KR20030026069A - Apoptosis-inducing cancer-cell-specific composition by combination with TNF family protein and flavopiridol - Google Patents
Apoptosis-inducing cancer-cell-specific composition by combination with TNF family protein and flavopiridol Download PDFInfo
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Abstract
Description
본 발명은 TNF계 단백질과 플라보피리돌의 조합에 의하여 암세포의 세포사멸이 상승적으로 일어날 수 있도록 한 세포사멸 유도 조성물에 관한 것이다.The present invention relates to an apoptosis inducing composition that allows apoptosis of cancer cells to occur synergistically by the combination of TNF-based protein and flavopyridol.
종양괴사인자인 TNF(tumor necrosis factor)는 많은 종류의 세포에서 생산되는 다양한 기능을 하는 사이토카인(cytokine)으로서 염증, 감염, 기타 환경적 자극에 대한 반응으로서 생성된다.Tumor necrosis factor (TNF) is a multifunctional cytokine produced by many types of cells and is produced in response to inflammation, infection and other environmental stimuli.
TNF는 개체 또는 세포의 반응을 유발하여 일반적으로, 열, 조직손상, 암의 괴사, 식욕부진, 다른 사이토카인의 분비, 세포의 생장, 분화, 세포사멸 등을 초래한다. 이러한 반응은 TNF의 결합에 의해 유도되는 세포 표면의 수용체, 즉 TNFR1(TNF receptor 1) 과 TNFR2(TNF receptor 2)의 삼량체화(trimerization)에 의해 유발된다. 대부분의 TNF에 의한 생물학적 활성들은 TNFR1에 의해 전달되지만, 많은 부분 역시 TNFR2에 의해 매개된다. 하지만 TNFR2는 TNF에 의한 세포사멸현상에는 영향을 미치지 못하는 것으로 추측된다.TNF triggers an individual or cell response and generally results in fever, tissue damage, cancer necrosis, anorexia, secretion of other cytokines, cell growth, differentiation, cell death, and the like. This response is triggered by trimerization of cell surface receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2) induced by the binding of TNF. Most biological activities by TNF are transmitted by TNFR1, but many are also mediated by TNFR2. However, TNFR2 is thought to have no effect on TNF apoptosis.
TNF에 의한 세포사멸에는 활성화된 TNF 수용체에 연속적으로 상호결합하는 일련의 단백질들에 의해 활성화된 캐스페이즈(caspase: cysteine-dependent aspartate-directed protease)들이 관여하여 세포사멸에 이르게 된다.Apoptosis by TNF involves cell cascades (cysteine-dependent aspartate-directed proteases) that are activated by a series of proteins that continuously bind to activated TNF receptors.
한편 TNF는 NF-κB(nuclear factor-kappaB)를 활성화시키는 것으로 알려져 있다. TNF에 민감하게 반응하는 세포에서 NF-κB를 활성화시키면, TNF에 노출되었을 때 일어나게 되는 세포사멸을 억제할 수 있다는 것이 여러 종류의 세포를 이용한 실험들을 통하여 알려져 있으며, 반대로 대부분의 경우 NF-κB의 활성을 억제하게 되면 TNF에 대한 세포의 민감도를 높일 수 있다고 알려져 있다.Meanwhile, TNF is known to activate nuclear factor-kappaB (NF-κB). Activating NF-κB in cells that are sensitive to TNF can inhibit apoptosis that occurs when exposed to TNF. Inhibition of activity is known to increase the sensitivity of cells to TNF.
예를 들면, 지질다당류(lipopolysaccharides)는 NF-κB를 활성화시켜서 골수(myeloid)세포의 TNF에 의한 세포사멸을 저해하며, 전립선암세포에 특이적으로 발현하는 Par-4 단백질은 NF-κB의 활성을 억제하여 TNF에 의한 세포사멸을 증가시킨다.For example, lipopolysaccharides activate NF-κB and inhibit apoptosis by TNF in myeloid cells. Par-4 protein, which is specifically expressed in prostate cancer cells, inhibits NF-κB activity. Inhibition increases apoptosis by TNF.
그러나 NF-κB가 어떻게 세포사멸을 저해하는가는 확실하지 않다. 한가지 가능성은 NF-κB에 의해 발현되는 유전자산물들이 세포사멸의 신호전달체계를 방해할 수 있을 것이라는 것이다. 예를 들면, NF-κB에 의해 조절되는 유전자 중에는 IAP(세포사멸저해제, inhibitor of apoptosis), TRAF1, 그리고 TRAF2 등이 포함되어 있으며, 이들은 캐스페이즈-8의 활성화를 억제하는 것으로 판단된다. 따라서 만약 NF-κB의 저해제와 TNF를 조합하여 사용하면 세균 또는 바이러스에 의한 감염증이나 암을 치료할 수 있을 것이다. TNF는 암 이외의 여러 종류의 질병(관절염, 골다공증, 천식 등을 포함)에도 관여하는 것으로 알려져 있다.However, it is not clear how NF-κB inhibits cell death. One possibility is that gene products expressed by NF-κB may interfere with the signaling system of apoptosis. For example, genes regulated by NF-κB include IAP (inhibitor of apoptosis), TRAF1, and TRAF2, which are thought to inhibit the activation of caspase-8. Therefore, if the combination of the inhibitor of NF-κB and TNF will be able to treat the infection or cancer caused by bacteria or viruses. TNF is known to be involved in a number of diseases other than cancer (including arthritis, osteoporosis, asthma, etc.).
TNF는 강력한 항암 활성을 가지고 있지만, 심한 전신독성(systemic toxicity) 때문에 항암제로서의 임상적 사용에 제한을 받는다. 하지만 최근에 있어, 알킬화제 항암제의 일종인 멜파란(melphalan)과 고용량의 TNF를 조합하여 단리된 사지관류(isolated limb perfusion) 또는 단리된 간 관류(isolated hepatic perfusion) 방법을 통하여 흑색종(melanoma), 육종(sarcoma) 그리고 간암(hepatic cancer)의 치료에 사용하여 상당한 반응율을 보이고 있다.TNF has strong anticancer activity but is limited in clinical use as an anticancer agent because of its severe systemic toxicity. Recently, however, melanolan, a type of alkylating agent anticancer agent, and high doses of TNF have been used to isolate melanoma, via isolated limb perfusion or isolated hepatic perfusion. Significant response rates have been used in the treatment of sarcoma and hepatic cancer.
TNF는 그 자체가 갖는 암세포에 대한 직접적인 독성에 의해서라기보다, 암조직과 관련된 혈관에 손상을 입히고, 또한 면역 기작을 활성화시키는 등의 간접적인 방법으로 항암효과를 나타내며, TNF와 감마인터페론(interferon-γ)과 멜파란(melphalan)의 조합적 처치에 의한 단리된 사지관류(isolated limb perfusion) 후에 암의 미세혈관과 거대 혈관이 상당히 손상을 입는다는 것이 밝혀져 있다. 이런 긍정적인 효과로 인해 TNF를 치료용량(therapeutic dose)으로 전신투약(systemic administration)을 하기 위해, TNF의 독성을 낮추려는 연구를 촉진되고 있다.TNF exerts anti-cancer effects by indirect methods such as damaging blood vessels related to cancer tissues and activating immune mechanisms, rather than by direct toxicity to cancer cells, and TNF and interferon- It has been found that the microvascular and giant vessels of cancer are significantly damaged after isolated limb perfusion by the combined treatment of γ) and melphalan. These positive effects are facilitating research to reduce the toxicity of TNF for systemic administration of TNF at therapeutic doses.
이러한 TNF를 이용한 질병치료방법에 대한 예로서는, 미합중국특허 5,750,495등이 있으며, 여기에서는 TNF-α을 이용하여 다낭신(polycystic kidney disease)을 치료하는 방법에 대하여 소개하고 있다.As an example of a method for treating a disease using TNF, there is a U.S. Patent 5,750,495, etc. Here, a method of treating polycystic kidney disease using TNF-α is introduced.
TRAIL은 2형 막횡단(type II transmenbrane) 단백질로서, TNF계 단백질에 속한다. 사람의 TRAIL은 281개의 아미노산으로 구성되어 있으며, FasL(TNF수용체와 관련된 세포표면 수용체)과 TNF와 각각 28%, 23%의 상동성(homology)을 갖는다. 최근까지 사람에 있어서, DR4, DR5(Apo-2, TRAIL-R2, TRICK2 또는 KILLER로도 불린다.), DcR1(TRID, TRAIL-R3, 또는 LIT로도 불린다.), DcR2(TRAIL-R4 또는 TRUNDD라고도 불린다.), TR1 등 5종류의 수용체가 알려져 있다. TRAIL과 DR4 또는 DR5의 결합은 수용체의 삼량체화(trimerization)를 유도하여, 이것은 더 나아가 TNF와 유사하게 신호전달의 말단에 있는 캐스페이즈들의 활성화를 통해 세포의 자기사멸을 활성화시킨다. 하지만 TNF나 FasL과는 달리 TRAIL은 정상세포에는 세포독성을 나타내지 않으며, 쥐에 주입했을 때도 전신독성(systemic toxicity)을 보이지 않고 항암효과를 나타낸다. 따라서 TRAIL은 상당히 유망한 암치료제로서의 유용성을 보인다.TRAIL is a type II transmenbrane protein and belongs to the TNF family of proteins. Human TRAIL consists of 281 amino acids and has 28% and 23% homology with FasL (cell surface receptors associated with TNF receptors) and TNF, respectively. Until recently, in humans, DR4, DR5 (also called Apo-2, TRAIL-R2, TRICK2 or KILLER), DcR1 (also called TRID, TRAIL-R3, or LIT), DcR2 (also called TRAIL-R4 or TRUNDD) And five kinds of receptors, such as TR1. Binding of TRAIL with DR4 or DR5 leads to trimerization of the receptor, which further activates cell death through activation of caspases at the end of signaling, similar to TNF. However, unlike TNF or FasL, TRAIL does not show cytotoxicity to normal cells and shows anticancer effects without systemic toxicity even when injected into mice. TRAIL is therefore useful as a promising cancer treatment.
진핵세포의 세포주기는 일련의 싸이클린(cyclin) 단백질들이 여러 종류의 싸이클린(cyclin) 의존성을 갖는 인산화효소(CDK, cyclin-dependent kinase)들을 활성화시킴으로써 진행된다. 세포 속에는 이러한 인산화효소들을 조절하는 작은 분자량의 단백질 저해제가 존재하는데, p21, p27, p15, p16등이 포함된다. 이러한 단백질 저해제들은 종종 사람의 암세포 내에서 돌연변이가 일어나 있거나, 조절이 비정상적으로 일어나고 있는 현상이 관찰된다.The cell cycle of eukaryotic cells proceeds by a series of cyclin proteins activating several cyclin-dependent kinase (CDK). Within the cell are small molecular weight protein inhibitors that regulate these kinases, including p21, p27, p15, and p16. These protein inhibitors are often observed to have mutations or abnormal regulation of human cancer cells.
이러한 실험결과들은 CDK들의 기능을 저해하는 합성화합물들이 세포주기를 방해함으로써 세포의 분열을 막아 항암작용을 할 수 있다는 가능성을 제시하였으며, 그 중에서 플라보피리돌은 임상시험이 진행중인 대표적인 CDK 저해제이다. 플라보피리돌은 특히 CDK2와 CDK4의 작용을 저해하여 세포로 하여금 G1 phase에서 멈추게 함으로써 세포의 과량증식을 막아 암을 치료할 수 있다(Carlson 등, 1996, Cancer Res. 56: 2973-2978).These results suggest that synthetic compounds that inhibit the functions of CDKs may interfere with the cell cycle and thus prevent cell division, thereby leading to anticancer activity. Flavopyridol is a representative CDK inhibitor in clinical trials. Flavopyridols can treat cancer by inhibiting the action of CDK2 and CDK4, thereby preventing cells from overproliferating by stopping cells in the G1 phase (Carlson et al., 1996, Cancer Res. 56: 2973-2978).
미합중국특허 6,087,366에서는 이러한 플라보피리돌 혹은 이의 염들을 이용하여 신경세포의 세포주기를 저해함으로써 알츠하이머나 파킨슨씨 병의 치료에 이용하는 방법이 나타나 있으며, 미합중국특허 6,225,473 에서는 이러한 플라보피리돌의 제조방법에 대하여 나타나 있다.US Pat. No. 6,087,366 discloses a method for treating Alzheimer's or Parkinson's disease by inhibiting the cell cycle of neurons using such flavopyridols or salts thereof, and US Pat. No. 6,225,473 discloses a method for preparing such flavopyridols. Is shown.
플라보피리돌은 쥐를 이용한 사람 암세포의 이종조직(xenograft)실험을 통해서도 암조직의 발달을 저해하는 효능이 있음이 입증되었다. 하지만 사람을 대상으로 한 임상실험에서 여러 가지 부작용을 수반하여 독성을 나타낸다.Flavopyridol has been shown to be effective in inhibiting cancer tissue development through xenograft experiments of human cancer cells in mice. However, in human clinical trials, it is toxic with various side effects.
위에서 언급한 바와 같이, TNF, TRAIL, 플라보피리돌등은 그 각각이 우수한 항암기능을 가지고 있으나, 개별적으로 임상에 쓰일 때, 고농도로 투약함에 따라 여러 가지 부작용을 수반하게 되는 문제점이 있었다.As mentioned above, TNF, TRAIL, flavopyridol, etc., each of which has excellent anticancer function, but when used individually in the clinic, there was a problem that accompanied by various side effects as a high dose.
따라서 본 발명이 해결하고자 하는 과제는 TNF-α나 TRAIL을 플라보피리돌과 조합 처리함으로써 암특이적 세포사멸을 촉진시키고 저농도에서 세포사멸을 극대화하여 고농도에서 단독처리시 나타날 수 있는 독성을 예방하고 나아가 보다 안전하고 효과적인 항암치료법을 제시하는 암세포 특이적인 세포사멸 유도 조성물을 제공하는 것을 목적으로 한다.Therefore, the problem to be solved by the present invention is to treat TNF-α or TRAIL in combination with flavopyridol to promote cancer-specific apoptosis and to maximize cell death at low concentrations to prevent toxicity that can occur when treated alone at high concentrations and Furthermore, it is an object of the present invention to provide a cancer cell-specific apoptosis inducing composition suggesting a safer and more effective anti-cancer therapy.
도 1은 다양한 사람의 암 세포주에서의 TNF-α와 플라보피리돌의 동시처리에 의한 상승적 세포사멸(synergistic apoptosis) 효과를 나타내는 그래프이다.1 is a graph showing the synergistic apoptosis effect by the simultaneous treatment of TNF-α and flavopyridol in various human cancer cell lines.
도 2는 사람의 폐암 세포주인 A549에서의 TNF-α와 플라보피리돌의 동시 처리시 상승적 세포사멸 유도에 대한 처리 시간별 효과를 나타낸 그래프이다.2 is a graph showing the effects of treatment time on the induction of synergistic apoptosis during simultaneous treatment of TNF-α and flavopyridol in human lung cancer cell line A549.
도 3은 TNF-α와 플라보피리돌의 동시 처리시 세포사멸 유도 효과와 플라보피리돌의 단독 처리시의 효과를 비교한 그래프이다.Figure 3 is a graph comparing the effect of apoptosis induction and the effect of the treatment of flavopyridol alone in the simultaneous treatment of TNF-α and flabopyridol.
도 4는 TNF-α와 플라보피리돌의 동시 처리시의 처리 농도에 따른 세포 사멸 유도 효과를 비교한 그래프이다.Figure 4 is a graph comparing the effect of inducing cell death according to the treatment concentration in the simultaneous treatment of TNF-α and flavopyridol.
도 5는 TNF-α와 플라보피리돌의 동시처리 후에 나타나는 세포내 단백질의 양적인 변화를 전기영동 패턴으로 나타낸 사진이다.Figure 5 is a photograph showing the electrophoretic pattern of the quantitative change of intracellular protein appearing after the simultaneous treatment of TNF-α and flavopyridol.
도 6은 TNF-α와 플라보피리돌의 동시 처리에 의한 NF-κB의 전사활성의 변화를 측정한 그래프이다.Figure 6 is a graph measuring the change in transcriptional activity of NF-κB by simultaneous treatment of TNF-α and flavopyridol.
도 7은 TNF-α와 플라보피리돌의 조합 처리시에 나타나는 DNA 절단구조(laddering)를 관찰한 전기영동 사진이다.FIG. 7 is an electrophoretic photograph of DNA cutting structure (laddering) appearing during the combination treatment of TNF-α and flavopyridol. FIG.
도 8은 정상 세포주인 래트-2(rat2)에서의 TNF-α와 플라보피리돌의 동시 처리시 비독성 효과를 나타낸 그래프이다.Figure 8 is a graph showing the non-toxic effect of the simultaneous treatment of TNF-α and flavopyridol in the normal cell line rat-2 (rat2).
상기와 같은 목적을 달성하기 위해서, 본 발명은 TNF계 단백질과 플라보피리돌을 혼합하여 사용함으로써, 단독으로 사용할 때보다 그 효과가 현저하게 상승되는 암세포 특이적인 세포사멸 유도용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for inducing cancer cell-specific apoptosis, the effect of which is significantly increased than when used alone by using a mixture of TNF-based protein and flavopyridol.
본 발명에 사용되는 TNF계 단백질로는 TNF-α, TRAIL 등이 사용될 수 있으며, 그밖에 이와 유사한 단백질들이 사용될 수 있으며, 본 발명의 플라보피리돌은 하기의 구조식에만 국한되는 것이 아니라 이들의 염이나 그밖에 이와 유사한 것까지 포함한다.As the TNF-based protein used in the present invention, TNF-α, TRAIL, etc. may be used, and other similar proteins may be used, and the flavopyridol of the present invention is not limited to the following structural formulas and salts thereof. And others like this.
본 발명의 상시와 같은 효과를 뒷받침하기 위해서 TNF-α, TRAIL, 플라보피리돌(flavopiridol: NSC649890, L86-8275, (-)cis-5,7-dihydroxy-2-(2-chlorophenyl) -8- [4-(3-hydroxy-1-methyl)-piperidinyl]-4H-1-benzopyran-4-one) 등을 단독으로 또는 조합하여 사람의 암세포 등을 포함한 여러 종류의 세포에 처리하여 세포독성을 FACScan 또는 DNA 단편(fragment) 관찰을 통하여 분석하였다. 약제가 처리된 세포에서의 여러 생물학적 변화를 이해하기 위해 웨스턴 블로팅(Western blot)방법이나 일시적 형질도입(transient transfection)방법을 이용하여 실험을 실시하였다.TNF-α, TRAIL, flavopyridol (flavopiridol: NSC649890, L86-8275, (-) cis-5,7-dihydroxy-2- (2-chlorophenyl) -8 to support the same effects as always -Cytotoxicity can be obtained by treating several types of cells including human cancer cells, etc., alone or in combination with [4- (3-hydroxy-1-methyl) -piperidinyl] -4H-1-benzopyran-4-one). Analysis was performed by FACScan or DNA fragment observation. In order to understand various biological changes in the cells treated with the drug, experiments were performed using Western blot or transient transfection.
본 발명의 조성물에는 세포사멸 효과를 향상시키기 위하여, 약제학적으로 첨가가 가능한 통상의 첨가제가 첨가될 수 있으며, TNF계 단백질은 1.0 내지 2000μg/㎖, 플리보피리돌은 1.0 내지 1000μg/㎖가 함유되는 것이 바람직하다.In order to enhance apoptosis effect, the composition of the present invention may be added with a conventional additive which can be added pharmaceutically, and contains 1.0 to 2000 μg / ml of TNF-based protein and 1.0 to 1000 μg / ml of flibopyridol. It is desirable to be.
하기 실시예 등을 통하여 본 발명은 더욱 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명이 이들만으로 한정되는 것은 아니다. 이하 본 발명을 상세히 설명하면 다음과 같다.Through the following examples, the present invention will be described in more detail. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto. Hereinafter, the present invention will be described in detail.
<실시예1>Example 1
다양한 사람의 암세포주에서의 TNF-α와 플라보피리돌 혹은 TRAIL과 플라보피리돌의 조합처리에 의한 상승적 세포사멸(synergistic apoptosis)Synergistic apoptosis by combination treatment of TNF-α and flabopyridol or TRAIL and flabopyridol in various human cancer cell lines
사람의 암세포주들은 5% 소태아혈청(FBS: fetal bovine serum)을 포함하는 RPMI1640 배지(1000units/㎖ 페니실린 지(penicillin G), 100㎍/㎖스트렙토마이신 지(streptomycin G), 0.25㎍/㎖ 암포테리신 비(Amphotericin B), 2mM 글루타민(Glutamine) 포함)로 세포배양기 내에서 5% CO2, 37℃조건 하에서 배양하였다. 사용한 사람의 암 세포주는 직장암 세포주 HCT-116, 방광암 세포주 T24, 직장암 세포주 HCT15, 폐암 세포주 A549 등이다. 암 세포에서 세포사멸 발생 여부를 측정하기 위해 FACScan(flow cytometric analysis)를 실시하였다. 그 구체적인 실험방법은 다음과 같다.Human cancer cell lines contained RPMI1640 medium (1000 units / ml penicillin G), 100 µg / ml streptomycin G, 0.25 µg / ml cancer cells containing 5% fetal bovine serum (FBS) It was incubated in a cell incubator at 5% CO 2 , 37 ° C. in a terrycin ratio (Amphotericin B), including 2 mM glutamine (Glutamine). Human cancer cell lines used are rectal cancer cell line HCT-116, bladder cancer cell line T24, rectal cancer cell line HCT15, and lung cancer cell line A549. FACScan (flow cytometric analysis) was performed to measure apoptosis in cancer cells. The specific experimental method is as follows.
먼저, 암 세포주를 직경이 60㎜인 배양접시(culture dish)에 5x105개를 접종하고 5㎖의 상기 세포 배지 내에서 하루동안 키운 뒤 TNF-α(Boehringer Mannheim사 제품, 10ng/㎖)또는 TRAIL (Serotec사 제품, 50ng/㎖)을 각각 플라보피리돌(0.5μM)과 조합하여 동시 처리하였다. 표시된 시간동안 처리후의 상층의 배지를 15㎖ falcon 튜브(Becton Dickinson사 제품)에 모으고 붙어있는 세포를 5㎖ PBS로 두 번 세척한 후 0.5㎖의 0.15% 트립신(trypsin) 처리하여 떼어낸 후 보관된 상층의 배지와 합하였다. 그 다음 튜브를 200xg(gravity)의 속도로 5분간 원심분리하여 세포를 가라앉힌 후, 상층액을 제거하고 가라앉은 세포들을 300㎕의 PBS로 다시 잘 현탁(suspension)시키고 난 후, 교반(vortexing)시키면서 4℃로 냉각된 75% 에탄올 5㎖를 서서히 첨가하여 세포를 고정시켰다. 이것을 4℃에서 최소한 3시간 이상 방치 후 다시 원심 분리하여 에탄올을 제거 후 0.1㎎/㎖농도의 RNase(Sigma사 제품)가 든 PBS를 세포 밀도가 1x106/㎖이 되도록 첨가한 후 여기에 다시 프로피디움 아이오다이드(propidium iodide, 1㎎/㎖,증류수)를 최종 농도 50㎍/㎖이 되도록첨가하여 30분간 상온으로 빛이 차단된 곳에 방치하여 염색하였다.First, 5x10 5 cancer cell lines were inoculated into a culture dish 60 mm in diameter and grown in 5 ml of the cell medium for one day, followed by TNF-α (Boehringer Mannheim, 10 ng / ml) or TRAIL. (50ng / ml from Serotec Co., Ltd.) were co-treated in combination with flavopyridol (0.5 μM), respectively. The supernatant medium after treatment for the indicated time was collected in 15 ml falcon tubes (Becton Dickinson) and the adhered cells were washed twice with 5 ml PBS, treated with 0.5 ml of 0.15% trypsin, removed and stored. Combined with the upper medium. The tubes were then centrifuged at 200xg (gravity) for 5 minutes to settle the cells, the supernatant was removed and the settled cells were again well suspended with 300 μl of PBS, followed by vortexing. The cells were fixed by slowly adding 5 ml of 75% ethanol cooled to 4 ° C. After standing at 4 ° C for at least 3 hours, centrifugation was carried out again to remove ethanol, and then PBS containing 0.1 mg / ml RNase (Sigma) was added so that the cell density was 1x10 6 / ml, and then the prop Diodium iodide (propidium iodide, 1mg / ㎖, distilled water) was added to a final concentration of 50㎍ / ㎖ and left for 30 minutes at room temperature where the light was blocked and stained.
그 후 염색된 세포를 488㎚의 여기(excitation)파장과 588㎚의 방사(emission)파장으로 FACSort(Becton Dickinson사 제품)를 이용하여 10,000개의 세포를 FACScan 분석하였다. 히스토그램의 수치분석은 모디피트 소프트웨어(Modifit software ; Veriety software house사, Maine주 Topsham 소재)로 분석하였다.The stained cells were then subjected to FACScan analysis of 10,000 cells using FACSort (Becton Dickinson, Inc.) at an excitation wavelength of 488 nm and an emission wavelength of 588 nm. Numerical analysis of the histogram was performed with Modifit software (Veriety software house, Topsham, Maine).
TNF-α와 플라보피리돌 또는 TRAIL과 플라보피리돌을 조합하여 동시에 처리하는 경우, 하기의 표 1에서 보는 바와 같이 현저한 상승적 세포사멸이 유도됨을 확인하였다. 이러한 결과는 도 1에서와 같이 다양한 종류의 세포를 통한 실험에서도 관찰되었다(도1 : TNF-α와 플라보피리돌을 각각 10ng/㎖와 0.5μM농도로 각각 혹은 동시 처리하여 24시간동안 배양하였다. C; control, T; TNF-α(10ng/㎖), F.P; 플라보피리돌(0.5μM), +;TNF-α와 플라보피리돌을 각각 10ng/㎖와 0.5μM 농도로 동시 처리).When TNF-α and flavopyridol or TRAIL and flavopyridol were simultaneously treated in combination, it was confirmed that marked synergistic apoptosis was induced as shown in Table 1 below. These results were also observed in experiments with various kinds of cells as shown in FIG. C; control, T; TNF-α (10 ng / ml), FP; flabopyridol (0.5 μM), +; simultaneous treatment of TNF-α and flabopyridol at concentrations of 10 ng / ml and 0.5 μM, respectively. .
(TNF-α, TRAIL, 플라보피리돌을 각각 10ng/㎖, 50ng/㎖, 0.5μM농도로 단독 혹은 동시 처리하였다.)(TNF-α, TRAIL, and flavopyridol were treated alone or simultaneously at 10 ng / ml, 50 ng / ml and 0.5 μM concentrations, respectively.)
<실시예2>Example 2
사람의 폐암 세포주인 A549에서의 TNF-α와 플라보피리돌의 동시처리 시 상승적 세포사멸 유도에 대한 처리 시간별 효과Effect of Treatment Time on Induction of Synergistic Apoptosis during Simultaneous Treatment of TNF-α and Flavopyridol in Human Lung Cancer Cell Line A549
A549 세포주를 실시예 1에서와 동일한 방법으로 배양한 후 TNF-α와 플라보피리돌을 각각 10ng/㎖, 0.5μM 농도로 동시 처리하여 각각 0, 3, 6, 12, 24시간 동안 배양처리 하였고, 이를 실시예 1에서와 동일한 방법으로 FACScan을 실시하였다.After culturing the A549 cell line in the same manner as in Example 1, TNF-α and flavopyridol were simultaneously treated at 10 ng / ml and 0.5 μM, respectively, and cultured for 0, 3, 6, 12, and 24 hours, respectively. FACScan was carried out in the same manner as in Example 1.
아래의 표 2 및 도 2에서와 같이 동일조건에서 시간이 경과함에 따라 세포사멸이 점차적으로 증가됨을 확인하였다.(도2)As shown in Table 2 and Figure 2 below, it was confirmed that apoptosis gradually increased over time under the same conditions.
<비교예1>Comparative Example 1
TNFα와 플라보피리돌의 처리순서가 세포사멸에 미치는 효과Effect of TNFα and Flavopyridol Sequence on Apoptosis
A549 세포주를 실시예 1에서와 동일한 방법으로 배양한 후 TNF-α를 10ng/㎖ 농도로 6시간 처리한 후 5㎖ PBS로 2번 세척한 후 다시 플라보피리돌을 0.5μM농도로 6시간 처리하거나, 혹은 그 반대 순서로 각각 6시간 처리하거나, 혹은 동시 처리한 후 각각 6시간 혹은 12시간 동안 처리하였고, 이를 실시예 1에서와 동일 방법으로 고정 후 FACScan을 실시하였다.After incubating the A549 cell line in the same manner as in Example 1, after treatment with TNF-α at a concentration of 10 ng / ml for 6 hours, washing twice with 5 ml PBS, and then again treating flavopyridol at a concentration of 0.5 μM for 6 hours. 6 hours or 6 hours or 12 hours after the simultaneous treatment or 6 hours or 12 hours, respectively, and the FACScan was fixed in the same manner as in Example 1.
하기의 표 3에서와 같이 TNF-α와 플라보피리돌의 동시 처리 시에만 현저하게 세포사멸이 유도됨을 확인함으로써, 동시에 두 약제에 세포가 노출되어야만 상승적 세포사멸이 유도됨을 알 수 있었다.As shown in Table 3 below, by confirming that apoptosis was remarkably induced only during simultaneous treatment of TNF-α and flavopyridol, synergistic apoptosis was induced only when cells were exposed to both agents at the same time.
<비교예2>Comparative Example 2
TNF-α와 플라보피리돌의 처리순서가 세포 사멸에 미치는 효과Effect of TNF-α and Flavopyridol Sequence on Cell Death
실시예 1과 동일한 방법으로 A549 세포주를 배양한 후, TNF-α를 10 ng/ml농도로 3시간 동안 처리한 후에 처리액을 제거한 후 추가로 TNF-α와 플라보피리돌을 각각 10 ng/ml, 0.5 μM로 6시간 처리하거나, 혹은 플라보피리돌을 0.5 μM 농도로 3시간 동안 처리 후, 처리액을 제거하고 추가로 TNF-α와 플라보피리돌을 각각 10 ng/ml, 0.5 μM로 6시간 처리하거나, 혹은 TNF-α만을 10 ng/ml 농도로 9시간, 혹은 플라보피리돌만을 0.5 μM농도로 9시간 처리한 후 각각을 실시예1에서와 같은 방법으로 고정한 후 FACScan을 실시하였다.After culturing the A549 cell line in the same manner as in Example 1, after treatment with TNF-α at a concentration of 10 ng / ml for 3 hours, the treatment solution was removed, followed by further addition of TNF-α and flavopyridol, respectively, 10 ng /. ml, 0.5 μM for 6 hours, or after treatment with flavopyridol for 3 hours at 0.5 μM concentration, the treatment solution was removed and additionally, 10 ng / ml and 0.5 μM of TNF-α and flabopyridol, respectively. Treatment for 6 hours or TNF-α alone at 10 ng / ml for 9 hours or flavopyridol at 0.5 μM for 9 hours, and then fixed in the same manner as in Example 1, followed by FACScan. It was.
하기 표 4에서와 같이 TNF-α와 플라보피리돌을 동시 처리 전에 TNF-α를 먼저 3시간 처리한 군에서는 세포사멸이 일어나지 않았고, 플라보피리돌을 먼저 처리한 군에서만 세포사멸이 강하게 일어났다.As shown in Table 4, apoptosis did not occur in the group treated with TNF-α for 3 hours prior to simultaneous treatment with TNF-α and flavopyridol, and apoptosis occurred only in the group treated with flavopyridol first. .
<실시예 3><Example 3>
TNF-α와 플라보피리돌의 동시 처리시의 세포사멸 유도효과의 효능(potency) 비교분석Comparative Analysis of Potency of Apoptosis-Inducing Effect During Simultaneous Treatment of TNF-α and Flavopyridol
폐암 세포주인 A549를 실시예 1에서와 동일한 방법으로 배양한 후 TNF-α와 플라보피리돌을 6시간 동안 각각 표시된 농도별로 따로 처리하거나 동시에 조합하여 처리한 후, 세포사멸 발생정도를 FACScan을 수행하여 비교하였다.After culturing the lung cancer cell line A549 in the same manner as in Example 1, TNF-α and flavopyridol were treated separately or simultaneously in combination for the indicated concentrations for 6 hours, followed by FACScan. By comparison.
단독처리 시에는 플라보피리돌의 경우 50μM에서도, TNF-α의 경우 50ng/㎖의 농도에서도 유의한 세포사멸은 관찰되지 않았으며(도3, 도4(A)), 따라서 각 약제를 동시처리하면 낮은 농도에서 유의한 세포사멸을 유도할 수 있었다. 그리고 TNF-α의 존재하에서는 세포사멸의 발생정도가 플라보피리돌의 농도에 의존성을 보임을 알 수 있었다(도4(B)).In the single treatment, no significant cell death was observed even at 50 μM for flavopyridol and at 50 ng / ml for TNF-α (Fig. 3, Fig. 4 (A)). Induced significant cell death at low concentrations. In the presence of TNF-α, the degree of apoptosis was shown to be dependent on the concentration of flavopyridol (FIG. 4 (B)).
<실시예 4><Example 4>
TNF-α와 플라보피리돌의 동시처리 시의 세포사멸 유도에 있어서의 캐스페이즈들의 역할 분석Analysis of the role of cascades in inducing apoptosis during simultaneous treatment of TNF-α with flabopyridol
A549를 실시예 1에서와 동일한 방법으로 배양한 후 여러 가지의 캐스페이즈들에 대한 펩티드성 저해제(peptide inhibitor, Alexis biochemical사 제품)들을 다양한 농도로 처리하고, 30분 후 TNF-α, 플라보피리돌을 동시 처리하여 24시간 동안 배양한 후 세포를 실시예 1에서와 동일한 방법으로 고정한 후 세포사멸의 발생정도를 FACScan을 통해 비교하였다.After incubating A549 in the same manner as in Example 1, the peptide inhibitors (peptide inhibitors, manufactured by Alexis biochemical) for various cascades were treated at various concentrations, and after 30 minutes, TNF-α and flabopyri After treatment with stones for 24 hours, the cells were fixed in the same manner as in Example 1, and then the incidence of apoptosis was compared by FACScan.
하기의 표 5에서 보는 바와 같이 캐스페이즈 저해제(caspase inhibitor)들에 의해 농도 의존성을 가지고 세포사멸 발생이 저해됨을 확인하였고, 특히 Z-YVAD-FMK가 상당히 세포사멸을 억제함을 알 수 있다. 이를 통해 TNF-α와 플라보피리돌의 동시처리에 의한 세포사멸 유도에 활성화된 다양한 캐스페이즈들이 관여함을 확인하였다.As shown in Table 5 below, it was confirmed that apoptosis was inhibited by the caspase inhibitors (caspase inhibitors) with a concentration dependency, and it can be seen that Z-YVAD-FMK significantly inhibited apoptosis. Through this, it was confirmed that various cascades activated to induce apoptosis by simultaneous treatment of TNF-α and flavopyridol were involved.
<실시예5>Example 5
TNF-α와 플라보피리돌의 동시 처리 후에 나타나는 세포내 단백질의 양적인 변화 측정Quantitative Changes in Intracellular Proteins Following Simultaneous Treatment of TNF-α and Flavopyridol
폐암 세포주인 A549 세포를 직경 100㎜인 배양접시에 4x106개를 상기 세포배양액에 깔아 하루동안 배양 한 뒤, TNF-α와 플라보피리돌을 각각 또는 합하여 표시된 농도로 24시간 동안 처리하였다. 그 후 세포를 10㎖ PBS로 조심해서 2번 세척 후 단백질분해효소 저해제 칵테일(protease inhibitor cocktail; Roche사 제품, completeTM-mini)이 든 PBS(1 tablet/50㎖ PBS)를 배양접시 당 1 ㎖씩 넣고 세포를 긁어모아 초음파처리로 세포를 파쇄시켰다. 이 샘플을 마이크로원심분리기로 12000 rpm으로 20분간 원심분리 후 상층액을 모아 브레드포드 염색시약(Bradford dye reagent, Bio-Rad사 제품)로 단백질을 정량한 후 40㎍의 단백질을 SDS-PAGE를 이용하여 젤(gel)상에서 전개하여 이를 다시 니트로셀룰로오스 멤브레인(nitrocellulose membrane, Bio-Rad사 제품)으로 옮긴 후 각각의 조사하고자 하는 단백질에 특이적인 일차 항체와 고추냉이 퍼옥시데이즈(HRP, horseradish peroxidase)가 붙은 2차 항체(Amersham 또는 Bio-Rad사 제품), 그리고 ECL 화학발광시약(chemiluminescence reagent, Amersham사 제품)를 사용하여 각각의 단백질의 양적 변화를 분석하였다.A549 cells, a lung cancer cell line, were placed in a culture dish of 100 mm in diameter, and 4 × 10 6 cells were incubated in the cell culture solution for one day, and then treated with TNF-α and flavopyridol, respectively, or in combination at the indicated concentration for 24 hours. The cells were then carefully washed twice with 10 ml PBS, followed by 1 ml per plate of PBS (1 tablet / 50 ml PBS) containing a protease inhibitor cocktail (Roche, complete TM- mini). The cells were scraped and scraped by sonication. Centrifuge the sample at 12000 rpm for 20 minutes using a microcentrifuge, collect the supernatant, and quantify the protein with Bradford dye reagent (Bio-Rad). The gel was developed on a gel and transferred back to a nitrocellulose membrane (Bio-Rad), followed by primary antibodies and horseradish peroxidase (HRP) specific to the protein to be investigated. Quantitative changes of each protein were analyzed using attached secondary antibodies (manufactured by Amersham or Bio-Rad) and ECL chemiluminescence reagent (manufactured by Amersham).
TNF-α와 플라보피리돌의 동시처리에 의해 캐스페이즈-3와 캐스페이즈-8이 활성화 됨을 확인하였고, 캐스페이즈의 기질인 Rb, PARP, 라민 비(Lamin B) 단백질이 분해됨을 확인하였다(도5 : C;control, T;TNF-α를 10ng/㎖ 농도로 처리, F.P; 플라보피리돌을 표시된 농도로 처리, +;TNF-α와 플라보피리돌을 각각 10ng/㎖, 0.5μM 농도로 동시 처리).It was confirmed that caspase-3 and caspase-8 were activated by simultaneous treatment of TNF-α and flabopyridol, and that the substrates of caspase Rb, PARP, and Lamin B were decomposed ( Figure 5: C; control, T; TNF-α treatment at 10 ng / ml concentration, FP; flabopyridol treatment at the indicated concentrations, +; TNF-α and flabopyridol 10 ng / ml, 0.5 μM, respectively. Simultaneous treatment with concentration).
<실시예 6><Example 6>
TNF-α와 플라보피리돌의 동시 처리에 의한 NF-κB의 전사활성(transcriptional activity)의 변화 측정Measurement of Transcriptional Activity Change of NF-κB by Simultaneous Treatment of TNF-α and Flavopyridol
폐암 세포주인 A549를 직경 100㎜인 배양접시 2개에 각각 4x106개씩 실시예 1에서와 동일한 배지에 깔고 난 후 24시간동안 배양하였다. 그 후 각 배양접시를 소태아혈청(FBS, fetal bovine serum)을 포함하지 않는 RPMI 배지 10㎖로 세척 후, 이 중 1개의 배양접시에는 pNF-κB-루시퍼레이즈 플라스미드(Luciferase plasmid, Stratagene사 제품, 카탈로그 No. 219078)를 리포펙트아민플러스 진핵세포 이입시스템(lipofectAMINE PLUSTMeukaryotic cell transfection system, Gibco-BRL사 제품)을 사용하여 제조회사의 지시대로 이입(transfection)시켰고, 다른 1개의 배양접시는 pNF-κB-루시퍼레이즈 플라스미드만을 제외한 대조 군으로 처리하였다. pNF-κB-루시퍼레이즈 플라스미드는 NF-κB 전사인자(transcription factor)에 의해서 발현이 조절될 수 있도록 프로모터 부위에 5개의 NF-κB결합인자를 포함하고 있으며, 표지 단백질인 루시퍼레이즈의 발현이 이 프로모터에 의존하도록 구성되어 있다. 자세히 기술하면 다음과 같다.A549, a lung cancer cell line, was placed in the same medium as in Example 1 with 4 × 10 6 cells in each of two culture plates 100 mm in diameter, and then cultured for 24 hours. Thereafter, each culture dish was washed with 10 ml of RPMI medium containing no fetal bovine serum (FBS), and one of the culture dishes contained pNF-κB-luciferase plasmid (Luciferase plasmid, product of Stratagene, Catalog No. 219078) was transfected using the lipofectAMINE PLUS ™ eukaryotic cell transfection system (manufactured by Gibco-BRL) and the other culture dish was pNF. Treatment was performed with the control group except for the -κB-luciferase plasmid. The pNF-κB-luciferase plasmid contains five NF-κB binding factors at the promoter site so that expression can be regulated by the NF-κB transcription factor and the expression of the luciferase marker protein It is configured to depend on. The details are as follows.
밑이 둥근 폴리스티렌 튜브(Becton Dickinson사 제품)에 9㎍의 플라스미드 DNA를, 대조군의 경우 동일 부피의 TE (10mM Tris, pH8.0, 1mM EDTA) 용액을, 54㎕의 PLUS 시약(Gibco-BRL사 제품)과 미리 섞은 후 FBS가 없는 RPMI1640 배지를 첨가하여 최종 900㎕로 만들었다. 그리고 다른 또 하나의 튜브(tube)에 36㎕의 리포펙트아민 시약(lipofectAMINE reagent, Gibco-BRL사 제품)에 소태아혈청(FBS)이 없는 RPMI1640 배지를 첨가하여 최종 900㎕로 만들었다. 그 후 상온에서 15분간 방치 후 각각을 서로 섞어 다시 상온에서 15분간 방치하였다. 그리고 나서 이 혼합액을 7.8㎖의 소태아혈청(FBS)이 없는 RPMI1640 배지로 교환된 A549 세포 배양 100㎜ 접시에 방울방울 떨어뜨려 이입(transfection)시켰다. 그리고 나서 37℃ CO2세포배양기에서 5시간 배양한 후 다시 5% 소태아혈청(FBS)이 든 RPMI1640 배지로 교환한 후 다시 1시간 동안 배양기 내에서 배양하였다. 그 후 각 배양접시를 1㎖의 0.1% 트립신(trypsin) 용액(Gibco-BRL사 제품)으로 처리하여 세포를 떼어낸 후 모든 세포를30㎖의 5% FBS를 포함하는 RPMI1640 배지로 옮겨 잘 섞은 후 12-웰 접시에 웰 당 2.5㎖씩 분주하였다. 그리고 나서 16시간 동안 배양기에서 배양한 후 TNF-α와 플라보피리돌을 각각 최종 농도 10ng/㎖, 0.5μM로 단독으로 혹은 조합하여 이중으로 2 웰 씩 처리하여 다시 6시간 동안 배양하였다.In a rounded polystyrene tube (Becton Dickinson), 9 μg of plasmid DNA was added to the control, in the same volume of TE (10 mM Tris, pH8.0, 1 mM EDTA) solution, and 54 μl of PLUS reagent (Gibco-BRL). Product) and pre-mixed with RPMI1640 medium without FBS to final 900 μl. In another tube, 36 μl of lipofectamine reagent (lipofectAMINE reagent, manufactured by Gibco-BRL) was added to RPMI1640 medium without fetal bovine serum (FBS) to make a final 900 μl. Thereafter, the mixture was left at room temperature for 15 minutes, and the mixtures were mixed with each other and allowed to stand at room temperature for 15 minutes. The mixed solution was then transfected by dropping onto a 100 mm dish of A549 cell culture exchanged with 7.8 mL fetal bovine serum (FBS) -free RPMI1640 medium. Then, after incubating for 5 hours in a 37 ℃ CO 2 cell incubator and exchanged again with RPMI1640 medium containing 5% fetal bovine serum (FBS) and incubated in the incubator again for 1 hour. Each dish was then treated with 1 ml of 0.1% trypsin solution (Gibco-BRL) to remove cells, and then all cells were transferred to RPMI1640 medium containing 30 ml of 5% FBS and mixed well. 2.5 ml per well were dispensed in a 12-well dish. Then, after incubating for 16 hours in the incubator, TNF-α and flabopyridol were treated with two wells, either alone or in combination, at a final concentration of 10 ng / ml and 0.5 μM, respectively, and incubated for another 6 hours.
그리고 나서 루시퍼레이즈 분석 시스템(luciferase assay system, Promega사 제품)을 제조회사의 지시대로 사용하여 세포 추출액 내의 루시퍼레이즈의 활성도를 측정하였다. 이 경우 빛(luciferase 활성도)의 세기는 루멧 모델 LB953 루미노미터(Lumat model LB953 Luminometer; Berthold사제품, Germany소재)를 사용하여 측정하였으며, 세포추출액 내의 단백질의 양은 중탄산염분석(bicarbonate assay, BCA)방법으로 소혈청단백질(bovine serum albumin, BSA)을 표준으로 하여 측정하였고, 이 값으로 활성도 측정값이 동일 단백질 양을 대변하도록 보정하였다.Then, the luciferase assay system (promega company) was used as directed by the manufacturer to measure the activity of luciferase in the cell extract. In this case, the intensity of light (luciferase activity) was measured using a Lumet model LB953 Luminometer (Berthold, Germany), and the amount of protein in the cell extract was determined by the bicarbonate assay (BCA) method. Bovine serum albumin (BSA) was measured as a standard, and the activity measurement was corrected to represent the same amount of protein.
TNF-α 단독으로 처리한 경우 루시퍼레이즈의 양이 증가하여 A549 세포가 TNF에 반응하는 것을 확인한 반면, 플라보피리돌은 루시퍼레이즈의 양적 변화에 영향을 미치지 못하였다. TNF-α와 플라보피리돌을 조합하여 처리하면 루시퍼레이즈양이 증가하지 않음을 알 수 있다(도6).When treated with TNF-α alone, the amount of luciferase increased, confirming that A549 cells responded to TNF, while flavopyridol did not affect the quantitative change of luciferase. Treatment with a combination of TNF-α and flavopyridol did not increase the amount of luciferase (Fig. 6).
<실시예 7><Example 7>
플라보피리돌과 TNF-α의 조합 처리시에 나타나는 DNA 절단구조(laddering) 관찰DNA Laddering in Combination Treatment with Flavopyridol and TNF-α
A549 세포(1x106cells)를 60㎜ 배양접시(culture dish)에 접종하고 하루동안 배양한 후, 플라보피리돌과 TNF-α를 표시된 농도로 단독으로 또는 조합하여 24시간동안 처리한 다음, DNA를 분리하기 위해 세포를 수거하였다. DNA 분리과정은 다음과 같다.A549 cells (1 × 10 6 cells) were inoculated in a 60 mm culture dish and incubated for one day, followed by treatment with flavopyridol and TNF-α alone or in combination at the indicated concentrations for 24 hours, followed by DNA Cells were harvested to isolate. DNA separation process is as follows.
배지에 떠 있는 세포와 바닥에 붙어 있는 세포를 수거하여 PBS(phosphate buffered saline) 1㎖로 세척하고, 마이크로 원심분리기에서 5000 rpm에서 5분간 원심분리하여 세포침전을 얻는다. 상층액은 버리고, 침전된 세포에 파쇄용액(lysis buffer)(1% NP-40, 20mM EDTA, 50mM Tris-HCl, pH 7.5)를 넣어 부드럽게 피펫팅(pipetting) 하면서 세포를 파쇄한다. 세포파쇄액(cell lysate)를 5000rpm에서 5분간 원심 분리하여 상층액을 새 튜브에 취한다. 여기에 NaCl과 SDS를 넣어 최종농도가 0.2M, 0.5%가 되게 한 다음, 상층액의 동량에 해당하는 PCI(phenol: chloroform: isoamylalchol=25:24:1)용액을 첨가하여 교반(vortexing)하였다. 이것을 14,000rpm에서 2내지 5분간 원심분리한 뒤 상층액을 취하여 페놀정제(phenol extraction)을 2회 더 반복한다. 상층액을 에탄올 침전하여 침전물을 100 ㎕의 물에 녹이고, RNase A를 넣어 37℃에서 10분간 반응시키면서 RNA를 제거하였다. 새로 만든 신선한 프로티네이즈 케이(proteinase K)로 37℃에서 10분간 반응시키면서 단백질을 제거하였다. 다시 페놀정제와 에탄올 침전법을 이용하여 DNA를 침전시킨 후, DNA를 1.5% 아가로오스 젤에서 전기영동하고, EtBr로 염색하여 자외선(UV) 하에서 확인하였다.The cells suspended in the medium and the cells attached to the bottom were collected, washed with 1 ml of PBS (phosphate buffered saline), and centrifuged at 5000 rpm in a microcentrifuge for 5 minutes to obtain cell precipitation. Discard the supernatant and crush the cells by gently pipetting the lysis buffer (1% NP-40, 20mM EDTA, 50mM Tris-HCl, pH 7.5) into the precipitated cells. The cell lysate is centrifuged at 5000 rpm for 5 minutes and the supernatant is taken into a new tube. NaCl and SDS were added to make final concentration of 0.2M and 0.5%, followed by stirring by adding a PCI (phenol: chloroform: isoamylalchol = 25: 24: 1) solution corresponding to the same amount of the supernatant. . After centrifugation at 14,000 rpm for 2 to 5 minutes, the supernatant is taken and phenol extraction is repeated two more times. The supernatant was ethanol precipitated, the precipitate was dissolved in 100 μl of water, and RNA was removed while RNase A was added and reacted at 37 ° C. for 10 minutes. Protein was removed by freshly prepared proteinase K while reacting at 37 ° C. for 10 minutes. After the DNA was precipitated by phenol purification and ethanol precipitation, the DNA was electrophoresed on 1.5% agarose gel, stained with EtBr, and confirmed under UV light (UV).
도 7에서 보는 바와 같이 플라보피리돌과 TNF-α를 동시에 처리한 세포들에서 세포사멸의 한 특징이 되는 DNA 절단구조(laddering)가 상당히 일어남을 관찰하였다(도7 : M; 1Kb DNA marker, C; control(0.1% DMSO), D; 1μM doxorubicun, T;TNF-α를 10ng/㎖ 농도로 처리, F.P; 플라보피리돌을 표시된 농도로 처리, +; TNF-α와 플라보피리돌을 각각 10ng/㎖, 0.5μM농도로 처리).As shown in FIG. 7, it was observed that DNA laddering, which is one characteristic of apoptosis, occurred in cells treated with flavopyridol and TNF-α simultaneously (FIG. 7: M; 1Kb DNA marker, C; control (0.1% DMSO), D; 1 μM doxorubicun, T; treated with TNF-α at a concentration of 10 ng / ml, FP; treated with flavopyridol at the indicated concentrations, +; TNF-α with flabopyridol Each at 10 ng / ml and 0.5 μM concentration).
<실시예 8><Example 8>
쥐의 정상 섬유아세포주 래트2(rat2)에서 TNF-α와 플라보피리돌의 동시 처리 시 비독성(non-cytotoxic)효과 확인Simultaneous treatment of TNF-α with flavopyridol in rat normal fibroblast line rat2 confirmed non-cytotoxic effects
쥐의 세포주인 래트2(rat2) 세포를 60㎜ 배양접시에 5x105개를 접종한 후 24시간동안 실시예 1에서와 같은 조건에서 배양 후 TNF-α와 플라보피리돌을 각각 10ng/㎖, 0.5μM 농도로 각각 단독으로 혹은 동시 처리하여 6시간동안 배양하였다. 그 후 실시예 1에서와 동일방법으로 세포를 떼어내 고정시켜 FACScan 분석을 하였다.After inoculating 5 × 10 5 rat 60 cells into a 60 mm culture dish and incubating the cells under the same conditions as in Example 1 for 24 hours, TNF-α and flavopyridol were respectively 10 ng / ml, Each was incubated for 6 hours, either alone or concurrently, at a concentration of 0.5 μM. Thereafter, cells were detached and fixed in the same manner as in Example 1 for FACScan analysis.
암세포주에서와는 달리 정상 세포주인 래트2(rat2)에서는, 도 8에서 보는 바와 같이 TNF-α와 플라보피리돌을 동시에 처리하였음에도 전혀 세포사멸을 관찰하지 못하였다. TNF-α와 플라보피리돌을 암세포에 동시처리 하였을 때에만 세포사멸이 일어나는 것으로 보아 항암 특이성을 지님을 알 수 있었다(도8).Unlike in cancer cell lines, rat 2 (rat2), which is a normal cell line, showed no cell death at all, even though TNF-α and flavopyridol were simultaneously treated as shown in FIG. 8. Apoptosis occurred only when TNF-α and flabopyridol were treated simultaneously with cancer cells, indicating that they had anticancer specificity (FIG. 8).
이상에서 본 바와 같이 본 발명은 TNF-α와 플라보피리돌 또는 TRAIL 과 플라보피리돌의 조합에 의해 암세포에서 특이적으로 상승적 세포사멸을 촉진하는 세포사멸 유도 조성물을 제공하는 유용한 발명인 것이다.As described above, the present invention is a useful invention for providing apoptosis inducing composition that specifically promotes synergistic apoptosis in cancer cells by the combination of TNF-α and flabopyridol or TRAIL and flabopyridol.
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| KR1020010059058A KR20030026069A (en) | 2001-09-24 | 2001-09-24 | Apoptosis-inducing cancer-cell-specific composition by combination with TNF family protein and flavopiridol |
| AU2002334433A AU2002334433A1 (en) | 2001-09-24 | 2002-09-19 | Method and pharmaceutical composition for inducing cancer cell-specific apoptosis by combination with tnf family proteins and flavopiridol |
| PCT/KR2002/001780 WO2003028001A2 (en) | 2001-09-24 | 2002-09-19 | Method and pharmaceutical composition for inducing cancer cell-specific apoptosis by combination with tnf family proteins and flavopiridol |
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| ES2356880B8 (en) | 2009-08-21 | 2012-10-30 | Universidad De Zaragoza | LIPOSOMES COVERED WITH THE EXTRACELLULAR DOMAIN OF THE APO2L / TRAIL PROTEIN. |
| WO2016073913A1 (en) * | 2014-11-07 | 2016-05-12 | Tolero Pharmaceuticals, Inc. | Methods to target transcriptional control at super-enhancer regions |
| US9901574B2 (en) | 2015-04-20 | 2018-02-27 | Tolero Pharmaceuticals, Inc. | Predicting response to alvocidib by mitochondrial profiling |
| EP3298021B1 (en) | 2015-05-18 | 2019-05-01 | Tolero Pharmaceuticals, Inc. | Alvocidib prodrugs having increased bioavailability |
| EP3331510A4 (en) | 2015-08-03 | 2019-04-03 | Tolero Pharmaceuticals, Inc. | Combination therapies for treatment of cancer |
| WO2018094275A1 (en) | 2016-11-18 | 2018-05-24 | Tolero Pharmaceuticals, Inc. | Alvocidib prodrugs and their use as protein kinase inhibitors |
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| WO2020191326A1 (en) | 2019-03-20 | 2020-09-24 | Sumitomo Dainippon Pharma Oncology, Inc. | Treatment of acute myeloid leukemia (aml) with venetoclax failure |
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| US5750495A (en) * | 1996-03-26 | 1998-05-12 | The Regents Of The University Of California | Treatment of cystic disease with TNF-α |
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- 2001-09-24 KR KR1020010059058A patent/KR20030026069A/en not_active Application Discontinuation
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| US4980160A (en) * | 1986-10-16 | 1990-12-25 | Biogen, Inc. | Combinations of tumor necrosis factors and anti-inflammatory agents and methods for treating malignant and non-malignant diseases |
| US6087366A (en) * | 1996-03-07 | 2000-07-11 | The Trustees Of Columbia University In The City Of New York | Use of flavopiridol or a pharmaceutically acceptable salt thereof for inhibiting cell damage or cell death |
| US5750495A (en) * | 1996-03-26 | 1998-05-12 | The Regents Of The University Of California | Treatment of cystic disease with TNF-α |
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| WO2003028001A8 (en) | 2003-05-08 |
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