KR20110115298A - Anticancer supplement composition comprising opa3 protein or polynucleotide encoding opa3 as an effective ingredient - Google Patents

Abstract

The present invention relates to an anticancer adjuvant composition containing an OPA3 protein or a polynucleotide encoding the protein as an active ingredient. Specifically, the OPA3 protein of the present invention regulates the cleavage and binding mechanism of mitochondria in a cell. , OPA3 protein or apoptosis in cancer cells overexpressed OPA3 is more sensitive to apoptotic stimulation of cancer cell death, which has a significant synergistic effect on chemotherapy caused by apoptosis induction compared to the use of anticancer drugs alone. By using the polynucleotide encoding the composition as an anticancer adjuvant in combination with an anticancer agent, not only can be very effectively used in anticancer treatment, but also the side effect of overdose of the anticancer agent can be reduced by minimizing the dose of the anticancer agent.

Description

Anti-cancer supplement composition comprising OPA3 protein or polynucleotide encoding OPA3 as an effective ingredient}

The present invention relates to an anticancer adjuvant composition using an OPA3 protein or a polynucleotide encoding the same.

Despite the development of modern science and technology along with the development of medical technology, biotechnology and genetic engineering, cancer has been recognized as a serious disease that cannot be cured yet. Many methods such as incision, chemotherapy, radiotherapy, and incision have been developed to treat these cancers, but radiation therapy and incisions are only effective when the initial diagnosis is made. It is of no use and can only be treated with chemotherapy. Since chemotherapy introduced in cancer treatment since the 1940s has the advantage of being relatively easy to apply regardless of the timing of cancer, a lot of attention is now focused, and various anti-cancer chemotherapy agents are being developed.

However, the anticancer agents have a disadvantage in that when repeated long-term administration or cancer recurs, cancer cells lose the therapeutic effect by obtaining resistance to the anticancer agent. In addition, most anticancer drugs are effective by inhibiting the synthesis of nucleic acid in the cell or by directly binding to the nucleic acid and impairing its function. These anticancer drugs not only act selectively on cancer cells but also on normal cells, especially tissue cells with active cell division. Because of the damage, there are many side effects such as decreased bone marrow function, damage to the gastrointestinal tract mucosa, and hair loss. In order to suppress such cancer cell resistance to anticancer drugs, combined chemotherapy using drugs that have no cross-resistance and different mechanisms of action is used, rather than using a single drug anticancer agent. In addition, as a method for suppressing side effects of an anticancer agent, a method of developing a new anticancer agent having no side effects or using a conventional anticancer agent at a minimum effective concentration has been proposed.

Recently, studies on the blocking of various genes and proteins involved in apoptosis as targets of anticancer drugs for chemotherapy have been conducted. These studies began with the discovery that chemotherapy drugs used in chemotherapy over the past few years damage cancer cell genes and cause apoptosis, or programmed cell death, to differentiate the cells that are not killed. The discovery led to the discovery of signaling regulatory molecules.

Apoptosis (programmed cell death, apoptosis) is a mechanism that plays a very important role in the various life activities of the cell, the excess or insufficient apoptosis ischemia, neurodegeneration, autoimmunity, viral infection It is closely related to various diseases such as infection and tumor. Signaling of apoptosis is largely divided into intrinsic and extrinsic pathways. In the intracellular pathway, the Bcl-2 family protein family acts as a major regulator, and mitochondria play a role as a hub for signaling and regulation.

Mitochondria have been found to play an important role in apoptosis since the major components have been found to play an important role in cell death since the 1990s. Mitochondria are composed of outer and inner membranes and are dynamic organelles that constantly move, fusion, and divide. Mitochondria form a tubular network connected to each other, and their morphology and number are precisely controlled by the mitochondrial fusion-fission machinery. When apoptosis is induced, various apoptosis-regulating proteins, ERs, calcium ions and related proteins in the cytoplasm move to mitochondria, and mitochondria form fragments by mitochondria-shaping proteins. ) Will happen. Thereafter, the loss of the mitochondrial membrane potential and mitochondria outer membrane permeabilization (mitochondrial outer membrane P) is triggered by the release of various proteins in the mitochondria into the cytoplasm, nucleus aggregation and DNA fragmentation, and finally the cells die.

As proteins involved in mitochondrial binding, mitofusin 1 (mitofusin 1; Mfn1), mitofusin 2 (mitofusin 2; Mfn2), and Opa1 have been identified, and proteins involved in mitochondrial cleavage have been identified as Drp1 and Fis1. Recently, dysfunction of mitochondria has been found to play a central role in the development of various degenerative neurological diseases such as Charcot-Marie-Tooth 2A (CMT2A), Parkinson's disease and hereditary optic neuropathy. ought. CMA2A is characterized by dendritic degeneration of sensory and motor neurons caused by mutations in mitofusin2 (Mfn2) that are involved in muscle weakness and mitochondrial binding, and genetic autosomal dominant atrophy Optic atrophy (ADOA) is associated with vision loss due to degeneration of optic nerve and retinal ganglion cells. ADOA is mainly caused by mutations in optic atrophy 1 (OPA1), an essential protein for mitochondrial binding.

The OPA3 gene is responsible for autosomal, dominant genetic optic atrophy and cataracts, as well as Coseff's optic neutropy syndrome, also known as 3-methylglutaconic aciduria (MGA) type III. Patients with the disease are found in the form of mutations. To date, the OPA3 protein is expected to be located in the mitochondrial lining based on mitochondrial lining (mitochondrial lining) proteomics and sequence analysis. OPA3 mutant mice induced with homogenous ENU (OPA3 L122P) are characterized by 3-methylglutaconic acidemia type 3, which shows decreased initiation function, loss of retinal ganglion cells, and optic nerve degeneration. Although OPA3 is expected to play an important role in optic nerve atrophy and neurodegeneration, the basic process of OPA3-linked development in the function and regulation of OPA3 protein has not been well described, and the effect of OPA3 on anticancer effects Research is not being done.

In this regard, the present inventors found that OPA3 protein is a binding protein of the mitochondrial outer membrane and plays an important role in regulating the division and binding mechanisms of mitochondria in cells, and through this mitochondrial regulation mechanism, apoptotic stimulation in OPA3 overexpressed cancer cells. OPA3 protein or polynucleotide encoding the anti-cancer therapy supplement composition of the anticancer therapy composition by confirming that the killing of cancer cells by the more sensitive to have a significant synergistic effect on chemotherapy due to apoptosis induction compared to the case of using an anticancer agent alone The present invention has been completed by revealing that it can be usefully used as an active ingredient.

It is an object of the present invention to provide an adjuvant composition for anticancer treatment, or a method for enhancing cell death of cancer cells of an anticancer agent using the adjuvant composition.

The present invention provides an anticancer adjuvant composition comprising an OPA3 (optic atrophy 3) protein or a polynucleotide encoding the protein as an active ingredient.

The present invention also provides a method for enhancing apoptosis of cancer cells of an anticancer agent, comprising administering an OPA3 protein or a polynucleotide encoding the protein together with an anticancer agent in a subject.

In the present invention, the OPA3 protein regulates the division and binding mechanism of the intracellular mitochondria, thereby making the death of cancer cells by apoptotic stimulation more sensitive to the OPA3 overexpressed cancer cells, when using an anticancer agent alone. Compared with the anticancer agent, OPA3 protein or polynucleotide encoding the same can be used in combination with an anticancer agent. By minimizing the dose, side effects from overdose of anticancer drugs can be reduced.

Figure 1 shows the results of confirming the effect of OPA3 in cervical cancer cells (HeLa cells) in the mitochondrial form:
A: Immunostaining of OPA3-myc and Tom 20 in mitochondria;
B: graph showing the distribution of mitochondrial morphology in control cells and cells transformed with OPA3-myc;
C: Figure showing mitochondrial fluorescent recovery effect before and after bleaching of control cells and OPA3-myc transformed cells in FRAP experiment;
D: Graph showing mitochondrial fluorescent recovery effect of control cells and OPA3-myc transformed cells in FRAP experiment;
E: Western blot results showing inhibition of expression of OPA3 protein by OPA3 siRNA treatment;
F: graph showing distribution of mitochondrial morphology by OPA3 siRNA treatment;
G: mitochondrial morphology by OPA3 siRNA treatment; And
H: Graph showing mitochondrial fluorescent recovery effect of control siRNA and OPA3 siRNA cells in FRAP experiment.
Figure 2 shows the results of correlation between mitochondrial cleavage factors (DRP1 and FIS1OPA3) and OPA3 in mitochondrial cleavage:
A: mitochondrial morphological changes through co-expression of DRP1 K38A and OPA3 protein;
B: graph showing the distribution of mitochondrial morphology in cells via co-expression of DRP1 K38A and OPA3 protein;
C: Western blot results showing inhibition of expression of Drp1 and Fis1 proteins intracellularly by Drp1 shRNA and Fis1 shRNA;
D: Figure showing changes in mitochondrial morphology in cells by Drp1 shRNA and Fis1 shRNA;
E: Figure showing intracellular mitochondrial morphology by control siRNA treatment; And
F: Figure showing changes in intracellular mitochondrial morphology by OPA3 siRNA treatment.
3 is a diagram showing the results of confirming the effect of apoptosis sensitivity on the staurosporin stimulation of OPA3 in cervical cancer cells:
A: A diagram showing cytochrome c release in mitochondria of cells transformed with OPA3 and OPA3 (G93S) without Staurosporine stimulation;
B: Figure showing cytochrome c release in mitochondria of cells transformed with OPA3 and OPA3 (G93S) under Staurosporine stimulation; And
C: Graph showing cytochrome c release in mitochondria of cells transformed with OPA3 and OPA3 (G93S) with and without Staurosporine stimulation.
Figure 4 shows the results of confirming the effect of enhancing the sensitivity of apoptosis to TRAIL stimulation of OPA3 and the effect of inhibiting cell death by OPA3 siRNA treatment in cervical cancer cells:
A: Figure showing mitochondrial morphology and cytochrome c release in TRAIL-stimulated state on OPA3;
B: graph comparing cytochrome c release in TRAIL stimulated state to OPA3;
C: Figure showing mitochondrial morphology change and Bax position change in TRAIL stimulated state in OPA3;
D: Graph showing the change in position of Bax in TRAIL stimulus state to OPA3;
E: Western blot analysis of protein cleavage of Caspase 3, PARP1 and actin in TRAIL-stimulated state on OPA3; And
F: Graph showing the effect of inhibition of cytochrome c release over time by OPA3 siRNA treatment.
5 is a diagram showing the results of confirming the position in the mitochondria of OPA3.
A: Western blot analysis showing the location of OPA3, Tom20, OPA1 and PLC-γ in the cytoplasm and mitochondria;
B: Western blot analysis showing the location of FLAG-OPA3, Tom20, OPA1 and Drp1 in the cytoplasm and mitochondria;
C: Figure showing the location of OPA3-myc and cytochrome c in HeLa cells and Cos7 cells;
D: Figure confirming OPA3, Tom 20, OPA1 protein degradation by Protease K treatment in HeLa cells by Western blot analysis;
E: OPA3-YFP, Tom 20, OPA1 protein degradation by proteinase K treatment in OPA3-YFP transformed cells was confirmed by Western blot analysis;
F: After trypsin treatment of mitochondria reacted with [ ³⁵ S] -labeled OPA3, the supernatant and pellets were separated and confirmed by Western blot analysis of 35S-labeled OPA3;
G: Western blot analysis of the locations of endogenous OPA3, FLAG-OPA3 and Drp1 in membrane fractions treated with 0.1 M sodium carbonate; And
H: Figure showing the results of the differential permeation test to confirm the position of OPA3 in the mitochondrial envelope.
Figure 6 shows the results of confirming mitochondrial cleavage by the hydrophobic portion of the OPA3 protein.
A: table showing the type and structure of deletion mutations of the OPA3 protein;
B: mitochondrial morphology change of deletion mutation of OPA3 protein;
C: graph showing hydrophobic portion of OPA3 protein amino acid;
D: graph comparing the distribution of mitochondrial forms of normal OPA3 and OPA3 hydrophobic partial deletion mutants;
E: mitochondrial morphology of normal OPA3 and OPA3 hydrophobic partial deletion mutants; And
F: Figure confirmed by Western blot analysis of the results of proteinase K treatment on normal OPA3 and OPA3 hydrophobic partial deletion mutants.
Figure 7 shows the results of comparing the morphological changes of mitochondria between OPA3 splicing variants.

Hereinafter, the present invention will be described in detail.

The present invention provides an anticancer adjuvant composition comprising an OPA3 (optic atrophy 3) protein or a polynucleotide encoding the protein as an active ingredient.

The OPA3 protein may be a protein originating from a mammal, preferably a human, most preferably a protein having the characteristics set forth in SEQ ID NO: 1, and the OPA3 protein used in the present invention may be genetically engineered from the sequence. (Park et al., J. Biol. Chem. 274: 16673-16676, 1999).

The OPA3 protein is preferably characterized in that it is administered with Staurosporine (Staurosporine) or TRAIL (TNF-related apoptosis-inducing ligand), but is not limited thereto, and in general, it is an anticancer agent having an anticancer effect due to cell death Either can be used.

The cancer may include lung cancer, stomach cancer, colon cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal muscle cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma Cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma And bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, solid cancer, such as brainstem glioma and pituitary adenoma, and more preferably May be any one selected from the group consisting of gastric cancer, lung cancer, uterine cancer, skin cancer, liver cancer, breast cancer, ovarian cancer and bladder cancer, and most preferably may be uterine cancer. Not limited.

In an embodiment of the present invention, a recombinant expression vector comprising the OPA3 gene is prepared by transforming OPA3 cDNA (NP-079412) described by the amino acid sequence of SEQ ID NO: 1 into a vector by PCR, and transforming the same into a cell. In order to confirm whether the OPA3 protein changes the mitochondrial morphology in cells, immunostaining was performed, and as a result, OPA3 (OPA3-myc), which was myc-tagged at the C-terminus expressed in HeLa cells, was a mitochondrial marker protein. HeLa cells, which were co-located with Tom 20 (see FIG. 1A) and transformed with OPA3-myc, frequently showed abnormal mitochondrial morphology following extensive mitochondrial cleavage (see Aii in FIG. 1), while myc- as a control. Most of the cells transformed with the vector had a mitochondria of normal form, confirming that each cell had a short or long form of mitochondria (FIG. 1B). It was confirmed that OPA3 protein induces cleavage of mitochondria.

In addition, fluorescence recovery after photobleaching (FRAP) was performed to quantify OPA3-induced mitochondrial cleavage in HeLa cells and to measure mitochondrial binding. As a result, the fluorescence of the mitochondrial protein (mito-YFP) in control cells was determined. Fast recovery in the bleached portion, while fluorescence recovery of the bleached portion in cells transformed with OPA3-myc was significantly inhibited (see C and D in FIG. 1), whereby the high expression of OPA3 protein resulted in cleavage of mitochondria. It reaffirmed that it induces anger.

In addition, to more clearly determine the effect of OPA3 on mitochondrial morphology, a specific small interfering RNA (siRNA; 156i, 157i and 158i) was devised and transformed to significantly reduce the amount of OPA3 protein resulting in OPA3. It was confirmed that siRNA inhibited the expression of endogenous OPA3 protein (see E in FIG. 1), and control siRNA showed a mitochondrial morphology elongated at ˜40% of mitochondria in transformed HeLa cells, and a small fraction (6.5 ± 2.2 %) Shows a short morphology (see F and G in FIG. 1), while knocking down the OPA3 protein confirms that most of the HeLa cells knocked down OPA3 have longer coronary mitochondria. It was confirmed that the morphology of the mitochondria was changed long.

In another embodiment of the present invention, to determine whether OPA3 induced mitochondrial cleavage is dependent on the activity of dynamin-related protein (DRP) 1, known as mitochondrial cleavage factor, OPA3 (OPA3-YFP). ) And DRP1 K38A (myc-DRP1 K38A) were co-transformed together, and the mitochondrial morphology was observed by immunostaining. As a result, mito-YFP transformed cells were compared with the mitochondria. High levels of linked mitochondria were observed and cells transformed with myc-DRP1 k38A did not induce mitochondrial cleavage, whereas cells transfected with OPA3-YFP had or without myc-DRP1 k38A. It was confirmed to induce (see A and B of Figure 2).

In addition, when knocking down DRP1 or Fission (FIS) 1 using RNA interference, the degree of protein expression and mitochondrial morphological changes were observed to determine whether there was an effect on mitochondrial cleavage by OPA3. As a result, when the Drp1 shRNA / Fis1 shRNA was introduced into the cells, it was confirmed by Western blot that the expression of each protein was inhibited (Fig. 2C), when DRP1 / Fis1 shRNA and OPA3-YFP cotransformed, It was confirmed that it did not affect the cleavage of mitochondria induced by OPA3 (see D in FIG. 2).

In addition, to confirm the effect of OPA3 knockdown on mitochondrial cleavage mediated by DRP1 / FIS1, cotransformation of OPA3 siRNA with YFP-DRP1 or YFP-FIS1 in HeLa cells, followed by morphological changes in mitochondria. As a result, when co-transformation of the control siRNA and DRP1 / FIS1, large spot-like YFP-DRP1 was increased at the cleavage site of mitochondria, and extensive mitochondrial cleavage was caused by overexpression of YFP-FIS1. Was observed (E of FIG. 2). In contrast, knocking down OPA3 by cotransforming OPA3 siRNA and DRP1 / FIS1 significantly induced cytoplasmic distribution of very small spot YFP-DRP1 and reduced mitochondrial cleavage induced by FIS1. It can be seen (see F of FIG. 2) that the OPA3 protein is a cleavage factor related to mitochondrial cleavage independently of DRP1 / FIS1.

In addition, in order to confirm that exogenous expression of OPA3 can lead to cell death with or without apoptotic stimulation, OPA3-YFP or mito-YFP were transformed into HeLa cells, and cytochrome c release and mitochondrial double lining The intercalation potential was confirmed, and as a result, cells with mitochondria cleaved due to OPA3 overexpression showed normal cytochrome c distribution (see FIG. 3A), and TMRE fluorescence intensity was concentrated, resulting in OPA3 Overexpression alone was not sufficient to induce spontaneous apoptotic cell death.

In addition, in a specific embodiment of the present invention, in order to confirm the apoptotic effect of OPA3 in the presence of apoptotic stimulation, the degree of apoptotic cell death was changed by treating OPA3 in the state of inducing apoptosis with staurosporine. As a result, when treated with staurosporine (STS) in the outpatient expression of OPA3, OPA3 compared to cells transfected with Mito-YFP (22.6 ± 2.1%, B and C in Fig. 3) In the cells transformed with -YFP (41.0 ± 1.2%), the redistribution of cytochrome c from the mitochondria to the cytoplasm was remarkably increased, indicating that the sensitivity of OPA3-induced mitochondrial cleavage when subjected to apoptotic stimulation with STS It was confirmed that is increased. However, cytochrome c was identified in the cytoplasm of HeLa cells transfected with OPA3 mutation, OPA3 (G93S) of the patient, even without external apoptotic stimulation (see A to C in FIG. 3), and overexpression of wild type OPA3. Compared to mitochondrial cleavage induced by, OPA3 (G93S) -YFP transformed cells induced a wide range of mitochondrial cleavage.

In addition, OPA3 was treated with TRAIL (TNF-related apoptosis-inducing ligand) to induce apoptosis and confirmed the degree of change in apoptotic cell death. ), The redistribution of cytochrome c into the cytoplasm in the mitochondria compared to mito-YFP-transformed cells (43.2 ± 1.2%; A and B in FIG. 4) in OPA3-YFP-transformed cells More was induced (85.8 ± 1.2%; see A and B in FIG. 4). Consistent results were also obtained from experiments confirming the migration of Bax protein (see C and D in FIG. 4), caspase-3 activity and cleavage of PARP (see E in FIG. 4), an apoptosis regulatory protein of mitochondria.

In addition, cytochrome c released by STS treatment in OPA3-knockdown HeLa cells was measured to demonstrate a clear role of OPA3 in apoptosis sensitivity. OPA3 knockdown significantly reduced the release of cytochrome c by STS, confirming that the destruction of OPA3 inhibited apoptotic cell death (see FIG. 4F).

Thus, OPA3 overexpression alone was not sufficient to induce spontaneous apoptotic cell death, but OPA3 was found to significantly increase the sensitivity of apoptosis by apoptotic stimulation such as STS or TRAIL.

In addition, in order to confirm the position of the OPA3 in the mitochondria, the organelles were separated, and as a result, both endogenous OPA3 or externally introduced OPA3 were identified in the mitochondrial fraction, but not in the cytoplasmic fraction. In addition, the position of OPA3-myc and FLAG-OPA3 in HeLa cells (C of FIG. 5, see above) and COS 7 cells (FIG. 5, see below) are mitochondrial proteins such as cytochrome c and Tom 20. It was confirmed that the.

In addition, in order to determine whether OPA3 is located in the mitochondrial outer membrane or the mitochondrial inner membrane, the mitochondrial fractions were identified by treatment with Protease K. As a result, the mitochondrial inner membrane protein OPA1 was produced only when osmotic shock was applied. While degraded by K, Tom 20, an endogenous OPA3 and mitochondrial outer membrane protein, was degraded by proteinase K even without osmotic shock, thereby confirming that OPA3 is located in the mitochondrial outer membrane (FIG. 5). See D).

In addition, mitochondrial transport experiments were performed to confirm the transport of mitochondria in the ATP regeneration system. As a result, [ ³⁵ S] was labeled when trypsin was treated to mitochondria reacted with OPA3 labeled [ ³⁵ S]. It was confirmed that OPA3 was not detected, and similar results were obtained in experiments using OPA3-YFP (see FIG. 5E), FLAG-OPA3 and OPA3-myc (see FIG. 5F).

In addition, in order to check whether OPA3 decomposed by Proteinase K (Sigma) is located at the periphery of the mitochondrial outer membrane and is interrupted, a high pH sodium carbonate that can extract proteins attached to the mitochondria is used. As a result, DRP1, known as a peripheral protein present on the surface of the mitochondrial outer membrane, was obtained by treating with alkali, while endogenous OPA3 and FLAG-OPA3 were treated with a control buffer solution and 0.1 M sodium carbonate (Na ₂ CO ₃ ) together. Precipitated in the fractions (see G in Figure 5), through which it was confirmed that OPA3 is an intramembrane protein located in the mitochondrial outer membrane.

In addition, it is important to understand the topology of OPA3 in the mitochondrial envelope to explain the role of OPA3 in its function in the mitochondria, for this purpose, digitonin, which selectively destroys various concentrations of plasma membrane and mitochondrial envelope, Differential permeabilization techniques were performed using Calbiochem, and as a result, in HeLa cells treated with 5 ug / ml of digittonin (FIG. 5H), Tom 20 exposed to the cytoplasm was immunostained, Cytochrome c located in the mitochondrial interstitial space was not detected. When HeLa cells were treated with 12 ug / ml of digitone, both cytochrome c and Tom 20 were detected. After treatment with 5 ug / ml of Digitonin, OPA3-myc protein in mitochondria was detected when using an antibody against a myc tag bound to the C-terminus, but at the C-terminus of OPA3 exposed to the cytoplasm under the same conditions. Identified cytochrome c was not detected. FLAG-OPA3 immunostaining and cytochrome c immunostaining by antibodies to FLAG bound to the N-terminus were positive only when treated with 12 ug / ml of digitotonin.

Thus, these results confirm that the N-terminus and C-terminus of OPA3 are intercalated into the mitochondrial outer membrane due to exposure to the intercellular space and cytoplasm.

In addition, in order to confirm the mitochondrial target sequence of OPA3, an experiment was performed to locate the NRIK deletion mutation of OPA3 in which YFP was bound to the N-terminus. It was confirmed that located in the mitochondria in HeLa cells using, OPA3 (△ NRIK) -YFP also was found to be located in the mitochondria unlike expected, through which, N-terminal NRIKE sequence is not important to the mitochondrial target of OPA3 It was confirmed.

In addition, to identify domains that target mitochondria in OPA3 proteins, the predictions according to TMHMM server (www.cbs.dtu.dk), DAS membrane potential prediction server (www.sbc.su.se) and Kyte-Doolittle hydropathy plot server Several deletion mutations were made, including mutations lacking a portion estimated to be hydrophobic, consistent with amino acids 83-102 represented by the resulting membrane-spanning fragments, which were transformed into cells and immunostained for the cells. As a result, C-terminally reduced OPA3-YFP mutants, including OPA3 (Δ83-102), OPA3 (1-30) and OPA3 (1-105), are located in the mitochondria, but PA3, OPA3 (20-179) And N-terminal deletion mutations in OPA3 (31-179) were identified in the cytoplasm (A and B in Figure 6). Thus, it was confirmed that 30 residues at the N-terminus of OPA3 are necessary parts for mitochondrial targeting.

In addition, in order to identify the functional domain of OPA3 in the mitochondrial form, the prepared mutations were transformed into cells and immunostaining was performed on the cells. As a result, overexpression of OPA3-YFP significantly induces mitochondrial cleavage, induces mitochondrial cleavage even when OPA3-YFP is expressed low, and the C-terminus of OPA3 in which the hydrophobic moiety (residues 83-102) remains intact Cells transformed with deleted mutants also exhibited excessive mitochondrial cleavage. In contrast, cells transformed with OPA3 (Δ83-102), an OPA3 deletion mutant lacking hydrophobic moieties, did not show morphological changes in mitochondria (see FIGS. 6D and 6E). Although OPA3 (Δ83-102) -YFP was located in the mitochondria, the protease K analysis revealed that OPA3 (Δ83-102) -YFP failed to be located in the mitochondrial outer membrane.

Thus, the results indicate that 83-102 residues of OPA3 are necessary for mitochondrial cleavage and mitochondrial outer membrane localization of OPA3.

In addition, human OPA3 has two splicing variants (v1, NP_001017989 and v2, NP_079412) having an amino acid identity of 81.7%, and the 93 residues (G93) of OPA3 variant 2 (NP_079412) used in the present invention. Is mutated in OPA3 of ADOA patients but not in OPA3 variant 1 (NP_001017989), so that expression of variant 2 (OPA3-v2) shows the same phenotype as that of OPA3 variant 1 (OPA3-v1). Immunostaining was performed. As a result, OPA3-v1-YFP appeared to be colocated with Tom 20 in the mitochondria and showed excessive mitochondrial cleavage (see FIG. 7).

Thus, the results confirm that mutation of the 93rd residue of OPA3 is not essential for mitochondrial cleavage.

The composition according to the invention may be formulated in the form of a single composition or the anticancer agent and the OPA3 protein in the form of a separate composition, preferably in the form of a separate composition. . In the composition comprising the OPA3 protein according to the present invention, the anticancer agent and the OPA3 protein of the present invention may be administered simultaneously, separately or sequentially. For example, if the components included in the composition of the present invention are in a single composition, they may be administered simultaneously. If the components are not in a single composition, one component may be administered before or after the other component is administered. And / or together with other ingredients. The order of administration of the composition according to the present invention, i.e., at which point in time, at the same time, individually or sequentially, may be determined by the physician or expert. This order of administration can vary depending on many factors. Preferably, for compositions of the invention that are not formulated in a single dosage unit, OPA3 protein, a composition of the invention, is administered first and the anticancer agent is administered at timed intervals. More preferably, the OPA3 protein is administered daily from the start of the administration, and the anticancer agent is administered at the start of the administration every two to three days after the start of administration. For example, OPA3 protein is administered once daily from the start of administration, and the anticancer agent is administered 5 times on the start of administration, 2 days, 5 days, 8 days and 11 days after administration.

The composition according to the present invention may be administered systemically or locally in the above-described order of administration, the route of administration may be parenteral administration, parenteral administration, for example, intravenous, intraperitoneal, subcutaneous It may be administered to the buccal, rectal, skin, nasal, tracheal and bronchus or inhaled into the lungs. Preferred modes of administration are parenteral administration, more preferably intravenous, intraperitoneal injection and subcutaneous administration. In addition, the compositions according to the invention can be formulated in a suitable form by further comprising a pharmaceutically acceptable carrier for administration in such a manner. Pharmaceutically acceptable carriers include inert diluents or fillers, water and various organic solvents. The composition may, if necessary, contain additional ingredients such as flavoring agents, binders, excipients, and the like. The composition according to the invention may comprise a carrier for oral administration, for example lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. Additionally, carriers for parenteral administration may include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutical carriers may be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). Preferably, the compositions according to the invention may be formulated in parenteral dosage forms, for example in the form of injectable preparations. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, it may be formulated for injection by dissolution in saline or buffer. In particular, the OPA3 protein included in the composition according to the present invention may be prepared as a fine emulsion by mixing lipids with PEG or olive oil or sunflower oil as disclosed in US Pat. No. 5,478,860. In addition, the OPA3 protein included in the composition according to the present invention may be formulated in the form of a general protein preparation. For example, the OPA3 protein solution may be formulated in a liquid form comprising freeze-dried and dissolved in a buffer solution and a stabilizer as described above. However, the formulation, route of administration, and method of administration are not particularly limited as long as the effects of the present invention are shown.

The OPA3 protein included in the composition according to the present invention may be administered once or several times a day at a dose of about 10 mg / kg to 50 mg / kg, and may be administered once or several times at the start of administration, and then from 2 days to the start of administration. It may be administered once or in divided doses every three days. Preferably, the OPA3 protein may be administered once daily at a dose of about 25 mg / kg / day to 50 mg / kg / day, and once daily at intervals of two to three days from the start of administration. Can be. However, the final dose and frequency of administration may be adjusted as necessary to the clinical judgment, taking into account the characteristics of the drug, its mode and route of administration, the patient's condition, age, weight, sex, the nature and severity of the symptoms, and the like. . On the other hand, when an anticancer agent and OPA3 protein, which is a composition of the present invention, are administered in combination, the dosage of each component is about the usual dosage when administered alone for the treatment of cancer in consideration of the synergistic effect. The amount can be reduced to 70 to 80%.

The present invention also provides a method for enhancing apoptosis of cancer cells of an anticancer agent, comprising administering an OPA3 protein or a polynucleotide encoding the protein together with an anticancer agent in a subject.

The OPA3 protein may be a protein originating from a mammal, preferably a human, most preferably a protein having the characteristics set forth in SEQ ID NO: 1, and the OPA3 protein used in the present invention may be genetically engineered from the sequence. It can be prepared by the method (Park et al., J. Biol. Chem. 274: 166673-166676, 1999).

The anticancer agent is preferably staurosporine or TRAIL (TNF-related apoptosis-inducing ligand), but is not limited thereto. In general, any anticancer agent having an anticancer effect due to cell death may be used.

The cancer cells are lung cancer, stomach cancer, colon cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal muscle cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma Cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma And bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, solid cancer, such as brainstem glioma and pituitary adenoma, and more preferably May be any one selected from the group consisting of gastric cancer, lung cancer, uterine cancer, skin cancer, liver cancer, breast cancer, ovarian cancer and bladder cancer, and most preferably may be uterine cancer It is not limited to this.

The present inventors found that OPA3 protein is a binding protein of the mitochondrial outer membrane (the mitochondrial outer membrane), and plays an important role in regulating the division and binding mechanisms of mitochondria in cells, and through these mitochondrial regulatory mechanisms, OPA3 is expressed in cancer cells overexpressed. It was confirmed that the death of cancer cells by apoptotic stimulation was more sensitive, resulting in a significant synergistic effect on chemotherapy due to apoptosis induction compared to the case of using each component alone.

Therefore, the OPA3 protein of the present invention or a polynucleotide encoding the protein can be administered together with an anticancer agent in an individual to enhance cell death of the anticancer agent against cancer cells.

 Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Preparation Examples.

< Example  1> OPA3  Preparation of Recombinant Expression Vector Containing Genes

OPA3 cDNA (NP-079412), described as amino acid sequence of SEQ ID NO: 1, was amplified by PCR, digested with Eco R1 and Sal 1 restriction enzymes, and then pFLAG-CMV-6A plasmid (purchased from Sigma; then FLAG- Ligation was performed on OPA3) and pCMV 3 Taq4B plasmid (purchased from Stratagene; then described as OPA3-myc). Wild type OPA3, OPA3 deletion mutant and OPA3 variant 1 (NP_001017989; hereinafter described as OPA3-v1) were amplified using specific primers, digested with restriction enzymes, and then digested with pEYFP-N1 plasmid (purchased from Clontech; then OPA3 -LFP and OPA3-YFP mutants) were then used to prepare recombinant expression vectors comprising the OPA3 gene. Wild-type and mutant OPA3 nucleic acid sequences inserted into the vectors were confirmed by DNA sequencing, and Mito-YFP and mito-DsRed (purchased from Clontech) were used as mitochondrial controls.

< Experimental Example  1> OPA3 Mitochondrial morphology induced by

<1-1> OPA3 Changes in Mitochondrial Cells Induced by Overexpression of

In order to confirm that OPA3 changes the intracellular mitochondrial morphology, HeLa, COS 7 and 293 cells were transformed with recombinant expression vectors containing the OPA3 gene, and then mitochondrial morphology was observed by immunostaining. Specifically, HeLa cells and COS-7 cells were cultured in DMEM medium added with 10% heat-inactivated fetal bovine serum, 100 u / ml penicillin and 100 ug / ml Streptomycin (purchased from Invitrogen), Intracellular transformation of the recombinant vector was performed using Effectene reagent (purchased from Qiagen) and FuGene 6 (purchased from Roche), and immunostaining was performed using 4% paraform of cells cultured on a 2-well chamber slide. Fix with aldehyde (paraformaldehyde) at room temperature for 15 minutes, permeate for 15 minutes at room temperature with buffer solution added 0.15% Triton X-100 to PBS, and then add 3% bovine serum albumin to PBS The solution was blocked for 45 minutes at room temperature. The slides were reacted with anti-Tom20 (purchased from BD science) or anti-myc antibody (purchased from Sigma) with the primary antibody, washed with PBS buffer solution, and goat anti-mouse IgG conjugated with Alexa Fluor 488 or 594. Antibody (purchased from Molecular probe) or goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 was reacted with secondary antibody (purchased from Molecular probe). The slides were then observed using a Zeiss LSM 510 confocal microscope, and the excitation wavelengths of YFP, Alexa Fluor 594 and Alexa Fluor 488 were measured at 514, 594 and 488 nm, respectively.

As a result, it was confirmed that myc-tagged OPA3 (OPA3-myc) at the C-terminus expressed in HeLa cells was co-located with the mitochondrial marker protein Tom 20 (FIG. 1A). In addition, HeLa cells transformed with OPA3-myc frequently showed abnormal mitochondrial morphology following extensive mitochondrial cleavage (FIG. 1Aii), while most of the cells transformed with myc-vector as a control showed normal mitochondria. It was confirmed that each cell had a short or long form of mitochondria (Fig. 1B). In addition, in the cells transformed with the control myc-vector, the cleavage of the mitochondria was confirmed to be less than 5%, whereas the cells transformed with the OPA3-myc vector were mostly cleaved mitochondria.

<1-2> OPA3 Induced by Truncated  Mitochondria's Combined Recovery Effect

OPA3-induced mitochondrial cleavage was quantified in HeLa cells, and fluorescence recovery after photobleaching (FRAP) was performed to measure mitochondrial binding. Specifically, each of myc-binding vectors or OPA3-myc vectors was transformed with mito-YFP in cells cultured on a two well chamber slide (Nunc). After 15 hours, each cell was imaged using an LSM510 Meta imaging station (Carl Zeiss Micro Imaging, Inc.). A small portion of the same size (white circle, ROI) was photobleached using a 30.0 mV argon laser set to mito-YFP-expressing cells until the fluorescence intensity disappeared at that site. YFP fluorescence recovery was monitored. Fluorescence intensity adjusted the intensity of the ROI in the first image of the series and measured the fluorescence intensity recovery rate. The results are expressed as the mean ± standard deviation of the experiment with 70 cells per treatment group.

As a result, the fluorescence of the yellow fluorescent protein (mito-YFP) targeted to the mitochondrial matrix in the control cells rapidly recovered in the bleached portion of the mitochondria, whereas the fluorescent recovery of the bleached portion was significantly inhibited in OPA3-myc transformed cells. (C and D in FIG. 1).

Therefore, it was confirmed that large expression of OPA3 protein induces cleavage of mitochondria.

<1-3> OPA3  Small interference RNA ( small interfering RNA Change in mitochondrial morphology

To more clearly identify the effect of OPA3 on mitochondrial morphology, three specific small interfering RNAs (siRNAs: 156i, 157i and 158i) were designed and knocked down on endogenous OPA3 protein. Later, morphological changes of mitochondria were observed. Specifically, the OPA3 siRNA and the negative control RNAi were prepared using the Invitrogen Stealth RNAi system, and the target sequences of the OPA3 siRNA prepared through the 5'-AGCAAGCCGCTTGCCAACCGTATTA-3 '(# 156) and 5'-GCCGAAGCGAGTTCTTCAAGACCTA-3' (# 157) and 5′-TCACTGGGTGGAGATGCGGACCAAG-3 ′ (# 158), the siRNAs were transformed into cells and after one day, the medium was exchanged and incubated for an additional 2 days before further experiments. Identification of the reduced OPA3 protein due to the siRNA was analyzed by Western blot using rabbit anti-OPA3 polyclonal antibody to the lysate of cells transformed with the recombinant vector containing the OPA3 gene, SDS for Western blot analysis -PAGE was carried out using a 4-12% polyacrylamide gradient gel (NuPAGE, Invitrogen), and the isolated protein was nitrocellulose or polyvinylidene fluoride (PVDF). Moved to the membrane.

As a result, compared with the control siRNA transformant, in the cells transformed with OPA3 siRNA, the amount of expressed OPA3 protein was significantly reduced, and it was confirmed that the expression of OPA3 was inhibited by OPA3 siRNA (Fig. 1). E). Control siRNA transformed HeLa cells showed a long mitochondrial morphology at ˜40% of the mitochondria, with a small fraction (6.5 ± 2.2%) showing a short morphology (F and G in FIG. 1), while most OPA3 knocked down. Knocked down HeLa cells had longer tubular mitochondria. In addition, fluorescence recovery in the bleached portion was more pronounced and faster in OPA3 RNAi cells than in the control siRNA transformants.

Therefore, knockdown of OPA3 protein was confirmed to change the morphology of mitochondria long.

< Experimental Example  2> mitochondria In truncated  Mitochondrial fission factor ( DRP1  And FIS1OPA3 ) And OPA3

<2-1> DRP1 K38A Wow OPA3  Mitochondrial Morphology Changes through Co-expression of Proteins

To determine if mitochondrial cleavage induced by OPA3 is dependent on the activity of dynamin-related protein (DRP) 1, known as mitochondrial cleavage factor, OPA3 (OPA3-YFP) tagged with YFP at the C-terminus And co-transformed DRP1 K38A (myc-DRP1 K38A), a dominant negative mutant myc tagged at the N-terminus, and then immunostained as in the method of Experimental Example <1-1>. The mitochondria were observed.

As a result, the mito-YFP transformed cells showed higher levels of interconnected mitochondria than the cleaved mitochondria, and the cells transformed with the dominant negative mutant myc-DRP1 k38A did not induce mitochondrial cleavage. In contrast, OPA3-YFP-transformed cells were found to induce mitochondrial cleavage, with or without myc-DRP1 k38A (FIGS. 2A and 2B).

<2-2> OPA3 in Mediated  Mitochondria In truncated DRP1 Of FIS1  Knockdown knockdown ) Effect

When knocking down DRP1 or Fissionn (FIS) 1 using RNA interference, co-transformation of OPA3-YFP and DRP1 / FIS1 shRNA was performed to determine whether it affected mitochondrial cleavage by OPA3. The degree of protein expression was compared by Western blot analysis, and immunostaining was performed in the same manner as in Experimental Example <1-1>, and the morphological changes of mitochondria were observed.

As a result, when the Drp1 shRNA / Fis1 shRNA was introduced into the cells, it was confirmed by Western blot that the expression of each protein was inhibited (Fig. 2C), when DRP1 / Fis1 shRNA and OPA3-YFP cotransformed, It was confirmed that it did not affect the cleavage of mitochondria induced by OPA3 (FIG. 2D).

<2-3> DRP1 Of FIS1 in Mediated  Mitochondria In truncated OPA3  Knockdown knockdown ) Effect

In order to confirm the effect of OPA3 knockdown on mitochondrial cleavage mediated by DRP1 / FIS1, co-transformation of OPA3 siRNA and YFP-DRP1 or YFP-FIS1 together in HeLa cells, the above Experimental Example <1- Immunostaining was performed as in the method of 1>, and the morphological changes of the mitochondria were observed.

As a result, when co-transforming the control siRNA with DRP1 / FIS1, YFP-DRP1 of large and small spots was increased at the cleavage site of mitochondria, and extensive mitochondrial cleavage occurred by overexpression of YFP-FIS1. Observation was made (E of FIG. 2). In contrast, knocking down OPA3 by cotransforming OPA3 siRNA and DRP1 / FIS1 significantly induced cytoplasmic distribution of very small spots of YFP-DRP1 and reduced mitochondrial cleavage induced by FIS1 ( 2 F).

Thus, it can be seen that OPA3 protein is a cleavage factor related to mitochondrial cleavage independently of DRP1 / FIS1.

< Experimental Example  3> OPA3 of Apoptotic  Sensitivity enhancement effect on stimuli

<3-1> Apoptotic  Without stimulation OPA3 of Apoptotic  effect

It is well known that morphological changes in mitochondria are closely associated with susceptibility to cell death. To confirm that extraneous expression of OPA3 can lead to cell death with or without apoptotic stimulation, OPA3-YFP or mito-YFP were transformed into HeLa cells, cytochrome c release and transmembrane intermembrane potential of mitochondria (potential) was confirmed by performing immunostaining in the same manner as in Experimental Example <1-1>.

As a result, cells with mitochondria cleaved due to OPA3 overexpression showed a normal cytochrome c distribution (A in FIG. 3), and TMRE fluorescence intensity was concentrated, and OPA3 overexpression alone induced spontaneous apoptotic cell death. It was confirmed that it was not enough.

<3-2> Apoptotic  With stimulus OPA3 of Apoptotic  effect

<3-2-1> Staurosporin ( staurosporine Treatment effect

In order to confirm the apoptotic effect of OPA3 in the presence of apoptotic stimulation, OPA3 was treated in the state of inducing apoptosis with staurosporine to confirm the degree of apoptotic cell death. Specifically, the cells were treated with or without 5 ug / ml cyclohexamide (CHX) for 3 hours and then treated with 1 uM of Staurosporine. Cell anti-cytochrome c (cytochrome c) or anti-immune were stained with Bex (Bax) antibodies was observed under confocal microscopy (confocal microscope). In addition, caspase-3 activity and poly (ADP-ribose) polymerase; PARP] cleavage, cells obtained through centrifugation were subjected to Western blot using anti-PARP antibody and anti-active caspease-3 antibody.

As a result, when treated with staurosporine (STS) in the outpatient expression of OPA3, Mito-YFP (22.6 ± 2.1%, B and C of Figure 3) compared to the cells transformed, OPA3-YFP ( 41.0 ± 1.2%) significantly increased the redistribution of cytochrome c from the mitochondria to the cytoplasm in cells transformed, indicating that the sensitivity of OPA3-induced mitochondrial cleavage increases with apoptotic stimulation with STS. Confirmed. However, cytochrome c was identified in the cytoplasm of HeLa cells transfected with OPA3 mutation, OPA3 (G93S) of the patient, even though no external apoptotic stimulation was performed (A to C in FIG. 3), and overexpression of wild type OPA3 was observed. Compared to the induced mitochondrial cleavage, cells transfected with OPA3 (G93S) -YFP induced a wide range of mitochondrial cleavage.

<3-2-2> TRAIL ( TNF - related apoptosis - inducing ligand Treatment effect

In order to confirm the apoptotic effect of OPA3 in the presence of apoptotic stimulation, OPA3 was treated with TRAIL (TNF-related apoptosis-inducing ligand) to determine the degree of apoptotic cell death. Specifically, the cells were treated with or without 5 ug / ml cyclohexamide (CHX) for 3 hours, and then treated with 300 ng / ml TRAIL. Cell anti-cytochrome c (cytochrome c) or anti-immune were stained with Bex (Bax) antibodies was observed under confocal microscopy (confocal microscope). In addition, caspase-3 activity and poly (ADP-ribose) polymerase; PARP] cleavage, cells obtained through centrifugation were subjected to Western blot using anti-PARP antibody and anti-active caspease-3 antibody.

As a result, when treated with TRAIL (TNF-related apoptosis-inducing ligand) in foreign expression of OPA3, mito-YFP-transformed cells (43.2 ± 1.2%; Fig. 4 of OPA3-YFP-transformed cells) Compared with A and B), redistribution of cytochrome c to the cytoplasm in the mitochondria was induced more (85.8 ± 1.2%; A and B in FIG. 4). Consistent results were also obtained from experiments confirming the migration of Bax protein (C and D in FIG. 4), caspase-3 activity and cleavage of PARP (E in FIG. 4), the apoptosis regulatory protein of mitochondria.

<3-3> Apoptotic  With stimulus OPA3 siRNA of Apoptotic  Inhibitory effect

To demonstrate the definite role of OPA3 in apoptosis sensitivity, cytochrome c released by STS treatment in OPA3-knockdown HeLa cells was measured by immunostaining in the same manner as in Experimental Example <1-1>. OPA3 knockdown significantly reduced the release of cytochrome c by STS, confirming that the destruction of OPA3 inhibited apoptotic cell death (FIG. 4F).

Thus, OPA3 overexpression alone was not sufficient to induce spontaneous apoptotic cell death, but OPA3 was found to significantly increase the sensitivity of apoptosis by apoptotic stimulation such as STS or TRAIL.

< Experimental Example  4> OPA3 Your location in Mitochondria

<4-1> OPA3 Intracellular placement of

In order to confirm the position in the mitochondria of OPA3, isolation of cell organs was performed. Specifically, cells were harvested and washed with cold PBS buffer, 10 mM HEPES / KOH (pH 7.4), 38 mM sodium chloride (NaCl), 1 μg / mL aprotinin, 1 μg / mL A buffer solution containing lepteptin and 1 mM phenylmethyl sulfonyl fluoride (PMSF) added with 250 mM sucrose were treated to 5 times the cell volume. Thereafter, the cells were homogenized by 50-strength treatment with a Dounce homogenizer, and cell lysate was separated by centrifugation. Mitochondria were harvested by centrifugation at 4 ° C., 10,000 rpm for 10 minutes, and mitochondrial extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-). 100, 0.3% NP-40, 1 mM PMSF, 1 μg / mL aprotinin, and 1 μg / mL leupeptin) and resuspended at high temperature centrifugation at 100,000 rpm for 30 minutes at 4 ° C to obtain cytosolic fractions. Was carried out to obtain a supernatant.

As a result, both endogenous OPA3 or externally introduced OPA3 were identified in the mitochondrial fraction, but not in the cytoplasmic fraction (A and B in FIG. 5). In addition, OPA3-myc and FLAG-OPA3 are in the same position as mitochondrial proteins such as cytochrome c and Tom 20 in HeLa cells (Fig. 5 C, top) and COS 7 cells (Fig. 5 C, below). It was confirmed.

<4-2> OPA3 Mitochondrial membrane of membrane ) Check my location

After confirming that OPA3 is located in the mitochondria, the mitochondrial fractions were treated with proteinase K to determine whether OPA3 is located in the mitochondrial outer membrane or the mitochondrial inner membrane. Specifically, mitochondrial fractions were washed with IB buffer (220 mM mannitol, 70 mM sucrose, 10 mM HEPES / KOH, pH 7.6, 1 mM MgCl 2, 1 mM DTT, and 1 mM EDTA), and 10 ug / ml of pro Tinase K (proteinase K) was added to the sample resuspended in IB buffer or swelling buffer (20 mM HEPES / KOH, pH 7.4), and the sample was then left on ice for 30 minutes, Protease activity was stopped by adding SDS-PAGE sample buffer solution.

As a result, OPA1, a mitochondrial inner membrane protein, was degraded by proteinase K only when osmotic shock was applied, whereas Tom 20, an endogenous OPA3 and mitochondrial outer membrane protein, was degraded by proteinase K even without osmotic shock. Through this, it was confirmed that OPA3 is located in the mitochondrial outer membrane (FIG. 5D).

In addition, mitochondrial transport experiments were performed to confirm the transport of mitochondria in the ATP regeneration system. Specifically, 1 mg / ml mitochondria were isolated from healthy cells, followed by an import buffer containing 220 mM mannitol, 70 mM sucrose, 10 mM Hepes / 1 mM ATP, 0.1 mM GTP and 5 mM NADH. KOH, pH 7.2, 1 mM MgCl 2, 1 mM EDTA and 1 mM dithiotheritol). The import reaction was initiated by adding 35S labeled OPA3 synthesized by in vitro transcription and translation to rabbit reticulite lysate (TNT coupled system, Promega), and after the reaction, the mitochondria were 4 ° C., 10,000. Recovery was by centrifugation at g for 10 minutes. After the import reaction, the separated mitochondria were treated with trypsin for 20 minutes at 4 ℃.

As a result, it was confirmed that the treatment of 35S-labeled OPA3 with trypsin did not detect 35S-labeled OPA3, and OPA3-YFP (FIG. 5E), FLAG-OPA3 and OPA3-myc. Similar results were obtained in the experiment using (FIG. 5F).

<4-3> OPA3 Mitochondrial envelope outer membrane ) Check my location

To determine whether OPA3 degraded by Proteinase K (Sigma) is located at the periphery of the mitochondrial outer membrane and is interrupted, high pH sodium carbonate can be extracted from proteins attached around the mitochondria. As a result, DRP1, known as a peripheral protein present on the mitochondrial outer membrane surface, was obtained by treatment with alkali, but endogenous OPA3 and FLAG-OPA3 were obtained from membrane fractions treated with a control buffer solution and 0.1 M sodium carbonate (Na ₂ CO ₃ ). It was precipitated (G in FIG. 5), and it was confirmed that OPA3 is an intramembrane protein located in the mitochondrial outer membrane.

<4-4> differential transmission test ( differential permeabilization technique In the mitochondrial envelope OPA3  Check location

To explain the role of OPA3 in the function of mitochondria, it is important to understand the topology of OPA3 in the mitochondrial envelope, so that digitonin (Calbiochem) selectively destroys the plasma membrane and mitochondrial envelope at various concentrations. Differential permeabilization technique was performed. Specifically, the plasmid was transformed into HeLa cells, and after 15 hours, fixed at room temperature for 15 minutes with 4% paraformaldehyde, and permeated with PBS while increasing the concentration of digitonin, and then anti- Labeled by immunofluorescence using FLAG antibody, anti-myc antibody, anti-Tom 20 antibody or anti-cytochrome c antibody.

As a result, in HeLa cells treated with 5 ug / ml of digtonin (FIG. 5H), Tom 20 exposed to the cytoplasm was immunostained, but cytochrome c located in the mitochondrial interstitial space was not detected. When HeLa cells were treated with 12 ug / ml of digitone, both cytochrome c and Tom 20 were detected. After treatment with 5 ug / ml of Digitonin, OPA3-myc protein in mitochondria was detected when using an antibody against a myc tag bound to the C-terminus, but at the C-terminus of OPA3 exposed to the cytoplasm under the same conditions. Identified cytochrome c was not detected. FLAG-OPA3 immunostaining and cytochrome c immunostaining by antibodies to FLAG bound to the N-terminus were positive only when treated with 12 ug / ml of digitonin (FIG. 5H).

Thus, these results confirm that the N-terminus and C-terminus of OPA3 are intercalated into the mitochondrial outer membrane due to exposure to the intercellular space and cytoplasm.

< Experimental Example  5> as mitochondrial target peptide OPA3 N-terminal of

To date, OPA3 amino acid residues 25-29 (NRIKE) have been proposed as mitochondrial target peptides that enter the mitochondrial matrix. In order to test the mitochondrial target sequence, an experiment was performed to determine the location of the NRIK deletion mutant of OPA3 having YFP attached to the N-terminus.

As a result, it was confirmed that the full-length OPA3-YFP is located in the mitochondria in HeLa cells using DsRed-Mito as a mitochondrial control, and OPA3 (△ NRIK) -YFP was also located in the mitochondria unlike expected, It was confirmed that the N-terminal NRIKE sequence was not important for the mitochondrial target of OPA3.

To identify domains that target mitochondria in OPA3 proteins, the expected membranes followed by TMHMM server (www.cbs.dtu.dk), DAS membrane potential prediction server (www.sbc.su.se) and Kyte-Doolittle hydropathy plot server. Several deletion mutations were made, including mutations lacking a presumed hydrophobic moiety consistent with amino acids 83-102 represented by spanning fragments, which were transformed into cells and immunostained for the cells. Specifically, for immunostaining, cells cultured in a 2-well chamber slide are fixed with 4% paraformaldehyde at room temperature for 15 minutes, and 0.15% Triton X-100 is added to PBS. The solution was permeated at room temperature for 15 minutes, and then blocked for 45 minutes at room temperature with a buffer solution in which 3% bovine serum albumin was added to PBS. After reacting the anti-Tom20 antibody or the anti-myc antibody with the primary antibody on the slide, washed with PBS buffer solution, goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 or 594 or goat anti-conjugated Alexa Fluor 488 Rabbit IgG antibodies were reacted with secondary antibodies. The slides were then observed using a Zeiss LSM 510 confocal microscope and the excitation wavelengths of YFP, Alexa Fluor 594, Alexa Fluor 488 and DsRed were 514, 594, 488, and 543 nm, respectively. It was measured by.

As a result, C-terminally reduced OPA3-YFP mutants, including OPA3 (Δ83-102), OPA3 (1-30) and OPA3 (1-105), are located in the mitochondria, while OPA3, OPA3 (20-179) And N-terminal deletion mutations in OPA3 (31-179) were identified in the cytoplasm (A and B in Figure 6). Thus, it was confirmed that 30 residues at the N-terminus of OPA3 are necessary parts for mitochondrial targeting.

< Experimental Example  6> OPA3  Mitochondria by hydrophobic portions of proteins Truncated

Expected Membrane with OPA3 TMHMM Server (www.cbs.dtu.dk), DAS Membrane Potential Estimation Server (www.sbc.su.se) and Kyte-Doolittle hydropathy plot server to identify functional domains of OPA3 in mitochondrial form. Several deletion mutations were made, including mutations lacking a presumed hydrophobic moiety consistent with amino acids 83-102 represented by spanning fragments, which were transformed into cells and immunostained for the cells.

Specifically, for immunostaining, cells cultured in a 2-well chamber slide are fixed with 4% paraformaldehyde at room temperature for 15 minutes, and 0.15% Triton X-100 is added to PBS. The solution was permeated at room temperature for 15 minutes, and then blocked for 45 minutes at room temperature with a buffer solution in which 3% bovine serum albumin was added to PBS. After reacting the anti-Tom20 antibody or the anti-myc antibody with the primary antibody on the slide, washed with PBS buffer solution, goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 or 594 or goat anti-conjugated Alexa Fluor 488 Rabbit IgG antibodies were reacted with secondary antibodies. The slides were then observed using a Zeiss LSM 510 confocal microscope and the excitation wavelengths of YFP, Alexa Fluor 594, Alexa Fluor 488 and DsRed were 514, 594, 488, and 543 nm, respectively. It was measured by.

 As a result, overexpression of OPA3-YFP significantly induces mitochondrial cleavage, induces mitochondrial cleavage even when OPA3-YFP is expressed low, and the C-terminus of OPA3 in which the hydrophobic moiety (residues 83-102) remains intact Cells transformed with deleted mutants also exhibited excessive mitochondrial cleavage. In contrast, cells transformed with OPA3 (Δ83-102), an OPA3 deletion mutant lacking a hydrophobic portion, did not show morphological changes in mitochondria (FIGS. 6D and 6E). Although OPA3 (Δ83-102) -YFP was located in the mitochondria, the results of the proteinase assay confirmed that OPA3 (Δ83-102) -YFP failed to be located in the mitochondrial outer membrane (FIG. 6F).

Thus, the results indicate that 83-102 residues of OPA3 are necessary for mitochondrial cleavage and mitochondrial outer membrane localization of OPA3.

< Experimental Example  7> OPA3 Splicing  Comparison of Phenotypes Between Variants

Human OPA3 has two splicing variants (v1, NP_001017989 and v2, NP_079412) with 81.7% amino acid identity and 93 residues (G93) of OPA3 variant 2 (NP_079412) used in the present invention are ADOA The patient was mutated in OPA3 but not in OPA3 variant 1 (NP_001017989). For this reason, immunostaining was performed to confirm that the expression of variant 2 (OPA3-v2) shows the same phenotype as the expression of OPA3 variant 1 (OPA3-v1). Specifically, for immunostaining, cells cultured in a 2-well chamber slide are fixed with 4% paraformaldehyde at room temperature for 15 minutes, and 0.15% Triton X-100 is added to PBS. The solution was permeated at room temperature for 15 minutes, and then blocked for 45 minutes at room temperature with a buffer solution in which 3% bovine serum albumin was added to PBS. After reacting the anti-Tom20 antibody with the primary antibody on the slide, washed with PBS buffer solution, goat anti-mouse IgG antibody Alexa Fluor 488 or 594 conjugated or goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 Reaction with secondary antibody. The slides were then observed using a Zeiss LSM 510 confocal microscope, and the excitation wavelengths of YFP, Alexa Fluor 594 and Alexa Fluor 488 were measured at 514, 594 and 488 nm, respectively.

As a result, OPA3-v1-YFP appeared to be colocated with Tom 20 in the mitochondria and showed excessive mitochondrial cleavage (FIG. 7).

Thus, the results confirm that mutation of the 93rd residue of OPA3 is not essential for mitochondrial cleavage.

< Manufacturing example  1> Preparation of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

OPA3 Protein One g, 0.6 g sodium chloride and 0.1 g ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

OPA3 protein ... 1 g

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ······················

As seen above, the OPA3 protein of the present invention or a polynucleotide encoding the same can be used as an anticancer adjuvant composition, and can be used in the development of an anticancer treatment method by co-administration with an anticancer agent.

<110> Korea Advanced Institute of Science and Technology <120> Anticancer supplement composition comprising OPA3 protein or          polynucleotide encoding OPA3 as an effective ingredient <130> 10p-03-38 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 179 <212> PRT <213> Homo sapiens <400> 1 Met Val Val Gly Ala Phe Pro Met Ala Lys Leu Leu Tyr Leu Gly Ile   1 5 10 15 Arg Gln Val Ser Lys Pro Leu Ala Asn Arg Ile Lys Glu Ala Ala Arg              20 25 30 Arg Ser Glu Phe Phe Lys Thr Tyr Ile Cys Leu Pro Pro Ala Gln Leu          35 40 45 Tyr His Trp Val Glu Met Arg Thr Lys Met Arg Ile Met Gly Phe Arg      50 55 60 Gly Thr Val Ile Lys Pro Leu Asn Glu Glu Ala Ala Ala Glu Leu Gly  65 70 75 80 Ala Glu Leu Leu Gly Glu Ala Thr Ile Phe Ile Val Gly Gly Gly Cys                  85 90 95 Leu Val Leu Glu Tyr Trp Arg His Gln Ala Gln Gln Arg His Lys Glu             100 105 110 Glu Glu Gln Arg Ala Ala Trp Asn Ala Leu Arg Asp Glu Val Gly His         115 120 125 Leu Ala Leu Ala Leu Glu Ala Leu Gln Ala Gln Val Gln Ala Ala Pro     130 135 140 Pro Gln Gly Ala Leu Glu Glu Leu Arg Thr Glu Leu Gln Glu Val Arg 145 150 155 160 Ala Gln Leu Cys Asn Pro Gly Arg Ser Ala Ser His Ala Val Pro Ala                 165 170 175 Ser Lys Lys            

Claims (8)

Anti-cancer adjuvant composition containing OPA3 (optic atrophy 3) protein or a polynucleotide encoding the protein as an active ingredient.
The anticancer adjuvant composition according to claim 1, wherein the OPA3 protein has an amino acid sequence as set forth in SEQ ID NO: 1.
The anticancer adjuvant composition according to claim 1, wherein the OPA3 protein is administered together with staurosporine or TRAIL (TNF-related apoptosis-inducing ligand).
According to claim 1, wherein the cancer is cancer cancer adjuvant composition, characterized in that any one selected from the group consisting of cancer, lung cancer, uterine cancer, skin cancer, liver cancer, breast cancer, ovarian cancer and bladder cancer.
A method for enhancing apoptosis of cancer cells of an anticancer agent, comprising administering an OPA3 protein or a polynucleotide encoding the protein together with an anticancer agent in a subject.
The method of claim 5, wherein the OPA3 protein has an amino acid sequence set forth in SEQ ID NO: 1.
The method of claim 5, wherein the anticancer agent is Staurosporine or TRAIL (TNF-related apoptosis-inducing ligand).
The method of claim 5, wherein the cancer cells are any one selected from the group consisting of gastric cancer, lung cancer, uterine cancer, skin cancer, liver cancer, breast cancer, ovarian cancer and bladder cancer.

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