1. Background
2. Methods
2.1 Materials
2.2 Cell lines
2.3 Viral vectors
2.4 Assessment of gene expression of Ad-vector transduced ovarian cancer cells
2.5 Assessment of cytoxicity of docetaxel and/or carboplatin to ovarian cancer cells
2.6 Evaluation of efficacy of combination of PNP-GDEPT with docetaxel and/or carboplatin
2.6.1 Clonogenic assays to assess cytotoxicity of different treatments
2.6.2 Evaluation of therapeutic interactions between modalities
2.7 Assessment of apoptosis in response to different treatments
2.8 Cell-cycle analysis of treated cells
2.9 Protein expression by Western blot analyses
2.10 Analysis of PNP-GDEPT treated samples by shot gun proteomics
2.10.1 Protein identification after mass spectrometry
3. Results
3.1 Efficiency of Ad-transduction in different ovarian cancer cell lines
3.2 Bystander effects of PNP-GDEPT correlate with the efficiency of gene transduction
3.3 Proteins implicated in actions of Ad-PNP GDEPT in ovarian cancer cells
Name of the Protein Identified | Hit Value (untreated/treated) | Ratio Transformed log (untreated:treated)1
|
---|---|---|
Keratin 5 | 13/0 | 19,306 |
Keratin 77 | 12/0 | 18,502 |
Cadherin 6 (K-cadherin) | 7/0 | 14,999 |
Desmoplakin | 6/0 | 14,037 |
Plakoglobin | 3/0 | 10,000 |
Spondin 1 | 3/0 | 10,000 |
Dynactin 1 | 2/0 | 7,925 |
Agrin | 2/0 | 7,925 |
Filaggrin | 2/0 | 7,925 |
Karyopherin alpha1 | 2/0 | 7,925 |
Antiquitin (ALDH7A1) | 2/0 | 7,925 |
Epoxide hydrolase | 2/0 | 7,925 |
Insulysin | 2/0 | 7,925 |
BRI3 binding protein (Cervical cancer 1 proto-oncogene-binding protein KG19) | 3/0 | 10,000 |
Ribosomal protein L4 | 5/0 | 12,924 |
Eukaryotic translation initiation factor 3 subunit 2 (eIF-3 beta) | 3/0 | 10,000 |
Mitochondrial trifunctional protein, beta subunit | 3/0 | 10,000 |
Polymerase (RNA) II (DNA directed) polypeptide E | 3/0 | 10,000 |
Dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate complex) | 5/0 | 12,924 |
v-ral simian leukemia viral oncogene homolog B (ras related; GTP binding protein) (RalB) | 2/0 | 7,925 |
Name of the Protein Identified | Hit Value (untreated/treated) | Ratio Transformed log (untreated:treated)1
|
---|---|---|
Purine nucleoside phosphorylase [E. coli]
| 0/37 | 26,239 |
Poly (ADP-ribose) polymerase (PARP) | 0/4 | 11,609 |
Progesterone receptor membrane component | 0/3 | 10,000 |
Angiotensinogen precursor (Serpin A8) | 0/3 | 10,000 |
Rab13 | 0/3 | 10,000 |
DEAD Box polypeptide | 0/2 | 7,925 |
3.4 Effects of docetaxel or carboplatin treatment on ovarian cancer cell lines
3.5 PNP-GDEPT, docetaxel and/or carboplatin act synergistically in ovarian cancer cells in vitro
Modalities | Cell Line | Combination Index (CI1) | Drug Alone (×IC50) | Dose Reduction Index (×IC50) | R3
| Effect4
| ||||
---|---|---|---|---|---|---|---|---|---|---|
IC2 50
| PNP | Doc | Car | PNP | Doc | Car | ||||
Docetaxel
Carboplatin
| SKOV5
| 0.96 | - | 5.01 | 1.34 | - | 2.7 | 1.68 | 0.98 | Additive
+
|
OVCAR6
| 0.89 | - | 2.76 | 2.16 | - | 2.1 | 2.44 | 0.99 | Additive
+
| |
PNP-GDEPT
Docetaxel
| SKOV | 0.43 | 8.22 | 5.01 | - | 6.12 | 3.7 | - | 0.98 | Synergy
+++
|
OVCAR | 0.21 | 2.03 | 2.76 | - | 7.97 | 10.82 | - | 0.93 | Strong Synergy
++++
| |
PNP-GDEPT
Carboplatin
| SKOV | 0.29 | 8.22 | - | 1.34 | 12.36 | - | 4.7 | 0.97 | Strong Synergy
++++
|
OVCAR | 0.28 | 2.03 | - | 2.16 | 5.66 | - | 9.08 | 0.96 | Strong Synergy ++++ | |
GDEPT Docetaxel
Carboplatin
| SKOV | 0.37 | 8.22 | 5.01 | 1.34 | 13.96 | 8.51 | 5.3 | 0.96 | Strong Synergy
++++
|
OVCAR | 0.11 | 2.03 | 2.76 | 2.16 | 19.89 | 27.02 | 31.9 | 0.94 | Very Strong Synergy
+++++
|