KR101592081B1 - Selective apoptosis of p53 deficient cancer cells or drug resistant cancer cells using non-thermal atmospheric pressure plasma - Google Patents

Abstract

The present invention relates to a selective cell death method for p53-deficient cancer cells or drug-resistant cancer cells using low-temperature atmospheric plasma, and more particularly, to a method for selectively removing apoptotic cells from p53-deficient cancer cells and drug- Because of the selective and effective induction of cell death compared to cancer cells with p53 function through the generation of species, it can be used as a new cancer treatment method because it can improve low therapeutic efficacy by existing chemotherapy.

Description

{Selective apoptosis of p53 deficient cancer cells or drug resistant cancer cells using non-thermal atmospheric pressure plasma}

The present invention relates to a selective cell death method of p53-deficient cancer cells or drug-resistant cancer cells using low-temperature atmospheric plasma.

Considering the selective induction of apoptosis in cancer cells as an ideal approach for cancer treatment, many anticancer agents with this mechanism have been developed. However, current approaches are still facing meaningful challenges to overcome drug resistance, low therapeutic efficacy and cancer cell selectivity. When cells are subjected to various genetic toxicities and cellular stresses, p53 is activated and its ubiquitin-dependent degradation is blocked, resulting in accumulation of active p53 transcription factors, cell cycle arrest, anti-oxidative and DNA repair activation, and cell death Target genes are regulated. P53-dependent induction of apoptosis in response to genotoxic damage is an important aspect of tumor suppression. Thus, loss of p53 or disruption of the pathway leading to p53 activation is mainly due to aggressive tumor behavior, often promoting the resistance of cancer cells to radiation and anti-cancer drugs. For example, irradiation with p53 ^{+ / +} mouse thymocytes results in apoptosis, whereas p53 ^{- / -} thymocyte cells show resistance. Similarly, p53 ^{+ / +} mouse embryonic fibroblasts transformed with adenovirus E1A protein and Ha-ras tumor gene undergo apoptosis in response to gamma-irradiation or anti-cancer agents, while p53 ^{- / -} Resistant. In addition, p53 mutation in cancer inhibits the function of p73 to induce apoptosis through a p53-independent mechanism. Thus, the general loss of p53 function in cancer cells represents a major limitation for chemotherapy.

Plasma is depicted as a quasi-neutral mixture of charged particles and radicals in a partially ionized gas. There are several types of NTAPPs, such as plasma needles, plasma jets, and dielectric barrier discharges (DBDs). The gas composition, field strength and pulse width determine the exact plasma composition. Recently, many studies have sought to obtain the low temperature advantages of nonthermal atmospheric pressure plasma (NTAPPs) for biomedical applications due to the ability to control plasma chemistry and kinetic. The effects of NTAPP on biotissues include sterilization, wound healing, and cell migration. Several other effects of plasma can be explained by their complex chemical composition and parameters in the plasma generation process.

Previous studies on the clinical application of NTAPP to mammalian cells have focused primarily on the effect of NTAPP on cell death. Several possible mechanisms have been reported related to NTAPP mediated cell death such as decreased cell adhesion. In particular, some researchers have demonstrated that NTAPP can induce apoptosis in certain types of cancer cells and be used in cancer therapy. There is further evidence that active oxygen species are a major player in NTAPP-induced apoptosis in vitro. The first step to develop NTAPP as a potential cancer therapy is to evaluate its selective efficacy against cancer cells.

Therefore, the present inventors have found that NTAPP can induce apoptosis in p53-deficient cancer cells but not in primary cells or tumor cells, as a result of trying to determine standardized plasma components and composition for selective cell death and cancer treatment of low temperature atmospheric plasma (NTAPP) And that NTAPP-mediated increase in intracellular ROS induces apoptotic cell death in a concentration-dependent manner, demonstrating that it can induce selective death even in drug-resistant cancer cells And thus the present invention has been completed by suggesting the potential of NTAPP as a new cancer treatment method.

Korea Open Patent No. 2012-0103293 (September 19, 2012) Japanese Laid-open Patent No. 2011-514814 (2011.05.12)

 Jae Koo et al. Jpn. J. Appl. Phys. 50 (2011.08.22)

It is an object of the present invention to provide a selective cell death method of cancer cells using low temperature atmospheric plasma.

In order to achieve the above object, the present invention includes a step of exposing p53-deficient cancer cells or drug-resistant cancer cells to a low-temperature atmospheric-pressure plasma generated in a low temperature atmospheric pressure plasma generator using a DBD (Dielectric Barrier Discharge) Thereby providing a selective cell death method of cancer cells.

The p53-deficient cancer cell means a cancer cell having a p53 gene mutation.

The drug-resistant cancer cell specifically refers to a cancer cell having resistance to an anti-cancer drug.

The low-temperature atmospheric plasma exposure of the p53-deficient cancer cell or the drug-resistant cancer cell is set at a distance of 2 to 5 cm from the plasma generation source of the low-temperature atmospheric plasma generator to the cancer cell, and the helium gas flow of 1 to 5 slm, Plasma treatment may be performed at an input voltage of 2 to 10 times for 30 seconds to 2 minutes every hour, or for 10 to 24 hours after the plasma exposure.

The low temperature atmospheric pressure plasma generator may include a He gas feeding system, an AC power source, and a DBD device covered with a plastic case to prevent active oxygen species (ROS) diffusion.

In the present invention, p53-deficient cancer cells or drug-resistant cancer cells are characterized by being killed by ROS of various species produced in the sprayed plasma.

Since the present invention selectively and effectively induces apoptosis in cancer cells that have p53 function through the production of reactive oxygen species in p53-deficient cancer cells and drug-resistant cancer cells without affecting the blastocysts or stem cells upon exposure to the cold atmospheric pressure plasma, It can be used as a new cancer treatment because it can improve the low therapeutic efficacy by chemotherapy.

FIG. 1 shows a low temperature atmospheric pressure plasma (NTAPP) generator used for selective cell death of cancer cells of the present invention.
Figure 2 shows the viability results of NTAPP exposed cells.
FIG. 3 shows results of NTAPP exposure conditions showing selective anti-proliferative effect by cell type. A, C and E show the results of NTAPP 10 repeated exposure of 5V input voltage and HeLa cell (A), ASC C, and IMR90 (E), and B, D and F show the expression of truncated caspase-3, its downstream activities PARP and gamma-H2AX, expressed in these cells .
FIG. 4 shows the anti-proliferative effect of NTAPP on cell lines derived from skin and oral epithelial cancer, wherein A and C show the effect of NTAPP 10 repeated exposure of 5 V input voltage and YD-9 cells (A) and G-361 (C), and B and D are the results of measuring the expression of truncated caspase-3, its downstream action, PARP and gamma-H2AX, expressed in these cells.
5 is NTAPP exposed p53 positive and negative tumor cells care anti-as a result of comparing the proliferative effect, (A) is a colon cancer cell line ^{HCT116 (p53 + / +),} (B) is ^{HCT116-E6 (p53 - / -} ) , (C), and (D), cancer cells (LoVo, MES-SA, HepG2 and RKO) with wild-type p53 and cancer cells without functional p53 (DLD-1, H1299, HT29 and HCT15) Proliferation of p53-negative HT29 cells transfected with HT29 cells, and the Western blot image shows the expression of transfected p53 in HT29 cells.
FIG. 6 shows the results of an anti-proliferation effect of intracellular and extracellular ROS by NTAPP exposure using an antioxidant and an ROS scavenger, (A) showing the results of monitoring intracellular ROS levels by NAC treatment after NTAPP exposure (B) shows the relative cell viability of HeLa cells according to NAC treatment concentration, (C) shows the expression of γ-H2AX, truncated caspase-3 and PARP by NAC treatment in NTAPP-exposed HeLa cells (D) shows the relative cell viability of NTAPP-exposed HeLa cells according to SP treatment concentration, (E) shows the result of monitoring intracellular ROS level by SP treatment after NTAPP exposure, (F) H2AX, truncated caspase-3 and PARP expression by SP treatment.
Fig. 7 shows the results of cytotoxicity test according to NAC treatment.
FIG. 8 shows the results of measurement of NO concentration in the medium after NTAPP exposure.
Figure 9 shows the results of evaluating the anti-proliferative effect of NO in the medium after NTAPP exposure.
Figure 10 shows the results of global modeling with variables of humidity and helium (He) moles fractions at an input power of 80 W, where (A) shows the ROS density in the plasma device for each He gas fraction in the discharge region , (B) shows the effect of humidity on the ROS density in the mixture of 99% He and 1% air in the discharge region, (C) shows the results of the arrival times of the species in the neutral gas region in contact with the culture dish , And (D) represent the number of moles of ROS reaching the culture dish per unit area and unit time in the neutral gas region in contact with the culture dish.
FIG. 11 shows anti-proliferative effect of doxorubicin-resistant cancer cells according to the additional incubation after NTAPP exposure, wherein (A) shows HCT15 / CLO2 cells and (B) shows the relative proportion of surviving cells in MES-SA / DX5 cells (C) shows HCT15 / CLO2 cells, and (D) shows the results of confirming the expression of γ-H2AX, truncated caspase-3 and PARP in MES-SA / DX5 cells.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a selective cell death method for cancer cells, which comprises culturing p53-deficient cancer cells or drug-resistant cancer cells by exposure to a low-temperature atmospheric pressure plasma generated in a low temperature atmospheric pressure plasma generator using a DBD (Dielectric Barrier Discharge) .

The selective cell death method of cancer cells of the present invention is characterized in that when cancer cells are exposed to a low-temperature atmospheric plasma, they exhibit a superior cell death effect against cancer cells having normal p53 gene, particularly p53-deficient cancer cells or drug-resistant cancer cells. This apoptosis effect is evidenced by increased levels of extracellular and extracellular reactive oxygen species by plasma exposure, and by a decrease in apoptotic effects upon antioxidant or ROS scavenging treatment.

The term " non-thermal atmospheric pressure plasma "referred to in the present specification means that the chemical reactivity to a target object is high without thermal change, and relatively energy is stable and acts only on the surface of the reacting substance. It has the advantage of not changing or damaging the state of the material.

The low-temperature atmospheric plasma generator used in the selective cell death method of cancer cells of the present invention includes a He gas feeding system, an AC power source, and a DBD device covered with a plastic case to prevent diffusion of ROS. It can be something to produce. This device has an annular-type dielectric barrier with a high-voltage sinusoidal wave or a special alternating current source for generating low-temperature atmospheric plasma under air or other types of gases when short pulse widths are applied between the electrodes, A dielectric barrier discharge (DBD) method (see FIG. 1).

The helium gas flow is provided at a rate of 1 to 5 standard liters / m (SLM) through the inlet of the low temperature atmospheric pressure plasma generator, and the gas flow rate is controlled by a mass flow controller. The AC voltage is applied to the internal electrodes of the DBD device at a peak applied for a peak voltage of 7 to 20 kV generated using a frequency of 11.4 kHz and a transformer circuit comprised of a rectifier diode and a transistor for bipolar power. The DC input voltage range is 5 to 12V. Thus, input voltages 5, 6, and 12V correspond to peak-to-peak output voltages 7.5, 8.9, and 18.8 kV, respectively.

In summary, the low temperature atmospheric pressure plasma generator is characterized by having the following applicability:

Distance from plasma source: 2-5 cm

Standard liter per minute (slm): 1-5 slm

Source: He gas

Frequency: 20 kHz

Input voltage: 5-12 V

Output voltage: 7-20 kV

The input voltage means a voltage applied to the low-temperature atmospheric plasma generator, specifically 5-12 volts.

The distance from the plasma generating source means a distance from the low temperature atmospheric plasma generator to the cultured cells. Preferably, the distance from the source can be 2-5 cm, more preferably 3 cm.

The plasma exposure condition means the number of times and time of plasma exposure to the cultured cells, and in the present invention, it is possible to expose it twice to 10 times every 30 seconds to 2 minutes.

Considering the above points, the selective cell death of the cancer cells of the present invention is performed by setting the distance from the plasma generation source of the low-temperature atmospheric pressure plasma generator to the cancer cells to 3 cm and adding 1 to 5 slm of helium gas Flow, 2 to 10 times plasma treatment for 30 seconds to 2 minutes every hour with an input voltage of 5 to 12 V, or culturing cancer cells for 10 to 24 hours after plasma exposure. These plasma exposure conditions can effectively and selectively induce apoptosis in cancer cells or drug-resistant cancer cells deficient in p53 function as compared to normal p53-functioning cancer cells, without inhibiting proliferation of normal cells and stem cells. .

Plasma generated in the low-temperature atmospheric pressure plasma apparatus generates various kinds of ROS in extracellular and intracellular cells, and cell death of p53-deficient cancer cells or drug-resistant cancer cells can be induced by the ROS through the cell apoptosis mechanism.

According to one embodiment, the extracellular ROS generated in a low temperature atmospheric pressure plasma generator is at least one of OH, H, H ₂ O ₂ , HNO ₂ , HNO ₃ , HO ₂ , or H ₂ , And the most dominant species in the discharge region is oxygen atoms or ozone, the most dominant species of ROS diffused into the air.

The p53-deficient cancer cell refers to a cancer cell that lacks p53 function through mutation of p53 gene, and any cancer cell with p53 gene mutation can be used without limitation. For example, it can be used for the treatment of cancer such as carcinoma, lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma, schwanoma, meningioma, Adenocarcinoma, melanoma, leukemia, lymphoid malignancy, squamous cell cancer, epithelial squamous cell cancer, lung cancer, small cell lung cancer, Small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the lung, peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, brain cancer, glioblastoma, cervical cancer, ovarian cancer, ovarian cancer, cancer, liver cancer, bladder cancer, Cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, endometrial carcinoma, endometrial or uterine carcinoma, Cancer, kidney and renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, It may be a cancer cell with p53 gene mutation in testicular cancer, esophageal cancer, biliary tract cancer or cancer of head and neck cancer.

The drug-resistant cancer cell is a cancer cell showing resistance to an anti-cancer drug. The drug-resistant cancer cell is a drug-resistant cancer cell in which a p53 gene mutation occurs and a p53 function is lost or a drug-resistant cancer cell in which a functional p53 is expressed by a wild-type p53 gene. . For example, there may be mentioned, for example, at least one compound selected from the group consisting of mechlorethamine, cyclophosphamide, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, But are not limited to, corticosteroids, corticosteroids, streptozocin, streptozocin, carboplatin, cisplatin, oxaliplatin, vincristine, vinblastine, binolelin, paclitaxel, docetaxel, etoposide, tenisopeed, irinotecan, topotecan, doxorubicin, Methotrexate, 5-fluorouracil, wormsidyne, cytarabine, capecitabine, gemcitabine, 6-fluorouracil, ditaminomycin, plicamycin, mitomycin, But are not limited to, mercaptopurine, mercaptopurine, 6-thioguanine, cladribine, fludarabine, nelarabine, pentostatin, ironotecan, topotecan, amsacrine, etoposide, etoposide phosphate, beauty Tan, may be asparaginase, the first page Gaspard, estradiol mu sustaining, Beck captured X, cancer cells that are resistant to anti-cancer drugs, such as isotretinoin or tretinoin (ATRA).

The selective cell death method of cancer cells of the present invention can be used directly as an anti-cancer therapy or induce apoptosis of cancer cells or drug-resistant cancer cells lacking p53 function effectively, so that the therapeutic efficacy can be improved by using them in combination with existing chemotherapy have.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Selective Cell Death of Cancer Cells Using Low Temperature Atmospheric Pressure Plasma (NTAPP) Exposure

First, an NTAPP generator for exposing NTAPP to living cells was designed (Fig. 1). Compared to direct plasma devices such as plasma jets, which transfer charged particles directly to the target, this device mainly produces ROS in plasma. The direction of the electric field is perpendicular to the direction of the gas flow, so that the generated ROS is widely diffused, but the charged particles are trapped within the plasma generation region. In addition, the NTAPP generator of FIG. 1 can be used to generate a high-voltage sinusoidal wave or, when a short pulse width is applied between at least two electrodes that are insulated, It is an annular type DBD (dielectric barrier discharge) system. The insulator suppresses current formation between the electrodes while generating an electrically safe plasma without substantial gas heating. In addition, the NTAPP generator is connected to a He gas-feeding system, an AC power supply, and a DBD device covered with a plastic case to reduce ROS diffusion. The He gas flow delivered to the inlet of this device is at a rate of 1 to 5 standard liters / m (SLM) and is controlled by a mass flow controller (MKP, MPR-2000). The AC voltage was applied to the inner electrode of the DBD device at a peak applied for a peak voltage of 7 to 20 kV generated using a transformer circuit (Ramsey Electronics, PG13 plasma generator kit) having a frequency of 11.4 kHz and a transistor for rectifying diode and bipolar power. . The DC input voltage range is 5-12 V, designed as a control parameter to change the voltage applied to the DBD device. For example, input voltages 5, 6, and 12V correspond to peak-to-peak output voltages 7.5, 8.9, and 18.8 kV, respectively.

HeLa cells (human cervical carcinoma) and Hep G2 (human liver carcinoma) cells used for selective cell death of cancer cells were treated with 10% (v / v) fetal bovine serum (FBS) and 10 mL / L penicillin-streptomycin The added Dulbecco? modified Eagle? medium (DMEM). G361 (melanoma), HCT116 (human colon carcinoma), HCT116 P53 ^{- / -} (human colon carcinoma), HCT15 (human colon carcinoma), MES-SA (human uterine sarcoma), H1299 (Non-kind small-cell lung cancer), (HCT15 / CL02, human colorectal carcinoma and MES-SA / dx5, human uterine sarcoma) were treated with 10% (v / v) / v) FBS and 10 mL / L penicillin-streptomycin-supplemented RPMI-1640. IMR90 (human lung fibroblasts) and RKO (human colon carcinoma) cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% (v / v) FBS and 10 mL / L penicillin-streptomycin. Adipose derived stem cells (ASCs) were cultured in DMEM / Ham's F-12 (DMEM / F12) supplemented with 10% (v / v) FBS and 10 mL / L penicillin-streptomycin . YD-9 (human oral carcinoma) cells were cultured in DMEM: DMEM / F12 (1: 1) supplemented with 10% (v / v) FBS and 10 mL / L penicillin-streptomycin.

All cells were maintained at 37 ° C under humidification conditions of 5% CO ₂ . To expose the cells to 1 × 10 ⁵ cells NTAPP seeded in 35-mm culture dishes and incubated for 24 hours. NTAPP at a specific dose (5 standard L / min [SLM], 5 V) was exposed to cells for up to 10 times every 30 seconds to 1 minute every hour. When necessary, NTAPP-exposed cells were further cultured for 15 hours before another experiment.

The Western blotting used for the analysis of intracellular p53 expression was performed as follows.

NTAPP-exposed cells were harvested, lysed as described (Kim et < RTI ID = 0.0 > al . , 2010b, Biochemical and biophysical research communications 400: 739-744) and histones were extracted with 0.6N HCl. Whole protein samples (50 μg) or histones were incubated with anti-caspase-3 (PARP), cell signaling technology (PARP), anti-actin (Sigma-Aldrich) And analyzed with anti-phospho-H2AX (gamma-H2AX) serum (Millipore). Western wasabi peroxidase-conjugated anti-mouse and anti-rabbit (Jackson Immuno Research) secondary antibodies were used, and the treated membranes were visualized using an ECL kit (Amersham Biosciences).

For intracellular ROS detection, HeLa cells (1 × 10 ⁵ ) were seeded in a 35-mm culture dish equipped with a glass cover slip and exposed to 5 times NTAPP for 30 seconds every hour for a total of 7 repetitions, Invitrogen) was used to assess intracellular ROS levels according to the manufacturer ' s instructions. Non-fluorescent carboxyl-H ₂ DCFDA is readily permeable to living cells and is oxidized by intracellular ROS to release a bright green fluorescent signal. Tert-butyl hydroperoxide (TBHP), which is known to induce intracellular ROS, was used as a positive control. N-acetyl cysteine (NAC) and sodium pyruvate (SP) were used as intracellular and extracellular ROS scavengers, respectively.

The accumulation of nitrite, a stable metabolite of nitric oxide secreted by the culture medium, was measured to detect the production of extracellular nitric oxide after NTAPP exposure.

To this end, the cells were exposed to NTAPP and then 50 ㎕ of culture medium was obtained and incubated with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2% phosphoric acid (phosphoric acid) and incubated at room temperature for 10 minutes. Absorbance was measured at 540 nm using a standardization curve in the range of 0-100 μM concentration of NaNO _{2 with} a microplate reader (SoftMax Pro 4.0, Molecular Devices). In the nitric oxide scavenging experiment, the cells were pretreated with carboxy-PTIO (Sigma-Aldrich) at 30, 50 or 100 μM concentrations stoichiometrically reacting with nitric oxide prior to exposure to NTAPP.

To demonstrate the inverse correlation between the function of p53 and the anti-proliferative effect of NTAPP in cancer cells, the number of viable cells after NTAPP exposure was compared in p53-expressing vector and vector-only transfected p53-negative HT29 cells as a control group.

2 μg of the plasmid pcDNA-p53-HA (obtained from J. Song, Yonsei University) was added to 6 μl of the linear L-PEI (polyethylenimine) for transfection of pcDNA-p53-HA and mixed with 150 mM NaCl Incubation was carried out at room temperature for 10 minutes. The mixture was treated with HT29 cells and incubated overnight.

Because it is difficult to experimentally measure all the species present in the NTAPP, plasma engineers can use time-dependent continuous equations for each species to determine the extent to which not only charged particles but also spatially normalized species (Liu et < RTI ID = 0.0 > al . , 2010, Plasma Sources Sci Technol 19: 025018; Sakiyama et al . , 2012, J Phys D: Appl Phys 45: 425201). The excitation domain is divided into two regions, a plasma source region and a neutral gas region without a plasma. The latter region is contacted with a culture dish containing cells. Except for the addition of the helium species used as the buffer gas for the NTAPP apparatus used in the experiment, the chemical reactions considered are as described in Sakiyama et al. ( J Phys D: Appl Phys 45: 425201). The total number of species is 68 and the total number of reactions is 826.

All data are expressed as mean ± standard deviation (SEM) of independent experiments at least 3 times. In addition, t-test was applied to evaluate statistically significant differences. p <0.05 (*) and p <0.01 (**) mean statistical significance compared with the control group.

<Experimental Example 1> Effect of NTAPP on human cell type

To determine the optimal exposure conditions of NTAPP for inhibiting cancer cell proliferation, the effect of NTAPP intensity on cell viability in normal cells and cancer cells was first evaluated. First, NTAPP was applied once per minute to cancer cell line HepG2, normal embryonic lung fibroblast cell line WI38 and ASCs at an input voltage (5? 10 V), then cells were incubated for 24 hours for MTT analysis to determine cell viability Respectively. In this experiment, the distance between the NTAPP device and the cell (3 cm) and the gas flow (5 SLM) were fixed.

As shown in Fig. 2, no difference in viability was observed in treated and untreated HepG2 cells, but WI38-VA13 and ASC cells were slightly increased compared to untreated control cells. Suggesting that NTAPP exposure would induce other physiological responses in non-cancerous and cancer cells.

Next, we attempted to identify suitable NTAPP conditions that inhibit proliferation in cancer cells but not in normal cells.

As shown in FIG. 3A, when HeLa cells were exposed to 5V NTAPP for 30 seconds every hour for a maximum of 9 hours (10 repeated NTAPP exposures), the relative proportion of viable cells compared to unexposed control cells (145%) But slightly increased in exposed cells (134%). In addition, differences in relative survival cells between NTAPP treated and untreated HeLa cells were more pronounced after 10 NTAPP exposure than when cells were further incubated for 15 hours. In other words, the relative percentage of surviving cells was an additional 15 hours incubation after 10 repeated NTAPP exposures compared to 200% in the untreated control (incubation time 0 is the start time of NTAPP exposure in FIG. 3 and incubation time 24 is the time after NTAPP exposure The incubation time was 15 hours, which is the same in the following figures).

Interestingly, relative cell counts were increased in NTAPP exposed cells compared to untreated control when NTAPP of the same conditions was applied to follicular IMR90 and ASCs (FIGS. 3C and E). The relative percentages of viable cells in 10 additional NTAPP exposures and additional incubated ASC and IMR90 cells for 15 hours were 178% and 168%, respectively, while those of untreated cells were approximately 150% (FIGS. 3C and E). This strongly suggests that the plasma exposure conditions selectively inhibit the proliferation of HeLa cells and the correct NTAPP exposure conditions (NTAPP repeated exposure and additional incubation at 5V input voltage) to activate proliferation in IMR90 cells and ASCs.

Then, to test whether reduced HeLa cell proliferation after repeated NTAPP exposure was due to apoptosis, the cleavage of caspase-3, which is activated by apoptosis, and its downstream action, PARP, was monitored.

3 and PARP were detected in HeLa cells but not in IMR90 or ASC cells when exposed to 10 repeated 5V NTAPP for 30 seconds, respectively (Fig. 3B). As shown in the relative cell numbers (Figures 3A, C and E), truncated caspase-3 and PARP were significantly increased by an additional 15 hour incubation in HeLa cells, but not in ASCs or IMR90 cells 3B, D and F). These results demonstrate that NTAPP treatment induces apoptosis in HeLa cells but not normal ASCs or IMR90 cells.

Considering that genetic instability caused by DNA damage is a major cause of apoptosis, we examined whether NTAPP exposure induces DNA damage in apoptotic HeLa cells. Phosphorylation of histone H2AX was one of the first cellular events to occur in response to DNA double strand breaks, thus confirming its expression.

Cancer cells, including HeLa cells, showed low basal level of γ-H2AX expression in the absence of any stress, but increased significantly in NTAPP-exposed HeLa cells (FIG. 3B). Surprisingly, no expression of gamma-H2AX was detected in ASCs or IMR90 blasts (Figures 3D and F). This suggests that NTAPP exposure selectively induces DNA damage leading to apoptosis of HeLa cells.

Because NTAPP exposure selectively induces apoptosis in HeLa cells but not IMR90 cells or ASCs, it includes melanoma-derived melanoma G361 and oral epithelial cancer YD-9 cells found on the surface of the body where plasma treatment can be applied immediately To test the effect of NTAPP on different types of cancer cells.

As shown in FIGS. 4A and 4C, 15 hour incubation after 10 repeated exposures of 5 V NTAPP resulted in an anti-proliferative effect on G-361 and YD-9 cells as compared to untreated control cells. NTAPP exposure also induced the expression of y-H2AX by DNA damage and apoptosis in G-361 and YD-9 cells (Fig. 4B and D). These results, as in HeLa cells, demonstrate that NTAPP exposure induces apoptotic cell death in melanoma and oral epithelial cancer cells, suggesting the potential for plasma exposure to skin and oral cancer.

Although all cell types eventually undergo apoptosis under the same NTAPP conditions, its anti-proliferative effect was greater in HeLa cells than in G-361 and YD-9 cells (Figures 3A and B). γ-H2AX phosphorylation and apoptosis were observed in HeLa cells when repeated NTAPP stimulation was applied, but were observed in G-361 and YD-9 cells only when further incubated for 15 hours after NTAPP exposure. HeLa, G-361, and YD-9 cells are all cancer cells, but HeLa cells are deficient in functional p53. Given that NTAPP induces apoptosis through DNA damage and that p53 is a major tumor suppressor in response to genotoxic stress, the presence or absence of functional p53 in HeLa, G-361 and YD-9 cells suggests that NTAPP- We could make a difference in the proliferative effect. (p53 ^{+ / +} ) and HCT116-E6 (p53 ^{- / -} ) with the exact same genetic background, except functional p53, to demonstrate the relationship between p53 function and the sensitivity of cancer cells to NTAPP Anti-proliferative effects were compared.

When HCT116 (p53 ^{+ / +} ) and HCT116-E6 (p53 ^{- / -} ) were further incubated for 15 hours after 10 repeats of 5V NTAPP stimulation, the relative ratios of viable cells, under the same conditions used in the preceding NTAPP treatment, (P53 ^&lt; ^{+ &gt;} ) and HCT116-E6 (p53 ^{- / -} ) as compared to cells (Fig. 5A and B). Interestingly, HCT116-E6 (p53 ^{- / -} ) cells showed hypersensitivity to NTAPP compared to HCT116 (p53 ^{+ / +} ). That is, the relative proportion of viable cells was 80% in HCT116-E6 cells and 140% in HCT116 cells compared to 180% in the untreated control (FIGS. 5A and B). These results are consistent with the NTAPP effect observed in HeLa, YD-9, and G361 cells, suggesting that cancer cells without functional p53 are significantly more sensitive to NTAPP than cancer cells with functional p53.

More than 50% of cancer cells have a p53 gene mutation that induces functional defects of p53. Since the deficiency of functional p53 often causes cancer cells to be resistant to gamma-ray or anticancer drugs, NTAPP's very special term for cancer cells without functional p53 - The proliferation effect is very interesting. To confirm the very specific anti-proliferative effect of NTAPP on cancerous cells without functional p53, the relative proportion of surviving cells was tested in various cancer cell types with or without p53 function after NTAPP treatment.

The number of viable cells was reduced by NTAPP exposure in both p53-positive and negative cells compared to cells not exposed to NTAPP (Fig. 5C). As expected, the anti-proliferative effect of NTAPP was much greater in cancer cells (DLD-1, H1299, HT29, and HCT15) without functional p53 than in cancer cells with wild-type p53 (LoVo, MES-SA, HepG2 and RKO) It was effective. That is, the relative proportion of surviving cells was approximately 60-80% in p53-negative cancer cells, while approximately 120-140% in p53-positive cancer cells (Fig. 5C). These results demonstrate that the anti-proliferative effect of NTAPP is highly selective for p53-negative cancer cells.

To further demonstrate the inverse correlation between the function of p53 and the anti-proliferative effect of NTAPP in cancer cells, the number of viable cells after NTAPP exposure was compared in the p53-expressing vector and the vector-only transfected p53-negative HT29 cells . Expression of transfected p53 in HT29 cells was confirmed by Western blot (Fig. 5D).

The HT29 cancer cell line did not contain functional p53 and its viability decreased sharply after NTAPP treatment (Figure 5C). However, the relative number of surviving cells is increased in HT29 cells expressing wild-type p53 to the level observed in other p53-proficient cancer cells (Fig. 5C), but in the vector-only transfected HT29 cells, There was no change (Fig. 5D). These results demonstrate that cell viability is restored in p53-transfected cancer cells. In short, this strongly suggests that the anti-proliferative effect of NTAPP is very favorable towards p53-deficient cancer cells, so that NTAPP is an effective treatment for cancer cell resistance to gamma-ray or anticancer drugs due to a deficiency of functional p53.

<Experimental Example 2> Anti-proliferative role of ROS caused by NTAPP

Previous studies by several research groups suggest that ROS is a major player in plasma-induced cellular responses. In the above Examples and Experimental Examples, NTAPP proved to have selective anti-proliferative effect by inducing DNA DSBs and apoptosis in cancer cells, particularly p53-deficient cancer cells. Therefore, we wanted to know whether the selective anti-proliferative effect of this NTAPP is due to ROS, and if so, intracellular or extracellular ROS mediates the biological effects of NTAPP. For this purpose, NAC, a well - known thiol antioxidant, and SP, a well known clearing agent for hydrogen peroxide (H ₂ O ₂ ), were used. First, intracellular ROS levels were monitored in HeLa cells exposed to NTAPP using DCFDA, an oxidation sensitive fluorescent dye. TBHP treated cells were used as a positive control for intracellular ROS.

As shown in FIG. 6A, intracellular ROS accumulated rapidly in HeLa cells after 7 repeated 30 second exposures (every hour exposure) with 5V NTAPP as compared to the untreated control. Conversely, treatment with 5 mM NAC effectively blocked intracellular ROS accumulation in HeLa cells exposed to the same NTAPP conditions. Before using NAC, the cytotoxicity of NAC was evaluated and it was confirmed that 5 mM NAC did not affect cell viability (FIG. 7).

To test the role of intracellular ROS in the anti-proliferative effect of NTAPP, the relative cell viability of HeLa cells treated with NTAPP or not in the presence of NAC concentrations (3 and 5 mM) was evaluated.

As shown in Figure 6B, HeLa cell viability was abruptly reduced by NTAPP exposure, but was restored by the presence of NAC in a concentration-dependent manner. In other words, the relative proportion of surviving cells (100%) to NTAPP untreated cells was 70% at 5 mM NAC, 60% at 3 mM NAC, and 40% without NAC treatment. These results demonstrate that intracellular ROS produced by NTAPP plays a major role in the anti-proliferative effect of NTAPP.

Taking this into consideration, γ-H2AX phosphorylation by DNA damage and apoptosis was delayed in the presence of NAC when comparing DNA damage and apoptosis in HeLa cells exposed to NTAPP with or without 5 mM NAC (FIG. 6C ). The anti - proliferative effect of intracellular ROS generated from NTAPP was further confirmed using SP, an intracellular ROS scavenger. When HeLa cells were exposed to NTAPP in the presence of SP, intracellular ROS decreased and the relative number of viable cells increased in a concentration-dependent manner compared to NTAPP-untreated cells not treated with SP (Figures 6D and 6D) E).

The relative percentage (100%) of surviving cells to the untreated control was 85% at 10 mM SP, 75% at 5 mM, 70% at 3 mM compared to only 50% in HeLa cells without SP 6E). Γ-H2AX phosphorylation by DNA damage and apoptosis was also delayed and decreased in the presence of SP as in NAC (FIG. 6F).

Overall, these results support the idea that intracellular ROS produced by NTAPP will play a major role in inducing DNA damage and apoptosis to block cancer cell proliferation.

NTAPP exposure increases both intracellular and extracellular ROS. Therefore, we tried to determine whether the increase in plasma in extracellular ROS directly affects the anti-proliferative effect of NTAPP in cancer cells. In particular, it was of interest that NO was easily produced when the plasma met N ₂ and O ₂ in air. In other words, NO is known to be an important cellular messenger substance involved in many physiological and signaling pathways in mammals. Therefore, cells were exposed to NTAPP and NO was detected in the medium using Griess reagent assay.

As shown in Fig. 8, the NO concentration in the medium after NTAPP treatment was increased by 7-8 times as compared with the untreated control, and the NO increased in the incubated medium for 15 hours after NTAPP exposure.

(4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO), which is a NO scavenger, was used to evaluate the anti- (Fang et &lt; RTI ID = 0.0 &gt; al . , 2006, Acta Anesthesiol Taiwan 44: 141-146). The relative viability of HeLa cells was tested after treatment with NTAPP or without treatment at the concentration of carboxy-PTIO in the medium (30, 50, or 100 μM).

As shown in NTAPP-exposed HeLa cells, the relative percentage of viable cells was reduced to about 60% by NTAPP exposure compared to untreated cells (100%) (Figure 9). However, there was no difference in viability after NTAPP exposure without addition of carboxy-PTIO (Fig. 9), suggesting that extracellular NO produced by NTAPP does not affect the anti-proliferative effect of NTAPP. In addition, these results suggest that intracellular ROS produced by NTAPP exposure plays an important role in inducing apoptosis and anti - proliferative effects in cancer cells, but not extracellular NO.

<Experimental Example 3> Characterization of composition and composition of NTAPP

Since the ROS produced by NTAPP plays an essential role in the selective anti-proliferative effect of NTAPP in cancer cells, it is necessary to measure the intensity and active species of the plasma generated from the NTAPP generator of the present invention. Plasma components and composition are essential information for reproduction, and the mechanism of action is needed to develop NTAPP as an anti-cancer therapy. However, many ROS do not emit light that can be detected in optical emission spectroscopy, and mass spectrometry is also difficult to use at this pressure, so it is important to measure each element generated at this plasma system, Challenges are underway. Therefore, global modeling is commonly used for the analysis of NTAPP reaction kinetic and plasma chemistry under given operating conditions. Thus, the inventors used global modeling to analyze the plasma chemistry of the device.

Figure 10 shows the results of global modeling with variables of humidity and helium (He) mole fraction at 80 W input power. The basis of the global model used here is very similar to that disclosed in the Sakiyama et al. In the case of dry air with a humidity of 0, the water-related reactions were excluded and the total number of species and the total number of reactions were reduced to 43 and 44, respectively. As Sakiyama et al. Showed two different stimulation regions for the discharge layer and the neutral gas domain, the stimulation domains of the discharge region and the neutral gas region were considered separately.

Figs. 10A and B are the results of the discharge region, and Figs. 10C and D are the results in the neutral gas region in contact with the culture dish. 10A shows the ROS density inside the plasma device for each He gas fraction that can be controlled by varying the gas flow rate. Since the critical energy for interaction with ROS is lower than that of He, the density of ROS is mainly determined by the amount of air, and the most dominant species within the discharge region due to the electron collision process of O ₂ and O ₃ are oxygen atoms to be. When the mixed air fraction is greater than 1%, the most species are oxygen atoms, nitrogen oxides and ozone.

Figure 10B shows the effect of humidity on ROS density in a mixture of 99% He and 1% air. The estimated molar fraction of water vapor is 1% and corresponds to 30% relative humidity at room temperature. The change in ROS density is not significant with the inclusion of the effect of humidity except that OH, H, H ₂ O ₂ , HNO ₂ , HNO ₃ , HO ₂ , and H ₂ are additionally present.

The ROS generated in the plasma apparatus diffuse into the air and, as in the graph of Fig. 10C, they were evaluated by time of arrival of the species from the plasma nozzle to the culture dish after a diffusion time of 0.1 ms. After the diffusion of ROS through the air, the predominant species is ozone instead of oxygen atom, and easily attached to the O ₂ generates O _3.

Finally, Figure 10D shows the number of moles of ROS reaching the culture dish per unit area and unit time. The total number of moles can be evaluated by multiplying the surface area of the dish by the total time of action. For example, if the dish diameter is 3.5 cm and the operating time is 30 seconds, the overall multiplication factor is 289. Therefore, the maximum number of moles of ozone delivered to the medium is approximately 30 mmol. The transport of ROS in the liquid medium is not well understood and is very difficult to calculate, but to some extent is referred to for the rate of reaction of hydrated electrons and hydroxyl radicals as well as the amount of transferring in aqueous solution. If we assume that 10-30% of the gaseous ROS is delivered to the liquid medium, the total molar number in the liquid is in the range of 3-9 mmol. This figure is related to the content of ROS scavenger used to control intracellular ROS in FIG.

Experimental Example 4 Anti-proliferative effect of NTAPP on doxorubicin-resistant cancer cells

A major hurdle in chemotherapy is overcoming the drug-resistance of cancer cells. Since NTAPP demonstrated selective anti-proliferative effect on cancer cells and could be a potent anti-cancer therapy, it was tested whether NTAPP exposure inhibited the proliferation of anti-cancer drug-resistant cancer cells. For this, MES-SA / dx5 cells originated from HCT15 / CL02 derived from two different doxorubicin-resistant cancer cells host colon cancer and human uterine sarcoma were used. Doxorubicin is a chemotherapy commonly used in the treatment of a wide range of cancers, including hematologic tumors, various types of carcinomas and soft-tissue sarcomas.

10 Repeat 30 sec. When exposed to NTAPP (1 exposures per hour) of 5V input voltage and further incubated for 15 h, at the same exposure conditions as those used in the other cancer cells above, the relative percentage of viable cells (200%), both significantly decreased (less than 150%) in HCT15 / CLO2 and MES-SA / DX5 cells (Figs. 11A and B). In addition, NTAPP exposure induced γ-H2AX phosphorylation by DNA damage and apoptosis in HCT15 / CLO2 and MES-SA / DX5 cells (FIGS. 11C and D). These results indicate that NTAPP also induces apoptosis and inhibits the proliferation of doxorubicin-resistant cancer cells, suggesting that NTAPP can be an effective chemotherapy even in cancer cells resistant to known drug therapies.

Claims (6)

controlling the exposure conditions of the low-temperature atmospheric-pressure plasma generated in the low-temperature atmospheric-pressure plasma generator of a DBD (Dielectric Barrier Discharge) method using an AC power source in culturing p53-deficient cancer cells or drug-resistant cancer cells,
The exposure conditions were set at a distance of 2 to 5 cm from the plasma generation source of the low-temperature atmospheric pressure plasma generator to the cancer cells, and a helium gas flow of 1 to 5 slm, an input voltage of 5 to 12 V for 30 seconds to 2 Wherein the cancer cells are cultured for 10 to 24 hours after 2 to 10 times of plasma exposure. The method according to claim 1,
The p53-deficient cancer cells may be selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma, schwanoma, meningioma meningioma, adenocarcinoma, melanoma, leukemia, lymphoid malignancy, squamous cell cancer, epithelial squamous cell cancer, lung cancer, Small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, brain cancer, glioblastoma, cervical cancer, ovarian cancer, ovarian cancer, liver cancer, bladder cancer (b laryngeal cancer, ladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary cancer, gland carcinoma, kidney and renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, cancer cells that are selected from the group consisting of pancreatic cancer, penile carcinoma, testicular cancer, esophageal cancer, biliary tract cancer, and head and neck cancer. A method for selective cell death of cancer cells in vitro. The method according to claim 1,
The drug resistant cancer cells may be selected from the group consisting of mechlorethamine, cyclophosphamide, chlorambucil, melphalan, isoparmid, thiotepa, hexamethylmelamine, hexadecanol, altretamine, procavagin, dacarbazine, temozolomide, Carnosine, carmustine, streptozocin, carboplatin, cisplatin, oxaliplatin, vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, tenisopeed, irinotecan, topotecan, doxorubicin, daunoru But are not limited to, bisphosphonates, bisphosphonates, epimersin, epinephrine, epinephrine, epinephrine, epinephrine, epinephrine, But are not limited to, 6-mercaptopurine, 6-thioguanine, cladribine, fludarabine, nelalabine, pentostatin, ironotecan, topotecan, amsacrine, etoposide, etoposide phosphate, Selective apoptosis of cancer cells in vitro, which is a cancer cell resistant to anti-cancer drugs selected from the group consisting of urea, mitotane, asparaginase, pegasperazine, estramustine, bexarotene, isotretinoin and tretinoin (ATRA) Way. delete The method according to claim 1,
A low temperature atmospheric plasma generation apparatus includes a He gas feeding system, an AC power source, and a DBD device covered with a plastic case to prevent diffusion of reactive oxygen species (ROS). The method according to claim 1,
wherein the p53-deficient cancer cell or the drug-resistant cancer cell is killed by ROS produced in the sprayed plasma.

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