-3에서는 변화가 없었다. Annexin-V-FITC 염색방법으로, OVCAR-8과 OVCAR-3에서 48시간 MIS 처치 후에 각각 8.05%와 3.67%의 세포자멸사가 관찰되었다. Western blot 분석으로, OVCAR-8에서 MIS에 의한 세포 증식 억제에 이은 세포자멸사는 p16단백 증가와 연관이 있다고 판단된다. 또한 MIS는 OVCAR-8에서 MISR II와 결합한 후 pRb 비의존적인 세포내 경로를 통해 간접적으로 E2F1 활성 증가로 세포자멸사를 유도할 가능성이 있다. 결론: 본 연구에서 MIS가 난소암 세포주에서 증식 억제와 세포자멸사를 일으키는 기전을 완전히 규명하지는 못하였지만, MIS가 MIS 수용체를 발현하는 난소암에 대하여 in vitro에서 효과적인 항종양능을 나타낸다는 사실을 확인할 수 있었다. 향후 더 많은 연구로 MIS가 MIS 수용체를 발현하는 종양의 생물학적 조절제 혹은 치료제로 개발될 것이라고 예상된다. Objective: In order to explore Mullerian inhibiting substance (MIS) effects on the ovarian neoplasia, the expression and localization of the MIS type II receptor (MISR II), the growth inhibitory effects of MIS, and the underlying molecular mechanisms were investigated in the ovarian cancer cell lines. Methods: Expression of MISR Ⅱ were studied in SKOV-3, OVCAR-3, and OVCAR-8 cell lines by immunohistochemical staining. The antiproliferative effects of MIS in these cell lines were investigated by methylthiazoletetrazolium (MTT) assay, fluorescence-activated cell sorting (FACS) analysis, annexin-V-FITC binding, and western blot analysis. Results: All cell lines showed strong specific staining for MISR II, although staining in OVCAR-8 cells was more intense than that in SKOV-3 and OVCAR-3. Treatment of OVCAR-8 cells with MIS led to a dose- and time-dependent inhibition of cell growth and survival was determined use by MTT assay. But OVCAR-3 cells exhibited growth inhibition at higher doses after 48 hours of treatment and SKOV-3 cells did not demonstrate response. Using FACS analysis, exposure of OVCAR-8 cells to MIS (71 nM) resulted in G1 arrest after 24 hours of treatment. This pattern was changed by time-dependent increase in the percentage of cells with a sub G0G1 DNA content, suggesting apoptosis, after 48 hours of treatment. These results suggested that cell death be preceded by cell cycle arrest. Time-related induction of apoptosis was also observed in this cell line as measured by annexin-V-FITC binding. In OVCAR-8 cells, the growth inhibitory effects of MIS were mediated through specific induction of CDKI p16 protein expression and via regulation of E2F1 in the absence of detectable levels of pRb. We estimated that OVCAR-3 cells were affected by MIS through p16-independent, alternative mechanistic pathways, since the growth inhibitory effects of MIS were minimal. SKOV-3 cells did not express p16 protein. Conclusion: We have demonstrated that ovarian cancer cells express the MISR II. Epithelial ovarian cancer cells respond to MIS by growth inhibition. Although the precise mechanisms of MIS mediated inhibition of ovarian cancer cell growth have not been fully defined, these data suggest that MIS has activity against ovarian cancers in vitro and may also be an effective targeted therapy for ovarian cancer.