Limits...
Efficacy of tomato concentrates in mouse models of dyslipidemia and cancer.

Chattopadhyay A, Grijalva V, Hough G, Su F, Mukherjee P, Farias-Eisner R, Anantharamaiah GM, Faull KF, Hwang LH, Navab M, Fogelman AM, Reddy ST - Pharmacol Res Perspect (2015)

Bottom Line: The same dose in a human would require three cups of tomato powder three times daily.To reduce the volume, we sought a method to concentrate 6F.Remarkably, extracting the transgenic freeze-dried tomato overnight in ethyl acetate with 5% acetic acid resulted in a 37-fold reduction in the amount of transgenic tomato needed for biologic activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Department of Medicine, David Geffen School of Medicine at UCLA Los Angeles, California, 90095-1736.

ABSTRACT
We previously reported that adding freeze-dried tomato powder from transgenic plants expressing the apolipoprotein A-I mimetic peptide 6F at 2.2% by weight to a Western diet (WD) ameliorated dyslipidemia and atherosclerosis in mice. The same dose in a human would require three cups of tomato powder three times daily. To reduce the volume, we sought a method to concentrate 6F. Remarkably, extracting the transgenic freeze-dried tomato overnight in ethyl acetate with 5% acetic acid resulted in a 37-fold reduction in the amount of transgenic tomato needed for biologic activity. In a mouse model of dyslipidemia, adding 0.06% by weight of the tomato concentrate expressing the 6F peptide (Tg6F) to a WD significantly reduced plasma total cholesterol and triglycerides (P < 0.0065). In a mouse model of colon cancer metastatic to the lungs, adding 0.06% of Tg6F, but not a control tomato concentrate (EV), to standard mouse chow reduced tumor-associated neutrophils by 94 ± 1.1% (P = 0.0052), and reduced tumor burden by two-thirds (P = 0.0371). Adding 0.06% of either EV or Tg6F by weight to standard mouse chow significantly reduced tumor burden in a mouse model of ovarian cancer; however, Tg6F was significantly more effective (35% reduction for EV vs. 53% reduction for Tg6F; P = 0.0069). Providing the same dose of tomato concentrate to humans would require only two tablespoons three times daily making this a practical approach for testing oral apoA-I mimetic therapy in the treatment of dyslipidemia and cancer.

No MeSH data available.


Related in: MedlinePlus

Ethyl acetate with 5% acetic acid (but not 60% ethanol) efficiently concentrates the 6F peptide produced in transgenic tomatoes. Freeze-dried transgenic tomatoes expressing β-glucuronidase (EV), or expressing the 6F peptide (Tg6F) were extracted overnight at room temperature in either ethyl acetate/acetic acid, or in aqueous ethanol as described in Materials and Methods. The ethyl acetate/acetic acid extract was dried under argon; the aqueous ethanol extract was dried under vacuum as described in Materials and Methods. The remaining solids were suspended in water and analyzed on 18% SDS PAGE gels as described in Materials and Methods. (A) Image of the Sypro Ruby-stained gel of the tomato concentrates prepared with ethyl acetate/acetic acid (100 μg or 200 μg of protein per lane). (B) Image of the Sypro Ruby-stained gel of tomato concentrates prepared with aqueous ethanol (100 μg protein per lane). Authentic chemically synthesized 6F peptide at 5, 10, or 20 μg per lane was included as standards in (A) and (B). (C) Summary of the results of LC/MS analysis of the trypsin digest of the authentic 6F standard (6F Standard, top of panel C, 5 μg), the trypsin digest of the corresponding band from the Tg6F lane (Tg6F, middle of panel C, 100 μg), and the trypsin digest of the corresponding band from the EV lane (EV, bottom of C, 100 μg) performed as described in Materials and Methods. To simplify data presentation, the spectra collected between 13 and 25 min from all three chromatograms were averaged. All the tryptic peptides eluted within this window. The arrows point to signals identified on the basis of molecular weight concordance as being derived from 6F peptide: EFF (residues 16–18), found m/z 442.2 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 442.197 Da (monoisotopic, mi); DWLK (residues 1–4), found m/z 561.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 561.303 Da (mi); FFEK (residues 10–13), found m/z 570.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 570.292 Da (mi); AFYDK (residues 5–9), found m/z 643.3 (detected in Tg6F, but not in 6F Standard or EV), calculated for MH+ 643.308 Da (mi); FKEFF (residues 14–18), found m/z 717.4 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 717.361 Da (mi); DWLKAFYDK (residues 1–9), found m/z 1185.6 and 1185.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1185.594 Da (mi); AFYDKFFEK (residues 5–13), found m/z 1194.6 and 1194.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1194.583 Da (mi). These partially overlapping tryptic peptides provided 100% sequence coverage for both the 6F standard and the Tg6F sample. These results are representative of two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492730&req=5

fig01: Ethyl acetate with 5% acetic acid (but not 60% ethanol) efficiently concentrates the 6F peptide produced in transgenic tomatoes. Freeze-dried transgenic tomatoes expressing β-glucuronidase (EV), or expressing the 6F peptide (Tg6F) were extracted overnight at room temperature in either ethyl acetate/acetic acid, or in aqueous ethanol as described in Materials and Methods. The ethyl acetate/acetic acid extract was dried under argon; the aqueous ethanol extract was dried under vacuum as described in Materials and Methods. The remaining solids were suspended in water and analyzed on 18% SDS PAGE gels as described in Materials and Methods. (A) Image of the Sypro Ruby-stained gel of the tomato concentrates prepared with ethyl acetate/acetic acid (100 μg or 200 μg of protein per lane). (B) Image of the Sypro Ruby-stained gel of tomato concentrates prepared with aqueous ethanol (100 μg protein per lane). Authentic chemically synthesized 6F peptide at 5, 10, or 20 μg per lane was included as standards in (A) and (B). (C) Summary of the results of LC/MS analysis of the trypsin digest of the authentic 6F standard (6F Standard, top of panel C, 5 μg), the trypsin digest of the corresponding band from the Tg6F lane (Tg6F, middle of panel C, 100 μg), and the trypsin digest of the corresponding band from the EV lane (EV, bottom of C, 100 μg) performed as described in Materials and Methods. To simplify data presentation, the spectra collected between 13 and 25 min from all three chromatograms were averaged. All the tryptic peptides eluted within this window. The arrows point to signals identified on the basis of molecular weight concordance as being derived from 6F peptide: EFF (residues 16–18), found m/z 442.2 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 442.197 Da (monoisotopic, mi); DWLK (residues 1–4), found m/z 561.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 561.303 Da (mi); FFEK (residues 10–13), found m/z 570.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 570.292 Da (mi); AFYDK (residues 5–9), found m/z 643.3 (detected in Tg6F, but not in 6F Standard or EV), calculated for MH+ 643.308 Da (mi); FKEFF (residues 14–18), found m/z 717.4 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 717.361 Da (mi); DWLKAFYDK (residues 1–9), found m/z 1185.6 and 1185.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1185.594 Da (mi); AFYDKFFEK (residues 5–13), found m/z 1194.6 and 1194.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1194.583 Da (mi). These partially overlapping tryptic peptides provided 100% sequence coverage for both the 6F standard and the Tg6F sample. These results are representative of two experiments.

Mentions: Twenty-three micrograms of 6F were found in 100 μg of tomato concentrate protein prepared with ethyl acetate with 5% acetic acid (Fig.1A). In contrast, concentrates prepared with 60% ethanol did not contain detectable amounts of 6F (Fig.1B). The presence of the 6F peptide was confirmed in the Tg6F concentrate (but not in the EV concentrate) by reverse-phase LC and mass spectrometry (Fig.1C). Based on these results, all subsequent experiments utilized tomato concentrates prepared with ethyl acetate with 5% acetic acid.


Efficacy of tomato concentrates in mouse models of dyslipidemia and cancer.

Chattopadhyay A, Grijalva V, Hough G, Su F, Mukherjee P, Farias-Eisner R, Anantharamaiah GM, Faull KF, Hwang LH, Navab M, Fogelman AM, Reddy ST - Pharmacol Res Perspect (2015)

Ethyl acetate with 5% acetic acid (but not 60% ethanol) efficiently concentrates the 6F peptide produced in transgenic tomatoes. Freeze-dried transgenic tomatoes expressing β-glucuronidase (EV), or expressing the 6F peptide (Tg6F) were extracted overnight at room temperature in either ethyl acetate/acetic acid, or in aqueous ethanol as described in Materials and Methods. The ethyl acetate/acetic acid extract was dried under argon; the aqueous ethanol extract was dried under vacuum as described in Materials and Methods. The remaining solids were suspended in water and analyzed on 18% SDS PAGE gels as described in Materials and Methods. (A) Image of the Sypro Ruby-stained gel of the tomato concentrates prepared with ethyl acetate/acetic acid (100 μg or 200 μg of protein per lane). (B) Image of the Sypro Ruby-stained gel of tomato concentrates prepared with aqueous ethanol (100 μg protein per lane). Authentic chemically synthesized 6F peptide at 5, 10, or 20 μg per lane was included as standards in (A) and (B). (C) Summary of the results of LC/MS analysis of the trypsin digest of the authentic 6F standard (6F Standard, top of panel C, 5 μg), the trypsin digest of the corresponding band from the Tg6F lane (Tg6F, middle of panel C, 100 μg), and the trypsin digest of the corresponding band from the EV lane (EV, bottom of C, 100 μg) performed as described in Materials and Methods. To simplify data presentation, the spectra collected between 13 and 25 min from all three chromatograms were averaged. All the tryptic peptides eluted within this window. The arrows point to signals identified on the basis of molecular weight concordance as being derived from 6F peptide: EFF (residues 16–18), found m/z 442.2 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 442.197 Da (monoisotopic, mi); DWLK (residues 1–4), found m/z 561.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 561.303 Da (mi); FFEK (residues 10–13), found m/z 570.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 570.292 Da (mi); AFYDK (residues 5–9), found m/z 643.3 (detected in Tg6F, but not in 6F Standard or EV), calculated for MH+ 643.308 Da (mi); FKEFF (residues 14–18), found m/z 717.4 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 717.361 Da (mi); DWLKAFYDK (residues 1–9), found m/z 1185.6 and 1185.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1185.594 Da (mi); AFYDKFFEK (residues 5–13), found m/z 1194.6 and 1194.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1194.583 Da (mi). These partially overlapping tryptic peptides provided 100% sequence coverage for both the 6F standard and the Tg6F sample. These results are representative of two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492730&req=5

fig01: Ethyl acetate with 5% acetic acid (but not 60% ethanol) efficiently concentrates the 6F peptide produced in transgenic tomatoes. Freeze-dried transgenic tomatoes expressing β-glucuronidase (EV), or expressing the 6F peptide (Tg6F) were extracted overnight at room temperature in either ethyl acetate/acetic acid, or in aqueous ethanol as described in Materials and Methods. The ethyl acetate/acetic acid extract was dried under argon; the aqueous ethanol extract was dried under vacuum as described in Materials and Methods. The remaining solids were suspended in water and analyzed on 18% SDS PAGE gels as described in Materials and Methods. (A) Image of the Sypro Ruby-stained gel of the tomato concentrates prepared with ethyl acetate/acetic acid (100 μg or 200 μg of protein per lane). (B) Image of the Sypro Ruby-stained gel of tomato concentrates prepared with aqueous ethanol (100 μg protein per lane). Authentic chemically synthesized 6F peptide at 5, 10, or 20 μg per lane was included as standards in (A) and (B). (C) Summary of the results of LC/MS analysis of the trypsin digest of the authentic 6F standard (6F Standard, top of panel C, 5 μg), the trypsin digest of the corresponding band from the Tg6F lane (Tg6F, middle of panel C, 100 μg), and the trypsin digest of the corresponding band from the EV lane (EV, bottom of C, 100 μg) performed as described in Materials and Methods. To simplify data presentation, the spectra collected between 13 and 25 min from all three chromatograms were averaged. All the tryptic peptides eluted within this window. The arrows point to signals identified on the basis of molecular weight concordance as being derived from 6F peptide: EFF (residues 16–18), found m/z 442.2 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 442.197 Da (monoisotopic, mi); DWLK (residues 1–4), found m/z 561.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 561.303 Da (mi); FFEK (residues 10–13), found m/z 570.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 570.292 Da (mi); AFYDK (residues 5–9), found m/z 643.3 (detected in Tg6F, but not in 6F Standard or EV), calculated for MH+ 643.308 Da (mi); FKEFF (residues 14–18), found m/z 717.4 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 717.361 Da (mi); DWLKAFYDK (residues 1–9), found m/z 1185.6 and 1185.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1185.594 Da (mi); AFYDKFFEK (residues 5–13), found m/z 1194.6 and 1194.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1194.583 Da (mi). These partially overlapping tryptic peptides provided 100% sequence coverage for both the 6F standard and the Tg6F sample. These results are representative of two experiments.
Mentions: Twenty-three micrograms of 6F were found in 100 μg of tomato concentrate protein prepared with ethyl acetate with 5% acetic acid (Fig.1A). In contrast, concentrates prepared with 60% ethanol did not contain detectable amounts of 6F (Fig.1B). The presence of the 6F peptide was confirmed in the Tg6F concentrate (but not in the EV concentrate) by reverse-phase LC and mass spectrometry (Fig.1C). Based on these results, all subsequent experiments utilized tomato concentrates prepared with ethyl acetate with 5% acetic acid.

Bottom Line: The same dose in a human would require three cups of tomato powder three times daily.To reduce the volume, we sought a method to concentrate 6F.Remarkably, extracting the transgenic freeze-dried tomato overnight in ethyl acetate with 5% acetic acid resulted in a 37-fold reduction in the amount of transgenic tomato needed for biologic activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Department of Medicine, David Geffen School of Medicine at UCLA Los Angeles, California, 90095-1736.

ABSTRACT
We previously reported that adding freeze-dried tomato powder from transgenic plants expressing the apolipoprotein A-I mimetic peptide 6F at 2.2% by weight to a Western diet (WD) ameliorated dyslipidemia and atherosclerosis in mice. The same dose in a human would require three cups of tomato powder three times daily. To reduce the volume, we sought a method to concentrate 6F. Remarkably, extracting the transgenic freeze-dried tomato overnight in ethyl acetate with 5% acetic acid resulted in a 37-fold reduction in the amount of transgenic tomato needed for biologic activity. In a mouse model of dyslipidemia, adding 0.06% by weight of the tomato concentrate expressing the 6F peptide (Tg6F) to a WD significantly reduced plasma total cholesterol and triglycerides (P < 0.0065). In a mouse model of colon cancer metastatic to the lungs, adding 0.06% of Tg6F, but not a control tomato concentrate (EV), to standard mouse chow reduced tumor-associated neutrophils by 94 ± 1.1% (P = 0.0052), and reduced tumor burden by two-thirds (P = 0.0371). Adding 0.06% of either EV or Tg6F by weight to standard mouse chow significantly reduced tumor burden in a mouse model of ovarian cancer; however, Tg6F was significantly more effective (35% reduction for EV vs. 53% reduction for Tg6F; P = 0.0069). Providing the same dose of tomato concentrate to humans would require only two tablespoons three times daily making this a practical approach for testing oral apoA-I mimetic therapy in the treatment of dyslipidemia and cancer.

No MeSH data available.


Related in: MedlinePlus