Shandong Gaomi Caihong Analytical Instruments Co., Ltd.
|Membership:||On ECPlaza since 2009|
|Business Type:||Manufacturer, Exporter|
|Main Item / Product:||chemistry analyzer/spectrophotometer|
|Keywords:||spectrophotometer, chemistry analyzer, analyzer|
|Main Target Region:||World Wide|
|Safety / Quality Approvals:||ISO/CE|
|Representative / CEO's Name:||Qiu Falin|
|Employees Total:||250 to 499|
Peptide structure and anticancer activity in vitro
We first examined the secondary structures of HPRP-A1 with or without co-administration of iRGD (concentration ratio HPRP-A1:iRGD = 1:1) using circular dichroism (CD) spectroscopy in benign condition as well as in an α-helix-inducing solvent in the presence of 50% trifluoroethyl alcohol (TFE), as described in Methods23. As shown in Fig. 1A, in benign condition, HPRP-A1 with or without iRGD co-administration and iRGD alone all exhibited a random coil structure. In contrast, in the presence of 50% TFE (Fig. 1B), iRGD exhibited a random coil structure, but HPRP-A1, both with or without iRGD, exhibited different degrees of an α-helical structure. However, the helical content of HPRP-A1 with iRGD was lower than HPRP-A1 peptide alone. The CD result of the peptides demonstrated that combination of iRGD could reduce the α-helical structure of HPRP-A1 peptide, suggesting a potential reducing toxicity against normal mammalian cells such as human erythrocytes3.
To study the anticancer activity and cancer cell selectivity of peptides, we used the A549 cell line, which exhibits overexpression of NRP-124. Human umbilical vein endothelial cells (HUVEC) have low NRP-1 receptor expression25 and were selected as a control. We first measured the cell viability of both HUVEC and A549 cells treated with peptides using MTT assays (Fig. 2A). The RGD peptide was used as a comparison of iRGD. We treated cells with 8 μM of HPRP-A1 with or without 64 μM or 125 μM of iRGD or RGD for 1 h, 24 h and 48 h. Both 64 μM and 125 μM iRGD decreased the cytotoxicity of the HPRP-A1 peptide against the HUVEC cell line. However, in the A549 cell line, the cytotoxicity of HPRP-A1 peptide was increased after co-administration with iRGD. Notably, compared with iRGD, co-administration of RGD did not noticeably impact the effects of HPRP-A1 on both HUVEC and A549 cells.
We calculated the half maximal inhibitory concentration (IC50) values of HPRP-A1 and HPRP-A1 co-administered with 32 μM, 64 μM or 125 μM iRGD in A549 cells (Fig. 2B). The IC50 values of HPRP-A1 combined with 64 μM and 125 μM iRGD at 1 h and 24 h were approximately half of the IC50 values of HPRP-A1 alone. These data indicate that HPRP-A1 can induce rapid cancer cell death and that iRGD can enhance the killing activity of HPRP-A1 at a co ncentration above 64 μM. Thus, we explored the use of iRGD as a homing peptide for co-administration with HPRP-A1 in the following experiments.
Apoptosis induction activity, cell cycle effect and caspase 3 activity of peptides
A previous report showed that HPRP-A1 disrupted the integrity of the cell membrane and induced apoptosis in HeLa cells3. Thus, we examined the effects of HPRP-A1 on inducing early and late apoptosis in A549 cells using a FITC Annexin V/PI apoptosis detection kit. A549 cells were cultured with 4 μM HPRP-A1 with or without 64 μM iRGD for 5 min, 30 min, 1 h and 24 h, and the percentages of apoptotic cells were examined by flow cytometry (Fig. 3A,B). HPRP-A1 co-administered with 64 μM iRGD for 30 and 60 min induced early apoptosis in 18.68% ± 0.51% and 36.63% ± 0.95% of cells, respectively, while HPRP-A1 alone only induced early apoptosis in 9.05% ± 0.16% and 30.74% ± 1.20% of cells, respectively (Fig. 3A,B). After 24 h, only a few early apoptotic cells were detected and the number of late apoptotic cells increased dramatically. HPRP-A1 co-administered with 64 μM iRGD induced late apoptosis in 73.99% ± 0.55% of cells, while HPRP-A1 alone induced late apoptosis in about 54.43% + 2.34% of cells. These results demonstrate that co-administration with iRGD could significantly improve the apoptotic ability of HPRP-A1 in A549 cancer cells (P < 0.01).
We also examined the effect of HPRP-A1 with or without iRGD on the cell cycle by flow cytometry. A549 cells were cultured with 4 μM HPRP-A1 with or without 64 μM iRGD for 24 h (Fig. 3C). Consistent with the apoptosis results in Fig. 3A, HPRP-A1 co-administered with iRGD resulted in much higher numbers of cells in sub-G1 (40.33% ± 0.57%) than treatment with HPRP-A1 alone (22.03% ± 0.50%). Furthermore, HPRP-A1 co-administered with iRGD induced a G1 arrest (87.95% ± 0.19%) compared with HPRP-A1 alone or controls (78.39% ± 0.56% and 75.24% ± 0.76%, respectively).
To further verify the apoptotic activity of peptides, caspase-3 activity was examined by western blotting (Fig. 3D). In cells treated with HPRP-A1 together with iRGD, much higher levels of cleaved caspase 3 levels and lower levels of pro-caspase-3 were observed compared with both the controls and cells treated with HPRP-A1 alone. These results suggest that co-administration with iRGD promotes the ability of HPRP-A1 to induce apoptosis by a caspase-dependent pathway.
Membrane destruction in A549 cells by peptides
HPRP-A1 is a membrane-active peptide and iRGD is a targeted penetrating peptide26,27. Previous studies showed that membrane-active peptides can interact with the cell membrane, penetrate the phospholipid bilayer and eventually cause cell death28. Thus, we next examined the membrane disruption activity of HPRP-A1 with or without iRGD by flow cytometry. Propidium iodide (PI) was used as a probe to assess the membrane integrity because PI can enter the cell and combine with DNA when the integrity of the cell membrane is altered. We treated A549 cells with various concentrations of HPRP-A1 with or without 64 μM iRGD and cultured cells for 1 h. The cells were then stained with PI and examined by flow cytometry (Fig. 4A and B). The results showed that 4 μM HPRP-A1 with or without 64 μM iRGD could induce around 3.03% ± 0.15% and 18.55% ± 6.49% PI uptake, (P < 0.01). Treatment with cells with 8 μM HPRP-A1 induced around 22.56% ± 0.83% PI uptake, and co-administration with iRGD caused the PI uptake rate to increase to around 39.18% ± 5.31%. However, upon treatment with cells with 16 μM HPRP-A1, the PI uptake rate was almost 90% in both the HPRP-A1 alone group and HPRP-A1 and iRGD co-administration group.
Cellular uptake of peptides
To explore the cellular uptake process of peptides, we performed laser scanning confocal microscope (LSCM) analysis (Fig. 5A). FITC-labeled HPRP-A1 at the concentration of 8 μM internalized into A549 cells from 300 s to 600 s, and the fluorescence intensity of cells with HPRP-A1 and iRGD co-administration was brighter than that of cells treated with HPRP-A1 alone. When the concentration of HPRP-A1 was increased to 16 μM, the fluorescence could be detected as early as 50 s and became very bright at 100 s. Figure 5B shows the quantitative values of fluorescence at each time point from Fig. 5A. The fluorescence intensity of the A549 cells, when treated with FITC-labelled HPRP-A1 peptide with co-administration of iRGD, was higher than the single peptide administration in 300 s, 600 s in 4 μM groups and 50 s, 100 s in 8 μM groups, revealing much more cellular uptake rates to the combination peptides compared with the single peptide. The fluorescence change rates of different groups are shown in Fig. 5C. The fluorescence change rates of cells treated with 4 μM FITC-labelled HPRP-A1 peptide showed a weak increase, while, that of cells treated with HPRP-A1 co-administration with iRGD increased sharply, and the final fluorescence potential was dramatically higher than the single HPRP-A1 group. The fluorescence change rate of cells treated with 8 μM FITC-labelled HPRP-A1 peptide with or without iRGD peptide exhibited similar increasing trend; in contrast, the increase rate and the final fluorescence intension in combination group was higher than the single peptide group. The result indicated that co-administration with iRGD could increase the rate and quantity of A549 cellular uptake. Together these results indicated that co-administration with iRGD significantly increased the uptake of HPRP-A1 in A549 cells. Supplementary video files show the cellular uptake process in more details (see Supplementary videos S1–S4).
Mitochondrial depolarization, ROS generation and co-localization of peptides
We next measured the mitochondrial membrane potential by flow cytometry using the JC-1 probe. JC-1 is a fluorescent dye that shows red fluorescence under aggregation conditions in normal mitochondria, but shows green fluorescence when the mitochondrial membrane potential decreases. Thus, the change of mitochondrial membrane potential can be determined by the ratio of green fluorescence and red fluorescence using JC-129. A549 cells were cultured with 4 μM or 8 μM HPRP-A1 with or without 64 μM iRGD for various time points, stained with JC-1 dye, and examined by flow cytometry (Fig. 6A). In the cells with HPRP-A1 co-administered with 64 μM iRGD, the mitochondrial membrane potential decreased much more than in cells treated with HPRP-A1 alone at both peptide concentrations (P < 0.01).
The increase of reactive oxygen species (ROS) generation can be one of the primary indicators of the disruption of the mitochondrial membrane30. We found that cells treated with HPRP-A1 co-administered with iRGD showed a higher level of ROS generation than cells treated with HPRP-A1 alone at 5 min and 15 min (Fig. 6B), which is consistent with the cell viability result showed in Fig. 2B.
The distribution of HPRP-A1 in cells by co-localization assays was investigated using LSCM (Fig. 6C). Interestingly, confocal images revealed that FITC-labelled HPRP-A1 and HPRP-A1 co-administration with iRGD could be taken up into A549 cells effectively and localized to mitochondria in a highly specific manner. In Fig. 6C, the merge images showed nearly complete overlapping between signals of FITC-labelled peptide and Mito-Tracker Green. The degree of overlapping was also demonstrated by Pearson’s correlation coefficient (Rr) using Image-Pro Plus software. The co-localization parameter was calculated using Rr, which describes the correlation of the intensity distribution between channels. The value of Rr ranges from −1.0 to 1.0, and 0 indicates no significant correlation and −1.0 indicates complete negative correlation31. The Rr vales in HPRP-A1-treated cells and cells treated with HPRP-A1 co-administered with iRGD were 0.620 and 0.778, respectively, which indicates that FITC-labeled HPRP-1 alone or co-administered with iRGD mainly co-localized at the mitochondrial membrane. Together these data revealed that HPRP-A1 alone or co-administered with iRGD localized to and disrupted the mitochondrial membrane to exert its anticancer activity.
Penetrability of FITC-labeled peptide in A549 3D-MCS
Previous studies showed that the homing peptide iRGD can enhance tumor-specific delivery of co-administered peptides9. We used an A549 3D cell model to further study the penetration ability of peptides. A549 3D MCS were cultured with 8 μM HPRP-A1 with or without 64 μM iRGD for 30 min, and the FITC-fluorescence was determined by LSCM using Z scan model (Fig. 7A). In the 15 μm layer of the MCS treated with HPRP-A1 alone, almost no green fluorescence was detected in the center of the MCS, indicating that HPRP-A1 may only penetrate into the 10th layer of the cell sphere. However, in the MCS with HPRP-A1 with iRGD, more green fluorescence was detected even in the 23 μm layer, showing that co-administration with iRGD markedly improved the penetration ability of FITC-labeled HPRP-A1 into 3D MCS. Figure 7B shows the changes of fluorescence intensity of the 23rd layer of MCS, from the center to the margin; the fluorescence intensity of the MCS with HPRP-A1 and iRGD was stronger than the MCS with HPRP-A1 alone. Figure 7C shows an image of the 23rd layer of MCS. Together these results indicate that HPRP-A1 co-administered with iRGD significantly enhanced the penetration ability of HPRP-A1 peptide in A549 3D MCS.
Effect of HPRP-A1 with iRGD on inhibiting tumor growth in nude xenograft mice
We next examined the effect of co-administration of HPRP-A1 and iRGD on the growth of tumors using nude xenograft mice in vivo. BALB/c nude mice were subcutaneously injected with A549 cells to create tumors. When tumors were about 120 mm3, the mice were randomly divided into three groups (n = 5 mice in each group). No statistically significant difference in tumor volume was detected among groups. HPRP-A1 peptide alone or with iRGD was directly injected into tumors once every 2 days. Saline solution was injected as the control. Tumor volume and body weight of mice in each group were recorded every 2 days. Both the tumor size and weight in the HPRP-A1 co-administered with iRGD group were dramatically smaller than those in the HPRP-A1 alone group and control group after intratumor injection for 16 days (Fig. 8A and Supplementary Fig. S1). In the control group, the tumor volume reached 400 mm3, while tumors were approximately 300 mm3 and 150 mm3 in the HPRP-A1 alone group and HPRP-A1 with iRGD group, respectively (Fig. 8C). These results showed statistical significance (P < 0.05, HPRP-A1 alone group compared with HPRP-A1 with iRGD group; P < 0.01, HPRP-A1 group compared with HPRP-A1 with control group). The inhibition rate in Fig. 8B clearly showed that mice treated with co-administration therapy experienced much greater tumor growth inhibition (65.0%) than animals treated with HPRP-A1 alone (36.47%). No difference in body weight among all groups was observed (Fig. 8D), indicating both the single HPRP-A1 and HPRP-A1 co-administration with iRGD did not reflect the body weight of the A549 xenograft mice.
We also performed hematoxylin-eosin staining (H&E), TUNEL assay and Ki 67 expression of the tumor tissue from all groups (Fig. 8E). The necrotic area in the tumor tissue from mice with HPRP-A1 co-administered with iRGD was much larger than that in mice with HPRP-A1 alone. TUNEL assays revealed much more apoptotic cells in the tissues from mice with HPRP-A1 co-administered with iRGD compared with tissues from mice with HPRP-A1 alone. Ki 67 protein expression in control group was much higher than that in HPRP-A1 alone or HPRP-A1 co-administration with iRGD. The expression content in HPRP-A1 peptide alone was higher than that in the combination peptide group, suggesting the proliferation inhibition of the combination peptides was stronger than that with HPRP-A1 peptide alone.
The potential toxicity of HPRP-A1 with or without iRGD to the primary organs (heat, liver, spleen, lung and kidney) was determined by H&E assay at the end of the experiment. As shown in Fig. 9, there were no obvious changes in these organs after treatment with HPRP-A1 alone or together with iRGD.