82
3 In-Tether Chiral Center Induced Helical Peptide Modulators …
3.2.5 Transcriptome Analysis of PhR-Treated Cells
To obtain an overview of the genes directly affected by activation of p53 pathway,
genome-wide mRNA microarrays were performed for PA-1 cells treated with PhR.
Compared with untreated cells, a total of 285 downregulated and 93 upregulated
genes were detected (fold change > 2) and clustered in PA-1 cells exhibiting the
activation of p53 pathway (Fig. 3.20a–d). Despite this result, gene ontology analysis revealed the number of genes with changed expression levels is involved in
different pathways, such as in the signaling pathway regulating the pluripotency
of stem cells, the p53 signaling pathway, the Ras signaling pathway, the PI3k-Akt
signaling pathway, and the Rap signaling pathway, and so on (Fig. 3.20e). These
genes play important roles in regulating the activity of cancer cells and the results
are summarized in Fig. 3.20f. Together, these results demonstrate that PhR peptides
act as potent modulators of p53-MDM2/X pathway and activate cell apoptosis.
3.2.6 PhR Suppresses Tumor Growth in Vivo in PA-1
Xenograft Model via Reactivation of the P53 Pathway
PhR was examined for its efficacy in a human cancer xenograft model carrying p53wt
and elevated levels of MDM2 proteins, namely the PA-1 human xenograft model. As
shown in Fig. 3.21, PhR resulted in statistically significant tumor growth inhibition
(TGI) in the MDM2-amplified human teratocarcinoma xenograft model PA-1. When
dosed for 3 weeks at 10 mg/Kg every other day with intratumoral injections, PhR
induced a TGI of 70% (Fig. 3.21a). In comparison, the selective MDM2 smallmolecule inhibitor nutlin-3a (administrated every other day at doses of 10 mg/Kg)
resulted in a TGI of 30%, which is much less effective compared to PhR. Actually, the
drug concentration in the tumor section is much higher for nutlin-3a than PhR for this
dosage [M.W.: PhR (1640) VS nutlin-3a (581)]. These results are confirmed by tumor
weight and tumor volume measurements (Fig. 3.21b, c). At the same time, the weight
of the mice showed no significant change (Fig. 3.21d), suggesting that our peptides
have little side effect with respect to mouse growth. Furthermore, the mortality of
the mice during the survival study was shown as survival curves (Fig. 3.21e), which
indicate that PhR was able to prolong the lives of the mice. Especially, no mice died
during the treatment period in PhR group while three in ten mice died in the nutlin-3a
group. To assess the pharmacokinetics and duration time of PhR within the tumor,
Cy3-labeled PhR was treated by intratumoral injections and imaged at different time
points (0 hour, 5 min, 30 min, 2 hours, 6 hours, and 24 hours) using the IVIS Lumina
II small animal in vivo optical imaging system. The optical signal intensity of the
PhR-Cy3 peptide remained consistently without apparent diminishing for 24 hours
(Fig. 3.21f and g). Furthermore, the ex vivo fluorescence results from tumors and
major organs were consistent with previous reports on the biodistribution of peptides
3 In-Tether Chiral Center Induced Helical Peptide Modulators …
3.2.5 Transcriptome Analysis of PhR-Treated Cells
To obtain an overview of the genes directly affected by activation of p53 pathway,
genome-wide mRNA microarrays were performed for PA-1 cells treated with PhR.
Compared with untreated cells, a total of 285 downregulated and 93 upregulated
genes were detected (fold change > 2) and clustered in PA-1 cells exhibiting the
activation of p53 pathway (Fig. 3.20a–d). Despite this result, gene ontology analysis revealed the number of genes with changed expression levels is involved in
different pathways, such as in the signaling pathway regulating the pluripotency
of stem cells, the p53 signaling pathway, the Ras signaling pathway, the PI3k-Akt
signaling pathway, and the Rap signaling pathway, and so on (Fig. 3.20e). These
genes play important roles in regulating the activity of cancer cells and the results
are summarized in Fig. 3.20f. Together, these results demonstrate that PhR peptides
act as potent modulators of p53-MDM2/X pathway and activate cell apoptosis.
3.2.6 PhR Suppresses Tumor Growth in Vivo in PA-1
Xenograft Model via Reactivation of the P53 Pathway
PhR was examined for its efficacy in a human cancer xenograft model carrying p53wt
and elevated levels of MDM2 proteins, namely the PA-1 human xenograft model. As
shown in Fig. 3.21, PhR resulted in statistically significant tumor growth inhibition
(TGI) in the MDM2-amplified human teratocarcinoma xenograft model PA-1. When
dosed for 3 weeks at 10 mg/Kg every other day with intratumoral injections, PhR
induced a TGI of 70% (Fig. 3.21a). In comparison, the selective MDM2 smallmolecule inhibitor nutlin-3a (administrated every other day at doses of 10 mg/Kg)
resulted in a TGI of 30%, which is much less effective compared to PhR. Actually, the
drug concentration in the tumor section is much higher for nutlin-3a than PhR for this
dosage [M.W.: PhR (1640) VS nutlin-3a (581)]. These results are confirmed by tumor
weight and tumor volume measurements (Fig. 3.21b, c). At the same time, the weight
of the mice showed no significant change (Fig. 3.21d), suggesting that our peptides
have little side effect with respect to mouse growth. Furthermore, the mortality of
the mice during the survival study was shown as survival curves (Fig. 3.21e), which
indicate that PhR was able to prolong the lives of the mice. Especially, no mice died
during the treatment period in PhR group while three in ten mice died in the nutlin-3a
group. To assess the pharmacokinetics and duration time of PhR within the tumor,
Cy3-labeled PhR was treated by intratumoral injections and imaged at different time
points (0 hour, 5 min, 30 min, 2 hours, 6 hours, and 24 hours) using the IVIS Lumina
II small animal in vivo optical imaging system. The optical signal intensity of the
PhR-Cy3 peptide remained consistently without apparent diminishing for 24 hours
(Fig. 3.21f and g). Furthermore, the ex vivo fluorescence results from tumors and
major organs were consistent with previous reports on the biodistribution of peptides