CN111467332B

CN111467332B - Application of low-temperature plasma and metformin in combination

Info

Publication number: CN111467332B
Application number: CN202010241315.0A
Authority: CN
Inventors: 孙敏轩; 杨帆; 周元帅; 杨姣; 黄煜伦; 李炎炎; 乔治·塞吉亚迪斯; 赵容川; 孟星君; 撒小涵; 欧阳楠
Original assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Current assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2021-08-24
Anticipated expiration: 2040-03-31
Also published as: CN111467332A

Abstract

The invention discloses an application of low-temperature plasma and metformin in combination, in particular to an application of low-temperature plasma and metformin in non-therapeutic induction of glioma cell death in vitro. The application of the combination of the low-temperature plasma and the metformin in non-therapeutic induction of glioma cell death in vitro shows that the combination of the low-temperature plasma and the metformin has obvious synergistic inhibition effect on glioma cell lines U251 and U87 cultured in vitro and can obviously induce cell death. The scheme of the application of the combination of the metformin and the low-temperature plasma in inducing glioma death can provide a new strategy for researching the application of the combination of the low-temperature plasma and the metformin in glioma treatment, so as to provide a new idea for designing a new product or a new scheme for treating glioma.

Description

Application of low-temperature plasma and metformin in combination

Technical Field

The invention relates to the field of new application of low-temperature plasma and metformin, in particular to application of the combination of the low-temperature plasma and the metformin.

Background

Gliomas are the most common primary brain tumors, with malignant Gliomas (GBMs) being the most malignant of the gliomas, with a five-year survival rate of about 5% and with malignant gliomas not having an effective improvement in survival over the last decades. The incidence of glioma in china is about 4/100000. The classification of tumors of the central nervous system in the 2016 world health organization classifies gliomas according to histopathology and mutational characteristics into peripheral gliomas (WHO, grade I) and invasive gliomas (WHO, grade II-IV), the latter including glioblastomas (60-70% of all malignant gliomas), anaplastic astrocytomas (10-15%), anaplastic oligodendrogliomas and anaplastic oligodendroastrocytomas (10%) and other less common subtypes. The treatment modalities for gliomas generally include surgical resection, chemotherapy, and radiation therapy. The surgery treatment is combined with chemotherapy, radiotherapy is a common treatment for treating brain glioma, and at present, the optimal median survival time for patients can be provided by surgical resection and synchronous radiotherapy and chemotherapy is 14 months. Therapeutic advances in malignant gliomas include novel surgical techniques to achieve the greatest degree of safe resection, specific radiation treatment regimens, and new systemic chemotherapy regimens. Temozolomide (TMZ), an alkylating agent, is prepared by reacting Temozolomide (TMZ), a diamine in the presence of O⁶The site-directed alkylation of guanine as well as the alkylation at the N3 and N7 positions of adenine kills gliomas, which represent the second-line drug of astrocytomas and the first-line drug of malignant glioblastomas. Although TMZ can provide positive therapeutic effects in patients with high grade gliomas, its role in low grade gliomas and some other subtypes of gliomas with genetic mutations is not well understood. For some subtypes, such as IDH mutant and low-grade astrocytomas, TMZ treatment will cause these tumors to exhibit highly mutated malignant properties. An increasing number of clinical cases indicate that some GBM patients develop resistance to TMZ and even that some patients do not respond to TMZ treatment. Thus, there is an urgent need for products or protocols for the treatment of gliomas.

Plasma in nature is the fourth form of material that forms at high temperatures, following solid, liquid, and gaseous states. Under laboratory conditions, engineers may generate plasmas at room temperature, and are therefore also referred to as low temperature plasmas (CAP). The plasma is composed of a variety of reactive radicals, the most prominent of which include free oxygen radicals and free nitrogen radicals (ROS/RNS). During the past decade, both in vitro and in vivo experiments, CAP or CAP-activated media (CAM) have exhibited a broad spectrum of anti-tumor effects. Multiple agents or combination cancer therapies can provide better therapeutic results for patients, while CAP/CAM has been used in the laboratory in combination with certain drugs to treat tumors. Tetsuo Adachi et al found that Histone Deacetylase (HDAC) inhibitors could enhance the killing effect of CAM on adenocarcinoma cells A549; the breast cancer cell line is pre-sensitized by HSP90 inhibitor (PU-H71), so that the killing effect of CAP on tumor cells can be enhanced; after treatment with CAP, GBM cells resistant to TMZ restored sensitivity to TMZ. In 2019, the FDA approved a low temperature plasma scalpel for clinical tumor treatment, which undoubtedly increases the possibility of CAP for clinical use.

Metformin (Met) is the most important drug for patients with type two diabetes mellitus (T2 DM). In recent years, metformin has been found to enhance the sensitivity of a variety of tumors to certain chemotherapeutic drugs. After TMZ and metformin are used together, compared with single-drug treatment, glioma cells can be obviously killed and killed; the combination of metformin and sorafenib can obviously inhibit the proliferation of hepatocellular carcinoma and induce autophagy; metformin increases the sensitivity of non-small cell lung cancer to catechins. Since metformin is inexpensive and has been approved for clinical use, its use in tumors, for example, has natural advantages over de novo development of new drugs and existing tumor-specific drugs.

Disclosure of Invention

The technical problem to be solved by the present invention is to provide an application of a combination of low temperature plasma and metformin for overcoming the above-mentioned disadvantages of the prior art.

It is an aspect of the present invention to provide the use of low temperature plasma in combination with metformin for the non-therapeutic induction of glioma cell death in vitro.

Preferably, the treatment is carried out by treating the glioma cells in vitro with metformin and then with a low temperature plasma.

In a second aspect of the invention, there is provided the use of low temperature plasma in combination with metformin to non-therapeutically induce U251 cell death in vitro.

Preferably, U251 cells cultured in vitro are treated with metformin, cultured for 6 to 24 hours, and then treated with low-temperature plasma for 5 to 120 seconds to induce U251 cell death.

In a third aspect of the invention, there is provided the use of low temperature plasma in combination with metformin to non-therapeutically induce U87 cell death in vitro.

Preferably, U251 cells are induced to die by treating U87 cells cultured in vitro with metformin followed by 6-24 hours of culture and then by treatment with low temperature plasma for 5-120 seconds.

The invention has the beneficial effects that: the application of the combination of the low-temperature plasma and the metformin in non-therapeutic induction of glioma cell death in vitro shows that the combination of the low-temperature plasma and the metformin has obvious synergistic inhibition effect on glioma cell lines U251 and U87 cultured in vitro and can obviously induce cell death. The scheme of the application of the combination of the metformin and the low-temperature plasma in inducing glioma death provided by the invention can provide a new strategy for researching the application of the combination of the low-temperature plasma and the metformin in glioma treatment, so as to provide a new idea for designing a new product or a new scheme for treating glioma.

Drawings

FIG. 1 Experimental results in example 1 of the present invention;

FIG. 2 experimental results in example 2 of the present invention;

FIG. 3 experimental results in example 3 of the present invention;

FIG. 4 experimental results in example 4 of the present invention.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The invention provides an application of low-temperature plasma and metformin in non-therapeutic induction of glioma cell death in vitro. The death of glioma cells cultured in vitro is induced by treating the glioma cells in vitro with metformin and then with low-temperature plasma. The invention is used for non-disease treatment, mainly used for in vitro research, and the application of the combination of the low-temperature plasma and the metformin in the aspect of glioma treatment can be researched through the scheme of the invention, so as to provide a new thought for designing a new product or a new scheme for treating glioma.

The experiments are performed in the following examples on U251 and U87 glioma cells to further illustrate the present invention, wherein common reagents and consumables related to molecular biology and cell biology are used.

The first embodiment is as follows: measurement of inhibitory Effect of Low temperature plasma (CAP) alone and in combination with Metformin (MET) on U251/U87 glioma cells

1. To determine whether the effect of the low temperature plasma and metformin alone has an effect on the cell viability of the glioma cell line, the effect of the low temperature plasma and metformin on the cell viability of the glioma cell line was first examined separately in this example. The U251 cell line was adjusted to 7.5 x 10⁴Cell line U87 5 x 10/ml⁴Density of/ml inoculated in 96-well plates, after 24 hours of culture:

(1) plasma single action experiment: after the treatment for 10, 20, 40 and 60 seconds of low-temperature plasma treatment time, respectively, after 24 hours and 48 hours of treatment, the cell viability was measured by the CellTiter Blue method, and as shown in fig. 1A-1B, it can be seen from the results shown in the figure that the low-temperature plasma can effectively inhibit the cell viability of glioma cells, and the inhibition effect is positively correlated with the time of exposing the cells to the plasma, but after the treatment for the low-temperature plasma, the low-temperature plasma is continuously cultured for 24 hours and 48 hours, and the inhibition of the cell viability by the low-temperature plasma has no significant difference.

The analysis was performed on FIGS. 1A-1B: after culturing the glioma cell lines U251 (FIG. 1A) and U87 (FIG. 1B) for 24 hours, treating the cells with CAP for 10, 20, 40 and 60 seconds respectively, further culturing the cells for 24 and 48 hours, and detecting the cell viability by using a CellTiter Blue kit, the inhibition effect of CAP on the cell viability is positively correlated with the time of CAP treating the tumor cells (the abscissa represents the time of CAP treatment, and the ordinate represents the cell viability (expressed by percentage)); after CAP treatment is finished, after the glioma cell line is cultured for 24 hours and 48 hours, the activity difference of the cells detected in the two time periods is small; meanwhile, the inhibition effect of CAP on U87 cells is more obvious than that of U251.

(2) Metformin alone effect experiment: the cells were treated with 2, 4, 8, 16 mmol/l metformin, and the cell viability was measured by CellTiter Blue after 24 hours and 48 hours, respectively, as shown in FIGS. 1C-1D, from which it can be seen that metformin has no significant inhibitory effect on cell viability. Thereafter, after culturing the cells in a 96-well plate for 12 hours, 2, 4, 8, and 16 mM of metformin were added, and the culture was continued for 12 hours, followed by treatment with low-temperature plasma for 10, 20, 40, and 60 seconds, respectively, as shown in FIGS. 1E to 1F, from which it was found that the inhibitory effect of low-temperature plasma on the viability of the cells was increased after the treatment with metformin. The results are shown in FIGS. 1G-1H, and it can be seen that the combination of low temperature plasma and metformin has a synergistic inhibitory effect.

In FIGS. 1C-1D, after culturing the glioma cell line U251 (FIG. 1C) and U87 (FIG. 1D) for 24 hours, the glioma cell line was treated with 2, 4, 8, 16 mM Met, respectively, and cell viability was determined after 24 hours and 48 hours of treatment, and it was found that Met had no significant inhibitory effect on the glioma cell line (Met concentration on the abscissa, cell viability on the ordinate, expressed as a percentage); cell viability was measured at 24 hours and 48 hours, with little difference between the cell viability at the same Met concentration for the two time periods (black bars for 24 hours, gray bars for 48 hours).

In FIGS. 1E-1F, after culturing the glioma cell line U251 (FIG. 1E) and U87 (FIG. 1F) for 12 hours, 2, 4, 8, 16 mM Met was added, and after further culturing for 12 hours, the cells were treated with CAP for 10, 20, 30 seconds, and 12 hours, respectively, and then the cell viability (concentration of Met on the abscissa, cell viability on the ordinate, expressed as a percentage) was measured. It was found that treatment with Met followed by treatment with CAP inhibited cell viability more significantly than Met alone or CAP alone.

In FIGS. 1G-1H, glioma cell line U251, FIG. 1G, U87 cell line, FIG. 1H, and the data in FIGS. 1E and 1F were analyzed by CompuSyn software (the software is the software commonly used in biology to analyze whether multiple drugs have synergistic effects), and it was found that Met and CAP have synergistic inhibitory effects (the circles of different colors in the figures represent combinations of different concentrations and different CAP treatment times, and the ordinate represents the CI value (synergy index), and when CI is less than 1, it means that both means have synergistic effects in such combinations, and when CI is greater than 1, it means none).

Example two: determination of the Effect of Low temperature plasma in combination with metformin on inducing U251/U87 brain glioma cell death

Culturing the U251 and U87 glioma cells in a 96-well plate for 12 hours, adding 16 mmol/L metformin, continuing to culture for 12 hours, treating the glioma cells with low-temperature plasma for 30 seconds, and continuing to culture for 12 hours, wherein the results are shown in FIGS. 2A-2B; as can be seen, the combination of low temperature plasma and metformin alters cell morphology, rounding the cells and presenting a death-like morphology. The combination did significantly induce cell death as detected by the cell death kit compared to the single treatment (see fig. 2C-2D, live cells in the circle, dead cells in the square; fig. 2E-2F, cell death statistics).

In FIGS. 2A-2B, after 12 hours of culture of the glioma cell line U251 (FIG. 2A) and U87 (FIG. 2B), 16 mM Met was added, after 12 hours of culture, the cells were further treated with CAP for 30 seconds, and after 12 hours, the cells were photographed under a bright field microscope to observe cell morphology (Control of the left panel, Met alone, CAP alone, and a combination of Met and CAP in the left panel), where Met alone had no effect on cell morphology (compared to the Control), and the treatment with CAP alone resulted in cell tentacles retraction, and the combination of Met and CAP significantly rounded cells, resulting in a cell morphology similar to death.

In FIGS. 2C-2D, after culturing the glioma cell line U251 (FIG. 2A) and U87 (FIG. 2B) for 12 hours, 16 mM Met was added, after culturing for another 12 hours, the cells were treated with CAP for 30 seconds, after 12 hours, the live and dead cells were detected with a cell live/dead reagent, and the results were observed under a fluorescence inverted microscope (Control of the left panel: Control, treatment of the left panel with Met alone: Met, treatment of the left panel with CAP alone: CAP, treatment of the left panel with Met and CAP in combination: Met + CAP), whereby it was found that the combination of Met and CAP significantly induced cell death, live cells within the circle and dead cells within the square.

In FIGS. 2E-2F, FIGS. 2E and 2F are quantitative statistical graphs of FIGS. 2C and 2D, respectively, and the numbers of live cells and dead cells in the 2C and 2D graphs were counted by ImageJ software, respectively, and the percentage of dead cells to the total number of cells was calculated, and the 2E and 2F graphs were plotted. Remarking: c, D are representative graphs because the experiment was done in triplicate, the other graphs are not shown, and E, F are data from triplicate.

Example three further analysis experiments on the mechanism of the synergistic inhibition effect of low-temperature plasma and metformin

After culturing the U251, U87 glioma cells in a 96-well plate for 24 hours, the cells were treated with low-temperature plasma for 30 seconds, and the levels of free oxygen radicals in the cells and hydrogen peroxide in the cell culture medium treated with low-temperature plasma were measured using an active oxygen measurement kit and a hydrogen peroxide measurement kit, respectively.

As shown in fig. 3A, after culturing the glioma cell line U251 (upper) and U87 (lower) for 12 hours, the cells were treated with CAP for 30 seconds, and the level of intracellular free oxygen radicals (intracellular reactive oxygen species in the round frame) was detected using a reactive oxygen species detection kit.

As shown in FIG. 3B, CAP treatment was detected using a Hydrogen peroxide kitAfter the hydrogen peroxide level in the cell culture medium (intracellular reactive oxygen species in the circle), it was found that CAP treatment significantly increased the H content in the medium₂O₂Horizontal (ordinate indicates H)₂O₂Change in fold of level).

As shown in FIGS. 3C to 3D, the glioma cell line U251 (FIG. 3C) and U87 (FIG. 3D) were cultured for 12 hours, then 16 mmol/L Met was added, and after further culturing for 12 hours, the following experiments were carried out, respectively: (CAP group in figure) was treated with CAP for 30 seconds, and cell viability was measured after 12 hours; (Cat group in the figure), 300U of catalase was added, and then CAP treatment was performed for 30 seconds, and cell viability was measured for 12 hours; (in the figure, H)₂O₂Group) addition of H to cells₂O₂Cell viability was measured after 12 hours. It was found that the synergistic inhibitory effect of Met and CAP disappeared after addition of Cat (catalase, capable of scavenging hydrogen peroxide). And Met and H₂O₂The inhibitory effect on cells is similar to that of Met and CAP, indicating that H produced by CAP₂O₂Plays an important role in the synergistic inhibitory effect of Met and CAP.

As shown in FIGS. 3E-3F, which are light field microscopic observations of the experiments of FIGS. 3C-3D, it was found that the effect of the combination of Met and CAP on cell morphology disappeared after the addition of catalase (CAP + Met + Cat group in FIGS. E-F); with Met and H₂O₂The combination clearly rounded the cells (Met + H in FIGS. E-F)₂O₂) Group, similar to Met in combination with CAP. H illustrating CAP production₂O₂Plays an important role in the synergistic inhibitory effect of Met and CAP.

The above experiment illustrates the generation of H by low temperature plasma₂O₂Plays an important role in the synergistic inhibition effect of low-temperature plasma and metformin. Through the experimental result, on one hand, the synergistic inhibition mechanism of the low-temperature plasma and the metformin can be researched, and on the other hand, reference can be provided for designing a glioma treatment scheme, a product or a device and the like based on the combination of the low-temperature plasma and the metformin. It is to be understood that although the above experiments illustrate the generation of H by CAP₂O₂Plays an important role in the synergistic inhibitory effect of Met and CAPThe research on the mechanism of the synergistic inhibition effect provides more references for the application of the inhibitor, but does not indicate that H can be directly adopted₂O₂Instead of CAP in combination with Met. The main reasons are that: h₂O₂The use in medicine is mainly directed to disinfection, which is rarely used for the treatment of diseases, partly due to H₂O₂The oxidizability of the compound can damage not only diseased cells but also normal cells, and CAP is proved to be capable of selectively killing tumor cells within a certain dosage and has no influence on the normal cells.

Example four further analysis experiments of the mechanism of the synergistic inhibitory effect of low temperature plasma and metformin were carried out

After culturing U251 and U87 glioma cells in a 96-well plate for 12 hours, 16 mmol of metformin is added, the culture is continued for 12 hours, then low-temperature plasma treatment is carried out for 30 seconds, and after the culture is continued for 12 hours, RNA and protein are collected respectively, and the experimental results are shown in fig. 4.

As shown in FIGS. 4A-4B, after culturing the glioma cell line U251 (FIG. 4A) and U87 (FIG. 4B) for 12 hours, 16 mM Met was added, the culture was continued for 12 hours, then CAP treatment was performed for 30 seconds, after 12 hours, RNA was extracted from the cells, and Q-PCR was used to detect the expression of the relevant gene. It was found that the combination of Met and CAP significantly increased the mRNA levels of HSPA6 and c-Fos (the abscissa of the graphs in 4A, 4B indicates the type of gene, the ordinate indicates the relative expression of the gene, Con indicates the control group, Met indicates the group treated with Met alone, CAP indicates the group treated with CAP alone, and Con indicates the group treated with Met and CAP in combination).

As shown in FIG. 4C, after culturing the glioma cell line U251 (left panel of FIG. 4C) and the U87 (right panel of FIG. 4C) for 12 hours, 16 mmol/L Met is added, after culturing for 12 hours, the glioma cell line U251 and U87 are treated with CAP for 30 seconds, and after 12 hours, the cells are collected to extract protein, so that the combination of Met and CAP can be found to improve the protein expression level of C-Fos, but the expression of HSPA6 and C-PARP is not influenced, and the experiment shows that the combination of Met and CAP can obviously improve the protein expression level of mRNA and C-Fos. (the darker the black bar in the figure, the thicker the bar, the higher the expression level, and the beta-actin the reference).

As shown in FIG. 4D, after culturing the glioma cell line U251 (left panel of FIG. 4D) and the U87 cell line (right panel of FIG. 4D) for 12 hours, 16 mmol/L Met was added, and after further culturing for 12 hours, the following experiments were performed, respectively: (Cat-, Met +, CAP + group in the figure) is treated with CAP for 30 seconds, and the expression level of the related protein is detected after 12 hours; (Cat +, Met +, CAP + group in the figure), 300U of catalase was added, and then CAP treatment was performed for 30 seconds for 12 hours to examine cell viability; the combination of Met and CAP was found to increase the protein expression level of c-Fos (Cat-, Met +, CAP + groups in the figure), whereas the c-Fos protein level was unchanged after the addition of Cat (catalase, abbreviated).

As shown in fig. 4E, after culturing the glioma cell line U251 (left panel of fig. 4E) and the U87 cell line (right panel of fig. 4E) for 12 hours, the following experiments were performed, respectively: (Met +, H in the figure)₂O₂+ group) 16 mM Met was added and after 12 hours of further culture, H was added to the cells₂O₂And protein was collected after 12 hours. And Met and H₂O₂The combination also improves the expression level of c-Fos, only adding Met and only adding H₂O₂The expression level of c-Fos was not changed.

Further elucidating the H production of CAP₂O₂Plays an important role in the combination of Met and CAP, and the expression level of c-Fos is up-regulated as a result of the combined effect. This suggests that the synergistic inhibitory effect of low temperature plasma and metformin on cells may be achieved through the c-Fos pathway. The results from this experiment may provide further reference for designing a glioma treatment regimen, product or device based on a combination of low temperature plasma and metformin.

The scale in FIGS. 1-4 represents 100 microns, where P is 0.01, P is 0.001, and P is 0.0001.

In the embodiment of the invention, the application of the combination of metformin and low-temperature plasma in inducing glioma death is researched by using glioma cell lines U251 and U87 as research objects, and the results of the first embodiment and the second embodiment can show that the combination of metformin and low-temperature plasma has obvious synergistic inhibition on glioma cell lines U251 and U87 cells cultured in vitroHas the function of remarkably inducing cell death. The pretreatment of the glioma cells by the metformin can enhance the sensitivity of the tumor cells to low-temperature plasmas, which is probably a potential treatment strategy for the glioma. The application of the combination of the low-temperature plasma and the metformin in glioma treatment can be researched through the scheme of the application of the combination of the metformin and the low-temperature plasma in glioma death induction, so that a new idea is provided for designing a new product or a new scheme for treating glioma. Further, it can be seen from the results of example three and example four that the low temperature plasma generated H₂O₂Plays an important role in synergistic inhibition; the c-FOS transcription and protein levels were significantly increased after the co-treatment with metformin and CAP; indicating that the synergistic inhibitory effect of low temperature plasma and metformin on cells may be achieved through the c-Fos pathway. The results from these experiments may provide further reference for designing a glioma treatment regimen, product or device based on a combination of low temperature plasma and metformin.

While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.

Claims

1. An application of low-temperature plasma and metformin in combination is characterized in that the low-temperature plasma and metformin in combination are applied to non-therapeutic induction of glioma cell death in vitro;

in vitro glioma cells were treated with metformin and then treated with low temperature plasma.

2. The use of low temperature plasma in combination with metformin according to claim 1, wherein the use of low temperature plasma in combination with metformin non-therapeutically induces U251 cell death in vitro.

3. The use of low temperature plasma in combination with metformin according to claim 2, wherein U251 cells cultured in vitro are treated with metformin at a concentration of 1-30 mmol/l, cultured for 6-24 hours and then treated with low temperature plasma for 5-120 seconds.

4. The use of low temperature plasma in combination with metformin according to claim 1, wherein the use of low temperature plasma in combination with metformin non-therapeutically induces U87 cell death in vitro.

5. The use of low temperature plasma in combination with metformin according to claim 4, wherein U87 cells cultured in vitro are treated with metformin at a concentration of 1-30 mmol/l, cultured for 6-24 hours and then treated with low temperature plasma for 5-120 seconds.