阅读说明：本技术 一种能抑制肿瘤细胞的组合物及其相关应用 (Composition capable of inhibiting tumor cells and related application thereof ) 是由 李劲 付珊珊 于 2021-08-04 设计创作，主要内容包括：本发明提供了一种能抑制肿瘤细胞的组合物及其相关应用,本发明的组合物包括2-苯甲酰胺基-1,4-萘醌和维生素C。本发明发现2-苯甲酰胺基-1,4-萘醌和维生素C联用可显著增加人肿瘤细胞内活性氧(ROS)的产生,能高效诱导膀胱癌、肺癌、乳腺癌、结肠癌、宫颈癌等肿瘤细胞的死亡。(The invention provides a composition capable of inhibiting tumor cells and related application thereof, wherein the composition comprises 2-benzamido-1, 4-naphthoquinone and vitamin C. The invention discovers that the combination of 2-benzamido-1, 4-naphthoquinone and vitamin C can obviously increase the generation of Reactive Oxygen Species (ROS) in human tumor cells, and can efficiently induce the death of tumor cells such as bladder cancer, lung cancer, breast cancer, colon cancer, cervical cancer and the like.)

1. A composition comprising 2-benzamido-1, 4-naphthoquinone and vitamin C.

2. The composition according to claim 1, wherein the ratio of 2-benzamido-1, 4-naphthoquinone to vitamin C is (1 μ M-5 μ M): (1mM-5 mM).

3. The composition of claim 1 or 2, wherein the 2-benzamido-1, 4-naphthoquinone is chemically synthesized.

4. The composition according to claim 1 or 2, wherein the vitamin C is naturally extracted or chemically synthesized.

5. Use of a composition according to any one of claims 1 to 4 for the preparation of a medicament and/or a formulation for inhibiting tumor cells, treating tumors and/or prolonging the survival of tumor patients.

6. The use of claim 5, wherein the inhibiting tumor cells, treating tumors, and/or extending survival of tumor patients comprises:

inhibiting the viability of tumor cells and/or inducing apoptosis of tumor cells.

7. The use according to claim 5 or 6, wherein the tumour is selected from one or more of bladder cancer, lung cancer, breast cancer, colon cancer, cervical cancer.

8. The use of claim 5 or 6, wherein said composition induces apoptosis of tumor cells by stimulating intracellular production of ROS.

9. The use of claim 5, wherein the treating a tumor comprises treating a tumor at a cellular level and/or an individual level.

10. The use of claim 5, wherein the composition has no significant viability-inhibiting effect on human normal liver cells.

Technical Field

The invention relates to a composition capable of inhibiting tumor cells and related application thereof, in particular to a technology for treating tumors by combining 2-benzamido-1, 4-naphthoquinone and vitamin C.

Background

The types of cancers are many, such as bladder cancer, lung cancer, breast cancer, colon cancer, cervical cancer and the like, the lethality rate is high, and the specific treatment needs to be performed according to the stage of the tumor. At present, the symptoms of cancer are mainly relieved clinically by means of surgical removal of cancerous tissues, chemotherapy, radiotherapy and immunotherapy. For early cancer patients, the purpose of radical treatment of diseases is mainly achieved by an operation method. In some patients in middle and advanced stages, the operation cannot be performed, and chemotherapy and radiotherapy are generally used to help kill cancer cells in the body so as to maintain the life health of the patient.

The structural formula of 2-benzamido-1, 4-naphthoquinone (2-benzamido-1, 4-naphthoquinone, also known as NQN1, PPM-18) is as follows:

the central structure of 2-benzamido-1, 4-naphthoquinone is a naphthoquinone ring, similar to many VK compounds. It has been reported that 2-benzamido-1, 4-naphthoquinone can inhibit the activation of NF- κ B in vitro and in vivo, and by this action, it inhibits the expression of iNOS (inducible nitric oxide synthase) in lipopolysaccharide-treated macrophages, thereby inhibiting the occurrence of inflammation and sepsis. At the same time, it is also an inhibitor of HDAC (histone deacetylase). In addition, 2-benzamido-1, 4-naphthoquinone can enhance electron transfer on a mitochondrial electron transfer chain by playing a role as an electron carrier on the mitochondrial respiratory chain (on the electron transfer chain), enhance mitochondrial aerobic respiration (oxygen consumption), and promote ATP synthesis of the mitochondrial respiratory chain, so that epilepsy of zebra fish and mice can be inhibited. CN 109172557A reports the effect of 2-benzamido-1, 4-naphthoquinone on inducing apoptosis of bladder cancer cells at higher dose.

There is still a need to develop more safe and effective drugs for treating tumors.

Disclosure of Invention

An object of the present invention is to develop a safe and highly effective anticancer composition against various tumors.

It is another object of the present invention to provide related uses of the compositions.

The inventor finds that the combined application of the 2-benzamido-1, 4-naphthoquinone and the vitamin C (vitamin C) has an obvious inhibiting effect on tumor cells of bladder cancer, lung cancer, breast cancer, colon cancer, cervical cancer and the like. The research on cell activity experiments and cell safety experiments of bladder cancer, lung cancer, breast cancer, colon cancer and cervical cancer shows that the 2-benzamido-1, 4-naphthoquinone and vitamin C are jointly applied, so that the cell activity inhibition effect on tumor cells of bladder cancer, lung cancer, breast cancer, colon cancer, cervical cancer and the like can be high-effectively realized under the condition of low concentration, and no obvious cytotoxicity effect is caused on normal human cells (such as liver cells).

Thus, in one aspect, the present invention provides a composition comprising 2-benzamido-1, 4-naphthoquinone and vitamin C.

The composition can inhibit tumor cells, and specifically comprises inhibiting the activity of the tumor cells and/or inducing the apoptosis of the tumor cells.

The composition of the invention, wherein the 2-benzamido-1, 4-naphthoquinone and the vitamin C can be used in combination in respective effective amounts, so as to achieve the effect of inhibiting tumor cells.

According to a specific embodiment of the present invention, the composition of the present invention comprises 2-benzamido-1, 4-naphthoquinone and vitamin C in a ratio of (1. mu.M-5. mu.M): (1mM-5 mM).

According to a specific embodiment of the present invention, in the composition of the present invention, the 2-benzamido-1, 4-naphthoquinone may be chemically synthesized.

According to a specific embodiment of the present invention, in the composition of the present invention, the vitamin C refers to threose-2, 3,4,5, 6-pentahydroxy-2-hexenoic acid-4-lactone, which may be naturally extracted or chemically synthesized.

According to a specific embodiment of the present invention, the composition of the present invention may further comprise one or more pharmaceutically acceptable excipients, such as disintegrants, diluents, excipients, etc.

According to a specific embodiment of the present invention, the composition of the present invention may be a reagent material for experiment, or a drug, and may be, for example, various dosage forms suitable for administration to a subject, such as an injection, etc.

In another aspect, the invention also provides the application of the composition in preparing medicines and/or preparations for inhibiting tumor cells, treating tumors and/or prolonging the life of tumor patients.

In the context of the present invention, the term "treating" includes ameliorating, preventing or reversing a disease or disorder or at least one overt symptom thereof, ameliorating, preventing or reversing at least one measurable physical parameter associated with the disease or disorder being treated, and/or physically (e.g., stabilization of overt symptoms) and/or physiologically (e.g., stabilization of physical parameters) inhibiting or slowing the progression of the disease or disorder.

According to a specific embodiment of the present invention, when the composition of the present invention is applied, 2-benzamido-1, 4-naphthoquinone and vitamin C may be administered to a subject separately or after mixing. When 2-benzamido-1, 4-naphthoquinone and vitamin C are administered separately to a subject, they may be administered to the subject at the same time period or at intervals.

According to a preferred embodiment of the present invention, the composition of the present invention is applied by administering 2-benzamido-1, 4-naphthoquinone and vitamin C to a subject in an effective amount. Experimental studies of the present invention have shown that an effective amount of 2-benzamido-1, 4-naphthoquinone to contact tumor cells can be 5. mu.M or less, e.g., 1. mu.M-5. mu.M; the effective amount of vitamin C contacted with the tumor cells may be 5mM or less, e.g., 1mM to 5 mM.

According to a specific embodiment of the present invention, the use of the composition of the present invention for inhibiting tumor cells, treating tumors and/or prolonging the survival of patients with tumors comprises: inhibiting the viability of tumor cells and/or inducing apoptosis of tumor cells. Namely, the invention also provides the application of the composition containing 2-benzamido-1, 4-naphthoquinone and vitamin C in preparing a preparation for inhibiting the activity of tumor cells and/or inducing the apoptosis of the tumor cells.

According to a particular embodiment of the invention, in the use of the composition of the invention, the tumor is selected from one or more of bladder cancer, lung cancer, breast cancer, colon cancer, cervical cancer.

According to a particular embodiment of the invention, the composition of the invention is used for inducing apoptosis of tumor cells by stimulating intracellular production of ROS.

According to a particular embodiment of the invention, the use of the composition of the invention for the treatment of tumors comprises the treatment of tumors at the cellular level and/or at the individual level.

According to a specific embodiment of the invention, the composition of the invention is used in applications where the composition has no significant inhibitory effect on the viability of normal human hepatocytes.

In some embodiments of the invention, the inventor of the present invention confirms the inhibition effect of the 2-benzamido-1, 4-naphthoquinone and vitamin C in the drug combination on bladder cancer, lung cancer, breast cancer, colon cancer and cervical cancer through in vitro experiments. The 2-benzamido-1, 4-naphthoquinone and vitamin C are combined to be used under the condition of low concentration, so that the compound has a more efficient cell activity inhibition effect on cells of bladder cancer, lung cancer, breast cancer, colon cancer and cervical cancer, and does not cause obvious cytotoxicity on normal cells (such as liver cells) of human. In addition, the addition of vitamin C can significantly reduce the dosage of 2-benzamido-1, 4-naphthoquinone during the process of inhibiting the activity of the human tumor cells and inducing the death of the human tumor cells, and reduce side effects and cost. In comprehensive consideration, the combination of 2-benzamido-1, 4-naphthoquinone and vitamin C is suitable for being used as a novel medicament for treating human lung cancer, breast cancer, colon cancer and cervical cancer including bladder cancer.

Drawings

FIGS. 1A and 1B show the results of experiments in which PPM-18 and Vitamin C were used in combination to inhibit the cell viability and colony formation of bladder cancer cells EJ and T24, but no significant cell viability inhibitory effect was observed on normal human cells L02. In FIG. 1A, the combined administration of PPM-18 and Vitamin C is shown to inhibit EJ and T24 cell viability, without significant inhibitory effect on L02. FIG. 1B shows the ability of PPM-18 in combination with Vitamin C to inhibit clonogenic activity of EJ, T24 cells. The results show that the single drug has no obvious inhibition effect.

FIGS. 2A and 2B show the results of the autophagy experiments induced by PPM-18 and Vitamin C in bladder cancer cells EJ and T24. FIG. 2A, electron micrograph, shows that PPM-18 and Vitamin C combination induce EJ autophagosome production. FIG. 2B, western felt detects the expression of the autophagy-related protein LC3B (β -actin as an internal reference).

FIGS. 3A and 3B show the results of experiments in which PPM-18 and Vitamin C were used in combination to induce bladder cancer cell death by generating intracellular ROS. Wherein, in FIG. 3A, the combination of PPM-18 and Vitamin C induces the production of ROS in bladder cancer cells T24, EJ; FIG. 3B, PPM-18 and Vitamin C in combination induce death of bladder cancer cells T24, EJ by ROS production.

FIG. 4A and FIG. 4B show that PPM-18 and Vitamin C are used in combination to inhibit the activity and clone formation of various cancer cell lines, such as non-small cell lung cancer A549 cells, triple negative breast cancer MDA-MB-231 cells, cervical cancer HeLa cells, and colorectal cancer HCT-116 cells. Wherein, FIG. 4A shows that PPM-18 and Vitamin C in combination inhibit cell viability of multiple cancer cell lines. FIG. 4B shows the clonogenic potency of PPM-18 in combination with Vitamin C against multiple cancer cell lines. The results show that the single drug has no obvious inhibition effect.

FIGS. 5A and 5B show the results of experiments in which PPM-18 and Vitamin C were used in combination to induce death of other cancer cell lines by generating intracellular ROS, as exemplified by non-small cell lung carcinoma A549 cells. Wherein, in FIG. 5A, PPM-18 and Vitamin C are administered in combination to induce non-small cell lung carcinoma A549 cell death by ROS production; FIG. 5B, PPM-18 and Vitamin C in combination inhibit the clonogenic capacity of non-small cell lung cancer cells by generating intracellular ROS.

FIGS. 6A and 6B show the results of the experiments that PPM-18 and Vitamin C have no significant cell viability inhibitory effect on human normal cell L02 when administered in combination. In FIG. 6A, the combined administration of PPM-18 and Vitamin C showed no significant inhibitory effect on L02 cell viability. FIG. 6B shows that the combination of PPM-18 and Vitamin C had no significant effect on L02 cell clonality.

In the results chart, P is abbreviated as 5. mu.M PPM-18, C is abbreviated as 5mM Vitamin C, and P + C is 5. mu.M PPM-18 and 5mM Vitamin C in combination, unless otherwise specified.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

PPM-18 and Vitamin C used in the examples are chemically synthesized drugs, wherein PPM-18 mother liquor (dissolved in DMSO) is stored at-80 ℃, the aqueous solution is ready for use, and the drugs are protected from light; the mother solution of Vitamin C (dissolved in sterile double distilled water) is stored at-80 deg.C, and the aqueous solution is ready for use, and is protected from light.

Example 1 combination of PPM-18 and Vitamin C inhibits bladder cancer cell viability and colony formation without significant viability inhibition of normal cells

In the embodiment, the experiment for inhibiting the activity of the bladder cancer cells by the PPM-18 and Vitamin C combined medicine mainly comprises the following steps:

(1-1) cell culture: in vitro culturing bladder cancer cells and human normal liver cells for experiments, and digesting and subculturing the cells when the cells grow to a logarithmic growth phase;

(1-2) kinds of 96-well plates: preparing a single cell suspension from bladder cancer cell lines EJ and T24 by using a culture solution containing 10% fetal calf serum, inoculating 10000 cells per hole into a 96-hole plate, wherein the hole volume is 100 mu l, and culturing until the abundance of the cells reaches 80-90% (generally culturing for 24 h);

(1-3) adding medicine treatment: after 24h of culture, gently sucking off the old culture solution, respectively treating the cultured cells with 5 μ M of PPM-18 and 5mM of Vitamin C combination and 5 μ M of PPM-18 and 5mM of Vitamin C single drug, and adding drugs for 24 h;

(1-4) MTT assay: after 24h of culture, 20 mu L of MTT detection reagent is added into each hole, the MTT detection reagent is incubated for 1-2h at 37 ℃ in the absence of light, then the 490nm wavelength is selected, the light absorption value of each hole is measured on a microplate reader, the result is recorded, and a cell activity detection result graph of the PPM-18 and Vitamin C combined drug is drawn according to the recorded result.

The experiment for inhibiting the clone formation of the bladder cancer cells by the combination of PPM-18 and vitamine C mainly comprises the following experimental steps:

(1-a) cell culture: in-vitro culturing the bladder cancer cells for experiments, and digesting and subculturing the cells when the cells grow to a logarithmic growth phase;

(1-b) cell harvesting: bladder cancer cells grown to logarithmic growth phase in culture flasks (cell number about 5 x 10)⁶) Washing with PBS, adding trypsin, digesting in 37 deg.C cell culture box for 2 min, adding MEM culture medium containing fetal calf serum to stop digestion, transferring into 15ml centrifuge tube, and centrifuging at 1500rpm for 5 min;

(1-c) dilution plate: at the end of centrifugation, the supernatant was discarded, the cell pellet was left, and 1ml of fresh MEM was added, gently and gently mixed by pipetting, and at the same time, 1ml of fresh MEM was added to each of 2 1.5ml EP tubes. And adding 100 mu L of cell suspension in a 15ml centrifuge tube into one of the 1.5ml EP tubes, gently blowing and stirring the cell suspension uniformly, taking 100 mu L of the cell suspension out of the centrifuge tube, adding the cell suspension into the other 1.5ml EP tube, and gently blowing and stirring the cell suspension uniformly again. Then, 100. mu.L of the mixture was taken out of the second 1.5ml EP tube and added to a 15ml centrifuge tube containing 12ml of fresh MEM culture medium, and the mixture was mixed into 12-well plates of 1ml per well. Dilution of the plates in this manner, approximately 200-500 cells per well in 12-well plates;

(1-d) adding medicine treatment: after the cells in the 12-well plate are cultured for 4-7 days in an adherent way, sucking out the old culture solution, adding 5 mu M of PPM-18 and 5mM of Vitamin C combined drug and 5 mu M of PPM-18 and 5mM of Vitamin C single drug, stimulating for 1h, then sucking out the culture medium containing the drug, and adding fresh MEM culture solution without the drug for continuous culture for 4-7 days;

(1-e) clone formation: after macroscopic cell clones had formed, the culture was terminated. And sucking the old culture solution in the plate, adding 1ml of PBS into each hole for rinsing once, then adding anhydrous methanol for fixing for 10 minutes, washing with the PBS for three times, adding crystal violet for dyeing for 30 minutes, rinsing for 3-4 times by the PBS after 30 minutes, drying the plate in the dark, photographing and keeping.

See fig. 1A and 1B for experimental results. The results show that: IC50 for PPM-18 and Vitamin C, respectively, on bladder cancer cells were calculated to screen for combined concentrations. As shown in FIG. 1A, the 6 concentrations of PPM-18 and 0mM, 5mM, 10mM, 15mM, 20mM were selected, and the 6 concentrations of Vitamin C were treated with drugs T24 for 24h to find PPM-18 and Vitaminn C all exhibit a concentration gradient dependent manner of inhibiting cancer cell viability and are calculated by calculating IC₅₀Obtaining the IC of PPM-18 to T24₅₀IC of 9.043 μ M, Vitamin C vs. T24₅₀8.652mM, and calculating the IC of the combination of PPM-18 and Vitamin C on T24 cells based on the optimal ratio of PPM-18 to Vitamin C of 1:1000₅₀2.422, wherein PPM-18: 2.422 μ M, Vitamin C: 2.422 mM. A combination of 5. mu.M PPM-18 and 5mM Vitamin C that did not kill bladder cancer cells was determined for subsequent studies. MTT experiment shows that the PPM-18 and Vitamin C combined medicine can effectively inhibit the activity of bladder cancer cells without obvious inhibition on normal human liver cells. MTT experiment can quickly verify the effect of the medicine on cells in a short term, and then cell cloning experiment is used for further verifying the effect of the PPM-18 and Vitamin C combined medicine on bladder cancer in long-term proliferation. As shown in FIG. 1B, the combined treatment of PPM-18 and Vitamin C showed significant inhibition of colony formation, but neither of the two drugs alone showed significant inhibition, indicating that the combination of 5. mu.M PPM-18 and 5mM Vitamin C showed synergistic killing of bladder cancer cells.

Example 2 PPM-18 and Vitamin C combination induce autophagy in bladder cancer cells

In this embodiment, the method for displaying autophagosome by taking a picture with a microscope mainly comprises the following experimental steps:

(2-1) cell culture: culturing the cells in the flask in a cell culture chamber at 37 ℃ and 5% CO2 until the cells are in logarithmic growth phase;

(2-2) cell plate: adding PBS (phosphate buffer solution) into cells to be passaged, rinsing, digesting with pancreatin, adding 12mL of fresh culture solution, gently blowing, beating and uniformly mixing, seeding a 6-hole plate, shaking up and down, left and right for 5 times by 2mL of cell suspension per hole, placing in a cell culture box for culturing for 48 hours, and changing the solution once when culturing for 24 hours.

(2-3) adding medicine treatment: after 48 hours of cell culture, when the cells are in 90-100% abundance, 5 mu M PPM-18 and 5mM Vitamin C are added for combined administration, and a control group without drug addition is set and treated for about 20 hours.

(2-4) cell harvesting: after 20h, the cells in the 6-well plate were collected in a 15ml centrifuge tube, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and then 1ml of PBS buffer was added to centrifuge and wash for 1 time.

(2-5) cell fixation: the cell pellet was fixed with 1mL of 2.5% glutaraldehyde electron microscope fixative and transferred to a 1.5mL centrifuge tube, sealed, and labeled for date, type of drug treatment, and cell line type.

(2-6) shooting by an electron microscope: the cells fixed with 2.5% glutaraldehyde electron microscope fixing solution were handed over to Google wuhanensis organisms for autophagosome shooting.

The method for detecting the autophagy of the cells by adopting western blot comprises the following steps:

(2-a) cell culture: culturing the cells in the flask in a cell culture chamber at 37 ℃ and 5% CO2 until the cells are in logarithmic growth phase;

(2-b) cell plate: adding PBS into cells in logarithmic phase for rinsing and digesting, adding 12ml of fresh culture solution, gently blowing and uniformly mixing, seeding a 6-hole plate, shaking up and down, left and right for 5 times with 2ml of cell suspension in each hole, placing the cell suspension in a cell culture box for culturing for 48 hours, and changing the solution once when culturing for 24 hours;

(2-c) adding medicine treatment: after 48h, the cells were in 90% -100% abundance, and then treated with 5 μ M PPM-18 and 5mM Vitamin C combination and 5 μ M PPM-18 and 5mM Vitamin C single drug for 20 h;

(2-d) extracting sample protein: removing the culture solution containing the medicine, and adding 1ml of PBS for washing once; adding lysis solution containing phosphatase inhibitor, and lysing on ice for 30 min; centrifuging at 12000rpm for 20min, and collecting supernatant; carrying out protein concentration determination; adding protein sample buffer, boiling at 100 deg.C for 5min to denature protein;

(2-e) electrophoresis: preparing 10% of separation gel and 5% of concentrated gel; determining the loading volume according to the protein concentration; running electrophoresis at initial voltage of 80V for 30min, and then regulating to voltage of 100V for 1.5 h; performing gel cutting operation according to the molecular weight of the target protein;

(2-f) transferring the film: the cut gel was placed on a rotating membrane plate and a sponge, filter paper and PVDF membrane were added according to the sandwich model. Setting current 300mA, and rotating the membrane for 1 h;

(2-g) blocking and incubating: placing the PVDF membrane containing the target protein in skimmed milk, and sealing for 3h in a shaking table at room temperature; primary antibody (1:2000 dilution) shaking table overnight at 4 ℃; diluting the secondary antibody (1: 10000), and incubating for 3 hours in a shaking table at room temperature in the dark; TBST washing for 3 times, each time for 30 min;

(2-h) developing.

The results are shown in FIGS. 2A and 2B. The results show that: the combination of PPM-18 and Vitamin C induces autophagy in bladder cancer cells. As shown in FIG. 2A, in the EJ cell electron micrograph, the production of autophagosomes was observed in the cells of the group treated with the combination, whereas it was not observed in the control group. In this example, the generation of autophagy was further verified by Western blot experiments, and the results are shown in fig. 2B. After the treatment of the PPM-18 and the Vitamin C in combination, the expression of the LC3BII protein in the bladder cancer T24 and EJ cells is greatly increased, which is 2.76 times and 2.98 times of that of a control group respectively.

Example 3 combination of PPM-18 and Vitamin C induces bladder cancer cell death by generating intracellular ROS

In this example, the combination of PPM-18 and Vitamin C to induce ROS production in bladder cancer cells comprises the following steps:

(3-1) cell culture: culturing the cells in the flask in a cell culture chamber at 37 ℃ and 5% CO2 until the cells are in logarithmic growth phase;

(3-2) cell plate: adding PBS (phosphate buffer solution) into cells in logarithmic phase for rinsing, digesting with pancreatin, adding 12ml of fresh culture solution, gently blowing, uniformly mixing, seeding a 6-hole plate, shaking up and down, left and right for 5 times with 2ml of cell suspension per hole, placing in a cell culture box for culturing for 48 hours, and changing the solution once when culturing for 24 hours;

(3-3) adding medicine treatment: after 48h, the cells are at 90% -100% abundance, and then the combination drug is added to induce ROS generation, wherein each group is a control group without drug, 5 mu M PPM-18 single drug, 5mM Vitamin C single drug, 5 mu M PPM-18 and 5mM Vitamin C combination drug, and the treatment is carried out for 6 h;

(3-4) cell harvesting: after 6h, collecting the cells in the 6-hole plate in a 15ml centrifuge tube, centrifuging for 5 minutes at the rotating speed of 1500rpm, removing the supernatant, adding 1ml of PBS buffer solution, centrifuging and washing for 1 time;

(3-5) adding ROS probe DCFH-DA: beijing prilley active oxygen detection kit (mother solution concentration is 10mM), according to the ratio of 1: 10000 of DCFH-DA dilution ratio by PBS, then 500 mu L of diluted DCFH-DA probe is added into each tube, the tubes are placed into a cell culture box to be incubated for 30 minutes, and the mixture is uniformly mixed every 10 minutes;

(3-6) detecting ROS level by using a flow analyzer: the incubated samples were washed twice with PBS, then resuspended in 500. mu.L of PBS per tube, and the ROS levels were measured on an up-flow analyzer.

The combination of PPM-18 and Vitamin C for inducing bladder cancer cell death by generating intracellular ROS comprises the steps of:

(3-a) cell culture: in-vitro culturing the bladder cancer cells for experiments, and digesting and subculturing the cells when the cells grow to a logarithmic growth phase;

(3-b) 96-well plates: preparing a single cell suspension from bladder cancer cell lines EJ and T24 by using a culture solution containing 10% fetal calf serum, inoculating 10000 cells per hole into a 96-hole plate, wherein the hole volume is 100 mu l, and culturing until the abundance of the cells reaches 80-90% (generally culturing for 24 h);

(3-c) adding medicine treatment: after 24h of incubation, gently aspirating the old medium, then adding PPM-18+ Vitamin C combination and NAC (to scavenge intracellular ROS) for co-treatment (control group, 6mM NAC group, 5. mu.M PPM-18 and 5mM Vitamin C combination +6mM NAC group, respectively), for 24 h;

(3-d) MTT assay: after 24h of culture, 20 mu L of MTT detection reagent is added into each well, the wells are incubated for 1-2h in the dark at 37 ℃, then the 490nm wavelength is selected, the light absorption value of each well is measured on a microplate reader, the result is recorded, and a graph of the detection result of the activity effect of the PPM-18+ Vitamin C combined drug and NAC (removal of intracellular ROS) co-processing cells is drawn according to the recorded result.

Intracellular ROS levels were measured in this example. FIGS. 3A and 3B show the results of experiments in which PPM-18 and Vitamin C were administered in combination to induce bladder cancer cell death by generating intracellular ROS in this example. As shown in FIG. 3A, when PPM-18 was added at a concentration of 5. mu.M and Vitamin C was added at a concentration of 5mM, the level of ROS showed a significantly elevated trend compared to the control group and the drug-treated group alone, demonstrating that the combination of PPM-18 and Vitamin C could indeed induce the production of ROS in bladder cancer cells T24 and EJ. Compared with the control group, the ROS level of the PPM-18 and Vitamin C combined treatment group reaches 2-3 times of that of the control group. Next, it was verified whether ROS is a factor in the induction of bladder cancer cell death by the combination of PPM-18 and Vitamin C. NAC (total intracellular ROS scavenging) scavenger of ROS was used in this example and was tested by MTT, and as shown in FIG. 3B, it was found that the combination of PPM-18 and Vitamin C induced significant cancer cell death when co-treated with NAC in combination with PPM-18 and Vitamin C.

As described above, PPM-18 can induce bladder cancer cell death by allowing intracellular production of ROS.

Example 4 combination of PPM-18 and Vitamin C inhibits cell viability and colony formation in multiple cancer cell lines (lung, breast, cervical, colon)

In the embodiment, the experiment for inhibiting the cell viability of the cancer cell lines of lung cancer, breast cancer, cervical cancer and colon cancer by using the PPM-18 and Vitamin C in a combined manner mainly comprises the following steps:

(4-1) cell culture: carrying out in-vitro culture on the cancer cells for experiments, and carrying out digestion and passage plating when the cells grow to a logarithmic growth phase;

(4-2) kinds of 96-well plates: preparing a single cell suspension from various cancer cell line cells by using a culture solution containing 10% fetal calf serum, inoculating 10000 cells per hole to a 96-hole plate, wherein the hole volume is 100 mu l, and culturing until the abundance of the cells reaches 80% -90% (generally culturing for 24 h);

(4-3) adding medicine treatment: after 24h of culture, gently sucking off the old culture solution, respectively treating the cultured cells with 5 μ M of PPM-18 and 5mM of Vitamin C combination and 5 μ M of PPM-18 and 5mM of Vitamin C single drug, and adding drugs for 24 h;

(4-4) MTT assay: after 24h of culture, 20 mu L of MTT detection reagent is added into each hole, the MTT detection reagent is incubated for 1-2h at 37 ℃ in the absence of light, then the 490nm wavelength is selected, the light absorption value of each hole is measured on a microplate reader, the result is recorded, and a cell activity detection result graph of the PPM-18 and Vitamin C combined drug is drawn according to the recorded result.

The experiment for inhibiting the cell clone formation of a plurality of cancer cell lines of lung cancer, breast cancer, cervical cancer and colon cancer cell lines by the PPM-18 and Vitamin C combined medicine mainly comprises the following experimental steps:

(4-a) cell culture: carrying out in-vitro culture on the cancer cells for experiments, and carrying out digestion and passage plating when the cells grow to a logarithmic growth phase;

(4-b) cell harvesting: cancer cells grown to logarithmic growth phase in culture flasks (cell number about 5 x 10)⁶) Washing with PBS, adding trypsin, digesting in 37 deg.C cell culture box for 2 min, adding MEM culture medium containing fetal calf serum to stop digestion, transferring into 15ml centrifuge tube, and centrifuging at 1500rpm for 5 min;

(4-c) dilution plate: at the end of centrifugation, the supernatant was discarded, the cell pellet was left, and 1ml of fresh MEM was added, gently and gently mixed by pipetting, and at the same time, 1ml of fresh MEM was added to each of 2 1.5ml EP tubes. And adding 100 mu L of cell suspension in a 15ml centrifuge tube into one of the 1.5ml EP tubes, gently blowing and stirring the cell suspension uniformly, taking 100 mu L of the cell suspension out of the centrifuge tube, adding the cell suspension into the other 1.5ml EP tube, and gently blowing and stirring the cell suspension uniformly again. Then, 100. mu.L of the mixture was taken out of the second 1.5ml EP tube and added to a 15ml centrifuge tube containing 12ml of fresh MEM culture medium, and the mixture was mixed into 12-well plates of 1ml per well. Dilution of the plates in this manner, approximately 200-500 cells per well in 12-well plates;

(4-d) adding medicine treatment: after the cells in the 12-well plate are cultured for 4-7 days in an adherent way, sucking out the old culture solution, adding 5 mu M of PPM-18 and 5mM of Vitamin C combined drug and 5 mu M of PPM-18 and 5mM of Vitamin C single drug, stimulating for 1h, sucking out the culture medium containing the drug, and adding fresh MEM culture solution without the drug for continuous culture for 4-7 days;

(4-e) clone formation: after macroscopic cell clones had formed, the culture was terminated. And sucking the old culture solution in the plate, adding 1ml of PBS into each hole for rinsing once, then adding anhydrous methanol for fixing for 10 minutes, washing with the PBS for three times, adding crystal violet for dyeing for 30 minutes, rinsing for 3-4 times by the PBS after 30 minutes, drying the plate in the dark, photographing and keeping.

See fig. 4A and 4B for experimental results. The results show that: the combination of PPM-18 and vitamine C can inhibit the cell activity and clone formation of various cancer cell lines. As shown in FIG. 4A, MTT assay showed that the combination of PPM-18 and Vitamin C was effective in inhibiting the activity of various cancer cell lines. MTT experiment can quickly verify the effect of the medicine on cells in a short term, and then cell cloning experiment is used for further verifying the effect of the combination of PPM-18 and Vitamin C on various cancer cells in long-term proliferation. As shown in FIG. 4B, the inhibitory effect on colony formation was significant when PPM-18 and Vitamin C were administered in combination. While neither of the two drugs alone had a significant inhibitory effect, indicating that the combination of 5. mu.M PPM-18 and 5mM Vitamin C had a synergistic effect in killing cells from a number of different cancer cell lines.

Example 5 combination of PPM-18 and Vitamin C induces non-Small cell Lung cancer cell death by generating intracellular ROS

The combination of PPM-18 and Vitamin C for inducing the death of non-small cell lung cancer A549 cells by activating the generation of intracellular ROS comprises the following steps:

(5-1) cell culture: in vitro culturing the experimental non-small cell lung cancer, and digesting and subculturing the cell when the cell grows to a logarithmic growth phase;

(5-2) kinds of 96-well plates: preparing a single cell suspension from a non-small cell lung cancer A549 cell by using a culture solution containing 10% fetal calf serum, inoculating 10000 cells per hole to a 96-hole plate, wherein the hole volume is 100 mu l per hole, and culturing until the abundance of the cells reaches 80-90% (generally culturing for 24 h);

(5-3) adding medicine treatment: after 24h of incubation, gently aspirating the old medium, then adding PPM-18+ Vitamin C combination and NAC (to scavenge intracellular ROS) for co-treatment (control group, 6mM NAC group, 5. mu.M PPM-18 and 5mM Vitamin C combination +6mM NAC group, respectively), for 24 h;

(5-4) MTT assay: after 24h of culture, 20 mu L of MTT detection reagent is added into each well, the wells are incubated for 1-2h in the dark at 37 ℃, then the 490nm wavelength is selected, the light absorption value of each well is measured on a microplate reader, the result is recorded, and a graph of the detection result of the activity effect of the PPM-18+ Vitamin C combined drug and NAC (removal of intracellular ROS) co-processing cells is drawn according to the recorded result.

The combination of PPM-18 and Vitamin C for inhibiting the clonogenic capacity of non-small cell lung cancer cells by generating intracellular ROS comprises the following steps:

(5-a) cell culture: in vitro culturing the experimental non-small cell lung cancer, and digesting and subculturing the cell when the cell grows to a logarithmic growth phase;

(5-b) cell harvesting: growth of non-small cell lung carcinoma to logarithmic growth phase in culture flasks (cell count of about 5 x 10)⁶) Washing with PBS, adding trypsin, digesting in 37 deg.C cell culture box for 2 min, adding MEM culture medium containing fetal calf serum to stop digestion, transferring into 15ml centrifuge tube, and centrifuging at 1500rpm for 5 min;

(5-c) dilution plate: at the end of centrifugation, the supernatant was discarded, the cell pellet was left, and 1ml of fresh MEM was added, gently and gently mixed by pipetting, and at the same time, 1ml of fresh MEM was added to each of 2 1.5ml EP tubes. And adding 100 mu L of cell suspension in a 15ml centrifuge tube into one of the 1.5ml EP tubes, gently blowing and stirring the cell suspension uniformly, taking 100 mu L of the cell suspension out of the centrifuge tube, adding the cell suspension into the other 1.5ml EP tube, and gently blowing and stirring the cell suspension uniformly again. Then, 100. mu.L of the mixture was taken out of the second 1.5ml EP tube and added to a 15ml centrifuge tube containing 12ml of fresh MEM culture medium, and the mixture was mixed into 12-well plates of 1ml per well. Dilution of the plates in this manner, approximately 200-500 cells per well in 12-well plates;

(5-d) adding medicine treatment: after the cells in the 12-well plate are cultured for 4-7 days in an adherent way, sucking out the old culture solution, adding 5 mu M of PPM-18 and 5mM of Vitamin C combined drug and 5 mu M of PPM-18 and 5mM of Vitamin C single drug, stimulating for 1h, then sucking out the culture medium containing the drug, and adding fresh MEM culture solution without the drug for continuous culture for 4-7 days;

(5-e) clone formation: after macroscopic cell clones had formed, the culture was terminated. And (3) sucking out the old culture solution in the plate, adding 1ml of PBS for rinsing once per hole, then adding anhydrous methanol for fixing for 10 minutes, washing with PBS for three times, adding crystal violet for dyeing for 30 minutes, rinsing for 3-4 times with PBS after 30 minutes, then drying the plate in the dark, photographing and keeping.

FIGS. 5A and 5B show the results of experiments in which PPM-18 and Vitamin C were administered in combination to induce cell death in other cancer cell lines by generating intracellular ROS in this example. As shown in FIG. 5A, it was verified whether ROS was a factor inducing non-small cell lung cancer A549 cell death by the combination of PPM-18 and Vitamin C. This example used NAC (total intracellular ROS scavenging) as a scavenger of ROS, and was tested by MTT, and as shown in FIG. 5B, it was found that when the combination of PPM-18 and Vitamin C and NAC were co-treated, cancer cell death induced by the combination of PPM-18 and Vitamin C could be significantly rescued.

Cell cloning experiments were then used to further verify that the combination of PPM-18 and Vitamin C produced an inhibitory effect on the long-term proliferation of a variety of cancer cells by producing intracellular ROS. As shown in FIG. 5B, the inhibitory effect on colony formation was significant when PPM-18 and Vitamin C were administered in combination. While neither of the two drugs alone had a significant inhibitory effect, indicating that the combination of 5. mu.M PPM-18 and 5mM Vitamin C had a synergistic effect in killing multiple cancer cell lines.

As described above, PPM-18 can induce cell death of other cancer cell lines by allowing intracellular production of ROS.

Example 6 combination of PPM-18 and Vitamin C No significant Activity inhibition on human Normal hepatocytes L02

In this example, the experiment for studying the effect of PPM-18 and Vitamin C combination on L02 cell viability mainly includes the following steps:

(6-1) cell culture: carrying out in-vitro culture on human normal hepatocytes for experiments, and carrying out digestion and subculture on the cells when the cells grow to a logarithmic growth phase;

(6-2) kinds of 96-well plates: preparing normal cells L02 into single cell suspension by using culture solution containing 10% fetal calf serum, inoculating 10000 cells per hole into a 96-hole plate, wherein the hole volume is 100 mu L, and culturing until the abundance of the cells reaches 80% -90% (generally culturing for 24 h);

(6-3) adding medicine treatment: after 24h of culture, gently sucking off the old culture solution, respectively treating the cultured cells with 5 μ M of PPM-18 and 5mM of Vitamin C combination and 5 μ M of PPM-18 and 5mM of Vitamin C single drug, and adding drugs for 24 h;

(6-4) MTT assay: after 24h of culture, 20 mu L of MTT detection reagent is added into each hole, the MTT detection reagent is incubated for 1-2h at 37 ℃ in the absence of light, then the 490nm wavelength is selected, the light absorption value of each hole is measured on a microplate reader, the result is recorded, and a cell activity detection result graph of the PPM-18 and Vitamin C combined drug is drawn according to the recorded result.

The experiment for researching the influence of the PPM-18 and the Vitamin C combination on the clone formation of the L02 cell mainly comprises the following experimental steps:

(6-a) cell culture: carrying out in-vitro culture on the normal cells for experiments, and carrying out digestion and passage plating when the cells grow to a logarithmic growth phase;

(6-b) cell harvesting: l02 cells grown to logarithmic growth phase in flasks (cell number approximately 5 x 10)⁶) Washing with PBS, adding trypsin, digesting in 37 deg.C cell culture box for 2 min, adding MEM culture medium containing fetal calf serum to stop digestion, transferring into 15ml centrifuge tube, and centrifuging at 1500rpm for 5 min;

(6-c) dilution plate: at the end of centrifugation, the supernatant was discarded, the cell pellet was left, and 1ml of fresh MEM was added, gently and gently mixed by pipetting, and at the same time, 1ml of fresh MEM was added to each of 2 1.5ml EP tubes. And adding 100 mu L of cell suspension in a 15ml centrifuge tube into one of the 1.5ml EP tubes, gently blowing and stirring the cell suspension uniformly, taking 100 mu L of the cell suspension out of the centrifuge tube, adding the cell suspension into the other 1.5ml EP tube, and gently blowing and stirring the cell suspension uniformly again. Then, 100. mu.L of the mixture was taken out of the second 1.5ml EP tube and added to a 15ml centrifuge tube containing 12ml of fresh MEM culture medium, and the mixture was mixed into 12-well plates of 1ml per well. Dilution of the plates in this manner, approximately 200-500 cells per well in 12-well plates;

(6-d) adding medicine treatment: after the cells in the 12-well plate are cultured for 4-7 days in an adherent way, sucking out the old culture solution, adding 5 mu M of PPM-18 and 5mM of Vitamin C combined drug and 5 mu M of PPM-18 and 5mM of Vitamin C single drug, stimulating for 1h, then sucking out the culture medium containing the drug, and adding fresh MEM culture solution without the drug for continuous culture for 4-7 days;

(6-e) clone formation: after macroscopic cell clones had formed, the culture was terminated. And sucking the old culture solution in the plate, adding 1ml of PBS into each hole for rinsing once, then adding anhydrous methanol for fixing for 10 minutes, washing with the PBS for three times, adding crystal violet for dyeing for 30 minutes, rinsing for 3-4 times by the PBS after 30 minutes, drying the plate in the dark, photographing and keeping.

See fig. 6A and 6B for experimental results. The results show that: the combination of PPM-18 and Vitamin C has no obvious effect on the cell activity and clone formation of human normal cells L02. As shown in FIG. 6A, MTT test showed that the combination of PPM-18 and Vitamin C had no significant effect on the activity of L02. Cell cloning experiments were used to further verify the effect of the combination of PPM-18 and Vitamin C on normal cells during long-term proliferation. As shown in FIG. 6B, PPM-18 and Vitamin C had no significant effect on the formation of normal cell clones. However, neither of the two drugs has an inhibitory effect, which indicates that 5. mu.M PPM-18 and 5mM Vitamin C have a selective tumor cell killing effect, but have no obvious effect on normal human cells, and indicates that 5. mu.M PPM-18 and 5mM Vitamin C have certain safety at a cellular level.