BEVACIZUMAB EFFECT ON TOPOTECAN PHARMACOKINETICS ...

non-favorable sites [109]. So developing new agents or new strategies to increase the
current agent efficacy is urgently needed for the treatment of RMS, especially ARMS.
1.3.2 Characteristics of Rh30 cell line
The Rh30 cell line we chose for the tumor xenograft model is highly complex in
genetic profile and overexpression of VEGF, VEGFR and MMP-2, which is associated
with more aggressive invasion behavior and is characterized as an ARMS subtype.
ARMS is genetically characterized by reciprocal translocations that generate the
fusion gene PAX3-FOXO1A (PF) or PAX7-FOXO1A. The Rh30 cell line is characterized
as ARMS cell line and positive for PAX3-FOXO1A fusion gene. By applying any of
several techniques such as spectral karyotyping, fluorescence in situ hybridization,
comparative genomic hybridization (CGH), and microarray CGH, Rodriguez-Perales et
al. [113] found Rh30 had a wide range of chromosome numbers, more than 50
chromosome rearrangements, amplification of the hybrid gene, 24 DNA changes and 21
gene copy changes. p53 is also mutated in the Rh30 cell line with a codon 280 A to T
transversion (arginine to serine) [114].
VEGF is overexpressed in the Rh30 cell line, which provides a rationale for our
selection of it for anti-VEGF therapy. Moreover, the overexpression of MMP-2 renders
Rh30 more invasive than ERMS cell lines [115]. Onisto et al. studied the MMP-2,
MT1-MMP, TIMP-2, VEGF and VEGF receptors in several ARMS, ERMS and
undifferentiated cell lines and tried to correlate the metastatic potential with the matrix
metalloproteinases (MMPs) and angiogenesis-related factors. The results showed that
Rh30 had increased expression levels of both VEGF 165 and VEGF 121 isoforms as well as
VEGFR-1 at mRNA and protein. Also the elevated expression of MMP-2 at mRNA and
protein levels correlated with the invasive behavior in vitro.
Comparing the ERMS cell line RD, Rh30 cell line is more sensitive to
camptothecin and its derivative TPT [116], and the effective schedules of exposure of
Rh30 xenografts to TPT have been identified [117]. By high-throughput screening, Zeng
et al. showed that camptothecin and TPT inhibited cell proliferation and induced cell
apoptosis more effectively in Rh30 than in RD (ERMS) cells. Ectopic expression of the
fusion protein PF in RD cells significantly increased their sensitivity to camptothecin and
TPT, whereas siRNA knockdown of PF decreased the sensitivity of Rh30 cells to
camptothecin and TPT [116]. Furthermore, Dr. Stewart and his collaborators showed that
TPT was highly schedule-dependent in Rh30 cells and that only daily exposure of TPT
achieved complete regressions of Rh30 xenografts [117]. The dose and schedule for
complete regression in Rh30 xenografts was to give TPT daily 0.6mg/kg for 2 weeks,
repeated every 21 days for three cycles. However, preclinical and clinical studies showed
that rhabdomyosarcoma is resistant to TPT [118, 119]. To overcome the drug resistant
and increase its efficacy, one of the approaches is to increase its intratumoral
concentration. Thus, we used Rh30 orthotopic rhabdomyosarcoma xenograft as a tumor
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