Noxa-derived, cell death-inducing peptide eMTD

Abstract

Disclosed herein is a cell death-inducing peptide that rapidly acts. The peptide is derived from Noxa protein and comprises 16 amino acid residues including MTD. The peptide is designated extended MTD (eMTD) because it contains the 10-mer MTD. eMTD rapidly exhibits potent necrotic cell death in various cell lines and, as such, can be applied to the treatment of various diseases including cancer when used in conjugation with peptides or materials for targeting specific cells.

Claims

1. A peptide comprising a sequence consisting of the amino acid sequence of SEQ ID NO: 1 or 2, wherein optionally the peptide further comprises a tumor-homing peptide (THP) conjugated to the N- or C-terminus of the peptide and the THP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 31.

2. The peptide of claim 1, wherein the peptide comprises said THP conjugated to the N- or C-terminus of the peptide.

3. A composition comprising the peptide of claim 1 or 2.

4. The peptide of claim 1, wherein the peptide comprises a sequence consisting of SEQ ID NO: 1, wherein optionally the peptide further comprises a tumor-homing peptide (THP) conjugated to the N- or C-terminus of the peptide and the THP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 31.

5. The peptide of claim 4, wherein the peptide comprises said THP further comprises a THP conjugated to the N- or C-terminus of the peptide.

6. The peptide of claim 1, wherein the peptide comprises a sequence consisting of SEQ ID NO: 2, wherein optionally the peptide further comprises a tumor-homing peptide (THP) conjugated to the N- or C-terminus of the peptide and the THP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 31.

7. The peptide of claim 6, wherein the peptide comprises said THP further comprises a THP conjugated to the N- or C-terminus of the peptide.

8. The peptide of claim 1, wherein the amino acid sequence of the peptide consists of the amino acid sequence of SEQ ID NO: 1.

9. The peptide of claim 1, wherein the amino acid sequence of the peptide consists of: (a) the amino acid sequence of SEQ ID NO: 1 and (ii) the amino acid sequence of a THP of an amino acid sequence selected from SEQ ID NOs: 5 to 31.

10. The peptide of claim 1, wherein the amino acid sequence of the peptide consists of the amino acid sequence of SEQ ID NO: 2.

11. The peptide of claim 1, wherein the amino acid sequence of the peptide consists of: (a) the amino acid sequence of SEQ ID NO: 2 and (ii) the amino acid sequence of a THP of an amino acid sequence selected from SEQ ID NOs: 5 to 31.

12. A polynucleotide coding for the peptide of claim 1 or 2.

13. A recombinant vector carrying the polynucleotide of claim 12.

14. A transformed cell transformed with the recombinant vector of claim 13.

15. A method for the Method for prevention or treatment of cancer comprising: administering to a subject a composition comprising the peptide of claim 1 or 2.

16. The method of claim 15, wherein the cancer is lung cancer, breast cancer, liver cancer, melanoma, stomach cancer, pancreatic cancer, colorectal cancer, ovarian cancer, renal cell carcinoma, prostate cancer, or brain tumor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows relative cell viability after treatment of HeLa cell line with eMTD (SEQ ID NO: 1) peptide or eMTDΔ4 (SEQ ID NO: 2) peptide according to an embodiment of the present disclosure, as measured by MTS assay;

(2) FIG. 2 shows microscopic images of HeLa cells after or before treatment with 20 μM of eMTDΔ4 (SEQ ID NO: 2) for 3 min;

(3) FIG. 3 shows relative cell viability after treatment of HeLa, CT26, and B16F10 cell lines with eMTDΔ4 (SEQ ID NO: 2) peptide according to an embodiment of the present disclosure, as measured by MTS assay;

(4) FIG. 4a shows time-lapse, confocal microscopic images of HeLa cells stained with the calcium indicator Fluo-4 and then treated with or without 20 μM of eMTDΔ4 (SEQ ID NO: 2) according to an embodiment of the present disclosure;

(5) FIG. 4b is a graph showing Fluo-4 intensities within ROI (region of interest) of time-lapse confocal microscopic images of HeLa cells stained with the calcium indicator Fluo-4 without eMTDΔ4 (SEQ ID NO: 2) treatment according to an embodiment of the present disclosure;

(6) FIG. 4c is a graph showing Fluo-4 intensities within ROI (region of interest) of time-lapse confocal microscopic images of HeLa cells stained with the calcium indicator Fluo-4 and then treated with 20 of eMTDΔ4 (SEQ ID NO: 2) according to an embodiment of the present disclosure;

(7) FIG. 5 shows confocal microscopic images of HeLa cells fixed 5 and 10 min after treatment with the peptide eMTDΔ4 (SEQ ID NO: 2) conjugated with a fluorescent (Fluorescein; FAM) according to an embodiment of the present disclosure;

(8) FIG. 6a shows mitochondrial swelling as analyzed by absorbance read at 540 nm after treatment of mitochondria isolated from livers of BalB/C mice with the peptide eMTDΔ4 (SEQ ID NO: 2) according to an embodiment of the present disclosure;

(9) FIG. 6b shows mitochondrial swelling as observed by TEM (transmission electron microscopy) after treatment of mitochondria isolated from livers of BalB/C mice with the peptide eMTDΔ4 (SEQ ID NO: 2) according to an embodiment of the present disclosure;

(10) FIG. 7a shows time-lapse, confocal microscopic images of HeLa cells to identify mitochondrial PTP opening after only mitochondria are stained with calcein AM and cobalt ions without treatment with the peptide eMTDΔ4 (SEQ ID NO: 2) according to an embodiment of the present disclosure;

(11) FIG. 7b shows time-lapse, confocal microscopic images of HeLa cells to identify mitochondrial PTP opening after only mitochondria are stained with calcein AM and cobalt ions and the cells are treated with the peptide eMTDΔ4 (SEQ ID NO: 2) according to an embodiment of the present disclosure;

(12) FIG. 8a shows time-lapse, confocal microscopic images of HeLa cells after the cells are stained with the mitochondrial reactive oxygen species indicator MitoSox and then treated with or without 20 of the peptide eMTDΔ4 (SEQ ID NO: 2) according to an embodiment of the present disclosure;

(13) FIG. 8b is a graph showing MitoSox intensities within ROI (region of interest) of time-lapse confocal microscopic images of HeLa cells stained with the mitochondrial reactive oxygen species indicator MitoSox without eMTDΔ4 (SEQ ID NO: 2) treatment according to an embodiment of the present disclosure;

(14) FIG. 8c is a graph showing MitoSox intensities within ROI (region of interest) of time-lapse confocal microscopic images of HeLa cells stained with the mitochondrial reactive oxygen species indicator MitoSox and then treated with 20 μM of eMTDΔ4 (SEQ ID NO: 2) according to an embodiment of the present disclosure;

(15) FIG. 9 shows time-lapse, confocal microscopic images of Hela cells treated with the peptide eMTDΔ4 (SEQ ID NO: 2) conjugated with a fluorescent (Fluorescein; FAM) according to an embodiment of the present embodiment;

(16) FIG. 10a shows degrees of damage on the lipid membrane of liposomes as analyzed by two-step assay according to an embodiment of the present disclosure.

(17) FIG. 10b shows the damage of the peptide eMTDΔ4 (SEQ ID NO: 2) on the cell membrane in time-lapse atomic force microscopic images according to an embodiment of the present disclosure;

(18) FIG. 10c shows the damage of the peptide eMTDΔ4 (SEQ ID NO: 2) on the cell membrane and the depth of the cell membrane damage in time-lapse atomic force microscopic images according to an embodiment of the present disclosure; and

(19) FIG. 10d shows statistical data of damage, photographically taken by atomic force microscopy, of the peptide eMTDΔ4 (SEQ ID NO: 2) on the cell membrane.

MODE FOR CARRYING OUT THE INVENTION

(20) A better understanding of the present disclosure may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present disclosure.

EXAMPLE 1: PEPTIDE SYNTHESIS

(21) Peptide synthesis was entrusted to Anygen in which peptides were synthesized using solid-phase peptide synthesis, purified by high-capacity HPLC, dissolved at a concentration of 1 mM in 50% aqueous DMSO (dimethyl sulfoxide) solution, and then stored at −20° C. Amino acids sequences of the peptides are given in Table 2, below.

(22) TABLE-US-00002 TABLE 2 SEQ ID NO: Name Sequencing List 1 eMTD KLNFRQKLLNLISKLFCSGT 2 eMTDΔ4 KLNFRQKLLNLISKLF 3 MTD KLLNLISKLF 4 Noxa MPGKKARKNAQPSPARAPAELEVECAT QLRRFGDKLNFRQKLLNLISKLFCSGT

EXAMPLE 2: CULTURE OF CANCER CELL LINE

(23) Individual cell lines (HeLa, CT26, and B16F10) were purchased from the Korean Cell Line Bank. Dulbecco's modified Eagle's medium (DMEM), trypsin-EDTA, fetal bovine serum (FBS), and Hank's balanced salt solution (HBSS) were products from Gibco (Thermo Fisher).

(24) Each of the cell lines was cultured in DMEM supplemented with 10% FBS in an incubator maintained at 37° C. and 5% 002.

EXAMPLE 3: ASSAY FOR CELL DEATH INDUCTION OF EMTD (SEQ ID NO: 1) PEPTIDE

(25) 3-1. Assay for Activity of Cell Death Induction in Uterine Cervical Cancer

(26) For use in assaying the peptides eMTD (SEQ ID NO: 1) and eMTDΔ4 (SEQ ID NO: 2) synthesized in Example 1 for activity of inducing cell death, the cell line HeLa was grown to about 90% confluency in 96-well plates. The cells were washed once with HBSS buffer before treatment with the peptides of the present disclosure eMTD (SEQ ID NO: 1) and eMTDΔ4 (SEQ ID NO: 2) at concentrations of 0, 20, and 40 for one hour. Thereafter, the cells were incubated with an MTS assay agent (Promega) for one hour, followed by reading absorbance to calculate relative viability of cells. The results are given in FIG. 1 and Table 3.

(27) TABLE-US-00003 TABLE 3 0 μM 20 μM 40 μM fl-Noxa (SEQ Viability (%) 100 101 106 ID NO: 4) Standard deviation 0.079 0.037 0.050 eMTD (SEQ Viability (%) 100 61 63 ID NO: 1) Standard deviation 0.079 0.007 0.023 eMTDΔ4 Viability (%) 100 53 40 (SEQ ID Standard deviation 0.079 0.082 0.044 NO: 2)

(28) As is understood from the data of FIG. 1 and Table 3, eMTD (SEQ ID NO: 1) and eMTDΔ4 (SEQ ID NO: 2) peptides exhibit potent cell death activity.

(29) 3-2. Microscopic Observation of Morphological Change in Cells

(30) In order to observe morphological changes in cells in detail, the cells were incubated with 20 μM of the peptide eMTDΔ4 (SEQ ID NO: 2) and observed with the aid of a microscope (Leica). The results are depicted in FIG. 2.

(31) As shown in FIG. 2, most of the cells were observed to undergo necrosis with the consequent rupture of cell membranes after 3 min of incubation with the peptide.

(32) 3-3. Assay for Activity of Cell Death Induction in Other Cancer Cell Lines

(33) Examination was made to see whether the peptide has the activity of inducing cell death in other cancer cell lines. In this regard, the cancer cell lines HeLa, CT26, and B16F10 were treated with eMTDΔ4 (SEQ ID NO: 2) at concentrations of 0, 20, and 40 μM. Cell viability was measured by MTS assay and the results are given in FIG. 3 and Table 4.

(34) TABLE-US-00004 TABLE 4 eMTDΔ4 (SEQ ID NO: 2) HeLa CT26 B16F10  0 μM Viability (%) 100 100 100 Standard 5.72 11.81 7.73 deviation 10 μM Viability (%) 47 69 68 Standard 5.07 13.08 5.71 deviation 20 μM Viability (%) 11 21 33 Standard 0.98 0.69 3.99 deviation

(35) As can be understood from the data of FIG. 3 and Table 4, the peptide eMTDΔ4 (SEQ ID NO: 2) was found to induce cell death in HeLa, which is a human uterine cervical cancer cell line as well as in CT26, which is derived from murine colon carcinoma, and B16F10, which is derived from murine melanoma cells.

EXAMPLE 4: INDUCTION OF CELL DEATH AND INTRACELLULAR CALCIUM LEVEL CHANGE BY PEPTIDE eMTDΔ4 (SEQ ID NO: 2)

(36) In order to examine the mechanism in which the peptide eMTD (SEQ ID NO: 1) induces cell death in cancer cell lines, a change in intracellular calcium level, which is known as an important factor for cell death, was observed using the calcium indicator Fluo-4 under a confocal microscope (Leica).

(37) In brief, HeLa cells were grown to about 70% confluency in a Lab-Tek chamber glass a day before the experiment. Immediately before the experiment, of the calcium indicator Fluo-4 was diluted to a concentration of 5 in HBSS. HeLa cells were incubated with the dilution for 10 min in an incubator and then treated with the peptide eMTDΔ4 (SEQ ID NO: 2). Results are given in FIGS. 4A and 4C.

(38) As shown in FIGS. 4A to 4C, almost no changes in intracellular calcium level were found in HeLa cells that had not been treated with the peptide. In contrast, the HeLa cells were observed to allow calcium influx into the cytosols thereof within less than one min after treatment with the peptide eMTDΔ4 (SEQ ID NO: 2). In this regard, the calcium levels are depicted in a saw-toothed pattern in a graph. When the cell membrane bubbles and is damaged, the calcium influx is reduced, with the cell contents being expelled. As a result, the intracellular calcium level becomes lower than the original level.

EXAMPLE 5: INTRACELLULAR MIGRATION OF PEPTIDE eMTDΔ4 (SEQ ID NO: 2) TO TARGET

(39) In order to investigate the source that allowed the intracellular calcium influx observed in Example 4, examination was made of the site at which the peptide eMTDΔ4 (SEQ ID NO: 2) acts. In this regard, the fluorescent tracer fluorescein was labeled to the C-terminal of the peptide eMTDΔ4 (SEQ ID NO: 2) (eMTDΔ4 (SEQ ID NO: 2)-FAM). Then, HeLa cells grown on Lab-Tek chamber glass were incubated with eMTDΔ4 (SEQ ID NO: 2)-FAM for 5 and 10 min before fixation with 1% paraformaldehyde.

(40) Mitochondria were visualized using an antibody to TOM20, which localizes to the outer membrane of the mitochondrion because MTD (SEQ ID NO: 3) is known to migrate to mitochondria, while nuclei were visualized using DAPI. Confocal micrographic images are shown in FIG. 5.

(41) As indicated by the images of FIG. 5, the localization of eMTDΔ4 (SEQ ID NO: 2)-FAM is consistent with the visualized positions of mitochondria. In addition, after treatment with eMTDΔ4 (SEQ ID NO: 2)-FAM, mitochondrial fragmentation was observed morphologically.

EXAMPLE 6: OBSERVATION OF eMTDΔ4 (SEQ ID NO: 2) ACTIVITY IN MITOCHONDRION

(42) 6-1. Mitochondrial Swelling

(43) In Example 5, eMTDΔ4 (SEQ ID NO: 2)-FAM was observed to localize to mitochondria and to induce mitochondrial fragmentation. Accordingly, examination was further made to see how eMTDΔ4 (SEQ ID NO: 2) practically works at mitochondria in greater detail.

(44) First, livers excised from 6-week-old BalB/C mice were immersed in buffer (250 mM mannitol, 70 mM EGTA, 5 mM HEPES, pH 7.4, 0.1 mM PMSF and 4 rotenone) and completely homogenized using a Teflon Potter-Elvehjem grinder (Sigma). The ground liver was centrifuged at 1000×g for 10 min at 4° C. and the supernatants were centrifuged at 10,000×g for 10 min at 4° C. The pellets thus obtained were suspended in a regeneration buffer (250 mM sucrose, 10 mM HEPES, pH 7.4, 5 mM sodium succinate, 2 mM potassium phosphate, 0.1 mM PMSF, 25 μM EGTA, and 4 rotenone).

(45) The mitochondria thus obtained were treated with 25 μM of eMTD (SEQ ID NO: 1) or eMTDΔ4 (SEQ ID NO: 2), followed by reading absorbance at 540 nm to determine mitochondrial swelling. The results are given in FIG. 6A and Table 5, below. For a positive control, the mitochondria were treated with 200 μM of Ca.sup.2+ while co-treatment with 200 μM of Ca.sup.2+ and 20 μM of CsA was conducted for a negative control.

(46) For direct identification, the mitochondria were treated with eMTDΔ4 (SEQ ID NO: 2) (25 μM), Ca.sup.2+ (200 μM), or Ca.sup.2+ (200 μM)+CsA (20 μM) and then observed by transmission electron microscopy. The results are given in FIG. 6B.

(47) TABLE-US-00005 TABLE 5 Minute 0 10 20 30 40 50 60 None 100% 98% 97% 95% 95% 95% 95% cNoxa del4 100% 79% 74% 70% 68% 66% 65% cNoxa 100% 86% 78% 71% 68% 68% 67% Ca 100% 77% 75% 73% 72% 70% 70% CsA 100% 98% 98% 97% 97% 96% 96% Ca + CsA 100% 98% 97% 96% 95% 94% 93% None  0%  0%  0%  0%  0%  0%  0% cNoxa del4  0%  0%  0%  0% 0.22%.sup.   0%  0% cNoxa  0%  4%  4%  6%  5%  5%  5% Ca  0%  0%  1%  1%  1%  1%  0% CsA  0%  0%  0%  0%  0%  0%  0% Ca + CsA  0%  0%  0%  0%  0%  0%  0%

(48) As indicated by the data of FIGS. 6A and 6B and Table 5, eMTDΔ4 (SEQ ID NO: 2) was observed to induce mitochondrial swelling.

(49) 6-2. Mitochondrial PTP

(50) Mitochondria are known to open permeability transition pores (PTP) to allow cytosolic calcium influx. The observation that eMTDΔ4 (SEQ ID NO: 2) migrates to mitochondria and induces cytosolic calcium influx led to investigating the association of the action of eMTDΔ4 (SEQ ID NO: 2) with PTP. To this end, PTP opening was monitored using calcein AM and cobalt ions.

(51) In brief, HeLa cells were grown on a Lab-Tek chamber glass slide one day before the experiment. Immediately before the experiment, the cells were stained with 1 μM of calcein AM and 2 mM of cobalt ions for 20 min and then with 0.1 μM of MitoTracker Red for 2 min. Time-lapse confocal images were taken and are given in FIGS. 7A and 7B. When PTP is opened, mitochondrial calcein AM meets cobalt ions so that the fluorescence of calcein AM is quenched.

(52) As is understood from the images of FIGS. 7A and 7B, the fluorescence of mitochondrial calcein AM was quenched upon treatment with eMTDΔ4 (SEQ ID NO: 2), implying that the eMTDΔ4 (SEQ ID NO: 2)-induced cytosolic calcium influx is conducted by PTP opening.

EXAMPLE 7: PEPTIDE eMTDΔ4 (SEQ ID NO: 2)-INDUCED CELL DEATH AND ROS GENERATION

(53) In addition to an increase in intracellular calcium level, reactive oxygen species (ROS) generation is an important factor for cell death. Examination was made of the role of ROS in the process of eMTDΔ4 (SEQ ID NO: 2)-induced cell death.

(54) In brief, HeLa cells were grown to 70% confluency on a Lab-Tek chamber glass slide one day before the experiment. The cells were stained for 10 min with 5 μM of the mitochondrial ROS indicator MitoSox (Invitrogen) in order to visualize ROS generation. Then, the cells were incubated with eMTDΔ4 (SEQ ID NO: 2) for 10 min during which confocal micrographic images were taken to monitor cell morphologies and ROS generation. The results are given in FIGS. 8A to 8C.

(55) As shown in FIGS. 8A to 8C, the non-treated HeLa cells scarcely changed in cell morphology and ROS level whereas eMTDΔ4 (SEQ ID NO: 2)-treated HeLa cells abruptly increased in ROS level just after the cell membrane bubbling. Although not demonstrating that ROS generated in mitochondria is a direct cause of cell death, this result indicates that the peptide eMTDΔ4 (SEQ ID NO: 2) of the present disclosure causes ROS generation in mitochondria in the light of the ROS generation just after the bubbling (cell membrane damage).

EXAMPLE 8: PEPTIDE eMTDΔ4 (SEQ ID NO: 2)-INDUCED CELL MEMBRANE DAMAGE

(56) 8-1. Confocal Microscopy

(57) In order to investigate the temporal order and cause relation between the introduction of eMTDΔ4 (SEQ ID NO: 2) and the damage of mitochondrial membrane and cell membrane, mitochondria were visualized by transfecting Mito-DsRed2 into HeLa cells one day before the experiment. Thereafter, eMTDΔ4 (SEQ ID NO: 2)-FAM was applied to the HeLa cells images of which were then taken every five seconds for 10 min under a confocal microscope. The results are given in FIG. 9.

(58) As shown in FIG. 9, eMTDΔ4 (SEQ ID NO: 2)-FAM diffused into the cytosol through the cell membrane bubbles from the time when the bubbles were formed and after eMTDΔ4 (SEQ ID NO: 2) reached thereto, the mitochondria started to undergo fragmentation.

(59) These data imply that eMTDΔ4 (SEQ ID NO: 2) is more likely to diffuse into the cytosol after cell membrane damage than to be introduced through channels or other receptors. The eMTDΔ4 (SEQ ID NO: 2) introduced by diffusion is considered to localize to mitochondria to introduce cell death.

(60) 8-2. Two-Step Assay

(61) The mechanism in which eMTDΔ4 (SEQ ID NO: 2) directly damages cell membranes was examined using a two-step assay.

(62) In brief, DOPS, DOPE, and DOPC (Avanti Polar Lipids) were dissolved at the ratio of 2:4:3 in chloroform, followed by vaporization. The residue was again dissolved at the concentration of 2 mg/mL in TbCl.sub.3 buffer (15 mM TbCl.sub.3, 50 mM sodium citrate, 20 mM HEPES, 150 mM NaCl, pH 7.4). The lipid mixture was extruded by Mini extruder (Avanti Polar Lipids) with a 400 nm to prepare liposomes which were washed with washing buffer (150 mM NaCl, 20 mM HEPES, pH 7.4) and then resuspended in an assay buffer (50 DPA, 150 M NaCl, 20 mM HEPES, pH 7.4). The suspension was plated into 96-well plates and treated with 0, 10, or 25 μM of eMTD (SEQ ID NO: 1) or eMTDΔ4 (SEQ ID NO: 2). After stimulation with 276 nm laser, fluorescence was read at 490 nm. The results are given in FIGS. 10A to 10C and Table 6.

(63) TABLE-US-00006 TABLE 6 eMTD eMTD Δ4 (SEQ ID (SEQ ID NO: 1) NO: 2)  0 μM Relative Fluorescence Intensity 0% 0% Standard Deviation 0% 0% 10 μM Relative Fluorescence Intensity 27%  25%  Standard Deviation 1% 1% 25 μM Relative Fluorescence Intensity 55%  51%  Standard Deviation 3% 1%

(64) As is understood from data of FIGS. 10A to 10C and Table 6, higher fluorescence intensity was detected upon treatment with eMTD (SEQ ID NO: 1) or eMTDΔ4 (SEQ ID NO: 2), indicating that TbCl.sub.3 was released through damaged portions of lipid membranes of the liposomes. Accordingly, it is concluded that eMTD (SEQ ID NO: 1) and eMTDΔ4 (SEQ ID NO: 2) directly act on cell membrane to damage the lipid structure of the cell membrane.

(65) This result was directly observed. In this regard, MDA-MB-231 cells were treated with eMTDΔ4 (SEQ ID NO: 2) and pores formed on the cell membrane were observed using an atomic force microscope. Measurements were statistically treated according time and the data thus obtained are given in FIG. 10D and Table 7.

(66) TABLE-US-00007 TABLE 7 Counts Counts Counts Counts Length t = 30 sec t = 2 min t = 5 min t = 40 min >0.625 1.408451 0 0 0 0.675~0.975 38.02817 20.63492 14.81481 2.5 1.025~1.325 43.66197 38.09524 39.50617 22.5 1.375~1.675 9.859155 20.63492 20.98765 22.5 1.725~2.025 4.225352 14.28571 13.58025 22.5 2.075~2.725 2.816901 6.349206 9.876543 22.5  2.775< 0 0 1.234568 7.5 S.D 18.47933 13.65448 13.38917 10.47957

(67) As can be seen in FIG. 10d and Table 7, larger pores were formed with time after treatment of MDA-MB-231 cells with eMTDΔ4 (SEQ ID NO: 2), demonstrating that eMTDΔ4 (SEQ ID NO: 2) gave damage to the surface of the MDA-MB-231 cell membrane.

(68) As described hitherto, the present disclosure pertains to a cell death-inducing peptide that rapidly acts and, more particularly, to a peptide, derived from Noxa protein (SEQ ID NO: 4), consisting of 16 amino acid residues including MTD (SEQ ID NO: 3). The peptide is designated extended MTD (eMTD; (SEQ ID NO: 1)) because it contains the 10-mer MTD. eMTD (SEQ ID NO: 1) rapidly exhibits potent necrotic cell death in various cell lines and, as such, can be applied to the treatment of various diseases including cancer when used in conjugation with peptides or materials for targeting specific cells.