Chemotherapy is a treatment that uses drugs to kill tumor cells, inhibit tumor cell growth and reproduction, and promote the differentiation of tumor cells. Longtime chemotherapy causes immunodeficiency and bone marrow suppression. It can decline the total number of leukocytes in peripheral blood, destroy the immune system function induced by a variety of infections, and affect the quality of life, treatment, and shortened survival. Therefore, this issue may have important implications in order to design therapeutic approaches to improve the anti-infectious or antitumoral resistance of patients undergoing bone marrow transplantation and/or anticancer treatment. Cyclophosphamide (CTX) is a widely used nitrogen mustard anticancer drug. Used for chemotherapy, it severely injures hematopoietic and lymphoid tissues which causes neutropenia and leads to infection and other serious consequences. Therefore, we used CTX to make a chemotherapy-damaged mice model for the research of recuperation of bone marrow hematopoietic function in our study.1–3
Heme oxygenase-1 (HO-1) is one of the most widespread cytoprotective enzymes. It is mostly expressed in a low level in the organizations, but there can be a variety of factors that can induce the expression of high level, including hemoglobin, thermal shock, ultraviolet irradiation, peroxide, heavy metal, low oxygen, and cell factor.4 It has been confirmed that under the condition of stress HO-1 high expression can relieve cell damage, reduce lipid peroxidation, and participate in the function of immune regulation.5–9 On the other hand, the cytoprotective effect of HO-1 might modify the endogenous balance between apoptosis and proliferation toward an antiapoptotic and proproliferative status, which is relevant to oncogenesis, maintenance, and resistance to chemotherapy.10 We tried to import HO-1 into a chemotherapy-damaged bone marrow via mouse mesenchymal stem cells (mMSCs) which can home to bone marrow to resist the damage and repair the hematopoietic function.11
Source of animals
Six to eight weeks old Balb/c mice were purchased from Guiyang Medical College (Guiyang, China). The mice were housed under specific pathogen-free conditions and allowed to acclimate for 2 weeks before the experiments.
Cell separation and culture
The normal mice were killed by cervical dislocation, and the tibias and femurs were isolated. The whole bone marrow was flushed from the femurs of Balb/c mice. The bone marrow cells (BMCs) were cultured in L-DMEM/F-12 (GIBCO, USA) with 20% fetal bovine serum (GIBCO) under the condition of 37°C with 5% CO2. Nonadherent cells were removed carefully after 3 hours and fresh medium was replaced. When primary cultures become almost confluent, the culture was treated with 0.5 ml of 0.25% trypsin containing 0.02% mmol/L ethylenediaminetetraacetic acid (GIBCO) for 2 minutes at room temperature (25°C). A purified population of mMSCs could be obtained 3 weeks after the initiation of culture.
Inducement of HO-1 expression in mMSCs
The normal mMSCs (2×106/cm2) were treated with a concentration of hemin (0.45 μg/L; Sigma, USA) for 3 days. Cell treatment was done in duplicate and the effects of these treatments on cell number, viability, proliferation, and apoptosis were assayed at the indicated time points.
Establishment of animal model
The mice in the control group were treated with an equal amount of normal saline administered intraperitoneally on the first 5 days and via a tail intravenous injection on the 6th day. The rest of 120 mice were bone marrow damaged by intraperitoneal injection of CTX (45 mg/kg; Jiangsu Hengrui, China) for 5 days. These mice were randomly divided into three groups equally. Forty of them were classified as the chemotherapy group, which were tail intravenously injected with an equal amount of normal saline after 5 days of chemotherapy. After the chemotherapy injury was performed, the mMSCs (2×106) were tail intravenously injected on the 6th day, which served as the mMSCs group. The HO-1 group mice consisting of 40 mice received tail intravenous injections of mMSCs (2×106) which expressed high HO-1 after chemotherapy.
RT-PCR analysis of the expression of HO-1 mRNA
BMCs were collected from the four groups. mRNA was abstracted using TRIzol reagent (Invitrogen, USA). For semiquantitative analysis, 2000 ng of RNA was reverse transcribed into cDNA with random hexamers and SuperScript III reverse transcriptase (Invitrogen) in 20 μl of reaction volume. An equal amount of mRNA was loaded and run in 5% SDS-PAGE gel. The level of gene expression was analyzed by amplifying 1 μl of reversetranscribed cDNA product with primers specific for HO-1 (sense 5′-CAGGCAGAGAATGCTGAGTTC-3′; antisense 5′-GATGTTGAGCAGGAACGCAGT-3′, 887 bp) and β-actin (sense 5′- GAGACCTTCAACACCCCAGC-3′; antisense 5′-ATGTCACGCACGATTTCCC-3′, 263 bp). Conditions for cDNA PCR reaction 40 seconds at 53°C and 58°C, 6 minutes at 94°C, then 30 cycles, each consisting of 40 seconds at 94°C and 50 seconds at 72°C. The relative expression of HO-1 mRNA was demonstrated by the ratio of gray scale between HO-1 and β-actin.
Western blotting analysis of the expression of HO-1 protein
BMCs in the four groups were harvested. After washing twice in phosphate-buffered saline, the cells were lyzed in lysis buffer and the lysate was transferred into EP tubes followed by centrifugation at 12 000 ×g for 10 minutes at 4°C. The supernatant was collected and mixed with loading buffer. The final solution was boiled for 10 minutes and aliquots were stored at -80°C for use. An equal amount of protein was loaded and run in 10% SDS-PAGE gel and transferred onto a nitrocellulose transfer membrane which was then blocked in 5% nonfat milk in Tris buffer at 4°C overnight. The membranes were treated with HO-1 mouse antibody (1:1 000) or β-actin mouse antibody (1:1 000) for 2 hours while shaking at room temperature. Subsequently, the membranes were washed in TBST 5 times (10 minutes each) and incubated with horseradish peroxidase-conjugated goat anti-mouse antibody for 2 hours at room temperature. After washing in TBST 5 times (10 minutes each), visualization was done. Protein bands were visualized on film by enhanced chemiluminescence (Applygen Technologies Inc., China) following the protocol of the manufacturer. Their optical density was analyzed with Quantity One software. The expression of HO-1 was normalized to that of β-actin. All data were obtained from independent experiments in triplicate. The mean value is presented as the experimental result.
MTT assay detects the cell proliferation of different groups
Cells in different groups were cultured separately in 96-well plates in five replicates. 5 000 cells/well were plated and half of the medium was changed twice a week. After cell attachment, cell proliferation was measured by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay in 0, 24, 48, and 72-hour culture. The medium was removed and MTT reagent (Sigma-Aldrich, St. Louis, MO, USA; 0.5 mg/ml in medium) was added to the cells and incubated for 2 hours at 37°C in 5% CO2 and 95% air. After incubation, the MTT solution was removed and 100 μl/well of dimethyl sulfoxide (Sigma-Aldrich) was added. The absorbance of the reduced form of MTT was measured at 550 nm and 650 nm (background) in a plate reader (Victor 2, Wallac Oy, Turku, Finland).
Observation of HO-1 on the recovery of the chemotherapy-induced hematopoietic damage
Changes in the hemogram in mice
Venous blood samples (500 μl) from the control group and the chemotherapy group were collected on days 0, +1, +3, +5, +7, +14, and +21 after intraperitoneal injection. Venous blood samples (500 μl) from the mMSCs group and the HO-1 group were collected on days 0, +1, +3, +5, +7, +14, and +21 after tail intravenous injection. Each blood sample was measured using a blood counter.
Observation of bone marrow pathological slices
In the chemotherapy group, the mMSCs group, and the HO-1 group, which had 1 week after injection, the femurs of mice were separated and soaked in the decalcified liquid (1 000 ml of 30% hydrochloric acid solution, 7 g of aluminum chloride, 815 ml of hydrochloric acid, 5 ml of formic acid, 15 ml of formaldehyde, 0.5 ml of TritonX-100, and 100 ml of distilled water) for a long time until the bones became soft. Then, the bones were soaked in 10% formalin for a week. A segment of the femurs centered on the injury site was immediately removed and preserved in 20% sucrose overnight at 4°C. The femurs were embedded in paraffin, serially sectioned on the horizontal plane, and stained with hematoxylin and eosin (HE).
Data were statistically analyzed using SPSS 11.5 software (SPSS Inc., USA). All data are presented as mean ± sandard deviation (SD). The statistical analyses were performed using analysis of variance and q-text. Optical density from Western blotting assay was quantified using Quantity One software (Bio-Rad, USA). P values of less than 0.05 were considered statistically significant.
Normal mMSCs culture
The mMSCs were collected from mice bones in this study. The number of nucleated cells was (1.23±0.58)×106. Nonadherent cells are removed carefully after 3 hours and fresh medium is replaced. When primary cultures become almost confluent, the culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid for 2 minutes at room temperature. A purified population of mMSCs can be obtained 3 weeks after the initiation of culture. The number of mMSCs purified was (1.95±0.65)×105. The hematopoietic stem cells were attached after 15 days (Figure 1).
Expression of HO-1 mRNA
The RT-PCR assay was used to measure HO-1 mRNA collected from different groups in vivo and in vitro. In vitro, 0.45 ìg/L of hemin induced the expression of HO-1 in mMSCs after 72 hours. Compared with normal mMSCs, the expression of HO-1 was significantly raised (Figure 2A). Therefore, we injected the mMSCs of HO-1 high expression into the chemotherapy mice. After a week injection, the BMCs of different groups were collected to abstracted mRNA. As shown in Figure 2, after injection of the HO-1 mMSCs, the HO-1 expression was found to be clearly higher than the other groups.
Protein expression of HO-1
The protein expression levels of BMCs which came from different groups were analyzed by Western blotting. The results just as the results of mRNA are shown in Figure 3. After the cell injection, HO-1 expressed the highest in the HO-1 group (Figure 3A and 3B). These data prove that HO-1 was expressed in the HO-1 group significantly.
Effects of CTX damage and HO-1 recovery of the hematopoietic function
First, we analyzed the changes in blood of mice which were in the chemotherapy group at 0–21 days. Intraperitoneal injection of CTX was performed for 5 days. On the first 7 days, as time passed by, there was a gradual decline in the number of white blood cells, red blood cells, and platelets. On the 7th day, the number of white blood cells (WBC), red blood cells (RBC), and platelets (PLT) was the lowest (Figure 4A). As a consequence, we tail intravenously infected the cells on the 7th day for the observation of recovery.
The cells were injected on the 7th day for a chemotherapy model. We counted the number of white blood cells, red blood cells, and platelets at 0–21 days after the cell injection. After 7 days, the number of white blood cells, red blood cells, and platelets was significantly increased (Figure 4B). The recovery of hemogram was obvious. However, the HO-1 group was more effective than the mMSCs group.
Observation of bone marrow pathological slices
The femurs of the mice were separated from different groups. We could see that there were some structural damages in the chemotherapy group (Figure 6A). The cell structure was not complete and the bone structure was clearly destroyed (Figure 6B). There was a significant recovery in the HO-1 group. The structure and the cell morphology were complete, tightly packed, and the color was fresh red. The mMSCs were better than the chemotherapy group, but not as good as the HO-1 group. Therefore, the function of the bone marrow of the HO-1 group was robust than the chemotherapy group and was alike as the control group.
HO-1 induces proliferation and decreases apoptosis
MTT analysis showed the survival rates of different groups. The cells of the HO-1 group were apoptosized slowly, even slower than the control group. The HO-1 group was the highest as time passed by. The mMSCs group was less than the HO-1 group, but was the highest than any other two groups. The chemotherapy group was the lowest (Figure 5).
Bone marrow suppression is a serious side effect of CTX.11,12 It is a difficulty for further treatments. It has been reported that MSCs could do a favor in reconstitution of the hematopoietic microenvironment.13 MSCs are considered to be a potential source for cell and gene therapy strategies.14 Under certain conditions, MSCs can differentiate into multiple tissue types, including bone, fat, and cartilage.15–18 Given the innate ability of these cells to promote hematopoiesis recovery and tissue repair,19,20 the effectiveness of MSCs in hematopoietic recovery, bone regeneration, and in the treatment of patients with osteogenesis imperfecta, infracted myocardium, or joint diseases has been well documented.21–27 However, it was not so effective and was targeted as gene modified. There had been many genes, such as the chemokine Mig11 and VCAM-1,28 which were used to modify the MSCs to restore the hematopoietic inductive microenvironment (HIM). It had improved that it is more effective than the MSCs. In this experiment, we stimulate the high expression of HO-1 which was considered as a protective gene in mMSCs.5–9
The bone marrow was damaged after 5 days of intraperitoneal injection of CTX. After injection, the normal mice became weak and the hematopoietic function was damaged. We could recognize it from the hemogram (Figure 4A). The number of white blood cells, red blood cells, and platelets declined obviously and was down to the bottom on the 7th day. As we saw in the bone marrow pathological slices, the cells and bone structures were destroyed and incomplete. In this study, we also proved that the model CTX construction of chemotherapy can cause damage to the inhibition of hematopoiesis.
HO-1 plays an important role in antioxidation and antiapoptosis, maintains the homeostasis, reduces the level of oxidative stress, and protects the cells.29–31 We tried to put HO-1 into the bone marrow via the ability of mMSCs which can homing to HIM. However, the mMSCs make a contribution to reconstitution of hematopoietic mivroenviroment.15,32–34 Therefore, we made mMSCs group in contrast to the function of HO-1 in recovery of chemotherapy-induced bone marrow suppression.
Although the HO-1 expression between the mMSCs group and the control group had no significant differences (Figures 2 and 3), the mMSCs group had an obviously better survival rate than the control group (Figure 5), because MSCs have proved with high self-renewal and multidifferentiation potential, which play an important role in tissue repair and regeneration. MSCs have been approved of their ability to support the expansion of HSCs by expressing hematopoietic cytokines and facilitate the reconstruction and stabilization of the hematopoietic microenvironment when transplanted together with HSCs.35 This was the reason why the survival rate of the mMSCs group was decided to be higher than the control group. Even though the bone marrow in the mMSCs group had been destroyed before, the mMSCs could secrete huge growth factors and stimulating factors which could promote the recovery of hematopoiesis and stability of HIM. Just as shown in the result (Figure 5), after the first 24 hours, the apoptosis rate of the mMSCs group was slower than the control group. Because the mMSCs are secreting the factors, the effective volume is not achieved and the cells did not recover completely. After 24 hours, the cells grew up and the factors worked. In contrast to the mMSCs group, the control group was dying and had no enough nutrition to support cell renewal and survival. As a consequence, mMSCs can reach the bone marrow and work for recovery.
After stimulating HO-1 high expression by hemin, which was detected by PT-PCR and Western blot (Figures 2 and 3), we tail intravenously injected the mMSCs to bring the HO-1 into the bone marrow of the chemotherapy model mice. After the cell injection, the hemogram was gradually increased. In contrast to the chemotherapy group, the number of WBC, RBC, and PLT rose and became normal as the control group (Figure 4). However, the tendency of the HO-1 group was obviously better than mMSCs. MTT assay showed that HO-1 can promote and increase hematopoietic cell proliferation (Figure 5). It may be one of the reasons that improved the hemogram. The structure of bone marrow became complete and the cells grew faster than the chemotherapy group. The mMSCs group could become better in bone marrow structure and cell growth as well.14–19 However, the effect of the mMSCs group was not as good as the HO-1 group. As a consequence, we could infer that the hematopoietic function was recovered in the mMSCs group and the HO-1 group and the HO-1 group can make a more powerful contribution than the mMSCs group (Figure 6).
Based on the above results, we draw attention to HO-1 which promotes hematopoietic and resumption of chemotherapy-induced bone marrow suppression. Compared with other genes, HO-1 not only can protect the cells from apoptosizing, but also can restore the hematopoietic function via some factors or pathways which need further research. Overall, these results suggest that HO-1-modified mMSCs have a stronger ability to repair HIM and promote the recovery of hematopoietic functions. HO-1 plays an important role in supporting hematopoiesis in the HIM.
These findings are helpful for investigating the effects of repairing HIM function on the rapid recovery of hematopoietic damage and its underlying mechanisms, which may provide a new route for finding more effective ancillary methods for treating hematopoietic damage. If we could short the time of hematopoietic recovery, it will reduce the bleeding and infection and the therapy will be more effective.
1. Guillaume T, Rubinstein DB, Symann M. Immune reconstitution and immunotherapy after autologous hematopoietic stem cell transplantation. Blood 1998; 92: 1471-1490.
2. Parkman R, Weinberg KI. Immunological reconstitution following bone marrow transplantation. Immunol Rev 1997; 157: 73-78.
3. Angulo I, de las Heras FG, García-Bustos JF, Gargallo D, Muñoz-Fernández MA, Fresno M. Nitric oxide-producing CD11b+Ly-6G(Gr-1)+CD31(ER-MP12)+ cells in the spleen of cyclophosphamide-treated mice: implications for T-cell responses in immunosuppressed mice. Blood 2000; 95: 212-220.
4. Ryter SW, Alam J, Choi AM. Heme oxygenase-1
/carbon monoxide: from basic science to therapeutic applications. Physiol Rev 2006; 86: 583-650.
5. Pae HO, Jeong SO, Koo BS, Ha HY, Lee KM, Chung HT. Tranilast, an orally active anti-allergic drug, up-regulates the anti-inflammatory heme oxygenase-1
expression but downregulates the pro-inflammatory cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW264.7 macrophages. Biochem Biophys Res Commun 2008; 371: 361-365.
6. Seidel P, Goulet S, Hostettler K, Tamm M, Roth M. DMF inhibits PDGF-BB induced airway smooth muscle cell proliferation through induction of heme-oxygenase-1. Respir Res 2010; 11: 145.
7. Lee BW, Chun SW, Kim SH, Lee Y, Kang ES, Cha BS, et al. Lithospermic acid B protects β-cells from cytokine-induced apoptosis by alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1. Toxicol Appl Pharmacol 2011; 252: 47-54.
8. Jozkowicz A, Was H, Dulak J. Heme oxygenase-1
in tumors: is it a false friend? Antioxid Redox Signal 2007; 9: 2099-2117.
9. Doi K, Akaike T, Fujii S, Tanaka S, Ikebe N, Beppu T, et al. Induction of haem oxygenase-1 nitric oxide and ischaemia in experimental solid tumours and implications for tumour growth. Br J Cancer 1999; 80: 1945-1954.
10. Lee SE, Yang H, Jeong SI, Jin YH, Park CS, Park YS. Induction of heme oxygenase-1
inhibits cell death in crotonaldehydestimulated HepG2 cells via the PKC-δ-p38-Nrf2 pathway. PLoS One 2012; 7: e41676.
11. Lu H, Zhu S, Qian L, Xiang D, Zhang W, Nie A, et al. Activated expression of the chemokine Mig after chemotherapy contributes to chemotherapy-induced bone marrow suppression and lethal toxicity. Blood 2012; 119: 4868-4877.
12. Siena S, Castro-Malaspina H, Gulati SC, Lu L, Colvin MO, Clarkson BD, et al. Effects of in vitro
purging with 4-hydroperoxycyclophosphamide on the hematopoietic and microenvironmental elements of human bone marrow. Blood 1985; 65: 655-662.
13. Muguruma Y, Yahata T, Miyatake H, Sato T, Uno T, Itoh J, et al. Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment. Blood 2006; 107: 1878-1887.
14. Blazsek I, Chagraoui J, Péault B. Ontogenic emergence of the hematon, a morphogenetic stromal unit that supports multipotential hematopoietic progenitors in mouse bone marrow. Blood 2000; 96: 3763-3771.
15. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 276: 71-74.
16. Dennis JE, Merriam A, Awadallah A, Yoo JU, Johnstone B, Caplan AI. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res 1999; 14: 700-709.
17. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143-147.
18. da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 2006; 119: 2204-2213.
19. Verfaillie CM. Adhesion receptors as regulators of the hematopoietic process. Blood 1998; 92: 2609-2612.
20. Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007; 131: 324-336.
21. Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004; 8: 301-316.
22. Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A 2002; 99: 8932-8937.
23. Koç ON, Gerson SL, Cooper BW, Dyhouse SM, Haynesworth SE, Caplan AI, et al. Rapid hematopoietic recovery after coinfusion of autologousblood stem cells and culture-expanded marrow mesenchymal stem cells
in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000; 18: 307-316.
24. Petite H, Viateau V, Bensaïd W, Meunier A, de Pollak C, Bourguignon M, et al. Tissue-engineered bone regeneration. Nat Biotechnol 2000; 18: 959-963.
25. Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, Lavroukov A, et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 2001; 344: 385-386.
26. Grinnemo KH, Månsson A, Dellgren G, Klingberg D, Wardell E, Drvota V, et al. Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infarcted rat myocardium. J Thorac Cardiovasc Surg 2004; 127: 1293-1300.
27. Barry FP. Mesenchymal stem cell therapy in joint disease. Novartis Found Symp 2003; 249: 86-96; discussion 96-102, 170-174, 239-241.
28. Liu Y, Chen XH, Si YJ, Li ZJ, Gao L, Gao L, et al. Reconstruction of hema topoietic inductive microenvironment after transplantation of VCAM-1-modified humanumbilical cord blood stromal cells. PLoS One 2012; 7: e31741.
29. Alam J, Cook JL. How many transcription factors does it take to turn on the heme oxygenase-1
gene? Am J Respir Cell Mol Biol 2007; 36: 166-174.
30. Miyazaki T, Kirino Y, Takeno M, Samukawa S, Hama M, Tanaka M, et al. Expression of heme oxygenase-1
in human leukemic cells and its regulation by transcriptional repressor Bach1. Cancer Sci 2010; 101: 1409-1416.
31. Ito K, Hirao A, Arai F, Matsuoka S, Takubo K, Hamaguchi I, et al. Regulation of oxidative stress by ATM is required for selfrenewal of hematopoietic stem cell. Nature 2004; 431: 997-1002.
32. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 1999; 181: 67-73.
33. Majumdar MK, Thiede MA, Mosca JD, Moorman M, Gerson SL. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol 1998; 176: 57-66.
34. Chai W, Ni M, Rui YF, Zhang KY, Zhang Q, Xu LL, et al. Effect of growth and differentiation factor 6 on the tenogenic differentiation of bone marrow-derived mesenchymal stem cells. Chin Med J 2013; 126: 1509-1516.
35. Han LY, Li YP, Ye MZ, Wang BW, Wang Q, Zhao SH, et al. Transduction of mesenchymal stem cells with multidrug resistance gene provides protection for bone marrow toxicity after being transplanted into a nude mice model. Chin Med J 2012; 125: 3246-3250.