Preclinical evidence of the enhanced effectiveness of combined rapamycin and AICAR in reducing kidney cancer


Abstract

Loss of Von Hippel-Lindau in renal carcinoma cells results in upregulation of the activity of hypoxia-inducible factor (HIF-α), a major transcription factor involved in kidney cancer. Rapamycin as mammalian target of rapamycin inhibitor and 5-aminoimidazole-4-carboxamide-riboside (AICAR) as AMPK activator are used separately to treat cancer patients. In the current study, the possible additive effect of drug combinations in reducing kidney tumorigenesis was investigated. Treatment with drug combinations significantly decreased cell proliferation, increased cell apoptosis, and abolished Akt phosphorylation and HIF-2α expression in renal cell carcinoma cells, including primary cells isolated from kidney cancer patients. Significant decreases in cell migration and invasion were detected using drug combinations. Drug combinations effectively abolished binding of HIF-2α to the Akt promoter and effected formation of the DNA-protein complex in nuclear extracts from 786-O cells, as demonstrated using electromobility shift assay and examination of Akt promoter activity. Importantly, we tested the effect of each drug and the combined drugs on kidney tumor size in the nude mouse model. Our data show that treatment with rapamycin, AICAR, and rapamycin+AICAR decreased tumor size by 38%, 36%, and 80%, respectively, suggesting that drug combinations have an additive effect in reducing tumor size compared with use of each drug alone. Drug combinations effectively decreased cell proliferation, increased apoptotic cells, and significantly decreased p-Akt, HIF-2α, and vascular endothelial growth factor expression in tumor kidney tissues from mice. These results show for the first time that drug combinations are more effective than single drugs in reducing kidney tumor progression. This study provides important evidence that may lead to the initiation of pre-clinical trials in patients with kidney cancer.

Keywords: 5-aminoimidazole-4-carboxamide-riboside (AICAR); Von Hippel-Lindau; hypoxia-inducible factor (HIF-α); kidney cancer; rapamycin.

Figures

Figure 1
Figure 1
Significant increase in number of apoptotic cells is dependent on drug concentration and time exposure in 786‐O cells. Serial concentrations of (A) rapamycin (0–100?nm), (B) AICAR (0–10?mm), and (C) drug combinations (0/0–2/20, 4/40 and 10/100,?mm/nm) show that increase in number of apoptotic cells is dose‐dependent using annexin V‐FITC conjugated to PI by flow cytometry. In addition, treatment of the cells with drug combinations for 24, 48, and 72?h (D) shows that increase in number of apoptotic cells is time‐dependent. Apoptotic data were confirmed in cells by measuring apoptotic protein expression. Lysates from cells treated with rapamycin (20?nm), AICAR (2?mm) or rapamycin+AICAR (20?nm/2?mm) for 72?h were subjected to Western blot analysis to measure PARP and caspase 3 cleavages. (E) Significant increase was detected in cleavage of PARP at 85 kDa and caspase 3 at 22 and 17?kDa in 786‐O cells treated with drug combinations compared with cells treated with each drug alone for 72?h. GAPDH was used as a loading control. Data represent mean?±?SEM (n?=?4). Significant difference from control tissues is indicated by *P?<?0.01.
Figure 2
Figure 2
Decrease in cell proliferation by drug combinations is dependent on the drug concentration and duration of exposure in 786‐O cells. Serial concentrations of (A) rapamycin (0–100?nm), (B) AICAR (0–10?mm), and (C) drug combinations (0/0–2/20, 4/40 and 10/100,?mm/nm) show that the decrease in cell proliferation is dose‐dependent using the 3H‐thymidine incorporation assay. In addition, treatment of the cells with drug combinations for 24, 48, and 72?h (D) show that the significant decrease in cell proliferation is time‐dependent. Cell proliferation data were confirmed in cells by measuring proliferative protein expression. Lysates from cells treated with rapamycin (20?nm), AICAR (2?mm) or rapamycin+AICAR (20?nm/2?mm) for 72?h were subjected to Western blot analysis to measure PCNA and cyclin D1. (E) A significant decrease in expression of PCNA and cyclin D1 was detected in cells treated with a single drug, whereas drug combinations abolished the expression of both proteins detected in cells, providing evidence of the additive effect of drug combinations in reducing cell proliferation. A combination of drugs abolished Akt survival kinase and blocked HIF‐2α and VEGF in 786‐O cells. Cells were treated with rapamycin (20?nm), AICAR (2?mm) or drug combination (20?nm rapamycin/2?mm AICAR) for 72?h. Cell lysates were subjected to Western blot analysis to measure p‐Akt, HIF‐2α, and VEGF expression. (F, G) A significant decrease in p‐Akt, HIF‐2α, and VEGF expression was seen in cells treated with single drug; however, the additive effect of drug combinations showed complete abolishment in HIF‐2α and VEGF as well as in phosphorylation of Akt at Ser473 expression compared with cells treated with single drug and control cells. GAPDH was used as a loading control. Further evidence of the effect of drug combinations on HIF‐2α function was measured by luciferase assay. (H) The additive effect of drug combinations caused a more than 90% decrease in the promoter activity of HIF‐2α compared with control cells using the Luciferase Reporter Assay System normalized to Renilla activity and measured by a luminometer. Data represent mean?±?SEM (n?=?4). Significant difference from control tissues is indicated by *P?<?0.01.
Figure 3
Figure 3
Combination of drugs reduces the binding of HIF‐2α to the Akt promoter element. (A) EMSA analysis of DNA probe corresponding to the putative HIF‐2α binding site in the Akt promoter was performed. Labeled probes were incubated with nuclear extracts isolated from 786‐O cells treated with rapamycin, AICAR or rapamycin+AICAR showed significant decrease binding of HIF‐2α to the Akt promoter. (B) Specificity of binding of HIF‐α to the Akt promoter was confirmed by adding HIF‐1α, HIF‐2α or IgG antibody to the reaction mixture. (C) Further confirmation of the binding site of HIF‐2 to Akt was obtained by incubation of the nuclear extract with mutated HIF‐2α probe. Data confirmed that HIF‐2α but not HIF‐1α was a part of the DNA/protein complex, and that the mutations in the binding sequence of HIF‐2 to Akt prevent the binding of HIF‐2 to the DNA/protein complex. These data provide new evidence that HIF2α is major transcription factor regulating cell survival kinase Akt.
Figure 4
Figure 4
Drug combinations significantly decreased cell migration and cell invasion of kidney cancer cells. 786‐O cells treated with rapamycin (20?nm), AICAR (2?mm) or a drug combination (20?nm rapamycin/2?mm AICAR) for 72?h were added into the upper well chamber of a 96‐well plate. (A, B) Cells treated with drug combination have a significantly low number of migrated cells and (C, D) invaded cells compared with cells treated with a single drug or control cells. The total number of migrated and invaded cells was counted using counting software and the images of migrated or invaded cells were taken using a Nikon light inverted microscope. Data represent mean?±?SEM (n?=?4). Significant difference from control tissues is indicated by *P?<?0.01.
Figure 5
Figure 5
Drug combination and single‐drug treatment caused no change in BW and no toxicity in treated mice. (A) No changes in BW were detected between four groups of mice during 4?weeks of treatment with single or a drug combination. (B) Renal injury was determined by measuring the urinary excretion of GST in mice following treatment with rapamycin, AICAR or combination of rapamycin+AICAR for 4?weeks. GST activity is represented as μmol/mL/min. There were no significant changes between four groups of mice, indicating the absence of toxicity of treatment with a single drug or drug combinations. An additive effect of drug combinations resulted in significant decreases in kidney tumor size. (C) One million 786‐O cells expressing luciferase were injected in the kidney capsule of nude mice. Ten minutes prior to imaging, animals were injected with luciferin and the bioluminescent image signals were recorded using the Xenogen IVIS System. Representative images show the tumor in each of four groups. (D) Total photon radiance (p/s/cm2/sr) in the tumor area was measured by bioluminescent imaging and blotted to show the differences in photon flux/s between four groups. Photon data were calculated and the percentage of changes in tumor sizes between the four groups was summarized. (E) Data show that treatment with drug combinations resulted in a decreased tumor radiance of 75%, compared with 32.2% and 38% in mice treated with rapamycin and AICAR, respectively. Values represent the mean?±?SEM (n?=?5). Significant difference from control mice is indicated by *P?<?0.01.
Figure 6
Figure 6
Drug combinations are more effective in reducing kidney tumorigenesis. A macroscopic view of all mice kidneys was examined and tumor size was measured along three axes as cm3 (length, width, and height). (A) Images of kidney from four groups of mice showing the tumor size in each group. (B) Data calculated from the tumor size from each group show that treatment with drug combination resulted in an 80% reduction in tumor size, treatment with rapamycin a 38% reduction, and treatment with AICAR a 36% reduction compared with control mice, further confirming the reduction of tumor size measured by bioluminescent imaging. Values represent the mean?±?SEM (n?=?5). Significant difference from control mice is indicated by *P?<?0.01.
Figure 7
Figure 7
The additive effect of drug combinations strongly increased cell apoptosis, decreased cell proliferation, and abolished p‐Akt. (A) TUNEL assay was performed in kidney sections of four groups of mice and pictures taken with a light microscope. (A, B) Positive‐stained nucleus for TUNEL shows a significant increase in the number of positive cells in mice treated with drug combinations and less in groups treated with a single drug, compared with control mice. In addition, tumor kidney homogenates from all four mice groups were subjected to Western blot analysis. (C) Apoptosis proteins including cleavages of PARP and caspase 3 were measured and confirmed TUNEL data showing a significant increase of apoptosis in mice treated with drug combinations compared with mice treated with a single drug and control mice. (D) Hematoxylin and eosin staining in a tumor kidney section shows that ccRCC formed within the tumor tissue in all four groups. (E, F) Staining of the proliferative protein Ki67 shows a significant decrease in the number of positive‐stained nuclei in kidney tumors from mice treated with drug combinations compared with mice treated with either drug alone. (G) Western blot analysis performed in tumor homogenates from four groups of mice shows abolishment of PCNA expression, with a significant decrease in cyclin D1 in mice treated with drug combinations than in mice treated with a single drug and control mice. The scale bar size used for all images is 20?μm. Values represent the mean?±?SEM (n?=?5). Significant difference from control mice is indicated by *P?<?0.01.
Figure 8
Figure 8
Additive effect of drug combinations strongly decreased HIF‐2α and VEGF expression. (A) Immunoperoxidase staining for VEGF and (B) immunofluorescence staining for HIF‐2α both show a significant decrease in protein staining in mice treated with drug combinations and mice treated with a single drug. (C) Western blot data show that drug combinations nearly abolished p‐Akt and significantly decreased expression of HIF‐2α and VEGF protein expression in tumor tissues compared with tumors from mice treated with a single drug and control mice. The scale bar size used for all images is 20?μm.
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