Patent Application
20200011852
The present invention provides a method for determining an optimal effective amount of vitamin C capable of exhibiting an anticancer effect without proliferating cancer cell, by predicting the vitamin C absorption level of cancer cell obtained from cancer patient, and a method for predicting response for vitamin C treatment.
Vitamin C, known as an antioxidant, acts as a pro-oxidant in cancer cells at a high dose and selectively kills cancer cells. Various studies have found that the anticancer effect of vitamin C inhibits cell proliferation and growth through a reactive oxygen species (ROS) production and hydrogen peroxide-mediated mechanism in an in vitro system.
ROS produced in cancer cells causes cell damage and induces oxidative stress in cancer cells with redox state and metabolism. In addition, the pharmaceutical dosage of vitamin C acting as an oxidation promoter showed an anticancer effect in an in vivo system together with production of ascorbate radicals. Many researchers have conducted studies to understand the mechanism of high-dose vitamin C treatment, and as a result, they have hound that the anticancer mechanism of vitamin C affects cytochrome c release in mitochondria and produces ROS to induce apoptosis.
Historically, vitamin C as a cancer therapeutic agent was first proposed by Linus Pauling and Ewan Cameron in 1976. Previous studies have shown that high-dose vitamin C treatment increases the mean survival time. However, other studies such as mayo clinic have shown that vitamin C treatment has no effect on cancer patients. The debate over this vitamin C cancer treatment began with a clash between clinical results of Linus Pauling's research and mayo clinic research.
To overcome this controversy, a number of studies have been conducted to elucidate the anticancer effect and mechanism of vitamin C. Some studies have found that the expression of vitamin C transporter family 2 (SVCT-2) is a crucial factor in cancer treatment of vitamin C, and have confirmed that a hypoxia-inducible factor-positive cell is sensitive to vitamin C treatment, and in addition, there are other studies for developing a vitamin C cancer therapy in combination with chemotherapeutic agents or other drugs.
Although a number of researches have revealed the anticancer mechanism of vitamin C, and have developed a more effective application of vitamin C cancer treatment, it has not to be found yet the reason why the result of mayo clinic showed a lower survival rate in patient groups treated with vitamin C than the placebo group.
The present inventors have tried to clarify the cause of the conflicting results, in which when treating vitamin C in an SVCT-2 expressing cancer cell line, not only the anticancer effect is shown in some patients while others do not exhibit the anticancer effect, but also the disease is rather worsened, and they have conducted the present study to clarify the criteria for screening cancer patients capable of vitamin C treatment.
One embodiment of the present invention relates to a method determining response of cancer patients for vitamin C treatment comprising (1) measuring the vitamin C absorption level of cancer cells obtained from the cancer patients; (2) classifying the cancer patients into a patient having a low absorption level to the vitamin C and a patient having a high absorption level to the vitamin C, by using the vitamin C absorption level of the cancer cells; and (3) determining whether the cancer patients have cancer deterioration or side effects by vitamin C, when the cancer patients are treated with the vitamin C of which the amount does not produce an amount of reactive oxygen species (ROS) being capable of inducing the cancer cell death effectively.
Another embodiment of the present invention relates to a method for screening of anticancer agent candidates comprising (a) measuring the vitamin C absorption level of cancer cells obtained from cancer patients; (b) treating the vitamin C of which the amount does not produce an amount of reactive oxygen species (ROS) being capable of inducing the cancer cell death to the cancer cell having a low absorption level to the vitamin C, and culturing them to facilitate the proliferation of cancer cells; (c) treating anticancer agent candidates to the cultured cancer cell and culturing them; (d) measuring the expression level of any one or more selected from the group consisting of Cyclin D1, CDK4, c-Myc, Ki-67, and E2F1 in the cancer cell treated with the anticancer agent candidates; and (e) determining the candidate as an anticancer agent, when the marker expression level of the cultured cancer cell which is treated with anticancer agent candidates is decreased compared to a cancer cell which is not treated with the anticancer agent candidates.
Other embodiment of the present invention relates to a cancer treatment method comprising measuring a vitamin C absorption level of cancer cells of a patient having a cancer disease; classifying the patient into a vitamin C insensitive group, when the measured vitamin C absorption is a standard value or less, and classifying the patient into a vitamin C sensitive group, when the measured vitamin C absorption is over the standard value; and administering vitamin C into the patient, wherein the vitamin C insensitive group is a case where the relative protein expression of SVCT-2 to the total protein of cancer cells is 0.04% or less, and wherein 1 mM or more of vitamin C is administered when the patient is the vitamin C insensitive group in the administering vitamin C.
Other embodiment of the present invention relates to a method for predicting an effect of vitamin C on cancer treatment, comprising measuring a vitamin C absorption level of a cancer cell of a patient having a cancer disease.
Other embodiment of the present invention relates to a kit for predicting cancer treatment response of vitamin C, comprising means for measuring expression of sodium-dependent vitamin C transporter 2 (SVCT-2) in a cancer cell.
A vitamin C (vitamin C, AA) high-dose therapy produces reactive oxygen species (ROS) and selectively damages cancer cells, thereby showing an anticancer effect. Such an anticancer effect of vitamin C is determined by a transporter of vitamin C, sodium-dependent vitamin C transporter 2 (SVCT-2).
The present inventors have demonstrated that when vitamin-C is treated to a cell line expressing SVCT-2 at a high level with different gradients of concentration (10 μM-2 mM), depending on this, an effective anticancer effect is shown. However, in a cell line expressing SVCT-2 at a low level, when treating a high dose of vitamin C (>1 mM), the anticancer effect was shown, but when treating a low dose of vitamin C (<10 μM), cancer cells were rather proliferated. In other words, the complete opposite conflicting results were shown depending on the concentration of vitamin C to be treated in cancer cells, and this was called the hormetic response.
As confirmed in the following experimental examples, the hormetic response was observed in the SVCT-2 high-expressing cell line treated with an SVCT-based inhibitor, and the hormetic response was not shown for low-dose of vitamin C. In other words, the present inventors have confirmed that the hormetic response occurred in a dose-dependent manner to vitamin C together with the expression level of SVCT-2 of cancer cells.
In addition, the hormetic response was shown in an SVCT-2 expressing cancer cell line producing insufficient ROS at an amount incapable of inducing death of cancer cells due to low vitamin C absorption. Additionally, through molecular analysis, it was confirmed that the expression of Ki-67 and other cancer proliferation markers increased in the hormetic response. Such results show that the anticancer effect of vitamin C is dependent on SVCT-2 expression and show that vitamin C plays two roles in cancer cells.
Previously, some clinical results in studies related to anticancer treatment using vitamin C still remained questionable. Specifically, (1) in the mayo clinic research, when administering a sufficient amount of vitamin C into various cancer patients, the survival rate of some patients administered with high-dose vitamin-C was lower than the placebo group, but the cause of this result remained unclear. (2) the cause of the anticancer activity of vitamin C which may or may not change, when the concentration of vitamin C administered in the body in plasma is reduced, and the concentration of vitamin C consistently remained low in blood for about 4 hours by constant oxidation of vitamin C in a short period of time, has not been revealed yet.
The present inventors have revealed that these questions can be explained by the hormetic response which is a dual dose response in a pharmaceutical concept, and can be explained by a U-shaped curve graph.
The present inventors have assumed that the reason, why these conflicting effects are shown in cancer cells treated with vitamin-C, is a change by hormetic proliferation in a dose-dependent manner of vitamin C together with an SVCT-2 expression level of cells when ROS is insufficiently produced in cells. It was predicted that since the insufficient ROS at a level incapable of inducing death of cancer cells facilitates proliferation of cancer cells through the activity of insulin-like growth factor-1 (IGF-1) and Ras genes, vitamin C insufficiently absorbed in cancer cells could not produce ROS in an amount capable of inducing death of cancer cells and may rather cause proliferation of cancer cells.
The present inventors have investigated the effect of vitamin C on cancer cells, after treating vitamin C to cancer cells with a pharmacokinetic concentration gradient (1 μM˜2 mM). As a result, it was confirmed that SVCT-2 acted as a transporter of vitamin C, and the expression of SVCT-2 in cells and the anticancer effect of vitamin C were proportional. In addition, the present inventors have tried to interpret the result of mayo clinics and the previous clinical result, and have revealed that vitamin C perform two kinds of different functions depending on the absorption level of vitamin C by SVCT-2 expression in cancer cells.
In other words, as the result of the experiment, when vitamin C is treated to cancer cells at a high concentration, it acts as an anticancer agent regardless of the expression level of SVCT-2 of cancer cells, but when vitamin C is treated at a low concentration, the hormetic cancer cell proliferation response has occurred in the SVCT-2 low-expression cell line. Specifically, in the SVCT-2 high-expression cell line, even in case of treating vitamin C at a low concentration, the cell growth inhibition and apoptosis response have occurred. This experimental result means that the vitamin-C treatment acts as an effective chemotherapy in the cancer cell line absorbing vitamin C sufficiently in cancer cells while it rather activates proliferation of cancer in the cancer cell absorbing vitamin C insufficiently.
As confirmed in the following examples and experimental examples, when treating vitamin C to the SVCT-2 low-expression cell line at a low concentration (10 μM), the relative expression of Cyclin D1 which is an important factor affecting proliferation and prognosis of cancer cells increased. Cyclin D1 plays an important role in attracting a transcriptional factor such as E2F1, and inhibits p300 to control transcription. From this, it has been confirmed that Cyclin D1 induced by vitamin C at a low concentration is an important factor in causing the hormetic proliferation response, and it has been confirmed that Cyclin D1 and CDK4 co-localization are important factors inducing cell proliferation (
In addition, as the result of cell viability analysis, it has been confirmed that the expression increases of c-Myc, Ki-67 and E2F1 functions as a Cyclin D1-related proliferation marker. c-Myc and Ki-67 bind to DNA to increase the cell proliferation activity.
From this result, it has been confirmed that vitamin C exhibits a characteristic as an effective chemotherapeutic agent in the SVCT-2 high-expression cancer cells and damages cancer cells sufficiently in the SVCT-2-low-expression cancer cells when treating vitamin C at a high dose, but the treatment of vitamin C at an insufficient dose stimulates cyclin-D1-mediated cancer cell proliferation.
In other words, the present inventors have demonstrated that the cancer therapy to treat a high dose of vitamin C is an effective therapy for SVCT-2 high-expression cancer cells, and it has few side effects in patients, and have revealed that not only it has a lower effect in SVCT-2 low-expression cancer patients, but also it may be a risk for cancer patients as it rather facilitates proliferation of cancer. In addition, the present inventors have demonstrated that the method for treating cancer by treating vitamin C requires a sufficient amount of vitamin C at a level incapable of inducing proliferation of cancer cells in SVCT-2 low-expression cell line, and requires careful vitamin C treatment concentration control and treatment. In addition, the present inventors have demonstrated that more effective cancer treatment is possible and the previous controversy over vitamin C cancer treatment can be overcome, by using the cancer treatment by high-dose vitamin C treatment, and SVCT-2 inducible agents or chemotherapeutic agents inducing a synergy effect in combination.
Hereinafter, the present invention will be described in more detail.
One embodiment of the present invention relates to a method for determining response of cancer patients for vitamin C treatment. The method for determining response of cancer patients for vitamin C treatment may comprise (1) measuring the vitamin C absorption level of cancer cells obtained from the cancer patients; (2) classifying the cancer patients into subgroup based on the measured vitamin C absorption level; and (3) determining whether the subgroup of cancer patients have a negative response by vitamin C, when the cancer patients are treated with the vitamin C of which the amount does not produce an amount of reactive oxygen species (ROS) being capable of inducing the cancer cell death effectively. It may further comprise administering 1 mM or more of vitamin C to the cancer patients, when the cancer patients are identified into subject cancer patients having a negative response by vitamin C. The negative response by vitamin C may be no treatment effect, cancer deterioration, or side effects by vitamin C. The cancer patients may be classified into a subgroup having cancer cells with a low absorption level to the vitamin C, and a subgroup having cancer cells with a high absorption level to the vitamin C in the step (2).
According to one embodiment of the present invention, the measuring the vitamin C absorption level of the step (1) may be measuring the relative protein expression of sodium-dependent vitamin C transporter 2 (SVCT-2) to the total protein of cancer cells, but the method is not particularly limited.
According to one specific embodiment, the step (2) may be classifying the cancer patients into a patient having a low absorption level to the vitamin C when the relative protein expression of SVCT-2 to the total protein of the cancer cells is in the range of 0.05(%) or less, 0.0001 to 0.05(%), 0.001 to 0.05(%), 0.01 to 0.05(%), 0.015 to 0.05(%), 0.02 to 0.05(%), 0.04(%) or less, 0.0001 to 0.04(%), 0.001 to 0.04(%), 0.01 to 0.04(%), 0.015 to 0.04(%), or 0.02 to 0.04(%), and may be classifying them into cancer cells having a low absorption level, for example, in case of the SVCT-2 low-expression cancer cell line in which the expression of SVCT-2 is 25 ng or less, 0.1 to 25 ng, 1 to 25 ng, 5 to 25 ng, 10 to 25 ng, 20 ng or less, 0.1 to 20 ng, 1 to 20 ng, 5 to 20 ng, or 10 to 20 ng of the cancer cell total protein 50 μg.
Meanwhile, the step (2) may be classifying the cancer patients into a patient having a high absorption level to the vitamin C when the relative protein expression of SVCT-2 to the total protein of the cancer cells is in the range of 0.04(%) or more, more than 0.04(%), more than 0.04 to 1.0(%), more than 0.04 to 0.1(%), more than 0.04 to 0.08(%), more than 0.04 to 0.076(%), 0.045(%) or more, more than 0.045(%), 0.045 to 1.0(%), 0.045 to 0.1(%), 0.045 to 0.08(%), 0.045 to 0.076(%), 0.05(%) or more, more than 0.05(%), 0.05 to 1.0(%), 0.05 to 0.10(%), 0.05 to 0.9(%), 0.05 to 0.08(%), or 0.05 to 0.076(%), and may be classifying them into cancer cells having a high absorption level, for example, in case of the SVCT-2 low-expression cancer cell line in which the expression of SVCT-2 is 20 ng or more, more than 20 ng, 20 to 50 ng, 20 to 45 ng, 20 to 40 ng, 25 ng or more, more than 25 ng, 25 to 50 ng, 25 to 45 ng, or 25 to 40 ng of the cancer cell total protein 50 μg.
The step (3) may be determining the subgroup having cancer cells with a low absorption level to the vitamin C as a cancer patient having negative response by the vitamin C
The step (3) may be determining the subgroup having cancer cells with a high absorption level to the vitamin C as a cancer patient having no cancer deterioration or side effects by the vitamin C.
The cancer patent having negative response by the vitamin C may be a patient treated with a small amount of vitamin C of which the amount does not produce reactive oxygen species (ROS) at a level at which an effective dose of vitamin C can induce death of cancer cells.
The meaning of the “a small amount of vitamin C” means the amount of vitamin C treatment that produces only insufficient reactive oxygen at a level incapable of reaching that cancer cells finally die by facilitating production of reactive oxygen in cancer cells, when treating sufficient vitamin C to cancer cells.
For example, when the expression of SVCT-2 of cancer cells extracted from cancer patients is lower than 20 ng, or lower than 25 ng of the total protein 50 μg, the amount of vitamin C concentration to be treated less than 2 mM at a level of 300 mL or less, 250 mL or less, 1 mL to 250 mL, 5 mL to 250 mL, or 10 mL to 250 mL may be considered as the small amount.
According to one specific embodiment, the type of the cancer is not particularly limited, but it may be one or more cancers selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer and brain tumor, and it may be preferably colorectal cancer or breast cancer, more preferably colorectal cancer.
According to one embodiment of the present invention, a kit for predicting cancer treatment response of vitamin C, comprising means for measuring expression of sodium-dependent vitamin C transporter 2 (SVCT-2) in cancer cells, may be provided.
The means for measuring expression of SVCT-2 may be one for measuring an expression level of mRNA of SVCT-2 gene or its protein, and methods commonly used in the related art may be used.
Specifically, as the result of measuring the relative protein expression of SVCT-2 to the total protein of cancer cells using the kit, when it is in a range of 0.0001 to 0.05, 0.001 to 0.05, 0.01 to 0.05, 0.02 to 0.05, 0.0001 to 0.04, 0.001 to 0.04, 0.01 to 0.04, or 0.02 to 0.04, cancer patients may be distinguished into a cancer patient having cancer cells having a low absorption level to vitamin C, and it may be predicted that the cancer patient rather has a risk of deterioration of prognosis of cancer or side effects by cancer cell proliferation when treating a small amount of vitamin C.
The meaning of the small amount of vitamin C means the content of vitamin C in a content incapable of producing reactive oxygen species (ROS) at a level capable of inducing death of cancer cells.
On the other hand, as the result of measuring the relative protein expression of SVCT-2 to the total protein of the cancer cells using the kit, when it is in a range of 0.045 to 1.0, preferably 0.05 to 0.10, more preferably 0.05 to 0.08, the most preferably 0.05 to 0.07, cancer patients may be distinguished into a cancer patient having cancer cells with a high absorption level to vitamin C, and it may be predicted that the cancer patient shows the anticancer effect in proportion to the treated concentration of vitamin C and has low possibility of side effects such as cancer cell proliferation and the like.
According to another embodiment of the present invention, provided is a method for screening of anticancer agent comprising (a) measuring the vitamin C absorption level of cancer cells obtained from cancer patients; (b) contacting the cancer cells having a low absorption level to the vitamin C with the vitamin C of which the amount does not produce an amount of reactive oxygen species (ROS) being capable of inducing the cancer cell death, and culturing them to facilitate the proliferation of cancer cells; (c) treating the cultured cancer cells with anticancer agent candidates and culturing them; (d) measuring the expression level of any one or more marker selected from the group consisting of Cyclin D1, CDK4, c-Myc, Ki-67, and E2F1 in the cancer cell treated with the anticancer agent candidates; and (e) determining the candidate as an anticancer agent, when the expression level of the marker in the cultured cancer cell which is treated with anticancer agent candidates is decreased compared to a cultured cancer cell which is not treated with the anticancer agent candidates.
The type of cancer and the step of measuring the absorption level to vitamin C which obtained cancer cells of cancer patients have are the same as described above.
As confirmed in the following examples, in case of the cancer cell determined as having a low absorption level to vitamin C, when treating a small amount of vitamin C, the expression of Cyclin D1, CDK4, c-Myc, Ki-67 and E2F1 that are cancer proliferation factors increased.
Thus, in case that the expression level of any one or more of Cyclin D1, CDK4, c-Myc, Ki-67, and E2F1, preferably all the markers is reduced, when treating an anticancer agent candidate to a cancer cell in which cancer proliferation is facilitated by vitamin C, the candidate may be determined as an anticancer agent.
According to one embodiment of the present invention, a cancer treatment method comprising measuring a vitamin C absorption level of cancer cells of a patient having a cancer disease; classifying the patient into a vitamin C sensitive group, or a vitamin C insensitive group; and administering vitamin C into the patient.
The vitamin C insensitive group is a case where the relative protein expression of SVCT-2 to the total protein of cancer cells may be 0.05% or less, 0.0001 to 0.05%, 0.001 to 0.05%, 0.01 to 0.05%, 0.02 to 0.05%, 0.04% or less, 0.0001 to 0.04%, 0.001 to 0.04%, 0.01 to 0.04%, or 0.02 to 0.04%. The vitamin C sensitive group may be cancer patients who are not classified into the vitamin C insensitive group.
In the step of administering vitamin C, when the patient is the vitamin C insensitive group, 1 mM or more of vitamin C may be administered. In the step of administering vitamin C, when the patient is the vitamin C sensitive group, vitamin C may be administered without dose limitations.
According to one embodiment of the present invention, provided is a method of treating cancer, comprising defining an insensitive subgroup and a sensitive subgroup of cancer patients by measuring a vitamin C absorption level of cancer cells obtained from a cancer patient and classifying the cancer patient into the insensitive subgroup or the sensitive subgroup based on the measured vitamin C absorption level; and administering vitamin C into the cancer patients, wherein the insensitive subgroup shows 0.05% or less of the relative protein expression of SVCT-2 to the total protein of cancer cells in cancer cells obtained from the patient, and wherein 1 mM or more of vitamin C is administered into the cancer patients of insensitive subgroup. The concentration of vitamin C being administered into the cancer patients of insensitive subgroup may be at a concentration such that the concentration of vitamin C reaching cancer cells is 1 mM or more.
The insensitive subgroup may be a group of patients showing a low absorption level to the vitamin C, or low relative protein expression of SVCT-2 to the total protein of cancer cells in cancer cells obtained from the patient, for example 0.05(%) or less of the relative protein expression of SVCT-2 to the total protein of the cancer cells.
The sensitive subgroup may be a group of patients who are not classified as the insensitive subgroup. Specifically, the sensitive subgroup may be a group of patients showing a high absorption level to the vitamin C, or high relative protein expression of SVCT-2 to the total protein of cancer cells in cancer cells obtained from the patient, for example more than 0.05(%) of the relative protein expression of SVCT-2 to the total protein of the cancer cells.
According to one embodiment of the present invention, provided is a method of treating a cancer, comprising administering 1 mM or more of vitamin C to a patient identified as a subject cancer patient having a negative response by vitamin C. The concentration of vitamin C being administered into the cancer patients of insensitive subgroup may be at a concentration such that the concentration of vitamin C reaching cancer cells is 1 mM or more.
According to one embodiment of the present invention, any of a variety of modes of administration may be used. For example, administration may be intravenous, topical, oral, intranasal, subcutaneous, intraperitoneal, intramuscular, intratumor, intradermal, mucosal, intrarectal, intravaginal, inhalation, or aerosol.
The vitamin C of the present invention may be administered to a subject by any of many different routes. For example, the vitamin C may be administered intravenously, intraperitonealy, subcutaneously, intranasally, orally, transdermally, intradermally, intramuscularly, intravaginally, intrarectally, and via aerosol for inhalation delivery. Suitable dosing regimes may be determined by taking into account factors well known in the art including, for example, the age, weight, sex, and medical condition of the subject; the route of administration; the desired effect; and the particular conjugate and formulation employed.
According to one embodiment of the present invention, vitamin C may be administered into a patient of the insensitive subgroup at a concentration such that the concentration of vitamin C reaching cancer cells is 1 mM or more.
The present invention provides a method for predicting a cancer treatment effect using vitamin-C by measurement and evaluation of expression of SVCT-2 of cancer cell, in cancer treatment using vitamin C as an anticancer agent. With the method, it is possible to determine the optimal effective amount of vitamin-C treatment for application of vitamin-C therapy to cancer patient, and minimize side effects, thereby alleviating the economic and physical pain of the patient.
Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are intended to illustrate the present invention only, but the scope of the present invention is not limited by these examples.
Human colorectal cancer cells were cultured in RPMI1640 medium Gilbco, Cergy Pontoise, France) containing 10% bovine fetal serum (PAN Biotech, Aidenbach, Germany) and 1% Penstrep (PAN Biotech) in a humidified incubator comprising 5% CO2 under the condition of 37° C. Vitamin C was purchased from BCWORLD PHARM (BCWORLD PHARM. CO, Seoul, Korea), and phloretin was purchased from sigma Aldrich (Sigma, St. Louis, Mo., USA).
The human colorectal cancer cell lines used in the experiment were Sw480, Sw620, HCT116, HCT15, SNU-C4, SNU-05, DLD-1, LoVo, and CoLo-205 cell lines commonly used in the related art. All the colorectal cancer cell lines used in the experiment of the present invention were received from Dr. Yu Byung-Chul of National Cancer Center.
The cell viability was measured by Neutral red (sigma) assay. Cells (1×104/each well) were inoculated on a 96-well plate and were cultured for 24 hours and were treated with vitamin C for 4 hours, and then were washed with PBS (Pan Biotech), and were further cultured in RPMI1640 without vitamin C for 20 hours. They were washed twice and were stained.
According to the method provided by the manufacturer, the total RNA of cells was extracted using TRI reagent (MRC; Molecular Research Center, Cincinnati, Ohio, USA). The RNA concentration was determined using a spectrophotometer reading the absorbance at 260 nm. cDNA was synthesized using mML-V reverse transcriptase (Bioneer Co, Daejeon, Republic of Korea) according to the protocol provided by the manufacturer. Then, using SYBR Premix Ex Taq (TaKaRa, Otsu, Shinga, Japan) and Rotor-Gene Q system (Qiagen, Chadstone, Victoria, Australia), quantitative real-time PCR was carried out. Data were analyzed using Rotor-Gene Q series software version 2.3.1 (Qiagen).
The following genes were amplified using primers shown in the following Table 1:
Protein was extracted from frozen tissue using PRO-PREP protein extraction kit according to the instruction of the manufacturer. The protein concentration was measured using Bradford assay (Bio-RAD, Munich, Germany)
After denaturalizing protein 30 μg in sample buffer at 95° C. for 6 minutes, samples were loaded on 12% SDS-polyacrylamide gel and were transferred to a nitrocellulose blotting membrane. Subsequently, the membrane was blocked with 5% skim milk in Tris-buffered saline. Then, after washing it with Tris-buffered saline-0.10% Tween 20 three times, the membrane was cultured at 4° C. overnight with anti-cyclin D1 (1:2500; NB600-584; Novus biologics), anti-c-Myc (1:2500; NB200-108; Novus biologics), anti-Bax (1:2000; 2774; Cell Signaling Technology, Beverly, Mass., USA), and anti-beta-actin (1:5000; ab20272; Abcam, Cambridge, Mass., USA) antibodies. Then, after washing it with Tris-buffered saline-0.10% Tween 20 4 times for 20 minutes, the membrane was cultured with a secondary anti-rabbit, anti-rat or anti-goat antibody for 1 hour. After additional washing, immunoreactive bands exposed to ECL substrate (Pierce, Rockford, Ill., USA) and X-ray film (Agfa-Gevaert N.V, Septestraat, Mortsel, Belgium) were detected.
Cells treated with vitamin C were immobilized with 4% paraformaldehyde for 10 minutes and were washed with PBS 3 times for 5 minutes, and then permeable buffer (Biolegend, San Diego, Calif., USA) was treated for 10 minutes. Then, cells were washed with PBS 3 times and were cultured with anti-c-Myc (1:500; NB200-108, Novus biologics) antibody at 4° C. overnight. Then, after washing with PBS 4 times, cells were cultured with a secondary anti-mouse-TRITC (1:1000; ab6786; Abcam) conjugated antibody. The stained cells were observed with a confocal microscope, and the image was treated by zen black edition program.
The present inventors measured the SVCT-2 expression, vitamin C absorption and cytotoxic effect of vitamin C in various kinds of colorectal cancer cell lines.
1-1. SVCT-2 Expression in Colorectal Cancer Cell Lines
The present inventors analyzed the SVCT-2 expression in colorectal cancer cell lines (Sw480, Sw620, HCT116, HCT15, SNU-C4, SNU-05, DLD-1, LoVo, CoLo-205) using the western blot method of Experimental example 4, and the result was shown in
As shown in
1-2. Quantitative Analysis of SVCT-2 Expression in Colorectal Cancer Cell Lines
ELISA analysis was carried out in order to quantitatively measure the amount of SVCT-2 to be expressed in each cancer cell line.
Specifically, after quantifying the total protein of cancer cells, the total protein 50 μg and a recombinant SVCT-2 protein (Novousbiologics, H00009962-P01) were under serial dilution, and 0.25 mg, 0.125 mg, 0.0625 mg and 0.03125 mg were put in wells, and after that, they were diluted to 1:200 in carbonate coating buffer (10 mM NaCO3, 35 mM NaHCO3, pH 9.6) and then 100 ul each was coated on each well in a 96 well plate. The antigen coating process was progressed at 25° C. for 4 hours. Then, after washing using TBS-T, an anti-SVCT2 antibody (Novous biologics, NBP2-1339) was diluted to 1:500 in PBS per well and 100 ul per well was added, and then it was reacted at 4° C. for 16 hours. After washing using TBS-T, HRP conjugated anti-rabbit igG antibody was diluted in PBS and the concentration was matched to 20 ug/ml, and 100 ul per well was added and it was reacted at 25° C. for 1 hours. Then, after washing using TBS-T, 100 ul of TBM solution was added to each well to progress color reaction, and in 15 minutes, 100 ul of 1M H2SO4 was added to stop the color reaction, and the color reaction was confirmed, and the result was shown in
Specific quantitative data of the graph of
1-2. Cell Viability Analysis
The cell viability of each colorectal cancer cell line was measured using the method of Experimental example 2, and the result was shown in
The black bar in
The relative SVCT2 expression of cell lines was shown as lower in the order of Sw480, Sw620, LoVo, SNU-C4, HCT116, SNU-05, CoLo-205, HCT15, and DLD-1, and as the result of cell viability analysis, it was confirmed that the cytotoxicity of vitamin C was proportional to SVCT-2 expression.
1-3. Measurement of Vitamin C Absorption of SVCT-2 Expressing Colorectal Cancer Cell Lines
In order to measure the vitamin C absorption of each colorectal cancer cell line, the absorption of vitamin C in cells was analyzed with High Performance Liquid Chromatography (HPLC), and the result was shown in
In order to research a role conducted by vitamin C treated at a high concentration or low concentration to cancer cell lines, after treating vitamin C at various concentrations according to a concentration gradient to each cell line, the cell viability analysis was conducted using the method of Experimental example 2.
2-1. Hormetic Response in SVCT-2 Low-Expression Cell Lines
After treating vitamin C to SVCT-2 low-expression cell liens (HCT116, CoLo-205, HCT15, DLD-1) according to a concentration gradient (0 μm to 2 mM), the cell viability was measured, and the result was shown in
In other words, it was confirmed that when treating vitamin C at a high dose (>1 mM), the anticancer effect was shown in SVCT-2 low-expression cell lines, but by contrast, when treating vitamin C at a low dose (<10 μM), the hormetic response occurred and the proliferation of cancer cells were rather induced.
2-2. Hormetic Response in SVCT-2 High-Expression Cell Lines
As same as the SVCT low-expression cell lines, after treating vitamin C to SVCT high-expression cell lines (Sw480, Sw620, LoVo, SNU-C4) according to a concentration gradient (0 μm to 2 mM), the cell viability was measured, and the result was shown in
In other words, it was confirmed that the SVCT-2 high-expression cell lines did not show the hormetic response, and all of them showed the anticancer effect when treating high-dose and low-dose vitamin C, and the anticancer effect increased in proportion to the vitamin C treatment concentration.
2-3. qRT-PCR Gene Expression Analysis in SVCT-2 Low-Expression Cell Lines and High-Expression Cell Lines
In order to confirm the apoptosis and hormetic proliferation response by vitamin C, the quantitative real-time polymerase chain reaction (qRT-PCR) gene-expression analysis of p53, Cyclin D1, E2F1 and Ki-67 genes was conducted using the method of Experimental example 3, and the result was shown in
As a result, the expression of p53 increased when treating 10 μM and 1 mM ascorbic acid in case of SVCT-2 high-expression cell lines, and they showed apoptosis response related to it, but on the other hand, in SVCT-2 low-expression cell lines, the expression of p53 was induced only in case of treatment of 1 mM vitamin C.
As shown in
In other words, when treating a low concentration of vitamin C to SVCT-2 low-expression cell lines, the hormetic response was induced, and specifically, the expression of cyclin D1, E2F1 and Ki-67 in SVCT-2 low-expression cell lines, DLD-1 and HCT15 increased. On the other hand, in the SVCT-2 high-expression cell lines, Sw620 and Sw480, when treating vitamin C 10 μM or 1 mM, the expression of cyclin D1, E2F1 and Ki-67 was reduced.
The above results show that vitamin C induces an anticancer effect at any concentration in SVCT-2 high-expression cell lines, but in SVCT-2 low-expression cell lines, it induces the anticancer effect as the result of molecular analysis, only when a high concentration of vitamin C is treated, but it induced cell proliferation in a low concentration of vitamin C
In order to investigate ROS production, when treating vitamin C of 10 μM or 1 mM to SVCT-2 low-expression cell lines (DLD1 and HCT15), after treating each dose of vitamin C to SVCT-2 low-expression cell lines, they were stained with DCF-Da, and the ROS measurement result was shown in
Specifically, after treating vitamin C to cancer cells for 15 minutes, cells were collected and cells were cultured in PBS solution containing 20 μMd DCF-DA for 10 minutes, and after a washing process with PBS, they were analyzed by FACs.
In the SVCT-2 low-expression cell lines, DLD-1 and HCT15, ROS was produced only in case of treating 1 mM vitamin C, and it was not produced in case of treating 10 μM. In other words, it was confirmed that when treating a high concentration 1 mM of vitamin C to SVCT-2 low-expression cell lines, the anticancer effect was induced, but when treating a low concentration 10 μM of vitamin C, the anticancer effect was not induced and rather the proliferation of cancer cells was facilitated.
4-1. Measurement of Apoptosis and Proliferation in SVCT-2 Low-Expression Cancer Cell Lines Treated With Vitamin C
In order to investigate the apoptosis and proliferation of cancer cells by vitamin C treatment in the protein expression level, a low concentration or high concentration of vitamin C was treated to SVCT-2 low-expression cell lines and the western blot analysis of Bax, c-Myc, and Cyclin D1 was conducted using the method of Experimental example 4, and the result was shown in
In HCT15 and DLD-1, the expression of cancer cell proliferation factors, c-Myc and cyclin D1 increased when treating a low concentration of 10 μM vitamin C, but the expression of the factors was reduced when treating a high concentration of 1 mM vitamin C. However, Bax expression did not significantly change in both vitamin C 1 mM and 10 μM.
4-2. Confirmation of c-Myc Localization
In order to investigate the expression of cancer proliferation markers in SVCT-2 low-expression cell lines and localization where c-Myc is present in cancer cell lines, after treating vitamin C of 1 mM or 10 μM to each SVCT-2 low-expression cell line, HCT15 and DLD-1 cell lines, the immunocytochemistry of Experimental example 5 was conducted, and the result was shown in
5-1. Vitamin C Absorption Change Measurement
In order to confirm whether the hormetic proliferation response is medicated from absorption of vitamin C through SVCT-2, the present inventors treated vitamin C 1 mM or vitamin C 10 μM, and an SVCT family inhibitor, phloretin together to SVCT-2 high-expression cell lines.
At first, the concentration of phloretin to be treated to exclude cytotoxicity was determined by cell viability assay, and the result was shown in
Then, after treating 1 mM vitamin C, or treating vitamin C 1 mM and phloretin 20 μM together to each cell line of the SVCT-2 high-expression cell lines, Sw480 and Sw620, the vitamin C absorption was measured, and the result was shown in
5-2. Confirmation of Hormetic Proliferation Response in SVCT-2 High-Expression Cell Lines
After treating vitamin C of 10 μM or 1 mM, or treating vitamin C and phloretin together, to the SVCT-2 high-expression cell lines, Sw480 and Sw620, the cell viability of each cell line was measured, and the result was shown in
In other words, it was confirmed that the SVCT-2 expression inhibition in SVCT-2 high-expression cell lines by phloretin treatment induced the hormetic proliferation when treating a low concentration (10 μM) of vitamin C.
In order to confirm ROS production in SVCT-2 high-expression cell lines in the apoptosis response and hormetic response, and more specifically confirm the cause of the result of Example 5, the present inventors investigated ROS production using DCF-Da staining in Sw620 cell line and Sw480 cell line, and the result was shown in
Specifically, after treating vitamin C to cancer cells for 15 minutes, cells were collected and cells were cultured in PBS solution containing 20 μMd DCF-DA for 10 minutes, and after a washing process with PBS, they were analyzed with FACs.
In other words, when treating 1 mM vitamin C or treating 10 μM vitamin C, ROS was produced respectively in SVCT-2 high-expression cell lines, Sw620 cell line and Sw480 cell line, but when inhibiting the expression of SVCT-2 by treating phloretin, ROS was not sufficiently produced.
In order to analyze the hormetic proliferation response induced by SVCT-2 inhibited by phloretin treatment in Example 5, the expression of cancer proliferation markers and BAX expression were analyzed using the qRT-PCR of Experimental example 3 and western blot method of Experimental example 4.
7-1. qRT-PCR Analysis of Cancer Proliferation Markers and BAX Expression
After treating vitamin C 10 μM and phloretin to SVCT-2 high-expression cell lines, Sw480 and Sw620 at the same time, the relative expression of Ki-67 and E2F1 used as cancer cell proliferation markers was measured using the qRT-PCR of Example 1, and the result was shown in
According to the above result, it could be confirmed that when treating vitamin C 10 μM and phloretin to Sw620 and Sw480 cell lines at the same time, the expression of Ki-67 and E2F1 increased, and that is, cancer cells were proliferated.
7-2. Localization of c-Myc in SVCT-2 High-Expression Cell Lines Under the Hormetic Response Conditions
In order to confirm the cell proliferation by the hormetic response in cancer cell lines, the cell proliferation confirmation experiment through c-Myc was conducted.
Specifically, after smearing cells on cover glass and culturing them, vitamin C was treated and after washing with PBS, an antigen with Cyclin D1 and CDK4 as antigens was attached to cells, and it was confirmed through fluorescence. The experimental result was shown in
As could be seen from the above result, the localization of c-Myc in SVCT-2 high-expression cells in which the expression of SVCT-2 was inhibited by treating phloretin together was present at the same location as c-Myc of SVCT-2 low-expression cell lines. In other words, in SVCT-2 high-expression cell lines, the increased localization of c-Myc was present in cytoplasm not a nucleus. From this, it could be seen that the cancer cell proliferation occurred actively when the hormetic response occurred.
7-3. Western Blot of C-Myc and Cyclin D1
After treating vitamin C 10 μM and phloretin to SVCT-2 high-expression cell lines, Sw480 and Sw620 at the same time, the western blot analysis of Bax, c-Myc and Cyclin D1 in cells was conducted, and the result was shown in
As shown in
From the results of
After treating 10 μM vitamin C and various concentrations (0 μM, 10 μM, 20 μM or 40 μM) of phloretin to SVCT-2 high-expression cell lines, Sw480 and Sw620 at the same time, the cell viability was measured, and the result was shown in
9-1: Analysis of Vitamin C Absorption of SVCT-2 High-Expression Cell Lines
The experimental results measured according to the method for measuring the vitamin C absorption of SVCT-2 expression cell lines of Example 1-3, after treating various concentrations (0 mM or 1 mM) of vitamin C and treating 1 mM vitamin C and 20 μM phloretin, for SVCT-2 high-expression cell lines, Sw480 and Sw620 were shown in
9-2: Confirmation of Localization of c-Myc of SVCT-2 Cell Lines
In order to confirm the expression of cancer proliferation markers, after treating vitamin C in SVCT-2 low-expression cell lines, the immunocytochemistry was conducted to confirm that, and the result was shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0079608 | Jul 2018 | KR | national |
