Study on the mechanism of CXCL12/CXCR4 axis mediated upregulation of IL-8 and IL-6 on the biological function of acute T lymphocyte leukaemia cells

doi:10.21203/rs.3.rs-2309964/v1

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Study on the mechanism of CXCL12/CXCR4 axis mediated upregulation of IL-8 and IL-6 on the biological function of acute T lymphocyte leukaemia cells

https://doi.org/10.21203/rs.3.rs-2309964/v1

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published 28 Oct, 2023

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Blocking the CXCL12/CXCR4 axis can alter the biological functions of leukaemia cells. We hypothesised that two cytokines, IL-8 and IL-6, play an important role in this process. We established a co-culture model of leukaemia cells and BMSCs. When AMD3100 and G-CSF blocked the CXCL12/CXCR4 axis, biological changes in the leukaemia cells and changes in IL-8 and IL-6 concentrations were observed. We assume this regulatory process may require the participation of certain signalling pathways. After stimulating the CXCL12/CXCR4 axis, we used specific pathway blockers to determine whether four candidate signalling pathways play regulatory roles in this process. ELISA results confirmed that MG132(10 µm) inhibits the expression of IL-8, confirming that the NF/κB signalling pathway is involved. Perifosine could inhibit the expression of IL-6, confirming that the PI3K/Akt signal system is involved in this process. Western blotting confirmed that changes in the NF/κB signalling pathway resulted in the inhibition of IL-8 expression. SP600125 and Perifosine treatments also played a role. It is possible that the JNK and PI3K/AKT signalling pathways would also inhibit the expression of IL-8, but their inhibition appeared later. IL-6 expression was lower in the Perifosine group. This shows that inhibiting the PI3K/AKT signalling pathway can reduce IL-6 expression. Hence, this process requires the participation of multiple signalling pathways to jointly regulate the expression of IL-8 and IL-6. This type of regulation may be a relatively sophisticated network in which there may be cross-effects. The pathogenesis of leukaemia may be affected by this. IL-6 and IL-8 are physiologically regulated by the CXCL12/CXCR4 axis, the NF/κB and JNK/AP-1 pathways are required for IL-8 expression in T-cell acute lymphoblastic leukaemia. By upregulating IL-8, the bone marrow microenvironment and CXCL12/CXCR4 axis may contribute to the pathogenesis of T-cell acute lymphoblastic leukaemia.

Interleukin-8

Interleukin-6

CXCL12/CXCR4 axis

regulation mechanism

NF/κB

PI3K/AKT

Acute lymphocytic leukaemia (ALL) is the most common malignancy in children. Because of the continuous improvement in its treatment plan, the cure rate of ALL has significantly improved. The event-free survival rate has reached 70–80%, but there remain 20–30% of children with leukaemia relapse, leading to treatment failure. Previous studies have confirmed that the bone marrow microenvironment plays an important role in drug resistance in leukaemia. Bone marrow stromal cells (BMSCs) can affect the survival of leukaemia cells through various mechanisms, such as physical contact with leukaemia cells and secretion of related cytokines(1). CXCL12 (chemokine 12) is mainly produced by BMSCs and immature osteoblasts and plays an important role in the immune, nervous, circulatory, and other systems (2). The CXCL12 chemokine receptor CXCR4 is expressed on many blood and bone marrow cells. CXCR4(CXC chemokine receptor 4) is abnormally expressed in many malignancies and haematopoietic tumours. It has been shown to play a key role in immune cell migration, tumour metastasis, and stem cell homing. It is also a prognostic marker in leukaemia, breast cancer, and other cancers(3). In recent years, it has been found that the CXCL12/CXCR4 biological axis plays an important role in the migration, homing, and activation of leukaemia stem cells. The CXCR4/CXCL12 biological axis can protect leukaemia cells from the cytotoxic effect of chemotherapeutic drugs and cause drug resistance and relapse of leukaemia (4). Therefore, an increasing number of researchers have begun to enhance the cytotoxic effect of chemotherapeutics by disrupting the interaction between leukaemia cells and stromal cells. In this study, a co-culture model of BMSC sand leukaemia cells was established in vitro to simulate the interaction between the bone marrow microenvironment and leukaemia cells and observe the effect of BMSCs on the biological effects in leukaemia cells.

AMD3100, an important tool in our research, is a CXCR4 antagonist, and its benefits have been confirmed in several clinical studies (5–7). Many studies have shown that the mechanism of AMD3100 is mainly dependent on the reduction of CXCR4 expression on the cell surface. Blocking the CXCL12/CXCR4 axis can reduce static leukaemia cells adhering to the bone marrow niche and enhance the cytotoxicity of cell cycle-dependent chemotherapeutics.This allows for the elimination of minimal residual disease (MRD) and reduces the recurrence of leukaemia. Sisoneet al. (8) also confirmed that the CXCL12/CXCR4 signal axis between leukaemia cells and stromal cells in the bone marrow microenvironment is conducive to the growth and proliferation of leukaemia cells. The CXCL12/CXCR4 axis is mainly involved in pathways such as phosphatidylinositol 3-kinases (P13K)/protein kinase B (PKB, also known as Akt) pathway, mitogen-activated protein kinase (MAPK) pathway, p42/44 mitogen-activated protein kinase (P42/44 MAPK) pathway (9).

Granulocyte-colony stimulating factor (G-CSF) has been widely used clinically in leukaemia chemotherapy. A high expression of CXCR4 on the surface of leukaemia cells is a sign of poor prognosis(10). However, G-CSF can inhibit the expression of CXCR4, thereby inhibiting the interaction of CXCL12/CXCR4. Ultimately, it promotes the release of leukaemia cells from the bone marrow, thereby enhancing the cytotoxic ability of chemotherapeutics. Zhong et al. (11) confirmed that G-CSF promotes cells in the quiescent phase to enter the S phase, thereby increasing the lethality of chemotherapeutics on leukaemia cells. Hence, the complete response rate can be achieved and MRD can be reduced. Kimet al.(12) reported that G-CSF can downregulate the expression of CXCL12 and CXCR4.

Recent studies on leukaemia suggest that BMSCs derived from patients with leukaemia can secrete various cytokines, such as interleukin-1(IL-1) β, IL-6, IL-8, IL-10, SDF-1, CXCL12, and basic fibroblast growth factor(bFGF). These cytokines can support the survival and proliferation of leukaemia cells and induce their resistance to apoptosis (13). As an important regulatory factor affecting the occurrence and development of tumours in the tumour microenvironment, IL-8 can affect the vascular formation of endothelial cells and promote the proliferation and survival of tumour cells and endothelial cells. Simultaneously, IL-8 can also enhance the motility of tumour cells, endothelial cells, or tumour infiltrating leukocytes (14). The concentration of IL-8 in the peripheral blood of patients with ALL was significantly higher than that in normal subjects. This may be because leukaemia cells and BMSCs can produce IL-8. Additionally, chemotherapy can affect the expression of IL-8 in patients with ALL. The level of IL-8 in the peripheral blood of patients with ALL is significantly higher than that of normal people. Serum IL-8 levels in patients with ALL who are in complete remission after chemotherapy can return to normal levels. Moreover, it can be used as an auxiliary index to monitor their therapeutic response. Therefore, it is speculated that IL-8 in the haematopoietic microenvironment and its cytokine network are closely related to the occurrence, development, and clinical manifestations of leukaemia (15).

The biological activities of IL-6 include inducing the proliferation and differentiation of B cells, active T cells, and natural killer cells; promoting the proliferation of haematopoietic progenitor cells; and participating in the development of autoimmune diseases and cancers(16). Some studies have shown that serum IL-6 concentration in children with ALL is significantly high, but the specific mechanism for this finding has not been fully elucidated (17). IL-6 can affect the tumour microenvironment to promote tumour development by inducing tumour angiogenesis and inhibiting cancer cell apoptosis(18). Currently, there are few studies on the mechanism of the CXCL12/CXCR4 biological axis underlying the expression, concentration, etc. of IL-8, IL-6, and other cytokines. Because the CXCL12/CXCR4 biological axis and IL-8, IL-6, and other cytokines play important roles in the pathogenesis of leukaemia, our study analysed that the relationship between CXCL12/CXCR4 biological axis and cytokines, represented by IL-8 and IL-6, is of great significance to the discovery of new therapeutic targets in the future.

We propose the following hypotheses based on previous studies: [1] the CXCL12/CXCR4 axis is known to promote the proliferation of ALL cells and prevent their apoptosis and [2]blocking the CXCL12/CXCR4 biological axis can alter the biological functions of leukaemia cells. The two cytokines IL-8 and IL-6 play an important role in this process. The confirmation of this function may require the participation of certain signalling pathways. We used specific pathway blockers to detect whether the four signal pathways, namely, NF/κB (nuclear factor-κB), JNK, PI3K/AKT, and P38MARK, play regulatory roles in this process, as well as study their specific mechanism. It has positive significance in the progress of clinical treatment of ALL.

BMSC culture

Patient samples: Six bone marrow samples were collected from patients without leukaemia (three from patients with immune thrombocytopenia, two from patients with anaemia, and one from a patient with juvenile rheumatoid arthritis) between January and June 2019 in the Paediatrics Department of the People’s Hospital of Zhangqiu District(Jinan, Shandong)According to the Declaration of Helsinki, informed consent was obtained, and the study was approved by the Ethics Committee of the People’s Hospital of Zhangqiu District, Jinan, Shandong. Two milliliters of bone marrow samples were collected and stored in heparin anticoagulation tubes, then they were diluted and mixed evenly with phosphate-buffered saline (PBS) of equal volume。Monocytes (MNCs) in PBS bone marrow mixture were separated using the Ficoll-Paque medium(density 1.077 ± 0.001 g/mL; HaoYang Company, Tianjin, China).The volume ratio of bone marrow fluid and separation fluid is 1:1 and centrifuged at 2500rpm for 20 minutes (Neofuge 15R, Heal Force Bio-meditech Holdings Limited, Shanghai, China). Isolated MNCs were collected and washed twice in PBS (Servicebio, Logan, Utah, USA), then centrifuged at 1000rpm for 5minutes. BM MNCs were blown and resuspended for counting. The mixed cells were suspended in in 10% DMEM-LM (Dulbecco's Modified Eagle's Limiting Medium) (Hyclone, Logan, Utah, USA) at a density of 1 × 10⁵ / mL inoculated in culture bottle. And they were cultured in a humidified incubator at 5% CO₂and 37°C(MCO-15AC, Sanyo, Osaka, Japan). The medium was replenished every 2–3 days. Following 7 days of culture, adherent fibroblast-like cells developed a confluent state. After 21–28 days, the fibroblast-like cells grow rapidly to cover the culture dish, and the cells were fusiform, closely arranged, and grew in a spiral or radial shape. Subsequently, cells were treated with 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) and passaged at 1×10⁴ cells/cm² in 10% DMEM-LM. Cells after the second and fourth passages were used in the experiment. During the process of cell culture, cell growth and morphology were observed under a microscope (magnification, ×200). (TE2000, Nikon Corporation, Tokyo, Japan).

Culture Of Jurkat Cells

The human ALL cell line Jurkat cells was purchased from from PROCEL(SWEXB11405,PROCEL, Wuhan, China). Jurkat cells were cultured in

25cm² cell culture bottle in RPMI1640(Hyclone, Logan, Utah, USA) medium with 10% foetal bovine serum (Sera Pro, SYSTECH GmbH, Germany)at 37°C in a humidified atmosphere of 5% CO₂ and 20% O₂(MCO-15AC, SANYO, Osaka, Japan). The medium was changed every 2–3 days. The growth of Jurkat cells were observed under microscope every day and counted. When the number of cells reached 5 × 10⁶, the cells in the logarithmic growth period were used for the experiment.

Establishment Of A Co-cultivation Model

Approximately 1 × 10⁵ BMSCs were seeded in 12-well plates. After the cell fusion reached 90% to form a monolayer under the microscope, PBS was used to wash and remove the non-adherent cells. Then, 1 × 10⁵ Jurkat cells (100 uL) in the logarithmic growth period and RPMI (Roswell Park Memorial Institute)-1640 medium were added to the 12-well plates for establish the co-culture model of BMSCs and Jurkat cells (Fig. 1B). These were divided into five groups(The cell suspension was 100 uL/well, and the same number): control group(control cells were cultured in the same conditions but in the absence of BMSCs),co-cultivation group(co culture of BMSCs and Jurkat cells), co-cultivation + AMD3100 group (10 µg/mL,group A), co-cultivation + G-CSF group(50 ng/mL, group G), and co-cultivation + G-CSF + AMD3100 group(group A + G). The cells of each group were cultured for 48 hours at 37°C in a humidified atmosphere of 5% CO₂. After 48 hours, all suspension cells were collected. The cells were centrifuged at 1800rpm for 10min, the supernatant was discarded and resuspended with 1ml RPMI 1640 for subsequent experiments.

Detection Of Cxcr4 Expression Using Flow Cytometry

Five groups of cells were cultured for 48 hours, centrifuged, and collected. After washing with cold PBS, the concentration of the cells was adjusted to 1× 10⁷ cells/mL, and 5 groups cell suspension(100 µL /group) were added to five 1 ml tubes These tubes were centrifuged at 1000 rpm for 5 minutes, after which the supernatant was discarded, the precipitate was washed twice with PBS, and centrifuged again at 1000 rpm for 5 minutes. The cells in the control group were resuspended in 120 µL of PBS. The cells of the other four groups were added to 100 µL of PBS and 20 µL of the PE-CD184 monoclonal antibody༈PROTEINTECH, Wuhan, China, Cat No. 60042-1-Ig༉, incubated at 4°C in dark for 15 minutes, washed twice with PBS, and resuspended in 100 µL of PBS. Finally, the expression of CXCR4 was detected using flow cytometry.

Cell Cycle Detection

After 48 hours of co-culture, the cells were separatedand collected. The supernatant was discarded, the precipitate was washed with 1 mL of PBS, and then digested with 1 mL of 0.25% trypsin. When the cells became round and some cells appeared suspended, PBS was added to terminate digestion. The cell suspension was transferred into a centrifuge tube and centrifuged at 1500 rpm for 5 minutes. The cells were resuspended in 3 mL of PBS precooled at 4°C, centrifuged again at 1500 rpm for 5 minutes, and the supernatant was discarded. Then, 75% ethanol precooled at -20°C was slowly added to the cell precipitation and the cells were incubated overnight at 4°C. After adding 2 mL of PBS, the cells were centrifuged at 1500 rpm for 5 minutes and then 100 µL PBS was added to resuspend the cells. Two microlitres of RNase were added, and the tubes were placed in a 37°C water bath for 40 minutes. Next, 100 uL of the propidium iodide (PI) staining solution were added to the tubes and allowed to stain for 20 minutes in dark. Then, flow cytometry detection was performed.

Cck-8 Detection Of Cell Viability

Five groups of cells were isolated and collected, and a cell suspension was prepared. Cells were seeded in 96-well plates at a density of 4 × 10³ cells per well, and each group was set up with four multiple wells; the cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C for 24 hours. Additionally, a background control well without cells was set up. Ten microlitres of the CCK-8 solution were added to all wells, and these were incubated for 2 hours. Absorbance A was detected at a wavelength of 450 nm using an enzyme reader. The cell proliferation rate was calculated as follows: (experimental group– blank control) / (negative control– blank control) × 100%.

Cell Apoptosis Detection

The cells were separated and collected after 48 hours of culture. According to the manufacturer’s instruction, the collected cells were rinsed with precooled PBS, after which 5×10⁵ cells were collected, resuspended in 500 µL binding buffer (Component C),the cells were stained with 5 µL PI ༈Component B༉and 5 µL FITC-labeled Annexin-V(Component B༉ using the Annexin V-FITC/PI Apoptosis Detection Kit (Elascience, Catalog #E-CK-A211, Huston, TX, USA). The cells were incubated in dark for 15 minutes at room temperature, and apoptosis was detected using flow cytometry within 1 hour.

First Concentration Analysis Of Il-8 And Il-6 Using Elisa

The concentrations of IL-8 and IL-6 in the culture supernatant of the five groups were detected using an ELISA (enzyme-linked immuno sorbent assay) kit(IL-6;Cloud-Clone Corp, #SEA079Hu, Wuhan, China༛IL-8: Cloud-Clone Corp, # SEA080Hu, Wuhan, China). Standard wells, sample wells to be tested, and blank wells were set. Five standard wells were added with 100 µL standard samples of different concentrations sequentially. 100 µL PBS were added to the blank well, and 100 µL of sample was added to be tested to other wells. The enzyme plate was covered with a membrane and incubated at 37°C for 1 hour; the cells were washed with lotion, 100 µL of detection solution A was added, followed by incubation at 37°C for 1 hour, and washed again. 100 µL of detection solution B was added, following which the cells were incubated at 37°C in dark for 30 minutes and washed by theWashBuffer (provided with the kit).The next step was to add 100 µL of the substrate solution and allow the development of colour at 37°C for 20 minutes in dark. When the standard wells turned blue, 50 µL of the stop solution was added. Thereafter, the colour changed from blue to yellow. Within 10 minutes of the colour change, the absorbance value of each well was measured at a wavelength of 450 nm, the standard curve was drawn, and the levels of IL-6 and IL-8 were calculated using the standard curve equation.

Second Concentration Analysis Of Il-8 And Il-6 Using Elisa

Jurkat cells in the logarithmic growth stage were suspended in RPMI 1640 complete medium, and the cell density was established at 5 × 10⁵ /mL. The cells were added to six-well plates, then PBS, 10 µM MG-132, 20 µM MG-132, 10 µMSP600125, 10 µM Perifosine, or 10 µM SB203580 was pretreated for 1 hour and CXCL12 (1µg/mL) was used to stimulate the samples for 5 hours. The cells were divided into six groups: control group, 10 µMMG-132 group (to block the NF/κB signal pathway), 20 µMMG-132 group, SP600125 group (to block the JNK signal pathway), Perifosine group (to block the PI3K/Akt signal pathway), and SB203580 group (to block the p38mark signal pathway). Finally, the concentrations of IL-8 and IL-6 in the supernatant of the six groups were detected using ELISA kits (Enzyme-linked Immunosorbent Assay Kit For IL-6 and Enzyme-linked Immunosorbent Assay Kit For IL-8, Your Ture-Life BioTech, Nanjing,China). The six groups of Jurkat cells without the supernatant were subjected to the following western blot experiments.

Western Blot Analysis: Detection Of The Expression Of Il-8 And Il-6 Protein

For total protein analysis, the six groups of Jurkat cells were lysed on ice in RIPA lysis buffer inhibitor cocktail(Servicebio,Wuhan,China) for 30 minutes. After centrifugation at 13400 × g for 10 minutes at 4°C, the supernatant containing the total protein was collected. The protein concentration of each sample was determined using a BCA protein concentration determination kit (Servicebio, Wuhan, China). The protein solution was added to protein buffer at a ratio of 4:1 and the proteins in it were denatured in boiling water for 5 minutes and cooled at 4°C. The electrophoresis tank was installed, and 5% concentrated gel and 10% separation gel were prepared according to the different molecular weights of the proteins. Next, the gel was poured, the sample was loaded, and electrophoresis was conducted. The polyvinylidene fluoride membrane was latersealed with 5% skim milk (0.5% TBST dilution) for 1 hour. The membranes were immunoblotted overnight at 4°C with primary antibodies against IL-8(cat.noab18672, AbcamCambridge,UK), IL-6(cat.no. GB11117, Servicebio,Wuhan,China),and GAPDH (Servicebio, Wuhan,China). Following washing with TBST for 5 minutes, the membranes were incubated with HRP-conjugated secondary antibody (Servicebio, Wuhan,China) for 30 minutesat room temperature. Thereafter, these were washed, visualised by employing enhanced chemiluminescence (ECL; Servicebio, Wuhan,China), and recorded using Kodak films. Finally, the Alpha Innotech software was used to analyse the results(alphaEaseFc).

Statistical analysis

Data were analysed using SPSS version 14.0 (SPSS Inc., Chicago, IL, USA). Quantitative data are presented as mean ± standard deviation. Two-way analysis of variance with Fisher's least significant difference as a post hoc analysis was used for comparisons among multiple groups. A P-value of < 0.05 was considered to indicate a statistically significant difference.

Microscopy

Following 7 days of culture, adherent fibroblast-like cells developed a confluent state. However, the cells were highly homogeneous, as shown in Fig. 1A. The co-cultivation model was established for 48 hours, as shown in Fig. 1B.

Expression Of Cxcr4 On The Surface Of Jurkat Cells

Expression of the CXCR4 receptor on the surface of Jurkat cells was detected using flow cytometry. Compared with the control group (88.4 ± 5.03%), the expression rate in the co-culture group was 98.5 ± 3.48%. Group A showed that after treatment with AMD3100, the positive rate of CXCR4 in Jurkat cells decreased to (40.2 ± 3.56%,)%. After group G was treated with G-CSF, CXCR4 decreased to (76.6 ± 4.81)%. In group A + G significantly the most to13.9 ± 2.09% (P < 0.05). The results showed that AMD3100 and G-CSF could reduce the expression of CXCR4 on the surface of Jurkat cells, especially when used together(Fig. 2).

Cell Cycle Analysis

Compared with the control group, the co-cultivation group significantly blocked the cell cycle, and the number of cells in G0/G1 phase increased from 51.49 ± 3.17% to 59.14 ± 2.69% (P < 0.05). The number of cells in the S phase decreased from 40.20 ± 3.17% to 29.07 ± 2.69(P < 0.05). The proportion of cells in the G0/G1 phase was low following the AMD3100 and G-CSF single or combined treatment. Compared with the control group (51.49 ± 3.17%),the proportion of cells in the G0/G1 phase in group A decreased to 49.24 ± 3.56%, in group G to 47.49 ± 1.81%, and in group A + G to 47.28 ± 3.77%. The corresponding S phase of the three groups increased to 43.16 ± 1.17%, (P > 0.05), 42.96 ± 2.0% (P > 0.05), and 45.44 ± 2.83% (P < 0.05)(Figs. 3 ).

Cck8 Detection Of The Cell Proliferation Rate

The cell proliferation of 5 groups of cells was observed under the microscope, as shown in Fig. 4(A-D). The CCK-8 kit was used to detect the proliferation rate of the five groups of leukaemia cells. The results showed that compared with the control group, the proliferation rate of the co-cultivation group was higher (1.44 ± 0.03 vs. 1.01 ± 0.12) (P < 0.05). The cell proliferation rates of groups A, G, and A + G were further reduced to 0.51 ± 0.04, 0.56 ± 0.03, and 0.27 ± 0.02, respectively, and the cell proliferation rate of group A + G significantly reduced (Fig. 5).

Apoptosis Of Jurkat Cells Detected Using Flow Cytometry

Compared with the control group with an apoptosis rate of 6.28 ± 0.84%, the apoptosis rate of the co-cultivation group was 5.12 ± 0.21%(P > 0.05). The apoptosis rate was 26.10 ± 3.51% in group A, 13.0 ± 1.79% in group G, and 36.14 ± 4.22% in group A + G, which makes it the highest among the five groups(P < 0.05). The differences were significant(Fig. 6). AMD3100 and G-CSF could induce the apoptosis rate of Jurkat cells, and the effect of the combination of AMD3100 and G-CSF was more obvious.

Initial Concentration Analysis Of Il-8 And Il-6 Using Elisa

The results showed that the concentration of IL-6 in the co-cultivation group was significantly higher than that in the control group (9999.92vs. 780.84 pg/mL, P < 0.05).The concentration of IL-6 in groups A (7899.65 pg/mL), G (8299.21 pg/mL), and A + G (5678.89 pg/mL) was significantly lower than that in the co-culture group (P < 0.05), with the most obvious decrease in group A + G. Additionally, there was no significant difference between groups A and G (P > 0.05).

Similarly, the concentration of IL-8 in the co-cultivation group was significantly higher than that in the control group(1579.14 vs 50.15 pg/mL, P < 0.05).The concentration of IL-8 in groups A (790.41 pg/mL), G (988.13 pg/mL), and A + G(544.03 pg/mL) was significantly lower than that in the co-culture group (P < 0.05), with the most obvious decrease in group A + G. Additionally, there was no significant difference between groups A and G (P > 0.05) (Fig. 7).Our experiments confirmed that CXCL12 can increase the production of IL-6 and IL-8 inleukaemia cells, whereas AMD3100 and C-GSF can reduce the production of IL-6 and IL-8 by inhibiting the CXCL12/CXCR4 signalling pathway. The combination of the two produces a stronger inhibitory effect.

Effects Of Four Signal Pathway Inhibitors On Il-8 And Il-6 Concentrations Were Determined Using Elisa

In the six groups of Jurkat cell culture supernatants, the concentrations of IL-8 and IL-6 were significantly lower than those in the control group (P < 0.05).

Comparison of IL-8 concentration among the six groups: In the first hour, compared with the IL-8 concentration in the control group(77.47 pg/mL), the IL-8 concentration in the 10 µMMG132 group (44.58 pg/mL), 20 µMMG132 group (50.36 pg/mL), SP600125 group (65.02 pg/mL), Perifosine group (52.98 pg/mL), and SB203580 (55.65 pg/mL) group was lower (P < 0.01and P < 0.05, respectively). In the third hour, compared with the IL-8 concentration in the control group (86.59 pg/mL), the IL-8 concentration in the 10µM MG132 group (30.42 pg/mL) was significantly lower (P < 0.01). There was no significant difference between the other groups and the control group ( P > 0.05). In the fifth hour, compared with the IL-8 concentration in the control group (70.12 pg/mL), the IL-8 concentration in the 10 µMMG132 group (40.91 pg/mL) was lower (P < 0.01). There was no significant difference between the other groups and the control group( P > 0.05). (Fig. 8A)The results confirmed that when the CXCL12/CXCR4 axis is activated, MG132 at a concentration of 10 µM can inhibit the expression of IL-8 in the first, third, and fifth hours. However, the concentration of the IL-8 protein in the 20 µM MG132, SP600125, Perifosine, and SB203580 groups significantly decreased only in the first hour. This confirmed that the NF/κB signalling pathway participates in this process. Additionally, the effect of 10 µMMG132 was better than that of 20 µM MG132.

Comparison of IL-6 concentration among the six groups: In the first hour, compared with the IL-6 concentration in the control group (15.07 pg/mL), only the IL-6 concentration in the Pifexin-treated group (5.51 pg/mL) was lower (P < 0.01). The other groups also showed a significant increase (P < 0.05). In the third hour, compared with the IL-6 concentration in the control group (24.90 pg/mL), the IL-6 concentration in the Perifosine group (15.90 pg/mL) was significantly lower than that in the other groups (P < 0.01).The IL-6 concentration in other groups was higher than that in the control group, especially in the 10 µM MG132 group (49.64 pg/mL).In the fifth hour, compared with the IL-6 concentration in the control group(17.93 pg /IL-6 concentration in the Perifosine group was not different (20.52 pg/mL) (P > 0.05) (Fig. 8B).The IL-6 concentration in other groups was significantly higher than that in the control group (P < 0.05).

The results showed that Perifosine could inhibit the concentration of IL-6 in the first and third hours when the CXCL12/CXCR4 axis was activated. It was confirmed that the PI3K/Akt signal system is involved in this process.

Effects Of Four Signal Pathway Inhibitors On The Protein Expression Of Il-8 And Il-6

To confirm whether the application of these four signalling pathway blockers affects the protein expression of IL-8 and IL-6, we performed western blot analysis. In the six groups of Jurkat cells, the expression of IL-8 or IL-6 was significantly lower than that in the control group (P < 0.05), and the results are as follows:

IL-8:In the first hour, compared with the expression of the IL-8 protein in the control group, there were no significant difference between the other groups (P > 0.05). In the third hour, compared with the expression of the IL-8 protein in the control group, the expression of the IL-8 protein in the 10 µM MG132 group was significantly lower( P < 0.01).There was no significant difference between the other groups and the control group( P > 0.05). In the fifth hour, compared with the expression of the IL-8 protein in the control group, the expression of the IL-8 protein in the 10 µM MG132 group, SP600125 group, and Perifosine group were significantly lower ( P < 0.05). There was no significant difference between the other groups and the control group( P > 0.05). (Fig. 9).

The results confirmed that when the CXCL12/CXCR4 axis was activated, 10 µM of MG132 inhibited the NF/κB signalling pathway in the third and fifth hours, resulting in the inhibition of the expression of the IL-8 protein. The SP600125 and Perifosine groups inhibited the expression of the IL-8 protein in the fifth hour. It is possible that blocking the JNK signal pathway and the PI3K/AKT signal pathway would also inhibit the expression of the IL-8 protein, but its effects may only be seen later.

IL-6: In the first hour, compared with the expression of the IL-6 protein in the control group, the expression of the IL-6 protein in the Perifosine group was significantly lower (P < 0.01). There was no significant difference between the other groups and the control group(P > 0.05). In the third hour, compared with the expression of the IL-6 protein in the control group, there were no significant difference between the other groups (P > 0.05). In the fifth hour, compared with the control group, there were no significant differences in the expression of the IL-6 protein among other groups (P > 0.05) (Fig. 9).

The expression of the IL-6 protein was lower in the Perifosine group in the first hour. This shows that inhibiting the PI3K/AKT signalling pathway can reduce IL-6 protein expression.

In this experiment, the following results were obtained:

The CXCLl2/CXCR4 signal axis has an important influence on the biological behaviour of leukaemia cells. It can influence the expression of CXCR4 in leukaemia cells, cell cycle, apoptosis rate and proliferation rate of leukaemia cells, as well as secretion of IL-8 and IL-6 by leukaemia cells. However, addition of AMD3100 or G-CSF can change these results, especially when used together, because both AMD3100 and G-CSF can inhibit the CXCLl2/CXCR4 signal axis.

ELISA confirmed that when the CXCL12/CXCR4 signal axis is activated by exogenous CXCL12, the concentration of IL-8 and IL-6 produced by leukaemia cells decreased with the addition of some type of blocker. Our experimental results confirm that NF/κB signalling pathway is involved in the regulation of IL-8, and PI3K/Akt signalling system is involved in the regulation of IL-6.

Western blot analysis confirmed that when the CXCL12/CXCR4 axis is activated, the NF/κB signalling pathway can downregulate the expression of the IL-8 protein. The PI3K/Akt signalling pathway can simultaneously regulate the protein expression of IL-8 and IL-6.

Regarding the problem of CXCL12/CXCR4 affecting the biological behaviour of leukaemia cells, we confirmed this using AMD3100 and G-CSF. In recent years, it has been found that the bone marrow microenvironment has an important influence on the biological behaviour of leukaemia cells. Stromal cells in the tumour microenvironment continuously secrete CXCL12 and enforce a variety of biological effects by binding with its CXCR4 receptor. Leukaemia cells with a high CXCR4 expression indicate that the bone marrow microenvironment has a strong protective effect and a worse prognosis (19).Inhibition of CXCL12/CXCR4 can induce the entry of leukaemia stem cells into the peripheral circulation and enhance the anti-leukaemia effect of chemotherapeutics(20). Some studies have shown that human primary leukaemia cells pretreated with antibodies neutralising the CXCR4 receptor were blocked from entering the bone marrow and spleen of transplanted mice(21).

We detected the expression of the CXCR4 receptor in Jurkat cells using flow cytometry. The results showed that the expression of the CXCR4 receptor in the Jurkat cell line was high, which was consistent with previous reports (22).In the co-cultivation group, the proportion of Jurkat cells in the G0/G1 phase increased and the proportion of cells in the Sphase decreased. It was suggested that BMCSs can induce leukaemia cells to enter the quiescent phase, which is consistent with a previous report by Shen et al.(23). However, the administration of AMD3100 and G-CSF increased the proportion of cells in the Sphase. AMD3100 and G-CSF could promote the entry of leukaemia cells into the cell cycle and enhanced the cytotoxic effect of chemotherapeutics. The possible reasons were that G-CSF decreased the level of CXCL12, inhibited the expression of CXCR4, and promoted stationary-phase leukaemia cells to enter the mitotic phase, enhancing the cytotoxic effect of chemotherapeutics. AMD3100 disrupted the interaction between the tumour and matrix, mobilises the leukaemia cells away from the protective microenvironment, and strengthens the cytotoxic effect of chemotherapeutics(24).

The CCK-8 test results showed that the proliferation rate of the co-cultivation group was higher than that of the control group. Sisonet al.(25)confirmed that the CXCL12/CXCR4 signal axis between leukaemia cells and stromal cells in the bone marrow microenvironment is conducive to the growth and proliferation of leukaemia cells. The experimental results were consistent with these results. When AMD3100 and G-CSF were added to the co-cultivation group, the viability of Jurkat cells decreased. It was considered that AMD3100 and G-CSF affected the CXCLl2/ CXCR4 signal axis, thereby inhibiting the proliferation of leukaemia cells. The inhibitory effect was more obvious when the two drugs were combined.

The interaction of CXCL12/CXCR4 can prevent the apoptosis of leukaemia cells induced by chemotherapy. Apoptosis of Jurkat cells was detected using flow cytometry. There was no difference between the control group and co-culture group. After adding AMD3100 and G-CSF, the apoptosis rate increased, and the combination of AMD3100 and G-CSF had the highest apoptosis rate.

The bone marrow microenvironment is rich in various cytokines that maintain and promote the growth of haematopoietic stem and progenitor cells. BMSCs produce high levels of IL-6, and the bone marrow microenvironment and tumour cells interact through paracrine soluble cell mediators to enhance the secretion of growth factors between the two. Scupoli et al.(26) found that BMSCs can upregulate the expression of IL-8 mRNA in acute lymphoblastic leukaemia by activating the CXCR4 receptor. When we detected the concentration of IL-8 and IL-6 using ELISA for the first time, we found that the concentrations in the co-cultivation group were significantly higher than those in the control group. The addition of AMD3100 or G-CSF reduced the concentrations of IL-6 and IL-8, especially when AMD3100 was combined with G-CSF. This may be because AMD3100 can block the CXCL12/CXCR4 biological axis, resulting in the reduced production of IL-8 and IL-6. The G-CSF group can also reduce the production of IL-8 and IL-6 because G-CSF can decrease the level of CXCL12 and inhibit the expression of CXCR4(27). Combined with the experimental results just now, the apoptosis rate of cells in group A and group G was different, and that in group G was higher, indicating that CSF has a more obvious effect on cell apoptosis. However, ELISA experiment showed that there was no difference in the concentration of IL-6 and IL-8. Considering that IL-6 and IL-8 were downstream cytokines, there may be other influencing factors at the same time, which needs further research and confirmation,

Thus, the addition of AMD3100 and G-CSF affects the CXCL12/CXCR4 signal axis, changes the biological characteristics of leukaemia cells, and reduces the proliferation rate of Jurkat cells. Furthermore, it increases the rate of apoptosis and changes the proportion of cells in the G0/G1 phase and S phase. More cells in the G0/G1 phase enter the S phase, which can enhance the cytotoxic effect of chemotherapeutics. In addition, in the co-cultivation model, the combined addition group had more significant effects than the single addition group. This indicates that AMD3100 and G-CSF have a synergistic inhibitory effect on CXCL12/CXCR4, which is consistent with the results of Shen et al. (28). Moreover, the CXCL12/CXCR4 signal axis can promote the secretion of IL-8 and IL-6, and this effect can be blocked using AMD3100 and G-CSF. In leukaemia cells alone, after adding specific signal pathway blockers, exogenous CXCL12 activated the CXCL12/CXCR4 signal axis, and it was found that the concentrations of IL-8 and IL-6 produced by leukaemia cells changed. During the second time, the concentrations of IL-8 and IL-6 in the supernatant of Jurkat cells cultured in the six groups were detected using ELISA. We selected four specific pathway blockers to block the four signalling pathways: NF-κB, JNK, PI3K/AKT, and P38MARK.Wethen added the exogenous CXCL12 factor to simulate the activation of the CXCL12/CXCR4 axis. The results showed that Perifosine can inhibit the production of IL-6, confirming that the PI3K/Akt signalling system is involved in the regulation of IL-6. Ten micromolar of MG132 inhibited the production of IL-8. This confirms that the NF/κB signalling pathway is involved in the regulation of IL-8.

Western blot analysis confirmed that when the CXCL12/CXCR4 axis is activated, the expression of IL-8 protein was affected by the regulation of the NF/κB signal pathway. The JNK and PI3K/AKT signalling pathways would also inhibit the expression of the IL-8 protein, but their effects would be observed later. The regulation of the PI3K/AKT signalling system affected the expression of the IL-6 protein, but only in the first hour. Therefore, the PI3K/AKT signalling system can also regulate the expression of IL-8 and IL-6 proteins. Our experiment showed that under the premise that MG132 works, although CXCL12/CXCR4 was activated, it still reduced IL-8 protein expression. This indicates that the NF/κB signalling pathway plays an important regulatory role in the expression of the IL-8 protein. Similarly, JNK and PI3K/AKT also have this inhibitory effect on the IL-8 protein expression. PI3K/AKT can also inhibit the expression of the IL-6 protein.

This experiment proved IL-8 and IL-6, which were important cytokines in the ALL and bone marrow microenvironment, when the CXCL12/CXCR4 axis was activated, NF/κB, JNK, and PI3K/Akt signalling systems were also activated, which can regulate the expression of IL-8 and IL-6 proteins. PI3K/Akt can regulate the expression of IL-8 and IL-6 simultaneously. It showed that IL-8 and IL-6 genes may share transcriptional regulation mechanisms. However, this process also requires the participation of multiple signalling pathways to jointly regulate the expression of IL-8 and IL-6. This type of regulation may be a relatively sophisticated network in which there may be cross-effects. This may play an important role in the pathogenesis of leukaemia. To clarify this detailed relationship and upstream and downstream adjustments, further research must be undertaken and the possible mechanism underlying this phenomenon be analysed. It is believed that this result has important basic clinical significance for the future discovery of new therapeutic targets. In this experiment, we selected IL-8 and IL-6 as the study cytokines. Unexpectedly, we found that both of them were regulated by the CXCL12/CXCR4 biological axis and PI3K/Akt signalling pathway at the same time, indicating that this regulatory network is very complex and related to each other. There may be many unknown signalling pathways and cytokines involved, which need to be further studied in the future.

CXCL12, chemokine 12

CR4, CXC chemokine receptor 4

ALL, acute lymphocytic leukaemia

NF/κB ,nuclear factor-κB

BMSCs, bone marrow stromal cells

ECL, enhanced chemiluminescence

G-CSF, granulocyte-colony stimulating factor

MAPK, mitogen-activated protein kinase

MNCs, mononuclear cells

MRD, minimal residual disease

P13K, phosphatidylinositol 3-kinases

P13K/Akt, phosphatidylinositol 3-kinases /protein kinase B (PKB, also known as Akt)

JNK/AP-1, C-Jun N-terminal kinase/ activator protein-1

MAPK, mitogen-activated protein kinase.

P42/44, mitogen-activated protein kinase

PBS, phosphate-buffered saline

MNCs，Monocytes

PI, propidium iodide (PI)

IL-1β,interleukin-1(IL-1) β

SDF-1, stromal cell derived factor-1

bFGF, basic fibroblast growth factor

IL-8, interleukin-8

IL-6, interleukin-6

IL-10, interleukin-10

ELISA ,enzyme-linked immuno sorbent assay

RPMI ,Roswell Park Memorial Institute

Acknowledgements

Not applicable.

Funding

The current study was supported by grants from Supporting Fund for Teachers' research of Jining Medical University (grant nos. JYFC2018FKJ027), the Science and technology development plan of Zhangqiu District in 2019 (grant no. 12), the Science and technology development plan of Zhangqiu people's Hospital of Jinan in 2018 (grant no. 2018ZY16)

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

LZ and GQ contributed to the study design. LZ, HZ, and JZ performed the experiments. LZ prepared the manuscript. LZ, LZ GQ and XZ performed the statistical analysis and interpreted the data. DL contributed to the experimental design and paper writing. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The current study was approved by the Ethics Committee of Zhangqiu people's Hospital (Jinan, China). Written informed consent was obtained from all donors.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Study on the mechanism of CXCL12/CXCR4 axis mediated upregulation of IL-8 and IL-6 on the biological function of acute T lymphocyte leukaemia cells

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

BMSC culture

Culture Of Jurkat Cells

Establishment Of A Co-cultivation Model

Detection Of Cxcr4 Expression Using Flow Cytometry

Cell Cycle Detection

Cck-8 Detection Of Cell Viability

Cell Apoptosis Detection

First Concentration Analysis Of Il-8 And Il-6 Using Elisa

Second Concentration Analysis Of Il-8 And Il-6 Using Elisa

Western Blot Analysis: Detection Of The Expression Of Il-8 And Il-6 Protein

Statistical analysis

Results

Microscopy

Expression Of Cxcr4 On The Surface Of Jurkat Cells

Cell Cycle Analysis

Cck8 Detection Of The Cell Proliferation Rate

Apoptosis Of Jurkat Cells Detected Using Flow Cytometry

Initial Concentration Analysis Of Il-8 And Il-6 Using Elisa

Effects Of Four Signal Pathway Inhibitors On Il-8 And Il-6 Concentrations Were Determined Using Elisa

Effects Of Four Signal Pathway Inhibitors On The Protein Expression Of Il-8 And Il-6

Discussion

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

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