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RGD-Modified Angiogenesis Inhibitor HM-3 Dose: Dual Function during Cancer Treatment Hanmei Xu,*,† Li Pan,† Yinling Ren,† Yongjing Yang,† Xiaofeng Huang,‡ and Zhendong Liu† † ‡
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, P. R. China Department of Oral Pathology, Nanjing Stamotology Hospital, Affiliated Medical School, Nanjing University, 210008, P. R. China ABSTRACT: In the present study, we have undertaken pharmacodynamic studies of HM-3 in vitro and in vivo. A dual function of HM-3 with various doses was observed. HM-3 at low dose revealed obvious anticancer activity. In contrast, HM-3 at high dose had a tendency to promote tumorigenesis and tumor metastasis. Microarray analysis demonstrated that HM-3 at high dose could up-regulate the transcription of AKT1 and MEK1, which resulted in the promotion of tumorigenesis and metastasis. Therefore, the dose of angiogenesis inhibitors plays a critical role in cancer treatment. In order to achieve the ideal effect of angiogenesis inhibitor drugs on cancer treatment, a ful exploration of administration dose, frequency, and period for this kind of drugs is highly desired.
’ INTRODUCTION Since Judah Folkman proposed the theory of tumor angiogenesis in 1971, the inhibition of tumor angiogenesis has become an attractive strategy for cancer therapy and has received tremendous attention by scientists. This cancer treatment strategy can effectively inhibit tumor angiogenesis, reduce the supply of nutrients and oxygen, and arrest tumor growth and metastasis. The angiogenesis inhibitors are characteristics of few side effects and less drug resistance. Currently, several angiogenesis inhibitor drugs have been developed for the clinical application, which include monoclonal anti-VEGF antibody drugs such as bevacizumab1,2 and the second-generation multitargeted receptor tyrosine kinase inhibitor drugs such as sunitinib3,4 and sorafenib.5 These investigations suggested that angiostatic therapies have been recognized and accepted. However, compared with traditional cancer treatments, angiogenesis inhibitor drugs still have unclear pharmacological mechanisms, undefined treatment efficacy, and debated views due to their short history in clinical application. In 2009, Ebos and Paez-Ribes6,7 reported that angiogenesis inhibitors could promote tumor metastasis. In previous experiments from Ebos’ research group,8 the mice treated with sunitinib at the doses of 60 and 120 mg/kg exhibited an increased trend for host (mouse) and tumor (human) VEGF levels. In contrast, plasma levels in mice treated with sunitinib at the doses of 15 and 30 mg/kg did not exhibit the obvious increase. However, based on clinical application, the effective dose in mice should be 10 12 mg/kg, which suggested that higher than effective dose could promote the development of tumors. Therefore, the administration dose, frequency, and period of angiogenesis inhibitor drugs can result in significant impact on treatment efficacy. The high dose of angiogenesis inhibitors may interrupt the antitumor effect and induce the generation of tumor metastasis factors. r 2011 American Chemical Society
In our laboratory, we found that if a comprehensive investigation of the dosage of angiogenesis inhibitors such as HM-3 was not undertaken, an objective evaluation of angiogenesis inhibitors could not be achieved. We synthesized HM-3 and explored its anticancer activity in vitro and in vivo. The HM-3,9 a polypeptide containing 18 amino acids, was designed and synthesized in our laboratory. During the design process of HM-3, RGD integrin ligand sequence was added to improve its target capability to tumor cells with highly expressed integrin Rvβ3. On the basis of the experimental results in vitro, HM-3 had no inhibitory effect on tumor cells, but significantly inhibited the migration of endothelial cells. Matrigel and aortic ring tests in rats demonstrated that HM-3 effectively inhibited angiogenesis, indicating that its antitumor effect was accomplished by inhibiting the migration of endothelial cells and angiogenesis. Meanwhile, the antitumor activity was dosedependent in the range 0.75 3 mg/kg. No obvious antitumor activity was observed when the dose of HM-3 was lower than 0.75 mg/kg or higher than 6 mg/kg. On the other hand, HM-3 at the dose higher than 6 mg/kg could promote the migration of endothelial cells and result in the overexpression of genes related to tumor metastasis. We hope that our investigations will provide a reference for further research of angiogenesis inhibitors: preclinical and clinical applications should consider administration dose, frequency, and period of angiogenesis inhibitors to achieve an objective evaluation.
’ EXPERIMENTAL PROCEDURES Drugs. HM-3 (synthesized by GL Biochem (Shanghai) Ltd.) was kept at 20 °C. Recombinant Human Endostar Injection Received: February 19, 2011 Revised: June 12, 2011 Published: June 13, 2011 1386
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Bioconjugate Chemistry (obtained from Simcere) was kept at 4 °C. Both of them were diluted to desired concentration using serum-free endothelial cell medium. Cell Lines. Human umbilical vein endothelial cells (HUVECs, obtained from American ScienCell Research Laboratory) were maintained in endothelial cell medium (ECM) with 5% fetal bovine serum (FBS) and 1% endothelial cell growth supplement (ECGS). Cells were incubated at 37 °C in 5% CO2 in a humidified incubator. HUVECs Migration Assay. Diluted matrigel (purchased from BD Biosciences) 1:3 using serum-free medium, mixed sufficiently, was coated evenly on the surface of the transwell bottom. HUVECs were detached with 0.2% EDTA and resuspended in serum free ECM at a density of 1 105 cells/mL, and then added to the matrigel-coated transwell (1 104 cells/well). HM-3 and Endostar were diluted to the indicated concentrations using serum-free ECM and added to the transwell to incubate HUVEC cells. An appropriate amount of ECM was added, which contained 5% FBS and 1% ECGS to a 24-well flat-bottom polyviny1 chloride plate (COSTAR). The transwells were then put to the 24-well plate, and cells were incubated at 37 °C in 5% CO2 in the humidified incubator for 24 h. After that, the medium was aspirated, and the migrated cells were fixed with ethanol for 30 min and then stained with 0.1% crystal violet for 10 min. Clear cells were migrated by cotton swab and photos taken under a microscope. Matrigel Experiment. 50 μL melting matrigel was applied to 96-well cell plates and incubated with synthetic rubber and polyethylene at 37 °C for 1 h. 1 104 cells per well were seeded in the gel, then HM-3 was administered at concentrations of 4 μg/mL, 8 μg/mL, 16 μg/mL, and 32 μg/mL, and the positive drug endostar 20 μg/mL, respectively. Cells were incubated at 37 °C, 5% CO2 for 6 h. The effects of HM-3 on the differentiation of HUVECs stimulated by ECGS were observed with microscope (Olympus). Culture of Murine Aortic Rings. Young (4 5 months old) female mice were obtained from the Shanghai Laboratory Animal Center of Chinese Academy of Sciences. To prepare aortic segments, mice were sacrificed by isoflurane inhalation, and thoracic aortae were removed and rinsed in PBS with 100 U/mL penicillin and 100 μg/mL streptomycin. The isolated aortae were cleaned of perivascular adipose tissue and cut into segments 1 mm in length. Fibrinogen was dissolved in serum-free culture medium DMEM; final concentration was 3 g/L. 20 μL of 50 U/mL of bovine thrombin solution was added into 1 mL solution of the fibrinogen. Then, the fibrinogen can form fibrin glue at room temperature in 30 s. After dispensing the collagen solution, the preparations were incubated at 37 °C for 30 min for collagen to gel. Subsequently, one aortic segment was placed onto the center of each collagen gel and each segment was then overlaid with 20 μL of collagen solution. After polymerization of the overlaid collagen, 96-well plates filled with 100 μL of DMEM, then administered with 0, 4, 8, 16, and 32 μg/mL of HM-3, and 20 μg/mL of Endostar. After 7 8 days of culture, the microvessellike structures were observed by inverted microscope. Tumor Implantation. Tumor cells were grown in culture medium. Cell lines were washed with phoshate buffered saline (PBS), dispersed in a 0.05% trypsin solution, and resuspended. After centrifugation at 1000 rpm for 5 min, the cell pellets were resuspended in PBS and adjusted to a concentration of 5 106 cells/mL. C57BL/6 female mice or nude mice (5 to 6 weeks old) were implanted subcutaneously (s.c.) on the mid-right side with
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5 105 B16F10 cells in 0.1 mL PBS or 1 106 human SMMC7721 hepatic carcinoma cells. C57BL/6 Female Mice Treatment. After the average tumor volume of B16F10 mouse melanoma had reached 50 100 mm3, mice were randomly divided into different groups with eight mice per group. Group 1 received PBS. Group 2 was injected with Paclitaxel (Taxol, provided by Taiji Pharmaceutical Co. Ltd., Chendu in Sichuan province of China) once daily at a dose of 10 mg/kg in PBS. Groups 3 to 7 received HM-3 twice a day at doses of 1.5, 3, 6, 12, and 24 mg/kg/day, respectively. All of the mice were injected subcutaneously far from the tumor sites. BALB/c Nude Mouse Treatment. After the mean SMMC7721 tumor volume reached 50 100 mm3, mice were randomly divided into two groups with three mice per treatment group. Group 1 received PBS. Group 2 was injected with HM-3, twice a day at a dose of 48 mg/kg/day. All nude mice were injected intravenously with 0.2 mL. Measurement of Tumor Growth. Tumors were measured individually with a vernier caliper. Volumes were determined using the formula: tumor volume = length width2 0.52. Therapeutic effects on tumor growth were expressed as mean tumor volume versus time, calculated as (1 T/C) 100%, where T = treated tumor volume and C = control tumor volume. For example, if the volume of the treated tumor was 40% that of the control on a given day, tumor suppression in the treated group was regarded as 60%. Immunohistochemistry. Mice were euthanized 2 weeks posttreatment, and tumor tissue was collected, fixed with 4% formaldehyde, embedded in paraffin, and sectioned for hematoxylin and eosin (H&E) staining and immunohistochemical staining for CD31. Vascular structures in tumors were evaluated by immunohistochemical staining of CD31 with rabbit antiCD31 polyclonal antibody (Boster Biological Company). Briefly, staining for CD31 was performed on sections using their specific primary antibodies and biotinylated goat antirabbit secondary antibody, incubated with horseradish peroxidase-labeled streptavidin, visualized with diaminobenzidine (DAB) chromogen, counterstained with hematoxylin, and observed with a microscope. The microvessel density (MVD) was determined as follows, and the most highly vascularized areas in the sections were identified and viewed at 40 original magnification; then, the number of microvessels was assessed in fifteen randomly selected fields in this area viewed at 200 magnification. The MVD of this section was obtained by counting the average number of the fifteen fields. Three different sections were selected per group. Human Tumor Metastasis PCR Array. Human tumor metastasis gene array (SABiosciences http://www.sabiosciences. com) containing 84 genes was performed. Cultured HUVECs in ECM with 5% FBS and 1% ECGS. Cells were then incubated with HM-3 at the concentration of 0 μg/mL (as control), 8 μg/mL, and 32 μg/mL at 37 °C for 24 h when reached 80% confluent. RNA was isolated and purified under the instruction of RNA extraction kit (OMEGA). After synthesizing the first strand of cDNA, Real-Time PCR was performed. The cycle duration temperature was set as follows: 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min, respectively; and the melting curve was analyzed through the ΔΔCt method. First, ΔCt was calculated for each pathway-focused gene in each treatment group. Then, ΔΔCt was calculated for each gene across two PCR arrays (or groups). Finally, change was calculated for each gene from group 1 to group 2 as 2 ΔΔCt. 1387
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Western Blot Analysis. HUVECs were seeded at 3 105 per
During the aortic ring experiments in rats, after the third day of the experiment, the migration and proliferation of a large number of endothelial cells around arterial rings were observed, and vascular buds from the arterial wall sprouted. The growth phase was the first 6 13 days. A peak with 114 newly generated microvessels was observed at the 14th day; however, no increase in microvessel growth was observed at the 15th day or later. HM-3 at the dose of 4 μg/mL exhibited the initial inhibition effect on angiogenesis with blood vessel number of 32; the most obvious effect on angiogenesis inhibition was observed at HM-3
well in 6-well culture plates and incubated for 24 h, treated by HM-3 for 24 h, harvested with 0.05% trysin (Hyclone, UT, USA)/0.53 mM EDTA (Hyclone, UT, USA), washed with PBS, and resuspended in 100 μL of Mammalian Protein Extraction Reagent (Shanghai Generay Biotech Co. Ltd., Shanghai, China). Concentrated protein were separated on 12% SDS-polyacrylamide gel (SDS-PAGE) for VEGF, PDGD-A, Akt1, Bcl-2, ERK1/ 2, MEK1, and 8% for integrin Rvβ3 and integrin R5β1 SDSpolyacrylamide gel (SDS-PAGE), respectively, and then the gel was transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, MIT, USA). Briefly, PVDF membrane was incubated with a 1:1000 dilution of VEGF, PDGD-A, Akt1, Bcl-2, ERK1/2, MEK1, integrin Rvβ3, integrin R5β1, and β-actin antibodies (Cell Signaling Technology, USA). After incubation with the appropriate secondary antibodies, blots were incubated with the ECL reagents (Beyetime, Jiangsu province, China) and exposed to photographic film to detect protein expression.
’ RESULTS HM-3 at Low Dose Inhibited Endothelial Cell Migration and Angiogenesis. In this study, the effects of HM-3 at various
doses on the migration of endothelial cells were explored. Compared with the negative control, antitumor peptide HM-3 exhibited an obvious inhibitory effect on the migration of ECGSinduced human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner in the concentration range 0.5 8 μg/mL, as shown in Figure 1a,b. Meanwhile, the peptide HM-3 at the dose of 8 μg/mL revealed the best inhibitory effect on cell migration. However, the further increased dose of HM-3 resulted in the gradual decrease in inhibitory activity of HM-3 on cell migration. HM-3 at the dose of 32 μg/mL or higher resulted in the loss of inhibitory activity on cell migration. During the evaluation of matrigel experiments, the ECGSinduced HUVECs differentiated into branched capillary-like division structures during the treatment of HM-3 for 6 48 h. HM-3 at low concentrations (4 μg/mL) began to reveal the inhibitory activity on cell differentiation. The most obvious blocking effect on the differentiation of HUVECs into tube-like structures in vitro was observed when the concentration of HM-3 was 8 μg/mL. Meanwhile, HM-3 at this dose level could significantly reduce the number of small tubes and attenuate the lumen formation during the differentiation of HUVECs (P < 0.01). Furthermore, most HUVECs exhibited a messy arrangement due to cell aggregation. In contrast, at the concentration up to 32 μg/mL, HM-3 did not exhibit a significant effect on the formation of small tubes, as shown in Table 1 and Figure 2a,b.
Figure 1. Effects of HM-3 on the migration of endothelial cells. HM-3 exhibited an obvious inhibitory effect on the migration of ECGS-induced human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner in the concentration range 0.5 8 μg/mL (a). The further increased dose of HM-3 (32 μg/mL) resulted in the gradual decrease in inhibitory activity of HM-3 on cell migration. The migrated cells of positive drug ES (endostar, 8 μg/mL), control, HM-3 (8 μg/mL), and HM-3 (32 μg/mL) are shown in (b). (n = 5, *P < 0.05, **P < 0.01).
Table 1. Results of Antitumor Peptide HM-3 Inhibiting HUVEC Differentiationa control
4 μg/mL
8 μg/mL
6h
37.80 ( 11.8
11.00 ( 3.4**
13.00 ( 3.2**
19.60 ( 7.2*
25.80 ( 4.6
18.40 ( 5.8
12 h
29.00 ( 8.68
8.00 ( 2.0*
8.60 ( 2.3**
19.00 ( 5.2
23.00 ( 4.8
15.00 ( 4.7* 13.00 ( 3.8
times
32 μg/mL
endostar
24 h
18.40 ( 2.4
4.33 ( 1.2*
7.40 ( 2.3**
12.50 ( 2.0*
15.80 ( 2.8*
48 h
11.33 ( 4.1
1.33 ( 0.6*
2.00 ( 0.5*
9.50 ( 1.3
10.50 ( 1.8
6.75 ( 1.7
72 h
9.00 ( 2.9
0.66 ( 0.2
1.33 ( 0.2*
6.50 ( 1.3
8.50 ( 1.2
2.33 ( 0.2
96 h
5.00 ( 1.5
0.66 ( 0.1
0.66 ( 0.2
3.00 ( 0.5
6.66 ( 0.6
1.00 ( 0.2
120 h
2.66 ( 0.5
0.66 ( 0.1
0.33 ( 0.1
2.00 ( 0.2
3.00 ( 0.3
0.66 ( 0.1
144 h a
16 μg/mL
0
0
0
0
0
0
(x ( SD) 40 (OHP), (n = 5, *P < 0.05, **P < 0.01). 1388
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Figure 2. (a) Antitumor peptide HM-3 could inhibit HUVEC differentiation. A and F display the control and the positive drug of endostar (20 μg/mL). B, C, D, and E display HM-3 at doses of 4, 8, 16, and 32 μg/mL. (b) The effects of HM-3 inhibiting HUVEC differentiation at 6 h. The HM-3 at low concentrations (4 μg/mL) began to reveal the inhibitory activity on cell differentiation. The most obvious blocking effect on the differentiation of HUVECs into tube-like structures in vitro was observed when the concentration of HM-3 was 8 μg/mL. At the concentration up to 32 μg/mL, HM-3 did not exhibit a significant effect on the formation of small tubes. Columns represented x ( SD of experiments, n = 5, * P < 0.05, **P < 0.01 vs control.
dose of 8 μg/mL, and 20 blood vessels were detected at this condition. However, when the concentration of HM-3 was up to 32 μg/mL, the total loss of angiogenesis inhibition effect was observed, which exhibited a similar number of blood vessels as the negative control group with 88 blood vessels, as shown in Figure 3a,b. HM-3 at Low Dose Significantly Inhibited Tumor Growth. As shown in Figure 4a, HM-3 with various doses exhibited an obvious tumor inhibition effect. Compared with the negative control, HM-3 revealed a clear inhibition effect on tumor growth after 9 days treatment, and the inhibition rates of tumor volume subjected to HM-3 treatments at the doses of 1.5, 3, 6, 12, and 24 mg/kg were 57.37%, 53.04%, 23.82%, 15.88%, and 9.47%, respectively. This result suggested that the inhibitory effect of HM-3 treatment on tumor growth was not dose-dependent. HM-3 at low dose exhibited an excellent antitumor effect; in contrast, the antitumor activity was decreased or lost at the dose higher than 6 mg/kg. Moreover, previous experiments also demonstrated that HM-3 at the dose of 3 mg/kg could effectively inhibit the growth of SMMC-7721 cell in liver cancer.9 However, when administrated at the dose of 48 mg/kg for 10 days, there was no significant difference in the tumor anatomical detection between the treatment and the control groups, as shown in Figure 4b.
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Figure 3. (a) Fourteenth day of microvessel growth under different treatments. A represents control, and F displays the positive drug of endostar, 20 μg/mL. B, C, D, and E display HM-3 at the doses of 4, 8, 16, and 32 μg/mL. (b) Microvascular growth curves of aortic ring-fibrin gel cultues in serum-free DMEM. HM-3 at the dose of 4 μg/mL exhibited the initial inhibition effect on angiogenesis with blood vessel number of 32; the most obvious effect on angiogenesis inhibition was observed at dose of 8 μg/mL and 20 blood vessels were detected at this condition. However, when the concentration of HM-3 was up to 32 μg/mL, the total loss of angiogenesis inhibition effect was observed, which exhibited a similar number of blood vessels as the negative control group with 88 blood vessels. (n = 5, *P < 0.05, **P < 0.01).
HM-3 at High Dose Promoted the Formation of Tumor Blood Vessels. In order to further confirm the effect of HM-3
at different doses on angiogenesis, tumor tissues from nude mice treated with HM-3 at two doses (3 and 48 mg/kg) were immunohistochemically analyzed for CD31 antibody. The average of 5.8 capillaries was detected in the tumor tissues treated with HM-3 at an effective dose of 3 mg/kg, which was much lower than that (16.8 capillaries) in the group treated with HM-3 at a high dose of 48 mg/kg. Interestingly, HM-3 at the high dose did not inhibit tumor growth; however, the amount of blood vessels within tumor tissues treated with HM-3 at high dose was higher than that in the group at the effective dose. These results suggested that HM-3 at the high dose could not inhibit the growth of blood vessels; in contrast, it exhibited a promotional role in angiogenesis (Figure 5). HM-3 at High Dose Was Associated with the Regulation Genes of Tumor Metastasis. In order to explore the effect of HM-3 at various doses on tumor metastasis, 84 tumor metastasisrelated genes were analyzed by using human tumor metastasis PCR array. Genes selected for this array encoded several classes of protein factors including cell adhesion, ECM components, cell cycle, cell growth and proliferation, apoptosis, transcription factors and regulators, and other genes related to tumor metastasis. After HM-3 at both doses (8 and 32 μg/mL) had been used 1389
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Figure 5. Blood vessel density within tumor tissue. A, B, and C represent tumor tissues from nude mice treated with PBS, HM-3 (48 mg/kg), and HM-3 (3 mg/kg), that have been immunohistochemically analyzed for CD31 antibody (top), the staining observed with light microscope (40 original magnifications). The blood vessels were counted (bottom graph), HM-3 at dose of 3 mg/kg with average of 5.8 capillaries, 16.8 capillaries were detected in the group treated with HM-3 at a high dose of 48 mg/kg. Results were expressed as mean (SD of 15 regions of three sections per group, *P < 0.05; **P < 0.01 vs control).
Figure 4. (a) Therapeutic effects of peptides on the growth of B16F10 mouse melanoma tumor. A represents control, and B displays the positive drug Taxol, 10 mg/kg, once a day. C, D, E, F, and G display HM-3 at the dose of 1.5, 3, 6, 12, and 24 mg/kg twice a day, and the inhibition rates of tumor volume were 57.37%, 53.04%, 23.82%, 15.88%, and 9.47%, respectively. Each point represents x ( SD of each group (n = 8, **P < 0.01, *P < 0.05 vs control). (b) Therapeutic effects of peptides at the dose of 48 mg/kg for 10 days on the growth of SMMC7721 hepatic carcinoma. Tumor could not be inhibited by 48 mg/kg HM-3 twice a day. Each point represents mean (SD of each group (n = 3, *P < 0.05, **P < 0.01 vs control).
to treat the HUVECs, HM-3 treatment at the dose of 8 μg/mL exhibited an obvious enhancement on gene transcription of CTSL1, KiSS-1, Syk, and SSTR2, while revealing an inhibition effect on gene transcription of ETV4 and Myc. On the other hand, HM-3 at the dose of 32 μg/mL exhibited a great impact on the genes of tumor metastasis, which included the increase of Hras, MMP10, SSTR2, and TCF20 and the reduction of NR4A3 (Nor1). These results indicated that HM-3 at a higher dose might promote tumor development and metastasis, as shown in Figure 6. HM-3 Down-Regulated AKT and ERK1/2 Pathway Activity by Integrin rvβ3. During the design of the peptide HM-3, the RGD sequence was included in HM-3 for targeting integrin receptor. Previous studies verified that HM-3 could bind to
Figure 6. HM-3 at the dose of 32 μg/mL exhibited a great impact on the genes of tumor metastasis. A and B represent tumor metastasis genes of HUVEC impacted by HM-3 (3 mg/kg) and HM-3 (32 mg/kg) treatments.
overexpressed integrin Rvβ3 in tumor cells.9 In the present study, the roles in intracellular signaling were further explored. Western blot results showed that HUVECs treated with HM-3 revealed a reduced expression level of VEGF, PDGF-A, Bcl-2, and AKT1 as well as the MEK1 in a dose-dependent mode, as shown in Figure 7a. Meanwhile, although HM-3 did not exhibit an obvious impact on integrin R5β1, it revealed a significant impact on the expression of integrin Rvβ3, indicating that HM-3 executed its biological activity through integrin Rvβ3. On one hand, HM-3 can regulate the transduction pathway through integrin Rvβ3 to result in the down-regulation of MEK1 and AKT1, thus inhibiting the migration of tumor vascular endothelial cells to accomplish its antitumor activity. On the other hand, HM-3 can directly induce endothelial cells to decrease the expression of VEGF and PDGF-A, thus in turn accomplishing 1390
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Figure 7. (a) Western blot analysis of the expression level of VEGF, PDGF-A, Bcl-2, AKT1, ERK1/2, MEK1, and integrin. Significant impact on the expression of VEGF, PDGF-A, Akt1, Bcl-2, MEK1, and integrin Rvβ3 was revealed in HUVECs after HM-3 treatments. (b) HM-3 may execute its biological activity through integrin Rvβ3.
tumor angiogenesis inhibition and executing antitumor activity, as shown in Figure 7b. Similarly, the dose of HM-3 plays a critical role in its antitumor activity. A possible explanation is that HM-3 at the high dose condition has the function of regulating two key proteins, which is due to the dramatically increased expression of AKT1 and
enhanced expression of MEK1 to some extent when treated with HM-3 at the dose of 64 μg/mL, as shown in Figure 7a.
’ DISCUSSION Human cancer is a disease that is difficult to eradicate. Due to the complex pathological mechanisms, a single treatment 1391
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Bioconjugate Chemistry method is still unable to achieve good results for cancer. Thus, a number of different strategies and means for cancer treatment have been developed in clinical applications. Angiogenesis inhibitors are newly developed anticancer drugs entering the market in recent years. Unlike traditional chemotherapy and radiation therapy, angiogenesis inhibitor drugs have the characteristic of high safety, and promote less suffering for patients during the treatment. Some angiogenesis inhibitors are endogenous inhibitors such as tumstatin and endostatin. Tumstatin and endostain as angiogenesis inhibitors have been derived from precursor human collagen molecules known as the R3 chain of type IV collagen and R1 chain of type XVIII collagen, respectively. In a normal person, there is a balanced state between the angiogenesis signal output and angiogenesis inhibition. Angiogenic signals can be provided by growth factor alkaline FGF, VEGF, IL-8, and PDGF as available. Endogenous angiogenesis inhibitors were distributed in different tissues or blood. These angiogenesis inhibitors include thrombosis contractile protein, tumstatin, canstatin, endostatin, angiostatin, and interferon.10 Some drugs such as avastin can inhibit angiogenesis factor VEGF to reduce the output signal of tumor angiogenesis and delay the onset of cancer. Another strategy is to improve the angiogenesis defense system, thus providing a natural angiogenesis inhibitor as part of the cancer treatment. In our laboratory, peptide HM-3 was designed and synthesized on the basis of adhesion molecule integrin receptor Rvβ3. Its antitumor activity was accomplished by improving the angiogenesis defense system, which was validated by pharmacodynamic and pharmacological tests in vitro and in vivo. During the pharmacodynamic study in vivo, peptide HM-3 at low dosage conditions (1.5 and 3 mg/kg) exhibited a significant tumor inhibition effect. Similarly, HM-3 at too high or too low dose proved to have no inhibitory effect on the cell migration or vascular activity in vitro. Through gene chip analysis, HM-3 at effective dosage can be seen to increase CTSL1, KiSS-1, Syk, and SSTR2 gene transcription and inhibit ETV4 and Myc gene transcription. TSLC1 as a tumor suppressor gene was detected in lung cancer. KISS1 as a metastasis suppressor gene plays an important role in a variety of tumor invasion and metastasis. Somatostatin or its analogues can inhibit the proliferation of tumor cells via the regulation of SSTR2. In addition, the c-Myc gene is highly associated with the development of many kinds of tumors. Whereas ETV4, ETV1, and ERG genes belonging to ETS gene family are correlated with normal division of cells, their integration with other genes can result in overexpression and abnormal activity. HM-3 at the condition of effective dose can inhibit tumor-related genes, whereas HM-3 treatment at high dose greatly impacts tumor metastasis genes. Interestingly, HM-3 at the dose of 32 μg/mL results in an increased number of transcription genes including FN1, IL1B, CXCR2, IGF1, KISS1, PLAUR, RORB, SRC, SYK, TIMP4, TRPM1, and TSHR. Moreover, the increased transcription of genes such as Hras, MMP10, SSTR2, and TCF20, as well as the reduced NR4A3 (Nor1), was related to tumor metastasis. For example, Nor1 is a tumor suppressor gene of myeloid leukemia. The transcriptional regulation activity of NR4A3 (Nor1) is dependent on cell-specific and stimulus-specific gene induction and protein phosphorylation, not the ligands. Overdose of HM-3 can result in excessive stimulation on cells, thus correspondingly leading to the decreased expression of the NR4A3 (Nor1) gene and interrupt the balance among cell proliferation, apoptosis, and
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Figure 8. Effects of HM-3 on the migration of endothelial cells at the dose range 64 256 μg/mL.
differentiation. At the considered protein level, HM-3 at the effective dose can result in a dramatic decrease in expression of AKT1 as well as MEK1. These results remind us of a cancer treatment strategy of “supporting righteousness and dispelling evil” using Chinese medicine. It is well-known that the human body is a balanced system with its own operation laws. Tumorigenesis results from damaged balance, less righteousness, or too much evil. The principle of “supporting righteousness and dispelling evil” is to restore the balance in the body and achieve coordination of the internal organs. Therefore, the cancer treatment should use drugs reasonably; overkill is also a taboo, which is consistent with the phrase, “overkill can not achieve the expectation” from Confucius, a Chinese ancient thinker and philosopher. Angiogenesis and angiogenesis inhibition remain a balanced process in normal organisms including animals and people. The process of tumorigenesis is due to the loss of balance. Therefore, the application of angiogenesis inhibitors for cancer treatment is to restore balance. Then, inadequate dose or administration of HM-3 is not enough to achieve the desired results; similarly, excessive dose of HM-3 can further result in the interruption of the balance and no expected treatment efficacy. Interestingly, based on the HUVEC migration, with HM-3 at the dose of 32 μg/mL, no inhibitory effect on cell migration was observed; however, with the increased HM-3 at the dose range 64 256 μg/mL, a new dose effect relationship was apparent, as shown in Figure 8. All of these investigations suggested that cells or the human body have the capability to adjust their own signal change according to external stimulation. In this study, HM-3 executes the antitumor effect through increasing angiogenesis as a defense system. In contrast, some drugs complete the antitumor effect by inhibiting angiogenesis factor VEGF to reduce the output signal of tumor angiogenesis. Regardless of the cancer treatment strategy, the appropriate treatment dose is important. In 2009, Ebos, Paez-Ribes, and their co-workers reported that the VEGF-target treatment could inhibit tumor growth and improve the survival period of patients. However, the promotion effect of angiogenesis inhibitors on tumor metastasis was also described. On the basis of our analysis of Ebos’ investigation, the treatment with sunitinib at high dose (120 mg/kg) increased tumor metastatsis and reduced the animal survival period. We found that, in previous studies,11 the optimal dose range of sunitinib was 40 mg/kg/day for HT-29 or 80 mg/kg/day (Colo205) with treatment duration of 100 days. Also, according to the calculation of clinical dose (60 mg daily), the effective dose of sunitinib was 10 12 mg/kg for mice. However, the cancer treatments with angiogenesis inhibitors at high doses and short treatment period promoted the metastasis of tumors and shortened the survival time. These trials include 1392
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Bioconjugate Chemistry sorafenib at the dose of 150 mg/kg/day and SU10944 at the dose of 225 mg/kg/day. For example, the recommended daily dose of sorafenib for human is 400 mg (2 200 mg tablets). According to the recommended daily dose and human body weight (400 mg/60 70 kg), the dose of sorafenib should be 75 85 mg/kg for mice; similarly, the appropriate dose of SU10944 should be 100 mg/kg/day in mice.12 Therefore, we believe that excessive angiogenesis inhibitors did not reduce the output signal of angiogenesis; in contrast, they could lead to the interruption of body balance and stimulate other signaling pathways under the body stress to produce more angiogenesis factors and express more tumor metastasis-associated genes. This hypothesis is consistent with previous experimental results in which the mice administered with sunitinib at doses of 60 and 120 mg/kg exhibited increased VEGF levels, while the plasma VEGF levels remained intact when treated with sunitinib at the doses of 15 or 30 mg/kg.8 Because angiogenesis and angiogenesis inhibition in the human body is a very delicate process, patient status, symptoms, and pathological mechanisms must be fully considered while using angiogenesis inhibitors. It is really necessary to understand the tumor mechanisms (whether the tumor is derived from increased tumor angiogenesis factor such as VEGF or decreased angiogenesis defense system); drug type, administration dose, treatment frequency, and period are the determinants for cancer treatments.
’ AUTHOR INFORMATION Corresponding Author
*Hanmei Xu, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, China. Tel: 86-2583271007. Fax: 86-25-83271007. E-mail:
[email protected].
’ ACKNOWLEDGMENT We would like to thank Wenhui Jiang (Department of immunity analyze, Nanjing oral medical hospital, P. R. China) for critical assistance on histochemistry and immunohistochemistry analysis. The authors thank Chunyan Pu, Chi Zhang, and Yongbing Li for assisting in implanting tumors. Funding was provided by China scientific and technological major special project — ‘significant creation of new drugs’ (Grant No. 2009ZX09102) and National Science Foundation of China (NSFC, No. 30873073).
ARTICLE
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