Chronic myeloid leukemia (CML) is a myeloproliferative hematological malignancy with a characteristic translocation of the Philadelphia chromosome.[1,2] Thanks to the discovery of the single oncogene BCR-ABL and the synthesis of the tyrosine protein kinase inhibitor, imatinib mesylate (IM), CML has become much easier to treat. Drug resistance and drug withdrawal are two emerging unsolved problems. Imatinib resistance can be divided into BCR-ABL-dependent and BCR-ABL-independent forms. The former is caused by the BCR-ABL mutation, and most patients choose next-generation targeted drugs for therapy. The latter is usually caused by abnormally activated minimal residual disease (MRD), which becomes difficult to treat.
The hematopoietic microenvironment (HM) appears to sustain and regulate the proliferation and differentiation of both hematopoietic stem/progenitor cells and leukemia cells. Bone marrow stromal cells (BMSCs) are the main functional components of the HM. Gap junction intercellular communication (GJIC) of BMSCs participates in the regulation and synchronization of hematopoietic cells. Previous studies on drug resistance and the generation of MRD mainly emphasized changes in the adhesion and secretion functions of BMSCs.[7,8] Currently, much evidence suggests that GJIC in HM is also an important factor that affects the proliferation, apoptosis, and drug sensitivity of leukemic cells.[9,10] Connexin 43 (Cx43), the most common and widespread connexin in humans, is a major structural protein expressed on the cell surface and in the nuclear membrane. It docks with similar molecules and forms gap junctions. Ions, small metabolites, second messengers, and micro ribonucleic acid (RNAs) can be directly delivered between closely adjacent cells. Our previous studies reported that Cx43-overexpressing human umbilical cord stem cells enhanced the effect of chemotherapeutics and eliminated minimal residual L615 cells in an animal model. However, the functions and detailed mechanisms of Cx43 in imatinib resistance in CML are poorly understood.
In this study, we investigated the effect of Cx43 in BMSCs on apoptosis, proliferation, and the cell cycle of K562 cells under IM treatment.
Isolation of BMSCs and treatment
Human BMSCs were isolated from bone marrow (BM) samples separated from healthy donors from the Medical Centre of Hematology, Xinqiao Hospital, as previously described. All tissue samples used in this study were collected after obtaining written informed consent from patients, and all procedures were conducted in accordance with the study protocol approved by Ethics Committee of Xinqiao Hospital (No. AMUWEC20211606). The study was performed in accordance with the Declaration of Helsinki and its later amendments. The cells were authenticated with a cytometry assay (FCASVerse, BD Biosciences, Franklin Lakes, NJ, USA). BMSCs were cultured in minimum essential medium alpha (MEM-α; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Waltham, Massachusetts, USA) and 1 ng/mL basic fibriblast growth factor (Sigma−Aldrich, St Louis, MO, USA) at 37°C in a humidified atmosphere with 5% CO2. BMSCs were treated with 100 μmol/L cobalt dichloride (CoCl2) (Sigma−Aldrich) for 72 h to prevent the degradation of hypoxia-inducible factor 1α (HIF-1α). The human leukemic cell line K562 (The Cell Bank of Chinese Academy of Science, Beijing, China) was cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640; HyClone) with 10% FBS.
Immunophenotypic identification and transfection of BMSCs
To identify BMSCs, fluorescently labeled anti-human and anti-mouse cluster of differentiation 73 (CD73), CD90, CD105, CD14, CD20, CD34, and CD45 monoclonal antibodies (BD Biosciences, Franklin Lakes, NJ, USA) were added to each tube, and the samples were mixed and incubated for 15 min in the dark. Next, 1 mL phosphate buffer saline (PBS) was added to the tubes, which were centrifuged at 1000 round per minute for 5 min (r=172 mm). Finally, the BMSCs were resuspended in PBS, detected by a BDVerse flow cytometer (BD Biosciences) and analyzed by FlowJo software (FlowJo, Ashland, OR, USA).
Adenovirus vectors containing different gene sequences were transfected into BMSCs as previously described. Adenovirus-short hairpin RNA of HIF-1α-green fluorescent protein (Ad-shHIF-1α-GFP), Ad-negative control-GFP (Ad-NC-GFP), Ad-Cx43 overexpression-GFP (Ad-overCx43-GFP), and Ad-shCx43-GFP were purchased from Heyuan Biotechnology (Shanghai, China). Expression of the target genes was measured 48 h after transfection by Western blotting.
Fluorescence recovery after photobleaching (FRAP) assay
Cx43-modified BMSCs were cultured on specialized glass and loaded with 1 μmol/L fluorescein diacetate (Sigma–Aldrich) for 5 to 10 min at 37°C. Laser confocal scanning microscopy (FluoView FV3000, Olympus, Tokyo, Japan) was used to photograph and bleach the target cells with 3−5 neighboring cells. The image was taken using a low laser intensity of acousto-optic tunable filter (AOTF; 0.1%). The laser intensity for photobleaching was increased 400 × (AOTF = 40%, 40.0 μs/pixel) for a total time of 1.126 s. Every image was scanned at intervals of 3.22 s for a total period of 1012 s.
Cell coculturing and transwell assay
BMSCs or Cx43-modified BMSCs were seeded at 1 × 105/well in a 6-well plate. Then, K562 cells were cocultured on top of the BMSC layer for 72 h with or without 1 μmol/L IM (Selleck, Houston, Texas, USA) in RPMI 1640 medium [Table 1].
Table 1 -
Five groups of cell co-culturing systems to investigate the effect of Cx43-modified BMSCs in K562 cells under IM treatment.
||Number of K562 cells
||7.5 × 105
||7.5 × 105
||7.5 × 105
||7.5 × 105
||7.5 × 105
BMSCs: Bone marrow stromal cells; Cx43: Connexin 43; IM: Imatinib mesylate; NC group: K562 cells cocultured with BMSCs-NC; overCx43 group: K562 cells cocultured with BMSCs-overCx43; shCx43 group: K562 cells cocultured with BMSCs-shCx43; NC: Negative control; overCx43: Cx43 overexpression; shCx43: Short hairpin RNA of Cx43; –: Not available.
To validate whether Cx43 mediates cell communication through cell–cell contact or other non-adjacent pathways, a transwell assay was performed to separate K562 cells and Cx43-modified BMSCs. K562 cells were placed in the lower chamber of the Transwell system (0.04 μm, pore filter, Corning, NY, USA), while BMSCs were placed in the upper chamber.
Cell proliferation, cell cycle, and apoptosis
Cell proliferation was assessed with a Cell Counting Kit-8 (CCK8; Dojindo Laboratories, Kumanoto, Japan) assay with at least three duplicate wells. K562 cells from each group were resuspended and incubated in 96-well plates at 37°C for 4 h before measuring the absorbance at 450 nm. For cell cycle analysis, K562 cells were fixed with 70% ethanol overnight at 4°C. The fixed cells were stained with propidium iodide (PI; Beyotime) at 37°C according to the manufacturer's protocol. For apoptosis analysis, K562 cells from each group were stained with annexin V and PI (BD Pharmingen) at room temperature for 15 min followed by flow cytometric testing.
Intracellular Ca2+, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) analysis
Flou-3-pentaacetoxymethyl (Fluo-3/AM; Beyotime) was used for intracellular Ca2+ testing. K562 cells from each group were collected and resuspended with 1 μmol/L Fluo-3/AM diluted in 1 mL D-Hank's balanced salt solution (Solarbio, Beijing, China) for 30 min. Then, the cells were incubated for another 30 min and analyzed by a C6 flow cytometer (BD Biosciences).
A 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) kit (Beyotime) was used for MMP measurement. K562 cells at 24 h after coculturing were stained with 0.5 mL JC-1 solution. All samples were incubated for 20 min and detected by a C6 flow cytometer.
2′,7′-Dichlorofluorescein diacetate (DCFH-DA, Beyotime) was used for ROS determination. Twenty-four hours after coculturing, K562 cells were stained with 10 μL of DCFH-DA for 20 min and resuspended in D-Hank's solution, and 2′,7′-Dichlorofluorescein (DCF) fluorescence was also detected by a C6 flow cytometer.
Luminescence assay for adenosine triphosphate (ATP) release
K562 cells (2 × 104) from each group were lysed with lysis buffer (Beyotime) on ice. Then, 100 μL ATP testing diluent (Beyotime) and 10 μL samples or reference were mixed and tested by a GloMax 20/20 Luminometer (Promega−GloMax, Madison, Wisconsin, USA).
The total cellular proteins of K562 cells and BMSCs were extracted by radio-immunoprecipitation assay. Forty micrograms of protein from each cell line was added to each lane, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in tris-buffer solution and tween (TBST) with 5% non-fat milk for 2 h at room temperature and washed for four times. Then, the membranes were incubated overnight at 4°C using the following primary antibodies: anti-Cx43 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HIF-1α (CST, Danvers, Massachusetts, USA), anti-Cytochrome C (CST), anti-B cell lymphoma 2 (anti-BCL-2; CST), anti-Cleaved Caspase-9 (CST), anti-Cleaved Caspase-3 (CST), anti-poly (ADP-ribose) polymerase-1 (anti-Cleaved PARP; CST), anti-Ca2+/calmodulin-dependent protein kenases II (anti-CaMKII; CST), anti-protein kinase B (anti-Akt; CST), anti-mammalian target of rapamycin (anti-mTOR; CST), anti-BCL2-associated X (anti-BAX; Boster, Wuhan, China), anti-BCR-ABL (Abcam, Cambridge, UK), anti-phosphorylated-CRK like protein (p-CrkL; CST), anti-p-CaMKII (CST), anti-p-Akt (CST), and anti-p-mTOR (Boster). The membranes were washed with TBST and incubated with anti-β-actin (Boster) for 1 h. Finally, Immobilon Western Chemiluminescent HRP Substrate (Millipore, Mass, Massachusetts, USA) was used to determine the target proteins.
Real-time quantitative polymerase chain reaction (RT-qPCR)
RNA was extracted from BMSCs and reversely transcribed into complementary DNA (cDNA) using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Quantification of Cx43 and HIF-1α relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed on the CFX ConnectTM Real Time PCR Detection System using Synergy Brands (SYBR) Green chemistry (Bio-Rad). The forward (F) and reversed (R) primer sequences were as follows:
In vivo experiments
Nude mice were purchased from the Laboratory Animal Center of Army Medical University (Chongqing, China) and fed in aseptic cages. All murine experiments were performed in accordance with protocols that were approved by the Animal Care and Use Committee of the Centre of Army Medical University (No. AMUWEC20211606). To establish the xenograft tumor model, nude mice aged 4−6 weeks were randomly divided into the K562 group, Cx43 group, and negative control (NC) group. The mice in the three groups received subcutaneous injections of K562 cells + Cx43-modified BMSCs or K562 cells only [Table 2]. Besides, they were intraperitoneally given with 200 μL PBS, 200 μL IM (80 mg/kg, diluted in PBS, Selleck), and 200 μL IM, respectively, every two days from the sixth day after bearing. The volume of the tumor was measured in two directions and calculated as follows: V = 1/2 × length × (width)2. All mice were sacrificed by cervical dislocation on the 24th day after cell injection.
Table 2 -
Three animal groups to in vivo
investigate the effect of Cx43-modified BMSCs in K562 cells under IM treatment.
||1 × 107
||8 × 106
||2 × 106 BMSCs-overCx43
||8 × 106
||2 × 106 BMSCs-NC
BMSCs: Bone marrow stromal cells; Cx43: Connexin 43; IM: Imatinib mesylate; NC: Negative control; overCx43: Cx43 overexpression; –: Not available.
Immunohistochemistry and immunofluorescence
Tissues were fixed in 10% neutral buffered formalin overnight at room temperature. The fixed tissues were embedded in paraffin and cut into 4 to 6 μm tissue sections. Then, the slides were stained with hematoxylin–eosin (HE), anti-HIF-1α (Abcam), anti-Cx43 (Santa Cruz), anti-BCR-ABL (Abcam), anti-BAX (Boster), and anti-Ki67 (Bioss, Beijing, China) and terminal dexoynucleotidyl transferase-mediated deoxyurinetriphate (dUTP)-digoxigenin nick end labeling (TUNEL; Beyotime). The slides were imaged under a light microscope, and Cx43 levels in human biopsy specimens were analyzed using ImageProPlus software (Media Cybernetics, Silver Springs, MD, USA).
Cx43-modified BMSCs were cultured in a 6-well plate at 1 × 105/well and fixed with 40 mg/L paraformaldehyde (Sigma) after cell adherence. Then, the cells were stained with anti-Cx43 (Santa Cruz) overnight at 4°C. After washing for three times, the cells were incubated with Anti-mouse Immunoglobin G (IgG; CST) for 30 min. Finally, the cells were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Boster) for 15 min, and the slides were sealed using 60% buffered glycerol (Sigma−Aldrich) and imaged under an immunofluorescence microscope (Olympus).
Statistical analyses were performed using SPSS software version 26.0 (IBM, Armonk, NY, USA). Statistical figures were drawn using GraphPad Prim 5.0 (GraphPad Software Inc., San Diego, CA, USA). Quantitative variables are expressed as the mean ± standard deviation. The unpaired two-tailed Student's t test was applied for comparing any two groups, and the one-way analysis of variance (ANOVA) test, and multiple comparisons test were used for comparison among multiple groups. P < 0.05 was considered statistically significant.
BM of CML patients shows relatively low levels of Cx43 and high levels of HIF-1α
As Cx43 is important for GJIC, we measured Cx43 expression in BM biopsy specimens of ten CML patients at first diagnosis and ten healthy donors by immunohistochemical assay. Patient information is listed in Table 3. The results showed that the mean integral optical intensity (IOD) of Cx43 in CML patients was statistically significantly higher than that in healthy people [Figure 1A]. Among the ten CML patients, four showed negative expression (IOD < 2000 was considered negative), while all BM specimens from healthy donors were positive [Figure 1B]. Cx43 expression was heterogeneous in BM specimens. However, it was decreased in patients who were diagnosed with CML.
Table 3 -
IOD (mean) of Cx43 in the biopsy of CML patients and healthy donors.
CML: Chronic myeloid leukemia; Cx43: Connexin 43; F: Female; IOD: Integral optical intensity; M: Male.
Hypoxia is common in many chronic tumor diseases. Recent studies revealed that physiological hypoxia in BM promotes communication deficiency between hematopoietic stem cells (HSCs) and BMSCs in some hematological malignancies. HIF-1α is a key transcription factor associated with hypoxia. HIF-1α and Cx43 were measured from biopsy specimens of healthy donors and CML patients [Figure 1C], and we found a negative correlation between HIF-1α level and Cx43 levels in both healthy donors and CML patients.
Hypoxia is negatively correlated with Cx43 expression in BMSCs
As BMSCs are the major component of BM, the physiological hypoxic state of BMSCs was induced with the hypoxia-inducing reagent CoCl2 (100 μmol/L) to prevent the degradation of HIF-1α. BMSCs were isolated from healthy donors and authenticated by flow cytometry [Figure 1D]. We found that HIF-1α expression was increased in CoCl2-treated BMSCs (BMSCs-CoCl2) and that Cx43 expression was decreased at the transcriptional and translational levels, whereas the results in untreated BMSCs were reversed [Figures 1E–1G].
To further validate that HIF-1α is a regulator of Cx43, HIF-1α was knocked down with Ad-shHIF-1α-GFP in BMSCs. As shown in Figure 1H, we found that CoCl2 treatment resulted in increased HIF-1α expression and decreased Cx43 expression in BMSCs transfected with Ad-NC-GFP (BMSCs-NC), but when HIF-1α was knocked down, the expression of Cx43 was recovered [Figure 1H]. This result indicated that HIF-1α is upstream of Cx43, and reversion of HIF-1α expression and hypoxia led to the recovery of Cx43 expression levels in BMSCs.
BMSCs-overCx43 had the strongest GJIC function
To observe the role of Cx43 in HM in vitro, P3–P5 BMSCs were transduced with Ad-overCx43-GFP, Ad-NC-GFP, and Ad-shCx43-GFP, which upregulate, do not change, and downregulate Cx43 expression in BMSCs, respectively. The three BMSCs were defined as BMSCs-overCx43, BMSCs-NC, and BMSCs-shCx43. The expression of Cx43 in the three BMSCs was measured by Western blotting and RT-qPCR [Figures 2A–2C].
GJIC function of the three Cx43-modified BMSCs was tested with the gap-FRAP assay [Figure 2D]. From the data of Figures 2E and 2F, the fluorescence intensity of normal BMSCs recovered to 22% in 300 s and 30% within 500 s, whereas that of BMSCs-over Cx43 recovered to 37% and 48%, and that of BMSCs-shCx43 recovered to 13% and 17% at 300 s and 500 s, respectively. After observation for 1000 s, BMSCs-overCx43 showed the highest recovered rate of fluorescence, with an average recovery percentage of 59%, compared to BMSCs-NC (43%), and BMSC-shCx43 (23%). Moreover, BMSCs-overCx43, BMSCs-NC, and BMSC-shCx43 took approximately 105 s, 140 s, and 759 s to reach 20% recovery, respectively. These results implied that BMSCs overexpressing Cx43 have the strongest GJIC function.
Cx43-modified BMSCs affect K562 apoptosis and the cell cycle but not proliferation under IM treatment
K562 is a hematopoietic progenitor cell line established from a human CML patient in the blast phase, which is classical and is recognized as representative when studying the disease. K562 cells can be killed by IM in a time- and concentration-dependent manner. Direct contact coculturing systems of Cx43-modified BMSCs and K562 cells were established to explore the effect of Cx43 on the survival state of K562 cells. Different treatments were administered to the five groups: the K562 group (only K562 cells), IM group (K562 cells treated with IM), overCx43 group (k562 cells cocultured with BMSCs-overCx43 and treated with IM), NC group (k562 cells cocultured with BMSCs-NC and treated with IM), and shCx43 group (k562 cells cocultured with BMSCs-shCx43 and treated with IM) [Table 1].
A CCK8 assay was used to detect K562 cell proliferation [Figure 3A]. We found no statistically significant difference in cell proliferation in any of the IM-treated groups at 72 h of coculturing. The cell cycle of K562 cells was detected [Figures 3B–3E], and results showed that the proportion in the G0/G1 phase was statistically significantly increased in the shCx43 group compared with the IM group [Figure 3B], while the opposite result was observed in the overCx43 group [Figures 3C]. Regarding apoptosis [Figures 3F–3I], we found that compared with the IM group, the NC group had a lower apoptosis rate, which confirmed the protective role of BM in leukemic cell viability [Figure 3F]. The apoptosis rate of K562 cells in the shCx43 group was significantly decreased compared with that in the IM group and even the NC group [Figures 3F and 3H]. The overCx43 group showed an apoptosis rate relatively equal to that of the IM group [Figures 3G and 3I]. Taken together, the results indicated that overexpression of Cx43 in BM plays an important role in promoting killing efficacy of IM.
Cx43 influences the efficacy of IM through cell–cell contact
Cx43 not only participates in GJIC, but also mediates communication between nonadjacent cells through tunneling nanotubes, extracellular vesicles, and even external secretion. To explore whether Cx43 plays its role through GJIC or other non-contact mechanisms, we employed a transwell plate (0.4 μm pores, 6-well) to separate BMSCs and K562 cells. The results showed no statistically significant difference in the apoptosis rate among the overCx43 group, shCx43 group, NC group, and even the IM group [Figure 4A], which suggested that cell–cell contact plays a dominant role in influencing the killing efficacy of IM.
After coculturing, Cx43 levels in K562 cells and BMSCs were measured. We found that K562 cells cocultured with BMSCs-overCx43 had enhanced Cx43 expression at both the transcriptional and translational levels [Figures 4B and 4C], while Cx43 was hardly expressed on the surface of untreated K562 cells. Interestingly, we observed that after coculturing, Cx43 expression in BMSCs-overCx43 was higher than that before coculturing in both the PCR and Western blotting experiments [Figures 4D and 4E]. Immunofluorescence assays of BMSCs before and after coculturing were performed and we found the stronger signal in BMSCs after coculturing [Figure 4F]. Moreover, we observed that the increased Cx43 was mainly expressed in BMSC cell and nuclear membranes. The results demonstrated that while HM affected the survival state of leukemic cells, the latter also remodeled the HM at the same time.
Ca2+ transfer via the Cx43 channel mediates the synergistic efficacy of IM in the Cx43-overexpressing HM
GJIC permits the rapid exchange of small molecules, including Ca2+ and ATP, into the cytoplasm. To explore whether ATP or Ca2+ plays a causal role in the drug resistance of CML cells, we detected the concentrations of intracellular ATP and Ca2+. No significant difference in ATP concentration was found among any IM-treated groups [Figure 5A], whereas the calcium fluorescence intensity in the overCx43 group was much higher than that in any IM-treated groups [Figure 5B].
To validate the hypothesis that Ca2+ transfer from BMSCs to K562 cells augments the pro-apoptotic function in the Cx43-overexpressing HM, Ca2+-related apoptotic indicators were tested. Ca2+ is tightly linked with mitochondrial signaling, and mitochondrion-mediated apoptosis is initiated by the loss of MMP. As MMP depolarization was considered a Ca2+-triggered early event of apoptosis, we also compared the MMP depolarization of K562 cells among every group. Consistent with our hypothesis, the MMP of the overCx43 group was significantly increased, while no difference was found between the overCx43 group and the IM group [Figures 5C and 5D]. Mitochondria are a major target of ROS, and accumulated ROS can lead to damage to the mitochondrial membrane and even cell death. The intracellular ROS level of K562 cells was tested by flow cytometry, and we found a statistically significantly increased ROS level in the overCx43 group [Figure 5E].
Ca2+ augments the killing effect through both the caspase and CaMKII–Akt–mTOR signaling pathways
Although the observation of Cx43 and Ca2+ transfer inducing apoptosis in K562 cells has been validated, the molecular mechanism remains elusive. Therefore, we investigated the classical and novel apoptotic pathways related to Ca2+ by Western blotting. The mitochondrial apoptotic pathway and caspase pathway are classical and most common when referring to apoptosis. As shown in Figure 5F, we observed decreased BCL-2, increased BAX, and increased cytochrome C (CytoC) expression in K562 cells cocultured with BMSCs-overCx43 compared with those cocultured with BMSCs-NC. Moreover, the expression levels of cleaved caspase-9, cleaved caspase-3, and cleaved PARP were obviously higher than those in NC group and K562 group.
CaMKII is a general integrator of Ca2+ signaling that regulates the development and activity of many cell types. CaMKII is tightly linked with cell growth-related pathways.[17,18] The CaMKII–Akt–mTOR pathway in K562 cells separated from the four groups (IM group, NC group, overCx43 group, and K562 group) was assessed by Western blotting, and the results showed increased p-CaMKII, decreased p-Akt, and decreased p-mTOR expression in the overCx43 group compared with the NC group [Figure 5G]. These bands indicated that the CaMKII–Akt–mTOR pathway was also involved in the Ca2+-mediated GJIC pathway.
We also measured the expression of BCR-ABL and its downstream molecule p-CrkL in K562 cells to test whether Cx43 could directly regulate the BCR–ABL pathway [Figure 5H]. However, no obvious difference was found between the Cx43 group and the NC group.
Cx43-modified BMSCs inhibit the tumorigenicity of K562 cells in vivo
A tumor-bearing model of nude mice was established to evaluate the synergistic effect of Cx43 on IM efficacy in K562 cells in vivo. Mice were randomly divided into three groups (n = 4). A total of 8 × 106 K562 cells plus 2 × 106 BMSCs-overCx43 (Cx43 group), 8 × 106 K562 cells plus BMSCs-NC (control group), and 1 × 107 K562 cells (K562 group) were subcutaneously injected into the right flank of mice in each group. From the sixth day, 200 μL diluent IM was intragastrically injected into the Cx43 group and control group every two days, while 200 μL water was given to the K562 group. The tumor volume was calculated every three days until the 24th day after inoculation. We observed that the tumor volumes of mice in the Cx43 group were consistently smaller with a slower growth rate compared with the control group [Figure 6A]. In addition, the latter showed relatively larger spleens than the former. Mice in the K562 group had the largest tumors and spleens [Figure 6B]. No significant difference was found in the average body weight of the three groups [Figure 6C].
Immunohistochemical staining was also performed. From HE staining results, we found a large number of tumor cells in Cx43 group under the microscope, which was much greater than that in the other two groups. In addition, inflammatory infiltration was observed in the Cx43 group [Figure 6D]. Cx43 was strongly positive in tumor tissues of the Cx43 group, while it was hardly stained in the K562 group. Other proliferative (Ki67, BCL-2, and BCR-ABL) and apoptotic (BAX and TUNEL) indicators were also stained, and the results displayed decreased levels of Ki67 and BCL-2 and increased levels of BAX and TUNEL in the Cx43 group compared with the control group, which suggested that the Cx43-overexpressing HM could inhibit tumorigenicity and synergize with the killing effect of IM in vivo. We found that Cx43 influences the killing effect of IM on K562 cells through a Ca2+-mediated pathway and further explored the possible mechanism. Our results might provide new ideas for reversing IM resistance.
Imatinib resistance is the major obstacle in the treatment of CML. The protective effect of BM on leukemia cells is a vital factor for the occurrence and relapse of leukemia and promotes the development of MRD and drug resistance. BM is a natural shelter in which leukemic cells can avoid chemotherapy. It has been reported that BMSCs protect CML cells from imatinib-induced apoptosis via the chemokine (C-X-C motif) receptor 4/C-X-C motif ligand 12 (CXCL12) axis. In this study, we observed that Cx43 expression in the CML microenvironment was decreased, and enhancing the level of Cx43 might be a novel strategy to reverse drug resistance and promote with the effect of IM.
A hypoxic state is common in many chronic tumors. It was reported that hypoxia can induce Cx43 dysregulation in H9c2 cardiomyocytes. In a rat model, hypoxia was shown to lead to cell apoptosis and decreased Cx43 expression in BMSCs. As HM serves as the sustenance for hematopoietic cells and leukemia cells, we detected the expression of Cx43 and HIF-1α in normal BMSCs and found a negative correlation between Cx43 and HIF-1α. Reversing the hypoxic state may be a good way to increase Cx43 expression in HM of patients and further improve treatment efficacy.
Cx43 is regarded as a tumor suppressor in many kinds of tumors due to its antitumor function. Previous studies on the formation of MRD and drug resistance in leukemia have mainly focused on the adjacent cells and secretory cytokines around leukemic cells.[7,8] In addition, some studies reported that exosomes secreted from mesenchymal stromal cells could also mediate the antileukemia process.[22,23] In this study, K562 cells were studied, as they are a classical and representative cell line established from human CML patients. We proved that Cx43-mediated apoptosis was gap junction dependent via a transwell assay and found that the lack of Cx43 can also arrest the cell cycle of K562 cells in G0/G1 phase, which is beneficial for escaping chemotherapy. Animal experiments showed that nude mice bearing K562 cells plus BMSCs-overCx43 manifested the smallest tumor volume under IM treatment, which indicated that increased Cx43 in HM can help enhance the killing effect of IM and delay the progression of diseases in vivo. Interestingly, we observed that the expression of Cx43 in K562 cells and BMSCs is reciprocal: Cx43 overexpression in BMSCs can improve the expression of Cx43 in K562 cells, and vice versa. This observation is suggestive and worth further exploration.
It has been reported that gap junctions can facilitate the direct cytoplasmic delivery of small molecules or metabolites and that Cx43 regulates Ca2+ entry and ATP release. ATP and Ca2+ are two major components that can be released from Cx43 channels and affect cellular function.[25,26] Ca2+ is known as a proapoptotic signaling molecule. In the present study, we found increased Ca2+ fluorescence intensity in K562 cells cocultured with BMSCs-overCx43, whereas no difference in ATP concentration was detected among the three IM-treated groups, which implies that Ca2+, but not ATP, plays a vital role in the regulation of K562 cells through GJIC.
CaMKII is a general integrator of Ca2+ signals, which are ubiquitously expressed in multiple systems and cell types. It has been reported that overexpression of CaMKII leads to disturbance of Ca2+ levels and promotes apoptosis of myocardial cells. Liu et al demonstrated that Ca2+–CaMKII signaling as well as ROS could mediate the inhibition of the Akt/mTOR pathways and finally lead to neural cell apoptosis. The downstream mTOR signal is involved in many metabolic processes of tumor cells, and its activated formation is recognized as a signal of cell proliferation. In this study, in addition to the classical mitochondrial-induced apoptotic pathway, the novel mitochondrial-independent CaMKII–Akt–mTOR pathway was assessed and established [Figure 7].
There are still some limitations in this study. We did not block the core molecule Ca2+ in K562 cells because Ca2+ is very important to cell survival, and chelating Ca2+ with the chelating agent ethylenebis (oxyethylenenitrilo) tetraacetic acid (EDTA) would lead to the death of both K562 cells and BMSCs. This study also gives us some new ideas. As Cx43-overexpressing BMSCs indeed improve the killing effect of IM and inhibit drug resistance to some degree, we can focus on repairing GJIC function in the HM for leukemia patients before chemotherapeutic resistance develops and even before transplantation. There are two ways to repair GJIC function in BM. All-trans retinoic acid (ATRA) has been used in the treatment of acute promyelocytic leukemia (APL) and other hematologic diseases based on the induction of differentiation and inhibition of growth. It has also been reported that ATRA treatment can increase Cx43 expression in leukemic BMSCs at both the mRNA and protein levels and result in intercellular communication. In addition, coinfusion of human umbilical mesenchymal stem cells after HSCT is another way to repair GJIC function, eliminate MRD, and inhibit relapse and drug resistance, as umbilical mesenchymal cell infusion has been widely used in clinical experiments with a variety of advantages.
In conclusion, this study demonstrated that hypoxia-induced downregulation of Cx43 is common in CML HM and benefits drug resistance. Cx43 overexpression in BMSCs weakened the protective effect of BMSCs and enhanced IM-induced apoptosis via Ca2+-dependent GJIC.
This work was supported by the National Key R&D Program of China (2022YFA1103300), the National Natural Science Foundation of China (81873424, 81570097), the Natural Science Foundation of Chongqing Innovation Group Science Program (cstc2021jcyj-cxttX0001), Clinical Medical Research Project of Army Medical University (2018XLC1006), and Translational Research Grant of NCRCH (2020ZKZC02).
Conflicts of interest
1. Mughal T, Corter J, Cross NC, Donato N, Hantschel O, Jabbour E, et al. Chronic myeloid leukemia— Some topical issues. Leukemia
2007; 21:1347–1352. doi: 10.1038/sj.leu.2404733.
2. Zheng YZ, Li J, Chen C, Zheng H, Fu DH, Hu JD. Long-term outcome of tyrosine kinase inhibitor treatment in children and adolescent with newly diagnosed chronic myeloid leukemia in chronic phase. Chin Med J
2021; 134:3009–3011. doi: 10.1097/CM9.0000000000001656.
3. Soverini S, Hochhaus A, Nicolini FE, Gruber F, Lange T, Saglio G, et al. BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet. Blood
2011; 118:1208–1215. doi: 10.1182/blood-2010-12-326405.
4. Cortes J, Lang F. Third-line therapy for chronic myeloid leukemia: current status and future directions. J Hematol Oncol
2021; 12:44doi: 10.1186/s13045-021-01055-9.
5. Saxena K, Jabbour E, Issa G, Sasaki K, Ravandi F, Maiti A, et al. Impact of frontline treatment approach on outcomes of myeloid blast phase CML. J Hematol Oncol
2021; 14:94doi: 10.1186/s13045-021-01106-1.
6. Vanegas NP, Vernot J. Loss of quiescence and self-renewal capacity of hematopoietic stem cell in an in vitro leukemic niche. Exp Hematol Oncol
2017; 6:2doi: 10.1186/s40164-016-0062-1.
7. Zhang B, Li M, McDonald T, Holyoake TL, Moon RT, Campana D, et al. Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signalling. Blood
2013; 121:1824–1838. doi: 10.1182/blood-2012-02-412890.
8. Jacamo R, Chen Y, Wang Z, Ma W, Zhang M, Spaeth EL, et al. Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation of NF-kappaB mediates chemoresistance. Blood
2013; 123:2691–2702. doi: 10.1182/blood-2013-06-511527.
9. Liu Y, Zhang X, Li ZJ, Chen XH. Up-regulation of Cx43 expression and GJIC function in acute leukemia bone marrow stromal cells post-chemotherapy. Leuk Res
2010; 34:631–640. doi: 10.1016/j.leukres.2009.10.013.
10. Liu Y, Wen Q, Chen XL, Yang SJ, Gao L, Zhang C, et al. All-trans retinoic acid arrests cell cycle in leukemic bone marrow stromal cells by increasing intercellular communication through connexin 43-mediated gap junction. J Hematol Oncol
2015; 8:110doi: 10.1186/s13045-015-0212-7.
11. Ribeiro-Rodrigues TM, Martins-Marques T, Morel S, Kwak B, Girão H. Role of connexin 43 in different forms of intercellular communication— gap junctions, extracellular vesicles and tunnelling nanotubes. J Cell Sci
2017; 130:619–630. doi: 10.1242/jcs.200667.
12. Yang S, Wen Q, Liu Y, Zhang C, Wang M, Chen G, et al. Increased expression of CX43 on stromal cells promotes leukemia apoptosis. Oncotarget
2015; 6:44323–44331. doi: 10.18632/oncotarget.6249.
13. Dong C, Zhang NJ, Zhang LJ. Oxidative stress in leukemia and antioxidant treatment. Chin Med J
2021; 134:1897–1907. doi: 10.1097/CM9.0000000000001628.
14. Wu X, Huang W, Luo G, Alain LA. Hypoxia induces connexin 43 dysregulation by modulating matrix metalloproteinases via MAPK signaling. Mol Cellular Biochem
2013; 384:155–162. doi: 10.1007/s11010-013-1793-5.
15. Zhang X, Tu H, Yang Y, Jiang X, Hu X, Luo Q, et al. Bone marrow-derived mesenchymal stromal cells promote resistance to tyrosine kinase inhibitors in chronic myeloid leukemia via the IL-7/JAK1/STAT5 pathway. J Biol Chem
2019; 294:12167–12179. doi: 10.1074/jbc.RA119.008037.
16. Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer
2002; 2:277–288. doi: 10.1038/nrc776.
17. Dong X, Qin J, Ma J, Zeng Q, Zhang Q, Zhang H, et al. BAFF inhibits autophagy promoting cell proliferation and survival by activating Ca2+-CaMKII-dependent Akt/mTOR signaling pathway in normal and neoplastic B-lymphoid cells. Cell Signal
2019; 53:68–79. doi: 10.1016/j.cellsig.2018.09.012.
18. Liu C, Ye Y, Zhou Q, Zhang R, Zhang H, Liu W, et al. Crosstalk between Ca2+ signaling and mitochondria H2O2 is required for rotenone inhibition of mTOR signaling leading to neuronal apoptosis. Oncotarget
2016; 7:7534–7549. doi: 10.18632/oncotarget.7183.
19. Vianello F, Villanova F, Tisato V, Lymperi S, Ho KK, Gomes AR, et al. Bone marrow mesenchymal stromal cells non-selectively protect chronic myeloid leukemia cells from imatinib-induced apoptosis via the CXCR4/CXCL12 axis. Haematologica
2010; 95:1081–1089. doi: 10.3324/haematol.2009.017178.
20. Ding XC, Wang LL, Zhang XD, Xu JL, Li PF, Liang H, et al. The relationship between expression of PD-L1 and HIF-1α in glioma cells under hypoxia. J Hematol Oncol
2021; 14:92doi: 10.1186/s13045-021-01102-5.
21. Jiang G, Dong S, Yu M, Han X, Zhang C, Zhu X. Influence of gap junction intercellular communication composed of connexin 43 on the antineoplastic effect of adriamycin in breast cancer cells. Oncol Lett
2017; 13:857–866. doi: 10.3892/ol.2016.5471.
22. Liu Y, Song B, Wei Y, Chen F, Chi Y, Fan H, et al. Exosomes from mesenchymal stromal cells enhance imatinib-induced apoptosis in human leukemia cells via activation of caspase signaling pathway. Cytotherapy
2018; 20:181–188. doi: 10.1016/j.jcyt.2017.11.006.
23. Yang C, Yang H, Liu J, Zhu L, Yu S, Zhang X, et al. Focus on exosomes: novel pathogenic components of leukemia. Am J Cancer Res
24. Bonacquisti EE, Nguyen J. Connexin 43 (Cx43) in cancer: implications for therapeutic approaches via gap junctions. Cancer Lett
2019; 442:439–444. doi: 10.1016/j.canlet.2018.10.043.
25. Kondratskyi A, Kondratska K, Skryma R, Prevarskaya N. Ion channels in the regulation of apoptosis. Biochim Biophys Acta
2015; 1848:2532–2546. doi: 10.1016/j.bbamem.2014.10.030.
26. Anguita E, Villalobo A. Ca2+ signaling and Src-kinases-controlled cellular functions. Arch Biochem Biophys
2018; 650:59–74. doi: 10.1016/j.abb.2018.05.005.
27. Chen X, Zhang X, Kubo H, Harris DM, Mills GD, Moyer J, et al. Ca2+ influx-induced sarcoplasmic reticulum Ca2+ overload causes mitochondrial-dependent apoptosis in ventricular myocytes. Circ Res
2005; 97:1009–1017. doi: 10.1161/01.RES.0000189270.72915.D1.
28. Gu R, Yang X, Wei H. Molecular landscape and targeted therapy of acute myeloid leukemia. Biomark Res
2018; 6:32doi: 10.1186/s40364-018-0146-7.
29. Gao L, Zhang Y, Hu B, Liu J, Kong P, Lou S, et al. Phase II multicenter, randomized, double-blind controlled study of efficacy and safety of umbilical cord-derived mesenchymal stromal cells in the prophylaxis of chronic graft-versus-host disease after HLA-haploidentical stem-cell transplantation. J Clin Oncol
2016; 34:2843–2850. doi: 10.1200/JCO.2015.65.3642.