Bortezomib과 JSI-124 병합 처리된 종양항원 부가 수지상세포의 항종양 효과

김춘희 전남대학교 대학원 2010 국내박사

Dendritic cell (DCs) based immunotherapy is emerging as a useful tool to treat multiple myeloma (MM). Although several tumor target antigens have been used, the clinical effectiveness of the vaccination is still limited in the MM and the improvement of immunotherapeutic approach is clearly needed. Recently, bortezomib is known to increase the expression of heat shock protein (HSP) 90 on the surface of tumor cells for enhancing the antitumor immunity against cancer and the ability of bortezomib to kill tumor cells could be improved by inhibition of signal transducer and activator of transcription 3 (STAT3). In this study, I investigated whether bortezomib and JSI-124, an STAT3 inhibitor, could be synergistically used to generate potent cytotoxic T lymphocytes (CTLs) by inducing HSP expression in necrotic tumor cells to loading onto DCs. The combination of bortezomib and JSI-124 showed to induce necrotic cells mostly from tumor cells and led to increase the expression of HSP90 compared to bortezomib or JSI-124 alone. The ability to uptake tumor antigens that induced by bortezomib, JSI-124 or both was comparable in DCs and there was no significant difference in the phenotypic expression of DCs. DCs loaded with the combination of bortezomib and JSI-124 treated tumor cells showed the increased production of IL-12p70 during maturation as well as after CD40L stimulation compared to bortezomib or JSI-124 alone. Necrotic tumor cells treated by bortezomib, JSI-124 or both were not increased the production of IL-10 by DCs. CTLs stimulated by both bortezomib plus JSI-124 treated tumor cells-loaded DCs displayed a greater number of IFN-r-secreting cells against autologous tumor cells than did those stimulated by either bortezomib or JSI-124 treated tumor cells-loaded DCs. In conclusion, these results indicate that DCs loaded with necrotic tumor cells that induced by the treatment of bortezomib and JSI-124 could elicit potent antitumor activity of CTLs. 수지상세포를 이용한 암면역치료가 다발성골수종의 유용한 치료법으로 각광받고 있다. 여러 종양 표적 항원들이 이용되었지만, 그 임상적인 유용성에 대해서는 여전히 제한점이 있어왔다. Bortezomib은 종양세포의 표면에 heat shock 단백질 (HSP) 90의 발현을 증진시켜서, 암세포에 대한 항종양 면역력을 증진시키는 것으로 알려져 있다. Bortezomib의 암세포를 살상하는 능력은 signal transducer and activator of transcription 3 (STAT3)의 억제에 의해서 더욱 증가할 수 있다. 저자는 bortezomib과 STAT3 억제제인 JSI-124를 조합하여 처리한 종양세포가 HSP90의 발현을 증가시키고, 이러한 종양세포로 부가된 수지상세포가 종양세포에 특이성을 보이는 강력한 세포독성 T 림프구를 유도할 수 있는지 알아보았다. Bortezomib과 JSI-124의 조합으로 처리된 종양세포는 대부분 괴사를 보였으며, bortezomib이나 JSI-124 단독으로 처리된 경우보다 HSP90의 발현이 증가하였다. Bortezomib과 JSI-124로 처리한 종양세포를 부가시킨 수지상세포가 bortezomib이나 JSI-124 단독으로 치료하여 제조한 수지상세포보다 IL-12p70의 생산이 증가하였으나, IL-10 생산능은 증가하지 않았다. Bortezomib과 JSI-124로 처리된 종양세포를 부가시킨 수지상세포에 의해서 제조된 T 림프구는 bortezomib과 JSI-124 단독으로 처리하여 제조한 세포독성 T 림프구보다 표적종양세포에 대해서 유의하게 INF-의 분비능이 증가하였다. 결론적으로 bortezomib과 JSI-124 병합처리한 종양세포를 부가시킨 수지상세포에 의해 항종양 효과를 보일 수 있는 세포독성 T 림프구의 제조가 가능함을 시사하고 있다.

Bortezomib을 포함하는 자가유화 약물전달시스템의 제제개발과 평가

홍언표 경희대학교 대학원 2017 국내박사

The purposes of the present study were to develop a self-micro emulsifying drug delivery system (SMEDDS) containing Bortezomib, a proteasome inhibitor. The solubility of the drug was evaluated in 15 pharmaceutical excipients. Combinations of oils, surfactants and cosurfactants were screened by drawing pseudo-ternary phase diagrams and exhibiting the largest region of microemulsion was considered optimal. Bortezomib SMEDDS spontaneously formed a microemulsion when diluted with an aqueous medium with a median droplet size of approximately 20–30nm. In vitro release studies showed that the SMEDDS had higher initial release rates for the drug when compared with the raw drug material alone. Measurement of the viscosity, size, and ion conductivity indicated that a phase inversion from W/O system to O/W system occurred when the weight ratio of the water exceeded 30% of the entire microemulsion system. The findings of this study imply that oral delivery of a Bortezomib and colloidal system containing Labrasol could be an effective strategy for the delivery of Bortezomib. 본 연구는 프로테아좀 저해제인 Bortezomib을 함유하는 자가유화 약물전달시 스템에 관한 것이다. 제약 원료로 사용되는 15종의 부형제에서 Bortezomib의 용해도를 평가하였다. 높은 용해도를 가지는 부형제들을 선정하여 pseudoternary phase diagram기법을 이용하여 오일, 계면활성제 및 공계면활성제의 최적화된 비율을 찾았으며, phase diagram 상에서 가장 넓은 면적을 나타내는 비율의 조합을 최종 조성으로 선정하였다. 선정된 Bortezomib-SMEDDS는 수상을 접촉할 경우 자발적으로 중앙값이 20-30 nm정도인 입자크기의 microemulsion을 형성하는 것을 알 수 있었다. 약물의 방출거동 평가에서, Bortezomib-SMEDDS는 약물로만 실시된 대조군에 비해 빠른 초기용출 양상을 나타내었다. 또한, 점도, 입자크기 및 이온 전도율 측정을 통해 microemulsion 시스템 내에서 수분량이 30%이상인 시점부터 W/O emulsion에서 O/W emulsion으로 상변이가 일어나는 것을 알 수 있었다. 본 연구를 통해, labrasol을 함유한 Bortezomib의 경구투여 가능성을 확인할 수 있었다.

암세포에서 bortezomib과 nutlin-3 병합처리를 통한 paraptosis 유도 기전 연구

이동민 아주대학교 2016 국내석사

The ubiquitin-proteasome system is a complex and tightly controlled system in charge of degrading 80-90% of proteins, and is central to regulating cellular function and keeping protein homeostasis. Since proteasome inhibition can lead to the accumulation of the misfolded proteins and subsequent proteotoxicity in tumor cells, the proteasome is considered a target for cancer therapies. Bortezomib, the first FAD approved proteasome inhibitor, is now in the clinic to effectively treat multiple myeloma and mantle cell lymphoma, but its efficacy in solid tumors is not satisfactory. In this study, we show that the nutlin-3, a small-molecule inhibitor of murine double minute 2 (MDM2), effectively overcomes the resistance of MDA-MB 435S breast cancer cells to bortezomib. While bortezomib treatment did not markedly alter cellular morphologies, nutlin-3 treatment induced mitochondrial dilation without induction of cell death. Alternatively, combined treatment with bortezomib and nutlin-3 effectively induced cell death, which was accompanied by the simultaneous dilation of mitochondria and the endoplasmic reticulum (ER). These results suggest that nutlin-3 may overcome the resistance of these breast cancer cells to bortezomib via induction of paraptosis-like cell death. We found that the combined treatment yielded in MCU channeled calcium increase. This is supported with inhibition of MCU by Ru360 or knockdown of MCU significantly attenuated the cell death by the combined treatment. We also found that nutlin-3 co-treatment remarkably potentiated the bortezomib-mediated upregulation of ER marker proteins, including ATF4 and CHOP. Knockdown of CHOP effectively inhibited the cellular vacuolation and subsequent cell death by the combined treatment, suggesting that upreugulation of CHOP plays an important role in this process. Taken together, our results suggest that MCU-mediated mitochondrial Ca²⁺ overload and CHOP-mediated ER dilation may critically contribute to paraptosis-like cell death induced by the combination of bortezomib and nutlin-3 in breast cancer cells.

Bortezomib Inhibits Proliferation, Migration, and Transforming Growth Factor-β1 Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells : 망막색소상피세포의 증식, 이동과 TGF-β1에 의해 유발된 상피-간엽 변형에 대한 Bortezomib의 억제효과

문건 계명대학교 대학원 2017 국내박사

Nuclear factor-kappa B (NF-κB) plays an important role in the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE). I investigated the effects of a proteasome inhibitor, bortezomib, on the EMT in RPE by regulating NF-κB expression. After treatment with various concentrations of bortezomib, cell population was analyzed by WST-8 assay, and cell-cycle regulation was evaluated by flow cytometry. Cell migration was monitored by in vitro wound healing and transwell migration assays. To induce fibroblastoid transformation, RPE were treated with recombinant human transforming growth factor (TGF)-β1 (10 ng/mL), and Western blot and immunocytochemical analyses were performed. The effect on RPE-collagen gel lattice contraction was determined by gel contraction assay. Bortezomib treatment decreased RPE population in a concentration-dependent manner and flow cytometry revealed that these effects were due to G2/M phase cell-cycle arrest. Bortezomib (20 nM) significantly inhibited RPE migration. Moreover, bortezomib treatment also significantly impeded TGF-β1-induced transdifferentiation of RPE. The effects on proliferation, migration, and EMT were mediated by regulation of NF-κB signaling pathway. Bortezomib prevented RPE-collagen gel lattice contraction. Bortezomib suppressed myofibroblastic transformation of RPE, and also inhibited RPE-collagen gel matrix contraction. Thus, bortezomib may be a candidate of putative therapeutic agent for management of fibrotic retinal diseases. 망막색소상피세포의 상피-간엽 변형에 있어 nuclear factor-kappa B (NF-κB)의 관련성이 보고되어 있다. 이에 본 연구에서는 프로테아좀(proteasome) 억제제인 bortezomib을 이용하여 망막색소상피세포의 증식과 이동 및 상피-간엽 변형의 억제효과에 대해 알아보고자 하였다. Bortezomib에 의한 세포 증식의 변화를 WST-8 분석법을 통해 확인하였고, 유동세포분석법을 통하여 bortezomib의 세포주기에 대한 효과를 확인하였다. 시험관내(in vitro) 상처치유 분석법 및 트랜스웰 이동 분석법을 통하여 망막색소상피세포의 이동에 대한 영향을 분석하였다. Transforming growth factor-β1 (TGF-β1)으로 상피-간엽 변형을 유발시킨 후, bortezomib을 처리한 후 상피-간엽 변형에 관여하는 다양한 표지자들의 발현변화를 분석하기 위하여 웨스턴 단백분석과 면역세포화학분석을 시행하였다. 또한, 막 수축에 대한 억제 효과를 알아보기 위하여 겔 수축분석(gel contraction assay)을 시행하였다. 20 nM농도의 bortezomib은 망막색소상피세포의 증식을 현저히 감소시키며 이는 G2/M기에서 세포주기를 정지시키는 것으로 확인되었다. Bortezomib은 망막색소상피세포의 이동과 TGF-β1으로 야기되는 상피-간엽 변형을 효과적으로 억제하였으며, 상피-간엽변형에 의한 막 수축도 효과적으로 억제하였다. Bortezomib은 망막색소상피세포에서 발현되는 NF-κB 세포신호전달기전을 하향조절 시킴으로써 망막색소상피세포가 근육아세포로 변형되는 것을 억제시키고 이를 통하여 망막색소상피세포-콜라겐 겔의 수축을 효과적으로 억제시켰다. 본 연구의 결과는 bortezomib이 섬유증식과 관련된 다양한 망막질환들에 있어서 치료제로서의 가능성을 갖고 있음을 제시하는 것이라 할 수 있겠다.

(The) mechanism of inhibitory effects of bortezomib on human colon cancer cell lines

홍용상 서울대학교 대학원 2012 국내박사

연구배경: Bortezomib은 proteasome 억제제로 현재 다발성 골수종에서 그 효과가 입증되어 있다. 대장암은 최근 그 발생이 증가하고 있으나 상대적으로 효과적인 약제가 적은 편으로 새로운 약제 개발에 대한 필요성이 제기되고 있다. 본 연구는 여러 대장암 세포주에 대한 bortezomib의 억제 효능을 확인하고 그 기전을 연구하고자 계획되었다. 연구방법: 대장암 세포주에서 bortezomib 처리한 후 세포 주기 제어에 관여하는 단백질의 발현 및 활성을 분석하고 이들 단백질과 활성 산소 간의 연관성 규명을 통해 bortezomib의 억제 기전을 규명하고자 하였다. 연구결과: Bortezomib 처리 후 대장암 세포주 모두에서 G2-M arrest가 공통적으로 유도되었다. G2-M arrest에 관여하는 것으로 잘 알려진 단백질인 ATM의 인산화가 bortezomib 처리 후 증가하였으며, 이로 인해 CHK1 단백질의 인산화 또한 증가됨을 확인하였다. CHK1 단백질의 하위 신호 단백질이면서 negative regulation 받는 cdc2 단백질의 활성이 CHK1 단백질의 활성으로 인해 억제됨을 확인하였다. 더욱이 bortezomib 처리에 따른 대장암 세포주에서 활성 산소 (reactive oxygen species, ROS)의 증가가 ATM 인산화를 유도한다는 사실을 확인하였다. ROS 증가, ATM-CHK1 경로 활성화를 통한 G2-M arrest는 대표적인 대장암 세포주에서 공통적으로 관찰되었으며, 이러한 현상은 특히 KRAS, p53의 유전자형과도 무관하게 관찰되었다. 특히 wild-type p53을 가지는 대장암 세포주에서는 bortezomib 처리 후 p53 활성이 증가하였으며, p53 증강제 (nutlin-3a)를 bortezomib과 병용하였을 때 세포 증식 억제에 synergy가 있음을 확인하였다. 결론: 대장암 세포주에서 bortezomib은 G2-M arrest을 통해 세포 증식 억제를 유발함이 관찰되었다. Bortezomib으로 세포 내 ROS 농도가 증가하여 대장암 세포 내 DNA 손상을 유발하고, 잇달아 ATM-CHK1 신호 전달 체계의 활성화 유도되는 것이 그 기전으로 추정된다. 또한 wild-type p53을 가지는 대장암 세포주에서 p53 증강제 (nutrlin-3a)를 bortezomib과 병용 투여 한 경우 세포 억제 효과의 synergy가 관찰되었는데, KRAS 돌연변이 여부와는 무관하였다. 대장암 세포의 다양성을 고려할 때, 관찰된 연구결과가 모든 대장암에 공통적으로 적용될 수 있다고는 아직 확정할 수 없으나, 기존의 항암제 치료에 잘 반응하지 않는 KRAS 돌연변이를 가지는 대장암 환자에 대한 새로운 치료법 개발의 가능성을 보여주는 것으로 사료된다. Colorectal cancer (CRC) is one of the most common cancer types, however, newer drug development has reached a plateau at present. Bortezomib (PS-341, Velcade®) is a proteasome inhibitor and approved in the treatment of hematologic malignancies including multiple myeloma. A few trials of bortezomib alone or combination chemotherapy for CRC patients have been reported, however, the results were not so satisfactory, and it might be due to the lack of understanding on the action mechanism. In this report, we suggest the possibility of using bortezomib as a proteasome inhibitor in human colon cancer cells. Although bortezomib has been known to induce apoptosis and cell cycle arrest in various human cancer cells, the inhibitory mechanism of it was not clear. First, bortezomib induced G2-M arrest in the human colon cancer cells. Its treatment causes an increase of intracellular reactive oxygen species (ROS) levels and activation of ATM-CHK1 pathway, but not ATR. Also, activation of that pathway led to inactivation of cdc2. Moreover, treatment with N-acetylcysteine (NAC), ROS scavenger, inhibited phosphorylation of ATM and subsequent to decrease of cell number at G2-M phase in response to bortezomib indicating that the increased level of ROS after exposure to bortezomib is a result of ATM phosphorylation. In addition, knockdown of endogenous ATM by RNA interference induced the decreased sensitivity to bortezomib. Therefore, these results suggest that bortezomib induces p53-independent G2-M arrest through ROS-inducible ATM phosphorylation and shows synergistic antitumor activity when combined with p53 inducer in human colon cancer cells with wild-type p53, implying a potential possibility of further investigations in the treatment of CRC patients.

Natural polyphenols antagonize the antimyeloma activity of proteasome inhibitor bortezomib by direct chemical interaction

김태영 서울대학교 대학원 2013 국내박사

Bortezomib is a therapeutic proteasome inhibitor with antimyeloma activity and polyphenols are well known compounds exerting antiproliferative effects against tumors. We attempted cotreatment of myeloma cells with bortezomib and polyphenols, anticipating a synergistic effect. However, the anticancer activity of bortezomib was blocked by the polyphenols. The structural features of the polyphenols correlated strikingly with their antagonistic effect; in particular, the presence or absence of a vicinal diol moiety was the key element for effective blockage of the anticancer function of bortezomib. We infer that the vicinal diols in the polyphenols interact with the boronic acid of bortezomib and convert the active triangular boronic acid of bortezomib to an inactive tetrahedral boronate, which abolishes the antimyeloma activity of bortezomib. We confirmed this hypothesis by 11B NMR spectroscopy and an in vitro assay on myeloma cell lines and primary myeloma cells from patients. Based on these findings, restriction of the intake of natural polyphenols in foods or vitamin supplements during bortezomib treatment in multiple myeloma patients should be considered.

(The) effect of bortezomib on expression of inflammatory cytokines and survival in an experimental sepsis model induced by cecal ligation and puncture

한상훈 Graduate School, Yonsei University 2013 국내박사

Bortezomib can modulate the inflammatory process through the nuclear factor-kappa B (NF-κB) signaling pathway. Although only a clinically used proteasome inhibitor, the immunomodulatory effect of pre-incubated bortezomib is not fully evaluated in inflammation caused by infectious agents. The effect of bortezomib on the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated cells was evaluated using an in vitro cell line experiment. Bortezomib was pre-applied 1 h before LPS stimulation in RAW 264.7 cells. Subsequently, cellular viability as well as various inflammatory cytokines or cellular adhesion molecule levels were measured by reverse transcription-polymerase chain reaction. In addition, nitric oxide (NO) concentration was measured. C57BL/6J mice were used for in vivo cecal ligation and puncture (CLP)-induced murine peritonitis sepsis model experiments. For the negative control, mice received neither surgery nor treatment, and were administered 1 mL of normal saline 1 h before sham surgery. The positive control mice received 1 mL of normal saline 1 h before CLP surgery. To evaluate the impact of bortezomib dose on survival, each group that received bortezomib 0.01 mg/kg or 0.1 mg/kg 1 h before CLP surgery was compared with the positive control. To evaluate the effect of delayed administration of bortezomib on survival, the mice that were administered bortezomib 0.01 mg/kg at 24 h after CLP surgery were compared with the positive control. The mice were assessed for survival up to seven days following surgery, and then mortality rates were compared between groups. All mice that were alive at day seven after surgery in each group were anesthetized to observe the histopathologic findings and measure pulmonary inflammatory score. Pre-incubation with bortezomib (25 or 50 nM) before LPS (50 or 100 ng/mL) stimulation significantly recovered the number of viable RAW 264.7 cells compared with no pre-incubation. The bortezomib decreased the inflammatory cytokines of TNF-α, IL-1β, IL-6, and IL-10, ICAM-1, as well as NO production in LPS-stimulated cells. The seven-day survival rate in mice administered bortezomib 0.01 mg/kg at 1 h before CLP surgery was significantly higher than in the mice treated with normal saline 1 h before CLP surgery. The administration of bortezomib 0.01 mg/kg 1 h before CLP surgery resulted in the significant decrease of lung parenchymal inflammation. In conclusion, pretreatment with bortezomib showed an increased survival rate and a change of inflammatory mediators. This study suggests the possibility of the pretreatment of bortezomib as the new therapeutic target for overwhelming inflammation characterized in severe sepsis.

Molecular mechanisms mediating anti-cancer activity of proteasome inhibitors

임소현 가천대학교 대학원 2015 국내석사

Both bortezomib and carfilzomib, small peptide derivatives that function as proteasome inhibitors, have been approved for the treatment of relapsed and refractory multiple myeloma. Recently, it was observed that bortezomib and carfilzomib reduced the cell viability in many other cancer cells, including B16-F1 melanoma cells, as judged by MTT assay. Carfilzomib was less cytotoxic but showed an additive effect on bortezomib-induced reduction in the viability of B16-F1 cells. The molecular mechanisms of the anti-cancer effects of these proteasome inhibitors were further investigated in B16-F1 cells. Flow cytometric analysis showed an increase in sub-G1 fraction and annexin V-FITC-labeled cells at 8-24 h after bortezomib and carfilzomib treatment, indicating proteasome inhibitor-induced apoptosis in B16-F1 cells. Apoptotic cell death was also proven by activation of caspase-3, -8, -9 and -12. Bortezomib treatment in serum free medium strongly induced expression of endoplasmic reticulum (ER) stress-associated proteins, including CCAAT/enhancer binding protein homologous protein and X-box binding protein 1, cleavage of activating transcription factor 6 and phosphorylation of eukaryotic initiation factor 2α. The effect of carfilzomib on apoptosis and ER stress-associated proteins were much lower than those of bortezomib. Two inhibitors also induced reactive oxygen species accumulation and mitochondrial membrane potential loss. Taken together, the results suggest that bortezomib and carfilzomib induce apoptosis by ER stress and mitochondrial dysfunction via reactive oxygen species accumulation in B16-F1 melanoma cells. Instead of increasing the dosage of bortezomib alone, supplementation with less cytotoxic carfilzomib might be beneficial for the treatment of melanoma Bortezomib과 carfilzomib은 세포 안에서 유비퀴틴이 부착된 단백질의 분해를 통해 단백질의 발현과 기능을 조절하고, 세포에서의 비정상적, 혹은 잘못 접힌 단백질을 제거하는 단백질 복합체인 proteasome의 기능을 억제하는 역할을 하는 물질로서 다발성 골수종 세포에서 그 기능이 입증되어 있다. 이 약물들은 MTT assay로 확인한 결과 B16-F1 흑색종 세포를 포함한 다양한 암세포주에서 세포의 생존률을 낮추는 것으로 밝혀졌다. Carfilzomib은 bortezomib보다 더 낮은 효과를 지닌 것으로 보이지만, bortezomib과 같이 사용할 경우 단일로 사용할 경우보다 좀 더 효과적인 것으로 나타났다. Proteasome 억제제의 항암 효과를 B16-F1 cell 에서 유세포 분석기를 통해 확인한 결과 세포의 죽음이 유도된다는 점이 증명되었으며, caspase -3, -8, -9, -12의 활성을 통해 이 세포 죽음이 apoptosis성 세포사멸임을 확인하였다. Bortezomib을 우혈청이 없는 조건하에서 처리할 경우 endoplasmic reticulum(ER) stress와 연관된 단백질인 CCAAT/enhancer binding protein homologous protein, X-box binding protein -1의 발현 증가, ATF6α의 분해, eIF2α의 인산화 등이 더욱 강하게 유도됨이 확인되었다. Carfilozomib은 bortezomib에 비하여 ER stress 관련 단백질의 발현량은 낮았다. 이 두 억제자는 또한 활성 산소종의 축적과 미토콘드리아 막 전위의 손실을 유도하였다. 이 결과들로 보아, bortezomib과 carfilzomib은 B16-F1 흑색종 세포에서 활성 산소종 축적을 통한 미토콘드리아 기능 장애와 ER stress 로 인한 apoptosis를 일으키는 것으로 보인다. 흑색종의 치료에 있어, 고농도의 bortezomib이 단일로 쓰이는 것 보다 carfilzomib이 병행됨이 더욱 유용할 것으로 생각된다.

K562세포에 대한 imatinib mesylate와 bortezomib의 시험관 내 부가적 항암효과

황지영 忠南大學校 大學院 2011 국내석사

Background Imatinib mesylate is a standard therapeutic agent for chronic myelogenous leukemia (CML). Bortezomib, a proteasome inhibitor, is mainly prescribed in patients with multiple myeloma at present time, but it has been shown that bortezomib also induces apoptosis and inhibits proliferation of CML cells in vitro. In this study, I investigated whether the combination of imatinib mesylate and bortezomib exerts better anti-tumor effects in CML in vitro. Materials and methods Effects of the combination of imatinib mesylate and bortezomib on proliferation and apoptosis of K562 cells were studied. After incubation of the cells in serum-free medium for up to 72 hours, cell proliferation was measured by a modified MTT assay (CCK-8 assay), and cell cycle and apoptosis was analyzed by flow cytometry, based on the checkerboard principles. Expression of apoptosis-related proteins and activation of pro-caspases were analyzed by Western blotting. Results Imatinib mesylate 0.3 nM, bortzomib 5 nM and their combination inhibited proliferation of K562 cells by 34.2%, 21.1% and 59.1% in 48-hour incubation, respectively, indicating that these two agent exert additive anti-proliferation effects. Decrease in S phase cell fraction in similar fashion was noticed. Imatinib mesylate 300 nM, bortezomib 5 nM, and their combination induced apoptosis of K562 cells by 63.0%, 48.7% and 79.1% in 48-hour incubation, respectively, indicating that these agents also exert additive effects in terms of apoptosis induction. Imatinib mesylate down-regulated the expression of Bcl-2, and the combination with bortezomib induced further down-regulation of this molecule. Imatinib mesylate and bortexomib, respectively, up-regulated the expression of Bad, and their combination induced further down-regulation of this molecule. Imatinib mesylate and bortezomib, respectively, activated pro-caspase-9 and pro-caspase-3, and their combination further enhanced the activation of these pro-caspases. Conclusion These results indicate that imatinib mesylate and bortezomib exert additive antitumor effects in K562 cells in vitro. 배경 : Imatinib mesylate는 만성골수성백혈병에 대한 표준적인 치료제이다. Bortezomib은 프로테아좀 억제제로, 주로 다발성골수종의 치료제로 사용되고 있으나 만성골수성백혈병 세포의 세포사멸을 유발하고 세포증식을 억제한다고 알려져 있다. 본 연구자는 이러한 두 가지 약제의 병용이 시험관내에서 만성골수성백혈병 세포에 대한 항종양효과를 증진시킬 수 있는지에 관하여 연구하였다. 대상 및 방법 : K562세포를 무 혈청배지를 사용하여 72시간 동안 imatinib mesylate와 bortezomib를 각각 단독적인 처리하거나 두 가지 약제를 동시에 처리하여 배양하였다. Imatinib mesylate와 bortezomib은 각각 여러 농도로 병용 처리하여 checkerboard 분석을 수행하였다. 세포 증식은 MTT 분석을 기초로 한 CCK-8 분석을 통하여 측정하였고, 유세포 분석기를 이용하여 세포사멸과 세포주기를 분석하였다. 세포사멸 관련 단백의 발현과 procaspase의 활성화는 Western blot으로 측정하였다. 결과 : K562 세포에 imatinib mesylate 0.3 nM 과 bortezomib 5 nM을 각각 또는 두 약제를 병용 처리한 후 48시간동안 배양하였을 때 각각 34.2%, 21.1% 및 59.1% 세포증식이 억제되었는데, 이는 imatinib 와 bortezomib을 병용으로 부가적인 범위 내에서 세포의 증식이 억제된다는 것을 의미한다. 이와 유사한 양상으로 세포합성단계(S phase)의 세포수가 감소하였다. K562세포에 300 nM 의 imanitib 와 5nM 의 bortezomib을 각각 처리하거나 동시에 처리하여 48시간동안 배양한 결과, 각각 63.0%, 48.7% 및 79.1%에서 세포사멸이 관찰되었다. 이러한 결과는 두 약제의 병용이 K562세포의 세포사멸의 증진에도 부가적인 효과를 나타낸다는 것을 의미한다. Imatinib은 Bcl-2 발현을 감소시켰으며, 이 단백의 발현은 bortezomib의 병용으로 더 감소하였다. Imatinib과 bortezomib은 각각 Bad의 발현을 증가시켰으며, 두 약제의 병용으로 이 단백의 발현은 더 증가하였다. Imatinib과 bortezomib은 각각 procaspase-9과 procaspase-3의 활성화를 유도하였으며, 두 약제의 병용으로 두 효소의 활성은 더 증가하였다. 결론 : Imatinib mesylate와 bortezomib은 시험관내에서 K562세포에 부가적인(additive) 항암효과를 나타낸다.

Drug repurposing screening identifies bortezomib and panobinostat as drugs targeting cancer associated fibroblasts (CAFs) by synergistic induction of apoptosis.

이학민 성균관대학교 일반대학원 2018 국내석사

It is clear that focusing only on the cancer cell cannot eradicate cancer perfectly. Without disruption of stromal factors supporting cancer cells, cancer can highly recur and develop drug resistance. Among the stromal cells, Cancer-associated fibroblast(CAF) is known for the important key factor of cancer microenvironment. In this study, we performed repurposed drug screening using our patient-derived CAF, with various types of drugs which are in clinical trials or FDA approved. As a result, Bortezomib, Carfilzomib, and Panobinostat were chosen. Bortezomib and Panobinostat are known for their anti-cancer effect to the Multiple myeloma cases. We expected this drug combination may affects not only cancer cells, but also surrounding stromal compartments, like CAFs. The synergistic effect between BTZ and PST on viability of CAFs was observed both in vitro CAF culture and in vivo mouse model. Furthermore, combination therapy with BTZ/PST and conventional anticancer compound docetaxel strongly inhibited tumor growth in xenografts of mouse breast cancer cells with mice CAFs. In conclusion, our present study revealed that BTZ and PST could significantly reduce the viability of CAFs. Therefore, a combination therapy with BTZ/PST and anticancer drugs might be considered as a new rational for the development of anticancer therapy.