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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 12  |  Page : 985-992

Role of dalteparin sodium on the growth of cancer cells and tumor-associated angiogenesis in A549 human lung cancer cell line and grafted mouse model


1 Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Bengbu Medical College, Lung Cancer Diagnosis and Treatment Center of Anhui Province, Anhui Provincial Key Laboratory of Clinical Basic Research on Respiratory Disease, Bengbu, China
2 Department of Respiration, Anhui Provincial Hospital, Hefei, China
3 Department of Respiration, The Third People's Hospital of Bengbu, Bengbu, China
4 Department of Respiration, The First Affiliated Hospital of Bengbu Medical College, Bengbu, China

Date of Web Publication11-Dec-2018

Correspondence Address:
Linian Huang
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Bengbu Medical College, Lung Cancer Diagnosis and Treatment Center of Anhui Province, Anhui Provincial Key Laboratory of Clinical Basic Research on Respiratory Disease, No. 287 Chang Huai Road, Bengbu 233004
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.192765

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 > Abstract 


Purpose: To investigate the effects of dalteparin sodium on the expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and hypoxia-inducible factor 1α (HIF-1α) in A549 human lung cancer (LC) cell line and a human A549-grafted nude mouse model.
Materials and Methods: A549 human lung adenocarcinoma cell line was divided into control group, treated using normal saline (NS); and dalteparin sodium groups, receiving 5, 15, 50, and 150 IU/ml of dalteparin sodium, respectively. Human A549-grafted nude mouse was induced through subcutaneous (SC) injection of A549 (5 × 106/0.2 ml) into the right armpit, and randomly assigned into control group (n = 6) receiving intraperitoneal (i.p.) injection of NS, cisplatin (DDP) group (n = 6, 3 mg/kg DDP alone, i.p., for 3 days), low molecular weight heparin (LMWH) group (n = 6) receiving SC injection of 1500 IU/kg dalteparin sodium for 35 days, and DDP plus LMWH group (n = 6, 3 mg/kg DDP, i.p., for 3 days, followed by SC injection of 1500 IU/kg dalteparin sodium for 35 days).
Results: Significant difference was noted in the messenger RNA expression of VEGF, VEGFR, and HIF-1α after treating with heparin with a concentration of 15, 50, or 150 IU/ml in the A549 cell line at 24 and 48 h, respectively. In the human A549-grafted nude mouse model, a significant reduction was noted in the expression of VEGF, VEGFR, and HIF-1α in the tumor mass harvested from the mice receiving administration of dalteparin sodium plus DDP.
Conclusion: Dalteparin sodium had the inhibitory effects on the growth of human LC A549 cells in vitro and A549 LC xenograft model, which could be enhanced when administrated together with DDP.

Keywords: Dalteparin sodium, hypoxia-inducible factor-1α, nonsmall cell lung cancer, nude mice, vascular endothelial growth factor


How to cite this article:
Rui Y, Wang D, Hu D, Huang L. Role of dalteparin sodium on the growth of cancer cells and tumor-associated angiogenesis in A549 human lung cancer cell line and grafted mouse model. J Can Res Ther 2018;14, Suppl S5:985-92

How to cite this URL:
Rui Y, Wang D, Hu D, Huang L. Role of dalteparin sodium on the growth of cancer cells and tumor-associated angiogenesis in A549 human lung cancer cell line and grafted mouse model. J Can Res Ther [serial online] 2018 [cited 2019 Sep 16];14:985-92. Available from: http://www.cancerjournal.net/text.asp?2018/14/12/985/192765




 > Introduction Top


Lung cancer (LC) has been considered as the leading cause for cancer-related death as it caused a mortality of over 1,000,000 annually.[1] Approximately, 75% of LC patients are in a state of local advance or metastasis. For the patients diagnosed with early-stage LC, relapse has been frequently noted, and the majority of them eventually die from metastasis.[2] To date, the therapeutic methods that have been commonly used in clinical practices are mainly included surgery, chemotherapy, and radiotherapy. Unfortunately, the overall 5-year survival rate of LC is still < 15%.[3] Although surgery is chosen as the first choice, approximately 70% of these patients are affected by advanced disease and are considered as the potential candidates for systemic treatment only.

The curative effects of mono-chemotherapy or radiotherapy are still limited due to a propensity to resistance.[4] In the setting of metastatic disease, chemotherapy is considered as the only available approach.[2],[5] To date, platinum-based chemotherapy is recommended as the frontline treatment for advanced nonsmall cell LC (NSCLC).[6] It has been acknowledged that cisplatin (DDP) is more effective than other platinum compounds; however, its application is limited due to the inducing of severe toxic side effects including myelosuppression, asthenia and gastrointestinal disorder, long-term cardiac, as well as renal and neurological consequences. These adverse events usually cause drug discontinuation, poor tolerance, and limited therapeutic efficacy.[7],[8] To solve these problems, preclinical and clinical trials are in progress to determine the efficiency of various dosing or scheduling strategies for chemotherapy to increase the treatment efficacy and decrease the toxicity.

Currently, it is well acknowledged that the growth, persistence, and metastasis of solid tumors are angiogenesis-dependent. Therefore, antiangiogenic therapy brings in hopes for treatment of solid tumors including NSCLC.[9] Vascular endothelial growth factor (VEGF), a primary stimulator of the vascularization in solid tumor, has been reported to play a pivotal role in the tumor angiogenesis in patients with carcinoma.[10] For the mechanism, VEGF could bind to its receptor (VEGFR), and then the VEGF-VEGFR complex stimulates tumor angiogenesis through paracrine and autocrine mechanisms,[11] based on which to promote the growth and metastasis of malignant tumors.

Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor controlling the expression of a battery of more than forty target genes that play crucial roles in the abovementioned processes such as oxygen delivery via angiogenesis and metabolic adaptation to hypoxia via glycolysis.[12] For example, HIF-1 contributes to the angiogenic switch and activates the transcription of VEGF required for tumor angiogenesis.[13] Meanwhile, loss of HIF-1 has been reported to cause dramatic negative effects on tumor growth, vascularization, and energy metabolism in xenograft assays.[13],[14]

Low molecular weight heparins (LMWHs) have been reported to confer a survival advantage in patients with carcinoma. For example, dalteparin sodium could inhibit the binding of heparin with the growth factors to their endothelial receptors, and the process is depending on the molecule's number of saccharide units.[15] To date, the majority of experimental data on the anticancer effects of heparins were obtained from in vitro studies.[16] In a previous study, intraperitoneal (i.p.) administration of VEGF-mediated angiogenesis is inhibited by a 5.0-kD heparin. To the best of our knowledge, no study has been carried out to determine the effects of LMWHs on the expression of VEGF and HIF-1 simultaneously. In this study, we studied the effects of the dalteparin sodium on tumor-associated angiogenesis in vitro and in vivo, using A549 cell line and a human A549-grafted nude mouse model.


 > Materials and Methods Top


Cell culture and treatment

Human lung adenocarcinoma cell line A549 was purchased from the Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (Life Technologies, Bedford, MA, USA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 U/ml streptomycin. All the cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. The cells were seeded in 6-well plates at a density of 3.0 × 105 cells per well and cultured after reaching 70–80% of confluence.

An aliquot of 1.0 × 107 cells were divided into two groups: control group, treating with normal saline and dalteparin sodium groups, which were subjected to 5, 15, 50, and 150 IU/ml of dalteparin sodium, respectively. Subsequently, the cells were cultured for 24 and 48 h, respectively.

Real-time polymerase chain reaction

Total RNA was extracted using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using M-MLV Reverse Transcriptase (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. Real-time polymerase chain reaction (PCR) was carried out using SYBR Green Supermix on 7500 Fast RT-PCR system (Applied Biosystems Inco., CA, USA). The messenger RNA (mRNA) levels of VEGF and HIF-1 were normalized by β-actin. The primers used for VEGF were 5'-GAGCCTTGCCTTGCTGCTGCTCTAC-3' and 5'-CACCAGGGTCTCGATTGGATG-3'. The primers used for VEGFR were 5'-AGCCAGCTCTGGATTTGTGGA-3' and 5'-CATGCCCTTAGCCACTTGGAA-3'. The primers used for HIF-1α were 5'-AGCCAGACGATCATGCAGCTACTA-3' and 5'-TGTGGTAATCCACTTTCATCCATTG-3. The cDNA of VEGF, VEGFR, and HIF-1α was amplified by PCR (predenaturation at 95°C for 30 s, followed by forty cycles of: 95°C, 30 s; 62°C, 30 s; and 72°C, 30 s) using 2 μL cDNA (10 × dilution) with strict adhesion to the manufacturer's introductions. The primers for β-actin were 5'-GGGAAGGTGAAGGTCGGAGTC-3' and 5'-AGCAGAGGGGGCAGAGAGATGAT-3'. The amplification results for real-time PCR were calculated as 2 (−ΔΔCt), according to the previous description.[17]

Animal tumor models and treatment

To rule out the contribution of host immune response, a nude mouse model was used in this study. Female BALB/c mice aged 4–6 weeks with a body weight of 16–18 g were purchased from the Laboratory Animal Center of Yangzhou University (Yangzhou, China). The animals were housed under controlled conditions with a room temperature of 25 ± 2°C and 12 h/12 h light/dark cycle. All the animals were allowed to access to food and water in specific pathogen-free conditions.

All animals (n = 18) were injected with human NSCLC cell A549 (5 × 106/0.2 ml) in the right armpit. One week later, the tumor size was speculated to reach a mean diameter of 3–5 mm, the animals were randomly assigned to control group (n = 6), which was subjected to i.p. injection of physiological saline; DDP group (n = 6), which was subjected to i.p. injection of 3 mg/kg DDP alone (days 1–3); LMWH group (n = 6), which was subjected to subcutaneous injection of 1500 IU/kg dalteparin sodium for (days 1–35); and DDP plus LMWH group (n = 6), which was subjected to i.p. injection of 3 mg/kg DDP (days 1–3) followed by SC injection of 1500 IU/kg dalteparin sodium (days 1–35). A caliper was used to measure the tumor size. The tumor volume was calculated using the formula: V = LW2 × 0.5236, where L stands for the greatest length and W stands for the perpendicular width as previously described.[18] In our study, the potential side effects were also observed including loss of weight, poor appetite, or altered behavior.

All animals were sacrificed on day 35 by ether anesthesia. All the protocols were approved by the Ethic Committee of the First Affiliated Hospital of Bengbu Medical College (Bengbu, China). During the studies, measures are taken to minimize the suffering of the animals. The tumors were excised and weighed. Tumor regression was calculated using the following formula: tumor suppression index = (V0V)/V0 ×100%, where V is the mean tumor volume of the treated group and V0 is the mean tumor volume of the control group.

Immunohistochemistry

The tumor specimens were fixed in 4% formaldehyde, embedded in paraffin, and cut into 4 μm sections for immunohistochemical analysis. Immunohistochemical analysis of VEGF, VEGFR, and HIF-1α expression was performed according to the procedure described elsewhere. The primary antibodies were mouse anti-human VEGF antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-human VEGFR antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and mouse HIF-1α antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively. The tumor sections were deparaffinized and rehydrated at first. Heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) at 120°C, and then endogenous peroxidase activity was blocked by 3% H2O2. The sections were treated with 10% normal goat or rabbit serum to reduce nonspecific staining and then incubated with the biotin-conjugated secondary antibody. The immunoreaction was observed using diaminobenzidine method.

The staining analysis was performed according to the degree of staining and percentage of stained cells as previously described.[19] The grade of the staining intensity was expressed in a score corresponding to the sum of both (a) staining intensity: 0 = no staining, 1 = weak staining in light yellow color, 2 = moderate staining in a yellow-brown color, and 3 = strong staining in a brown color and (b) percentage of positive staining cells randomly selected under five high power field: 0 = ≤5% positive cells, 1 = 6–25% positive cells, 2 = 26–50%, and 3 = ≥51% positive cells. In general, a score of 0–1 is defined as negative (−), a score of 2–3 is defined as weakly positive (+), a score of 4–5 is defined as moderately positive (++), and a score of >5 is defined as strongly positive (+++).

Hematoxylin and eosin staining

To evaluate the underlying side effects and toxicity of the combination therapy, animal weight was monitored every 4 days, based on which to determine the correlative indices including anorexia, diarrhea, skin ulceration, and mortality. Upon the organs including liver, spleen, kidney, and brain were harvested, hematoxylin and eosin staining was performed as described previously.[20] The images were captured using the ImageXpress Micro Imaging System (Molecular Devices Corporation, Sunnyvale, CA USA) and analyzed with MetaXpress Software (Molecular Devices Corporation, Sunnyvale, CA, USA).

Statistical analysis

All data were expressed as a mean ± standard deviation. Statistical tests were performed using SPSS software (version 13.0, SPSS Inc., Chicago, IL, USA). Student's t-test was used for the intergroup comparison. One-way analysis of variance and the rank-sum test were used to compare the difference among the groups. P < 0.05 was considered statistically significant.


 > Results Top


The messenger RNA expression of vascular endothelial growth factor, vascular endothelial growth factor receptor, and hypoxia-inducible factor-1α in vitro

The mRNA expression of VEGF, VEGFR, and HIF-1α was determined using real-time PCR. [Table 1] displayed the mRNA expression of VEGF, VEGFR, and HIF-1α in LC A549 cells after treating with dalteparin sodium. Compared with the control group, no statistical difference was noted in the mRNA expression of VEGF, VEGFR, and HIF-1α 24 h after the interference of 5 IU/ml dalteparin sodium (P > 0.05). After treating with heparin with a concentration of 15, 50, or 150 IU/ml, significantly decreased was noted in the mRNA expression of VEGF, VEGFR, and HIF-1α at 24 and 48 h, respectively (P < 0.05). Different concentrations (0 IU/ml, 15 IU/ml, 50 IU/ml, 150 IU/ml) of dalteparin sodium acting on the A549 cells 24 and 48 h, compared with the control group, the mRNA expression of HIF-1α level was significantly decreased (P < 0.05). Dalteparin sodium can depress the mRNA expression of HIF-1α level.
Table 1: Messenger RNA expression of vascular endothelial growth factor, vascular endothelial growth factor receptor, and hypoxia-inducible factor 1α

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To evaluate the correlation between the heparin interference and the mRNA expression in VEGFR and VEGF, ΔCT values for each group were analyzed using linear correlation statistical analysis. The results indicated that the mRNA expression of VEGFR and VEGF was positively correlated with the interference of heparin (24 h: r = 0.8725, P < 0.05; 48 h: r = 0.8614, P < 0.05). Meanwhile, a positive correlation was identified between the mRNA expression of HIF-1α and VEGF at 24 and 48 h, respectively (24 h: r = 0.8143, P < 0.05; 48 h: r = 0.8963, P < 0.05).

Tumor growth

In our study, tumor growth including the tumor volume and weight of the tumor mass was determined after administration of dalteparin sodium and DDP. Compared with control group, obvious inhibiting response to tumor was noted in LMWH group and DDP group, respectively. For the combination of LMWH and DDP, a superior suppression of the tumor volume and weight was noted compared with single administration of LMWH or DDP [Figure 1] and [Figure 2]. To be exact, the combined group showed marked decrease of tumor volume on day 35 compared with that of the control group (289.83 ± 56.57 mm3 vs. 945.09 ± 180.75 mm3), LMWH group (289.83 ± 56.57 mm3 vs. 497.83 ± 89.95 mm3), or DDP group (289.83 ± 56.57 mm3 vs. 688.53 ± 123.28 mm3). For the tumor weight, the LMWH group and LMWH plus DDP group resulted in a significant decrease compared with the control group (P < 0.05) [Table 2]. However, no statistical difference was noted in the DDP group compared with the control group (P > 0.05).
Figure 1: Tumor volume after the treatment of low molecular weight heparin and/or cisplatin monotherapy in nude mice grafted with human nonsmall cell carcinoma A549 cells

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Figure 2: Tumor weight changes after the treatment of low molecular weight heparin and/or cisplatin monotherapy in nude mice grafted with human nonsmall cell carcinoma A549 cells

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Table 2: Characterization of intraperitoneal xenografts of A549 cell line in nude mice

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Expression of vascular endothelial growth factor, vascular endothelial growth factor receptor, and hypoxia-inducible factor-1α in vivo

An i.p. xenograft model of human LC was established to determine the anticancer efficacy of dalteparin sodium (LMWH) through determining the expression of VEGF, VEGFR, and HIF-1α in vivo. First, we evaluated the expression of VEGF in the harvested tumor mass. As shown in [Figure 3]a, the expression of VEGF showed a remarkable reduction in the tumor mass harvested from the mice treated with dalteparin sodium or DDP alone compared with the normal group. Nevertheless, the most significant reduction in VEGF expression was noted in the tumor mass harvested from the mice receiving administration of dalteparin sodium plus DDP (P < 0.05) [Table 3]. Compared with the expression of VEGF in dalteparin sodium group, no statistical difference was noted in that of DDP group (P > 0.05). For the evaluation of VEGFR expression, as shown in [Figure 3]b, the expression of VEGFR was reduced apparently in the tumor mass harvested from the mice treated with dalteparin sodium or DDP alone compared with the normal control. The most significant reduction in VEGFR was observed in the tumor mass harvested from the mice receiving administration of dalteparin sodium plus DDP (P < 0.05) [Table 4]. Compared with the expression of VEGFR in dalteparin sodium group, no statistical difference was noted in that of DDP group (P > 0.05). [Figure 3]c showed the expression of HIF-1α in tumor mass, and the results demonstrated that the expression of HIF-1α was reduced apparently in the tumor mass harvested from the mice treated with dalteparin sodium or DDP alone compared with control group. The most significant reduction in VEGFR expression was noted in the tumor mass harvested from the mice receiving administration of dalteparin sodium plus DDP (P < 0.05) [Table 5]. Compared with the expression of HIF-1α in dalteparin sodium group, no statistical difference was noted in that of DDP group (P > 0.05).
Figure 3: Expression of vascular endothelial growth factor, vascular endothelial growth factor receptor, and hypoxia-inducible factor-1α (red arrow) in the animals treated using normal saline, cisplatin, low molecular weight heparin, and low molecular weight heparin plus cisplatin, respectively. The expression of vascular endothelial growth factor (a), vascular endothelial growth factor receptor (b), and hypoxia-inducible factor-1α (c) in vivo using a fluorescent microscope (Elivision Incorporation) with a magnification of ×400

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Table 3: Expression of vascular endothelial growth factor in each group transplanted tumor tissue

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Table 4: Expression of vascular endothelial growth factor receptor in each group transplanted tumor tissue

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Table 5: Expression of hypoxia-inducible factor-1a in each group transplanted tumor tissue

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In our study, rank correlation analysis was carried out to investigate the potential correlation among the expression of VEGF, VEGFR, and HIF-1α. A positive correlation was noted between the expression of VEGF and VEGFR (rs= 0.803, P < 0.05). In addition, the expression of HIF-1α and VEGF was also positively correlated (rs= 0.464, P < 0.05).

To evaluate treatment-related toxicity, body weight was used as a surrogate for the evaluation of the general health status of the mice. Temporal loss of body weight was observed in the DDP group and LMWH plus DDP group. In general, no significant difference was noted in the body weight among the control group, DDP group, dalteparin sodium group, and dalteparin sodium plus DDP group [Figure 4]. No adverse consequences such as strange behaviors or toxic deaths were observed in any group. In the control group, infiltration of xenograft was noticed in the surrounding skin tissues, whereas there is no local infiltration in the other mice. Furthermore, no pathologic lesions were observed in liver, spleen, kidney, and brain.
Figure 4: Loss of body weight in mice treated using normal saline, cisplatin, low molecular weight heparin, and cisplatin plus low molecular weight heparin. No significant differences in body weight among these groups (P > 0.05) even though minor weight loss was noticed in the cisplatin group and cisplatin plus low molecular weight heparin group, respectively

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 > Discussion Top


Recently, antiangiogenesis therapy provides a new way for the clinical treatment of LC. Several factors or protein has been considered to be involved in the tumor angiogenesis. In our study, we want to explore the effects of dalteparin sodium on tumor-associated angiogenesis in vitro and in vivo, using A549 cell line and a human A549-grafted nude mouse model. Our study indicated that dalteparin sodium had the inhibitory effects on the expression of VEGF, VEGFR, and HIF-1α in vitro and in vivo.

VEGF is a key regulator in tumor angiogenesis, which contributes to the tumor growth and metastasis. In a previous study, significant upregulation was noted in the expression of VEGF in patients with LC tissues.[21] Meanwhile, its expression has been reported to be associated with the prognosis of patients.[22] In the past, paracrine has been speculated to be involved in the expression of VEGF in tumor cells, which is capable to specifically combine with VEGFs and its receptors (e.g., Flt and KDR). The combination (VEGF-VEGFR) induced receptor dimers and their interaction phosphorylation, activation of downstream signaling effect proteins, thereby caused endothelial cell migration, proliferation, and angiogenesis. Besides, many recent studies have reported that expression of VEGFR in tumor cell surface can occultly combine with its receptor and trigger its biological effects through autocrine.[23] For instance, VEGF-1 and -2 was expressed in a large number of tumor cells, through which VEGF could directly affect the proliferation of tumor cell proliferation by the autocrine.[24]

The HIF-1 transcription factor was first identified based on its ability to activate the erythropoietin gene in response to hypoxia.[25] Since then, it has been shown to be activated by hypoxia, based on which to induce hypoxia-responsive target genes including VEGF and Glut1.[26] As the major solid tumor inducing regulator of VEGF, the expression of HIF-1α gene can directly induce a significant increase of the stability and transcriptional activity of VEGF. According to the previous reports, HIF-1α contributes to the activation and induction of VEGF.[26],[27] Meanwhile, in animal models with the administration of LY294002, the gastric adenocarcinoma PI3K/HIF-1α signaling pathway was inhibited, resulting in reduced expression of HIF-1α and downregulation of VEGF secretion. Thus, it seems that the expression of VEGF may be reduced by means of inhibiting the expression of HIF-1α, which functioned to inhibit the tumor growth and metastasis.[28]

Dalteparin sodium is commonly used as an anticoagulant in clinical practices as it has several advantages such as high bioavailability, long half-life in vivo, satisfactory antithrombotic effect, and fewer side effects.[29],[30] Besides, LMWH has been reported to impede the proliferation of tumor cells by inhibiting the expression of the cell cycle regulating factors such as cyclin A, B, and E.[31],[32] Recently, LMWH has been revealed to involve in anticoagulation and the process of antitumor angiogenesis,[30],[33] and the inhibitory effects on malignant cell proliferation.[34] Dalteparin alone was reported to cause a proangiogenic effect on tumor growth.[35] In this study, we investigated the potential antiangiogenic mechanism of dalteparin sodium. Compared with control group, the expression of VEGF, VEGFR, and HIF-1α showed a significant decrease in A549 LC cells after treating with dalteparin sodium. Meanwhile, a dose-dependent and time-dependent manner was noted in the expression of VEGF, VEGFR, and HIF-1α. Further, significant reduction was noticed in the mean tumor volume and tumor weight in the treatment group compared with the control group, especially in the dalteparin sodium group plus DDP group. Compared with the control group, a significant decrease was noticed in the expression of VEGF, VEGFR, and HIF-1α in dalteparin sodium group, whereas a more significant was noted in the dalteparin sodium plus DDP group. A positive correlation was noted between the expression of VEGFR and VEGF, and that of HIF-1α and VEGF in vitro and in vivo. Our results indicate that there is an autocrine mechanism for VEGF/VEGFR in LC line A549 cells. In addition, dalteparin sodium involves in reducing the expression of VEGF and VEGFR in vivo and in vitro. We speculate this process may be associated with the destruction of VEGF/VEGFR autocrine loop, which finally resulted in inhibition of tumor angiogenesis. Further, our results demonstrated that dalteparin sodium also involved in the downregulation of HIF-1α. All these demonstrated that dalteparin sodium played a biological role in the antiangiogenesis.

To date, the mechanism of how LMWH plus DDP can enhance the antitumor activity is not well defined. We speculate that it may partially be associated with the increased induction of the apoptosis as well as inhibition of angiogenesis. Interestingly, DDP has been reported to inhibit the DNA synthesis in human umbilical endothelial cells in a dose-dependent fashion.[36] Taken together, we conclude that the enhanced antitumor efficacy in the present study may be partly resulted from the increased apoptosis as well as the inhibition of angiogenesis in the combined administration of DDP and dalteparin sodium.

To investigate whether dalteparin sodium can inhibit tumor growth by inhibiting the mutant gene of solid tumors, A549 cells with a mutant active Ras oncogene were used in our study.[37],[38] Our data suggest that dalteparin sodium showed antitumor activity against human LC in xenograft model of A549 LC, which could be enhanced when administrated together with DDP. It has also been reported that the mutant K-ras blocks the efficacy of targeted EGFR inhibitors such as Iressa and Tarceva.[39],[40],[41] Therefore, whether or not dalteparin sodium could sensitize A549 cells to these agents is an intriguing question to be further explored.


 > Conclusion Top


Our study demonstrated that dalteparin sodium had the inhibitory effects on the growth of human LC A549 cells in vitro and A549 LC xenograft model, which could be enhanced when administrated together with DDP. Thus, we speculate that monotherapy using dalteparin sodium or the dalteparin sodium plus DDP may be applicable in the treatment of lung carcinoma in clinical practices.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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