JP2013142070A - Overcoming of trail resistivity using mitochondria division inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To overcome TRAIL resistivity in a cancer cell, particularly, in a malignant tumor.SOLUTION: The fact that a mitochondria division inhibitor can be utilized for overcoming the TRAIL resistivity of a cancer cell has been found. TRAIL induced apoptosis can be reinforced in a tumor showing TRAIL resistivity by using a mitochondria division inhibitor together with TRAIL. The agent and method effectively kill a cancer cell with TRAIL resistivity, but does not show cytotoxicity to a normal cell at all or shows the same only little. Thus, by using the agent or method, the prognosis of a cancer patient is improved, or the QOL of a TRAIL therapy subject can be improved.

Description

  The present invention belongs to the field of pharmaceuticals that enhance the sensitivity to anticancer agents containing TRAIL.

  Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as APO-2L, is a member of the tumor necrosis factor superfamily and exhibits antitumor activity against a wide variety of cancer cells. In recent years, it has attracted attention because it does not show cytotoxicity to most normal cells and tissues (Non-patent Document 1). TRAIL is a member of the death ligand, binds to the death receptor on the target cell, and induces apoptosis of the target cell via the so-called death receptor pathway. Death receptor 4 (death receptor 4 (DR4), also known as TRAIL-R1; TNFRSF10A) and death receptor 5 (death receptor 5 (DR5), also known as TRAIL-R2; TNFRSF10B) are identified as death receptors that specifically bind to TRAIL Has been.

  However, malignant tumors such as chronic lymphocytic leukemia, astrocytoma, meningioma, pancreatic cancer, melanoma, neuroblastoma, etc. are not affected by TRAIL resistance (TRAIL resistance / resistance to TRAIL). Moreover, it is known that when TRAIL protein is continuously administered, TRAIL-resistant cancer cells are selected and amplified, and eventually the subject acquires TRAIL resistance (Non-patent Document 2). Therefore, overcoming the TRAIL resistance of these cancer cells is a very important issue for clinical application of TRAIL.

  As drugs to overcome TRAIL resistance, DNA cross-linking agent oxaliplatin (non-patent document 3), PI3K inhibitor PI103 (non-patent document 4), proteasome inhibitor MG132 (non-patent document 5), DNA cross-linking Cisplatin (non-patent document 6), mangrove-derived compound (non-patent document 7), HDAC inhibitor MS-275 (non-patent document 8), Raf kinase inhibitor sorafenib (non-patent document 9) , Plant phenylpropanoid intermediate metabolite naringenin (Non-Patent Document 10), proteasome NF-κB inhibitor bortezomib (Non-patent Document 11), histone acetylase p300 inhibitor Garcinol (Garcinol) (Non-patent document 12), adenosine receptor A3 agonist Cl-IB-MECA (non-patent document 13), HDAC inhibitor trichostatin A (trichostatin A) (non-patent document 14), hedgehog signal-specific inhibitor KAAD- Cyclopamine (KAAD-Cyclopamine) (Non-patent Document 15) HDAC inhibitor apicidin (Non-patent document 16), HDAC inhibitor KD5170 (Non-patent document 17), antibiotic taurolidine (Non-patent document 18), Frigigocandin B (Non-patent document 19) ), Proteasome inhibitor benzyloxycarbonylleucyl-leucyl-leucine aldehyde, MG-132 (Non-patent document 20), HDAC inhibitor valproic acid (Non-patent document 21), Golden Flower (Scutellaria baicalensis) -derived flavonoid Wogonin (Non-patent document 22, Non-patent document 23), curry dye curcumin (non-patent document 24), BIR domain binding oligopeptide (BIR Domain Binding oligopeptide) (Patent Document 1) has been reported.

  Anti-cancer drugs such as DNA cross-linking agents, HDAC inhibitors, and proteasome inhibitors have side effects on normal cells and tissues, but show synergism when coadministered with TRAIL and exert anticancer effects with acceptable side effects There is a possibility. However, the TRAIL sensitivity enhancing action by these compounds has low selectivity for cancer cells, and is not an ideal TRAIL sensitivity enhancing agent.

  Fas et al. Show that ougonin enhances TRAIL-induced apoptosis in Jurkat cell lines (human leukemia T-cell line), but not TRAIL-induced apoptosis in freshly isolated peripheral blood T cells. (Non-patent Document 22). In addition, Lee et al. Reported similar cancer cell selective effects in a system using LNCaP cells (cultured cell lines derived from human prostate cancer cells) and YPEN cell lines (derived from rat prostate endothelial cells) ( Non-patent document 23). Lee et al. Further show that coadministration of ougonin and TRAIL leads to the cleavage of inducible caspases caspase 8 and caspase 9, and caspase 3, an executioner caspase, and ougonin produces reactive oxygen species. This suggests that the reactive oxygen species cause DNA damage and that p53 is up-regulated by the DNA damage (Non-patent Document 23).

  The death receptor pathway described above is called the extrinsic apoptotic program or the receptor-activated apoptotic pathway and is one of two major apoptotic pathways. The other pathway is called the intrinsic apoptotic program or the stress-activated apoptotic pathway, and it is between mitochondrial membranes via mitochondrial outer membrane permeabilization (MOMP). With the release of cytochrome c from the cavity into the cytoplasm. Cytoplasmic cytochrome c forms an apoptosome with Apaf-1 and caspase 9, and activates caspases 3, 6, and 7, which are operative caspases.

  These extrinsic and intrinsic apoptotic pathways are known to cross-talk with each other through activation of caspase 8 by death receptors, followed by activation of executive caspases and BID.

  Cassidy-Stone et al. Show that the mitochondrial mitotic inhibitor mdivi-1 selectively inhibits mitochondrial dynamin Drp1, and mdivi-1 attenuates apoptosis by inhibiting Bax / Bak-dependent MOMP. Indicated. (Non-patent document 25). Due to this apoptosis inhibitory effect, it has attracted attention as a therapeutic agent for diseases involving abnormal cell death such as stroke, myocardial infarction, and neurodegenerative diseases.

  Surprisingly, the present inventors have found that mdivi-1 having an apoptosis inhibitory effect enhances TRAIL-induced apoptosis of cancer cells when used in combination with TRAIL. When TRAIL and mdivi-1 were used in combination, activation of caspase 8, which is a feature of apoptosis via the death receptor pathway, was not observed, and endoplasmic reticulum stress and activation of endoplasmic reticulum-bound caspase 12 were induced, and the endoplasmic reticulum pathway It is suggested that apoptosis is induced via the pathway via the present invention (unpublished data of the present inventors, data not shown).

[Explanation of abbreviations]
TRAIL: tumor necrosis factor-related apoptosis-inducing ligand
APO-2L: Apo-2 ligand, another name for TRAIL
DR4: death receptor 4
DR5: death receptor 5
TRAIL-R1: TNF-related apoptosis inducing ligand receptor 1; another name for TRAIL receptor 1, DR4
TRAIL-R2: TNF-related apoptosis inducing ligand receptor 2; another name for TRAIL receptor 2, DR5
TNFRSF10A: tumor necrosis factor receptor superfamily member 10A, also known as DR4
TNFRSF10B: tumor necrosis factor receptor superfamily member 10B, also known as DR5
HDAC: histone deacetylase
QOL: quality of life
MOMP: mitochondrial outer membrane permeabilization

  The problem to be solved by the present invention is to overcome TRAIL resistance in cancer cells, particularly malignant tumors.

  The inventors have found that mitochondrial mitotic inhibitors can be used to overcome the TRAIL resistance of cancer cells. By using a mitochondrial mitotic inhibitor in combination with TRAIL, TRAIL-induced apoptosis can be enhanced in tumors exhibiting TRAIL resistance.

Specifically, aspects of the present invention include the following.
[Embodiment 1]
A medicament for increasing selective TRAIL-induced apoptosis of cancer cells, comprising an effective amount of an inhibitor of mitochondrial fission:
Here, the medicament is administered simultaneously or sequentially with the TRAIL polypeptide.
[Embodiment 2]
The medicament according to embodiment 1, wherein the mitochondrial division inhibitor is represented by the following general formula:
Where R ₁ is selected from the group consisting of Cl and CF ₃ ;
R ₂ is selected from the group consisting of H and Cl;
R ₃ is selected from the group consisting of H, OCH ₃ , and OCH (CH ₃ ) ₂ ;
R ₄ is selected from the group consisting of H and CH ₃ and
R ₅ is selected from the group consisting of H and Cl.
[Embodiment 3]
The medicament according to embodiment 1, wherein the mitochondrial division inhibitor is mdivi-1.
[Embodiment 4]
From embodiment 1, wherein the cancer cells are cancer cells derived from non-small cell lung cancer, glioblastoma, chronic lymphocytic leukemia, astrocytoma, meningioma, pancreatic cancer, melanoma, or neuroblastoma 4. The medicine according to any one of 3.
[Embodiment 5]
5. The medicament according to any one of embodiments 1 to 4, wherein the administration is application, intravenous injection, intramuscular injection, or oral administration.
[Embodiment 6]
The medicament according to any one of embodiments 1 to 5, wherein the amino acid sequence of the TRAIL polypeptide is the same as that of a human-derived TRAIL polypeptide.
[Embodiment 7]
Embodiment 7. The medicament according to any one of embodiments 1 to 6, further comprising an effective amount of a TRAIL polypeptide.
[Embodiment 8]
A pharmaceutical kit for increasing selective TRAIL-induced apoptosis of cancer cells comprising the following components:
An effective amount of an inhibitor of mitochondrial fission, and
A statement describing that simultaneous or sequential administration with a TRAIL polypeptide increases selective TRAIL-induced apoptosis of the cancer cells.
[Embodiment 9]
The kit according to embodiment 8, wherein the mitochondrial division inhibitor is represented by the following general formula:
Where R ₁ is selected from the group consisting of Cl and CF ₃ ;
R ₂ is selected from the group consisting of H and Cl;
R ₃ is selected from the group consisting of H, OCH ₃ , and OCH (CH ₃ ) ₂ ;
R ₄ is selected from the group consisting of H and CH ₃ and
R ₅ is selected from the group consisting of H and Cl.
[Embodiment 10]
The kit according to embodiment 8, wherein the mitochondrial division inhibitor is mdivi-1.
[Embodiment 11]
Embodiment 8 from which the cancer cell is a cancer cell derived from non-small cell lung cancer, glioblastoma, chronic lymphocytic leukemia, astrocytoma, meningioma, pancreatic cancer, melanoma, or neuroblastoma The kit according to any one of 10 above.
[Embodiment 12]
The kit according to any one of embodiments 8 to 11, wherein the administration is application, intravenous injection, intramuscular injection, or oral administration.
[Embodiment 13]
The kit according to any one of embodiments 8 to 12, wherein the amino acid sequence of the TRAIL polypeptide is the same as that of a human-derived TRAIL polypeptide.
[Embodiment 14]
14. The kit according to any one of embodiments 8 to 13, further comprising an effective amount of a TRAIL polypeptide.
[Embodiment 15]
A method of increasing selective TRAIL-induced apoptosis of cancer cells comprising the following steps:
contacting the cancer cell with an effective amount of a mitochondrial division inhibitor in vivo or ex vivo;
Here, the cancer cells are treated simultaneously or sequentially with the TRAIL polypeptide.
[Embodiment 16]
The method of embodiment 15, wherein the mitochondrial division inhibitor is represented by the general formula:
Where R ₁ is selected from the group consisting of Cl and CF ₃ ;
R ₂ is selected from the group consisting of H and Cl;
R ₃ is selected from the group consisting of H, OCH ₃ , and OCH (CH ₃ ) ₂ ;
R ₄ is selected from the group consisting of H and CH ₃ and
R ₅ is selected from the group consisting of H and Cl.
[Embodiment 17]
Embodiment 16. The method of embodiment 15, wherein the mitochondrial division inhibitor is mdivi-1.
[Embodiment 18]
From embodiment 15, wherein said cancer cell is a cancer cell derived from non-small cell lung cancer, glioblastoma, chronic lymphocytic leukemia, astrocytoma, meningioma, pancreatic cancer, melanoma, or neuroblastoma 18. The method according to any one of items 17.
[Embodiment 19]
The method according to any one of embodiments 15 to 18, wherein the contacting step is application, intravenous injection, intramuscular injection, or oral administration.
[Embodiment 20]
The method according to any one of embodiments 15 to 19, wherein the amino acid sequence of the TRAIL polypeptide is identical to a human-derived TRAIL polypeptide.

  Do drugs and methods for enhancing TRAIL-induced apoptosis of cancer cells using mitochondrial mitotic inhibitors effectively kill TRAIL-resistant cancer cells while showing no cytotoxicity against normal cells? Or show only a few. Therefore, the prognosis of cancer patients can be improved and the QOL of TRAIL therapy subjects can be improved by using the drug or method.

It is the figure which observed the apoptosis induction effect of TRAIL, mdivi-1, and these combination with respect to the human melanoma cell A375 with the fluorescence microscope. Live: Calcein staining. Dead: Ethidium bromide homodimer staining. The left panel is a bright field image. It is the figure which observed the apoptosis induction effect of TRAIL, mdivi-1, and these combination with respect to normal melanocyte with the fluorescence microscope. Live: Calcein staining. Dead: Ethidium bromide homodimer staining. The left panel is a bright field image. It is a bar graph which shows the ratio (%) of an apoptotic cell at the time of processing human melanoma cell A375 with TRAIL, mdivi-1, and these combination. A: When treated with a final concentration of 25 ng / mL TRAIL protein. B: When treated with TRAIL protein at a final concentration of 100 ng / mL. vehicle: No mdivi-1. 25 μM Mdivi-1: When treated with 25 μM mdivi-1. 50 μM Mdivi-1: When treated with 50 μM mdivi-1. ** represents p <0.01, *** represents p <0.001 (Anova and Tukey post hoc test).

  [Definition]

  Effective amount: The effective amount (final concentration) of the mitochondrial division inhibitor is, for example, 1 to 999 μM, 1 to 607 μM, 1 to 368 μM, 1 to 223 μM, 1 to 135 μM, 1 to 82 μM, 1 to 50 μM, 2 to 999 μM, 2-607 μM, 2-368 μM, 2-223 μM, 2-135 μM, 2-82 μM, 2-50 μM, 3-999 μM, 3-607 μM, 3-368 μM, 3-223 μM, 3-135 μM, 3-82 μM, 3- 50 μM, 5-999 μM, 5-607 μM, 5-368 μM, 5-223 μM, 5-135 μM, 5-82 μM, 5-50 μM, 9-999 μM, 9-607 μM, 9-368 μM, 9-223 μM, 9-135 μM, 9-82 μM, 9-50 μM, 15-999 μM, 15-607 μM, 15-368 μM, 15-223 μM, 15-135 μM, 15-82 μM, 15-50 μM, 25-999 μM, 25-607 μM, 25-368 μM, 25- 223 μM, 25-135 μM, 25-82 μM, 25-50 μM, preferably 25-50 μM (8.8-17.6 μg / ml in the case of mdivi-1).

Mitochondrial Mitosis Inhibitor: In one embodiment, mitochondrial fission inhibitor refers to a compound represented by the general formula:
Where R ₁ is selected from the group consisting of Cl and CF ₃ ;
R ₂ is selected from the group consisting of H and Cl;
R ₃ is selected from the group consisting of H, OCH ₃ , and OCH (CH ₃ ) ₂ ;
R ₄ is selected from the group consisting of H and CH ₃ and
R ₅ is selected from the group consisting of H and Cl.

Examples of mitochondrial mitotic inhibitors particularly suitable for the present invention include compounds represented by the following chemical formula (Non-patent Document 25).

mdivi-1: A compound represented by the following chemical formula: IUPAC name: 3- (2,4-dichloro-5-methoxyphenyl) -2-sulfanylidene-1H-quinazolin-4-one.

  Cancer cell: In the present specification, the term “cancer cell” means any cancer cell. In one embodiment of the invention, the term cancer cell refers to any cancer cell that exhibits TRAIL resistance. In other embodiments of the invention, the term cancer cell is derived from non-small cell lung cancer, glioblastoma, chronic lymphocytic leukemia, astrocytoma, meningioma, pancreatic cancer, melanoma, or neuroblastoma Means cancer cells.

  Selective: As used herein, “selective” TRAIL-induced apoptosis of cancer cells means that TRAIL-induced apoptosis occurs more frequently in cancer cells compared to normal cells.

  TRAIL-induced apoptosis: As used herein, “TRAIL-induced apoptosis” refers to apoptosis induced by TRAIL protein on cancer cells.

  Increase: In this specification, when `` increasing '' TRAIL-induced apoptosis, when a mitochondrial mitosis inhibitor is used in combination with TRAIL, it is more frequently observed in cancer cells compared to TRAIL alone. It means that induced apoptosis occurs.

  TRAIL polypeptide: In the present specification, the term “TRAIL polypeptide” means a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). From the viewpoint of effect, the TRAIL polypeptide used in the present invention preferably has an amino acid sequence derived from the same species as the subject. Further, from the viewpoint of safety and economy, the TRAIL polypeptide is preferably recombinant.

  Simultaneous: As used herein, “simultaneous” administration simply refers to the combined use of a mitochondrial inhibitor and a TRAIL polypeptide, and need not be exactly simultaneous.

  Sequential: As used herein, “sequential” administration means that the mitochondrial mitotic inhibitor and the TRAIL polypeptide are intentionally administered with a certain time difference.

  Administration: Mitochondrial mitosis inhibitor or TRAIL polypeptide is preferably applied by application, intravenous, intramuscular, or oral for skin cancer such as melanoma, and intravenous, intramuscular, or oral for other cancers. preferable.

  [Experimental materials and experimental methods]

<Mitochondrial division inhibitor>
Mdivi-1 was purchased from Enzo Life Sciences, Inc. (Cat. No. BML-CM127-0050).

<Cells and culture conditions>
Human melanoma cells (A375 cells) were cultured at 37 ° C. in 5% CO ₂ /95% air using 10% FBS / DMEM (High glucose). Human leukemia cells (Jurkat cells) were cultured in 10% FBS / DRPMI1640 at 37 ° C. in 5% CO ₂ /95% air.

<TRAIL protein>
Soluble human recombinant TRAIL (500 μg / ml) (Killer TRAIL ^™ , Cat. No. ALX-201-073-3020, Enzo Life Sciences, Inc.) was stored at −80 ° C. This was diluted to a final concentration of 6.3 to 100 ng / ml at the time of use.

<Apoptosis assay>
A375 cells adhered to 24-well plates or suspended Jurkat cells (2 × 10 ⁵ cells / well) were mixed with Mdivi-1 (final concentration: 8.8 to 17.6 μg / ml) and TRAIL (final concentration: 25 to 100 ng). / ml) was added alone or in combination and incubated at 37 ° C. for 24 hours. After washing the cells, the percentage of apoptotic (Annexin V ⁺ ) cells stained with FITC-Annexin V Apoptosis Detection Kit I (BD Pharmingen) was measured with a flow cytometer FACSCalibur (BD Bioscience, San Jose, CA). The data was analyzed with CellQuest software (BD Bioscience).

<Fluorescence microscope observation>
Adivided on A375 cells (1 × 10 ⁴ cells) adhered to 8-chamber cover glass (Asahi Glass Co., Tokyo, Japan) with Mdivi-1 (final concentration: 8.8 to 17.6 μg / ml) and TRAIL (final concentration: 25 to 100 ng / ml) was added alone or in combination. After cell washing, cell death was examined using LIVE / DEAD (registered trademark) Viability / Cytotoxicity Kit (Invitrogen). That is, cells were stained at 37 ° C. for 30 minutes with 4 μM calcein-AM ethidium bromide homodimer, and then observed with a fluorescence microscope (Olympus IX71 inverted microscope, Olympus, Tokyo, Japan). Analysis was performed using LuminaVision software (Mitani Corporation, Fukui, Japan).

[Example 1]
The effects of human recombinant TRAIL on human melanoma cells A375 and normal melanocytes are shown in FIGS. 1 and 2, respectively. Live cells are stained green with calcein and dead cells are stained red with ethidium bromide homodimer. When TRAIL (100 ng / ml) alone was treated for 20 hours, almost no cell death was observed, confirming that human melanoma cell A375 was TRAIL resistant (FIG. 1, TRAIL).

  Mitochondrial mitosis inhibitor mdivi-1 (final concentration 17.6 μg / ml (50 μM)) alone did not cause cell death (Fig. 1, Mdivi), but significantly enhanced the apoptosis-inducing effect of TRAIL (Fig.1, TRAIL + Mdivi).

  On the other hand, TRAIL alone, mdivi-1 alone, or a combination of both showed little cytotoxicity against normal melanocytes (FIG. 2).

[Example 2]
In the same experiment as in Example 1, cells stained with annexin V-FITC and propidium iodide (PI) were analyzed with a flow cytometer and shown as a percentage of the total cell number in FIG. Human melanoma cell A375 was resistant to 25 ng / mL or 100 ng / mL TRAIL (vehicle), but co-administration of 25 μM or 50 μM mdivi-1 induced apoptosis in a concentration-dependent manner (25 μM Mdivi-1, 50 μM Mdivi-1). Between 25 and 100 ng / mL, TRAIL showed some concentration dependence, and 100 ng / mL induced apoptosis in more cells.

  A medicine containing a mitochondrial mitotic inhibitor can be used in the pharmaceutical industry, the medical industry, the research reagent manufacturing industry, and the like.

WO / 2004/072641

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Claims (20)

A medicament for increasing selective TRAIL-induced apoptosis of cancer cells, comprising an effective amount of an inhibitor of mitochondrial fission:
Here, the medicament is administered simultaneously or sequentially with the TRAIL polypeptide. The medicament according to claim 1, wherein the mitochondrial division inhibitor is represented by the following general formula:
Where R ₁ is selected from the group consisting of Cl and CF ₃ ;
R ₂ is selected from the group consisting of H and Cl;
R ₃ is selected from the group consisting of H, OCH ₃ , and OCH (CH ₃ ) ₂ ;
R ₄ is selected from the group consisting of H and CH ₃ and
R ₅ is selected from the group consisting of H and Cl.   2. The medicament according to claim 1, wherein the mitochondrial division inhibitor is mdivi-1.   The cancer cell is a cancer cell derived from non-small cell lung cancer, glioblastoma, chronic lymphocytic leukemia, astrocytoma, meningioma, pancreatic cancer, melanoma, or neuroblastoma. 4. The medicine according to any one of 3.   5. The medicament according to any one of claims 1 to 4, wherein the administration is application, intravenous injection, intramuscular injection, or oral administration.   6. The medicament according to any one of claims 1 to 5, wherein the amino acid sequence of the TRAIL polypeptide is the same as that of a human-derived TRAIL polypeptide.   The medicament according to any one of claims 1 to 6, further comprising an effective amount of a TRAIL polypeptide. A pharmaceutical kit for increasing selective TRAIL-induced apoptosis of cancer cells comprising the following components:
An effective amount of an inhibitor of mitochondrial fission, and
A statement describing that simultaneous or sequential administration with a TRAIL polypeptide increases selective TRAIL-induced apoptosis of the cancer cells. The kit according to claim 8, wherein the mitochondrial division inhibitor is represented by the following general formula:
Where R ₁ is selected from the group consisting of Cl and CF ₃ ;
R ₂ is selected from the group consisting of H and Cl;
R ₃ is selected from the group consisting of H, OCH ₃ , and OCH (CH ₃ ) ₂ ;
R ₄ is selected from the group consisting of H and CH ₃ and
R ₅ is selected from the group consisting of H and Cl.   9. The kit according to claim 8, wherein the mitochondrial division inhibitor is mdivi-1.   The cancer cell is a cancer cell derived from non-small cell lung cancer, glioblastoma, chronic lymphocytic leukemia, astrocytoma, meningioma, pancreatic cancer, melanoma, or neuroblastoma. The kit according to any one of 10 above.   12. The kit according to any one of claims 8 to 11, wherein the administration is application, intravenous injection, intramuscular injection, or oral administration.   The kit according to any one of claims 8 to 12, wherein the amino acid sequence of the TRAIL polypeptide is the same as that of a human-derived TRAIL polypeptide.   14. The kit according to any one of claims 8 to 13, further comprising an effective amount of a TRAIL polypeptide. A method of increasing selective TRAIL-induced apoptosis of cancer cells comprising the following steps:
contacting the cancer cell with an effective amount of a mitochondrial division inhibitor in vivo or ex vivo;
Here, the cancer cells are treated simultaneously or sequentially with the TRAIL polypeptide. The method of claim 15, wherein the mitochondrial mitotic inhibitor is represented by the general formula:
Where R ₁ is selected from the group consisting of Cl and CF ₃ ;
R ₂ is selected from the group consisting of H and Cl;
R ₃ is selected from the group consisting of H, OCH ₃ , and OCH (CH ₃ ) ₂ ;
R ₄ is selected from the group consisting of H and CH ₃ and
R ₅ is selected from the group consisting of H and Cl.   16. The method according to claim 15, wherein the mitochondrial division inhibitor is mdivi-1.   The cancer cell is a cancer cell derived from non-small cell lung cancer, glioblastoma, chronic lymphocytic leukemia, astrocytoma, meningioma, pancreatic cancer, melanoma, or neuroblastoma. 18. The method according to any one of items 17.   The method according to any one of claims 15 to 18, wherein the contacting step is application, intravenous injection, intramuscular injection, or oral administration.   20. The method according to any one of claims 15 to 19, wherein the amino acid sequence of the TRAIL polypeptide is the same as that of a human-derived TRAIL polypeptide.